Welcome message

On behalf of the Brazilian Society for Cell Biology and the Scientific Committee, it is our pleasure to welcome you to São Paulo, at the XIV Meeting of the Brazilian Society for Cell Biology – XIV Congresso da Sociedade Brasileira de Biologia Celular. The 2008 Meeting offers a panoramic view of the research in cell biology: evolution and development, neuroscience, microbiology, stem cells, protein structure and function, extracellular matrix, cell motility, tumor cell biology, cell signaling and chromosome organization. Additionally, important aspects related to translational research in reproductive biology, degenerative diseases, cancer and transplants are discussed. The program also includes round tables in hot topics such as politics in research and development in cell biology and biotechnology & biosecurity, which will give the participants the opportunity to discuss with scientists committed with State and Federal Funding Agencies. We are also fortunate to have Professor Mina Bissell from Life Sciences Division, Berkeley, California as our Honorable Speaker in the closing ceremony and Professor Rafael Linden from Universidade Federal do Rio de Janeiro in the opening conference. The Meetings organized by the Brazilian Society of Cell Biology are always frequented by a large number of undergraduate and graduate students. In a special attention to them we offer eleven pre meeting courses in diverse basic topics in cell biology where they will have the opportunity to meet specialists in each one of these areas. In complement to that we stimulate debates on education in Cell Biology with the effort to develop new teaching methodologies and achieve the excellence in this field of the knowledge. We appreciate the efforts of our scientific community for selecting and attracting top quality scientists to participate in this meeting. We are grateful to the speakers and all the participants. We also thank the scientific committee for helping to select good quality studies for poster and oral presentations. Particularly, we would like to thank our commercial sponsors and the funding agencies that are supporting the Meeting. Finally, the organizing committee and sponsors have provided the funding, the venue and the environment for the XIV SBBC Meeting, it is now up to all of you to participate learn, interact and establish contacts with other researchers, and so, to ensure that the Meeting will be an outstanding success. Enjoy the Cell Biology in São Paulo!

Vilma Martins and Estela Bevilacqua

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livro.indd 1 7/7/2008 16:33:16 XIV Congresso da Sociedade Brasileira de Biologia Celular SBBC 27 a 30 de julho de 2008 Bourbon Convention Ibirapuera São Paulo, SP Brasil

REALIZAÇÃO Sociedade Brasileira de Biologia Celular

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livro.indd 2 7/7/2008 16:33:21 Comissão Organizadora

Profa Dra Estela Bevilacqua, USP (Presidente) Dra Vilma Regina Martins, Instituto Ludwig, SP (Presidente) Prof Dr Hernandes F Carvalho, UNICAMP (Presidente SBBC) Prof Dr Roger Chammas, USP Prof Dr Vivaldo Moura Neto, UFRJ Profa Dra Marimélia Porcionatto, UNIFESP Profa Dra Patrícia Gama, USP Profa Dra Chao Yun Irene Yan, USP

Comissão Científica

Profa Dra Estela Bevilacqua, USP Dra Vilma Regina Martins, Instituto Ludwig Prof Dr Hernandes F Carvalho, UNICAMP Prof Dr Roger Chammas, USP Profa Dra Marimélia Porcionatto, UNIFESP Profa Dra Patrícia Gama, USP Profa Dra Chao Yun Irene Yan, USP Profa Dra Silvana Allodi, UFRJ Prof Dr Luiz Eurico Nasciutti, UFRJ Profa Dra Telma M T Zorn, USP Profa Dra Marinilce F Santos, USP Profa Dra Edna T Kimura, USP Profa Dra Edna F Haapaleinen, UNIFESP Prof Dr Niels O C Saraiva, USP Profa Dr José Garcia Abreu, UFRJ Prof Dr Ivarne Tersariol, UNIFESP Profa Dra Enilza Espreafico, USP

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livro.indd 3 7/7/2008 16:33:21 Apoio Financeiro

Apoio Institucional e Financeiro

Apoio Institucional

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livro.indd 4 7/7/2008 16:33:24 Expositores e Patrocinadores Categoria Diamante

Categoria Esmeralda

Categoria Safira

Categoria Quartzo

Outros Patrocinadores

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livro.indd 5 7/7/2008 16:33:24 Program at a glance 07 Detailed program 09 Abstracts 30 Lectures 31 Round tables. 36 Posters A – Biology of reproduction 60 B – Cancer 91 C – Cell biophysics 116 D – Developmental biology 117 E – Education 130 F – Fundamental cell biology 131 G – Host parasite Interaction 173 H – Immune system 183 I – Metabolic diseases 190 J – Neurobiology 192 K – Plant cell biology 203 L – Stem cells 205 Index 214

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livro.indd 6 7/7/2008 16:33:24 1. 7

livro.indd 7 7/7/2008 16:33:25 1. 8

livro.indd 8 7/7/2008 16:33:26 Program 27/07/2008 ______Concurrent Pre meeting courses (at the meeting location)

10h00-13h00; 14h00-16h00

1.Evolution and development (Room Macuco)(5 h) Morning session: The invertebrates constitute an animal group with a great diversity of models in the embryo development with cellular and molecular mechanisms of control sometimes conservative, sometimes species- specific. This course has as goal the analysis in the main models of development, of the characteristics ofthe triblastic invertebrates, under an evolutionary perspective. The primary themes of the course will include: (1) the origin and differentiation of the systems; (2) the organization and space distribution of these systems in some invertebrate models, such as flatworms, mollusks, crustaceans and insects and; (3) the effect of some physical and chemical factors (ultraviolet radiation, detergents of domestic use and bisphenol) on the development of crustaceans. The course will use morphologic tools such as 3D-embryo analysis, serial sections at light microscopy and fluorescent and transmission electron micrographs. The afternoon session will broach the basic principles in Developmental Biology of vertebrates. Specifically, we will explore main signaling pathways involved in the embryogenesis of birds and mammals.

Shankar Srinivas Wellcome Trust Research Fellow & University Lecturer Department of Physiology Anatomy and Genetics, University of Oxford, London, UK [email protected]

Chao Yun Irene Yan Instituto de Ciências Biomédicas, Universidade de São Paulo, SP [email protected]

Cláudio Simon Departamento de Ciências Biológicas, Embriologia, Universidade Federal do Triangulo Mineiro, Uberlândia, MG [email protected]

Silvana Allodi Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, RJ [email protected]

Evelise Maria Nazari Laboratório de Reprodução e Desenvolvimento Animal, Universidade Federal de Santa Catarina, SC [email protected]

Pablo Elias Martinez Universidade Federal do Rio Grande do Sul, RS [email protected]

Dib Ammar Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, RJ [email protected]

10h00-12h00; 14h00-16h00

2.Scientific writing(Room Canário) The course will include: I. Introduction, Materials and Methods, Results and Discussion - what these chapters must contain; what should not be done. II. Writing and revising a text - better ways to compose and review a text; what should not be done.

Paulo Abrahamsohn Instituto de Ciências Biomédicas, Universidade de São Paulo, SP - [email protected] 1. 9

livro.indd 9 7/7/2008 16:33:26 3.Radioautography for soluble molecules (Room Sabiá) Autoradiography or Radioautography is a method for localization of chemical substances based on labeling with radioactive isotopes without changing the chemical properties of the labeled molecules. Paths of delivery, sites of deposition, specific binding in tissues and cells, metabolism and excretion can be identified and monitored. The goal of this course is to present the fundaments of this technique applied to detection of soluble compounds in tissues and cells.

Telma Zorn Instituto de Ciências Biomédicas, Universidade de São Paulo, SP [email protected]

Mauricio Soto-Suazo Escuela de Medicina, Universidad de Santiago de Chile [email protected]

4.Bioinformatics (Room Colibri) Helena Paula Brentani Laboratório de BioInformática – Hospital do Câncer LBHC, SP [email protected]

5.Epigenetics (Room Rouxinol 2) The main topics that will be discussed are: mechanisms and models; concept of epigenetics; main mechanisms of epigenetic regulation: DNA methylation, covalent modifications of histones, and polycomb-trithorax complex; transgeneration carcinogenesis; the epigentics inheritance; parental imprinting: mechanisms, models, and applications.

Hernandes F Carvalho Instituto de Biologia, Universidade Estadual de Campinas, SP [email protected]

6.Virus and cancer (Room Rouxinol 1) Ana Paula Lepique & Enrique Boccardo Instituto Ludwig de Pesquisa sobre o Câncer, SP [email protected]

7. Regulation of gene expression: iRNA and microRNA (Room Gaivota 3) In the last years an important role for non-coding small RNAs, including the microRNA (miRNA) and interference RNA (iRNA) family, as post transcriptional regulators for gene expression has been revealed. While the exogenous iRNA function in transcript silencing is recognized, the growing miRNA family is naturally present in cells. We will discuss in this course the biogenesis and expression of miRNA and the implications of its alterations as well as the roles of iRNA and miRNA in mammal cells, exploring the main technical procedures that involve these molecules.

Edna Kimura Instituto de Ciências Biomédicas, Universidade de São Paulo [email protected]

Murilo Vieira Geraldo Instituto de Ciências Biomédicas, Universidade de São Paulo [email protected]

8. Stem cells (Room Gaivota 2) The purpose of the course is to provide update information on biological properties of adult stem cells. Control and regulation of cell differentiation, immunomodulatory actions and clinical approaches of stem cells will be addressed as follows:

Immunomodulation roles of mesenchymal stem cells Niels Olsen Saraiva Câmara Instituto de Ciências Biomédicas, Universidade de São Paulo, SP niels@icb usp.br

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livro.indd 10 7/7/2008 16:33:26 The biology of a stem cell Patrícia Semedo Universidade Federal de São Paulo, UNIFESP, SP [email protected]

Regulation of the stem cell differentiation Fernando Henrique Lojudice da Silva Instituto de Química, Universidade de São Paulo, SP [email protected]

Stem cells and neurological disorders Beatriz Longo Monteiro Depto. de Fisiologia, Universidade Federal de São Paulo, SP [email protected]

9. Cytoskeleton and cell adhesion (Room Gaivota 1) The adhesion of cells to their surrounding extracellular matrix (ECM) and to other cells has vital roles in embryonic development and adult tissue homeostasis. Cells attach to extracellular matrix by specific cell-surface receptors and intracellular cytoskeleton and signaling molecules. The complex interactions of cells with ECM and with other cells involve bi-directional signaling and the organization of cytoskeleton elements, such as microfilaments, microtubules and intermediate filaments. Actin, tubulin, intermediate filament-proteins and their associated proteins are key players in the cell adhesion process.

Claudia dos Santos Mermelstein Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, RJ [email protected]

______Pre Meeting Courses

10. Strategies for ultrastructural immunolabeling The aim of this course is to suggest the appropriate application of techniques used in immunocytochemistry for transmission electron microscopy.

Edna Haapalainen [email protected] Centro de Microscopia, Universidade Federal de São Paulo, SP Date: July 21st to 25th, 30 h (from 9h00-12h00; 14h00-17h00)

11. Image processing using the public domain software Image J The goal of the course is to familiarize scientists and students with the Internet free access software IMAGE J. This software is a powerful tool to carry out image analysis, processing, morphometry, gel densitometry, and volumetric reconstruction. A comprehensive set of free modules (Plugins) can be installed in the software, allowing time lapse acquisition, microscope control, co-localization analysis and quantitative fluorescence.

Ruy Jaeger [email protected] Instituto de Ciências Biomédicas, Universidade de São Paulo, SP Date: July 24th and 25th, 12 h (from 9h00-12h00; 14h00-17h00)

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livro.indd 11 7/7/2008 16:33:26 Program 27/07/2008

Room Gaivota

18h00 Opening Ceremony

Homage Emeritus Member: Prof Dr Michel Rabinovitch Universidade Federal de São Paulo (UNIFESP)

Brazilian music- Grupo Língua Brasileira

Homage Luis Carlos Uchoa Junqueira (In memorian) Conceição Machado (In memorian)

Opening conference Prion diseases and the prion protein: From bedside to bench Rafael Linden Instituto de Biofísica, Universidade Federal do Rio de Janeiro, RJ rlinden@biof ufrj.br

Chairperson: Ricardo Brentani Hospital do Câncer, SP Diretor Presidente do Conselho Técnico-Administrativo da FAPESP rbrentani@hcancer org.br

20h30 Cocktail (sponsored by BD Bioscience)

Program 28/07/2008

Room Gaivota 1

08h00-09h30

Forum # 1: Políticas em pesquisa e desenvolvimento em Biologia Celular Chairperson: Hernandes F. Carvalho Instituto de Biologia, Universidade Estadual de Campinas, SP

Demanda e qualificação de projetos e pesquisadores no Comitê MF-CNPq Hernandes F Carvalho Instituto de Biologia, Universidade Estadual de Campinas, SP [email protected]

A Pós-Graduação em Biologia Celular – Classificação e a dispersão nos comitês da CAPES - ações necessárias Adalberto Ramon Vieyra Instituto de Biofisica Carlos Chagas Fo, Universidade Federal do Rio de Janeiro, RJ avieyra@biof ufrj.br

Demanda e qualificação de projetos em Biologia Celular na FAPESP Walter Colli Instituto de Química, USP, SP [email protected] 1. 12

livro.indd 12 7/7/2008 16:33:26 Demanda e qualificação de projetos em Biologia Celular na FAPERJ Jérson Lima e Silva Universidade Federal do Rio de Janeiro, Instituto de Bioquímica Médica. jerson@bioqmed ufrj.br

09h30-11h00

Forum # 2: Biotechnology & Biosecurity In the last decade, we watched a big revolution in the biology with promising perspectives for the improvement of life quality of the man. This, however, came across with dogmas, life concepts and obviously laws that control its use, but nevertheless, should allow conquer benefits. Nowadays, researchers and citizens deeply debate the “new” scientific challenge: how to control the biotechnology without restricting the perspectives that it can show us?

Chairperson: Ricardo Brentani Presidente do Hospital do Câncer AC. Camargo, SP Diretor Presidente do Conselho Técnico-Administrativo da FAPESP rbrentani@hcancer org.br

Walter Colli Instituto de Química, USP, SP [email protected]

Maria Lúcia Zaidan Dagli Faculdade de Medicina Veterinária e Zootecnia, USP, São Paulo - [email protected] Luiz Antonio Barreto de Castro Ministério de Ciência e Tecnologia [email protected]

Room Rouxinol 1

08h00-09h00

BD Biosciences: New hydrogel of synthetic peptide for 3D cell culture Marshall J. Kosovsky, Ph.D BD Biosciences - USA

09h00-10h00

Zeiss: LSM 710 QUASAR: A New dimension in confocal microscopy Bruno Vale Product Application Specialist, Carl Zeiss do Brasil Ltda [email protected]

10h00-11h00

BD Biosciences: Cell therapy and tissue bioengineering using stem cells Nance Nardi Instituto de Biociências, Universidade Federal do Rio Grande do Sul

Room Rouxinol 2

08h00-09h00

Biomechanics of single molecules and single cells Oscar Nassif Mesquita Instituto de Ciências Exatas, Universidade Federal de Minas Gerais

09h00-10h00

Prodimol: Expanding applications of bioluminescence technology: Cell-based assays to assess apoptosis, 1. 13

livro.indd 13 7/7/2008 16:33:27 cell viability, cytotoxicity, and cell signaling Sarah Shultz R&D Integrated Solutions, Promega Corporation

10h00-11h00

AOTEC (Leica): Innovations in confocal systems: from microscopy to nanoscopy Robert Dunakin Leica Microsystems

11h00 - Coffee break

Room Gaivota 1

11h30-13h00

Round Table # 1 Cell Biology and novel therapeutic approaches to degenerative diseases Chairperson: Rafael Linden Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro [email protected]

Human pancreatic islet cell transplantation: alternative approaches Mari Sogayar Instituto de Química, Universidade de São Paulo, SP [email protected]

Gene therapy and DNA repair Carlos Frederico Martins Menck Instituto de Ciências Biomédicas, Universidade de São Paulo, SP [email protected]

Stem cell transplantation for autoimmune diseases Julio Cesar Voltarelli Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, SP [email protected]

14h00-15h00

Lecture #1 Immune-neuro-endocrine cross-talk: different cells, shared receptors, other messages Adriana Del Rey Medical Faculty of Philipps University Marburg, Germany [email protected]

Chairperson: Wilson Savino Fundação Oswaldo Cruz, RJ savino@fiocruz br

15h00-16h30

Round Table #2 Immuno-neuro-endocrine interactions Chairperson: Wilson Savino Fundação Oswaldo Cruz, RJ savino@fiocruz br

Cell signaling specificity in hormone and cytokine communication networks Eduardo Arzt Laboratorio de Fisiología y Biología Molecular de la Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina [email protected].

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livro.indd 14 7/7/2008 16:33:27 A particular neuro-immune synapsis: noradrenergic nerves and lymphocytes Adriana Del Rey Medical Faculty of Philipps University Marburg, Alemanha [email protected]

Neural and behavioral correlates of experimental murine allergy Frederico Costa Pinto Faculdade de Medicina Veterinária, Universidade de São Paulo, SP [email protected]

Cellular and molecular bases of intrathymic immunoneuroendocrine interactions Wilson Savino Fundação Oswaldo Cruz, Rio de Janeiro, RJ savino@fiocruz br

16h30 Coffee break

17h00-18h30

Round Table #3 Topics in neuroscience Chairpersons: Stevens Kastrup Rehen Universidade Federal do Rio de Janeiro, RJ

Bettina Malnic Universidade de São Paulo, SP

Neuronal mechanisms of memory consolidation Sidarta Ribeiro Instituto Internacional de Neurociências de Natal Edmond e Lily Safra, RN [email protected]

Plasticity in human motor cortex after hand allograft Claudia Domingues Vargas Instituto de Biofísica Carlos Chagas Fo., Federal do Rio de Janeiro, RJ cdvargas@biof ufrj.br

Molecular mechanisms of odor recognition Bettina Malnic Instituto de Química, Universidade de São Paulo, SP [email protected]

Neurogenesis induced by non-conventional conditions in embryonic stem cells Stevens Kastrup Rehen Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, RJ [email protected]

18h30-19h30

Lecture # 2 Persistence of the memory Ivan Antônio Izquierdo Pontifícia Universidade Católica do Rio Grande do Sul, Faculdade de Medicina, Instituto de Ciências Biológicas Centro de Memória. [email protected]

Room Gaivota 2

11h30-13h00

Round Table #4 Hypoxia-induced mechanisms Chairperson: Aureo Yamada 1. 15

livro.indd 15 7/7/2008 16:33:27 Universidade Estadual de Campinas, Unicamp, SP [email protected]

Placenta-mediated fetal hypoglycemia is the cause of altitude-associated reduction in fetal growth Stacy Zamudio Department of Obstetrics, Gynecology and Women’s Health, New Jersey Medical School, New Jersey, USA [email protected]

Regulation of mitochondrial oxygen consumption in primary human syncytial cells under conditions of hypoxia Nicholas P. Illsley Department of Obstetrics, Gynecology and Women’s Health, New Jersey Medical School, New Jersey, USA [email protected]

Hypoxia and preeclampsia changes alano signaling pathway in human placenta endothelium Luis Sobrevia Faculty of Medicine, Pontifícia Universidad Católica de Chile, Santiago, Chile [email protected]

Magnesium sulfate as an antioxidant of full term placental explants under hypoxia Reinaldo Marin Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela [email protected]

14h00-15h00

Lecture #3 Turnover and lengh regulation of actin Bechara Kachar National Institutes of Health - National Institute on Deafness and other Communication Disorders, Bethesda, MD USA [email protected]

Chairperson: Manoel Luis Costa Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, RJ [email protected]

15h00-16h30

Round Table #5 Hormonal disruptors Chairperson: Wilma Kempinas Universidade Estadual Paulista Júlio de Mesquita Fo, Botucatu, SP kempinas@ibb unesp.br

Impact of steroid and endocrine disruptors on the morphophysiology of Mongolia gerbil prostate Sebastião R. Taboga Instituto de Biociências Letras e Ciências Exatas de São José do Rio Preto, Universidade Estadual Paulista Júlio de Mesquita Filho, São José do Rio Preto, SP taboga@ibilce unesp.br

Estrogen imprinting: from morphology to behavior Hernandes F. Carvalho Instituto de Biologia, Universidade Estadual de Campinas, SP [email protected] Disruption of cellular signaling in the epididymis by bacterial infection: role of antimicrobial genes Maria Christina Werneck Avellar Departamento de Farmacologia, Setor de Endocrinologia Experimental, Universidade Federal de São Paulo, SP avellar@farm epm.br

The epididymis as a target organ for endocrine disruptors Wilma Kempinas 1. 16

livro.indd 16 7/7/2008 16:33:27 Instituto de Biociências, Universidade Estadual Paulista Júlio de Mesquita Fo, Botucatu, SP kempinas@ibb unesp.br

16h30 Coffee break

17h00-18h30

Round Table #6 Implantation and decidualization: normal and pathological aspects Chairperson: Telma Zorn Instituto de Ciências Biomédicas, Universidade de São Paulo, SP [email protected]

Acquisition and regulation of an invasive phenotype in non-malignant cells Paul Bischof Clinic of Infertility and Gynaecologic Endocrinology, Department of Obstetrics and Gynaecology, University of Geneva, Maternité, Switzerland [email protected]

Placental morphogenesis in normal and pathological condition Sebastian San Martin Facultad de Medicina, Universidad de Valparaiso, Chile [email protected]

Endometrial extracellular matrix during the perimplantation period Telma Zorn Instituto de Ciências Biomédicas, Universidade de São Paulo, SP [email protected]

18h30-19h30

Lecture #4 Germ cells transplantation and testis graft: current status Luis Renato de França Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, MG lrfranca@icb ufmg.br

Chairperson: Sebastião Taboga Universidade Estadual Paulista, São José do Rio Preto, SP taboga@ibilce unesp.br

Room Gaivota 3

11h30-13h00

Round Table #7 Stem cells Chairperson: Radovan Borojevic Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, RJ [email protected]

Biology and therapeutic potential of mesenchymal stem cells Nance B. Nardi Instituto de Biociências, Universidade Federal do Rio Grande do Sul, RS [email protected]

Stem cells in a tissue context Radovan Borojevic Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, RJ [email protected]

Cellular therapy in animal models of neurological diseases Rosália Mendez-Otero 1. 17

livro.indd 17 7/7/2008 16:33:27 Instituto de Biofísica Carlos Chagas Fo, Universidade Federal do Rio de Janeiro, RJ [email protected]

14h00-15h00

Lecture #5 Stephen Ferguson (Sponsored by: AOTEC Equipamentos Científicos LTDA) University of Western Ontario, London, Canada [email protected] Chairperson: Marco Antonio Maximo Prado Departamento de Farmacologia, Universidade Federal de Minas Gerais, MG [email protected]

15h00-16h30

Round Table #8 Leica Symposium on Protein motion in cell physiology and disease Chairperson: Marco Antonio Maximo Prado Universidade Federal de Minas Gerais, MG [email protected]

Phosphorylated nuclear myosin VA distributes from speckles to nucleoli depending upon transcription activity Roy E. Larson Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, SP [email protected]

The spindle assembly checkpoint proteins and their roles in progression through mitosis Cláudio Sunkel Instituto de Biologia Molecular e Celular, Porto, Portugal [email protected]

Illuminating prion protein trafficking and function Marco Antonio Maximo Prado Departamento de Farmacologia, Universidade Federal de Minas Gerais, MG [email protected]

16h30 Coffee break

17h00-18h30

Round Table #9 The role of ubiquitin-proteasome system in degenerative processes Chairperson: Cinthya Guimarães-Sternberg Instituto Nacional do Câncer, RJ [email protected]

Redox modulation of the proteasome 20S Marilene Demasi Instituto Butantã, SP [email protected]

FBXO25- associated nuclear domains (FANDs): a novel subnuclear structure Marcelo Damário Gomes Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, SP [email protected]

Functional analysis of a novel NKX2-5 mutation unravels a new role for ubiquitin-proteasome system in cardiomyopathies Mauro Weyne da Costa Universidade Federal do Rio de Janeiro, RJ [email protected] 1. 18

livro.indd 18 7/7/2008 16:33:27 Ubiquitin-mediated degradation of XIAP: Do we know enough to target it for therapies? Cinthya Guimarães-Sternberg Instituto Nacional do Câncer, RJ [email protected]

18h30-19h30

Lecture #6 Virus and cancer: therapy, diagnosis and prophylaxis Luisa Villa Instituto Ludwig de Pesquisa sobre o Câncer, SP [email protected]

Chairperson: Vilma Martins Instituto Ludwig de Pesquisa sobre o Câncer, SP [email protected]

Room Rouxinol 1

11h30-13h00

Round Table # 10 Interactive teaching of Biology, Cell and Tissue Biology and Development Chairperson: Paulo Abrahamsohn Instituto de Ciências Biomédicas, Universidade de São Paulo, SP [email protected]

Learning biology at distance Marlene Benchimol Universidade Santa Ursula, Rio de Janeiro, Brazil [email protected]

Interactive basic histology: an electronic atlas Maria Gabriela Tavares Rheingantz & Iria Guimarães Machado Instituto de Biologia, Universidade Federal de Pelotas, RS [email protected]; [email protected]

MOL- An interactive guide of Histology Paulo Abrahamsohn Instituto de Ciências Biomédicas, Universidade de São Paulo, SP [email protected]

Interactive teaching and learning of embryology Luis Violin Pereira Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP [email protected]

14h00-15h00

Lecture #7 Gene regulation in protozoons Sergio Schenkman Depto de Microbiologia, Imunobiologia e Parasitologia, Universidade Federal de São Paulo [email protected]

Chairperson: Edna Kimura Instituto de Ciências Biomédicas, Universidade de São Paulo, SP [email protected]

15h00-16h30

Round Table #11 Parasite-host interaction Chairperson: Marlene Benchimol 1. 19

livro.indd 19 7/7/2008 16:33:27 Universidade Santa Ursula, Rio de Janeiro, Brazil [email protected]

Trichomonas vaginalis kills and eats – evidence for phagocytic activity as cytophatic effect Marlene Benchimol Universidade Santa Ursula, Rio de Janeiro, RJ [email protected]

Mammalian cell invasion by Trypanosoma cruzi infective forms Renato Mortara Universidade Federal de São Paulo, SP [email protected]

Proteolytic pathways leading to cooperative activation of G-protein coupled receptors during host cell invasion by Trypanosoma cruzi Julio Scharfstein Instituto de Biofísica Carlos Chagas Fo, Universidade Federal do Rio de Janeiro, RJ [email protected]

Plasmodium functional genome Celia Regina da Silva Garcia Instituto de Biociências, Universidade de São Paulo [email protected]

16h30 Coffee break

17h00-18h30

Round Table #12 Teaching Cell Biology Chairperson: Hernandes Carvalho Instituto de Biologia, Universidade Estadual de Campinas, SP [email protected]

The use of cooperative games in the teaching of the cellular biology Marco A. F. Randi Depto de Biologia Celular, Universidade Federal do Paraná, Curitiba, PR mafrandi@ufpr Br

Bayardo Torres Instituto de Química, Universidade de São Paulo [email protected]

Teaching of cellular biology through computers Eduardo Galembeck Departamento de Bioquímica, Universidade Estadual de Campinas [email protected]

18h30-19h30

Lecture #8 Telomeres in health and disease Maria Isabel Cano Departamento de Genética, Universidade Julio de Mesquita Filho, Botucatu, SP [email protected]

Chairperson: Hernandes F. Carvalho Instituto de Biologia, Universidade Estadual de Campinas, SP [email protected]

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livro.indd 20 7/7/2008 16:33:27 Room Rouxinol 2

11h15-12h30

Round Table #13 Extracellular matrix Chairperson: Helena Bonciani Nader Departamento de Bioquímica, Universidade Federal de São Paulo, SP [email protected]

Lysosomal cysteine proteinases are implicated in development and progression of human carious dentin lesions Ivarne Luis dos Santos Tersariol Centro Interdisciplinar de Investigações Bioquímicas, Universidade de Mogi das Cruzes, SP [email protected]; [email protected]

P-selectin as a therapeutic target in thrombosis, inflammation and cancer: the role of heparin analogues. Mauro Sérgio Gonçalves Pavão Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, RJ - mpavao@hucff ufrj.br

Helena Bonciani Nader Departamento de Bioquímica, Universidade Federal de São Paulo, SP [email protected]

14h00-15h00

Lecture #9 Cell movements and fate during early mammalian embryogenesis Shankar Srinivas Wellcome Trust Research Fellow & University Lecturer Department of Physiology Anatomy and Genetics, University of Oxford, London, UK [email protected]

Chairperson: Irene Yan Instituto de Ciencias Biomédicas, Universidade de São Paulo, SP ireneyan@usp,br

15h00-16h30

Round Table #14 Molecular processes of the development in Drosophila Chairperson: Ricardo G. P. Ramos Faculdade de Medicina, Universidade de São Paulo, SP [email protected]

Pair-rule striped pattern regulation in the segmentation cascade of Drosophila melanogaster Luiz Paulo Moura Andrioli Instituto de Biociências, Universidade de São Paulo, SP [email protected]

Insights in developmentally regulated mechanisms in Drosophila: lessons from a heterologous sciarid promoter Nadia Monesi Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, SP [email protected]

Molecular and functional characterization of JAZIGO: exploring new roles of formins during development Cláudio Simon Universidade Federal do Triangulo Mineiro, Uberlândia, MG [email protected]

1. 21

livro.indd 21 7/7/2008 16:33:27 The Neph/Rst family of cell adhesion molecules: evolutionary and functional insights from Drosophila and vertebrates Ricardo G. P. Ramos Faculdade de Medicina, Universidade de São Paulo, SP [email protected]

16h30 Coffee break

17h00-18h30

Round Table #15 Zebrafish - animal model for biological studies Chairperson: Mônica Ryff Moreira Vianna Faculdade de Biociências, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS monica.vianna@ pucrs.br

Purinergic system in zebrafish: toxicological implications Carla Denise Bonan Faculdade de Biociências, Pontifícia Universidade Católica, Porto Alegre, RS [email protected]

Zebrafish as a model for adenosinergic system studies Mauricio Bogo Faculdade de Biociências, Pontifícia Universidade Católica, Porto Alegre, RS [email protected]

Using zebrafih as a model for studying the cell biology of the myogenesis Manoel Luis Costa Universidade Federal do Rio de Janeiro, UFRJ [email protected]

Zebrafish as a model to study evolution and development of cardiac chambers José Xavier Neto Instituto do Coração, Universidade de São Paulo, SP [email protected]

18h30-19h30

Lecture #10 Intracellular pathways of the muscular regeneration Anselmo Moriscot Instituto de Ciências Biomédicas, Universidade de São Paulo, SP [email protected]

Chairperson: Marinilce dos Santos Fagundes Instituto de Ciências Biomédicas, Universidade de São Paulo, SP [email protected]

Program 29/07/2008

Outdoors activity

6h30-8h30

Cytocinetic Walk (3km) Ibirapuera Park– MAM’s Parking Area Sponsorship & awards: BD Biosciences Organization and support: PN Treinamento Assessoria Esportiva 1. 22

livro.indd 22 7/7/2008 16:33:27 Room Gaivota 1

9h00-10h00

Lecture #11 Molecular Mechanisms of Melanoma Progression and Chemoresistance Maria Soengas Cancer Center Dermatology Department, University of Michigan, USA [email protected]

Chairperson: Silvya Stuchi Maria-Engler Depto Análises Clínicas e Toxicológicas, Universidade de São Paulo, SP [email protected]

10h00 Coffee break

10h30-12h00

Round Table #16 Cancer Chairperson: Roger Chammas Faculdade de Medicina, Universidade de São Paulo, SP [email protected]

Fibroblast Growth Factor 2 (FGF2): classical and novel mechanisms in cell cycle control Hugo Armelin Instituto de Química, Universidade de São Paulo, SP [email protected]

Functional changes associated with cell-cell and cell-ECM interactions in melanomas Luiz Fernando Lima Reis Hospital AC Camargo/Fundação Antônio Prudente, SP luiz.reis@hcancer org.br

Matricellular proteins in angiogenesis and cancer Verônica Morandi Depto Biologia Celular e Genética, Centro Biomédico, Universidade do Estado do Rio de Janeiro, RJ [email protected]; verônica@uerj br

12h00-13h30

General Assembly of the Brazilian Society for Cellular Biology

14h00-15h00

Lecture #12 Imaging epithelial mesenchymal transition Marcelo Ehrlich Department of Cell Research and Immunology, Tel-Aviv University, Israel [email protected]

Chairperson: Roger Chammas Faculdade de Medicina, Universidade de São Paulo, SP [email protected]

15h00-16h30

Round Table # 17 Biology of tumoral cell Chairperson: José Andrés Morgado Diaz Centro de Pesquisas – INCa, RJ jmorgado @inca.gov.br 1. 23

livro.indd 23 7/7/2008 16:33:27 Cytoskeleton as target to anticancer drugs Glaucia M.M. Santelli Instituto de Ciências Biomédicas, Universidade de São Paulo [email protected]

Laminin peptides in tumoral biology Ruy Jager Instituto de Ciências Biomédicas, Universidade de São Paulo [email protected]

Cell-cell adhesion in epithelial cells and cancer José Andrés Morgado Diaz Grupo de Biologia estrutural, Centro de Pesquisas – INCa, RJ jmorgado @inca.gov.br

Room Gaivota 2

9h00

Lecture # 13 Arginine deprivation and its differential effects on malignant versus normal cells Denys Wheatley International Federation for Cell Biology –IFCB-Secretary General, Aberdeen, UK [email protected]

Chairperson: Patrícia Gama Instituto de Ciências Biomédicas, Universidade de São Paulo, SP [email protected]

10h00 Coffee break

10h30-12h00

Round Table #18 Cytoskeleton Chairperson: Enilza M Espreafico Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, SP [email protected]

Modulation of cell adhesion and survival by actin-based molecular motor Enilza M Espreafico [email protected]

Myosin-10 and actin filaments are essential for mitotic spindle function Sarah Woolner Dept. of Zoology, University of Wisconsin-Madison, Madison, USA [email protected]

Filopodia ensure accurate epithelial fusion during Drosophila dorsal closure Thomas H Millard The Healing Foundation Centre, Faculty Life Sciences, University of Manchester, UK [email protected]

14h00-15h00

Lecture #14 Molecular machinery for the transport of neurotransmitter receptors at synapses José Esteban Department of Pharmacology, University of Michigan, Ann Arbor, Michigan, USA [email protected]

Chairperson: Vivaldo Moura Neto Universidade Federal do Rio de Janeiro, RJ - [email protected] 1. 24

livro.indd 24 7/7/2008 16:33:27 15h00-16h30

Round Table #19 Cellular migration Chairperson: Marimélia Porcionatto Departamento de Bioquímica, Universidade Federal de São Paulo, SP [email protected]

Fibroblast migration in three-dimensional collagen gels: different mechanisms for different conditions Marinilce Fagundes dos Santos Instituto de Ciências Biomédicas, Universidade de São Paulo, SP [email protected]

A multivectorial model for intrathymic T cell migration Wilson Savino Fundação Oswaldo Cruz, RJ savino@fiocruz br

ADAM23: a new metastasis supressor gene? Anamaria Aranha Camargo Instituto Ludwig de Pesquisa sobre o Câncer, SP anamaria@ludwig org.br

16h30 Coffee break

Room Gaivota 3

9h00-10h00

Lecture # 15 Translating proteomics and developing predictive capacity Gregory Rice Perinatal Research Centre, Department of Perinatal Medicine, Royal Women’s Hospital, Carlton, Australia [email protected]

Chairperson: Stacy Zamudio Department of Obstetrics, Gynecology, and Women’s Health, New Jersey Medical School, New Jersey, USA [email protected]

10h00 Coffee break

10h30-12h00

Round Table #20 Cytokine cell signaling Chairperson: Estela Bevilacqua

Participation of inflammatory agents in a model of septic pregnancy loss induced by LPS Ana Franchi Universidad de Buenos Aires, Argentina [email protected]

Cytokines and uterine-natural killer cells at the maternal-fetal interface Áureo Yamada Universidade Estadual de Campinas, UNICAMP [email protected]

Is macrophage migration inhibitory factor (MIF) implicated in the placental defense against Toxoplasma gondii? Eloísa Amália Vieira Ferro Universidade federal de Uberlândia, Minas Gerais [email protected]

1. 25

livro.indd 25 7/7/2008 16:33:27 14h00-15h00

Lecture #16 Mechanisms of centromere and kinetochore formation Aaron Straight Stanford University School of Medicine, Department of Biochemistry [email protected]

Chairperson: Frederico Gueiros Instituto de Química, Universidade de São Paulo, SP [email protected]

15h00-16h30

Round Table #21 Bacterial cell biology Chairperson: Frederico Gueiros Instituto de Química, Universidade de São Paulo, SP [email protected]

Spatial organization of a replicating bacterial chromosome Sigal Ben-Yehuda Hadassah School, Jerusalem, Israel [email protected]

Small molecule synthesis by a dedicated enzymatic megacomplex Paul Straight Texas A&M University [email protected]

Molecular mechanism of cytokinesis in Bacillus subtilis Frederico Gueiros Instituto de Química, Universidade de São Paulo, SP [email protected]

16h30 Coffee break

Room Rouxinol 1/2

9h00-10h00

Lecture #17 Guidance cues for development of the vascular tree Anne Eichmann Institut National de la Santé et de la Recherche Médicale, INSERM, Paris, France [email protected]

Chairperson: Luiz Eurico Nasciutti Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro [email protected]

10h00 Coffee break

10h30-12h00

Round Table # 22 Angiogenesis Chairperson: Luiz Eurico Nasciutti Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, RJ luiz.nasciutti@histo ufrj.br

VEGF-C / VEGFR-3: new-comers in the neurovascular cross-talk Jean-Léon Thomas Unité Mixte de Recherche INSERM, Université Pierre & Marie Curie, Paris, France - [email protected] 1. 26

livro.indd 26 7/7/2008 16:33:27 Angiogenic genes for the treatment of ischemic diseases Sang Won Han Centro Interdisciplinar de Terapia Gênica, Universidade Federal de São Paulo [email protected]

Anti-angiogenesis therapeutic development in an experimental model of endometriosis Luiz Eurico Nasciutti Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, RJ luiz.nasciutti@histo ufrj.br

Room Rouxinol 1

14h00-15h00

Lecture #18 Mechanisms of cellular signaling and remodeling in myocard Kleber G. Franchini Faculdade de Ciências Médicas, Universidade Estadual de Campinas franchin@unicamp br

Chairperson: Maria Luiza Morais Barreto de Chaves Instituto de Ciências Biomédicas, Universidade de São Paulo, [email protected]

15h00-16h30

Round Table #23 Tissue repair Chairperson: Luis Cristovão Sobrino Porto Centro de Ciências Médicas, Universidade do Estado do Rio de Janeiro, RJ lcporto@uerj br

Tissue repair in liver Alexis Desmoulière Faculty of Pharmacy, University of Limoges, Limoges, France [email protected]

Tissue Repair: Repair in the skin and kidney Timothy D. Hewitson School / Work Unit Medical Sciences, University of Melbourne, Melbourne, Australia [email protected]

Adrenoceptors and rat cutaneous wound healing Andréa Monte Alto Costa Centro de Ciências Médicas, Universidade do Estado do Rio de Janeiro, RJ [email protected]

Distinct effects of allopurinol N-(2-Mercaptopropionyl))glycine and N-acetylcysteine in mice exposed to either short-term or long-term cigarette smoke Luis Cristovão Sobrino Porto Centro de Ciências Médicas, Universidade do Estado do Rio de Janeiro, RJ lcporto@uerj br

Room Rouxinol 2

14h00-15h00

Lecture #19 Calcium channels, neurotransmitter release and synaptic vesicle recycling Osvaldo D. Uchitel Institute of Physiology, Molecular Biology and Neurosciences 1. 27

livro.indd 27 7/7/2008 16:33:27 Faculty of Exact and Natural Sciences, University of Buenos Aires, Argentina

15h00-16h30

Round Table #24 Chromosomal organization and physiology Chairperson: Maria Luiza Silveira Mello [email protected]

Holocentric chromosomal structure in triatomines Maria Tercília Vilela de Azeredo Oliveira IBILCE, Universidade Estadual Paulista, São José do Rio Preto, SP [email protected]

Chromatin organization in the mouse hepatocyte Alberto da Silva Moraes Instituto de Biologia, Universidade Estadual de Campinas, SP [email protected]

Chromosome painting in the context of cell biology Júlio César Pieczarka Universidade Federal do Pará, Belém, PA [email protected]

16h30 Coffee break

17h00-18h30 Poster Session I

19h30-21h00 Poster Session II

Program 30/07/2008

Room Gaivota 2

8h00

Oral presentation: selected abstracts: Young Scientist Award Category IC (undergraduates) and PD (Post-Docs)

Vivaldo Moura Neto Universidade Federal do Rio de Janeiro [email protected]

Luiz Eurico Nasciuttti Universidade Federal do Rio de Janeiro, RJ [email protected]

José Garcia Abreu Universidade Federal do Rio de Janeiro, RJ [email protected]

Sérgio Schenkman Universidade Federal de São Paulo [email protected]

1. 28

livro.indd 28 7/7/2008 16:33:28 Room Gaivota 3

8h00-9h00

Oral presentation: selected abstracts: Young Scientist Award Category ME-DR (Graduate students)

Roger Chammas Universidade de São Paulo, SP [email protected]

Rafael Linden Universidade Federal do Rio de Janeiro, RJ rlinden@biof ufrj br Hugo Aguirre Armelin Universidade de São Paulo, SP haarmeli@iq usp.br

Edna Haapalainen Universidade Federal de São Paulo, SP [email protected]

Room Rouxinol 1/2

8h00-9h00

Lecture #20 - Nanomedicine

Room Gaivota 1

9h00-10h00

Closing lecture Of extracellular matrix, the genome and the microenvironment in breast cancer. No cell is an island. Dr Mina Bissell, Honorable Speaker Distinguished Scientist Life Sciences Division, Berkeley, CA, USA [email protected]

Chairperson: Vilma Martins Instituto Ludwig de Pesquisa sobre o Câncer, SP [email protected]

10h00 Coffee break

10h30

Closing Session Presentation of the Young Scientists Awards Closing remarks from the organizers of the XIV SBBC Congress Introduction and Greeting of the new SBBC’s Board of Directors

1. 29

livro.indd 29 7/7/2008 16:33:28 Abstracts

1. 30

livro.indd 30 7/7/2008 16:33:28 Lectures

OPENING CONFERENCE PRION DISEASES AND THE PRION PROTEIN: FROM BEDSIDE TO BENCH. Rafael Linden Instituto de Biofísica da UFRJ

Prion diseases are transmissible spongiform encephalopathies (TSEs), attributed to conformational conversion of the cellular prion protein (PrPC) into an abnormal conformer that accumulates in the brain. Understanding the pathogenesis of TSEs requires the identification of functional properties of PrPC. Here we examine the physiological functions of PrPC at the systemic, cellular and molecular level. Current data show that both the expression and the engagement of PrPC with a variety of ligands modulate: (a) Functions of the nervous and immune systems, including memory and inflammatory reactions; (b) Cell proliferation, differentiation, and sensitivity to programmed cell death both in the nervous and immune systems, as well as in various cell lines; (c) The activity of numerous signal transduction pathways, including cAMP/protein kinase A, MAP kinase, PI3-K/Akt pathways, as well as soluble non-receptor tyrosine kinases; (d) Trafficking of PrPC both laterally among distinct plasma membrane domains, and along endocytic pathways, on top of continuous, rapid recycling. A unified view of these functional properties indicates that the prion protein is a dynamic cell surface platform for the assembly of signaling modules, based on which selective interactions with many ligands and transmembrane signaling pathways translate into wide-range consequences upon both physiology and behavior. [email protected]

CLOSING LECTURE OF EXTRACELLULAR MATRIX, THE GENOME AND THE MICROENVIRONMENT IN BREAST CANCER. NO CELL IS AN ISLAND.

Dr Mina Bissell Honorable Speaker Distinguished Scientist Life Sciences Division, Berkeley, CA, USA

[email protected]

1. 31

livro.indd 31 7/7/2008 16:33:28 L-1 simultaneoulsy with other inputs. As a whole, this IMMUNE-NEURO-ENDOCRINE CROSS-TALK: knowledge has lead to the proposal of the existance DIFFERENT CELLS, SHARED RECEPTORS, OTHER of a network of immune-neuro-endocrine interactions. MESSAGES We know at present that the operation of this network Adriana del Rey is relevant not only for immunoregulation but also for Dept. of Immunophysiology, Inst. of general host homeostasis, and that disturbances in its Physiology and Pathophysiology, Medical Faculty, functioning can contribute to different types of diseases. Marburg, Germany The actual state of knowledge in this field of research will be illustrated using examples from those immune- At the beginning of the 90’, most efforts were directed neuro-endocrine circuits that are better understood at at understanding the molecular and cellular basis present. of the immune response and the mechanisms [email protected] of acquisition of immunological diversity and self tolerance. Now, the structure of the main molecules L-2 that allow recognition of antigens is known, as well PERSISTENCE OF THE MEMORY as the types and subtypes of cells that participate Ivan Antônio Izquierdo in an immune response. The genetic basis of the Pontifícia Universidade Católica do Rio Grande do Sul, differentiation and diversification of immunological cells Faculdade de Medicina, Instituto de Ciências Biológicas is largely understood. Furthermore, the biochemical Centro de Memória. and molecular mechanisms of immune cell activation [email protected] and of how these cells interact and receive information from other immune cells has been clarified to a great L-3 extent. The existence of autoregulatory mechanisms TURNOVER AND LENGTH REGULATION OF ACTIN which can control the immune response has also CYTOSKELETON IN STEREOCILIA been established. In contrast, much less is known Bechara Kachar and Uri Manor about how immunological cells and their products Structural Cell Biology Section, NIDCD, NIH, Bethesda, interact with other bodily systems, and about the MD 20892 consequences of such interactions for mechanisms intrinsic and extrinsic to the immune system. Thus, Perhaps one of the most striking examples of highly the understanding of the organization of the immune regulated cellular and tissue organization is the inner system itself has raised essential questions concerning ear sensory epithelia, home of the enigmatic hair cells, its physiological functioning within the whole organism. which are the most sensitive mechanical transducers The need to provide answers to these questions is in the human body. Hair cells are specialized epithelial becoming increasingly a priority, and the research of cells named for their bundles of 50-100 tightly packed many laboratories is oriented now to understand the stereocilia (“hairs”) found on their apical cellular surface. functional and molecular basis of interactions between Stereocilia are the key players in the transduction the immune system and integrative neuro-endocrine of sound waves or motion into electrical signals that mechanisms. It is at present well-established that underlie our senses of hearing and balance. Sometimes receptors for immune-derived cytokines are expressed referred to as “giant microvilli,” stereocilia are rigid in endocrine and neural cells, and, conversely, that membrane protrusions that contain hundreds of receptors for hormones, neurotransmitters and crosslinked parallel actin filaments that run the entire neuropeptides are present in immune cells. Not only length of the protrusion with the barbed ends of the the receptors, but some of the ligands themselves are actin filaments pointing away from the cell body. Each also found in both systems. A large body of evidence hair cell stereocilia bundle has a tightly regulated attests for the fact that hormones, neurotransmitters length that follows a distinct pattern based on the cell’s and neuropeptides can affect immune functions, and location within the tissue. For example, the vertebrate that immune cell products can affect neuro-endocrine auditory epithelium displays a tonotopic gradient of mechanisms. An interesting concept is that the immune stereocilia bundles with lengths inversely proportional system, acting as a diffuse, sensorial receptor organ, to the frequency of sound the cell is tuned to detect. can convey information derived from stimuli from The overall organization bears semblance to an array outside or inside the organism to the brain or to brain- of tuning forks organized like a piano with a systematic controlled structures, and receive, in turn, brain-derived increase in ‘string’ length from one end of the instrument messages. Thus, the outcome of the biochemical to the other. Likewise, within a single hair cell each events initiated in a lymphocyte following recognition stereocilium, and therefore each actin filament within of an antigen or induced by a cytokine would, in fact, the stereocilium, displays a tightly regulated length depend on other intracellular signals resulting from the that is dependent on its location within each individual simultaneous binding of neuroendocrine ligands that cell. Although stereocilia are exquisitely sensitive to occur under physiological or pathological conditions. mechanical vibration, and easily damaged by over- Conversely, brain-controlled structures would respond stimulation, they are maintained in proper working order differently if immune-derived messages are received for a lifetime. Recent studies on actin incorporation and 1. 32

livro.indd 32 7/7/2008 16:33:28 turnover in stereocilia show that they are dynamically recipients, and the quality of germ cell development maintained and undergo continuous renewal. These after transplant. Also, although germ cells from rabbits, observations, together with the identification of several dogs, large domestic animals (boar, bull and horse), novel proteins, including myosin motors and other actin- primate (baboon) and humans into nude mice were binding proteins, which when mutated cause deafness able to colonize the testis at different degrees, no through stereocilia disruption, provide new insights into spermatogenesis of the donors occurred. These results the mechanisms of turnover and length regulation of suggest that the success of transplantation might be actin protrusions. positively related to the degree of phylogenetic proximity [email protected] of species during evolution. To bypass this limitation, recent investigation grafting small pieces of testis tissue L-4 from several species into nude mice hosts showed GERM CELL TRANSPLANTATION AND TESTIS that male germ cells could be propagated and produce GRAFT: CURRENT STATUS fertile spermatozoa. This approach created a totally Luiz R. França new scenario in this field allowing, for instance, the Laboratory of Cellular Biology, Department of production of transgenic domestic animals. However, Morphology, ICB, Federal University of Minas Gerais, regardless of its limitation germ cell transplantation Belo Horizonte, MG, Brazil has been proved to be an extraordinary and powerful technique to investigate reproductive biology and stem Spermatogonial transplantation involves the removal cell biology in mammals and, more recently, in other of stem cells from a donor testis and their replacement vertebrates such as fish. into a recipient largely deprived or lacking endogenous Financial support: CNPq and FAPEMIG. spermatogenesis, where these transplanted cells grow [email protected] to form mature fertile sperm with genetic characteristics of the donor. Since spermatogonial transplantation L-5 from mouse-to-mouse was first reported by Brinster TO BE ANNOUNCED and colleagues in 1994, many important developments Stephen Ferguson in this fascinating methodology such as interspecies University of Western Ontario, London, Canada transplants, transplants from cryopreserved and (Sponsored by: AOTEC Equipamentos Científicos cultured spermatogonial stem cells have been made. In LTDA) this regard, this technique has been shown a powerful [email protected] approach to studying the biology of spermatogonial stem cells and their microenvironment, the stem cell L-6 niche. Also, several important functional questions VIRUS AND CANCER, THERAPY, DIAGNOSIS AND regarding Sertoli-germ cell interactions and the role of PROPHYLAXIS the Sertoli cell and germ cells during spermatogenesis Luisa Villa have now been answered. Transplantation of cultured Instituto Ludwig de Pesquisa sobre o Câncer, SP spermatogonial stem cells is now opening exciting [email protected] possibilities for in vitro multiplication of male germ cells and transfection or retroviral transduction has shown L-7 that it is now possible to produce transgenic mice. GENE REGULATION IN By freezing and storing testicular tissue, it should be PROTOZOONS possible to preserve indefinitely the genetic stocks of Sergio Schenkman valuable farm animals, endangered species and unique Depto de Microbiologia, Imunobiologia e Parasitologia, experimental animals, until a suitable recipient can be Universidade Federal de São Paulo, SP found that will maintain the germ line. Also, because [email protected] many cases of male infertility have proved intractable to therapy, spermatogonial transplantation has also L-8 potential clinical applications to address human TELOMERES IN HEALTH AND DISEASE infertility. For instance, if the testis of infertile individuals Maria Isabel Nogueira Cano contains at least spermatogonial stem cells, it might be Depto. de Genética, IBB, UNESP- Botucatu, São paulo, possible to transplant these cells into a host testis of the Brazil same or different species to obtain sufficient sperm of donor origin to achieve a pregnancy using ICSI. Another Telomeres are stretches of repetitive DNA (5’TTAGGG­ potential clinical use of spermatogonial transplantation ­3’ in vertebrates and protozoa) and associated technique is the replacement of the germ line in patients proteins that cap the ends of linear chromosomes and whose endogenous stem cells had been eliminated protect them from degradation or fusion by DNA­repair (or damaged) as a result of gonadotoxic chemo- processes. Due to the inherent mechanism of DNA or radiotherapeutic treatment. The spermatogonial replication (the end-replication problem), the ends of transplantation efficiency is still relatively poor and chromosomes remain single stranded and telomeres presents limitations related to the preparation of donors, shorten gradually with each round of cell division. 1. 33

livro.indd 33 7/7/2008 16:33:28 Dysfunctional telomeres trigger a classical DNA­damage cells meeting at a point). Rosettes are observed more response that enables double­strand breaks and cell­ frequently in the VE epithelium during AVE migration cycle arrest. Telomerase is the enzyme responsible and are non-randomly distributed within the VE. In to maintain telomeres. In humans, most normal cells different mutants where AVE migration fails, we see do not express telomerase or express it at levels that a substantial reduction in the number of rosettes, and are too low to prevent telomere shortening. In such those that do form are made up of fewer cells than cells, telomere lengths decline with each cell cycle and rosettes in wild type embryos. I will present these and eventually, one or a few telomeres become sufficiently other data relating to rosettes in the VE, and discuss short and loose the protective protein–DNA structure, their bearing on our understanding of cell movements to which cells respond by undergoing senescence. This within epithelial sheets. process of “replicative senescence” is now believed [email protected] to play an important role in age-related diseases (e.g. cardiovascular diseases, stroke, macular degeneration, L-10 osteoporosis, and joint disease), and in conditions INTRACELLULAR PATHWAYS OF THE MUSCULAR such as viral infections or chronic stress (e.g. AIDS, REGENERATION liver diseases, and skin ulcers). On the other hand, Anselmo Moriscot germline cells and many cancer cells express high Instituto de Ciências Biomédicas, Universidade de São levels of telomerase, which confers immortality to these Paulo, SP cells, but does not cause tumorogenesis. Since multiple [email protected] studies on human populations show an association between shortened telomeres and disease, telomerase L-11 activation has potential to improve cell or tissue function MOLECULAR MECHANISMS OF MELANOMA and repair. For many pathogenic unicellular organisms, PROGRESSION AND CHEMORESISTANCE such as trypanosomes and Plasmodium, telomeres are Maria Soengas conserved structures also maintained by telomerase. Cancer Center Dermatology Department, University of However, they are involved in homologous and ectopic Michigan, USA recombination events and acquisition of heterologous [email protected] gene sequences to diversify, as well as regulate the expression of genes involved in virulence. Thus, as L-12 telomeres are essential for genome stability, in this IMAGING EPITHELIAL particular case, perturbation of its structure and function MESENCHYMAL TRANSITION should be used as the key for the design of future drugs Marcelo Ehrlich and therapy. Department of Cell Research and Immunology, Tel-Aviv [email protected] University, Israel [email protected] L-9 CELL MIGRATION WITHIN THE EPITHELIUM OF L-13 THE EMBRYONIC ARGININE DEPRIVATION AND ITS DIFFERENTIAL VISCERAL ENDODERM IS MEDIATED BY EFFECTS ON MALIGNANT VERSUS NORMAL MULTICELLULAR ROSETTES CELLS Shankar Srinivas Dennis Wheatley Wellcome Trust Research Fellow & University Lecturer, International Federation for Cell Biology-IFCB- University of Oxford, Department of Physiology Secretary General, Aberdeen, UK Anatomy and Genetics [email protected]

In mammalian embryos, the visceral endoderm (VE) is L-14 an extra-embryonic epithelial tissue that lies adjacent to MOLECULAR MACHINERY FOR THE TRANSPORT the epiblast, the tissue that develops into the fetus. The OF NEUROTRANSMITTER RECEPTORS AT anterior visceral endoderm (AVE) is a specialized group SYNAPSES of cells within the VE that is responsible for inducing Jose Esteban anterior pattern in the abutting epiblast. AVE cells show Department of Pharmacology, University of Michigan, a stereotypic migration from their site of induction to the Ann Arbor, Michigan, USA presumptive anterior. Though this migratory behavior is essential for normal embryonic development – in Synaptic connections in the brain are mutants where this migration fails, embryonic anterior is continuously remodeled in response to neuronal activity. induced in the incorrect location, resulting in lethality – This process, known as synaptic plasticity, is widely little is known about the cellular mechanism driving AVE accepted as the cellular process underlying learning migration. Recent work from my group suggests that and memory. In fact, disruption of synaptic plasticity AVE migration may be driven by a convergent extension is known to be associated with multiple cognitive mechanism mediated by cellular rosettes (5 or more disorders, such as Alzheimer’s disease, autism and 1. 34

livro.indd 34 7/7/2008 16:33:28 several forms of mental retardation. We now know that and to disease onset, progression and treatment an important contributor to synaptic plasticity in the response. Indeed, the identification of peptide hippocampus and other brain regions is the signatures or motifs within biological samples for the regulated addition and removal of glutamate receptors purpose of donor classification is a burgeoning area at excitatory synapses. In particular, AMPA-type within the domain of diagnostic and predictive medicine. glutamate receptors (AMPARs) can be transported The premise upon which such initiatives are based in and out of the synaptic membrane in a regulated is that: (i) the expression of specific proteins and/or manner, resulting in long-lasting changes in synaptic their metabolites is altered by and informative of the strength. Despite the importance of AMPAR trafficking attendant pathophysiology; and (ii) the measurement for the regulation of synaptic function and plasticity, very and combination of multiple biomarkers of disease little is known of the membrane trafficking machinery increases the diagnostic efficiency. In this brief that controls the intracellular sorting and targeting of commentary, recent applications in proteomic profiling AMPARs at synaptic terminals. During this presentation, (including, mass spectrometry-based profiling and I will describe our latest results that have led us to protein array platforms) and the opportunities they identify an intricate network of distinct endosomal afford researchers to develop new system-based compartments and motor proteins that mediate the insights into biological processes will be considered. In bidirectional movement of AMPARs in and out of the particular, we have recently employed protein solution synaptic membrane during plasticity. and membrane array techniques to simultaneously [email protected] monitor labour-associated changes in more than 80 nuclear transcription factors and their interactions within L-15 human gestational tissues. The data obtained implicate TRANSLATING PROTEOMICS AND novel pathways that may provide insight into and help DEVELOPING PREDICTIVE define integrated systems behaviour at the time of CAPACITY human labour and delivery. Gregory E Rice [email protected] Translational Proteomics, Baker Medical Research Institute, Melbourne, Australia L-16 MECHANISMS OF CENTROMERE AND Over the past decade, we have experienced a KINETOCHORE FORMATION transformation in the zeitgeist of scientific investigation. Aaron Straight A transformation that has been, not so much, a move Department of Biochemistry, away from reductionism but a greater liberalism Stanford University and acceptance of more holistic (systems biology) approaches to investigating complex biological systems. The kinetochore is a unique chromosomal locus that Traditionally, the objective of scientific reductionism binds to the mitotic spindle so that chromosomes has been to isolate elements or subsets of related can segregate in mitosis. Kinetochore assembly observations or phenomena that account for the requires the specialized chromatin of the centromere phenotype and circumstance in a given domain of that is distinguished by the replacement of histone nature. The intellectual attraction of reducing complex H3 with the histone H3-variant centromere protein systems to smaller and more basic components A (CENP-A). CENP-A is thought to epigenetically resides in the ease of conceptualization, experimental mark the centromere and is required for kinetochore design and hypothesis testing. Limitations of this assembly but it is not known how this epigenetic mark approach have often emerged with attempts to is recognized to initiate kinetochore assembly. We have reconstruct complex systems based upon insights explored the molecular basis for centromere recognition thus generated. The discipline of proteomics, in part, by developing a simple and rapid in vitro assay represents a response to these limitations. Proteomics designed to identify human kinetochore components [i.e. the systematic, reproducible, differential and that directly and specifically interact with reconstituted quantitative measure of the protein expression in CENP-A nucleosomes. This approach led to discovery samples from a defined biological domain] is one of a that centromere protein N (CENP-N) is a DNA- number of systems biology approaches that has as a sequence independent CENP-A-nucleosome binding core paradigm the study of the interactions between protein. Nucleosomes reconstituted with a chimeric the components of complex biological systems histone H3 that contains only the centromere-targeting and how these interactions give rise to the function domain of CENP-A were sufficient for CENP-N binding. and behaviour of that system. Proteomics, thus, Point mutations that disrupted binding of CENP-N to is an integrative rather than reductionist approach CENP-A nucleosomes in vitro blocked localization to understanding how systems rather than their of CENP-N to the centromere in vivo, indicating that individual elements respond. Consistent with this nucleosome binding is required for the centromere- thesis, proteomic platforms now provided opportunity specific localization of CENP-N. Thus CENP-N to monitor protein and peptide response profiles to recognition of centromeric chromatin is an essential challenges to homoeostasis, experimental manipulation, step in the initiation of centromere and kinetochore 1. 35

livro.indd 35 7/7/2008 16:33:28 assembly. Round Tables [email protected] RT-01.1 L-17 HUMAN PANCREATIC ISLET CELL GUIDANCE CUES FOR DEVELOPMENT OF THE TRANSPLANTATION: ALTERNATIVE APPROACHES VASCULAR TREE Mari Sogayar Anne Eichmann Instituto de Química, Universidade de São Paulo, SP Institut National de la Santé et de la Recherche [email protected] Médicale, INSERM, Paris, France [email protected] RT-01.2 GENE THERAPY AND DNA REPAIR L-18 Carlos Frederico Martins Menck MECHANISMS OF CELLULAR Instituto de Ciências Biomédicas, Universidade de São SIGNALING AND REMODELING IN MYOCARD Paulo, SP Kleber G. Franchini [email protected] Faculdade de Ciências Médicas, Universidade Estadual de Campinas RT-01.3 [email protected] STEM CELL TRANSPLANTATION IN AUTOIMMUNE DISEASES L-19 Julio C. Voltarelli CALCIUM CHANNELS, Faculdade de Medicina de Ribeirão Preto, Universidade NEUROTRANSMITTER RELEASE AND SYNAPTIC de São Paulo, SP VESICLE RECYCLING Hematopoietic stem cell transplantation (HSCT) Osvaldo D. Uchitel has been performed since 1996 in more than 1,000 Institute of Physiology, Molecular Biology and patients with autoimmune diseases (AID) around the Neurosciences world, specially for systemic lupus erythematosus, Faculty of Exact and Natural Sciences, University of multiple sclerosis, systemic sclerosis and juvenile Buenos Aires, Argentina idiopathic arthritis. In these diseases, more than 50% of the patients with severe and refractory forms of the C-1 disease achieve durable clinical remission. In Brazil, BIOMECHANICS OF SINGLE MOLECULES AND a multicentric cooperative trial started in June/2001 SINGLE CELLS and about 150 patients have been transplanted for the Oscar Nassif Mesquita diseases mentioned above and also for early onset Departamento de Física, Instituto de diabetes mellitus (N= 21) which was a pioneer work in Ciências Exatas the world. In our presentation, we will review the results Universidade Federal de Minas Gerais, of HSCT for AID in Brazil and in the rest of the world, Belo Horizonte, MG the possible mechanisms mediating clinical benefits in those patients and the prospectives of using other We use optical tweezers, light scattering and defocusing modalities of stem cell therapy in AID. microscopy to study mechanical properties (static [email protected] and dynamic) of single molecules and single cells. With optical tweezers and light scattering we perform RT-02.4 stretching experiments on single DNA molecules to CELL SIGNALING SPECIFICITY IN HORMONE AND study interaction of DNA and drugs of pharmaceutical CYTOKINE COMMUNICATION NETWORKS interest. Using our recently developed defocusing Eduardo Arzt microscopy we study living cell motility. We show that a Laboratorio de Fisiologia y Biologia Molecular, defocused microscope is a phase contrast microscope Universtity of Buenos Aires, and CONICET, Argentina. with advantages over standard Zernike´s phase contrast microscope. With defocusing microscopy we are able The interaction between the immune and to study fluctuations of membrane and cytoskeleton of neuroendocrine systems involves the participation living cell surfaces, and distinguish fluctuations from the of he hypothalamic-pituitary-adrenal axis (HPA). upper and lower (in contact with a substrate) surfaces. Cytokines activate the HPA axis and induce a rise in It allows us to obtain biomechanical information from glucocorticoid levels, which are instrumental in order the free surface separately from that of the surface in to control immune-cytokine overreactions, acting on contact with substrates, or other media. Therefore, one T lymphocytes and other ctyokine/target cells. The can have access to adhesion parameters together with specificity of hormonal and cytokine signals on their other mechanical parameters, important to quantitatively target cells is crucial to provide specificity to their characterize living cell motility. We will present results actions. The cross-talk between cytokine-induced on macrophages and red blood cells. transcription factors such as the Th1 and Th2 Tbet, 1. 36

livro.indd 36 7/7/2008 16:33:28 and GATA-3, and steroid (i.e. glucocorticoid) receptors mechanisms. The combination of multielectrode involve both genomic and non-genomic actions, recordings of neuronal activity and in situ hybridization and constitutes the mechanism for fine tuning both for immediate early gene (IEG) expression shows hormone and cytokine responses. A further level of that the two phases of sleep cooperate to promote mutual regulation may be the ubiquitin-proteasome the propagation of memories from their entry point and sumoylation systems, which regulate GR (hippocampus) to their final destination (cortex). transactivation. Corticotrophin releasing hormone Investigating rats, we found that memories of novel (CRH) is the key mediator of the HPA axis at the objects fade within minutes in the hippocampus, but central nervous systemt. The final outcome of CRH/ persist reverberating in the cortex during sleep many CRH1 signaling depends on the context of specific hours after the end of object exploration. The non- cells and ligands, cross-talk of signaling pathways dreaming phase of sleep (SWS) reverberates recently and the effector actions of the pathways once they acquired changes in neuronal activity, maintaining are activated. This specificity bears consequences memories “alive” in the cortex for several hours after at the CNS level where CRH activates through the the offset of novel experience. The dreaming phase same receptor (CRHR1) different signaling pathways of sleep (REM) switches on the cortical expression of depending on neuroanatomical context. All these IEG related to memory stabilization and propagation. molecular interactions represent a key step for Altogether, the results indicate that novel experience understanding, at the cellular and genetic level, the is ensued by multiple waves of cortical – but not specificity and ultimate response of physiological hippocampal - plasticity as sleep cycles recur. As a neuroendocrine-immune interactions. consequence, memories become more dependent on [email protected] cortex than hippocampus as sleep goes on, migrating from their original entry circuits to deeper cortical RT-02.5 networks. A PARTICULAR NEURO-IMMUNE SYNAPSIS: [email protected] NORADRENERGIC NERVES AND LYMPHOCYTES Adriana Del Rey RT-03.9 Medical Faculty of Philipps University Marburg, PLASTICITY IN HUMAN MOTOR CORTEX AFTER Alemanha HAND ALLOGRAFT [email protected] Claudia D. Vargas Instituto de Biofísica Carlos Chagas Fo., Federal do Rio RT-02.6 de Janeiro, RJ NEURAL AND BEHAVIORAL CORRELATES OF EXPERIMENTAL The human motor cortex (M1) undergoes considerable MURINE ALLERGY reorganization in response to traumatic upper limb Frederico Costa Pinto amputation. The representation of the unaffected arm Departamento de Patologia, Faculdade de Medicina muscles expands, invading portions of M1 previously Veterinária, dedicated to the hand, suggesting that former hand Universidade de São Paulo, SP neurons are reassigned to the control of proximal [email protected] upper limb movements. Hand allograft to replace the amputated body part offers a unique opportunity RT-02.7 to study the reversibility of such long-term cortical CELLULAR AND MOLECULAR BASES OF changes. In this talk I will present evidence for cortical INTRATHYMIC IMMUNONEUROENDOCRINE reorganization in the human primary motor cortex (M1) INTERACTIONS of one bilateral hand allograft recipient (BY), tested Wilson Savino pre and post operatively. TMS pulses were applied at Fundação Oswaldo Cruz, scalp points overlying the primary motor cortex (M1) Rio de Janeiro, RJ with a Magstim stimulator via a 70mm figure-of-eight savino@fiocruz br coil. Each grid node was stimulated 6 times at 110% of the recorded muscle’s motor threshold (MT). The same RT-03.8 MT values were used pre and post graft. An estimate NEURONAL MECHANISMS OF of TMS pulse localization into the magnetic resonance MEMORY CONSOLIDATION image (MRI) of the subject’s brain was obtained Sidarta Ribeiro online by means of co-registration: each TMS site was Instituto Internacional de Neurociências de Natal localized using a 3D tracking system (Polhemus, Inc.) Edmond e Lily Safra, RN and co-registered on the subject’s MRI. Motor evoked potentials (MEP) were simultaneously recorded by Memories of places, things and events involve two means of Ag-AgCl surface electrodes from the abductor different brain portions: The hippocampus acts as a digiti minimi (ADM), opponens pollicis (OP), first short-term buffer but memories eventually move to interosseous dorsalis (FDI), biceps brachii (BB) and the cortex, a phenomenon with unknown underlying flexor digitorum superficialis (FDS). EMG sweeps were 1. 37

livro.indd 37 7/7/2008 16:33:28 band pass filtered (20-1000 Hz), digitized and recorded blood gases were used to quantify fetal blood flow, for successive off-line analysis. A map of the MEP oxygen delivery/consumption at elective Cesarean response throughout M1 was computed for each muscle delivery (n=70 at 3600, 78 at 400 m). Plasma venous of interest and plotted into the subject’s anatomical and arterial glucose concentrations were measured in MRI. Results in the transplanted patient showed that mothers and in cord blood from the doubly clamped intrinsic newly transplanted muscles acquired a full cord using a hexokinase-G6PDH coupled enzyme functional representation in the patient’s motor cortex assay. Data were analyzed by unpaired at ten months for the left hand and at twenty six months t-tests or ANOVA, as appropriate. after the graft for the right hand. The cortical emergence Results: Birth weight was decreased by >300 grams of transplanted hand muscles co-occurred with a shrink at 3600 m (p<0.0001) despite similarity in placental of stump muscles representation. The temporal gap in weight, gestational age and maternal attributes. While gaining a cortical status between the two hands was umbilical blood flow was lower at 3600 m, umbilical echoed by improved motor recovery for the left hand venous oxygen delivery was similar (0.46 ± 0.03 vs. compared to the right one. Our results show that newly 0.50 ± 0.03 mol/min/kg, mean SEM, p=0.40), as was transplanted muscles are recognized and integrated fetal oxygen consumption (0.26 ± 0.02 versus 0.25 ± in the patient’s brain in order to build novel limb motor 0.02 mol/min/kg). Maternal A-V glucose extraction was synergies. The mechanisms underlying these plastic increased at high altitude (1.25 versus 0.9 mM, p<0.05), phenomena will be discussed. but this did not translate into increased fetal glucose cdvargas@biof ufrj.br concentrations. High altitude neonates had reduced glucose concentrations in the umbilical vein (2.9 ± 0.01 RT-03.10 versus 3.60 ± 0.01 mM, p<0.0001) and artery (2.30 ± MOLECULAR MECHANISMS OF ODOR 0.01 versus 2.80 ± 0.01 mM, p<0.0001). Fetal glucose RECOGNITION consumption was reduced at high altitude (49.8 ± 6.0 Bettina Malnic mol/min/kg versus 69.0 ± 5.6 mol/min/kg at 400 Instituto de Química, Universidade de São Paulo, SP m, p<0.05). In fetuses at the lowest quartile of oxygen [email protected] consumption at 3600 m the glucose/oxygen quotient (mol of glucose consumed per mol of oxygen) was RT-03.11 increased (2.40 ± 0.7 vs. 1.00 ± 0.7, p<.005), indicating NEUROGENESIS INDUCED BY NON- increased anaerobic glucose consumption. CONVENTIONAL CONDITIONS IN EMBRYONIC Conclusion: These data suggest that the placenta is STEM CELLS consuming glucose, perhaps thereby sparing oxygen for Stevens Kastrup Rehen the fetus, but at the cost of reduced placental transport Instituto de Ciências Biomédicas, Universidade Federal of substrate for fetal growth. do Rio de Janeiro, RJ Support: NIH HD 04237, 046982, NSF BCS0309142 [email protected] [email protected]

RT-04.12 RT-04.13 PLACENTA-MEDIATED FETAL HYPOGLYCEMIA REGULATION OF MITOCHONDRIAL OXYGEN IS THE CAUSE OF ALTITUDE-ASSOCIATED CONSUMPTION IN PRIMARY HUMAN SYNCYTIAL REDUCTION IN FETAL GROWTH CELLS UNDER CONDITIONS OF HYPOXIA. Stacy Zamudio, PhD1, Tatiana Torricos, MD2, Maria M Nicholas P. Illsley, Oyola1, Lourdes Echalar, Lic2, Enrique Vargas, MD2 Ewa Fik-Rymarkiewicz, Stacy Zamudio. and Nicholas P Illsley, D Phil1. 1Obstetrics, Gynecology Department of Obstetrics, Gynecology and Women’s & Women’s Health, UMD-New Jersey Medical School, Health, UMDNJ-New Jersey Newark, NJ, United States, 07103 and 2Instituto Medical School, Newark, NJ, USA Boliviano de Biología de Altura, Universidad de San Andreas Mayor, La Paz, Bolivia. In a number of cell types hypoxic conditions have been shown, paradoxically, to inhibit mitochondrial O2 Introduction: Human residence at >3000 m is consumption, thus increasing cellular O2 availability. associated with diminished fetal growth, averaging Several inhibitory mechanisms have been described ~100 grams/1000 m gain in altitude. Placental hypoxia ranging from diversion of oxidative substrates to direct is present, as evidenced by up-regulation of HIF-1 inhibition of electron transport chain activity. While and HIF-regulated gene products. But deficits in this “oxygen sparing” phenomenon has several effects maternal oxygen delivery are not causally associated including decreased apoptosis in severely hypoxic with altitude-associated IUGR, nor do they contribute cells, we believe it may also have an important to placental hypoxia. We asked whether glucose, the role in the supply of substrates to the fetus. We most important fetal substrate, might be reduced or speculate that under hypoxic conditions, decreased preferentially consumed anaerobically, thereby sparing placental O2 consumption and the consequently oxygen, but limiting energy available for growth. increased glucose consumption may alter the mixture Methods: Doppler/imaging ultrasound, hematology and of substrates available for transfer to the fetus. To 1. 38

livro.indd 38 7/7/2008 16:33:28 test these possibilities, we have investigated rates increased hENT2 expression and activity. Thus, a of O2 consumption in trophoblast cells in which the potential role for adenosine as a differential modulator hypoxia response has been stimulated. The hypoxia of the ALANO pathway and hENT2 membrane response was stimulated using dimethyloxalylglycine transporters in HUVEC and hPMEC is proposed. (DMOG), a prolyl hydroxylase inhibitor which inhibits Support: FONDECYT 1070865/7070249, CONICYT HIF-1 degradation, thus elevating intracellular HIF-1 24071039/23070213. MECESUP/PUC PhD fellowships levels. In both BeWo choriocarcinoma cells and (CE), CONICYT PhD thesis support (CE, MG, MF), primary cultured syncytiotrophoblast cells, treatment PUC PhD fellowships (CP, JLV), CONICYT PhD with DMOG produced a decrease in O2 consumption fellowships (CP, BK, JLV, MG, CP, MF). in a dose-dependent manner, under conditions which [email protected] simultaneously showed a significant increase in HIF-1 and a significant increase in glucose consumption and RT-04.15 lactate production. Because these experiments were MAGNESIUM SULFATE AS AN ANTIOXIDANT performed under normoxic conditions, we conclude OF FULL TERM PLACENTAL EXPLANTS UNDER that the decrease in O2 consumption is not due to HYPOXIA. an inadequate O2 supply but rather as a result of Reinaldo Marín; Cilia Abad; Delia Indira Chiarello; the hypoxia response stimulated by increased HIF-1. Teresa Proverbio; These data demonstrate that the hypoxia response in Sandy Piñero; Fulgencio Proverbio trophoblast cells leads to an alteration in the available Laboratorio de Bioenergética Celular, Centro de mix of O2, glucose and lactate, key substrates Biofísica y Bioquímica, Instituto Venezolano de fueling fetal energy supply, with potentially significant Investigaciones Científicas, Caracas,Venezuela consequences for fetal growth. [email protected] Preeclampsia is an important complication affecting approximately 7% to 10% of pregnant women that RT-04.14 appears after the twentieth week of pregnancy, being HYPOXIA AND PREECLAMPSIA CHANGES ALANO far more common in the third trimester. This disorder SIGNALLING PATHWAY IN HUMAN PLACENTA is characterized by vascular endothelial damage, ENDOTHELIUM. abnormalities in plasma volume, hypertension, Casanello P, Prieto C, Krause B, Vega JL, González M, proteinuria, edema and generalized arteriolar Puebla C, Farías M, Escudero C, Sobrevia L. vasospasm. It can progress to eclampsia (seizures), Cellular and Molecular Physiology Laboratory (CMPL) renal failure, stroke, liver failure, pulmonary edema, and Perinatology Research Laboratory (PRL), and coagulopathy. Parenteral MgSO4 is widely used Department of Obstetrics and Gynecology, Medical to treat severe preeclampsia in order to prevent the Research Centre (CIM), School of Medicine, Faculty recurrent seizures of eclampsia and for tocolysis in of Medicine, Pontificia Universidad Católica de preterm labor. Even when MgSO4 has been in use for a Chile,Santiago, Chile. long time, its mechanism of action at a molecular level in preeclamptic/eclamptic pregnant women, remains Hypoxia and preeclampsia (PE), a disease associated an enigma. In previous works we have shown that the with restricted placental blood flow and placental Ca-ATPase activity of maternal and neonatal red cell hypoxia, are conditions where extracellular adenosine ghosts, myometrium and syncytiotrophoblast basal concentration is increased. Adenosine induces nitric (fetal facing) plasma membranes of pregnant women oxide (NO) synthesis and L-arginine transport (ie. with preeclampsia, is diminished by about 50%, as ALANO pathway) in human umbilical vein (HUVEC) and compared to the ATPase activity of the same tissues placenta microvascular (hPMEC) endothelium. Since from normotensive pregnant women. In addition, red extracellular adenosine concentration is maintained cell membranes from preeclamptic women show higher by an efficient uptake by the endothelium, we studied levels of lipid peroxidation than those from normotensive the potential role of human equilibrative nucleoside pregnant women. These results have been considered transporter 1 (hENT1) or hENT2 in the alterations to suggest a close relationship between the level of lipid induced by hypoxia and PE in HUVEC and hPMEC, peroxidation of the plasma membranes and the activity respectively. Primary cultured cells from normal or PE of the Ca-ATPase. In the present work, the effect of pregnancies were cultured (24 h) in normoxia (5-7% magnesium sulfate (MgSO4) on the Ca-ATPase activity O2) or hypoxia (2% O2). Hypoxia increased hENT1 and the level of lipid peroxidation of plasma membranes expression and activity, but reduced endothelial NO of syncytiotrophoblast from full term placental explants synthase (eNOS) and human cationic amino acid under hypoxia were examined. Explants of full term transporters 1 (hCAT-1) activity and expression in placentas from normotensive pregnant women HUVEC. However, hypoxia did not alter PE-increased were prepared. The explants were incubated under extracellular adenosine concentration. PE was hypoxia for 18 h at 37°C in absence and presence of associated with reduced activity of the inducible MgSO4. Syncytiotrophoblast plasma membranes were NOS (iNOS), hCAT-1 mediated L-arginine transport isolated in order to determine Ca- and Na,K-ATPase and hENT1 mediated adenosine transport, but with activities and level of lipid peroxidation. It was found 1. 39

livro.indd 39 7/7/2008 16:33:29 that placental explants incubated under hypoxia in the for the studies of steroidal disruptors due de fact that presence of MgSO4 show higher Ca- and Na-K-ATPase this gland to respond, in short time at low dosages, activities than those found in explants incubated under consistent modifications in morphopysiology. Financial hypoxia but in the absence of MgSO4. The diminution Support: FAPESP, CAPES and CNPq. of the level of lipid peroxidation by MgSO4, can account [email protected] for the increase in both Ca- and Na.K-ATPase activities in the placental explants under hypoxia. RT-05.17 [email protected] / [email protected] ESTROGEN IMPRINTING: FROM MORPHOLOGY TO BEHAVIOR RT-05.16 Hernandes F. Carvalho IMPACT OF STEROIDS AND ENDOCRINE Instituto de Biologia, DISRUPTORS ON THE MORPHOPHISIOLOGY OF Universidade Estadual de Campinas, SP MONGOLIAN GERBIL PROSTATE [email protected] Sebastião Roberto Taboga São Paulo State University - UNESP, Institute of RT-05.18 Biosciences, Humanities and Exact Sciences - IBILCE, DISRUPTION OF CELLULAR SIGNALING IN THE São José do Rio Preto, SP, Brazil EPIDIDYMIS BY BACTERIAL INFECTION: ROLE OF ANTIMICROBIAL GENES. The endocrine system is a complex network of Maria Christina W. Avellar glands and hormones that regulates many of the Department of Pharmacology, Section of Experimental body’s functions, including growth, development and Endocrinology, UNIFESP-Escola Paulista de Medicina, maturation, as well as the way various organs operate. São Paulo, SP Endocrine disruptors are exogenous molecules that are capable to promote the synthesis, liberation or Host defense proteins of the male reproductive tract accumulation, transport and metabolism of nature have become a subject of investigation in the last hormones that are important to the homeostasis and years in part because of their potential importance development. The prostate is an important target in sexually transmitted diseases. There are several of these molecules and its functional maturity and antimicrobial proteins reported in the male reproductive development is influenced by the steroids levels. In tract, including members of the antimicrobial β-defensin this sense, the studies about the effects of steroids family such as the SPAG11 gene, a gene highly and some steroids disruptors are fundamentally expressed along epididymis from different species. important for elicitation of the role of the steroids in Lipopolysaccharide (LPS), an endotoxin found in the prostate structure and physiology. Our research the outer membrane of Gram negative bacteria, is group has been evaluating the potential effects of the responsible for the activation of the innate immune steroidal agents on the Mongolian gerbil (Meriones response in a variety of mammalian cell types leading unguiculatus) male and female prostate using different to sepsis syndrome. It is known that LPS activates, morphological and immunohistochemical methods. through interaction with Toll-like receptor 4 (TLR4), an The principal agents used in our researches are intracellular pathway that culminates in the activation of cyproterone acetate (progestin), 17-beta-estradiol nuclear factor kappa-B (NFKB) transcription factor in a and etinil-estradiol, letrozole, tamoxifen, flutamide, variety of cells. finasteride, dehydroepiandrosterone (DHEA) and Our recent studies have focused on the characterization testosterone cypionate. All agents promote changes of the disruption of cellular signaling by LPS from E. into the normal pattern of the male and female coli, using rat epididymis as an experimental model. prostate histology, ultrastructure and steroid receptors Our work has provided the first evidence that this tissue imunohistochemistry. Major histopathological is fully capable of initiating an inflammatory response alterations were evaluated and quantified, such as to LPS through activation of TLR4. Constitutive TLR4 epithelial transient dysplasias and atypias, proliferative was immunolocalized in some, but not all, epididymal inflammatory atrophies, prostatic intraepithelial epithelial cells and in interstitial cells, some identified neoplasias and adenocarcinomas. In the prostatic as macrophages. TLR4 functionality was demonstrated stroma some alterations were observed such as, by in vivo LPS challenge (0.01 and 1 mg/kg, i.v.) which desmoplasias, fibroblast increase and smooth muscle induced a time- (0-24 h) and dose-dependent NFKB cells phenotype alterations. Ultrastructurally, these activation in caput and cauda epididymis. Within 2 h events are characterized by increase of secretion of LPS treatment, NFKB activation in epididymis was organelles, mitochondrial modifications and increase of accompanied by up-regulation of Il1b, Nfkbia and endosome-lysosomal system, including accumulation of Cd14, but not Tlr4, mRNA. These effects, however, ceramide-like myelinic bodies. Immunohistochemistry were not sustained after 24 h LPS treatment. No for androgen and alpha- and beta-estradiol receptors change in TLR4 immunostaining pattern was observed reveals that these receptors alter its expressions when epididymides from control and in vivo LPS- differently each treatment. In conclusion, our results treated rats were compared. Significant activation reveal that the gerbil prostate may be an important tool of NFKB was also achieved within 1 min of in vitro 1. 40

livro.indd 40 7/7/2008 16:33:29 incubation of caput epididymis with LPS (0.01-5 µg/ provoking increased sperm reserves in this region, but ml), confirming that the components for TLR4 signaling the sperm quality was not compromised, as shown cascade activation are fully active in this tissue. We by in utero artificial insemination. Animals exposed to have also shown that products of the rat Spag11 DES showed an acceleration of sperm transit time in antimicrobial gene (SPAG11C and SPAG11E) are not the epididymis, and consequently decreased sperm only expressed in epididymal epithelial cells, but also density, and diminished sperm motility. In both cases in smooth muscle cells surrounding epididymal tubules sperm production was not altered. Testosterone and blood vessels, in few interstitial cells and in luminal supplementation was able to restore the transit time sperm. Immunohistochemistry has indicated that in to values close to normality, since they were higher vivo LPS treatment induces changes in the distribution than in the control rats. The same occurred in relation and abundance of SPAG11E- and SPAG11C-positive to sperm motility. Rats exposed to DES presented elements along rat epididymis depending on protein lower fertility after in utero artificial insemination using isoform and region of the epididymis (caput and cauda) sperm collected from the proximal cauda epididymidis. analysed, suggesting that SPAG11 gene products aid Therefore, it was concluded that the acceleration in the defense of the male reproductive tract against of rat sperm transit time appeared to harm normal infection. Taken together the results contribute to a sperm maturation, thus decreasing sperm quality and better understanding of the innate immune response in fertility capacity, in an androgen-dependent way. In this the epididymis and its importance in physiological and presentation the general definitions about endocrine pathological processes. disruptors will be provided, and emphasis will be given Financial Support: FAPESP, CAPES, CNPq, TW on the effects on the epididymis and the process of Fogarty International Center. sperm maturation. Financial support: FUNDUNESP, [email protected] CAPES, CNPq, FAPES [email protected] RT-05.19 THE EPIDIDYMIS AS A TARGET ORGAN FOR RT-06.21 ENDOCRINE DISRUPTORS. PLACENTAL MORPHOGENESIS IN NORMAL AND Wilma De Grava Kempinas PATHOLOGICAL CONDITION Departamento de Morfologia, Instituto de Biociências/ (1)S. San Martin, (2)Tostes R, (2)Fortes Z and (2)Zorn TM. UNESP, Botucatu, SP (1) Reproductive Biology Research Centre, School of Medicine, Universidad de Valparaiso-Chile. (2) Institute The epididymis is the organ in which sperm mature of Biomedical Sciences, University of São Paulo, Brazil. by developing the capacity for progressive motion and fertilizing ability. Much of the maturation The placenta plays an essential role in the process appears to involve an interaction between maintenance of pregnancy and foetal growth. the epididymal epithelium, luminal fluid, and sperm. During the morphogenesis and development of this Sperm maturation occurs in an orderly fashion, with organ, a coordinated process of proliferation and the acquisition of the capacity for progressive motility differentiation of trophoblast cells are require. In this preceding the ability to fertilize, which in turn, precedes context, adequate trophoblast invasion is essential for the ability of the fertilized egg to develop normally. embryo implantation and placentation in mammals. Testosterone and dihydrotestosterone are involved in The basic phases of trophoblast differentiation the maturation of sperm within the epididymis as well as and placenta development are common among the transit of sperm through the duct, but the manner in species that have haemochorial placentation, such which these events are regulated is poorly understood. as rodents, non-human primates, and humans. In Endocrine disruptors, by definition, are exogenous rodents, placentation begins with trophoblast cell agents that interfere with the synthesis, storage/release, proliferation, which forms the ectoplacental cone. transport, metabolism, binding, action or elimination The cone-derived cells then differentiate to form the of natural blood-borne hormones responsible for labyrinth, the spongiotrophoblast layer, and the giant the regulation of homeostasis and the regulation of cell compartment. These structures constitute the developmental processes. Recent studies demonstrated fetal interface with the maternal tissues. Reproductive that some chemicals exert deleterious effects on disorders, such as diabetes, during the pregnancy the epididymis, independent of the testis (ex. alpha- are related to abnormal placental development and chlorohydrin, methyl chloride, cyclophosphamide). In morbidity of offspring in human and animal models. the past 10 years our lab has been involved in studying This pathology is associated with an increased risk the influence of altered sperm transit time through the of fetal death, macrosomia, growth retardation and epididymis on sperm parameters and fertility of rats. increase of placental weight. Placentas from women For this, adult rats received either diethylstilbestrol - with gestational diabetes have been reported to have an DES (with or without testosterone replacement), or abnormal syncytiotrophoblastic layer, cytotrophoblastic guanethidine sulfate - GUA, to accelerate and delay the hyperplasia, focal thickening of trophoblastic basement sperm transit time, respectively. GUA treatment delayed membranes with both amorphous and fibrillary the sperm transit time through the epididymal cauda, components, and narrowing of small vessels. In a 1. 41

livro.indd 41 7/7/2008 16:33:29 diabetic rat, alterations of morphology and extracellular pathway. Genotoxic stresses that induce DNA breaks matrix protein distribution were found in placenta. induce p53 phosphorylations. This phosphorylated and These observations suggest that diabetes may induce thus stabilised protein acts as an onco-supressor by alteration in cellular function of human and animal inducing either cell cycle arrest (allowing the DNA to models that affect placental development and alter be repaired) or apoptosis. This important mechanism gas exchange and nutrient transfer to the offspring. thus avoids DNA damage to be carried over to daughter Single injections of alloxan (40 mg/kg) were used to cells. If the p53 gene is mutated or if the p53 pathway is induce diabetes on day 2 of pregnancy in Wistar rats. defective, then p53 is no longer protective and becomes Placentas were collected on days 14, 17, and 20 of oncogenic. It has been shown that p53 mediates pregnancy. We used immunoperoxidase techniques activation of metalloproteinases (MMP) MMP-2 and to identify members of the extracellular matrix potently inhibits expression of MMP-1 and MMP-13 protein and cell proliferation protein (Ki67) in various gene transcription. Given that human MMP-9 promoter compartments of the rat placenta and to determine has several features in common with the promoters of whether experimentally induced diabetes affects these MMPs and since it was shown that MMP-9 over- placental morphology and alters the distribution of these expression is strongly associated with the functional molecules during pregnancy when compare with control loss of p53 in different carcinoma cells, we hypothesized animals. Prior to the immunoreaction, some samples that MMP-9 expression could also be regulated by wild were stained with haematoxylin and eosin for the type p53 (wt-p53) in the human trophoblast. To test this morphological and morphometric studies. hypothesis, transcription assays were performed with Diabetes induced in rats is a suitable model of human chloramphenicol acetyl transferase reporter driven by type I diabetes and is potentially useful for clarify serial deletion of hMMP-9 promoter, co-transfected with some of diabetes outstanding questions. The present expression vector of p53 in HT1080 cells and human results showed that the diabetes model employed cytotrophoblastic cells (CTB). The results demonstrate in this study promotes morphological changes in the that wt-p53 down-regulates the –670, but neither the size and organization of the different compartment of –531 nor the -90 promoters activity of hMMP-9. On the placenta, in the cell proliferation and in the distribution other hand, the tested p53 mutants loose partially their of extracellular matrix glycoprotein such as fibronectin. repressive activity towards MMP-9. Experiments with Immunolocalization in conjunction with a comprehensive mutated hMMP-9 promoters revealed that NF-kB at morphological analysis as performed in this study position -600 and Ets at position –541 are involved in allow us to detect specific microenvironments within p53 trans-repression of MMP-9. Moreover, results from the placenta which was identified by the presence of band shift assays demonstrated that MMP-9 promoter specific ECM components. In addition, we showed that was inhibited by p53, through a NF-kB dependent but a diabetes promote several morphological changes that NF-kB binding independent mechanism. Taken together, may affect the normal development of placenta and these observations indicate that the hMMP-9 gene is a offspring. Supported by grants from DIPUV (Universidad target gene for p53 repression. de Valparaiso, Valparaiso, Chile; grant no. 24/2004; In experiments designed to test the effects of 12/2006). endogenous trophoblastic p53, we observed that [email protected] inhibition of endogenous p53 with pifithrin alpha or p53 siRNA did not increase the activity or expression of RT-06.20 MMP-9 in CTB. This indicates that in CTB p53 is indeed ACQUISITION AND REGULATION OF AN INVASIVE expressed and decreased by p53siRNA, but that it is PHENOTYPE IN NON-MALIGNANT CELLS. functionally incompetent (as has been described for Paul Bischof tumour cells) since it cannot modify MMP-9 expression. Department of Obstetrics and Our surprise was even greater when we performed Gynaecology, University of Geneva, Geneva, immunohistochemistry on first trimester trophoblast Switzerland with antibodies raised to p53. With the antibody Ab 5, recognising a wild type conformation of p53, no The establishment of an invasive phenotype in staining could be observed in trophoblast whereas it the placenta (trophoblast) as well as in tumours was positive in decidua. In contrast, antibody Ab 3, involves a host of cellular processes and an array of recognising a mutated conformation of p53, showed expression and/or repression of several genes such an abundant cytoplasmic localisation of p53 in villous as those involved in cell adhesion, composition of the and extra-villous trophoblast. We speculate that extra-cellular matrix, matrix digestion, angiogenesis, the functional incompetence and the cytoplasmic apoptosis or cell cycle arrest. It is now commonly localisation of p53 are linked and that this particular admitted that trophoblast invasion and tumour status of trophoblastic p53 could be instrumental to the progression depend on the same biochemical and invasive behaviour of CTB and of the embryo. molecular mediators The transcription factor and [email protected] tumour suppressor p53 plays a key role in malignant transformation. Almost all invasive human tumours have RT-06.22 either an inactivated p53 gene or a defect in the p53 ENDOMETRIAL EXTRACELLULAR MATRIX DURING 1. 42

livro.indd 42 7/7/2008 16:33:29 THE and V. In addition, using immunofluorescence we have PERIMPLANTATION PERIOD showed that collagen type III was chiefly expressed Telma Zorn in the three regions of pregnant endometrium being Laboratory of Reproductive and Extracellular Matrix the only collagen to be present in the materno-fetal Biology, Institute of Biomedical Sciences, University of interface on the day of embryo implantation suggesting São Paulo, Brazil that collagen type III may offer an adequate substratum for trophoblast invasion and embryo implantation. After conception, extensive morphological and Fibrilli-1 is a constitutive molecule in the pregnant and functional changes that take place in the uterus can non-pregnant endometrium, although some changes be followed along the pre-implantation, implantation, in its organization occur after the embryo implantation. and post-implantation stages. Proliferation of Supported by FAPESP and CNPq. endometrial fibroblasts as well as remodeling of [email protected] components of the extracellular matrix occur during early pregnancy in most mammals. Following either RT-07.23 blastocyst implantation or proper uterine stimulation, BIOLOGY AND THERAPEUTIC POTENTIAL OF the endometrium undergoes decidual differentiation. MESENCHYMAL STEM CELLS Decidualization is a singular phenomenon that occurs Nance B. Nardi during pregnancy in several species of rodents as well Universidade Federal do Rio Grande do Sul, Instituto de in humans, in which fibroblasts of the endometrium Biociências, Porto Alegre, RS redifferentiate under physiological conditions after their [email protected] exposure to a specific hormonal sequence. These redifferentiated fibroblasts are called decidual cells and RT-07.24 can be considered as a new cell type because of their STEM CELLS IN A TISSUE CONTEXT modified morphology and acquisition of new synthetic Radovan Borojevic capabilities. Decidualization can be considered as Instituto de Ciências Biomédicas, Universidade Federal an organogenesis, taking place, however, within the do Rio de Janeiro, RJ confines of an organ - the uterus- and consequently [email protected] limited by it. Collagen, glycosaminoglycans , proteoglycans and multiadhesive glycoproteins RT-07.25 undergo modifications in their structure, organization CELLULAR THERAPY IN ANIMAL MODELS OF and molecular composition during decidualization in the NEUROLOGICAL mouse indicating that remodeling of extracellular matrix DISEASES components is required for successful implantation and Rosália Mendez-Otero development in the mouse. In mice proteoglycans are Instituto de Biofísica Carlos Chagas Fo, Universidade temporal and spatially expressed in the endometrium . Federal do Rio de Janeiro, RJ Members of the family of small leucine-rich proteoglycan [email protected] (SLRPs) show a dynamic balance in the mouse endometrium during pregnancy. Decorin was present RT-08.26 together with lesser amount of lumican in the stroma PHOSPHORYLATED NUCLEAR MYOSIN VA before the onset of decidualization, whereas biglycan DISTRIBUTES FROM SPECKLES TO NUCLEOLI and fibromodulin were expressed in decidualized DEPENDING UPON TRANSCRIPTION ACTIVITY regions of the endometrium, but decorin was absent. Roy E. Larson, Munira M. A. Baqui and Claudia Perlecan, was identified in basement membranes Tavares dos Santos. during the pre-implantation. Contrary, the expression Departamento de Biologia Celular e of syndecan-4 was quite weak. In the periimplantation Molecular e Bioagentes Patogênicos, Faculdade de period perlecan was no longer observed in the stroma Medicina de Ribeirão Preto, Universidade de São whereas syndecan-4 increases markedly after the onset Paulo, Ribeirão Preto. of decidualization. The coordinate balance between pre- and post-implantation periods indicates a role of these Nuclear actin and nuclear myosins have been recently molecules in the embryo implantation and development. implicated in the regulation of gene expression in Very thick collagen fibrils (reach up 500nm in diameter) vertebrate cells. Myosin V is a class of actin-based appear around mouse decidual cells as soon as motor proteins involved in cytoplasmic vesicle decidualization is triggered. We hypothesized that the transport and anchorage, spindle-pole alignment interposition of thick collagen fibrils between decidual and mRNA translocation. Recently we have shown cells may act as barrier that helps to block leukocyte by immunofluorescence microscopy that myosin- migration within the decidua, participating in this way Va, phosphorylated on a conserved serine in its tail in preventing immunological recognition and rejecting domain (phospho-ser MVa), is localized to nuclear of embryos tissues. Recently, using double immnogold compartments referred to as “speckles”, which are electron microcopy we have demonstrated that the thick rich in splicing factors responsible for the processing collagen fibrils are formed at least by collagen type I, III of pre-mRNA. In the present study we applied 1. 43

livro.indd 43 7/7/2008 16:33:29 immunofluorescence techniques with confocal RT-08.28 microscopy to determine the localization of phospho- ILLUMINATING PRION PROTEIN TRAFFICKING AND ser MVa and other factors, such as the splicing FUNCTION factor SC35 that earmarks nuclear speckles, in the Marco Antonio Maximo Prado nucleus of HeLa cells treated with drugs that inhibit Departamento de Farmacologia, UFMG. transcription and phosphorylation. Our data show that phospho-ser MVa is associated mainly with speckles Conversion of the cellular prion protein (PrPc) into an in normal transcribing cells, but when transcription is abnormal, protease resistant isoform (PrPsc) has been inhibited by actinomicin D or prematurely aborted by suggested to participate in the pathogenesis of prion 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DRB), diseases. This conversion occurs at the cell surface or there is a relocalization of phospho-ser MVa to the in intracellular compartments just after endocytosis. In nucleoli, as well as a separation of a subpopulation of order to understand the trafficking of PrPC and PrPSc, phospho-ser MVa from SC35 into near-neighboring we have developed fluorescent proteins that allowed particles. These observations indicate a novel role for for investigation of prion protein trafficking in living myosin Va in nuclear compartmentalization and offer cells. We found that PrPC and PrPSc follow distinct a new lead towards the understanding of actomyosin- intracellular pathways in cells. The cellular functions based gene regulation. Support: FAPESP, CNPq, of PrPC are not completely understood, but previous FAEPA work has shown that recombinant PrPC interacts with [email protected] STI-1 promoting neuritogenesis and neuroprotection by distinct signaling pathways. STI-1 is a co-chaperonin RT-08.27 that function as a secreted neurotrophic factor. We THE SPINDLE ASSEMBLY CHECKPOINT PROTEINS found that STI1, but not a STI1 mutant unable to bind AND THEIR ROLES IN PROGRESSION THROUGH PrPC, can evoke PrPC endocytosis. STI1 induced MITOSIS signaling does not occur in cells devoid of endogenous Claudio. E. Sunkel PrPC, however heterologous expression of PrPC Instituto de Biologia Molecular e Celular, Universidade reconstituted both PKA and ERK1/2 activation. In do Porto, Portugal contrast, a PrPC mutant which lacks endocytic activity is unable to promote ERK1/2 signaling induced by The Spindle assembly checkpoint (SAC) monitors STI1, but it can reconstitute PKA activity in the same correct attachment of chromosomes to spindle condition, suggesting a key role of endocytosis in the microtubules. The SAC delays mitotic exit when former process. The activation of ERK1/2 by STI1 is chromosomes fail to attach correctly and activates transient and appears to depend on the interaction correction mechanisms. Previous work has shown of the two proteins at the cell surface or short after that BubR1, Mad2 and Bub3 are key components of internalization, as fluorescent STI1 and PrPC show this checkpoint, however, there still many unanswered distinct patterns of intracellular localization in living questions. Previously we showed that SAC proteins cells. Moreover, inhibition of dynamin activity by are already required during the G2-M transition for expression of a dominant-negative mutant causes the the proper accumulation of cyclins and progression accumulation and colocalization of PrPC and STI1 at through mitosis (Lopes, C.S., Sampaio, P., Williams, the plasma membrane, suggesting that both proteins B., Goldberg, M. and Sunkel, C.E. (2005). J.Cell Sci. use a dynamin-dependent internalization pathway. 118, 187-198). More recently work from our lab has These results uncover potential compartments involved shown that Mad2 plays an essential role in providing with PrPC conversion and illuminate mechanisms time during early stages of prometaphase to allow full of cellular signaling dependent on PrPC, suggesting engagement of the SAC (Orr, B., Bousbaa, H. and that trafficking of these proteins can influence their Sunkel, C.E. (2007). Mol. Biol. Cell 18, 850-863) and physiological functions. that BubR1 and the kinetochore motor protein CENP-E Supported by: CNPq and FAPESP. have cooperative roles in ensuring proper kinetochore [email protected] microtubule attachment (Maia, A.F., Lopes, C.S. and Sunkel, C.E. (2007) Cell Cycle. 6, 1367-1378). These RT-09.29 results argue that at least some of the SAC components REDOX MODULATION OF THE probably play multiple roles during progression through PROTEASOME 20S mitosis. Currently we have continued to study the role Marilene Demasi of these proteins during progression through mitosis by Instituto Butantã, SP investigation how kinetochore components contribute to [email protected] the SAC. Here, I will describe some our recent work in which have studied how mitotic progression and SAC RT-09.30 activity occur in the absence of different kinetochore FBXO25- ASSOCIATED NUCLEAR DOMAINS components. (FANDS): A NOVEL [email protected] SUBNUCLEAR STRUCTURE Marcelo Damário Gomes 1. 44

livro.indd 44 7/7/2008 16:33:29 Faculdade de Medicina de Ribeirão Preto, Universidade of mutant NKX2-5 protein with known transcriptional de São Paulo, SP co-factors, but reduced DNA binding and transcriptional [email protected] activation. Levels of the mutant protein were increased 3-fold due to reduced degradation by the ubiquitin- Skp1, Cul1, Rbx1 and the FBXO25 protein form a proteasome system (UPS). Increased protein levels functional ubiquitin ligase complex. Here we investigate were also observed for other reported human NKX2-5 the cellular distribution of FBXO25 and its colocalization mutations located within the homeodomain but not for with some nuclear proteins using immunochemical mutations outside this region. Interestingly, prolonged and biochemical approaches. FBXO25 was monitored overexpression of these mutations caused a secondary with affinity-purified antibodies raised against the impairment of the UPS, leading to accumulation of recombinant fragment spanning residues 2 to 62 of the ubiquitinated proteins in the cellOur data provides FBXO25 sequence. FBXO25 protein was expressed evidence that impaired transcriptional activation in all mouse tissues tested except striated muscle, as and DNA binding coupled to a progressive defective indicated by immunoblot analysis. Confocal analysis ubiquitin-mediated degradation contributes to the revealed that the endogenous FBXO25 was partially pathogenesis of DCM and suggest that in addition to concentrated in a novel dot-like nuclear domain that is transactivation defects, protein aggregate formation distinct from clastosomes and other well-characterized may contribute to myocardial dysfunction in NKX2- structures. These nuclear compartments contain a 5-related cardiac pathologies. These observations high concentration of ubiquitin conjugates and at least highlight the importance of NKX2-5 not only in cardiac two other components of the ubiquitin-proteasome development but also in the maintenance of normal system: 20S proteasome and Skp1. We propose adult heart function, and suggest that changes in to name these compartments FANDs for FBXO25 post-translational protein regulation may potentiate the associated nuclear domains. Interestingly, inhibition of effects of inherited gene mutations and contribute to transcription by actinomycin D or heat shock treatment disease pathogenesis. drastically affected the nuclear organization of FBXO25- [email protected] containing structures, indicating that they are dynamic compartments influenced by the transcriptional activity RT-09.32 of the cell. Also, we present evidences that an FBXO25- UBIQUITIN-MEDIATED DEGRADATION OF XIAP - DO dependent ubiquitin ligase activity prevents aggregation WE KNOW ENOUGH TO TARGET IT FOR of recombinant polyglutamine-containing huntingtin THERAPIES? protein in the nucleus of HEK293 cells, suggesting that Cinthya Sternberg this protein can be a target for the nuclear FBXO25 (Instituto Nacional de Câncer - INCa) mediated ubiquitination. Manipulation of anti-apoptotic factors may constitute an RT-09.31 important approach to treat degenerative diseases in FUNCTIONAL ANALYSIS OF A NOVEL the future. The XIAP (X-linked inhhibitor of apoptosis) NKX2-5 MUTATION UNRAVELS A NEW ROLE protein - which is a member of the ubiquitin ligase family FOR UBIQUITIN-PROTEASOME SYSTEM IN - is expressed in the nervous system and directly binds CARDIOMYOPATHIES and inhibits caspases, therefore inhibiting apoptosis. Costa MW1, Guo G2, Wolstein O2, Castro M2, Li D2, XIAP and XIAP-like proteins are downregulated in Harvey RP2 & Fatkin D2. several degenerative diseases and this low expression 1 Instituto de Biofísica Carlos Chagas Filho, UFRJ, might reflect an increased degradation of these Brazil. 2 Developmental Biology and Sr Bernice proteins by the ubiquitin-proteasome system (UPS), Research Program in Inherited Heart Diseases, Victor culminating in increased susceptibility of neurons to Chang Cardiac Research Institute, Sydney, Australia. cell death. Ubiquitination is a tightly controlled process of protein degradation that involves multiple enzymes Mutations in the cardiac transcription factor NKX2-5 are of the UPS as well as many modifying and ancillary associated with diverse congenital heart defects as well proteins. Aberrations in the UPS have been implicated, as progressive impairment of cardiac function in mice. either as a primary cause or secondary consequence, Mutation screening of the protein-coding sequence in the pathogenesis of both inherited and acquired of the NKX2-5 was performed in probands from 90 degenerative diseases. XIAP is targeted to degradation families with familial dilated cardiomyopathy (DCM) via the UPS, probably via its autoubiquitination. This linked or not to conduction abnormalities (CD) and/ activity is stimulated following its interaction with Smac, or congenital heart disease. One novel heterozygous which is released from mitochondria during apoptosis. missense mutation in the homeodomain region of Adenovirus-mediated delivery of XIAP is currently in the NKX2-5 gene was identified in one family. The test, but a degradation-resistant XIAP could increase clinical phenotype of affected family members was the efficacy of this type of therapy, protecting XIAP characterised by adult-onset DCM and CD and sporadic from endogenous antagonists. XIAP bears several congenital heart disease association. Functional domains that interact with multiple regulators and/or molecular studies demonstrated preserved interactions substrates. Identification and mapping of such regions 1. 45

livro.indd 45 7/7/2008 16:33:29 and determination of sites of ubiquitination are valuable accompanied by the respective description. The slides tools to develop therapy-suitable DNA constructs with belong to the Histology courses of the Department enhanced efficacy. of Morphology of the Institute of Biology, Federal [email protected] University of Pelotas (UFPel), Brazil. It is therefore an illustrated syllabus of slides studied during lab RT-10.33 classes in this University. As the syllabus was aimed LEARNING BIOLOGY AT DISTANCE to our students, the slides are identified by letters and Marlene Benchimol numbers according to the slides collections that the CEDER-Centro de Ensino a Distância do Estado do Rio students use during lab sessions and is organized by de Janeiro, Universidade Santa Ursula, Rio de Janeiro tissues and organs. The students can easily choose [email protected] subjects from a content list. By generating this study material we wish to stimulate the interest of the students The Cederj is a center where Biology and other courses towards learning Histology and to contribute to improve are teach. We have developed an approach to teach the quality of pre-clinical teaching, which is fundamental Biology using animations, videos, images and a short for obtaining competent professionals. text where the student can follow a complete class [email protected]; without the necessity of a professor. The academic [email protected] staff includes PhDs and masters in different areas of Biology. They are in charge of preparing the protocols RT-10.35 and direction of art of the class. Each one receives MOL - UM GUIA INTERATIVO DE every month a subject and prepares the class, frame HISTOLOGIA by frame. The second group in the staff is responsible Paulo A. Abrahamsohn for the artistic part of the class. They work with (ICB/USP) different programs of animations, such as the use of the advanced Flash and 3-D Studio. The third group The study of cells, tissues and organs under the prepares the illustrations. The forth group prepare the light microscope is considered a very difficult task by exercises and for this develops new programs, since undergraduate students. Many of the complaining that the exercises are in form of games. The fifth group is they can not recognize or identify structures, perceive made by language reviewers. The sixth group is made their main components and features and, worst of by web-designers that make the class upload. The all, differentiate between similar tissues or organs. seventh group classifies and stores the animations in an Being essentially based on images, this activity is Image Bank, for future use. Until the present moment very appropriate to be learned on a computer. We are we have about of 30,000 images and animations. We developing a guide for the study of cells, tissues and have already prepared about 300 classes in Biology, organs entitled Mol - Microscopy Online. It intensively including Cell Biology, Biochemistry, Molecular Biology, uses tools of the World Wide Web to precisely point Genetics, Zoology, Botanic, Immunology, among others out and highlight features of the images. Each image that are in preparation. These classes are also stored is accompanied by a text that presents the principal in CDs and DVDs and we intend to distribute them for components of the image and sometimes contains those people interested. a brief theoretical background. The purpose of this guide is to present in a very straightforward way the RT-10.34 components and the morphology of cells, tissues and BASIC INTERACTIVE HISTOLOGY: AN ELECTRONIC organs. Important c ATLAS omponents stand out by the touch of the cursor, or in Maria Gabriela Tavares Rheingantz & Iria Guimarães most cases, by setting the cursor over the image or Machado over words indicative of the components. The guide Instituto de Biologia, allows free back and forth navigation among chapters. Universidade Federal de Pelotas, RS Features of a tissue or organ that had been presented earlier in the guide have links for fast recollection of Learning Histology as takes place during laboratory past topics by the student. classes depends on analysis and understanding [email protected] histological slides. To use the slides collection the students are, however, limited in most cases to the RT-10.36 schedule of their classes as the Histology labs are INTERACTIVE TEACHING AND LEARNING OF usually used by many other students from other EMBRYOLOGY courses. Histology textbooks and printed atlases are Pereira, LAV sometimes not available for all students as many of Department of Histology and them need to use the books at the same time. Embryology, Institute of Biology, State University of For these reasons we decided to organize the contents Campinas (UNICAMP) of a Histology lab course as an interactive CD-ROM. This CD-ROM contains images of histological slides In the traditional Human Embryology courses the 1. 46

livro.indd 46 7/7/2008 16:33:30 students have difficulties in understanding and in isolate was used. The cells monolayers were damaged, creating three-dimensional mental images of the showing several signs of cell death by apoptosis, processes involved in normal development and in birth such as membrane blebbing, vacuolization, DNA defects. In this speech we will show our experience fragmentation, apoptotic bodies, etc. Cells were also in using multimedia and multidisciplinary approach detached from the substrate and lysed. Phagocytosis to stimulate the acquisition of knowledge and create occurred with trichomonads avidly eating large portions possible solutions to the referred difficulties. Our of epithelial cells. Modifications of the epithelial cell teaching material included clinical histories, movies, membranes as well junction formation between the animations, digital documentation of the ultrasound, trichomonad and the epithelium was seen. The lytic radiograph images, autopsies and digital quizzes – all capacity of the trichomonas appears to be the result of of them related to embryos, fetus and neonates. These mechanical disturbance on the membrane of the target teaching tools were used in the Human Morpho- cell aided by intense parasite movement below the cells Physiology discipline of the medical curriculum at the monolayer. Epithelial cell debris were ingested and State University of Campinas. The teaching materials rapidly digested in lysosomes. The cytopathic action were also organized in educational softwares used by of T. vaginalis on cell monolayers can be divided in the the students as a complement to the lectures. At the following stages: (1) recognition; (2) attachment; (3) end of the discipline, the material and methodology cytolysis; (4) phagocytosis; (5) intracellular digestion. were evaluated via an attitudinal measuring scale instrument, interview and knowledge examination. RT-11.38 Most of the students approved of the method and MAMMALIAN CELL INVASION BY emphasized the importance of integration between Trypanosoma Cruzi INFECTIVE FORMS basic and clinical disciplines and the use of digital Renato A. Mortara, Claudio V. da Silva, Maria Cecília videos, computer animations and interactive software Fernandes, Emanuelle B. Gaspar, Miriam P. Oliveira that facilitates the comprehension of normal and and Solange da Silva. abnormal human development. Departamento de Microbiologia, Imunologia e [email protected] Parasitologia, UNIFESP, Escola Paulista de Medicina, São Paulo, SP RT-11.37 Trichomonas vaginalis KILLS AND EATS – In the mammalian host Trypanosoma cruzi EVIDENCE FOR PHAGOCYTIC trypomastigotes have to invade cells in order to grow ACTIVITY AS CYTOPHATIC EFFECT and multiply and the flagellated trypomastigotes Marlene Benchimol have been implicated in this process. The parasite Universidade Santa Ursula, occurs as different strains or isolates grouped in two Rio de Janeiro, RJ major phylogenetic lineages: T. cruzi I associated to [email protected] the sylvatic cycle and T. cruzi II, linked to the human disease. Several parasite surface components and Tritrichomonas foetus and T. vaginalis are extracellular few host cell receptors with which they interact parasites of the reproductive tract in cattle and humans, have been identified. Our work mainly focused respectively. The mechanism by which trichomonas on how amastigotes, usually found growing in causes abortion is not completely known. There are no the cytoplasm, can invade mammalian cells with studies of infection in the oviducts, and how host-cells infectivities comparable to that of trypomastigotes. interacts with ciliated cells, since almost all published We found differences in cellular responses induced papers are related to vagina infection and few articles by amastigotes and trypomastigotes regarding focusing on the uterus. The aim of the present study cytoskeletal components and actin-rich projections. was to establish a working model of bovine oviduct Extracellularly generated amastigotes (EA) of T. cruzi I epithelial cells and submit these cells to trichomonas strains may display greater infectivity than metacyclic interactions. Twenty bovine oviducts were obtained trypomastigotes (MT) towards cultured cell lines as well from cows at a commercial abattoir and T. foetus as target cells that have modified expression of different was injected through the isthmus into the oviduct classes of cellular components. We also observed that lumen, and after interaction, cells were analyzed EA of T. cruzi I strains were more infective towards using calorimetric assays, detection of cell viability cultured cells than parasites of T. cruzi II group, a assays ,fluorescence microscopy, videomicroscopy, behavior opposite to that of the classic trypomastigote and scanning and transmission electron microscopy. forms. We then hypothesized that the expression of the This study reports structural mechanisms of cytopathic carbohydrate epitope defined by Mab 1D9 on Ssp-4, a effect of Trichomonas vaginalis on primary culture major amastigote surface antigen, was related to the bovine monolayers. Trophozoites of different degrees greater infectivity of T. cruzi I amastigotes, particularly of virulence were co-incubated with the monolayers of the G strain. By examining T. cruzi II isolates from for a maximum of 24 h. Injury was observed as early chagasic patients with distinct pathologies, we then as 6 h. After 12 h of incubation, the cell monolayer verified that, although carbohydrate epitopes defined was severely damaged when a fresh trichomonad by Mabs 1D9 and 2B7 were involved in EA invasion, 1. 47

livro.indd 47 7/7/2008 16:33:30 their abundance on the surface of the parasite was RT-12.41 not directly related to their infectivity. In the search of THE USE OF COOPERATIVE GAMES IN THE additional parasite components that might be related TEACHING OF THE CELLULAR BIOLOGY to cell invasion, a DNA microarray approach has been Marco A. F. Randi1, Hernandes F. Carvalho2 undertaken by screening components that, being more 1Depto. de Biologia Celular / Setor de Ciências abundant in G strain amastigotes, in comparison to the Biológicas / UFPR; 2 Depto. de Biologia Celular / less infective CL strain parasites, could be associated to Instituto de Biologia / Unicamp this phenotype. A hypothetical protein of 21 kDa with a signal peptide was selected and cloned. P21 is secreted Active and collaborative learning are important aspects by amastigotes and is ubiquitous among T. cruzi strains in discussing teaching methodology nowadays. Take and developmental stages. Although the recombinant the students off his passive position and bringing them protein (His6-P21) binds in a dose-dependent manner to the responsibility upon their own learning was the to cells, it does not trigger Ca2+ transients. Both His6- fundamental basis for this research. Thus, a different P21 and an anti- His6-P21 Mab inhibit cell invasion by approach of class was created, where the students G strain parasites [C.V. Silva et al., in preparation]. We participate in a game where they are characters in are currently examining the potential signaling pathways a story and must resolve problems based in cellular that we envisaged are triggered by secreted P21 at biology concepts that they need to obtain by themselves the local micro-environment after attachment of the through reading textbooks, assisted by a master (the amastigote forms to the host cell. story teller, usually played by the teacher). The classes In another approach aiming at understanding the role were applied to undergraduate students of two public of cholesterol and membrane rafts in mammalian universities and one private institution. The concepts cell invasion by T. cruzi infective forms, we used the studied were ATP synthesis, endocytosis and digestion following tools: the cholesterol depleting compound and the cell cycle. To evaluate the validity of this methyl--cyclodextrin, filipin a cholesterol-binding method, students were stimulated to respond a Likert antibiotic and cholera toxin subunit B (CTX-B) that based survey test or to construct concept maps before binds to ganglioside GM1, a sphingolipid enriched and after the class; also, his testimonies were taken in membrane rafts. Depletion of host cell cholesterol anonymously as a form of qualitative measure. Analysis decreased both MT and EA invasion (of either G or of the results lead to the following conclusions: (1) CL strain), that were restored by replacing cholesterol. cooperative games are efficient in learning cell biology; MT and to a lesser extent EA ergosterol removal also (2) apparently, this type of teaching tool increases the reduced their infectivity. Cholesterol recruitment to the student self-confidence in the subjects learned; (3) the sites of EA and MT invasion was disclosed by filipin students considered this type of class dynamic and labeling recently-infected cells. Moreover, CTX-B not pleasurable. only inhibited cell invasion by both infective forms [email protected] but was also found in parasitophorous vacuoles (PV) of recently-internalized parasites, thus unveiling that RT-12.42 both cholesterol and membrane rafts participate in TO BE ANNOUNCED mammalian cell invasion by T. cruzi infective forms. Bayardo Torres Financial support: FAPESP, CNPq and CAPES. Instituto de Química, Universidade de São Paulo [email protected] [email protected]

RT-11.39 RT-12.43 PROTEOLYTIC PATHWAYS LEADING TO ENSINO DE BIOLOGIA MEDIADO POR COOPERATIVE ACTIVATION OF COMPUTADOR G-PROTEIN COUPLED RECEPTORS DURING HOST Eduardo Galembeck CELL INVASION BY Depto de Bioquímica Trypanosoma cruzi Laboratório de Tecnologia Educacional do IB, Unicamp, Julio Scharfstein CAMPINAS, SP Instituto de Biofísica Carlos Chagas Fo, Universidade Federal do Rio de Janeiro, RJ O uso de computadores no ensino de Biologia oferece [email protected] recursoscompatíveis com diversas metodologias de ensino, o que inclui métodostradicionais e métodos RT-11.40 inovadores. A observação, o conhecimento de PLASMODIUM FUNCTIONAL fenômenos biológicos, da nomenclatura científica, a GENOME elaboração de explicações sobre processos biológicos Celia Regina da Silva Garcia e o seu confronto com explicações científicas são Instituto de Biociências, Universidade de São Paulo algumas das habilidades fundamentais ao ensino [email protected] de Biologia e demandam diferentes metodologias e estratégias de ensino. Em relação à observação de fenômenos biológicos, por exemplo, na maioria dos 1. 48

livro.indd 48 7/7/2008 16:33:30 casos não conseguimos proporcionar aos estudantes o provides a reservoir of bioactive molecules for the acompanhamento dos mesmos, quer seja na natureza, signaling of tertiary dentinogenesis. This pool of latent quer seja “in vitro”. O uso de imagens, vídeos e TGF-beta present in dentin matrix can be activated representações externas (modelos, gráficos, esquemas, by the proteolytic cascade initiated by extracellular etc.) são uma alternativa para mostrar e ilustrar cathepsin B. Since MMPs have been indicated fenômenos biológicos sendo que os computadores to participate in dentinal caries development and proporcionam um excelente ambiente para uso progression, the aim of this study was to examine the integrado de tais mídias. Serão apresentadas algumas cysteine protease expression in human odontoblasts experiências do Laboratório de Tecnologia Educacional and cysteine activity in dentinal caries lesions in relation do IB, Unicamp, no desenvolvimento, utilização e to the lesion depth and activity, and to evaluate the avaliação de mídias no ensino de Biologia. potential sources of the enzymes. The hypothesis [email protected] was set that if odontoblasts would express cysteine proteinases, they would be present in caries lesions; RT-13.44 and that salivary cysteine proteinases would contribute LYSOSOMAL CYSTEINE PROTEINASES to the lesion progression. (Supported by CAPES, CNPq ARE IMPLICATED IN DEVELOPMENT AND and FAPESP) PROGRESSION OF HUMAN CARIOUS DENTIN [email protected]; [email protected] LESIONS Minciotti1, C.L., Nascimento2, F.D., Trevelian3, M.M., RT-13.45 Geraldeli4, S., Tersariol1, I.L.S* and Tjäderhane5, L.* P-SELECTIN AS A THERAPEUTIC TARGET IN 1Centro Interdisciplinar de Investigação Bioquímica, THROMBOSIS, INFLAMMATION AND CANCER: THE UMC, SP; 2Depto de Bioquímica, UNIFESP, SP, 3Depto ROLE OF HEPARIN ANALOGUES. de Patologia Bucal, Faculdade de Odontologia, USP- Mauro S.G. Pavão SP; 4Dows Institute for Dental Research and Operative Laboratório de Tecido Conjuntivo, Hospital Universitário Dentistry, University of Iowa, USA; 5Institute of Dentistry, e Instituto de Bioquímica Médica – UFRJ University of Helsinki, Finland. The interface among different biological processes The triple helical collagens, in particular type I and III is beneficial but in many cases contributes to the collagens, are highly resistant to general proteolysis pathogeneses of diseases. This is particularly and require specific proteases for their degradation. relevant in the case of coagulation, inflammation The mammalian enzymes with collagenolytic activities and cancer. The inter-relationship among these include members of the matrix metalloprotease processes is due to mechanisms that lead to the (MMP) family such as MMP-1, -2, -8, -13, and -14, production of fundamental molecules common to the the serine protease, human neutrophil elastase, and three diseases. P-selectin and PSGL-1 are vascular thiol-dependent cathepsins. Enhanced collagenolytic adhesion molecules that have important roles in the activity had been observed in carious dentin. The interactions between cells. In coagulation, P-selectin presence of MMP2, MMP8, and MMP9 was identified from activated platelets is responsible for the capture in demineralized dentinal lesions in vitro. The latent of leucocytes and pro-coagulant microparticles during purified forms of these enzymes were activated at thrombogenesis by a PSGL-1-involving mechanism. acidic pH, followed by neutralization. These enzymes In inflammation, interaction of leukocyte PSGL-1 were not found in samples of cariogenic bacteria. The and endothelial P-selectin is essential for leukocyte lysosomal cysteine proteinases cathepsins can degrade rolling and transmigration. Similarly, platelet P-selectin extracellular matrix proteins such as type I collagen, and Sialy lewis-containing oligosaccharides from laminin, fibronectin and proteoglycans. This class of tumor cells mediate tumor cell-platelet interaction enzyme has been implicated in a variety of pathological during hematogeneous metastasis. Therefore, conditions, especially in diseases involving tissue inhibition of P-selectin and or PSGL-1 can attenuate remodeling states. It has been shown that cathepsins B thrombosis, inflammation and metastasis. Heparin is an and L cleave in the non-helical telopeptide extensions of antithrombotic agent commonly used in the treatment collagens and cathepsin K cleaves the collagen at the of thrombosis. Recently, it has been shown that in triple helical region. Cathepsin B can be linked to the addition to its anticoagulant effect, heparin inhibits activation of procollagenase, prostromelysin and pro- P-and L-selectin and as a consequence it attenuates uPA, enzymes involved in the cell surface proteolytic inflammation and metastasis. However, the use of cascade observed in focal degradation of osteoid and heparin as an anti-selectin agent is restricted by its tumoral matrix. TGF-beta1 and other members of this potent hemorrhagic effect and the search of alternative family of growth factors (BMP) have been implicated in anti-selectin compounds has increased recently. tooth development and dental tissue repair. Members During the last years we have found unique sulfated of the TGF-beta family of growth factors are important glycans in marine invertebrates. We have isolated novel in modulation of odontoblast secretory activity during molecules with interesting characteristics that have not dental tissue repair. The presence of endogenous been previously described and showed unequivocally pools of TGF-beta in the dentin matrix and pulpal cells that these glycans can inhibit the binding of selectins 1. 49

livro.indd 49 7/7/2008 16:33:30 to their natural ligands. In addition, recent results from gene regulatory regions or intronic regions and 28 our laboratory have shown that the invertebrate glycans sequences are associated to CGs expressed in the inhibit thrombosis, inflammation and metastasis in salivary gland. The observation that we have identified experimental animals without significant bleeding effect. a set of genes that share cis-regulatory elements mpavao@hucff ufrj.br] suggests that they are regulated by common sets of transcription factors and therefore are members of the RT-13.46 same node in a transcriptional regulatory network.The TO BE ANNOUNCED second group of results to be presented constitutes Helena Bonciani Nader the characterization of the BhC4-1 proximal repressor Departamento de Bioquímica, region (-1293/-254), previously reported to restrict Universidade Federal de São Paulo, SP BhC4-1 expression in the ring gland to the second [email protected] larval instar. Preliminary results in lines transformed with two constructs that constitute deletions of this RT-14.47 repressor region reveal BhC4-1-lacZ expression in the PAIR-RULE STRIPED PATTERN REGULATION IN ring gland, during the larvae to prepupae transition.The THE third group of results comprises those obtained from the SEGMENTATION CASCADE OF functional characterization of the BhC4-1 core promoter. Drosophila melanogaster By employing the UAS-GAL4 system we tested the Luiz Paulo Moura Andrioli ability of this core promoter to function in different Instituto de Biociências, Universidade de São Paulo, SP developmental stages and distinct tissues. Our results [email protected] indicate that the BhC4-1 core promoter is permissive and enables gene expression in different larval tissues, RT-14.48 as well in the germ line. In this context, our laboratory INSIGHTS IN DEVELOPMENTALLY REGULATED has been contributing both with the generation of new MECHANISMS IN DROSOPHILA: LESSONS FROM A tools for functional assays in Drosophila and unraveling HETEROLOGOUS SCIARID PROMOTER new information on gene regulatory mechanisms in Nadia Monesi insects. Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, FCFRP-USP, SP, Brazil RT-14.49 MOLECULAR AND FUNCTIONAL DNA puffs are a feature of sciarids and are formed CHARACTERIZATION OF JAZIGO: EXPLORING at discrete sites in the salivary gland polytene NEW ROLES OF chromosomes prior to the pupal molt. The BhC4-1 FORMINS DURING DEVELOPMENT OF Drosophila DNA puff gene of Bradysia hygida is amplified and Cláudio Simon abundantly transcribed in the salivary gland when DNA Universidade Federal do Triangulo puff C4 is formed. BhC4-1 amplification and expression Mineiro, Uberlândia, MG are both induced as a late response to the increase of ecdysone titers that triggers metamorphosis. The A previous genetic screen looking for mutations existence of a transformation system, coupled to afecting salivary gland programmed cell death during genomic information and the availability of mutant Drosophila development lead to theidentification of collections in Drosophila melanogaster, prompted jazigo. A locus that encodes a protein sharing high us to perform functional assays of DNA puff genes homology to components of the Formin family of in this model system. Overall, previous results from proteins. Several reportshave shown that formins are our laboratory strongly suggest that the mechanisms active players in cytoskeleton remodeling in different that control BhC4-1 expression are largely conserved biological phenomena such as cell division, vesicular in Drosophila, and revealed that the cis-regulatory trafic and cell polarity. In addition, formins were elements which control BhC4-1 expression are evolutionarily conserved in different organisms, such recognized by Drosophila trans-activating factors. as yeast and mammals. Our studies have shown that The first set of results to be presented concerns jazigo is expressed in different tissues undergoing those derived from the bioinformatics and functional cell death and that, mutations obtained by imprecise studies which resulted in the characterization of both P-element excisions, cause drastic cell death defective a salivary gland and a ring gland enhancer, located phenotypes such as cell death arrest and salivary gland within the BhC4-1 proximal promoter. The functional persistency disrupting tissue responses to the presence assays revealed that the sciarid enhancers drive of ecdysone. In Drosophila, two distinct pulses of developmentally regulated patterns of gene expression ecdysone (steroid hormone) trigger stage and tissue- either in the ring gland or in the salivary gland, specific responses through the activation of a cascade regardless of their orientation. The bioinformatics of early and late responsive genes to the presence of analyses led to the identification of 162 sequences the hormone. Among these responses we can highlight similar to the sciarid promoter within the Drosophila the programmed cell death of larval salivary glands genome. Of these, 121 sequences are located in either and midgut which occur in distinct periods. Jazigo 1. 50

livro.indd 50 7/7/2008 16:33:30 transcription is selectively regulated by ecdysone during de Rst em Drosophila e os de seus homólogos em development in a similar fashion to other cell deah outros organismos, especialmente vertebrados, e quais genes, which respond to the presence of ecdysone. Our a suas implicações para o entendimento da evolução data constitutes the first evidence of formin-like activity dos modos de desenvolvimento embrionário em on cell death processes in Drosophila, and we are now metazoários. validating jazigo´s function as a formin-like protein using [email protected] different approaches, including: purification of polyclonal antibodies; confocal analysys of the cytoskeleton RT-15.51 remodeling during cell death in wild type and mutant PURINERGIC SYSTEM AND ZEBRAFISH: flies; and production of transgenic flies for functional “in TOXICOLOGICAL IMPLICATIONS vivo” RNAi mediated gene knockout; Herewe present Carla Denise Bonan1, Eduardo Pacheco Rico3 Mario our last progresses towards the understanding of Roberto Senger3, Denis Broock Rosemberg3 Renato Jazigo´s function during development Dutra Dias1, Mauricio Reis Bogo2 [email protected] 1Laboratório de Neuroquímica e Psicofarmacologia e 2Laboratório de Biologia Genômica e Molecular, Depto. RT-14.50 Biologia Celular e Molecular, Faculdade de Biociências, FUNÇÃO E EVOLUÇÃO DA FAMÍLIA NEPH/RST DE PUCRS, Porto Alegre, RS, Brasil.3Departamento de MOLÉCULAS DE ADESÃO CELULAR EM Drosophila Bioquímica, Instituto de Ciências Básicas da Saúde, E UFRGS, Porto Alegre, RS, Brasil. VERTEBRADOS Ricardo P.G.Ramos Zebrafish (Danio rerio) is a small freshwater teleost Depto De Biologia Celular e Molecular e Bioagentes belonging to Cyprinidae family. Due its characteristics, Patogênicos, Faculdade de Medicina de Ribeirão Preto, the zebrafish is a model system in studies involving Universidade de São Paulo developmental biology, toxicology, transgenic research, vertebrate genome evolution, teratology and A especificação do destino celular durante o neuroscience. Several neurotransmitter systems have desenvolvimento de um organismo depende been already described in zebrafish. Among them, the freqüentemente de interações celulares precisamente purinergic system, in which ATP acts as an important orquestradas no espaço e no tempo, as quais por sua neurotransmitter and neuromodulator. Extracellular vez são mediadas por uma variedade de moléculas ATP evokes responses through the ionotropic P2X and de adesão. Nosso grupo de pesquisa vem já há vários the metabotropic P2Y receptors. The neurotransmitter anos estudando o papel da proteína Roughest (Rst) ATP can be inactivated through hydrolysis by enzymes de Drosophila melanogaster , o membro prototípico da known as ectonucleotidases. Nucleoside 5´-tri and família Neph-Rst de glicoproteínas transmembranares diphosphates may be hydrolyzed by NTPDase da superfamília das imunoglobulinas. Membros desta (nucleoside triphosphate diphosphohydrolase) família foram identificados desde em nematódeos family whereas nucleosides 5´-monophosphates até humanos, sempre envolvidos em processos de are subjected to hydrolysis by ecto-5´-nucleotidase. adesão e sinalização durante o desenvolvimento Therefore, the final product of ATP degradation embrionário e pós-embrionário. Em Drosophila, a promoted by nucleotidases is adenosine, an important integridade estrutural de Rst e sua correta regulação neuromodulator. Our laboratory has characterized the no espaço e no tempo são necessários, entras outras presence of NTPDase and 5´-nucleotidase activities in funções, para o desenvolvimento dos sistemas zebrafish brain membranes. Considering the important nervoso, visual e olfatório, da musculatura somática, role played by the purinergic system, we evaluated da histólise das glândulas salivares larvais, além this neurotransmitter system as a potential target of de interferir com a celularização inicial do embrião neurotoxic compounds. Thus, we analyzed the effect quando expresso inapropriadamente nos estágios of pesticides, heavy metals, and organic compounds iniciais da embriogênese. Na presente palestra on nucleotidase activities. Among the pesticides, serão discutidos resultados recentes obtidos em the exposure in vitro and in vivo to carbofuran and nosso laboratório relacionados com a caracterização malathion on nucleotidase activities was investigated estrutural e funcional de Rst e sua regulação espacial e in central nervous system (CNS) of zebrafish. After temporal, visando: a)Entender as complexas relações carbofuran exposure (50 and 500 µg/L) during 7 existentes entre os diferentes domínios estruturais days, ADP hydrolysis was decreased (19 and 24%, das proteínas da família Neph-Rst e as suas múltiplas respectively). Malathion, at 500 µg/L during 7 days, was funções durante o desenvolvimento; b) Definir e able to inhibit ADP and AMP hydrolysis (28 and 58%, caracterizar contextualmente as funções gênicas e respectively). This study has shown that nucleotidases vias de transdução de sinais capazes de modular from zebrafish brain membranes can be targets to the diretamente a atividade do gene rst tanto em nível neurotoxicity promoted by pesticides. Furthermore, the transcricional como pós transcricional e c) Estabelecer effect of lead, mercury, and copper was investigated on o grau de conservação e divergência evolutiva entre nucleotidases from zebrafish CNS. Lead and mercury os mecanismos moleculares subjacentes às funções promoted changes in ATP, ADP, and AMP hydrolysis 1. 51

livro.indd 51 7/7/2008 16:33:30 after 96h and 30 days of treatment whereas there shown that ADA might be located in both intracellular were no changes in NTPDases and 5´-nucleotidase and membranes, acting as an ecto-ADA. Considering gene expression. Copper is a divalent cation with that the adenyl-deaminase family groups distinct physiological relevance, since the copper deficiency ADA-related subfamilies and that the adenosinergic can induce neurological disorders. The treatment with system plays a key role in the signaling within tissues, copper sulphate (15 µg/L during 24 h) inhibited ATP the aim of the present study was to identify and to hydrolysis whereas ADP and AMP hydrolysis were not map the expression profile of different ADA-related altered. The exposure to copper during 96h significantly genes in zebrafish tissues. Furthermore, a kinetic altered ATP (31%), ADP (42%), and AMP hydrolysis characterization of ADO deamination activity in the brain (40%). NTPDase1, NTPDase2_mg, and NTPDase2_ of this teleost was also performed. mv mRNA transcript levels decreased after copper A phylogenetic analysis confirmed the presence of exposure during 24 and 96h. Copper treatment during different ADA-related genes (ADA1, ADAL and two 96h also reduced the gene expression for NTPDase2_ ADA2 orthologous). Using optimized semi-quantitative mq and 5´-nucleotidase. Organic compounds, such as RT-PCR assays, the relative amount of transcripts methanol, are widely used in industry and can induce was determined in brain, gills, heart, liver, skeletal damage to CNS. After acute treatment with 0.5 and 1% muscle and kidney. The ADA1 gene presented a similar methanol (during 1 h), there was a significant decrease expression pattern in the studied tissues, whereas in ATP and ADP hydrolysis, but there were no changes ADAL was less expressed in the heart. The highest in ecto-5´-nucleotidase activity. Moreover, methanol relative amount of ADA2-1 transcripts was detected in was able to decrease gene expression of NTPDase1 brain, liver and gills, being less expressed in the heart. and NTPDase2. Our results suggest that nucleotidases The other ADA2 form (ADA2-2) was expressed at can be altered differentially by the action of pesticides, comparable levels within the studied tissues. Besides, heavy metals and organic compounds in zebrafish CNS. the adopted strategy also allowed the identification Therefore, these enzymes may be important neurotoxic of a truncated alternative splice isoform of ADA2-1 targets of these compounds, influencing the purinergic (ADA2-1/T), which was expressed in distinct intensities. signaling. Supported by: CNPq, CAPES, FAPERGS, Concerning that ADO is an important neuromodulator FINEP (IBN-Net # 01.06.0842-00) in CNS and that different ADA-related genes are [email protected] present in zebrafish, the ADO deamination activity was characterized in both soluble and brain membrane RT-15.52 fractions. The optimal pH of ADA activity was verified in ZEBRAFISH AS A MODEL FOR ADENOSINERGIC the range of 6.0-7.0 in soluble fraction and reached 5.0 SYSTEM STUDIES in membranes. A significant decrease of 31.3% on ADA Maurício R. Bogoa, Denis B. Rosembergb, Eduardo P. activity in membranes was obtained in the presence Ricob, Diogo O. of 5 mM Zn2+, which was prevented by 5 mM EDTA. Souzab, Carla Bonanc. The apparent KM for ADO deamination were 0.22 + a Laboratório de Biologia Genômica e Molecular, 0.03 and 0.19 + 0.04 mM for soluble and membrane Faculdade de Biociências, Pontifícia Universidade fractions, respectively. The apparent Vmax for soluble Católica do Rio Grande do Sul. ADA activity was 12.3 + 0.73 nmol NH3. min-1. mg-1, b Departamento de Bioquímica, Universidade Federal whereas the calculated Vmax for membrane-bound do Rio Grande do Sul. ADA was 17.5 + 0,51 nmol NH3. min-1. mg-1. In both c Laboratório de Neuroquímica e Psicofarmacologia, cellular fractions, ADO and 2’-deoxyadenosine were Faculdade de Biociências, Pontifícia Universidade deaminated in higher proportions when compared to Católica do Rio Grande do Sul guanine-derivate nucleosides. A significant inhibition on soluble and membrane ADA activity was observed Zebrafish (Danio rerio) has several advantageous in the presence of 0.1 mM EHNA, a classical ADA1 characteristics that make it useful as a prominent inhibitor. The existence of different ADA-related genes vertebrate model for developmental biology, genetics and the ADO deamination activity in zebrafish brain lead and other biomedical areas studies. This animal shares us to suggest that this animal is an emergent model for the relevance of a vertebrate with the scalability of adenosinergic system studies. an invertebrate organism. The knowledge from the [email protected] “Zebrafish Genome Project” associated to its ability to quickly absorb chemical substances directly from RT-15.53 water is attractive for using this model in toxicological USING ZEBRAFISH AS A MODEL FOR STUDYING and human diseases studies.n the CNS and in different THE CELL BIOLOGY OF THE MYOGENESIS tissues, adenosine (ADO) is an important signaling Manoel Luís Costa molecule which plays its effects via specific P1 Lab. Diferenciação Muscular e Citoesqueleto purinoreceptors. The irreversible hydrolytic deamination Instituto de Ciências Biomédicas of ADO into inosine promoted by adenosine deaminase Universidade Federal do Rio de Janeiro (ADA) is one of the mechanisms responsible for controlling the levels of this nucleoside. Studies have The detailed study of myofibrillogenesis used to require 1. 52

livro.indd 52 7/7/2008 16:33:30 cell cultures systems, which do are a three-dimensional, [email protected]; complex verônica@uerj br environment. To overcome this limitation, we have been studying cytoskeletal and cell adhesion structures and RT-17.58 their interactions during zebrafish muscle development. CYTOSKELETON AS TARGET TO ANTICANCER Since there are no easily available pre-assembled fish DRUGS raising systems, we constructed our own system, using Glaucia M.M. Santelli common aquaria parts, such as UV lamps, porous Instituto de Ciências Biomédicas, ceramics and activated charcoal. We use antibodies Universidade de São Paulo and fluorescent probes to follow specific proteins and [email protected] molecules in zebrafish embryos, using conventional and laser-scanning confocal optical microscopy. We RT-17.59 also use biochemical analysis, including one and two- LAMININ PEPTIDES IN TUMORAL BIOLOGY dimensional electrophoresis, and electron microscopy. Ruy Jager After a considerable struggle, we are now have a Instituto de Ciências Biomédicas, Universidade de São steady production of eggs. The remarkable embryo Paulo transparency allow us to follow specific proteins with [email protected] high definition, and, therefore to follow specific cellular components: actin, alpha-actinin, troponin, etc, (markers RT-17.60 of myofibrils), paxillin and vinculin (intracellular adhesion CELL-CELL ADHESION IN EPITHELIAL CELLS AND complexes), laminin and fibronectin (extracellular CANCER matrix), and desmin and plectin (intermediate filaments). José Andrés Morgado Diaz The use of a heat shock to disrupt somites provides us Grupo de Biologia estrutural, Centro de Pesquisas – a more physiological approach. The growing demand INCa, RJ for zebrafish as a research model led us to establish several collaborations. We have been able to describe Barrier function and polarity of epithelial cells depend novel protein structures at a sub-cellular level, and to on specialized intracellular structures referred as the propose a model of when and where these structures apical junctional complex (AJC). The most apical tight are formed in the zebrafish myogenesis junction (TJ) and its subjacent adherens junction (AJ) [email protected] constitute the AJC. Both TJs and AJs are multiprotein complex composed of transmembrane proteins that RT-15.54 affiliate with cytoplasmic plaque proteins. The former ZEBRAFISH AS A MODEL TO STUDY EVOLUTION proteins mediate cell-cell adhesion, whereas the AND DEVELOPMENT OF CARDIAC CHAMBERS latter link TJs and AJs to the actin cytoskeleton and José Xavier Neto participate in intracellular signaling. The AJC is a highly Instituto do Coração, Universidade de São Paulo, SP dynamic structure and may rapidly and reversibly [email protected] disassembled in different physiological and pathological circumstances as in epithelial-mesenchimal transition RT-16.55 (EMT). EMT is a key process in tissue development FIBROBLAST GROWTH FACTOR 2 (FGF2): and carcinogenesis in which cells acquire a mobile CLASSICAL AND NOVEL MECHANISMS IN CELL phenotype characterized by loss of cell polarity and cell- CYCLE CONTROL cell adhesion, and actin cytoskeleton rearrangement, Hugo Armelin however the cell signaling pathways regulating this Instituto de Química, Universidade de São Paulo, SP events still remain to be defined. The main focus of [email protected] our Group is to elucidate the cellular mechanisms involved in the loss of cell-cell adhesion transforming RT-16.56 a non-migratory, non-invasive epithelial cell into a FUNCTIONAL CHANGES ASSOCIATED WITH migratory invasive mesenchimal cell. In a previous CELL-CELL AND CELL-ECM INTERACTIONS IN study we used chemical activation of PKC and EGFR MELANOMAS with 12- O-tetradecanoylphorbol-13-acetate (TPA) and Luiz Fernando Lima Reis pretreatment with protein kinase inhibitors to analyze Hospital AC Camargo/Fundação Antônio Prudente, SP the effect of the phorbol ester on the distribution of AJC [email protected] proteins in Caco-2 cells. TPA treatment resulted in AJs disassembly, but the TJ structure and function remained RT-16.57 unaltered. We observed that the major effect caused MATRICELLULAR PROTEINS IN ANGIOGENESIS by TPA was a decrease of the cytoskeleton-linked AND CANCER E-cadherin. EGFR and PKC inhibitors significantly Verônica Morandi blocked the effect on AJs, and inhibition of the TPA Depto Biologia Celular e Genética, Centro Biomédico, effect by the PD98059 MAPK inhibitor suggests that Universidade do Estado do Rio de Janeiro, RJ this kinase was the final event in the modulation of 1. 53

livro.indd 53 7/7/2008 16:33:30 cadherin-dependent cell-cell adhesion. Pretreatment of other hand, during the life of a cell, molecular motors cells with Calphostin C before EGF treatment, blocked appear to antagonize intracellular death factors keeping the redistribution of E-cadherin, strongly indicating them under check, until specific extracellular cues or that both EGFR and PKC are involved in TPA-induced insults prompt their release to trigger apoptosis. Class V cell signaling for modulation of cadherin-dependent myosins as well as dynein were proposed to sequester cell-cell adhesion. A recent research in our laboratory proapoptotic BH3-only members of the Bcl2 superfamily using Caco-2 cells and the well-known extracellular of proteins, major sensors of cell damage, including Ca2+ depletion model identified a regulatory cascade loss of cell adhesion, that trigger the mitochondria- that mediate AJC disassembly and actin cytoskeleton dependent cell death by provoking its outer membrane organization. Pharmacological inhibition of PKA with permeabilization and release of cytochrome c and H-89 prevented AJC protein redistribution and loss of other death factors in the cytosol. Recent data from our TJ functionality caused by calcium depletion, as well laboratory suggest that myosin-Va enhances melanoma as actin disorganization at apical and medial regions cell adhesion on fibronectin matrix and this role may of the cell volume. Rho inhibition using Toxin A caused involve an interaction with the focal adhesion kinase. AJC disruption and actin cytoskeleton reorganization. We have also demonstrated that interference in the Y-27632, a ROCK inhibitor, prevented redistribution function of myosin-Va by expressing a small fragment of of E-cadherin, but not of tight junction proteins, as this protein in tumor cells can induce them to undergo a well as the actin cytoskeleton disorganization caused rapid apoptotic response, marked by classic alterations by the calcium depletion. The low calcium medium that characterize this condition, such as, cytochrome and Forskolin treatment caused activation of Rho and c realease in the cytosol, caspase 9 and caspase 3 Rac as evidenced by their translocation to the plasma activation, active cell blebbing, phosphatidylserine membrane fraction, and this effect was reverted by externalization detected by Annexin V staining of H-89. Thus, in this study our findings demonstrate a the cell surface, TUNEL positive DNA fragmentation, central role of a regulatory cascade that integrates PKA and the cell rupture into apoptotic bodies. We also and Rho-family GTPases in the AJC disassembly and discuss the molecular mechanisms that account for the actin cytoskeleton organization in tumor epithelial cells. response triggered by this myosin fragment as well as Identification of this and others cell signaling pathways future directions and possible applications in cancer and its mechanism of action in epithelial cancer are the therapy. current focus in this area of research in our laboratory Financial support: CNPq, FAPESP, CAPES, FAEPA jmorgado @inca.gov.br [email protected]

RT-18.61 RT-18.62 MODULATION OF CELL ADHESION AND MYOSIN-10 AND ACTIN FILAMENTS ARE APOPTOSIS BY THE ACTIN-BASED MOLECULAR ESSENTIAL FOR MITOTIC SPINDLE FUNCTION MOTOR MYOSIN-VA Sarah Woolner1, Lori O’Brien2, Christiane Wiese2 and ENILZA M ESPREAFICO Bill Bement1 Departamento de Biologia Celular e Molecular e Dept. of Zoology, University of Wisconsin-Madison, Bioagentes Patogênicos, Faculdade de Medicina de Madison, USA.Dept. of Biochemistry, University of Ribeirão Preto, University of São Paulo Wisconsin-Madison, Madison, USA

Molecular motors, mechanoenzymes that use The role of actomyosin in the mitotic spindle has the energy of ATP hydrolysis to generate force long been a subject of controversy. Here we present on cytoskeleton polymers, comprise three protein evidence that Myosin-10 (Myo10), an actin-based motor superfamilies, which include dyneins and kinesins as that binds microtubules, participates with F-actin to microtubule-based motors, and myosins as F-actin- regulate mitotic spindle function. Using Xenopus laevis based motors. In addition to the conserved motor early embryos as an in vivo system to study mitosis, we domain, all molecular motors are built with extra show that Myo10 localizes to the mitotic spindle where domains that confer specific and multiple roles to each it concentrates at the spindle poles from prometaphase member of these protein superfamilies, evolved to through to the end of telophase. Knockdown of Myo10 play a diverse array of cellular functions, including a levels using an anti-sense morpholino approach reveals plethora of forms of cell motility and intracellular motility that Myo10 is essential for 4 aspects of mitotic spindle and anchorage. Molecular motors are required to function: proper spindle anchoring, spindle length organize and distribute components, such as vesicles, maintenance, spindle pole integrity, and progression organelles, oligomeric structures and macromolecules, through metaphase. Furthermore, by using live imaging in complex eukaryotic cells. One of the most dramatic we show for the first time that highly dynamic F-actin manifestations of motility during the existence of a cell cables are present in the mitotic spindle and that these happens when it undergoes an active form of death cables both surround the spindle and extend between known as apoptosis. The dynamic blebbing in the cell the spindle and the cortex. Remarkably, we find that surface that marks the execution phase of apoptosis although proper anchoring depends on both F-actin requires actomyosin function in the cell cortex. On the and Myo10, the requirement for Myo10 in spindle 1. 54

livro.indd 54 7/7/2008 16:33:30 pole integrity is F-actin independent, while F-actin and Wilson Savino Myo10 actually play antagonistic roles in maintenance Fundação Oswaldo Cruz, RJ of spindle length. Our results show that Myo10 and savino@fiocruz br F-actin play critical roles in mitotic spindles and that Myo10 and F-actin have unexpected interdependencies. RT-19.66 [email protected] ADAM23: A NEW METASTASIS SUPRESSOR GENE? RT-18.63 Anamaria Aranha Camargo FILOPODIA ENSURE ACCURATE EPITHELIAL Instituto Ludwig de Pesquisa sobre o Câncer, SP FUSION DURING DROSOPHILA DORSAL CLOSURE anamaria@ludwig org.br Thomas H. Millard The Healing Foundation Centre, Faculty of Life RT-20.67 Sciences, Michael Smith Building, Faculty of Life PARTICIPATION OF INFLAMMATORY AGENTS IN A Sciences, University of Manchester, Oxford Road, MODEL OF SEPTIC PREGNANCY LOSS INDUCED Manchester, M13 9PT, U.K. BY LPS J.Aisemberg, C. Vercelli, S. Billi, M.L. Ribeiro, Dorsal closure is a morphogenetic movement that A.M.Franchi occurs during Drosophila embryogenesis in which two Center of Pharmacological and Botanical Studies. epithelial sheets fuse with one another at the dorsal Buenos Aires, Argentina midline, thus forming a continuous epidermis. Dynamic filopodia are generated by the leading edge cells during Although there has been some progress in the dorsal closure and these drive fusion by ‘zippering’ understanding of the cause of early pregnancy loss together the two epithelial sheets. Since the epidermis due to chromosomal abnormalities, there is a dearth of is segmentally patterned prior to dorsal closure, fusion knowledge of the causes of isolated and recurrent loss of the epithelial sheets must occur extremely accurately in euploid conceptuses. so that this patterning is continuous across the seam. Genital tract bacterial infections could induce abortion In order to investigate the mechanisms responsible for and are some of the most common complications of ensuring the accuracy of fusion we generated a fly line pregnancy; however, the mechanisms remain unclear. in which the posterior compartment of each segment In the early pregnancy, low doses of LPS that was labelled with RFP, while the anterior compartment reproduce most of the systemic and cellular effects of was labelled with GFP, and imaged the behaviour sepsis, without affecting maternal survival, produce of leading edge cells during dorsal closure in live high percentage of embryonic resorption (ER). We embryos. We find that leading edge filopodia are able investigated the role of several inflammatory agents to recognise and specifically fuse with matching cells in the mechanism of LPS-induced pregnancy loss in a in the opposing epithelial sheet. Filopodial interaction mouse model. between matching cells lead to the formation of tethers We have demonstrated that nitric oxide (NO) and between them and these can pull misaligned sheets into prostaglandins (PG) are involved in the ER, observing perfect alignment. We also observe filopodial matching augmented production of these molecules and that during healing of wounds in the ventral epithelium, inhibitors of their synthesis could prevent ER. LPS also indicating that the molecular mechanisms that underlie caused an increase in the oxidative damage, evidenced matching are not exclusive to the dorsal closure leading by nitration of tyrosine proteins, due to the peroxynitrite edge. This work provides clear evidence that filopodia anion. perform a sensory function and demonstrates how We have also observed that LPS was able to increase precision can be achieved in a complex morphogenetic in vitro uterine and decidual synthesis of PGE, NO and movement. TNFa and this effect was abrogated by pro gesterone [email protected] (P). The serum levels of this hormone were diminished in the LPS treated animals.The non-rejection of the RT-19.64 fetus in normal pregnancies has been attributed to FIBROBLAST MIGRATION IN a number of mechanisms, one of which may be the THREE-DIMENSIONAL COLLAGEN GELS: presence of immunomodulatory molecules induced by DIFFERENT MECHANISMS FOR DIFFERENT P within the feto-placental unit. So we characterized CONDITIONS the uterine and decidual production of two of these Marinilce Fagundes dos Santos molecules: PP14 (Progestagen-dependent endometrial Instituto de Ciências Biomédicas, Universidade de São protein) and LIF (Leukemia inhibitory factor). PP14 Paulo, SP is expressed in maternal reproductive tracts and is [email protected] highly abundant during early pregnancy, and it has anti- inflammatory effects. RT-19.65 LIF, a pro-inflammatory cytokine, is one of the A MULTIVECTORIAL MODEL FOR INTRATHYMIC T molecules known to be indispensable for mouse CELL MIGRATION blastocyst implantation. PP14 protein levels in LPS- 1. 55

livro.indd 55 7/7/2008 16:33:31 treated animals were significantly lower than in control Increasing of gene expression of cytolytic proteins group while LIF mRNA expression was increased. P perforin and granzyme-A and Th1 type cytokine TNF-α increased LIF mRNA expression in normal pregnant confirms the mobilization of cytotoxic skill of uNK cells mice in vitro and it blocked LIF increase stimulated by under changes of maternal–fetal interface homeostasis LPS.Anandamide (AEA), the major endocannabinoid after embryo lesion but it is not dependent of IL-2. On studied so far, has a role in implantation and embryo the other hand, the decreasing of all transcriptomic development. However, high levels of this molecule expression after 24 h coincides with the tissue collapse correlate with fetal weight loss and abortion. We seen at maternal–fetal interface similar to necrosis evaluated if AEA participates in the mechanism of and resorption of entire embryo developing site at LPS-induced NO production on early murine pregnancy. this time after ESL, probably due to the participation LPS treatment augmented uterine AEA synthesis and of cytotoxic activity of uNK cells. However, it does not decreased its degradation.We also observed that seem dependent only from perforin/Gzyme or Fas/FasL antagonists of cannabinoid receptors blocked the pathway. The uNK cells express constitutively the iNOS augmentation of NO due to LPS suggesting that AEA isoforms though out the gestation at the maternal-fetal could have an anti-inflammatory effect. interface and continuously produce NO (nitric oxide), These results suggest that several inflammatory which level increase 20% after 30 min of ESL and than, molecules could participate in the mechanism of LPS- gradually decrease reaching less than 2% after 24h, if induced resorption and that modulation could be useful compared to normal pregnancy. The default level of NO tools to prevent early pregnancy loss. produced by uNK cells during normal pregnancy is a [email protected] physiological response of these cells and requires the expression of HSP72/73 (heat shock protein), a proper RT-20.68 cytoprotective mechanism against stress conditions by CYTOKINES AND UTERINE-NATURAL KILLER uNK cells. Nevertheless, quick increasing of NO under CELLS AT THE MATERNAL-FETAL INTERFACE. stress induced by ESL drastically change the uterine AT Yamada, KY Degaki, ELO Lippe, PDA Lima. environment and affects uNK cell bearing cytokine Department of Histology and unbalance at the maternal-fetal interface. Embryology, Institute of Biology, State University of [email protected] Campinas, 13083-970, Campinas, SP., Brazil RT-20.69 Pregnant uterus is a complex environment bearing IS MACROPHAGE MIGRATION INHIBITORY FACTOR structural and physiological changes from early (MIF) IMPLICATED IN DEFENCE ON PLACENTA peri-implantation stage to parturition in a unique INFECTED BY Toxoplasma gondii? stressing environment. The high accumulation of Eloisa Amália Vieira Ferro cytokine producers uterine-specific subset natural Universidade Federal de Uberlândia, Minas Gerais killer (uNK) cells contribute to homeostasis of maternal–fetal interface and successful pregnancy. Macrophage Migration Inhibitory Factor (MIF) is a However, in humans, some of recurrent pregnancy cytokine originally identified for its capacity to inhibit loss of unknown etiology has been attributed to innate the random migration of macrophages in vitro. MIF immune type-response of uNK cells, but not proved is a key regulator of the inflammatory and immune experimentally. Surgical lesion of embryo (LSE) in the responses which exhibits a protective immune pregnant mouse uterus used as experimental model response to intracellular infectious agents. We recently of abnormal pregnancy in our laboratory induced demonstrated that MIF is abundantly expressed by uNK cell response with loss of cytolytic contents of human tissues in early pregnancy. However the specific secretory-lysosome granules, which suggest direct role of MIF in physiological and pathological conditions involvement of these cells affecting the homeostasis of during pregnancy remains to be elucidated. In order to maternal–fetal interface of pregnant uterus. The gene explore the possible involvement of MIF in Toxoplasma expression evaluated by RT-PCR of pro-inflammatory gondii infection in human pregnancy, first trimester cytokines related to cytotoxicity (TNF-α, IFN-γ, IL-2, chorionic villous explant cultures were stimulated with IL-15, IL-18 and Fas/FasL) of uNK cells in the mRNA T. gondii antigen, IFN-y, antigen plus IFN-y and/or rMIF. obtained from homogenates of LSE endometrium Then, explants were co-coltured with the monocyte cell confirms the absence of IL-2, while IL-15 gradually line THP1 and assessed for monocyte adhesiveness. increase after LSE if compared to normal pregnancy. We found that a significantly higher concentration of TNF-α decreased the expression up to 2h after LSE MIF was produced by explant cultures treated by T. and than increased with peak expression at 6 h and gondii antigen or antigen plus IFN-y. In contrast, no decreased again after 12h. IL18 increased just 30 effect was observed after treatment with IFN-y alone. min after ESL and seems to up regulate the IFN-y Addition of rMIF significantly increased THP1 cell expression, which increased after 1h, reaching a peak adhesiveness to villous tissues.The high production of expression at 12h. The perforin and granzyme-A MIF by villous explant after stimulation is favourable to gradually increased after embryo lesion till 12 h adhesion on villous of the monocytes, the principal cell which drastically reduced after 24 h of embryo lesion. type responsible for the dissemination of T. gondii.. 1. 56

livro.indd 56 7/7/2008 16:33:31 Our findings suggest that MIF, locally secreted after relies on continuous interactions between neural stimulation with T. gondii antigen, plays an important and endothelial cells, including the exchange of role as an autocrine/paracrine mediator in defence common signals such as members of the VEGF mechanism against T. gondii in placenta. family of vascular growth factors implicated in neural [email protected] development and neurodegenerative diseases. We are studying the VEGF-C (vascular endothelial growth RT-21.70 factor-C) that was first identified as a specific regulator SPATIAL ORGANIZATION OF A of lymphangiogenesis through its VEGFR-3 tyrosine REPLICATING BACTERIAL kinase receptor. This VEGFR-3 expression is largely CHROMOSOME restricted to the lymphatic endothelial cells that are Idit Anna Berlatzky, Alex Rouvinski, and Sigal Ben- excluded from the CNS. Recent investigations from Yehuda our group, using in vitro and in vivo studies, have Department of Molecular Biology, Institute of Medical shown that VEGFC promotes the proliferation of Research, The Hebrew University of Jerusalem, subpopulations of ventricular neural progenitor cells and Jerusalem, Israel neuronal precursors during embryonic development, a function that is not mimicked by the related growth The biochemistry of the DNA replication process in factors VEGF-A or PDGF-A. VEGF-C has thus a novel bacteria is relatively well understood, but little is known function as a growth factor selectively required by pools about the spatial organization of the global mass of of neural progenitor cells which express VEGFR-3 newly synthesized DNA. To visualize replicated DNA receptor. The implication of VEGFR-3 in embryonic within living cells, we developed an experimental neurogenesis, as well as the role of VEGF-C and system in the bacterium Bacillus subtilis whereby the VEGFR-3 signaling during postnatal and adult fluorescently labeled nucleotides are incorporated into neurogenesis remain to be investigated. To this aim, the chromosome as it is being replicated. Our data we have examined the expression of VEGFR-3 and its reveal that at the start of replication, newly synthesized co-receptor NPN-2 in the developing and adult brain, DNA is translocated via a helical structure from the and are now exploring the functional contribution of mid-cell towards the poles, where it accumulates. Next, VEGFR-3 during neurogenesis, both in vivo by loss- additionally synthesized DNA forms a second, visually of-function approaches in murine and non-mammalian distinct helix that interweaves with the original one. In models, and in vitro by neurosphere assays. The most the final stage of replication, the space between the recent findings will be presented. two helices is filled up with the very last synthesized [email protected] DNA. This striking geometry provides new insight into the three-dimensional conformation of the replicating RT-22.74 chromosome, and suggests that distal genes can ANGIOGENIC GENES FOR THE TREATMENT OF associate spatially to coordinate their regulation. ISCHEMIC [email protected] DISEASES Sang Won Han RT-21.71 Centro Interdisciplinar de Terapia SMALL MOLECULE SYNTHESIS BY A DEDICATED Gênica, Universidade Federal de São Paulo ENZYMATIC [email protected] MEGACOMPLEX Paul Straight RT-22.75 Texas A&M University ANTI-ANGIOGENESIS THERAPEUTIC [email protected] DEVELOPMENT IN AN EXPERIMENTAL MODEL OF ENDOMETRIOSIS RT-21.72 Daniel E. Machado, Plínio T. Berardo, Christina M. MOLECULAR MECHANISM OF Takiya, Maurício S. Abrão and Luiz E. Nasciutti CYTOKINESIS IN Bacillus subtilis Instituto de Ciências Biomédicas, Universidade Federal Frederico Gueiros do Rio de Janeiro, RJ Instituto de Química, Universidade de São Paulo, SP [email protected] Endometriosis, defined as the presence of functional endometrium outside the uterine cavity, is a common RT-22.73 disease, causing abdominal pain, dysmenorrhea, VEGF-C / VEGFR-3: NEW-COMERS IN THE dyspareunia and infertility in about 10% of the female NEUROVASCULAR CROSS-TALK population of reproductive age. One of the most Thomas J-L. widely accepted hypothesis for the development of Unite Mixte de Recherche INSERM U-711; UPMC endometriosis is the retrograde flow of exfoliated Hôpital de la Salpêtrière, Paris, France endometrium during menstruation, where new blood vessels are essential for the survival of the endometrial The development of the central nervous system implant. Our previous results showed that angiogenesis 1. 57

livro.indd 57 7/7/2008 16:33:31 is predominantly found in rectal endometriosis effectively and fibrosis is the main complication of the compared with bladder and ovarian endometriosis and many known chronic liver diseases. The endpoint of normal tissues. Although rectal and bladder lesions liver fibrosis, defined as the abnormal accumulation are examples of deeply infiltrating endometriosis, the of extracellular matrix in the liver, is cirrhosis, which vascularization and the expression of VEGF and its is responsible for a significant morbidity and mortality. receptor Flk-1 is significantly higher in cases affecting Cirrhosis is an advanced stage of fibrosis, characterised the rectum, suggesting that endometriotic lesions are by the formation of regenerative nodules of liver heterogeneous. Rectal endometriosis is an infiltrative parenchyma separated by fibrotic septa. It is accepted disease, characterized by the severity of its symptoms, that liver fibrosis is reversible, whereas cirrhosis is concomitant infiltration of pelvic organs, with possibility generally irreversible. Extracellular matrix deposition is of bowel obstruction resulting from the progression of consecutive to the activity of fibrogenic cells known as the disease and the technical difficulties of its surgical liver myofibroblasts. Myofibroblasts are almost absent removal. Many studies raise doubts about whether from normal liver; they derive from the activation of deeply infiltrating endometriosis can still be considered precursor cells, the best studied being hepatic stellate a clinically benign disease because it shares some of cells. We and others have put forward the concept the characteristics of malignancy such as abnormal of heterogeneity of liver fibrogenic cells, by showing morphology, deregulated cell growth, cellular invasion, that portal fibroblasts present in portal tracts also and neoangiogenesis. However, in the treatment of play a major role in liver fibrogenesis. Myofibroblasts endometriosis patients, prevention of endometriotic are also a major component of the stroma reaction lesions development only will not be sufficient as a which develops around hepatocellular carcinomas and therapy. Treatments options, aimed to interfere with cholangiocarcinomas. All these findings suggest that established lesions have to be developed. In our it is necessary to reevaluate the role of the different recent study, we have established an experimental fibroblastic cells involved in the development of liver model of endometriosis to evaluate whether inhibition fibrosis. We have also shown that apoptosis mediates of angiogenesis by angiostatic therapy is effective in the decrease in cellularity during the transition between antagonizing the sustentation of endometriosis. In this skin granulation tissue and scar. This observation has model, we are analyzing the relationship between the now been extended to liver. Moreover, it is thought progression of the lesion and the angiogenesis process, that the canals of Hering which have a highly strategic through the study of VEGF and its receptor Flk-1 distribution in the liver, and the surrounding stromal distribution and effects in experimental endometriosis, cells may constitute a niche for hepatic stem cells. All treated with angiostatics agents and inhibitors of these data can generate new therapeutic strategies for COX-2. These results are being analyzed. liver repopulation. luiz.nasciutti@histo ufrj.br [email protected]

RT-23.76 RT-23.77 TISSUE REPAIR IN LIVER TISSUE REPAIR: REPAIR IN THE SKIN AND KIDNEY Alexis Desmoulière Timothy D. Hewitson Faculty of Pharmacy, University of Limoges, Limoges, School / Work Unit Medical Sciences, University of France Melbourne, Melbourne, Australia [email protected] After injury, the processes of liver repair and of fibrogenesis resemble a wound healing process. When RT-23.78 injury and the associated acute inflammation response ADRENOCEPTORS AND RAT result in moderate cell necrosis and limited extracellular CUTANEOUS WOUND HEALING matrix damage, tissue repair normally takes place. In Andréa Monte Alto Costa this situation, dead cells are replaced by normal tissue, Centro de Ciências Médicas, Universidade do Estado with regeneration of specialised cells, formation of a do Rio de Janeiro, RJ granulation tissue, and tissue remodelling with scar formation. The specific regenerative capacities of the The skin is equipped with a dense network of sensory liver generally allow it to reconstitute itself entirely and sympathetic nerve branches distributed in all following acute, moderate lesions. The process of cutaneous layers. Sympathetic postganglionic fibres regeneration in the liver is observed in donor and maintain cutaneous homeostasis by regulating transplanted livers, after hepatic resection, and after vasomotor functions, pilomotor activities, and glandular liver injury. After partial hepatectomy, in rats and mice, secretion. Wound healing is a dynamic process that the original liver mass is restored to approximately includes inflammation, granulation tissue formation, 100% in 7-10 days. In humans, there is a very rapid contraction, re-epithelialization, and tissue remodelling; increase in liver mass during the first 7 days after partial recently, studies demonstrated that sympathetic liver transplantation, leading to complete restoration by nervous system play an important role in wound 3 months. However, chronic injuries to the liver (e.g. healing. Initial studies showed that sympathetic alcohol abuse, viral hepatitis) do not always heal as denervation accelerates wound contraction, and 1. 58

livro.indd 58 7/7/2008 16:33:31 increases myofibroblast density, but impairs re- antioxidant treatment only impeded inflammatory epithelialization and disturbs inflammatory phase cells influx in short-term CS exposure. All antioxidant and mast cell accumulation in rat excisional wounds. enzymes activities were increased in CS-ST (p<0.05) Recent evidences propose that those effects are group but were significantly decreased in CS-LT group mediated by the blockade of b-adrenergic receptors (p<0.05). Long-term NAC administration kept GPx that disturbs wound healing. b-Adrenoceptors are activity similar to control group. Oxidative damage was present in numerous cells that participate on wound prevented by all non-enzymatic antioxidant drugs in ST healing such as fibroblasts, keratinocytes, mast cells, cigarette exposure, whereas long-term NAC treatment polymorphonuclear leucocytes, endothelial cells. resulted in values similar to control group. Conclusion: Subsequently, it was shown that oral administration Our present results indicate that the treatment with of propranolol (b1- and b2-antagonist) delays wound N-acetilcysteine impeded acute and chronic lung contraction and re-epithelialization, prolongs the injury induced by cigarette smoke in mice by oxidative inflammatory phase and delays the formation of damage indicated by preserved antioxidant enzymes granulation tissue in rat excisional wound healing. To activity. Therefore, NAC antioxidants properties may be better understand the effects of adrenoceptors the role favorable to DPOC treatment. of –adrenoceptors was investigated and we showed [email protected] that a1- and a2-adrenoceptor blockade only affected leucocyte migration, epithelial and connective cell RT-24.80 proliferation, and pro-MM9 levels. In conclusion b-1 and HOLOCENTRIC CHROMOSOMAL STRUCTURE IN b-2, but not a-1 and a-2, adrenoceptor blockade delays TRIATOMINES cutaneous wound healing. Maria Tercília Vilela de Azeredo Oliveira [email protected] IBILCE, Universidade Estadual Paulista, São José do Rio Preto, SP RT-23.79 [email protected] DISTINCT EFFECTS OF ALLOPURINOL, RT-24.81 N-(2-MERCAPTOPROPIONYL) CHROMATIN ORGANIZATION IN MOUSE GLYCINE AND N-ACETYLCYSTEINE IN MICE HEPATOCYTES FROM AGED MICE EXPOSED TO EITHER SHORT-TERM OR LONG- Alberto da Silva Moraes (Universidade Estadual TERM CIGARETTE SMOKE de Campinas), Mateus Mondin (ESALQ/USP), Ana Luis Cristovão Sobrino Porto, Frank Bezerra, Samuel S Maria Aparecida Guaraldo (Universidade Estadual de Valença. Campinas), Benedicto de Campos Vidal (Universidade Laboratório de Reparo Tecidual, Centro Biomédico, Estadual de Campinas), Margarida Lopes Aguiar- Universidade do Estado do Rio de Janeiro, RJ Perecin (ESALQ/USP), Shelagh Boyle (4Medical Research Council Human Genetics Unit), Nick Gilbert Background: Cigarette smoke exposure leads to major (The University of Edinburgh Cancer Research Centre), damage into lung parenchyma due to the unbalance Maria Luiza Silveira Mello (Universidade Estadual de between oxidants and antioxidants. Methods: We Campinas) investigated the effects of non-enzymatic antioxidant drugs on both acute and chronic lung inflammation Cellular senescence is an extremely stable form of cell induced by cigarette smoke exposure on inflammatory cycle arrest that limits the proliferation of damaged cells cells influx, lipid peroxidation, and superoxide and may act as a natural barrier to cancer progression, dismutase (SOD), glutathione peroxidase (GPx) and being often considered as a cellular counterpart of catalase (CAT) activities. Male C57Bl/6 mice were aging. This feature has been studied typically in cultured exposed either to 6 commercial filtered cigarettes per cells, but its occurrence in vivo has been shown only in day for 5 days (Short-term) or 12 commercial filtered some mammalian tissues. Although, not all cells in an cigarettes per day for 60 days (Long-term). Groups aging organism are subjected to cellular senescence, were divided as: exposed to smoke only (CS), CS + it is expected that changes in the chromatin structure N-acetyl-cysteine (CS+N); CS +n-(2-mercaptopropionyl) and organization still occur. Screening for alterations glycine (CS+M) and CS + allopurinol (CS+A). All drugs in the chromatin organization in mouse hepatocyte were administered by oral gavages (100nM.kg-1. nuclei in aged mice, we observed an increase in the day-1). Animals exposed to ambient air were used as viscoelastic properties of the chromatin along aging, control. BAL fluid was collected for macrophages and according to changes in the ability of this chromatin to neutrophils counts. Lipid peroxidation was measured form extended chromatin fibers after a lysis treatment through TBARS. Lung homogenates were used to in liver imprints on histological slides. These differences determine SOD, GPx and CAT activities. Results: were accompanied by a decrease in the overall packing Long-term NAC and MPG treatments kept lung states of the chromatin along aging. Further, it was seen parenchyma conformation similar to control group. that most of this viscoelasticity of the chromatin are Both macrophages and neutrophils count were higher dependent on its interactions with the nuclear matrix. in CS-ST and CS-LT groups (p<0.05). Non-enzymatic Finally, we observed accumulation of heterochromatic 1. 59

livro.indd 59 7/7/2008 16:33:31 marks and proteins, as well decreased histone Biology of Reproduction modifications associated with transcription in hepatocyte nuclei of aged mice. All these changes are related to altered RNA synthesis observed in aged animals and A-001 are and evidence of the strong relationship between HISTOMORPHOLOGY OF THE INITIAL chromatin organization and control of gene expression. DEVELOPMENT GONADAL IN ASTYANAX [email protected] ALTIPARANAE (PISCES, CHARACIDAE) Jaqueline Cristina de Bem (Univesidade Estadual RT-24.82 Paulista- UNESP), Sebastião Roberto Taboga CHROMOSOME PAINTING IN THE CONTEXT OF (Universidade Estadual Paulista-UNESP), José Augusto CELL BIOLOGY Senhorini (CEPTA-Instituto Chico Mendes), Patricia Júlio César Pieczarka Pasquali Parise Maltempi (Universidade Estadual Universidade Federal do Pará, Belém, PA Paulista-UNESP) [email protected] The A. altiparanae, lambari, is gaining the attention of researchers and producers, a high rate of survival, fast growth, easy acceptance of artificial food, tasty meat and high demand as live bait. Females have higher growth rates than males making its culture better. The process of sexual differentiation can be controlled by the administration of sex hormones in fish sexually undifferentiated, changing the course of differentiation in the direction of the desired sex. In this study, the initial gonadal development was accompanied in an attempt to ascertain the time of sexual differentiation and the best stage for implementation of female hormone. Ten specimens were fixed daily in Karnovsky, included in historesin, and stained with HE. The results revealed that soon after the hatching of the larvae, the gonocytes are already allocated, indicating that the initial formation of the gonad give up during embryogenesis. By the third day, the gonads are undifferentiated and formed by cystic cells. Probable germ cells are observed with large nuclei showing only a small nucleolus and vacuoles occupying the entire cytoplasm. Attached to these cells, are supposed Leydig cells that have characteristic aspect of cells that synthetize steroids. The increase of cells in different stages and changes in the size and shape of gonadal cells observed from the fourth day indicate a likely beginning of gonadal differentiation. These results, combined with tests of the effectiveness of female hormone dosages, will contribute to the consolidation of a sex reverse technical in lambaris. CAPES, FUNDUNESP. [email protected]

A-002 ULTRASTRUCTURAL ALTERATIONS PROMOVED BY DOXAZOSIN IN RAT PROSTATIC LOBES Flávia Karina Delella (UNICAMP), Sérgio Luis Felisbino (UNESP - Botucatu)

Doxazosin (Dox) has been widelly used to treat benign prostate hyperplasia by relaxation of the dynamic contraction of the smooth muscle component of the prostate and to relieve the lower urinary tract symptoms. In previous work, we showed that Dox alters rat prostates morphology and increases stromal collagen fibers. Here, we investigate the effect of Dox 1. 60

livro.indd 60 7/7/2008 16:33:31 on different rat prostatic lobes, anterior (AP), dorsal A-004 (DP) and ventral (VP) prostate at ultrastructural level, AQUAPORIN 9 (AQP9) AND with special attention to the epithelial and stromal cells ANDROGEN RECEPTOR (AR) morphophysiology and collagen and elastic fibers IMMUNORREACTIVITY IN THE DIABETIC MALE amount and organization. Adult male Wistar rats were RAT EPIDIDYMIS: A POSSIBLE ROLE IN SPERM treated with Dox (25mg/kg/day) dissolved in corn oil, by QUALITY oral gavage, during 30 days. The VP, DP and AP had Davi Abeid Pontes (IB/PPGBCE, UNICAMP, Campinas, been dissected and evaluated by trasmission electron SP), Wellerson Rodrigo Scarano (IB/UNESP, Botucatu, microscopy. The ultrastructural analyses showed that all SP), Raquel Fantin Domeniconi (IB/UNESP, Botucatu, Dox treated prostatic lobes presented reduced epithelial SP), Silvana Gisele Pegorin de Campos (IBILCE/ cells height and protein synthesis organelles. Apoptotic UNESP, S.J. Rio Preto, SP), Wilma De Grava Kempinas epithelial cells were also found in prostatic lobes. (IB/UNESP, Botucatu, SP) Fibroblasts were found more activated in the treated group in all three lobes, presenting loose chromatin Diabetes is one of the most widespread diseases, and great amount of Rough Endoplasmic Reticulum rendering many people hyperglycemic and infertile, and Golgi Complex. Bundles of collagen fibrils and most of them still of fertile age. Reabsorption of elastic fibers were also found thicker and in greater fluids and solutes across the epididymal epithelium amount than in control group. In conclusion, althought is important for sperm maturation, concentration and Dox treatment acts ratherly in the smooth muscle cells, storage, processes in which AQP9 is essential. AQP9 it also alters fibroblast behavior, which increases the expression appears to be modulated by androgens. extracellular matrix fibers deposition and organization. The present study aimed to evaluate the AQP9 [email protected] and AR immunoreactivity in the epididymis of male rats experimentally rendered diabetic. Male Wistar A-003 rats, 90 days old and weighing around 300g, were COMBINED EFFECTS OF FINASTERIDE AND randomly allocated into 3 experimental groups: diabetic DOXAZOSIN ON RAT PROSTATE MORPHOLOGY, (streptozotocin 40mg/Kg BW), diabetic+testosterone EPITHELIAL CELL PROLIFERATION AND DEATH (same protocol, receiving a capsule implant of Luis Antonio Justulin Junior (UNICAMP), Sérgio Luis testosterone for 2 weeks) and control (vehicle). Three Felisbino (UNESP) weeks after diabetes induction, the rats were killed, the blood was collected for glucose and testosterone Although clinical data have demonstrated that dosages and the epididymis was processed for AQP9 combination therapy with Finasteride (Fin) and and AR immunohistochemical assays. Diabetic rats Doxazosin (Dox) reduced the long-term risk of acute had a reduction in plasma testosterone levels (ng/dL, urinary retention and the need for Benign Prostate control = 1.91±0.53; diabetic = 0.32±0.09*; diabetic+ Hyperplasia (BPH) invasive therapys than either drug testosterone = 1.42±0.21; * p<0.05), that was correlated alone, little is kwon about the effects of this combination with a less intense reactivity for AQP9 and androgen therapy on prostate tissue morphophysiology. Here, receptor in the caput and cauda epididymidis compared we investigate the combination effect of Fin+Dox on rat to the other groups. These data demonstrated that prostatic lobes morphology, epithelail cell proliferation diabetes affects AR and AQP9 reactivities in the and death. Adult Wistar rats were treated with Dox epididymis, which can account for the impairment of and Fin (25 mg/kg per day), and the ventral (VP), sperm quality in the rat. dorsal (DP), and anterior prostatic (AP) lobes were [email protected] excised at days 7 and 30 of treatment. The prostatic lobes were dissected out, weighted and immersed in A-005 fixative and submitted to histological, immunostaining LONG TERM EFFECTS OF DEVELOPMENTAL (PCNA) and TUNEL procedures. Fin+Dox treatment EXPOSURE TO DI-N-BUTIL-PHTHALATE (DBP) IN reduces prostatic lobes weight, epithelial cell height and THE PROSTATE OF RATS epithelial cell proliferation. Moreover, Fin+Dox treatment Wellerson Rodrigo Scarano (IB/UNESP, Botucatu, also increased apoptosis of epithelial cells, collagen SP.), Fabíola Choqueta de Toledo (IB/PPGBCE, fibers volume fraction and reticular fibers in the three UNICAMP, Campinas, SP.), Marina Trevisan Guerra prostatic lobes in all period of treatment. These results (IB, PPGBGA, UNESP, Botucatu, SP.), Silvana Gisele show that combined effects of Fin+Dox on prostates do Pegorin de Campos (IBILCE/UNESP, S.J. Rio Preto, not differ those observed in the Fin or Dox treatment SP), Sebastião Roberto Taboga (IBILCE/UNESP, S.J. alone. These observations are in accordance with Fin Rio Preto, SP.), Wilma De Grava Kempinas (IB/UNESP, and Dox prostate tissue target, since Fin acts mainly Botucatu, SP) on epithelial cells and Dox on smooth muscle cells, and probably their effects do not overlay. Financial Support: Phthalates constitute a class of industrial chemicals FAPESP, CNPq. used as plasticizers to impart flexibility to plastics. [email protected] The rat has been explored for its developmental susceptibility to male reproductive tract insults 1. 61

livro.indd 61 7/7/2008 16:33:31 following phthalate exposure. In the present study we antibody, and then incubated with streptavidin-FITC evaluated the toxic effects on the prostate of male or streptavidin- peroxidase complex. Laminin were adult rats exposed to DBP during fetal and lactational immunolocalized at the basement membrane of both periods. Pregnant females were obtained through germinal and secretory epithelium, whereas type I natural mating, being distributed in two experimental collagen predominated over the interstitial space during groups: Control (C) and Treated (T). The females of regression stages of testis. The presence of type I the T group received DBP (100 mg/Kg - LOAEL, by collagen during gonadal phases in which the testis is gavage) from gestation day 12 to postnatal day 21, in regression suggests an interstitial rearrangement while the C group received the vehicle (corn oil). In required for a new spermatogenic cycle. adulthood (90 days old), the animals were euthanized. [email protected] The serum testosterone levels were measured. The ventral prostate was removed, weighed, fixed and A-007 processed for histochemical staining. Stereological COMBINED EFFECTS OF THE CADMIUM AND and histopathological analyses were performed. CAFFEINE ON RAT PROSTATE MORPHOLOGY Serum testosterone levels and prostate weight were DURING PUBERTY comparable among groups. In the T group the relative Lívia Maria Lacorte (UNESP), Flavia Karina Dellela proportions (%) of epithelium (C=32.86;T=42.04*) (UNESP), Antonio Francisco Godinho (UNESP), Sergio and stromal (C=21.61;T=27.88*) compartments Luis Felisbino (UNESP) were increased, while the luminal compartment was decreased (C=45.54;T=30.08*),*p<0.05. In T, Male puberty presents an increases in serum disseminated inflammatory infiltrate in the stroma testosterone level, which induces marked growth in the associated or not with epithelial dysplasia and PIN prostate gland. However, in the puberty, many youngers (Prostatic Intraepithelial Neoplasia) was observed. male are exposed to environments chemicals, such Accumulation of the collagen fibrils was observed as cadmium and caffeine, by smoke and energetic adjacent to the epithelium in some T animals. These beverage, respectively. Here, we investigated the results suggested that phthalates could play a role in effects of cadmium and caffeine combined on rat the proliferative and inflammatory disorders in the rat prostate morphology and lesions appearing, during prostate. pubertal growth age. Pubere male Wistar rats (60 days- [email protected] old) received by drink water 10 ppm of cadmium (Cd), 10 mg/l of caffeine (Cf), cadmium more caffeine (CC) A-006 or tap water (Ct), during 30 days. The ventral (VP) and IMMUNOHISTOCHEMICAL DETECTION OF TYPE I dorsolateral (DLP) prostatic lobes were dissected out, COLLAGEN AND LAMININ IN TESTIS OF LAMBARI weighted and submitted to the histological procedures. ASTYANAX ALTIPARANAE Resin sections were stained by Hematoxylin- Fabiano Gonçalves Costa (UENP/FFALM e ICB/USP), Eosin and Paraplast sections were submitted to Mateus Contar Adolfi (ICB/USP), Celia Guadalupe the immunohistochemistry for Proliferating Cellular Tardeli de Jesus Andrade (UEL), Cruz Alberto Mendoza Nuclear Antigen (PCNA). The incidence of proliferative Rigonati (ICB/USP), Maria Inês Borella (ICB/USP) intraepithelial neoplasias (PIN) and adenocarcinomas (AC) was also determined. Analysis showed that Cd, Teleost species show a great diversity of gonads. in the VP and DLP, increased the proliferation index of Currently, it was not found any work describing epithelial cells. However, any neoplasic lesion we found the morphology of the testis of lambari (Astyanax in the VP, but in DLP, the frequency of PIN was higher altiparanae, Characidae), a South American fish than control ones and also to the AC. Caffeine alone did largely distributed along Paraná River basin. Moreover, not change PCNA index. In the group CC, Cf seems to works that describe the distribution extracellular matrix antagonize the proliferative effects of cadmium, but do components (EM) in testis of this species were not not alter the lesions incidence in the DLP. In conclusion found. Type I collagen is an important compound of EM Cd can represent an important initiator or promoter of that gives resistance and support for tissues. Laminin is prostatic lesions in puberty, and DLP, homologe with a glycoprotein frequently found in basement membrane. human prostate peripheric zone, is more affected by Cd The aim of the present work was to describe the testis effects. Support: FAPESP. morphology and the distribution of type I collagen and [email protected] laminin throughout the gonadal cycle of this species. From July 2006 to December 2007 male fishes were A-008 collected from an aquaculture station in Andirá, PR, REMODELING OF MOUSE PUBIC SYMPHYSIS ON Brazil. These animals were anesthetized and the MULTIPAROUS AND SENESCENT testes were fixed in 4% paraformaldehyde, Bouin or FEMALES Methacarn, and processed for optical microscopy. For Sílvio Roberto Consonni (Department of Histology and immunohistochemical reaction, testes sections were Embryology, State University of Campinas (Unicamp)), incubated with primary antibody (anti-type I collagen Renata Giardini Rosa (Department of Histology and or anti-laminin), incubated with biotinyleted secondary Embryology, State University of Campinas (Unicamp)), 1. 62

livro.indd 62 7/7/2008 16:33:31 Maria Amália Cavinato (Department of Histology and Undifferentiated gonads present the precursors of Embryology, State University of Campinas (Unicamp)), the germ cells (gonocytes) and of the somatic cells. Cristiane Mendes Vinagre (Multidisciplinary Center for Along the gonad, germ cells (gonocytes) form a Biological Investigation, State University of Campinas continuous cord, which are limited by somatic cells (Unicamp)), Paulo Pinto Joazeiro (Department (pre-Sertoli cells). Differentiation starts with the gonadal of Histology and Embryology, State University of reorganization: germ cells are gradually individualized in Campinas (Unicamp)) cyst, loosing the cord organization and forming clusters. At the same time, interstitial components arisen from The pubic symphysis (PS) is part of the muscle-skeleton the capsule surround the clusters, and afterward a system that supports pelvic organs. In some mammals, lumen appears into the cell clusters. With the cyst PS remodels during pregnancy under hormonal action formation, gonocytes differentiate into spermatogonia exhibiting: 1) the “separation” of joint as results in (undifferentiated type), which are now completely the fibrocartilage “transformation” in an interpubic surrounded by Sertoli cells. In the differentiated ligament (IpL); and 2) the IpL “relaxation” before gonads, spermatogenesis begins within the cysts. As parturition. In this work, we intended to characterize spermiogenesis occurs, the sperm are released into the cellular and extracellular matrix phenotypes of the lumen, and the testicular tubules become anastomosed, IpL on multiparous and senescent (MS) female culminating also with the formation of the sperm duct mouse articulation (animals with more than seven system. pregnancies and one year old). This aim could help [email protected] the understanding of the weakness of the connective tissues on pelvic organs. Thus virgin, primiparous and A-010 MS female mouse groups were studied using light, ANGIOGENESIS STUDY IN AN EXPERIMENTAL transmission electron microscopy and morphometric MODEL OF ENDOMETRIOSIS analyses. IpL compounds showed the remodeling Daniel Escorsim Machado (Universidade Federal do processes during and after pregnancy on primiparous Rio de Janeiro), Plínio Tostes Berardo (Universidade mice. In the MS group, the PS gives place to an IpL with Federal do Rio de Janeiro), Christina Maeda Takiya a similar arrangement as 24h postpartum tissues, with (Universidade Federal do Rio de Janeiro), Maurício collagen fibers arranged compactly, decrease of “crimp” Simões Abrao (Universidade de São Paulo - USP), and extended phenotype fibroblast-like cell. Differences Luiz Eurico Nasciutti (Universidade Federal do Rio de were observed on central and peripheral collagen Janeiro) fibrils of IpL in all groups suggesting that biomechanical adaptations during pregnancy might occur. The absence Endometriosis, defined as the presence of functional of eosinophil infiltration on the entire IpL of all studied endometrium outside the uterine cavity, is a common groups suggested that the modulation at postpartum disease, causing pelvic pain, dysmenorrhea, and is dependent on fibroblasts-like cells. Thus the infertility in about 10% of the female population of morphological features of MS IpL may be associated reproductive age. Assuming that new blood vessels are with the loss of tissue remodeling capacity under stimuli essential for the survival of the endometrial implant, as multiparity and/or senescence. our previous results showed that angiogenesis is [email protected] predominantly found in human endometriosis compared with normal endometrium. In this study, we have A-009 established an experimental model of endometriosis MORPHOLOGICAL CHANGES OF GERM AND to evaluate angiogenesis process and to compare SOMATIC CELLS DURING THE GONADAL with endometrium. We have investigated the vascular DIFFERENTIATION AND FIRST MATURATION IN density, VEGF and its receptor Flk-1 distribution, by MALE OF CYPRINUS CARPIO immunohistochemistry and RT-PCR. The preliminary (TELEOSTEI: CYPRINIFORMES) results showed that the autotransplantation of Talita Sarah Mazzoni (Institute of Biology - UNICAMP - endometrium pieces into the peritoneal cavity is a well- Campinas - SP), Irani Quagio-Grassiotto (Department established method for induction of endometriosis in rat. of Morphology - UNESP - Botucatu - SP) The lesions were cystic, vascularized and demonstrated histological hallmarks of human pathology, as In Teleostei, the gonadal differentiation involves endometrial glands and stroma. The vascular density changes in somatic and germ cells. It includes and the presence of VEGF and Flk-1 were significantly gonocytes divisions and differentiation, proliferation higher in the endometriotic lesions than in the of somatic cells, germinal epithelium differentiation, endometrium, and confirmed the angiogenic potential and formation of the testicular tubules. In order to of these lesions. We also investigated the expression study the morphological changes of germ and somatic of COX-2 that is directly correlated with the disease cells during the gonadal differentiation and the first progression and we observed an increase of COX-2 in maturation, gonads of juvenile males of Cyprinus carpio the endometriotic lesions, showing a positive correlation were processed for inclusion in historesin, and the with VEGF. These results suggest that the treatment obtained sections submitted to different stain methods. with selective COX-2 inhibitors may be effective to 1. 63

livro.indd 63 7/7/2008 16:33:31 suppress the maintenance of endometriosis through and castrated that received the drug vehicle (corn their antiangiogenic activity. oil). Additionally, the experimental groups received [email protected] Flutamide (antiandrogen, 10mg/Kg/day) and/or Tamoxifen (antiestrogen, 1mg/Kg/48h) doses during A-011 1, 3, 7 and 30 days post-castration. Methodologies EFFECTS OF ESTROGEN AND ANDROGEN involved morphological (HE), immunohistochemical SUPPRESSION ON THE PROSTATE (PCNA and androgen receptor-AR) analysis and EPITHELIUM OF OLD GERBILS apoptotic cell detection (TUNEL). Hormonal ablation Silvana Gisele Pegorin de Campos (IBILCE/UNESP promoted a decrease in the frequency of prostatic São José do Rio Preto), Sebastião Roberto Taboga lesions after 30 days. However, some animals showed (IBILCE/UNESP São José do Rio Preto) injuries such as prostatic intraepithelial neoplasias (PIN), carcinomas and adenocarcinomas. An increase In this study was evaluated the response of luminal cells of atypical epithelial cells (but not characterizing in the prostate epithelium of old gerbils (18 months) PIN), acinar hyperplasia, intra-luminal calculi and submitted to the surgical castration associated or not inflammation appeared as commom response to to steroidal blockade. Animals were divided in 6 groups therapies. The lesions exhibited few PCNA-positive and all of them were surgically castrated, excluding the cells similar to what occurs in intact regions of the control group composed by intact animals. The others gland, while an increase of immunostaining was two control groups were formed by castrated animals observed in epithelium associated with inflammatory and castrated that received the drug vehicle (corn oil). sites. Few apoptotic and AR-positives cells were found. In the experimental groups additionally were applied Treatments involving surgical and steroidal blockades doses of Flutamide (antiandrogen, 10mg/Kg/day) and/ decreased the incidence of prostatic lesions in old or Tamoxifen (antiestrogen, 1mg/Kg/48h) during 1, gerbils, but epithelial cells that remained post-therapies 3, 7 and 30 days after-castration. The metodologies showed great independence of hormone ablation. involved quantitative and statistic analyses of body [email protected] and prostate weights; apoptotic cells (TUNEL) and immunohistochemical detection of androgen receptor A-013 (AR) and cytokeratins 8/18 (CK). The castration CASE REPORT: HISTOLOGICAL associated or not to anti-steroidal drugs by short or DIFFERENCES OBSERVED IN TWO HUMAN long-term promoted a decrease in wet and relative SISTERS PLACENTAS ONE FROM NORMAL weights of the prostate. The apoptotic index increased GESTATION AND THE OTHER FROM FETAL significantly after 24 and 72h, declining 7 days post- SUFFERING GESTATION castration. The immuoreactivity for AR diminished Otávio Augusto Oliveira de Carvalho (UFAM), Marcelo gretaly after 72h of hormonal ablation, coinciding with de Castro Santos (UFAM), Oscar Tadeu F. Costa the highest levels of apoptosis. The majority of cells in (UFAM), Maria Lúcia Góes de Araújo (UFAM) all groups expressed CK 8/18 post-therapies, indicating that they remained differentiated. The prostate of The placenta is a fetomaternal organ, discoid in old gerbil presented itself as good model to assess shape, with a villous region formed by trophoblast, epithelial behavior front steroidal ablation and the extraembryonary mesenchyma and capillary molecular markers utilized may help to understand the endothelium. The components of trophoblast, the events that lead to emergence of treatment-resistant syncytiotrophoblast (ST) and cytotrophoblast (CT) are cells. part of the respiratory diffusion barrier of the placenta. [email protected] Modifications in the width of the diffusion barrier can compromise hematosis, possibly leading to a fetal A-012 suffering, by hypoxia. The objective of this study was SURGICAL CASTRATION AND STEROIDAL to observe the placenta histological features related BLOCKADES IN THE TREATMENT OF PROSTATIC with morphological causes of fetal suffering. Samples LESIONS OF OLD GERBILS from many regions of both human placentas (CAAE - Silvana Gisele Pegorin de Campos (IBILCE/UNESP), 0141.0.115.000-07) were fixed in formaldehyde 10%, Wellerson Rodrigo Scarano (UNESP - Botucatu), Luiz and routinely processed for histological analysis. Light Roberto Falleiros Junior (IBILCE/UNESP), Sebastião microscopic examination showed evident subchorionic Roberto Taboga (IBILCE/UNESP) fibrin deposits, fibrinoid necrosis, and more extenses areas of infarctation, placental calcification and intervilli The present study was conducted to assess the thrombosis in the placenta with fetal suffering than therapeutic action of surgical castration associated or placenta from normal gestation. The calcified areas not to steroidal blockades in prostatic lesions of old were found in villous extremities. The heteroformed gerbils (18 months). Animals were divided in 6 groups cotyledons observed in placenta with fetal suffering and all of them were surgically castrated, excluding support these histological findings. the control group composed by intact animals. The [email protected] other two groups were formed by castrated animals 1. 64

livro.indd 64 7/7/2008 16:33:32 A-014 Sertoli cells. Spermatogonia, after several mitotic CYTOPROTECTIVE CHAPERONE HSP72/73 divisions differentiate into spermatocytes, which ISOFORMS EXPRESSION IN THE UTERINE enter in meiosis. At this time, germinal epithelium NATURAL KILLER CELLS becomes pseudo stratified, due the increase in size Patrícia Daniele Azevedo Lima (UNICAMP), Karina of the cysts, characterizing the early maturation class. Yumi Degaki (UNICAMP), Marcia Crisitina Bizinotto Cysts of spermatocytes proceed on spermatogenesis (UNICAMP), Áureo Tatsumi Yamada (UNICAMP) produce spermatids, and spermatozoa. The mid maturation class, ephithelium presents all germ cell The low oxygen concentration and drastic shift of types and spermatozoa in the luminal compartment. cytokine profile at maternal-fetal interface during early The epithelium stays continuous. Progressively more stage of pregnancy might be a stressing environment, cysts advance on spermatogenesis, the quantity of but any attempts have been made to evaluate the the spermatozoa increase, and at the same time mechanism necessary for cell surviving in such spermatogonia self-renovation reduces. Thus in the late conditions. The present work evaluates the heat-shock maturation class, epithelium becomes discontinuous. proteins HSP72/73 chaperone isoforms expression in By the end of the reproductive period, in the regression the cells present at the maternal-interface of pregnant class, epithelium is discontinuous, and has mainly uterus. Normal pregnant mice uteri from gestational spermatogonia. Cysts with the other cell types are day (gd) 6 to 17 and uteri from mice on gd 9 submitted scarce. Many macrophages cells in the luminal and to surgical lesion of the embryo (SLE) were used to interstital compartments are also present in this class. evaluate the protein and gene expression of HSP72/73 [email protected] isoforms and also the proportions of proliferating and apoptotic cells. Constant labeling of HSP72/73 was A-016 detected in the cytoplasm of uterine natural killer (uNK) LATE INSULIN REPLACEMENT RESTORES cells through the gestation and by immunelectron THE EFFECT OF STZ-INDUCED DIABETES ON microscopy identified conspicuous labeling in their GLANDULAR WEIGHT AND EPITHELIAL CELL mitochondria. The PCR confirmed the gene expression PROLIFERATION OF THE RAT PROSTATIC LOBES of both HSP72 and 73 in the uNK cells. The SLE Sérgio Alexandre Alcantara dos Santos, Elaine Manoela induced a time-dependent decreasing of HSP72/73 Porto, Sérgio Luis Felisbino (UNESP) expression, mainly for HSP73 isoforms in the uNK cells and mitochondrial labeling as soon as 30min after Streptozotocin (STZ)-induced diabetes in rats provides the SLE. The proportion of apoptotic uNK cells nuclei a relevant model to study reproductive dysfunction increased after 6h of SLE while the proliferating nuclei under diabetic conditions, as they exhibit a number of became negative. These constitutive expression of deficits in reproductive function that resemble those HSP72/73 isoforms in the uNK cells is a new insight seen in humans. Here we investigated the impact of for innate immune responsive effectors leucocytes and STZ-induced diabetes and insulin replacement on the the cytoprotective chaperone requirement namely the epithelial cell proliferation of the rat prostatic lobes. HSP73 in the mitochondria seems to down-regulate the Prepubertal male Wistar rats (40 days-old) were used. apoptotic signaling of uNK cells. Diabetes was induced through administration of a single [email protected] dose of streptozotocin (STZ; 40mg/kg body weight) at 40-days old. Three (simultaneous) or twenty days A-015 (late) after STZ-administration, insulin was replaced REPRODUCTIVE CLASSES IN MALE OF THE (3U/100g) for 17 and 20 days, respectivelly. Diabetic, GYMNOTUS SP. (TELEOSTEI, diabetic plus insulin (simultaneous and late) and age- GYMNOTIFORMES) matched controls animals were killed by overdoses of Gisleine Fernanda França (Instituto de Biologia - pentobarbital after 20 days and 40 days of beginning of UNICAMP), Irani Quagio-Grassiotto (Instituto de the experimental period. Prostatic lobes were dissected Biociências - UNESP) out, weighted and processed for immunohistochemistry to PCNA and a morphometric analysis were applied In fish, spermatogenesis occurs in cysts. Cysts, and to determine the proliferation index. The experimental consequently germinal epithelium, undergo several diabetes caused a significant reduction in prostatic changes during the reproductive cycle. The source weight and also reduces the epithelial cell proliferation of these changes is the alternation of the germinal index in the all prostatic lobes. Both simultaneous and epithelium between continuous and discontinuous, late insulin replacement restores the prostate weight and the developmental stage of germ cells. The and proliferation index. These findings indicate that reproductive classes, during male cycle, were described late replacement of insulin can restores the diabetes- from testis, of adults of Gymnotus sp., processed induced prostate damages. This fact may have according to the usual methods for light microscopy. important clinical relevance, since most of the male In the regressed class the germinal epithelium that diabetic patients are negligent in the diagnosis and bordered testicular tubules is simple and continuous treatment. Financial Support: FAPESP. and contain only spermatogonia associated with [email protected] 1. 65

livro.indd 65 7/7/2008 16:33:32 involve the activity of some enzymes. However, the A-017 role of matrix metalloproteinases (MMPs) in this TIMP-2 EXPRESSION ON RAT PROSTATIC LOBES phenomenon remains unclear. In this work, we used DURING POST-NATAL Immunochemistry and zymography to investigate the MORPHOGENESIS AND GROWTH. expression of MMP-8 (collagenase) and to assess the Carolina Sarobo (UNESP - Botucatu), Flávia Karina expression and activity of MMP-2 and -9 (gelatinases) Delella (UNICAMP), Sérgio Luis Felisbino (UNESP - in virgin, late pregnant and post-partum mice. Ligth Botucatu) microscopy showed extensive remodeling of the ECM around well-preserved fibroblasts in late pregnancy Matrix metalloproteinases (MMPs) and their tissue and detection of MMP-8, -2 and -9 immunoexpression inhibitors (TIMPs) play a major role in extracellular in cells of virgin and in fibroblast-like cells of IpL in the matrix component degradation in several normal study groups. Western blot confirmed the presence and abnormal tissue situations; they are also found of inactive and active forms of MMP-8. Zymography in human seminal plasma. MMPs have been found revealed proenzyme and mature forms of gelatinases in rat prostate secretions and are nearly lobe in the PS and IpL, with some differences among the specific in expression pattern. In previous study groups. No inflammatory cells were found. These we demonstrated that TIMP-2, like other semen findings indicate that there is extensive remodeling components, is expressed differently among the of the extracellular matrix in the PS of pregnant and rat prostatic lobes epithelial cells in the following post-partum mice. This remodeling is accompanied by sequence: LP>AP>DP>VP in both young and adult alterations in the expression and activities of MMP-2, rats. In this study, we investigated when the TIMP-2 MMP-8 and MMP-9, but without an inflammatory expression began in the prostatic lobe during response. post-natal morphogenesis and growth. For this, [email protected] immunohistochemistry staining was performed in Paraplast section from 1 to 6 weeks post-natal prostatic A-019 lobes: ventral (VP), lateral (LP), dorsal (DP), and UTERINE-NATURAL KILLER CELLS ARE AFFECTED anterior (AP). The results showed that epithelial cells BY AMNIOTIC FLUID from lateral lobe starts to express TIMP-2 at second JUARES EDNALDO ROMERO BIANCO week and after lumen formation. The others lobes (UNICAMP), PATRICIA DANIELE AZEVEDO LIMA are negative untill the sixth week. Thus, lobe-specific (UNICAMP), KARINA YUMI DEGAKI (UNICAMP), TIMP-2 expression appear earlier in the epithelial cells AUREO TATSUMI YAMADA (UNICAMP) form lateral lobe. The role of early TIMP-2 expression in the lateral lobe still remains to be determinated. Uterine-Natural Killer (uNK) cells strongly influence and However, TIMP-2 may take places in the regulation regulate the homeostasis of maternal-fetal interface of MMP activity in seminal plasma and glandular and despite the quiescence of cytotoxic activity in homeostase. Further studies should address whether the normal pregnancy it is predicted the participation TIMP-2 expression is related to the highest incidence on miscarriages and recurrent pregnancy loss in a of prostatitis and adenocarcinoma in the LP of the older unknown mechanism or the cytotoxic pathway. The rats. present study evaluated the possibility of amniotic [email protected] fluid contents releasing in the pregnant uterus could affect the uNK response. It was used pseudopregnant A-018 mouse uterus (PPU) induced by mineral oil injection MATRIX METALLOPROTEINASE EXPRESSION AND and amniotic fluid (AF) collected from syngeneic ACTIVITY DURING REMODELING OF THE MOUSE pregnant mice on 10thgd. The PPU samples were PUBIC SYMPHYSIS IN collected after 0.5, 1, 2 and 6 hours of AF injected in PREGNANCY AND AFTER BIRTH the anti-mesometrial side of PPU and same volume Renata Giardini Rosa (Universidade Estadual de of saline was injected in the control animals. The local Campinas), Christiane Aparecida Badin Tarsitano of AF injection showed hyperemic reaction as soon (Universidade Estadual de Campinas), Stephen Hyslop as after 0.5 hr, with large blood vessels containing (Universidade Estadual de Campinas), Paulo Pinto blood cloths not removed by perfusion, rupture of Joazeiro (Universidade Estadual de Campinas) epithelial-like assembled decidual cells with apoptotic nucleus, which was confirmed by TUNEL reaction. A hormonally controlled mechanism that increases The uNK cells showed vacuolization of the cytoplasm, the Pubic Symphysis (PS), flexibility during pregnancy discontinuous labeling of cell surface and loosing of in mammals is a well defined phenomenum in mice granules contents by DBA lectin. The PPU injected and guinea pigs. Immediately after parturition, the with saline did not show any extensive damage in the interpubic ligament (IpL) of pregnant mice undergoes deciduas or in the uNK cell morphology. These results rapid remodeling to restore the original morphology. indicate that amniotic fluid contents strongly and quickly Alterations associated with cervical ripening, affects locally the homeostasis of uterine tissue in the including relaxation of the mouse PS and IpL, may PPU, which suggest the amniotic fluid leakage into the 1. 66

livro.indd 66 7/7/2008 16:33:32 maternal-fetal interface during early stage of pregnancy rats (40 days-old) were used. Diabetes was induced could induce miscarriage with uNK cells commitment. through administration of a single dose of streptozotocin [email protected] (STZ; 40mg/kg body weight) at 40-days old. Three (simultaneous) or twenty days (late) after STZ- A-020 administration, insulin was replaced (3U/100g) for 17 INFLUENCE OF DIABETES AND and 20 days, respectivelly. Diabetic, diabetic plus insulin INSULIN REPLACEMENT ON MATRIX (simultaneous and late) and age-matched controls METALLOPROTEINASES -2 AND -9 ACITVITIES animals were killed by overdoses of pentobarbital. IN RAT PROSTATIC LOBES DURING PUBERTAL Prostatic lobes were dissected out, weighted, processed GROWTH for histological procedures and by picrosirius, Gomori’s Elaine Manoela Porto , Sergio Alexandre Alcantara dos reticulin and Masson’s trichrome staining. Results Santos, Sergio Luis Felisbino (Unesp) showed that diabetes induced an atrophy of the glandular parenchyma and these atrophy is followed Previous works have showed that MMP-2 and MMP-9 by modifications in the prostatic stroma, which includes activities take place during post-natal prostate ductal an accumulation and thickening of collagen fibers and growth and branching. Here, we invetigated if the STZ- reticular fibers. Insulin replacement (simultaneous and induced diabetes interfere with MMP-2 and MMP-9 lately) prevented the detrimental effects of diabetes acitivities during prostate pubertal growth and the in the prostate. In conclusion, diabetes disturbs the effect of an, simultaneous or late, insulin replacement. structure of the prostatic parenchyma and led to an Prepubertal male Wistar rats (40 days-old) were used. effective remodeling of the glandular stroma. The late Diabetes was induced through administration of a single insulin treatment restored the prostate parenchyma and dose of streptozotocin (STZ; 40mg/kg body weight) stroma organization, showing that adverse effects of at 40-days old. Three (simultaneous) or twenty days these disease in the prostate are reversible. Support: (late) after STZ-administration, insulin was replaced FAPESP. (3U/100g) for 17 and 20 days, respectivelly. Diabetic, [email protected] diabetic plus insulin (simultaneous and late) and age- matched controls animals were killed by overdoses of A-022 pentobarbital after 20 days and 40 days of beginning of MIXTURE OF PESTICIDES: A STUDY OF the experimental period. Prostatic lobes were dissected REPRODUCTIVE TOXICITY IN MALE RATS out, weighted and processed for biochemical analysis of Juliana Elaine Perobelli (Programa de Pós- gelatin-zymography. The zymography analysis showed Graduação em Biologia Celular e Estrutural, Instituto clear bands of MMP-2 and MMP-9 in control, diabetic de Biologia, UNICAMP), Meire França Martinez and diabetic plus insulin replacement. The densitometric (Departamento de Patologia da Faculdade de Medicina, analysis of the bands showed that prostatic lobes from UNESP, Botucatu), Carla Adriene da Silva Franchi diabetic animals presented reduced activities of MMP-2 (Departamento de Patologia da Faculdade de Medicina, and -9. Simultaneous and late replacement of insulin UNESP, Botucatu), João Lauro Viana de Camargo restores the MMP-2 and -9 activities to the control (Departamento de Patologia da Faculdade de Medicina, levels. In conclusion, diabetes disturbs the structure of UNESP, Botucatu), Wilma De Grava Kempinas the prostate growth and led to an effective reduction (Departamento de Morfologia do Instituto de Biociências in the MMPs activities. The insulin replacement, even de Botucatu, UNESP, Botucatu) delayed, restores the MMPs activities, showing that adverse effects of these disease in the prostate are The Brazilian National Agency of Sanitary Vigilance reversible. Financial support: FAPESP. indicated that the population has been constantly [email protected] exposed to residues from different pesticide mixtures. The aim of this work was to evaluate reproductive A-021 toxicity in rats exposed to the following pesticides, EFFECT OF STZ-INDUCED DIABETES AND INSULIN individually or in a mixture: dicofol, dichlorvos, REPLACEMENT ON COLLAGEN AND RETICULAR permethrin, endosulfan and dieldrin. For this, sixty male FIBERS DEPOSITION IN THE PROSTATIC LOBES Lewis rats (6 weeks old, 200g), were divided into 8 DURING PUBERTAL GROWTH groups: 1- negative control (n=8), received basal chow; Elaine Manoela Porto, Sergio Alexandre Alcantara dos 2 - low dose (n=10), received the pesticide mixture Santos, Sergio Luis Felisbino (UNESP) added to basal chow, each pesticide at NOEL dose for rats; 3 - effective dose (n=12), received mixture Stroma influences epithelial function by producing added to basal chow, at LOAEL dose to dieldrin and growth factors and by providing a structurally and endosulfan, LOEL to dicofol and LEL to dichlorvos compositionally adequate extracellular matrix. In this and permethrin; 4 (divided into a, b, c, d, e; 6 rats per work we characterized the collagen fiber reorganization subgroup) - positive control, each subgroup received a in the rat prostatic lobes from diabetic, diabetic plus chow containing each pesticide individually, at effective simultaneous insulin-replacement and diabetic plus doses. After 8 weeks of treatment the animals were late insulin-replacement. Prepubertal male Wistar sacrificed and reproductive parameters were evaluated. 1. 67

livro.indd 67 7/7/2008 16:33:32 Sperm morphology, daily sperm production (DSP), PREPUBERTAL PHASE sperm transit time through the epididymis, hormonal Fatima Kazue Okada, Taiza Stumpp Teixeira, levels and histopathological evaluation of testis and Sandra Maria Miraglia Valdeolivas epididymis did not present significant difference (Universidade Federal de São Paulo) among the groups. On the other hand, sperm motility decreased (p<0.05) in animals of groups 2, 3, 4a and Carnitine is an important amino acid to spermatogenic 4e when compared to negative control. These results function. It has antiapoptotic action against the suggest impairment of sperm quality and fertility damage provoked by radio and chemotherapy. potential in rats exposed to low doses of the studied There is little information about carnitine protective pesticides, in spite of negative effects on the DSP and action on seminiferous epithelium. Etoposide is a spermatogenesis. chemotherapeutic agent used against cancer and is [email protected] known to increase germ cell apoptosis, histopathological and morphometric testicular alterations and temporary A-023 or permanent infertility. The aim of this research BIOMETRIC AND MORPHOMETRIC TESTICULAR was to investigate whether carnitine protects the DATA OF ADULTS RATS TREATED WITH seminiferous epithelium against the damage caused ETHANOLIC EXTRACT OF ARCTIUM LAPPA AND by etoposide. One hundred-twenty 25 day-old rats CADMIUM CHLORIDE were distributed into experimental groups: Etoposide; Fabrícia de Souza Predes (Universidade Estadual Carnitine+Etoposide; Carnitine and Control; the groups de Campinas), Maria Aparecida de Silva Diamante were divided according to the sacrifice ages: 32, 64 (Universidade Paulista), Mary Anne Heidi Dolder and 127 days. The testes were dissected for TUNEL, (Universidade Estadual de Campinas) Fas, FasL and p53 immunostaining. PAS+H method was also used for histopathological and morphometric Several studies have shown that scavengers of free analyses. The Etoposide and Carnitine+Etoposide radicals and antioxidants protect various animals groups showed accentuated testicular morphometric from damage by cadmium. The aim of this study is to alterations when they were 64 days old and an increase investigate the effect of Arctium lappa ethanolic extract of the numerical density of TUNEL-positive cells and (AlE) after cadmium chloride (CdCl2) treatment on the cells with abnormal nuclear morphology. The volume male reproductive system of adult Wistar rats. Four density of p53, Fas and FasL immunostaining was groups of 12 animals received respectively water, higher in the testes of 127 day-old rats than in the CdCl2, AlE and AlE+CdCl2. The AlE was administrated 64 day-old in all groups. Under the conditions of this in a dose of 300 mg/Kg BW daily by gavage and 1mg/ experiment, carnitine did not protect the seminiferous Kg BW of CdCl2 was injected intraperitoneally as a epithelium against etoposide. Other protocols must be single dose. Six animals of each group were sacrificed utilized to better investigate its possible cytoprotective on the 7th and 56th days of experiment. Rats were effect on the testis. The interaction between the two fixed by whole-body perfusion in Karnovsky fixative. drugs must be also studied. One testis and the reproductive acessory glands were [email protected] weighed. Fragments of testis tissue were embedded in glicol methacrylate using standart techniques. A-025 The histological sections were examined with light DIFFERENTIAL EXPRESSION OF GENES RELATED microscope for testis morphometry study. The tubular TO CYTOTOXIC PATHWAY IN THE SUBSETS diameter and epithelium height was obtained in Image OF MOUSE UTERINE-NATURAL KILLER CELLS Pro Plus program. No change in the average corporal ISOLATED WITH LASER CAPTURE MICROSCOPY and testicular weights was observed in the study. Karina Yumi Degaki (Instituto de Biologia - The weight of epididymis, seminal vesicle, prostate UNICAMP), Nicola F. Linton (Biomedical and coagulation gland did not alter significantly. The Sciences- , Ontário Veterinary College-University of epithelium height increased significantly in AlE and Guelph), Theri Leica Degaki (Instituto de Química - AlE+CdCl2 - treated groups with 7 days of treatment. USP), Gonçalo Amarante Guimarães Pereira (Instituto In the groups treated for 56 days no alteration was de Biologia - UNICAMP), observed in the tubular diameter and epithelium height. Chandrakant Tayade (Biomedical Sciences, With these preliminary results it was not possible to Ontário Veterinary College - University of Guelph), ascertain the effect of the plant on the possible damage Aureo Tatsumi Yamada (Instituto de Biologia - caused by cadmium, since this was not detected by the UNICAMP) parameters studied. [email protected] The uterine-natural killer (uNK) cells modulate the homeostasis of maternal-fetal interface in the pregnant A-024 uterus. However, the supposed quiescence of cytotoxic EFFECT OF CARNITINE ON THE activity of these cells during normal pregnancy is SEMINIFEROUS EPITHELIUM OF ALBINO RATS still a paradox of immunology of reproduction to TREATED WITH ETOPOSIDE DURING THE be elucidated. The present work first evaluated the 1. 68

livro.indd 68 7/7/2008 16:33:32 expression of IFN-g, TNF-α, FasL, IL-6, perforin, to structural disturbance in the prostate and the lack granzymes A (GzmA), DAP12 genes related to innate of DG receptors pointed to diabetes as a risk factor of immune response of NK cells by semi-quantitative developing prostatic malignancy, considering that DG is RT-PCR. The RNA were obtained from uterine tissue as an important cell and matrix extracellular attachment. homogenates of normal pregnant mice and their [email protected] possible changes after surgical lesion of embryo (SLE). Two of these genes IFN-g and GzmA were A-027 further evaluated with quantitative RT-PCR using MORPHOMETRICAL AND HORMONE RNA isolated from uNK cells dissected with laser ANALYSIS OF THE EFFECTS OF THE INFUSION OF capture microdissection (LCM). The PCR confirmed de “NÓ-DE-CACHORRO” expression of all the genes evaluated and the semi- (HETEROPTERYS APHRODISIACA, O.MACH.) ON quantitative analysis revealed consistent increasing TRAINED WISTAR RATS 1hr after SLE. On the other hand, the qRT-PCR of Marcos de Lucca Morera Gomes, Juliana Castro dissected uNK cells showed high expression of IFN-g Monteiro, Mariana Mendes Sbervelheri, in the agranular rather than uNK containing granules in Heidi Dolder (Unicamp) the cytoplasm, which was down regulated after 30 min of SLE. The Gzm A showed higher expression in the Throughout the last decades, an increased search granules containing uNK cells than agranular forms and for substances of plant origin was observed, which similarly to IFN-g, the expression of Gzm A decreased could act on the reproductive system assisting in 30min after LSE and recovered after 12 h in the the performance of the sexual functions, mainly granular uNK cells. These LCM-qRT-PCR data confirms of males. In this context, the roots of Heteropterys the differences on gene expression between granular aphrodisiaca are used as an aphrodisiac, a stimulant and agranular subpopulations of mouse uNK cells and and to strengthen muscle and bone. In this research, both of them, are responsive to changes of homeostasis the plant infusion’s effect was studied in Wistar rats, in the evaluating possible corporal biometric changes, as maternal-fetal interface induced by embryo lesion. well as testicular morphometric parameters, glandular [email protected] weights, and hormone concentrations. Forty rats were divided into groups: GI-control, sedentary; GII-H. A-026 aphrodisiaca, sedentary; GIII-control, trained; GIV-H. DYSTROGLYCAN FEATURE ON THE PROSTATE aphrodisiaca, trained. The training protocol consisted OF NON-OBESE DIABETIC MICE SUBMITTED TO in running on a motorized treadmill, five times a week, GLYCEMIC CONTROL with weekly increase in treadmill velocity and duration. Valéria Helena Alves Cagnon Quitete, Wagner José Water (sham) or H. aphrodisiaca was administered Fávaro (Unicamp) daily by gavage (35mg/animal/day) for 8 weeks. The testosterone concentration was determined by Dystroglycan (DG) is an adhesion protein which plays radioimmunoassay. Both sedentary and trained animals a crucial role in epithelial cell development; formation that received the plant infusion showed higher testicular of basal membrane and maintenance of tissue and prostatic weights (ventral prostate). However, there integrity. Also, diabetes has been pointed out as a was no significant difference in the tubule/interstice pathology which causes harmful effects on the prostate proportion among groups. Also, the trained animals homeostasis. Therefore, the main objective of this work showed an increase in the seminal vesicle weights. was to verify DG and structure features in diabetic Despite the decrease in testosterone concentrations mice with and without glycemic control, and as well as in trained rats, there were no alterations in testicular to verify the relation of these parameters on prostate parenchyma weights and seminiferous tubule volumes. pathogenesis. 30 mice (Nod and BALB/c) were divided This behavior was probably due to the fact that the rats into three groups, after twenty days of diabetic state: the were submitted to a long, hard treadmill training. control group received a 5 mL/Kg dose of physiological [email protected] saline daily for 20 days; the diabetic group had the same treatment as the control group; the diabetic- A-028 Insulin group received 4-5 IU doses of NPH insulin DYSTROGLYCAN AND STRUCTURAL daily for 20 days. After 20 days of treatment, all animals CHARACTERISTICS OF THE VENTRAL LOBE OF were sacrificed and samples from the ventral prostate THE PROSTATE OF MDX MICE were processed for immunological and light microscopy Leslie Cristina Pinto, Wagner José Fávaro, Valéria analyses. The results showed diminished b and a DG Helena Alves Cagnon Quitete (Unicamp) receptors in the diabetic group. However, there was a recovery of both b and a DG receptor immulocalization Dystroglycan is an adhesion molecule, showing an after administrating insulin. The epithelial and stromal important role in prostate structural maintenance. morphological changes were verified in the diabetic Diminished dystroglycan expression has been group which also presented recovery after insulin associated to tumor progression in prostate treatment. Thus, it could be concluded that diabetes led cancer. First, dystroglycan was studied in muscle 1. 69

livro.indd 69 7/7/2008 16:33:32 physiopathology such as Duchenne’s muscular Willcoxon tests. Insulin addition to fresh semen caused dystrophy. MDX mice have been studied due to a decrease (P<0,01) in both parameters from 100min presenting this type of muscular disorder, which of incubation. Insulin addition to diluted semen caused involves dystroglycan dystrophin complex degeneration. a decrease in spermatic motility (P<0,01) and vigour Thus, the aim of this work was to characterize epithelial (P<0,05) from 120min. In the experimental conditions and stromal structural desorganization in MDX mice, utilized, the addition of insulin did not improve the to locate IGF-1, a, b dystroglycan receptors and to seminal parameters evaluated. Future studies on this relate these results to proliferation and cellular death issue are suggested by the authors. processes as well as to verify the viability of this [email protected] experimental model in prostatic disease studies. Thirty male mice (C57 BL10 and MDX) were divided into A-030 two groups; control and MDX. Samples from ventral ESTROGEN IMPRINTING: EFFECTS ON ANDROGEN prostate were collected for immunological and light RECEPTOR EXPRESSION IN THE HYPOTHALAMUS microscopical analyses. Estradiol and testosterone OF WISTAR RATS measurements were verified. The results showed Elusa Cristina de Oliveira (UNICAMP), Jackson Cioni diminished testosterone and increased estradiol Bittencourt (USP), Hernandes Faustino de Carvalho levels in the MDX group. Atrophic cells, stromal (UNICAMP) hypertrophy, and prostatic intraepithelial neoplasia and inflammatory cells were verified in the MDX In male rats, complete reproduction function is critically mice. Weak a, b dystroglycan immunolocalization dependent on adequate estrogen action at different was verified in the MDX group. However, intense levels of the hypothalamic-pituitary-gonadal axis. The IGF-1 receptor localization was identified in the MDX administration of high doses during the early postnatal animals. Thus, it was concluded that MDX animals period results in irreversible alterations in the male presented changes in the molecular and structural reproduction system and promotes a mechanism integrity and proliferation signals, leading to glandular known as estrogenic imprinting. One of the estrogenic pathogenesis, compromising the prostate homeostasis imprinting effects caused by high doses of the estrogen and reproduction. process. is the negative regulation of the expression of androgen [email protected] receptor (AR) in the prostate and reduced response to androgen exposure. This study investigated the A-029 influence of estrogen imprinting on the AR in the EFFECT OF THE ADDITION OF INSULIN ON hypothalamus of Wistar rats. For this, male rats were SPERM MOTILITY AND VIGOUR IN THE SEMEN treated in the early neonatal period with 17b-estradiol OF RABBITS (ORICTOLAGUS CUNICULUS LINEU, (E2) (three doses of 15mg/kg) and the effects were 1758), IN FRESH AND DILUTED IN TRIS-GLUCOSE analyzed in the adulthood in comparison with control Lázaro Wender Oliveira de Jesus, Silvio Luiz de rats and with intact females. AR distribution was Oliveira, Paula Fernanda de Souza Braga, Maria analyzed by immunohistochemistry, the total content Raquel Isnard Moulin, Samuel Mariano-da-Silva, was determined by Western blotting and its mRNA Edismair Garcia Carvalho (UFG - Campus Jataí) by RT-PCR. The neonatal estrogenization promoted decreased expression of the AR and its mRNA. Counts The development of artificial insemination techniques of AR-positive nuclei revealed that the ventromedial, in rabbits has prompted many studies on energy anterior medial preoptic and paraventricular nuclei substrates and diluents associations to the semen. and preoptic area were altered as compared to control Diluents protect the spermatozoa from critical effects males and exhibited a pattern similar to those of from semen processing and harvesting and should females. The data indicate that E2 is able to induce contain nutrients for sperm metabolism. Knowing the estrogen imprint in the hypothalamus and to alter the insulin action on animal metabolism, this work aimed to AR expression, with possible effects on sexual behavior. evaluate the effect of the addition of insulin on sperm [email protected] motility and vigour in fresh and in diluted semen of rabbits. Semen of three rabbits Orictolagus cuniculus A-031 (Lineu, 1758) (Chinchilla x New Zealand) was collected RADIATION UVB INDUCES APOPTOSIS IN THE in the same flask. To evaluate the fresh semen, a tube HEPATOPANCREAS OF MACROBRACHIUM containing 0,5ml de semen was added 100µl of mixed OLFERSI (DECAPODA, PALAEMONIDAE) insulin (NPH IOLIN® 100U/ml). Treatment tube and a Francielle Regina Ceccato (Universidade Federal de control tube containing only semen were both kept at Santa Catarina), Evelise Maria Nazari (Universidade 37°C. To evaluate the diluted semen, total semen was Federal do Rio de Janeiro), Yara Maria Rauh Müler diluted 1:1 in tris-glucose and treated as described (Universidade Federal de Santa Catarina) above. Samples of semen were taken from each tube and sperm motility and vigour were analysed under The decrease of ozone layer increases the incidence light microscope at each 20 minutes during two hours. of ultraviolet radiation on living organisms and this has The results were analysed by Kruskall-Wallis and by been associated with structural damages. The prawn 1. 70

livro.indd 70 7/7/2008 16:33:32 Macrobrachium olfersi lives in freshwater streams and groups M, MT and in areas of PIN and microinvasives are daily exposed to this radiation. In these animals the sites. Collagen fibers decreased in neoplastic sites, digestive gland (hepatopancreas) has also absorptive since its bundle arrangement around of the acini and detoxificative functions being formed by tubules was lost. The stromal compartment emerged as an with proliferative cells (distal region) and mature cells active participant in the establishment of proliferative (proximal region). To assess the effects of UVB radiation lesions creating a microenvironment favorable to the on the hepatopancreas cells, adults of M.olfersi were development of prostate cancer. collected and kept in aquaria at 24°C, controlled [email protected] photoperiod, constant aeration and daily food. After acclimatation, animals were exposed to UVB radiation A-033 (30min) and kept on dark for 4 days. In order to identify EXPERIMENTAL CRYPTORCHIDISM AND apoptotic process, the animals were anaesthetized ORCHIDOPEXY IN IMMATURE MICE: (4°C) to remove the hepatopancreas which were fixed, STEREOLOGICAL ANALYSES OF EPIDIDYMAL embedded in paraffin, sectioned (3um) and submitted CAPUT to TUNEL method and immunohistochemistry using Patrick Vianna Garcia (UNICAMP), Kélen Fabiola anti-Bcl2. Reactive cells were count in five fields of Arrotéia (UNICAMP), Paulo Pinto Joazeiro (UNICAMP), each slice randomly chosen. Control animals had few Suzana de Fátima Paccola Mesquita (UEL), Luís apoptotic cells (0.6%) observed only in the proximal Antonio Violin Pereira (UNICAMP) region by TUNEL method. This same method showed that irradiated animals present an increase of apoptotic Background: Cryptorchidism is a disease in which the cells (3.1%) in both proximal and distal regions of testes are retained in the abdominal cavity, resulting tubules. However, Bcl2 reactive cells corresponding to in testicular atrophy. Orchidopexy restores testicular 0.7% in control and 17,9% in irradiated hepatopancreas. spermatogenesis in clinical procedures, but it is As expected, the UVB radiation seems trigger apoptosis still unclear whether the histological changes in the in the hepatopancreas, but the increase of Bcl-2 protein epididymis caused by cryptorchidism can be reverted immunodetection indicates the occurrence of a cell by orchidopexy. Methods: The aim of this study was to survival mechanism to prevent the apoptosis. investigate stereological alterations, expression of the [email protected] antigen recognized by mAb TRA54, SM alpha-actin and SM-MHC in immature mice following cryptorchidism A-032 and orchidopexy. Results: In criptorquidic epididymis QUANTITATIVE EVALUATION OF STROMAL was observed a significant reduction in all stereological COMPONENTS ASSOCIATED TO PROSTATIC parameters that were analyzed and a small but TUMORS EXPERIMENTALLY INDUCED IN GERBIL significant increase in the SM alpha-actin expression. Bianca Facchim Gonçalves, Sebastião Roberto Taboga, After orchidopexy the epididymis recovered all Silvana Gisele Pegorin de Campos (Instituto de parameters that were analyzed. The dosage of Biociências Letras e Ciências Exatas - UNESP) testosterone serum and the expression of TRA54 antigen remains unaltered suggesting a compensatory In this work it was performed the quantitative evaluation mechanism in the activities of Leydig cells in of stromal and glandular components of gerbil’s cryptorchidism. Conclusions: These findings indicate prostatic tumors chemically induced. Adult gerbils that orchidopexy restores the histological alterations (3 months) were divided in four groups: MT and M caused in the epididymis by cryptorchidism. received previously a dose of carcinogen N-methyl-N- [email protected] nitrosurea (MNU, 30mg/Kg). During the experiment, MT and T groups received weekly dosage of testosterone A-034 propionate (T, 2mg/Kg) while M and O received weekly ANDROGEN AND IGF-1 RECEPTOR FEATURES doses of oil. After 3, 6, 9 e 12 months of therapy, IN THE COAGULATING GLAND OF RATS (UCHB) the prostates were removed and processed for light SUBMITTED TO EXPERIMENTAL CHRONIC microscopy. Histopathological lesions images were ALCOHOLISM submitted to Weibel’s and Tukey’s honest statistical Fábio Montico (UNICAMP), Eduardo Marcelo Cândido test for analysis of the relative frequency of prostatic (UNICAMP), Wagner José Fávaro components. The carcinogen and hormone promoted (UNICAMP), Francisco Eduardo Martinez high incidence of histopathological alterations as (UNESP - Botucatu), Valéria Helena Alves Prostatic intraepithelial neoplasia (PIN), Microinvasive Cagnon Quitete ( UNICAMP) carcinoma and Adenocarcinoma. Inflammatory focuses were commonly associated with these injuries. Alcoholism induces harmful effects on the accessory T, M and MT groups showed increased volume of sex glands. The aim of this work was to analyze epithelium which was also observed in areas of PIN and morphological and immunohistochemical changes in microinvasives lesions. However, in these groups the the coagulating gland of rats from long-term alcohol secretory activity was significantly reduced mainly in treatment and the possibility of tissue recovery. Thirty neoplastic foci. Reticular fibers increased significantly in rats (10 Wistar, 20 UChB) were divided ito three groups: 1. 71

livro.indd 71 7/7/2008 16:33:32 control group (10 Wistar) received tap water; alcoholic enzymatic complex. The current results, associated group (10 UChB) received 10% etanol and abstinent with gene and protein expression of NAD(P)H-oxidase group (10 UChB) received 10% ethanol for 120 days components obtained in our laboratory, reinforce the and then tap water like the control group for another close association between trophoblast phagocytic 30 days. After 150 days, all the animals were sacrificed activation and ROS mostly via NAD(P)H-oxidase and coagulating gland samples were collected for light regulatory mechanisms.FAPESP,CAPES,CNPq. microscopy and AR e IGF-1R immunolocalization. Both [email protected] alcoholic and abstinent rats showed diminished mucosa folding, atrophied secretory epithelium and stromal A-036 hypertrophy. Intense AR immunolocalization was CYTOGENETIC STUDY OF THE NUCLEOLAR verified in epithelial, luminal and basal cells from the CYCLE IN THE SEMINIFEROUS EPITHELIUM OF control group. However, this same receptor presented THE LEPTODACTYLUS PODICIPINUS (AMPHIBIA, weak immunolozalization in basal and stromal cells ANURA) in both alcoholic and abstinent groups. The IGF-1R Maristela Previato (UNESP/IBILCE, Department demonstrated intense stromal immunolocalization in of Biology), Rita Luiza Peruquetti (UNESP/IBILCE, the alcoholic and abstinent groups in comparison to the Genetic Graduate Program), Maria Tercília Vilela de control. Thus, it could be concluded that chronic alcohol Azeredo Oliveira (UNESP/IBILCE, Department of use led to morphological and molecular alterations Biology) in the coagulating gland, showing an inverse ratio of the AR and IGF-1 receptor immunolocalization of the The nucleolus is nuclear territory related to the alcoholic and abstinent groups in relation to the control compartmentalization of nuclear functions. The one. This result pointed alcohol as being a stimulating nucleolus formation is a dynamic process in eukaryotic factor of a proliferative process, leading to coagulating cell cycle denominated nucleologenesis. Some authors gland pathogenesis. believe that the chromatoid body (CB), a cytoplasmic [email protected] structure of spermatogenic cells that has an important role in the spermiogenesis, is formed by extrusion of A-035 nucleolar material from the nucleus to the cytoplasm. PRODUCTION OF O2- AND H2O2 BY PMA- The aim of the present study was to follow the nucleolar STIMULATED TROPHOBLAST CELLS cycle during spermatogenesis of Leptodactylus Aline Rodrigues Lorenzon, Juliana Siviero Vieira, podicipinus (Amphibia, Anura). The testes was treated Clarissa Rocha, Sara Zago Gomes, Estela Bevilacqua with a hypotonic solution of KCl and fixed in just- (Universidade de São Paulo) preparing methanol: acid acetic. The material had been transferred to excavated plate and it was fragmented in The production of reactive oxygen species (ROS) order to get a cellular suspension. Three drops of this allows phagocytes to play important cytolytic functions. suspension was placed on a warm slide. The slides Previous studies showed that phagocytic trophoblast were submitted to cytochemical procedures: Giemsa cells are able to produce and release ROS, in a similar stain and silver ion impregnation (AgNOR). The results manner to phagocytes. They also suggested that of cytogenetic analyses were: spermatogonium with NAD(P)H oxidase organized nucleolus; primary spermatocytes (leptotene, enzymatic complex may be involved in this production/ zygotene and pachytene) with fragmented nucleolar activation. Herein, we investigated the production material distributed around of the chromosomes; of ROS by cultured trophoblast cells upon different primary spermatocytes (diplotene, diakinesis e phagocytic stimuli and metaphase I) with absence of organized nucleolar NADH-oxidase and xanthine-oxidase specific inhibitors. material; earlier and later spermatids with a single/ The production of O2- and H2O2 was monitored by central nucleolus reorganized, however it presented a alterations in the florescence arising from the oxidation lesser volume. In conclusion, data demonstrated that of dihydroethydium (DHE), which in the presence of there is disintegration of the nucleolus and, probably, O2- is oxidized yielding oxyethydium and with H2O2 a fraction of this nucleolar material migrates to the and others ROS is oxidized yielding ethydium. These cytoplasm, where a specific structure is formed, known molecules specifically bind to DNA to produce a bright as chromatoid body (CB). red fluorescence under fluorescent microscopy and [email protected] specific light waves under confocal microscopy. We used NAD(P)H- and xanthine-oxidase A-037 substrates and inhibitors and mitochondrial chain CYTOCHEMICAL STUDY OF THE inhibitor. O2- and H2O2 was detected by NUCLEOLAR CYCLE IN THE SEMINIFEROUS incubating trophoblast cultures with NAD(P)H- and EPITHELIUM OF THE PHRYNOPS xanthine-oxidase substrates, associated or not with GEOFFROANUS (REPTILIA, TESTUDINES) PMA and all-trans-retinal stimuli, but always more Rita Luiza Peruquetti (São Paulo State University – intensively upon these stimuli and NAD(P)H-oxidase UNESP/IBILCE, Genetic Graduate Program), Silvia substrate. NAD(P)H-oxidase inhibition blocked both Regina Pegorin Cabral (São Paulo State University 1. 72

livro.indd 72 7/7/2008 16:33:32 – UNESP/IBILCE, Animal Biology Graduate Program), presented dispersed cells in between dense colagen Classius de Oliveira (São Paulo State University – fibrils showing an evident disorganization. PGs were UNESP/IBILCE, Department of Biology), Maristela classifyed as P1 in the basement membrane, P2 Previato (São Paulo State University – UNESP/IBILCE, associated to collagen fibrils, P3 in the intersticial Department of Biology), Maria Tercilia Vilela de Azeredo stroma and P4 large PGs distributed througout stroma. Oliveira (São Paulo State University – UNESP/IBILCE, Diabetes promoted a disarrangement in P1 and an Department of Biology) increase in P3 and P4. There were a significative increase in CS (33%, mainly in points where P4 was The nucleolus is a particular nuclear territory related accumulated) and collagen (44%) in the prostatic to the compartmentalization of nuclear functions. The stroma of diabetic rats. FN expression was not nucleolus formation is a dynamic process in eukaryotic changed. These data suggest that P4 could be CS, cell cycle denominated nucleologenesis. Some authors because of their large size and similar distriution. In believe that the chromatoid body (CB), a cytoplasmic a previous work it was described malignant lesions in structure of spermatogenic cells that has a role in the prostate of diabetic rats. Thus, beside the epithelial the RNA and protein reserve during spermiogenesis, changes, such increase in stromal elements caused by is formed by extrusion of nucleolar material from diabetes could impair epithelium-stroma interaction as the nucleus to the cytoplasm. The aim of this study well as stimulate a malignant process in the rat prostate. was to follow the nucleolar cycle in the seminiferous [email protected] epithelium of the Phrynops geoffroanus (Reptilia, Testudines). The testes was removed and fixed by A-039 immersion in Karnovsky’s fixative solution. The material THE EMBRYO LESION INDUCES QUICK was embedded in glycol-metacrylate historesin. Thick INCREASING OF NITRIC OXIDE CONCENTRATION sections were obtained in Leica RM 2155 microtome AT THE MATERNAL-FETAL INTERFACE OF and submitted to the following cytochemical procedures: PREGNANT MOUSE UTERUS Hematoxylin-eosin (HE), Toluidine blue (TB), modified Eliana Mara Oliveira Lippe, Selma Giorgio, Renata Critical Eletrolyte Concentration (CEC), silver ion Miliani Martinez, Aureo Tatsumi Yamada (UNICAMP) impregnation (AgNOR) and Feulgen reaction. HE stain was used to general analysis of seminiferous Several factors to affect the homeostasis of pregnant tubules and the other cytochemical techniques (AT, uterus bearing the miscarriage, but most of these CEC, Feulgen reaction and AgNOR) demonstrated the proposed mechanisms have not been experimentally fragmentation in the primary spermatocytes nuclei, the confirmed. Previous study showed hyperemia and distribution of this material inside the nucleus and a hemorrhage in the pregnant uterus as consequence reduction of the nucleolar volume in earlier spermatids. of embryo lesion, suggesting alterations of vascular In conclusion, these data demonstrated that there is permeability involving releasing of cytolytic components disintegration of the nucleolus and, probably, a fraction of uNK cells granules. The aimed to evaluate the of this nucleolar material migrates to the cytoplasm, participation of nitric oxide synthase (NOS) and nitric where the structure known as the chromatoid body (CB) oxide (NO) production in the abnormal pregnancy is formed. Support: CNPq / FAPESP. induced by surgical lesion of mouse embryo (SLE) and [email protected] compared with normal pregnancy. The expression of isoforms iNOS, eNOS and nNOS were evaluate by A-038 immunocytochemistry and Western-blot, as well as the DIABETES INCREASES COMPONENTS OF nitric oxide concentration was determined (nitrate and EXTRACELLULAR MATRIX IN THE STROMA OF RAT nitrite forms) in the uterine samples after 30 min, 1h, PROSTATE 2h and 6h of SLE. The immunocytochemistry detected Daniele Lisboa Ribeiro (Insituto de Biologia- Unicamp), the isoforms in the uNK cells of normal pregnancy. Sebastião Roberto Taboga (Ibilce- Unesp), Rejane Interestingly, the iNOS expression decreased after 1 Maira Góes (Ibilce-Unesp) and 2h of SLE and partially recovered after 6h, while the NO showed a peak concentration at 30 min after Objective: To evaluate the extracellular matrix (EM) SLE and than decreased level at 6h. These data in the prostatic stroma of diabetic rats and its relation suggest the relation between hemorrhage after SLE with development of malignant lesions. Diabetes was and increased proportion of the highly cytotoxic nitrite induced in male adult Wistar rats with alloxan (45 form of NO affecting the vascular permeability or even mg/kg bw). 90 days after diabetes onset, rats were integrity of blood vessels. These data also suggest a killed and prostate fragments were processed for quick consumption of iNOS in the uNK cells probably immunohistochemistry, citochemistry using Cuprolinic due to the increased mobilization of this enzyme Blue to identify proteoglycans and transmission to produce harmful local concentration of NO as electronic microscopy. We studied the expression of consequence of stressing changes on maternal-fetal fibronectin (FN), chondroitin sulphate (CS) and collagen interface homeostasis induced by embryo failure. as well as different types of proteoglicans (PG) based [email protected] on their size and distribution. Stromal compartment 1. 73

livro.indd 73 7/7/2008 16:33:33 A-040 showed that the process of gonocyte (Gn) relocation GENE EXPRESSION OF INFLAMMATORY to the basement membrane starts at 1 d and it is CYTOKINES BY THE TROPHOBLAST CELLS observed up to 14 d. A small percentage of relocated Mara Sandra Hoshida (Dept Cellular and Gn was found during early postnatal days, suggesting Developmental Biology, Institute of Biomedical that this process is followed by its rapid phenotypical Sciences, University of São Paulo), Renata Gorjão transformation into cells that exhibit transient features (Dept Physiology and Biophysics, Institute of Biomedical between Gn and A spermatogonia, here designated pre- Sciences, University of São Paulo), Rui Curi (Dept spermatogonia (PSp). A close association between PSp Physiology and Biophysics, Institute of Biomedical and central Gn was verified, but specific cell junctions Sciences, University of São Paulo), Estela Bevilacqua were not observed. High amounts of non-relocated (Dept Cellular and Developmental Biology, Institute of apoptotic Gn were found at first week of age. On the Biomedical Sciences, University of São Paulo) other hand, the peak of germ cell proliferation was detected 14d forward. The differentiation of male germ Chemokines are a critical component of basal leukocyte cells of gerbil in neonatal days involves the progressive trafficking, essential for immune system development processes of relocation, transformation into PSp and and immune surveillance. They also participate in subsequent proliferation. The onset of differentiation is leukocyte growth, differentiation and activation beside not synchronic in all seminiferous tubules and probably to stimulate effector functions on these cells, such as: several successive waves of gonocyte differentiation activation of integrin pathway signaling, chemotaxis seem to occur within the same tubule. and superoxide radical production. In the gestational [email protected] scenario and particularly at the maternal-placental interface in which numerous immune cells play A-042 relevant functions lymphocyte homing may be critical PREGNANT MICE VACCINED AGAINST for pregnancy success. In this study we examined the YELLOW FEVER HAVE INCREASED expression of inflammatory cytokines on ectoplacental ABORTION RATES, INTRAUTERINE GROWTH cone and placenta of CD-1 mice. Ectoplacental cones RESTRICTION AND VIRUS PARTICLES AT THE were obtained during the postimplantation period on MATERNAL-PLACENTAL INTERFACE gestational day (gd) 7.5 and placentas isolated on gd Fernanda Carini da Silva (Instituto de Ciências 13.5 from CD-1 mice. Gene expression was determined Biomédicas da Universidade de São Paulo), Fernanda by cDNA macroarray and semiquantitative RT-PCR. Milani Magaldi (Instituto de Ciências Biomédicas da Among the 96 genes investigated (SuperArray, MM- Universidade de São Paulo), Helena Saito (Programa O15) chiefly receptor for IL10 and IL25 were highly de Imunização da Secretaria de Saúde do Estado de expressed in trophoblast cells and placenta. Expression São Paulo - Brasil), Estela Bevilacqua (Instituto de of IL25 is closely related to pro-inflammatory activity Ciências Biomédicas da Universidade de São Paulo) favoring Th2-type immune response. These findings suggest that specific factors expressed by trophoblast The hypothetical risk of transplacental infection of the related to maternal immune response can be of 17DD virus reinforces the general recommendation great value in the interpretation and understanding of that anti-yellow fever vaccine must be administered trophoblast adaptation of the uterine environment, and during pregnancy only when epidemiologically justified. be of help in the knowledge of pregnancy-associated Objectives: To evaluate the consequences of yellow diseases. Financial support: FAPESP, CNPq and fever (YF) vaccination during gestation. Methods: CAPES. Mice females were vaccinated on day 5.5 of gestation [email protected] and sacrificed 12 day after. Rates of fetal losses (MR) were estimated as well as the morphology of the A-041 implantation sites and placentas. Immunohistochemistry NEONATAL RELOCATION AND APOPTOSIS reactions investigated the presence of the viruses at OF MALE GERM CELLS DURING TESTES the maternal liver, maternal-placental interface and DEVELOPMENT PRECEDES THE PROLIFERATION in the fetuses (normal appearance or reabsorbed). IN MONGOLIAN GERBIL Results: Vaccination significantly increased MR. In Lívia Silva Botta (IBILCE/UNESP), Maria Etelvina the vaccinated group, no morphological changes Pinto (UNICAMP), Sebastião Roberto Taboga (IBILCE/ were detected in the placenta of normal fetus, UNESP), Rejane Maira Góes (IBILCE/UNESP) although fetal weight was lower than in controls. Virus particles were immunolocalized at the maternal liver, The aim of this study was to examine the postnatal maternal-placental interface and in some fetuses. In differentiation of germ cells during postnatal reabsorbed fetuses tissues a strongly reaction were development Mongolian gerbil testes. The testes always observed. Conclusions: As in other vaccination from 1, 5, 7, 14, 21 and 28 days old (d) gerbils were programs, increased MR and intrauterine growth conventionally processed to historresin embedding and restriction may be a consequence of the immune/ transmission electron microscopy (TEM). The analysis inflammatory maternal response to the virus. However, of high resolution tick sections in association with TEM virus particles into the fetuses are, obviously, correlated 1. 74

livro.indd 74 7/7/2008 16:33:33 to a transplacental transmission. These preliminary de Ponta Grossa), Claudia Feijó Ortolani Machado data suggest that anti-yellow fever vaccination may (Universidade Federal do Paraná) alter the reproductive physiology and, support the recommendation of avoiding vaccination in pregnant Anatomical variations of the male genital tract heve women. Financial support: CNPq, FAPESP, CAPES. been described for different species of spiders, mainly [email protected] concerning to the spermatic channels. Loxosceles intermedia testes consist of a long pair of cylindrical A-043 bodies, connected to a long and coiled vas deferens A NEW MOUSE EPIDIDYMAL ANTIGEN by a thin duct and an enlarged ampulla. At the final INVOLVED IN FERTILIZATION vas deferens a secretion sheath is deposited around Kélen Fabiola Arrotéia (State University of Campinas each synspermium unit, and the conjunct synspermium (UNICAMP)), Gustavo H. M. F. Souza (State University more secretion is released at copulation. This secretion of Campinas (UNICAMP)), Stephen Hyslop (State has certainly a function of maintain the synspermium University of Campinas (UNICAMP)), Marcos N. Eberlin integrity (Costa-Ayub 2006). The aim of the present (State University of Campinas (UNICAMP)), Hiromitsu work was to investigate the duct morphology and Tanaka (Osaka University), Lucia Elvira Alvares (State ampulla secretion at light (HE stained sections and University of Campinas (UNICAMP)), Luis Antonio Violin PAS reaction) and scanning electron microscopy levels. Pereira (State University of Campinas (UNICAMP) Synspermia leave testes, within a thin secretion, pass through the thin duct, and gain the enlarged ampulla Fertilization is the union of spermatozoa and where they are covered by a great quantity of PAS oocyte in order to reconstitute a diploid cell with the positive secretion. Scanning electron microscopy potential to become a new individual. Mammalian analysis showed that the internal epithelium of ampulla spermatozoa leaving the testis are unable to fertilize is composed by cell with apical surface depressions that eggs, but they are made competent for fertilization indicate a high secretory activity. It was also observed after multiple interactions with proteins present in that there is a clear difference in appearance between the luminal fluid of the epididymis. Besides that, little secretions of the ampulla (more granulated) and final is known about the most of epididymal molecules vas deferens (more compacted). The non secretory involved in the spermatozoa maturation. We have epithelia of the duct could act as a selective gate for previously described a novel pattern of expression synspermia that allows a slow flux to the ampulla for an antigen in epididymal epithelial cells that reservatoir and their interaction with the local secretion. reacts with the monoclonal antibody (mAb) TRA 54. [email protected] Immunohistochemical analyses suggested that this antigen was released into the epididymal lumen and A-045 subsequently bound to luminal spermatozoa. Now, IMMUNOLOCALIZATION OF MIF AT THE the purification and structural characterization of this MATERNAL-PLACENTAL INTERFACE IN molecule were done. Sequencing of tryptic fragments LIPOPOLYSACCHARIDE (LPS)-TREATED MICE obtained from the purified protein by MALDI-TOF-MS Karollina Ferreira do Nascimento, Mara Sandra and LC-nano-ESI/MS/MS mass spectrometry showed Hoshida, Miriam Rubio Faria, Estela Bevilacqua that the antigen corresponded to an unnamed product (Instituto de Ciências Biomédicas USP) (BAC 34360) previously identified in the mouse genome. The protein is originated from a cDNA related Background: Data from the literature show that to the mouse albumin-1 gene. In vitro fertilization serum MIF concentration is positively correlated assays indicate that the protein is important during with the severity of bacteria infection in humans. the sperm acrosomal reaction, being strongly involved Moreover, deletion of the MIF gene in mice seems in penetration of the oocyte zona pellucida. The to confer protection against lethal endotoxemia. epididymal antigen recognized by mAb TRA 54 plays MIF has also been demonstrated at maternal fetal an important role not only for epididymal maturation interface in several species where it is supposed to of the spermatozoa but mainly for the success of the counter regulate the anti-inflammatory glucocorticoid fertilization process itself. activities. Objectives: In this study, we investigated [email protected] the presence of MIF at maternal-placental interface in pregnant females in which inflammation has been A-044 induced by administration of LPS on gestation day LIGHT AND SCANNING ELECTRON MICROSCOPY 6.5 in CD-1 mice. We specifically aimed to determine ANALYSIS OF THE DUCT AND AMPULLA SEGMENTS whether the inflammatory conditions change the cell OF LOXOSCELES INTEMEDIA MELLO-LEITÃO (1934) population producing MIF at the implantation sites in (ARANEAE: SICARIIDAE) MALE GENITAL TRACT. fetuses that have survived to the infection. Material and Aline Margraf (Universidade Estadual de Ponta methods: Immunolocalization of MIF was performed Grossa), Maria Albertina de Miranda Soares in implantation sites from day 12.5 of gestation. They (Universidade Estadual de Ponta Grossa), Cristina were selected from pregnant females that received Lucia Sant’Ana Costa Ayub (Universidade Estadual 4microg/100microL LPS, but still showed fetus with 1. 75

livro.indd 75 7/7/2008 16:33:33 health signs on day 12.5 of gestation. Results: In A-047 comparison with non-treated animals, implantation sites ULTRASTRUCTURE OF PROSTATE AFTER of LPS-treated animals had less reactive cells to MIF. SURGICAL AND CHEMICAL CASTRATION Trophoblast cells when reactive were weakly stained FOLLOWED BY HORMONAL REPLACEMENT: as well as leukocytes and decidual cells. Conclusions: MORPHOFUNCTIONAL CHANGES IN THE STROMA The fetal survival in LPS-treated pregnant females AND SMOTH MUSCLE CELLS (SMC) is certainly a consequence of multiple mechanisms Sérgio Marcelino de Oliveira (UNICAMP), Sebastião of defense triggered after infection. However, our Roberto Taboga (IBILCE-UNESP) findings lead us to suppose that the reduction of MIF at maternal-placental interface may play a role in this This work compared the behavior of the prostatic tissue intricate survival mechanism. FAPESP-CNPq. after two hormonal blockages, following by testosterone [email protected] (T) replacement. Experimental groups: ExG01 were surgical castrated, and after 30 days it received A-046 doses of 0,25ml of testosterone cipionate (25mg/Kg) SPERMATOZOAN MORPHOLOGY AND ELECTRON in intervals of 48 hours, during 21 days, and animals MICROSCOPIC DEMONSTRATION OF NUCLEAR were sacrificed after 0, 7, 14 and 21 days of the begin ACID PHOSPHATASE DURING SPERMIOGENESIS of treatment; ExG02 received weekly alternate doses OF ANOMALOCARDIA BRASILIANA AND TIVELA of 0.3ml of anti-androgens ciproterone acetate (25mg/ MACTROIDES (BIVALVIA,VENERIDAE) Kg) and flutamide (25mgKg) during 30 days. After Gisele Orlandi Introíni (Departamento de Biologia this, the treatemente was suspended and the animals Celular, UNICAMP), Alexandre Lobo da Cunha (Instituto were sacrificed in intervals of 0, 7, 14 and 21 days. de Ciências Biomédicas de Abel Salazar, Universidade Five intact adult animals were employed as control do Porto), Mário Manuel da Silva Leite Sousa (Instituto group (CG). After sacrifices the animal’s sample blood de Ciências Biomédicas de Abel Salazar, Universidade were collected to measurements of T and LH levels. do Porto), Shirlei Maria Recco-Pimentel (Departamento Prostate fragments were processed and analyzed de Biologia Celular, UNICAMP) by transmission electron microscopy. A makeable regression of the weight of the prostate was observed in In this work, we investigated the ultrastructural both castration after 30 days. Hormonal levels showed characteristics of the spermatozoa of two bivalve a reduction of circulating T, and LH showed an increase species, Anomalocardia brasiliana and Tivela during the surgical castration and a decrease in the mactroides and examined the relationship between chemical castratrion. After 30 days of castration, in both spermatozoan features and environmental conditions. groups, SMC assumed an altered spinous phenotype. In addition, the improved Gomori-chloride cytochemical After T replacement beginning prostatic epithelium procedure for acid phosphatase (ACPase) was showed a recovery of its typical height with presence performed during spermiogenesis. The sperm cells of secretory vesicles and a enlargement of the ER of both species were of the primitive type or ect- cisterns. Besides, hormonal replacement leads to a aquasperm form. The nucleus was slightly curved modulation of contract to synthetic phenotype of SMC in A. brasiliana and long, prominently curved in T. noted by presence of lipofuscin bodies and an increase mactroides. The mitochondria were distributed equally of secretion organelles, such as ER and GC. (FAPESP, around the centrioles in the midpiece of A. brasiliana Capes, CNPq). spermatozoa, but asymmetrically in the midpiece of T. [email protected] mactroides spermatozoa. There were six mitochondria and glycogen clusters in the middlepiece of the T. A-048 mactroides sperm cell. These findings suggest that T. SECRETORY PATTERN OF THE GERBIL mactroides spermatozoa have some features that are (MERIONES UNGUICULATUS) FEMALE PROSTATE adapted to an environment with constant wave activity ALONG THE ESTROUS CYCLE and a large amount of suspended material whereas Ricardo Alexandre Fochi (UNICAMP), Ana Paula A. brasiliana spermatozoa are more adapted for Silva Perez (UNICAMP), Sabrina Santos Rochel Maia calm water along beaches with little wave movement. (UNICAMP), Rejane Maira Góes (IBILCE/UNESP), During spermiogenesis, the chromatin filaments have Patricia Simone Leite Vilamaior (UNIRP), Fernanda aggregated into lamellar structures and finally into a Cristina Alcantara dos Santos (UNICAMP), Sebastião homogeneous and compact DNA arrangement. In A. Roberto Taboga (IBILCE/UNESP) brasiliana and T. mactroides gonads, a nuclear acid phosphatase was detected in mid and late spermatids. The objective of this work was to understand the In the present analysis, the nuclear acid phosphatase female prostate morphophysiology during the gerbil activity in both bivalve species has followed a very estrous cycle (proestrus, estrus, diestrus I and diestrus specific time and spatial-course pattern related to II), since the female organism is naturally subject to spermatid chromatin condensation. hormonal oscillations. Therefore, hormonal dosage [email protected] and immunohistochemical techniques for androgen receptor (AR) and serine protease reactive to human 1. 76

livro.indd 76 7/7/2008 16:33:33 antibody anti-PSA (PSA) were used. There was a A-050 peak of estrogen in the estrus and progesterone in the EFFECTS OF NANDROLONE DECANOATE ON diestrus II stage. In estrus, the concentration of PSA SPERM HEAD MORPHOMETRY OF WISTAR RATS was 3 times higher than in other stages, possibly due Leonardo Bruno Figueiredo (Universidade Federal de to the intense activity of this protein in such stage. PSA Uberlândia), Romualdo Morandi Filho (Universidade immunoreaction was intense at the glandular lumen and Federal de Uberlândia), Silveli Suzuki (Universidade apex of epithelium in the proestrus stage, while in other Federal de Uberlândia), Rosilene Aparecida Reis phases the reaction was intense in the supranuclear Rodrigues dos Santos (Universidade Federal de cytoplasm of isolated cells. AR expression was noted Uberlândia), Ana Flávia Gontijo Pimenta (Universidade in epithelium and stroma in all cycle stages. This AR Federal de Alfenas), Miguel Maurício Diaz Gomez positivity was more intense in the stroma and basal (Universidade Federal de Uberlândia), Foued Salmen cells at the estrus phase, while in other stages was Espíndola (Universidade Federal de Uberlândia), intense in basal and secretory cells of the epithelium. Marcelo Emílio Beletti (Universidade Federal de This AR positivity of the basal cells is probably Uberlândia) necessary for maintenance of epithelial secretion in the estrous cycle. Thus, it is important to go deeply in the Nandrolone Decanoate (DECA), even in a small dose, studies of hormonal interactions in the female prostate, might cause alterations in the male reproductive since the unbalance between these hormones has been system. However, its related effects on the sperm associated with prostate disorders in elapsing of aging. morphometry have yet to be known. The aim of this [email protected] study was to evaluate the sperm head morphometry of Wistar rats treated with DECA. Spermatozoa from A-049 20 Wistar rats divided into two groups: treated with IMMUNOCYTOCHEMICAL AND TEMPORAL DECA (T) (n=10), and control group (C) (n=10) were CHARACTERIZATION OF ADULT LEYDIG CELL assessed. Smears of spermatozoa removed from LINEAGES IN MONGOLIAN GERBIL the epididymis were fixed in ethanol and acetic acid Maria Etelvina Pinto (UNICAMP), Sebastião Roberto solution (3:1), and ethanol 70%. After hydrolyzing the Taboga (IBILCE-UNESP), Rejane Maira Góes (IBILCE- samples in clorhidric acid, toluidine blue staining was UNESP) used at 0,025%, pH 4 %. Fifty digital images, which were used to segmentation 100 sperm heads, were The aim of the present study is to trace the taken from each sperm smears. The following sperm development of adult Leydig cells (ALC) in the gerbil head morphometric characteristics were assessed: (Meriones unguiculatus), as well as to characterize the area (A), perimeter (P), width (W), length (L), length/ main subtypes of this cell lineage. Testes from 1, 7, 14, width (L/W), ellipticity (E), shape factor (SF), lateral 21, 28, 40, 60 e 90 days age (d) were evaluated using symmetry (LS), anterior-posterior symmetry (APS), and thick sections and transmission electron microscopy. Fourier descriptor from zero to 2 amplitude (F0, F1, F2). Immunocytochemistry for steredoigenic enzymes 3beta- Significant statistical differences between groups (test t, hidroxysteroid dehydrogenase (3b-HSD) and 11beta- p≤0.05) were observed for all assessed characteristics hidroxysteroid steroid dehydrogenase 1 (11b-HSD1) with the exception of FO, F1, and APS. Results show were also employed as marker of cell subtypes. Five that DECA administration influences sperm head cell types previously described during differentiation morphometry in Wistar rats. of ALC lineage to other mammals species were also [email protected] recognized to gerbil: mesenchymal precursor (PC), progenitor (PG), newly formed (NF), immature (IM), A-051 and mature Leydig cells (M). A peak of PC proliferation COMPUTATIONAL SPERM HEAD MORPHOMETRY was detected during first and second week of age and TO EVALUATE RAT SEMEN it precedes its conversion into PGs. The first PGs were Leonardo Bruno Figueiredo, Silveli Suzuki, Romualdo noted in 21d animals. They were differentiated from Morandi Filho, Thiago Jose Xavier Costa, Danilo Borges PC on the basis of 3b-HSD expression, specific for Paulino, Foued Salmen Espíndola, Marcelo Emílio Leydig cell lineage. The first NFs exhibiting phenotype Beletti typical of ALC, such as polygonal shape, steroidogenic (Universidade Federal de Uberlândia) organelles and nuclear aspect, were detected at 40d. As soon as NFs differentiate into IMs they express The sperm morphological evaluation is typically 11b-HSD1, and these later were identified in 60d. In performed through human visual inspection, which is this age some Ms were observed, however they were characterized by a high degree of subjectivity and bias. more abundant at 90d. Our data point to morphological The sperm morphometric evaluation by computational similarities in ALC lineage between gerbil and other image analysis has been considered as an attempt to rodents but indicates that the establishment of adult reduce such problems. The aim of this study was tested population is probably more delayed. for rat semen a morphometric analysis commonly used [email protected] for bull semen. Smears of spermatozoa removed from the epididymis of 20 Wistar rats were fixed in ethanol 1. 77

livro.indd 77 7/7/2008 16:33:33 and acetic acid solution (3:1), and ethanol 70%. After also suggest that the balancing VEGF/VEGF-Rs is hydrolyzing the samples in clorhidric acid, toluidine altered in MH placentas, which seems to favor an blue staining was done at 0,025%, pH 4 %. Fifty digital angiogenic specific signaling at placental territories and images, which were used to segmentation 100 sperm to explain the induced-villous hypercapillarization in this heads, were taken from each sperm smears. The gestational disturb. Financial support: CAPES. following sperm head morphometric characteristics [email protected] were evaluated: area (A), perimeter (P), width (W), length (L), length/width (L/W), ellipticity (E), shape A-053 factor (SF), lateral symmetry (LS), anterior-posterior MORPHOLOGICAL CARACTERIZATION OF symmetry (APS), and the three first Fourier values (F0, TESTIS OF LAMBARI (ASTYANAX ALTIPARANAE): F1, F2). The visual validation was made, as well as, the IMMUNOFLUORESCENCE DETECTION OF correlation coefficients between all characteristic were CYTOKERATIN AND ACTIN calculated. The visual validation showed that the results Mateus Contar Adolfi, Fabiano Gonçalves Costa, for A and P were coherent and W and L were influenced Chayrra Chehade Gomes, Cruz Alberto Mendoza by head curvature. This was confirmed by the ratio Rigonati, Maria Inês Borella L/W and the negative correlation between W and L. (Instituto de Ciências Biomédicas, USP) The shape of rat sperm head is naturally asymmetric, what influenced the other characteristics. We conclude Teleost species show great morphological diversity of that the rat sperm head morphometry can be used for gonads. As there were not found works describing the evaluated the rat semen, however the interpretation morphology of testis of lambari (Astyanax altiparanae), must be different of that one used for bull. and considering the importance of this species, the [email protected] present work intended to describe the testis of this species. The results will improve the knowledge about A-052 the gonadal and reproductive cycle of this animal. VASCULAR ENDOTHELIAL GROWTH FACTOR Testes of adult lambaris were collected and routinely (VEGF) IN PLACENTAS OF HYPERGLYCEMIA- processed to optical microscopy. The parafin and ASSOCIATED PREGNANCY histological resin slides were stained by H.E. method, Luciana Pietro (USP), Silvia Daher (UNIFESP), PAS histochemical reaction and Metanil-Yellow. For Marilza Vieira Cunha Rudge (UNESP), Iracema de the immunofluorescence reaction, the sections were Mattos Paranhos Calderon (UNESP), Débora Cristina incubated with anti-pan cytokeratin or anti-actin, Damasceno (UNESP), Yuri K Sinzato (UNESP), Andrea submitted to biotin conjugate antibody anti-IgG, and Albieri-Borges (UFRJ), Carla L Bandeira (USP), Estela then incubated with FITC-streptavidin. The nucleus Bevilacqua (USP) of the cell was evidenced with propidium iodide. The results show that the testis of this species is classified Hyperglycemia dysfunctions may include different as anastomosing tubule type, where the cysts are conditions such as overt diabetes (D), gestational located within tubular structures involved by diabetes (GD) and also mild hyperglycemia (MH) germinal epithelium. Also, it was found a portion of a classified according to changes in OGTT and glucose secretory epithelium showing apocrine secretion not profiles. Morphologically, the placenta of women with labeled by PAS reaction. The gonads show different MH is characterized by a relevant increase in the sites of maturation in the same testis, which suggests number of terminal villi and capillaries, which has been that the releasing of spermatozoa occurs several times attributed to a compensatory condition for maintenance during the year. The Sertoli cells are stronger labeled of homeostasis at maternal-fetal interface. This study for cytokeratin at early than late maturation. Actin analyzes the participation of VEGF and receptors was detected in apical and basal surface of secretor (VEGFRs) 1 (Flt-1) and 2 (KDR) in placentas from epithelium cells. women diagnosed as MH comparatively to those mateus.adolfi@usp. obtained from normoglycemic and, from GD and D. In the MH group an expressive immune reaction for A-054 VEGF and VEGF-R2 in vascular and trophoblast cells DIFFERENTIAL EXPRESSION OF MMPS IN THE was seen. For VEGF-R1 the immunolocalization was PROSTATE OF MALE AND FEMALE GERBIL UNDER discreet and limited to the trophoblast. The pattern HORMONAL MANIPULATION of VEGF and receptors reactivity in D placentas was Sabrina Santos Rochel Maia (UNICAMP), Luis Antonio similar to those observed in the normoglycemic group. Justulin Junior (Unesp), Fernanda Cristina Alcantara The expression of these proteins was also confirmed dos Santos (Unesp), Silvana Gisele Pegorin de Campos by Western blotting. In the decidual compartment (Unesp), Rejane Maira Góes (Unesp), Sergio Luis only extravillous trophoblast reacted to all antibodies. Felisbino (Unesp), Sebastião Roberto Taboga (Unesp) Morphological analysis also showed that collagen deposition occurs around large arterial vessels in all The female prostate of Mongolian gerbil (Meriones groups with glycemia disturbs, being apparently the unguiculatus) is a functional organ homologue to the most affected the GD group. Altogether these results male prostate, and it has been widely investigated 1. 78

livro.indd 78 7/7/2008 16:33:33 nowadays. Among the male prostatic lobes, the ventral which didn’t express AQP9, and was observed an is the one that presents better similarity to the female evident epithelial regression too. The decrease organ, both morphologically and ultrastructurally. in AQP9 could be attributed to morphological and The proper functioning of the prostate, both in males physiological alterations in epithelial and peritubular and females depends on epithelial and especially tissues of epididymis. Therefore the vasectomy cause stromal factors, such as Matrix Metalloproteinases morphological alterations accompanied by decreased in (MMPs). In order to determine possible alterations on AQP9-expression. the expression of these proteases front to hormonal [email protected] alterations caused by castration of the male and administration f Testosterone (T) to the female, paraffin A-056 sections of control and treated animals were submitted CHANGES IN EXPRESSION OF ESTROGEN to immunocytochemical reactions to MMP-2. In the RECEPTOR ERALPHA IN THE TESTIS AND MALE ventral prostate of male controls the immunoreaction EXCURRENT DUCTS OF BIG FRUIT-EATING BAT occurs in the apical region of the epithelial cells, on the ARTIBEUS LITURATUS DURING REPRODUCTIVE endothelium and in sparse fibroblasts. In the prostate AND REGRESSION PHASES of the females, the reaction to MMP-2 was positive Regiana Lucia de Oliveira, André Gustavo Oliveira, mainly in the perinuclear region of the epithelial cells José Carlos Nogueira, Germán Arturo Bohorquéz cytoplasm, while the stroma presented a greater Mahecha, Cleida Aparecida Oliveira (Universidade number of positive cells. After 7 days of treatment, both Federal de Minas Gerais) prostates of castrated males or females treated with T showed a less intense immunocytochemical reaction The aromatization of androgen to estrogen plays an all over the organ. The results indicate a hormonal essential role in the maintenance of the male fertility. interference on the regulation of the MMPs, but with The actions of estrogens are mediated by the receptors distinct regulatory mechanisms between males and ERa and ERb. Studies regarding the distribution of ERa females. have been restricted to few species of domestic animals [email protected] and rarely in wild species, and, to our knowledge, there are no studies in Chiroptera. Therefore, the aim A-055 of the present study was to compare the expression ALTERATIONS IN EXPRESSION OF THE AQP9 and immunohistochemical distribution of ERa in the AFTER VASECTOMY male reproductive tract of adult big fruit-eating bat A. Sergio Pereira, Raquel Fantin Domeniconi, lituratus, during reproductive or regression phases. In Patrícia Fernanda Felipe Pinheiro, bats during the reproductive phase, ERa expression Wílson de Mello Júnior was found predominantly in the nuclei of the non- (Departamento de Anatomia - Instituto de Biociências - ciliated cells of the efferent ductules (ED) epithelia. UNESP/Botucatu) A slight positivity for ERa was also observed in some ED ciliated cells and basal and principal cells in the Vasectomy affects epididymal morphology and water initial segment, caput and corpus of epididymis (EP), reabsorption in the initial segment of the epididymis. as well as peritubular and some connective cells in Aquaporins (AQPs) are transmembrane water channels both ED and EP. During the regression phase, there proteins present in tissues involved in fluid transport. was a wider ERa distribution. In the testis, Leydig Among the AQPs detected in male reproductive and myoid cells were intermittently positive, whereas tract, the AQP9 is the major apical water channel in the ED and EP there was a larger number of ERa expressed. Studies related to the regulation of AQP9 positive cells in both epithelia and connective tissue. EP were concentrating in initial regions of epididymis and cauda epithelium was negative for ERa in both phases there is rare information about expression of AQP9 studied. Concluding, there was a differential expression after vasectomy. The work target was study possible of ERa along the male tract of A. Lituratus during alterations in morphology and AQP9-immunolocalization reproductive and regression phases; however, strongest in epithelial cells from corpus and cauda in gerbil immunoexpression of this receptor was always found in epididymis after vasectomy. The fragments of the the ED, further highlighting the importance of estrogen corpus and cauda of the epididymis were obtained from action in this segment. ten gerbils: five vasectomized and five sham operated [email protected] animals. The surgeries were performed in aged 120 days and the fragments were collected in aged 360 A-057 days. Histological analisis and immunohistochemistry EXPRESSION OF CRIPTO-1 DURING MOUSE to AQP9 were used to epididymal evaluation. The PLACENTAL DEVELOPMENT results showed a decreased in AQP9 accompanied Carla Letícia dos Santos Bandeira (Dept of Cellular and by luminal distension and increased of peritubular Developmental Biology, Institute of Biomedical Sciences tissue in corpus and cauda of vasectomized gerbils- University of São Paulo) epididymis. In epididymal cauda was observed an inflammatory infiltration in major part of tubule sections Cripto-1 (Cr-1) is an EGF-like multifunctional 1. 79

livro.indd 79 7/7/2008 16:33:33 modulatory factor playing important roles in early Discontinuos labeling was detected in postovulatory embryogenesis, cellular differentiation and induction follicles with 48 and 96 hours after spawning. The of endothelial cell migration in tumoral angiogenesis. MMP-9 was immunolocalized to the peripheral ooplasm, The current study aims to determine whether Cr-1 is zona radiata and micropylar cell of vitellogenic follicles. expressed and participate of the trophoblast biology in In postovulatory follicles, MMP-9 was detected on mice pregnancy. The gene and protein expression of granulocytes, theca and follicular cells, increasing Cr-1 were determined by rt-PCR and Western Blotting the intensity 48 hours after spawning. The results respectively using CD-1 mice ectoplacental cones (day suggest that molecules of the basement membrane 7.5 of gestation) and placentas (days 10.5, 13.5 and are important to survival of the follicular cells and the 17.5 of gestation). Our preliminary results revealed the MMP-9 participates in the ovulatory process and in the presence of the protein and mRNA for Cr-1 in all studied postovulatory follicle involution. pregnancy stages. However, by rt-PCR semiquantitative [email protected] analyses Cr-1 expression showed a relevant increase in the days 7.5 and 13.5 of gestation in comparison to A-059 the lower expression found in the days 10.5 and 17.5. CELLULAR AND REGIONAL DISTRIBUTION OF The presence of Cr-1 during the gestation in trophoblast COMPONENTS OF THE UBIQUITIN-PROTEASOMAL cells suggests this factor may be playing a role in the AND ENDOCYTOTIC PATHWAYS ALONG THE RAT physiology of trophoblast. In addition, the variations in EFFERENT DUCTULES AND INITIAL SEGMENT OF the Cr-1 expression may also be correlated to specific THE EPIDIDYMIS functions of this cell population. Interestingly, on day 7.5 Anna Bolivar Victor Costa (Departamento de of gestation trophoblast reaches its maximum invasive Morfologia, UFMG), Rex A. Hess (Department of activity for anchoring the embryo into the endometrium Veterinary Biosciences, University of Illinois), Cleida and the 13rd day of gestation characterizes the Aparecida Oliveira (Departamento de Morfologia, maturation of the maternal-placental circulation. In this UFMG) context, Cr-1 might be participating of the vascular placental adjustments for placental completion. Intracellular protein degradation may occur through Financial Support: CNPq and FAPESP. lysosomal and ubiquitin/proteasomal routes. Studies [email protected] characterizing immunocitochemically the components of both pathways in the efferent ductule (ED) are A-058 scarce or even inexistent. In this study, the expression OVARIAN FOLLICULAR REMODELING IN of proteasome (P20S), ubiquitin (Ub) and markers CURIMATÃ-PACU, PROCHILODUS of endocytotic apparatus (early endosome antigen1- ARGENTEUS, IMPORTANT FISH SPECIES OF THE EEA1, sulphated glycoprotein-SGP2 and cathepsin SÃO FRANCISCO BASIN D) was examined in adult rat ED and initial segment Ralph Gruppi Thomé (Universidade Federal de Minas of epididymis (IS). The effect of the antiestrogen ICI Gerais), Hélio Batista dos Santos (Universidade Federal 182,780 (10mg/week/73days) on the expression of de Minas Gerais), Yoshimi Sato (CODEVASF), Elizete these proteins was also studied. In the ED, P20S and Rizzo (Universidade Federal de Minas Gerais), Nilo Ub were expressed in the nuclei of nonciliated cells, Bazzoli (Programa de Pós-graduação em Zoologia de whereas in the IS these proteins were detected in the Vertebrados PUCminas) nuclei (apical, narrow, basal and principal cells) as well as in the cytoplasm (principal cells) of the epithelial Ovarian function is dependent on extensive cyclical cells. EEA1 was detected in large vesicles in cytoplasm remodeling, that involves proliferation and cell death of ED nonciliated cells and IS epithelium. SPG2 was besides extracellular matrix alterations. The disruption restricted to vesicles of different sizes in non-ciliated of cell-matrix interactions can induce apoptosis. cells of ED, as well as luminal sperm. Cathepsin D was The matrix metalloproteinases (MMPs) are enzimes localized in supranuclear granules of nonciliated cells able to cleave proteins of extracellular matrix. In the and some ciliated cells of ED, and apical and narrow present study was used immunohistochesmistry to cells of the IS. A gradient of P20S, Ub, EEA1 and SPG2 localize laminin B2 and MMP-9, and TUNEL in situ staining was seen in the ED, which decreased 32%, technique to quantify apoptosis during the ovarian 41%, 30% and 40%, respectively, from the proximal follicle development and postovulatory follicle involution to terminal region suggesting differential regulation of in ovaries of the Prochilodus argenteus. The TUNEL- protein degradation/turnover. ICI treatment resulted positive reaction associated with morphological features in decreased P20S (15%), EEA1 (25%) and SPG2 such as chromatin condensation, cytoplasmic retraction (42%) expression in the ED, indicating that estrogen and cell fragmentation in apoptotic bodies, indicated action has a role in maintaining the cell degradation follicular apoptosis. The apoptosis rate increased of machinery. preovulatory follicles until postovulatory follicles with 48 [email protected] hours after spawning. Continuos labeling was detected for laminin B2 on basement membrane in the ovarian A-060 follicles and in recently spawned postovulatory follicles. IFN-GAMA INDUCES ACTIN POLIMERIZATION 1. 80

livro.indd 80 7/7/2008 16:33:33 OF MOUSE TROPHOBLASTIC CELLS DURING dimetilsulfoxide(DMSO)/15min] and Vitrification solution PHAGOCYTIC ACTIVITY [15% EG, 15% DMSO + 0.5% sucrose (suc)/90sec], in Patrícia Cabral de Vasconcelos (UNIFAL-MG), Paulo a metal container and immersed in liquid nitrogen. For Sérgio de Souza Prizmic Kimar (UNIFAL-MG), Estela warming, the containers were exposed to air (10sec) Bevilacqua (USP), Andréa Mollica do Amarante Pafaro and water (37°C/10sec), the gonads were put into 1M (UNIFAL-MG) and 0.5M suc solution and fixed in Bouin’s fixative. Controls were fixed soon after collection.Results:Total In early pregnancy the mouse trophoblast cells exhibits of 1822 primary and primordial follicles were analyzed. intense phagocytic activity, important for embryo Immediate, 2h and 4h vitrified gonads had 100% intact nutrition, defense and development. Trophoblast follicles. Ooplasm was intact in 89%, 96% and 94% phagocytosis resembles to the macrophage in immediate, 2h and 4h vitrification, respectively. And phagocytosis and seens to be treggered by the oocyte nucleus was intact in 89%, 98% and 95%, same stimulus. In the current study, ectoplacental respectively. In controls 100% of follicles were normal cones on 7.5 day of pregnancy were cultured in and 98% and 99% of the ooplasms and nuclei were D-MEM supplemented-medium for 48h. Zymozan- normal, respectively. No difference was observed Texas-red, opsonized or not with C3, was added, among vitrified and control groups. Conclusion: Study remaining in culture for 1, 5, 15, 30 or 45 min., with shows successful gonad vitrification up to 4h post- or whithout IFN-gama stimulus. The cultures were collection. fixed in paraformaldehyde (4 %), treated with NH4Cl [email protected] and incubed with falloydin. We have observed that trophoblast cells were able to polymerize actin very fast A-062 for engolph and phagocytic zymosan particles. Plasma VITAMINS C AND E REDUCE OXIDATIVE STRESS membrane projections showing actin filaments were AND REPRODUCTIVE DYSFUNCTION IN seen in direction to zymosan particles and the number HYPERGLYCEMIC ADULT MALE RATS of these increases by opsonization. After the phagocytic Glaura Scantamburlo Alves Fernandes (IB - PPGBCE, vacuole with the zymosan particles inside was evident, UNICAMP, Campinas, SP), Thaiane Amanda de the actin filaments were depolymerizated or were Assumpção (IB - PPGBCE, UNICAMP, Campinas, SP), restrict surround them. When culture received IFN- Carla Dal Bianco Fernandez (IB - PPGBCE, UNICAMP, gama treatment we observed a relevant increase in the Campinas, SP), Kleber Eduardo de Campos (Dep. de number of cells with actin filaments for all the cytoplasm Ginecologia e Obstetrícia, Fac. de Medicina - UNESP, during phagocitic process. Cells that were not cultivated Botucatu, SP), Débora Cristina Damasceno (Dep. de with zymosan under IFN-gama stimulus showed also Ginecologia e Obstetrícia, Fac. de Medicina - UNESP, actin polymerization. These data suggest that IFN-gama Botucatu, SP), Wilma De Grava Kempinas (Dep. de is a very important stimulus for actin polimerization Morfologia, Instituto de Biociências - UNESP, Botucatu, during phagocytosis of trophoblastic cells. Supported SP) by CNPq. [email protected] Oxidative stress caused by hyperglycemia can lead to impairment of the male reproductive system. The A-061 objective of this work was to evaluate whether the EFFECT OF THE TRANSPORT PERIOD ON treatment with antioxidants (vit. C and/or E), reducing GONADAL TISSUE VIABILITY oxidative stress, can ameliorate the reproductive insults POST-VITRIFICATION in hyperglycemic male rats. Adult male rats (90 days Nívia Lothhammer, Káren Regina Silva de Souza, old) received a single dose of streptozotocin (40 mg/ Mariana Meister, Antônia Pesente, Kg BW) and were divided into four groups (n=10 per Adriana Bos-Mikich group): hyperglycemic (HG); HG treated with 150 mg of (Universidade Federal do Rio Grande do Sul) vit. C diluted in water; HG treated with 100 mg of vit. E diluted in corn oil; HG treated with vit. C and E (same Introduction: Survival rates for cancers that occur in mentioned doses). The control group (normoglycemic) young girls and women have improved in recent years. received the vehicles. A mean glycemic level higher However, the treatment has side-effects as premature than 120 mg/dL was registered throughout the 30-day menopause. Therefore, preservation of fertility in these experiment. At the end the rats were anesthetized women is a major concern. The cryopreservation of and sacrificed, and sperm parameters and oxidative ovarian tissue is an alternative and vitrification is one stress biomarkers (TBARS, SOD, GSHt e GSH-Px) method that has been showing promissing results in were evaluated. Both vitamins reduced oxidative this field. Objective: Histological study of ovaries vitrified stress in relation to HG group, and the results were after 3 time intervals at 4°C. Methods: Mouse ovaries comparable to the control group. Moreover, vit. C and were vitrified immediately, 2h and 4h post-collection. E reduced the number of malformed sperm in relation Gonads were kept in TCM199 + fetal calf serum (FCS, to the HG group, while the former was more efficient. 20%; 4°C), exposed to Equilibrium solution [TCM- There was also an improvement of sperm motility in 199 + 20% FCS + 7.5% etilineglycol (EG) + 7.5% the rats treated with vit. C, besides an increase in the 1. 81

livro.indd 81 7/7/2008 16:33:33 sperm number in caput/corpus epididymidis in the HG A-064 groups treated with vit. C and E, isolated, compared EFFECTS OF IFN-g ON IN VITRO INTERACTION OF to the other groups. It was concluded that vitamins C TROPHOBLAST CELLS AND DECIDUAL CELLS and E, due to their antioxidant properties, isolated or Alexandre Urban Borbely, Sara Zago Gomes, in combination, can ameliorate some of the negative Luciana Oliveira Silva, effects of hyperglycemia on the reproductive function of Estela Maris Forell Bevilacqua, male rats. Sérgio Ferreira de Oliveira [email protected] (Department of Developmental and Cell Biology, ICB, University of Sao Paulo, Sao Paulo, Brazil) A-063 SIGMODONTINAE PLACENTA During early pregnancy many growth factors and (RODENTIA-CRICETIDAE): COMPARATIVE cytokines are expressed at the maternal-fetal interface ASPECTS including interferon g (IFN-g), and which may be Phelipe Oliveira Favaron (Sao Paulo University, Sao involved in the tissue remodeling, decidual integrity Paulo, SP, Brazil), Anthony Michael Carter (Department and maintenance, and trophoblast invasiviness. In the of Physiology and Pharmacology, University of present study we aimed to analyze the effects of IFNg Southern Denmark, Odense, Denmark), Andrea Mess on cell death-associated invasion processes using (Department of Research, Museum of Natural History the TUNEL assay in the co-culture system of mouse Berlin, Humboldt-University Berlin, Germany), Ana ectoplacental cone cells (EC), and isolated mesometrial Flávia de Carvalho (Faculty of Veterinary Medicine decidual cells. TUNEL staining in mesometrial decidual Sao Joao da Boa Vista, Brazil.), Adriana Caroprezo cells decreased in relation to time of cell culture, 12.0% Morini (Sao Paulo University, Sao Paulo, SP, Brazil), at 24h to 4.7% at 96h. In the presence of 100 UI/mL Carlos Eduardo Ambrósio (Sao Paulo University, Sao IFNg for 24h, the 96h decidual cell cultures increased Paulo, SP, Brazil), Maria Angélica Miglino (Sao Paulo the apoptotic index in 26,5%. TUNEL staining in 48h University, Sao Paulo, SP) of co-cultures showed that the presence of EC cells decreased the apoptotic index in the decidual cells in Sigmodontinae subfamily includes the rodents know 2%. However, in the co-cultures treated with 100 UI/ as New World rats and mice. Among other rodents, mL IFNg for 24h, the apoptotic index increased in 6% sigmodonts are an important group to investigate compared to the control co-cultures. These preliminary the placentation and placenta, the functional results suggest that the presence of the trophoblast organ for materno-fetal exchange. Placentae from may be essential for decidual maintenance in an IFN- 5 Sigmodontinae species (Necromys lasiurus, gamma-containing maternal-fetal interface. Financial Oligoryzomys sp., Oryzomys subflavus, Oryzomys Support: FAPESP- No.05/54517-8R. megacephalus and Oryzomys sp.), were obtained [email protected] from the Collection of Zoological Museum of Sao Paulo University, Brazil. Four placentas from each A-065 species, fixed in 10% formaldehyde, were subjected EXPRESSION OF DECORIN AND BIGLYCAN to histological standard processing for staining with: IN HUMAN TERM PLACENTA IN NORMAL AND H&E, Masson’s Trichrome, and Periodic acid Schiff INVASIVENESS-CHANGED TROPHOBLAST (PAS). The placenta had a labyrinth and spongy zone CONDITIONS as in other muroid rodents. In placentas from early Alexandre Urban Borbely (Department of pregnancy, the yolk sac was more developed and Developmental and Cellular Biology, ICB, University of formed by an extensive villous area. In Oligoryzomys Sao Paulo, Sao Paulo), Marcus Borges (Department of sp., it covers the placental disc. In Oryzomys subflavus Obstetrics, Federal University of Sao Paulo, Sao Paulo), the yolk sac cells were positive to PAS and show an Marcia Marcelino Ishigai (Department of Pathology, extensive haemophagous activity. The yolk sac cells Federal University of Sao Paulo, Sao Paulo), Silvia tend towards to cuboid shape, whereas in the others Daher (Department of Obstetrics, Federal University of species these cells are cylindrical. Giant cells in N. Sao Paulo, Sao Paulo), Rosiane Mattar (Department lasiurus and Oligoryzomys sp. were dispersed around of Obstetrics, Federal University of Sao Paulo, Sao the spongio zone and next to maternal blood vessels Paulo), Sue Yazaki Sun (Department of Obstetrics, in decidua. In O. spp. these cells accumulated at Federal University of Sao Paulo, Sao Paulo), Estela the placental extremities forming a continuous band Maris Forell Bevilacqua (Department of Developmental between the decidua and spongio zone. Spongio zone and Cellular Biology, ICB, University of Sao Paulo, were lobulated but if it becomes a continuous band Sao Paulo), Sérgio Ferreira de Oliveira (Department of during the gestation. In all species examined, the Developmental and Cellular Biology, ICB, University of decidua was positive to PAS, thereby suggesting that Sao Paulo, Sao Paulo, Brazil) this structure may be function as an important energetic reservoir of glycogen. Small leucine-rich proteoglycans (SLRPs) such as [email protected] decorin and biglycan are major components of the extracellular matrix involved in tissue remodeling 1. 82

livro.indd 82 7/7/2008 16:33:33 and growth factors storage. In human maternal-fetal of seminiferous tubule diameter and volume density. interface they are produced by decidual cells playing Histological alterations such as germ cell depletion and roles as anti-proliferative and anti-invasive to the displacement of Sertoli cell nucleus were also observed. extravillous trophoblast. A few biochemical studies The histological alterations and the reduction of have also detected SLRPs in the villi but the tissue morphometric and stereological parameters were more expression and roles remain unknown. This study aims accentuated in the rats treated with etoposide+ApoTf to immunolocalized decorin and biglycan proteoglycans than in those treated only with Etoposide, indicating that in term human placenta and in placentas with invasive exogenous transferrin potentiates etoposide action in trophoblastic changes (hydatiform mole, invasive the testis. mole and accreta placenta). In normal and accreta [email protected] placentas, decorin immunostaining was moderate in the syncytiotrophoblast (SC) and in syncytial knots A-067 (SK). In the invasive mole, decorin was intense in HISTOLOGICAL DESCRIPTION OF THE HEMIPENIS the cytotrophoblast (CT), SC and SK, but was faint OF LIZARD CERCOSAURA in the hydatiform mole. In normal term placenta, the OCELLATA WAGNER, 1830 (SQUAMATA, immunolocalization of biglycan showed a remarkable GYMNOPHTHALMIDAE) labeling in the SC, SK, endothelial cells and in the Mônica Caroline Pavan Cassel, Adelina Ferreira villous extracellular matrix. In the accreta placenta, (Universidade Federal de Mato Grosso) biglycan was moderately expressed at all villi structures but intensely reactive to the fibrinoid. In the invasive Referring literature to the study of hemipenis in lizards mole, biglycan reaction was intense in the SC, CT and and serpents is limited to the description of anatomical SK, however, in the hydatiform mole only the fibrinoid characters. With the objective to describe histologicaly showed a faint staining between the edematous villi. the hemipenis of the lizard Cercosaura ocellata, this These preliminary results suggest that the expression was dehydrated in alcohol, enclosed in plastic resin of decorin and biglycan correlate the invasive and the cuts colored for 1% Toluidine blue. The organ characteristic of the trophoblast pathologies increasing has a rough texture in the dorsal face and smooth in in the invasive mole and decreasing in the hydatiform the ventral face. The epithelium in the ventral face mole. Financial Support: CNPq. presents itself high, stratified, well evident cellular limits [email protected] and the presence of dense points. The basal cells are cylindrical with similar organization to a pseudostratified A-066 epithelium. Cells with one clearly halo around the EFFECT OF TRANSFERRIN ON THE TESTES OF nucleus occurs. The apical cells possess rounded ALBINO RATS TREATED WITH ETOPOSIDE DURING nucleus and appearance of spindle to the cytoplasm. PREPUBERTAL PHASE Few squamous cells. The keratin distribution is not Taiza Stumpp, Fernanda Rocha, uniform and when presents possess similar form to a Sandra Maria Miralgia spider web. In the dorsal face the epithelium is low, (Federal University of Sao Paulo) with formation of papillaes, stratified and irregular. The keratin layer is not perceivable. The connective tissue Etoposide is a chemotherapeutic drug used in different is much irrigated in both faces, flabby and with many types of cancers. It causes an increase of germ cell cells in the ventral face; and dense not shaped and apoptosis and Sertoli cell alterations, including severe colagenous fibre increase in the dorsal face. In this impairment of transferrin production by these cells. face, associated to the region of the connective tissue, Transferrin is the most abundant protein in Sertoli there are structures composed by cells with rounded cells and is esponsible for the transport of iron to the nucleus and dense chromatin, partially involved for a germ cells located in the adluminal compartment of structure dense and destitute of nucleus that follows the the seminiferous epithelium. In addition, transferrin projection of papillaes. In such a way, we conclude that protects cardiomyocites from apoptosis caused by the hemipenis of lizard C. ocellata presents a different chemotherapeutic drugs. Thus, the aim of this study structure of what traditionally it is known for copulatory was to investigate whether transferrin administration organ. protects germ cells from death caused by etoposide. [email protected] Prepubertal male rats were distributed into four groups: Control, Etoposide (40mg/Kg), Apotransferrin A-068 (ApoTf - 1mg/rat) and Etoposide+ApoTf. Both drugs CELLULAR ORGANIZATION OF THE TESTIS IN were administered intraperitoneally. The rats were ANURA killed at 64 days post-partum. The testes were Ana Bárbara dos Santos Rosa (UFMT), Adelina Ferreira dissected, weighed, fixed in Bouin’s liquid and (UFMT) PAS+H-stained. Histopathological, histomorphometric and stereological analyses were performed. The The objective of this work is to make a comparative rats treated with etoposide and those treated with analysis of the organization of germ cells at different Etoposide+ApoTf showed reduction of testis weight and stages of cellular differentiation in adult males of 1. 83

livro.indd 83 7/7/2008 16:33:34 Dendropsophus nanus (Boulenger, 1889), Pseudis in stromal cells, what differs from control animals, limellum (Cope, 1862), Pseudis paradoxa (Linnaeus, which mostly presented epithelial AR. These results 1758), and Scinax acuminatus (Cope, 1862) (Hylidae), are similar to those observed in mice and rats, in which and Leptodactylus chaquensis Cei, 1950 and L. the exposure to EE during the prostatic morphogenesis podicipinus (Cope, 1862) (Leptodactylidae) collected directs the AR expression from the epithelium to the in Southern Pantanal Matogrossense and in Serra da stroma. Thus, the histophysiological changes observed Bodoquena, Brazil. The testis were removed and fixed, with this work can be associated with the alteration of dehydrated in a graded series of alcohol and embedded the AR expression pattern, wich was induced by EE in plastic resin. The sections were stained and observed during the VP morphogenesis. under the microscope light. We observed that the [email protected] anurans of Hylidae family show, throughout the year, the spermatogenesis characterized by the presence of A-070 all the germ cell types, however, the anurans of family VERY HIGH GLYCEMIC AND LOW Leptodactylidae only show it during the rainfall period. TESTOSTERONE LEVELS LEAD TO DISTAL TIPS The variations of characteristics in the organization ATROPHY OF VENTRAL PROSTATE IN SHORT- of seminiferous epithelium, as well as the evident TERM DIABETIC RATS difference in the amount of spermatozoa inside the Marina Guimarães Gobbo (Ibilce-Unesp), Fanny tubules, leads us to believe that these anurans show Oliveira Arcolino (Ibilce-Unesp), Daniele Lisboa Ribeiro different forms of development of spermatogenesis (Instituto de Biologia- Unicamp), Sebastião Roberto throughout the year, so that the cycle is continuous for Taboga (Ibilce-Unesp), Rejane Maira Góes (Ibilce- the family Hylidae, and discontinuous with explosive Unesp) release of spermatozoa for the family Leptodactylidae. [email protected] Stromal remodeling and epithelial atrophy are usual prostatic responses to diabetes. Although these A-069 alterations are primarily influenced by testosterone INTRAUTERINE EXPOSURE TO decrease, this issue is still controversial due the ETHINYLESTRADIOL ALTERS THE complexity of innumerous metabolic interactions. In HISTOPHYSIOLOGY OF ADULT GERBIL this work it was evaluated the short-term histological VENTRAL PROSTATE response of ventral prostate (VP) in a group of Luiz Roberto Falleiros Júnior (IBILCE/UNESP), Ana diabetic rats with very high glycemic serum levels Paula Silva Perez (UNICAMP), João Augusto Salviano (HG). Diabetes was induced in 30 adult male Wistar Sant’ana (Centro Universitário de Rio Preto - UNIRP), rats using alloxan (45 mg/kg b.w.) injection. After 7 Patrícia Simone Leite Vilmaior (Centro Universitário days of diabetes onset, animals were killed and VP de Rio Preto - UNIRP), Rejane Maira Góes (IBILCE/ was processed for glucose and testosterone levels UNESP), Sebastião Roberto Taboga (IBILCE/UNESP), measurement, analysis at light microscopy and Fernanda Cristina Alcantara dos Santos (IBILCE/ immunocytochemical for androgen receptor (AR) and UNESP) PCNA. Most of diabetic rats exhibited glycemic levels between 250 and 400 mg/dl, however 35% showed Considering the fact that intrauterine exposure very high levels (>550 mg/dl). In this HG group it was to chemical disrupters causes permanent observed an inverse correlation between hyperglycemia morphophysiological alterations in the reproductive and testosterone concentration. Testosterone of system of rodents, the objective of this work HG was 12% of control rats (p< 0.05). VP atrophy was to investigate if the intrauterine exposure to (40%) was detected to HG based on wet and relative ethinylestradiol (EE) could influence the histophysiology weight. The atrophy affected mainly the distal portion of the ventral prostate (VP) of adult gerbils. Pregnant of tubuloacinar glands that showed a high degree of females received oral doses of EE (0.5mg/kg/day), shrinkage, epithelial disorganization and cell death. from day 10 to 23 of gestation. The young males were Diabetic animals also showed a decrease of 5% in the sacrificed with 90 days old, and the collected VPs were number of AR expressing cells and reduced proliferating subjected to morphological and immunocytochemical cells in distal regions. In conclusion, the hyperglycemia analyses. The results showed that the EE exposure in association with low testosterone levels causes, at caused significant changes in the VP tissue short-term a drastic atrophy of VP in diabetic rats which composition. These alterations including reduction is directed by loss of epithelial counterparts in distal of the epithelial (control = 23.3%, EE = 16.3%) and ductal tips. stromal (control = 31.3%, EE = 19%) compartments, [email protected] and luminal enlargement (control = 45.4%, EE = 64.7%). Besides the fact that all the glands analyzed A-071 showed epithelial and stromal inflammatory foci, they ULTRASTRUCTURE AND IMMUNOCYTOCHEMISTRY also presented epithelial dysplasia and the formation OF THE RAT FEMALE PROSTATE FOLLOWING of microacinar structures. In animals exposed to EE, SHORT-TERM TESTOSTERONE THERAPY the androgenic receptor (AR) expression was intense MANOEL FRANCISCO BIANCARDI (UNESP/ 1. 84

livro.indd 84 7/7/2008 16:33:34 IBILCE), FERNANDA CRISTINA ALCANTARA DOS and progesterone levels were high in the estrus stage SANTOS (UNESP/IBILCE), PATRÍCIA SIMONE LEITE . Through morphometric and morphologic analysis we VILAMAIOR (CENTRO UNIVERSITÁRIO DE RIO observed a minor quantity of prostatic alveolus well PRETO), REJANE MAIRA GÓES (UNESP/IBILCE), developed in the estrus stage (12,07± 0,96 units). Mean SEBASTIÃO ROBERTO TABOGA (UNESP/IBILCE) height of epithelium and of muscle layer that surrounds alveolus during this phase was high, being respectively The rat female prostate is an organ located around the 16,05 ± 0,37um and 9,40 ± 0,22um. Still on estrus bladder neck and it has an intimate contact with urethral phase, we verified the occurrence of great amount of wall. As this gland is hormone-dependent, females secretion into the prostatic lumen. On diestrus I phase, exposed to androgen are subject to develop severe we reported a smaller mean on epithelium height morphofunctional alterations. The aim of this work (9,40 ± 0,23um) and muscle layer (5,93 ± 0,17um). was to evaluate the effects of short-term testosterone On diestrus II phase, we observed high amounts of (T) administration on the epithelial and stromal less developed alveolus (23,43 ± 1,02 units) and a compartments of adult Wistar rats (Rattus norvegicus) lower amount of secretion into the lumen. The data female prostate. To this, the prostatic glands of the emphasize that estrus phase is a period of high animals treated for 7, 14 and 21 days with T (1mg/Kg secretory activity of the female prostate, while diestrus every 48h) were processed to detection of androgen II phase exhibits low secretory activity. Serological data receptor (AR) and for scanning and transmission revealed that hormonal oscillations result in higher electron microscopy. Immunocytochemical data showed activity of the gland during the sexual receptivity period higher cytoplasmic reaction and lack of fibroblasts with of the animal and a minor development and glandular positive marked to AR in the control glands, while higher activity on diestrus I and II phases. marked epithelial cells and fibroblasts were observed [email protected] on the treated one. Ultrastructurally, the prostatic epithelial cells of the control group showed low synthetic A-073 activity, while on the treated groups there was a higher URBAN AIR POLLUTION PROMOTES OXIDATIVE development of the secretory epithelium, together with STRESS AND THE IMBALANCE OF ENDOGENOUS a development of the rough edoplasmic reticulum. The REGULATOR OF VASCULAR TONE IN UMBILICAL glandular stromal compartment of the treated animals VESSELS OF MICE FETUS EXPOSED DURING showed several fibers of the extracellular matrix, mainly GESTATIONAL PERIOD collagen. Thus, the results indicate that androgen Mariana Matera Veras (Lab. Biologia Celular administration promotes a stimulatory effect on the FMUSP), Nilsa Regina Damaceno-Rodrigues (Lab. rat female prostate, inducing luminal development, Biologia Celular FMUSP), Angela Batista Gomes epithelial cell proliferation and higher secretory dos Santos (Lab. Biologia Celular FMUSP), Paulo activity. Moreover, adult female rats, in the presence of Hilario Nascimento Saldiva (Lab. Poluição Atmosférica androgens, present glands with similar secretory activity Experimental FMUSP), Marisa Dolhnikoff (Lab. Poluição to that observed in adult male rats. Atmosférica Experimental FMUSP), Elia Garcia Caldini [email protected] (Lab. Biologia Celular FMUSP)

A-072 It is known that low birth weight and histophysiological INFLUENCE OF THE ESTROUS CYCLE ON THE changes of umbilical cord are associated to gestational MORPHOPHYSIOLOGY OF THE GERBIL FEMALE exposure to air pollution. It is also known that PROSTATE: THE CELL POPULATION IN THE progressive decrease in the fetoplacental vascular SECRETORY EPITHELIUM resistance normally occurs during gestation, leading Ana Paula Silva Perez (Unicamp), Carlos Vitor Bianchi to an increase in the blood flow in order to support Sobrinho (IBILCE/UNESP), Ricardo Alexandre Fochi the fetal growth. As the umbilical vessels (UV) lack (Unicamp), Sabrina Santos Rochel Maia (Unicamp), innervation, the vascular tone modulation necessary Patricia Simone Leite Vilamaior (UNIRP), Rejane Maira to cope with the blood flow is controlled by vasoactive Góes (IBILCE/UNESP), Fernanda Cristina Alcântara substances such as endothelin-1 (ET-1). In addition Santos (IBILCE/UNESP), Sebastião Roberto Taboga to the perturbation of vascular homeostasis, the (IBILCE/UNESP) increased oxidative stress is related to adverse effects of air pollution. Based on these observations, The female prostate exhibits a paraurethral set of it was thought that would be of interest to study, using numerous glands and ducts that are inserted into a immunohistochemistry, the levels of ET-1 receptor (as fibromuscular stroma. The emphasis of the present indicator of vascular tone dysfunction) and also the study was to characterize the female prostate of levels of isoprostane (as indicator of oxidative stress) in adult gerbil (90 days) in different stages of estrous UV of mice fetus exposed during gestational period in cycle (proestrus, estrus, diestrus I and diestrus II) a clean chamber (C, with filtered air) and in a polluted that oscillates from 4 to 6 days. Serological analysis chamber (P, with urban air of Sao Paulo city, Brazil). showed high significant levels of estrogens in the The immunoreactive areal fractions of allantoic artery, diestrus II stage, while prostate-specific antigen (PSA) allantoic vein, vitelline artery, and vitelline vein were 1. 85

livro.indd 85 7/7/2008 16:33:34 quantified by morphometrical analysis. The statistical the body, in addition to molecular data. It is known analysis showed that gestational exposure enhances that sexual characteristics can have phylogenetic the immunoreactivity for both ET-1 receptor and significance and be useful in cladistic analyses. In order isoprostane in all UV (p<0.045 in all comparisons). In to study the spermiogenesis and sperm ultrastructure conclusion, the imbalance of endogenous regulators of Trachydoras paraguayensis, a member of the of vascular tone and oxidative stress in UV could be family Doradidae, fragments of testis were fixed in a involved in the basic mechanisms of fetal tissue injury Karnovsky modified solution and prepared as the usual due to exposure to urban air pollution. methods on TEM. In the spermatids, the centrioles [email protected] are originally perpendicular to the central axis of the nucleus, centriolar migration and nuclear rotation do not A-074 occur. Sperm of T. paraguayensis has spherical nuclei IN SITU HYBRIDIZATION OF VIMENTIN MRNA with homogeneous, highly condensed chromatin and IN MICE TROPHOBLASTIC GIANT CELLS AT some small lucent areas. Nuclear fossa is absent, the MIDGESTATION proximal centriole is slightly anterior and perpendicular Pedro Luiz Andrade Scherholz, Renato Borges Tesser, to the distal and both lie close to the central axis of Sima Godosevicius Katz (Federal University of São the nucleus. The midpiece is slightly asymmetric and Paulo - School of Medicine) contains several elongate mitochondria in its anterior third, around the axoneme beginning. Numerous large At midgestation mice trophoblastic giant cells (TGCs) vesicles surround the cytoplasmic canal in the two exhibit rapid growth and large proportions and its posterior thirds of midpiece. The single flagellum has a cytoskeleton shows an extensive network of cytokeratin classic 9+2 axoneme and no fins. Despite Doradidae and vimentin (VM) intermediate filaments. VM in TGCs is considered monophyletic. The available data show from the 9th-11th days of pregnancy (dop) was detected that sperm ultrastructure is very variable in this family, in our laboratory, by immunolocalization, however the indicating that Doradidae can be paraphyletic. origin of VM was not determined. In the present report [email protected] we studied the expression of VM by in situ hybridization (ISH) in TGCs from the 9th-13th dop. Samples were A-076 processed for detection of VM mRNA and antigen. For ULTRASTRUCTURAL BEHAVIOR OF ADULT GERBIL immunoperoxidase reaction the fixative was buffered PROSTATIC CELLULAR AND EXTRACELLULAR 10% formalin; the material was subsequently embedded COMPONENTS FOLLOWING THE LONG-TERM in paraffin. The primary polyclonal antibody was a goat INHIBITION OF 5 ALPHA-REDUCTASE AND antibody to VM and negative controls were lacking AROMATASE ENZYMES primary antibody. For ISH, tissues were fixed in buffered Lara Silvia Corradi (Department of Cell Biology - IB - paraformaldehyde 4%. Detection of VM mRNA was UNICAMP), Sebastião Roberto Taboga (Department of carried out in cryostat sections using biotinylated VM Biology - São Paulo State University - IBILCE/UNESP) oligoprobes. Each hybridization experiment comprised an antisense probe and both a sense probe and no- Prostate contains steroid-metabolizing enzymes, as probe controls. The TGCs showed strong expression 5-alpha reductase (5a-r) and Aromatase (Aro), required of VM except in cells undergoing degeneration in which for local formation of active steroids within the gland. the hybridization signal decreased or was absent. This study aimed to investigate what the long-term (30 Labyrinth cells expressed VM. Hence, TGCs from the days) inhibition of these enzymes provoked to prostatic 9th-13th dop synthesize VM and this is probably related compartments. Animals were sacrificed 1 and 21 to TGCs cellular architecture, signal transduction and days after the last administration of Finasteride (5a-r organization of cytoplasmatic organelles. Support: inhibitor) and Letrozole (Aro inhibitor). Ventral prostate CAPES. was processed for Transmission Electron Microscopy. [email protected] In both post-treatment stages, prostatic epithelial cells showed fewer secretory blebs and decreased A-075 cytoplasmic volume, besides enlarged cistern of rough SPERMIOGENESIS AND SPERM endoplasmic reticulum (RER) in the basal perinuclear ULTRASTRUCTURE OF TRACHYDORAS region. In stroma, disorganized collagen fibrils had PARAGUAYENSIS accumulated in the epithelial base and among smooth (SILURIFORMES: DORADIDAE) muscle cells (SMCs). There were also increased Rinaldo José Ortiz, Claudio de Oliveira, deposits of amorphous granular material. SMCs Irani Quagio-Grassiotto (Unesp) presented reduced diameter and condensed cytoplasm, and some had a highly irregular external contour. Doradidae is a family of freshwater catfish of South Various fibroblasts acquired an activated phenotype, America in which the inter-generic relationships are besides an establishement of close contact with other still not well-defined. Studies on the relationships fibroblasts´prolongations. Concluding, the inhibition of among the different genera and species have generally 5a-r and Aro enzymes affected, in a persistent manner, been based on bony and/or soft characteristics of the ultrastructural morphology of the prostate, what 1. 86

livro.indd 86 7/7/2008 16:33:34 gives to these enzymes a status of crucial importance Spermiogenesis in the harvestman, Iporangaia to the maintenance of this gland. In an effort to correlate pustulosa, originates an aflagellated spermatozoon the prostatic morphology and actived hormones, which presents a U-shaped nucleus, a cytoplasmic these data bring more light to the complex issue of the invagination, a pair of centrioles, some mitochondria mechanisms of local steroid metabolism and prostatic and a complex acrosome. This study detected the histology. Funding by FAPESP. presence of basic proteins during the spermiogenesis of [email protected] I. pustulosa. Testes were fixed with 2,5% glutaraldehyde and 4% paraformaldehyde in phosphate buffer; A-077 washed in the same buffer, dehydrated, stained with EFFECTS OF ATRAZINE EXPOSURE IN THE 2% ethanolic phosphotungstic acid (E-PTA) and STRUCTURE AND ANDROGEN RECEPTOR finally, included in epoxy resin. In early spermatids, EXPRESSION IN THE RAT TESTIS chromatin is positively stained by the E-PTA method. André Gustavo Oliveira (Departamento de Morfologia, With progressive condensation, the chromatin UFMG), Anna Bolivar Victor Costa (Departamento de appeared negatively stained. This loss of stainability Morfologia, UFMG), Simone Miranda Carozzi Bandeira is due to chromatin condensation, in which the basic (Departamento de Morfologia, UFMG), Germán Arturo proteins gradually become inaccessible to the large Bohórquez Mahecha (Departamento de Morfologia, PTA molecule. The cytoplasmic invagination’s opening UFMG), Cleida Aparecida Oliveira (Departamento de and mitochondria are E-PTA negative, because Morfologia, UFMG) the membranes do not permit the passage of the stain molecule. Also, in the mitochondria, the matrix Atrazine (ATZ) is an herbicide widely used worldwide, density could exclude the stain. The centriole at the which is considered as a potent xenoestrogen. Potential invagination’s opening can hardly be detected, since risks on health include effects in male reproductive only the centriolar proteins and not the microtubules system, such as reduction of testosterone levels and are stained. The acrosome is negative with the E-PTA in the number and motility of spermatozoa, which treatment while the subacrosomic compartment is suggests alterations in testicular and epididymal strongly E-PTA positive. This staining pattern is due to function. Considering that spermatogenesis is a process different condensation levels between the acrosome highly dependent on androgen levels, we investigated and its subacrosomal compartment. This technique the impact of androgen reduction on testicular allowed the identification and behavior of basic morphology and androgen receptor (AR) in the testis proteins during the spermiogenesis in Opiliones and of ATZ treated-animals. Adult rats were treated with it represents the first cytochemical study in Opiliones ATZ at 200mg/Kg for 15 or 40 days or at 300mg/Kg sperm. for 7 days. There was a transient increase in testicular [email protected] weight at the dosages of 200mg/Kg/15d and 300mg/ Kg/7d, followed by a decrease in this organ weight A-079 after exposure for 40 days. At the dosages of 200mg/ GLYPHOSATE-ROUNDUP AFFECTS THE UTERUS Kg/40d and 300mg/Kg/7d, there were remarkable AND EMBRYO IMPLANTATION changes in the testicular structure, including loss of Camila Fernandes Moro (Universidade Estadual spermatogenesis and seminiferous tubules atrophy. de Londrina), Maria Amália Cavinato Nascimento These changes paralleled a drastic reduction in plasma (Universidade Estadual de Campinas), Juca testosterone levels and AR expression in Sertoli cells Abramo Barrera San Martin (Universidade Estadual in both groups. On the other hand, the AR expression de Londrina), Juares Ednaldo Romero Bianco was decreased in Leydig cells of rats treated with ATZ (Universidade Estadual de Campinas), Celia Guadalupe 300mg/Kg, whereas an increase in AR protein was Tardeli de Jesus Andrade (Universidade Estadual de found in 200mg/Kg/40d treated animals. Despite the Londrina) drastic reduction in testosterone levels, no evident alterations were observed in these parameters in Glyphosate is the active ingredient of Roundup®. animals treated with ATZ at 200mg/Kg/15d. These Many studies described the effects of this herbicide on results suggest that ATZ may act as an endocrine quickly and longer exposition. However, few studies disruptor resulting in acute and chronic histopathological had investigated the effects on pregnancy. This work alterations in the testis. aims the objective to evaluate the effects of glyphosate [email protected] ingestion on endometrial stroma of pregnant mice. For this propose mice on 5th and 7th day of pregnancy A-078 (DP) received glyphosate solution by gavage between PROTEIN DETECTION IN THE SPERMIOGENESIS the 1st and 5th DP. Control animals received the same OF IPORANGAIA PUSTULOSA (ARACHNIDA: volume of water. Animals were sacrificed on 5th and OPILIONES) 7th DP, fixed by vascular perfusion and uteri were Juliana Siqueira Moya, Karina Carvalho Mancini, Mary processed for light and electron microscopy. The results Anne Heidi Dolder (Departamento de Biologia Celular, demonstrated that implantation did not occur on the IB, UNICAMP) 5th DP. The blastocysts still had zona pellucida and 1. 87

livro.indd 87 7/7/2008 16:33:34 were free in uterine lumen. Stromal cells exhibited a disturbance and statistically different from the genotype thin decidual cells layer with infiltrated leucocytes. On profile found in normoglycemic women. Conclusion: the 7th DP uterus showed large decidual reaction. The This study suggests that VEGF 634 polymorphism can uterine lumen was healthy on the mesometrial side be related with the vascular placental alterations seen and the embryo was attached on the antimesometrial in mild hyperglycemic pregnant women. FINANCIAL side. The uNKs cells, without morphological alterations, SUPPORT: CAPES, CNPq. were present on subtypes 1 and 2. Trabeculas and [email protected] mesometrial vessels were filled of blood and a high number of leucocytes. The embryos were attached A-081 and it was possible to observe the ectoplacental cone. MICE TROPHOBLAST CELLS EXPRESS ALL These results demonstrated that glyphosate ingestion SUBUNITS OF THE MACROPHAGE NAD(P) promoted a delay of implantation, but did not affect the H-OXIDASE COMPLEX behavior of the decidual and uNK cells. Moreover, the Sara Maria Zago Gomes, Estela Bevilacqua exposition induced the lymphocyte migration to the (Universidade de São Paulo) uterus which may be associated to an inflammation reaction and/or influence on embryo implantation and The complex NAD(P)H-oxidase catalyzes the development. production of reactive oxygen species (ROS). This [email protected] plasma membrane-cell enzyme is formed by the membrane-bound components gp91-phox and p22- A-080 phox, the flavocytochrome b558 and, the cytosolic VASCULAR ENDOTHELIAL GROWTH components p47-phox, p67-phox, Rac1 and p40- FACTOR POLYMORPHISM AND phox. ROS unbalanced production in human is also HYPERGLYCEMIC GESTATIONAL DISORDER implied in gestational disorders, like preeclampsia. Erica Giovana Barreiro (Universidade de São Paulo), Aim: To investigated the protein and gene expression Marilza Vieira Cunha Rudge (Universidade Estadual of NADP(H)-oxidase subunits in the mouse Paulista Júlio de Mesquita Filho - UNESP), Iracema trophoblast. Methods: Trophoblast cells obtained de Mattos Paranhos Calderon (Universidade Estadual from ectoplacental cones of CD-1 mouse embryos at Paulista Júlio de Mesquita Filho - UNESP), Debora gestation day 7.5 were cultivated by 48 h, stimulated Cristina Damasceno (Universidade Estadual Paulista with PMA and analyzed by rT-PCR, Western Blot Júlio de Mesquita Filho - UNESP), Yuri Karen Sinzato and immunohistochemistry. Results: Rt-PCR semi- (Universidade Estadual Paulista Júlio de Mesquita quantitative analyses showed increase expression of Filho - UNESP), Silvia Daher (Universidade Federal de p22-phox, gp91-phox, p47-phox, p67-phox, p40-phox São Paulo), Estela Maris Andrade Forell Bevilacqua and Rac1 in PMA-treated ectoplacental cones. The (Universidade de São Paulo) expression of gp91-phox, p47-phox and p67-phox were confirmed by Western blot (also increased in Vascular endothelial growth factor (VEGF) is a mediator the presence of PMA). These subunits were mostly of vascular permeability and angiogenesis, which located in the trophoblast giant cells associated to the may be crucial in mediating the development and phagocytic process at the maternal-placental interface. progression of diabetic vasculopathies. Our hypothesis Conclusions: We provided the first evidence that is that placental vascular alterations in hyperglycemic trophoblast cells express the critical components of pregnant women can be related to an atypical VEGF superoxide-generating NADPH oxidase complex. These production as a consequence of genetic polymorphisms. findings indicate ROS production by trophoblast cells Objective: To investigate VEGF gene polymorphisms during embryo implantation and, open new interests in placentas of hyperglycemic gestational disorders. on NADPH-oxidase regulatory processes and its likely Method: The study group consisted of pregnant women involvement in defense functions in both healthy and diagnosed as: overt diabetes (abnormal 100gƒ{OGTT pathological processes that can determine the failure [100 g-oral glucose tolerance test] and glycemic of the gestation. *Supported by FAPESP, CAPES and profile) and mild hyperglycemia (normal 100gƒ{OGTT CNPq. but abnormal glycemic profile). All pregnant women [email protected] attended the Diabetes & Pregnancy Services of Botucatu Medical School. Placental fragments A-082 were collected from normoglycemic (n = 34); mild SPERM MORPHOLOGY OF THE STONEFLY hyperglycemic (n = 14) and overt diabetes (n = 27), LEUCTRA HIPPOPUS followed by DNA extraction and VEGF 634C/G and (INSECTA: PLECOPTERA) 936C/T polymorphisms genotyping. Results: Our data Karina Carvalho Mancini (Unicamp), David Mercati show a different pattern in genotype frequencies of (Universidade de Siena), Heidi Dolder (Unicamp), VEGF 936C allele polymorphism in women with mild Romano Dallai (Universidade de Siena) hyperglycemia in comparison to the other groups. The genotype frequencies of VEGF 634 polymorphism This study describes Leuctra hippopus sperm were similar among the specimens with glycemic ultrastructure in order to obtain more information 1. 88

livro.indd 88 7/7/2008 16:33:34 about the genus, contributing to the phylogeny of VEGF-R1 and R2 mRNA expression level decreased Plecoptera. Testes were processed according to Dallai significantly (p < 0.05). Conclusions: Clearly, these & Afzelius (1990). The spermatozoon possesses an findings indicate that stress can modulate VEGF and acrosome, nucleus, axoneme, centriolar adjunct, two its receptors in the rat placenta. VEGF has been mitochondrial derivatives and two accessory bodies. considered a potent angiogenic factor, but also relevant The acrosome extends along the nuclear length and in increasing vascular permeability. The increase in presents an acrosomal vesicle and a perforatorium. VEGF seen in this study may therefore be directly The acrosomal vesicle is a very complex structure associated to a compensatory response due to the showing varied morphology along its length. Its anterior maternal hypertension. tip is triangular, in cross section, while in the medium [email protected] and posterior levels it is divided into two portions united by a thin bridge. The nucleus is elongated and A-084 presents compact chromatin. Its anterior tip is oval, in MOUSE TROPHOBLAST CELLS PRODUCE cross section, and at lower levels, it has a singular star MACROPHAGE MIGRATION INHIBITORY FACTOR shape. The centriole is composed of nine accessory (MIF) DURING ALL GESTATION STEPS microtubules and nine doublets. It forms the axoneme, Miriam Rubio Faria (Institute of Biomedical Sciences, which is composed of nine accessory microtubules, University of São Paulo), Andrea Albieri (State nine doublets and a central pair of microtubules. The University of Rio de Janeiro), Mara Sandra Hoshida accessory microtubules contain 16 protofilaments. The (Institute of Biomedical Sciences, University of São centriolar adjunct is located in the nucleus flagellum Paulo), Eloisa A. V. Ferro (Federal University of transition region and is divided in three triangular Uberlândia), Luana Paulesu (University of Siena), portions. Two portions originate the accessory bodies. Estela Bevilacqua (Institute of Biomedical Sciences, The mitochondrial derivatives present a paracrystalline University of São Paulo) region and a small clear amorphous portion. These data confirm and add to the sperm features previously Macrophage migration inhibitory factor (MIF) was described in Leuctra genus. Moreover, the study has originally identified for its capacity to inhibit the random found some morphological differences that allow the migration of macrophages in vitro. This cytokine plays species identification. special roles as pro-inflammatory factor affecting mainly [email protected] functions of macrophages and lymphocytes and as counter-regulator of the glucocortocoid effects on the A-083 immune response. In spite of being a pro-inflammatory PLACENTAL VEGF AND VEGF RECEPTORS IN cytokine recent evidences also suggest a critical role STRESSED PREGNANT RATS for MIF during human implantation and early embryonic Isis Paloppi Corrêa (Laboratory of Obstetric Physiology development. Therefore, the overall objective of this (LIM57), Obstetrics Department, University of São study has been to determine whether trophoblast cells Paulo, Brazil), Rodrigo Ruano (Laboratory of Obstetric are involved in the MIF production at the maternal- Physiology (LIM57), Obstetrics Department, University placental interface, in mice. The protein expression of of São Paulo, Brazil), Estela Bevilacqua (Dept. MIF has been performed through immunohistochemistry of Cellular and Developmental Biology, Institute of and Western blotting and, gene expression by reverse Biomedical Science, University of São Paulo), Marcelo transcription-polymerase chain reaction. Assessment Zugaib (Laboratory of Obstetric Physiology (LIM57), of MIF is being performed at implantation sites on Obstetrics Department, University of São Paulo) gestation days 5.5, 7.5 and 10.5 and placentas at gd. 13.5 and 17.5. Our initial immunohistochemical Background: Experimental studies have demostrated results and Western Blotting analysis have confirmed a great similarity between hypertension-associated the production of MIF by trophoblast cells. Initially, pregnancies and stressed-induced pregnancies in rats. the expression of MIF seems to be concentrated at Both conditions include changes in arterial pressure, implanting giant cells, but as gestation progresses proteinuria, intrauterine growth restriction, atypical other trophoblast cells appear to also participate in this trophoblast invasion, increased cathecolamine levels, production. Quantitative analyses are in development fibrin deposits, vascular congestion and decrease to estimate the temporal differences among gestational in insulin levels. The degree in which the vascular MIF production by trophoblast cells to serve as base compartment is committed is pivotal to determine the for examining the potential functional effects of this gestational prognostic. Objectives: This study aims to cytokine at maternal fetal interface. Supported by analyze the gene expression of VEGF and its receptors CNPq. VEGF-R1 and R2 in the rat placenta submitted to [email protected] stress thought RNA isolation and quantitative real- time polymerase chain reaction. Results: VEGF A-085 mRNA expression level were significantly increased NITRIC OXIDE PRODUCTION AND EFFECTS OF in stressed-rat placental tissue compared to normal L-NAME IN THE MATERNAL-FETAL INTERFACE placental tissue (p < 0.05), whereas the receptors OF MOUSE UTERUS DURING NORMAL AND 1. 89

livro.indd 89 7/7/2008 16:33:34 ABNORMAL PREGNANCY byglican and decorin, and quantified through computer Renata Miliani Martinez, Eliana Mara Oliveira Lippe , assisted image analyses. The diabetic condition was Aureo Tatsumi Yamada (UNICAMP) characterized by: high glycemic levels, glycosuria, The hypoxia in the early stage of pregnancy is a increased food and water consumption and decreased physiological condition and is believed to induce body mass weight. No cetonuria was detected. the oxidative stress generating the reactive oxygen Diabetes promotes the reduction in the number substances (ROS) in the maternal-fetal interface (MFI), and dimension of implantation sites and affects the but the mechanism controlling this stress conditions ECM. Decreased deposition of fibrillar collagens and is controversial. The present work evaluated the augmented deposition of biglycan in the decidua were cells expressing the isoforms of nitric oxide synthase observed in diabetic female mice. In addition, diabetes enzymes (iNOS, eNOS, nNOS) to establish the cells affects the myometrium morphology and its ECM. Our responsive to produce nitric oxide (NO) and the effects experimental model shows physiological parameters of of L-NAME (L-nitro-arginine-methyl-ester), the inhibitor diabetes type I, allowing the observation of its negative of NO production by NOS in the normal pregnant impact on the cytoarchitecture of the uterine tissues mice uterus and abnormal pregnancy induced by and, consequently, on the development of pregnancy. surgical lesion of the embryos (SLE). The DBA lectin Supported by FAPESP. cytochemisty and immunocytochemistry showed all [email protected] NOS isoforms expression by uterine-natural killer (uNK) cells and the concentration of NO in the normal A-087 pregnant uterus which increased significantly after 0.5h THE ESTROUS CYCLE MODULATES THE SMALL of LSE. This is time coincident with the hemorrhages LEUCINE-RICH PROTEOGLYCANS IN THE UTERINE localized in the mesometrial side of the uterus and lost TISSUES OF MICE of secretory-lysosome granules contents by uNK cells. Renato de Mayrinck Salgado (Universidade de São The NO concentration decreased to less than 10% after Paulo), Rodolfo Favaro Ribeiro (Universidade de 6h of LSE. The L-NAME reduced the NO concentration São Paulo), Sebastian San Martin (Universidad de to less than 50% when compared either to normal and Valparaíso), Telma Maria Tenório Zorn (Universidade de after LSE, but did not prevent the uNK cells damage, São Paulo) or the hemorrhage and degeneration of uterine tissues and embryo resorption. These data suggest the NO Previously we have shown that extracellular matrix concentration is critical to support the homeostasis of molecules, including members of the small leucine-rich pregnant uterus and its increasing in the MFI could proteoglycans (SLRPs) family, are deeply remodeled be related to miscarriage. Grants: (*) undergraduate in the pregnant mouse uterus. Remodeling was fellowship student from SAE/UNICAMP. observed a few hours after fertilization, suggesting an [email protected] action of ovarian hormone levels in the synthesis and degradation of these molecules. In the present we A-086 analyzed four members (decorin, biglycan, lumican and DIABETES MELLITUS ALTERS DEPOSITION OF fibromodulin) of the SLRP family in the uterus along EXTRACELLULAR MOLECULES IN THE PREGNANT the four phases of the estrous cycle. Uteri of mice were MOUSE UTERUS collected in proestrus, estrus, metaestrus and diestrus Rodolfo Favaro Ribeiro, Priscila Ribeiro Raspantini, after vaginal smear and Shorr technique analysis. Munick Cristina Fulquim, Renato de Mayrinck Salgado, Paraffin sections were submitted to peroxidase Zuleica Bruno Fortes, immunocytochemistry. Proteoglycans are differentially Telma Maria Tenório Zorn distributed in the uterus according to the estrous cycle (USP - São Paulo) phase. Fibromodulin is essentially expressed in the myometrium, whereas the other proteoglycans studied During pregnancy the endometrium undergoes a set herein are present in both stroma and myometrium. of adaptations that support embryo implantation and Except biglycan, all of these molecules predominate in development. This process is known as decidualization the estrus phase. Only biglycan and fibromodulin were and involves the transdifferentiation of endometrial expressed in the luminal and glandular epithelium. fibroblasts into decidual cells. The impact of diabetes All four proteoglycans were also found in cytoplasmic in early pregnant uterus is poorly understood. The aim granules of mononucleated cells. These results show of this work was to investigate the effects of diabetes that the profile of the immunoreaction in the uterine on the decidual extracellular matrix (ECM) composition ECM alters with the cyclicity of the sexual hormones in early pregnancy. Diabetes was induced in female that happens during the estrous cycle. Differences on Swiss mice by intravenous injection of alloxan (40mg/ the deposition of proteoglycans between superficial Kg) after 16 hours of food deprivation. Diabetic females and deep stroma may identify two subpopulations of (glycemia >400mg/dl) were mated with normal males endometrial fibroblasts, which possess different ability 85-100 days after alloxan administration. 168 hour to respond to decidual stimuli. Supported by FAPESP. implantation sites were submitted to picrosirius staining [email protected] and immunoperoxidase method for detection of 1. 90

livro.indd 90 7/7/2008 16:33:34 A-088 Plant Cell Biology MORPHOLOGICAL CHANGES IN THE UTERINE TISSUES OF MICE DURING THE ESTROUS CYCLE Renato de Mayrinck Salgado, Rodolfo Favaro Ribeiro, B-001 Fernanda Fernandes Farias, Fernanda Ângela Correa THE END OF THE TUNNEL: MCF-7 CELLS DEATH Barrence, Telma Maria AND PROLIFERATION INSIDE Tenório Zorn (Universidade de São Paulo) SPHEROIDS Jônatas Bussador do Amaral, Gláucia Maria Machado- The estrous cycle of rodents presents recurring Santelli changes in the reproductive organs that are (Instituto de Ciências Biomédicas, Depto de orchestrated by the profile of the ovarian sexual Biologia Celular e do Desenvolvimento -USP) hormones. The short length of only 4.5 days of the mouse estrous cycle has allowed several laboratories Spherical aggregates represent in vitro models of to elect this animal an ideal model to study hormone- tumor microregions and of an early, avascular stage dependent processes. The stages of the estrous cycle of tumor growth. The similarities between the original can be identified by a colpocitologic examination. The tumor and the respective spheroids include: cellular present is a comprehensive morphological study on heterogeneity, proliferation gradients, quiescence the mouse uterine tissues during the estrous cycle. and cell death. Due to these characteristics, in this For that, samples were fixed in Methacarn, embedded work MCF-7 cells spheroids with different ages were in Paraplast, and stained with haematoxilin-eosin. In analyzed, showing the proliferation and cell death proestrus, the connective tissue was highly vascularized inside of distinct cell layers. Around 106 cells were and homogeneously distributed in the endometrial transferred to liquid suspension on a non-adhesive, stroma. The myometrium presented two narrow layers. non-toxic agarose surface. The 3D culture was grown In estrus, edema was apparent in the deep stroma for 75 days at 37 ºC, with 5% of CO2. For transmission and glands were filled by secretion. Leukocytes were electron microscopy, spheroids were fixed with 2.5% scattered in the stroma. The myometrium layers grew. glutaraldehyde in 0.2 M caccodylate buffer, post- In metaestrus, the stroma was more compact. Mitosis fixation with 1% osmium tetroxide and were uranyl-lead was frequent in the uterine glands and leukocytes contrasted. For proliferation analysis, spheroids were were observed in the luminal epithelium. The whole incubated with BrdU and after this were fixed with myometrium was thinner than in estrous. In diestrus, 3,7% formaldehyde and labeled by propidium iodide the endometrial stroma was compartmentalized in (nuclear morphology) and Cy5 anti-BrdU monoclonal superficial and deep regions. The superficial stroma antibody. The spheroids formation was the result of contained round-shaped cells, tightly arranged and in cell aggregation and cellular proliferation (show by the deep stroma, the fibroblasts were elongated and BrdU label cells). The central region of spheroid when more scattered. The endometrial glands were filled by analyzed by transmission electron microscopy indicated secretion. The myometrium was similar to proestrus. cells with different morphologies, showing necrotic, Our data show detailed morphological information, autophagic and apoptotic cells. The shell viable cells which could be useful to establish baseline data for surround central region become more similar of the experimental studies on reproductive phenomena. luminal space, showing cell polarization with traffic FAPESP. vesicles. The relationship between cell death and [email protected] cellular differentiation were not limited of metabolites decrease, as well as cellular signalization for lumen formation. [email protected]

B-002 PROTECTIVE EFFECTS OF GREEN TEA ON CHEMICALLY INDUCED RAT HEPATOCARCINOGENESIS Kelly Silva Furtado, Juliana Ferreira da Silva, Luis Fernando Barbisan (UNESP Botucatu) The habitual use of green tea, an infusion of dried and minced leaves of Camellia sinensis, has been related to a decrease in incidence of some human cancers. Besides, experimental studies point to chemopreventive properties of crude green tea or their specific compounds in various rodent chemically- induced carcinogenesis assays. Thus, the present study evaluated the potential protective effects of green tea in a medium-term liver tumorigenesis assay. Male Wistar 1. 91

livro.indd 91 7/7/2008 16:33:34 rats were allocated into four groups: G1- treated once a B-004 week with diethylnitrosamine (DEN, 8 x 80 mg/kg, i.p), WNT PATHWAY ANALYSIS AND ITS G2- DEN–treated plus green tea infusion (20g leaves/L) INVOLVEMENT IN THE COLORECTAL during eight weeks and G3- green tea infusion (20g TUMORIGENESIS leaves/L) during eight weeks and G4- non-treated. The Flávia Castello Branco Vidal, animals were killed at weeks 8 and 20 and liver samples José Andrés Morgado Diaz (INCa) were colleted to analyze development of preneoplastic foci of altered hepatocytes expressing GST-P or tumor Wnt pathway controls cell differentiation, proliferation development, respectively. Green tea treatment during and apoptosis. Disorders in Wnt signaling cause human the DEN-initiation stage reduced the number and size degenerative diseases as well as cancer. The activation of GST-P-positive foci and the incidence and multiplicity of the Wnt pathway leads to the inactivation of the of liver tumors. After twenty four hours the last dose of kinase GSK3-beta and consequently, translocation DEN, samples from peripheral blood were collected to of the protein beta-catenin to the nucleus. Nuclear comet assay. Mate and green tea treatments reduced beta-catenin interacts with transcription factors and DEN-induced DNA damage when compared to the activates transcription of different target genes such as control (G1 group). Therefore, the results suggest that cyclin D1 and c-myc. Studies have shown that some green tea presented inhibitory actions during initiation signaling pathways such as MAPK when activated phase of rat liver carcinogenesis induced by DEN. by external signals can inhibit GSK3-beta, promote [email protected] Wnt signaling and in some cases, induce epithelial mesenchymal transition (EMT). In this study we B-003 sough to understand the effects of epidermal growth RHODIUM (II) CITRATE INDUCED CYTOTOXICITY IN factor (EGF) and LiCl (an inhibitor of GSK3-beta) on HUMAN MCF-7 BREAST CANCER CELL the activation of Wnt pathway, and on the induction Marcella Lemos Brettas Carneiro (UnB), Elaine of EMT. Human adenocarcinoma cell line, Caco-2, Paulúcio Porfírio (UnB), Raphael C. A Peixoto (UnB), was treated with 100ng/ml EGF and 50mM LiCl. Luiza Helena Madia Lourenço (UnB), Jaqueline da Silva After treatment, localization of junctional proteins was (UnB), Aparecido Ribeiro de Souza (UFG), Ricardo analyzed by immunofluorescence and immunoblotting. Bentes Azevedo (UnB), Zulmira Guerrero Marques Treatments induced loss of the normal localization at Lacava (UnB), Sônia Nair Báo (UnB) the cell membrane of ZO-1, p120 and beta-catenin. Claudin-1 and E-cadherin were unaffected. PD98059, Breast cancer represents a major cause of death to an inhibitor of the MAPK pathway, was unable to Western countries women. This fact demands for reverse the effects caused by EGF. On the other hand, a continuing development of new anticancer drugs PD98059 reversed the effects caused by LiCl. We are that may effectively treat breast cancer patients. The performing experiments using other inhibitors to identify antitumor activity of the inorganic complex cisplatin has the pathway responsible by the effects of EGF on the led to the development of other types of non-organic junction proteins. cytostatic drugs. Rhodium carboxylate compounds [email protected] have been shown to be effective against animal model tumors as well as tumors in man. Therefore B-005 we investigated the cytotoxicity, carcinostaticity, and ALTERED GLYCOSYLATION OF E-CADHERIN apoptosis of rhodium (II) citrate in human MCF-7 AND ITS RELATIONSHIP WITH THE STABILITY OF breast cancer and in human FHN skin fibroblasts cell ADHERENS JUNCTIONS IN COLORECTAL CANCER lines. Cells were treated with different concentrations CELLS of rhodium (II) citrate for 24, 48, and 72 hours. Cell Julio Cesar Madureira de Freitas Junior, viability was assessed by MTT colorimetric assay and José Andrés Morgado Díaz Tripan Blue exclusion method. The cell cycle distribution (Instituto Nacional de Câncer) of MCF-7 was analyzed through flow cytometry. The observed IC50 values for MCF-7 cells were 337 (±1.03), E-cadherin is a transmembrane glycoprotein that 251 (±1.04), and 250 (±1.03) µM, respectively at 24, function as the main mediator of cell-cell adhesion. It 48, and 72 h while they were 1.587 (±1.49), 802 (±1.23) is anchored to the actin cytoskeleton via catenin family and 513 (±1.52) µM for FHN cells. Our results showed proteins in a multiprotein complex named adherent in vitro cytotoxicity of rhodium (II) citrate in a dose junction (AJ). Post-translational modifications of dependent manner in both tested cell types. However, E-cadherin, like phosphorylation and glycosylation, only FHN cells presented a time dependent cytotoxicity. can modulate the stability and assembly of AJs. The MCF-7 cells growth inhibition induced by rhodium Many studies have suggested that cell invasion (II) citrate may be attributed to cell-cycle arrest and and cancer metastasis can be associated to glycan apoptosis. The data suggest that rhodium (II) citrate patterns on cell surface. In colorectal cancer the role might be investigated as a potential tool for the human of altered glycosylation of E-cadherin protein is not breast cancer chemotherapy. clear. In this study we investigated the cross-talk [email protected] between glycosylation of E-cadherin and stability of 1. 92

livro.indd 92 7/7/2008 16:33:35 AJs in colorectal cancer cells. We used two colon PROGRESSION IN EPITHELIAL CANCER CELLS adenocarcinoma cell lines with different metastatic Fernanda Leve, José Andrés Morgado Díaz (INCA) potential, Caco-2 and HCT-116, models of stable and unstable AJs respectively. Immunoprecipitation assays Epithelial-to-mesenchymal transition (EMT) occurs in demonstrated that in HCT-116, the most invasive cell normal development and pathologies such as cancer. line, E-cadherin presents a decrease of the glycans EMT is characterized by loss of cell-cell, cell-matrix recognized by HPA and WGA lectins, which are specific adhesion and actin for N-acetylgalactosamine and sialic acid, respectively. cytoskeleton rearrangement, forming membrane Furthermore, immunofluorescence assays showed that protrusions. In this process, some studies suggest the the presence of â1,6-branched N-acetylglucosamine, participation of Rho GTPases (Rho, Rac and Cdc42) recognized by L-PHA lectin, in HCT-116, is intense and their effectors (Rock, IQGAP), and some kinase on cell contact regions, as compared with Caco-2 proteins, like PKA, Src and focal adhesion kinase cells where the labeling was observed on apical (FAK). The aim of this study was to identify signaling region. These findings suggest that altered subcellular pathways that mediate alterations of the cell motility localization of these glycans can be an important event as consequence of cell-cell adhesion lost. We used associated to loss of stability of AJs in colorectal cancer. transformed and non-transformed epithelial cell lines [email protected] (Caco-2 and MDCK II, respectively) and the calcium depletion model to disrupt the junctional complex, B-006 as well as specific kinase inhibitors. We observed by INVOLVEMENT OF PI3K PATHWAY IN CELL confocal microscopy that low calcium medium (LC) DIFFERENTIATION AND APICAL JUNCTIONAL caused cell rounding showing a thick F-actin labeling COMPLEX FORMATION IN A COLON at the cell periphery, stress fiber disruption and CARCINOMA CELL LINE, HCT-116 lamelipodia formation. Pharmacological inhibition of Wallace Martins de Araújo, José Andrés Morgado Díaz PKA with H-89, prevented the LC effect on the actin (Instituto Nacional de Câncer) cytoskeleton at the apical and medial sides, but not reestablished the stress fibers. Both, Rock (Y27632) Phosphatidylinositol 3-kinase is an important mediator and Src (PP2) inhibitors caused more intense stress of intracellular signal transduction, proliferation, fibers disruption, however lamelipodia formation was differentiation, and apoptosis, the role of PI3K in not observed. Taken together, our findings show that intestinal cell differentiation with junctional complex in calcium-depleted cells occur a spatial modulation of formation is not known. The objective this study was the actin cytoskeleton, whereas PKA modulates actin to verify the participation of the PI3K pathway in the cytoskeleton at apical and medial region, Rock and Src enterocyte differentiation and its involvement in the activation should contribute to the lamelipodia formation formation of junctional complex. To investigate this and acquisition of a mobile phenotype. event HCT-116 cells were treated for 10 days with [email protected] LY294002 (8 mM), a specific PI3K inhibitor. We verified that HCT-116 cells treated with the PI3K inhibitor B-008 showed an increase of the alkaline phosphatase INVOLVEMENT OF SRC IN THE EGFR-RAS-MAPK enzymatic activity, a well-known cellular differentiation PATHWAY MODULATES LOSS OF CELL-CELL marker. They grow in monolayers and developed ADHESION E-CADHERIN-MEDIATED AND MOTILITY cell polarity with apical junctional complex formation. IN CACO-2 CELLS Analysis by immunoblotting and immunofluorescence Waldemir Fernandes de Souza, José Andrés Morgado showed an increase of the adheren junction proteins, Díaz (Instituto Nacional de Câncer) E-cadherin and b-catenin, and of the tight junction proteins, claudin-3 and ZO-1 at the cell-cell contacts in During tumour progression various events are critical differentiated cells, as compared with undifferentiated to induce metastasis. Cell-cell adhesion disassembly cells. Moreover, the technique of ruthenium red and and enhance of cell motility with alterations at the actin electron microscopy showed that tight junctions formed cytoskeleton, occur in a process known as epithelial- by the treatment with PI3K inhibitor were functional. mesenquimal transition. E-cadherin plays an important Further studies using the retroviral expression vector role in the organization of cell-cell contacts. The pBabe-purol-PTEN to overexpress PTEN, a negative involvement of EGFR and MAPK in the loss of cell-cell regulator of PI3K, is in progress to confirm these results. adhesion E-cadherin-mediated induced by TPA, has In conclusion, our results show that PI3K inhibition been proposed. However, the molecular mechanisms induces differentiation and biogenesis of the junctional that modulate this signal transduction still remain to complex in HCT-116 cells. be defined. In the present study we verified the role [email protected] of Src and ERK1/2 proteins during the loss of cell-cell adhesion E-cadherin-mediated in Caco-2 cells. TPA B-007 and EGF treatment induced redistribution of E-cadherin EVIDENCE OF A SPATIAL MODULATION OF THE from cell-cell contact to cytoplasm as observed by ACTIN CYTOSKELETON DYNAMIC DURING TUMOR immunofluorescence. This effect was blocked when 1. 93

livro.indd 93 7/7/2008 16:33:35 cells were pretreated with Src inhibitor, PP1. It was also Paloma Silva de Souza, André Luiz de Souza Cruz, possible to observe by light microscopy that Src protein João Paulo de Biaso Viola, José Andrés Morgado Díaz is involved in the morphological alterations caused by (INCa) TPA or EGF. Furthermore, immunoblotting analysis showed that TPA and EGF can modulate ERK1/2 Claudin proteins form a large family of integral activity. Wound-healing assay showed that enhance of membrane proteins crucial for formation and function cell migration caused by TPA and EGF were prevented of tight junctions in epithelial cells. Claudins play a by treatment with PP1. The present study shows that role in selective paracellular permeability, as well as in Src protein is involved in the loss of cell-cell adhesion, the establishment and maintenance of epithelial cell morphologic changes and increase of cell motility polarity. Recent studies have shown that in epithelial induced by TPA and EGF in Caco-2 cells. Together, tumorigenesis, these proteins are down- or up-regulated our results show the participation of Src in the EGFR- depending of the tumor type. The aim of this study was Ras-MAPK pathway to modulate cell-cell adhesion establish a methodology to overexpress claudin-1 in E-cadherin-mediated. epithelial cells and analyze their phenotypic behavior. [email protected] HEK 239T cells, which endogenously do not express claudin-1, were transiently transfected using the B-009 pCCL1 vector and the calcium phosphate technique. CELL SIGNALING PATHWAYS INVOLVED IN We observe that after transfection, HEK 293T cells ALTERATIONS OF THE CELL-CELL CONTACT AND were able to express high levels of claudin-1 protein ACTIN CYTOSKELETON INDUCED BY IONIZING as analyzed by immunoblotting analysis. The protein RADIATION IN CACO-2 CELLS localized exclusively at cell-cell contact, as seen Lílian Gonçalves dos Reis Bastos, Fernanda Leve, José by immunofluorescence. Claudin-1 overexpression Andrés Morgado Díaz (INCa) caused an increase of hyperphosphorylated occludin and reduced the expression of ZO-2. Furthermore, Epithelial-mesenchimal transitions (EMT) occur at the reduced cell viability and cell death by apoptosis beginning of epithelial cancer.This process involves was observed in cells overexpressing claudin-1, but apical junctional complex disassembly, down regulation the cell growth was not altered. Our results showed of epithelial markers and cytoskeleton reorganization. that claudin-1 overexpression in non-tumoral cells, The colorectal cancer is one of the most common which endogenously no express this protein, induces tumours among population and the ionizing radiation alterations of the behavior phenotype. Induced (IR) is used as first line of treatment, however the overexpression studies of Claudin-1 in tumor cells may molecular mechanisms underlying IR treatment, to contribute to understand the role that this protein are no defined. Here we investigate the signaling plays in the epithelial tumorigenesis. pathways involved in the cell-cell lost and cytoskeleton [email protected] disorganization caused by IR. Caco-2 monolayer were treated with a series of signaling pathways inhibitors B-011 before IR (5 Gy): 100nM PD15035 (EGFR), 10mM SUBCELLULAR LOCALIZATION OF LY294002 (PI3K), 50µM PD98059 (MAPK), and 100nM PROHIBITIN IN CISPLATIN-TREATED HUMAN PP2 (Src). Imunofluorescense microscopy showed that MELANOMA CELLS IR caused a significant cytosolic labeling of E-cadherin, Tharcísio Citrângulo Tortelli Jr, Andréia Hanada Otake, β-catenin, and claudin-1, and this effect was reverted Natália Schwarz, Roger Chammas (FMUSP) by Src, EGFR and MAPK inhibitors. Immunoblotting analysis showed that IR induced β-catenin and Melanoma incidence is increasing worldwide and E-cadherin translocation to the Triton X-100 soluble represents a clinical challenge, as its treatment outcome fraction, which was abrogated by pretreatment is still poor. A possible cause to the failure of melanoma with PP2, PD15035 and PD98059 inhibitors. We treatment is the development of chemoresistance, further observed that IR caused actin cytoskeleton whose molecular bases are still poorly understood. disorganization at basal and medial regions of the cell Our laboratory conducted a proteomic approach volume, as analyzed by confocal microscopy and this to identify differences in protein expression or effect was abrogated by pretreatment with PP2 and accumulation using cisplatin in a human melanoma PD15035. Our results suggest the involvement of Src, cell line. Prohibitin (PHB), a mitochondrial chaperone EGFR and MAPK in the cell-cell contacts disassembly, and an E2F inhibitor, was among the molecules wich as well as Src and EGFR at the actin disorganization accumulated upon cisplatin treatment. Here, we have caused by IR in caco-2 cells. Supported by: CNPq, investigated prohibitin accumulation and subcellular FAPERJ, FAF and MS. compartmentalization in cisplatin-induced cell death [email protected] in human metastatic melanoma cell lines (Mel85, SKMel37 and LB373). PHB was overexpressed in all of B-010 them, as seen by western blot. We also showed a direct PHENOTYPIC ANALYSIS OF HEK 293T CELLS correlation between PHB overexpression and resistance OVEREXPRESSING CLAUDIN-1 to cisplatin-induced cell death. PHB was found in the 1. 94

livro.indd 94 7/7/2008 16:33:35 cytoplasm, associated to mitochondria, and within Abnormal DNA content can be considered a marker of nuclear compartments as seen by confocal microscopy. malignancy and a relevant factor in clinical evaluation in In cisplatin-resistant cells, PHB was found around the patients with breast cancer. It would be associated with nucleolus while in cisplatin-sensitive cells, PHB was increased aggressiveness of the tumor. The antimitotic found spread over the nucleus. PHB knock-down by agents vincristine (VCR) and paclitaxel (PTX), drugs siRNA has sensitized cells to cisplatin. Our data suggest used in cancer therapy, may cause genetic instability that Prohibitin may be involved in melanoma resistance, at low concentrations. The aim of the present study not only for its overexpression, but also due to its was to evaluate this drugs effects on the induction nuclear compartimentalization. Nuclear reorganization, of aneuploid cell subpopulation. Normal and tumoral as evidenced by Prohibitin compartimentalization, breast cancer cell lines were treated with 10nM of VCR accompanies the celular response to cisplatin towards and PTX for 24 hrs. The effect of VCR on mitotic spindle survival. Supported by FAPESP and CNPq. was confirmed by immunofluorescence and the G2/M [email protected] arrest was also observed by flow cytometry. There was an increased in the frequencies of binucleated, B-012 multinucleated and cells with nuclear DNA content PROSTAGLANDIN E2-EP1 AND EP2 RECEPTORS higher than 4C. PTX also act in mitotic cells on tubulin SIGNALING MEDIATES TIGHT JUNCTION spindles, but was not effective in blockade the cells ALTERATION OF HUMAN COLORECTAL CANCER in mitosis. The flow cytometry analysis showed that CELLS, CACO-2 cells were predominantly in G1, with distribution similar Marcelo Neves Tanaka (Instituto Nacional de Câncer), to controls cells. The number of binucleated and Bruno Lourenço Diaz (Instituto de Biofísica Carlos multinucleated cells did not increased and the DNA Chagas Filho), Pedro Henrique Schumann Lima quantification by image analysis (CIRES) did not show (Instituto Nacional de Câncer), José Andrés Morgado difference in DNA content when we compare control Díaz (Instituto Nacional de Câncer) cells with treated cells. In conclusion, only VCR was effective in induce aneuploidy in these breast cell lines. Prostaglandins play a significant role in the [email protected] pathophysiology of the digestive system, affecting water and electrolyte transport and cell motility, however B-014 cell-signaling pathways governing this process are yet CROTAMINE, MIOTOXIN DERIVED FROM THE poorly understood. Here, we investigated the effects of VENOM OF THE RATTLESNAKE CROTALUS prostaglandin E2 (PGE2) treatment on the structure and DURISSUS TERRIFICUS, SHOWS MELANOMA function of the tight junction (TJ) proteins using Caco-2 MURINO CELL NUCLEUS UPTAKE IN VITRO AND cells, an epithelial cell line derived from human colon IN VIVO adenocarcinoma. Using prostaglandin E2 receptor (EP) Alexandre Pereira (Laboratório de Genética, Instituto subtype agonists, we verified the involvemente of EP1 Butantan, São Paulo), Mirian AF Hayashi (Centro and EP2 receptors in the modulation of the TJ proteins de Toxinologia Aplicada CAT CEPID Instituto redistribution, as evidenced by immunofluorescence Butantan, São Paulo), Ivarne LS Tersariol (Centro and immunoblotting analysis. We observe that Interdisciplinarde Investigação Bioquímica (CHB), sulprostone and butaproste agonists, both EP1 and Universidade de Mogi das Cruzes, São Paulo), Gandhi EP2 agonist receptors respectively promote increase Rádis-Baptista (Universidade de São Paulo, São of the paracellular permeability, noted by a decrease of Paulo; Instituto Butantan, São Paulo), Eduardo B. the transepithelial electric resistance and permeability Oliveira (Departmento de Bioquímica e Imunologia, to ruthenium red marker. Furthermore, pharmacological Universidade de São Paulo, Ribeirão Preto), inhibition of protein kinase-C prevented the PGE2 Tetsuo Yamane (Laboratório de Genética, Instituto effects on the TJ alteration. Other inhibitors of cell Butantan, São Paulo), Alexandre Kerkis (Clinica e signaling pathways, such as Src, EGFR and MAPK, Centro de Pesquisaem Reprodução Humana “Roger seem are not involved in this events. Our findings Abdelmassih”, São Paulo), Irina Kerkis (Laboratório de support the hypothesis of a central role of PGE2-EP1 Genética, Instituto Butantan, São Paulo, Brasil) and EP2 receptors signaling that mediates the loss of cell-cell adhesion, a pivotal event at the beginning of the Introduction: Cell penetrating peptides (CPP) is a small colorectal tumorigenesis. molecular weight peptides, rich in basic amino acids [email protected] like arginine and the lysine, which have a capacity to penetrate in the cells. Previously, we demonstrate that B-013 the crotamine, which belongs to the family of CPP, NUCLEAR ALTERATIONS CAUSED BY present preferential uptake into actively proliferating ANTIMITOTIC AGENTS cells. Objectives: These properties of the crotamine led Beatriz Brandão Vaz de Lima, Gláucia Maria Machado us to investigate its uptake into B16-F10 cells in vitro Santelli (Dept. Biologia Celular e do Desenvolvimento, aiming also at its possible interference on melanomas Inst. Ciências Biomédicas, USP, São Paulo) development in vivo. Materials and Methods: In vitro the cellular viability in response of crotamine application 1. 95

livro.indd 95 7/7/2008 16:33:35 was evaluated by XTT, while its uptake into B16-F10 B-016 cells by immunocytochemistry. Animals with melanomas EVALUATION OF SUPERANTIGEN have received 5 ug of Cy3-crotamine by intraperitoneal STAPHYLOCOCCAL ENTEROTOXIN B (SEB) inoculation. In vivo crotamine localization in melanomas VERSUS BACILLUS CALMETTE-GUERIN (BCG) was evaluated 24 hours after the injection on frozen IMMUNOTHERAPY FOR SUPERFICIAL BLADDER cuts. Results: In vitro strong fluorescent signal was CANCER (SBC) observed within B16-F10 indicating Cy3-crotamine Leonardo Oliveira Reis, Wagner José Fávaro, Valéria localization in the cytoplasm and in the nuclei of dividing Helena Alves Cagnon Quitete, Wagner Eduardo cells. In vivo crotamine presence was evidenced in Matheus, Athanase Billis, developing melanomas, while other tissues were Ubirajara Ferreira (UNICAMP) crotamine free. Our data suggest that crotamine was able to penetrate into the cytoplasm and nuclei of B16- Intravesical BCG therapy is considered the most F10 cells in vitro and in vivo after the tumor formation. successful treatment for superficial bladder cancer. Preferential localization of crotamine in developing However, undesirable effects of BCG were noted such tumors suggests its potential use as a tumor cell marker as irritative voiding symptoms. Authors have evaluated in vivo. Remarkably, it was not toxic for mice organism the SEB as a potential candidate to intravesical therapy regarding the doses, which was applied in present of bladder cancer, showing no local or systemic toxicity, study. despite having doubts about this topic. Thus, the aim of [email protected] this work was to compare the BCG and SEB treatments in the superficial urinary bladder cancer of rats, induced B-015 experimentally. 20 rats were anesthetized and 5 of C16, A LAMININ GAMMA-1 DERIVED them received 0.2 ml dose of saline (control group). PEPTIDE, INTERACTS WITH INTEGRINS AND Other 15 rats received 1.5 mg/kg dose of n-methyl- REGULATES MIGRATION, INVASION AND n-nitrosourea (MNU), intravesically every other week PROTEASE ACTIVITY OF A HUMAN SALIVARY for 7 weeks. After MNU treatment, the 15 rats were GLAND ADENOID CYSTIC CARCINOMA CELL LINE divided into 3 groups: MNU group received the above Leticia Nogueira da Gama de Souza, proceeding; SEB group received 10 mg/ml dose of SEB Ruy Gastaldoni Jaeger (Institute of Biomedical intravesically for 8 weeks; BCG group received 106 Sciences, University of São Paulo) UFC dose of BCG for 8 weeks. After 15 weeks, all rats were anesthetized and bladders were processed for Our laboratory studies effects of laminin and its derived structural and immunological analyses and staged.The peptides in tumor biology. Here we analyzed induction results showed undifferentiated tumor, characterizing of migration, invasion and protease activity by laminin- carcinoma in situ and papillary carcinoma in MNU derived peptide C16 (KAFDITYVRLKF, g1 chain) in a group. The SEB group showed urothelial hyperplasia cell line (CAC2) derived from adenoid cystic carcinoma. and BCG group presented bladder papiloma. Apoptosis Laminin g1 was immunolocalized in adenoid cystic and cellular proliferation were increased in MNU, BCG carcinoma cells in vivo. To study migration, were used and SEB groups. However, these processes showed wound assay and Transwell chambers. C16 enhanced diminishing in the SEB and BCG groups. The SEB CAC2 cell migration in both assays compared to control. group showed better recovery of cancer state than that This peptide stimulated cell invasion in Transwell observed in BCG group.Thus, SEB treatment should chambers coated with Matrigel. Invasion could be be the first choice in superficial bladder cancer in related to protease activity. Zymograms were carried comparison to BCG one. out and showed that C16 increased secretion of MMP9 [email protected] in CAC2 cells. To study putative receptors of C16 we used affinity chromatography. Membrane preparations B-017 of CAC2 cells were run through C16-affinity columns. LAMININ-DERIVED PEPTIDES AG73 AND C16 A 40kDa band was eluted and analyzed by mass INDUCE MIGRATION, INVASION AND MMP spectrometry (LC-MS/MS) identifying a collagen a1 SECRETION IN AN ORAL SQUAMOUS CELL chain. Processing of collagen by MMPs is common in CARCINOMA CELL LINE cancer and results in fragments, such as the 40kDa Adriane Sousa de Siqueira, Letícia Nogueira da Gama observed. We suggest that the collagen fragment eluted de Souza, Ruy Gastaldoni Jaeger form C16 column would be part of a protein complex (Department of Cell and Developmental Biology, ICB - involving C16. Integrins are collagen receptors. We USP, São Paulo, Brazil) decided to study whether integrin would be involved in C16 effetcs in CAC2 cells. Silencing of b1 integrin Oral squamous cell carcinoma is a prevalent head and by siRNA decreased CAC2 cell migration. We suggest neck tumor, related to high mortality rates. Its growth that signal generated by C16 is transduced by collagen and invasiveness is stimulated by interactions with and integrins resulting in increase of migration, invasion extracellular matrix and basement membrane. Laminin and protease activity. Support: FAPESP (06/57079-4, is prominently expressed in basement membrane and 05/55602-9), CNPq (470622/2007-5). regulates tumor biology. This molecule also harbors [email protected] 1. 96

livro.indd 96 7/7/2008 16:33:35 cryptic sites with important roles upon release by Matrigel. Cells transfected with “scrambled” siRNA proteolytic enzymes such as matrix metalloproteinases served as controls. CAC2 cells with b1 integrin silenced (MMPs). MMP-mediated breakdown of basement showed decreased invasion compared to control cells. membrane is a critical step in tumor progression that Therefore, b1 integrin is a putative receptor of AG73 involves laminin cleavage and release of peptides which and probably associated to the invasive behavior may influence carcinoma´s behavior. Our Laboratory of CAC2 cells. Support: FAPESP (06/57079-4, has been studying the effects of laminin-derived 05/55601-2) and CNPq (470622/2007-5). peptides in tumor biology. Here we studied whether [email protected] these peptides would induce migration and invasion in a oral squamous cell carcinoma cell line (OSCC). Two B-019 laminin-peptides were used: AG73 (RKRLQVQLSIRT, PSEUDOCYSTS, HALLMARKS OF ADENOID alpha 1 chain) and C16 (KAFDITYVRLKF, gamma 1 CYSTIC CARCINOMA IN VIVO, ARE RELATED TO chain). Migratory activity was analyzed in monolayer SIKVAV-INDUCED PROTEASE ACTIVITY wound assay and in Neuroprobe chambers. Invasive Karen Piedade Cruz, Elaine Cyreno Oliveira, Leticia behavior was observed in Boyden chambers coated Nogueira da Gama de Souza, Vanessa Morais Freitas, by Matrigel. Our results showed that AG73 and C16 Ruy Gastaldoni Jaeger induced cell migration and invasion compared to (Department of Cell and Developmental Biology, ICB- controls. We also adressed whether these peptides USP, Sao Paulo, Brazil) would stimulate MMP secretion. Media from invasion assays were subjected to zymography. Both peptides Our Laboratory has been studying the role played by increased secretion of MMPs 2 and 9. Our results laminin-111 and its derived peptide SIKVAV (alpha1 suggested that laminin-derived peptides AG73 and C16 chain) regulating behavior of salivary gland neoplasms. stimulate migration, invasion and protease activity in We have demonstrated that this peptide regulates oral carcinoma cells. Support: FAPESP (06/57079-4, morphology and protease activity of an adenoid cystic 07/51950-8) and CNPq (470622/2007-5). carcinoma cell line (CAC2, Freitas et al., 2007). In [email protected] three-dimensional cultures, this peptide induces pseudocystic spaces, recapitulating tumor phenotype B-018 in vivo. Pseudocystic spaces are hallmarks of adenoid CHARACTERIZATION OF RECEPTORS AND cystic carcinoma. SIKVAV induces protease activity and INVASION BEHAVIOR RELATED TO LAMININ- is related to the formation of pseudocystic structures in DERIVED PEPTIDE AG73 IN CELL LINE DERIVED 3D culture. We then decided to study whether SIKVAV- FROM ADENOID CYSTIC CARCINOMA induced pseudocysts would be the result of protease Elaine Cyreno Oliveira, Letícia Nogueira da Gama de activity. Three experimental groups were used 1) CAC2 Souza, Ruy Gastaldoni Jaeger (Institute of Biomedical cells grown in laminin 3D; 2) Cells cultured inside Sciences, University of Sao Paulo) SIKVAV-enriched laminin; 3) CAC2 cells pretreated with a broad range matrix metalloproteinase inhibitor Our laboratory has been studying the role played (GM6001), followed by growth within SIKVAV-enriched by laminin-111 and its derived peptide AG73 laminin. Samples were studied by light microscopy. (RKRLQVQLSIRT, LG4 domain) regulating the behavior CAC2 cells grown in laminin 3D formed closely packed of salivary gland neoplasms. We demonstrated that aggregates. Cells grown within SIKVAV-enriched laminin syndecan-1 and b1 integrin signaling downstream formed pseudocystic spaces. The protease inhibitor of AG73 regulate adhesion and MMP production GM6001 decreased the effect of SIKVAV in CAC2 in a cell line (CAC2) derived from adenoid cystic cells. Cells treated by this drug showed a decrease of carcinoma (Gama-de-Souza et al., 2007). Here, we pseudocystics spaces, creating architecture similar to further investigated AG73 receptors in CAC2 cells. the laminin group. Our results suggest that formation Affinity columns were coupled with either AG73 or a of pseudocystic spaces in adenoid cystic carcinoma scrambled peptide control. Membrane preparations of cells could be the result of SIKVAV-mediated protease CAC2 cells were passed through these columns and activity. Support: PIBIC-CNPq, FAPESP (02/04208-0) eluted with EDTA 20mM to isolate putative ligands. and CNPq (470622/2007-5). These receptors were resolved in electrophoresis gel, [email protected] and submitted to mass spectrometry (LC-MS/MS) and peptide “fingerprint” (Proteomics Resource Center, B-020 Rockefeller University, NY, USA). Our results showed ANTIPROLIFERATIVE ACTIVITY OF PSIDIUM different potential receptors for AG73, such as integrin GUAJAVA L., A PLANT POPULARLY USED IN subunits b1 and aV. We then addressed whether TREATMENT OF PARASITIC DISEASES integrins would be involved in AG73-related invasive Débora Barbosa Vendramini Costa (Programa de Pós- activity. CAC2 cells had b1 integrin silenced by siRNA. Graduação em Biologia Celular e Estrutural, Instituto Silencing efficiencywas confirmed by immunoblot. Cells de Biologia - UNICAMP), Ana Lúcia Tasca Gois Ruiz with reduced expression of receptor were submitted (Divisão de Farmacologia e Toxicologia - CPQBA - to invasion assay in Boyden chambers coated with UNICAMP), Mary Ann Foglio (Divisão de Fitoquímica 1. 97

livro.indd 97 7/7/2008 16:33:35 - CPQBA - UNICAMP), João Ernesto de Carvalho days, the arising nodules had a medium tumoral volume (Divisão de Farmacologia e Toxicologia - CPQBA - over 1.500 mm3. The tumoral cells (MCA-GFP) were UNICAMP) isolated through mechanical dissociation and enzymatic digestion. These cells were also characterized by About 60% of anticancer drugs are provided from tumorigenic potential and by immunophenotypic, natural sources. As antiparasitic drugs also belong morphologic, histologic analyses. Histopathological to chemotherapy group, this work investigated analysis confirmed the presence of atypical cells with in vitro anticancer activity of crude extracts and oval nuclei that contained coarsely granular chromatin fractions (F) from Psidium guajava L., popularly and a fairly evident nucleolus; several blood vessels; used as antiparasitic. P. guajava leaves were and necrotic foci within the nodule. The MCA-GFPs successively extracted with dichloromethane providing cells presented spindle-shaped morphology with dichloromethanic crude extract (DCE) and ethanol long cytoplasmatic projections and multinuclear 95%, providing ethanolic crude extract (ECE). DCE giant cells. Flow cytometry analysis demonstrated was fractionated by column chromatography, providing negative expression for hematopoietic markers (CD34; 7 fractions. Antiproliferative activity was evaluated in CD45 and CD117), cell cycle deregulation, and ki-67 human tumor cell lines of breast (MCF-7), lung (NCI- overexpression (71.17%). Cytogenetic analysis reported H460), melanoma (UACC-62), prostate (PC-3), kidney polyploidy; culture kinetics showed a proliferative rate (786-0), colon (HT-29), ovarian (OVCAR-3) and ovarian 1.62x higher than the control cells. Finally, secondary expressing multiple drugs resistance phenotype (NCI/ hosts demonstrated the tumorigenic potential which ADR-RES). Sample concentrations were from 0.25 to formed the previously mentioned tumoral nodules in 250 µg/mL and doxorubicin the positive control. After 5-10 days after the MCA-GFP cells (5x106) infusion. 48h, cell growth was evaluated by sulforhodamine B Thus, our work assures the establishment of a new assay and total growth inhibition (TGI) was calculated experimental fibrosarcoma model based on GFP+ by non linear regression. ECE was inactive; DCE was tumoral cells. active for colon, ovarian and ovarian expressing multiple [email protected] drugs resistance phenotype. After DCE purification process, the compounds responsible for DCE activity B-022 were found in F2, active for colon; F5, 6 and 7 for GPR56 ROLE ON CHEMOTHERAPY ovarian; F4 for ovarian expressing multiple drugs RESISTANCE ON ACUTE LYMPHOBLASTIC resistance phenotype. F7 was also active for lung, LEUKEMIA (ALL) suggesting that DCE minority molecules are responsible André Bortolini Silveira, Angelo Brunelli Albertoni for this activity. Further purification processes will Laranjeira, Silvia Regina Brandalise, José Andres yunes make possible the identification of active principles, (Centro Infantil Boldrini) mechanism of action and in vivo studies. [email protected] Interaction of acute lymphoblastic leukemia (ALL) cells with bone marrow (BM) stroma enhances their B-021 resistance to chemotherapy. We found that culture ISOLATION AND CHARACTERIZATION of ALL with BM stroma cells causes downregulation OF A TUMORAL CELL OBTAINED FROM of G protein-coupled receptor 56 (GPR56), a gene THE FIBROSARCOMA INDUCED WITH reported to have lower expression levels on ALL cases CARCINOGENIC CHEMICAL SOLUTION OF more sensitive to L-asparaginase chemotherapy. 3-METHYLCHOLANTHRENE IN VIVO We hypothesize that downregulation of GPR56 Danilo Candido de Almeida1, Aparecida Maria Fontes1, may somehow directly induce ALL resistance to Ricardo Bonfim Silva1, Lucas Eduardo Botelho de L-asparaginase. To verify if the modulation of GPR56 by Souza1, Maristela Delgado Orellana1, Patricia Viana stromal contact is specific or shared by other adhesion Bonini Palma1, Elvis Cristian Cueva Mateo2, Luiz G protein-coupled receptors, we analyzed the effect Gonzaga Tone2, Sérgio Brito Garcia2, Dimas Tadeu of culturing ALL cells on BM stromal layers on the Covas1 expression of 4 genes of this family (GPR56, GPR124, 1Centro de Terapia Celular CTC - Centro Regional LPHN1 and CD97). The expression levels of 17 ALL de Hemoterapia de Ribeirão Preto FMRP/USP; 2 patients’ cells were quantified by Real-Time PCR after Faculdade de Medicina de Ribeirão Preto FMRP/USP 0h or 6h of culture. GPR56 was downregulated after 6h of culture (p=0,0037). GPR124 was practically The abnormal progressive growth of tissues defines undetectable on cells deprived of contact with stroma Neoplasia, a major cause of death of our time. The (0h) and was upregulated after culture (p<0,0001). long-term research for efficient therapies for this GPR124 codes a glycosaminoglycans receptor pathology requires an adequate experimental model. responsible for cell-cell and cell-matrix contact survival This work aims at featuring a tumor fibrosarcoma signaling on endothelial cells. We speculate that this model, and to isolate these tumor cells. Systematically, protein acts likewise on ALL cells. LPHN1 and CD97 6 FVB-GFP+ mice were administered a 3- were not differentially expressed (p=0,352 and p=0,582, methylcholanthrene solution in tricapylin; after 76±6.9 respectively). The invariable expression of these genes 1. 98

livro.indd 98 7/7/2008 16:33:35 supports the hypothesis that downregulation of GPR56 knowledge of such cancer cells response to proteins and upregulation of GPR124 are responses specifically which can act as mitogens or autocrine factors. Stress- driven by ALL adhesion to BM stromal layers. inducible protein 1 (STI1), a phosphoprotein with [email protected] co-chaperone activity, was described by our group as a ligand for cellular prion protein (PrPC). This protein is B-023 secreted by primary astrocyte cultures, binds to PrPC POTENCIAL EFFECT OF 4-NEROLYDILCATECHOL at the neuronal surface and elicits neuroprotection ON INHIBITION OF MELANOMA CELL DEATH AND and neuritogenesis. More recently, STI1 was observed INVASIVENESS to induce PrPC-dependent proliferation on human Carla Abdo Brohem (FCF-USP), Tânia Cristina Higashi glioblastoma cell lines. Aiming to find new possible Sawada (FCF-USP), Renato Ramos Massaro (FCF- therapeutic strategies for this issue, we proposed USP), Rebeca Leite de Almeida (FCF-USP), Diogo the hypothesis of a STI1-PrPC induced proliferation Pineda Rivelli (FCF-USP), Cristina Dislich Ropke (FCF- blockage by the addition of a STI1 competitor peptide USP), Vanessa Vitoriano da Silva (FCF-USP), Thaís (STI1230-245) that mimics the PrPC binding site. By Martins de Lima (ICB-USP), Rui Curi (ICB-USP), Silvia using in vitro cell proliferation assays based on BrdU Berlanga de Moraes Barros (FCF-USP), Silvya Stuchi incorporation and immunofluorescence positivity, we Maria-Engler (FCF-USP) observed a marked proliferation induced by STI1 treatment on A172 or U87MG human glioblastoma Melanoma is the most aggressive skin cancer cell lines (respectively, 1.8 and 2.4 times higher than and is resistant to all current cancer therapy. untreated cells). Conversely, while peptide has no 4-nerolydilcatechol (4-NC), a compound from the effect by itself, STI1230-245 peptide markedly impaired Brazilian plant Pothomorphe umbellata L. Miq exerts STI1-PrPC induced cellular proliferation (2.5 fold for a potent antioxidant activity and was described as in A172 and 5 fold for U87MG). Additional evidences to vitro inhibitor of matrix metalloproteinase (MMP-2 and these findings will allow us to lead such tests for in vivo MMP-9) activities. Invasion of tumor-surrounding tissues models. by cancer cells is mediated by proteolytic enzymes [email protected] such as MMPs. Here, we investigated the cytotoxicity of 4-NC on different tumor cell lines as melanoma B-025 (Sk-Mel-28, 103 and 147), cervical carcinoma (SiHa INHIBITION OF CELL INVASION IN AN MMP- and HeLa) and glioma (A172 and T98G) as well as INDEPENDENT MANNER IN THE T98G HUMAN a non-malignant human dermal fibroblast and the GLIOBLASTOMA CELL LINE OVEREXPRESSING inhibitory effects on MMP activities. 4-NC showed THE RECK GENE cytotoxicity for all cancer cell lines (IC50 from 20-40µM Tatiana Caroline Silveira Corrêa (Faculdade de Ciências after 24 hours of treatment/for tumoral cell lines and Farmacêuticas - USP), Renato Ramos Massaro 50µM for dermal fibroblast). Apoptosis induction was (Faculdade de Ciências Farmacêuticas - USP), Carla observed in melanoma cell lines. Inhibithory effect of Abdo Brohem (Faculdade de Ciências Farmacêuticas cell invasiveness, associated to the capacity to lead - USP), Mari Cleide Sogayar (Instituto de Química - cells to arrest in G1 cell cycle and to inhibit MMP-2 USP), Sebastião Roberto Taboga (IBILCE - UNESP), activity in melanoma cell lines, Sk-Mel-147, when Silvya Stuchi Maria Engler (Faculdade de Ciências exposed a 30µM of 4-NC. These results show that 4-NC Farmacêuticas-USP) is a candidate for further experiments on cell death induction in melanoma therapy. Gliomas represent a great challenge in view of their [email protected] poor clinical outcome, with low patient survival rate and great resistance to chemo and radio therapy. RECK, an B-024 important tumor suppressor gene, is known to regulate CELLULAR PRION INTERACTION WITH STI1 AS different MMPs, namely: MMP-2, MMP-9 and MT1- A TARGET FOR THERAPEUTIC INTERVENTION IN MMP, with its anti-tumoral activity being associated GLIAL TUMORS to an anti-MMPs activity. T98G cells, derived from a Nicolle Gilda Teixeira de Queiroz (Inst. Ludwig de human multiform glioblastoma, were described as a Pesquisa sobre o Câncer e Hospital A. C. Camargo), highly invasive glioma cell line, which displayshigh Marilene Hohmuth Lopes (Inst. Ludwig de Pesquisa levels of MMPs 2 and 9. The RECK gene, cloned sobre o Câncer), Vilma Regina Martins (Inst. Ludwig de into the pCXN2 vector was overexpressed in T98G Pesquisa sobre o Câncer e Hospital A. C. Camargo) cells(T98G/RECK+). The expression of RECK, MMP-2, MMP-9 and MT1-MMP was evaluated by qPCR and by Glial cells can acquire a pathogenic character and rise western blotting in T98G/RECK+, control cells (cloned as tumors called gliomas. Glioblastoma multiforme with empty vector) and wild type cells. Surprisingly, (GBM) represents the worse form of glioma. GBM no alteration on MMPs gene expression at the mRNA is poorly responsive to most of the currently known level (qPCR) was observed in T98G/RECK+, neither therapies and the prognosis is intimately related to on protein levels nor on protein activity (western its proliferative activity. Therefore, it is crucial the blotting and gelatin zymogram, respectively). On the 1. 99

livro.indd 99 7/7/2008 16:33:35 other hand, the invasion capacity of T98G/RECK+ CARCINOMA cells was inhibited in transwell assay. In addition, Camila Fernandes Nascimento, Letícia Nogueira images of autophagic vacuoles were visualized upon da Gama de Souza, Vanessa Morais Freitas, electron microscopy of T98G/RECK+ cells, cultured Ruy Gastaldoni Jaeger (Department of Cell and onto collagen, but not on the control cells. The volume Developmental Biology, ICB-USP, São Paulo) of these autophagic vacuoles were considered to be higher for T98G/RECK+ cells, when compared to the Our laboratory studies different processes in tumor control cells, as evaluated by a computer-assisted biology, such as migration, invasion and protease stereological approach. Since autophagy represents secretion. In this study, we used cells derived from an important anti-tumoral cell behavior strategy, it is adenoid cystic carcinoma (CAC2), a frequently important to evaluate whether RECK is involved in this occurring malignant salivary gland neoplasm, with process, in addition to its capability to inhibit invasion. high level of recurrence and distant metastasis. [email protected] Metastatic tumor cells that actively migrate and invade surrounding tissues rely on invadopodia to degrade B-026 extracellular matrix (ECM) barriers. Invadopodia CCL2 AND IL8 GENE EXPRESSION AND are membrane protrusions that localize enzymes FUNCTION ANALYSIS IN CHILDREN WITH ACUTE required for ECM degradation. Studies identified that LYMPHOBLASTIC LEUKEMIA organization of invadopodia structure requires cortactin Jaíra Ferreira de Vasconcellos (Centro Infantil Boldrini and membrane type 1 matrix metalloproteinase / Universidade Estadual de Campinas), Silvia Regina (MT1-MMP) as key invadopodial enzyme responsible Brandalise (Centro Infantil Boldrini), José Andrés Yunes for matrix degradation. Here we characterized the (Centro Infantil Boldrini) invadopodia in CAC2 cells. To study structural and functional aspects of this protrusion, we carried out Acute Lymphoblastic Leukemia (ALL) is the most fluorescent-Matrigel degradation assay complemented common cancer in childhood. Little is known about by immunofluorescence. Cells were plated on Matrigel- the expression and function of CCL2, IL8 and their FITC and fixed at different time points (1 hour, 3 and receptors (CCR2A/B to CCL2; CXCR1/2 to IL8) in ALL. 24 hours). After fixation, CAC2 cells grown on Matrigel- Our aim was to analyze the prognostic significance FITC were double-labeled to actin and cortactin. Cells of CCL2, IL8, CCR2A/B and CXCR1/2 expression, cultured on Matrigel-FITC were also double-stained and their function in ALL cells in vitro. ALL diagnostic to actin and MT1-MMP. Samples were studied by samples were analyzed to CCL2 (n=58), CCR2A/B confocal microscopy. In all time points CAC2 showed (n=106), IL8 (n=57), CXCR1/2 (n=50) by RT-PCR. actin, cortactin and MT1-MMP colocalized at matrix CCR2B was also analyzed by Q-PCR. Primary ALL degradation spots in fluorescent Matrigel. Our results cells were co-cultured with human mesenchymal stem suggest that invasiveness of CAC2 cells is mediated cell and treated with CCL2 (1-50ng/ml), IL8 (500ng/ by colocalization of actin, cortactin and MT1-MMP ml) or their neutralizing antibodies (5µg/ml). Cell at invadopodia. Support: FAPESP (06/57079-4, proliferation was measured by FACS. The ALL cases 07/05447-2), CNPq (470622/2007-5). expressed CCL2 (33/58 cases), CCR2A (10/106), [email protected] CCR2B (64/106), IL8 (42/57), CXCR1 (40/50) and CXCR2 (38/50). ALL that expressed at least one B-028 myeloid marker (CD13, CD15 and CD33) had an SELECTION OF A SET OF RELIABLE REFERENCE increased expression of CCR2B (p=0.02). No other GENES FOR VALIDATION OF DIFFERENTIAL DOWN association was found between expression of CCL2/ REGULATED GENE EXPRESSION IN CARCINOMA- CCR2 and IL8/CXCR1/2 and clinical/biological features ASSOCIATED FIBROBLASTS (CAF) AND NORMAL- of patients. Proliferation and survival of primary ALL ASSOCIATED FIBROBLASTS (NAF) cells in vitro were not affected by addition of CCL2, Natália Bromberg, Patrícia Bortman Rozenchan, Maria IL8 or neutralizing antibodies to these chemokines. Mitzi Brentani (Faculdade de Medicina - USP) We are currently investigating if these chemokines could be implicated in leukemia migration. It is known An increasing body of research indicates that stroma that CCL2 and IL8 have suppressive effects in normal surrounding cancer cells plays an important role in hematopoiesis. The fact that ALL cells are unaffected the development and subsequent behavior of the by IL8 or CCL2 is an interesting finding and might help tumor. The evaluation of differential gene expression in to elucidate the survival/proliferation advantage of ALL carcinoma-associated fibroblasts (CAF) and normal- cells compared to normal bone marrow cells. CNPq, associated fibroblasts (NAF) from breast cancer FAPESP. patients followed by SAGE, microarray and codelink [email protected] characterization generated an interesting gene expression profile. The validation of the differential down B-027 regulated genes found in that study required a proper CHARACTERIZATION OF INVADOPODIA IN internal control gene for the Real-Time RT-PCR method CELL LINE DERIVED FROM ADENOID CYSTIC used. Here, we analysed 6 commonly used reference 1. 100

livro.indd 100 7/7/2008 16:33:35 genes (ACTB, TFRC, GAPDH, RPLPO, GUS and PPIA) Vanessa Morais Freitas (Department of Cell and in CAF and NAF from breast cancer patients maintained Developmental Biology, ICB-USP, São Paulo), in cell culture to normalize genes of interest expression Paula Rezende Teixeira (Department of Cell and levels. Total RNA were isolated from samples using the Developmental Biology, ICB-USP, São Paulo), Gláucia TRIzol Reagent and RT-PCR assays were conducted Maria Machado Santelli (Department of Cell and using SYBR green system. To identify the most stable Developmental Biology, ICB-USP, São Paulo), Bechara reference genes we applied the GeNorm program. The Kachar (Laboratory of Cellular Biology, Section on ranking of the candidate reference genes according Cell Structure, NIDCD, NIH, Bethesda, MD, USA), to their stability measure (M) in each cell sample was Ruy Gastaldoni Jaeger (Department of Cell and ACTB (1.068), TFRC (0.976), GAPDH (0.645), RPLPO Developmental Biology, ICB-USP, São Paulo, Brazil) (0.479), GUS and PPIA (0.433). RPLPO, GUS and PPIA showed the most stable expression pattern. We Podosomes and invadopodia are actin-rich adhesions concluded that the normalization strategy by multiple that establish contact to substratum and degrade housekeeping genes instead of one is a prerequisite for extracellular matrix. They resemble each other in accurate RT-PCR expression profiling, which, among molecular organization, both consisting of actin other things, opens up the possibility of studying the core surrounded by ring of actin-binding proteins. biological relevance of small expression differences. Podosomes and invadopodia are easily distinguished Supported by FAPESP, CNPq. from dash-like focal adhesions. By contrast, few [email protected] defining features discriminate podosomes from invadopodia. Podosomes may form on two-dimensional B-029 substrates whereas invadopodia develop in matrix- INCREASED ALFA 2-6 SIALYLATION IS embedded cells. On the other hand podossomes could FOLLOWED BY DECREASE OF GALECTIN-3 be initial step of invadopodia formation. Here we studied LIGANDS IN A MURINE MELANOMA CELL LINE dynamics of these structures in malignant mammary TRANSFECTED WITH THE GALECTIN-3 GENE gland cells (Hs578T). Cells were cultured on Matrigel- Gabriella Malheiros Moraes (Faculdade de Medicina- FITC and labeled with actin probe rhodamine-phalloidin. Supported by CNPq/Brazil), Andréia H. Otake Confocal microscopy showed thin invadopodia forming (Faculdade de Medicina), Roger Chammas (Faculdade tracks in degraded fluorescent matrix. Hs578T cells de Medicina) were transfected with fluorescently-tagged proteins related to podosomes, such as GFP-vinculin and Galectin-3 is a beta-galactoside-binding lectin. Cherry-actin. Podosome and invadopodia formation Exogenous galectin-3 induces apoptosis by interacting was assessed by 4D confocal microscopy, with with cell surface glycans. However, endogenous z-sections taken overtime. Hs578T cells grown on galectin-3 does not lead to cell death. Aberrant glass formed podossomes after 8 min. Cells grown glycosylation is a commonly observed feature in tumor on Matrigel presented dots of colocalized vinculin development which may lead to several modifications and actin. To study whether podossomes would have in surface molecules and signaling alterations. In this evolved to invadopodia a series of 10 confocal sections work, we observed the difference of glycosylation were collected sequentially for each time point and between two clones from TM1M, a murine melanoma 3D reconstructed. Podossomes form tubules invading line. TM1MG3 was transfected with the human Matrigel. These preliminary results suggest that galectin-3 gene and the TM1MN3 was transfected with podossomes are initial steps of invadopodia formation. an empty plasmid as a control. Western blot reactions Support: FAPESP (06/57079-4, 07/05166-3), CNPq were performed using protein extracts of TM1MG3 (304868/2006-0). and TM1MN3 to analyze alterations in glycosylation. [email protected] We used Gal-3-FA (galectin-3 conjugated with alkaline phosphatase) which recognizes galectin-3 ligands, B-031 and SNA (Sambucus nigra), a lectin which recognizes THE ROLE PLAYED BY ADAMTS-1 ON MIGRATION sialic acids linked to galectin in a alfa(2-6) configuration. AND INVASION OF MALIGNANT MAMMARY GLAND We have shown that TM1MG3 increased alfa(2-6) CELLS IN 3D CULTURE sialylation and decreased the expressions of galectin-3 Vanessa Morais Freitas (Department of Cell and ligands as compared to TM1MN3. We propose that Developmental Biology, ICB-USP, São Paulo), TM1MG3 cells adapted to the presence of apoptogenic Ruy Gastaldoni Jaeger (Department of Cell and galectin-3 by increasing the specific sialylation of their Developmental Biology, ICB-USP, São Paulo), Bechara glycoproteins, thereby decreasing galectin-3 binding. Kachar (Laboratory of Cellular Biology, Section on Cell This hypothesis will be now specifically addressed. Structure, NIDCD, NIH, Bethesda, MD, USA), Gláucia [email protected] Maria Machado Santelli (Department of Cell and Developmental Biology, ICB-USP, São Paulo, Brazil) B-030 PODOSOMES AND INVADOPODIA DYNAMICS IN A ADAMTS-1 (a disintegrin and metalloprotease with MAMMARY GLAND CELL LINE thrombospondin motifs) is a member of the ADAMTS 1. 101

livro.indd 101 7/7/2008 16:33:35 family of metalloproteases. This enzyme regulates showed that BTCI is a promising anticarcinogenic physiological and pathological processes. Here we molecule. Further studies concerning BTCI mechanism studied the role played by ADAMTS-1 on three- of action and its use as a complement or alternative dimensional migration of a malignant mammary gland anticarcinogenic agent are underway. cell line (Hs578T). Cells had ADAMTS-1 silenced by [email protected] siRNA. Cells transfected with scrambled siRNA served as control. Immunoblot confirmed silencing efficiency. B-033 Treated and control cells were grown on Matrigel-FITC. MODULATION OF ENDOTHELIAL APOPTOSIS AND To follow migration cells were loaded with Cell Trace Far DIFFERENTIATION IN THE CONTEXT OF HUMAN Red. Growth of cells in fluorescent Matrigel was used GLIOMAS: ROLE OF TUMOR EXTRACELLULAR to image invasion spots. Hs578T migratory activity in MATRIX Matrigel 3D was assessed by 4D confocal microscopy Tercia Rodrigues Alves (Universidade do Estado do Rio (UltraView Yokogawa spinning confocal, Perkin Elmer, de Janeiro(UERJ);Fiocruz), Jane Faria (Universidade Waltham, MA, USA), with z-sections taken at different Federal do Rio de Janeiro (UFRJ) - Dep. de Anatomia), time points. A series of 10 confocal sections were Nathan Bessa Viana (Universidade Federal do Rio collected sequentially for each time point for a total de Janeiro - Instituto de Física), Sara Santana Nunes of 30 minutes and three-dimensionally reconstructed (Universidade do Estado do Rio de Janeiro (UERJ) - by the Volocity software (Improvision, Waltham, MA, Dep. de Biologia Celular e Genética), Christina Takiya USA). Our results showed that control cells spread (Universidade Federal do Rio de Janeiro (UFRJ) - Dep. and digested Matrigel after 15 min in culture. On the de Histologia e Embriologia), Jorge Marcondes de other hand cells with silenced ADAMTS-1 focally Souza (Universidade Federal do Rio de Janeiro (UFRJ) digested Matrigel, but failed to spread even after 30min - Serviço de Neurocirurgia/HUCFF), Vivaldo Moura in culture. Our preliminary results suggested that Neto (Universidade Federal do Rio de Janeiro (UFRJ) ADAMTS-1 play a role in three-dimensional migration of - Dep. de Anatomia), Verônica Morandi (Universidade Hs578T cells. Support: FAPESP (2006/54963-0). do Estado do Rio de Janeiro (UERJ) - Dep. de Biologia [email protected] Celular e Genética)

B-032 Gliomas, in particular astrocitomas, account for more INDUCTION OF APOPTOSIS ASSOCIATED WITH than 40 % of all neoplasms of the central nervous AUTOPHAGY BY A BOWMAN-BIRK INHIBITOR system. Extremely infiltrative and lethal, these tumors (BTCI) ON BREAST CANCER CELLS bear an aberrant vascular network. In this work, we Graziella Anselmo Joanitti, Sonia Maria de Freitas, focused on their secreted extracellular matrix (ECM), Ricardo Bentes de Azevedo (Universidade de Brasília) which contains high levels of tenascin-C (TN-C). TN-C is not expressed by quiescent brain endothelial Breast cancer is the type of cancer with the highest cells, but instead, it is strongly upregulated around incidence and mortality rate among women. Nowadays, angiogenic vessels inside the tumor mass. TN-C there is constant research for new and efficient disorganizes focal adhesions induced by fibronectin, anticarcinogenic treatments and molecules. Several thus resulting in glioma proliferation. Here we show purified compounds of plants and their anticarcinogenic that glioma ECM induces apoptosis of an endothelial effects have been reported in the literature. Among population in progress through the cell cycle (S-G2/M), these compounds, Bowman-Birk protease inhibitor by deadhesion (anoikis). Anoikis was also observed in family stands out. The aim of the present work was co-culture systems with glioma cell lines or explants to analyze the effects of a Bowman-Birk protease from human primary gliomas. However, endothelial inhibitor named BTCI, extracted from Vigna unguiculata cells that survived to anoikis became more proliferative, seeds, on breast cancer cells (MCF-7) in vitro. After 72 although less efficient to form tube-like structures in hours BTCI treatment, the number, plasma membrane vitro, when compared to cells plated on autologous integrity, viability, and morphology of the cells were ECM or onto the matrix secreted by fibroblasts. The analyzed by Neubauer chamber, tripan blue assay, study of the composition of glioma ECM from different MTT assay, and transmission electron microscopy, cell lines showed an increased TN/FN ratio, as respectively. Cell cycle, DNA fragmentation, and compared to ECM from the other non-tumoral sources. mitochondrial membrane potential were analyzed by The EGF-like domain of TN-C was involved in the flow cytometry. BTCI induced significant effects on impairment of tubulogenesis, but the physical contact approximately 70% (p<0.01) of the treated cells, such between surviving and apoptotic endothelial cells as: mitochondrial membrane potential, cell viability and rescued cells from the tubulogenesis defect. Thus, we proliferation reduction; DNA fragmentation increase; suggest that these effects of tumor ECM on endothelial and alterations in cell cycle, plasma membrane integrity tubulogenesis and proliferation would match with the and morphology. Taken together, the cytotoxic and aberrant pattern of angioarchitecture of the glioma mass cytostatic effects observed in BTCI treatment suggested in vivo. the induction of apoptosis cell death associated with [email protected] autophagy activation. In conclusion, the present work 1. 102

livro.indd 102 7/7/2008 16:33:36 B-034 responsiveness to signals mediated by cell-cell and EFFECTS OF RAPAMYCIN ON CELL CYCLE cell-extracellular matrix interactions which are critical OF ERBB2-OVEREXPRESSING BREAST CELL for maintenance of cellular homeostasis. Although LINES AND BREAST CANCER-ASSOCIATED some of these cell communications are well known, FIBROBLASTS there are furthermore to find out; mainly the underlying Adriana Priscila Trapé, Maria Lúcia Hirata Katayama, mechanisms of melanoma cell aggressiveness. In an Rosimeire Aparecida Roela, Tharcísio Citrângulo Tortelli effort to point out pathways that might predict the clinical Júnior, Graziela Rosa Ravacci, Maria Mitzi Brentani and biological behavior of melanoma, we analyzed the (Faculdade de Medicina da Universidade de São Paulo) expression profile of 64 human melanomas. To identify biological processes involved in melanoma progression ErbB2 overexpression occurs in about 30% of we first identified functional modules with significant breast cancers and correlates with more aggressive alteration when primary and metastasis melanomas disease and poor prognosis. The PI3K/AKT/mTOR were compared, and for genes belonging to altered pathway is an important mediator of ErbB2 effects on modules we generate relevance networks. Among cell proliferation, which might explain the increased the pair of genes whose correlation was significantly response of ERbB2-overexpressing cells to the altered we detached several pertaining to epidermis treatment with mTOR inhibitors, such as rapamycin. delevopment and hKLK pathway. We demonstrate, for Otherwise, breast epithelial cancer is not a cell- the first time, that mRNA levels of KLKs (6, 7, 8 and single disease, and its interaction with stromal cells 13) are significantly reduced in metastasis and, among plays an important role in tumor promotion, which primary lesions, named as group B, its diminished points to cancer-associated fibroblasts (CAFs) as expression was significantly associated to advanced another potential target to the antiproliferative effects Clark index and ulceration. Taken together, our data of rapamycin. Purpose: To investigate the effects of provides evidences for the role of kallikreins on rapamycin on cell cycle of ErbB2- overexpressing cell melanoma invasion, and as well points to a set of genes lines (HB4AC5.2a and SKBR3) and CAFs. Methods: whose altered expression may contribute to metastasis The HB4AC5.2A cell line is an ErbB2-overexpressing phenotype of melanoma cells. Financial Support: variant derived from HB4A cells (established from FAPESP/CEPID; LUDWIG. normal breast luminal cells), through stable transfection [email protected] with full-length human c-erbB2 cDNA. The SKBR3 cell line exhibits naturally an amplification of the ErbB2 B-036 gene. CAFs were obtained from primary culture of PRIMARY CULTURES AS TOOLS FOR breast carcinoma. Cells were treated with 20 nM MORPHOLOGICAL AND FUNCTIONAL rapamycin for 24 hs. Rapamycin effects were analyzed CHARACTERIZATION OF HUMAN TUMOR CELLS by flow citometry, considering the proportion of cells Isabela Mundim Palhares (Laboratory of Cell Biology/ arrested in G1 phase. Results and Conclusions: CAFs Fundação Ezequiel Dias, Belo Horizonte - M.G.), seem to be more sensitive to the rapamycin among the Márcia da Silva Hermógenes (Laboratory of Cell cells tested since the G1-cell cycle arrest was about Biology/Fundação Ezequiel Dias, Belo Horizonte 10% for these cells compared to 20% for epithelial cells. - M.G.), Karine Sampaio Lima (Alberto Cavalcanti Quantitative RT_PCR reactions are being carried out Hospital), Marco Antônio Dias Filho (Alberto Cavalcanti for gene expression analysis of targets clearly related to Hospital), Alberto Julius Wainstein (Alberto Cavalcanti cell cycle. Supported by FAPESP and CNPq. Hospital), Consuelo Latorre Fortes-Dias (Laboratory of [email protected] Cell Biology/Fundação Ezequiel Dias, Belo Horizonte - M.G.), Luciana Maria Silva (Laboratory of Cell Biology/ B-035 Fundação Ezequiel Dias, Belo Horizonte - M.G.) ALTERED EXPRESSION OF KALLIKREINS AND GENES RELATED TO EPIDERMIS DEVELOPMENT Cancer is a world wide distributed disease with high IN PRIMARY AND METASTATIC MELANOMAS morbidity and mortality. In Brazil, more than 450.000 Waleska Kerllen Martins (Hospital A.C. Camargo, São new cases of cancer have been estimated for 2008 by Paulo; Ludwig Institute for Cancer Research, São the National Institute of Cancer. Presently, a research Paulo), Gustavo H. Esteves (Instituto de Matemática and development project is in progress with partnership e Estatística da USP, São Paulo), Otávio M. Almeida of two public institutions of the state of Minas Gerais (Hospital A.C. Camargo, São Paulo), Gilles Landman with the main objective of starting a Depository Bank (Hospital A.C. Camargo, São Paulo), Rogério I. Neves of Human Tumor Tissues. The samples are collected (Hospital A.C. Camargo, São Paulo), Alex F. Carvalho from surgical patients at the Alberto Cavalcanti Hospital/ (Ludwig Institute for Cancer Research, São Paulo), FHEMIG, after authorization by signing a Consent Eduardo J. Neves (Instituto de Matemática e Estatística Form previously approved by the Ethical Committee da USP, São Paulo), Luiz F. L. Reis (Hospital A.C. for Human Research. A Data Bank to ensure proper Camargo, São Paulo, Brazil) sample identification is also under development. Adequate samples, which quality has been assessed During melanoma progression, cells might loose by examination by a pathologist, are sent to the Cell 1. 103

livro.indd 103 7/7/2008 16:33:36 Biology Laboratory/FUNED for the preparation of tissue B-038 explants and storage. Our first successful result was EFFECT OF POLYSACCHARIDES ISOLATED FROM obtained with ovarian tumor cells. Different culture MARINE INVERTEBRATES IN THE INTERACTION media with varying supplements were tested and OF PROSTATE CARCINOMA WITH ENDOTHELIAL the growing of different cell types evaluated by daily CELLS examination of the cultures. After two weeks, three Camila Castro Figueiredo (Universidade do Estado morphological cell types were observed: fibroblast-, do Rio de Janeiro (UERJ) Departamento de Biologia epithelial- and cuboid-like cells. These cells grew in Celular e Genética (DBCG)), Tercia Rodrigues Alves culture for up to two months and yielded a total number (Universidade do Estado do Rio de Janeiro (UERJ) of 29 aliquots that were frozen for storage at -80oC. Departamento de Biologia Celular e Genética (DBCG)), Our next steps will be devoted to immortalization trials, Viviane W. Mignone Dantas (Universidade do Estado aiming to establish new cell lines that might be used do Rio de Janeiro (UERJ) Departamento de Biologia in morphological, functional and pharmacogenomical Celular e Genética (DBCG)), Aline Oliveira da Silva studies. Financial support: FAPEMIG (PP/SUS), (Universidade do Estado do Rio de Janeiro (UERJ) FUNED, FHEMIG. Departamento de Biologia Celular e Genética (DBCG)), [email protected] Paulo A. S. Mourão (Universidade Federal do Rio de Janeiro (UFRJ) Institudo de Bioquímica Médica), B-037 Verônica Morandi (Universidade do Estado do Rio de A QUANTITATIVE ANALYSIS OF BREAST CANCER Janeiro (UERJ) Departamento de Biologia Celular e CELLS CULTURED IN THREE-DIMENSIONAL Genética (DBCG)) ENVIRONMENT THROUGH FLOW CYTOMETRY Letícia Franco Bisson, Evandro Luís de Oliveira Niero, Metastatic tumor cells may interact with both endothelial Gláucia Maria Machado Santelli (Depto de Biologia cells and components of the subendothelial matrix. Celular e do Desenvolvimento, Instituto de Ciências Many cancer cells exhibit altered glycosilation Biomédicas, USP, Brasil) patterns, often containing highly branched or sialylated oligosaccharides, especially fucosylated glycans Multicellular tumor spheroids (MTS) are a realistic such as sialyl-Lewisx, which act as ligands for model to investigate many aspects of tumor biology selectins. These receptors are normally involved in the because they recapitulate several aspects of the in inflammatory response, but they were also implicated vivo tissue, like morphologic and functional features in the hematogenous metastasis. Polysaccharides as well as the microenvironment, with cell-to-cell isolated from marine organisms have been identified as and cell-to-extracellular matrix interactions. Despite potential modulators of tumor growth, angiogenesis and of the increasingly use of three-dimensional (3D) metastasis. In the present work, we studied the effect cultures, some methods for analysis are not properly of the fucosylated chondroitin sulfate (FucCS), isolated standardized and some aspects regarding to MTS from sea cucumber L. grisea and a sulfated fucan development remain controversial, like cell proliferation (Fuc Sulf I), isolated from sea urchin L. variegatus, and apoptosis. Therefore, it was necessary to in the interaction of prostate cancer cell lines (PC-3 standardize a protocol for quantitative analysis of and DU-145) with primary endothelial cells (HUVECs) spheroid cells through flow cytometry and to establish monolayers, activated by TNF-alpha (10 ng/ml). We parameters relative to their growth and the cellular also tested their ability to modulate angiogenesis in processes involved. Breast tumor cell lines MCF-7 and vitro (tubulogenesis matrigel assay). We found that the T-47D were grown in liquid overlay cultures and some treatment of HUVECs with TNF-alpha increased both conditions for cell dissociation were tested. Samples adhesion and endothelial transmigration of tumor cells of MTS were harvested after 10, 20 and 30 days of (between 30 to 100%, depending on the cell line). The 3D culture and the cells were dissociated with trypsin treatment of monolayers with Fuc Sulf I (10 to 100 µg/ according to the established protocol, fixed in methanol ml) reversed the interaction induced by TNF-alpha. This and stained with propidium iodide. DNA values were polyssacharide also inhibited endothelial tubulogenesis quantified in a flow cytometer in order to determine (1 to 100 µg/ml), whereas FucCS was partially effective the percentage of cells in each cell cycle compartment only in the highest concentration tested. These findings and cell death. The data indicated a gradual increasing suggest that Fuc Sulf I may be a potential candidate for in apoptosis rate until 30 days of culture, while G2/M an antimetastatic drug. remains constant, for both cell lines. Our results [email protected] demonstrate that the protocol is effective in analyzing cells grown as spheroids through flow cytometry B-039 and that mitosis and apoptosis are involved in MTS THE IMPACT OF GADD45A SUPPRESSION IN growth, but these processes’ rate does not seem to be INDUCED CELL OXIDATIVE STRESS correlationed. Milene Pereira Moreira (Centro Universitário [email protected] UNA; BioCib/FUNED, Belo Horizonte), Sabrina S. Campolina (BioCib/FUNED, Belo Horizonte), Márcia S. Hermógenes (BioCib/FUNED; Pontifícia Universidade 1. 104

livro.indd 104 7/7/2008 16:33:36 Católica de Minas Gerais, Belo Horizonte), Luciana transwell system caused a marked increased of Caco-2 M. Silva (BioCib/FUNED, Belo Horizonte), Waleska K. cell proliferation has shown by MTT assay, not observed Martins (BioCib/FUNED, Belo Horizonte, Brazil) on fibroblasts. An increase of GAGs biosynthesis was observed in both cell lines whereas a prominent GADD45A is a growth arrest-associated and p53- increase of chondroitin sulfate (CS) was noticed mainly regulated gene which has been considered as an in stromal cells, as determined by incorporation of [S35] effective indicator of poor prognosis or malignant sulfate. Confocal mycroscopy showed an increase potential in some human tumors. The expression of of versican production by fibroblasts cells. TGF-B GADD45A is known to be regulated by the NFkB/ was also tested exhibiting a significant increased on IkB signaling pathway and by some kinase(s). GAGs synthesis only on fibroblasts cells, producing a Nevertheless, this expression might be induced by strong CS-stimulating response. These results suggest oxidative stress but the role of its activation in cell that this growth factor may be responsible for the CS death is not fully understood. In an attempt to elucidate increase observed in stromal cells. CAPES. about the GADD45A suppression in apoptosis induced [email protected] by oxidative stress, we treated RKO-AS45-1 - a human cell line that contains an antisense GADD45 B-041 expression (SMITH et al. 1995) - with Menadione COUPLING THE ENDOPLASMIC RETICULUM (MEN) and Hydrogen Peroxide (HP). These substances STRESS RESPONSE TO THE CELLULAR are known to induce oxidative stress, affecting cell RESPONSE TO CISPLATIN IN HUMAN MELANOMA proliferation and survival leading to apoptosis. The CELL LINES, A PRELIMINARY REPORT effect of MEN and HP on cellular viability was evaluated Renata de Freitas Saito, Andréia Hanada Otake, Roger by a colorimetric MTT assay. To determine the impact Chammas (Faculdade de Medicina da USP) of GADD45A suppression in HP and MEN-mediated cellular responses, RKO-AS45-1 cell line was treated Malignant melanoma is a potentially fatal skin cancer with varying doses of MEN and HP for 30 minutes at that is increasing in incidence at high rates. Although 37°C. After obtaining the IC50 dose, we performed early-stage disease is often curable, the survival assays using NFkB antibodies and the kit Live/dead rate for advanced disease is low, and it is in part due (Molecular probes) for analysis of apoptosis. According to its poor response to different chemotherapeutic to our preliminary results, the GADD45A-suppression agents. Previous data from our group indicated that might alter cell resistance to apoptosis induced by both melanoma progression and chemoresistance are oxidative stress in a dose and time-dependent MEN associated with cellular adaptation to oxidative stress. and HP treatment. Financial support: CNPq; FAPEMIG; Oxidative stress leads to endoplasmic reticulum (ER) Fundação Ezequiel Dias (FUNED). stress, thereby activating cell death mechanisms. [email protected] Melanoma cells survive despite their pro-oxidant state, as they adapt to ER stress. To better understand this B-040 phenomenon, malignant metastatic melanoma cells, MODULATION OF EXTRACELLULAR MATRIX LB373 and SKmel37, were treated with tunicamycin, PRODUCTION BY TUMOR AND STROMAL CELLS IN which inhibits protein N-glycosylation and induces CULTURE ER stress. We showed that SKmel37 cells were Ritchelli Ricci (Depto Clínica Médica-UNIFESP), more sensitive to cell death induced by tunicamycin Carolina Meloni Vicente (Depto de Bioquímica- than LB373 cells, as evaluated by the percentage of UNIFESP), Maria Aparecida da Silva Pinhal (Depto hypodiploid cells. Additionally, SKmel37 cells were Clínica Médica-UNIFESP), Jacques Weisberg (Depto also more sensitive to cisplatin-induced cell death than Clínica Médica-UNIFESP), Helena Bonciani Nader LB373 cells. Tunicamycin treatment led to decreased (Depto de Bioquímica-UNIFESP), Leny Toma (Depto overall N-glycosylation in both cell lines. The induction Clínica Médica-UNIFESP) of stress was accompanied by the increased expression of the chaperone GRP78 in SKmel37 cells, as Proteoglycans (PG) are complex macromolecules determined by Western blot experiments. Otherwise, we composed of linear polysaccharide chains, the showed no alterations in GRP78 levels in LB373 treated glycosaminoglycans (GAGs), covalently attached to cells. The systematic comparison between these two a core protein. These GAGs chains contain a sulfate cell lines may reveal aspects of the molecular basis of groups at various positions, giving rise to specific stress adaptation and chemoresistance. domains, which allows them to interact with extracellular [email protected] matrix molecules, including various growth factors. In this study, we have used a transwell coculture system B-042 to analyse the interaction between human fibroblasts BONE MARROW SUBENDOSTEAL NICHE-LIKE 3D stromal cells and human colonic carcinoma cell line CULTURE AS MODEL TO STUDY BREAST CANCER (Caco-2) and investigated the effects of this interaction INVASION on cell proliferation and GAGs synthesis. Soluble Ana Paula Dantas Nunes de Barros (Departamento components exchanged between the cell lines in the de Histologia e Embriologia-UFRJ), Leandro de Souza 1. 105

livro.indd 105 7/7/2008 16:33:36 Thiago (Departamento de Histologia e Embriologia- and remodeling of the extracellular matrix. We showed UFRJ), Christina Maeda Takiya (Departamento de that neurons remodeled glioma cell laminin and that Histologia e Embriologia-UFRJ), Mona Lisa Leal they were also able to modulate CTGF expression in (Instituto de Biofísica-UFRJ), Luciana B Chiarini glioblastoma. RT-PCR shows that Gbm cells express (Instituto de Biofísica-UFRJ), Maria Nazareth Meirelles CTGF as well as the astrocyte marker GFAP. CTGF (Fiocruz-RJ), Hélio dos Santos Dutra (Departamento immunoreactivity and mRNA levels in Gbm cells de Histologia e Embriologia-UFRJ), Radovan Borojevic are dramatically decreased when these cells are (Departamento de Histologia e Embriologia-UFRJ), co-cultured with neonatal neurons. Increasing the Maria Isabel Doria Rossi (Departamento de Histologia e number of neonatal neurons cocultured onto Gbm Embriologia-UFRJ) cells inhibit the reporter luciferase activity under control of the CTGF promoter, suggesting inhibition at the Maintenance of hematopoietic stem cells (HSC) transcription level. This inhibition seems to be contact- depends of a 3D bone marrow (BM) microenvironment mediated since conditioned medium from embryonic within the endosteal region. In order to establish a 3D in and neonatal neurons do not affect CTGF expression vitro model that mimics the human subendosteal niche, in Gbm cells. The inhibition of CTGF expression in osteo-induced BM mesenchymal stem cells (MSC) Gbm/neuronal co-cultures is not related to an inhibition were cultured as spheroid aggregates with non-induced of SMAD2/3 phosphorylation, but levels of phospho- MSC, forming mixed spheroids with segregation of p44/42 MAPK are decreased in co-cultured Gbm cells. both cell populations. Location of extracellullar matrix Previous data of laminin and these results showing proteins, and gene expression profile were investigated. that CTGF is downregulated in glioblastoma cocultured Cord blood (CB) or BM CD34+ cells migrated into with neonatal neurons point out an interesting view the spheroids reaching a plateau that was due to to understand interactions of the tumor and cerebral recirculation of these cells. After 72h they localized microenvironment. at the interface between the two cell layers. A higher [email protected] percentage of quiescent cells were observed by BrdU incorporation in mixed spheroids comparing to simple B-044 MSC ones. Therefore, this in vitro mixed mimics the SMURFS, TGFB UBIQUITINATION RELATED subendosteal niche. We propose that HSC niche also PROTEINS, ARE HIGHLY EXPRESSED IN maintain breast cancer micrometastasis. To address THYROID CARCINOMA this, invasive and non-invasive breast carcinoma SABRINA GONÇALVES RAIMUNDO, GABRIELLA cell lines, MCF7 and MDA435, respectively, were RAIMUNDO BLANCO, ANA PAULA ZEN PETISCO co-cultured with mixed and human skin fibroblast FIORE, JULIO CEZAR MARQUES RICARTE-FILHO, (hSF) spheroids. MDA435 invaded continuously CESAR SEIGI FUZIWARA, ELOIZA DE REZENDE, both spheroids. However, their migration into BM EDNA TERUKO KIMURA (INSTITUTO DE CIENCIAS spheroids were more effective. Since Wnt signaling are BIOMEDICAS, UNIVERSIDADE DE SÃO PAULO) supposed to be involved in breast cancer metastasis we transfected the cell lines with constitutively active TGFb is a potent inhibitor factor of thyroid follicular cells beta-catenin. Curiously, transfected cell lines effectively proliferation, which acts through the phosphorilation invaded BM spheroids, but their capacity to invade hSF of the Smads proteins. Smad7, Smurf1 and Smurf2 did not change. These data suggest that activation of can negatively control the TGFb pathway. Interestingly, canonic Wnt pathway are an important event in BM TGFb expression is increased in thyroid tumors. invasion by breast carcinoma cells. Thyroid benign tumors are related to the increased [email protected] releasing of Thyroid Stimulating Hormone (TSH), while malignant tumors are related to the activation B-043 of the MAPK pathway by several oncogenes such CONNECTIVE TISSUE GROWTH FACTOR (CTGF) as RET/PTC, RAS and BRAF. The aim of this study IS NEGATIVELY REGULATED DURING NEURON- was to analyze the expression of Smurfs in RET/ GLIOBLASTOMA INTERACTION PTC induced cells and in thyroid goiter and cancer. Luciana Romao, Fabio Mendes, Jane Cristina Faria, METHODS: We have grown PTC3-5 cells, PCCl3 cells Juliana M Coelho, Jorge P de Souza, Jose Garcia harboring a RET/PTC3 oncogene under a doxycycline Abreu, Vivaldo Moura Neto (UFRJ) responsive induction (DX) for 24, 48 and 72 hours. Smurf1 and Smurf2 mRNA expression were evaluated Connective-tissue growth factor (CTGF) is a by quantitative RT-PCR. Smurf1 and Smurf2 protein matricellular secreted protein involved in complex expression was analyzed by immunohistochemistry processes such as wound healing, angiogenesis, in paraffin embedded PTC tissues samples and in fibrosis and metastasis by regulating cell proliferation, rat pharmacological goiter. RESULTS: The induction migration and extracellular matrix remodeling. of RET/PTC3 oncogene showed an increased in Glioblastoma Multiform (Gbm) is the major malignant Smurf1 (24h:+64%, 48h:+76%, 72h:+85%) and Smurf2 primary brain tumor and its adaptation to the central (24h:+75%, 48h:+42%, 72h:87%) mRNA expression. nervous system microenvironment requires production Moreover, the protein analysis revealed that Smurf1 and 1. 106

livro.indd 106 7/7/2008 16:33:36 Smurf2 presented a high immunostaining in PTC, while epitope able to label these structures. Their functions fainted in normal and pharmacological goiter tissues. are still to be clarified. There is a protein in common CONCLUSION: Smurf1 and Smurf2 can be related to to the Cajal bodies and nucleolus, the fibrillarin that the impairment of TGFb inhibitory pathway and can takes part in the processing of rRNA. The Cajal bodies contribute to the PTC oncogenesis through the MAPK can be found at the periphery of the nucleoli or even signaling. physically connected to them. It is believed that the [email protected] CBs may have role in the synthesis of rRNA and maturation, transport and association of ribosome B-045 subunits. Because of these, this work aims to study the CYTOTOXIC ACTIVITY OF CISPLATIN DERIVATIVES interrelationship between these structures in normal IN NORMAL AND CANCER CELLS cells and their respective tumor cell line in culture Renata Ramalho Oliveira, Marcelo José Vilela, Marcelo before and after treatments with actinomycin D, which in Ribeiro Leite de Oliveira, Raquel dos Santos Amim low concentrations, blocks the transcription by the RNA (Universidade Federal de Viçosa) polymerase I and II and á-amanitin, which is responsible for blocking the transcription by the RNA polymerase The cytotoxic activity of chemical compounds using II. The confocal microscope enabled the study of these platin is traditionally used in cancer therapy, a disease structures in three-dimensional analysis and the AgNOR responsible for millions of deaths annually. Six enabled the study of nucleoli in the light microscopy. complexes of platina (II) with dithiocarbamate anions After treatment with inhibitors, both Cajal bodies as with the groups R: ethyl(2), phenyl(3), 4-fluorophenyl(4), nucleoli, made quite clear morphological changes, 4-iodophenyl(5), 4-clorophenyl(6) e 4-bromophenyl(7); sometimes giving large accumulation of proteins in were evaluated for their cytotoxic capacity in normal organelles and sometimes appeared disorganized in the and cancer cells “in vitro”. The cell lines MDCK nucleoplasm. Changes in the size and shape were also (normal canin), MCF-7 (cancerous human) and HeLa highlighted. (cancerous human) were used. The complexes of platin [email protected] (II) were solubilized in dimethyl sulphoxyde (DMSO) and tested at concentrations of 500, 100, 50, and B-047 10mM; and the selected were tested at concentrations A PANEL OF PEPTIDES SELECTED TO THE of 80, 60 and 40mM. Control groups: DMEM+10%FCS RECEPTOR HER2 BRING NEW INSIGHTS IN and DMEM+10%FCS+DMSO. The cisplatin, at the BREAST CANCER TREATMENT same conditions, was used as a reference. 1x105cells/ Eloah Rabello Suarez (Universidade Federal de well were seeded in 6 wells plate. After 24 hours, the São Paulo), Thérèse Rachell Theodoro (Faculdade compounds were added and remained for 24 hours. de Medicina do ABC), Carolina Martinez Bonaldi After that, 2000cells treated/well were re-seeded on (Faculdade de Medicina do ABC), Clarice Silvia Taemi coverslipes (diameter: 13mm) in 24 wells plates (well Origassa (Universidade Federal de São Paulo), Helena diameter: 16mm) in DMEM+10%FCS and incubated Bonciani Nader (Universidade Federal de São Paulo), for 144 hours. The colonies were stained with HE Maria Aparecida da Silva Pinhal (Universidade Federal and the ones with at least 50 cells were counted. The de São Paulo) compounds 4, 5 and 6 showed a cytotoxic selective effect. The compound 7 showed greater cytotoxic effect HER2 is a member of the epidermal growth than that of cisplatin, however this effect was present factor receptor family of tirosine kinases and it is both in cancer cells and in normal ones. The compound overexpressed in about 30% of breast cancers. ErbB2 4 was selected for new tests as a possible anti-tumoral, is often associated with an unfavorable prognosis. because it showed significant anti-proliferative effect in A monoclonal antibody against HER2, trastuzumab, cancer cells. is currently in use as a treatment for breast cancer; [email protected] however it can develop a high rate of cardiac failure. As an alternative to trastuzumab we have selected B-046 specific peptides to HER2 using a phage display CAJAL BODIES AND NUCLEOLI BEHAVIOR AFTER technology. A cyclic 7 aminoacids random peptide INTERFERENCE WITH INHIBITORS OF SYNTHESIS library had been panned using an external domain os OF RNA IN NORMAL AND TUMORAL CELL LINES recombinant HER2. Specific peptides were dislodged Stefania Morisco Tasca Pinheiro, Glaucia Maria and selected using trastuzumab. After each round of Machado Santelli (Universidade de São Paulo) binding assays, peptides were selected, sequenced and analyzed by ClustalW program. These peptides Nucleus is a structure that has sub-compartments were assayed using different breast cancer cell lines which can be called nuclear organelles. These nuclear in comparison with trastuzumab. It was observed that compartments are dynamic structures, maintained by one of the selected peptides (CXBBXXXXC), where C association and stock of macromolecules involved represents cysteine, X non charged aminoacids and in gene expression. The main protein found in the B positive charged aminoacids, had shown inhibitory Cajal bodies is a p-80-coilin and therefore the main proliferation effect in these breast cancer cells. This 1. 107

livro.indd 107 7/7/2008 16:33:36 peptide showed the highest effect, about 30%, over Priscila Afonso de Faria, Iseli Lourenço Nantes, Tiago MCF-7 cells. Immunocytochemistry analysis of HER2 Rodrigues (Universidade de Mogi das Cruzes) expression at all tested cells confirmed the obtained responses with selected peptides and trastuzumab Phenothiazines (PTZ) are anti-schizophrenic drugs, in cells with high levels of this receptor. In addition, but recent studies showed that they exhibit also pro- confocal microscopy confirmed co-localizatoin of HER2 apoptotic and potential antitumoral activity. In this study, and selected peptides. The data suggest a potential use we investigated the effects of five structure-related of these peptides as an anti-tumoral therapy for breast PTZ derivatives chlorpromazine (CPZ), fluphenazine cancer treatment. (FP), thioridazine (TR), triflupromazine (TFPZ), and [email protected] trifluoperazine (TFP) on the viability of HTC (hepatoma, tissue culture) cells correlated with their effects on B-048 mitochondrial function. All PTZ tested were able to PHENOTHIAZINE-INDUCED CELL DEATH decrease the viability of HTC cells in a concentration- INVOLVES LYSOSOMAL AND MITOCHONDRIAL and incubation time-dependent manner. For all probed PERMEABILIZATION IN HUMAN LEUKEMIC K562 drugs, the minimal IC50 values, from 37 to 67 µM, were CELLS obtained with 24 h incubation. According to the drug Vivian Matsukura dos Santos (Universidade de Mogi structure, increasing cytotoxicity was observed in the das Cruzes (UMC), Fabio D. Nascimento (Universidade following order: TR > TFP > FP > CPZ > TFPZ. The Federal de São Paulo (UNIFESP), Cláudia B. Trindade decrease of the incubation time to 6 and 12 h increased (Universidade de Mogi das Cruzes (UMC), Tiago the IC50 values of TR, TFP and FP up to 80 µM while Rodrigues (Universidade de Mogi das Cruzes (UMC), led CPZ and TFPZ to become unable to decrease Ivarne Luis dos the cell viability to values > 50%. Similarly to that was Santos Tersariol (Universidade de Mogi das Cruzes previously observed for isolated rat liver mitochondria, (UMC) the addition of 40 µM PTZ to suspensions of digitonin- treated cells resulted in immediate dissipation of the Phenothiazines (PTZ) are drugs widely used in the mitochondrial transmembrane potential whose intensity treatment of schizophrenia. Recent studies have varied according to the drug structure in the same order demonstrated the pro-apoptotic activity of these drugs observed in the viability assays. However, to elucidate however their mechanisms of action are not clarified. the mechanism responsible for the antitumoral activity The presence of hydrophobic and proton trapping of PTZ, it is necessary to conduct further studies basic amino groups in the structure of PTZ indicates establishing the role played by mitochondria in the that they may display lysosomotropic properties. HTC cell death induced by these drugs. Supported by In this work we investigate the involvement of the FAPESP, CNPq and FAEP-UMC. lysosomal and mitochondrial permeabilization in the [email protected] PTZ- induced cell death. The incubation of leukemic K562 cells with thioridazine (TR), trifluoperazine (TFP) B-050 and fluphenazine (FP) for 24h promoted a decrease ANTITUMORAL ACTIVITY OF THE LECTIN KM+ TO of the cell viability assessed by MTT method, with THE GROWTH AND ESTABLISHMENT OF WALKER the IC50 values ranging from 15 to 50 µM depending CARCINOMA on the structure of the phenothiazine derivative. This Adriana Cristina Baréa (Pós-Graduação em Ciências cytotoxic effect was partially prevented by the pre- da Saúde - PUCPR), Michelle Simone Herdoiza Baran incubation of cells with E-64, a specific irreversible (PUC-PR), Marcieli Elisa Pereira Razera (PUC-PR), inhibitor of cysteine cathepsin. In parallel, PTZ disrupt Andréa Novais Moreno (Pós-Graduação em Ciências the mitochondrial transmembrane potential, assessed da Saúde - PUC-PR) with rhodamine 123, and the pre-incubation with E-64 also prevented this effect. It was not observed lipid INTRODUCTION: KM+, a lectin that binds to oxidation induced by PTZ, suggesting that free radicals D-mannose, induces macrophages activation with may not be involved in the organelles permeabilization. release of IL-12, which is related with antitumoral These results indicate that PTZ are able to induce a activity. OBJECTIVES: Evaluate the antitumoral cathepsin-dependent cell death in human leukemic activation induced by the lectin KM+ against K562 with involvement of the lysosomal membrane the Walker tumor cells. METHODS: The in vivo permeabilization upstream to the dissipation of antitumoral activity was evaluated using the Walker mitochondrial transmembrane potential, evidencing the carcinoma. Intraperitoneal administration of KM+ chemotherapeutic potential of these drugs. (50 mg/mL) or PBS (negative control), began 24 h [email protected] after tumor cell inoculation. The antitumoral activity was assessed 7, 14 and 21 days after treatment B-049 by measuring the tumor mass; observing the cell PHENOTHIAZINE DERIVATIVES INDUCE CELL infiltration by immunohistochemistry; analyzing the DEATH IN HTC HEPATOMA CELLS ASSOCIATED TO nitrite oxide production by peritoneal cells obtained MITOCHONDRIAL DYSFUNCTION from the experimental animals. RESULTS: The 1. 108

livro.indd 108 7/7/2008 16:33:36 results demonstrated that KM+ inhibits the tumoral B-052 growth when compared with the control animals. The CYTOTOXICITY OF LECTINS IN BREAST CANCER characterization of the migrating cells into the tumor CELLS: INFLUENCE OF FETAL BOVINE SERUM revealed the presence of macrophages, NK cells, T CONCENTRATION helper cell and cytotoxic T cell in animals treated with Glaucia Veríssimo Faheina (LTF-UFPB), Bruno KM+. The lectin increases the ability of the peritoneal Chaussê de Freitas (LTF-UFPB), Alethéia Lacerda cells to produce NO. CONCLUSION: Together, these da Silveira (LTF-UFPB), Márcio Viana Ramos (UFC), results suggested that the antitumoral activity of KM+ Demetrius Antônio Machado de Araújo (LTF-UFPB) on Walker tumor cells is due to the efficient immune response activation. Supported by CAPES; CNPq and Plant lectins have been related to antitumoral activity. In PUC-PR. this study, we analyzed the cytotoxicity of lectins from [email protected] Canavalia ensiformis (ConA), Canavalia brasiliensis (Conbr) and Cratylia floribunda (CFL), using MCF-7 B-051 human breast cancer cell line. The interaction between PROTEIN PROFILING OF T98G AND U87MG HUMAN these lectins (in native form or denatured by heat) and MALIGNANT GLIOMA CELL LINES BY PROTEOMIC different fetal bovine serum (FBS) concentrations used APPROACH in culture medium was evaluated. The MCF-7 cells were Anelisa Ramão (Centro de Química de Proteínas e grown for 24 h and then incubated with each lectin at Depto de Biologia Celular e Molecular - FMRP - USP), concentrations which ranged from 10 to 400 µg/ml using Marcela Gimenez (Centro de Química de Proteínas e RPMI medium supplemented with 1% or 20% FBS. In Depto de Biologia Celular e Molecular - FMRP - USP), 1% FBS cultures, the values of IC50 were 62, 46 and Maristela Delgado Orellana (Centro Terapia Celular – 86 µg/ml for ConA, Conbr and CFL, respectively. We Centro Regional de Hemoterapia de Ribeirão Preto), did not observe any cytotoxic effect in the 20% FBS Sueli Oba-Shinjo (Faculdade de Medicina de São Paulo cultures. There was also no cytotoxic activity of ConA, – USP), Suely Kazue Nagahashi Marie (Faculdade Conbr and CFL when these proteins were denatured de Medicina de São Paulo – USP), José César Rosa for 1 hour at 100°C, indicating that three-dimensional (Centro de Química de Proteínas e Depto de Biologia structure has fundamental importance in the activity of Celular e Molecular - FMRP - USP) these lectins. We conclude that high FBS concentration produced inhibition of the cytotoxic activities of the Glioblastoma is the deadliest brain tumor that remains lectins, suggesting that FBS contains glycoproteins or incurable. T98G and U87MG are two human malignant glycolipids which interact with these lectins. FINANCIAL cell lines derived from glioblastoma multiforme (GBM). SUPPORT: CNPq and CAPES. U87MG cells (p53 wild-type) are tumorigenic in nude [email protected] mice while T98G cells (p53 mutant) are not. However, both cell lines have infinite life span in vitro and B-053 their characteristics remain the same after so many A STUDY OF GALECTIN-3 AND ITS LIGANDS IN A doublings. Proliferation rate of T98G and U87MG cell HYBRID GLIOMA CELL LINE, NG97HT lines was measured and it showed little difference Rafael Yamashita Ikemori (UNICAMP - Dept of between them until 96hs. After this time, U87MG cells Immunology), Camila Maria Longo Machado (UNICAMP continued growing exponentially, but T98G did not. In - Dept of Immunology), Roger Chammas (FMUSP - order to identify differential protein expression between Dept of Experimental Oncology LIM24), Liana Maria T98G and U87MG during proliferation, we harvested Cardoso Verinaud (UNICAMP - Dept of Immunology) the cells when they reached 90% confluence and prepared protein extract to submit to 2D gel. Gels were Galectin-3 belongs to a family of proteins that share stained, image captured and analysed by ImageMaster a common carbohydrate recognition domain and is 2D Platinum v.5.0 software. Selected 2D protein spots associated with different cellular aspects of cancer were hydrolysed by trypsin and proteins were identified progression, like angiogenesis, cell survival and by mass spectrometry (ESI-CID-MS/MS). The number motility, as well as cell death depending where of spots on 2D gel was 355 for U87MG and 323 for galectin-3 is localized. Recent studies have shown T98G. We detected that 189 spots were matched that galectin-3 is expressed in vivo in specialized and 15 protein spots were differentially expressed. tumor tissue compartments, such as the perinecrotic We identified 10 spots that were super-expressed pseudopalisades commonly found in glioblastomas, in U87MG compared to T98G. Seven proteins were which are supposedly formed by cells migrating identified: nucleophosmin, tubulin, annexin A5, galectin from a necrotic/hypoxic environment towards blood 1, lactate dehydrogenase B chain, protein dissulfide vessels. In these experiments we investigated the isomerase and tropomyosin. Most of them have production of galectin-3 and its ligands both in vitro and already been implicated in cancer development and in vivo using a hybrid human/mouse glioma cell line, will be discussed about them in the light of glioma NG97ht. Galectin-3 was assessed with the M3/38 rat development. Financial support: CTC-CEPID-FAPESP, monoclonal antibody and its ligands by the galectin-3 CNPq and FAEPA. - [email protected] conjugated with alkaline phosphatase in vitro and in 1. 109

livro.indd 109 7/7/2008 16:33:36 vivo in tumoral masses derived from the inoculation Prudente) of NG97ht cells in the flank of nude mice. Western blot results showed a distribution of galectin-3 in the Galectin-3 (Gal3) is a pleiotropic protein nucleus and in the cytoplasm, but cytochemistry and expressed in a variety of tissues. It has diverse western blots demonstrated that its ligands are present activities that are contingent upon its subcellular in the cytoplasm and membrane. In vivo, galectin-3 localization. Previous studies have demonstrated expression in the perinecrotic pseudopalides but that Gal3 expression and subcellular distribution is not in the rest of the tumor tissue implies a silencing correlated to melanoma progression. Here, the effects mechanism that needs to be more explored and of Gal3 depletion were investigated in melanoma cells gene promoter methylation is a promising alternative. derived from different stages of tumor progression Also, galectin-3 ligands are expressed in some cells using lentiviral shRNA. Gal3-deficient (shGal3) vertical within the pseudopalisade, which may sustain the growth phase (VGP) melanoma cells displayed altered promigratory activity ascribed to galectin-3. Financial cell morphology and decreased rates of proliferation. support: CNPq. These cells also showed impaired growth in both [email protected] serum-reduced conditions and soft agar, as well as reduced invasiveness. Impaired Akt activation and up B-054 regulation of pro-apoptotic proteins were detected. ARE CHRYSOTILE FIBERS ASSOCIATED WITH Immunofluorescence analyses demonstrated that Gal3 ANEUPLOIDY? and β-catenin co-localize, but Gal3 knockdown caused Beatriz de Araujo Cortez (Instituto de Biociências, decreased nuclear β-catenin localization, suggesting Universidade de São Paulo), Glaucia Maria Machado a possible role for nuclear β-catenin in malignant Santelli (Instituto de Ciências Biomédicas, Universidade phenotype. Interestingly, shGal3 caused no phenotypic de São Paulo) changes in metastatic melanoma cells. Together, these data demonstrate that Gal3 has a crucial role in Chrysotile is considered less harmful to human health VGP melanoma. Furthermore, Gal3 depletion is not than other types of asbestos fibers. Its clearance from sufficient to affect metastatic cells, which likely develop the lung is faster and, in comparison to amphibole forms complementary survival strategies to escape tumor of asbestos, chrysotile asbestos fail to accumulate suppressive mechanisms. in the lung tissue due to a mechanism involving [email protected] fibers fragmentation in short pieces. Short exposure to chrysotile has not been associated with any B-056 histopathological alteration of lung tissue. In this work ALTERNAGIN-C, AN ECD-DISINTEGRIN FROM chrysotile could be visualized by its autofluorescence BOTHROPS ALTERNATUS, INHIBITS THE in laser scanning microscopy, and using this technique ADHESION OF BREAST ADENOCARCINOMA were found fibers and short pieces of chrysotile inside CELLS TO THE VASCULAR human lung cells in culture after 48h of chrysotile SUBENDOTHELIUM UNDER STATIC AND exposure. Short pieces were found also inside the DYNAMIC FLOW CONDITIONS nucleus, evidenced by immunofluorescence with Juliana Uema Ribeiro (Universidade Federal de São anti-laminin antibody. Long fibers appear to interfere Carlos), Ibtissem Samira Djaafri (U553, INSERM), on mitosis, and this could be one of the mechanisms Françoise Fauvel-Lafève (U553, INSERM), Michel of aneuploid cells formation. The present study aimed Crépin (U553, INSERM), Heloísa Sobreiro Selistre de to verify the DNA ploidy changes in chrysotile treated Araújo (Universidade Federal de São Carlos) cells. HK2 cell line (NSCL) was treated with 3 chrysotile concentration during 48h and then recovered in fiber Tumor cell adhesion to the subendothelial matrix is free-medium for 24h, 48h and 72h. Data of DNA content an important step of metastatic progression and it is quantified by image analysis showed that aneuploid enhanced by platelets. Alternagin-C (ALT-C), an ECD- cells frequencies increased progressively from 0.2 to disintegrin, is a potent inhibitor of binding of tumor 10%, in dose –dependent way. Longer times of recovery cells to collagen under static conditions by interactions in fiber-free medium (4 and 7 days) will indicate if the with their integrins receptors. This work analyzed the aneuploid cell population persists in cell culture or not. effects of ALT-C on the adhesion of breast cancer cells [email protected] MDA-MB-231 to the subendothelial matrix produced by HUVEC under static and dynamic conditions. Under B-055 static conditions, tumoral cells were incubated with GALECTIN-3 SUPPORTS THE MALIGNANT ALT-C (1 to 10000nM) and seeded in 96 well plates PHENOTYPE OF VERTICAL GROWTH PHASE coated with subendothelial matrix. Adherent cells were MELANOMAS fixed with 4% PFA, stained with 0.5% crystal violet, Patricia Abrão Possik (Fundação Antônio Prudente), dissolved in 1% SDS and spectrophotometrically Susan Zabierowski (The Wistar Institute), John T Lee measured at 540 nm. For the flow assay, cell tracker (The Wistar Institute), Meenhard Herlyn (The Wistar red-labeled MDA-MB-231 cells were incubated with Institute), Luiz Fernando Lima Reis (Fundação Antônio ALT-C (10 to 1000nM) or PBS (control) and added to 1. 110

livro.indd 110 7/7/2008 16:33:36 whole blood with green calcein-labeled platelets. Blood CHANGED BY DHA BUT NOT EPA was perfused in a flow chamber with matrix on glass Graziela Rosa Ravacci, Tarcisio Tortelli, Adriana slides at 50 sec-1. The adhesion of tumor cells and Priscila Trapé, Fellipe Santos Bronze, Raquel Suzana platelets was estimated using the Image J software. Torrinhas, Maria Mitzi Brentani, Dan Linetzky Waitzberg Only 10000nM ALT-C inhibited tumoral cell adhesion (Faculdade de Medicina da USP) to the HUVEC matrix under static conditions. Under flow, tumoral and platelet adhesion were inhibited by Here, we investigate the effects of DHA and EPA on the 10nM ALT-C, about 40% and 60%, respectively, 100nM cell death and cell cycle of different human mammary ALT-C increased cell adhesion but at 1000nM ALT-C, epithelial cells (HB4a, HB4aC5.2 and MDA-MB-231) cell adhesion was completely inhibited. The activities and verify their action mechanism. These cell lines of ALT-C may be helpful for the study of breast cancer were treated for 72 hours with EPA or DHA at 100µM metastasis as well as in the design of new therapeutic and DHA + EPA (50µM+50µM) or ethanol. The specific agent targeting tumor cell extravasation and invasion. effect on cell cycle phases and apoptosis was studied FAPESP, CNPq. by Flow Cytometry. These cell lines were stained with [email protected] Alexa Fluor conjugated with cholera toxin B subunit to lipid raft visualization by Fluorescence and Confocal B-057 Microscope Imaging. Statistical analysis was performed ANTIPROLIFERATIVE EFFECT OF SODIUM using the Oneway Anova and Bonferroni post-test. BUTYRATE IN NON-SMALL CELL LUNG CANCER DHA induced cell death and apoptosis with a higher CELL LINES percentage in HB4aC5.2 than HB4a and MDA-MB Paula Pezzuto, Nívea Dias Amoedo, Ana Lúcia Moraes 231 (p<0.001, respectively). In addition, DHA also Giannini, Franklin David Rumjanek delayed HB4aC5.2 progression in the cell cycle with (Universidade Federal do Rio de Janeiro) the majority of cells into G1 phase (30%) against 6.5% into G2/M phase (p<0.001). However, when DHA Histone deacetylases are enzymes that promote was supplemented with EPA the observed effects modifications of chromatin structure causing and decreased. This combination only induced cell death in thereby regulate several genes involved in the cell HB4aC5.2 (p<0.001). In contrast, there was no effect cycle, survival, differentiation and apoptosis. In on cell death and cell cycle phases when the cell lines the present work, we have analyzed the effects of were supplemented only with EPA. HB4aC5.2 cell line Sodium Butyrate(Nab) and Trichostatin A(TSA), two show better lipid rafts stained and lipid rafts grouping histone deacetylase inhibitors(HDACi), that affect cell increase. Furthermore, DHA treatment promoted lipid proliferation and morphology in two different non-small rafts composition alterated or destructiveness only cell lung cancer, A549 and H460 cell lines. These in HB4aC5.2. DHA treatment promote apoptosis and cell lines were treated with Nab and TSA in different arrest cell cycle in HB4aC5.2 by lipid rafts composition concentrations for 24, 48 or 72 hours. At the end of alterated or destructiveness. Our results indicate each time point, cell proliferation and viability were that DHA modify the lipid rafts structure in a way that measured. We also analyzed apoptosis by AnexinV decreases the growth of breast tumors. staining and TUNEL assay. Our results show that Nab [email protected] and TSA produce a time and dose dependent negative effect on growth of both cell types. Only a small B-059 number of apoptotic cells could be observed after Nab IMMUNOHISTOCHEMICAL LOCALIZATION OF treatment. Flow cytometry analysis showed a decrease CYCLOOXIGENASE-2, TUMOR-ASSOCIATED of cells in S phase. The reduction in S phase cells MACROPHAGES, MICROVASCULAR resulted in change in G1 and G2/M phase cells, when DENSITY AND KI-67 IN ORAL SQUAMOUS CELL treated with Nab and TSA. Morphological changes CARCINOMA and cytoskeletal actin rearrangement, evaluated Deise Souza Vilas Boas (Dept of Histology and staining with phalloidin, were detected only in H460 Embryology, Instituto de Ciências Biomédicas, UFRJ), cell line suggesting induction of cell differentiation. To Jean Nunes dos Santos (Laboratory of Oral Surgical understand the mechanisms of Nab action, we have Pathology, Faculdade de Odontologia, UFBA), performed immunoblotting and real time PCR analyses Tatiana Lobo Coelho de Sampaio (Dept of Histology to access the levels and activation state of proteins and Embryology, Instituto de Ciências Biomédicas, of and Epidermal Growth Factor Receptor(EGFR) UFRJ), Eduardo Antônio Gonçalves Ramos (Centro signal transduction pathway. We have observed that de Pesquisa Gonçalo Moniz (Fiocruz-BA), Márcia Nab and TSA cause an up-regulation of EGFR at the Grillo Cabral (Dept of Pathology and Oral Diagnostic, transcriptional level. CNPq, FAF-ONCO, FAPERJ. Faculdade de Odontologia, UFRJ), Leonardo Campos [email protected] Monção Ribeiro (Dept of Histology and Embryology, Instituto de Ciências Biomédicas, UFRJ), Clarissa B-058 Araújo Silva Gurgel (Centro de Pesquisa Gonçalo APOPTOSIS AND PROLIFERATION OF HUMAN Moniz (Fiocruz-BA), Christina Maeda Takiya (Dept CANCER MAMMARY EPITHELIAL CELLS ARE of Histology and Embryology, Instituto de Ciências 1. 111

livro.indd 111 7/7/2008 16:33:37 Biomédicas, UFRJ, Brazil) phenotype upon 4OHT treatment. 40 clonal HaCat/ ER-RasV12 sublines were selected with G418, isolated Cyclooxygenase-2 (COX-2), tumor-associated and screened for response to 4OHT and/or FGF2. In macrophages (TAMs) and angiogenesis are important clonogenic assays, 6 HaCat/ER-RasV12 sublines grew components in tumor growth, including the oral well with 4OHT or FGF2, but did not develop colonies squamous cell carcinoma (OSCC). The aim of this in the presence of both 4OHT and FGF2. These results study was to detect by immunohistochemistry the suggest that FGF2 is cytotoxic for human keratinocytes expression of COX-2 and Ki-67, and the presence of transformed by the rasV12 oncogene even in the TAMs and tumor microvessels in OSCC exhibiting absence of a functional p53 pathway. Supported by histological grading in accordance with the World Health FAPESP, CNPq and CAPES. Organization. The study was carried out in 23 cases of [email protected] OSCC and 4 cases of “in situ” carcinoma (Dept Oral Pathology, UFBA, Brazil). Immunohistochemistry was B-061 performed using formalin-fixed and paraffin embedded GENOTOXIC RISK TO WORKERS EXPOSED TO tissue sections. COX-2 was expressed by epithelial, PETROLEUM DERIVATIVES: endothelial and stromal cells in OSCC . Chi-square BIOMONITORING EXFOLIATED BUCCAL analysis indicated no significant association between EPITHELIAL CELLS epithelial COX-2 histoscore (intensity and proportion) Bráulio Fernandes de Carvalho (Universidade Federal and histological degree of malignancy (p=0.89). do Piauí), Paula Andrea Sampaio dos Vasconcelos ANOVA showed that proliferative index determined (Universidade Federal do Piauí), Hamanda Soares by Ki-67 was associated with the histological degree (Universidade Federal do Piauí), Renara Morais of malignancy (p<0.01). Microvascular density (MVD) (Universidade Federal do Piauí), Aracelli de Sousa assessed by Factor- VIII labeling was statistically Leite (Universidade Luterana do Brasil), Dorival signifcant between OSCCs exhibiting different degrees Mendes Rodrigues Junior (Universidade Federal of malignancy (p<0.05). TAMs were identified by do Piauí), Sergio Ewerton Meneses dos Santos CD68 immunodetection and were found with a high (Universidade Federal do Piauí), Lhais Suelen Soares density in both OSCC and “in situ” carcinoma (p=0.51). Leal (Universidade Federal do Piauí), Sandra Maria de In conclusion, Ki-67 and MVD are accordance with Mendes Moura Dantas (Universidade Federal do Piauí), the histological degree of malignancy of OSCC, and Ana Amélia de C. Melo Cavalcante (Universidade might be important as prognostic factors. COX-2 Federal do Piauí) overexpression in OSCC is independent of the histological degree of the lesion. Furthermore, it is There is evidence of a relationship between possible that COX-2 and TAMs may be involved in micronucleus induction and cancer onset. This study diverse stages of oral carcinogenesis. was carried out with a group of workers (20) in a filling- [email protected] station in Teresina (PI, Brazil) and a control matched- group (20) that had smoking habit and consumed B-060 alcohol. Our aim was to investigate the genotoxic risk INDUCTION OF THE H-RASV12 ONCOGENE of occupational exposure to petroleum derivatives by RENDERS THE HUMAN KERATINOCYTE CELL LINE analyzing the micronuclei (MN) frequency and other HACAT SENSITIVE TO FGF-2 nuclear abnormalities as binucleus (BN), karyorrhexis CYTOTOXICITY (KR) and karyolysis (KL). Two thousand cells per data Julianna Dias Zeidler, Hugo Aguirre Armelin point were analyzed for the frequency of MN per 1.000 (Departamento de Bioquímica, Instituto de Química, cells. Slides were stained with Giemsa. MN frequencies Universidade de São Paulo) were higher in workers exposed (2.8 ± 2.1) in relation to control group (0.2 ± 0.4). The mean values for BN, KR We recently reported that exogenous recombinant and KL in workers were 0.8 ± 0.3; 2.0 ± 1.4; 3.8 ± 0.8, FGF2 (Fibroblast Growth Factor 2) irreversibly inhibits whereas in the control group we found 0.4 ± 0.8; 0.2 proliferation by inducing senescence in Ras-dependent ± 0.7; 1.8 ± 1.2, respectively. All parameters obtained mouse malignant cells, but not in immortalized non- from workers were statistically different (P<0.001) from tumorigenic cell lines. To further analyze this FGF2 the control group. No significance was detected when stress response we are using the immortalized human smoking habit was considered, however differences keratinocyte cell line HaCaT, which is not tumorigenic were observed in relation to alcohol consumption. but displays a functionally impaired p53 pathway. We Results may be correlated with the different composition infected HaCaT cells with a viral vector carrying a of petroleum derivates. Therefore, we suggest that cDNA construction which encodes the fusion protein long-term exposure to petroleum derivatives might be ER-RasV12 (estrogen receptor binding domain fused to genotoxic to buccal epithelial cells due to DNA damage. the constitutively activated RasV12 oncogene) plus the [email protected] neomycin marker gene. ER-RasV12 protein is activated by 4OHT (4-hidroxitamoxifen, an estrogen analog) and B-062 HaCaT/ER-RasV12 cells could exhibit a malignant PREVALENCE OF HPV INFECTION AND 1. 112

livro.indd 112 7/7/2008 16:33:37 GENOTYPING OF CERVICAL SCRAPES approach to the rapid analysis of sequence variation OBTAINED FROM ASYMPTOMATIC WOMEN in gene-sized DNA fragments. LSSP-PCR has already Soraya Lobato Chamon (ICB/UFMG), Clarice Marcolino been used to genetically typing some HPV genotypes (EE/UFMG), Dora Mendez del Castillo (NUPAD/UFMG), directly from cloned genomes of the viruses. The Paula Ávila Fernandes (FAFAR/UFMG), Annamaria main aim of this study was to investigate the utility of Ravara Vago (ICB/UFMG) LSSP-PCR in the molecular typing of HPV directly from human cervical samples. As previously described, Human papillomavirus (HPV) infection has LSSP-PCR was performed on the 450 bp DNA been identified as a major risk factor for cervical fragments of the L1 region of HPV genome, which were precancerous lesions (CPL) and invasive cervical previously PCR-amplified from the cervical scrapes with cancer (CC). Prophylactic vaccines against HPV are the set of primers MY09/MY11. By using MY11 primer currently underway, and one of them was recently in the LSSP-PCR reaction, we were able to obtain very approved by the FDA – USA. The knowledge of HPV informative and reproducible gene signatures specific types incidence in a target population to be vaccinated to each HPV analyzed type, which allowed us the is the first step to achieve the design of an appropriate identification of the low-risk (HPV 6) and high-risk HPV and effective HPV vaccine. In order to investigate the (16, 18, 31, 33, 52 e 58), directly to human cervical HPV prevalence and HR types (16, 18, 31, 33, 35, 52 samples. Thus, the LSSP-PCR technique represents and 58) in women with normal cervical cytology, which a useful and simple molecular approach to genetically would represent a fraction of the normal population typing HPV directly from biological samples. women, we PCR-analyzed 94 cervical-scrape samples [email protected] obtained from asymptomatic women attended at a Public Service of Health from Minas Gerais State. By B-064 using the generic (MY09/MY11; GP5/GP6+) and type- THE GRP-R ANTAGONIST, RC-3095, ELICITS A specific primers, we observed an overall frequency of PROLIFERATIVE EFFECT IN NEURO-2A CELLS HPV-DNA of 57.4% in the population under study. In our WHICH IS REVERTED BY TREATMENT WITH analysis, HPV16 was the most prevalent type (38.3%) SODIUM BUTYRATE followed by HPV18 (30.8%). Concerning other high- Caroline Brunetto de Farias (Instituto do Câncer risk HPV types analyzed, we observed a prevalence Infantil do RS), Viviane Rösner de Almeida (Pontifícia of 9.6% for HPV52, of 5.3% for HPV31 and 35, and Universidade Católica do Rio Grande do Sul), Gilberto of 1.06% for HPV33 and 58. Our results show a high Schwartsmann (Fundação SOAD), Algemir Lunardi prevalence of HPV infection in patients with normal Brunetto (Instituto do Câncer Infantil do RS), Ana Lucia cytology, indicating that those women group should Abujamra (Instituto do Câncer Infantil do RS), Rafael be followed up more frequently than the HPV negative Roesler (Universidade Federal do Rio Grande do Sul) cases in order to prevent a persistent infection. Besides, a relevant presence of high-risk types among the Introduction: Neuroblastoma is the most common asymptomatic women suggests that the development extracranial solid tumor in infants and children, of effective vaccines should considerer the main HPV accounting for more than 8 – 10% of all childhood types co-habiting in distinct geographic regions. cancers. Gastrin releasing peptide (GRP) acts as a [email protected] potent mitogen for neoplastic tissues by activating its receptor (GRP-R). The selective inhibitor of GRP-R, B-063 RC-3095, has been widely studied as a potential LOW-STRINGENCY SINGLE SPECIFIC PRIMER-PCR antitumor agent because of its anti-proliferative (LSSP-PCR) TECHNIQUE FOR GENOTYPING OF activities in prostate, lung and colorectal cancer HPV DIRECTLY FROM cell lines, but has not yet been evaluated with other HUMAN SAMPLES adjuvants for chemotherapy. Sodium butyrate (NaB), a Soraya Lobato Chamon (ICB/UFMG), Clarice Marcolino potent histone deacetylase inhibitor, induces cell cycle (EE/UFMG), Dora Mendez del Castillo (NUPAD/UFMG), arrest and apoptosis in malignant cells. We proposed to Nara de Oliveira Carvalho (NUPAD/UFMG), Annamaria study the effects of RC-3095 alone or with NaB in the Ravara Vago (ICB/UFMG) murine neuroblastoma cell line, Neuro-2A. Methods: Neuro-2A cells were seeded in 24- or 96-well plates and Human papillomaviruses (HPV) are responsible for treated with sodium butyrate (1mM), RC-3095 (100nM, both benign warts and malignant lesions, particularly 10nM, 1nM, 0.1nM), or a combination of both for 48h. of the anogenital tract. Presently, more than 100 HPV At this point cells were submitted to the trypan blue genotypes have been identified, and about 20 of them exclusion test or MTT assay in order to evaluate cellular have been strongly implicated in the development of proliferation and viability. Conclusion: RC-3095 elicits anogenital cancer. HPV 16 and 18 types are considered a proliferative effect at a final concentration of 100nM to be high risk for cervical carcinogenesis. The most in Neuro-2A cells. Cotreatment of 100nM RC-3095 and rigorous mean of identifying HPVs once detected is by 1mM sodium butyrate prevented this effect. sequencing of PCR products. Low-stringency Single [email protected] Specific Primer-PCR (LSSP-PCR) technique is an 1. 113

livro.indd 113 7/7/2008 16:33:37 B-065 of Sherbrooke, University of Sherbrooke), Bruno THE COTREATMENT BETWEEN RC-3095 AND Battistini (Institute of Pharmacology of Sherbrooke, CHEMOTHERAPICS: A NEW ANTITUMORAL University of Sherbrooke), Pierre Sirois (Institute STRATEGYE TO COLORECTAL CANCER IN VITRO of Pharmacology of Sherbrooke, University of Caroline Brunetto de Farias (Instituto do Câncer Sherbrooke), Roger Chammas (Faculdade de Medicina Infantil), Rodrigo Cruz de Lima (Universidade Federal da USP) do Rio Grande do Sul), Tiago Elias Heinen (Fundação Faculdade Federal de Ciências Médicas de Porto Tumor microenvironment has been considered an Alegre), Laura Roesler Nery (Pontifícia universidade important target for anticancer therapies. In a previous Católica do Rio Grande do Sul), André Lubian report, we had shown a delay in melanoma engraftment (Universidade Luterana do Brasil), Ana Lúcia Abujamra in bradykinin receptor 1 (B1R) knockout mice. Here we (Instituto do Câncer Infantil do RS), Algemir Lunardi investigated the effects of the B1R antagonist R-954 Brunetto (Instituto do CÂncer Infantil do RS), Gilberto and its combination with the chemotherapeutic agent Schwartsmann (Fundação SOAD), Rafael Roesler dacarbazine (DTIC) in melanoma. B16-F10 melanoma (Universidade Federal do Rio Grande do Sul) cells were injected subcutaneously in C57bl-6 mice, which were treated with DTIC (2mg/kg) every 3 days, The human gastrin-releasing peptide receptor appears R-954 (1mg/kg) daily, DTIC combined with R-954 or to be involved in the growth of many types of cancers. PBS, as control. Animals were sacrificed at day 12 GRPR is aberrantly expressed in colorectal neoplasms of treatment, tumors were excised and processed and mediates signals of cellular proliferation. Colorectal for both histological analysis and RNA extraction. cancers are major international health problems and Treatments with R-954, DTIC or R-954 plus DTIC did result in more than a million deaths worldwide each not reduced tumor mass. However, these treatments year. The therapies for these malignancies must caused significant decrease in microvascular density. be improved. Conventional chemotherapy has not Microarray experiments were performed using produced dramatic improvements in response rates GeneChip Mouse 430 (Affymetrix) according to or patient survival, and new treatment modalities manufacturer’s protocols. Data was normalized using are urgently needed. GRPR has emerged as a new the RMA method. Two different methods were employed molecular target for the development of new anticancer for selection of differentially expressed genes (DEGs): drugs. In this study, we evaluated the antitumoral effects the Student’s T-test and the Limma package. A total of the synergism between chemotherapeutic agents and of 322 different DEGs were common to both analysis. RC-3095, a GRP antagonist, in cancer colorectal human Some interesting genes identified, with fold change cell lines. HT-29 and SW620 cell lines were seeded at ¡Ý2, are involved in immune response (CD24a, CXCL3, 7x10³ cells per well in RPMI-supplemented medium. CXCR-7), chemoresistance (transglutaminase-2, HSP- After 24h, the cells were incubated with RC-3095 (10–9 1b) and angiogenesis (neuropilin-2, Serpine 1). These ìM to 1ìM) alone or in addition to irinotecan (1nM) or results point to signatures that may synthesize the to cisplatin (1nM) and cell viability was measured after intricate interactions of tumor and host cells, leading to 48h by MTT assay. Moreover, we analyzed, using a tumor response to chemotherapeutic agents and will be reverse-transcription-polymerase-chain-reaction (RT- useful for understanding treatment failure. PCR) technique and by immunohistochemistry, the [email protected] presence of GRPR in those cell lines. Our preliminaries results shows that RC-3095 inhibits the proliferation of B-067 HT-29 and SW620 human colorectal cancer cell lines GENES UP AND DOWN REGULATED BY and that the effect was improved in addition to the DERMCIDIN IN BREAST CANCER: A MICROARRAY chemotherapics in vitro. Data are shown as mean+SEM ANALYSIS of 3 different experiments performed in sextuple wells Dayson Friaça Moreira (Department of Pharmacology, each and differences between mean values were Institute of Biomedical Sciences, University of São evaluated by ANOVA test. Paulo, SP), Bryan Eric Strauss (Laboratory of Genetics [email protected] and Molecular Cardiology/LIM-13, School of Medicine, University of São Paulo), Eduard Vannier (Tufts B-066 University-New England Medical Center, Boston, TUMOR RESPONSES TO CHEMOTHERAPY: MA, USA), José Ernesto Belizário (Department of SEARCHING FOR GENE EXPRESSION Pharmacology, Institute of Biomedical Sciences, SIGNATURES THAT MAY EXPLAIN WHY University of São Paulo, SP, Brazil) TREATMENT OFTEN FAILS Patrícia Luiza Nunes da Costa (Faculdade de Dermcidin (DCD) is a human gene mapped to Medicina da USP), Fernando José Bigaton (Faculdade chromosome 12q13 region that is co-amplified with de Medicina da USP), Daniele Yumi Sunaga various oncogenes with well established role on breast (Departamento de Genética e Biologia Evolutiva do cancer. We will show a DNA microarray study that Instituto de Biociências, USP), Suely Nonogaki (Intituto identified the genes that are up- and down-regulated Adolf Lutz), François Nantel (Institute of Pharmacology in a human MDA-361 pLKO control clone and three 1. 114

livro.indd 114 7/7/2008 16:33:37 clones expressing short hairpin RNA against DCD B-069 mRNA. This analysis indicated the gene expression FUNCTIONAL CHARACTERIZATION OF GENES of 208 was reduced and of 27 was increased in the DIFFERENTIALLY EXPRESSED IN THE CYTOKINE- three DCD-RNAi clones compared to pLKO control CYTOKINE RECEPTOR INTERACTION PATHWAY IN (>3 fold-change and p < 0.005). The expression of PROSTATE CANCER USING SAGE LIBRARIES 77 genes (37%) encoding for enzymes involved in Melline Fontes Noronha (Laboratório de Genômica the amino acid metabolism, glucose metabolism, the e Expressão - UNICAMP), Murilo Castro Cervato oxidoreductase activity and several genes required for (Laboratório Nacional de Computação Científica), Israel cellular survival and DNA repair was decreased. The Tojal da Silva (Laboratório de Genética Molecular e expression EGFR/ErbB1 gene, an important predictor Bioinfórmatica - USP-RP) of outcome in breast cancer, was reduced together with the genes for betacellulin and amphiregulin, two known Prostate cancer is a neoplasic disease heavily studied ligands of EGFR/ErbBs receptors. Among of 27 genes by researchers mainly because it is the second largest up-regulated with known function, we identified the cause of death among men. Cytokines are proteins calcium-calmodulin-dependent protein kinase-II delta that regulate the immune and hematopoietic systems. and calcineurin A alpha. We compared 132 up-regulated Based on this, we analyzed the Cytokine receptor and 12 down-regulated genes with those genes up- and interaction pathway in light of Prostate Cancer. Using down-regulated by inhibitors targeting various signaling bioinformatics tools, we compared data from two, pathways. The analysis showed that the genes in the previously published [1], SAGE libraries: one obtained DCD pathway are aligned with those influenced by the using normal prostate tissue and a second from cancer drugs sirolimus, LY-294002 and wortmannin. Therefore, prostate tissue. We selected genes involved in the DCD may exert its function by activating the PI3K/AKT/ cytokine pathway and applied the Audic Claverie’s mTOR signaling pathway. Together these data suggest method using p-value < 0.05 as a threshold. Then, we the involvement of DCD in the regulation of genes for compared our results with KEGG libraries and then they breast cancer cell metabolism, proliferation and survival. were separated in two groups of differentially expressed [email protected] genes, the up-regulated genes: CCL18, TNFRSF14, CCL17, CXCL9, LIFR, IL23A, CX3CL1, TGFB1 B-068 and IL17RB, and the down-regulated genes: CSF1, FGF2 BLOCKS H-RASV12-DEPENDENT MET, IL8, IL4R, CCL5, TNFSF13, IL7, CXCL1, HGF, PROLIFERATION OF BALB3T3 MOUSE CXCL3 and PDGFRA. We observed that the down- FIBROBLASTS regulated genes are mainly involved in the regulation Marianna Mainardi Koga, Hugo Aguirre Armelin of the immune system. Some of them are chemokines (Departamento de Bioquímica, Instituto de Química - involved in monocytes and macrophages control. IL8 USP) is related to the inflammatory and angiogenic factor response. HGF, MET and PDGPRA are involved in the We recently reported that recombinant FGF2 acts as a control of the cell cycle by the activation of tyrosine classical mitogen in Balb3T3 mouse fibroblasts, but is kinase signaling pathway. Most of up-regulated genes toxic for Balb3T3 clones transformed with the activated are involved in regulation processes of the immune H-RasV12 oncogene. To further characterize this FGF2- response through T-cells, B-cells and other types of stress response and its molecular mechanisms, we lymphocytes. These preliminary results show that this transformed Balb3T3 fibroblasts with a cDNA chimera “in silico” study of the cytokine pathway can help us to that encodes the 4-OHT (4-hydroxytamoxifen)-activated understand the changes taken place in prostate cancer. ER:H-RasV12 fusion protein comprised of a mutant [email protected] form of the estrogen receptor ligand binding domain and oncogenic Ras. Balb3T3 fibroblasts were infected with a B-070 retroviral vector containing the cDNA sequence and the GROWTH INHIBITION AND INCREASE IN neomycin marker gene. A number of 3T3-ER:H-RasV12 ADHESION CAPACITY OF clonal sublines were selected with G418, isolated and HEPATOCARCINOMA CELLS TREATED WITH screened for response to 4-OHT and/or FGF2. All viable RETINOIC ACID AND CAMP 3T3-ER:H-Ras sublines displayed 4-OHT-dependent Raquel Bernadeth de Almeida, Vanessa Morais Freitas, malignant phenotype characterized by morphological Gláucia Maria Machado Santelli, Marisa Ionta (Dept of changes and high saturation densities. FGF2 caused Cell and Developmental Biology, Biomedical Science a moderate growth stimulation in 3T3-ER:H-RasV12 Institute, Univ of Sao Paulo, Brazil) sublines. On the other hand, treatment of 3T3-ER:H- RasV12 sublines with both 4-OHT and FGF2 led to The hepatocarcinoma (HCC) is one of the most drastic inhibiton of proliferation. We concluded that common malignant tumors. Classical cytotoxic therapy ectopic expression of the activated H-RasV12 oncogene has been minimally useful in the treatment of HCC is sufficient to render Balb3T3 fibroblasts sensitive to and so, alternative therapeutic approaches must be FGF2 cytotoxicity. developed. Retinoic Acid (RA) is a natural derivative [email protected] of the vitamin A that can modulate cell proliferation 1. 115

livro.indd 115 7/7/2008 16:33:37 and differentiation. Likewise, the cAMP acts as second Cell Biophysics messenger in signal transduction pathway of growth cell regulation. This study aimed to investigate the effects of the RA and cAMP on the phenotype of C-001 hepatocarcinoma cells. Cell proliferation was evaluated BIOMECHANICAL RESPONSE OF RAT TENDON by BrdU incorporation. The cell-to-substrate interaction TO MECHANICAL LOAD EXERCISE AND was analyzed through adhesion assay and actin NANDROLONE DECANOATE filaments distribution by Confocal microscopy. The Rita de Cássia Marqueti (Universidade Federal de results showed that hepatocarcinoma cells treated São Carlos), Oscar Henrique Pereira Ramos (Instituto for 48h with RA [0.1 µM] and cAMP [1mM] presented Butantan), Heloisa Sobreiro Selistre de Araujo lower proliferation rate (37% and 13%, respectively) (Universidade Federal de São Carlos) and increased strongly the adhesion capacity to laminin (70% to RA and 84% to cAMP). Growth inhibition Tendon rupture has been linked with androgenic and increased adhesiveness in treated cells were anabolic steroids (AAS) abuse. The aim of this study accompanied by reorganization of the actin filaments was to avaluate the biomechanical proprerties of rat (stress fiber formation) and change in cell shape. deep flexor tendon (DFT) in AAS-treated animals Taken together the results suggest that RA and cAMP associated with training. Forty Wistar rats were used: contributed to reversion of the transformed phenotype. Sedentary (S), trained (T), AAS-treated (5mg/kg, twice [email protected] a week) sedentary rats (AAS) and AAS-treated and trained animals (AAST). Trained groups carried out B-071 jumps in water: 4 series of 10 jumps each, 30 second COLLAGEN AND DECORIN IN UTERINE interval between series, for 7 weeks, with 50-80% CERVICAL CANCER overload of animal weigth. The DFT was removed and a Sebastian San Martin (Universidad de Valparaiso- biomechanical test was carried out at room temperature Chile), Humberto Vallejos (Hospital Carlos Van using physiological saline to prevent the fibers from Buren-Chile), Makarena Gonzalez (Universidad de drying. Maximum displacement, displacement at Valparaiso-Chile), Carolina Yañez (Universidad de maximum load, maximum load, cross sectional-area, Valparaiso-Chile), Daniela Asencio (Universidad de maximum stress strength and maximum strain were Valparaiso-Chile), Gregory Cordova (Universidad de calculed. Statistical analysis was performed by two- Valparaiso-Chile), Javier Egaña (Hospital Carlos Van way analysis of variance (ANOVA) and p<0,05 was Buren-Chile) considered statistically significant. The displacement at maximum load, as well as maximum load, were Collagen and proteoglycans, as decorin, are members significantly lower for the AAST group compared to of the extracellular matrix (ECM) that contribute to S, T and AAS groups. The cross sectional-area was molecular organization of connective tissues and to the significantly higher for the S group compared with T, control of cell proliferation. Cell and ECM interaction AAS and AAST groups and higher for AAS compared is a crucial step for tumor progression. In this work we with T and AAST groups. The maximum stress strength investigate the presence and distribution of collagen was sigificantly increased in the T group compared and decorin from biopsies of uterine cervix with the with other groups. These results suggest that training diagnosis of different stages of malignant neoplasia. increase tensile strength, but when associated with Histochemistry and immunohistochemistry were used AAS the biomechanical properties of DFT are impaired. to analyze the presence and distribution of collagen Support: Fapesp. and decorin respectively in healthy cervical tissues and [email protected] in different stages of cervical malignant neoplasia. We found that collagen and decorin were abundant in both C-002 the stromas of normal cervical tissues and low grade INTERNALIZATION OF SUPER PARAMAGNETIC intraepithelial displasia. However, in more advanced IRON OXIDE NANOPARTICLES (SPION) BY J774 stages such as high grade intraepithelial displasia MACROPHAGES a decrease in the presence of collagen and decorin Rita de Cássia Ruiz (Lab. de Bacteriologia - Instituto were observed. In invasive squamous cell carcinoma, Butantan), Sylvia Mendes Carneiro (Lab. de Biologia collagen was observed only in the perivascular regions Celular - Instituto Butantan), Keyde Cristina Martins and decorin was no longer present. The decrease in de Melo (Lab. de Bacteriologia - Instituto Butantan), the presence of collagen and decorin in high grade Lionel Fernel Gamarra (Instituto de Ensino e Pesquisa - intraepithelial displasia, together with their abscence in Hospital Israelita Albert Einstein) invasive squamous cell carcinoma shows an intense remodeling of the cervical stroma in pathological Recent advances in nanotechnology, such as the conditions. This remodeling of the extracellular matrix development of Super Paramagnetic Iron Oxide could be related with the degree of invasiveness of the Nanoparticles (SPION), are providing new options for cervical cancer. medicine and science. A biocompatible SPION, coated [email protected] with dextran, (Endorem ® - Guebert), associated with 1. 116

livro.indd 116 7/7/2008 16:33:37 magnetic resonance imaging (MRI), has been used for Developmental Biology the study of cell distribution and fate, but conditions haven’t yet been established on uptake, action and elimination in cells other than hepatocytes. In this work D-001 we analyze Endoren uptake both qualitatively and SKELETAL MUSCLE GROWTH AND MYOD GENE quantitatively, as well as the survival and proliferation EXPRESSION IN PIRARUCU of macrophages. The macrophages were exposed to (ARAPAIMA GIGAS CUVIER, 1817) Endoren at 100, 200, 350, 500, 750 e 1000 mg/ml, for Fernanda Regina Carani, Fernanda Losi Alves de 45 min, washed and counted at different incubation Almeida, Carlos Roberto Padovani, Maeli Dal Pai Silva times. The presence of SPIONs in cells was still (Universidade Estadual Paulista - campus de Botucatu) detected by Prussian blue staining at day 10, even when exposed to the highest concentrations tested. In most fish skeletal muscle growth occurs by two SPIONs were detected by Ferromagnetic Resonance mechanisms: hypertrophy and/or hyperplasia of muscle in supernatants from cultures incubated with 350, fibers, through satellite cells or myoblasts. These 500, 750 and 1000 µg/ml, after 7, 5, 3 and 1 day processes are controlled by the Myogenic Regulatory respectively. Cells incubated with 100, 200 and 500 Factors (MRFs). During fish growth, MRF expression µg/ml proliferated like the control groups. Professional may control muscle celullarity and growth mechanisms, phagocytes not only can phagocyte huge amounts of which directly influence fillet quality. The aim of this SPION, but they also keep proliferating for at least 10 work was to evaluate in pirarucu (Arapaima gigas) days thereafter. Elimination of SPION appears to be the growth characteristics of skeletal muscle, and dose dependent. To be clarified: 1) if SPION elimination also the MyoD gene expression and its possible role is due to exocytosis or to cell death; 2) the behavior of in regulating muscle growth. Alevins (n=5; 50 days SPION in non professional phagocytes. old) and juveniles (n=4; 1 year old) were sacrificed [email protected] and white muscle samples were collected from dorsal region. Histological frozen sections (10 mm) C-003 were stained with HE (Haematoxilin-Eosin) and the THE INSOLUBLE MATRIX OF A MOLLUSCAN SHELL morphometric analysis was performed by measuring Marina Silveira (Instituto de Fisica USP), Silvia Maria de the smallest diameter fiber. MyoD gene expression was Paula (Instituto de Quimica, USP) determined by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and the semi-quantitative analysis Molluscs are able to synthesize robust shells, consisting was done by densitometry. Hyperplasia muscle growth solely of calcium carbonate crystals firmly bound by thin predominated in alevin and, hypertrophy, in juvenile. layers of biological matter. The biological component MyoD gene expression was significantly higher in alevin or matrix, represents less than 5% of the whole shell; than juvenile. The differential expression of MyoD gene it is synthesized by the mantle epithelium, admittedly observed between the two growth phases indicates driving the mineralization process. Two fractions are that skeletal muscle growth is initially regulated by recognised, the soluble and insoluble moieties. The MyoD expression, which controls satellite cells (or matrix is known to contain acidic-glycoproteins, beta- myoblasts) activation and fusion to form new myofibers chitin, and a gel-like phase of silk fibroin. Mineralizing (hyperplasia). These results may help fish farmers to proteins would likely attach to the chitin. Investigations understand the molecular mechanisms that control by Levi-Kalisman et al. (2001); Weiss and Schönitzer skeletal muscle growth in pirarucu, an (2006) suggest that the polysaccharide chitin and acidic economically important large (giant) fish. macromolecules play important formative and functional [email protected] roles during shell growth, providing a framework upon which minerals are deposited. The insoluble fraction D-002 of both prismatic and nacreous layers were isolated MATERNAL PROTEIN RESTRICTION AND LEFT from shells of a gastropode, P. cubensis, and a VENTRICLE HYPERTROPHY IN RATS: ROLE OF bivalve, A. trapesialis, following dissolution of crystals, OVEREXPRESSION OF AT1 and studied by scanning and transmission electron RECEPTORS AND TIMP2 microscopies, histochemistry and infrared spectroscopy. Brisolla R (UNIARARAS), Mesquita, FF (UNICAMP), Thin sectioned nacreous matrix of A. trapesialis exhibit Juliani CC (UNESP), Justulin Jr, LA (UNESP), Felisbino, a periodic layering spaced at 25nm. SEM of fixed, SL (UNESP), Dal Pai Silva, M (UNESP), Gontijo, JAR tannin impregnated and critical-point dried matrices (UNICAMP), Boer, PA (UNESP) preserved the prismatic, 3D profile of crystals, whereas those from nacreous layer formed a continuous Maternal protein restriction leads to a reduction sheath. In both matrices of the bivalve, a system of of cardiomyocytes number at birth, which may fine, PAS-positive fibrils, is found, interpreted as chitin. compromise the heart functionality with increased Infrared spectroscopy essays are underway, to further risk of heart failure and hypertrophy in adulthood. The characterize the biochemical nature of the filamentous renin-angiotensin-system (RAS) plays an important structures. - [email protected] role in the development of cardiac hypertrophy and in 1. 117

livro.indd 117 7/7/2008 16:33:37 vascular structures. In heart, all the RAS components, pairs of myotomes are formed. The Pharyngula Period including angiotensin II (Ang II) type 1 (ATR1) and type (1 stage) is the stage of organogenesis. This period 2 (ATR2) receptors are expressed locally during genesis lasted about 2h (between 10 and 12 hpf). The first of cardiomyocytes. ATR1 stimulate the production embryos started to hatching at 11 hpf and 50% of them of metalloproteinases (MMPs) and their specific had already hatched at 13 hpf. The quick development inhibitors (TIMPs) associated with fibrosis. Here, we of this species is probably an adaptation to minimize investigated if gestational dietary protein restriction predation risk, allowing survival in ecosystems with high alters the amount and sites of AngII receptors, MMP2 animal diversity and low food availability. and TIMP2 expression associated with cardiac [email protected] hypertrophy in left ventricle of 16 weeks old male offspring. Pregnant rats were submitted to a normal D-004 (NP) or a low protein (LP) diet. The pups blood pressure MATERNAL MALNUTRITION DURING LACTATION increases significantly in LP than in NP rats from 6 to 16 PROGRAMS THE EXPRESSION OF GROWTH weeks of age. The mass and left ventricle volume are FACTORS VEGF AND BFGF IN THE OVARY OF THE significantly greater in LP 16 weeks old male offspring OFFSPRING compared to NP. Furthermore, the immunoblotting Rafaela Veiga Ferreira, Flávia Meireles Gombar, and immunohistochemical analysis of current study Fernanda da Silveira Cavalcante, Francisco Jose showed a significantly increased expression of ATR1 Barcellos Sampaio, Cristiane da Fonte Ramos (Uerj) and TIMP2 around the vasculature and epicardium in LP rats accompanied with unchanged ATR2 and Introduction: Early malnutrition may change the original MMP2 expression. These data suggest that AT1 programming of organs, resulting in metabolism overexpression and change in the AT1R/AT2R rate and long-term changes. Vascular endothelial growth factor in MMP2/TIMP2 balance could be involved in cardiac (VEGF) and basic fibroblast growth factor (bFGF) fibrosis and left ventricular hypertrophy in this maternal are important for normal ovarian function. Objective: underfed model. Study supported by: FAPESP. Evaluate if maternal food restriction during lactation can Luis Antonio Justulin Junior, program bFGF and VEGF expression in the ovary of [email protected] the offspring. Methodology: At parturition, dams were randomly assigned to the following groups: (C) control D-003 group, with free access to a standard laboratory diet QUICK EMBRYONIC DEVELOPMENT OF THE containing 23% protein; (PR) protein-restricted group, AMAZONIC FISH COLOSSOMA MACROPOMUM with free access to an isoenergy and protein-restricted (TAMBAQUI) diet containing 8% protein; and (ER) energy-restricted Alana Marielle Rodrigues Galdino (Universidade group, receiving standard laboratory diet in restricted Federal do Paraná), Lucélia Donatti (Universidade quantities. After weaning, the pups had free access Federal do Paraná), Paulo César Falanghe Carneiro to standard laboratory diet until day 90. Only rats (Embrapa Tabuleiros Costeiros), Flavia Sant’Anna Rios in proestrous stage were used. Statistics: One-way (Universidade Federal do Paraná) analysis of variance followed by Newman Keuls test. Results: When compared to C, the expression of bFGF The tambaqui (Colossoma macropomum) is an Amazon (C=0.33±0.10; PER=0.38±0.07, ER=0.63±0.10) and fish with high commercial value and ecological interest. VEGF (C=0.80±0.10; PER=0.86±0.23; ER=1.27±0.45) The embryonic stages were described using light was significantly (p<0.05) increased only in the and scanning electron microscopy. The embryonic ER group. Conclusion: Maternal energy restriction development is similar to the other teleost fish, however programs the expression of both VEGF and bFGF in the development of this species is characterized the ovary of the offspring. Financial Support: FAPERJ, by the short time between the stages. At 26oC, the CNPq. embryonic development last about 13 hours, divided in [email protected] 26 stages. The first cleavage occurs about 20 minutes after fertilization. The Cleavage Period presented 6 D-005 stages (6 cell cycles) and lasted only about 1h. At the MATERNAL MALNUTRITION DURING end of the Blastula Period (4 stages), the blastoderm LACTATION PROGRAMS THE LEPTIN are compacted and covered about 30% the yolk cell. RECEPTOR EXPRESSION IN THE OVARY OF THE The Gastrulation (3 stages), which is usually a long OFFSPRING time period in the most fish species, begins about Fernanda da Silveira Cavalcante, Flavia Meireles 3,5-4 hpf and completes itself in approximately 3h (6,5 Gombar, Rafaela Veiga Ferreira, Francisco Jose hpf). After epiboly the blastoderm completely covers Barcellos Sampaio, Cristiane da Fonte Ramos (Uerj) the yolk plug, closing the blastopore (6,5 hpf). The formation of the first somitic furrow marks the beginning Introduction: Leptin, the product of the obese (ob) of Segmentation Period (6 stages, 7 hpf). The somites gene, is an important satiety and reproductive form at a rate of about 7,5 pairs per hour. Miotomes hormone. There are six known splice variants of the were visible 1h after the beginning of somites. About 35 leptin receptor (OB-R), the longest one (OB-Rb) is the 1. 118

livro.indd 118 7/7/2008 16:33:37 main form present in the ovary. Objective: Evaluate differentiation. In conclusion, DMSO could be blocking if maternal malnutrition during lactation programs the normal angiogenesis, thus preventing the normal expression of the leptin receptor in the ovary of the organogenesis. Experiments are underway to answer offspring. Methodology: At parturition, dams were this question. randomly assigned to the following groups: (C) control [email protected] group, with free access to a standard laboratory diet containing 23protein; (PR) protein-restricted group, D-007 with free access to an isoenergy and protein-restricted CELL PROLIFERATION AND CELL DEATH ON diet containing 8protein; and (ER) energy-restricted SPINAL CORD AND group, receiving standard laboratory diet in restricted NON-NEURAL ADJACENT TISSUES IN quantities. After weaning, the pups had free access GALLUS DOMESTICUS EMBRYOS to standard laboratory diet until day 90. Only rats Heloisa Ghizoni (Universidade Federal de Santa in proestrous stage were used. Statistics: One-way Catarina), Eliane Maria Goldfeder (Universidade analysis of variance followed by Newman Keuls Federal de Santa Catarina), Evelise Maria Nazari test. Results: Transcripts of both Ob-R (C=1.7±0.3; (Universidade Federal do Rio de Janeiro), Dib Ammar PER=0.46±0.09; ER=1.1±0.1) and OB-Rb (C=179±19; (Universidade Federal do Rio de Janeiro), Yara Maria PER=98±3.7; ER=89±15) were significantly reduced Rauh Müller (Universidade Federal de Santa Catarina) (p<0.05) in both PER and ER groups. Conclusion:The maternal malnutrition during lactation programs the Folic acid (FA) deficiency and high homocysteine expression of leptin receptors in the ovary of the (Hcy) might trigger alterations in cell proliferation offspring.Financial support: FAPERJ, CNPq. and cell death during the embryogenesis. In order to [email protected] investigate the effects of the FA and Hcy on spinal cord and non-neural adjacent tissues, fertile eggs (n=33) D-006 were incubated at 38ºC and divided in 3 Groups, ANTI-ANGIOGENIC EFFECTS OF DIMETHYL according pretreatment (24h) and treatment (44h): SULFOXIDE (DMSO) ON RETINA’S GI (25µl saline and 10µmol Hcy), GII (0.5µg FA and DEVELOPMENT AT GALLUS GALLUS 10µmol Hcy) and GIII (25µl saline and 25µl saline). To EMBRYOS quantify cell proliferation and death (1000X), embryos Andréa Cavalho de Almeida (UniRio), Aline Caetano on 8° incubation day were anaesthetized (4°C), Carvalhal (UFRJ), Cíntia Monteiro de Barros (UFRJ), tissues were fixed and sectioned (6µm) to perform André Luiz Freire Portes (USP), Nádia Campos de immunohistochemistry (anti-phosphoHistone H3) and Oliveira Miguel (UFRJ) TUNEL methods. Three alternate slices were selected in each microscope slides (n= 9 slices) to measure Dimethyl sulfoxide (DMSO) is a widely used solvent the cell proliferation and death per group. Differences for industrialized products and therapeutics. Its anti- were evaluated by ANOVA, followed by Duncan’s tests. angiogenic properties have been investigated along Proliferative process on spinal cord was assessing by 9 the last years as treatment for some pathologies mitotic cells in GI, while GIII presented 6 cells and GII 5 related to excessive neovascularization [1]. Vascular mitotic cells (p<0,05). However, on non-neural tissues, endothelial growth factor (VEGF) has been shown GII showed 6 mitotic cells, followed by GIII (3 cells) and to be the major factor responsible for physiological GI (2 cells) (p<0,05). Apoptosis was more expressive and pathological angiogenesis, although it is not on spinal cord of GII (9 cells), followed by GI (4 cells) the only molecule involved in this process [2]. and GIII (3 cells) (p<0,05). The non-neural tissues Retinal vascular diseases are tightly linked to VEGF showed a similar number of apoptotic cells in GI (12) overexpression [3]. In this study, we investigate the and GII (11 cells) in contrast with GIII (3 cells) (p<0,05). effects of DMSO on retinal development. For this, we Hcy treatment causes a decrease on cell proliferation injected DMSO (100%), or not (as control group), at associated with an increase on cell death, mainly in the air space of fertilized chicken eggs. At the day 9 non-neural tissues. The FA pretreatment seems to of embryonic development we analyzed the retina reduce the harmful effect of Hcy in embryos. helo. [4]. The results showed an arrested development [email protected] in DMSO-treated eggs in comparison to the control group, as visualized in stereoscopic microscopy. No D-008 evidence of vascularization and no change at retinal ASSOCIATION OF CELLULAR PRION PROTEIN cellular differentiation were observed in controlembryo WITH STRESS INDUCIBLE PROTEIN 1 IN NEURAL sections stained with hematoxylin and eosin. We STEM CELL BIOLOGY also verified by imunohistochemistry that VEGF is Tiago Góss dos Santos, Marilene Hohmuth Lopes, expressed in several embryonic tissues of the control Vilma Regina Martins (Instituto Ludwig de Pesquisa groups, including brain, heart, kidney and skeleton. sobre o Câncer) Interestingly, we verified VEGF expression in DMSO- injected embryos, however, the organogenesis in these Cellular Prion Protein (PrPC) is associated to brain embryos is completely disorganized without proper development and function. Since the engagement 1. 119

livro.indd 119 7/7/2008 16:33:37 of PrPC with STI1 (Stress inducible protein 1) is control mice, or non-tolerant mice immunized with Ova. involved in neuronal survival and neuritogenesis, we Conclusion: These results suggest that injection of a investigated the PrPC/STI1 roles in neural stem cell tolerated antigen on the day of skin lesion can improve biology. Neurospheres cultures, which contain a mix of skin regeneration and reduce scar formation. Supported multipotent and fully committed progenitor populations, by: FAPEMIG. were derived from mice telencephalon and from [email protected] subventricular zone of wild-type and PrPC-null mice. Neurospheres were maintained for several passages D-010 in the presence of growth factors without apparent loss FOLIC ACID AND HOMOCYSTEINE DISTURB of self-renewal and multipotenciality, with continuous NEURAL AND NON-NEURAL TISSUES capacity to generate astrocytes, oligodendrocytes and DEVELOPMENT IN CHICK EMBRYO neurons. The absence of PrPC significantly affected Karoline Kobus (UFSC), Evelise Maria Nazari (UFRJ), the capacity of cells to form new neurospheres, when Dib Ammar (UFRJ), Yara Maria Rauh Müller (UFSC) compared to their wild-type counterparts, suggesting a potential role of PrPC in self-renewal. STI1 as well Folic acid (FA) nutritional deficiency produce an as nestin, a neural stem cell marker, were uniformly increase in homocysteine (Hcy) levels, that tends to distributed with similar levels in neurospheres accumulate intracellulary and it cause DNA damages. derived from both wild-type and PrPC-null mice. STI1 Moreover, FA plays an important role in the regulation significantly increases the number of spheres in the of neurogenesis and apoptosis. The aim of this study wild-type population when compared to the untreated was to analyze the neural and non-neural tissues group, conversely, in PrPC-null neurospheres STI1 development in the trunk region of Gallus domesticus had no effect. These data suggest that STI1 could be embryos treated with FA and Hcy. Eggs (N=12) were involved in pivotal events in neural stem cell biology, like incubated (38°C and 65% humidity) and past 32h it proliferation, survival or growth factor resposiveness, were submitted to three treatments using 25µl saline through its binding to PrPC on the cell surface. vehicle in yolk sac: 0,5µg FA; 10 µmol D,L-Hcy; 0,5µg Indeed, we expeculate that PrPC-STI1 complex may FA+10 µmol D,L-Hcy. Control received only saline. In represent a novel therapeutic target for the treatment of the 4th embryonic day, embryos were anaesthetized neurodegenerative illness including prion diseases. at 4ºC and fixed for light microscopic analyses. [email protected] Immunohistochemistry using anti-phospho-histone H3 and anti-NCAM antibodies were performed. Spinal cord D-009 (SC) layers thickness were measured and proliferative INJECTION OF OVALBUMIN INTO TOLERANT MICE mesoderm and neural cells were counted (400X). REDUCES SCARS OF SKIN WOUNDS FA treatment and control exhibited similar means for Raquel Alves Costa, Geraldo Magela de Azevedo SC layers thickness (56.65 and 60.72mm), while the Júnior, Valéria Ruiz de Souza, Nelson Monteiro Vaz, simultaneous treatment parttially reverts (45.21mm) Cláudia Rocha Carvalho (UFMG) the increase on SC layers thickness caused by Hcy (65.92mm). A significant reduction on mesoderm Introduction: In orally tolerant organisms, injections of proliferative cells (21.25; p<0.00001) was observed the tolerated antigen inhibit primary immune responses in Hcy treatment. Spinal cord proliferative cells were to unrelated antigens and associated inflammatory reduced by Hcy (p<0.01) an FA reverts this status on reactions. Inflammation plays complex roles in wound mantle layer (p<0.05). NCAM immunodetection was healing. We investigated if i.p. injection of an orally obtained in all treatments, but reactive cells were most tolerated antigen (Ovalbumin, Ova) affects dorsal evident in control embryos. Hcy caused disruptions on skin healing in mice. Methods and Results: C57BL/6 neural and non-neural tissues of trunk region and FA mice (n=5) were rendered orally tolerant by ingestion treatment sometimes reverts these effects. of 20 mg Ova for 3 consecutive days; control mice [email protected] (n=5) drank water. 7days later, under anesthesia, all animals received a 1 cm cut in the dorsal back skin D-011 and were injected i.p. with 10 µg Ova + Al(OH)3. Mice LIPIDOMIC ANALYSIS OF MEMBRANE were sacrificed 1 – 3 – 5 - 40 days thereafter, skin MICRODOMAINS IN THE DEVELOPMENT OF tissue samples were fixed in formalin, embedded in XENOPUS LEAVIS paraffin, sectioned at 5 micrometres and stained with Mariana Pinto Louza, Alice Helena dos Reis, Hematoxiline-eosine or Gomori trichrome. Cells were Mirna Abreu Soares, Karla Loureiro Almeida, Marcelo quantified by counting in 4 randomly selected sections Einicker-Lamas, Georgia Correa Atella, using a grid and the wound area was measured through Jose Garcia Abreu (UFRJ) the software Image Tool v. 3.0. As compared to control mice, orally tolerant mice showed a reduction of Our laboratory has recently shown that cholesterol inflammatory cells, fibroblasts and collagen deposition. depletion interferes in the development of the anterior The healing area and the scar formed after healing was region of Xenopus embryo, suggesting a correlation much less pronounced in orally tolerant mice than in between the membrane microdomains and signaling 1. 120

livro.indd 120 7/7/2008 16:33:37 pathway necessary for head formation. This work affected. Taken together, these results indicate that lens aimed to analyze the lipid composition in cholesterol- placode invagination could share with other epithelia the rich membrane microdomains during early Xenopus common strategy of driving invagination through apical development and its relation with the formation of the cell constriction, but in contrast to other epithelia, this anterior embryonic region. Stage 8 Xenopus embryos process is not regulated by GTPase RhoA. (blastula) were microinjected with 40 mM M-beta- [email protected] CD (metil-beta-ciclodextrin) or H2O and stopped at gastrula. The extract was passed trough a sucrose D-013 density gradient that isolates the detergent resistant CLONING AND EXPRESSION OF SCP2, OSBP membrane microdomains also known as lipid rafts (LR). AND STOMATIN DURING THE DEVELOPMENT OF We performed cholesterol measurement to identify in XENOPUS LEAVIS whole embryo extracts LR fraction. Analysis of protein Nathália da Graça Amado, Débora Malta concentration and SDS-page revealed no drastic Cerqueira, José Garcia Abreu (UFRJ) differences between the fractions with M-beta-CD or H2O. Thin-layer chromatography for neutral lipids Recently we showed that the depletion of cholesterol revealed differences between the two extracts when interfere in the development of anterior region in the microinjection leads to an increase of fatty-acids Xenopus, suggesting a correlation between the and decrease of triacylglycerol. These results showed membrane microdomains and the signaling pathway differences in lipid content upon LR disruption by necessary for the head formation. Lipid rafts are cholesterol depletion and revealed that this depletion membrane microdomains serving as platforms for in the membrane microdomains may be involved in cell the organization of signaling complex and the traffic signaling during development of Xenopus leavis. of molecules. Sterol Carrier Protein-2 (SCP-2) and [email protected] Stomatin are related with the composition, structure and function of the lipid rafts and Oxysterol Binding Protein D-012 (OSBP) is related with cholesterol biosynthesis, but APICAL CELL CONSTRICTION AND LENS their roles during embryonic development are unknown. PLACODE INVAGINATION IN CHICK This work aimed to identify and characterize the SCP2, EMBRYOS OSBP and Stomatin expression pattern during the Ricardo Moraes Borges (Department of Cell and embryonic developmental in Xenopus. The cDNAs Developmental Biology, Institute of Biomedical containing the ESTs (expression sequence tag) of the Sciences, USP), Marinilce Fagundes Santos human homologues of SCP2, OSBP and Stomatin (Department of Cell and Developmental Biology, were obtained at NIBB database. These sequences Institute of Biomedical Sciences, USP), Enilza Maria showed high degree of identity with human, zebra-fish, Espreafico (Department of Cellular and Molecular rat and mouse, and are therefore a possible candidate Biology and Pathogenic Bioagents, Faculty of Medicine, for orthologues in Xenopus. In situ hybridization was USP), Chao Yun Irene Yan (Department of Cell performed in different stages of Xenopus embryos. We and Developmental Biology, Institute of Biomedical observed expression of SCP2, OSBP and Stomatin in Sciences, USP) the animal pole (stage 4 cells and gastrula). Expression along antero-posterio axis, branchial arches, optics Vertebrate lens derives from invagination of the and optical vesicles, olfactory plates, in pronephric lens placode that overlies optic vesicles. Apical cell region and in the somites were found in latter stages. constriction is a common developmental process that These results showed the expression in restricted drives epithelial invagination in many model systems. areas and specific embryonic stages, suggesting that Constriction is driven by contraction of apically the ortologues of the SCP2, OSBP and Stomatin in located actin filaments mediated by non muscle Xenopus can play a significant role during development. myosin II, thus creating a basal-ward bending event [email protected] that initiates the invagination of the epithelium. In a variety of previously characterized scenarios, myosin D-014 localization and activation is regulated by GTPase CHOLESTEROL-ENRICHED MEMBRANE RhoA. Here, we investigate the possibility that lens MICRODOMAINS PARTICIPATE IN XENOPUS placode invagination could also be driven by apical LAEVIS HEAD DEVELOPMENT cell constriction and controlled by RhoA. We show Alice Helena dos Reis, Leonardo Rodrigues Andrade, that filamentous-actin and myosin II are expressed at Karla Loureiro Almeida, Mirna Santos Abreu, apical side of lens placode prior to invagination and José Garcia Abreu during lens placode invagination. Moreover, if actin polymerization is blocked by Cytochalasin D, lens During vertebrate development, cell signaling placode fail to invaginate, suggesting that lens placode interactions are extremely important to the correct invagination could be driven by apical cell constriction. formation of the antero-posterior and dorso-ventral axis. However, when we overexpressed dominant-negative Although, many different signaling mechanisms have RhoA in the pre-lens ectoderm, invagination was not been studied during early embryonic development, 1. 121

livro.indd 121 7/7/2008 16:33:38 it is unclear how cells respond differentially to these showing details of subcellular distribution of actin and its signals in order to achieve correct fate. In this respect isoforms and desmin. cell membrane machinery play pivotal role in capturing [email protected] and transduction cell signaling. In this study, we addressed the membrane morphology and components D-016 of lipid raft (LR) during anterior structures formation. YOLK SAC IS THE MAJOR SITE OF Phenotype analysis by Scaning electron microscopy HEMATOPOIETIC EXPANSION IN CHICKEN as well as histology showed that cholesterol depletion EMBRYOS by methyl-β-cyclodextrin (MâCD) caused anterior Priscila Tavares Guedes (Laboratory of Pathology, defects in Xenopus embryo. Imunostaining and dot- Instituto Oswaldo Cruz, Fiocruz), Barbara Cristina blotting analysis showed affected contents of lipid Euzebio Pereira Dias de Oliveira (Laboratory of raft markers such as GM1 ganglioside, caveolin1 and Pathology, Instituto Oswaldo Cruz, Fiocruz), Pedro flotillin. Negative staining of MβCD treated embryonic Paulo de Abreu de Manso (Laboratory of Pathology, LR isolated by sucrose gradient centrifugation was not Instituto Oswaldo Cruz, Fiocruz), Luzia Fátima able to form organized micelles. However transmission Gonçalves Caputo (Laboratory of Pathology, Instituto electron microscopy confirmed that the overall cell Oswaldo Cruz, Fiocruz), Gerson Cotta-Pereira architecture including membrane organization and (Laboratory of Immunochemistry and Histochemistry, intracellular organelles were not affected by MâCD Santa Casa da Misericórdia do Rio de Janeiro), Marcelo treatment. These results strongly support that Pelajo Machado (Laboratory of Pathology, Instituto cholesterol-enriched membrane microdomains play Oswaldo Cruz, Fiocruz, Brazil.) crucial role during Xenopus anterior development. Support: CNPq, CAPES, MCT-PRONEX/FAPERJ. Although bone marrow (BM) is the major hematopoietic [email protected] organ after birth, during ontogeny the hematopoietic cells travel to one site to another, expanding and D-015 becoming committed to hematopoietic lineages. RELATIONSHIP BETWEEN CYTOSKELETAL AND Chicken embryos are largely used in studying the ADHESION PROTEINS IN THE ontogeny events responsible for the hematopoietic ZEBRAFISH (DANIO RERIO) MYOGENESIS establishment. However, there are few data concerning Maria Luisa Lima Marques, Ingrid Antunes da Silva, the morphology of the transient hematopoietic organs. Cláudia dos Santos Mermelstein, Manoel Luís Costa To study them, 18 to 46 HH chicken embryos and (Universidade Federal do Rio de Janeiro) respective extra-embryonic membranes were collected, processed and embedded in paraffin. Sections were We decided to use zebrafish embryos to study stained by HE, Giemsa and Sirius Red pH 10.2. muscle differentiation in situ, in a three-dimensional, At stages 19-20, intra-aortic clusters were seen in complex environment. We have been studying aorta-gonads-mesonephrons (AGM) region and cytoskeletal components, membrane and extracellular erythropoiesis was observed in Yolk Sac (YS). During matrix, characterizing the distribution and time of stages 25-26, basophilic cells were seen in para-aortic appearance of marker proteins during zebrafish muscle foci and YS was constituted by erythropoietic activity development. In particular, we have been following and precursors inside its vessels. Expansion and the distribution of the cytolinker protein plectin, which differentiation of erythrocytic and granulocytic lineages connects all three major cytoskeletal components. We were gradually enhanced in YS embryos between studied, using conventional and laser-scanning confocal stages 31 to 40 and 36 to 40 respectively. At stages optical microscopy, antibodies and fluorescent probes 43-44, when BM became functional, YS hematopoiesis in 24 h zebrafish embryos. To test the physiological has been reduced, but still proliferative. At stage 46, interactions between cell components, we used a heat hematopoiesis was no longer detected in chicken YS. shock to disrupt somitogenesis and microinjection of In all stages, hematopoiesis was practically inexistent drugs and nucleic acids. We describe the full range inside the liver. Our results suggest that during chicken of desmin distribution in zebrafish development, from embryo development, YS is an important site of perinuclear to striated, until its deposition around the expansion and differentiation of hematopoietic cells. intersomite septa in older somites. We show that actin It seems that during phylogeny, the mammalian YS is initially located in the intersomite septum region while involution after AGM phases determine hematopoietic it is confined to the myofibrils later on. The adhesion progenitors’ migration to the liver. Supported by Fiocruz, complex proteins vinculin, paxillin, talin, dystrophin, CNPq. laminin and fibronectin always appear exclusively at [email protected] the intersomite septa, and appear to be co-distributed. The cytoskeletal linker plectin has a distribution similar D-017 to desmin and not to actin.We propose a model were EXPRESSION OF SCRATCH2 IN THE CHICKEN each skeletal compartment has a specific sequence EARLY EMBRYO of change in distribution along zebrafish myogenesis. Felipe Monteleone Vieceli, Chao Yun Irene Yan We are also describing novel adhesion structures and (Instituto de Ciências Biomédicas - USP) 1. 122

livro.indd 122 7/7/2008 16:33:38 The gene Scratch (Scrt) is a member of the already expected, we could not observe desmin in the Snail superfamily of transcription factors, which intersomit septa. This is interesting, because were able plays evolutionary conserved roles in mesoderm to show that changes in desmin distribution in zebrafish development, neural differentiation and neural crest embryos mark specific stages of development. These formation. Functional interference studies carried out are the first results about the distribution of the main in invertebrate embryos and human cells showed sarcomeric and adhesion proteins in mature zebrafish that Scrt induces neural differentiation. In vertebrate skeletal muscle. embryos no functional analyses have been carried out, [email protected] but its expression was verified in several regions of the developing nervous system in mice, including the D-019 dorsal root ganglia (DRGs). The DRGs are part of the APOPTOSIS AND HSP70 IN EMBRYOS OF peripheral nervous system, which is entirely derived PROCHILODUS LINEATUS EXPOSED TO THERMAL from the neural crest. This work intends to investigate STRESS the conservation of Scrt function in the chicken embryo. Flávia Silveira Lemos (UFMG), Ralph Gruppi Thomé Initially, a homologue to Scrt2 was identified in the (UFMG), Elizete Rizzo(UFMG), Nilo Bazzoli (Programa chicken genome (cScrt2) and two internal fragments de Pós-graduação em Zoologia de Vertebrados were cloned based on this sequence. In order to PUCminas) assess cScrt2 expression in the early embryo, both fragments were used as probes for whole mount in The early development of fish embryos is susceptible situ hybridization. We obtained strong signal with to changes of temperature. During morphogenesis, both probes in the whole extension of the neural tube cells that are no longer required are removed by from developmental stages HH 13 to HH 23, besides apoptosis. Heat shock proteins (HSPs) are associated some weak expression in other structures, such as to cellular stress, preventing the protein denaturing the somites in HH 19 to HH 21. Detailed analysis of and inhibiting apoptosis. In the present study, histology these embryos after sectioning showed also that Scrt and immunohistochemistry techniques were used to is expressed bilaterally between the somites and the detect apoptosis and HSP70 in Prochilodus lineatus neural tube, in a group of cells reminiscent of the embryos. Embryo samples in gastrulation (75% ventrally migrating neural crest cells that give rise epiboly) were submitted to thermal stress for 1hour at to DRGs. Thus, our results are in agreement with a 20ºC (1º group), 25ºC (2° group) and 30°C (3° group). proposed conserved role for Scrt in neural development. They were collected immediately after treatment and [email protected] 8 hours later. Morphological features of the apoptosis such as chromatin condensation, cytoplasmic D-018 retraction and apoptotic bodies during embryogenesis CYTOSKELETAL AND ADHESION PROTEINS IN THE were detected in each time in all groups. Caspase-3 ZEBRAFISH (DANIO RERIO) ADULT MUSCLE immunostaining was observed in apoptotic cells. HSP70 Laise Monteiro Campos (UFRJ) was constitutively expressed during embryogenesis in the cytoplasm of all blastomeres at each time and While zebrafish has become a paramount model for temperature. At 8 hours after thermal stress HSP70 was studies of vertebrate biology, only a few studies have expressed in the nucleus of some cells. This nuclear attempted to specifically localize proteins in adult staining could be involved in the control of nuclear zebrafish muscle. Our objective is to fully appreciate activity associated with important developmental events the distribution of cytoskeletal and adhesion proteins, such as cleavage and transport of proteins. Results which is critical for the contractile function of skeletal indicated that HSP70 under thermal stress could play muscle, and therefore to better correlate the adult a role in the modulation of apoptosis during embryo muscle with its myogenesis. We used antibodies to development. study specific proteins in adult zebrafish cryosections [email protected] of skeletal muscle, using optical microscopy. We also used conventional transmission electron microscopy, D-020 staining ultra-thin sections (80nm-thick) with 2% uranyl IMMUNOLOCALIZATION OF CD44, A acetate and 1% lead citrate. Images were processed MACROPHAGE MIGRATION INHIBITORY for analysis and display using Image J. We observed FACTOR (MIF) CO-RECEPTOR AT THE MATERNAL- striations that stained for desmin, alpha-actinin, myosin, PLACENTAL INTERFACE IN MICE titin, troponin, tropomyosinand nebulin. Using Fourier Mariane Ferracin Martucci, Mara Sandra transform, we could show that their periodicity is 1.7um Hoshida, Estela Bevilacqua on average. We also observed that the intersomite (Instituto de Ciencias Biomédicas I - USP) septa stained for the adhesion proteins alpha-actinin, vinculin and paxilin, and the extracellular matrix proteins The maternal-fetal interaction involves a complex laminin and fibronectin. Collagen fibers were observed dialogue between regulatory molecules crucial to the in the septa in trasmission electron micrographs. pregnancy success. One of these molecules is the While most of these proteins distributions were Macrophage Migration Inhibitory Factor (MIF), a pro- 1. 123

livro.indd 123 7/7/2008 16:33:38 inflammatory cytokine produced by leukocytes and synergistically activates transcription of several cardiac immune-competent cells, expressed during pregnancy, specific promoters. One of these sites was localized particularly by trophoblast cells in different species. to residue K51 in a consensus SUMOylation region, The intracellular MIF signaling pathway is triggered by while the second was positioned in a yet unidentified binding to its membrane receptor CD74 and co-receptor non-canonical region. Mutation analysis of K51 site CD44. An effective intracellular kinase response and fails to impair Nkx2-5 transcriptional synergism, induction of MIF-dependent gene expressions seems to nuclear localisation or DNA binding affinity. In order to depend on simultaneously binding to CD74 and CD44. understand the mechanism of transcriptional synergism Knowing the target cells to MIF during pregnancy will we are currently undertaking experiments to identify the facilitate the understanding of the downstream cascade second SUMOylation site and co-activators of Nkx2-5. and roles played during pregnancy. To determine [email protected] the expression of the MIF co-receptor CD44 at the maternal-placental interface. Material and Methods: D-022 Immunohistochemistry reactions for MIF CD44 co- CHARACTERIZATION OF MEMBERS OF THE receptor localization in implantation sites and placentas ROUGHEST-NEPH PROTEIN FAMILY IN CHICK of females at days 7.5, 9.5, 13.5 and 17.5 of gestation. EMBRYOS Results: CD44 was immunolocalized exclusively at José Eduardo Baroneza, José Henrique Ferreira Alves, the decidua along the gestational studied periods. Mara Silvia Alexandre Costa, Ricardo Guelerman Conclusions: Altogether previous data that MIF is chiefly Pinheiro Ramos (USP) produced by the mice trophoblast cells at the maternal- placental interface, our results suggest that decidua Immunoglobulin superfamily proteins are essential for is the target for the paracrine MIF secretion exhibited the regulation of early developmental events both in trophoblast cells. The gene and protein expression of invertebrate and vertebrate embryos. In vertebrates, for both CD44 and CD74 and in vitro studies are right now example, immunoglobulins Neph1, Neph2 and Neph3 in progress to evaluate the exact signaling role of MIF at show high similarity to D. melanogaster Roughest, are maternal-placental interface.Financial support: FAPESP, part of the urine filtration apparatus in the slit diaphragm CNPq, CAPES. of glomerular podocytes in the kidney and, in both mice [email protected] and humans, their absence can cause proteinuria, congenital nephrotic syndrome and perinatal death. D-021 Here we report the identification of two putative TRANSCRIPTIONAL ACTIVITY OF THE Roughest homologues genes in the recently available CARDIAC FACTOR NKX2-5 IS REGULATED BY chicken genome complete sequence, chkNeph1 and SUMOYLATION chkNeph2, their partial isolation and their expression Camila Guerra Martinez (IBCCF, UFRJ), Milena Bastos pattern in different chicken organs by RT-PCR, Northern Furtado (ICB, UFRJ), Duncan Sparrow (VCCRI, Blot and in situ hybridization. Both chkNeph proteins Australia), Richard P. Harvey (VCCRI, Australia), are highly conserved and, in filogenetic trees, localized Eleonora Kurtenbach (IBCCF, UFRJ), Mauro Eduardo in the same branch that other vertebrate Nephs as Weyne Ferreira da Costa (IBCCF, UFRJ, Brazil) well as the Drosophila homologues Roughest and Kirre. RT-PCR and Norther Blot analyses of chkNeph1 Reversible pos-translational modifications regulate expression in adult tissues showed its presence in gene expression in a strict manner, adding another heart, kidney, testis, ovarium, retina and pancreas, while layer of complexity to cardiac transcriptional regulation. by in situ hybridization of whole mount embryos, we Recently, members of the Small Ubiquitin-Related detected that this gene began to be expressed in HH-22 Modifier (SUMO) family of proteins have been developmental stage in nervous system. ChkNeph2 is implicated with important roles in eukaryotic gene expressed in adults in heart, liver, kidney, testis, brain regulation. Nkx2-5/CSX is a homeobox protein and retina and its expression starts in HH-24 stage expressed in cardiac progenitor cells that is essential of development. These results contribute to a better for heart specification, spatial definition of chamber understanding of the molecular evolution and functional myocardium and formation/maintenance of elements divergence of genes that code for immunoglobulin of the conduction system. In order to understand [email protected] the importance of SUMOylation during embryonic development, we analysed the spatial expression D-023 profile of SUMO components in mouse embryos EFFECTS OF UVB RADIATION ON FRESHWATER by in situ hybridisation. We detected ubiquitous PRAWN EMBRYOS expression of several components during early mouse Evelise Maria Nazari (UFRJ), Francielle Regina development and uncovered a later spatial restriction Ceccato (UFSC), Dib Ammar (UFRJ), Yara Maria Rauh of pathway components, in particular to forming limb Muller(UFSC), Silvana Allodi(UFRJ) buds and forebrain regions. Through biochemical assays, we demonstrated that Nkx2-5 is SUMOylated Solar UVB radiation is an important environmental at 2 sites at the N-terminal region. This modification factor known to induce cell damages in terrestrial and 1. 124

livro.indd 124 7/7/2008 16:33:38 aquatic organisms. Currently, few works focus on the although they did not inhibit Smad-2 phosphorylation, effects of this radiation on freshwater embryos, which suggesting that TGF-b1-induced GFAP gene activation in many cases, live at 1 meter depth and are exposed might involve cooperation between the canonical and to an intensity of solar radiation similar to that which noncanonical TGF-b pathways. Together, our results reaches the surface. In order to evaluate the effects suggest that glial metabotropic glutamate 2/3 receptor of UVB radiation on freshwater prawn embryos, activation by neurons induces TGF-b1 secretion, ovigerous females of Macrobrachium olfersi with eggs leading to GFAP gene activation and astrocyte on E1-E3(group I) and E4-E6(group II) were irradiated differentiation and involves Smad and MAPK/PI3K for 30min with a 6V UVB lamp. Females were kept pathways. in the dark for 4 days and the eggs were removed [email protected] either at E5-E7(group I) or at E8-E10(group II), fixed and submitted to procedures for light microscopy. In D-025 both groups, irradiated embryos showed a significant SMAD1 IS A REPRESSOR OF THE LEFT-RIGHT decrease on their egg volume (0.04mm3 ±0.004) PATHWAY IN THE MOUSE compared to control embryos (0.05mm3 ±0.006) Milena B. Furtado (Instituto de Ciências Biomédicas - (p<0.001). Irradiated embryos (4% group I and 73% UFRJ), Mark Solloway (Genentech - USA), Mauro W. group II) presented external morphological alterations, Costa (IBCCF - UFRJ), Vanessa Franklin (Children’s which difficult to recognize the typical embryonic Medical Research Institute - Australia), Patrick Tam structures of the analyzed stages. In order to investigate (Children’s Medical Research Institute - Australia), if anomalous embryonic features are related to cell Christine Biben (Victor Chang Cardiac Research proliferation and death, the immunohistochemical Institute - Australia), Richard P. Harvey (Victor Chang technique with anti-phospho histoneH3 was used. It Cardiac Research Institute - Australia) revealed that in irradiated embryos the proliferative zones changed their original positions. The TUNEL Despite the apparent external bilateral symmetry method showed that these embryos presented an displayed by vertebrates, internal organs are irregular number of apoptotic cells in the appendages asymmetric in regards to their anatomy and position and optic lobes. These preliminary findings suggest that within the body cavities, a rearrangement known the UVB radiation causes harmful consequences in the as situs solitus. Disruption of this process leads to morphogenesis of freshwater prawns. conditions known as situs inversus and heterotaxia, [email protected] which are normally lethal during embryonic life. Left- right (LR) axis is determined at gastrulation and can D-024 be sub-divided into four integrated steps: 1. breaking GLUTAMATE ACTIVATES GFAP GENE PROMOTER of molecular symmetry at the node, when the TGFß FROM CULTURED ASTROCYTES THROUGH TGF- super-family member Nodal becomes enhanced on BETA1 PATHWAYS the left side of this structure; 2. transfer of asymmetric Luciana Romao, Vivian de Oliveira Sousa, Vivaldo Nodal expression to the lateral plate mesoderm (LPM), Moura Neto, Flávia Carvalho Alcantara Gomes (UFRJ) from which most viscera will form; 3. propagation of asymmetric cues throughout LPM and 4. conversion Glial cells are currently viewed as active partners of this information into organ morphogenesis. The goal of neurons in synapse formation. We recently of this work is to investigate the mechanism involved demonstrated that cortical neurons activate the gene in initial maintenance of asymmetric gene expression promoter of the astrocyte maturation marker, GFAP in LPM in the mouse. We undertook knockout and of cerebral cortex astrocytes by inducing TGF-b1 transgenic analyses to understand how the LR secretion in vitro. To access the effect of glutamate pathway is silenced on the right side of the embryo on GFAP gene, we used transgenic mice bearing and demonstrated that bone morphogenetic proteins the b-Galactosidase (b-Gal) reporter gene under the (BMPs), via the cytoplasmic transducer Smad1, are regulation of the GFAP gene promoter. We report default repressors of Nodal. In addition, Smad1 was that glutamate activates the GFAP gene promoter of able to repress a Nodal responsive element in vitro. astrocytes from cerebral cortex revealed by a significant We have further examined these two distinct TGFß increase in the number of b-Gal positive astrocytes. signalling pathways (Nodal x BMP) and uncovered Neutralizing antibodies against TGF-b completely an intracellular competition step converging on the prevented glutamate and neuronal-induction of GFAP common Smad4. We therefore concluded that Smad1 gene, thus indicating that this event is mediated by buffers biological noise in LPM to allow specific left- TGF-b1. The antagonist of the metabotropic glutamate sided Nodal activation by limiting amounts of available receptor, MCPG, inhibited neuronal effect suggesting Smad4. that neuronal activation of GFAP gene promoter [email protected] involves glutamate metabotropic receptors. MAPK and PI3K inhibitors fully prevented activation of GFAP gene D-026 promoter by all treatments. Surprisingly, these inhibitors THE SUPPLEMENTATION WITH DIFFERENTS OILS also abrogated TGF-b1 direct action on GFAP gene ON CUTANEOUS WOUND HEALING 1. 125

livro.indd 125 7/7/2008 16:33:38 Marcela Otranto de Souza e Mello, Adriana Paulino do from the embryos and seeded on Sl4m220 feeder Nascimento, Thaís Porte Amadeu, Andréa Monte Alto cells. PGC were maintained for 7 days in DMEM Costa (UERJ) supplemented with LIF, retinoic acid, SCF, FGF2 and forskolin. After 7 days the PGC were identified by Fatty acids are bioactive molecules, but their effects on alkaline phosphatase staining. The results obtained to cutaneous wound repair are not well elucidated. We date showed that PGC number is progressively reduced investigated the effects caused by supplementation with during their embryonic development, suggesting differents oils in cutaneous wound healing. Male Wistar that Dazl plays important role in PGC development (150-200g) rats were separated in four groups. Thirty before and after their entry into the gonads. In vitro days before wounding, animals received daily (g.o.), 1.5 analyses are being performed to address Dazl KO PGC ml/kg of linseed, fish (n-3) or sunflower (n-6) oils and proliferation. The mechanisms by which Dazl influences continued supplementation until euthanasia. In d0 an PGC numbers are to be explored. excisional wound (1 cm2) was performed in the back of [email protected] each animal. Wound contraction and re-epithelialization were evaluated. When animals were sacrificed the D-028 wound and adjacent normal skin were collected. Wound DISTRIBUTION OF OSTEONECTIN (A BONE- closure was significantly higher in the Control group SPECIFIC PROTEIN) DURING SPINAL CORD when compared to other groups, in d7 an d14. Linseed DEVELOPMENT and Fish groups showed a higher wound area in d7 and Sandra Iara Lopes Seixas, Fabio Franceschini Mitri d14. The amount of inflammatory cells was moderate Luiz, Yuri Salles Lutz, Marcelle Gonçalves Zuchelli, in Control, Linseed and Fish groups, while Sunflower Danillo Gonçalves de Barros, Caroline de Souza group was discrete. Large number of myofibroblasts Fortuna Nogueira, Raniery França Monteiro, Tiago was observed in Control and Sunflower groups. Linseed Fajardo Póvoa, Gabriella Santos de Moura, Terezinha and Fish groups had high density of dilated blood de Jesus Sirotheau Corrêa (Universidade Federal vessels. Control and Sunflower groups presented blood Fluminense) vessels occluded and concentrated in the deep region of granulation tissue. Control and Sunflower groups During the dorsoventral patterning of the spinal showed moderate density of collagen fibers in the deep cord, axons projections are guided to their targets by region of granulation tissue, and in superficial region molecular cues in their local environment. The floor there was in light amount. However, in Linseed and Fish plate is a source of long-and-shot range guidance cues groups was observed an intense amount of collagen for many axons whose main function may be to direct fibers in the deep region of the granulation tissue. The later-extending commissural axons that must migrate supplementation with different types of oils can increase through the complex environment of the developing the quality of the scars. motor column. Osteonectin, is a unique matricellular [email protected] glycoprotein that is expressed by many different types of cells is associated with development remodeling, cell D-027 turnover, and tissue repair. In the present investigation, PRE AND POST-MIGRATION DEFECT IN immunohistochemistry is used to compare the pattern PRIMORDIAL GERM CELLS FROM DAZL of the expression of this glycoprotein during the KNOCKOUT MICE development of spinal cord. Chick embryos have Taiza Stumpp (Universidade Federal de Sao Paulo), been collected from stage 15 through 40, fixed in Nicola Reynolds (Medical Research Council - Human Bouins solution, embedded in paraffin. The slides Genetics Unit), Howard J Cooke (Medical Research were prepared for immunohistochemical staining Council - Human Genetics Unit) using polyclonal primary antibodies (Chemicon) anti-osteonectin plus ExtrAvidin-biotin-peroxidase Dazl is a protein required for both male and female labeled secondary antibodies (Sigma). The basement germ cell function in different species. During mouse membrane around the spinal cord and notochord were development, the expression of this protein is first enriched with, osteonectin, especially in the region reported at 11.5 days post coitum (dpc), when the of the floor plate where commissural “C” cells axons primordial germ cells (PGC) reach the gonad. In first differentiate. A positive staining is found in the adults, Dazl is expressed in spermatogonia, primary ventricular zone, commisural axonal segment and in the spermatocytes and in the oocyte periphery. It has been floor plate. The pial surface shows a stronger pattern of shown that Dazl knockout male and female mice are immunostaining. These data indicate that interneurons infertile. More recently, it was suggested that Dazl may use multiple molecules during axonal pathway protein is involved in PGC proliferation. Thus, the formation, depending on cell type, pathway position, aim of the present study was to count PGC in mouse and developing [email protected] embryos at different ages and isolate them to check their proliferation in vitro. PGC from Dazl knockout and D-029 wild type embryos were counted at 8.5, 9.5, 10.5 and KIDNEY EXPRESSION OF 11.5dpc. At the same ages, these cells were isolated RENIN-ANGIOTENSIN SYSTEM PROTEINS IN 1. 126

livro.indd 126 7/7/2008 16:33:38 MALES OFFSPRING AFTER MATERNAL LOW rats. We not found differences between groups in both PROTEIN DIET: KIDNEY FUNCTION AND BLOOD birth weight, ano-genital distance and onset of puberty. PRESSURE There were no differences between BP and right and Flávia Fernandes Mesquita (Unicamp), Amanda left ovarian weight. In the LP group the right ovarian Roberta de Almeida (Unicamp), José Antonio Rocha have reduced weight when compared to NP (0,0202 ± Gontijo (Unicamp), Patrícia Aline Boer (Unesp) 0.001 vs. 0,0327 ± 0,008 P = 0.02).By Western Blot we observed significant differences between the NP and LP Low maternal dietary protein during pregnancy is groups in the PCNA (NP 4144.26 ± 124 vs. NP 4481.8 associated with retardation of fetal growth, renal ± 254; p<0.05) as well as in the HSP90 expression alterations and adult hypertension. We investigated (NP 737.17 ± 176.43 vs. LP 1005.14 ± 96.89; p<0.03). whether low protein diet intake during pregnancy Immunocytochemical labeling of HSP90 and PCNA altered kidney expression of angiotensin II type 1 confirmed these data so that positive reaction for (ATR1) and type 2 (ATR2) receptors, their pathways HSP90 has been observed preferentially in the follicular and if the modified expression of the RAS compounds cells where the nucleus are positive to PCNA. This is associated with changes in urinary sodium and in fact indicate that HSP90 is probably working in an arterial blood pressure in 16 week-old males offspring of anti-apoptotic way, which might direct or indirectly the underfed group. The pregnancy dams were divided activate the follicular cell proliferation, whose adaptation in two groups: with normal protein diet [pups named response could guarantee the function of the ovary from NP] or low protein diet [pups LP] during all pregnancy. females submitted to maternal protein restriction during The present data confirm a significant enhance in their fetal [email protected] arterial pressure in the LP group. By immunoblotting and immunohistochemical analysis the study showed D-031 a significantly decreased expression of RAS pathway EXPRESSION OF ADRENAL RENIN-ANGIOTENSIN protein and Ang II receptors in the kidney of LP rats. SYSTEM SIGNALING IN MATERNAL PROTEIN- The present investigation also showed, by lithium RESTRICTED OFFSPRING: EFFECT ON clearance, a pronounced decrease in fractional urinary SYMPATHOADRENAL LINEAGE DIFFERENTIATION sodium excretion in LP, compared with the NP age- AND BLOOD PRESSURE matched group. This occurred despite unchanged Juliana Cristina Longo Frederico (UNESP), Flávia creatinine clearance. The current data led us to Fernandes Mesquita (UNICAMP), Sebastião Roberto hypothesize that under conditions of protein restriction, Taboga (UNESP), José Antônio Rocha Gontijo the fetus responds, in the short-term, by making (UNICAMP), Patrícia Aline Boer (UNESP) suitable adaptations to accommodate the disturbance in intrauterine substrate supply. After birth, under normal Low maternal dietary protein during pregnancy is a protein diet intake, the long-term outcome of these classical model of programming adult blood pressure adaptations is represented as an absolute and relative (BP). Fetal overexposure to glucocorticoids is thought small urinary sodium excretion capacity associated to be central to the nutritional programming of BP. with increased blood pressure and a relative propensity Progenitor sympathoadrenal sublineage of the neural toward a diseased state in adult life. crest in developing rat embryos, can be differentiated [email protected] in chromaffin cells by glucocorticoid induction. Adrenal renin-angiotensin system (ARAS) signaling expression D-030 are also important during development and are involved PROTEIN RESTRICTION DURING RAT FETAL in the control of cardiovascular, renal and adrenal DEVELOPMENT AND OVARIAN RESPONSE IN function governing body fluid and electrolyte balance, ADULT FEMALE PROGENY as well as BP. The purpose of the present study was Camila de Campos Juliani (Unesp), Luis Antonio to determine if protein restriction during pregnancy Justulin Junior (Unesp), Elaine Cristina Silva-Zacarin alters the number of chromaffin adrenal cells and (UFScar), Daniela Carvalho dos Santos (Unesp), José the expression and localization ARAS proteins, in 16 Antonio Rocha Gontijo (Unicamp), Patrícia Aline Boer weeks old male offspring. Pregnant rats were submitted (Unesp) to a normal protein (NP), or a low protein (LP) diet during all pregnancy. The pups systemic BP increase Nutrient restriction during pregnancy impairs overall significantly in LP than in NP rats from 6 to 16 weeks fetal growth and development. We analyze the effects of age. Western blot analysis showed that expression of maternal protein restriction on the 90kDa heat shock of ARAS signaling proteins were significantly greater protein (HSP90) and PCNA in order to evaluate the in the adrenal of LP offspring. Immunohistochemical role played by this protein in the adult ovary. Rats were analysis showed a significantly enhancement of ARAS fed either normal protein diet (NP) or a low protein diet compounds expression in whole adrenal regions (LP) during pregnancy. Pup weight and ano-genital accompanied by increased tyrosine hydroxilase distance was recorded at birth. Onset of puberty was immunorreactivity in the adrenal medullar cells of LP defined as the time of vaginal opening. The blood rats. Speculatively, it seems that adrenal inappropriate pressure (BP) was measured in conscious 17-week-old AngII pathways expression may result in increased 1. 127

livro.indd 127 7/7/2008 16:33:38 adrenal number of chromaffin cells, contributing to the ubiquitous overexpression of a transgene encoding adult development of hypertension in low maternal the Rst ectodomain (ECDRst), under the control of protein intake model. an inducible heat shock promoter, can either block [email protected] blastoderm cellularization or prevent myoblast fusion, depending on the developmental time it is induced D-032 during embryogenesis. Recently we observed that SPACE-TIME RELATIONSHIP AND DISTRIBUTION ECDRst induction for 2 hours in 3 hour old embryos OF COLLAGEN TYPES I, III AND IV IN THE often lead to longitudinal separation around the EMBRYOLOGIC DEVELOPMENT OF THE INNER, midline of the normally fused two simetric halves of MIDDLE AND EXTERNAL EAR the ventral cord and abnormalities in comissures of Mauricio Soto-Suazo (Escuela de Medicina-Facultad nervous system. This defect of neural organization de Ciencias Médicas-Universidad de Santiago de affects more often the two or three most proximal Chile), Gonzalo Leiva Rojas (Escuela de Obstetricia- abdominal parasegments. Additionally, we showed Facultad de Ciencias Médicas-Universidad de Santiago that this phenotype is strongly enhanced by a de Chile), Samir Zara Zara (Escuela de Obstetricía- chromosomal deficiency that removes the hibris gene: Facultad de Ciencias Médicas-Universidad de Santiago flies heterozygotes for the deficiency produce embryos de Chile), Telma Maria Tenorio Zorn (Departamento de with nervous system defects, such as expansion of the Biologia do Desenvolvimento-USP-Brasil) area occupied by the CNS and abnormal ventral cord formation when crossed with ECDRst transgene. Both The collagens play a role in assembling the extracellular types of defects are present at 25ºC, but their relative matrix, promoting cell adhesion, cell growth, and percentage varies as the temperature is upshifted, permitting cell differentiation. The aim of this work with CNS expansion being more common at lower was to establish the distribution and space-time temperatures. Finally we show that Roughest and Hibris relationship of collagen types I, III and IV in the ear are both present at midline at the same time, suggesting embryologic development. Mouse embryos samples that these proteins might bind each other during ventral aged 9, 10, 11, 12, 13, 14 and 15-days of development cord midline formation. were fixed, dehydrated, embedded in paraffin and [email protected] cut in 5 µm-thick serial sections. The sections were incubated with antibodies against collagen types I, D-034 III and IV. The immunoreactions were visualized by EXPRESSION PATTERN OF JAZIGO DURING THE the peroxidase-antiperoxidase method. Collagen PROGRAMMED CELL DEATH OF THE DROSOPHILA types I and IV were observed in the basal membrane MELANOGASTER LARVAL of the inner ear during all the development phases. SALIVARY GLAND However, collagen type III was expressed only in Ana Paula Saita (USP), Lucas Anhezini Araujo (USP), the early phases. Otherwise, collagen types III and Mara Silvia Alexandre Costa (USP), Ricardo Guelerman IV were located in the mesenchyme of the inner ear Pinheiro Ramos (USP), Claudio Roberto Simon during all the development phases. Moreover, type I (Universidade Federal do Triângulo Mineiro) collagen was expressed only in the later phases. In the development of middle ear the collagen types I, III and In a genetic screen we have identified a mutant IV were observed during all the phases. Ectoderm basal showing a drastic defective phenotype of larval membrane of the auricle expressed collagen types I, salivary gland programmed cell death. This mutation III and IV and only type III was observed in the inner causes the persistency of this tissue beyond its normal mesenchyme of the auricle. Our results showed that developmental fate, which is to be eliminated during collagen types I, III and IV are differentially expressed in the transition of the pre-pupal to pupal stage of the time and distribution correlated with ear morphogenesis, Drosophila melanogaster development. This mutation suggesting that these molecules have a role on the is caused by a P-element insertion flanking the coding development this organ. region of the gene. We have named this gene jazigo [email protected] and bioinformatics analyses reveled great homology, at the gene and protein level, to components of the family D-033 of proteins formins. These proteins are conserved ROUGHEST AND HIBRIS ARE BOTH REQUIRED throughout eukaryotes and its most prominent role has FOR VENTRAL CORD FORMATION IN DROSOPHILA been related to the remodeling of cytoskeleton, mainly Lívia Maria Rosatto Moda, Mara Silvia Alexandre Costa, by functioning as Ricardo Guelerman Pinheiro Ramos (Departamento bridging factors for the actin/microtubule assembling Biologia Celular e Molecular e Bioagentes Patogênicos machinery, during mitosis, establishment of cell polarity FMRP-USP) and embryonic development. Based on both the drastic cell death phenotype (lethal during metamorphoses) Roughest (rst) encodes a phylogenetically conserved and presumptive important function, our major goal is IgCAM that has multiple functions during Drosophila the genetic characterization of the jazigo mutants along development. We have previously shown that the with the biochemical charaterization of Jaz protein. 1. 128

livro.indd 128 7/7/2008 16:33:38 Here we show the progress toward the expression from healthy individuals (PBC). For transcript analysis, and purification of a Jaz recombinant peptide named reverse PCR was used, and for protein detection, Jaz-rec II used for the immunization e purification of a immunoprecipitation, followed by SDS-PAGE and polyclonal antibody against Jazigo and its use to identify Western Blot. RESULTS: Transcripts for HER1 were the expression pattern of Jaz during the programmed seen in 3/4 B-lymphoid cell lines representative cell death of Drosophila melanogaster larval salivary of Burkitt Lymphomas (Daudi, Ramos, Raji), and gland. Supported by CAPES and FAPESP. the erythroid K562. For HER4, only Raji, while for [email protected] HER2, 12/14 cell lines were positive, besides, and unexpectedly, 5/5 PBC. None of the lines showed D-035 the canonical HER1 transcript, suggesting alternative RSTD: AN UNSTABLE MUTATION IN THE RST GENE processing. We could not obtain HER1 peptides from REGULATORY REGION INTERFERING WITH ITS Daudi cell extracts, but for HER2 we obtained the EXPRESSION PATTERN DURING DROSOPHILA EYE expected 185 kDa band, plus a smaller one, which DEVELOPMENT identities are under investigation. Transcripts for EGF, Shirlei Octacilio da Silva, Maiaro Cabral Rosa Machado, TGF-a, and HB-EGF, were broadly expressed in Mara Silvia Alexandre Costa, Ricardo Guelerman these cell lines, as well as in PBC that, however, did Pinheiro Ramos (FMRP - USP) not bear HER1/4. CONCLUSIONS: The presence of transcripts for HER1 in Burkitt Lymphomas, and their The roughest (rst) gene is pleiotropic during D. absence in Pre-B cell lines (0/6) and PBC (0/5) suggest melanogaster development. It was observed that downregulation during B-lymphoid development, with mutations affecting the transcriptional and regulatory a return in malignancy, as a secondary and relatively portions of this gene generate different phenotypes. late event. The meaning of HER1 transcripts alternative In both cases, molecular alterations cause defects processing remains as a question, as well as the ligand in the eye development; the former is related to peptides presence in both PBC and leukemic cells, lack of programmed cell death and the eyes have and the putative biologic function of HER2 in leukocyte a “rough” appearance; the second, to problems in subpopulations. the specification of secondary and tertiary pigment [email protected] cells and the eyes have a “glassy” appearance. rstD regulatory mutation is unstable and frequently reverts to individuals with wild type or lack-of-function- like eyes. Here we show that the revertants have phenotypic behaviors clearly different from each other, from the wild type and from the parental rstD, being a consequence of secondary rearrangements over the rstD mutation, and not a true reversion to the wild type state. Sequence analyses of this altered region were also performed showing that the mutant fragments appear similar to the wild type sequence deposited in the database, so the rstD mutation and the other revertants remain to be completely defined. Finally, quantitative real time PCR showed that rst is regulated at transcriptional level during retinal development as previously suggested by its protein expression pattern and that the rstD regulatory pattern differs from the wild type. So, alterations in 5´ region of the rst gene affects its transcriptional, as well as, translational regulation. [email protected]

D-036 EXPRESSION OF HER FAMILY RECEPTORS AND LIGANDS BY HUMAN LEUKEMIC CELL LINES Gabriel Padron Rios, Camila Santos de Moraes, Claudia Sondermann Freitas (INCA)

OBJECTIVE AND METHODS: Beyond development, HER-family receptors and their ligands (EGF-like growth factors) show ubiquitous expression, except for mature blood cells. We searched for transcripts and proteins of these families in 14 human leukemic cell lines, compared to mature, peripheral blood cells 1. 129

livro.indd 129 7/7/2008 16:33:38 Education congenital defects. They consist in two parts: 1) a theoretical contents with texts, animations and the macro- and microscopical digital documentation of E-001 embryos, fetuses and neonates and 2) one or more HOW DOES A TEACHER TEACH AND EVALUATE multiple choice questions. The digital quizzes were THE UNDERSTANDING OF THE CELL BIOLOGY evaluated by students at the first year of UNICAMP CONTENT IN HIGH SCHOOL? Medicine’s course in 2006, through the use of Mestrando Julio Sergio dos Santos, Prof. Dr. Angelo quantitative and qualitative instruments. About 98.8% Luiz Cortelazzo of the students had had an attitude at least positive in (Instituto de Biologia - Unicamp) relation to digital quizzes. They had emphasized that the theoretical part of the quizzes allow them to solve all In this study we analysed the assessments used by the multiple choice questions; stimulate the reasoning; biology teachers in high schools, in relation to the add new information; put together the basic to the clinic subject of cell biology. We draw a parallel between the areas; deal with relevant concepts to the professional assessments and the teaching methods used in two formation and develop abilities in the resolution of central and two suburban schools. We analysed the clinical problems. These results allow us to conclude textbook content, exercises and the class planning that this method constitute an adequate complementary for cell biology teaching. Also, we proposed several tool for the education of Human Embryology. questions to analyze the teacher’s view of teaching, [email protected] learning and how the assessment is inserted in this process. We used two questioning methods. In the E-003 first case, we employed a partly structured interview MICROCOPY MODELS: REAL INCLUSION with fifteen biology teachers. The second consisted OF DEFICIENT VISUAL OF COLLEGE IN THE in a questionnaire using the Likert scale, which was MORPHOLOGIC DISCIPLINES answered by fifty-nine teachers that administered Michele Waltz Comarú dos Passos (Fiocruz), Renata several different subjects. In this research, we found Santos de Oliveira (Faculdade Estácio de Sá - Vitória), that the textbook contents and exercises interfere Tatiana Ferreira Pimentel Santana (Faculdade Estácio strongly in the planning of assessments. This influence de Sá - Vitória), Claudia Mara Lara Melo Coutinho leads to the teacher’s questioning the exact textbook (Fiocruz) content, which is fragmented. In the central schools, we noticed that the contents were more profound than Morphologic disciplines are ministered in every in suburban schools. In this case study, we noticed graduations of Health area. In cytology and histology, that the assessment of cell biology was based on is very important the visualization of structures fragmented contents and there is a lack of dynamic in microscopy practical classes. Visual deficient learning, in which cellular functions and structures students do not have accessibility to this education would be integrated. Also, these courses often do not methodology. In agreement with the School Sense promote constructive learning and we found very little accomplished by ministry of education in 2004, there evidence of effective course renewal and of efforts to were 5392 registrations of students with special needs give a better formation in cell biology. in Brazilian colleges. These new demands of students [email protected] require adaptations in the process teaching-learning of several disciplines, specialty morphologic disciplines. E-002 Our work is based on providing inclusion to these DIGITAL QUIZZES FOR TEACHING HUMAN students, therefore, to allow the permanency of them EMBRYOLOGY. A DEVELOPING AND EVALUATION in the same class with the others, giving the same OF A METHODOLOGY knowledge and, being graded too. For this were made Ana Gabriela Esteves Duarte, Suzana Guimarães microscopy models to histological images in several Moraes, Luis Antonio Violin Pereira (UNICAMP) materials, such as pin, special papers, beads, on wood boards, measuring a sheet size A4 (210X297mm), The congenital anomalies are responsible for the obeying the similarity of the tissue. The structures were majority of the deaths in the first infancy. In the standardized for the same structure to be seen equality traditional Human Embryology courses the students in every models. A practical class test was made have difficulties in understanding and in creating with the students in order to evaluate the quality and three-dimensional mental images of the processes applicability of the models. The results showed the blind involved in congenital anomalies. The use of multimedia students in classroom equality learned (grade 4) as stimulates the knowledge and the creativity and makes other students using the models. We concluded that it possible solutions to proposed problems. In this study is possible to teach for blind students the same content an education methodology for Human Embryology with different methodology without compromising quality based on digital quizzes has been developed. The of the class and creating a real inclusion. quizzes correlated concepts of Embryology with the [email protected] genesis, diagnosis and treatment of the more frequent 1. 130

livro.indd 130 7/7/2008 16:33:39 E-004 Fundamental Cell Biology THE INVESTIGATIVE POSTURE OF THE CELL BIOLOGY’S TEACHING: REDRAWING CRITICAL AND INTERACTIVE ISSUES BETWEEN TEACHERS AND HIGHER F-001 EDUCATION STUDENTS TGFALPHA AND EGFR ROLES IN THE Priscilla Motta Ramos Lara, Fábio Camargo PROLIFERATION AND DIFFERENTIATION OF THE Abdalla, Fabiana Gimenes, Hylio Laganá Fernandes GASTRIC EPITHELIUM IN DEVELOPING RATS (Universidade Federal de São Carlos) Luciana Harumi Osaki, Marco Aurélio Fauni Curi, Priscila Moreira Figueiredo, Cruz Alberto Mendonça The interactions among individuals may help the Rigonati, Eliana Parisi Alvares, Patrícia Gama (USP) construction of the individuality. Once the individuality and the self-perception of an individual is full The development of the gastric mucosa is controlled by constructed, it permits the one naturally develops the hormones, growth factors and feeding behavior. Early critical posture inside a multicultural society, specifically weaning (EW), that means the abrupt interruption of in education. To stimulate share and knowledge suckling, increases proliferation and differentiation in development, it is necessary arouse the desire to the rat gastric epithelium, in parallel with the over- comprehend. The desire is fundamental in the learning expression of Transforming Growth Factor alpha process. In the educational scope, this is a challenge (TGFalpha) and its receptor EGFR. To study how for all professors. The present investigation focus on they can control gastric growth we evaluated EGFR this relation and how it can be ruptured to construct activation and signaling through ERK, Akt and Src an education of better quality, and more: with great pathways. 15-d-old Wistar rats were split into 2 satisfaction by both students and professor. We present groups: suckling (control) and EW, to which pups were two dynamics with the ingress-graduating students of separated from the dam and fed with powdered chow. biological sciences utilizing bi and three-dimensional Sacrifices occurred on days 15, 16, 17 and 18th. We dynamics to construct their own concept of cells (by also tested the effects of EGFR inhibition by treating build cell tool made of various magnetized organelles pups with AG1478. The proteins involved in EGFR that could be attached on a metal plaque and by play- signaling and cell cycle were accessed by Western blot. doh modeling cell organelles). During the activities, the Mucous neck cells maturation was followed in samples students had the stained with PAS-AB or BSII lectin. Cell proliferation opportunity to reflect about their previous concepts and was studied by DNA (SI) and mitotic indices (MI) after to compare their prior knowledge with the scientifically AG1478 use. EW induced high levels of signaling accepted; building closer proper concept that is in proteins: p-ERK1/2 and p-Src at 17d (p<0.05), and did fact the cell. During the process the professor, in not alter Akt. Mucous neck cells increased throughout an investigation posture, exploring the potential of EW, and differences to controls were pronounced at students (their individualities), deconstructing mistaken 17d (p<0.05). At 18d, AG1478 use decreased SI, MI concepts and enhancing the learning process, once the and the number of mucous neck cells when compared curiosity and the endeavor to find answers of emergent to controls (p<0.05). We showed that feeding pattern questionings provoke the interesting for the knowledge does influence the proliferation and differentiation of by the students, which are shared among them. the gastric epithelium and suggest that TGFapha/ [email protected] EGFR can regulate these processes during postnatal development, probably by activating MAPK and Src signaling pathways. Support: FAPESP. [email protected]

F-002 DIFFERENTIAL EFFECTS OF FASTING ON CORTICOSTERONE LEVELS IN SUCKLING AND ADULT RATS: CONSEQUENCES TO THE GASTRIC EPITHELIUM Daniela Ogias (USP), Carolina Botini Prates (USP), Ariane Kasai (USP), Eliana Parisi Alvares (USP), Eunice Ribeiro de Andrade Sá (USP), Marie-Pierre Moisan (Inserm), Patrícia Gama (USP)

The growth of the stomach is controlled by an interaction of milk- borne hormones and growth factors, and feeding behavior. Among these elements, glucocorticoids (GCs) play a special role. We have shown that fasting increases corticosterone and stimulates cell proliferation in the gastric epithelium 1. 131

livro.indd 131 7/7/2008 16:33:39 of pups, whereas in adults, it is inhibitory. Because papain, respectively. These results show that the fasting triggers opposite responses in the stomach, mechanical properties of chicken tendons are altered and GCs may control growth, we studied GCs behavior by enzymatic treatment, and that collagenous and towards fasting in suckling and adult rats. Animals non-collagenous components are important for proper were fasted according to age. Blood was analyzed by functioning of this organ. RIA, microfiltration and binding assays to respectively [email protected] determine total and free corticosterone, and CBG. GCs receptor (GR) was identified in the gastric mucosa F-004 by immunohistochemistry and measured in scraped DIFFERENT WAYS OF CELL DEATH INDUCED IN samples by Western blot. We found that long-term TRYPANOSOMA CRUZI BY fasting increased total and free corticosterone by 7 CHEMOTHERAPIC AGENTS: AN and 20 times in pups and adults, respectively, when ULTRASTRUCTURAL STUDY compared to fed controls (p<0.05). CBG binding Rubem Figueiredo Sadok Menna-Barreto (Instituto increased 12.5 times in fasted pups, whereas in adults Oswaldo Cruz, Fiocruz), Kelly Salomão (Instituto it augmented only 2 times, indicating that the avidity Oswaldo Cruz, Fiocruz), Andréia Pires Dantas (Instituto of CBG for corticosterone is higher in suckling fasted Oswaldo Cruz, Fiocruz), Ricardo Mattos Santa-Rita rats. GR was found in the cytoplasm and/or nucleus (Instituto Oswaldo Cruz, Fiocruz), Maurilio José Soares of epithelial cells and levels augmented (p<0.05) (Instituto Carlos Chagas, Fiocruz), Helene Santos after short-term fasting at both ages. We suggest that Barbosa (Instituto Oswaldo Cruz, Fiocruz), Solange although fasting stimulates GCs secretion in pups and Lisboa de Castro (Instituto Oswaldo Cruz, Fiocruz) adults, corticosterone is higher and seems to be more available in adults, due to the lower CBG binding. Such The unsatisfactory treatment of Chagas disease differences might account for the opposite proliferative determines the search for alternative drugs, with responses detected at distinct ages in the rat gastric an intense investigation of potential targets in mucosa. Support: FAPESP, CAPES, CNPq. Trypanosoma cruzi such as specific molecules, [email protected] metabolic pathways and organelles. In this context, electron microscopy represents a powerful tool for F-003 morphological evaluation. Programmed cell death THE ROLE OF NON-COLLAGENOUS COMPONENTS (PCD) is fundamental in the homeostasis of multicellular IN THE MECHANICAL PROPERTIES OF CHICKEN organisms, playing a role in morphogenesis, physiology TENDONS and host defense against pathogens. Apoptosis-like, Flávia de Paoli (Universidade Estadual de Campinas autophagy and necrosis led to ATP depletion, acting and Centro Universitário Serra dos Órgãos), Tatiana the mitochondria as a central checkpoint. PCD induced Carla Tomiosso (Universidade Estadual de Campinas), by drugs have been described in trypanosomatids Edson Rosa Pimentel (Universidade Estadual de such as Leishmania spp. Our morphological evidences Campinas), Benedicto de Campos Vidal (Universidade show that treatment of Trypanosoma cruzi with Estadual de Campinas) different classes of compounds can lead to dramatic mitochondrial alterations and autophagic phenotypes. Mechanical functions of the tendons depend on It is well-known that a single stimulus can trigger an composition, organization and precise interaction alternative pathway when the original one is inhibited. of their extracellular components. In this work, we The intense cross-talking between the three types of hypothesized, according to previous reports, that the PCD strongly suggests the involvement of different non-collagenous components have an important role mechanisms leading to the protozoan death. in the mechanical properties of tendons. In order to [email protected] investigate this hypothesis, we used chicken flexor tendons treated with testicular hyaluronidase or papain, F-005 which were compared with non-treated tendons. C-ALLYL LAWSONE DERIVATIVES INDUCED After incubation with these enzymes, the tendons ROS PRODUCTION AND MITOCHONDRIAL were processed for biochemical analysis, or were DYSFUNCTION IN TRYPANOSOMA CRUZI immediately fixed and processed for histochemical EPIMASTIGOTES and morphological analyses. Tendons treated with Rubem Figueiredo Sadok Menna-Barreto (Instituto hyaluronidase or papain showed a decrease in Oswaldo Cruz, Fiocruz), Renata L. S. Gonçalves their ultimate tensile, elastic energy and elasticity (IInstituto de Bioquímica Médica, UFRJ), Raphael S. modulus values. In contrast, the ultimate strain values F. Silva (Núcleo de Pesquisas em Produtos Naturais, were highest after treatment with hyaluronidase, UFRJ), Antonio Ventura Pinto (Núcleo de Pesquisas em indicating that the tendons lost their capacity to resist Produtos Naturais, UFRJ), Marcus F. Oliveira (Instituto tensile forces after incubation with this enzyme. de Bioquímica Médica, UFRJ), Solange Lisboa de Histochemical and biochemical analyses confirmed that Castro (Instituto Oswaldo Cruz, Fiocruz) glycosaminoglycans and non-collagenous/collagenous components were removed by hyaluronidase and The chemotherapy for Chagas’ disease using nifurtimox 1. 132

livro.indd 132 7/7/2008 16:33:39 or benznidazole is unsatisfactory, mainly due to severe Chagas’ disease. side effects and the controversy about the treatment [email protected] of chronic patients. Our group has been assaying natural quinones isolated from Brazilian flora and its F-007 derivatives against Trypanosoma cruzi. From C-allyl STIMULATION OF BOTH ALPHA- AND BETA- lawsone were obtained three naphthofuranquinones ADRENOCEPTORS ACTIVATES TRANSCRIPTION active against trypomastigotes and epimastigotes. In FACTORS IN VENOM GLAND OF BOTHROPS the present work, further investigations were performed JARARACA SNAKE to elucidate machinery involved in their trypanocidal Milene Schmidt Amaral Luna (Instituto Butantan), activity. Ultrastructural and flow cytometry analysis of Zulma Silva Ferreira (Instituto de Biociências), Norma treated epimastigotes pointed to the mitochondrion, as Yamanouye (Instituto Butantan) a primary target organelle. Biochemical assays showed an increase in hydrogen peroxide generation in treated Activation of noradrenergic innervation triggers the parasites, compromising the activity of mitochondrial venom production cycle in Bothrops jararaca venom respiratory chain complexes I-III and oxygen uptake. gland by regulating synthesis of proteins of venom Our data, suggests that the mode of action of gland that probably is essential to activate the gland naphthofuranquinones is associated with oxidative (Luna et al., Comp. Biochem. Physiol, S107, 2007). In stress and mitochondrial impairment, and consequently this study we verify whether stimulation of both a- and leading to energetic collapse and death of the parasite. b-adrenoceptors with noradrenaline (NA), phenylephrine [email protected] (PHE) or isoprenaline (ISO) is able to activate NFkB and AP-1 transcription factors. Dispersed quiescent F-006 secretory cells were incubated with NA (10-4M), PHE, ANTI-TRYPANOSOMA CRUZI ACTIVITY OF NEW (3X10-4M) or ISO (3X10-4M) for 30 or 60 min at 30°C DERIVATIVES FROM NOR-LAPACHONES AND and the activation of transcription factors were analyzed LAPACHONES by electrophoretic mobility shift assay. NA and ISO Michelle Casal Fernandes (IOC, Fiocruz), Rubem F. (30 min) increased activation of NFkB (24.60 ± 8.83%, S. Menna-Barreto (IOC, Fiocruz), Eufrânio N. da Silva n=6; 19.31 ± 5.29%, n=6) and AP-1 (12.01 ± 4.21%, Júnior (Instituto de Química, Universidade de Brasília), n=3, 25.49 ± 7.73%, n=3). PHE (30 min) increased the Maria do Carmo F. R. Pinto (NPPN, UFRJ), Francisco activation of NFkB (30.35 ± 4.58%, n=8), but decreased de Assis Lopes (NPPN, UFRJ), Antônio Ventura Pinto the activation of AP-1 (-12.67 ± 2.94%, n=9). After 60 (NPPN, UFRJ), Vitor F. Ferreira (Instituto de Química, min of incubation, NA, ISO and PHE increased the UFF), Solange Lisboa De Castro (IOC, Fiocruz) AP-1 activation (19.19 ± 5.92%, n=4; 25.52 ± 12.30%, n=5 and 13.93 ± 6.81%, n=6, respectively). Inhibitors of Currently, the treatment of Chagas’ disease is phopholipase C, U73122 (10mM), protein kinase C, H89 unsatisfactory, being limited to two nitroheterocycles, (90mM) or staurosporine (100nM) and protein kinase benznidazole and nifurtimox. In this context, our group A, PKI (10mM), completely inhibited NA responses. In is involved in the synthesis and the evaluation of the conclusion, noradrenaline, released after stimulation trypanocidal activity of new naphthoquinones prepared of noradrenergic innervation, triggers the venom from nor-lapachones and lapachones. Stock solutions production cycle by activating transcription factors via of the compounds were prepared in dimethylsulfoxide phospholipase C/protein kinase C and protein kinase A. (DMSO), with the final concentration of the solvent Supported by: FAPESP. never exceeding 0.1%. The assays were performed [email protected] with trypomastigote forms from the Y strain (107 cells/ ml) in the presence of 5% of mouse blood. Compounds F-008 15 and 20 presented values of IC50/24 h of 86.3 ± ORGANIZATION OF THE DIGESTIVE SISTEM OF 4.6 and 88.2 ± 6.7 µM, respectively, both being more THE STICK BUG PHIBALOSOMA active than benznidazole (Bz). Compounds 7, 10 and PHYLLINUM (PHASMIDA, PHASMATIDAE): A 14 showed activity similar to Bz, in the range of 140 MORPHOLOGICAL AND BIOCHEMICAL to 180 µM. For 13, 17, 18, 19 and 22, this parameter APPROACH was situated between 300 and 1000 µM, and the Emiliano Carneiro Monteiro (Departamento de other derivatives were considered inactive. The 1,2- Genética e Biologia Evolutiva IBUSP), Walter Ribeiro and 1,4-naphthoquinones are considered privileged Terra (Departamento de Bioquímica do IQUSP), structures in medicinal chemistry, being the easiness Alberto Augusto Gonçalves de Freitas Castro Ribeiro of reduction-oxidation of the quinoidal moiety the basis (Departamento de Genética e Biologia Evolutiva do for their participation in electron transport and oxidative- IBUSP) phosphorylation processes. Ours results showed that the most of the derivatives assayed were more active The digestive process in Phasmids species is than the original quinones, suggesting that this type of completly unknown, what motivated the study of the compounds are a good starting point for a medicinal digestive system of P.phyllinum, histologically and chemistry investigation aiming the chemotherapy of ultrastructurally. For histological observation, the 1. 133

livro.indd 133 7/7/2008 16:33:39 whole gut was fixed in Bouin’s solution and embedded Preliminary results of the distribution of digestive in historesin. For ultrastructural analysis using the enzymes revealed the presence of amylase and trypsin transmission electron microscope, the midgut was fixed that occur in the luminal contents; aminopeptidase and in 3% glutaraldehyde solution in sodium cacodilate maltase is found in the epitelium along the ventriculus. buffer 0,1M (pH 7,4), postfixed in 1% osmium tetroxide Supported by CNPq and FAPESP. and embedded in Spurr resin. The digestive system of [email protected] P.phyllinum is made up of 3 regions: foregut, midgut and hindgut. The foregut epithelium is composed of F-010 a squamous epithelium covered with a cuticle. The USE OF RT-PCR TO IDENTIFY GENES midgut is formed by a tube (ventriculus), where 3 main SELECTIVELY MODULATED BY CHEMICAL TOXIC cell types are present: the columnar cells (enterocytes), IN RECONSTITUDED HUMAN EPIDERMIS the endocrine and regenerative cells. The enterocytes Maria Carolina Braga de Azeredo, Paula Padilha, are tall polarized cells with apical microvilli. The Natália Haddad, Radovan Borojevic (UFRJ) cytoplasm is rich in Golgi areas and stacks of rough endoplasmatic reticulum, mainly near the central The development of in vitro reconstructed human nucleus. The basal plasma membrane is infolded with epidermis represents a suitable alternative to the use associated mitochondria. Secretory vesicles are seen of animals for pharmacological and pharmaceutical along the ventricular epithelium. In the anterior and research, due to the different properties of human skin middle ventricular regions these vesicles are eliminated when compared to animal skin. In addition, several by exocytosis, whereas in the posterior region they scientific and ethical issues against the use of animal are extruded through a microapocrine mechanism. A models have been raised. We have already developed complex system of ventricular tubules, whose function our epidermal in vitro model and established the best is still unknouwn, connects the posterior ventriculus to culture conditions. In order to better characterize the the region of insertion of the Malpighian tubules. The model’s toxicological responses, gene expression of hindgut is made up of a simple cuboidal epithelium cytokines TGF-â1, IL-1 á and IL-8 was determined. covered by cuticle. Suported by CAPES and FAPESP. Human keratinocytes were cultured on a 3T3 feeder [email protected] layer until confluency was reached, being subsequently inoculated on a polycarbonate filter membrane coated F-009 with porcine gelatin. The system was then raised to MORPHOLOGICAL AND BIOCHEMICAL ASPECTS the air-liquid interface until a stratified epidermis was OF THE DIGESTIVE SYSTEM OF GRYLLODES formed. The model was later exposed to two different SUPPLICANS (ORTHOPTERA, GRYLLIDAE) substances whose effects in skin were previously Fernanda Pompilio Biagio (UNIFESP), Walter Ribeiro known, the Sodium Dodecyl Sulfate (SDS) and Tween- Terra (USP), Alberto de Freitas Ribeiro (USP) 20. Following the 24 hour exposure, we performed a semiquantitative RT-PCR analysis of mRNA levels from The digestive system of the cricket Gryllodes supplicans cytokines TGF-â1, IL-1 á and IL-8. When exposed to was analyzed histologically and ultrastructurally. For the irritating substance, mRNA levels from all analysed histological observation, the gut was fixed in Bouin’s cytokines were detected in increased amounts. When solution and embedded in historesin. For ultrastructural exposed to the non irritating substance, however, analysis by transmission electron microscopy, the the cytokines mRNA levels were low or absent, midgut was fixed in 3% glutaraldehyde solution in corresponding with the expected results and with data sodium cacodylate buffer 0,1M, postfixed in 1% already shown in the literature. Thus, these results osmium tetroxide and embedded in Spurr resin. The reinforce the realibility of our model in discriminating digestive system of G. supplicans has 3 regions: irritating and non irritating substances. foregut, midgut and hindgut. The foregut is composed [email protected] by the pharynx, esophagus, crop and gizzard. The midgut consists of a ventriculus and 2 anterior caeca. F-011 The hindgut is composed by the ileum, colon and SELECTIVE INDUCTION OF INFLAMMATORY rectum. Histologically, the midgut is composed by 2 MEDIATORS IN RECONSTRUCTED HUMAN cells types: the columnar and the regenerative cells. EPIDERMIS Ultrastructurally, the midgut present basal plasma Maria Carolina Braga de Azeredo, Paula Padilha, membrane invaginations in the columnar cells of Natália Haddad, Radovan Brojevic (UFRJ) the basal plasma membrane. The apical surface is modified into microvilli and it shows secretory vesicles The utilization of classical animal models in toxicological with different aspects according of the region. Golgi assays has been increasingly restricted by laws and areas and stacks of rough endoplasmatic reticulum regulamentations. These models are constantly being can be seen around central nucleus. A presence of questioned as to their validity, due to the different a peritrophic membrane in the ventricular lumen was properties of human skin when compared to animal confirmed by fluorescence confocal microscopy using skin. In addition, several ethical issues against the WGA-FITC under special experimental conditions. use of animals in testing have been raised. Therefore, 1. 134

livro.indd 134 7/7/2008 16:33:39 the development of in vitro human epidermal models fasciculata. TcKAPs are small and highly basic proteins, represents a suitable alternative to this use. After presenting a cleavable pre-sequence potentially establishing the best culture conditions for our involved in the mitochondrial import. Additionally, they epidermal in vitro model, we proceeded to evaluate are differentially distributed within the kinetoplast of its toxicological responses by measuring the pro- epimastigote, amastigote and trypomastigote forms. inflammatory mediators TGF-β1 and IL-1α. Human Based on our results, we suggest that TcKAPs may be keratinocytes were cultured on a 3T3 feeder layer until involved in charge neutralization and condensation of confluency was reached, being subsequently inoculated the kDNA network, as well as in the maintenance of the on a polycarbonate filter membrane coated with porcine topological organization of the kinetoplast during T. cruzi gelatin. The system was then raised to the air-liquid differentiation. interface until a stratified epidermis was formed. The [email protected] model was then exposed to two different substances whose features were previously known, the Sodium F-013 Dodecyl Sulfate (SDS) and Tween-20. After 24 hours CAN ESTROGEN INDUCE IN VITRO TGF-B1 SIGNAL exposure, the extracellular levels of pro-inflammatory FROM STROMAL TO FOLLICULAR THYROID mediators TGF-β1 and IL-1α were measured by CELLS? ELISA method. Our results demonstrated a significant Michel Alexandre Villani Gantus, Elaine Crsitina Lima increase in the extracellular levels of both cytokines de Souza, Joice Stipursky, Flávia Alcantara Gomes, when exposed to the irritating substance. Hence, Leandro Miranda Alves, Ana Maria Blanco Martinez, our epidermal in vitro model has demonstrated itself Luiz Eurico Nasciutti (UFRJ) capable of discriminating irritating and non irritating substances, indicating to be a promising functional The prevalence of thyroid disease on women suggests model for in vitro toxicology tests. that estrogen (E2) might be involved on thyroid [email protected] cell growth and function. TS7 thyroid stromal cells, previously isolated and characterized in our laboratory, F-012 showed the ability to synthesize basement membrane IDENTIFICATION OF KINETOPLAST-ASSOCIATED components and to express TGF-b1. In FRTL5 cells, a PROTEINS IN THE DIFFERENT DEVELOPMENTAL follicular thyroid cell line, it was observed a decrease of STAGES OF TRYPANOSOMA CRUZI iodide uptake when cultivated on TS7 cells substrate, Danielle Pereira Cavalcanti (Instituto de Biofísica Carlos suggesting the involvement of the stroma in thyroid Chagas Filho- UFRJ), Márcia Kiyoe Shimada (Instituto function. Our proposition is that the effects of E2 on de Biologia Molecular do Paraná), Wanderley de Souza follicular thyroid cells could be related to the inhibition (Instituto de Biofísica Carlos Chagas Filho- UFRJ), of TGF-b1 synthesis and secretion by stromal cells Samuel Goldenberg (Instituto de Biologia Molecular do (paracrine action). In this study, we have investigated Paraná/ FIOCRUZ), Stênio Perdigão Fragoso (Instituto the in vitro effect of E2 on TGF-b1 synthesis and its de Biologia Molecular do Paraná/ FIOCRUZ), Maria signaling pathway in TS7 cells and in a follicular cell Cristina Machado Motta (Instituto de Biofísica Carlos line, PCCL3 cells. By RT-PCR it was showed that Chagas Filho- UFRJ) only TS7 cells express estrogen receptor a (ERa). In addition, using RT-PCR, immunocytochemistry and The kinetoplast DNA (kDNA) of trypanosomatids Western blotting assays, it was demonstrated that TS7 consists of an unusual arrangement of circular cells express TGF-b1 and its receptor TGFRII, and that molecules catenated into a single network. The E2 10-8M was able to increase TGF-b1 expression and diameter of the isolated kDNA network is similar to its Smad2 phosphorilation signaling pathway, but not that of the entire cell. However, within the kinetoplast TGFRII expression. In contrast, cell viability and iodide matrix, the kDNA is highly condensed. Studies in uptake were not modified by the presence of E2 or Crithidia fasciculata showed that kinetoplast-associated TGF-b1 in the culture medium of PCCL3 cells cultivated proteins (KAPs) are capable of condensing the kDNA with or without TS7 cells substrate. These results network. However, little is known about the KAPs indicated that E2 could be able to modulate TGF-b1 of Trypanosoma cruzi, a parasitic protozoan that activity on thyroid stromal cells, however it was unclear shows distinct patterns of kDNA condensation during what is the relationship between this TGF-b1 signaling its complex morphogenetic development. In T. cruzi and the follicular cell functions. replicating forms (epimastigotes and amastigotes), [email protected] the kDNA fibers are tightly packed into a bar-shaped structure, whereas non-replicating trypomastigotes F-014 present a more relaxed kDNA arrangement contained SHP2 NEGATIVELY REGULATES GROWTH IN into a globular kinetoplast. As yet, the conversion of CARDIAC MYOCYTES BY CONTROLLING the bar-shaped kinetoplast into a globular structure FOCAL ADHESION KINASE AND THE MTOR/S6 remains unclear, but probably involves the participation KINASE PATHWAYS of KAPs. We identified two kinetoplast-associated Talita Miguel Marin, Carolina FMZ Clemente, Paty Karol proteins in T. cruzi (TcKAPs), homologous to KAPs of C. Picardi, Vinicius DB Pascoal, Íscia Lopes-Cendes, 1. 135

livro.indd 135 7/7/2008 16:33:40 Mário JA Saad, Kleber Gomes Franchini (Unicamp) stained with HE for morphological and morphometric analysis. Smallest fiber diameter was calculated and Focal adhesion kinase (FAK) has been assigned a role grouped into three classes (<20, 20-50, and >50mm). in cardiomyocyte hypertrophy. We investigate whether MyoD gene expression was determined by semi- the Src homology 2 domain-containing phosphotyrosine quantitative RT-PCR. The skeletal muscle morphology phosphatase (Shp2) controls FAK activity and its was similar in all growth phases. The large number of effect on hypertrophy of cardiomyocytes.Neonatal <20µm diameter muscle fibers observed in fish with rat ventricular myocytes (NRVMs) were stretched in 10, 45 and 60 days confirms active hyperplasia. In silicone plates.Phosphatase activity was assessed in 180 days fish the >50 diameter fibers predominance the anti-Shp2 immune complex and in samples of characterized intense muscle fiber hypertrophy. MyoD Shp2-PTP domain.FAK/Shp2 association was detected mRNA expression level was high in fish with 10, by co-immunoprecipitation.Shp2 was depleted with 45, 60 and 90 days, decreasing significantly in 180 siRNA.Hypertrophy of NRVMs was assessed by days. The high MyoD mRNA expression observed in morphometry and the expression of bmyosin heavy pacu white muscle is possibly related to an intense chain (b-MHC) transcripts with RT-PCR.FAK was cell proliferation that occurs during muscle growth most non-phosphorylated in non-stretched NRVMs, hyperplastic mechanism. The low MyoD mRNA coinciding with relatively high basal association with expression in 180 days fish can be associated to the Shp2 and Shp2 tyrosine phosphatase activity.Cyclic muscle fiber hypertrophy, characterized by a decreased stretch enhanced FAK phosphorylation at Tyr397 cell proliferation and intense cell differentiation. eureka_ (2.3-fold) and reduced its association with Shp2 [email protected] (~85%) and the phosphatase activity of the anti-Shp2 immune complex (~40%). Shp2-PTP domain was F-016 shown to dephosphorylated FAK immunoprecipitated BIOENGINEERING OF BLOOD VESSELS USING of NRVMs.Depletion of Shp2 by siRNA enhanced FAK PORCINE XENOGENIC IMPLANTS phosphorylation in non-stretched NVRMs (2.1-fold), Sara Spinola Clemente, Leonardo da Cunha increased b-MHC transcript and induced hypertrophic Boldrini Pereira, André Marchiori, Luciene Bottentuit morphology (3-fold) in non-stretched NRVMs.These López Balottin, Eliene de Oliveira Koslowski, Radovan effects were accompanied by increased p70S6 kinase Borojevic, Christina Maeda Takiya (UFRJ) phosphorylation at Thr389.Treatment with PP2 or rapamycin abolished the hypertrophy induced by Shp2 Large-diameter vascular grafts have been successfully silencing.These data indicate that Shp2 controls basal developed from polymers such as Dacron or e-PTFE. FAK activity in cardiomyocytes, acting as a counter- However, it has been difficult to develop small-caliber regulatory mechanism to antagonize hypertrophy grafts (inner diameter < 6mm) because of biological induced by FAK and mTOR/S6K pathways. reactions at the blood-material and tissue-material [email protected] interface, such as acute thrombogenicity, intimal hyperplasia and formation of aneurisms. Autologous F-015 vein grafts require a removal of a vein from another GROWTH CHARACTERISTICS AND part of the body, when available. Synthetic grafts EXPRESSION OF MYOGENIC REGULATORY pose long-term health risks and are not successful for FACTOR MYOD IN THE PACU (PIARACTUS small diameter graft applications. Here we developed MESOPOTAMICUS HOLMBERG 1887: a mechanically stable vascular graft using intact iliac SERRASALMINAE, CHARACIDAE, arteries of pig decellularized by using sodium dodecyl TELEOSTEI) SKELETAL MUSCLE sulfate (SDS). Components of extracellular matrix Fernanda Losi Alves de Almeida (UNICAMP- involved with the mechanical support and elastic Campinas), Nabila Scabine Pessotti (UNESP - properties (elastin, collagen, chondroitin sulfate, Botucatu), Rachel Colauto Milanezi (UNESP-Botucatu), laminin and fibronectin) were evaluated, before and Robson Francisco Carvalho (UNESP- Botucatu), Carlos after treatment, by biochemical and histochemical Roberto Padovani (UNESP-Botucatu), Maeli Dal Pai- methods. No differences were observed. Iliac arteries Silva (UNESP-Botucatu) of pig rendered acellular with SDS has well-preserved extracellular matrix and basement membrane structure. The differential expression of Myogenic Regulatory Our results indicate that we were able to produce a Factors (MRFs) regulates the skeletal muscle growth vascular graft with optimal cell seeding characteristics. hyperplastic and hypertrophic mechanisms. The aim of These properties suggest proof of concept for its use as this study was to analyze hyperplasia and hypertrophy a scaffold for further vascular tissue engineering. and MyoD mRNA expression in skeletal muscle of pacu [email protected] (Piaractus mesopotamicus). Fish with 10, 45, 60, 90 and 180 days after hatching (n=8) were anaesthetized, F-017 sacrificed and total length (cm) determined. White PROTEIN RESTRICTION DURING DEVELOPMENT dorsal region muscle samples were collected and frozen INDUCES REDUCED PROLIFERATION IN THE RAT in liquid nitrogen. Transverse sections (10µm) were STOMACH WITH ENHANCED GASTRIC GHRELIN 1. 136

livro.indd 136 7/7/2008 16:33:40 LEVELS The posterior part has tubules with larger lumen than Ariane Kasai, Cruz Alberto Mendoza Rigonati, Patrícia the anterior part, and contains serous cells with low Gama, Eliana Parisi Alvares reactivity for PAS and Alcian Blue. The epithelium (Instituto de Ciências Biomédicas -USP) of anterior part has two kinds of mucosecretory cells with secretory vesicles with different size and Diet is an important factor during gastrointestinal electrodensities. In the AG from milked snakes large postnatal development, especially in the rat stomach vesicle exocytes like as a bulk while small vesicle that undergoes morphophysiological changes during exocytes one by one. The PD is folded and has a the first month of life. Studies from our laboratory pseudostratified epithelium with negative reactivity for showed that protein restriction during pregnancy and PAS and Alcian Blue, horizontal cells and mitochondria- lactation decreases animal and stomach weights as rich cells. Secretory cells contain vesicles with well as metaphasic index (MI) of stomach and small different electrodensities. The number of vesicle with intestine in suckling rats. We also observed that chow low electrodensity is higher in PD from milked than intake was similar in normal protein (NP) and restrict unmilked snake. Our data show that the AG and the protein (RP) diet groups but higher in RP if related to PD follow the modifications of main venom gland after body weight. Ghrelin - a hormone produced mainly in venom extraction. Data from milked snake suggest the stomach - is related to the control of food intake the participation of AG and PD in the whole venom. and secretion of growth hormone. This study aimed Supported by: FAPESP. to evaluate the effect of protein restriction on stomach [email protected] development and tissue ghrelin levels in 14 and 30-d- old Wistar rats. Pregnant female rats were fed a 20% F-019 protein diet (NP) or 8% protein diet (RP) during all MORPHOLOGICAL AND AUTORADIOGRAPHIC pregnancy and lactation of the pups. 14 and 30-d-old STUDIES ON THE CORNEAL AND LIMBAL pups were sacrificed and the stomachs were collected. EPITHELIUM OF RABBITS We analysed the MI, DNA synthesis index (SI) and Flávia Leão Barbosa (FMRP-USP), Rejane Maira Góes immunohistochemistry for ghrelin in stomachs of 14 (IBILCE-UNESP), Sidney Júlio Faria e Sousa (FMRP- and 30-d-old rats. We observed that MI and SI were USP), Antonio Haddad (FMRP-USP) decreased in RP 14 and 30-d-old animals (p<0.05). The immunostaining for ghrelin was significantly higher The investigation was centered on the morphological in RP 30-d-old animals than in NP animals (p<0.05) features of the limbus of the rabbit eye and the and no difference was found in 14-d-old animals. RP proliferative behavior of its epithelium. The eyes were diet diminished cell proliferation in both ages. Our data processed for examination with light and electron suggest that the increase in ghrelin expression induced microscopy, as well as for autoradiography after by long-term RP diet may be an adaptative survival intravitreal injection of 3H-thymidine (3H-TdR). At the strategy in response to reduced body weight. Suported sites of extraocular muscle insertion the vascularization by Fapesp and CNPq. of the stroma extended to the peripheral cornea, and [email protected] the limbal epithelium was thin with its basal stratum was made up by clear cuboidal cells. At the light F-018 microscope level the basement membrane was distinct ACCESSORY GLAND AND PRIMARY DUCT OF in the cornea but not in the limbus or the conjunctiva. BOTHROPS JARARACA SNAKE DURING THE Autoradiographs demonstrated that at the limbus the VENOM PRODUCTION CYCLE: basal cells migrated very quickly to the suprabasal MORPHOLOGICAL STUDY region and remained there up to the 28-day interval. Fernanda Sakai, Karina Cristina Giannotti, Sylvia Labeled cells were identified in all epithelial layers of the Mendes Carneiro, Norma Yamanouye cornea, including the basal one, at 21 and 28 days but (Instituto Butantan) not in the limbal basal clear cells. The rate of renewal of conjunctival epithelium was similar to the one Venom gland of viperid snakes has a central lumen observed for the transition with scarce clear cells. The where venom is stored. A new cycle of venom synthesis high resolution autoradiographs demonstrated that the starts when the amount of the venom decreases. There basal cuboidal clear limbal cells exhibit a quick renewal are no studies about the accessory gland (AG) and the and that they are not label-retaining cells. These latter primary duct (PD) during venom production cycle. This ones were detected all over the corneal epithelium and study aimed the morphological analysis of the AG and in the suprabasal layers of the limbus up to 28 days, in the PD of B. jararaca during venom production cycle. physiological conditions, without the need of stimulation The AG and PD from unmilked and 4 days milked by damage to corneal epithelium. snakes were fixed and processed for light or electron [email protected] microscopy. The AG has a simple secretory epithelium, horizontal cells and dark cells. The anterior part has F-020 long tubules with a narrow lumen and mucosecretory BIOMONITORING OCCUPATIONAL BY THE cells with positive reactivity for PAS and Alcian Blue. MICRONUCLEUS TEST IN CELLS OF THE ORAL 1. 137

livro.indd 137 7/7/2008 16:33:40 MUCOSA OF PROFESSIONALS EXPOSED TO concentrations and periods of time and submitted to ANTINEOPLASTIC DRUGS MTT cell viability assay, showed a statistically significant Bruna Antunes Más, Julio Boschini Filho, reduction in viability comparing to controls.BPH tissue Gilson Luchezi Delgado, Camila Figueira Mendes was also processed for organ culture, treated with OSE, (Pontifícia Universidade Católica de São Paulo) morphologically characterized and immunolabeled for proliferation cell nuclear antigen (PCNA). A significant Workers in the health area, while working in hospitals reduction in PCNA immunoreactivity was observed in and laboratories are potentially exposed to different treated cultures, indicating an important decrease of occupational risks. In cancer chemotherapy services, cell proliferation. These results showed that OSE was the increasing use of anticancer drugs has been able to inhibit BPH cells viability/proliferation, raising the considered an important genotoxic risk for professionals possibility for a new therapeutic approach for BPH. involved in their preparation and administration. [email protected] Through the micronucleus test it is possible to biomonitor the genotoxicity degree of these drugs. The F-022 micronucleus is composed of in a small piece of nuclear NOT INVOLVEMENT OF L-TYPE CALCIUM mass bounded by a nuclear membrane and separated CHANNEL AND TYROSINE KINASES IN from the main nucleus, resulting either from acentric ANGIOTENSIN II (ANGII) VASOCONSTRICTION chromosomal fragments or whole chromosomes that RESPONSE OF BRAZILIAN SNAKES were not included in the principal core. In our study, Paula Luize Burckhardt, Amanda Paz Perussi, Carlos both in Sorocaba, we assessed the exposure of 13 Augusto Esteves, Maria Cristina Breno (Instituto professionals from two services, oncology, being Butantan) 9 in a public Hospital and 4 in a private Oncology service, dressed according to the rules of the National Renin-angiotensin system produces its effects through Health Surveillance Agency, DRC-Resolution No.220, the interaction of AngII with AT1 and AT2 receptors and September 21,2004, during 10 months, compared in some vertebrates, like the Brazilian snakes Bothrops with 13 professionals in the control group. The results jararaca (Bj) and Crotalus durissus terrificus (Cdt), with showed that the average frequency of cells in the an atypical angiotensin AT receptor. The majority of micronucleus buccal mucosa of the porfessional AngII responses are attributed to activation of the AT1 exposed in the public Hospital (0.30) was higher when receptor, that is coupled to several intracellular signaling compared to the control group (0.22), with statistical pathways, like phospholipases C, D, A2; voltage- significance for (P>0001). dependent Ca+2 channel; [email protected] adenylylcyclase and tyrosine kinases. We have demonstrated that the PLC and adenylylcyclase F-021 pathways were not coupled with the atypical AT IN VITRO EFFECTS OF A NEW HEXANIC PLANT receptor in the heart of Bj. To evaluate the signal EXTRACT ON HUMAN BENIGN transduction pathway activated by AngII in these PROSTATIC HYPERPLASIA (BPH) CELL snakes, we examined the participation of L-type calcium PROLIFERATION channel and the tyrosine kinases in the angiotensin Pedro Augusto Reis de Souza (UFRJ), Antonio vasoconstriction response. The isometric tension Palumbo Júnior (UFRJ), Christina Maeda Takiya induced by cumulative AngII (10-10-10-6M) and KCl (UFRJ), Lúcio Mendes Cabral (UFRJ), Fábio de Sousa (10-2–10-1M) concentrations on aorta rings from Bj Menezes (Trinity College Dublin), Luiz Eurico Nasciutti and Cdt were obtained in the absence and presence of (UFRJ) nifedipine, diltiazen (L-type Ca+2 channel blockers) and genistein (tyrosine kinase inhibitor). Although nifedipine BPH is one of the most common diseases and diltiazen (10-5M, n=7-15) were effective to reduce affecting quality of life in older men, characterized KCl effect, they failed to modify AngII response in both by nonmalignant enlargement of the prostate. snakes species. Genistein (10-5M, n=5) was also not Dissatisfaction with side effects from conventional able to modify AngII and KCl effect in the Bj. These treatments has led to an increased popularity of results suggest that, unlike AT1 receptor from other self-medication with herbal medicines, but there is a vertebrates, L-type calcium channel and tyrosine dearth of information on their mode of action. In this kinases are not involved with AngII response in these study, we have investigated the in vitro effect of a snakes. Others new lipid/sterol extract (OSE) from a Brazilian native intracellular signaling pathways should be investigated. plant on human BPH primary cell and organ cultures FUNDAP, CNPq, FAPESP. obtained from men undergoing prostate surgery. OSE [email protected] exhibited anti-proliferative effects on leukemia cell lines and analgesic/anti-inflammatory activities in different F-023 animal models. Cultured cells were characterized COMPARATIVE ANALYISIS OF ANGIOGENIC by immunostaining of cytoskeleton proteins and POTENTIAL OF BONE MARROW AND ADIPOSE extracellular matrix (ECM) components. It was obtained TISSUE MESENCHYMAL STEM CELLS IN MOUSE a homogeneous fibroblastoid stromal cell population HINDLIMB ISCHEMIA MODEL that expressed ECM components in a similar way to Carolina da Silva Gouveia Pedrosa (UFRJ), Paulo that observed in vivo. Cells treated with different OSE Eduardo Ocke Reis (UNIFESP), Eduardo Gallatti 1. 138

livro.indd 138 7/7/2008 16:33:40 Yasumura (UNIFESP), Leonardo Pinto de Carvalho showed no morphological changes. Nevertheless, for (UNIFESP), Daiana Vieira Lopes (UFRJ), Christina EA90d liver samples, morphological changes in the Maeda Takiya (UFRJ), Radovan Borojevic (UFRJ), tissue structure and Disse space were detected. In all Sang Won Han (UNIFESP), Maria Isabel Doria Rossi analyses performed, the DMSA-MF was internalized (UFRJ) in cytoplasmic vesicles; however, for EA90d liver samples, secondary lysosomes were also observed. Peripheral vascular disease is a major health care Morphometrical tests of hepatic mitochondria were problem in an aging society. In the late stages, the performed and analyzed for all animals. Apparently, progression of tissue hypoperfusion results in ischemic DMSA-MF was not proven to be completely harmless, ulceration and gangrene. The convencional treatements due to the fact that hepatic alterations were attributed are not eficient; so many alternatives therapeutic to the MF presence in the tissue. With this, the present protocols were created. A possible candidate for cell study demonstrated the possibility of using maghemite- therapy were bone marrow mesenchymal stem cells based magnetic fluid stabilized with DMSA coating as a (BM-MSC) who possess multipotential differentiation promissory tool for diagnostic and therapeutic ends in capacity, including to endothelial and vascular smooth primates, including human beings. muscle cells. BM-MSC transplantation has been [email protected] shown to induce neovascularization in a rat model of hindlimb ischemia. Adipose tissue mesenchymal stem F-025 cells (AT-MSC), like BM-MSC, possess multipotential REGULATION OF ACTIVITY AND capacity and have been proposed as an alternative EXPRESSION OF MMP2/ 9 INDUCED BY STRETCH sourse of MSC. The aim of this study is to compare IS MEDIADED BY FAK the angiogenuc potential of BM-MSC and AT-MSC in Ana Paula Dalla Costa (Unicamp), Aline Mara dos mouse model of hindlimb ischemia. Right femoral artery Santos (Unicamp), Carolina Fernanda Manfredi occlusion of three month old Balb/c mice was followed Zambon Clemente (Unicamp), Deborah Schechtman by intramuscular injection of 5x105 BM-MSC (BM (INCOR USP-SP), Kleber Gomes Franchini (Unicamp) group) or AT-MSC (AT group). Mice injected only with vehicle (control group) and not opered (not ischemic Dynamic changes occur in the interstice contribute group) were used for comparation. Four weeks after directly to myocardial remodeling due to heart transplantation, the AT group presented less weight diseases such as myocardial infarction, hypertensive and tensory force in the ischemic muscle than the heart disease and cardiomyopathies. The Matrix control and not ischemic groups. Morphological analysis Metalloproteinases, (MMPs) have important role showed mature fat cells in the middle of muscle fibers in remodeling in these diseases. Mechanical and similarly to the fat infiltration process. The BM group is neurohumoral stimulus regulate the expression and under examination. These preliminary results suggest activity of MMPs through balance of MMP/TIMP (tissue that most murine AT-MSC are primarily committed to the inhibitors of metalloproteinases). In our laboratory adipogenic lineage. demonstrated that FAK silencing in the left ventricle in [email protected] mice, inhibits the development of fibrosis and reduces MMP2 activity and expression. This study assess F-024 the role of FAK control in MMPs 2/ 9 expression and ULTRASTRUCTURAL EFFECTS OF LIVER, SPLEEN, activity in rat cardiac fibroblasts. Neonatal Rat Cardiac AND LYMPH NODES OF MONKEY (CEBUS APELLA) Fibroblasts (NRCFs) were grown on silicon plates and BY MAGNETIC FLUID MAGHEMITE-BASED subjected to cyclical stretch (10%) by up to 8 hours. The STABILIZED WITH DMSA COATING FAK expression and activity were evaluated through Shélida Vasconcelos Braz, Victoria Monge Fuentes , imunoblotting with specific antibodies (FAK/phospho- Raphael Canido Apolinario Peixoto, Paulo Cesar de FAK Tyr397, respectively). The expression of MMP2/9 Morais , Sônia Nair Báo, Ricardo Bentes de Azevedo, was evaluated with specific antibody in imunoblotting Silene Paulino Lozzi (UnB) and activity through zimography. NRCFs were also treated (2 hs) with FAK activators peptides merged To our knowledge, the present work represents the with TAT sequence, designed on the basis of specific first study to research the effects of a type of magnetic regions of the FAK N-terminal domain. Both the stretch fluid (MF) in an experimental model other than culture as the peptides activators stimulated phosphorylation of cells or in vivo rodent models. The aim of the present FAK in Tyr397, and increased the expression of MMP study was to assess any morphological alterations in 9 and the activity of MMPs 2 and 9 in the supernatant liver, spleen, and lymph nodes after single intravenous culture. The results indicate that the activation of FAK injection with maghemite (Fe2O4) -based magnetic fluid may have a critical role in the activation of MMPs in stabilized with DMSA (2,3-dimercaptosuccinic acid) in cardiac fibroblasts undergoing mechanical stretch. monkey (Cebus apella). Samples of spleen, liver and FAPESP; CNPq. lymph node from control animal (CA), 12 h post injection [email protected] animal (EA12h) and 90 days experimental subject (EA90d) were observed ultra-structural changes, F-026 by transmission electron microscopy to locate MNP INFLUENCE OF TYPE I DIABETES IN BONE in cellular structures. Samples of spleen and lymph RESORPTION DURING THE EXPERIMENTAL nodes 12 h and 90 days after DMSA-MF administration PERIODONTAL DISEASE (EPD) IN RATS 1. 139

livro.indd 139 7/7/2008 16:33:40 Juliete Aparecida Francisco da Silva, Luis Antonio and 22.55; ALT-C60: 42.17, 61.89, 56.78 and 59.52; Peroni, Hernandes F. Carvalho, Dagmar Ruth Stach- ALT-C100: 21.80, 88.60, 19.32 and 61.67 at 1, 3, 5 Machado (UNICAMP) and 7d of treatment respectively. ALT-C stimulated type I collagen synthesis, which is important to increased Introduction: Diabetes Mellitus intensifies the severity of wound strength. The results of the present study EPD inducing bone resorption and tooth loss mediated indicate the potential cicatrizing action of this protein. by RANKL. During the disease there is a progressive Financial support: CNPq, FAPESP. degradation of extracellular matrix components due to [email protected] imbalance between matrix metallopeptidases and their inhibitors (TIMPs and RECK). Objectives: Evaluate the F-028 expression of molecules involved in bone resorption GROWTH FACTOR MODULATION BY during progression of EPD. Methods: Wistar male ALTERNAGIN-C, A DISINTEGRIN-LIKE rats were examined 5, 15 or 30 days after ligature PROTEIN FROM BOTHROPS ALTERNATUS SNAKE (employed to induce EPD). Replicates were done using VENOM streptozotocin-treated animals. The animals were killed, Estela Maria Correia Sant’Ana (Universidade Federal the jaws were removed and it soft tissues were taken de São Carlos), Cibele Marli Cação Paiva Gouvêa out while it hard tissues were stained with 1% toluidine (Universidade Federal de Alfenas), Heloisa Sobreiro blue to measure the exposed tooth root. The gene Selistre-de-Araujo (Universidade Federal de São expression was analyzed by Real time RT-PCR with Carlos) SYBR Green Master Mix. Results: Bone resorption was increased 15 and 30 days after ligature and shown to Alternagin-C (ALT-C), a disintegrin-like protein from be more intense in diabetic animals. The increase of the venom of Bothrops alternatus induces in vivo MMP-13 and MMP-14 expression occurred at 5 days in angiogenesis. It is known that skin wound repair is diabetic animals with ligature. In non-diabetic animals, mediated by angiogenesis induction and several growth only MMP-13 was increased at 5 days. RANKL was factors that are strictly released. This study investigated increased in non-diabetic animals at 15 days of ligature, the effects of ALT-C on growth factors expression in a while Diabetes inhibited its expression during the time rat wounded skin model. The rats were anaesthetized line of this study. TIMP-1 expression increased at 5 and two cutaneous excisions (4 mm diameter) were days in response to ligature and was more intense in made on the back of each animal, close to the cervical diabetics. TIMP-2 and RECK expression did not show area. Animals were then divided into 4 groups (three variation after ligature, except by a slightly increase in rats/group): control (treated with vehicle) and locally RECK expression at 15 days in non-diabetic animals. treated with 10, 60 and 100 ng ALT-C. After 24h the Conclusions: Diabetes Mellitus influenced the bone wounded skin was removed, proteins were extracted, resorption mediators and highligthed periodontal separated by SDS-PAGE, transferred to blots and disease effects. reacted with antibodies. The results showed that [email protected] ALT-C treatment modulated growth factor expression, mostly TGFa, -B1, IGF1 and VEGF. The values in F-027 percentages of increasing (+) or decreasing (-) growth ALTERNAGIN-C, A DISINTEGRIN-LIKE PROTEIN factor expression in rat wounded skin, compared to FROM BOTHROPS ALTERNATUS SNAKE VENOM, control, was: TGFa: -46.6 , -46.6 and +35.0; TGFa1: INDUCED COLLAGEN SYNTHESIS -57.9, 0.0 and +26.3; IGF1: 0.0, -13.6 and +9.1; PDGF: Estela Maria Correia Sant´Ana (Universidade Federal -48.2, +10.3 and -62.1; FGF1: -28.6, +42.8 and 0.0; de São Carlos), Cibele Marli Cação Paiva Gouvêa VEGF: -5.3, +1.3 and +7.9 in 10, 60 and 100 ng of (Universidade Federal de Alfenas), Heloisa Sobreiro ALT-C treated animals respectively. Besides being Selistre de Araújo (Universidade Federal de São Carlos) angiogenic, ALT-C 100 ng stimulated TGFa, -B1, IGF1 and VEGF, important factors to ptomote cell motility, Alternagin-C (ALT-C), a disintegrin-like protein from the proliferation and reepithelization, indicating a potencial venom of Bothrops alternatus induces in vivo growth cicatrizing action of this protein. Financial support: factors releasing. It is known that in early wounded CNPq, FAPESP. healing process the amount of collagen is increased [email protected] (about 30%). This study investigated the effects of ALT-C on the collagen in a rat wounded skin model. F-029 The rats were anaesthetized; a cutaneous excision (4 INFLUENCE OF FASTING ON CELL CYCLE mm diameter) was made on the back of each animal. PROTEINS BEHAVIOR IN THE GASTRIC Animals were then divided into 4 groups (three rats/ EPITHELIUM OF PUPS AND ADULT RATS group): control (treated with vehicle); locally treated Carolina Bottini Prates, Daniela Ogias, Eliana Parisi with 10, 60 and 100 ng ALT-C for 1, 3, 5, and 7 days. Alvares, Patrícia Gama (USP) After that the wounded skin was removed histologically processed and used for collagen quantification by The interaction of hormones, growth factors, and morphometry. The results showed that ALT-C treatment feeding behavior is essential to control the renewal increased collagen synthesis in all experimental of the gastric epithelium. Among these elements, groups, compared to control. Collagen increased in a any modification in dietary pattern influences cell rat wounded skin was (%): ALT-C10: 6.68, 72.15, 1.03 proliferation and affects growth. We have shown that 1. 140

livro.indd 140 7/7/2008 16:33:40 fasting inhibits cell suggest that TGFbeta1 may be involved in the control of proliferation in the gastric epithelium of young adult rats, gastric cell proliferation, while TGFbeta3 may be related whereas it is stimulatory in pups. Such opposite effects to differentiation events. The involvement of TGFalpha can be associated to the presence and availability is unclear and more studies are needed. of corticosterone and growth factors, but might be [email protected] reflected in cell control. In the current study we aimed to evaluate the behavior of cell cycle proteins in the F-031 gastric mucosa of pups and young adult rats submitted ANALYSIS OF CELLULAR BEHAVIOR IN CULTURE to fasting. Wistar rats were fasted for different ON MULTI-WALLED CARBON NANOTUBE (MWCNT) periods according to age. Cyclin D1, Cdk4, cyclin E, Marcus Alexandre Finzi Corat (CEMIB - UNICAMP), Cdk2, p27 and p21 were measured by Western blot in Anderson de Oliveira Lobo (ITA/INPE), Erica Freire samples scraped from the gastric mucosa. We found Antunes (ITA/INPE), Rovilson Gilioli (CEMIB/ that whereas Cdk 2 and Cdk4 remain constant, cyclins UNICAMP), Luiz Augusto Corrêa Passos (CEMIB/ D and E are differently altered by the progress of fasting UNICAMP), Evaldo José Corat (INPE) in pups and adult rats. Interestingly, we observed that p27, which can retard cell cycle as a CKI, increases The nanometric characteristics of carbon significantly prior to the inhibition of cell proliferation nanotubes (CNTs) are supposed to have mimetic in the adult mucosa, whereas it does not change for properties of extra-cellular matrix and systematic in vitro pups. We suggest that cyclins D and E and p27 are and in vivo investigations using biologically compatible diversely affected by fasting in pups and adult rats and CNT are necessary. In this way, this work shows the such differences might account for the opposite effects behavior of cultivated mouse embryonic fibroblast cells triggered by food restriction at distinct developmental with expression of green fluorescent protein (GFP) on stages. Supported by FAPESP, CNPq and CAPES. aligned MWCNTs surface. The adherence, proliferation [email protected] and cellular survival were observed by images of fluorescence and scanning electron microscopy (SEM). F-030 The fluorescent cells were used due to opacity of MATERNAL PROTEIN MALNUTRITION MWCNTs. Despite the results have showed adherence, EFFECTS ON CELL PROLIFERATION AND survival and increasing of the number of the cells on GROWTH FACTORS EXPRESSION IN THE MWCNTs at the first 48h, after 72—144h those cells GASTROINTESTINAL EPITHELIUM OF started to die. Cell death did not happen when we SUCKLING RATS increased the number of the seeded cells from 50000 Larissa Candido Alves, Cruz Alberto Mendoza Rigonati, to 100000. The biocompatibility of cell-to-MWCNTs Patrícia Gama, Eliana Parisi Alvares (Instituto de showed to be dependent on number of cells adhered Ciências Biomédicas - USP) on MWCNTs surface. The structure of MWCNTs is hydrophobic which may induce weak adherence of Maternal malnutrition during pregnancy and cells in aqueous medium environment. Cell death in lactation causes significant changes in the this condition may suggest anoikis. The fluorescence gastrointestinal epithelium of the offspring. Data from observation corroborates this assumption: the actin was our laboratory showed a decrease on not so straight and in some cases completely disrupted metaphase indices of stomach and small after intervals of 48h into cultivated cells.Although intestine of undernourished suckling rats. The aim anoikis hypothesis might be better investigated using of this study is to check if protein restriction (8% specific markers and overcoming the fixation of protein) during pregnancy and lactation causes cell MWCNTs, we have showed that the biocompatibility proliferation and growth factors expression changes in to MWCNTs is possible and dependent of number the gastrointestinal epithelium of 14 d-old suckling rats. of the cells. Further studies are necessary to better Female Wistar rats received control (20%) or restricted understand the interaction cell-CNTs using biomaterials. (8%) protein diet [email protected] during all pregnancy and lactating period. Animals were sacrificed at 14 days. Stomachs and pieces of intestines F-032 were collected for cell proliferation and histological EVALUATION OF CYTOTOXICITY, analyses. We analyzed intestine cell proliferation CYTOCOMPATIBILITY AND CELL’S by means of metaphase and crypt fission indices. PROLIFERATION OF INTERACTION THE Immunoreaction for TGFbeta1 and 3 and TGFalpha VERTICALLY ALIGNED MWCNT WITH HUMAN was performed in the gastric tissue. Metaphase OSTEOBLASTS CELL’S index of large intestine was not different between Anderson de Oliveira Lobo (ITA/INPE), Marcus the groups, while crypt fission index of intestine was Alexandre Finzi Corat (CEMIB/UNICAMP), Mariana significantly reduced in the undernourished group. Bernardes Silva Palma (UNIVAP/INPE), Erica Freire Immunohistochemistry analysis showed an increase in Antunes (ITA/INPE), Cristina Pacheco-Soares the number of TGFbeta1 positive cells and a decrease (UNIVAP), Evaldo José Corat (INPE) of TGFbeta3 positive cells in the gastric glands of undernourished rats, while there was no difference The very important role of nanotechnology in the regarding TGFalpha analysis. As cell proliferation biological scene is becoming clear in the last few years was decreased in intestine of undernourished rats we with several papers studying the interaction of multi- 1. 141

livro.indd 141 7/7/2008 16:33:41 walled carbon nanotubes (MWCNT) with biological that RT with low period of recovery between sessions systems. CNTs are added to other biomaterials to promoted CSA reduction in the skeletal muscle obtain composites, and these CNT composites are fibers and the modulation occurred in hybrid fibers proven to be suitable for cell growth and increasing predominantly. enzyme activity. Although previous works have [email protected] shown that CNTs are in principle ideal candidates for scaffolds/matrices in tissue engineering, it is still F-034 early to establish a general toxicological profile for EFFECT OF A SHORT AND A LONG TERM OF this material and more systematic in vitro and in vivo FASTING ON THE CELL PROLIFERATION OF THE investigations using biologically compatible CNT are JEJUNAL EPITHELIUM OF RATS AT WEANING necessary. The objective of this work was evaluated PHASE IN DIFFERENT PERIODS OF THE DAY the cytotoxicity, cytocompatibility and proliferation of Maria Albertina de Miranda Soares, Larissa F Z Moreira, osteoblasts cell on vertically aligned MWCNT. The Liliane Galvão, Monica A Okada, Cristina L S C Ayub, MWCNT films were obtained by microwave plasma José R Gomes (UEPG) process. The biocompatibility tests were performed on raw-MWCNT. The biocompatibility “in vitro” tests used Effects of a short (5.00 h) and a long term were: 1) cytotoxicity, using MTT (cell viability) and LDH (25.00 h) of fasting on the metaphase index (MI) in (cell proliferation) assays (3days) and cellular adhesion the jejunal epithelium of young rats were investigated (7 days) by scanning electron microscopy (SEM). The at 09.00 h, 15.00 h, 21.00 h and 02.00 h using the results of high cell viability, possible cell’s proliferation arrested metaphases method. Previous studies (>20%) and exceptional cell adhesion showed that the demonstrated that during the growth of small intestine first cell monolayer spreads flat on the surface of the there is a reduction in MI in the morning period. In the vertically aligned MWCNTs after 7 days. We present present study it was demonstrated that both 5h and 25 results of high viability, proliferation and exceptional cell h of fasting reduced the MI in the morning period (09.00 adhesion on vertically aligned MWCNTs, indicating the h) and that 25h of fasting also reduced MI at 21:00h. possible application for preparation of scaffold/matrices It suggests that a short or a long term of fasting can in tissue engineering. accentuate the reduction of MI of intestinal epithelium [email protected] at specific times of the day. Based on the results presented it can be concluded that food could be an F-033 important external factor regulating the cell proliferation HIGH-INTENSITY RESISTANCE TRAINING AND LOW during the growth of small intestine. PERIOD OF RECOVERY CAUSE MODULATION AND [email protected] CROSS-SECTIONAL AREA REDUCTION IN RATS SKELETAL MUSCLE FIBERS F-035 Rodrigo Wagner Alves de Souza, Alan Dantas Santos THE PROLONGED EFFECT OF VINBLASTINE ON Felisberto, Andreo Fernando Aguiar, THE INCISOR RAT TOOTH ERUPTION Maeli Dal Pai Silva (Unesp - IBB) Pedro Duarte Novaes (FOP/UNICAMP), FlÁvia Maria De Moraes Ramos (FOP/UNICAMP), Eliene Aparecida Physical training can promote benefic adjustments Orsini Narvaes (FOP/UNICAMP), Nádia Fayez Omar in muscle fibers phenotype. However, high-intensity (FOP/UNICAMP), Juliana dos Santos Neves (FOP/ training associated with an insufficient period of UNICAMP), José Rosa Gomes (UEPG) recovery may lead to an “overtraining syndrome,” which results in the fall of performance. We investigated the The vinblastine drug reduces the eruption rate in incisor effects of the high-intensity resistance training with low rodent tooth and there is not information about how long period of recovery between sessions on the skeletal this reduced effect persists. Thus the aim of this study muscle morphology. Male Wistar rats (200-250g) were was to investigate the prolonged effect of vinblastine in divided into four groups: trained 8 weeks(T8), control a hipofunctional condition eruption. Rats were divided in 8 weeks(C8), trained 12 weeks(T12) and control 12 a normofunctional vinblastine (group I); normofunctional weeks(C12). T8 and T12 underwent high-intensity control (group II); hypofunctional vinblastine (group III), resistance training (RT), with low period of recovery and hypofunctional control (group IV). Groups I and II between sessions(5 days/ week). During the RT, had their incisors teeth intact and a reference mark was animals realized jumps in a vat of water, with the made on the right tooth using a diamond high-speed overload adjusted by a vest coupled to the animal. At rotating instrument and the groups III and IV had their the end of RT, plantar muscle was withdrawn. Muscle left inferior incisors shortened at intergingival level by fibers types were classified by using myofibrillar ATPase the same instrument each two days to produce the and the cross-sectional area(CSA) were measured hypofunctional condition. Groups I and III received a using an image analyzer system. There was a decrease single intraperitoneal vinblastine injection (2mg/Kg) in CSA of muscle fiber IIC and IIAD in T8, and IIA and while groups II and IV received a salina injection on IID in T12 in relation to their respective controls. In T8, the 8 day respectively. The eruption rates (mm) were there was a reduction in IIA and IIBD and an increase measured by distances from the gingival margin to in IIB fiber frequency, compared to C8. T12 showed an the marks done on the right teeth and up to the end increase in IIBD and decreased IID fibers frequency, of the left incisor teeth using a calibrated grid ocular comparing with C12. In conclusion, our results show under a microscope eyepiece at 10x magnification 1. 142

livro.indd 142 7/7/2008 16:33:41 each two days during 18 days. The decrease in APHRODISIACA (O. MACH.) INFUSION eruption rate was observed only in group IV 2 days Juliana Castro Monteiro (Universidade Estadual after vinblastine injection and it continued for the next de Campinas), Marcos de Lucca Moreira Gomes 8 days when it returned to normal levels. The results (Universidade Estadual de Campinas), Tatiana Carla suggest a prolonged citotoxic effect of vinblastine on the Tomiosso (Universidade Estadual de Campinas), proliferative cells of enamel organ and/or in fibroblast Débora Amador Lima de Almeida (Universidade Federal cell compartment of the periodontal ligament which de Viçosa), Edson Rosa Pimentel (Universidade it could be disrupted the mitotic spindles reducing Estadual de Campinas), Mary Anne Heidi Dolder although the eruption [email protected] (Universidade Estadual de Campinas)

F-036 Heteropterys aphrodisiaca roots are used as ENDURANCE TRAINING IMPROVE VENTRICULAR aphrodisiac, stimulant and to strengthen muscle FUNCTION WITHOUT ALTER VENTRICULAR and bone. The plant infusion effect was studied in HYPERTROPHY IN HEART FAILURE Wistar rats, evaluating tendon biochemistry and Joyce Reissler (Departamento de Morfologia, IB, morphology. Forty rats were divided into groups: GI- UNESP, Botucatu - SP), Francis da Silva Lopes control sedentary; GII- H. aphrodisiaca sedentary; (Faculdade de Fisioterapia, UNOESTE, Presidente GIII–control trained; GIV- H. aphrodisiaca trained. The Prudente - SP), Katashi Okoshi (Faculdade de training protocol consisted in running on a motorized Medicina, UNESP, Botucatu - SP), Dijon Henrique treadmill, five times a week, with weekly increase in Salomé de Campos (Faculdade de Medicina, treadmill velocity and duration. Water (sham) or H. UNESP, Botucatu - SP), Robson Francisco Carvalho aphrodisiaca was administered daily by gavage (dose: (Departamento de Morfologia, IB, UNESP, Botucatu 35 mg/animal/day) for 8 weeks. The rat´s tendons - SP), Carlos Roberto Padovani (Departamento de were either frozen for biochemical analysis or fixed in Bioestatística, IB, UNESP, Botucatu - SP), Antônio Karnovsky and paraffin included. Longitudinal sections Carlos Cicogna (Faculdade de Medicina, UNESP, (7 μm) were stained with HE and toluidine blue. The Botucatu - SP), Maeli Dal Pai Silva (Departamento de tendon’s compression region of GII showed cells Morfologia, IB, UNESP, Botucatu - SP) arranged in groups and more rounded than those of GI. In trained animals, they were even more rounded, Heart failure(HF) is characterized by cardiac aligned between collagen fibers, and apparently remodeling(CR), intolerance to the exercise, and the in greater number when compared with sedentary endurance training(ET) cause cardiac benefit. There groups. SDS-PAGE of proteins showed, comparatively, are few studies that have evaluated the ET in the CR that treatment resulted in an expressive increase in cardiopaties with constant periphery resistance, as of collagen and non-collagenic proteins. This was in aortic stenosis(As). The purpose of this paper was to confirmed by quantification of total proteins and sulfated determine if ET alters clinical signs of HF e CR during glycosaminoglycans according to the micro-methods of transition between left ventricular dysfunction and HF. Bradford and DMMB, respectively. An anabolic effect Four groups were used: AsET(8), AsSedentary(6), for the infusion associated with physical training cannot ShamSedentary(9) and ShamET(4). After 18 weeks be affirmed with these partial results. However, the of As and ventricular dysfunction, rats were submitted undesirable side effects described for tendons with the to the ET(treadmill), for 10 weeks with progression of use of anabolic steroids, including micro-lesions and velocity and duration. The HF was analyzed by clinical inflammation, were not observed here. signs, the CR by cardiomyocyte morphological and [email protected] morphometric analysis and echocardiogram. Compared with the AsSedentary, clinical signs were decreased F-038 in the AsET. Compared with the ShamSedentary, the THE ROLE OF NF-KB PATHWAY IN RAT SKELETAL cardiomyocyte area was increased in the AsET. On the MUSCLE ATROPHY WITH MONOCROTALINE other hand the ET did not show any modification in the INDUCED HEART FAILURE cardiac hypertrophy in the AsET. The variables atrium Robson Francisco Carvalho (Departamento de and right ventricle/body weight, left atrium diameter/ Morfologia, IB, UNESP, Botucatu-SP), Eduardo aortic diameter, diastolic and systolic diameters and Paulino Castan (Departamento de Morfologia, IB, waves E/A decreased, the endocardial fractional UNESP, Botucatu-SP), Cesar Augusto Oliveira Coelho shortening, posterior wall shortening velocity and the (Departamento de Morfologia, IB, UNESP, Botucatu- isovolumetric relaxing time of the left ventricle increased SP), Célia Regina Nogueira (Departamento de in the AsET. The ET attenuated and delayed this Clínica Médica, FM, UNESP, Botucatu-SP), Antônio dysfunction and show the beneficial effect of endurance Carlos Cicogna (Departamento de Clínica Médica, training on left ventricular functional and remodeling. FMB, UNESP, Botucatu-SP), Maeli Dal Pai Silva The ET can be a temporary therapeutic procedure (Departamento de Morfologia, IB, UNESP, Botucatu-SP) recommended until the surgical act.joyce_reissler@ hotmail.com Heart failure (HF) is characterized by a skeletal muscle myopathy with increased expression of fast myosin F-037 heavy chains (MHC), change expression of myogenic ALTERATIONS IN THE CALCANEAL TENDON OF regulatory factors (MRFs) and muscle atrophy. The TRAINED RATS THAT RECEIVED HETEROPTERYS pathophysiological mechanisms responsible for muscle 1. 143

livro.indd 143 7/7/2008 16:33:41 changes during heart failure are not defined. Muscle MORPHOLOGICAL AND IMMUNOFLUORESCENCE atrophy may be induced by several cytokine activation, STUDY mainly TNF-a that increase the transcriptional activity of Cristiani Baldo (Laboratório de Imunopatologia- NF-kB. This fact can activate the ubiquitin-proteasome Instituto Butantan), Norma Yamanouye (Laboratório pathway, that can mediate skeletal muscle atrophy. de Farmacologia-Instituto Butantan), Ana Maria In this study we evalueted the genes expression of Moura da Silva (Laboratório de Imunopatologia- the transcription factor NF-kB, the ubiquitin protein Instituto Butantan), Colin Jamora (Section of Cell and ligases MuRF1 and MAFbx, the MRF MyoD, the Developmental Biology-University of California) MyHC, and TNF-a level in wistar rat skeletal muscle with monocrotaline induced HF. We studied extensor Snake venom metalloproteinases (SVMPs) are digitorum longus (EDL) muscles from both HF and responsible for local and systemic hemorrhage following control Wistar rats. NF-kB, MuRF1, MAFbx, MyoD a snake bite. However, the mechanisms of SVMPs- and MyHC contents were determined by using qRT- induced hemorrhage are not completely clarified. In PCR. TNF-a was measured by Elisa. The mRNA this study, jararhagin, a hemorrhagic P-III SVMP, and relative expression of MuRF-1, MaFbx and NF-kB BnP1, a weakly hemorrhagic P-I SVMP, were compared were significantly increased; the MyoD was reduced in a skin model using primary mouse keratinocytes whereas MyHC-2B and TNF-a level were not changed. and intradermal injection in mice. Keratinocytes The increase in the mRNA relative expression treated with 800nM of jararhagin or BnP1 exhibited of MuRF-1, MaFbx e NF-kB in EDL muscle was a change of morphology and cell detachment. This associated with atrophy in response to HF without alter was accompanied by a rearrangement of the actin the TNF-a. Taken together, our results show a potential network (F-actin staining), but the microtubules (tubulin role for MRFs and the ubiquitin-proteasome pathway staining) and adherens junctions (E-cadherin staining) in rat skeletal muscle myopathy with monocrotaline were unaffected. Sections of mouse skin, stained induced HF. FAPESP, Proc. 2006/54419-9 e CNPq, with haematoxilin-eosin, 1 hour after injection of Proc.151981/2006-0. 1mg jararhagin, presented a profound loss of dermal [email protected] cellularity. This effect was not observed in samples injected with control or 0,5ug of BnP1, but was evident F-039 when a hemorrhagic dose of BnP1 (50mg) was used. FREQUENCY OF MICRONUCLEOUS CELLS OF THE Keratinocytes of the epidermis and hair follicle as SQUAMOCOLUMNAR JUNCTION IN HIV-1 PATIENTS well as the basement membrane were not affected by AND ASYMPTOMATIC TO THE ONCOLOGIC injection of toxins when analyzed by haematoxilin-eosin COLPOCYTOLOGY TEST staining or by immunofluorescence using markers Camila Figueira Mendes, Júlio Boschini Filho, Luiz to epidermis (keratin 5) and basement membrane Ferraz de Sampaio Neto, Bruna Antunes Más (PUC-SP) (laminin 5). These data show the effect of SVMPs on keratinocyte cell and dermal extracellular matrix. The Cytogenetic tests of the micronucleous are the most effect on keratinocytes was similar for hemorrhagic and used ones when assessing genotoxicity degree in non hemorrhagic doses of toxins while the degradation the different human tissues. The micronucleation of dermis occurred only when a hemorrhagic dose was frequency indicates the presence of structural or used, indicating a role of dermal extracellular proteins numerical alterations in chromosomes during the for setting SVMP-induced hemorrhage. cristianibaldo@ cellular reproduction. In our investigation carried out at butantan.gov.br the Research an Environmental Mutagenesis Center (NuPEMA) and approved by the local Ethics Committee, F-041 we evaluated the frequency of micronucleous cells in STEREOLOGICAL ANALYSIS OF the squamocolumnar junction of 40 patients, being SUBCELLULAR STRUCTURES OF 20 of them HIV-1 positive, treated in the Gynecology STANNIUS CORPUSCLES OF FRESH WATER Clinic of the CCMB-PUC-SP and the other 20, control FISH HYPOSTOMUS PUNCTATUS DURING THE patients, treated in the School Health Center of the City GONADAL CYCLE, SUBMITTED TO HIGH CALCIUM of Sorocaba. The Mann WIthney statistical analysis CONCENTRATION of the results demonstrated that the frequency of Yur Maria e Souza Tedesco (Universidade Presbiteriana micronucleous cells (0.41) of HIV-1 positive patients Mackenzie), Sylvia Mendes Carneiro (Instituto was higher than that of the control group (0.20), with Butantan) statistical significance of p<0.001. It is concluded, therefore, that the soropositivity for HIV-1 is related to The Stannius Corpuscles (SC) are endocrine glands, the increase of the micronucleation rate in the cells found mainly in teleosts fishes, which of the squamocolumnar junction, in compliance with produce stanniocalcin, a hypocalcemic what was gotten by Shimura et al., 1999, in lymphocyte hormone also detected in some human organs. culture. This work intends to analyse, both quantitatively [email protected] and qualitatively, the SC ultrastructure, in adults Hypostomus punctatus, during the gonadal cycle, F-040 exposed to a calcium concentration 100% greater than ACTION OF SNAKE VENOM that of normal environment. SC from anesthetized METALLOPROTEINASES ON SKIN: A animals were collected and prepared for transmission 1. 144

livro.indd 144 7/7/2008 16:33:41 electron microscopy. Experimental groups, composed [email protected] by females and males in the resting, maturation and F-043 mature gonadal stages, were submitted to exogenous WNT SIGNALING ACTIVATION DURING Calcium (10.2 mM/l pH 6.8) for 10 days, and compared OSTEOBLASTIC DIFFERENTIATION OF HUMAN to control groups (n=3/group). Volume density (Vv) and BONE MARROW MESENCHYMAL STEM CELL IS cell volume of granular and agranular cells in the SC, CELL DENSITY DEPENDENT the Vv of granular cell organelles, and the number of Danielle Cabral Bonfim, Débora Morueco granules per cell were evaluated for each animal by Portilho, Eliane Ribeiro Martins, Radovan Borojevic, the point-counting method using a Freere-Weibel type Claudia dos Santos Mermelstein, test-system on electron micrographs. In the treated SC Maria Isabel Doria Rossi (Universidade Federal do Rio cells, peripheral chromatin condensation, numerous de Janeiro) vacuolated mitochondria and thicker interlobular The mechanisms that control stem cell differentiation connective tissue were observed. Apparently, the are not fully elucidated, although non-canonical Wnt increase in Ca 2+ concentration in the water affects signalling pathway is thought to have a pivotal role mainly the nuclear volume in the mature stage and the in osteogenic differentiation of MSC. Glucocorticoids mitochondrial compartment. The volume density, total are used to promote osteoblastic differentiation of volume and number of secretory granules are greater MSC in vitro. However, long term administration of in the animals at the mature stage in the control and dexamethasone (dex) induces osteoporosis due to experimental conditions. These data reinforce the inhibition of Wnt signalling in osteoblasts. Besides, dex importance of SC in calcium metabolism as well as its regulates the expression of cadherins in osteoblasts interaction with the gonads. but the effects of dex on the differentiation of MSC are [email protected] not clear. We propose that canonical Wnt pathway and cell adhesion, mediated by cadherins, influence F-042 osteoblastic differentiation of MSC in vitro, and are MACROPHAGE ACCUMULATION IN FAT TISSUE OF modulated by dex. Bone marrow MSC were isolated EX-OBESE PATIENTS AND THE ROLE OF ADIPOSE from volunteered donors and maintained in different TISSUE-MESENCHYMAL STROMAL CELLS IN THE densities. Osteoblastic differentiation was induced DIFFERENTATION OF MONOCYTES in the presence of 10-8M or 10-6 M of dex, Wnt 3a Daiana Vieira Lopes, Karina Ribeiro da Silva, Carolina or 5a conditioned medium, or 10mM of LiCl. Mineral da Silva Golveia Pedrosa, Leandra deposition was evaluated by Von Kossa or Alizarin Santos Baptista, Marcelo Carlos Aniceto de Souza, Red staining. Cadherin and bcatenin expression were Cezar Silveira Cláudio-da-Silva, analised by immunofluorescence and Western Blot. Radovan Borojevic, Christyna Maeda Takiya, Maria High dose of dex inhibited osteoblastic differentiation Isabel Doria Rossi (UFRJ) of MSC maintained at low density. This effect was not observed when Wnt3a, but not Wnt5a, was added. The incidence of obesity and related diseases are The expression of cadherins and bcatenin was down- increasing in the modern world. Obesity is associated modulated by dex treatment. Taken together, the with a state of chronic, low-grade inflammation with results suggest a possible role of glucocorticoids on abnormal citokine production and macrophage (MØ) the regulation of Wnt signalling during osteoblastic infiltration that contributes to insulin resistance. differentiation, through the negative modulation of Adipocytes are thought to be the major source of pro- cadherin-catenin complexes. inflammatory cytokines but a role for Adipose tissue- [email protected] derived mesenchymal stem cells (AT-MSC) has been proposed. AT-MSC are an alternative source of MSC F-044 for cell therapy. They share with bone marrow-derived STRUCTURAL ANALYSIS OF THE ARYTENOID MSC (BM-MSC) the capacity to support myelopoiesis CARTILAGE OF BULLFROG (RANA in vitro, although favoring the differentiation towards the CATESBEIANA) DURING AGING mono-macrophage lineage. The changes that occur in Kris Daiana de Oliveira (Centro Universitário Hermínio AT-MSC during obesity, especially their proinflammatory Ometto - UNIARARAS), Laurecir Gomes (Depto potential, are unknown. In this regard, we evaluated Biologia Celular/IB/UNICAMP), Marcelo Augusto the capacity of BM-MSC and AT-MSC, isolated from Marretto Esquisatto (Centro Universitário Hermínio normal and ex-obese (one year after bariatric surgery) Ometto - UNIARARAS) patients, to induce the differentiation of MØ. FACS analysis showed that the mean fluorescence intensity The arytenoids cartilages are a pair of small three-sided of CD14 was higher in hematopoietic cells cocultured pyramids which form part of the larynx to which the for 14 days with BM-MSC compared to those from AT- vocal cords are attached. The structural aspects of the MSC cocultures. Around 50% of the CD14Hi cells were arytenoids CD16+. Morphological analysis of fat tissues showed cartilages in Bullfrog larynx were analyzed in the presence of small aggregates of CD68+ monocytes animals with 07, 90, 180, 540 and 1080 days old after between the adipocytes of ex-obese patients. These metamorphosis. The samples were fixed and the results suggest that AT-MSC modulate the differentiation sections stained with Toluidine blue; of monocytes and that fat tissue inflammatory process Picrossirius; Van Kossa and Weigert methods. The could be active even one year after bariatric surgery. basophilia indicating the presence of 1. 145

livro.indd 145 7/7/2008 16:33:41 glycosaminoglycans was widely distributed for all extracellular matrix during aging has strong contribution extracellular matrix. During the aging the of mechanical forces. basophilia restricted to the territorial matrix [email protected] became faint. The collagen fibers were distributed perpendicularly to the longest axis of the F-046 cartilaginous plate and were thicker in the older. No THE EFFECT OF IMMOBILIZATION FOLLOWED BY difference was observed in the quantity of the elastic STRETCHING ON THE CALCANEAL TENDON OF fibers present in the perichondrium and territorial matrix RATS in all ages. Some little areas of calcification in the Andrea Aparecida de Aro (UNICAMP), Benedicto de periphery of the cartilaginous stroma were detected in Campos Vidal (UNICAMP), Tatiana Carla Tomiosso the old animals. The ultrastructural analysis showed (UNICAMP), Laurecir Gomes (UNICAMP), Stela reduction in the amount of rough endoplasmic reticulum, Matiello-Rosa (UFSCAr), Edson Rosa Pimentel Golgi complex and other organelles. Also proteoglycans (UNICAMP) seemed less abundant in cartilage of older frogs. The highest birefringence observed in section of cartilage Little is known about the effect of stretching on tendons of older animals, indicates that in those animals there submitted to immobilization. So rats wistar were is larger aggregation of collagen bundles, probably due separeted into groups: GI- control of immobilized rats; a larger number of cross-links. Our conclusion is that GII- immobilized; GIII- control of immobilized rats and the arytenoids cartilage exhibits important structural afterward stretched or allowed free cage activity; GIV- alterations with the aging and that the distribution of the immobilized and stretched; and GV- immobilized and ECM components is similar to mammals. Supported by allowed free cage activity. For biochemical analysis, FAPESP (05/56339-0). glycosaminoglycans (GAG) and hidroxyproline (HO- [email protected] Pro) were quantified. For morphology the sections were stained with toluidine blue (TB) and ponceau SS (PSS). F-045 Analysis of HO-Pro in the different groups, showed no PHYSICOCHEMICAL AND STRUCTURAL ANALYSIS differences in the tension region, but in the compression OF THE PLANTARIS LONGUS region, a larger concentration (mg/g tissue) was TENDON OF BULLFROGS IN DIFFERENT AGES observed in GII (50in relation to the control GI (38), Jymenez de Morais (Centro Universitário Hermínio whereas in GIV and GV were detected lesser HO-Pro Ometto - UNIARARAS), Laurecir Gomes (Depto (around 32) than in the control GIII (45). With respect Biologia Celular/IB/UNICAMP), Marcelo Augusto to GAG, a remarkable presence was found in the Maretto Esquisatto (Centro Universitário Hermínio compression region of GIV (4.2) in relation to the control Ometto - UNIARARAS) GIII (2.7). Observations of sections stained with PSS, indicated that the organization of the collagen bundles In order to determine the physicochemical and structural in immobilized tendons decreased. Analysis of Linear alterations that occur in tendons of animals of different Dichroism of sections stained with TB, showed that ages, the plantaris longus tendon (PT) of bullfrogs immobilization diminished the alignment of the GAG in (Rana catesbeiana) aged seven, 90, 180, 540 and 1080 relation to the collagen bundles in immobilized tendons, days after metamorphosis were analyzed. Tendons however this situation was reverted when remobilization were submitted to swelling (water and acetic acid) test. was allowed. Our results suggest a revision in the The values were analyzed using ANOVA (p<0.5). The stretching procedures, in terms of duration and swelling properties of the different ages of PT presented periodicity of the sessions. noticeable modification. When the tendons were [email protected] transferred from PBS to water a significative increase in wet weight was observed in animals with 180 and 540 F-047 days in relation to others. When tendons were soaked INTERACTION AND REGULATION OF FOCAL in 3% acetic acid, the wet weight was most prominent ADHESION KINASE BY CARDIAC MYOSIN for the animals with 1080 days old. The higher content Aline Mara dos Santos, Rosana Y. Inoue, Ítalo J. C. of water was detected with 180 days (84.2±3.6%) and Rigotti, Antonio R. Calixto, Kleber Gomes Franchini the smaller in animals with 1080 days (79.2±2.8%). The (Universidade Estadual de Campinas) PT has been shown to wrap around the distal tibio- fibular joint, extending into an Aim: Focal Adesion Kinase (FAK) has been implicated aponeurosis. The region under the joint exhibits a in the signaling of cardiac myocytes to mechanical large cartilaginous pad. It presents an arrangement of stress. In this study, we investigated whether FAK convoluted collagen bundles and fibers associated with physically interacts with the C-terminal region of glycosaminoglycans. This region was highly cellular and sacomeric myosin heavy chain (MHC) and whether this weakly basophilic in animals with seven and 90 days. interaction is important to regulation of FAK activity in However, in old animals a large basophilic area was neonatal rat ventricular myocytes (NRVMs). Methods: observed on that region. The cell population observed The pGEX2T construction encoding N-terminal FERM, along the age varied from a fibroblastic phenotype in Kinase and C-terminal domains of FAK tagged to GST, tensional regions to condrocyte-like in compression and the pGEX5X2 encoding the C-terminal region of region of mature animals. In conclusion, our results MHC (Myosin-GST) were expressed in E. coli. MHC confirm that structural adaptation of was purified from neonatal rat ventricles. FAK/MHC 1. 146

livro.indd 146 7/7/2008 16:33:41 association was detected by Pull Down Assay and Far Western Blotting. In vitro Kinase Assay was performed F-049 with FL(full length)-FAK and Myosin-GST. NRVMs were MYOGENIC REGULATORY FACTOR stretched in silicone plates (1 Hz, 10%). Laser confocal MYOGENIN EXPRESSION IN PACU SKELETAL analysis of NRVMs was performed to examine the MUSCLE (PIARACTUS MESOPOTAMICUS) DURING distribution of FAK. Results: Pull Down assays indicated GROWTH that FAK physically interacts with the C-terminal Nabila Scabine Pessotti (UNESP, Institute of region of MHC through its FERM domain. Myosin-GST Biosciences, Department of Morphology, Botucatu), inhibits FL-FAK activation. Autophosphorylation of Fernanda Losi Alves de Almeida (UNESP, Institute of FAK reduces its ability to bind to Myosin-GST. In non- Biosciences, Department of Morphology, Botucatu), stretched NRVMs, FAK coincided with cardiac myosin. Rachel Colauto Milanezi (UNESP, Institute of Cyclic stretch reduced FAK/Myosin association in Biosciences, Department of Morphology, Botucatu), parallel with increasing FAK phosphorylation at Tyr397. Robson Francisco Carvalho (UNESP, Institute of Inhibition of FAK phosphorylation by PP2 attenuated Biosciences, Department of Morphology, Botucatu), the stretch-induced FAK/Myosin dissociation in NMRVs. Danillo Pinhal (UNESP, Institute of Biosciences, Conclusion: Our results indicate that the interaction of Department of Morphology, Botucatu), Carlos Roberto FAK with the C-terminal region MHC is mediated by Padovani (UNESP, Institute of Biosciences, Department FAK FERM domain and may contribute to the control of of Biostatistics, Botucatu), César Martins (UNESP, FAK activity in cardiac myocytes. Institute of Biosciences, Department of Morphology, [email protected] Botucatu), Maeli Dal Pai Silva (UNESP, Institute of Biosciences, Department of Morphology, Botucatu) F-048 TISSUE LOCALIZATION AND ANTICOAGULANT Skeletal muscle growth in fish occurs by ACTIVITY OF A HEPARAN SULFATE FROM hypertrophy and hyperplasia, process regulated by the MOLLUSK differential expression of myogenic Angélica Maciel Gomes (Instituto de Bioquímica Médica regulatory factors (MRFs). The aim of this study was to (UFRJ), Eliene Oliveira Kozlowski de Farias (Instituto analyze Myogenin mRNA expression in white skeletal de Bioquímica Médica (UFRJ), Cintia Monteiro de muscle of pacu (Piaractus Barros (Instituto de Ciências Biomédicas (UFRJ), Vitor mesopotamicus). Fish with 10, 45, 60, 90 and 180 days Hugo Pomin (Instituto de Bioquímica Médica (UFRJ), after hatching were sacrificed and muscle samples were Mauro Sergio Gonçalves Pavão (Instituto de Bioquímica collected and frozen in liquid nitrogen. Myogenin mRNA Médica (UFRJ) expression was determined by semi-quantitative RT- PCR. All PCR products were cloned and sequenced. Extracellular matrix (ECM) is a complex network A consensus partial sequence for Myogenin gene of macromolecules, which forms a barrier and a (832 base pairs) was obtained. The pacu Myogenin scaffold on which tissues are built. It is composed of nucleotide sequence displayed high similarity among glycosaminoglycans and different proteins. Heparan several vertebrates, including teleosts. Myogenin mRNA sulfate (HS) is a glycosaminoglycan present in expression level was significantly high in 45 days after both cells and ECM. Besides other roles, HS has hatching. This significative expression can be related anticoagulant activity by binding to antithrombin and to an intense cell differentiation that occurs during inhibiting thrombin and factor Xa. In the present work hypertrophy process in pacu skeletal muscle. In the we performed a histological study aiming to access other phases, the smaller and continuous Myogenin the distribution of sulfated glycosaminoglycans in a mRNA expression level shows that cell differentiation bivalve mollusk. In addition, we analyzed a heparan and muscle fiber hypertrophy also occurs in this period. sulfate-like glycan extracted from the mollusk tissues. [email protected] Histochemistry performed with alcian blue before and after degradation with specific enzymes and F-050 nitrous acid, indicated that HS was the main sulfated XAF EXPRESSION CORRELATES WITH XIAP glycosaminoglycan present in the mollusk tissues. DEGRADATION DURING MEGAKARYOCYTIC Preliminary structural analysis revealed that the glycan DIFFERENTIATION OF THE HUMAN CHRONIC resists incubation with heparinase I and III and is MYELOGENOUS CELL LINE K562 partially degraded by deaminative cleavage with nitrous Érika Carvalho Silva, João de Séllos R. Laclette, acid. Further structural features were determined Gabriel Wajnberg, Carlos G. M. Ferreira, Cinthya by 1D 1H NMR spectrum and 2D COSY and HSQC Guimarães Sternberg (Instituto Nacional de Câncer) spectra, showing NMR signals of glucuronic acid. The mollusk HS showed a 4-folds lower anticoagulant The human chronic myelogenous cell line K562 activity (43.2 units/mL) than bovine heparin (180 units/ behaves as a pluripotent hematopoietic precursor mL), as measured by the aPTT assay. This HS was and can undergo megakaryocytic differentiation upon also highly able to inhibit factor Xa (IC50= 0.835 µg/ treatment with Phorbol 12-myristate 13-acetate (PMA). mL) and thrombin (IC50= 9.3 ng/mL), in the presence of PMA-treated cells displayed several known hallmarks antithrombin. of differentiation such as expression of CD41, CD61, [email protected] increased DNA ploidy and adhesion. Activation of caspase-3 was recently shown to be a marker of 1. 147

livro.indd 147 7/7/2008 16:33:42 megakaryocytic differentiation, although originally EXPRESSION associated to apoptosis. We Therefore sough to Elenice Monte Alvarenga, Maria Luiza Silveira Mello investigate the fate of other apoptosis-related proteins (Universidade Estadual de Campinas) during megakaryocytic differentiation. PMA treatment of K562 cells induced XIAP degradation, as well as the Proteins of the LOX family are copper-dependent lysyl appearance of caspase-3 activity related markers, such oxidases involved with the initiation of as fodrin cleavage. The degradation of XIAP appeared cross-links in collagens and elastin and in interactions to be dependent on proteasome activity, since MG132 with histone H1 in the cell nuclei. It has recently been was able to block XIAP degradation. We also observed demonstrated that transfection of COS-7 cells with lox actin overexpression, previously reported to occur induces interaction between H1 and recombinant LOX during megakaryocyte differentiation. XAF is a pro- followed by chromatin decondensation. On assuming apoptotic protein, which overexpression is associated that changes in interphase chromatin organization to XIAP degradation. Indeed, XAF mRNA increases induced by LOX could also affect the organization of over differentiation of K562 cells, correlating to XIAP chromosomes, we examined cell division images in degradation and suggesting that this event might not toluidine blue-stained COS-7 cells transfected with occur by the canonical pathway involving Smac release a lox gene, and NRK-49F cells transfected with lox from mitochondria. antisense constructs. The cells were kindly provided [email protected] by Dr. A. Di Donato. A decrease in frequency of anomalous metaphases and of chromosome bridge F-051 formation was detected in the lox-transfected COS-7 VALPROIC ACID CAUSES DECONDENSATION OF cells in comparison to the untreated control. Mitosis INTERPHASE CHROMATIN IN 3T3 CELLS abnormalities were very scarce in NRK-49F cells; Marina Barreto Felisbino; Maria Sílvia Viccari Gatti; practically no change in frequency of chromosome Maria Luiza Silveira Mello bridge and tripolar metaphase formation was (Universidade Estadual de Campinas) detected with transfection of the cells with antisense lox constructs. It is suggested that for cells usually Histone hypoacetylation promoted by histone displaying abundant cell division abnormalities (COS-7 deacetylases (HDACs) is generally associated with transformed kidney fibroblasts), the repression of gene expression. Valproic acid (VPA), a chromatin organization affected by LOX-H1 potent anticonvulsant, has been interactions may reduce the abnormal signals demonstrated to inhibit HDACs at therapeutic responsible for chromosome-mitotic spindle concentrations (< 0.7 mM) in some human cell lines. aberrant relationships. For cells with scarce Here we investigated the association of a putative mitosis defects (NRK-49F normal kidney HDAC inhibition by VPA with change in chromatin fibroblasts), reduction in frequency of LOX-H1 supraorganization in 3T3 cell nuclei, a model with interactions possibly does not affect chromatin or conspicuous condensed chromatin bodies. Cells chromosome organization. (Supported by CNPq and cultivated for 24 h and then treated with 0.5 and 1.0 FAPESP). mM VPA for 1 and 2 h were subjected to the Feulgen [email protected] reaction and investigated with a Zeiss scanning microspectrophotometer image analyser. Data were F-053 plotted in a scatter diagram relating the percentage of BLOOD EXAMINATION X WATER POLLUTION nuclear areas covered by condensed chromatin (Sc%) Bruno Fiorelini Pereira, Patricia Pasquali Parise and the contrast between condensed and Maltempi, Flávio Henrique Caetano noncondensed chromatin (AAR) (Vidal BC, Biol Cell (Departamento de Biologia - Universidade 50: 137,1984). Only the 1 mM VPA concentration Estadual Paulista - Campus de Rio Claro) significantly induced a decrease in Sc% simultaneous to an increase in AAR for the 1 h-treatment. Other All research studies regarding the effects of water image parameters indicated in this case chromatin pollution on fish analyze many different variables to decondensation. After extending the treatment period demonstrate and prove an environmental problem. On to 2 h, the increase in AAR values was maintained this study we propose a simple, new and faster method while a light increase in condensed chromatin areas to verify these effects that consists on analyzing and was verified. The results indicate that the interphase comparing the number of fish white cells between a chromatin (including condensed chromatin) in 3T3 cells treated group and a control group. The experiment is susceptible to decondensation as affected by VPA used thirty females of Astyanax scabripinnis abtained in a time – and concentration–dependent manner and in CEPTA - Instituto Chico Mendes, that were divided in possibly resulting from an induced HDAC inhibition. two groups. The control group was exposed to the pure The longer above-cited VPA exposition may trigger a water of UNESP Campus de Rio Claro’s well, and the cytotoxic effect. (Supported by CNPq and FAPESP). treated group exposed to the water from Lago Azul - Rio [email protected] Claro - SP, considered polluted by research studies from SABESP (Companhia F-052 de Saneamento Básico do Estado de São Paulo) CELL DIVISION ABNORMALITIES IN CELLS and the city’s Town Hall. Blood samples from both ELICITED TO HIGH AND LOW LOX fish groups were obtained by cardiac puncture with 1. 148

livro.indd 148 7/7/2008 16:33:42 heparinized syringes, stained with Leishman and Tendons have the main function of transmitting force photographed on Leica optic microscope. The analysis from muscles to bones. The extracellular matrix (ECM) showed a difference on the number of white cells is composed mainly of type I collagen, proteoglycans between these two groups. A considerable increase on and non-collagenous proteins. The biomechanical the number of white cells was noticed on the treated properties of the tendon are directly related with the group, while the control group had no changes. This distribution of collagen fibrils and with their interaction increase is a reaction to the water pollution, easily with other ECM components. Today the obesity is noticed by this experiment. The results presented an considered a mundial epidemy, but little is known interesting relationship between fish white cells and about its influence on the tendon. The analysis of water pollution and can be easily used to detect an the distribution of fibrils showed that the deep digital environmental problem before starting other complex flexor tendon (DDFT) of Zucker rats (fa/fa), genetically research studies. obese, presented unimodal distribution of collagen, [email protected] and fibrils with smaller mean diameters. The content of glycosaminoglycans of obese animals was smaller F-054 (1.31 ± 0.46 mg/g) than the lean rats (2.45 ± 0.6 CELL CULTURE OF PERNA PERNA MUSSEL GILL mg/g), but with respect to non-collagenous proteins VERSUS WHOLE ORGANISM no difference was found. SDS-PAGE of extract of both EXPOSURE FOR TESTING POLLUTANT ON THE groups exhibited the same bands, and no alterations MULTIXENOBIOTIC RESISTANCE MECHANISM were observed in reducing and non-reducing conditions. (MXR) Our results show in short, the obesity and overweight Ana Carolina Barni de Azevedo, Fernanda cause alterations in the diameter and distribution of Emmert, Francine Rodrigues Machado, Edileusa collagen fibrils, as well as in the glycosaminoglycans Cristina Marques Gerhardt, Marcos Luiz Pessatti content. The reduced mobility observed in the fat rats, (Universidade do Vale do Itajaí) and consequent absence of stimulus, may be the reason of small diameter fibres and low content of The multixenobiotic resistance mechanism (MXR), glycosaminoglycans. (FAPESP Processo 04/14358-5). mediated by proteins like Pgp and MRP, control the cell [email protected] permeability against various cytotoxic agents, conferring cellular adaptation to contaminated environments. This F-056 mechanism has itself been revealed as a universal THE EFFECTS OF CADMIUM CHLORIDE ON THE defense biologic system. To test the effects of pollutants NUCLEAR PHENOTYPE IN THE SILVER CATFISH on the MXR mechanism, organisms in aquarium and (RHAMDIA SP.) cells in culture were exposed to lead (0,025 and 0,25 DIEGO FRAGA SILVEIRA, MARI ALINE TODERO mg/L) and tributyltin (5 and 25 ng/L) for until 48 hours. WINCK, MÔNICA SAUDADE, CARLOS AUGUSTO Gills sections of exposed organisms or 104 cells were BORBA MEYER NORMANN, VALESCA VEIGA incubated in rhodamine B CARDOSO CASALI (fluorophore substrate of Pgp and MRP) 2 uM for (Centro Universitário Metodista IPA) 60 min in absence or presence of the contaminants. The efflux activity (0-60 min) was measured using Cadmium chloride is known to cause adverse effects microplate reader (Ex 540nm and Em 590nm). The cells in biological processes. This study investigates the when exposed to lead 0,25 mg/L and TBT 25 ng/L had effects of CdCl2 on red blood cells of the silver catfish, a significant loss of the cellular viability. When exposed Rhamdia quellen. Juvenile specimens with 10 ±1,5 cm to 0,05 mg/L and 5 ng/L it was verified an induction body lenght, raised in pools, were acquired by EMATER of mechanism MXR in 24 hours of exposure. The in Lomba Grande, Novo Hamburgo-RS. Fishes were exposure of animals in aquarium to lead 0,25 mg/L and maintained in aquaria, pH 6,7, photoperiod 14/10 light/ to TBT 25 ng/L promoted an increase of the dark, 24±1,2°C, receiving flocculated food ad libidum expression of mechanism MXR in 24 and 48 hours. twice a day. 40 fishes werer used; 10 animals per Taken in set, the results evidence the similarity of aquarium. In two aquaria, we added CdCl2, q.s.p. 9 mechanism MXR between isolated cells and animals ppm. The fish were killed after periods of 24, 48, 72 (gill tissue), although isolated cells were more sensible and 96 hours of exposure. After ice anesthesia and than gill tissue front the contaminants. In view of the decapitation, blood was collected, smears were fixed cost and practice, gill tissue would have to be prioritized in methanol and stained with May Grüenwald/Giemsa. for transport activity assays while the cells would have 2000 cells were counted per slide. Blebbed nuclear to be protected for specific applications. morphology and micronuclei were found in cadmium- [email protected] exposed fishes. The number of micronuclei increased while the number of blebbed erythrocytes decreased F-055 during the experimental exposition of the fishes. Cations ALTERATION IN THE DIAMETER OF are competitors for nuclear histones attaching to DNA COLLAGEN FIBRILS IN TENDONS OF OBESE phosphate radicals which may have caused the nuclear ZUCKER RATS (FA/FA) alterations observeds. Breaks in genetic material Adriano Biancalana, Lúcio Algusto Veloso, yielding to micronuclei reflect cytogenetic abnormalities, Laurecir Gomes (UNICAMP) increasing with exposure, in hematopoietic stem cells of the fish, induced by 9 ppm Cd2+. Possibly, the 1. 149

livro.indd 149 7/7/2008 16:33:42 metallothionein expression in fish tissues reduced the the present study was to extend the initial impact of Cd2+ leading to decreased number of analysis of signaling pathways and biological effects like blebbed erythrocytes in the smears. The results confirm viability, proliferation, senescence and death promoted clastogenic and morphological nuclear alterations as by sustained-3-days stimulation. G and F/R cells were a result of Cd2+ contamination in red blood cells of R. pre-treated with [email protected] pharmacological inhibitors, and then stimulated with 0.001-100nM ACTH, 1-10ng/ml FGF2 or both and F-057 assayed by immunoblotting, dye exclusion assay, MTS INVESTIGATION OF A POTENTIAL LINK BETWEEN assay, Anexina V and SABGal assay. We found that MYOSIN-VA AND FOCAL ADHESION KINASE IN THE ACTH-treatments: promote an ADHESION DYNAMICS anti-proliferative effect characterized by Guilherme Pedreira de Freitas Nader (USP), Kleber G. accentuated apoptosis but not necrosis or senescence; Franchini (UNICAMP), Enilza Maria Espreafico (USP) induce strong PKA-dependent JunB expression; inhibit Akt, JNK and c-Jun Myosins constitute a family of molecular motors phosphorilation; and activate p38-MAPK. However, responsible for traffic of biomolecules and cell migration. FGF-2-treatments: present opposite Apoptosis is a programmed cell death, which plays a biological effects increasing viability and crucial role in the maintenance of tissue homeostasis. proliferation; strongly phosphorilate c-Jun protein Evidence from protein interaction implicates class V through JNK pathway; and present differential kinetics myosin with the regulation of pro-apoptotic molecules. of Ser-Akt (and not Thr) phosphorilation in G and F/R Disturbs in the regulation of this process may lead to cells, in 15 and 5 minutes, respectively. Taken together, diseases, such as cancer. Integrins and focal adhesion these results support the notion that ACTH and FGF2 kinase (FAK) are essential mediators of the drive contrasting responses in normal adrenocortical adhesion process, controlling cell survival and cells in proliferation. Since interaction between myosin II vitro: ACTH induces apoptosis by p38 and JunB and FAK have already been confirmed, we proposed activation, while FGF2 leads to proliferation by ERK, to investigate a possible role for myosin-Va (MVa) JNK, c-Jun and Akt phosphorilation. in adhesion process and if it interacts with FAK or Supported by FAPESP, CAPES, CNPq and PRPq-USP. modulates its activity. Assays of adhesion [email protected] dynamics at fibronectin matrix showed an increase in phosphorylation at serine residue of MVa in cells that F-059 are in suspension in comparison with cells that had just IS IT POSSIBLE TO EXTEND THE SURVIVAL OF adhered. Blocking adhesion of melanoma cells led to ARTIFICIAL ENUCLEATED CELLS? redistribution of phosphorylated MVa toward the cell Ketna Guilhermino Khusal, Michel Rabinovitch periphery. Interestingly, when MVa is transfected in (UNIFESP) MYO5A mutant cells, the adhesive capacity of these cells raises and MVa localizes at the filopodia. Enucleated cells (cytoplasts) obtained by Immunoprecipitation using cells cotransfected with centrifugation of cells monolayers or suspension, in the MVa and FAK, and pull down assay, using recombinant presence of cytochalasin, have a FAK and purified MVa, revealed a physical interaction necessarily limited rate survival. On our between these proteins. In addition, colocalization of laboratory, we develop studies on the infection of FAK and MVa in focal contacts during cell adhesion cytoplasts with non viral pathogens, like Shigella dynamics was flexneri, to evaluate the participation of the host cell observed using confocal microscopy. This nucleus at the different steps of the research suggests a functional link between MVa and intracellular infection. Among other applications, the FAK in the control of cell adhesion. extension of survival of cytoplasts would permit the [email protected] study of intracellular infections by long “double-time pathogens”. Our objective is to search for interventions F-058 capable of extending cytoplasts survival. In our DISTINCT BIOLOGICAL EFFECTS AND SIGNALING laboratory, the mild hypothermia (32-34ºC) was able PATHWAYS OF ACTH AND FGF2 IN PRIMARY RAT to prolong the survival of cytoplasts of L929 cells, ADRENOCORTICAL CELLS elevating its half-life from 37h to 65h and allowing the Gabriele Ebling Mattos, Claudimara Ferini Pacicco Lotfi infection, differentiation and multiplication of (Anatomy Department, Institute of Biomedical Science - Trypanosoma cruzi (Coimbra et al, 2006). Cytoplasts of University of São Paulo) Vero cells survive far less (about 18h at 37ºC and 30h at 34ºC). Nevertheless, the treatment for both cells was In previous work we reported that the only successful when performed after the enucleation, anti-mitogenic effect of ACTH (corticotropin) is mediated which excludes genetic modulation in this case. by PKA/CREB but not by ERK pathway in Glomerulosa Treatments with other sulfydril molecules reported as (G) and Fasciculata/Reticularis (F/R) adrenocortical cytoprotective were also assayed. Therefore, we verified primary culture cells. In that in the presence of 50mM of 2-mercaptoethanol the contrast, FGF2 (Fibroblast Growth Factor 2) number of cytoplasts was constant at 37ºC for more elicits mitogenic response in both cell types. The aim of than 5 days. However, it reduced or abolished the 1. 150

livro.indd 150 7/7/2008 16:33:42 staining of the cytoplasts with neutral red. On the other stained with toluidine blue and observed under the light hand, N-acetyl-L-cysteine and d-b-hidroxybutirate didn´t microscope. For biochemical analysis, the cartilage present toxicity for the cytoplasts, but its effects upon extracts were fractionated by ultracentrifugation in CsCl. cell longevity is still unclear. The top fraction (D4) was used for dosages of [email protected] proteins, glycosaminoglycans, and for chromatography in DEAE-Sephacel. Samples of the F-060 fractions eluted from DEAE were analyzed in SDS- EFFECT OF DIFFERENT FEEDER LAYERS ON PAGE. The analysis of the sections stained with THE PROLIFERATION OF PRIMARY ACUTE toluidine blue showed a basophilia widely distributed in LYMPHOBLASTIC LEUKEMIA the ECM of cartilage of 30 days old chickens, while for Jaíra Ferreira de Vasconcellos (Centro Infantil Boldrini ages of 90 and 150 days, the basophilia was restricted / Universidade Estadual de Campinas), Silvia Regina to the territorial matrix. Our biochemical results Brandalise (Centro Infantil Boldrini), José Andrés Yunes showed no significant alterations in the concentration (Centro Infantil Boldrini) of proteins (around 13 mg/g tissue); increase in the concentration of glycosaminoglycans (0.304±0.016; Background: Acute Lymphoblastic Leukemia (ALL) 0.341±0.001; 0.377±0.011 mg/g tissue), in the ages is the most frequent childhood malignancy. ALL cells of 30, 90 and 150 days respectively, were observed. rapidly undergo apoptosis in culture, with a hundred Analysis in SDS-PAGE showed a protein with 200 kDa, percent of cell dead after 2-3 days. The survival and, in remarkably present in older chickens. In presence of some cases, mercaptoethanol, this protein migrated as a 50 kDa proliferation can be achieved if the ALL is protein. The ensemble of our results indicates that the co-cultured with bone marrow (BM) feeder layer cells. tracheal cartilage is quite sensible to ageing and that Objectives: The aim of this study was to compare the the oligomeric protein with 200 kDa may be a marker proliferation of primary ALL cells in co-culture with protein for ageing. primary human BM stroma (BMS), mesenchymal (MSC) [email protected] or endothelial (BMEC) cells. Methods: BMS, MSC and F-062 BMEC cells were KININOGEN INTERACTION WITH CELL SURFACE isolated from normal BM mononuclear cell samples. RESULTS IN ENDOCYTOSIS MEDIATED BY BMS and MSC cells were obtained by direct culture of PROTEOGLYCANS BM mononuclear cells in DMEM+10%FCS and MSCGM Kátia Regina Brasil Melo (UNIFESP/ EPM), Fábio (Cambrex) D. Nascimento (UNIFESP/EPM), Mariana S. Araújo medium, respectively. BMEC were derived by culturing (UNIFESP/EPM), Misako U. Sampaio (UNIFESP/ immunomagnetic selected CD105+ cells (Miltenyi) EPM), Helena B. Nader (UNIFESP/EPM), Ivarne Luis S. in EGM-2MV (Cambrex) medium. Primary ALL cells Tersariol (UMC), Guacyara da Motta (UNIFESP/EPM) were co-cultured with the three feeder-layers in AIM-V (Invitrogen) medium. In some cases medium was Human high molecular weight kininogen (HK) is a supplemented with soluble CD40-ligand (CD40L). Co- multifunctional plasma protein involved in both cell cultures were incubated for 48h and cell proliferation and vascular biology. It is also a potent local regulator was measured by FACS analysis. Results: ALL cells of blood pressure and vascular permeability through survived better in contact with the MSC or BMEC bradykinin delivery. Some proteins and proteoglycans than with BMS cells. The CD40L did not confer any have been described as HK receptors on cell surface. survival advantage to the ALL cells. Only one ALL case The aim of the present work is to analyze the HK proliferated in co-culture with MSC. Conclusion: MSC or interaction with cell surface.We used rabbit aorta BMEC did not confer differences as supportive cells to endothelial cells (RAECs) and Chinese hamster ovary primary ALL survival and should be used in co-culture cell lines wild type (CHO-K1) and defective in studies. glycosaminoglycans biosynthesis (CHO-745). HK Supported by: CNPq and FAPESP. binding to CHO-K1 was disturbed by heparinn heparan [email protected] sulfate and chondroitin 4-sulfate. HK bound to RAECs colocalized with heparan sulfate on cell surface and F-061 was visualized in acid endosomal vesicles in both ANALYSIS OF THE EXTRACELLULAR MATRIX RAECs and CHO-K1 but not in CHO-745. In CHO-K1 OF TRACHEAL CARTILAGE OF CHICKEN WITH HK internalization was blocked by chloroquine, methyl- DIFFERENT AGES beta-cyclodextrine, 2-deoxy-D-glucose and FCCP, and Laurecir Gomes, Frances Lilian Lanhellas HK did not colocalize with transferrin-Cy5. HK Gonçalves, Edson Rosa Pimentel (UNICAMP) assembled on CHO-K1 was cleaved and chloroquine blocked the proteolysis. kinins were Several studies have analyzed the morphological and detected in incubation buffer of CHO-K1 and RAECs, biochemical alterations in articular cartilage of animals the amout was much higher at pH 7.35 comparing of different ages, but few similar to pH 5.5 in CHO-K1 and both serine and cysteine studies have been carried out with tracheal peptidases were involved in this process. HK-receptor cartilage. Here, the extracellular matrix (ECM) of the complex assembled on cell surface enters target cells tracheal cartilage of chickens with 30, 90 and 150 by caveolae-mediated andocytosis and heparan sulfate days, was analyzed. Sections of tracheal cartilage were proteoglycan contribute for the uptake. HK interaction 1. 151

livro.indd 151 7/7/2008 16:33:42 with tumor cells can be a source of kinins on cell surface. Financial support: FAPESP, CNPq, CAPES. Brown spider venom are capable to induce [email protected] clinical manifestations frequently associated with dermonecrosis, intravascular hemolysis, and renal F-063 failure. The venom is a complex mixture of proteins, MECHANISTIC RELATIONSHIP OF including phospholipases-D (PLD). PLD functional PHOSPHOLIPASIC ACTIVITY AND TOXICITY OF characterization showed that toxin has ability to DERMONECROTIC TOXIN FROM BROWN SPIDER trigger complement-dependent hemolysis. We aimed VENOM to evaluate “in vitro” hemolytic effect directly induced Olga Meiri Chaim (UNIFESP), Dilza Trevisan Silva by the venom, recombinant PLD (LiRecDT1), and (Universidade Federal do Paraná), Valéria Pereira a site-directed mutated isoform which has a drastic Ferrer (Universidade Federal do Paraná), Daniele reduction of the phospholipasic activity. Human Chaves Moreira (Universidade Federal do Paraná), erythrocytes lysis assays were performed incubating Rafael Bertoni da Silveira (UEPG), Waldemiro Gremski concentrations (0.025-25 µg/mL) of the venom and (Pontifícia Universidade Católica do Paraná), Silvio both toxins with a Tris-sucrose buffered-suspension Sanches Veiga (Universidade Federal do Paraná), of erythrocytes (serum free, 108 cells/mL) for 4h-24h Helena Bonciani Nader (UNIFESP) at 37°C. LiRecDT1 induced hemolysis in time and concentration dependent manner and mutant toxin Dermonecrotic toxins are the most well-characterized triggered no hemolytic effect. LiRecDT1 -induced biochemical constituents of Loxosceles spp. venom. hemolysis decreased when the venom and LiRecDT1 Recombinant forms are capable of reproducing most all were previously exposed to metal divalent chelators. cutaneous and systemic manifestations. They belong to Morphological changes in inverted microscope and a toxin family containing 30-35 kDa proteins identified interaction of toxins on the cell surface as “planted as phospholipase-D. There is currently no direct antigens” by confocal immunofluorescence analysis confirmation for a relationship between dermonecrosis were observed. The intensity of hemolysis changed and inflammatory response induced by DTs and their when incubated with erythrocytes from different animal PLD activity. We modified a toxin isoform by site- models which show distinct sphingomyelin quantities directed mutagenesis to elucidate if phospholipasic on cell membrane. We suggest that LiRecDT1 can activity is directly related to the biological effects. cause direct hemolysis which efforts the idea of catalytic Mutated toxin has a His 12 (conserved catalytic domain) activity involvement on hemolytic effects. These results has been replaced with Ala. H12A SMase activity was corroborates with the comprehension of molecular drastically reduced and CD analysis demonstrated mechanism of the Loxoscelism. [email protected] similar spectra for both isoforms, confirming the mutation did not change general secondary structures F-065 of the molecule or its stability. Venom and DT1 antisera CHANGES IN VINCULIN AND PAXILLIN showed antigenic cross-reactivity to both toxins by DISTRIBUTION AND EXPRESSION IN ELISA and WB. Dermonecrosis was abolished by MYOGENIC CELLS AFTER MEMBRANE mutation, whereas rabbit skin revealed a decreased CHOLESTEROL DEPLETION inflammatory response for H12A. Residual PLD activity Eliane Ribeiro Martins, Carolina Pontes Soares, Manoel was confirmed increasing concentrations of H12A by Luis Costa, Claudia dos Santos Mermelstein (UFRJ) dermonecrosis and fluorometric measurement in vitro. Lipid array showed toxins have an affinity for the same Muscle differentiation is a multistep process that lipids and were both toxins detected on cell surface involves the proliferation of myoblasts and its of treated RAEC cells. Confocal analysis suggested subsequent fusion, culminating with the formation of redistribution of lipid rafts on the RAEC membrane multinucleated and striated myotubes. Cell-cell and only after exposure to DT1. Choline release in vitro in cell-extracellular matrix (ECM) adhesion are key events RAEC membrane detergent extract and morphological in this process. Particularly important for myogenesis changes corroborate with a phospholipasic-dependent is the cell-ECM adhesion, since it can regulates both toxic mechanism. adhesion and movement of myoblasts during the [email protected] pre-fusion step. Many proteins have been described as being involved in the adhesion of myoblasts to its F-064 surrounding substrate, among them are actin, integrin, BROWN SPIDER (LOXOSCELES vinculin and paxillin. Cell-ECM adhesion sites have INTERMEDIA) VENOM AND RECOMBINANT been found to be present within cholesterol PHOSPHOLIPASE – D SHOWS DIRECT enriched-membrane micro-domains. Recently, our HEMOLYTIC ACTIVITY group has shown that membrane cholesterol depletion Daniele Chaves Moreira (Universidade Federal do by methyl-beta-cyclodextrin (MCD, 4 mM) induces the Paraná), Olga Meiri Chaim (UNIFESP), Youssef Bacila activation of myogenic Sade (Universidade Federal do Paraná), Juliana proliferation and differentiation. Here we further Ferreira de Moura (Universidade Federal do Paraná), studied the effects of MCD on cell-ECM adhesion in Waldemiro Gremski (Pontifícia Universidade Católica do embryonic chick skeletal muscle cells cultures. We used Paraná), Silvio Sanches Veiga (Universidade Federal immunofluorescence do Paraná) microscopy and immunoblotting to study the distribution 1. 152

livro.indd 152 7/7/2008 16:33:43 and expression of the proteins actin, vinculin and The inhibition of COX activity by NSAIDs has been paxillin. After cholesterol depletion, vinculin expression widely used. NSAIDs commonly inhibit both COX is up-regulated and the protein is found concentrated in isoforms (COX-1 and COX-2), also lines in the dorsal part of the sarcolemma. Expression inhibiting the prostaglandins synthesis. The COX-2 of paxillin is highly enhanced, as well as its selective inhibitors have been an phosphorylation state. We suggest that alternative option in a variety of diseases cholesterol-enriched membrane micro-domains treatments with antiinflammatory effects so potent as might be involved in the regulation of adhesion and the traditional NSAIDs. This study investigated the movements of myoblast prior to the cell fusion event effect of two different doses of aspirin (25 and 100 mg/ that leads to a complete myogenic differentiation. kg/day) and celecoxib (5 and 10 mg/kg/day) in the [email protected] excisional cutaneous wound healing in males Balb/c mice. Wounds were performed two days after the F-066 beginning of treatment. Fourteen days after wounding, LEUCUROLYSIN-A, A METALLOPROTEINASE wounds with adjacent skin were removed. Wound FROM BOTHROPS LEUCURUS SNAKE VENOM contraction, WITH CYTOTOXIC, THROMBOLYTIC AND reepithelialization, inflammatory infiltrate and collagen EDEMATOGENIC ACTIVITIES fibers were analyzed and myofibroblasts and blood Luiza Helena Gremski (UNIFESP), Olga Meiri Chaim vessels were evaluated by stereology. The animals (UNIFESP), Katia Sabrina Paludo (UNIFESP), Youssef treated with low-dose of aspirin and celecoxib presented Bacila Sade (Universidade Federal do Paraná), Michel a similar pattern of wound contraction, reepithelialization Fleith Otuki (Universidade Federal do Paraná), Michael and inflammatory infiltrate with the control animals. Richardson (Fundação Ezequiel Dias), Waldemiro Nonetheless, these groups showed a reduced collagen Gremski (Pontifícia Universidade Católica do Paraná), fibers density and an elevated density of blood vessels. Eladio Flores Sanchez (Fundação Ezequiel Dias), Silvio The animals treated with high-dose of aspirin and Sanches Veiga (Universidade Federal do Paraná) celecoxib showed a delay in wound contraction and reepithelialization, a reduced collagen fibers density Leucurolysin-a (leuc-a), a 23kDa non-hemorrhagic and an elevated inflammatory infiltrate. These groups metalloproteinase, is found in venom of the viper also presented the density of blood vessels reduced. Bothrops leucurus. Here, we examine the biological These findings demonstrated that high-dose of aspirin activities of leuc-a, including thrombolytic activity, direct and celecoxib delayed the cutaneous wound healing in effects on endothelial cells in culture and edematogenic mice, highlighting the importance of both COX isoforms activity in vivo. We demonstrate fibrinolytic activity of during wound repair, not only in the inflammatory phase. leuc-a, in which the protease specifically degrades [email protected] alpha, beta, and gamma-gamma chains. While not causing hemorrhaging, leuc-a does cause thrombolytic F-068 activities in whole blood clots. Endothelial cells are LOW DOSE OF PROPRANOLOL IMPROVES highly resistant to leuc-a in culture. Cell viability suffered CUTANEOUS WOUND HEALING OF THERMICAL only when cells were exposed to large quantities of INJURED RATS the protease. Nevertheless, leuc-a induces changes in Bruna Romana-Souza, Adriana Paulino do cell morphology. The impact of leuc-a on cell adhesion Nascimento, Andréa Monte-Alto-Costa (UERJ) was confirmed by an adhesion assay, in which cell adhesion to fibronectin decreased due to leuc-a. This Severe burns stimulate a hypermetabolic mild cellular impact is unlike that of crude venom, response which causes systemic complications to where lower concentrations triggered cell death and patients. Propranolol administration, a a greater reduction in cell adhesion. Also, leuc-a non-selective beta-blocker, reduces this response and increased microvessel permeability with marked edema increases patients’ survival. in mice peritoneum and foot pads. These effects are Nevertheless, few studies showed effects of propranolol similar to those of other P-I class SVPMs. These in vivo on cutaneous wound healing of severe burns. effects were weaker when crude venom was tested. In Therefore, this study macro and microscopically conclusion, albeit not showing significant hemorrhagic evaluated effects of propranolol administration on activity, leuc-a can induce a prominent edema which cutaneous wound healing of thermical injured rats. A appears to be significant in the local effects observed third-degree burn was created in female Wistar rats. after B. leucurus venom accidents. One week after burning, animals were daily treated [email protected] with propranolol (6 mg/kg) dissolved in water until euthanasia, while control group only received water. F-067 Wound area was HIGH-DOSES OF ASPIRIN AND CELECOXIB measured weekly and animals were killed 63 days after DELAYS MICE CUTANEOUS WOUND burning. Lesions and adjacent skin were formalin-fixed HEALING and paraffin-embedded. Wound area was greaterin the Jeanine Salles dos Santos, Adriana Paulino do control group than in the propranolol-treated group 21, Nascimento, Thaís Porto Amadeu, 53 and 63 days after burning. All propranolol-treated Andréa Monte-Alto-Costa (UERJ) animals presented more than 70% of re-epithelialized wound area 63 days after burning, while control animals 1. 153

livro.indd 153 7/7/2008 16:33:43 did not. Inflammatory cells amount and blood vessels F-070 density were greater in the control group than in the PROCESSING OF KININOGEN ASSEMBLED ON propranolol-treated group. CELL SURFACE Cellular proliferation, myofibroblasts density, collagen Camila Lopes Veronez (UNIFESP/EPM), Katia Regina deposition, active MMP-2 levels were reduced in the Brasil Melo (UNIFESP/EPM), Veronica Carvalho control group compared to the propranolol-treated Rezende Pacheco (UNIFESP/EPM), Misako Uemura group. In conclusion, Sampaio (UNIFESP/EPM), Helena Bonciani Nader propranolol administration (low dose) improves (UNIFESP/EPM), Ivarne Luis dos Santos Tersariol cutaneous wound healing of thermical injured rats (UMC), Guacyara da Motta (UNIFESP/EPM) reducing local inflammatory response and improving subsequent phases of the reparative process. Human kininogens are intravascular proteins and high [email protected] molecular weight kininogen (HK) is a F-069 multifunctional glycoprotein and circulates as a single COMPARATIVE STUDY OF TWO RECOMBINANT polypeptide chain. HK hydrolysis by DERMONECROTIC TOXINS OBTAINED FROM plasma kallikrein releases bradykinin (BK), a potent BROW SPIDER (LOXOSCELES INTERMEDIA) local regulator of blood pressure, and produces cleaved VENOM HK (HKa). BK promotes angiogenesis and HKa is Youssef Bacila Sade (Universidade Federal do Paraná), anti-angiogenic. In spite of HK is a cystatin cathepsins Josiana Kusma (Universidade Federal do Paraná), B and L have been characterized as kininogenases. Valéria Pereira Ferrer (Universidade Federal do The aim of the present work is to verify if intact HK can Paraná), Dilza Trevisan Silva (Universidade Federal do be proteolyzed after assemble on cell surface. Our Paraná), Luiza Gremski (UNIFESP), Rafael Bertoni da cell model was rabbit aorta endothelial cells (RAECs). Silveira (UEPG), Andréa Senff-Ribeiro (Universidade HK assembled on cell was visualized by spectrometry Federal do Paraná), Waldemiro Gremski (Pontifícia and confocal microscopy. The HK proteolysis after Universidade Católica do Paraná), Silvio Sanches Veiga interaction with cell surface or lysate was detected (Universidade Federal do Paraná) by changes in structure using polyclonal antibody anti-HK in immunoblotting. HK bound to RAECs was Accidents involving brown spider L. intermedia conduce disturbed by a peptide sequence of HK domain 5 and to a clinical status known as Loxoscelism, which BK. It colocalized with cathepsin B on cell surface and includes hematological disturbs and renal damage. In after assemble HK was cleaved and the process was order to investigate kidney injury, we compared two inhibited by E-64 and an specific cathepsin B inhibitor. recombinant Cell lysates prepared either in pH 5.5 or pH 7.35 were dermonecrotic toxins obtained from the spider incubated over time with intact HK and only in pH 5.5 a venom. One of them is the active toxin (LiRecDT1) fragment corresponding to cleaved HK was detected. In and the other is the toxin mutated in its probably the presence of PMSF, E-64, antipain, aprotinin and catalytical site, where a His was substituted by an Ala o-phenantroline HK proteolysis was not inhibited. Our (LiRecDT1H12A). To preliminary data suggests an enzyme different from evaluate the citotoxicity of those toxins we used a plasma kallikrein present in cells which can process cell renal cell line (MDCK) and mice were used for in vivo associated HK. Financial support FAPESP, CNPq and investigation. Analysis of DT1 treated cells by Light CAPES. Microscopy showed cytoplasmatic [email protected] vacuolization and culture substract detachment, signs of cell suffering, while SEM showed F-071 morphological changes such as the appearance MORPHOLOGICAL STUDY OF THE LEAD ACETATE of globular structures and a higher number of EFFECTS IN HEPATIC CELLS OF FETUSES FROM microvilosities in cell surface. Analysis of renal biopsies WISTAR RATS by Light Microscopy matched the previous results, Vera Lúcia Corrêa Feitosa (Federal University of showing tubular and Sergipe), Ricardo Scher (Federal University of Sergipe), glomerular edema only in animals treated with DT1. Martha Rebeca Barreto Silva (Federal University of Direct binding of both toxins at MDCK cells and at renal Sergipe), José Aderval Aragão (Federal University of biopsies of treated mice were Sergipe), José Arnaldo Vasconcelos Palmeira (Science observed by Confocal Imunofluorescense and Technology Institute² of Aracaju/SE), Marcos Prado Microscopy. Cell viability were assessed by Neutral Red Nunes (Science and Technology Institute² of Aracaju/ Uptake assay, showing reduction directly proportional SE), Francisco Prado Reis (Science and Technology to time and concentration in cells treated with protein Institute² of Aracaju/SE) DT1. Mice treated with DT1 presented increased values of protein and hemoglobin in urine and also a rise of Lead is among the main heavy metals employed in creatinine and urea in serum, what was not verified the industry. Nowadays, the most important form of in mice treated with H12A. We suggest that histidine intoxication is related with the prolonged exposition to residues are essential for phospolipase activity of the lead and to its compounds. The Lead Poisoning is an dermonecrotic toxin and its effects. occupational disease knew for many years. In addition [email protected] to its effects in the Central Nervous System, lead could affect others organic tissues as the digestive system, 1. 154

livro.indd 154 7/7/2008 16:33:43 where this metal can injury the liver. In this case, the [email protected] effect of lead can damage on the heme radical and a probable reduction in the functional capacity of F-073 cytochrome P-450 drug metabolism. In this study, we SIMILARITIES ON STRUCTURE, COMPOSITION have used a morphological and functional approach in IN INTRACYTOPLASMIC GRANULES PRESENT IN order to evaluate the Lead Acetate effects on the liver TELEOSTEI HEPATOCYTE AND MACROPHAGE tissue from rats fetuses exposed to this metal during AGGREGATES embryonic development. We have been studied 20 Heder José Ribeiro (Departamento de Morfologia - fetuses from Wistar Rats, which received 12.5, 25.0 Instituto de Ciências Biológicas - UFMG), Marcela and 50.0 mg/kg of lead acetate during the gestational dos Santos Procópio (Departamento de Morfologia period. Our results showed that the lead impregnation in - Instituto de Ciências Biológicas - UFMG), Elizete the liver tissue to alterations in the visceral peritoneum, Rizzo (Departamento de Morfologia - Instituto de destruction of the hepatic ligaments and to the lead Ciências Biológicas - UFMG), Yoshimi Sato (Estação accumulation in areas of vacuolization. It was also de Hidrobiologia e Piscicultura de Três Marias/ observed an inverse correlation between the amount CODEVASF), Karla Balzuweit (Departamento de of glycogen in hepatic cells and the doses of lead Física - Instituto de Ciências Exatas - UFMG), Luciana evaluated. So, we can conclude that the ingestion of Moro (Departamento de Patologia Geral - Instituto de lead during pregnancy can affect the liver tissue of Ciências Biológicas - UFMG), José Dias Corrêa Junior fetuses at a morphological and functional level. Key- (Departamento de Morfologia - Instituto de Ciências words: Lead Acetate; Hepatic Cells; Rats Fetuses. Biológicas -UFMG) [email protected] Intracytoplasmic granules (IG) occurred in organisms F-072 of all trophic levels. Their formation is associated in TELEOST CURIMATÃ-PACU, PROCHILODUS organism homeostasis process, generally involving ARGENTEUS (PISCES, PROCHILODONTIDADE) anabolic or catabolic pathways. In the teleost fish liver, GILL EPHITELIUM FINE STRUCTURE some authors have showed the occurrence of IG in Marcela dos Santos Procópio (Departamento de hepatocytes and aggregated macrophages; however Morfologia - Instituto de Ciências Biológicas - UFMG), studies that demonstrated the correlation among the Heder José Ribeiro (Departamento de Morfologia occurrence and the granules composition among these - Instituto de Ciências Biológicas - UFMG), Elizete two cell types are lacking in literature. The hepatic Rizzo (Departamento de Morfologia - Instituto de IG from females of Prochilodus argenteus in gonadal Ciências Biológicas - UFMG), Yoshimi Sato (Estação resting were analyzed by routine de Hidrobiologia e Piscicultura de Três Marias/ hematoxilin-eosin (HE) and Perls, von Kossa CODEVASF), José Dias Corrêa Junior (Departamento histochemistry techniques. Routine transmission de Morfologia - Instituto de Ciências Biológicas - electron microscopy and scanning electron UFMG) microscopy associated to analytical electron microscopy with back scattered electrons and x-ray microanalysis Teleost gill plays several functions, among them also were applied. In liver HE stained, either hepatocyte gas exchange, ionic changes, acid-base regulation as aggregated and metabolic waste excretion. The gill epithelium macrophage IG display similar morphological aspect. plays fundamental role in these functions and also By light microscopy these IG presents regions with presents morphological and physiological responses hemosiderin and calcium deposits. to environmental changes. These characteristics allow Ultrastructural data showed that these the gill epithelium to be studied with the purpose to granules contain typical degradation products. They evaluate the aquatic environmental quality. The teleostei presented areas rich on structures curimatã-pacu, Prochilodus argenteus is an endemic resembling ferritin, low electron dense areas with species of São Francisco River and represents almost hydrophobic aspect and membrane fragments. The 50% of fishery in region of Três Marias dam. However most electron-dense regions were to the best of our knowledge, no studies on gill fine phosphorous, calcium and iron rich in agreement with structure in this species have been published. For this the light microscopy findings. These results suggest that purpose, specimens were catched in two sites of São hepatic IG of P. argenteus presents similar metabolic Francisco River, near Três Marias dam. Gill fragments dynamic in both cellular types, where their genesis are were fixed in Karnovsky’s fixative and processed associated to catabolic pathway associated to turn over for routine transmission electron microscopy. Semi- cellular thin and ultra-thin sections were stained in toluidine processes. Financial support: CNPq, Fapemig. blue and uranyl acetate/lead citrate, respectively. P. [email protected] argenteus gill epithelium presents, mainly, of four cell types: pavement, chloride, mucous and, pillar. Other F-074 cells as macrophages and rodlet were also found. Their BIOCHEMICAL AND HEMATOLOGICAL VARIATIONS distribution, and fine structure described in this work IN ARMOURED CATFISHES (PTERYGOLICHTHYS is discussed focusing their role in metabolism and the ANISITSI) AFTER potential use on evaluation of potential contaminants in CONTROLLED EXPOSURE TO HGCL2 the river. Financial support:Fapemig, CNPq. Leide Daiana de Castro, Carlos Eduardo Saranz Zago, 1. 155

livro.indd 155 7/7/2008 16:33:43 Cláudia Regina Bonini Domingos, Eduardo Alves de and redish-colored collagen fibers in EXE and EXL Almeida (IBILCE - UNESP) groups than in CG. Hydroxyproline content was higher in EXE and EXL groups than in CG (p<0.05). Exercise In this work, the activities of catalase (CAT), increased collagen amount and improved microscopical glutathione S-transferase (GST), glutathione arrangement in wound [email protected] peroxidase (GPx), acetylcholinesterase (AChE) and carboxylesterase (CbE), as well as the levels of lipid F-076 peroxidation (LP) were evaluated in liver of armoured PHOTORECEPTORS CHARACTERIZATION OF THE catfishes (Pterygolichthys anisitsi), after controlled RETINA OF TRAHIRA, HOPLIAS MALABARICUS exposure for 2 and 10 days to 100 ìg/L of HgCl2. Some (BLOCK, 1794) THROUGH STRUCTURAL AND hematological parameters were also evaluated. The ULTRASTRUCTURAL ANALYSIS activities of CAT, GPx, GST and LP were unchanged Maritana Mela (Universidade Federal do Paraná), Dora during the whole experiment, indicating that exposed Selma Fix Ventura (USP), Sonia Grötzner (Universidade animals are not dealing with an oxidative challenge. Federal do Paraná), Marco Antonio Ferreira Randi AChE activity was slightly increased and CbE (Universidade Federal do Paraná), Ciro Alberto de decreased after 2 exposure days, but no differences Oliveira Ribeiro (Universidade Federal do Paraná) were observed in these enzymes after 10 days compared to control animals. HgCl2 also caused an in Most fish present a retina with both rods and cones vitro dose-response inhibition of AChE activity, which (duplex theory of vision), which can be differentiated was less evidenced for CbE. Animals exposed to by their morphology and the spectral sensitivity of HgCl2 had also changes in hematological parameters, their visual pigments. In this study, different types presenting lower levels of hemoglobin, hematocrit of photoreceptors of Hoplias malabaricus were and erythrocyte, which were recovered to control characterized by light (LM) and electron microscopy values after 10 exposure days. These data indicates (EM). The eyes were enucleated and immersed in that HgCl2 exposure can alter significantly esterase Bouin’s fixative for 12 h (LM) or in glutaraldehyde activities and hematological parameters after acute for 2 h (EM). We noted that the retina of this species exposure periods, at the concentration tested, indicating consists of several cell types arranged in three nuclear these parameters as good indicators of Hg exposure in layers separated by two plexiform layers. The rods this species. are the more numerous photoreceptors and, as in [email protected] most teleosts, the retina of Hoplias malabaricus has more than one class of single cones (long and short F-075 single cones). Double cones are a prominent feature, EXERCISE INCREASES COLLAGEN DEPOSITION IN contributing to about 50% of the total number of MICE CUTANEOUS WOUND HEALING cones. EM revealed that the plasma membranes of Fernanda Gurgel Zogaib, these cones are juxtaposed at the level of the inner Andréa Monte-Alto-Costa (UERJ) segments up to the ellipsoids; they run parallel and are separated by an extracellular space suggesting Cutaneous wound healing is an organic response that double cones, due to the elliptical cross section to injury involving cells and mediators that can be of the two inner segments that acts as a birefringent, influenced by physical training. This study aims to dielectric waveguide, are responsible for polarized observe macro and microscopic alterations induced vision in teleosts. With this characterization we see by moderate-intensity exercise in cutaneous wound that the retina of Trahira has the basic morphological healing of mice. Male Swiss mice (n=18) were trained characteristics already described for teleosts. These in a motorized treadmill for 8 weeks (EXL: exercised characteristics may be related to eating behavior (at until the day of excisional lesion) or 10 weeks (EXE: dusk or at night) and the habitat (turbid waters) of this exercised for 2 weeks more after excisional lesion until species. With this morphological description we shown euthanasia), 5 times/week, with gradual increases new data which can be used in future work with the until 45 minutes of duration and 14m/min velocity (CG: retina of this species. control group - 3 times/week, 8 weeks, 5 minutes, 5m/ [email protected] min). Mice were anaestethized, and a full-thickness excisional wound (1cm2) was made in the 8th week. F-077 Wound areas were measured 4, 7 and 14 days after ARTIFICIAL LAMININ POLYMERS ASSEMBLED IN wounding to evaluate wound contraction and re- ACIDIC PH AFFECT PCCL3 EPITHELIAL THYROID epithelialization. Fragments of lesion and adjacent CELL MORPHOLOGY AND ACTIVITY normal skin were collected, formol fixed and paraffin CELIA YELIMAR PALMERO QUINTANA1, LEANDRO embedded. Sections were stained with haematoxylin- MIRANDA ALVES1, ELAINE CRISTINA SOUZA2, eosin (HE) and sirius-red. Hydroxyproline dosage was VANIA COSTA2, DENISSE CARVALHO2, MADALENA used to quantify collagen. No statistical differences MARTINS SANT’ ANA BARROSO3, TATIANA COELHO- were found among the groups in wound contraction SAMPAIO3, LUIZ EURICO NASCIUTTI1 and re-epithelialization. Microscopical analysis of HE 1Laboratório de Interações Celulares, Instituto de sections showed a reduced number of inflammatory Ciências Biomédicas, UFRJ; 2Laboratório de Fisiologia cells in exercised groups compared to CG. Sirius-red Endócrina, Instituto de Biofísica Carlos Chagas Filho, sections showed a greater number of organized, yellow UFRJ; 3Laboratório de Biologia da Matriz Extracelular, 1. 156

livro.indd 156 7/7/2008 16:33:43 Instituto de Ciências Biomédicas, UFRJ compounds. The both experiments are in execution Although the main factor regulating the thyroid activity phase in our laboratory. is the thyroid stimulating hormone (TSH), there are [email protected] evidences showing that some insoluble extracellular matrix factors contribute to this regulation. PCCL3 F-079 cells are differentiated rat thyroid follicular cells that MORPHOLOGICAL ANALYSIS OF THE are able to express TSH receptor, capture iodide and EXTRACELLULAR MATRIX OF THE LONG HEAD secrete thyroglobulin. . In this study, we observed TENDON FROM BICEPS BRACHII IN HUMANS that laminin (LN) polymers assembled in acidic (4.0) FETUSES and neutral (7.0) pHs affect PCCL3 cells morphology Francisco Prado Reis (Science and Technology by cytoskeleton reorganization after 24, 48 and 72 h Institute² of Aracaju/SE), José Aderval Aragão (Science of culture in the presence or absence of TSH. After and Technology Institute² of Aracaju/SE), Marcos Prado 24h of culture in medium with TSH, the cells were Nunes (Science and Technology Institute² of Aracaju/ adherent and confluent in all experimental conditions. SE), José Arnaldo Vasconcelos Palmeira (Science and Cells cultivated on LN pH 7.0 showed shape-spindle Technology Institute² of Aracaju/SE), Eric Állan Nunes and actin filaments dispersed all over the cytoplasm; Carvalho (Federal University of Sergipe), Vera Lúcia nevertheless, cells cultivated on LN pH 4.0 were Corrêa Feitosa (Federal University of Sergipe) rounded and smaller than that cultivated on pH 7.0, and presented parallels beams of actin filaments localized The biceps brachii is the most superficial of the front of associated to the cellular membrane. PCCL3 cells the arm and an of the main forearm flexor muscle. The cultivated without LN substrate and without TSH were biceps typically arises by two heads: long and short. Its also rounded but showed few actin filaments. Cell head long arises from adjacent gleinoidal labrum. This survival was measured by MTT assays, and showed relationship has been enough emphasized because that the cells cultivated on acid matrix presented a high the possible role of the long tendon of the biceps level of survival when compared with the cells cultivated in the development of traumatic and degenerative on neutral matrix or without laminin substrates. These pathologies. Each tendon was divided in three regions: preliminary results indicate that the conditions of laminin the distal corresponding the passage of the tendon assembly in basal membrane of thyroid follicles could through the intertubercular bicipital groove; the modulate cell adhesion and proliferation and probably intermediary is surrounding the head of the humerus; hormone secretion. and a proximal corresponding the arises of the tendon [email protected] in the gleinoidal labrum. Tendons fragments were fixed in 4% parafolmaldehyde in Milloning’s buffer, F-078 embedded in Paraffin and stained with hematoxylin- CELLULAR PHOTODAMAGE INDUCED BY CRUDE eosin, picrosirius red with hematoxylin, Masson’s EXTRACT OF BIDENS SULPHUREA trichrome and Weigert’s fuchsin-resorcin. Morphological Erick Guimarães França, Renata Oliveira Santos, analysis showed an extracellular matrix, in the proximal Alexandre Dias Araújo Neto, Thiago Oliveira da Costa, region rich in collagen bundles more thickness, less Lucas Ferreira de Paula, João Fernando Neves Martins, organized comparatively the other regions. The Marselha Pereira Ceolin, Bruno Pereira Garcês, birefringence analysis showed a higher molecular José Eduardo Buiatte, Carlos Alberto de Oliveira order and aggregation degree of the collagen bundles (Universidade Federal de Uberlândia) in areas where the tension was more prominent. Elastic fibers were disposed in parallel to the collagen A photodynamic event occurs when a colorant is bundles in the region under tension, while they were excited by light and generates reactive oxygen species. disposed in different directions in the regions subjected Alcoholic crude extract obtained from Bidens sulphurea to compressive forces. Key-Words: Tendon; Collagen contains several colorants that absorbs in different Bundles; Birefringence; Elastic Fibers. wavelengths going from ultraviolet to near-infrared. [email protected] Some of them had antioxidants properties and others could have a potential application in photodynamic F-080 studies. In this work we showed, by the first time, APOPTOSIS INDUCED BY DENGUE-2 VIRUS: that crude extracts of Bidens sulphurea posses a INVESTIGATION OF MITOCHONDRIAL PATHWAY significant photosensitizer activity in vitro, using the Daniel Sanches1, Samir Pereira da Costa Campos1, photohemolysis assay. The extract was able to induce Diego Ribeiro Martins1, Luciane Pinto Gaspar2, hemolysis of sheep and human erythrocytes in a very Marcos da Silva Freire2, Luciana Barreto Chiarini3, high aqueous dilution. A photodynamic event occurs Claire Fernandes Kubelka4, Marcos Henrique Ferreira only with an irradiation step using a red light source. Sorgine1, Jerson Lima Silva1, Andre Marco de Oliveira The result points generate of reactive oxygen species Gomes1, Andréa Cheble Oliveira1 that causes damage in cellular membranes. Comparing 1Universidade Federal do Rio de Janeiro – IBqM; with 100% control in Tx100, the extract causes 82% 2Departamento de Desenvolvimento Tecnológico, of photohemolysis. It‘s necessary a chromatographic Instituto de Tecnologia em Imunobiológicos – separation experiments to isolate the active compound FIOCRUZ; 3 Universidade Federal do Rio de Janeiro – that causes the cell death and an experiment using IBCCF; 4Departamento de Virologia, Instituto Oswaldo others cell models to define the activity of this Cruz – FIOCRUZ 1. 157

livro.indd 157 7/7/2008 16:33:43 Fever-induced cell death process. With this aim, we Flaviviruses cause diseases like Dengue and Yellow infected Vero cells with Yellow Fever virus using a fever. These viruses are transmitted by mosquitoes multiplicity of infection (MOI) 1. To follow the process mainly in South America, Central America and Asiatic induced by infection, we analyzed the PS exposure southeast, where they have a particular importance and the Äym through fluorescence microscopy, for public health. Virus-induced apoptosis is known as using Fluorescein (FITC) – annexin V conjugate a consequence of an infection by flaviviruses. During and Dioc6, respectively. We also analyzed the DNA apoptosis, some cellular mechanisms occur, such as condensation through fluorescence microscopy. The activation of messengers of the apoptotic pathways. present data show that Yellow Fever virus is able to Once the mitochondrial pathway is activated, loss of induce programmed cell death from the fifth day of mitochondrial membrane potential (Äym) and release infection and that the apoptotic mitochondrial pathway of pro-apoptotic messengers through the voltage- is probably being activated, contributing partially for dependent anion channel (VDAC) occur. The process the cell death process induced by these flaviviruses. by which Dengue virus induces apoptosis remains [email protected] not clearly understood. Here, we investigate the Dengue-induced apoptosis process. With this aim, F-082 we infected Vero cells with DENV2 and analyzed the EFFECTS OF HYPEROXIA ON WISTAR RAT LUNGS DNA fragmentation by TUNEL and the effect of the Akinori Cardozo Nagato (Laboratório de Reparo pancaspases inhibitor BAF. We also analyzed the Äym Tecidual/DHE-UERJ), Milena Leonarde Kloss (FAFIS- through fluorescence microscopy. Apoptosis is observed IAENE), Frank Silva Bezerra (Laboratório de Reparo on the fifth day of infection with loss of Äym. We also Tecidual/DHE-UERJ), Manuella Lanzetti (Laboratório investigated the importance of the mitochondrial de Reparo Tecidual/DHE-UERJ), Fabiano Leichsenring pathway by VDAC inhibition. We also observed that Silva (FAFIS-IAENE), Luís Cristóvão Porto (Laboratório viral RNA replication is controlled by an understood de Reparo Tecidual/DHE-UERJ), Samuel Santos mechanism. Our real time rt-PCR results showed an Valenca (Laboratório de Reparo Tecidual/DHE-UERJ) increased viral RNA replication rate from the fourth day, explaining the infection effects observed only from The supplementation of oxygen is very important the fifth day post infection. The present data show that for patients with chronic lung diseases and heart Dengue virus is able to induce apoptosis from the fifth failure. However the effects of hyperoxia is unclear. day of infection and that the mitochondrial pathway Our objective was to study the effects of short-term is activated, contributing partially for the cell death exposure to hyperoxia on Wistar rat lungs. The animals process induced by these flaviviruses. were divided in 3 groups: exposed to hyperoxia 10 [email protected] min (O10), 30 min (O30) and 90 min (O90), together with a control group (exposed to ambient air). BAL was F-081 performed and the lungs were removed for histological STUDY OF YELLOW FEVER VIRUS-INDUCED CELL or biochemical analysis. In the O10, O30 and O90 we DEATH observed an increase in the numbers of macrophages Daniel Sanches (UFRJ - IBqM), Samir Pereira da Costa (2169.9±118.0, 1560.5±107.0, and 1467.6±39.0) and Campos (UFRJ - IBqM), Diego Martins Ribeiro (UFRJ - neutrophils (396.3±35.4, 338.4±17.3 and 388.7±11.7) IBqM), Luciane Pinto Gaspar (FIOCRUZ - RJ), Marcos when compared to control group (781.3±78.3 and Da Silva Freire (FIOCRUZ - RJ), Luciana Barreto 61.6±4.2, respectively). Lipid peroxidation was Chiarini (UFRJ - IBCCF), Jerson Lima Silva (UFRJ - increase in O10, O30 and O90 (143±7.8%, 180±5.6% IBqM), Andre Marco de Oliveira Gomes (UFRJ - IBqM), and 235±13.7%) when compared to control group Andréa Cheble de Oliveira (UFRJ - IBqM) 100±1.7%. The histological and stereological analyses revealed normal alveoli and alveolar septa in the Flaviviruses cause diseases like Yellow fever and control (83.5±1.2% and 15±1.2%), in the O10 group Dengue. These arboviruses have a particular (81.3±0.5% and 16.6±0.7%) and in the O30 group importance for public health in South America, Central (78.7±0.5% and 17.7±0.2%). However, in the O90 America and Asiatic southeast. Virus-induced cell group, inflammatory cell infiltration was observed in the death is related to a cytopathological consequence of alveoli and alveolar septa. Red blood cells extravasated an infection by flaviviruses, in vivo or in vitro. During from capillaries to the alveoli (59.0±1.2%), with programmed cell death, some cellular mechanisms evidence of congestion, hemorrhage, and septal edema occur, such as phosphatidylserine (PS) exposure and (35.1±0.6%). Hyperoxia for 90 min caused injury of the DNA condensation. If the programmed cell death type lung parenchyma, resulting in oxidative damage and 1 (Apoptosis) is activated, occur DNA fragmentation, inflammatory cell infiltration. caspases activation and release of messengers of the [email protected] apoptotic pathways. Once the mitochondrial pathway is activated, loss of mitochondrial membrane potential F-083 (Äym) occurs and caspase-9 is activated by release LONG-TERM EXPOSURE TO CIGARETTE SMOKE of pro-apoptotic messengers through the voltage- DECREASES LUNG FUNCTION AND INCREASES dependent anion channel (VDAC). The process by HMGB-1 EXPRESSION which Yellow Fever virus induces cell death remains Frank Silva Bezerra (Laboratório de Reparo Tecidual/ not clearly understood. Here, we investigate the Yellow DHE-UERJ), Samuel Santos Valenca (LRT/DHE- 1. 158

livro.indd 158 7/7/2008 16:33:44 UERJ), Karla Maria Pereira Pires (DFP-UERJ), b) cells with lesser condensed chromatin, suggesting Manuella Lanzetti (LRT/DHE-UERJ), Wagner Alves a larger transcriptional activity, c) metachromasy in Pimenta (LRT/DHE-UERJ), Rojane Oliveira Paiva (LRT/ the compression regions indicating accumulation DHE-UERJ), Aline Schmith (CCS/IBCCF-UFRJ), Walter of proteoglycans in that region. An enhancement of Araújo Zin (CCS/IBCCF-UFRJ), Luís Cristóvão Porto collagen bundles aggregation (PSH) was observed (LRT/DHE-UERJ) in trained rats. Biomechanical test using a constant traction force, showed higher relative tension in trained Cigarette smoke (CS) induced emphysema is one of the rats (15.77±2.77 N/mm2) in relation to the control major causes of COPD. The alterations of antioxidant (8.98±3.84 N/mm2). These differences were due to the defenses and inflammatory cytokines expression in CS smaller diameter observed in trained animals tendons. smoke induced-emphysema remain largely unknown. differences in the maximum force were not observed. This study aimed to describe the effects of long-term Conclusion: The eccentric exercise promotes a larger CS smoke exposure on gasometrical parameters, organization of the collagen bundles and probably alveolar cell recruitment, lung mechanics (lung static stimulates metabolic activities in tendons. Supported by elastance ¡V Est, and elastic recoil variance ¡V DL), CAPES and CNPq. catalase (CAT), superoxide dismutase (SOD) and [email protected] glutathione peroxidase (GPx) activities and MMP-2, MMP-12 and HMGB-1 expression. C57Bl/6 mice F-085 (n=15) were exposed to 12 cigarettes-a-day smoke HMG-COA REDUCTASE INHIBITORS PRESENT for 60 consecutive days. Animals exposed to ambient DIFFERENT EFFECTS ON RAT CUTANEOUS air were used as controls (n=15). After euthanasia WOUND REPAIR bronchoalveolar lavage fluid (BALF), histological Tatiana Almeida Pádua (UERJ), Thaís Porto Amadeu and biochemical analysis were performed. PCO2, (Fundação Oswaldo Cruz), Andréa Monte Alto Costa hematocrit and hemoglobin values were increased and (UERJ) PO2 was decreased in CS group (p<0.05). CS showed significantly increased influx of alveolar macrophages Statins are HMG-CoA reductase inhibitors that have in BALF (p<0.001). Est and ƒ´L were higher in CS immunomodulatory activities and pleiotropic effects (p<0.05) group. SOD and CAT activities were reduced, but their role on wound healing is unclear. This study but GPx activity remained unchanged. Although MMP-2 investigated the effects of three statins on cutaneous expression did not change between groups, MMP- wound repair. Rats were separated into control 12 and HMGB-1 presented a 2-fold increase in their (received water only), simvastatin, pravastatin and expression (p<0.01) in CS. Conclusion: Our results atorvastatin groups (20mg/kg/day). Statins were indicate that long-term cigarette exposure leads to administrated by gavage 10 days preoperatively until impaired lung function and decreased antioxidant the end of the experiment. Eleven days after the defenses with involvement of HMGB-1 protein. beginning of the treatment, an excisional wound was [email protected] performed. The wound area was measured to evaluate wound contraction and reepithelialization. Fourteen F-084 days after wounding, the animals were sacrificed. MORPHO-FUNCTIONAL ALTERATIONS IN THE Hematoxylin-eosin, toluidine blue, hydroxyproline CALCANEAL TENDON OF RATS SUBMITTED TO A analysis and determination of NO2- in serum were PROGRAM OF ECCENTRIC EXERCISE done. The wound contraction was higher in simvastatin Bernhard Jordan (Department of Cell Biology- Institute than pravastatin, atorvastatin and control groups 7 days of Biology- UNICAMP), Sebastião Roberto Taboga after wounding and in pravastatin than simvastatin, (Department of Biology-ibilce-UNESP, SJ Rio Preto), control and atorvastatin groups 14 days after wounding. Laurecir Gomes (Department of Cell Biology- Institute of The reepithelialization and NO2- level was higher in Biology- UNICAMP), Edson Rosa Pimentel (Department both simvastatin and pravastatin than control and of Cell Biology- Institute of Biology- UNICAMP) atorvastatin groups. Atorvastatin presented higher and pravastatin presented lower amount of inflammatory The eccentric contraction is the phase of the motion cells in granulation tissue compared to the other where there is, despite the contraction, an enlargement groups. Hydroxyproline concentration was lower in the length of the muscle. Under these conditions, in simvastatin and atorvastatin than control group. muscles exhibit maximum voluntary contraction force. The number of mast cells was higher in simvastatin The purpose of this work was to analyze the effect compared to control group and in atorvastatin compared of the tension, arisen from eccentric contraction, to pravastatin and control groups. Results showed that on the organization of the extracellular matrix and statins present different effects on cutaneous wound on some biomechanical properties of the tendon. repair. Supported by CAPES and CNPq. Rats wistar 90 days old and weighting 300g were [email protected] submitted to four weeks eccentric exercise protocol. For morphology 6µm sections were stained with toluidine F-086 blue (TB) or with picrosirius-hematoxylin (PSH) for IMMUNOEXPRESSION OF GALECTIN-3 IN MOUSE polarized light microscopy. Intact tendons were used PUBIC SYMPHYSIS AND INTERPUBIC LIGAMENT for biomechanical test. The analysis of TB stained DURING PREGNANCY AND POST PARTUM sections of trained rats showed increments of: a) cells, MARIA AMÁLIA CAVINATO NASCIMENTO, RENATA GIARDINI ROSA, SILVIO ROBERTO CONSONI, 1. 159

livro.indd 159 7/7/2008 16:33:44 PAULO PINTO JOAZEIRO (UNIVERSIDADE agarose gel electrophoresis. The groups was compared ESTADUAL DE CAMPINAS) by the Student’s t test. Our results had shown that The pubic symphysis (PS) of mice is a non synovial the gene expression of decorin was 0,9 times bigger amphiarthrodial joint situated between the two (-10,4%, p=0,233) and versican´s was 1,3 times bigger pubic bones. This region passes through several (23,5%, p=0,259) that ones found in the normal skin. modifications during pregnancy. At the beginning We concluded that significant differences of the gene of gestational period the joint is composed by a expression for the proteoglycans of extracellular matrix fibrocartilaginous disc situated between two caps of between the two groups was not observed.biodsvb@ hyaline cartilage. During pregnancy, the interpubic uol.com.br ligament (IP), a tissue composed by myofibroblast-like cells, collagen and elastic fibers, gradually substitutes F-088 the fibrocartilaginous tissue. The region also goes MODULATION OF RHODOPSIN EXPRESSION IN through a softening process, and the ligament is A FISH AND A MAMMALIAN PIGMENT CELL LINE: many times longer than the PS. After delivery these MECHANISMS OF ENDOTHELIN SIGNALING tissues go through a new remodeling process. All of Gláucia Jansen Da Re Lopes, Maria Aparecida Visconti, these processes are regulated by hormones and other Ana Maria de Lauro Castrucci (USP) molecules, and also by the balance between cell death and proliferation. Galectin-3, an animal lectin with Endothelins (ETs) and sarafotoxins (SRTXs) are a multiple functions, is one of the molecules that can family of vasoconstrictive peptides, which can regulate be involved in these mechanisms. We investigated its pigment migration and/or production in pigment expression in PS of mice in different days of pregnancy cells. In teleostean fish, ETs/SRTXs induce pigment (DOP). The immunohistochemistry showed that this migration. In human melanocytes, ETs promote lectin is present in all types of cells of the PS in all melanogenesis and mitogenesis. ETs can also regulate groups studied. It is mainly found in the cytoplasm of gene transcription of several molecules. These effects cells located at the transitional region between the are mediated by different intracellular signaling cartilage and the connective tissue. The blotting assay mechanisms, such as the PLC pathway, the activation revealed that the expression of galectin-3 increases of PKC and the MAPK pathway. Since ETs are able until 18th DOP, decrease in 19th DOP and increases to induce responses in pigment cells and GEM-81 again by the 3rd day post partum. This pattern is Carassius auratus erythrophoroma cells and B16 Mus associated with the peacks of remodeling that occur musculus melanocytes express rhodopsin as well as in these tissues, suggesting that galectin-3 is probably ET receptors (ETB and ETA, respectively), we decided involved in this process. to investigate: 1) whether rhodopsin expression could [email protected] be regulated by ETs; 2) the signaling mechanisms involved in this modulation. Using real-time PCR, we F-087 were able to demonstrate that SRTX S6c and ET-1 GENE EXPRESSION OF PROTEOGLYCANS modulate rhodopsin mRNA levels in a dose-dependent DECORIN AND VERSICAN OF EXTRACELLULAR way in GEM-81 and B16 cells, respectively. In GEM- MATRIX IN KELOID 81 cells, the modulation of rhodopsin expression Daniel Siquieroli Vilas Boas, Monique Matsuda, Mônica involves the activation of a PKC, cRaf, MEK and ERK. Valéria Marquezini (USP) In B16 cells, it involves PLC, Ca2+ as an intracellular messenger, calmodulin and activation of Ca2+/CaM Keloid is a cicatricial process, with frequency increased kinase and PKC. These findings suggest that even in regions with bigger tension in the skin or where the though the ET-system role in intravascular pressure skin is thicker, characterized for exceeding beyond control is evolutionarily more recent, it is in fact, ancient the limits of the injury that originated it and for the in terms of its relationship with vertebrate pigment cells, tendency to relapse after its ressection. Its occurs and seems to have been preserved during vertebrate in both genders, with bigger incidence between the phylogeny. Financial support: FAPESP and CNPq. first and the third decade of life and in individuals of GJDL is a fellow of FAPESP (process # 06/53291-9). black ethnia. The familial relation is consistent with an [email protected] autosomal dominant inheritance. The keloid presents distinct molecular characteristics of the normal skin F-089 involving a variety of still little understood signallings THE ENIGMATIC FAT BODY OF ASPISOMA and an increase of the expression of components LINEATUM (COLEOPTERA: LAMPYRIDAE): of the extracellular matrix, as the collagen, the CLUES ON THE ANATOMICAL ORIGIN OF glicosaminoglycans and the proteoglycans. This study BIOLUMINESCENCE analyzed the gene expression of the proteoglycans of Fernanda Miyuki Okawachi, Amanda Pocol Carniato, extracellular matrix decorin and versican in the keloid Vadim R. Viviani, Fábio Camargo Abdalla (UFSCar tissue from not treated individuals in comparison with Sorocaba) the normal skin. Tissue samples was obtained from 10 individuals with keloid (Q group) and 10 individuals The Aspisoma lineatum larvae emit a low level diffuse with normal skin (N group). The gene expression of the bioluminescence throughout the body. By CCD camera proteoglycans was amplified by reverse transcription imaging, we found that this basal bioluminescence polymerase chain reaction and analyzed through arises from the fat body, abundant in this phase. 1. 160

livro.indd 160 7/7/2008 16:33:44 Therefore, a morphological analysis of the tissue was meristematic cells. In this way, the azo dye interferes carried out. Larvae were collected, dissected and in the cellular viability of the tested organism and the fat body was analyzed under light microscopy represents, therefore, a potential danger for all exposed (HE, PAS and Bromophenol Blue) and transmission organisms to this chemical. electron microscopy. The visceral fat body is pinkish [email protected] and is formed by globular units constituted by many trophocytes involved by a thin connective layer, which F-091 also penetrates into the unit involving each trophocyte. POTENTIAL ROLE OF FOCAL ADHESION KINASE These cells presented two types of secretion: lipid IN THE EXPRESSION OF PROINFLAMMATORY droplets and protein vesicles. The protein vesicles MOLECULES IN ENDOTHELIAL CELLS SUBJECTED reacted to the techniques PAS and Bromophenol TO MECHANICAL STRETCH blue, indicating that these proteins could be alkaline Maruska do Rocio Neufert Fernandes, Kleber Gomes glycoproteins, additionally they also was stained by Franchini, Wilson Nadruz Júnior (Unicamp) eosin. The cellular apparatus of well developed rough endoplasmic reticulum (RER) near to the nucleus INTRODUCTION: Mechanical stretch is an important seems to be responsible by the synthesis of the protein stimulus for the development of proatherogenic vesicles. We also noticed that in a globular unit the phenotype in vascular cells. However, little is known synthesis and migration of the protein vesicles are about the molecular mechanisms involved in this synchronized and continuous, being produced at the process. OBJECTIVE: To investigate the role of basal portion of the trophocytes, where dilated cisterns Focal Adhesion Kinase (FAK) in the expression of RER were observed, and released in the apical of proinflammatory molecules in endothelial cells portion. Few mitochondria and rare Golgi apparatus subjected to mechanical stretch in vitro. MATERIAL were observed. The lipids droplets are produced by the and METHODS: Rabbit aorta endothelial cells (RAEC) trophocytes since the smooth endoplasmic reticulum is were subjected to cyclic stretching (1 Hz) for several associated to the lipids. Financial Support: FAPESP. periods of time and studied by western blot. In order to [email protected] evaluate role of FAK in this process, cells were treated with the pharmacological inhibitor PP2 (1 mol/mL). F-090 RESULTS: Mechanical stretch induced a precocious CYTOTOXIC, GENOTOXIC AND MUTAGENIC activation of FAK (as early as 10 min) evaluated by its EFFECTS OF AN AZO DYE IN ALLIUM CEPA phosphorylation on tyrosine residue 397. Stretching MERISTEMATIC CELLS, DETECTED BY THE for 6 hours promoted a significant increase of Toll- CHROMOSOME ABERRATIONS TEST AND Like Receptors (TLRs) 2 and 4 expression, but not CHROMOSOME BANDING ASSAYS of the adapter protein Myd88. Furthermore, this (C, NOR AND CMA/DAPI) stimulus induced an increase of E-selectin and VCAM Bruna de Campos Ventura, Maria Aparecida Marin- expression. On the other hand, treatment of endothelial Morales (Universidade Estadual Paulista) cells with PP2 inhibited the stretch-induced expression of TLR2, TLR4, E-selectin and VCAM. CONCLUSIONS: Azo dyes are chemicals extremely used by textile Cyclic mechanical stretch stimulates the expression of industries and may cause environmental damage adhesion molecules and proinflammatory proteins in by the potentiality to induce harmful changes in the endothelial cells in vitro. Moreover, signaling pathways DNA of exposed organisms. The objective of this regulated by FAK might play an important role in this study was to evaluate the cytotoxicity, genotoxicity process. and mutagenicity of an azo dye (BDCP), by means [email protected] the chromosome aberrations test and chromosome banding assays (C, NOR e CMA/DAPI), using Allium F-092 cepa as test system. One hundred Allium cepa seeds DIFFERENTIAL CELL LABELLING BY ALT-C, A were germinated in the azo dye concentrations (1 DISINTEGRIN-LIKE PROTEIN FROM BOTHROPS mg/L, 10 mg/L, 100 mg/L e 1000 mg/L). The negative ALTERNATUS VENOM control was performed with only milli-Q water and Aline Zamboni Machado (Dep. Ciências Fisiológicas, the positive control with MMS (methyl methane- UFSCar), Charlotte L. Ownby (Microscopy Laboratory, sulphonate). After 20 h of exposition to the azo dye and Oklahoma State University), Edward Shaw (Dep. to the MMS, roots were then submitted to the assays Microbiology and Molecular Genetics, Oklahoma State of chromosome aberrations and chromosome banding. University), Heloisa Sobreiro Selistre de Araújo (Dep. By the chromosome aberrations test, it was possible Ciências Fisiológicas, UFSCar) to observe chromosome adherences, bridges, breaks and losses, beyond micronucleated cells and in process Integrins are cytoadhesive receptors expressed on of cellular death. The chromosome banding assays cell surfaces essential for cell-cell interaction and showed specific chromosome regions more susceptible cell adhesion to the extracellular matrix. Alternagin-C to the azo dye action, as well as cellular morphological (ALT-C), a disintegrin-like protein purified from the changes and alterations in the number of nucleolus and venom of the Brazilian snake Bothrops alternatus, micronuclei of many cells. The statistical tests showed interacts with the major collagen I receptor, the that the higher the azo dye concentration, higher is the integrin, inhibiting collagen binding. In this work ALT-C cytological and genetic degradation of the Allium cepa was used to identify collagen receptors by confocal 1. 161

livro.indd 161 7/7/2008 16:33:44 microscopy in order to identify binding differences Interações Celulares, Instituto de Ciências Biomédicas, between normal and tumor cells. Alexa Fluor 546- UFRJ), LEANDRO VAIRO (Laboratório de Cardiologia labelled ALT-C (100-1000nM) was incubated with HeLa Celular e Molecular, IBCCF, UFRJ), LEANDRO (human ovary carcinoma) and L929 (mouse fibroblasts) MIRANDA ALVES (Laboratório de Interações Celulares, cells for different times (5-60 minutes). The slides Instituto de Ciências Biomédicas, UFRJ), REGINA were prepared in duplicate and analyzed by confocal COELI DOS SANTOS GOLDENBERG (Laboratório microscopy. Labeling was mostly found on the surface de Cardiologia Celular e Molecular, IBCCF, UFRJ), of HeLa cells incubated with 1000nM ALT-C for 15 min, LUIZ EURICO NASCIUTTI (Laboratório de Interações disappearing after 30 min incubation. In contrast, ALT-C Celulares, Instituto de Ciências Biomédicas, UFRJ) (1000nM) was exclusively found in the cytoplasm of the fibroblasts, after 5-15 min incubation. These results Endocrine cells of adenohypophysis are known to indicate that ALT-C binds distinctly to these two cell communicate with each other and with non-endocrine types, being rapidly internalized by fibroblasts. Since cells via gap junctions, which are formed by proteins a b both cells express high levels of 2 1 integrin, these called connexins. In the present study we evaluated the results can not be explained by the ALT-C binding to in vitro expression of some connexins and the possibility this receptor only. Other receptors are probably involved of communication through channels formed by them which could result in different cell responses after in rat adenohypophysis cell lines, GH3B6 tumoral binding to the same disintegrin. li_zamboni@hotmail. prolactin cells and APS9 stromal cells. RT-PCR assays com performed in adenohypophysis primary culture, as control, demonstrated the expression of connexins 26, F-093 32 and 43. However, the cell lines cultivated alone and CHROMATOPHORES DISTRIBUTION IN THE in a co-culture system for a period of 7 days, showed OCELLATED SPOTS OF CERCOSAURA that only APS9 cells express connexin 43. GH3B6 OCELLATA WAGNER, 1830 (SQUAMATA, cells did not show any connexins isoform expression. GYMNOPHTHALMIDAE) To evaluate a possible communication between the Mônica Caroline Pavan Cassel, Adelina Ferreira cell lines, microinjections with Lucifer Yellow were (Universidade Federal de Mato Grosso) performed and, after 5 minutes, the dye-coupled cells were counted. The dye transfer was observed between This work had as objective the description of the APS9 cells but not between GH3B6 cells. Nevertheless, distribution of chromatophores in the ocellated spots in the co-culture system, the dye transfer was observed of Cercosaura ocellata Wagner, 1830 (Squamata, between both cell lines, suggesting that the interactions Gymnophthalmidae), through histological analysis of between GH3B6 and APS9 cells could form gap the skin. For this, one fragment was dehydrated in junctions by the expression of connexin 43 in GH3B6 increasing alcohol concentrations, enclosed in plastic cells or the existence of other connexin not examined in resin and the cuts colored for 1% Toluidine blue. In the this study, which could be expressed in both cell lines. prepared was possible to observe a high epithelium, The role of these interactions on prolactin secretion are stratified, squamous, with formation of papillaes being analyzed. and keratin in little amount and weakly colored. The [email protected] underlying conjunctive tissue is rich in fibroblasts and colagenous fibres, associate it has one skeletal striated F-095 muscular tissue, being both good vascularizated. NO DONNOR APPLICATION ACCELERATES RAT The chromatophores are present in derm and after IMPAIRED HEALING the muscular blocks. Being that, after the muscular Janaina de Lima Georgii (UERJ), Thaís Porto Amadeu tissue, the cells are small, with granules rounded off (FIOCRUZ), Amedea Barozzi Seabra (UNICAMP), and chestnut well clearly, with regular distribution and Marcelo Ganzarolli de Oliveira (UNICAMP), Andréa delimited well. Already the dermal chromatophores Monte Alto Costa (UERJ) are bigger cells, with irregular format and without visible cellular limits, coloration dark chestnut and Nitric oxide (NO) is an important molecule involved irregular distribution, which occurs because of the in wound repair. Since we reported that hydrogel aspect of the ocellated spots. In the regions where the containing S-nitrosoglutathione (GSNO), a NO donor, chromatophores are more numerous, the pigments improves cutaneous wound repair, the aim of this are spread for the cells of the epidermis, also in the study was to evaluate the effect of topical application keratin. The standard of the keratin, the distribution of of GSNO-containing hydrogel on ischemic wounds. the chromatophores, and the pigment presence in the Young rats were separated in control group, in which an keratin and epidermis of C. ocellata are facts unknown hydrogel without GSNO was applied on wound beds, in literature. and GSNO group, in which an hydrogel with GSNO [email protected] (200mM) was applied during 7 days consecutively. For ischemia, two parallel incisions were done to separate F-094 the skin of the underlying tissue and were sutured, and CONNEXINS EXPRESSION AND GAP an excisional wound was done between the incisions. JUNCTION COMMUNICATION ANALYSIS IN The wound contraction and reepithelialization were ADENOHYPOPHYSIS CELL LINES measured during the experiment. Wound and skin NATALIA GONÇALVES MAÇÃO (Laboratório de were collected, formalin fixed and paraffin-embedded. 1. 162

livro.indd 162 7/7/2008 16:33:44 Sections were stained with HE and Sirius Red, and an Paulo Hilário Nascimento Saldiva (Lab. de Poluição immunohistochemistry to a-SMactin was also done. Atmosférica FMUSP), Elia Garcia Caldini (Lab. Biologia GNSO group presented smaller wound area 12 and 14 Celular FMUSP), Elnara Márcia Negri (Lab. Biologia days after wounding compared to control group. GSNO Celular FMUSP) also promotes a higher reepithelialization 12 and 14 days after wounding. Control group presented a high It is known that hospital admissions increased due to amount of inflammatory cells compared to GSNO group. respiratory and cardiovascular causes in cities with Control group showed thin red-yellowish collagen fibers high atmospheric particulate matter levels (PM10). disarranged, while GSNO group presented organized Furthermore, increased vascular oxidative stress thick red-yellowish collagen fibers. Control group has been associated to the development of various showed a higher amount of blood vessels compared to cardiovascular disorders. To investigate whether the GSNO group. Topical application of GSNO-containing exposure to air pollution can enhance the reactive hydrogel improves the wound contraction and oxygen species in the aorta, adult mice were placed reepithelialization, besides to promote organization of in clean chamber (C, with filtered urban air) and in a granulation tissue. polluted chamber (P, with urban air of Sao Paulo city, [email protected] Brazil) for 1, 2, 4, 8, or 16 weeks (Groups C or P, 1, 2, 4, 8 or 16, respectively). Daily records of atmospheric F-096 PM10 levels were obtained from the Air Pollution CYTOCHEMISTRY AND ULTRASTRUCTURAL Controlling Agency. After sacrifice, a fragment of ANALYSIS OF PARAWIXIA BISTRIATA VENOM the thoracic aorta was assigned for quantification of GLAND O2• by chemiluminescence and another to evaluate Hellen Maria Soares, Cynthia Renata de Oliveira Jacob, nitric oxide synthase levels (iNOS and eNOS), by Lilian Mari Marcondes César Tognoli, Roberta Cornélio immunohistochemistry. Analysis of variance showed Ferreira Nocelli, Mario Sérgio Palma, Osmar Malaspina a statistically significant increase in the O2• levels (UNESP - Rio Claro) in aorta only in P8 group when compared to C8 group (3007±513 x 967±190) and P1 (820±151), P2 Histological analysis showed that the venom glands (1274±268) and P4 (1522±139) groups; no difference of the Parawixia bistriata is formed, externally, by two was observed when compared to P16 (1841 ± 251). muscle layers, being the most external thicker with A multiple regression method showed significant fibers arranged in spiral and the more internal thinner associations between PM10 concentrations and aortic and related with basal membrane. The secretory O2• levels. Aorta of P8 group showed a higher iNOS epithelium has cylindrical cells supported on the basal immunoreactivity than C8 group. No difference was membrane which release a secretion within the gland noted when comparing P8 and C8 groups for eNOS. by the holocrine system. The secretion has two very The results of this study showed an association different parts: a fibrous in appearance and the other between oxidative stress in aorta and short-term PM10 with vesicular aspect, filled with homogeneous material. changes in the urban air. In order to identify the characteristics of the material [email protected] inside the vesicles there were applied cytochemistry techniques such as PAS, Xilidinae Ponceau and Sudan F-098 Black and transmission electron microscopy (TEM) DIFFERENTIAL DISTRIBUTION OF ELASTIC routine. The content of the vesicles were positive for SYSTEM FIBRES IN THE VENTRICULAR WALL OF the three cytochemistry techniques indicating the THE NORMAL RAT HEART existence of carbohydrates, proteins and lipids within Gisele Miozzo Fink (Lab. Biologia Celular FMUSP), the vesicle. The comments of TEM showed vesicular Nilsa Regina Damaceno-Rodrigues (Lab. Biologia structures through which it could be observed several Celular FMUSP), Marcelo Alves Ferreira (Lab. Biologia microvesicules with different electron densities. These Celular FMUSP), Aline Borges dos Reis (Lab. Biologia results indicate that the cells of the secretory epithelium Celular FMUSP), Juliana Braga Furtado (Lab. Biologia produce different compounds of venom separately and Celular FMUSP), Maria Ires Amorim-Mendes (Lab. they are attending the larger vesicles, which are found Biologia Celular FMUSP), Olga Maria S. Toledo (Depto. in different stages of maturation. However, the mode of Morfologia UNIFESP), Cláudia Naves Battlehner (Lab. maturation of the substances found in vesicles has not Biologia Celular FMUSP), Elia Garcia Caldini (Lab. been elucidated yet. Financial support: FAPESP, CNPq. Biologia Celular FMUSP) [email protected] The connective tissue elasticity has a protective F-097 function acting as a tensile spring during muscular work. INCREASED OXIDATIVE STRESS IN AORTA Nevertheless, few is known about the distribution of the OF NORMAL MICE EXPOSED TO URBAN AIR elastic system fibers (ESF) in the heart. Considering: POLLUTION a) that the study of the distribution of the ESF may help Nilsa Regina Damaceno-Rodrigues (Lab. Biologia understand the cardiac mechanics, and b) rat models Celular FMUSP), Mariana Matera Veras (Lab. Biologia are used to study cardiac dysfunctions, our aim is to Celular FMUSP), Hermes Vieira Barbeiro (Lab. de investigate the distribution of ESF in the ventricular Emergências Clínicas FMUSP), Francisco Garcia endocardium, epicardium and myocardium of normal Soriano (Lab. de Emergências Clínicas FMUSP), rats. Longitudinal and transversal tissue sections of 1. 163

livro.indd 163 7/7/2008 16:33:45 10 hearts (obtained from adult rats) were studied by TOLUENE AND XILENE (BTX) the comparative association of the selective staining Eloísa Aparecida Ribeiro (Centro Universitário methods of light microscopy with the ultrastructural Campos de Andrade), Edith Susana Elisabeth Fanta observation after tannic acid-glutaraldehyde fixation, (Universidade Federal do Paraná), Lucélia Donatti which provided a reliable means to identify the elastic (Universidade Federal do Paraná), Helena Gonçalves system fibers, as oxytalan (OX), elaunin (ELN) or Kawall (Centro Universitário Campos de Andrade) elastic fibers (EL). A differential distribution of the ESF is noted in the different connective tissue compartments Estuarine regions show a great diversity of fish of the ventricles, as follows: 1) abundant EL and rare and they are important sites for reproduction and ELN fibers are present in the endo- and epicardium; in development of many species. Anthropogenic impacts both locations they are arranged in two orthogonally are frequent in these regions. Juvenile of Sphoeroides disposed sheets; 2) The perimysium of the myocardium testudineus were exposed experimentally to a mixture has a few ELN fibers and abundant OX fibers; 3) The of polyaromatic hydrocarbons (BTX) at sub lethal doses endomysium of the myocardium is rich in OX fibers of 4.386 mL/L of benzene, 2.215 mL/L of toluene and and, at this location, EL and ELN fibers are found only 0.445 mL/L of xylene. After 24, 48, 96 and 168 hours in proximity of the vessels. In spite of the functional of contamination, liver and gills were extracted and implications being speculative, the existence of a prepared for morphological analyses by optical and differential distribution of the ESF suggests that the transmission electronic microscopy. The liver was differences in elasticity provide biomechanical versatility affected by BTX at all times studied after exposure, to the entire [email protected] showing an increased number of lipid vacuoles, abnormal and lateral nuclei and swollen mitochondria. F-099 Morphometry of the hepatocytes showed a significant VERAPAMIL EFFECTS IN CYTOSKELETON OF increase in size after 168 hours of exposure. The HUMAN FIBROBLASTS number of lesions was counted in ten lamellae and at all RICARDO FROTA BOGGIO, VANESSA MORAIS times gills presented a higher level of severe epithelial FREITAS, MARCEL SHINITI URABAYASHI, GLAUCIA detachment at secondary lamellae, if compared to the MARIA MACHADO SANTELLI (INSTITUTO DE control fish. After 168 hours, oedema was observed. CIÊNCIAS BIOMÉDICAS - ICB/USP) Lamellar fusion, hypertrophy and hyperplasia were not significantly higher in experimental fish. The number of Skin injuries initiate a complex series of biological goblet cells decreased steadily along the experimental events that result in wound healing. Any process failure times. Results indicated that even in sublethal levels, can result in the development of pathological scars. BTX causes histopathologies on liver and gills, which One of the drugs suggested to improve this abnormal are important organs for the metabolism and physiology process is the verapamil. So, this research aimed a of fish. This shows the need for more studies of the study about effects of the verapamil on short-term effects of exposure of native fish to xenobiotics in the monolayer culture of human fibroblasts. Cell viability environment, such as derivates of petrol. was evaluated in 50mM of verapamil-treated fibroblasts [email protected] for 48 hours by trypan blue and immunofluorescence. The control and treated cells showed respectively F-101 92.31% and 91.01% of living cells. In the cytoskeleton USE OF THE CRITICAL ELECTROLYTE study the fibroblasts were labeled with the primary CONCENTRATION METHOD (CEC) IN ALLIUM CEPA anti-bodies for vimentin and a-tubulin. The actin was TO INVESTIGATE GENOTOXICITY stained with Phalloidin. Verapamil-treated cells changed Renata Caritá, Dania Elisa Christofoletti Mazzeo, Maria their spindle-like shape to stellate. Microfilament and Aparecida Marin Morales (Unesp - Campus Rio Claro) vimentin organization was changed and microtubules kept the normal pattern of distribution. Verapamil Using CEC method it is possible to identify anions was also effective in inhibiting the extracellular matrix compounds of chromatin and cytoplasm based on contraction in the 3D collagen assay. Collagen I and competition of inorganic salts with thiazin dyes. collagenase were reduced and increased respectively in Applied in A. cepa this methodology can provide treated cells, when they were analyzed by immunoblot. details on the structural and molecular alterations that The cytoskeleton study showed the dynamics of couldn’t be distinguishable with the only application those three fibers observed during the treatment and of toluidine blue colour. The concentration of Mg+2, an interaction cell-matrix in collagen assay. Those which abolishes the DNA or RNA metachromasy in changes in the morphology of the cytoskeleton and a complex of nuclear-protein, is known as its point of the fibroblasts shape may also be related to the effects CEC. In this study, seeds of A. cepa were submitted of the verapamil. These changes could be a clue to to germinate in Ribeirão Claro water (Rio Claro–SP), a better knowledgement of the pathological scars. in ultra pure water (negative control) and trifluralin [email protected] herbicide - 0,84ppm (positive control). After fixation, meristems were subjected to the CEC methodology, F-100 according to Mello et al.,1993. For this technique was HISTOLOGICAL ALTERATION IN LIVER AND GILL possible to see differences in metachromasy, which OF THE FISH SPHOEROIDES TESTUDINEUS AFTER allowed to view nucleoli in the A. cepa cells subjected SUBLETHAL CONTAMINATION WITH BENZENE, to the treatment. In cells in division process, the DNA 1. 164

livro.indd 164 7/7/2008 16:33:45 showed bluish-green colour and the ribonucleoprotein The Fasciculation and Elongation protein Zeta1 (FEZ1) complex and RNA staining purple. Micronuclei had is the mammalian orthologue of the C. elegans protein bluish-green coloration. The analysis of cells allowed UNC-76, necessary for neuronal development in this the identification of nucleoli and the verification of worm. FEZ1 acts with JIP1 in the formation of cargo differences in the nucleoli quantity and morphometry, complex transported by kinesin, and in the polarization due to good contrast in colour. Seeds exposed to of neuronal cells. It promotes mitochondrial transport germination in Ribeirão Claro water showed high and also cooperates with PKC during the differentiation indexes of micronucleus, which had bluish-green of PC12cells in neurons. We over-expressed GFP-FEZ1 colour similar to that presented by DNA. This way, the in human HEK293 cells and observed that over 40% use of CEC methodology in A. cepa was efficient in of the transfected cells develop “flower-like” nuclei. We characterization of the micronucleus material induced further demonstrated that GFP-FEZ1 localizes to both by exposure to the contaminated river water.renata_ the cytoplasmatic and the nuclear fractions, and that the [email protected] appearance of the “flower-like” nuclei depends on intact microtubules. Interestingly, the GFP-FEZ1 transfected F-102 cells do not show disturbance of the phases of the cell EVALUATION OF THE GENOTOXIC AND cycle, by FACS analysis, and a FISH probe show the MUTAGENIC ACTION OF MUD SEWERAGE chromosomes inside of the individual nuclear lobules. TREATMENT BY ALLIUM CEPA SYSTEM ASSAYS Finally, we show that FEZ1 co-localizes and interacts Renata Caritá, Maria Aparecida Marin Morales with alpha and gamma tubulin, at a centrosome (Unesp - Campus Rio Claro) like structure at the center of the nuclear lobules. Concluding, our data suggest that FEZ1 seems to have Numerous chemicals have been studied because an important centrosomal function and supplies new they can cause inheritable changes in the genetic mechanistic insights to the formation of “flower-like” material. Compounds present in mud from sewerage nuclei, which are a phenotypical hallmark of human treatment can cause damage in exposed organisms leukemic cells. and its application in alternative ways as in agriculture [email protected] can also be dangerous. With use of nuclear and chromosomal aberration assays it is possible to obtain F-104 a good evaluation of mud’s damage potential. In this HNRNP Q INTERACTS WITH HSP70/BIP AND CO- study seeds of A. cepa were submitted to germinate LOCALIZES WITH IT UPON PMA AND STRESS in several concentrations of mud solution (1.2, 2.5, CONDITIONS IN THE ENDOPLASMIC RETICULUM 5, 10 and 100%) obtained from sewerage treatment (ER) of a city of São Paulo metropolitan region (ETE 4), Alexandre José Christino Quaresma (Laboratório in ultra pure water (negative control), forest soil’s Nacional de Luz Síncrotron - LNLS), Gustavo Costa aqueous solution (environmental control) and methyl Bressan (Laboratório Nacional de Luz Síncrotron methansulphonate (positive control). After fixation, e Instituto de Biologia - Unicamp), Lizandra Gava meristems were submitted to Feulgen reaction. About (Instituto de Química e Instituto de Biologia - Unicamp), 5000 cells were analyzed from each assay. Our Daniel Ferreira Lanza (Laboratório Nacional de Luz studies showed that this mud sample had substances Síncrotron e Instituto de Biologia - Unicamp), Carlos capable of promoting significant alterations in the Henrique Inácio Ramos (Instituto de Química - most evaluated concentrations. Analysing the mitotic Unicamp), Jörg Kobarg (Laboratório Nacional de Luz index (citotoxicity), results weren’t significant for all Síncrotron - LNLS) concentrations. Mutagenicity assays were significant for all concentrations mainly 5 and 100%, these Eukaryotic gene expression is regulated on different samples showed high indexes of micronucleus. levels ranging from pre-mRNA processing to Analyzing genotoxicity, the sample showed significant translation. One of the most known families of RNA- indexes of nuclear and chromosomal alterations for binding protein is the group of hnRNPs (heterogenous all analyzed concentrations. Significant alterations nuclear ribonucleoproteins). Members this family play frequency observed suggest that this mud sample can important roles in gene expression control and mRNAs have genotoxic substances for A. cepa. The significant metabolism. In the cytoplasm, several hnRNPs proteins mutagenicity indexes observed can also alert us about are involved in RNA-related processes and they can be the hazards of the use of mud in agriculture. frequently found in two specialized structures, known [email protected] as GW-bodies (or processing bodies, PBs) - and stress granules (SGs), which may be formed in response to F-103 specific stimuli. GW bodies (GWbs) have been early OVER-EXPRESSION OF GFP-FEZ1 CAUSES reported to be involved in the mRNA decay process, GENERATION OF MULTI-LOBULATED NUCLEI acting as a site of mRNA degradation. In a similar MEDIATED BY MICROTUBULES IN HEK293 AND way, stress granules (SGs) have been described as HELA HUMAN CELLS cytoplasmic aggregates, which contain accumulated Daniel Carlos Ferreira Lanza, Daniel Maranho Trindade, mRNAs in cells under stress conditions and present Eliana Assmann, Jörg Kobarg reduced translation. Briefly, our main goal was to (Laboratório Nacional de Luz Síncrotron) characterize the hnRNP Q localization after different stress conditions. HnRNP Q is a nuclear protein that 1. 165

livro.indd 165 7/7/2008 16:33:45 exhibits a modular organization and several RNA- Larissa P A Martins (UFSCar - Campus Sorocaba), Ana related functions. Our data suggest that the nuclear Carolina Batista (UFSCar- Campus Sorocaba), Samuel localization of hnRNP Q might be modified after Espinosa Dal Medico (UFSCar - Campus Sorocaba), different treatments, such as: PMA, Thapsigargin, Ana Lúcia Kalinin (UFSCar - Campus São Carlos), Arsenite and 42oC (heat shock). Further, under different Diana A Monteiro (UFSCar - Campus São Carlos), stress conditions, hnRNP Q can fully co-localize with Monica Jones Costa (UFSCar - Campus Sorocaba), BiP (a specific ER chaperone). However, under stress, Elaine Cristina Mathias da Silva Zacarin (UFSCar - this protein only co-localizes partially with the proteins: Campus Sorocaba), Fábio Camargo Abdalla (UFSCar GW182 (a protein that can be found in GWbs) and - Campus Sorocaba) TIA-1 (an important protein in the SG´s formation). Financial support: FAPESP, CNPq & LNLS. In recent years, the quantity of xenobiotic compounds [email protected] has grown alarmingly in the aquatics ecosystems as a result of very damaging human activity on the F-105 environment, contributing to a large reduction of the MORPHOLOGICAL ANALYSIS OF THE CADMIUM amphibian population in the world. Cadmium is a heavy CHLORIDE EFFECT ON BULLFROG TADPOLES’ metal largely used for battery and pigment manufacture. LIVER (LITHOBATES CATESBEIANA) Since cadmium is the element that has presented some Ana Carolina Batista (UFSCar - Campus Sorocaba), of the highest elevation rates in the environment over Larissa P A Martins (UFSCar - Campus Sorocaba), the last two decades, this work proposed to characterize Samuel Espinosa Dal Medico (UFSCar - Campus the morphological effects of acute exposure over a 48h Sorocaba), Ana L Kalinin (UFSCar - Campus São period to 1ppb of cadmium chloride on the gonads of Carlos), Diana A Monteiro (UFSCar - Campus São tadpoles of Lithobates catesbeiana. For ecotoxicological Carlos), Monica Jones Costa (UFSCar- Campus tests, the animals were divided into two experimental Sorocaba), Fábio Camargo Abdalla (UFSCar- Campus groups: control group (C, n=12) and group exposed to Sorocaba), Elaine Cristina Mathias da Silva Zacarin cadmium chloride (E, n=12), and these groups were (UFSCar - Campus Sorocaba) tested in three replicates. The tadpoles analyzed were males. They presented well-developed testicles and A worldwide decline in amphibian populations has in experimental groups testicles could be observed been observed, due to contamination by xenobiotics positioned ventrally to the kidneys, and were slightly and global warming. Their highly permeable skin lobular. As preliminary results, we observed that the and peculiar life cycle make amphibians very animals exposed to cadmium presented germ cells vulnerable to environmental conditions. We performed in a more advanced stage of spermatogenesis than a morphological analysis on the livers of bullfrog the control group, as cadmium is known as a strong tadpoles, Lithobates catesbeiana, after acute exposure androgen disruptor. The literature reports that it also over a period of 48h to 1ppb cadmium chloride may produce multi-testicles and other mutagenic (a safe environmental concentration proposed by anomalies. The effect of cadmium on the gonads of bull- CONAMA/2005 for class 1 and 2 rivers). For the frog tadpoles presented here are preliminary results, ecotoxicological tests, 12 bullfrog tadpoles (stage more replicates of the experiment should be done, and 25) for each treatment were selected: a control group with an exposure of 96h for more conclusive results. (CG, n=12) and a group exposed to 1ppb of CdCl2 Financial support: CNPq-PIBIC/ FAPESP. (EG, n=12). These tests were conducted with triple [email protected] replications. Tissue samples were submitted to the Hematoxilin-Eosin staining technique associated F-107 with the “Instant Prov Kit”. The results showed that EFFECT OF DERMATAN SULFATE IN NEOINTIMA the cadmium modified the quantity of defense cells, PROLIFERATION AFTER ARTERIAL INJURY IN but it did not affect the histology of the tadpole livers ATEROSCLEROTIC MICE nor the hepatocytes. The HE staining was combined Juliana Aparecida Preto de Godoy, Cristina Pontes with the hematological kit to identify different types Vicente (Departamento de Biologia Celular - IB - of leukocytes. In the group treated with cadmium, Unicamp - Campinas - SP) eosinophils appeared in larger quantities than in the control group, forming an epithelioid arrangement Atherosclerosis is one of the most lethal diseases in lining the outer wall of the vessels. This result could the industrialized countries; it is characterized by the indicate the production of eosinophils for the moderation formation of atherosclerotic plaques that protrude of possible inflammatory processes. These results to the lumen of the vessels causing its occlusion. suggest that exposure to an environmentally relevant Percutaneous coronary angioplasty is the main cadmium concentration exerts an impact on the defense treatment used in this disease. This technique may response of bullfrog tadpoles. Financial Support: CNPq- cause endothelial lesion, leading to the formation of PIBIC/ FAPESP. neointima and re-occlusion of the vessel. Dermatan [email protected] Sulfate (DS) activates HCII (Heparin Cofactor II) that inhibits thrombin. Thrombin activates macrophages F-106 and smooth muscle cell proliferation, after endothelial EFFECT OF CHLORIDE CADMIUM ON GONADS OF lesion. We studied the effect of DS in endothelial cells, BULL-FROGS (LITHOBATES CATESBEIANA) macrophages and smooth muscle cells proliferation 1. 166

livro.indd 166 7/7/2008 16:33:45 after arterial injury in the left commom carotid of Marcos de Assis Moura, Marialice Ferreira-da-Silva, C57BL06 mice, in the presence or absence (control) Helene Santos Barbosa (Instituto Oswaldo Cruz/ of DS. We used immunohistochemistry to analyse FIOCRUZ) neointima formation, endothelial cells growth and to detect macrophages proliferation. Our results showed Primary cultures of the feline intestinal epithelial cell in the DS treated group the presence of smooth muscle (FEIC) have importance for the study of the normal cells and macrophages was much lower than observed development and differentiation of intestinal epithelium. in the control group. We also observed inhibition Many mechanisms involved in the pathogenesis of of thrombus formation in DS treated group when chronic enteropathies or host-pathogen interactions compared to control (0µm2 vs 30 000µm2, P=.003). in feline intestine have not been elucidated yet. This These data show that DS administration in the first 24 cellular model potentially applies to the investigation hours after angioplasty inhibits, significantly, thrombus of the infection process provoked by protozoan and formation, neointima growth, and inflamation. The virus. The dynamic and epithelial nature of the cells in mechanism of the antithrombotic effect of DS remains culture was confirmed by morphological criteria using poorly understood. The striking inhibitory effect of DS on confocal and light microscopy and also scanning and neointima formation may have therapeutic implications transmission electron microscopy showing: (i) cellular in the setting of vascular surgery, angioplasty or stent replication; (ii) cell substrate adhesion; (iii) cellular placement. migration; (iv) cell alignment as demonstrated by the [email protected] nucleus position indicative of cellular polarization, similar intestinal epithelium; (v) enterocyte-like F-108 phenotype exhibition by revelation of cytokeratin TREATMENT OF MUSCLE DISEASE IN MDX and alkaline phosphatase expression. The functional MICE WITH LMP-420: AN INHIBITOR OF TNF properties of the FEIC were revealed by demonstrating TRANSCRIPTION its endocytic activity monitored through the Daniela Dover de Araujo (FMRP-USP/AADM- incorporation of cationized ferritin. The establishment UNAERP), Aline Cristina Pimenta (Curso de Medicina- of a primary feline intestinal epithelial cell culture UNAERP), Ana Carolina Toschi (Curso de Medicina- is potentially important for the investigation of the UNAERP), Lílian Cataldi Rodrigues (AADM/UNAERP), complex intestinal physiology and pathology, especially George J. Cianciolo (University Medical Center, as a model during infectious processes provoked by Durham), João Carlos da Silva Bizario (Medicina- enteropathogenous. Additionally, these cells can be AADM/UNAERP), Maria Cristina Ramos Costa (FMRP- applied to the study of various events in cell biology USP/AADM-UNAERP) including endocytic activity through intestinal cells, as well as for screening pharmacological effects of new The mdx mouse, a Duchenne Muscular Dystrophy drugs. model, shows susceptibility to muscle injury and high [email protected] levels of serum creatine kinase (CK); a phenotype intensified by exercise. Muscular degeneration is F-110 exacerbated by a chronic inflammatory process in SHORT TERM SPECIALIZED ENTERAL DIET which tumor necrosis factor alpha (TNF) has a central REFEEDING IS NOT ENOUGH TO RESTORE role. TNF blockade, with either specific antibodies GROWTH FACTOR AND COLLAGEN GENE or soluble receptors, reduces necrosis in mdx mice. EXPRESSION IN WOUND HEALING OF LMP-420, a purine nucleoside analog, is an orally- MALNOURISHED RATS active inhibitor of TNF transcription that could be more Claudia C. Alves, Ricardo Giorgi, Maria Mitzi Brentani, easily administered than TNF antagonists requiring Daniel Giannella, Dan L. Waitzberg injection. The effect of 25 mg/kg/day of LMP-420 in 4 (Faculdade de Medicina da USP) week-old mdx mice submitted to a treadmill running program for 5 weeks was evaluated using the following Malnutrition delays wound healing by increase parameters: quantitative histopathological analyses of the inflammatory phase, reducing the fibroblasts gastrocnemius and diaphragm and CK levels. LMP- proliferation and collagen synthesis. Aim - To verify 420 treated mice showed significantly-decreased CK the influence of specialized enteral nutrition upon levels and amelioration of muscular degeneration, growth factors and collagen expression on cutaneous fewer inflammatory infiltrates, decreased degeneration/ wound healing tissue in malnourished rats. Method - regeneration ratios and Evans blue dye infiltrated Lewis male adult isogenic rats (N=57) eutrophic (E) myofibers. Our data suggest that the inhibition of TNF (n=30) and malnourished (M) (n=27) were submitted production by a small molecule transcriptional inhibitor to gastrostomy (G) and standardized dorsal skin is beneficial for retarding dystrophinopathy and may wound. All animals were fed with oral diet AIN-93M lead to an option for future clinical trials for DMD. and received saline or enteral diet through G. E and [email protected] M rats were subdivided in 3 groups: S - saline [E(S) and M(S)]; STD - standard enteral diet [E(STD) and F-109 M(STD)]; SPD - enteral diet supplemented with arginine ESTABLISHMENT AND CHARACTERIZATION OF A and antioxidants [E(SPD) and M(SPD)]. At 7th day PRIMARY FELINE INTESTINAL EPITHELIAL CELL post trauma, wound granulation tissue was sampled CULTURE for growth factors and collagen gene expression 1. 167

livro.indd 167 7/7/2008 16:33:45 analyzed by real time PCR. Variance analysis was belong to Curimatidae family in two lakes of Federal done, considering p<0.05. Results - M(S) rats had University of Acre. Six collets were made in each lake lower TGF-beta and collagen III than E(S) rats. during the period from September to July 2006 and M(SPD) rats had lower VEGF, KGF-7 and collagen III 2007. Each sample was weighed, measured and the than E(SPD). M(S) rats had higher collagen III when ovaries and liver were taken and fixed in alcoholic compared to M and M(SPD). E(S) rats had higher Bouin. The material was subjected to routine techniques collagen III when compared to E(STD) and E(SPD). for the inclusion in Historesin. For that, it was applied Conclusion - Refeeding of M rats has not influence to the color techniques on each sample: Hematoxilin and restore the growth factor and collagen expression at the Eosin (HE), Toluidine Blue (TB), Bromophenol Blue granulation wound tissue independent of the standard (BB) and Periodic acid Schiff with Alcian Blue (PAS). or supplemented enteral diet. The total length average specimen in both lakes was [email protected] 107,875 mm, the standard length was 76,312 mm and the weight was 20g. It was found differences between F-111 young and old fishing cells of genus Steindachnerina CYTOCHEMICAL STUDIES IN inside each lake and also differences between the two TETRAGONOPTERÍNAE FISHES lakes studied. Besides, in Viveiro Lake, younger fishes (CHARACIFORMES: TETRAGONOPTERINAE) IN had better protein synthesis than other younger fishes TWO LAKES AT UFAC’S ZOOBOTANICO PARK from Horta Lake, according to the color techniques Joana Figueiredo da Mata, Rusleyd Maria Magalhães made. None of the fishes showed uniformity about the de Abreu (Univerdidade Federal do Acre) color technique results. Specimens in both lakes had shown protein synthesis, lipids, and polysaccharides The methodological difficulties surrounding research in all the liver cells analized. Viveiro Lake’s individuals about small fishes has resulted in scarce literature had cell metabolism more intense in ovaries and about the topic. The main aim of this paper was liver cells. It Occurred protein synthesis, lipids and to compare development and reproductive cycles polysaccharides in all the ovary cells of the samples based on cytochemical studies of Tetragonopterinae studied, but in Viveiro Lake specimens had acid and specimens collected from three lakes on the campus basic polysaccharides and in Horta Lake had only basic of the Federal University of Acre. Six samples of ones. Tetragonopterinae specimens were collected from [email protected] each lake on the University of Acre´s campus, between November 2005 and June 2006. The liver and ovaries F-113 from each fish were removed. The material was fixed BONE FORMATION AFTER IRRADIATION WITH in alcoholic Bouin, conserved 70% in alcohol and LOW INTENSITY LASER subjected to routine techniques from there incluson in Rejane Daniele Reginato (UNIFESP), Eduardo paraffin. Histological sections were transferred to glass Katchburian (UNIFESP), Andressa Cristina Morretto slides and used for further cytochemistry analyses. (UNIFESP-FIP), Raphael Tiago de Sousa (UNIFESP- To do this, it was applied the following staining FIP), Luiz Antonio de Sousa Merli (UNIFESP), Flavio techniques on each sample: Hematoxilin and Eosin Faloppa (UNIFESP), Queila Vasselo (UNIFESP), (HE), Toluidine Blue (TB), and Bromephenol Blue (BB). Helena Bonciani Nader (UNIFESP) The analysis showed slight differences between fishes cells’ that belong the same lake and the ones belong Bone is a mineralized tissue that is under influence of to different lakes.On the other hand, it was observed several systemic, local and environmental factors and that fishes belong to Vivieiro Lake showed according depends upon the combined activities of osteoblasts, to the staining techniques applied above better RNA osteoclasts and osteocytes. In addition, cellular synthesis and also protein synthesis than specimens activity in bone, following injury, has been shown to be provided from Piaba Lake and Horta Lake. According interfered by the aplication of low-level laser therapy. to the staining techniques analysis of the cells in all We studied the effect of low-level Ga-Al-As laser specimens, Our results suggest that Viviero Lake therapy ( Gallium-aluminum-arsenide – 3J/cm2) in bone promotes better conditions for the development of tissue after injury. Femurs of male Rattus novergicus Tetragonopterinae cell specimens. albinus (Wistar lineage), 3 months old, whose body [email protected] weights ranged from 250 to 300g were used. A small hole (3cm diameter) was drilled on the left/right femurs. F-112 Femurs of left paws were irradiated with laser (irradiated MORPHOMETRICAL AND CYTOCHEMICAL group). Femurs of right paws were not irradiated STUDIES IN CURIMATIDAE FISHES (control group). The animals were sacrificed on the (CHARACIFORMES: CURIMATIDAE) IN THREE 0th, 4th, 8th, 14 th after surgery. After dissection of the LAKES AT UFAC’S ZOOBOTANICO PARK femur, the fragments were fixed in 4% formaldehyde Joana Figueiredo da Mata, Rusleyd Maria Magalhães (derived from paraformaldehyde) in 0,1 phosphate de Abreu (Universidade Federal do Acre) buffered saline, pH 7,4, at room temperature for 16 hours. After decalcification in formic acid 25% pH 2,0, The main objective of this paper was to evaluate the samples were dehydrated, embedded in paraffin, and compare protein synthesis process and the sectioned and stained with H.E. Preliminary results development cycle of Steindachnerina genus that indicate that there is increased bone formation in the 1. 168

livro.indd 168 7/7/2008 16:33:45 irradiated group, when compared with the control group. Naila Francis Paulo de Oliveira, Ana Paula de Souza Although most of the bone present in the lesion is Pardo, Gilcy Raimundo Damn (UNICAMP) primary bone, some lamellar bone was also observed. Chronic periodontitis is an inflammatory disorder We are currently carrying out a quantitative analysis of that affects the tissues that suport the tooth, often our results. culminating in tooth loss. The presence of the DNA [email protected] methylation in regions of gene promoters rich in CpG islands can promote lifting or removal the levels of F-114 transcription and may even completely silence the THE HUMAN REGULATORY PROTEIN KI-1/57 gene. It has been shown that smoking causes increase INTERACTS WITH PRE-MRNA SPLICING- in the synthesis of some inflammatory cytokines, ASSOCIATED PROTEINS AND RELOCALIZES FROM including the chemokine interleukin-8 (IL-8). The A DIFFUSE PATTERN TO DOT-LIKE STRUCTURES objective of this study was to analyse the pattern of UPON ADOX TREATMENT DNA methylation in the promoter region of IL-8 gene Gustavo Costa Bressan (Laboratorio Nacional de in oral and blood cells from healthy subjects and Luz Sincrontron), Alexandre José Cristino Quaresma with chronic periodontitis to associate the pattern of (Laboratorio Nacional de Luz Sincrontron), Eduardo methylation in these cells with inflammation. DNA of Cruz Moraes (Laboratorio Nacional de Luz Sincrontron), healthy subjects and patients with chronic periodontitis Adriana Oliveira Manfiolli (Faculdade de Medicina smokers and nonsmokers was purified and then de Ribeirão Preto-USP), Dario Oliveira dos Passos modified by sodium bisulphite, followed by Methylation (Laboratorio Nacional de Luz Sincrontron), Tais Mayumi Specific PCR. Samples of PCR were visualized by Kuniyoshi (Laboratorio Nacional de Luz Sincrontron), electrophoresis on 10% polyacrylamide gels and stain Marcelo Damário Gomes (Faculdade de Medicina by SYBR Gold. The statistical analysis was performed de Ribeirão Preto-USP), Jörg Kobarg (Laboratorio using the test of Qui-Quadrado and variance test of Nacional de Luz Sincrontron) Friedman with significance level of 5%. The results indicate that individuals with chronic periodontitis The cytoplasmic and nuclear protein Ki-1/57 was firstly independent of smoking have a higher percentage identified in malignant Hodgkin and Sternberg-Reed of hypomethylation of IL-8 gene than those controls. cells in Hodgkin’s lymphoma. Ki-1/57 is phosphorylated The methylation pattern of DNA leukocytes showed a by protein kinase C upon PMA cellular activation, tendency of hypomethylation in the smokers group. Our which redistributes it to the cytoplasm and abolishes its results suggest that inflammation in the oral mucosa interaction with the scaffold/signaling protein RACK1. can promote changes in the DNA methylation pattern Several p53-associated proteins and p53 itself also of IL-8 gene in buccal cells while smoking may promote have been described as Ki-1/57 interacting partners. DNA systemic changes. Furthermore, Ki-1/57 is methylated by PRMT1, an [email protected] arginine methyltransferase that methylates several RGG box-containing RNA binding proteins. Here, we present F-116 the interaction of the pre-mRNA splicing-associated GENOTOXIC POTENTIAL OF CHRYSIN IN HUMAN proteins SFRS9, SF2p32, YB-1 and hnRNPQ with DERMAL FIBROBLASTS Ki-1/57. In vitro and in vivo experiments confirmed the Evandro Luís de Oliveira Niero, Ricardo Frota Boggio, protein-protein associations. By using electrophoretic Gláucia Maria Machado-Santelli (Dept. of Cell and mobility shifty assays, we found that Ki-1/57 directly Developmental Biology, Inst. of Biomedical Sciences, binds to RNA oligonucleotides. This interaction occurs USP) preferentially with U-rich probes and mainly through its C-terminal RGG box-containing portion. We analyzed Flavonoids are compounds widely present in fruits, the subcellular localization of several deletions vegetables and beverages that protect against constructs fused to EGFP and observed the importance chemically induced cancer. Chrysin is supposed to have of the Ki-1/57 N-terminal to its nuclear localization at one of the most pronounced protection against drugs several subnuclear structures upon Adox treatment, like benzo(a)pyrene in HepG2 cells. According to recent an inhibitor of protein arginine-methylation. Further studies chrysin could have the ability to accelerate confocal analysis in Adox-treated HEK293T permitted wound healing, control scar formation and prevent the us to observe the colocalization of the EGFP-Ki-1/57 process of skin aging by collagen stimulating activity. with nuclear structures involved in transcription and Nevertheless, depending on their concentrations, some splicing of pre-mRNA. Taken together, our findings flavonoids could be cytotoxic. Thus, the present work suggest that Ki-1/57 may be a novel human protein aimed to analyze the genotoxic potential of chrysin in involved with RNA processing mechanisms controled by human dermal fibroblasts after 48 hours of treatment. protein-methylation. The cell viability test (MTT) showed a discrete increase [email protected] (over 10%) in the proliferation at concentrations between 0.5 and 2.5mcg/mL of the drug. At 5.0mcg/ F-115 mL, that effect was not observed. The DNA index was DNA METHYLATION PATTERN OF IL 8 GENE IN not altered after the treatment, but the cells treated with BUCCAL EPITHELIAL CELLS AND PERIPHERAL chrysin at 5.0mcg/mL showed an increased frequency BLOOD OF PACIENTS WITH CHRONIC (over 100%) of micronucleated cells compared to PERIODONTITIS SMOKERS AND NON-SMOKERS control group. The frequency of nuclear buds was also 1. 169

livro.indd 169 7/7/2008 16:33:46 higher at that concentration. Other nuclear aberrations, like binucleation and multinucleation, were not observed We have described a subset of T. cruzi calpain-like with any concentration of the drug. The cytoskeleton cysteine peptidases associated with 68-amino acids analysis did not show microtubules or microfilaments repeats which are tandemly arranged in the central reorganization. The results allow us to conclude that domain of the molecule. The repeats were previously despite of the absence of cytotoxic effects in human found in an immunodominant antigen which is involved dermal fibroblasts, chrysin had genotoxic activity at in the attachment of the flagellum to the cell body of the 5.0mcg/mL and its effects should be better investigated. parasite. We called this protein H49/calpain-like protein. [email protected] We present further characterization of the structure and cellular location of H49/calpain-like protein. To F-117 study the occurrence and subcellular distribution of DIFFERENTIAL EXPRESSION OF GAL-3 IN this protein in epimastigotes, we have cloned and EPIDERMAL OF MICE HAIRLESS AFTER UV-B overexpressed different domains of the calpain and the EXPOSURE repeats in bacteria, purified the proteins, and employed Silvina Odete Bustos (Faculdade de Medicina), V.V da them to immunize rabbits and mice. Antibodies raised Silva (Faculdade de Ciências Farmacêuticas da USP), against the calpain domains and repeats reacted, by C.D.Ropke (Faculdade de Ciências Farmacêuticas immunoblot analysis, with a >240 kDa in the whole da USP), S.B.M.Barros (Faculdade de Ciências epimastigote extracts. Anti-calpain antibodies reacted Farmacêuticas da USP), R,Chammas (Faculdade de with additional bands of ~180 and ~60 kDa which may Medicina) correspond to the calpain proteins without repeats. Immunofluorescence analysis showed that H49/calpain- The cancer of the skin is one of the most common like protein is only localized in the region of adhesion type of cancer and its incidence has been increasing between the cell body and flagellum. Calpain was lately. The ultraviolet radiation (UV) is a factor of risk present not only in the flagella, but it was also localized for this neoplasia. Several data have shown that the in the cytoplasm. Further, the cytoskeleton-associated UV can promote tumor progression due a generation H49/calpain-like was colocalized with markers of the of -galactoside-binding lectinbreactive oxygen flagellar attachment zone. We describe here a novel species. Galectin-3 (gal-3) is associated with tumoral family of cytoskeleton-associated proteins in T. cruzi. progression and metastasis. In this work, we analyzed These proteins are characterized by their similarity to the expression of gal-3 in different epidermal layers of the catalytic region of calpain, and also by the presence mice exposed to UVB. Mice hairless were irradiated of tandemly arranged amino acid repeats. or not with a dose of 11mJ/cm2 and sacrificied after 2, [email protected] 48 and 96 hours. Sections of skin were analyzed for imunohistochemical for the expression of gal-3. We F-119 realized the morphometric analyses considering the UTILIZATION OF FISH (FLUORECENSE IN SITU presence/absence of gal-3, its subcellular localization HIBRIDIZATION) WITH TELOMERIC PROBES and its expression in the basal or suprabasal layers. FOR AVALIATION OF HUMAN LINFOCITES WITH We showed that the number of pyknotic cells in the MICRONUCLEUS RADIATED WITH Y60CO suprabasal layer increased after the exposure of UVB. Antonio Augusto Adami Pires (Instituto de pesquisas In the basal layer we showed a decrease, however, energéticas e nucleares IPEN), Frederico Henning after 48 hours the number of pyknotics cells increased. (Universidade de São Paulo; Istituto de Biociencias IB- We also observed an increase in hyperkeratosis areas. USP), Kayo Okazaki (Instituto de pesquisas energéticas We showed a significant statistically decrease in gal-3 e nucleares IPEN) positive cells, and we could note a slight increase at 96 hours after irradiation. We observed an increased in Micronuclei are chromosome fragments or entire positive gal-3 cells in suprabasal layer when compared chromosomes that do not incorporate in the main to basal layer. Besides that a small fraction of cells nucleus of cell, remaining in the cytoplasm after presented nuclear expression of gal-3. We showed that mitosis. Micronucleus test is usually used to inquire the expression and localization of gal-3 was altered after damage caused in DNA by different external factors, stimulus of UV-B suggesting a role of gal-3 in different like for exemple ionizing radiation in proficionals layers of epidermis. exposed. In this view we have used the FISH technique [email protected] (Fluorecense in situ hibridization) in radiated human linfocites. We collected 4 ml of blood and it was radiated F-118 with y 60Co in a dose of 5 Gy and after that it was kept CHARACTERIZATION OF A FAMILY OF in celular culture by 72 hours. The cells were treated CYTOSKELETON-ASSOCIATED CALPAIN-LIKE with isotonic solution and fixed to prepare the slides. PROTEINS IN TRYPANOSOMA CRUZI The telomeric probes were created by polymerase chain Alexandra Galetovic Carabantes (UNIFESP), Márcia Reaction (PCR), flanked with the primers (TTAGGG)5 Regina Machado dos Santos (UniBan), Esteban and (CCCTAA)5 and this was marked with dUTP- Mauricio Cordero Veas (UNIFESP), Jaime Santana fluorecein according with Roche® online protocol. After (UnB), Jerônimo C Ruiz (Centro de Pesquisa Rene that, was made the process of In Situ Hibridization. The Rachou), Renata Souza (UNIFESP), Renato A. Mortara slides were treated with RNAse, pepsin and formamide, (UNIFESP), José Franco da Silveira (UNIFESP) the probes were desnaturated and the solution of 1. 170

livro.indd 170 7/7/2008 16:33:46 probes was put together with the slides and was colored divisome formation, but how this complex assembles is with vectorshield DAPI. The results of this pilot test still unclear. In this work, we have studied the temporal observed in a fluorecence microscopy can show us sequence of assembly of different division proteins into that the dose of 5 Gy was sufficient to cause damege the Bacillus subtilis divisome by dual color fluorescence in DNA like for exemple micronucleus, binuclear cells. microscopy. We fused FtsZ to the red reporter mCherry The micronucleus with all telomeres region come from and four other division proteins (ZapA, EzrA, DivIVA, entire chromosomes (aneugenic micronucleus) while YpsB) to green reporter (GFP). Because Z-ring the micronucleus that do not have all telomeres region formation is the first step in divisome assembly, we come from chromosomes fragments (clastogenic reasoned that the frequency of colocalization between micronucleus). the Z-ring and a division protein will provide a measure [email protected] of how long it takes for this protein to associate with the divisome. Our results revealed that ZapA, EzrA, DivIVA F-120 and YpsB colocalized with the Z-ring in 95%, 96%, GENETIC STUDY OF THE INTERACTION BETWEEN 72% and 50% of the cells, respectively. This suggests THE TUBULIN-LIKE PROTEIN FTSZ AND DIVISION that ZapA and EzrA associate with the Z-ring almost MODULATORS IN BACILLUS SUBTILIS immediately after its formation. DivIVA and YpsB, on the Alexandre Wilson Bisson Filho, Frederico José Gueiros other hand, seem to exhibit a delay in their association. Filho (Instituto de Química, USP) These results suggest that the divisome complex does not assemble instantaneously after division is initiated. The tubulin-like protein FtsZ is the main molecule of We are using the colocalization approach with the bacterial cell division and self-associates into a ring remaining division proteins with the goal of generating a structure (Z ring). The Z ring recruits other division detailed view of the dynamics of divisome assembly in proteins to the place where division will occur. Some of B. subtilis. these division proteins interact with FtsZ and modulate [email protected] its polymerization properties, promoting the spatial- temporal control of Z ring formation. Our laboratory is F-122 interested in characterizing the interaction between ALTERNATIVE PROCESSING OF TRANSCRIPTS FtsZ and ZapA, a positive Z ring modulator. Here, FOR EPIDERMAL GROWTH FACTOR IN we sought to identify FtsZ mutants that have lost the HUMAN AND MOUSE, NORMAL OR LEUKEMIC, ability to interact with ZapA. We created a mutagenized LEUKOCYTES ftsZ plasmid library by error prone PCR. This library Camila Santos de Moraes, Gabriel Padron Rios, Miguel contained 100.000 transformants and exhibited a Angelo Moreira, Claudia Sondermann Freitas (INCA) mutation rate of one substitution every 1400bp. The mutagenized library was transformed into a modified The Epidermal Growth Factor (EGF) family of cell Bacillus subtilis strain to select for mutants that lost growth factors are synthesized as precursor peptides the ability to interact with ZapA. In this strain, the addressed to the cell membranes, where they are interaction between FtsZ and over-expressed ZapA- assembled as transmembrane peptides. From there, MTS (a membrane associated version of ZapA) causes the soluble, autocrine or paracrinally active forms, are lethality. Thus, bacteria carrying interaction-defective released by proteolysis, leaving a free intracellular FtsZ will be easily isolated as these are the only ones moiety that can act intracrinally, for example, modulating that survive. Using this genetic selection we have, the cell cycle, as described for HB-EGF (Heparin so far, found several FtsZ mutants that are not killed Binding-EGF). Although quantitatively varying among by ZapA-MTS overexpression (one mutant every tissues, the ubiquitously expressed EGF precursor itself 10.000 transformants). Several of these mutants have shows an unusually large transcript and transmembrane substitutions in the N-terminal region of the protein. We peptide, for which some tissue- specific processing are currently investigating their properties. Financial was reported in literature, and whose functions remain Support: FAPESP and CNPq. to be fully understood. When studying EGF transcript [email protected] expression in human and mouse, either normal or leukemic, blood cells, by cloning and sequencing F-121 PCR products, we could observe two still undescribed USING DUAL COLOR GFP MICROSCOPY TO STUDY alternative splice patterns, one of which seems THE ASSEMBLY OF THE CELL DIVISION COMPLEX exclusive of hematopoetic tissues. At present, we OF BACILLUS SUBTILIS investigate their putative implications in EGF peptide Guilherme Louzada Silva Meira, Alessandra Roque synthesis, subcelular fate, and eventual signaling Pancetti, Frederico José Gueiros Filho (USP) function in leukocyte biology. [email protected] Cell division in bacteria is orchestrated by FtsZ, a bacterial protein homologous to tubulin that self- F-123 assembles into a structure denominated Z-ring. PHOTOSENSITIZATION WITH ALUMINIUM Positioning of the Z-ring in the middle of the cell PHTHALOCYANINE TETRASULFONATE PROMOTES triggers the association of other division proteins into ACTIN FILAMENTS REORGANIZATION a macromolecular complex known as the divisome. Maira Maftoum-Costa, Aline Helena de Araujo There are at least fifteen proteins that participate in Machado, Karen Cristiane Martinez de Moraes, Cristina 1. 171

livro.indd 171 7/7/2008 16:33:46 Pacheco Soares (Universidade do Vale do Paraíba) prepared for transmission electron microscopy (a Zeiss EM 900 microscope) using uranyl acetate and lead Photodynamic therapy (PDT) of cancer is a therapeutic citrate. Our results presented an extensive cytoplasm modality for treatment of tumours in which visible light vacuolization and retraction of the cells after PDT is used to activate a photosensitizer (JUARRANZ et and the ultrastructural analysis shown cell death. al., 2001). The interaction of light with photosensitizer [email protected] molecules in the presence of molecular oxygen catalyses the formation of reactive oxygen species (ROS) within cells to produce the cytotoxic effect (MAGI et al., 2004). In this work effects of PDT on actin organization of human laryngeal carcinoma cells were investigated. HEp-2 cells (human laryngeal carcinoma) were incubated with 10μM aluminium phthalocyanine tetrasulfonate (AlPcS4) for 1h, and irradiated with a diode laser (working at 660 nm; energy density of 4.5 J/cm2 and power of 35mW). After PDT the culture was stained with 250μg/mL phalloidin-TRITC for 1h. Actin was found in the ruffles at the perimeter of the cells and diffusely over the body of the cells with reduction at the number of stress fibers 6h after photosensibilization. 24h after the PDT-AlPcS4 a significant proportion of the cells (30%) seemed to be retracted and exhibiting punctuate plalloidin-TRICT staining at the cell-substrate interface. Oxidation of β-actin during PDT oxidative stress has been reported (MAGI et al., 2004); this event may affect actin filaments architecture and lead to disorganization of the cytoskeleton. Therefore, exposure to AlPcS4 and Laser caused actin filaments to reorganize from typical stress fibers to substrate- associated aggregates of actin. [email protected]

F-124 ULTRASTRUCTURAL EFFECTS OF ZNPCBR8 ON L929 CELLS CULTURE AFTER PHOTODYNAMIC THERAPY ALINE HELENA ARAUJO MACHADO, CRISTINA PACHECO SOARES, MAÍRA MAFTOUM-COSTA, MILTON BELTRAME JUNIOR, Newton Soares da Silva (Universidade do Vale do Paraíba)

Photodynamic therapy (PDT) is a kind of promising treatment for neoplastic and non-neoplastic injured cells based on photosensitizer activation by visible light. In the presence of oxygen, the interaction of light with a photosensitizer molecules catalyses the formation of reactive oxygen species (ROS) which can destroy the cells. Considering the novelty of this research area too many different classes of new sensitizers have been developed focusing on clinical trials. Phthalocyanines, a second generation of photosensitizers have received particular attention and in this work we described the ultrastructural changes in L929 cells after PDT using for that Octal-bromide Zinc Phthalocyanine (ZnPcBr8). After they had being incubated with 1μM of ZnPcBr8 for 1h, the cells were irradiated with a InGaAlP semiconductor laser (685 nm for 15 minutes; 4.5 J/cm2 and 35mW) and subsequently incubated in Minimum Essential Medium (MEM) for 24 hours. The cells were then fixed in a solution containing 2.5% glutaraldehyde and 4% formaldehyde, post fixed in 1% osmium tetroxide, dehydrated in acetone and embedded in Epon. Once we have sectioned the cells, they were 1. 172

livro.indd 172 7/7/2008 16:33:46 Host Parasite Interaction larvae midgut. The chitin in PM was specifically visualized in a confocal fluorescence microscope, using a WGA-FITC (wheat germ agglutinin- fluorescein isothiocyanate) conjugated in the presence of excess G-001 free N-acetylglucosamine. Fragments of the anterior VIRAL INFECTION OF THE MIDGUT EPITHELIUM OF and posterior midgut regions from parasitized and non- DIATRAEA SACCHARALIS (LEPIDOPTERA) parasitized larvae (6 days after parasitism) were fixed, Daniela de Oliveira Pinheiro, Fernando Luis Cônsoli embedded in Paraffin resin, processed and analyzed in (Esalq-USP-Piracicaba) confocal fluorescence microscope. The chitin content of the PM is higher at the posterior midgut region in Columnar cells are responsible for the production of both non-parasitized and parasitized D. saccharalis enzymes and absorption of digestive products. An larvae, suggesting regional differences in the PM along analysis of Diatraea saccharalis midgut epithelium the midgut lenght; however, the WGA-FITC labeling indicated these cells were heavily infected by a is diminished in the parasitized larvae. Our results symptomless, cytoplasmic virus. To understand the showed that the parasitism by C. flavipes affect the viral role on the host biology and its possible interaction morphological structure of the PM in the sugarcane with Cotesia flavipes, further studies on the virus borer midgut. distribution during the last instar of D. saccharalis [email protected] and the parasitism role may have on virus infection abundance were developed. The possible mode of G-003 viral transmission was investigated, as transovarial TOXOPLASMA GONDII EGRESS: A STRUCTURAL transmission seems to fix this infection. D. saccharalis APPROACH were stung once by C. flavipes females, and reared Lúcio Ayres Caldas, Márcia Attias, Wanderley de Souza on an artificial diet under controlled conditions. Midgut (Instituto de Biofísica Carlos Chagas Filho - UFRJ) fragments were processed for transmission electron microscopy. Larval and adult ovaries and midgut of Toxoplasma gondii is the obligate intracellular parasite D. saccharalis were processed for negative staining. responsible for toxoplasmosis, a major cause of death Our results showed that columnar epithelial cell and in immunocompromised patients. Despite recent lumen content have viral inclusion. The cytoplasmic advances in T. gondii cell cycle research, its egress polyedrose virus was observed into the cytoplasm and from host cells is still a poorly understood process. If in protrusions of columnar cells. Independent of the the mechanisms involved in egress were the same midgut region, variations in the size and form of the viral as in invasion, as argued by some authors, the inclusion bodies were the same. Viral inclusions were parasitophorous vacuole membrane would fuse with confirmed by negative stained. Virus inclusion bodies the host cell plasma membrane in order to release the were more abundant in parasitized larvae. The ovary parasites. In this study we induce T. gondii egress from analysis of both larvae and adult showed viral inclusion, LLC-MK2 cells at different hours post-infection. Host confirming the hypothesis that this infection passes cells were grown for 24h over glass coverslips in RPMI through all developmental stages following a vertical medium and infected with tachyzoite forms of the RH pathway. We believe that control and parasitized larvae strain of T. gondii. 2 or 24h later, egress was induced behaviors are not affected by this viral infection. with 10µM calcium ionophore A23187 for 10 min. [email protected] Coverslips were fixed with 2.5% glutaraldehyde in 0.1M

cacodylate buffer, pH 7.2, post fixed with 1% OsO4 in G-002 0,1M cacodylate buffer, pH 7.2, plus 0.8% potassium PERITROPHIC MEMBRANE IN THE MIDGUT ferrocyanide, dehydrated in ethanol, critical point dried EPITHELIUM OF DIATRAEA SACCHARALIS in CO2. Egress of T. gondii from host cells is usually (LEPIDOPTERA) PARASITIZED BY COTESIA associated with host cell burst. Our observations of FLAVIPES (HYMENOPTERA) calcium ionophore treated and non-treated infected cells Daniela O. Pinheiro (Esalq-USP-Piracicaba), Alberto suggest, otherwise, that host cell membrane rupture F. Ribeiro (IB-USP-São Paulo), Walter R. Terra (IQ- occurs only when there is a great number of egressing USP-São Paulo), Márcio V. Cruz (IB-USP-São Paulo), parasites. Some monolayers had their cell membranes Waldir Caldeira (IB-USP-São Paulo), Elisa A. Gregório breached by adhesive ribbon and were observed by (IB-UNESP-Botucatu) field emission scanning electron microscopy. Our observations indicate that the parasite glides though Diatraea saccharalis, main pest for the sugarcane host cell cytosol towards extracellular medium. crop, has been widely controlled by Cotesia flavipes; [email protected] little is known about the effect of the parasitism in

the host midgut. The columnar cell is responsible for G-004 processing the diet, secretion of the digestive enzymes PARTIAL STRUCTURAL CHARACTERIZATION OF and the peritrophic membrane (PM), and for the uptake PARACOCCIN, A CHITIN-BINDING PROTEIN FROM of the final digested product. This work studies the PARACOCCIDIOIDES BRASILIENSIS ASSOCIATED effect of parasitism in the PM of the D. saccharalis 1. 173

livro.indd 173 7/7/2008 16:33:46 WITH HOST IMMUNE RESPONSE the human pathogen Paracoccidioides brasiliensis. Fausto Bruno dos Reis Almeida (USP), Ebert Seixas Electron microscopy shows that P. brasiliensis cells Hanna (USP; Invent Biotecnologia Ltda; Universidade treated with farnesol exhibited a fully cytoplasm Estadual de Londrina), Vânia Sanmartino Mariano degeneration. However, the cell wall remained intact, (USP), Leandro Licursi de Oliveira (USP), José César and the cell permeability was not affected. Our results Rosa (USP), Marcelo Valle de Sousa (UnB), Maria clearly showed the in vitro antimicrobial activity of Cristina Roque-Barreira (USP) farnesol against P. brasiliensis. However, additional studies involving animal models of experimental The dimorphic fungus Paracoccidioides brasiliensis paracoccidioidomycosis need to be performed to assess is the causative agent of paracoccidioidomycosis, their potential use as an adjuvant chemotherapeutic the most frequent systemic mycosis in Latin America. agent. Various fungal components are associated with [email protected] its virulence, such as lipids, polysaccharides and glycoproteins. Paracoccin, a P. brasiliensis lectin G-006 with MM 70 kDa purified by affinity to immobilized AMASTIN, A TRYPANOSOMA CRUZI AMASTIGOTE N-acetylglucosamine, promotes fungal adhesion to SPECIFIC PROTEIN, IS INVOLVED IN HOST CELL extracellular matrix components and induces TNF-a INVASION AND INTRACELLULAR MULTIPLICATION and of nitric oxide production by macrophages. Anti- Mário Costa Cruz (UNIFESP), Claudio Vieira da Silva paracoccin murine antibody represents an important (UNIFESP), Wanderson Duarte da Rocha (UFMG), tool to study paracoccin, allowing verifying that the Santuza Maria Ribeiro Teixeira (UFMG), Renato Arruda lectin is abundant on the surface and within the fungal Mortara (UNIFESP) cell, a fact that contrasts with the low yield provided by the paracoccin purification procedures. In order to Extracellular amastigotes (EA) forms of Trypanosoma reach higher yields in the purification we substituted the cruzi from G strain (T. cruzi I) show high infectivity affinity procedure carried out on a N-acetylglucosamine towards host cells in vitro when compared to the column by a procedure on a particulate chitin column. traditionally more infectious CL strain (T. cruzi II). We Electrophoresis and Western blot analysis revealed have performed a DNA microarray from EA from G and similarity between N-acetylglucosamine- and chitin- CL strain, in order to identify components that would bound materials, which was confirmed by MALDI-TOF- modulate infectivity of these strains. It was identified a MS of trypsinic peptides. Furthermore, the spectra of 21 kDa protein that upregulates cell invasion by EA from peptide mass fingerprint overlapped putative sequences G strain. On the other hand, we have not investigated with a hypothetical protein from Neurospora crassa. We the mechanisms beneath the low infection rate of EA are presently producing anti-paracoccin IgY antibodies, from CL strain. The possibility of negative modulators which will enable us to construct platforms for improving was considered once previous studies showed the paracoccin yield, as well as to immunoscreen a that poorly infectious metacyclic trypomastigotes cDNA library from P. brasiliensis. express high levels of gp90, a stage-specific surface [email protected] glycoprotein that modulates negatively cell invasion. We observed in the microarray data that amastin, a G-005 stage specific surface protein, was 21 times more ULTRASTRUCTURAL EFFECTS OF FARNESOL ON expressed in EA from CL strain. In order to study the PARACOCCIDIOIDES BRASILIENSIS YEAST FORM putative involvement of amastin in cell invasion, we Lorena da Silveira Derengowski, Shélida Vasconcelos used amastin transfected clone of CL-Brener strain Braz, Calliandra de Souza Silva, Thiago Machado Mello expressing high levels of the protein. Preliminary results de Souza, Sônia Nair Báo, Cyntia Maria Kyaw, ldinete showed that EA from parasites expressing high levels Silva Pereira (Universidade de Brasília) of amastin had a significant decrease in cell invasion when compared to the wild-type clone. Furthermore, Farnesol is a sesquiterpenoid found in several when the number of intracellular amastigotes was essential oils, possibly protecting plants from parasitic assessed 48 hours after the invasion, it was significantly induced damages. Recently, this compound has higher in cells infected with parasites expressing high been demonstrated to inhibit the growth of some levels of the protein. These results indicated that microorganisms, such as the human pathogens amastin downregutates cell invasion in the same way Staphylococcus aureus and Streptococcus mutans, and as it upregulates intracellular parasite multiplication. the plant pathogenic fungus Fusarium graminearum. [email protected] A recent study has shown that farnesol also mediates apoptosis in the filamentous fungus Aspergillus G-007 nidulans. Aditionally, farnesol enhances microbial TOXOPLASMA GONDII INHIBITS THE IN VITRO susceptibility to antibiotics, indicating a putative MYOGENESIS OF SKELETAL MUSCLE CELLS application as an adjuvant therapeutic agent. These Alessandra Ferreira Gomes (Instituto Oswaldo Cruz/ results point to its potential use as an antimicrobial FIOCRUZ), Erick Vaz Guimarães (Instituto Oswaldo agent. This work reports the farnesol activity against Cruz/FIOCRUZ), Juliana Leite Pinto (Instituto Oswaldo 1. 174

livro.indd 174 7/7/2008 16:33:47 Cruz/FIOCRUZ), Laís de Carvalho (UERJ), Helene the apical and posterior regions or associated with Santos Barbosa (Instituto Oswaldo Cruz/FIOCRUZ) SkMC membrane expansions. Migration of the rough endoplasmic reticulum (RER) profiles to the parasite The Toxoplasma gondii, belongs to a group of parasitic invasion site was observed. PVM alterations were protozoa that essentially lead obligate intracellular observed with 48 hr PI and cystogenesis complete lifestyles. We employed primary culture of skeletal with 96 hr PI. After 31 days PI, RER profiles and muscle cells (SkMC) as a model for experimental mitochondria were not observed in association with the toxoplasmosis studies. So, we decided to investigate cyst wall. These results show that the in vitro T. gondii the infective capacity of T. gondii tachyzoites in SkMC, model and SkMC are potential tools for the study of the influence of the infection on the myogenesis and cystogenesis using molecular approaches and the drug the possible role of the parasite on the modulation of screening action on tissue cysts and Bz. This work cadherin during this process. Our results showed that was supported by CAPES, CNPq, FAPERJ and IOC/ myoblasts were more infective than myotubes indicating FIOCRUZ. differences in the ability of tachyzoites to invade the cell [email protected] types presents in the culture during the differentiation stage. The analysis of 24-hours-old SkMC infected with tachyzoites for 24 h showed that myotubes are 1.6-fold G-009 less infected than myoblasts. It was observed that the STATINS INHIBIT TOXOPLASMA GONDII parasites are able to interfere molecularly the host MULTIPLICATION IN MACROPHAGES cell, preventing membranes fusion and consequently Erika Afonso Costa Cortez, Mariana de Freitas Oliveira, affecting the myogenesis process. Infected myocytes Juliana Cristina Tobar da Silva, Manoela Lopes presented low expression of the cadherin and were Carvalho, Ana Carolina Stumbo, Laís de Carvalho incapable to form myotubes. Cultures infected for 24 (Laboratório Cultura de Células - UERJ) hours presented an inhibition of 75% in formation of myotubes. Our results demonstrated that in low ratios Toxoplasma gondii is an obligate intracellular parasite of infection, as for example, 1:1 (parasite-host cell) and that replicates inside a host cell in a compartment culture only presenting 43% (± 0.02) of infected cells, called parasitophorous vacuole. Although cholesterol the parasited myocytes did not fuse either with infected is not synthesized by T. gondii, there is evidence for or non-infected cells. These data suggest that T. gondii isoprenoids synthesis. This pathway is essential for is able to module negatively the cadherin expression various cellular functions as control of cell growth interfering as with the myogenesis process. This work and tRNA synthesis, revealing an attractive area for was supported by CAPES, CNPq, FAPERJ and IOC/ therapeutic intervention. The isoprenoid pathway is FIOCRUZ. highly regulated at the level of two enzymes involved [email protected] in the synthesis of mevalonate: HMG-CoA synthase and reductase. Simvastatin (SV), Rosuvastatin (RV) G-008 and Atorvastatin (AV) are drugs of the Statins family TOXOPLASMA GONDII-SKELETAL MUSCLE CELLS available as hypocholesterolemic agent in humans. INTERACTION AND CYSTOGENESIS IN VITRO These drugs are HMG-CoA reductase inhibitors, Erick Vaz Guimarães, Alessandra Ferreira Gomes, preventing the synthesis of isoprenoid intermediates of Helene Santos Barbosa (Instituto Oswaldo Cruz/ the cholesterol biosynthetic pathway. Our aim was to FIOCRUZ) establish whether the statins SV, RV and AV are able to inhibit T. gondii multiplication. Macrophages from Toxoplasma gondii is a protozoan pathogen of birds mice peritoneal cavities were plated and infected with T. and mammals, including humans. The bradyzoite (Bz) gondii tachyzoites for 1h and then were independently lives within cysts, occurring predominantly in the central treated with different concentrations of each statin nervous system and muscles, characterizing the chronic (5 – 60 mg.ml-1) for 24h, fixed and stained. The phase of toxoplasmosis. We employed for the first time parasite multiplication was determined by examining primary culture of skeletal muscle cells (SkMC) infected 200 infected cells and statistical analysis was done by with Bz, as a cellular model for cystogenesis. The ANOVA followed by Tukey test. The results showed interconversion bradyzoite-tachyzoite was analyzed by that the statins possess an important antitoxoplasmic immunofluorescence using 2 stage-specific antibodies. activity, but only SV caused more than 50% inhibition With 24 hr post infection (PI) only bradyzoites forms of multiplication. So, we demonstrated that statins were multiplying. After 48 hr PI, 2 types of vacuoles inhibit T. gondii multiplication in a dose-dependent way, (BAG1+, SAG1+) were seen, indicating the presence suggesting an important pathway for drug therapy. of Bz and their interconversion to tachyzoites. After [email protected] 96 hr PI, BAG1+ vacuoles presented a higher number of parasites when compared to 48 hr, indicating G-010 multiplication of Bz without interconversion. The ALPHAV-BETA3 INTEGRIN PARTICIPATION IN ultrastructural analysis of parasite-host cell interaction TOXOPLASMA GONDII HOST CELL INVASION showed Bz to adhere the cell membranes via both Erika Afonso Costa Cortez (Laboratório Cultura de 1. 175

livro.indd 175 7/7/2008 16:33:47 Células- Universidade do Estado do Rio de Janeiro by reactive oxygen intermediates (ROI) inside Mf. - UERJ), Juliana Cristina Tobar da Silva (Laboratório Leishmania could also inhibit ROI production by those Cultura de Células- UERJ), Mariana de Freitas Oliveira cells. Our proteomics analyses of CBA/J Mf infected (Laboratório Cultura de Células- UERJ), Ana Carolina by L. major compared to L. amazonensis identified Stumbo (Laboratório Cultura de Células- UERJ), higher expression of catalase and anti-oxidative Christina Barja-Fidalgo (Departamento de Farmacologia enzymes, such as glutathione-S-transferase, in - UERJ), Lais de Carvalho (Laboratório Cultura de response to L. major. We hypothesized that although Células- UERJ) CBA/J Mf liberate ROI in response to either L. major or L. amazonensis infection, only L. amazonensis is Toxoplasma gondii is a ubiquitous protozoan parasite resistant to these radicals. Using Pick’s technique capable of invading and surviving within nearly all and cytochrome c oxidation, we were unable to detect nucleated cells. The parasite invades host cell by differences in H2O2 and superoxide production active penetration, beginning with proteins secretion by infected cells. Due to low sensibility of those of apical organelles called micronemes, which contain techniques, it was used a more sensitive approach a variety of adhesive domains including integrin-like. based on photon counts of lucigenin/luminol-enhanced Studies have focused on the ligands and receptors chemiluminescence. It was observed that L. major mediating parasite attachment to target cells. Herein, induced a higher superoxide production when compared we investigated the role of host cell integrins on the to L. amazonensis-infected cells. In addition, evaluation parasite invasion capacity by using the antagonist of of H2O2 production also showed that L. major induces integrins, RGD peptide, and a specific antagonist of higher levels of H2O2 by CBA/J Mf when compared to a b v 3 integrin, the disintegrin Kistrin (a protein isolated L. amazonensis-infected cells. These results suggest from the venom of snake). Resident macrophages from that L. major destruction occurs in dependence of ROI mice peritoneal cavities were plated and incubated production in CBA/J Mf. Current experiments goals to with 1 mM RGD peptide or with 0.5 µM Kistrin for 2h. evaluate the mechanisms related to ROI effects on L. Then, T. gondii tachyzoites were incubated for 1h in the major and L. amazonensis survival inside Mf. presence of macrophages. After interaction, the cultures [email protected] were fixed and stained. The quantification of infected host cells was determined by examining 300 cells and G-012 the statistical analysis was done by Student’s t-test. The GROUP B STREPTOCOCCI INDUCES TYROSINE results showed that the blockage of integrins with RGD PHOSPHORYLATION OF ANNEXIN V AND peptide or with Kistrin significantly inhibited T. gondii GLUTATHIONE S-TRANSFERASE DURING invasion by 35,2% and 35.6%, respectively. In order INTERACTION WITH ENDOTHELIAL CELL to eliminate the possibility of T. gondii phagocytosis Gabriela S. Santos, Carla Verônica Loureiro Y Penha, by macrophages we also performed experiments with Andréia Ferreira Eduardo da Costa, Leila Maria Lopes- human endothelial cell incubated with Kistrin, that Bezerra, Prescilla Emy Nagao Ferreira (UERJ) showed a significant inhibition of 33,8% on T. gondii invasion. These results suggest an important role for Group B streptococcus (GBS), a human pathogen that a b integrins, specially the v 3 integrin, during host cell causes infection and invasive disease in newborns, invasion by T. gondii. pregnant women and immunocompromised adults, has [email protected] been shown to invade human umbilical vein endothelial cells (HUVEC). The objective of this study was to G-011 investigate the molecular mechanisms underlying REACTIVE OXYGEN INTERMEDIATES GBS-HUVEC interaction, focusing specifically the PRODUCTION DURING INNATE IMMUNE responsiveness of host protein tyrosine kinase (PTK). RESPONSE OF CBA/J MICE MACROPHAGES The involvement of actin filaments on GBS-HUVEC INFECTED BY LEISHMANIA MAJOR OR interaction was evaluated by treatment of HUVEC with LEISHMANIA AMAZONENSIS IN VITRO cytochalasin E and labeling filaments with phalloidin- Taís Fontoura de Almeida (Fundação Oswaldo Cruz), FITC. PTK involvement was evaluated using Genistein Lucas Castro Mendez (Fundação Oswaldo Cruz), and by electroblotting with 4G10 anti-phosphotyrosine Juliana Perrone Bezerra de Menezes (Fundação monoclonal antibody. We found that GBS induced Oswaldo Cruz), Ivana Nunes Gomes (Fundação actin reorganization and formation of stress fibers into Oswaldo Cruz), Alberto A. Noronha Dutra (University of HUVEC. Since rearrangements of the actin cytoskeleton London), Patrícia Sampaio Tavares Veras (Fundação into eukaryotic cells are usually associated to the Oswaldo Cruz) activation of PTK, we decided to follow the expression of this class of kinases in the course of the interaction. We previously demonstrated that CBA/J macrophages Unexpectedly, treatment of HUVEC with genistein (Mf) control L. major infection and are permissive to increased both adherence and intracellular viability. L. amazonensis, indicating an important role of Mf on GBS increased tyrosine phosphorylation of two proteins determination of Leishmania infection outcome. It is with an apparent molecular mass of 35 and 23kDa well known that some Leishmania spp. are destroyed in HUVEC.. Mass spectra analysis identified these 1. 176

livro.indd 176 7/7/2008 16:33:47 proteins as annexin V and glutathione S-transferase. the North and Northeast regions. Although the rabies Our findings suggest that actin reorganization of is under control in the urban areas of our country, due microfilaments is important for GBS invasion of HUVEC. to a massive canine vaccination as part of a national GBS seems to co-opt host cells by a mechanism prophylaxis program, the profile of virus transmission in which an activation of PTK is involved. Studies has changed to an airborne cycle. Nowadays, bats are in progress to identify the role of annexin V and are being considered as the main source of infection glutathione S-transferase on GBS-HUVEC interaction. and the annual demand of anti-rabies serum reaches [email protected] 180,000 ampoules per year. The objective of this study was to compare the viral replication and potency in two G-013 cell lines, Vero and BHK-21, which are currently used in STREPTOCOCCUS AGALACTIAE rabies prophylaxis and therapeutics, respectively. The INTERNALIZATION AND PERSISTENCE WITHIN introduction of a filtration step during the preparation of HUMAN EPITHELIAL RESPIRATORY CELLS the seed lot led to an increase in the viral titers assayed Andréia Ferreira Eduardo da Costa, Geórgia Cristina in mice. This simple modofication in the methodology Tavolaro Soares, Prescilla Emy Nagao Ferreira (UERJ) improved the yield of antigen production that might enhance the serum production. Aditional tests are in Streptococcus agalactiae or group B streptococcus progress aiming to compare the interference of that (GBS), mainly serotypes III and V, are a leading cause modification in the viral assays, in vivo and in vitro. of invasive bacterial diseases in neonates, pregnant [email protected] women, and non-pregnant adults with underlying medical conditions. Virulence potential of GBS strains G-015 may determine the outcome of host colonization or ACTIVITY OF HETEROCYCLIC CATIONS ON infection. Because the lung constitutes a first step in TRYPANOSOMA CRUZI GBS systemic invasion processes, we investigated the Anissa Daliry (Fundação Oswaldo Cruz), Marcos adherence and the intracellular viability of GBS strains Meuser (Fundação Oswaldo Cruz), Richard Tidwell to A549 cells, a human alveolar epithelial carcinoma cell (University of North Carolina at Chapel Hill), Maria de line. Interaction of GBS isolates serotype III (90356- Nazaré Correia Soeiro (Fundação Oswaldo Cruz) liquor and 80340-vagina) and serotype V (90186-blood and 88641-vagina) with A549 cells was quantified by Trypanosomes are unicellular protozoan that cause technique of exclusion to penicillin and gentamicin. serious public health problems such as Chagas Interaction of GBS-A549 cells showed that the most Disease in Latin America. Their available therapies are isolates exhibited a more efficient adherence after 6 acknowledged inadequate, justifying the development h incubation (P<0.001). Only 90186-V strain, isolated and screening of new compounds. Trypanosomes from blood, exhibited progressive adherence rate until exhibit biochemical peculiarities, several of which of 8 h incubation. All GBS strains showed intracellular pharmacological importance. They present a single viability during first 4 h incubation. Except for 88641-V mitochondrion genome organized into a network, called vagina isolate that survived only for 6 h, all strains Kinetoplast DNA (kDNA), which is considered a highly tested were found to survive 8 h into epithelial cells. selective target for the design of new antitrypanosomal The GBS 90356-III and 90186-V isolates from patients compounds. In fact, heterocyclic cations are DNA exhibited a higher intracellular survival ability (P≤0.01) binders that display considerable biological activity compared with asymptomatic carriers (80340-III and with low toxicity against several microorganisms. In 88641-V). These results showed that GBS strains our work we aim to determine the mechanisms of serotypes III and V were able to persist viable within the action, selectivity and in vitro and in vivo activity of intracellular environment of human alveolar epithelial heterocyclic cations. Testing on clinically relevant cells, may favor the development of invasive disease. forms of Trypanosoma cruzi (bloodstream forms and [email protected] intracellular amastigotes), we are determining their in vitro tripanocidal activity besides evaluating the G-014 toxicity against mammalian cells, such as embryonic EVALUATION OF A MODIFIED CELL CULTURE cardiomyocytes. Up to now, our results compiled from PROCESSING IN ANTI-RABIES SERUM the activity of 11 different compounds upon bloodstream PRODUCTION trypomastigotes (Y strain) and cardiac cells in vitro Brígida Gomes de Almeida (Puc- Minas), Luciana allowed subdivide these drugs in two major groups Maria Silva (FUNED), Consuelo Latorre Fortes- Dias regarding their respective selectivity index profiles (IS). (FUNED), Sophie Yvette Leclerq (FUNED), Sabrina In the first group we have drugs with IS ranging from 30 Sidney Campolina (FUNED) to 180 and on the second, between <3 up to 6. Further in vitro analyses are under way and the early data Rabies is an important disease that is 100% fatal in the suggest that the first group behave as good candidates affected patients after the symptoms appearance. In for further in vivo analysis. Brazil, 743 cases of humam rabies cases were reported [email protected] between 1986- 2005, the great majority of which in 1. 177

livro.indd 177 7/7/2008 16:33:47 G-016 with different host proteins. On the other hand, the PROINFLAMMATORY CYTOKINES MODULATE mechanisms that may protect GBS from host defenses ADHERENCE, SURVIVAL AND SURFACE PROTEINS remain poorly defined. In the present study, GBS EXPRESSION IN HUMAN ENDOTHELIAL CELLS endocytosis in human umbilical vein endothelial cells INFECTED BY GROUP B STREPTOCOCCUS (HUVECs) after stimulation by tumor necrosis factor- Michelle Hanthequeste Bittencourt dos Santos alpha (TNF-a) and interferon-gamma (INF-g) was (UERJ), Viviane de Oliveira Freitas Lione(UERJ), investigated. HUVEC were treated with TNF-a or INF-g Marcelo Aranha Gardel (Instituto Fernandes Figueira and infected with GBS strains. Fusion of HUVEC - FIOCRUZ, Rio de Janeiro), Maria Aparecida Ferreira lysosomes with bacteria-containing phagocytic vacuoles Tristão (Instituto Fernandes Figueira - FIOCRUZ, Rio de was observed in endothelial cells treated with Lucifer Janeiro), Willian Douglas Santos (Instituto Fernandes yellow as well as by localization of acid phosfhatase Figueira - FIOCRUZ, Rio de Janeiro), Prescilla Emy for all strains. In addition, intracellular Rab7 and Nagao Ferreira (UERJ) LAMP-1 staining were analyzed and quantified by immunofluorescence and confocal laser microscopy, Group B Streptococcus (GBS) is the most important respectively. Lysosome-phagosome fusion was clearly cause of neonatal sepsis, which is mediated in part indicated by presence of many labeled vacuoles that by cytokines. Neonatal sepsis has been associated contained bacteria. Acid phosphatase was detected with increases plasma concentrations of tumor within many but not all phagocytic vacuoles. Following necrosis factor-alfa, (TNF-a), interleukin 1Beta (IL-1B), internalization, the ingested GBS were dischaged interleukin 6 (IL-6), and interferon-gamma (IFN-g). In into RAB-7 and Lamp-1-positive acidic vesicles. this work, we studied the interaction between GBS Interestingly, treatment of HUVEC with INF-g promoted and human umbilical vein endothelial cells (HUVECs) enhancement in the proportion of bacteria-containning after stimulation with cytokines. GBS serotypes III LAMP-1 positive vesicles after 1 h and 2 h incubation. (80340-vagina and 90356-liquor) and V (88641-vagina TNF-a treatment, however, induced increases only after and 90186-blood) were tested for adhesive properties 2 h. GBS is able to survive within acidic compartments with HUVECs untreated or treated with cytokines by endothelial, and cytokines increase phagosome exclusion penicillin and gentamicin method. All GBS maturation time-dependent. Together, these data help strains were able to adhere to HUVECs. The ability of to clarify some aspects of cellular events operanting GBS to enter into HUVECs was observed for 90186 in phagocytosis and pathogeneses of GBS diseases. strain that presented the major rate of invasiveness. [email protected] TNF-a seems to promote surveillance of 80340 isolate within HUVECs. The effects of IL-6 and IL-1B were G-018 restricted to both cytoadhesion and invasion performed GENETIC BACKGROUND AND BASIC EXPRESSION by serotype V in HUVECs. Only treatment of HUVECs LEVEL OF MURINE PERITONEAL MACROPHAGES with IFN-g reduced adherence and inhibited the INFLUENCE THE RESPONSE TO LEISHMANIA invasion for all GBS analyzed. When HUVECs were AMAZONENSIS INFECTION treated with cytokines, a significant alteration on profile Christian Macagnam Probst (Instituto Carlos Chagas/ of surface proteins was observed. The analysis of the FIOCRUZ), Tais Fontoura de Almeida (Instituto de endothelial surface proteins after treatment with TNF-a, Pesquisas Gonçalo Moniz/FIOCRUZ), Ivana Nunes IL-6 or INF-g revealed enhance of expression of five Gomes (Instituto de Pesquisas Gonçalo Moniz/ major molecules, with Mr of ~24-, 27-, 37-, 63-, and 98- FIOCRUZ), Rodrigo Araújo (Instituto de Pesquisas kDa. Cytokines induced alterations on HUVECs surface Gonçalo Moniz/FIOCRUZ), Andréia Cristine Dallabona molecules migth be involved in the adherence, survival (Instituto Carlos Chagas/FIOCRUZ), Luiz Shozo Ozaki and an earlier onset of innate immune responses during (Virginia Commonwealth University), Gregory A. Buck GBS infections. (Virginia Commonwealth University), Daniela Parada [email protected] Pavoni (Instituto Carlos Chagas/FIOCRUZ), Marco Aurélio Krieger (Instituto de Carlos Chagas/FIOCRUZ), G-017 Patrícia Sampaio Tavares Veras (Instituto de Pesquisas INTERFERON-GAMMA AND TUMOR NECROSIS Gonçalo Moniz/FIOCRUZ) FACTOR-ALPHA CONTRIBUTE TO VESICLE MATURATION CONTAINING GROUP B CBA/J mice are highly susceptible to L. amazonensis STREPTOCOCCUS IN ENDOTHELIAL CELLS and C57BL/6 mice once infected with L. amazonensis Viviane de Oliveira Freitas Lione (UERJ and UNIFESP), develop a chronicle disease. In addition, CBA/J Michelle Hanthequeste Bittencourt dos Santos (UERJ), peritoneal macrophages (Mf) present twice the Marcelle Marcolino de França (UERJ), Renato Arruda percentage of L. amazonensis infection compared Mortara (UNIFESP), Prescilla Emy Nagao Ferreira to C57BL/6 Mf. We hypothesized that the mouse (UERJ) genotype and basic expression level of either CBA/J or C57BL/6 Mf influence their response to L. amazonensis Group B Streptococcus (GBS) colonizes and invades infection. DNAmicroarray experiments were used to a number of host compartments, thereby interacting determine CBA/J or C57BL/6 Mf differences in gene 1. 178

livro.indd 178 7/7/2008 16:33:47 expression before and after Leishmania infection. Non- [email protected] infected C57BL/6 Mf differentially expressed 3 times more genes compared to CBA/J cells. After infection, G-020 C57BL/6 Mf modulated 6 times more genes compared TOXOPLASMA GONDII IN SKELETAL MUSCLE to CBA/J cells in response to L. amazonensis. One CELLS IN VITRO: STAGE CONVERSION IN THE important finding was a higher stability in gene ABSENCE OF EXOGENOUS STRESS FACTORS expression of C57BL/6 Mf when compared to CBA/J Marialice Ferreira-da-Silva (Instituto Oswaldo Cruz/ cells. These data could explain the differences in the FIOCRUZ), Helene Santos Barbosa (Instituto Oswaldo number of modulated genes in response to infection Cruz/FIOCRUZ), Uwe Gross (Institute for Medical and justify the limit number of modulated genes in Microbiology, Georg-August-University), Carsten Lüder the CBA/J model and also that the CBA/J genotype (Institute for Medical Microbiology, Georg-August- represents a more complex model than C57BL/6. In University) addition, despite the differences observed between these two mouse models, we were able to identify slight Lifelong persistence of T. gondii may rely on the differences in gene expression by these Mf in response conversion from the proliferative tachyzoite stage into to the same Leishmania specie. Differentially expressed quiescent encysted bradyzoites. Although persistence genes were confirmed by qRT-PCR. In conclusion, within muscle tissue is critical for parasite transmission Leishmania infection produces subtle alterations in to humans via raw or undercooked meat, little attention gene expression and the caos level of gene expression has been paid to skeletal muscle cells (SkMC) as by macrophages can influence our ability to identify a cell type to study Toxoplasma stage conversion modulations induced by Leishmania infection. Support (Guimarães et al., 2008). In the present work, owing to by CNPq/FAPESB. investigate in vitro differentiation of Toxoplasma, SkMC [email protected] primary cultures were maintained in vitro to mature myotubes and the parasite stage conversion analysed G-019 till 6 days post infection. The differentiation started SETTING STANDART OF ANTI-TRYPANOSOMA spontaneously at 1 day post infection (dpi), when CRUZI DRUGS SCREENING METHODOLOGY parasites displayed proteins specific to the bradyzoite Carolina Terumi Tsuchiya, Vanessa Cristina Coimbra, stage by immunoassays (14, 2 ± 0, 8 %). At 2 and 4 Rodrigo Luiz Oliveira Rodrigues Cunha, Luiz Juliano dpi, vacuoles containing bradyzoites and rosettes of Neto, Sergio Schenkman (UNIFESP) Toxoplasma in asynchronous stage conversion were detected. Until 6 dpi, the muscle cells harboured high Most of researchs related to development of Chagas percentages of bradyzoite-containing vacuoles (38, Disease treatment drugs is well-founded in models 6 ± 1, 1 %). In addition, when analysed by real time using Trypanosoma cruzi epimastigotes forms in RT-PCR, total RNA from SkMC contained increasing axenic culture, corresponding a present forms in the amounts of bradyzoite-specific ENO1 transcripts until 6 midgut of triatomine bugs. Only in recent studies dpi. Our data show that high rates of Toxoplasma stage is possible to observe the use of mammalian cells conversion takes place in SkMC, without exogenous infected with trypomastigotes, wich is closer to a real stress factors being required as drugs, changes in infection. However, those assays are too toilsome, temperature or pH. We believe that SkMC might and the amount of substances that can be tested be an appropriate cell model to study the intriguing simultaneously is limited. Inhibitors of cysteine process of tachyzoite to bradyzoite stage conversion of proteases seem to be able to block T. cruzi cellular Toxoplasma in vitro. This work was supported by DAAD, invasion. To establish and set standart of a more CAPES, CNPq, FAPERJ and IOC/FIOCRUZ. efficient assay, the antiparasitary properties of the [email protected] organic tellurium compounds were both tested in cellular invasion and in intracellular proliferation, in L6 G-021 culture cells originated of rat myoblast, since these IDENTIFICATION OF CIRCOVIRUS IN SMALL showed inhibitory activity of T. cruzi cysteine proteases INTESTINE OF ULTRAMARINE-GROSBEAK in low concentrations. After cytotoxicity test by MTT (CYANOCOMPSA BRISSONII) BY TRANSMISSION assay, five non-toxic compounds had been selected ELECTRON MICROSCOPY in concentration until 50uM at 48h of incubation. Two Marcia Helena Braga Catroxo (Instituto Biológico de compounds significantly decreased the trypomastigotes São Paulo/UNIFESP), Nataly Alves Melo (Instituto cellular invasion and four compounds retarded the Biológico de São Paulo), Aline Cristina Oliveira (Instituto intracellular replication with a dose of 5uM. Those Biológico de São Paulo), Liliane Milanelo (Parque results will allow (i) to set a standart of an automatic Ecológico do Tietê), Marcia Maria Rebouças (Instituto intracellular parasites counting methodology, in order to Biológico de São Paulo), Ana Maria Cristina Rabelo facilitate the screening of anti-T. cruzi new drugs; (ii) to Pinto da Fonseca Martins (Instituto Biológico de São verify how effective is the treatment of infected animals Paulo), Selma Petrella (Instituto Adolfo Lutz) administering organotelluric compounds; (iii) to check a possible synergic effect associating those drugs and Avian circoviruses of the genus Circovirus has been benznidazole, applied in Chagas Disease treatment. 1. 179

livro.indd 179 7/7/2008 16:33:47 detected in free-ranging and captive birds worldwide. for health public authorities since these areas could be The transmission occurs through the combined a way for the introduction of arthropod vectors carrying oral, intracloacal and intranasal or faecal routes. different arboviruses potentially pathogenic for humans Avian circovirus infections cause different clinical and also for the dissemination of arbovirus diseases. manifestations ranging from severe chronic feather, [email protected] beak and claw abnormalities to non-specific ill-thrift, lethargy and poor performance, but a common G-023 feature shared is lymphoid depletion and associated ENTOMOLOGICAL STUDY AND ARBOVIRUS immunossupression that exposes the infected SURVEILLANCE IN THE MUNICIPALITIES OF birds to secondary opportunistic infections. Other MARCELÃNDIA, MATO GROSSO STATE, BRAZIL symptoms as enteritis, sinusitis, rhinitis, traqueitis, Elpidia do Socorro de Sousa Costa (Instituto Evandro bronchopneumonia, myocarditis, nephritis, splenitis, Chagas) dyspnoea, anorexia, depression and a very mortality were also related. Virus can be detected in birds that The current work was carried out in the municipality are clinically and histopathologically normal, suggesting of Marcelândia, and in the village of Santa Rita, Mato that many circoviruses infections may be subclinical. Grosso state in 2006 aiming the determination of In the period of the 2006 to 2008, during illegal the hematophagous insect’s fauna, as well as the commercialization of Brazilian birds 29 ultramarine- arbovirus surveillance in the region. The insects were grosbeak were apprehended and being forwarded to captures in the ground and canopy respectively, the Titê Ecological Park. After sudden death the birds and were immediately stored in liquid nitrogen and were sent to EML of Biological Institute of São Paulo, transported to the laboratory for identification and SP, Brazil, to investigate viral agents. During necropsy separation in different pools. For virus isolation one bird show hemorrhage in the intestines. Fragments attempt, polls of insects were inoculated in newborn of small intestine were processed for transmission mice and C6/36 cells. A total of 6.821 insects were electron microscopy by negative staining and a great identified. Of these, 62,5% were classified as number of the circovirus particles, non-enveloped, members of the family Culicidae 33%, which were spherical, isometric, characterized as “complete” and grouped in 219 pools. Importantly, were captured “empty”, measuring between 17 and 20 nm of diameter mosquitoes from the genera Haemagogus and was visualized. This is the first report of circovirus in Sabethes (vectors for the transmission of silvatic Cyanocompsa brissonii. yellow fever), Ochlerotatus (secondary vector for [email protected] the yellow fever), the phlebotomines associated with the transmission of arboviruses as well as with the G-022 cutaneous leishmaniasis. In addition a the Culicoides ISOLATION OF VIRUS DEN IN C6/36 CELLS FROM paraensis (Ceratopognidae family) a midge related to LOTS OF MOSQUITOES CAPTURED IN PORT the transmission of the Oropouche virus in Amazonian AREAS OF BELÉM , SÃO LUIS AND MANAUS, urban areas, was also captured. Only one arbovirus BRAZIL was isolated from a pool of Culex (Mel.) pedroi (AR Francisco Correa Castro (Instituto Evandro Chagas) 718935) and was serologically identified as a group A virus (Alphavirus) by cross reactivity with a group A The arboviruses are transmitted by arthropods and polyclonal antibody by the CF test. No specific reactions constitute the largest group of viruses with a worldwide were observed with any know alphavirus which geographic distribution mainly in tropical and subtropical circulates in the Amazon region. Further serological and areas. Mosquitoes of the family Culicidae, as well as molecular studies are under progress for the complete other hematophagous arthropods (mainly ticks) are antigenic and genetic characterization of this alphavirus. of great interest on arbovirology studies, since it is [email protected] associated to the natural maintenance cycles of these viruses. Thus, the identification of current and possible G-024 new vectors for arboviruses should be helpful for the ENZOOTIC CIRCULATION OF EASTERN EQUINE establishment of efficient control measure of arbovirus ENCEPHALITIS VIRUS IN PORTEL, AMAZON diseases. In the current study, a total of 261 polls of REGION, BRAZIL mosquitoes captured in three different Brazilian ports Jonas Raimundo dos Santos Moraes (Instituto Evandro located in the cities of Belém, São Luiz and Manaus Chagas) were used for virus isolation attempt in C6/36 cells. Two polls of mosquitoes were positive for dengue virus type INTRODUCTION: Entomologic studies were developed 2 (DENV-2). Our findings corroborates previous data, in the municipality of Portel, Pará state, between 24 since all the three serotypes of DENV (DENV-1, DEN-2 March and 2 April of 2004 (10 days). MATERIAL and and DENV-3) have been detected in samples (blood or METHODS: Insects were collected at ground level serum) collected from patients residents in Belém and and at forest canopy at 12-15 meters between 9:00 - clinically suspected of dengue infection. Finally, periodic 14:30. RESULTS: A total of 3,995 haematophagous surveillance in different Brazilian ports is of importance insects were captured belonging to forty-four Culicidae 1. 180

livro.indd 180 7/7/2008 16:33:48 species 74.5% of them at ground and 25.5% in MUNICIPALITY OF PASTOS BONS/MARANHÃO- the canopy that represented 30 and 77 lots for STATE/BRAZIL inoculation, respectively. The most prevalent species Ercilia de Jesus Gonçalves (Instituto Evandro Chagas) were Haemagogus leucocelaenus 545 (13.9%), Hg. janthinomys 521 (13%), Ochlerotatus argyrothorax 435 The area for investigation is located in “Saco Seco” (11%), Oc. serratus 396 (10%), Sabethes chloropterus in Pastos Bons municipality State of Maranhão. 298 (8%), Wyeomyia sp. 297 (7%), Oc. fulvus 294 Mosquitoes captured, identified and conserved in liquid (7%), Psorophora albipes 258 (6%), Trichoprosopon nitrogen until identification in the Lab. of Entomology, digitatum 150 (4%) and Limatus durhamii 137 (3%). afterwards were pooled by species, dates, and All mosquito pools were inoculated intracerebrally inoculated for attempts of virus isolation. In 1994 into newborn mice. Three strains of Easten Equine were identified 3118 Culicid belonging in 9 genera Encephalitis virus (EEEV) were isolated, two from lots and 19 species. The most prevalent species was Hg. of Culex (Mel.) sp. and another from Coquillettidia janthinomys with 79%, followed by Sa. chloropterus arribalzagae. CONCLUSIONS: This is the first time 12%. In 2005 were obtained 169 mosquitoes belonging that EEEV was isolated from mosquitoes of genus in 8 genera and 18 species. The most prevalent species Coquilletidia and is the first report of this virus in Portel, were Hg. janthinomys 22%, Sa. chloropterus 15%, Wy. Marajó Island. Further studies at molecular level must sp. 20%), and Li. durhamii 13%. The recurrent species be performed in order to characterize strains isolated. 2005 (1994) they were Oc. scapularis 2(2), Oc. species [email protected] 2(1), Hg. janthinomys 37(2451), Psorophora ferox 1(2), Culex declarator 7(3), Cq. venezuelensis 2(11), Li. G-025 durhamii 22 (12), Sa. albiprivus 11(61), Sa. belisarioi ENTOMOLOGICAL STUDY AND ARBOVIRUS 1(14), Sa. chloropterus 25 (368), Sa. glaucodaemon SURVEILLANCE IN THREE MUNICIPALITIES OF 8(112) and Wy. species 34(29). A strain of Tucunduba THE MATO GROSSO STATE, BRAZIL, 2008 virus was isolated from a pool of Wyeomyia sp. colleted Maria de Nazaré de Oliveira Segura (Instituto Evandro in 2005. No virus strain was isolated from mosquito Chagas) captured in 1994. The study developed Pastos Bons showed a large variety of Culicidae with an important The anthropic actions have been direct affected the presence of Hg. janthinomys the main vector of yellow region causing different ecological impacts, such as fever virus and general other arboviruses such or the emerging and reemerging of different arboviruses, Mayaro virus, Tacaiuma and others potential vectors of which have been associated with human diseases arbovirus were else obtained. A remarkable found was (Yellow fever virus, Oropouche virus, Mayaro virus). the isolation of a Tucunduba virus strain from Wyeomyia The current was carried out in order to determine the mosquito. This virus is frequently isolated of these entomological fauna (hematophagous insects), as mosquitoes and has been responsible for a encephalitis well as the arbovirus surveillance in the region. The case in Belém. work was conducted in three different municipalities [email protected] (Anastásio, Aquidauana, and Águas Claras) in January of 2008. The insects were captures in the ground and G-027 canopy, respectively, and were immediately stored in OCCURRENCE OF AVIANPOXVIRUS EM liquid nitrogen and transported to the laboratory for SALTADOR SIMILIS BY TRANSMISSION ELECTRON identification and separation in different pools. For virus MICROSCOPY isolation attempt, polls of insects were inoculated in Aline Cristina Oliveira (Instituto Biológico de São Paulo), newborn mice and C6/36 cells. Only one arbovirus was Nataly Alves Melo (Instituto Biológico de São Paulo), isolated from a pool of Psorophora ferox AR 742353 Liliane Milanelo (Parque Ecológico do Tietê), Marcia in the municipality of Anastasia and was serologically Maria Rebouças (Instituto Biológico de São Paulo), identified as a member of the Bunyawera group Ana Maria Cristina Rabelo Pinto da Fonseca Martins polyclonal antibody by the CF test. No specific reactions (Instituto Biológico de São Paulo), Selma Petrella were observed with any know Bunyamwera virus which (Instituto Adolfo Lutz), Marcia Helena Braga Catroxo circulates in the Amazon region. The knowledgement (Instituto Biológico de São Paulo) of the entomological fauna is important for further ecoepidemiological studies, and also to provide a better The poxviruses which infect birds belong to the understanding regarding the dynamics of arbovirus genus Avianpoxvirus of the Poxviridae family. The emerging and reemerging in the area. Finally, additional transmissible disease is spread by biting arthropods, studies on serology and molecular biology are under aerosols generated from infected brids, or the progress for the complete antigenic and genetic ingestion of contamined food or water. The disease is characterization of this possible new bunyawera virus. characterized by three forms: cutaneous, diphtheroid [email protected] or septicemic. The cutaneous form or dry pox is represented by nodular, proliferative skin lesions on G-026 the unfeathered parts of the body: legs, feet, base ENTOMOLOGIC FAUNA OF CULICIDAE IN THE of the beak and eyelids. In the diphtheroid form the 1. 181

livro.indd 181 7/7/2008 16:33:48 lesions that occur on the mucosa of the tongue, Bollinger, sorrounded by membrane, constituted by pharynx and larynx cause fibrinous lesions. An acute filamentous material that giving arise to immature of ruffled plumage, somnolence, cyanosis, anorexia virions; inclusion body B where mature virus particles and high mortality characterize the septicemic form. budded into extensive groupings and fibril inclusion. In the period of the 2006 to 2008, during illegal Viral antigenic sites were labeled with specific antibody commercialization of Brazilian birds, 49 Saltator similis followed by colloidal gold conjugated with protein A. were apprehended and being forwarded to the Tietê [email protected] Ecological Park. After sudden death the birds were sent to EML of Biological Institute of São Paulo, SP, Brazil, to investigate viral agents. Eight birds presented cutaneous lesions around to the beak and legs. During necropsy were removed fragments of skin lesions which were processed by negative staining technique. On the transmission electron microscopy a great number of the poxvirus particles, some enveloped in a brick-shaped and irregular disposition of tubules on the external membrane measuring 320 x 215 nm on the average were visualized. This is the first report of Avianpoxvirus in Saltator similis. The technique utilized is greatly effective for a rapid diagnosis of this important disease. [email protected]

G-028 ISOLATION OF POXVIRUS IN CUTANEOUS LESIONS FO THE COWLED-CARDINAL (PAROARIA DOMINICANA) BY TRANSMISSION ELECTRON MICROSCOPY TECHNIQUES Nataly Alves Melo (Instituto Biológico de São Paulo), Liliane Milanelo (Parque Ecológico do Tietê), Marcia Maria Rebouças (Instituto Biológico), Aline Cristina Oliveira (Instituto Biológico de São Paulo), Ana Maria Cristina Rabello Pinto da Fonseca Martins (Instituto Biológico de São Paulo), Selma Petrella (Instituto Adolfo Lutz), Marcia Helena Braga Catroxo (Instituto Biológico de São Paulo)

Poxviruses members of the Poxviridae family and genus Avianpoxvirus, cause a variety of diseases in birds. Poxvirus infection occurs in 3 forms: the cutaneous form is characterized by nodular proliferative lesions in the eyelids, beak, legs, and feet. In the diphtheric form fibronecrotic lesions occur in the membranes of the oropharynx, upper respiratory tract ans esophagus, causing high mortality. In the septicemic form occur ruffled plumage, somnolence, cyanosis, anorexia and death. Transmission is by direct and indirect contact or through mechanical transfer by biting insets. During illegal commercialization of Brazilian birds in january 2006 to april 2008, 86 cowled-cardinal were apprehended and being forwarded to the Tietê Ecological Park. After sudden death the birds were sent to EML of Biological Institute of São Paulo, SP, Brazil, to investigate viral agentes. 28 birds presented cutaneous nodular lesions around the beak, eyelids and legs. Fragments of lesions were processed for TEM using resin embedding and immunocitochemistry techniques. Ultrathin sections revealed the presence of oval particles where a biconcave nucleus and two lateral bodies could be observed. Three types of cytoplasmic inclusion bodies are visualized: inclusion body A or 1. 182

livro.indd 182 7/7/2008 16:33:48 Immune System (UFRJ) Galectin-3 (gal-3) has been reported to regulate the differentiation of B lymphocytes into plasma cells in H-001 secondary lymphoid organs. The aim of this work was EXPRESSION OF EXTRACELLULAR MATRIX to investigate if B lymphopoiesis in the bone marrow LIGANDS AND RECEPTORS IN THYMIC of gal-3 knockout mice (Gal-3-/-) was altered. The ENDOTHELIAL CELLS AND THEIR ROLE IN THE steps of B cell development was analyzed by flow THYMOCYTE MIGRATION cytometry, using specific markers: B220, c-Kit, CD19 Andréa Aragão Francelino (Laboratory of and IL-7R. The number of B precursors was quantified immunohistology, Federal University of Alagoas/ in methyl cellulose culture system. The interaction of B CESMAC), Salete Smaniotto (Laboratory of progenitors and stromal cells was done by co-culture Immunohistology, Federal University of Alagoas), systtem, using mononuclear cells from Gal-3-/- and Fernando Wagner da Silva Ramos (Laboratory of control mice and the stromal cell line OP9. The Immunohistology, Federal University of Alagoas/ expression of IL-7 was evaluated by Real-Time PCR CESMAC), Theresinha Carvalho Calado (Laboratory in stromal cells from the bone marrow of Gal-3-/- and of Immunohistology, Federal University of Alagoas), control mice maintened in vitro for 7 and 14 days. We Wilson Savino (Laboratory on Thymus Research, described a significant increase in B cell precursors Oswaldo Cruz Institute,Oswaldo Cruz Foundation, Rio in the bone marrow of the Gal-3-/- mice, both in vivo de Janeiro, Brazi), Silvana Ayres Martins (Laboratoty of and in vitro, compared with the control mice. The Immunohistology, Federal University of Alagoas) presence of gal-3 in the stromal cell line OP9 was not sufficient to reverse the increase on B cell precursors The thymus provides a variety of specialized in the co-cultures with Gal-3-/- mononuclear cells. The microenvironments that supports yhe migration, expression of IL-7 in stromal cells from Gal-3-/- mice is proliferation and differentiation of T-cell.The extracellular diminished, compared with the expression of the control matrix(ECM) is distributed heterogeneously within cells. The increase in B cell precursors in the absence the thymic parenchyma, being produced by distinct of gal-3 was not, probably, due to IL-7, and that the microenvironmental cells, such as various thymic presence of gal-3 in stromal cell did not rescue the epithelial cells, fibroblasts and phagocytic cells. It is increase of B precursors in vitro. Others mechanisms known that selected populations of lymphoid cells may be associated with the absence of gal-3 in the migrate into andfrom of the thymus through blood regulation of the B cell compartment in the bone vessels st the cortico-medullary junction and in the marrow, such as Wnt/b-catenin signalling. medulla. The production of ECM glycoproteins an [email protected] expression of ECM receptors, as well as the role of these molecules and their interaction with the H-003 thymocytes, was evaluated in a thymic endothelial EFFECTS OF GROWTH HORMONE ON cell line, tEnd.1. We first showed the production of MACROPHAGES ECM glycoproteins fibronectin and laminin, and the Maria Danielma dos Santos Reis, Maíra Estanislau expression of their respectivereceptors VLA5 and Saores de Almeida, Adalberto Alves Martins Neto, VLA6 by immunofluorescence, immunoperoxidase Theresinha Carvalho Calado, Salete Smaniotto and cytofluorimetry in cultured tEnd.1 cells. The use of (Universidade Federal de Alagoas) antibodies anti-VLA5 and anti-VLA6 partially inhibited the adhesion between thymocites/tEnd.1. Lastly, we Growth hormone (GH) play a physiological role in showed transendothelial migration of thymocites in immune system. Studies were performed to determine transwell migration chambers was lower whem the the effects of GH on macrophages (mo) in vivo and thymocites werw pre-treated with anti-VLA5 and anti- in vitro. For in vivo studies, spleen from 2-3 months- VLA6. Our data demonstrated that the tEnd.1 cells are old GH-transgenic mice (GH-Tg) C57BL/6 and able to producefibronectin and laminin, and express corresponding C57BL/6 wild-type mice were used. their respective receptors VLA5 and VLA6, and it’s These organs were fixed in formalin and processed suggested the participation oft these molecules in the according to routine protocols. After staining with processes of transendothelial migration. HE or toluidine blue/eosin tissue sections were [email protected] examined to analyze the mo. Some specimens were frozen and cryostat sections were subjected to H-002 immunofluorescence. Primary cultures of peritoneal mo GALECTIN-3 INTERFERES ON B CELL harvested from 3-4 months-old Swiss mice following COMPARTMENT IN THE BONE MARROW 200 or 400 ng/ml GH-tratament for 24 h, were also Lauremília Ricon Gomes Rodrigues da Costa (UFRJ), used. These cells were immunostained with laminin Felipe Leite de Oliveira (UFRJ), Marise Fermino Lopes (LN) and fibronectin (FN) specific antibodies. The (USP/Ribeirão Preto), Eme rson Soares Bernardes proliferation assays were carried out with cultured (USP/Ribeirão Preto), Roger Chammas (USP), J-774 mo cell line treated or not with GH 100, 200 or Radovan Borojevic (UFRJ), Márcia Cury El-Cheikh 400 ng/ml for 24 or 48 h. It was observed under opitical 1. 183

livro.indd 183 7/7/2008 16:33:48 microscopy an increase in mo numbers (2,90+-0,26) of the spleens from GH-Tg mice when compared with Ascidians occupy a phylogenetic position immediately controls (1,80+-0,19). It was also noticed that high before vertebrates. They belong to the phylum levels of circulating GH increase the deposition of FN Chordata, sub-phylum Urochordata. These primitive and LN in the splenic microenvironment of the white and chordates have been used as models for understanding red pulp. In contrast, the GH decreased the production ancient immunological mechanisms through the study of FN and LN by peritoneal mo. However, we did not of individual cells, such as hemocytes. Nitric oxide (NO) observe signifcant changes in the proliferation of J-774 is a widespread intracellular and intercellular signaling GH-treated when compared with controls. Our studies molecule in mammals with a variety of functions in show that GH may exert modulatory effects on immnue the nervous, cardiovascular and immune systems. In system, increasing mo number and ECM production ascidians, the production of NO was reported in the in spleen and inhibiting ECM production in GH-treated muscle cells of ascidian larvae tail. In this work we peritoneal mo. intended to investigate the presence of NO synthase [email protected] (NOS) and the production of NO by the hemocytes of the ascidian Styela plicata in response to the stimulation H-004 by LPS and heterologous serum. In addition, we THE HEMOCYTES FROM ASCIDIAN STYELA intended to look for the morphology of the hemocytes PLICATA (CHORDATA-TUNICATA) CONTAINS that produced NO. The hemocytes were isolated and HEPARIN PROTEOGYCAN AND HISTAMINE INSIDE prepared for the NADPH-diaphorase cytochemistry BASOPHIL-LIKE CELLS that revealed the presence of NOS. Confirming this Cintia Monteiro de Barros, Silvana Allodi, Mauro Sérgio result, the immunoelectronmicroscopy showed that only Gonçalves Pavão (UFRJ) one type of hemocytes was labeled with anti-iNOS, the lymphocyte-like cell. After, to verify which stimulus Ascidians (Subphylum Urochordata) are sessile marine has the capacity to induce the NO production by these invertebrates and occupy a peculiar phylogenetic hemocytes, we incubated theses cells with LPS from position forerunner of vertebrates. They probably share E.coli and heterologous serum and then we measured immunological characteristics with both vertebrates the amounts of nitrite and nitrate, the sub-products of and invertebrates. The hemolymph of ascidians NO. In conclusion, we found that lymphocyte-like cells contains different types of circulating blood cells. In express NOS but these cells do not respond to LPS and mammals, heparin is synthesized on a specific protein heterologuous serum stimuli. core, forming the serglycin proteoglycan (PGs). [email protected] These PGs occur in secretory granules of some immunologic cells, such as mast cells ans basophils. H-006 Considering the phylogenetic position of ascidians IMMUNE RESPONSE AGAINST EHEC O157:H7 and taking account that a heparin-containing cell, AFTER ORAL IMMUNIZATION WITH A similar to mammalian mast cells, was detected in RECOMBINANT SALMONELLA VACCINE STRAIN the tissues of S.plicata, it is possible that a basophil- EXPRESSING THE INTIMIN GAMMA like cell containing intracellular glycosaminoglycans Fausto Bruno dos Reis Almeida (USP), Marcelo Brocchi (GAGs) circulates in the hemolymph of this animal. To (Universidade Estadual de Campinas), Maria Cristina investigate this hypothesis, we classified the hemocytes Roque-Barreira (USP), Sandro Gomes Soares (USP; by electronmicroscopy. After, the hemocytes were Invent Biotecnologia Ltda), Leandro Licursi de Oliveira submitted to proteolytic digestion and the GAGs were (USP), Aline Ferreira de Oliveira (USP), André Pitondo analyzed by agarose gel electrophoresis. The PGs da Silva (USP), Antonio Fernando Pestana de Castro inside hemocytes were extracted by 4M of guanidine (USP), Ebert Seixas Hanna (USP; Invent Biotecnologia hydrochloride and analyzed by polyacrylamide gel. In Ltda; Universidade Estadual de Londrina) addition, immunocytochemical studies were carried out to determinate which hemocytes express heparin and Enterohaemorrhagic Escherichia coli (EHEC) has been histamine. Theses results showed that a heparin was described as an emerging human pathogen which found in a PG form co-localized with histamine in only adheres and colonizes the intestinal epithelium causing one type of the cell, the granulocyte. bloody diarrhoea. Cattle are most frequently identified [email protected] as the primary source of infection, and its vaccination represents an attractive strategy for reducing the H-005 incidence of human disease. E. coli serotype O157:H7 DETECTION OF NITRIC OXIDE IN HEMOCYTES expresses intimin gamma (Intg), one type of adhesin OF THE ASCIDIAN STYELA PLICATA (CHORDATA- associated with attaching and effacing (A/E) lesions on TUNICATA): DOES IT HELP TO BETTER enterocyte. In this work, we assessed the ability of the UNDERSTAND THEIR INNATE IMMUNITY? attenuated strain of S. enterica expressing the Intg to Cintia Monteiro de Barros, Danielle Ronald de Carvalho, induce mucosal as well as systemic immunity against Leonardo Rodrigues de Andrade, Mauro Sérgio EHEC O157:H7. In this way, the gene encoding the Gonçalves Pavão, Silvana Allodi (UFRJ) Intg was cloned into the asd-containg plasmid pYA3137 1. 184

livro.indd 184 7/7/2008 16:33:48 and electroporated into the c3987 strain of Salmonella type (WT) and null mice for ANXA1 (ANXA1null) asd. Immune responses were evaluated by detection received i.p. injection of zymosan (1.0 mg) and were of specific antibodies in serum and fecal samples. This sacrificed after 0, 4, 24 and 36h. Pharmacological recombinant strain may be a useful component in an treatment was performed with peptide Ac2-26 (ANXA1 oral vaccine to reduce E. coli O157:H7 prevalence in N-terminal; 100 mg) before zymosan administration. cattle. [email protected] Mesentery and blood were processed for light and electron microscopy analysis. Expressions of ANXA1, H-007 CD11b and CD62L were monitored in neutrophils GROWTH HORMONE MODULATES (PMN) and endothelial cells (EC) of tissues by TRANSENDOTHELIAL MIGRATION OF immunocytochemical analysis. Zymosan peritonitis THYMOCYTES provoked mesentery inflammation with a significant Adalberto Alves Martins Neto (Universidade Federal de increase in intravascular PMN at 4h, and transmigrated Alagoas), Silvana Ayres Martins (Universiade Federal PMN at 24h in ANXA1null mice compared to WT. de Alagoas), Wilson Savino (Instituto Oswaldo Cruz- At 36h, few PMNs were observed in mesenteries. Fiocruz/RJ), Salete Smanitto (Universidade Federal de Pharmacological treatment with peptide produced a Alagoas) significant reduction of PMN recruitment at 4h. In this early phase, immunogold staining showed an increase It is known that growth hormone (GH) modulates of ANXA1 expression in transmigrated PMN and its intrathymic T cell migration, and that extracellular matrix col-localization with CD11b and CD62L in plasma and chemokines may act in combination during this membrane of PMN and EC. In the ANXA1null mice, process. Herein, we evaluated the effect of GH on the the lack of ANXA1 resulted in a diminished expression transendothelial migration of thymocytes. We applied of CD11b in PMN and EC during the transmigration the thymic endothelial cell line (tEnd.1) and thymocytes process in both genotypes of mice that was not from C57BL/6 mice aged 4-6 weeks.Transendothelial observed for CD62L. Our results suggest an important thymocyte migration was performed in transwell culture mechanism of anti-inflammatory action of ANXA1 inserts after treatment of tEnd.1 and/or thymocytes with through modulation of CD11b on PMN recruitment, GH [100 ng/ml]. Thymocytes were allowed to migrate and might have an impact on the development of new for 18 h, and migrating cells were harvested, counted, strategies for inflammatory diseases. and phenotyped by flow cytometry. When the tEnd.1 [email protected] alone were GH for 8 h, we observed a significant reduction in the absolute numbers of migrating H-009 thymocytes, as compared to untreated controls. This THE ANTI-INFLAMMATORY PROTEINS decline was mainly seen in CD4+CD8+ cells. Differently, ANNEXIN-A1 AND GALECTIN-1: EFFECT OF when we treated thymocytes with GH for 1 h, or when PHARMACOLOGICAL TREATMENT ON INNATE thymocytes and tEnd.1 were treated with the hormone IMMUNE CELLS DURING MOUSE PERITONITIS (for 1 and 8 h, respectively) the numbers of migrating Caio Eduardo Gullo (FAMERP), Cássia Fernanda cells increased significantly as compared with the GH Estofolete (FAMERP), Sonia Maria Oliani (IBILCE- treatment of tEnd.1 alone. The relative number of CD4+ UNESP), Cristiane Damas Gil (FAMERP) migrating cells increased significantly after the treatment of thymocytes or thymocytes and tEnd.1 with GH, as In this study we investigated the effect of exogenous compared with those migration chambers in which only annexin-A1 (ANXA1) and galectin-1 (Gal-1) on the tEnd.1 were treated with the hormone. As regards acute inflammatory process focusing mast cell (MC), expression of VLA-4 and CXCR4 on the total thymic cell leukocyte (PMN) and monocytes that represents the population, as well as in migrating thymocyte subsets, first line of host defense. Pharmacological treatment we did not find significant differences among the was performed in C57BL/6 mice that received experimental groups. Our results reinforce the idea that intraperitoneal injection of peptide Ac2-26 (ANXA1 GH is relevant in thymus physiology. N-terminal; 100 mg) or human recombinant Gal-1 (0.3 [email protected] mg) 10 min before zymosan administration (1.0 mg) and were sacrificed after 0, 4, and 24h. Blood and peritoneal H-008 fluids were collected and processed for statistical THE MECHANISM OF ANTI-INFLAMMATORY analysis of PMN and monocytes at light microscopy. ACTION OF ANNEXIN-A1: MODULATION OF Flat-mount preparations of mesenteries were performed ADHESION MOLECULES DURING LEUKOCYTE to PMN and MCs morphological analysis. Zymosan TRANSMIGRATION administration produced visible signs of inflammation Cristiane Damas Gil (FAMERP), Sonia Maria Oliani at 4h with high number of PMN (blood, peritoneal fluid (IBILCE-UNESP) and mesentery) and mesentery degranulated MCs. At 24h, all tissues analyzed demonstrated a phase of In this study we focused the effect of annexin-A1 resolution of inflammation with downregulation of these (ANXA1) on expression of integrin (CD11b) and cell types. Pharmacological treatment with proteins L-selectin (CD62L) during leukocyte migration. Wild- produced a significant reduction of PMN recruitment 1. 185

livro.indd 185 7/7/2008 16:33:48 (4h), but provoked an increase in peritoneal monocytes Barreira (Depto de Biologia Celular e Molecular e (4 and 24h). MC population was upregulated by ANXA1 Bioagentes Patogênicos, Faculdade de Medicina administration at 4h, but not by Gal-1. These results de Ribeirão Preto), Gabriela Silva Bisson (Depto de suggest an important role of ANXA1 and Gal-1 in Enfermagem Materno-Infantil e Saúde Pública, Escola orchestrating inflammatory response through regulation de Enfermagem de Ribeirão Preto) of MC activation, PMN and monocyte migration to tissues, and indicate these proteins as potent anti- Galectin-3 (Gal-3) is probably the most studied inflammatory drugs to future clinical application. member of the galectin family. This lectin is composed [email protected] of a C-terminal carbohydrate recognition domain, which is responsible for the specific recognition of H-010 beta-galactosides, and an N-terminal domain. Due to INFLUENCE OF PHYSICAL EXERCISE IN IMMUNE its capacity to compose dimers through the amino- RESPONSE OF RATS SUBJECTED TO THE terminal nonlectin domain, it has the potential to RESTRAINT CHRONIC STRESS cross-link appropriate cell surface glycoproteins, Juliana Malta, Rodolfo Duarte Nascimento, David which gives Gal-3 the ability to mediate cell-cell and Teixeira Guilherme Pereira, Guilherme José Pereira, cell-extracellular matrix interactions. Also, Gal-3 Rodrigo José Carraro Sobrinho, Rosane Gomes de promotes the adhesion of neutrophils to laminin and Oliveira, Lamara Laguárdia Valente Rocha (Centro activates various types of cells, including monocytes/ Universitário de Caratinga) macrophages mast cells, neutrophils and lymphocytes. In this study we confirmed the interaction between gal-3 Was evaluated the benefit of physical activity in the and the surface of human monocytes and investigated immune response of rats wistar adults, submitted to the identity of gal-3 possible ligands involved in the restraint chronic stress. It was used 30 animals of such interaction. By using confocal microscopy we both sexes, divided in groups - I: sedentary and not demonstrated that rGal-3 interacts with the surface of stressed, II: trained and not stressed, III: trained in the monocytes through its C-terminal domain since this morning and stressed in the afternoon; IV: stressed in interaction is inhibited by lactose. Using western blot the morning and trained in the afternoon V: stressed analysis of cell lysates,we identified candidates for and not trained. The rats was subject to swimming gal-3 receptors on the monocyte membrane and in the protocol without load, one hour a day, five days a cytoplasm. The correspondent bands in SDS-PAGE week during the period of six weeks. Last week, the were sent for protein sequencing. claudiadp@hotmail. groups III, IV and V were placed in glass containers com with to the restraint chronic stress. After sacrifice with a lethal dose of anesthetic, 5mL of blood were collected H-012 for leukocytes total and differential counting. It also SIGNALING PATHWAYS INDUCED BY LECTIN analyzed changes in weight, which showed significant MNCF ON NEUTROPHILS DURING DEGRALUTION differences between the group III (2,46 + ou - 21,22) PROCESS and I (3,28 + ou - 1,14), II (5,27 + ou - 10,59) e V (0,83 Karina Alves de Toledo (INSERM U845, Necker + ou - 20,54) groups. These differences must involve Hospital, Paris, France), CLAUDIA DANELLA POLLI change in the levels of corticosterone and the greater (Depto de Biologia Celular e Molecular e Bioagentes activation of the lipolytic track influenced by circadian Patogênicos, Faculdade de Medicina de Ribeirão cycle. Leukocytes total and differential counting Preto), Maria Cristina Roque-Barreira (Depto de obtained are significant differences only for the number Biologia Celular e Molecular e Bioagentes Patogênicos, of monocytes and neutrophils of V (216 + ou - 52,13 Faculdade de Medicina de Ribeirão Preto), Lise and 768,16 + ou - 132,82) and III (340 + ou - 132,04 Halbwachs-Mecarelli (INSERM U845, Necker Hospital, and 1488,16 + ou - 706,75) groups. In group III, light Paris, France) physical training shows beneficial effect in reversing the immunosuppression induced by the restraint chronic MNCF, a galactose-binding lectin, has been described stress, stimulating the innate immune response. as an agonist and chemoattractant for neutrophils, both [email protected] in vivo and in vitro, and resistant to dexamethasone actions which awakes great interest in understanding H-011 the mechanism of action on polymorphonuclears by this GALECTIN-3 BINDS TO HUMAN MONOCYTES lectin. Previous results suggest that the maintenance PROTEIN of cytokines and chemokines, induced by MNCF, in CLAUDIA DANELLA POLLI (Depto de Biologia Celular the inflammatory environment, could explain, even e Molecular e Bioagentes Patogênicos, Faculdade de partially, the break in the resistance to glucocorticoids Medicina de Ribeirão Preto), Karina Alves de Toledo actions by this lectin. To better study the neutrophil (INSERM U845, Necker Hospital, Paris, France), activation induced by MNCF we analyzed the neutrophil Emerson Soares Bernardes (Depto de Biologia Celular degranulation evaluating their surface markers such e Molecular e Bioagentes Patogênicos, Faculdade de as CD11b, CD66b, CD35 and CD63. By using flow Medicina de Ribeirão Preto), Maria Cristina Roque- cytometry we demonstrated that MNCF degranulates 1. 186

livro.indd 186 7/7/2008 16:33:49 secretory vesicles and secondary granules, but not H-014 azurophilic granules. We also used the inhibitory drugs NOVEL PATHWAYS FOR ANNEXIN 1 EFFECTS IN to tyrosine kinases (genistein), MAP kinases (PD98059 RHEUMATOID ARTHRITIS and SB203580), Src (PP2) and PKC (staurosporin) Kallyne Kioko Mimura (USP, IBILCE, Department of during the degranulation assays. All drugs were able Biology, São José do Rio Preto, SP.), Tatiana Aparecida to inhibit the increase of CD11b expression induced by Pimentel (UNIFESP, UNIFESP-EPM, Post-Graduation MNCF, except staurosporin, indicating that the signaling in Morphology, São Paulo, SP.), Sonia Maria Oliani induced by MNCF does not cross PKC. Finally, we (USP, IBILCE, Dept of Biology / Post-Graduation in demonstrated by Western blot analysis that MNCF Morphology, UNIFESP-EPM, SP.) induces tyrosine and MAP kinases phosphorylation, through NFkB translocation. All results here described Annexin 1 (ANXA1) is an anti-inflammatory protein are independent of neutrophil priming induced by LPS. that plays a key role in innate immunity by modulating Together, these results confirm the agonist activity of several types of cells. This study investigated the MNCF on neutrophils and add information concerning role of ANXA1 in a collagen induced arthritis (CIA) the pathway signaling induced by this lectin, which can model. Bovine type II collagen was injected on day be used to further investigations. claudiadp@hotmail. 0 and human recombinant ANXA1 (hrANXA1) were com administered daily from day 22 to 32. Disease was assessed on days 32 and 42 by histologic examination H-013 of the knee joints. Analysis of aspects of CIA revealed SURVIVAL AND ACTIN AND FIBRONECTIN THYMIC a significant exacerbation of inflammation in the EXPRESSIONS, IN SEPSIS TREATED WITH THYMUS hrANXA1 treated mice compared with non-treated CULTURE SUPERNATANT (NT). However, our results showed that degradation of Miguel Ferreira Mouta Junior (HC- FMUSP), Regina collagen fibers occurred under pathological conditions Maria Catarino (INSTITUTO ADOLFO LUTZ-SP), Luíz (NT) and the birefringence showed a different pattern Vicente Rizzo (HC - FMUSP), Kleber Espirito Santo in comparison with that presented by the collagen Simões (INSTITUTO ADOLFO LUTZ - SP) molecules in hrANXA1 treated tissues (day 32). Administration of hrANXA1 significantly reduced the OBJECTIVES: Sepsis causes lymphoid apoptosis, number of neutrophils in the knee synovial tissues (day related to the increase in transglutaminase, greater 32) and in the joint space even 10 days after the end expression of interstitial actin and decrease in of treatment. Mast cells degranulation was reduced plasmatic fibronectin. We have studied the mortality and the major cells were intact after treatment (day 32) and the expression of interstitial actin and fibronectin and on day 42, in relation to NT. Our studies indicated through the immunohistochemical technique in that the hrANXA1 exerted inhibition of mast cells adult Wistar rats with sepsis by cecal ligature with degranulation and neutrophils transmigration. Besides puncture, treated with supernatant from the culture of the anti-inflammatory effects no significant reduction thymuses from healthy newly born rats. Procedures: was observed in the features of arthritis. This might Groups (n=20 rats/group): 1-Control: Sepsis by cecal indicate that other leukocyte types are participating in ligature with puncture, according to Wichterman the pathogenesis of arthritis. Further studies will provide (1980). 2-Sepsis treated with culture medium only. better understanding which may help us in the quest for 3- Sepsis treated with culture supernate. Thymus of new drugs to treat inflammatory diseases. newly born was cultivated in DMEM without serum. [email protected] After 48 hours, 1,0 ml of supernate or medium only were applied, intraperitonially, up to 2 hours after H-015 cecal ligature, or after 0,01ml of glutaraldehyde 2% GALECTIN-3 INTERFERES IN THE DYNAMIC OF IM ( to dismiss unspecific anti-inflammatory action). PERITONEAL B1 LYMPHOCYTES Immunohistochemical was analyzed by a single Felipe Leite de Oliveira (UFRJ), Lauremília Ricon pathologist. RESULTS: The greatest survival occurred da Costa (UFRJ), Roger Chammas (USP), Dan Hu in the group with sepsis treated with supernate, in 5 Su (Universidade de San Diego (USA), Fu Tong Liu days after cecal puncture (55%). There was significant (Universidade de San Diego (USA), Radovan Borojevic difference amongst the probabilities of survival in (UFRJ), Marcia Cury El-Cheikh (UFRJ) the 4 groups, with p(log-rank): 0,0037. Thymuses of those treated with supernate presented lower Galectin-3 (Gal-3) is β-galactoside binding protein expressions of actin and fibronectin. The supernate, expressed by several cells regulating the activation, or medium, treatment did not act on IM inflammation. migration and differentiation. Previously, using Gal-3-/- CONCLUSION: The data suggest an action promoting mice we described a remarkable acceleration in the B cellular survival, through treatment with supernatant cell differentiation into plasma cells in the secondary from the culture of newly born thymus, in experimental lymphoid tissues such spleen and mesenteric lymph sepsis, and the possibility of cell therapy in this nodes. Here, we investigated if the absence of Gal-3 pathology. [email protected] could interfere in the relation between the peritoneal B1a, B1b and B2 lymphocytes. In the peritoneal fluid 1. 187

livro.indd 187 7/7/2008 16:33:49 of Gal-3-/- mice, we observed a decreased of the total cells increase in the initial phase of the regeneration number of B1 lymphocytes. Although the B1a cells process, that is, 24 hours after the injection of poison of were significantly reduced in this fluid, these cells B. moojeni. presented up-modulation in surface B220 expression [email protected] and down-modulation in surface CD43 expression, comparing with wild type mice. The same alterations in H-017 B220 and CD43 expression were observed in B1b cells. 3D ALLERGIC LUNG MYOFIBROBLAST CULTURE Interesting, corroborating with the increase in pertioneal SYSTEM: NEW MODEL TO STUDY FIBROSIS AND cavity, the number of their bone marrow progenitor REMODELING cells (CD19+B220-) were significantly increased in Diana Dalzy Viveiros (Fundação Oswaldo Cruz), Gal-3-/- mice, probably trying to maintain the peritoneal Patricia Machado Rodrigues e Silva Martins (Fundação equilibrium. The inhibition of exogenous Gal-3 altered Oswaldo Cruz), Luciana Garzoni (Fundação Oswaldo the adherence of peritoneal cells in mesothelial-like Cruz), Edna Vallota (Fundação Oswaldo Cruz), stromal cells and histological analysis revealed a Radovan Borojevic (UFRJ), Marco Aurelio Martins significant modification in distribution of peritoneal (Fundação Oswaldo Cruz), Sandra Aurora Chavez lymphocytes and plasma cells in the mesenteric Perez Rodrigues (Fundação Oswaldo Cruz) membrane of gal-3-/- mice. Then, we suggest a new role to Gal-3 in the physiology peritoneal B1a and B1b Background: Airway remodeling is a feature of asthma lymphocytes, accelerating their activation and migration that is identified by peribronchial and subepithelial from peritoneal cavity and the differentiation into plasma fibrosis. The neogenesis of lung fibrosis observed in cells in effector sites. asthma is ill understood mainly because of limited [email protected] experimental models that mimics of what occurs in living organisms. Aim: To establish a 3D culture model H-016 with lung myofibroblast obtained from allergic mice and LOW POTENCY LASER EFFECT IN SATELLITE to analyze the effect of rmIL-13 in this system. Methods: CELLS IN THE INITIAL REGENERATION PROCESS Balb/c mice were immunized with a suspension of OF GASTROCNEMIC MUSCLE OF SWISS MICE ovalbumin (OA) (50 ug) and Al (OH)3 (5 mg) on days INJECTED WITH BOTROP POISON 0 (sc) and 14 (ip). At day 28, OA challenges (50 ug/25 Doroty Mesquita Dourado, Ana Paula Machado, uL, in) were performed 3 times a week during 2 weeks. Rosemary Matias Coelho, Paulo Tarso Camillo de Myofibroblast from naive or allergic lung were grown Carvalho (Universidade para o Desenvolvimento do after lung dissociation and multicellular spheroids were Estado e da Região do Pantanal) formed using myofibroblast plated in agarose-coated 96 U-well plates. Analyses were performed by phase and This study had as objective to evaluate the irradiation confocal microscopy. Results: Naive and allergic lung effects of gallium arseniet laser (904nm) and aluminum myofibroblasts were able to reaggregate into cellular arseniet gallium (650 nm), in the satellite cells of the spheres and spontaneously generated fibronection and gastrocnemic muscle of Swiss mice after rough poison collagen, as attested by confocal microscopy. Though inoculation of Botrops moojeni (in the right gastrocnemic spheroids generated from allergic lung appeared higher muscle). For that, 15 Swiss mice were used with in volume as compared to the controls, they grew even average weigh of 20g., divides in three groups more following exposure to rmIL-13. This cytokine up- –positive control group inoculated with rough poison, regulated fibronectin production in naive cell spheres. treated group with AsGa laser wave length of 904nm Conclusion: Multicellular spheroids sensitive to IL-13 and treated group with laser AsAlGa wave length of and able to produce components of extracellular matrix 650nm. The treatment with laser was done with an can be generated from lung myofibroblast. This 3D unique application after the inoculation of the poison model seems to provide an appropriate enviroment for in the group 904 and 650nm with the energy density of the study of lung fibrosis in allergic conditions. 2 J/cm2. To determine the number of satellite cells in [email protected] the areas of muscle lesion, it was done procedure of immunohistochemistry anti-MyoD was performed. The H-018 resulting data were submitted to the test of variance PHOTODYNAMIC ACTIVITY OF CRUDE analysis ANOVA and “post hoc test” of Tukey. In the EXTRACT OF BIDENS SULPHUREA IN statistic analysis by the ANOVA test was obtained POLYMORPHONUCLEAR LEUKOCYTES p<0.0001, that is, there is difference among the studied Erick Guimarães França, Renata Oliveira Santos, João averages, the comparison among the groups was done Fernando Neves Martins, Alexandre Dias Araújo Neto, by the Tukey test obtaining p < 0.001 for the crossings Lucas Ferreira de Paula, Marselha Pereira Ceolin, in the group Cpv 24 hours (41,75 ±8,3) and 24 hours Flávia de Sousa Ferreira, Bruno Pereira Garcês, José 904 nm (25,87±9,8) and 24 hours 650 nm (17,25 ± Eduardo Buiatte, Carlos Alberto de Oliveira 6,2). But in the two treated groups with laser there was (Universidade Federal de Uberlândia) no statistic difference p > 0.05. This study suggests that the low potency laser with 2 J/cm², gave satellite A way to inactivate cells or microorganisms is 1. 188

livro.indd 188 7/7/2008 16:33:49 generating reactive oxygen species (ROS) in a process MURINE SPLENIC LYMPHOCYTES called photo-inactivation. This event involves the Flavia F Bloise (Instituto de Biofísica Carlos Chagas combination of a colorant, light and molecular oxygen Filho/ UFRJ), Adriana Cordeiro (Instituto de Biofísica to promote ROS. In contact with cells, ROS generated Carlos Chagas Filho/ UFRJ), Valeria de Mello Coelho via photoreaction cause damages in organelles and (Instituto de Ciências Biomédicas/ UFRJ), Vivian membranes leading to the cell death. Using an alcoholic Rumjanek (Instituto de Bioquímica Médica/ UFRJ), crude extract obtained from Bidens sulphurea, we Carmen Pazos Moura (Instituto de Biofísica Carlos evaluated the photo-inactivation of polymorphonuclear Chagas Filho/ UFRJ) leukocytes, obtained from human peripheric blood, using a LED irradiation system constructed in our Effects of thyroid hormones on the immune system laboratory. The initial results indicated that the photo- are still not completely understood. In this work, we inactivation is dependent of the pre-incubation time investigated the influence of T3 on murine splenocytes of these cells with the extract. In a comparison with and thymocytes. For this, male and female C57/Bl6 a synthetic compound used like a model, the extract mice were treated with T3 (5mg/10g body weight) for shows more than 2 times of death effects. This initial 15 days. T3-treated male mice showed an increase result shows the great potential of the Bidens Sulphurea in the size of spleen (control: 4,2±0,3mg/g P.C., T3: crude extract in photo-inactivation of cells and 7,8±4,2mg/g P.C.; p=0,0003) and thymus (control: microorganisms and reaffirms the possibility of natural 2,9±0,2mg/g P.C., T3: 4,3±0,5mg/g P.C.; p=0,0315). colorant applications in this process. Female mice showed a tendency to increase the spleen [email protected] size (control: 4,3±0,2mg/g P.C., T3: 5,4±0,8mg/g P.C.). However, no changes were observed in the splenic or H-019 thymic density (total cellularity/ organ weight) between DETECTION OF MNCF LECTIN IN MAST CELL the T3-treated and control in both male and female Fernando Lourenço Pereira, Emerson Soares mice groups. Furthermore, we analyzed the profile of Bernardes, Maria Célia Jamur, Constance Oliver, Maria thymic T cell differentiation as well as the population of Cristina Roque Barreira (USP) helper T cells, cytotoxic T cells and B lymphocytes in the spleen. Following T3 treatment in male and female We have previously described that activated mast mice, no changes were detected in the subpopulations cells produce a lectin denoted Macrophage-derived of thymocytes or in CD4+ or CD8+ mature T cell Neutrophil Chemotactic Factor (MNCF), which exerts population in the spleen, as compared to control groups. attractant activity on neutrophils. The objective of this On the other hand, FACS analysis revealed a reduction study was to characterize the MNCF localization in of 10% in the population of B cells (B220+) in the mast cell by fluorescence microscopy. Rat basophilic spleen of males treated with T3 (p=0,0077) in relation leukaemia (RBL-2H3) mast cells were grown on glass to the control. The same result was observed to female coverslips and stimulated or not with LPS (10 µg/mL) mice although the result was not statistically significant. for 30 minutes, and immediately fixed or cultured for 1 Our data show that T3-treatment does not interfere additional hour. Cells were permeabilized or not with significantly in the T cell population but influences the saponin, and then incubated with biotinylated affinity- physiology of the spleen at least promoting a reduction purified IgY anti-MNCF (concentration 2mg/ml). After in the splenic resident B lymphocyte population. rinsing in PBS, cells were stained with streptavidin-FITC [email protected] (green) and counterstained with phalloidin (red) and analyzed by fluorescence microscopy. In unstimulated H-021 mast cells, MNCF was strongly detected in intracellular IMPACT OF VASCULAR ENDOTHELIAL GROWTH compartments and barely detected on the cell surface. FACTOR ON MONOCYTES DERIVED DENDRITIC When mast cells were stimulated with LPS for 30 CELLS MATURATION “IN VITRO” min and immediately fixed, we observed mast cell Luciana Cavalheiro Marti (Instituto Israelita de Ensino polarization and an intense punctuate intracellular e Pesquisa Albert Einstein), Patricia Severino (Instituto labeling, even though MNCF still could not be detected Israelita de Ensino e Pesquisa Albert Einstein), Daiane on the cell surface. Otherwise, in the LPS-stimulated Donna Guilhen (Instituto Israelita de Ensino e Pesquisa mast cells that were cultured for one additional hour Albert Einstein), Carlos Alberto Moreira-Filho (Instituto MNCF was strongly detected on the cell surface, Israelita de Ensino e Pesquisa Albert Einstein e suggesting that the lectin was secreted by mast cells in Departamento de Imunologia - USP) a time-dependent manner. These results suggest that intracellularly stored MNCF is secreted after mast cell Dendritic cells (DCs) are in charge of orchestrating the stimulation and may contribute to leucocyte recruitment adaptive immune response through T cells activation. to sites of inflammation. Supported by FAPESP. DCs are the main target in cancer cell immunotherapy. [email protected] However, DCs inadequate maturation interferes in the outcome of the immune response. In these study, were H-020 analyzed the efects of vascular endothelial growth IN VIVO EFFECTS OF TRIIODOTHYRONINE ON factor (VEGF) on DCs maturation. DCs matured in the 1. 189

livro.indd 189 7/7/2008 16:33:49 presence of VEGF, showed morphologic alterations Metabolic Diseases such as reduced number of dendrites and basophilic cytoplasm by cytochemistry. Global gene expression I-001 assessed by DNA microarrays demonstrated broad MYOSTATIN GENE EXPRESSION AND ATROPHY IN variation in the expression of cell adhesion and RAT SKELETAL MUSCLE WITH MONOCROTALINE cytoskeleton regulation-related genes. All together INDUCED HF these results suggest that cells exposed to VEGF are Cesar Augusto de Oliveira Coelho (Departamento less differentiated, a possible mechanism involved in de Morfologia, IB, UNESP, Botucatu-SP), Robson the immune suppresion caused by VEGF secreting Francisco de Carvalho (Applied Biosystems), Eduardo tumors. Paulino Castan (Departamento de Morfologia, IB, [email protected] UNESP, Botucatu - SP), Antonio Carlos Cicogna (Departamento de Clínica Médica, FMB, UNESP, H-022 Botucatu - SP), Maeli Dal Pai Silva (Departamento de NOVEL THALIDOMIDE ANALOG: LASSBIO-542 A Morfologia, IB, UNESP, Botucatu - SP) CYTOKINE AND ANGIOGENESIS MODULATOR Daniel Serra Carvalho (Fundação Oswaldo Cruz), Heart Failure (HF) is associated with peripheral skeletal Tercia Rodrigues Alves (Fundação Oswaldo Cruz), muscle myopathy, which is characterized by an increase Lídia M Lima (UFRJ), Eliezer Jesus Barreiro (UFRJ), in type II fibers (fast contraction), a decrease in type I Verônica Maria Morandi da Silva (UERJ), Euzenir fibers (slow contraction), and a loss of muscle mass. Nunes Sarno (Fundação Oswaldo Cruz), Elizabeth The physiopathological mechanisms responsible for Pereira Sampaio (Fundação Oswaldo Cruz) the muscle changes during HF are not defined. It has been established that Myostatin, a TGF-â super family Thalidomide (THAL) is known by its and anti- member, is a negative regulator of muscle growth and angiogenic action. Novel THAL analogs may have is associated with atrophic conditions. In this study aselective anti-TNF different immune activities, which we evaluated muscle fiber types area and Myostatin can be important for treatment of diseases. Human gene expression in Wistar rats skeletal muscle with PBMC were treated with THAL, LASSBio-542 (30µM) monocrotaline induced HF. We studied extensor or DMSO (0,01%) 30 min before LPS stimulation (10ng/ digitorum longus (EDL) muscle from both HF and ml). Cells were harvested after 3h for RNA extraction control Wistar rats. Muscle fibers types were classified and supernatants were collect after 18 hours. Cytokines by using myofibrillar ATPase(PH 4.6) and the area were accessed by ELISA are show as percent of cytokine measured using an image analyzer system. Myostatin production ± SD in comparison to (61,4%±19,6, n=14 gene expression was determined by using qRT-PCR. anda“LPS+DMSO” (100%). THAL and LASSBio-542 Muscle fiber types area decreased, but only the type IIa inhibit TNF 65,3%±10,3, n=8, respectively), but the IL- area was significant. The mRNA relative expression of 10 production demonstrate a difference between drugs, Myostatin was significantly decreased in HF. Decreased THAL promote a slightly augment in IL-10 production muscle fibers area was associated to muscle atrophy (134%±12,3, n=7) when LASSBio-542 reduces it in HF and the decreased content of Myostatin indicates (57,4%±14,3, n=6). Real-time RT-PCR confirms these that atrophy in rat skeletal muscle with monocrotaline alterations caused by THAL and LASSBio-542 at mRNA induced HF does not require Myostatin. levels. In the angiogenesis context, THAL and LASSBio- [email protected] 542 reduced angiogenic differentiation on human umbilical vein endothelial cells (HUVECs) seeded I-002 onto matrigel, in a dose-dependent manner (0.01-30 ULTRASTRUCTURAL ANALISYS OF MOUSE µM), without inducing citotoxicity, since propidium PANCREATIC ISLETS IN VITRO AFTER A HIGH- iodide (PI) incorporation was similar in all conditions. GLUCOSE CHALLENGE In 24h, only LASSBio-542 inhibited HUVEC migration. Alessandra Alves Thole (Laboratório Cultura de Moreover, THAL or LASSBio-542 did not inhibit HUVEC Células - LCC - Universidade do Estado do Rio proliferation after 72h. LASSBio-542 shows a dissimilar de Janeiro - UERJ), Simone Nunes de Carvalho profile of action demonstrated by a different modulation (Laboratório Cultura de Células - LCC - UERJ), Ana of IL-10 and impairments of endothelial cell migration Carolina Stumbo (Laboratório Cultura de Células - and alignment, which may provide a new therapy option LCC - UERJ), Helene Santos Barbosa (Laboratório for diseases that have singular alterations on cytokine de Biologia Ultraestrutural - Fundação Oswaldo Cruz), network and angiogenesis dependency. Aníbal Sanchez Moura (Laboratório de Fisiologia da [email protected] Nutrição e Desenvolvimento - UERJ), Laís de Carvalho (Laboratório Cultura de Células - LCC - UERJ)

There are several studies showing that increased glucose levels are somehow deleterious for beta- cells, which leads to a defective insulin response to glucose observed in early phases of diabetes 1. 190

livro.indd 190 7/7/2008 16:33:49 mellitus. The aim of the present study was to evaluate morphological analyses as sensitive biomarkers of OP whether in vitro exposure of mouse pancreatic islets exposure in P. anisitsi, opening new perspectives for the to high-glucose begins to impair the function of the use of this species in aquatic environmental monitoring beta-cells. Pancreatic islets were isolated from male studies concerned to the contamination by OPs. swiss mouse by microdissection. Batches of 20 islets [email protected] were hand-picked and cultured in RPMI 1640 medium containing SFB plus 5.6 mmol/L glucose. High-glucose I-004 challenge was performed by the replacement of RPMI HIGH GLUCOSE CONCENTRATION IMPAIRS CELL 1640 complete medium plus 16.8 mmol/L glucose MIGRATION for 30 minutes. For ultrastructural study pancreatic MARCELO LAZZARON LAMERS (USP), ALAN islets were colleted, fixed in 2,5% glutharaldeyde and FREDERICK HORWTZ (UNIVERSITY OF VIRGINIA), included in Epon. The ultrasctruture of beta-cells at MARINILCE FAGUNDES DOS SANTOS (USP) culture with basal glucose (5.6 mmol/L) showed insulin granules with tipical eletrondense core and round Most of Diabetes Mellitus complications, including shape nucleous. After 16.8 mmol/L glucose exposure wound healing impairment, are associated with chronic beta-cells were poorly granulated and we could identify hyperglycemia. Because cell migration is essential for apoptotic cells with condensation and margination of wound healing, we hypothesized that high glucose chromatin, dilatation of the endoplasmic reticulum and concentrations could play a direct role suppressing the myelin figures. These results suggest that high-glucose migration process. Using CHO-K1 cells and NIH-3T3 concentration induces remarkable changes in beta-cells fibroblasts cultivated for 3 days in control medium (C), soon after the glucose exposure in vitro. medium containing 25 mM D-glucose (HG) or 25mM [email protected] L-glucose (HC, hyperosmotic), we observed that HG increased in 200% the number of cells with a low I-003 migratory rate (p<0.01) and/or a random migration DIAZINON-INDUCED BIOCHEMICAL AND pattern (p<0.01), when compared to C or HC. HG also MORPHOLOGICAL ALTERATIONS IN ARMOURED promoted a 3-fold increase in the number of protrusions CATFISH (PTERYGOPLICHTHYS ANISITSI), in CHO-K1 cells when compared to C and HC groups AS BIOMARKERS OF ENVIRONMENTAL (p<0.01). Protrusion rates, analyzed by kimography, CONTAMINATION BY ORGANOPHOSPHOROUS remained unchanged among the different groups; PESTICIDE nonetheless, HG lead to a 30% decrease in protrusion Aline Cristina Ferreira Rodrigues, Marcus Alexandre persistence and stability, possibly correlated to a 40% Mendes Luz, Camila Pereira Tridico, Eduardo Alves de reduction of adhesion maturation. Cell polarity was Almeida (UNESP) strongly affected by HG; 50% of the cell population became spread and rounded shaped. Using a pull Environmental contaminants are ecotoxicologically down assay, we observed that these phenotypic relevant due to their cytotoxic and carcinogenic effects. changes were accompanied by a 30% increase in the The analysis of biochemical biomarkers can be useful to activity of Rac1, a small GTPase that regulates the predict the impact of contaminants in the environment, actin cytoskeleton and cell migration. In conclusion, providing data about the effect of these compounds in high glucose directly impairs cell polarity, migration resident animals without the need of their identification velocity/directionality, the persistence and stability in the environment. In this work, several biochemical of cell protrusions, as well as adhesion maturation. and morphological parameters were evaluated in brain, These effects may be at least partially attributed to the liver and gills of armoured catfish Pterygoplichthys activation of Rac1. Financial support: FAPESP, CNPq, anisitsi after organophosphate pesticide (OP) exposure. CAPES, NIH. Acetylcholinesterase (AChE) was measured in [email protected] brain, while the activities of carboxylesterase (CbE), glutathione S-transferase (GST) and glutathione I-005 peroxidase (GPx) were evaluated in liver and gills. EFFECT OF LYCOPENE IN SMOOTH MUSCLE Fishes were exposed to 2 mg/L of the OP Diazinon CELLS AND ENDOTHELIAL CELLS IN for 2 and 7 days (n=6). Liver morphology was studied CHOLESTEROL PRESENCE in animals exposed for 7 days to the pesticide, after Ronaldo José Farias Corrêa do Amaral, Anderson paraffin inclusion and HE staining. Significant increase Junger Teodoro, Luciene Bottentuit Lopez, Radovan in nucleus diameter was observed in fish liver after 7 Borojevic (Dept of Histology and Embriology, Instituto exposure days and hypercromatosis was observed, but de Ciências Biomédicas, UFRJ) it was not statistically significant. Diazinon exposure caused strong AChE and CbE inhibition in all tissues Atherosclerosis, main cause of cardiovascular diseases analyzed, and a significant GST increase in liver, in the world, starts with the endothelium dysfunction, after 2 and 7 days of exposure. GPx activity was caused by LDL oxidation and others. Traditionally, the unchanged throughout the experiment. These results atheromatous foam cells are considered to be derived indicate the utility of AChE, CbE and GST activities and from monocytes recruited to the site of developing 1. 191

livro.indd 191 7/7/2008 16:33:49 atheroma. However, the ability of smooth muscle cells Neurobiology (SMCs) to give origin to foam cells was confirmed by several reports in vitro. Lycopene is a carotenoid that has been described as a powerful antioxidant. It J-001 is believed that it can prevent atherosclerosis by not FEASIBILITY OF NEURONS FROM THE BRAINSTEM allowing LDL oxidation and other factors. The objective OF EMBRYO AND NEONATE RATS IN PRIMARY of this work was to verify the effect of lycopene over CULTURE. A COMPARATIVE STUDY the proliferation of endothelial cells and SMCs and lipid Daniela Valente Andrade (Universidade Federal de accumulation of SMCs in the presence of cholesterol. Viçosa), Sergio Marinho da Silva (USP), Merari de The SMCs and human umbilical vein endothelial cells Fátima Ramires Ferrari (USP) (HUVECs) were obtained by collagenase digestion (0,2% for 15 minutes, and 0,1% for 10 minutes) of The brainstem is one of the major sites responsible human umbilical cord vein. The proliferation was for survival functions such as breathing, digestion, evaluated by the MTT assay and the lipid accumulation cardiovascular control, alert responses, etc. The study by Oil Red O staining in the presence of 0,62-40uM of this area is important for a better comprehension of lycopene and 0,62-5ug/ml cholesterol for SMCs and the pathologies related to the brainstem. Cell culture is HUVECs. Preliminary data showed that cholesterol a useful tool for studying the physiology of isolated cells enhanced SMCs proliferation and decreased HUVECs. to analyze the differentiation stages. Since we know that When these cells were treated with lycopene, SMCs the cell differentiation is directly related to the organism proliferation was normalized and HUVECs proliferation development, the aim of the present study is to compare was enhanced. Beside that, lycopene was also able the feasibility and adatability of brainstem neurons from to decrease lipid accumulation by SMCs treated embryos (16 days of gestation) and neonate(1 day-old) with cholesterol. These results suggest an effect of rats in primary culture. Brainstem neurons were isolated lycopene in the metabolism of cholesterol. Future and plated at a density of 1800 cells/mm2 in serum studies can explain mechanisms of lycopene actions in free culture medium under a controlled atmosphere of atherosclerosis and other pathologies. 95% air and 5% CO2 ,37°C for 9 days prior to analysis. [email protected] The percentage of neurons in relation to the total cells was estimated through MAP2( a specific neuronal protein) immunoreactivity and DAPI labeling. Data were evaluated by the Student´s T-test (p<0.05). We observed that the percentage of neurons in cultures from embryos was higher than from neonate rats(85% vs. 75% respectively). Furthermore, neurons from embyos developed more neurites than neurons from postnatal rats. In conclusion, we have demonstrated that neurons from the brainstem of embryos are more feasible and adaptable for culture conditions that from neonate rats, which constitute a new approach to study brainstem cells. [email protected]

J-002 PLASMA MEMBRANE CHOLESTEROL DEPLETION INTERFERES WITH GLUTAMATE RELEASE FROM RAT BRAIN SYNAPTOSOMES Graziele Teixeira da Silva, Ernani Amaral, Marcus Vinícius Gomez, Cristina Guatimosim (UFMG)

Lipid rafts are specialized membrane microdomains that serve as organizing centers for assembly of signaling molecules, influence membrane fluidity and trafficking of membrane proteins, and regulate different cellular processes. The lipid composition of these microdomains consists primarily in cholesterol and sphingolipids and in the present work we have investigated the influence of cholesterol depletion by 10mM methyl- beta-cyclodextrin (MBCD) treatment on glutamate release from rat brain synaptosomes. Synaptosomes were incubated with MBCD for different time periods (5-30 minutes) and glutamate release was evoked by 1. 192

livro.indd 192 7/7/2008 16:33:50 depolarizing stimuli KCl 33mM. Incubation with MBCD FADA-UNIFESP. during periods of 15 and 30 minutes led to a significant [email protected] decrease in glutamate release induced by KCl 33mM suggesting that lipid rafts may participate in stimuli- J-004 dependent glutamate release. We therefore asked if EXPRESSION OF NUCLEAR FACTOR OF cholesterol depletion would interfere with spontaneous ACTIVATED T-CELLS (NFAT) IN RETINA glutamate release, that is, that one which occurs without Gabriel Wajnberg, João Paulo de Biaso Viola, Cinthya the presence of KCl. Preliminary data reveal that the Guimarães Sternberg (Instituto Nacional de Câncer) treatment of synaptosomes with MBCD increases the spontaneous glutamate release. These data The NFAT family transcription factors was first suggests that cholesterol depletion may disorganize described in lymphocytes, as a signaling intermediate the lipids rafts and that there is cholesterol-dependency between the TCR and IL-2. Five members compose this in the organization of the exocytose machinery in transcription factors family (NFAT 1 through 5). Theses nerve terminals. Considering that altered cholesterol transcription factors associate with other proteins to metabolism and/or lipid rafts play a critical role in bind DNA and induce genes responsible for cell-cell the pathophysiology of multiple CNS disorders, this interactions and are expressed in several different types study may help to understand the role o lipid rafts on of tissues and systems, such as muscle and diverse neurotransmitter release in nerve terminals. types of nervous system cells, like astrocytes, neurons [email protected] from hypothalamus and cerebellum. Studies in retinal microglial and retinoblastoma cell lines have implicated J-003 NFAT in the control of proliferation, but NFAT family was ATAXIA AND ABNORMAL CEREBELLAR never studied in the normal retina in vivo. The retina DEVELOPMENT IS CAUSED BY A MUTATION constitutes a model of Central Nervous System, where ON THE CENTROMERIC EDGE OF MOUSE the easy access allowing precise dissection and layered CHROMOSOME 17 structure are the main advantages. Our aim in this Carolina Batista Ariza (Depto. de Bioquímica - study is to analyze the expression of the NFAT family UNIFESP), Silvia Maria Gomes Massironi (ICB - members throughout the development of the mice retina USP), Valderez Bastos Valero-Lapchik (Laboratório (post natal days 1, 7, 14 and 21). We observed that de Experimentação Animal - UNIFESP), Marimélia NFAT1, 2 and 4 mRNAs are differentially expressed Porcionatto (Depto. de Bioquímica - UNIFESP) during the development of the retina. We confirmed the expression of the proteins NFAT1 and NFAT2 by Several human diseases currently known are of immunohistochemistry. NFAT1 is indeed expressed genetic cause. One way to understand the molecular by retinal cells, but NFAT2 seems to be expressed by alterations that lead to a given disease is through the endothelial cells only. We believe the expression of analysis of gene dysfunction, which can be done using NFAT in the retina is associated to proliferation and animal models that recapitulate such pathologies. differentiation processes and it is our intention to further Spontaneous or chemically induced mutations are dissect the role of this transcription factor in those useful to elucidate how a mutated gene gives rise to a events occurring in the retina. given illness phenotype. Through an ENU mutagenesis [email protected] project, we obtained a recessive mutant, named equilibrio (eqlb), which presents balance problems J-005 and lack of motor coordination, phenotype that led us MEMBRANE CHOLESTEROL REGULATES to hypothesize that the mutation could have affected SYNAPTIC VESICLE CYCLE AT THE cerebellar development. Histological analysis of eqlb NEUROMUSCULAR JUNCTION mice cerebella showed abnormal formation of the Ernani Aloysio Amaral (Departamento de Morfologia cellular layers, with disorganization of the Purkinje (ICB-UFMG)), Marco Antônio Máximo Prado cells, and a thicker external granule cell layer in eqlb (Departamento de Farmacologia (ICB-UFMG)), Marcus mutant mice, when compared to same age wild-type Vinícius Gomez (Núcleo Santa Casa de Pós-Graduação mice cerebellum. [3H]-Thymidine incorporation by (Belo Horizonte-MG)), Cristina Guatimosim Fonseca neuronal precursors showed that the proliferation rate (Departamento de Morfologia (ICB-UFMG) is significantly higher in eqlb mutants. Mapping of the mutation, using polymorphic microsatelite markers, Lipid rafts are membrane microdomains with high located the mutation on mouse chromosome 17, in a amounts of cholesterol associated to several proteins sub region between the marker D17Mit267 (3.31Mb) including SNARE proteins. It has been suggested and the centromeric edge. This chromosomal region that lipid rafts play a role in the synaptic vesicle cycle. presents synteny with a region of human chromosome To investigate how membrane cholesterol acts in 6, where recessive ataxias have been mapped. We are the exo/endocytosis cycle, we used a fluorescent presently sequencing candidate genes that are located probe called FM1-43 which is up taken by recycling at the region pointed by genetic mapping in order to vesicles during endocytosis and released during find out the mutated gene. Granted by FAPESP, CNPq, exocytosis. To modify the cholesterol content on the 1. 193

livro.indd 193 7/7/2008 16:33:50 presynaptic terminal, we used methyl-b-cyclodextrin effects of this anesthetic on neurotransmistter release at (MbCD) which removes cholesterol from membranes CNS. Our next step will be to examine the importance leading to lipid rafts disruption. Frog neuromuscular of voltage-dependent calcium and sodium channel on junctions stained with FM1-43 through stimulation with ketamine effect and other putative presynaptic targets K+ were incubated with MbCD which caused vesicle of this anesthetic. release and consequent FM1-43 destaining in a dose [email protected] dependent way. Electrophysiological findings indicated that MbCD induced a significant increase in MEPPs J-007 frequency and promoted a drop of membrane potential. SCIATIC NERVE REGENERATION IS ENHANCED IN However, MbCD inhibited the exocytosis evoked by the GALECTIN-3 KNOCK-OUT MICE depolarizing high K+ Ringer. Pre-incubation with MbCD Bruno de Siqueira Mietto, Marcelo Sampaio Narciso, before stimulus with K+ also inhibited FM1-43 uptake Suelen Adriane Marques, Marcia Cury El-Cheikh, Ana indicating endocytosis impairment. Hydroxy-propyl- Maria Blanco Martinez (UFRJ) MgCDcyclodextrin (HgCD) has low affinity for cholesterol and did not influence FM1-43 uptake or destaining, Success of peripheral nerve regeneration depends confirming that the effects of bM CD over synaptic mainly on a permissive environment; although the vesicle cycle were resultant from cholesterol removal mechanisms underlying its molecular events are still from presynaptic membrane. In conclusion, cholesterol not elucidated. Schwann cells are considered one of seems to play a regulatory action over the synaptic the best candidates for this success. These cells and vesicle cycle, grouping the machinery involved in exo/ invading macrophages are responsible for myelin/ endocytosis in proper domains. axonal debris clearance, creating an appropriate route [email protected] for a successful regeneration. It is known that, after injury, Schwann cells express galectin-3 and this has J-006 been correlated with phagocytosis; in the presence THE PRESYNAPTIC EFFECTS OF THE GENERAL of galectin-3, there is an inhibition of Schwann cells ANESTHETIC KETAMINE ON SYNAPTIC VESICLES proliferation in vitro. In the present study we explored EXOCYTOSIS in vivo the effects of the absence of galectin-3 in Monalise Costa Batista (1Departamento de Morfologia, Wallerian degeneration and nerve fiber regeneration. ICB- UFMG), Renato Santiago Gomez (Departamento For that, we crushed sciatic nerves of galectin-3 de Cirurgia, FM-UFMG), Marcus Vinícius Gomez knock-out and wild-type mice and followed the pattern (Núcleo Santa Casa de Pós-Graduação), Cristina of degeneration and regeneration from 48 hours up Guatimosim Fonseca (1Departamento de Morfologia, to 3 weeks. We analyzed the number of myelinated ICB- UFMG) fibers, axon area, fiber area, myelin area, G-ratio and immunofluorescence for macrophages in DAPI Ketamine is an intravenous anesthetic used for human counterstained sections. In our results, knock-out mice anesthesia for more than 30 years. Multiple sites presented a much faster regeneration when compared of action in the central nervous system (CNS) are to wild-type animals. We concluded that the absence of supposed to account for the different clinical effects galectin-3 allowed a better regenerataion of the crushed of this compound. General anesthetics have potent nerve. These results are discussed in terms of an presynaptic and postsynaptic effects neurotransmission increase on Schwann cell proliferation, combined with a by altering neurotransmitter release or by modulating possible increase in the release of neurotrophic factors, their responses to neurotransmitters. To investigate which would favor neuron survival and axon sprouting, the presynaptic effects of ketamine at the CNS, we followed by faster myelination, culminating in a better stained isolated rat cortical nerve terminals with morphological and functional outcome. the fluorescent probe FM2-10 which is taken up by [email protected] recycling synaptic vesicles. Our experiments indicated that sub clinical ketamine concentration (1µM) do not J-008 elicited significant exocytosis of synaptic vesicle. On CHARACTERIZATION OF A NEW CONNECTION the other hand, clinical concentrations (10-30µM) of this NEURAL NETWORK ASSOCIATED WITH THE anesthetic induce a decrease of FM2-10 fluorescence OLFACTORY LOBES IN THE FRESHWATER PRAWN that corresponds to vesicle exocystosis. In addition, MACROBRACHIUM GENUS exocytosis evoked by concentrations of ketamine Dib Ammar (UNOESC), Evelise Maria Nazari (UFSC), (50,100 and 200µM) that are not clinically relevant Yara Maria Rauh Müller (UFSC), Silvana Allodi (UFRJ) induced a fluorescence decrease higher than that observed with clinically relevant concentrations and Crustaceans display a great variety of body forms and at concentrations of 500µM, the decrease of FM2- complex behavior patterns, being suitable for studies 10 fluorescence was quite similar to that evoked by of landmarks in the evolution of the nervous system. depolarizing agent KCl 30mM. Our results indicate In this work we analyzed a specific brain region, the that ketamine may interfere with synaptic vesicles olfactory lobes (OL), of freshwater prawn species. exocytosis and this may account for some presynaptic The brains of the Macrobrachium acanthurus, M. 1. 194

livro.indd 194 7/7/2008 16:33:50 olfersi and M. potiuna were dissected and prepared These findings showed a reduced number of Purkinje for morphological and immunohystochemical. The cells in MeHg-treated mice this phenomenon was brains were sectioned at 7 mm, stained with Mallory reversed by DMPS treatment. trichromic, and submitted to immunohystochemical [email protected] detection of synorf 1, 5HT, NF 200, NF 160, and NOS. The microanatomy of the OL showed that it is internally J-010 subdivided into two parts: an anterior and a posterior RESCUE SIGNALING PATHWAYS IN PRPC-NULL region, linked by a common medullar region formed by NEURONS EXPRESSING EXOGENOUS PRPC fibers of the olfactory globular tract (OGT), and a new Cleiton Fagundes Machado, Marilene Hohmuth Lopes, prospective deutocerebral commissure (PDC), as well Vilma Regina Martins (Ludwig Istitute for Cancer as by neurites from cluster 10 (C10) and the new cluster Research) 9 (C9) neurons. This structural organization appears to be a real difference in the Macrobrachium species Cellular prion protein (PrPC) is a GPI anchored protein compared with the basic crustacean brain model and mostly expressed on the surface of neurons and glia evidences an increase in the complexity of the OL. The cells. Our group demonstrated that PrPC engagement appearance of a C9 associated with the OL implies to the Stress-inducible-Protein 1 (STI1), a c-chaperonin, that the addition of a new level of olfactory processing triggered activation of PKA and ERK1/2 signaling involving local interneurons, which send processes promoting neuroprotection and neuritogenesis, around the OL, must have happened, and that there respectively. It is well known that mutations at the PrPC appeared a new tract of axons that crosses the brain gene are involved in neurodegenerative disorders and projects to the posterior part of the OL, which called genetic prion diseases. It is believed that PrPC together indicates that Macrobrachium species may be loss-of-function can be responsible for at least part of an important model for studies of the crustacean brain the pathogenesis of these diseases. Understanding evolution. how individual pathogenic mutations affect the signaling [email protected] pathway of the PrPC could provide important clues regarding the molecular basis of these diseases. J-009 Here we investigated whether PrPC-null immortalized EVALUATION CEREBELLAR LAYERS TREATED neurons (CF10) reconstitute the functions of wild- WITH METHYL MERCURY AND CHELATING type PrPC. CF10 cells were transfected with the THERAPY vector pCDNA3-PrPC which express the wild-type Márcia Cristina de Carvalho, Marcelo Farina, PrPC using a liposome-mediated method. The PrPC Yara Maria Rauh Müller (UFSC) expression was confirmed by flow cytometry and western blot assays. CF10 and CF10 cells expressing The animals (14 per group) were submitted to the exogenous PrPC (CF10-PrPC) were treated with experimental groups during 17 days: group I received STI1 and evaluated for ERK1/2 and PKA activation. only water ad libitum; groups II and III were exposed to ERK1/2 activation was achieved by immunobloting 40mg MeHg/L, in drinking water ad libitum. In the last using specific antibodies and PKA activity measured by 3 days (15-17), the group III received DMPS injections g[P32]-ATP incorporation to a PKA-specific substrate. (150 mg/kg, i.p.; once a day) in order to reverse MeHg- Our data demonstrate that STI1 treatment increased induced neurotoxicity. The groups I and II received PKA and ERK1/2 activity in CF10-PrPC, while any effect only saline (1ml/kg). After transcardiac perfusion (day was observed in CF10 cells. Indeed, the expression of 18), cerebellum slices (6mm) are stained with cresyl wild-type PrPC in CF10 neurons is able to reconstitute violet and submitted to autometalography method PrPC/STI1-dependent signaling. counterstained with hematoxylin. The Purkinje cells [email protected] analysis was performed by direct counting (40x) and thickness of the cerebellar layers was made with ocular J-011 micrometric (10x). Mercury deposition was evalueted INTRACELLULAR CALCIUM INCREASE by the presence of the granules in cerebellar cortex. DEPENDENT ON BOTH STI1/PRPC AND THE The lowest thickness of the molecular and Purkinje NICOTINIC ACETYLCHOLINE RECEPTOR INDUCES layers [8.99 (±1.05) and (1.30 (±0,03)] were observed ACTIVATION OF ERK AND PKC in the group II, followed by groups I [ 10.95 (±0.93) and Flavio henrique beraldo (Ludwig Institute for Cancer 1.36 (±0.14)] and III [9.88 (±0.93) and 1.35 (±0.11)] Research), Camila Pinto Arantes (Ludwig Institute for respectively. The group II showed a reduced number Cancer Research), cleiton Fagundes Machado (Ludwig of Purkinje cells [5.90 (±0,28)] in comparison to groups Institute for cancer Research), Rafael Linden (Instituto I [8.20 (±0.25)] and group III [8.10 (±0.28)]. A marked de Biofísica , Centro de Ciências da Saúde, UFRJ), Hg-deposition was observed in the group II, which Marco Antonio Maximo Prado (Departamento de the granular layer present intense Hg-impregnation, Farmacologia, ICB-UFMG), Regina Pelkeman Markus followed by the Purkinje and molecular layers. The (Departamento de Fisiologia, IB-USP), Vilmar Regina granular layer of group III presented a moderate and Martins (Ludwig Institute for Cancer Research) Purkinje and molecular layers are mild impregnation. 1. 195

livro.indd 195 7/7/2008 16:33:50 The cellular prion protein (PrPC) is a highly conserved of tissue disagregation. The grey matter presented cell surface glycoprotein expressed in the Central a dysmorphic pattern with visible magnification of Nervous System. Its conformationally modified isoform neurites, of the somas diametrum and the number is associated with prion diseases. Recently, multiple of Nissl bodies. The morphology of the grey matter and diverse functions of PrPC have been reported. sugest a tissular reorganization, with neuronal loss. Our Our group identified the Stress Inducible Protein 1 findings indicate that 3-butyl-1-phenyl-2- (phenyltelluro) (STI1), which promotes neuronal differentiation via oct-en-1-one shows kaotropic proprieties to the cortical ERK phosphorylation, in wild-type but not in PrPC-null histology in all concentrations tested. Therefore, the neurons (Lopes et al., J. Neurosc. 2005). Our data brain, specially the temporal lobe and hippocampus, are showed that, in hippocampal neurons, STI1-PrPC potential targets to the alpha-beta-unsaturated ketones. interaction induced an increase in intracellular Ca2+ [email protected] levels through extracellular Ca2+ influx. Conversely, there was no effect on intracellular calcium increase J-013 when neurons were treated with a STI1 molecule which PRION DISEASES SURVEILLANCE IN BRAZIL: lacks the PrPC binding domain (STI1D230-245) or 2005-2007 when PrPC-null cells were treated with STI1. Moreover, Michele Christine Landemberger (Ludwig Institute for PrPC-STI1 interaction was also able to induce ERK Cancer Research), Hélio Rodrigues Gomes (Centro phosphorylation and PKC activation in a Ca2+ influx- de Investigação em Neurologia and Faculdade de dependent matter. These data indicate that calcium Medicina da USP), Leila Chimelli (Departamento de influx mediated by STI-PrPC complex is upstream to Patologia and Faculdade de Medicina da UFRJ), Sergio ERK1/2 and PKC activation, and the former is promoted Rosemberg (Departamento de Patologia da Faculdade by a calcium channel at the plasma membrane. de Medicina da USP), Vilma Regina Martins (Ludwig Inhibitors for store operated (SKF) and voltage calcium Institute for Cancer Research) channels (nifedipine) had no effect on STI1-induced calcium signaling. Remarkably, when we blocked Transmissible Spongiform Encephalophaties (TSE) are the ligand-gated channel type nicotinic acetylcholine a group of infectious neurodegenerative diseases. The receptor (AChR) with á-bungarotoxin, STI1 was unable Creutzfeldt-Jakob disease (CJD) is the most prevalent to induce Ca2+ increase as well ERK and/or PKC human prion disease and possesses sporadic, activation. Thus, our results indicate that interaction of genetic or acquired forms. The transmission of bovine PrPC/STI1/AChR promotes Ca2+ influx activating ERK spongiform encephalopathy to humans by ingestion of and/or PKC, inducing neuritogenesis. contaminated meet caused a new variant of Creutzfeldt- [email protected] Jakob disease (vCJD). Additionally, the risk of iatrogenic transmission of CJD (iCJD), mainly by blood transfusion, is a cause of major concern due to the lack J-012 of both comprehensive screening and treatment. Since MORPHOLOGICAL ALTERATIONS IN IMMATURE 2005 the notification of prion diseases is compulsory RATS ENCEPHALI INDUCTED BY 3-BUTYL-1- in Brazil. We evaluated 35 cases suspected of CJD PHENYL-2-(PHENYLTELLURO)OCT-EN-1-ONE by biochemical and/or genetic tests. According to the CARLOS AUGUSTO BORBA MEYER NORMANN, World Heath Organization (WHO) criteria, 74% of them KARINA R ANDRÉ, ROBSON BRUM GUERRA, PAULA were considered as possible sporadic CJD (sCJD), CILENE PEREIRA DOS SANTOS, PAULA RIGON DA and from these, 51% fulfill the criteria for probable LUZ SOSTER, ALESSANDRA PERES, CLÁUDIA DA sCJD. Two cases fulfill criteria for possible vCJD but SILVA FUNCHAL (Centro Universitário Metodista IPA) not for probable disease. None of the cases were associated to genetic forms of disease. Although some Organic forms of tellurium (Te) are used in organic polymorphisms within cellular prion gene have been sinthesys reactions. Nevertheless, the exposition associated to a higher sensitivity to sCJD and vCJD, to organotellurium has a great health potential risk. our data do not show any correlation for the probable The present work aim to investigate the effects of sCJD cases. Three patients had brain tissue analysed, the alpha-beta unsaturated ketone 3-butyl-1-phenyl- two classified as definite sCJD and one as negative. 2phenyltelluro-oct-en-1-one on the brain histology of Thus, the notification of these diseases has been 10-days-old Wistar rats. Brains of 10-days-old Wistar satisfactorily implanted, however their definite diagnosis rats were incubated for 1 hour in presence or not of needs improvement. Financial Support: Howard Hughes the organotellurium at 1, 10 or 30 µM concentrations. Medical Institute. The tissue was fixed in 10% formalin for 24 hours, [email protected] embeeded in paraffin wax and sectioned in microtome. The 7 µm sections were stained with HE or 0,025%, J-014 pH 4,5 toluidin blue. Morphological analisys revealed STRESS INDUCIBLE PROTEIN 1 NEUROTROPHIC that, in controls, the hippocampus and temporal lobe of FUNCTIONS INVOLVE PROTEIN SYNTHESIS brain correspond to the normal histological pattern. In STIMULATION VIA THE MTOR PATHWAY the treated brains, we verified a dose-dependent effect Martin Roffe (Departamento de Microbiologia, 1. 196

livro.indd 196 7/7/2008 16:33:50 Imunologia e Parasitologia - UNIFESP), Flávio Henrique it lacks a secretory signal sequence. STI1 secretion Beraldo (Ludwig Institute for Cancer Research), Gabriel is independent on the classical secretory pathway Mancini (Ludwig Institute for Cancer Research), Beatriz mediated by the Golgi apparatus, since astrocyte Castilho (Departamento de Microbiologia, Imunologia treatment with Brefeldin A and Monensin had no e Parasitologia - UNIFESP), Vilma Martins (Ludwig blocking effect. STI1 is present in a soluble form and in Institute for Cancer Research), Glaucia Hajj (Ludwig unidentified vesicles/aggregates that can be precipitated Institute for Cancer Research) by ultracentrifugation. STI1 secretion by exosomes was ruled out since very few of those vesicles could be It is known that the control of protein synthesis is observed in astrocyte conditioned medium by electron essential for neuronal development and that some microscopy. Transfected GFP-STI1 is also secreted in neurotrophic factors, exert their functions through the same manner of endogenous STI1 and we could translational regulation. We have recently described show that neurons are able to bind and internalize that Stress Inducible Protein 1 (STI1) promotes it. Additionally, we show that astrocyte-secreted STI neuritogenesis and protection against cell death in activates the MAPK signaling pathway in wild-type neurons when associated with its cell surface receptor, hippocampal neurons but not in PrPC knockout ones Cellular Prion Protein (PrPC) (J Neurosci. 2005, and anti-STI1 antibodies block this activation. We also 25(49):11330). Because protein synthesis modulation demonstrated that the protein content of wild-type is essential for these phenomena, we studied the astrocyte conditioned medium is significantly lower than ability of STI1 to control the translational machinery. PrPc knockout ones. Taken together these findings STI1 increased protein synthesis in a dose dependent indicate that astrocyte secreted STI1 is able to modulate manner in wild-type neurons but not in PrPC knockout a signaling pathway previously associated to neuronal ones. Strikingly, these STI1 effects were blocked by differentiation. Moreover, PrPc expression in astrocytes PI3K and mTOR inhibitors. In accordance, we observed can modulate protein secretion. Supported by FAPESP AKT activation mediated by STI1 only in wild-type and HHMI. neurons. Confirming mTOR pathway activation, STI1 [email protected] stimulated p70S6 Kinase phosphorylation, which could be blocked by both mTOR and PI3K inhibitors. On J-016 the other hand, we observed eIF4E phosphorylation CONSTRUCTION OF A RETROVIRAL VECTOR FOR upon STI1 treatment. Moreover, the neuritogenic and CONTROLLED EXPRESSION OF CHONDROITINASE neuroprotective effects of STI1 were also abolished AC IN CENTRAL NERVOUS SYSTEM INJURIES by PI3K and mTOR inhibitors, suggesting that protein Caroline Mônaco Moreira, Yvette Coulson-Thomas, synthesis is a critical step for these processes. In Vanessa Dionisio Cantagalli, Sang Won Han, Marimélia conclusion, we demonstrated that the stimulation Porcionatto (UNIFESP) of protein synthesis is behind STI1-mediated neuritogenesis and neuroprotection. These STI1 effects Adult mammalian central nervous system is unable are dependent upon its engagement with PrPC and to regenerate axons and to reestablish functional are mediated by the PI3K-AKT-mTOR pathway. Thus connections after an injury. The lack of regenerative suggesting that STI1, could act as a neurotrophic factor capacity is, in part, due to a glial scar formed on the and PrPC may represent a new neurotrophin receptor. injury site. Glial cells produce high levels of molecules [email protected] that inhibit axonal growth, including chondroitin sulfate proteoglycans. Chondroitin sulfate chains can be degraded by bacterial enzymes named chondroitinases, J-015 and it has been shown that its degradation promotes ASTROCYTES SECRETE STI1 MODULATING axonal regeneration after spinal cord and nigrostriatal NEURONAL MAPK IN A CELLULAR PRION tract injuries. The aim of this work was to clone the DEPENDENT MANNER chondroitinase AC (cAC) gene into the retroviral system Camila Arantes (Ludwig Institute for Cancer Research), BD RevTetOn to be used to express the active enzyme Glaucia Hajj (Ludwig Institute for Cancer Research), into central nervous system injuries. This retroviral Marilene Hohmuth Lopes (Ludwig Institute for Cancer system consists of a regulatory vector, pRevTetOn, Research), Flávia Lima (UFRJ), Isabel Porto-Carreiro and an expression one, pRevTRE. The regulatory (UFRJ), Rafael Linden (UFRJ), Marco Antônio Prado vector produces a protein that acts on the expression (UFMG), Vilmas Martins (Ludwig Institute for Cancer vector promoting gene transcription in the presence of Research) doxycycline. The present work describes the strategy used to produce the vectors that were used to produce The physiological functions of cellular prion protein the retroviruses containing chondroitinase AC gene (PrPC) are under intense investigation. In neurons, by transfecting packaging cell lines, PE501 and PT67. the association of PrPC with its ligand, the co- So far, we have succeeded in producing the viruses chaperone Stress Inducible Protein 1 (STI1) induces with titles of 7x105 cfu/mL (RevTRE-cAC) and 5x105 neuritogenesis via MAPK. In this work, we observed cfu/mL (RevTetOn) to be used to transduce bone that STI1 can be secreted by astrocytes, even though marrow-derived cells to be transplanted into mice motor 1. 197

livro.indd 197 7/7/2008 16:33:51 cortex injuries in order to produce the active enzyme, STI1, a PrPc ligand, is secreted by glial cells and on a controlled manner, degrade chondroitin sulfate promotes glioblastoma (GBM) proliferation (Lima et and promote axonal regeneration. Granted by CNPq, al., 2007; Erlich et al., 2007). In this context, we intend FAPESP, FADA-UNIFESP. to verify the influence of microglial secreted factors, [email protected] particularly STI1, on the proliferation and migration of GBM cells. Highly pure cultures of microglial cells and J-017 tumor cells from GBM95 cell line were obtained as IMPACT INHIBITS GCN2 ACTIVATION AND described previously (Lima et al., 2007; Erlich et al., STIMULATES PROTEIN SYNTHESIS IN NEURONS 2007). We have verified by immunocitochemistry that Martin Roffe (Departamento de Microbiologia, microglial cells express STI1. In GBM cultures, the Imunologia e Parasitologia - UNIFESP), Glaucia [3H]-thymidine incorporation assays have showed that Hajj (Ludwig Institute for Cancer Research), Beatriz proliferation increased 100% when when GBM cells Castilho (Departamento de Microbiologia, Imunologia e where cultivated with microglial conditioned medium Parasitologia - UNIFESP) (MCM), compared to the control condition (fresh medium). However, this effect was reverted when anti- One mechanism that regulates translation in neurons STI1 antibody was added to the MCM. In the migration is the phosphorylation of eukaryotic Initiation Factor 2 assays by scratch, both MCM and MCM added of (eIF2), which reduces general translation but increases anti-STI1 antibody promote cell migration in a similar translation of a specific class of mRNAs, such as ATF4. manner. Our initial results suggest that STI1 is a soluble GCN2 is a stress activated eIF2 kinase that responds factor produced by microglia and favors growth but not to amino acid deprivation and recently was related to tumoral invasion. Supported by CNPQ, FAPERJ and learning and memory. Activation of GCN2 requires its CAPES. binding to GCN1. IMPACT is a protein preferentially [email protected] expressed in the nervous system that binds GCN1 in the same region as GCN2 does. We thus postulated J-019 IMPACT as an inhibitor of GCN2 activation. Using GDNF EXPRESSION IN SKELETAL MUSCLE OF siRNA to inhibit IMPACT expression in a neuronal cell TRYPANOSOMA CRUZI-INFECTED RATS line we demonstrated that GCN2 became prematurely Elizabeth Ribeiro da Silva, Conceição Ribeiro da Silva activated upon leucine deprivation, an event that Machado, Vinícius Alves Lara dos Santos, Patrícia resulted in a premature eIF2a phosphorylation and Massara Martinelli (UFMG) ATF4 expression. In addition, lack of IMPACT was enough to reduce polysome/monosome relationship, Glial Cell Derived Neurotrophic Factor (GDNF) is reflecting decreased protein synthesis. This relationship expressed in skeletal muscle of both humans and was even more reduced upon stress. We found IMPACT rodents, and it is suggested to influence homeostasis associated with polysomes and this is in agreement with of the adult neuromuscular junction. After muscle a translational regulatory protein. Surprisingly, IMPACT denervation, there is an up-regulation of the GDNF forms large granules along neuronal processes, which expression. In patients with Chagas’ disease, muscular partially co-localize with FMRP-containing neuronal pain and weakness are frequent symptoms, and granules (NG). NG allows mRNA transport and muscular ultrastructural changes have been described. local translational control in neurons. In conclusion, In rodents, the experimental infection provokes myofibre IMPACT is a protein that positively controls translation parasitism and myositis and there are evidences of in neurons and at least part of its action depends on motor denervation. Our group has demonstrated the GCN2 inhibition. Its localization in NG and in polysomes occurrence of segmental regeneration of myofibres opens the question of whether IMPACT, in non-stressed and the activation of satellite cells in T. cruzi-infected conditions, activates translation of mRNAs that are rats. Now we aimed to verify the GDNF expression silenced in NG. in skeletal muscles of the same experimental model. [email protected] Samples of diaphragm and soleus of T. cruzi-infected (day 20) and control animals were used. Through ELISA we observed higher GDNF levels in the diaphragm J-018 of 20-day-infected (13,000 ± 1,850 pg/mg of total THE ROLE OF STI1 IN THE MICROGLIA- protein) compared to uninfected (7,087 ± 1,561 pg/ GLIOBLASTOMA INTERACTION mg of total protein) rats. Immunocytochemistry showed Anna Carolina Carvalho da Fonseca (UFRJ), Vilma GDNF expression in the diaphragm and soleus of Regina Martins (Instituto Ludwig de Pesquisa sobre o both uninfected and infected animals, but is was Câncer), Flavia Regina Souza Li (UFRJ), Vivaldo Moura clearly stronger in the latter. In muscle sections from Neto (UFRJ) infected animals, a high GDNF positive-fluorescence was seen in the cytoplasm of satellite cells double There is evidence that microglial behavior is controlled labeled for GDNF and Myo D, a transcription factor by tumor cells, supporting their progression and characteristic of proliferative myoblasts. We are now infiltration. Our group demonstrated previously that investigating the role of neurotrophic factors in the 1. 198

livro.indd 198 7/7/2008 16:33:51 regenerative phenomena observed in the acute phase granular neurons during early post natal development of experimental trypanosomiasis. on an age-dependent manner. Based on that, the [email protected] aim of the present work was to find candidate genes involved in the control of axonal outgrowth during J-020 cerebellar development using Representational MANGANESE OXIDATION STATE MEDIATES Differential Analysis (RDA). We have compared the TOXICITY IN PRIMARY NEURONAL CORTICAL gene expression profile of neuron and glia from 3 days CULTURE: ROLE OF PROTECTOR AGENTS SUCH old (P3) with 9 days old pups (P9). RNA extracted from AS ASCORBIC ACID AND D,L-LACTATE P3 and P9 neurons and glia were reverse-transcribed Raúl Bonne Hernández ((IIBB)-CSIC-IDIBAPS e IQ- into cDNA using the SMART PCR cDNA Synthesis kit. USP), Marcelo Farina ((IIBB)-CSIC-IDIBAPS), Breno The cDNAs were digested using Bsp143I and the DNA Pannia Espósito (IQ-USP), Cristina Suñol ((IIBB)-CSIC- fragments were called “representations”. Using the IDIBAPS) cohesive ends of the representations, adaptors were added on each end of the fragments and subtractive Several metals, such as manganese (Mn) are essential hybridizations were performed. By the end of the third trace elements but can induce neurodegeneration subtractive hybridization, the genes were cloned into at high level. Evidence suggests one action of these a pGEM-T easy plasmid and DH5-a bacteria were metals, when concentrated beyond physiological levels, transformed with vectors. The bacterial clones were may be to inhibit cellular energy production, ultimately selected and their products, sequenced. From the leading to increased radical formation. On the other sequencing, we found two candidate genes so far: hand, the role of the manganese (Mn) oxidation state stathimin1, involved in the organization of microtubules on toxicity is not well understood. For that reason, our and found to be more expressed in P3 neurons; and study was carried out to investigate the toxic effects of septin11, involved in cytokinesis and found to be more Mn(II)-Chloride and Mn(III)-Citrate in undifferentiated expressed in P9 neurons. Supported by FAPESP, CNPq cultured neocortical neurons, which were exposed to 0 - and FADA-UNIFESP. 1 mM, for 120 h, with or without ascorbic acid (AA) and [email protected] d,l-lactate (L). Cell viability (MTT), cytotoxicity (lactate dehydrogenase, LDH) and GSHt were measured. J-022 Mn(II) induced higher toxicity than Mn (III), according PRION PROTEIN AND STRESS-INDUCIBLE to the MTT and LDH assays (EC50 values were 0,179 PROTEIN 1 TRAFFICKING: THE ROLE OF mM and 0,588 mM, respectively). Both metal species ENDOCYTOSIS IN SIGNALING increased GSHt levels by 35 and 45 % respectivaly, Fabiana Andrade Caetano (UFMG), Marilene H Lopes at concentrations producing mild cytotoxicity (< 20 % (Ludwig Institute for Cancer Research, São Paulo,), of cell death); thereafter, GSHt levels were reduced Glaucia N Hajj (Ludwig Institute for Cancer Research, at higher metal concentrations. The co-treatment with São Paulo), Cleiton F Machado (Ludwig Institute for AA offered protection to both metal species, however Cancer Research, São Paulo), Camila Pinto Arantes Lactate treatment presented protection against Mn(II) (Ludwig Institute for Cancer Research, São Paulo), only. This result suggest that Mn(II) induced injured Vania Ferreira Prado (UFMG), Rafael Linden (UFRJ), cell by multiple pathways: inhibition of cellular energy Vilma Regina Martins (Ludwig Institute for Cancer production, oxidative stress and other not evaluated in Research, São Paulo), Marco Antonio Maximo Prado this work; and Mn(III) induced neurotoxicity basically by (UFMG) oxidative stress. Additionally, these results imply that the oxidation state of manganese plays an important role in The interaction between cellular prion protein (PrPC) mediating its cytotoxicity. and its ligand Stress-inducible protein 1 (STI1) [email protected] promotes neuroprotection and neuritogenesis by distinct signaling pathways. Here we investigated the J-021 role of endocytosis in PrPC-dependent signaling. We STATHMIN1 AND SEPTIN11, TWO CANDIDATE determine whether STI1 could affect PrPC trafficking. GENES INVOLVED IN AXONAL OUTGROWTH BY SN56 cells expressing GFP-PrPC were analyzed by CEREBELLAR NEURONAL PRECURSORS DURING confocal microscopy before and after STI1 treatment. EARLY POSTNATAL DEVELOPMENT STI1, but not STI1delta230-245 a deletion mutant that Maria Emilia de Oliveira Brenha Ribeiro, Yvette May does not bind PrPC induced GFP-PrPC internalization. Coulson-Thomas , Marimelia Porcionatto (UNIFESP) To evaluate the role of PrPC endocytosis in signaling, we used a CF-10 cells expressing a mutant PrPC The role played by glial cells in the development of the whose internalization is impaired (N-PrP3F4). STI1 central nervous system is not completely understood evoked PKA-activation in cells expressing, PrP3F4 and its interactions with neurons are extremely or N-PrP3F4. In contrast, transient activation of important to the correct development of the brain. ERK1/2 by STI1 did not occur in N-PrP3F4. STI1 was Our previous results showed that glial cells secrete also internalized by cells and we evaluated if STI1 factors that influence axonal outgrowth of cerebellar endocytosis is PrPC dependent using a fluorescent 1. 199

livro.indd 199 7/7/2008 16:33:51 recombinant STI1 and STI1delta230-245. STI1 and the J-024 mutant STI1delta230-245 were effectively internalized IDENTIFICATION OF NEW PUTATIVE PRPC by SN56 cells. These observations were confirmed in LIGANDS BY PHAGE DISPLAY APPROACH PrP null CF-10 cells and CF-10 cells expressing PrP3F4 Tatiana Alves Americo (IBCCF-UFRJ), Margaret treated with fluorescent STI1. Interesting STI1 and Haiganouch Magdesian (IBqMed-UFRJ), Rafael PrPC colocalized predominantly at the cell surface and Linden (IBCCF-UFRJ) shortly after internalization, but downstream trafficking was different for these two proteins. The results show Cellular prion protein (PrPC) is a GPI-achored protein, that STI1 interacts with PrPC at the cell surface, triggers the physiological functions of which are still under PrPC endocytosis and this seems to be a critical discussion (Linden et al, 2008). Various PrPC ligands process for ERK1/2 signaling.Supported by: CNPq, were described, including hop/STI1 (Zanata et al, FINEP, Millennium Institute, PRONEX-MG, Howard 2002). The interaction between these two proteins Hughes Medical Institute and FAPESP. triggers various signaling pathways such as AMPc/PKA, [email protected] ERK and PI3K pathways. The AMPc/PKA pathway is related to neuroprotection and the ERK pathway J-023 to neuritogenesis and proliferation. How the signal IDENTIFICATION OF NEW LIGANDS FOR THE transduction triggered by these proteins occurs through STRESS INDUCIBLE PROTEIN 1 (STI1) the cellular membrane is still unclear. In order to identify Iaci Nunes Soares (UFMG), Grace Schenatto Pereira new PrPC ligands, presumably involved in a signaling (UFMG), Marilene H. Lopes (Ludwig Institute for plataform with the hop/STI1 protein, we have used a Cancer Research Sao Paulo), Fabiana Andrade phage-display approach. A random heptapeptide phage Caetano(UFMG), Nancy Scardua Binda(UFMG), Nicolle display library (Complexity ~2.8 x 10e9 transformants) G. T. Queiroz (Ludwig Institute for Cancer Research was used to isolate phages which binds to PrPC after Sao Paulo), Cristiane Menezes (UFMG), Vânia Ferreira five biopanning rounds. The specifity of the binding of Prado(UFMG), Vilma R. Martins (Ludwig Institute for PrPC to the selected clones was tested by ELISA. The Cancer Research Sao Paulo), Marco Antônio Máximo isolated clones were sequenced and the sequence Prado (UFMG) was analysed using the BLAST algorithm (psi-blast) to search for homologies to known membrane proteins in The Stress Inducible Protein 1 (STI1) is a specific the non-redundant protein sequences database (NCBI). ligand for the Prion protein (PrPC) and this interaction One of the clones showed homology to a region in the promotes neuroprotection and neuritogenesis through extracellular domain of Metabotropic Glutamatergic different signaling pathways. In order to understand Receptor 1 (mGluR1). This homology region is located possible physiological functions of STI1 and eventually next to a putative hop/STI-1 binding site (Magdesian PrPC we used a yeast two hybrid assay to identify et al, unpublished data). The interaction among hop/ STI1 ligands. We performed two screenings using the STI1, PrPC and mGluR1 has yet to be validated by N-terminus region of STI1 as bait using a human or biochemical methods. a murine brain cDNA libraries. We isolated the DNA [email protected] from interacting clones, amplified them in bacteria, sequenced and did bioinformatics analyses. Amongst J-025 the various clones interacting with STI1, we identified NEUROTRANSMITTERS AND SYNAPTIC PROTEINS Ubc9 (ubiquitin-conjugating enzyme E2I) and PIAS ARE MODIFIED IN THE MEDIAL POSTERO-DORSAL (Protein Inhibitor of Activated STAT, a SUMO E3 ligase), AMYGDALA IN FEMALE RATS ALONG THE ESTRUS both involved with the post-translational modification CYCLE IN BOTH BRAIN HEMISPHERES pathway named SUMOylation. We confirmed the Janaína Brusco (Departamento de Biologia Celular e interaction between STI1 and the different identified Molecular e Bioagentes Patogênicos, FMRP-USP), clones by a second round of yeast two hybrid assays. Daniel G Gitaí (Departamento de Biologia Celular e HA and MYC-tagged STI1 and PIAS expressed in Molecular e Bioagentes Patogênicos, FMRP-USP), HEK 293 cells were also co-immunoprecipitated. Maria Luiza Paçó-Larson (Departamento de Biologia Additionally, SUMOylation assays revealed that STI1 is Celular e Molecular e Bioagentes Patogênicos, SUMOylated. These data suggest that STI1 interacts FMRP-USP), Alberto A. Rasia-Filho (Departamento with members of the SUMOylation pathway and is de Ciências Fisiológicas, UFCSPA), Jorge E. Moreira a substrate for SUMOylation. Future experiments (Departamento de Biologia Celular e Molecular e will address the role of SUMOylation in STI1-PrPC Bioagentes Patogênicos, FMRP-USP) interaction and signaling functions. Support: FAPESP, PRODOC/CAPES, CNPq, FINEP, Millennium Institute, The function of the medial postero-dorsal nuclei of PRONEX-FAPEMIG and Howard Hughes Medical the amygdala (MePD) is related mainly to sexual institute. behavior mediated by the receptors for estrogens and [email protected] androgens. The activation of these steroid receptors are capable to alter gene expression in specific sites. To study the synaptic plasticity is useful to understand 1. 200

livro.indd 200 7/7/2008 16:33:51 the molecular mechanisms that direct the function of the DAPI (0.13ul DAPI in 6.25ml PBS) for 3 min. Anti- nervous system. Electrophysiological results suggest fading mounting medium was used with a coverslip that MePD is different between male and female rats in and observed under confocal microscopy. Powdered both brain hemisferes. The aim of the present work is to DiI labeling is a quick and reliable procedure for spine understand the brain laterality in the sexual dimorphism quantification with advantages over full DiI crystal along the estrous cycle. The mRNA is quantified for applications allowing a reliable 3D study of dendritic the subunits NR1 and NR2B of the glutamatergic spines. NMDA receptor, Narp docking protein for AMPA, and [email protected] the enzymes GAD65 and 67 that synthesize GABA in the MePD of adult male and female rats. Using real J-027 time PCR (qRT-PCR), it was possible to identify lower ENDOPLASMIC RETICULUM STRESS-INDUCED mRNA for the NR1 subunit in the right hemisphere of CELL DEATH INVOLVES AUTOPHAGY IN RETINAL males and females in proestrus than in estrus. In the TISSUE left hemisphere, females in estrus had more mRNA for Mona Lisa Leal Ferreira, Thaís de Medeiros Gameiro, the NR1 subunit than males. On the right MePD, males Bruno de Souza Gonçalves, Rafael Linden, Luciana show higher mRNA for NR2B than females in estrus Barreto Chiarini (IBCCF - UFRJ) and also higher than on the left MePD of females in proestrus. Interesting enough, MePDs of males, and Endoplasmic reticulum (ER) stress has been associated females in estrus showed more mRNA expression for with cell death in various degenerative pathologies both NR1 and NR2B in the right than in the left side. in which accumulation of mutated proteins occur, The present report provides findings relevant to the including retinitis pigmentosa. Recently, autophagy knowledge of this forebrain area, and to understand the was shown to be linked to ER stress. However, its role of excitatory and inhibitory components in the local role in ER stress has not been fully established. ER modulation and projective circuits in males and females. stress-induced autophagy has been equated with [email protected] both protection from and induction of cell death. Previously, we showed that tunicamycin, an inhibitor J-026 of N-glycosylation, also described as inducer of ER EXTRACELLULAR POWDERED DII AND CONFOCAL stress, triggers cell death in the retina. Here we studied MICROSCOPY FOR DEPICTING DENSITY AND the role of autophagy in tunicamycin-induced cell death MORPHOLOGY OF DENDRITIC SPINES in retinal tissue. Retinal explants from 6 day-old rats Alberto A. Rasia-Filho (Universidade Federal de were maintained for 24 hours in vitro with tunicamycin Ciências da Saúde, Porto Alegre), Janaína Brusco (1µg/mL), in the presence or not of either salubrinal (Departamento de Biologia Celular e Molecular e (1-10µM), an inhibitor of ER stress-induced cell death, Bioagentes Patogênicos, FMRP-USP), Lenaldo Branco or 3-methyladenine (3MA, 1-10mM), an inhibitor of Rocha (Departamento de Biologia Celular e Molecular autophagy. Cell death was estimated by counting e Bioagentes Patogênicos, FMRP-USP), Jorge E. condensed profiles stained with neutral red. Protein Moreira (Departamento de Biologia Celular e Molecular content was examined by western blot. We found that e Bioagentes Patogênicos, FMRP-USP) tunicamycin increased the level of CHOP/GADD153, a transcription factor implicated in cell death induced Dendritic spines are specialized postsynaptic by ER stress. Salubrinal partially blocked tunicamycin- compartments with neurotransmitter receptors to induced cell death. Finally, we observed that treatment alter local biophysical properties and sequences of with 3MA prevented cell death in this model. Our data intracellular biochemical events. The shape and density show that tunicamycin-induced cell death in retinal of spines change upon synaptic demands and can show tissue involves both induction of ER stress and the dynamic modifications following different physiological participation of autophagy. Finantial support: CNPq, or pathological conditions. The aim of this work is to FAPERJ. describe the steps to apply sonicated fine powdered [email protected] carbocyanine dye DiI on adult rat brain slices to reveal aspects of dendritic spines under confocal microscopy. J-028 The classic Golgi silver-impregnation was done to serve HUMAN DENTAL PULP CELLS INFLUENCE as comparison. For the DiI method, male rats were POSITIVELY FUNCTIONAL RECOVERY AFTER perfused through the heart breefly with saline followed CHRONIC SPINAL CORD INJURY by 1.5% paraformadehyde in phosphate buffer solution Fernanda Martins de Almeida (UFRJ), Suelen Adriani (PBS) 0.1M, pH 7.4. On coronal sections, we placed the Marques(UFRJ), Rafaela Fintelman Rodrigues (UFRJ), powered DiI on the axonal efferent pathway that would Bruna dos Santos Ramalho (UFRJ), Daniel Veloso migrate retrogradelly. The DiI tissue sections were kept Cadilhe(UFRJ), Fabio Silva Lima da Conceição (UFRJ), on glass slides covered with PBS at room temperature Irina Kerkis (Instituto Butantan), Lygia da Veiga Pereira for approximately 16h. After that, the PBS was rinsed (USP), Stevens Kastrup Rehen (UFRJ), Ana Maria and a post fixation was done with 4% paraformaldehyde Blanco Martinez(UFRJ) in PBS for 30 min. The slices were counterstained with 1. 201

livro.indd 201 7/7/2008 16:33:51 Functional deficits following spinal cord injury result reductions in all layers at nine (P9) and twenty-one from damage to axons, loss of neurons and glia and post natal days (P21) leading to a smaller cerebellum demyelination. Cell transplantation is a therapeutic area characterized by severe foliation defects. BrdU strategy that promotes axonal growth and replaces lost incorporation and imunohistochemistry assays for cells.The aim of this study was to evaluate the efficacy GFAP, a BG marker, suggested that both EGL precursor of the transplant of human dental pulp cells (HDPC) proliferation and migration are affected. Further, the on the recovery of chronic spinal cord injury in C57/ analysis revealed a reduction of the BG fibers and a Black6 mice. After anesthesia, animals were submitted drastic alteration in their morphology. The adult mutant to laminectomy at T9 level followed by spinal cord cerebellum presented severe deficits in Purkinje cells compression with a 30g vascular clip (KENT Scientific arborization and organization, although no apparent Corporation) for 1 minute. The animals were divided alteration in its morphology. Together, our data suggest into two groups:HDPC-treated animals and untreated that the action of TH via TRb can regulate important animals. Cells were injected into the lesion epicenter events on the cerebellar ontogenesis including EGL (8 x 10e5) in 4µL of culture medium (DMEM)four precursor proliferation, GC migration, BG maturation weeks after injury. Behavior testing (BMS and global and cerebellar foliation, contributing to a better mobility)was performed weekly up to 8 weeks after understanding of some neuroendocrine disorders. transplantation. After this period, the spinal cords were Financial support: CNPq, FAPERJ, CAPES and SBEM. processed for fluorescence microscopy to evaluate [email protected] the survival of transplanted cells into the host tissue. Our results showed that HPDC were present in the host tissue after 2 months. At 8 weeks, HPDC-treated animals reached the intermediate phase of locomotor recovery, whereas the untreated control group achieved the initial phase, according to BMS score. Video analysis of the global mobility test showed that treated animals presented higher speed in locomotion than untreated animals.Our results suggest that HDPC transplantation into the spinal cord improves locomotor recovery and also that these cells are able to survive in the host tissue and may contribute to the regenerative process. [email protected]

J-029 DEFICIT IN CEREBELLAR DEVELOPMENT OF TRB MUTANT MICE: EFFECTS ON FOLIATION, NEURONAL PRECURSOR PROLIFERATION AND GLIAL DEVELOPMENT Aline Cristina Portella Pereira (Instituto de Ciências Biomédicas, UFRJ), Fernando Carvalho Rodrigues Pereira (Instituto de Ciências Biomédicas, UFRJ), Larissa Costa Faustino (Instituto de Biofísica Carlos Chagas Filho, UFRJ), Tania Maria Ortiga-Carvalho (Instituto de Biofísica Carlos Chagas Filho, UFRJ), Flávia Carvalho Alcantara Gomes (Instituto de Ciências Biomédicas, UFRJ)

Cerebellar development on the post-natal period is mainly characterized by an intense cellular proliferation in the external granular layer (EGL) followed by migration of granular cells (GC) through the Bergmann glia (BG) fibers, in order to form the granular layer in the adult. All these events are drastically affected by thyroid hormone (TH) levels. Most of the biological effects of TH are mediated by the binding of T3 to the nuclear receptors type alpha (TRa) and beta (TRb). In this work, we analyzed the cerebellar development of D337T TRb mutant mice, a mutation which impairs T3 binding to its receptor. In vivo and in vitro studies demonstrated an alteration in the mutant cerebellar morphology with 1. 202

livro.indd 202 7/7/2008 16:33:51 Plant Cell Biology share of cells with heteromorphic-paired nucleoli) in interphase nuclei of cells of Allium cepa growing in the presence of cadmium (Cd) solution. Onion seeds K-001 were germinated in Petri plates containing Milli-Q INDUCTION OF CHROMOSOME DOUBLING OF water being the negative control (NC) and cadmium ONION (ALLIUM CEPA L.) THROUGH IN VITRO (Cd) in concentrations of 0.017mg/L and 0.007mg/L. GYNOGENESIS After treatment roots were fixed in 3:1 ethanol/glacial Ana Elisa de Oliveira e Longo (IAC), Walter José acetic acid (Carnoy solution) for 24 h. To prepare the Siqueira (IAC), Ilene Ribeiro da Silva Passos (IAC), slides, the meristematic regions were covered with a Marta Dias Soares Scott (IAC), Olga Satie Suzuki coverslip and carefully squashed into a drop of 45% (SAKATA), Cicero Beserra Menezes (SAKATA) acetic acid solution. The coverslip was removed with liquid nitrogen and the slides stained with 50% AgNO3 One of the great barriers of hybrid onion varieties is to solution. The number of nucleoli and their dimensions identify and select male sterile lines. The maintainer were measured in 100 cells per one root for 10 roots lines possess generation times of years. The specifc of each experimental series using immersion objective. cabability to obtain superior hybrids depends on the Treatment with cadmium (0.017mg/L and 0.007mg/L) pollinating homozygous lines. In vitro ovule and ovary caused an increase in the number of cells with one, tissue culture techniques have made it possible in two (homomorphic and heteromorphic), three and four two years. The objective of this work was to induce nucleoli and changes in the nucleolar size. Diminution chromosome doubling in haploid plants regenerated in the number of cells with one nucleolus was seen through in vitro gynogenesis. A total of 440 flower especially in concentration 0.017mg/L. In conclusion, buds from 15 genotypes supplied by SAKATA SEED our results seem to support also other studies opinion SUDAMERICA LTDA were cultured on B5 medium that nucleoli may be useful for the demonstration of Gamborg et al.1968 and 0.74% regenerated into metal toxicity. plantlets. Chromosome counting of root tip cells was [email protected] used to evaluate ploidy levels of regenerants. Growing root tips meristems qere excised and pretreated with 8 K-003 hydroxyquinoline for 4.5 h at room temperature. After THE USE OF NUCLEOLAR MORPHOLOGICAL this, root tips were fixed in 3:1 ethanol : glacial acetic CHARACTERISTICS IN CELLS OF ALLIUM CEPA acid for at least 30 min. Fixed roots were washed in FOR THE ASSESSMENT OF THE LEAD (PB) distilled water and hydrolyzed in 20% pectinase in TOXICITY sodium citrate buffer for 30 min at 370C. The roots Maysa Succi, Lucilene Regina Maschio, Maria Tercília were then washed in distilled water and fixed in 45% Vilela de Azeredo Oliveira acetic acid, root tips were subsequently squashed (UNESP/IBILCE, Department of Biology) under a coverslip in a drop of 45% acetic acid. Staining was done with 2% Giemsa in phosphate buffer, for The nucleolus is a cell component that reflects the 5 min. Up to now, chromosome counting has shown state of cell activity. The size of the nucleolus usually that 4 regenerants are diploids or doubled haploids. indicates its overall activity but may also reflect the Regenerants will still be submitted to esterase isozyme degree of coordination of all processes affecting electrophoresis analysis and SSRs microsatellitesto the accumulation of ribosomal half units within the establish the frequency of homozygous gameticor nucleolus. The aim of this study was to assess heterozygous somaticorigin. the citotoxicity of lead on quantitative nucleolar [email protected] characteristics (number and size of nucleoli, share of cells with heteromorphic-paired nucleoli) on interphase K-002 nuclei of cells of Allium cepa. Onion seeds were STUDY OF THE NUCLEOLAR ALTERATIONS IN germinated in Petri plates containing Milli-Q water being CELLS OF ALLIUM CEPA AFTER TREATMENT WITH the negative control (NC) and lead at the concentrations CADMIUM HEAVY METAL of 0.025mg/L and 0.034mg/L. After treatment roots were Tamires Fernanda Vilas Boas Cordeiro Correia fixed in 3:1 ethanol/glacial acetic acid (Carnoy solution) (UNESP/IBILCE, Department of Biology), Lucilene for 24 h. To prepare the slides, the meristematic Regina Maschio (UNESP/IBILCE, Genetic Graduate regions were covered with a coverslip and carefully Program), Maria Tercília Vilela de Azeredo Oliveira squashed into a drop of 45% acetic acid solution. The (UNESP/IBILCE, Department of Biology) coverslip was removed with liquid nitrogen and the slides stained with 50% AgNO3 solution. The number The nucleolus is very sensitive to various stresses of nucleoli and their dimensions were measured in 100 e.g., ambient atmosphere, temperature and drugs. In cells per one root for 10 roots of each experimental stress conditions a disorganization of structure and series using immersion objective. Treatment with lead disturbances in the activity of nucleolus takes place. (0.025mg/L and 0.034mg/L) caused an increase in The aim of this study was to investigate the quantitative the number of cells with one, two (homomorphic and nucleolar characteristics (number and size of nucleoli, heteromorphic), three and four nucleoli and changes 1. 203

livro.indd 203 7/7/2008 16:33:52 in the nucleolar size. Diminution in the number of cells Flow cytometry has been used for somaclonal variation with one nucleolus was seen especially in concentration detection in plant tissue culture.This application is 0.034mg/L. In conclusion, the effectiveness of our important because this phenomenon can affect the approach of using nucleolar parameters as a means for in vitro morfogenic processes.This research was assessment of lead cytotoxicity was confirmed. conducted in order to verify the somaclonal variation [email protected] occurrence in cell aggregate suspension cultures of Coffea arabica (4x=44 chromosomes).Friable C. arabica K-004 callus were established and maintained in embryogenic CHROMOSOME ABERRATION AND medium.In the regular subcultures every 15 days, MICRONUCLEUS FREQUENCIES IN ALLIUM samples of cell aggregates were collected.Nuclei were CEPA CELLS EXPOSED TO WATER SAMPLES isolated and stained from the 16 fixed-samples.The CONTAMINATED BY POLLUTANTS nuclei suspensions DAPI-stained were analyzed by Lucilene Regina Maschio (UNESP/IBILCE, Genetic flow cytometer. Three independent repetitions were Graduate Program), Maysa Succi (UNESP/IBILCE, performed for all samples,considering that more than Department of Biology), Tamires Fernanda Vilas 10,000 nuclei were analyzed in each. FlowMax software Boas Cordeiro Correia (UNESP/IBILCE, Department (Partec) was used for data analyses.The flow cytometry of Biology), Maria Tercilia Vilela de Azeredo Oliveira procedures supplied histograms showing G0/G1 peaks (UNESP/IBILCE, Department of Biology), Maria with coefficient of variation below 3. This result indicates Aparecida Marin Morales that the suspensions contained sufficient isolated and (UNESP/IB, Department of Biology) intact DAPI-stained nuclei. G0/G1 peaks, generated by tetraploid (4x) cells, were observed in all histograms. Cell alterations and micronuclei can be spontaneously In the same histograms were also looked small originated; however, their induction is commonly used peaks provided by cells with numerical chromosomic to detect genetic damage derived from exposure to a alterations. Samples collected from the fourth to twelfth mutagenic agent. In the present study, the potential subculture also produced G0G1 peaks of octaploid for micronucleus (M) and chromosome aberration (8x) cells.This study evidenced the occurrence of (CA) induction in cells of Allium cepa after exposure somaclonal variation during the in vitro tissue culture of to water samples from the Preto river, localized in the the C. arabica cell aggregates. city of São José do Rio Preto-SP, Brazil was evaluated. [email protected] Samples were collected in six river sites and chemical analyses carried out. Onion seeds were germinated in K-006 several Petri plates, each plate covered with filter paper ECOLOGICAL LEAF ANATOMY OF OCHROMA and individually poured with a distinct water sample. PYRAMIDALE (CAV. EX LAM.) URB. IN SOUTH- After treatment roots were fixed in 3:1 ethanol/glacial WEST AMAZON. acetic acid (Carnoy) for 24 h, carefully squashed and Marcelo Pereira da Costa, Paulo Cesar Poeta hydrolysed with 1 N HCl at 60 °C for 8 min, washed Fermino Junior, Rusleyd Maria Magalhães de Abreu with distilled water and Schiff stained for 2 h in the (Universidade Federal do Acre) dark. Pollutant-free Milli-Q water was used as negative control (NC), and trifluralin solution was used as positive The Amazon forest is considered the last major area control (PC). All the categories were analyzed by of wet relatively intact tropical forest in the world. counting 5,000 cells per treatment, being 500 cells per Among the tree species with potential for use for slide, comprising a total of 10 slides. The frequencies of reforestation stands out Ochroma pyramidale (Cav. Ex CA and MN were found to be higher than those found Lam.) Urb. The objective this work was to describe the in NC. The CA observed were C-metaphases; stick anatomical features leaf and relate to environmental chromosomes, anaphase irregularities; chromosome conditions. Were used leaves of adult plants, collected breaks and binucleate cells. Some cells showed loss of during seasonal dry. Observations were made in light chromosomes, which persisted up to telophase, such microscopy through frontal views of the leaf blade losses probably resulting in micronucleated interphase surfaces. The epidermis is single layer, covered with cells. The results indicate that pollutants encountered cuticle thickened in cross section. On the adaxial in the water samples present mutagenic activity, due to face, the epidermal cells present format multifaceted, ability to promote cellular alterations. irregular sizes, and straight anticlinal walls and [email protected] thickened. The trichomes are the type star, distributed in the abaxial face. The leaves are anfihipoestomatous. K-005 The stomata are paracitic type. The average media OCCURRENCE OF SOMACLONAL VARIATION IN in the length of the cell-guard of stomata in the face COFFEA ARABICA CELL AGGREGATE CULTURES abaxial is 18.16mm, and in the adaxial face is 16,81mm. SCREENED BY FLOW CYTOMETRY The width of the cell-guard is 5.14mm.The density of Thaís Cristina Ribeiro da Silva, Wellington Ronildo stomata in the adaxial face is 5.14 st/mm2. On the Clarindo, Carlos Roberto de Carvalho face abaxial, stomatal density showed higher values, (Universidade Federal de Viçosa) with 322.27 st./mm2. It was concluded that the high 1. 204

livro.indd 204 7/7/2008 16:33:52 density stomatal together with the great development Stem Cells of the cuticle and epidemis suggest xeromorphic characteristics for O.pyramidale. [email protected] L-001 MAGNETICALLY LABELED EMBRYONIC STEM CELLS MAINTAIN PLURIPOTEN CHARACTERISTICS FOLLOWING LONG TERM CULTURE EXPANSION Erika Regina Leal de Freitas (Departamento de Bioquimica e Biologia Molecular - Instituto de Ciências Biológicas), Paula Roberta Otaviano Soares (Departamento de Bioquímica e Biologia Molecular - Instituto de Ciências Biológicas), Regiane Lopes dos Santos (Instituto de Química - UFG), Emilia Celma de Oliveira Lima ( Instituto de Química - UFG), Lidia Andreu Guillo (Instituto de Ciências Biológicas - Universidade Federal de Goiás)

INTRODUCTION: Embryonic stem cells (ESC) are originally grown on a feeder of primary mouse embryo fibroblasts (MEF) cells. In order to exploit the clinical potential of embryonic stem cells (ESC), new methods for in vitro expansion need to be developed. Iron-oxide nanoparticles have been clinically used as contrast agents for magnetic resonance imaging (MRI). Recently this powerful technique has been tested to monitor the traffic of labeled ex vivo adult stem cells. METHODS: Control cells were seeded on a feeder of mouse embryo fibroblasts (MEF) for comparative purposes. Nanoparticles-treated cells were grown on 25 cm2 culture flasks and placed above a neodymium magnet (4000 Gauss). The ensemble was put on a humidified incubator at 37oC and 5% CO2. Cultures were passaged every 48, 72or 96 hours over three weeks. RESULTS AND CONCLUSIONS: Comparative analysis including immunofluorescence for SSEA-1 and Oct-4 protein expression, RT-PCR for OCT-4 and Nanog gene expression, demonstrated that magnetic nanoparticles can support prolonged self-renewal of ESC. Average population-doubling times recorded for nanoparticles- treated cells (20.7 + 0.7 hours) were not significantly different from their respective controls (16.2 + 0.9 hours). Nanoparticles-treated cells showed a normal karyotype. So, we have demonstrated that iron oxide anionic nanoparticles can maintain the proliferation potential of embryonic stem cells in vitro without any feeders, providing a valuable tool for both the in vitro expansion of feeder-free ESC and for in vivo tracking in animal models studies. [email protected]

L-002 DISTINCT SUPPORT OF B LYMPHOCYTES GENERATION BY OSTEOBLASTS AND RETICULOCYTES FROM MURINE BONE MARROW Evelyn Wilkens Eder (UFRJ), Alex Balduino (INTO), Radovan Borojevic (UFRJ)

Heamatopoetic stem cells (HSCs) are able to self- renew and to differentiate into all blood cell types. They dwell in the subendosteal/endosteal niche of 1. 205

livro.indd 205 7/7/2008 16:33:52 bone marrow (BM), which is composed of two stroma- CNPase (oligodendrocytes) and GFAP (astrocytes) cell populations: subendosteal reticulocytes and were also upregulated (24 and 55 fold respectively, osteoblasts. Our group isolated these populations 21d p<0.05), which was confirmed by confocal and named reticulocytes from subendosteal niche as immunolocalization. In conclusion, murine derived SKPs FRET and osteoblast from endosteal niche as FOST. were capable of in vitro expansion and both neuronal When co-cultured with bone marrow mononuclear cells and glial differentiation, paving the way for isolation, (BMMC), FOST stroma induces low proliferation and characterization and therapeutical application of human high maintenance of early haemopoietic progenitors, skin SKPs. while FRET stroma induces high proliferation and [email protected] differentiation. Besides HSCs support, this niche also constitute B cell progenitor niche as indicated by L-004 imuno-localization assays in which B precursors are TRANSPERICARDIAL INFUSION OF BMMC IN in close association with reticulocytes otherwise near ACUTE INFARCTION to osteoblast. To investigate the role of each stroma Érika Branco (FMVZ/USP), Carlos Alberto Palmeira population on B lymphopoiesis, we expanded FRET Sarmento (FMVZ/USP), Emerson Ticona Fioretto and FOST and performed BMMC co-cultured on (FMVZ/USP), Angelo João Stopiglia (FMVZ/USP), both stromas. We also performed phenotypical and Paulo César Maiorka (FMVZ/USP), Carlos A. Campos molecular characterization (flow cytometry and PCR) of (Incor/SP), Celso Takimura (Incor/SP), Pedro Alves these expanded stromas. Our results showed that there Lemos (Incor/SP), José Antônio Franchini Ramires was more relative generation of B cells in FOST than (Incor/SP), Maria Angelica Miglino (FMVZ/USP) FRET co-culture. Moreover, FRET and FOST showed different expression profile of positive and negative Stem cells therapy (SCT) has emerged as a potential B cell precursor regulators and adhesion molecules treatment for cardiovascular diseases because of involved in lymphopoiesis. Thus, in this present study, possible capability to induct neovascularization osteoblasts have higher potential for B cell generation/ and tissue formation. SCT has been applied using maintenance than reticulocytes. intracoronary,intramyocardic and retrogade infusion [email protected] techniques.Another infusion technique is the transpericardic (TC),which has not yet been tested in L-003 STC, even though the application in drugs therapeutics. NEURONAL AND GLIAL DIFFERENTIATION OF We present TC as an alternative route for infusion of MURINE SKIN-DERIVED SKP STEM CELLS cells in pigs submitted to acute myocardim infarction Maria Fernanda P A D Forni (Instituto de Química (AMI).Bone marrow was sampled from iliac crest with da USP), Aline Ramos Maia (Instituto de Química), a bone punction needle.Mononuclear cells (BMMC) Alexandre Hamilton Pereira Ferreira (Allergisa Pesquisa were separated using Ficoll-Paque Plus.Animals were Dermato Cosmética), Mari Cleide Sogayar (Instituto de infarcted by femoral artery catheterization using a Bioquímica da USP) balloon catheter displaced at the first interventricular coronary artery.Epicardic space was accessed by pre The skin is continuously undergoing self-renewal to umbilical region at median portion using an epidural repair damaged tissue and replace old cells, relying needle and 10E8 of bone marrow mononuclear on stem cell populations to maintain homeostasis. In cells (BMMC) Hoechst stained were injected (Group the dermis follicle papillae, a stem cell named SKP 1).Control group was composed by BMMC injection in for “skin precursor”, displays differentiation potential animals without infarction.Groups were evaluated 21 into mesodermal and neuronal lineages.We isolated days after BMMC injection and histological investigated SKP floating spheres from murine Balb/c skin and under microscopy.Macroscopic orservation revealed maintained them in vitro up to the 10th passage. By pericardium without fluid and adherences including the qPCR, we show that upon culturing the mRNA levels punction region and the infarct area was observed. for nestin and fibronectin, two positive SKP markers, Microscopic evaluation showed 109494+-46505 increased by 31 and 4.7 fold respectively (4th passage cells/myocardial area in both groups. Nevertheless, p<0.001, GAPDH and HPRT). The same could be there were significant more cells/area in group of observed at the protein level by Western blotting. The AMI model(p<0.01). We believe the transpericardic negative SKP marker, vimentin, decreased 1,5 fold technique is an alternative for cell therapy in cases of at the mRNA level after the 1st passage, remaining acute myocardial injury. constant upon further subculturing, as confirmed by [email protected] WB. By immunolocalization, we found that nestin and fibronectin are expressed at high levels in the clusters L-005 core while vimentin is expressed by a few peripheral OSTEOGENIC POTENTIAL OF EX-VIVO ADIPOSE cells. SKP cells were capable of neuronal differentiation. TISSUE-DERIVED MESENCHYMAL STROMAL The relative mRNA levels for bIIItubulin, a neuron CELLS marker, increased 36, 42 and 44 fold (upon 7, 14 and Natália Silva Araujo dos Santos (Instituto de Ciências 21 days culturing, p<0.001). Glial markers such as Biomédicas, UFRJ), Karina Ribeiro da Silva (Instituto de 1. 206

livro.indd 206 7/7/2008 16:33:52 Ciências Biomédicas, UFRJ), Marcelo Henrique Prado P3, P6, P9 and P12). A total of 106 cells/mL were da Silva (Instituto Militar de Engenharia do Rio de cultivated in DMEM/F12 medium containing 10% FBS. Janeiro), Alexandre Malta Rossi (Centro Brasileiro de Cell proliferation was evaluated 24h later by [3H]- Pesquisas Físicas), Marcos Farina de Souza (Instituto thymidine incorporation. Kinetics of population growth de Ciências Biomédicas, UFRJ), Cesar Silveira Cláudio- was determined by cell counts every 72h for 12 days. da-Silva (Departamento de Clínica Médica, Faculdade Daily analysis of cell morphology was performed by de Medicina, UFRJ), Marcelo Carlos Aniceto de Souza phase contrast microscopy. The proliferative capacity (UFRJ), Radovan Borojevic (Instituto de Ciências of neuroprogenitors peaked at P3 and was significantly Biomédicas, UFRJ), Leonardo Rodrigues de Andrade lower at P9 and P12. Analysis of 12 days in culture (Instituto de Ciências Biomédicas, UFRJ), Maria Isabel showed that cell populations tend to decline during the Doria Rossi (Instituto de Ciências Biomédicas, UFRJ) first 3 days in vitro and became stable thereafter. This effect could be related to spontaneous differentiation Bone marrow (BM) stromal cells are the progeny of of neuroprogenitors since early changes in cell multipotent and self-renewable mesenchymal stromal morphology were detected in vitro, with evidences cells (MSC). BMMSC have been used, in association of cell differentiation already observed after 1 d.i.v.. with biomaterials, in bone tissue engineering, but the Altogether, these findings indicate that neuroprogenitor need of high numbers of BMMSC is critical. Recently, cells can be isolated from the cerebellum of the presence of MSC in adipose tissue (AT) was shown, neonatal rats. Although their proliferative capacity and because of its vast distribution and accessibility decreases significantly within the first 12 days of age, they have been proposed as an alternative source neuroprogenitors isolated up to P3 display the highest for cell therapy. However, subtle differences in the proliferation rates and could be used as an alternative potential to differentiate towards the osteogenic lineage source of cells for therapeutic purposes. were observed. We proposed that these differences [email protected] are due to the presence of adipogenic progenitor cells among the ATMSC. To address this question, L-007 adherent cells were obtained by collagenase digestion IN VIVO DISTIBUTION OF HUMAN IMMATURE of subcutaneous AT from patients submitted to plastic DENTAL PULP STEM CELLS AFTER INFUSION INTO surgery at HUCFF-UFRJ, and cultured with a mixture HEALTHY MICE (vol/vol) of osteogenic and adipogenic culture medium, Maranduba, C. M. C. (Genetic Laboratory of which allows the differentiation towards both lineages. Butantan Institute), Sant’Anna, O. A (Laboratório Freshly isolated and osteoinduced ATMSC and de Imunoquímica of Butantan Institute), Lizier N. F. BMMSC were plated in hydroxyapatite (HA) scaffolds (Morphology Department of Federal University of São and maintained for up to 2 weeks. Although most AT Paulo), Monteiro BG (Genetic Laboratory of Butantan adherent cells differentiated into mature adipocytes, Istitute), Carvalho, L. V. (Morphology Department of some foci of mineralization were also seen when cells Federal University of São Paulo), Fonseca, S.A.S were cultured with the mixed medium, suggesting that (Genetic Laboratory of Butantan Institute), Alexandre freshly isolated cells were heterogeneous. Furthermore, Pereira (Genetic Laboratory of Butantan Institute), in contact with HA, ATMSC, like BMMSC, were able to Humberto Cerruti Filho (Clinic and Center of Odontology adhere, spread, and proliferate, covering most of the Research – CERA), Kerkis, A. (Applied Genetics, surface of the HA scaffolds. Our preliminary results Veterinary Activities Ltda. Center of Innovation suggest that cells with osteogenic potential can be Technologies, Sao Paulo, Brazil.), Kerkis, I (Genetic isolated from AT, and are a promising source of MSC for Laboratory of Butantan Institute) bone engineering. [email protected] Mesenchymal stem cells (MSC) can be isolated from an adult organism and are now at the center of the L-006 current stem cell therapy research. Their administration PROLIFERATIVE CAPACITY OF is a promising option due to their ability to regenerate NEUROPROGENITORS IN THE POSTNATAL RAT damaged organs. Nevertheless, the great number CEREBELLUM of failures of cellular therapies could be attributed to DANIELA EMI SUZUKI, CAROLINA BATISTA ARIZA, many reasons such as the type of stem cell, dose MARIMÉLIA APARECIDA PORCIONATTO, OSWALDO of cellular therapeutic, dosing regime, and route of KEITH OKAMOTO (UNIVERSIDADE FEDERAL DE delivery. Knowledge of such MSC biodistribution in SÃO PAULO - UNIFESP) the recipient organism is also important in order to prevent remote organ engraftment of stem cells and Advances in cell therapy protocols may benefit to overcome potential collateral effects, which can patients with neurodegenerative disease, although be produced by stem cell therapy in patients. In the the choice and availability of the cell type to transplant present study, fluorescently labeled human immature is still an open question. For this application, we dental pulp stem cells (hIDPSC), which co-express the have evaluated neuroprogenitors isolated from rat markers of both human embryonic stem (ES) cells and cerebellum (n=4) at different postnatal days (P1, MSC were infused into a mouse using different routes, 1. 207

livro.indd 207 7/7/2008 16:33:52 including the intravenous (iv), subcutaneous (sc), or Thais Raquel Martins Filippo, Yvette May Coulson- intraperitoneal (ip). Our results indicate multiple homing Thomas, Marimélia Aparecida Porcionatto (UNIFESP) sites for injected hIDPSC, since their homing was detected in the heart, liver, spleen and bone marrow of Injury to the vertebrate central nervous system (CNS) BALB/c mice at different time intervals (24 hours 7, 14, leads to the formation of a glial scar that contains 21 and 28 days) after having used all three injection four axonal growth inhibitory molecules: MAG, routes. The estimated levels of engraftment of hIDPSC OMgp, Nogo and chondroitin sulfate proteoglycans. in these tissues was assessed by Flow Cytometry and Various studies demonstrate that functional recovery ranged from 0.03 and 0, 1 %. In this study, the iv route is possible by degrading chondroitin sulphate using was the best since higher number of labeled hIDPSCs chondroitinases. We have cloned the F. heparinum were detected when compared with other routes of gene for chondroitinase AC into the expression vector administration. pcDNA3.1(+) and shown that it is expressed by adult [email protected] bone marrow-derived mononuclear cells transplanted at injury sites in the motor cortex (Coulson-Thomas et al, L-008 J. Neurosci. Meth, 171:19-29, 2008). We also showed CLONAL ANALYSIS OF ADIPOSE TISSUE that degradation of chondroitin sulfate enhances the MESENCHYMAL STEM CELLS DERIVED FROM expression of growth-associated protein GAP-43. In the LEAN AND EX-OBESE PATIENTS present work we have analyzed the expression of GAP- Karina Ribeiro da Silva, Leandra Santos Baptista, 43 and synaptophysin as well as functional recovery Marcelo Carlos Aniceto de Souza, Cesar Silveira of animals submitted to motor cortex injury followed by Cláudio-da-Silva, Radovan Borojevic, Maria Isabel bone marrow-derived cells expressing chondroitinase Doria Rossi (Universidade Federal do Rio de Janeiro) AC transplantation. Using immunohistochemistry we observed that GAP-43 and synaptophysin Adipose tissue (AT) is a source of cells that share were expressed close to injury sites in all animals with bone marrow mesenchymal stem cells (MSC) the that received chondroitinase AC-expressing cells. capacity to differentiate towards mesodermal lineages. Spontaneous partial functional recovery was observed Although AT-derived plastic adherent cells differentiated in animals that received vehicle but not in those that in vitro into osteoblasts, they expressed the adipogenic received cells, regardless of expressing chondroitinase transcriptor factor PPARg2 and differentiated more or not. Therefore, chondroitinase AC expressed by adult efficiently into unilocular fat cells, suggesting that it bone marrow-derived mononuclear cells transplanted at contains commited adipogenic progenitor cells, wich CNS injury sites results in axonal regeneration but does could jeopardize its use for cell therapy. Identification of not promote functional recovery. Granted by CNPq., clonogenic multipotent MSC in AT are then important. FAPESP, FADA-UNIFESP. AT-MSC were isolated from the subcutaneous of lean [email protected] and ex-obese patients undergoing plastic surgery at the HUCFF-UFRJ. Clones were obtained by limiting L-010 dilution: 10-25 cells derived from collagenase digested ANALYSIS OF THE PROLIFFERATIVE POTENTIAL fat tissue were plated and wells with a single cell were OF HUMAN MESENCHYMAL STEM CELLS (HMSC), identified. 45 (12,5%) clones from lean and 20 (8,3%) OBTAINED FROM FETAL (FHMSC) AND ADULT from ex-obese patients were selected. Differences TISSUES (AHMSC) in proliferation and differentiation potential towards Danilo Candido de Almeida, Aparecida Maria Fontes, adipogenic and osteogenic lineages were observed in Fernanda Ursoli Ferreira, Marcela Correia de Freitas, clones from lean patients. Out of the 45 clones, 4 clones Maristela Delgado Orellana, Patricia Viana Bonini were able to reach confluence after passage. From Palma, Dimas Tadeu Covas (Centro de Terapia Celular these clones, about 50% were pure adipogenic and only CTC - Centro Regional de Hemoterapia de Ribeirão 25% were bipotent. Some multilocular preadipocyte Preto FMRP/USP) clones were identified in both patients. In conclusion, the AT adherent fraction is heterogeneous, containing Spindle-shaped hMSCs have self renewing ability cells with limited proliferation potential commited and potential differentiation along multiple tissue cells to the adipogenic lineage, and higly proliferative lineages including, osteoblasts (Os), adipocytes (Ad), bipotent cells. In order to distinguish and separate chondrocytes (Ch), myocytes, and marrow stromal these subpopulations, molecular and phenotipic cells. The research for a better understanding of characterizations are in progress. hMSC`s biological features leads to several paths. This [email protected] study aims to determine the growth kinetics and the proliferation potential of purified ahMSCs and fhMSCs L-009 during extensive subcultivation. We established primary CHONDROITINASE AC EXPRESSING BONE cultures of hMSCs from bone marrow aspirates toward MARROW-DERIVED CELLS TRANSPLANTED adult tissue (AT) and collagenase digestion toward INTO MOTOR CORTEX INJURY SITE PROMOTES several fetal tissues (FT). Sistematicaly, these cells yet EXPRESSION OF GAP-43 AND SYNAPTOPHYSIN were characterized immunofenotipically, analyzed to 1. 208

livro.indd 208 7/7/2008 16:33:52 their proliferation capacity [population doubling (PD)] method to bone regeneration of critical bone defects. and differentiation potentials into three mesenchymal [email protected] lineages in vitro (Os, Ad and Ch). Multipotential hMSCs in culture exhibited a nearly 5-day growth lag phase L-012 to both two cells types, followed by an approximate MORPHOLOGICAL ANALYSIS OF THE 38-day log phase to ahMSCs and a 90-day log phase HEMATOPOIETIC TISSUE IN THE BONE MARROW to fhMSC. Passage cultures proceeded through OF LITHOBATES CATESBEIANUS (ANURA) the different stages and the cells obtained from AT Pedro Paulo de Abreu Manso (Instituto Oswaldo Cruz), reached no more than 5 passages (61±8,5 days) with Lycia de Brito Gitirana (UFRJ), Priscila Tavares Guedes the average number of PD to be 26 ±5,6 while cells (Instituto Oswaldo Cruz), Barbara Cristina Euzebio obtained from FT reached 12 passages (101±11,04 Pereira Dias de Oliveira (Instituto Oswaldo Cruz), Luzia days) with a PD of the 61±10,1. Finally the cells Fátima Gonçalves Caputo (Instituto Oswaldo Cruz), cultures resulted into a stationary growth phase. The Marcelo Pelajo Machado (Instituto Oswaldo Cruz) results of our experiments proved that the fhMSC are highly proliferative in vitro and the PD can become an Hematopoiesis is the process of blood cell formation attractive useful tool to analyze the expansion potential during both the embryonic and adult stages of an of the stem cells in culture. organism. Amphibian is the oldest model used to study [email protected] the cascade of events leading to embryonic patterning. Conversely, the adult amphibian hematopoiesis is L-011 related only in some species, and information is yet ADIPOSE TISSUE MESENCHYMAL CELLS confused and scarce. Considering that the bone CULTURED IN BIOCERAMICS BASED MATERIALS marrow is the main site of hematopoiesis in other TO BONE HEALING vertebrates, this work was carried out in order to Husten da Silva Carvalho (UFRJ), Karina R. da Silva describe the hematopoiesis in the bone marrow of (UFRJ), Natália S.A. Santos (UFRJ), Leandra S. Lithobates catesbeianus, using light microscopy Baptista (UFRJ), Marcelo H. Prado da Silva (Instituto and immunohistochemical methods under confocal Militar de Engenharia), Alexandre M. Rossi (Centro laser microscopy. The bone marrow of the vertebrae, Brasileiro de Pesquisas Físicas), Marcos Farina the femur and the finger consists of hematopoietic (UFRJ), Radovan Borojevic (UFRJ), Maria Isabel D. stroma, supporting different cell types such as Rossi (UFRJ), Leonardo R. Andrade (UFRJ) hematopoietic cells, fat cells, pigment cells and stromal cells. The stroma is formed mainly by scarce thin Bioengineering is a new scientific area that concerns and delicate reticular fibers organized as a network. biomedical and engineering disciplines that develops Groups of CD34+ cells were observed intermingled different biomaterials that can be used as tissue with hematopoietic cells in different stages of its substitutes. Bioceramics composed by hydroxy-apatite/ development. Osteoblasts show positive c-kit labeling, beta-Tricalcium phosphate (HA/TCP) have been studied besides few cells with rounded eccentric nucleus as bone mimetic material due to its biocompatibility and intermingled with hematopoietic cells. EpoR and osteoconductive ability. Bone marrow mesenchymal G-CSFR positive cells occur as islets of rounded cells stem cells (BMSC) and adipose tissue derived stem near venous sinus and the endosteum of the bone cells (ASC) can be differentiated in vitro into adipocytes, marrow. Then, this study indicates that the bone marrow chondrocytes, myoblasts and osteoblasts. In this work, is an important site of hematopoietic activity in anuran. human BMSC harvested of iliac crest and ASC isolated [email protected] by liposuction from abdominal region were proliferated in culture flasks in DMEM culture medium during 02 L-013 weeks. An osteoinductive medium was added to DMEM, BONE MARROW MONONUCLEAR CELL offered to cells during one and two weeks. Those TRANSPLANTATION IN HEPATECTOMIZED RATS: cells were then cultured in porous HA/TCP (60/40) LAMININ EXPRESSION ANALYSIS during 07 and 14 days and examined by scanning and Simone Nunes de Carvalho (Laboratório Cultura transmission electron microscopy. Osteopontin, an de Células - UERJ), Erika Afonso Costa Cortez important molecule in bone phenotype, was studied (Laboratório Cultura de Células - UERJ), Ana Carolina by immunofluorescence assays. Preliminary results Stumbo (Laboratório Cultura de Células - UERJ), have shown that HA/TCP provided cell adhesion and Alessandra Alves Thole (Laboratório Cultura de proliferation. BMSC and ASC cultured during 02 weeks Células - UERJ), Manoela Lopes Carvalho (Laboratório under the biomaterial shown a general morphology Cultura de Células - UERJ), Carlos Eduardo Caetano similar to osteoblasts. Round cells that could indicate (Laboratório de Cirurgia Experimental - UERJ), Ruy lipid storage cells (adipoblasts) were not seen. The Garcia Marques (Laboratório de Cirurgia Experimental expression of osteopontin was detected in both cells - UERJ), Marcelo Pelajo-Machado (Departamento de culture indicating an osteoblast differentiation. BMSC Patologia - Instituto Oswaldo Cruz), Luis Cristóvão and principally ASC isolated from proper patient and Porto (Laboratório Cultura de Células - UERJ), Laís cultured in this biomaterial for 14 days can be a new Carvalho (Laboratório Cultura de Células - UERJ) 1. 209

livro.indd 209 7/7/2008 16:33:52 The adult bone marrow retains two populations of stem hybridized with a cDNA microarray (4,500 sequences). cells with emerging importance for the treatment of An irrelevant cDNA was Cy5 labeled and used as diverse liver diseases: hematopoietic and mesenchymal reference. The differential gene expression during stem cells. However, the mechanisms that control liver differentiation was analysed by the SAM method (FDR regeneration after bone marrow cells transplantation 5%). The number of modulated genes increased toward are still controversial. Liver regeneration after partial osteoblast lineage commitment, achieving 20% of hepatectomy is a complex process that requires them after 7 days. As early as 24 hs were observed the proliferation of all hepatic cells. Growth factors, genes involved with osteoblast phenotype, including cytokines and extracellular matrix molecules are IGFBP4, FN1, ITGA5, BGN and HTRA1. These findings key elements in this process. Laminins are a family revealed that the initial differentiation process and the of extracellular matrix proteins with adhesive and emergency of a specific phenotype is accompanied by chemotactic functions. The aim of this study was to an increasing changes in the gene expression. investigate laminin expression during liver regeneration [email protected] induced by partial hepatectomy followed by bone marrow mononuclear cells (BMMNCs) transplantation. L-015 Rat BMMNCs were isolated by Ficoll-gradient LABELING OF HUMAN MESENCHYMAL STEM centrifugation, stained with DAPI and injected into CELLS WITH QUANTUM DOT recently hepatectomyzed rats via portal vein. Liver Daniela Mara de Oliveira, Lorena F. Pavon, Luciana C. sections obtained 1 day and 3 days after the surgery Marti, Tatiana T. Sibov, Lionel Gamarra, Edson Amaro were immunolabeled with anti-rat laminin primary Jr., Alexandre H. Campos (Instituto Israelita de Ensino e antibody and observed under a Laser Scanning Pesquisa Albert Einstein) Confocal Microscope. Results showed that 1 and 3 days after hepatectomy, laminin expression increases Mesenchymal stem cells (MSC) are a rare population in portal vessels and centrolobular veins. Moreover, of self-renewing, multipotent cells found in the bone the transient increase in laminin expression in the marrow, and more recently also identified in several proliferating hepatic sinusoids is correlated with the other tissues, including fat and umbilical cord (blood localization of the transplanted BMMNCs near this and vessel wall). There is an increasing interest in the laminin-rich area. These results strongly suggest that use of human MSC therapy in regenerative medicine laminin might be an important extracellular matrix due its multi-differentiation potential, high expansion component for bone marrow cell attachment and capability and low immunogenicity. Labeling cells migration in the injured liver. and tracking their fate in vivo is a crucial tool in the [email protected] study of the potential of stem cells in regenerative medicine. Traditional tracking agents such as green L-014 fluorescent protein and fluorescent dyes fail when MOLECULAR CHANGES DURING IN VITRO they are used to track delivered stem cells in vivo due OSTEOBLASTIC DIFFERENTIATION OF the autofluorescence of host tissue and limitations of MESENCHYMAL STEM CELLS existing labeling techniques. In this study, we report Vanessa Fontana, Adriane Feijó Evangelista, Janaína the efficacy of labeling human MSC using intracellular de Andrea Dernowsek, Cristina Moraes Junta, Glauce quantum dots. Quantum dots are small highly Crivelaro do Nascimento, Rayana Longo Bighetti, fluorescent photostable particles. We observed that Márcia Maria Chiquitelli Marques, Karina Fittipaldi MSCs can uptake quantum dots by a passive load Bombonato Prado, Adalberto Luiz Rosa, Geraldo Aleixo approach. In spite of the high proliferative nature of da Silva Passos Junior (Universidade de São Paulo) MSCs, the labeling of the cells was maintained for the tested 50 days. In addition, during the differentiation of Mesenchymal stem cells (MSC) are multipotent stem MSCs into chondrogenic, osteogenic and adipogenic cells that under proper stimuli can differentiate in cells, quantum dots remain to exist in the cells without several cell types of mesodermal origin. The stem cell significantly disturbing their differentiation behavior. lineage determination is accompanied by molecular These findings suggest that quantum dots are a useful changes, specially in the gene expression. Our aim probe for labeling and further tracking MSCs during was to evaluate the differential gene expression by differentiation in multiple lineages. This work was cDNA microarrays using a model-system of osteogenic suported by FAPESP and IIEP Albert Einstein. DMO is a differentiation of human MSC. These cells was isolated FAPESP fellow. from the bone marrow of healthy donors and cultured [email protected] 24 h to 21 days in alfa-MEM or in the differentiation medium (alfa-MEM supplemented with betaglycerol L-016 phosphate, ascorbic acid and dexametasone). The THE ROLE OF PKCS IN THE DIFFERENTIATION osteoblast penotype was confirmed by cell morphology, OF MURINE EMBRYONIC STEM CELLS IN alizarin red staining and alkaline phosphatase activity. CARDIOMYOCYTES Total RNA was obtained from undifferentiated and Talita Glaser, Deborah Schechtman, José Eduardo differentiated cells. Fluorescent Cy3 cDNA probes were Krieger (InCor HC/FMUSP) 1. 210

livro.indd 210 7/7/2008 16:33:53 Embryonic stem cells (ESC) are capable of proliferating reveals negative by PCNA in livers in this stages and self-renewing. To understand the role of of gestation. Fetal liver of dogs showed an intense differentiation of these cells to cardiomyocytes we can proliferative cell activity, it is justified by hematopoietic selectively interfere in specific signalling pathways. stem cell reservoir functional action, over there are The protein cinase C (PKC) family has 10 enzymes in development. These results imply that canine fetal which phosphorylate serine and threonine residues and liver is an excellent model for stem cell study, cells in it’s divided into 3 subfamilies, we used the classical phase of higher proliferative capacity, discarding the (cPKC), which contain a, bI, bII and gisoforms, and use of techniques for induction of hepatic proliferation novel (nPKC) include the d, e, h and q isoforms. cPKC by carcinogenic and noncarcinogenic injury. Financial and nPKC are known to be important in physiological Support: FAPESP. and pathological process of the heart. In this study [email protected] we investigated the role of cPKC and PKCe in ESC differentiation to cardiomyocytes using PKC isoenzyme L-018 specific peptides modulators. First of all we used BONE MARROW-DERIVED MESENCHYMAL the Hanging Drop method which consists of forming STEM CELLS (MSCS) CAN PREVENT ACUTE embryoid bodies (EB) by suspension drops and plating GRAFT-VERSUS-HOST DISEASE (GVHD) AFTER some days later. Specific PKC modulators (activator ALLOGENEIC BONE MARROW TRANSPLANTATION or inhibitor) were added daily to a final concentration (ABMT) of 500nM from the 6th day. Then EB were monitored Jorge Luiz Curado Siufi (FMRP-USP), Carolina Caliari daily by fase microscopy for beating foci. There was an Oliveira (FMRP-USP), Sérgio Britto Garcia (FMRP- increase in beating foci/EB upon treatment with PKCe USP), Patrícia Viana Bonini Palma (Centro Regional de activator peptide. Similar results were also obtained Hemoterapia), Dimas Tadeu Covas (FMRP-USP) with cPKC inhibitor peptide indicating that cPKC may display opposing effects compared with PKCe. We ABMT is an effective therapy in malignant diseases confirmed that our beating foci were cardiomyocytes by but the appearance of GVHD decrease the successful colocalization of sarcomeric myosin with the transcriptor of transplants. MSCs can expand extensively in vitro factor GATA4. We determined subcelular localization of and posses immunoregulatory proprieties. The aim of different PKC isoenzymes by confocal microscopy. this study was to evaluate the preventive therapeutic [email protected] potential of in vitro expanded MSCs to decrease the effects of GVHD. C57BL/6 mice were used as donor L-017 of ABMT, splenocytes (sple) and MSCs; and Balb/c PROLIFERATING CELL NUCLEAR ANTIGEN (n=79) and FVB GFP + (n=41) as receptors. Mice EXPRESSION IN DOGS FETAL LIVERS were distributed in following groups: 1- normal control; Cristiane Valverde Wenceslau (USP), Daniele dos 2- irradiated control; 3- singenic BMT; 4- ABMT; 5- Santos Martins (USP), Carlos Eduardo Ambrósio (USP), ABMT+sple; 6- ABMT+MSCs; 7- ABMT+sple+MSCs; 8- Phelipe Oliveira Favaron (USP), André Luis Rezende ABMT+sple+MSCs (2 infusions D0 and D+3). Animals Franciolli (USP), Adriana Caroprezo Morini (USP), were evaluated daily and clinical scoring system was Eduardo Vieira (Faculty of Veterinary Medicine, Sao used to assess the degree of GVHD. Probability of Joao da Boa Vista), Maria Angélica Miglino (USP) survival was evaluated by Kaplan-Meier method. Histological analyses of intestine, liver and spleen were Liver present high capacity of regeneration by mature performed on the day of death. Prolonged survival cells, highly differentiated, called hepatocytes, however was observed in groups that received co-infusion of in the normal adult liver the proliferation of these cells is MSCs. However, MSCs preventive treatments do not very small. In addition, reply the severe damages, the inhibit totally the clinical and histopathological signs of hepatocytes are able to fast proliferate, restoring the disease. Groups that don’t received preventive MSCs organ. The aim was to analyze the fetal liver in order to treatment died within 30 days after transplantation. check the potential proliferative of hepatic progenitors Animals that received MSCs and sple to increase the cells by endogenous cells replication related to specific incidence of GVHD (n=29/3; 89,7%) showed mortality marker to proliferanting cell nuclear antingen antibody rates higher than those that do not received sple (PCNA). Six samples from canine fetal liver with 30 and (n=14/9; 36%). Flow cytometry analysis of chimerism 60 days of gestation were fixed on parafolmaldeyde showed long-term engraftment with more than 90% of 3.5%, and embedded in paraffin. Tissues were cut in donor cells in peripheral blood of survived receptors. sections (5 µm) and submitted to immunohistochemical These results indicate the ability of MSCs to maintain techniques by PCNA antibody (1:800). Positive immunoregulatoy effects. Support: CTC, CEPID, staining for PCNA was based on cellular distribution FAPESP. and intensity red reaction of nucleus cells, by light [email protected] microscopy. PCNA positive reaction showed intense for hepatocytes and cells biliary ducts. High number L-019 of mononuclear cellular types of the fetal liver were GERM CELLS DERIVED FROM MOUSE positive by PCNA. And a small number of hepatocytes REPROGRAMMED SOMATIC CELLS 1. 211

livro.indd 211 7/7/2008 16:33:53 Thais de Mello Cintra Lavagnolli (Instituto Butantan), into bone, cartilage, fat and muscle. In this study, Simone Aparecida Siqueira da Fonseca (Clínica we characterized UCB-MSCs by their morphologic e Centro de Pesquisa em Reprodução Humana and immunophenotypic characteristics, cytochemical Roger Abdelmassih), Rui Cosme Serafim (Clínica e properties, and by their capacity of differentiation into Centro de Pesquisa em Reprodução Humana Roger adipogenic, chondrogenic and osteogenic lineages. Abdelmassih), Virgínia de Souza Pereira (Clínica We also identified the presence of genes related to e Centro de Pesquisa em Reprodução Humana committed lineage cells in undifferentiated UCB-MSCs. Roger Abdelmassih), Alexandre Kerkis (Clínica e We were able to detect by RT-PCR (Real-Time PCR) Centro de Pesquisa em Reprodução Humana Roger that undifferentiated MSCs express specific markers of Abdelmassih), Irina Kerkis (Instituto Butantan) committed lineage cells, like PPAR gama, LPL, SOX9, Osteocalcin and Osteopontin. Our results indicate Embryonic stem (ES) cells hold the ability to trigger that UCB is a good alternative source for obtaining nuclear reprogramming of a differentiated cell through MSCs. These cells can differentiate into adipocytes, cell fusion. Gametogenesis is a specialized regulated osteoblasts and chondrocytes and undifferentiated series of cell cycle events, involving mitosis and meiosis MSCs spontaneously express specific markers of as well as cell differentiation. Since this requires committed lineage cells. Financial support: FAPESP, unique morphologial changes, we evaluated if mouse ITAÚ and SBIBHAE. somatic hybrid cells (SHC) were able to differentiate [email protected] into germ cells (GC) in vitro. SHC were obtained by the fusion of HPRT-deficient ES cells and adult L-021 spleen cells. To induce GC differentiation we formed OPTIMIZATION OF EXPANSION AND embryoid bodies (EB) and added retinoic acid to the CHARACTERIZATION OF HUMAN ENDOTHELIAL suspension. Morphological, immunocytochemistry (IC), PROGENITOR CELLS (EPCS) ISOLATED FROM RT-PCR and electron microscopy (EM) analyses were PERIPHERAL BLOOD performed to evaluate SHC pluripotency and to confirm Francisco Vardiero Morais, Marianna Augusta Ferrari their differentiation into GC. SHC were positive for do Outeiro-Bernstein, Juliana Vieira Dias, Sara Santana Oct4, Nanog and SSEA1 antibodies and for Sox2 and Nunes, Verônica Morandi (Universidade do Estado Nanog expression, confirming their pluripotent capacity. do Rio de Janeiro (UERJ) / Dep. de Biologia Celular e Primordial GC (PGC) were observed migrating from Genética (DBCG)) the EB surface as well as cells resembling round and elongated spermatids and oocytes. RT-PCR revealed Endothelial precursor cells (EPCs) isolated from the expression of 12 GC-specific genes: Stella, Dazl, adult peripheral blood promote de novo vessel Piwil2, Text14, Bmp8b, Tdrd1, Haprin, Rnf17, Acrosin formation (adult vasculogenesis). Thus, EPCs became and Stra8, which are expressed in early and late stages a major target for therapeutic revascularization. of male GC development, and ZP2 and ZP3, markers Syndecan-4 (S4), a cell surface heparan-sulfate of mouse oocyte. These findings were confirmed by proteoglycan that regulates cell-matrix interactions, IC for Fragilis, Vasa, Dazl and EMA. EM revealed the binds to thrombospondin-1 (TSP-1) and modulates presence of round and elongated spermatid-like cells. angiogenesis. Our aim was to optimize the expansion The presented data demonstrate that somatic cells of EPCs and characterize their phenotype in vitro. were reprogrammed by the ES cells genome, allowing Buffy coats were fractionated on Histopaque 1077 their differentiation into GC in vitro. for isolation of the mononuclear fraction (PBMC), [email protected] which was seeded on fibronectin and maintained in a modified EGM-2 medium (EGM-2/ENR), developed L-020 in our laboratory. Immunocytochemistry staining was EXPRESSION OF GENES RELATED TO COMMITTED used for detecting the expression of the hematopoietic LINEAGE CELLS IN HUMAN UMBILICAL CORD transcriptional factor Gata-2 and the endothelial BLOOD UNDIFFERENTIATED MESENCHYMAL markers von Willebrand factor (vWf) and VE-caderin. STEM CELLS The expression analysis of syndecan-2 (S2) and S4 Tatiana Taís Sibov (Programa Interunidades em was performed by flow cytometry. EPCs tubulogenesis Biotecnologia, USP-IPT-Butantan, IIEP Albert Einstein), was quantified in Matrigel assays. All parameters were Lorena Favaro Pavon (IIEP Albert Einstein), Luciana compared to those obtained for human umbilical vein Cavalheiro Marti (Programa Interunidades em endothelial cells (HUVECs), considered as mature Biotecnologia, USP-IPT-Butantan, IIEP Albert Einstein), endothelial cells. EPCs showed positively staining Carlos Alberto Moreira-Filho (Programa Interunidades for Gata-2, whereas HUVECs were negative. EPCs em Biotecnologia, USP-IPT-Butantan, IIEP Albert express the endothelial markers VWf and VE-cadherin, Einstein, Imunologia ICB-USP.) together with S2 and S4. These markers were maintained after more than 8 passages in culture. EPCs Umbilical cord blood (UCB) has been used as an were also able to form networks of tube-like structures alternative source for obtaining mesenchymal stem cells in vitro. The use of the modified EGM-2/ENR medium (MSCs). UCB-MSCs have been shown to differentiate allowed the obtention of high amounts of EPCs for 1. 212

livro.indd 212 7/7/2008 16:33:53 further studies, including their modulation by S4 ligands. expression of neural markers was analyzed through Sponsored by CNPq. imunocitochemistry. In relation the morphology of [email protected] these cells, after 7 days of treatment, MSCs showed differences in the morphology and organization in L-022 different experimental conditions. Expression of beta- DISTINCTIVE CHANGES IN INTRACELLULAR CA2+ tubulin and nestin were changed from 1 to 20 ng/ CONCENTRATION PRODUCED BY CYTOKINES ml FGF-2 and EGF treatment: both neural markers AND P2 AGONISTS INDUCE PROLIFERATION OR diminished with the increase of FGF-2 or EGF DIFFERENTIATION IN MURINE HEMATOPOIETIC concentration. Treatment with EGF had effects in the CELLS potential of neural differentiation with morphological Edgar Julian Parededs Gamero (UNIFESP), Carlos differences in 10 ng/ml treatment of MSCs with passage Márcio Moura Ponce de Leon (UNIFESP), Radovan 10. We observed morphologic changes in 10 ng/ml EGF Borojevic (UFRJ), Maria Etsuko Myamoto Oshiro treated MSCs with beta-tubulin III positive cells had (UNFESP), Alice Teixeira Ferreira (UNIFESP) longer processes. GFAP expression was negative in our experimental conditions. Conclusion: The presence The role of intracellular Ca2+ (Ca2+i) on hematopoiesis of growth factors influenced in the neural potentiality of was investigated in long-term bone marrow cultures the CTMs, resulting in changes in the expression of the using cytokines and agonists of P2 receptors. Cytokines neural markers as well as morphologic changes. IL-3 and GM-CSF promoted a modest increase in [email protected] [Ca2+]i, with activation of PLCg, MEK1/2 and Ca2+/ Calmodulin kinase II (CaMKII). Involvement of PKC was restricted to stimulation with IL-3. In addition, these cytokines promoted proliferation (20 times) and increase in Gr-1-Mac-1+ population, with participation of gap junctions (GJ). Nevertheless, ATP, ADP and UTP promoted a large increase in [Ca2+]i, moderate proliferation (6 times), reduction in the primitive Gr- 1-Mac-1-c-Kit+ population and differentiation into macrophages, without participation of GJ. It is likely that Ca2+i participates as a regulator of hematopoietic signaling: moderate increases in [Ca2+]i would be related to cytokine-dependent proliferation, with participation of GJ, whereas high increases in [Ca2+]i would be related to macrophage differentiation, without maintenance of the primitive population. Financial Support: FAPESP. [email protected]

L-023 EFFECT OF EGF AND FGF-2 IN THE POTENTIALITY OF NEURAL DIFFERENTIATION OF THE MESENCHYMAL STEM CELLS IN HUMAN PLACENTA Talita da Silva Jeremias, Lucas Felipe Fernandes Bittencourt, Márcio Alvarez Silva (Universidade Federal de Santa Catarina)

Placenta and umbilical cord are tissues known as sources of mesenchymal stem cells (MSCs). In the present study we isolated a population cell derived from human placenta with morphological characteristics similar to those of MSCs isolated from bone marrow. Objectives: analyze the effect of EGF and FGF-2 on the potentiality of MSCs. Placenta was obtained from pregnant health donors in Hospital Universitário/ UFSC, Brazil. Tissue was enzymatic dissociated and explanted into culture dishes in IMDM supplemented with FBS 10%. We expanded MSCs in different in vitro passage (1-10) to test different concentrations (1, 10 and 20 ng/ml) of EGF and FGF-2 during 7 days. The 1. 213

livro.indd 213 7/7/2008 16:33:53 Index Arantes, CP J-011, J-022 Araújo Neto, AD F-078, H-018 Abad, C RT-04.15 Araújo, DAM B-052 Abdalla, FC E-004, F-089, F-106 Araújo, DD F-108 Abrahamsohn, PA RT-10.35 Araujo, HSS B-056,C-001,F-027,F-028,F-092 Abrão, MS A-010, RT-22.75 Araujo, LA D-034 Abreu, JG B-043, D-011, D-013, D-014 Araújo, MLG A-013 Abreu, RMM F-111, F-112, K-006 Araújo, MS F-062 Abujamra, AL B-064, B-065 Araújo, R G-018 Adolfi, MC A-006, A-053 Araújo, WM B-006 Aguiar, AF F-033 Arcolino, FO A-070 Aguiar-Perecin, ML RT-24.81 Ariza, CB J-003, L-006 Aisemberg, J RT-20.67 Armelin, HA B-060, B-068, RT-16.55 Albieri-Borges, A A-052, A-084 Aro, AA F-046 Allodi, S D-023, H-004, H-005, J-008 Arroteia, KF A-033, A-043 Almeida, AC D-006 Arzt, E RT-02.4 Almeida, AR D-029 Asencio, D B-071 Almeida, BG G-014 Assmann, E F-103 Almeida, DAL F-037 Assumpção, TA A-062 Almeida, DC B-021, L-010 Atella, GC D-011 Almeida, DEA F-074 Attias, M G-003 Almeida, EA I-003 Avellar, MCW RT-05.18 Almeida, FBR G-004, H-006 Ayub, ALSC A-044, F-034 Almeida, FLA D-001, F-015, F-049 Azeredo, MCB F-010, F-011 Almeida, FM J-028 Azevedo Jr, GM D-009 Almeida, KL D-011, D-014 Azevedo, ACB F-054 Almeida, MES H-003 Azevedo, RB B-003, B-032, F-024 Almeida, OM B-035 Baldo, C F-040 Almeida, RB B-070 Balduino, A L-002 Almeida, RL B-023 Balottin, LBL F-016 Almeida, TF G-011, G-018 Balzuweit, K F-073 Almeida, VR B-064 Bandeira, CL A-052, A-057 Alvarenga, EM F-052 Bandeira, SMC A-077 Alvares, EP F-001,F-002,F-017,F-029,F-030 Báo, SN B-003, F-024, G-005 Álvares, LE A-043 Baptista, LS F-042, L-011, L-008 Alves, CC F-110 Baqui, MMA RT-08.26 Alves, JHF D-022 Baran, MSH B-050 Alves, LC F-030 Barbeiro, HV F-097 Alves, LM F-013, F-077, F-094 Barbisan, LF B-002 Alves, TR B-033, B-038, H-022 Barbosa, FL F-019 Amadeu, TP D-024, F-067, F-085, F-095 Barbosa, HS F-004, F-109, G-007, G-008, Amado, NG D-013 G-020, I-002 Amaral, EA J-002, J-005 Baréa, AC B-050 Amaral, JB B-001 Barja-Fidalgo, C G-010 Amaral, RJFC I-005 Baroneza, JE D-022 Amaro Jr, E L-015 Barreira, MCR H-006, H-011, H-012, H-019 Ambrósio, CE A-063, L-017 Barreira, MCR G-004 Americo, TA J-024 Barreiro, EG A-080 Amim, RS B-045 Barreiro, EJ H-022 Ammar, D D-007, D-010, D-023, J-008 Barrence, FÂC A-088 Amoedo, ND B-057 Barros, APDN B-042 A-Mendes, MI F-098 Barros, CM D-006, F-048, H-004, H-005 Andrade, CGTJ A-006, A-079 Barros, DG D-028 Andrade, DV J-001 Barros, SBM F-117 Andrade, LR D-014, H-005, L-011, L-005 Barros, SBM B-023 André, KR J-012 Barroso, MMS F-077 Andrioli, LPM RT-14.47 Bastos, LGR B-009 Antunes, EF F-031, F-032 Batista, AC F-105, F-106 Aragão, JÁ F-071, F-079 Batista, MC J-006 Arantes, C J-015 Battistini, B B-066 1. 214

livro.indd 214 7/7/2008 16:33:53 Battlehner, CN F-098 Brandalise, SR B-022, B-026, F-060 Bazzoli, N A-058 Braz, SV F-024, G-005 Beletti, ME A-050, A-051 Breno, MC F-022 Belizário, JE B-067 Brentani, MM B-028, B-034, B-058, F-110 Beltrame Junior, M F-124 Bressan, GC F-104, F-114 Bem, JC A-001 Brisolla, R D-002 Bement, B RT-18.62 Brocchi, M H-006 Benchimol, M RT-10.33, RT-11.37 Brohem, CA B-023, B-025 Ben-Yehuda, S RT-21.70 Bromberg, N B-028 Beraldo, FH J-011, J-014 Bronze, FS B-058 Berardo, PT A-010, RT-22.75 Brunetto, AL B-064, B-065 Berlatzky, IA RT-21.70 Brusco, J J-025, J-026 Bernardes, ES H-002, H-011, H-019 Buck, GA G-018 Bernstein, MAFO L-021 Buiatte, JE F-078, H-018 Bevilacqua, E A-035, A-040, A-042, A-045, A-052, Burckhardt, L F-022 A-060, A-064, A-065, A-080, A-081, Bustos, SO F-117 A-083, A-084, D-020 Cabral, LM F-021 Bezerra, F F-082, F-083, RT-23.79 Cabral, MG B-059 Biagio, FP F-009 Cabral, SRP A-037 Biancalana, A F-055 Cadilhe, DV J-028 Biancardi, MF A-071 Caetano, CE L-013 Bianco, JER A-019, A-079 Caetano, FA J-022, J-023 Biben, C D-025 Caetano, FH F-053 Bigaton, FJ B-066 Calado, TC H-001, H-003 Bighetti, RL L-014 Caldas, LA G-003 Billi, S RT-20.67 Caldeira, W G-002 Billis, A B-016 Calderon, IMP A-052, A-080 Binda, NS J-023 Caldini, EG A-073, F-097, F-098 Bischof, P RT-06.20 Calixto, AR F-047 Bissell, M CLOSING CONFERENCE Camargo, AA RT-19.66 Bisson Filho, AW F-120 Camargo, JLV A-022 Bisson, GS H-011 Campolina, SS B-039 Bisson, LF B-037 Campolina, SS G-014 Bittencourt, JC A-030 Campos, AH L-015 Bittencourt, LFF L-023 Campos, CA L-004 Bizario, JCS F-108 Campos, DHS F-036 Bizinotto, MC A-014 Campos, KE A-062 Blanco, GR B-044 Campos, LM D-018 Bloise, FF H-020 Campos, SGP A-004, A-005, A-011, A-012, A-032, Boer, PA D-002, D-029, D-030, D-031 A-054 Boggio, RF F-099, F-116 Campos, SPC F-080, F-081 Bogo, MR RT-15.51, RT-15.52 Cândido, EM A-034 Bonaldi, CM B-047 Cano, MIN Lec-08 Bonan, CD RT-15.51, RT-15.52 Cantagalli, VD J-016 Bonfim, DC F-043 Caputo, LFG D-016, L-012 Bonini, CR F-074 Carabantes, AG F-118 Borbely, AU A-064, A-065 Carani, FR D-001 Borella, MI A-006, A-053 Caritá, R F-101, F-102 Borges, M A-065 Carneiro, MLB B-003 Borges, RM D-012 Carneiro, PCF D-003 Borojevic, R B-042, F-010, F-016, F-023, F-042, Carneiro, SM C-002, F-018, F-041 F-043, F-011, H-002, H-015, H-017, Carniato, AP F-089 I-005, L-002, L-005, L-008, L-011, Carter, AM A-063 L-022, RT-07.24 Carvalhal, AC D-006 Boschini Filho, J F-020, F-039 Carvalho, AF B-035 Bos-Mikich, A A-061 Carvalho, AF A-063 Botta, LS A-041 Carvalho, BF B-061 Boyle, S RT-24.81 Carvalho, CR K-005 Braga, PFS A-029 Carvalho, CR D-009 Branco, E L-004 Carvalho, D F-077 1. 215

livro.indd 215 7/7/2008 16:33:54 Carvalho, DR H-005 Oliver, C H-019 Carvalho, DS H-022 Cooke, HJ D-027 Carvalho, EAN F-079 Corat, EJ F-031, F-032 Carvalho, EG A-029 Corat, MAF F-031, F-032 Carvalho, HF A-030,F-026,RT-05.17,RT-12.41 Cordeiro, A H-020 Carvalho, HS L-011 Cordova, G B-071 Carvalho, JE B-020 Corradi, LS A-076 Carvalho, L G-010,G-007,G-009,I-002,L013 Corrêa Junior, JD F-072, F-073 Carvalho, LP F-023 Corrêa, IP A-083 Carvalho, LV L-007 Corrêa, TCS B-025 Carvalho, MC J-009 Corrêa, TJS D-028 Carvalho, ML G-009, L-013 Correia, TFVBC K-002, K-004 Carvalho, NO B-063 Cortelazzo, AL E-001 Carvalho, OAO A-013 Cortez, BA B-054 Carvalho, PTC H-016 Cortez, EFC G-009, G-010, L-013 Carvalho, RF F-015,F-036,F-038,F-049,I-001 Costa, ABV A-059, A-077 Carvalho, SN I-002, L-013 Costa, AFE G-013, G-012 Casali, VVC F-056 Costa, AMA D-024, F-067, F-068, F-075, F-085, Casanello, P RT-04.14 F-095, RT-23.78 Cassel, MCP A-067, F-093 Costa, APD F-025 Castan, EP F-038, I-001 Costa, DBV B-020 Castilho, B J-014, J-017 Costa, ESS G-023 Castro, AFP H-006 Costa, FG A-006, A-053 Castro, FC G-022 Costa, L R H-002, H-015 Castro, LD F-074 Costa, MCR F-108 Castro, M RT-09.31 Costa, MEWF D-021, D-025, RT-09.31 Castro, SL F-004, F-005, F-006 Costa, MJ F-105, F-106 Castrucci, AML F-088 Costa, ML D-015, F-065, RT-15.53 Catarino, RM H-013 Costa, MP K-006 Catroxo, MHB G-021, G-027, G-028 Costa, MAS D-002, D-033, D-034 Cavalcante, AACM B-061 Costa, OTF A-013 Cavalcante, FS D-004, D-005 Costa, PLN B-066 Cavalcanti, DP F-012 Costa, RA D-009 Ceccato, FR A-031, D-023 Costa, TJX A-051 Ceolin, MP F-078, H-018 Costa, TO F-078 Cerqueira, DM D-013 Costa, V F-077 Cerruti Filho, H L-007 Cotta-Pereira, G D-016 Cervato, MC B-069 C-Thomas, YM J-021, L-009 Chaim, OM F-063, F-064, F-066 Coutinho, CMLM E-003 Chammas, R B-011, B-029, B-041, B-053, B-066, Covas, DT B-021, L-010, L-018 H-002, H-015 Crépin, M B-056 Chamon, SL B-062, B-063 Cruz, ALS B-010 Chiarello, DI RT-04.15 Cruz, KP B-019 Chiarini, LB B-042, F-080, F-081, J-027 Cruz, MC G-006 Chimelli, L J-013 Cruz, MV G-002 Cianciolo, GJ F-108 Cunha, AL A-046 Cicogna, AC F-036, F-038, I-001 Cunha, RLOR G-019 Clarindo, WR K-005 Curi, MAF F-001 C-da-Silva, CS F-042, L-005, L-008 Curi, R A-040, B-023 Clemente, CFMZ F-014, F-025 Da Silva, NW F-124 Clemente, SS F-016 Daher, S A-052, A-065, A-080 Coelho, CAO I-001, F-038 Daliry, A G-015 Coelho, JM B-043 Dallabona, AC G-018 Coelho, RM H-016 Dallai, R A-082 Coelho, VM H-020 Damasceno, DC A-052, A-062, A-080 Coelho-Sampaio, T B-059, F-077 Damn, GR F-115 Coimbra, VC G-019 Dantas, AP F-004 Conceição, FSL J-028 Dantas, SMMM B-061 Cônsoli, FL G-001 Dantas, VWM B-038 Consonni, SR A-008, F-086 Degaki, KY A-014, A-019, A-025, RT-20.68 1. 216

livro.indd 216 7/7/2008 16:33:54 Degaki, TL A-025 Felisberto, ADS F-033 del Castillo, DM B-062, B-063 Felisbino, MB F-051 del Rey, A Lec-01, RT-02.5 Felisbino, SL A-002, A-003, A-007, A-016, A-017, Delella, FK A-002, A-007, A-017 A-020, A-021, A-054, D-002 Delgado, GL F-020 Ferguson, S Lec-05 Demasi, M RT-09.29 Fermino Jr, PCP K-006 Derengowski, LS G-005 Fernandes, GSA A-062 Dernowsek, JÁ L-014 Fernandes, HL E-004 Desmoulière, A RT-23.76 Fernandes, MC RT-11.38 Diamante, MAS A-023 Fernandes, MC F-006 Dias Filho, MA B-036 Fernandes, MRN F-091 Dias, JV L-021 Fernandes, PA B-062 Dias, RD RT-15.51 Fernandez, CDB A-062 Diaz, BL B-012 Ferrari, MFR J-001 Diaz, JAM B-004, B-005, B-006, B-009, B-007, Ferreira, A A-067, A-068, F-093 B-008, B-010, B-012, RT-17.60 Ferreira, AHP L-003 Djaafri, IS B-056 Ferreira, AT L-022 Dolder, H A-027, A-082 Ferreira, CGM F-050 Dolder, MAH A-023, A-078, F-037 Ferreira, FS H-018 Dolhnikoff, M A-073 Ferreira, FU L-010 Domeniconi, RF A-004, A-055 Ferreira, MA F-098 Donatti, L D-003, F-100 Ferreira, MLL B-042, J-027 Dourado, DM H-016 Ferreira, PEN G-012, G-013, G-016, G-017 Duarte, AGE E-002 Ferreira, RV D-004, D-005 Dutra, AAN G-011 Ferreira, U B-016 Dutra, HS B-042 Ferreira, VF F-006 Echalar, L RT-04.12 Ferreira, ZS F-007 Eder, EW L-002 F-da-Silva, M F-109, G-020 Egaña, J B-071 Ferrer, VP F-063, F-069 Ehrlich, M Lec-12 Ferro, EAV RT-20.69 Eichmann, A Lec-17 Figueiredo, CC B-038 Einicker-Lamas, M D-011 Figueiredo, LB A-050, A-051 El-Cheikh, MC H-015, H-002, J-007 Figueiredo, PM F-001 Emmert, F F-054 F-Rymarkiewicz, E RT-04.13 Escudero, C RT-04.14 Filippo, TRM L-009 Espósito, BP J-020 Fink, GM F-098 Espreafico, EM D-012, F-057, RT-18.61 Fiore, APZP B-044 Esquisatto, MAM F-044, F-045 Fioretto, ET L-004 Esteban, J Lec-14 Fochi, RA A-048, A-072 Esteves, CA F-022 Foglio, MA B-020 Estofolete, CF H-009 Fonseca, ACC J-018 Evangelista, AF L-014 Fonseca, CG J-002, J-005, J-006 Faheina, GV B-052 Fonseca, SAS L-019 Falleiros Jr, LR A-012, A-069 Fonseca, SAS L-007 Faloppa, F F-113 Fontana, V L-014 Fanta, ESE F-100 Fontes, AM B-021, L-010 Faria e Sousa, SJ F-019 Forni, MFPAD L-003 Faria, JC B-033, B-043 Fortes, ZB A-086, RT-06.21 Faria, MR A-045, A-084 Fortes-Dias, CL B-036, G-014 Faria, PA B-049 Fragoso, SP F-012 Farias, CB B-064, B-065 França, EG F-078, H-018 Farias, FF A-088 França, GF A-015 Farías, M RT-04.14 França, LR Lec-04 Farina, M J-009, J-020 França, MM G-017 Fatkin, D RT-09.31 Francelino, AA H-001 Faustino, LC J-029 Franchi, AM RT-20.67 Fauvel-Lafève, F B-056 Franchi, CAS A-022 Fávaro, WJ A-026, A-028, A-034, B-016 Franchini, KG F-057, F-014, F-025, F-047, F-091, Favaron, PO A-063, L-017 Lec-18 Feitosa, VLC F-071, F-079 Franciolli, ALR L-017 1. 217

livro.indd 217 7/7/2008 16:33:54 Franklin, V D-025 Goldfeder, EM D-007 Frederico, JCL D-031 Gombar, FM D-004, D-005 Freire, MS F-080, F-081 Gomes, AF G-007, G-008 Freitas Jr, JCM B-005 Gomes, AM F-048 Freitas, BC B-052 Gomes, AMO F-080, F-081 Freitas, CS D-036, F-122 Gomes, CC A-053 Freitas, ERL L-001 Gomes, FA D-024, F-013, J-029 Freitas, MC L-010 Gomes, HR J-013 Freitas, SM B-032 Gomes, IN G-011, G-018 Freitas, VM B-019, B-027, B-030, B-031, B-070, Gomes, JR F-034, F-035 F-099 Gomes, L F-044, F-045, F-046, F-055, F-061, Fuentes, VM F-024 F-084 Fulquim, MC A-086 Gomes, MD F-114, RT-09.30 Funchal, CS J-012 Gomes, MLM A-027, F-037 Furtado, J F-098 Gomes, SMZ A-035, A-064, A-081 Furtado, KS B-002 Gomez, MMD A-050 Furtado, MBF D-021, D-025 Gómez, MV J-002, J-005, J-006 Fuziwara, CS B-044 Gómez, RS J-006 Galdino, AMR D-003 Gonçalves, BF A-032 Galembeck, E RT-12.43 Gonçalves, BS J-027 Galvão, L F-034 Gonçalves, EJ G-026 Gama, P F-001, F-002, F-017, F-029, F-030 Gonçalves, FLL F-061 Gamarra, L L-015 Gonçalves, RLS F-005 Gamarra, LF C-002 Gontijo, JAR D-002, D-029, D-030, D-031 Gamero, EJP L-022 Gonzalez, M B-071 Gantus, MAV F-013 González, M RT-04.14 Garcês, BP F-078, H-018 Gorjão, R A-040 Garcia, CRS RT-11.40 Gouvêa, CMCP F-028, F-027 Garcia, PV A-033 Gregório, EA G-002 Garcia, SB B-021, L-018 Gremski, LHM B-003, F-066, F-069 Gardel, MA G-016 Gremski, W F-063, F-064, F-066, F-069 Garzoni, L H-017 Gross, U G-020 Gaspar, EB RT-11.38 Grötzner, S F-076 Gaspar, LP F-080, F-081 Guaraldo, AMA RT-24.81 Gatti, MSV F-051 Guedes, PT D-016, L-012 Gava, L F-104 Gueiros, F F-120, F-121, RT-21.72 Georgii, JL F-095 Guerra, MT A-005 Geraldeli, S RT-13.44 Guerra, RB J-012 Gerhardt, ECM F-054 Guilhen, DD H-021 Ghizoni, H D-007 Guillo, LA L-001 Giannella, D F-110 Guimarães, EV G-007, G-008 Giannini, ALM B-057 Gullo, CE H-009 Giannotti, KC F-018 Guo, G RT-09.31 Gil, CD H-008, H-009 Gurgel, CAS B-059 Gilbert, N RT-24.81 Haddad, A F-019 Gilioli, R F-031 Haddad, N F-010, F-011 Gimenes, F E-004 Hajj, G J-014, J-015, J-017, J-022 Gimenez, M B-051 H-Mecarelli, L H-012 Giorgi, R F-110 Han, SW F-023, J-016, RT-22.74 Giorgio, S A-039 Hanna, ES G-004, H-006 Gitaí, DG J-025 Harvey, RP RT-09.31 Gitirana, LB L-012 Harvey, RP D-021, D-025 Glaser, T L-016 Hayashi, MAF B-014 Gobbo, MG A-070 Heinen, TE B-065 Godinho, AF A-007 Henning, F F-119 Godoy, JAP F-107 Herlyn, M B-055 Góes, RM A-038, A-041, A-048, A-049, A-054, Hermógenes, MS B-036, B-039 A-069, A-070, A-071, A-072, A-019 Hernández, RB J-020 Goldenberg, RCS F-094 Hess, RA A-059 Goldenberg, S F-012 Hewitson, TD RT-23.77 1. 218

livro.indd 218 7/7/2008 16:33:54 Horwtz, AF I-004 Lanzetti, M F-082, F-083 Hoshida, MS A-040, A-045, A-084, D-020 Lara, PMR E-004 Hyslop, S A-018, A-043 Laranjeira, ABA B-022 Ikemori, RY B-053 Larson, RE RT-08.26 Illsley, NP RT-04.12, RT-04.13 Lavagnolli, TMC L-019 Inoue, RY F-047 Leal, LSS B-061 Introíni, GO A-046 Leclerq, SY G-014 Ionta, M B-070 Lee, JT B-055 Ishigai, MM A-065 Leite, AS B-061 Izquierdo, IA Lec-02 Lemos, FS D-019 Jacob, CRO F-096 Lemos, PA L-004 Jaeger, RG B-015, B-017, B-018, B -019, Leon, CMMP L-022 B-027, B-030, B-031, RT-17.59 Leve, F B-007, B-009 Jamora, C F-040 Li, D RT-09.31 Jamur, MC H-019 Lima, BBV B-013 Jeremias, TS L-023 Lima, ECO L-001 Jesus, LWO A-029 Lima, F J-015 Joanitti, GA B-032 Lima, FRS J-018 Joazeiro, PP A-008, A-018, A-033, F-086 Lima, KS B-036 Jordan, B F-084 Lima, LM H-022 Juliani, CC D-002 Lima, PDA A-019, A-014, RT-20.68 Juliani, CC D-030 Lima, PHS B-012 Juliano Neto, L G-019 Lima, RC B-065 Junta, CM L-014 Lima, TM B-023 Justulin Jr, LA A-003, A-054, D-002, D-030 Linden, R J-011, J-015, J-022, J-024, J-027, OPENING Kachar, B B-030, B-031, Lec-03 CONFERENCE Kalinin, AL F-105, F-106 Linton, NF A-025 Kasai, A F-002, F-017 Lione, VOF G-016, G-017 Katayama, MLH B-034 Lippe, EMO A-039, A-085, RT-20.68 Katchburian, E F-113 Liu, FT H-015 Katz, SG A-074 Lizier, NF L-007 Kawall, HG F-100 Lobo, AO F-031, F-032 Kempinas, WG A-004, A-005, A-022, A-062, RT- Lopes, DV F-023, F-042 05.19 Lopes, FA F-006 Kerkis, A B-014, L-007, L-019 Lopes, FS F-036 Kerkis, I B-014, J-028, L-007, L-019 Lopes, GJR F-088 Khusal, KG F-059 Lopes, MF H-002 Kimar, PSSP A-060 Lopes, MH B-024, D-008, J-010, J-015, J-022, Kimura, ET B-044 J-023 Kloss, ML F-082 Lopes-Bezerra, LM G-012 Kobarg, J F-103, F-104, F-114 Lopes-Cendes, I F-014 Kobus, K D-010 Lopez, LB I-005 Koga, MM B-068 Lorenzon, AR A-035 Koslowski, EO F-016, F-048 Lotfi, CFP F-058 Krause, B RT-04.14 Lothhammer, N A-061 Krieger, JE L-016 Louza, MP D-011 Krieger, MA G-018 Lozzi, SP F-024 Kubelka, CF F-080 Lubian, A B-065 Kuniyoshi, TM F-114 Lüder, C G-020 Kurtenbach, E D-021 Luiz, FFM D-028 Kusma, J F-069 Luna, MSA F-007 Kyaw, CM G-005 Lutz, YS D-028 Laçava, ZGM B-003 Luz, MAM I-003 Laclette, JSR F-050 Mação, NG F-094 Lacorte, LM A-007 Machado, AHA F-123, F-124 Lamers, ML I-004 Machado, AP H-016 Landemberger, MC J-013 Machado, AZ F-092 Landman, G B-035 Machado, CF J-022, J-010, J-011 Lanza, DCF F-103 Machado, CFO A-044 Lanza, DF F-104 Machado, CML B-053 1. 219

livro.indd 219 7/7/2008 16:33:54 Machado, CRS J-019 Martucci, MF D-020 Machado, DE RT-22.75 Más, BA F-020, F-039 Machado, DE A-010 Maschio, LR K-002, K-003, K-004 Machado, FR F-054 Massaro, RR B-023, B-025 Machado, IG RT-10.34 Massironi, SMG J-003 Machado, MCR D-035 Mata, JF F-111, F-112 Machado, MP D-016, L-012, L-013 Mateo, ECC B-021 Maftoum-Costa, M F-123, F-124 Matheus, WE B-016 Magaldi, FM A-042 Matiello-Rosa, S F-046 Magdesian, MH J-024 Matsuda, M F-087 Mahecha, GAB A-056, A-077 Mattar, R A-065 Maia, AR L-003 Mattos, GE F-058 Maia, SSR A-048, A-054, A-072 Mazzeo, DEC F-101 Maiorka, PC L-004 Mazzoni, TS A-009 Malaspina, O F-096 M Gameiro, TM J-027 Malnic, B RT-03.10 Medico, SED F-105, F-106 Malta, J H-010 Meira, GLS F-121 Maltempi, PPP A-001, F-053 Meirelles, MN B-042 Mancini, G J-014 Meister, M A-061 Mancini, KC A-078, A-082 Mela, M F-076 Manfiolli, AO F-114 Mello Júnior, W A-055 Manor, U Lec-03 Mello, MLS F-051, F-052, RT-24.81 Manso, PPA D-016, L-012 Melo, KCM C-002 Maranduba, CMC L-007 Melo, KRB F-062, F-070 Marchiori, A F-016 Melo, NA G-021, G-027, G-028 Marcolino, C B-062, B-063 Menck, CFM RT-01.2 Margraf, A A-044 Mendes, CF F-020, F-039 Maria Engler, SS B-023, B-025 Mendes, F B-043 Mariano, VS G-004 Mendez, LC G-011 M-da-Silva, S A-029 Mendez-Otero, R RT-07.25 Marie, SKN B-051 Menezes, C J-023 Marín, R RT-04.15 Menezes, CB K-001 Marin, TM F-014 Menezes, FS F-021 Markus, RP J-011 Menezes, JPB G-011 Marques, MLL D-015 M-Barreto, RFS F-006, F-004, F-005 Marques, MMC L-014 Mercati, D A-082 Marques, RG L-013 Merli, LAS F-113 Marques, SA J-007, J-028 Mermelstein, CS D-015, F-043F-065 Marqueti, RC C-001 Mesquita, FF D-002 Marquezini, MV F-087 Mesquita, FF D-029, D-031 Marti, LC H-021, L-015, L-020 Mesquita, O Cf-1 Martinelli, PM J-019 Mesquita, SFP A-033 Martinez, AMB F-013, J-007, J-028 Mess,A A-063 Martinez, CG D-021 Meuser, M G-015 Martinez, FE A-034 Mietto, BS J-007 Martinez, MF A-022 Miglino, MA A-063, L-004, L-017 Martinez, RM A-039, A-085 Miguel, NCO D-006 Martins Neto, AA H-003, H-007 Milanelo, L G-021, G-027, G-028 Martins, AMCRPF G-021, G-027, G-028 Milanezi, RC F-015, F-049 Martins, C F-049 Millard, TH RT-18.63 Martins, DS L-017 Mimura, KK H-014 Martins, ER F-043, F-065 Minciotti, CL RT-13.44 Martins, JFN F-078, H-018 Moda, LMR D-033 Martins, LPA F-105 Moisan, M-P F-002 Martins, MA H-017 Mondin, M RT-24.81 Martins, PMRS H-017 Monesi, N RT-14.48 Martins, AS H-001, H-007 Monteiro, BG L-007 Martins, VR B-024, D-008, J-010, J-011, J-013, Monteiro, DA F-105, F-106 J-014, J-015, J-018, J-022, J-023 Monteiro, EC F-008 Martins, WK B-035, B-039 Monteiro, JC A-027, F-037 1. 220

livro.indd 220 7/7/2008 16:33:55 Monteiro, RF D-028 Nascimento, MAC A-008, A-079, F-086 Montico, F A-034 Nascimento, RD H-010 Moraes, AS RT-24.81 Nasciutti, LE A-010, F-013, F-021, F-077, F-094, Moraes, CS D-036, F-122 RT-22.75 Moraes, EC F-114 Nazari, EM A-031,D-007,D-010,D-023,J-008 Moraes, GM B-029 Negri, EM F-097 Moraes, JRS G-024 Nery, LR B-065 Moraes, KCM F-123 Neves, EJ B-035 Moraes, SG E-002 Neves, JS F-035 Morais, FV L-021 Neves, RI B-035 Morais, J F-045 Niero, ELO B-037, F-116 Morais, PC F-024 Nocelli, RCF F-096 Morais, R B-061 Nogueira, CR F-038 Morales, MAM F-101, F- 102, F-090, K-004 Nogueira, CSF D-028 Morandi Filho, R A-050, A-051 Nogueira, JC A-056 Moreira, CM J-016 Nonogaki, S B-066 Moreira, DC F-063, F-064 Normann, CABM F-056, J-012 Moreira, DF B-067 Noronha, MF B-069 Moreira, JE J-025, J-026 Novaes, PD F-035 Moreira, LFZ F-034 Nunes, MP F-071, F-079 Moreira, MA F-122 Nunes, SS B-033, L-021 Moreira, MP B-039 O’Brien, L RT-18.62 Moreira-Filho, CA H-021, L-020 Oba-Shinjo, S B-051 Moreno, NA B-050 Ogias, D F-002, F-029 Morini, AC A-063, L-017 Okada, FK A-024 Moriscot, A Lec-10 Okada, MA F-034 Moro, CF A-079 Okamoto, OK L-006 Moro, L F-073 Okawachi, FM F-089 Morretto, AC F-113 Okazaki, K F-119 Mortara, RA F-118, G-006, G-017, RT-11.38 Okoshi, K F-036 Motta, CM F-013 Oliani, SM H-008, H-009, H-014 Motta, G F-062, F-070 Oliveira e Longo, AE K-001 Motta, MCM F-012 Oliveira, AC G-021, G-027, G-028 Moulin, MRI A-029 Oliveira, AC F-080, F-081 Moura Neto, V B-033, B-043, D-024, J-018 Oliveira, AF H-006 Moura, AS I-002 Oliveira, AG A-056, A-077 Moura, CP H-020 Oliveira, BCEPD D-016, L-012 Moura, GS D-028 Oliveira, C A-037 Moura, JF F-064 Oliveira, C A-075 Moura, MA F-109 Oliveira, CA F-078, H-018 Mourão, PAS B-038 Oliveira, CA A-056, A-059, A-077 Mouta Jr, MF H-013 Oliveira, CC L-018 Moya, JS A-078 Oliveira, DM L-015 Müler, YMR A-031, D-007, D-010, D-023, J-008, Oliveira, EB B-014 J-009, J-016 Oliveira, EC B-018, B-019 Nader, GPF F-057 Oliveira, EC A-030 Nader, HB B-040, B -047, F-062, F-063, Oliveira, FL H-002, H-015 F-070, F-113, RT-13.46 Oliveira, KD F-044 Nadruz Júnior, W F-091 Oliveira, LL G-004, H-006 Nagato, AC F-082 Oliveira, MF F-005 Nantel, F B-066 Oliveira, MFO G-009, G-010 Nantes, IL B-049 Oliveira, MG F-095 Narciso, MS J-007 Oliveira, MP RT-11.38 Nardi, NB RT-07.23 Oliveira, MRL B-045 Narvaes, EAO F-035 Oliveira, MTVA A-036, A-037, K-004, K-002, K-003, Nascimento, AP D-026, F-067, F-068 RT-24.80 Nascimento, CF B-027 Oliveira, NFP F-115 Nascimento, FD B-048, F-062, RT-13.44 Oliveira, RG H-010 Nascimento, GC L-014 Oliveira, RL A-056 Nascimento, KF A-045 Oliveira, RR B-045, E-003 1. 221

livro.indd 221 7/7/2008 16:33:55 Oliveira, SF A-064, A-065 Pereira, LCB F-016 Oliveira, SL A-029 Pereira, LV J-028 Oliveira, SM A-047 Pereira, S A-055 Omar, NF F-035 Pereira, VS L-019 Orellana, MD B-021, B-051, L-010 Peres, A J-012 Origassa, CST B-047 Perez, APS A-048, A-069, A-072 O-Carvalho, TM J-029 Perobelli, JE A-022 Ortiz, RJ A-075 Peroni, LA F-026 Osaki, LH F-001 Peruquetti, RL A-036, A-037 Oshiro, MEM L-022 Perussi, AP F-022 Otake, AH B-011, B-029, B-041 Pesente, A A-061 Otuki, MF F -066 Pessatti, ML F-054 Ownby, CL F-092 Pessotti, NS F-015, F-049 Oyola, MM RT-04.12 Petrella, S G-021, G-027, G-028 Ozaki, LS G-018 Pezzuto, P B-057 Pacheco, VCR F-070 Picardi, PK F-014 Paçó-Larson, ML J-025 Pieczarka, JC RT-24.82 Padilha, P F-010, F-011 Pietro, L A-052 Padovani, CR D-001, F-015, F-016, F-036, F-049 Pimenta, AC F-108 Pádua, TA F-085 Pimenta, AFG A-050 Pafaro, AMA A-060 Pimenta, WA F-083 Paiva, RO F-083 Pimentel, ER F-003,F-037,F-046,F-061,F-084 Palhares, IM B-036 Pimentel, TA H-014 Palma, MBS F-032 Piñero, S RT-04.15 Palma, MS F-096 Pinhal, D F-049 Palma, PVB B-021, L-010, L-018 Pinhal, MAS B-040, B-047 Palmeira, JAV F-071, F-079 Pinheiro, DO G-001, G-002 Paludo, KS F-066 Pinheiro, PFF A-055 Palumbo Jr, A F-021 Pinheiro, SMT B-046 Pancetti, AR F-121 Pinto, AV F-005, F-006 Paoli, F F-003 Pinto, FC RT-02.6 Pardo, APS F-115 Pinto, JP G-007 Pascoal, VDB F-014 Pinto, LC A-028 Passos Jr, GAS L-014 Pinto, MCFR F-006 Passos, DO F-114 Pinto, ME A-041, A-049 Passos, IRS K-001 Pires, AAA F-119 Passos, LAC F-031 Pires, KMP F-083 Passos, MWC E-003 Polli, CD H-011, H-012 Paula, LF F-078, H-018 Pomin, VH F-048 Paula, SM C-003 Pontes, DA A-004 Paulesu, L A-084 Porcionatto, MA J-021, J-003, J-016, L-006, L-009 Paulino, DB A-051 Porfírio, EP B-003 Pavão, MSG F-048, H-004, H-005, RT-13.45 Portes, ALF D-006 Pavon, LF L-015, L-020 Portilho, DM F-043 Pavoni, DP G-018 Porto, EM A-016, A-020, A-021 Pedrosa, CSG F-023, F-042 Porto, LCS F-082, F-083, L-013, RT-23.79 Peixoto, RCA B-003, F-024 Porto-Carreiro, I J-015 Penha, CVLY G-012 Possik, PA B-055 Pereira, A B-014, L-007 Póvoa, TF D-028 Pereira, ACP J-029 Prado, KFB L-014 Pereira, BF F-053 Prado, MAM J-005, J-011, J-015, J-022, J-023, Pereira, DTG H-010 RT-08.28 Pereira, FCR J-029 Prado, VF J-022, J-023 Pereira, FL H-019 Prates, CB F-002, F-029 Pereira, GAG A-025 Predes, FS A-023 Pereira, GJ H-010 Previato, M A-036, A-037 Pereira, GS J-023 Prieto, C RT-04.14 Pereira, IS G-005 Probst, CM G-018 Pereira, LAV RT-10.36 Procópio, MS F-072, F-073 Pereira, LAV A-033, A-043, E-002 Proverbio, T RT-04.15 1. 222

livro.indd 222 7/7/2008 16:33:55 Puebla, C RT-04.14 Rios, GP D-036, F-122 Quagio-Grassiotto, I A-009, A-015, A-075 Rivelli, DP B-023 Quaresma, AJC F-104, F-114 Rizzo, E A-058, D-019, F-072, F-073 Queiroz, NGT B-024, J-023 Rizzo, LV H-013 Quintana, CYP F-077 Rocha, C A-035 Quitete, VHAC A-026, A-028, A-034, B-016 Rocha, F A-066 Rabinovitch, M F-059 Rocha, LB J-026 Rádis-Baptista, G B-014 Rocha, LLV H-010 Raimundo, SG B-044 Rocha, WD G-006 Ramalho, BS J-028 Rodrigues Jr, DM B-061 Ramão, A B-051 Rodrigues, ACF I-003 Ramires, JAF L-004 Rodrigues, LC F-108 Ramos, CF D-004, D-005 Rodrigues, NRD A-073, F-097, F-098 Ramos, CHI F-104 Rodrigues, RF J-028 Ramos, EAG B-059 Rodrigues, SACP H-017 Ramos, FMM F-035 Rodrigues, T B-048, B-049 Ramos, FWS H-001 Roela, RA B-034 Ramos, MV B-052 Roesler, R B-064, B-065 Ramos, OHP C-001 Roffe, M J-014, J-017 Ramos, RGP D-022, D-033, D-034, RT-14.50 Rojas, GL D-032 Randi, MAF F-076, RT-12.41 Romana-Souza, B F-068, F-068 Rasia-Filho, AA J-025, J-026 Romão, L B-043, D-024 Raspantini, PR A-086 Ropke, CD F-117 Ravacci, GR B-034, B-058 Ropke, CD B-023 Razera, MEP B-050 Rosa, ABS A-068 Rebouças, MM G-021, G-027, G-028 Rosa, AL L-014 Recco-Pimentel, SM A-046 Rosa, JC B-051, G-004 Reginato, RD F-113 Rosa, RG A-008, A-018, F-086 Rehen, SK J-028, RT-03.11 Rosemberg, DB RT-15.51, RT-15.52 Reis, AB F-098 Rosemberg, S J-013 Reis, AH D-011, D-014 Rossi, AM L-005, L-011 Reis, FP F-071, F-079 Rossi, MID B-042, F-023, F-042, F-043, L-011, Reis, LFL B-035, B-055, RT-16.56 L-005, L-008 Reis, LO B-016 Rouvinski, A RT-21.70 Reis, MDS H-003 Rozenchan, PB B-028 Reis, PEO F-023 Ruano, R A-083 Reissler, J F-036 Rudge, MVC A-052, A-080 Reynolds, N D-027 Ruiz, ALTG B-020 Rezende, E B-044 Ruiz, JC F-118 Rheingantz, MGT RT-10.34 Ruiz, RC C-002 Ribeiro, AAGFC F-008, F-009, G-002 Rumjanek, FD B-057 Ribeiro, CAO F-076 Rumjanek, V H-020 Ribeiro, DL A-038, A-070 Sá, ERA F-002 Ribeiro, DM F-081, F-080 Saad, MJA F-014 Ribeiro, EA F-100 Sade, YB F-064, F-066, F-069 Ribeiro, HJ F-072, F-073 Saita, AP D-034 Ribeiro, JU B-056 Saito, H A-042 Ribeiro, LCM B-059 Saito, RF B-041 Ribeiro, MEOB J-021 Sakai, F F-018 Ribeiro, ML RT-20.67 Saldiva, PHN A-073, F-097 Ribeiro, RF A-086, A-087, A-088 Salgado, RM A-086, A-087, A-088 Ribeiro, S RT-03.8 Salmen Espíndola, FS A-050, A-051 Ricarte-Filho, JCM B-044 Salomão, K F-004 Ricci, R B-040 Sampaio Neto, LF F-039 Rice, GE Lec-15 Sampaio, EP H-022 Richardson, M F-066 Sampaio, FJB D-004, D-005 Rico, EP RT-15.51, RT-15.52 Sampaio, MU F-062, F-070 Rigonati, CAM A-006,A-053,F-017,F-030,F-001 San Martín, JAB A-079 Rigotti, IJC F-047 San Martin, S A-087, B-071, RT-06.21 Rios, FSA D-003 Sanches, D F-080, F-081 1. 223

livro.indd 223 7/7/2008 16:33:55 Sanchez, EF F-066 Severino, P H-021 Sant´Ana, EMC F-027, F-028 Shaw, E F-092 Sant’Anna, OA L-007 Shimada, MK F-012 Santana, J F-118 Sibov, TT L-015, L-020 Sant’ana, JAS A-069 Silva Júnior, EN F-006 Santana, TFP E-003 Silva, AMM F-040 Santa-Rita, RM F-004 Silva, AO B-038 Santelli, GMM B-001, B-013, B-030, B-031, B-037, Silva, AP H-006 B-046, B-054, B-070, F-099, Silva, CS G-005 F-116, RT-17.58 Silva, CV G-006, RT-11.38 Santos, ABG A-073 Silva, DT F-063, F-069 Santos, AM F-025, F-047 Silva, EC F-050 Santos, CT RT-08.26 Silva, ER J-019 Santos, DC D-030 Silva, FCS A-042 Santos, FCA A-048,A-054,A-069,A-071,A-072 Silva, FL F-082 Santos, GS G-012 Silva, GT J-002 Santos, HB A-058 Silva, IA D-015 Santos, JN B-059 Silva, IT B-069 Santos, JS F-067 Silva, J B-003 Santos, JS E-001 Silva, JAF F-026 Santos, MC A-013 Silva, JCT G-009, G-010 Santos, MF D-012, I-004, RT-19.64 Silva, JF B-002 Santos, MHB G-016, G-017 Silva, JL F-080, F-081 Santos, MRM F-118 Silva, KR F-042, L-011, L-005, L-008 Santos, NSA L-005, L-011 Silva, LM B-036, B-039, G-014 Santos, PCP J-012 Silva, LO A-064 Santos, RARR A-050 Silva, MA L-023 Santos, RL L-001 Silva, MDP D-001, D-002, F-033, F-036, F-038, Santos, RO F-078, H-018 F-049, F-015, I-001 Santos, SAA A-020, A-016, A-021 Silva, MHP L-011, L-005 Santos, SEM B-061 Silva, MRB F-071 Santos, TG D-008 Silva, RB B-021 Santos, VAL J-019 Silva, RSF F-005 Santos, VM B-048 Silva, S RT-11.38 Santos, WD G-016 Silva, SM J-001 Sarmento, CAP L-004 Silva, SO D-035 Sarno, EN H-022 Silva, TCR K-005 Sarobo, C A-017 Silva, VMM B-033, B-038, H-022, L-021, RT- Sato, Y A-058, F-072, F-073 16.57 Saudade, M F-056 Silva, VV F-117 Savino, W H-001,H-007,RT-02.7,RT-19.65 Silva, VV B-023 Sawada, TCH B-023 Silveira, AB B-022 Sbervelheri, MM A-027 Silveira, AL B-052 Scarano, WR A-004, A-005, A-012 Silveira, DF F-056 Scharfstein, J RT-11.39 Silveira, JF F-118 Schechtman, D F-025, L-016 Silveira, M C-003 Schenkman, S G-019, Lec-07 Silveira, RB F-063, F-069 Scher, R F-071 Simões, KES H-013 Scherholz, PLA A-074 Simon, CR D-034, RT-14.49 Schmith, A F-083 Sinzato, YK A-052, A-080 Schwartsmann, G B-064, B-065 Siqueira, AS B-017 Schwarz, N B-011 Siqueira, WJ K-001 Scott, MDS K-001 Sirois, P B-066 Seabra, AB F-095 Siufi, JLC L-018 Segura, MNO G-025 Smaniotto, S H-001, H-003, H-007 Seixas, SIL D-028 Soares, CP F-065 Senff-Ribeiro, A F-069 Soares, CP F-032, F-123, F-124 Senger, MR RT-15.51 Soares, GCT G-013 Senhorini, JA A-001 Soares, H B-061 Serafim, RC L-019 Soares, HM F-096 1. 224

livro.indd 224 7/7/2008 16:33:55 Soares, IN J-023 Suzuki, OS K-001 Soares, MA D-011, D-014 Suzuki, S A-050, A-051 Soares, MAM A-044, F-034 Taboga, SR A-005, A-001, A-011, A-012, A-032, Soares, MJ F-004 A-038, A-041, A-047, A-048, A-049, A-054, A-069, Soares, PRO L-001 A-070, A-071, A-072, A-076, B-025, D-031, F-084, RT- Soares, SG H-006 05.16 Sobrevia, L RT-04.14 Takimura, C L-004 Sobrinho, CVB A-072 Takiya, CM A-010, B-033, B-042, B-059, F-016, Sobrinho, RJC H-010 F-021, F-023, F-042, RT-22.75 Soeiro, MNC G-015 Tam, P D-025 Soengas, M Lec-11 Tanaka, H A-043 Sogayar, MC B-025, L-003, RT-01.1 Tanaka, MN B-012 Solloway, M D-025 Tarsitano, CAB A-018 Sorgine, MHF F-080 Tayade, C A-025 Soriano, FG F-097 Tedesco, YMS F-041 Soster, PRL J-012 Teixeira, PR B-030 Soto-Suazo, M D-032 Teixeira, SMR G-006 Sousa, MMSL A-046 Teixeira, TS A-024, A-066, D-027 Sousa, MV G-004 Teodoro, AJ I-005 Sousa, RT F-113 Terra, WR F-008, F-009, G-002 Sousa, VO D-024 Tersariol, ILS B-014, B-048, F-062, F-070, RT- Souza e Mello, MA D-026 13.44 Souza, AR B-003 Tesser, RB A-074 Souza, DO RT-15.52 Theodoro, TR B-047 Souza, EC F-013, F-077 Thiago, LS B-042 Souza, GHMF A-043 Thole, AA I-002, L-013 Souza, JM B-033 Thomas, J-L RT-22.73 Souza, JP B-043 Thomé, RG A-058, D-019 Souza, KRS A-061 Tidwell, R G-015 Souza, LEB B-021 Tjäderhane, L RT-13.44 Souza, LNG B-015,B-017,B-018,B-019,B-027 Tognoli, LMMC F-096 Souza, MCA F-042, L-005, L-008 Toledo, FC A-005 Souza, MF L-011, L-005 Toledo, KA H-011, H-012 Souza, PAR F-021 Toledo, OMS F-098 Souza, PS B-010 Toma, L B-040 Souza, R F-118 Tomiosso, TC F-003, F-037, F-046 Souza, RWA F-033 Tone, LG B-021 Souza, TMM G-005 Torres, B RT-12.42 Souza, VR D-009 Torricos, T RT-04.12 Souza, W F-012, G-003 Torrinhas, RS B-058 Souza, WF B-008 Tortelli Jr, TC B-011, B-034, B-058 Sparrow, D D-021 Toschi, AC F-108 Srinivas, S Lec-09 Tostes, R RT-06.21 Stach-Machado, DR F-026 Trapé, AP B-034, B-058 Sternberg, CG F-050, J-004, RT-09.32 Trevelian, MM RT-13.44 steves, GH B-035 Tridico, CP I-003 Stipursky, J F-013 Trindade, CB B-048 Stopiglia, AJ L-004 Trindade, DM F-103 Straight, A Lec-16 Tristão, MAF G-016 Straight, P RT-21.71 Tsuchiya, CT G-019 Strauss, BE B-067 Uchitel, OD Lec-19 Stumbo, AC G-009, G-010, I-002, L-013 Urabayashi, MS F-099 Su, DH H-015 Vago, AR B-062, B-063 Suarez, ER B-047 Vairo, L F-094 Succi, M K-003, K-004 Valdeolivas, SMM A-024, A-066 Sun, SY A-065 Valenca, SS F-082, F-083, RT-23.79 Sunaga, DY B-066 Valero-Lapchik, VB J-003 Sunkel, CE RT-08.27 Vallejos, H B-071 Suñol, C J-020 Vallota, E H-017 Suzuki, DE L-006 Vannier, E B-067 1. 225

livro.indd 225 7/7/2008 16:33:56 Vargas, CD RT-03.9 Zara, SZ D-032 Vargas, E RT-04.12 Zeidler, JD B-060 Vasconcellos, JF B-026, F-060 Zin, WA F-083 Vasconcelos, PAS B-061 Zogaib, FG F-075 Vasconcelos, PC A-060 Zorn, TMT A-086, A-087, A-088, D-032, RT- Vasselo, Q F-113 06.21, RT-06.22 Vaz, NM D-009 Zuchelli, MG D-028 Veas, EMC F-118 Zugaib, M A-083 Veiga, SS F-063, F-064, F-066, F-069 Veja, JL RT-04.14 Veloso, LA F-055 Ventura, BC F-090 Ventura, DSF F-076 Veras, MMV A-073, F-097 Veras, PST G-011, G-018 Vercelli, C RT-20.67 Verinaud, LMC B-053 Veronez, CL F-070 Viana, NB B-033 Vicente, CM B-040 Vicente, CP F-107 Vidal, BC F-003, F-046, RT-24.81 Vidal, FCB B-004 Vieceli, FM D-017 Vieira, E L-017 Vieira, JS A-035 Vilamaior, PSL A-048, A-069, A-071, A-072 Vilas Boas, DS F-087 Vilas Boas, DS B-059 Vilela, MJ B-045 Villa, L Lec-06 Vinagre, CM A-008 Viola, JPB B-010, J-004 Visconti, MA F-088 Viveiros, DD H-017 Viviani, VR F-089 Voltarelli, JC RT-01.3 Wainstein, AJ B-036 Waitzberg, DL B-058, F-110 Wajnberg, G F-050, J-004 Weisberg, J B-040 Wenceslau, CV L-017 Wheatley, D Lec-13 Wiese , C RT-18.62 Winck, MA F-056 Wolstein, O RT-09.31 Woolner, S RT-18.62 Xavier Neto, J RT-15.54 Yamada, AT A-014, A-019, A-025, A-039, A-085, RT-20.68 Yamane, T B-014 Yamanouye, N F-007, F-018, F-040 Yan, CYI D-012, D-017 Yañez, C B-071 Yasumura, EG F-023 Yunes, JA B-022, B-026, F-060 Zabierowski, S B-055 Zacarin, ECMS D-030, F-105, F-106 Zago, CES F-074 Zamudio, S RT-04.12, RT-04.13 1. 226

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