National Institute of Science and Tecnology in Stem Cells and Cell Therapy in Cancer

National Institutes of Science and Tecnology Edital N. 16/2014 MCTI/CNPq/ /CAPES/FAPs

INCTC

Coordinator: Dimas Tadeu Covas Vice-Coordinator: Eduardo Magalhães Rego

Headquarters: Hemocentro Foundation and Ribeirão Preto Medical School

SUMMARY

A) Headquarters 4 B) Institutions, Laboratories and Associated Research Groups 4 C) Management Committee 4 D) Principal Investigators 5 D.1) Detailed description of the proposing group 5 D.2) International Collaborators 14 D.3) Associated Researchers 14 D.4) Technical Team and post-graduate students associated with the project 15 D.5) ) Technical Team and post-graduate students associated with PIs 16 E) Organization and Functional Chart of the Institute 17 F) Mechanisms that will be used to promote the interaction among groups and forms 18 of interaction with groups abroad G) Specification of the activities to be developed by team members 20 H) Definition of specific tasks of every participant entity 22 I) Description of the Program of the Institute 22 J) Detailing the Research Program 30 J.1) Description of the scientific project 31 J.2) Subprojects, objectives and goals 49 J.3) Methodology 62 J.4) Follow-up Indicators of the project 66 K) Detailing the program of qualified professional training 67 L) Detailing the actions of knowledge transfer to society 69 M) Detailing the actions of knowledge transfer to business sector or formulation of 72 public policies N) Potential for generating patents 75 O) Scientific contributions and comparative analysis between the current and expected 77 situation P) Justified Budget 78 Q) Institutional counterpart 83 R) Description of the infrastructure 84 S) Implementation schedule 99 Appendix I – Formal agreement of institutions 100 Appendix II – Formal agreement of international collaborators 115 Appendix III – Team 126 Appendix IV – Qualified professional training (completed) 128 Appendix V – Qualified professional training (ongoing) 136 Appendix VI – Projects approved in the last 5 years 143 Reference 149

A) Headquarters

• Hemocentro Foundation of Ribeirão Preto • Ribeirão Preto Medical School – HCFMRP- USP

B) Associated Institutions, Laboratories or Research Groups

• Ribeirão Preto Medical School – HCFMRP- USP

• School of Animal Husbandry and Food Engeneering of Pirassununga – USP

• School of Veterinary Medicine – USP

• Regional Blood Center of Ribeirão Preto – HCFMRP-USP

• Primatology Center of the Evandro Chagas Institute of Belém (PA)

• Institute of Bioscience – USP

• São Carlos Institute of Physics – USP

• Institute of Biomedical Sciences – USP

C) Management Committee

The management committee will be integrated by the project coordinator (Prof. Dr. Dimas Tadeu Covas) and one representative from each of the five groups associated in this project:

• School of Veterinary Medicine of São Paulo – Prof. Dr. Maria Angélica Miglino

• School of Animal Husbandry of Pirassununga – Prof. Dr. Flávio Meirelles

• Institute of Biomedical Sciences – Prof. Dr. José Alexandre Marzagão Barbuto

• Ribeirão Preto Medical School – Prof. Dr. Eduardo Magalhães Rego

• São Carlos Institute of Physics – Prof. Dr. Valtencir Zucolotto

D) Principal Investigators

• Belinda Pinto Simões – Ribeirão Preto Medical School/USP • Dimas Tadeu Covas – Hemocentro Foundation of Ribeirão Preto • Eduardo Antonio Donadi – Ribeirão Preto Medical School/USP • Eduardo Magalhães Rego – Ribeirão Preto Medical School/USP • Flavio Vieira Meirelles – School of Animal Husbandry and Food Engeneering/USP • José Alexandre Marzagão Barbuto – Institute of Biomedical Sciences /USP • Klena Sarges Marruaz da Silva – Center of Primates of the Institute Evandro Chagas – Belém/PA • Lewis Joel Greene – Ribeirão Preto Medical School/USP • Lygia da Veiga Pereira – Institute of Bioscience/USP • Marco Antonio Zago – Ribeirão Preto Medical School/USP • Maria Angélica Miglino – School of Veterinary Medicine and Animal Husbandry/USP • Roberto Passetto Falcão – Ribeirão Preto Medical School/USP • Rodrigo Tocantins Calado – Ribeirão Preto Medical School/USP • Valtencir Zucolotto – São Carlos Institute of Physics/USP • Wilson Araújo da Silva Jr. – Ribeirão Preto Medical School/USP

D.1) Detailed Description of the Proposing Group

The Project will be coordinated by Prof. Dr. Dimas Tadeu Covas – CNPq researcher level 1B, graduated in Medicine at University of São Paulo (USP) in (1981). Master’s (1986) and doctorate (1993) degrees in Medicine by USP, and he obtained livre-docência at the same University in 1999. Currently, Dr. Covas is a full Professor at the Ribeirão Preto Medical School/USP, President-Director of the Hemocentro Foundation of Ribeirão Preto, Coordinator of the Center for Cell-based Therapy (CTC-CEPID-FAPESP), and president of the Brazilian Association of Hematology, Hemotherapy and Cell Therapy (ABHH). He is a hematologist and hemotherapist, and develops research on the following subjects: molecular and cellular biology, stem cells, erythrocytic and platelet antigens, viruses (HIV and HTLV), protein heterologous expression in cellular systems in vitro. He is Associate Editor of Revista Brasileira de Hematologia e Hemoterapia, and associate editor of the journal PLosOne; he was one of the founders of the Escola Brasileira de Hematologia. In addition, Dr. 5

Covas is member of Academia de Ciências de Ribeirão Preto. He is the coordinator of the Postgraduate Program on Clinical Oncology, Stem Cells and Cell Therapy of the FMRP/USP. He has also promoted the dissemination of science and education, and organized postgraduate programs for teachers and elementary and high school students. He has edited two books, and one of these was awarded Prêmio Jabuti in 2007. Dr. Covas has published 158 articles in specialized periodicals, 790 works in congresses including complete studies and abstracts, 16 book chapters, and 10 books. He has participated in 231 meetings in Brazil and abroad. His activities as advisor resulted in 22 Master's dissertations, 12 doctorate and 12 post-doctoral theses. In addition, Dr. Covas has advised 7 scientific initiation works, and has received 23 awards and/or honors. Lattes CV: http://lattes.cnpq.br/5244926449437566

The following researchers also participate in this proposal as principal investigators:

Prof. Dr. Belinda Pinto Simões – She received her doctorate in Medicine (Hematology) by the University of São Paulo (1998) and her post-doctoral by the Ludwig-Maximilians- Universität München (2003). Currently she is a professor at the Ribeirão Preto Medical School of the University of São Paulo (FMRP-USP). She is the coordinator and supervisor of the Unit of Bone Marrow Transplantation of the Hospital of the FMRP-USP and was elected president of the Brazilian Society of Bone Marrow Transplantation. She has experience in Medicine, with emphasis on hematology, acute leukemias, bone marrow transplantation, and sickle cell disease. She has published 161 complete studies in journals and congresses and one book. She has participated in 22 scientific meetings and supervised 6 master’s dissertations, 1 doctorate thesis and she has received 5 awards. Lattes CV: http://lattes.cnpq.br/3238550237127398

Prof. Dr. Eduardo Antônio Donadi – Researcher level 1A of CNPq. Dr. Donadi was graduated in Medicine by the Ribeirão Preto Medical School (1976), he has received his master’s degree by the Ribeirão Preto Medical School (1982) and his doctorate in Medical Practice by the same institution (1986). He conducted his post-doctoral activities in the Virginia Mason Research Center, in Seattle, in the years of 1990 and 1991. He concluded his livre-docência in 1997 and currently he is a professor at the Division of Clinical Immunology of the Department of Medical Practice of the Ribeirão Preto Medical School- USP. He has experience in Medicine, in the field of Clinical Immunology, by researching, teaching and

6 treating patients with diseases related to immune system disorders, such as autoimmune diseases. His current research lines are related to the study of the etiopathogenic aspects of autoimmune diseases, transplantations, neoplastic and infeccious diseases, focusing on the evaluations of differential and particular gene expression of candidate genes, polymorphisms of genes involved in innate and acquired immune response, and also association of genetic polymorphisms with gene expressions. Dr. Donadi has a particular interest in the study of mechanisms that control the expression of genes of the major histocompatibility complex. He supervises postgraduate students in the Programs of Basic and Applied Immunology and Medical Practice, both in the Ribeirão Preto Medical School of University of São Paulo. He has published 268 complete articles in journals, 1 book, 12 book chapters. He has participated in 63 national and international events. He has supervised 22 master’s dissertations, 30 doctorate and 30 post-doctoral theses. Lattes CV: http://lattes.cnpq.br/8778713382135771

Prof. Dr. Eduardo Magalhães Rego – CNPq researcher level 1C. He graduated in Medicine at Medicine Faculty of Ribeirão Preto, University of São Paulo, in 1988. He obtained his doctoral degree in Medical Clinic from the same institution (1997), and he did a post-doc at Memorial Sloan Kettering Cancer Center in New York, USA. Currently, Dr. Rego is Associate Professor at University of São Paulo, and he works in the field of Hematology. His research line focuses on acute leukemias, with special emphasis on Acute Promyelocytic Leukemia. He has published studies that demonstrate that the hybrid genes PML/RARa, PLZF/RARa, NPM/RARa and NuMA/RARa are able to induce leukemia in transgenic mice. These transgenic models have been used for the study of the leukemia molecular bases and for research on new therapeutic approaches. He is one of the founders and coordinator in Brazil of the International Consortium in Acute Promyelocytic Leukemia, which is a multinational innitiative for the establishment and development of a network of institutions in Latin America, USA and Europe with the aim of improving the clinical outcome of patients with acute leukemias and contributing to the understanding of the genesis and evolution of these diseases. Dr. Rego also studies the relationship between ribosome function and cancer by analyzing mice that carry the mutations in the gene DKC1. His most important works are: a) Rego EM et al. Improving acute promyelocytic leukemia (APL) outcome in developing countries through networking, results of the International Consortium on APL. Blood 2013; 121(11):1935-43; b) Yoon A et al. Impaired control of IRES-mediated translation in X-linked

7 dyskeratosis congenita. Science. 2006, 312(5775):902-6; c) Rego EM et al. Leukemia with distinct phenotypes in transgenic mice expressing PML/RAR alpha, PLZF/RAR alpha or NPM/RAR alpha. Oncogene. 2006, 25(13):1974-9; d) Rego EM et al. Role of promyelocytic leukemia (PML) protein in tumor suppression.J Exp Med. 2001, 193(4):521-29; e) Rego EM et al. Retinoic acid (RA) and As2O3 treatment in transgenic models of acute promyelocytic leukemia (APL) unravel the distinct nature of the leukemogenic process induced by the PML- RARalpha and PLZF-RARalpha oncoproteins. Proc Natl Acad Sci U S A. 2000, 97(18):10173-8. Dr. Rego has published 99 articles in specialized periodicals, 251 works in meeting proceedings including complete studies and abstracts, and 19 book chapters. He has participated in 169 meetings in Brazil and abroad. His activities as advisor resulted in 8 Master's dissertations and 14 doctorate and 8 post-doctoral theses. In addition, Dr. Rego has advised 7 scientific initiation works, and has received 15 awards and/or honors. Lattes CV: http://lattes.cnpq.br/1543544998729361

Prof. Dr. Flávio Vieira Meirelles – CNPq researcher level 1A. He graduated in Veterinary Science in Universidade Estadual Paulista Júlio de Mesquita Filho (1993), and received his Master’s degree in Sciences Vétérinaires Option Reproduction from Université de Montréal (1997). He received his Ph.D. in Genetics from Universidade de São Paulo (1999). Currently, he is Associate Professor III (livre docente) at University of São Paulo; ad hoc consultant for São Paulo Research Foundation (FAPESP), associate editor of Genetics and Molecular Research, Sociedade Brasileira de Tecnologia de Embriões, and consultant for the journals Heredity, Animal Genetics, and Genetics and Molecular Biology. Dr. Meirelles has experience in the field of Genetics, with emphasis on Cytoplasmic Inheritance, and is involved mainly with the following subjects: mtDNA, Bos indicus, cattle, embryos and apoptosis. Lattes CV: http://lattes.cnpq.br/6698246156573680

Prof. Dr. José Alexandre Marzagão Barbuto - CNPq researcher level 2. He graduated in Medicine at the University of São Paulo (1981), he received his master’s degree in Microbiology and Immunology by the Federal University of São Paulo (1983) and his doctorate degree in Immunology by the University of São Paulo (1988). Currently he is an Associate Professor (ms-5) of the University of São Paulo. He has experience in the field of Immunology, with emphasis on Cell Immunology, working mainly on the following subjects: cancer, immunotherapy, dendritic cells, macrophages. He has published 54 articles in

8 periodicals and 18 book chapters. He has participated in 87 national and international events. He has supervised 19 master’s dissertations, 12 doctorate theses and two post- doctoral theses. He has received 10 awards. Lattes CV: http://lattes.cnpq.br/0400432783033740

Klena Sarges Marruaz da Silva – Veterinarian. She has received her master’s degree in Animal Science by the Universidade Federal do Pará (2005). Currently she is a researcher at the Evandro Chagas Institute (Ministry of Health), INCTC and technical responsible for the Escola Experimental de Primatas (Universidade Federal do Pará). She has experience in the field of Veterinary Medicine, working mainly on the following subjects: primates, pathology, laboratory animals, wild animals, and Parkinson Disease. She has published 10 articles in specialized periodicals and 38 abstracts in congresses. She has participated in 41 events in Brazil and abroad and has supervised to graduation works and six scientific initiations. Lattes CV: http://lattes.cnpq.br/9546515243950901

Prof. Dr. Lewis Joel Greene – CNPq researcher level 1A. He is full voluntary professor at the Ribeirão Preto Medical School, University of São Paulo, in the Department of Cellular Biology, Molecular and Pathogenic Bioagents. Dr. Greene is the supervisor of Protein Chemistry Center at the Regional Blood Center of Ribeirão Preto, where he develops studies concerning the chemical, functional and structural characterization of proteins, using traditional protein chemistry approaches and proteomic analysis. The research lines developed by him comprise: a) Studies on the functional and structural characterization of proteins during the proliferation and cellular differentiation of hematopoietic progenitor cells and human tumor cells, using a proteomic approach; and b) Chemical and biological characterization of biologically active proteins using traditional protein chemistry methods. Dr. Greene has published 130 articles in specialized periodicals, 243 works in congresses including complete studies and abstracts, and 3 book chapters. His activities as advisor have resulted in 37 Master’s dissertations and 58 doctorate theses. In addition, he has advised 2 scientific initiation works. Lattes CV: http://lattes.cnpq.br/7810652486138159

Prof. Dr. Lygia Veiga Pereira – CNPq researcher level 1D, Ph.D., obtained her Bachelor of Physics from Pontifícia Catholic University of Rio de Janeiro in 1988. She received her Master’s degree in Biological Sciences (Biophysics) from Federal University of Rio de Janeiro

9 in 1990, and her Ph.D. in Biomedical Sciences from the Mount Sinai School of Medicine, City University of New York in 1994. Currently, she is Associate Professor MS5 (livre docente) at University of São Paulo, member of Science Academy of São Paulo State, and she is the head of Molecular Genetic Lab of Bioscience Institute, USP. She has experience in the field of Genetics, with emphasis on Human Molecular Genetics, and she is involved in the following subjects: animal models of genetic disease, embryonic stem cells, epigenetic inheritance, and X chromosome inactivation. She is the author of two scientific dissemination books: “Seqüenciaram o Genoma Humano... E Agora?” and “Clonagem: da Ovelha Dolly às Células- Tronco”, Moderna publishing house. Dr. Pereira has published 54 articles in specialized periodicals, 23 works in congresses including complete studies and abstracts, 21 book chapters and 6 books. She has published 55 articles in magazines/newspapers. Her activities as advisor resulted in 7 Master’s dissertations and 23 doctorate theses. She has received 9 awards and/or honors. Lattes CV: http://lattes.cnpq.br/1550542923772116

Prof. Dr. Marco Antonio Zago – CNPq researcher level 1A. President of the University of São Paulo. He graduated in Medicine in the Ribeirão Preto Medical School of the University of São Paulo (1970). He received his master’s degree in Clinical Practice by the Ribeirão Preto Medical School of the University of São Paulo (1973) and his doctorate degree by the same institution (1975). He is a full Professor at the University of São Paulo, full member of the Academia Brasileira de Ciências, he was the president CNPq (Conselho Nacional do Desenvolvimento Científico e Tecnológico). He has experience in the field of Medicine, with emphasis on Hematology. He was the president and scientific director of the Hemocentro Foundation of Ribeirão Preto, and clinical director of the Clinical Hospital of Ribeirão Preto. He has published 208 articles in specialized periodicals, two books and 18 book chapters. He has supervised five master’s dissertations and 21 doctorate theses. He has received four awards or titles. Lattes CV: http://lattes.cnpq.br/3234638489546052

Prof. Dr. Maria Angélica Miglino – CNPq researcher level 1A. Maria Angélica Miglino has received her PhD in Sciences (funcional anatomy: structure and ultra-structure) from Universidade de São Paulo in 1985. Currently, Dr. Miglino is a full professor at University of São Paulo. She has published 460 articles in specialized periodicals and 478 works in meeting proceedings. She has published 24 book chapters and 2 books. She has 1 technique process and other 28 technical production items. Dr. Miglino has participated in 194 meetings 10 abroad and in Brazil. Her activities as advisor resulted in 46 Master's dissertations, 55 PhD theses. She has been a co-advisor in 1 Master's dissertation, and in 2 PhD theses. In addition, she has advised 71 scientific initiation works in the Veterinary Medicine and Morphology fields. She has been granted 8 awards and/or honors. She has coordinated 36 research projects between 1993 and 2004. Currently, she is engaged in 24 research projects and coordinates 22 of these. Dr. Miglino develops activities in Veterinary Medicine, with emphasis on animal reproduction. During her professional activities, she has interacted with 941 collaborators in co-authorships of scientific works. The most frequent terms used to contextualize her scientific, technological and artistic-cultural in her Curriculum Lattes are: placenta, anatomy, morphology, bovines, study, ruminants, dogs, arterial, plaque, and vascularization. Lattes CV: http://lattes.cnpq.br/0806064137922471

Prof. Dr. Roberto Passetto Falcão – CNPq researcher level 1A and full professor at University of São Paulo, who has the following qualifications: graduation in Medicine at University of São Paulo, Ribeirão Preto (1966), doctorate in Medical Practice by the Ribeirão Preto Medical School at University of São Paulo, Ribeirão Preto (1972), post-doctoral at Oxford University (1975 – 1976) livre-docência by USP (1980). He was the head of the Department of Medical Practice of the Ribeirão Preto Medical School-USP from 1997 to 2001, president of the Colégio Brasileiro de Hematologia from 1997 to 2005, and representative of the Brazilian Society for Science Progress in the National Health Council. Currently, Dr. Falcão is a full professor of the Ribeirão Preto Medical School, University of São Paulo and coordinator of the National Institute of Science and Technology in Stem Cells and Cell Therapy. He has experience in Medicine, with emphasis on Medical Practice, Hematology and Immunology, and he is involved mainly in the following subjects: aplastic anemia, lymphocytes, myeloid and lymphoproliferative diseases He has supervised 10 master dissertations and 12 doctorate theses. He is a co-editor of the journal Revista Brasileira de Hematologia e Hemoterapia and member of the editorial board of the journal Lancet Haematology. Lattes CV: http://lattes.cnpq.br/0739840296573834

Prof. Dr. Rodrigo do Tocantins Calado De Saloma Rodrigues – He graduated in Medicine by the Ribeirão Preto Medical School (FMRP) of the University of São Paulo (USP) (1997), medical residence in Hematology and Hemotherapy (2001) by the Clinic Hospital of Ribeirão Preto and doctorate in Medical Practice by the Ribeirão Preto Medical School of the

University of São Paulo (2003). He did a post-doctoral in the National Institutes of Health, Bethesda, Maryland, EUA, where he was from the Scientist Staff of the Hematology Branch. Currently he is an Associate Professor of the Department of Clinical Practice of the FMRP- USP, coordinator of the Program of Professional Master on Hemotherapy and Biotechnology and coordinator of the Hematology Laboratory "Cássio Bottura" of the Hospital of the FMRP- USP. He has experience in the field of Medicine, with emphasis on Hematology, working mainly on the following subjects: aplastic anemia, bone marrow failure, myelodysplasia, idiopathic pulmonary fibrosis, and hepatic cirrhosis, nodular regenerative hyperplasia of the liver, telomere diseases, telomeres, telomerase, and chromosomal instability. He is an affiliated member of the Academia Brasileira de Ciências, member of the Education Committee of the American Society of Hematology (ASH), associate editor of the journal Revista Brasileira de Hematologia e Hemoterapia and member of the editorial board of the journal Blood. He has published 57 articles in periodicals, one book, and 10 book chapters. He has supervised 12 master’s dissertations, 3 doctorate theses and 2 post-doctoral theses. He has received 8 awards. Lattes CV: http://lattes.cnpq.br/5187902541390363

Prof. Dr. Valtencir Zucolotto – Affiliated member of the Academia Brasileira de Ciências (ABC). He is an Associate Professor at the São Carlos Physics Institute – IFSC of the University of São Paulo (USP), where he coordinates the Group Nanomedicine and Nanotoxicology GNano/IFSC/USP. He is the president of the Culture and Extension Comission (CCEx) of the IFSC/USP. He graduated in Material Engineering by the Federal University of São Paulo (1997), he received his master’s degree in Science and Material Engineering by the Federal University of São Paulo (1999) and his doctorate degree in Science and Material Engineering by the University of São Paulo (2003). He received training at the University of Massachusetts at Lowell, USA (2000-2001). He was a visiting professor at the University Joseph Fourier, Grenoble, France, in 2010. He has experience in the field of Nanotechnology and Development and Application of Nanomaterials in Medicine. Currently he develops research on the use of nanomaterial in medicine and studies the toxicity effects of nanoproducts in living cells and tissues. He has developed and taught the Postgraduate Course on Techniques of Scientific Writing in English. He has developed and taught several mini-courses on scientific writing and how to write scientific articles in many universities in Brazil and abroad. He has published more that 125 articles on international journals and 9 book chapters. He has more than 1,800 citations and factor H 25. He is associate editor of

12 the Journal of Biomedical Nanotechnology (Impact Factor 7.578) and editor of the series of books Nanomedicine and Nanotoxicology (Springer). Lattes CV: http://lattes.cnpq.br/5768000922241088

Prof. Dr. Wilson Araújo da Silva Jr. – CNPq researcher level 1C. He graduated in Biology, Medical Modality (Biomedicine) at Pará Federal University 1989, and obtained his Master's degree in Genetics from Medicine Faculty of Ribeirão Preto, University of São Paulo in 1993. He received his Ph.D. in Genetics from the Ribeirão Preto Medical School, University of São Paulo, in 1999. Dr. da Silva did a post-doc in Cancer Genetics at Ludwig Institute, Memorial Sloan-Kattering Cancer Center in New York City, NY, USA. He is Associate Professor of Genetic Department of the Ribeirão Preto Medical School– USP. He develops his research in the field of Human and Medical Genetics, working mainly with the following subjects: DNA polymorphisms, Bioinformatics, and Cancer Genetics. Dr. da Silva has published 141 articles in specialized periodicals and 80 works in meeting proceedings including complete studies and abstracts. His activities as advisor have resulted in 16 Master’s dissertations, 18 doctorate theses, and he has advised 3 scientific initiation students. He has received 9 awards and/or honors. Lattes CV: http://lattes.cnpq.br/8655277884027052

D.2) International Collaborators

Collaborator Position Institution Country

Group Leader International Centre for Genetic Luiz F Zerbini, Ph.D. South Africa Cancer Genomics Engineering and Biotechnology

Professor of University Hospitals Medicine Seidman Cancer Center United Marcos de Lima, MD Director Stem Cell Case Medical Center States- Transplant Cleveland Program University United Colorado State University George E. Seidel Distinguished States- Department of Biomedical Sciences Professor Colorado Gerrit J. Bouma, Ph.D. United Associate Professor Colorado State University States- Department of Biomedical Sciences Colorado Lawrence C. Smith Research University of Montreal Canada PhD Professor Faculté de Médecine Vétérinaire Michel C. Investigator Howard Hughes Medical Institute New York Nussenzweig, PhD Miguel Mano, PhD Group Leader University of Coimbra Portugal United Quinton Associate Colorado State University States- Winger Professor Department of Biomedical Sciences Colorado Associate Professor of Medicine Towia A. Libermann, Beth Israel Deaconess Medical United Director Genomics PhD Center States- and Proteomics Harvard Medical School Boston Center and Cancer Proteomics Core Professor and W. Allan King, PhD University of Guelph Canada Tier 1 Canada Departament of Biomedical Sciences Research Chair Hans-Jochem Kolb Senior Consultant Technische Universitat München Germany

D.3) Associated Researchers

• Anelisa Ramão • Carlos Eduardo Ambrósio • Carlos Gilberto Carlotti Jr. • Cleidson de Pádua Alves • Daniela Pretti da Cunha Tirapelli • Daniele dos Santos Martins

• David Livingstone Alves Figueiredo • Durvanei Augusto Maria • Elisa Maria de Sousa Russo • Erika Maria Terra • Fabíola Attié de Castro • Fabíola Traina • Felipe Perecin • Josane de Freitas Sousa • Julia Maria Matera • Kamila Chagas Peronni • Kamilla Swiech • Kelen Cristina Ribeiro Malmegrim de Farias • Lorena Lôbo de Figueiredo Pontes • Luciano Neder Serafini • Marcia Rita Fernandes Machado • Maria Carolina de Oliveira Rodrigues • Marisa Ramos Barbieri • Maristela Delgado Orellana • Mirela Tinucci Costa • Najeh Maissar Khalil • Patricia Cristina Baleeiro Beltrão Braga • Patricia Vianna Bonini Palma • Rodrigo Alexandre Panepucci • Rubiana Mara Mainardes • Simone Kashima Haddad • Sonja Ellen Lobo • Tais Harumi de Castro Sasahara • Valeria Valente

• Virginia Picanço e Castro

D.4) Technical team and post-graduate students associated with the project (Appendix I)

The technical team and post-graduate students associated with the project are described in Appendix I. 15

D.5) Technical team and post-graduate students associated with PIs

E) Organization and Functional Chart of the Institute

The Institute will be administered in accordance with the organization chart below.

Project Coordinator

Management International Committee Advisory Committee

Research Education and Technology Internationalization Management Coordinator Diffusion Transfer Coordinator Coordinator Coordinator Coordinator

Principal CEDUC Innovation Principal Nucleus of Investigators Agency Investigators Projects and Programs House of Science Technological Park Quality Management MuLEC

TV Incubator

Library

Teaching Laboratory

Project Coordinator – Prof. Dr. Dimas Tadeu Covas Vice-Coordinator – Prof. Dr. Eduardo Magalhães Rego Research Coordinator – Prof. Dr. Eduardo Magalhães Rego Education and Diffusion Coordinator – Prof. Dr. Marisa Ramos Barbieri Technology Transfer Coordinator – Prof. Dr. Flávio Meirelles Internationalization Coordinator – Prof. Dr. Rodrigo Calado Management Coordinator – Elaine Teresinha Faria de Sousa

F) Management and mechanisms that will be used to promote the interaction among groups and forms of interaction with groups abroad

INCTC management will be performed by the technical and administrative areas of FUNDHERP (Hemocentro Foundation of Ribeirão Preto). The INCTC Coordinator and Vice- Coordinator, supported by the management committee, will be in charge of following the achievement of the goals related to research, education, technology transfer and financial and administrative management.

The management of resources will be centralized in the Management Coordination, responsible for following technical and financial development of the project, including the establishment of priorities, according to the schedule for the release of resources jointly with the Management Committee, also responsible for the centralization of the information to be used for the elaboration of scientific reports.

In 2013, FUNDHERP acquired an integrated management system (Benner) that includes a module of Project Management and integrates all departments involved (project management, costs, material, equipment, purchase, finance, accounting, engineering, and quality). This integration assures the follow-up of the development managing financial and physical budget, monitoring the results, and integrating the cash flow of every project, in real time, besides the background register.

This monitoring system enables to control costs, deviations and to simulate by reports, facilitating thus to take actions for the accomplishment of goals, optimizing costs and deadlines. The system allows the researcher to register and follow the purchase resquests. Since its implementation, in September 2013, 500 orders were registered, totaling R$1,298,606.51 and USD$ 1,736,188.64.

The Center of Education will be responsible for the management of the educational projects, and will enable the interaction between students and researchers, apart from organizing and guiding the activities in the Laboratory of Studies, in the House of Science, in the Museum and Laboratory for Science Teaching and in other places where practical activities with basic education students and postgraduate fellows will be conducted, for instance in the Celularium. I will also coordinate the activities of the Library and Hemocentro TV.

In the innovation aspect, the Technology Transfer Coordinator will set up a data base in order to register all transfer and research initiatives that may generate products of technological interest or value. The Technological Park of Ribeirão Preto will offer all the support needed for the establishment of partnership with companies both from the incubator and from the local production sector that work in the segments of biotechnology, pharmacology, cosmetics and animal health. It will also support the studies on economic viability, business plans, establishment of small companies, intellectual property, etc.

The first general meeting for the evaluation of the Project and design of the activities for the beginning of the development of projects will be held 30 days after the project approval. Every semester meetings will be held in order to evaluate results, follow the goals and define strategies.

The groups will also interact in the programs of education and knowledge transfer to society, with effective participation in interdisciplinary postgraduate course on Clinical Oncology, Stem Cells and Cell Therapy.

The International Advisory Committee will be invited at least once in every two years to conduct a critical analysis of the performance of the Institute and recommend or propose new action plans on the scientific, technical and management aspect.

G) Specification of the activities to be developed by Team Members

Subprojects

Investigators Goal 1 Goal 2 Goal 3 (PI)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Belinda Pinto Simões Dimas Tadeu Covas Eduardo Antonio Donadi Eduardo Magalhães Rego Flavio Vieira Meirelles José Alexandre Marzagão Barbuto Klena Sarges Marruaz da Silva Lewis Joel Greene Lygia da Veiga Pereira Marco Antonio Zago Maria Angélica Miglino Roberto Passetto Falcão Rodrigo Tocantins Calado Valtencir Zucolotto Wilson Araújo da Silva Jr.

Figure 1. Specification of the activities to be developed by team members.

H) Definition of Specific Tasks of each participating entity

Unit General Responsible Subprojects

Center of Primates of the Evandro Chagas Klena Sarges Marruaz da Silva 26 Institute – Belém/PA Ribeirão Preto Medical School/USP Rodrigo Tocantins Calado 5 and 26 Ribeirão Preto Medical School/USP Belinda Pinto Simões 22, 23, 24 and 26 Ribeirão Preto Medical School/USP Eduardo Antonio Donadi 4 and 26 Ribeirão Preto Medical School/USP Eduardo Magalhães Rego 6 and 26 Ribeirão Preto Medical School/USP Lewis Joel Greene 26 Ribeirão Preto Medical School/USP Marco Antonio Zago 2, 9 and 26 Ribeirão Preto Medical School /USP Roberto Passetto Falcão 26 Ribeirão Preto Medical School /USP Wilson Araújo da Silva Jr 1 and 26 School of Veterinary Medicine and Animal Maria Angélica Miglino 3, 10, 20, 25 Husbandry/USP and 26 School of Animal Husbandry and Food Flavio Vieira Meirelles 16, 17, 18 and Engineering/USP 26 Ribeirão Preto Medical School/USP Dimas Tadeu Covas 7, 11, 12, 14, Hemocentro Foundation of Ribeirão Preto 15, 18, 21, 26 Institute of Bioscience/USP Lygia da Veiga Pereira 26 Institute of Biomedical Sciences/USP José Alexandre Marzagão Barbuto 13 and 26 São Carlos Physics Institute/USP Valtencir Zucolotto 19 and 26

I) Description of the Institute Program

The present proposal establishes the continuity and expansion of the activities of the National Institute of Science and Technology in Stem Cells and Cell Therapy, one of the National Institutes of Science and Technology created by CNPq/FAPESP in 2008. In that opportunity, we proposed the development of an extensive program of basic and clinical research to understand, isolate, culture and therapeutically use somatic and pluripotent

22 stem cells, both in animal and human models. That research program also predicted the study on neoplastic stem cells, especially the ones associated with leukemias and lymphomas. Considering the evolution of scientific knowledge of neoplastic stem cells that culminated in the proposal of a new oncogenesis model (1,2), according to which there are multiple subpopulations of cancer stem cells (CSCs) that exhibit higher or lower predominance in tumor mass throughout the evolution of the disease, we decided in this proposal to isolate, culture, characterize single tumor cells (single cell analysis), and study tumor microenvironment with the aim of understanding the intrinsic and extrinsic mechanisms that lead to cancer genesis. Additionally, we intend to make a great effort to develop innovative therapies against cancer, including immunotherapy and nanomedicine, which might originate pre-clinical and clinical studies in a broad range of neoplasias. To carry out this ambitious research plan, we have assembled experts and institutions with acknowledged scientific experience in the areas involved: cell and molecular biology, genetics, immunology, hematology, oncology, systems biology and bioinformatics, protein chemistry, chemical and material engineering, and veterinary. The participation of principal investigators (PIs) and collaborators will be fostered in an integrated and complementary way, fruit of the understanding that cancer is a complex issue that could only be effectively approached in a multidisciplinary way. The strongest argument in favor of our group to assure that we will be able to move forward with this project in a successful way is to demonstrate what we have done and what we have been doing so far. Eleven of the 15 PIs of this proposal have participated in the previous project (INCTC- National Institute of Science and Technology in Stem Cells and Cell Therapy in Cancer), as well as in the CEPID-FAPESP (CTC – Center for Cell-based Therapy). The scientific production of these investigators on the subject “cell therapy”, during the period of development of the project (2009-2014), accounts for more than 470 articles published in refereed journals, representing an increase of more than 50% when compared to the period of five years before INCTC. They have also published 7 books and 11 book chapters and applied for 17 patents. Sixty- six master dissertations, 72 doctorate and 34 post-doctoral theses have been completed. Using the project resources, 28 junior post-doctoral fellowships and 59 Technological and Industrial Development scholarships (DTI) have been granted. The scholarships granted throughout the project were essential for the success of the proposals. It is worthy to stand out that without these scholarships the project would not have succeeded.

This proposal gathers 15 PIs from 7 institutions, with expressive academic production, highlighting the publication of 1,358 articles in refereed journals and 14,084 citations and index h equals to 56 (Figure 1).

Início do INCTC Início do INCTC

Figure 1. Evolution of the number of works and citations of 15 principal investigators within this proposal.

This wide academic experience – has certainly constituted on of the best groups of research on stem cells, cell therapy and cancer in the country – brought together in an integrated way to address a challenging issue, such as cancer, will enable a great scientific advance which will be able to put the country in a prominent position in the international scene.

Why to study cancer?

The World Health Organization estimated the existence of about 33 million people living with cancer in the world, in 2012. In the same year, 14.1 million new cases were diagnosed and 8.2 million people died from the disease, totaling 13% of deaths per year worldwide. In the last decades, cancer has become a world public health problem. In Brazil, neoplasias are the second leading cause of mortality, accounting for 17% of the total of deaths in 2011. The National Institute of Cancer has

24 projected for 2014 the occurrence of 576,000 new cases of cancer, what represents an increase of 20% in the incidence in the last decade. In general, two in every three people will be diagnosed with cancer throughout their lives. Cancer is a complex biological phenomenon that has been challenging science for decades. Despite numerous projects aiming at the elucidation of the genome of several types of cancer have been successfully concluded, even among our group that actively participates in this process (M. A. Zago was the coordinator of the project clinical genome of cancer funded by FAPESP) few specific therapies (also known as “target”) have emerged, reflecting the paradigm that genetic mutations are necessary, but not enough for full cancer development. Therefore studies on basic oncogenic mechanisms are absolutely essential. Part of the difficulties to advance in the area results from the use of misconceptions or outdated paradigms that have driven the planning of most part of the studies conducted until the end of the last century. Perhaps the biggest conceptual mistake in which one has incurred was to consider that cancer was the result of a disorderly proliferation of genetically altered somatic cells that lost the capacity of replicative control and originated abnormal clonal tissue. In this regard, to study tumor cell mass and its genome would be enough to produce biological information needed to understand tumor biology once that cancer would be nothing but a clonal evolution of a genetically altered tumor cell due to the action of endogenous and exogenous carcinogens. One of the most convincing proofs of the limitation of this model comes from the studies in transgenic animals, including those generated by our group, in which only a fraction of the animals developed the disease, although 100% of the individuals have the same mutation associated with oncogenesis in humans (3). More recently, a new paradigm has risen in relation to the biology of cancer: the concept that cancer is a specialized body tissue that develops from a limited number of cancer stem cells (CSCs) which are responsible for the origin and maintenance of all tumor progeny. Therefore, cancer would be a tissue adapted to the internal economy of the organism similarly to normal tissues, and naturally specialized due to its origin, in survival. This new concept brings deeper implications for the understanding of tumor biology, as well as for the understanding of rationality of the therapeutic effort. In this conception, full comprehension of oncogenic mechanisms will only be achieved with the isolation and characterization of CSCs what faces numerous methodological challenges to be implemented. On the other hand, curative therapies for cancer will only be developed when directed towards the eradication of CSCs.

Thus, the focus of this project is to develop a broad set of experimental and clinical approaches, based on this new paradigm that shows cancer, in general, as product of the emergence and development of cancer stem cells. To understand and fight these cells summarizes our INCTC continuity proposal.

The Scope of the Scientific Project

The scope of the scientific project can be seen in Figure 2. In general, the set of studies in the area of basic research involves the isolation and characterization of tumor cells and cancer stem cells which will be studied in a detailed level by tools grouped as “OMICs”: transcriptome, genomics, epigenomics, cytomics and proteomics that allow the integration of large scale biology and approach to biological systems. In the second group, new therapeutic approaches will be studied focusing on immunotherapy, hyperthermia, and nanomedicine. In the third segment, the application of cells and therapeutic tools in animal and human models will be performed.

Figura 3. Scope of the scientific project.

In general, the scientific proposal covers three main areas: 1) Basic studies for understanding the biology of cancer; 2) Development of new therapeutic approaches with emphasis on immunotherapy; and 3) Pre-clinical and clinical trials. In item C, we present the Goals and Objectives of the 26 subprojects that we intend to develop and the detailed description of the integrated scientific project.

Educational Project

In this new INCTC phase, we aim to consolidate and expand the educational activities already developed in the ongoing Project and that can be considered as a successful example. INCTC educational activities are coordinated by the Center of Education (CEDUC) that relies on a team formed by 4 employees (1 translator, 1 biologist, and 2 secretaries) with full-time dedication to the project. The physical structure of CEDUC is composed of four classrooms with capacity of 200 people, two amphitheaters with capacity of 170 and 40 seats respectively, all equipped with multimedia projectors and Wi-Fi network. Additionally, it has a research and teaching laboratory with a total area of 34 m2 equipped with appropriate infrastructure for experimental activities in molecular and cell biology and where teachers and high school students develop scientific initiation projects and preparation for practical classes. The House of Science and the Museum and Laboratory for Science Teaching (MuLEC) also integrate the educational project. The House of Science is multipurpose space of 184 m2 designed to host and coordinate the activities of science teaching and dissemination to students and teachers of elementary and high school education, and to the general public. The House is coordinated by Prof. Dr. Marisa Ramos Barbieri, a retired professor at USP and a renowned educator. The activities developed by the House are divulged main on its educational portal (http://ead.hemocentro.fmrp.usp.br/joomla) which allows interactive access to the educational material developed by teachers, students and postgraduate students involved in the project. In 2013, the House portal reached more 160 mil visits from more than 100 countries. The Museum and Laboratory for Science Teaching (MuLEC) was established in 2014 to serve as a space for the permanent exhibition of the educational material produced during the activities conducted in the House of Science. It occupies a building of 276 m2 of the University of São Paulo, located in the campus of Ribeirão Preto. It is open to public visitation. The educational activities also have the support of Hemocentro TV, which is a center for media production, composed of a television studio and an editing suite. Two journalists and two image and sound technicians work at Hemocentro TV, they are responsible for the production of all digital material which includes videoclasses, documentaries, interviews, video record of lectures and courses, maintenance of the YouTube channel and dissemination of events.

Other educational resource produced in INCTC, which proved to be effective to divulge and popularize science, is the “Celularium”, an inflatable dome of 22 m2, with a full dome digital technology projector, which has been used to exhibit exhibition of the movie “A trip through the cell” that leads the visitor though an amazing trip inside an animal cell, exploring the morphology and function of every cell organelle. The “Celularium” has been exhibited in Scientific Congresses, such as Congresso Brasileiro de Genética, held in Águas de Lindóia, in 2013. It also travels to many cities in the State of São Paulo, opportunities in which it is exhibited to the general public in schools and open spaces. In 2013, 1,500 students from Ribeirão Preto and its surrounding towns had the chance to watch this movie produced by researchers linked to INCTC. In addition to these resources, the area of education and science dissemination keeps many training and scientific initiation programs designed to teachers and students of elementary and high schools, such as the program “Adopt a Scientist” where postgraduate students are tutors of small groups of students of basic education with the aim of discussing and studying biology subjects corresponding to the themes of the research carried out by the Institute. This program complements another one named “Little Scientist”, when students of elementary and high schools develop simple scientific initiation programs. The numerous educational activities developed within the Educational Program of the Institute can be visualized on the House of Science website, as mentioned before. Lato and stricto sensu postgraduate education has also been developed as part of the program of knowledge dissemination and specialized professional training in charge of the INCTC. In 2010, two postgraduate courses were organized; they are linked to the Department of Medical Practice, but administered and taught by INCTC investigators. The academic master and doctorate program named “Clinical Oncology, Stem Cells and Cell Therapy” and the professional master course “Professional Master in Hemotherapy and Biotechnology”. In the first case, the course is focused on, as the title shows, the theme addressed in this proposal, which is the development of research programs in the areas of oncology, stem cells and cell therapies. Currently, the course has 21 students (12 in the master and 9 in the doctorate). In the second case, the aim is the specialized training of professionals to work at the hematology, hemotherapy and biotechnology centers in the country. Currently, 36 students are enrolled in the master in Hematology and five in the area of Biotechnology. The educational activities that will be added to the ongoing activities, which will be developed in this new INCTC phase, are described in item K.

Innovation Activities

The innovation activities will also be amplified with the support of the Innovation Agency of the University of São Paulo and of SUPERA Innovation and Technology Park of Ribeirão Preto, inaugurated on March 26, 2014. Since 2005, 15 companies have been incubated in the unit of the Regional Blood Center. Recently, both units of the SUPERA have been incorporated to the Technological Park. Therefore, the four resident companies linked to INCTC were transferred to the Park: 1) Cross Reality (software of virtual reality); 2) Figlabs (tracking systems XYZ); 3) ProRadis (administration software); and 4) Sevna (a set of innovation companies) which receive technical, administrative, management and technological support, in addition to services of conservation and maintenance. Additionally, new actions will be developed aiming at the technological development and its transfer to the production sector as described in detail in the item M.

Internationalization Activities

Finally, the internationalization activities, jointly with the program of research, science dissemination and diffusion and technology transfer, form the INCTC foundation. This proposal relies on eleven international collaborators, committed to the team and to the development of activities in collaboration, according to letters attached to item F. This proposal intends to keep a strong connection with partner institutions overseas, conducting local and virtual meetings, and to strengthen the exchange among researchers, as described in detail in item F.

J) Detailing the Research Program

INCTC research program is composed of 26 subprojects that will be presented below, jointly with the objectives and goals of each one. In the second part of item J (J.2), these subprojects will be described in summary as part of a rational organization of the program, indicating the integrating character of this proposal. This rationality is indicated in Figure 1 of item G of the project (Specification of the Activities to be developed by the members of the Team) and

30 a table demonstrates the integrated participation of the diverse research groups involved in these subprojects (the groups are represented by the coordinator’s name).

J.1) DESCRIPTION OF THE SCIENTIFIC PROJECT

“Drugs can only repress symptoms: they cannot eradicate disease. The true remedy for all diseases is Nature’s remedy.... There is at bottom only one genuinely scientific treatment for all diseases, and that is to stimulate the phagocytes. Stimulate the phagocytes. Drugs are a delusion”.

In The Doctor’s Dilemma, George Bernard Shaw (1906)

In the last 12 years, we have consolidated a strong research group in the field of stem cells and cell therapy. This was only possible due to two innovative funding research programs: CEPID-FAPESP and INCT-CNPq. In the first case, we have instituted the Center for Cell-based Therapy (CTC-CEPID- FAPESP), started in 2001, and, in the second case, the National Institute of Science and Technology in Stem Cells and Cell Therapy (INCTC-CNPq-FAPESP) from 2008. Although both belonged to the same general theme, these two projects have differentiated as they have developed complementary, but not superimposed, investigation programs, besides being composed of different teams of researchers. Recently, part of the group of researchers has been granted with a new CEPID-FAPESP whose implementation took place in 2013. The theme of this new Center continues to be “Stem Cells and Cell Therapy”, but with a different research program centered on basic studies, focusing on understanding the biology of stem cells (SC), in addition to clinical studies based on the use of hematopoietic SC and mesenchymal SC. The main types of stem cells investigated are embryonic, induced pluripotency (iPS), and somatic SC (hematopoietic SC and mesenchymal SC).

Near the completion of the INCTC Project, we propose its continuity, now with a new focus on the study on tumors, neoplastic stem cells and innovative therapies for cancer based on

31 immunotherapy and on nanomedicine. To face the scientific challenge represented by cancer, we have assembled a team of researchers that include physicians, immunologists, geneticists, cell and molecular biologists, veterinarians, and chemical and material engineers. This proposal comprises three broad areas: 1) basic studies for understanding the biology of cancer; 2) development of new therapeutic approaches focusing on immunotherapy and nanomedicine; 3) pre-clinical and clinical studies for cancer treatment.

1. Biology of cancer: tumor cells and microenvironment Recently we have testified an essential modification in the understanding of the biology of cancer which has brought profound implications for its therapy. Until the end of the last century, the prevailing view was that any somatic or germ cell, target of cumulative genetic alterations, could originate cancer. This view has been replaced by the concept that cancer only begins with a restrict number of cells, the ones called cancer stem cells (CSC). These CSC have specific characteristics that facilitate their isolation and characterization: a) they are the only one capable of originating tumor; b) they have capacity for self-renewal, it means, for keeping tumor; c) they originate all heterogeneous cell progeny that constitutes tumor; d) when transplanted in immunodeficient mice, these cells – and only them – are capable of recapitulating the original tumor with all their functional and morphological characteristics (2). Although the concept of CSC may be attributed to Rudolph Virchow in the 19th century, the effective demonstration of its existence only appeared in 1994 when Lapidot and collaborators demonstrated that only CD34+/CD38- cells, in acute myeloid leukemia, were capable of reproducing leukemia in NOD/SCID mice, whereas CD34+/CD38+ were not. Since then, CSCs have been demonstrated in diverse tumor types such as breast, prostate, liver, colon, lung, skin, pancreas and brain. With the consolidation of this concept, the challenge for understanding the biology of cancer is to isolate and characterize CSCs and no longer the tumor. The same can be applied to treatment, which is only effective when it is able to eradicate CSCs. Tumors, as a whole, are heterogeneous masses of normal and neoplastic cells, associated with the extracellular matrix in a way to constitute a microenvironment appropriate to CSCs maintenance and proliferation and respective progeny. Understanding the biology of cancer, in our conception, means to separate and analyze these several components with the aim of evaluating the specific contribution of every element to the whole set.

Based upon this consideration, we propose an innovative methodological approach in our environment, in which we will conduct cytomic, genomic and proteomic analyses of individual cells (single cell analysis) isolated from many tumor compartments (cancer SC, cancer stroma cells, tumor infiltrating T cells) in addition to the components of the tumor matrix. To meet this proposal, we have planned a set of subprojects which are briefly indicated below.

1.1 Isolation, identification, and characterization of Cancer Stem Cells CSCs isolation will be performed by classical methodologies, which include flow cytometry analyses (side population, Hoechst 33342 efflux), cell sorting based on the expression of surface molecules and capacity to form spheres in culture. Additionally, these CSCs will be individually sorted in the platform C1™ Single-Cell Auto Prep System (Fluidigm Corporation) for the subsequent preparation of RNA-Seq libraries and exomes corresponding to 96 single cells from the population selected. In Subproject 1 entitled “Genetic heterogeneity of tumor cells: identification and functional characterization of biomarkers for therapeutic purposes in cancer”, coordinated by Prof. Wilson Araújo Silva Júnior, we intend, apart from the analysis of genetic and epigenetic alterations obtained by new generation sequencing (NGS), to study non-coding RNAs that play a role in the regulation of oncogenic processes. Small (miRNAs, piRNAs), medium (PASRs) or long (lncRNAs) non-coding RNAs regulate diverse molecular, genetic and cellular processes linked to tumor progression that include: chromosome dosage compensation, modification of chromatin structure, transcription and translation, splicing, cell differentiation, integrity maintenance of cell structures, control of cell cycle and intracellular trafficking, stem cell reprogramming and heat-shock response. This strategy will be used to identify new non-coding RNAs in aggressive tumors such as melanoma, glioblastoma multiforme and gastric cancer that may be applied to cancer diagnosis and prognosis, as well as, in the future, become new therapeutic targets. A second methodological approach, quite innovative among us, will be the application of High Content Screening (HCS) to the functional evaluation of the subpopulations of isolated CSCs, as mentioned before. HCS is an automated process of computational acquisition and analysis of fluorescence microscopy images in cells disposed on plates that allow qualitative and quantitative evaluation of a large number of morphological and functional parameters. Therefore, it is possible to perform functional analyses in large scale. In Subproject 2 “Functional analysis in large scale for the identification of microRNAs with antitumor and antimetastatic potential in head and neck cancer”,

33 coordinated by Prof. Marco Antonio Zago, we will identify microRNAs or anti-miRs with anti- proliferative, pro-apoptotic and anti-metastatic activity involved in the processes of Epithelial- Mesenchymal Transition (EMT) or in the reverse process Mesenchymal-Epithelial Transition (MET) that may be used in the development of new therapeutic approaches for the treatment of carcinoma in general. In previous studies on functional genomics of brain and neck tumors, our group (Process 559809/2009-3 - Rede GENOPROT, coordinated by Prof. Wilson Araújo da Silva Júnior) has identified several microRNAs and mRNAs with altered expression and possibly implicated in the pathogenesis of these neoplasias, constituting new potential therapeutic targets. In the same line, we intend in Subproject 3 “Identification and characterization of neoplastic stem cells in prostate cancer”, coordinated by Prof. Maria Angélica Miglino, to quantify and characterize CSCs in prostate cancer, which is the second mortality cause by neoplasia in men. Samples of tumors in different stages of progression and aggressiveness will be collected to characterize CSCs, applying some of the methodologies already cited. The hypothesis to be tested is that the number of CSCs and their biological and functional characteristics correlate with disease severity and with its metastatic power. CSCs will be isolated by cell sorting and analyzed in isolated cells in relation to their cytome and exome; in sequence, they will be cultured, characterized once more and implemented in NOD/SCID mice for studies on tumor reconstitution. Still in the context of the characterization of the biological properties of tumor cells, we also propose Subproject 4 “Study on HLA-G expression and other immunoregulatory molecules in selected tumors”, coordinated by Prof. Eduardo A. Donadi. Genes of the Main Histocompatibility Complex (MHC) are directly involved in immune surveillance, preventing the development and progression of neoplasias. In humans, the complex is known as Human Leukocyte Antigens (HLA). The HLA-G gene is a non-classical class I HLA locus and its polymorphisms and expression pattern have been studied in diverse pathological conditions, including tumors. Tumor cells seek to develop evasion mechanism of immune response. Several studies have demonstrated that HLA-G molecules can be expressed in an aberrant way in tumor cells and that this expression may be associated with a possible role in the immune surveillance escape by inhibiting the action of natural killer and T cytotoxic cells. However, most aspects related to the HLA-G gene expression mechanism still remain unknown. The promoter region of HLA-G gene is highly polymorphic, being identified 28 SNPs (single- nucleotide polymorphism) which are organized in haplotypes and may be related to distinct promoter activities, resulting in high or low HLA-G expression. The evaluation of the polymorphisms

34 of the promoter region in tumors is a fundamental component to elucidate HLA-G expression mechanisms, once it is demonstrated that HLA-G presents immunosupressive properties and its expression is associated with a great variety of neoplasias. The hypothesis that SNPs, found in the promoter region of the HLA-G gene, influence on its expression, and then on the susceptibility and progression of tumors. These data will allow to evaluate the applicability of HLA-G gene polymorphisms based on predictive tests related to the prognosis of these tumors. The group proposes to conduct complete gene typing (promoter region, coding and non-translated 3') of classical (HLA- A, B and C) and non-classical (E, F and G) histocompatibility molecules, and the analysis of the expression of immunoregulatory molecules (HLA-G, PD-1, CTLA-4) in sarcomas, grade IV melanoma or lymphomas, before and after chemotherapy treatment associated with hyperthermia and immunotherapy. In the study of tumor cells in myeloid neoplasias, Subproject 5 “Telomere dynamics and genomic instability in tumor cell of myelodysplasia”, coordinated by Prof. Rodrigo T. Calado, has as objective telomere dynamics and genomic instability in the tumor cell of myelodysplasia. This subproject has the aim of evaluating the effect of telomere shortening in genomic instability in precursor cells (CD34+) of patients with myelodysplastic syndrome (MDS). MDS is a myeloid neoplasia characterized by ineffective hematopoiesis, cytopenias in peripheral blood and evolution to acute myeloid leukemia. Alterations in tumor cell are MDS characteristics, particularly cytogenetic alterations and mutations. Telomere shortening is the major molecular event of the pathophysiology of some bone marrow failure syndromes that can evolve to MDS. CD34+CD38- cells will be obtained by cell sorting (JSAN flow cytometer) from the bone marrow of patients with MDS diagnosis. Telomere length will be determined by flow-FISH, a technique that combines fluorescent hybridization in situ with flow cytometry and genomic instability will be assessed by SNP-array.

1.2. Tumor Microenvironment

Tumor cells are indispensable to neoplastic initiation and progression, but are highly dependent on their micro-environment. A diversified repertoire of non-tumorigenic cells (e.g. fibroblasts, macrophages, endothelial cells) is recruited from surrounding and/or distant sites to constitute the stroma of the tumor microenvironment. These recruited cells, along with the extracellular matrix, are capable of providing the main characteristics of cancer, as escape from

35 immune surveillance, angiogenesis activation, invasion and metastasis, apart from supporting therapeutic resistance. Therefore, the comprehension of the role of tumor stroma and its communication with neoplastic cells may lead to the identification of targets in antineoplastic therapy. Among cell components of tumor stroma, our interest is in tumor associated macrophages and fibroblasts (TAMs and TAFs, respectively). In response to the cytokines present in the tumor environment, TAMs undergo a polarization process, assuming an immunosuppressive phenotype named M2. Besides contributing to immune surveillance evasion through this mechanism, molecules secreted by TAMs promote the proliferation of tumor cells, angiogenesis, lymphangiogenesis, and metastasis. Therefore, we intend to study the role of TAMs in the tumor microenvironment in acute promyelocytic leukemia and in melanoma in Subproject 6 “Macrophages associated with tumor: isolation, characterization, and functional study in animal models”, coordinated by Prof. Eduardo M. Rego. The first disease displays particular genetic, morphological and clinical features which are in great part reproduced in hCG-PML-RARA transgenic murine model, in a way that it became one of the most used models for the study on leukemogenesis. In addition to studies on the murine transgenic model, we will study samples of cohort of more than 200 patients with acute promyelocytic leukemia that participate in the International Consortium in Acute Promyelocytic Leukemia, whose database covers characteristics of diagnosis, relevant clinical outcomes and molecular monitoring of the response to common treatment. TAFs consist in a heterogeneous population of fibroblastic cells which virtually sustain all cancer properties. Remarkably, it is postulated that TAFs facilitate the invasion and colonization processes of tumor cells. Those are two limiting steps of metastasis, the leading cause of death in patients with cancer. Therefore, we will study the role of TAFs in the invasion and metastatic colonization of melanoma cells in Subproject 7 “Studies on the role of fibroblasts associated with tumor during invasion and metastatic colonization of melanoma”, coordinated by Prof. Dimas T. Covas. Melanoma will be used as model, once this class of neoplasia shows great propensity for metastasis and non-invasive, invasive, and metastatic subtypes are easily identified after clinical inspection. Still within oncohematological diseases, Subproject 8 "Investigation of signaling pathways involved in the interaction between hematopoietic stem cells and cells from bone marrow niche in myeloid neoplasias”, coordinated by Prof. Roberto P. Falcão, has the objective of studying the relation between CSCs and their regulatory niche, interaction that maintains uncontrolled

36 myeloproliferation, with the purpose of identifying new therapeutic targets that reach this complex interaction in myeloid neoplasias and tumor microenvironment. The classification of WHO (World Health Organization) distinguishes four groups of myeloid neoplasias: chronic myeloproliferative neoplasias (MPN), myelodysplastic syndromes (MDS), MPN/MDS and acute myeloid leukemia (AML), each one with their specific characteristics. The different cell populations will be isolated by sorting with specific markers; hematopoietic cancer stem cell will be identified as CD34+, mesenchymal stroma cell (MSC) will be identified as negative CD45, CD34 and CD31 and positive CD44, CD73, CD90 and CD105. Additionally, we will conduct co-culture experiments of primary neoplastic hematopoietic cells with MSC obtained from patients with myeloid neoplasias. After co-culture, HSC and MSC will be isolated for proteomic analysis and HSC will be functionally analyzed in relation to cell proliferation, apoptosis, differentiation in vitro and capacity of hematopoietic reconstitution in vivo by xenotransplantation. The samples will be subjected to proteomic analysis by liquid chromatography coupled to high resolution mass spectrometry. Once identified, the targets will be subjected to functional analysis in cell lines by pharmacological inhibition or the use of gene therapy tools. The targets validated in lines considered to be relevant to phenotypic alterations will be subjected to experiments using primary cells. Other population of cells present in the tumor microenvironment is T regulatory lymphocytes (Tregs), which act in a fundamental way in the peripheral control of homeostasis of the immune system and are involved in the control of autoimmune diseases, in the tolerance to transplantation, as well as in the immune escape of tumors (4,5). The regulatory role of these cells offers them therapeutic potential, once their administration could benefit patients with autoimmune diseases or patients subjected to transplantation, and on the other hand, the inhibition of their generation or function could benefit patients with tumors, allowing them to act in the immune system against tumor cells (6). Thus, understanding the processes responsible for the generation of these cells could contribute to the development of therapeutic approaches in both senses. In this regard, in the Subproject 9 “Study of regulatory mechanisms involved in the process of generation in vitro of induced T regulatory cells (iTreg) from T naïve cells”, coordinated by Prof. Marco Antonio Zago, we propose to evaluate different regulation mechanisms during the process of induction and generation in vitro of Treg lymphocytes, including transcriptional regulation mediated by different NF-kB subunits (by ChIP-Seq), post- transcriptional regulation mediated by microRNAs, and the role of signaling by adenosine and its receptors.

The extracellular matrix components (EMC) also modulate molecular circuits in tumor cells by the interaction with receptors present in plasma membrane. It is known that in some neoplasias EMC favors the proliferation of tumor cells, stimulating the production of reactive oxygen species (ROS). Thus, we will evaluate EMC constitution and its effect in ROS production in different stages of osteosarcoma progression. Moreover, we will study the effect of immunotherapy and hyperthermia on the modulation of EMC composition and on ROS synthesis in isolated cells of osteosarcomas at different progression stages in Subproject 10 “Evaluation of extracellular matrix and reactive oxygen species in the tumor microenvironment”, coordinated by Prof. Maria Angélica Miglino.

1.3. Tumor Reconstitution

Cancer originates from CSCs, a small number of cells with self-renewal properties and maintenance of all progeny of mature cells that compose tumor tissue. The processes that originate CSC are not completely unknown and two major hypotheses have been proposed: 1) CSC originate from normal stem cells that, due their greater longevity and consequent exposition to agents that damage DNA, accumulate gene and epigenetic modifications that end in originating CSCs. The similarities between normal SC and CSC found in several types of cancer reinforce this hypothesis (7- 11); 2) The second hypothesis postulates that differentiated somatic cells can be undifferentiated or reprogrammed to acquire initially metaplastic or neoplastic characteristics. This hypothesis has the support of experimental evidence. Scaffidi and collaborators have demonstrated that mature fibroblasts can be induced in vitro to acquire CSC characteristics, including the ability to self-renew and differentiate into several lines. Similarly, fibroblasts transduced with SSEA1 antigen can originate and maintain tumors, and normal astrocytes from central nervous system (CNS) transduced with oncogenes can originate gliomas. This second hypothesis also is supported by the fact that differentiated cells can be turned into pluripotent stem cells (12) by the insertion of four pluripotency genes (Sox, Oct4, C-Myc and Klf4) which originate teratomas when injected in immunodeficient mice. With the objective of exploring the process of CSC origin, we propose to use the cell reprogramming technology in tumor cells in Subproject 11 “Tumor Reconstitution from tumor cells reprogramming”, coordinated by Prof. Dimas T. Covas. A question which we intend to answer is whether gene reprogramming of tumor cells, using pluripotency genes (SOX, NANOG and OCT4), reverts the abnormal epigenetic pattern accumulated during oncogenic development. To answer this

38 question, the objective consists in testing the classical reprogramming protocol (OCT4, SOX2, KLF4 and MYC) in different tumor cell lines and to evaluate: i) pluripotency acquisition; ii) recapitulation of cell differentiation hierarchy; iii) epigenetic reprogramming capacity; and iv) possible reversion of tumorigenicity.

2. Development of new approaches in cancer immunotherapy Conventional treatments for cancer (chemotherapy and radiotherapy) display limitations: they are not selective or specific and affect both tumor and normal cells; they are not effective, in general, in eliminating CSCs and often they are only palliative; they weaken the natural defenses of the organism against cancer, facilitating its maintenance and spread. In the last years, the emergence of selective molecules, capable of blocking specific pathways involved tumor growth regulation, meant a great progress for treatment, however cure potential still depends on the association with conventional therapies. We intend to develop, in this project, innovative therapies that enable significant advance in cancer treatment. Among the therapies, we will prioritize immunotherapy (both cell and humoral) and the use of nanocompounds, to increase antitumor selectivity of pharmacological therapies and to make compounds, which can modulate or even destroy tumor growth, get to tumor. Immunotherapy for cancer treatment is nowadays the most promising therapy, because of its selectivity, curative potential, and low toxicity. This type of therapy was described as the greatest scientific event of 2013 by the journal Science (13), highlighting the therapy with monoclonal antibodies (anti-CTLA-4 and anti-PD1) and T cells genetically modified with chimeric antigen receptors (CAR T cells) which were capable of inducing potent antitumor response. We intend, within this context, develop cell lines with antitumor activity and, in sequence, develop bioprocesses for their expansion and production in large scale. We also intend to produce human recombinant antibodies with antitumor activity and to produce and evaluate the effectiveness of nanocompounds as a weapon in cancer treatment. Several immune system cell types have been used for cancer treatment in pre-clinical studies and phase I/II clinical trials, such as: total lymphocytes, dendritic cells (DC), natural killer cells (NK), T regulatory cells (Tregs), genetically modified T cells (14,15). Specifically, we intend to develop protocols for expansion and generation of lines of immunocompetent cells that exhibit antitumor effect, including T lymphocytes, T CD8+ lymphocytes, NK lymphocytes, Treg lymphocytes, and dendritic cells. These cells can be obtained from the differentiation of HSCs obtained from bone

39 marrow, peripheral blood or umbilical cord blood, or even from the differentiation of pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells (iPS). The translation of experimental immunotherapy for clinical practice requires the development of bioprocesses of cell expansion which are effective, rapid, robust and reproducible. Once the cells are the product, the culture system must minimize the variability in cell population, assure the maintenance of effector functions of cells, allow the collection and formulation without harming cells and incorporate process to guarantee feasibility during storage, transportation and administration in a safe and low cost way. For that, the technology for expanding cells must be based on ideally closed and disposable systems that allow expansion in accordance with good manipulation practices (GMP) guidelines. In Subproject 12 “Expansion of lymphocytes for immunotherapy of neoplastic diseases”, coordinated by Prof. Dimas Tadeu Covas, we aim to develop bioprocesses for the expansion of T and NK cells compatible with GMP guidelines to be used in pre-clinical and clinical trials. Effective antitumoral vaccination has been really hard to get (16). During cancer development, neoplastic tissue and organism (specifically the immune system in this context) reach a balance that allows the survival of neoplastic cells in one individual (17). This balance depends on diverse mechanisms of tumor escape (18-20), among them the alteration of antigenic presentation (21,22) represents a highly effective strategy : without antigenic presentation there is no immune response. However, DCs are capable of breaking immune tolerance states already established, and since it has become possible to differentiate this cell type from blood precursors (23), one can design therapeutic strategies that explore them, in diverse clinical situations, in cancer (24). Nevertheless, DCs are an extremely heterogeneous population and are very sensitive to the environmental variations in the individual. In cancer patients, both the ones present in tumor (22) and the ones derived from circulating monocytes (16, 25) exhibit diverse functional alterations which can often explain the evolution of the disease or patients’ clinical response to different treatments. Therefore, the proposal of Subproject 13 “Dendritic cells in cancer immunotherapy”, coordinated by Prof. José Alexandre Marzagão Barbuto, is to evaluate, both in experimental models and in patients with neoplasias, dendritic cells integrating neoplastic tissues or derived from monocytes. An alternative source of immune system cells for immunotherapeutic applications is cells differentiated from pluripotent cells. The development of the technology for generating induced pluripotent stem cells (iPS) (12) has significantly contributed to several fields of medicine, including immunotherapy. In the last years, our group has established two distinct protocols for iPS generation that allow the generation of patient-specific pluripotent cells. iPS will be generated from different

40 types of somatic cells (skin fibroblasts, whole blood monocytes, among others) and after they will be differentiated into NK cells and lymphocytes (Subproject 14 "Generation of T lymphocytes and NK cells from pluripotent cells”, coordinated by Prof. Dimas Tadeu Covas). iPS differentiation into NK cells will be done by a methodology that involves two steps: formation of embryoid bodies using defined conditions, followed by the expansion of NK cells by the co-culture with mbIL-21 artificial antigen-presenting cells. This methodology allows the production mature and functional NK cells (26). For the differentiation into T cells, iPS cells will be co-cultured with OP9-DL1 stromal cells to obtain T progenitor cells (27). Final maturation for T cell line requires their introduction in fetal thymus cultures in order to offer an appropriate microenvironment for the rearrangement of TCR genes and subsequent positive selection of a diversity of CD4+ and CD8+ cells. However, T cells generated in vitro thus show an unpredictable receptor repertoire of T cells (TCR) what limits their therapeutic use. To overcome this difficulty and allow the use of these cells independently of endogenous TCR, we will produce cells with chimeric antigen receptor (CAR). Cell therapy using genetically modified T cells to express a chimeric antigen receptor (CAR) against an antigen associated with tumor is one emerging immunotherapeutic approach for a variety of neoplastic diseases, including lymphomas and leukemias. This technology involves gene modification of lymphocytes, by adding a surface molecule (CAR) which allows these lymphocytes to recognize molecules present on the surface of the cells of the tumor independently of MHC system, making antitumor response more effective. The objective of the Subproject 15 “Generation of anti-CD20 CAR (chimeric antigenic receptor) T lymphocytes”, coordinated by Prof. Dimas Tadeu Covas, is to develop T cells derived from iPS and to modify them using anti-CD20 CAR. CD20 is an antigen present on the surface of normal B line lymphocytes and also on neoplastic lymphocytes found in many types of leukemia and B lymphomas. The therapy with monoclonal antibodies with anti-CD20 activity showed to be effective in the treatment of these neoplasias. However, the therapy with monoclonal antibodies does not lead to a memory immune response. Immunotherapy with modified CAR anti- CD20 T cells is more potent and effective than humoral therapy. Immunotherapy with T cells has been considered the most promising alternative to cancer treatment since the emergence of the first chemotherapy drugs in the late 1940s (28). In general lines, T-CAR cells are T cells genetically modified to express on their surface a hybrid molecule of TCR receptor associated with N-terminal domain of a single-chain variable fragment (scFv) specific to a determined tumor antigen; additionally they can associate with the construction of molecules or fragments of co-stimulatory

41 molecules like CD28 and CD137. The experimental strategy includes in the first place the introduction of CAR construction in iPS cells followed by their differentiation into T lymphocytes with the aim of obtaining a stable line with high replicative capacity and thereafter, to test the performance of the produced cells in the treatment of animals and humans with B neoplasias. This Subproject articulates with Subproject 12, which predicts the expansion of T lymphocytes in large scale; in this case CAR will be used to direct lymphocytes’ specificity. Within the area of cell reprogramming, new methodologies to generate iPS will be tested. Current methodologies still demonstrate low effectiveness in obtaining an appropriate nuclear reprogramming and the iPS lines obtained by these methodologies show a series of epigenetic alterations that produce aberrant gene expression patterns, and culminate with failure in obtaining safe lines for cell therapy. One of the strategies to increase the effectiveness of nuclear reprogramming has been the use of chromatin-modifying molecules as inhibitors of histone deacetylases which alter cell metabolism and epigenome. The mechanisms of action of these molecules are still elusive, and their comprehension will lead to the optimization of nuclear reprogramming processes. Subproject 16 “Study on the inhibition of histone deacetylases in cell reprogramming by nucleus transfer or gene induction to pluripotency (iPS cells)”, coordinated by Prof. Flávio Meireles, will investigate the inhibitory effect of histone desacetylases β-hydroxybutyric acid during the nuclear reprogramming process. Subproject 17 “Study on the function of miRNAs associated with AGO2 in the differentiation process in the culture of stem cells”, coordinated by Prof. Flávio Meireles, aims to study the process of differentiation in vitro of pluripotent cells, which is a laborious and often ineffective process. Our group will study the role of cell microvesicles in this process of differentiation in vitro. Vesicles secreted by cells (exosomes and microvesicles) are mediators of cell communication in different tissues and cell processes, delivering contents of a donor cells to a receptor target cell. Exosomes and microvesicles are carriers of miRNA, mRNAs, DNA and proteins. Microvesicle content can also be used as diagnosis and therapeutics. One of the proteins of this content is the Argonaute 2 (AGO2) protein which is part of a protein complex responsible for base pairing between miRNA and target mRNA and facilitates the delivery of miRNAs ready to act by silencing target mRNAs. In this subproject, we will test the hypothesis that vesicles secreted by monolayers of fetal mice fibroblasts exposed to differentiation medium induce cell differentiation by the transfer of miRNAs linked to AGO2 protein.

In addition to cell therapy, we intend to develop a platform for the production of human antibodies (Subproject 18 “Identification of anti-CD20 autoantibodies for the production of therapeutic human monoclonal antibodies”, coordinated by Prof. Dimas Tadeu Covas). Cancer patients may produce antibodies against antigens on the surface of their neoplastic cells due to two mechanisms basically: 1) expression of modified antigens or 2) immune response against their own antigens. Patients with B-cell neoplasias often produce autoantibodies, which can be a source of immunoglobulin for the production of libraries of antibodies directed against antigens associated with tumors (29). The antibodies against CD20 are potential therapeutic agents for B-cell malignancies. Currently, humanized chimeric monoclonal antibodies are used in the treatment of B- cell lymphomas and leukemias [36]. Monoclonal antibodies (mAbs) represent effective therapeutic agents. However, the use of chimeric mAbs in clinical settings has been complicated by some technical difficulties, including immunogenic responses. This type of response against therapeutic antibodies can affect both safety and pharmacokinetic properties that can impact the use and effectiveness of medications. Even humanized antibodies show adverse reactions. We are proposing the production of human antibodies that show lower risk to induce immune responses. However, we intend to determine whether patients with B cell tumors can enable a good source of material for the isolation of human antibodies directed against CD20 antigen. CD20 antigen of B cells is a specific surface molecule whose expression comprises pre-B cells until the stages of mature B cells. About 95% of B-cell non- Hodgkin’s lymphoma and in B cells, in the cases of lymphoma relapse, express CD20, in spite of the repetitive exposition to anti-CD20 monoclonal antibody. All these characteristics make CD20 an excellent tumor associated target for therapies based on new antibodies and for T cell therapies. This Subproject will be developed in partnership with Prof Dr. Michael C. Nussenzweig from Rockefeller University, New York, USA, who has vast experience in developing and producing human recombinant antibodies. Subprojects 19 (“Nanostructured systems for controlled release of nucleic and antitumor acids and for hyperthermia against cancer”) and 20 (“Evaluation of the use of Nanoparticles and Electrochemotherapy in the treatment of feline sarcoma application”), coordinated by Prof. Valtencir Zuculotto and Prof. Maria Angélica Miglino, aim to study, among the new strategies recently applied to cancer issue, the use of nanomaterials – area named Nanomedicine – that has attracted considerable attention, not only for the innovation of materials and methodologies, but specially for its high efficacy in treatments, already proved in clinical studies at all phases.

The idea of this new approach is to develop nanomaterials (nanoparticles and nanocapsules) that might be combined with therapeutic or biological agents in order to facilitate their entrance into the target cell or tissue. Once they are incorporated to cells, these materials can be used passively as contrast agents in MRIs and CTs with high effectiveness or actively by releasing antitumor agents and nucleic acids. These systems offer advantages over conventional drugs, including the dosage decrease of the active, minimization of adverse effects, drug protection against degradation and maintenance of their stability. In the therapy against cancer, several nanomaterials have been used as active agents in the processes of localized hyperthermia. This approach allows that nanoparticles initially localized in tumors or specific regions are stimulated by non-invasive techniques (magnetic field or light), causing highly temperature rise locally in tumor/tissue. With this approach, damages to healthy cells surrounding tumor are minimized. In the case of photo-hyperthermia, radiation absorption can be converted into heating, provoking localized temperature rise. This concept can be applied as a minimally invasive strategy to induce cell hyperthermia and to selectively eliminate cancer cells. In this project, several nanomaterials, including polylactic acid (PLA) and poly(D,L-lactide-co- glycolide) (PLG) nanoparticles, liposomal systems, gold nanorods (AuNRs) and superparamagnetic nanoparticles (FeFe2O4), will be designed and developed for application in the therapy against cancer. Nanomaterials will be applied in many fronts and will serve as innovative tools in different subprojects, including the encapsulation and selective delivery α tocopherol acid succinate (α-TOS) for the treatment of monocytic leukemia. This synthetic molecule has been studied by our group in the transgenic model of acute promyelocytic leukemia and it demonstrated high anti-leukemic activity. The study of the action mechanism showed that α-TOS inhibits the complex II of mitochondrial respiratory chain of leukemic cells. Carrying nanoparticles will be also applied in gene therapy, transporting RNAs (miRNAs, piRNAs, PASRs or (long non-coding RNAs) - lncRNAs), as predicted in Subproject 1 or in the form of phosphoethanolamine liposomal nanoparticles for application in felines with soft tissue sarcoma (Subproject 20). Metallic (AuNRs) and magnetic (FeFe2O4) particles will be used as promoter agents of photo and magnetohyperthermia, respectively, in T lymphocytes and their subpopulations (CD4+, CD8+), in monocytes and in dendritic cells derived from monocytes, as detailed in Subproject 13.

3. Pre-clinical and clinical studies for cancer treatment

The studies aiming at cancer immunotherapy described in this proposal comprise the establishment of methodologies and the study on important conceptual and practical issues. Together they form the foundation for the development of the technical and scientific know-how needed for the establishment of cell immunotherapy, as a future reality in the Brazilian medicine. However, the effective practical application of the potential of immucompetent cells which will be developed by the group will necessarily depend on pre-clinical trials in animals with characteristics similar to the human organism. The previous proposals will be therapeutically tested in animal models and in selected human diseases. Animal models are essential in cancer studies. Such investigations in these models involve the convergence of diverse areas of knowledge, in order to clarify the mechanisms involved in the disease, to evaluate the impacts of the therapeutic innovations used and planned for its treatment. Therefore, we described the proposals of pre-clinical and clinical studies that will be concentrated on the area of cell immunotherapy and hyperthermia.

3.1 Immunotherapy To evaluate therapeutic safety and efficacy of human NK expanded in vitro (Subproject 21 “Development of humanized murine models of leukemias and lymphomas for the evaluation of therapeutic safety and efficacy of human NK expanded in vitro”, coordinated by Prof. Dimas Tadeu Covas), we will use humanized murine models of hematological neoplasias (such as B-cell lymphoma and acute myeloid leukemia). These pre-clinical models will be developed using a new generation of immunodeficient mice, named NSG mice (NOD scid gamma or NOD/SCID/IL2Rgnull or NOD.Cg- Prkdcscid Il2rgtm1Wjl/SzJ, Jackson Laboratories). These mice show severe defects in innate and adaptive immunity; therefore they allow the effective engraftment of human cells and tissue, providing a platform for innovative xenogeneic models for the study on the pathophysiology of diseases and development of immunotherapies. We will perform an adoptive transfer of cells from tumor lines to the establishment of the disease in animals and later, they will be treated with infusions of human NK cells expanded in vitro. This pre-clinical study has the general objective of evaluating therapeutic safety and efficacy of human NK expanded in vitro under conditions compatible with GMP guidelines, in humanized murine models of hematological neoplasias (B-cell lymphoma and acute myeloid leukemia). After the evaluation, in these pre-clinical models, we will use these cells in phase I/II clinical trials.

Adoptive immunotherapy with immune system cells, such as T lymphocytes and NK cells, has been used for many years in combination with allogeneic bone marrow transplantation (BMT). The most employed approach to date has been the infusion of donor’s lymphocytes, already well- established for the treatment of post-BMT relapsed patients. Several tumors express antigens that can be targets for immune approach, both for T lymphocytes and NK cells. The primary function of NK cells is the recognition and elimination of cells that exhibit decrease of the expression of classical histocompatibility molecules, and thereby escape from the cytotoxic action of CD8+ T cells. According to the missing self theory, NK cells recognize and eliminate the cells that do not express the same classical histocompatibility molecules that they express. Many hematological neoplasias have their BMT response related to donor’s and patient’s KIR (killer inhibitory receptor) genotype. Recently it has been described that patients with acute myeloid leukemia (AML) that received HSC engraftment from donors with KIR2DS1 genotype showed lower rate of post-BMT relapse. Adoptive transfer of NK cells has already been described in a small group of patients with encouraging results. In this context, Subproject 22 “Functional evaluation of autologous and allogeneic NK cells against cells of patients with acute leukemias”, coordinated by Prof. Belinda P. Simões, has the objective of evaluating antitumor activities of autologous and allogeneic human NK cells in eliminating leukemic cells of patients with acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL) in vitro, aiming at the application of autologous and allogeneic human NK cells, which will be developed in Subproject 12, in high risk patients with AML and ALL (phase I/II study on viability and safety). The new study of the international Consortium in Acute Promyelocytic Leukemia, renamed International Consortium in Acute Leukemias, will compare the use of autologous transplantation versus conventional chemotherapy as a way of consolidating the treatment of patients with low and intermediate risk acute myeloid leukemia. This study is part of the CEPID/CTC project and is not part of this proposal, however the study does not cover high risk patients. AML is the most prevailing leukemia in adults comprising approximately 80% of cases in this group. The mean age of diagnosis is 67 years. In addition, we propose to develop another immunotherapeutic approach for AML treatment. The Brazilian AML treatment protocol (IC-AML) has the aim of standardizing the treatment of low risk and intermediate risk patients, but it does not cover the treatment of high risk patients. Despite BMT is considered the treatment choice in this situation, it still has high rate of relapse. The use of preventive adoptive therapy, with the purpose of reducing relapse rates, has already been successfully made. This therapeutic approach poses as risk the development of graft versus host disease. Therefore, we intend to compare the preemptive

46 strategy of infusions of donor’s T lymphocytes in post-BMT high risk AML patients with a group of patients without intervention (Subproject 23 “Preemptive adoptive immunotherapy with donor’s T lymphocytes post-BMT in high risk AML patients”, coordinated by Prof. Belinda P. Simões).

3.2 Hyperthermia

The protective effect of fever against tumor growth, described by Ehrlich, has been poorly recognized until now when hyperthermia started to be investigated in cancer control. Hyperthermia has great influence on the effect of chemotherapy and immunotherapy (31). By depleting glutathione, it potentiates the cytotoxic effects of chemotherapy agents. The beneficial effect of hyperthermia has been demonstrated in random studies comparing neoadjuvant chemotherapy with or without associated hyperthermia (32). Local hyperthermia can also amplify the effect of chemotherapy when it is delivered as liposomal preparation (33). This principle has been verified to be effective in rat sarcomas (34). This beneficial effect of hyperthermia can be even improved by adding immunotherapy. Sristava and collaborators evaluated the role of heat shock proteins (HSP) that tend to increase their expression post-hyperthermia. HSPs work as chaperones for tumor antigens, presenting them to NK cells. In one case, we have noticed dramatic clinical response in child treated with hyperthermia and infusion of haploidentical lymphocytes from the mother. The incidence of soft tissue sarcomas is frequent in childhood. Taking into account the experimental character of the proposal and the presence of very similar tumor in dogs, we aim to study this therapy in canine model. Initially, we will perform neoadjuvant chemotherapy with or without hyperthermia followed by immunotherapy with previously collected lymphocytes (Subproject 24 "Treatment of canine sarcoma with chemotherapy and hyperthermia followed by immunotherapy with lymphocytes”, coordinated by Prof. Belinda P. Simões). After this procedure, dogs will be subjected to conventional surgical treatment. Some open questions must be answered with this approach, such as the number and composition of infused cells, if we should previously activate NK lymphocytes with IL-2, if autologous cells will be able to exert effect or if we need allogeneic cells. To develop our pre-clinical trials, we will rely on Prof. Maria Angélica Miglino’s collaboration that has diverse animal models for cancer studies. Among the most prevailing neoplasias, both in humans and animals, we can highlight mammary neoplasias of carnivores, sarcomas of felines and canines, osteosarcomas and lymphomas of dogs. Mastocytomas are common skin neoplasias in

47 dogs, they can be focal or multifocal in skin, and/or involve spleen, liver, and intestine. Other tumors, such as sertolioma can be produced in animals to serve as a basis for the investigation in humans, once the number of cases in humans has been highly increased in the last years. The proposal in animal models (Subproject 25 “Clinical study of the impact of therapeutic combinations in microenvironment and in tumor cells of dogs with cancer”, coordinated by Prof. Maria Angélica Miglino) involves studies on the impact of therapeutic combinations in microenvironment and in tumor cells of dogs with lymphomas, sarcomas and mammary neoplasias, research on the anti-proliferative and pro-apoptotic effect in canine osteosarcoma, and the use of nanoparticles in the treatment of feline sarcoma and canine mastocytoma. On the other hand, it aims to chemically induce sertolioma in pacas (Cuniculus paca), hystricomorph rodent exclusive to Latin America, as a new experimental model for the study of this tumor in humans. Aside from that, we consider the evaluation of the mechanisms involved in the interaction between stem cells and osteosarcomas in dogs as a form of treating this disease. In most cases, murine model is the most used in pre-clinical trials, due to their relative low cost and easiness to maintain and manipulate. This model, however, places several limitations. From the experimental point of view, some techniques are limited by animal size, hampering the serial collection of samples in appropriate volume. From the genetic point of view, murine model differs from human in several aspects, what leads to a need to validate determined results in an organism phylogenetically closer to man in order to conduct pre-clinical research. This project also counts on Prof. Klena Sarges Marruaz da Silva’s collaboration from the National Center of Primates of Belém-PA, who will establish protocols of toxicity/pharmacokinetic pre-clinical trials in Neotropical primates. The advantage of performing tests in primates is their similarity to human immune system. The wide spectrum of the scientific activities proposed creates the need for storage of biological material that range from nucleic acids to tumor biopsies which will be obtained in the moment of analyses. Therefore, this proposal contemplates the creation of biobank with cataloged and systematic storage that will be indispensable to the progress of the research activities, and it will also serve as support for other projects and partner research groups. This project is described in Subproject 26 “Creation of a bank for storage of biological samples for research purposes in health sciences”, under the coordination of Prof. Dimas Tadeu Covas. In summary, we intend to develop in this new INCT an extensive research program, allying basic knowledge of cancer to translation for clinical practice. The comprehension of cell and

48 molecular processes associated with malignant transformation, the use of modern tools of investigation, the identification of tumor markers and pathways essential for tumorigenesis, will allow the development of new therapeutic strategies against cancer.

J.2) SUBPROJECTS, OBJECTIVES AND GOALS

GOAL 1: Basic studies for understanding the biology of cancer and cancer stem cells.

Subproject 1 Genetic heterogeneity of tumor cells: identification and functional characterization of biomarkers for therapeutic purposes in cancer.

Coordinator: Wilson Araújo Silva Júnior

Objectives To analyze the heterogeneity of the tumor cells, characterizing the populations expression regarding the profile of coding and non-coding RNAs, with special attention to regulatory microRNAs and long RNAs. The functional characterization of these molecules will contribute to the identification of potential therapeutic targets.

Goals 1. To identify mRNAs, lncRNAs and miRNAs enriched in tumor tissues and tumor cell lines; 2. To characterize the genetic and epigenetic heterogeneity of tumor cell subpopulations ; 3. To carry out a functional study. The transcripts selected as a potential therapeutic target will be functionally characterized using tumor cell lines with approaches of siRNA, shRNA and CRISPR/Cas9, followed by the evaluation of biological processes such as cell proliferation, apoptosis, migration, invasion and repair of DNA damage; 4. To develop therapeutic approaches. After functional validation, in the case of lncRNA, we can use the approach of AntagoNAT (Natural Antisense Transcripts - NATs) transcripts or ATFs (Artificial Transcription Factors). To the miRNAs, the antagomir is the most appropriate approach to cancer treatment. The three types of targets will be tested in animal model and tumor cell lines. Once proven the efficiency of the target, the next step will be the development of nanostructures for its controlled and selective release in animal model. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta1/sub1.pdf

Subproject 2 Large-Scale functional analysis for the identification of microRNAs with antitumor and anti-metastatic potential in head and neck cancers.

Coordinator: Marco Antonio Zago

Objectives To identify microRNAs or microRNA inhibitors with antitumor and anti-metastatic therapeutic potential for the treatment of head and neck cancers, and to investigate the potential molecular mechanisms involved.

Goals 1. To standardize the transfection assays of the cell lines to be used; 2. To standardize labeling with antibodies specific to proteins involved in the processes of EMT and MET; 3. To carry out a microRNA Functional Screen to evaluate proliferation and cell viability using the FADU HNC lineage; 4. To carry out a microRNA Functional Screen to identify modulators of microRNAs in EMT and MET process ; 5. To carry out secondary focused microRNA Functional Screens to evaluate the activity of selected microRNAs on cell migration;

6. To carry additional microRNA Functional Screen to evaluate proliferation and cell viability in distinct HNC cell lines (UM-SCC-14, UM-SCC-28 and UM-SCC-38); 7. To analyze data derived from screenings using bioinformatics tools; 8. To carry out a large-scale target-transcript identification of selected microRNAs; and the signaling pathways potentially involved in the functional responses observed in the biological screenings. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta1/sub2.pdf

Subproject 3 Identification and characterization of neoplastic stem cells in prostate cancer.

Coordinator: Maria Angélica Miglino

Objectives To identify and quantify stem cells in tumor samples in different stages of the disease. Our hypothesis is that tumors in advanced stages have higher percentage of stem cells. Moreover, cancer stem cells are related to the acquisition of resistance to chemotherapy drugs. The development of the project will also serve as foundation for new diagnosis methods, besides offering scientific basis for better diagnosis and prognosis of the disease.

Goals 1. To analyze the expression of stem cell markers in tumors in different stages; 2. To correlate the proportion of stem cells with tumor aggressiveness and ethnicity of the patient; 3. To isolate and culture stem cells from tumors in different stages; 4. To conduct the molecular characterization of cancer stem cells obtained from tumors in different stages by exoma sequencing. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta1/sub3.pdf

Subproject 4 Study on HLA-G expression and other immunoregulatory molecules in selected tumors.

Coordinator: Eduardo Antonio Donadi

Objectives To perform the complete typing (promoter region, coding and 3’UTR) of classical histocompatibility (HLA-A, B and C) and non-classical (E, F, G) genes, and analyze the expression of immunoregulatory molecules (HLA-G, PD-1, CTLA-4) in several types of tumors, including sarcomas, lymphomas or stage IV melanoma before and after treatment (chemotherapy, immunotherapy and hyperthermia).

Goals

1. To perform the complete gene typing (promoter region, coding and 3’UTR) of classical and non-classical histocompatibility molecules in patients with sarcomas, stage IV melanoma or lymphomas using next- generation sequencing; 2. Based on the differentially expressed transcripts, described in subproject 1, to perform in vitro and in silico studies to assess the differential action of transcription factors and microRNAs, targeting the gene control regions; i. e., promoter and 3'UTR, respectively; 3. To evaluate differential gene expression and the expression of immunoregulatory molecules (HLA-E, F, G, PD-1, Foxp3, and CTLA-4) on tumor cell lines undergoing hyperthermia; 4. To assess the expression of classical histocompatibility molecules (HLA-A, B and C) and non-classical (E, F, G) on tumor stem cells and in tumor niches; ; 5. To evaluate the differential gene expression and antigenic expression of immunoregulatory molecules (HLA-G, PD-1, CTLA-4) in tumor biopsies from patients with sarcoma, melanoma or lymphoma grade IV, before and after treatment; 6. To assess the concentration of soluble HLA-G (sHLA-G) molecule in plasma of patients with sarcoma, melanoma or lymphoma grade IV, before and after treatment.. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta1/sub4.pdf

Subproject 5 Telomere dynamics and genomic instability in myelodysplasia tumor cell.

Coordinator: Rodrigo Calado

Objectives To investigate the influence of telomere lenght on the genomic instability of neoplastic myeloid cell and evaluate the relation between hematopoietic niche and phenotype of neoplastic myeloid cell.

Goals 1. To investigate the association between telomere lenght in the genomic instability of cancer (stem) cell in myeloid neoplasia; 2. To identify transcripts/protein implicated in the interaction between tumor cell and hematopoietic niche in myeloid neoplasias; 3. To investigate the functional participation of targets selected in the neoplastic cell and hematopoietic niche. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta1/sub5.pdf

Subproject 6 Tumor associated macrophages: isolation, characterization, and functional study in animal models.

Coordinator: Eduardo Magalhães Rego

Objectives To study TAMs and tumor microenvironment in acute promyelocytic leucemia (APL) and in melanoma.

Goals 1. To develop a melanoma model in vivo with and without mutation in the BRAF gene for clinical assays; 2. To investigate if there is promotion of tumor growth or initiation associated with the increase of TAMs induced by clinically relevant conditions and previously associated with the increase in Tregs activity;

3. To investigate TGF-β participation in the possible induction to tumor growth, also evaluating its role in the possible increase of TAMs induced by Tregs. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta1/sub6.pdf

Subproject 7 Study on the role of tumor associated fibroblasts (TAFs) during invasion and metastatic colonization of melanoma.

Coordinator: Dimas Tadeu Covas

Objectives To investigate the role of tumor-associated fibroblasts (TAFs) during the acquisition of invasive properties and during the metastatic colonization of human melanoma.

Goals 1. To compare gene expression profile of TAFs isolated from melanoma samples in different clinical stages of progression; 2. To evaluate the interaction between TAFs and melanoma cells during the acquisition of invasive properties in vitro; 3. To evaluate the interaction between TAFs and melanoma cells during metastatic dissemination; 4. To characterize lung stromal cells during the formation of melanoma metastases in an experimental model of colonization; 5. To functionally evaluate molecular targets identified on TAFs isolated from melanoma metastases. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta1/sub7.pdf

Subproject 8 Investigation of signaling pathways implicated in the interaction between myeloid malignant cell and hematopoietic microenvironment.

Coordinator: Roberto Passeto Falcão

Objectives

To investigate the role of bone marrow microenviroment in the malignant phenotype of myeloid malignant cells. Goals 1. To perform co-culture experiments of myeloid malignant cells with human stromal cells submitted or not to treatment with specific inhibitors; 2. To perform cell sorting and proteomic studies; 3. To conduct proliferation, apoptosis and differentiation studies of the myeloid malignant cells submitted to co-culture with stromal cells; 4. To perform silencing or hyperexpression studies of proteins of interest in the myeloid malignant cells or stromal cells. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta1/sub8.pdf

Subproject 9 Study of regulatory mechanisms involved in the process of generation in vitro of induced T regulatory cells (iTreg) from T naïve cells.

Coordinator: Marco Antonio Zago

Objectives To functionally assess the specific roles of different regulatory mechanisms during the process of induction and in vitro generation of regulatory T-lymphocytes, including: the transcriptional regulation mediated by different NF-kB subunits (RelA, RelB, cRel, NFKB1 and NFKB2); the post-transcriptional regulation mediated by different miRNAs identified in our previous studies; and adenosine signaling trough its receptors.

Goals 1. To conduct functional assessment of the microRNA roles during the generation of induced regulatory T cells (iTregs); 2. To identify the role of different NF-kB pathways in the generation in vitro of Treg and Th17 Lymphocytes from naive CD4 T lymphocytes of Umbilical Cord Blood; 3. To identify generation mechanisms of T regulatory cells from naive T cells: signaling role of adenosine; 4. To carry out assays in vivo of Treg cells obtained by selected approaches. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta1/sub9.pdf

Subproject 10 Evaluation of the extracellular matrix and reactive oxygen species in the tumor microenvironment.

Coordinator: Maria Angélica Miglino

Objectives To evaluate the extracellular matrix and reactive oxygen species in the tumor microenvironment in a murine model of osteosarcomas. Furthermore, using a new tool for the reconstruction of tissues affected by tumors through vascular biological scaffolds derived from decellularized placentas.

Goals 1. To evaluate the constitutional changes of extracellular matrix along the different stages of the disease, as well as after different treatments in vivo; 2. To nalyze the correlation of the matrix composition with the phenotypes of cells isolated from tumors with and without treatment; 3. To determine intracellular ROS levels by flow cytometry, of isolated cells from tumors at different stages of evolution, with and without treatment; 4. To perform the repopulation of the acellular biological biomaterial derived from placenta, with cells of the yolk sac and bone marrow and evaluation of its osteogenic differentiation; 5. To evaluate the behavior in vivo in murine models, of bone regeneration from the acellular biomaterial and cells described. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta1/sub10.pdf

Subproject 11 Tumor Reconstitution from tumor cells reprogramming.

Coordinator: Dimas Tadeu Covas

Objectives

To test the classical reprogramming protocol (OCT4, SOX2, KLF4 and MYC) in different tumor cell lines and to evaluate: i) pluripotency acquisition; ii) recapitulation of cell differentiation hierarchy; iii) epigenetic reprogramming capacity; and iv) possible reversion of tumorigenicity.

Goals 1. To reprogram tumor cells by lentiviral and episomal vectors in isolated tumor cells and/or different tumor cell lines, available in our cell bank; 2. To characterize reprogrammed cells by transcriptome and methylome analysis (NGS) of cells with the aim of evaluating the alterations resulted from the reprogramming process; 3. To evaluate the capacity of reprogrammed cells to terminally differentiate in vitro; 4. To evaluate the possible reversion of tumorigenesis by in vivo assays. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta1/sub11.pdf

GOAL 2: Experimental development of new therapeutic approaches focusing on immunotherapy and nanomedicine.

Subproject 12 Expansion of lymphocytes for immunotherapy of neoplastic diseases.

Coordinator: Dimas Tadeu Covas

Objectives To develop bioprocesses compatible with GMP guidelines for the expansion of immunocompetent cells that will be used in pre-clinical and clinicar assays.

Goals 1. To isolate and characterize subpopulations of human T and NK cells; 2. To develop bioprocesses under conditions compatible with GMP guidelines for large scale expansion of T and NK cells in bioreactors; 3. To characterize the immunophenotype and functionally expanded T cells; 4. To characterize the immunophenotype, evaluate the expression of genes related and the production of cytokines, and functionally NK cells expanded in vitro in toxicity assays by flow cytometry and degranulation assays of NK cells; 5. To develop protocols for cryopreservation of human T and NK cells expanded in vitro for clinical application in several infusions; 6. To develop protocols for quality control of cell products for clinical application. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta2/sub1.pdf

Subproject 13 Dendritic cells in cancer immunotherapy.

Coordinator: José Alexandre Marzagão Barbuto

Objectives To evaluate, both in experimental models and in cancer patients, DCs within tumors and those derived from blood monocytes.

Goals

1. To evaluate DCs in the tumor infiltrate under the various treatment as: their membrane phenotype; their cytokine secretion pattern, spontaneous or induced by different stimuli, including tumor extracts; and their ability to stimulate T lymphocytes, considering their response patterns (Th1, Th2, Th17, Treg); 2. To evaluate tumor infiltrating macrophages in the same conditions (considering the close proximity of these cell types) as to the same parameters above described, aiming mainly, at the characterization of the macrophages as to the M1 and M2 patterns; 3. Evaluate, before and after their inclusion in the different treatments, in the patients’ blood: a) the frequency of the various leukocyte subpopulations (CD4+, CD8+, FoxP3+ T lymphocytes, NK cells, B lymphocytes, dendritic cells, monocytes and granulocytes); and b) the differentiation capacity of monocytes in vitro (evaluating, phenotypically and functionally the dendritic cells from them derived); 4. To study in vitro the effects of the various treatments over monocytes and monocyte-derived dendritic cells; 5. To analyze global gene expression of mRNAs and microRNAs (microarrays or NGS) and protein expression; 6. To evaluate cell function (preferential stimulation of subpopulations of lymphocytes, production of cytokines, immunoregulatory/immunostimulatory potential). http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta2/sub2.pdf

Subproject 14 Generation of T lymphocytes and NK cells from pluripotent cells.

Coordinator: Dimas Tadeu Covas

Objectives Differentiation of iPS cells into NK cells and T lymphocytes, the phenotypic and functional characterization of these cells in vitro and the assessment of safety and therapeutic efficacy in preclinical models. After the establishment of lymphocyte differentiation, lymphocytes generated will be modified with anti-CD20 CAR.

Goals 1. To generate iPS from skin biopsies and blood cells; 2. To characterize the pluripotency of the cells generated by gene expression, immunocytochemistry, and formation of embryoid bodies formation of teratomas; 3. To establish the protocol for differentiation of iPS cells into NK cells and T lymphocytes; 4. To perform morphological, phenotypic and cell expression characterization of the differentiated cell lines.; 5. To develop protocols for cryopreservation of differentiated cells; 6. To evaluate effectiveness in vitro and in vivo. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta2/sub3.pdf

Subproject 15 Generation of anti-CD20 CAR (chimeric antigenic receptor).

Coordinator: Dimas Tadeu Covas

Objectives To build anti CD20 CAR that can be used in therapies against malignancies of B cell.

Goals 1. To assess the efficiency of transduction with lentiviral vectors derived T lymphocytes and T lymphocytes pluripotent cells; 2. To draw primers for amplification.; 3. To conduct the overlap Amplification (overlap extension PCR multistep) for mounting the CAR; 4. To generate T lymphocytes expressing anti-CD20 CARs. 5. To evaluate the activation of T cells transduced with CARs; 6. To conduct specificity assays of the generated CAR. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta2/sub4.pdf

Subproject 16 Analysis of histone desacetylase inhibition on nuclear reprogramming mediated by nuclear transfer or induced pluripotency.

Coordinator: Flávio Vieira Meirelles

Objectives To study histone desacetylase inhibition in cell reprogramming by nuclear transfer or gene induction to pluripotency (iPS cells).

Goals 1. To evaluate the effects of β–hidroxybutyric acid on developmental rates in cloned embryos produced by SCNT and derivation of induced pluripotent stem cells (iPS cells); 2. To analyze gene expression of genes involved in epigenetic process or in controlling cellular metabolism in cloned embryos or iPS cells; 3. To investigate the epigenetic alterations occurring in imprinted genes such as IGF2R and H19 during the nuclear reprogramming process mediated by SCNT or induced pluripotency; 4. To increase the efficiency of nuclear reprogramming process achieved by SCNT or induced pluripotency; 5. To produce safe lines of stem cells aiming cell therapy with low incidence of metabolic or epigenetic alterations. 6. To generate cloned embryos with low incidence of metabolic or epigenetic alterations. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta2/sub5.pdf

Subproject 17 The role of miRNAs associated to AGO2 during differentiation of stem cells.

Coordinator: Flávio Vieira Meirelles

Objectives To determine the function of miRNAs associated to AGO2 protein, present in extracellular vesicles. Initially, we will determine miRNA analysis to identify miRNA-mRNA complexes associated to AGO2 and then the targets, based on bioinformatics. In sequence, we will determine epigenetic modulators and pluripotency factors.

Goals

1. To dentify the profile of miRNAs associated to AGO2 in extracellular vesicles secreted by fetal mice fibroblasts after culture in Stem Cells differentiation media; 2. To determine the miRNA-target mRNA interaction using co-immuneprecipitation; 3. To determine the effects of inhibitors of miRNAs associated to AGO2 following treatment of Stem Cells with extracellular vesicles secreted by fetal mice fibroblasts. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta2/sub6.pdf

Subproject 18 Identification of anti-CD20 antibodies for the production of human monoclonal antibodies.

Coordinator: Dimas Tadeu Covas

Objectives To detect anti CD20 antibodies in patients with autoimmune diseases, to isolate clones producing, sequence and recombinantly produce anti-human CD20 antibodies.

Goals

1. To separate groups of patients likely to produce autoantibodies; 2. To detect anti-CD20 antibodies in patients; 3. To assess the affinity of antibodies detected; 4. To isolate and expand clones of B cells of patients who produce anti-CD20 antibodies with high affinity in vitro; 5. To sequence clones of B cells; 6. To sequence the genes of immunoglobulins (anti-CD20); 7. To clone variable and constant immunoglobulin regions into expression plasmids; 8. To produce the immunoglobulins in 293T cells. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta2/sub7.pdf

Subproject 19 Nanomedicine applied to the controlled release of nucleic acids and antitumor drugs, and to hyperthermia for cancer treatment.

Coordinator: Valtencir Zucolotto

Objectives To develop, optimize and evaluate the in vitro and in vivo efficacy of polymeric nanoparticles for controlled delivery and release of antitumor agents, and for photo and magneto hyperthermia.

In the controlled release area, we plan to develop polymeric nanoparticles to deliver two antitumor agents. 1)RNA (miRNAs, piRNAs, PASRs and lncRNAs (long non-coding RNAs)) delivery in tumor models that reproduce the cell metabolic pathways, including gene specific therapy. 2) Antitumor drug delivery, including α tocopherol acid succinate (α-TOS), to cells derived from acute monocytic leukemia. Other antitumor agents may be included.

In the hyperthermia area, we plan to develop two systems to induce anticancer hyperthermia: gold nanorods and iron oxide superparamagnetic nanoparticles. The temperature increase is caused by infrared light

57 exposure (photohyperthermia) and by application of an alternating magnetic field (magnetohyperthermia), respectively.

Goals Controlled release area: 1. To conduct the design and fabrication of the nanoparticles containing the therapeutic agents; 2. To analyze and optimize drug binding to the nanoparticles and the kinetics of the drug release; 3. To conduct the functionalization of nanoparticles with specific receptors/antibodies to increase the targeting efficiency; 4. To evaluate the toxicity caused by the nanoparticles in healthy cells; 5. To assess of the release efficacy and therapeutic potential of the nanoparticles through in vitro and in vivo assays.

Hyperthermia área against cancer: 1. To synthesize and characterize gold nanorods and iron oxide superparamagnetic nanoparticles; 2. To perform Functionalization of the nanomaterials with specific receptors/antibodies to increase the targeting efficiency; 3. To evaluate the stability and toxicity caused by the nanomaterials in healthy cells; 4. To apply nanomaterials as active agents in photo and magneto hyperthermia procedures in vitro and in vivo models; 5. To optimize physical parameters (laser power, magnetic field magnitude, exposure time, etc.) and efficacy assessment using tumor models and cell cultures, such as lymphocytes and dendritic cells (to be defined). http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta2/sub8.pdf

Subproject 20 Evaluation of nanoparticles and electrochemotherapy in the treatment of feline sarcoma application.

Coordinator: Maria Angélica Miglino

Objectives To study the progress and the therapeutic effect pre and post-application of liposomal nanoparticle and phosphoethanolamine and electrochemotherapy on cats soft-tissue sarcoma.

Goals Using aspiration cytology of the formation, analyzing infrared images captured through thermo graphic camera, computed tomography, and quantification of apoptotic and necrotic index:

1. To determine the clinical characteristics of the patient and each tumor; 2. To determine the clinical stage of each patient; 3. To determine the histological grade of the tumors. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta2/sub9.pdf

GOAL 3: Pre-clinical and Clinical Studies

Subproject 21 Development of humanized murine models of leukemias and lymphomas for the evaluation of therapeutic safety and efficacy of human NK expanded in vitro.

Coordinator: Dimas Tadeu Covas

Objectives To evaluate the safety and therapeutic efficacy of expanded in vitro expanded human NK cells infusions in humanized murine models of B-cell lymphoma and acute myeloid leukemia, aiming at future clinical applications in patients with hematologic malignancies and solid tumors.

Goals 1. To develop and characterize humanized models of B-cell lymphoma in immune deficient mice NSG (NOD scid gamma); 2. To develop and characterize humanized models of acute myeloid leukemia in NSG immune deficient mice (NOD scid gamma); 3. To evaluate functionally the human NK cells, in vitro expanded under conditions compatible with GMP standards, in cytotoxicity and NK cell degranulation in vitro assays; 4. To standardize the amount of cells, administration route, number of infusions of NK cells to be used; 5. To evaluate the safety and therapeutic efficacy of in vitro expanded NK cells infusions in humanized models of acute myeloid leukemia in NSG immune deficient mice (NOD scid gamma); 6. To assess the safety and therapeutic efficacy of in vitro expanded NK cells infusions humanized model of B-cell lymphoma in immune deficient mice NSG (NOD scid gamma); 7. To develop and characterize other humanized mouse models of neoplastic diseases for evaluation of safety and therapeutic efficacy of other immune competent cell types to be developed in the project scope. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta3/sub1.pdf

Subproject 22 Functional evaluation of autologous and allogeneic NK cells from patients with acute leukemia.

Coordinator: Belinda Pinto Simões

Objectives To evaluate the antitumor activity of autologous and allogeneic human NK cells in eliminating leukemic cells from patients with acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) in vitro, aiming at clinical application of autologous and allogeneic human NK cells in patients with AML and high-risk ALL (phase I / II safety and feasibility clinical trial).

Goals 1. To isolate and characterize human NK cells from patients with acute leukemia (AML or ALL) and haploidentical relatives regarding the immunophenotype and KIR genotype; 2. To isolate and freeze leukemic cells from patients with AML and ALL before the treatment; 3. To evaluate functionally in vitro expanded human NK cells (Subproject "Expansion of immune competent cells') against allogeneic and autologous leukemic cells in in vitro cytotoxicity and NK cell degranulation assays;

4. To design a phase I / II clinical trial of adoptive transfer of autologous and allogeneic NK cells in high risk AML and ALL patients; 5. To recruit patients for clinical study. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta3/sub2.pdf

Subproject 23 Preemptive adoptive immunotherapy with donor T lymphocytes in high-risk AML patients undergoing bone marrow transplantation.

Coordinator: Belinda Simões

Objectives To compare the preemptive strategy of donor T lymphocytes infusions of in patients with high-risk AML after BMT with a group of patients without intervention.

Goals 1. To conclude project writing and submit to research ethics committee; 2. To include patients in the study; 3. To analyze the results; 4. To perform statistical analysis. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta3/sub3.pdf

Subproject 24 Treatment of canine sarcoma with chemotherapy and hyperthermia followed by immunotherapy with lymphocytes.

Coordinator: Belinda Simões

Objectives To evaluate the therapeutic efficacy and mechanisms of the treatment of canine sarcoma with chemotherapy and hyperthermia followed by immunotherapy with lymphocytes.

Goals 1. To treat canine sarcoma with neoadjuvant chemotherapy with or without hyperthermia, followed by immunotherapy with previously collected lymphocytes. After these procedures, the dogs will be submitted to conventional surgical treatment; 2. To evaluate the efficacy of the therapeutic approach; 3. To standardize the infusion of lymphocytes collected before chemotherapy (to determine the number and composition of infused lymphocytes, to assess whether activation of NK-cells with IL-2 is required; to assess whether autologous cells will be effective or allogeneic cells will be necessary. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta3/sub4.pdf

Subproject 25 Clinical study of the impact of combination therapies in microenvironment and in tumor cells of dogs with cancer.

Coordinator: Maria Angélica Miglino

Objectives To evaluate the cellular microenvironment and tumor response before and after different therapeutic modalities, which involve nanoparticles, iHDAC and hyperthermia in dogs affected with breast tumors, lymphoma and soft tissue sarcomas.

Goals 1 To examine the microenvironment of breast tumors in dogs and evaluate primary cell cultures treated with nanoparticles containing doxorubicin 2 To evaluate the microenvironment and tumor cells in lymph nodes of dogs with multicentric lymphoma before and after treatment with nanoparticles loaded with fosfoetanolamida. 3 To evaluate the tumor microenvironment as well as the cellular effect of an inhibitor of histone deacetylases (iHDAC) in dogs with cutaneous lymphoma. 4 To assess the cellular microenvironment and tumor response in dogs with soft tissue sarcomas undergoing treatment by hyperthermia. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta3/sub5.pdf

Subprojeto 26 Creation of a bank for storage of biological samples for research purposes in health sciences. Coordenador: Dimas Tadeu Covas

Objectives To create a biobank and an institutional biorepository for managing biological samples (whole blood, nucleic acids, cells, tissues, among others) in order to meet the activities developed in this proposal.

Goals 1. To plan the creation of the institutional biobank based on the current Brazilian regulations (Ordinance MS nº 441/2011 and Resolution CNS nº 441/221) by constituting an internal Institutional committee; 2. To organize the institutional infrastructure for the biobank; 3. To install hardware and apply information technology to manage the biospecimens; 4. To provide high quality biospecimens for institutional projects and research group partners. http://lgmb.fmrp.usp.br/inctc/filesaug/proposta/meta3/sub6.pdf

J.3) Methodology

A great set of tools dominated by our researchers will be employed in these studies, including: genomic, proteomic, cytogenetic, genetic, epigenetic, immunology, embryology, cell biology and culture and systems biology (also known as Systeomics) methodologies.

Ethical aspects

All research projects will be submitted for consideration by the Research Ethics Committees of the Institutions, where the projects will be performed.

Facilities

During the term of our INCT- 2008 several facilities were consolidated and now our group has 11 facilities for carrying out research activities (Item R: Infrastructure).

Below are described the main methodologies that will be used by our group.

Next Generation Sequencing (NGS): this methodology will be use to investigate the genetic and epigenetic alterations. The epigenomics signatures will be obtained by large-scale sequencing (next generation sequencing). The cells or tissues will be subjected to extraction of DNA and RNAs by AllPrep DNA/RNA/miRNA Universal Kit (Quiagen). The RNA-seq libraries will be done with TruSeq RNA Sample Prep Kit v2 (Illumina) and the libraries of Methil-seq will be prepare with kit SureSelect Methyl-Seq (Agilent Technologies). All libraries will be sequenced in the equipment available in our institution. Briefly, analysis of the generated data will be divided into three parts: 1) quality control / filter 2 ) identification of statistically significant regions of interest and 3) integration with publicly available data, such as ENCODE, NIH Roadmap and TCGA. Each step of the analysis will be performed using a high-performance computer with Linux. We emphasize that our group has experience with analysis of data generated by large-scale sequencing in cancer and is currently developing a new bioinformatics tool, named biOMICS (Biologically integrating Omics), which aims to integrate project information: ENCODE, NIH Roadmap and the Cancer Genome Atlas (TCGA), with new data from large-scale sequencing.

System CRISPR/Cas for gene editing: systems CRISPR/Cas edition will be used to edit genes of interest. This technology is established in Professor Peggy Farnham (University of Southern California, Los Angeles, USA), our collaborator, which have possibility to train someone of our group.

High Content Screening (HCS): this technique has recently emerged as a powerful tool in the functional screening of genomic libraries. HCS methods are based on the automation of image acquisition of fluorescence microscopy of cells arranged in plates (96 or 384 wells), as well as, processing and

62 computational analysis of the images; allowing a qualitative and quantitative assessment of a large number of morphometric parameters and experimental conditions. Recently our group has acquired HCS equipment, in order to study the role of microRNAs in maintaining the pluripotent state and in the differentiation of stem cells.

Flow Cytometry: Through the technique of flow cytometry we are able to evaluate cell viability and characterize cell populations labeled with specific antibodies. It is a technique used for counting, examining and sorting cells. Allows the analysis of multiple parameters simultaneously (multiparametric).

Cell cultivation: Cultivation is carried out according to the characteristics of each cell type cultured with specific culture medium. The cells are maintained in incubator (with 85% relative humidity, temperature of 37 ° C and 5% CO2). The cell lines are cryopreserved in FBS containing 10% DMSO and kept in liquid nitrogen. Some analytical methods that we use to monitor our cell culture:

Analysis of cell concentration and viability: For analysis of viability and cell concentration the dye exclusion method with trypan blue hemacytometer will be used.

Analysis of metabolites and substrates: Glucose, lactate and ammonia levels will determined using Biolyser Analyzer following centrifugation of the sample at 200 g for 5 minutes to remove cells. The concentration of amino acids will be analyzed by the system using AccQ.Tag HPLC colum.

Flow-FISH: a technique that combines fluorescent in situ hybridization, flow cytometry and evaluated by SNP-array genomic instability. This is a cytogenetic technique that will be used by our group to quantify the number of copies of specific repetitive elements in genomic DNA from whole cell populations by combining flow cytometry staining with fluorescence in situ hybridization protocols. Flow-FISH is more commonly used to quantify the length of telomeres, which are stretches of repetitive DNA (TTAGGG hexameric repeats) in the distal ends of chromosomes in white blood cells, and it is a semi-automatic method.

Analysis of Gene Expression by Real-Time PCR: Gene expression is evaluated by real-time PCR. The quantitative expression analysis methodology is performed by TaqMan (Applied Biosystems). The primers and probes are acquired by the system AssayOnDemand. For normalization of samples geometric mean Cts of endogenous GAPDH and b-actin genes, obtained by (Applied Biosystems) PDAR system will be used whose amplification efficiency is the same of target genes, which allowed the analysis methodology by 10000 / 2 ^ DC, this formula requires no gene calibrator for analysis.

Cell reprogramming using lentiviral vectors and episomal: iPS cells will be obtained using a plasmid system EBNA1/enhanced OriP containing 5 reprogramming factors (Oct4, Sox2, Klf4, c-Myc, and Lin28) in the same molecule separated by 2A sequences, C5-called PEB (Addgene). iPS are also generated using lentiviral

63 vecto. The vectors used for insertion of 4FT are: the cre-lentiviral vector polycistronic excisable hSTEMCCA EF1α-LoxP-3 vectors encoding the capsid proteins Gag-Pol, Rev and Tat (HDM-Hgpm2, RC-CMV-REV and HDM -TAT) and the vector for the HDM-VSV-G virus envelope glycoprotein coding for vesicular stomatitis virus. The iPS lines will be established according to protocols described in the literature with some variations. Colonies with similar morphology to human human embryonic stem cells will be isolated, expanded and evaluated for their pluripotent characteristics and differentiation potential.

Production of viral vectors: 293T cells are often used for the production of lentiviral particles. For virus production is important that the cell line (293T cells) stably expressing the gene for the SV40 large T antigen. In this method the use of a vector containing the transgene, and two auxiliary vectors, which contain the SV40 origin of replication so that the plasmid after transfection into cells able to replicate, which increases the transcription of the transgene is necessary and the production viral proteins and the viral particles eventually will be secreted into the culture medium. For the production of the viral particle transfection protocol used is the calcium phosphate. We use the triple co-transfection with the plasmids of the transgene, capsid (pCMVΔR8.91) and envelope (VSVG PMD2 (envoltótio encoding the VSV-G).

Culture and differentiation of pluripotent cells: pluripotent cell lines will be maintained in culture in an undifferentiated state, the cells will be cultured in 100mm3 plates treated with 0.1% gelatin and coated with OP9 mouse stromal cells in medium Minimum Essential Medium Alpha Medium (α-MEM) supplemented with 10% Fetal Bovine Serum (FBS), monoatilcilglicerol 100μM (Roche Diagnostics) for nine days, exchanging half the medium from the culture plates on days 4:06 without adding cytokines. After nine days, cells are collected from the plates. For this, they are treated with the enzyme trypsin-EDTA (0.5%) (Mediatech) for 10 minutes at 37, collected in conical tube and centrifuged at 1300rpm for 10 minutes. The supernatant is discarded and the cell pellet formed is treated again with the enzyme trypsin-EDTA (0.25%) for 15 minutes to distinguish cells. After this time, the individual cells will be centrifuged at 1300rpm for 10 minutes, resuspended in Dulbecco's Modified Eagle Medium F12 (DMEM / F12) (Sigma) and counted in a Neubauer chamber.

The repique of colonies of pluripotent stem cells will occur through the transfer of fragments of colonies to a new plate. Manual replating of colonies is used, using a biological Chapel horizontal laminar flow containing an inverted microscope. After replacing the culture medium with fresh medium, and observed under a microscope, the colonies will be split with the aid of a sterile pipette tip into smaller sizes. Then the supernatant containing the fragments of colonies suspension will be diluted to the desired density and transferred to new plates. The individualization of pluripotent cells is not desirable because it facilitates cell death or differentiation.

The colonies of stem cells (hESC and HIPS) will be characterized by flow cytometry and fluorescence microscopy using antibodies against the following antigens: SSEA-1, SSEA-3, SSEA-4, TRA-1-60, PRO- 1-80, NANOG, SOX2 and OCT4. Also be evaluated for alkaline phosphatase activity and by G banding karyotype according to protocols described in the literature.

Differentiation into embryoid bodies: To obain embryoid bodies (EB), the colonies of pluripotent cells (hES and hIPS) will be digested with dispase enzyme (0.5 mg / mL) for 10 minutes and the clumps will be grown in suspension in low adhesion plates in DMEM 10% FCS. In this culture condition, pluripotent cells tend to differentiate spontaneously. These samples will be used for immunohistochemical characterization and for the extraction of DNA and RNA.

Extraction of DNA, RNA and microRNA: we will use AllPrep DNA / RNA / miRNA Universal Kit (QIAGEN) kit for the extraction of DNA, RNA and microRNA samples, following the manufacturer's instructions. The principle of the kit is to integrate the technology of isolation of double stranded DNA by specific binding with the RNeasy RNA extraction technology. Biological samples are first lysed and homogenized in a buffer of guanidine isothiocyanate high denaturation, which inactivates RNases and DNases to ensure the isolation of RNA and DNA integrity. The lysate is passed through AllPrep DNA Mini spin column, which in combination with a buffer with a high salt concentration allows efficient and selective binding of genomic DNA. Then the column is washed and eluted is pure and ready for use DNA. The column wash eluate AllPrep DNA is digested mini spin proteinase K in the presence of ethanol. This optimal digestion and the subsequent addition of ethanol allows efficient binding of total RNA, including microRNA RNeasy mini spin column. Digestion with DNase I will ensure high concentrations of RNA free of DNA. The contaminants are efficiently removed and RNA is eluted in high quality.

Analysis of RNA integrity: the quality of extracted RNA will be analyzed by the equipment Bioanalizer 2100 (Agilent Technologies) as follows: 1) the extracted RNA will quantified by spectrophotometry (NanoDrop, Thermo Scientific) and diluted to a concentration of 5 ng / ul. After dilution, the sample is denatured by incubation at 70 ° C for 2 minutes and immediately placed on ice. 2) the samples will applied in chip kit Eukaryote Total RNA Pico (Agilent), following the manufacturer's instructions for preparation of the chip. 3) Then, the chip is placed in Bioanalizer unit 2100 for analysis. 4) After the race, the values of RIN (RNA integrity number) and the corresponding gel for each sample will be generated.

Large-scale expansion of T lymphocytes and NK cells: T cells and NK will be properly insolated and will be inoculated at a concentration of 1x106 cells / mL in permeable culture bags (VueLife) containing culture medium preheated. Once you reach the appropriate number of cells, the same will be transferred to the bags the Wave Bioreactor (approximately 1x108 cells for 2L bags and bags of 2x108 cells to 10L). Cultures will be held in perfusion

J.4) Follow-up indicators of the Project

Indicator 2015 2016 2017 2018 2019 2020

Articles published 20 20 30 30 40 40

Theses and dissertations defended 10 10 20 20 20 20

Patents deposited - - - 1 1 2 News or reports divulged by written or 12 12 12 12 12 12 electronic media Postgraduate students participating 20 20 20 20 20 20

Undergraduate students participating 10 10 10 10 10 10

Elementary students participating 200 300 400 500 500 500

Animal models developed 0 0 1 0 1 0

Vectors produced 0 2 1 1 1 1

Concluded subprojects - - 1 1 2 22

Expositions or presentations held 1 2 2 2 2 2

Number of acess to INCTC pages 500 500 1.000 1.000 2.000 2.000

Scientific meetings held 2 2 2 2 2 2

Workshops held 1 2 2 2 2 2

K) Detailing the Program of Qualified Professional Training

This proposal aims specially at the amplification of the educational activities related to basic education; postgraduate students training so they can be able to interact with researchers involved in several laboratories of the Institute and to enable to exchange with international collaborators.

Therefore, we intend to consolidate the program of scientific education, with individuals capable of accomplishing knowledge diffusion by the production of material and products.

Thus spaces for scientific dissemination will be constituted by the partner institutions or the community, coordinated by the Center of Education of the Regional Blood Center, particularly, the activities dedicated to basic education, managed by the House of Science, with the participation of postgraduate students and researchers, teachers, and students from basic education. Aiming at the scientific training of biology and science teachers, with specific interest in the areas of the Institute, we propose to strengthen the interaction with elementary and high schools, using the material and the experience of the House of Science.

In addition to the material produced since 2001, throughout INCTC/CNPq and CTC/FAPESP, the complete collection of the Laboratory of Science Teaching of the School of Philosophy, Sciences and Modern Languages of Ribeirão Preto-USP will also be used, relying on more than 30 years of experience in this field. This material is cataloged and available for consultation in the PIPOC (Point of Information, Research and Organization in Sciences) of the House of Science.

The Laboratory of Research and Teaching, with a total area of 34 m2, available to postgraduate students, teachers and students of basic education is equipped with infrastructure for research on molecular and cell biology.

The current educational activities of the INCTC/CNPq and CTC/FAPESP will be kept and amplified, within this proposal. The Journal of Sciences will be bimonthly edited, incorporating the experiences and competences of the diverse groups participating in INCTC and, in addition to the 2,500 copies distributed, and it will be available on the website of the House of Science (http://ead.hemocentro.fmrp.usp.br/joomla/).

For postgraduate students training, the Institute relies on two postgraduate programs of the University of São Paulo, approved by Capes in 2012, with the faculty formed by the Principal Investigators of this proposal. The Program of Clinical Oncology, Stem Cells and Cell Therapy, academic master and doctorate, aims to train high level personnel to work both in the academic field and in the private and governmental sector.

The disciplines are directed towards the development of skills in clinical oncology, in the area of cell therapy and stem cells and towards the development of a critical spirit involving scientific writing, argumentation skills and communication techniques. Currently 25 students are enrolled, 16 in the master program and nine in the doctorate program.

The Professional Master on Hemotherapy and Biotechnology aims to specialize professionals capable of participating in health development and promotion in Brazil, enhancing their technical and scientific knowledge. The basic and applied disciplines have a multidisciplinary character with the objective of training professional with integrated view on the application of hemotherapy and biotechnology. Currently, 41 students are enrolled, 36 in the area of hemotherapy and five in biotechnology. These students develop professional activities in the states of Amapá, Rondônia, Ceará, Piauí, Bahia, Tocantins, São Paulo, Minas Gerais, Rio de Janeiro, Paraná, Santa Catarina, and Distrito Federal.

For graduate students, the traditional summer courses will be kept: “Genome, Proteome and Cell Universe” and the course of Bioinformatics. The courses take the students to research laboratories, where they can access different technological approaches and enable the integration among postgraduate and graduate students, national and international researchers, allowing the exchange with international collaborators. In 2014, 100 students took part in the course of Bioinformatics, with 12 foreign students, apart from two researchers from United Stated and one from Canada.

The courses of Transfusion Medicine, Applied Hemotherapy and Update on Hemotherapy, will also be kept and annually offered to hemotherapists or professionals that work directly with hemotherapy, pharmacists, biologists, biomedical professionals, nurses and social workers, as well as graduate and postgraduate students.

With the purpose of strengthening the action of knowledge dissemination, courses and mini-courses on Scientific Writing will be offered, coordinated by Prof. Zucoloto and specially developed to qualify scientists, researchers and postgraduate students for processing and producing high impact scientific articles. These courses will be held in a distance learning format, with videos available on escritacientifica.com. Classroom workshops and courses on this subject will also be held.

With the course of scientific journalism, we intend to reinforce the production techniques of reports, knowledge of science history and basic information about science with the aim of training new professionals for science dissemination. The public will be graduate and postgraduate students from different fields who are interested in becoming a science disseminator and journalists.

The experience of the Principal Investigators in Qualified Professional Training is demonstrated in Appendixes II and III.

L) Detailing the Actions of Knowledge Transfer to Society

The Institute seeks to establish a solid human resources training, a network among researchers and to promote scientific dissemination of the knowledge produced to the population. Therefore, the portal of the Institute will be updated and it will keep newsletters, interviews, distance learning courses, and other educational materials produced (http://lgmb.fmrp.usp.br/inctc/).

The educational programs coordinated by the House of Science aim to encourage young researchers to participate in projects or initiatives of knowledge diffusion that include students of basic education.

The plan of science dissemination and popularization involves the participation in international and national events, like the National Week of Science and Technology, National Week of Museums among others, and also in regional events such as Science Fairs, the Book Fair of de Ribeirão Preto, itinerant exhibitions with the Celularium and permanent exhibitions and murals in the MuLEC (Museum and Laboratory of Science Teaching) and in the House of Science.

The National Strategy for Science, Technology and Innovation - MCTI (2012-2015) stands out the relevance of the social acquisition of knowledge in permanent training for citizenship and in the increase of scientific and technological qualification of society. This proposal intends to analyze the relation between the knowledge produced in the university and informal learning through instruments to measure and identify social repercussion, using as the main tool the educational portal of the House of Science with questionnaires and polls. Every year workshops will be held with the purpose of evaluating the activities of knowledge diffusion, enabling the opportunity of producing innovative teaching material to postgraduate students, scholars and researchers.

One of the goals of this proposal is to intensify the presence of the researchers in the media, by TV programs, radio, social networks, Hemocentro TV and radio USP.

In the world today, demonstrating is as important as accomplishing. But in the case of research, despite the efforts of some, communication restrains to equals. Changing this paradigm is the effort of the integrating members of INCTC. According to Roald Hoffmann, Nobel Prize of Chemistry 1981, the scientists have the responsibility to teach science to people, informing the public in general, because scientific knowledge helps in the comprehension of decisions, corroborating to a democratic society.

As a tool for knowledge diffusion, we predict in this INCTC the implementation of a SCHOOL OF SCIENTIST TRAINING. The initiative has the objective of improving and consolidating the skills needed for the scientific life of Brazilian researchers and professors in basic and/or applied research. Initially, the School will be offered to the groups integrating the INCTC, but we hope to promote actions soon so the school can be extended to several research and HR training centers in the country. We hope that researchers are able to optimize their potential for research concerning the production of 1) National and international research projects and obtaining financial resources; 2) high impact international scientific articles; 3) Protection of knowledge, patent writing, and technology transfer.

The course is formatted to cover all knowledge areas. The exact science/engineering and biological/biomedical areas are those which will be rapidly benefited.

With this proposal, we intend that the information network among researchers enables INCTC scientific dissemination and the discussion of what is interesting to disseminate, when and how; amplifying the number of openings in the programs of HR training to diffuse knowledge; offering extension latu senso courses designed to science and biology teachers of elementary and high schools, postgraduate students and journalists; amplifying the opportunities for scientific initiation to students from basic education; and contributing with educational authorities to the implementation of public policies related to the promotion of scientific education of the population as a factor of economic and social development.

The project of knowledge diffusion and education in science will be developed throughout all the period of this proposal, in accordance with the schedule below:

1 2 3 4 5 Goal 1st 2nd 3rd 4th 5th 6th year year year year year year

Updating the INCTC and the House of Science websites. Organizing a portal, developed by the researchers to disseminate the knowledge produced in an interdisciplinary way. Producing diffusion material: science kits, informative poster, articles for newspapers and magazines, interviews.

Holding interactive scientific exhibitions, fairs and events. Increasing the number of openings offered in the academic and professional postgraduate programs.

Implementing the School of Scientist Training.

Offering summer courses. Offering extension courses to Biology and Science teachers, postgraduate students and journalists.

Identifying young talents to develop scientific initiation. Organizing workshop with the aim of evaluating the activities of knowledge dissemination.

M) Detailing the Actions of Knowledge Transfer to business sector and formation of public policies

Since its establishment in 2008, the Institute has been developing an ensemble of works, aiming the generation of solid scientific knowledge, as well as interesting knowledge to society for the application in the public and business sector. Because it is an institute of the medical area, these actions are slower than in other sectors, once they implicate in several trials, previously regulated, starting from pre-clinical trials and ending in clinical trials before this technology can be transferred to society. Therefore, to apply a new therapy or a new product, we need at least ten years of work. In summary, throughout the last five years, the Institute has been successful in developing treatments, at stage of clinical studies, based on cell therapy in immune-mediated diseases using autologous and heterologous mesenchymal stem cells. It has also developed new protocols of treatment for acute promyelocytic leukemia that have been already employed in all Latin America, has succeeded in cloning and expressing human XIII and IX coagulation factors, both with proved activity in cell and animal models, has developed mechanisms of nuclear reprogramming and produced various human and animal iPS cell lines for the use in medication trials or pre-clinical trials, among others. As a whole, these activities show team’s commitment not only to the development of basic science but also to the application of the knowledge produced in the Institute. More than that, an innovation strategy has been developed, which is summarized in Figure 4, beginning from the conception of this culture in the team (researchers and students), mediated by the establishment and alignment of priorities in meetings every six months, professional master courses, specific subjects of entrepreneurship and intellectual protection, aiming to recognize and protect potential technological knowledge. Finally, it was possible to create an innovation management system, with the support of the USP Innovation Agency which is part of our innovation council. In respect of the innovation processes: the products generated by the Institute throughout the years have been linked to the process of rupture and incremental reality, processes that take longer therefore they must be developed by research institutes. We understand that a research and innovation institution funded by public resources must prioritize the processes of rupture and incremental reality, and depending on the interest of the Healthy

System, it can make adjustments following the current regulations and propose modications in the treatment protocols.

Figure 4. Strategy for Innovation and Technology Transfer.

In this proposal, the actions of knowledge transfer continue to be the main axis, technology transfer to public sector (given the characteristic of our group), but also to business sector, aiming to promote activities of technological extension for productive and social inclusion. The USP Innovation Agency will take part in the actions mainly designed to the diffusion of the activities of technological extension, with emphasis on the identification and support to the development of innovation initiatives.

It is also part of the priorities, in this field, to strengthen the mechanisms of management, cooperation, infrastructure and technological services.

Therefore we aim to: 1) contribute to educational authorities in the implementation of public policies designed to the promotion of scientific education of the population as a factor of economic and social development; 2) train qualified human resources; 3) enable the articulation among the university, research centers and companies; 4) stimulate the development of

73 technological innovation; 5) produce medication, drugs, blood derivatives, diagnostic kits and equipment; and 6) promote the generation of knowledge and development of products, processes, and services.

The USP Innovation Agency will be responsible for transferring the technology generated from the research developed in the INCTC. Its main activities are: protection of intellectual property, support to researchers in the establishment of agreements/partnerships with the business sector, promotion of entrepreneurship, transfer of the technology generated within USP and institutional relationship with technological parks and incubators linked to USP.

The area of protection to intellectual property acts in two steps by: advising, receiving the request, writing, depositing/registering, following the process and defending them with competent organs. Specifically in the case of patents, it relies on the support of an outsourced office, hired by a public bidding process to make previous search and to write national patents, after the application, it follow and manage the process in the Instituto Nacional de Propriedade Industrial, conducted by an internal team.

The area of Technology Transfer is structured to take a proactive action concerning licenses. Therefore, the Committee of Technical and Commercial Analysis (CATC) was created to evaluate the market potential of the inventions. The Committee analyzes the technology received in order to classify them. Every classification corresponds to a type of priority treatment/effort of team work designed to that technology. In the analysis, they consider: characterization, application, similar technologies, development stage for market access, market study, innovation level, clearness about the advantages of the technology and potential interested companies. According to the classification done for every technology, better actions will be defined to search for partners and/or licensing institutions.

In summary, we intend to develop in the new INCT an extensive research program that can be, broadly speaking, named as “Translational Medicine in Cancer”. This proposal comprises innovative basic, applied and clinical studies designed to understand, accurately diagnose, identify prognostic factors and develop more effective treatments for some types of neoplasias, using cell therapy, hyperthermia and phototherapy associated with nanomedicine.

Specifically in the field of nanomedicine, covered in this project, there is a great potential for the immediate application of new nanomaterials and methodologies in diagnoses and therapies against cancer, highlighting the group of Nanomedicine and Nanotoxicology, GNano/IFSC/USP, which has been incorporated to the Institue in this new version and has developed and patented diverse products and processes related to the application of nanomaterials in medicine over the last years (www.nanomed.ind.br).

N) Potential for the Generation of Patents

Processes or Patents deposited/obtained by the proposing group.

Process Tittle Year of deposit Principal Investigators Covas, Large scale and stable production of D.T. ; CARBOLANTE, E M P.I. 11053178 human FVIII in the human cell line Sk- 2012 S R ; Picanço-Castro, Hep-1 Virgínia Factor VIII Human Blood Coagulation Covas, D.T. ; Fontes, EP 2 180 009 2010 Recombinant Protein Aparecida Maria Covas, Dimas T. ; da Silva PI11048751 Process of Isolation of Pericytes 2011 Meirelles L ; da Silva Meirelles L Human blood coagulation factor IX recombinant protein, use of a factor IX recombinant protein, use of a COVAS, Dimas BR2009/000240 composition, method of obtaining 2009 Tadeu ; Fontes, human blood coagulation factor IX Aparecida Maria recombinant protein and use of the factor IX recombinant protein MIGLINO, M. A. ; RUSSO, Processo de obtenção e cultivo de F. B. ; Graciela C. 018110038009 células epiteliais da mucosa oral 2011 Pignatari ; Isabella humana Fernandes ; Beltrão- Braga P.C.B. Guimarães, F. G.; Microdispositivos detectores do vírus ZUCOLOTTO, V.; da dengue; processo de produção de Figueiredo, A.; Vieira, BR 10 2013 024319-1 microdispositivos detectores do vírus 2013 N.C.S.; Arakaki, H. ; da dengue; kit contendo o mesmo e Lovato, R.L.; Aoki, S. M.; método de identificaçâo da dengue Peitl Junior, P. Figueiredo, B.; Mascarenhas, Y.M.; Processo de detecção de anticorpos Mascarenhas, P. R.; anti-virus da febre aftosa utilizando BR 10 2013 006812-8 2013 Frigieri, G.; Mangerona, medidas elétricas e kit de detecção N.J.; Vieira, N.C.S.; baseado nesse método Santos, F. A.; ZUCOLOTTO, V.

Process Tittle Year of deposit Principal Investigators Nanocomplexos para reconhecimento de células tumorais e produto de Marangoni, V.S.; Paíno, BR 10 2012 009598 0 diagnóstico ou terapêutico derivado 2012 I.M.M.; Zucolotto, V deste para aplicação em nanomedicina Processo de síntese e incorporação de nanopartículas de prata em matrizes Berni Neto, E.; Zucolotto, P.I. 1.101.028-2 poliméricas hidrofílicas e uso de 2012 V. nanopartículas de prata. Pedido de Patente Bioanodo para biocélulas a Forti, J. C.; Aquino Neto, combustível etanol/o2 e processo S.; Zucolotto, V.; 018130020870 2012 para preparar o mesmo Ciancaglini, P.; Andrade, A. R Filmes LbLs contendo PPV ancorado Santos, V.; Wohnrath, K.; ao Híbrido Silano-Pt utilizados na Pessoa, C. A.; Garcia, J. PI BR 2012 006538 0 2012 determinação simultânea de R.; Santos, M.; Zucolotto, dopamina e ácido ascórbico V. Perinotto, A. C.; Santos, Biossensor tendo eletrodos F.R.; Colhone, M. C.; interdigitados para aplicação em Ferraz, D. B.; Daghastanli PI 1.104.815-8 2012 nanomedicina na detecção e K.R.P.; Stabeli R. G.; diagnóstico Ciancaglini, P.; Oliveira Jr, O. N.; Zucolotto, V. Fernandes, E. G.; Garcia, Biossensor, seu uso e métodos para A. F.; Vieira, N. C. S.; P.I. 1.102.880-7 2011 detecção de íons cálcio Guimarães, F.E.G; Araújo, A. P. U.; ZUCOLOTTO, V Biossensor para detecção de Zucolotto, V.; Polikarpov, P.I. 1.101.437-7 hormônios tireoidianos e aplicações 2011 I.; Bendo, L.; Figueira, A. em nanomedicina C. M Filme nanoestruturado contendo polissacarídeo de origem algal P.I. 1.101.127-0 2011 Nordi, C. F.; Zucolotto, V imobilizado em conjunto com polieletrólito Processo de preparo de nanopartículas metálicas de ouro por Casanova, M.; Crespilho, P.I. 018130018374 uma rota de um só passo e 2011 F.; Machado, S.S.; nanopartículas de ouro estabilizadas Zucolotto V para aplicação em nanomedicina Crespilho, F. N.; Processo de obtenção de um produto Pavinatto, F. J.; Róz, A.; à base de nanopartículas metálicas e Zucolotto, V.; Barioto, V. P.I.: 0.802.649-1 polímeros para tecidos autolimpantes 2009 L.; Gasparotto, L. H. S.; e auto esterilizantes e produtos Avansi Jr., W.; Oliveira Jr, resultantes O. N Da Róz, A.L.; Pereiro, L.V.; PRODUTO A BASE DE QUITOSANA E Pavinatto, F.J.; Crespilho, P.I.: 0.802.290-9 PROCESSO DE IMPREGNAÇÃO DO 2008 F.N.; Zucolotto, V.; MESMO EM TÊXTEIS Carvalho, A.J.F.; Oliveira Jr., O.N. Membranas eletroativas Crespilho, F. N.; nanoestruturadas (mens), processo de P.I.: 0.705.295-2 2007 Zucolotto V.; Oliveira Jr., preparo das mesmas e dispositivos N.; Brett C.M.A. contendo as mesmas

O) Scientific Contribution and Comparative Analysis between the Current and Expected Situation

Considering the recent change of the paradigms of oncogenesis, which reinforce the importance of the diversity of subtypes of malignant cells and cellular and acellular of tumor microenvironment for neoplasia initiation and progress, the studies listed in this proposal will contribute to genomic, proteomic, and functional characterization of these protagonists, and will especially explore new forms of therapy against cancer, using pre-clinical models and clinical assays. These studies are original, based on the experience gained by the group in cell therapy in the last 12 years, and were possible because of the infrastructure for ex vivo manipulation and reinfusion of cell subpopulations in humans existing in the Regional Blood Center of Ribeirão Preto. We expect a great advance in understanding about conditions intrinsic and extrinsic to neoplastic stem cells associated with the emergence and progression of the disease, as well as the development of innovative therapies that may result in the cure or in deceleration of hematopoietic neoplasias, melanomas and sarcomas. From the educational point of view, the proposal will offer the opportunity to approximately 200 students of public elementary and high schools for attending theoretical and practical classes in the House of Science, in the INCTC researchers’ laboratories and in the classrooms of the Blood Center. They will also participate in playful activities aiming at the development of logical reasoning and critical spirit essential to science. Therefore, as an example of what has happened in the CEPID/CTC project, we intend to reinforce their basic knowledge of life sciences, foster the appreciation for this area, and identify and promote the development of these young talented students in their early stages of education. Apart from them, several academic and professional post-graduate students, and even post-doctoral fellows, will take part in the project, developing part of their projects according to the theme proposed. In regard to technology transfer, methodologies at the forefront, not widely available in the country, will be used as tools for several studies proposed, in order to identify molecular patterns, profile of functional markers with potential use in diagnosis, and in new therapeutic approaches, highlighting the use of nanoparticles and immunotherapy. From these achievements, it is predicted the development of patents, software for analysis and management, as well as public policies in health for the population.

P) Justified Budget

General

Item description Total Amount (R$) Consumable Material 3,100,000.00 Permanent Material 2,134,756.67 Travel Tickets 300,000.00 Scholarships 3,353,846.40 Travel Support 400,000.00 Outsourced Services and Import Expenses 700,000.00 Total 9,988,603.07

Consumable Material

Item Value (R$) Antibodies 1,000,000.00 Office Material 50,000.00 Collection Material 50,000.00 Laboratory Plasticware 300,000.00 Kits 700,000.00 Laboratory Reagents 1,000,000.00 Solvents 50,000.00 Laboratory Glassware 50,000.00 Total 3,200,000.00

Permant Material

Value (R$, Item Permanent Material Quant. EUR, US$, Value (R$) CHF)

564,179.62 1 Confocal system C2 inverted microscope Ti-E 1 249,637.00

2 Orbital-shaker Incubator 1 31,944.00 79,860.00

3 BD Canto Cytometer 1 195,945.00 442,835.70

12,502.32 4 Refrigerator 1 5,532.00

5 Freezer - 30°C 2 8,719.00 19,704.94

6 Ultrafreezer 2 22,719.00 102,689.88

7 Image scanner 1 29,810.31 65,582.68

8 C1 Single-Cell Auto Prep System 1 214,068.00 483,793.68

40,855.80 9 Multidrop combi reagent dispenser 1 13,710.00

10 Nucleofector 1 15,100.01 34,126.02 Variable volume multi-channel pipette of 30-300uL 4,099.64 11 and 8 positions, model Eppendorf Research Plus 1 1,814.00 Variable volume pipette pack, model Eppendorf 3,493.96 12 Research Plus,(2-20uL, 20-200uL e 100-1000uL) 2 773.00 Variable volume multi-channel pipette of 15-300ul, 8 channels, brand Eppendorf, model Xplorer, with 4,099.64 13 load adapter 1 1,814.00

14 Orca Bioreactor 1 52,581.00 118,833.06 Nano ZS90 for the measurement of size, molecular 94,016.00 15 weight, 1 41,600.00

16 Microtome 1 13,201.76 39,341.24

17 ReadyKart with three shelves for bag transportation 1 10,948.00 24,742.48

Total: 2,134,756.67

1) Confocal Microscope: The subprojects that focus on nanomedicine, coordinated by Prof. Valtencir Zucolotto, will develop nanomaterials for diagnostic and therapeutic use (drug delivery and hyperthermia). In both applications, nanoparticles and other nanomaterials will be projected and designed to interact directly with the target cells and tissue. For these therapies it is assumed

79 that the nanoparticles will be internalized into tumor cells or in the models that will be evaluated. Thus, the Nikon Confocal Microscope, as requested, is an essential hardware to monitor, quantify and optimize the process of internalization of nanomaterials into the cells. This equipment will be used in subprojects 1, 13, 20, 24, and 25.

2) Orbital shaking incubator: it will be employed in lymphocyte and non-adherent cells expansion at medium-scale directed to therapy. This equipment will be used the subproject 12.

3) Flow cytometer: BD FacsCanto II Cytometer is equipped with three lasers (blue, red and violet) what enables the analysis of eight colors at the same time, in addition to size and complexity parameters. Several approaches including hematopoietic cells from murine models and humans (donor and receptor cells for immunotherapy) could be evaluated by a combination of 6 to 8 markers using this technique. In addition, this cytometer has a fluidic system that shows streamline work (by the rapid acquisition), saves time, and improves performance what is essential for the high number of analysis of samples that will be conducted. It is important to notice that the current flow cytometry sorter (2 lasers) that is installed in the Center is not adequate for the multiple cell analysis and the throughput of analysis is limited because the velocity of acquisition. This equipment will be used in subprojects 5, 6, 8, 22, 23, and 24.

4; 5 e 6) Refrigerator; Freezer -30° C e Ultra Freezer: equipment designed to the storage of sample and consumables for cell culture under controlled and safe conditions. This temperature control is included at Good manufacturing practices guidelines and is mandatory to cell products designed to human use. This equipment will be used subproject 25.

7) Image Scanner: Consists of a sensitive and robust system for recording images of chemiluminescence with high resolution to be use in assays for proteins and DNA in gels and membranes, this equipment allows simultaneous images of a sample of chemiluminescent and of molecular weight marker. It is an ideal instrument for multiuser laboratories in a research center as INCTC. This equipment will be used subproject 1 and 2.

8) C1 Single-Cell Auto Prep System: an innovative platform based on microfluidic technology that enables the rapid and reliable study of individual single cell for genomic analysis. This equipment is essential for the current proposal since several questions concerning the structural and functional heterogeneity of cancer cells and cancer stem cell subpopulations are addressed. We aim to

80 detect genetic markers for cancer diagnosis and therapy. This equipment will be used in subprojects 1, 2, 4, 5, 6, 7, 12, 13, 14, and 26.

9) Multidrop Combi Reagent Dispenser (Thermo Scientific): HCS (High Content Screening) methods are based on the acquisition and automated computational analysis of fluorescence microscopy images of cells arranged on plates (96 or 384 wells), allowing quantitative evaluation of a large set of morphometric and functional measurements under a high number of experimental conditions, in parallel. The subproject entitled “Large-Scale Functional Analysis for the Identification of microRNAs with Antitumor and Anti-metastatic Potential in Head and Neck Cancers” will apply this approach to functionally evaluate 2,048 mimetic RNA synthetic molecules of human microRNAs (Human miRIDIAN miRNA Mimic Library, GE Dharmacon CS-001030), and other 2,052 inhibitor RNA synthetic molecules of human microRNAs (Human miRIDIAN miRNA Inhibitor Library, GE Dharmacon IH-001030), every one arranged on 26 plates of 96 wells. To conduct these cell assays in a reproducible way (crucial condition to reliable quantification of the parameter evaluated), these molecules will be transfected in parallel in cells arranged on plates. Therefore the use of the equipment is necessary to dispense reagents and cells, at the stages reverse transfection of the cells, by dispensing transfection reagent on well plates already with RNA synthetic molecules, followed by the deposition of cells in the same wells. In addition, after the transfection and culture of these cells for a determined period, cells will be subjected to the labeling of regions (nucleus, cytoplasm, etc.) and protein markers to be evaluated (using fluorescent dyes and antibodies). The processing in parallel, using automated dispensers, besides enabling the screening of more than 52 plates per experiment, allows the fluorescence intensity of different marks to be quantitatively compatible among the wells evaluated; enabling proper acquisition and analysis of images. It will be very useful to subprojects 1, 7, 8, 17 and 19.

10) Nucleofector: this technology is based on the creation of membranes pores by the application of electric pulse. Different from other methodologies and equipment, this one allows the delivery of nucleic acids not only to cell cytoplasm, but also through nuclear membrane to the host genome. This technology allows high transfection rate, independently of cell proliferation and viral integration. Thus, the equipment will be used in all the projects that predict protein or gene expression modification, with emphasis on the projects of induced cell reprogramming: generation of T lymphocytes and NK cells from pluripotent cells, generation of anti-CD20 CAR (chimeric antigenic receptor) T lymphocytes, study on the inhibition of histones desacetylases in cell

81 reprogramming by nuclear transfer or gene induction to pluripotency (iPS cells), study on the function of miRNAs associated with AGO2 in the differentiation process in bovine stem cells culture, study on the regulatory mechanisms involved in the process of generation in vitro of T induced regulatory cells (iTregs) from T naïve cells, and tumor reconstitution from tumor cells reprogramming. The equipment will be used in subprojects 10, 12, 15, 16, 17 and 18.

11; 12 and 13) Pipettes: designed to imunohistochemistry analysis. This technique will be employed in several subprojects. High quality, precision, wide and a reliable range are essential features for a consistent and accurate analysis. Thus, because they are aliquots with very small volumes, especially for measuring buffer volumes, for the dilution of antibodies and application of chromogens, pipettes are essential to ensure accuracy. The result of this technique will offer information about microenvironment as well as about the neoplasia researched. The equipment will be used in subprojects 26

14) Orca Bioreactor: this equipment is very important to perform decellularization and repopulation of the tissues proposed in the project. The equipment will control the input and output of the media needed to the decellularization of organs and tissues. It will be very important for subprojects that use cellular matrix as biological scaffold. The equipment will be used in subproject 10.

15) Nano ZS90: is a flexible analytical tool important to characterize the physical properties of nanoparticles. This equipment will be used in the analysis of particle size performed by dynamic light scattering (DLS). Depending on the physical properties of the samples and formulation, the determined dynamic range will be from 0.3 nm to 8 µm. The equipment will be used in suproject 20.

16) Microtome: Equipment essential to cut material (in paraffin, methacrylate, paraplast) to be studied and prepared for other techniques and observation at optical microscope. Indispensable to histological techniques, morphometric studies, and immunohistochemical analyses. The equipment will be used in subproject 3.

17) ReadyKart with three shelves for bag transportation: equipment designed to the wash and concentration of the cells produced in large scale, as well as aseptic transfer to the bags where cells will be cryopreserved. It will be used in subproject 12.

Q) Institutional Counterpart The participant institutions offer as counterpart their equipment park existing in their laboratories and human resources needed to develop the proposal.

Counterpart

Human Laboratory Institution Total Resources Infrastructure

Institute of 198,000.00 2,500,000.00 2,698,000.00 Bioscience/USP

FMVZ/USP 600,000.00 3,500,000.00 4,100,000.00

FZEA/USP 472,440.00 2,050,000.00 2,522,440.00

GNANO/IFSC/USP 1,380,000.00 2,110,000.00 3,490,000.00

Hemocentro Foundation 1,853,838.00 11,640,073.49 13,493,911.49 of Ribeirão Preto (*)

Total 4,504,278.00 21,800,073.49 26,304,351.49

• 10 hours/week of dedication to the project.

R) Description of the Infrastructure

Most of the activities will be developed in the Regional Blood Center of Ribeirão Preto, headquarters of the Institute. The Hemocentro Foundation of Ribeirão Preto has independent administration, but is supported by the University of São Paulo, the Hospital of the Ribeirão Preto Medical School and the São Paulo Secretariat of Health. The Regional Blood Center has 11.804 m2 of construction area, 5.217 m2 of this area will be used for INCTC activities. In this area, research laboratories, areas designed to educational activities (House of Science, MuLEC, TV Hemocentro, classrooms) and areas for administrative support are included. The Regional Blood Center also offers support for the costs of routine expenses such as cleaning, water, telephone, power energy, and safety. In addition, it has in its human resources chart: 6 researchers, 96 specialized laboratory technicians, and 8 scholarships for technical training. For administrative and educational activities, 12 professionals have been hired, including managers, teachers, journalists, translators, technicians, and assistants. It also offers the Laboratory for Animal Breeding and Manipulation with 1.411m2. Currently it possesses four classrooms and two amphitheaters, all air condiotioned and equipped with multimedia system: Green Room (72 m²), with capacity of 80 people; Yellow Room (22 m²), with capacity of 30 people; Block F Classrooms, with capacity of 32 people; Red Amphitheater (146 m²), with capacity of 180 people; and Blue Amphitheater (62 m²), with capacity of 50 people. For the first semester of 2015, four new classrooms will be made available, with capacity of approximately 120 people. The detailed description of the areas, including pictures and plans, are available on the address: http://lgmb.fmrp.usp.br/inctc/index.php?option=com_content&view=article&id=19&Itemid=27&l ang=pt

Regional Blood Center of Ribeirão Preto

Clinical Hospital - Ribeirão Preto Medical School/USP

Classrooms:

• Green classroom - (72 m²): unit with controlled temperature, it has modulated tables with individual chairs, capacity of 80 people, equipped with internet and multimedia system. • Yellow classroom – (22 m²): unit with controlled temperature, it has individual school chairs, capacity of 30 people, equipped with mobile multimedia system. • Classroom 1 Block F (33.93 m²): unit with controlled temperature, it has individual school chairs, capacity of 16 people, equipped with mobile multimedia system. • Classroom 1 Block F (31.30 m²): unit with controlled temperature, it has individual school chairs, capacity of 16 people, equipped with mobile multimedia system.

Amphitheaters:

• Red Amphitheater – (146 m²): with controlled temperature, it has individual chairs with mobile arms, capacity of 180 people, equipped with multimedia system, internet and sound system. • Blue Amphitheater – (62 m²): with controlled temperature, it has individual chairs with mobile arms, capacity of 50 people, equipped with multimedia system, internet and sound system.

Informatics Center

• (47 m²) Informatic central unit, it has: 1) Central Server, with an optic disc unit to record and read, 16 DIMM memory module , HP U320 SCSI 73 GB two Hard drives, 12 disc units of 72,8 GB, one unit of magnetic tape Ultrium 448, HPXV v1 – Model HP RP 4440 operational system; 2) PA-RISC serie 9000 server, with 2 memory modules of 512 MB, with unit of magnetic tape Leasg 16-00, HP-UX V.11 – Model HP L2000 operational system; 3) PA-RISC serie 9000 server, with disc unit of 2 GB, two external dics of 2 GB – HP K210/128 model;

4) Data bank: a) Oracle 10G Database Standard, platform HP-UXPA-RISC; b) “Progress” system; 5) 2 Servers with 1024 MB memory, disc unit of 72 GB, HP DL 380 model; 6) Net system Hub – Model 3 COM Super Stack; 7) No-Break, nominal power de 10 KVA automatic; personal computer, scanners and printers, among others.

Laboratories:

• Cryobiology I Laboratory – (71 m²): this unit has support, hematopoietic progenitor cell freezing and storage areas (umbilical cord blood). It has the following equipment: sterile connection device, microcomputer, pipettes, preservation chamber with embryo freezer accessory to blood bags storage, storage system cryoplus, dielectric manual sealer, hemoseal automatic sealer for blood bags, laminar flow, automatic cell counter, -20C freezer, -86C freezer, 25 kg high pressure cylinder, water purification system to produce

type 1 water, CO2 incubator, phmeter, optic inverted microscopy, thermal hydrometer, cryogenic container for transportation, conservation chamber, analytical balance, plasma extractor, frezenius universal sealer, storage system “bioarchive”, low speed refrigerator centrifuge, among others.

• Cryobiology II Laboratory – (41 m²): laboratorial area. It has basically the following equipment: inverted microscopy, colored monitors, micro pipettes, precision pipettes, electrophoresis chambers, vacuum pampers, automatic electrophoresis system, refrigerator, biological safety laminar flows, centrifuges, incubators, printers, stabilizer, colored table scanner, angle rotor, freezer, video digital, duplex refrigerator, magnetic stirrer, automatic thermo cycler, phmeter, incubator, biological stirrer, advanced filtration system, microcomputers and printers, among others.

• Quality Control Laboratory: (44 m²): laboratorial area. It has basically the following equipment: coagulometer, precision pipettes, refrigerators, microscopes, phmeters, water baths, freezer, centrifuges, spectrophotometer, laminar flow, among others.

• H.L.A. Laboratory – (22 m²): laboratorial area. It has basically the following equipment: thermo cycler, flow cytometer, platform, feed pump, microscope, water-bath, centrifuge, pipettes, digital camera, electrophoresis chamber, scale, stirrer tubes, freezers, refrigerators, biosafety laminar flow, incubator, electrophoresis system, microcomputer and printer.

• Cell Culture Laboratory – (68 m²): laboratorial and room culture area. It has basically the following equipment: binocular optic microscope, CO2 container, precision pipettes, pipettor, freezer (-86°), phase contrast inverted microscope, vortex, CO2 incubator, water bath, refrigerated centrifuge, semi automatic photo-micrography system, N2 container, biological safety laminar flow, binocular microscope, phmeter, refrigerators, inverted microscope, analytical scale, water distiller, electrophoresis system, bar code reader, laminar flow, among others.

• Molecular Biology Laboratory: (94 m²): laboratorial area. It has the following basic equipment: electrophoresis power supply, water distiller, water bath, video copy processor, micro plates homogenizator, pipettes, vacuum pump, microscopic image acquisition system, transilluminator to detect DNA fluorescence, laminar flow, incubator, centrifuge, duplex refrigerator, automatic thermo cycler, sample concentrator, freezer, spectrophotometer, speed vac with high capacity components, microcomputers and printers, among others.

• Cell Biology Laboratory: (67 m²): laboratorial area. It has the following basic equipment: refrigerators, electrophoresis system, printers, pipettes, chambers, spectrophotometer, cellmax pump station, scintillations counter and micro plates, freezer, centrifuges, microscope, water bath, laminar flow, microcomputers, printers, among others.

• Flow Cytometry Laboratory: (47 m²): laboratorial area. It has the following basic equipment: flow cytometry, pipettes, refrigerators, vacuum pump, facstation power macintosh station, facsorting interface, micro pipettes, centrifuges, microcomputers, printers, among others.

• Molecular Genetics Laboratory: (47 m²): laboratorial area. This laboratory has the following basic equipment: multimedia projector, automatic thermocyclers, freezers, VDS imaging system, vertical laminar flow, pipettes, refrigerators, water bath, micro centrifuges, magnetic stirrers, plate sealers, incubator for bacterial culture, DNA genetic analyzer, multi-screen mini protean, micro plates systems, sonicator, dry thermo blocks, vertical and horizontal electrophoresis apparatus, automatic sequencers, scanner HP Scanjet, micropipettes, digital thermo mixer, personal computers, notebooks, printers, among others.

• Bioinformatics Laboratory: (25 m²): informatics area. This laboratory has the following basic equipment: personal computers, desk scanners, printers, multimedia projector, among others.

• Research and Study Laboratory: (34 m²): area of informatics research. This laboratory has the following basic equipments: personal computers, printers, etc.

• CTC Library: (42 m²): this unit has 2,024 books, 4,000 periodic and journals, 19 references sets (printed and digitalized), theses, dissertations and monographs, guides, 11 electronic subscriptions, 02 journals subscriptions, annals and collection of scientific production of the group of professors, etc.

• Audiovisual Resources: (33 m²): area for elaboration and production of bibliographic material and multimedia images. This area has the following basic equipment: personal computers, desk scanners, printers, plotter, camera camcorder, CD and DVD recorder machine, portable projectors, etc.

• Nursing services – (76 m²) – areas for nursing management, administrative support, aphaeresis, transfusion room, medical offices, screening of donors and blood collection. This area has the basic equipment: computers, printers, sphignomanometer, aphaeresis

systems, blood bag sealers, chairs for aphaeresis, infusion pumps, urgency cars, vacuum clean surgical, etc.

• Social Work – (21 m²) –areas for social support to donors and outpatients. It has the basic equipment: computers, printers, scanner, video camera, photo camera, television and DVD player, among others.

• Laboratory of Hematology – (622 m2): this unit is composed of administration area, laboratory and cell culture room. It has the basic equipment: refrigerators, freezers –20 and –80 C, centrifuges with and without refrigeration, speed-vac centrifuge, cytocentrifuge, microwave, hybridization oven, hybridization oven for microarray, analytical scale, pHmeter, geneQuant, spectrophotometer, ice machine, machine for cell freeze, bench refrigerate incubator, thermo cycle, Real Time PCR system, dry bath, water bath, water purification system, laminar flow, negative isolator rack, CO2 incubator, horizontal stirrer, tubes stirrer, magnetic stirrer with and without heating, power supply for electrophoresis chamber, vertical and horizontal electrophoresis chambers, tissue drill, scanner, photo documentation system, autoclave, cell counter/analyzed, pipettes, computers, and printers.

• Biocenter – (1,006 m²) area for HLA and biotechnology laboratories and hereditary anemias, Cell therapy, Cytogenetics and Immunofluorescency, House of Science and support rooms.

• Laboratory of Biotechnology and Hereditary anemias – It has the basic equipment: microwaves, stirrers, water bath, incubator and analytical scale.

• Laboratory of Cell therapy (296.83 m²) - It has the basic equipment: microscopy, pippetors, refrigerated centrifuge, freezer, water bath, Laminar-flow class 2, cell counter, CO2 incubator, Nitrogen liquid container, electrophoresis chambers, phmetrer, analytical scale, electrophoresis power and freezer -86C.

• Laboratory of Cytogenetic and Immunofluorescence (86.24 m²) – This laboratory has the following basic equipment: microscopy, fluorescence source, incubator for culture, safety cabin, vortex, cell counters.

• House of Science and Museum and Laboratory of Science Education – area for educational activities. It has the basic equipment: computers, printers, plotter, scanner, DVD player, cassette player, video cameras, photo cameras, multimedia system, televisions, microscopy, electronic keyboard, notebook and refrigerator.

• Laboratory of Animal Studies -1,587.47 m² – The Laboratory of Experimental Studies in Animals (LEEA) comprises an area designed to the production of rodents for scientific research composed of a large room with 3 double racks with positive pressure (Tecniplast) for housing mice couples, making a total of 13 lines, composed of knockout, transgenic and spontaneous mutant animals and two Tecniplast exchange stations. There are still in the sanitary barrier 4 rooms, one room for quarantine of newcomer animals to the institution (Alesco double rack with positive pressure), room for murine embryos cryopreservation equipped with stereomicroscope, one microforge and a CO2 incubator, room for murine embryos manipulation (transgenia) with inverted microscope and two micromanipulators and a room reserved for breeding rats. The LEEA comprises an área designed to experimentation, composed of 4 rooms, one of them to keep the animals under experimentation, where there are two Alesco racks and an exchange flow of cages (Veco), a refrigerator and 2 steel shelves. Other rooms is designed to experimental protocols equipped with 3 biological safety cabinets, 2 refrigerated centrifuges, 1 inhalation anesthesia machine for mice, 2 CO2 euthanasia chambers, semi-analytical scale, water bath, vortex, two refrigerators, in vivo reading equipment of bio and chemiluminescence (IVIS), in addition to a simple incubator and one dental autoclave. The other two rooms will be equipped to be a culture room and image laboratory. The hygiene and sterilization area relies on steam/pressure sterilization system with 2 horizontal autoclaves of 570 liters each, on an immersion tank, a cleaning and disinfection tank, on reverse osmosis

equipment and a sonicator (for cleaning nozzle of the water bottles). The other environments are designed to store feed bags, animal bedding, and other products and material for use in the LEEA routine. The building has restrooms (a conventional one and other for disabled people), in addition to a small kitchen, reception, and freezer room (1 freezer -80C, 2 horizontal freezers, 1 Crioplus 4).

• Protein Chemistry Center – (460 m²) – area for sample preparation, electrophoresis, administration offices and meeting rooms for support. It has the basic equipment: tube stirrer, magnetic stirrer, electrophoresis power, refrigerator, hypercassete, phmeter, and electrophoresis evaluation system, production system of ultrapure water and pre- treatment of water.

Evandro Chagas Institute

• National Center of Primates: every section has physical space where the laboratories that develop each research line are allocated. In addition, the physical structure of the campus of Ananindeua has: Mangement, Administration Services, Budget and Finance, Warehouse, Purchase, Informatics, Maintenance, Material and Property, Transportation, Human Resources, Register, Human Resources Development, Payment, Employee’s Health, Library, Geoprocessing Laboratory, Communication consultants, Scientific and Academic Development, Planning, Institutional Program of Scientific Initiation Scholarships, Biosafety Comission, Ethics Committee of Research in Human Beings, Ethics Committee of Research in Animals, Scientific and Technical Council, Quality Management. It relies on this basic equipment: surgery, autoclaves, embebbed material, equipment for paraffin embedding and preparation of slides: equipment for inclusion, vacuum incubator, microtome, stain material and equipment, light microscope, Freezer -70°C, thermocycler. Equipment for other examination: PCR, ELISA, serum biochemistry, 4D ultrasound, stereotactic to non- human primates, non-invasive telemetry equipment, and digital radiography equipment.

• Laboratory of Cell Culture and Cytogenetics of the Environment Section. Equipment: CO2 incubators, incubators, flow cytometer, laminar flow, freezer -70º, freezer -120ºC, refrigeration chamber, refrigerators, optical microscopes, inverted microscope, fluorescence microscope.

University of São Paulo

Bioscience Institute / Department of Genetics and Evolutionary Biology

• Cell culture room: is equipped with the following basic equipment: laminar flow, CO2 incubators, Refrigerate Centrifuge, Inverted microscope, magnifying glass, warm water baths, microcentrifuge, freezer –70C, nitrogen liquid containers, electrophoreses apparatus with agarose and polyacrilamide gel, 2 PCR machines (24 and 96 wells), scales, incubators, pHmeters, stirrers, electronic pippettes, microwave, computers with internet access, scanner and printers.

• Animal Facility Laboratory: This sector is located at the Immunology Department and has already been working for 8 years. This structure gives support to keep mice and rats in pathogen-free conditions. The laboratory possesses central air conditioning with 100% air renew, area with wash machines for cages and water dispensers, ultrasound system for nozzle cleaner, 2 high volume autoclave and wash and dry machine for clothes, uniforms for the employees and users. All the material (cages, lids, drinking trough and food) is autoclaved. The rooms for animal keeping have 21 +- 2 °C temperature, with 12 light and 12 of dark. The animal facility relies on a laboratory structure for routine genetic and sanitary controls (virology, parasitology and bacteriology).

University of São Paulo

Laboratory of Molecular Morphophysiology (LMMD) of the Department of Basic Sciences of the School of Animal Husbandry and Food Engineering

• As infrastructure, the LMMD laboratory presents a built area of 280 m2 equipped with cell culture room with incubators, microscopes and also molecular biology equipments, including Real time PCR, laser image reader for different application as “differential display” PCR and macro-array. The LMMD also has another area denominated Animal Neonatology Unit (200 m2) that is currently under construction (FINEP grant) and is designated to the large scale production of transgenic mammals.

University of São Paulo

School of Veterinary Medicine and Animal Husbandry

• Laboratory of Microscopic Anatomy and Immunohistochemistry - This laboratory is equipped with distiller, oven for drying materials, dispenser for paraffin, sputter coating machine, critical point, microtomes, water bath, refrigerators for samples storage, microscopes, ultrasonic cleaner, computers and a fume hood. It offers technological conditions to support research in the area of organic light-microscopy, preparation of tissues, molds, models and several staining techniques. It develops microvascular techniques, preparation of molds and vascular models, confection and analyses. It is provided with an imaging system for microscopy with 10 trinocular microscopes, one interconnected with dual observation system (Leica® industries Mod DME system) and images acquisition system for television, using the trinocular tube for Leica microscopy, with adapter C-Mount cameras for the video Color 1 / 3 "CCD Samsung® SDC-313 with higher resolution of 480 lines-TV. It offers services to the general public such as: preparation and staining of tissue for histological and immunohistochemistry research; whole mount preparations, confection of molds and vascular models for electron microscopy; preparation of stains and fixative solutions for tissues; confection and preparation of semi-thin and mesoscopic sections. The Laboratory was used to support the course of practical specialization in Biology of Development and Embryonic stem cells in 2006.

• Laboratory of Cell Culture - Equipped with CO2 incubator, Leica® microscope adapted in laminar flow (MINIFLOW) to analyze embryos of different species with guarantee of no contamination of biological material, laminar flow (Veco®), inverted microscope connected to an image system, refrigerator, water bath, centrifuge and -80°C freezer, providing conditions to developing research involving cell culture, and counts with materials such as bottles, falcon tubes (15 and 50ml), tips, disposable pipettes, plates of various sizes, filters, culture medias and supplements.

• Laboratory of Electron Microscopy - Equipped with a Scanning Electron Microscope (Leo - 435 VPZeiss), and a Transmission Electron Microscope (MET) model Magni 268D (FEI Company - Philips), equipped with a system for image analysis SIS DOCU MET, digital camera 268, working kilovoltage between 40 and 100 KV, which increases vary from 25 to 280.000X. It is also equipped with water cooling unit, especially developed for biological sciences, in addition to two Ultra microtomes (Leica), sputter coating machine (Balzers), a unit of Critical Point and other equipments needed for sample preparing. It is appropriate for the analysis of different types of tissues and samples. The scanning electron microscope has the ability to work with samples dehydrated or not, metallic or not, ensuring speed, efficiency and low cost of processing. It performs the preparation and analysis of electron micrographs; development and enhancements of electron micrographs; preparation and ultra cuts for transmission electron microscopy; preparation and analysis of the material for scanning electron microscopy; preparation of material for transmission electron microscopy.

• Surgery Center: It offers to the researchers the possibility of pre-clinical tests on different types of animals. Equipped with surgical table, cabinets for medicines, surgical focus, oxygen cylinder, trichotomy machine, and anesthetic equipment HB Hospital (Galanti).

The INCTC/CNPq and CTC/FAPESP also keep some facilities that support the research activities, including laboratories of cell therapy, molecular biology, gene transfer, molecular

94 genetics, flow cytometry, bioinformatics, protein chemistry, and HLA genotyping. These facilities are coordinated by researchers highly experienced in the technology offered.

• Flow Cytometry:

By the flow cytometry technique, the laboratory performs tests such as subpopulation quantification of lymphocytes in patients with HIV, quantification of progenitor stem cells for autologous or allogeneic transplantation, subpopulation quantification of peripheral blood and other methodologies. This technique allows to measure chemical and physical characteristics, in particles or in cells in suspension. The laboratory also supports research conducted with mesenchymal, endothelial, and dendritic cells.

• Cell Therapy:

The objective is providing mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSC) to clinical research conducted in the INCTC and CTC. We have experience to manipulate and cryopreserve HSCs for transplantation since 1997. In our service, the products that contain hematopoietic stem cells (HSC), such as bone marrow, umbilical cord blood and mobilized peripheral blood are processed in order to maintain their biological function, up to the moment of use, what can happen years after harvesting. Also large scale MSC expansion in the culture system, using T flasks. MSCs are tested concerning safety (sterility, mycoplasma, and accidental virus), identity/purity (morphology, antigenic phenotype and cell viability by flow cytometry), the potency to inhibit T lymphocyte proliferation and differentiating into adipocytes and osteocytes, in purity (endotoxin) and stability (consistent composition, stable karyotype [G-bands] and capacity to differentiate and to inhibit T lymphocyte proliferation) after long term culture. Our next step is the establishment of an allogenic bank of MSC cells, when cryopreserved cells will be previously tested and offered for off-the-shelf clinical use.

• Animal Manipulation:

This unit is specific pathogen free. It is composed of three rooms for mice and rats, equipped with ventilated rack, shelves for keeping about 300 couples of matrixes of mice under expansion and autoclaves with capacity to provide the scientific community with 500 mice a month and a total of 40 couples (matrixes) of Wistar rats.

• Protein Chemistry Center:

The Protein Chemistry Center offers scientific and technical support for proteomic analysis and mass spectrometry. It is equipped with three mass spectrometers (ESI-Triplo Quadrupolo, ESI- Q-TOF e MALDI-TOF / TOF) and complete systems for online and offline protein separation line/ peptide by capillary liquid chromatography, with specialized software for protein quantification and identification using local data bases or on the internet.

• Bioinformatics:

The Bioinformatics team (BIT) offers scientific and technical support to molecular biology, genomics, proteomics and other approaches. The Laboratory is equipped with high performance server, specialized software and advanced systems of data bases for genomic analysis.

• DNA Sequencing:

It offers access to DNA sequencing automated technology. The services provided include experimental consultations, library preparation, data export and analysis of primary and secondary data.

• Confocal Microscopy:

It was conceived to serve as a center of services to the researchers that need primary stem cells and cancer cells from human or murine analyses. It is specialized in the characterization of morphology, detection of specific proteins by immunofluorescence and detection of cells marked

96 with fluorescence. The laboratory is equipped with Laser Scanning Microscopy (LSM710, Carl Zeiss).

• Microarray Laboratory:

Equipped with a high density Agilent auto-loader Microarray Scanner, an Axon GenePix 4000B (Molecular Devices), and hardware of additional processing; the specialists are trained in the installation to generate data derived from Agilent or any other type of microarrays (including antibodies or microarray proteins). Among possibilities, the expression levels of more than 27,958 genes (mRNAs and 7,419 lincRNAs) can be evaluated simultaneously or alternately (miRbase microarrays 8x60K). Moreover, DNA gains or losses can be evaluated until a resolution of about 2 KB, throughout all the genome (1x1m microarrays CGH). Finally the use of promoter or CpG focused microarrays, jointly with experiments of chromatin immunoprecipitation, allows the study of the occupying promoter by transcription factors (chip-chip) or the epigenomic profile of DNA modifications (Metil-chip).

• HLA Genotyping:

The laboratory offers histocompatibility exams for kidney and bone marrow transplantations, it performs compatibility tests for: 1) all cadaver donors of the State of São Paulo; 2) bone marrow and kidney donors (relatives) for transplantation in the Hospital of the Ribeirão Preto Medical School of the University of São Paulo; 3) banks of bone marrow and umbilical cord blood voluntary donors, REDOME and Brasilcord respectively, which are centralized at the Instituto Nacional do Câncer in Rio de Janeiro.

• Embryonic Micromanipulation:

The Laboratory of Embryonic Micromanipulation has a complete structure for the production of embryos in vitro in a wide range of domestic species, including equipment for gamete micromanipulation, inverted epifluorescence microscope, Narishige microinjectors,

FemtoJet® microinjector, electrofusion system, Piezo drill and in vitro systems with controlled atmosphere.

• Experimental Hospital of Animals

It was created to serve as a laboratory for teaching and for researching on innovative therapies (Finep support). This experimental facility has 3,000 m2 of infrastructure for small and large domestic animal, clinic of image and laboratory. It also performs analysis and surgery. In addition, 143 m2 will be available for production and manipulation of germ cells in free conditions.

S) Schedule

Subproject 1st year 2nd year 3rd year 4th year 5th year 6th year 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

Appendix I – Formal Agreement of Institutions Involved

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Appendix II - Formal Agreement of International Collaborators

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Appendix III - Team

Name Position Edson Giuliani Ramos Fernandes Post-doctoral fellow Fabiana Fernandes Bressan Post-doctoral fellow Idelma Aparecida Alves Terra Post-doctoral fellow Iêda Maria Martinez Paino Post-doctoral fellow Jaqueline Pérola de Souza Post-doctoral fellow Juliana Cancino Bernardi Post-doctoral fellow Patrícia Franklin Mayrink Nogueira Post-doctoral fellow Raquel de Melo Alves Paiva Post-doctoral fellow Roberta Machado Ferreira Saran Post-doctoral fellow Tathiane Maistro Malta Post-doctoral fellow Thiers Massami Uehara Post-doctoral fellow Virgínia Mara de Deus Wagatsuma Post-doctoral fellow Bruna Rodrigues Muys Doctorate student Bruno Braga Sangiorgi Doctorate student Camilo Arturo Suarez Ballesteros Doctorate student Danuta Carolina Sastre Pacheco Doctorate student Fabrício Aparecido dos Santos Doctorate student Fernanda Gutierrez Rodrigues Doctorate student Flávia Sacilotto Donaires Doctorate student Helder Teixeira de Freitas Doctorate student Henrique Antonio Mendonça Faria Doctorate student Ildercílio Mota de Souza Lima Doctorate student Júlia Teixeira Cottas de Azevedo Doctorate student Juliano Rodrigues Sangalli Doctorate student Leonardo Campos Zanelatto Doctorate student Lilian Maria Pessôa da Cruz Centurion Doctorate student Lucas Coelho Marliére Arruda Doctorate student Lucas Eduardo Botelho de Souza Doctorate student Luciana Ribeiro Jarduli Doctorate student Luciana Senna Simões Doctorate student Luíza Ferreira de Araújo Doctorate student Pedro Henrique Padilha Doctorate student Pedro Ratto Lisboa Pires Doctorate student

126

Name Position Rafael Vilar Sampaio Doctorate student Rodolfo Bortolozo Serafim Doctorate student Rodrigo da Silva Nunes Barreto Doctorate student Valéria Spolon Marangoni Doctorate student Vitor Leão Doctorate student Ádamo Davi Diogenes Siena Master student Adriana Queiroz Arantes Master student Ana Júlia Bandeira Machry Master student Ana Paula Nunes Rodrigues Alves Master student Carolina Habermann Macabelli Master student Cristina Tavares Leal Master student Fernanda Borges da Silva Master student Gustavo Borges Master student Isabela Ichihara de Barros Master student Jaqueline Cristina Fernandes Master student Jessica Rodrigues Plaça Master student Juliana Bernardes Elias Dias Master student Laís Canniatti Brazaca Master student Laís Ribovski Master student Maria Florencia Tellechea Master student Renata Scopim Ribeiro Master student Adilson Kleber Ferreira Other Ana Carolina Franco Ferreira Other Bárbara Amélia Aparecida Santana-Lemos Other Celso Teixeira Mendes Junior Other Cristina Ribeiro de Barros Cardoso Other Elaine Teresinha Faria de Sousa Other Emerson Takeshi Urushibata Other Erick da Cruz Castelli Other Fabrício César Dias Other Israel Tojal Da Silva Other Michel Platini Caldas de Souza Other Patricia Cruz Bergami-Santos Other Ricardo Alexandre de Azevedo Other Salomão Dória Jorge Other

127

Name Position Simon Gert Coetzee Other Thaís Cristine Arns Other Thaís Sarraf Sabedot Other Vinicius Moreno Godoi Other Adriana Aparecida Marques Laboratory Technician Amelia Goes de Araujo Laboratory Technician Anemari Ramos Dinarte dos Santos Laboratory Technician Greice Andreotti de Molfetta Laboratory Technician Josiane Lilian dos Santos Schiavinato Laboratory Technician Marcelo Gomes de Paula Laboratory Technician

Appendix IV – Experience in Qualified Professional Training – Supervision concluded during the term of INCTC (2008-2014)

MASTER Year of Supervisor Student Current Position Current Institution Country completion Carlos Eduardo Ambrósio Atanasio Serafim Vidane 2012 Doctorate student CAPES Brazil Carlos Eduardo Ambrósio Fabio Sergio Cury 2012 Doctorate student FAPESP Brazil School of Veterinary Carlos Eduardo Ambrósio Helena Debiazi Zomer 2013 Medicine and Animal USP/Pirassununga Brazil Husbandry Carlos Eduardo Ambrósio Juliana Barbosa Casals 2011 Doctorate student FZEA (USP) Brazil Natalia Juliana Nardelli Carlos Eduardo Ambrósio 2011 Doctorate student FAPESP Brazil Gonçalves Everton de Brito Oliveira Dimas Tadeu Covas 2012 Doctorate student CAPES Brazil Costa Dimas Tadeu Covas Lilian Figueiredo Moreira 2009 Doctorate student CAPES Brazil Lucas Eduardo Botelho de Doctorate student Dimas Tadeu Covas 2012 CAPES Brazil Souza Eduardo Antonio Donadi Daiani Cristina Ciliao Alves 2010 Doctorate student FAPESP Brazil Ana Paula Alencar de Lima Doctorate student Eduardo Magalhães Rego 2011 FAPESP Brazil Lange Mariana Tereza de Lira Doctorate student Eduardo Magalhães Rego 2011 CAPES Brazil Benicio Eduardo Magalhães Rego Patricia Aparecida de Assis 2010 Doctorate student FAPESP Brazil Faculdade de Gislane Lelis Vilela de Fabiola Attié de Castro 2008 Professor Coordinator Ciências da Saúde- Brazil Oliveira Barretos Works in Cell and Fabiola Attié de Castro Livia Gonzaga Moura 2012 FCFRP-USP Brazil molecular Biology Fabiola Attié de Castro Sandra Mara Burin 2011 Doctorate student CAPES Brazil João Agostinho Machado Doctorate student Fabíola Traina 2011 FAPESP Brazil Neto Fabíola Traina Renata Scopim Ribeiro 2014 Doctorate student CAPES Brazil Selection process for Fabíola Traina Ana Paula Nunes Alves 2014 CAPES Brazil doctorate Gabriella Mamede Doctorate student Flavio Vieira Meirelles 2014 FZEA-USP Brazil Andrade

128

Flavio Vieira Meirelles Juliano Rodrigues Sangalli 2012 Doctorate student FZEA-USP Brazil Lais Vicari de Figueiredo Doctorate student Flavio Vieira Meirelles 2012 FZEA-USP Brazil Pessoa Rodrigo da Silva Nunes Doctorate student Flavio Vieira Meirelles 2011 CAPES Brazil Barreto José Alexandre Marzagão Doctorate student Brazil Rodrigo Nalio Ramos 2009 CNPQ Barbuto Kamilla Swietch Antonietto Rafael Tagé Biaggio 2014 Doctorate student FAPESP Brazil Kelen Cristina Ribeiro Doctorate student Luciana Ribeiro Jarduli 2013 FAPESP Brazil Malmegrim de Farias Kelen Cristina Ribeiro Doctorate student Ana Elisa Teofilo Saturi 2011 FAPESP Brazil Malmegrin de Farias Kelen Cristina Ribeiro Julia Teixeira Cottas de Doctorate student 2013 USP/RP Brazil Malmegrin de Farias Azevedo Doctorate student Department of Kelen Cristina Ribeiro Kalil William Alves de Lima 2013 Pharmacology Brazil Malmegrin de Farias FMRP/USP Kelen Cristina Ribeiro Lucas Coelho Marliére Doctorate student 2013 FAPESP Brazil Malmegrin de Farias Arruda Lewis Joel Greene Ana Carolina Humanes 2010 Doctorate student CNPq Brazil Protein Chemistry Center – Blood Lewis Joel Greene Germano Aguiar Ferreira 2008 Researcher Brazil Center/Hospital Das Clínicas of FMRP-USP Lygia da Veiga Pereira Joana Carvalho M. Mello 2010 Doctorate student FAPESP Brazil

MASTER Year of Supervisor Student Current Position Current Institution Country completion Alvaro Carlos Galdos Professor at Faculdade Pharmacy Course- Maria Angélica Miglino 2009 Brazil Riveros Anhanguera Brasília-DF Doctorate student Maria Angélica Miglino Dayane Alcântara 2010 FMVZ-USP Brazil

Marcio Nogueira Doctorate student Maria Angélica Miglino 2009 FMVZ-USP Brazil Rodrigues

Maria Angélica Miglino Carolina Costola de Souza 2010 Doctorate student CAPES Brazil Faculdade de Ensino e Cultura Pio X e Carlos Alberto Palmeira Coordenador da Maria Angélica Miglino 2009 Professor Brazil Sarmento Vigilância Sanitária do Município de Lagarto-SE Patricia Cristina Baleeiro Fabiele Baldino Russo 2010 Doctorate student FAPESP Brazil Beltrão Braga Hospital do Câncer - Rodrigo Alexandre Panepucci Carolina Dias Carlos 2011 Biomedical scientist Brazil Barretos Ildercilio Mota de Souza Doctorate student Rodrigo Alexandre Panepucci 2013 FAPESP Brazil Lima Josiane Lilian dos Santos Doctorate student Rodrigo Alexandre Panepucci 2011 FAPESP Brazil Schiavinato Rodrigo do Tocantins Calado Fernanda Gutierrez Doctorate student 2014 CAPES Brazil Saloma Rodrigues Rodrigues Rodrigo do Tocantins Calado Leonardo Campos Doctorate student 2013 Capes Brazil Saloma Rodrigues Zanelatto Simone Kashima Haddad Katia Kaori Otaguiri 2013 Doctorate student CAPES Brazil Simone Kashima Haddad Mariana Tomazini Pinto 2011 Doctorate student FAPESP Brazil Tathiane Maistro Malta Simone Kashima Haddad 2009 Post-doctoral fellow FAPESP Brazil Pereira Valtencir Zucolotto Doctorate student Brazil Valéria Spolon Marangoni 2010 FAPESP Valtencir Zucolotto Doctorate student Brazil Camilo Suarez 2012 CAPES Valtencir Zucolotto Jaciara Cássia de Carvalho Doctorate student Brazil 2012 Capes Santos Wilson Araújo da Silva Júnior Bruna Rodrigues Muys 2013 Doctorate student FAPESP Brazil

129

Wilson Araújo da Silva Júnior Carolina Arruda de Faria 2011 Doctorate student CNPq Brazil Wilson Araújo da Silva Júnior Leonardo Rippel Salgado 2009 Doctorate student CNPq Brazil Wilson Araújo da Silva Júnior Luiza Ferreira de Araújo 2013 Doctorate student CAPES Brazil Wilson Araújo da Silva Júnior Nathalia Moreno Cury 2012 Doctorate student CAPES Brazil Rodrigo Guarischi Mattos Doctorate student Wilson Araújo da Silva Júnior 2012 FAPESP Brazil Amaral de Sousa

130

DOCTORATE Year of Supervisor Student Current Position Current Institution Country completion Andrielle de Castilho Dimas Tadeu Covas 2011 Post-doctoral fellow CAPES Brazil Fernandes Faculdade Estácio de Dimas Tadeu Covas Jorge Luiz Curado Siufi 2009 Professor Sá/member of Brazil professor nucleus Hemocentro Maristela Delgado Dimas Tadeu Covas 2014 Employee Foundation of Brazil Orellana Ribeirão Preto Gislane Lelis Vilela de Faculdade de Ciências Eduardo Antonio Donadi 2013 Professor Coordinator Brazil Oliveira da Saúde - Barretos Juliana Navarro Ueda Eduardo Antonio Donadi 2014 Doctorate student FMRP-USP Brazil Yaochite Antonio Roberto Lucena Eduardo Magalhães Rego 2012 Post-doctoral fellow FAPESP Brazil de Araújo Guilherme Augusto Silva Eduardo Magalhães Rego 2009 Professor of Medicine III Unaerp Brazil dos Santos Hamilton Luiz Gimenes Eduardo Magalhães Rego 2009 Post-doctoral fellow FCFRP-USP Brazil Teixeira Mirela de Barros Invent Biotecnologia - Eduardo Magalhães Rego 2009 Scholarship Researcher Brazil Tamarozzi Supera Incubator Eduardo Magalhães Rego Priscila Santos Scheucher 2010 Post-doctoral fellow FAPESP Brazil Member of the International Consortium of Acute Promyelocytic Leukemia (IC-APL) and Eduardo Magalhães Rego Rafael Henriques Jacomo 2010 Member and Supervisor supervisor of the post- Brazil graduate program in medical sciences Faculdade de Medicina - UnB Fabiola Attié de Castro Aline Fernanda Ferreira 2012 Jr. Post-doctoral fellow CNPq Brazil Universidade Federal de Juiz de Fora,/Campus Avançado Governador Fabiola Attié de Castro Raquel Tognon Ribeiro 2011 Associate Professor Brazil Valadares - Departamento de Farmácia Fabíola Traina Marina Pereira Colella 2013 Employee/Physician UNICAMP/Campinas Brazil Fabiana Fernandes Post-doctoral fellow Flávio Vieira Meirelles 2013 FZEA-USP Brazil Bressan Jose Rodrigo Valim Post-doctoral fellow Flávio Vieira Meirelles 2010 FZEA-USP Brazil Pimentel Alana Maria Cerqueira de Post-doctoral fellow Lewis Joel Greene 2011 CNPq Brazil Oliveira Lewis Joel Greene Carolina Hassibe Thome 2011 Post-doctoral fellow FAPESP Brazil Protein Chemistry Lewis Joel Greene Germano Aguiar Ferreira 2013 Researcher Brazil Center Lewis Joel Greene Gisele Guicardi Tomazella 2009 Post-doctoral fellow University of Oslo Norway Protein Chemistry Lewis Joel Greene Lucas Oliveira Sousa 2013 Researcher Brazil Center Fundação Antônio Lygia da Veiga Pereira Gustavo Ribeiro Fernandes 2013 Employee Prudente, Hospital AC Brazil Camargo Marco Antonio Zago Ana Cristina Silva Pinto 2011 Employee/Physician Fundherp Brazil

DOCTORATE Year of Supervisor Student Current Position Current Institution Country completion Curso de Farmácia da Marco Antonio Zago Felipe Saldanha de Araujo 2010 Associate Professor - II Universidade de Brazil Brasília Marco Antonio Zago Francisco de Paula Careta 2011 Post-doctoral fellow FAPESP Brazil

131

Lucila Habib Bourguignon Faculdade de Ciências Marco Antonio Zago 2013 Professor Brazil Oliveira Médicas da Paraíba NESCA (Departamento de Marco Antonio Zago Manuela Ramos Barbieri 2009 Employee Pediatria e Brazil Puericultura da UFRGS e FMRP) Universidade de Associate Professor I Brasília UNB/FCE e do Marco Antonio Zago Rodrigo Haddad 2009 Brazil and Coordinator Curso de Farmácia da UNB/FCE

André Luiz Rezende Maria Angélica Miglino 2012 Anatomy Technician FMVZ-USP Brazil Franciolli

Faculdade de Ensino e Cultura Pio X e Carlos Alberto Palmeira Coordenador da Maria Angélica Miglino 2009 Professor Brazil Sarmento Vigilância Sanitária do Município de Lagarto- SE

Cristiane Valverde Maria Angélica Miglino 2010 Post-doctoral fellow Instituto Butantã Brazil Wenceslau

Fundação Universidade Federal do Tocantins, Campus Maria Angélica Miglino Erika Toledo da Fonseca 2010 Professor Universitário de Brazil Araguaína

Universidade Federal Maria Angélica Miglino Flávio Ribeiro Alves 2009 Associate Professor I do Piauí Brazil

Instituto Brasileiro de Greyson Vitor Zanatta Collaborator Professor Terapias e Ensino - Maria Angélica Miglino 2009 Brazil Esper Veterinary Acupunture IBRATE

Juliana Passos Alves dos Clínica Veterinária Maria Angélica Miglino 2009 Brazil Santos Particular

Centro de Hematologia Roberto Passetto Falcão Daniel Mazza Matos 2009 Employee e Hemoterapia do Brazil Ceará Tathiane Maistro Malta Post-doctoral fellow Simone Kashima Haddad 2013 FAPESP Brazil Pereira Valtencir Zucolotto Post-doctoral fellow Instituto de Física de São Carlos - Juliana Cancino Bernardi 2011 Brazil Universidade de São Paulo Valtencir Zucolotto Edson Giuliani Ramos Post-doctoral fellow 2012 IFSC/USP Brazil Fernandes Full Professor at Wilson Araújo da Silva Júnior Alynne Oya Chiromatzo 2010 Universidade Paulista UNIP - RP Brazil (UNIP). Universidade Wilson Araújo da Silva Júnior Carla Martins Kaneto 2011 Associate Professor Estadual de Santa Brazil Cruz, Ilhéus

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DOCTORATE Year of Supervisor Student Current Position Current Institution Country completion Gislaine da Silva Pimentel Wilson Araújo da Silva Júnior 2009 FMRP-USP Brazil Pereira Researcher Wilson Araújo da Silva Júnior Julio César Cetrulo Lorenzi 2013 Post-doctoral fellow CNPq Brazil Thiago Yukio Kikuchi Post-doctoral fellow Wilson Araújo da Silva Júnior 2013 Rockefeller University USA Oliveira

POST–DOCTORAL AND JUNIOR POST-DOCTORAL Year of Supervisor Student Current Position Current Institution Country completion Centro Universitário da Celina Almeida Furlanetto Full Professor and Fundação de Ensino Carlos Eduardo Ambrósio 2013 Brazil Mançanares Researcher Octávio Bastos- São João da Boa Vista, SP Programa de Pós- Graduação Stricto Professor and Carlos Eduardo Ambrósio Silvio Henrique de Freitas 2013 Sensu - Mestrado em Brazil Researcher Biociência Animal /FMVET-UNIC Instituto Federal de Educação, Ciência e Tecnologia de São Dimas Tadeu Covas André Perticarrari 2011 Professor Brazil Paulo, campus São Paulo, Biological Sciences Dimas Tadeu Covas Kamilla Swiech 2011 Professor FCFRP - USP Brazil Adjunto na Universidade Luterana Dimas Tadeu Covas Lindolfo da Silva Meirelles 2011 Associate Professor Brazil do Brazil (ULBRA)- Canoas-RS Dimas Tadeu Covas Svetoslav Nanev Slavov 2013 Post-doctoral fellow CAPES Brazil Fundação Hemocentro Dimas Tadeu Covas Vírginia Picanço e Castro 2011 Researcher Brazil de Ribeirão Preto Instituto Federal de Educação, Ciência e Dimas Tadeu Covas Andre Perticarrari 2011 Professor Brazil Tecnologia de São Paulo Associate Professor and Universidade de Dimas Tadeu Covas Rodrigo Haddad 2011 Brazil Coordinator I Brasília UNB/FCE Fundação Hemocentro Dimas Tadeu Covas Virginia Picanço e Castro 2013 Researcher Brazil de Ribeirão Preto Universidade Federal Eduardo Magalhães Rego Alexandre Krause 2010 Associate Professor Brazil de Santa Maria - RS Barbara Amelia Aparecida Laboratório Eduardo Magalhães Rego 2010 Employee Brazil Santana Lemos Hematologia-HC-RP Guilherme Augusto Silva Eduardo Magalhães Rego 2013 Professor of Medicine III Unaerp Brazil dos Santos Centro Universitário Luciana Maria Fontanari Eduardo Magalhães Rego 2010 Associate Professor Franciscano (Unifra) - Brazil Krause Santa Maria/ RS Hamilton Luiz Gimenes Eduardo Magalhães Rego 2011 Post-doctoral fellow FCFRP-USP Brazil Teixeira

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POST–DOCTORAL AND JUNIOR POST-DOCTORAL Year of Supervisor Student Current Position Current Institution Country completion Universidade Federal de Juiz de Fora,/Campus Fabiola Attié de Castro Raquel Tognon Ribeiro 2012 Associate Professor Avançado Governador Brazil Valadares - Department of Pharmacy Fabiana Fernandes Post-doctoral fellow Flávio Vieira Meirelles 2013 FZEA Brazil Bressan Jose Rodrigo Valim Post-doctoral fellow Flávio Vieira Meirelles 2012 FZEA-USP Brazil Pimentel University of São Paulo, Institute of José Alexandre Marzagão Ana Carolina Franco 2009 Researcher Biomedical Sciences, Brazil Barbuto Ferreira Department of Immunology Farmacore Lewis Joel Greene Idalete da Silva 2009 Employee Brazil Biotecnologia JP Indústria Lewis Joel Greene Patricia Pereira Macaroff 2011 Employee Brazil Farmacêutica Lewis Joel Greene Aline Poersch 2014 Scholarship -DTI-2 INCTC/CNPq Brazil Alana Maria Cerqueira Post-doctoral fellow Lewis Joel Greene 2012 CNPq Brazil Catalan Lewis Joel Greene Helen Julie Laure 2010 Post-doctoral fellow FMRP-USP Brazil Fundação Antônio Gustavo Ribeiro Lygia da Veiga Pereira 2013 Employee Prudente, Hospital AC Brazil Fernandes Camargo Curso de Farmácia da Marco Antonio Zago Felipe Saldanha de Araujo 2012 Associate Professor -II Universidade de Brazil Brasília

Municipal Health Maria Angélica Miglino Janaina M. Monteiro 2010 São Paulo Brazil Surveillance

Maria Angélica Miglino Paula Fratini 2010 Post-doctoral fellow FMVZ-USP Brazil

Universidade Federal da Paraíba, Centro de Maria Angélica Miglino Ricardo Romão Guerra 2010 Associate Professor Brazil Ciências Agrárias

Electronic microscopy Maria Angélica Miglino Rose Eli Grassi Rici Azarias 2010 (PROCONTES) Brazil technician Supervisor of the Post- graduate Program on Maria Angélica Miglino Rose Eli Grassi Rici Azarias 2010 FMVZ-USP Brazil Domestic and Wild Animal Anatomy Patricia Cristina Baleeiro Graciela Conceicao 2007 Scholarship -PNPB CAPES Brazil Beltrão Braga Pignatari Patricia Cristina Baleeiro Graciela Conceicao 2010 Scholarship -PNPB CAPES Brazil Beltrão Braga Pignatari Rodrigo T. Calado de Saloma Raquel de Melo Alves Post-doctoral fellow 2011 FMRP-USP Brazil Rodrigues Paiva Maria Augusta Sartori da Post-doctoral fellow Department of Molecular Simone Kashima Haddad 2012 Genetics – Univeristy of Canada Silva Toronto

134

POST–DOCTORAL AND JUNIOR POST-DOCTORAL Year of Supervisor Student Current Position Current Institution Country completion Valtencir Zucolotto Associate Professor and Universidade Estadual Brazil Luiz Carlos Salay 2009 Researcher de Santa Cruz (UESC) Vice-coordinator of local committee of UFTM José Roberto Siqueira Jr 2010 Minas Gerais Valtencir Zucolotto Post-graduate Program Brazil on Chemistry Centro de Pesquisa em Oncologia Molecular at Wilson Araújo da Silva Júnior Daniel Onofre Vidal 2012 Researcher Hospital do Câncer de Brazil Barretos, área of pediatric tumors Harvard Medical Wilson Araújo da Silva Júnior Cleidson de Padua Alves 2012 Scholarship Researcher USA School Centro de Pesquisa em Oncologia Molecular no Wilson Araújo da Silva Júnior Daniel Onofre Vidal 2010 Researcher Hospital do Câncer de Brazil Barretos, na área de tumores pediátricos

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Appendix V – Experience in Qualified Professional Training – Ongoing Supervisions

MASTER

Supervisor Student Project Title Institution Beginning INCTC/CAPES 2014 Avaliação do efeito citotóxico dos quimioterápicos radiossensibilizadores Belinda Pinto Simões Nayara Rezende Cisplatina e Temozolomida veiculados em sistemas de liberação de fármacos Avaliação do tratamento experimental de cães infectados naturalmente pelo Alessandra de Oliveira Carlos Eduardo Ambrósio vírus da cinomose canina na fase CAPES 2013 Pinheiro neurológica com o uso de células - tronco de epitélio olfatório fetal canino Comparação molecular entre células- Carlos Eduardo Ambrósio Mariana Três Cardoso tronco mesenquimais de membrana CAPES 2013 amniótica canina e felina Geração de uma linhagem celular Daianne Maciely Alves de humana portadora do FVIII sintético Dimas Tadeu Covas CAPES 2012 Carvalho com mutações nos domínios A1 e A2 utilizando o sistema lentiviral Estudo da relação transcricional entre Dimas Tadeu Covas Felipe Canto de Souza DIDO e Aurora-quinases A e B na FMRP-USP 2014 Leucemia Linfoide Crônica Estudo da transição endotélio- Fernanda Ursoli Ferreira Dimas Tadeu Covas mesenquimal (ENDMT) na biologia de FMRP-USP 2012 Melo células endoteliais Legislação em Hemoterapia – Tendência evolutiva da legislação referente aos Dimas Tadeu Covas Helder Teixeira Melo critérios para seleção e triagem de FMRP-USP 2012 doadores , comparada à evolução tecnológica da hemoterapia Isolamento e caracterização de Liziane Raquel Dimas Tadeu Covas subpopulações celulares da fração CAPES 2012 Beckenkamp vascular estromal do tecido adiposo Avaliação do efeito citotóxico dos quimioterápicos radiossensibilizadores Dimas Tadeu Covas Nayara Rezende FMRP-USP 2014 Cisplatina e Temozolomida veiculados em sistemas de liberação de fármacos Estudo do cultivo de células Thais Valeria Costa de mesenquimais multipotentes em meio Dimas Tadeu Covas INCTC/CAPES 2012 Andrade Pimentel de cultura contendo fatores pró- angiogênicos Avaliação do efeito da mitoquinona-10 Katarina Holanda Lúcia de (MitoQ) em tratamento combinado com Eduardo Magalhães Rego CAPES 2012 Melo o trióxido de arsênico (ATO) em modelos in vitro e in vivo de melanoma Estudo do efeito da administração de células tronco mesenquimais na lesão Eduardo Magalhães Rego Larissa Ananias Candido CNPQ 2013 aguda pulmonar induzida por transfusão sanguínea INCTC/CAPES 2014 Juliana Poltronieri de Análise Funcional da Proteína MLLS na Eduardo Magalhães Rego Oliveira Leucemia Promielocítica Aguda

Uso do Single Nucletide Polymorphism Array (SNP-A) na investigação de Fabíola Traina Fernanda Borges da Silva FMRP-USP 2014 alterações citogenéticas em pacientes com síndromes mielodisplásicas Investigação da participação do IRS1 na Jaqueline Cristina Fabíola Traina via de sinalização da ß-catenina na FMRP-USP 2014 Fernandes leucemia linfoide aguda Avaliação da Interação entre Células José Alexandre Marzagão Instituto de Ciências Cecília Pessoa Rodrigues Dendríticas e Mastócitos sensibilizados 2013 Barbuto Biomédicas - USP com Células Tumorais MASTER

136

Supervisor Student Project Title Institution Beginning Enriquecimento antigênico de linhagens José Alexandre Marzagão Mariana Pereira Pinho tumorais: estratégia para abordagens FAPESP 2012 Barbuto imunoterapêuticas personalizadas Inibição de STAT3 por Timosina alfa-1: estratégia eficaz para recuperação José Alexandre Marzagão Cristiano Jacob de Moraes funcional de células dendríticas FAPESP 2011 Barbuto derivadas de monócitos de pacientes com câncer Estabelecimento de uma plataforma de Kamilla Swietch produção de proteínas recombinantes Angelo Luis Caron CAPES 2014 Antonietto terapêuticas em células humanas Ana Carolina Martins dos Caracterização das células do epitélio Maria Angélica Miglino CAPES 2013 Santos coclear de fetos de cão

Isolamento e Caracterização de células Maria Angélica Miglino Lara Carolina Mario FAPESP 2013 tronco embrionárias do gênero Aedes sp Células-Tronco Mesenquimais de Medula Óssea e de Polpa Dentaria Maria Angélica Miglino Márcio Aparecido Pereira CAPES 2013 Canina: Potencial Neurogênico para Terapia Celular em Cães Isolamento e caracterização de células- Maria Angélica Miglino Rennan Lopes Olio tronco derivadas do tecido ósseo CAPES 2013 normal Células mesenquemais estromais multipotentes da medula óssea no Roberto Passetto Falcão Diego Villa Clé FMRP-USP 2010 tratamento da anemia aplastica refreatária Associação do comprimento telomérico dos leucócitos do sangue periférico de Rodrigo do Tocantins Juliana Bernardes Elias doadores e receptores de transplante Calado de Saloma FMRP-USP 2012 Dias de medula óssea para anemia aplástica Rodrigues grave com resultados clínicos após o transplante Geração de células pluripotentes Rodrigo do Tocantins Maria Florencia Tellechea induzidas de pacientes com anemia CAPES 2013 Calado Saloma Rodrigues aplástica adquirida Avaliação de disfunção telomérica em Rodrigo do Tocantins Natália Ferreira Scatena pacientes com carcinoma hepatocelular FMRP-USP 2012 Calado Saloma Rodrigues secundário à cirrose hepática Análise Mutacional do gene GATA-2 e Rodrigo do Tocantins seu Perfil Imunológico Associado em Calado de Saloma Gustavo Borges INCTC/CAPES 2014 pacientes com Mielodisplasias e Rodrigues Síndromes de Falência Medular Desenvolvimento de uma Plataforma Mayra Dorigan de Simone Kashima Haddad Molecular para Detecção de FCFRP-USP 2012 Macedo Mycoplasma spp em Culturas Celulares Purificação e Processamento de Anticorpos conjugados com Valtencir Zucolotto Verônica Pereira Lebre CNPQ 2009 Nanopartículas Metálicas para Aplicação em Biossensores para Febre Aftosa Desenvolvimento de Genossensores Valtencir Zucolotto Wagner Rafael Correr para Diagnóstico Precoce do Papiloma CAPES 2011 Virus Humano (HPV). Pesquisa de Biomarcadores de Câncer Wilson Araújo da Silva Adamo Davi Diogenes de Próstata presentes na urina de CAPES 2014 Júnior Siena pacientes doHCFMRP-USP

137

DOCTORATE

Supervisor Student Project Title Institution Beginning Modelo pré-clinico do uso de células Atanasio Serafim tronco mesenquimais da membrana Carlos Eduardo Ambrósio CAPES 2013 Vidane amniótica para o tratamento de insuficiência renal crônica em gatos Desenvolvimento placentário e descrição morfológica do órgão reprodutor feminino Carlos Eduardo Ambrósio Fabio Sergio Cury FAPESP 2013 em Coendou prehensilis (Porco-espinho Caixeiro Descrição e caracterização das células localizadas na cinta placentária de Carlos Eduardo Ambrósio Juliana Barbosa Casals FZEA-USP 2012 carnívoros (Canis familiaris e Felis domesticus) Geração de células tronco pluripotentes Natalia Juliana Nardelli Carlos Eduardo Ambrósio caninas através de mecanismos in vivo e in FAPESP 2011 Gonçalves vitro Expansão in vitro de células estromais mesenquimais e caracterização do Dimas Tadeu Covas Amanda Mizukami FAPESP 2012 secretoma: Aplicações Terapêuticas e Biotecnológicas Caracterização Ontogênica, Biológica e Everton de Brito Funcional das Células Eritróides obtidas de Dimas Tadeu Covas CAPES 2012 Oliveira Costa Células-Tronco Embrionárias Humanas IN VITRO Análise proteômica de células estromais Lilian Figueiredo OP9 durante a diferenciação endotelial e Dimas Tadeu Covas CAPES 2010 Moreira hematopoética in vitro a partir de células- tronco embrionárias humanas Geração de tecido adiposo ectópico como Lucas Eduardo Botelho veículo Dimas Tadeu Covas CAPES 2013 de Souza secretor do fator VIII humano da coagulação sanguínea Clonagem e expressão do fator VII de Marcela Cristina Correia Dimas Tadeu Covas coagulação sanguínea em linhagens FAPESP 2011 de Freitas celulares humanas Avaliação do potencial regenerativo de exossomos derivados de célulastronco mesenquimais de cordão umbilical Priscilla Carnavale Dimas Tadeu Covas cultivadas em garrafas estáticas e em CAPES 2014 Gomes Ferreira biorreator em um modelo murino de lesão crônica de pele in vivo Tratamento de queimaduras graves com células mesenquimais estromais Eduardo Antonio Donadi Carolina Caliári Oliveira multipotentes (CMs) em modelo pré- USP/RP 2010 clínico - Desenvolvimento de biocurativo associando CMs à biomembranas Perfil de Expressão de mRNAS, miRNAS e Daiani Cristina Ciliao Eduardo Antonio Donadi de Moléculas Regulatórias da resposta FAPESP 2010 Alves Imune em Aloenxertos Renais Avaliação dos mecanismos imunológicos e hematológicos envolvidos na resposta Julia Teixeira Cottas de terapêutica de pacientes com anemia Eduardo Antonio Donadi CAPES 2013 Azevedo falciforme submetidos ao transplante alogênico de células-tronco hematopoéticas Estudo dos mecanismos imunológicos envolvidos na resposta terapêutica de Lucas Coelho Marliére Eduardo Antonio Donadi pacientes com esclerose sistêmica ao CNPQ 2013 Arruda transplante autólogo de células-tronco Hematopoéticas Estudo in vivo do papel da hipoexpressão Ana Paula Alencar de do gene CCAAT Enhancer Binding Protein Eduardo Magalhães Rego FAPESP 2011 Lima Lange alfa (CEBPA) na leucemogênese induzida pela proteína híbrida CALM/AF10 Avaliação de vias de sinalização em Mariana Tereza de Lira Eduardo Magalhães Rego células-tronco da Leucemia Mielóide CAPES 2011 Benicio Aguda de novo

138

DOCTORATE

Supervisor Student Project Title Institution Beginning Análise do mitocan alfa-tocoferol no Silvia Elena Sánchez Eduardo Magalhães Rego modelo animal de leucemia mielóide CAPES 2014 Mendoza aguda associada à mutação FLT3-ITD Efeito do veneno bruto e da L-aminoácido oxidase de Bothrops moojeni e Calloselasma rhodostoma na maquinaria Fabiola Attié de Castro Sandra Mara Burin CAPES 2011 apoptótica, expressão de miRNAs e metilação do DNA na Leucemia Mielóide Crônica Investigação do efeito do silenciamento do IRS1/IRS2 no fenótipo de células Fabíola Traína Renata Scopim Ribeiro INCTC/CAPES 2014 hematopoiéticas primárias normais CD34+ e leucêmicas BCR-ABL+ Estudo funcional de ANKHD1 na João Agostinho Fabíola Traina proliferação, apoptose e ciclo celular em FAPESP 2011 Machado Neto neoplasias hematológicas Juliano Rodrigues Reprogramação núclear em bovinos, um Flávio Vieira Meirelles FZEA-USP 2012 Sangalli modelo usando agentes demetilantes Controle e modelo do número de copias Laís Vicari de Figueiredo Flávio Vieira Meirelles de DNA mitocondrial em células bovinas: FZEA-USP 2013 Pessôa um modelo baseado na depleção Desenvolvimento de pesquisa nas áreas de Biotecnologias da Reprodução, Terapia Flávio Vieira Meirelles Pedro Ratto Lisboa Pires celular, Indução da pluripotência em FZEA-USP 2011 células mesenquimais (iPS) e diferenciação celular Relação da 5-Metilcitosina e 5- Flávio Vieira Meirelles Rafael Vilar Sampaio Hidroximetilcitosina na Reprogramação CAPES 2011 Nuclear de Bovinos Níveis de metilação, hidroximetilação e Rodrigo da Silva Nunes Flávio Vieira Meirelles modificações de histona na placenta de CAPES 2011 Barreto bovinos clonados e normais Interações celulares em ambiente tridimensional entre células híbridas José Alexandre Marzagão Karen Steponavicius dendríticas-tumorais e linfócitos humanos: CNPQ 2010 Barbuto Piedade Cruz em busca de estratégias de aprimoramento de vacina antitumoral. Estudo do efeito das células Natural Killer José Alexandre Marzagão Maria Alejandra Clavijo sobre a diferenciação in vitro de FAPESP 2010 Barbuto Salomón monócitos em células dendríticas em pacientes com câncer Efeito da melatonina sobre a José Alexandre Marzagão Roberto Pereira diferenciação, ativação e função de células FAPESP 2010 Barbuto Gonzalez dendríticas José Alexandre Marzagão Comprometimento funcional de células Barbuto dendríticas derivadas de monócitos de Bruna Zelante Barbosa pacientes com câncer: envolvimento da FAPESP 2011 via de sinalização da p38MAPK e da proteína de choque térmico HSP27 José Alexandre Marzagão Suppressors of cytokines signaling (SOCS) Barbuto na modulação funcional de células Patricia Argenta Toniolo FAPESP 2011 dendríticas derivadas de pacientes com câncer José Alexandre Marzagão Avaliação funcional de NF-kB em células Barbuto Isabella Katz Migliori dendríticas de pacientes com câncer de FAPESP 2011 mama José Alexandre Marzagão Estudo de mecanismos envolvidos na Barbuto modulação da resposta de linfócitos T Rodrigo Nalio Ramos contra antígenos tumorais por células FAPESP 2011 dendríticas derivadas de monócitos de pacientes com câncer Avaliação do potencial de crescimento e produção de proteínas recombinantes de Kamilla Swietch Antonietto Rafael Tagé Biaggio células humanas adaptadas para FAPESP 2014 crescimento em suspensão e meios de cultura livres de soro fetal bovino

139

DOCTORATE

Supervisor Student Project Title Institution Beginning Kelen Cristina Ribeiro Avaliação da Função Tímica em pacientes Luciana Ribeiro Jarduli CAPES 2014 Malmegrim de Farias com Anemia Falciforme Estado Epigenético do Cromossomo X em Joana Carvalho Moreira Lygia da Veiga Pereira Tecidos Extra Embrionários Humanos e FAPESP 2010 de Mello Bovinos Manipulação de vias inibitórias da indução Marco Antonio Zago Mariane Serra Fraguas de pluripotência visando o aumento de FAPESP 2010 eficiência no processo de geração de iPSs Interação entre células-tronco de polpa Maria Angélica Miglino Dayane Alcantara dentária imatura e o osteossarcoma FAPESP 2011 canino

Morfologia e ultra-estrutura dos órgãos Fernanda Menezes de Maria Angélica Miglino linfáticos e hematopoiéticos de cetáceos FAPESP 2012 Oliveira e Silva (Ordem Cetacea, Subordem Odontoceti).

Horácio Luis Pinto Utilização de células tronco nos processos Maria Angélica Miglino CAPES 2011 Tommasi Júnior de reparação tecidual em cães

Potencial terapêutico de células derivadas Marcio Nogueira Maria Angélica Miglino do órgão vômeronasal de coelhos da raça FAPESP 2011 Rodrigues Nova Zelândia

Anosmia: Recuperação da função olfatória Maria Angélica Miglino Rafael Cardoso Carvalho FAPEMA 2010 por terapia celular

Terapia Celular aplicada à reconstituição Maria Angélica Miglino Valdir Pavanelo Junior FAPESP 2012 da calota craniana em modelos primatas

Geração de células pluripotentes induzidas Patricia Cristina Baleeiro a partir de células epiteliais de mucosa Fabiele Baldino Russo FAPESP 2011 Beltrão Braga oral humana de pacientes com desordem do espectro autista Papel da sinalização da adenosina na Rodrigo Alexandre Helder Teixeira de geração de células T regulatórias a partir CAPES 2014 Panepucci Freitas de células T naive O papel dos microRNAs na proliferação e Rodrigo Alexandre Ildercilio Mota de Souza diferenciação celular da linhagem CAPES 2013 Panepucci Lima pluripotente de carcinoma embrionário humano NTera-2 Análise genômica do papel dos mecanismos epigenéticos de metilação de Rodrigo Alexandre Josiane Lilian dos regiões promotoras durante a geração in FAPESP 2011 Panepucci Santos Schiavinato vitro de células T regulatórias induzidas (iTreg) a partir de células T naive de sangue de cordão umbilical Células mesenquimais estromais Rodrigo do Tocantins Calado Diego Villa Clé multipotentes da medula óssea no FMRP-USP 2010 Saloma Rodrigues tratamento da anemia aplástica refratária Identificação de Moduladores Genético da Rodrigo do Tocantins Calado Fernanda Gutierrez Resposta a Imunossupressão na anemia CAPES 2014 Saloma Rodrigues Rodrigues aplástica por sequenciamento de nova geração Diferenciação em sangue, hepatócitos e Rodrigo do Tocantins Calado Flávia Sacilotto neurônios de células-tronco pluripotentes FAPESP 2011 Saloma Rodrigues Donaires induzidas (iPS) de pacientes com doenças dos telômeros Desenvolvimento de células-tronco Rodrigo do Tocantins Calado Leonardo Campos pluripotentes induzidas (iPSCs) para o FAPESP 2014 Saloma Rodrigues Zanelatto estudo da etiologia e tratamento da anemia de Fanconi Análise do comprimento telomérico em Rodrigo do Tocantins Calado Pedro Henrique Padilha cromossomos de pacientes com Síndrome FMRP-USP 2013 Saloma Rodrigues Mielodisplásicas com mutação nos genes

140

DOCTORATE

Supervisor Student Project Title Institution Beginning Avaliação do efeito dos exossomos na Simone Kashima Haddad Katia Kaori Otaguiri infecção pelo vírus linfotrópico humano de CAPES 2014 células T do tipo 1 (HTLV-1) Avaliação dos fatores de transcrição indutores da transição epitélio- Simone Kashima Haddad Mariana Tomazini Pinto FAPESP 2012 mesenquimal (EMT) na Biologia das Células Endoteliais Desenvolvimento de uma plataforma Mauricio Cristiano Simone Kashima Haddad molecular in house para o Diagnóstico FAPESP 2011 Rocha Júnior Confirmatório da infecção pelo HTLV Valtencir Zucolotto Estudo da Interação de Nanotubos e Thiers Massami Uehara Nanopartículas com Membranas e CAPES 2010 Modelos de Membranas Celulares Valtencir Zucolotto Imobilização de nanotubos de carbono e Lilian Maria Pessoa da metaloftalocianinas em filmes Instituto de Física 2010 Cruz Centurion nanoestruturados: caracterização e de São Carlos aplicação em sensores Valtencir Zucolotto Estudo da imobilização de enzimas em Jaciara Cássia de filmes nanoestruturados para aplicação CAPES 2012 Carvalho Santos em biossensores: caracterização óptica, estrutural e eletroquímica Valtencir Zucolotto Desenvolvimento de Nanopartículas Camilo Arturo Superparamagnéticas para Aplicações em CAPES 2012 Ballestero Suarez Nanomedicina Valtencir Zucolotto Nanomaterias complexados com Proteínas Valeria Spolon para Aplicação em Diagnóstico e Terapia CAPES 2012 Marangoni em Nanomedicina Identificação de marcadores genéticos de Wilson Araújo da Silva Júnior Aline Simoneti Fonseca diagnóstico e prognóstico aplicados ao CAPES 2010 carcinoma colorretal Wilson Araújo da Silva Júnior Bruna Rodrigues Muys Genética molecular do câncer CAPES 2013 Análise do perfil genotípico de pacientes Wilson Araújo da Silva Júnior Daniel Fantozzi Garcia com galactosemia clássica e estudo da USP/RP 2010 relação do genótipo-fenótipo Estudo do metabolismo energético na Wilson Araújo da Silva Júnior Luiza Ferreira de Araujo progressão do melanoma com base na CAPES 2013 instabilidade do genoma mitocondrial Assinatura de expressão gênica durante o Rafaela de Barros e Wilson Araújo da Silva Júnior desenvolvimento do carcinoma CNPQ 2010 Lima Bueno espinocelular de laringe

POST–DOCTORAL AND JUNIOR POST-DOCTORAL

Supervisor Student Project Title Institution Beginning Desenvolvimento de plataforma molecular para o diagnóstico Dimas Tadeu Covas Svetoslav Nanev Slavov CAPES 2014 confirmatório e discriminatório da infecção pelo HTLV-1/2 Correção fenotípica da hemofilia A humana utilizando terapia celular baseada Dimas Tadeu Covas Aline Fernanda Ferreira INCTC/CNPQ 2014 em células-tronco pluripotentes induzidas (iPS) Análise funcional e proteômica das células Antonio Roberto Lucena progenitoras Eduardo Magalhães Rego FAPESP 2013 de Araujo hematopoiéticas do modelo animal de disceratose congênita Hamilton Luiz Gimenes Estudo da via mitocondrial alternativa no Eduardo Magalhães Rego FCFRP- USP 2011 Teixeira fungo Aspergillus fumigatus Desenvolvimento de técnica por Priscila Santos citometria de fluxo baseada em FRET para Eduardo Magalhães Rego FAPESP 2011 Scheucher diagnóstico de Leucemia Promielocítica Aguda Fabiana Fernandes Atuação na área de biotecnologia, biologia Flávio Vieira Meirelles FZEA-USP 2013 Bressan celular e biologia molecular

141

POST–DOCTORAL AND JUNIOR POST-DOCTORAL

Supervisor Student Project Title Institution Beginning Reprodução Animal, Biotecnologia da Jose Rodrigo Valim Reprodução, Clones bovinos, Flávio Vieira Meirelles CNPq 2012 Pimentel sincronização do estro, obstetricia e melhoramento genético Efeito do alquilfosfolipídio, perifosine sobre composição e quantidade relativa Germano Aguiar Lewis Joel Greene de proteínas palmitoiladas em frações CNPQ 2014 Ferreira rafts de células da linhagem Granta-519 de linfoma de células do manto Reprogramação de células sanguíneas Maximiliano Carlos Lygia da Veiga Pereira humanas periféricas em células-tronco FAPESP 2013 Dasso pluripotentes induzidas, Pesquisa com avaliação de atividade de Francisco de Paula biológica de extratos vegetais em cultivo Marco Antonio Zago FAPESP 2012 Careta de células e com identificação molecular por análise de DNA Utilização de células de saco vitelino canino com VEGF para o tratamento in vitro da Distrofia Muscular de Duchenne Maria Angélica Miglino Paula Fratini CAPES 2013 no modelo GRMD (Golden Retriever Muscular Dystrophy)

Padrões de expressão gênica em tecidos Maria Angélica Miglino Phelipe Oliveira Favaron placentários e fetais durante a gestação de FAPESP 2013 Galea spixii (Rodentia, Caviomorpha) Patricia Cristina Baleeiro Graciela Conceição Constituição de banco público de células CAPES 2014 Beltrão Braga Pignatari de pluripotência induzida Rodrigo do Tocantins Calado Raquel de Melo Alves Faculdade de Medicina de Ribeirao Preto CNPq 2012 de Saloma Rodrigues Paiva da Universidade de São Paulo Valtencir Zucolotto Marcia Regina Moura Aplicação de nanopartículas em filmes FAPESP 2009 Auoada utilizados para embalagens em alimentos Caracterização de Miosina Va como um Harvard Medical Wilson Araújo da Silva Júnior Cleidson de Pádua Alves novo alvo de MITF com envolvimento em 2012 School tumorigenese e invasão David Santos Marco Pesquisa em Genomica Funcional do Pró-Reitoria de Wilson Araújo da Silva Júnior 2013 Antonio câncer com ênfase em Bioinformática Pesquisa-USP Alteração do perfil de expressão gênica Wilson Araújo da Silva Júnior Dalila Luciola Zanette induzida pela expansão in vitro de células CAPES 2013 precursoras hematopoéticas Análise da expressão de miRNAs em CNPq - Rockefeller Julio César Cetrulo Wilson Araújo da Silva Júnior subpopulações de linfócitos T em University- Estados 2014 Lorenzi pacientes com Esclerose Múltipla Unidos Thiago Yukio Kikuchi Biologia Computacional, Genética Rockefeller Wilson Araújo da Silva Júnior 2014 Oliveira Molecular e Evolução Molecular University-NY

142

Appendix VI – Relation of the Projects Funded in the last 5 years

VALUE VALIDITY SOURCE COORDINATOR PROJECT AGREEMENT (R$)

Isolamento, Caracterização, Cultura, Expansão e Avaliação do Potencial Vasculogênico in vitro e in vivo de 01/03/2008 CNPq DIMAS TADEU COVAS 558137/2008-3 54.158,80 Células Tronco Pluripotenciais do 01/03/2009 Adulto com Capacidade de Diferenciação 01/01/2009 FAPESP DIMAS TADEU COVAS Sala Retrô - "O Básico nas Ciências" 2008/55162-7 8.750,00 31/03/2009 Clonagem e Expressão dos fatores 21/11/2005 FINEP DIMAS TADEU COVAS VIII e IX de Coagulação Sanguínea 2556/2005 370.000,00 21/05/2009 em Células de Mamífero Análise Biologica e Molecular de Células Tronco Somáticas 04/12/2007 FINEP DIMAS TADEU COVAS 0156/07 434.050,00 Pluripotenciais no Reparo Tecidual 04/12/2009 CTCPLURI Avaliação da Expressão Gênica de Células Progenitoras Endoteliais CNPq DIMAS TADEU COVAS 480770/2007-7 56.000,00 20/12/2009 Isoladas a partir de Sangue de Cordão Umbilical e Medula Óssea Proteômica Estrutural e Funcional 14/12/2007 FINEP DIMAS TADEU COVAS 0875/07 720.000,00 aplicada à Área Biomédica 14/06/2010 Avaliação da Expressão Gênica de Células Progenitoras Endoteliais 01/08/2008 CNPq DIMAS TADEU COVAS 501541/2008-0 11.592,24 Isoladas a partir de Sangue de 01/08/2010 Cordão Umbilical e Medula óssea Consolidação do Laboratório de Cultura Celular para Estudos 26/08/2005 FINEP DIMAS TADEU COVAS 0777/05 522.082,00 Moleculares e Proteômicos de 26/08/2010 Células Dendríticas e Células Tronco Infra Estrutura para Geração e Manipulação de Modelos Animais 25/09/2006 FINEP DIMAS TADEU COVAS 1081/06 742.800,00 Geneticamente Modificados no 25/09/2010 Estudo de Doenças Humanas Geração de Células Tronco Pluripotentes Induzidas (IPS) 18/02/2008 CNPq DIMAS TADEU COVAS 573763/2008-9 180.000,00 Humanas utilizando Vetores 17/02/2012 Lentivirais e Adenovirais Banco de Sangue de Cordão 13/11/2008 BNDES DIMAS TADEU COVAS Umbilical - Aquisição de 08.2.0752-1 1.094.220,00 13/11/2012 Equipamentos Consolidação do Laboratório de Experimentação Animal para 18/12/2008 FINEP DIMAS TADEU COVAS 0238/08 532.613,30 Estudos Funcionais das Células 18/12/2012 Tronco Mesenquimais e Pericitos DISSEMINAÇÃO E DIFUSÃO DE UM SISTEMA MINIMAMENTE INVASIVO 23/02/2011 OPAS DIMAS TADEU COVAS /1100014.001 440.000,00 PARA MONITORAMENTO DE 28/12/2012 PARÂMETROS MÉDICOS Overespression Of EMT Genes in Endothelial Cells During Endothelial 01/01/2011 FAPESP DIMAS TADEU COVAS 2010/51962-9 28.480,00 Msesenchymal Transition ( 31/12/2012 Fapesp/MIT) Escalonamento da Produção dos Fatores VIII e IX de Recombinantes 19/12/2007 FINEP DIMAS TADEU COVAS em Biorreatores e Ensaios Pré- 0927/07 2.787.122,38 19/12/2013 Clínicos em Camundongos Hemofílicos Estudo da transição endotélio- 10/02/2012 CNPq DIMAS TADEU COVAS mesenquimal (ENDMT) na Biologia 480521/2011-5 106.107,00 09/02/2014 de Células Endoteliais e no Câncer

143

VALUE VALIDITY SOURCE COORDINATOR PROJECT AGREEMENT (R$)

TRATAMENTO DA DOENÇA DO ENXERTO-CONTRA-O-HOSPEDEIRO AGUDA, CÓRTICO-REFRATÁRIA 03/12/2012 CNPq DIMAS TADEU COVAS 404622/2012-7 760.000,00 COM CÉLULAS ESTROMAIS 02/12/2014 MESENQUIMAIS OBTIDAS DO CORDÃO UMBILICAL PÓS DOC SUS 1989/2009- PROJETO 036/2009 - EDITAL 12/2009 - ANÁLISE FUNCIONAL E DA EXPRESSÃO GÊNICA EM LARGA ESCALA DE CÉLULAS-TRONCO 27/11/2012 CAPES DIMAS TADEU COVAS HEMATOPOÉTICAS E 036/2009 300.000,00 09/12/2014 MESENQUIMAIS DE PACIENTES COM DIABETES MELLITUS TIPO 1 E ESCLEROSE MÚLTIPLA SUBMETIDOS AO TRANSPLANTE DE CÉLULA- TRONCO HEMATOPOÉTICAS Anemia Falciforme: bases 08/01/2014 CAPES DIMAS TADEU COVAS 795/2014 26.200,00 genéticas da variabilidade 31/12/2015 fenotípica ESTRUTURAÇÃO DE REDE DE 18/03/2010 FINEP DIMAS TADEU COVAS LABORATÓRIOS DE SANGUE E 01.10.0124.00 4.499.703,00 18/03/2015 HEMODERIVADOS DESENVOLVIMENTO DE PLATAFORMA MOLECULAR PARA O 16/05/2012 FINEP DIMAS TADEU COVAS DIAGNÓSTICO CONFIRMATÓRIO E 01.12.0160.00 1.016.200,00 15/05/2015 DISCRIMINATÓRIO DA INFECÇÃO PELO HTLV-1/2" PRODUÇÃO DE CÉLULAS TRONCO MESENQUIMAIS DA MEDULA 23/12/2008 FINEP DIMAS TADEU COVAS ÓSSEA E DA VEIA UMBILICAL EM 01.08.0591.00 325.451,16 23/12/2015 LARGA ESCALA PARA FINS TERAPÊUTICOS PRODUÇÃO DE CÉLULAS TRONCO MESENQUIMAIS DA MEDULA 23/12/2009 BNDES DIMAS TADEU COVAS ÓSSEA E DA VEIA UMBILICAL EM 09.2.0706.1 3.574.645,00 23/12/2015 LARGA ESCALA PARA FINS TERAPÊUTICOS VALIDAÇÃO E UTLIZAÇÃO DOS MÉTODOS MINIMAMENTE OPAS 29/11/2013 OPAS DIMAS TADEU COVAS INVASIVOS E NÃO INVASIVOS PARA 1.404.900,00 BR/LOA/1300067.001 29/10/2015 MONITORAR A PRESSÃO INTRACRANIANA Diversidade genética de HLA-G e 2008 correlação dos polimorfismos com CNPQ Eduardo Antonio Donadi 558476/2008-2 281.300,00 expressão de HLA-G no carcinoma 2013 urotelial de bexiga 1.146.000,00 2009 Controle do transcriptoma no FAPESP Eduardo Antonio Donadi 08/56594-8 diabetes 2013 Associação entre a expressão de 2009 CAPES/ Eduardo Antonio Donadi HLA-G e seu polimorfismo em 653/09 490.000,00 COFECUB cancerologia e transplantes 2013 2011 Regulação transcricional e pós- CNPQ Eduardo Antonio Donadi 105.000,00 transcricional do gene HLA-G. 2013 Marcadores transcricionais de 2012 780.000,00. CNPQ Eduardo Antonio Donadi predição de complicações clínicas 563731/2010-9 no diabetes mellitus 2014

144

VALUE VALIDITY SOURCE COORDINATOR PROJECT AGREEMENT (R$)

Avaliação de genes de histocompatibilidade HLA-DRB1, DQB1, DQA1, HLA-A, -B, -C, -E, -F e 105.000,00. 2014 CNPQ Eduardo Antonio Donadi –G e de seus elementos de 4760/2013-5 regulação pós-transcricional em 2017 pacientes com diabetes mellitus tipo 1 e diabetes gestacional Polimorfismos e mecanismos pós- transcricionais de regulação dos 2013 CNPQ Eduardo Antonio Donadi genes HLA-G e BRAF associados 457231/2013-9 469.000,00 com a malignidade do carcinoma 2014 papilífero de tireoide Contribuição de moléculas imunorregulatórias HLA-G, HLA-E, 2014 CAPES/PROCA Foxp-3, PD-1 e IL-17 no câncer, 750.000,00. Eduardo Antonio Donadi 177382/2014 D polimorfismos gênicos, regulação 2017 de microRNAs e perfis da resposta imune 2014 CNPQ Eduardo Antonio Donadi Philippe Moreau 406594/2013 172.000,00 2016 Sequenciador Next generation CAPES Eduardo Antonio Donadi 176.000,00 2011 (Illumina) ESPCA - Escola São Paulo de Ciência 17/02/2014 FAPESP EDUARDO MAGALHÃES REGO 2013/50552-0 450.600,00 Avançada - Oncogenese 30/05/2014 TERAPIA DO DIABETES MELLITUS COM CÉLULAS-TRONCO 24/05/2013 CNPq EDUARDO MAGALHÃES REGO MESENQUIMAIS: AVALIAÇÃO DA 550642/2012-9 62.657,70 24/12/2014 RESPOSTA CLÍNICA E INVESTIGAÇÃO DOS MECANISMOS DE AÇÃO INFRA-USP/2012-2013- ADEQUAÇÃO DO CENTRO DE 01/04/2013 USP EDUARDO MAGALHÃES REGO 360/2012 210.000,00 ONCOLOGIA, CÉLULAS-TRONCO E 31/03/2016 TERAPIA CELULAR PESQUISA DE MUTAÇÕES ADICIONAIS AO REARRANJO PML/RaRa POR MEIO DE ANÁLISE DO TRANSCRIPTOMA, EXOMA 12/11/2013 CNPq EDUARDO MAGALHÃES REGO COMPLETO E METILOMA EM 481949/2013-5 80.000,00 11/11/2016 AMOSTRAS DE LEUCEMIA PROMIELOCITICA AGUDA NO DIAGNÓSTICO, REMISSÃO MOLECULAR E RECAÍDA EFEITO DA QUANTIDADE DE MITOCONDRIAS E DE DNA 01/02/2007 MITOCONDRIAL SOBRE O FAPESP FLÁVIO MEIRELLES 2006/59074-0 191.095,42 DESENVOLVIMENTO EMBRIONARIO 31/01/2009 BOVINO: DOIS MODELOS ORIGINAIS. Células dendríticas: elementos 01/06/2010 FAPESP José Alexandre M. Barbuto integradores do sistema imune – 2009/54599-5 1.328.730,00 enfoque aplicado 31/05/2014 Planejamento racional e desenvolvimento de novos protótipos derivados de fosfolipídios antitumorais como 01/11/2013 inibidores potenciais da enzima FAPESP José Alexandre M. Barbuto 2013/072773-2 434.000,00 CTP:fosfoetanolamina 31/10/2015 citidililtransferase e agentes antitumorais em carcinoma de pulmão do tipo não pequenas células. Análise da expressão gênica em KELEN CRISTINA MALMEGRIM larga escla de células tronco 26/11/2008 CNPq 477678/2008-4 16.000,00 FARIAS mesenquimais de pacientes com 26/12/2011 esclerose múltipla

145

VALUE VALIDITY SOURCE COORDINATOR PROJECT AGREEMENT (R$)

Avaliação do potencial terapêutico FAPESP LYGIA PEREIRA de Ramipril em um modelo murino 2011/01477-0 270.000,00 30/4/2014 da Síndrome de Marfan. Validação de microRNAs candidatos 1/7/2013 FAPESP LYGIA PEREIRA à regulação da expressão da DNA 2013/05583-4 150.000,00 30/6/2015 metiltransferase 3B (DNMT3B) ESTUDO DOS MECANISMOS PATOGENICOS DA EPILEPSIA EM 1/12/2012 CNPq LYGIA PEREIRA UM MODELO IN VITRO DE CÉLULAS 401820/2012-2 300.000,00 30/11/2015 TRONCO PLURIPOTENTES HUMANAS (hiPSCs). Analysing Interindividual Differences In Drug Exposure Using 1/3/2014 CNPq LYGIA PEREIRA 405494/2013-0 339.860,59 Human Induced Pluripotent Stem 28/2/2017 Cells Constituição de um Banco Nacional de Células Tronco de Pluripotência 1/11/2013 CNPq LYGIA PEREIRA 420139/2013-3 1.500.000,00 Induzida Paciente-Específico - grupo 30/10/2015 São Paulo Laboratório Nacional de Células 1/3/2010 BNDES LYGIA PEREIRA 2.800.000,00 Tronco Pluripotentes 1/7/2015 RTI/Parcela de Reserva Técnica para Custos de Infra-Estrutura 01/08/2007 FAPESP MARCO ANTONIO ZAGO Institucional para pesquisa- 2007/54816-0 129.955,01 31/05/2010 Exercício 2007 - Adequação da área de Ensino da Casa da Ciência. Parcela de Reserva Técnica Institucional para Conectividade à 01/12/2009 FAPESP MARCO ANTONIO ZAGO 2009/51396-6 21.536,00 Rede ANSP - Adequação da Rede de 30/11/2010 Informática RTI/ Parcela da Reserva Técnica para Custos com Infra-Estrutura Intitucional para Pesquisa - 01/11/2008 FAPESP MARCO ANTONIO ZAGO 2008/57506-5 251.179,00 Exercício 2008 - Adequação dos 31/03/2011 laboratórios de pesquisa (pisos/bancadas e pintura) RTI - Parcela da Reserva Técnica 01/12/2009 FAPESP MARCO ANTONIO ZAGO para Infra-Estrutura Institucional da 2009/51531-0 211.652,00 31/05/2011 Pesquisa - Exercício 2009 RTI - Parcela da Reserva Técnica para Conectividade à Rede Ansp - 01/01/2011 FAPESP MARCO ANTONIO ZAGO 2010/52556-4 27.927,00 Exercício 2010 - (Adequação da 31/12/2011 Rede de Informática) RTI/ Parcela da Reserva Técnica para Custos com Infra-Estrutura Intitucional para Pesquisa - 01/02/2011 FAPESP MARCO ANTONIO ZAGO 2010/52555-8 265.411,00 Exercício 2010 - Adequação dos 31/07/2012 laboratórios de pesquisa e modernização da Rede do CPD 01/10/2000 FAPESP MARCO ANTONIO ZAGO CTC - Centro de Terapia Celular 1998/14247-6 34.713.144,59 31/12/2012 RTI - Parcela da Reserva Técnica para Conectividade à Rede Ansp - 01/02/2012 FAPESP MARCO ANTONIO ZAGO 2011/51892-3 36.486,00 Exercício 2011 - (Adequação da 31/01/2013 Rede de Informática) RTI - Parcela da Reserva Técnica para Infra-Estrutura Institucional de Pesquisa - Exercício 2011 - 01/03/2012 FAPESP MARCO ANTONIO ZAGO 2011/51891-7 335.655,00 (Adequação dos Laboratórios de 31/07/2013 Pesquisa: Serviços de Instalações Elétricas) Caracterização do papel do locus 24/03/2011 CNPq MARCO ANTONIO ZAGO Dido na diferenciação de células- 560884/2010-9 157.000,00 23/03/2014 tronco e em neoplasias mieloides

146

VALUE VALIDITY SOURCE COORDINATOR PROJECT AGREEMENT (R$)

RT ANSP - 2008 - Adequação da 01/04/2013 FAPESP MARCO ANTONIO ZAGO 2012/51561-0 25.118,00 Rede de Informática 31/03/2014 CTC-CENTRO DE TERAPIA CELULAR - 01/06/2013 FAPESP MARCO ANTONIO ZAGO 2013/08135-2 23.456.276,90 CEPID 31/05/2018 INCTC- INSTITUTO NACIONAL DE CIÊNCIAS E TECNOLOGIA EM 01/03/2009 FAPESP ROBERTO PASSETTO FALCÃO 2008/57877-3 3.575.321,75 CÉLULAS TRONCO E TERAPIA 28/02/2015 CELULAR INCTC- INSTITUTO NACIONAL DE CIÊNCIAS E TECNOLOGIA EM 17/04/2009 CNPq ROBERTO PASSETTO FALCÃO 573754/2008-0 4.877.284,18 CÉLULAS TRONCO E TERAPIA 16/04/2015 CELULAR Avaliação Funcional do Papel dos microRNAs Durante a Geração de 2014 CNPq Rodrigo Alexandre Panepucci 484152/2013-0 30.000,00 Células T Regulatórias Induzidas 2016 (iTregs) Estudo de Mecanismos Regulatórios no Processo de Geração in-vitro de Células T Regulatórias Induzidas 2011 CNPq Rodrigo Alexandre Panepucci (iTreg) a partir de Células Naive de 484319/2010-8 45.000,00 2013 Sangue de Cordão Umbilical: Papel da Via Canônica e Não-Canônica de NF-kB. RODRIGO DO TOCANTINS HORMÔNIO MASCULINO NO 24/09/2013 CNPq CALADO DE SALOMA TRATAMENTO DE DOENÇAS DOS 401446/2013-1 90.136,00 23/09/2016 RODRIGUES TELÔMEROS DISFUNÇÃO TELOMÉRICA NA RODRIGO DO TOCANTINS CIRROSE HEPÁTICA E 11/11/2013 CNPq CALADO DE SALOMA 481389/2013-0 60.000,00 HEPATOCARCINOMA - EDITAL Nº 10/11/2016 RODRIGUES 14/2013 Função das Células Mesenquimais 05/02/2010 CNPq SIMONE KASHIMA HADDAD Estromais Humanas em Supressão 477126/2009-0 37.500,00 25/11/2011 de Genes Virais do HTLV-1 PPSUS - DESENVOLVIMENTO DE PLATAFORMA MOLECULAR PARA 01/07/2013 FAPESP SIMONE KASHIMA HADDAD DETECÇÃO DO VIRUS DA HEPATITE 2012/51735-8 270.890,00 30/06/2015 B (HBV) PARA TRIAGEM DE DOADORES DE SANGUE Desenvolvimento e Estudo das Propriedades de Materiais CNPq 2010 Valtencir Zucolotto Nanoestruturados conjgados com 504402/2010-2 8.640,00 2012 Sistemas Biológicos: Aplicações em Nanomedicina. Estudo da Interação entre Materiais Nanoestruturados e Sistemas 05/2009 FAPESP Valtencir Zucolotto Biológicos: Aplicações ao Estudo de 2008/08639-2 311.238,58 05/2012 NanoToxicidade e Desenvolvimento de Sensores para Diagnóstico Desenvolvimento de Biossensores de Altas Seletividade e Sensibilidade para Detecção e CNPq 03/2009 Valtencir Zucolotto Diagnóstico da Febre Aftosa no 578591/2008-1 360.000,00 03/2012 Brazil, e Possibilidade de Monitoramento do Processo de Vacinação Desenvolvimento de Nanocompósitos contendo 03/2009 CNPq Valtencir Zucolotto Materiais Nanoestruturados e 575288/2008-6 87.000,00 03/2012 Biomoléculas: Estudos Fundamentais e Aplicações Interações entre Nanomateriais e Filmes Finos contendo 03/2009 CNPq Valtencir Zucolotto 478074/2008-5 38.723,28 Biomoléculas: Estudos 03/2011 Fundamentais e Aplicações

147

VALUE VALIDITY SOURCE COORDINATOR PROJECT AGREEMENT (R$)

Estudo da Imobilização de Biomoléculas em Nanocompósitos 03/2007 FAPESP Valtencir Zucolotto 2007/00607-1 150.000,00 Poliméricos: Controle Molecular e 03/2009 Aplicações em Biossensores Incorporação de Nanotubos de 2007 CNPq Valtencir Zucolotto Carbono e Ftalocianinas Metálicas 503111/2007-4 10.800,00 2010 em Filmes Finos Automontados Estudo da Interação de 03/2013 Nanopartículas com Moléculas CNPq Valtencir Zucolotto 477525/2012-1 108.000,00 biológicas e Células Humanas: 03/2015 Aplicação em Nanomedicina Avaliando a Interação entre 01/2011 Nanopartículas e Sistemas Biológicos: Estudo da CAPES Valtencir Zucolotto 2952/2010 774.000,00 01/2016 Nanotoxicidade e Possíveis Aplicações Terapêuticas em

Nanomedicina. Avaliação da Toxicidade de Nanomateriais Aplicados em 12/2011 Medicina e Agricultura: CNPq Valtencir Zucolotto 552124/2011-6 502.000,00 Desenvolvimento de Estudos in 12/2014 vivo, in vitro e em Modelos de Membrana Efeito do Hormônio Adiponectina no Risco do Desenvolvimento do 12/2011 Diabetes mellitus tipo 2: Avaliação CNPq Valtencir Zucolotto 563836/2010-5 611.000,00 clínico-laboratorial e Determinação 12/2014 do Diagnóstico Precoce por dispositivos de Biossensoriamento Novos Nanomateriais para 05/2013 Aplicação no Diagnóstico e Terapia INFRA-USP Valtencir Zucolotto 520.850,00 do Câncer e Estudos de 05/2016 Nanotoxicidade Avanços, Benefícios e Riscos da 09/2009 CAPES Valtencir Zucolotto Nanobiotecnologia Aplicada à 705/2009 2.400.000,00 Saúde 09/2014 Nanopartículas de prata aplicadas ao desenvolvimento de cateteres 09/2009 intraluminais: avaliação in vitro da CAPES Valtencir Zucolotto colonização de microrganismos e 858/2009 2.400.000,00 09/2014 estudo do impacto no choque séptico em modelo experimental in vivo Centro de Inovação em Diagnóstico 2011 e Terapias: Convergência de PRP/USP Valtencir Zucolotto 3.000.000,00 Ciências da Vida, Física e 2014 Engenharias Análise da Expressão Gênica durante a diferenciação 04/12/2008 CNPq WILSON ARAUJO SILVA JUNIOR Osteogênica em Amostras de 481393/2008-0 89.820,00 03/12/2010 Pacientes Portadores de Osteogênese Imperfeita Análise Anatomo-Funcional do 12/12/2008 CNPq WILSON ARAUJO SILVA JUNIOR Genoma de uma linhagem de 577600/2008-7 190.111,00 10/06/2012 carcinoma ductal de mama PROSUL - Cooperação Internacional em Ciência, Tecnologia e Inovação - 17/11/2009 CNPq WILSON ARAUJO SILVA JUNIOR 490748/2008-2 641.911,60 Sequenciamento do Transcriptoma 30/09/2012 da Hevea Braziliensis

TOTAL: 111.788.885,48

148

REFERENCE

1. Sharma SV, Lee DY, Li B, Quinlan MP, Takahashi F, Maheswaran S, et al. A chromatin- mediated reversible drug-tolerant state in cancer cell subpopulations. Cell. 2010;141(1):69- 80. 2. Wang JC, Dick JE. Cancer stem cells: lessons from leukemia. Trends in cell biology. 2005;15(9):494-501. 3. Trumpp A, Wiestler OD. Mechanisms of Disease: cancer stem cells--targeting the evil twin. Nature clinical practice Oncology. 2008;5(6):337-47. 4. Shevach EM. Regulatory T cells in autoimmmunity*. Annual review of immunology. 2000;18:423-49. 5. Maloy KJ, Powrie F. Regulatory T cells in the control of immune pathology. Nature immunology. 2001;2(9):816-22. 6. Riley JL, June CH, Blazar BR. Human T regulatory cell therapy: take a billion or so and call me in the morning. Immunity. 2009;30(5):656-65. 7. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nature medicine. 1997;3(7):730-7. 8. Kim CF, Jackson EL, Woolfenden AE, Lawrence S, Babar I, Vogel S, et al. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell. 2005;121(6):823-35. 9. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 1994;367(6464):645-8. 10. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001;414(6859):105-11. 11. Scaffidi P, Misteli T. In vitro generation of human cells with cancer stem cell properties. Nature cell biology. 2011;13(9):1051-61. 12. Noushmehr H, Weisenberger DJ, Diefes K, Phillips HS, Pujara K, Berman BP, et al. Identification of a CpG island methylator phenotype that defines a distinct subgroup of glioma. Cancer cell. 2010;17(5):510-22. 13. Couzin-Frankel J. Breakthrough of the year 2013. Cancer immunotherapy. Science. 2013;342(6165):1432-3. 14. Wood JH, Partrick DA, Johnston RB, Jr. The inflammatory response to injury in children. Current opinion in pediatrics. 2010;22(3):315-20. 15. Restifo NP, Dudley ME, Rosenberg SA. Adoptive immunotherapy for cancer: harnessing the T cell response. Nature reviews Immunology. 2012;12(4):269-81. 16. Ramos RN, de Moraes CJ, Zelante B, Barbuto JA. What are the molecules involved in regulatory T-cells induction by dendritic cells in cancer? Clinical & developmental immunology. 2013;2013:806025. 17. Dunn SE, Bhat R, Straus DS, Sobel RA, Axtell R, Johnson A, et al. Peroxisome proliferator-activated receptor delta limits the expansion of pathogenic Th cells during central nervous system autoimmunity. The Journal of experimental medicine. 2010;207(8):1599-608. 18. Ohm JE, Carbone DP. VEGF as a mediator of tumor-associated immunodeficiency. Immunologic research. 2001;23(2-3):263-72. 19. Lissoni P, Fumagalli L, Paolorossi F, Mandala M. Changes in lymphocyte number during cancer chemotherapy and their relation to clinical response. The International journal of biological markers. 1999;14(2):115-7. 20. Kusmartsev S, Gabrilovich DI. Immature myeloid cells and cancer-associated immune suppression. Cancer immunology, immunotherapy : CII. 2002;51(6):293-8.

149

21. Baleeiro RB, Barbuto JA. Local secretion/shedding of tumor-derived CD83 molecules as a novel tumor escape mechanism. Molecular immunology. 2008;45(12):3502-4. 22. Baleeiro RB, Bergami-Santos PC, Tomiyoshi MY, Gross JL, Haddad F, Pinto CA, et al. Expression of a dendritic cell maturation marker CD83 on tumor cells from lung cancer patients and several human tumor cell lines: is there a biological meaning behind it? Cancer immunology, immunotherapy : CII. 2008;57(2):265-70. 23. Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. The Journal of experimental medicine. 1994;179(4):1109-18. 24. Barbuto JA, Ensina LF, Neves AR, Bergami-Santos P, Leite KR, Marques R, et al. Dendritic cell-tumor cell hybrid vaccination for metastatic cancer. Cancer immunology, immunotherapy : CII. 2004;53(12):1111-8. 25. Ramos RN, Chin LS, Dos Santos AP, Bergami-Santos PC, Laginha F, Barbuto JA. Monocyte-derived dendritic cells from breast cancer patients are biased to induce CD4+CD25+Foxp3+ regulatory T cells. Journal of leukocyte biology. 2012;92(3):673-82. 26. Knorr DA, Ni Z, Hermanson D, Hexum MK, Bendzick L, Cooper LJ, et al. Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy. Stem cells translational medicine. 2013;2(4):274-83. 27. Schmitt TM, de Pooter RF, Gronski MA, Cho SK, Ohashi PS, Zuniga-Pflucker JC. Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro. Nature immunology. 2004;5(4):410-7. 28. June CH, Maus MV, Plesa G, Johnson LA, Zhao Y, Levine BL, et al. Engineered T cells for cancer therapy. Cancer immunology, immunotherapy : CII. 2014;63(9):969-75. 29. Adamson PJ, Zola H, Nicholson IC, Pilkington G, Hohmann A. Antibody against CD20 in patients with B cell malignancy. Leukemia research. 2001;25(12):1047-50. 30. Leget GA, Czuczman MS. Use of rituximab, the new FDA-approved antibody. Current opinion in oncology. 1998;10(6):548-51. 31. Multhoff G, Botzler C, Wiesnet M, Eissner G, Issels R. CD3- large granular lymphocytes recognize a heat-inducible immunogenic determinant associated with the 72-kD heat shock protein on human sarcoma cells. Blood. 1995;86(4):1374-82. 32. Issels RD, Lindner LH, Verweij J, Wust P, Reichardt P, Schem BC, et al. Neo-adjuvant chemotherapy alone or with regional hyperthermia for localised high-risk soft-tissue sarcoma: a randomised phase 3 multicentre study. The Lancet Oncology. 2010;11(6):561-70. 33. Hossann M, Wang T, Wiggenhorn M, Schmidt R, Zengerle A, Winter G, et al. Size of thermosensitive liposomes influences content release. Journal of controlled release : official journal of the Controlled Release Society. 2010;147(3):436-43. 34. Limmer S, Hahn J, Schmidt R, Wachholz K, Zengerle A, Lechner K, et al. Gemcitabine Treatment of Rat Soft Tissue Sarcoma with Phosphatidyldiglycerol-Based Thermosensitive Liposomes. Pharmaceutical research. 2014.

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