Scientific Program

1 he Annual Meeting of the Brazilian Biophysics Society, one of the largest T and most traditional gathering of Biophysicists in Latin America, occurs during the second semester of each year. Now in its 43rd edition, the 2018 meeting will be held again at the Mendes Plaza Hotel, in the city of Santos (SP), from September 27th to 30th. A rich program of 16 symposia, 4 plenary talks, and 2 poster sessions covering a broad range of topics in Biophysics will guarantee the appropriate environment for productive discussions about developments regarding Biophysics and its interfaces with Biochemistry, Medicine, Pharmacy, among other disciplines.

World leaders in several areas of Biophysics, from at least 12 different countries have already confirmed participation in our meeting this year! We will host researchers from Argentina, Belgium, Canada, Denmark, England, France, Germany, Israel, Portugal, United States, Uruguay and, of course, from Brazil, which will be represented by colleagues from 10 different states. This shows our broad representation in both national and international scenarios.

We believe our meeting is the perfect opportunity for researchers interested in Biophysics and related fields to present their latest results to a very distinguished audience. Therefore, we invite you to participate and to submit an abstract for the poster sessions.

Looking forward to seeing you in Santos!

Antonio Costa-Filho President of SBBf

2 3 Index Iseli L. Nantes Cardoso...... 56 Tayana Mazin Tsubone...... 83 Juliana S Yoneda ...... 57 Yan M. H. Gonçalves...... 84 Organizing Committee...... 7 Katia Regina Perez...... 33 Lucas Rodrigues de Mello...... 57 Yeny Y. P. Valencia...... 84 Scientific Committee...... 7 Maria Elena de Lima...... 33 Lucivaldo R. Menezes...... 58 Virginia Sara Grancieri do Amaral...... 85 Sponsors...... 7 SY8 - Photobiology and its application Mario de Oliveira Neto...... 58 Biomembranes...... 86 Congress Venue...... 8 in Health Sciences...... 35 Maurício S. Baptista...... 59 Ana Paula R. Povinelli...... 86 Criação e diagramção:...... 8 Renato E. de Araujo...... 35 Rafael G. Carvalho...... 59 Anacleto Silva de Souza...... 86 Program...... 9 Yoshimura, T. M...... 35 Rhiannon W. Harries...... 59 Anderson F. Sepulveda...... 86 Plenary Lectures...... 13 Tayana Tsubone...... 36 Vinicius Carrascosa...... 60 Balan, A...... 87 Gronenborn, A. M...... 14 João Paulo Tardivo...... 36 Biomedical Application...... 61 Aryane A. Vigato...... 87 Benoît Roux ...... 14 Beate Röder...... 37 Raissa L. Oblitas ...... 61 Raphael Dias de Castro...... 88 Aníbal E. Vercesi...... 14 SY9 - Structural and Unstructural Biology...... 38 André L.S. Santos...... 61 Amantino C. F...... 88 J. A. Bouwstra...... 15 Garegin A. Papoian...... 38 Angela D. B. de Brito ...... 62 Lígia N. M. Ribeiro...... 89 Vladimir N. Uversky...... 38 Camila Ramos Silva...... 62 Fabiana V. Diasa...... 89 Pre-symposium...... 16 Yraima Cordeiro...... 38 Catarina Cataldi...... 63 Franciele Garcia Baveloni...... 90 PSY1 - Sirius and the new opportunities Pinheiro, Glaucia M.S...... 39 Débora C. K. Codognato...... 63 Gabriel Zazeri...... 90 in biophysics...... 17 SY10 - Biophysics of the heart...... 40 Cilli, E. M...... 64 Gianella Facchin1...... 90 Carla Polo...... 17 Danilo Roman Campos...... 40 Cabral, F. V...... 64 Giovana Firpo...... 91 Ana Carolina de Mattos Zeri...... 17 Rosana A. Bassani...... 40 Haroldo de Lima P. Cravo...... 65 Gonçalves G. E. G ...... 91 Maria Cristina Nonato...... 18 Micaela Lopez Alarcón ...... 41 Juliana Guerra Pinto...... 65 Isabele Ap. S. de Campos...... 92 Emiliano Medei...... 41 Letícia Corrêa Fontana...... 66 João Hermínio Martins da Silva...... 92 Symposium...... 19 SY11 - Methods in Biophysics...... 42 L. M. Rebelo...... 66 Juliana Damasceno Oliveira...... 93 SY1 - Cell and model membranes Biophysics...... 20 Paul C. Whitford...... 42 Luciana Maria Cortez Marcolino ...... 66 Kelli Cristina Freitas Mariano...... 94 Adriana Fontes ...... 20 Marin van Heel...... 42 Natalia K. Gushiken...... 67 Larissa D. da Silva...... 94 Koji Kinoshita...... 20 Fabio Cesar Gozzo ...... 42 Nayara S. Alcântara Contessoto...... 67 Luciana Guimarães Munhoz...... 95 Nuno C. Santos...... 20 Roberto K. Salinas...... 43 Nicole M. A. Chaparro ...... 68 Matheus P. Pinheiro...... 95 Luis A. Bagatolli...... 21 SY12 - Nanostructures...... 44 Sa. Arsalani...... 68 Mayk C. Ramos...... 96 SY2 - Central Nervous System Electrophysiology..... 22 Alan B. Dalton...... 44 Soudabeh. Arsalani...... 69 Mayra C. G. Lotierzo ...... 96 Leonardo Cirqueira ...... 22 Leandro Ramos Souza Barbosa...... 44 Taline S. Almeida...... 69 Nascimento M. H. M...... 96 Melina Pires da Silva...... 22 Frédéric Frézard...... 45 Taline S. Almeida...... 70 Monique Lemos...... 97 Christopher Kushmerick...... 23 Daniele Ribeiro de Araujo...... 45 Juliana Ferreira Strixino...... 70 Naially C. de Faria...... 97 Ricardo M. Leão...... 23 SY13 - Molecular Modeling and Dynamics...... 46 Guilherme S. Ramos...... 71 Pablo V. M. Reis...... 98 SY3 - Lipid-protein interactions in Guilherme Menegon Arantes...... 46 Biomembranes...... 72 Cordeiro Lima P. F ...... 98 mimetic systems...... 24 Werner Treptow...... 46 Andresa Messias da Silva...... 72 Bárbara Malheiros...... 99 Thierry Granjon...... 24 M. Pickholz ...... 46 Bruna Renata Casadei...... 72 Sandra B. N. Agostini...... 99 Ambrosio ...... 24 Sergio Pantano...... 47 Favarin, B. F...... 73 Sandra M. G. Dias...... 100 M. Prieto ...... 25 SY14 - Photodynamic Therapy...... 48 C.R. SIMÕES...... 73 Castro, Simone R. de...... 100 M. Bolean...... 25 Juliana Ferreira Strixino...... 48 C. R. Ferreira...... 74 Viviane Corrêa Santos...... 101 SY4 - Nanostructures and Drug Delivery...... 26 Fabio Parra Sellera...... 48 VESCHI E. A...... 74 Amantino C. F...... 101 Renata F. V. Lopez...... 26 Renata Aparecida Belotto...... 48 Fernando Freitas de Lima...... 75 Molecular Mechanisms of Disease...... 102 Marcelo Morales ...... 26 Silvia Cristina Nunez...... 49 Frederico J. S. Pontes...... 75 Andreia Laura Prates Rodrigues...... 102 Omar Mertins...... 26 SY15 - Biophysics of Proteins...... 50 Gabriel S. Vignoli Muniz...... 76 Artur S. Miranda...... 102 Lígia N. M. Ribeiro...... 27 Carlos Henrique I. Ramos...... 50 Sebinelli, H. G...... 76 Marinonio L. Cornélio...... 103 SY5 - Protein Complexes...... 28 Chuck S. Farah...... 50 Helena Couto Junqueira...... 76 Osias B. S. Filho...... 104 Ana Paula Valente ...... 28 Jorge F. B. Pereira...... 50 Isabela Moreira-Silva...... 77 Paula Rhana...... 104 Elio A. Cino...... 28 Chehín, Rosana Nieves...... 51 Jefferson C Rodrigues ...... 77 Victor Barbosa...... 105 Rachel Nechushtai...... 28 SY16 - Cellular Biophysics...... 52 Kenneth M. F. Miasaki...... 78 Diogo B. Peruchetti...... 105 Carla Mattos...... 29 Celso Caruso Neves...... 52 Ludmilla D. Moura...... 78 Ainhoa Rodriguez de Yurre...... 106 SY6 - Modern Methods in Computational Fernando Abdulkader...... 52 L. H. Andrilli...... 79 Nucleic acids structure and functions...... 107 Biophysics...... 30 Felipe de Souza Leite ...... 53 Maressa Donato...... 79 Erik de Oliveira Martins...... 107 Kaline Coutinho...... 30 Adriano M. Alencar...... 53 Maria C. Oliveira ...... 80 Izabela F da Silva...... 107 Gerald Weber...... 30 Natalia Alvarez...... 80 Mateus Rodrigues Leal ...... 107 Rafaela S. Ferreira ...... 30 Posters...... 54 Natália B. Leite...... 81 Pâmella Miranda ...... 108 Roberto A. Chica...... 31 Biomaterials...... 55 Natália F. de Oliveira...... 81 Vinícius Fernandes...... 108 SY7 - Antimicrobial peptides...... 32 Alex H. Miller...... 55 Strazza Junior PS ...... 82 Vinícius G. Contessoto...... 109 Catarina Cataldi...... 32 Aline Amorim Graf...... 55 Priscilla Freddi...... 82 Vivianne Basílio Barbosa...... 109 Yeny Y. P. Valencia...... 32 Denys E. S. Santos...... 55 Raphael de A. N. Gomes ...... 83 Protein Folding Misfolding and Unfolding...... 111 Joao Ruggiero Neto...... 33 Elisângela Belleti ...... 56 Romildo A. Nogueira ...... 83 Dayanne P. Rosa...... 111

4 5 Fernando B. da Silva ...... 111 Lilia Iriarte...... 135 Organizing Committee Gabriel G. Slade...... 112 Luan C. Marques...... 135 Guilherme G. Costa...... 112 Lucas Bleicher...... 136 • Antonio José da Costa - USP • Rosangela Itri - USP Scanavachi, G...... 113 Lucas Carrijo de Oliveira...... 136 • Eneida de Paula - UNICAMP • Cynthia Bando - Secretária Geral Luciano Censoni ...... 113 Lucianna Helene Santos...... 137 • Leandro R S Barbosa - USP Mariana Chaves Micheletto...... 113 Luis Felipe Santos Mendes...... 137 Freire Ribeiro, M...... 114 Luiz Fernando de C. Rodrigues...... 138 Mariana P. Cali ...... 114 Bertozo L. C...... 138 Nelson A. Alves...... 115 Marcelo Querino Lima Afonso ...... 139 Scientific Committee Paulo Ricardo Mouro...... 115 Mariana A. Ajalla Aleixo ...... 139 Rose M Carlos ...... 115 Batista, M. R. B...... 139 • Alessandro Nascimento - USP • Leandro R. S. Barbosa - USP Yulli M. F. Passos...... 116 Marilia L. Cirqueira...... 140 • Antonio José da Costa Filho - USP • Lucas Bleicher - UFMG Protein Structure dynamics and Functions...... 117 Marina G. Fontes...... 140 • Christopher Kushmerick - UFMG • Paulo Bisch - UFRJ Agnes Magri ...... 117 Marjorie C. L. C. Freire...... 141 • Emiliano Medei - UFRJ • Pietro Ciancaglini - USP Anderson S. Pinheiro...... 117 Miguel de S. Andrade...... 141 • Eneida de Paula - UNICAMP • Rosangela Itri - USP André Anversa Oliveira Reis...... 118 Naiá Porã Santos...... 141 • Ernesto Caffarena - FIOCRUZ Angie Dávalos...... 118 Natália A. Fontana...... 142 Antonio Victor B. Vasconcelos...... 118 Neli Fonseca...... 142 Atílio Tomazini Júnior...... 119 Olívia Teixeira...... 143 Penna, B. R...... 119 Paola Silva ...... 143 Sponsors Bianca Rizo...... 120 Paulo S. L. Beirão ...... 143 Bruno P. O. Santos...... 120 Luccas, P. H...... 144 Camila A. Yamada...... 121 Priscila dos S. Bury...... 144 Camila F. T. Pontes ...... 121 Rafael E. O. Rocha...... 145 Carmen Domene...... 121 Rafael F. Soares...... 145 Carolina T. A. Ferreira...... 122 Raissa Ferreira Gutierrez...... 146 Catharina dos S. Silva...... 122 Ricardo A. P. Pádua...... 146 Claudemir Oliveira Souza...... 123 Beserra S. dos S. Author ...... 147 Fabio C. L. Almeida...... 123 Tábata Renée Doratioto...... 147 Felipe A. Otsuka...... 124 Thainá Miranda...... 147 Fernanda Paiva...... 124 Thamires Q. Froes...... 148 Fernando T. Tanouye ...... 124 Thirupathi R. Soudherpally...... 148 Fredderico C. Machado...... 125 Ximenes V. F ...... 149 Gabriel Ernesto Jara...... 125 Vanessa S. Silva...... 149 Gabriel R. Sousa...... 125 Víctor U. Antunes...... 150 Giovana C. Guimarães...... 126 Victor Lopes Rangel...... 150 Glauce M. Barbosa...... 126 Victoria Oakes...... 151 Helder V. Ribeiro Filho...... 127 Vinícius M. Oliveira...... 151 Iara Aimê Cardoso...... 127 Vitor B. Machado...... 151 Ingrid B. S. Martins...... 128 Pedro, R. P...... 152 Isabella O. L...... 128 Marcus V. C. Cardoso ...... 153 Isis Sebastião...... 129 Vitor B. P. Leite...... 153 Sarmento J. O ...... 129 Miscellaneous...... 154 Jéssica A. Tedesco...... 129 Felipe Curtolo...... 154 Jéssica M. de Sá...... 130 Marlon Augusto P. De Almeida ...... 154 José Antonio Fiorote...... 130 Nataly Herrera...... 154 Juliana Raw...... 131 Rafael C. Marchi...... 155 Juscemácia N. Araujo...... 131 Rômulo P. Tenório...... 155 Karoline Sanches...... 131 Amanda Schukarucha Gomes...... 156 Larissa G. Maciel...... 132 Ana Carolina M. Figueira...... 156 Bartkevihi, L...... 132 Telma Lisbôa Nascimento...... 157 Leonardo Boechi...... 133 R. V. Maximiano...... 157 Leonardo Cirqueira, ...... 133 Fernando de Mesquita Júnior...... 158 Letícia Stock...... 134 Alphabet Index...... 159 Ramos, L...... 134

6 7 Congress Venue Program

THURSDAY – Sep 27th PSY1 - Pre-symposium: Sirius and the new opportunities in biophysics Chair: Chuck Farah (USP) PSY1.1 – Carla Polo (LNLS): From macromolecules to cells: 3D imaging using coherent X-ray diffraction at Cateretê beamline (Sirius) 16:00 - 17:30 PSY1.2 – Ana Carolina de Mattos Zeri (LNLS): Macromolecular Crystallography at Sirius – frontiers and challenges for data collection and sample preparation at Manacá beamline PSY1.3 – Maria Cristina Nonato (USP): X-ray crystallography fragment screening. Opportunities for Manacá beamline (Sirius) 16:30 - 18:00 Registration PL01 – Opening lecture: Angela Gronenborn (Univ. Pittsburgh): “Not so crystal clear – crystallins and 18:00 - 19:00 cataract” Chair: Antonio José Costa Filho (USP) 19:00 – 21:00 Poster session FRIDAY Sep 28th TIME ROOM 1 ROOM 2 Mendes Plaza Hotel Complexo Diamante 8:00 - 9:00 Registration Av. Marechal Floriano Peixoto 42 SY1 – Cell and model membranes Biophysics SY2 – Central Nervous System Electrophysiology Gonzaga, Santos, SP Chair: Karin Riske (UNIFESP) Chair: Jader Cruz (UFMG) Phone: + 55 13 3208-6400 SY2.1B – Leonardo Cirqueira Pimentel (UNB): www.mendesplaza.com.br SY1.1 – Adriana Fontes (UFPE): Optical Tweezers: State-dependant affinity for general anesthetics Shining Light to Cell Biology dictates channel sensitivity

Criação e diagramção: SY1.2 – Koji Kinoshita (Univ. South. Denmark): Red Blood Cell Membrane: Mechanical Property SY2.2 – Melina Pires (USP): Nitrergic Modulation Change and Cytoskeletal Network Reformation of Magnocellular Neurons from the Supraoptic 8:30 - 10:30 Under Photosensitizer-mediated Oxidative Stress of Nucleus DigitalMindbr Cis-Porphyrin http://www.digitalmindbr.com.br/ SY1.3 – Nuno Santos (Univ. Lisbon): Arterial hyperten- SY2.3 – Christopher Kushmerick (UFMG): Ionotropic sion and stroke patients present Higher erythrocyte actions of Metabotropic Glutamate Receptors adhesion forces, contributing to cardiovascular risk SY2.4 – Ricardo Leão (USP): Creating rest and SY1.4 – Luis Bagatolli (Univ. Nac.Córdoba): The unrest: distinct ways to create stable and unstable cell as a gel: materials for a conceptual discussion membrane potential in neurons 10:30 - 11:00 Coffee Break SY3 – Lipid-protein interactions in mimetic SY4 – Nanostructures and Drug Delivery systems Chair: Daniele R. Araújo (UFABC) Chair: Pietro Ciancaglini (USP) SY3.1 – Thierry Granjon (Univ. Lyon): Cardiolipin SY4.1 – Renata Lopes (USP): Iontophoresis impact and mitochondrial proteolipidic platforms: toward a on topical delivery of nanoparticles mitochondrial raft-like organization?

11:00 - 13:00 SY3.2 – André Luis Ambrosio (USP): Human MPC2 SY4.2 – Marcelo Morales (UFRJ): Nanoparticle- as an autonomous membrane transporter mediated gene delivery for the lung

SY3.3 – Manuel Prieto (Univ. Lisbon): Surface SY4.3 – Omar Mertins (UNIFESP): Development of crowding and membrane remodeling by IAPP functional Nanoparticles for delivery of bioactives

SY4.4 – Ligia Nunes de Morais Ribeiro (UNICAMP): SY3.4 – Mayte Bolean (USP): Biophysical aspects Stability study of Drug Delivery Systems Based on of biomineralization Nanoparticle Tracking Analysis

8 9 13:00 - 15:00 Lunch 10:30 - 11:00 Coffee Break 14:30 – 15:00 Bruker Space SY9 – Structural and Unstructural Biology SY10 – Biophysics of the heart Chair: Rosangela Itri (USP) Chair: Emiliano Medei (UFRJ) SY6 – Modern Methods in Computational SY5 – Protein Complexes Biophysics SY9.1 – Garegin Papoian (Univ. Maryland): SY10.1 – Danilo Campos (UNIFESP): Keep your Chair: Fábio Almeida (UFRJ) Chair: Lucas Bleicher (UFMG) Assembly and Dynamics of Histone Oligomers and heart stressed: impaired superoxide production and Nucleosomes pathogenesis of Chagas disease SY5.1 – Ana Paula Valente (UFRJ): Conformational SY6.1 – Kaline Coutinho (USP): Theoretical Studies diversity in protein complexes: NMR studies of SY9.2 – Vladimir Uversky (Univ. South Florida): SY10.2 – Rosana Bassani (UNICAMP): of Electronic Structure of Molecules Interacting Allergens Bet v 1 and Fag s 1 with ligands and DIII Unusual Biophysics and Strange Biology of Proarrhyhmic effects of Doxorubicin in the with Phospholipid Bilayers of Dengue virus E protein with antibodies 11:00 - 13:00 Intrinsic Disorder myocardium SY9.3 – Yraima Cordeiro (UFRJ): Prion protein and SY5.2 – Elio Anthony Cino (UFMG): Role of SY6.2 – Gerald Weber (UFMG): Mesoscopic models SY10.3 – Micaela Lopez Alarcón (UFABC): The role alpha-synuclein aggregation in vitro: characteriza- Hydrogen Bonds in P53 Core Domain Stability and as a Tool for Probing molecular interactions in DNA of NLRP3/Casp1/IL-1β axis in renal ischemia/reper- 15:00 - 17:00 tion of therapeutic compounds and cofactors for Aggregation and RNA fusion-induced cardiac electrical disturbances protein misfolding SY5.3 – Rachel Nechushtai (Hebrew Univ.): The SY6.3 – Rafaela Ferreira (UFMG): Rational Drug role played by the labile 2Fe-2S clusters of neet SY9.4 – Glaucia Pinheiro (UNICAMP): NMR struc- SY10.4 – Emiliano Medei (UFRJ): Cardiac electrical Design Towards New Treatments proteins in controling their structure, function and ture of the J-domain of co-chaperone Sis1 from and mechanical study on a type 2 diabetes experi- For Neglected And Emerging Diseases folding Saccharomyces cerevisiae mental model SY5.4 – Carla Mattos (Northeastern Univ.): Ras 13:00 - 15:00 Lunch SY6.4 – Roberto Chica (Univ. Ottawa): Rational dimerization and allosteric modulation of GTP Design of Proteins that Exchange on Functional hydrolysis linked in the presence of Raf-RBD and 14:30 – 15:00 NANOTEMPER: Binding Characterization trough MicroScale Thermophoresis… in any buffer! Timescales the membrane SY11 – Methods in Biophysics SY12 – Nanostructures 17:00 - 17:30 Coffee Break Chair: Paulo Bisch (UFRJ) Chair: Luciana M. Rebelo Alencar (UFMA) SY12.1 – Alan Dalton (Univ. Sussex): Biological PL02 -Plenary – Benoit Roux (Univ. Chicago): Molecular Dynamics Studies of P-type ATPase Ion Pumps SY11.1 – Paul Whitford (Northeastern Univ.): 17:30 - 18:30 functionalization of synthetic nanoparticles using Chair: Carla Mattos (Northeastern Univ) Quantifying collective dynamics in the ribosome novel 2D peptide assemblies 18:30 - 19:30 SBBf General Assembly SY11.2 – Marin van Heel (Imp. College): Dynamic SY12.2 – Leandro RS Barbosa (USP): Cubosomes th 15:00 - 17:00 Structures of Biological Nano-Machines by Single- SATURDAY Sep 29 as delivery for many drugs Particle Cryo-EM TIME ROOM 1 ROOM 2 SY11.3 – Fabio Gozzo (UNICAMP): Advances in SY12.3 – Frederic Frezard (UFMG): Targeting the 8:00 - 9:00 Registration Mass Spectrometry as a Structural Biology Tool host-cell for improved therapy of leishmaniasis SY8 – Photobiology and its application in Health SY12.4 – Daniele Araújo (UFABC): Thermosensitive SY7 – Antimicrobial peptides SY11.4 – Roberto Salinas (USP): Solution NMR Sciences hydrogels and organogels: from supramolecular Chair: Maria Lucia Bianconi (UFRJ) spectroscopy studies of the Na+/Ca2+exchanger Chair: Silvia Nunez (Un. Brasil) structure to new drug-delivery systems SY7.1A – Catarina Cataldi: Zika NS2B epitope as a 17:00 - 17:30 Coffee Break candidate for diffential diagnosis – from Poster SY8.1 – Renato Araújo (UFPE): Optical thera- PL03 – Plenary – Anibal Vercesi (UNICAMP): The redox nature of the mitochondrial permeability SY7.1B – Yeny Yaneth Pillco Valencia: py and medical diagnosis assisted by metallic 17:30 - 18:30 transition Computational simulation of popg aggregates in nanoparticles Chair: Mauricio da S. Baptista (USP) the presence of antimicrobial peptide LL-37 – from Poster 18:30 - 20:30 Poster Session SY8.2A – Tania M Yoshimura (IPEN): Biochemical 8:30 - 10:30 SY7.2 – João Ruggiero (UNESP): Lipid-packing changes in serum of obese mice related to photoac- perturbation induced by pH-responsive peptide in tivation of brown adipose tissue lipid membranes: effect of pH and peptide chemical modification. SY8.2B – Tayana Tsubone (USP): How does cell membrane respond to PDT? SY8.3 – Joao Paulo Tardivo (Faculdade de SY7.3 – Katia Perez (UNIFESP): Using biomimetical Medicina do ABC): Is Photodynamic Therapy membranes to study the mode of action of peptides Efficient to Treat Osteomyelitis in Diabetic Foot? SY7.4 – Maria Elena Lima (UFMG): A synthetic pep- SY8.4 – Beate Roder (Univ. Humbolt): tide derived from a toxin of the spider Phoneutria Photodynamic inactivation (PDI) of biofilm forming nigriventer: a promising candidate to a therapeutic microorganisms drug to treat erectile dysfunction

10 11 SUNDAY Sep 30th TIME ROOM 1 ROOM 2 SY13 – Molecular Modeling and Dynamics SY14 – Photodynamic Therapy Chair: Ernesto Caffarena (FioCruz) Chair: Martha Ribeiro (IPEN) SY13.1 – Guilherme Arantes (USP): Unfolding SY14.1 – Juliana Ferreira Strixino (UNIVAP): metalloproteins with force: From simple models to Photodynamic therapy in cutaneous Leishmaniasis quantum mechanical calculations SY13.2 – Werner Treptow (UnB): Binding of Small SY14.2 – Fabio Parra Sellera (USP): Antimicrobial Ligands to Two State Membrane Proteins photodynamic therapy in Veterinary Medicine 8:30 - 10:30 SY14.3 – Renata Belotto (Hospital Perola SY13.3 – Monica Pickholz (Univ. B. Aires): Byington): Can the photodynamic therapy be an Contributions of Simulations to the Understanding alternative to treat high grade cervical intraepitheli- of Drug Delivery Systems al neoplasia? SY13.4 – Sergio Pantano (Pasteur): Towards SY14.4 – Silvia Nunez (Univ. Brasil): Antimicrobial a Complete Coarse-Grained Force Field for Photodynamic Therapy Biosimulations 10:30 - 11:00 Coffee Break PL04 – Plenary – Joke Bouwstra (Leiden): Lipid organization in the skin barrier in health and disease 11:00 - 12:00 Chair: Eneida de Paula (UNICAMP) 12:00 - 14:00 Lunch SY15 – Biophysics of Proteins SY16 – Cellular Biophysics Chair: Nuno Santos (IMM) Chair: Gilberto Weismuller (UFRJ) SY16.1 – Celso Caruso (UFRJ): albumin handling SY15.1 – Carlos Ramos (UNICAMP): Chaperone- in renal proximal tubule cells: central role in the assisted protein aggregate reactivation progression of renal disease SY15.2 – Chuck Farah (USP): Structural and SY16.2 – Fernando Abdulkader (USP): Ion channels Functional Studies on the Xanthomonadaceae Type 14:00 - 16:00 in the amplifying pathway of insulin secretion IV Secretion System – a Bacteria Killing Machine SY16.3 – Felipe Leite (USP): post-translational ar- SY15.3 – Jorge Pereira (UNESP): Are Ionic Liquids ginylation and inter-sarcomere dynamics in skeletal Good Protein Stabilizers? muscle myofibrils SY15.4 – Rosana Chehin (CONICET-UNT): SY16.4 – Adriano Mesquita (USP): Dynamics of Biophysical characterization of doxycycline poten- cardiomyocytes obtained by the method of traction tial neuroprotective mechanism force microscopy Plenary Lectures 16:00 - 16:30 Closing and awards ceremony 16:30 Refreshments

12 13 PL01 - Not so crystal clear - crystallins and E1-E2-type ATPase). The Na/K pump transports either by apoptosis or necrosis. A large body of evidence why these changes in lipid organization were observed, + + cataract three Na out of the cell by two K into the cell at indicates that mitochondrial redox imbalance and MPT simultaneously studies were performed studies using the expense of the hydrolysis of one molecule of ATP. are responsible for the development and progression lipid model systems(4). These studies revealed that we Gronenborn, A. M. The sarcoplasmic reticulum Ca2+-ATPase (SERCA) of a series of pathologies such as cancer, diabetes, are able to form the LPP as well as the SPP in these 2+ Xi, Z., Whitley, M.J., pumps two Ca from the cytosol of muscle cells to dyslipidemias, inflammatory diseases, hypertension, lipid model systems and that the lipids mainly form a the sarcoplasmic reticulum by exchanging two H+. ischemia/reperfusion injury, neurodegenerative dense orthorhombic lateral packing. When changing the 1 Department of Structural Biology, University of From crystallography, we have a remarkable series diseases, muscular dystrophy, drug toxicity and aging. ceramide and/or fatty acid composition it was shown Pittsburgh School of Medicine, Pittsburgh, PA, of snapshots showing how these enzymes look at Here, we focus on the contributions of our laboratory that long chain free fatty acids play an important role in United States different states of their transport cycle. Using molecular to the understanding of the redox nature of MPT and the formation of the dense orthorhombic lateral packing dynamics simulation, the string method with swarms- the participation of PTP opening in mitochondrial and that a reduction in the free fatty acid chain length βγ-Crystallins are long-lived eye lens proteins that are of-trajectories, and free energy methods, we seek to dysfunction in hypercholesterolemic mice and statins or an increase in the degree of unsaturation reduced crucial for lens transparency and refractive power. Each βγ- understand the conformational dynamics involved as toxicity. the lipid fraction adopting an orthorhombic packing crystallin comprises two homologous domains, which are such pump transits through conformational states (5). These changes in the lipid composition increased connected by a short linker. γ-Crystallins are monomeric, revealed by x-ray crystallography, the nature of the This work was supported by Fundação de Amparo à also the permeability of these lipid membranes. These while β-crystallins crystallize as dimers and multimers. In coupling between the binding of ATP, phosphorylation, Pesquisa do Estado de São Paulo (FAPESP # 17/17728- changes in lipid organization and permeability induced the crystal, human βB2-crystallin is a domain-swapped and the movements of charged species across the core 8), Conselho Nacional Desenvolvimento Científico e by changes in lipid composition explained very well dimer while the N-terminally truncated βB1-crystallin of the protein, the stepwise voltage-sensitive steps, and Tecnológico, Coordenação de Aperfeiçoamento de the deviation in lipid organization and barrier reduction forms a face-en-face dimer. Combining and integrating the origin of the ion binding specificity associated with Pessoal de Nível Superior and Fundação de Apoio ao in atopic eczema skin. Finally using neutron, X-ray data from multi-angle light scattering, nuclear magnetic different conformational states.1-8 A special attention Ensino, à Pesquisa e à Extensão (UNICAMP/FAEPEX). diffraction and infrared spectroscopy studies detailed resonance, and small-angle X-ray scattering of full-length is given to the protonation state of ionizable residues analysis of the arrangement of the lipids in the SPP 9 and terminally truncated human βB2-crystallin in solution, during the pumping cycle. PL04 - Lipid organization in the skin barrier in and LPP has been examined and it was observed we show that both these βB2-crystallin proteins are health and disease that that the lipids in the LPP have a very exceptional dimeric, possess C2 symmetry, and are more compact than References: 1.H. Yu, I. M. Ratheal, P. Artigas & B. arrangement with small lipid droplets in a fluid phase(6, domain-swapped dimers. Importantly, no inter-molecular Roux. Protonation of key acidic residues is critical for J. A. Bouwstra 7). In conclusion the lipids in the stratum corneum have paramagnetic relaxation enhancement effects compatible the K-selectivity of the Na/K pump. Nat. Struct. & Mol. 1Leiden Academic Centre for Drug Research, Leiden a very exceptional arrangement and play a prominent 2. with domain swapping were detected. Our collective Biol. 18, 1159-1163, (2011). PMC3190665. J. P. University, Einsteinweg 55, 2333 RC, Leiden, The role in the reduced skin barrier in inflammatory skin experimental results unambiguously demonstrate that, in Castillo, H. Rui, D. Basilio, A. Das, B. Roux, R. Latorre, Netherlands diseases. solution, human βB2-crystallin is not domain swapped F. Bezanilla & M. Holmgren. Mechanism of potassium and exhibits a face-en-face dimer structure similar to the ion uptake by the Na(+)/K(+)-ATPase. Nat. Comm. References: 1. Bouwstra J A, Gooris G S, van der Spek crystal structure of truncated βB1-crystallin 6, 7622, (2015). 3.H. Rui, P. Artigas & B. Roux. The The skin barrier function is primarily located in the J A, et al. Structural investigations of human stratum selectivity of the Na(+)/K(+)-pump is controlled by stratum corneum (SC) comprised of corneocytes corneum by small-angle X-ray scattering. J Invest PL02 - Molecular Dynamics Studies of P-type binding site protonation and self-correcting occlusion. (dead cells) and intercellular lipids. The main lipid Dermatol 1991: 97: 1005-1012. 2. Bouwstra J A, 4. ATPase Ion Pumps eLife 5, (2016). A. Das, H. Rui, R. Nakamoto & B. classes in the stratum corneum are ceramides, free Gooris G S, Salomonsdevries M A, et al. Structure of Roux. Conformational transitions and alternating- fatty acids, and cholesterol. These lipids form two Human Stratum-Corneum as a Function of Temperature Benoît Roux1 access mechanism in the sarcoplasmic reticulum crystalline lipid lamellar phases, one with a repeat and Hydration - a Wide-Angle X-Ray-Diffraction Study. 5. Huan Rui1, Avisek Das1, Francisco Bezanilla1, M. calcium pump. J. Mol. Biol., (2017). Y. Chen & B. distance of 13 nm (The long periodicity phase, LPP) International Journal of Pharmaceutics 1992: 84: 205- Holmgren2, Pablo Artigas3, Robert Nakamoto4. Roux. Constant-pH Hybrid Nonequilibrium Molecular and the other with a repeat distance of 6 nm (The short 216. 3.van Smeden J, Janssens M, Kaye E C, et al. The Dynamics-Monte Carlo Simulation Method. J. Chem. periodicity phase, SPP)(1). The most prevalent lateral importance of free fatty acid chain length for the skin 1 Department of Biochemistry and Molecular Theo. Comp. 11, 3919-3931, (2015). packing is orthorhombic(2). The composition and barrier function in atopic eczema patients. Exp Dermatol Biology, The University of Chicago, 929 organization of these lipids are crucial for a proper skin 2014: 23: 45-52. 4. van Smeden J, Janssens M, Gooris 2 E57th St, Chicago, IL 60637, USA. Molecular barrier function. Especially the LPP is considered to be G S, et al. The important role of stratum corneum lipids Neurophysiology Section, Porter Neuroscience PL03 - THE REDOX NATURE OF THE MITOCHONDRIAL PERMEABILITY TRANSITION crucial for the skin barrier function. In our group we for the cutaneous barrier function. Biochim Biophys Research Center, National Institute of Neurological are interested in the lipid composition and organization Acta 2014: 1841: 295-313. 5.Mojumdar E H, Helder Disorders and Stroke, National Institutes of Health, Aníbal E. Vercesi1 and how this is related to the skin barrier, especially R W, Gooris G S, et al. Monounsaturated fatty acids Bethesda, Maryland 20892, USA. 3Department of 1Department of Clinical Pathology, State University to understand the impaired skin barrier function in reduce the barrier of stratum corneum lipid membranes Cell Physiology and Molecular Biophysics, Texas of Campinas, São Paulo, SP - email: anibal@ inflammatory skin diseases. When summarizing by enhancing the formation of a hexagonal lateral Tech University Health Sciences Center, Lubbock, the deviation in lipid composition in this tissue, we packing. Langmuir 2014: 30: 6534-6543. 6.Paz Ramos 4 unicamp.br United States Department of Molecular Physiology observed in atopic eczema (most prevalent skin A, Gooris G, Bouwstra J, et al. Evidence of hydrocarbon and Biological Physics, University of Virginia School disease in Europe) a reduced chain length of free fatty nanodrops in highly ordered stratum corneum of Medicine, PO Box 800886, 480Ray C. Hunt Drive, In addition to be the cell’s powerhouse, mitochondria acids and of ceramides and an increase in the level model membranes. J Lipid Res 2018: 59: 137-143. Charlottesville, VA 22908, USA also contain a cell death machinery that include of unsaturation(3). These changes in lipid composition 7.Mojumdar E H, Gooris G S, Groen D, et al. Stratum highly regulated processes such as the membrane resulted in an altered lipid lamellar organization and corneum lipid matrix: Location of acyl ceramide and P-class ATPases ion pumps constitute a superfamily of permeability transition (MPT) and reactive oxygen a lower lipid fraction of adopting an orthorhombic cholesterol in the unit cell of the long periodicity phase. cation transport enzymes, present both in prokaryote species (ROS) generation. High levels of matrix Ca2+ packing. Therefore the lipid properties substantially Biochim Biophys Acta 2016: 1858: 1926-1934 and eukaryote, whose members mediate membrane stimulate ROS production and opening of a nonspecific deviate from normal human SC. In order to understand flux of all common biologically relevant cations. inner membrane pore, the permeability transition P-class pumps use ATP to transport ions against pore (PTP). These conditions disrupt energy-linked their electrochemical potential (they are also called mitochondrial functions and may promote cell death

14 15 PSY1 - Sirius and the new opportunities in biophysics

PSY1.1 - FROM MACROMOLECULES TO state-of-the-art macromolecular microcrystallography CELLS: 3D IMAGING USING COHERENT X-RAY employing current methods, with a beam focused in DIFFRACTION AT CATERETÊ BEAMLINE (SIRIUS) the low tens of micrometers, and nanocrystallography, with a submicron focus beam and sub miliradian 1 Carla Polo divergence. The beamline will operate with X-ray Tiago Kalile1, Harry Westfahl Jr1, Florian Meneau1 energies between 5 to 20 keV with large emphasis on membrane proteins and supramacromolecular protein 1 Laboratório Nacional de Luz Síncrotron (LNLS- complexes (megadalton size), such as receptors, CNPEM), Campinas-SP, Brazil e-mail: carla.polo@ proteasomes, translation complexes (protein and lnls.br DNA/RNA components), bacterial cell division machinery and eukaryotic transcription complexes, The Cateretê beamline at Sirius, the new Brazilian and will enable a range of automated screening synchrotron light source will be dedicated to assays for molecular fragments and natural products. coherent and time-resolved scattering experiments. The applications cover areas such as pharmaceutics, It will provide unique capabilities with cutting edge agriculture and cosmetics, and will enable the use and research tools that are non-existent today in Brazil, development of new techniques for the investigation like 3D imaging with nanometer resolution. In this of biological materials at the atomic level. Accelerated talk an overview of the main characteristics and new methods for small molecule ligand screening are scientific potentialities of coherent diffraction imaging paramount for the development of new bio-catalysts (CXDI), X-ray photon correlation spectroscopy (XPCS) and drug candidates, and the proposed methods of and ultra-small angle X-ray scattering (USAXS) for data collection and data analysis for single or multiple studying biomaterials will be provided as well as the crystals in both experimental stations of the Manaca status of the Cateretê project and the bio-experimental beamline will contribute to advances in Biotechnology, devices. We will present how CXDI studies allows to akin to what h been done in current third- generation investigate structures in a wide range of length scales. sources. Atomic details of the active sites of the This will be illustrated by the imaging characterization proteins under study and potential new important of morphological features in different lignocellulosic ligand binding pockets, and their interactions with materials which shed light to biomolecular deposition/ several ligand candidates must be obtained in a fast deconstruction mechanisms that are important to manner, for ever smaller crystals. Frequently, proteins plant biology and fuel production. are obtained in small quantities, derived from natural sources, and yield microcrystals that must be screened PSY1.2 - Macromolecular Crystallography under many different conditions. Methods of serial at Sirius - frontiers and challenges for data data collection, employing micro and sub-micron collection and sample preparation at Manacá beams, with crystals in plates or micro-chips are a beamline reality in microfocus beamlines in 3rd and 4th genera Pre-symposium SR sources, and will be available at the Microfocus Ana Carolina de Mattos Zeri station of Manaca. New methods such as the acoustic drop levitation, to be implemented in the Nanofocus station, will also be important for the understanding of these enzymes. Current platforms for data collection, Sirius is the new brazilian Synchrotron light source, storage and distribution are being tested at the UVX to under construction in Campinas, São Paulo state. It will simulate the needs of the new beam lines. Software be a 4th generation source, with various applications tools for data treatment are being developed and the in Biophysics. Advanced imaging techniques will be team is contributing to the international MXCube data available alongside new in-operando techniques. collection software consortium. The open source The MANACÁ beamline will be installed in a high- collaborative platforms MXCube and ISPyB are beta straight section of the Sirius storage ring and its employed and maintained by a group of Synchrotron main goal is to bridge the gap between conventional Laboratories staff members, and is aimed at speeding crystallography, which uses large single crystals, up and making the data acquisition and treatment, up and single-molecule X-ray diffraction performed to structure solution, a more straightforward process in free electron lasers. The beamline will have for the users. two experimental stations, for the performance of

16 17 PSY1.3 - X-ray crystallography fragment and disadvantages in terms of sample consumption, screening. Opportunities for Manacá beamline degree of automation, and assay complexity. However, (Sirius) because the affinities of the initial fragments are often low, detection methods are pushed to their limits, Maria Cristina Nonato leading to a variety of artifacts, false positives, and false Departamento de Física e Química, Faculdade de negatives that too often go unrecognized. Investigators Ciências Farmacêuticas de Ribeirão Preto, USP, in industry and academia have overcome these Ribeirão Preto, SP ([email protected]) challenges by taking advantage X-ray crystallography techniques, which provide structural information that enables rapid and efficient assessment of hits. Also, Fragment screening has emerged as a modern recent technical developments enabled screening on approach to identify novel lead compounds for drug a timescale comparable to other techniques allowing development. Moreover, fragment screening can its routine use in drug discovery. In this present work, identify secondary sites in proteins that may have a we will discuss the details (hardware/software) of biological function. This approach entails screening High throughput X-ray Crystallography Fragment libraries of very small molecules that follow the “rule Screening (HTXCFS), illustrated with examples of our of three” criteria. The smaller and less complex nature latest experiments at XCHEM facility at Diamond Light of fragments increases the probability of binding Source, and will bring to attention of future users of to a target protein, resulting in higher hit rates and Sirius our efforts/ambitions to build at the Manacá an efficient search of diverse chemical space. Hits beamline a world leader facility for the application of identified from fragment screening can be efficiently HTXCFS. developed by growing, merging, or linking them to produce drug leads. Today, an array of biophysical This work was supported by Fundação de Apoio methods has been developed to rapidly identify weakly à Pesquisa do Estado de São Paulo - FAPESP binding fragments to a target protein: SPR, DSF, NMR, (2016/22929-0), (2017/26559-5) among many others. Each method has advantages

Symposium

18 19 SY1 - Cell and model membranes Biophysics in both groups when higher fibrinogen concentrations mass action kinetics and dilute solution theory, can be are present. Fibrinogen-erythrocyte (un)binding forces mechanistically depicted using an alternative theory were also higher for patients, when compared with called the Association-Induction Hypothesis, proposed (4) SY1.1 - OPTICAL TWEEZERS: SHINING LIGHT ON oxidative stress of cis-porphyrin (CisDiMPyP) could the control group, despite a lower binding frequency. by G. N. Ling in the 60’s . Additionally, in the same CELL BIOLOGY induce a continuous change of RBC deformations, The results can be associated with changes in blood oscillating cells we also measured temporal oscillations depending on irradiation degree, until finally causing flow, due to transient bridging of two erythrocytes by in thermodynamic variables such as temperature, heat Adriana Fontes1 hemolysis. To understand the signalling for the RBC fibrinogen, representing an important cardiovascular risk flux and volume. Oscillations in these variables have 1Department of Biophysics and Radiobiology, morphological change under the condition, RBC factor. γ’ fibrinogen may contribute for the increased risk the same frequency as oscillations in the activity of Federal University of Pernambuco, Recife, PE - shear modulus was measured by using micropipette in both diseases, as we demonstrate that its levels are intracellular metabolites, suggesting strong coupling ([email protected]) manipulation techniques. The obtained results showed increased in these patients’ blood. Our results may be between them. These results can be interpreted in the increasing of RBC shear modulus from 6.7 x 10-6 to relevant for potential future drug interventions to reduce light of a recently proposed theoretical formalism by T. 11 x 10-6 N/m dynamically or statistically depend on the erythrocyte aggregation and enhance microcirculatory Heimburg(5) in which isentropic thermodynamic systems Advances in the field of biophotonics have led to the photosensitizer effect. We hypothesize that cytoskeletal flow conditions in cardiovascular patients. can display coupled oscillations in all extensive and development of new optical tools able to be applied network reformation with the oxidative stress could cause intensive variables, reminiscent of adiabatic waves. to study diverse biological events. The dual nature of these behaviors. Therefore, the formation of cytoskeletal Guedes et al. (2016) Nature Nanotechnol. 11, 687; This interpretation suggests that oscillations may be light has allowed extracting important morphological, network of band 4.1R was also investigated by using Guedes et al. (2017) Nanoscale 12, 14897. a consequence of the requirement of living cells for rheological, and chemical information related to the the Stimulated Emission Depletion (STED) microscopy, a constant low entropy state while simultaneously cellular and molecular world of biological systems. and successfully visualized the band 4.1R positions of SY1.4 - The cell as a gel: materials for a performing biochemical transformations, i.e., remaining The optical tweezer, as a biophotonic tool, has been 169 ± 13 and 224 ± 10 nm distance (corresponded conceptual discussion metabolically active. This hypothesis, which is in line used not only to capture and move small particles with a reference result of contour length of spectrin with the view of the cellular interior as a highly structured (from micrometers to nanometers in size) but also to dimers between anchoring positions). We consider that Luis A. Bagatolli and near equilibrium system where energy inputs can measure its mechanical properties. The optical tweezer this further characterization of the RBC membrane with Instituto de Investigación Médica Mercedes y be low and sustain regular oscillatory regimes, challenge is a sensitive tool capable of: (i) measuring molecular the signaling will help to understand more complicated Martín Ferreyra (INIMEC-CONICET-Universidad the notion that biological processes are essentially and cellular biophysical parameters, without mechanical cytoskeletal network of immune, cancer or even neuron Nacional de Córdoba), Friuli 2434, 5016- Córdoba, dissipative. contact; (ii) detecting small variations in biological cells for therapeutic development in the future. Argentina. properties, and (iii) analyzing cells and biomolecules References: 1) Thoke HS, et al. (2015) Tight coupling of one by one providing individual measurements, rather SY1.3 - Arterial hypertension and stroke patients metabolic oscillations and intracellular water dynamics in than just average values, which may help to identify present higher erythrocyte adhesion forces, In recent studies we established that glycolytic Saccharomyces cerevisiae. PLoS One 10(2):e0117308. signatures of many biological conditions. In this talk, contributing to cardiovascular risk oscillations in non-dividing yeasts are tightly coupled 2) Bagatolli, L.A. and Stock, R. P. 2016. "The cell as a gel: we will present the basic principles related to the optical with the global state of intracellular water, being this material for a conceptual discussion" Physiological Mini 1 tweezer and illustrate its potential detailing biophotonic Nuno C. Santos phenomenon scale independent. Specifically, we Reviews 9(5): 38-49 3) Thoke HS, Thorsteinsson S, Stock RP, applications, such as those developed for the study of Filomena A. Carvalho1, Ana Filipa Guedes1, demonstrated that an optimum dynamic state of the Bagatolli LA, & Olsen LF (2017) The dynamics of intracellular red blood cells and protozoa. Carlos Moreira2, Ana Catarina Fonseca1,2, José B. major component of the cell cytosol, water, modulated water constrains glycolytic oscillations in Saccharomyces Nogueira2, José M. Ferro1,2 by optimal levels of ATP and an optimally organized actin cerevisiae. Sci Rep 7(1):16250 4) Ling GN (2001) Life at the This work was supported by CNPq, CAPES, and FACEPE. network is crucial to the emergence of the oscillations cell and below cell level. The hidden history of a fundamental 1 It is also linked to the National Institute of Science in Instituto de Medicina Molecular, Faculdade de (1-3), supporting the view of a highly coherent and revolution in biology (Pacific press). 5) Heimburg T (2017) Photonics (INCT-INFo). Medicina, Universidade de Lisboa, Lisbon, Portugal ordered cellular interior with properties similar to a Linear nonequilibrium thermodynamics of reversible periodic 2 ([email protected]) and Hospital Santa Maria, responsive hydrogel. These results, which are difficult processes and chemical oscillations. Phys Chem Chem Phys Centro Hospitalar Lisboa Norte, Lisbon, Portugal SY1.2 - Red Blood Cell Membrane: Mechanical to conceptualize using canonical cell models based in 19(26):17331-17341 Property Change and Cytoskeletal Network Reformation Under Photosensitizer-mediated The increase of erythrocyte aggregation by high Oxidative Stress of Cis-Porphyrin fibrinogen levels may be an indicator of cardiovascular risk. γ’ fibrinogen variant has been considered as a Koji Kinoshita possible player in enhancing aggregation. Here, we Department of Molecular Medicine, Faculity of assessed, at the single-cell level, the influence of Health Science, University of Southern Denmark, fibrinogen on erythrocyte aggregation in essential Odense, Denmark arterial hypertension (EAH) and stroke, as well as how it constitutes a cardiovascular risk factor for these patients. We also aimed at understanding how γ’ fibrinogen is Red blood cell (RBC) membrane has highly deformable altered on these patients. Differences on fibrinogen- and resilient property, due to a cytoskeletal network erythrocyte interaction and cell-cell adhesion forces consisting of proteins such as band 4.1R, spectrin, and were evaluated by atomic force microscopy (AFM)- actin. However, the connection between the cytoskeleton based force spectroscopy, with cells from EAH patients, network and the mechanical properties of RBC stroke patients and healthy blood donors. Results were membranes – i.e., “signaling” – is still not well understood. correlated with patients’ clinical profiles. AFM data show This is because of lack of comprehensive study to that the work and force necessary for erythrocyte- understand the complex of the signaling. Recently, our erythrocyte detachment is higher for patients than for research group found that a photosensitizer-mediated healthy donors, with these parameters further increasing

20 21 SY2 - Central Nervous System Electrophysiology SY2.3 - Ionotropic actions of metabotropic SY2.4 - Creating rest and unrest: distinct ways to glutamate receptors. create stable and unstable membrane potential in neurons. Christopher Kushmerick (1,2) - Nitrergic Modulation of Magnocellular SY2.1B - State-dependant affinity for general SY2.2 Ricardo M. Leão anesthetics dictates channel sensitivity Neurons from the Supraoptic Nucleus Everton dos Santos e Alhadas(1), Ana Maria Bernal Correia(1), Jennifer Diniz Soares Guimarães(1) 1Department of Physiology-FMRP, University of São Leonardo Cirqueira1 Melina Pires da Silva1 1) Paulo, Ribeirão Preto, SP ([email protected]). 1 1 1 1 Programa de Pós-Graduação em Fisiologia e Letícia Stock , Caio Souza and Werner Treptow Davi José de Almeida Moraes , André de Souza Farmacologia – ICB – UFMG. 2) Departamento de Mecawi2, José Antunes Rodrigues1 and Wamberto 1Department of Cell Biology, Biology Institute, Fisiologia e Biofísica – ICB – UFMG Antonio Varanda1 Central neurons usually have a stable resting membrane University of Brasília, DF ([email protected]) potential, firing action potentials only in response to 1 Department of Physiology, School of Medicine Metabotropic glutamate receptors are G-protein coupled synaptic stimulation. However, some neurons have Since their discovery, general anesthetics allowed a big of Ribeirão Preto – University of São Paulo, SP receptors widely expressed in the central nervous unstable membrane potentials leading to the spontaneous 2 change in medicine, making possible some procedures ([email protected]). Federal Rural University system. Binding of ligand to these receptors activates firing of action potentials. But some neuronal types that were unconceived without this class of drugs. of Rio de Janeiro, Department of Physiological signaling pathways leading to changes in neuronal present both types of behavior, a silent or quiet mode, Despite the wide pharmacological descriptions, major Sciences, Seropedica, Brazil. function. Here, we describe electrophysiological actions with a stable resting membrane potential, and an active advances in anesthesiology, the general anesthesia of mGluR receptors that increase neuronal excitability mode, which fires action potentials spontaneously. mechanism remains unknown. Electrophysiology The control of the excitability in magnocellular resulting in functional changes to neurotransmission Fusiform and cartwheel neurons from the dorsal studies show that the main targets of general neurosecretory cells (MNCs) of the supraoptic nucleus between the neurons involved. cochlear nucleus present such behavior, with 50% of anesthetics(GA) are the brain ion channels. There are has been attributed mainly to synaptic inputs from fusiform neurons present an active behavior and more two main points of view that concern the mechanism, circunventricular organs. However, nitric oxide (NO), than 80% of cartwheel neurons firing spontaneous action the indirect hypothesis, which states that change in a gaseous messenger produced in this nucleus during potentials at rest. Moreover, cartwheel neurons can membrane properties affects channels, and the direct- isotonic and short-term hypertonic conditions, is an present biostability, a situation where a neuron transits site hypothesis, which assumes that direct and specific example of a modulator that can act directly on MNCs spontaneously between 2 states. Here I will present the interactions between anesthetics and ion channels to modulate their firing rate. NO inhibits the electrical ionic mechanisms that these neurons use to produce modulate their behavior. Through direct-site point of excitability of MNCs, leading to a decrease in the release the quiet and active states, and show that specific ion view, modulation of a protein can be seen as differential of vasopressin and oxytocin. Although the effects of channels have distinct effects on the creation of quiet interaction between the two states, which suggest that NO on MNCs are well established, the mechanism by and active neurons in a neuronal population. there must be differences in most probable positions which this gas produces its effect is, so far, unknown. and in the number of ligands that neighbor the protein. Because NO acts independently of synaptic inputs, This work was supported by FAPESP (2012/09421-6, The molecular target used here is the mammalian we hypothesized that ion channels present in MNCs 2016/01607-4) voltage-gated potassium channel Kv1.2, which has are the targets of NO. To investigate this hypothesis, well characterized both open and closed states atomic we used the patch-clamp technique in vitro and in structures and is positively modulated by GA. Using situ to measure currents carried by hyperpolarization- the atomic models, there were performed extended activated and nucleotide-gated cation (HCN) channels (> 200ns) molecular dynamics simulations of both and establish their role in determining the electrical ion channel conformations embed in a membranous excitability of MNCs in rats. Our results show that system with fixed sevoflurane, isoflurane or propofol blockade of HCN channels by ZD7288 decreases concentrations. From each simulation, the spatial MNC firing rate with significant consequences on the mean probabilities of occurrence were acquired. In release of OT and VP, measured by radioimmunoassay. positive modulators (sevoflurane and isoflurane) there NO induced a significant reduction in HCN currents by were differences between the open and closed states. binding to cysteine residues and forming S-nitrosothiol In addition, simulations with propofol, an insensitive complexes. These findings shed new light on the ligand, will check if the different interaction hypothesis mechanisms that control the electrical excitability also explain the insensitive cases. These results offer a of MNCs via the nitrergic system and strengthen new and simpler way to see ligand-modulated protein the importance of HCN channels in the control of activation, and helps to explain a single component of a hydroelectrolyte homeostasis. more complex event: the general anesthesia. This work was supported by CAPES, FAPESP and CNPq This work was supported by CNPq, FAPDF and CAPES.

22 23 SY3 - Lipid-protein interactions in mimetic systems SY3.3 - Surface crowding and membrane SY3.4 - BIOPHYSICAL ASPECTS OF remodeling by IAPP BIOMINERALIZATION M. Prieto1 M. Bolean1 SY3.1 - CARDIOLIPIN AND MITOCHONDRIAL SY3.2 - HUMAN MPC2 AS AN AUTONOMOUS PROTEOLIPIDIC PLATFORMS: TOWARD A MEMBRANE TRANSPORTER J.C. Ricardo1, G. Scanavachi2, F. Fernandes1, R. Itri2 B.Z. Favarin1, H.G. Sebinelli1, E.A. Veschi1, M. and A. Coutinho1,3 Bottini2,4, M.F. Hoylaerts3. J.L. Millan4, P.Ciancaglini1. MITOCHONDRIAL RAFT-LIKE ORGANIZATION? Ambrosio1 1CQFM-IN and IBB, Instituto Superior Técnico, Univ. 1University of São Paulo, SP, Brazil (maytebolean@ Thierry Granjon R. Nagampalli1, J.E. Quesñay1, D. Adamoski1, Lisboa, Lisboa, Portugal (manuel.prieto@tecnico. usp.br) 2University of Rome Tor Vergata, Rome, Italy, Department of Chemistry and Biochemistry, Z. Islam1, J. Birch2, H. Sebinelli3, R. Girard4, C. ulisboa.pt), 2 Dept of Applied Physics, Univ. Sao 3University of Leuven, Belgium, 4Sanford Burnham University of Lyon, ICBMS UMR 5246-CNRS, Ascenção1, A. Fala1, B. Pauletti1, S. Consonni1, J. Paulo, Sao Paulo, Brazil, and 3Dept of Chemistry and Prebys Medical Discovery Institute, CA, USA. France Oliveira1, A. Silva1, K. Franchini1, A. Paes Leme1, A. Biochemistry, Fac. Ciências, Univ. Lisboa, Lisboa, Silber4, P. Ciancaglini3, I. Moraes2, S. Dias1 & A. Portugal During the process of endochondral bone formation, 1 Aim/hypothesis - Cardiolipin (CL) is a mitochondrial LNBio, CNPEM, Brazil(andre.ambrosio@lnbio. osteoblasts mineralize their extracellular matrix (ECM) 2 phospholipid predominantly found in the inner cnpem.br). MPL, DLS, England; 3FFCLRP, USP, Membrane-catalyzed amyloid fibril formation of human by promoting the synthesis of hydroxyapatite (HA) seed mitochondrial membrane and known to be crucial in Brazil; 4ICB, USP, Brazil islet amyloid polypeptide (hIAPP) or amylin has been crystals in the sheltered interior of membrane-limited several metabolism processes and in apoptosis. The implicated as a mechanism by which hIAPP exerts matrix vesicles (MVs). Several lipids and proteins present existence of functional CL platforms formed at the inner The active transport of glycolytic pyruvate across the its toxicity in type 2 diabetes. On the other hand, the in the membrane of the MVs mediate the interactions mitochondrial membrane under specific protein binding inner mitochondrial membrane is thought to involve membrane-bound aggregates of the rat variant of IAPP of MVs with the ECM and regulate the initial mineral was often evocated and discussed. We hypothesise that two mitochondrial pyruvate carrier subunits, MPC1 and (rIAPP) are unable to progress into fibrillar structures deposition and posterior propagation. Among the proteins the binding of two mitochondrial proteins, involved in MPC2, assembled as a 150 kDa heterotypic oligomer. since this is a non-amyloidogenic peptide. Here, we of MV membranes, ion transporters control the availability the metabolism processes and known to be associated Here, the recombinant production of human MPC combined steady-state and time-resolved fluorescence of phosphate and calcium needed for initial HA deposition. with CL, the mitochondrial isoforms of creatine kinase through a co-expression strategy is first described; methods and microscopy techniques to perform a Phosphatases play a crucial role in controlling the (mtCK) and of nucleoside diphosphate kinase (NDPKD) however, substantial complex formation was not comparative study on how anionic lipid membranes inorganic pyrophosphate/inorganic phosphate ratio that leads to such proteolipidic platforms. observed, and predominantly individual subunits were control the self-assembly of these two peptides. Our allows MV-mediated initiation of mineralization. The lipid purified. In contrast to MPC1, which co-purifies with a studies confirmed that membrane-catalyzed fibrillation microenvironment can help in the nucleation process of Research design and Methods - Simplified biomimetic host chaperone, we demonstrated that MPC2 homo- of hIAPP results in fibril growth into the solution. first crystals and also plays a crucial physiological role in systems made of CL-containing liposomes or monolayers oligomers promote efficient pyruvate transport into However, the membrane-mediated self-assembly of the function of MV-associated enzymes and transporters. were prepared to test mtCK and NDPKD binding effect proteoliposomes. The derived functional requirements rIAPP is confined to the lipid bilayer. To evaluate the The whole process is mediated and regulated by the on membrane fluidity, lipid organisation and protein and kinetic features of MPC2 resemble those impact of unlabeled rIAPP/lipid concentration on the action of several molecules and steps, which make the structure. Protein structural modifications upon previously demonstrated for MPC in the literature. membrane surface coverage reached by the peptide, process complex and highly regulated. Liposomes and membrane binding and effects on liposome membrane Distinctly, chemical inhibition of transport is observed FRET-based measurements of membrane binding proteoliposomes, as models of biological membranes, fluidity were analysed by combining both intrinsic or only for a thiazolidinedione derivative. The autonomous of a tracer amount of fluorescently-labeled Atto488- facilitate the understanding of lipid–protein interactions extrinsic fluorescence (using Laurdan as fluorescent transport role for MPC2 is validated in cells when the rIAPP were combined with fluorescence anisotropy with emphasis on the properties of physicochemical probe) and infrared spectroscopy. CL organisation within ectopic expression of human MPC2 in yeast lacking measurements of its membrane-bound oligomerization and biochemical processes. Here, we discuss the use monolayers after interaction with proteins was visualised endogenous MPC stimulated growth and increased state. We found that progressive membrane saturation of proteoliposomes as multiple protein carrier systems with Brewster angle microscopy. oxygen consumption. Multiple oligomeric species of with rIAPP correlated with its ability in reducing the intended to mimic the various functions of MVs during MPC2 across mitochondrial isolates, purified protein average size of anionic liposomes. Furthermore, a time- the initiation and propagation of mineral growth in the Results - Fluorescence and infrared spectroscopy and artificial lipid bilayers suggest functional high- resolved emission spectra (TRES) study of Laurdan course of biomineralization. We focus on studies applying approaches on liposomes indicated changes both in order complexes. Significant changes in the secondary revealed that membrane binding and oligomerization biophysical tools to characterize the biomimetic models in protein structure and phospholipid physical state upon structure content of MPC2, as probed by synchrotron of rIAPP produced an increased rigidity and surface order to gain an understanding of the importance of lipid– the protein-lipid interaction. Laurdan fluorescence radiation circular dichroism, further supports the dehydration in the vicinity of the probe that ultimately protein and lipid–lipid interfaces throughout the process. measurements indicated that protein binding to interaction between the protein and ligands. Our results might be responsible for rIAPP ability in remodeling the liposomes changed the phospholipid liquid-crystalline provide the initial framework for the independent lipid membranes. Acknowledgements: FAPESP (2016/21236-0), CNPq state to a more rigid state. Brewster angle microscopy role of MPC2 in homeostasis and diseases related to (304021/2017-2,167497/2017-0) and CAPES. investigations provide, for the first time, images dysregulated pyruvate metabolism. This work was supported by the Bilateral Project FAPESP indicating that both mtCK and NDPKD binding induced - FCT (FAPESP/20107/2014) and PPBI-POCI-01-0145- cluster formation on CL monolayers consolidating Funded by grants and scholarships from FAPESP and The FEDER-022122. JCR acknowledges grant SFRH/ and changing the morphology of the interfacial film. Wellcome Trust. BD/95856/2013 from FCT. (Portugal) Altogether, those findings support the hypothesis of the existence of proteolipidic platform in the inner mitochondrial membrane and suggest a major role of CL in the structuring of the mitochondrion.

This work was supported by University Lyon 1, CNRS and French Ministery of Research

24 25 SY4 - Nanostructures and Drug Delivery SY4.4 - STABILITY STUDY OF DRUG DELIVERY characterization. Based on its particle-by-particle SYSTEMS BASED ON NANOPARTICLE TRACKING approach, NTA provides size distribution and nanoparticle ANALYSIS concentration data in real time. Lately, several works SY4.1 - Iontophoresis impact on topical delivery lung inflammation, collagen deposition and smooth have demonstrated important relationship between Lígia N. M. Ribeiro1 of nanoparticles muscle hypertrophy in the lungs of a murine model nanoparticle concentration and biological performance of ovalbumin-challenged allergic asthma, leading Verônica M. Couto1, Leonardo F. Fraceto2, and of colloids. Given the importance of the nanoparticle Renata F. V. Lopez (USP) to improved lung mechanics. In the present model Eneida de Paula1 concentration elucidation, we propose its use as of chronic allergic asthma, highly compacted DNA mandatory analytical parameter in physicochemical 1 nanoparticles using thymulin analog gene modulated Department of Biochemistry and Tissue stability of colloidal systems used in drug-delivery. The the inflammatory and remodeling processes improving Biology, University of Campinas, Campinas, monitoring of nanoparticle concentration over time can 2 The application of a constant electric current of lung mechanics. SP ([email protected]) and Department also prematurely predict the degradation, erosion or low intensity, or iontophoresis, is traditionally used of Environmental Engineering, São Paulo State disruption of the nanoparticles, which directly affect University, Sorocaba, SP. to administer drugs through the skin aiming their SY4.3 - DEVELOPMENT OF FUNCTIONAL the colloid quality, and cannot be followed by changes systemic action. With the advent of nanoparticles, NANOPARTICLES FOR DELIVERY OF BIOACTIVES in size distribution, as currently measured by Dynamic the potential of iontophoresis to aid in the treatment Evaluating the physicochemical stability of Ligth Scattering. Nanoparticle concentration is the of dermatological disorders, such as skin tumors and Omar Mertins nanostructured colloids as drug delivery systems is a parameter that bridges the abyss between the in vitro inflammation, has been explored in association with the Patrick D. Mathews, Ana C.M.F. Patta, Bianca B.M. requirement to ensure their successfully application. characterization and biological performance of colloidal nanoparticles. Nanoparticles can modify the movement Garcia. However, assessing the structural properties of colloidal systems, and therefore, should be monitored in the of charged and non-charged hydrophilic molecules systems is difficult due to their intrinsic dynamism. stability study of colloidal formulations. through the skin under the influence of iontophoresis Department of Biophysics, Federal University of Sao Moreover, there is also a lack of in vitro techniques that enabling the targeting of drugs to specific regions of Paulo, São Paulo, SP. ([email protected]) can be directly correlated to the biological performance This work was supported by the FAPESP (#14/25372-0 the skin. Iontophoresis may also facilitate the uptake of colloids. Nanoparticle Tracking Analysis (NTA) and #14/14457-5). of nanoparticles by tumor cells. Results will be Micro and nanoparticles are currently designed for has been exponentially applied in the in vitro colloidal presented regarding the influence of iontophoresis on plethora of biomedical, biological and environmental the penetration of solid lipid nanoparticles, liposomes applications. The combination between polymers of and immunoliposomes in skin and in skin tumors. different properties or between polymer and lipids has Some surface properties of nanoparticles and their prompted the production of nano-delivery systems of impact on iontophoresis and cellular internalization will specific physicochemical characteristics, which enable be discussed. Finally, the effectiveness of treatment of its structure stability during route of administration skin tumors with nanoparticles by iontophoresis and by besides interaction to the expected target site, thus subcutaneous injection will be compared. improving delivery and performance of transported bioactive compounds. In this sense, pH-responsive SY4.2 - Nanoparticle-mediated gene delivery for nanoparticles, composed by polysaccharides chitosan the lung and alginate, where the two macromolecules respond differently to pH of application media thereby allowing Marcelo Morales both structural and surface charge modifications Instituto de Biofísica Carlos Chagas Filho - UFRJ of the nanoparticles, actually improve delivery and pharmacological effect of transported drugs. Moreover, chitosan modified with arginine and associated to Thymulin has been shown to present anti-inflammatory cationic liposomes has proven functional characteristics and anti-fibrotic properties in experimental lung in interaction and complexation of plasmid-DNA, diseases. We hypothesized that a biologically active stability in cell culture media, besides promoting high thymulin analog gene, methionine serum thymus factor, transfection yields. Hence, this functional lipopolyplex delivered by highly compacted DNA nanoparticles boosts DNA delivery, which is under technologic composed of single molecule of plasmid DNA scrutiny for genetic therapy. Indeed, the development compacted with block copolymers of poly-L-lysine and of functional nanoparticles as drug and gene carriers polyethylene glycol (CK30PEG) that have been found is an emerging area, and further promising to medical safe in a human phase I clinical trial, may prevent lung applications. inflammation and remodeling in a mouse model of allergic asthma. Thymulin plasmids were detected in This work was supported by the Sao Paulo Research the lungs of ovalbumin-challenged asthmatic mice up Foundation (FAPESP: 2015/23948-5; 2016/13368-4). to 27 days after administration of DNA nanoparticles carrying thymulin plasmids. A single dose of DNA nanoparticles carrying thymulin plasmids prevented

26 27 SY5 - Protein Complexes of the cluster is highly important to prevent Fe/2Fe-2S SY5.4 - Ras dimerization and allosteric and ROS accumulation and prevent damage to cells modulation of GTP hydrolysis linked in the and organs. Moreover, we also showed that long-term presence of Raf-RBD and the membrane susceptible to pressure denaturation. The aggregation dynamic changes in the NEET multi-domain proteins’ SY5.1 - Conformational diversity in protein Carla Mattos1 complexes: NMR studies of Allergens Bet v 1 and tendencies of p53C, p63C, and p73C were strongly backbone have significant effects on the functional Fag s 1 with ligands and DIII of Dengue virus E correlated with their thermal and pressure denaturation properties of their 2Fe-2S clusters. The involvement of Jillian A. Parker1, Morgan Packer1, Kendra Marcus1, protein with antibodies midpoints. Molecular Dynamics simulations indicated NEET proteins in metabolic diseases and in cancer was Lee Makowski1, Jay T. Groves2, Jean K. Chung2, specific regions of structural heterogeneity unique to shown. In the neurological Wolfram Syndrome type 2 Young Kwang Lee2 Ana Paula Valente1 p53C, which may be promoted by elevated incidence (WFS-2) disease, a monogenic autosomal recessive 1Department of Chemistry and Chemical Biology, 1Department of Structural Biology, Federal of exposed backbone hydrogen bonds (BHBs), reduced disorder, point mutation in the cisd2 gene (G109C), BHB strength, and increased water interaction with leads to exon skipping, frameshift and premature Northeastern University, 360 Huntington Ave, University of Rio de Janeiro, Rio de Janeiro, RJ, Boston, MA 02115. 2Departments of Chemistry, Brazil ([email protected]) BHBs compared to p63C and p73C. Based on the stop codon causing its encoded protein NAF-1 to be higher sensitivity of p53C to pressure denaturation, absent. In a cellular system of fibroblasts taken from University of California Berkeley, Berkeley, CA 94720. and elevated number of vulnerable BHBs, we propose WFS-2 patients, we show that the absence of NAF-1 We studied the structure and molecular dynamics of that water infiltration into the core of the p53C structure results in misdistribution of cellular iron, mitochondrial Ras dimerization is necessary for Raf activation on DIII, highlighting their role in the molecular mechanisms is a root cause of its instability. Using 3D sequence iron accumulation and oxidative damage with ensuing the membrane, yet the mechanism through which of Dengue virus infection and antibody recognition. alignments, we have identified regions of structural autophagy and apoptosis. These disorders can this occurs has been elusive. To date, attempts to We observed conformational exchange in the isolated vulnerability in p53C, suggesting new targetable sites be partially repaired by conservative iron chelator detect Ras dimers on supported membranes have DIII, in regions important for the packing of E protein for modulating p53C stability and aggregation, which is treatment. led to the conclusion that Ras does not dimerize to dimers on the virus surface. Antibody binding not only a potential approach to cancer therapy. the extent necessary to produce robust signaling removed the exchange regime in the epitope region, This work was supported by USA-ISRAEL Binational outcomes. Computational studies have identified in a process reminiscent of conformational selection, SY5.3 - THE ROLE PLAYED BY THE LABILE NSF-BSF program (BSF Grant awarded to RN No. multiple dimer interfaces, all of which are expected to but, somewhat surprisingly, also caused exchange in 2Fe-2S CLUSTERS OF NEET PROTEINS IN 2015831 and NSF grant awarded to RM No 1613462) have low dimerization affinity. Here we show that Raf other parts of DIII through allosteric effects. We also CONTROLING THEIR STRUCTURE, FUNCTION is a key element in Ras dimerization in the presence of studied the modulation of dynamics in allergens from AND FOLDING membrane phospholipid head group mimics in solution Bet v 1 family observed upon ligand binding and the and that the addition of Raf produces robust levels of importance for antibody-epitope complex formation. Rachel Nechushtai1 dimers on supported membranes containing Ras. Raf Our data suggest that the ligand-binding cavity of the Jose’ N. Onuchic2, Patricia A. Jennings3, Paolo binding to Ras and dimerization on the membrane allergen and the related changes in structural dynamics, Carloni4 and Ron Mittler5 appear to be concerted events that lead to a high-affinity as a key structural element for allergenicity. signaling complex. The emerging mechanism is one in 1 Alexander Silberman Life Science Institute, The which Ras dimerization and allosteric modulation of This work was supported by Conselho Nac. Des. Cient. Hebrew University, Jerusalem (rachel@maul. GTP hydrolysis in the presence of Raf are linked in the 2 Tecnológico (CNPq # 303785/2014-4; 426265) and by huji.ac.il) and Center for Theoretical Biological signaling cycle through the Ras/Raf/MEK/ERK pathway. 3 the Fundação de Amparo a Pesquisa do Estado do Rio Physics, Rice University, Houstone, Department de Janeiro (E-26/202.902/2015; E-26/201.314/2016). of Chemistry and Biochemistry, UCSD, La Jolla and 4Department of Physics RWTH- University of Aachen, Aachen 5University of Missouri School of SY5.2 - Role of Hydrogen Bonds in P53 Core Medicine, Columbia MO Domain Stability and Aggregation Elio A. Cino The human NEET protein family comprise of three and Igor M. Lima members: mitoNEET (mNT), NAF-1 and MiNT, encoded by cisd1, cisd2 and cisd3, respectively. While mNT Department of Biochemistry and Immunology, and NAF-1 are homodimeric membrane proteins, the Federal University of Minas Gerais, Belo Horizonte, most ancient MiNT protein is monomeric. All three MG ([email protected]) proteins share the novel ‘NEET-fold’ and a 3Cys:1His cluster-coordinating structure which conveys the The functionality of tumor suppressor p53 is altered clusters labile nature. NEET proteins were shown to be in over 50% of human cancers, and many patients important players in key cellular processes including exhibit amyloid-like buildups of aggregated p53. apoptosis, autophagy, Fe/2Fe-2S and reactive oxygen Understanding the initial steps that trigger pathogenic species (ROS) homeostasis. In a recent simulation amyloid conversion of p53 is required for further study, we demonstrated a key role for the cluster development of cancer therapies. Despite having coordinating histidine (H87 in mNT and H114 in NAF- ~60% sequence identity, out results from Thioflavin-T 1) and its de-protonated state in the stabilization of kinetics experiments showed that the p53 core domain the [2Fe-2S] cluster of the NEET proteins; The latter (p53C) aggregated faster, and to a greater extent than ensures the NEET proteins’ proper fold and prevents that of family members p63C and p73C, and was more the release of its clusters. Controlling the stabilization

28 29 SY6 - Modern Methods in Computational Biophysics further insight into enzyme selectivity for parasitic focused on the creation of sequences that stably adopt protease inhibitors. Altogether, by employing an a single target structure, ignoring the potential impact of interdisciplinary approach, our group has contributed protein dynamics in function. Although computational SY6.1 - THEORETICAL STUDIES OF STRUCTURAL hidden treasure of information lies in the combinatorial with new leads for drug discovery and improved protein design has enjoyed considerable success in AND ELECTRONIC PROPERTIES OF MOLECULES nature of DNA sequences, that is, the fact that base understanding of molecular interactions which play an creating new proteins using this approach, most have INTERACTING WITH PHOSPHOLIPID BILAYERS pairs can be arranged in so many ways and that each important role towards their potency. failed to match the efficiencies that are found in nature arrangement results in a different melting temperature. because standard methods do not allow for the design of Kaline Coutinho Combine this with a computationally efficient physical This work was supported by Conselho Nac. Des. Cient. exchange between necessary conformational states on a Instituto de Física, Universidade de São Paulo, model and a wealth of new information can be Tecnologico (CNPq # 306606/2017-8), FAPEMIG, functionally-relevant timescale. We developed a broadly- Brazil ([email protected]) uncovered. Here, we review how we obtained detailed CAPES and L’Oréal-UNESCO For Women In Science applicable computational method to engineer protein information about microscopic flexibilities by using a Program. dynamics that we term meta-multistate design. We model capable of dealing separately with hydrogen used this methodology to design spontaneous exchange The interaction of a pharmacophore with cell membranes bonds and stacking interactions. We will show how SY6.4 - Rational Design of Proteins that Exchange between two novel conformations introduced into the is a topic of great interest in biology and pharmacology we independently confirmed NMR measurements that on Functional Timescales global fold of Streptococcal protein G domain β1. The due to the possible relevance in the pathway of action. controversially suggested stronger hydrogen bonds designed proteins, named DANCERs, for Dynamic And There are several molecular features that govern the for AU in RNA. We will review some of the software Roberto A. Chica Native Conformational ExchangeRs, are stably folded behavior of a drug in cell membranes such as size, shape, that made all this possible and which is now freely Department of Chemistry and Biomolecular and exchange between predicted conformational states solubility, hydrophilicity, li- pophilicity, and pKa, among available. Furthermore, we will show how we were Sciences, University of Ottawa, Ottawa, Canada. on the millisecond timescale, as evidenced by nuclear others. In particular, studies based on drug partitioning able to improve the traditional Gibbs free energies with ([email protected]) magnetic resonance structures and ZZ-exchange in lipid bilayers and the thermodynamics of drug/lipid these same methods. Finally, we will present some experiments. The successful introduction of defined interaction have great importance in understanding the of the current projects of our group and discuss the dynamics on functional timescales paves the way to new reaction mechanisms of antitumor drugs and to design exciting possibilities revealed by those humble melting Proteins are the molecular machines of life, carrying out applications requiring a protein to spontaneously access new cell membrane-targeted drugs. In this work, we temperatures. complex physical and chemical processes that often multiple conformational states. report theoretical studies of the location, interaction require concerted motions of local protein structural and perturbing effects of some molecules of biological This work was supported by CNPq and Fapemig. elements. Previous efforts to design new proteins for This work was supported by the Natural Sciences interest on the model membrane, such as fluorescent applications in research, industry, and medicine have and Engineering Research Council of Canada probe [1], drug [2] and peptide [3]. SY6.3 - RATIONAL DRUG DESIGN TOWARDS NEW (RGPIN-2016-04831). TREATMENTS FOR NEGLECTED AND EMERGING This work was partially supported by CNPq, CAPES, DISEASES FAPESP, INCT-FCx, NAP-FCx(USP) and BioMol (Brazil). Rafaela S. Ferreira1 References: [1] W. Nitschke, C. Vequi-Suplicy, K. 1Department of Biochemistry and Immunology, Coutinho, H. Stassen, J. Phys. Chem. B, 116 (2012) 2713. Federal University of Minas Gerais, Belo Horizonte, [2] A. R. da Cunha, E. L. Duarte, H. Stassen, M. T. Lamy, K. MG ([email protected]) Coutinho, Biophys. Rev., 9, (2017) 729. [3] G. C. A. Hora, N. L. Archilha, J. L. S. Lopes, D. M. Müller, K. Coutinho, R. Itri, T. A. Soares, Soft Matter, 12, 8807 (2016). Better medicines are needed for several neglected and emerging diseases, for which currently available treatments have limited efficacy and safety. Our group employs structure-based drug design approaches to SY6.2 - MESOSCOPIC MODELS AS A TOOL FOR discover and develop protease inhibitors as new leads PROBING MOLECULAR INTERACTIONS IN DNA for treatment of diseases such as Chagas disease, AND RNA Human African Trypanosomiasis (HAT), and Zika virus Gerald Weber infection. By combining experimental screening and Departmento de Física, Universidade Federal de computational techniques, such as structure-based Minas Gerais, Belo Horizonte - MG. (gweberbh@ virtual screening, molecular docking, and molecular gmail.com) dynamics simulations, we have recently described novel series of inhibitors for the cysteine proteases cruzain and rhodesain, validated drug targets for Measuring melting temperatures is a routine task Chagas disease and HAT, respectively. These included for chemical scientist involved with the synthesis of compounds with nanomolar affinities to the enzymes. canonical or modified oligonucleotides. Yet, despite To aid compound optimization, we have also employed its importance, little practical information is gathered microsecond molecular dynamics simulations and from those melting temperatures. The information free energy calculations, being able to recapitulate retrieved is mostly limited to Gibbs free energies relative binding free energies in compound series, with which reveal almost nothing about the intramolecular RMSE up to 1.1 kcal/mol from the experimental values. interactions. However, we discovered that those melting Additionally, simulations revealed the most stable temperatures can reveal much more. The key to a interactions among potent compounds and provided

30 31 SY7 - Antimicrobial peptides SY7.2 - Lipid-packing perturbation induced by pathogens to its survival. The use of antimicrobial pH-responsive peptide in lipid membranes: effect biomolecules that act in a nonspecific way against of pH and peptide chemical modification. the microorganism causing an important change SY7.1A - ZIKA NS2B EPITOPE AS A CANDIDATE SY7.1B - COMPUTATIONAL SIMULATION OF and destabilization of the membrane structure is an Joao Ruggiero Neto FOR DIFFENTIAL DIAGNOSIS POPG AGGREGATES IN THE PRESENCE OF interesting alternative. Antimicrobial peptides are ANTIMICROBIAL PEPTIDE LL-37 Dept. of Physics - UNESP – S. Jose do Rio Preto immune system components of the fauna and flora Catarina Cataldi1 with proven action against many pathogens, mainly Yeny Y. P. Valencia1 Bruno Leite1 , Felix Loeffler2 , Nico Fischer3 , Danilo bacteria and fungi. The antimicrobial peptides are Coelho1 , Tereza Magalhães4, Marli Tenório1 , Gabriel C. A. da Hora1 and Thereza A. Soares1 Lytic peptides with antimicrobial properties are, in amphiphilic molecules with positive net charge 1,5 6 general, rich in cationic, hydrophobic and non-polar what confers to them a high affinity for membranes Ernesto Marques , Thomas Jaenisch , Isabelle 1 Department of Fundamental Chemistry, CCEN, Viana1 , Roberto Lins1 residues. The positive net charge warrants their of microorganisms, rich in anionic lipids. The Federal University of Pernambuco, Recife-PE preference for bacterial anionic plasma membranes, activity of antimicrobial peptides generally depends 1 Department of Virology, Aggeu Magalhães ([email protected]) and consequently the peptide net charge is correlated on a destabilization/rupture of the microorganism Institute, Oswaldo Cruz Foundation, Recife, PE with its affinity to phospholipid bilayer surface. Besides membrane. The mechanism of action of these peptides 2 ([email protected]) Max Planck Institute Antimicrobial peptides (AMPs) are biological compounds the importance of these electrostatic effects on the is commonly divided into two distinct modes of action: 3 of Colloids and Interfaces, Germany, University with broad-spectrum activity against bacteria, viruses peptide-membrane interaction, higher affinity does not pore formation and carpet mode. The mechanism of 4 of Tübingen, Germany Colorado State University, and fungi. Once in contact with the target microbial always result in lytic efficiency. Some peptides bearing action of antimicrobial peptides has been studied by 5 United States University of Pittsburgh, United membrane, the peptide can cause the cells to rupture both acidic and basic residues in their sequences can our group using biomimetic membrane systems. Here 6 States , Heidelberg University Hospital, Germany. through several hypothetical mechanisms (e.g. barrel have their net charges regulated by both the solution will be presented the studies on mode of action of the model, aggregate, carpet and toroidal pore). The AMP pH and the membrane electrostatic potential. The effect peptides Ocelatin PT7 and Esculentin 1b (1-18) using The major public health risks associated with the Zika virus LL-37 is derived from human cathelicidin and displays an of these two factors can result in smaller net charge biophysical tools as isothermal titration calorimetry, (ZIKV) pandemic infection are neurological disorders, such amphipathic alpha-helical structure at physiological pH for the peptide that sometimes is accompanied by differential scanning calorimetry, circular dichroism, as Guillain-Barré syndrome and congenital abnormalities. and a charge of +6. It consists of a chain of 37 residues higher lytic activity. This communication will focus fluorescence, microscopy of giant unilamelar vesicles, Due to the lack of vaccines and antivirals, the treatment (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) on this type peptide and on the effect of charge dynamic light scattering, zeta potential and electronic remains non-specific. Efficient diagnostic tools can help and acts on the first line of defense against local modulation by chemical modification and by the paramagnetic resonance. The results show that each stopping the virus spread and guide prophylactic and infection and systemic invasion of pathogens at sites solution pH. The association of several experimental antimicrobial peptide has a particular mechanism to therapeutic interventions, however, the main in the field of inflammation. Small-angle X-ray scattering (SAXS) techniques: visualization of giant vesicles, fluorescence interact with the membrane suggesting that is not trivial is the structural conservation among Flaviviruses, which and electronic microscopy measurements suggest spectroscopy, Langmuir monolayers and differential to classify the mode of action of peptide according to results in high cross-reactivity in serological tests. We that LL-37 destabilizes POPG bilayers. LL-37 induces scanning calorimetry allowed exploring correlations its structure. postulate that the identification of ZIKV specific epitopes the disappearance of multilamellar bilayers and the between lytic activity and changes in the membrane is crucial for the development of effective differential appearance of micelles in a concentration-dependent lipid-packing. These correlations have been shown that, This work was supported by FAPESP and CNPq diagnostic assays. To this aim, a high-density peptide manner. We have performed atomistic and coarse- despite the lower peptide net charge, some peptides array was synthesized by printing the whole proteome of grained MD simulations to bring higher resolution induce higher perturbation on the lipid- packing SY7.4 - A synthetic peptide derived from a toxin ZIKV, under the form of 15-mers peptides, on a microchip structural information into the experimental picture. We resulting in higher lytic efficiency. of the spider Phoneutria nigriventer: a potential and tested against 192 serum samples from ZIKVinfected have carried out simulations of pure POPG and pure therapeutic drug to treat erectile dysfunction individuals with and without Dengue – DENV background. POPC bilayer at different concentrations of the peptide Financial support: FAPESP and CNPq. The analysis of the IgG antibody profiles allowed the to investigate the process of LL-37 inducing transition in Maria Elena de Lima identification of a peptide sequence corresponding to the POPG but not POPC bilayers. Our simulations show that Instituto de Ensino e Pesquisa da Santa Casa ZIKV NS2b protein specifically recognized by 71% of the LL-37 binds to POPG bilayer in a horizontal orientation. SY7.3 - "USING BIOMIMETICAL MEMBRANES TO de Belo Horizonte. R. Domingos Vieira, 590 - ZIKV positive sera. The immunoreactivity of this epitope At low peptide concentrations (lipid:peptide mass ratio STUDY THE MODE OF ACTION OF PEPTIDES" Santa Efigênia, 30150-240-Belo Horizonte, MG. was further confirmed by ELISA, which showed that the = 15:1), LL-37 remains at the lipid headgroups area [email protected] Departamento identified peptide can partially discriminate ZIKV from throughout 3µs. Increasing the peptide concentration Katia Regina Perez de Bioquímica e Imunologia, Instituto de Ciências DENV infections. Quantitative analyses using Microscale (9:1), we observe a slow migration of the peptides to 1Department of Biophysics, Paulista Medical Biológicas, Universidade Federal de Minas Gerias Thermophoresis (MST) was used to determine the the hydrophobic region of the bilayer. Interestingly, the School, Federal University of Sao Paulo, Sao Paulo, (UFMG). Av. Antônio Carlos, 6627. 31.270 901 – antibody binding affinity of specific IgG antibodies to process appears to take place faster at the atomistic SP ([email protected]; perez.katiaregina@ Belo Horizonte, M.G, Brazil. [email protected]. the NS2b peptide. The MST data showed that ZIKV- than at the coarse-grained level, but further sampling gmail.com) br, [email protected] specific antibodies recognize the NS2b peptide with is required to verify this claim. We argue that the higher affinity (at least two orders of magnitude higher), POPG bilayer is destabilized by LL-37 via a carpet-like when compared to cross-reacting antibodies from mechanism consistent with experimental structural data. In recent decades the resistance of pathogens to the Accidents caused by the armed spider Phoneutria DENV-infected individuals. Future work will involve the conventional antibiotics has grown considerably and nigriventer may cause priapism, an involuntary/ engineering of this epitope in its native-like structure into This work was supported by PAEC OAS-GCUB, CAPES became an alarming public health problem. Therefore, painful erection in men and it has also been observed a scaffold protein aiming to improve its recognition by (Biocomp/BioMol), CNPq, FACEPE. the search for new drugs or antimicrobial strategies in animals. PnTx2-6 (or δ-CNTX-Pn2a) is a very specific antibodies. Ethical Committee approval numbers: with a lower propensity for resistance has been toxic peptide (48 amino acid residues, 10 Cysteines) 15580013.5.1001.5534 and 28309414.9.3001.5201. strongly encouraged. Conventional antimicrobial drugs purified from the venom of this spider and this toxin act mainly by inhibiting essential biomolecules in the is one of the molecules responsible for priapism. This work was supported by ZIKAlliance-EU, CAPES, CNPq and FACEPE.

32 33 To study the mode of action of PnTx2-6 could In addition, PnPP-19 showed no apparent toxicity, ve SY8 - Photobiology and its application in Health Sciences contribute to better understand erectile dysfunction r y low immunogenicity to mice and, different to the (ED). Erectile dysfunction is a growing world health native toxin, it did not target any sodium channel – the problem, especially in patients affected by vascular main targets of PnPTx2-6, neither affected rat heart. SY8.1 - Optical therapy and medical diagnosis it an interesting target for treating metabolic syndrome diseases, including diabetes and hypertension. In Compared to the native toxin, PnPP-19 shows several assisted by metallic nanoparticles (MS). On the other hand, photobiomodulation (PBM) addition, some people cannot use, or do not respond advantages, as a higher specificity, lower toxicity has proven to be beneficial to manage the chronic to the conventional drugs to treat ED. So, based on and immunogenicity, besides a simpler synthesis. Renato E. de Araujo inflammatory component of obesity and hyperglycemia the properties of PnTx2-6 to cause erection and on its The technology involving PnPP-19 was transferred to Laboratory of Biomedical Optics and Imaging, by irradiating WAT. However, PBM has still not amino acid sequence, we designed a smaller peptide, Biozeus company and so far, several pre-clinical tests Federal University of Pernambuco, Recife-PE been used to activate BAT. In this work, obese and called PnPP-19 (Phoneutria nigriventer potentiator have been done. In a first exploratory clinical trial in hyperglycemic mice were treated with PBM, and their peptide, 19 amino acid residues), in an attempt to healthy subjects, PnPP-19 showed to be safe when BAT was irradiated. The mouse serum was collected optimize a new compound, i.e. decreasing the toxicity topically administered. In conclusion, PnPP-19 could Metallic nanostructures are receiving increased and submitted to attenuated total reflection (ATR)- and retaining the potentiation of erectile function. The be indicated to patients not responding or not indicated attention as an important material for medical therapy Fourier transform infrared (FTIR) spectroscopy to experimental results, in vivo and ex vivo, showed to receive the conventional drugs used to treat ED. and medical diagnosis. In medical therapies, metallic evaluate the biochemical changes promoted by PBM. that PnPP-19 is able to potentiate erectile function, nanoparticles (NP) have been exploited in photothermal Five animals were fed with standard food (normal diet in normotensive rats and mice, besides to restore Supported by: CAPES, CNPq, FAPEMIG, INCTTOX and therapy applications. Light energy absorbed by the – ND) and used as negative control. The remaining 10 erectile function in hypertensive and diabetic animals. BIOZEUS. metallic NP can be converted to heat that dissipates animals were submitted to a hyperlipidic diet (high fat – Our studies have shown that action mechanism of to the environment, increasing the temperature of the HF) and their body mass, Lee index and glycemia were PnPP-19 involves the via of nitric oxide (NO)/cGMP. NP surrounding medium, thereby potentially destroying measured weekly during 15 weeks to induce MS. After cells or tissues. Various nanoparticle shapes, such as MS establishment, the HF animals were divided into two spheres, rods, shells and cages, show robust potential groups: HF and HF660. The HF660 group was exposed for thermal related therapy. Likewise, Photodynamic to six irradiation sessions using a 662 ± 20 nm LED. Therapy (PDT) has emerged as an important therapeutic The radiant exposure was 5.7 Jcm-2 at 19 mWcm-2 option, for numerous disease treatments. PDT per session, delivered at 300 s, on days 1, 3, 7, 10, combines photosensitizing drugs and light to induce 14 and 21. The HF group was sham-irradiated. After selective damage on a target tissue or microorganism. the treatment ended, the mouse serum was collected Metallic NP could improve PDT action by enhancing from cardiac puncture under deep anesthesia. Serum the generation of oxygen singlet by a photosensitized samples were prepared and analyzed by ATR-FTIR molecule. On medical diagnosis, metallic nanoparticles spectroscopy. The lipid absorptions were responsible have been used as platforms for biosensors. Several for the clearest differences among the IR spectra. examples of nanostructured optical biosensors were An intense C=O stretching absorption at 1742 cm-1

demonstrated, aiming the identification of cancer- was noticed only for HF group. Acyl CH2 stretching related biomolecules, Alzheimer disease, influenza absorptions at 2853 and 2925 cm−1 were also more virus, HIV-1 virus, Dengue virus, hepatitis B virus, and pronounced in HF group. After PBM, these major preeclampsia. In this presentation the light interaction lipid peaks decreased their vibrational modes. In fact, of light with metallic nanoparticles will be described, hierarchical cluster analysis identified the similarities and the use of NP on optical therapy and medical between the spectra and grouped ND and HF660 diagnosis will be reveled. groups into a same cluster. Taking together, our findings suggest that PBM applied to the BAT is able to promote SY8.2A - Biochemical changes in serum of obese biochemical changes in serum of obese mice mainly in mice related to photoactivation of brown adipose lipid bands. tissue This work was supported by FAPESP (grant Yoshimura, T. M.1 #2014/02564-1) and CNPq. Baptista, M. S.2; Ribeiro, M.S.1 1Centro de Lasers e Aplicações, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, 05508-000, São Paulo, SP, Brazil. 2Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Brazil. ([email protected])

Obesity is closely related to the development of insulin resistance and type-2 diabetes. Mammal fat consists of white and brown adipose tissues (WAT and BAT). Although most fat is energy-storing WAT, brown adipocytes dissipate energy as heat. Thus, the thermogenic capacity of BAT and its activation makes

34 35 [1] SY8.2B - How does cell membrane respond to PDT? References: I. Bacellar, M.C. Oliveira, L. Dantas, Anchieta and Municipal Hospitals showed a decrease be photodynamical inactivated with aforementioned E. Costa, H.C. Junqueira, W.K. Martins, A.M. of 80% of minor amputation in diabetic patients treated PS over a period of three weeks with light doses in the Tayana Tsubone (USP) Durantini, G. Cosa, P. Di Mascio, M. Wainwright, R. with PDT. PDT is efficient to treat osteomyelitis in scale of natural sunlight and in an alternating light/dark Miotto, R.M. Cordeiro, S. Miyamoto, M.S. Baptista, diabetic foot. regi me [ 6 ] . Comparison of the two PSs shows, that the Photosensitized membrane permeabilization requires efficiency of PDI largely depends on their localization [ 7 ] contact-dependent reactions between photosensitizer SY8.4 - Photodynamic inactivation (PDI) of in - and outside the cells . The algicidal effect of For several decades, Photodynamic Therapy (PDT) and lipids, J. Am. Chem. Soc. (2018). doi:10.1021/ biofilm forming microorganisms PDI is not limited to planktonic cultures but extends has been the focus of research and development to jacs.8b05014. [2]C. Pavani, Y. Iamamoto, M.S. Baptista, towards biofil ms. During formation of biofilms, facilitate medical field application. Despite advantages Mechanism and efficiency of cell death of type II Beate Röder microorganisms secrete EPS to form a highly resistant of PDT for a variety of applications, it has not photosensitizers: Effect of zinc chelation, Photochem. A. Preuß, J.Pohl. N. Shamali and T. Bornhütter protective film, making them impervious to most achieved an equally prominent position in clinical Photobiol. 88 (2012) 774–781. doi:10.1111/j.1751- conservative removal strategies. On model cultures of practice. A critical aspect during PDT treatments is 1097.2012.01102.x. [3]I.O.L. Bacellar, C. Pavani, H umboldt - Universität zu Berlin, Institut für Physik, green algae, cyanobacteria and samples from naturally the PDT efficacy and the determination of accurate E.M. Sales, R. Itri, M. Wainwright, M.S. Baptista, AG Photobiophysik occurring bi ofilms, we show that the secretion of this treatment protocols. One of the main strategy Membrane damage efficiency of phenothiazinium EPS can be reduced through PDI and that a successful used to increase PDT efficiency by several authors photosensitizers, Photochem. Photobiol. 90 (2014) Microorganisms like algae or mold fungi forming inactivation is possible even in biofilms. is based on photosensitizers (PS) that interacts 801–813. doi:10.1111/php.12264. [4]C.K. Haluska, biofilms on inside and outside walls are the main strongly with membranes, since lipid membrane M.S. Baptista, A.U. Fernandes, A.P. Schroder, C.M. cause for respiratory diseases in human and REFERENCES: 1. Th. Warscheid, J. Braams, International are important target of photosensitized oxidations. Marques, R. Itri, Photo-activated phase separation biofouling and biodeterioration of building materials Biodeterioration & Biodegradation , 2000, 343-368. 2. M. [1] “ISSN”:”15205126”,”abstract”:”Although the in giant vesicles made from different lipid mixtures, [1, 2] . This may result in substantial and irreversible Hoppert et al , Environmental Geology , 2004, 421-428. general mechanisms of lipid oxidation are known, the Biochim. Biophys. Acta - Biomembr. 1818 (2012) damages to historic and contemporary constructions 3. R.G. Welton et al. , Environmental Geochemistry and chemical steps through which photosensitizers and 666–672. doi:10.1016/j.bbamem.2011.11.025. [2 - 4] . Growth inhibition of phototroph ic organisms Health , 2003 , 139-145. 4. T . C . Dakal, S.S. Cameotra, light permeabilize lipid membranes are still poorly using Photodynamic Inactivation (PDI) could pose Environmental Sciences Europe , 2012, 36. 5. A. Krieger understood. Herein we characterized the products of - Title: Is Photodynamic Therapy Efficient an alternative to the use of environmentally harmful - Liszkay, Journal of Experimental Botany 56 , 2005, 337 SY8.3 6. lipid photooxidation and their effects on lipid bilayers, to Treat Osteomyelitis in Diabetic Foot? biocides. Here we present the successful PDI of – 346. J. Pohl, I. Saltsman, A. Mahammed, Z. Gross, also giving insight into their formation pathways. different microorganisms, including pathogenic and B. Röder, J Appl Microbiol 118 , 2015, 305 – 312. 7. T. Our experimental system was designed to allow two João Paulo Tardivo mold fungi, algae and cyanobacteria, using the well Bornhütter, J. Pohl, C. Fischer, I. Saltsman, A. Mahammed, phenothiazinium-based photosensitizers (methylene ABC Medicine School, São Paulo, Brazil - known photosensitizer (PS) TMPyP and a novel Z. Gross, B. Röder, Molecules 21 , 2016, 485 blue, MB, and DO15 In PDT, it is well known that the cationic phosphocorrol PCor + . Microorganisms role of membrane interaction with photosensitizers performing photosynthesis are confronted with singlet is crucial to the extension of photo-induced damage Antibiotic therapy and debridement are the most used oxygen naturally being generated in their photosystem[5] and consequently to the efficiency of cell death.[2,3] It practices to manage infectious diabetic foot, which and thus possess efficient quenching systems as a because the primary product of the photo-oxidation usually culminates with amputation. Presently there protection. Despite th is evolutionary advantage, plank (lipid hydroperoxides) depends on reaction between is no effective method that can be used to provide tonic cultures of green algae and cyanobacteria could 1 singlet oxygen ( O2) and unsaturated lipids. Although an efficient cure. It is necessary to develop novel the general consequences of lipid oxidation during treatment strategies. Photodynamic therapy (PDT) is PDT is well known, the impact that photosensitization a treatment modality that uses light to generate in situ may trigger on rafts membrane microdomains are reactive oxygen species, which can cause bacteria still poorly understood. We have previously reported death. Foot infections that do not respond well to that photosensitized oxidation reactions promote antibiotics can be treated with PDT. It is an efficient lipid rafts of a model membrane composed of POPC/ process, even against resistant microorganisms and DPPC/Cholesterol and this effect was interpreted as avoid the development of resistance. An inexpensive an enhancement in the hydrophobic mismatch due PDT protocol was developed and applied to diabetic to the formation of hydroperoxide lipid (POPCOOH) outpatients with osteomyelitis, in the Anchieta Hospital that remains immersed in the bilayer[4]. However, we of ABC Medicine School. In order to make the best did not observe the continuity of oxidation reactions choice between surgical management or PDT, an transforming POPCOOH lipids into cleaved tail lipids algorithm was created. It is called Tardivo Algorithm in order to understand the role of truncated lipid on and gives a predictive amputation score. The result rafts membrane. Therefore, we investigate the role gives confidence to adopt either PDT treatment or of different structures of oxidized lipids on rafts surgery. The score is based on three factors: Wagner microdomains by using an experimental approach classification, clinical signs of peripheral arterial based on fluorescence microscopy of giant unilamellar disease and the ulcers location. The final score is vesicles (GUV). Herein, I will present some data that obtained by multiplying the value of each factor. After reveal the consequences of different levels of lipid statistical analysis it was established that patients with photo-oxidation on rafts membrane microdomains, scores below 12 have better chances with PDT. While also giving insight into their possible effects on cell patients with scores equal or higher than 12, have more signaling and cell death. probability to a surgical approach. A clinical trial in the

36 37 SY9 - Structural and Unstructural Biology identification of organic compounds able to inhibit this for instance the binding to partially folded proteins to conversion is a promising strategy towards therapy. I is be delivered to Hsp70. However, the presence of the not possible to work with PrP infectious (mammalian J-domain characterizes Hsp40s or, by this reason, as disordered regions. The functional repertoire of IDPs is scrapie) material at UFRJ. Therefore, we established a J-proteins. The J-domain can be isolated maintaining SY9.1 - Assembly and Dynamics of Histone complementary to that of ordered proteins, with IDPs safer, faster, and more economical cell-free protocol for it function of Hsp70 ATPase stimulator. Apparently, Oligomers and Nucleosomes being commonly involved in regulation, signaling and in vitro aggregation of mammalian PrP in comparison it also maintains the same conformation as in the Garegin A. Papoian1 control pathways, where binding to multiple partners and with published methods. We used this protocol as whole protein although structural information on full 1 1 high-specificity/low-affinity interactions play a crucial basis to adapt a new in vitro aggregation protocol for J-proteins is still missing. This work reports the H, Department of Chemistry and Biochemistry, 15 13 Institute for Physical Science and Technology, role. It is suggested that functions of IDPs may arise alpha-synuclein. This protocol is useful to screen and N and C resonance assignments of the J-domain of University of Maryland, Colege Park, MD, USA from the specific disorder form, from inter-conversion select drug candidates against TSEs and PD and to a Hsp40 from S. cerevisiae, named Sis1. Secondary ([email protected]) of disordered forms, or from transitions between investigate their mechanism of action. We developed structure and order parameter prediction from chemical disordered and ordered conformations. The choice a PrP conversion protocol using concentrations as low shifts are also reported. Altogether, the data show that between these conformations is determined by the as 2 μM that reached plateau within 8 to 12 hours using Sis1 J-domain is highly structured and predominantly DNA is strongly compacted in cells of higher peculiarities of the protein environment, and many IDPs previously formed PrPSc-like seeds at 1%. The alpha- formed by alpha-helices, results that are in very good organisms, aided by the positively charged proteins, possess an exceptional ability to be highly responsive synuclein aggregation protocol was standardized at 10 agreement with those previously reported for the called histones, where the latter help to fold DNA into to change in their environment and to fold in a template- to 25 mM protein (much lower than the usual 140 mM crystallographic structure. dense superstructures, called chromatin. Flexible dependent manner. All this requires a close attention concentration). We verified that our most promising tails protruding from histones mediate and maintain to the odd biophysics of IDPs. In this talk, some key antiprion compound, a trimethoxychalcone, which KEYWORDS: HSP40 type II, co-chaperone, J-domain, nucleosomal packaging in chromatin fibers and, thus, biophysical features of IDPs will be covered. In addition efficacy was previously verified in scrapie-infected cell NMR significantly contribute to chromosomal remodeling and to the peculiar sequence characteristics these unusual lines, significantly decreased rPrP conversion in this gene activation processes. The physical mechanisms biophysical features include sequential, structural, new method. Besides, this same compound seems to behind chromatin assembly and dynamics are still and spatiotemporal heterogeneity of IDPs; their rough be effective against alpha-synuclein aggregation. Thus, not fully understood. To shed light on these problems, and relatively flat energy landscapes; their ability to this method has the potential to provide a simplified, we used atomistic and coarse-grained simulations to undergo both induced folding and induced unfolding; the safe, and economical approach to search for antiprion investigate the conformational dynamics of regular ability to interact specifically with structurally unrelated and anti-PD compounds and assess their underlying nucleosomes as well as ones where the H3 histone is partners; the ability to gain different structures at binding mechanism of action. substituted by its centromeric variant, CENP-A. Despite to different partners; and the ability to keep essential close structural similarity, we found profound differences amount of disorder even in the bound form. IDPs are in the dynamics of these nucleosomes. In particular, also characterized by the “turned-out” response to the SY9.4 - NMR structure of the J-domain of co- we discovered that the CENP-A nucleosomes are more changes in their environment. It is proposed that the chaperone Sis1 from Saccharomyces cerevisiae structurally distortable in the context of dimeric and heterogeneous spatiotemporal structure of IDPs/IDPRs octameric complexes, but more compact and rigid in the can be described as a set of foldons, inducible foldons, Pinheiro, Glaucia M.S.1* tetramer state. Our studies shed light on the structural semi-foldons and non-foldons. They may lose their Amorim, Gisele C.2,3*; Iqbal, Anwar2, Ramos, C.H.I.1 frustrations as histone complexes oligomerize to higher function when folded, and activation of some IDPs is and Almeida, Fabio C.L.2*, order structures, with this frustrated landscape having associated with the awaking of the dormant disorder. important functional implications. Finally, we used 1Institute of Chemistry, University of Campinas coarse-grained simulations to study how the H1 linker UNICAMP, Campinas SP. Brazil. 2 Institute of Medical Biochemistry and Nucleus for Structural Biology histone interacts with nucleosomal DNA and other SY9.3 - Prion protein and alpha-synuclein and Bioimaging (CENABIO) - Federal University histones. aggregation in vitro: high-throughput method as of Rio de Janeiro, Rio de Janeiro RJ. Brazil. 3 tool to select and study therapeutic compounds Numpex-Bio - Federal University of Rio de Janeiro, SY9.2 - Unusual Biophysics and Strange Biology against neurodegenerative disease Duque de Caxias RJ. Brazil. correspondent authors: of Intrinsic Disorder Yraima Cordeiro [email protected], falmeida@cnrmn. Vladimir N. Uversky bioqmed.ufrj * authors contributed equally in the Faculdade de Farmácia, Universidade Federal do abstract body Department of Molecular Medicine, University Rio de Janeiro (UFRJ), RJ, Brazil. of South Florida, Tampa, Florida 33612, USA; Laboratory of New Methods in Biology, Institute for Biological Instrumentation of the Russian Academy of The conversion of the cellular prion protein (PrPC) into Protein folding in the cell is usually aided by molecular Sciences Pushchino, Moscow region 142290 Russia; the aggregated scrapie form (PrPSc) is the molecular chaperones, from which the Hsp70 (Hsp=heat shock basis for a group of incurable neurodegenerative protein) family has many important roles, such as diseases named transmissible spongiform encepha- aiding nascent folding and participating in translocation. Intrinsically disordered proteins (IDPs) lack stable lopathies (TSEs). Alpha-synuclein aggregation (a-syn) Hsp70 has ATPase activity which is stimulated by tertiary and/or secondary structure under physiological is a hallmark of Parkinson´s disease (PD) and the binding to the J-domain present in co-chaperones from conditions in vitro, often resembling ‘protein clouds’. pathogenesis of PD shares characteristics with TSEs. the Hsp40 family. Hsp40s have many functions, as Computational studies revealed that IDPs are highly In both neurodegenerative diseases, abnormally abundant in nature, as ~25-30% of eukaryotic proteins folded proteins can act as seeds to induce native are mostly disordered, and >50% of eukaryotic protein (PrPC or a-syn) conformational conversion into proteins and > 70% of signaling proteins have long more abnormal and aggregated species. Thus, the

38 39 SY10 - Biophysics of the heart SY10.3 - The role of NLRP3/Casp1/IL-1β axis SY10.4 - Cardiac electrical and mechanical study in renal ischemia/reperfusion-induced cardiac on a type 2 diabetes experimental model. electrical disturbances Emiliano Medei SY10.1 - Keep your heart stressed: impaired SY10.2 - PROARRHYHMIC EFFECTS OF Micaela Lopez Alarcón1 superoxide production and pathogenesis of DOXORUBICIN IN THE MYOCARDIUM Laboratory of Cardioimmunology – Federal 2 1 Chagas disease 1 Mayra Trentin-Sonoda, Claudia Paiva Neto, University of Rio de Janeiro - Brazil. Rosana A. Bassani 1 2 1 Danilo Roman Campos2 Fabiano Ferreira, Karine Panico, Emiliano Medei, Natália F Oshiyama1, Alexandre C Pinto2 and José 2Marcela Sorelli Carneiro-Ramos. Artur Santos-Miranda1a, Julliane Joviano1b, Petter 1,3 Type 2 diabetes (T2D) is the most prevalent form of WM Bassani 1 2 1a 1a Universidade Federal do Rio de Janeiro. Universidade diabetes and represents about 90% of the diabetic cases Guerra , Grazielle Alves Ribeiro Ana Flávia 1 1c 1a Center for Biomedical Engineering (rosana@ Federal do ABC. ([email protected]) all over the world. As a consequence of the lifestyle Mchado botelho , Leda Quércia Vieira , Jader 3 Santos Cruz1a. ceb.unicamp.br) and School of Electrical and and feeding, this syndrome has turned into one of the Computer Engineering, University of Campinas, Patients with chronic renal ischemia have a high largest health problem worldwide and it is associated 1aDepartment Biochemistry, 1bDepartment of 2 Campinas, SP; and Ribeirão Preto Medical School, risk to develop cardiovascular diseases. Therefore, with an increase of premature appearance of several Morphology, 1cDepartment of Veterinary Clinical University of São Paulo understanding the pathogenic links between renal disorders such as cardiovascular complications, and Surgery, Federal University of Minas Gerais; ischemia and cardiovascular disease is of utmost which can evoke cardiac electrical disturbances as 2Department of Biophysics, Federal University of Doxorubicin (DOX), a potent, widely used anti-neoplastic importance. Previous work of our group showed a arrhythmias. Thus, the aim of the present study was São Paulo ([email protected]) agent, may lead to acute development of arrhythmias, systemic peak of IL-1β on the day 8 after renal ischemia- to investigate the mechanisms underlying the cardiac in addition to chronic cardiomyopathy. Several reperfusion (IR) in mice. This finding was associated with electrical and mechanical remodeling in a T2D mice Chagas disease, caused by the triatominae Trypanosoma mechanisms have been implicated in DOX cardiotoxicity, prolonged QJ on the EKG, an arrhythmogenic marker, model which combines a high fat diet (HFD; 45% of cruzi, is one of the leading causes of heart malfunctioning including production of oxidative stress and cytosolic sustained up to day 15. We have also demonstrated the fat) and low doses of streptozotocin (STZ). Blood in Latin America. The cardiac phenotype is observed Ca2+ overload. In ventricular myocytes, DOX decreased important role played by resident heart macrophages glucose levels, intraperitoneal glucose tolerance test in 20-30% of infected people 10-40 years after their NAD(P)H fluorescence emission, which is a hallmark in IL-1β release upon NLRP3 inflammasome activation (IPGTT), intraperitoneal insulin tolerance test (IPITT) primary infection. The cardiac complications during of oxidative stress. DOX also increase diastolic [Ca2+] and in cardiac electrical remodeling. Thus, in this work and serum insulin levels were used to confirm T2D. In Chagas disease range from cardiac arrhythmias to heart i, which was accompanied by reduction in sarcoplasmic we tested the hypothesis that the activation of NLRP3/ vivoelectrocardiogram recordings showed prolonged QJ failure. Using a murine model of Chagas disease, in the reticulum (SR) Ca2+ content and enhancement of SR Casp1/IL-1β axis is involved in cardiac electrical interval on T2D mice, which can be explained at least 2+ last years, our group demonstrated that the altered heart Ca leak. Additionally, DOX augmented the density disturbances promoted by renal IR. Our data showed that partially, by transient outward potassium current (Ito) function is associated with a profound electromechanical of the current carried by the sarcolemmal Na+/Ca2+ the genetic ablation of NLRP3 or Caspase-1 prevented decrease. In addition, when T2D mice were submitted remodeling of cardiomyocyte cells during the time exchanger (NCX). The functional impact of this change either the QJ prolongation and the renal IR-induced to an arrhythmia protocol combining caffeine and course of infection. Proper therapy to altered cardiac was confirmed in intact myocytes by the observed systemic peak of IL-1β on day 8 (measured by ELISA). dobutamine, not only more susceptibility but also dysfunction in Chagas disease is still missing. There are increase in the estimated NCX-mediated Ca2+ flux and In fact, renal IR failed to prolong the QJ interval in IL- arrhythmia severity was observed in T2D animals. some evidences in literature that antioxidant therapy may in the NCX relative contribution to [Ca2+]i decay during 1r-/- mice. The depletion of macrophages (by Clodronate Furthermore, ventricular function was studied by be of benefit for cardiac function during the time course of a twich. Most changes were attenuated by pretreatment Liposomes) on day 7 post- renal IR was able to prevent magnetic resonance imaging (MRI-7T), which showed disease. In cardiomyocytes there are a range of production with the antioxidant N-acetyl-cysteine. Thus, DOX may the IR-induced longer QJ observed at day 15 post-IR. lower ejection fraction (%) on diabetic mice. Collectively sources of Reactive Oxygen Species, including the acutely predispose the myocarium to generation of Finally, the daily treatment with IL-1r-antagonist from day these results indicate that a combination of a HFD and NADPH oxidase 2 (NOX2). Thus, we probe the function Ca2+-dependent ectopic activity by stimulating two 8 to day 15 reverted the IR-induced longer QJ. The data low doses of STZ recapitulate the cardiac T2D classical of NOX2 (gp91 PHOX knockout mice) in heart function proarrhythmic mechanisms that are serially connected: presented here demonstrates that NLRP3/Casp1/IL-1β electrical and mechanical events. during the acute phase of Chagas disease. Ablation of diastolic SR Ca2+ leak and NCX-mediated Ca2+ efflux. axis is involved on the induction of prolonged cardiac QJ NOX2 lead to profound cardiomyocyte electromechanical These effects appear to involve the DOX-induced by renal ischemia/reperfusion. Additionally, the block of dysfunction when compared to infected control mice generation of oxygen reactive species. the IL-1r was found to be a potential therapeutic approach and it was associated with increased nitric oxide and to revert the IR-induces cardiac changes. reduced superoxide production. Reduction of nitric oxide This work was supported by Conselho Nac. Des. Cient. production in isolated cardiomyocytes restored most of Tecnologico (CNPq # 302996/2011-7). This work was supported by Conselho Nac. Des. Cient. electrical properties of cardiomyocytes, suggesting that Tecnologico (CNPq # 306004/2015-1) and by the complete abrogation of superoxide production is not a FAPESP (2008/10175-4 e 2015/19107-5). good terapeutical option for Chagasic patients.

This work was supported by São Paulo Research Foundation (2014/09861-1), CAPES, CNPq and FAPEMIG-Pronex

40 41 SY11 - Methods in Biophysics in conformation or dynamics and can be coupled to regulation of the exchanger activity. Binding of Ca2+ to XLMS to better describe the structure and dynamics CBD12, the exchanger calcium-sensor domain that is of proteins and complexes. In this talk, the current located at the intracelular loop, activates the mammlian SY11.1 - Quantifying collective dynamics in the structure determination by cryo-EM alone has proved status of XLMS and HDX will be presented along with exchanger and inhibits the Drosophila exchanger. We 2+ ribosome feasible. For example, the resolution levels achieved interesting exaxmples on how these techniques have characterized the energetics of Ca -binding to the in determining the structure of the bacterial ribosome, been applied in the context of integrative approach. Drosophila CBD12 domain using isothermal titration 1 Paul C. Whitford already exceed those achieved by conventional X-ray calorimetry (ITC), and followed changes in the Jeffrey K. Noel2 and Mariana Levi3 crystallography. It is now even possible to obtain This work was supported by Fapesp (2014/17264-2). protein backbone dynamics due to Ca2+-binding by sets of atomic-resolution 3D reconstructions of measurements of 15N relaxation rates. We found that 1 Department of Physics, Northeastern University, 2+ macromolecular machinery in action from very large, SY11.4 - Solution NMR spectroscopy studies of the binding of Ca to CBD12 is highly cooperative as Boston, MA, USA ([email protected]) and 2+ noisy EM data sets (movies of 3D structures, or ‘4D’ + 2+ expected for a biological switch. The binding of Ca to 2 the Na /Ca exchanger DMax Delbruck Center for Molecular Medicine, structures). The improvements we have seen over the CBD12 decreases inter-domain motions as observed 3 Berlin, Germany and Department of Physics, last decade include: better handling of the cryogenic Roberto K. Salinas previously for the canine exchanger, and this provides Northeastern University, Boston, MA, USA 2+ specimens; computer-control of ever better electron Department of Biochemistry, University of São clues about how Ca regulates the exchanger activity. microscopes; novel data-processing approaches Paulo, São Paulo, SP ([email protected]) The functional dynamics of molecular assemblies often running on powerful new computers; and last-but-not- This work was supported by the Brazilian National involve large-scale collective rearrangements. While least, the direct recording of electrons in very sensitive Research Council (CNPq #420490/2016-7) and by the these motions occur in a high-dimensional space, new digital cameras. At the resolution level of around The Na+/Ca2+ exchanger is a key player in the control São Paulo Research Foundation (2016/07490-1). experiments are typically only able to simultaneously 3-4Å cryo-EM reaches an important threshold: we of intracellular Ca2+ homeostasis. Altered functioning measure a small number of interatomic distances. now start seeing side chains of proteins allowing for of this exchanger is related to various pathologies, Accordingly, a major challenge in single-molecule a better insight in their functioning. The 2017 Wiley such as stroke and arrythimias. The exchanger studies is to identify kinetically-relevant degrees of Prize, and the Nobel Prize for Chemistry 2017 were consists of a transmembrane domain that catalyzes freedom. To address this issue for the ribosome, both awarded to cryo-EM in recognition of the decades the transport of ions across the cell membrane, and we use a range of theoretical models and molecular of developments that led to this current 4D visualization a large intracellular loop that is responsible for the dynamics simulations in order to simulate hundreds of revolution. spontaneous large-scale (~30-50 Å) conformational transitions at various points of the elongation cycle. This research was financed in part by grants from the With these large data sets, we are assessing the Dutch Ministry of Economic Affairs (Cyttron II FES-0908), ability of experimentally-accessible coordinates to the BBSRC (grant BB/G015236/1), and the Brazilian capture the rate-limiting free-energy barriers. This Science Foundation CNPq (grants CNPq-152746/2012- analysis suggests design strategies for next-general 9 and CNPq-400796/2012-0). experiments, as well as helps rationalize controversial/ contradictory experimental observations. Finally, these SY11.3 - Advances in Mass Spectrometry as a calculations provide a quantitative foundation that is Structural Biology Tool allowing us to study the precise relationship between structure and dynamics in the ribosome. Fabio Cesar Gozzo1 1Institute of chemistry, University of Campinas, This work was supported by the National Science Campinas, SP (email:[email protected]) Foundation (grant MCB-1350312).

SY11.2 - Dynamic Structures of Biological Nano- Mass spectrometry (MS) is currently the most powerful Machines by Single-Particle Cryo-EM tool for large scale protein identification, quantitation and determination of post-translational modifications. Marin van Heel 1 2 3 Recent developments in instrumentation, software 1LNNano-CNPEM Campinas SP (marin.vanheel@ and chemistry, however, have expanded the use of gmail.com) 2Biology Dept. Leiden University, MS to study structure, conformation and dynamics The Netherlands 3Imperial College London, UK of proteins and protein complexes, making it a ([email protected]) useful complementary technique. Different MS based techniques exists, giving different information and, together, can expand our understanding of structural Molecular nano-machines – better known as aspects of a system. Chemical cross-linking/mass macromolecular complexes – perform most biological spectrometry (XLMS) give us distance constraints processes in all walks of life. In recent years electron between residues in a protein complex and can be microscopy has gone through a “resolution revolution” used to model protein-protein complexes. Recently, which has made cryogenic electron microscopy to a the set of distance constraints was used to solve technique capable of elucidating the three-dimensional the structure of protein for the first time. Hydrogen/ (3D) structure of large macromolecular complexes Deuterium exchange (HDX) experiments, another with an ever-improving level of detail. A de-novo MS based technique, is very sensitive to changes

42 43 SY12 - Nanostructures SY12.3 - TARGETING THE HOST-CELL FOR SY12.4 - Thermosensitive hydrogels and IMPROVED THERAPY OF LEISHMANIASIS organogels: from supramolecular structure to new drug-delivery systems SY12.2 - CUBOSOMES AS DELIVERY FOR MANY Frédéric Frézard1 SY12.1 - Biological Functionalisation of DRUGS Daniele Ribeiro de Araujo1 Synthetic Nanoparticles Using Novel 2D Peptide Cynthia Demicheli2, Ricardo T. Fujiwara3, Luiz O. 1 Assemblies Leandro Ramos Souza Barbosa Ladeira4, Sydnei M. Da Silva5, Philippe M. Loiseau6, 1Human and Natural Sciences Center, Federal 6 1,6, 1Department of Physics, University of São Paulo, Sébastien Pomel , Juliane S. Lanza Guilherme S. University of ABC. ([email protected]) Alan B. Dalton 1 1 São Paulo, SP ([email protected]) Ramos , Virginia M. Carregal Cristiano C. P. Dos School of Mathematics and Physics, Department of Santos1 and Arshad Islam1 Poloxamers (PLs) are thermosensitive triblock Physics, University of Sussex 1 Nanomedicine is one of the research areas with Department of Physiology and Biophysics copolymers, composed of polyethylene oxide (PEO) and 2 increasing interesting due to its fundamental role in ([email protected]), Department of Chemistry, polypropylene oxide (PPO) units, able to self-assemble 3 4 Here we present a route for non-covalent several areas, mainly those related to drug delivery Department of Parasitology, Department of in micelles as a result of the PPO dehydration groups functionalization of graphene and related materials with and its applications. There are several different Physics, Universidade Federal de Minas Gerais, and micellar aggregation, forming hydrogels at a critical 5 novel two-dimensional peptide assemblies. We show possibilities to produce the nanoparticle, depending Belo Horizonte - MG, Brazil, Department of gelation temperature. Those properties allow the PL that self-assembled amino-terminated biantennary and on the use and on its application. Commonly we Immunology, Microbiology and Parasitology, application in the chemical, cosmetic and pharmaceutical tetraantennary oligoglycine peptides (referred to as find: metallic nanoparticles (Au, Ag and Si), micelles, Universidade Federal de Uberlândia, Uberlândia – fields. The incorporation of different components (drugs, 6 tectomers) effectively coat and strongly interact with liposomes, proteasomes, solid lipid nanoparticles, MG, Brazil and UMR 8076 CNRS, Université Paris- surfactants, oils, polymers, nanoparticles, organic graphene oxide due to electrostatic interactions and polymersomes, proteasomes and cubosomes, among Saclay, Chatenay-Malabry, France solvents, solubilizers) into PL-based formulations has hydrogen bonding as the driving force, respectively. others. Cubosomes have a quite interesting perspective directed investigations regarding to their influence The resulting hybrids can be made into free-standing since due its peculiar inner cubic structure can have Current treatments of leishmaniasis have several on supramolecular structure and biopharmaceutical conducting composites or applied in the form of thin, around ~50% of hydrophobic volume, much larger drawbacks including undesired side effects, resistance properties. Our research group has presented some pH-switchable bioadhesive coatings onto graphene than the ~4% of a common liposome, for instance. development and high cost, requiring new more results about PL hydrogels (unique and binary oxide fibers. Monitoring of cell viability of pancreatic Here we present the production and characterization of effective antileishmanial approaches. Considering systems, with different hydrophilic-lipophilic balances- cell lines, seeded on the hybrids, show that they can cubosomes composed by monoolein or phytantriol, in that Leishmania is an obligate intracellular parasite HLB) phase organization and the influence on drug be used as two- and three-dimensional scaffolds to the absence and presence of several target molecules, of cells of the macrophage-dendritic cell lineage, release mechanisms by intra-articular, subcutaneous, tissue engineer tumour models for studying ex vivo the like: miltefosine and cationic ionic liquid. On one hand, approaches based on targeting the host-cell have been intramuscular and intracolonic routes. More recently, we tumour development and response to treatment. This miltefosine is a alkyl-based amphiphilic and zwitterionic explored. Here, we report the antileishmanial targeting have also devoted to the study of PL-based organogels, highly versatile method in producing pH-responsive molecule, used in the treatment of some cancers and strategies under investigation by our group and the associating different oil phases (lecithin, lanolin), as hybrids and coatings offers an attractive platform also was found to have an interesting effect against most promising results. These strategies comprise skin-delivery systems. The association of PL with for a variety of biomedical applications and for the Leishmaniosis. On the other hand, cationic-based those aimed at enhancing the host microbicidal activity natural polymers (such as hyaluronic acid, cellulose development of functional materials such as smart nanoparticles are being used to the transfection of some and carrier-mediated drug delivery. In this context, we derivatives) or other nanocarries (such as polymeric textiles, sensors and bioelectronic devices. We also nucleic acids, like DNAs and RNAs. Herein we report a demonstrated that the immunomodulator fullerol is a nanoparticles) can modulate the temperatures relative show that the tectomer sheets have a strong affinity for systematic physical-chemical characterization of those potent antileishmanial agent, through in vitro and in to micellization/gellation and stabilize the hydrogels silver nanowires through a charge-transfer interaction drug-containing nanoparticles using small angle X-ray vivo studies in experimental models of leishmaniasis. and organogels rheological properties. The most as evidenced by X-ray photoelectron spectroscopy. scattering (SAXS), cryogenic transmission electron Its incorporation in liposomes further enhanced its pronounced structural changes were observed on drug We show that extended assemblies of metal-peptide microscopy (cryo-TEM), dynamic light scattering antileishmanial activity in a murine model of visceral release and permeation profiles, since slower release/ hybrids offer additional augmentative functionalities, (DLS), and ζ-potential. According to SAXS results, leishmaniasis. We also found that amphiphilic Sb(V) premeation constants were obtained according to the for instance, the tectomer sheets are hydrophobic in phytantriol, in the absence of any external molecule, complexes improved the uptake of Sb by the host-cell hydrophobic PL or polymeric nanoparticles insertion, as nature and act as a protective layer preventing oxidation the cell parameter was 6.75(07) nm and the inner and and were much more active by the oral route, when a result of their different phase organization transitions. and degradation of the nanowires when exposed to Pn3m cubic structure. Such inner structure was also compared to conventional antimonial drug. Finally, we In addition, the incoporation of lanolin and lecithin as oil atmospheric conditions. Moreover, for silver nanowire confirmed by cryo-TEM measurements. DLS indicates developed different liposome formulations of antimonial phases enhanced the drug permeation across artificial percolating networks the presence of the peptide that the cubosomes have an effective hydrodynamic drug and established their therapeutic efficacy and membrane skin-model, point out the application of both markedly increases the overall electrical conductivity diameter of ~200nm with a polidispersive index of 0.07 safety in naturally infected dogs. systems as new drug nanocarries. through mechanical squeezing of wire-wire junctions after extrusion. Interestingly, the presence of external in the network. The peptide-metal interface can be molecules changes significantly such parameters, as it Financial support: from the Brazilian agencies CNPq, This work was supported by Conselho Nac. Des. Cient. controlled by pH stimulus thus potentially offering new will be shown during the presentation. CAPES and FAPEMIG and Programme d’Investissements Tecnologico (CNPq # 309207/2016-9, 402838/2016- directions where silver nanowire assemblies are used d’Avenir de l’ANR. 5) and by the Fundação de Amparo à Pesquisa do Estado for transparent electrodes ranging from antimicrobial This work was supported by CNPq, FAPESP and CAPES. de São Paulo (2014/26200-9; 2014/14457-5). coatings to biosensors.

44 45 SY13 - Molecular Modeling and Dynamics SY13.4 - Towards a Complete Coarse-Grained simulations provide a cost-effective way to overcome Force Field for Biosimulations such limitations, although resigning atomic details. However, the current availability of complete CG force Sergio Pantano 1 SY13.1 - Unfolding metalloproteins with force: measurements. Illustration of the approach will be fields to simulate the wide and chemically complex From simple models to quantum mechanical presented in the context of anesthetic binding to 1Dgroup of Biomolecular Simulaitions, Institut biomolecular diversity in a consistent manner is still calculations ion channels, highlighting how such calculations in Pasteur de Montevideo, Uruguay. (spantano@ quite limited. I will illustrate how the use of a classical combination with measurements have been applied to pasteur.edu.uy) Hamiltonian for molecular dynamics in combination Guilherme Menegon Arantes investigate anesthetic action. with pair-specific van der Waals interactions allows for Instituto de Química, Universidade de São Paulo a rather accurate CG representation for biomolecules. This work was supported by Conselho Nac. Des. Cient. The accuracy attained by molecular dynamics CG molecular topologies for popular packages as Amber Tecnologico (CNPq # 305008/2015-3), Coordenação simulations using state-of-the-art force fields have made and Gromacs are currently implemented in a force field Mechanical tension may induce unfolding and chemical de Aperfeiçoamento de Pessoal de Nível Superior of simulation tools a reliable alternative in biomolecular called SIRAH (South American Initiative for a Rapid and reactions in a macromolecule. One rich example (CAPES # 23038.010052/2013-95) and Fundação de sciences. Nevertheless, despite the dazing advances Accurate Hamiltonian) including DNA, proteins, explicit recently studied by atomic force microscopy is the Apoio à Pesquisa do DF (FAPDF # 193.001.202/2016). in hardware and algorithmic implementations, the electrolytic solvent, lipids, glycans and metallic ions unfolding of the metalloprotein rubredoxin coupled elevated computational cost poses severe challenges playing structural roles in proteins. to dissociation of iron-sulfur bonds. Here, we will SY13.3 - Contributions of Simulations to the for molecular simulations to reach biologically present multi-scale models used to understand these Understanding of Drug Delivery Systems relevant size and/or time scales. Coarse-grained (CG) processes in molecular detail. We developed an approximate molecular dynamics scheme to simulate M. Pickholz1 unfolding trajectories coupled to bond dissociation J.M.R. Albano1 and we employ hybrid quantum chemical molecular 1 mechanical (QC/MM) potentials to describe the Physics Institute of Buenos Aires (IFIBA, UBA- mechanism of Fe-S rupture in stretched rubredoxin. CONICET) and Physics Department of the University of Buenos Aires, Buenos Aires, Argentina. SY13.2 - Binding of Small Ligands to Two State Membrane Proteins Nowadays, it is a challenge to developed drug delivery systems (DDS) for a given application. In this context, Werner Treptow1 computer simulation could help understanding drug/ Leticia Stock1, Caio Souza1, Juliana Hosoume1, lipid bilayer interaction and in the search of a suitable Leonardo Cirqueira1 DDS, among others. Relevant information on the system can be obtained using Molecular Dynamics 1 Department of Cell Biology, University of Brasília, (MD) simulations at very different length and time Brasília, DF ([email protected]) scales: moving from continuous to the atomistic level. During the past decades, wide range of drug delivery Understanding how lipophilic small ligands impact system have been developed, such as liposomes, lipid membrane proteins requires knowledge on the molecular solid, and metallic nanoparticles. In this work, we go structure of ligand binding, a reasoning that has driven over different application of this methodology to Drug relentless efforts in drug discovery and translational delivery systems (DDS) carried out in the group. DDS - research. Binding of such ligands appears however a formulation or a device that enables the introduction highly complex involving interactions to multiple of a therapeutic substance in the body and improves transmembrane protein sites featuring single or multiple its efficacy and safety by controlling the rate, time, and occupancy states in a concentration-dependent manner place of release of drugs - are an important component - a process that might depend further on chemotypes, of drug development and therapeutics. Biocompatible protein types and conformations. Looking for new nanoparticles are materials in the nanoscale emerged developments in the field, we will present and discuss as important players in this direction, improving efficacy a statistical mechanical formulation of the equilibrium of approved drugs, for example. In this direction, the properties of ligand binding to membrane proteins, molecular understanding of the encapsulation process clarifying the impact of multiple binding events on the could be very helpful to guide the nanocarrier for a energetics of two-state membrane proteins intrinsically specific system. Here we discuss different applications driven by a variety of thermodynamical conjugates. of drug delivery carriers, such as liposomes, polymeric The theory in combination with docking or flooding micelles and polymersomes using atomistic and CG MD may be of special interest by establishing a molecular dynamics simulations. microscopic framework to be linked with macroscopic

46 47 SY14 - Photodynamic Therapy study with 40 women with histological diagnosis of practice. Antimicrobial photodynamic therapy (aPDT) HCIN (CIN 2), whom were collected TP and PCR, pre has been intensively studied in the last few years, and post-treatment and followed for 18 months. The and substantial evidence has been collected to prove SY14.1 - PHOTODYNAMIC THERAPY IN SY14.2 - Antimicrobial photodynamic therapy in patients were allocated into 2 groups (ETZ and PDT) its efficiency against a broad spectrum of microbes (2), (3) (4) CUTANEOUS LEISHMANIASIS Veterinary Medicine with 20 women in each. ETZ was performed by an including bacteria fungi and viruses in vitro. electrosurgical loop excision procedure electrode in Antimicrobial resistance is considered a health threat Juliana Ferreira Strixino Fabio Parra Sellera order to remove part of the affected uterine cervix. PDT: across the globe (5) and despite the potential for the Juliana Guerra Pinto, Luciana M. C. Marcolino, Faculdade de Medicina Veterinária, USP Methyl amino levulinate cream 20% (MAL) was used use of aPDT we still cannot substitute antimicrobials André H. C. Pereira 10 hours before the procedure and then, a single photo due to the lack of predictable therapy results. Much treatment with a LED tip was applied. TP and PCR were effort has been done to develop new photosensitizers Laboratory of Photodynamic Therapy, Institute Photodynamic therapy (PDT) is an emerging light- collected pre and post treatment, every six months for and different light sources but in order to promote an of Research and Development, University of Vale based technology that uses light in combination 18 months. Results: In the ETZ group was followed effective clinical use of aPDT the oxidative stress effect do Paraiba, Sao Jose dos Campos, SP, Brazil with a photosensitizer (PS) to treat localized tumors and the TP remained positive in 15% patients as well over complex biofilm has to be completely understand. ([email protected]) and infections. Regarding antimicrobial PDT, main as in 20% patients´ DNA-HPV. On the other hand, in Furthermore the environmental effect enclosing pH, advantages are the wide spectrum of action (bacteria, the PDT group 35% patients remained TP positive as body fluids interaction, and natural antioxidative Leishmaniasis in an infecctious an non-contagius yeasts, viruses, and parasitic protozoa), the outcome same as in 40% patients´ DNA HPV. Conclusion: After defenses have to be evaluated to understand the full disease caused by protozoa of the genus is independent of the antibiotic resistance pattern of the 18 months of follow up the PDT results demonstrated potential of this therapy. aPDT is a reasonable, easy to Leishmania. They present two main evolutionary microbial strain, the wide-ranging decrease in pathogens remission in circa of 65% patients and probably a perform therapy that presents enough compliance to forms: Promastigote ou flagellate and aflagellate with minimal damage to the host tissue, the absence second application could be necessary, even though be used in contained infections. With further research or amastigote. The cutaneous Leishmaniasis (CL) of selection of “photoresistant” strains after multiple such results signalized an alternative treatment for effort, it may become the first choice treatment for presents with skin lesions, ulcerated or not and limited treatments, and the lack of mutagenicity. In addition, HCIN. localized infections leaving antimicrobial prescriptions to the site inoculation. It is considered by World Health antimicrobial PDT is a low-cost and minimally invasive reserved for widespread infections where light and PS Organization (WHO) to be one of the six most important localized therapy with many advantages in veterinary Key words: High grade cervical intraepithelial neoplasia, are not an easy option. infectious diseases, due to its high detection coefficient clinical practice. Photodynamic Therapy (PDT), Excision of the and capacity for causing deformities. It is still found in Transformation Zone, HPV-DNA, Thin prep. References: Baptista MS et al. (2017) Type I 85 countries. In 2014, Epidemiological Vigilance data SY14.3 - CAN THE PHOTODYNAMIC THERAPY and Type II Photosensitized Oxidation Reactions: shows cases of Clin all regions of Brazil, especially BE AN ALTERNATIVE TO TREAT HIGH GRADE SY14.4 - Antimicrobial Photodynamic Therapy Guidelines and Mechanistic Pathways. Photochem in the north where more than 15% of the cases were CERVICAL INTRAEPITHELIAL NEOPLASIA? Photobiol.;93(4):912-919. 1). 1) Ribeiro MS et al. recorded. Globally, the disease endangers 350 million Silvia Cristina Nunez (2018) Photodynamic inactivation assisted by localized people in 98 countries, mostly developing countries. Renata Aparecida Belotto1,2 surface plasmon resonance of silver nanoparticles: In 2 Instituto de Ciências e Tecnologia da Universidade In Latin American, the disease has been found at Maria Cristina Chavantes , Fernanda Mansano Brasil – Universidade Brasil, Jardim Santana, Sao vitro evaluation on Escherichia coli and Streptococcus least in 12 countries and 90% of the cases occur in 3 3 mutans. Photodiagnosis Photodyn Ther. Jun;22:191- Carbinatto Cynthia Aparecida1 de Castro , Raquel Paulo, SP- Brazil. silvia.nunez@universidadebrasil. Brazil where is the only place to gather a large number , 196. 2) Nuñez SC et al (2015). Urea enhances Civolani3 Marques Fernandes , Natalia3 Mayumi edu.br of the three forms of Leishmaniasis : cutaneous. Inada , Vanderlei Salvador Bagnato . the photodynamic efficiency of methylene blue. J Mucocutaneous and visceral leishmaniasis. The effects Photochem Photobiol B;150:31–7. 3) Zhang W et al of Photodynamic Therapy (PDT) in CL treatments 1- Perola Byington Hospital. 2- Postgraduate Photodynamic therapy is a therapeutic approach (2018). Efficacy and safety of photodynamic therapy in vitro and in vivo have been the subject of studies Program in Medicine – UNINOVE. 3- Sao Carlos that combines a chemical compound known as a for cervical intraepithelial neoplasia and human with several photosensitizers (FS) and FS precursors, Institute of Physics – University of São Paulo. photosensitizer (PS) and light to produce oxidative papilloma virus infection: A systematic review and such phytalocianines, aminolevulinic acid (5-ALA) stress at the target area. For more than 100 years the meta-analysis of randomized clinical trials. Medicine and derivates, methylene blue, chlorines, among Background and Objectives: High grade cervical antimicrobial potential of employing photosensitizers (Baltimore);97(21):e10864. 4)Alanis AJ. (2005). others. There is a great variety in literature on the intraepithelial neoplasia (HCIN) can progress to cervix associated with an appropriate wavelength of light is Resistance to antibiotics: are we in the post-antibiotic concentration of FS, applications forms and different cell carcinoma from 5 to 22%. HCIN is a HPV infection known(1). Although the effect is well known, we have era? Arch Med Res;36(6):697–705. irradiation parameters. The in vivo and in vitro studies and the Excision of the Transformation Zone (ETZ) is the not yet found a widespread use of this therapy in clinical demonstrate the efficacy of PDT, decreasing the viability standard treatment. Photodynamic therapy promotes of parasites and even the effective cure for cutaneous photoinduced cell death and stimulates local immune lesions in case studies. effect, justifying its application in HCIN. The aim of this study was to evaluate Thin prep (TP) and PCR for high- This work was supported by Fundação de Amparo à risk HPV screening, in HCIN (CIN 2) patients, treated Pesquisa do Estado de São Paulo (FAPESP 2013/12284- with PDT and ETZ, followed by 18 months. Study 3 and 2016/12211-4) (CNPq # XXXXX). Design and Methods: Controlled and randomized

48 49 SY15 - Biophysics of Proteins nontoxic compounds. Anyway, recently, new classes SY15.4 - Biophysical characterization of of ILs have been synthesized using bio-precursors doxycycline potential neuroprotective mechanism through sustainable and easy procedures, resulting Chehín, Rosana Nieves¹ SY15.1 - CHAPERONE-ASSITED PROTEIN conjugation that often leads to antibiotic resistance in compounds with lower toxicities, high rate of AGGREGATE REACTIVATION as well as the transfer of virulence factors that cause biodegradation, and particularly, higher biocompatibility Medina, L¹, Vera, C¹, Gonzalez Lizarraga, F, Avila disease in humans. Many bacteria of the family than conventional ILs and solvents. In addition, CL¹, Socias, B¹ ,Ploper D¹, Itri, R., Ramos Souza Carlos Henrique I. Ramos Xanthomonadaceae carry a Type IV Secretion System considering the wide range of possible cation and anion Barbosa, L². Raisman-Vozari, R³.. Department of Chemistry, University of Campinas (T4SS) with several characteristics that distinguishes combinations, and corresponding interesting physic- it from other T4SSs, including a VirD4 protein that chemical characteristics and behaviors, ILs have been ¹Instituto Superior de Investigaciones Biológicas – UNICAMP, Campinas, SP ([email protected]) 2 interacts with a set of proteins (XVIPs) that contain used as distinctive substances in the development of CONICET-UNT. Instituto de Física - Universidade 3 a conserved C-terminal domain termed XVIPCD several biological processes. Among these properties, de São Paulo, Brasil. l’Institut du cerveau et de Proteins reach a stable and lower favorable free energy (XVIP-conserved domain). Many XVIPs are predicted ILs exhibit a huge array of overall interactions with la moelle épinière-ICM - Université Pierre et Marie level in the funneling process of folding. However, to act within the periplasm as peptidoglycan (PG) proteins and other bio-based systems or products. Curie, Paris, France especially inside the cell, the folding process is not glycohydrolases, peptidases or lipases and their Particularly, there are already many studies that without complications, and misfolding can not only genes are found downstream genes that code for claim that ILs are good protein stabilizers, but due to Amyloid aggregation of specific proteins seems to be cause loss of function, but also lead to aggregation, proteins lipoprotein signal peptides. Three purified the large number of ILs, these generalizations fail in the common biological event involved in neuronal death which is strongly correlated with several diseases. X. citri XVIP proteins are able to degrade PG and certain extent. The ILs’ interactions with proteins are in different neurodegenerative diseases. Interfering with The risk of protein aggregation is heightened by lyse B. subtilis cells and lytic activity is inhibited by very dependent of the ILs’ type and concentration, this abnormal aggregation could slow down or stop stress and age, and by mutations that disrupt folding. their cognate lipoproteins. We therefore now refer to as well the intrinsic characteristics of the protein. neuronal death. However, despite of the huge effort Therefore, cells need robust mechanisms to prevent the XVIPs and their cognate lipoproteins as X-Tfes Properties like IL alkyl chain length, ion composition, invested, traditional drug discovery strategies have aggregation, or to clear aggregates once they have and X-Tfis (Xanthomonadaceae-T4SS effectors and hydrophobicity/hydrophilicity balance and non-covalent failed. Thus, the exploration of new uses for approved formed. Many chaperones have the ability to bind non- immunity proteins). X. citri colonies do not secrete interactions can play a decisive role in the interaction drugs provides a useful alternative to fill the gap native polypeptide substrates and prevent aberrant X-TfeXAC2609. However, X-TfeXAC2609 is secreted in between ILs and proteins, resulting in their stabilization between the increasing incidence of neurodegenerative interactions leading to aggregation. Clearance of mixed colonies of WT X. citri and E. coli cells but or destabilization. Herein, an overview of the most diseases and the long-term assessment of classical aggregates is more complex, and still poorly understood. not when using Xac strains lacking a functional relevant aspects of the interactions between IL and drug discovery technologies. Drug re-profiling is The solubilization process in humans, as opposed to T4SS. Using time-lapse microscopy, we frequently proteins, as well as deliberate how different properties currently the quickest transition from bench to clinic. In yeast or bacteria model organisms, has only recently observed the rapid lysis of E. coli cells that were can impact the synergy among them, will be presented. this context, we have explored the ability of doxycycline, been addressed. Reconstitution experiments showed in direct physical contact with X. citri WT cells but As case study, novel work involving the effects of ILs a second-generation tetracycline which prevents that Hsp70 (70 kDa heat shock protein) chaperones did not observe lysis when the ΔvirB7 and ΔvirD4 on the stability of Green Fluorescent Protein (protein neurodegeneration in animal models, to prevent the have disaggregation activity when a specific set of strains were used. We also present the results of biomarker and biosensor), Lipase (industrial enzyme) abnormal aggregation of specific neurotoxic protein regulatory co-chaperones is present. We are using cryo-electron microscopy studies on the periplasmic and Asparaginase (therapeutic enzyme) will be also species. By combining biophysics techniques, we have biophysical, biochemical and cell biological approaches portion of the X. citri T4SS. discussed. been able to demonstrate the ability of this antibiotic to explore the characterization of Hsp70 and small Hsp reshapes α-synuclein oligomers into off-pathway, non- chaperones and DnaJ and Hsp110 co-chaperones This work was supported by the Fundação de Amparo Acknowledgement: Jorge F. B. Pereira is grateful to toxic species losing the ability to destabilize biological from several organisms and to understand how these a Pesquisa do Estado de São Paulo (2011/07777-5 and the financial support from young investigator project membranes and thus. This mechanism could explain proteins cooperate to promote aggregate solubilization. 2017/17303-7). (2014/16424-7) financed by FAPESP (Sao Paulo the ability of doxycycline to preserve dopaminergic Mechanistic models and future perspectives on utilizing Research Foundation Brazil), and the project co- cell viability. We have extended these studies to Tau, proteostasis maintenance to develop novel therapeutic SY15.3 - Are Ionic Liquids Good Protein financed by FAPESP and FCT through the project FCT/ whose aggregation and/or phosphorylation have been strategies for the treatment of diseases caused by Stabilizers? FAPESP 2014/19793-3. involved in neurodegeneration in Alzheimer’s disease. aggregation will be discussed. According with our results, that heparin can induce Jorge F. B. Pereiraa* tau aggregation with a classical sigmoidal behaviour. This work was supported by CNPq (305018/2015-9) Nathalia V. dos Santos,a Paloma A. M. Nascimento,a The presence of doxycycline strongly inhibits the and by FAPESP (2012/50161-8). Agnes Magri,a Valéria. C. Santos-Ebinuma,a amyloid fibrils formation but oligomeric species are still formed. Doxycycline does not inhibit the GSK3-β a School of Pharmaceutical Sciences, Department activity, suggesting that the antibiotic could not affect SY15.2 - STRUCTURAL AND FUNCTIONAL of Bioprocesses and Biotechnology, São Paulo STUDIES ON THE XANTHOMONADACEAE TYPE the phosphorylation pattern. Since results suggest that State University (UNESP), Araraquara, SP, Brazil, doxycycline may form different tau oligomeric species, IV SECRETION SYSTEM - A BACTERIA KILLING 14800-903 - [email protected] MACHINE we are evaluating its effects in a C. elegans model expressing human tau, and dopaminergic cells. We Chuck S. Farah1 Ionic liquids (ILs) are relatively modern solvents that, have also test the doxycycline effect in other protein 1Department of Biochemistry, University of São due to their tunable physical properties, low vapor aggregation models without the effects obtained with Paulo, São Paulo, SP ([email protected]) pressure, negligible volatility, and, in general, low alpha-synuclein and tau. Taking together our results, toxicity, have been suggested as potential substitutes of doxycycline could be a selective inhibitor of pathologic more traditional organic solvents for the development of aggregation of the main neurotoxic proteins. This fact Type IV secretion system (T4SSs) are complex greener and more biocompatible industrial processes. together with it well-known safe, low toxicity in long- nanomachines that mediate the transfer of proteins Although most ILs were originally considered as term treatments and good bioavailability in central or protein-DNA complexes from bacteria into other green solvents, recent studies have shown that nervous system became it as a good candidate to be cells. In this way, they are responsible for bacterial some should not be considered as sustainable and repurposed as neuroprotector.

50 51 SY16 - Cellular Biophysics SY16.3 - POST-TRANSLATIONAL ARGINYLATION SY16.4 - DYNAMICS OF CARDIOMYOCYTES AND INTER-SARCOMERE DYNAMICS IN OBTAINED BY THE METHOD OF TRACTION FORCE SKELETAL MUSCLE MYOFIBRILS MICROSCOPY SY16.1 - ALBUMIN HANDLING IN RENAL SY16.2 - ION CHANNELS IN THE AMPLIFYING Felipe de Souza Leite1,4 Adriano M. Alencar1 PROXIMAL TUBULE CELLS: CENTRAL ROLE IN PATHWAY OF INSULIN SECRETION THE PROGRESSION OF RENAL DISEASE Dilson Rassier1,2,3 Mariana S. Ferraz1, Vinicius Bassaneze2, José E. Fernando Abdulkader1 Krieger2 Celso Caruso Neves 1Department of Kinesiology,2Department of 1Department of Physiology and Biophysics, Institute Physiology,3Department of Physics, Mcgill 1Institute of Physics, University of São Paulo, São Diogo B. Peruchetti, Rodrigo P. Silva-Aguiar of Biomedical Sciences, University of São Paulo, University, Canada. 4Institute of Biosciencies, USP, Paulo, SP ([email protected]). 2Institute of Heart São Paulo, SP ([email protected]) Instituto de Biofísica Carlos Chagas Filho, Federal São Paulo, SP ([email protected]). (InCor), University of São Paulo, São Paulo, SP University of Rio de Janeiro, RJ ([email protected]) In this lecture I will discuss the current knowledge on Myofibrils are organized in functional units named Cultured neonatal cardiomyocytes are instrumental for Renal proximal tubule (PT) cells play a central role in the the mechanisms underlying the amplifying mechanisms sarcomeres. We studied two important factors that disease modeling, allowing, for example, the investigation genesis and progression of renal disease. PT cells have of insulin secretion by pancreatic beta-cells, with a modulate the work produced by sarcomeres. First, of genetic diseases or drug testing. However, often, the a remarkable ability to reabsorb albumin filtrated in the focus on the possible role of ion channels in these we investigated how the myofibril inter-sarcomere phenotype characterization, specifically the mechanical glomerulus. Albumin reabsorption in these cells involves mechanisms. The stimulus-secretion coupling in these dynamics affect its mechanical function. We developed differences among cells are faint for most of the receptor-mediated endocytosis, with megalin as a endocrine cells is triggered by glucose-derived ATP a new experimental system that allowed us to control experimental techniques. Traction Force Microscopy central receptor. Changes in the albumin endocytosis synthesis and Kir6.1 channel closure by the consequent individual sarcomeres within myofibrils. We observed (TFM) is a recent method that often used only two images machinery induce a proinflammatory phenotype in the rise in the [ATP]/[ADP] ratio that leads to increased that the cooperatively work of active and passive forces to map the force a cell makes in a substrate marked with 2+ PT environment leading to tubule-interstitial injury. This [Ca ]ic and activation of the exocytotic machinery . transmit the motion of one sarcomere to the other fluorescent beads. Cysteine And Glycine Rich Protein 3 process could be triggered by variations in albumin The amplifying effects are considered to be additional sarcomeres in series, creating a cooperative model (CSRP3) is a gene that participates in the organization concentration in the lumen of PT what depends on stimuli derived from glucose metabolism that enhance for force development. Second, post-translational of sarcomeres in cardiomyocytes. When abrogated in the albumin filtration in the glomerulus. Our group has insulin secretion via Kir6.1-independent pathways. In arginylation is mediated by the enzyme arginyl-tRNA- mice severely affects heart function, leading to a dilated shown that these changes in albumin concentration this context I will present unpublished data from the transferase (ATE1) that transfers arginine residues from heart. The effect of the absence of expression of this

modulate PT cell function determining either the tubule laboratory that substantiate a role for Kv channels, and tRNA onto proteins. We used KO mice (Ckmm-Ate1) and gene on the behavior of cardiomyocytes in vitro is still remodeling or tubule-interstitial injury. It was observed a lack thereof for Cl- channels, in the amplification of advanced techniques to study the effects of arginylation unknown. Here we aim to test the hypothesis that is that lower albumin concentration increases cell survival glucose-triggered insulin secretion. Additionally I shall on skeletal muscle molecular function. We found a possible to detect differential alteration in the forces and albumin endocytosis. On the other hand, higher discuss the possible implications of these findings for significant reduction in active force in single myofibrils knockout CSRP3 neonatal cardiomyocytes exerts in a albumin concentration decreases megalin expression diabetes mellitus progression and treatment. and isolated myosin filaments that lacked arginylation. substrate by using TFM. Measuring the displacements and albumin endocytosis being these effects associated Next, we observed that the mechanical work of titin – of the beads between images, at rate of 145 fps, while with cell death and proinflammatory phenotype. These This work was supported by Conselho Nac. Des. a gigantic elastic protein responsible for passive force the cardiomyocytes are spontaneously or stimulated effects involve a complex network with the participation Cient. Tecnológico (CNPq 490320/2011-2) and by development – is affected by arginylation, likely due beating, we can construct a dynamic map of forces. At of phosphoinositide 3-kinase (PI3K) / protein kinase B the São Paulo State Research Foundation (FAPESP to its effects on titin- protein interactions. Thus, post- this rate, we could evaluate the mean and the fluctuation (PKB) and mechanistic target of rapamycin (mTOR) 2013/08769-1). translational arginylation is a global regulator of force of the total force, contraction power, relaxation power, pathways. Interestingly, we observed that deleterious development in skeletal muscles, affecting several the time interval between beats, torque, and spatial effect of higher albumin concentration on PT cells proteins responsible for sarcomere function. distribution of forces. These parameters were also depends on the secretion of angiotensin II by these cells. measured in response to acute isoproterenol challenge These results open new possibilities to understanding This work was supported by the Canadian Institutes of (10µM). Here we present our latest results regarding the molecular mechanisms involved in the modulation Health Research and Natural Science and Engineering the instrumentation, methods, and analyses of neonate of PT albumin endocytosis and progression of renal Research Council of Canada. F.S.L. was a recipient of a cardiomyocytes using TFM. The comparison of the disease. CNPq scholarship. FAPESP (2016/22318-0). neonatal cardiomyocytes beating behavior when wild- type versus with a genetic mutation of the gene KO- This work was supported by Conselho Nacional de CSRP3 might lead to a better understanding of inherited Desenvolvimento Científico e Tecnológico (CNPq # human diseases. 456997/2014-8) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro This work was supported by Fundação de Amparo (FAPERJ # E-26/110.085/2014, E-26/201.197/2014, a Pesquisa do Estado de São Paulo (FAPESP # E-26/202.950/2016). 2013/17368-0).

52 53 Biomaterials

A01 - Laccase from Aspergillus sp immobilized A02 - DESIGNING NANOMATERIAL FIBRES FOR onto Cu2+ ion-exchanged nanocrystalline BIOLOGICAL APPLICATIONS faujasite zeolite as catalyst for glycerol oxidation Aline Amorim Graf1 1 Alex H. Miller Matthew J. Large1, Sean P. Ogilvie1, Lisa Prof. Dr. José Geraldo Nery1 Woodbine2, Alice A. K. King1 and Alan B. Dalton1 1Department of Physics, São Paulo State University, 1Department of Physics and Astronomy, University São Paulo, SP. ([email protected]) of Sussex, Brighton, United Kingdom (a.amorim- [email protected]) and 2Genome Damage and The worldwide increasing on biodiesel production has Stability Centre, University of Sussex, Brighton, UK. concomitantly increased the glycerol stocks. Glycerol is a building block molecule that under appropriate Polymer composites are one common way of harnessing reaction conditions (right catalyst, temperature, the enhanced properties of 2D materials in a macroscale pressure, pH, etc.) can be transformed into high-cost and controlled structure. One way of achieving it is by and industrially attractive compounds. The main goal of using electrospinning, a fibre production technique which this work was to prepare a clean and green catalyst for relies on applying an electric field to a polymer melt or glycerol oxidation. The research project approaches the solution. This process is being studied in depth recently immobilization of different laccases onto nano-sized due to the high level of uniformity obtained on the fibres zeolites, and the complexes application to glycerol and the variety of polymer and solvents that can be used. oxidation in a laccase-mediated system. In order to Tuning properties of fibres including electronic, optical oxidize glycerol in a liquid phase, it was applied the and mechanical is critical for applications for electrospun mediator 4-Amino-2,2,6,6-tetramethylpiperidine-1- fibres such as tissue engineering scaffolds, drug delivery, oxyl (TEMPO), which can be oxidized by immobilized wound healing dressings, filtration, biosensors, protective laccase, and then its reduced form oxidize glycerol. clothing and electrodes. Following the recent advances in The laccase from Aspergillus sp was successfully the technique, particularly regarding the control over the immobilized onto Cu2+ ion-exchanged nanocrystalline fibre deposition and coaxial electrospinning, applications faujasite zeolite, according to spectroscopic assays of patterned electrospun fibres as tissue engineering using 2,2′-Azino-bis(3- ethylbenzthiazoline-6-sulfonic scaffolds are showing great promise. Using these fibres, acid)(ABTS) as probe. The support was characterized it is possible to mimic the natural tissue environment and by XRD, FTIR, HRTEM and SEM. The free enzyme and release bioactive agents to specific places. The future complex activities were analyzed under several assay of the field lies in multidisciplinary research, integrating conditions (pH, temperature, etc.). After all, both free material physics, biology, chemistry, engineering and and immobilized enzyme where applied to glycerol computational modelling. oxidation, under similar conditions. The enzyme on Posters its free form successfully catalyzed the TEMPO- A03 - SuAVE: a new computational perspective mediated glycerol oxidation (significant amounts of for analyzing biological systems glycerol products were yielded). When the immobilized enzymes were used, the reaction rates were a way Denys E. S. Santos1 slower, and fewer amounts of products obtained. These Frederico J. S. Pontes1, Kaline Coutinho2, Roberto results are not yet understood, and further research are D. Lins3, Thereza A.Soares1 in progress. 1Department of Fundamental Chemistry. Federal This work was supported by “Fundação Amp. Pesq. Est. University of Pernambuco. Recife, PE, (e-mail: 2 de S. Paulo/FAPESP” (Proc.:2016/24303-0). [email protected]), Physics Institute, University of São Paulo, São Paulo, SP and 3Oswaldo Cruz Foundation, Recife, PE.

Data analysis is an inevitable and necessary step for engineering predictive models for a studied system. The success of the model depends directly on how precise, and preferably, efficient are the analyses in measuring properties of interest. The program SuAVE (Surface Assessment Via grid Evaluation) is a program developed to analyze geometrical and physical properties of

54 55 surfaces taking into account the morphology of the functionalization was confirmed by FTIR. Luciferases in A06 - Supramolecular G-Quadruplex Hydrogel as A07 - Self-assembled peptides nanostructures: systems. This ensemble of numerical routines relies the native form and associated to MNPs were assayed a Promising Drug Carrier How shape and structure impact cytotoxicity on a surface-fitting algorithm established over the with the substrates and the luminescence monitored. basis of differential and computational geometry. The In the assay with MNPs, the bioluminescence was Juliana S Yoneda Lucas Rodrigues de Mello1 program has been developed to handle any organic, detected earlier than for the native enzyme. The Rosangela Itri 1Renata Yoshiko Yamada, 1Jane Zveiter de Moraes, inorganic, or biological system containing a surface luminescence spectra in the presence of MNPs did 2 3 1 Dept. of Applied Physics, Physics Institute, Ian Hamley, Wendel Andrade Alves and Emerson or an interface. It reads atomic coordinates trajectory not differ significantly from those obtained with native Rodrigo da Silva files generated through molecular dynamics or Monte enzyme. Taking together, these results shows that the University of São Paulo [email protected] 1 Carlos simulations as well as any other numerical activity of PyLuc and RELuc, can be modulated by the Universidade Federal de São Paulo, Departamendo methodology, and calculates area and volume association with nanoparticles and has the potential for Self-assembly of guanosine is an efficient way to de Biofísica, 04023-062, São Paulo, Brazil; 2 per molecule composing an interface, membrane a diversity of nanotechnological applications. form biomaterials, such as supramolecular hydrogel University of Reading, United Kingdom, 3 thickness, surface topology maps, density profiles, with promising applications. Guanosine form cyclic Universidade Federal do ABC, Centro de Ciências curvature order parameters and Gaussian curvatures. (FAPESP 2010/05426-8, 2015/17688-0; 2017/02317- planar units, called G-quartets, through hydrogen Naturais e Humanascontact: lucasr.mello@hotmail. It is also possible to analyze thermodynamic variables 2, CNPq 401867/2016-1; 401827/2016-1) bonds (Hoogsteen). In presence of monovalent cation, com; [email protected] and evaluate the energy associated to the generation G-quartets stack through π-π interactions resulting of curvature on a flat surface. The accuracy of the A05 - Gold Nanoparticles Decorated Hematite in columns: the G-quadruplex. Depending on the The capacity of short peptide sequences to spontaneously numerical methodology implemented in SuAVE to Microwires for cell growing and Thiol conditions, G- quadruplex is able to make a 3D-network self-assembly into major supramolecular structures has assess the aforementioned properties for curved Functionalization with high capacity to entrap water (Carducci et al., 2018). been extensively investigated for developing potential surfaces has been validated via the analysis of four Our study has focused in getting stable hydrogels by new drug delivery systems. Since peptides can be easily bilayers composed of different Lipid A chemotypes, Iseli L. Nantes Cardoso the self-association of two precursors: Guanosine (G) synthesized using solid-phase methods, it is possible to each one adopting distinct membrane polymorphism. Lucivaldo R. Menezes, Guilherme Sombrio, Tiago and Guanosine 5’-monophosphate (GMP), varying the rationally design sequences wich will self-organized into Comparisons of the implemented methodology against Rodrigues, José A. Souza proportion between them. The ability of these gels to particular structures such as nanovesicles, nanotubes, conventional ones underscore the need to take into trap and release bioactive molecules as Methylene Blue nanofibers, hydrogels and micelle- like structures. These account membrane curvature to correctly estimate Centro de Ciências Naturais e Humanas, (MB) was analyzed. A range of G:GMP proportion was structures are naturally biocompatible and can be used structural properties of lipid aggregates. Therefore, Universidade Federal do ABC, Santo André, SP, Brazil. able to form hydrogel. Three (1:6; 1:2 and 1:1) were as carriers or controlled-release systems for drugs, SuAVE enables the accurate quantification of structural ([email protected], and [email protected]) used for the following experiments. Small Angle X-Ray biomaterials, or even delivery of nucleic acids in gene and energetic properties of pliable soft matter with Scattering (SAXS) was performed to infer about the therapy. In this work, we designed two octapeptides with application to the analysis of a wide range of systems. Hierarchically structured iron/magnetite/ hematite structure. Higher structural factor peak was observed the very same aminoacid composition: four arginines microwires and antiferromagnetic and ferrimagnetic to lower proportion of GMP, indicating that 1:1 gel (R) and four phenylalanines (F). The primary structure of This work was supported by CNPq, CAPES (BioComp/ magnetite hematite microtubes, both decorated with presents the strongest lateral interaction between the these sequences was chosen to produce either a block BioMol), FACEPE and STINT-Sweden. hematite nanowires were obtained by the thermal oxidation fibers. The MB solution was deposited on the top of the or a bolamphiphile peptide, with composition NH2-R-R- process. The photocatalytic reduction property of the gel to check its diffusion into G-quadruplex network. R-R-F-F-F-F-COOH (RF8A) and NH2-R-R-F-F-F-F-R-R- A04 - Association of Metallic Nanoparticles with hematite surface of microwires and microtubes was used The MB was able to penetrate homogeneously into COOH (RF8B). Through a range of techniques including Green and Red Emitting Luciferases: Potential for the reduction of cytochrome c, and synthesis of gold hydrogel without mechanical stress. The velocity atomic force microscopy, cryo-electron microscopy and Nanotechnological Applications nanoparticles (GNPs) from the HAuCl4 solution under was proportional to GMP (negative charge). The total small-angle X-ray scattering, it was observed that RF8A simulated solar light irradiation. GNP decorated hematite penetration was achieved with 270 min to 1:6; 390 min self-assemble into sphere-like nanostructures whereas Elisângela Belleti 2 microwires, and microtubes were also used as a substrate to 1:2 and 600 min to 1:1. Different patterns of fibers RF8B is organized into flat nanotapes. After observing Vanessa Bevilaqua1 , Adrianne Brito2, Vadim Viviani1, for adherence and proliferation of aortic smooth muscle were seen on the Atomic Force Microscopy (AFM) that RF8A and RF8B aggregate into different polymorphs, 3, *, Iseli L. Nantes-Cardoso2. cells and keratinocytes. Cell viability assays indicated that images for different proportion G:GMP. In presence we investigated how these structures interact with living the cells remained viable, except in the wire extremities of MB, no significant changes were detected on the cells by MTT assay, a colorimetric assay for measuring 1 Graduate School of Evolutive Genetics and where iron core exposure caused extensive oxidation and aggregation type seen by SAXS and AFM. Hydrogel cell metabolic activity. Surprisingly, MTT assays in SK- Molecular Biology (UFSCar), São Carlos, SP, died cells adhered on its surface. In this regard, hematite G- quadruplex-based seems to be a promising drug MEL-28 cells revealed a threefold cytotoxicity increase of 2 Brazil (UFSCar); Universidade Federal do ABC microtubes did not present extensive oxidation since the iron delivery system to be used in the future. RF8A in comparison with RF8B. A possible mechanism 3 (UFABC), Santo André, SP, Brazil; Graduate School in the core of the material was consumed during synthesis for explaining such a difference on cytotoxicity caused by of Biotechnology and Environmental Monitoring by ion diffusion due to Kirkendall effect. Also, GNPs attached Acknowledgment: This work was supported by peptides with same composition relies on the chemical (UFSCar), Sorocaba, SP, Brazil (UFSCar). on the surface of microwires, and microtubes allowed the CNPq (grant No. 150515/2016-2), FAPESP, CAPES. groups available at the interacting interfaces of each functionalization of the material surfaces with fluorescein- References: Carducci F, Yoneda JS, Itri R, Mariani P. On the polymorph, which likely demonstrates a clear influence of Luciferases are enzymes that catalyze bioluminescent labeled polyethylene glycol thiol derivative through the structural stability of guanosine-based supramolecular the nanoscopic organization of the peptide superstructures reactions used for high-throughput screening, Au−S bond. The anchoring of thiol compounds facilitated hydrogels. Soft Matter. 2018; 14(15):2938-2948. on biological activity. Due to the innate potential of self- particularly the bioluminescence-based assays. In by GNPs creates an efficient charge transfer channel for the assemble peptides as carriers, our findings may help in the present study, gold and silver nanoparticles were material with a diversity of potential applications. the design of less toxic peptide nanostructures. produced by using HS-PEG-SH as reducing and stabilized agents. In this condition, silver nanoprisms This work was supported by Conselho Nac. Des. Acknowledgements: This work was supported by FAPESP (SNP) and spherical gold nanoparticles (GNP) were Cient. Tecnologico (CNPq: 309247/2017-9) and by the under Grant n° 2016/24409-3 and by PROEX-CAPES produced. Recombinant luciferases from Pyrearinus Fundação de Amparo à Pesquisa do Estado de São Paulo fellowship. termitilluminans click beetle green emitting (PyLuc) (Regular 2015/17688-0 and Temático 2017/02317-2). and Phrixothrix hirtus (RELuc) were added to metallic nanoparticles (MNPs) suspensions and the

56 57 A08 - Hematite microwires and microtubes A09 - NON-PRODUCTIVE ADSORPTION OF A10 - Polymeric and nanostructured dentin tissues remains unclear. Thus, the present study investigated the interaction of P11-4 with inorganic and as biologically compatible materials for the BACTERIAL β-GLUCOSIDASES ON LIGNINS AND photosensitizers for PDT association with proteins and cells SMALL PHENOLICS. organic components of dentin as well as the effect of Maurício S. Baptista the self-assembling peptide on the proteolytic activity Mario de Oliveira Neto1 Lucivaldo R. Menezes Department of Biochemistry, Instituto de Química, and mechanical properties of the bonding interface to Guilherme Sombrio, Tiago Rodrigues, José A. Mariana A. Barbosa1 Mariana Barduco1, Thiago B.B Universidade de São Paulo, Brazil. E-mail: artificial caries-affected dentin. Intrinsic fluorescence Souza and Iseli L. Nantes-Cardoso Zuccari1, Viviam M. da Silva2, Fabio M. Squina3, [email protected] analysis, dynamical light scattering with pH monitoring Wanius Garcia2. and Atomic Force Microscopy (AFM) showed P11- Centro de Ciências Naturais e Humanas, 4 induces hydroxyapatite (HAP) crystal nucleation 1 Universidade Federal do ABC, Santo André, SP, Departamento de Física e Biofísica, Instituto In Photodynamic Therapy (PDT) synthetic process improving the structural match among HAP Brazil. ([email protected], and ilnantes@ de Biociências, UNESP-Univ Estadual Paulista, photosensitizers (PS) and light are used to efficiently crystallite aggregates. Surface Plasmon Resonance 2 gmail.com) Botucatu, SP. ([email protected]) Centro induce photosensitized reactions in specific tissues. (SPR) and AFM evidenced P11-4 binds to collagen de Ciências Naturais e Humanas (CCNH), We aim to increase the efficiency of PS by tailoring I fibers, increasing its diameter from 214 ± 4 nm Hierarchically layered antiferromagnetic and Universidade Federal do ABC (UFABC), Santo them to execute damage in well defined cellular targets to 308 ± 5 nm (P< 0.0001). P11-4 also increased 3 ferrimagnetic magnetite/hematite microtubes and André, SP. Programa de Processos Tecnológicos and consequently to induce specific mechanisms the resistance of collagen type I fibers against the iron/magnetite/hematite microwires, both decorated e Ambientais, Universidade de Sorocaba (UNISO), of regulated cell death. Several molecular-based proteolytic activity of collagenase. The immediate with hematite nanowires were obtained by the thermal Sorocaba, SP. and nanostructured PS will be used to alter cell treatment of artificial caries-affected dentin with P11-4 oxidation. The photocatalytic property of the hematite localization and intracellular release of the PS including enhanced the µTBs of bonding interface (P < 0.0001) surface allowed the synthesis of gold nanoparticles Non-productive adsorption of cellulases onto lignins functionalized nanosilica, metal-based PS and PS reaching the values of sound dentin, additionally to

from the HAuCl4 solution that remained associated is an important mechanism that negatively affects adsorbed in biopolymers. Examples will be shown in decrease the proteolytic activity at hybrid layer (HL); but with the material surface. The hematite microwires the enzymatic hydrolysis of lignocellulose biomass. which small damages in cytoplasmic membrane cause such effects impaired after 6 months of water storage and microtubes were incubated with cultured aortic We examined the non-productive adsorption of two necrotic cell death, damages in mitochondria cause (P< 0.05). In conclusion, the present study observed smooth muscle cells and keratinocytes. Both, the bacterial-glucosidases (TpBgl1 and TpBgl3) on lignins mainly apoptotic cell death, while parallel damages in that P11-4 induces the nucleation of hydroxyapatite pristine and gold-decorated microstructures were and small phenolic structures. The results showed that mitochondia and in lysosome cause autophagic cell and interacts with collagen type I. In addition, P11-4 used as a substrate for the cell adherence and growth. β-glucosidases can adsorb to lignins through different death. We will also show how a polymer made of poly- improves immediately the stability of the hybrid layer Cell viability assays were carried out with Hoechst mechanisms. TpBgl1 adsorption onto lignins was lysine modified with protoporphyrin IX can be used to formed by artificial caries-affected dentin. 33342/PI kit that stains the nucleus of all cells with found to be strongly pH-dependent, suggesting that the modulate cell adhesion to substrates. We propose to the blue-fluorescence dye. The kit also contains adsorption is electrostatically modulated. For TpBgl3, discuss the development of nanostructured and target- This work was supported by FAPESP, CNPq and CAPES Propidium iodide (PI), a red-fluorescence dye that the results suggested that the fibronectin type III-like specific PS aiming to improve the efficiency of PDT simultaneously stains only apoptotic, and dead cell domain interacts with lignins throughelectrostatic protocols against cancer and infection diseases. A12 - TUNABLE SYNTHETIC NANOMATERIAL populations. The cells remained viable on the surface and hydrophobic interactions that can partially, or SUBSTRATES FOR CELLULAR GROWTH of hematite microtubes and microwires, except in the completely, overcome repulsive electrostatic forces A11 - THE SELF-ASSEMBLING PEPTIDE P11-4 Rhiannon W. Harries1 wire extremities where iron core exposure caused between the catalytic domain and lignins. Finally, the PREVENTS COLLAGEN TYPE I PROTEOLYTIC extensive oxidation and cell death. In this regard, increase of temperature did not result in the increase DEGRADATION, IMPROVING BIOMECHANICAL Matthew J. Large1, Sean P. Ogilvie1, Thomas hematite microtubes did not present extensive of β-glucosidases adsorption, probably because there PROPERTIES OF RESIN/DENTIN INTERFACE Simon2, Georgios Giamas2, Lisa Woodbine3, Alice oxidation since the iron in the core of the material is no significant increase inhydrophobic regions in the 1 1 1 A. K. King and Alan B. Dalton was consumed during synthesis. The hematite β-glucosidases structures. The data provided here can Rafael G. Carvalho 1Department of Physics and Astronomy, microtubes and microwires were also compatible be useful for biotechnological applications, especially in Jossaria P. de Sousa2, Luiz Filipe Barbosa Martins2, University of Sussex, Brighton, UK; 2Department for the association with cytochrome c and promoted the field of plant structural polysaccharides conversion Ricardo J. S. Torquato1, Kátia C. U. Mugnol3, Fábio of Biochemistry and Biomedicine, University of photoreduction of heme iron. into bioenergyand bioproducts. D. Nascimento3, Regina M. Puppin-Rontani2, Ivarne Sussex, Brighton, UK; 3Genome Damage and L. S. Tersariol1 This work was supported by Conselho Nac. Des. This work was supported by Conselho Nac. Des. Cient. Stability Centre, University of Sussex, Brighton, UK. 1 Cient. Tecnologico (CNPq: 309247/2017-9) and by Tecnólogico and by FAPESP. Department of Biochemisty, Federal University ([email protected]) the Fundação de Amparo à Pesquisa do Estado de of São Paulo, São Paulo, SP, Brazil (rafaelguc@ 2 São Paulo (Regular 2015/17688-0 and Temático hotmail.com) ; Department of Pediatric Dentistry, The use of synthetic 2D nanomaterial substrates for 2017/02317-2). Piracicaba Dental School, University of Campinas cell growth remains an exciting area of interdisciplinary - UNICAMP, Campinas, SP, Piracicaba, São Paulo, research, with cells sensing and responding to 3 Brazil; Interdisciplinary Center of Biochemistry their substrate during movement, proliferation and Investigation, University of Mogi das Cruzes, Mogi interaction with the colony. Langmuir film creation is das Cruzes, SP, Brazil. a powerful tool for obtaining monolayer nanomaterial films, with fine control over the surface coverage, A true regenerative approach that recover and selection of the nanomaterial dictating chemistry, hydroxyapatite crystals within the carious lesion elasticity, etc. Using various liquid-phase exfoliated remains the major goals of operative dentistry. nanomaterials, the pressure-area isotherm has been Recently, a rationally designed self-assembling peptide used to select repeatable surface coverage, thereby P11-4 was introduced to enhance remineralization tuning the substrate properties to identify cellular on initial caries lesions; however, its applicability on responses to substrate cues. For example, the size

58 59 of graphene platelets affects the proliferation of The tripeptide RGD is largely present in fibronectin Biomedical Application glioblastoma multiforme (GBM) cells. Three different segments responsible for adhesion proprerties to cell thicknesses of graphene were produced by size- membranes. This work aim to prepare and characterize selection via liquid cascade centrifugation, with cell hydrogel matrices from a bola- amphiphile peptide based B01 - PARTICLE ADHESION ANALYSIS OF B02 - Metal-based drugs as promising anti- colonies performing best on the smallest size flakes. on the fibronectin epitope RGD and also investigate POLLUTANTS IN HAIR FIBER Leishmania braziliensis agents Molybdenum disulfide (MoS ) Langmuir films used as a the behavior of these gels regarding living cells. The 2 1 1, substrate is shown to be uptaken into the endoplasmic peptide has composition NH2- RGDLLLLRGD-COOH Raissa L. Oblitas André L.S. Santos * reticulum, a significantly different response than (R: arginine, G: glycine, D: aspartic acid). Through Flavio B. Camargo Jr.2 , Marcos Roberto Rossan2 Marta H. Branquinha1, Patrícia M.L. Dutra2, Ana when introduced in the media only. The endoplasmic atomic force microscopy, fluorescence assays and and Maria Cecília Salvadori1 Karina C. Lima2, Michael Devereux3, Malachy reticulum has become important in the understanding thermal stability assays we have demonstrated that the 4 1 1 McCann , Simone S.C. Oliveira of diseases such as Alzheimer’s, and so nanoparticles RGD peptide forms hydrogel matrices highly stable at Department of Applied Physics, University of 1 that selectively locate here could prove useful in the temperature as high as 85°C with an inner structure São Paulo, São Paulo, SP (email:raissa.oblitas@ Microbiology Institute Paulo de Góes, General 2 treatment of these conditions. composed of nanoscopic peptide aggregated into usp.br) Research and Development Department, Microbiology Department, Federal University of Rio 2 fibers. Cells assays are in progress to probe cytotoxicity Chemyunion Ltda, Sorocaba, SP de Janeiro, Brazil. Parasitology Department, State 3 of the hydrogels. University of Rio de Janeiro, Brazil. The Center A13 - Development and caracterization of a new for Biomimetic and Therapeutic Research, Focas drug release system based in a thermostable The hair not only works as an esthetic ally, but also Acknowledgements: This work was supported by shapes and improves the face; however, it also works Research Institute, Dublin Institute of Technology, peptide hydrogel 4 Programa Institucional de Bolsas de Iniciação Científica as a thermal insulator, protecting the head against solar Ireland. Chemistry Department, Maynooth Vinicius Carrascosa1 (PIBIC), and by Universidade Federal de São Paulo – radiation impacts and mechanical abrasion. A hair University, National University of Ireland, Ireland 1 Lucas Rodrigues de Mello, 1Eduardo Rebelato and Escola Paulista de Medicina. fiber, also referred to as α-keratin fiber, is a micro- and 1Emerson Rodrigo da Silva nanostructured composite, consisting mainly of keratin According to the WHO, cutaneous and visceral protein (≈ 91%). It has 6 to 8 overlapping outer layers of leishmaniases are included in the list of neglected 1 Universidade Federal de São Paulo, Departamendo structures called cuticles, which are hydrophobic when tropical diseases, being considered a major global de Biofísica, 04023-062, São Paulo, Brazil; healthy, giving protection to the fiber internal structures. threat for health. Despite some few options, treatment [email protected] There is worldwide awareness on damages caused by of leishmaniasis is far from satisfactory due to the pollution and its effect on our health. Hair can have its increases in drug resistance and relapses, and Hydrogel compounds are able to retain between 70 structure damaged and mechanical properties changed toxicity of compounds to the host. As a consequence, and 99% of water regarding its own mass. Currently, due to daily exposure to the most varied pollutants, the development of new leishmanicidal drugs and most of hydrogels reported in literature are based which added to sun light exposure and inadequate the search of new targets in the parasite biology on synthetic polymers. Peptides are an interesting hair treatments loses strength, ease of combing, shine are important goals. In recent years, the field of alternative because they provide multiple applications and color. In order to analyze a formulation efficacy on bioinorganic, metal-based chemotherapeutics has in both clinics and pharmacology. These systems are reducing the adhesion of pollutant particles in hair fibers, undergone a renaissance, and particular attention a promising strategy for producing new drugs able images were taken by Scanning Electron Microscopy of has been paid to the development of potential drug to adapt to different tissues and promote controlled the fibers after exposition to coal particles. From the candidates comprising organic heterocyclic ligands release. In this work, we produced a hydrogel matrix processed images, the percentage area occupied by chelated to a central transition metal ion. In this based on the bolaamphiphile sequence RGDL4RGD the coal particles was measured on fibers. The analysis sense, 1,10-phenanthroline-5,6-dione (phendione) (R: arginine, G: glycine, D: aspartic acid, L: leucine). was performed in fibers without treatment (control is one of the most extensively studied heterocyclic group) and in fibers treated with formulations added or chelating ligands, and several published papers have not (placebo group) with a cosmetic active composed shown that phendione-based drugs possess potent of plant polysaccharides (Trehalose, Galactoarabinan antibacterial and antifungal properties. In an effort and Salvia hispanica seed extract) associated with to create new phendione metal complex compounds green tea, panthenol and xylitol. It was verified that the having increased antimicrobial activity and reduced fibers treated using the mentioned product reduced the cytotoxicity to host cells, our group synthesized several

adhesion of coal particles in comparison to the fibers compounds, among which were [Ag(phendione)2] treated with placebo, according to statistical analysis ClO4 and [Cu(phendione)3](ClO4)2.4H2O. In vivo tests by single factor ANOVA, with significance level of 5%. showed that these complexes were well tolerated by the insect larvae of Galleria mellonella and also by This work was supported by FAPESP, CNPq, CAPES and Swiss mice. Additionally, blood samples taken from by Chemyunion Ltda. the Swiss mice revealed that levels of the two hepatic enzymes, AST and ALT, were unaffected by treatment with the phendione complexes. Also, these compounds have presented potent activity against promastigote

form of different Leishmania species, displaying IC50 values at the sub-micromolar range. Subsequently, we investigated the possible mechanisms of action and the death pathway induced by these phendione-based

60 61 compounds on L. braziliensis promastigote forms. Our electron microscopy (SEM), dynamic light scattering B05 - ZIKA NS2B EPITOPE AS A CANDIDATE FOR B06 – SERUM ALBUMIN AS CARRIER results revealed that the test compounds were able to (DLS), thermal gravimetric analysis (TGA), and DIFFENTIAL DIAGNOSIS OF PROTOPORPHYRIN IX USED IN (i) inhibit the zinc-metallopeptidase (gp63) activity, one vibrating sample magnetometry (VSM) analyses. 1 PHOTOCHEMOTHERAPY of the main virulence factor of Leishmania; (ii) bind X-ray Diffraction (XRD) confirmed the formation of Catarina Cataldi Débora C. K. Codognato1 to parasite DNA, inducing alteration in its cell cycle; single phase cobalt ferrite nanoparticles with a size Bruno Leite1 , Felix Loeffler2 , Nico Fischer3 , Danilo (iii) arrest the parasite motility, as a consequence of distribution of 18 nm approximately. The obtained cobalt Coelho1 , Tereza Magalhães4, Marli Tenório1 , Edmyr R. dos Reis2, Iouri Bosissevitch3,4, Ana Paula reducing the activity of mitochondrial dehydrogenases ferrite nanoparticles exhibit a ferrimagnetic behaviour Ernesto Marques1,5, Thomas Jaenisch6 , Isabelle Ramos1 and depolarization of the mitochondrial membrane; (iv) at room temperature with a saturation magnetization Viana1 , Roberto Lins1 1 induce several morphological changes, as observed of 38,1 emu/g and a coercivity field of 1034Oe. Physical-Chemistry of Colloids and Surfaces, by optical and scanning electron microscopies; (v) Subsequently, the nanoparticles of CoFe O coated with 1Department of Virology, Aggeu Magalhães FFCLRP, University of São Paulo, Ribeirão Preto, 2 4 2 trigger intracellular oxidative stress, culminating in chitosan will be synthesized incorporating rhodamine Institute, Oswaldo Cruz Foundation, Recife, PE SP ([email protected]), Laser Laboratory, parasite death by apoptotic-like pathway. Moreover, and encapsulated in giant liposomal vesicles (GUVs). ([email protected]) 2Max Planck Institute Center for Experimental Medicine and Campinas 3 the pre-treatment of promastigotes with sub-inhibitory Through the latter procedure it is intended to detect the of Colloids and Interfaces, Germany, 3 University State University, Campinas, SP, Department of concentration of the phendione-derived compounds location and distribution of fluorescent nanoparticles of Tübingen, Germany 4 Colorado State University, Physics-FFCLRP, University of São Paulo, Ribeirão 4 blocked their interaction with hamster macrophages, by fluorescence and confocal microscopy. United States 5 University of Pittsburgh, United Preto, SP Institute of Physics, Federal University of while the treatment of infected macrophages significantly States ,6 Heidelberg University Hospital, Germany. Goiania, Goiania,GO, reduced the number of intracellular amastigote. To This work was supported by Federal University of Lavras finalize, hamsters infected with promastigotes were (UFLA), and by FAPESP. The major public health risks associated with the Photochemotherapy is a technique used in the treatment cured after the treatment with the test compounds, Zika virus (ZIKV) pandemic infection are neurological of cancer by applying light to induce photosensitizers particularly the copper-derivative that considerably B04 - LOW LEVEL LIGHT THERAPY ON BREAST disorders, such as Guillain-Barré syndrome and (PS) to produce reactions that lead to death of the cell reduced the feet’s lesions and the number of parasites TUMOR. IN VITRO AND IN VIVO STUDIES congenital abnormalities. Due to the lack of vaccines onto which PS are attached. The use of a carriers to bring in the feet and lymph nodes. Coordination of phendione and antivirals, the treatment remains non-specific. PS to diseased tissues would improve their selectivity. to silver and copper represents a new promising group Camila Ramos Silva1 Efficient diagnostic tools can help stopping the In this sense, serum albumin can be an option as a of anti-infective agents, which revealed a potent anti- Saulo Pereira1 Carolina Contatori1 and Martha virus spread and guide prophylactic and therapeutic carrier once it is abundant in the living tissue and has a Leishmania action. Simões Ribeiro1 interventions, however, the main in the field is the affinity to these cancer cells . In the present study, we structural conservation among Flaviviruses, which studied the interaction between bovine serum albumin 1 Financial Support: CNPq, CAPES, FAPERJ, SFI and RBI. Center for Lasers and Applications, IPEN/ CNEN, results in high cross-reactivity in serological tests. (BSA) and two types of Protoporphyrin XI (PpIX) used São Paulo, SP ([email protected]) We postulate that the identification of ZIKV specific as PS: synthetic and endogenous extracted from B03 - Preparation and characterization of epitopes is crucial for the development of effective Harderian glands of rats. The use of BSA is justified by magnetoliposomes based on cobalt ferrite Low Level Light Therapy (LLLT) has been gaining differential diagnostic assays. To this aim, a high- its similarity with Human Serum Albumin (HSA). PpIX nanoparticles prominence in health areas due to search for less density peptide array was synthesized by printing the is an intrinsic photosensitizer of the human body, emits invasive and cost-effective treatments. However, its whole proteome of ZIKV, under the form of 15-mers strong fluorescence, has effective production of singlet Angela D. B. de Brito1 use is not indicated to treat patients with cancer due peptides, on a microchip and tested against 192 oxygen which makes it an efficient PS. Changes in the Rosangela Itri2 to a few studies in vivo. The aim of this work was to serum samples from ZIKVinfected individuals with and profile of optical absorption spectra and fluorescence evaluate the effects of LLLT on breast tumor 4T1 cells without Dengue – DENV background. The analysis of emission and decay of lifetime show that BSA binds to 1 Department of Physics, Federal University of Lavras, in vitro and in a murine model. For the in vitro assay, the IgG antibody profiles allowed the identification of PpIXs, but these photophysical changes do not impair 2 Minas Gerais. ([email protected]) and Institute 4T1 cells were submitted to a single session of LLLT a peptide sequence corresponding to the ZIKV NS2b efficiency. Particle size measures at pH 4.5 and 7.3 of Physics, University of Sao Paulo, São Paulo. with red light (660 ± 20 nm) delivering energies of protein specifically recognized by 71% of the ZIKV were carried out. The results showed the formation of 1.2 J and 6 J. The cell viability was measured by MTT positive sera. The immunoreactivity of this epitope BSA/PpIX complexes depending on BSA concentration Magnetoliposomes result from the encapsulation of assay. For in vivo study, nine BALB/c female mice was further confirmed by ELISA, which showed with sizes ranging from 5-1000 nm, for both PS. The magnetic nanoparticles into liposomes. These system (6 weeks old) received 4T1 cells transfected with that the identified peptide can partially discriminate formation of the complex was favored at pH 4.5, close combine the amazing physical properties of these two luciferase (4T1-Luc) into the mammary fat pad. After ZIKV from DENV infections. Quantitative analyses to the BSA isoelectric point, a WUNDER, A.; MÜLLER- types of particles and preserve the magnetic properties two weeks, the animals were divided into control (n=3) using Microscale Thermophoresis (MST) was used LADNER, U.; STELZER, E. H. K.; et al. Albumin-based of the magnetic nanoparticles. In this work, magnetic and test groups were submitted to LLLT with same to determine the antibody binding affinity of specific drug delivery as novel therapeutic approach for

nanoparticles of cobalt ferrite (CoFe2O4) have been parameters described above. The tumor progression IgG antibodies to the NS2b peptide. The MST data rheumatoid arthritis. Journal of immunology (Baltimore, synthesized by the coprecipitation method. Then was monitored by a caliper and bioluminescence. The showed that ZIKV-specific antibodies recognize the Md.: 1950) the nanoparticles were coated with chitosan using anti-fatigue effect of LLLT was assessed by a forced NS2b peptide with higher affinity (at least two orders of a chemical cross-linking method. In this method; swimming test. Our results showed that LLLT was not magnitude higher), when compared to cross-reacting This work was supported by FAPESP (2017/08892-9) during the adsorption of cationic chitosan molecules able to increase cell viability regardless the energy antibodies from DENV-infected individuals. Future and CAPES. onto the surface of anionic magnetic nanoparticles used. In vivo, the lower energy promoted an increase work will involve the engineering of this epitope in its with electrostatic interactions, tripolyphosphate of the tumor volume compared to control group. On the native-like structure into a scaffold protein aiming to (TPP) is added for ionic cross-linking of the chitosan other hand, the higher energy was able to arrest tumor improve its recognition by specific antibodies. Ethical molecules with each other. The characterization of progression as well as increase the swimming time. Committee approval numbers: 15580013.5.1001.5534 synthesized nanoparticles with and without chitosan Taken together, our results suggest that LLLT triggers and 28309414.9.3001.5201. was performed by X-ray diffraction (XRD), Fourier opposite effects on breast tumor depending on the type transform infrared spectroscopy (FTIR), Scanning of assay. This work was supported by ZIKAlliance-EU, CAPES, CNPq and FACEPE.

62 63 B07 - PEPTIDES AS A PROMISING TOOL FOR B08 - EVALUATION OF COMBINED B09 - PORE FORMATION MECHANISMS STUDIES B10 - Comparison of the cytotoxic and SELF-ASSEMBLED MONOLAYERS IN CAPACITIVE PHOTODYNAMIC INACTIVATION AND NO BY ANTIMICROBIAL PEPTIDE phototoxic effects of curcumin and chlorin e6 DIAGNOSTICS RELEASING CHITOSAN NANOPARTICLES ON Ctx(Ile21)-Ha USING ELETRONIC PARAMAGNETIC on J774 macrophages infected with Leishmania CUTANEOUS LEISHMANIASIS RESONANCE Cilli, E. M.1 promastigotes Cabral, F. V1 Haroldo de Lima P. Cravo1 1 Piccoli, J.P.1; Soares, A.C.2; Oliveira Jr, O.N. 2; Juliana Guerra Pinto Bueno, P. B 1. Pelegrino, M.T2; Dimmer. J. A3; Seabra, A.B2; Eduardo Festozo Vicente2, Eduardo Maffud Cilli3, Luciana Maria Cortez Marcolino¹, André Henrique 1 1 1 1 1 Ribeiro, M.S. Antonio José C. Filho Correia Pereira ; Letícia Correa Fontana ; Juliana Institute of Chemistry, São Paulo State University 1 (UNESP) ([email protected]). 2São Carlos 1Centro de Lasers e Aplicações, Instituto de 1Department of Physics, University of São Paulo, Ferreira-Strixino Institute of Physics, University of São Paulo (USP). Pesquisas Energéticas e Nucleares, IPEN-CNEN/ SP ([email protected]), 2 School of Science and 1Laboratory of Photodynamic Therapy, University SP, 05508-000, São Paulo, SP, Brazil. 2Centro de Engineering, Tupã, 3Institute of Chemistry, UNESP, of Vale do Paraíba, São José dos Campos, SP Electrochemical capacitance spectroscopy (ECS) Ciências Naturais e Humanas, Universidade Federal Araraquara ([email protected]) and 2Department of Cell has been introduced as impedance based approach do ABC, 09210-580, Santo André, SP, Brazil. Compartments, University of Vale do Paraíba, São 3 for assays as sensitive and technique capable to IMBIV, CONICET, Dpto. Farmacia, Fac. Cs. Qcas. Over the past decades, a large number of antimicrobial José dos Campos, SP indicate interfacial change, such as target binding at an Universidad Nacional Córdoba. CP: X5000HUA. peptides (AMPs) have been identified from a variety of appropriate receptor. Herein, we integrated synthesis of Córdoba, Argentina. [email protected] vertebrate and invertebrate species, such as magainin, Cutaneous Leishmaniasis is an infectious disease redox-tagged peptide with self-assembling capability, aurein, cecropin, ceratotoxin, among others peptides. caused by protozoa of the genus Leishmania. Brazil is for C Reactive Protein (CRP) detection (a biomarker Cutaneous leishmaniasis (CL) is a chronic disease The permeabilizing properties destroying bacterial considered an intense transmission area, with 47.4% of risk of diabetes, hypertension, and cardiovascular developed by parasites of the genus Leishmania that membranes with consequent cell death, it makes of the population in transmission areas. Conventional disease). Peptides containing ferrocene (Fc) were promotes destructive lesions. The available treatments these peptides the main focus in the development of treatment is toxic and, in many cases, requires patient synthesized by solid phase peptide synthesis: Fc-E-A- are limited because of side effects, resistance and new antibiotics. In particular, the antimicrobial peptide hospitalization, which increases the cost of treatment. 21 A-C-NH2 and Fc-E-A-A-A-C-NH2. Ferrocene molecule toxicity. New strategies against CL have been studied Ctx (Ile )-Ha, extracted and isolated from the Brazilian Photodynamic therapy (PDT) has been studied as an is related to the electron transfer that occurred in the such as Photodynamic inactivation (PDI) and exogenous frog Hypsiboas albopunctatus, has been shown to alternative therapy for the local treatment of cutaneous system as confirmed by redox capacitance. Peptides NO donors. The aim of this work was to explore the effects be a very promising molecule for this purpose due lesions, minimizing systemic side effects. The objective were used for generating self-assembled monolayers of methylene blue (MB)-mediated PDI in association to its biological activities against fungi, bacteria and of this study was to compare the effect of PDT with over gold surfaces and it was used for capacitance- with encapsulated NO donors in chitosan nanoparticles its moderate toxicity against human cells. This work curcumin (500, 250, 125 and 62.5 μg / ml) and chlorin based impedance spectroscopy. The platforms were (NPNO) on Leishmania amazonensis. NPNOs were tested proposes a structural, conformational and topological e6 (400, 200, 100 and 50 μg / ml) on macrophages able to detect CRP with a limit of detection (LOD) of in vitro with L. amazonensis transgenic line expressing studies through biophysical techniques in order to infected with L. major and L. braziliensis, after one 0.30 and 0.24 nM. The difference found on LOD and luciferase at increasing concentrations (25-200µM) clarify the Ctx(Ile21)-Ha peptide oligomerization pore hour of incubation with different concentrations of both sensitivity are attributed to the peptides sequences and inhibitory concentrations (IC50 and IC90 ) were formation, since it is not known specifically how this photosensitizers. Mitochondrial activity and cell viability and their probe redox distance. Therefore, we have calculated. Based on inhibitory concentrations results, mechanism works in the interaction with membranes were assessed by MTT and Trypan Blue tests. Although monitored immunosensor construction with PM- twelve BALB/c mice were infected in the left footpad at a molecular level. The EPR and DEER techniques the MTT test demonstrated change in mitochondrial cell IRRAS. The spectra showed bands at 1550 cm-1 and and randomly assigned to experimental groups (n=4): provided information regarding the spin distance activity after treatment with curcumin in the dark and 1655 cm-1 assigned to amide II and amide I groups Control (non-treated), G1 (two PDI sessions), G2 (two markers contained in the peptide monomers in the after PDT, the trypan test showed no toxicity in the dark, present in the peptides, antibody and antigen. The area PDI sessions and 80 µM of NPNO, immediately after absence and presence of membrane mimetics, by while PDT triggered 100% death at all concentrations and intensity of these bands indicated the organization PDI) and G3 (only 80 µM NPNO). PDI was performed insertion of paramagnetic compounds such as TOAC tested. Chlorine e6 also promoted significant changes of self-assembled monolayers. The immunosensors using a red LED (λ= 660±22 nm) at 150 J/cm² fluence and MTSSL into the peptide chain. In addition to in mitochondrial activity and, unlike that observed in are highlighted for the robustness and potential to be and MB at 100 µM. Parasite burden was obtained by these techniques, the studies were complemented curcumin treatment, chlorine showed dark toxicity at used in real samples, once they showed specificity bioluminescence every day, in the first 96 h and for using circular dichroism, in order to obtain important the highest concentrations (400 and 200 μg / ml) and response to the target, these systems could be used the next 4 weeks, once a week. Test groups presented data about structure and function membrane-peptide all concentrations resulted in 100% death after PDT. for any relevant biomarkers diagnostics. significant reduction in parasite load compared to interactions, as well as to suggest a mechanism of Both photosensitizers have shown promising results control during all experimental period. In the first 24 h pore formation by measuring the distances between and can be used in the future for in vivo tests. CNPq, Fapesp and CAPES supported this work. after treatments, parasite burden was significant lower paramagnetic centers. This information is essential in for G2. After 96 h, all test groups were similar. Following an attempt to improve the modulation of its activity and This work was supported by Fundação de Amparo 4 weeks, statistically significant differences were broaden the understanding of the peptide mechanism à Pesquisa no Estado de São Paulo (FAPESP noticed when test groups were compared to control but action. 2016/12211-4). parasite burden was similar among all treated groups. Under conditions used in this study, our results show This work was supported by FAPESP. that NPNOs were not able to sustain the parasite killing promoted by MB-mediated PDI on CL induced in mice 24 h after treatments.

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).

64 65 B11 - COMPARISON OF FOTOENTICINE AND B12 - Investigating Blood Tissue with Atomic assessed shortly after irradiation using the trypan these substrates so that they can be used in sensing, PHOTODITHAZINE CHLORINS IN PHOTODYNAMIC Force Microscopy and Raman Spectroscopy blue exclusion test. For morphological analysis the since there is a great interest in the analysis by SERS THERAPY IN GLIOSARCOMA LINEAGE cells were fixed and prepared for visualization in the in applications for biochemistry, biophysics and L. M. Rebelo* Scanning Electron Microscope (SEM). In the exclusion biomedicine. Letícia Corrêa Fontana1 R. P. de Oliveira*, C. C. dos Santos*, F. F. Maia tests, the reduction in cell viability was observed in the André Henrique Correia Pereira1; Juliana Guerra Jr.**, E. F. da Costa*** treated groups when compared to the control group and This work was supported by Conselho Nac. Des. Cient. Pinto1; Cristina Pacheco Soares2; Juliana dark in their respective concentration, Pinto et al., 2016 Tecnológico (CNPq), Fund. de Amparo à Pesquisa do * Physics Department,, Federal University of Ferreira-Strixino1 performed viability tests with an 18-hour interval and Estado de São Paulo (FAPESP) and Coordenação de Maranhão, Maranhão, Brazil; ** Physics Department, also obtained similar results. Morphological changes Aperfeiçoamento de Pessoal de Nível Superior (CAPES). 1 Department of Photodynamic Therapy, University Federal University of the Semi-Arid, Brazil; *** were observed in all groups treated with PDT, whereas of Vale do Paraíba, São José dos Campos, SP Physics Department, Federal Institute of Education, in the control groups there were no changes in their ([email protected]) and 2Department Science and Technology of Bahia, Bahia, Brazil. B15 - INVESTIGATING THE EFFECT OF ANTI- morphology. Andrade et al., 2018 also observed such CANCER COMPOUND (PIPERLONGUMINE) ON of Cell Compartments, University of Vale do morphological changes in L. braziliensis promastigotes Paraíba, São José dos Campos, SP THE PHYSICAL PROPERTIES OF CANCER CELLS The ability of the Atomic Force Microscope (AFM) and after PDT. Curcumin presented favorable results, USING ATOMIC FORCE MICROSCOPY Raman Spectroscopy (RS) to investigate in situ all suggesting that it is a photosensitizer with potential Photodynamic therapy (PDT) is a rising technique in components of blood tissue, from cells (erythrocytes, for future applications in the treatment of cutaneous Nayara S. Alcântara Contessoto1,2 the treatment of brain tumors, being an alternative white cells and platelets) to plasma, is of great leishmaniasis by PDT. D. P. Bezerra3, J. M. Barbosa-Filho4, I. Lian5, M. L. with potential of better prognosis to the patients. It importance for hematology studies. This investigation Cornélio1 and C.-H. Kiang2 is based on the interaction of a photosensitive drug occurs with such a resolution that we can evaluate the This work was supported by Fundação de Amparo à 1 (PS), light and molecular oxygen, generating reactive structures of the cells that make up this tissue besides Pesquisa no Estado de São Paulo (FAPESP 2016/12211-4). Physics Department, São Paulo State University, 2 oxygen species, through photochemical reactions. In characteristics that go beyond the structural, such as São José do Rio Preto, SP, Brazil. Department of Physics & Astronomy, Rice University, Houston, TX, order to improve PDT, the objective of this work was to the molecular, tribological (friction and roughness) and B14 – PRODUCING SERS SUBSTRATES evaluate the action of PDT with Fotoenticine (FTC) and mechanical (viscoelasticity) properties at nanometric USA. ([email protected]). THROUGHT GOLD ION IMPLANTATION IN PMMA 3 Photoditazine (PDZ) chlorins, in 9L/lacZ gliosarcoma levels. In this work we observe via AFM (in its different Centro de Pesquisas Gonçalo Moniz, FIOCRUZ, 4 line, maintained in DMEM medium supplemented with modes of operation) and RS processes, structures, Natalia K. Gushiken1 Salvador, BA, Brazil. Lab. de Tec. Farmacêutica, Federal University of Paraíba , João Pessoa, PB, 10% Fetal Bovine Serum and 1% of antibiotic. Initially, molecular changes and properties of blood cells not Giordano T. Paganoto2, Márcia L. A. Temperini2 and Brazil. 5Department of Biology, Lamar University, fluorescence microscopy analysis was performed to accessible by means of conventional microscopy Maria C. B. S. Salvadori3 observe the cellular internalization of both chlorins, with techniques. The results demonstrate the ability of AFM Beaumont, TX, USA. 1 a concentration of 200 g/ml and 1h of incubation. It and RS to bring new information that can compose Escola Politécnica of the Univ. of São Paulo, Univ. μ 2 has been observed that both PSs are internalized by clinical parameters that in the future may complement of São Paulo ([email protected]); Dept. of Natural compounds have shown a great potential for the cells. FTC accumulates in the cytoplasm, but not the analyzes currently performed. Fundamental Chemistry, Inst. of Chemistry, Univ. cancer treatment. Piperlongumine (PL), a natural 3 in the cell nucleus, however the PDZ has its location of São Paulo and Dept. of Appl. Physics, Inst. of anti-tumor compound derived from pepper species, Physics, Univ. of São Paulo ([email protected]) in the cytoplasm and in the nuclear or perinuclear B13 – EVALUATION OF PHOTODYNAMIC has been demonstrated anti-cancer effect on Hela region. Cell viability was assessed by Trypan test, PSs THERAPY IN PROMASTIGOTAS Leishmania cells, and toxicity of PL against Hela cells has been were tested at different concentrations, 200 μg/ml at braziliensis In this work, we formed a thin subsurface composite investigated. Here we focus on understanding the 6.25 μg/ml, serial dilution 1:2, with 1h incubation, material formed by gold nanoparticles (AuNPs) in the mechanical properties of Hela cells under PL treatment, irradiated by Biotable IrradLED 660 nm (10 J/cm²; 25 Luciana Maria Cortez Marcolino1 polymer polymethylmethacrylate (PMMA). This material using Atomic Force Microscopy (AFM) based single- mW/cm²). There was a significant (p<0.01) decrease Juliana Guerra Pinto1, André Henrique Correia was tailored for producing SERS (Surface-Enhanced cell manipulation technique. We used AFM to pull single in cell viability for both chlorins in PDT treated groups Pereira1, Juliana Ferreira Strixino1 Raman Spectroscopy) substrates. The AuNPs were Hela cells and acquired the force-distance curve. The compared to the control group, with 97.3% death at produced by low energy (49 eV) ion implantation characteristic force curves showed stepwise patterns, 1 the highest concentration and 75% at the lowest Laboratory of Photodynamic Therapy at the of gold in PMMA, using cathodic arc plasma. In this and we analyzed the step force and found that, that concentration for the Fotoenticine and 100% cell University of Vale do Paraíba, São José dos Campos, approach, the nanoparticles were formed about 10 under treatment of PL, the most probable step force death at all concentrations for the PDZ. Both chlorins SP (e-mail: lumaria.cortez @ hotmail, com) nm below the polymer surface with mean nanoparticle shifted to higher values compared to the non-treated presented as a promising alternative in the use of PDT. diameter of 4.3 nm. This composite material was tested cells. This change was observed in cells on substrates American Cutaneous Leishmaniasis (ACL) is a non- as a SERS substrate using the analyte Rhodamine 6G of different stiffness. These effects may be due to the This work was supported by Fundação de Amparo contagious infectious disease caused by protozoa of the (R6G), with concentration of 10 μM, and a laser with change in cytoskeleton structure of Hela cells after PL à Pesquisa no Estado de São Paulo (FAPESP genus Leishmania. It is considered by WHO a neglected wavelength of 633 nm as a source of excitation. In the treatment, possibly due to microtubule destabilization 2016/20022-7). disease present in several countries inculcating Brazil. obtained spectra, we have verified the presence of the by PL. Therefore, understanding the PL effects on the The treatments adopted are aggressive and have characteristic peaks of R6G and no signal was obseved cellular physical properties beyond the biochemical several side effects. Photodynamic therapy (PDT) when using a PMMA without gold implantation. It was mechanisms of PL’s anti-cancer effects, may help in has been studied as a therapeutic modality for ACL. also observed an increase of the SERS signal intensity the development of effective therapeutic drugs against The objective of this work was to test the effect of with the increase of the implantation dose. SERS cancers. PDT on the viability and morphology of Leishmania signal was also observed when the 10 nm of PMMA braziliensis promastigotes using curcumin as was removed from its top. But the better result was This work was supported by Conselho Nac. Des. Cient. FS. The promastigotes were incubated with FS at observed when the PMMA layer was maintained over Tecnologico (CNPq # 141714/2017-4), Prog. Pós- concentrations of 500μg/ml; 125μg/ml; 31,25μg/ the composite layer, i.e without removing the polymer Graduação Unesp (PROPG-Unesp), and the Welch ml for a 1h under light. Then irradiated at 10 J/cm2. layer above the nanoparticles. More experiments must Foundation (C-1632). Control groups received only FS or light. Viability was be carried out in order to improve the performance of

66 67 B16 - EFFECT OF CHRONIC TREATMENT B17 - Studying on size of magnetic nanoparticles B18 - Ag@Au Core-Shell Nanoparticles for DNA B19 - STUDY OF THE ANTITUMOR POTENTIAL OF WITH ENALAPRIL OR HYDRALAZINE ON THE in Shear Wave Dispersion Magneto Motive Detection THE LEAF EXTRACT OF Passiflora alata CURTIS AUTONOMICAL BALANCE OF SPONTANEOUSLY Ultrasound Soudabeh. Arsalani1 Taline S. Almeida2 HYPERTENSIVE RATS Sa. Arsalani Tayebeh. Ghodselahi2, Tiam. Neishaboorynejad2, Ricardo G. Amaral1, Sara A. Santos1, Luciana N. Nicole M. A. Chaparro1 Su. Arsalani, Y. Hadadian, D.R.T Sampaio, Baffa, Oswaldo. Baffa1 Andrade2, Silvana V. F. Gomes2, Rafael S. Santos2, 1 2 2 2 Luiz F. Rodrigues. Jr , José G. Mill , José H. M. Oswaldo, T. Z. Pavan, A.A.O. Carneiro 1 Diego M. Batista , Patrícia Severino , and Adriana A. 1 Departamento de Física, Universidade de São 1 Nascimento Departamento de Fisica, FFCLRP, Universidade de Paulo, SP, Brazil. Email: [email protected]. 2School Carvalho 1Carlos Chagas Filho Institute of Biophysics, Federal Sao Paulo Ribeiro Preto, SP, Brazil. [email protected] of Physics, Institute for Research in Fundamental 1Department of Physiology, University of Sergipe, University of Rio de Janeiro, Rio de Janeiro, RJ Sciences, Tehran, Iran São Cristóvão, SE and 2Institute of Tecnology 2 ([email protected]) and Department Elastography has been considered as an appropriate and Research, University Tiradentes, Aracaju of Physiological Sciences, Federal University of imaging technique since changing elasticity of soft tissue Bimetallic Ag@Au core-shell, a type of composite ([email protected]) Epírito Santo, VItória, ES. usually are associated with pathology. Stand-alone nanoparticle consisting of a spherical Ag core covered elastography has some limitations for lesions located in by Au shell, due to their optical properties have been Cancer is a set of diseases of cellular origin, responsible Hypertension is associated to ventricular autonomic deep. Therefore, some ultrasound based elastography used as active material for biomolecular analytic for one of the most significant causes of death in the dysfunction evidenced by reduced heart rate variability techniques have been developed to address this issue detection based on surface plasmon resonance (SPR) world, with an estimate of new cases and increasing (HRV). However, it is still unclear whether therapy with including sonoelasticity, transient elastography, and spectroscopy. LSPR is a kind of electromagnetic mortality. These data demonstrate the need for social- different antihypertensive drug classes can restore HRV. Shear Wave Dispersion Magneto Motive Ultrasound resonances that exist when there is an interface scientific engagement in the search for more effective Therefore, the objective of this study was to evaluate (SDMMUS). The modality of SDMMUS evaluates between metal and dielectric material and this system anticancer agents. In this context, it was evaluated the the effects of angiotensin converting enzyme inhibitors elasticity and viscosity of a medium labeled with have been use for sensing various biomolecules. In this cytotoxic potential of leaf extract from 16 Passiflora and vasodilatators on the HRV of spontaneously superparamagnetic nanoparticles (Fe3O4) via created work, Ag@Au bimetallic nanoparticles (BNPs) on the species cultivated in Brazil, obtained by accelerated hypertensive rats (SHR). 18 SHR were divided in SHR shear wave magnetically by an external magnetic field. carbon thin film were synthesize by co-deposition of solvent extraction (ASE) and detected that the extract control group (SHR, N = 6), SHR treated with 10 mg/ In this study, two different magnetite nanoparticles, RF-Sputtering and RF-plasma enhanced chemical vapor of the leaves of Passiflora alata (ELPA). Therefore, the kg/day of enalapril (ENALAPRIL, N = 4), SHR treated synthesized by co-precipitation method, were used as deposition method. Two thin film of gold nanoparticles present study aimed to evaluate the antitumor activity with 20 mg/kg/day of hydralazine (HYDRALAZINE, N = a contrast agent in SDMMUS. In this work, the induced in different sizes (small and bigger) were deposited on in vivo of ELPA using mice inoculated with sarcoma 5). Wistar Kyoto were used as normotensive control displacement within structure of the sample labeling with a thin film of Ag nanoparticles. Ag@Au BNPs were 180. In this assay, the ELPA showed antitumor activity (CONTROL, N = 8). After 6 weeks of treatment, two magnetite nanoparticles, sample 1 (14 nm) and 2 characterized by several techniques. The average size in the treatments performed for 7 days i.p. (36.75% blood pressure (BP) (by tail plethysmography) and (30 nm), generated using an oscillating remote magnetic and morphology of Ag@Au bimetallic nanoparticles and 44.99% at the doses of 100 and 150 mg/ kg/ D2 lead electrocardiogram (ECG) was recorded. HRV force. These displacements detected inside the sample by was estimated by scanning electron microscope day, respectively). By oral route, the ELPA showed parameters were obtained from ECG through LabChart a linear ultrasound transducer connected to a diagnostics (SEM) and Atomic force microscopy (AFM) images. no antitumor activity in vivo at the doses tested. 7.0 software. One-way ANOVA and Kruskal-Wallis tests ultrasound. To perform SDMMUS measurements, we X-ray diffraction (XRD) patterns showed Ag and Au When the toxicological parameters of the animals with post-tests Holm-Sidak's and Dunn's, respectively, made gelatin tissue mimicking phantoms of agar (4 nanoparticles with face-centered cubic (FCC) structure submitted to i.p. treatment was evaluated, there was were used for comparing variables between groups. percentage of Bovine gelatin, Bloom 250) homogenously were formed in our samples. The ageing of biosensor no significant change in toxicological endpoints: body BP was increased (P<0.0001) in the SHR group, labeled with 1 percentage of nanoparticles as inclusions. chips of Ag@Au BNPs by localized surface plasmon mass (liver and kidneys), biochemical analyzes (AST, compared to CONTROL. Heart rate (HR) increased The setup of SDMMUS made up of a coil, included a resonance (LSPR) spectroscopy was investigated up ALT, AF, uric acid, urea, and creatinine), erythrocyte in both ENALAPRIL (P<0.001) and HYDRALAZINE ferrite core. To produce the magnetic field, the coil was to four months. The UV–visible ageing of the prepared hematology (hemocyte, hemoglobin, and hematocrit) (P<0.0001) compared to CONTROL and SHR, driven by a function generator connected to a power samples indicated Ag NPs covered by small gold and histopathological analysis (liver and kidneys). The

reducing the mean RR (MNN). The HYDRALAZINE amplifier. Mechanical properties of gelatin phantom were nanoparticles (Ag@Au1) are more sensitive rather toxicological alterations were: reduced body mass group presented lower VLF (P <0.05) and LF (n.u.) estimated by Kelvin-Voigt Model. Induced displacements than Ag@Au2 NPs. Moreover, we investigated the associated with reduced food intake, increased spleen (P <0.05) compared to CONTROL. Our data suggests in the range of micro were observed by processing a change in SPR absorption peak of Ag@Au1 BNPs and mass related to the histopathological increase of white that, despite the higher BP observed SHR, HRV is not developed code in Matlab. As it was expected, oscillation bare Ag NPs in presence of DNA primer decamer at spleen pulp, and increased total leukocyte count with

altered compared to normotensive rats. Also, although, frequency of magnetic nanoparticles is two times larger pM concentrations. The result demonstrated Ag@Au1 a change in percentage relation between lymphocytes both enalapril and hydralazine reduced BP to the same than the excitation frequency. Regarding to the obtained BNPs are more sensitive biosensor chip in vicinity of and neutrophils. As the results showed that ELPA has levels of normotensive rats, only hydralazine treatment results, however samples have different sizes, both DNA in a short response time compare to bare Ag NPs. antitumor activity, with few toxicological alterations, reduced sympathetic tonus. It is necessary to increase samples reported almost the same displacements it was proposed to evaluate the benefit of the ip sample size to better observe those effects. because of having the same magnetization saturation. association between ELPA (100 and 150 mg/kg/day) and 5-Fluorouracil in a dose (10 mg/kg/day), however, This work was supported by Conselho Nac. Des. Cient. This work was supported by CNPq, CAPES, FINEP, and the association was lethal at both doses. Tecnologico (CNPq). FAPESP. This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

68 69 B20 - EVALUATION OF TOXICOLOGICAL some adverse effects of antineoplastic drug such B22 - THERAPEUTIC EFFICACY OF A MIXED pegylated liposomes, compared to either conventional PARAMETERS INVOLVING THE ASSOCIATION as weight loss, diarrhea and changes in heart FORMULATION OF MEGLUMINE ANTIMONIATE IN or pegylated liposomes. Based on these promising OF Matricaria recutita (CHAMOMILE) AND mass without leukopenia and thrombocytopenia. CONVENTIONAL AND PEGYLATED LIPOSOMES results, the formulation of MA in a mixture of ANTINEOPLASIC 5-FLUOROURACIL IN THE These findings indicate the existence of possible IN ASSOCIATION WITH ALLOPURINOL IN DOGS conventional and pegylated liposomes (LIPO-2) has TREATMENT OF MICE WITH SARCOMA 180 interactions due to associations between medicinal WITH VISCERAL LEISHMANIASIS been investigated here for the treatment of canine VL and chemotherapeutic plants. in association with allopurinol. Comparison was done Taline S. Almeida2 Guilherme S. Ramos1 to treatment with a formulation of MA in conventional Sara A. Santos1, Ricardo G. Amaral1, Robério This work was supported by Coordenação de Cristiano C. P. dos Santos1, Renata C. de Paula2, liposomes (LIPO-1). The study consisted in 4 groups O. Menezes Filho1; Ariel S. Graça1; Luciana N. Aperfeiçoamento de Pessoal de Nível Superior Maria N. Melo3, Nelder F. Gontijo3, Wagner L. of 7-11 infected mongrel dogs: G1 receiving LIPO- Andrade2, Silvana V. F. Gomes2, Patrícia Severino2, (CAPES) and Fundação de Apoio à Pesquisa e Inovação Tafuri4, Cynthia Demicheli5, Raul R. Ribeiro6, Erly G. 1 (two cycles of 6 doses of 6.5mg Sb/kg/4days i.v.) Sandra L. Santos1, and Adriana A. Carvalho1 Tecnológica do Estado de Sergipe (FAPITEC/SE). Azevedo7, Sydnei M. da Silva2, Frédéric Frézard1. plus allopurinol (60mg/kg/day p.o.) during 4.3 months; G2 receiving LIPO-2 (two cycles of 6 doses of 6.5mg 1 Department of Physiology, University of Sergipe, 1-Depart. Fisiologia e Biofísica-ICB, Universidade Sb/kg/4days i.v) plus allopurinol during 4.3 months; 2 B21 - ANALYSIS OF PHOTOENTICINE IN São Cristóvão, SE and Institute of Tecnology Federal de Minas Gerais (UFMG), Belo Horizonte- G3 treated with allopurinol only; G4 as non-treated and Research, University Tiradentes, Aracaju THE TREATMENT OF GLIOSSARCOMA BY MG, Brazil ([email protected]); 2-Instituto PHOTODYNAMIC THERAPY control group. Parasite load was evaluated in the liver, ([email protected]) de Ciências Biomédicas, Universidade Federal spleen, bone marrow and skin just after the end of Juliana Ferreira Strixino de Uberlândia, Uberlândia-MG, Brazil; 3-Depart. the treatment and 4 months later, using quantitative Parasitologia-ICB, UFMG, Brazil; 4-Depart. Infusions of the medicinal plant Matricaria Rainara Moreno Sanches Almeida, Letícia Corrêa PCR. Treatment with both liposome formulations in Patologia-ICB, UFMG, Brazil; 5- Depart. Quimica- recutita (Chamomile) have been popularly used in Fontana, Gabrielle dos Santos Vitorio, Juliana association with allopurinol was effective in reducing ICEX, UFMG, Brazil; 6- Depart. Medicina Veterinária, association with chemotherapy in cancer treatment. Guerra Pinto the parasite load in all evaluated tissues. LIPO-1 and The objective of this study was to evaluate the Universidade Federal de Juiz de Fora (UFJF), Juiz LIPO-2 showed distinct therapeutic profiles, with long- effect of Aqueous Extract of Matricaria recutita Instituto de Pesquisa e Desenvolvimento, de Fora, MG, Brazil; 7- Depart. Farmácia, UFJF, term antileishmanial actions either in the liver (LIPO- (AEMR) associated with 5-fluorouracil (5-FU) on Laboratório de Terapia Fotodinâmica Governador Valadares, MG, Brazil. 2) or in the skin (LIPO-1). Regarding the therapeutic toxicological parameters of animals transplanted ([email protected]), University of Paraíba efficacy in the bone marrow, the mixed and conventional with tumor Sarcoma 180 (S180). Animals Valley, São Paulo In a previous work using a murine model of formulations showed no difference, in contrast to our transplanted with S180 (2 x 106 cells/0.5 mL, s.c.) visceral leishmaniasis (VL), the antileishmanial study in the mouse model (single dose), suggesting a were used. Mice were separated in six groups Despite advances in conventional treatments for drug meglumine antimoniate (MA) showed higher possible interference of Accelerated Blood Clearance (n=7/group): vehicle (distilled water, v.o), standard brain cancers, the prognosis of the patients is still therapeutic efficacy and broader distribution among (ABC) phenomenon of PEGylated liposomes. This work control (5-FU 25 mg/kg/day/i.p.), AEMR (100 mg/ unfavorable, once the treatments are not effective. As tissues of the mononuclear phagocyte system, when was supported by CNPq, FAPEMIG and CAPES. kg/day/v.o), AEMR (200 mg/kg/day/v.o.), 5-FU-25 an alternative, photodynamic therapy (PDT), which given encapsulated in a mixture of conventional and mg + AEMR (100 mg/kg/day/v.o.) and 5-FU 25 consists of the action of a photosensitizer (PS), mg + AEMR (200 mg/kg/day/v.o.), treated and light and molecular oxygen, leads to photochemical weighed daily (1x/day) for 7 days. After 24 hours reactions leading to the death of tumor cells. The of the last day of treatment, biochemical analyzes objective of this work was to evaluate the cellular were realized (Aspartate transaminase, AST, Alanine viability of the Fotoenticine in 9L/LacZ gliosarcoma transaminase, ALT, urea and creatinine), complete cells submitted to PDT. For this, cytotoxicity analyzes blood count and organ weighing were performed. were performed by the Trypan test, MTT and There was a reduction in body mass from the 3rd morphology. The Trypan test showed absence of day of treatment in the associated groups (-1.2 ± cytotoxicity in the dark at all concentrations and 100% 0.3; -1.3 ± 0.3 g, respectively), change in heart cell death at the highest concentrations after PDT, mass also in the associated groups 100 and confirmed by MTT, which showed a reduction of 93% 200 (0.50 ± 0.01; 0.51 ± 0.02 g, respectively); in mitochondrial activity at the highest concentration splenic atrophy in the groups 5-FU-25 mg, 5-FU- after irradiation. In addition, it was possible to observe 25 mg + AEMR (100 mg) and 5-FU-25 mg + morphological analysis of cytoplasmic destruction and AEMR (200 mg) (0.23 ± 0.02; 0.31 ± 0.03; 0,21 loss of structure, corroborating with previous results. ± 0.02 g, respectively) and reduction of hepatic Thus, PDT with Fotoenticine showed promise with mass in the group 5-FU-25 mg + AEMR (200 decreased cell viability, pointing to its potential use in mg), but without elevation of hepatic enzymes. future applications. There was no potentiation of myelosuppression (leukopenia and thrombocytopenia) in the This work was supported by Fundação de Amparo à associated groups. The results suggest that the Pesquisa no Estado de São Paulo (FAPESP 2016/12211- association between AEMR and 5-FU potentiated 4 and 2017/01469-3).

70 71 Biomembranes C03 - CORRELATION OF MINERAL PROPAGATION C04 - THE EFFECT OF COLLAGEN AND AND PHOSPHOMONOHYDROLASE ACTIVITY OF PROTEOLIPOSOMES HARBORING ANNEXIN V IN ALKALINE PHOSPHATASE RECONSTITUTED IN THE MINERALIZATION STUDIES - Polymorphism of Mono and - Lipids and protein influence on LIPID RAFT MODELS. C01 C02 C.R. SIMÕES¹ Diphosphorylated Lipid A Bilayers in Presence of phase separation and lysis from hereditary Favarin, B. F.¹ M. Bolean¹; P. Ciancaglini¹ Different Concentrations of NaCl and AlCl3 salts spherocytosis membranes Bolean, M.¹; Millán, J.L.2; Ciancaglini, P¹. Andresa Messias da Silva1 Bruna Renata Casadei1 1Department of Chemistry, FFCLRP - USP, Ribeirão

1 1 1 1 ¹Depto. Química, FFCLRP-USP, SP, Brazil and ²Sanford Preto, Brazil. ([email protected]) Frederico J. S. Pontes and Thereza A. Soares Daniel F. P. Perez , Rafaela R. M. Cavalcanti , Valeria Burnham Prebys Medical Discovery Institute, La Jolla, de Freitas Dutra2, Maria Stella Figueiredo2, Eneida 1Department of Fundamental Chemistry,Federal USA ([email protected]) de Paula3, Karin A. Riske1 The mineralization process initiates inside the specific University of Pernambuco, Recife, PE 1 vesicles called matrix vesicles (MVs). MVs arise ([email protected]). Biophysics Department, Universidade Federal de Osteoblasts are responsible for the bone by budding from osteoblast cells surface and are São Paulo, São Paulo, SP, Brazil. (brucasadei@ biomineralization and the process is mediated by secreted in the specific site where biomineralization 2 Lipid A is the endotoxic principle of lipopolysaccharide yahoo.com.br), Hematology Division, Department release of matrix vesicles (MVs). The MVs membrane begins. These vesicles have the ability of accumulating of Experimental and Clinical Oncology, Universidade 2+ molecules which are the major constituent of has high levels of Tissue Nonspecific Alkaline high concentrations of Ca and Pi ions, providing formulation of vaccines relies on the use of Federal de São Paulo, São Paulo, SP, Brazil; Phosphatase (TNAP), Cholesterol (Chol) as well as an adequate microenvironment for the initial 3 monophosphorilated Lipid A and aluminum salts as Biochemistry and Tissue Biology Department, Sphingomyelin (SM), when compared to the plasma formation and propagation of apatite crystals on the vaccines adjuvants [1]. Previous experimental studies Institute of Biology, University of Campinas, membrane. TNAP is a phosphomonohydrolase capable collagenous extracellular matrix. Annexin V (AnxA5) Campinas, SP, Brazil. 2+ established a relationship between chemical structure, of generating inorganic phosphate (Pi) through the ATP is responsible for the Ca -channels formation in the aggregate arrangement and endotoxicity of Lipid A hydrolysis taking to the beginning of the mineralization. MVs’ membrane. Here, we have studied the effect [2]. Furthermore, we have previously shown that Hereditary spherocytosis (HS) is an inherited hemolytic In this study, we evaluated the incorporation of TNAP of proteoliposomes composed of 1,2-dipalmitoyl- specific cations can stabilized or induce lamellar to disease caused by a deficiency in cytoskeletal or integral into liposome constituted by lipids mixture that sn-glycero-3-phosphocholine and harboring AnxA5 nonlamellar transitions in lipopolysaccharide bilayers. proteins of erythrocyte membranes. To understand the forming “rafts”, in order to study the kinetic properties on the mineralization assay in vitro, investigating if Therefore, we have performed atomistic molecular complex behavior of the composing lipids and protein in of TNAP and the ability of these proteoliposomes these vesicles have the ability to induce or modulate dynamics (MD) simulations to investigate the influence the stability and organization of the membrane structure, to induce the biomineralization in vitro. TNAP was the mineral deposition in the presence of collagen of aluminum cations on Lipid A bilayers. We have we have used optical microscopy of giant unilamellar reconstituted into liposomes constituted by neat matrix. Preliminary results were performed incubating 2+ performed simulations of mono- and diphosphorylated vesicles (GUVs) and turbidimetry in large unilamellar Dipalmitoylglycerophosphocholine (DPPC), DPPC:Chol proteoliposomes with Ca (CaCl2 2 mM) and Pi 3+ Lipid A bilayers from E. coli in presence of Al at vesicles (LUVs) to assess the response of membranes (9:1), DPPC:SM (9:1) , DPPC:Chol:SM (8:1:1), (Na2HPO4 8 mM) solutions in microplates coated different salt regimes: (a) Al3+ were added exclusively to the detergent Triton X-100. GUVs and LUVs were dimyristoylglycerophosphocholine(DMPC), DMPC:Chol or not with collagen matrix, during 20 h at 25 ºC. as counter-ions to neutralize the system total charge, composed of integral or lipid extract from erythrocyte (9:1) , DMPC:SM (9:1) and DMPC:Chol:SM (8:1:1) Mineral formation was measured by turbidity variation

(b) ionic concentration of 150 mM NaCl and (c) ionic membranes of healthy individuals (NI), HS patients and (molar ratios) as previously described by Favarin et (final minus initial) at Abs340nm and empty liposomes concentration of 150 mM AlCl3. Every system was biomimetics composed of mixtures of 1-palmitoyl-2- al. (2017). The presence of SM or Chol:SM in DPPC- were used as control. There was an increase in the simulated in replica using GROMACS version 4.65 oleyl-sn-glycero- phosphatidylcholine (POPC), egg proteoliposomes did not alter significantly the values mineral deposition in the presence of proteoliposomes

and 4.03, starting from different configurations of (chicken) sphingomyelin (SM), 1-palmitoyl-2-oleyl- of catalytic efficiency for ATP hydrolysis. However, (∆Abs=0.110) comparing to liposomes (∆Abs=0.076) the bilayer system and using different equilibration sn-glycero- phosphatidylserine (POPS), di-oleyl-sn- these proteoliposomes increased the mineral formation indicating the effect of the protein in the mineralization. protocols. The systems at salt concentration near to 0 glycero-phosphatidylserine (DOPS) and cholesterol by about 4.5 and 8-fold, respectively, compared to We have also performed the assay in the presence mM keep their lamellar arrangement with accentuated (chol). The solubilization profile of GUVs-NI, GUVs-HS DPPC proteoliposomes. The increase in catalytic of two nucleators, amorphous calcium phosphate membrane curvature of the diphosphorylated Lipid A were very similar: Triton X-100 first induced phase efficiency of proteoliposomes containing DMPC:SM (ACP) and calcium–phosphate–lipid complexes (PS-

bilayer. Lipid A bilayers in presence of 150 mM of AlCl3 separation (liquid-ordered/liquid-disordered phase, Lo/ and DMPC:Chol:SM was observed. However, in vitro CPLX), incubated in a synthetic cartilage lymph (SCL) or NaCl transition from a lamellar to a non-lamellar Ld) followed by partial solubilization of the Ld phase, mineralization assays revealed an increase of 7.2 carefully constructed to contain physiological levels arrangement. During the structural transition, ions Na however the fraction Lo/Ld was lower in GUVs-HS, and 4-fold compared to DPPC proteoliposomes, of electrolytes and substrates. These are essential replace Al3+ bound to the phosphate groups of Lipid reflecting differences in their acyl chain composition. respectively. Thus, regarding to the propagation of in components present in the nucleational core that drives A concomitant with changes in the hydration patterns In parallel, the solubilization extent of LUVs by turbidity vitro mineralization, DMPC proteoliposomes were more mineral formation by native MVs. Proteoliposomes of the latter. These simulations suggest that Lipid A measurements showed that the solubilization is efficient when compared to DPPC proteoliposomes. induced mineral formation when incubated with ACP

bilayers exhibit selective affinity for cations, which highly influenced by the type of phospholipids, not Taking together the kinetics parameters and in vitro when compared with liposome (∆Abs=0.103 and influence directly its aggregate phase. [1] MacLeod by protein (mainly in HS, that the type of protein mineralization results, it is possible to observe that 0.073, respectively). The same behavior was observed

et al. Proc. Natl., 2011, 7914–7919. [2] Brandenburg. defect in the extracted RBC membrane proteins was the ternary rafts proteoliposomes composed of when PS-CPLX was used as nucleator (∆Abs=0.098 Biophys., 1993, 1215-1231. confirmed by sodium dodecyl sulfate-polyacrylamide DPPC:Chol:SM and DMPC:Chol:SM had higher values and 0.050, respectively). In the absence of collagen gel electrophoresis). A biophysical characterization of of catalytic efficiency and they were more efficient in matrix, it was not detected significantly differences in This work was supported by FACEPE, BioMol Project/ the Triton X-100 influence in erythrocytes membranes the mineral propagation in comparison with DPPC and the absorbance. CAPES, STINT (Sweden) leads to the determination of the lipid composition, DMPC proteoliposomes. which is essential to membrane solubilization. Acknowledgements: FAPESP (2016/21236-0), CNPq This work was supported by CAPES, CNPq (167497/2017-0, 304021/2017-2), CAPES. This work was supported by Fapesp (# 2015/09948-2). (167497/2017-0, 304021/2017-2) and FAPESP (2016/21236-0).

72 73 C05 - CHARACTERIZATION OF LIPOSOMES AS C06 - THE EFFECT OF LIPID COMPOSITION AND C07 - GEL FORMATION FROM HSPC C08 - Reprodutibility of Molecular Dynamics LIPID RAFTS MODELS BY AFM CALCIUM IONS ON THE RECONSTITUTION OF PLUS TETRACAINE, ABOVE ITS MICELLE Simulations of Lipopolyssacharides Membranes ANNEXIN VI INTO LIPOSOMES C. R. Ferreira¹ CONCENTRATION using Different Integrators and Long-Range Electrostatic Treatments. VESCHI E. A.1 Fernando Freitas de Lima1 M. Bolean¹; P. Ciancaglini¹ 1 Bolean M.1; Pikula S.2; Buchet R.3, Granjon T.3 and 1 Frederico J. S. Pontes 1Department of Chemistry, FFCLRP, University Juliana Damasceno Oliveira , Viviane Ap. Guilherme Ciancaglini P.1 1 1 2 1 of São Paulo, Ribeirão Preto, SP (claudio.reis. Damasio , Eneida de Paula . Michele Cascella and Thereza A. Soares [email protected]) 1Department of Chemistry, FFCLRP, University 1Department of Biochemistry and Tissue Biology, 1Department of Fundamental Chemistry,Federal of São Paulo, Ribeirão Preto, SP, Brazil; 2Nencki Institute of Biology / University of Campinas University of Pernambuco, Recife, PE Matrix vesicles (MVs) are specific vesicles which serve Institute of Experimental Biology, Poland and (Unicamp), Campinas, SP, Brazil. (flfernando_@ ([email protected]). 2Department of 3 as the initial sites for mineralization process. In this University of Lyon, Lyon – France. (ekeveliny. hotmail.com). Chemistry, Oslo University, Oslo, Norway. study, we used liposomes in order to mimic the lipid [email protected]) microenvironment present in the membranes of MVs. One of the first synthetic local anesthetic, tetracaine Lipopolisaccharides (LPS) are the major components The MVs’ membrane contains acid palmitic (16:0) as the Matrix vesicles (MVs) provides a suitable (TTC), was launched in 1930 and it is still used, mainly in the outer membranes of Gram-negative bacterias, saturated fatty acid predominant, reaching to 35 mol%. microenvironment for mediation of bone mineralization. for topical (mucosal) and ophthalmological anesthesia1. responsible for bacterial defense and endotoxicity. Additionally, MVs are also enriched in Cholesterol (Chol) Annexin VI (AnxA6) is a protein found in three distinct TTC has aggregative properties (critical micelle Cations can affect LPS aggregation phase. Previous and Sphingomyelin (SM), resembling the lipid content regions of the MVs. This protein belongs to the annexins’ concentration ~70 mM)2. In an attempt to develop computational studies [1] point to a combination of of rafts in the membrane. We have explored here the family that are acidic phospholipid-dependent Ca2+ ionic-gradient liposomes for the encapsulation of TTC, multiple cation properties (ionic radius, charge and differences in morphology and mechanical properties binding proteins. The present study was aimed to we verified that the anesthetic at clinical concentrations hydration) as the driving initiator of structural changes of viscosity and elasticity by liposomes composed of evaluate if AnxA6 interacts with liposomes as MVs’ (0.5% or 16.6 mM) destabilized hydrogenated soy in these membranes. It was Recently [2] it was 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), biomimetic composed of dipalmitoylphosphatidylcholine phosphatidylcholine (HSPC, 15mM) vesicles. But investigated the effect of the implementation of multiple 9:1 DPPC:Chol, 9:1 DPPC:SM and 8:1:1 DPPC:Chol:SM (DPPC), dipalmitoylphosphatidylserine (DPPS) and curiously, at higher concentrations (66 mM), and in timestep algorithms on the accuracy of phospholipid (molar ratios) employing the tapping mode Atomic cholesterol (Chol). For this, it was prepared lipids the presence of HSPC and salt, a termosensitive gel membranes, raised questions about potential artifacts Force Microscopy (AFM) technique to observe lipid- mixtures composed of neat DPPC, 9:1 DPPC:DPPS, was formed. Here we show rheological, SAXS, and on membrane simulations with the new implementation. lipid interaction in liposomes. The scan rate was 6:4 DPPC:Chol and 5:4:1 DPPC:Chol:DPPS (molar EPR results on the characterization of this peculiar In this work, we have tested how different algorithm adequately reduced to 0.2 – 0.3 Hz and the integral gain ratios), in the absence and presence of 2mM Ca2+. gel. Gel formation was tested with different lipids implementations, long-range electrostatic treatments was obtained with the lowest possible value in order to Through the protein quantification by means Hartree (eggPC, soyPC, dipalmitoylPC, palmitoyloleoylPC, (Generalized Reaction-Field and Particle Mesh Ewald), prevent tip induced vesicles deformation, as described methodology, it was verified the incorporation of AnxA6 distearoylPC), TTC concentrations (from 30 to 78 code architecture (CPU and GPU) availiable in different by Bolean et al. 2017 (Biochimica et Biophysica Acta into different liposome (100 nm) compositions, resulting mM), salt types/concentrations (e.g. NaCl from 77 GROMACS versions [3], could influence aggregation, 1859, 1911–1920). AFM analysis for DPPC-liposomes in DPPC 59 µg/mL and 18 µg/mL, DPPC:DPPS 43 µg/ to 308 mM) and pH (below and above the pKa of the ordering and structure on LPS membranes. We have showed intact and spherical vesicles, with a smooth mL and 17 µg/mL, DPPC:Chol 13 µg/mL and 0,6 µg/ anesthetic). The rheological behavior of the gel was run atomistic molecular dynamics simulations of surface and neither topographic irregularities nor mL and DPPC:Chol:DPPS 54 µg/mL and 19 µg/mL, measured at different temperatures (10, 17, 25, 37, 42 LPS:DPPE bilayers in presence of Ca2+, K+ and Na+ significant visco-elastic alterations appeared. Phase with and without Ca2+, respectively. The presence of and 50 °C) and shear forces. The viscosity of the gel at 300K, 1 bar and semi-isotropic conditions. We imaging revealed that DPPC:Chol composition results in Ca2+ resulted in an increase of the AnxA6 reconstitution varied a lot as a function of pH (8.5 < 5.7 < 4.7) and have estimated area per lipid, order parameters and domains with higher phase angle shift values (brighter in all the lipid compositions used. By means Dynamic temperature (with a drastic decrease at temperatures curvature of all systems. Our results show agreement regions) than those of other regions on the liposomes Light Scattering (DLS) measurements no significant from 42 to 50 °C, that was recovered upon cooling). between the identical systems independent of long surface. Spots with higher phase angle shift values in changes were observed in the mean diameter and The molecular arrangement in the hydrogel is under range electrostatic treatments and architecture on older AFM phase images correspond to regions with a higher polydispersity index in the presence of Ca2+, remaining study, since reversible gelification occurs in a narrow GROMACS versions. However, in more recent versions stiffness values. The images obtained for DPPC:SM below 0.2. Differential Scanning Calorimetry (DSC) also range of (TTC, lipid and salt) concentrations, specific of the code, we observe differences in the membrane show spherical vesicles but with wrinkled surface. confirmed the protein interaction into the bilayers through pH and temperature. We propose the gel to be used as microstructure. [1] Soares et al. Chem. Commun., The domains with higher stiffness values were also of the changes in the thermodynamic parameters of the a drug-delivery system for tetracaine. 2014, 50, 231. [2] Mark et al. J. Chem. Theory observed, however are domains smaller than those liposomes. The presence of AnxA6 into proteoliposomes Comput., 2017, 13, 2367. [3] Kutzner et al. J. Comput. observed for DPPC:Chol composition. The ternary provokes a decrease in the phase transition enthalpy Acknowledgement: FAPESP (# 14/14457-5 / # Chem., 2015, 36, 1990. composition resulted in domains even more segregated values (ΔH) when compared with the respectively 17/20832-1). 1. de JONG R. H. Local Anesthetics. and complex. Thus, it can be suggested that these lipids liposome composition. The presence of Chol increased Springfield, Illinois: C.C. Thomas, 1994. 2. Matsuki, This work was supported by FACEPE, CAPES (Biocomp/ composition induce the formation of these distinct the phase transition cooperativity. Thus, it was possible H., Hashimoto, S., & Kaneshina, S. Incorporation BioMol), CNPq, STINT/SE, Oslo University. microdomains with different mechanical properties and observed interaction between AnxA6 and liposomes, of micelle-forming Local Anesthetics into Surface- also with different chemical composition. with best yields of proteins incorporation in the presence Adsorbed Films and Micelles of Decylammonium of 2 mM Ca2+. Chloride, Langmuir 7463, 2687–2693, 1997. Acknowledgements: FAPESP (2016/21236-0), CNPq (167497/2017-0, 304021/2017-2), CAPES. Acknowledgements: FAPESP (2016/21236-0), CNPq (167497/2017-0, 304021/2017-2), CAPES (1738449).

74 75 Ca2+ and P ions that along with several proteins/enzymes goals of this study were the binary (POPC/Chol) and and the aggregation is stronger in fluid phase of the C09 - THERMAL AND FLUORESCENCE STUDIES i OF LEVOFLOXACIN COPPER (II) COMPLEXES: generate a very particular and proper microenvironment ternary systems (POPC/PSM/Chol) of membranes. membrane. It is possible to conclude that Esc 1b (1-18) INTERACTION WITH ANIONIC MEMBRANES. for the correct initiation and propagation of the In the binary systems, the results of anisotropy interacts better with negatively charged membranes on hydroxyapatite crystals. Some of the proteins present in showed that the presence of hydroperoxide keeps the fluid phase. Gabriel S. Vignoli Muniz this process have a more important role and demand membrane in the liquid-disordered phase (ld), and Evandro L. Duarte, M. Teresa Lamy a special attention. The Na,K-ATPase (NKA) has been just in high concentrations of cholesterol (above 40%) Acknowledgment: CNPq, Fapesp, Capes and INCTx. recently reported by proteome studies to be present in the membrane shows an increase in the anisotropy, Institute of Physics, University of São Paulo, São MVs. Its multifunctional characteristics are well described suggesting that the membrane becomes more ordered, Paulo, SP ([email protected]) C13 - Interactions of compounds with bioactive in the literature including its specific phosphatase activity corroborating with the literature. In the ternary system, properties with models of membranes at the air- producing P from ATP hydrolysis. The objective is to we replaced POPC by hydroperoxide. Results show i water interface Levofloxacin (LVX) is a naturally fluorescent evaluate if NKA is able to propagate mineralization and that in both cases the anisotropy increases with the fluoroquinolone antibiotic commonly used for medical the effects of Tris buffer concentration, from 0.8mM concentration of cholesterol, but in the presence of Jefferson C Rodrigues¹ treatment. Recent studies indicate that the complexation to 3.5mM. Therefore, proteoliposomes harboring NKA hydroperoxide all the anisotropy values are higher than Luciano Caseli2 of fluoroquinolone with divalent cations has a strong were prepared by co-solubilization method with two similar ones without hydroperoxide. We conclude the 1 impact on its interaction with amphiphilic structures different lipid compositions: 1,2-dipalmitoyl- sn-glycero- hydroperoxide can change the phase of membrane Department of Chemistry, Federal University of São and membranes thus having important consequences 3-phosphocholine (PC) and DPPC: (1,2-dipalmitoyl-sn- in binary system, suggesting that in ternary systems Paulo, Diadema, SP ([email protected]) 2 for fluoroquinolones solubility, pharmacokinetics, and glycero-3-phosphoethanolamine) (PE) (1:1). The mineral the changes in membrane are more pronounced by and Department of Chemistry , Federal University bioavailability. The complexation also involves the propagation was measured by turbidity at 340nm after cholesterol. of São Paulo, Diadema, SP. mechanism of this class of antibiotics and is proposed incubation at 37°C for 48h, in triplicates and liposomes as a strategy to overcome bacteria resistance. Our were used as controls. Results were normalized This work was supported by FCT- FAPESP (2014/20107- Interactions of bactericidal and antiprotozoal drugs focus is to study the interaction of LVX and its copper according to the protein concentration. The solubilized 7) and FAPESP (2017/18521-8) with cell membranes could be better understood (II) complex with anionic membranes. Large unilamellar NKA had the most effective activity and enhanced the at the molecular level if nanostructured systems vesicles (LUVs) of dipalmitoil fosfatidil glicerol (DPPG) mineral propagation by 51.9 Abs.mg-1. PC:PE-NKA and are employed. This study aimed to study the -1 C12 - USING GEL AND FLUID PHASES OF THE was used to simulate a bacteria membrane. By using PC-NKA had turbidity of 51.4 and 38.3 Abs.mg . When MEMBRANE TO STUDY THE MODE OF ACTION OF action of the drug Metronidazole in Langmuir differential calorimetry scanning (DSC), time-resolved, the concentration of buffer was increased to 3.5mM, ANTIMICROBIAL PEPTIDE ESC 1B (1-18) films of phospholipids, which are used as simple and steady-state fluorescence spectroscopy, we the turbidities decreased to 5.0, 1.1 and 0.6 Abs.mg-1. models of cell membranes. The phospholipids Isabela Moreira-Silva¹* investigated the interaction of LVX in the presence Although the Pi produced from ATP hydrolysis by NKA used were dipalmitoylphosphatidylcholine (DPPC), of an equimolar concentration of CuCl2 or dichloro propagated the growth of calcium phosphate in the Tiago Ribeiro de Oliveira², Maria Teresa Lamy³, dipalmitoylfosfatil serine (DPPS) and dipalmitoylfosfatil (1,10-phenanthroline) copper(II) (Cuphen) with LUVs mineralization process, a higher concentration of Tris Katia Regina Perez¹ glycerol (DPPG), cholesterol (COL), 1,2 Dioleoyl-sn- of DPPG at pH 7.4. Fluorescence data shows that pure buffer inhibited the mineral propagation. glycero-3-phosphocoline (DOPC), and combinations LVX has low affinity to LUVs. The tritation of LVX in the ¹ Universidade Federal de São Paulo, São Paulo of glycoproteins with dipalmitoylphosphatidylcholine – SP, Brasil. ²Universidade Federal do ABC, Santo presence of CuCl2 or Cuphen with LUVs, suggests that Acknowledgements: FAPESP (2016/21236-0), CNPq (GLICOP +DPPC), peptidoglycan with DPPG the majority of LVX molecules remains associated to (167497/2017-0, 304021/2017-2), CAPES. André – SP, Brasil. ³Universidade de São Paulo, São (Pep+DPPG) and lipopolysaccharides with DPPG copper ions. DSC profiles show that the association Paulo – SP, Brasil. *[email protected] (LPS+DPPG). The lipids were spread on the air- 2+ of LVX with Cu or Cuphen profoundly disturb lipid C11 - Changes in lateral organization of water interface, and the drug injected into the transition. This disturbance resolutely indicates that the membranes containing hydroperoxided lipid The indiscriminate use of antibiotics becomes aqueous subphase. The adsorption of the drug was antibiotic copper complex incorporates itself into lipid necessary the search for new antimicrobial compounds. evaluated by surface pressure measurements, infrared bilayer. These techniques attest that the association Helena Couto Junqueira1 The antimicrobial peptide Esculentin 1b presents spectroscopy and Brewster angle microscopy (BAM). of copper (II) ions with LVX has a synergic effect on its Manuel Prieto2 and Rosangela Itri3 48 amino acids residues but the region composed The drug causes pronounced effects on pressure- interaction with anionic LUVs, indicating an impact on of the first 18 amino acids residues (Esc 1b (1-18)) area isotherms for DPPC, DPPS, DPPG, Cholesterol, 1 membrane diffusion possibly influencing bioavailability, Department of Biochemistry, Institute of Chemistry, have the same antimicrobial activity as entire peptide Glycoproteins + DPPC, Lipopolysaccharides + DPPG and its mechanism of action. University of São Paulo, São Paulo, SP. (helena@ without hemolytic activity. This work aims to study and Peptidoglycans+DPPG condensing the monolayer, 2 iq.usp.br). Centro de Química-Física Molecular, the interaction of antimicrobial peptide Esc 1b (1-18) but expanding DPPG and DPPE monolayers. Infrared Instituto Superior Técnico, University of Lisbon. using model membranes composed of neutral and spectroscopy shows significant changes in the bands C10 - THE INFLUENCE OF TRIS BUFFER 3 Department of Applied Physics, Institute of negatively charged phospholipids bearing saturated related to the stretch of methyl, increasing the molecular CONCENTRATION IN MINERAL PROPAGATION Physics, University of São Paulo, São Paulo PROMOTED BY THE Na,K-ATPASE and unsaturated fatty acyl chains. Calorimetric and order for DPPS, DOPC, DPPE monolayers, and mixtures Spectroscopic measurements were used to verify the of DPPC with glycoproteins, whereas for DPPG, DPPG Sebinelli, H. G. The oxidative stress can generate reactive species that interaction of Esc 1b (1-18) with gel and fluid phases + LPS and DPPG + Pep the molecular order has a Favarin, B.F.; Bolean, M.; Ciancaglini, P. cause damage in cell membranes, leading to cell death. of the membrane. ITC shows the interaction of Esc 1b decrease, can also be observed probable interactions in The first product that is produced in lipid oxidation is (1-18) with the membranes involves an endothermic the hydrophilic regions of the film when in the presence Dept. Chemistry, FFCLRP-USP, SP, Brazil. the hydroperoxide. The lipid hydroperoxide can change heat variation with both gel and fluid phases. DSC of mixed monolayers (LPS + DPPG and Pep + DPPG). ([email protected]) the lateral organization of membranes, and altering indicates that the peptide can change the gel-fluid BAM images show that the surface morphology of the membrane properties, as fluidity and permeability. In bilayer transition of the negatively charged membranes, lipid monolayers also changed with the presence of the The biomineralization process consists of mineral previous works of our group, we observed that the reducing the medium transition temperature, and drug, causing molecular aggregation, but with different growth mediated by matrix vesicles (MVs) liberated from hydroperoxide lipid can be responsible for separation decreasing the cooperativity. EPR shows the peptide morphological configurations for each film. This shows osteoblasts. These vesicles leak from cells surface in the of lipids in the membrane. In this work we studied the enters in the membrane, increasing the rigidity of gel that the interaction of Metronidazole in the monolayer specific localization where the biomineralization starts in changes in lateral organization of membranes containing and fluid phases of the membrane. And light scattering is modulated by the lipid composition at the air-water matrix of bone tissue. MVs have high concentrations of oxidized lipids by time resolved anisotropy studies. Our shows aggregation of negatively charged membranes, interface, as well as the drug-lipid interactions. We

76 77 hope that these results have a relevant impact on C15 - THE EFFECT OF SIZE DISTRIBUTION ON C16 - THE INFLUENCE OF pH AND ZETA C17 - Effects of Pore-forming toxins (PFTs) on the understanding of how bioactive compounds act THE CYTOTOXICITY OF LIPID NANOPARTICLES POTENTIAL IN THE ANNEXIN V INCORPORATION oxidized model membranes represented by Giant in microbial cells and in the pursuit for proposals of INTO LIPOSOMES Unilamellar Vesicles (GUVs) mechanisms at the molecular level. Ludmilla D. Moura1 Maressa Donato1e2 Lígia N. Ribeiro1, Eneida de Paula1 L. H. Andrilli¹ Carlos Alvarez3, Martha Simões Ribeiro2, C14 - PE/PG DEMIXING INDUCED BY A 1Department of Biochemistry, University of Campinas, M. Bolean¹; A.P. Ramos¹; P. Ciancaglini¹ Rosangela Itri1 SYNTHETIC MASTOPARAN-LIKE PEPTIDE IN São Paulo, SP. ([email protected]) 1Department of Chemistry, FFCLRP, USP, Ribeirão MODEL MEMBRANES Preto, SP. ([email protected]) 1Institute of Physics University of São Paulo 2 Kenneth M. F. Miasaki A nanobiotechnology has contributed to the promising (IFUSP), National Institute of Nuclear Energy Research(IPEN), 3 Center for Protein Studies, Dayane S. Alvares, and João Ruggiero Neto path in the development of new clinical treatments. The The mineralization process is mediated by osteoblasts dynamism of colloidal systems becomes useful and, that control the deposition of the extracellular matrix University of Havana, Havana, Cuba Department of Physics, São Paulo State University, especially, due to the lack of physical techniques, which and the release of matrix vesicles (MVs), which IBILCE, Brazil ([email protected]) can be correlated with their biological performance, serve as the initial sites for hydroxyapatite formation. Sticholysin I (St1) and II (St2) are polypeptides isolated emphasizing the size of nanoparticles are directly Annexin V (AnxA5) is an acidic phospholipid- from the Caribbean sea anemone Stichodactyla L1A, a synthetic peptide, is a potent Gram-negative related to biodistribution and cellular interaction. dependent Ca2+-binding protein, which acts as Ca2+- Helianthus, with a molecular mass of 19401 and antibacterial without being hemolytic. We have shown Thus, the objective of this work was to develop lipid channel in the MVs’ membrane. Herein, we describe 19290 Da, with hemolytic and pore-forming properties that the N-terminus acetylation of L1A (ac-L1A) nanocarriers based on nanostructured lipid carriers the preparation of proteoliposomes composed in membranes (LANIO et al., 2000). It has been enhanced the lytic activity in anionic vesicle compared (NLC) and nanoemulsion (NE) formulations composed of dipalmitoylphosphatidylcholine (DPPC) and previously determined that the activity of membrane to L1A which was correlated to its capability to insert of copaiba oil (Copaifera duckei), which presents dipalmitoylphosphatidylserine (DPPS) harboring AnxA5. pore formation is related to the N-terminal insertion into and disturb lipid packing of model membranes. anti-inflammatory, antibacterial, antifungal and The aim is understanding the influence of pH and zeta in the bilayer and lipid phase coexistence must play a We have evaluated the impact of L1A and ac-L1A, analgesic properties1. These systems present different potential (from 3 to 9) in the incorporation of AnxA5 role too (ROS et al., 2013). As PFTs, both toxins are on model membrane that mimic the cytoplasm average particle sizes, and this parameter was used into DPPC:DPPS 9:1 (molar ratio) liposomes (100 considered to have potential appliance in parasitic membrane of gram-negative bacteria, e.g. E. Coli, that to investigate its effect on the in vitro cytotoxicity. nm, 1.5 mg/mL). For pH range from 3 to 6, liposomes and tumor diseases (MARTÍNEZ et al., 2007). In this contain mainly phosphatidylethanolamine (PE) and The structural characterization was carried out by and proteoliposomes were produced in 100 mmol/L work, we have investigated GUVs composed of non- phosphatidylglycerol (PG) using a variety of techniques. Dynamic light scattering (DLS) in terms of size (nm), Acetate buffer. For pH from 7 to 9 vesicles were formed oxidized POPC and POPC hydroperoxide (POPC-OOH) Circular dichroism experiments showed that the polydispersity index (PDI) and Zeta potential (mV); in 100 mmol/L Tris-HCl buffer. AnxA5 has incubated interacting with ST1 and ST2 by optical microscopy. reduction of N-terminus charge did not influence its Nanoparticle tracking analysis (NTA) was employed with liposomes (1:100 protein:lipid ratio) during 20h, The results did not reveal a significant PFT-membrane affinity to 3POPE:1DOPG large vesicles; however, the to obtain the nanoparticle concentration (part/mL) as at 25 ºC under gentle stirring. Then, the mixture was interaction such that no membrane destabilization percentage of alpha-helix was higher for ac-L1A than well as the particle size distribution of the formulations; ultracentrifuged at 100,000xg during 1 h, at 4 ºC. The was observed over incubation time of 20 min. On the for L1A. Further we employed differential scanning and pH of the lipid nanoparticles was also quantified. pellet containing proteoliposomes was resuspended other hand, when GUVs were made of mixtures of calorimetry to explore the thermotropic changes NLC formulations showed particle size around 207.8 in the initial volume of each buffer. The best yields POPC or POPC-OOH and Sphingomyellin (SM), optical induced by these peptides. Thermograms of pure nm (DLS) and 141.4 nm (NTA), PDI close to 0.19 and of protein incorporation were obtained at pH 5 and 4 contrast fading was noticed indicating an increase in mixed lipids MLV, undergoes a gel to liquid-crystalline Zeta values of -27.1 mV. NE formulations presented (113.8 and 16.6 μg/mL, respectively). In the other pHs, lipid bilayer permeability due to pore formation. Of transition at 15 oC with a shoulder at ~17 oC. In the size around 128.7 nm (DLS) and 99.4 nm (NTA), no more than 10 μg/mL of protein was incorporated. note, neither micron-sized pores were observed nor presence of both peptides, the transition peak was shift PDI close to 0.18 and Zeta values of -20.5 mV. The Zeta potential analysis were acquired to investigate the membrane disruption. Further, the results also pointed to higher temperature indicating that the incorporation amount of lipid nanoparticles was 5.17x1013 for NLC isoelectric point (IP) of the purified protein, DPPC:DPPS- out vesicles composed of oxidized lipids and SM have of peptides induced lipid perturbation. ac-L1A was, and 1.75x1014 for NE. Finally, the pH of NE was found liposome and DPPC:DPPS-proteoliposome harboring a much faster pore forming capacity. Therefore, our however, able to induce higher phase separation with more acid (3.35) than of NLC (5.65). Considering these AnxA5. The analysis was performed in the same results thus suggest that membranes containing -OOH two symmetric phase transitions. Ac-L1A more deeply clear differences in the structural properties of such range of pH. AnxA5 has showed the IP at pH 3.1 and and SM promote the insertion of toxins due to their inserted into monolayers at constant area compared copaiba-based nanostructured systems, cell viability DPPC:DPPS-liposome and proteoliposomes exhibited great fluidity, facilitating the insertion of the TFPs and with L1A, inducing surface pressure changes that assays will be performed for the analysis of cytotoxicity IP of 3.5 and 4.1, respectively. It can be observed that their differentiation, leading to the formation of pores. surpassed the lateral pressure of vesicles. Visualization in fibroblast and mammary adenocarcinoma cells, in when AnxA5 is present in the vesicles there is a Zeta of morphological domains change of lipid monolayer order to elucidate the size distribution effect on the Potential displacement to more negative values, from Acknowledgement: IUBMB, IUPAB, FAPESP e CNPQ. by fluorescence microscopy showed that the presence further therapeutic action of these formulations. -30 mV for liposomes to – 40mV for proteoliposomes. of both peptides disordered the hydrophobic chains This set of data indicate that AnxA5 binds at positives References: LANIO, M. E. et al. Purification and preventing the formation of stiff films. All the results References: 1RIBEIRO, L. N. M.; BREITKREITZ, M. C.; charge of liposomes, resulting in proteoliposomes with characterization of two hemolysins from Stichodactyla agree in that both peptides disrupt the lipid packing GUILHERME, V. A.; SILVA, G. H. R.; COUTO, V. M.; more negative Zeta Potential. helianthus. Toxicon, v. 39, n. 2–3, p. 187–194, 2000. and that ac-L1A was able to cluster anionic lipids more CASTRO, S. R.; PAULA, B.O.; MACHADO D.; DE PAULA, MARTÍNEZ, D. et al. Effect of sphingomyelin and efficiently. ENEIDA. Natural lipids-based NLC containing lidocaine: Acknowledgements: FAPESP (2016/21236-0, 2017/ cholesterol on the interaction of St II with lipidic interfaces. from pre-formulation to in vivo studies. European 25475-2), CNPq (167497/2017-0, 304021/2017-2), Toxicon, v. 49, n. 1, p. 68–81, 2007. ROS, U. et al. The This work was supported by FAPESP, CAPES, CNPq Journal Of Pharmaceutical Sciences, v. 106, p. 102- CAPES. sticholysin family of pore-forming toxins induces the (Fapesp #2015/25619-9 and #2015/25620-7). 112, 2017. Acknowledgments: CAPES, FAPESP, CNPq, mixing of lipids in membrane domains. Biochimica et UNICAMP Biophysica Acta - Biomembranes, v. 1828, n. 11, p. 2757–2762, 2013.

78 79 C18 - MOLECULAR SIMULATIONS OF further simulations to investigate pore formation in C20 - The role of cholesterol on the interaction of This research was supported by Fundação de Amparo OXIDIZED LIPID MEMBRANES UNDER lipid mixtures and in phase-separated membranes with Curcumin and Quercetin with model membranes a Pesquisa do Estado de São Paulo – FAPESP Nos. oxidized domains. 2012/24259-0, 2014/08372-7, 2014/06713-1, MECHANICAL STRESS 1,2 Natália B. Leite 2014/11877-3, and UFTM. 1 Maria C. Oliveira This work was supported by Federal University of ABC. Danubia B. Martins3, Vinicius E. Fazani3, Maira R. Rodrigo M. Cordeiro2 Vieira2, Marcia P. dos Santos Cabrera2,3 C21 - The study of the interaction of amphiphilic References:[1] I. O. L. Bacellar, M. C. Oliveira, et al. 1,2ABCSim Group – Simulation and Modeling of 1Departamento de Física, Universidade Federal ionic liquids with model membranes: a structural Journal of the American Chemical Society. (2018) and spectroscopic approach Atoms, Molecules and Condensed Matter, Federal DOI:10.1021/jacs.8b05014. [2] M. Yusupov, D. Y. Yan, do Triângulo Mineiro (UFTM), Instituto de University of ABC, Santo André, SP (cecilia. et al. Biochimica Et Biophysica Acta. 1861 (2017) 839. Ciências Naturais, Exatas e Educação (ICENE), Natália F. de Oliveira1 [email protected]) Univerdecidade, Uberaba, MG, Brazil (e-mail: Luiz Fernando De C. Rodrigues1, Gustavo [email protected]). 2Departamento de Carretero2, Greice Kelle Viegas2, Iolanda Cuccovia2 C19 - MOLECULAR INTERACTIONS OF Química e Ciências Ambientais and 3Departamento Membrane lipids are susceptible to non-enzymatic PHENANTHROLINE AND ITS COPPER(II) and Leandro R. S. Barbosa1 oxidation caused by free radicals, generating a de Física, Universidade Estadual Paulista (Unesp), COMPLEX WITH MODEL MEMBRANES 1 mixture of several lipid oxidation products which Instituto de Biociências Letras e Ciências Exatas Department of General Physics, University of may cause inflammatory and neurodegenerative Natalia Alvarez1 (Ibilce), Câmpus São José do Rio Preto, SP, Brazil. São Paulo, São Paulo, SP (email:[email protected]). [1] 2Department of Biochemistry, University of São diseases . Experimental and theoretical studies have Priscilla Freddi2, Rafael P. Barroso2, Antonio J. Paulo, São Paulo, SP demonstrated that different products have different Costa-Filho2 and Gianella Facchin1 Curcumin and Quercetin present a wide range of effects over biological membranes. For instance, biological activities as antioxidant, anticancer, anti- 1 oxidation products such as lipid aldehydes are known Inorganic Chemistry, Facultad de Química, inflammatory and antimicrobial. Their biological effects Ionic Liquids (ILs) are salts composed of an organic to facilitate the formation of membrane pores, but this Universidad de la República, Mdeo., Uruguay have been explained by binding and/or interference ion and an inorganic counterion, which is found in 2 mechanism is still poorly understood[2]. In order to ([email protected]), Biophysics Laboratory, with enzymes, receptors, transporters and signal the liquid state at temperatures smaller than 100°C. unravel the underlying molecular mechanisms to pore Physics Department, Universidade de São Paulo, transduction systems. Once that these important It has both academic and industrial interest due to formation on biological membranes, we have performed Ribeirão Preto, SP, Brasil. mechanisms mostly occur in membrane environments, its potential applications in several areas, including non-equilibrium molecular dynamics simulations it is worth to investigate these compounds interactions the green chemistry, the pharmacology, the bio- of membrane stretching. We studied membrane Inorganic Medicinal Chemistry has made great progress with lipid membranes of biological interest. Mammalian medicine and bio-nanotechnology. Nevertheless, models composed of native POPC (1-palmitoyl-2- in the development of antineoplasic drugs. In this work cell membranes characteristically contain 25-50% recent results show that the toxicity of ILs is greater oleoyl-sn-glycero-3-phosphocholine) lipid and its we describe the interaction of phenanthroline (phen) cholesterol/phospholipid ratio; however, most studies than previously believed, particularly with systems of oxidation products containing alcohol (Ox-alcohol), and [Cu(phen)]2+ cation with simple lipidic bilayer involving lipid bilayers and these antioxidants consider biological relevance. The main goal of this project is hydroperoxide (Ox-hydroperoxide), and ketone (Ox- membrane models. Phen is a commonly used ligand in pure phosphatidylcholine. In this sense, we investigated to get more information on the interaction of an ionic ketone) functional groups. inorganic chemistry due to its strong chelating ability. curcumin and quercetin interactions with lipid bilayers liquid: 1- tetradecyl-3-methylimidazolium chloride

Several phen metal complexes present high cytotoxic containing cholesterol, by mimicking mammalian cells, ([C14MIM][Cl]) on biomimetic membrane systems, activity. In particular, the [Cu(phen)]2+ complex has through spectroscopy techniques to determine partition which are increasingly being used to simulate cellular been used as a building block for numerous compounds coefficients and rigidity parameters. Additionally, phase plasmatic membrane behavior. To do so, we utilize with potent antitumor activity. Although many studies contrast microscopy was used to observe antioxidants two lipids, DPPC (1, 2-dipalmitoyl-sn-glyecro-3- have been focused on the interaction of phenanthrolinic lytic activity and possible morfological effects over phosphatidylcholine) and DPPG (1, 2-dipalmitoyl- ligands and their metal complexes with DNA as a the membranes structure. We found that curcumin sn- glyecro-3-phosphatidylglycerol). The interactions biomolecular target molecule for cytotoxic action, little and quercetin partitions into different lipid bilayers, between these lipids systems and the IL in question attention has been paid to the interaction with lipidic containing cholesterol or not, and in the presence of have been studied through fluorescent probe leakage, membranes. The interaction of the chosen molecules sphingomyelin or phosphatidylserine; where curcumin utilizing the fluorophore PTS (sodium 1,3,6,8 pirene with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine partition coefficients showed to be higher than tetrasulfonate) in presence of methyl viologen (MV).

(DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phospho- those found for quercetin. Besides this, curcumin Based on the results, we observed that [C14MIM][Cl] (1’-rac-glycerol) (DPPG) has been studied through decreases rigidity in all tested compositions, except alters the internal arrangement of vesicles composed of differential scanning calorimetry (DSC) and EPR using that containing 40% cholesterol in which it increases anionic lipids. The IL was able to rupture 100% of the spin labels at the head group, C5 and C16 of the alkyl the lipid packing order while quercetin did not exhibited DPPG membranes, as the concentration of IL increased chain. Phen introduced only subtle changes at a deep this effect. Also, both curcumin and quercetin induced the vesicles are destroyed and, interestingly, failed to Figure 1. Stretching simulation of native POPC and oxidized level of both model membranes. For [Cu(phen)]2+ the leakage of giant unilamellar vesicles internal content permeabilize DPPC membranes, the percentages of POPC at 0.025 m/s. effect on DPPC was similar than that observed for on a cholesterol concentration dependent way; an leakage remained low and close independent of IL phen, whereas with DPPG a stronger interaction was effect most pronounced for curcumin. In addition concentration. In addition to the mixed vesicles, the We calculated the areal strain, i.e., the area variation evidenced at a superficial level due to the anionic to our results suggesting that quercetin presents a IL also was capable of permeabilize, however the required for pore formation. As shown in Figure 1, charge of the latter. more superficial activity in membrane environments, maximum percentage of leakage obtained was 60%. lipid oxidation cause pores to form at lower values when compared to curcumin; they also showed that of areal strain. Our preliminary data suggest that lipid This work was supported by FAPESP, CAPES and the antioxidants interaction with model membranes is This work was supported by Fundação de Amparo à oxidation products might contribute to cellular damage PEDECIBA (Uruguay). modulated by the cholesterol content, which may help Pesquisa do Estado de São Paulo (FAPESP#2016/ due to increased membrane leakiness. We will perform researchers in the analysis of their biological effects by 05019-0, 2015/15822-1). calling the attention to the role of cholesterol content on their mechanism of action.

80 81 C22 - CONTROL OF FIRING OF DORSAL This work was supported by Conselho Nacional C24 - Photoactive Ruthenium Complexes: solution/ αHL-nanopore system. This investigation is COCHLEAR NUCLEUS CARTWHEEL NEURONS BY Desenvolvimento Cientifico e Tecnologico (CNPq). Efficiency and Mechanism of Damage in relevant to characterize the alpha-hemolysin (αHL) ATP-SENSITIVE K+ CHANNELS Membrane Models nanopore as an analyte biosensor. Three different C23 - STUDIES OF THE INTERACTION OF THE concentrations of PEG were used (400 µM, 1mM Strazza Junior PS Raphael de A. N. Gomes1 DENGUE FUSION PEPTIDE AND LIPID BILAYERS and 2mM) and several voltage clamp values were Leão RM Maurício da S. Baptista1 applied to the nanopore (20-100 mV). The results Priscilla Freddi1 disclose that none significant difference in the LZC Department of Physiology, University of São Paulo, 1Department of Biochemistry, University of São Eduardo F. Vicente2 and Antonio J. Costa-Filho1 mean values was noted (all very close to 1), neither São Paulo, SP ([email protected]) Paulo, São Paulo, SP ([email protected]) in the three analyte concentrations nor to different 1Laboratory of Molecular Biophysics - Faculty of voltage values applied in each concentration. Our Glycinergic cartwheel neurons from the dorsal cochlear Philosophy, Sciences and Letters of Ribeirão Preto, Photodynamic therapy (PDT) comprises the combinatorial results show that under the conditions studied the nucleus (DCN) provide a strong inhibitory force on the University of São Paulo ([email protected]) use of a photosensitizer (PS), light and oxygen to biosensing with alpha-hemolysin nanopores to the 2 glutamatergic DCN fusiform neuron. Most cartwheel and Faculty of Science and Engineering - Campus promote cell death, and is being extensively researched PEG behaved as a stochastic process both for the neurons present spontaneous action potential firing of Tupã, State University of São Paulo (UNESP) and tested to treat different types of diseases, such as time of arrival of the analyte at the mouth of the contributing to most of inhibitory post-synaptic currents cancer, leishmaniosis, malaria, among others. Ruthenium nanopore and for its residence time inside the pore. on fusiform neurons. Several evidences suggest that Dengue disease is endemic in more than 100 countries complexes are promising compounds to function as a decrease in the inhibitory drive in the DCN could and its caused by a virus member of the Flaviviridae PDT optimized drugs (PS), because they present high This work was supported by Coordination for the be related to the increased in the firing of fusiform family, a group of enveloped virus. The infection starts absorption in the UV-Vis region of the spectrum and long Improvement of Higher Education Personnel (CAPES), neurons observed in animal models of tinnitus. We by the fusion of the virus with the plasma membrane, lifetimes of triplet states. The objective of this project is to Foundation for Science and Technology Support in performed whole-cell patch-clamp recordings of DCN mediated by a small hydrophobic protein domain, known characterize the photochemical damage mechanisms of Pernambuco (FACEPE) and the National Council for cartwheel neurons of young rats (p18-22) in order to as fusion peptide, that is highly conserved on the other ruthenium complexes in lipids and in membrane models, Scientific and Technological Development (CNPq). investigate the ion channels influencing spontaneous virus of the family, like Zika virus. Due to conformational and to analyse their interactions with these membrane firing of cartwheel neurons, and consequently the changes that occur depending on the environment, models. The polypyridyl ruthenium complexes, that have C26 - Specific roles of truncated and tonic inhibition on fusiform neurons. Most of cartwheel this domain interacts with cell membranes, leading been tested, act either by generating 1O (singlet oxygen) or 2 hydroperoxized lipids in membrane neurons (>80%) fired action potentials spontaneously to a fusion of both membranes. Once linked, the viral by releasing a label ligand, allowing ruthenium to covalently microdomains at rest (active) while the other present a stable resting nucleocapsid can be released into the host cell, allowing bond with biomolecules. The membrane models consisted membrane potential (quiet). The spontaneous firing the virus replication. Understanding the interaction of unilamellar and multilamellar liposomes, mainly Tayana Mazin Tsubone1 was not abolished by perfusion of glutamatergic or mechanisms between the Dengue virus and its host are composed of POPC, POPG and/or soy lecithin. The main Helena Couto Junqueira2, Maurício S. Baptista2 and glycinergic synaptic blockers. Active neurons had bigger of great interest and importance for therapeutic, since effects studied were the oxidation of lipids constituent of Rosangela Itri1 membrane input resistances and longer membrane time this information may assist the development of new the membrane, the permeabilization of the membrane and 1 constants. Application of barium chloride (0.1 mM) drugs or of new vaccines. Here we focus on the basics the aggregation of the ruthenium complexes. These data Institute of Physics - University of Sao Paulo, São a blocker of inwardly rectifying potassium channels of these interactions that occur at the molecular level. will help to characterize the photochemical mechanisms Paulo - SP (e-mail: [email protected]) and 2Institute of Chemistry - University of Sao Paulo, (Kir), which controls spontaneous firing of fusiform To do so we synthesized dengue fusion peptides of by which ruthenium complexes damage the membranes. neurons, depolarized the membrane of both active and serotypes 1 and 3 with their flanking regions and their São Paulo - SP. quiet cartwheel neurons, and induced firing of quiet mutants containing cysteine-serine substitutions. We This work was supported by CNPq (CNPq # neurons. Interestingly, the current blocked by barium evaluated by fluorescence spectroscopy and circular 134170/2017-2). Oxidized lipids are usually associated in numerous had a similar conductance in the non-rectifying part, dichroism (CD) the interaction between the Dengue diseases such as artherosclerosis, cancer, diabetes, but in quiet neurons we observed a smaller rectification. fusion peptide and membrane mimetics. The CD - LEMPEL-ZIV COMPLEXITY APPLIED IN cardiovascular and Alzheimer’s since lipid oxidation + C25 ATP-sensitive K channels (KATP) are formed by the results showed that the peptides maintained structures NANOBIOSENSING disturbs several functions of cellular membrane. Various K subunits and have small rectification. Application disordered, even in the presence of liposomes. To signaling pathways are initiated in response to oxidative ir6 Romildo A. Nogueira1* of the KATP blocker tolbutamide (0.2 mM) depolarized monitor the lipid mixing induced by the peptides was stress in membranes that require the localization or quiet neurons, leading to spontaneous firing, and used Fluorescence Resonance Energy Transfer (FRET) Gesilda F. Neves1, Dijanah C. Machado2, Carlos binding of proteins on lipid rafts. However, it is still inhibited a non-rectifying potassium conductance. with different molar ratios in pH 5 and 7,4, endosomal M. M. Carneiro2, Luiz H. A. Consoni2, Cláudio G. unclear the contribution of oxidized lipids to the formation In active neurons tolbutamide did not have any effect and physiological pH, respectively, which showed us Rodrigues2. or disruption of membrane domains at molecular level. on both firing and membrane currents. On the other that the lipid mixing is higher for the serotype 1 peptide, Then, we investigated the phase separation behavior of 1 hand, the K activator diazoxide hyperpolarized the being higher for the native sequence on the acid pH. Departamento de Morfologia e Fisiologia Animal, two oxidized lipids bearing different shapes, POPCOOH ATP 2 membrane potential of both quiet and active neurons, The binding experiments showed that the peptides bind UFRPE ([email protected]). Departamento de as the cylindrical and PazePC the conical one, in ternary Biofísica e Radiobiologia, UFPE and silenced active neurons. Thus, KATP channels have with the vesicles in the fusogenic pH. mixtures-containing membranes. The role of each an active role in controlling the spontaneous firing of oxidized lipid on phase separation was compared to

DCN cartwheel neurons. Because KATP channels couple This work was supported by Coordination for the The Lempel-Ziv Complexity (LZC) is a method that those of the corresponding ternary mixtures membranes membrane potential with the energetic status of the cell Improvement of Higher Education Personnel (CAPES), allows estimates the complexity in a time series containing the respective non-oxidized lipid, i.e. POPC and continuous action potential firing is energetically by The São Paulo Research Foundation (FAPESP), and without the need of very much data. Here, time by using fluorescence microscopy of giant unilamellar demanding, these channels can be an important by the National Research Council (CNPq). series were obtained by the recording of ionic vesicles (GUV). The results show in an unprecedented mechanism to decrease tonic firing in cartwheel neurons current blockage by polyethylene glycol (PEG) in way that hydroperoxide lipid (POPCOOH) promotes in response to decreased ATP levels during situations of an alpha-hemolysin (αHL) nanopore inserted in a formation of micro-sized lipid rafts membranes while high intensity firing. planar lipid bilayer, bathed by 4 M KCl in both sides. the truncated oxidized lipid (PazePC) disarrange the lipid In this context we use the LZC in the analysis the micro-domains, up to 18% mol fractions. The distinctive dynamics of the partitioning of the analyte PEG in the impact of each oxidized lipid on domains membrane due

82 83 to molecular geometry, might be a key for the choice of agreement with the experimental data. This study also and Erg individually or in simultaneously enriched C29 - Psd2 pea defensin shows preference solani of selective association or disassociation of essential reveals two potential issues with the GROMACS software, membranes. The K obtained for these vesicles for mimetic membrane rafts enriched with p proteins signaling on recovery or elimination of cells. related to an incorrect calculation of the pressure glucosylceramide and ergosterol indicated a good interaction with Psd2 that takes Our findings provide insights about the behavior of lipid when using LINCS in version 4.0.7, and an inadequate place in the interface aqueous-membrane bilayer as rafts, mediated by oxidative stress, contributing to better implementation of the twin-range scheme in version 5.1.2. Virginia Sara Grancieri do Amaral 1 showed by quenching assays using acrylamide and 5 comprehension of its at molecular level. Caroline Mota Fernandes2, Mário R. Felício3, Aline and 16-doxyl-stearic acid probes. Furthermore, using This work was supported by CNPQ, CAPES and FAPERJ. Sol Valle1, Paula G. Quintana1, Caroline Correa mutants strains in the GlcCer synthesis pathway we This work was supported by São Paulo Research Foundation de Almeida1, Eliana Barreto-Bergter4, Sónia showed that the presence of C8-C9 double bonds and (FAPESP) with thematic grant number #2012/50680- C28 - COMPUTATIONAL SIMULATION OF Gonçalves3, Nuno C. Santos3, Eleonora Kurtenbach1 a methyl group at position C9 of the sphingoid base 5 and postdoctoral fellowships #2011/00963-8 and backbone in GlcCer were relevant for Psd2 Aspergillus POPG AGGREGATES IN THE PRESENCE OF 1Instituto de Biofísica Carlos Chagas Filho, #2016/23071-9. The authors also acknowledge Conselho ANTIMICROBIAL PEPTIDE LL-37 nidulans antifungal activity. In addition, the absence Nacional de Pesquisa (CNPq) for the financial support to Universidade Federal do Rio de Janeiro, RJ of GlcCer compromised the Psd2 ability to inhibit the 2 research and scholarship number #150561/2017-2. Yeny Y. P. Valencia1 ([email protected]), Department of Molecular growth of A. nidulans, Candida albicans and C. glabrata. 1 1 Genetics and Microbiology, Stony Brook University, These results are in agreement with the selectivity Gabriel C. A. da Hora and Thereza A. Soares 3 NY, Instituto de Medicina Molecular, Faculdade de of Psd2 against fungi and its lack of activity against C27 - Influence of the treatment of non-bonded 1 Department of Fundamental Chemistry, CCEN, Medicina, Universidade de Lisboa, Lisbon, 4Instituto interactions on MD simulations of pure liquids mammalian human erythrocytes. Psd2 represents a Federal University of Pernambuco, Recife-PE de Microbiologia Paulo de Goés, Universidade promising natural compound for the treatment of fungal and of a lipid bilayer performed with the 2016H66 ([email protected]) Federal do Rio de Janeiro, RJ force field diseases. Yan M. H. Gonçalves1 Antimicrobial peptides (AMPs) are biological compounds Psd2 is a pea defensin with 47 amino acid residues that This work was supported by Conselho Nacional de Caroline Senac2, Patrick F. J. Fuchs3,4, Philippe H. with broad-spectrum activity against bacteria, viruses inhibits the growth of fungal species by an uncharacterized Desenvolvimento Científico e Tecnológico (CNPq, Hünenberger5 and Bruno A. C. Horta1 and fungi. Once in contact with the target microbial mechanism. In this work, the Psd2 interaction with Brazil), Fundação de Amparo a Pesquisa do Rio membrane, the peptide can cause the cells to rupture model membranes that mimic the lipid composition of de Janeiro (FAPERJ, Brazil) and Comissão de 1 Instituto de Química, Universidade Federal do Rio through several hypothetical mechanisms (e.g. barrel different organisms was evaluated. Protein-lipid overlay Aperfeiçoamento de Pessoal do Nível Superior (CAPES, de Janeiro, Rio de Janeiro, Brazil (yanmarques@ model, aggregate, carpet and toroidal pore). The AMP assays indicated that Psd2 recognizes Fusarium solani Brazil) for funding, including fellowship to V.S.G.A. gmail.com). 2Sorbonne Universite, CNRS, INSERM, LL-37 is derived from human cathelicidin and displays an glucosylceramide (GlcCerF.solani) and ergosterol (Erg) Authors also acknowledge Fundação para a Ciência Laboratoire d’Imagerie Biomedicale (LIB), Paris, amphipathic alpha-helical structure at physiological pH in addition to phosphatidylcholine (POPC) and some e a Tecnologia – Ministério da Ciência, Tecnologia e 3 France, Sorbonne Universite, École normale and a charge of +6. It consists of a chain of 37 residues phosphatidylinositol species, such as PtdIns (3)P, (5) Ensino Superior (FCT-MCTES, Portugal) for funding, superieure, PSL University, CNRS, Laboratoire des (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) P and (3,5)P2, suggesting that these lipids may play including fellowship SFRH/BD/100517/2014 to 4 biomolecules (LBM), Paris, France, Universite and acts on the first line of defense against local an important role as Psd2 target. Surface plasmon M.R.F., Marie Sklodowska-Curie Research and 5 Paris Diderot, Paris, France, Laboratory of Physical infection and systemic invasion of pathogens at sites resonance analysis showed that Psd2 interacts with Innovation Staff Exchange (MSCA-RISE, European Chemistry, ETH Zürich, Zürich, Switzerland of inflammation. Small-angle X-ray scattering (SAXS) higher affinity with pure POPC and POPC-based vesicles Union) project INPACT (call H2020-MSCA-RISE-2014, and electronic microscopy measurements suggest containing GlcCer and Erg in a 70:30 proportion than grant agreement 644167), andFP7- PEAPLE IRSES The effect of different treatments of the non-bonded that LL-37 destabilizes POPG bilayers. LL-37 induces with those containing Chol. Partition experiments by (International Research Staff Exchange Scheme, interactions in simulations employing the recently the disappearance of multilamellar bilayers and the fluorescence spectroscopy showed a decrease of the European Union. 42 introduced GROMOS-compatible 2016H66 force field appearance of micelles in a concentration-dependent Trp quantum yield of Psd2 in the presence of GlcCerF. is evaluated based on simulations carried out with the manner. We have performed atomistic and coarse- GROMACS software. This is done considering four grained MD simulations to bring higher resolution thermodynamic and transport properties (pure liquid structural information into the experimental picture. We density, vaporization enthalpy, surface-tension coefficient have carried out simulations of pure POPG and pure and self-diffusion constant) of 57 organic liquids POPC bilayer at different concentrations of the peptide representative of the chemical groups alcohol, ether, to investigate the process of LL-37 inducing transition in aldehyde, ketone, carboxylic acid, ester, amine, amide, POPG but not POPC bilayers. Our simulations show that thiol, sulfide, disulfide and aromatic compounds, along LL-37 binds to POPG bilayer in a horizontal orientation. with water (SPC model). A dipalmitoylphosphatidylcholine At low peptide concentrations (lipid:peptide mass ratio bilayer system is considered as well. The simulated = 15:1), LL-37 remains at the lipid headgroups area properties are seen to be very sensitive to the treatment throughout 3µs. Increasing the peptide concentration of the long-range dispersion interactions, notably for the (9:1), we observe a slow migration of the peptides to least polar systems. In general, the treatment of the long- the hydrophobic region of the bilayer. Interestingly, the range electrostatic or Lennard-Jones interactions using process appears to take place faster at the atomistic homogeneous correction terms or lattice-sum approaches than at the coarse-grained level, but further sampling yield similar results, with punctual discrepancies. The is required to verify this claim. We argue that the combination of a lattice-sum approach for the electrostatic POPG bilayer is destabilized by LL-37 via a carpet-like interactions with a straight cutoff truncation of the mechanism consistent with experimental structural data. Lennard-Jones interactions at a distance of at least 1.2 nm is found to represent a good compromise setup within This work was supported by PAEC OAS-GCUB, CAPES GROMACS for achieving compatibility with the reference (Biocomp/BioMol), CNPq, FACEPE. results obtained using GROMOS, and a comparable level

84 85 Biomembranes hydrogels with different compositions and additives, fragments, we submitted SubI to thermal shift analysis. such as the antimigraine drug, sumatriptan succinate. The results revealed 17 fragments that significantly The program, in Python language, fits a Gaussian curve increased the thermal stability of the protein. The binding D01 - EXPERIMENTAL AND COMPUTATIONAL Organization (WHO), 8 million people are infected with under the first intensity peak being possible to compare of the fragments was avaluated by tryptophan intrinsic APPROACHES IN THE CHARACTERIZATION OF the parasite and 25 million live under the risk of infection. the intensity peak values and the distances among that fluroescence, NMR, and virtual docking. From the 17, PIPERLONGUMINE AND RAT ALBUMIN BINDING The drugs available for the treatment of the disease – identified. SAXS data obtained from PL407 18% or 20% 4 fragments were located inside the active site and 3 nifurtimox and benznidazole – are characterized by low with or without sumatriptan, at 25°C or 37°C, showed a others close to the ligand-binding site. The crystallization 1 Ana Paula R. Povinelli efficacy in the chronic phase of the disease and high cubic organization phase, with edges varying from 19 of SubI in the presence of the fragments is under way Gabriel Zazeri1, Marcelo F. Lima2 and Marinonio toxicity. Given this context, there is an urgent need for the nm to 23 nm. However, in the presence of PL-L81 at and the data obtained until the moment suggest that we Lopes Cornelio1 development of safe and effective drugs for the treatment 2.6 % or 3.2 %, the systems were organized in different have at least 4 lead molecules. In addition, the protein 1 of the disease. The enzyme cruzain is the major cysteine cubic structures (Pm3n, lm3m and Pn3m cubics), with imunogenicity and ability to induce imunogenic response Department of physics, Universidade Estadual protease of T. cruzi and is a validated target for Chagas sides ranging from 19 nm to 50 nm, approximately. For was tested suggesting the protein is capable to be tested Paulista (Unesp), São José do Rio Preto, S.P. disease drug discovery. Recent studies have shown all the cases, it was seen that mean scattering vector as a dignostic marker. 2 ([email protected]) and Department of -1 that the activity of cruzain inhibitors are pH-dependent. was qpeak=(0.32 ±0.05)nm . The next steps are (1) Chemistry, Universidade Estadual Paulista (Unesp), Herein, we report the influence of pH change in aqueous to make a second Gaussian fitting under the first peak Finantial support: FAPESP, CNPq São José do Rio Preto, S.P. environment (from 5.5 to 8.0) on the stability of the and then to determine if there is a second structure for cruzain-K1777 complex using molecular dynamics the system, and (2) to create an interactive and feasible D05 - LIPID PHASE INFLUENCE ON POLOXAMER- The search for metabolites with pharmacological potential simulations of 300 ns. This information was integrated interface to the user. ORGANOGELS AS SKIN DELIVERY SYSTEMS from plants and microorganisms has been growing to artificial neural networks, which were able to point out in the scientific community. Recent studies show that key interactions in the enzyme-inhibitor complex related This work was supported by FAPESP (2014/26200-9), Aryane A. Vigato1 piperlongumine, a molecule extracted from several pepper to the protonation state of four histidine residues. This CNPq (402838/2016-5, 309207/2016-9). Samir Q. Machado1, Naially C. de Faria1, Eneida de species, is an anti-inflammatory agent that acts similarly approach was useful to identify structural features that Paula2, Daniele R. Araujo1 to commercially available drugs such as sulfasalazine are important to guide future efforts in the design of D04 - SubI of Mycobacterium tuberculosis – new 1 and lornoxicam, inhibiting the interaction of NF- B protein novel cruzain inhibitors. Department of Natural and Human Sciences, κ target for drug development and diagnosis 2 with DNA and, consequently, blocking the transcription University of ABC, Santo André, SP. Institute of Biology, of cytokine genes and other pro-inflammatory proteins. Balan, A.1 State University of Campinas, Campinas, SP (aryane. D03 - Developing a software for analysis [email protected] / [email protected]) Considering that the first experiments in vivo are performed of supramolecular structures formed by Cerone, A. N.1,2; Nascimento, A.3; Moreno, C.R.4; in rat, this work, aimed at characterizing the rat serum THERMOSENSITIVE triblock copolymers Hyvonen, M.5; Rocco, S.A.6; Sforça, M. 6; albumin binding sites for piperlongumine, since this protein 1 Topical and transdermal routes are important strategies is able to bind and transport many exogenous compouds. Anderson F. Sepulveda1 Microbiology Departament, Biology Institute, for drug administration, since they are non-invasive, Universisty of São Paulo, São Paulo, Brazil; 2LNBio, For the characterization, it was performed the steady Margareth K. K. D. Franco2, Fabiano Yokaichiya3, avoid first-pass biotransformation and enable the use of CNPEM, Campinas, São Paulo, Brazil. state fluorescence spectroscopy technique to determine Daniele R. de Araujo4 self-administered pharmaceutical forms. However, the the mechanism of suppression of the interaction using clinical efficacy depends on the drug physico-chemical 1 the Stern-Volmer ratio, the number of sites in the protein Human and Natural Sciences Centre, Mycobacterium tuberculosis is the bacterium responsible and pharmacological properties, which is limited by the and the affinity constants through the double-logarithmic Federal University of ABC, Santo André, SP for human tuberculosis (TB) being responsable for 2 low stratum corneum permeability. Organogels are semi- relationship, besides the thermodynamic parameters ([email protected], daniele. million deaths per year and latent infection in one third solid systems, composed of an organic phase immobilized 2 through the Gibbs free energy equation. For a complete [email protected]) Helmholtz-Zentrum Berlin of the world population. Futhermore, there is a serious by a three-dimensional self- assembled network, as 3 description of the interactions, the experimental data für Materialien und Energie GmbH, Nuclear and concern about the multi drug- resistant (MDR) and aqueous phase. Lipid and poloxamer organogels (LPL- 4 was associated to theoretical data obtained by molecular Energy Research Institute and Human and Natural extensively drug-resistant (XDR)-TB cases among newly Org) form a two- phase system which can be effectively docking. Sciences Centre, Federal University of ABC, Santo diagnosed patients. The structural basis for inhibition of used as skin-delivery systems for drugs, favoring their André, SP this disease is poorly defined and the search for new permeation across the stratum corneum. This work D02 - STRUCTURAL STUDIES OF CRUZAIN-K1777 molecules that might specifically interact with unique aimed to study the influence of different lipid phases COMPLEX IN pH 5.5 AND 8.0: MOLECULAR Poloxamers (PL) are copolymers formed by poly(ethylene targets without cause damage to the host is a mandatory on development of new LPL-Org and to evaluate their oxide) -poly(propylene oxide) -poly(ethylene oxide) and goal. Bacterial ATP-Binding Cassette (ABC) transporters properties as skin-delivery systems for the drug model DYNAMICS COMBINED WITH ARTIFICIAL x y x INTELLIGENCE values of x and y confer different physical-chemical dedicated to ions transport play critical roles in nutrient lidocaine. LPL-Org were prepared associating poloxamer properties such as hydrophilic-lipophilic balances, acquisition and are potential antibacterial targets. In this 407 (PL407-20% w/v) aqueous phase with different lipid Anacleto Silva de Souza1 micellization and sol-gel transition temperatures. sense, we worked with SubI, a sulfate-binding protein phases: isopropyl miristate (IPM), isopropyl palmitate Leonardo L. G. Ferreira1 and Adriano D. Andricopulo1 When a micellar system reaches the gelification from the ABC transporter CysWTA1, which belongs to an (IPP), oleic acid (OAc) and lanolin (LAN). Lidocaine 1 temperature, micelles tend to self-assemble, forming important and essential pathway of sulfur assimilation. concentration was 2%. The incorporation of OAc-LAN Laboratório de Química Medicinal e supramolecular structures with different organization SubI was expressed in E. coli BL21 (DE3) strain and evoked shifts on PL407 rheological parameters, reducing Computacional, Centro de Pesquisa e Inovação em phase (cubic, hexagonal, lamellar etc). In order to purified by immobilized metal affinity chromatography G’/G” relationship (~20x, G’=8170Pa), possibly due to Biodiversidade e Fármacos, Instituto de Física de study those structures, Small-Angle X-ray Scattering followed by size exclusion chromatography. Circular the high lidocaine partition coefficient value (log P=1.2) São Carlos, Universidade de São Paulo, São Carlos, (SAXS) and Small-Angle Neutron Scattering (SANS) dichroism, Dynamic Light Scattering and analytical between OAc/water phase. Permeation assays, across Brazil ([email protected]) assays provide information shape, size and effects of Size Exclusion Chromatography were performed to STRAT-M® membranes, revealed similar flux values for drugs incorporation. However, a computer program is show that the protein is a soluble monomer in solution. both commercial cream 4% and 2% LDC (J=125µg. Chagas disease, or American trypanosomiasis, is a necessary to analyze the data given by those techniques. The three-dimensional structure of SubI was solved cm-2.h-1). However, IPP, IPM and OAc reduced the flux neglected tropical disease caused by the parasitic For this reason, a software was developed to study by crystallography at 2.2. Å resolution in the presence from 83 to 50 µg.cm-2.h-1. The association OAc-LAN protozoan Trypanosoma cruzi. According to World Health the supramolecular structure observed for PL-based of sulfate in the active-site. Using a library of 400 enhanced lidocaine permeation (J=117µg.cm-2.h-1),

86 87 showing similar profiles to commercial creams with D07 - DEVELOPMENT AND CHARACTERIZATION D08 - NANO-HYBRID PRE-ANESTHETICS D09 - Polymeric nanoparticles-loaded higher lidocaine concentration. Those results pointed OF NANOESTRUTURED FORMULATIONS DESIGNED FOR DENTISTRY APPLICATION thermosensitive hydrogels: an in vitro skin out the influence of esters, free fatty acids and wax on FORENCAPSULATION OF RED DYE FROM 1 permeation using synthetic membrane organogels rheology and their relationships with drug Lígia N. M. Ribeiro BIOTECHNOLOGICAL ORIGIN a permeation profiles. 2 1 Fabiana V. Dias 1 Michelle Franz-Montan , Simone R. Castro , Amantino C. F Gustavo H. Rodrigues da Silva1, Viviane A. Samyr M. Querobinoa, Carlos Alberto-Silvaa, This work was supported by FAPESP (2014/14457-5), Munhoz L. G1, Tedesco A.C3, Badino Jr A.C2, Guilherme1, and Eneida de Paula1 Leonardo F. Fracetob, Eneida de Paulac, Daniele R. CNPq (#402838/2016-5, 309207/2016-9) and CAPES. 1 1* de Araujoa, Renato Grillod* Baptista Neto A. , Primo F.L 1Department of Biochemistry and Tissue Biology, 1São Paulo State University (UNESP), School of University of Campinas, Campinas, SP (depaula@ a Human and Natural Sciences Center, Federal D06 - Preparation and characterization of cationic Pharmaceutical Sciences, Araraquara-SP (camila. unicamp.br) and 2 Department of Physiological University of ABC (UFABC), Santo André, SP, Brazil; cubosomes [email protected]; [email protected]*), Sciences, Piracicaba Dental School, University of bSão Paulo State University (UNESP), Environmental Raphael Dias de Castro1,2 2 Federal University of São Carlos, Department of Campinas – UNICAMP, Piracicaba, São Paulo, Brazil. Engineering, Sorocaba, SP, Brazil; cInstitute of Chemical Engineering, São Carlos - SP. 3Department Biology, UNICAMP, Campinas, Brazil dSão Paulo State Bárbara Malheiros1,2, Mayra Lotierzo1,2, Giovana of Chemistry, FFCLRP - University of São Paulo, University (UNESP), School of Engineering, Ilha Firpo Rodrigues2, Leandro Ramos Souza Barbosa2 Pre-anesthetics are widely used in Dentistry to minimize Ribeirão Preto – SP. the pain related to procedures such as local anesthetics Solteira, SP, Brazil. (E-mail: [email protected]) 1 - Department of Pharmaceutical and Biochemical injection, orthodontic bands insertion and primary Technology - Pharmaceutical Sciences Faculty – Nanostructured materials have different potential tooth exodontia1. However, there are not commercially Hybrid drug delivery systems, such as nanoparticles 2 - USP. Biosystems Laboratory – Biophysics Group compared to other materials in larger scale resulting in available formulations able to effectively eliminate loaded hydrogels, have received considerable – General Physics Department, Physics Institute different characteristics and properties. Nanocapsules these induced aches at the oral mucosa2. Therefore, attention for skin-drug delivery in the last years. USP. email: [email protected] (NC) can increase bioavailability, protection against in this work we developed nano-hybrid pharmaceutical Poloxamer (PL)-based hydrogels have been studied degradation, and controlled release of lipophilic forms processed as bioadhesives and hydrogels, as drug-delivery systems due to their ability to Cubosomes are nanostructures formed by the self- bioactivity compounds, increase “shelf life” protecting composed of nanostructured lipid carriers encapsulating modulate the release rate according to the PL assembly of a lipid in an aqueous solution, under them from volatilization and oxidation. The NC lidocaine-prilocaine (5%) and different biopolymeric concentration and temperature. In this context, we certain experimental circumstances. Nanoparticles were prepared containing a red dye (RD) derivative matrices (chitosan, pectin, alginate and xanthan). developed a hybrid system containing benzocaine- have been widely used as carriers of hydrophobic from anthraquinone, produced by the culture of the These nanodevices were characterized in terms of loaded polymeric nanoparticles incorporated into drugs, acting as a delivery method with the potential to fungus Talaromyces minioluteus on rotary incubator visual inspection, differential scanning calorimetry, PL407-based hydrogel, as well as studied the drug solve problems related to the drug itself, such as low table. The RD/NC showed satisfactory physical- electron microscopy and in vitro lidocaine-prilocaine permeation in a transdermal diffusion membrane solubility and instability. Theses benefits are specially chemical characteristics, with particle size 233.4 nm, release test. Viability tests in 3T3 and HaCat cells and (Strat-M®). Also, the nanoparticles permeation was related to the cubic crystalline structure of these polydispersity index 0.25 and Zeta potential of -20.6 analgesic efficacy trough the tail-flick model in mice studied by microscopy and analytical techniques. The particles, in particular their large usable hydrophobic mV. The analyses of accelerated stability showed a were performed for the most appropriate bioadhesive local anesthetic benzocaine (BZC) was used as drug volume. The production of cubosomes relies on the 5 month as shelf life. Absorption and fluorescence and hydrogel compositions. It was observed a synergic model and 16:0 Liss Rhod as a label for fluorescence self-assembly of lipids in excess of water, aided by emission analyzes were performed from UV-visible interaction between the lipid and biopolymeric excipients analysis in the permeation study. BZC-loaded poly- the presence of a polymer acting as an emulsifier. absorption spectroscopy which did not show changes which exhibited a sustained release profile for both e-caprolactone nanoparticles (PCL-NPs:BZC) were The physicochemical properties of such particles in the photophysical properties at the stationary state anesthetics during 24 h. The pectin-based bioadhesive prepared by coprecipitation method, their physico- can be characterized mainly by scattering techniques, of free RD (λ = 500 and 515 nm) compared to RD/ and xanthan-based hydrogels were the most compatible chemical properties and encapsulation efficiency namely small angle x-ray scattering, dynamic light NC (λ = 505 and 512 nm). Analyses of fluorescence forms; they were found no toxic to the cells and their measured. PCL-NPs:BZC were incorporated into the scattering, conventional and cryogenic transmission lifetime were carried out, and we have observed multi- anesthetic efficacy was optimized (7-8 h) in comparison thermosensitive hydrogel and viscosity measurements electron microscopy and ζ (zeta) potential. This work exponential fit for RD/DMSO_ACN (t1 = 3.14 ns/ to the commercially available form (2 h). In face of the were performed by rheological analysis. Results aimed the investigation of cubosomes production and 34.9%, RD/NC (t1 = 3.59 ns /58.6 %). In this way, exciting results, these nano-hybrid pharmaceutical forms showed high BZC encapsulation efficiency into PCL- characterization in the presence of two different cationic was possible to determine the main physicochemical are now prompt to be tested in clinical trials. NPs (93%) and adequate physico-chemical stability substances, in order to produce cationic nanoparticles characteristics of the nanomaterial, in addition to before and after hydrogel incorporation. Cumulative that might be suitable for gene therapy, as in the initiating the in vitro assays in monolayer cell culture References: 1. FRANZ-MONTAN, M.; RIBEIRO, L. N.M., BZC permeated concentrations, drug flux and case of DNA transfection. Cubosomes were prepared for determination of biological compatibility. VOLPATO, M. C.; CEREDA, C. M.; GROPPO, F. C.; TOFOLI, permeability coefficient values were lower for NPs- with phytantriol and stabilized with Puronic F127 in G. R.; DE ARAÚJO, D. R.; SANTI, P.; PADULA, C.; DE hydrogels (21.6 ± 1.8 μg.cm−2.h−1 and 4.32x10-3 -1 excess of water. Ionic liquid [C14mim][Cl] (1-methyl- PAULA, E. Recent advances and perspectives in topical cm.h ) than that observed for plain hydrogels (49.6 3-tetradecylimidazolium cloride) and surfactant TTAB oral anestesia. Expert Opinion on Drug Delivery. v.14, ± 2.1 μg.cm−2.h−1 and 9.92x10-3 cm.h-1). Also, (tetradecyltrimethylammonium bromide) were added p.673-684,2017. 2. RIBEIRO, L.N.M.; FRANZ-MONTAN, NPs were observed into the receptor diffusion cells to these formulations and it was verified that the final M.; BREITKREITZ, M.C.; ALCANTARA, A.C.S.; CASTRO, compartment after the in vitro permeation study. dispersions contained cationic cubosomes with size S. R.; GUILHERME, V.A.; BARBOSA, R.M.; de PAULA, E. These results contribute to a better understanding the range between 260 nm to 310 nm and Pn3m internal Nanostructured lipid carriers as robust systems for topical interaction between drug, controlled release hybrid structure, demonstraded by x-ray scattering and cryo- lidocaine-prilocaine release in dentistry. European Journal systems and synthetic membrane, as well as open EM, without any severe alteration of the physical of Pharmaceutical Science, v.93, p. 192-202, 2016. perspectives for the development of new drug delivery properties of the nanoparticles related to the presence systems for the skin. of the cationic substances up to 0.5% w/w. Acknowledgment: FAPESP (#14/14457-5; #14/25372-0). Acknowledgment: FAPESP (#2014/14457-5 and This work was supported by Conselho Nac. Des. Cient. #2015/26189-8), CAPES and CNPq. Tecnologico (CNPq) and by FAPESP.

88 89 D10 - STUDY OF THE ANTITUMOR POTENTIAL OF D11 - STUDY OF THE INTERACTION BETWEEN end, new copper complexes are synthesized and hydrophobic and amphiphilic molecules as well. A CORDIALINA A IN A NANOEMULSIFIED SYSTEM PIPERINE AND RAT SERUM ALBUMIN BY characterized. certain lipid, called Phytantriol, was used, as well as, FOR THE TREATMENT OF HUMAN CERVICAL FLUORESCENCE AND MOLECULAR MODELING water and a polymer that has the function of stabilizing the molecule. This study aims to do the physical- TUMOR CELL LINES. 1 Gabriel Zazeri chemical characterization of cubosomes encapsulating 1 Franciele Garcia Baveloni Ana Paula R. Povinelli1, Marcelo F. Lima2 and essential oils. The essential oils are vital aromatic Kayla Petronila Medina Alarcon1, Juhan Augusto Marinonio Lopes Cornelio1 substances that can be found in flowers, herbs, fruits and 1 1 spices and have many uses in the industry (cosmetics, Scardelato Pereira , André Gonzaga dos Santos , 1Department of physics, Universidade Estadual 1 1 beverages and food) and in the herbal medicine. Marlus Chorilli , Valéria Valente , Christiane Pienna Paulista (Unesp), São José do Rio Preto, S.P. 1 These oils are very non polar, so the cubosome is a Soares . ([email protected]) and 2Department of 1 good nanoparticle for the encapsulation because of Universidade Estadual Paulista "Júlio de Mesquita Chemistry, Universidade Estadual Paulista (Unesp), its hydrophobic volume. For the characterization of Filho" - UNESP. Faculdade de Ciências Farmacêuticas São José do Rio Preto, S.P. these cubosomes, biophysical techniques were used, - Araraquara / SP. E-mail: francielebaveloni@gmail. such as Dynamic Light Scattering (DLS), Small Angle com and [email protected]. Currently, the search for metabolites with pharmacological X-ray Scattering (SAXS) and Transmission Electronic potential originated from plants and microorganisms Microscopy (TEM). These techniques make it possible Cervical cancer is one of the most frequent tumors has been growing in the scientific community. Recent A key point in characterization of metal complexes is to know which is the internal structure of the cubosome, in women in Brazil, being developed, majority, by the studies show that piperine, which is extracted from natural to have information about their structure in aqueous in other words, allows to know how the water channels Human Papillomavirus (HPV). In face, it has sought products, has anti-inflammatory effects. These molecules solution, especially the coordination sphere, where inside the cubosome are arranged and whether or not alternative treatments from natural products, such as inhibit NF-κβ protein activation through several molecular they exhibit their activity. EPR has been widely used the cubosomes were formed within the oils. cordialina A, isolated from the plant Cordia verbenacea mechanisms during the pathway of activation, as a to achieve that goal. Classic “Peisach plots” (Peisach DC, which has strong antineoplastic activity, as consequence, cytokine genes and of pro- inflammatory & Blumberg, Arch Biochem Biophys, 165 (1974) 691) This work was supported by Conselho Nac. Des. Cient.

well as insolubility in aqueous medium. Thus, Lipid proteins are not transcribed. Considering that the first correlate A// and g// parameters with the donor atoms of Tecnológico (CNPq), FAPESP and Capes. Nanoemulsified Systems (LNS) have been explored as experiments in vivo are performed in rat, this work, aimed complexes with ligands only in the equatorial plane and th th tools to increase the solubility of bioactive, few polar in at characterizing the rat serum albumin binding sites for water in the 5 and 6 positions. In this report, we try to D14 - Interaction of polygodial with membrane aqueous medium. Objective: To evaluate and compare piperine, since this protein is able to bind and transport define zones in the plot for compounds with ligands in models of microorganisms at the air-water the cytotoxic effect of cordialina A with cordialina many exogenous compouds. For the characterizations, it the apical position, i.e. pentacoordinated copper centres, interface A + LNS in cervical cancer cells developed by HPV. was performed the steady-state fluorescence spectroscopy as presented in the Figure. Methodology: Sulforrodamine B (SRB) assay, in vitro, technique to characterize the mechanism of suppression of Gonçalves G. E. G1 used as cell lines for cervical cancer HPV-16 (SiHa), the interaction using the Stern- Volmer ratio, the number of This work was supported by CAPES, FAPESP and Barrosa K.H.¹; Morais T.R.1; Lago J.H.G.2; Caseli L.1 HPV negative (C33A) and normal lines of human sites of the ligands in the protein and the affinity constants PEDECIBA. 1 keratinocytes (HaCaT). Results: In treatment with applying the double-logarithmic relationship, besides the Department of Chemistry, Federal University of São cordialina A the inhibitory concentration 50% (IC ) thermodynamic parameters through the Gibbs free energy Paulo, Diadema, SP ([email protected]) and 50 D13 - ENCAPSULATION OF ESSENTIAL OILS IN ²Center for Natural and Human Sciences, Federal was 106.58 μM, 83.55 μM and 55.9 μM, whereas for equation. For a completed description of the interactions, CUBOSOMES: STRUCTURAL CHARACTERIZATION the cordialina A + LNS the IC was 0.89 μM, 0.186 the experimental data was associated to theoretical University of ABC, Santo André, SP 50 1 μM and 0.35 μM in the SiHa, C-33A and HaCaT lines, modeling obtained by molecular docking. Giovana Firpo respectively. The selectivity index (SI) for treatment Bárbara Malheiros1,2, Raphael Dias de Castro1,2, Polygodial and epi-polygodial, isomers with with cordialina A was 1.9 and 1.49 and the cordialina A D12 - DEVELOPMENT OF “PEISACH PLOTS” FOR José Henrique Leal Cardoso3 and Leandro Ramos antiparasitic activity, extracted and isolated from leaves + LNS the SI was 2.54 and 0.53 for SiHa and C-33A, PENTACOORDINATED COPPER COMPLEXES Souza Barbosa1,2, of D. brasiliensis, were studied in Langmuir monolayers respectively. Conclusion: The cordialina A showed of different lipids. Tensiometric techniques, vibrational 1 high cytotoxicity in tumor cells, when incorporated into Gianella Facchin1 Department of Physics, University of São Paulo, spectroscopy and Brewster angle microscopy (BAM) São Paulo, SP ([email protected]);2Biochemical the LNS, representing a promising therapy in the fight Natalia Alvarez1, Luis F. S. Mendes2, María H. Torre1, were employed to characterize these monolayers. and Pharmaceutical Technologies Department - against cervical cancer. Key words: Cervical cancer, Javier Ellena3, Antonio J. Costa-Filho2. Comparisons using DPPC (dipalmitoylphosphatidylcho- Cordia verbenacea DC, nanoemulsion. Pharmaceutical Sciences Faculty - University of line); DMPC (dimyristoylphosphocholine); DOPC 1 3 Facultad de Química, General Flores 2124, São Paulo, SP; Universidade Estadual do Ceará, (dioleoylphosphocholine); DPPE (dipalmitoilfos- Financial support: Coordination of Improvement of Universidad de la República, Montevideo, Uruguay Fortaleza, CE. fatidiletalonoamina); DPPS (dipalmitoylphosphati- 2 Higher Level Personnel - CAPES. ([email protected]), Faculdade de Filosofia, dylserine); DPPG (dipalmitoylphosphatidylglycerol); Ciências e Letras de Ribeirão Preto, Universidade de The nanotechnology has been widely used because DODAB (dioctadecyldimethylammonium bromide) and São Paulo, Av. Bandeirantes, 14040-901, Ribeirão of its potential and the diversity of applications, as cholesterol were performed for polygodial and showed 3 Preto (SP), Brazil and Instituto de Física de São in the nano medicine. A nanoparticle that has been that the monolayer of DPPG is the only to undergo a Carlos, Universidade de São Paulo, C.P. 369, 13560- studied and has important qualities is the cubosome, condensation in the presence of the compound. On the 970, São Carlos (SP), Brazil a particle formed by the addition of a specific lipid in other hand, for the study of epi- polygodial, selected an aqueous solution. It is believed that it has the ability lipids commonly found in protozoan membranes Our research is focused on the development of inorganic to be a drug carrier, because of its internal structure, (DPPG, DPPS and DPPE) were used, whose results coordination complexes with antitumor activity. To that a cubic crystalline structure that is 50% (fifty percent) were compared to those presented by polygodial. hydrophobic and 50% (fifty percent) hydrophilic, what Alteration have been observed mainly for DPPS, where, can be of great use because allows the cubosome through vibrational spectroscopy, it was possible to incorporate not only hydrophilic molecules, but to detect a state of disorder of the monolayer in the

90 91 presence of epi-polygodial, relatively to the changes increased π–π intermolecular interactions that lead to ID: 5A2J) and a different antibody fragment bound to a of drug into their inner aqueous compartments[2]. in the relative intensity values of the methylene stretch the generation of a pPDIp•- moiety in DMSO, pPDIp•- human P-cadherin (PDB ID: 5JYL). Then we compared the We have designed an IGL system to prolong the bands. BAM showed changes in the morphology of the (518 nm) and pPDIp2- (420 nm) in the cell culture results with those estimated by PDBePISA (for 5A2J) and delivery of the local anesthetic etidocaine (EDC). DPPS monolayer in the presence of both: polygodial medium RPMI, and 3*pPDIp in buffer (pH 7.4) and starch with those obtained through Surface Plasmon Resonance Large unilamellar vesicles (LUV) composed of and its isomer. BAM also showed an increase in the films. The complex activates singlet oxygen in buffer (for 5JYL). We used the distance between the two centers hydrogenated soy phosphatidylcholine:cholesterol number and domains for the DODAB monolayer in the solution under green light irradiation (dose=0.41J/cm2; of mass in each complex as the reaction coordinate (6:4 mol%) plus 250 mM ammonium sulfate gradient, 1 -1 2+ presence of the polygodial and different behaviors of the [ O2]=0.03–0.47 mmolL for [Ru(phen)2(pPDIp)] and divided the dissociation trajectory in a series of 2 were prepared for the upload of 0.5% EDC. Different DPPE monolayer in the presence of each compound. =10– 150 mmolL-1). No cytotoxic effects on B16F10 wide windows to improve convergence. Additionally, methodological approaches (Dynamic light scattering Therefore, it can be concluded that the interactions murine melanoma cell viability were observed up to a we searched for hydrogen bonds and salt bridges that - DLS, nanotracking analysis, transmission electron of the polygodial with the different monolayers are concentration of 10 mmolL-1 of complex. However, under areÅ important for the binding of the two proteins in 5JYL microscopy-NTA, differential scanning calorimetry - modulated according to the lipid composition, which green LED illumination (dose=0.41 J/cm2) the complex complex. Hence, as the dissociation was carried out, DSC, and nuclear magnetic resonance- RMN) helped also regulated the fluidity of the monolayer. The change exhibits a strong photocytotoxic effect, displaying we found four pairs of residues that form non-bonded to characterize the drug delivery system. The in -1 o in the configuration of the stereogenic center of the IC50=1.2 mmolL . In order to facilitate the application interactions that come at play only when the antibody vitro release of EDC, at 37 C, was measured using compound also seems to contribute to changes in the and control as an antitumor agent, the complex was recognizes the antigen. These residues are potential hot Franz diffusion cells. The prepared IGL were able to interactions lipid-compound. immobilized on a cassava starch- based polymer film. spots that could be mutated in order to create stronger increase EDC upload (41% encapsulation efficiency); Spectroscopic assays demonstrated that the complex intermolecular interactions and thus increase antibody the vesicles were stable over a period of 6 months at This work was supported by Coordenação de adhered to the p→π* aggregate form when immobilized affinity. Finally, we obtained the following values for 40ºC ± 3, with average diameter (470.1 nm ± 3.2), Aperfeiçoamento de Pessoal de Nível Superior (CAPES) on the film, maintaining its luminescent properties and the binding free energies of the complexes: -0.2 Kcal/ polidispersity (<0.2), mean particle concentration 1 13 and by the Fundação de Amparo à Pesquisa do Estado activation of O2 with exposure to light. These results mol and -8.5 Kcal/mol for 5A2J and 5JYL, respectively, (4.3 ± 0.9.10 /mL) and zeta values (-25 mV). The de São Paulo (2018/23446-0) demonstrate that complex is a promising candidate for which are in good agreement with the reference values of size and spherical morphology of the vesicles were the application of photosensitization in PDT. -0.9 Kcal/mol and -10 Kcal/mol, respectively. Thus, we confirmed by transmission electron microscopy. D15 - PERSPECTIVES ON THE APPLICATION OF conclude that ABF is an accurate method for measuring The use of NMR spectra were important to find the RUTHENIUM COMPLEXES IN PHOTODYNAMIC This work was supported by the Fundação de Amparo binding affinities in silico, even in complex antibody- association constants (DOSY-NMR) and the saturation THERAPY FOR MELANOMA TREATMENT à Pesquisa do Estado de São Paulo (FAPESP, Proc. no. antigen systems. In addition, we aim to study the binding transfer difference (STD) NMR was used to study for 2017/00839-1, 2018/03424-0) and by the Coordenação free energy of these antibody-antigen complexes through IGL- ligand interactions. The DSC results showed that Isabele Ap. S. de Campos1 de Aperfeiçoamento de Pessoal de Nível Superior other computational tools, like MM-PBSA, to further the EDC change the main phase transition temperature Elaine G. Rodrigues2, Wallance M. Pazin3, Amando (CAPES). explore the protein-protein association process. of lipossome. EDC are able to enter de membrane. At o S. Ito4, Vinicius T. Santana5, Otaciro R. Nascimento5 37 C the time for total release of the anesthetic (free and Rose Maria Carlos1 D16 - THE ADAPTIVE BIASING FORCE METHOD Palvravras-chave: Energia Livre; Adaptive Biasing EDC = 3 h) was extended to ca. 24h in the IGLEDC Force; Anticorpos. Agradecimentos: Coordenação de formulation. The alleged toxicity of EDC led the FDA to 1Department of Chemistry, Federal University of São APPLIED TO STUDY THE ANTIBODY-ANTIGEN MOLECULAR INTERACTIONS Aperfeiçoamento de Pessoal de Nível Superior (CAPES), ban its clinical use, in 2008. But here we have shown Carlos, São Carlos, SP (isaabele- soares@hotmail. Fundação de Amparo à Pesquisa do Estado do Rio de that IGL can successfully promote the sustained 2 com), Department of Microbiology, Immunology João Hermínio Martins da Silva3 Janeiro (FAPERJ) e Fundação Oswaldo Cruz (FIOCRUZ). release of etidocaine. In that way IGL may serve to and Parasitology, Federal University of São Paulo, EDC Artur Hermano Sampaio Dias1, Alexandre Suman de reintroduce EDC in the clinics, for surgical procedures, São Paulo, SP. 3Department of Physics, Paulista Araújo2, Ernesto Raúl Caffarena1 post-operative and chronic pain treatment. State University Júlio de Mesquita, Presidente D17 - STABILITY OF IONIC-GRADIENT 4 1 LIPOSOMES, DESIGNED AS DRUG-DELIVERY Prudente, SP, Department of Physics, University of Programa de Computação Científica, Instituto Acknowledgement: FAPESP (# 14/14457-5) and CNPq 5 2 SYSTEM FOR ETIDOCAINE São Paulo (FFLRP/USP), Ribeirão Preto, SP, São Oswaldo Cruz, Rio de Janeiro-RJ. IBILCE-UNESP, (J.D.O fellowship). [1]de Paula E, Cereda CMS, Fraceto 3 Carlos Institute of Physics, University of São Paulo, São José do Rio Preto-SP. Fundação Oswaldo Cruz, Juliana Damasceno Oliveira1 LF, de Araújo DR, Franz-Montan M, Tofoli GR, Ranali São Carlos, SP. Eusébio-CE. ([email protected]) Bruna Renata Casadei2, Lígia Nunes Morais J, Volpato MC, Groppo, FC.Micro and nanosystems Ribeiro1,Karin A. Riske2, Luis Fernando Cabeça3, for delivering local anesthetics. Expert Opin Drug [2] Cutaneous melanoma is one of the most feared cancers Protein-protein interactions play a significant role when Eneida de Paula1. Deliv. 2012;9(12):1505–1524. Grant GJ, Barenholz worldwide due to resistance to treatments and the high it comes to designing new pharmaceuticals, since 1 Y, Bolotin EM, Bansinath M, Turndorf H, Piskoun B, capacity to develop metastases. Photodynamic therapy biomolecules like antibodies can bind to their targets Department of Biochemistry and Tissue Biology, Davidson EM. A novel liposomal bupivacaine formulation (PDT) is a promising treatment strategy for malignant with considerable affinity if properly designed. The Institute of Biology / State University of Campinas to produce ultralong-acting analgesia. Anesthesiology. (Unicamp), Campinas, SP, Brazil. (damasceno. and non-malignant lesions. Ru(II) polypyridine association of biomolecules can be studied through 2 2004;101(1):133–137. complexes are interesting photosensitizers for PDT free energy calculations. However, such computational [email protected]). Department of Biophysics, Federal University of São Paulo – UNIFESP, São due to their unique spectroscopic and anticancer simulations are a hard endeavor, mainly because of the 3 properties. This study reports the influence of the soaring number of possible side-chain interactions that Paulo,São SP, Brazil. Department of Chemistry, Federal medium on the aggregation processes of the complex account for many degrees of freedom, which makes it Technological University of Parana, Londrina, PR, Brazil. 2+ [Ru(phen)2(pPDIp)] (phen = 1,10-phenanthroline; even harder to assess the thermodynamics of binding pPDIp= bis-phenanthroline-substituted perylene affinities. The Adaptive Biasing Force (ABF) method is Liposomes are versatile drug delivery systems. Local diimide) and its consequences for the photochemical a tool for calculating free energy in molecular dynamics anesthetics interact with liposomes, partitioning in- and photophysical properties of the system. Photolysis simulations which has been reported to yield accurate between the lipids and also occupying the vesicles leads, initially, to emission of both chromophores: results, compared to experimental data. In this work, aqueous core. Encapsulation into liposomes prolong Triplet metal-to-ligand charge transfer (3*MLCT; Ru, we applied the ABF method to the binding free energy nerve blockade and reduces the toxicity of local d→phen, π*) and singlet intraligand charge transfer calculation of two antibody-antigen complexes: an anesthetics[1]. Ionic gradient liposomes (IGL) are (1*ILCT; pPDIp, p→π*). However, the medium induces APDTRP peptide bound to an antibody fragment (PDB modified vesicles, proposed to upload higher amounts

92 93 D18 - THERMOSENSITIVE BINARY POLOXAMER D19 - Design of new antagonists for the enzyme D20 - Development of cationic nanostructures D21 – DESIGN OF NEW GLUTAMINASE 407/338 HYDROGEL AS DRUG DELIVERY FOR 3-hydroxykynurenine transaminase from Aedes for controlled released of chlorin-e6 and derivate INHIBITORS WITH POTENTIAL ANTITUMOR LOCAL ANESTHESIA aegypti: a new larvicide prototype photosensitizers from biotechnological origin to ACTIVITY APDT application Kelli Cristina Freitas Mariano1 Larissa D. da Silva1 Matheus P. Pinheiro1 Luciana Guimarães Munhoz1 Eneida de Paula2, Giovana R. Tófoli2, Daniele Denys E. S. Santos1, Larissa G. Maciel1, Janaína V. Jean C. H. Campos1 and Sandra M. G. Dias1 1* 1 1 Camila Fernanda Amantino¹ Fernando Lucas Ribeiro de Araujo dos Anjos , Thereza A. Soares . 1Brazilian Biosciences National Laboratory (LNBio), Primo¹* 1 Human and Natural Sciences Center, ABC Federal 1Departament of Fundamental Chemistry, Brazilian Center for Research in Energy and University, Santo André, SP, Brazil; 2 Faculty of Federal University of Pernambuco, Recife, PE. 1Department of Bioprocess and Biotechnology, Materials (CNPEM), Campinas, SP, Brazil (matheus. Dentistry São Leopoldo Mandic, Campinas, São ([email protected]) State University “Julio de Mesquita Filho”, [email protected]) Paulo, Brazil. *Corresponding author: daniele. Araraquara, SP ([email protected]; [email protected] The mosquito Aedes aegypti is the vector of the [email protected]*) Glutamine is an abundant nutrient used to support arbovirus causing the chikungunya, zika and dengue the elevated bioenergetic and biosynthetic demands The development of new formulations for controlling fevers. An efficient and economically-viable alternative At the present time the abusive use of antibiotics required by many tumor cells. Glutaminase, a key drug release after regional blockades offers a to curb these diseases is vector control. We have has resulted in increasingly resistant infections enzyme in the conversion of glutamine to glutamate, technological strategy for prolonging the therapeutic previously shown that 1,2,4-oxadiazole derivatives generating enormous difficulty for the eradication plays a vital role in up-regulating cell metabolism for effects of local anesthetics, such as ropivacaine exhibit larvicidal activity against Ae. aegypti where of microorganisms. Thus, according to the current tumor cell growth and is considered to be an attractive (RVC). Poloxamers (PL) are copolymers composed of the enzyme 3-hydroxykynurenine transaminase situation has been necessary the development of therapeutic target for cancer treatment. Given this ethylene oxide and propylene oxide blocks, able to form (HKT) would be a potential molecular target for new techniques that result in a better bactericidal important function of glutaminase in cancer, there micellar thermosensitive hydrogels being investigated inhibition. The inhibition by competitive antagonists or fungicidal activity. The research aims to develop has been interest in developing inhibitors targeting as delivery systems for different molecules. In this of HKT results in the accumulation of the substrate new photoactive nanomaterials for the use of glutaminase. Although the compound CB-839, a work, we present the design, preparation and physico- 3-hydroxykynurenine, a potential neurotoxic agent antimicrobial photodynamic therapy (APDT). glutaminase inhibitor, is currently being evaluated chemical characterization of PL-hydrogels for delivering for the Ae. aegypti. In this work, molecular dynamics This inactivation has a mechanism that is based in phase 1 and 2 clinical trials, many efforts to RVC. Hydrogels were prepared by dispersing PL407 (MD) simulations were performed for the holoenzyme on photochemical and photophysical controlled develop a new class of glutaminase inhibitors that (20, 25, 30 % m/v) in water, alone or associated to HKT free in solution and bound to different inhibitors. processes. Photodynamic mechanism induces target glutamine metabolism have been done in both 5 or 10 % PL338. Polymers were kept at 4 °C under Atomic parameters and novel topologies have been the production of reactive oxygen species in a academia and industry. After a High Throughput stirring (100 rpm), until the complete dissolution. RVC assigned to the cofactor pyridoxal phosphate (PLP) biological environment, leading to death of the Screening (HTS) campaign performed in our group hydrochloride (0.5 % m/v) was incorporated into the and inhibitors 4-(2-aminophenyl)-4-oxobutanoic (4OB) microorganisms. The photosensitizing agent with a Screening Library of 30,000 compounds, from hydrogels. Rheological analysis was performed for and 1,2,4-oxadiazole derivatives (compounds 1a, 3a, used in the development of this research was ChemBridge, several hits were identified using the determining the elastic (G′) and viscous (G″) module, 3b). Since 4OB has been co-crystalized with HKT, it chlorin-e6 (Ce-6) from biotechnological origin. isoform glutaminase C (GAC) as target. The most viscosity (η) and sol-gel temperature (Tsolgel). G′ has been used as a compound reference to test the The administration of the Ce-6 unload shows to be promising compounds and analogues were acquired values were higher than G″ (~40x), revealing a protocols applied to our target inhibitors. Analysis ineffective in relation to its functionality, therefore and evaluated by combining thermal shift assay, viscoelastic behavior, even after RVC incorporation, of the simulated systems show that the new atomic it is proposed a delivery mechanism which is dynamic light scattering and in vitro inhibition assay.

with differences of Tsol-gel observed for both PL systems parameters and topologies reproduce the structural based on a Ce-6 cationic encapsulation strategy. Here, we set out to identify a novel class of inhibitors (PL407 = ~28-30ºC and PL338= ~32-34ºC). DLS dynamics and the crystallographic data adequately. This encapsulation occurs in a nanostructured able to inhibit the enzymatic activity of GAC with low analysis at 25ºC for both systems presented bimodal The simulations of HKT and HKT-inhibitors converge to cationic nanoemulsion (NEC). The physical- micromolar potency. In addition, these results allowed distributions. PL 407 presented micellar diameters of an equilibrium conformation after 30 ns probed through chemical analysis shows that the Ce-6/NEC us to implement strategies of ligand-based design to 37.4 ± 0.3 nm and a minnor size population of ~ 5 nm. a total of of 100 ns. The holoenzyme is fairly rigid, both have particles size of 176 nm, the Zeta potential propose analogues of these promising GAC inhibitors At 37 °C, it was observed a unimodal distribution with in the absence and presence of the inhibitors (RMSD is equal to +65,3 mV and polydispersity index that are being synthesized for evaluation aiming at lower hydrodynamic diameters values (21.71± 0.74 below 0.25 nm). Regions of greater atomic fluctuations (PdI) of 0,29. Physical chemistry stability shows increasing the potency of these inhibitors. and 30.4 ± 3.5 nm). SEM analysis showed RVC with (RMSF ranging from 0.54 to 0.67 nm) vary between the a colloidal stability of 61 days, under 7 oC and 25 morphology elongated, sharp and rectangular. PL 407 simulated systems, suggestive of different substrate oC of storage temperature. Spectroscopic studies This work was supported by FAPESP. had a layered morphology while and PL338 presented a inducing different HKT conformational dynamics. Free were carried out to Ce-6/EtOH and Ce-6/NEC, porous structure, with homogeneous features after RVC energy calculations are being performed to estimate the shows maximum absorption band at 670 nm; 666 incorporation. PL407/338 prolonged RVC release when inhibition constants of derivatives of 1,2,4-oxadiazoles nm and fluorescence emission at 678 nm; 670 compared to RVC free at 24h, following the Higuchi with two goals: to establish HKT as the molecular nm (lexc. = 640 nm) respectively. In this way model (0.92>R2>0.98).Those results pointed to the target for these larvicides, and to propose chemical it was possible to carry out the initial stages of application of thermoresensitive hydrogels for RVC modifications to improve the specificity and larvicide development of a nanostructured formulation with release, as possible innovative drug release systems. activity against HKT. specific characteristics for use in APDT.

This work was supported by FAPESP (2014/14457-5), This work was supported by CNPq, FACEPE, Stint- This work was supported by Conselho Nac. Des. Cient. CNPq (#402838/2016-5, 309207/2016-9) and CAPES Sweden and CAPES(BioComp/BioMol) Tecnologico (CNPq PIBIC 2017/2018) and Universal project (CNPq 403025/2016-8).

94 95 D22 - Molecular Dynamics simulations of intervention that has multiple adverse effects and 1%) showed the highest SFN incorporation (88%) drug CMT ≈ 23 °C and 29 °C in saline and saline/glycoside parasitic resistance. The search for new applications loading (0.43%) percentages and release constant (K mixture. The enthalpy ( H ) of the mixed vesicles and PAMAM and PPI dendrimers with the GROMOS- kp Δ m compatible 2016H66 force field for drugs already approved by regulatory agencies (also 31.6) Tsol-gel at ~32 °C and G’>G” (8 times). Cell micelles increases by approximately 20 kJ/mol in saline called drug repositioning) is important for the treatment viability assays showed that SFN (2.5 μM to 20 μM), or saline/glycoside mixture. While it remains roughly the Mayk C. Ramos1 of neglected parasitic diseases, as it reduces costs copolymers PL 407/108 (0.02 and 0.1 m/v%) and HA same in glycoside. In conclusion, the thermal behavior Vitor A. C. Horta2 and Bruno A. C. Horta1 and time for pharmaceutical development, since these (0.001, 0.5 and 1%) did not evoked cytotoxic effect to of the DODAB/F127 mixed system is little affected by drugs are known and studied. In addition, another area SW1353 cell line, being possible to validate the in vitro DOX in saline and/or in glycoside, indicating that it can 1 Institute of Chemistry, UFRJ, Rio de Janeiro-RJ, and research that has been gaining a lot of attention in OA model detecting COX-2 after IL-1β stimulus. These be solubilized in the micelle or vesicle structures in 2 Brazil ([email protected]). Department of recent years is nanomedicine, with particular attention results pointed the tendency to self-assembling of PL/HA saline and/or glucose, having potential application as Computational Science, UFJF, Juiz de Fora-MG, Brazil. to systems with controlled drug delivery. Among the systems and its possible application as intra-articular pharmaceutical formulations. various particles used for this purpose are the systems delivery systems for SFN. Dendrimers are highly branched synthetic macromolecules formed by lipids and polymers, such as liposomes This work was supported by CAPES (ML) and CNPq (EF). with various technological applications. Among these, and cubosomes. In this work we are investigating the This work was supported by FAPESP (2015-14763- a notorious application is drug delivery. Molecular structural influence of Cinarizine (CNZ) on monosteen 1, 2014/ 14457-5), CAPES, CNPq (309207/2016-9, D26 - PREPARATION AND CHARACTERIZATION dynamics simulations have contributed significantly and phytantriol cubes, together with nonionic polymers, 402838/2016-5) and Multiuser Central Facilities UFABC. OF NANOESTRUTURED LIPID-ORGANOGELS AS to provide structural and dynamical information of as well as its parasitoxic effect on Leishmania cells. To SKIN-DELIVERY SYSTEMS FOR SULFORAPHANE dendrimers. This work aims at validating the 2016H66 do so, we constructed a system based on cubosomes D25 - DODAB/PLURONIC F127/DOXORUBICIN Naially C. de Faria1 force field [1], which was calibrated on the basis of for the encapsulation of the drug, which will be analyzed ASSEMBLY STRUCTURES IN SALINE AND/ thermodynamic properties of organic liquids, to be used through biophysical techniques such as low-angle X-ray OR GLYCOSIDE STUDIED BY DIFFERENTIAL Mônica H. M. Nascimento1, Samyr M. Querobino1, in the context of dendrimer simulations. The advantage (SAXS) and dynamic light scattering (DLS). SCANNING CALORIMETRY Eneida de Paula2, Daniele R. de Araújo1 of using such a force field relies on its thermodynamics 1 consistency. For instance, it accurately reproduces the This work was supported by Conselho Nac. Des. Cient. Monique Lemos1 CCNH, ABC Federal University, São Paulo, Brazil and 2Institute of Biology. (naiallycardoso@gmail. partition coefficient between polar and non-polar solvents Tecnologico (CNPq) and by the Foundation for Research Eloi Feitosa1 com, [email protected]) for a variety of small molecules, a property that is crucial Support of the State of São Paulo (Fapesp). 1 for correctly modeling biomolecular processes. Among São Paulo State University, Department of the existing dendrimers, we have chosen the poly(amido Physics, Sao Jose do Rio Preto, SP, Brazil (moniq. Inflammation is a physiological phenomenon in D24 - DEVELOPMENT OF POLOXAMER- [email protected]) amine) (PAMAM) and the poly(propylene imine) (PPI) HYALURONIC ACID HYDROGELS AS response to the occurrence of a tissue or cell injury, that since they are commercially available and well studied. SULFORAPHANE DELIVERY SYSTEMS FOR may be associated with the development of pathologies Our results indicate that the force field leads to good OSTEOARTHRITIS TREATMENT Aqueous systems comprising of cationic lipids, such as arthritis, degenerative diseases, dermatitis and results, in agreement with the available experimental and as dioctadecyldimethylammonium bromide (DODAB), others. The development of new nanostructured simulation data. As a continuation of this project, we will Nascimento M. H. M1 and triblock copolymers, such as Pluronic F127 (F127), carrier systems are strategies for delivering new investigate the formation of drug/dendrimer complexes. Querobino S. M1.; Alberto-Silva C1.; de Paula E.2; are interesting because they can assemble as stabilized molecules, such as sulforaphane (SFN), an anti- A variety of different organic molecules representative Lombello C.B.3; de Araujo D.R.1 vesicle or micelle structures, depending on composition, inflammatory drug. This study aimed to prepare, to of several chemical functions will be considered and in order to host small molecules of pharmaceutical perform the physico-chemical characterization and to 1 their free energy of complexation will be estimated. The CCNH, Universidade Federal do ABC, São Paulo, interest, for example, doxorubicin (DOX), known in evaluate the in vitro permeation, of SFN from different objective is to better understand molecular properties Brasil ([email protected], daniele.araujo@ chemotherapy medication. The thermal behavior of organogels formulations. Organogels were prepared 2 that contribute to improve dendrimer-drug interaction. ufabc.edu.br); Departamento de Bioquímica, DODAB/F127/DOX mixtures was investigated under using an oil:aqueous phases proportion of 1:4 (v/v), Instituto de Biologia, Universidade Estadual de different solvent conditions, such as saline (0.9 wt% by a mixture of poloxamer 407 (PL407, 20% m/v) with 3 [1]J. Chem. Theory Comput. 12, 3825-3850 (2016). This Campinas, São Paulo, Brasil; CECS, Universidade NaCl) and/or glycoside (5 wt% glucose). Here we have oleic acid-isopropyl myristate (OA-IPM) or oleic acid- work was supported by CAPES (master fellowship) and Federal do ABC, São Paulo, Brasil investigated by differential scanning calorimetry (DSC) isopropyl palmitate (OA-IPP) at 50:50 (v/v %), oleic FAPERJ. the thermal behavior of the assembled structures formed acid-lanolin (OA- LAN 1%) or bee wax (OA-BW 1%) Osteoarthritis (OA) is a chronic and degenerative by mixing DODAB 1 mM with F127 0.2 mM and DOX (m/v), copaiba oil (COP) and grape seed oil (GSO). D23 - STRUCTURAL STUDY OF NON-IONIC disease, that affects the joints, where the treatment 0.5 mM in saline and/or glycoside and, for comparison, SFN concentration was 0.1 % (m/v). Formulations were CUBOSOMES ENCAPSULATED WITH CINARIZINE aims the relief of pain and inflammation. Thus, the in water, in which DODAB displays mainly three well- evaluated by partition coefficient (P) determination, association of thermoreversible poloxamers (PL) and known transitions at Ts, Tm and Tp ≈ 36, 45 and 53 °C, rheology and in vitro permeation assays, across Mayra C. G. Lotierzo1 the biocompatibility of hyaluronic acid (HA), support respectively. In these solvents or water, F127 inhibits STRAT-M® membranes. The P values were from 1.34 Leandro R. S. Barbosa2 the development of ostheotropic pharmaceutical the pre- and post-transitions, and the thermogram to 1.85, with the highest P was observed for OA- formulations for new drugs, such as sulforaphane displays two peaks characteristic of F127/DODAB mixed LAN. All formulations showed viscoelastic behavior 1 Department of Pharmacy, University of São (SFN). Therefore, this study aimed to prepare, to micelles and vesicles, respectively around CMT ≈ 25 with G’>G” (~ 10 x). SNF incorporation provided an Paulo, São Paulo, SP ([email protected]) and characterize and to evaluate the biocompatibility and the °C and T ≈ 45 °C. The addition of DOX to this sample increase on viscosity (η, at 32.5°C), from 604 to 730 2 m Department of General Physics , University of São in vitro efficacy of PL-HA hybrid hydrogels as delivery preserves the mixed micelle and vesicles structures, as mPa.s, especially for COP. The flux permeation values Paulo, São Paulo systems for SFN intra-articular release for OA treatment. the thermograms retain the two transition peaks, shifted presented a decreasing profile according to the oil PL-HA polymeric hydrogels were prepared by dispersing a little bit to larger (in saline or saline/glycoside) or lower phase components IPP>IPM>LAN>GSO>BW=COP Leishmaniasis is a parasitic disease that presents different HA (0.05 to 1 m/v %) in isolated PL 407 or PL 108 at (in glycoside) temperatures, therefore indicating that (from 24.8 to 0.1 µg.cm-2.h-1) with SFN concentrations forms of infection in humans. Due to its high severity 20% and their association PL407-108 (18-2 and 10-10 the DOX molecules are solubilized either in the mixed permeated ranged from 492.6 to 570.6 µg.h-1, at and prevalence, this disease is considered a priority by m/v%). SFN concentration was 0.1 m/v %. Hydrogels micelles or in the mixed vesicles in spite of NaCl and/ 24 h. Those results showed the potential use of the World Health Organization (WHO), however, infection were evaluated by DSC, MEV, Rheology, in vitro release or glucose affecting somehow the thermal behavior of nanostructured systems lipid-poloxamer as possible control depends mainly on the chemotherapeutic and cytotoxicity assays. PL407-108 + HA (10-10% + the F127/DODAB aqueous systems. In glycoside the skin-delivery systems for SFN.

96 97 Supported by FAPESP (2018/02482-6, 2014/14457-5), used against clinically strains and also as template for D29 - Biophysical study of phytantriol cubosomes D30 - CHARACTERIZATION OF POLYELECTROLYTE CNPq 309207/2016-9, 402838/2016-5). the development of new antibiotics. with a drug, miltefosine, in different aqueous COMPLEXES BASED ON CHITOSAN BY POWDER media, pH and temperatures. X-RAY DIFFRACTION, INFRARED SPECTROSCOPY D27 - LyeTxI-b a synthetic peptide active on This work was supported by CAPES, CNPq and FAPEMIG AND THERMAL ANALYSIS Bárbara Malheiros1,2 membranes shows potential action on bacteria, in Sandra B. N. Agostini1 planktonic and biofilm conditions. D28 - FACTORIAL DESIGN APPLIED TO Raphael Dias de Castro1,2, Mayra Lotierzo1,2, 2 1 1 1 DEVELOPMENT OF LIPID NANOCAPSULES Giovana Firpo Rodrigues , Leandro Ramos Souza Jennifer T.J. Freitas and Flávia C. Carvalho Pablo V. M. Reis Barbosa2 Cordeiro Lima P. F1 1Department of Drug and Food, Federal University Vinícius M. Lima1; Kelton R. Souza6; Gabriele 1Department of Pharmaceutical and Biochemical of Alfenas, Alfenas, MG. ([email protected]) A. Cardoso6; Marcella N. Melo- Braga1; Jarbas de Paula E.1 Technology - Pharmaceutical Sciences Faculty – M. Resende2; Daniel M. S. 4; Rodrigo M. Verly 6; 1Department of Biochemistry and Tissue Biology, USP. 2Biosystems Laboratory – Biophysics Group – Adriano M.C. Pimenta1; Vera L. Santos3; Maria Polyelectrolyte complexes (PECs) have been developed University of Campinas (Unicamp), Campinas, SP General Physics Department, Physics Institute USP. Elena de Lima1 as potential carriers to drug delivery. This kind of ([email protected]) [email protected] particles is obtained from polysaccharide polymers by 1 Departamento de Bioquímica e Imunologia do ICB easy, fast, and eco-friendly technique. The aim of this 2 ([email protected]), Departamento de Química Lipid nanocapsules (LNC) is a novel nanoparticle Nanomedicine is one of the most promising fields in work was to characterize PECs based on chitosan and 3 do ICEx and Departamento de Microbiologia do employed in Drug Delivery System (DDS). The purpose nanotechnology nowadays. The use of nanoparticles hypromellose loaded with methotrexate (PEC-MTX) or 4 ICB - UFMG, Belo Horizonte, MG, Brazil. Fundação of this study was to optimize the development of LNC as carriers aims to improve efficiency of drugs that without the drug (empty PEC). In order to verify the 5 Ezequiel Dias, Belo Horizonte, MG, Brazil. for the anesthetics lidocaine and prilocaine (ALs), possess low solubility in aqueous environment (very success of the polyelectrolyte complexation, PEC-MTX, Programa de Pós-graduação em Ciências da using chemometry. A F4 factorial design experiment hydrophobic molecules). In this way, nanoparticles empty PEC, and the raw materials were analysed by Saúde, Biomedicina e Medicina, Instituto de Ensino was proposed, with variable amounts of lipid (16.5 to offer both a protection for the molecules and a carrying Powder X-ray Diffraction (PXRD), Infrared Spectroscopy e Pesquisa da Santa Casa de Belo Horizonte, Grupo 23.5%), co-surfactant (1 to 2.4%), and drugs (0 to 5%). vehicle. On this ground, cubosomes are nanoparticles (IR), Thermogravimetry (TG) and Differential Scanning Santa Casa de Belo Horizonte, Belo Horizonte, The amount of surfactant was kept the same (17%). capable of storing both hydrophilic, hydrophobic Calorimetry (DSC). The powder diffraction patterns of 6 MG, Brazil. Departamento de Química - FACET The number of - 60-85 °C - temperature cycles (3, 5 and amphiphilic molecules within its structure. Also, PEC-MTX and empty PEC were different from those Universidade Federal dos Vales do Jequitinhonha e or 7) was also varied. LNCs were prepared with phase- cubosomes have approximately 50% hydrophilic of the polymers and drug, but were the same one to Mucuri, Diamantina, MG, Brazil. inversion temperature (Heurtault et al, Phar. Research and hydrophobic surface areas, therefore, they can each other. Both samples were amorphous. In the IR vol:19(6), 875-880, 2002). The nanoparticles size, carry more molecules than liposomes for instance. spectrum the formation of the particle was indicated A potent antimicrobial peptide (LyeTxI) isolated from the polydispersity index (PDI), zeta potential (ZP), and In particular, cubosomes are quite easy to fabricate by the increase of absorption at 3296 cm-1 what can

venom of the spider Lycosa erythrognatha was used encapsulation efficiency (%EE) were measured. due to its base product, lipids like monoolein (GMO) be attributed to –NH2 groups probably exhibited on as a template to obtain the derived peptide LyeTxI-b, Results revealed an increase in the nanoparticles size or phytantriol (PHY) that self-assembly in water the surface of the PECs, and by the appearance of which showed activity in vitro (planktonic and biofilm of as a function of lipid concentration (from 41.6 ± 0.4 media. A model drug, miltefosine (MILT), was chosen absorption at 1418 cm-1 what indicates the presence + - bacteria) as well as in vivo (septic arthritis in mouse). The to 81.3 ± 0.1 nm) while the co-surfactant (lecithin) to study the interaction of such nanosystem with a of –NH3 and –COO . The difference between PEC- aim of this work was to elucidate the secondary structure had an opposite effect, decreasing the LNC size. The guest molecule. In this project the aim was to obtain MTX and empty PEC IR spectra was observed in the of LyeTxI-b by NMR technique, its interaction with anesthetics presence didn´t affect the size of the cubosomes in sizes smaller than 500 nm. PHY was displacement of the wavenumber to higher values. artificial membranes by ITC, DLS and SPR-techniques LNC, but the absolute value of ZP (from 9.1 ± 0.8 to the chosen lipid,and cubosomes were reproduced by a By DSC, it was noticed both PECs showed similar and to test its antimicrobial activity in clinically isolated 54.8 ± 3.7 mV). PDI values were always extremely well stablished bottom up protocol. SAXS experiments endothermic and exothermic events, but with differents

strains in planktonic and biofilm conditions. LyeTxI-b low (< 0.18) revealing the homogeneity of LNC size were performed in water, 2.25% glycerol, PBS and Tonset. In addition, PEC-MTX thermogram showed one did not show a clear partition between hydrophobic/ distribution. The number of temperature cycles had AcPhBo (pH4.5) buffer media and 22 °C, 40 °C, 50 °C, exothermic event a more than the empty PEC. There hydrophilic faces, similarly to LyeTxI. However, LyeTxI-b no significant effect on the particle size response and 65 °C. TEM and cryo-EM revealed individual particles was an initial mass loss of . ~7.5% in the PEC-MTX showed higher structural stability near N-terminus, the system was found stable when stored at 2 °C. of ~400 nm with internal structure. DLS is compatible sample up to the temperature of 123°C what was less compared to the native peptide, possibly due to the Transmission Electron Microscopy and Nanotracking with micrsocopy, MILT does not affect particle size. pronounced in the empty PEC sample (~2.3%). In terminal amine acetylation. When evaluating interactions analysis revealed spherical nanocapsules with 1.86 Extrusion in a 0.05 µm filter was also implemented conclusion, all the characterization techniques proved of the peptides with artificial membranes, LyeTxI-b ± 0.26 x 1014 particles/mL. The %EE of the lipid in order to make a monodisperse solution, even after the obtention of the particle, and IR and thermal analysis showed a greater interaction compared to the original nanoparticulated system was very good: 83 ± 4% for extrusion the nanoparticles remained cubic, with sizes distinguished PEC-MTX from empty PEC. peptide. The minimum inhibitory concentration (MIC) lidocaine and 78 ± 5% for prilocaine. In conclusion, ~190 nm showing extraordinary malleability. was determined in 12 different clinically isolated strains stable LNCs could be prepared by the phase-inversion This work was supported by CAPES (Grant#1241/2014); and, LyeTxI-b was two folds more active than LyeTxI in technique. Factorial Design allowed the selection of This work was supported by Conselho Nac. Des. Cient. CNPQ (Grant#454679/2014-9); FAPEMIG A. baumanni, E. coli, P. aeruginosa and S. aureus. The an optimized formulation (particle size < 50 nm, PDI Tecnologico (CNPq) and by FAPESP. activities on the other tested strains were similar for both < 0.1, positive ZP), containing lipid (<20%) lecithin peptides, although two strains of Serratia sp were not (>1%), surfactant (17%) and local anesthetics (2.5%). susceptive to them. In a S. aureus death curve assay The low sizes of the LNCs should favor the topical we found that the bacterial killing time depends on the delivery of lidocaine and prilocaine, for odontology peptide concentration indicating more than one action purposes. mechanism to these bacteria. In the S. aureus strain biofilm assay, LyeTxI-b activity was more potent than the This work was supported by CNPq (fellowship C-D.P.F, native peptide. However, both peptides were unable to E.P) and Fapesp (# 14/14457-5). reduce biofilm from Gram negative A. baumannii. These results showed that LyeTx-b could be a good drug to be

98 99 D31 - DESIGN OF NEW GLUTAMINASE D32 - Study of miscibility of lipid components D33 - SEARCHING FOR NEW CRUZAIN D34 - DEVELOPMENT AND CHARACTERIZATION OF INHIBITORS WITH POTENTIAL ANTITUMOR through Raman Spectroscopy and its INHIBITORS WITH DOCKING AND MOLECULAR POLYMERIC NANOPARTICLES CONTAINING ZINC ACTIVITY applicability in the physico-chemical stability of DYNAMICS SIMULATIONS PHENIL-TIO-PHTALOCYANINE FOR ANTIMICROBIAL PHOTODYNAMIC THERAPY - APDT Sandra M. G. Dias1 nanostructured lipid carriers (NLC) Viviane Corrêa Santos*1 1 Amantino C. F1 Matheus P. Pinheiro1, Jean C. H. Campos1 Castro, Simone R. de Lucianna Helene Santos2, Pedro Geraldo Pascutti3 2 2 1 1 2 1* 1 Mitsutake, Hery , Breitkreitz, Márcia C. , de Paula, and Rafaela Salgado Ferreira Evangelista RP , Tedesco AC and Primo FL Brazilian Biosciences National Laboratory (LNBio), 1 Brazilian Center for Research in Energy and Eneida 1Department of Biochemistry and Immunology, 1UNESP - Universidade Estadual Paulista, Campus Materials (CNPEM), Campinas, SP, Brazil (matheus. 1Department of Biochemistry and Tissue Biology, Federal University of Minas Gerais, MG;2 Programa de Araraquara, Department of Bioprocesses [email protected]) State University of Campinas, Campinas, SP de Pós-graduação em Bioinformática, Federal and Biotechnology, Araraquara, SP (e-mail: ([email protected]) and 2Chemistry University of Minas Gerais, MG;3 Instituto de [email protected]; [email protected]*) 2 Glutamine is an abundant nutrient used to support Institute, State University of Campinas, Campinas, SP Biofísica Carlos Chagas Filho, Federal University of and Department of Chemistry-FFCLRP, University the elevated bioenergetic and biosynthetic demands Rio de Janeiro, RJ. *[email protected] of São Paulo- USP, Ribeirão Preto, SP required by many tumor cells. Glutaminase, a key The NLC are particles produced from a mixture of enzyme in the conversion of glutamine to glutamate, solid lithium and a liquid lithium (oil), resulting in The only medicine available for Chagas disease causes Phthalocyanines are one of the photosensitizers (PS) plays a vital role in up-regulating cell metabolism for a core of the particles with many imperfections1. several side effects. Cruzain is an established target classes and stand out by the intense absorption in the near tumor cell growth and is considered to be an attractive Raman spectroscopy has been discreetly used to in Trypanosoma cruzi, thus discovering new inhibitor infrared region (650-800 nm) in which there is greater therapeutic target for cancer treatment. Given this investigate the distribution of drugs and lipids in lipid scaffolds can be useful in the development of new tissue penetration of light. Nanostructured drug delivery important function of glutaminase in cancer, there nanoparticle systems2. Therefore, in this work we medicines to treat this condition. We did a validation systems compared to free drugs present as advantages: has been interest in developing inhibitors targeting investigated the applicability of the lipid miscibility study with 66 known cruzain inhibitors and 3,772 possibility of targeting specific tissues; better solubility glutaminase. Although the compound CB-839, a study by RAMAN on the physico-chemical stability decoys to design the virtual screening workflow we of drugs increasing their bioavailability; overcoming glutaminase inhibitor, is currently being evaluated of NLCs prepared with different lipids. Preparation of would apply in the search for new cruzain inhibitors. the resistance offered by physiological barriers (due in phase 1 and 2 clinical trials, many efforts to lipid mixtures was achieved by heating the solid lipids Then, we screened about 300,000 Leads Now to the reduction of particle size); decreased toxicity; develop a new class of glutaminase inhibitors that (CP, MM), and adding to the liquid lipid (TRANSC, DK) molecules from ZincDB in a hierarchical approach. better therapeutic index, among others. The objective target glutamine metabolism have been done in both under stirring until a visually homogeneous mixture. Firstly, we did an HTVS docking with Glide. Secondly, of the present work was to develop and characterize academia and industry. After a High Throughput The samples were cooled to room temperature in top 10% results were submitted to Glide SP docking. polymeric nanocapsules (NC) containing Zinc phenyl-tio- Screening (HTS) campaign performed in our group an aluminum cell and was mapped using a Raman Thirdly, top 10% results were filtered based on the phthalocyanine (ZnS4Pc) from a physical-chemical and with a Screening Library of 30,000 compounds, from Station 400 spectrometer. The nanostructured lipid interactions with the receptor. Molecules predicted to photophysical point of view. Polymer interfacial deposition ChemBridge, several hits were identified using the carriers were obtained by the emulsification-sonication interact with S2 pocket and making hydrogen bonds method was used to prepared NC/ZnS4Pc (0.05 mg/ isoform glutaminase C (GAC) as target. The most technique and analyzed by DLS. From the analysis of with G66 were kept for the next step. Then, LogS mL) and followed by the investigation of the particle size, promising compounds and analogues were acquired the RAMAN images, we can conclude that the three and PAINs filters were applied. Subsequently, poses polydispersity index (PdI) and zeta potential using dynamic and evaluated by combining thermal shift assay, mixtures presented different degrees of miscibility and were visually inspected, eighteen compounds were light scattering technique (DLS). The mean of 96-days dynamic light scattering and in vitro inhibition assay. in relation to the homogeneity profile. Such samples selected to molecular dynamics simulation step. These follow-up values were 252.9 nm, 0.465 and -24,89 mV Here, we set out to identify a novel class of inhibitors may be cited as: CP-DK> MM-DK> CP-TRANS. This simulations are ongoing in AMBER. The molecules respectively, thus showing satisfactory parameters. able to inhibit the enzymatic activity of GAC with low result corroborated the macroscopic issues observed: with binding stability will be purchased and tested in Absorption and emission spectroscopy analyzes in the micromolar potency. In addition, these results allowed whereas CP-TRANS showed phase separation after two enzymatic assays against cruzain. In doing so, we found UV-visible region were performed, and no changes were us to implement strategies of ligand-based design to months, the other two were stable for a period of more that performing the docking with C25, G66 and H162 found in the photophysical properties at the steady state of propose analogues of these promising GAC inhibitors than 360 days. Size and PDI analyzes also showed a in two protonation states (all neutral or all charged) NC/ZnS4Pc relative to free ZnS4Pc. In addition, a spectral that are being synthesized for evaluation aiming at significant increase in these parameters and a reduction can enhance the chemical diversity of the “hits”. Thus, analytical method was established for the quantification increasing the potency of these inhibitors. in the value of ZP, which may have contributed to the docking and molecular dynamics simulations were of the active in the formulation through a correlation instability of the CP-TRANS system in the long term. It executed with these variations. Perspectives of this curve between maximum absorption and corresponding This work was supported by FAPESP. is concluded that a brief study of lipid miscibility by the work include calculating MM-GB/SA free energies for concentration in the range of 0.25 - 2.04 μg/mL, which RAMAN technique can be a fast and important tool for these compounds, performing the enzymatic assays allowed the determination of a ZnS4Pc quantification of obtaining a lipid carrier product with long-term stability and verifying whether the approach was successful in 42.73 % in the formulation of NC / ZnS4Pc, with potential desirable. correctly predicting the activity of the compounds. to future application in APDT in vitro assays

This work was supported by Conselho Nac. Des. Cient. This work was supported by CNPq, CAPES, FAPEMIG Technologic (CNPq) and by the São Paulo Research and L’Oréal-UNESCO-ABC. Foundation – FAPESP ((# 14/14457-5).

100 101 Molecular Mechanisms of Disease with antioxidants have provided promising alternatives to E04 - PHYSICAL AND BIOLOGICAL APPROACHES prevent and/or revert heart dysfunction in CD. However, OF ANTI-INFLAMMATORY ACTIONS IN less understood is the role of individual ROS sources ENDOTOXIN-INDUCED UVEITS MODELS: metastatic stages whereas PDE4 is more active in early in the progression or resolution of CD. In this study, PIPERLONGUMINE AND AC EXPERIMENTAL E01 - CAMP AND VOLTAGE GATED CHANNELS IN PHOX 2-26 BREAST TUMOR DEVELOPMENT. stages of cancer development. To further investigate our we used a gp91 knockout mice model (PHOXKO), AND MOLECULAR DYNAMIC STUDIES initial findings, we will now evaluate the level of expression lacking functional NOX2 on plasma membrane, to Andreia Laura Prates Rodrigues1 of different phosphodiesterases in these cells. Was not investigate the effects of absence production of NOX2- Marinonio L. Cornélio3 Andreas Koschinski2, Paula Rhana2, Bruna Lima1, observed in any of the strains input calcium currents but derived ROS on the outcome of cardiac dysfunction Ana Paula Girol1, Caroline F. Zanon2, Sonia Maria Flavia Aguiar1, Jader S. Cruz3, Manuela Zaccolo2 voltage-gated Na + channels are expressed in primary during the acute phase of CD. Whole-cell patch-clamp, Oliani2, Ícaro P. Caruso3 breast tumor, presenting inward currents of -12.68 ± confocal microscopy, calcium fluorescence and cell- 1 1 Laboratório de Câncer de Mama, Canais Iônicos 1.29 pA/pF and -13.40 ± 2.35 pA/pF at 0 mV for MACL- contraction assays were performed on cardiomyocytes Integrated College Padre Albino Foundation 2 e AMPc, FUMEC University, Belo Horizonte, Minas 1 (n=21) and MGSO-3 (n=15), respectively and mainly isolated from C57Bl6/N (WT) and PHOXKO mice, non- (FIPA), Catanduva, SP. Department of Biology, and 3 Gerais, Brazil ([email protected]), 2Department of composed by TTX-resistant isoforms. These cells also infected or 15 days post-infection with T. cruzi Y-strain. Department of Physics, Universidade Estadual Physiology, Anatomy and Genetics, Oxford University, showed the presence of an outflow stream, which could We found that infected PHOXKO-cardiomyocytes has Paulista (Unesp), São José do Rio Preto, SP South Parks Road, Oxford OX1 3QX, 3Laboratório be a chain of potassium. Tests with external solution to an overall pro-arrhythmic phenotype, notably with ([email protected]) de Membranas Excitáveis e Biologia Cardiovascular, block potassium channels with TEA and cesium, showed higher appearance of early after depolarizations (EAD) Department of Biochemistry and Immunology, a decrease in this current MgSO-3 cells. In MACL-1 cells and 2.5- fold increase in action potential (AP) duration Uveits is one of the leading causes of blindness worldwide Biological Sciences Institute, Federal University of also decreased, but not as sharp. Comparing the results, alternans, compared to other groups. Further, infected which requests the search for new therapeutics Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. cells MgSO 3 shows the current density at the potential PHOXKO-cells display increased diastolic [Ca2+], alternatives. Here we studied the interactions between of 60 mV in solution with potassium channel blocker increased Ca2+- transients disturbances and reduced Piperlongumine (PL) and the Annexin A1 (AnxA1)- 2+ Breast cancer remains the second most common cause decreased by 51.9% (SEM = 7.59, n = 3) compared Ca -transient amplitude. Cardiomyocytes contraction derived peptide, Ac2-26 and the effect of PL administration of death for women. A commonly used cell model to the current density measured in the control condition. For is 20-30% reduced in infected WT and PHOXKO mice, singly or associated with Ac2-26, in endotoxin induced investigate breast cancer are metastatic cells. Metastatic MACL-1 cell line that reduction was on average 24.99% however, with comparable extent. Nevertheless, uveits (EIU). The approach consisted of investigating the cells represent an advanced stage of the pathology that is (SEM = 3.249, n = 3). In a next step, we investigated the only infected PHOXKO-ventricular myocytes display interaction using a combination of cellular assays and often associated with poor prognosis. The study of non- involvement of chloride channels. In both lines there was significant increase in non-triggered extra contractions biophysical methods, in order to understand the molecular metastatic stages of breast cancer might offer the possibility a significant decrease in current, and in the third MgSO (appearing in ~75% of cells). Electro-mechanical mechanism of uveits. In vivo, studies showed a decreased to uncover new mechanistic insights into the development this reduction was an average of 31.67% (SEM = 16.31, remodeling of infected PHOXKO-cardiomyocytes is of leukocytes influx, as well as inflammatory mediator of breast cancer. cAMP and calcium are known as two n = 3), and MACL-1 of 64.1% (SEM = 12.86, n = 3). associated with increase in ·NO and mitochondria- levels and COX-2 expression, which confirmed the main important second messengers, characterizing The results show that there is no input currents, sodium derived ROS production. Interestingly, EADs and AP peptide anti-inflammatory action and pointed protective them from non-tumor cells to tumor from primary and or calcium, and the efflux stream with the participation duration alternans were reverted by pre-incubation with effects of PL at 400µg. However, when combined PL and

metastatic stage might also elucidated new pathways. of both potassium and chloride channels in the strains NOS inhibitors. We conclude that lack of NOX2-derived Ac2-26 affected the control of inflammation. The antagonist Phosphodiesterase (PDEs) play a role in cell proliferation tested. To further investigate our initial findings, we will ROS promoted a pro-arrhythmic phenotype in the heart, response showed that the conserved sequence of Ac2- and regulation of cell cycle progression. Although their now evaluate the level of cAMPi, the expression of different in which the crosstalk between ROS and ·NO could 26 has to preserve its conformation independent of the involvement in cancer has been recognized, little is known phosphodiesterases, as well the expression of the voltage play an important role in modulation of cardiomyocytes protein frame, and needs the cellular environment to keep about the role of PDE in different stages of breast cancer channels in these cells. electro-mechanical function. its biological activity. development. PDEs hydrolyze the cyclic nucleotides adenosine 3’, 5’-cyclic monophosphate (cAMP) and E02 - NADPH-OXIDASE2 DERIVED ROS IN THE This work was supported by Conselho Nacional de This work was supported by FAPESP (2017/08834-9) guanosine 3’, 5’-cyclic monophosphate (cGMP) to their OUTCOME OF CHAGASIC CARDIOMYOPATHY: Desenvolvimento Científico e Tecnológico (CNPq), inactive form (5’nucleotide). Thus, they play an essential NEW INSIGHTS INTO ROS/NO INTERPLAY Coordenação de Aperfeiçoamento de Pessoal de Nível role in the regulation of these cyclic nucleotides at global Superior (CAPES), Fundação de Amparo à Pesquisa as well as at compartmental or microdomain-levels. Artur S. Miranda1 do Estado de Minas Gerais (FAPEMIG) and Fundação Therefore, they are able to modulate crucial intracellular Julliane V. J. Santos2, Graziell A. Ribeiro1, Petter do Amparo a Pesquisa do Estado de São Paulo signaling pathways that might promote the progression of Guerra1, Jader S. Cruz1, Leda Q. Vieira1, Danilo R. (2014/09861-1). non-metastatic into metastatic cancer cells. To investigate Campos3 the activity of phosphodiesterases in different stages of 1 breast cancer development we analyzed three different Department of Biochemistry and Immunology, cell lines derived from a metastatic breast cancer (MDA- Federal Universityty of Minas Gerais, MG 2 231 cells), non-metastatic breast cancer cells (MACL-1 ([email protected]), Department of and MGSO-3-cells)1 and non-cancer mammary epithelial Morphology, Federal Univesity of Minas Gerais, MG, 3 cells (MCF-10). To monitor PDE activity and cAMP in these Department of Biophysics, Federal University of different cell lines, we established stable clones expressing São Paulo, SP the cAMP-reporter EPAC1_camps2 and generated the respective monoclonal lines (MDA-231 EPAC C4, MACL-1 Chagas’ Disease (CD) is a leading cause of heart EPAC F12 F10, MGSO-3 EPAC D9 and MCF-10 EPAC C10) failure and sudden death in Latin America. CD- and used FRET technique. Our results show a lower level triggered inflammation is associated with increased of basal cAMP and higher phosphodiesterases activity in oxidative stress in which NADPH-oxidase2 (NOX2), metastatic cells as compared to non-metastatic and to mitochondrial-derived reactive oxygen species (ROS) non-cancer cells. PDE8 activity appears to be higher in and nitric oxide (·NO) play an important role. Treatments

102 103 E05 - A COMPUTER TOOL TO IMPLEMENT E06 - VOLTAGE-GATED Na+ CHANNELS ACT IN E09 - INSIGHTS INTO Klebsiella pneumoniae E10 - HIGH GLUCOSE REDUCES MEGALIN- AND COMPARE MODELS OF CONTRACTION IN TUMOR DEVELOPMENT. TYPE VI SECRETION SYSTEM TRANSCRIPTIONAL MEDIATED ALBUMIN ENDOCYTOSIS IN RENAL CARDIAC MYOCYTES REGULATION PROXIMAL TUBULE CELLS THROUGH PROTEIN Paula Rhana1,3 KINASE B O-GLCNACYLATION Osias B. S. Filho1 Victor Barbosa Tatiany Luiza Silveira2, Tatiane de Oliveira Barreto Diogo B. Peruchetti1 Rosana A. Bassani2; José W. M. Bassani2 and Lopes1, Ênio Ferreira2, Jader Santos Cruz1, Andréia Leticia MS Lery Robson R. Silva1,2 3 Rodrigo P. Silva-Aguiar1, Gabriela M. Siqueira1, Laura Prates Rodrigues . FIOCRUZ, Rio de Janeiro, RJ (barbosa.a.victor@ Wagner B. Dias1, and Celso Caruso-Neves1,2 1Research and Technology Center, University of 1Department of Biochemistry and Immunology, gmail.com) Mogi das Cruzes, São Paulo, SP (osiasbap@gmail. Biological Sciences Institute, Federal University 1Instituto de Biofísica Carlos Chagas Filho, Federal com) and 2Center for Biomedical Engineering, of Minas Gerais, Belo Horizonte, Minas Gerais, Klebsiella pneumoniae (KP) is an opportunistic University of Rio de Janeiro, Rio de Janeiro, RJ 2 2 University of Campinas, Campinas. Brazil ([email protected]), Department pathogen that mainly causes infections in the ([email protected]); Instituto Nacional de of Pathology, Biological Sciences Institute, Federal respiratory and urinary tract. The frequent Ciência e Tecnologia em Medicina Regenerativa, Computer models are useful tools in the study of University of Minas Gerais, Belo Horizonte, occurrence of these virulent isolates with resistance INCT-Regenera, CNPq/MCT, Rio de Janeiro, RJ 3 physiological phenomena. However, as the available Minas Gerais, Brazil and University FUMEC, Belo to multiple drugsled WHO to include this species in models present differences in assumptions and Horizonte, Minas Gerais, Brazil. the list of priorities for the search for therapeutic It has been pointed out an active role of tubular mathematical formulations, discrepancy in the resulting alternatives. Understanding the mechanisms dysfunction in the development of early stage diabetic simulations is not uncommon. Among the models for Besides the great advance in diagnosis and treatment, underlying KP virulence is necessary and may lead kidney disease. The possible mechanism proposed contraction in cardiomyocytes based on Huxley´s we still do not have a cure for breast cancer. One to the development of more efficient and specific is the inhibitory effect of high glucose concentration crossbridge model, those proposed by Negroni and promising drug target is voltage-gated Na+ channels, drugs. One of these virulence factors is the Type (HG) on megalin-mediated albumin endocytosis in Lascano (NL model) and Rice et al. (RWH model) are pore-forming transmembrane proteins responsible VI Secretion System (T6SS), which provides proximal tubule (PT) cells. The aim of this work is to the most frequently used. The objective of this study for initiation and propagation of the action potential in competitive and adaptative advantages for the identify the molecular mechanisms underlying this was to develop in Python a computational tool, ForceLAB excitable cells. However, these proteins are abnormally bacteria that possess it. Recent work has shown process. For that, we used LLC-PK1 cells, a model simulator, which allows implementing different models expressed in several types of tumors and have been that Type VI Secretion System (T6SS) genes are of PT cells. HG inhibited albumin-FITC uptake (n=9) of contraction, and comparing their performance for an associated with increased aggressiveness. Until now, expressed by KP during the infectious process and and megalin expression (n=3) in 40% and 50%, equal input (i.e., Ca2+ influx and intracellular release). work have been done using cell lineages of non-tumor are important for bacterial competition. This work respectively. The simultaneous incubation of cells with As an application, the NL and the RWH models were or metastatic breast tumor, not representing all stages aims to identify the mechanisms of transcriptional HG and 100µM phlorizin (PLZ), SGLT inhibitor, or 10 compared in the simulation of a single twitch contraction. of tumorigenesis. In this work, we use patch-clamp, regulation of T6SS genes expression in KP through µM DON, GFAT inhibitor, blocked the HG effects on the Initially, to check the impact of different Ca2+-troponin C western-blot and immunohistochemistry assays in two computational predictions and experimental albumin-FITC uptake (n=6) and megalin expression

apparent dissociation constants (Kd= 0.77 and 0.60 µM different cell lineages (MACL-1 and MGSO-3) to evaluate validations. We analyzed the genome of Kp52.145 (n=3). Interestingly, MK-2206 10nM (PKB inhibitor) for NL and RWH models, respectively) on the steady- the expression of Nav1.5, Nav1.6 and Nav1.7 in initial strain to perform a broad prediction of T6SS genes for 48h inhibited albumin-FITC uptake (n=5) and state Ca2+-myofilament interaction, force-pCa curves stages of tumor development. Our results demonstrated with their promoters through comparative analyses, megalin expression (n=3), mimicking HG effect. In were simulated. Although the Hill coefficientes were that voltage-gated Na+ channels are expressed in similarity searches and linear discriminant analysis. addition, incubation of the cells with FBS, activator similar (~5% differerence), the Ca2+ concentration primary breast tumor, presenting inward currents of 45 T6SS genes were found and re-annotated and of PKB, abolished the inhibitory effect of HG on required for half-maximal contraction activation was -12.68 ± 1.29 pA/pF and - 13.40 ± 2.35 pA/pF at 0 mV 23 promoter regions dependent on sigma 70 factor albumin-FITC uptake (n=5) and megalin expression ~65% greater with NL model (780 vs. 470 nM, both for MACL-1 (n=21) and MGSO-3 (n=15), respectively. were identified. Using Virtual Footprint, binding (n=3). HG inhibited PKB activity in 60%, measured within the physiological range). Then a twitch simulation The addition of 100nM of tetrodotoxin (TTX) inhibited sites of 15 transcriptional regulators were found. by its phosphorylation at S473 and T308 (n=4). was performed, revealing that, whereas contraction approximately 100% of INa+ in MACL-1 (n=7) and Six of these regulators have sites in regions that These inhibitory effects of HG on PKB activation and Ca2+ transient amplitudes were similar (~1% about 80% in MGSO-3 (n=7), indicating that voltage- promote the genes encoding proteins related to were completely reversed by DON and PLZ (n=4). difference), both phases of the Ca2+ transient were gated Na+ channels are mainly composed by TTX- the complex assembly platform; nine related to the Furthermore, HG increased PKB O-GlcNAcylation ≥25% slower with the NL model, although identical resistant isoforms. Western-blot results confirmed the tubular complex and 13 to the recycling process (n=3). Taken together, our results indicate that HG cytosolic Ca2+ removal functions were set, and the higher expression of Nav1.5 (which is TTX-resistant) for system. These data corroborate to the hypothesis increases PKB O-GlcNAcylation leading to the inhibition contraction timecourse was less affected (≤10%). This both cells (0.30 ± 0.02 for MACL-1 and 0.13 ± 0.03 that transcription regulation of these components of its kinase activity, megalin expression and megalin- result stresses that differences in troponin C affinity for for MGSO-3; protein levels were normalized to β-actin can occur in the form of a regulatory cascade from mediated albumin endocytosis in PT cells. These Ca2+ can shape the Ca2+ transient even in the absence of levels), but Nav1.7 was also expressed (0.06 ± 0.04 environmental stimuli sensed by the bacterium. results open new perspectives for the understanding of changes in Ca2+ transport. and 0.04 ± 0.01 for MACL-1 and MGSO-3, respectively). Such layered regulation may be important to the molecular mechanisms underlying the development However, immunohistochemistry demonstrated the maintain different levels of expression of the of early stage diabetic kidney disease. This work was supported by São Paulo Research presence of Nav1.5, Nav1.6 and Nav1.7 in both cells, components that can be secreted or recycled Foundation (FAPESP, grant#2016/18422-7). and surprisingly, not only in the cell membrane but also internally. inside the cytoplasm, indicating that these ion channels can act in other cellular processes important for tumor This work was supported by IOC, CAPES, CNPq, FAPERJ development.

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico and Fundação de Amparo à Pesquisa do Estado de Minas Gerais.

104 105 E11 - CARDIAC ELECTRICAL AND MECHANICAL to investigate the mechanisms underlying the cardiac Nucleic acids structure and functions REMODELLING STUDY ON A TYPE 2 DIABETES electrical remodeling on type 2 diabetes (T2D) mice EXPERIMENTAL MODEL model which combines a high fat diet (HFD) and low doses of streptozotocin (STZ). Blood glucose F01 - Mesoscopic parametrization of DNA-RNA may occur which are not fully understood. Linear models Ainhoa Rodriguez de Yurre1 levels, intraperitoneal glucose tolerance test (IPGTT), hybrids based in Gibbs free energies address these interactions Maria Micaela Lopez Alarcón1, Isalira Peroba intraperitoneal insulin tolerance test (IPITT) and serum by adding simple contributions to the total sequence Erik de Oliveira Martins1,2 Ramos1, Oscar Casis Sáenz3 and Emiliano Medei1 insulin levels were used to confirm T2D. In vivo entropy. Nevertheless, for non-linear models such as the electrocardiogram recordings showed prolonged QJ 1 1 Peyrard- Bishop (PB) mesoscopic model it is not clear 1 Vivianne Basílio Barbosa , Gerald Weber Universidade Federal do Rio de Janeiro and interval on T2D mice which can be explained at least how such effects should be taken into account. One 3 1 Universidad del País Vasco partially, by shorter APD30 and transient outward Departamento de Física, Universidade Federal de important advantage of the PB model over linear Gibbs Minas Gerais, Belo Horizonte-MG. 2Departamento potassium current (Ito) decrease. In addition, when free energy schemes is the ability to distinguish base Type 2 diabetes (T2D) is the most prevalent form of T2D mice were submitted to an arrhythmia protocol de Formação Geral, Centro Federal de Educação pair hydrogen bonds from nearest neighbour stacking diabetes and represents about 90% of the diabetic cases combining caffeine and dobutamine, not only more Tecnológica de Minas Gerais, Timóteo-MG Funding: interactions. To find out the behavior of these interactions all over the world. As a consequence of the lifestyle susceptibility but also arrhythmia severity was observed Fapemig, CNPq and Capes at the end of the helix, we modelled RNA sequences by and feeding, this syndrome has turned into one of the in T2D animals. Furthermore, ventricular function was allowing the Morse potentials of the Peyrard-Bishop largest health problems worldwide and it is associated studied by magnetic resonance imaging (MRI-7T), DNA/RNA hybridization has important role in model, which represent the hydrogen bonds, to take with an increase of premature appearance of several which showed ejection fraction percentage diminution biotechnological applications such as in CRISPR/Cas9 different values for terminal base pairs. We then used the disorders such as cardiovascular complications, on diabetic mice. This indicate that a combination genome editing. Differently from double helices like DNA/ temperature minimization technique developed by our which can evoke cardiac electrical disturbances as of a HFD and low doses of STZ recapitulate the T2D DNA or RNA/RNA, which can be self- complementary, groupto obtain those new potentials. For RNA we found arrhythmias. Thus, the aim of the present study was classical electrical and mechanical events. sequences of DNA/RNA hybrids have a strongly that generally, the terminal Morse potentials increase in asymmetrical structure due the different compositions comparison to internal base pairs. This is in contrast the backbone. Minor groove width, helical rise, the unique to our previous results for DNA where we found that desolvation pattern, conformational sampling of the DNA terminal Morse potentials that are marginally smaller, backbone, intrinsic flexibility and deformability have been indicating a destabilization of the helix. For DNA, where proposed as possible factors to describe the structure of a detailed experimental data are available for a range of hybrid molecule. The quantity of deoxypyrimidine (dPy) has salt concentrations we found this destabilizing trend a direct role in these structures. Here, we use the Peyrard- for all concentrations. In a subsequent analysis we Bishop model (PB) parameterize the DNA/RNA from also allowed the study of the stacking interactions. Our experimental melting temperatures. The PB model was preliminary results of the terminal stacking interactions previously applied to DNA/DNA and RNA/RNA structures showed that they usually depend on the strand direction and the prediction of hydrogen bonds were found to be for both RNA. generally consistent with NMR and x-ray measurements. One of the key elements of the PB model is that the stacking This work was supported by CNPq, Capes, Fapemig. interaction of both strands is assumed to be the same, a condition that can not be justified for the hybrid DNA/RNA F03 - THREE-DIMENSIONAL HELICOIDAL duplex. However, we have shown recently that the model MESOSCOPIC MODEL FOR DNA can be adapted to highly asymmetric situations such as bulges in RNA. Here, we take advantage of this adaptation Mateus Rodrigues Leal and perform the missing parametrization for DNA/RNA. Gerald Weber We found that the stacking interactions follow closely that of RNA and that the average strand displacements are Department of Physics, Federal University of Minas strongly dependent on the deoxypyrimidine content. Gerais, Belo Horizonte, MG (mateusfisica87@ gmail.com) Funding: CNPq, Capes, and Fapemig. The Peyrard-Bishop model is a two-dimensional F02 - TERMINAL EFFECTS IN MESOSCOPIC physical statistics method proposed in 1989 for MODELS OF RNA calculating thermodynamic properties of DNA and RNA molecules. Currently, it is widely used with several Izabela F da Silva1 theoretical techniques such as molecular dynamics Tauanne D Amarante2 and Gerald Weber1 and path integrals, but its main application still lies within its original method which is the transfer-integral 1 Departamento de Física, UFMG, Belo Horizonte- technique. It also used to predict hydrogen bonds and 2 MG and. ([email protected]). Department of stacking interaction in oligonucleotides from melting Medical Genetics, University of Cambridge, UK temperature experiments, complementing experimental techniques such as NRM and X-ray diffraction. One The RNA double helix is primarily stabilized by hydrogen of the key simplifications of the model is to ignore bonds of the base pairs and by stacking interactions. the helical structure of DNA, nevertheless it has been However, at the end of the helix additional interactions highly successful in predicting melting temperatures

106 107 and other important properties of DNA. Here, we add used our PB model for cyanine dyes and correlated the F06 - EXPLORING THE ENERGY LANDSCAPE F07 - EVALUATING THE THERMAL STABILITY OF the missing helical torsion to the original 2D Hamiltonian average opening with the fluorescence intensities. For OF CHROMOSOMES: TRANSITIONS BETWEEN SUB-OPTIMAL RNA FOLDING ASSOCIATED TO while maintaining the existing framework of numerical Cy5 we obtained Pearson correlation coefficient of 0.8 INTERPHASE AND MITOTIC PHASE RET-MEN2 ONCOGENE techniques for solving the classical partition function. indicating that the average opening may be used as a We introduce some approximations to avoid an predictor for fluorescence intensities. Vinícius G. Contessoto1,2,3 Vivianne Basílio Barbosa¹ excessive increase in degrees of freedom which would Di Pierro M1, Cheng RR1, Wolynes PG1, Onuchuc JN1 Rodolfo Vieira Maximiano² and Gerald Weber¹ make the problem intractable. This allows us to use the Our work was financial supported by Fapemig, CNPq 1 same transfer integral technique for both the 2D and 3D and Capes. Center for Theoretical Biological Physics, Rice ¹Department of Physics, Federal University of Minas models. The 3D model shows similar anti-crossings in University, Houston - TX ([email protected]). Gerais, Belo Horizonte, MG (viviannebasilio@ 2 the eigenvalue spectra as the 2D models leading to very Brazilian Bioethanol Science and Technology gmail.com) and ²Department of General Formation, F05 - FAT NUCLEOSOME: ROLE OF LIPIDS ON Laboratory, Campinas - SP, Brazil 3Department of Federal Center of Technological Education of Minas sharp increases in the average strand displacement. The CHROMATIN approximations introduced in the 3D model were validated Physics, Institute of Biosciences, Letters and Exact Gerais, Contagem Unit, Contagem, MG by comparing them with the numerical integration of the Vinícius Fernandes1,2 Sciences, São Paulo State University, São José do Rio Preto - SP, Brazil exact 3D Hamiltonian. With this new 3D approach we Kaian A. Teles2, Camyla Ribeiro2, Werner Treptow1 & Mutations to the proto-oncogene RET (rearranged are now able to use mesoscopic models to investigate Guilherme M. Santos2 during transfection) is associated to the development structural properties of DNA such as the helical rise Understanding the genome architecture and how of various types of human cancer such as medullary 1 distance and torsion angle from melting temperatures, Laboratório de Biologia Teórica e Computacional, the chromosomes are organized during the different thyroid carcinoma (MTC) and multiple endocrine in the same way as we obtained hydrogen bonds from Departamento de Biologia Celular, Universidade phases of the cell is a significant challenge which neoplasias of type 2 (MEN2). Synonymous variantes 2 the 2D model. de Brasília, Brasília, DF and Laboratório de involves scientists from diverse areas of knowledge. of single nucleotide polymorphisms (SNPs) appear Farmacologia Molecular, Departamento de Recent studies using a physical model (MiChroM) for to be associated to the agressiveness of MTC. In Funding: Capes, CNPq and Fapemig. Farmácia, Universidade de Brasília, Brasília, DF the chromatin folding described with high accuracy the our previous work we established that this was ([email protected]) structures from chromosomes in interphase. These related to the thermal stability of the messenger RNA F04 - MODELING DATA OF FLUORESCENCE studies also get the chromatin types of annotation (mRNA). The hypothesis is that the a more stable OF CYANINE DYE WITH PEYRARD-BISHOP Structural changes in chromatin regulate gene based on its epigenetic markers using a neural RNA increases protein production which in turn may MESOSCOPIC MODEL expression and define phenotypic outcomes. Most network method for extracting the data from chromatin explain the MTC aggressiveness. However, the mRNA recently, the formation of condensed chromatin regions immunoprecipitation experiments (MEGABASE). Here, sequence associated to RET is around 5000 bases long Pâmella Miranda based on phase-separation in the cell, a basic physical using these recently developed models and tools, we and changes of just a few nucleotides do not cause Gerald Weber Department of Physics, Federal mechanism, was proposed. Increased understanding investigate the liquid-like nature of chromatins and significant changes to the Gibbs free energy. In other University of Minas Gerais, of the mechanisms of interaction between chromatin how this is related to the formation of chromosome words, a simple energy calculation does not allow to and lipids suggest that small lipid molecules, such as territories and compartments. Additionally, using draw conclusions regarding the thermal stability of Minas Gerais, MG ([email protected]) cholesterol and short-chain fatty acids, can regulate molecular dynamics simulations, we study the folding- the various sequences. To overcome this problem, important nuclear functions. New biophysical data has unfolding dynamics of chromosomal structures, more we generated an ensemble of suboptimal secondary Cyanine dyes are fluorescent markers which have been suggested that cholesterol interacts with nucleosome precisely the transition of the chromosome structures structures and counted how often we had a more stable successful applied in biotechnology, biophysics and through multiple binding sites and affects chromatin between the interphase to mitotic phase, also known structure than the wildtype sequence. This resulted in biochemistry. They have been used in many techniques structure in vitro. Regardless of the mechanism of as chromosome condensation process. The simulation a positive correlation of five polymorphic variants and as real- time PCR, microarrays, Forster Resonance how lipids bind to chromatin, there is currently little results indicate that the genome structural organization their respective aggressiveness. Here we extend this Energy Transfer (FRET), biomedical imaging and awareness that lipids may be stored in chromatin and during the mitotic phase might be related to the analysis to the 195 known variants of the MEN2 gene Fluorescence in situ Hybridization (FISH). The most influence its state. Herein, we review the main impacts modulation of non-specific long-range interactions. currently available in the MEN2 public database. We commonly used cyanine dyes are Cy3 and Cy5 which of lipids on the nuclear environment, emphasizing This modulation leads the chromosome structure to also analyse the impact regarding the recent changes fluoresce in green and yellow, respectively. Both of them its role on chromatin architecture. We postulate that lose some compartments associated with the long in the human genome reference sequences. are formed by a main polymethine chain (three atoms of lipids that bind to nucleosomes and affect chromatin genomic distance contacts. These preliminaries finding carbon for Cy3 and five atoms for Cy5), two indole rings states are likely to be worth investigating as tools suggest that the local (short genomic distances) Our work was financial supported by Fapemig, CNPq and two nitrogen atoms. The fluorescence intensities of to modify disease phenotypes at a molecular level. interactions play an essential role in the condensation and Capes. these markers can be analyzed applying the Peyrard- Moreover, focusing on lipids that bind to nuclear process which more studies are necessary for a better Bishop (PB) mesoscopic model. Using this model, we receptors, clinically relevant transcription factors, comprehension of this transition. can estimate the stacking interactions and hydrogen we are exploring the interactions of the nucleosome bonds parameters for these cyanine dyes attached to with steroid hormones, bile acids and fatty acids. This work was supported by the Center for Theoretical sequences of DNA or RNA. The PB model considers Preliminary molecular docking results suggests that Biological Physics and funded by the National Science the stacking interactions and the hydrogen bonds other lipids chemotypes may also impact chromatin Foundation (PHY-1427654). VGC acknowledges the São separately. In that way, we believe that it is possible to structure through binding to common cholesterol sites Paulo Research Foundation – FAPESP (2016/13998-8 verify and improve existing techniques such as DNA on the nucleosome. and 2017/09662-7). microarrays using the PB model and data sets collected experimentally. In our work, we used a published data set This work was supported by Conselho Nac. Des. Cient. of fluorescence intensities both for Cy3 and Cy5 attached Tecnologico (CNPq) to 5’ terminal of 1024 distinct sequences of DNA. We

108 109 F08 - Structure and Function of RNA substrate RNA. We also show that Mettl16, another Protein Folding Misfolding and Unfolding Methyltransferases m6A writer, uses a distinct mechanism to recognize and bind the RNA substrates. We determine the Yunsun Nam molecular basis for why the RNA methyltransferases G01 - PHYSICAL-CHEMICAL STUDY OF G02 - Speeding Up Protein Folding by Non-Native manifest distinct substrate specificities. Moreover, β-TRYPSIN IN AQUEOUS-ORGANIC MEDIA Cooperative Effects N6-methyladenosine (m6A) is an abundant, we show that a polypeptide in the catalytic domain reversible chemical modification that regulates of METTL16 can undergo a conformational change, Dayanne P. Rosa; Fernando B. da Silva1 function and stability of many types of RNAs. and that it can act as an autoregulatory switch for Mariana T. Q. Magalhães; Alexandre M. C. Santos Vinícius G. Contessoto,1,2 Vinícius M. de Oliveira,1 We use biochemical and structural methods to the methyltransferase activity. Furthermore, we 3 4 5 Postgraduate Program in Biochemistry and Jane Clarke, Luis A. Campos, Víctor Muñoz and elucidate how m6A marks are generated by RNA reveal how disease mutations of m6A writers lead Vitor B. P. Leite1 methyltransferases. We show that Mettl3 and Mettl14 to aberrant methyltransferase activity. Together, Immunology, UFMG, Belo Horizonte, MG. 1 cooperate to recognize and catalyze modification our study shows that each m6A writer enzyme ([email protected] and alexandre. IBILCE, São Paulo State University, S. J. Rio 2 of target RNAs. Mettl3 is the catalytically active has evolved to modify a specific set of RNAs with [email protected]) Preto, Brazil ([email protected]). Brazilian. enzyme, while Mettl14 has a structural role to controlled efficiency. Bioethanol Science and Technology Laboratory, 3 support the necessary conformation and to bind the The study of water-soluble proteins in organic media is Campinas, Brazil. Department of Chemistry, 4 an important area of molecular biophysics. The transfer University of Cambridge, Cambridge, UK. National 5 of proteins for media containing some amount of Biotechnology Center, Madrid, Spain. Univeristy of organic solvent brings some advantages, such as better California, Merced, USA. understanding of folding and unfolding processes, improvement of substrate specificity of enzymes, Recent experimental studies have shown large folding which are some of the crucial aspects for an efficient time variations in very similar protein homologs. These industrial process. In this context, we are searching changes have also been observed in specific protein a better understanding of influence of organic solvent mutations. Such results have been attributed to the on β-trypsin, an isoform that is well characterized variation of internal friction during the folding process, only in aqueous media. The β-trypsin isoform was but these friction effects are still unclear. In the present obtained by purification of commercial bovine trypsin study, we address this question using a Structure- by ion exchange bench top chromatography. The Based Model (SBM) with an addition of electrostatic enzymatic activity assays were performed with five and hydrophobic non-native interactions. The first solvents: methanol, ethanol, n-propanol, acetonitrile, set of proteins investigated was R15, R16, and R17 and DMSO. The thermodynamic assay was performed of α-spectrin, which have been studied by Clarke using fluorescence spectroscopy. The activity assay group. Experimental results indicate that R15 folds indicates that, between the solvents, the best would be and unfolds three orders of magnitude faster than its ethanol. The fluorescence results demonstrate that has homologs (R16 and R17). Our computational results occurred an increase of the emission intensity and a have shown a qualitative agreement in folding time with displacement of wavelength for the infrared region with the experimental ones, which suggest that the origin of the ethanol concentration increase. Thus, our results this anomalous observation arises from a cooperativity suggest a process of unfolding followed of protein effects of non-native interactions. We have applied the aggregation over some concentrations of organic same model to the Chymotrypsin inhibitor 2 (CI2) and solvent. its mutants. Experimental results have shown that the mutated CI2 is able to fold much faster than the wild- This work was supported by Cons. Nac. Des. Cient. type one. These CI2 mutations lost thermal stability Tecnológico (CNPq). when they are compared to the wild-type. Again, we have been able to obtain a good agreement with the experimental results. This agreement emphasizes the importance of hydrophobic and electrostatic non- native interactions in protein folding playing a crucial role in the folding time.

This work was supported by Conselho Nac. Des. Cient. Tecnologico (CNPq # 141715/2017-0)

110 111 G03 - De Novo Design and Characterization of G04 – INITIAL CHARACTERIZATION OF CLASS C G05 - Unveiling the role of surfactants on G06 - PREDICTION OF KINETICS OF PROTEIN HIV-1 Antibody Epitope Grafted into the Top7 DNAJ FROM Aedes aegypti amyloid-like protein self-assembling FOLDING WITH NON-REDUNDANT CONTACT Protein Scaffold INFORMATION Guilherme G. Costa1 Scanavachi, G.¹ Gabriel G. Slade1* Luciano Censoni1 Glaucia M. S. Pinheiro1 and Carlos H. I. Ramos1 Espinosa, Y. R.²; Ruso, J.³; Itri, R.¹ Isabelle F. T. Viana2, Danilo Coêlho2, Roberto Lins2 Leandro Martínez1 1Institute of Chemistry, University of Campinas, ¹Crystallography Laboratory, Department of Applied and Ronaldo J. Oliveira1 Campinas, SP ([email protected]) Physics, University of Sao Paulo, Sao Paulo, Brazil. 1Institute of Chemistry, University of Campinas, 1Laboratório de Biofísica Teórica, Departamento de ²Institute of Physics of Liquids and Biological Campinas, SP ([email protected]) Física, Universidade Federal do Triângulo Mineiro, The highly conserved Hsp70 system plays essential Systems (CONICETUNLP), La Plata, Argentina. 2 Uberaba, MG, Instituto Aggeu Magalhães, FIOCRUZ, roles in diverse cellular functions, such as protein ³Soft Matter and Molecular Biophysics Group, In protein structures, the majority of the distances Recife, PE, Brazil. (*[email protected]) translocation, proteolysis and prevention of misfolding Department of Applied Physics, University of between residues are correlated given a fixed topology. or induced aggregation by cellular stress situations. Santiago de Compostela, Santiago de Compostela, We exploit this notion in order to develop a model that The designed Top7 protein is a suitable scaffold to Additionally, this system delivers client-proteins to Spain. [email protected] predicts structures’ experimental folding rates, which provide the required structural stability to predetermined other molecular chaperones that together participate are known to vary over several orders of magnitude and target shapes. There have been many attempts to graft in a complex crucial for proteostasis. Hsp70 main co- Some proteins can undergo structural changes that depend on the fold’s complexity, while considering only a functional sequences into stable protein scaffolds with chaperones, which belong to the Hsp40 family, act by may trigger an aggregation process where they self- small, uncorrelated subset of the structure’s inter-residue the resulting molecule working as novel recognition recognizing and delivering client-proteins to it while assembly into highly ordered aggregates called amyloid contacts. To estimate the complexity of a given structure’s modules, inhibitors, enzymes or immunogens. In this stimulating its ATPase activity. Since details related fibers. In vivo, these amyloid fibers are related to more fold, we fit the protein chain to a self-avoiding random walk work, the HIV-1 epitope (E2F5) was grafted into Top7 to the function and interaction between Hsp70 and than 25 different diseases, some of them are lethal as model, and derive a probability distribution for the distance scaffold resulting in a protein with enhanced stability Hsp40 remain unclear, studies targeting its structural Creutzfeldt-Jakob disease and others can lead a person between pairs of amino acid residues as a function of their and high affinity for a broadly neutralizing antibody. It and functional investigation are of great interest in the to incapacities, as diabetes type II, Alzheimer’s and separation along the sequence. We define an expression was also created biotinylated Top7’s by attaching Biotin area of proteostasis. The Hsp40/DnaJ co-chaperones Parkinson’s diseases. With the aim of understanding for the probabilistic Information content associated to in the Lysine residues in which biotinylation provided are classified in three distinct classes: A, B and C, the conditions and mechanisms by which proteins the relative position of a residue pair, and then show that additional structural stability. Within this context, Top7 according to the position of their J-domain (a signature form amyloid fibers, we mixed bovine serum albumin the average information content of all residue pairs in a and their variants (Top7-E2F5 and Top7-E2F5-Biotin) of this class of proteins). Whilst classes A and B DnaJs (BSA) at pH 3.7 with sodium dodecyl sulfate (SDS) structure, taken as an estimate of its complexity, is well had their folding mechanisms and thermodynamic have a J-domain located at the N-terminus, class C and sodium perfluorooctanoate (SPFO) to induce the correlated to the logarithm of its folding rate. Furthermore, stabilization characterized by employing the structure- DnaJs would have a J-domain located either at the amyloid fibers formation. BSA conformational changes we show that the same information content measure may based model (SBM). SBM’s are largely employed to center or at the C-terminus. Although extensive data were followed in order to suggest a possible pathway be exploited to rank, identify, and discard a large set of study the dynamical properties of the ensemble of have been published, class C DnaJ proteins still lack of aggregation. Dynamic Light Scattering (DLS) and redundant observations, allowing the prediction of the structures of many biological problems such as protein further characterization, which reasoned our selection Thioflavin T fluorescence data revealed, respectively, structure’s folding rate with similar accuracy while taking folding, functional changes and protein diffusion of the Aedes aegypti DnaJC8 protein. DnaJC8 was the presence of large aggregates and the formation into account less than 5% of its contacts. effects. Besides that, one important feature to the Top7 expressed in Escherichia coli BL21(DE3) strain, and of amyloid-like fibers as the surfactant concentration stabilization and antibody interaction is electrostatics. a purification methodology involving affinity and gel increased, whereas circular dichroism (CD) showed This work was supported by FAPESP (grants Electrostatics plays a key role in biomolecule recognition, filtration chromatography was developed, hence that BSA second structure changes from α-helix to 2010/16947-9, 2013/05475-7 and 2013/08293-7), thermostabilization and in silico protein. Thus, a fast obtaining the protein with high-level of purity. As β-sheet. Transmission electron microscopy (TEM) CNPq (grant 470374/2013-6) and graduate fellowships algorithm was used to obtain the electrostatic energy of verified by circular dichroism spectropolarimetry, permitted us to obtain images of fibers and aggregates from CAPES. each charged residue for the whole protein electrostatic the protein was produced folded and stable up to in the micrometers scale. Further, small angle x-ray energy stabilization. Electrostatic were calculated 42°C, with a secondary structure composition of scattering (SAXS) measurements provided information G07 - Effects of Ionizing Radiation on Fluorescent by implementing the Tanford-Kirkwood model with about 34% of α-helix. SEC-MALS analysis indicated about the protein’s quaternary structure as a function Proteins a Solvent Accessibility (TKSA) methodology. The that the protein was a monomer. Further structural of surfactant concentration and a more detailed difference in stability, flexibility and antibody recognition characterization by other biophysical techniques, analysis allowed us to suggest a pathway of fibrillation Mariana Chaves Micheletto1 of Top7 and their variants was also evaluated by the such as dynamic light scattering and fluorescence process. Lastly, we performed molecular dynamics Eder José Guidelli2, Antonio Jose Costa Filho1 techniques circular dichroism, ELISA assays, flow assays, as well as the measurement of the chaperone (MD) simulations to obtain an all atom structure of BSA 1 2 cytometry and Molecular Dynamics. The results show activity, are underway. at pH 3.7 to study the influence of SDS and SPFO in the Laboratório de Biofísica Molecular, Laboratório that theory allied with experimental techniques is able BSA conformation and we also used the MD structures de Ressonância Magnética. Faculdade de Filosofia, to engineer vaccine antigens with enhanced protective This work was supported by FAPESP (2012/50161-8), to modeling the SAXS experimental curve Ciências e Letras de Ribeirão Preto. Universidade antibody response. CNPq and Coordination for the Improvement of Higher de São Paulo Education Personnel (CAPES #1766206). This work was supported by CAPES and by the Santander This work was supported by CNPq and FAPEMIG. Mobilidade. Photodynamic Therapy (PDT) has gained relevance due to its applications in cancer treatments and has now stood out for its variety of applications, such as in the fight against neoplasic diseases, inflammatory diseases, microbial infections and aging. The discovery

112 113 and development of phototoxic proteins capable of the parameters choice, such as acceptance of new This work was supported by Fundação de Amparo à physics. The failure of a protein to achieve the correct producing reactive oxygen species aligned with the conformations, qA, visitation, that controls the search in Pesquisa do Estado de São Paulo (FAPESP 00839-1) folded native state can cause a series of pathological development of Optogenetics makes it possible to use the hypersurface, qV, thermalization, responsible for the conditions like neurodegenerative disorders. Recent genetically encoded photosensitizers, increasing the cooling, qT and the artificial temperature, T.We performed G10 - In silico comparative study of human and studies have associated the absence of bound specificity of the treatment. However, when it comes a parameter sweep in order to obtain the ideal values porcine amylin metals (Apo protein) in the Cu-Zn Human Superoxide to in vivo use, optical techniques are limited by the for the antimicrobial peptides used in the study. The Dismutase (SOD1) with Amyotrophic Lateral Sclerosis low penetration of UV-visible light into biological structures were evaluated by DOPE score and g-factor Nelson A. Alves† (ALS) disease, suggesting mechanisms of SOD1 tissues. To overcome this limitation, the use of X-rays and then the best ones were selected and refined using Rafael B. Frigori‡ aggregation. Using the Ca structure based-model and has been proposed as energy source of excitation of molecular dynamics. The structures obtained presented modifying the energy of interaction between the amino † photosensitizers, due to their high penetrability in soft good agreement with the experimentally solved structure Departamento de Física, FFCLRP, Universidade de acids that are in the metal-binding site, it was possible tissues. When X-rays are used as a source of energy presenting RMSD values below 3 Å. The GSA showed São Paulo, Avenida Bandeirantes, 3900. Ribeirão to observe some differences between the folding ‡ in PDT and Optogenetics, these techniques are called efficiency in problems of PSP and in the decrease the Preto 14040-901, SP, Brazil. Universidade process of the apo and holo protein. Our results suggest XE-PDT (X-ray excited PDT) and X-optogenetics, computational time in the search for the global minimum Tecnológica Federal do Paraná, Rua Cristo Rei that difficulting the metal binding site formation can respectively. Considering the use of proteins as energy conformation and in the optimization of the 19. Toledo 85902-490, PR, Brazil.alves@ffclrp. change the SOD1 thermodynamic stability, the folding photosensitizing molecules also in the application of molecular geometry. usp.br, [email protected] pathway and the SOD1 folding kinetics, hence SOD1 these innovative techniques, this work presents a study becomes less stable and propense to form secondaries of the effects of ionizing radiation on photosensitive This work was supported by: CAPES, CNPq, FAPERJ Islet transplantation is a promising treatment for type 2 structures that are related with aggregation. We also proteins, GFP, KillerOrange and KillerRed. For this diabetes, however its success is impaired by progressive performed simulations hindering the formation of purpose, the structure, stability and quantum yields of G09 - LUMINESCENT PROBE CANDIDATE TO graft loss, likely due to cytotoxic aggregation of the C57-C146 disulfide bond. The disulfide bond, as fluorescence and generation of reactive oxygen species MONITOR THE AGGREGATION OF THE AMYLOID hormone human islet amyloid polypeptide (hIAPP), or well as the metal binding site, is responsible for the were evaluated after undergoing radiation. Strategies BETA PEPTIDE FOR EARLY DIAGNOSIS OF amylin, secreted by the endocrine pancreas. Alternatively, decrease of the native stability, but it has less effect of increasing the interaction of ionizing radiation and ALZHEIMER’S DISEASE the effectiveness of porcine xenotransplantations might on the monomeric SOD1 kinetics and folding pathway. these biomolecules aiming to improve the efficiency of be explained by the fibrillization-resistance of the porcine Furthermore, including all-atom simulation and the the techniques above will be discussed. Mariana P. Cali1 mutant (pIAPP). To better elucidate such molecular adaptive biasing force, we search to elucidate some Rose M. Carlos1 mechanisms, we performed comparative replica- questions about SOD1 dimerization process, and how This work was supported by FAPESP, CNPQ and CAPES exchange molecular dynamics (REMD) simulations it can be affected by the absence of metals. 1 Department of Chemistry, Federal University of both IAPP isoforms. The accurate force field of São Carlos, São Carlos, SP (marianapcali@ Charmm22* with explicit aqueous solvation TIP4P/Ew This work was supported by Coordenação de G08 - APPLICATION OF THE AB INITIO METHOD hotmail.com) GENERALIZED SIMULATED ANNEALING IN ensure a minimal structural bias around physiological Aperfeiçoamento de Pessoal de Nível Superior (CAPES) PROBLEMS OF 3D STRUCTURE PREDICTION OF temperatures. Along which, the peptides are shown to ANTIMICROBIAL PEPTIDES Alzheimer’s disease is the most common cause of present no structural-phase transition of folding from G12 - LUMINESCENT RU(II) COMPLEXES AS dementia in elderly people and it is responsible for a microcanonical thermodynamics perspective. We SENSITIVE PROBES FOR Aβ AGGREGATION Freire Ribeiro, M¹ theprogressive decline of their cognitive and motor observe a direct α-helix → β-sheet thermal conversion Rose M Carlos1 Fernandes T. V. A.¹, Pascutti, P. G.¹ functions. Once the disease has progressed, amyloid during the folding of hIAPP, which is absent in pIAPP. The beta aggregates are found in the patient’s brain and are amyloidogenic segment 20-29 in hIAPP, which hosts Mariana P. Cali1 ¹Instituto de Biofísica Carlos Chagas Filho, known to disturb the normal brain function. With that 5 out of the 10 overall mutations found in pIAPP, is 1 Universidade Federal do Rio de Janeiro, Rio de in mind, a ruthenium metal complex was developed strongly depleted of β-sheet structures in such isoform. Department of Chemistry, Federal University of São Janeiro, RJ ([email protected]) to, with its different sets of ligands, act as a probe to Hydrogen bond analysis revealed a predominant Carlos, São Carlos, SP ([email protected]) monitor in real time the aggregation process of amyloid frequency of contacts in the residue range 17-22 for Experimental methods such as x-ray diffraction and beta through its luminescence. The initial luminescence pIAPP, in which two mutations occur, whereas the same Alzheimer’s disease (AD) is a devastating nuclear magnetic resonance (NMR) are, currently, the studies showed that our ruthenium complex does hIAPP protofilament showed a β-turn. These features of neurodegenerative disease that affects mainly older standard to determine protein structure. Despite being in fact bind to the amyloid beta protein from early pIAPP anticorrelate with the presence of a well-known people. It is demonstrated that soluble monomer the best to generate a 3D structure of a protein, they have stages of aggregation to fibril formation. The intrinsic β-sheet rich monomeric state that in hIAPP acts as an of amyloid-beta (Ab) aggregates into low-ordered limitations, high financial costs and are time consuming, fluorescence of the peptide is increased significantly in intermediate inducing oligomerization. structures form pore-like structures. These oligomers which justifies the development of computational the presence of ruthenium complex. The aggregation bind to the membrane of the neuron, acting as methods of protein structure prediction (PSP). The process of many fragments of the Ab1-40 peptide– Ab1- G11 - STUDY OF THE SUPEROXIDE DISMUTASE secondary ion channels, causing Ca(II) homeostasis Generalized Simulated Annealing (GSA) is a ab initio 28, Ab15-21, Ab11-22, Ab25-35 and Ab29-40 were tested FOLDING PROCESS USING MOLECULAR that lead to synaptic loss and toxicity. Subsequent method of PSP that consists in a stochastic algorithm with the complex. Our preliminary studies through DYNAMIC TECHNIQUES reports have described roles of larger oligomers that that uses physical models that only considers the forces emission lifetime have shown that there the complex is aggregates to form protofibrills and have implications 1 that act in the interactions between the proteins atoms able to interact more significantly with the Ab11-22 and Paulo Ricardo Mouro on neurotoxicity and memory loss in AD. Whereas the in order to optimize the molecular geometry. The goal of Ab1-28 and less significantly with Ab25-35 and Ab29-40. It Vitor B. P. Leite1 and Jorge Chahine1 importance of these results has renewed the interest in the present study was to use the GSA method alongside is possible to see that the aggregates increase in size the solution-phase of Ab, it has also brought significant 1 with molecular dynamics to obtain the 3D structure of with the incubation time and tend form plaques. Fibrils Department of Physics, São Paulo State University, challenges to researches. Consequently, strategies antimicrobial peptides. The GSA is based on the Monte could be seen prominently in 120 minutes of incubation São José do Rio Preto, S.P. ([email protected]) to follow the early stages of Ab aggregation must be Carlo method and in the Tsallis thermostatistics. It whereas in 240 minutes the fibrils tend to form fibril- very sensitive. A recent study from our laboratory has involves thermodynamics and optimization process, plaques. When the complex was added in the 1:1 The understanding of the underlying folding mechanism addressed the development of Ru(II) complexes with where, from the gradual cooling of the system, the proportion is possible to see a significant difference in of a protein to its functional compact three- dimensional theranostic properties such as antioxidant and anti- global energy minimum is reached. It is sensitive to the size of aggregates in 240 minutes of incubation. structure is one of the greatest challenges of biological acetilcholinesterase activity, and they were a sensitive

114 115 probe to early stages of Ab aggregation. These findings PrP (PrPC) is a cell surface glycoprotein; however, it Protein Structure dynamics and Functions motivated us to use the luminescent Ru(II) complex may undergo structural changes, being converted into and the TCSPC-FLIM technique in combination PrP scrapie (PrPSc), which is pathogenic, infectious with TEM and DC to capture real-time images of the and is associated with TSEs development. Although the H01 - STRUCTURAL AND FUNCTIONAL STABILITY C Sc H02 - RETINOIC ACID BINDING SUPPRESSES structural changes produced in the early stages of PrP to PrP conversion mechanism is not completely OF ASPARAGINASE BY IONIC LIQUIDS CRABP2 MICRO-MILLISECOND DYNAMICS: the aggregation process of Ab1-40 and its fragments: understood, it is proposed that an accessory molecule Agnes Magri1 IMPLICATIONS TO THE MECHANISM OF LIGAND Ab1-28 from N-terminus, Ab29-40 from C-terminus, and is required to modulate the conversion. Nucleic acids ENTRY INTO THE CAVITY 1 Ab11-22 from central region. Our study contributes to and copper [Cu (II)] ions have been reported to Jorge F. B. Pereira understanding of the aggregation and toxicity of Ab affect PrP aggregation when tested separately. PrPC Anderson S. Pinheiro1 1Department of Bioprocess, School of by targeting, in real time, its N- and C- terminal and coordinates Cu(II) in vivo and may interact with nucleic Carolina Lixa1, Anwar Iqbal2, Fabio C. L. Almeida2 central- regions in the early stages. This knowledge can acids in physiological conditions. As interaction with Pharmaceutical Science, State University of São 1 facilitate the development of luminescent probes for the the isolated ligands (Cu(II) and nucleic acids, both RNA Paulo, Araraquara, SP ([email protected]) Department of Biochemistry, Institute of diagnosis and treatment of AD. and DNA) has been deeply investigated, we aimed to Chemistry, Federal University of Rio de Janeiro, Rio 2 evaluate whether the combination of both ligands, DNA L-Asparaginase (ASNase) is an antileukemic enzyme de Janeiro-RJ ([email protected]) and Institute of This work was supported by CNPq 306005/2017-4 and and Cu(II), could affect PrP conversion to scrapie- that present an effective antileukemic activity commonly Medical Biochemistry, Federal University of Rio de FAPESP (2017/00839-1). like forms and the study may help understanding the applied to ALL treatment in children. In fact, one of major Janeiro, Rio de Janeiro-RJ conversion mechanisms. We used as a model murine barriers to pharmaceutical industry is the structural G13 - EVALUATION OF PRION PROTEIN recombinant PrP (rPrP) and a nucleic acid that has modifications in proteins applied as therapeutic agents. Cellular Retinoic Acid Binding Protein 2 (CRABP2) INTERACTION WITH CU(II) AND NUCLEIC ACIDS high affinity for rPrP binding (21-mer DNA). The effects Consider this, and due to proteins are sometimes binds and delivers retinoic acid (RA) to Retinoic Acid of both DNA and Cu(II) in the secondary structure, unstable when handled in vitro, stabilizing agents are Receptors, activating transcription of anti-oncogenic Yulli M. F. Passos1 oligomerization profile and rPrP aggregate morphology extremely necessary component to ensure their long- genes. Crystallographic structures of CRABP2 reveal Natalia C. Ferreira, Bruno Macedo, Mariana P. B. were investigated by FTIR, CD and TEM. Our results term stability. In these context, Ionic liquids (ILs) exhibit minimal differences between its apo (ligand-free) and Gomes and Yraima Cordeiro1 indicate that rPrP aggregation is induced by DNA and a high potential as a solvent media for biocatalysis and holo (ligand-bound) forms, suggesting that dynamics Cu(II) and the heterogeneous aggregates formed are a stabilization processes due to their unique features, as must play an important role in the ligand recognition 1 Faculty of Pharmacy, Federal University of Rio de result of both ligands combined interaction. It was also high selectivity, high solvation capability and, recently, process. Here, we set out to characterize both apo- and Janeiro, Rio de Janeiro, RJ, Brazil (yullipassos@ seen content changes on secondary structure of rPrP. protein super activity ability. Considering that the holo-CRABP2 dynamics, at different time scales, to gmail.com) Although we can provide preliminary evidence that DNA maintaining of native protein fold is essential for their shed light into the mechanism of RA entry into CRABP2 and Cu(II) interact at the same time with rPrP, we still enzymatic function, the use of ILs can be interesting, cavity. Using heteronuclear multidimensional NMR, we Transmissible spongiform encephalopathies need to prove the formation of PrP:Cu(II):DNA ternary maintain intact its structure and catalytic function. Thus, unambiguously assigned 90% and 95% of apo- and (TSEs), also known as prion diseases, form a complexes. this work proposed to evaluate the stability capability holo-CRABP2 backbone resonances, respectively. group of neurodegenerative and fatal diseases that of cholinium-based IL on ASNase activity, varying CRABP2 interacts with RA extensively, through residues affect mammals. Its pathology is associated with This work was supported by FAPERJ and INBEB-CNPq. the anion chain length (chloride, acetate, propanoate, located in sheets βC and βD, α1-loop-α2 domain, and conformational changes in prion protein (PrP). Cellular butanoate and hexanoate). ASNase remained active in loops forming the portal entrance to the cavity (βC- in all compounds evaluated, and the anion alkyl chain βD, βE-βF, βG-βH, and βI-βJ), as probed by NMR length increase as well as the increase in temperature chemical shift mapping. We show by 15N auto-correlated were not favor ASNase activity. [Ch]Cl have induced relaxation measurements that residues Asn14 (βA- the highest ASNase activity with stabilization of alpha- α1), Arg59 (βC-βD), Lys101-Gly104 (βG-βH), and helix secondary structure and different mechanisms Glu137 (C-terminal) undergo fast backbone dynamics,

of stabilization from the concentration of 0.025 mol[Ch] both in the free and RA-bound states, suggesting that -1 Cl.moltotal was observed. The molecular docking RA binding does not lead to changes in CRABP2 ps- analysis provided support to these data and have ns motions. 15N CPMG-type relaxation dispersion shown that [Ch]Cl influences the ASNase structure experiments for apo-CRABP2 identified residues in at all concentration of [Ch]Cl by ligation of anion in sheets βC and βD, α1-loop-α2 domain, and in the special regions in ANSase that expose the active site. βE-βF loop as undergoing conformational exchange. The series of cholinium-based IL seems to be effective Interestingly, RA binding suppressed CRABP2 µs- alternative solvents for further biopharmaceuticals ms dynamics. These results provide evidence for the stabilization. existence of a low-populated, higher energy state for free CRABP2, which is RA-binding competent. This work was developed within the scope of the project Young Researcher Project (FAPESP 2014/16424-7). A. This work was supported by FAPERJ, CNPq, CAPES, Magri acknowledge the funding from CNPq. and Brown University.

116 117 Ct H03 - COMBINING FREE-ENERGY CALCULATIONS into the structure and dynamics of IDPs. VirB9 is a interact with charged side chains (aspartate) where broth (TB), auto-induction medium and high-density AND NMR EXPERIMENTS TO DETERMINE component of the type IV secretion system (T4SS) from the presence of the ion can alter the activity/structural medium (HDM)). The best yield of soluble protein was Ct TRANSIENT INTERACTIONS IN CDC25B the phytopathogen Xanthomonas citri. VirB9 behaves as stability of the protein. At higher concentrations the achieved in BL21(DE3)+pRARE2 in HDM. The protein PHOSPHATASES an IDP in solution, however, it participates in the assembly ions interact strongly with the protein shell hydration, sample quality (monodisperse, polydispersity<30%) of the T4SS making important contacts with its also IDP therefore unstable it. At higher concentrations the high was evaluated by dynamic light scattering (DLS) André Anversa Oliveira Reis1 partner VirB7Nt, and forming a stable and rigid complex. charge density anions compete for water with the protein and submitted to crystallization trials. Crystallization Raphael Santa Rosa Sayegh2 and Guilherme Here we used CEST NMR experiments to investigate as it unfolds increasing the Gibbs free energy required to conditions to Pyrearinus termitilluminans (lithium Menegon Arantes3 the transient conformations that VirB9Ct populates when hydrate the newly exposed core of the protein therefore sulfate and Hepes) and Phrixotrix hirtus (polyethylene alone in solution. Such a state is diminished in the stabilizing the structure. glycol and tri-potassium citrate) were obtained and its 1,2,3 Department of Biochemistry, University of São presence of VirB7Nt as observed in our CEST profiles, optimization in presence of luciferin analogs or metals 1 Paulo, São Paulo, SP ( [email protected] and where the addition of VirB7Nt leads to a decrease in the This work was supported by UFES, CAPES, CNPQ and for X-ray diffraction are being evaluated. 3 [email protected]) ligand-free state signal. Structure information derived FAPES. from different techniques (fluorescence and circular This work was supported by The São Paulo Research Tyrosine phosphatases CDC25B act on cell cycle dichroism spectroscopies, and differential scanning - Structural studies of luciferases from Foundation, FAPESP (Grant number# 18/02538-1). Ct H06 checkpoints and have been considered potencial calorimetry) showed that VirB9 has secondary and beetles and related enzymes targets for drug development since their deregulation tertiary structures, suggesting that it does not behave H07 - Structural determination of NS2B protein Atílio Tomazini Júnior1,2 has been linked to cancer progression. Previous as a random coil polymer but rather as a molten globule. Zika virus and interaction studies with NS3 kinetic experiments with CDC25B have shown that CEST experiments recorded at sub-stoichiometric Vadim Ravara Viviani1 and Mário Tyago Murakami2. viral protease: The search for new antiviral the disordered C-terminal region is involved in binding molar ratios of VirB9CT and VirB7NT, yielded 15N and 13CO 1 compounds of competitive inhibitors. Here, free-energy profiles chemical shift information on the free state of VirB9Ct, Centro de Ciências e Tecnologias para a determined from enhanced sampling of molecular as well as exchange rates between the free and bound Sustentabilidade (CCTS). Universidade Federal de Penna, B. R.1 São Carlos (UFSCAR). Sorocaba, SP and 2Centro dynamics simulations suggested transient cation- forms. The introduction of amino acid substitutions Anobom, C. D.2; Valente, A.P.1 Nacional de Pesquisa em Energia e Materiais pi interactions that may be important for CDC25B indicated that VirB9’s intrinsic flexibility is important for 1 structure and inhibitor binding, in particular between binding to VirB7. Constructs of VirB9 designed to be less (CNPEM), Laboratório Nacional de Ciência e Institute Medical Biochemistry, Federal University residues W550 in the C-terminus and R482, in the flexible than the wild type protein, displayed lower affinity Tecnologia do Bioetanol (CTBE), Campinas, SP of Rio de Janeiro, RJ (pennabeatriz@gmail. 2 protein core. Chemical shift perturbations of 1H-15N for VirB7Nt according to isothermal titration calorimetry ([email protected]) com) and Departament of Biochemistry, Federal – HSQC NMR spectra were measured in W550A and experiments. University of Rio de Janeiro, RJ R482A point-mutants leading us to experimentally In beetle luciferases, the structural determinants of confirm the importance of this transient interaction, in Acknowledgments: This work was supported by FAPESP bioluminescence spectra and pH sensitivity are still The Zika virus (ZIKV) became an important global agreement with the free-energy calculations. 2011/07777-5. A.D. receives a CNPq PhD fellowship. unclear. The understanding of the molecular mechanisms public health concern due to the Zika outbreak in that allow the bioluminescence and the development of 2015. ZIKV infection can cause severe neurological This work was supported by Conselho Nacional de H05 - ENZIMATIC STABILITY OF β-TRIPSIN AS A potential biotechnological and biomedical applications diseases such as, fetal microcephaly and Guillain-Barré Desenvolvimento Cienífico e Tecnológico (CNPq # FUNCTION OF ION CONCENTRATION can be effectively increased through the combined syndrome in adults. In response, the structural virology 141683/2016-3) and by the Fundação de Amparo à use of biophysical techniques (such as circular field has explored the characteristics of ZIKV providing Pesquisa do Estado de São Paulo (FAPESP # 16/24096-5). Antonio Victor B. Vasconcelos1* dichroism spectroscopy, small angle X-ray scattering significant information about the pathogenesis and in Jamil S. de Oliveira2, Dayanne P. Rosa1, Evaldo V. and macromolecular crystallography) and enzymology. the identification of novel targets for the design of new H04 - Biophysical studies of the T4SS subunit Pereira1, Fabiano T. Cruz2, Juliana B. B. Coitinho3 Within this context, we have been characterizing distinct drugs. The ZIKV NS2B-NS3 protease has a crucial role VirB9 by NMR spectroscopy and Alexandre M. C. Santos1. luciferases including the green light-emitting from in virus replication which make it an attractive target for Pyrearinus termitilluminans, the red light-emitting from antiviral drugs. Despite great efforts, several structural 1 1 Angie Dávalos Postgraduate at Biotecnology, Federal University of Phrixotrix hirtus and the pH-sensitive from Macrolampis components of ZIKV, and other flaviviruses, remains Espirito Santo, Espírito Santo, ES. 2Postgraduate at 1Marcus V. C Cardoso, 2Denize Fávaro, 3José D. sp in the presence of luciferin analogs or metals. unknown, especially, the transmembrane domains of Biochemistry and Imunology, Federal University of Rivera, 1Caroline Dutra, 1Gustavo Carretero, 4Roberto The objective of this research is to contribute to the the non-structural proteins, such as, the NS2B protein, Minas Gerais, Minas Gerais, MG. And 3Postgraduate D. Lins, 4Carlos Cruz, 1Chuck S. Farah and 1Roberto K. molecular elucidation of bioluminescence of luciferases the target of this study. All structural information related at Biochemistry, Federal University of Espirito Salinas [email protected], [email protected]. from beetles and related enzymes. It is expected that by to these transmembrane domains will be of great Santo, ES. *[email protected] 1 comparing the three-dimensional structures obtained in relevance for understanding the pathogenesis of ZIKV Department of Biochemistry, Institute of Chemistry, this study and those described in the literature we can and other flaviviruses. Thus, the aim of this project is University of São Paulo, São Paulo, 05508-000, Brazil. Bovine beta trypsin was purified by cationic bench top identify the residues and catalytic groups responsible the structural determination of transmembrane protein 2Institute of Chemistry, State University of Campinas, 3 chromatography and purity was confirmed by mass for oxygen activity and how they influence the emitted NS2B free and bound to NS3 protease domain of ZIKV São Paulo, 13083-970, Brazil. Institute of Astronomy, spectrometry. The effect of salts of hoffmeister series light spectrum. The ultimate goal is to rationally apply using Nuclear Magnetic Resonance (NMR) in solution. Geophysics and atmospheric sciences, University of São 4 was investigate on enzyme activity and structural this knowledge to modulate the catalytic properties of The structural studies of membrane proteins by NMR Paulo, São Paulo, Brazil. Aggeu Magalhães Research stability. Anion and cation effects on the enzymatic and these enzymes for biotechnological and biomedical are still a challenge, however, recently the use of smaller Center, Fiocruz, Pernambuco, Brazil structural stability of trypsin isoform were performed by applications. The heterologous expression of the phospholipid nanodiscs allied to specific isotopic UV and fluorescence spectroscopy. Our results show luciferases and protoluciferase were performed in labeling strategies and NMR optimization became this Intrinsically disordered proteins (IDPs) display high that at low concentrations (<50 mM) anions and cations cold shock expression system pCold II (Takara Bio strategy possible. We expressed NS2B ZIKV in E. coli degree of flexibility and lack a defined 3D structure in alter the activity/stability of trypsin but they do not follow Inc.) using four different Escherichia coli expression BL21(DE3) and refolding and purification protocols contrast with globular proteins. Due to their large flexibility, the Hofmeister series. At higher ions concentrations, strains BL21(DE3)+pRARE2, Rosetta-gami, Shuffle has been performed to initiate the structural studies. IDPs are ideally suited to be studied by solution NMR protein stabilization follows the well‐established and C41(DE3). The transformed cells were cultivated in The interaction and dynamics studies of NS2B/NS3 spectroscopy. NMR has indeed provided critical insights Hofmeister series. At low concentrations, the cations different culture medium (lysogeny broth (LB), Terrific will allow the screening of leading compounds through

118 119 fragment libraries and phage display searching to the that interacts directly with VEGFR3-D2, as well, the H10 - Protein-nanodisc model: A new perspective model can be discriminated from alternative models development of new antiviral compounds. region of the protein that interacts with this inhibitor. to study membrane active peptides derived from artificial MSA with disrupted synchronism These studies could be of great importance to the of compensatory mutations. If our hypothesis is true, we Camila A. Yamada* H08 - STRUCTURAL DETERMINATION OF design of new anti-angiogenic drugs. would then be able to propose a coevolution model for VEGFR-3 DOMAIN 2 AND ITS INTERACTION Yala Sampaio*, Gladstony O. Souza, Mariana T. Q. proteins coevolving on separate genomes. To achieve WITH VEGF INHIBITOR PEPTIDES BY NUCLEAR H09 - AMPs DESIGN BASED ON THE de Magalhães1, this goal, we developed a sophisticated theoretical approach. We apply the Envelop Theorem to quantify MAGNETIC RESSONANCE SCHISTOSOMA MANSONI SmKI-1 KUNITZ Federal University of Minas Gerais, Belo Horizonte, INHIBITOR the dependency of the maximum entropy values with Bianca Rizo1 MG, Department of Biochemistry and Immunology the set of joint amino acids distributions parametrically Bruno P. O. Santos1,2 Cristiane D. Anobom2, Fábio C.L. Almeida3, Ricardo described in terms of the coevolution models. The result J. Giordano4 Carolina S. Ferreira3, Talita L. Santos3, Eliane S. F. Nanodiscs are a synthetic nanostructures that mimetizes translates itself in the gradient or shadow price of the 1 Alves4, Luciano M. Lião4, Rodrigo M. Verly3, Sérgio the membrane environment of a cell. They consist of entropy distributions. For three independent cases, we Department of Biochemistry, Federal University of C. Oliveira1,2 and Mariana T. Q. de Magalhães1,2 lipid domains encased within two- amphipathic proteins observed that coevolution-like models present the higher Rio de Janeiro, Rio de Janeiro, RJ (biancarizov@ derivative of Apolipoprotein A-1 (Apo-1), known as shadow price values compared to alternative models as gmail.com), 2Department of Biochemistry, Federal 1Bioinformatics Pos Graduation Program, Federal 3 membrane scaffolding proteins (MSP). This system is a consequence of an increasingly heterogeneity in the University of Rio de Janeiro, RJ, Institute of Medical University of Minas Gerais, Belo Horizonte, MG capable of solubilize and stabilize membrane proteins joint amino acids frequencies distributions. Moreover, Biochemistry, Federal University of Rio de Janeiro, ([email protected]), 2Department of 4 providing a novel platform for understanding their coevolution-like models present lower values of entropy Rio de Janeiro, RJ and Department of Biochemistry, Biochemistry and Immunology, Federal University of function. In contrast to others cell-mimetic, it is possible when compared to alternative models, characterizing 3 University of São Paulo, São Paulo, SP Minas Gerais, Belo Horizonte, MG, Department of to control the ratio of the nanodics by editing the length a more orderly structure. These results indicate the Chemistry, Federal University of Jequitinhonha and of MSP and it can be used in NMR or others biophysical possibility of an heuristic approach to find an optimized 4 Vascular endothelial growth factor (VEGF) and its receptor Mucuri Valleys, Diamantina, MG and Institute of techniques without causing strong interferences. Here coevolution MSA following a entropy minimization/ (VEGFR) are key drivers not only in physiological but Chemistry, Federal University of Goiás, Goiânia, GO we propose to study membrane active peptides using shadow price maximization criterion. also in most pathological angiogenesis. VEGF receptor an efficient method with nanodiscs formed from natural consist of an intracellular protein-tyrosine kinase domain, The SmKI-1 is a protein present in Schistosoma sources. The main idea is to make these structures This work was supported by CAPES and FAPDF a single transmembrane segment and an extracellular mansoni with an important role in the parasite with natural phospholipids extracted from the model (#07/2016) component containing seven immunoglobulin-like survival inside the host. Morais et al. (2018) studied microorganisms to study their interaction with membrane domains, while domain 2 plays the major role for ligand the recombinant protein in inflammatory diseases active peptides. The environment provided will be more H12 - Role of zinc and magnesium ions in the binding. The binding of growth factors to receptors (eg. goat, arthritis) and observed its ability to inhibit similar to a real cell membrane allowing us to a better modulation of phosphoryl transfer in protein initiates the angiogenesis signaling pathways. Available neutrophils, being a promising anti-inflammatory drug. understand of membrane active peptides. Moreover, tyrosine phosphatase 1B anti-angiogenic therapies focus either on neutralization The full-length protein contains two domains: the they are going to be used as a versatile tool among of VEGF binding or paths activated by these factors. Kunitz-domain, highly conserved and the C-terminal other model membrane systems for future studies using Carmen Domeneb,c,d* However, small specific inhibitory molecules design domain, with no homology outside the genera, peptides and as drug delivery systems Elisa Bellomo,a Asma Abro,b Christer Hogstrand,a has been a challenge in this field. Previous studies, which seems to be cytotoxic. Due to this evaluation, Wolfgang Mareta,* using VEGF inhibitor peptides, suggested that VEGFR we performed in silico analysis to design potential H11 - THE SHADOW PRICE OF COEVOLVING aDepartments of Biochemistry and Nutritional family members share a common binding site, which is AMPs. Properties like basic hydrophobic momentum, PROTEINS extremely important for drug development. Therefore, positive carried isoelectric point, molecular mass and Sciences, King’s College London, 150 Stamford b structural studies of the interaction mechanism the probability to form helix-wheel were evaluated. Camila F. T. Pontes1 Street, London, SE1 9NH, UK, Department of between peptides and VEGFRs are highly important Three peptide were designed: smki-pep1, smki-pep2 Chemistry, King’s College London, Britannia House, Miguel S. Andrade¹ and Werner Treptow¹ c to understand VEGFs signaling pathway and drugs and smki-pep3. These peptides were synthetized 7 Trinity Street, London SE1 1DB, UK, Chemistry 1 design as well. Thus, the goal of this work is to in solid phase, by using fmoc strategy and purified Laboratório de Biologia Teórica e Computacional, Research Laboratory, Mansfield Road, University d determine VEGFR3-D2 structure and its interaction by reverse phase chromatography. The purity of the Departamento de Biologia Celular, Universidade de of Oxford, Oxford OX1 3TA, UK, Department of with VEGF inhibitory peptides using Nuclear Magnetic peptides were evaluated by mass spectrometry and Brasília, Brasília, DF ([email protected]) Chemistry, University of Bath, 1 South Building, Resonance (NMR). VEGFR3-D2 was expressed in E. the sequence confirmed by de novo sequencing. The Claverton Down, Bath BA2 7AY, UK coli BL21 (DE3) strains for heterologous production. peptides had their biological activity positively detected The molecular coevolution process is characterized by Different expression conditions were tested. Cell lysis in antimicrobial assays against both bacteria and fungi. a series of compensatory substitutions on the interface Whilst the majority of phosphatases are metalloenzymes, was achieved through sonication, however, the protein Their interaction with mimetic membrane models were of interacting proteins across time. As a result, an the prevailing model for the reactions catalyzed by protein remained in inclusion bodies. Therefore, refolding studied by a series of biophysical techniques, that ensemble of protein variants which store the memory tyrosine phosphatases does not involve any metal protocols associated with two steps of Nickel affinity allowed the dissection of these interactions and the of this process is produced. The information encoded in ion. Yet, both metal cations and oxoanions affect their chromatography was implemented in order to obtain extraction of thermodynamical parameters (Kp, Kapp) such ensemble is known to effectively recover structural enzymatic activity. Mg2+ and Zn2+ activate and inhibit, the structured protein. All experiments were monitored from their adsorption into phospholipid vesicles. Finally, information. Some theoretical approaches based on respectively, protein tyrosine phosphatase 1B (PTP1B). by SDS-PAGE 15%. 1H-1D NMR spectra showed that the peptides structures were solved by using NMR protein coevolution apply the entropy minimization Molecular dynamics simulations, metadynamics and VEGFR3 domain was structured and the NMR structural spectroscopy. Together, our results give new insights principle to infer a global statistical Boltzmann model quantum chemical calculations in combination with experiments will be performed immediately in order to into AMPs design and demonstrate the biotechnological in which the single- and double-site amino acids experimental investigations demonstrate that Mg2+ and calculate the structure and dynamics of this protein. potential of the SmKI-1 protein. distributions correspond to the respective empirical Zn2+ compete for the same binding site in the active The assignment of the inhibitor peptide PCAIWF has distributions extracted from an MSA (Multiple Sequence site only in the closed conformation of the enzyme in already been performed and the interaction studies of This work was supported by Conselho Nac. Des. Alignment) of interacting proteins. Inspired by these its phosphorylated state. The two cations have different this peptide and VEGFR3 will be performed by NMR, Cient. Tecnológico (GRANTS# 302660/2015-1 and studies and by the advances in the theoretical field, our effects on the arrangements and activities of water which will allow the recognition of the peptide residues 465229/2014-0) and by UFMG (GRANT# 23853*40) group has been investigating if a coherent coevolution molecules that are necessary for the hydrolysis of the

120 121 phosphocysteine intermediate in the second catalytic H14 - STRUCTURAL BASIS OF THE CONTROL H15 - SENSITIVITY OF DETECTION OF COPPER H16 - Role of conformational equilibrium in step of the reaction. Remarkable differences between the OF CELLULAR ELONGATION AND DIVISION IN AND CADMIUM USING ANNELID HEMOGLOBIN molecular recognition and capsid assembly: the established structural enzymology of PTP1B investigated MYCOBACTERIUM TUBERCULOSIS case of flavivirus capsid proteins ex vivo and the function of PTP1B in vivo become Claudemir Oliveira Souza1,2 Catharina dos S. Silva1 Fabio C. L. Almeida1 evident. Different reaction pathways are viable when the Lierge Ramos1,2; Isis Sebastião1,2; Marli Leite de presence of metal ions and their cellular concentrations Gerardo A. Libreros-Zünigã1,2,3, Marcio V. Bertacine Moraes3; Patrícia Soares Santiago2 Glauce M. Barbosa1, Maria A. Morando2, Christine are considered. The findings suggest that the substrate 1 1 1 Dias 1 Cruz-Oliveira , Nathane C. Mebus , Thais C.N. delivers the inhibitory Zn2+ ion to the active site. The Instituto de Química, UNESP/Araraquara, São 1 1 1 2 Oliveira , Andrea T. Da Poian inhibition and activation can be ascribed to the different Department of Microbiology, University of Paulo ([email protected]) and UNESP/ 3 1 2+ 2+ São Paulo, São Paulo, SP Brazil. (catharina_ Registro, São Paulo and ICT-UNIFESP/São José Instituto de Bioquímica Médica, Universidade coordination chemistry of Zn and Mg ions and the 2 orientation of the metal-coordinated water molecules. [email protected]). IBILCE, São Paulo State dos Campos, São Paulo. Federal do Rio de Janeiro, Rio de Janeiro, RJ. University (UNESP), São José do Rio Preto, SP, ([email protected]) and 2Centro de Metallochemistry adds an additional dimension to the 3 regulation of PTP1B, and presumably other members of Brazil. Department of Microbiology, University of Biosensors are widely used in hospitals, pharmacies, Desenvolvimento de Tecnologia de Saúde, this enzyme family. Valle, Cali, Colombia. industries and in our life in general, helping in the most FIOCRUZ, Rio de Janeiro, RJ) varied daily tasks due to their sensitivity and selectivity References: [1] E Bellomo, A Abro, C Hogstrand, W In spite of being one of the oldest and further studied in addition to the low cost employee. These devices Proteins are dynamic entities able to move in a wide range Maret, C Domene, J Am Chem Soc, 2018, 140 (12), disease of mankind, tuberculosis follows as leader in use biological molecules, such as enzymes, to aid of timescales that goes from picoseconds to seconds. 4446-4454. both incidence and mortality rates among bacterial or detect the analyte in very low concentrations. The Dengue and Zika are major arthropod-borne human viral infections. Current studies have contributed to the use of hemoglobins in biosensors is an alternative to disease, for which no specific treatment is available. The H13 - PIVOT-SAXS: EFFICIENTLY COMBINING identification of novel targets for antitubercular drug the use of purified enzymes that are costly and have a flavivirus capsid protein is the trigger of virus assembly, EXPERIMENT AND THEORY FOR MOLECULAR design. In particular, researches on mycobacterial relatively short shelf life. These hemoproteins oxidize in they are located at the cytoplasm bound to lipid droplets CHARACTERIZATION. cell wall biosynthesis are becoming prominent as the presence of certain concentrations of environmental (LD) or hydrophobic membrane interfaces. In Dengue, such cellular structure has a role on virulence and pollutants, allowing their use for the detection of these binding to LDs is essential for virus assembly. We Carolina T. A. Ferreira¹ acquired resistance. Recently, researchers discovered molecules. Thus, the objective of this study was to showed that the positively charged N-terminal region of Leandro C. Oliveira¹ a synergistic interaction between the endopeptidase compare the sensibility of initial detection of cadmium Dengue virus capsid protein prompts the interaction with RipA and the glycosylase RpfB. The complex formed and copper in the biosensor with Amynthas gracilis negatively charged LDs, after which a conformational ¹São Paulo State University (Unesp), Institute of (RipA:RpfB) coordinates the cell division and appear hemoglobin (HbAg) immobilized in polyethyleneimine rearrangement enables the access of the central Biosciences, Humanities and Exact Sciences, São to be inhibited by a penicillin-binding protein named (PEI). The technique of assembly of the films was hydrophobic patch to the LD surface. We also showed José do Rio Preto PonA1. Apparently, PonA1 and RpfB compete for the the Layer-by-Layer (LBL) with 10 PEI/HbAg bilayers the participation of the intrinsically disordered region same interaction site on RipA. Thus, it implies the grown, and for the detection of copper and cadmium in binding and possible regulation of capsid assembly. It is well known by the scientific community the importance existence of a post-translational regulatory mechanism at concentrations of 0 to 70μmol.L-1 was used cyclic We probed the structure and dynamics of Dengue virus of combining experimental and computational techniques for peptidoglycan synthesis and hydrolysis. Yet, voltammetry. Both metals were sensitive in the test and Zika virus capsid proteins (DENVC and ZIKVC) by and this is even more enhanced in a complex biological there is no structural data available to aim the that obtained electrode saturation of 10μmol.L-1 of nuclear magnetic resonance. They bind lipid droplets system. The prediction of biological molecules structure comprehension of both intermolecular interaction and metal concentration. The biosensor in the presence (LD) in the cytoplasm, which mediates virus assembly in solution is a problem that has been challenging the regulation.Therefore, the present study aims to obtain of cadmium was 2.23 times less sensitive than the in an unknown way. We showed that the dynamics experimentalists and theoreticals alike. The Small and structurally analyze both complexes RipA:RpfB copper metal, this value corresponds to the difference of the capsid proteins are intrinsically involved in the Angle X-ray Scattering (SAXS) is an low resolution and RipA:PonA1, in order to improve the knowledge of the angular coefficients of each detection curve. mechanism of hydrophobic surface recognition, binding experiment that provides only quantitative information, about the physiological processes in which those Therefore, it is suggested that HbAg can be used as a to LD, RNA and virus assembly. We also probed the such as gyration radius and pair distance. Nevertheless, proteins act. To do that, truncated protein constructs base in a biosensor, with a higher sensitivity for copper assembly using small angle x-ray scattering and atomic SAXS is capable of addressing this systems and when were cloned into pET28a or pGAT systems to produce as compared as with cadmium heavy metal. force microscopy. The understanding of the participation combined with a computation technique, it turns into a individual domains by heterologous superexpression in of the intrinsically disordered N-terminal region and its powerful characterization tool for macromolecules in E. coli strains. Protocols for soluble protein production This work was supported by CAPES and by FAPESP dynamics helped us propose a mechanism for Dengue solution. In line with this, we have developed a software, and purification are being standardized to allow further (2015/11447-1). and Zika virus assembly and to develop a peptide with the Pivot-SAXS, for the theoretical protein structure co-crystallization and X-ray diffraction experiments. the potential to block virus assembly. prediction. Pivot-SAXS considers the information from the experimental profile, the secondary structure and the This work is being supported by the Foundation for This work was supported by CNPq, FAPERJ and INBEB/ hydrogen bond that can contribute to the final structure Research Support of the State of São Paulo (FAPESP) CNPq. to minimize the conformational space to be evaluated by grant 2016/18721-4. a Metropolis criteria while the new conformations are generated using a pivot algorithm. The software, even though it is still in process of adjustments, has achieved satisfactory results and will be available as an online server for the scientific community.

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

122 123 H17 - QUATERNARY STRUCTURE AND ENZYME characteristic benzoxazole moiety is a heterocyclic system. Continuing the theoretical and computational H21 - Product dissociation process in Edema ACTIVITY CORRELATION IN THE b-GLYCOSIDASE benzene-fused oxazole ring that forms the benzoxazole approaches developed recently in our research group, Factor of Anthrax FROM SPODOPTERA FRUGIPERDA motif. The biosynthesis of benzoxazole-family we will seek to understand the behavior of the system compounds has not been explored in detail and its based on the analysis of the scattering data and the aid Gabriel Ernesto Jara Felipe A. Otsuka enzymes are poorly studied. In biosynthesis of AJI9561 of adjusted potential functions. Leandro Martínez Vitor M. Almeida, Maira A. Frutuoso, Rafael S. was identified a gene for a putative ATP-dependent Instituto de Química/UNICAMP, Rua Josué de Castro Chagas and Sandro R. Marana ligase (3-HA adenylation and dimerization enzyme) that This work was supported by Coord. de Aperfeiçoamento encode for the enzyme AjiA1. The aiming of this study de Pessoal de Nível Superior (Capes). s/n, Cidade Universitária “Zeferino Vaz", Barão Institute of Chemistry, University of São Paulo, São is to deepen into the understanding the mechanisms Geraldo 13083-861 Campinas, São Paulo, Brasil; Paulo, SP ([email protected]) [email protected] of formation of benzoxazole ring in AJI9561 and, H20 - CHARGE REGULATION IN BSA PROTEIN therefore, solve the three-dimensional structure of Enzymes are powerful catalysts whose function depends AjiA1 by X-ray crystallography. ajiA1 was cloned into Fredderico C. Machado1 The Edema Factor (EF) is one of three major toxins on their spatial structure and organization. It is known that expression vectors from pET system and was used to Leandro R. S. Barbosa2 involved in the disruption of cellular functions resulting β-glycosidases from GH1 family display a characteristic transform bacterial expression strains. The expressed from infection of a host organism by anthrax. EF is 1 tertiary structure (a β/α8 barrel) which permits the protein was purified according to a two steps protocol Department of General Physics, University of São an adenylyl cyclases (AC) and, as such, catalyzes catalytic hydrolysis of O-glycosidic bond, but few studies involving an Immobilized Metal Affinity Chromatography Paulo, São Paulo, SP (fredderico.machado@usp. the production of cyclic-AMP (cAMP) from ATP. In 2 evaluate the influence of the quaternary structure on their (IMAC) followed by a Size Exclusion Chromatography br) and Department of General Physics, University eukaryotes, cAMP is a key signaling molecule, and the catalytic efficiency and function. Thus, the goal of this (SEC). Crystallization screening were performed using of São Paulo, São Paulo, SP infection by EF induces its overproduction, leading to project is to characterize the quaternary structure of the the automated crystallization platform of OryX4 robot cell death. Recently, we have published a computational β-glycosidase GH1 from Spodoptera frugiperda (Sfβgly; (LBEA-USP) and then these crystals were optimized. The Charge regulation is the process in which the charge on a study of the mechanism of the reaction catalyzed by EF O61594) with emphasis on its dimer interface. Mutants obtained crystals were exposed to a beam of X-rays from surface of a protein is changed. For instance, the overall (J. Phys. Chem. B 2016, 120, 6504–6514.). However, (F155N, F155A, and a sextuple mutant) were designed a Synchrotron source for diffraction. We solve the AjiA1 protein surface charge can be altered by approximating the catalytic reaction is just one step of the whole targeting the residues within the Sfβgly dimer interface structure using Molecular replacement. The collected another protein close enough to this, or changing in pH catalytic cycle. A complete description of the catalysis aiming to prevent its formation. So, it is expected that data was processed using crystallographic software of the medium, as well as protein concentration or even mechanism only can be accomplished by understanding the mutant enzymes will be monomers and the wild-type XDS, CCP4 and Phenix suite. AjiA1 is member of the the presence of an external electric field. In the present all the other stepsof the catalytic cycle, e.g. products dimers. Then, biophysical assays (SEC-MALS, DLS and adenylate-forming enzyme (AFE) family PFAM00501. study the effect of charge regulation was tested in the release. To our knowledge, no model of the product SAXS) will be used to dissect the oligomer state of these We have determined the crystal structure of AjiA1 at BSA protein. To this aim, Small Angle X-ray Scattering release exists for EF. An experimental characterization proteins, whereas kinetic enzymatic assays will be made 2.6 Å resolution that crystallized as an intimate dimer in (SAXS) was used to calculate the protein-protein of product release from a mammalian AC was reported to evaluate their enzyme activity. The wild-type Sfβgly was space group P3121. A deep analysis of AjiA1 structure Structure factor S(q), which is directly related to the by Dessauer et al. (J. Biol. Chem. 1997, 272, 27787– produced as recombinant protein in bacteria and purified will reveal insights into the reaction mechanism of Fourier transform of the radial distribution function, 27795). A similar experimental description is missing for using affinity and ionic exchange chromatography. The AJI9561 benzoxazole ring biosynthesis. To further g(r). This latter function depends on the total correlation EF. Thus, the product release of EF could be described relative molecular weight of the wild-type Sfβgly was explore the catalytic mechanism of AjiA1, experiments among the scattering particles, which can be calculated by different alternatives. Here, we present preliminary evaluated in two different ionic strengths using SEC. In of crystallization of this enzyme in complex with ATP and analytically (for a few cases) or numerically using results of free energy profiles of dissociation for product, addition, steady-state kinetic parameters for 4 different substrate are in progress. the Ornstein-Zernike equation. Herein, we solved the considering different alternatives of the catalytic steps. substrates were also determined for the wild-type enzyme. Ornstein-Zernick equation using RPA (Random Phase The simulations were performed using Umbrella The production of the mutant Sfβgly as recombinant H19 - Protein-protein interactions at high Approximation) as a closure relation. Such closure Sampling (US) method. protein is in progress. densities using Small Angle X-ray Scattering relation supposes that the protein-protein interaction potential can be described as a reference potential This work was supported by Fundación de Apoyo a la This work was supported by Coordenação de Fernando T. Tanouye1 (hard sphere model, as described in the Perkus- Investigación del Estado de São Paulo (FASEP): Grants Aperfeiçoamento de Pessoal de Nível Superior (CAPES / Rosangela Itri1 Yevich approximation) modulated by a “perturbation 2010/16947-9, 2013/08293-7 and 2013/22360-9. PROEX 1888/2016), Fundação de Amparo à Pesquisa do potential”, like the Coulomb or the Kirkwood ones. As 1 Estado de São Paulo (FAPESP) and Conselho Nac. Des. Department of Applied Physics, University of São Kirkwood showed in his studies, the charge regulation Paulo, São Paulo, SP ([email protected]) H22 - EFFECT OF pH AND TEMPERATURE IN THE Cient. Tecnologico (CNPq). effect induces an attractive contribution on the protein- BOVINE CARBONIC ANHYDRASE II STRUCTURE protein overall interaction potential. According to initial Gabriel R. Sousa H18 - STRUCTURAL STUDY OF AJIA1 FROM THE Proteins are linear polymers of amino acids involved simulations, the charge regulation effect can be important BENZOXAZOLE AJI9561 GENE CLUSTER in virtually all the metabolic functions of living things. on the protein-protein interaction potential, mainly for pH Gustavo Scanavachi, Juliana Sakamoto, Rosangela Itri In their natural context of action they are immersed in values near the protein isoelectric point, pI. It’s also Fernanda Paiva1 a highly complex and diverse environment, containing showed by simulations that protein concentration may Physics Institute, University of São Paulo, São Paulo, SP ([email protected]) Karen Chan2, Peter Leadlay2 and Marcio Dias1 high protein concentrations (around 50-400 g/L inside be important since its increase lead to an approximation cells), in addition to other organic and inorganic of the scattering particles. 1 Department of Microbiology, University of São molecules. However, most of the small angle scattering Carbonic anhydrase (CA), is a metalloenzyme, which Paulo, São Paulo, SP ([email protected]) and studies (SAS) which propose models for the effective contains a zinc atom. Its most important function is the 2 Department of Biochemistry, University of potential of interaction between proteins are limited hydration of the carbon dioxide and the dehydration of Cambridge, Cambridge, United Kingdom to very diluted solutions, thus far from the conditions the carbonic acid generating residual carbon dioxide found in vivo. We propose here to investigate highly The benzoxazole family of compounds includes concentrated protein solutions by means of the natural products with cytotoxic activity, such as UK-1 SAXS technique, using binary mixtures of bovine and AJI9561 produced by Streptomyces spp. The serum albumin (BSA) and lysozyme as the model

124 125 molecule. Several isozymes with the same catalytic study consists of characterizing the interaction between obtain information about position of IDR N-terminal, PPARγ and CDK5 and identified essential residues for activity are known in humans and the differences are the recombinant core domain of M2-1 protein (dgM2-1) a1/a1’ and a2/a2’ helices, the solvent-exposure this interaction, allowing a rational and more specific related to the substrate specificity, inhibition patterns, and RNA by using NMR spectroscopy techniques such of each backbone amide was measured by solvent design of ligands to impair S245 phosphorylation and catalytic efficiency, and tissue distribution. The higher as chemical shift perturbation (CSP) Δδ (1H,15N) and paramagnetic relaxation enhancement (sPRE). The improve insulin sensitivity. concentration of CA is found in the red blood cells, nuclear spin relaxation experiments. CSP experiments thermal susceptibility of the amide chemical shifts and in which three isozymes (CAI, CAII and CAIII) are were used to determine the protein residues involved SAXS technique were also assessed. DENVC presents a This work was supported by Coordenação de recognized. Further, it is well known that denaturation in the interaction with nucleic acid and the nuclear spin pH-dependent stability (∆G0 = 17 kcal/mol, pH 7.5; ∆G0 Aperfeiçoamento de Pessoal de Nível Superior (CAPES 15 and/or aggregates of CAII is related to some disorders in relaxation measurements ( N R1, R2 e hetNOE) were used = 10 kcal/mol, pH 6.0), which is not observed for D1- #23038.006737/2012-56) and by Fundação de vivo, as neurons degeneration, pulmonary emphysema, to characterize the main chain dynamics of dgM2-1 in 19. D1-19 structure is been calculated by Aria software. Amparo à Pesquisa do Estado de São Paulo (FAPESP hypothyroidism. Then, the study of destabilization of the the absence and presence of RNA. The most significant Dynamics, sPRE and thermal susceptibility are in #2016/22246-0) native conformational of CA II it is important to understand chemical shift perturbations Δδ (1H, 15N) were found analysis. Previous results suggest a partially protection the mechanisms of those disorders and this is the aim in the residues Leu74, Val76, Val79, Lys92, Gln93, Ala98, of a2/a2’ in DENVC, probably conferred by residues in H26 - BIOCHEMICAL AND STRUCTURAL of this work. Here, we are investigating the role of pH Arg151, Leu152, Asp155, and Thr169. The nuclear spin IDR N- terminal and/or a1/a1’, in agreement with SAXS CHARACTERIZATION OF THE ENZYME (between 3 and 9) and temperature (20ºC up to 90ºC) relaxation experiments showed an increase in the R1/R2 ratio of results. Schistosoma mansoni FUMARATE HYDRATASE in the secondary, tertiary and quaternary structure of the protein in the presence of nucleic acid. The outcomes of the bovine CA II (more than 80% homologue to the human). present work provide a detailed understanding of the molecular This work was supported by CNPq, FAPERJ and INBEB/ Iara Aimê Cardoso1 For doing so we are performing circular dichroism (CD), mechanism of interaction of the core domain of hRSV M2-1 CNPq. Aline Kusumota Luiz de Souza¹, Maria Cristina fluorescence of tryptophan (there are 7), and small protein with one of its major molecular targets, RNA. Nonato1 angle X-ray scattering (SAXS). Preliminary results show H25 - EXPLORING THE MECHANISM OF PPARγ 1Department of Physics and Chemistry, School a thermal denaturation of CA II around 67ºC as observed This work was supported by: FAPESP, CNPq. PIBIC PHOSPHORYLATION MEDIATED BY CDK5 by CD and SAXS and a conformational change it was of Pharmaceutical Sciences of Ribeirão Preto, observed between pH 3 and pH 5 but not observed Helder V. Ribeiro Filho1,2 University of São Paulo, Ribeirão Preto, SP, Brazil H24 - ROLE OF INTRINSICALLY DISORDERED ([email protected]) between pH 5 and pH 9, even though the CA II has +13 REGION IN STRUCTURE AND DYNAMICS OF Fernanda A. H. Batista1,2, Paulo S. L. de Oliveira1,2 and -6 charges respectively. Other experiments will be DENGUE VIRUS CAPSID PROTEIN and Ana C.M. Figueira1,2 performed in order to get more information and confirm Schistosomiasis is a neglected disease caused by six 1 these previous results. Fluorescence of tryptophan will Glauce M. Barbosa1 Brazilian Biosciences National Laboratory (LNBio), species of the trematode flatworm Schistosoma. The Brazilian Center for Research in Energy and be analyzed depending on pH and temperature. Also, Maria A. Morando1,2.3; Christine Cruz-Oliveira1.2; disease has been reported from 78 countries, prevalent Materials (CNPEM), and 2University of Campinas secondary structure by CD will be determined in different Fabio C. L. Almeida1.2; Andrea T. Da Poian1 in tropical and sub- tropical areas, and affects almost pH to corroborate with SAXS results. (UNICAMP), Campinas, SP (helder.ribeiro@lnbio. 240 million people worldwide. The cytosolic isoform of 1 Instituto de Bioquímica Médica Leopoldo De Meis, cnpem.br) class II fumarate hydratase of Schistosoma mansoni Programa de Biologia Estrutural, Universidade (SmFH) catalyzes the reversible hydration of fumarate H23 - Study of the Interaction of the Globular Federal do Rio de Janeiro - UFRJ; 2Centro Nacional Domain of the M2-1 Protein from hRSV with RNA. PPARγ is a target of CDK5 kinase which to L-malate and is suggested being involved in DNA Biologia Estrutural e Bioimagem (CENABIO), Centro hyperphosphorylates this nuclear receptor at S245 repair mechanisms and urea cycle. As a first step Giovana C. Guimarães1,2 Nacional de Ressonância Magnética Nuclear under obese condition thereby inducing insulin towards evaluating SmFH as a drug target against (CNRMN), Universidade Federal do Rio de Janeiro Vitor B. Machado1,2; Marcelo A. Fossey2,3, Fátima P. resistance. A remarkable effort has been carried out to schistosomiasis, we focus on its biophysical and - UFRJ; 3Centro de Desenvolvimento de Tecnologia de Souza2,3; Ícaro P. Caruso2,3 find PPARγ ligands that inhibit S245 phosphorylation, biochemical characterization. In the present work, em Saúde, Fundação Oswaldo Cruz (FIOCRUZ), Rio but this process is structurally and mechanistically SmFH was successfully expressed, and soluble 1 Biology Department, IBILCE/UNESP, São José do de Janeiro. [email protected] poorly understood. Thus, here we used computational protein was purified with a good yield. Differential 2 Rio Preto, SP ([email protected]); Centro approaches and comparative structural information scanning fluorimetry allowed us to evaluate protein Multiusuário de Inovação Biomolecular (CMIB), Dengue virus (DENV) is responsible for one of the most to propose a model of interaction between PPARγ stability under different physico-chemical conditions 3 IBILCE/UNESP, São José do Rio Preto, SP; Physics important arboviroses. DENV capsid protein (DENVC) and CDK5. Employing site-directed mutagenesis and and demonstrated to be useful technique for ligand Department, IBILCE/UNESP, São José do Rio Preto, SP is a stable homodimer in solution. Each DENVC biophysical techniques, we validate our model and a screening. The optimal pH determined for SmFH activity monomer has 100 residues organized in 4 α-helices possible mechanism of interaction. The docking model was 7-7.5 using fumarate as substrate, and pH 8 using The human respiratory syncytial virus (hRSV) is and an intrinsically disordered N-terminal region (IDR and structural analysis allowed to (i) identify three L-malate. Enzyme kinetics using both substrates was the main cause of severe respiratory infections as N-terminal), comprising the first 20 residues. Previous main regions in the PPARγ-CDK5 interface, (ii) select used to determine the steady-state kinetic constants, pneumonia and bronchiolitis in children, elderly and studies have shown that the interaction between DENVC PPARγ residues K240, K263 and K265 as possible and the results reveal Km of 0.55 mM and 0.15 mM for immunocompromised people. The M2-1 protein is an and lipid droplets (LDs) is essential for viral replication anchor residues, and (iii) propose a non-continuous L-malate and fumarate, respectively. Comparing with important factor that prevents the premature dissociation and occurs through residues present in hydrophobic mechanism of PPARγ recognition by CDK5 through the the human fumarase (HsFH), the Km values were found of the polymerase from the RNA, allowing the equal cleft (a2/a2’ helices) and IDR N-terminal. The main goal residue K261. Single- point mutations of these lysines to be pretty similar, although the kcat values were much transcription of all mRNAs. This protein is mainly of this study is to understand the role of IDR N-terminal were sufficient to reduce (K240) or inhibit (K261, 2K63 higher for HsFH than SmFH. Moreover, we established composed of α-helix and its three-dimensional structure in function, structure and dynamics of DENVC. The and K265) the affinity of PPARγ for CDK5 in fluorescence a reproducible crystallization protocol, and data X-ray is divided in four domains: a zinc finger domain which is structural stability of DENVC and the mutant protein with anisotropy assay. Moreover, all the mutants, reduced diffraction of recombinant SmFH at 2.39 Å resolution composed of N-terminal residues which bind to a zinc atom the deletion of IDR N-terminal (D1-19) were evaluated the phosphorylation levels of PPARγ. The structural allowed us to solve the first structure of S. mansoni and assist in the transcription process; the oligomerization by fluorescence spectroscopy. In order to solve the 3D stability and the biophysical characterization of the fumarase. domain is responsible for the tetrameric arrangement; the structure, the D1-19 assignment was performed by mutants in comparison to the wild-type proteins were core domain that acts directly on the interaction with the viral triple resonance experiments. 15N-backbone relaxation performed and validated using circular dichroism This work was supported by CNPq (306208/2015-6).

phosphoprotein and RNA; and the unstructured C-terminal parameters (R1, R2, Het-NOE) were collected to determine and dynamic light scattering. In conclusion, we domain with low degree of conservation. The present the dynamics in pico-nanoseconds timescale. To characterized the interface of interaction between

126 127 H27 - ANTIMICROBIAL PEPTIDES – LIPID H28 - INTERACTION BETWEEN FLAVONOID H29 - PURIFICATION AND ESTIMATION OF THE H30 – Implication of free intracellular calcium BILAYER INTERACTION STUDY BY CONSTANT PH DERIVED CHRYSIN WITH hRSV NS1 PROTEIN ISOELECTRIC POINT (pI) OF THE MONOMER concentration and spontaneous arrhythmic MOLECULAR DYNAMICS SIMULATIONS D OF THE EXTRACELLULAR HEMOGLOBIN OF activity in isolated cardiomyocytes Isabella O. L.1 1 Amynthas gracilis (HbAg) 1 Ingrid B. S. Martins 2 3 4 Sarmento J. O Evelyn P.T. ; Carolina S.L. ; Ícaro P.C. ; Luis Octávio 1 5 6 7 Isis Sebastião 1 Sidney J. de Carvalho1 and Alexandre S. de R. ; Fátima P.S. ; Marcelo A.F. Roman-Campos D. Author Araujo1 Jonathan Brito Souza de Oliveira1, Márcia Cristina 1,4,6,7Departamento de Física, IBILCE-UNESP, Department of Biophysics, Federal University of Ohya2, Claudemir Oliveira Souza1, Lierge Ramos1, 1Department of Physics, São Paulo State Rio Preto, SP ([email protected]), São Paulo1, SP ([email protected]) Patrícia Soares Santiago2,1 University, São José do Rio Preto, SP. (ingrid. 2Departamento de Biologia, IBILCE-UNESP, Rio 3,5 1 [email protected]) Preto, SP, Departamento de Química e Ciências Instituto de Quimíca, University of State of São According to the World Health Organization (WHO), Ambientais, IBILCE-UNESP, Rio Preto, SP Paulo-UNESP, São Paulo, SP (isisebastiao@gmail. heart diseases are the leading cause of death, for both 2 The pH is a physical-chemical property that com) and University of State of São Paulo-UNESP, men and women, worldwide. Also, three-quarters control several biological processes that happens According to the WHO, hRSV accounts for more than São Paulo, SP occur in low- and middle-income countries. Among all in a cell, due to the influence of the pH on the 30 million new episodes of acute lower respiratory forms of heart disease, arrhythmias are the main cause protonation state of the molecules. Antimicrobial tract infections (LRTI) in children under five and more The HbAg is an extracellular hemoglobin from annelid of death. In this context, a better cellular and molecular peptides (AMPs) are short amino acid sequences than 3.4 million hospital admissions linked to severe Amynthas gracilis. That biomolecule presents a understanding of how arrhythmias occur may help to with titratable residues, and amphipathicity, that disease by RSV every year around the world, there is high molecular weight around 3,6 MDa and each prevent them and, at the end, reduce premature deaths. interact with anionic lipid bilayers such as the still no licensed vaccine. The RSV viral genome consists biomolecule consists of an assembly of polypeptide In the context of heart physiology concentration of cell membrane of prokaryote organisms, lysing in a negative and non-segmented single-stranded RNA chains including functional chains and linkers chains. cytoplasmic calcium ([Ca2+]i) plays a pivotal role. the cell. Their mechanism of action are regulated of 15.2 kb organized into 10 genes that encoding 11 The oligomeric dissociation is interesting because Increase in [Ca2+]i often leads to arrhythmias by a by pH, once it modulates the charge of both the proteins. The NS1 protein contributes to the success it allows to evaluate the biotechnological potential of range of mechanisms. [Ca2+]i may augment after a AMP and the cell membrane. There are several of viral replication by inhibiting the induction of type I isolated monomers from HbAg. The aim of this work train of action potential which occurs during ventricular experimental evidences about the study of interferon (IFN) and genes stimulated by IFN. The study of was to separate and purify the monomer d from HbAg tachycardia. Following, at rest, increased [Ca2+]i AMP's/bilayer interactions, but these works only the interaction between NS1 with ligands is important to and estimate its isoelectric point (pI) by Scattered Light could facilitate occurrence of Early and Delayed After investigate which amino acids are involved in the promote the mechanism of infection inhibition, of which Intensity (LSI). Separation and purification of the HbAg Depolarization (EAD and DAD) in cardiomyocytes. interaction and how their protonation state can flavonoids have been described as effective suppressors subunits followed the described to HbGp.1,5 mL of Thus, in order to evaluate them we used isolated affect this interaction. So, a theoretical approach with the antiviral function. The aim of this work was whole HbAg, 30 mg.mL-1 at pH 7.0, was used in the cardiomyocytes from healthy adult male guinea pig and of AMP’s mechanism of action is relevant to to express and to purify the NS1 protein. Then, it was process. The SDS-PAGE electrophoresis was used patch-clamp technique. In our experiments we took understand how the lyse of the cell happens. analyzed the interaction between NS1 and derived. The for confirmation the purity of the samples obtained. advantage of two types of internal solution: one with Molecular Dynamics simulations are widely used NS1 expression was with 0.3 mM IPTG for 4 h at 37° C Scattered Light Intensity (LSI) of the pure monomer at high [EGTA] and another one with low [EGTA] (a slow to study diverse biological systems due to the in Rosetta-gami 2(DE3) E. coli cells. The protein elution different values of pHs was perform to estimate the pI Ca2+ chelator). A tachycardia preconditioning test was reliability of its results. However, they are not was performed by imidazole gradient purified by Cobalt- of the monomeric chain. The separation and purification applied in order to verify its relevance in the arising efficient to describe systems that are sensitive to pH affinity chromatography and a final purification using a of monomer d was successfully obtained. These results of spontaneous activity (EAD and DAD). [EGTA] had changes because the protonation state do not vary Superdex 75 column (GE). The protein-ligand interaction were confirmed by chromatogram of dissociated no impact on action potential waveform, measured at throughout the simulation. The protonation state was analyzed in three different temperatures with titration analyses and SDS-PAGE. The chromatogram of 0.2 of stimulation. Important, EAD and DAD occurred

is fixed based on the pKa of these isolated groups of derived chrysin by spectroscopy fluorescence. The dissociated HbAg showed four major peaks: at 45 only when tachycardia preconditioning test was applied and pH's solution. This is a severe limitation since results showed that Stern-Volmer constants increase and 92 mL associated respectively, whole HbAg and along with the low [EGTA] internal solution. Thus, we the processes that are modulated by the change in with increment of temperature suggesting a dynamic monomer d. The fractions had their concentration can conclude that [Ca2+]i participates in the formation the protonation can not be observed. So, a more quenching process. The thermodynamic parameters determined, obtaining monomer d of 3.56 mg.mL-1. of spontaneous activity following tachycardia. realistic approach is to run simulations where the indicated the binding reaction is spontaneous and the The SDS-PAGE profile confirm purity of monomer d protonation state of the system components may hydrophobic interaction is the main contribution to obtained by existence of a single band stained around H31 - Dynamics of FGFR2-Grb2 Interaction by vary with time, in other words, the Constant pH the complex formation. From double-log analysis, the 12-16 KDa. The LSI data indicated a higher scattering 5 -1 TROSY-based NMR essay Molecular Dynamics (CpHMD). In this study we binding constant (Kb) ranging from 1.09x10 M to at pH 4.6 suggesting that pI values are in the range analyse the interaction between the MP-1 and 1.73x105 M-1 and the number of binding sites equal to 1 from pH 4,0 to 6,0. The result is quite similar to related Jéssica A. Tedesco1 HMP-1 antimicrobial peptides and POPC/POPG were determined for the complex. The results point out pI of monomer d from HbGp (~6,2) and HbLt (~5,74), Karoline Sanches1, Fábio C. L. Almeida2 and lipid bilayers through CpHMD simulations. the modified chrysin as a candidate for verification test obtained from other biophysical techniques. Fernando A. de Melo1. to block infection caused by hRSV. 1 This work was supported by Coordenação de This work was supported by Fundação de Amparo à Department of Molecular Biophysics, São Paulo Aperfeiçoamento de Pessoal de Nível Superior. This work was supported by CNPq and FAPESP. Pesquisa do Estado de São Paulo (FAPESP) – Process: State University, São José do Rio Preto campus 2 2015/11447-1 ([email protected]) and National Center of Nuclear Magnetic Resonance, University of Rio de Janeiro, Rio de Janeiro.

FGFR2 (Fibroblast Growth Factor Receptor 2) is a receptor protein playing a crucial role in regulating cellular metabolism, genetic expression, growth, division and cellular differentiation. Signaling upstream starts when

128 129 growth factors bind to receptors localized on the activated and molecular exclusion chromatography. The western H34 - STUDY ON THE INTERACTION OF IONIC (Fcs) and enoyl- CoA hydratase/aldolase (Ech) in surface of FGFR2 which induce its phosphorylation blotting technique was performed and revealed a LIQUIDS WITH MODEL PROTEINS IN AQUEOUS various microorganisms. In this study, we report the followed by recruitment of protein partners from cytosol. band of approximately 26 kDa, corresponding to the SOLUTION expression and purification of Fcs and Ech identified One of these partners is the adaptor protein Grb2 (Growth molecular mass of the amino terminal domain of by metagenomics methodology. Subsequently, Juliana Raw1* Factor Receptor Bound Protein 2) which interaction the hRSV N protein. The interaction between N32–252 the enzymes were characterized biophysically and mechanism between those proteins is fundamental for protein and flavonoid quercetin was investigated by Leandro R. S. Barbosa1 biochemically. The results obtained demonstrate the health operation of MAPK (Mitogen Activated Protein fluorescence quenching method, which indicated a 1 that Fcs forms dimers in solution, while Ech forms 4 -1 Institute of Physics, University of São Paulo. Kinase). Elucidating such mechanism is crucial for the binding on order of 10 M with stoichiometry of 1:1, * hexamers. Both enzymes were stable and biologically study of that signaling pathway and its aberrant behavior. i.e. one ligand by protein. [email protected] active under neutral and alkaline conditions. Fcs was TROSY-based studies of transverse relaxation rates able to form the complex feruloyl-CoA at pH 7 and showed a loss of signal related to SH3 domains in the This work was supported by CAPES, FAPESP and CNPq. In this work we studied the interaction of ionic liquids 30oC. Biotransformation studies of FA to vanillin using compared spectrums of 15N isotopic labeled Grb2 and the (ILs) with model proteins. The effect of the ILs 1-methyl- Fcs and Ech in bacteria cells will be conducted in the 3-tetradecylimidazolium chloride, ([C mim][Cl]), 1- mixture of labeled Grb2 and cytosolic FGFR2. This signal H33 - RANKING DOCKING MODELS BY 14 future. dodecyl-3-methylimidazolium chloride, ([C mim][Cl]) loss demonstrates that both N- and C- terminal Grb2-SH3 COEVOLUTION ANALYSIS 12 domains take place on the interaction with FGFR2 while and 1-decyl-methylimidazolium chloride, ([C10mim] This study was supported by Fundação de Amparo à Grb2-SH2 domain remains dynamic. This result adds a José Antonio Fiorote1 [Cl]) on the structure of bovine serum albumin (BSA) Pesquisa do Estado de São Paulo (FAPESP), Processo new figure to the FGFR2-Grb2 interaction tale which early Werner Treptow² and human serum albumin (HSA) was investigated by No 2017/16976-8. was thought to be a role exclusively done by C-terminus fluorescence spectroscopic, UV-Vis, absorbance and 1Department of Cell Biology, University of Brasilia, Grb2-SH3 domain. small angle X-ray scattering (SAXS). Concerning the H36 - Grb2-SH2 domain dynamics and Brasilia, DF ([email protected]). ²Department of fluorescence measurements, we observed a blue-shift Cell Biology, University of Brasilia, Brasilia, DF interaction: a fluorescence and NMR This work was supported by PROPG/UNESP, CAPES, and a fluorescence quenching as IL concentrations spectroscopy approach. CNPq and FAPESP. increases in solution. SAXS results suggest that the Protein-protein interactions are critical for diverse proteins underwent a partially unfolding, evidenced Karoline Sanches1 H32 - Fluorescence studies of the interaction of cell processes. Docking is one of the most used by an increase in the radius of gyration (Rg). Taking Ícaro P. Caruso1, Jéssica A. Tedesco1, Fábio C. L. human Respiratory Syncytial Virus nucleoprotein approaches to resolve protein-protein interactions at together, these results indicates that the interaction of de Almeida2, Fernando A. Melo1 the atomic level. The generation of false positive hits BSA and HSA with IL are divided into three stages, as N-terminus with quercetin 1 is however a well documented drawback of docking follows: the first stage is characterized by the protein on Department of Physics - Institute of Biosciences, Jéssica M. de Sá1 algorithms as a result of limitations of the scoring its native form and the interaction is predominantly due Humanities and Exact Sciences (IBILCE) São Paulo State University “Júlio de Mesquita Filho” (UNESP), Fátima P. de Souza1, Marcelo A. Fossey1, Giovana function in describing solvation energies and protein to the electrostatic forces, provided by the opposing São José do Rio Preto/SP, Brazil. 2National Center C. Guimarães1, Ícaro P. Caruso1. flexibility. Looking for new developments in the field, charges on the surface of the proteins and the polar we have created a software, the Docking Score Module head of the ILs. In the second stage, higher of IL of Nuclear Magnetic Resonance (CNRMN), Federal 1 Centro Multiusuário de Inovação Biomolecular, (DSM), that combines docking calculations and protein concentrations induce the unfolding of the proteins and University Federal of Rio de Janeiro (UFRJ) IBILCE/UNESP, São José do Rio Preto- SP coevolutionary information to improve protein-protein approximation of IL’s alkyl chain to protein’s tryptophan. ([email protected]). interactions modeling. Specifically, DSM takes as In the last stage IL micelles start to form and, therefore, In regular functioning cells in any tissues, the input 3D models generated from docking programs interaction with protein reaches a saturation point. We information stream is mediated by innumerable The human Respiratory Syncytial Virus (hRSV) is and a multiple sequence alignment based on these believe this work provides new information about the signaling pathways that, normally, are found to be a major cause of lower respiratory tract infections models. At the heart of the algorithm, a new alignment interaction of ILs with model proteins, indicating its regulated in a precise way to control its own survive, in children under five years of age, elderly and exclusively containing amino acid positions wich are in ability to alter the conformation of the same. growth, differentiation and apoptosis. However, during immunocompromised people. Among the 11 proteins protein-protein interface is generated for each docking the cell proliferation process, it is also susceptible encoded by the virus, the nucleoprotein (N) stands out model. For each new alignment, DSM calculates H35 - Biophysical characterization of feruloil-CoA to aberrant signaling causing the uncontrolled cell for its has importance in the viral replication due to its mutual information (MI), as defined from Shannon’s synthetase and enoyl-CoA hydratase/aldolase growth that can turn into a tumor. The MAP Kinase is participation in the formation of the ribonucleoprotein information entropies, and correlation index (r), which involved in the conversion of ferulic acid to vanillin an important signaling pathway related to uncontrolled (RNP) complex via interaction with viral RNA, reports phylogenetic similarity of protein sequences in growth where the adaptor protein Grb2 plays an phosphoprotein and RNA-dependent RNA polymerase. MSA. The output consists of a plot, in wich models are Juscemácia N. Araujo1 important role by mediating this pathway through its Recent studies showed that N-terminal domain of N weighted by MI or r. For a case study, we docked chains Fabio M. Squina2 and Wanius Garcia1 SH2, C-SH3 and N-SH3 domains. In this study we have protein presents a hydrophobic pocket responsible for A and B of the carbamoyl phosphate synthetase using 1 used fluorescence and NMR spectroscopy to evaluate the interaction with a phenylalanine of the C-terminal different docking programs. Across a space of docking Center for Natural and Human Sciences, University the interaction and dynamics of Grb2-SH2 domain in the Federal of ABC (UFABC), Santo André, Brazil. of phosphoprotein. This hydrophobic pocket has been solutions and taking into consideration the value of MI 2 presence of Coumarin. This small molecule is known a target of investigations for drug design that block the an r, best ranked models in DSM contain the biggest Universidade de Sorocaba (UNISO), Sorocaba, by its antitumor properties despite a lack of information binding between N and P protein in the formation of RNP number of true contacts and are closer to the native Brazil ([email protected]) at a molecular level about its antitumor mechanism. complex. The present work consists in investigating complex. Besides that, the dynamics studies of the Grb2-SH2 the interaction of N-terminal domain (residues 32– Vanillin is one of the main flavoring molecules used in domain in the presence of Coumarin are important to 252) of N protein with quercetin. These polyphenols This work was supported by CAPES and FAPDF the world. The price of natural vanillin varies between evaluate possible induced conformational changes as are hydrophobic molecules that present interesting (#07/2016). US$ 1,200 and US$ 4,000 per Kg, in contrast to the well as different folding states that could affect Grb2- pharmacologic properties as antiviral activity and they price of synthetic vanillin at under US$15 per Kg. Ferulic SH2 domain function on MAP Kinase pathway. could act as potential therapeutic agents against hRSV acid (FA) is one of the potential vanillin precursors in by binding of N protein. The recombinant N-terminus the biotransformation process. FA can be converted This work was supported by FAPESP (2014/17630-0), (32–252) of N protein was obtained by Ni2+ affinity to vanillin by the enzymes feruloyl-CoA synthetase CNPq (442951/2014-0) and PROPG/UNESP (12/2017).

130 131 H37 - CRYSTALLOGRAPHIC STRUCTURE OF H39 - Screening of New Compounds for H40 - EFFECTIVE USE OF THE JARZYNSKI H41 - STATE-DEPENDANT AFFINITY FOR 3-HYDROXYKYNURENINE TRANSAMINASE FROM Tuberculosis and Trypanosomiasis: Structural EQUATION THROUGH THE CHARACTERIZATION GENERAL ANESTHETICS DICTATES CHANNEL AEDES AEGYPTI and Dynamics Studies of Ribose-5-phosphate OF THE UNDERLYING WORK DISTRIBUTION SENSITIVITY isomerase of Mycobacterium tuberculosis and Larissa G. Maciel1 Leonardo Boechi2 Leonardo Cirqueira,1 Trypanosoma cruzi Andrew A. Oliveira2, Rafael V. C. Guido2, Janaína V. Mehrnoosh Arrar1, Fernando Martin Boubeta1, Maria Letícia Stock1, Caio Souza1 and Werner Treptow1 Bartkevihi, L.1,2 dos Anjos1, Thereza A. Soares1 Eugenia Szretter2, Mariela Sued2, Daniela Rodriguez2 1Department of Cell Biology, Biology Institute, Martins, B.3; Oliveira, D.M.P.3; Pires, J. R.2; 1 1Department of Fundamental Chemistry, Department of Chemistry, University of Buenos University of Brasília, DF ([email protected]) Anobom, C. D.3 and Almeida, F. C. L.1,2 2 Federal University of Pernambuco, Recife, PE Aires, Argentina and Instituto de Calculo, 2 1 ([email protected]) and São Carlos Centro Nacional de Ressonância Magnética University of Buenos Aires, Argentina. (lboechi@ Since their discovery, general anesthetics allowed a big Institute of Physics, University of São Paulo, São Nuclear Jiri Jonas, UFRJ, Rio de Janeiro, ic.fcen.uba.ar) change in medicine, making possible some procedures 2 Carlos, SP. Brazil. Instituto de Bioquímica Médica, UFRJ, that were unconceived without this class of drugs. Rio de Janeiro, Brazil ([email protected]). The Jarzynski equality is one of the most widely Despite the wide pharmacological descriptions, major 3 Aedes aegypti mosquito is the vector of arboviruses Departamento de Bioquímica, Instituto de Química, celebrated and scrutinized theorems of the last advances in anesthesiology, the general anesthesia such as Dengue and Zika fever, major public health UFRJ, Rio de Janeiro, Brazil 30 years, relating free energy to the external work mechanism remains unknown. Electrophysiology threats. Chemical control remains one of the performed in non- equilibrium transitions. In practice, studies show that the main targets of general current strategies to decrease mosquito population. The neglected diseases are a serious public health the required ensemble average of the Boltzmann anesthetics(GA) are the brain ion channels. There are One potential target for new insecticides is the problem generally found among the poorest populations weights of infinite non-equilibrium transitions is two main points of view that concern the mechanism, enzyme 3-hydroxykynurenine transaminase (HKT), in the world. These diseases affect 149 countries and estimated as a finite sample average, resulting in the the indirect hypothesis, which states that change in a detoxification enzyme of kynurenine pathway. are caused by infectious agents and parasites endemic so-called Jarzynski estimator. Alternatively, the second- membrane properties affects channels, and the direct- Substances containing the 1,2,4-oxadiazole scaffold in tropical and subtropical regions. Although they affect order approximation of the Jarzynski equality, though site hypothesis, which assumes that direct and specific have shown larvicide activity with evidence that these over one billion people, the pharmaceutical industry seldom invoked, is exact for Gaussian distributions interactions between anesthetics and ion channels molecules are potential HKT inhibitors. The inhibitory does not have interest on this research area. Due to and gives rise to the Fluctuation-Dissipation estimator. modulate their behavior. Through direct-site point of activity of eleven 1,2,4-oxadiazole derivatives against limited resources, only 1.3% of the drugs produced Here we derive the parametric maximum-likelihood view, modulation of a protein can be seen as differential recombinant HKT was further evaluated via an enzymatic between 1975 and 2004 targeted neglected diseases estimator of the free energy considering unidirectional interaction between the two states, which suggest that assay with HPLC detection. Therefore, we aim to and tuberculosis. The ribose 5-phosphate isomerase work distributions belonging to Gaussian or Gamma there must be differences in most probable positions investigate the potential of HKT as a molecular target (Rpi) catalyzes the interconversion of D-ribulose-5- families, and compare this estimator to the so-called and in the number of ligands that neighbor the protein. for 1,2,4-oxadiazoles. Towards this end, HKT gene was phosphate and D-ribose-5-phosphate. This enzyme Jarzynski estimator. We further consider bidirectional The molecular target used here is the mammalian cloned into pET-Trx plasmid and expressed by E. coli is important for cellular anabolism, which leads to work distributions belonging to the same families, and voltage-gated potassium channel Kv1.2, which has Rosetta 2 (DE3) strain. Purification steps resulted in a the synthesis of molecules such as nucleotides and compare the corresponding bidirectional maximum- well characterized both open and closed states atomic highly purified protein with approximately 43 kDa. The cofactors. There are two families of Rpi, called type likelihood estimator of the free energy to the Bennett structures and is positively modulated by GA. Using screening of more than 1000 crystallization conditions A and type B. The RpiA is present in most eukaryotes, acceptance ratio estimator. We show that, for Gaussian the atomic models, there were performed extended yielded the collection of 4 HKT crystals dataset from while RpiB is found, almost exclusively, in prokaryotes unidirectional work distributions, Fluctuation-Dissipation (> 200ns) molecular dynamics simulations of both HKT RecLab lineage, collected in Recife-PE. 3D and some basal eukaryotes and fungi. Thus, the enzyme estimator is in fact the parametric maximum-likelihood ion channel conformations embed in a membranous structure has been determined by X-ray crystallography RpiB is a good target for drugs, since it is essential estimator of the free energy, and as such, the most system with fixed sevoflurane, isoflurane or propofol with 2.5 resolution and co-crystalized with its co- to cell growth and there is no human homologue. The efficient estimator for this statistical family. We observe concentrations. From each simulation, the spatial factor PLP. This structure is in its final refinement purpose of this project is the identification of ligands that the maximum-likelihood estimator performs better mean probabilities of occurrence were acquired. In steps, showingÅ R-work and R-free about 18% and for proteins RpiB of Mycobacterium tuberculosis than the so-called Jarzynski estimator and Bennett positive modulators (sevoflurane and isoflurane) there 23%, respectively. Aedes aegypti HKT is very similar (MtRpiB) and Trypanosoma cruzi (TcRpiB) and analyze acceptance ratio estimator, for unidirectional and were differences between the open and closed states. to its homologue Anopheles gambiae HKT, showing the protein-ligand interaction by NMR techniques bidirectional distributions. These results illustrate that In addition, simulations with propofol, an insensitive RMSD of 0.4 and 79% identity. However, Aedes such as chemical shift mapping. Both enzymes were the characterization of the underlying work distribution ligand, will check if the different interaction hypothesis aegypti HKT and its human homologue kynurenine heterologous expressed in Escherichia coli and purified permits an optimal use of the Jarzynski equality. also explain the insensitive cases. These results offer a aminotransferaseÅ II share only 21% identity and show from cell lysate in a two-step process of nickel affinity new and simpler way to see ligand-modulated protein RMSD of 10.4 , revealing great differences between and gel filtration chromatography. Isotopically labeled This work was supported by CONICET, ANPCyT, CeCAR activation, and helps to explain a single component of a these two enzymes. These structural comparisons samples were produced and NMR data was acquired. and Bunge & Born Foundation. more complex event: the general anesthesia. will be importantÅ in improvement steps of these HKT Currently, MtRpiB backbone resonances were assigned probable inhibitors. Molecular docking calculations and TcRpiB experiments are being acquired. In parallel, This work was supported by CNPq, FAPDF and CAPES. are in progress to evaluate the most promising HKT some screening experiments were performed and ligand inhibitors between the eleven previously tested. candidates were selected. The structural, dynamic and interaction data obtained by NMR will be essential to This work was supported by CNPq, FACEPE, BioMol- generate information for design of high affinity and CAPES and Cibfar-FAPESP. specificity inhibitors.

132 133 H42 - OPTIMIZED FLOODING MOLECULAR H43 - Biophysical Characterization of H44 - Biophysical studies of Substrate H45 - Electrostatic interactions between DYNAMICS ANALYSIS a Freshwater Shrimp Hemocyanin of Binding Protein (SBP) PotF of Xanthomonas polyelectrolytes and proteins 1 Macrobrachium acanthurus (HcMa) citri Letícia Stock Luan C. Marques1 1,2 1,2 1 1 Ramos, L. Lilia Iriarte Leonardo Cirqueira and Werner Treptow Daniel L. Z. Caetano1 and Sidney J. Carvalho1 Faria, G.H.A.2; Souza, C.O.1,2; Oliveira, J.B.S.1,2; Aline Sampaio1, Cristiano Pinto de Oliveira3, Gabriel Laboratório de Biologia Teórica e Computacional, 1 Santiago, P.S.2 S. Vignoli Muniz4, M. Teresa Lamy-Freund4, Andrea Department of Physics, Sao Paulo State University Departmento de Biologia Celular, Universidade de (UNESP), Institute of Biosciences, Humanities and 1 Balan1,2 Brasília, Brasília-DF, Brazil ([email protected]) Chemistry Institute of Araraquara, UNESP, SP, Exact Sciences (Ibilce), Campus Sao Jose do Rio Brasil; 2Biomolecules Laboratory, Experimental 1Applied Structural Biology Laboratory LBEA, Preto ([email protected]) Understanding how small molecules modulate protein Campus of Registro, UNESP, SP, Brasil (lrg.ramos@ Department of Microbiology, Biomedical Sciences receptors requires knowledge of their molecular bound- hotmail.com) Institute, University of São Paulo ([email protected]), It is known that several biological processes are 2 structure. Nonetheless, such characterization is far from Postgraduate Program in Genetics and Molecular mediated by interactions between polyelectrolytes trivial given modulation frequently involves binding to Hemocyanins are considered giant proteins because Biology, Institute of Biology, State University and proteins, such as the interaction between histone 3 multiple protein sites. The process can depend further they have a molecular weight in the order of 0.45 to of Campinas; Complex Fluids Group, Physics proteins and DNA molecule in the nucleosome. 4 on receptor conformation and ligand chemotype. Front 3.0 MDa which dioxygen carriers in the hemolymph Institute, University of São Paulo; Biophysics Another important example is the aggregation of of this complexity, flooding molecular dynamics (MD) of several crustaceans. The aim of the present work Group, Physics Institute, University of São Paulo. glycosaminoglycans (GAGs) into proteins, such as the simulation are being increasingly employed to characterize is the biophysical characterization of this hemocyanin complex formed by heparin (Hp) and bovine serum ligand-receptor interactions in molecular detail. In a typical by Analytical Ultracentrifugation (AUC), Matrix- Xanthomonas citri is a gram-negative phytopatho- albumin (BSA). In this case, the aggregation is mainly flooding-MD, one simulates a molecular system comprised assisted laser desorption/ionization time-of-flight mass genic bacterium responsible for the citrus canker. given by electrostatic interactions as observed in of a protein receptor embedded in bulk volume containing spectrometry (MALDI TOF MS), optical absorption, In the putative putrescine ABC transporter PotFGHI experimental studies which contacted the dependence a fixed ligand concentration. As simulation proceeds, fluorescence and light scattering (LSI). It was of X. citri, the PotF is the substrate binding protein, of the formation of the Hp-BSA complex with the pH ligands can partition into protein sites in a binding-affinity- utilized 60 shrimp to extraction, the hemolymph was which is associated to the specificity and affinity of and ionic strength of the solution. This study aims to oriented process. From spacial probability densities, high centrifuged at 3,000 x g by 15 min, dialyzed for 12 h the transport. In this work we produced PotF from investigate the interaction between the polyelectrolytes -1 -1 ligand probabilities are delimited in the vicity of the protein, in 100 mmol.L Tris-HCl + 20 mmol.L CaCl2 buffer E. coli DH5α cells, purified it using the immobilized and macromolecules with heterogeneous charge thus solving ligand binding to specific sites and respective at pH 7.0. Followed of ultracentrifugation at 250,000 metal affinity chromatography followed by size- distributions, like proteins, by means of Metropolis binding-affinities. While flooding-MD offers straightforward x g by 5 h. AUC experiments were performed in 100 exclusion. To identify its ligand, protein was used Monte Carlo simulations. The protein was represented implementation, limited computational power imposes mmol.L-1 Tris– HCl + 50 mmol.L-1 NaCl at pH 7.0 and for a set of biophysical assays. Small angle X-ray as a spherical cavity with an arbitrary point-like charge these simulations are carried out at high, non-physiological 20 °C with the protein concentrations of 100, 200 and scattering (SAXS) showed the protein suffered distribution. The polyelectrolyte was modeled by a chain ligand concentrations as a way of accelerating sampling 300 μg.mL-1. The spectroscopies measurements the structural changes in the presence of the poliamines of charged hard spheres connected by a harmonic convergence. Hence, a naturally emergent question HcMa concentration was 1.63 mg.mL-1, prepared in 30 putrescine and spermidine getting a more stable potential. The electrolyte solution was represented is how does results obtained at such extreme, if very mmol.L-1 acetate-phosphate-borate buffer at pH range structure. The Kratky plot and the Porod-Debye implicitly through the Debye-Hückel theory, where useful, conditions compare to physiologically-relevant of 3.7 to 10.2, equilibrated for 2 h. It was obtained 3.0 plot indicated change in the flexibility of PotF when the electrostatic interaction among the monomers of concentrations. We thus wish to put forth an optimized mL of HcMa (428 mg.mL-1). The AUC data showed that in the presence of ligands, making it more rigid. the polyelectrolyte is given by the screened Coulomb flooding-MD analysis to assess concent ration- dependent HcMa presents sedimentation coefficient of 19S and Putrescine-PotF interaction was also measured potential. The interaction between the polyelectrolyte ligand interaction to multiple, saturable receptor sites. The molecular mass (MM) of 450 kDa. MALDI TOF MS data using UV-Vis absorption spectroscopy, steady- and the protein is calculated using the model proposed work builds on previous contribution dealing with ligand- suggest that monomers of 75 kDa form the polypeptide state and time-resolved fluorimetry as a function by Kirkwood. The effects of salt concentration, charge binding to membrane proteins in the limit of dilute bulk, via chain. The absorption spectra showed an absorption of putrescine concentration. Tryptophan intrinsic distribution of the protein and polyelectrolyte length docking-FEP approach. Presently, by adopting an innovative intensity maximum in wavelength scan (λ) the 340 fluorescence of PotF in presence of increasing on the energetic and conformational properties of the coarse-graining procedure, we avoided arbitrary binding nm in the pH range from 4.5 to 9.0. Fluorescence amounts of putrescine showed an increasing polymer were investigated. Analyzing the dependence site definition to resolve ligand interactions to functionally data presented three HcMa forms and were revealed of fluorescence and indicating that PotF binds of the mean binding energy and the radius of gyration distinguishable protein regions from flooding-MD densities. changes in the tertiary and quaternary structure. It is putrescine with high affinity. We are working to with the ionic strength, it was possible to identify Affinity constants thus solved are then corrected for non- worth mentioning that in the pH range from 4.0 to 4.8 get affinity parameters of PotF in the presence of discontinuous transitions between bound and unbound dilute conditions and used to extrapolate space- and state- an increase was observed followed by the decrease other ligands. Altogether, these results provide the states, with critical ionic strength dependence on the dependent ligand density distributions over a range of bulk of the LSI. The HcMa presented MM value similar the basis for further structural and functional studies chain length. Furthermore, the influence of different concentrations, clarifying the stability of multiple protein others shrimp hemocyanin (1 x 6 or 1 hexamer), it of polyamine signal importation system in X. citri charge distributions was also verified. occupancy states at the individual site level. was not possible to accurately determine the pI of the and other species of Xanthomonas genus, since the protein, and according to the results presented a HcMa protein is highly conserved. This work was supported by National Council for This work was supported by Coordenação de isoform shows a pI around 4.5 and other forms with a Scientific and Technological Development (CNPq), Aperfeicoamento de Pessoal de Nível Superior (Capes edital pI between 8.0-10.0. This work was supported by COLCIENCIAS (Colombia) by the Sao Paulo Research Foundation (FAPESP # 51/2013) and by Conselho Nacional de Desenvolvimento and FAPESP. Key words: Xanthomonas citri, ATP-Binding 2018/01841-2) and by resources supplied by the Center Científico e Tecnológico (CNPq 140845/2014-3) This work was supported by Biomolecules Laboratory, Cassette Transporters, PotF protein, SAXS. Grant: for Scientific Computing (NCC/GridUNESP) of the São UNESP. FAPESP, University of Sao Paulo, Colciencias Paulo State University (UNESP).

134 135 H46 - BEYOND GLPG: COEVOLUTION AND H47 - CHARACTERIZATION OF RECONSTRUCTED H48 - INVESTIGATION OF THE DYNAMIC AND H49 - UNDERSTANDING THE STRUCTURAL PHYLOGENETIC ANALYSES OF THE RHOMBOID ANCESTRAL PROTEINS OF VERTEBRATE ENERGY BEHAVIOR OF BENZIMIDAZOLES CONSERVATION AND FEATURES OF THE PROTEIN FAMILY 5-HYDROXYISOURATE HYDROLASES / INHIBITORS AGAINST TRYPANOSOMAL GRASP DOMAIN IN “GOLGI REASSEMBLY AND Lucas Bleicher1 TRANSTHYRETINS CYSTEINE PROTEASES STACKING PROTEIN’ FROM DIFFERENT EUKARYA 1 1 COMPLEXITY 1 2 Lucas Carrijo de Oliveira Lucianna Helene Santos* Paloma Corrêa , Laila Alves Nahum 1 1 2 3 4 Luis Felipe Santos Mendes 1Department of Biochemistry and Immunology, Mariana A. F. Costa , Ana Carolina M. Figueira , Birgit J. Waldner , Julian E. Fuchs , Glaécia A. N. Fernanda A. H. Batista2, Priscila S. F. C. Gomes3 Pereira5, Klaus R Liedl3, Ernesto R. Caffarena2, and Natalia A. Fontana1; Marjorie C.L.C Freire2; Carolina Universidade Federal de Minas Gerais, Belo 1 1 1 1 Horizonte, MG ([email protected]) and 2Instituto and Lucas Bleicher Rafaela S. Ferreira G. Oliveira ; Antonio J. Costa-Filho . René Rachou (IRR), Fundação Oswaldo Cruz 1 Department of Biochemistry and Immunology, 1Depto de Bioquímica e Imunologia, ICB/UFMG, 1Laboratório de Biofísica Molecular. Faculdade (FIOCRUZ), Belo Horizonte, 30190-009, Brazil Universidade Federal de Minas Gerais, Belo Horizonte, 2Grupo de Biofísica Computacional e Modelagem de Filosofia, Ciências e Letras de Ribeirão Preto. Brazil ([email protected]), 2LNBio, Molecular, PROCC/FIOCRUZ, RJ, 3Institute of Universidade de São Paulo. Av. Bandeirantes 3900, 3 Rhomboids are intra-membrane proteases which use Campinas, Brazil and Université de Strasbourg, France General, Inorganic and Theoretical Chemistry, Ribeirão Preto – SP- Brazil. Postal Code: 14040- 4 2 a Ser-His catalytic dyad to cleave proteins inside the University of Innsbruck, Austria, Department of 901. Centro de Pesquisas Aggeu Magalhães - 5 hydrophobic moieties in membranes, which are part of Transthyretin (TTR) is the main carrier of thyroid hormones Medicinal Chemistry, Vienna, Austria, Sys2Diag Fundação Oswaldo Cruz (FIOCRUZ- PE) a homolog family that also includes iRhoms, derlins, in the brain. Evidence suggests the TTR gene arose during UNR, Montpellier, France. *[email protected] and unclassified rhomboid-like proteins; Despite the emergence of vertebrates from a duplication of the The Golgi complex is part of the endomembrane system the fact rhomboids show remarkable diversity at the gene for 5-hydroxyisourate hydrolase (HIUase). Although Chagas disease and Human African Trypanosomiasis are and is responsible for receiving transport cargo from sequence and functional levels, virtually all structure- firstly described in eutherian mammals as a carrier of parasitic diseases caused respectively by Trypanosoma the endoplasmic reticulum to sort and target it to their functional studies available use E. coli GlpG or its close thyroxine (T4), TTR has more affinity to triiodothyronine cruzi and Trypanosoma brucei spp., which lack adequate final destination. In order to achieve such an expressive homolog from Haemophilus influenzae. We investigated (T3) in most vertebrates. At the present work we have treatment. Two cysteine proteases stand out as validated number of functionalities in higher eukaryotic cells, the rhomboid family using Decomposition of Residue computationally reconstructed ancestral sequences therapeutic targets for these diseases, cruzain in T. the Golgi needs to be correctly assembled as a flatted Coevolution Networks used to identify family-wide and for three nodes of the phylogenetic tree generated from cruzi and rhodesain in T. brucei. Here we investigate a membrane sandwich kept by a protein matrix. The class- specific residues potentially related to structure representative vertebrate HIUase/TTR sequeces. In order benzimidazole series that was previously characterized disruption of this assembly ultimately leads to failure and function, and phylogenetic analysis was performed to assess the history of TTR function, we are applying ITC as potent non-covalent competitive cruzain and in correct protein glycosylation and sorting, along with by the Maximum Likelihood method. We identified 12 and doing molecular dynamics simulations to evaluate T3 rhodesain inhibitors with activity against both enzymes. protein secretion impairment. The correct mechanism highly conserved residues and three sets of correlated and T4 binding to the transthyretin ancestors. Moreover, we The reported binding mode of the lead compound 1 was controlling the Golgi cisternae assembly is currently residues, suggesting that there are collectively have already obtained crystals for all three reconstructed used as the initial structure in cruzain, while molecular unknown, but it is already understood the pivotal role conserved patterns related to protein stability and proteins, which will be solved by x-ray diffraction, and docking was used to determine its putative binding mode of the Golgi Reassembly and Stacking Protein (GRASP) ligand recognition and that the mechanism of water we will detect residual HIUase activity using enzymatic in rhodesain. We employed microsecond molecular as main part of the Golgi protein matrix. GRASPs are retention may be class-specific or at least variable. assays. With such experiments, we aim to understand dynamics simulations, MMPB/SA and free energy observed in all eukaryotic cells with the exception of The phylogenetic tree of sequences from five taxa how hormone binding function evolved from a protein calculations to understand qualitative structure activity plants, where no obvious homologues were observed (two bacteria, yeast, human, and the causative agent family involved in uric acid metabolism. relationship trends of the series and to quantitatively so far. Interestingly, the presence of two GRASP genes of malaria) showed that most relationships are well recapitulate them. Simulations revealed that compound is observed in vertebrates, with lower eukaryotic cells resolved. Among the three sets of coevolving residues, This work is supported by CAPES, CNPq and FAPEMIG. 1 is most likely in a protonated state, showing the most having only one and where the correct Golgi cisternae one is a set of three polar residues (two serines and an stable protein-ligand interactions and energy behavior. assembly is not a common feature. In this study, we aspartate) which are very close to the catalytic residues The protonated ligand facilitated electrostatic interactions performed a set of biophysical studies to get insights (suggesting a role in specificity) and are present in due to its additional hydrogen in the benzimidazole on both human GRASP55 and GRASP65 and compare important rhomboids such as human PARL, yeast ring and positive charge in an overall negatively it with two examples of single GRASPs from lower PCP1 and E. coli GluP, but not on the model rhomboid charged binding site, while in its absence interactions eukaryotes (S. cerevisiae and C. neoformans). Our data GlpG. This strongly suggests that other rhomboids may with solvent molecules were mostly generated. The suggest that both human GRASPs are essentially different have different features in catalysis which may not be calculated absolute free energies from MMPB/SA also from each other and GRASP65 is more similar to the captured by experimental studies using GlpG. showed an advantage of the protonated state against subgroup of GRASPs from lower eukaryotes. GRASP55 the neutral state for binding to both enzymes. Relative is present mainly in the Golgi medial and trans faces, This work is supported by CNPq, CAPES and FAPEMIG. binding free energies of analogs of 1, calculated through which are absent in both funguses, while GRASP65 is thermodynamic integration, exhibited deviations of located in the cis part. We suggest that the GRASP65 1.1 kcal/mol and 2.2 kcal/mol from the experimental gene is a more ancient one and the paralogue GRASP55 values for cruzain and rhodesain, respectively. These might have appeared latter in evolution, together with the data encourage prospective thermodynamic integration medial and trans Golgi faces. studies to optimize this series and facilitate the prioritization of compounds for synthesis. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (Proc. 2017/24669-8) This work was supported by CAPES, CNPq, FAPEMIG and L’Oréal-UNESCO-ABC.

136 137 H50 - Determination of structure and oligomeric H51 - THE OXIDATION OF AMINO ACIDS H52 - ANALYSIS OF THE NETWORK OF response. Heterozygous HsFH mutations are associated state of sugarcane small Heat shock proteins by RESIDUES AT SITE I OF HUMAN SERUM COEVOLVED RESIDUES IN THE LOW MOLECULAR with a predisposition to autosomal dominant syndrome means of Small Angle X-Ray Scattering (SAXS) ALBUMIN INCREASES THE ASSOCIATION WEIGHT PHOSPHATASE FOLD PROTEIN FAMILY of multiple cutaneous and uterine leiomyomatosis CONSTANT WITH PHENYLBUTAZONE: (MCUL). Renal cell cancer can also form a component Luiz Fernando de C. Rodrigues1 Marcelo Querino Lima Afonso1 EXPERIMENTAL AND TEORICAL EVIDENCES of the complex, and as such it has been described as 2, 2 hereditary leiomyomatosis and renal cell cancer (HLRCC) Glaucia M. S. Pinheiro Carlos H. I. Ramos and 1 Lucas Bleicher¹ 1 Bertozo L. C syndrome. In the present work, we characterized the Leandro R. S. Barbosa 1 2 2 1 Department of Biochemistry and Immunology, 1 Tavares Neto E Oliveira LC and Ximenes VF N107T mutant HsFH (N107THsFH)properties, which was Institute of Physics, University of São Paulo, Federal University of Minas Gerais, MG identified in MCUL patients. Biophysical and biochemical São Paulo, SP (luiz.camargo.rodrigues@usp. 1Department of Chemistry, Faculty of Sciences, ([email protected]) 2 characterization of N107THsFH will shed light over the br) and Institute of Chemistry, State University of UNESP - Bauru, SP (luiza_bertozo@yahoo. relationship between the mutation and the disease. The 2 Campinas, Campinas com.br) and Department of Physics - Institute The Low Molecular Weight Phosphatase fold protein mutant was produced by QuikChange™ site-directed of Biosciences, Humanities and Exact Sciences, family has importance in various eukaryotic and mutagenesis protocol using the native enzyme as model. Molecular chaperones compose a large and diverse UNESP - São José do Rio Preto, SP, Brazil. prokaryotic cellular signalling networks. Its proteins The protein was purified by affinity chromatography and family of proteins which prevents and corrects protein influence various diseases such as cancer, diabetes and its homogeneity was evaluated with Dynamic Light misfolding, and the small heat shock protein (sHsp) class Human serum albumin (HSA) is constantly exposed to tuberculosis. Many enzymatic functions are displayed Scattering (DLS) and size exclusion chromatography. acts particularly on cell response to high temperatures. an oxidative stress environment. It has been shown that by this protein family, the most important being the N107THsFH was crystallized usingPEG 10000 as the sHsps are a diversified class of chaperones that modification of amino acid residues in HSA by oxidation dephosphorylation of Tyrosines, Arginines, Ribulosamines precipitant agent. The crystal structure was solved at acts as holdases, stabilizing the substrate protein, may influence the ability of the protein as a drug carrier, and Erythrulosamines, and the reduction of the Arsenate 2.3 Å resolution and DSF experiments showed that the preventing aggregation, for it to be refolded by another particularly in multi-therapies. In this work, the amino ion to Arsenite. Mutiple characterizations, structures and mutant protein melting temperature is 10 °C lower than class of chaperones, called foldases. sHsps may form acid residues Trp-214 and Lys-199, which are located at site-directed mutagenesis informations related to this the native enzyme. Although this mutation is found in the oligomers of varying size and may bind to a wide array site I of HSA, were experimentally and computationally protein family can be found in the literature. In this work, active site, the mutant enzyme is still active and its kinetic of different peptides. Although structurally different, this oxidized, and the effect in the binding constant with we aimed to describe the biological functions related to parameters were determined based on the reversible class of chaperones has in common an α-crystallin phenylbutazone measured. HSA was submitted to the correlated and anticorrelated residue sets extracted reaction mechanism of catalysis where the effect of domain between the C-terminal and the N-terminal, two mild oxidizing reagents, taurine monochloramine from the Pfam database Multiple Sequence Alignment for product on forward velocity is taken into account.

being the latter responsible for binding with target (Tau-NHCl) and taurine dibromamine (Tau-NBr2). The this family. We found a clear pattern of residues related proteins. In sugarcane the sHsp17.2 and sHsp17.9 are oxidation of Trp-214 provoked spectroscopic alterations to the Tyrosine Phosphatase and Arsenate Reductase This work was supported by Coordenação de expressed the most and the current hypothesis is that in the protein which is consistent with the formation classes in the network and describe here new possible Aperfeiçoamento de Pessoal de Nível Superior (Capes) their mechanism of action is given by these proteins’ of N′-formylkynurenine. It was found that the oxidation important positions to be explored in future experimental , oligomers being dissociated in dimers, which would be of HSA by Tau-NBr2 provoked significant increase studies. By comparing regions from the structures of H54 - EXPLORING CONFORMATIONAL more efficient in substrate binding. It is known that most in the association constant with phenylbutazone. Arsenate Reductases to the corresponded regions in the TRANSITIONS AND FREE ENERGY PROFILE sHsps form quite large oligomers, but little information These experimental findings were corroborated by structures of Low Molecular Weight Phosphatases, we OF PROTON COUPLED OLIGOPEPTIDE on their structure and mechanism of substrate binding is computational simulation. The energy of interaction of found structural differences possibly related to the different TRANSPORTERS available. For a better understanding on how this class phenylbutazone decrease of -21.3 kJ/mol to -28.4 kJ/mol catalytical mechanisms displayed by these two enzyme of chaperones works and aid in the cell proteostasis in a and to -33.9 kJ/mol when the protein was modeled with classes. In the future we plan to mutate residues from Co- Batista, M. R. B.1 structural level, the present work uses SAXS for it, giving Trp-214 and Lys-199 altered, respectively. In summary, evolution Sets conserved in LMW-PTPs still unexplored by Costa-Filho, A. J.1 tridimensional information on protein structure and also this is a direct evidence of experimental and theoretical the literature in order to describe their function. 1 on protein-substrate dynamics by examining oligomer oxidative alterations of amino acid residues at site I of Department of Physics, Faculty of Philosophy, formation and dissociation in solution. HSA and the consequent effect in its binding efficacy. Sciences, and Letters of RibeirãoPreto, University of H53 - CHARACTERIZATING THE RECOMBINANT São Paulo, São Paulo, SP ([email protected]) These results strength the proposal that oxidative stress N107T MUTANT HUMAN FUMARATE HYDRATASE This work was supported by Conselho de may alter the physiological properties of HSA. Aperfeiçoamento de Pessoal de Nível Superior (PROEX Mariana A. Ajalla Aleixo1 Proteins involved in peptide uptake and transport belong – 0487) and by FAPESP (2012/50161-8). This work was supported by FAPESP (Fundação de Amparo Maria Cristina Nonato1 to the proton-coupled oligopeptide transporter (POT) à Pesquisa do Estado de São Paulo, #2016/20549-5; 1 family. Crystal structures of POT family members #2016/22014-1 and INCT.Bio.Nat #2014/50926-0), Department of Physics and Chemistry, School reveal a common fold consisting of two domains of six CNPq (Conselho Nacional de Desenvolvimento Científico of Pharmaceutical Sciences of Ribeirão Preto, transmenbrane α helices that come together to form a “V” e Tecnológico (#302793/2016-0 and #442352/2014- University of São Paulo, Ribeirão Preto - SP, Brazil shaped transporter with a central substrate binding site. 0) and UNESP-PROPe (Primeiros Projetos - 09/2016). ([email protected]) Proton coupled oligopeptide transporters operate through an alternate access mechanism. This mechanism involves Fumarate hydratases (FH) are proteins responsible for at least three states: an outward-facing state, where the the stereospecific hydration of fumarate into L-malate. ligand binding site is exposed to the extracellular side, an Fumarases are usually classified into two distinct classes occluded state, and finally an inward-facing state, where according to oligomeric state, sensitivity to oxygen and the ligand and ion dissociate into de cell. Conformational metal-dependence. Human fumarase (HsFH) belongs to transitions are promoted by proton and ligand binding, class II and in mitochondria is part of the tricarboxylic however, due to the absence of crystallographic models acid (TCA) cycle, and in the cytosol can act in the of outward-open state, the role of H+ and ligands are metabolism of amino acids and is required for protection still incomplete. To provide a comprehensive picture of from double strand breaks, as part of the DNA damage the POT conformational equilibrium, conventional and

138 139 o enhanced sampling molecular dynamics simulations thermostable with a melting temperature 6 C higher. H57 - ENGINEERING ARTIFICIAL ZIKV EPITOPE same genome interacting proteins, the memory of their of PepTso and PepTst in the presence or absence of By DLS the construct 72-312 displays around 15% of BASED MONOBODY AS A POTENTIAL VACCINE stability-driven process may be readly known once the ligand and protons were performed. Free energy polydispersitivity, therefore suitable for crystallization CANDIDATE multiple-sequence alignment (MSA) concatenation is profile of the conformational variability of POT proteins assays (under development). DSC experiments showed given by the genome. In nature, however, there is a class were obtained from microseconds of adaptive biasing possible interaction within TcNTR and liposomes due to Marjorie C. L. C. Freire1 of protein systems that coevolves in separate genomes force (ABF) simulations. Our results demonstrated changes in the phase transition enthalpy of the lipid that Carlos H. B. Cruz1, Catarina M. C. S. de Araújo1, under the same energy constraints, e.g.: pathogen and that both, proton and ligand, change significantly the showed to be correlated to incubation time. Bruno H. de S. Leite1, Isabelle F. T. Viana1, Natália host-cell proteins. For those system, the synchronism conformational free energy profile. The wider free energy A. Fontana2, Luis Felipe S. Mendes, Antônio J. da of compensatory mutations is unknown. Here, we well for His61 protonated PepTso indicates a greater This work was supported by CAPES, FAPESP and CNPq. Costa Filho2, Roberto D. Lins1. investigate whether the molecular coevolution process of conformational variability relative to the Apo system. In two interacting proteins, A and B, can be recovered from 1 room temperatures, for deprotonated transporters, the Aggeu Magalhães Institute, Oswaldo Cruz their MSAs. Specifically we are interested in dissecting H56 - INTERACTIONS OF SCHISTOSOMA Foundation, Recife, PE and 2Laboratory of only accessible conformation is the inward-open. After MANSONI SEPTINS WITH MEMBRANE MODELS the information in terms of compensatory mutations at proton binding to His61, the transporter is able to shift Molecular Biophysics, Department of Physics, physically-coupled and uncoupled amino acids from between all three conformations. Similar effect was Marina G. Fontes1 University of São Paulo, Ribeirão Preto, SP. proteins A and B. For this purpose, we calculate proteins observed after ligand binding. Ana Eliza Zeraik1, Ricardo De Marco1, Amy S. A and B mutual information (MI) between coupled (amino Gladfelter2 and Ana Paula U. de Araújo1 Zika virus (ZIKV) is a flavivirus that belongs to the acids within 8Å) and uncoupled (farther than 8Å) amino Flaviviridae family, transmitted by Aedes sp. mosquitoes, acids for three case study systems. On average, the H55 – BIOPHYSICAL STUDIES OF TRYPANOSOMA 1São Carlos Institute of Physics, University of São CRUZI NITROREDUCTASE ENZYME: A POTENTIAL and closely related to dengue virus (DENV). Given the coupled amino acids MI is bigger than that of uncoupled Paulo, São Carlos ([email protected]) and recent outbreak of Zika virus (ZIKV) in the Americas in ones, nonetheless both coupled and uncoupled amino ANTICHAGASIC DRUG TARGET 2 Biology Department, University of North Carolina, 2015 and the association with microcephaly cases and acid’s MI degeneracy are smaller than the coupled Marilia L. Cirqueira1 Chapel Hill. neurological disorders, ZIKV was considered a public amino acids’ MI. Using MI maximization simulations, Mariana A. A. Aleixo1, Pedro H. Luccas1, Luiz H. S. health emergency of international concern by the World we show that both coevolutive (coupled) and evolutive Andrilli2, Maytê Bolean2, Pietro Ciancaglini2, Shane Septins are GTP-binding proteins that can form hetero- Health Organization (WHO). Up to date there is neither (uncoupled) sources of information are required to R. Wilkinsson3, Antônio J. Costa-Filho2, M. Cristina oligomeric polymers. Even though these proteins prophylactic treatment nor vaccine available against improve accuracy in determining coevolutionary Nonato1. are involved in numerous cellular processes, little is ZIKV infection. In this context, B cell epitope sequences protein-protein interactions starting from arbitrary MSA known about the molecular mechanisms that drive from ZIKV E protein have been identified using in silico concatenations. In conclusion, a maximum MI criteria, 1 Faculdade de Ciências Farmacêuticas de Ribeirão polymerization and membrane binding, especially in non- epitope prediction tools. The E1 epitope corresponding considering all amino acids of proteins A and B, could 2 Preto, S.P., Brazil. Faculdade de Filosofia Ciências human septins. Four septin genes have been described to the viral fusion loop, a highly conserved region among be used to resolve the coevolution process for protein 3 e Letras de Ribeirão Preto, S.P., Brazil. School of in Schistosoma mansoni, one of the causative agents Flavivirus, was used to construct the chimeric protein systems in which it is unknown. Biological and Chemical Sciences, Queen Mary, of schistosomiasis. Previous results from our research Monobody 1, based on domain III of human fibronectin. University of London, London, United Kingdom. group have shown that septins of S. mansoni are This protein was expressed in bacterial system, purified ([email protected]) H59 - INVESTIGATION OF AN ALLOSTERIC SITE capable of binding membrane models. Here, we aimed and biophysical assays were performed for the evaluation ON ZIKA VIRUS NS2B/NS3 PROTEASE to investigate if the membrane phospholipid content of its stability. The results showed that Monobody 1 Benznidazole and Nifurtimox are currently the only drugs and curvature are critical to determine the binding of presented a profile of high structural stability against the Naiá Porã Santos1 available for the treatment of Chagas disease. They have septins. A comprehensive approach comparing the evaluation conditions and this opens possibilities for the Jian Lei2, Rolf Hilgenfeld2, Rafaela Salgado Ferreira1 been used for more than 50 years and are characterized behavior of S. mansoni septins with those from the use of this chimeric protein and this type of strategy for and Lucas Bleicher1 by low effectiveness in the chronic phase of the disease, invertebrate chordate Ciona intestinalis was performed. vaccination purposes for the prevention of ZIKV infection. 1 high toxicity, and cases of resistance have been reported. Data from confocal fluorescence microscopy showed Department of Biochemistry and Immunology, The drugs are nitroheterociclics considered prodrugs the binding of all different septins tested was dependent This work was supported by Coordenação de Universidade Federal de Minas Gerais, Belo 2 and believed to be activated bythe nitroreductase enzyme on the membrane curvature, with optimum binding Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), Horizonte, MG ([email protected]) and (NTR), absent in the human host. The Trypanosoma occurring in membranes attached to spheres of Biomol-CAPES, FAPESP, CuraZika Foundation and Institute of Biochemistry, Center for Structural and cruzi NTR (TcNTR) is a type I nitroreductase protein, 0.96 μm diameter. In contrast, it was verified that C. Laboratório Nacional de Computação Científica (LNCC). Cell Biology in Medicine, University of Lübeck, mitochondrial, oxygen insensitive that have a flavin intestinalis septin complex binds mostly to membranes Lübeck, Germany. while S. mansoni septins mononucleotide as prostetic group and uses NADH as an composed of 10 mol% POPS, H58 - DECOMPOSITION ANALYSIS OF mainly interact with membranes containing PI(4,5)P . electron donor. Little information about TcNTR structure 2 MUTUAL INFORMATION IN PROTEIN-PROTEIN Since the 2015 outbreaks of Zika virus infection This data is available, although necessary for the understanding of suggest that septin binding to membranes INTERACTIONS worldwide, the academic community has turned the mechanism of activation of nitroheterociclic drugs. is a intricate process that is strongly dependent on attention to the better understanding of the effects of this Our goal is the characterization of two gene constructs membrane characteristics. Miguel de S. Andrade1 flavivirus infection, which, although nonsymptomatic of TcNTR (from residues 80-312 and 72-312) using Camila Pontes1 and Werner Treptow1 in 80% of the cases, was proven to be associated with biophysical techniques such as ThermoFMN, a thermal This work was supported by São Paulo Research severe outcomes such as Guillain-Barré syndrome and shift assay monitoring the fluorescence of the cofactor Foundation (FAPESP 2016/13961-7). Department of Cell Biology, University of Brasília, fetal neuronal abnormalities, especially microcephaly. Flavin mononucleotide (FMN), and Dynamic Light Brasília, DF ([email protected]) Our research group has been investigating amino acid Scattering (DLS). And Differential Scanning Calorimetry positions of structural and functional importance for (DSC) were used in order to evaluate a possible Protein-protein interactions are critical for many cell flaviviral NS2B/NS3 proteases with the main goal of interaction of the enzyme with liposomes composed processes. Pairwise-interaction proteins have coevolved targeting this enzyme for Zika virus inhibition due to its of 1,2-dipalmitoyl-sn-glycero-3- phosphocholine to maintain their bound free-energy stability generating a crucial role on viral infectivity. In this work, our efforts are (DPPC, 2,95 mg/mL). The construct designed by us, series of primary-sequence variants with compensatory focused on the investigation of a hypothetic allosteric site comprising the residues 72 to 312, shows itself more substitutions at physically-coupled amino acids. For that could negatively modulate enzyme activity. Therefore,

140 141 our work is based on cysteine-tethering- based-covalent- the intrinsic fluorescence of fibrils, and the technique of all the HIUases and transthyretin from the Swiss-Prot, H63 - Structure and mechanism of action of compound binding targeting a cavity on Zika virus`s FLIM, to prove that Grh1 does fibrillate in E. coli, when and obtained an average F1 Score of 0.85 from the Brazilian Klebsiella carbapenemase - BKC-1 NS2B/NS3 formed by amino acids that have conserved the cells are heated. Furthermore, we saw the same GPCRs classification. This approach was still applied their identity and structure within flaviviral NS2B/NS3 type fluorescence in yeast when submitted to stress to deorphanize sequences. We are also presenting Paola Silva1 proteases, and which has been shown to be an allosteric situations. Although these data does not confirm that the a software to apply and visualize the results of this Adriana Giannini Nicoletti3, Ricardo José Soares site in the Dengue virus homolog. The Ala125 residue fibrillation in yeast is due to Grh1, we now know that methodology. Torquato2, Pedro José Barbosa Pereira4, Gales, was chosen to be mutated for a cysteine, thus enabling there is a fibrillation process occurring when the yeast A.C3, Vitor Oliveira1 and Marcelo F.M. Marcondes1. covalent binding by cysteine-reactive ligands and starts to use UPS routes. This alone is a major break H62 - KINETIC CHARACTERIZATION AND 1 2 evaluation of enzymatic activity under possible allosteric through, and can potentially help understanding several Department of Biophysics; Department of CRYSTALLIZATION OF LEISHMANIA Biochemistry; 3Laboratório Alerta Division of influence. We expressed and purified both WT and open issues. We are now working with Grh1 partners, BRAZILIENSISTRYPANOTHIONE REDUCTASE. A125C mutant proteases and evaluated activity effects and trying in vivo experiments, hoping to get more insight Infectious Diseases - Universidade Federal de São 4 of covalent-cysteine-binding ligands. There was up to into Grh1 functions. Olívia Teixeira1 Paulo (UNIFESP), São Paulo and IBMC - Instituto de Biologia Molecular e Celular, Universidade do 88% of enzymatic activity inhibition of A125C NS2B/NS3 Marcelo Castilho2, Gean Marcelo Costa2, Humberto Porto. ([email protected]) and no effect on WT, assessed by a fluorimetric assay. This work was supported by CNPq and FAPESP Fonseca Freitas2, M. Cristina Nonato1 Even though further experiments are required (such as 1Faculdade de Ciências Farmacêuticas de Ribeirão investigation of conservation of native structure of A125C H61 - Modeling and Application of Residue Co- Multidrug-resistant Gram-negative bacteria represents a mutant and the confirmation of covalent bond with Preto, Ribeirão Preto, S.P., Brazil.(Olivia.teixeira@ major public health problem worldwide especially those Evolutionary Networks for Specificity Determinant usp.br). 2Universidade Federal da Bahia, Salvador, ligands), our results already suggest that the Zika NS2B/ Sites Detection with carbapenem resistance, since carbapenems are NS3 may possess a promising allosteric site which may BA, Brazil considered the last resort to treatment these infections. 1 be a target for non-competitive inhibitors. Neli Fonseca BKC-1 is novel β-lactamase identified in clinical samples Marcelo Querino¹, Lucas Carrijo¹ and Lucas Leishmaniasis is a group of parasiticdiseases which of Klebsiella pneumoniae at two hospitals in the city of This work is supported by CNPq, CAPES, FAPEMIG and Bleicher1 puts350 million individuals at risk. It is caused by São Paulo in the year 2008. This enzyme belongs to L’Oréal-UNESCO-ABC. parasites of the genus Leishmania.Treatment of class A of Ambler and exhibit an extended spectrum of 1 Department of Biochemistry and Immunology, Leishmaniasis is unsatisfactory. Most available drugs action, including the ability to hydrolyze the carbapenems Federal University of Minas Gerais, Belo Horizonte, are costly, require long treatment regimes and are compounds. The main objective of our work was to study H60 - S. cerevisae Golgi Reassembly and MG ([email protected]) Stacking Protein: an unusual behavior for unusual becoming more and more ineffective.Trypanothione the mechanisms of action of BKC-1 through structural processes reductase, considered a therapeutic target against analysis, for this purpose single crystals of BKC WT in Computational studies of molecular evolution are leishmaniais, is a homodimeric flavoprotein that complex with different ligands were obtained thought Natália A. Fontana1 usually performed from a multiple alignment of presents approximately 53 kDa per subunit. It catalyzes soaking methodology, and subsequent X-ray diffraction Raquel F. Maldonado2, Luis F.S. Mendes1 and homologous sequences, on which sequences resulting two substrates according to the mechanism of double data were collected on LNLS - Campinas, SP. Three- Antonio J. da Costa-Filho1 from a common ancestor are aligned so that equivalent exchange or ping-pong,and its first reaction consists dimensional structure of BKC-1 shows that this enzyme residues are placed in the same position. Multiple in the formation of an enzyme-substrate complex with has a structure like other β-lactamases of class A of Ambler. 1 Laboratory of Molecular Biophysics, Department of sequence alignments frequency patterns can be highly NADPH, which donates its electron to the FAD domain The absence of the Cys238 residue and the insertion of a Physics,University of São Paulo, Ribeirão Preto, SP useful for suggesting positions under selection. Almost (creating the FADH anion), with NADP + being its first Tyr residue in the Ω loop suggest that a major degree of 2 ([email protected]) and São Paulo Federal Institute all methods proposed to identify specificity determinant product. The enzyme then, modified into its original protein flexibility may allow conformational changes and of Science and Technology, Jacareí, SP - Brazil sites requires a priori classification of the subfamilies. form, catalyzes the reduction of its second substrate, / or changes in the catalytic pocket, which can explain In addition, many of these are impractical for very trypanothione,comcomitant with the flavinreoxidation. the carbapenemase activity. No ligands could be observed GRASPs are proteins related to the maintenance of the large alignments, such as those obtained from Pfam. The cloned enzyme fused with the NusA protein was in the structures obtained from the diffraction data of the Golgi complex, keeping the several stacks together. In this paper we apply the complexes networks theory expressed in E.coli/Roseta(DE3).Its purification was complexes. Our group are working to obtain the inactive While in other organisms this role is very clear given the to map groups of functional related residues through performed in affinity chromatography with imidazole mutant for co-crystallization trials, and the expression of anatomy of the organelle, in Saccharomyces cereviseae the co-variation model. Physicochemical and structural gradient followed by the cleavage of the Nus A protein new mutants of BKC-1, for kinetic and structural studies. this function has no place, since this yeast has the properties are also considered by adding super with the TEV enzyme and a subsequent purification step Golgi vesicles disperse in the cytoplasm. We have been nodes to the network. This methodology was firstly based on molecular exclusion chromatography.The This work was supported by CNPq and FAPESP. studying Grh1 (the yeast GRASP) and the findings are evaluated in simulated environments and then applied trypanothione reductase kinetics assay was perfomed very interesting. A biophyisical characterization showed to the Transthyretin/HIUase and class A GPCRs protein by monitoring NADPH consumption, stoichiometrically H64 - MOLECULAR MODELS OF HUMAN that the protein is not globular and fully folded, but a families, in which several specificity determinant sets equivalent to the consumption of trypanothione and SODIUM CHANNEL Na 1.4 molten-globule with regions of intrinsic disorder, which and functional motifs were successfully detected. We measured on the Spectra Max Plus 384 microplate V confers higher flexibility. More surprisingly, though, was have identified groups of residues specific of HIUases reader (Molecular devices). The determination of Paulo S. L. Beirão the behavior of Grh1 in certain conditions. We have and of Transthyretins surrounding the T3/T4 binding the kinetic parameters Km and kcat were obtained by João R. C. Fernandes, Lucas Bleicher proved that in vitro, when submitted to high temperatures site, probably relating to the ligand specificity. In the adjusting the data to the ping-pong equation using or different dieletric constants, Grh1 forms amyloid-like GPCR study, we identified specificity determinant SigmaPlot 11 software. The crystallization assays were Program on Bioinformatics, Department of fibrils. Moreover, the conditions that trigger fibrillation are groups for aminergic, alicarboxylic and sensory ligand performed through vapor diffusion, sitting drop, using Biochemistry and Immunology, ICB-UFMG, the same that lead to different routes of protein secretion types, in addition to prostanoid and glycoprotein the sparse matrix protocol, implemented in commercial Universidade Federal de Minas Gerais, Belo in the yeast, and papers have shown the participation receptors. Given the hypothesis that a restricted group crystallization kits Crystal Screen and Crystal Screen II Horizonte, MG - [email protected] of Grh1 in two processes of Unconventional Protein of residues is sufficiently informative to determine (Hampton Research). Secretion (UPS). Given that, our efforts were focused functional subclass of a protein family, a SVM machine Sodium channels play a crucial role in the generation on proving that the fibrillation of Grh1 is a natural was trained in order to test and measure the fitness of and propagation of vertebrate action potentials. Human occurrence, rather than just an in vitro artifact. We used this methodology. We were able to correctly classify sodium channels are composed by one alpha subunit,

142 143 + which contains the Na conducting pore and the voltage- interference by the nitrocompounds. This project aims to group P21 and GenB4 in the space group P212121. Both analysis suggests better preservation of the substrate sensing mechanism, associated with beta subunits. develop a fluorometric assay as a tool for evaluating new enzymes have two molecules in the asymmetric unit and allocability at the active site under heating for both Despite its importance, the structure of human sodium nitroheterocyclic substrates, kinetically characterize their each monomer is divided into 3 domains: N-terminal, dynamically modified point mutants. We expect that channel alpha subunit remains unsolved. Recently the mechanism and compare its efficacy with benznidazole. PLP binding domain and C-terminal domain. These the results be useful in future studies of thermostable structure of cockroach (NavPaS) and eel (EeNav1.4) TcNTR was expressed by E. coli Rosetta (DE3) with a enzymes perform the same function in the gentamicin GH1 engineering for industrial and biotechnological sodium channels have been determined by EM- polyhistidine tag and purified by affinity chromatography pathway, however the substrates are different, possibly applications. cryomicroscopy. In order to obtain a model of the human using Ni-NTA resin (Quiagen). The kinetics parameters GenB3 catalyzes the removal of hydroxylates in a muscle Nav1.4 channel, we used EeNav1.4 and NavPaS have been determined by monitoring NADH oxidation at phosphorphylated substrate, which is not reported for This work was supported by CNPq, CAPES, FAPEMIG structures as templates, using Swiss-Model. Two models 360 and 460nm as excitation and emission wavelengths, any other PLP-dependent enzyme. GenB3 and GenB4 in and L’Oréal-UNESCO-ABC. were obtained with characteristics of closed (hNav1.4/ respectively, in presence of different nitro compounds. binary complex with PLP provides us useful information PaS) and open (hNav1.4/Eel) channel, respectively. In the Calibration curve, sensitivity and concentration range on protein folding and cofactor binding. At moment, H68 - An in silico alternative find and understand open model, the outward displacement of ~8 charges were determined for NADH. Kinetic data for benznidazole we are performing experiments to obtain complexes important residues of catalytic binding sites in across the gating pore could be identified, along with were used as reference. The reaction was started with the with substrates or analogues to elucidate the catalytic Ribose-5-Phosphate Isomerase B from T.cruzi unrestrained conducting pore. Both models were probed addition of enzyme to the mixture of NADH, buffer (Tris mechanism and substrate specificity. with Ramachandran, TM-align, ProSa, ProCheck, with 50mM, pH 7.5) and substrate. The data were adjusted Rafael F. Soares1 results similar to the original templates. hNav1.4/PaS was according Michaelis-Menten model of enzymatic This work was supported by Fundação de Amparo Ana Carolina R. Guimarães2; Ernesto R. Caffarena1 inserted into a model palmitoyl oleyl phosphatidylcholine catalysis. Kinetic assays identified new substrates which à Pesquisa do Estado de São Paulo FAPESP membrane and submitted to molecular dynamics (MD) are currently being tested regarding their trypanocidal (2014/07843-6). Computational Biophysics and Molecular Modeling for 30 ns. We successfully achieved the docking of activity. Group, PROCC, Fiocruz, RJ (rafael.ferreira@ioc. fiocruz.br), 2Laboratory of Functional Genomics and the peptide toxin AaHII to the channel using Haddock H67 - Computational studies of thermostabilizing webserver. considering residues 15, 18, 38 and 64 This work was supported by FAPESP (Project Bioinformatics, Oswaldo Cruz institute, Fiocruz, RJ. mutations in GH1 β-glucosidases as active docking residues. On hNav1.4/PaS, residues 2016/26196-7). between 1430-1445 were involved in the docking. Twelve Rafael E. O. Rocha1 Diseases caused by protozoan members of the hydrogen bonds were identified linking both structures. H66 - STRUCTURAL STUDY OF PLP- Diego C. B. Mariano1, Tiago S. Almeida2, Carlos H. Trypanosomatidae family are one of the major public The stability of the docked complex was confirmed with DEPENDENT ENZYMES FROM THE GENTAMICIN da Silveira3, Lucas Bleicher1, Raquel C. De Melo- health problems in developing countries, in tropical MD for 15 ns. The generated models can account for the PATHWAY THAT PERFORM AN UNUSUAL Minardi1, Leonardo H. F. de Lima2. and subtropical regions of our planet and particularly known properties of the human Nav1.4 channels. in Brazil. According to data from the World Health DEHYDROXYLATION 1 Department of Biochemistry and Immunology, Organization, Trypanosoma cruzi, the etiologic agent Supported by CAPES-Toxinologia and CNPq. Priscila dos S. Bury1 Federal University of Minas Gerais, Belo Horizonte, of Chagas’ disease, infects around 10 million people MG, 2Department of Physical and Biological Fanglu Huang2, Peter F. Leadlay2, Marcio V. B. Dias 1 worldwide, mainly in Latin America. Unfortunately, Sciences, Federal University of São João Del-Rei, H65 - DEVELOPMENT OF A FLUOROMETRIC 1 there are no vaccines to control this disease and the Department of Microbiology, University of São Sete Lagoas, MG ([email protected]) and two currently available drugs are inadequate for several KINETIC ASSAY FOR T. CRUZI NITROREDUCTASE 3 Paulo, São Paulo, SP ([email protected]) Advanced Campus of Itabira, Federal University of reasons: toxicity, acting only in the acute phase of 2 Luccas, P. H.1 and Department of Biochemistry , University of Itajubá, Itabira, MG infection and resistance developed by some strains Cambridge, Cambridge, UK Cirqueira, M.L.1; Aleixo, M.A.A.1; Naal, R.M.Z.G.²; of the parasite. The enzyme ribose 5-phosphate Boechat, N.3; Nonato, M.C.1 β-glucosidases play a key role in the saccharification isomerase (R5PI) becomes a good candidate for a Pyridoxal 5'-phosphate (PLP) dependent enzymes step of second-generation biofuel production. molecular target since the enzyme present in T. cruzi 1 Laboratório de Cristalografia de Proteínas, are involved in various reactions in nature, such as in Thermal and conformational stability are desirable (TcR5PI) is considered analogous to that of Homo Faculdade de Ciências Farmacêuticas de Ribeirão cellular processes, amino acid biosynthesis and amino for these enzymes due the industrial environment. sapiens (HsR5PI), performing the same enzymatic Preto (FCFRP), Universidade de São Paulo (USP), compounds. The PLP is a cofactor, which provides In this work, we investigated the molecular bases of activity despite different primary and tertiary structures. Ribeirão Preto, SP, Brazil ([email protected]); to these enzymes to have a large catalytic versatility thermostabilization by point mutations described by the In this work, we discuss an alternative to describe the 2 Departamento de Química Geral e Inorgânica, because its unique property of electron dissipating. literature in β-glucosidases from family GH1 by a set of possible residues with importance in the catalysis in 3 FCFRP, USP, Ribeirão Preto, SP, Brazil; Fundação Gentamicin is an aminoglycoside that in its clinical computational studies. We carried molecular dynamics R5PI protein through the Networks Analysis. Using Osvaldo Cruz, Rio de Janeiro, RJ, Brazil. form is composed of five biosynthesis intermediates, in simulations (MD) at 300 and 363 K for the Bacillus measures of network’s centrality (Betweenness and which they have several methylations and aminations. In polymyxa β-glucosidase A and two thermostable point degree) it was possible to find significance for C69 Chagas’ disease is caused by the parasite Trypanosoma gentamicin gene cluster were identified several enzymes mutants: E96K (TR1) and M416I (TR2). Clusterization, residue and correlate these data with the protein cruzi and affects about 7 million people worldwide. dependent on PLP, which catalyze different reactions. PCA and dynamic correlation analysis from the MD mutation experiments described by Kaur et al., 2016, The disease was restricted to low-income population, Currently, two PLP enzymes, GenB3 and GenB4, are results suggested that both mutants established a new revealing the potential of the technique for predicting mainly in emergent tropical countries, as Brazil. Due identified in the gentamicin pathway that are responsible dynamic behavior, indicating a higher distribution of important residues in catalytic binding site. To built to globalization, it is now spreading around the world. to remove two hydroxylates from the garosamine ring of their mobility in collective modes of higher amplitude. the networks, the R5PI crystal of T.cruzi (PDB-id In Brazil, the only treatment available is benznidazole, the biosynthetic intermediates. Due to this, the objective Poisson-Boltzmann surface analysis (PBSA) suggested 3K7S) (Stern, Ana L et al., 2011) was used in apo and a nitroheterocyclic prodrug with limited action, high of the work was to perform a structural study of the that, for TR1, this difference in dynamic behavior is due holo states with different states of protonation of the toxicity, activated when a nitro group is reduced through enzymes GenB3 and GenB4 to unravel the catalytic to a higher stabilization of the electrostatic surface residues Cys69, His11, His102, totalizing 8μs of MD the action of the nitroreductase enzyme (TcNTR). There is mechanism and the substrate specificity. Thus, these potential, what in turn results in motions with lower simulations and 16 networks for analysis in triplicate. lack of data regarding NTR mechanism, structure, and an enzymes were overexpressed and the crystals obtained, vibrational internal energy. For TR2, the reason seems urgent need to find compounds with trypanocidal activity. that diffracted in x-rays sources. The obtained data were to be due to a more flexible packaging of the residue 416 This work was supported by Oswaldo Cruz Foundation Unfortunately, the only available colorimetric kinetic assay processed and the calculation of the phase occurred by in the hydrophobic core, leading to a majorly entropic (Fiocruz),by Coordenação de Aperfeiçoamento de available for TcNTR has shown low reproducibility due to MR using online Softwares. GenB3 crystallized in space stabilization of its vibrational dynamics. Virtual docking Pessoal de Nível Superior (CAPES).

144 145 H69 - BIOPHYSICAL CHARACTERIZATION OF H70 - HOW THE ALLOSTERIC DRUG INHIBITION H71 - AMIODARONE ACTION IN ISOLATED H72 - BIOPHYSICAL STUDY OF PPARγ AND EFTENA PROTEIN OF SHP2 PHOSPHASTASE OVERACTIVE CARDIOMYOCYTES: LESSONS FROM A CORREGULATOR INTERACTIONS AND THE ROLE MUTANTS WORKS CELLULAR MODEL OF THE LONG QT SYNDROME OF PPAR ’S PHOSPHORYLATION Raissa Ferreira Gutierrez1 γ TYPE 3 Ricardo A. P. Pádua Tábata Renée Doratioto1,2 Carsten Wrenger2 and Alessandro S. Nascimento1 Beserra S. dos S. Author1 Yizhi Sun Ingrid Marko, Warintra Pitsawong, John Helder V. Ribeiro Filho1,2, Fernanda A. H. Batista 1São Carlos Institute of Physics, USP, São Carlos, , B. Stiller, Renee Otten and Dorothee Kern Roman-Campos D. Author2 and Ana C.M. Figueira1,2 SP ([email protected]) and 2Institute of Biomedical Sciences, USP, São Paulo, SP Howard Hughes Medical Institute, Department of Department of Biophysics, Federal University of 1Brazilian Biosciences National Laboratory (LNBio), Biochemistry, Brandeis University, Waltham, 02454 São Paulo1,2, SP ([email protected]) Brazilian Center for Research in Energy and 2 Bacterial resistance is one of the greatest threats to ([email protected]) Materials (CNPEM), and University of Campinas global health according to the World Health Organization Amiodarone (AMIO) has been widely applied to prevent (UNICAMP), Campinas, SP (tabata.doratioto@ (WHO). The misuse of antibiotics in veterinary and The protein tyrosine phosphatase SHP2 is a key ventricular tachycardia in several types of arrhythmia. lnbio.cnpem.br) human medicine after World War II resulted in an regulator of cell cycle control. SHP2 is a multidomain This drug has multiple effects, including the inactivation important selective pressure for the emergence of protein containing two regulatory SH2 domains (N- and of the long-lasting Ca+2 channel and the adrenergic Peroxisome proliferator-activated receptor γ (PPAR γ) is multidrug- resistant pathogens to known antibiotics. C-SH2) located N-terminally to the catalytic phosphatase β-receptors in a non-competitive manner. The present a protein member of the nuclear receptor superfamily In addition to public policies to contain infections, the domain. Occasionally, its finely tuned activity is work aims to determine the effects of AMIO in the involved in the regulation of genes related to metabolism. research and the discovery of new molecular targets to disturbed by mutations which causes several diseases. contractile properties of isolated left ventricle myocyte PPARγ plays an important role in regulating glucose and treat infections caused by multi- resistant pathogens By dissecting the energy landscape of both wild-type (LVM) in healthy 8 weeks old male C57BL/6 mice lipid metabolism and is therefore related to metabolic is necessary and urgent. Enterococcus faecalis is SHP2 and the cancer-associated mutation E76K we (Mus musculus). First we access AMIO action (0.1, diseases such as diabetes and obesity. Studies show a Gram-positive commensal bacterium present in provide a comprehensive picture of this activation 1 and 10 µM (at 25 and 35°C) in LVM paced at 1, 3 that the phosphorylation of PPARγ in serine 273 on its the normal flora of the human gastrointestinal tract process. Using NMR and X-ray crystallography, we and 5 Hz. Next, we evaluate the antiarrhythmic effect ligand binding domain (LBD) interferes in the regulation and it is associated with nosocomial infections such show that wild-type SHP2 exchanges between a closed, of AMIO (10 µM) (at 25 and 35°C) in LVM exposed of these pathways by changing the interaction of PPAR as bacteremia, endocarditis, urinary tract infections inactive population (89.6%), and an open, activated to toxin ATXII, which induces late sodium current. We with corepressors. To evaluate biophysically the effects and surgical wounds. Our study aims to characterize conformation (10.4%). In the closed conformation the evaluated: percentage of sarcomere shortening, time to of phosphorylation on the activation and repression of the TenA protein of E. faecalis (EfTenA), which is N-SH2 docks in the active site and blocks catalysis. of 50% contraction (T50C) and relaxation (T50R). AMIO PPARγ, fluorescence anisotropy assays were performed annotated as part of the biosynthesis pathway of The oncogenic E76K mutation inverts this equilibrium produced a significant reduction in the percentage of to verify the interaction of PPARγ and its two mutants vitamin B1 (thiamine). The focus of the project towards the open state (95.8%) whose structure was sarcomere shortening in all preparations in comparison that mimic phosphorylation, with different domains involves the validation of this enzyme as a molecular previously unknown. Activation involves a drastic 120° to control groups, especially at 10 µM. A minor effect of interaction (ID) of different co-regulatory proteins. target for antimicrobials. The EfTenA coding sequence rotation of the C-terminal SH2 domain which, in turn, was found for T50C. Also, some groups presented This assay allowed the measurement of differences was amplified from the E. faecalis genomic DNA drags the N- SH2 far from the active site enabling increased in T50R at 10 µM. These results demonstrate in affinity between PPARγ and co-regulators under (DSM20478) and cloned into the pET-TRX-LIC/1a catalysis. Interestingly, binding of the allosteric inhibitor that decreasing in sarcomere shortening is probably conditions mimicking or not phosphorylation, showing expression vector. Preliminary expression tests in E. SHP099 to E76K, despite much weaker, results in an due to reduction in L-type calcium current leading to that phosphorylation may interfere in the process of coli Rosetta (DE3) and purification by nickel affinity identical structure as the wild-type inhibitor complex. lower calcium-induced calcium release mechanism. activation and repression of PPARγ. chromatography showed that the TRX-EfTenA fusion We show that SHP099 cannot bind to the active Moreover, although there was a reduction in arrhythimc protein was super expressed and it is extremely soluble conformation. The reduced affinity is a consequence of a phenotype following ATX-II exposition, at 5 Hz in both This work was supported by Fundação de Amparo in 50 mM Tris-HCl, 150 mM NaCl, pH 8. Biophysical conformational selection mechanism where the binding temperatures, AMIO could not completely eliminate the à Pesquisa do Estado de São Paulo (FAPESP characterization assays, such as SEC-MALS, thermal competent state is only 4.2% populated for E76K. This arrhythmogenic effects produced by the toxin. Thus, our #2016/02348-2). stability and spectroscopic characterization of the weakened affinity combined with E76K’s much higher results suggest that AMIO could have limited potential enzyme confirmed the expression and characterization activity require a very high SHP099 concentration to antiarrhythmic effect in diseases related to late sodium H73 - FUCTIONAL, STRUCTURAL AND EVOLUTIVE of the folded state of the enzyme and defined the best restore WT activity levels. We hope that the differences current augmentation, however drug action could CHARACTERIZATION OF METALLOPROTEINASE conditions for further structural studies. in the structural ensembles and drug-binding kinetics depend on the type of arrhythmia. Additional studies DOMAINS THROUGH CO-VARIATION AND of the cancer-associated SHP2 forms we describe are necessary to confirm our hypotesis. STRUCTURAL ANALYSIS This work was supported by CAPES and FAPESP here will encourage the development of more potent (2015/26722-8). inhibitors for activated SHP2 mutants found in patients. This work was supported by CAPES and by the FAPESP Thainá Miranda1 (2014/09861-1). Marcelo Querino¹, Néli Fonseca¹, Lucas Carrijo¹ This work was supported by CNPq (PDE 233809/2014- and Lucas Bleicher1 7) and by HHMI. 1 Department of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, MG ([email protected])

Metalloproteases are proteolytic enzymes that use a metal for its catalytic mechanism, in which the most common is zinc. Its architecture is commonly composed by three domains: a reprolysin catalytic domain, also called adamalysins, a disintegrin domain and a reprolysin family propeptide domain. Reprolysins are

146 147 endopeptidases dependents of zinc and a subfamily of interaction with the SAM ribose ring. Docking studies the tryptophans in the ordered region of the protein This work was supported by FAPESP (Fundação the M12 metalloproteinases found in snake venoms. The suggest that TYR133 and SER178 hold the phenazine would become exposed to the aqueous environment de Amparo à Pesquisa do Estado de São Paulo, reprolysin family propeptide domain is the propeptide ring of the substrate in place for the methyl transfer. upon binding to membranes. To the best of our #2016/20549-5; #2016/22014-1 and CNPq (Conselho of peptidase family M12B. Desintegrins, also present in Interestingly, an MCSA search (https://www.ebi.ac.uk/ knowledge, out of all GRASPs, only human full-length Nacional de Desenvolvimento Científico e Tecnológico snake venom, consist of a small domain of low molecular thornton-srv/m-csa/search/?s=2.1.1) shows that GRASP55 interacts with membranes. These results (#302793/2016-0). weight proteins. They possess an integrin-binding motif in several N-methytranferases (e.g. PDB ID= 1H3I) would contribute to understanding the proper function located near the C-terminus, commonly composed by a tyrosine residue, positioned similarly to TYR133 in of this protein in the secretory pathway. H77 - Analysis in silico between NF-kB and Arg-Gly-Asp, that determines the integrin specificity. our structure, activates the phenazine nitrogen atom Thiophenoacetamide In this project, a study was carried out with the aim of for a nucleophilic attack on the SAM methyl moiety, This work was supported by FAPESP (2017/12146-0, 1 find specificity determinants and functional related sites suggesting a similar SN2 catalytic mechanism for the 2015/50366-7). Vanessa S. Silva on the metalloprotease proteins families. The domains PhzM-SAM complex. Kinetic studies will be carried out Fatima M. Vergara2,3,4; Maria G. Henriques2,3; families alignments were obtained from Pfam (PF00200, with the Try133Phe PhzM mutant to test this proposed H76 - INTERACTION BETWEEN Ernesto R. Caffarena1 PF01421 and PF01552) and filtered by occupancy and mechanism. 1-PYRENESULFONIC ACID AND ALBUMIN: Computational Biophysics and Molecular Modeling max identity in order to reduce bias. A co-variation MOVING INSIDE THE PROTEIN analysis was conduct with the PFstats software and the This work was supported by Conselho Nac. Des. Cient. Group, PROCC, Fiocruz, RJ (vanessa.silva@ioc. 2 results were interpreted through an extensive literature Tecnologico (CNPq # 310138/2017-5). Ximenes V. F1 fiocruz.br), Applied Pharmacology Laboratory, Farmanguinhos, Fiocruz, RJ, 3National Institute search followed by structural analysis. We identified Bertozo LC1 Philot EA2 Lima AN2 Lara PTR2 Scott communities of residues specificity related to ligands, 2 for Science and Technology on Innovation on H75 - HUMAN FULL-LENGTH GRASP55: AL 4 taxons and to the functional desintegrin motiff. These Neglected Diseases (INCT-IDPN) and Center for STRUCTURE, DYNAMICS, AND MEMBRANE 1Department of Chemistry, Faculty of Sciences, Technological Development in Health (CDTS); findings are going to be further evaluated by molecular INTERACTIONS EXPLORED BY BIOPHYSICAL dynamics and mutagenesis experiments. UNESP - Bauru, SP ([email protected]) Fiocruz, Rio de Janeiro, RJ. STUDIES and 2Laboratory of Computational Biology and This work is supported by CNPq, CAPES and FAPEMIG. Thirupathi R. Soudherpally Bioinformatics, UFABC - Federal University of ABC, Nuclear factor kappa B (NF-κB) is a nuclear factor that 09210-580, Santo André, SP, Brazil. Antonio J. Costa Filho regulates gene expression and binds to a particular H74 - THE CO-FACTOR BOUND STRUCTURE DNA region and plays critical roles in the regulation Department of Physics, University of São Paulo, Due to the high sensitivity to alterations in of many pathophysiological processes, including OF PSEUDOMONAS AERUGINOSA PHENAZINE Ribeirão Preto, SP ([email protected]) M SUGGESTS A CATALYTIC TYROSINE IN ITS microenvironment polarity of macromolecules, pyrene inflammation and immune responses, cell growth METHYLTRANSFERASE ACTIVITY and its derivatives have long been applied in biosciences. and apoptosis. This DNA-binding protein receptor is In mammalian cells, the Golgi apparatus is a central Human serum albumin (HSA), besides its numerous considered an important molecular target to treat many Thamires Q. Froes1,2 hub for intracellular trafficking and post-translational physiological functions, is the main responsible by diseases through host-directed therapy. In this line, Marcelo S. Castilho1, Carla Mattos2 modifications of protein and lipids. The structural transport of endogenous and exogenous compounds several drugs containing thiophene cores have been architecture of the Golgi apparatus depends on the in the circulatory system. Here, a comprehensive study extensively evaluated due to their ability to interfere 1 Department of Pharmacy, Federal University of laterally linked stacks (ribbon-like structures) of was carry out to understand the interaction between on NF-κB translocation to the nucleus. Notably, the Bahia, Rua Barão de Jeremoabo, s/n - Campus flattened cisternae. The Golgi reassembly and stacking HSA and the pyrene derivative 1-pyrenesulfonic acid parent compound N-(Aryl)-2-thiophen-2-ylacetamide Universitário de Ondina, Ondina, 40170-115, proteins (GRASPs) play a pivotal role to maintain the (PMS), which showed a singular behavior when bound aka thiophenacetamide (TAA) has shown the ability Salvador, Brazil ([email protected]), intact architecture of the Golgi and its function in the to this protein. The complexation of PMS with HSA was to inhibit DNA-binding to the NF- B receptor. In this 2 κ Department of Chemistry & Chemical Biology, secretory pathway. However, the structure, dynamics, studied by steady state, time-resolved and anisotropy work, we investigated TAA abilities to act bind to NF-κB Northeastern University, 102 Hurtig Hall, 360 function, membrane interactions of human full-length fluorescence, induction of circular dichroism (ICD) using in silico methodologies. We have identified some Huntington Ave, Boston, MA 02115, USA. GRASPs are still elusive in solution. The lacuna of this and molecular docking. The fluorescence quenching putative interaction sites in NF-κB where TAA could prompted us to explore the complete biophysical and of PMS by HSA was abnormal, being stronger at lower bind. Dynamical behavior of the NF-κB-TAA complex Phenazine M (PhzM) is a class I methyltransferase biochemical properties of human full-length GRASP55 concentration of the quencher. Similar behavior was along with the stability of TAA in these putative binding essential for the biosynthesis of pyocyanin, a virulence in solution. In this context, we have investigated the obtained by measuring the ICD signal and fluorescence sites was analyzed by MD simulations. factor from Pseudomonas aeruginosa. It is currently an secondary structure, thermal stability, and tryptophan lifetime of PMS complexed in HSA. The displacement unexplored drug target for anti- virulence drug discovery. fluorescence of GRASP55 utilizing circular dichroism, of PMS by site-specific drugs showed that this probe This work was supported by Oswaldo Cruz Foundation The previously available apo crystal structure (PDB ID differential scanning calorimetry, and fluorescence occupied both sites, but with higher affinity for site II. (Fiocruz), by the Coordination of Improvement of Higher 2IP2) does not allow the design of transition state spectroscopy, respectively. These results revealed that The movement of PMS between these main binding Level Personnel (CAPES) and by the Research Support analogs, as the co-factor S-adenosylmethionine (SAM) the structure of GRASP55 altered from the ordered sites was responsible by the abnormal effect. Using Foundation for the State of Rio de Janeiro (FAPERJ). donates its methyl group to the substrate PCA and thus to a disordered state in a highly cooperative manner the holo (PDB: ID 1A06) and apo (PDB: ID 1E7A) HSA is an essential component in the reaction. In order to with the addition of the chemical agent or increasing structures, the experimental results were corroborated overcome this limitation we have crystallized phzM in temperature. GRASP55 is a Golgi peripheral membrane by molecular docking simulation. The abnormal complex with SAM (Resolution: 2.2 ). Comparison of protein and plays a significant role in the formation spectroscopic behavior of PMS is related to its binding the apo and holo forms of phzM shows that the Arg 241 of Golgi stacks by interacting with membranes of in different regions in the protein. The movement of side chain, which takes on two conformationsÅ in the apo adjacent cisternae. Interestingly, we have shown that PMS into the protein can be traced by alteration in the structure, is found in a single conformation in the holo GRASP55 interacts with model membranes (such as spectroscopic signals. These findings bring a new 3 -1 protein, where it forms a salt bridge with the carboxylic POPC) with a binding constant (Kb) of 1.4x10 M . point of view about the use of fluorescence quenching acid group of the SAM molecule. Furthermore, Asp198 Surprisingly, GRASP55 exhibited the reduction in the to characterize the interaction between albumin and

is rotated by approximately 90 degrees about c1 in the tryptophan fluorescence with the addition of increasing ligands. holo relative to the apo structure, optimizing H-bonding concentrations of POPC membranes, indicating that

148 149 H78 - OXA-231 a step back in the evolution of H79 - The new crystal form for human prion H80 - Regulation of KcsA by Anionic works have extensively studied not only dynamic CHDL OXA-143? protein: a structural model for fragment Phospholipids properties and interactions that lead proteins to different screening configurations, but also how these changes are related Víctor U. Antunes1 Victoria Oakes1 to function. One protein that is known to have a disperse Victor Lopes Rangel Edgar E. Liontop2, Fernanda Vasconcelos3, Stefanya Simone Furini2, and Carmen Domene1,3 set of accessible configurations is the estrogen receptor V. Gomez4 and Denize C. Favaro1 Francesca A. Pavlovici, Leandro Oliveira Bortot, and (ER), which regulates function in several different tissues 1Department of Chemistry, University of Bath, 1 of both men and women. ER undergoes extensive 1 Maria Cristina Nonato Department of Organic Chemistry, State University South Bldg., Claverton Down, Bath, BA2 7AY, UK. remodeling as it interacts with different agonists and of Campinas, SP ([email protected]), Protein Crystallography Laboratory, School 2Department of Medical Biotechnologies, University antagonists, as well as transcription activation and 2 Department of Biochemistry, University of Sao of Pharmaceutical Sciences of Ribeirão of Siena, Siena, Italy. 3Department of Chemistry, repression factors. Moreover, breast cancer tumors 3 Paulo, SP, Citrus Research and Education Center, Preto, University of Sao Paulo, Ribeirão Preto, University of Oxford, Oxford, OX1 3TA, UK resistant to hormone therapy have been associated with 4 University of Florida, FL and Department of SP ([email protected]) the imbalance between the active and the non-active ER Chemistry, Federal University of Minas Gerais, MG. Membrane lipids are known to modulate the structure states. The balance between these states is affected by The prion disease belongs to an invariably fatal group and function of integral membrane proteins. This changes in pH and by mutations in ionizable residues. Class-D β-lactamases, also known as Oxacilinases of neurodegenerative disorders that affect humans effect can be achieved directly, by specific binding In line with this, we have used simplified computational (OXAs), are enzymes characterized by highly structural and animals. The critical event in prion diseases is the sites on the protein surface, or indirectly, by changing models to characterize conformational transitions similarity but very different substrate spectrum and conversion of the cellular prion protein PrPC into PrPSc, the physical properties of the membrane bilayer. In between the transcriptional active (agonist PDB-1ERE) efficiency. Of particular concern are the carbapenem- a self-replicating isoform (prion) that accumulates in the the former, binding sites at the interface between two and non-active (antagonist PDB-1ERR) form of ER. Our hydrolyzing class D (CHDL), because these OXA central nervous system of affected individuals resulting protein subunits, known as non-annular sites, have results indicate that cancer-activating mutations and enzymes confer carbapenem resistance in the in neuronal death and amyloidogenesis. Despite been implicated. Anionic lipids have been shown to small pH variations promotes an imbalance between Acinetobacter baumannii, a Gram-negative pathogen. tremendous efforts, PrP physiological function and its modulate various functional properties of the KcsA the ER states, thus favoring the active configuration. In this work the structural and kinetic parameters of mechanism of conversion to PrPSc remains elusive. K+-channel in different manners, and are required Electrostatic interaction seems to be responsible by two CHDL natural variants, OXA-143 and OXA-231 Screening of compounds against human PrP, currently for channel function. Here, we present molecular this imbalance and we were able to identify the most (OXA-143 D224A), were evaluated against ampicillin stigmatized as an “undrugable” target, can benefit from dynamics simulations of KcsA with anionic and important residues involved in ER functional transition. and cloxacillin, both penicillins, and meropenem a the fragment screening strategy, enhancing the chances zwitterionic lipids bound in non-annular sites, in order to Also, the preferential routes that lead ER from a state carbapenem. Intriguingly, the kinetic assays carried of finding ligands to stabilize its native form therefore deduce the molecular mechanism of action of membrane to another have been characterized. Finally, we present out by UV-Vis spectroscopy and Nuclear Magnetic offering an alternative route to quest a treatment to prion lipids in the prototypical K+-channel. possible targets for the development of new ER inhibitors. Resonance showed that this simple mutation turned diseases. We propose to explore X-ray crystallography OXA-231 in a better oxacillinase compared to OXA- fragment screening as a tool to unprecedentedly identify [1] Raja, M., R.E. Spelbrink, K.B. de, and J.A. Killian. This work is suported by CNPq (proc. 167374/2017-6) 143. Although the enzymes presented a similar pockets for ligand interaction on human PrP leading to 2007. Phosphatidic acid plays a special role in stabilizing and Fapesp (proc. 2014/50739-5) turnover number (Kcat) towards cloxacillin, around 45 protein stabilization. To do so, we managed to crystallize and folding of the tetrameric potassium channel KcsA. s-1, OXA-231 showed a significant higher catalytic the globular fragment of recombinant PrP, residues FEBS Lett. 581:5715–5722. [2] Marius, P., M. Zagnoni, H82 - Biophysical characterization of the efficiency (K /Km) 162 mM-1s-1 against 9.7 mM- 1 -1 cat s of comprising 90 to 231, in a new space group with high M.E. Sandison, J.M. East, H. Morgan, and A.G. Lee. 2008. interaction between human respiratory syncytial the OXA-143. So, to analyze if this was a reproducible solvent content (62%), which facilitates fragment access Binding of anionic lipids to at least three nonannular sites virus M2- 1 protein core domain and RNA by behavior against all the penicillins, kinetic assays were to the protein. Moreover, the higher level of symmetry on the potassium channel KcsA is required for channel using fluorescence quenching techniques performed using ampicillin. Nevertheless, towards this (I4 22) reduces the number of images necessary to opening. Biophys.J. 3194:1689–1698. [3] Triano, I., 1 1,3 β-lactam, OXA-143 exhibited a higher kcat/Km1133 achieve data full completeness therefore speeding up the F.N. Barrera, M.L. Renart, M.L. Molina, G. Fernandez- Vitor B. Machado -1 -1 -1 -1 mM s against 229 mM s of the OXA-231. For process of data collection and processing and reducing Ballester, J.A. Poveda, A.M. Fernandez, J.A. Encinar, A. V Giovana C. Guimarães1,3, Marcelo A. Fossey1, 2, meropenem, both enzymes presented a non Michaelis- radiation damage on cadmium sites. Crystals are grown Ferrer-Montiel, D. Otzen, and J.M. Gonzalez-Ros. 2010. Fátima P. de Souza1, 2, and Ícaro P. Caruso1, 2 Menten behavior at the tested conditions and OXA-143 in presence of sodium chloride as precipitant agent and Occupancy of nonannular lipid binding sites on KcsA 1 were the most active one. Besides, circular dichroism cadmium chloride as additive. The best data set was greatly increases the stability of the tetrameric protein. Multiuser Center of Biomolecular Innovation experiments showed a huge difference (8.5 oC) in the collected at I04-1 beamline at Diamond Synchrotron Biochemistry. 49:5397–5404. (CMIB), São Paulo State University (UNESP), 2 thermal stability between the two enzymes and a small Oxfordshire, UK. The structure was refined at 2.3 Å São José do Rio Preto, SP-Brazil Department change in the percentages of alpha helix (34.6 % for resolution. The final model comprises 725 protein atoms, of Physics, São Paulo State University (UNESP), H81 - CHARACTERIZATION OF POSSIBLE São José do Rio Preto, SP-Brazil 3Department OXA-143 and 31.0% for OXA-231). So, our results 1 Cl, 3 Cd and 18 water molecules, with Rfactor 19.2%, FUNCTIONAL TRANSITION ROUTES OF of Biology, São Paulo State University indicate that the modification of an aspartic acid Rfree of 23.5%. Coordinates were deposited at Protein ESTROGEN RECEPTORS residue by an alanine at position 224 leads a change in Data Bank under the code 6DU9. This crystal form is (UNESP), São José do Rio Preto, SP-Brazil protein secondary structure that reflects in the thermal currently being used as a model for X-ray crystallography Vinícius M. Oliveira¹ ([email protected]) stability differences and, possibly, in the enzymatic fragment screening. Paul C. Whitford² and Vitor B. P. Leite¹ activity. Hence, our results indicate that OXA-231 is a Human respiratory syncytial virus (hRSV) is the step back in the evolution of CHDL OXA-143 enzyme. This work was supported by Brazilian National Council ¹Department of Physics, IBILCE, São Paulo major causative agent of chronic lower respiratory for Scientific and Technological Development (CNPq State University, SJ do Rio Preto, SP (vinicius. tract diseases in children and elderly worldwide. RSV This work was supported by the 23853 UFMG/REITORIA/ 141945/2018-4) and by the FAPESP (2016/22929-0 [email protected]) and ²Department of Physics, belongs to the Pneumoviridae family and encodes PRPQ, CAPES, FAPESP and FAEPEX(519.292:3095/17) and 2017/26559-5). Northeastern University, Boston, USA for eleven proteins, among them the transcription antitermination factor protein M2-1, which is essential Flexibility and conformational changes are fundamental for hRSV viral cycle maintenance. M2-1 is composed aspects for understanding how proteins perform their mostly of α-helices and presents a three-dimensional biological functions. Experimental and computational structure divided into four domains: the finger zinc

150 151 2+ domain, composed by the first N-terminus amino acid E83 - Biophysical studies of the Interaction H84 - Calcium binding to the CALX Ca2+-sensor ratios. Binding of Ca to CBD1 stabilizes the structure residues which bind to a zinc atom and supports the between Grb2 cSH3 domain and coumarin domain studied by 15N relaxation and calorimetry increasing the Tm by ~15 ˚C as indicated by DSC. transcription process; the oligomerization domain, experiments responsible for the tetrameric arrangement formation; Pedro, R. P.1 2 Referências: (1)Giladi, M.; Boyman, L.; Mikhasenko, H.; Marcus V. C. Cardoso the core domain, directly linked to viral phosphoprotein Sanches, K.1,2; Dias, R. V. R.1; Caruso, I. P.1,2; Hiller, R.; Khananshvili, D. Essential Role of the CBD1-CBD2 2+ and RNA; and the unstructured C-terminal domain with Oliveira, L. C.1; Fossey, M. A.1,2; Souza, F. P.1,2 and Phelipe A. M. Vitale, Roberto K. Salinas Linker in Slow Dissociation of Ca from the Regulatory Two- low degree of conservation. The present study aims to Melo, F. A.1,2 Domain Tandem of NCX1. J. Biol. Chem. 2010, 285 (36), characterize the interaction between the recombinant Department of Biochemistry, Institute of Chemistry, 28117–28125.2 (2)Abiko, L. A.; Vitale, P. M.; Favaro, D. C.; 1 M2-1 protein core domain (cdM2-1) and RNA by using Instituto de Biociências, Letras e Ciências Exatas University of Sao Paulo. Brazil Hauk, P.; Li, D. W.; Yuan, J.; Bruschweiler-Li, L.; Salinas, R. K.; 2 fluorescence spectroscopy techniques. To achieve this, - UNESP/São Jose do Rio Preto – SP. Centro Brüschweiler, R. Model for the Allosteric Regulation of the Na+/ the fluorescence suppression of cdM2-1 by RNA was Multiusuário de Inovação Biomolecular - CMIB The Na+/Ca2+ exchanger present in photoreceptor cells of Ca2+ Exchanger NCX. Proteins Struct. Funct. Bioinforma. 2016, measured in different temperature (15, 25 and 35 °C) Drosophila melanogaster, CALX, is an integral membrane 84 (5), 580–590. and ionic strength conditions (0, 150 and 350 mM Cell signaling is regulated by enzymatic activities protein formed by 10 transmembrane α-helices and a NaCl), allowing to characterize the thermodynamic through reversible phosphorylation of amino acids large cytosolic loop of approximately 500 residues. While - ELViM: A method to visualize pathways and + H85 parameters and the non-covalent interactions involved side chains where Receptor Tyrosine Kinases the transmembrane domain is responsible for Na and metastable states of biomolecules in the formation and stabilization of the cdM2-1/RNA (RTKs) rule the regulation upon cell proliferation. Ca2+ translocation across the membrane, the large loop is complex. The determined dissociation constant for the Once phosphorylated, RTKs interact with proteins involved in the exchanger ionic regulation. It contains two Vitor B. P. Leite1 -6 complex was on the order of 10 M and the binding partners to build up complexes known as ESCs that calcium-binding domains, CBD1 and CBD2, covalently Antonio B. Oliveira Jr.,1 Huan Yang,2 Paul C. stoichiometry was of two proteins per RNA molecule. activate specific signaling pathways inside cells. linked to each other by a short segment of three residues. Whitford,2 2+ -6 1 The effect of temperature revealed a negative binding FGFR2 is a key RTK in many signaling process CBD1 binds four Ca with high affinity ( Κd ~ 10 M) As 1 enthalpy change, whereas the increase in saline and aberrance activity from this protein causes a a consequence of Ca2+-binding, the CBD1-CBD2 tandem Departamento de Física, Instituto de Biociências, concentration resulted in a decrease in the affinity variety of human cancer. Previous studies have adopts a rigid inter-domain arrangement.2 It is proposed Letras e Ciências Exatas, Universidade Estadual between cdM2-1 and RNA. The results also indicate shown that Grb2 is an important regulator of that this process triggers the allosteric inhibition of CALX. Paulista, São José do Rio Preto, SP, Brazil . 2 that the interaction is spontaneous, with hydrogen FGFR2 by preventing kinase activities before extra- Here we used 15N chemical exchange saturation transfer Department of Physics, Northeastern University, bonds and van der Waals forces playing a key role on cellular stimuli. Therefore, Grb2 is a constitutive (CEST) and CPMG-relaxation dispersion experiments Boston, MA. [email protected] complex stabilization. These findings are important controller over the mutually dependent activities of to investigate the chemical exchange process between for understanding the mechanism of the interaction FGFR2 and a potential target to drug development. free and Ca2+-bound CBD1 under quasi-stoichiometric Protein folding occurs in a very high dimensional between hRSV M2-1 protein with its major molecular Another study using cancer cell culture states that conditions. The backbone dynamics of Ca2+-bound CBD1 phase space, with the representation of their complex target, RNA. coumarin presents antitumor properties. This study in fast time scales was investigated by 15N CEST derived energy landscape being non-trivial. The overall 1 15 is qualitative and there is a lack of information of R2/R1 ratios and the { H}- N NOE. To obtain information behavior is usually represented in terms of one This work was supported by Conselho Nacional de how that molecule could be acting in a molecular on the chemical exchange at ms timescale, the Bloch- effective reaction coordinate, in which the role of each Desenvolvimento Científico e Tecnológico (CNPq) and level to kill cancer cells. In order to address this McConnell equation was numerically integrated for CEST local minimum is not statistically taken into account. by Fundação de Amparo a Pesquisa do Estado de São question, we have used a biophysical approach (15N) profiles of residues Asn456 e Val518, which are at There have been many attempts to obtain a more 2+ Paulo (FAPESP). to study in vitro the interaction between coumarin the Ca binding site. Exchange parameters (κex = κon detailed representation of all available conformations and full length Grb2 protein as well as the Grb2 + κoff), the fractions of population of each state (ρb, e for a given system. In this work, we address this separately Grb2 domains. In this study we have used ρa), and the relaxation rates of the bound state were problem using a metric based on internal distances fluorescence spectroscopy to show the interaction determined from 15N CEST. The populations found for between amino acids to describe the differences between coumarin and Grb2 cSH3 domain happens the low-populated (calcium-free) state were between between any two conformations. Using an effective in a micromolar range and it is entropic driven which 5.8 and 6.5% and the interconverting pseudo first-order projection method, we are able to go beyond the usual -1 -1 is characteristic of hydrophobic forces taking place exchange rates were 442 s < κex < 500 s at 35 ˚C. one-dimensional representation and thus visualize as the main interaction mechanism. Computational The majority of residues, however, showed CEST profile landscapes in two dimensions. We refer to this simulations give us a theoretical model of how this with only one dip, in which some of them presented method as Energy Landscape Visualization Method interaction could be happening. The role of this strong line-broadening. This last effect indicates that (ELViM). We have applied this methodology using Cα interaction still remains unknown but help us to for most residues|ωa - ωb| < 442 Hz, but it allows the structure-based model (SBM) to study the folding of get basic information about the interaction of small location of residues whose either 15N chemical shifts or two well-known proteins: SH3 domain and Protein-A; molecules, known for its antitumor properties, with relaxation rates (or both) change upon Ca2+-binding. This distinct funnels have been generated according to the key proteins inside cell. information is relevant since most residues located near major motifs in each protein (β-sheet or/and α-helix). the Ca2+-binding sites of CBD1 are not observed in the 15N More precisely, our visualization method yields a

HSQC experiment. The mean isotropic tumbling time, 〈tc〉 detailed description of the folding process, making iso, was (8.7±0.5) ns and (9.4±0.6) ns, determined using possible the identification of transition state regions, 15N CEST or 15N relaxation experiments, respectively, at and establishing the paths that lead to the native state. 2+ excess of Ca over CBD1. Therefore, the 〈tc〉iso of CBD1 In addition, we have analyzed structural differences in 2+ in the Ca -bound state calculated from the mean R2/R1 the dominant route of SH3 protein, the competition ratios obtained from CEST at excess of Ca2+(16:1), are in between the native and mirror structures in protein good agreement with values obtained by direct measures A. Additionally the method is applied to discuss the 15 of N R2 and R1. This shows that CEST measurements conformational changes of the Elongation Factor could be an alternative approach to obtain reliable R2/R1 Thermally unstable (EF-Tu).

152 153 Miscellaneous molecule that alters the adhesive and migratory behavior I06 - DIFFUSION MAPS OF Bacillus subtilis of tumor cells, allowing a comparative evaluation. In order BIOFILMS VIA MAGNETIC RESONANCE IMAGING to characterize the mechanical properties of these cells, Rômulo P. Tenório1 I01 HYBRID QC/MM SIMULATIONS OF have a cure or a reliable early diagnosis method, limiting we used atomic force microscopy (AFM) indentation, ENZYMATIC ELECTRON TRANSFER REACTIONS the treatment to symptomatic relief. Amyloid cascade is the which results in force curves as a function of the Ramon V. Galdino1,2, Clayton A. Benevides1 most widespread hypothesis for AD. It states that amyloid indentation depth in the sample. Then these curves were 1 Felipe Curtolo precursor protein (APP) is cleaved by β-secretase enzime, analyzed by the Hertzian model for pyramidal tip. We Centro Regional de Ciências Nucleares generating -Amyloid peptide that aggregates forming obtained a relative value of elasticity for each biological do Nordeste (CRCN-NE/CNEN), Recife, PE Guilherme Menegon Arantes β 2 several different neurotoxic morfologies, since soluble system. In addition, we built elasticity maps that can be ([email protected]) and Departamento Department of Biochemistry, Instituto de Química, oligomers until unsoluble fibrils deposited over the neurons, compared directly with the topographic images of the de Farmácia, Universidade Federal de Pernambuco Universidade de São Paulo, São Paulo, Brazil leading to an inflammatory response and neuronal death. sample and in this way clearly identifying the variations (UFPE), Recife, PE. ([email protected], [email protected]) Evidence support that Ru(II) polypyridinic complexes of the modulus of elasticity. are capable of bind amyloid aggregates, enableing real We report the use of high-resolution magnetic resonance Mitochondrial complex II catalyzes succinate oxidation time mapping of the aggregation process. However, the This work was supported by CNPq, FAPESP e CAPES. imaging to obtain self-diffusion coefficient maps of the when two C-H bonds are broken by elimination of a proton complex itself is unable to cross blood brain barrier (BBB). Bacillus subtilis biofilm surface. Some strains of B. and a hydride equivalent (H + 2e ). Hydride is transfered To overcome this issue, we suggest a novel [Ru(phen) ]2+ subtilis develops wrinkles on surface that has been 3 I05 - Mg(II) LUMINESCENT COMPLEXES: A TOOL to an adjacent flavin cofactor (FAD) which is later complex bearing a β-D thioglucose derivative to assess FOR ECOLOGICAL MONITORING INTERACTIONS proposed to participate in fluid transport along the biofilm reoxidized passing electrons⁺ to iron-sulfur⁻ clusters and BBB crossing trhough GLUT1 transporter. The complex BETWEEN THE PLANT AND THE SUBSTRATES surface. Here, we would like to check this theory making ubiquinone. Here, we modeled this enzymatic oxidation showed intense and broad emission band, with λmax = measurements of the diffusion coefficient (D) in the 1 reaction with hybrid quantum chemical/molecular 605nm, extended until λ=705nm, covering background Rafael C. Marchi biofilm using the technique of Diffusion-Weight-Nuclear -1 mechanics simulations (QC/MM). The complex II analogue emission, Stokes Shift of 5000cm , good solubility in Danielle F. da Silva1 and Rose M. Carlos1 Magnetic Resonance Microimaging to highlight the role flavocytochrome c3 (Fcc3) was used as a model system. water and pH = 7,4 buffer solution and the capability of of wrinkled channels in the diffusion processes. The 1 A quantum (QC) region composed of active-site residues, binding all the amyloid forms and to the GLUT1 transporter, Department of Chemistry, Federal University of experiments were conducted with the B. subtilis growing water molecules, succinate and flavin was defined and being a good candidate for an early diagnosis platform. São Carlos, São Carlos, SP ([email protected]) in a MSgg media, in a glass tube, and incubated at 33oC treated with density functional theory by the functional/ during 14 days. Micro-MRI experiments were performed basis M06-L/def2-SVP. The remaining of the system This work was supported by FAPESP (2017/00839) and Crop protection is a worldwide precaution due to its in VARIAN UNMRS 400 MHz (9.4 T) equipped with a was treated with the CHARMM36 force field. Reaction by CAPES. impact in any economy from any country. The search microimaging probe, using a fast spin echo diffusion- profiles were determined using the nudge elastic band for a bio-pesticide is now more urgent then never to weighted sequence. The diffusion coefficients measured method. Three possible oxidation mechanisms in Fcc3 I04 - MECHANICAL PROPERTIES control pests (e.g. leaf-cutter ants, thrips, spodopetera in the x and y direction (Dx and Dy) are in the plane of the were tested: one concerted and two with intermediates, CHARACTERIZATION OF MELANOMA AND frugiperda) in different plant cultures (e.g. citrus, soy, biofilm, whereas the diffusion coefficient in z direction a carbocation or a carbanion. We find that the carbanion KERATINOCYTES CELLS BY AFM maize, tomato). This work reports the synthesis and (Dz) reflects the self-diffusion values perpendicular to intermediate is more stable than the carbocation and that characterization of Mg(II) metal complexes containing the plane of the biofilm. Our results shows an anisotropy the electron transfer to FAD is facilitated when a proton Nataly Herrera1 a natural product (hesperidin) in its coordination sphere and a heterogeneous diffusion behavior dependent on is abstracted from succinate first by a basic residue in Roger Chammas2 and M.C. Salvadori1 and the luminescent properties. These complexes have the direction and presence of the channels. Finally the the active site. These results help to elucidate in detail two absorptions, one at 280 nm and other at 330 nm due method is noninvasive and the contrast can be tuned for 1 the molecular mechanisms involved in biological electron Institute of Physics, University of São Paulo, São to π→π* transition. Also, the absorption spectra have specific regions or even completely suppressed. 2 transfer reactions. Paulo, SP ([email protected]) and Instituto de slightly differences depending on the solvent. That same Câncer do Estado de São Paulo, Faculdade de behavior is observed for the emission spectra which This work has been supported by FAPESP and CNPq Medicina de São Paulo, São Paulo, SP. goes from a maximum at 468 nm for DMSO to 490 nm when in water, showing that the solvents play a notable I02 - A NOVEL RU(II) POLYPYRIDINIC COMPLEX Cancer transformation introduces significant changes interaction with the complex molecule either on the in cell structure and behavior. Alterations in cell growth, ground state or on the excited state. The emission, in all BEARING A β-D-THIOGLUCOSE TO CROSS -1 BLOOD BRAIN BARRIER THROUGH GLUT1 morphology, cell-to-cell interaction, and in cell membrane cases, is broad and have a Stoke Shift equal 6000 cm . TRANSPORTER and organization of the cytoskeleton have been reported. Using confocal fluorescence microscopy, the complexes These differences may also induce changes in mechanical are analyzed through their emission at 450 nm. Using 1 Marlon Augusto P. De Almeida properties of the cell, these properties can be tested this technique, it is possible to access the interaction Optical microscopy Dz-Map MRI Rose Maria Carlos2 through the cell stiffness, which seems to be a global plant-complexes and define if it is in the phloem or on the 1 parameter describing the overall changes occurring in leaves. Monitoring this interaction is possible to target Department of Chemistry, Federal University São the cell structure. In this work we characterized confluent which type of insect can be controlled because each one Carlos, São Paulo, SP (marlon.quimica@gmail. cultures of keratinocytes co-cultured with melanoma has a specific way to eat the plant. 2 com) and Department of Chemistry, Federal cells to test the hypothesis that the presence of tumor University of São Carlos, São Paulo cells will modify the cytoskeletal arrangement of This work was supported by Conselho Nac. Des. Cient. neighboring normal epithelial cells. Another aspect is to Tecnologico (CNPq 166303/2017-8) and by Fundação Alzheimer’s disease (AD) is one of the most devastating verify the possibility of obtain a biomarker of the disease de Amparo à Pesquisa do Estado de São Paulo (FAPESP, neurodegenerative conditions that affect the elderly around malignancy. For this purpose, we used two lineages, 2016/21722-2) the globe. AD is a progressive and multifactorial disease; keratinocytes (HaCaT) and melanoma cell (WM1366

which first symptoms are usually ignored and pointed out shSCR). Also, we used melanoma cells silenced for Dy-Map MRI Dx-Map MRI as an age effect. Despite the numbers, AD still does not the galectin-3 gene (WM1366 shGAL3), a matricellular

154 155 This work was supported by Fundação de Amparo à I08 - Protein disulfide isomerase modulates I09 - Cancer Stem cells dependent on the I09 - INFECTION - A SCIENCE EDUCATION Ciência e Tecnologia de Pernambuco (FACEPE APQ- activation of Thyroid hormone receptors concentration of the superoxide in the TABLETOP GAME 1245-1.06/15) and CNPq (432279/2016-4). metastasis to the sentinel lymph node - murine Ana Carolina M. Figueira1,2 R. V. Maximiano melanoma model 1 1,2 I07 - EFFECT OF HYPERGLYCEMIA IN THE Fernanda A. H. Batista ; Tabata R. Doratioto ; 1 L L Ribeiro, G R Gonçalves Natalia B. Videira1; Helder V. Ribeiro Filho1,2 Telma Lisbôa Nascimento ACTIVITY OF GLYCINE RECEPTORS IN INSULIN Department of General Formation, Federal Center Alice T. Ferreira1 SECRETING CELLS 1Laboratory of Spectroscopy and Calorimetry, of Technological Education Of Minas Gerais, Amanda Schukarucha Gomes1 1Brazilian Biosciences National Laboratory 1Department of Biophysics, Federal University of Contagem Unit ([email protected]) (LNBio), Brazilian Center for Research in Energy Sao Paulo, SP ([email protected]) Silvana Bordin2 and Fernando Abdulkader2 and Materials (CNPEM), Campinas, Sao Paulo, SP, Gamification theory is one of the most successful 1, 2 2 Department of Physiology and Biophysics, Brazil. ([email protected]); Graduation Superoxide (O -.) is a reactive species of oxygen techniques used in schools to improve learning rates. 1 2 University of São Paulo, São Paulo, SP program of Biosciences and Bioactive Products (ROS) that participates in cellular biological processes Transforming day-to-day tasks, like science classes ([email protected]). Technology, Institute of Biology, State University of such as proliferation, differentiation and survival. and math homework, into a fun activity helps enhance Campinas (Unicamp), Campinas, Sao Paulo, Brazil - Studies show that depending on the levels of the (O2 the commitment of the students to these tasks. In this Pancreatic β cells secrete insulin, an essential hormone .), modifications occur in cellular signals of healthy work we have created a game that you can play in any for glucose homeostasis. Its secretion is triggered by Thyroid hormone receptors (TRs) are responsible for cells and cancer stem cells (CSCs). CSCs are cells context, either during classes or within leisure time. By action potentials, and it can be influenced by various mediating thyroid hormones (T3 and T4) actions at that initiates the tumor and participates in their playing it, you’ll be exposed to the main mechanisms neurotransmitters found in the pancreatic islets. cellular level. They belong to the nuclear receptor (NR) survival, proliferation and metastasis. Metastasis is associated with amino acids production and cell Among them is glycine, who acts through its ionotropic superfamily and execute their main function inside the the spread of the tumor to distant regions, forming metabolism, and can even use the game components receptors (GlyRs), which are ligand-gated ion channels cell nuclei, as hormone-regulated transcription factors. secondary tumors. However, in order for this spread themselves as an educational tool. We could teach selectively permeable to Cl-.These receptors have been These receptors also exhibit so-called ‘nonclassic’ to occur, the first ganglion to receive the metastatic our students about the subject without having to rely recently reported to participate in a positive feedback actions, for which other cellular proteins, apart from cells is the sentinel lymph node (SLF), which serves as on conventional educational methods by establishing loop during insulin secretion in human β cells. coregulators inside the nuclei, regulate their activity. portal of dissemination for these cells. The metastatic win conditions that depend on realistic scientific However, this feedback is absent in islets from diabetic Aiming to find alternative pathways of TR modulation, process is complex, and studies show that oxidative chemical reactions and process. After playing a few donors, in which the glycine receptors are less active. we found PDIA1 interaction with TRβ in yeast two- stress can modify the signals that act in the epithelial- rounds the students had understood the biophysical Our study aims to investigate if the exposure to high hybrid screening assay. Functional implications of mesenchymal transition (EMT), aiding in the migration and biochemical concepts presented in the game, glucose concentrations, a main feature of diabetes, PDIA1 for TR are unclear, but our results show that of cancer cells by the extracellular matrix until they without classes on the subject being given prior to the is responsible for this difference in the physiological PDI bind both TR isoforms in vitro, through Co-IP and reach the vessels lymph or blood to the development gameplay. We also compare the results of students role of the GlyRs, through changes in its expression fluorescence assays, and T3 played no important role the cancer in other organs. In this work, we use the that did play the game and had classes on the subject or its activity. For that we used the β cell line INS-1E, in these interactions. In cell assays, PDIA1 was able to lineage of murine melanoma (B16F10), after long with those who did not, and check for improvements and cultivated these cells for 24h in 3mM, 11.1mM or regulate transcription of TRα and TRβ-mediated genes, periods of culture, under specific conditions giving rise on learning rates and excitement with the lectures. -. 30mM glucose. We found that the genetic expression in different ways, depending on the promoter region to the CSCs of melanoma. In vitro low levels of (O2 ) of the GlyRs subunits α1, α3 and β in these cells was and TR isoform involved. Although PDIA1 promotes stimulated the survival and quiescence of the CSCs. This work was supported by Centro Federal de Educação -. not influenced by the different glucose concentrations, TR activation in gene reporter experiments, it binds In vivo, the levels of (O2 ) increased significantly Tecnológica (CEFET) and by Fundação de Amparo a while the expression of the α2 and α4 subunits was to a TR surface that does not interfere in coactivator assisting in the proliferation and the metastatic process Pesquisa de Minas Gerais (FAPEMIG). too low to be quantified by qPCR. We identified the binding. Moreover, the TR:PDIA1 activation is different of the tumors formed by the CSCs for the (SLF). It expression of the glycine transporters 1 (GlyT1) and depending on the TR isoform. Such differences may was observed that lesions formed by CSCs showed 2 (GlyT2), which are responsible for the cellular uptake reflect the structural organization of the PDIA1:TR greater tumor growth and an aggressive metastatic of glycine, what suggests that INS-1E cells accumulate complex, as shown by structural models, depicting an behavior compared with the control tumors. Metabolic this neurotransmitter and may secrete it. Moreover, interaction interface with exposed cysteines from both singularities that distinguish the CSCs of the other cells the expression of these transporters was modulated proteins, suggesting that PDIA1 might modulate TR by of the tumor population, need deep research, because by different glucose concentrations; GlyT1 expression its thiol reductase/ isomerase activity. the better knowledge these cellular pathways have was higher in 3mM glucose, while GlyT2 expression great potential in the development of future therapies. increases accordingly to glucose concentrations. This work was supported by FAPESP (#2011/23659-2, We will continue to investigate our hypothesis by #2013/08743-2, #2014/22215-1, #2016/22246-0); Financial Support: CAPES, CNPq and FAPESP. measuring the electric current of the GlyRs and their and CAPES (# 23038.006737/2012-56). protein expression in these cells.

This work was supported by Conselho Nac. Des. Cient. Tecnológico (CNPq # 160314/2017-8)

156 157 I11 - FIRST BRAZILIAN BIOPHYSICS LEAGUE Alphabet Index strategies to follow: classes, research and extension. Fernando de Mesquita Júnior1 Inicially, it has built a partnership with the Physical Thiago Alves de Souza2, Dalton Cristofer de Course in the University, which will be very useful to Contents Chehín, Rosana Nieves...... 51 Campos2, Lucas Silva Dias2, Gabriel Tibúrcio Castro introduce the League in the subject, by attending several Adriana Fontes ...... 20 Christopher Kushmerick...... 23 Zulke2, Guilherme Takeshi Matsuoka², Pádua Maia classes with PhDs in the area. In addition, the League will Adriano M. Alencar...... 53 Chuck S. Farah...... 50 Araújo², Yohan Alves Victor de Matos², Vinícius develop courses on the physiology of systems, classes Agnes Magri ...... 117 Cilli, E. M...... 64 Soares do Espírito Santo2, Luisa Bertoldi Aguilar2, about complementary exams, such as electrocardiogram Ainhoa Rodriguez de Yurre...... 106 Claudemir Oliveira Souza...... 123 Lucas Figueira Andrade 2 and electroencephalogram, and pratical classes, both Alan B. Dalton...... 44 Cordeiro Lima P. F ...... 98 relevant for the medical practice, with especialists. Alex H. Miller...... 55 C. R. Ferreira...... 74 1 Department of medical sciences, University of Another point to consider is that LABio has permission to Aline Amorim Graf...... 55 C.R. SIMÕES...... 73 Mato Grosso, Cuiabá, MT (fernando.mesquita.jr@ research in the Physiology Lab, as well as use its devices Amanda Schukarucha Gomes...... 156 Daniele Ribeiro de Araujo...... 45 2 gmail.com) and Medical students and biophysics to perform practical lessons. Furthermore, the students Amantino C. F...... 88 Danilo Roman Campos...... 40 league’s members, University of Mato Grosso, plan to offer monitoring programs to the colleagues, to Amantino C. F...... 101 Dayanne P. Rosa...... 111 Cuiabá, MT ([email protected]) encourage and help them to learn Biophysics, especially Ambrosio ...... 24 Débora C. K. Codognato...... 63 in a physiological approach. Another goal is to promote Ana Carolina de Mattos Zeri...... 17 Denys E. S. Santos...... 55 The Academic Biophysics LEAGUE (LABio) consists an extension program visit with high school students, Ana Carolina M. Figueira...... 156 Diogo B. Peruchetti...... 105 in a group of medical students that has officially been so they can know the Lab and learn more about the Anacleto Silva de Souza...... 86 Elio A. Cino...... 28 approved in July of 2018 by the Federal University of theme. Finally, the group intends to be prepared to future Ana Paula R. Povinelli...... 86 Elisângela Belleti ...... 56 Mato Grosso (UFMT), placed in Cuiabá. Being the first congresses and symposia, therefore it will occur weekly Ana Paula Valente ...... 28 Emiliano Medei...... 41 project of it’s type in the country, LABio counts on ten meetings to discuss the literature in a very pertinent way. Anderson F. Sepulveda...... 86 Erik de Oliveira Martins...... 107 students and an advisor: Professor Fernando Mesquita Anderson S. Pinheiro...... 117 Fabiana V. Diasa...... 89 Jr, M.D and specialist in the theme. Thus, there are three This work was supported by University of Mato Grosso. André Anversa Oliveira Reis...... 118 Fabio Cesar Gozzo ...... 42 Andreia Laura Prates Rodrigues...... 102 Fabio C. L. Almeida...... 123 André L.S. Santos...... 61 Fabio Parra Sellera...... 48 Andresa Messias da Silva...... 72 Favarin, B. F...... 73 Angela D. B. de Brito ...... 62 Felipe A. Otsuka...... 124 Angie Dávalos...... 118 Felipe Curtolo...... 154 Aníbal E. Vercesi...... 14 Felipe de Souza Leite...... 53 Antonio Victor B. Vasconcelos...... 118 Fernanda Paiva...... 124 Artur S. Miranda...... 102 Fernando Abdulkader...... 52 Aryane A. Vigato...... 87 Fernando B. da Silva ...... 111 Atílio Tomazini Júnior...... 119 Fernando de Mesquita Júnior...... 158 Balan, A...... 87 Fernando Freitas de Lima...... 75 Bárbara Malheiros...... 99 Fernando T. Tanouye ...... 124 Bartkevihi, L...... 132 Franciele Garcia Baveloni...... 90 Batista, M. R. B...... 139 Fredderico C. Machado...... 125 Beate Röder...... 37 Frédéric Frézard...... 45 Benoît Roux ...... 14 Frederico J. S. Pontes...... 75 Bertozo L. C...... 138 Freire Ribeiro, M...... 114 Beserra S. dos S. Author ...... 147 Gabriel Ernesto Jara...... 125 Bianca Rizo...... 120 Gabriel G. Slade...... 112 Bruna Renata Casadei...... 72 Gabriel R. Sousa...... 125 Bruno P. O. Santos...... 120 Gabriel S. Vignoli Muniz...... 76 Cabral, F. V...... 64 Gabriel Zazeri...... 90 Camila A. Yamada...... 121 Garegin A. Papoian...... 38 Camila F. T. Pontes ...... 121 Gerald Weber...... 30 Camila Ramos Silva...... 62 Gianella Facchin...... 90 Carla Mattos...... 29 Giovana C. Guimarães...... 126 Carla Polo...... 17 Giovana Firpo...... 91 Carlos Henrique I. Ramos...... 50 Glauce M. Barbosa...... 126 Carmen Domene...... 121 Gonçalves G. E. G ...... 91 Carolina T. A. Ferreira...... 122 Gronenborn, A. M...... 14 Castro, Simone R. de...... 100 Guilherme G. Costa...... 112 Catarina Cataldi...... 32 Guilherme Menegon Arantes...... 46 Catarina Cataldi...... 63 Guilherme S. Ramos...... 71 Catharina dos S. Silva...... 122 Haroldo de Lima P. Cravo...... 65 Celso Caruso Neves...... 52 Helder V. Ribeiro Filho...... 127

158 159 Helena Couto Junqueira...... 76 Marcelo Morales ...... 26 Rachel Nechushtai...... 28 Tábata Renée Doratioto...... 147 Iara Aimê Cardoso...... 127 Marcelo Querino Lima Afonso ...... 139 Rafaela S. Ferreira ...... 30 Taline S. Almeida...... 69 Ingrid B. S. Martins...... 128 Marcus V. C. Cardoso ...... 153 Rafael C. Marchi...... 155 Taline S. Almeida...... 70 Isabela Moreira-Silva...... 77 Maressa Donato...... 79 Rafael E. O. Rocha...... 145 Tayana Mazin Tsubone...... 83 Isabele Ap. S. de Campos...... 92 Maria C. Oliveira ...... 80 Rafael F. Soares...... 145 Tayana Tsubone...... 36 Isabella O. L...... 128 Maria Cristina Nonato...... 18 Rafael G. Carvalho...... 59 Telma Lisbôa Nascimento...... 157 Iseli L. Nantes Cardoso...... 56 Maria Elena de Lima...... 33 Raissa Ferreira Gutierrez...... 146 Thainá Miranda...... 147 Isis Sebastião...... 129 Mariana A. Ajalla Aleixo ...... 139 Raissa L. Oblitas ...... 61 Thamires Q. Froes...... 148 Izabela F da Silva...... 107 Mariana Chaves Micheletto...... 113 Ramos, L...... 134 Thierry Granjon...... 24 J. A. Bouwstra...... 15 Mariana P. Cali ...... 114 Raphael de A. N. Gomes ...... 83 Thirupathi R. Soudherpally...... 148 Jefferson C Rodrigues ...... 77 Marilia L. Cirqueira...... 140 Raphael Dias de Castro...... 88 Vanessa S. Silva...... 149 Jéssica A. Tedesco...... 129 Marina G. Fontes...... 140 Renata Aparecida Belotto...... 48 VESCHI E. A...... 74 Jéssica M. de Sá...... 130 Marinonio L. Cornélio...... 103 Renata F. V. Lopez...... 26 Vitor B. P. Leite...... 153 João Hermínio Martins da Silva...... 92 Marin van Heel...... 42 Renato E. de Araujo...... 35 Victor Barbosa...... 105 João Paulo Tardivo...... 36 Mario de Oliveira Neto...... 58 Rhiannon W. Harries...... 59 Victoria Oakes...... 151 Joao Ruggiero Neto...... 33 Marjorie C. L. C. Freire...... 141 Ricardo A. P. Pádua...... 146 Victor Lopes Rangel...... 150 Jorge F. B. Pereiraa...... 50 Marlon Augusto P. De Almeida ...... 154 Ricardo M. Leão...... 23 Víctor U. Antunes...... 150 José Antonio Fiorote...... 130 Mateus Rodrigues Leal ...... 107 Roberto A. Chica...... 31 Vinicius Carrascosa...... 60 Juliana Damasceno Oliveira...... 93 Matheus P. Pinheiro...... 95 Roberto K. Salinas...... 43 Vinícius Fernandes...... 108 Juliana Ferreira Strixino...... 48 Maurício S. Baptista...... 59 Romildo A. Nogueira ...... 83 Vinícius G. Contessoto...... 109 Juliana Ferreira Strixino...... 70 Mayk C. Ramos...... 96 Rômulo P. Tenório...... 155 Vinícius M. Oliveira...... 151 Juliana Guerra Pinto...... 65 Mayra C. G. Lotierzo ...... 96 Rosana A. Bassani...... 40 Virginia Sara Grancieri do Amaral...... 85 Juliana Raw...... 131 M. Bolean...... 25 Rose M Carlos ...... 115 Vitor B. Machado...... 151 Juliana S Yoneda ...... 57 Melina Pires da Silva...... 22 R. V. Maximiano...... 157 Viviane Corrêa Santos...... 101 Juscemácia N. Araujo...... 131 Micaela Lopez Alarcón ...... 41 Sa. Arsalani...... 68 Vivianne Basílio Barbosa...... 109 Kaline Coutinho...... 30 Miguel de S. Andrade...... 141 Sandra B. N. Agostini...... 99 Vladimir N. Uversky...... 38 Karoline Sanches...... 131 Monique Lemos ...... 97 Sandra M. G. Dias...... 100 Werner Treptow...... 46 Katia Regina Perez...... 33 M. Pickholz ...... 46 Sarmento J. O ...... 129 Ximenes V. F ...... 149 Kelli Cristina Freitas Mariano...... 94 M. Prieto ...... 25 Scanavachi, G...... 113 Yan M. H. Gonçalves...... 84 Kenneth M. F. Miasaki...... 78 Naially C. de Faria...... 97 Sebinelli, H. G...... 76 Yeny Y. P. Valencia...... 84 Koji Kinoshita...... 20 Naiá Porã Santos...... 141 Sergio Pantano...... 47 Yeny Y. P. Valencia...... 32 Larissa D. da Silva...... 94 Nascimento M. H. M...... 96 Silvia Cristina Nunez...... 49 Yoshimura, T. M...... 35 Larissa G. Maciel...... 132 Natália A. Fontana...... 142 Soudabeh. Arsalani...... 69 Yraima Cordeiro...... 38 Leandro Ramos Souza Barbosa...... 44 Natalia Alvarez...... 80 Strazza Junior PS ...... 82 Yulli M. F. Passos...... 116 Leonardo Boechi...... 133 Natália B. Leite...... 81 Leonardo Cirqueira ...... 133 Natália F. de Oliveira...... 81 Leonardo Cirqueira ...... 22 Natalia K. Gushiken...... 67 Letícia Corrêa Fontana...... 66 Nataly Herrera...... 154 Letícia Stock...... 134 Nayara S. Alcântara Contessoto...... 67 L. H. Andrilli...... 79 Neli Fonseca...... 142 Lígia N. M. Ribeiro...... 27 Nelson A. Alves...... 115 Lígia N. M. Ribeiro...... 89 Nicole M. A. Chaparro ...... 68 Lilia Iriarte...... 135 Nuno C. Santos...... 20 L. M. Rebelo...... 66 Olívia Teixeira...... 143 Luan C. Marques...... 135 Omar Mertins...... 26 Lucas Bleicher...... 136 Osias B. S. Filho...... 104 Lucas Carrijo de Oliveira...... 136 Pablo V. M. Reis...... 98 Lucas Rodrigues de Mello...... 57 Pâmella Miranda ...... 108 Luccas, P. H...... 144 Paola Silva ...... 143 Luciana Guimarães Munhoz...... 95 Paula Rhana...... 104 Luciana Maria Cortez Marcolino ...... 66 Paul C. Whitford...... 42 Lucianna Helene Santos...... 137 Paulo Ricardo Mouro...... 115 Luciano Censoni ...... 113 Paulo S. L. Beirão ...... 143 Lucivaldo R. Menezes...... 58 Pedro, R. P...... 152 Ludmilla D. Moura...... 78 Penna, B. R...... 119 Luis A. Bagatolli...... 21 Pinheiro, Glaucia M.S...... 39 Luis Felipe Santos Mendes...... 137 Priscila dos S. Bury...... 144 Luiz Fernando de C. Rodrigues...... 138 Priscilla Freddi...... 82

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