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Boettger, 1892) Faculdade de Medicina Programa de Pós-Graduação em Patologia Molecular LUZ ELENA TRIANA VIDAL TESE DE DOUTORADO Composição proteica e caracterização funcional da secreção cutânea de Dendropsophus columbianus (Boettger, 1892) Brasília, Março de 2017 Faculdade de Medicina Programa de Pós-Graduação em Patologia Molecular LUZ ELENA TRIANA VIDAL TESE DE DOUTORADO Composição proteica e caracterização funcional da secreção cutânea de Dendropsophus columbianus (Boettger, 1892) Tese apresentada ao Programa de Pós-Graduação em Patologia Molecular da Universidade de Brasília, como requisito para obtenção do título de Doutor em Patologia Molecular. Orientador: Prof. Dr. Sébastien Olivier Charneau Laboratório de Bioquímica e Química de Proteínas – Departamento de Biologia Celular - UnB Brasília, Março de 2017 i Dedico este trabalho ao povo brasileiro por ter realizado meu sonho de estudar ii Agradecimentos Apresento os meus mais sinceros agradecimentos à meu orientador Sébastien Olivier Charneau, aos professores Wagner Fontes e Mariana Castro, ao laboratório de bioquímica de proteínas, ao programa de Pós-Graduação em Patologia Molecular da faculdade de Medicina, à Universidade de Brasília (UnB), à Universidad del Cauca, aos professores Carlos André Ricart, Consuelo Lima, Philippe Grellier, Osmindo Rodrigues Pires Júnior, Sonia Maria de Freitas, Jimmy Alexander Guerrero Vargas, Izabela Dourado Bastos, Renato Caparroz, aos biólogos Jorge Alberto Zúñiga e Alice Cunha Morales Álvares, à Deus, minha família, meus amigos e colegas. Este projeto foi desenvolvido graças ao apoio financeiro da Universidade de Brasília (UnB) e das agências brasileiras de fomento à pesquisa, CAPES/Incentivo à Pesquisa em Parasitologia Básica [23038.005298/2011-83], Cooperação Franco-Brasileira CAPES-COFECUB (723/11), Rede Pró-Centro- Oeste (FAPEG / CNPq 563998/2010-4 e 563998/2010-5), FAPDF [193.000.987], Finep, Fundação Hemocentro de Brasília, Muséum National d'Histoire Naturelle de Paris na França. iii SÚMARIO iv Resumo ....................................................................................................................................... 1 Palavras-chave ......................................................................................................................... 3 Abstract ...................................................................................................................................... 4 Keywords ................................................................................................................................... 6 Lista de abreviaturas .............................................................................................................. 7 1. Introdução ............................................................................................................................ 9 1.1. Secreção cutânea de anfíbios .......................................................................................... 10 1.1.1. Peptídeos antimicrobianos (AMPs): conceitos gerais ........................................11 1.1.2. Seletividade, conformação, carga e hidrofobicidade dos AMP ....................13 1.1.3. Mecanismos de ação dos AMPs ...............................................................................................17 1.1.3.1. Mecanismo por agregação ............................................................................................................... 17 1.1.3.2. Mecanismo de formação de poro toroidal .................................................................................. 17 1.1.3.3. Mecanismo de barril ............................................................................................................................ 18 1.1.3.4. Mecanismo Carpet-like ....................................................................................................................... 19 1.2. Potencial farmacológico dos AMPs ........................................................................... 21 1.2.1. Atividade antibacteriana dos AMPs ...................................................................................21 1.2.2. Atividade antiparasitária dos AMPs ..................................................................................22 1.2.3. Atividade antifúngica dos AMPs ..........................................................................................23 1.2.4. Atividade imunomoduladora dos AMPs .........................................................................24 1.3. O processamento enzimático de AMPs e a atividade proteolítica da secreção cutânea de anuros ..................................................................................................... 25 1.4. Estudos proteômicos do tipo shotgun das secreções cutâneas de anuros .... 26 1.4. Dendropsophus columbianus (Boettger, 1892) ..................................................... 27 2. Justificativa ........................................................................................................................ 29 3. Objetivo .............................................................................................................................. 31 4. Materiais e Métodos ........................................................................................................ 33 4.1. Etapa I, busca de peptídeos antimicrobianos da secreção cutânea de D. columbianus ................................................................................................................................... 36 4.1.1. Obtenção da secreção cutânea ...................................................................................................36 4.1.2. Perfil proteico: Eletroforese em Gel de Poliacrilamida (SDS-PAGE) ..............36 4.1.3. Fracionamento da secreção bruta ............................................................................................36 4.1.5. Screening antimicrobian ..................................................................................................................37 4.1.6. Purificação do peptídeo de interesse .....................................................................................38 4.1.7. Determinação da estrutura primária de peptídeo ..........................................................38 4.1.8. Desenho racional de análogos e síntese de peptídeos ............................................38 4.1.9. Dicroísmo circular .................................................................................................................................39 4.2. Ensaios biológicos ............................................................................................................... 40 4.2.1. Avaliação da atividade antibacteriana e antifúngica .................................................40 4.2.2. Determinação da atividade antiparasitária .........................................................................40 4.2.2.1. Ensaio In vitro contra Plasmodium falciparum ........................................................................ 41 v 4.2.2.2. Ensaio In vitro contra Trypanosoma brucei brucei ................................................................. 42 4.2.2.3. Ensaio in vitro contra Trypanosoma cruzi .................................................................................. 42 4.2.2.4. Atividade in vitro contra Giardia lamblia ...................................................................................... 43 4.3. Atividade hemolítica in vitro.............................................................................................. 43 4.4. Atividade citotóxica in vitro ............................................................................................... 43 4.5. Etapa II, análise peptidômica e proteômica da secreção cutânea de D. columbianus por shotgun .......................................................................................................... 44 4.5.1. Identificação de proteínas presentes na secreção cutânea de D. columbianus.............................................................................................................................................................44 4.5.1.1. Digestão tríptica em solução ............................................................................................................ 44 4.5.1.2. Cromatografia liquida acoplada a espectrometria de massas (LC-MS / MS) ............. 45 4.5.1.3. Identificação de proteínas ................................................................................................................. 45 4.6. Identificação das enzimas proteolíticas ........................................................................ 46 4.6.1. Zimografia ....................................................................................................................................................46 4.6.2. Digestão em gel SDS-PAGE ...........................................................................................................46 4.6.3. Analise por (LC-MS / MS) .................................................................................................................47 4.7. Identificação dos peptídeos não trípticos presentes na secreção cutâneo de D. columbianus .............................................................................................................................. 48 4.8. Avaliação da ativação de neutrófilos ............................................................................. 49 5. Resultados.........................................................................................................................
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