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Identificaçào De Sequâncias Gânicas De TELMA ALVES MONEZI IDENTIFICAÇÃO DE SEQUÊNCIAS GÊNICAS DE CHELONID ALPHAHERPESVIRUS 5 (ChHV5) EM TECIDOS TUMORAIS CARACTERIZADOS HISTOLOGICAMENTE E SECREÇÕES DE CHELONIA MYDAS CAPTURADAS NO LITORAL NORTE DO ESTADO DE SÃO PAULO NO PERÍODO DE 2001 A 2012 Tese apresentada ao Programa de Pós- Graduação em Biologia Celular e Tecidual do Instituto de Ciências Biomédicas da Universidade de São Paulo, para obtenção do Título de Doutor em Ciências. São Paulo 2016 TELMA ALVES MONEZI IDENTIFICAÇÃO DE SEQUÊNCIAS GÊNICAS DE CHELONID ALPHAHERPESVIRUS 5 (ChHV5) EM TECIDOS TUMORAIS CARACTERIZADOS HISTOLOGICAMENTE E SECREÇÕES DE CHELONIA MYDAS CAPTURADAS NO LITORAL NORTE DO ESTADO DE SÃO PAULO NO PERÍODO DE 2001 A 2012 Tese apresentada ao Departamento de Biologia Celular e do Desenvolvimento do Instituto de Ciências Biomédicas da Universidade de São Paulo, para obtenção do Título de Doutor em Ciências. Área de concentração: Biologia Celular e Tecidual Orientadora: Profa. Dra. Maria Inês Borella Versão original São Paulo 2016 DADOS DE CATALOGAÇÃO NA PUBLICAÇÃO (CIP) Serviço de Biblioteca e Informação Biomédica do Instituto de Ciências Biomédicas da Universidade de São Paulo © reprodução total Monezi, Telma Alves. Identificação de sequências gênicas de Chelonid alphaherpesvirus 5 (ChHV5) em tecidos tumorais caracterizados histologicamente e secreções de Chelonia mydas capturadas no litoral norte do Estado de São Paulo no período de 2001 a 2012 / Telma Alves Monezi. -- São Paulo, 2016. Orientador: Maria Inês Borella. Tese (Doutorado) – Universidade de São Paulo. Instituto de Ciências Biomédicas. Departamento de Biologia Celular e do Desenvolvimento. Área de concentração: Biologia Celular e Tecidual. Linha de pesquisa: Estudo etiológico de fibropapilomatose de tartarugas marinhas. Versão do título para o inglês: Identification of Chelonid alphaherpesvirus 5 (ChHV5) gene sequences in tumor tissues histologically characterized and secretions from green turtles Chelonia mydas captured off the coast of São Paulo State in the period 2001- 2012. 1. Herpesvirus 2. ChHV5 3. Fibropapilomatose 4. Chelonia mydas 5. Reação em cadeia pela polimerase 6. Análise filogenética I. Borella, Maria Inês II. Universidade de São Paulo. Instituto de Ciências Biomédicas. Programa de Pós-Graduação em Biologia Celular e Tecidual III. Título. ICB/SBIB0107/2016 UNIVERSIDADE DE SÃO PAULO INSTITUTO DE CIÊNCIAS BIOMÉDICAS ______________________________________________________________________________________________________________ Candidato(a): Telma Alves Monezi. Título da Tese: Identificação de sequências gênicas de Chelonid alphaherpesvirus 5 (ChHV5) em tecidos tumorais caracterizados histologicamente e secreções de Chelonia mydas capturadas no litoral norte do Estado de São Paulo no período de 2001 a 2012. Orientador(a): Maria Inês Borella. A Comissão Julgadora dos trabalhos de Defesa da Tese de Doutorado, em sessão pública realizada a ................./................./................., considerou ( ) Aprovado(a) ( ) Reprovado(a) Examinador(a): Assinatura: ............................................................................................... Nome: ....................................................................................................... Instituição: ................................................................................................ Examinador(a): Assinatura: ................................................................................................ Nome: ....................................................................................................... Instituição: ................................................................................................ Examinador(a): Assinatura: ................................................................................................ Nome: ....................................................................................................... Instituição: ................................................................................................ Examinador(a): Assinatura: ................................................................................................ Nome: ....................................................................................................... Instituição: ................................................................................................ Presidente: Assinatura: ................................................................................................ Nome: ....................................................................................................... Instituição: ................................................................................................ A toda minha família, razão do meu viver! AGRADECIMENTOS ESPECIAIS A Profa. Dra. Maria Inês Borella, pela orientação do presente trabalho e por ter me recebido de braços abertos em seu laboratório. Pela amizade e pelo jeito meigo e doce que sempre me recebeu, muito obrigada! A Profa. Dra. Dolores Ursula Mehnert, por termos iniciado esse projeto juntas, há anos atrás, e me permitir continuar a desenvolvê-lo em seu laboratório. Pela ajuda, pelos incentivos para realização deste trabalho e pela amizade de todos esses anos. A minha filha tão amada Ananda Alves Monezi, meu “tudo”, e ao Paulo Monezi que conviveu comigo todos esses anos e que tem se tornado uma pessoa muito melhor. Parabéns! Aos meus pais queridos e sábios, que me deram a oportunidade de estar aqui e me tornar a pessoa que sou. AGRADECIMENTOS Ao Projeto TAMAR-ICMBio, especialmente a Base de Ubatuba (SP), por ter permitido a realização deste trabalho, e ter concedido todas as amostras, desde o início do projeto. Especialmente a Cecília Baptistotte, Berenice M. G. Gallo, José Henrique Becker, Max Rondon Werneck e Renato Velloso. A todos os estagiários e equipe do TAMAR, fundamental para a coleta de dados. Ao Instituto Chico Mendes de Conservação da Biodiversidade (IBAMA/ICMBio), do Ministério do Meio Ambiente, pela licença concedida para conduzir as pesquisas e trabalhar com esses animais fantásticos e, ainda intrigantes, as tartarugas marinhas. A FAPESP, pela ajuda financeira concedida nos primeiros anos do estudo com as tartarugas marinhas (2004/13218-5). A Profa. Dra. Eliana R. Matushima, da FMVZ da USP, por nos ter convidado a participar deste projeto incrível. Ao Centro de Pesquisas sobre o Genoma Humano e Células-Tronco, IB-USP, pelo Sequenciamento de DNA e, especialmente, a Vanessa Naomi, pela atenção e disponibilidade a mim dispensadas, além das dicas preciosas e “macetes”, fundamentais pa ra os resultados alcançados. Ao Cruz Alberto Mendoza Rigonati, pelo seu exímio trabalho e por ter me ensinado as técnicas histológicas, que nunca encontraria nos livros e pela oportunidade de convívio no laboratório, tornando nossa convivência muito alegre, obrigada! A MSc. Elisabeth Mendes Martins de Moura, toda a minha gratidão pelo auxílio concedido em várias etapas desse trabalho, mas principalmente nas etapas inicias das análises filogenéticas, cuja ajuda foi crucial para que eu persistisse. Por nos tornarmos amigas, compartilhando alegrias, angústias, cafezinhos, lanchinhos e guloseimas. Ao Prof. Dr. Paolo Zanotto, do Laboratório de Evolução Viral e Bioinfomática do Departamento de Microbiologia, por permitir o processamento de dados para as análises filogenéticas nos “poderosos” equipamentos de seu laboratório. e ao Pós -Doc Dr. Caio Freire pela ajuda fundamental e indispensável na análise dos dados que muito contribuíram para a nossa publicação em revista científica, meu muito obrigada! Ao Prof. Dr. Enrique Mario Boccardo Pierulivo, do Laboratório de Oncovirologia, do Departamento de Microbiologia, ICB-USP, pelos incentivos constantes para a realização deste trabalho e do paper , e por compartilharmos o café da virologia todas as manhãs, tornando nossos dias mais alegres e descontraídos. Ao Prof. Dr. Mario Julio A. Campos, então chefe do Depto de Micro, por ter permitido que eu realizasse minha tese no Depto de BioCel. Ao Prof. Dr. Flavio Alterthum, do Depto de Micro, por sua atenção, generosidade, apoio e incentivos constantes para a realização deste trabalho, além da parceria na autoria de alguns livros e capítulos. Obrigada pelas discussões e dicas preciosas na elaboração da tese. Muito obrigada mesmo! Aos alunos de Pós-Doc do Departamento de Microbiologia, Dr. Jansen de Araújo e Dra. Angélica Cristine Campos, do Laboratório de Virologia Clinica e Molecular, pela ajuda e sugestões concedida nas análises filogenéticas para a publicação científica do presente trabalho, fundamental para que se concretizasse. Ao Dr. Gustavo Dutra, do Aquário de Santos, por ter gentilmente coletado e cedido algumas amostras clínicas dos animais deste estudo. Ao Prof. Dr. Carlos F. Menck, do Departamento de Microbiologia, ICB-USP, por ter permitido a utilização dos equipamentos de seu laboratório, principalmente para a captação das imagens. A Natascha M. G. Müller, minha grande amiga, por ter compartilhado comigo muito dos anos iniciais desse projeto, por termos descoberto juntas as aventuras das coletas em Ubatuba, os sacrifícios e maravilhas de trabalhar no laboratório. Amiga, você sabe, é peça fundamental na minha vida! A Patrícia Garrafa, por tantos anos de convívio no laboratório, amizade, por sempre acreditar em mim e torcer para minha felicidade! Obrigada Paty! A Dra. Veridiana Munford, por toda ajuda concedida no manuseio do equipamento de fotografia digital, além da amizade de todos esses anos, que não são
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