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UNIVERSIDADE DE SÃO PAULO INSTITUTO DE BIOCIÊNCIAS Programa De Pós-Graduação Em Ciências Biológica (Biologia/Genética)

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UNIVERSIDADE DE SÃO PAULO INSTITUTO DE BIOCIÊNCIAS Programa De Pós-Graduação Em Ciências Biológica (Biologia/Genética) UNIVERSIDADE DE SÃO PAULO INSTITUTO DE BIOCIÊNCIAS Programa de Pós-Graduação em Ciências Biológica (Biologia/Genética) CAROLINA MANCINI VALL BASTOS Análise da expressão gênica diferencial das glândulas de veneno de Bothrops jararaca (Serpentes: Viperidae). Analysis of differential gene expression of the venom gland of Bothrops jararaca (Serpentes: Viperidae) São Paulo 2011 CAROLINA MANCINI VALL BASTOS Análise da expressão gênica diferencial das glândulas de veneno de Bothrops jararaca (Serpentes: Viperidae). Analysis of differential gene expression of the venom gland of Bothrops jararaca (Serpentes: Viperidae). Tese apresentada ao Instituto de Biociências da Universidade de São Paulo, para a obtenção de Título de Doutor em Ciências Biológicas (Biologia/Genética), na Área de Biologia (Genética). Orientador(a): Dr. Inácio de Loiola Meirelles Junqueira de Azevedo São Paulo 2011 Bastos, Carolina Mancini Vall Análise da expressão gênica diferencial das glândulas de veneno de Bothrops jararaca (Serpentes: Viperidae). 157p Tese (Doutorado) - Instituto de Biociências da Universidade de São Paulo. Departamento de Genética e Biologia Evolutiva 1. Bothrops jararaca 2. Glândula de veneno 3. RNA-seq I. Universidade de São Paulo. Instituto de Biociências. Departamento de Genética e Biologia Evolutiva. Comissão Julgadora: ________________________ _____ _______________________ Prof(a). Dr(a). Prof(a). Dr(a). _________________________ ____________________________ Prof(a). Dr(a). Prof(a). Dr(a). Prof(a). Dr(a). Orientador(a) With a little help from my friends.... Em memória do meu avô, Paulo Mancini. You gain strength, courage and confidence by every experience in which you really stop to look fear in the face. You are able to say to yourself, 'I have lived through this horror. I can take the next thing that comes along.' You must do the thing you think you cannot do. Eleanor Roosevelt US diplomat & reformer (1884 - 1962) Agradecimentos Gostaria de agradecer ao meu orientador, Dr. Inácio, pela confiança, paciência, apoio e pela oportunidade de trabalhar nesse projeto que exigiu muito de mim e me mostrou que ciência se faz com trabalho duro, muito estudo e um pouco de teimosia; Ao Dr. Paulo Lee Ho que me recebeu em seu laboratório e sempre foi muito participativo, dando sugestões, broncas e muito incentivo; À Dra. Norma Yamanouye, colaboradora e uma das idealizadoras desse projeto, que me ajudou com as serpentes e contribuiu com valiosas discussões; Ao Dr. Marcelo Laia, que me ajudou muito nas análises de bioinformática, me recebeu em Jaboticabal, com quem troquei dúzias de e-mails e que sempre foi muito atencioso, solícito, interessado e sem ele, com certeza, esse trabalho teria sido muito mais difícil; À Dra Maria Leonor de Oliveira e à Dra. Eliane Miyaji pelas conversas, muitas vezes informais mas que me fizeram aprender muito sobre ciência e sobre o meio acadêmico, e por me inspirarem a ser uma profissional melhor; À todos os meus colegas do laboratório de biotecnologia molecular I pela companhia diária, troca de idéias e apoio nos momentos difíceis; À todo pessoal do CAT que me recebeu muito bem e fez com que me sentisse em casa desde o primeiro dia, e principalmente, aos meus colegas de laboratório, ao Ivan e a Mari que em pouco tempo conquistaram meu respeito, admiração e ganharam minha amizade; Aos amigos do laboratório de biotecnologia molecular IV pela atenção e disponibilidade sempre que precisei usar algum equipamento deles e pela companhia sempre bem vinda na happy hour pós expediente; Aos queridíssimos Luciane Schons e o Henrique Rofatto, pelas ótimas discussões sobre RT- PCR que foram muito proveitosas para o meu trabalho; As amigas, Adriana, Luciane, Liliane, Vivian, Fernanda, Patrícia, Regiane e Swiany, por acreditarem em mim quando nem eu acreditava mais, pela parceria, e por serem responsáveis pelas melhores lembranças que vou guardar desses anos; Aos meus pais por toda dedicação, por estarem sempre ao meu lado apoiando minhas escolhas e decisões; Às minhas irmãs, Catarina e Camila, pela paciência, pelo apoio e por tornarem tudo mais leve, me mostrarando o que realmente importa; Às minhas melhores amigas Mariana e Marcella por serem minhas melhores amigas, mesmo com toda distância, a falta de tempo, e as obrigações que temos, ainda conseguimos manter nossa amizade e no fim das contas só isso importa; Ao Patrick, que há 3 anos me fez criar coragem e sair pelo mundo, graças a ele fui mais longe do que imaginei ir em toda minha vida, e nos últimos meses ele foi meu melhor e mais paciente amigo, mesmo a 12.000km de distância; Ao meu assessor(a) FAPESP e à Fundação de Amparo a Pesquisa do Estado de São Paulo, FAPESP, (processo 2006/07029-0) pela concessão da bolsa de Doutorado Direto. Índice 1. Introdução .......................................................................................................... 01 1.1. Serpentes ......................................................................................................... 01 1.2. Veneno e Envenenamento .............................................................................. 04 1.3. Glândula de veneno ......................................................................................... 09 1.4. Análise transcriptômica e biologia de sistemas ............................................... 14 2. Objetivos .............................................................................................................. 21 3. Justificativa .......................................................................................................... 21 4. Desenho experimental ...................................................................................... 22 5. Materiais e Métodos ......................................................................................... 24 5.1. Procedimentos básicos .................................................................................... 24 5.1.1. Animais .................................................................................................. 24 5.1.2. Extração da glândula de veneno ........................................................... 24 5.1.3. Obtenção do RNA .................................................................................. 25 5.1.4. Extração de DNA plasmidial (miniprep de placa) .................................. 26 5.1.5. Sequenciamento dos clones .................................................................. 26 5.2. Construção da biblioteca de cDNA para confecção das membranas de macroarranjo.................................................................................................... 27 5.2.1. Construção da biblioteca de cDNA ........................................................ 27 5.2.2. Transformação e eletroporação da biblioteca em E.coli ....................... 29 5.2.3. Validação da biblioteca ......................................................................... 30 5.2.4. Seleção de clones para construção dos macroarranjos ........................ 30 5.3. Construção das membranas de macroarranjo ................................................ 31 5.3.1. Transferência das colônias bacterianas para a membrana de náilon......................................................................................................... 31 5.3.2. Crescimento das colônias bacterianas na membrana ........................... 33 5.3.3. Processamento da membrana após o crescimento das colônias bacterianas................................................................................................ 33 5.4. Análise temporal da expressão gênica na glândula de veneno de Bothrops jararaca ........................................................................................................... 34 5.4.1. Síntese do cDNA .................................................................................... 34 5.4.2. Marcação das sondas de cDNA ............................................................. 35 5.4.3. Hibridização da membrana com a sonda de cDNA ............................... 35 5.4.4. Pós-Hibridização e revelação das membranas ...................................... 36 5.4.5. Captura e análise das imagens .............................................................. 36 5.4.6. Análises estatísticas ............................................................................... 37 5.4.7. Análise temporal do perfil de expressão gênica .................................... 37 5.4.8. Análise das sequências .......................................................................... 38 5.4.9. Anotação dos clones .............................................................................. 38 5.5. Análise da expressão gênica diferencial em resposta a tratamentos farmacológicos ................................................................................................. 39 5.5.1. Tratamentos e extração da glândula de veneno.................................... 39 5.5.2. Identificação e anotação funcional dos clones diferencialmente expressos................................................................................................... 39 5.6. Análise da expressão gênica por PCR quantitativo em tempo real (qPCR)............................................................................................................... 40 5.6.1. Escolha do gene de referência para análise da expressão gênica na glândula de veneno................................................................................... 40 5.6.2. Escolha e construção dos oligonucleotídeos iniciadores dos genes de estudo
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