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Marcadores Moleculares Para Identificação De Carrapatos Vetores De Agentes Rickettsiais

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Marcadores Moleculares Para Identificação De Carrapatos Vetores De Agentes Rickettsiais MARLOS DA MATTA AGOSTINI MARCADORES MOLECULARES PARA IDENTIFICAÇÃO DE CARRAPATOS VETORES DE AGENTES RICKETTSIAIS Dissertação apresentada à Universidade Federal de Viçosa, como parte das exigências do Programa de Pós-Graduação em Bioquímica Agrícola, para obtenção do título de Magister Scientiae. VIÇOSA MINAS GERAIS - BRASIL 2008 Ficha catalográfica preparada pela Seção de Catalogação e Classificação da Biblioteca Central da UFV T Agostini, Marlos da Matta, 1980- A275m Marcadores moleculares para identificação de carrapatos 2008 vetores de agentes rickettsiais / Marlos da Matta Agostini. – Viçosa, MG, 2008. xiii, 86f.: il. (algumas col.) ; 29cm. Inclui apêndices. Orientador: Cláudio Lísias Mafra de Siqueira. Dissertação (mestrado) - Universidade Federal de Viçosa. Referências bibliográficas: f. 62-74. 1. Desenho de primers. 2. Reação em cadeia de polimera- se. 3. Marcadores genéticos. 4. Identificação molecular. 5. Carrapato como transmissor de doenças. I. Universidade Federal de Viçosa. II.Título. CDD 22.ed. 572.86 MARLOS DA MATTA AGOSTINI MARCADORES MOLECULARES PARA IDENTIFICAÇÃO DE CARRAPATOS VETORES DE AGENTES RICKETTSIAIS Dissertação apresentada à Universidade Federal de Viçosa, como parte das exigências do Programa de Pós-Graduação em Bioquímica Agrícola, para obtenção do título de Magister Scientiae. APROVADA: 26 de fevereiro de 2008. Prof. Jorge Abdala Dergam dos Profª. Márcia Rogéria de Santos Almeida Lamêgo (Co-Orientador) (Co-Orientadora) Profª. Juliana Lopes Rangel Fietto Prof. Sérgio Oliveira de Paula Prof. Cláudio Lísias Mafra de Siqueira (Orientador) “Ao meu pai, José Vasco, pelo exemplo de vida, dedicação, incentivo e apoio”. “À minha querida Mãe, Santinha, pelo exemplo de vida, amor incondicional, renúncia e dedicação”. In memoriam ii “A percepção do desconhecido é a mais fascinante das experiências. O homem que não tem os olhos abertos para o misterioso passará pela vida sem ver nada.” Albert Einstein iii AGRADECIMENTOS À Universidade Federal de Viçosa pela oportunidade. Ao Prof. Cláudio Mafra, pela orientação, amizade, companheirismo e confiança, fundamentais para o desenvolvimento deste trabalho. À Prof.ª Márcia Rogéria, pela co-orientação, por disponibilizar o Laboratório de Infectologia Molecular Animal (LIMA) e pelo carinho de sempre. Ao Prof. Jorge Dergam, pela co-orientação, disponibilidade, amizade e ensinamentos valiosos. Ao Prof. Sérgio de Paula, pelos ensinamentos, empréstimo de material e amizade. À Prof.ª Juliana Fietto, pelos ensinamentos, sugestões no desenho dos primers e correções. Aos Professores Marcelo Labruna (USP) e Matias Szabó (UFU), pela gentileza e dedicação no envio das amostras. Aos queridos amigos Edvaldo, Karla, Luíza, Luciana, Thiago e Wiliane, do Laboratório de Bioquímica e Biologia Molecular de Agentes Infecciosos e Parasitários pela excelente convivência, pela força e grande colaboração. A todos do Departamento de Bioquímica e Biologia Molecular, professores e funcionários, e em especial ao Eduardo, Reginaldo, Serafim e Adenilson, por toda a ajuda. Aos colegas do LIMA pela ajuda durante a realização dos experimentos. Ao Prof. José Lino e ao Vinícius, por disponibilizarem o laboratório para a obtenção das fotografias. Ao Dr. David H. Walker do Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston - EUA, pelo apoio financeiro e científico. Aos colegas do Laboratório de Bioquímica Animal, Anderson e Elisa, por sempre estarem dispostos a ajudar. iv Aos grandes amigos Bruno, Christiano, Juninho, Juliano, Balta, Liquin, Michel, Paulão e Sérgio, por facilitarem a vida em Viçosa e pelos inúmeros momentos de alegria. À amiga Kênia, pela força, carinho e companheirismo de sempre. As amigas “antigas” da Bioquímica, Cassiana, Fernanda, Hebréia, Rejane e Thaís, pela torcida de sempre. Aos amigos da república, da “antiga” e da “nova” geração, por terem sido minha família em Viçosa. A todos os colegas das disciplinas da pós-graduação, pelos ensinamentos, amizade e companheirismo. Ao meu pai, José Vasco, e minha querida mãe, Santinha, pelo exemplo de vida, amor incondicional, renúncia e dedicação. Sem o apoio de vocês nada disso seria possível. Ao Magno, Mozart e Fabiane, pelo apoio, amizade, amor e por sempre zelarem por mim. Aos meus anjinhos, Giuliano e Gabriela, inspiração para o que eu faço. À Beatriz, pelo carinho, paciência, incentivo, compreensão, pela importância em minha vida e pelos sonhos que compartilhamos. A todos os meus familiares e amigos que mesmo distantes, contribuíram de alguma forma para a realização deste trabalho. Acima de tudo, agradeço todas as bênçãos recebidas ao longo da minha vida, as oportunidades concedidas por Deus para realização dos meus objetivos e a força para chegar até aqui. v BIOGRAFIA MARLOS DA MATTA AGOSTINI, filho de José Vasco Agostini e Maria do Amparo Matta Agostini, nasceu em 06 de dezembro de 1980, em Muriaé, Minas Gerais. Em janeiro de 2005 graduou-se em Bioquímica, pela Universidade Federal de Viçosa, Minas Gerais – Brasil. Trabalhou por um período na Monsanto do Brasil Ltda em Uberlândia, Minas Gerais. Em maio de 2006 ingressou no curso de Mestrado em Bioquímica Agrícola da Universidade Federal de Viçosa. Durante o curso de mestrado foi monitor da disciplina Bioquímica Prática, defendendo a dissertação em Fevereiro de 2008. vi SUMÁRIO LISTA DE TABELAS ....................................................................................................IX LISTA DE FIGURAS ......................................................................................................X ABREVIATURAS ..........................................................................................................XI RESUMO.......................................................................................................................XII ABSTRACT .................................................................................................................XIII 1 INTRODUÇÃO .......................................................................................................1 2 REVISÃO DE LITERATURA.................................................................................5 2.1 O Carrapato .......................................................................................................... 5 2.1.1 Aspectos gerais da biologia, classificação e diversidade ......................... 5 2.1.2 O carrapato como vetor.................................................................................. 8 2.1.3 Filogenia, evolução e zoogeografia.............................................................. 9 2.1.4 Dificuldades na identificação ....................................................................... 11 2.2 Marcadores moleculares aplicados a acarologia.......................................... 13 2.2.1 Aloenzimas ........................................................................................................ 13 2.2.2 Análise direta de proteínas ............................................................................. 14 2.2.3 Amplificação de DNA utilizando a PCR ........................................................ 14 2.2.4 Amplificação aleatória de DNA polimórfico (RAPD) ................................... 15 2.2.5 Polimorfismo do comprimento de fragmentos de restrição (RFLP).......... 15 2.2.6 Microsatélites .................................................................................................... 16 2.2.7 Polimorfismo de comprimento de fragmentos amplificados (AFLP) ........ 16 2.2.8 Sequenciamento de fragmentos de DNA ..................................................... 16 2.2.9 DNA mitocondrial.............................................................................................. 17 2.2.10 DNA ribossomal nuclear................................................................................ 21 2.3 Armazenamento de espécimes ....................................................................... 24 2.4 Extração de DNA em carrapatos..................................................................... 24 2.5 Amplificação de DNA em carrapatos.............................................................. 26 2.6 Panorama atual da filogenia e taxonomia molecular em carrapatos......... 27 3 OBJETIVOS .........................................................................................................28 3.1 Objetivo geral ..................................................................................................... 28 3.2 Objetivos específicos ........................................................................................ 28 4 MATERIAL E MÉTODOS....................................................................................29 4.1 Amostras ............................................................................................................. 29 4.2 Extração de DNA ............................................................................................... 29 4.3 Amplificação pela PCR ..................................................................................... 32 4.4 Purificação dos produtos da PCR ................................................................... 33 4.5 Clonagem dos produtos purificados ............................................................... 33 4.5.1 Preparo de células competentes................................................................. 33 4.5.2 Transformação de células competentes.................................................... 34 4.5.3 Triagem das colônias recombinantes........................................................
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