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Tese Final.Pdf PRISCYLLA TATIANA CHALFUN GUIMARÃES CARACTERIZAÇÃO MOLECULAR E IMUNOLÓGICA DO VENENO DE Tityus fasciolatus E SUA AÇÃO SOBRE CAMUNDONGOS Tese apresentada na Escola de Veterinária da Universidade Federal de Minas Gerais, como requisito parcial para obtenção do título de Doutor em Ciência Animal Área : Toxicologia e Plantas tóxicas Orientadora : Profa. Marília Martins Melo Belo Horizonte Escola de Veterinária da UFMG 2009 G936c Guimarães, Priscylla Tatiana Chalfun, 1974- Caracterização molecular e imunológica do veneno de Tityus fasciolatus e sua ação sobre camundongos / Priscylla Tatiana Chalfun Guimarães. – 2009. 130 p. : il. Orientadora: Marília Martins Melo Tese (doutorado) – Universidade Federal de Minas Gerais, Escola de Veterinária Inclui bibliografia 1. Camundongo como animal de laboratório – Teses. 2. Escorpião – Veneno – Teses. 3. Envenenamento – Teses. 4. Hematologia – Teses. I. Melo, Marília Martins. II. Universidade Federal de Minas Gerais. Escola de Veterinária. III. Título. CDD – 615.918 2 3 4 AGRADECIMENTOS À Deus, que me fortalece com sua benção, abrindo meus caminhos. Com Ele, ninguém nos detem. À meu pai e minha mãe, a razão de tudo que faço em minha vida, é por eles que luto e enfrento todos os obstáculos com muito prazer e satisfação. À minha família, pelo amor e apoio, em especial a minha avó Colete Haddad que me ensinou os princípios de uma vida honesta. Obrigada pelo amor. À D. Marília, um anjo que Deus colocou em minha vida, obrigada pelo amor e atenção de mãe. Obrigada por me deixar entrar na sua família e fazer parte dela. À minha orientadora Prof(a). Dr(a). Marília Martins Melo pela dedicação constante para minha vitória nesse doutorado. Obrigada pela oportunidade, paciência, confiança, amizade e principalmente o grande aprendizado na vida profissional e pessoal. À Prof(a) Dr(a). Zélia pela paciência, ensinamentos e co-orientação para a obtenção de ótimos resultados Ao Prof. Dr. Jenner pelas valiosas sugestões e atenção. Ao Prof. Dr. Evanguedes Kalapothakis do Laboratorio de Biotecnologia e Marcadores Moleculares-ICB pela oportunidade e aprendizado na área de Biologia Molecular. Ao Prof. Dr. Carlos Delfin Chaves Olortegui e sua equipe pela oportunidade e auxílio para a realização deste trabalho A toda equipe do laboratório de Toxicologia da Veterinária-UFMG, Eduardo, Durval, Márcia, Luana, Carla, Stella, Ana Flavia, Victoria e Felipe muito obrigada pela atenção, carinho e em especial a Mariana pela grande dedicação para alcançarmos excelentes resultados. Aos amigos do Laboratorio de Biotecnologia e Marcadores Moleculares-ICB, Arthur, Higgor, Ana Luiza, Ana Paula, Carolina, Ana Carolina, Gabriel, Juliana, André e Bárbara pela ajuda, amizade e companherismo durante o tempo que passamos juntos. À Thais, o outro anjo que Deus colocou na minha vida. Pela valiosa ajuda me enriquecendo com os conhecimento na área de biologia molecular e principalmente por nunca me negar uma ajuda pessoal e profissional. À Thalita e Sabrina, minhas amigas e irmãs, obrigada pela paciência, carinho e compreensão durante esses 4 anos. 5 Aos funcionários da Escola de Veterinária-UFMG pelo carinho e auxilio para realização deste trabalho. Aos funcionários da Pós-graduação da Escola de Veterinaria-UFMG pelo atenção e apoio durante esses 4 anos. Aos membros da banca examinadora pelos elogios e críticas construtivas. Ao CNPq e FAPEMIG pelo suporte financeiro para realização deste trabalho. A equipe da FUNED pelas sugestões e auxilio para o desenvolvimento deste projeto. Ao centro de Zoonoses de Ituiutaba pela doação das espécies de Tityus Aos meus amigos de Liverpool que me fizeram sentir em casa me recebendo com todo carinho e atenção. Em especial ao Dr. Robert Harrison por ter sido um orientador e amigo me ensinando e apoiando nos momentos felizes e difíceis e sua equipe, Dr. David Theakston, Dr. Gavin, Dr. Darren e Dr. Simon pelo apoio durante os 15 meses que permaneci no grupo. Obrigada pela oportunidade de crescimento profissional e pessoal. Aos animais meu respeito e amor. Sem vocês esse trabalho não seria possível. Minha eterna gratidão. 6 SUMÁRIO RESUMO .............................................................................................................................. 17 ABSTRACT .......................................................................................................................... 18 1. INTRODUÇÃO ................................................................................................................ 19 2. REVISÃO DE LITERATURA ......................................................................................... 20 2.1. Escorpiões ...................................................................................................................... 20 2.1.1. Distribuição geográfica e classificação dos escorpiões .............................................. 20 2.1.2. Aspectos epidemiologicos .......................................................................................... 22 2.2. Manifestações clínicas .................................................................................................. 23 2.2.1. Alterações sanguíneas ................................................................................................ 25 2.3. Diagnóstico .................................................................................................................... 27 2.4. Composição do veneno .................................................................................................. 29 2.4.1. Toxinas escorpiônicas e seus mecanismos de ação .................................................... 29 2.4.2. Estrutura molecular das toxinas .................................................................................. 31 2.4.3. Toxinas que atuam nos canais de sódio ..................................................................... 32 2.4.4. Toxinas que atuam nos canais de potássio .................................................................. 33 2.4.5. Toxinas que atuam nos canais de cloro ..................................................................... 33 2.4.6. Toxinas que atuam nos canais de cálcio .................................................................... 33 2.4.7. Toxinas sem ponte dissulfeto ...................................................................................... 33 2.5. Resposta imune (Soroterapia e vacinas) ........................................................................ 34 2.6. Nomenclatura das toxinas .............................................................................................. 39 2.7. Veneno do Tityus fasciolatus ......................................................................................... 40 2.8. Veneno do Tityus serrulatus .......................................................................................... 41 2.9. Homologia entre os venenos .......................................................................................... 43 3. OBJETIVOS ..................................................................................................................... 43 3.1. Objetivo geral ................................................................................................................ 43 3.2. Objetivos específicos ..................................................................................................... 43 4. MATERIAL E MÉTODOS .............................................................................................. 43 4.1. Veneno e sua extração .................................................................................................. 43 4.1.1. Tityus fasciolatus ....................................................................................................... 43 4.1.2. Tityus serrulatus ........................................................................................................ 45 4.2. Caracterização clínica do veneno................................................................................... 46 4.2.1. Determinação da DL 50 ................................................................................................ 46 4.2.2. Avaliação anátomo-histológica ................................................................................... 46 4..2.3. Avaliação hematológica, bioquímica sérica e plasmática ........................................ 46 4..2.4. Exames laboratoriais .................................................................................................. 47 4.3. Caracterização molecular do veneno ............................................................................. 47 4.3.1. Extração e purificação do RNA (Ácido ribonucleico) ................................................ 49 4.3.2. Construção do cDNA da glândula de veneno ............................................................. 50 4.3.3. Padronização da RT-PCR ........................................................................................... 50 4.3.4. Purificação do cDNA a partir do gel de agarose 1% .................................................. 51 4.3.5 Ligação do amplicon no vetor de clonagem ............................................................... 51 4.3.6. Preparação de bactérias competentes .......................................................................... 52 4.3.7. Transformação da linhagem E. coli XL1 Blue ............................................................ 53 4.3.8. Purificação do plasmídeo ........................................................................................... 53 7 4.3.9.
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