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Methylglyoxal Effects on Protein Aggregation and Amyloid Formation

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Methylglyoxal Effects on Protein Aggregation and Amyloid Formation UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS DEPARTAMENTO DE QUÍMICA E BIOQUÍMICA Methylglyoxal effects on protein aggregation and amyloid formation Luís Miguel Araújo da Silva Oliveira Doutoramento em Bioquímica (Especialidade: Biofísica Molecular) Tese orientada por: Prof. Doutor Alexandre Luís de Matos Botica Côrtes Quintas Doutor Carlos Alberto Alves Cordeiro 2009 De acordo com o disposto no artigo n°. 40 do Regulamento de Estudos Pós‐Graduados da Universidade de Lisboa, Deliberação n° 961/2003, publicada no Diário da República – II Série n°. 153 – 5 de Julho de 2003, foram incluídos nesta dissertação os resultados dos seguintes artigos: Luís M. A. Oliveira, Carlos Cordeiro, Ana Ponces Freire, Carla Ascenso, Alexandre Quintas. 2007. Unveiling heme proteins conformational stability through a UV absorbance ratio method Anal Biochem. 371: 253‐5 Alexandre Quintas, Luís M. A. Oliveira. 2008. Protein hierarchy levels of structural organization for amyloidogenesis: Biochemical and biophysical aspects underlying misfolding and disease. Protein Misfolding in Biology and Disease T. F. Outeiro, Research Signpost: 1‐34. Ricardo A. Gomes, Luís M. A. Oliveira, Mariana Silva, Carla Ascenso, Alexandre Quintas, Gonçalo Costa, Ana V. Coelho, Marta Sousa Silva, António E. N. Ferreira, Ana Ponces Freire, Carlos Cordeiro. 2008. Protein glycation in vivo: Functional and structural effects on yeast enolase. Biochem J. 416: 317‐26 Aknowledgments/Agradecimentos Aknowledgments/Agradecimentos Chegada a conclusão desta etapa com mais de quatro anos, não a poderia deixar terminar sem agradecer a todos os que, directa ou indirectamente, contribuíram para a realização deste trabalho. Em primeiro lugar quero expressar o meu mais profundo agradecimento ao Professor Doutor Alexandre Quintas, orientador principal deste trabalho, por todo o seu apoio, amizade, compreensão e disponibilidade. Desde que começámos a trabalhar juntos que sempre manteve a nossa relação numa base de confiança, transmitindo‐me a ideia de que somos uma equipa. E uma equipa luta junta pelos seus objectivos. As suas qualidades humanas e científicas que sempre marcaram presença são uma referência. Estou certo que no futuro ainda muito mais irei aprender com ele. Por tudo, o meu mais sincero obrigado. Ao Doutor Carlos Cordeiro, segundo orientador deste trabalho, agradeço a preocupação e ensinamentos que me fizeram crescer como investigador desde o primeiro dia que integrei o grupo de enzimologia. A sua serenidade, ponderação e conhecimento contribuíram de forma extremamente positiva para o desenrolar deste trabalho e para a minha aprendizagem como investigador. À Professora Doutora Ana Ponces, uma palavra especial por tudo o que representa. Desde os tempos de licenciatura que é uma referência minha pelos ensinamentos, pela sua capacidade invulgar de gerar motivação, pelo apoio, amizade e postura perante a vida. Uma pessoa que admiro profundamente que me ensinou e preparou a ser o investigador que sou. Aos amigos e colegas do Laboratório de Patologia Molecular, onde passei a maior parte do meu doutoramento, quero agradecer a simpatia, amizade, companheirismo e boa disposição que sempre pautou a nossa investigação. O meu destaque à Professora Carla Ascenso pelo apoio, ao Carlos Família, com quem partilho o laboratório desde o início, e à Lena pela amizade. Carlos fica a frase: “Oi pessoal tudo bem? Vai um café?”. À Ana e ao Miguel pela amizade, companheirismo e espírito de grupo criado. Não esquecerei a entreajuda estabelecida convosco bem como as Aknowledgments/Agradecimentos aventuras por Lausanne e Copenhaga. Espero sinceramente que não fiquem por aqui. À Joana pela alegria, amizade e bom espírito. É bom ter‐te no laboratório. Ao Gonçalo pelo espírito e amizade. À Catarina que foi marcante pelo furacão de alegria que sempre trazia consigo quando entrava pelo laboratório. Uma amiga que ficou. E finalmente, aos colegas que, não fazendo parte do nosso grupo, estão tão perto: à Teresa e à Susana pela amizade e carinho e ao Henrique, à Inês e ao Paulo pelo bom espírito e profissionalismo que trazem sempre consigo. You make my day!!! Agradeço também a todas as pessoas do Grupo de Enzimologia, o grupo que inicialmente me acolheu e formou. Foi muito o que aprendi junto de vós. O meu agradecimento especial ao Professor António Ferreira pelos conhecimentos e disponibilidade que sempre mostrou. Ao Ricardo Gomes por ser um colega espectacular. Diversão, capacidade de trabalho, entreajuda, amizade, espírito empreendedor, sempre foram qualidades que estiveram presentes. Ao Nuno Lages por ser um grande amigo com quem partilhei bons e maus momentos ao longo destes anos. Ao Hugo Miranda pela longa amizade e por todos os desafios que superámos em conjunto ainda desde a licenciatura. À sempre sorridente Lídia pela amizade e boa disposição. E finalmente à Mariana, ao Gonçalo e à Marta pela amizade e apoio. Uma palavra também para o Doutor Tiago Outeiro que desde o início se revelou ser uma pessoa inspiradora que me leva sempre a tentar ser um investigador cada vez melhor. Admiro muito a sua capacidade de comunicação, trabalho e toda a ciência que produz. O meu obrigado por toda disponibilidade, serenidade e confiança que sempre esteve presente na nossa relação, bem como a ajuda fornecida para que fosse possível trabalhar no laboratório do Professor Doutor Hilal Lashuel. Ao Professor Doutor Hilal Lashuel o meu muito obrigado por me ter acolhido no seu laboratório. As suas qualidades científicas são do melhor que tive a possibilidade de presenciar. Apesar do pouco tempo que estive no seu laboratório, foram inúmeros os conhecimentos que obtive e a aprendizagem que fiz. Para isso contribuiu também sem dúvida a orientação mais estreita da Doutora Katrina Paleologou com quem foi um prazer enorme trabalhar. A todos o meu muito obrigado em especial aos colegas Abid, Asad, Margot e Mirva. Aos amigos, que estão sempre lá, para a ciência e para tudo o resto. Não há palavras para descrever a vossa importância na minha vida. Sinto‐me um privilegiado por poder chamar amigo a Aknowledgments/Agradecimentos muita gente, mas não consigo deixar de vos destacar. Obrigado João Luís, Sónia Pais, Margarida Neves de Sousa, Ana Rita Moreira, João Lopes, Sara Gomes e a todos os Scramble por fazerem parte da minha vida. Por último o meu agradecimento a toda a família… aos avós, aos padrinhos, aos tios e primos que, embora longe, são tão importantes e sabem estar tão perto; e duas palavras de grande carinho: para com a minha mãe por tudo o que me proporcionou desde que nasci. Este meu ínfimo sucesso é sem dúvida fruto do seu amor, trabalho e dedicação… e eu não sou fácil de aturar!!! É impossível verbalizar a importância que tens na minha vida; e para com o meu pai a quem quero muito dedicar este trabalho. A tua partida a meio deste caminho deixa‐me uma mágoa inigualável. Era capaz de escrever outra tese sobre a pessoa, o exemplo e a referência que és e como te tenho presente nos meus dias, nas minhas atitudes e decisões. Sei que ninguém estaria mais orgulhoso que tu por me ver chegar aqui e por isso sei que o que fiz também é teu. Aknowledgments/Agradecimentos Aknowledgments/Agradecimentos Table of Contents AKNOWLEDGMENTS/AGRADECIMENTOS...........................................................................................V TABLE OF CONTENTS...............................................................................................................................IX SUMMARY..................................................................................................................................................XIII RESUMO .......................................................................................................................................................XV ABBREVIATIONS ..................................................................................................................................... XIX CHAPTER I...................................................................................................................................................... 2 1. PROTEIN STRUCTURE .................................................................................................................................. 3 1.1 Secondary structure............................................................................................................................. 5 1.1.1 Helical structures ........................................................................................................................................... 5 1.1.2 Beta structures ............................................................................................................................................... 6 1.1.3 Non-repetitive secondary structure................................................................................................................ 7 1.2 Tertiary structure................................................................................................................................. 7 1.3 Protein dynamics and flexibility .........................................................................................................10 1.4 Natively unfolded proteins..................................................................................................................11 2. THE PROTEIN FOLDING PROBLEM ...............................................................................................................14 2.1 The folding code .................................................................................................................................14
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