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Protothecosis: Agent Characterization and Pathogenesis

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Protothecosis: Agent Characterization and Pathogenesis PROTOTHECOSIS: AGENT CHARACTERIZATION AND PATHOGENESIS SARA ANDREIA DE BARROS COSTA MARQUES Tese de doutoramento em Ciências Veterinárias 2010 SARA ANDREIA DE BARROS COSTA MARQUES PROTOTHECOSIS: AGENT CHARACTERIZATION AND PATHOGENESIS Tese de Candidatura ao grau de Doutor em Ciências Veterinárias submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Orientador – Doutora Gertrude Averil Baker Thompson Categoria – Professor Associado Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Co-orientador – Doutor Arnaldo António de Moura Silvestre Videira Categoria – Professor Catedrático Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Co-orientador – Doutor Volker A. R. Huss Categoria – Professor Associado Afiliação – Molekulare Pflanzenphysiologie, Friedrich-Alexander – Universität Erlangen – Nürnberg, Germany. Os resultados dos trabalhos experimentais incluídos na presente dissertação fazem parte dos seguintes artigos científicos: The results obtained from the experimental work included in this thesis became from several research articles published: Marques S., Silva E., Kraft C., Carvalheira J., Videira A., Huss V., Thompson G. 2008. Bovine mastitis associated with Prototheca blaschkeae. Journal of Clinical Microbiology. 46(6):1941-1945. Thompson, G., E. Silva, S. Marques, A. Müller and J. Carvalheira. 2008. Algaemia in a dairy cow by Prototheca blaschkeae. Medical Mycology. 47(5): 527-531. Marques S., Silva E., Carvalheira J., Thompson G. 2010. Phenotypic characterization of mastitic Prototheca spp. isolates. Research in Veterinary Science. 89(1): 5-9. Marques S., Silva E., Carvalheira J., Thompson G. 2010. In vitro susceptibility of Prototheca to pH and salt concentration. Mycopathologia. 169(4): 297-302. Marques S., Silva E., Carvalheira J., Thompson G. 2010. Short communication: Temperature sensibility of Prototheca blaschkeae strains isolated from bovine mastitic milk. Journal of Dairy Science. 93 (11): 5110-5113 Marques S., Silva E., Thompson G., Huss V. Preliminary studies on the nuclear internal transcribed spacer and the plastid ribosomal RNA operon for phylogenetic analyses of pathogenic Prototheca strains. Manuscript in preparation Marques S., Silva E., Carvalheira J., Thompson G. In vitro algaecide effect of borate on Prototheca strains isolated from bovine mastitic milk. Manuscript submitted Marques S., Silva E., Osório H., Videira A., Thompson G. Identification of immunogenic proteins associated with Prototheca. Manuscript submitted O trabalho apresentado nesta tese foi realizado no Laboratório de Doenças Infecciosas do Departamento de Clínicas Veterinárias do Mestrado Integrado em Medicina Veterinária do Instituto de Ciências Biomédicas Abel Salazar (ICBAS) da Universidade do Porto, Vila do Conde, Portugal; no Laboratório Mitocôndria da divisão da Biologia Estrutural e Molecular do Instituto de Biologia Molecular e Celular (IBMC) da Universidade do Porto, Porto, Portugal; e na Unidade de Proteómica do Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Porto, Portugal. O programa de Doutoramento foi realizado com o apoio de uma Bolsa de Doutoramento Mista (SFRH / BD / 28892 / 2006) financiada pela Fundação para a Ciência e Tecnologia (FCT), a qual permitiu a realização de parte do trabalho de tese na Universidade Friedrich-Alexander Erlangen-Nuremberga, Alemanha, no grupo de Fisiologia Molecular de Plantas. The research presented in this thesis was conducted in the Laboratory of Infectious Diseases of the Department of Veterinary Clinics of the Instituto de Ciências Biomédicas Abel Salazar (ICBAS) of the University of Porto, Vila do Conde, Portugal; at the Laboratory of Mitochondria of the Structural and Molecular Biology research group of Institute for Molecular and Cell Biology (IBMC) of the University of Porto, Porto, Portugal; and at the Proteomics Unit of the Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal. The PhD program was supported by a scholarship (SFRH / BD / 28892 / 2006) awarded by Fundação para a Ciência e Tecnologia (FCT) that financed the development of part of the thesis research at the Friedrich-Alexander University of Erlangen-Nuremberg, Germany in the Molecular Plant Physiology Department. Em memória de Fernanda Barros pelo seu amor e carinho sempre presentes AGRADECIMENTOS / ACKNOWLEDGEMENTS Nesta fase de finalização do meu Doutoramento gostaria de agradecer a todas as pessoas sem as quais a concretização deste projecto não teria sido possível, não só pelo seu contributo directo neste trabalho como também por todo o apoio e amizade que sempre me transmitiram. A todos eles o meu sincero muito obrigado. Mas de uma forma especial quero dirigir especial agradecimento: À Professora Doutora Gertrude Thompson pela sua orientação, pela transmissão dos seus conhecimentos, pelo seu incondicional apoio e amizade, disponibilidade e contribuição científica em todos os trabalhos. Gostaria de realçar o seu incentivo permanente durante todo o percurso deste projecto e ter acreditado sempre em mim e no projecto em todos os momentos, especialmente nos mais difíceis. Estou-lhe para sempre agradecida. Ao Professor Doutor Arnaldo Videira pela sua prontidão e disponibilidade em ser co- orientador desta dissertação e pela sua infinita paciência e disponibilidade. Também gostaria de agradecer todo o apoio prestado, a amizade, as agradáveis e produtivas discussões científicas que auxiliaram bastante a progressão do projecto. Thank you, Volker Huss for having me in your laboratory. Thank you very much for all your intellectual input, for your time and availability, for everything that you taught me, for all your support and guidance. It was an honour to be part of your lab and to learn from you and everyone in the MPP. Thank you very much for your friendship and help with everything. I will always be grateful. I would also like to thank you and your family for receiving me so well and for making it easy to be away from my home. Ao Professor Júlio Carvalheira, que apesar de não ser meu co-orientador, auxiliou positivamente a discussão cientifica de vários trabalhos deste projecto, e reviu atentamente vários manuscritos submetidos. O meu sincero agradecimento pela sua paciência, disponibilidade, disciplina e amizade constantes durante todos estes anos. À Eliane pela sua sincera e enorme amizade, pela sua força e incondicional apoio. Obrigada por teres sempre acreditado em mim e no trabalho! Aos meus restantes colegas de Doutoramento pelo apoio e discussões cientificas que enriqueceram-me durante todos estes anos. À Silvia por toda a sua sincera amizade e força transmitidas ao longo destes anos que tornaram sempre o maus momentos muito mais leves! À Margarida Duarte do IBMC por toda a sua simpatia, ajuda e fornecimento de meios para a realização de uma parte deste projecto. Ao Hugo Osório e Dr. Celso Reis pela ajuda e prontidão. Muito obrigada pelas discussões científicas, pelas “aulas” sobre espectrometria de massa e ajuda na interpretação dos resultados. Thanks to Christine Kraft for all your help and support during my stay at MPP in Erlangen. Thank you very much for everything you taught me and for the patience you had. Also, your friendship was extremely important for my survival in Erlangen, thank you very much for everything! I would also like to thank Daggi, Kiene, Patrick and Andreas for all your friendship and support during my stay in Erlangen. All of you will always be in my heart. À Isabel Santos, Teresa Pena e Joana Correia pelo fornecimento de parte das amostras utilizadas neste projecto, pela amizade e apoio durante o decorrer deste tempo. Ao Luís Pinho e Pedro Meireles dos Serviços Veterinários Associados (SVA) pelo fornecimento de parte das amostras utilizadas neste projecto. À Teresa do Bar pelo magnífico café sempre pronto e pela sua amizade e apoio constantes. À D. Goreti pela ajuda na parte de secretariado e logística e pela sua enorme amizade. Ao senhor Bernardino por toda a ajuda e disponibilidade constante, pelo apoio e amizade sempre presentes. Às instituições e aos grupos científicos que me acolheram, Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Unidade Multidisciplinar de Investigação Biomédica (UMIB), Associação para Promoção e Gestão de Campos Agrários de Vairão e Instituto de Ciências Agrárias de Vairão da Universidade do Porto (ACAV-ICAV), Centro de Investigação em Biodiversidade e Recursos Genéticos (CIBIO), Instituto de Biologia Molecular e Celular da Universidade do Porto (IBMC), Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP) e Molecular Plant Physiology Erlangen University (MPP), o meu sincero agradecimento pelo fornecimento de todos os meios para poder realizar este projecto. À Fundação para a Ciência e Tecnologia (FCT), pelo apoio financeiro prestado para o trabalho realizado na Alemanha e para as deslocações a reuniões internacionais, permitindo partilhar com a comunidade científica internacional o meu trabalho. Um especial e grandioso agradecimento vai para os meus pais, irmão, Teófilo e avó! Todas as palavras para vos expressar a minha profunda gratidão serão sempre insuficientes… Muito obrigada aos meus Pais pelo apoio incondicional, força, paciência, amizade, carinho, amor e por me terem ensinado que tudo é possível com muito trabalho, disciplina e dedicação e por me terem sempre dado tudo para poder chegar onde cheguei. Muito obrigada, sem vocês,
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