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Study of the Secretome of Leishmania Involved in the Infection Nuno Pedro Moreira Santarém Study of the secretome of Leishmania involved in the infection Tese do 3º Ciclo de Estudos Conducente ao Grau de Doutoramento em Ciências Farmacêuticas ­ Especialidade de Bioquimica (Dissertation thesis for the degree of Doctor of Philosophy in Biochemistry by the Faculty of Pharmacy in Porto University and Molecular and Cellular Biology by the Faculty of Medicine in University Laval) With the supervision of: Professor Doutora Anabela Cordeiro da Silva (Associate professor with Aggregation in the Faculdade de Farmácia da Universidade do Porto) Professor Marc Ouellette (Scientific Director Institute of Infection and Immunity CIHR. Burroughs Wellcome Fund Scholar in Molecular parasitology Canada Research Chair in Antimicrobial resistance Centre de Recherche en Infectiologie CHUQ et Université Laval) December, 2011 Declaração de reprodução É AUTORIZADA A REPRODUÇÃO INTEGRAL DESTA TESE APENAS PARA EFEITOS DE. INVESTIGAÇÃO, MEDIANTE DECLARAÇÃO ESCRITA DO INTERESSADO QUE A TAL SE COMPROMETE. ii Faculty of Farmacy of Porto University & Faculty Medicine University Laval Study of the secretome of Leishmania involved in the infection Nuno Pedro Moreira Santarém iii The candidate performed the experimental work with a doctoral fellowship (SFRH / BD / 37352 / 2007) supported by the “Fundação para a Ciência e a Tecnologia” (FCT; Portugal) and CIHR operating grants from the Centre de Recherche en Infectiologie du Centre de Recherche du CHUL (Quebec, Canada). FCT also also participated with grants to attend international meetings and for the graphical execution of this thesis. The Faculty of Pharmacy of the University of Porto (FFUP; Portugal), the Institute for Molecular and Cell Biology (IBMC; Portugal), and Centre de Recherche en Infectiologie du Centre de Recherche du CHUL provided the facilities and logistic support. iv AUTHOR’S DECLARATION Under the terms of the Decree­Law nº 216/92, of October 13th, is hereby declared that the following original articles were prepared in the scope of this dissertation. PUBLICATIONS Articles in international peer­reviewed journals In the scope of this dissertation I. Impact of continuous axenic cultivation in Leishmania infantum virulence (Acepted for publication in Plos Neglected Tropical Diseases) II. The development of a semi­defined media for growth of Leishmania infantum (To be Submited) III. Exoproteome dynamics in L. infantum (To be submitted to Journal of Proteomic Research) IV. Activation bone marrow derived dendritic cells by the exoproteome from Leishmania infantum (To be Submited) Participation in other publications in related fields I. Neves, B.M., Silvestre, R., Resende, M., Ouaissi, A., Cunha, J., Tavares, J., Loureiro, I., Santarem, N., Silva, A.M., Lopes, M.C., Cruz, M.T., Cordeiro da Silva, A., 2010, Activation of phosphatidylinositol 3­kinase/Akt and impairment of nuclear factor­ kappaB: molecular mechanisms behind the arrested maturation/activation state of Leishmania infantum­infected dendritic cells. Am J Pathol 177, 2898­2911. II. Santarem, N.*, Silvestre, R.*, Cardoso, L., Schallig, H., Reed, S.G., Cordeiro­da­ Silva, A., 2010, Application of an improved enzyme­linked immunosorbent assay method for serological diagnosis of canine leishmaniasis. J Clin Microbiol 48, 1866­1874. * The authors have equally contributed to the work v III. Silvestre, R.*, Santarem, N.*, Teixeira, L., Cunha, J., Schallig, H., Cordeiro­da­ Silva, A., 2009, Evaluation of Leishmania species reactivity in human serologic diagnosis of leishmaniasis. Am J Trop Med Hyg 81, 202­208. * The authors have equally contributed to the work IV. Carvalho, S., Cruz, T., Santarem, N., Castro, H., Costa, V., Tomas, A.M., 2008, Heme as a source of iron to Leishmania infantum amastigotes. Acta Trop 109, 131­135. V. Silvestre, R*., Santarem, N*., Cunha, J., Cardoso, L., Nieto, J., Carrillo, E., Moreno, J., Cordeiro­da­Silva, A., 2008, Serological evaluation of experimentally infected dogs by LicTXNPx­ELISA and amastigote­flow cytometry. Vet Parasitol 158, 23­30. * The authors have equally contributed to the work VI. Tavares, J., Ouaissi, A., Santarem, N., Sereno, D., Vergnes, B., Sampaio, P., Cordeiro­da­Silva, A., 2008, The Leishmania infantum cytosolic SIR2­related protein 1 (LiSIR2RP1) is an NAD+ ­dependent deacetylase and ADP­ribosyltransferase. Biochem J 415, 377­386. VII. Borges, O., Cordeiro­da­Silva, A., Tavares, J., Santarem, N., de Sousa, A., Borchard, G., Junginger, H.E., 2008, Immune response by nasal delivery of hepatitis B surface antigen and codelivery of a CpG ODN in alginate coated chitosan nanoparticles. Eur J Pharm Biopharm 69, 405­416. Under the terms of the referred Decree­Law, the author declares that he afforded a major contribution to the conceptual design and technical execution of the work, interpretation of the results and manuscript preparation of the published articles included in this dissertation. vi “Far better is it to dare mighty things, to win glorious triumphs, even though checked by failure...than to rank with those poor spirits who neither enjoy much nor suffer much, because they live in a gray twilight that knows not victory nor defeat.” ­ Theodore Roosevelt vii viii ACKNOWLEDGMENTS It is difficult to find words to translate my gratitude to all that contributed to make this thesis possible. First, I must acknowledge my supervisors, the Professors Anabela Cordeiro­da­Silva and Marc Ouellette. To both I thank for the advice, guidance, and support. It was truly an honor and a privilege to have both of you as my supervisors. It would be ungrateful of me to let pass the opportunity to publically thank to Professora Anabela for always believing and supporting me throughout my academic trajectory. I will never forget. To Professor Marc Ouellette I must confess that when I arrived in your laboratory I was feeling a bit scared because I was afraid that I might be outside of my depth, far from the comfort zone of my laboratory in Portugal. It was truly important for my confidence and self believe the time I spent in “Mou team”. I will also like to thank to Professor Claudio Sunkel, Director of the IBMC, and Michel G. Bergeron MD, the director of the CRI for opportunity of developing my research project at their institutes, in which all the necessary conditions for the success of my work were available. I also extend my gratitude to the directors of the doctoral programs, Professor José Costa Lima in the University of Pharmacy and Jacques Landry in Laval University for the help in solving all the burocratic problems during these four years. To all the co­authors of the work developed during this thesis, thank you for your contribution, it would not have been possible to have this thesis without your help. To the Parasite Disease group I must thank you all for the continuous support and friendship. Ricardo Silvestre (A veteran from many scientific battles, always there for a discussion, a joke, a word of encouragement, never without an advice), Mariana and Diana (What can I say! There would not be a PD as we know it without you both! Mariana is the “P” – Public Relations ­ and Diana is the “D”­ Dénia! In fact not only PD would be different but also the IBMC, at least there would no need of canteen in the IBMC without Diana!!! To you both I thank the friendship and cooperation and the research – MEGASOMAAAAA!!!). To Sofia Lima, Inês, Suzana, Vasco (Vascão!!!!!), Joana Tavares, Susana, Marta and all trainees that have passed in the laboratory during these years (Daniela, Simão, Ana Luisa, “Seu” Jorge… just to name a few, you know who you are!!!), a big thank you for enduring four years of bad jokes and “excellent” music and all of the constant vesicle theories!!! And of course, Dona Casimira from the Biochemistry department in the FFUP!!!! I did not forget!!! ix For my friends from CRI, I have a special word of acknowledgment for receiving me and always being there when I needed. Suzanne Avoine, Gaétan Roy (the best thing I can say from Suzanne and Gaétan is that I will always remember them… and miss them), Gina Racine (my proteomic partner, always available to help with a smile), Marie­Claude Laffitte (So funny… petit enfant!), Wilfred Moreira (Miss mostly the discussions in the Lab meetings) and Adriano Coelho (A true friend, always there, will see you again for sure!! To you a “simple thanks” is not enough, even if all had failed in Quebec I would still consider my self fortunate for having found a true friend like you), Rubens do Monte (Pão Bola!!! Biiixxxxoooo), the South American connection (Andres, Mariana, Naika, Alejandro, Julie and the adopted Alma), Andree and Phill (Thank you for the help with the crazy burocracy), and all others that I will not name for being afraid to single someone out… Many thanks for all small and big gestures gestures… Et au revoir! As there is life beyond the lab, I must also thank my friends that contributed to the maintenance of my sanity in the most difficult circumstances. Pedro (and obviously Zé and Vitor), Vicente, Mário, André, Mosca, Milde, Andreia, Esquerdo, Hugo, Kury, the guys from Clube Carpe Diem… thank you very much!!! Always that I needed some R&R you were there. And at last, but not least, my family! Their confidence and support, especially when on the other side of the ocean, was instrumental for all my efforts. And to close the biggest “thank you” goes to my life partner Ângela Neves. You were the smile on my face when I just felt overwhelmed by the distance, your love and attention made all the difference in these four years… Thank you all… x ABSTRACT Protozoa parasites of the genus Leishmania are the microbial agents responsible for a group of diseases known as leishmaniasis. The infection is associated with the capacity of these parasites to survive in the phagolysosomes of infected macrophages.
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