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TOWARDS a WIDE GENETIC APPROACH for the EUROPEAN WILDCAT (Felis Silvestris Silvestris) CONSERVATION

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TOWARDS a WIDE GENETIC APPROACH for the EUROPEAN WILDCAT (Felis Silvestris Silvestris) CONSERVATION TOWARDS A WIDE GENETIC APPROACH FOR THE EUROPEAN WILDCAT (Felis silvestris silvestris) CONSERVATION: IMPROVING NONINVASIVE MOLECULAR TECHNIQUES, POPULATION ANALYSIS AND ADMIXTURE INFERENCES Rita Isabel Ribeiro Miranda de Oliveira Doutoramento em Biologia Departamento de Biologia 2012 Orientador Paulo Célio Alves, Professor Auxiliar, Faculdade de Ciências da Universidade do Porto Coorientador Ettore Randi, Professor, Università di Bologna ! DECLARAÇÃO Na elaboração desta dissertação, e nos termos do número 2 do Artigo 4º do Regulamento Geral dos Terceiros Ciclos de Estudos da Universidade do Porto e do Artigo 31º do D.L. 74/2006, de 24 de Março, com a nova redação introduzida pelo D.L. 230/2009, de 14 de Setembro, foi efetuado o aproveitamento total de um conjunto coerente de trabalhos de investigação já publicados ou submetidos para publicação em revistas internacionais indexadas e com arbitragem científica, os quais integram alguns dos capítulos da presente tese. Tendo em conta que os referidos trabalhos foram realizados com a colaboração de outros autores, o candidato esclarece que, em todos eles, participou ativamente na sua conceção, na obtenção, análise e discussão de resultados, bem como na elaboração da sua forma publicada. A Faculdade de Ciências da Universidade do Porto foi a instituição de origem do candidato, tendo o trabalho sido realizado sob orientação do Doutor Paulo Célio Alves, Professor Auxiliar da Faculdade de Ciências da Universidade do Porto e investigador do Centro de Investigação em Biodiversidade e Recursos Genéticos (CIBIO); e co-orientação do Doutor Ettore Randi, professor na Universidade de Bologna e director do departamento de genética da instituição de acolhimento do candidato, o Istituto Superiore per la Protezzione e la Ricerca Ambientale (ISPRA) em Itália. O trabalho laboratorial foi realizado no Centro de Testagem Molecular (CTM), unidade integrante do CIBIO, no departamento de genética do ISPRA e ainda na School of Veterinary Medicine (SVM), department of Population Health & Reproduction (PHR), University of California, em colaboração com a Doutora Leslie A. Lyons. Este doutoramento foi apoiado fianceiramente pela Fundação para a Ciência e a Tecnologia (FCT) através da atribuição de uma bolsa individual com a referência SFRH/BD/2361/2005 e do projeto PTDC/CVT/71683/2006. STATEMENT In this dissertation, and in compliance with the number 2 of article 4º of the General Regulation of Third cycles of Studies in the University of Porto and with the article 31º from the DL 74/2006 from 24th March, with the new writing introduced by DL 230/2009, from 14th September, the results of published or submitted works were totally used and included in some of the Chapters of this thesis. In all these works, the candidate participated in obtaining, interpreting, analysing, discussing the results and writing the published forms. The Faculdade de Ciências da Universidade do Porto was the home institution of the candidate, and the work was directed by Dr. Paulo Célio Alves, Auxiliary Professor of the Faculdade de Ciências da Universidade do Porto and researcher at Centro de Investigação em Biodiversidade e Recursos Genéticos (CIBIO); and co-directed by Dr. Ettore Randi, Director of the Genetics Department of the Istituto Superiore per la Protezzione e la Ricerca Ambientale (ISPRA), in Italy. The laboratory work was performed in Centro de Testagem Molecular (CTM), a unit from CIBIO, in the genetics department of ISPRA and at the School of Veterinary Medicine (SVM), department of Population Health & Reproduction (PHR), University of California, in collaboration with Dra. Leslie A. Lyons. This work was financially supported by Fundação para a Ciência e a Tecnologia through a PhD grant with reference SFRH/BD/24361/2005 and the project PTDC/CVT/71683/2006. ! Aos meus pais ! “In our age, when the term 'nature conservation' has become part of everyday life, and humanity is struggling with species' extinctions going faster than we can catalogue them, one can only look back with astonishment at the fact, that it has taken man more than two thousand years to realize that one is part of nature too, and, what is more, depends on it.” Andrea Grill “Anche il più piccolo dei felini, il gatto, è un capolavoro.” Leonardo da Vinci ! AGRADECIMENTOS / RINGRAZIAMENTI / ACKNOWLEDGEMENTS My PhD thesis was, without any doubt, a long journey full of ups and downs. And because the years dealing with cat DNA accompanied the most demanding years of my personal life, I was particularly fortunate not to travel alone. In the following lines I acknowledge all of you that, with great dedication to the cause and/or the causer, supported me in this important step. If, by a lapse of memory (that characterize me so well!), I unfairly omitted your name, I leave you here my sincerest apologies… I would like to start by acknowledging my supervisors Paulo and Ettore. Without you this thesis would have never been possible. Em primeiro lugar agradeço ao Paulo, meu orientador e, acima de tudo, amigo. Obrigada por me teres introduzido no mundo da genética da conservação, quando há muitos anos te disse que o que eu queria era ajudar a conservar as espécies! Obrigada por me teres acompanhado todos estes anos com enorme disponibilidade e generosidade e por teres, desde muito cedo, acreditado nas minhas capacidades e no trabalho que desenvolvemos juntos. Com a tua enorme compreensão, as tuas palavras encorajadoras e o teu forte entusiasmo e dedicação dos últimos tempos és, juntamente com a minha família, a razão pela qual me encontro neste momento a escrever as últimas linhas desta tese. Muito obrigada. To Ettore, my co-supervisor, I acknowledge for being so welcoming and for the great support you gave me when I first arrived in Bologna. Thank you for the opportunity of working in your great facilities at ISPRA, with you I used for the first time the sequencer and the extraction robot! Thank you for the total support and confidence in my work. I would like also to thank Leslie Lyons, for welcoming me at your department in UC Davis and for sharing with me all your knowledge and experience in domestic cat’s research. Thank you for making me staying in your house, for sharing your car, for making me feel at home. Thank you for your supporting words! À Raquel Godinho, uma espécie de orientadora não oficial, agradeço a grande competência científica com que sempre acompanhaste e discutiste o meu trabalho, o inestimável apoio, a disponibilidade e a amizade que caracterizam os anos em que trabalhámos juntas. Obrigada pela grande consideração que sempre demonstraste por mim e pelo meu esforço. I also express my sincere gratitude to all the institutions that supported and made this work possible: FCT, for the attribution of my PhD fellowship (SFRH/BD/24361/2005) and for supporting a research project that partially contributed to the work developed in this thesis (PTDC/CVT/71683/2006); CIBIO and Professor Nuno Ferrand for the opportunity of developing this study and learning so much with the group of outstanding researchers that work in this extraordinary research centre; ISPRA for welcoming me so well every 6 months and UC Davis, particularly the School of Veterinary Medicine at CCAH, for receiving me during 2,5 months of laboratory work. All the molecular work presented in this thesis would not have been accomplished without the contribution of a great number of people or institutions that provided wild and domestic cat samples. I acknowledge Adriano De Faveri, ACENVA - Asociación para la Conservación y Estudio de la Naturaleza de Valladolid, Angelo Giuliani, Ângela Ribeiro, Andrea Sforzi, Aritz Ruiz-González and the University of Bask Country, Bernardino Ragni, BTVS/ICNB – Banco de Tecidos de Vertebrados Selvagens (in particular the effort from Armando Loureiro and Nuno Santos), Biró Zsolt, Claudio Monti, Direcção das clínicas veterinárias de Matosinhos, da Boa Nova e S. Francisco de Assis, Dagny Knauze, Francisco Álvares, Franz Suchentrunk, Francesca Vercillo, Gerardo Dominguez Penafiel, Giancarlo Cagnolati, Hubert Potocnik, Pablo Ferreras, Iris Eckert, João Rodrigues, José Carlos Brito, José Godoy and the EBD - Estación Biológica de Doñana, José Maria Fernández, José Luis Robles, José Vicente López-Bao, Juan Luis Ortega Arranz, Karsten Hupe, Lolita Bizzarri, Luca Lapini, Luigi Ricci, Manuela Malsaña, Martin Liberek, Matteo Visceglia, Mathias Herrmann, Marc Moes, Ellena Ballesteros-Duperón, Marcos Moleón, Emilio Virgós, José Miguel Barea-Azcón, José María Gil-Sánchez, Mauro Fabbri, Pedro Monterroso, Pirlot Romy, Sara Rocha and all anonymous people that contributed with any kind of cat samples to this thesis. I also acknowledge José Godoy and his team, especially Laura Soriano, from EBD for kindly teaching us their non-invasive procedures to deal with carnivore’s scats. I would like to thank all colleagues I met during my stay in the research laboratory of Leslie at UC DAVIS, in particular Monika Lipinski, Jennifer Kurushima, Robert Grahn, Jennifer Grahn and Leslie Bach for your support. It was a pleasure meeting you all. Dedico um agradecimento especial às meninas Portuguesas que conheci em Davis: Catarina, Helena e Maria. Obrigada pelos excelentes momentos passados na vossa companhia e pela hospitalidade com que sempre me receberam. Nel laboratorio di “Ozzangeles” ci sono passate tante persone e tante di queste sono diventate, oltre a splendidi colleghi di lavoro (grazie per l’aiuto in ogni cosa!), amici che tuttora fanno parte della mia vita. Ringrazio con enorme affetto gli amici che ho fatto durante gli anni passati a Bologna, una città meravigliosa che mi è rimasta nel cuore: Andrea Grill, Alice, Aritz, Barbara, Chiara, Claudia, De Faveri, Elena, Federica (grazie mille per il lavoro sul gatto che hai fatto negli ultimi mesi!), Ferri, Francesca, Ilaria, Mario, Marco, Marta, Max, Nadia, Romolo, Severino e Viglino. Romolix e Vitulano grazie per avermi coinvolto e fatto tanto ridere (col vostro migliore inglese!) nelle prime settimane passate ad Ozzano.
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