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Felipe Jun Fuzita Molecular Physiology of Digestion In FELIPE JUN FUZITA MOLECULAR PHYSIOLOGY OF DIGESTION IN ARACHNIDA: FUNCTIONAL AND COMPARATIVE-EVOLUTIONARY APPROACHES Thesis presented to the Programa de Pós-Graduação Interunidades em Biotecnologia USP/Instituto Butantan/IPT, to obtain the Title of Doctor in Biotechnology. São Paulo 2014 FELIPE JUN FUZITA MOLECULAR PHYSIOLOGY OF DIGESTION IN ARACHNIDA: FUNCTIONAL AND COMPARATIVE-EVOLUTIONARY APPROACHES Thesis presented to the Programa de Pós-Graduação Interunidades em Biotecnologia USP/Instituto Butantan/IPT, to obtain the Title of Doctor in Biotechnology. Concentration area: Biotechnology Advisor: Dr. Adriana Rios Lopes Rocha Corrected version. The original electronic version is available either in the library of the Institute of Biomedical Sciences and in the Digital Library of Theses and Dissertations of the University of Sao Paulo (BDTD). São Paulo 2014 DADOS DE CATALOGAÇÃO NA PUBLICAÇÃO (CIP) Serviço de Biblioteca e Informação Biomédica do Instituto de Ciências Biomédicas da Universidade de São Paulo © reprodução total Fuzita, Felipe Jun. Molecular physiology of digestion in Arachnida: functional and comparative-evolutionary approaches / Felipe Jun Fuzita. -- São Paulo, 2014. Orientador: Profa. Dra. Adriana Rios Lopes Rocha. Tese (Doutorado) – Universidade de São Paulo. Instituto de Ciências Biomédicas. Programa de Pós-Graduação Interunidades em Biotecnologia USP/IPT/Instituto Butantan. Área de concentração: Biotecnologia. Linha de pesquisa: Bioquímica, biologia molecular, espectrometria de massa. Versão do título para o português: Fisiologia molecular da digestão em Arachnida: abordagens funcional e comparativo-evolutiva. 1. Digestão 2. Aranha 3. Escorpião 4. Enzimologia 5. Proteoma 6. Transcriptoma I. Rocha, Profa. Dra. Adriana Rios Lopes I. Universidade de São Paulo. Instituto de Ciências Biomédicas. Programa de Pós-Graduação Interunidades em Biotecnologia USP/IPT/Instituto Butantan III. Título. ICB/SBIB07/2014 UNIVERSIDADE DE SÃO PAULO Programa de Pós-Graduação Interunidades em Biotecnologia Universidade de São Paulo, Instituto Butantan, Instituto de Pesquisas Tecnológicas _____________________________________________________________________________________________________________ Candidato(a): Felipe Jun Fuzita. Título da Tese: Molecular physiology of digestion in Arachnida: functional and comparative-evolutionary approaches. Orientador(a): Profa. Dra. Adriana Rios Lopes Rocha. A Comissão Julgadora dos trabalhos de Defesa da Tese de Doutorado, em sessão pública realizada a ................./................./................., considerou ( ) Aprovado(a) ( ) Reprovado(a) Examinador(a): Assinatura: .............................................................................................. Nome: ...................................................................................................... Instituição: ............................................................................................... Examinador(a): Assinatura: .............................................................................................. Nome: ...................................................................................................... Instituição: ............................................................................................... Examinador(a): Assinatura: .............................................................................................. Nome: ...................................................................................................... Instituição: ............................................................................................... Examinador(a): Assinatura: .............................................................................................. Nome: ...................................................................................................... Instituição: ............................................................................................... Presidente: Assinatura: .............................................................................................. Nome: ...................................................................................................... Instituição: ............................................................................................... À memoria do meu pai, Ernesto Junji Fuzita, que desde cedo me ensinou a importância de ser útil ao próximo e à sociedade ACKNOWLEDGMENTS I am grateful to many people who helped me to produce this thesis: My mother Sonia and my sister Yumi who provided me the basis for becoming a scientist in many aspects. Together we could overcome the natural obstacles of life. My girlfriend Anna Lindinha for supporting me in the last years of this doctorate. My advisor, Dr. Adriana Lopes, which is an open-minded person who not only taught me the laboratory work but also heard my opinions about how could we test our hypothesis. Carlos, Nati, Antônio, Ciça, Pedro, Aline, Rodrigo, Carla, Angela and recently Mariana. It is very nice to be part of a united research group that it is worried about helping each other. Also for the nice moments at SBBq. The Analytical Biotechnology Group from the Delft University of Technology at The Netherlands for receiving me as an integrant of their group. Dr. Peter Verhaert for giving to me the opportunity of working there and Dr. Martijn Pinkse for daily assisting me with the sample analysis. Yuanjie Yu for the chats, technical support, meeting company and coffee-breaks. Mervin Pieterse and Johan for the nice chats and technical support. Also Matthijs for the bioinformatics support and good talking. I felt glad for being part of a group with such nice people. All the researches from the Laboratório de Bioquímica e Biofísica from Instituto Butantan, Drs. Marilene Demasi, Daniel Pimenta, Ivo Lebrun, Ana Chudzinski- Tavassi, Ana Olívia de Souza, Isabel Correia, Janice Onuki, Fernanda Faria, Luziane Chaguri and Sonia Aparecida Andrade. In many aspects, including equipment usage and protocol discussion, these researchers contributed a lot for my thesis. Also the employees for technical support and funny moments, Pati, Silvana, Toninha, Bia, Marcia, Valdeli, Rafael, Júlio, João and Heleusa. Drs. Walter Terra and Clélia Ferreira from the Departamento de Bioquímica at the Universidade de São Paulo for opening the doors of their laboratory always that I needed. Their help was fundamental for developing this work. To the students from this group for assisting me with the equipment usage and for the friendship, Dani, Thaís, Marcelo, Vanvan, André, Tici, Nati, Pererê, Val, Rebola and Fabi. Also to the employees Cris, Nilde, Giliard and Luísa for the technical support and good conversation. Dr Pedro Ismael from Instituto Butantan for equipment usage and nice talks about arachnid biology. Dr. Rogério Bertani and Dr. Irene Knysak from Instituto Butantan for providing me with Tityus serrulatus. All my friends from the biology bachelor course of Universidade de São Paulo. At the student´s “living space” I could meet all kind of different people and opinions, in discussions ranging from biology or musical taste to politics and religion. I can say that I learned a lot of cultural things there. CAPES, CNPq (process number 237706/2012-1) and FAPESP for the financial support. “I believe it is worthwhile trying to discover more about the world, even if this only teaches us how little we know.” Karl Popper ABSTRACT Fuzita FJ. Molecular physiology of digestion in Arachnida: functional and comparative-evolutionary approaches. [Ph. D. thesis (Biotechnology)]. São Paulo: Instituto de Ciências Biomédicas, Universidade de São Paulo; 2014. Spiders and scorpions are efficient predator arachnids which can consume preys larger than themselves. This is only feasible due to a particular mechanism which combines extra-oral and intracellular digestion associated to extremely branched midgut glands and efficient digestive enzymes. Chelicerata is a basal arthropod group and scorpions passed to few changes during millions of years. Thus scorpions and other Chelicerata, such as spiders, are interesting animals for comparative-evolutionary studies. Few studies reported the molecular mechanisms of digestion in predator arachnids and until now there wasn´t a single complete sequence of digestive enzymes from this group of animals in the public databases. Hence, this work describes a biochemical, transcriptomic and proteomic characterization of the midgut and midgut glands (MMG) from the spider Nephilengys cruentata and from the scorpion Tityus serrulatus. Animals under two different physiological conditions, fasting and fed, were dissected and their MMG removed, homogenized and used as sample source. The digestive juice (DJ) of spiders was also collected and analyzed. Enzymatic activity assays focused on endopeptidases identification using different substrates and inhibitors and purification of some peptidases involved in protein digestion in scorpions and spiders. A transcriptome followed by a shotgun proteome was performed using the MMG of fasting and fed animals as well as the spider digestive juice proteome. The enzymological data showed, for the first time, that predator arachnids possess cysteine cathepsins (L, B and F) as digestive enzymes acting mainly intracellularly under acidic conditions and can be found as zymogens, which can be activated in vitro after acidification. In the proteomic label-free quantitative analysis cathepsin L is one of the most abundant enzymes in the MMG of both animals, but
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