Samina Arshid Avaliação do efeito do précondicionamento isquêmico no proteoma e fosfoproteoma de neutrófilos de ratos após isquemia/reperfusão SÃO PAULO 2016 Versão corrigida Resolução CoPGr 6018/11, 01/11/2011 Samina Arshid Avaliação do efeito do précondicionamento isquêmico no proteoma e fosfoproteoma de neutrófilos de ratos após isquemia/reperfusão Tese apresentada à Faculdade de Medicina da Universidade de São Paulo para obtenção do título de Doutor em Ciências Programa de: Clínica Cirúrgica Orientador: Prof. Dr. Belchor Fontes Coorientador: Prof. Dr. Wagner Fontes SÃO PAULO 2016 Dados Internacionais de Catalogação na Publicação (CIP) Preparada pela Biblioteca da Faculdade de Medicina da Universidade de São Paulo ãreprodução autorizada pelo autor Ar s h id, Samina Avaliação do efeito do pré-condicionamento isquêmico no proteoma e fosfoproteoma de neutrófilos de ratos após isquemia/reperfusão / Samina Arshid. -- São Paulo, 2016. Tese(doutorado)--Faculdade de Medicina da Universidade de São Paulo. Programa de Clínica Cirúrgica. Orientador: Belchor Fontes. Coorientador: Wagner Fontes. Descritores: 1.Isquemia 2.Traumatismo por reperfusão 3.Precondicionamento isquêmico 4.Ativação de neutrófilo 5.Síndrome de resposta inflamatória sistêmica 6.Proteoma USP/FM/DBD-260/16 DEDICATION DEDICATION To my husband Muhammad Tahir, who has always been there for me in good and bad times, with patience and care, giving all the necessary support, unconditional love, patience and encouragement so that together we reached this point that is very important in our lives. To my children Ayan Tahir and Ahmad Tahir, who are the greatest achievements of my life, for always making me smile and understanding times when I was busy. Without you no achievement is worth. To my father Arshid Mehmood and Father in-law Taj Merin who have always served me as supporting persons with dedication and honesty. To my mother Farzana Iqbal (Late), who dedicated her entire life for me and whose faith in me taught me to have faith in myself. To all of my other family members without whom no event is special and complete in my life. They always guide and encourage me during every moment of my life. ACKNOWLEDGMENT ACKNOWLEDGMENT To Prof. Dr. Belchor Fontes, for his kind supervision, useful suggestions, consistent encouragement, friendly behavior and dynamic supervision enabled me to perform my research work in ensuring my academic, professional and moral well-being. To Prof. Dr. Wagner Fontes, as a co-supervisor, for his co-operation in providing me all possible facilities during my research at Laboratory of Biochemistry and Protein chemistry (LBQP), University of Brasilia (UnB). With all the help offered, availability, incredible scientific spirit, guidance and knowledge offered. Besides a great example of life, professionalism and dedication, he is also a great friend. To Prof. Dr. Edna Montero for her support during research at LIM 62 and for all the opportunities offered for the surgical teaching procedures carried for this work. To Prof. Dr. Peter Roepstorff, for all the opportunities offered at Southern University Denmark for the proteomics analysis and other professional support and help, who for me, will always serve as an example. To Prof. Veit Schwämmle at Southern University Denmark, who guided in the analysis of the data sets provided his bioinformatics experience and expertise. To Dr. Simone Sidoli at Southern University Denmark, who also helped in statisctical analysis of proteomic data. To the Prof. Dr. Vilma for providing access to her lab to use the sonicator facility. To the biologist Dr. Muhammad Tahir, for all the help offered, availability, incredible scientific spirit and all the guidance and knowledge offered. Without whom the success of this project was not possible. To biologist Ana Maria Heimbecker, for providing practical experience as a research assistant, for the patience ad efforts in the surgical experiments from the beginning. To Marco Luna, for his key role in enabling the experiments, impeccable organization and careful observation of animals. To Biologist Mario Itinoshi, for all the help and availability from part of practice until the paperwork involving the project. To pharmaceutical Lucí Takasaka, who kindly took turns in supporting surgical procedures and collection of materials. To Eliane Falconi Monico Gazetto, in the office of graduate for the care, organization and who always answered my numerous questions with patience and kindness. To CAPES, CNPq and FAPDF for financial support to run the project. Esta tese está de acordo com as seguintes normas em vigor no momento da sua impressão: Referências: Adaptado de International Commitee of Medical Journals Editors (Vancouver). Universidade de São Paulo. Faculdade de Medicina. Serviço de Biblioteca e Documentação. Guia de apresentação de dissertações, teses e monografias. Elaborado por Anneliese Carneiro da Cunha, Maria Júlia de A. L. Freddi, Maria F. Crestana, Marinalva de Souza Aragão, Suely Campos Cardoso, Valéria Vilhena. 2ª Ed. São Paulo: Serviço de Biblioteca e Documentação; 2005. Abreviaturas dos títulos dos periódicos de acordo com Listo f Journals Indexed in Index Medicus. LIST OF ABBREVIATIONS Et al And others Fig Figure List of Symbols Da Dalton g Gravity kg Kilogram mg Milligram ml Milliliter mM Millimolar rpm Rotation per minute List of Acronyms 1DE One-dimensional gel electrophoresis 2DE Two-dimensional gel electrophoresis 6PGDH 6-phosphogluconate dehydrogenase AA Arachidonic acid ACN Acetonitrile ADORA2B Receptor, adenosine A2B ALI Acute lung injury ARDS Respiratory distress syndrome, adult ATP Adenosine triphosphate BAL Bronchoalveolar lavage C3a Complement C3 CAMKS Calcium-calmodulin-dependent protein kinases CD11B Antigens, cd11b CID Collision-induced dissociation CINC-1 Cytokine-induced neutrophil chemoattractant 1 CXCR Cxc chemokine receptors DB3 Dilution buffer 3 DNA Deoxyribonucleic acid DOCK2 Dedicator of cytokinesis 2 ECS Endothelial cells EIF4B Eukaryotic translation initiation factor 4b ERK1/2 Extracellular signal-regulated protein kinase-1/2 ESI Electrospray ionization FAK Focal adhesion kinase FGR Gardner-rasheed feline FMLP N-formylmethionyl-leucyl-phenylalanine GCSF Granulocyte-colony stimulating factor GM-CSF Granulocyte-macrophage colony-stimulating factor GPS Group-based prediction system GRK2 G protein-coupled receptor kinase 2 H2O2 Hydrogen peroxide HCD Higher-energy collisional dissociation HILIC Hydrophilic interaction chromatography HO-1 Heme-oxygenase-1 HPLC High performance liquid chromatography HPLC High performance liquid chromatography IGPS Gps algorithm with the interaction filter ILS Interlukeins IMAC Immobilized metal affinity chromatography INPP5C Inositol polyphosphate 5-phosphatase INPP5D Inositol polyphosphate-5-phosphatase D IPC Ischemic preconditioning IR Ischemic reperfusion IRAK-4 Interleukin-1 receptor-associated kinase-4 ITRAQ Isobaric tags for relative and absolute quantitation KEGG Kyoto encyclopedia of genes and genomes LC-MS/MS Liquid chromatography–mass spectrometry LPS Lipopolysaccharide LIM-62 Laboratory of Surgical Physiopathology, department of Surgery of FMUSP LBQP Laboratory of Biochemistry and Protein Chemistry of UnB LTB4 Leukotriene B4 MALDI-MS Matrix-assisted laser desorption/ionization MAPK Mitogen-activated protein kinases MAPKAPK2 MAPK activated protein kinase 2 MCM Mini-chromosome maintenance proteins MOAC Metal oxide affinity chromatography MOF Multiple organ failure MPV Mean platelet volume MS Mass spectrometer MS/MS Tandem mass spectrometry NADPH Nicotinamide adenine dinucleotide phosphate NDR1 Non-race specific disease resistance 1 NF-KB Nuclear factor kappa B OXSR1 Oxidative stress responsive 1 PAF Platelet-activating factor PAK P21 protein activated kinase PCBP1 Poly(rc)-binding protein 1 PDCD4 Programmed cell death protein 4 PDW Platelet distribution width PECAM-1 Platelet endothelial cell adhesion molecule PIP2 Phosphatidylinositol biphosphate PK Protein kinase PKCδ Protein kinase c delta PKN1 Serine/threonine-protein kinase n1 PMNS Polymorphonuclear neutrophils PR3 Proteinase 3 PSGL-1 P-selectin glycoprotein ligand 1 PSSM Position-specific scoring matrices PTB Phosphotyrosine-binding PTM Post translational modification PTP Phosphotyrosine phosphatase PTPN6 Tyrosine-protein phosphatase non-receptor type 6 PTPRC Receptor-type tyrosine-protein phosphatase C RAC2 Ras-related c3 botulinum toxin substrate 2 RHOA Ras homolog gene family, member A RNA Ribonucleic acid ROS Reactive oxygen species RPS6 Ribosomal protein s6 RPS6KA2 Ribosomal protein s6 kinase alpha-2 RSK1 Ribosomal s6 kinase 1 SCOP Structural classification of proteins SDS Sodium dodecyl sulfate SDSPAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SDU University of Southern Denmark SHIP Sh2-containing inositol-5'-phosphatase 1 SHP1 Src homology region 2 domain-containing phosphatase-1 SILAC Stable isotope labeling with amino acids in cell culture SIMAC Sequential elution from IMAC SIRS Systemic inflammatory response syndrome SLK Ste20 like kinase SMAO Acute superior mesenteric artery occlusion SOD Superoxide dismutase SPH Serine phosphorylation SPT16 Component of the fact complex STK38 Serine/threonine-protein kinase 38 STY Serine, threonine or tyrosine residues SYK Spleen tyrosine kinase, TEAB Triethylammonium bicarbonate TEC Tyrosine-protein kinase TIO2 Titanium dioxide TLR Toll-like receptors TNF Tumor necrosis factor TOF Time of flight U2AF2 U2 small nuclear auxiliary factor 2 i LIST OF FIGURES Figure 1 Structure of the iTRAQ