It is the aim of this work to study the role, if any, of C'4 in the immune response against Staphylococcus aureus infections.

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Yau Wing Yiu, Winifred. / On t.p. "alpha"s appear as the Greek letter. / Thesis submitted in: October 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 91-98). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese version) --- p.iv / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Abbreviations --- p.xii / List of Figures --- p.xiv / List of Tables --- p.xvi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Peroxisome proliferator-activated receptors (PPARs) --- p.1 / Chapter 1.1.1 --- What are PPARs? --- p.1 / Chapter 1.1.2 --- PPAR ligands - peroxisome proliferators --- p.1 / Chapter 1.1.3 --- PPAR isoforms --- p.2 / Chapter 1.2 --- Biological roles of PPARα --- p.3 / Chapter 1.2.1 --- Lipid metabolism --- p.3 / Chapter 1.2.2 --- Glucose metabolism --- p.4 / Chapter 1.2.3 --- Inflammation --- p.5 / Chapter 1.2.4 --- Oxidative stress --- p.5 / Chapter 1.2.5 --- Cell proliferation and apoptosis --- p.6 / Chapter 1.3 --- PPARα in health and diseases --- p.6 / Chapter 1.3.1 --- Wound-healing --- p.6 / Chapter 1.3.2 --- Anti-atherogenesis --- p.7 / Chapter 1.3.3 --- Neuroprotection --- p.7 / Chapter 1.3.4 --- Carcineogenesis --- p.7 / Chapter 1.4 --- PPARα-regulated and starvation inducible gene (PPSIG) --- p.8 / Chapter 1.4.1 --- PPSIG is a PPARα target gene --- p.8 / Chapter 1.4.2 --- Computer-assisted predictions on PPSIG --- p.9 / Chapter 1.4.3 --- Current characterization of PPSIG --- p.10 / Chapter 1.5 --- Objectives of the present study --- p.11 / Chapter Chapter 2 --- Materials and Methods --- p.12 / Chapter 2.1 --- Materials --- p.12 / Chapter 2.2 --- Animals and treatment --- p.13 / Chapter 2.3 --- Cloning of PPSIG into pThioHis and pTYB expression vectors --- p.13 / Chapter 2.3.1 --- PCR amplification of PPSIG cDNA insert --- p.13 / Chapter 2.3.1.1 --- PPSIG cDNA insert for pThioHis vector --- p.13 / Chapter 2.3.1.2 --- PPSIG cDNA insert for pTYB vector --- p.15 / Chapter 2.3.2 --- Restriction enzyme digestion of PPSIG cDNA insert and pThioHis vector --- p.18 / Chapter 2.3.3 --- Restriction enzyme digestion of PPSIG cDNA insert and pTYB vector --- p.20 / Chapter 2.3.4 --- Ligation and transformation --- p.20 / Chapter 2.3.5 --- Screening for recombinants by phenol/chloroform method --- p.21 / Chapter 2.3.6 --- Confirmation of recombinant plasmid by restriction enzyme digestion --- p.22 / Chapter 2.3.6.1 --- Digestion of pThioHis-PPSIG plasmid with Xba I and Sac II --- p.22 / Chapter 2.3.6.2 --- Digestion of pTYB-PPSIG plasmid with EcoR V --- p.22 / Chapter 2.3.7 --- Transformation into expression E. coli strains --- p.23 / Chapter 2.4 --- Over expression of PPSIG proteins in E. coli --- p.23 / Chapter 2.5 --- Semi-purification of PPSIG fusion proteins by preparative SDS-PAGE --- p.24 / Chapter 2.6 --- Rabbit immunization --- p.25 / Chapter 2.7 --- Northern blotting analysis --- p.26 / Chapter 2.7.1 --- Probe preparation --- p.26 / Chapter 2.7.2 --- "Formaldehyde-agarose gel electrophoresis, blotting of RNA and hybridization" --- p.26 / Chapter 2.8 --- Subcellular fractionation --- p.29 / Chapter 2.9 --- Western blotting of liver microsomes --- p.31 / Chapter 2.10 --- Immunoprecipitation --- p.32 / Chapter 2.11 --- Mass spectrometry --- p.33 / Chapter 2.11.1 --- Trypsin digestion and peptide extraction --- p.33 / Chapter 2.11.2 --- Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry --- p.34 / Chapter Chapter 3 --- Results --- p.36 / Chapter 3.1 --- Cloning of PPSIG into pThioHis and pTYB vectors --- p.36 / Chapter 3.1.1 --- Cloning of PPSIG into pThioHis vector --- p.36 / Chapter 3.1.2 --- Cloning of PPSIG into pTYB vector --- p.36 / Chapter 3.2 --- Protein expression of Thio-PPSIG and Intein-PPSIG --- p.41 / Chapter 3.3 --- Identification of recombinant Thio-PPSIG and Intein-PPSIG by mass spectrometry --- p.49 / Chapter 3.4 --- Preparation and characterization of Thio-PPSIG and Intein-PPSIG antisera --- p.61 / Chapter 3.5 --- Identification of native PPSIG and its induction pattern --- p.65 / Chapter 3.5.1 --- PPSIG was highly inducible upon 72-h starvation in a PPARα dependent manner --- p.65 / Chapter 3.5.2 --- "PPSIG showed slight induction upon 2-wk Wy-14,643 treatment" --- p.71 / Chapter 3.6 --- Confirmation of the specificity of PPSIG antiserum --- p.74 / Chapter Chapter 4 --- Discussion --- p.81 / References --- p.91 / Appendix A Deduced amino acid sequences of PPSIG fusion proteins --- p.99 / Chapter A1 --- Deduced amino acid sequence of Thio-PPSIG from pThioHis-PPSIG plasmid --- p.99 / Chapter A2 --- Deduced amino acid sequence of Intein-PPSIG from pTYB-PPSIG plasmid --- p.101 / Appendix B Mass spectra of trypsin digested native PPSIG --- p.104 / Chapter B1 --- Mass spectrum of trypsin digested native PPSIG immunoprecipitated from liver microsomes from PPARα wild-type mice fed with normal diet (starvation experiment) --- p.104 / Chapter B2 --- Mass spectrum of trypsin digested native PPSIG immunoprecipitated from liver microsomes from PPARα wild-type mice starved for 72 hours (starvation experiment) --- p.105 / Chapter B3 --- "Mass spectrum of trypsin digested native PPSIG immunoprecipitated from liver microsomes from PPARα wild-type mice fed with control diet (Wy-14,643 feeding experiment)" --- p.106 / Chapter B4 --- "Mass spectrum of trypsin digested native PPSIG immunoprecipitated from liver microsomes from PPARα wild-type mice fed with 0.1% (w/w) Wy-14,643 for 2 weeks (Wy-14,643 feeding experiment)" --- p.107

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Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2016-04-08T13:26:34Z No. of bitstreams: 0 / Made available in DSpace on 2016-04-08T13:26:34Z (GMT). No. of bitstreams: 0 / Dissertação (Mestrado em Tecnologia Nuclear) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:28:20Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:56:38Z (GMT). No. of bitstreams: 0 / No presente estudo valores de referência para Br (0,0008 - 0,0056 gL-1), Ca (0,089 - 0,369 gL-1), Cl (2,10 - 3,26 gL-1), Fe (0,381 - 0,689 gL-1), I (0,00018 - 0,00266 gL-1), K (1,14 - 2,74 gL-1), Mg (0,030 - 0,074 gL-1), Na (1,36 - 2,80 gL-1), P (<1,99 gL-1), S (0,99 - 2,79 gL-1) e Zn (0,0012 - 0,0048 gL-1) bem como a matriz de correlação em sangue de eqüinos da Raça Crioula foram determinados utilizando metodologia nuclear (técnica de Análise por Ativação com Nêutrons). Estes dados permitiram identificar alterações fisiológicas relacionadas ao gênero e regime de exercício em que se enquadram estes animais (produção de soros hiperimunes no Instituto Butantan, São Paulo, Brasil). Para realização dessas análises foram utilizados 20 cavalos adultos (8 machos e 12 fêmeas) sadios, na faixa etária de 1 a 3 anos e peso médio de 350 kg, mantidos na Fazenda São Joaquim do Instituto Butantan (São Paulo). Outro grupo recém imunizados, composto por 6 cavalos machos (mesmo peso e idade) foram também analisados. Estes dados auxiliaram na interpretação das funções fisiológicas desses elementos no sangue destes animais durante o processo de imunização para produção de soros. / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP