Chan, Pui Ting. / Thesis submitted in: May 2007. / On t.p. "alpha" appears as the Greek letter. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 110-118). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Abbreviations --- p.xi / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Peroxisome proliferater-activated receptors (PPARs) --- p.1 / Chapter 1.1.1 --- What are PPARs? --- p.1 / Chapter 1.1.2 --- PPAR isoforms --- p.1 / Chapter 1.1.3 --- PPARα ligands --- p.2 / Chapter 1.2 --- Biological role of PPARα --- p.3 / Chapter 1.2.1 --- Lipid metabolism --- p.3 / Chapter 1.2.2 --- Glucose metabolism --- p.5 / Chapter 1.2.3 --- Oxidative stress and carcinogenesis --- p.6 / Chapter 1.3 --- Discovery of PPARα-regulated and starvation-induced gene (PPSIG) --- p.7 / Chapter 1.4 --- Objectives of the present study --- p.9 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.10 / Chapter 2.1 --- Cloning of PPSIG cDNA into a pCMV-Tag epitope tagging mammalian expression vector --- p.10 / Chapter 2.1.1 --- Materials --- p.10 / Chapter 2.1.2 --- Methods --- p.10 / Chapter 2.2 --- Transient transfection of PPSIG cDNA into CHO-K1 and AML-12 cells --- p.16 / Chapter 2.2.1 --- Cell culture and transfection --- p.16 / Chapter 2.2.1.1 --- Materials --- p.16 / Chapter 2.2.1.2 --- Methods --- p.19 / Chapter 2.2.2 --- Western blot analysis --- p.20 / Chapter 2.2.2.1 --- Materials --- p.20 / Chapter 2.2.2.2 --- Methods --- p.20 / Chapter 2.3 --- Stable transfection of PPSIG cDNA into CHO-K1 and AML-12 cells --- p.22 / Chapter 2.3.1 --- Linearization of the pCMVT4B-PPSIG construct --- p.22 / Chapter 2.3.1.1 --- Materials --- p.22 / Chapter 2.3.1.2 --- Methods --- p.22 / Chapter 2.3.2 --- Cell culture and stable transfection --- p.23 / Chapter 2.3.2.1 --- Materials --- p.23 / Chapter 2.3.2.2 --- Methods --- p.23 / Chapter 2.3.3 --- Selection of the G418-resistant clones --- p.26 / Chapter 2.3.3.1 --- Materials --- p.26 / Chapter 2.3.3.2 --- Methods --- p.29 / Chapter 2.3.4 --- Picking and expanding the G418-resistant clones --- p.30 / Chapter 2.3.4.1 --- Materials --- p.30 / Chapter 2.3.4.2 --- Methods --- p.30 / Chapter 2.3.5 --- Screening and confirmation of the stable transfectants --- p.31 / Chapter 2.3.5.1 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.31 / Chapter 2.3.5.1.1 --- Materials --- p.31 / Chapter 2.3.5.1.2 --- Methods --- p.31 / Chapter 2.3.5.2 --- Northern blot analysis --- p.35 / Chapter 2.3.5.2.1 --- Materials --- p.35 / Chapter 2.3.5.2.2 --- Methods --- p.35 / Chapter 2.3.5.3 --- Western blot analysis --- p.37 / Chapter 2.3.5.3.1 --- Materials --- p.37 / Chapter 2.3.5.3.2 --- Methods --- p.37 / Chapter 2.3.5.4 --- Immunoprecipitation --- p.37 / Chapter 2.3.5.4.1 --- Materials --- p.37 / Chapter 2.3.5.4.2 --- Methods --- p.38 / Chapter 2.3.5.5 --- Matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry analysis --- p.39 / Chapter 2.3.5.5.1 --- Materials --- p.39 / Chapter 2.3.5.5.2 --- Methods --- p.39 / Chapter 2.4 --- "Analysis of the all-trans-13,14-dihydroretinol saturase (RetSat) activity by high-performance liquid chromatography (HPLC) analysis" --- p.41 / Chapter 2.4.1 --- Materials --- p.41 / Chapter 2.4.2 --- Methods --- p.42 / Chapter 2.4.2.1 --- Preparation of all-trans-retinol --- p.42 / Chapter 2.4.2.2 --- Treatment of PPSIG-transfected cells with all-trans-retinol --- p.42 / Chapter 2.4.2.3 --- Retinoid analysis --- p.43 / Chapter 2.5 --- Analysis of fatty acid compositions by gas chromatography-mass spectrometry (GC-MS) --- p.43 / Chapter 2.5.1 --- Materials --- p.43 / Chapter 2.5.2 --- Methods --- p.44 / Chapter 2.5.2.1 --- Preparation of fatty acid-BSA complex --- p.44 / Chapter 2.5.2.2 --- Treatment of PPSIG-transfected cells with fatty acid-BSA complex --- p.44 / Chapter 2.5.2.3 --- Extraction of fatty acids --- p.45 / Chapter 2.5.2.4 --- Methylation of the fatty acids --- p.45 / Chapter 2.5.2.5 --- GC-MS analysis --- p.46 / Chapter 2.5.2.6 --- Statistical analysis --- p.47 / Chapter CHAPTER 3 --- RESULTS --- p.48 / Chapter 3.1 --- The PPSIG cDNA was subcloned into a pCMV-Tag epitope tagging mammalian expression vector --- p.48 / Chapter 3.2 --- The pCMVT4B-PPSIG expression construct was transiently transfected into CHO-K1 and AML-12 cells --- p.54 / Chapter 3.3 --- Stable transfection of the pCMVT4B-PPSIG expression construct into CHO-K1 and AML-12 cells --- p.54 / Chapter 3.3.1 --- PPSIG-transfected CHO-K1 and AML-12 cells were obtained after G418 selection --- p.54 / Chapter 3.3.2 --- PPSIG-transfected CHO-K1 and AML-12 cells had high PPSIG mRNA expression --- p.58 / Chapter 3.3.3 --- PPSIG-FLAG fusion protein was over-expressed in the PPSIG- transfected CHO-K1 and AML-12 cells --- p.61 / Chapter 3.3.4 --- The stable transfectants were immunoprecipitated and identified as PPSIG protein by the mass spectrometry analysis --- p.64 / Chapter 3.4 --- PPSIG protein posseses saturase activity towards all-trans-retinol --- p.66 / Chapter 3.5 --- PPSIG protein is not a fatty acid transporter --- p.78 / Chapter CHAPTER 4 --- DISCUSSION --- p.101 / FUTURE STUDIES --- p.107 / REFERENCES --- p.110 / Appendix A: Prediction of the molecular weight of pCMVT4B- PPSIG protein --- p.119 / Appendix B: Theoretical tryptic peptides of PPSIG --- p.120 / Appendix C: Protein-peptide mass reports --- p.122 / Chapter C1. --- Peptide mass summary of trypsin-digested PPSIG immunoprecipitated protein from clone L2H4B18 --- p.122 / Chapter C2. --- Peptide mass summary of trypsin-digested PPSIG immunoprecipitated protein from clone AL2L7 --- p.123 / Appendix D: HPLC spectrum of the RetSat activity towards all- trans retinol --- p.124 / Chapter D1. --- RetSat activity towards all-trans retinol according to the Moise's group study ((Moise et al. 2004) --- p.124

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

Yuen, Yee Lok. / On t.p. "alpha" appears as the Greek letter. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 165-177). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgement --- p.vii / Table of Contents --- p.viii / List of Abbreviations --- p.xiii / List of Figures --- p.xv / List of Tables --- p.xx / Chapter Chapter 1 --- Literature review --- p.1 / Chapter 1.1 --- Peroxisome proliferator-activated receptor (PPAR) --- p.1 / Chapter 1.1.1 --- PPARα isoforms --- p.1 / Chapter 1.2 --- PPARα ligands --- p.2 / Chapter 1.3 --- Biological roles of PPARα --- p.3 / Chapter 1.3.1 --- Lipid metabolism --- p.3 / Chapter 1.3.2 --- Bile acid metabolism --- p.4 / Chapter 1.3.3 --- Biotransformation --- p.6 / Chapter 1.4 --- Roles of PPARα in hepatocarcinogenesis --- p.7 / Chapter 1.4.1 --- Cell proliferation and apoptosis --- p.7 / Chapter 1.4.2 --- Oxidative stress --- p.8 / Chapter 1.5 --- Discovery of novel PPARα target genes --- p.9 / Chapter 1.5.1 --- Identification of a novel PPARα-regulated gene L5#55 by fluorescent differential mRNA display (FDD) analysis --- p.9 / Chapter 1.6 --- Sulfotransferase (SULT) --- p.15 / Chapter 1.7 --- Objective of the present study --- p.16 / Chapter Chapter 2 --- Molecular cloning and characterization of mouse liver sulfotransferase-like (mL-STL) gene --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and methods --- p.17 / Chapter 2.2.1 --- Animals --- p.17 / Chapter 2.2.2 --- Treatments --- p.18 / Chapter 2.2.3 --- Total RNA extraction --- p.18 / Chapter 2.2.3.1 --- Materials --- p.18 / Chapter 2.2.3.2 --- Methods --- p.19 / Chapter 2.2.4 --- Rapid amplification of cDNA ends (RACE) --- p.19 / Chapter 2.2.4.1 --- Materials --- p.19 / Chapter 2.2.4.2 --- Methods --- p.20 / Chapter 2.2.4.2.1 --- Primer design --- p.20 / Chapter 2.2.4.2.2 --- Rapid amplification of 5'- and 3'-cDNA ends --- p.20 / Chapter 2.2.5 --- Cloning of the 5'- and 3' RACE products --- p.25 / Chapter 2.2.5.1 --- Materials --- p.25 / Chapter 2.2.5.2 --- Methods --- p.25 / Chapter 2.2.6 --- Northern blot analysis --- p.28 / Chapter 2.2.6.1 --- Materials --- p.28 / Chapter 2.2.6.2 --- Methods --- p.28 / Chapter 2.2.6.2.1 --- Formaldehyde-agarose gel electrophoresis and blotting of RNA --- p.31 / Chapter 2.2.6.2.2 --- PCR DIG-labeling --- p.31 / Chapter 2.2.6.2.3 --- Hybridization and signal detection --- p.32 / Chapter 2.2.7 --- Reverse transcription (RT)-PCR --- p.34 / Chapter 2.2.7.1 --- Materials --- p.34 / Chapter 2.2.7.2 --- Methods --- p.34 / Chapter 2.3 --- Results and discussion --- p.37 / Chapter 2.3.1 --- Cloning of the full-length mL-STL cDNA --- p.37 / Chapter 2.3.2 --- In silico analysis of the mL-STL cDNAs --- p.50 / Chapter 2.3.3 --- Genomic organization of the mL-STL gene --- p.61 / Chapter 2.3.4 --- Tissue distribution of mL-STL mRNA transcript --- p.68 / Chapter 2.3.5 --- "PPARα-dependent regulation of mL-STL mRNA expression by fasting and Wy-14,643 treatment" --- p.74 / Chapter Chapter 3 --- Identification of the native mL-STL protein in mouse liver --- p.86 / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.2 --- Materials and methods --- p.87 / Chapter 3.2.1 --- Animal and treatments --- p.87 / Chapter 3.2.2 --- Cloning of the mL-STL cDNA into a modified pRSET (mpRSET) expression vector --- p.88 / Chapter 3.2.2.1 --- Materials --- p.88 / Chapter 3.2.2.2 --- Methods --- p.88 / Chapter 3.2.2.2.1 --- Amplification of mL-STL cDNA fragments --- p.88 / Chapter 3.2.2.2.2 --- Preparation of mpRSET expression vector --- p.92 / Chapter 3.2.2.2.3 --- "Ligation, transformation, and screening of recombinants" --- p.92 / Chapter 3.2.3 --- Over-expression of the mL-STL recombinant proteins in E coli strains --- p.94 / Chapter 3.2.3.1 --- Materials --- p.94 / Chapter 3.2.3.2 --- Methods --- p.94 / Chapter 3.2.4 --- Mass spectrometry analysis of the mL-STL recombinant proteins --- p.95 / Chapter 3.2.4.1 --- Materials --- p.96 / Chapter 3.2.4.2 --- Methods --- p.96 / Chapter 3.2.4.2.1 --- Trypsin digestion and peptide extraction --- p.96 / Chapter 3.2.4.2.2 --- Matrix-assisted laser desorption/ionization time-of- flight (MALDI-TOF) mass spectrometry --- p.97 / Chapter 3.2.5 --- Purification of the mL-STL recombinant proteins --- p.98 / Chapter 3.2.5.1 --- Materials --- p.98 / Chapter 3.2.5.2 --- Methods --- p.98 / Chapter 3.2.5.2.1 --- Semi-purification of the mL-STL recombinant proteins by preparative SDS-PAGE --- p.98 / Chapter 3.2.5.2.2 --- Purification of mL-STL recombinant proteins by column chromatography --- p.99 / Chapter 3.2.6 --- Rabbit immunization using purified mL-STL recombinant proteins --- p.101 / Chapter 3.2.7 --- Subcellular fractionation of mouse liver by ultracentrifugation --- p.101 / Chapter 3.2.7.1 --- Materials --- p.101 / Chapter 3.2.7.2 --- Methods --- p.102 / Chapter 3.2.8 --- Western blot analysis of the native mL-STL protein --- p.104 / Chapter 3.2.8.1 --- Materials --- p.104 / Chapter 3.2.8.2 --- Methods --- p.104 / Chapter 3.2.8.2.1 --- SDS-PAGE and electro-blotting of proteins --- p.104 / Chapter 3.2.8.2.2 --- Immunostaining and signal detection --- p.105 / Chapter 3.3 --- Results and discussion --- p.106 / Chapter 3.3.1 --- Cloning of the mL-STLl and mL-STL2 cDNAs into a modified pRSET (mpRSET) vector --- p.106 / Chapter 3.3.2 --- IPTG induction of the mpRSET-mL-STL protein expression --- p.106 / Chapter 3.3.3 --- Confirmation of mL-STL recombinant proteins by mass spectrometry --- p.118 / Chapter 3.3.4 --- Purification of mL-STL recombinant proteins for rabbit immunization and polyclonal antisera production --- p.130 / Chapter 3.3.5 --- Antigenicity of mL-STL antisera --- p.134 / Chapter 3.3.6 --- Identification of mL-STL native protein and its induction pattern in mouse liver --- p.139 / Chapter 3.3.7 --- "Time-course of fasting and Wy-14,643 treatment on the mL- STLl native protein expression" --- p.147 / Chapter Chapter 4 --- Overall discussion --- p.153 / Future study --- p.163 / References --- p.165 / "Appendix A. Alignment of nucleotide sequences of mouse chromosome 7，Riken2810007J24, mL-STLl, and mL-STL2 cDNA sequences" --- p.178 / Appendix Bl. Theoretical tryptic peptide masses of mpRSET- mL-STLl protein --- p.217 / Appendix B2. Raw data from mass spectrometry analysis of mpRSET-mL-STLl protein --- p.218 / Appendix C1. Residue molecular mass of amino acids --- p.219 / Appendix C2. Di-peptide table --- p.220 / Appendix D1. Theoretical tryptic peptide masses of mpRSET- mL-STL2 protein --- p.221 / Appendix D2. Raw data from mass spectrometry analysis of mpRSET-mL-STL2 protein --- p.222

Xu, Jian. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 238-255). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016).