Ng Lui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 243-257). / Abstracts in English and Chinese. / Abstract --- p.i_iii / Abstract (Chinese version) --- p.iv-v / Acknowledgements --- p.vi / Table of Contents --- p.vii-xvii / List of Abbreviations --- p.xviii-xx / List of Figures --- p.xxi-xxvi / List of Tables --- p.xxvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Peroxisome Proliferators (PPs) --- p.1 / Chapter 1.2 --- Peroxisome proliferator-activated receptors (PPARs) --- p.3 / Chapter 1.2.1 --- What are PPARs? --- p.3 / Chapter 1.2.2 --- PPAR isoforms --- p.3 / Chapter 1.2.2.1 --- PPARp/δ --- p.3 / Chapter 1.2.2.2 --- PPARγ --- p.4 / Chapter 1.2.2.3 --- PPARα --- p.5 / Chapter 1.2.3 --- PPARα target genes --- p.5 / Chapter 1.2.3.1 --- Transcriptional regulation --- p.5 / Chapter 1.2.3.2 --- PPRE --- p.6 / Chapter 1.2.4 --- Physiological roles --- p.9 / Chapter 1.2.4.1 --- Lipid metabolism --- p.9 / Chapter 1.2.4.1.1 --- Cellular fatty acid uptake and fatty acid activation --- p.9 / Chapter 1.2.4.1.2 --- Intracellular fatty acid transport --- p.11 / Chapter 1.2.4.1.3 --- Mitochondrial fatty acid uptake --- p.12 / Chapter 1.2.4.1.4 --- Mitochondrial fatty-acid P-oxidation / Chapter 1.2.4.1.5 --- Peroxisomal fatty acid uptake --- p.13 / Chapter 1.2.4.1.6 --- Peroxisomal fatty acid oxidation --- p.13 / Chapter 1.2.4.1.7 --- Micorsomal co-hydroxylation of fatty acids --- p.14 / Chapter 1.2.4.1.8 --- Ketogenesis --- p.15 / Chapter 1.2.4.1.9 --- Bile acid metabolism --- p.15 / Chapter 1.2.4.1.10 --- Lipoprotein metabolism --- p.17 / Chapter 1.2.4.1.11 --- Hepatic lipogenesis --- p.18 / Chapter 1.2.4.2 --- Glucose metabolism --- p.19 / Chapter 1.2.4.2.1 --- Glycogenolysis --- p.19 / Chapter 1.2.4.2.2 --- Glycolysis --- p.20 / Chapter 1.2.4.2.3 --- Gluconeogenesis --- p.20 / Chapter 1.2.4.3 --- Urea cycle --- p.21 / Chapter 1.2.4.4 --- Biotransformation --- p.22 / Chapter 1.2.4.5 --- Inflammation --- p.23 / Chapter 1.2.4.6 --- Acute phase response --- p.23 / Chapter 1.2.5 --- Toxicological roles --- p.24 / Chapter 1.2.5.1 --- PPs induce hepatocarcinoma formation through PPARα --- p.24 / Chapter 1.2.5.2 --- Mechanism of PPARa-mediated PP-induced hepatocarcinoma --- p.25 / Chapter 1.2.5.2.1 --- Oxidative stress --- p.25 / Chapter 1.2.5.2.2 --- Hepatocellular proliferation and inhibition of apoptosis --- p.26 / Chapter 1.3 --- Discovery of novel PPARα target genes --- p.27 / Chapter 1.3.1 --- Peroxisome proliferator and starvation-induced gene (PPSIG) --- p.28 / Chapter 1.3.1.1 --- PPSIG is a putative PPARa target gene --- p.28 / Chapter 1.3.1.2 --- Examination of PPSIG FDD fragment cDNA sequence --- p.28 / Chapter 1.4 --- Objectives --- p.32 / Chapter Chapter 2 --- Materials and Methods --- p.38 / Chapter 2.1 --- Cloning of the full-length mouse PPSIG cDNA --- p.38 / Chapter 2.1.1 --- Rapid amplification of cDNA ends (RACE) --- p.38 / Chapter 2.1.1.1 --- Total RNA extraction --- p.38 / Chapter 2.1.1.1.1 --- Materials --- p.38 / Chapter 2.1.1.1.2 --- Methods --- p.38 / Chapter 2.1.1.2 --- Primers design --- p.39 / Chapter 2.1.1.3 --- 5' and 3' cDNA ends amplification --- p.42 / Chapter 2.1.1.3.1 --- Materials --- p.42 / Chapter 2.1.1.3.2 --- Methods --- p.42 / Chapter 2.1.2 --- Subcloning of 5' and 3'RACED products --- p.45 / Chapter 2.1.2.1 --- Ligation and transformation --- p.45 / Chapter 2.1.2.1.1 --- Materials --- p.45 / Chapter 2.1.2.1.2 --- Methods --- p.46 / Chapter 2.1.2.2 --- Screening of the recombinants --- p.48 / Chapter 2.1.2.2.1 --- PhenoI:chloroform test --- p.48 / Chapter 2.1.2.2.1.1 --- Materials --- p.48 / Chapter 2.1.2.2.1.2 --- Methods --- p.48 / Chapter 2.1.2.2.2 --- Restriction enzyme digestion --- p.48 / Chapter 2.1.2.2.2.1 --- Materials --- p.48 / Chapter 2.1.2.2.2.2 --- Methods --- p.49 / Chapter 2.1.3 --- DNA sequencing of the 5'and 3'RACED subclones --- p.49 / Chapter 2.1.4 --- Northern blot analysis using PPSIG 5' and 3' RACED cDNA as probes --- p.52 / Chapter 2.1.4.1 --- RNA sample preparation --- p.52 / Chapter 2.1.4.1.1 --- Materials --- p.52 / Chapter 2.1.4.1.2 --- Methods --- p.52 / Chapter 2.1.4.2 --- Formaldehyde-agarose gel electrophoresis and blotting of RNA --- p.52 / Chapter 2.1.4.2.1 --- Materials --- p.52 / Chapter 2.1.4.2.2 --- Methods --- p.53 / Chapter 2.1.4.3 --- Probe preparation --- p.55 / Chapter 2.1.4.3.1 --- DIG labeling of RNA probe from 5'RACED PPSIG cDN A subclone 5'#32 --- p.55 / Chapter 2.1.4.3.1.1 --- Materials --- p.55 / Chapter 2.1.4.3.1.2 --- Methods --- p.55 / Chapter 2.1.4.3.2 --- PCR DIG labeling of 3´ة RACED PPSIG cDNA subclone 3' #12 --- p.56 / Chapter 2.1.4.3.2.1 --- Materials --- p.56 / Chapter 2.1.4.3.2.2 --- Methods --- p.57 / Chapter 2.1.4.4 --- Hybridization --- p.57 / Chapter 2.1.4.4.1 --- Materials --- p.57 / Chapter 2.1.4.4.2 --- Methods --- p.57 / Chapter 2.1.4.5 --- Post-hybridization washing and colour development --- p.59 / Chapter 2.1.4.5.1 --- Materials --- p.59 / Chapter 2.1.4.5.2 --- Methods --- p.59 / Chapter 2.2 --- Cloning of the PPSIG genomic DNA --- p.61 / Chapter 2.2.1 --- Screening of bacterial artificial chromosome (BAC) clones --- p.61 / Chapter 2.2.1.1 --- Screening of a mouse genomic library --- p.61 / Chapter 2.2.1.2 --- "Purification of BAC DNA by solution I, II,III" --- p.61 / Chapter 2.2.1.2.1 --- Materials --- p.61 / Chapter 2.2.1.2.2 --- Methods --- p.61 / Chapter 2.2.2 --- Confirmation of PPSIG genomic BAC clones --- p.64 / Chapter 2.2.2.1 --- Genomic Southern blot analysis --- p.64 / Chapter 2.2.2.1.1 --- Agarose gel electrophoresis and blotting of BAC DNA --- p.64 / Chapter 2.2.2.1.1.1 --- Materials --- p.64 / Chapter 2.2.2.1.1.2 --- Methods --- p.64 / Chapter 2.2.2.1.2 --- DIG labeling of DNA probe by random priming --- p.65 / Chapter 2.2.2.1.2.1 --- Materials --- p.65 / Chapter 2.2.2.1.2.2 --- Methods --- p.65 / Chapter 2.2.2.1.3 --- Hybridization --- p.66 / Chapter 2.2.2.1.4 --- Post-hybridization washing and colour development --- p.66 / Chapter 2.2.2.2 --- EcoR I digestion --- p.67 / Chapter 2.2.2.2.1 --- Materials --- p.67 / Chapter 2.2.2.2.2 --- Methods --- p.67 / Chapter 2.2.2.3 --- Large scale preparation of BAC DNA --- p.67 / Chapter 2.2.2.3.1 --- Materials --- p.67 / Chapter 2.2.2.3.2 --- Methods --- p.68 / Chapter 2.2.3 --- Determination of PPSIG genomic sequences --- p.68 / Chapter 2.2.3.1 --- Primers design --- p.68 / Chapter 2.2.3.2 --- PCR --- p.73 / Chapter 2.2.3.2.1 --- Materials --- p.73 / Chapter 2.2.3.2.2 --- Methods --- p.73 / Chapter 2.2.3.3 --- Subcloning of the PPSIG genomic fragments --- p.73 / Chapter 2.2.3.3.1 --- Ligation and transformation --- p.73 / Chapter 2.2.3.3.2 --- PCR screening --- p.74 / Chapter 2.2.3.3.2.1 --- Materials --- p.74 / Chapter 2.2.3.3.2.2 --- Methods --- p.74 / Chapter 2.2.3.4 --- DNA sequencing --- p.75 / Chapter 2.3 --- Cloning of PPSIG-promoter reporter constructs --- p.75 / Chapter 2.3.1 --- Amplification of PPSIG 5'-flanking fragment by PCR --- p.75 / Chapter 2.3.1.1 --- Materials --- p.75 / Chapter 2.3.1.2 --- Methods --- p.75 / Chapter 2.3.2 --- Preparation of pGL3-Basic vector DNA --- p.81 / Chapter 2.3.2.1 --- Materials --- p.81 / Chapter 2.3.2.2 --- Methods --- p.81 / Chapter 2.3.3 --- Ligation and transformation --- p.84 / Chapter 2.3.3.1 --- Materials --- p.84 / Chapter 2.3.3.2 --- Methods --- p.84 / Chapter 2.3.4 --- Screening and confirmation of recombinants --- p.85 / Chapter 2.3.4.1 --- Materials --- p.85 / Chapter 2.3.4.2 --- Methods --- p.85 / Chapter 2.4 --- Cloning of PPSIG 5'-deletion promoter constructs --- p.87 / Chapter 2.4.1 --- Deletion of target fragments by restriction enzyme digestion --- p.87 / Chapter 2.4.1.1 --- Materials --- p.87 / Chapter 2.4.1.2 --- Methods --- p.88 / Chapter 2.4.2 --- Ligation and transformation --- p.90 / Chapter 2.4.2.1 --- Materials --- p.90 / Chapter 2.4.2.2 --- Methods --- p.90 / Chapter 2.4.3 --- Screening and confirmation of recombinants --- p.91 / Chapter 2.5 --- Cloning of PPSIG-PPRE reporter constructs --- p.91 / Chapter 2.5.1 --- Amplification of PPSIG-PPRE fragments --- p.91 / Chapter 2.5.1.1 --- Materials --- p.91 / Chapter 2.5.1.2 --- Methods --- p.93 / Chapter 2.5.2 --- Preparation of pGL3-Basic vector DNA --- p.96 / Chapter 2.5.2.1 --- Materials --- p.96 / Chapter 2.5.2.2 --- Methods --- p.96 / Chapter 2.5.3 --- Ligation and transformation --- p.97 / Chapter 2.5.3.1 --- Materials --- p.97 / Chapter 2.5.3.2 --- Methods --- p.97 / Chapter 2.5.4 --- Screening and confirmation of recombinants --- p.97 / Chapter 2.6 --- Cloning of PPSIG-PPRE deletion construct --- p.101 / Chapter 2.6.1 --- Deletion of PPRE fragment by Stu I/Xho I digestion --- p.101 / Chapter 2.6.1.1 --- Materials --- p.101 / Chapter 2.6.1.2 --- Methods --- p.101 / Chapter 2.6.2 --- "Ligation, transformation, screening and confirmation of recombinants" --- p.103 / Chapter 2.7 --- Construction of PPSIG-PPRE-deletion and PPSIG- PPRE-mutation constructs by site-directed mutagenesis --- p.105 / Chapter 2.7.1 --- Primers design --- p.105 / Chapter 2.7.2 --- Amplification of the left and right halves of the PPRE-deletion and PPRE-mutation constructs by PCR --- p.109 / Chapter 2.7.2.1 --- Materials --- p.109 / Chapter 2.7.2.2 --- Methods --- p.109 / Chapter 2.7.3 --- "Ligation, Dpn I digestion and transformation" --- p.110 / Chapter 2.7.3.1 --- Materials --- p.110 / Chapter 2.7.3.2 --- Methods --- p.110 / Chapter 2.7.4 --- Screening and confirmation of recombinants --- p.111 / Chapter 2.7.4.1 --- Materials --- p.111 / Chapter 2.7.4.2 --- Methods --- p.111 / Chapter 2.8 --- Cloning of mouse malonyl-CoA decarboxylase (MCD) and rat acyl-CoA binding protein (ACBP) PPRE reporter constructs --- p.112 / Chapter 2.8.1 --- Preparation of mouse and rat genomic DNA --- p.112 / Chapter 2.8.1.1 --- Materials --- p.112 / Chapter 2.8.1.2 --- Methods --- p.113 / Chapter 2.8.2 --- Amplification of MCD and ACBP PPRE fragments by PCR --- p.113 / Chapter 2.8.2.1 --- Materials --- p.113 / Chapter 2.8.2.2 --- Methods --- p.114 / Chapter 2.8.3 --- Ligation and transformation --- p.117 / Chapter 2.8.4 --- Screening and confirmation of recombinants --- p.117 / Chapter 2.9 --- Cloning of mPPARα and mRXRα expression plasmids --- p.119 / Chapter 2.9.1 --- RT-PCR of mouse PPARα and RXRa cDNAs --- p.119 / Chapter 2.9.1.1 --- Materials --- p.119 / Chapter 2.9.1.2 --- Methods --- p.119 / Chapter 2.9.2 --- Preparation of pSG5 vector DNA --- p.123 / Chapter 2.9.2.1 --- Materials --- p.123 / Chapter 2.9.2.2 --- Methods --- p.123 / Chapter 2.9.3 --- Ligation and transformation --- p.125 / Chapter 2.9.3.1 --- Materials --- p.125 / Chapter 2.9.3.2 --- Methods --- p.125 / Chapter 2.9.4 --- Screening and confirmation of recombinants --- p.125 / Chapter 2.9.4.1 --- Materials --- p.125 / Chapter 2.9.4.2 --- Methods --- p.126 / Chapter 2.10 --- Transient transfection and reporter assays --- p.128 / Chapter 2.10.1 --- Cell culture and transient transfection --- p.128 / Chapter 2.10.1.1 --- Materials --- p.128 / Chapter 2.10.1.2 --- Methods --- p.128 / Chapter 2.10.2 --- Assay for reporter construct luciferase activity --- p.131 / Chapter 2.10.2.1 --- Materials --- p.131 / Chapter 2.10.2.2 --- Methods --- p.131 / Chapter 2.11 --- Electrophoretic mobility-shift assay (EMSA) --- p.133 / Chapter 2.11.1 --- In vitro transcription/translation --- p.133 / Chapter 2.11.1.1 --- Materials --- p.133 / Chapter 2.11.1.2 --- Methods --- p.133 / Chapter 2.11.2 --- Preparation of AML-12 nuclear extract --- p.134 / Chapter 2.11.3 --- Preparation of DIG-labeled PPSIG-PPRE oligonucleotides --- p.136 / Chapter 2.11.3.1 --- Oligonucleotides design --- p.136 / Chapter 2.11.3.2 --- Annealing of single-stranded oligonucleotides to form double- stranded oligonucleotides --- p.136 / Chapter 2.11.3.2.1 --- Materials --- p.136 / Chapter 2.11.3.2.2 --- Methods --- p.138 / Chapter 2.11.3.3 --- 3' end labeling of the double-stranded oligonucleotides --- p.138 / Chapter 2.11.3.3.1 --- Materials --- p.138 / Chapter 2.11.3.3.2 --- Methods --- p.138 / Chapter 2.11.3.4 --- Testing the labeling efficiency of the double-stranded oligonucleoides --- p.139 / Chapter 2.11.3.4.1 --- Materials --- p.139 / Chapter 2.11.3.4.2 --- Methods --- p.139 / Chapter 2.11.4 --- Preparation of unlabeled oligonucleotides as competitors --- p.140 / Chapter 2.11.5 --- Binding reactions --- p.142 / Chapter 2.11.5.1 --- Perform with in vitro transcribed/translated proteins --- p.142 / Chapter 2.11.5.1.1 --- Materials --- p.142 / Chapter 2.11.5.1.2 --- Methods --- p.142 / Chapter 2.11.5.2 --- Perform with AML-12 nuclear extracts --- p.144 / Chapter 2.11.5.2.1 --- Materials --- p.144 / Chapter 2.11.5.2.2 --- Methods --- p.144 / Chapter 2.11.6 --- Detection of shift-up pattern --- p.145 / Chapter 2.11.6.1 --- Materials --- p.145 / Chapter 2.11.6.2 --- Methods --- p.145 / Chapter 2.12 --- Statistical analysis --- p.146 / Chapter Chapter 3 --- Results --- p.147 / Chapter 3.1 --- PPSIG cDNA sequence analysis --- p.147 / Chapter 3.1.1 --- Cloning of PPSIG full-length cDNA sequence --- p.147 / Chapter 3.1.2 --- Northern blot analysis of PPSIG --- p.160 / Chapter 3.1.3 --- "Comparison of PPSIG, Riken cDNA 0610039N19 and all-trans-13'14-dihydroretinol saturase cDNA sequences" --- p.163 / Chapter 3.2 --- PPSIG genomic sequence analysis --- p.166 / Chapter 3.2.1 --- Screening of the PPSIG BAC clone --- p.166 / Chapter 3.2.2 --- Cloning of PPSIG genomic fragments --- p.167 / Chapter 3.2.3 --- Examination of PPSIG genomic organization --- p.170 / Chapter 3.2.3.1 --- "Comparison of PPSIG, Riken cDNA 0610039N19 and all-trans-13'14-dihydroretinol saturase genomic sequence" --- p.177 / Chapter 3.3 --- Characterization of the 5'-flanking region of PPSIG --- p.184 / Chapter 3.4 --- Identification of a functional PPRE in the intron 1 of PPSIG gene --- p.201 / Chapter 3.5 --- Gel shift analysis of PPARa/RXRa heterodimer to PPSIG-PPRE --- p.222 / Chapter Chapter 4 --- Discussion --- p.234 / Chapter Chapter 5 --- Future studies --- p.241 / References --- p.243 / Appendix A Seating plan of transfection experiments (24-wells) / Chapter A1 --- Transfection experiment to study PPSIG-promoter reporter constructs --- p.258 / Chapter A2 --- Transfection experiment to study the PPSIG- promoter deletion constructs --- p.259 / Chapter A3 --- Transfection experiment to study the PPSIG-PPRE reporter constructs --- p.260 / Chapter A4 --- Transfection experiment to study PPSIG-PPRE- deletion and PPSIG-PPRE-mutation constructs --- p.262 / Appendix B Alignment result of RACE clone DNAs --- p.265 / Chapter B1 --- Alignment result of 5´ة#7 --- p.265 / Chapter B2 --- Alignment result of 5'#11 --- p.267 / Chapter B3 --- Alignment result of 5'#12 --- p.269 / Chapter B4 --- Alignment result of 5´ة#16 --- p.271 / Chapter B5 --- Alignment result of 5´ة#20 --- p.274 / Chapter B6 --- Alignment result of 5´ة#31 --- p.276 / Chapter B7 --- Alignment result of 5´ة#32 --- p.278 / Chapter B8 --- Consensus sequence of each 5'RACED clone --- p.280 / Chapter B9 --- Alignment result of all 5'RACE clones consensus sequence --- p.287 / Chapter B10 --- Alignment result of 3´ة#2 --- p.290 / Chapter B11 --- Alignment result of 3´ة#3 --- p.291 / Chapter B12 --- Alignment result of 3´ة#14 --- p.292 / Chapter B13 --- Alignment result of 3´ة#5 --- p.293 / Chapter B14 --- Alignment result of 3´ة#6 --- p.294 / Chapter B15 --- Alignment result of 3´ة#8 --- p.295 / Chapter B16 --- Alignment result of 3´ة#10 --- p.297 / Chapter B17 --- Alignment result of 3´ة#11 --- p.298 / Chapter B18 --- Alignment result of 3´ة#12 --- p.299 / Chapter B19 --- Alignment result of 3´ة#16 --- p.301 / Chapter B20 --- Alignment result of 3´ة#22 --- p.302 / Chapter B21 --- Alignment result of 3´ة#25 --- p.303 / Chapter B22 --- Consensus sequence of each 3'RACED clone --- p.305 / Chapter B23 --- Alignment result of all 3' RACE clones consensus sequence --- p.310 / Appendix C DNA sequencing and alignment result of PPSIG genomic fragments --- p.312 / Chapter C1 --- Exon 1 to exon 2 --- p.312 / Chapter C2 --- Exon 2 to exon 3 --- p.315 / Chapter C3 --- Exon 3 to exon 4 --- p.316 / Chapter C4 --- Exon 4 to exon 5 --- p.318 / Chapter C5 --- Exon 5 to exon 6 --- p.319 / Chapter C6 --- Exon 6 to exon 7 --- p.321 / Chapter C7 --- Exon 7 to exon 8 --- p.322 / Chapter C8 --- Exon 8 to exon 9 --- p.323 / Chapter C9 --- Exon 9 to exon 10 --- p.324 / Chapter C10 --- Exon 10 to exon 11 --- p.325 / Chapter C11 --- Exon 11 to downstream --- p.326 / Chapter C12 --- Consensus sequence of each BAC genomic DNA fragment --- p.328 / Chapter C13 --- The alignment result of all the PPSIG genomic sequence --- p.335 / Appendix D DNA sequencing and alignment result of constructs --- p.347 / Chapter D1 --- "pGL3-PPSIG (-2936/+119), pGL3-PPSIG (-1534/+119), pGL3-PPSIG (-879/+119) and pGL3- PPSIG (-375/+119) reporter constructs DNA sequencing and alignment result" --- p.347 / Chapter D2 --- pSG5-PPARa expression plasmid DNA sequencing and alignment result --- p.351 / Chapter D3 --- pSG5-RXRa expression plasmid DNA sequencing and alignment result --- p.353 / Chapter D4 --- pGL3-MCD reporter constructs DNA sequencing and alignment result --- p.355 / Chapter D5 --- pGL3-PPSIG (-229/+435) reporter construct DNA sequencing and alignment result --- p.356 / Chapter D6 --- pGL3-PPSIG (+94/+435) and pGL3-PPSIG (+94/+190) reporter constructs DNA sequencing and alignment result --- p.357 / Chapter D7 --- pGL3-PPSIG (-229/+3031) reporter construct DNA sequencing and alignment result --- p.358 / Chapter D8 --- pGL3-PPSIG (+94/+3031) reporter construct DNA sequencing and alignment result --- p.360 / Chapter D9 --- pGL3-ACBP reporter construct DNA sequencing and alignment result --- p.362 / Chapter D10 --- PPSIG-PPRE-deletion and PPSIG-PPRE-mutation constructs DNA sequencing and alignment result --- p.363
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325483 |
| Date | January 2006 |
| Contributors | Ng, Lui., Chinese University of Hong Kong Graduate School. Division of Environmental Science. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xxviii, 365 leaves : ill. (some col.) ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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