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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326250 |
| Date | January 2008 |
| Contributors | Chan, Pui Ting., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xvii, 124 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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