Lui, Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 163-171). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.v / Acknowledgements --- p.vii / Abbreviations --- p.ix / Table of Content --- p.x / Chapter Chapter 1. --- Introduction / Chapter 1.1 --- Overview and Endocrinology of hormones and hormone receptors --- p.1 / Chapter 1.2 --- Hormone receptors: membrane bounded receptors --- p.3 / Chapter 1.3 --- Hormone receptors: steroid nuclear receptors --- p.4 / Chapter 1.4 --- "Estrogen, estrogen receptor alpha and beta (ERa, ERβ) and prostate gland" --- p.6 / Chapter 1.5 --- Orphan nuclear receptors --- p.10 / Chapter 1.6 --- The first orphan receptors identified-estrogen receptor related receptors --- p.12 / Chapter 1.6.1 --- Estrogen receptor related receptor alpha (ERRα) --- p.13 / Chapter 1.6.2 --- Estrogen receptor related receptor alpha (ERRβ) --- p.17 / Chapter 1.6.3 --- Estrogen receptor related receptor alpha (ERRγ) --- p.19 / Chapter 1.7 --- Aim of study --- p.21 / Figure 1.1 Mechanism of activation of classical nuclear receptor by ligand --- p.23 / Figure 1.2 Distribution of ERa and ERβ in human body --- p.24 / Chapter Chapter 2. --- Methods and Materials / Chapter 2.1 --- Origin and supply of Noble rats --- p.25 / Chapter 2.2 --- Cell culture / Chapter 2.2.1 --- Cell lines and culture media --- p.26 / Chapter 2.2.2 --- Cell culture onto cover slips for immunohistochemistry --- p.27 / Chapter 2.3 --- RNA preparation / Chapter 2.3.1 --- Total RNA extraction --- p.27 / Chapter 2.3.2 --- mRNA extraction by Oligote´xёØ procedure --- p.29 / Chapter 2.3.3 --- mRNA extraction by Fast Track 2.0 procedure --- p.30 / Chapter 2.4 --- Molecular cloning by Rapid Amplification of cDNA Ends (RACE) / Chapter 2.4.1 --- Molecular cloning of rERRα --- p.31 / Chapter 2.4.2 --- Molecular cloning of rERRβ --- p.36 / Chapter 2.4.3 --- Molecular cloning of rERRγ --- p.42 / Chapter 2.5 --- Molecular cloning into pCRII TOPO cloning vector --- p.47 / Chapter 2.6 --- Sequencing analysis of DNA sequence by dRodamine® or BigDye® --- p.47 / Chapter 2.7 --- DNA sequence analysis --- p.49 / Chapter 2.8 --- Reverse transcription and RT-PCR --- p.49 / Chapter 2.9 --- Southern blotting analysis / Chapter 2.9.1 --- Preparation of DNA blot membrane --- p.51 / Chapter 2.9.2 --- Purification of DNA fragment from agarose gel for DIG-DNA labeling --- p.52 / Chapter 2.9.3 --- Preparation of the DIG-labeled DNA probe --- p.53 / Chapter 2.9.4 --- Membrane hybridization and colorimetric detection --- p.53 / Chapter 2.10 --- In-situ hybridization histochemistry / Chapter 2.10.1 --- Linearization of DNA plasmid --- p.55 / Chapter 2.10.2 --- Synthesis of riboprobe --- p.56 / Chapter 2.10.3 --- Hybridization and detection --- p.56 / Chapter 2.11 --- Western blotting analysis / Chapter 2.11.1 --- Protein extraction --- p.59 / Chapter 2.11.2 --- Casting of SDS-PAGE electrophoresis --- p.59 / Chapter 2.11.3 --- Polyacrylamide gel electrophoresis --- p.61 / Chapter 2.11.4 --- Protein blotting analysis --- p.61 / Chapter 2.12.1 --- Immunohistochemistry / Chapter 2.12.1 --- Histological preparation --- p.63 / Chapter 2.12.2 --- Immunohistochemistry --- p.64 / Table 1. List of culture media --- p.66 / Table 2. Primer sequences for RACE-PCR --- p.67 / Table 3. PCR conditions for RT-PCR --- p.68 / Table 4. Primer sequences for RT-PCR --- p.68 / Table 5. Reagent mixtures for linearization of the plasmid DNA --- p.69 / Table 6. Riboprobe synthesis by in-vitro transcription --- p.70 / Chapter Chapter 3. --- Results / Chapter 3.1 --- Cloning of full-length cDNA of rERRs by RACE-PCR --- p.71 / Chapter 3.2 --- Cloning of full-length cDNA of rERRα from rat ovary cDNA library --- p.72 / Chapter 3.3 --- Cloning of full-length cDNA of rERRβ from rat ventral prostate --- p.76 / Chapter 3.4 --- Cloning of full-length cDNA of rERRγ from rat prostate --- p.80 / Chapter 3.5 --- Expression distribution of ERRs detected by RT-PCR --- p.83 / Chapter 3.6 --- mRNA expression of ERRs detected by in-situ hybridization --- p.86 / Chapter 3.7 --- Protein expression of ERRa and ERRγ detected by western blotting --- p.87 / Chapter 3.8 --- Expression of ERRa and ERRγ detected by immunohistochemistry --- p.88 / Figure 3.1 Full-length DNA sequence of rERRα --- p.92 / Figure 3.2 Predicted amino acid sequence of rERRα --- p.93 / "Figure 3.3 DNA sequence alignment of rat, mouse and human ERRα" --- p.94 / "Figure 3.4 Amino acid sequence alignment analysis of rat, mouse and human ERRα" --- p.95 / Figure 3.5 Full-length DNA sequence of rERRβ --- p.96 / Figure 3.6 Predicted amino acid sequence of rERRβ --- p.97 / "Figure 3.7 DNA sequence alignment of rat, mouse and human ERRβ" --- p.98 / "Figure 3.8 Amino acid sequence alignment analysis of rat, mouse and human ERRβ" --- p.99 / Figure 3.9 Full-length DNA sequence of rERRγ --- p.100 / Figure 3.10 Predicted amino acid sequence of rERRγ --- p.101 / "Figure 3.11 DNA sequence alignment of rat, mouse and human ERRγ" --- p.102 / "Figure 3.12 Amino acid sequence alignment analysis of rat, mouse and human ERRγ" --- p.103 / Figure 3.13 Restriction enzyme cutting of full-length plasmids --- p.104 / Figure 3.14 Expression pattern of rERRα in male sex accessory sex glands by RT-PCR --- p.105 / Figure 3.15 Expression pattern of rERRα in urinary system and female sex organs by RT-PCR --- p.106 / Figure 3.16 Tissue expression of rERRα by RT-PCR --- p.107 / Figure 3.17 In-situ hybridization of ERRα in ovary --- p.108 / Figure 3.18 Western blotting of ERRα --- p.109 / Figure 3.19 Immunohistochemistry of ERRα in ovary --- p.110 / Figure 3.20 Expression pattern of rERRβ in male sex accessory sex glands by RT-PCR --- p.111 / Figure 3.21 Expression pattern of rERRβ in urinary system and female sex organs by RT-PCR --- p.112 / Figure 3.22 Tissue expression of rERRβ by RT-PCR --- p.113 / Figure 3.23 In-situ hybridization of ERRβ in rat prostate --- p.114 / Figure 3.24 Negative control of in-situ hybridization of ERRβ in rat prostate --- p.115 / Figure 3.25 Expression pattern of rERRγ in male sex accessory sex glands by RT-PCR --- p.116 / Figure 3.26 Expression pattern of rERRy in urinary system and female sex organs by RT-PCR --- p.117 / Figure 3.27 Tissue expression of rERRγ by RT-PCR --- p.118 / Figure 3.28 Expression pattern of rERRγ in different prostatic cancer cell lines and xenografts by RT-PCR --- p.119 / Figure 3.29 In-situ hybridization of ERRγ in rat prostate --- p.120 / Figure 3.30 Negative control of in-situ hybridization of ERRβ in rat prostate --- p.121 / Figure 3.31 Western blotting of ERRγ --- p.122 / Figure 3.32 Immunohistochemistry of ERRγ in ERRy-transfected MCF-7 cells --- p.123 / Figure 3.33 Immunohistochemistry of ERRγ in ventral prostate of rat --- p.124 / Figure 3.34 Immunohistochemistry of ERRγ in lateral prostate of rat --- p.125 / Figure 3.35 Immunohistochemistry of ERRγ in dorsal prostate of rat --- p.126 / Figure 3.36 Immunohistochemistry of ERRγ in testis of rat --- p.127 / Figure 3.37 Immunohistochemistry of ERRγ in epididymis of rat --- p.128 / Figure 3.38 Immunohistochemistry of ERRγ in brown adipose tissues of rat --- p.129 / Figure 3.39 Immunohistochemistry of ERRγ in brain of rat --- p.130 / Figure 3.40 Immunohistochemistry of ERRγ in brain of rat --- p.131 / Chapter Chapter 4. --- Discussion / Chapter 4.1 --- Sequence analysis of the full-length cDNA sequences of the rat estrogen receptor-related receptors (ERRs) --- p.132 / Chapter 4.2 --- Ligand independence and constitutive self-activation of estrogen receptor-related receptors --- p.133 / Chapter 4.3 --- Board expression pattern of estrogen receptor-related receptors --- p.138 / Chapter 4.3.1 --- Board expression pattern of estrogen receptor-related receptor alpha --- p.138 / Chapter 4.3.2 --- Board expression pattern of estrogen receptor-related receptor beta --- p.140 / Chapter 4.3.3 --- Board expression pattern of estrogen receptor-related receptor gamma --- p.141 / Chapter 4.4 --- Expression of ERRs in the prostate gland --- p.143 / Chapter 4.5 --- Expression of ERRs in the prostatic cell lines and cancer xenografts --- p.147 / Chapter 4.6 --- Expression of ERRs in the ERRγ-transfected MCF-7 cells --- p.149 / Chapter 4.7 --- Expression of ERRs in the testis and epididymis --- p.149 / Chapter 4.8 --- Expression of ERRs in the adipose tissue --- p.150 / Chapter 4.9 --- Expression of ERRs in the ovary --- p.151 / Chapter 4.10 --- Expression of ERRs in the brain --- p.153 / Figure 5.1 Map of full-length clone of rERRα --- p.155 / Figure 5.2 Map of full-length clone of rERRβ --- p.156 / Figure 5.3 Map of full-length clone of rERRα --- p.157 / Figure 5.4 Comparison of the homology of amino acid sequences amongst ERs and ERRs --- p.158 / Figure 5.5 Phylogeny tree of nuclear receptors --- p.159 / Figure 5.6 Relationship of different prostatic cell lines and xenografts --- p.160 / Chapter Chapter 5. --- Summary --- p.161 / References --- p.163-171

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 228-272). Also available on the Internet.

Yuen Yee Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 108-125). / Abstracts in English and Chinese. / TITLE PAGE --- p.1 / ACKNOWLEGDEMENTS --- p.2 / ABSTRACT --- p.3 / 摘要 --- p.5 / table of contents --- p.7 / list of figures and tables --- p.13 / CHAPTER 1 GENERAL INTRODUCTION --- p.16 / Chapter 1.1 --- role of PHYTOESTROGENS in soy and red WINE the PREVENTION OF CARDIOVASCULAR DISEASES (CVD) --- p.17 / Chapter 1.1.1 --- INTRoduction and Classification of Phytoestrogens --- p.17 / Chapter 1.1.2 --- estrogenic1ty of phytoestrogens and theIr abundancesin Plasma --- p.18 / Chapter 1.1.3 --- phytoestrogens as one of the active components In cvd Protection --- p.21 / Chapter 1.1.4 --- effects of Phytoestrogens on LDL Receptor and Apolipoprotein A-1 --- p.22 / Chapter 1.2 --- role of estrogen receptors (ers) in gene regulation --- p.24 / Chapter 1.2.1 --- "structure, Classification and tissue distribution of ERS" --- p.24 / Chapter 1.2.2 --- ligands for ERS --- p.25 / Chapter 1.2.3 --- mechaniSMS OF LIgands-ERS complex in GENE regulation --- p.27 / Chapter 1.2.4 --- ligand-independent ER activation --- p.28 / Chapter 1.3 --- aims and scopes of investigation --- p.29 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.30 / Chapter 2.1 --- chemicals and materials --- p.30 / Chapter 2.1.1 --- Chemicals --- p.30 / Chapter 2.1.2 --- Plasmids --- p.30 / Chapter 2.2 --- mammalian cell culture maintainence --- p.30 / Chapter 2.2.1 --- Maintenance of Cells --- p.31 / Chapter 2.2.2 --- Preparation of Cell Stock --- p.31 / Chapter 2.2.3 --- Cell Recovery from Liquid Nitrogen Stock --- p.31 / Chapter 2.3 --- manipulation of dna --- p.31 / Chapter 2.3.1 --- isolation of HEPG2 cells genonmic DNA --- p.31 / Chapter 2.3.2 --- separation and purification of dna from agarose gel --- p.31 / Chapter 2.3.3 --- Restriction digestionof DNA --- p.32 / Chapter 2.3.4 --- Ligation of DNA Fragments --- p.32 / Chapter 2.3.5 --- Transformation of --- p.32 / Chapter 2.3.6 --- Small Scale Plasmids Purification from DH5a --- p.32 / Chapter 2.4 --- construction of expression and reporter plasmids --- p.33 / Chapter 2.4.1 --- Construction of Estrogen Receptorα (Erα) Expression Vectors --- p.33 / Chapter 2.4.2 --- construction of reporter vectors of LDLR promoter and the Respective Mutants --- p.33 / Chapter 2.4.3 --- Construction of Reporter Vectors of APOAI Promoter and the Respective Mutants --- p.33 / Chapter 2.5 --- determination of promoter transcrtiption activities --- p.34 / Chapter 2.5.1 --- Transient Transfection of Cell with ERa Expression Vector and Promoter Reporter using Lipofectamine PLUS Reagent --- p.34 / Chapter 2.5.2 --- Dual Luciferase Assay --- p.34 / Chapter 2.6 --- semi-quantitative and quantitative rt-pcr assay --- p.34 / Chapter 2.6.1 --- Transient transfection of Cell with ERa Expression Vector Using Lipofectamine PLUS Reagent --- p.34 / Chapter 2.6.2 --- "Isolation of RNA using TRIzol® Reagent (Life Technology, USA)" --- p.35 / Chapter 2.6.3 --- Quantitation of RNA --- p.35 / Chapter 2.6.4 --- First Strand cDNA Synthesis --- p.35 / Chapter 2.6.5 --- Sem卜Quantitative PCR Reactions --- p.35 / Chapter 2.6.6 --- Quantitative PCR Reactions --- p.36 / Chapter 2.7 --- western blotting analysis --- p.36 / Chapter 2.8 --- statistical methods --- p.36 / Chapter CHAPTER 3 --- REGULATION BY PHYSIOLOGICAL LEVEL OF 17B-ESTRADIOL ON APOLIPOPROTEIN A-I AND LOW-DENSITY- LIPOPROTEIN RECEPTOR IN HEPG2 CELLS --- p.37 / Chapter 3.1 --- introduction --- p.37 / Chapter 3.2 --- results --- p.39 / Chapter 3.2.1 --- Determination of transient transfection functionality of estrogen receptors in hepg2 cells --- p.39 / Chapter 3.2.2 --- Effect of 17β-Estradiolon LDLR promoter transcription activity --- p.39 / Chapter 3.2.3 --- Effect of 17β-Estradiol on apoai promoter transcription activity --- p.40 / Chapter 3.2 --- discussion --- p.47 / Chapter CHAPTER 4 --- SOY ISOFLAVONES AND RESVERATROL DISPLAY DIFFERENT MECHANISM IN THE UP-REGULATION OF LOVV-DENSITY-LIPOPROTEIN RECEPTOR IN HEPG2 CELLS --- p.49 / Chapter 4.1 --- introduction --- p.49 / Chapter 4.2 --- results --- p.54 / Chapter 4.2.1 --- Association of ERα and isoflavones or resveratrol on LDLR promoter transcription activity --- p.54 / Chapter 4.2.2 --- Association of ERβ and isoflavones or resveratrol on LDLR promoter transcription activity --- p.54 / Chapter 4.2.3 --- "Role of MAP Kinase, PKA and PKC in isoflavones and resveratrol induced LDLR promoter transcription" --- p.55 / Chapter 4.2.4 --- Identification of promoter regions responsible for induction of LDLR transcription by isoflavones in the presence OF ERα --- p.55 / Chapter 4.2.5 --- Identification of promoter regions responsible for induction of LDLR TRANSCRIPTION BY resveratrol IN THE ABSENCE OF ERα --- p.56 / Chapter 4.3 --- DISCUSSION --- p.75 / Chapter CHAPTER 5 --- SOY ISOFLAVONES AND RESVERATROL UP-REGULATE APOLIPOPROTEIN A-I SIMILAR TO 17B-ESTRADIOL IN HEPG2 CELLS --- p.80 / Chapter 5.1 --- INTRODUCTION --- p.80 / Chapter 5.2 --- RESULTS --- p.84 / Chapter 5.2.1 --- Association of ERα phytoestrogens on APCAI gene expression --- p.84 / Chapter 5.2.2 --- Association of ERβ and isoflavones or resveratrol on APOAI promoter transcription activity --- p.85 / Chapter 5.2.3 --- "Role of MAP Kinase, PKA and PKC in isoflavones and resveratrol in APOAI promoter transcription in the presence of ERα" --- p.85 / Chapter 5.2.4 --- Identification of promoter regions responsible for induction of APOAI transcription by isoflavones and resveratrol in the presence of ERα --- p.85 / Chapter 5.3 --- DISCUSSION --- p.100 / Chapter CHAPTER 6 --- GENERAL DISCUSSION --- p.103 / Chapter CHAPTER 7 --- SUMMARY --- p.106 / BIBLIOGRAPHY --- p.108 / APPENDIX 1 ABBREVIATIONS --- p.126 / APPENDIX 2 MATERIALS AND METHODS --- p.129 / APPENDIX 3 PRIMER LISTS --- p.145 / APPENDIX 4 REAGENTS AND BUFFERS --- p.147