Tang Wai-man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 142-147). / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgement --- p.v / Lists of Abbreviations --- p.vi / Table of Contents --- p.vii / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Isolation of Mas Oncogene --- p.1 / Chapter 1.2 --- Distribution of Mas Oncogene..........…… --- p.3 / Chapter 1.3 --- Developmental Expression of Mas Oncogene --- p.5 / Chapter 1.4 --- Study of Mas-deficient Mice --- p.7 / Chapter 1.5 --- Signal Transduction of Mas Oncogene --- p.8 / Chapter 1.6 --- Other Family Member of Mas Oncogene --- p.9 / Chapter 1.7 --- Mas and Angiotensin Receptor --- p.11 / Chapter 1.8 --- Angiotensin Receptors / Chapter 1.8.1 --- Classification of Angiotensin AT1 Receptor --- p.14 / Chapter 1.8.2 --- Cloning of Angiotensin Receptor --- p.15 / Chapter 1.9 --- Expression of Angiotensin Receptor / Chapter 1.9.1 --- Physiological Factors --- p.17 / Chapter 1.9.2 --- Cis-regulatory Elements / Chapter 1.9.2.1 --- Organization and Regulatory Elements of AT1 Receptor --- p.19 / Chapter 1.9.2.2 --- Expression of AT1a Receptor Promoter was Induced by AP-1 and GATA-4 in Pressure Overload Model --- p.20 / Chapter 1.9.2.3 --- AT1a Receptor Reveals Three Glucocorticoid Responsive Elements --- p.22 / Chapter 1.10 --- Signal Transduction of Angiotensin Receptor --- p.22 / Chapter 1.11 --- Aim of Project --- p.25 / Chapter Chapter 2: --- Mas Oncogene in AR4-2J cells / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods / Chapter 2.2.1 --- Materials / Chapter 2.2.1.1 --- Reagents --- p.27 / Chapter 2.2.1.2 --- Enzymes --- p.27 / Chapter 2.2.1.3 --- DNA Purification Kits --- p.28 / Chapter 2.2.1.4 --- Materials and Antibodies for Western Blot --- p.28 / Chapter 2.2.1.5 --- Others --- p.28 / Chapter 2.2.2 --- Restriction Enzyme Digestion --- p.29 / Chapter 2.2.3 --- Agarose Gel Electrophoresis --- p.29 / Chapter 2.2.4 --- DNA Extraction and Purification --- p.29 / Chapter 2.2.5 --- Plasmid Vector Modification and DNA Ligation --- p.30 / Chapter 2.2.6 --- Bacterial Transformation --- p.31 / Chapter 2.2.7 --- Preparation of Plasmid DNA / Chapter 2.2.7.1 --- Minipreps --- p.32 / Chapter 2.2.7.2 --- Midipreps and Maxipreps --- p.33 / Chapter 2.2.8 --- Genomic DNA Extraction From Tissue and Cell Culture --- p.34 / Chapter 2.2.9 --- RT-PCR Cloning of Mas Oncogene --- p.35 / Chapter 2.2.10 --- Construction of Full Length Mas cDNA into pBluescript® II SK Vector --- p.38 / Chapter 2.2.11 --- Southern Blot Analysis / Chapter 2.2.11.1 --- Preparation of DIG-labeled Mas Probe --- p.38 / Chapter 2.2.11.2 --- Enzyme Restriction of Genomic DNA --- p.39 / Chapter 2.2.11.3 --- Transferring DNA to Nylon Membrane --- p.40 / Chapter 2.2.11.4 --- Prehybridization and Hybridization --- p.40 / Chapter 2.2.11.5 --- Post-hybridization Washes and Blocking --- p.41 / Chapter 2.2.11.6 --- Detection --- p.41 / Chapter 2.2.12 --- DNA Sequencing / Chapter 2.2.12.1 --- Manual Sequencing --- p.42 / Chapter 2.2.12.2 --- Autosequencing --- p.43 / Chapter 2.2.12.3 --- Sequencing Primers --- p.44 / Chapter 2.2.13 --- Cell Culture --- p.45 / Chapter 2.2.14 --- Protein Assay by Modified Lowery --- p.46 / Chapter 2.2.15 --- SDS-PAGE and Western Blot Analysis --- p.47 / Chapter 2.3 --- Results --- p.49 / Chapter 2.4 --- Discussion --- p.60 / Chapter Chapter 3: --- Analysis of Transfected Mas Cell Lines / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Materials and Methods / Chapter 3.2.1 --- Materials --- p.62 / Chapter 3.2.2 --- Cell Culture and Transfection / Chapter 3.2.2.1 --- Cell Culture --- p.62 / Chapter 3.2.2.2 --- Transfection Optimization --- p.62 / Chapter 3.2.2.3 --- Fluorescent SEAP Assay --- p.63 / Chapter 3.2.2.4 --- Transient Transfection --- p.64 / Chapter 3.2.2.5 --- Stable Cell Line Construction --- p.64 / Chapter 3.2.3 --- Protein Assay ESL --- p.65 / Chapter 3.2.4 --- SDS-PAGE and Western Blot Analysis --- p.65 / Chapter 3.2.5 --- Preparation of an AT1a Receptor Internal Standard for Quantitative RT-PCR Analysis / Chapter 3.2.5.1 --- Preparation of an AT1a Receptor cDNA by RT-PC --- p.66 / Chapter 3.2.5.2 --- Cloning of AT1A Receptor cDNA into pBluescript® II SK Vector --- p.67 / Chapter 3.2.5.3 --- Autosequence of pBluescript® II SK Vector/AT1AR --- p.68 / Chapter 3.2.5.4 --- Preparation of 100 bp Deleted AT1a Receptor cDNA by RT- PCR --- p.68 / Chapter 3.2.5.5 --- Cloning of Deleted AT1a R cDNA into pCAPs Vector --- p.71 / Chapter 3.2.6 --- Construction of Full Length Mas cDNA into pOPRSVI/MCS Operator Vector --- p.71 / Chapter 3.2.7 --- Preparation of an Mas Internal Standard for Quantitative RT-PCR Analysis / Chapter 3.2.7.1 --- Preparation of 100 bp Deleted Mas cDNA by RT- PCR --- p.72 / Chapter 3.2.7.2 --- Cloning of 100 bp Deleted Mas cDNA into pCAPs Vector (Mas/pCAPs) --- p.73 / Chapter 3.2.8 --- Quantitative RT-PCR Analysis of AT1A R Expression --- p.74 / Chapter 3.2.9 --- Quantitative RT-PCR Analysis for the Expression of Mas --- p.74 / Chapter 3.3 --- Results --- p.76 / Chapter 3.4 --- Discussions --- p.100 / Chapter Chapter 4: --- Cloning of AT1A Receptor Promoter / Chapter 4.1 --- Introduction --- p.104 / Chapter 4.2 --- Materials and Methods / Chapter 4.2.1 --- Materials --- p.105 / Chapter 4.2.2 --- Genomic DNA Extraction From Rat Pancreas --- p.105 / Chapter 4.2.3 --- "Nest PCR Amplification of 3.2, 2.8 and 1.4kb AT1a Receptor Promoter" --- p.105 / Chapter 4.2.4 --- PCR Amplification of 2.2 kb Aproximal Portion of AT1a Receptor Promoter --- p.107 / Chapter 4.2.5 --- Construction of PCR Fragment of Angiotensin Receptor Promoter into Various Vector --- p.108 / Chapter 4.2.5.1 --- pSEAP2-Basic --- p.108 / Chapter 4.2.5.2 --- pBluescript® II SK Vector --- p.109 / Chapter 4.2.5.3 --- PCR Cloning Kit (pCAPs vector) --- p.109 / Chapter 4.2.5.4 --- PCR-TRAP Cloning System --- p.109 / Chapter 4.2.6 --- Direct PCR Analysis --- p.110 / Chapter 4.2.7 --- Autosequencing of PCR Fragment of AT1A Receptor Promoter --- p.111 / Chapter 4.3 --- Results --- p.114 / Chapter 4.4 --- Discussions --- p.130 / Chapter Chapter 5: --- General Discussion --- p.131 / Chapter Appendix 1 --- Composition of Solutions --- p.133 / Chapter Appendix 2 --- Published Abstract --- p.141 / References --- p.142

Lam Kwok Lim. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 107-126). / Abstract also in Chinese. / Acknowledgments --- p.i / Presentations Derived from the Present Thesis Work --- p.ii / Abstract --- p.iv / Abbreviations --- p.vii / Abbreviation Table for Amino Acids --- p.ix / List of Figures --- p.x / List of Tables --- p.xii / Contents --- p.xiii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Metallothionein (MT) --- p.1 / Chapter 1.1.1 --- Classification of MT --- p.1 / Chapter 1.1.2 --- Structure of MT --- p.2 / Chapter 1.1.3. --- Structure of MT Genes --- p.4 / Chapter 1.1.4 --- Function of MT --- p.5 / Chapter 1.1.5 --- Regulation of MT Expression --- p.7 / Chapter 1.1.6 --- Fish MT --- p.9 / Chapter 1.1.7. --- Aims and Rationale of the Present Study --- p.12 / Chapter 2 --- MT mRNA Induction of Tilapia After Intraperitoneal Injection of Metal --- p.18 / Chapter 2.1 --- Introduction --- p.18 / Chapter 2.1.1. --- Specific Aims of This Chapter --- p.19 / Chapter 2.2 --- Materials and Methods --- p.20 / Chapter 2.2.1 --- Regents --- p.20 / Chapter 2.2.1.1 --- Purification of Total RNA --- p.20 / Chapter 2.2.1.2 --- Denaturing Gel and Vacuum Blotting of RNA (Northern Blotting) --- p.20 / Chapter 2.2.1.3 --- Hybridization --- p.21 / Chapter 2.2.2 --- Methods --- p.21 / Chapter 2.2.2.1 --- Purification of Total RNA --- p.21 / Chapter 2.2.2.2 --- Vacuum Blotting of Total RNA (Northern Blotting) --- p.22 / Chapter 2.2.2.3 --- Radioactive Labeling of Nucleic Acid Probes --- p.22 / Chapter 2.2.2.4 --- Hybridization --- p.22 / Chapter 2.2.2.5 --- Densitometric Analysis --- p.23 / Chapter 2.2.2.6 --- Calculation of MT mRNA Levels and Analysis of Results --- p.23 / Chapter 2.2.3 --- Endogenous MT mRNA Expression of Juvenile Tilapia and Carp --- p.23 / Chapter 2.2.4 --- Induction of MT mRNA Juvenile Tilapia and Carp Injected with Metals --- p.24 / Chapter 2.3 --- Results --- p.25 / Chapter 2.3.1 --- Endogenous Levels of MT mRNA in Tilapias in Normal Conditions --- p.25 / Chapter 2.3.2 --- Induction of MT mRNA Levels in Juvenile Tilapia Injected with Metals --- p.25 / Chapter 2.3.1.1 --- Copper Injection --- p.25 / Chapter 2.3.1.2 --- Zinc Injection --- p.25 / Chapter 2.3.1.3 --- Cadmium Injection --- p.26 / Chapter 2.3.3 --- Induction of MT mRNA Levels in Juvenile Carp with Zinc Injection --- p.26 / Chapter 2.4 --- Discussion --- p.26 / Chapter 2.4.1 --- MT mRNA Expression of Tilapia and Carp Injected with Metals --- p.26 / Chapter 2.5 --- Conclusions --- p.29 / Chapter 3 --- Induction Level of MT mRNA in Tilapia After Aqueous Exposure to Metals --- p.35 / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.1.1 --- Specific aims of this chapter --- p.36 / Chapter 3.2 --- Material s and Methods --- p.36 / Chapter 3.2.1 --- 96hours LC-50 values for zinc and copper --- p.36 / Chapter 3.2.2 --- Induction of MT mRNA in Juvenile Tiapias under Metal Aqueous Exposures --- p.37 / Chapter 3.2.3 --- Calculation of Fold Induction of MT mRNA and Analysis of Results --- p.38 / Chapter 3.2.4 --- Metal Analysis --- p.38 / Chapter 3.3 --- Results --- p.38 / Chapter 3.3.1 --- LC-50 values of metals for Juvenile Tilapia --- p.38 / Chapter 3.3.2 --- Induction of MT mRNA in Juvenile Tilapia under Metal Aqueous Exposures --- p.39 / Chapter 3.3.2.1 --- Aqueous Exposure to Copper --- p.39 / Chapter 3.3.2.2 --- Aqueous Exposure to Zinc --- p.40 / Chapter 3.3.2.3 --- Aqueous Exposure to Cadmium --- p.41 / Chapter 3.3.3 --- Induction of MT mRNA in Juvenile Carp after Aqueous Exposures to Metal --- p.41 / Chapter 3.3.3.1 --- Aqueous Exposure to Cadmium --- p.41 / Chapter 3.3.4 --- Metal Concentrations of Water Samples from the Aquaria in the Metal Exposure Test of Tilapia and Carp --- p.42 / Chapter 3.4 --- Discussion --- p.42 / Chapter 3.4.1 --- LC-50 values of Metals for Tilapia --- p.42 / Chapter 3.4.2 --- MT mRNA Expression of Tilapias under Metal Aqueous Exposure --- p.44 / Chapter 3.4.3 --- Normalization of the Signals of Northern Blot Analysis --- p.47 / Chapter 3.5 --- Conclusions --- p.48 / Chapter 4 --- Field Study --- p.58 / Chapter 4.1 --- Introduction --- p.58 / Chapter 4.1.1 --- Specific Aims of this Chapter --- p.59 / Chapter 4.2 --- Materials and Methods --- p.59 / Chapter 4.2.1 --- Sampling Sites --- p.59 / Chapter 4.2.2 --- Data Analysis --- p.60 / Chapter 4.2.3 --- Harvest of Feral Tilapia --- p.60 / Chapter 4.2.4 --- Determination of Metal Concentration of Metal Concentration in the Tissues of Feral Tilapia --- p.60 / Chapter 4.2.5 --- Endogenous MT mRNA Levels Using Northern Blot Analysis --- p.61 / Chapter 4.2.6 --- Calculation of MT mRNA Levels and Analysis of Results --- p.61 / Chapter 4.3 --- Results --- p.62 / Chapter 4.3.1 --- Metal Concentrations in the Tissues of Feral Tilapia --- p.62 / Chapter 4.3.2 --- Comparison of Metal Concentrations Among Different Tissues of Feral Tilapia --- p.62 / Chapter 4.3.3 --- MT mRNA Levels in the Tissues of Feral Tilapia --- p.63 / Chapter 4.3.4 --- Correlation Between Metal Concentrations and Endogenous MT mRNA Levels in the Tissues of Feral Tilapia --- p.63 / Chapter 4.4 --- Discussion --- p.64 / Chapter 4.4.1 --- Bioaccumulation of Metals --- p.64 / Chapter 4.4.2 --- Endogenous Levels of MT mRNA in the Feral Tilapia --- p.67 / Chapter 4.5 --- Conclusions --- p.68 / Chapter 5 --- Cloning of Tilapia MT Genes --- p.86 / Chapter 5.1 --- Specific Aims of This Chapter 、 --- p.86 / Chapter 5.2 --- Materials and Methods --- p.87 / Chapter 5.2.1 --- Regents --- p.87 / Chapter 5.2.1.1 --- Preparation of Plasmid DNA --- p.87 / Chapter 5.2.1.2 --- Preparation of Genomic DNA --- p.87 / Chapter 5.2.1.3 --- Restriction Enzyme Digestion --- p.88 / Chapter 5.2.1.4 --- Vacuum Blotting of DNA (Southern Blotting) --- p.88 / Chapter 5.2.1.5 --- Polymerase Chain Reaction --- p.89 / Chapter 5.2.1.6 --- Transformation of E.coli Competent Cells --- p.89 / Chapter 5.2.1.7 --- Nucleotide Sequence Determination --- p.89 / Chapter 5.2.1.8 --- List of Primers --- p.90 / Chapter 5.2.1.8.1 --- Primers for Nucleotide Sequence Determination --- p.90 / Chapter 5.2.1.8.2 --- Tilapia MT Specific Primers for PCR --- p.90 / Chapter 5.2.2 --- Methods --- p.91 / Chapter 5.2.2.1 --- Preparation of Plasmid --- p.91 / Chapter 5.2.2.2 --- Preparation of Genomic DNA --- p.91 / Chapter 5.2.2.3 --- Preparation of Enzyme Digestion --- p.92 / Chapter 5.2.2.4 --- Vacuum Blotting of Genomic DNA (Southern Blotting) --- p.92 / Chapter 5.2.2.5 --- Radioactive Labeling of Nucleic Acid Probes --- p.92 / Chapter 5.2.2.6 --- Hybridization --- p.93 / Chapter 5.2.2.7 --- Polymerase Chain Reaction --- p.93 / Chapter 5.2.3 --- Southern Blot Analysis of Tilapia Genomic DNA --- p.93 / Chapter 5.2.4 --- Analysis of the Sequences of Tilapia MT Genes --- p.94 / Chapter 5.2.4.1 --- Amplification of MT Genes Using PCR --- p.94 / Chapter 5.2.4.2 --- Cloning of the MT Genes --- p.94 / Chapter 5.2.4.3 --- Transformation of E.coli Competent Cell --- p.94 / Chapter 5.2.4.4 --- Nucleotide Sequence Determination --- p.95 / Chapter 5.3 --- Results --- p.95 / Chapter 5.3.1 --- Southern Blot Analysis of Tilapia Genomic DNA --- p.95 / Chapter 5.3.2 --- Amplification of MT Gene Fragments Using PCR --- p.95 / Chapter 5.3.3 --- Analysis of the Sequences of Tilapia MT Genes --- p.96 / Chapter 5.4 --- Discussion --- p.96 / Chapter 5.4.1 --- Fish MT Genes --- p.96 / Chapter 5.5 --- Conclusions --- p.98 / Chapter 6 --- General Discussion --- p.104 / References --- p.107

Shiu Ka Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 176-182). / Abstract also in Chinese. / Acknowledgments --- p.i / Presentations Derived from the Present Thesis Work --- p.ii / Chinese Abstract --- p.iii / English Abstract --- p.iv / List of Abbreviations --- p.v / Abbreviation for Amino Acids and Nucleotides --- p.vii / List of Figures --- p.viii / List of Tables --- p.vi / Contents / Chapter Chapter.1 --- Literature Review --- p.1 / Chapter 1.1 --- Transcriptional Regulation of Gene Expression --- p.1 / Chapter 1.2 --- MT: A Brief Review --- p.4 / Chapter 1.3 --- Transcriptional Regulation of MT --- p.15 / Chapter 1.4 --- MT Promoter Organization and Function --- p.18 / Chapter 1.5 --- Fish MT Genes --- p.29 / Chapter 1.6 --- Aim and Rationale of Present Studies --- p.32 / Chapter Chapter 2 --- PCR Cloning of Common Carp MT Gene --- p.34 / Chapter 2.1 --- Introduction --- p.34 / Chapter 2.1.1 --- The Biology of Common Carp --- p.34 / Chapter 2.1.2 --- The Study of Common Carp MT --- p.35 / Chapter 2.2 --- Materials and Methods --- p.39 / Chapter 2.2.1 --- Materials --- p.39 / Chapter 2.2.1.1 --- Polymerase Chain Reaction (PCR) --- p.39 / Chapter 2.2.1.2 --- Agarose Gel Electrophoresis --- p.39 / Chapter 2.2.1.3 --- Gene Clean by Sephaglas´ёØ BandPrep Kit (Pharmacia) --- p.40 / Chapter 2.2.1.4 --- TA Cloning --- p.40 / Chapter 2.2.1.5 --- Transformation of Plasmid Vector into Competent Cell (Heat Shock Method) --- p.41 / Chapter 2.2.1.6 --- Preparation of Plasmid DNA --- p.41 / Chapter 2.2.1.7 --- DNA Sequencing --- p.42 / Chapter 2.2.1.7.1 --- Template Denaturation and Primer Annealing --- p.42 / Chapter 2.2.1.7.2 --- Labeling and Termination Reaction --- p.42 / Chapter 2.2.1.7.3 --- DNA Sequencing Electrophoresis --- p.43 / Chapter 2.2.1.8 --- Total RNA Extraction --- p.43 / Chapter 2.2.1.9 --- PolyA RNA Extraction --- p.44 / Chapter 2.2.1.10 --- Micro Bio-Spin Chromatography --- p.44 / Chapter 2.2.1.11 --- Analysis of the Transcription Start Site --- p.45 / Chapter 2.2.2 --- Methods --- p.46 / Chapter 2.2.2.1 --- Polymerase Chain Reaction (PCR) --- p.46 / Chapter 2.2.2.2 --- Gene Clean by Sephaglas ´ёØ BandPrep Kit (Pharmacia) --- p.46 / Chapter 2.2.2.3 --- TA Cloning --- p.47 / Chapter 2.2.2.4 --- Transformation of Plasmid Vector into Competent Cell (Heat Shock Method) --- p.47 / Chapter 2.2.2.5 --- Transformation of Plasmid Vector into Competent Cell (Heat Shock Method) --- p.48 / Chapter 2.2.2.6 --- Preparation of Plasmid DNA --- p.48 / Chapter 2.2.2.6.1 --- Small Scale Alkali Preparation of Plasmid DNA --- p.48 / Chapter 2.2.2.6.2 --- Large Scale Preparation of Plasmid DNA using Wizard Maxiprep Kit (Promega) --- p.49 / Chapter 2.2.7 --- DNA Sequencing --- p.50 / Chapter 2.2.2.7.1 --- Template Denaturation and Primer Annealing --- p.50 / Chapter 2.2.2.7.2 --- Labeling and Termination Reaction --- p.51 / Chapter 2.2.2.7.3 --- DNA Sequencing Electrophoresis --- p.51 / Chapter 2.2.2.8 --- Total RNA Extraction --- p.52 / Chapter 2.2.2.9 --- PolyA RNA Extraction --- p.53 / Chapter 2.2.2.10 --- Analysis of the Transcription Start Site --- p.55 / Chapter 2.3 --- Results --- p.56 / Chapter 2.3.1 --- PCR Cloning of the MT Gene --- p.56 / Chapter 2.3.2 --- Identification of the Transcriptional Start Site --- p.57 / Chapter 2.4 --- Discussion --- p.60 / Chapter 2.4.1 --- PCR Cloning of the MT Gene --- p.60 / Chapter 2.4.2 --- Comparison of Common Carp MT Promoter with Other --- p.60 / Chapter 2.4.3 --- Identification of the Transcriptional Start Site --- p.62 / Chapter 2.5 --- Conclusion --- p.63 / Chapter Chapter 3. --- Functional Assay of Common Carp MT Promoter --- p.64 / Chapter 3.1 --- Introduction --- p.64 / Chapter 3.1.1 --- Fish MT Promoters --- p.64 / Chapter 3.2 --- Materials and Methods --- p.68 / Chapter 3.2.1 --- Materials --- p.68 / Chapter 3.2.1.2 --- Micro Bio-Spin Chromatography --- p.68 / Chapter 3.2.1.3 --- Construction of Deletion Mutants --- p.68 / Chapter 3.2.1.4 --- Isolation of Hepatocytes --- p.69 / Chapter 3.2.1.5 --- Determination of LC50 Values for Common Carp Hepatocytes --- p.69 / Chapter 3.2.1.6 --- Transfection by LipofectAMINE´ёØ (Gibco) --- p.70 / Chapter 3.2.1.9 --- Determination of the Amount of Protein by BCA Protein Assay --- p.70 / Chapter 3.2.1.10 --- β-galactosidase Analysis --- p.71 / Chapter 3.2.2 --- Methods --- p.72 / Chapter 3.2.2.1 --- Subcloning of 5' Flanking Region of Common Carp MT Gene into Reporter Gene --- p.72 / Chapter 3.2.2.2 --- Micro Bio-Spin Chromatography (Bio-rad) --- p.72 / Chapter 3.2.2.3 --- Creating Deletion Mutants --- p.73 / Chapter 3.2.2.4 --- Isolation of Hepatocytes --- p.73 / Chapter 3.2.2.5 --- Determination ofLC50 Values for Common Carp Hepatocytes --- p.74 / Chapter 3.2.2.6 --- Transfection with LipofectAMINE´ёØ (Gibco BRL) --- p.75 / Chapter 3.2.2.7 --- Optimization of Incubation Time of Cells with LipofectAMINE´ёØ --- p.75 / Chapter 3.2.2.8 --- Optimization of Amount of DNA for Transfection --- p.76 / Chapter 3.2.2.9 --- Determination of Protein Concentration by --- p.76 / Chapter 3 2.2.10 --- β-galactosidase Analysis --- p.77 / Chapter 3.2.2.11 --- Fluorescence Measurement --- p.77 / Chapter 3.2.2.12 --- Dose-Response Curve of Different Metals on Transfected Cells --- p.77 / Chapter 3.2.2.13 --- "Fold-Induction of Different Metals, LPS and H202" --- p.78 / Chapter 3.3. --- Result --- p.79 / Chapter 3.3.1 --- Deletion Mutants --- p.79 / Chapter 3.3.2 --- LC50 of Common Carp Hepatocytes --- p.80 / Chapter 3.3.3 --- Optimization of Transfection --- p.81 / Chapter 3.3.4 --- Dose Response Curve --- p.85 / Chapter 3.3.5 --- Deletion Mutants with Different Treatments --- p.95 / Chapter 3.4 --- Discussion --- p.109 / Chapter 3.4.1 --- LC50 Values of Metal Toxicity in Different in vitro Fish Cells Studies --- p.109 / Chapter 3.4.2 --- Dose Response Curve (Figure 3.9 to 3.16) --- p.110 / Chapter 3.4.3 --- Fold Induction in Deletion Mutants --- p.111 / Chapter 3.5 --- Conclusion --- p.128 / Chapter Chapter 4. --- MRE-Binding Proteins --- p.129 / Chapter 4.1 --- Introduction --- p.129 / Chapter 4.1.1 --- MTF-1 --- p.129 / Chapter 4.1.1.1 --- Structure of MTF-1 --- p.129 / Chapter 4.1.1.2 --- MTF-1 is a Zinc Dependent Factor --- p.130 / Chapter 4.1.1.3 --- Band-shift Assay of MTF-1 --- p.132 / Chapter 4.1.1.4 --- MTF-1 is Essential for Both Basal and Metal-Induced MT Transcription --- p.133 / Chapter 4.1.2 --- MBP-l --- p.134 / Chapter 4.1.3 --- MBF-l l --- p.35 / Chapter 4.1.4 --- Rat Zinc Activated Protein --- p.135 / Chapter 4.1.5 --- MREBF-1 and MREBF-2 --- p.136 / Chapter 4.1.6 --- Human Zinc Regulatory Factor --- p.136 / Chapter 4.1.7 --- MREBP --- p.137 / Chapter 4.1.8 --- Aim of This Chapter --- p.138 / Chapter 4.2 --- Materials and Methods --- p.139 / Chapter 4.2.1 --- Materials --- p.139 / Chapter 4.2.1.1 --- Preparation of Nuclear Extract from Common Carp Liver Tissue --- p.139 / Chapter 4.2.1.2 --- Preparation of the Double-Stranded Oligonucleotides --- p.139 / Chapter 4.2.1.3 --- Binding Reaction of Protein and DNA --- p.141 / Chapter 4.2.1.4 --- Gel-Shift Mobility Electrophoresis --- p.142 / Chapter 4.2.1.5 --- Screening of Expression Library --- p.142 / Chapter 4.2.1.5.1 --- Preparation of Labeled DNA Probe --- p.142 / Chapter 4.2.1.5.2 --- Plating of the Library --- p.142 / Chapter 4.2.1.6. --- Isolation of Positive Clones In Vivo Excision --- p.143 / Chapter 4.2.2 --- Methods --- p.144 / Chapter 4.2.2.1 --- Gel Mobility-Shift Assays --- p.144 / Chapter 4.2.2.1.1 --- Preparation of Nuclear Extract from Common Carp Liver Tissue --- p.145 / Chapter 4.2.2.1.2 --- Preparation of the Double-Stranded Oligonucleotides --- p.145 / Chapter 4.2.2.1.3 --- Binding Reaction of Protein and DNA --- p.146 / Chapter 4.2.2.1.4 --- Gel-Shift Mobility Electrophoresis --- p.146 / Chapter 4.2.2.2 --- Screening of Expression Library --- p.146 / Chapter 4.2.2.2.1 --- Preparation of Labeled DNA Probe --- p.147 / Chapter 4.2.2.2.2 --- Plating of the Library --- p.148 / Chapter 4.2.2.2.3 --- Isolation of Positive Clones --- p.150 / Chapter 4.3 --- Results --- p.150 / Chapter 4.3.1 --- Gel Mobility-Shift Assays --- p.150 / Chapter 4.3.2 --- Expression Library Screening --- p.163 / Chapter 4.4 --- Discussion --- p.166 / Chapter 4.4.1 --- Gel Mobility-Shift Assays --- p.166 / Chapter 4.4.2 --- Expression Library Screening --- p.171 / Chapter 4.5 --- Conclusion --- p.172 / Chapter Chapter 5 --- Conclusion --- p.173 / Chapter 5.1 --- Conclusion --- p.173 / Chapter 5 2 --- Model of MT Gene Transcription --- p.174 / Chapter 5.3 --- Future Direction --- p.175 / references --- p.176

Cheung Chun Pan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 192-227). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abstract (English) --- p.II / Abstract (Chinese) --- p.VI / Contents --- p.VIII / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Nuclear hormone receptor: a general review --- p.1 / Chapter 1.1.1 --- Classification of nuclear hormone receptors --- p.1 / Chapter 1.1.2 --- Mechanism of action --- p.2 / Chapter 1.1.3 --- Domains structure and functions --- p.3 / Chapter 1.1.4 --- Orphan nuclear receptors --- p.4 / Chapter 1.2 --- Prostate gland - a male accessory reproductive organ --- p.6 / Chapter 1.2.1 --- "Anatomy, histology and physiology of the prostate gland" --- p.6 / Chapter 1.2.2 --- Endocrinology of the prostate gland --- p.8 / Chapter 1.2.3 --- Pathogenesis of the prostate gland --- p.8 / Chapter 1.3 --- The role of estrogen receptors in the prostate gland and prostate cancer --- p.9 / Chapter 1.3.1 --- Estrogens in male --- p.10 / Chapter 1.3.2 --- Effects of estrogens in the prostate gland --- p.11 / Chapter 1.3.3 --- Estrogen receptors - two isoforms --- p.13 / Chapter 1.3.4 --- Expression of ERs in the prostate gland --- p.14 / Chapter 1.3.5 --- Estrogen-modulated transgenic mice 226}0ؤ functional studies of ERs --- p.16 / Chapter 1.4 --- Estrogen receptor-related receptors: orphan receptors --- p.18 / Chapter 1.4.1 --- Estrogen receptor-related receptors: Three isoforms --- p.18 / Chapter 1.4.2 --- Expression of ERRs in different tissues --- p.20 / Chapter 1.4.3 --- Promoter binding and genes regulated by of ERRs --- p.22 / Chapter 1.4.4 --- Coregulators of ERRs --- p.24 / Chapter 1.4.5 --- Ligand of ERRs --- p.25 / Chapter 1.4.6 --- Functional roles of ERRs --- p.29 / Chapter 1.4.7 --- Cross talk between ERRs and ERs --- p.31 / Table 11 --- p.33 / Figure 1.1 - 15 --- p.35 / Chapter Chapter 2 --- Aims of the Study --- p.40 / Chapter Chapter 3 --- Methods and Materials / Chapter 3.1 --- "Expression patterns of ERRs and steroid hormone receptors in the human prostate cell lines, tumor xenografts and prostatic tissues" --- p.41 / Chapter 3.1.1 --- Human prostatic tissues --- p.41 / Chapter 3.1.2 --- Cell cultures --- p.41 / Chapter 3.1.3 --- Human prostate cancer xenografts --- p.42 / Chapter 3.1.4 --- Full length clones of ERR isoforms --- p.42 / Chapter 3.1.5 --- Reverse transcription-polymerase chain reactions (RT-PCR) --- p.43 / Chapter 3.1.6 --- Semi-quantitative RT-PCR analysis --- p.45 / Chapter 3.1.7 --- Southern blot analysis --- p.46 / Chapter 3.1.8 --- Generation and characterization of polyclonal antibodies --- p.49 / Chapter 3.1.9 --- Western blot analysis --- p.55 / Chapter 3.1.10 --- Immunohistochemistry --- p.56 / Chapter 3.2 --- Relationship of ERR and ER expressions in the prostatic cells --- p.57 / Chapter 3.2.1 --- "Expression vectors of ERRa, ERRγ and ERa" --- p.57 / Chapter 3.2.2 --- "Transient transfection of ERRa, ERRγ and ERa expression vectors in PC-3 cells" --- p.58 / Chapter 3.2.3 --- Semi-quantitative RT-PCR analysis --- p.59 / Chapter 3.3 --- Intracellular trafficking and transcriptional activity of GFP-tagged ERRs in the prostatic cells --- p.59 / Chapter 3.3.1 --- Construction of GFP-tagged ERR fusion plasmids --- p.59 / Chapter 3.3.2 --- Examination of transcriptional activity of GFP-tagged ERRs by luciferase assay --- p.61 / Chapter 3.3.3 --- Subcellular localization of GFP-tagged ERRs in the living prostatic cells --- p.63 / Chapter 3.3.4 --- Immunofluorescent staining GFP-tagged ERRs --- p.63 / Chapter 3.4 --- The Role of ERRs in the growth of the prostatic cells --- p.64 / Chapter 3.4.1 --- Evaluation the transfection efficiencies of PC-3 and PNT2 cells --- p.64 / Chapter 3.4.2 --- Cells proliferation assays in ERRs transient transfected prostatic cells --- p.66 / Chapter 3.4.3 --- Flow cytometry of ERRs transient transfected PC-3 cells --- p.66 / Chapter 3.4.4 --- RT-PCR of cell cycle-related genes in ERRs transient transfected PC-3 cells --- p.67 / Chapter 3.4.5 --- Generation of PNT2 and DU145 cells stably transfected with ERRy --- p.68 / Chapter 3.4.6 --- Cell proliferation assay of ERRy stable-transfected PNT2 and DU 145cells --- p.72 / Chapter 3. 4.7 --- Anchorage independent growth assay of ERRy stable-transfected PNT2 and DU 145 cells --- p.72 / Chapter 3.4.8 --- Flow cytometry of ERRγ stable-transfected PNT2 and DU145 cells --- p.74 / Chapter 3.4.9 --- RT-PCR of cell cycle-related genes in ERRγ stable-transfected PNT2 and DU 145 cells --- p.74 / Chapter 3.4.10 --- Western blot analysis of p21 in ERRγ stable-transfected PNT2 cells --- p.75 / Chapter 3.5 --- Statistical analysis --- p.75 / Table 3.1 - 3.2，Figure 3.1 - 35 --- p.76 / Chapter Chapter 4 --- Results / Chapter 4.1 --- "Expression patterns of ERRs and steroid hormone receptors in the human prostate cell lines, tumor xenografts and prostatic tissues" --- p.93 / Chapter 4.1.1 --- "mRNA expression patterns of ERR isoforms in the prostatic cell lines, prostate cancer xenografts and human prostatic tissues" --- p.93 / Chapter 4.1.2 --- mRNA expression patterns of steroid hormone receptors and prostatic differentiation markers in the prostatic cell lines and xenografts --- p.95 / Chapter 4.1.3 --- Characterization of antisera against human ERRs by ERR recombinant proteins --- p.97 / Chapter 4.1.4 --- Protein expression of ERR isoforms in the human prostatic cell lines --- p.98 / Chapter 4.1.5 --- "Immunolocalization of ERR isoforms in the normal, dysplastic and neoplastic prostates" --- p.98 / Chapter 4.2 --- Interrelationship of ERR and ER expression in PC-3 prostate cancer cells --- p.100 / Chapter 4.2.1 --- "Expressions of ERRγ, ERa and ERβ in the ERRa transient transfected PC-3 cells" --- p.100 / Chapter 4.2.2 --- Expression of ERRa in the ERRγ and ERa transient transfected PC-3 cells --- p.101 / Chapter 4.3 --- Intracellular trafficking and transcriptional activities of ERRs in the prostatic cells with fused green fluorescence protein 一 ERRs --- p.102 / Chapter 4.3.1 --- Trans activation of ERE response element 226}0ؤ driven reporter by ERR isoforms in the PC-3 cells in the presence or absence of serum --- p.102 / Chapter 4.3.2 --- Trans activation of SF-1 response element driven reporter by ERR isoforms in the PC-3 cells in the presence or absence of serum --- p.104 / Chapter 4.3.3 --- Subcellular localizations of three ERR isoforms in the PC-3 cells in the presence or absence of serum --- p.105 / Chapter 4.4 --- The role of ERRs in the growth of prostatic cells --- p.106 / Chapter 4.4.1 --- "The growth of ERRs transient transfected PC-3, PNT2 prostatic cells" --- p.107 / Chapter 4.4.2 --- Cell cycle analysis of ERRs transient transfected PC-3 cells --- p.108 / Chapter 4.4.3 --- Expression of cyclin-dependent kinase (CDK) inhibitors and p53 in the ERRs transient transfected PC-3 cells --- p.108 / Chapter 4.4.4 --- Establishment of ERRγ stable-transfected PNT2 and DU145 cells --- p.109 / Chapter 4.4.5 --- Transcriptional activation of ERE response element in ERRγ stable-transfected PNT2 cells --- p.111 / Chapter 4.4.6 --- Effect of over-expression of ERRγ on the growth of PNT2 and DU145 stable-transfected cells --- p.112 / Chapter 4.4.7 --- Efficiencies of colony formation of ERRγ stable-transfected PNT2 and DU145 cells --- p.113 / Chapter 4.4.8 --- Cell cycle analysis of ERRγ stable-transfected PNT2and DU 145 cells --- p.114 / Chapter 4.4.9 --- Expression of cell cycle-related genes in the ERRy stable-transfected PNT2 and DU 145 cells --- p.116 / Figure 4.1 - 4.38，Table 4.1 - 43 --- p.119 / Chapter Chapter 5 --- Discussion / Chapter 5.1 --- "Expression study in human prostatic cells, tumor xenografts" --- p.159 / Chapter 5.1.1 --- "Differential expression patterns of ERRs in prostatic cells, cancer xenografts and tissues" --- p.160 / Chapter 5.1.2 --- Co-localization of ERRs and ERβ in the human prostate --- p.166 / Chapter 5.1.3 --- Differential expression patterns of steroid hormone receptors and prostatic specific markers in prostatic cells and xenografts --- p.168 / Chapter 5.2 --- ERRα acts as a expression repressor of ERRγ and ERα in PC-3 cells --- p.173 / Chapter 5.3 --- ERRs are nuclear localized and constitutively active in PC-3 cells --- p.176 / Chapter 5.4 --- ERRs acts as the negative growth regulators in the prostatic cells --- p.179 / Chapter 5.4.1 --- Cell cycle control of mammalian cells --- p.180 / Chapter 5.4.2 --- The roles of AR and ERs in the cell cycle regulation --- p.181 / Chapter 5.4.3 --- Inhibition of cell proliferation in ERRs transient transfected PC-3 cells and ERRγ stable-transfected PNT2 and DU145 cells --- p.184 / Chapter 5.4.4 --- Inhibition of anchorage independent growth in ERRγ stable-transfected PNT2 and DU 145 cells --- p.188 / Chapter Chapter 6 --- Conclusion --- p.191 / Chapter Chapter 7 --- References --- p.192 / Chapter Chapter 8 --- Publications --- p.227

Interest in the mechanisms of homeo domain specificity led to a screen that identified Drosophila proteins able to bind a consensus homeo domain site. One clone isolated in this screen produced no homeo domain and was selected for further characterisation as a protein with an unknown DNA binding domain and the potential to interact with homeo domain proteins on the DNA. This thesis describes the characterisation of the Drosophila gene dead ringer ( dri ) corresponding to this clone. Isolation of overlapping cDNA clones and sequence analysis allowed the identification of a complete open reading frame in the dri message that gave a predicted protein of 901 amino acids. Database searches and multiple sequence alignment revealed a widely conserved motif in the Dri sequence that is found in proteins from organisms as diverse as yeast, nematodes, flies and humans. Biochemical analysis of the properties of this conserved motif revealed that it could function as a DNA binding domain when expressed in a fusion protein. The in vitro specificity of the Dri DNA binding domain was determined by selection and sequencing of target sites. The Dri consensus site obtained was strikingly similar to that of the Qfo class of homeo domains, although the sequence and predicted secondary structure of the Dri DNA binding domain do not resemble a homeo domain. Analysis of the developmental expression pattern of dri showed a ubiquitous maternal deposit gradually refined to localisation in the mesoderm at germ band extension, then further restriction to a diverse set of tissues including the salivary gland ducts, parts of the gut and a subset of the central nervous system. The phenotype of P - element insertion and deletion mutations of dri were identified as causing embryonic lethality preceded by a disruption of the hindgut and loss of Dri expression in the ring gland. The identification of the novel, conserved DNA - binding domain in Dead ringer offers an explanation for the regulatory activity of several important related proteins and presents an opportunity to use the advantages of the Drosophila model system to clarify the role of these proteins in transcriptional control. / Thesis (Ph.D.)--Departments of Biochemistry and Genetics, 1996.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 119-141).

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "May 2001." Typescript. Vita. Includes bibliographical references (leaves 180-198). Also available on the Internet.

Dissertation (Ph.D.)--University of Toledo, 2009. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Science." Title from title page of PDF document. Bibliography: p. 87-96.

Thesis (M.S.)--University of Toledo, 2009. / "In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Sciences (Bioinformatics and Proteomics-Genomics)." Title from title page of PDF document. Bibliography: p. 52-57.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 114-136).