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by Liang Yiu-hon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 104-117). / ACKNOWLEDGMENTS --- p.i / ABSTRACT --- p.ii / ABBREVIATIONS --- p.iii / AMINO ACIDS SHORTHAND --- p.v / TABLE OF CONTENTS --- p.vi-x / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- The Discovery of IGFs --- p.1 / Chapter 1.3 --- The Growth Promoting Actions of IGFs --- p.3 / Chapter 1.4 --- Molecular Biology of IGFs in Mammals --- p.6 / Chapter 1.4.1 --- IGF Genes and Transcripts --- p.6 / Chapter 1.4.2 --- Regulation of IGF Gene Expression --- p.8 / Chapter 1.5 --- IGF Binding Proteins --- p.11 / Chapter 1.5.1 --- Regulation of IGF Action by IGF Binding Proteins --- p.11 / Chapter 1.6 --- The Insulin and IGF Receptors --- p.13 / Chapter 1.6.1 --- IGF-I Receptor --- p.13 / Chapter 1.6.2 --- IGF-II Receptor --- p.13 / Chapter 1.6.3 --- Insulin/IGF-I Hybrid Receptor --- p.15 / Chapter 1.7 --- IGF in Mammalian Fetal Growth --- p.17 / Chapter 1.8 --- The Role of IGFs in Fish --- p.19 / Chapter 1.9 --- Aims of the Present Study --- p.26 / Chapter CHAPTER 2 --- GENERAL METHODOLOGY / Chapter 2.1 --- Materials --- p.27 / Chapter 2.2 --- Methods --- p.32 / Chapter 2.2.1 --- Gene Clean --- p.32 / Chapter 2.2.1A --- Gene Clean by Glassmilk Method --- p.32 / Chapter 2.2.1B --- Gene Clean by Sephaglas´ёØ BandPrep Kit --- p.32 / Chapter 2.2.2 --- Preparation of Radioactive Nucleic Acid Probes --- p.33 / Chapter 2.2.3 --- Sephadex G-50 Spun-column Chromatography --- p.33 / Chapter 2.2.4 --- Small Scale Alkali Preparation of Plasmid DNA --- p.34 / Chapter 2.2.5 --- Large Scale Preparation of Plasmid DNA 36 - using Wizard Maxiprep Kit (Promega) / Chapter 2.2.6 --- DNA Sequencing using T7 DNA Polymerase Sequencing Kit (Pharmacia) --- p.37 / Chapter 2.2.7 --- Restriction Enzyme Digestion --- p.38 / Chapter 2.2.8 --- Agarose Gel Electrophoresis --- p.39 / Chapter 2.2.9 --- Dephosphorylation of Linearized Plasmid DNA --- p.39 / Chapter 2.2.10 --- Ligation of Foreign DNA with Linearized Plasmid --- p.40 / Chapter 2.2.11 --- Transformation of Plasmid Vector into Competent Cell (Heat Shock Method) --- p.40 / Chapter 2.2.12 --- Blotting : Transfer of DNA to Nylon Membrane --- p.41 / Chapter 2.2.12A --- Capillary Transfer of DNA to Nylon Membrane in 10X SSC --- p.41 / Chapter 2.2.12B --- Capillary Transfer of DNA to Nylon Membrane under Alkaline Condition --- p.42 / Chapter CHAPTER 3 --- SCREENING OF COMMON CARP LIVER cDNA LIBRARY / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods --- p.45 / Chapter 3.2.1 --- Materials --- p.45 / Chapter 3.2.2 --- Methods --- p.48 / Chapter 3.2.2.1 --- Preparation of the Plating Host --- p.48 / Chapter 3.2.2.2 --- Phage Titering --- p.48 / Chapter 3.2.2.3 --- Primary Screening of Common Carp Liver cDNA Library --- p.48 / Chapter 3.2.2.4 --- Purification of the Positive Clone --- p.49 / Chapter 3.2.2.5 --- Checking the Insert Size of the Positive Clone --- p.50 / Chapter 2.2.2.6 --- In vivo Excision to Release Phagemid from the Phage vector --- p.51 / Chapter 3.2.2.7 --- Plasmid Minipreparation of the Positive Clone --- p.52 / Chapter 3.2.2.8 --- Restriction Enzyme Digestion to Release the Insert --- p.52 / Chapter 3.2.2.9 --- Large Scale Plasmid Preparation of the Positive Clone --- p.53 / Chapter 3.2.2.10 --- DNA Sequencing of the Positive Clone --- p.53 / Chapter 3.2.2.11 --- Restriction Mapping of the Positive Clone --- p.53 / Chapter 3.2.2.12 --- Subcloning of the Positive Clone into Plasmid Vectors --- p.53 / Chapter 3.2.2.13 --- DNA Sequencing of the Subclones --- p.54 / Chapter 3.3 --- Results and Discussion --- p.55 / Chapter CHAPTER 4 --- RNA ASSAY USING REVERSE TRANSCRIPTION- POLYMERASE CHAIN REACTION / Chapter 4.1 --- Introduction --- p.70 / Chapter 4.2 --- Materials and Methods --- p.71 / Chapter 4.2.1 --- Materials --- p.71 / Chapter 4.2.2 --- Methods --- p.72 / Chapter 4.2.2.1 --- Tissue Preparation --- p.72 / Chapter 4.2.2.2 --- Total RNA Extraction --- p.72 / Chapter 4.2.2.3 --- Electrophoresis of RNA in Agarose Gel Containing Formaldehyde --- p.73 / Chapter 4.2.2.4 --- First Strand cDNA Synthesis --- p.74 / Chapter 4.2.2.5 --- IGF-I Specific PCR --- p.75 / Chapter 4.2.2.6 --- Preparation of Carp IGF Conserve Region --- p.75 / Chapter 4.2.2.7 --- Southern Hybridization of PCR Products --- p.76 / Chapter 4.3 --- Results and Discussion --- p.76 / Chapter CHAPTER 5 --- GENOMIC SOUTHERN ANALYSIS / Chapter 5.1 --- Introduction --- p.80 / Chapter 5.2 --- Materials and Methods --- p.81 / Chapter 5.2.1 --- Materials --- p.81 / Chapter 5.2.2 --- Methods --- p.82 / Chapter 5.2.2.1 --- Preparation of Genomic DNA from Carp Testis --- p.82 / Chapter 5.2.2.2 --- Restriction Enzyme Digestion of Genomic DNA --- p.82 / Chapter 5.2.2.3 --- Southern Blotting of the Digested Genomic DNA --- p.83 / Chapter 5.2.2.4 --- Preparation of the Carp IGF-I Specific Probe --- p.83 / Chapter 5.2.2.5 --- Genomic Southern Hybridization --- p.83 / Chapter 5.3 --- Results and Discussion --- p.84 / Chapter CHAPTER 6 --- THE SEARCH OF OTHER IGF cDNA SUBTYPES IN COMMON CARP / Chapter 6.1 --- Introduction --- p.88 / Chapter 6.2 --- Materials and Methods --- p.89 / Chapter 6.2.1 --- Materials --- p.89 / Chapter 6.2.2 --- Methods --- p.91 / Chapter 6.2.2.1 --- Screening using a Conserve Region cDNA Probe of Carp IGF-I --- p.91 / Chapter 6.2.2.2 --- PCR using IGF-I Specific Primers --- p.92 / Chapter 6.2.2.3 --- PCR Using T3 and T7 Primers --- p.92 / Chapter 6.2.2.4 --- Southern Blot Analysis of T3 and T7 PCR Products of cDNA Insert --- p.93 / Chapter 6.2.2.5 --- DNA Sequencing of Positive Clones --- p.94 / Chapter 6.3 --- Results and Discussion --- p.94 / Chapter CHAPTER 7 --- GENERAL DISCUSSION AND CONCLUSION --- p.99 / REFERENCES --- p.104-117

by Lau Kwok Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 126-134). / Acknowledgments --- p.i / Abstract --- p.ii / Contents --- p.iv / Abbreviations --- p.ix / List of Figures --- p.x / List of Tables --- p.xii / List of Primers --- p.xiii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- A Brief Introduction of GH --- p.1 / Chapter 1.2 --- Growth Hormone Receptor (GHR) --- p.3 / Chapter 1.2.1 --- Tissue Distribution of GHR --- p.4 / Chapter 1.2.2 --- GHR Biosynthesis and Degradation --- p.7 / Chapter 1.2.3 --- Regulation of GHR level --- p.8 / Chapter 1.2.4 --- Structure of GHR --- p.10 / Chapter 1.2.5 --- Possible Signal Transduction Pathways of GHR --- p.13 / Chapter 1.2.6 --- GHR Related Dwarfism --- p.15 / Chapter 1.2.7 --- Significance of Cloning of GHR cDNA --- p.16 / Chapter 1.3 --- Objectives of the Present Study --- p.17 / Chapter Chapter 2 --- General Materials and Methods / Chapter 2.1 --- Ethanol Precipitation of DNA and RNA --- p.19 / Chapter 2.2 --- Spectrophotometric Determination of DNA and RNA --- p.19 / Chapter 2.3 --- Minipreparation of Plasmid DNA --- p.19 / Chapter 2.4 --- Preparation of Plasmid DNA using Magic´ёØ Minipreps DNA Purification Kit from Promega --- p.20 / Chapter 2.5 --- Preparation of Plasmid DNA using QIAGEN-tip100 --- p.21 / Chapter 2.6 --- Preparation and Transformation of Escherichia coli Competent Cell --- p.22 / Chapter 2.7 --- Rapid Screening for the Presence of Desired Plasmid --- p.23 / Chapter 2.8 --- Agarose Gel Electrophoresis --- p.23 / Chapter 2.9 --- Formaldehyde / Agarose Gel Electrophoresis --- p.24 / Chapter 2.10 --- Restriction Digestion of DNA --- p.25 / Chapter 2.11 --- Linearization and Dephosphorylation of Plasmid Vector --- p.25 / Chapter 2.12 --- Purification of DNA form Agarose Gel Using GENECLEAN II® Kit --- p.25 / Chapter 2.13 --- Purification of DNA by Phenol / Chloroform Extraction --- p.26 / Chapter 2.14 --- DNA Radiolabelling --- p.26 / Chapter 2.15 --- Spun-Column Chromatography --- p.27 / Chapter 2.16 --- Capillary Transfer of DNA/RNA to a Nylon Membrane --- p.27 / Chapter 2.16.1 --- DNA Denaturation --- p.27 / Chapter 2.16.2 --- Capillary Transfer --- p.28 / Chapter 2.17 --- Hybridization of DNA/RNA --- p.28 / Chapter 2.18 --- Autoradiography --- p.29 / Chapter 2.19 --- Preparation of Ribonuclease Free Reagents and Apparatus --- p.29 / Chapter 2.20 --- Total RNA Isolation --- p.30 / Chapter 2.21 --- mRNA Isolation --- p.31 / Chapter 2.22 --- First Strand cDNA Synthesis --- p.32 / Chapter 2.23 --- Polymerase Chain Reaction --- p.32 / Chapter 2.24 --- 3'End Modification of PCR Amplified DNA --- p.33 / Chapter 2.25 --- Ligation of DNA Fragments --- p.34 / Chapter 2.26 --- DNA Sequencing --- p.34 / Chapter 2.26.1 --- DNA Sequencing Reaction --- p.34 / Chapter 2.26.2 --- DNA Sequencing Electrophoresis --- p.35 / Chapter 2.27 --- Reagents and Buffers --- p.38 / Chapter 2.27.1 --- Media for Bacterial Culture --- p.38 / Chapter 2.27.2 --- Reagents for Preparation of Plasmid DNA --- p.38 / Chapter 2.27.3 --- Buffers for Agarose Gel Electrophoresis --- p.40 / Chapter 2.27.4 --- Buffers for Formaldehyde Gel Electrophoresis --- p.40 / Chapter 2.27.5 --- Buffers for Preparation Competent Cells --- p.41 / Chapter 2.27.6 --- Buffers for Capillary Transfer and Hybridization --- p.42 / Chapter 2.27.7 --- Buffers for Total RNA Extraction --- p.43 / Chapter 2.27.8 --- 10X CIP Buffers --- p.43 / Chapter 2.28 --- Size of DNA/RNA Molecular Weight Markers --- p.44 / Chapter Chapter 3 --- Molecular Studies on Chicken Growth Hormone Receptor / Chapter 3.1 --- Introduction --- p.45 / Chapter 3.2 --- Material and Methods --- p.46 / Chapter 3.2.1 --- Molecular Cloning of Chicken GHR cDNA by PCR --- p.46 / Chapter 3.2.1.1 --- Animals and Tissue --- p.46 / Chapter 3.2.1.2 --- Reverse Transcrbed-Polymerase Chain Reaction (RT-PCR) --- p.46 / Chapter 3.2.1.3 --- Subcloning of PCR Amplified DNA Fragments --- p.47 / Chapter 3.2.2 --- Ontogeny of GHR mRNA Expression in Chicken Liver and Brain --- p.48 / Chapter 3.2.2.1 --- Animals and Tissues --- p.48 / Chapter 3.2.2.2 --- Northern Analysis --- p.48 / Chapter 3.2.2.3 --- Quantification of GHR mRNA level --- p.49 / Chapter 3.2.3 --- Prokaryotic Expression of Chicken GHR cDNA --- p.49 / Chapter 3.2.3.1 --- Subcloning of Chicken GHR cDNA into a Prokaryotic Expression Vector --- p.49 / Chapter 3.2.3.2 --- Expression of Chicken GHR cDNAin E.coli --- p.50 / Chapter 3.2.3.3 --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.50 / Chapter 3.2.4 --- Reagents and Buffers / Chapter 3.2.4.1 --- Medium for Bacterial Culture --- p.53 / Chapter 3.2.4.2 --- Reagents for SDS-PAGE --- p.53 / Chapter 3.2.5 --- Size of Protein Molecular Weight Markers --- p.54 / Chapter 3.3 --- Results --- p.55 / Chapter 3.3.1 --- Molecular Cloning of Chicken GHR cDNA by PCR --- p.55 / Chapter 3.3.1.1 --- RT-PCR --- p.55 / Chapter 3.3.1.2 --- Subcloning --- p.56 / Chapter 3.3.1.3 --- Nucleotide Sequence Analysis --- p.57 / Chapter 3.3.2 --- Ontogeny of GHR mRNA Expression in Chicken Liver and Brain --- p.59 / Chapter 3.3.3 --- Prokaryotic Expression of Chicken GHR cDNA --- p.64 / Chapter 3.3.3.1 --- Subcloning --- p.64 / Chapter 3.3.3.2 --- Nucleotide Sequence Analysis --- p.65 / Chapter 3.3.3.3 --- Prokaryotic Expression --- p.66 / Chapter 3.4 --- Discussion --- p.68 / Chapter 3.4.1 --- Molecular Cloning of Chicken GHR cDNA by PCR --- p.68 / Chapter 3.4.2 --- Ontogeny of GHR mRNA Expression in Chicken Liver and Brain --- p.70 / Chapter 3.4.3 --- Prokaryotic Expression of Chicken GHR cDNA --- p.71 / Chapter Chapter 4 --- Molecular Cloning of Pigeon Growth Hormone Receptor Complementary DNA by Polymerase Chain Reaction and Sequence Analysis / Chapter 4.1 --- Introduction --- p.74 / Chapter 4.2 --- Materials and Methods --- p.75 / Chapter 4.2.1 --- Animals and Tissues --- p.75 / Chapter 4.2.2 --- Cloning of Pigeon GHR cDNA Main Core by PCR --- p.75 / Chapter 4.2.2.1 --- RT-PCR --- p.75 / Chapter 4.2.2.2 --- Southern Analysis of PCR Amplified Product --- p.76 / Chapter 4.2.2.3 --- Subcloning of PCR Amplified DNA Fragment --- p.76 / Chapter 4.2.3 --- Determination of 3' End Coding Sequence of Pigeon GHR cDNA --- p.76 / Chapter 4.2.4 --- Determination of 5' End Coding Sequence of Pigeon GHR cDNA --- p.79 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Cloning of Pigeon GHR cDNA Main Core by PCR --- p.82 / Chapter 4.3.1.1 --- RT-PCR --- p.82 / Chapter 4.3.1.2 --- Southern Analysis --- p.83 / Chapter 4.3.1.3 --- Subcloning of Fragment M --- p.83 / Chapter 4.3.1.4 --- Restriction Digestion of Plasmid --- p.85 / Chapter 4.3.1.5 --- Nucleotide Sequence Analysis --- p.86 / Chapter 4.3.2 --- Determination of 3' End and 5' End coding Sequences of Pigeon GHR cDNA --- p.88 / Chapter 4.3.2.1 --- Random Primer Initiated RNA-PCR --- p.88 / Chapter 4.3.2.2 --- AmpliFINDER RACE --- p.88 / Chapter 4.3.2.3 --- Subcloning of Fragment 3' and Fragment 5' --- p.90 / Chapter 4.3.2.4 --- Nucleotide Sequence Analysis --- p.92 / Chapter 4.3.3 --- Nucleotide Sequence and Predicted Amino Acid Sequence of Pigeon GHR --- p.93 / Chapter 4.4 --- Discussion --- p.100 / Chapter Chapter 5 --- Attempts on Molecular Cloning of Fish Growth Hormone Receptor Complementary DNA / Chapter 5.1 --- Introduction --- p.106 / Chapter 5.2 --- Materials and Methods --- p.107 / Chapter 5.2.1 --- Animals and Tissues --- p.107 / Chapter 5.2.2 --- Design of PCR primers --- p.107 / Chapter 5.2.3 --- RT-PCR and Subcloning of PCR Amplified DNA --- p.108 / Chapter 5.2.4 --- Northern Analysis of Dace Liver RNA --- p.110 / Chapter 5.3 --- Results / Chapter 5.3.1 --- PCR --- p.111 / Chapter 5.3.2 --- Subcloning --- p.112 / Chapter 5.3.3 --- Nucleotide Sequence Analysis --- p.114 / Chapter 5.3.4 --- Northern Analysis --- p.117 / Chapter 5.4 --- Discussion --- p.119 / Chapter Chapter 6 --- General Discussion --- p.123 / References --- p.126 / Appendix --- p.135

Thesis (Ph.D.)--City University of Hong Kong, 2009. / "Submitted to Department of Biology and Chemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy." Includes bibliographical references (leaves 133-147)

In recent years, the general public has recognized the dangers of a high fat diet and are demanding meat with lower fat content. This demand has stimulated research in the growth and regulation of adipocytes. However, despite much effort, no adipocyte-specific plasma membrane markers from any species are available as an aid to accurately distinguish adipocytes from non-adipocytes. One potential candidate for such a marker in porcine adipocytes has been identified by Killefer and Hu (1990b). Characterization of the cDNA for this protein, designated porcine adipocyte membrane protein (PAMP), is presented here. Sequence for the 910 by clone is 80% similar to an internal region of a rat prostaglandin F[subscript 2��] receptor regulator protein (FPRP) described by Orlickey (1996). Western blot analysis suggests that the pig protein is a homotetramer held together with disulfide bonds which form very close to the transmembrane region making the tetramer extremely difficult to reduce to monomeric units. Oligonucleotide primers were designed to amplify a genomic fragment by the polymerase chain reaction (PCR) and for a reverse transcriptase PCR (RT-PCR) assay to study the expression of the mRNA. A 2114 bp genomic clone revealed one intron in the coding region. A serum-free primary cell culture system was used to study the expression of the mRNA. Although message was detected every day over a ten day period, it appeared to peak between 6 to 8 days after plating. The PAMP protein is clearly of the same family as the rat FPRP but its size and conformation are quite different so it is not clear what function it performs in porcine adipocytes. Further experiments should focus on attaining full length cDNA's, confirming the molecular conformation of the protein, and assessing its function in a serum-free primary cell culture system. / Graduation date: 1997

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 239-251).

published_or_final_version / Medicine / Master / Master of Philosophy

Keung Yim Mei. / "October 2001." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 187-195). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Thesis (M.Phil.)--University of Hong Kong, 2003. / Includes bibliographical references (leaves 65-71) Also available in print.