In the tissue distribution study, the expression of GHR2 is significantly higher than GHR1 in many tissues of the seabream including the gonad, kidney, muscle, pituitary and spleen. In vivo hormone treatment data indicated that cortisol and testosterone have differential expression regulation between GHR1 and GHR2. On the other hand, hepatic expression of both GHR1 and GHR2 in seabream was decreased by estradiol treatment. In primary cultures of seabream hepatocytes, the expression patterns after treatment by the various concentrations of hormones were consistent with the in vivo results. / To study the actions of environmental estrogens on the somatotropic axis, a transgenic yeast system was developed for estrogenicity screening. The fish estrogen receptor (gfER) and a reporter vector containing the estrogen responsive element (ERE) were expressed in yeast cells as a means to identify potential estrogens. Using this system, more than fifty chemicals including pesticides, herbicides, industrial chemicals and phytoestrogens were screened. Ten compounds including dibutyl phthalate (DBP) and bisphenol A (BPA) were demonstrated to exhibit estrogenic activities. And a compound (malachite green, MG) with novel anti-estrogenenic activities was identified. Then BPA and MG were focused to explore the disrupting effects of environmental estrogens on the two GHRs. Through the method of real-time PCR, both compounds could attenuate the gene expression level of GHRs in seabream hepatocytes. Using the method of luciferase assay, the signal transduction of the two GHRs was found to be desensitized by both BPA and MG. / Two genomic contigs of putative growth hormone receptor (GHR) were identified in fugu and zebrafish genomes by in silico analysis, suggesting the presence of two GHR subtypes in a single teleost species. This hypothesis was tested by cloning the full-length cDNA sequence of a second GHR subtype from the black seabream in which the first GHR subtype has been previously reported. Phylogenetic analysis of known GHR sequences from various vertebrates revealed that fish GHRs cluster into two distinct clades, viz. GHR1 and GHR2. The biological activities of both GHR subtypes from seabream had been examined using the reporter transcription assays in cultured eukaryotic cells. It was demonstrated that both of them have differential signal transduction upon Spi 2.1, beta-casein and c-fos promoter activities. / by Jiao, Baowei. / "December 2006." / Adviser: Christopher H. K. Cheng. / Source: Dissertation Abstracts International, Volume: 68-09, Section: B, page: 5662. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 150-180). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

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

by Lee Tsz On. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 148-155). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgments --- p.v / Contents --- p.vi / List of figures --- p.xii / List of table --- p.xiv / Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction / Chapter 1.1. --- Growth hormone (GH) --- p.1 / Chapter 1.1.1. --- Introduction to GH --- p.1 / Chapter 1.1.2. --- Actions of GH --- p.2 / Chapter 1.1.3. --- Structure of GH --- p.3 / Chapter 1.1.4. --- The sequence of GH --- p.5 / Chapter 1.2. --- Growth hormone receptor (GHR) --- p.6 / Chapter 1.2.1 --- Introduction to GHR --- p.6 / Chapter 1.2.2. --- Structure of the extracellular domain of GHR --- p.9 / Chapter 1.2.3. --- The regulation of GHR --- p.12 / Chapter 1.2.4. --- GHR biosynthesis --- p.13 / Chapter 1.2.5. --- Tissue distribution of GHR --- p.14 / Chapter 1.3. --- Signal transduction mechanisms of GHR --- p.15 / Chapter 1.3.1. --- Dimerization of GH and GHR complex --- p.15 / Chapter 1.3.2. --- The Jak and Stat pathway --- p.18 / Chapter 1.3.3. --- The ras and other signaling pathways --- p.20 / Chapter 1.4. --- Project aim --- p.22 / Chapter Chapter 2 --- Material and Methods / Chapter 2.1. --- Preparation of ribonuclease free reagents and apparatus --- p.23 / Chapter 2.2. --- Isolation of total RNA --- p.23 / Chapter 2.3. --- Isolation of mRNA --- p.24 / Chapter 2.4. --- Spectrophotometric quantification and qualification of DNA and RNA --- p.24 / Chapter 2.5. --- First strand cDNA synthesis --- p.25 / Chapter 2.6. --- Agarose gel electrophoresis of DNA --- p.25 / Chapter 2.7. --- Formaldehyde agarose gel electrophoresis of RNA --- p.26 / Chapter 2.8. --- Vacuum transfer of DNA to a nylon membrane --- p.26 / Chapter 2.9. --- Nucleic acids purification by MicroSpin (S-200HR) columns --- p.27 / Chapter 2.10. --- DNA radioactive labeling by nick translation --- p.27 / Chapter 2.11. --- Southern blot analysis --- p.28 / Chapter 2.12. --- Autoradiography and molecular imager --- p.28 / Chapter 2.13 . --- Linearization and dephosphorylation of plasmid DNA --- p.29 / Chapter 2.14. --- Purification of DNA from agarose using QIAEX II kit --- p.29 / Chapter 2.15. --- 3'End modification of PCR amplified DNA --- p.30 / Chapter 2.16. --- Ligation of DNA fragments to linearized vector --- p.30 / Chapter 2.17. --- Preparation of Escherichia coli competent cells --- p.31 / Chapter 2.18. --- Transformation --- p.31 / Chapter 2.19. --- Mini preparation of plasmid DNA --- p.32 / Chapter 2.20. --- Maxi preparation of plasmid DNA --- p.34 / Chapter 2.21 . --- PCR sequencing --- p.35 / Chapter 2.22. --- cDNA library screening --- p.36 / Chapter 2.23. --- Preparation and sterilization of culture medium --- p.38 / Chapter 2.24. --- Preparation of frozen stock of culture cells --- p.39 / Chapter 2.25. --- Cell passage of CHO-Kl --- p.39 / Chapter 2.26. --- Counting of cells --- p.40 / Chapter 2.27. --- Proliferation assay performed on CHO-K1 cells (MTT method) --- p.40 / Chapter 2.28. --- Luciferase assay --- p.41 / Chapter 2.29. --- SDS-PAGE preparation --- p.42 / Chapter 2.30. --- SDS-PAGE analysis of proteins --- p.42 / Chapter 2.31 . --- Recombinant protein expression --- p.43 / Chapter 2.32. --- Small scale purification of recombinant proteins --- p.44 / Chapter 2.33. --- Restriction digestion of DNA --- p.45 / Chapter 2.34. --- Purification of PCR product using QIAquick PCR purification kit --- p.45 / Chapter 2.35. --- TA cloning of PCR fragment --- p.45 / Chapter 2.36. --- Transfection of plasmid into CHO-K1 cells --- p.46 / Chapter 2.37. --- Sources of hormones --- p.46 / Chapter 2.38. --- Buffer and reagents --- p.47 / Chapter Chapter 3 --- "Cloning, expression and tissue distribution of Xenopus laevis GHR" / Chapter 3.1. --- Introduction --- p.50 / Chapter 3.2. --- Materials and methods --- p.51 / Chapter 3.2.1. --- Molecular cloning of xGHR cDNA / Chapter 3.2.1.1. --- Animals and tissues --- p.51 / Chapter 3.2.1.2. --- Reverse transcribed´ؤpolymerase chain reaction (RT-PCR) --- p.51 / Chapter 3.2.1.3. --- Subcloning of PCR amplified DNA fragment --- p.53 / Chapter 3.2.1.4. --- Library screening of xGHR --- p.53 / Chapter 3.2.1.5. --- 5 'Rapid amplification of cDNA end (5' RACE) --- p.55 / Chapter 3.2.2. --- Tissue distribution of xGHR / Chapter 3.2.2.1. --- Animals and tissues --- p.56 / Chapter 3.2.2.2. --- RT-PCR and Southern blot --- p.56 / Chapter 3.2.3. --- Eukarytoic expression of xGHR and functional assay of xGHR / Chapter 3.2.3.1. --- Subcloning ofxGHR into pRc/CMV --- p.57 / Chapter 3.2.3.2. --- Expression of xGHR in CHO-K1 cell --- p.58 / Chapter 3.2.3.3. --- Proliferation assay --- p.58 / Chapter 3.3. --- Results --- p.60 / Chapter 3.3.1. --- RT-PCR of the partial fragment --- p.60 / Chapter 3.3.2. --- Library screening of xGHR cDNA library --- p.61 / Chapter 3.3.3. --- 5' RACE --- p.64 / Chapter 3.3.4. --- The full-length cDNA sequence of xGHR --- p.65 / Chapter 3.3.5. --- Tissue distribution of xGHR mRNA --- p.69 / Chapter 3.3.6. --- Functional assay of xGHR in CHO-K1 cells --- p.71 / Chapter 3.4. --- Discussion --- p.74 / Chapter Chapter 4 --- Cloning and expression of Xenopus laevis GH-A and GH-B / Chapter 4.1. --- Introduction --- p.78 / Chapter 4.2. --- Materials and Methods --- p.79 / Chapter 4.2.1. --- PCR amplification of xGH-A and xGH-B partial fragments --- p.79 / Chapter 4.2.2. --- cDNA library screening of xGH-A and xGH-B --- p.80 / Chapter 4.2.3. --- Rapid amplification of cDNA ends of xGH-B / Chapter 4.2.3.1. --- 3'RACE --- p.80 / Chapter 4.2.3.2. --- 5'RACE --- p.81 / Chapter 4.2.4. --- Expression of xGH-A and xGH-B / Chapter 4.2.4.1 --- Construction of the expression vector --- p.84 / Chapter 4.2.4.2. --- Protein expression of xGH-A and xGH-B --- p.85 / Chapter 4.2.5. --- Purification of recombinant xGH-A and xGH-B --- p.85 / Chapter 4.3. --- Results --- p.87 / Chapter 4.3.1. --- PCRof xGH-A and xGH-B partial fragment --- p.87 / Chapter 4.3.2. --- Library screening of xGH-A --- p.87 / Chapter 4.3.3. --- 5' RACE and 3' RACE of xGH-B --- p.91 / Chapter 4.3.4. --- Sequence analysis of xGH-A and xGH-B --- p.93 / Chapter 4.3.5. --- Protein expression and purification of recombinant xGH-A and xGH-B --- p.100 / Chapter 4.4. --- Discussion --- p.102 / Chapter Chapter 5 --- Molecular cloning and function expression of goldfish GHR / Chapter 5.1. --- Introduction --- p.105 / Chapter 5.2. --- Materials and methods --- p.106 / Chapter 5.2.1. --- Molecular cloning of the partial fragment of gfGHR / Chapter 5.2.1.1. --- Primer design --- p.106 / Chapter 5.2.1.2. --- Library PCR of gfGHR partial fragment --- p.108 / Chapter 5.2.2. --- Library PCR of gfGHR cDNA sequence --- p.110 / Chapter 5.2.3. --- Determination of 3' End and 5' End sequences of gfGHR cDNA --- p.112 / Chapter 5.2.4. --- Tissue distribution of gfGHR / Chapter 5.2.4.1. --- Animals and tissues --- p.115 / Chapter 5.2.4.2. --- Semi-quantitative R T-PCR --- p.115 / Chapter 5.2.5. --- Functional expression of gfGHR in CHO-K1 cell / Chapter 5.2.5.1. --- Construction of an expression vector containing gfGHR --- p.116 / Chapter 5.2.5.2. --- Functional assay of gfGHR expression on CHO-K1 cells --- p.117 / Chapter 5.2.5.3. --- Proliferation assay --- p.118 / Chapter 5.2.5.4. --- Spi luciferase assay --- p.118 / Chapter 5.3. --- Results --- p.120 / Chapter 5.3.1. --- PCR amplification of the partial sequence of gfGHR --- p.120 / Chapter 5.3.2. --- The library PCR of gfGHR cDNA sequence --- p.122 / Chapter 5.3.3. --- The sequence of gfGHR --- p.124 / Chapter 5.3.4. --- Tissue distribution of gfGHR --- p.131 / Chapter 5.3.5. --- Proliferation assay --- p.133 / Chapter 5.3.6. --- Spi luciferase assay --- p.135 / Chapter 5.4. --- Discussion --- p.137 / Chapter Chapter 6 --- General discussion and future works --- p.145 / References --- p.148 / Appendix --- p.156

by Yam Kwok Fai. / Year shown on spine: 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 141-149). / Acknowledgments --- p.i / List of Contents --- p.ii / List of Figures --- p.viii / List of Tables --- p.xii / List of Primers --- p.xiii / Abbreviations --- p.xiv / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Growth Hormone (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 --- Biosynthesis and Degradation of GHR --- p.6 / Chapter 1.2.3 --- Regulation of GHR Level --- p.7 / Chapter 1.2.4 --- The Structure of GHR --- p.9 / Chapter 1.2.5 --- The Structure of GHR Gene --- p.13 / Chapter 1.2.6 --- Growth Hormone Binding Protein (GHBP) --- p.14 / Chapter 1.2.7 --- The GH/Prolactin/Cytokine/Erythropoietin Receptor Superfamily --- p.15 / Chapter 1.2.8 --- Proposed Signal Transduction Pathway --- p.17 / Chapter 1.2.9 --- GHR Related Dwarfism --- p.22 / Chapter i). --- Substitution of certain amino acid residues in the extracellular domain --- p.22 / Chapter ii). --- Deletion of the extracellular domain --- p.23 / Chapter a). --- deletion of a small portion of the binding protein / Chapter b). --- deletion of a large portion of the binding protein / Chapter c). --- deletion of a large portion of the binding domain and the whole transmembrane domain / Chapter iii). --- Associated with normal GHBP --- p.24 / Chapter 1.3 --- Objectives of Cloning Vertebrate GHR cDNAs --- p.24 / Chapter Chapter 2 --- General Experimental Methods / Chapter 2.1 --- Preparation of Ribonuclease Free Reagents and Apparatus --- p.26 / Chapter 2.2 --- Isolation of Total RNA --- p.26 / Chapter 2.3 --- Isolation of mRNA --- p.26 / Chapter a). --- directly from tissue / Chapter b). --- from isolated total RNA / Chapter 2.4 --- Spectrophotometric Quantification and Qualification of DNA and RNA --- p.29 / Chapter 2.5 --- First Strand cDNA Synthesis --- p.29 / Chapter 2.6 --- Polymerase Chain Reaction (PCR) --- p.30 / Chapter 2.7 --- Agarose Gel Electrophoresis --- p.31 / Chapter 2.8 --- Formaldehyde Agarose Gel Electrophoresis of RNA --- p.31 / Chapter 2.9 --- Capillary Transfer of DNA/RNA to a Nylon Membrane (Southern/Northern Blotting) --- p.32 / Chapter a). --- DNA denaturing / Chapter b). --- Capillary transfer / Chapter 2.10 --- DNA Radiolabelling --- p.33 / Chapter a). --- By random primer translation / Chapter b). --- By nick translation / Chapter 2.11 --- Spuncolumn Chromatography --- p.34 / Chapter 2.12 --- Hybridization of Southern/Northern Blot --- p.35 / Chapter 2.13 --- Autoradiography --- p.35 / Chapter 2.14 --- Linearization and Dephosphorylation of Plasmid DNA --- p.36 / Chapter 2.15 --- Restriction Digestion of DNA --- p.36 / Chapter 2.16 --- Purification of DNA from Agarose Gel using GENECLEAN® Kit --- p.36 / Chapter 2.17 --- 3' End Modification of PCR Amplified DNA --- p.37 / Chapter 2.18 --- Ligation of DNA Fragments to Linearized Vector --- p.37 / Chapter 2.19 --- Preparation of Escherichia coli Competent Cells --- p.38 / Chapter 2.20 --- Transformation of the Escherichia coli Strain DH5a --- p.38 / Chapter 2.21 --- Minipreparation of Plasmid DNA --- p.39 / Chapter 2.22 --- DNA Purification by Phenol/Chloroform Extraction --- p.39 / Chapter 2.23 --- Ethanol Precipitation of DNA and RNA --- p.40 / Chapter 2.24 --- Preparation of Plasmid DNA using Wizard´ёØ Minipreps DNA Purification Kit from Promega --- p.40 / Chapter 2.25 --- Preparation of Plasmid DNA using QIAGEN-tip100 --- p.41 / Chapter 2.26 --- DNA Sequencing --- p.42 / Chapter 2.26.1 --- DNA Sequencing Reaction / Chapter a). --- T7 sequencing / Chapter b). --- PCR sequencing / Chapter 2.26.2 --- DNA Sequencing Electrophoresis --- p.44 / Chapter i). --- Preparation of 8% polyacrylamide gel solution / Chapter ii). --- Casting the gel / Chapter iii). --- Electrophoresis / Chapter Chapter 3 --- Molecular Cloning of Golden Hamster (Mesocricetus auratus) GHR cDNA / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.2 --- Experimental Methods / Chapter 3.2.1 --- Animals and Tissues --- p.47 / Chapter 3.2.2 --- PCR Cloning of GHR cDNA Fragments in the Cytoplasmic Domain --- p.47 / Chapter 3.2.2.1 --- Primer design and PCR strategy --- p.47 / Chapter 3.2.2.2 --- PCR studies on the hamster liver and kidney first strand cDNA --- p.49 / Chapter 3.2.2.3 --- Southern analysis of the PCR products --- p.50 / Chapter 3.2.2.4 --- Subcloning and sequencing of PCR amplified cDNA fragments --- p.50 / Chapter 3.2.3 --- Screening of a Hamster Liver cDNA Library --- p.51 / Chapter 3.2.3.1 --- Preparation of the plating bacteria --- p.51 / Chapter 3.2.3.2 --- Phage titering of the λ ZAP library --- p.51 / Chapter 3.2.3.3 --- Primary screening of the amplified hamster liver cDNA library --- p.52 / Chapter 3.2.3.4 --- Plaque uplifting and hybridization with hamster GHR cDNA fragment --- p.52 / Chapter 3.2.3.5 --- Purification of putative clones from primary screening --- p.53 / Chapter 3.2.3.6 --- Checking the size of the DNA insert --- p.53 / Chapter 3.2.3.7 --- In vitro excision to release phagemid from the phage vector --- p.54 / Chapter 3.2.3.8 --- Plasmid minipreparation of the putative clones --- p.56 / Chapter 3.2.3.9 --- Nucleotide sequencing of the DNA inserts of different clones --- p.56 / Chapter 3.2.4 --- Tissue Distribution of GHR in Hamster Tissues and the Relative Expression Level of GHR mRNAin these tissues --- p.58 / Chapter 3.2.5 --- Cloning of the Full-length GHR cDNA into a Mammalian Vector --- p.59 / Chapter 3.2.5.1 --- PCR amplification of the full-length hamster GHR cDNA --- p.59 / Chapter 3.2.5.2 --- Preparation of the hamster GHR cDNA insert for ligation --- p.60 / Chapter 3.2.5.3 --- Linearization of pRc/CMV expression vector --- p.60 / Chapter 3.2.5.4 --- Ligation of the linearized expression vector with the full-length hamster GHR cDNA --- p.61 / Chapter 3.3 --- Results / Chapter 3.3.1 --- PCR Amplification of Hamster GHR cDNA Fragments --- p.61 / Chapter 3.3.1.1 --- RT-PCR --- p.61 / Chapter 3.3.1.2 --- Southern blot analysis --- p.62 / Chapter 3.3.1.3 --- Subcloning and nucleotide sequencing of PCR amplified hamster GHR cDNA fragments --- p.64 / Chapter 3.3.2 --- Screening of an Amplified λZAP Hamster Liver cDNA Library --- p.70 / Chapter 3.3.2.1 --- Preparation of the cDNA probe and phage titering --- p.70 / Chapter 3.3.2.2 --- Screening of the cDNA library --- p.70 / Chapter 3.3.2.3 --- PCR study of the 5' and 3' regions of the DNA insert of the clones selected for secondary screening --- p.72 / Chapter 3.2.3.4 --- Nucleotide sequencing of the full-length hamster GHR cDNA --- p.73 / Chapter 3.2.3.5 --- Tissue distribution of GHR in hamster and the relative expression level of the GHR mRNA in these tissues --- p.73 / Chapter 3.2.3.6 --- Cloning of the full-length hamster GHR cDNA into a mammalian expression vector --- p.79 / Chapter 3.4 --- Discussion / Chapter 3.4.1 --- Cloning of the Full-length hamster GHR cDNA --- p.81 / Chapter 3.4.2 --- Comparison of the Nucleotide and the Predicted Amino Acid Sequences of the Hamster GHR with other Cloned GHRs --- p.82 / Chapter 3.4.3 --- Tissue Distribution of GHR in Hamster and the Relative Expression Level of the GHR mRNA in these Tissues --- p.89 / Chapter 3.4.4 --- Further Studies on Hamster GHR --- p.90 / Chapter Chapter 4 --- Molecular Cloning of Chinese Bullfrog (Rana tigria rigulosa) GHR cDNA from Adult Frog Liver / Chapter 4.1 --- Introduction --- p.92 / Chapter 4.2 --- Experimental Methods / Chapter 4.2.1 --- Animal and Tissues --- p.93 / Chapter 4.2.2 --- Cloning of the Cytoplasmic Domain of Frog GHR cDNA by PCR --- p.93 / Chapter 4.2.2.1 --- RT-PCR --- p.93 / Chapter 4.2.2.2 --- Southern blot analysis of PCR amplified products --- p.95 / Chapter 4.2.2.3 --- Subcloning and sequencing of PCR amplified DNA fragments --- p.95 / Chapter 4.2.2.4 --- Restriction analysis of GHR cDNA fragment between GHR p1 and GHR p2 --- p.95 / Chapter 4.2.2.5 --- PCR cloning of other portions of frog GHR cDNA --- p.96 / Chapter 4.2.2.6 --- Subcloning and sequencing of PCR amplified GHR cDNA fragment using primers other than GHR p1 and GHR p2 --- p.97 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Cloning of the Intracellular Domain of Frog GHR cDNA by RT-PCR --- p.97 / Chapter 4.3.1.1 --- RT-PCR --- p.97 / Chapter 4.3.1.2 --- Southern blot analysis --- p.98 / Chapter 4.3.1.3 --- Subcloning and sequencing of PCR amplified DNA fragments --- p.98 / Chapter 4.3.1.4 --- Restriction enzyme analysis of GHR cDNA fragments --- p.102 / Chapter 4.3.1.5 --- PCR cloning of other portions of frog GHR cDNA --- p.103 / Chapter 4.3.1.6 --- Subcloning and sequencing of PCR products from other portions of frog GHR cDNA --- p.103 / Chapter 4.4 --- Discussion / Chapter 4.4.1 --- Cloning of the Full-length frog GHR cDNA --- p.109 / Chapter 4.4.2 --- Further Studies on Frog GHR --- p.117 / Chapter Chapter 5 --- Attempts on the Molecular Cloning of Teleost GHR cDNA / Chapter 5.1 --- Introduction --- p.119 / Chapter 5.2 --- Experimental Methods / Chapter 5.2.1 --- Animals and Tissues --- p.120 / Chapter 5.2.2 --- PCR Cloning of Teleost GHR cDNA fragments --- p.120 / Chapter 5.2.2.1 --- Design of PCR primers --- p.120 / Chapter 5.2.2.2 --- Preparation of mRNA and synthesis of first strand cDNA --- p.122 / Chapter 5.2.2.3 --- PCR studies on dace and snakehead fish liver first strand cDNA --- p.122 / Chapter 5.2.2.3.1 --- PCR studies on dace liver first strand cDNA --- p.122 / Chapter 5.2.2.3.2 --- PCR studies on snakehead fish liver first strand cDNA --- p.122 / Chapter 5.2.3 --- "Northern Analysis on Dace, Snakehead fish and Eel mRNA" --- p.123 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Molecular Studies on Dace GHR cDNA --- p.123 / Chapter 5.3.1.1 --- PCR studies on dace first strand cDNA --- p.123 / Chapter 5.3.2 --- PCR Studies on Teleost First Strand cDNA --- p.128 / Chapter 5.3.3 --- Northern Analysis on Teleost mRNA --- p.128 / Chapter 5.4 --- Discussion --- p.130 / Chapter 5.4.1 --- PCR Studies on Teleost GHR cDNA --- p.130 / Chapter 5.4.2 --- Northern Analysis on Teleost mRNA --- p.131 / Chapter Chapter 6 --- General Discussion / Chapter 6.1 --- Achievement of this Project --- p.134 / Chapter 6.1.1 --- Hamster GHR --- p.134 / Chapter 6.1.2 --- Frog GHR --- p.135 / Chapter 6.1.3 --- Teleost GHR --- p.136 / Chapter 6.2 --- Postulation on Cloned GHRs at the Molecular Level --- p.136 / Bibliography --- p.141 / Appendices --- p.150

by Simon Chan Siu Hoi. / Spine title varies. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 130-148). / Acknowledgments --- p.i / Table of Contents --- p.ii / List of Abbreviations --- p.ix / List of Figures --- p.xiii / List of Tables --- p.xvi / Page / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Growth Hormone --- p.1 / Chapter 1.2 --- Growth Hormone Receptor --- p.3 / Chapter 1.2.1 --- Cytokine/Hematopoietin Receptor Superfamily --- p.3 / Chapter 1.2.2 --- Tissue Distribution of GHR --- p.6 / Chapter 1.2.3 --- Biosynthesis and Degradation of GHR --- p.7 / Chapter 1.2.4 --- Regulation of GHR Level --- p.8 / Chapter 1.2.5 --- The GHR Protein --- p.10 / Chapter 1.2.6 --- The GHR Gene --- p.15 / Chapter 1.2.7 --- GHR Dimerization --- p.16 / Chapter 1.2.8 --- Mechanism of Signaling by GHR --- p.19 / Chapter 1.2.9 --- GH Binding Protein --- p.21 / Chapter 1.2.10 --- GHR Related Dwarfism --- p.23 / Chapter 1.3 --- Objectives of the Present Investigation --- p.25 / Chapter Chapter 2 --- Materials and Methods --- p.27 / Chapter 2.1 --- Fish Growth Hormone Radioactive Labeling --- p.27 / Chapter 2.1.1 --- Preparation of Iodogen-Coated Tubes --- p.27 / Chapter 2.1.2 --- Packing of the Sephadex G-75 Column --- p.28 / Chapter 2.1.3 --- Iodination of brGH and Purification of the Iodinated brGH --- p.28 / Chapter 2.1.4 --- Determination of the Specific Radioactivity and Percentage of 125I Incorporation --- p.29 / Chapter 2.1.5 --- Reagents and Buffers Used --- p.30 / Chapter 2.2 --- Integrity of 125I-brGH --- p.30 / Chapter 2.2.1 --- HPLC of brGH --- p.31 / Chapter 2.2.2 --- HPLC of 125I-brGH after Iodination --- p.31 / Chapter 2.2.3 --- HPLC of 125I-brGH after Receptor Binding --- p.31 / Chapter 2.3 --- Preparation of Membranes from Fish Tissues --- p.32 / Chapter 2.3.1 --- Preparation of Snakehead Fish Liver Membranes --- p.32 / Chapter 2.3.2 --- Reagents and Buffers Used --- p.33 / Chapter 2.4 --- Protein Determination of Membrane Preparations --- p.34 / Chapter 2.4.1 --- The BCA Protein Reaction Scheme --- p.34 / Chapter 2.4.2 --- BCA Protein Determination Protocol --- p.34 / Chapter 2.5 --- Receptor Binding Studies --- p.35 / Chapter 2.5.1 --- Association and Dissociation Studies --- p.36 / Chapter 2.5.2 --- pH Dependence Study --- p.36 / Chapter 2.5.3 --- Membrane Protein Dependence Study --- p.37 / Chapter 2.5.4 --- Ca2+ Dependence Study --- p.37 / Chapter 2.5.5 --- Tissue Distribution Study --- p.37 / Chapter 2.5.6 --- Displacement and Specificity Studies --- p.38 / Chapter 2.5.7 --- Dithiothreitol (DTT) Dependence Study --- p.39 / Chapter 2.5.8 --- p-Chloromercuribenzene Sulfonate (PCMBS) Pretreatment: Dose Dependence Study --- p.39 / Chapter 2.5.9 --- Scatchard Analysis of the PCMBS Pretreated and Control Snakehead Fish Liver Membranes --- p.40 / Chapter 2.5.10 --- Reversibility of the PCMBS Effect --- p.40 / Chapter 2.5.11 --- Reagents and Buffers Used --- p.41 / Chapter 2.6 --- Crosslinking Studies --- p.41 / Chapter 2.6.1 --- Crosslinking Performed on Snakehead Fish Liver Membranes --- p.41 / Chapter 2.6.2 --- Crosslinking Performed on Solubilized Snakehead Fish Liver Membranes --- p.42 / Chapter 2.6.3 --- Gel Filtration Chromatography of the Crosslinked Comp)lexes --- p.43 / Chapter 2.6.4 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of the Crosslinked Complexes --- p.43 / Chapter 2.6.5 --- Reagents and Buffers Used --- p.45 / Chapter 2.7 --- Western Blot Analysis of Snakehead Fish Liver GHR --- p.46 / Chapter 2.7.1 --- SDS-PAGE of Snakehead Fish Liver Membranes --- p.46 / Chapter 2.7.2 --- Transfer of Proteins onto Polyvinylidene Fluoride (PVDF) Membrane --- p.46 / Chapter 2.7.3 --- Antibody Development of PVDF Membrane --- p.47 / Chapter 2.7.4 --- Reagents and Buffers Used --- p.48 / Chapter 2.8 --- Solubilization of Snakehead Fish Liver Membranes and Solubilized Receptor Binding Studies --- p.48 / Chapter 2.8.1 --- Solubilization of Snakehead Fish Liver Membranes --- p.49 / Chapter 2.8.2 --- Solubilized Receptor Binding Assay --- p.49 / Chapter 2.8.3 --- "Solubilization of Snakehead Fish Liver Membranes: Detergent Concentration, pH, Temperature and Time Dependence" --- p.50 / Chapter 2.8.4 --- Solubilized Receptor Binding Study: Interference of Detergent --- p.50 / Chapter 2.8.5 --- Reagents and Buffers Used --- p.51 / Chapter 2.9 --- Purification of Snakehead Fish Liver GHR by Affinity Chromatography --- p.51 / Chapter 2.9.1 --- Affinity Column Preparation --- p.52 / Chapter 2.9.2 --- Snakehead Fish Liver GHR Purification --- p.52 / Chapter 2.9.3 --- Reagents and Buffers Used --- p.53 / Chapter Chapter 3 --- Results: fGH Labeling and Integrity Determination --- p.54 / Chapter 3.1 --- Introduction --- p.54 / Chapter 3.2 --- Experimental Results --- p.55 / Chapter 3.2.1 --- Iodination of fGH --- p.55 / Chapter 3.2.2 --- Integrity of 125I-fGH --- p.55 / Chapter 3.3 --- Discussion --- p.61 / Chapter Chapter 4 --- Results: Membrane Receptor Binding Studies --- p.62 / Chapter 4.1 --- Introduction --- p.62 / Chapter 4.2 --- Experimental Results --- p.63 / Chapter 4.2.1 --- Optimal Conditions for Snakehead Fish Liver Membrane GHR Binding --- p.64 / Chapter 4.2.1.1 --- Association and Dissociation Studies --- p.64 / Chapter 4.2.1.2 --- pH Dependence Study --- p.67 / Chapter 4.2.1.3 --- Membrane Protein Dependence Study --- p.70 / Chapter 4.2.1.4 --- Ca2+ Dependence Study --- p.73 / Chapter 4.2.2 --- Localization and Specificity of Snakehead Fish GHR --- p.76 / Chapter 4.2.2.1 --- Tissue Distribution Study --- p.76 / Chapter 4.2.2.2 --- Displacement and Specificity Studies --- p.78 / Chapter 4.2.3 --- Effects of Sulfhydryl Group Reducing and Oxidizing Agents on GHR Binding --- p.81 / Chapter 4.2.3.1 --- Effect of DTT: Concentration Dependence Study --- p.81 / Chapter 4.2.3.2 --- Effect of PCMBS: Concentration Dependence Study --- p.84 / Chapter 4.2.3.3 --- Scatchard Analysis of Control and PCMBS- pretreated Membranes --- p.86 / Chapter 4.2.3.4 --- Reversibility of the PCMBS Effect --- p.88 / Chapter 4.3 --- Discussion --- p.90 / Chapter 4.3.1 --- Optimal Conditions for Snakehead Fish Liver Membrane GHR Binding --- p.90 / Chapter 4.3.2 --- Localization and Specificity of Snakehead Fish GHR --- p.93 / Chapter 4.3.3 --- Effects of Sulfhydryl Group Reducing and Oxidizing Agents on GHR Binding --- p.96 / Chapter Chapter 5 --- Results: Crosslinking and Western Blot Analysis --- p.101 / Chapter 5.1 --- Introduction --- p.101 / Chapter 5.1.1 --- Crosslinking Studies --- p.101 / Chapter 5.1.2 --- Western Blot Analysis --- p.103 / Chapter 5.2 --- Experimental Results --- p.104 / Chapter 5.2.1 --- Crosslinking Studies --- p.104 / Chapter 5.2.2 --- Western Blot Analysis --- p.105 / Chapter 5.3 --- Discussion --- p.112 / Chapter Chapter 6 --- Results: Affinity Purification of Snakehead Fish Liver GHR --- p.115 / Chapter 6.1 --- Introduction --- p.115 / Chapter 6.1.1 --- Membrane Solubilization and Solubilized GHR Binding Studies --- p.115 / Chapter 6.1.2 --- Affinity Purification of Solubilized Snakehead Fish Liver GHR --- p.116 / Chapter 6.2 --- Exp erimental Results --- p.117 / Chapter 6.2.1 --- Solubilization of Snakehead Fish Liver Membranes --- p.117 / Chapter 6.2.2 --- Interference of Detergents in the Solubilized Receptor Binding Assay --- p.118 / Chapter 6.2.3 --- Affinity Purification of Solubilized Snakehead Fish Liver GHR --- p.120 / Chapter 6.3 --- Discussion --- p.122 / Chapter Chapter 7 --- General Discussion --- p.125 / References --- p.130

Chicken genomic DNA containing 5 kb of the 5$ sp prime$ end of the growth hormone (GH) receptor gene and 12 kb of the region up-stream was cloned and a restriction map was constructed. Using subcloned fragments as probes, a HindIII polymorphism was detected in both egg layer and in meat-type chickens. This polymorphic site was mapped at 7 kb up-stream of the coding region of the GH-receptor gene and a PCR assay for the polymorphism was developed to facilitate genotyping of large numbers of chickens. / Alleles of the GH-receptor gene and the GH gene were analyzed for association with traits in chicken strains of different genetic origins. In egg layers, association was significant for juvenile body weight, egg weight, feed consumption and feed efficiency for egg mass (P $<$ 0.05). In meat-type chickens, the GH-receptor allele associated with high juvenile body weight in egg layers was co-selected with leanness. A comparison of the genotype classes revealed that for several traits there was significant interaction between the GH and GH-receptor genotype. The results indicated that there are variants of the genes of the GH-axis which affect traits in White Leghorns and that the effect of a genetic variation in one gene may depend on the variation in another gene.

No description available.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

生長滯後是包括炎症性腸病在內的炎症疾病引起的併發症。實驗表明，炎症使肝臟對生長激素(GH)的作用變得不敏感或引起生長激素抵抗。生長激素抵抗會引起胰島素生長因子-1 (IGF-I)的表達下降，並且會啟動一系列的代謝反應。多年來的研究證明炎症因子白介素-6 (IL-6)，腫瘤壞死因子 -α (TNF-α)和白介素-1β(IL-1β)參與肝臟生長激素抵抗的病理過程。然而這些炎症因子調控生長激素通路的具體機理尚不清楚。通過用人肝癌細胞系Huh-7和慢性炎症及急性炎症兩種老鼠模型，我們發現: 1) TNF-α和IL-1β抑制生長激素受體(GHR)的表達; 2) IL-6誘導細胞因子信號轉導抑制因子-3 (SOCS3)的高表達; 3) IL-6-SOCS3途徑對GH-IGF-I信號通路的抑制作用依賴于GHR的表達量，當TNF-α及IL-1β升高而使GHR的表達量下降後，IL-6就不再對GH-IGF-I信號通路有抑制作用。以上結果表明IL-6， TNF-α和IL-1β抑制肝臟生長激素信號通路的機制是不一樣的，這些結果或許對臨床上治療青少年中炎症引起的生長激素抵抗疾病有一定的指導意義。 / 成纖維細胞生長因子(FGF) 通過結合和啟動成纖維細胞生長因子受體(FGFR)而參與許多生理過程。FGF19屬於FGF15/19亞家族，這個亞家族還包括FGF21和FGF23。FGF19調節肝臟中膽汁酸的穩態及蛋白和糖原的合成。FGF19通過與FGFR4及共受體β-klotho結合來啟動信號通路。研究表明，TNF-α通過抑制共受體β-klotho的表達來抑制脂肪細胞中的FGF21信號通路。然而IL-6，TNF-α和IL-1β在調節肝臟FGF19信號通路中的作用尚不清楚。我們的體外細胞和體內動物實驗結果表明，IL-1β通過JNK和NF-κB通路抑制肝臟中β-klotho的表達。IL-6與TNF-α不調節Huh-7細胞中β-klotho的表達。 / 綜上所述，IL-6，TNF-α及IL-1β在肝臟生長激素及FGF19通路中起不同的調節作用。 / Growth failure is a major complication of inflammatory diseases including inflammatory bowel disease. Evidence suggests that during inflammation, the liver becomes resistant to growth hormone (GH) actions, leading to downregulation of the anabolic gene IGF-I and the activation of catabolic processes. Decades of studies demonstrated that pro-inflammatory cytokines IL-6, TNF-α and IL-1β are involved in the pathogenesis of hepatic GH resistance. However, the exact mechanisms used by these individual cytokines to regulate GH signaling are not defined. Using Huh-7 human hepatoma cells and mouse models of chronic and acute inflammation, we show that TNF-α and IL-1β but not IL-6 inhibited hepatic GH receptor (GHR) expression, and that IL-6 but not TNF-α and IL-1β stimulated expression of suppressor of cytokine signaling-3 (SOCS3). TNF-α/IL-1β and IL-6 acted primarily at GHR and SOCS3 respectively to inhibit the GH-IGF-I pathway. While TNF-α/IL-1β exerted a tonic inhibition on hepatic GH signaling, IL-6 activity is dependent on the active GH pathway. IL-6 lost its inhibition on the GH-IGF-I pathway when GHR expression was blocked as the inflammation progressed. These results reveal previously undefined distinct mechanisms used by TNF-α/IL-1β and IL-6 to inhibit the hepatic GH pathway. Our results may provide a new guidance for clinical practice in treating pediatric infammation-induced GH resistance. / Fibroblast growth factors (FGFs) play critical roles in many physiological processes by binding to and activating FGF receptor (FGFR) family. FGF19 belongs to FGF15/19 subfamily of FGFs that includes FGF15/19, FGF21 and FGF23. FGF19 has been shown to regulate bile acid homeostasis, and protein and glycogen synthesis in the liver. FGF19 binds FGFR4 and the co-receptor β-klotho to initiate signaling. Studies have shown that proinflammatory cytokines such as TNF-α can impair FGF21 signaling in adipose cells by repressing the expression of β-klotho. However, little is known about the effects of IL-6, TNF-α and IL-1β on regulating hepatic FGF19 signaling. In the present study, we found that IL-1β inhibited β-klotho expression both in vitro and in vivo, and this inhibition required JNK and NF-κB pathways. IL-6 and TNF-α did not inhibit β-klotho expression in Huh-7 cells. / Taken together, our results demonstrate that IL-6, TNF-α and IL-1β play different roles in regulating the GH and FGF-19 pathways in the liver. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhao, Yueshui. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 147-182). / Abstracts also in Chinese.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed on June 8, 2009). Includes bibliographical references.