by Lee Sau Yat. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 89-94). / Acknowledgements --- p.I / Abbreviation --- p.II / Abstract --- p.III / Table of content --- p.IV / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Historical background in the studies of LDL and LDLR --- p.1 / Chapter 1.2 --- Homeostasis of Cholesterol in Man --- p.4 / Chapter 1.2.1 --- Origin and catabolism of low density lipoprotein --- p.4 / Chapter 1.2.2 --- The LDL receptor --- p.6 / Chapter 1.2.3 --- LDL pathway --- p.7 / Chapter 1.2.4 --- Feedback regulatory action of LDL receptor --- p.10 / Chapter 1.3 --- Gene structure of LDL receptor promoter --- p.11 / Chapter 1.3.1 --- The LDL receptor promoter --- p.11 / Chapter 1.3.2 --- The responsive element in LDL receptor promoter --- p.13 / Chapter 1.4 --- Eukaryotic transcription factor --- p.15 / Chapter 1.5 --- Role of Gel-shifted assay in studying DNA binding protein --- p.17 / Chapter 1.6 --- Objective of the present thesis --- p.20 / Chapter Chapter 2 --- Materials and Methods --- p.21 / Chapter 2.1 --- Oligonucleotide synthesis and purification --- p.21 / Chapter 2.1.1 --- Primer construction --- p.21 / Chapter 2.1.2 --- Purification of oligonucleotides --- p.22 / Chapter 2.2 --- Recombinant plasmid construction --- p.24 / Chapter 2.2.1 --- Preparation of competent cell --- p.24 / Chapter 2.2.2 --- Preparation of phage DNA --- p.24 / Chapter 2.2.3 --- Amplification and purification of LDLR-promoter by PCR techniques --- p.26 / Chapter 2.2.3.1 --- Amplification and restriction site construction of LDLR- promoter --- p.26 / Chapter 2.2.3.2 --- Purification of the PCR product --- p.27 / Chapter 2.2.4 --- Preparation of plasmid pGCAT-A --- p.27 / Chapter 2.2.5 --- Recombinant plasmid pLDLRP-GCAT- A construction --- p.29 / Chapter 2.2.6 --- Transformation of DNA to competent cell --- p.29 / Chapter 2.2.7 --- Screening of positive clone pLDLRP- GCAT-A --- p.30 / Chapter 2.3 --- DNA sequencing --- p.31 / Chapter 2.3.1 --- Denaturing the double strand template --- p.31 / Chapter 2.3.2 --- Annealing reaction --- p.31 / Chapter 2.3.3 --- Labeling reaction --- p.32 / Chapter 2.3.4 --- Termination reaction --- p.32 / Chapter 2.3.5 --- Running and fixing the gel --- p.32 / Chapter 2.4 --- Cell culture and passage of different cell lines --- p.34 / Chapter 2.4.1 --- "Routine subculture of HepG 2, CHO, FSF, Cos-7 and FSF" --- p.34 / Chapter 2.4.2 --- "BMN, RTGH-1" --- p.34 / Chapter 2.5 --- Preparation of human LDL and LPDS --- p.36 / Chapter 2.5.1 --- Purification of LDL --- p.36 / Chapter 2.5.2 --- Purification of LPDS --- p.37 / Chapter 2.6 --- DNA transfection and CAT assay --- p.38 / Chapter 2.6.1 --- Transfection of recombinant plasmid to eukaryotic cells --- p.38 / Chapter 2.6.2 --- CAT assay --- p.39 / Chapter 2.7 --- 125I-LDL binding assay --- p.41 / Chapter 2.7.1 --- Radioactive iodination of LDL --- p.41 / Chapter 2.7.2 --- Purification of iodinated LDL --- p.41 / Chapter 2.7.3 --- Down regulation of LDL receptorin different cell lines --- p.41 / Chapter 2.7.4 --- Different Drugs treatment in HepG2 Cell line --- p.42 / Chapter 2.7.5 --- 125I-LDL binding assay --- p.43 / Chapter 2.8 --- Gel-shift mobility assay --- p.44 / Chapter 2.8.1 --- Extraction of crude nuclear extracts --- p.44 / Chapter 2.8.2 --- 5'end-labeling of synthetic oligonucleotides --- p.45 / Chapter 2.8.3 --- Purification of labeled oligonucleotides --- p.45 / Chapter 2.8.4 --- Nuclear protein and DNA binding reaction --- p.46 / Chapter 2.8.5 --- Gel-shift mobility electrophoresis by PhastSystem --- p.46 / Chapter 2.9 --- Construction of λ gt 11 cDNA library of HepG 2cell --- p.48 / Chapter 2.9.1 --- Purification of mRNA from HepG 2 --- p.48 / Chapter 2.9.2 --- cDNA preparation --- p.48 / Chapter 2.9.3 --- In-vitro packaging of phage --- p.48 / Chapter 2.9.3 --- Screening the expression library --- p.49 / Chapter Chapter 3 --- Results --- p.50 / Chapter 3.1 --- Construction of recombinant plasmid --- p.50 / Chapter 3.1.1 --- hLDLR-promoter λ 34 clone --- p.50 / Chapter 3.1.2 --- Restriction site generation in LDLR- promoter by PCR --- p.52 / Chapter 3.1.3 --- Preparation of pGCAT-A reporter plasmid --- p.55 / Chapter 3.1.4 --- Screening of pGCAT-A-LDLRP recombinant --- p.55 / Chapter 3.1.5 --- Sequencing of pGCAT-A-LDLR- promoter recombinant --- p.57 / Chapter 3.2 --- CAT assay of recombinant plasmid on transfected HepG 2 cell --- p.57 / Chapter 3.3 --- 125I-LDL binding assay --- p.57 / Chapter 3.3.1 --- 125 I - LDL binding assay of different cell lines --- p.57 / Chapter 3.3.2 --- Characterization of cell surface receptor of HepG 2 cell by different drugs treatment --- p.61 / Chapter 3.4 --- Gel shift mobility assay --- p.63 / Chapter 3.4.1 --- Binding effect of Repeat 2 to different cell lines --- p.63 / Chapter 3.4.2 --- Optimizing the binding reactionin HepG 2 cell by poly(dI.dC) --- p.63 / Chapter 3.4.3 --- Specificity of Repeat 2 in binding to HepG 2 cell nuclear protein --- p.66 / Chapter 3.4.4 --- LDL dose response treatment in HepG2 cell --- p.68 / Chapter 3.4.4.1 --- Binding of Repeat 2 to specific nuclear protein --- p.68 / Chapter 3.4.4.2 --- Binding of Repeat 2 to a non- specific cell nuclear protein from cells treated with LDL --- p.68 / Chapter 3.4.5 --- Effect of different drugs on the binding of Repeat 2 to nuclear proteins in HepG 2 cell --- p.71 / Chapter 3.5 --- HepG 2 cell cDNA library screening --- p.73 / Chapter Chapter 4 --- Discussion --- p.77 / Chapter 4.1 --- Strategy on construction of reporter plasmid pLDLRP-GC AT-A --- p.77 / Chapter 4.2 --- The expression of CAT in HepG 2 cell --- p.78 / Chapter 4.3 --- Identification of a DNA binding protein for Repeat 2 in HepG 2 cell --- p.80 / Chapter 4.3.1 --- Binding effect of nuclear protein to Repeat 2 --- p.82 / Chapter 4.3.1.1 --- LDL dose response relationships --- p.82 / Chapter 4.3.1.2 --- Effect of protein inhibitors --- p.83 / Chapter 4.3.1.3 --- Effect of Shanzha --- p.86 / Chapter 4.3.2 --- Screening of the cDNA library of HepG 2 cells --- p.86 / Chapter Chapter 5 --- Further Studies --- p.88 / References --- p.89 / Appendix --- p.95

by Zhen Yang. / On t.p. "1" is subscript following A in the title. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 138-139). / Acknowledgements --- p.i / List of Nomenclature --- p.ii-ix / "Abstracts (for Chapter 2,Chapter 3,and Chapter 4)" --- p.x / Chapter Chapter 1. --- General Introduction --- p.1-8 / Chapter Chapter 2. --- Synthesis of 5-(3-Hydroxypropyl)-7-methoxy-2-(3'-methoxy-4'- hydroxyphenyl)-benzo[b]furan-3-carbaldehyde (XH14) (18) / Chapter 2.1. --- Introduction --- p.9-12 / Chapter 2.2. --- Isolation and Structure Elucidation of XH14 (18) / Chapter 2.2.1. --- Material and method --- p.13-17 / Chapter 2.2.2. --- Structure elucidation --- p.18-23 / Chapter 2.3. --- Results and Discussion / Chapter 2.3.1. --- Total synthesis of XH14 (18) --- p.24-52 / Chapter 2.3.2. --- Chemical modification of XH14 (18) / Chapter (a) --- Synthesis of 3-hydroxymethyl-5-(3-hydroxypropyl)- 7-methoxy-2-(3'-methoxy-4'-hydroxy phenyl)-benzo[b] furan(49) and 3-hydroxymethyl-5-(2-methoxycarbonyl ethyl)-7-methoxy-2-(3'-methoxy-4'-hydroxy phenyl)- benzo[b]furan (66) --- p.53-54 / Chapter (b) --- Synthesis of 5-(2-carboxyethyl)-7-methoxy-2-(3'- methoxy-4'-hydroxyphenyl)-benzo[b]furan-3- carbaldehyde (67) --- p.54 / Chapter (c) --- Synthesis of 5-(3-Hydroxypropyl)-7-methoxy-3- methyl-2-(3'-methoxy-4'-hydroxyphenyl)- benzo[b]furan (68) and 5-(2-methoxycarbonylethyl)-7- methoxy-3-methyl-2-(3'-methoxy-4'-hydroxyphenyl)- benzo[b]furan (69) --- p.54-55 / Chapter (d) --- Synthesis of 5-(2-carboxy-trans-ethenyl)-7- methoxy-2-(3'-methoxy-4'- hydroxy phenyl) benzo[b]furan-3-carbaldehyde (70) --- p.55-56 / Chapter (e) --- Synthesis of 4-bromo-5-(3-hydroxypropyI)-7- methoxy-2-(3'-methoxy-4'- hydroxy phenyl)- benzo[b]furan-3-carbaldehyde (72) --- p.56-57 / Chapter (f) --- Synthesis of 4-acetyl-5-(3-hydroxypropyl)-7- methoxy-2-(3'-methoxy-4'-hydroxyphenyl)-benzo [b]furan (73) and 3-acetyI-5-(3-hydroxypropyl)-7- methoxy-2-(3'-methoxy-4'-hydroxy phenyl)- benzo[b]furan (75) --- p.57-61 / Chapter (g) --- Synthesis of 3-nitro-5-(3-hydroxypropyl)-7- methoxy -2-(3'-methoxy-4'-hydroxyphenyl)- benzo[b]furan (77) and 4-nitro-5-(3-hydroxyphenyl)-7- methoxy-2-(3'-methoxy-4'-hydroxyphenyl)- benzo[b]furan (78) --- p.61-64 / Chapter (h) --- Synthesis of 3-(α-hydroxyethyl)-5-(3-hydroxy propyl) -7-methoxy-2- (3'-methoxy-4' -hydroxyphenyl) - benzo[b]furan (81) --- p.64 / Chapter (i) --- Synthesis of 5-(3-hydroxypropyl)-7-methoxy-2-(3'- methoxy-4'-benzyloxyphenyl )-benzo[b]furan-4- carbaldehyde (82) --- p.64 / Chapter 2.4. --- Structure-activity Relationship of A1 Antagonists --- p.65-71 / Chapter 2.5. --- Conclusion --- p.72 / Chapter 2.6. --- Experimental Section --- p.73-97 / Chapter 2.7. --- References --- p.98-103 / Chapter Chapter 3. --- "Synthesis of 9,10-Dihydro-10,10-dimethoxyanthracen-9-one" / Chapter 3.1. --- Introduction --- p.104 / Chapter 3.2 --- Results and Discussion --- p.105-118 / Chapter 3.3. --- Experimental Section --- p.119-124 / Chapter 3.4. --- References --- p.125-128 / Chapter Chapter 4. --- "Synthesis of 10,11-Dimethoxydibenz[b,f]oxep in" / Chapter 3.1. --- Introduction --- p.129 / Chapter 3.2. --- Results and Discussion --- p.130-133 / Chapter 3.3. --- Experimental Section --- p.134-137 / Chapter 3.4. --- References --- p.138-139 / Appendix / Spectra --- p.140-145

by Poon Chuen Wai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 161-176). / Abstract --- p.i / Acknowledgements --- p.vi / Abbreviations --- p.viii / Introduction --- p.1 / Review of the literature --- p.2 / Incidence and historical background --- p.3 / Theories on etiology of emesis/hyperemesis gavidarum --- p.5 / Mechanism of vomiting --- p.7 / Temporal association between hCG and emesis/hyperemesis gravidarum --- p.9 / Thyroid function and emesis/hyperemesis gravidarum --- p.13 / Steroid hormone and emesis/hyperemesis gravidarum --- p.15 / Hyperemesis caused by corpus luteum in right ovary --- p.15 / "Formulation of a new hypothesis for emesis/hyperemesis gravidarum : “hCG stimulated electrolyte and fluid secretion in the UGIT, leading to pregnancy associated vomiting 226}0ح" --- p.16 / Is hCG a humoural secretory agent ？ --- p.18 / Can hCG stimulate the secretion of fluid and electrolytes from the GIT ？ --- p.18 / Are LH-CGR present in the GIT ？ --- p.20 / Can electrolyte and fluid secretion in the UGIT lead to vomiting during pregnancy ？ --- p.24 / HCG and regulation of electrolyte and fluid movement --- p.24 / Gut- gonad axis --- p.25 / Objectives --- p.27 / Section 1 hCG and in vitro secretion of anions from cells with functional LH-CGR - A cell model --- p.28 / Introduction --- p.28 / Materials and Methods / Materials --- p.30 / Methods / Chapter 1. --- Test for the presence of LH-CGR in human gastrointestinal cell lines --- p.31 / Chapter 2. --- Establishment of cells expressing functional receptor for hCG --- p.35 / Chapter 3. --- Effect of hCG on [125I] iodide efflux from COS-1 cells transiently expressing MC1+2 LH-CG/TSH chimera receptor --- p.38 / Chapter 4. --- Anion secretion from MLTC-1 cells with wild type LH-CGR --- p.38 / Chapter 5. --- Statistics --- p.41 / Results --- p.42 / Discussion --- p.55 / Section 2 Presence of LH-CGR in the upper gastrointestinal tract of rat --- p.61 / Introduction --- p.61 / Materials and Methods / Materials --- p.62 / Methods / Chapter 1. --- Screening for hCG binding sites along the rat GIT --- p.63 / Chapter 2. --- Detection of Triton X-100 soluble plasma membrane hCG binding protein along the rat GIT --- p.64 / Chapter 3. --- Measurement of affinity of the hCG binding proteins by Scatchard analysis --- p.66 / Chapter 4. --- Determination of the isoelectric points for the duodenal hCG binding protein and ovarian LH-CGR --- p.67 / Chapter 5. --- Developement of antibodies to LH-CGR --- p.69 / Chapter 6. --- Western blotting to determine the molecular weight of the LH-CGR --- p.78 / Chapter 7. --- Immunohistochemical localization of LH-CGR in the UGIT --- p.80 / Chapter 8. --- Statistics --- p.81 / Results --- p.82 / Discussion --- p.109 / Section 3 hCG and in vivo secretion of electrolytes from pancreas / duodenal region in rats --- p.119 / Introduction --- p.119 / Materials and Methods / Materials --- p.121 / Methods --- p.121 / Chapter 1. --- The effect of hCG on the secretion of electrolytes into bile and pancreatic/duodenal effluent --- p.121 / Chapter 2. --- Effect of ovariectomy on pancreatic content and electrolyte levels in blood --- p.125 / Chapter 3. --- Statistics --- p.128 / Results --- p.129 / Discussion --- p.151 / General discussion --- p.155 / Reference --- p.161 / Original data --- p.176

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No description available.

催乳素是一種負責廣鹽性硬骨魚低滲環境適應的重要激素。最近發現的催乳素釋放肽被報導在魚類生理活動中參與了刺激催乳素釋放和滲透調節過程。本研究以黃錫鯛為實驗動物，旨在詮釋催乳素和催乳素釋放肽在廣鹽性硬骨魚中的滲透調節作用和調控機制。 / 實驗第一部份將黃錫鯛催乳素在大腸桿菌中過量表達並用鎳離子親和層析法將之純化。此重組黃錫鯛催乳素可被抗黃錫鯛催乳素抗體特異識別。將此重組催乳素注射入活體紅箭魚可導致其血漿鈉離子濃度和滲透壓顯著提高，從而證明了此催乳素的生物活性。在第二部份的研究中，黃錫鯛催乳素受體和催乳素釋放肽受體的全長編碼基因被克隆和分析。黃錫鯛催乳素受體和硬骨魚二型催乳素受體蛋白質相似性較高（可達47%至94%），但同魚類一型催乳素受體及其他高等脊椎動物催乳素受體同源性較低，相似性只有22%至37%。此催乳素受體廣泛表達在各種組織器官中，包括腦組織、腮、心臟、腸、腎、肌肉及性腺。黃錫鯛催乳素釋放肽受體屬於G-蛋白偶聯受體家族，它具備該家族成員的各種保守序列特徵。並且它含有同其他催乳素釋放肽受體一樣的PDZ域結合位點。組織表達分佈結果顯示此受體在中樞神經系統各部份及腮、心臟、肝臟和性腺中有表達。本研究最後一部份以黃錫鯛原代培養腮細胞為實驗對象，分別探究了腮細胞對於不同滲透壓力、催乳素和催乳素釋放肽處理的反應。在這些實驗中，我們檢測了腮細胞的死亡及凋亡狀況，并用實時定量PCR分析了一些滲透調節相關基因的表達狀況。另外，我們還採用了蛋白免疫印跡法檢測了腮細胞處於不同滲透壓條件下，其熱休克蛋白70的蛋白質豐度變化。我們的結果顯示：低滲壓力會引起腮細胞死亡包括誘導凋亡。催乳素和催乳素釋放肽處理均可以顯著降低低滲壓力所誘導的細胞死亡，並且催乳素還可以阻止一部份細胞進行凋亡。腮細胞中鈉鉀泵兩個亞基、熱休克同族蛋白70及催乳素受體的基因表達均被催乳素和催乳素釋放肽處理上調。但是水通道蛋白3的基因表達并未出現明顯的變化。當腮細胞暴露于高滲透壓力環境時，水通道蛋白3、囊性纤维化跨膜转运调节因子、鈉鉀泵兩個亞基和熱休克同族蛋白70的基因表達都明顯上調。與之相反，這些基因的表達在處於低滲環境的腮細胞中均被下調。 / 本研究提供了許多關於催乳素和催乳素釋放肽在魚類腮滲透調節過程中的詳細作用，并且首次報導了催乳素釋放肽可以直接作用于魚類滲透調節組織發揮功能從而參與滲透調節過程，而不只是如以往被認為的通過旁分泌或者自分泌方式刺激催乳素的表達來發揮生理功能。本研究的探索和發現使我們更加深刻地理解催乳素和催乳素釋放肽在廣鹽性硬骨魚鹽度適應過程中的調控機制。 / Prolactin (PRL) is well known as a crucial hormone responsible for fresh (hypoosmotic) water acclimation in euryhaline teleosts. The recently discovered prolactin-releasing peptide (PrRP) has been reported to take part in stimulating PRL release and affecting osmoregulatory processes in fish. This study aims to investigate the osmoregulatory effects and regulatory mechanisms of PRL and PrRP in a euryhaline teleost, silver sea bream (Sparus sarba). / First, silver sea bream prolactin (ssPRL) was over-expressed in E. coli by IPTG induction and purified by Ni-based immobilized metal ion affinity chromatography. This recombinant silver sea bream prolactin (rssPRL) was recognized by specific antibodies against ssPRL. Subsequently, its bioactivity was confirmed by in vivo injection to swordtails, which resulted in significant increase in plasma Na⁺ level and osmolality. In the second part, full-length cDNAs of silver sea bream PRL receptor (ssPRLR) and PrRP receptor (ssPrRPR) were cloned and characterized. ssPRLR shares high amino acid identities (47% to 94%) with teleost PRLR2s but low identities (22% to 37%) with piscine PRLR1s and higher vertebrate PRLRs. It is widely distributed in brain, gill, heart, gut, kidney, muscle and gonad. ssPrRPR belongs to G protein-coupled receptor (GPCR) family with all the conserved features of GPCRs, and possesses the special PDZ domain-binding motifs of other PrRPRs. Its expression was detected in the central nervous system, gill, heart, liver and gonad. Lastly, a primary gill cell culture of silver sea bream was developed and used as a tool for studying responses of gill cells following exposure to media of different osmotic stress, rssPRL and ssPrRP, respectively. Then, cytotoxicity and apoptosis assays were performed, and effects of these treatments on expression profiles of osmoregulatory genes were analyzed by real time PCR. Influence of osmotic stresses on protein abundance of heat shock protein 70s was examined by Western blot. The results showed that hypoosmotic challenge could induce cell death including apoptosis. Both PRL and PrRP treatment markedly decreased the induced cell death, and PRL treatment prevented some gill cells from apoptosis. Expression levels of Na⁺-K⁺-ATPase alpha (NKA-α) and beta (NKA-β) subunits, heat shock cognate 70 (HSC70) and PRLR were up-regulated in PRL- and PrRP- treated gill cells, however no significant effect on expression of aquaporin3 (AQP3) mRNA was apparent. After hyperosmotic exposure, expression levels of AQP3, cystic fibrosis transmembrane conductance regulator (CFTR), NKA-α, NKA-β, and HSC70 were significantly increased. In contrast, hypoosmotic exposure considerably down-regulated expression levels of these genes. / The present study represented considerable addition of details about the precise actions of PRL and PrRP in branchial osmoregulation. Furthermore, it first reported that PrRP can exert direct effects on fish osmoregulatory epithelia, instead of its oft presumed autocrine or paracrine action. All these efforts provide new insights into the control mechanisms of PRL and PrRP in euryhaline teleost during salinity acclimation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Qu, Zhe. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 108-142). / Abstracts also Chinese. / Title page --- p.i / Thesis committee --- p.ii / Acknowledgement --- p.iii / Abstract --- p.v / Abstract-Chinese version --- p.viii / Table of contents --- p.x / List of figures --- p.xiv / List of abbreviations --- p.xvi / Chapter Chapter 1 --- General introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.6 / Chapter 2.1 --- The role of gill in teleost osmoregulation --- p.7 / Chapter 2.1.1 --- An overview of fish osmoregulation --- p.7 / Chapter 2.1.2 --- The gill structure and epithelial ionocytes --- p.8 / Chapter 2.1.3 --- Osmoregulatory molecules in gill cells --- p.10 / Chapter 2.2 --- Piscine prolactin --- p.16 / Chapter 2.2.1 --- General features and distribution --- p.16 / Chapter 2.2.2 --- Prolactin receptor --- p.17 / Chapter 2.2.3 --- Prolactin and teleost osmoregulation --- p.18 / Chapter 2.3 --- Prolactin-releasing peptide in teleost --- p.21 / Chapter 2.3.1 --- Discovery and characteristics --- p.21 / Chapter 2.3.2 --- Distribution and its receptor --- p.22 / Chapter 2.3.3 --- Functions of PrRP --- p.23 / Chapter A. --- PrRP and prolactin release --- p.23 / Chapter B. --- PrRP and neuroendocrine stress response --- p.24 / Chapter C. --- Effects on food intake and energy metabolism --- p.26 / Chapter D. --- PrRP and cardiovascular system --- p.27 / Chapter Chapter 3 --- Production and characterization of recombinant silver sea bream (Sparus sarba) prolactin --- p.29 / Chapter Abstract --- p.30 / Chapter 3.1 --- Introduction --- p.31 / Chapter 3.2 --- Materials and methods --- p.33 / Chapter 3.2.1 --- Experimental animals --- p.33 / Chapter 3.2.2 --- Construction of recombinant silver sea bream prolactin expression vector --- p.33 / Chapter 3.2.3 --- Large-scale production and purification of rssPRL --- p.34 / Chapter 3.2.4 --- SDS polyacrylamide gel electrophoresis and Western blotting analysis of rssPRL --- p.35 / Chapter 3.2.5 --- In vivo bioassay of rssPRL --- p.36 / Chapter 3.2.6 --- Statistical analysis --- p.37 / Chapter 3.3 --- Results --- p.38 / Chapter 3.3.1 --- Overexpression and purification of rssPRL --- p.38 / Chapter 3.3.2 --- Biological activity assay of rssPRL in vivo --- p.38 / Chapter 3.4 --- Discussion --- p.43 / Chapter Chapter 4 --- Molecular cloning and characterization of prolactin receptor (PRLR) and prolactin-releasing peptide receptor (PrRPR) in silver sea bream (Sparus sarba) --- p.46 / Chapter Abstract --- p.47 / Chapter 4.1 --- Introduction --- p.48 / Chapter 4.2 --- Materials and methods --- p.51 / Chapter 4.2.1 --- Fish and tissue samples --- p.51 / Chapter 4.2.2 --- Molecular cloning of full-length ssPRLR and ssPrRPR cDNA --- p.51 / Chapter 4.2.3 --- Multiple sequence alignments and phylogenetic analysis --- p.53 / Chapter 4.2.4 --- Tissue distribution of ssPRLR and ssPrRPR --- p.53 / Chapter 4.3 --- Results --- p.54 / Chapter 4.3.1 --- Cloning and characterization of silver sea bream PRLR and PrRPR cDNA --- p.54 / Chapter 4.3.2 --- Tissue distribution of ssPRLR and ssPrRPR mRNA --- p.56 / Chapter 4.4 --- Discussion --- p.65 / Chapter Chapter 5 --- Influences of osmotic stress and hormones on gill cell of silver sea bream (Sparus sarba) --- p.69 / Chapter Abstract --- p.70 / Chapter 5.1 --- Introduction --- p.72 / Chapter 5.2 --- Methods and materials --- p.77 / Chapter 5.2.1 --- Fish and primary gill cell culture --- p.77 / Chapter 5.2.2 --- Experiments using gill cell culture --- p.78 / Chapter 5.2.3 --- Cell survival assay and apoptosis relevant detections --- p.79 / Chapter 5.2.4 --- Real time PCR analysis --- p.80 / Chapter 5.2.5 --- Western blot analysis of HSP70 --- p.82 / Chapter 5.2.6 --- Data processing and statistical analysis --- p.82 / Chapter 5.3 --- Results --- p.83 / Chapter 5.3.1 --- Hypoosmotic stress induced cell death --- p.83 / Chapter 5.3.2 --- Influences of PRL and PrRP on hypoosmotic challenge induced cell death --- p.84 / Chapter 5.3.3 --- Effects of hormones and osmotic stresses on expression levels of branchial genes --- p.84 / Chapter 5.3.4 --- Osmotic stresses induced changes in branchial protein amount of HSP70s --- p.85 / Chapter 5.4 --- Discussion --- p.94 / Chapter Chapter 6 --- General discussion and conclusions --- p.102 / References --- p.108

by Zhi-Hui Xie. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 214-244). / Abstract --- p.i / Acknowledgments --- p.iv / Publications --- p.v / Abbreviations --- p.vi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Neutrophils --- p.1 / Chapter 1.1.1 --- General --- p.1 / Chapter 1.1.2 --- HL-60 cells as model of neutrophils --- p.3 / Chapter 1.1.3 --- Physiological role --- p.6 / Chapter 1.1.4 --- Intracellular signalling --- p.12 / Chapter 1.1.4.1 --- General --- p.12 / Chapter 1.1.4.2 --- Intracellular signalling linked by FMLP receptors --- p.13 / Chapter 1.1.4.3 --- Intracellular signalling of neutrophil functions --- p.17 / Chapter 1.2 --- Neutrophil aggregation --- p.21 / Chapter 1.2.1 --- General --- p.21 / Chapter 1.2.2 --- Measurement of aggregation --- p.24 / Chapter 1.3 --- Prostaglandins/Prostanoids --- p.25 / Chapter 1.3.1 --- General --- p.25 / Chapter 1.3.2 --- Prostanoid receptors --- p.27 / Chapter 1.3.2.1 --- Agonists and antagonists --- p.28 / Chapter 1.3.2.2 --- EP1 receptors --- p.32 / Chapter 1.3.2.3 --- EP2 receptors --- p.33 / Chapter 1.3.2.4 --- EP3 receptors --- p.34 / Chapter 1.3.2.5 --- EP4 receptors --- p.37 / Chapter 1.3.2.6 --- DP receptors --- p.38 / Chapter 1.3.2.7 --- IP receptors --- p.39 / Chapter 1.3.2.8 --- TP receptors --- p.40 / Chapter 1.3.2.9 --- FP receptors --- p.40 / Chapter 1.4 --- Regulation of neutrophil functions by prostanoids --- p.42 / Chapter 1.4.1 --- Inhibition --- p.42 / Chapter 1.4.2 --- Activation --- p.46 / Chapter 1.4.3 --- Role of cyclic AMP --- p.48 / Chapter 1.5 --- Aim of this study --- p.51 / Chapter Chapter 2 --- Materials and methods --- p.53 / Chapter 2.1 --- Materials and solutions --- p.53 / Chapter 2.1.1 --- Materials --- p.53 / Chapter 2.1.2 --- "Buffers, solutions and cell culture medium" --- p.62 / Chapter 2.2 --- Methods --- p.64 / Chapter 2.2.1 --- Culture of HL-60 cells --- p.64 / Chapter 2.2.2 --- Differentiation of HL-60 cells --- p.65 / Chapter 2.2.3 --- Preparation of neutrophils --- p.65 / Chapter 2.2.4 --- Measurement of neutrophil aggregation --- p.67 / Chapter 2.2.5 --- Measurement of [Ca2+]i --- p.69 / Chapter 2.2.6 --- Microscopic observation --- p.70 / Chapter 2.2.7 --- Measurement of [3H]-cyclic AMP accumulation --- p.72 / Chapter 2.2.8 --- Measurement of [3H]-PGE2 receptor binding --- p.73 / Chapter 2.3 --- Data analysis --- p.76 / Chapter Chapter 3 --- Differentiation of HL-60 cells --- p.77 / Chapter 3.1 --- Introduction --- p.77 / Chapter 3.2 --- Results and discussion --- p.78 / Chapter 3.2.1 --- The observation of cell proliferation and morphology --- p.78 / Chapter 3.2.2 --- The response of HL-60 cells to FMLP --- p.80 / Chapter 3.2.3 --- The response of HL-60 cells to ATP --- p.83 / Chapter 3.3 --- Conclusion --- p.84 / Chapter Chapter 4 --- Activation of dHL-60 cells by PGE2 --- p.90 / Chapter 4.1 --- Effect of PGE2 on cell aggregation and [Ca2+]i --- p.90 / Chapter 4.1.1 --- Introduction --- p.90 / Chapter 4.1.2 --- Results --- p.90 / Chapter 4.1.2.1 --- Effect of PGE2 on cell aggregation --- p.90 / Chapter 4.1.2.2 --- Effect of PGE2 on [Ca2+]i --- p.91 / Chapter 4.1.2.3 --- Effect of PGE2 on human neutrophils --- p.93 / Chapter 4.1.2.4 --- Do PGE2 and PGE1 act at the same receptor? --- p.94 / Chapter 4.1.3 --- Discussion --- p.95 / Chapter 4.1.3.1 --- Activation of dHL-60 cells by PGE2 --- p.95 / Chapter 4.1.3.2 --- Effect of PGE2 on human neutrophils --- p.99 / Chapter 4.2 --- What does the aggregation response mean? --- p.101 / Chapter 4.2.1 --- Introduction --- p.101 / Chapter 4.2.2 --- Results --- p.101 / Chapter 4.2.2.1 --- Observation by light microscopy --- p.101 / Chapter 4.2.2.2 --- Observation by Scanning Electron Microscope --- p.102 / Chapter 4.2.3 --- Discussion --- p.103 / Chapter 4.3 --- Conclusion --- p.106 / Chapter Chapter 5 --- Characterisation of prostanoid receptors on dHL-60 cells --- p.121 / Chapter 5.1 --- Introduction --- p.121 / Chapter 5.2 --- Results --- p.121 / Chapter 5.2.1 --- Effect of prostanoid receptor agonists and antagonists on cell aggregation --- p.121 / Chapter 5.2.2 --- Effect of prostanoid receptor agonists on [Ca2+]i --- p.122 / Chapter 5.3 --- Discussion --- p.123 / Chapter 5.3.1 --- Involvement of prostanoid receptors --- p.123 / Chapter 5.3.2 --- Involvement of EP receptors --- p.126 / Chapter 5.4 --- Conclusion --- p.130 / Chapter Chapter 6 --- Binding studies of [3H]-PGE2 on rat neutrophils and dHL-60 cells --- p.137 / Chapter 6.1 --- Introduction --- p.137 / Chapter 6.2 --- Results --- p.138 / Chapter 6.3 --- Discussion --- p.140 / Chapter Chapter 7 --- The mechanism of action of PGE2 in activating dHL-60 cells --- p.146 / Chapter 7.1 --- Introduction --- p.146 / Chapter 7.2 --- Methods --- p.147 / Chapter 7.2.1 --- Pretreatment with enzyme inhibitors --- p.147 / Chapter 7.2.2 --- Pretreatment with toxins --- p.148 / Chapter 7.2.3 --- Pretreatment with PMA --- p.148 / Chapter 7.3 --- Results and discussion --- p.148 / Chapter 7.3.1 --- The role of Ca2+ --- p.149 / Chapter 7.3.2 --- The role of cyclic AMP --- p.152 / Chapter 7.3.3 --- The role of PI3-kinase --- p.159 / Chapter 7.3.4 --- The role of PLD --- p.165 / Chapter 7.3.5 --- The role of PLA2 --- p.169 / Chapter 7.3.6 --- The role of tyrosine kinase --- p.171 / Chapter 7.3.7 --- The role of PKC --- p.173 / Chapter 7.3.8 --- The role of G protein --- p.180 / Chapter 7.3.9 --- The role of cyclic GMP --- p.185 / Chapter 7.3.10 --- The role of KATP channels --- p.186 / Chapter 7.4 --- Conclusion --- p.187 / Chapter Chapter 8 --- General discussion and conclusion --- p.209 / References --- p.214

Genetic variability within both the HIV-1 coreceptor, CCR5, and its ligand, CCL3L, has been shown to contribute towards differences between individuals in their susceptibility to HIV-1 infection and rate of disease progression. In this study we investigated the extent of genetic variation within the CCR5 gene as well as CCL3L, CCL3La and CCL3Lb gene copy number distribution in two healthy HIV-1 uninfected South African populations, South African Africans (SAA) and South African Caucasians (SAC). The impact of variations within these genes on the expression of CCR5 and CCL3 was subsequently assessed. Furthermore, CCR5 genetic variability, CCL3L gene copy number distribution and the expression of CCR5 and CCL3, was assessed in a similar way in a small cohort of HIV-1 infected long term nonprogressors (LTNPs). Genotyping of the CCR5 gene in SAA (n=41) and SAC (n=46) HIV-1 uninfected individuals revealed a high degree of genetic variation between the two population groups, both in terms of single nucleotide polymorphism (SNP) profiles and CCR5 haplotype distribution. Seven complex putative haplotypes spanning the length of the sequenced region were identified with only one of the identified haplotypes, SAA/C-HHC, common to both study populations. The effect of genetic variability on promoter activity of four different CCR5 promoter regions for three CCR5 haplotypes,SAA-HHA, SAA/C-HHC and SAC-HHE, were evaluated. Results showed variability in (i) promoter activity between different promoter regions tested, (ii) results obtained with different cells used for analysis, and (iii) the haplotype being analysed, thereby highlighting that both the cellular environment as well as genetic variability within the promoter region, have the capacity to influence the efficiency of a promoter and consequently CCR5 expression levels. Haplotype-specific promoter analysis demonstrated the SAA-HHA haplotype to have the strongest promoter activity in THP-1 and K562 cells for both P1A (downstream) and P2 (upstream) promoter regions, while in the other cell lines tested (Jurkat and U937), HHA demonstrated intermediate promoter strength.Differences seen between the haplotypes tested in this study and other published studies may be attributable to additional SNPs being tested in the promoter constructs used in this study. The two population groups differed significantly with regards to cell activation levels, as measured by HLA-DR expression, in CD4+ T cell (P=0.002) and CD56+ NK cell subsets (P<0.001). CCR5 expression, determined both as the number of CCR5 molecules per cell (density) and the percentage of CCR5-expressing cells, was found to differ between SAA and SAC individuals across all peripheral blood cell types. SAA individuals had larger proportions of CCR5-expressing natural killer (NK) cell subsets (P<0.01) but lower CCR5 molecules per cell density on CCR5+CD8+ T cell and CCR5+ NK cell subsets (CD56+, CD16+CD56+ and CD56dim) (all P<0.05) compared to SAC individuals. These differences were maintained even after CCR3D32 heterozygous SAC individuals were included in the analyses. Furthermore, the previously described haplotypes, HHA and HHC, associated with differences in CCR5 expression on different cell subsets between individuals within the same population group. SAA individuals with the HHA haplotype had significantly lower percentages of CCR5-expressing CD8+ T cells compared to SAA individuals that lacked the haplotype (P=0.001). SAC individuals with the HHC haplotype had significantly higher density on NK (CD56+) and CD16+CD56+ NK cell subsets (P=0.030 and P=0.024, respectively) compared to SAC individuals without this haplotype. The latter observation suggests that the protective effect of the HHC haplotype in Caucasians might be explained by higher density of CCR5 expression on NK cells that is not evident in HHC+ SAA individuals, thus highlighting the potential role of CCR5-expressing cells other than CD4+ T cells in protection from HIV-1 acquisition and disease progression. Despite significant differences in CCL3La (CCL3L chemokine coding) and CCL3Lb (nonchemokine coding) copy number between SAA and SAC populations, no difference in CCL3 production by peripheral blood mononuclear cells (PBMCs) was noted between the two study populations. Assuming equal contribution of CCL3 and each copy of CCL3La to CCL3 production,we found that SAC individuals produced higher levels of CCL3 per functional copy of CCL3La compared to SAA individuals (P<0.001). Although, when SAA and SAC individuals with comparable CCL3La and CCL3Lb gene copy numbers were compared, there was no difference in production per functional copy between the two groups (P=0.974). We also determined CCL3La and CCL3Lb gene copy number for a previously established cohort of HIV-1 intrapartum-infected (IP) and exposed uninfected (EU) infants and found that differences previously seen in cord blood mononuclear cell (CBMC) CCL3 production between IP and EU infants with comparable CCL3L copy numbers could not be attributed to differences in CCL3Lb copy number. The potential role of differences in CCR5 genotype, CCR5 expression, CCL3 genotypes and CCL3 production levels in the control of HIV-1 infection was then examined by comparing a small group (10 SAA and 4 SAC) of LTNPs to the respective background population. No polymorphisms in the CCR5 open reading frame were detected in these LTNP individuals. However, the HHA haplotype frequency was significantly higher in SAC LTNP individuals compared to SAC control individuals (P=0.010). Interestingly, CCR5 density on CD4+ T cells and monocytes was significantly lower in SAA LTNP individuals (P=0.025 and P=0.022, respectively) with a trend towards a similar relationship in CD8+ T cells (P=0.058), while the proportions of CCR5-expressing CD8+ T cells were elevated compared to SAA controls (P=0.043). This latter finding reflects the increased immune activation in these individuals compared to uninfected individuals, as evidenced by increased proportions of HLA-DR-expressing T cells (CD8+ and CD4+, P<0.0001). In addition,PHA-induced CCL3 production by PBMCs was significantly lower in LTNP (SAA and SAC combined) compared to control individuals (P=0.004). SAA LTNP individuals had higher proportions of CD8+ T cells (P<0.0001) and lower proportions of natural killer cells (CD56+,P=0.002) compared to control SAA individuals. Thus, CCL3 production differences may be partially explained by differences in the distribution of immune cell subsets between the two study groups.Furthermore, PBMCs of LTNP individuals with low viral loads (<400 copies/ml) produced CCL3 at lower levels than those from individuals with higher viral loads, irrespective of whether or not the cells were stimulated (P=0.005 and P=0.035, respectively). In summary, this study demonstrates that: (i) two ethnically divergent populations show marked differences in CCR5 genetic variability, cell activation and CCR5 expression which are likely to impact on both susceptibility to HIV-1 infection and the rate of HIV-1 disease progression, (ii) both CCR5 genotypic differences and differences in baseline cellular activation levels appear to be contributing towards the observed differences in CCR5 protein expression, and (iii) the two study populations do not differ with respect to CCL3 production by PBMC cultures which suggests that either the two copy per diploid genome gene, CCL3, may play a significant role in CCL3 production and/or that as yet undefined mechanisms regulate production of CCL3 from variable CCL3L copy number. In addition, a pilot study conducted in a small group of LTNP individuals demonstrates that two major determinants of HIV-1 disease progression, CCR5 and CCL3, are both expressed at lower levels in LTNPs individuals compared to healthy uninfected controls and has identified CCR5 haplotypes which are potentially associated with disease progression.

Abstract not available

"January 2002" Bibliography: leaves 204-230.