by Chan Yuk Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 120-137). / Acknowledgments --- p.i / Table of Content --- p.ii / List of Tables --- p.vi / List of Figures --- p.viii / List of Abbreviations --- p.xi / Chapter CHAPTER 1 --- Literature Review Page / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- "Overview of the Structures of GH, PRL and SL" --- p.2 / Chapter 1.3 --- Physiological Significance of the Hormones in Teleost --- p.12 / Chapter 1.3.1 --- Growth Hormone --- p.12 / Chapter 1.3.2 --- Prolactin --- p.12 / Chapter 1.3.3 --- Somatolactin --- p.13 / Chapter 1.4 --- Structure - Function Relationship and Receptor Binding Activities --- p.15 / Chapter 1.5 --- Aims of Thesis --- p.23 / Chapter CHAPTER 2 --- General Methodology / Chapter 2.1 --- Materials --- p.25 / Chapter 2.2 --- DNA manipulation methods --- p.28 / Chapter 2.2.1 --- Polymerase Chain Reaction (PCR) --- p.28 / Chapter 2.2.2 --- Ethanol Precipitation of DNA --- p.29 / Chapter 2.2.3 --- Agarose Gel Electrophoresis of DNA --- p.29 / Chapter 2.2.4 --- Sephadex G-50 Spun Column --- p.30 / Chapter 2.2.5 --- Nick Translation --- p.30 / Chapter 2.2.6 --- Small Scale Plasmid Preparation by Alkaline Lysis Method --- p.31 / Chapter 2.2.7 --- Large Scale Plasmid Preparation Using Magic´ёØ Maxipreps DNA Purification System (Promega) --- p.32 / Chapter 2.3 --- DNA Cloning Methods --- p.33 / Chapter 2.3.1 --- Blunt-end Ligation of PCR Product to pUC18 Vector --- p.33 / Chapter 2.3.1.1 --- Preparation and Transformation of E. coli (JM109) Competent Cell --- p.33 / Chapter 2.3.1.2 --- Recovery of DNA from Agarose Gel Using the Sephaglas Bandprep Kit (Pharmacia Biotech) --- p.34 / Chapter 2.3.1.3 --- Kinasing Reaction --- p.34 / Chapter 2.3.1.4 --- Klenow Fill-in Reaction and Ligation of DNA Fragments --- p.35 / Chapter 2.3.2 --- Screening of Lambda Phage cDNA Library --- p.35 / Chapter 2.3.2.1 --- Preparation of Host Cell for Screening of Lambda Phage cDNA Library --- p.35 / Chapter 2.3.2.2 --- Phage Stock Tittering --- p.36 / Chapter 2.3.2.3 --- Plaque Lifting and Fixation on Nylon Membranes --- p.36 / Chapter 2.3.2.4 --- Library Screening by Hybridization --- p.37 / Chapter 2.3.2.5 --- In vivo Excision --- p.37 / Chapter 2.4 --- Nucleotide Sequence Determination using Dideoxy Nucleotide Chain Termination Method --- p.38 / Chapter 2.5 --- Protein methods --- p.39 / Chapter 2.5.1 --- Bicinchoninic Acid (BCA) Assay --- p.39 / Chapter 2.5.2 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.40 / Chapter CHAPTER 3 --- Isolation and Characterization of cDNA Clones for Goldfish Prolactin / Chapter 3.1 --- Introduction --- p.41 / Chapter 3.2 --- Methods --- p.43 / Chapter 3.2.1 --- Preparation of Hormone Specific DNA Probes by PCR Cloning --- p.43 / Chapter 3.2.2 --- Construction of Goldfish Pituitary cDNA Library --- p.44 / Chapter 3.2.3 --- cDNA Library Screening for Hormone Specific cDNAs --- p.44 / Chapter 3.2.4 --- Restriction Enzyme Digestion of the cDNA Clones and Subcloning of the Restriction Fragments --- p.45 / Chapter 3.2.5 --- Nucleotide Sequences Determination using Dideoxy Nucleotide Chain Termination Method --- p.46 / Chapter 3.2.6 --- Southern Analysis of the Goldfish Genomic DNA --- p.47 / Chapter 3.2.7 --- Northern Blot Analyses of Goldfish Pituitary Total RNA --- p.47 / Chapter 3.3 --- Results --- p.49 / Chapter 3.3.1 --- Screening of the Library and Characterization of the Clones --- p.49 / Chapter 3.3.1.1 --- Analyses of the cDNA Clones --- p.49 / Chapter 3.3.1.2 --- Cross Hybridization Reactivity --- p.57 / Chapter 3.3.2 --- Genomic Southern Blot Analyses --- p.58 / Chapter 3.3.3 --- Gene Expression of the Pituitary Hormones - Northern Blot Analyses --- p.61 / Chapter 3.4 --- Discussion --- p.64 / Chapter 3.4.1 --- Cross Hybridization Reavtivities of the Probes to the Hormone cDNA Clones --- p.64 / Chapter 3.4.2 --- Structural Analyses of the Hormone cDNA Clones --- p.65 / Chapter 3.4.2.1 --- Growth Hormone --- p.65 / Chapter 3.4.2.2 --- Prolactin --- p.68 / Chapter 3.4.2.3 --- Somatolactin --- p.70 / Chapter 3.4.3 --- Secondary Structure Prediction --- p.72 / Chapter 3.4.4 --- Genomic Southern Analyses --- p.78 / Chapter 3.4.5 --- Pituitary Expression of Goldfish Hormone mRNAs --- p.79 / Chapter 3.5 --- Conclusion --- p.81 / Chapter CHAPTER 4 --- "Expression of Recombinant Goldfish Growth Hormone, Prolactin and Somatolactin in Escherichia coli (E. coli)" / Chapter 4.1 --- Introduction --- p.82 / Chapter 4.2 --- Materials and Methods --- p.83 / Chapter 4.2.1 --- Materials --- p.83 / Chapter 4.2.2 --- Construction of Expression Vectors Carrying the Hormone Coding Regions --- p.84 / Chapter 4.2.3 --- Pilot Expression Experiment to Determine Kinetics of Expression --- p.86 / Chapter 4.2.4 --- "Large Scale Expression and Purification of Recombinant Hormones by Ni2+-NTA Affinity Column (ProBond´ёØ column, Invitrogen)" --- p.86 / Chapter 4.2.5 --- Western Blotting of the Recombinant Hormones on Polyvinylidene Fluoride (PVDF) Membrane --- p.87 / Chapter 4.2.6 --- Dot Blot Immobilisation of the Recombinant Hormones on Nitrocellulose Membrane --- p.88 / Chapter 4.2.7 --- Detection of the Blotted Protein by Enzyme-linked Immunodetaction Method --- p.88 / Chapter 4.3 --- Results --- p.90 / Chapter 4.3.1 --- Construction of the Expression Vectors --- p.90 / Chapter 4.3.2 --- Expression of the Recombinant Hormones --- p.97 / Chapter 4.3.3 --- Partial Purification and Analyses of the recombinant Hormones --- p.97 / Chapter 4.4 --- Discussion --- p.105 / Chapter 4.4.1 --- Construction of the Expression Vectors --- p.105 / Chapter 4.4.2 --- Expression of the Recombinant Hormones --- p.108 / Chapter 4.4.3 --- Partial Purification and Characterization of the Recombinant Hormones --- p.109 / Chapter 4.5 --- Conclusion --- p.114 / Chapter CHAPTER 5 --- General Discussion --- p.115 / References --- p.120

Chan, Yuk-Hang. / "June 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 162-190). / 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.

Helen Wei Ling Lai. / "December 2000." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2000. / includes bibliographical references (p. 97-102). / 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.

Reorganization of the intermediate filament (IF) network during axonal regeneration is accompanied by changes in the expression of various IF proteins. An increase in expression of the neuronal IF subunit gefiltin in goldfish retinal ganglion cells (RGCs) has been linked to the unique ability of the goldfish optic nerve to regenerate following injury. Evidence suggests that the optic tectum may regulate the expression of gefiltin during regeneration. The goal of this thesis was to determine the function of the tectum in the regulation of gefiltin mRNA expression during optic fiber regeneration in the goldfish. It was found that gefiltin mRNA levels in the RGCs of animals that received an optic nerve crush (ONC group) increased by 10 days, peaked from 20 to 38 days at around 5.5-fold over normal, and declined to near normal by 115 days. In animals that had the entire tectum removed and an optic nerve crush (ETR group), gefiltin mRNA levels increased by 10 days, peaked at 20 days at around 5.5- to 6.5-fold over normal, and although they dropped slightly thereafter, they remained elevated at around 5-fold over normal for at least 115 days. When axons regenerated to the ipsilateral tectal lobe as a result of a left tectal lobe removal and left eye removal surgery the expression pattern of gefiltin mRNA paralleled that of the ONC group. It was also found that the abundance of gefiltin subunits in the retina was elevated at 30 days of regeneration in ONC and ETR animals, and that levels in the nerve were reconstituted to 80% of normal by 30 days. These results demonstrate that increases in gefiltin mRNA and protein levels during optic nerve regeneration are independent of the tectum, whereas the downregulation of gefiltin mRNA levels is entirely dependent upon the tectum. (Abstract shortened by UMI.)

No description available.

published_or_final_version / Zoology / Master / Master of Philosophy

This study aimed to better understand the clearance of myelin debris during Wallerian degeneration in the goldfish visual paths. Myelin debris was first examined immunohistochemically in the presence or absence of regenerating axons. From these preliminary experiments it was apparent that the clearance of myelin debris was not affected by regenerating axons and that the debris was removed in a differential pattern along the visual pathway. Specifically, in the distal stump of the nerve as well as in the optic tract, myelin debris had been effectively cleared by one-month postoperative, while in the cranial segment of the nerve debris persisted for at least 6 weeks after injury. The differential pattern of myelin debris in the optic nerve and tract was then analyzed qualitatively and quantitatively using thick and thin plastic sections at various time points during regeneration. The results suggested that highly activated peripheral macrophages were responsible for the effective clearance of myelin in the distal nerve stump. In the optic tract a number of cellular properties, including their unique population of astrocytes may have enhanced the rate of debris clearance. By contrast, in the cranial segment of the nerve persistent debris was found both intracellularly in phagosomes and extracellularly, suggesting that the resident phagocytes were deficient in effecting both phagocytosis and emigration. Deficient phagocytosis may be a result of the production of anti-inflammatory cytokines in this region, while the failure to emigrate is most likely due to the rigid network of astrocytes in the nerve.

No description available.

Cannulae were implanted into forebrain loci of goldfish (Carassius auratus; 45-90 g) to determine (i) the effects and site of action of intracranial norepinephrine (NE) injections on behavioral thermoregulation and (ii) the mechanism and the types of adrenoreceptors involved in the thermoregulatory effect of NE. After 30 min in a thermal gradient, implanted fish were injected with norepinephrinebitartrate salt (2.5-500 ng NE) in a total volume of 0.2 ul (carrier was 0.7% NaCl). Injections of 5, 10, 25, and 50 ng NE into the anterior aspect of the nucleus preopticus periventricularis (NPP1 Peter and Gill 1975) led to consistent, dose-dependent decreases in selected temperature. No effect on temperature selection was observed following injections of 2.5 ng NE or control injections of 100 ng tartaric acid. The effects of injections into other loci, including intraventricular injections, were dependent upon the dose and proximity to the anterior NPP1 at sites adjacent to the anterior NPP, larger doses were required, and the effects became inconsistent. At sites further removed, no effect on selected temperature was observed1 included in this category were more caudal sites within the NPP and the nucleus preopticus.

No description available.