by Yu Wai Fu, Jason. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 112-120). / ACKNOWLEDGMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iii / Chapter CHAPTER 1 --- INTORDUCTION --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Historical Overview --- p.3 / Chapter 1.2.1 --- Insulin-Like Growth Factors I and II --- p.3 / Chapter 1.2.2 --- IGF Receptors --- p.5 / Chapter 1.2.3 --- IGF Binding Proteins --- p.7 / Chapter 1.3 --- Origin and Production of IGFs --- p.7 / Chapter 1.3.1 --- The Hypothalamo-Pituitary-GH-IGF-I Axis --- p.7 / Chapter 1.3.2 --- Factors Regulating IGF production --- p.9 / Chapter 1.3.3 --- Expression of IGFs in the Central Nervous System --- p.11 / Chapter 1.4 --- Actions of IGFs --- p.12 / Chapter 1.4.1 --- Insulin-like Metabolic Effects --- p.12 / Chapter 1.4.2 --- Mitogenic Effects --- p.13 / Chapter 1.4.3 --- Effects on Differentiation --- p.13 / Chapter 1.4.4 --- IGFs in Reproductive System --- p.14 / Chapter 1.4.5 --- IGF Actions in the Central Nervous System --- p.14 / Chapter 1.5 --- Transgenic and Knockout Animal Models for IGFs --- p.15 / Chapter 1.6 --- Molecular Biology of IGFs --- p.17 / Chapter 1.6.1 --- Structure of the IGF Genes --- p.17 / Chapter 1.6.2 --- Expression of IGF Genes --- p.21 / Chapter 1.6.3 --- Regulation of IGF Gene Expression --- p.23 / Chapter 1.7 --- IGF Receptors --- p.24 / Chapter 1.7.1 --- IGF-I Receptor --- p.24 / Chapter 1.7.2 --- IGF-II Receptor --- p.26 / Chapter 1.8 --- IGFBPs --- p.26 / Chapter 1.9 --- Teleost IGFs --- p.28 / Chapter 1.9.1 --- The GH-IGF-Axis in Teleost --- p.28 / Chapter 1.9.2 --- Osmoregulation and Other Biological Actions of IGFin Teleost --- p.29 / Chapter 1.9.3 --- Molecular Biology of IGFs in Teleost --- p.30 / Chapter 1.9.4 --- IGFBPs and IGF Receptors in Teleost --- p.31 / Chapter 1.10 --- Rationale and Aim of the Present Study --- p.32 / Chapter CHAPTER 2 --- SEARCH OF IGF-I PROMOTER BY GENOMIC DNA POLYMERASE CHAIN REACTION --- p.34 / Chapter 2.1 --- Introduction --- p.34 / Chapter 2.2 --- Materials --- p.35 / Chapter 2.3 --- Methods --- p.39 / Chapter 2.3.1 --- Preparation of Genomic DNA from Carp Testis --- p.39 / Chapter 2.3.2 --- Restriction Enzyme Digestion of Genomic DNA --- p.40 / Chapter 2.3.3 --- Polymerase Chain Reaction --- p.40 / Chapter 2.3.3.1 --- Ligation of the Cassette to Digested Genomic DNA --- p.40 / Chapter 2.3.3.2 --- Amplification by PCR --- p.40 / Chapter 2.3.4 --- Agarose Gel Electrophoresis --- p.42 / Chapter 2.3.5 --- Gene Clean Using Sephaglas´ёØ BandPrep Kit (Pharamica) --- p.42 / Chapter 2.3.6 --- Cloning of PCR Products --- p.43 / Chapter 2.3.7 --- Transformation of Competent Cell (Heat Shock Method) --- p.43 / Chapter 2.3.8 --- Small Scale Alkaline Preparation of Plasmid DNA --- p.44 / Chapter 2.3.9 --- Restriction Enzyme Digestion to Release the Insert --- p.45 / Chapter 2.3.10 --- Large Scale Plasmid Preparation of the Positive Clone --- p.45 / Chapter 2.3.11 --- DNA Sequencing of the Positive Clone Using the T7 DNA Polymerase Sequencing Kit (Pharmacia) --- p.46 / Chapter 2.4 --- Results and Discussion --- p.49 / Chapter CHAPTER 3 --- ISOLATION OF GENOMIC CLONES CARRYING THE IGF-I GENE --- p.55 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Materials --- p.56 / Chapter 3.3 --- Methods --- p.58 / Chapter 3.3.1 --- Preparation of the Plating Host Cells --- p.58 / Chapter 3.3.2 --- Phage Titering --- p.58 / Chapter 3.3.3 --- Primary Screening of Common Carp Genomic Library --- p.59 / Chapter 3.3.4 --- Preparation of Radioactive Nucleic Acid Probes --- p.60 / Chapter 3.3.5 --- Purification of the Positive Clones --- p.60 / Chapter 3.3.6 --- Purification of DNA from Lambda Phage Using Sephaglas´ёØ PhagePrep Kit (Pharmacia) --- p.61 / Chapter 3.3.7 --- Restriction Enzyme Digestion Release of Inserts --- p.62 / Chapter 3.3.8 --- Capillary Transfer of DNA to Nylon Membrane Under Alkaline Condition --- p.62 / Chapter 3.3.9 --- Southern Analysis of the 10 Positive Clones --- p.63 / Chapter 3.3.10 --- Restriction Mapping of the Clone P1 --- p.64 / Chapter 3.3.11 --- Subcloning of the Fragments of the Clone PI into Plasmid Vector --- p.64 / Chapter 3.3.12 --- IGF-I Specific PCR --- p.64 / Chapter 3.3.13 --- Amplification of Introns from the Clone P1 Using PCR --- p.67 / Chapter 3.4 --- Results and Discussion --- p.70 / Chapter CHAPTER 4 --- RNA ASSAY USING REVERSE TRANSCRIPTION- POLYMERASE CHAIN REACTION --- p.83 / Chapter 4.1 --- Introduction --- p.83 / Chapter 4.2 --- Materials --- p.85 / Chapter 4.3 --- Methods --- p.86 / Chapter 4.3.1 --- Administration of Hormones --- p.86 / Chapter 4.3.1.1 --- Injection Time Course1 --- p.86 / Chapter 4.3.1.2 --- Injection Time Course2 --- p.86 / Chapter 4.3.2 --- Total RNA Extraction --- p.87 / Chapter 4.3.2.1 --- Rapid RNA Isolation --- p.87 / Chapter 4.3.3 --- Electrophoresis of RNA in Agarose Gel Containing Formaldehyde --- p.88 / Chapter 4.3.4 --- Rapid Isolation of PolyA+ mRNA from Total RNA --- p.89 / Chapter 4.3.5 --- IGF-I Specific RT-PCR --- p.90 / Chapter 4.4 --- Results and Discussion --- p.92 / Chapter CHAPTER 5 --- SEARCH FOR IGF-II GENE USING GENOMIC SOUTHERN BLOT ANALYSIS --- p.101 / Chapter 5.1 --- Introduction --- p.101 / Chapter 5.2 --- Materials --- p.103 / Chapter 5.3 --- Methods --- p.104 / Chapter 5.3.1 --- Preparation of Genomic DNA from Carp Testis --- p.104 / Chapter 5.3.2 --- Restriction Enzyme Digestion of Genomic DNA --- p.104 / Chapter 5.3.3 --- Southern Blotting of the Digested Genomic DNA --- p.104 / Chapter 5.3.4 --- Preparation of the Trout IGF-II Specific Probe --- p.104 / Chapter 5.3.5 --- Genomic Southern Hybridization --- p.105 / Chapter 5.4 --- Results and Discussion --- p.106 / Chapter CHAPTER 6 --- GENERAL DISCUSSION AND CONCLUSION --- p.109 / REFERENCES --- p.112

Thèse d'agrégation de l'enseignement supérieur--Liege. / "Ce travail paraîtra dans les Archives internationales de physiologie et de biochemie, 1955, vol. LXIII." Bibliography: p. 145-151.

Leptin, the protein product of obese gene, is a 16-kD adipokine with regulatory functions on food intake and energy metabolism. At present, limited information is available on leptin functions and regulation in lower vertebrates mainly due to the fact that the primary structure of leptin is highly diversified from fish to mammals. Leptin in teleost fish is even more complicated as leptin isoforms have been reported presumably as a result of whole-genome duplication that occurred during the evolution of modern-day bony fish. Using grass carp (Ctenopharyngodon idella) as a model, I sought to investigate the physiological functions and endocrine regulation of leptin in bony fishes. As a first step, the structural identities of two leptins, namely leptin A and B, were established by 5’/3’RACE. The two isoforms share low levels of amino acid sequence homology with mammalian leptins but their deduced 3D-protein models are highly comparable to that of the human counterpart. In grass carp, leptin A and B are widely expressed with highest levels of expression detected in the liver with leptin A as the dominant form. To study the biological actions of grass carp leptins, recombinant proteins of leptin A and B were produced in Escherichia coli and found to inhibit both basal and NPY-stimulated food consumption and feeding behavior in goldfish by both intraperitoneal and intracerebroventricular injection. In addition to the anorexic effects observed, the effects of leptin on pituitary hormone secretion and synthesis were also examined in primary culture of carp pituitary cells. Using reverse transcription-polymerase chain reaction coupled to laser captured microdissection, leptin receptor expression was detected in somatotrophs, gonadotrophs and lactotrophs. Furthermore, leptin A and B were both effective in increasing basal secretion, cell content and transcript expression of growth hormone, luteinizing hormone and prolactin in carp pituitary cells. In the same study, parallel rises in somatolactin α and β mRNA levels without major changes in transcript expression of other pituitary hormones were also noted. These stimulatory effects were mediated by differential coupling with Janus kinase-2 (JAK2)/ signal transducers and activators of transcription (STATs), mitogen-activated protein kinase (MAPK) and/or phosphoinositide 3-kinase (PI3K)/Akt pathways. Although leptin A and B exhibited similar effects on feeding and pituitary hormone expression, their endocrine regulation appears to be quite different. In primary culture of carp hepatocytes, insulin could reduce leptin A but not leptin B mRNA levels through MAPK but not PI3K/Akt pathway. Glucagon, in contrast, could trigger leptin A but not leptin B mRNA expression via the cAMP/PKA cascades and this stimulatory effect could be negated by co-treatment with insulin. At the hepatic level, SLα could also induce leptin A but not leptin B mRNA expression via JAK2 activation of PI3K/Akt cascades. Parallel treatment with SLβ, however, was found to up-regulate leptin B but not leptin A transcription by MAPK coupling to JAK2. These results suggest that the two leptin isoforms identified in grass carp are responsible for similar biological functions but under differential regulation by various endocrine factors coupled to a number of post-receptor signaling mechanisms. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

Protective immunity of carp to ichthyophthiriasis has been confirmed, and demonstrated for the first time in juvenile carp, 10-12 weeks old. Standard immunisation procedures were developed here using the theront in preference to cysts. Immunisation included exposure of fish on 3 separate occasions of 14 day intervals to doses of approximately 2,000 theronts per fish, 80/ cm³. Procedures were controlled so that infections were not allowed to continue beyond the primary stage pH (7.0-7.2 ) and temperature (20±2° C) were maintained throughout experimental periods. Four weeks after third immunising dose, fish were exposed to a potentially lethal challenge, approximately 8,000 theronts per fish, 320/cm³. Following immunisation , fish showed total protection up to 1 month and decreasing protection up to 3 months during which period, mortalities were recorded on challenge. Humoral antibody was monitored at specific stages of experimental infections, peak response 6-8 weeks following exposure with detectable levels of antibody persisting for at least 12 weeks. Immunosuppression was demonstrated following intraperitoneal administration of synthetic corticosteroid Triamcinolone acetonide, doses of 200 µg, 100 µg and 10 µg gˉ¹ body weight, and corticosteroid Hydrocortisone 21-hemisuccinate, doses of 100 µg and 10 µg gˉ¹ body weight, given 14 days after challenge. Immunosuppression was not associated with any significant fall in antibody titre. Studies in cross immunity between Tetrahymena pyriformis (CCAP 1630/W Claff, 1939 (w)) and Ichthyophthirius multifiliis showed no evidence of the former conferring protection to ichthyophthiriasis. Methods of administration of T. pyriformis to juvenile carp included intraperitoneal injection of freeze dried cilia and whole, live T. pyriformis. The kinetics of the humoral response were measured over 12 weeks, peak antibody titres occurring 6-8 weeks following antigen administration. Proliferative responses measured by autoradiography were recorded prior to peak antibody production. Overall results are discussed in relation to immunosuppression, mechanisms of immunity and control and treatment of the disease.

Typescript. / Thesis (Ph. D.)--University of Hawaii, 1966. / Bibliography: leaves [103]-107. / viii, 107 l illus., tables

Thesis (M.S.)--University of North Texas, May, 2008. / Title from title page display. Includes bibliographical references.

Thèse d'agrégation de l'enseignement supérieur--Liege. / "Ce travail paraîtra dans les Archives internationales de physiologie et de biochemie, 1955, vol. LXIII." Bibliography: p. 145-151.

Thesis (M. Phil.)--University of Hong Kong, 1998. / Includes bibliographical references (leaf 69-83).

Inaug.-diss.--Berlin. / Vita.