Thesis (M.S.)--University of Michigan, 1991.

Thesis (M.S.)--University of Michigan, 1991.

The allocation of energetic resources into competing physiological systems is crucial for the survival and fitness of an organism. Maintenance of active growth and effective immune function is energetically costly and therefore, trade-offs should have evolved to optimise the allocation of resources into these systems according to physiological status. While recent studies have begun to elucidate the mechanisms by which cross-talk exists between these two systems, more work is needed to characterise their interactions. A high-throughput proteomics approach was developed to investigate the molecular mechanisms underpinning fast growth and molecular cross-talk between growth and immune function. This approach revealed unique routes to fast growth in two different growth-accelerated coho salmon strains, including increased protein synthesis in fish overexpressing growth hormone (GH) through a focussed up-regulation of translation machinery. Conversely, selectively-bred fish showed more complex alterations in multiple metabolic pathways potentially underlying increased growth and domestication. Additionally, the liver proteome response of rainbow trout to a bacterial challenge unveiled host defence and immune proteins upregulated during the acute phase response (APR), along with candidate proteins involved in re-allocation of energetic resources during an immune response. Additionally, novel genetic expansions of salmonid complement C3 proteins upregulated during bacterial challenge were identified and characterised. Finally, the AMP-activated kinase (AMPK) system was investigated due to its role in managing energetic status, making it an ideal system to investigate crosstalk between growth and immunity. Novel salmonid-specific genetic expansions in AMPK subunits (α, β and γ) were demonstrated and their mRNA level expression analysed in fast-growing fish strains exposed to immune stimuli, where an increase in expression of several subunits was observed for fish overexpressing GH. However, a significant downregulation in expression of several AMPK subunit genes occurred in response to immune stimuli. Overall, this project provides insights into links between growth and immune function in salmonids, which are relevant to the aquaculture industry, where the aim is to maximise fish growth while retaining strong immunocompetence.

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

No description available.

No description available.

Bibliography: leaves 116-129.

Bibliography: leaves 116-129 / iv, 129 leaves, [23] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1987

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 55-63).

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 88-110).