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published_or_final_version / abstract / toc / Biochemistry / Master / Master of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

by Tsang Yuen Shan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 98-110). / ACKNOWLEDGMENTS --- p.I / ABSTRACT --- p.II / CONTENTS --- p.IV / ABBREVIATIONS --- p.VII / LIST OF FIGURES --- p.IX / LIST OF TABLES --- p.XII / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 1.1 --- Biochemistry of zinc --- p.1 / Chapter 1.2 --- Zinc within the body --- p.2 / Chapter 1.2.1 --- Roles of zinc in general biochemical processes --- p.4 / Chapter 1.2.2 --- Zinc and zinc-binding proteins (ZnBPs) --- p.9 / Chapter 1.3 --- Zinc in brain --- p.12 / Chapter 1.3.1 --- Distribution of zinc in brain --- p.13 / Chapter 1.3.2 --- Roles of zinc in brain 、 --- p.14 / Chapter 1.4 --- Summary --- p.18 / Chapter 1.5 --- Aim of Study --- p.20 / Chapter 2. --- MATERIALS AND METHODS --- p.22 / Chapter 2.1 --- Detection of zinc binding proteins --- p.22 / Chapter 2.1.1 --- Experimental animal --- p.22 / Chapter 2.1.2 --- Subcellular fractionation of porcine brain --- p.22 / Chapter 2.1.2 --- Determination of protein concentration --- p.24 / Chapter 2.1.3 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) --- p.24 / Chapter 2.1.4 --- Two dimensional polyacrylmide gel electrophoresis (2D-PAGE) --- p.25 / Chapter 2.1.5 --- "Fixing, staining and destaining" --- p.28 / Chapter 2.1.6 --- Western blotting for SDS-PAGE --- p.29 / Chapter 2.1.7 --- 65Zn binding --- p.30 / Chapter 2.1.8 --- Autoradiography --- p.30 / Chapter 2.1.9 --- Data quantification --- p.31 / Chapter 2.2 --- Identification of the 21 kD ZnBP --- p.31 / Chapter 2.2.1 --- N-terminal sequencing of the 21 kD ZnBP --- p.31 / Chapter 2.2.2 --- In situ cyanogen bromide (CNBr) cleavage --- p.32 / Chapter 2.2.3 --- Tricine SDS-PAGE --- p.33 / Chapter 2.2.4 --- Isolation of myelin --- p.34 / Chapter 2.2.5 --- Extraction of the 21 kD ZnBP --- p.35 / Chapter 2.2.6 --- Comparison of the mobility of the 21 kD ZnBP with that of bovine myelin basic protein (MBP) in three different gel electrophoresis system --- p.35 / Chapter 2.3 --- Characterization of the zinc binding properties of MBP --- p.38 / Chapter 2.3.1 --- Concentration-dependence of MBP on 65Zn binding --- p.38 / Chapter 2.3.2 --- Effect of other divalent cations on 65Zn binding to the 21 kD ZnBP --- p.38 / Chapter 2.3.3 --- Effect of pH on 65Zn binding to the 21 kD ZnBP --- p.39 / Chapter 2.3.4 --- Effect of modification of histidine residues in MBP on 65Zn binding --- p.39 / Chapter 2.4 --- Effect of zinc on the interaction between MBP and membrane- lipid --- p.40 / Chapter 2.4.1 --- Effect of zinc on MBP-induced phospholipid vesicle aggregation --- p.40 / Chapter 2.4.2 --- Effect of some divalent cations on MBP-induced phospholipid vesicle aggregation --- p.41 / Chapter 3. --- RESULTS --- p.42 / Chapter 3.1 --- Distribution of ZnBPs in porcine brain --- p.42 / Chapter 3.1.1 --- Subcellular distribution of ZnBPs in porcine brain --- p.42 / Chapter 3.1.2 --- "Comparison of the subcellular distribution of ZnBPs in brain, heart and liver" --- p.45 / Chapter 3.1.3 --- Regional distribution of ZnBPs in porcine brain --- p.48 / Chapter 3.1.4 --- Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of total brain homogenates --- p.51 / Chapter 3.2 --- Identification of the 21 kD ZnBP --- p.58 / Chapter 3.2.1 --- N-terminal sequencing of the 21 kD ZnBP --- p.58 / Chapter 3.2.2 --- CNBr cleavage of the 21 kD ZnBP and the N-terminal sequencing of CNBr cleaved fragment --- p.59 / Chapter 3.2.3 --- Detection of the 21 kD ZnBP in myelin fraction and extraction of the 21 kD ZnBP by chloroform-methanol extraction --- p.59 / Chapter 3.2.4 --- Comparison of properties of the 21 kD ZnBP with bovine MBP on three electrophoresis systems --- p.61 / Chapter 3.2.5 --- CNBr cleavage patterns of the 21 kD ZnBP and the bovine MBP --- p.64 / Chapter 3.3 --- Characterization of the zinc binding properties of MBP --- p.69 / Chapter 3.3.1 --- Concentration-dependence of MBP on 65Zn binding --- p.69 / Chapter 3.3.2 --- Effect of other divalent cations on 65Zn binding to the 21 kD ZnBP --- p.69 / Chapter 3.3.3 --- Effect of pH on 65Zn binding to the 21 kD ZnBP --- p.71 / Chapter 3.3.4 --- Effect of modification of histidine residues in MBP on 65Zn binding --- p.73 / Chapter 3.4 --- Effect of zinc on the interaction between MBP and membrane- lipid --- p.76 / Chapter 3.4.1 --- Effect of zinc on MBP-induced phospholipid vesicle aggregation --- p.76 / Chapter 3.4.2 --- Effect of other divalent cations on MBP-induced phospholipid vesicle aggregation --- p.81 / Chapter 4. --- DISCUSSION --- p.83 / Chapter 5. --- CONCLUSIONS --- p.96 / Chapter 6. --- REFERENCES --- p.98

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 118 pages. Includes Vita. Includes bibliographical references.

The role that various myelin membrane proteins play during development and disease processes is not well understood. To better understand their role in vivo we have crossed transgenic mice possessing a single truncated pmp22 gene with mice expressing yellow fluorescent protein in the cytoplasm of their neurons. The resulting double transgenic mice were examined by a combination of confocal microscopy, transmission electron microscopy, and immunohistochemistry to determine if pmp22 insufficiency alters the structural integrity of myelin, glial cells, axons, or the subcellular milieu of these various components. Axons from mice with pmp22 insufficiency developed sprouts and debris localized to nodes with no signs of degeneration of a Wallerian type. Ultrastructurally, the nodes accumulated tubovesicular structures as well as disrupted cytoskeleton that did not appear to alter axon transport. Together, these results suggest that pmp22 insufficiency leads to a non-lethal axonopathy that is restricted to nodes. / Department of Physiology and Health Science

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 154-192).

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 123 pages. Includes Vita. Includes bibliographical references.

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Regrowth in the lesioned central nervous system is impeded by inhibitory molecules including myelin-associated inhibitors (MAIs) and chondroitin sulfate proteoglycans (CSPGs). Inhibitory molecules engage neuronal cell surface receptors and activate the small GTPase RhoA in injured neurons to mediate neurite outgrowth inhibition through targeted modifications to the cytoskeleton. Inhibition of RhoA with the ribosyltransferase C3 attenuates neurite outgrowth inhibition in vitro and in vivo but the ubiquitous expression and multifunctionality of RhoA may limit the specificity of therapeutic RhoA antagonists. The hypothesis of the thesis is that molecules that functionally interact with RhoA to mediate myelin-dependent inhibition may represent more specific targets for therapeutic intervention. We have explored the contribution of two RhoA interacting proteins to the neurite outgrowth inhibitory effects of MAIs. In Chapter 2 we describe the contribution of the rho effector, Rho kinase (ROCK) to MAI responses in neurons. In Chapter 3 we identify the cytosolic phosphoprotein CRMP4b (Collapsin Response Mediator Protein 4b) as a novel RhoA binding partner that mediates neuronal responses to CNS inhibitors. By structure function analysis we have developed a molecular antagonist of CRMP4b-RhoA binding that promotes neurite outgrowth on inhibitory substrates in vitro and has the potential to be a potent and specific molecular therapeutic for spinal cord injury. In Chapter 4 we identify glycogen sythase kinase 3b (GSK3b) as an important kinase in the MAI pathway that regulates protein interactions with RhoA. This thesis provides insights into the signal transduction machinery that is engaged in response to CNS inhibitors and suggests several novel therapeutic targets to promote axon regeneration following CNS injury.