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Lau San Shing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 113-120). / Abstracts in English and Chinese. / Title Page --- p.1 / List of Thesis Committee --- p.2 / Declaration Page --- p.3 / Acknowledgements --- p.4 / Table of Contents --- p.5 / Abstract --- p.10 / 論文撰要 --- p.12 / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Drug Metabolism --- p.14 / Chapter 1.2 --- Glucuronidation --- p.16 / Chapter 1.3 --- UDP-glucuronosyltransferase (UGTs) / Chapter 1.3.1 --- Nomenclature --- p.18 / Chapter 1.3.2 --- Tissue Distributions of UGTs --- p.20 / Chapter 1.3.3 --- Genetics --- p.26 / Chapter 1.3.4 --- Evolution of UGTs --- p.28 / Chapter 1.4 --- UDP-glucuronosyltransferase related Human Diseases --- p.33 / Chapter 1.4.1 --- Hyperbilirubinemia --- p.33 / Chapter 1.4.2 --- Cancer --- p.37 / Chapter 1.5 --- Rattus norvrgicus UDP-glucuronosyltransferase 1A8 --- p.38 / Chapter 1.6 --- Aims of the Project --- p.42 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials / Chapter 1. --- Rat liver mRNA Extraction --- p.43 / Chapter 2. --- RT-PCR of rat liver mRNA --- p.43 / Chapter 3. --- Amplification of UGT1A8 gene from the cDNA library --- p.43 / Chapter 4. --- Construction of bacterial expression vector --- p.43 / Chapter 5. --- Expression of recombinant protein in E.coli --- p.44 / Chapter 6. --- Purification of protein with Ni column --- p.44 / Chapter 7. --- Purification of protein with gel filtration column --- p.44 / Chapter 8. --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44 / Chapter 9. --- Concentration and Desalting of protein --- p.45 / Chapter 10. --- Enzyme activity of glucuronidation --- p.45 / Chapter 11. --- Near UV and far UV circular dichroism (CD) spectroscopy --- p.45 / Chapter 12. --- Fluorescent properties studies --- p.45 / Chapter 13. --- Western Blotting --- p.46 / Chapter 14. --- 3D modeling of UGT1A8 and interactions with ligands --- p.46 / Chapter 2.2 --- Methods / Chapter 1. --- Rat liver mRNA extraction --- p.46 / Chapter 2. --- RT-PCR of rat liver mRNA --- p.47 / Chapter 3. --- Amplification of UGT1A8 gene from the cDNA library --- p.48 / Chapter 4. --- Cloning of UGT1A8 PCR product into expression vector pRSet B --- p.49 / Chapter 5. --- Confirmation of the presence of insert in the plasmid --- p.51 / Chapter 6. --- Sequence checking for UGT1A8 gene in the pRSet B vector --- p.52 / Chapter 7. --- Expression of recombinant protein in E.coli JM109(DE3) cell strain --- p.52 / Chapter 8. --- Purification of recombinant protein by Ni-column --- p.53 / Chapter 9. --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.53 / Chapter 10. --- Recombinant protein purification by gel filtration column --- p.54 / Chapter 11. --- Concentration or Desalting of Purified Protein --- p.54 / Chapter 12. --- Determination of Protein Concentration --- p.55 / Chapter 13. --- Far- UV Circular dichroism spectroscopy --- p.55 / Chapter 14. --- Intrinsic Fluorescence Studies of Proteins --- p.57 / Chapter 15. --- Chemical denaturation stability studies --- p.58 / Chapter 16. --- Glucuronidation protein activity assay --- p.59 / Chapter 17. --- Mutagenesis --- p.60 / Chapter 18. --- Western Blotting for the presence of protein --- p.61 / Chapter 19. --- Protein Modeling with Insight II / Chapter 19.1 --- Construction of substrate 1-napthol structure --- p.62 / Chapter 19.2 --- Obtaining UDP-glucuronic acid in PDB file --- p.63 / Chapter 19.3 --- Obtaining rat UGT1A8 model structure in PDB file --- p.63 / Chapter 19.4 --- Optimization of rat UGT1A8 structure --- p.63 / Chapter 19.5 --- Docking studies of interaction between ligands and protein / Chapter 19.5.1 --- Setting up a Grid --- p.66 / Chapter 19.5.2 --- Docking of 1-napthol to UGT1A8 --- p.67 / Chapter 19.5.3 --- Docking of UDP-glucuronic acid in the complex of UGT1A8 and1- napthol --- p.68 / Chapter 19.5.4 --- Definition of Subsets --- p.68 / Chapter Chapter 3 --- Results --- p.70 / Figure 3.1 The extracted RNA from rat liver tissue --- p.76 / Figure 3.2 DNA gel of PCR amplified gene product --- p.77 / Figure 3.3 Colony PCR of UGT1 A8-pRSetB transformed DH5 a bacteria --- p.78 / Figure 3.4 The alignment of amplified gene sequence with the rat UGT1A8 sequence on NCBI database --- p.79 / Figure 3.5 SDS-PAGE of cell lysates with different expression temperature and time duration --- p.82 / Figure 3.6 SDS-PAGE of bacterial cell lysates --- p.83 / Figure 3.7 SDS-PAGE of Ni-column eluted protein --- p.84 / Figure 3.8 Elution Profile of Gel Filtration Chromatography --- p.85 / Figure 3.9 SDS-PAGE analysis of UGT1A8 fractions from Ni-column and gel filtration column --- p.86 / Figure 3.10 Sequence Alignment of UGTs in the rat UGT1A family and 2D structure prediction of UGT1A8 --- p.88 / Figure 3.11 Circular Dichroism (CD) measurements on rat UGT1A8 --- p.89 / Figure 3.12 Western Blotting of UGT1A8 wild-type and mutant proteins --- p.91 / Table 3.1 The specific activity of wild-type and mutated proteins --- p.92 / Figure 3.13 Fluorescence spectrum of wild type and two charged-residue mutants ofUGTlA --- p.93 / Figure 3.14 Fluorescence spectrum of wild type and Trp mutants of UGT1A8 --- p.94 / Figure 3.15 Chemical denaturation of wild type and Trp-mutated UGT1A8 proteins --- p.95 / Figure 3.16 Resolved Stern-Volmer plot of UGT1A8 on acrylamide quenching --- p.96 / Figure 3.17 The 3D modeling structure of rat UGT1A8 --- p.97 / Figure 3.18 Modeling simulated the interaction between UDP-glucuronic acid and UGT1A8 --- p.98 / "Figure 3.19 Modeling simulated the interaction between UDP-glucuronic acid, 1-napthol and UGT1A8" --- p.99 / Chapter Chapter 4 --- Discussion / Chapter 1. --- Successful Expression of Rat UGT1A8 --- p.100 / Chapter 2. --- The recombinant rat UGT1A8 protein was properly folded and enzymatic functioning --- p.102 / Chapter 3. --- Purified recombinant rat UGT1A8 protein contained well-ordered structure --- p.103 / Chapter 4. --- "Relative positions of Trp38, Trp64, Trp98 and Trp208 in the protein" --- p.105 / Chapter 5. --- Contribution of Trp residues in the folding and stability of the protein --- p.106 / Chapter 6. --- Probing of substrate coupling region by mutagenesis --- p.108 / Chapter 7. --- Interaction studies of substrates and UDP-glucuronic acid with UGT1A8 by computer modeling and docking simulation --- p.109 / Chapter Chapter 5 --- Conclusion --- p.111 / Chapter Chapter 6 --- References --- p.113

Heart failure has a significant impact on mortality and morbidity. Dilated cardiomyopathy (DCM) is the third most common cause of heart failure and the most common reason for heart transplantation. Familial DCM is known to be caused by mutations in the LMNA gene encoding lamins A and C. New methods to enhance cardiac contractility would be beneficial in the treatment or prevention of heart failure. The focus of this thesis was to evaluate the mechanisms of altered contractility in two mouse models: the LMNA knockout model (homozygous, Lmna-/-; heterozygous, Lmna+/-) generated by targeted deletion of the lmna gene, and the model of enhanced contractility due to cardiac alpha1A-adrenergic receptor (???1A-AR) overexpression (A1A1). Previous studies have found altered nuclear-desmin connections in lamin A/C deficient mice. It was proposed that these alterations result in ???defective force transmission???, which leads to DCM. Studies in this thesis have supported this hypothesis. Studies of isolated single cardiomyocytes from mice aged 4-6 weeks demonstrated abnormal cell morphology and contractile dysfunction in Lmna-/- cardiomyocytes, while Lmna+/- cells showed no overt phenotype. Excitation-contraction coupling experiments and forcecalcium studies in skinned fibers excluded altered calcium kinetics as a primary cause of DCM in this model, but there was evidence of reduced sarcomere numbers and reduced sarcomere lengths as a contributor to reduce force generation in Lmna-/- and Lmna+/- mice. Previous in vivo studies showed that A1A1 mice had enhanced contractility with the absence of hypertrophy. Studies on isolated single cardiomyocytes from A1A1 mice aged 8-12 weeks showed reduced contractility in the absence of ???1A-AR stimulation, but an exaggerated response to ???1A-AR stimulation. In contrast isolated isovolumic Langendorff perfused A1A1 hearts without ???1A-AR stimulation replicated the enhanced contractility observed in vivo. These studies are consistent with down-regulation of contractility due to the hyperactivity of the overexpressed ???1A-AR in vivo, which only becomes evident in isolated cells without ???1A-AR stimulation due to the loss of functional receptor numbers during isolation. Sufficient spontaneously active ???1A-ARs are preserved in the isolated Langendorff heart preparation to ensure maximum contractility driven by increase calcium release.

Contact Lens Acute Red Eye (CLARE) and Infiltrative Keratitis (IK) are inflammatory responses of the eye associated with extended wear of soft contact lenses. Bacterial colonization of contact lenses with Gram-negative bacteria such as Pseudomonas aeruginosa is an important risk factor for the development of these adverse responses. Strategies that control the bacterial colonization of contact lenses may help prevent the occurrence of adverse responses. This thesis aimed to develop an animal model of CLARE/IK to test this hypothesis and to test the effectiveness of contact lenses containing antimicrobial compounds, namely silver and furanone compounds, in controlling corneal inflammation caused by Pseudomonas aeruginosa. A guinea pig model of contact lens wear was developed for the study and it was observed that the ocular responses to contact lens wear in the guinea pig were similar to those seen in human eyes wearing contact lenses. Also, three different models for CLARE/IK were developed and tested in the guinea pig eye. The pathological features of CLARE/IK in the guinea pig were virtually identical to those observed in human eyes. Bacterial contamination of contact lenses was confirmed to be a major risk factor for the development of CLARE/IK. Contact lenses containing nano-particles of silver demonstrated very good antibacterial activity against Pseudomonas aeruginosa in-vitro. The silver lenses were able to control the development of CLARE/IK responses in one of the models for CLARE/IK. Silver lenses might be most effective if used to prevent the establishment of a biofilm of bacteria on a lens such as might occur during storage in a contact lens case. Contact lenses were coated with different concentrations of the furanone compounds by physical adsorption and demonstrated good antibacterial activity at higher concentrations. However these concentrations were cytotoxic in-vitro and lower concentrations of furanones did not possess adequate antibacterial activity to control CLARE/IK responses in-vivo. This thesis has successfully demonstrated that guinea pigs can be used to test the effects of extended wear of contact lenses and developed models to test the pathogenesis of adverse responses such as CLARE/IK. The CLARE/IK models developed could be used to further our understanding of the pathogenesis of these inflammatory conditions and explore the activity of other antimicrobials.

Thesis (M.A.)--University of North Carolina at Greensboro, 2006. / Title from PDF title page screen. Advisor: Walter Salinger; submitted to the Dept. of Psychology. Includes bibliographical references (p. 76-86).

Thesis (M.A.)--University of North Carolina at Greensboro, 2006. / Title from PDF title page screen. Advisor: Walter Salinger; submitted to the Dept. of Psychology. Includes bibliographical references (p. 76-86).

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 179-205). Available also in a digital version from Dissertation Abstracts.

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 27-31).

Thesis--Ph. D., University of Hong Kong, 1984.