published_or_final_version / Pathology / Master / Master of Philosophy

The first half of this dissertation describes the synthesis and biological activities of a series of amino aldehydes; which were derivatives of the basic amino acids, arginine, lysine and ornithine. The synthesis of the amino aldehydes was complicated by the difficulty of producing an intermediate oxidation state (the aldehyde) in the presence of two other functional groups (the α-amino, and the side chain functionality). The amino aldehydes were of biological interest due to the fact that they were inhibitors of the proteolytic enzymes called kallikreins. The kallikreins are known to be involved with the renin-angiotensin system, arginine vasopressin, and the prostaglandins, in the regulation of blood pressure. The aldehydes were assayed for their ability to inhibit the kallikrein-mediated production of kinins, and by the inhibition of the cleavage of Nᵅ-tosyl arginine methyl ester (TAME) to the carboxylic acid. Two of the amino aldehydes (Nᵅ-t-Boc-Nᴳ-nitro-L-argininal and Nᵅ-t-Boc-Nᴳ-tosyl-L-argininal) were effective inhibitors in both bioassays at micromolar concentrations. The second part of the dissertation details the development of two syntheses of β-substituted analogues of cysteine. The first method was based on sulfenylation of Nᵅ-formyl-α, β-dehydro amino acid esters, followed by protection of the sulfhydryl group as the benzyl or para-methylbenzyl thioether. The Nᵅ-formyl and ester groups were cleaved by acidic hydrolysis, and the amino group was then blocked as the t-butyloxycarbonyl derivative. This procedure gave cysteine analogues which were suitable for direct use in solid phase peptide synthesis. A second, more efficient preparation of the cysteine analogues was based on the conjugate addition of lithium benzylthiolate (or lithium para-methylbenzylthiolate) to the Nᵅ-formyl-α, β-dehydroamino acid esters. This synthesis was more efficient since the cysteine analogues were generated directly in S-protected form. The fully protected intermediates were deprotected at the amino and carboxyl groups, followed by treatment with di-tert-butyl dicarbonate. The Nᵅ- t-Boc-β-S-benzyl cysteine analogues (or Nᵅ -t-Boc-β-S-para-methylbenzyl) also were suitable for direct use in solid phase peptide synthesis.

This study compared auditory behavioral and physiologic measures in normal control subjects and subjects prescribed with a selective serotonin reuptake inhibitor (SSRI) who were yet to take the drug and those currently taking an SSRI. Test measures used were pure tone averages (PTA), acoustic reflex thresholds, uncomfortable loudness levels (UCL), otoacoustic emissions, masking level difference, temporal integration, amplitude resolution, and Beck Depression Inventory-II (BDI-II) scores. Results indicated that there was a significant difference in the amplitude resolution of the unmedicated group when compared to the medicated and the control group. There was also a significant positive correlation between dynamic range (difference between UCL and PTA) and amplitude resolution. The BDI-II revealed a significant difference between the scores of the unmedicated and the control group as well as the medicated and the control group. Although other test measures indicated differences between the groups, the differences were not statistically significant.

Fung Pik Ha. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 116-121). / Acknowledgments --- p.i / Table of Contents --- p.ii / List of Figures --- p.v / List of Tables --- p.x / Abstract --- p.xi / Chapter Chapter I --- Introduction --- p.1 / Chapter Chapter II --- Literature Reviews / Chapter II.l --- Glycosidase --- p.3 / Chapter II.2 --- Biosynthesis of N-linked Glycoprotein --- p.4 / Chapter II.3 --- Mechanism of Enzyme Catalysed Reaction --- p.8 / Chapter II.4 --- Types of Glycosidase Inhibitors --- p.12 / Chapter II.5 --- Cyclophellitol and Aminocyclitols / Chapter II.5.1 --- General background on cyclophellitol --- p.17 / Chapter II.5.2 --- Mode of inhibition of cyclophellitol --- p.20 / Chapter II.5.3 --- General background on aminocyclitols --- p.24 / Chapter Chapter III --- Characterization of Synthetic Glycosidase Inhibitors / Chapter III.1 --- Covalent-based Inactivator (Cyclophellitol and its Analogues) / Chapter III.1.1 --- Introduction --- p.28 / Chapter III.1.2 --- Materials --- p.32 / Chapter III.1.3 --- Methods / Chapter III.1.3.1 --- Inhibitory assay of commercially available glycosidases --- p.33 / Chapter III.1.3.2 --- Partial purification of β-D-mannosidase from A. oryzae --- p.34 / Chapter III.1.3.3 --- Protein assay in purification of β-D-mannosidase --- p.38 / Chapter III.1.3.4 --- Inhibitory assay for partially purified β-D- mannosidase (A . oryzae) --- p.38 / Chapter III.1.3.5 --- Influence of dialysis on glycosidase inhibition --- p.39 / Chapter III.1.3.6 --- Inactivation experiment on glycosidases --- p.39 / Chapter III.1.4 --- Results / Chapter III.1.4.1 --- Inhibitory activities of cyclophellitol and its analogues against glycosidases --- p.41 / Chapter III.1.4.2 --- Effect of dialysis on glycosidase inhibition --- p.44 / Chapter III.1.4.3 --- The kinetic studies of glycosidase inactivation --- p.47 / Chapter III. 1.5 --- Discussion --- p.50 / Chapter III.1.6 --- Further studies --- p.55 / Chapter III.2 --- Reversible Competitive Inhibitors (Aminocyclitols) / Chapter III.2.1 --- Introduction --- p.56 / Chapter III.2.2 --- Materials --- p.58 / Chapter III.2.3 --- Methods / Chapter III.2.3.1 --- Assay of glucoside hydrolase inhibition activity --- p.60 / Chapter III.2.3.2 --- Glucose oxidase method for determination of released D-glucose --- p.60 / Chapter III.2.3.3 --- Inhibitory assay of aminocyclitols on other glycosidases --- p.61 / Chapter III.2.3.4 --- Influence of dialysis on the glycosidase inhibition --- p.62 / Chapter III.2.3.5 --- Lineweaver-Burk plot --- p.63 / Chapter III.2.4 --- Results / Chapter III.2.4.1 --- Inhibitory activities of valiolamine and related aminocyclitols against six glycosidases --- p.64 / Chapter III.2.4.2 --- Characterization the aminocyclitols as reversible competitive inhibitors --- p.69 / Chapter III.2.5 --- Discussion --- p.80 / Chapter Chapter IV --- Isolation of the Naturally Occurring Glycosidase Inhibitor from Mushrooms / Chapter IV.1 --- Introduction --- p.83 / Chapter IV.2 --- Materials --- p.84 / Chapter IV.3 --- Methods / Chapter IV.3.1 --- Preparation of Ganoderma lucidum --- p.86 / Chapter IV.3.2 --- Preparation of V. volvacea --- p.86 / Chapter IV.3.3 --- Inhibitory assay of aqueous extract of mushrooms on glycosidases --- p.87 / Chapter IV.3.4 --- Anthrone method for determination of reducing sugars --- p.87 / Chapter IV.3.5 --- Flash liquid chromatography for purification of putative inhibitors in G. lucidum --- p.88 / Chapter IV.4 --- Results / Chapter IV.4.1 --- Prescreening of Inhibitory effects of Various Fungal Extracts --- p.90 / Chapter IV.4.2 --- Inhibitory Effects of Partially Purified G. lucidum Extract on Glycosidase --- p.92 / Chapter IV.4.3 --- Effect of Endogenous Substrates on Glycosidase Activities --- p.93 / Chapter IV.4.4 --- Results of Liquid Column Chromatography --- p.93 / Chapter IV.4.5 --- Structure Determination and Characterization of purified compounds --- p.95 / Chapter IV.4.6 --- Inhibitory Activities of Compounds A and B against Brewers yeast a- glucosidase --- p.96 / Chapter IV.5 --- Discussion --- p.98 / Chapter Chapter V --- Conclusions --- p.113 / References --- p.116

癌病是人類的主要死亡原因之一，所以有必要研發出一個有效的癌症治療辦法。大多數癌病是由細胞無限增殖所引起，而端粒酶活性和端粒長度的維持是細胞永生化和轉型的關鍵。超過 85的永生化腫瘤細胞株表達高水平端粒酶。因此，端粒酶的調控機理成為研究和治療癌病的一個主要目標。 / 這項端粒酶的調控機制研究，集中在調查端粒酶抑製蛋白PinX1及其相互作用的蛋白質。透過牽出試驗和質譜鑑定發現45個潛在與PinX1有相互作用的蛋白。其中Nucleophosmin（NPM）被選定作為進一步研究的對象。通過牽出試驗與免疫共沉澱的方法證明NPM與PinX1可在细胞内和外作直接的相互作用。NPM、PinX1和hTERT在細胞內形成復合體，而PinX1是連接NPM和hTERT之間的連接蛋白。PinX1招聘NPM至端粒酶可以減輕 PinX1對端粒酶的抑制作用，表明PinX1/NPM的相互作用可能參與端粒酶的激活過程。此外，NPM和hTERT被發現在細胞周期的S-早期共定位於核仁，而此發現與以往研究中的端粒酶激活的時間相匹配。所有提供的證據表明，PinX1/NPM相互作用在端粒酶激活過程中扮演重要角色。 / 此外，研究證明PinX1參與在端粒酶的募集過程，通過siRNA下調PinX1的表達導致在細胞週期的不同階段中減少端粒酶在端粒的定位。這項研究顯示出PinX1在端粒酶激活和募集過程方面的重要性。 / Cancer is always one of the leading causes of death in humankind and an effective approach for cancer therapy is needed. Most cancers are caused by unlimited proliferation of cells. Telomerase activation and telomere maintenance are found to be critical in cellular immortalization and transformation. Over 85% of the immortal cancer cell lines express high level of telomerase which is essential for telomere maintenance. Therefore, studies on the telomerase regulatory pathway become one of the major targets in cancer research for cancer therapy. / This study focused on investigating a telomerase inhibitor, PinX1 and its interacting proteins for understanding the telomerase regulation. 45 potential PinX1 interacting proteins were identified by pull-down assay coupled with mass spectrometry. Out of these potential partners, Nucleophosmin (NPM) was chosen for further studies and confirmed to have direct interaction with PinX1 through in vitro pull down assay and co-immunoprecipitation. NPM, PinX1 and hTERT form complex inside the cell and PinX1 acts as the linker to bridge the association between NPM and hTERT. The recruitment of NPM by PinX1 to the telomerase can attenuate the PinX1 inhibition on telomerase activity, indicating that PinX1/NPM interaction may involve in telomerase activation. Moreover, NPM and hTERT were found to co-localize in nucleolus during early S-phase which matched the timing of telomerase activation in previous studies. All these provided evidence that PinX1/NPM interaction is implicated in telomerase activation. / Besides, PinX1 was shown to be involved in the telomerase recruitment to telomere, as down-regulation of PinX1 led to reduction of hTERT localization to telomere at different stages of cell cycle. This study revealed the importance of PinX1 in telomerase regulation in terms of its activation and recruitment. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheung, Hang Cheong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 119-135). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 摘要 --- p.v / Table of Contents --- p.vi / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Overview of Telomere and Telomerase in Cancer --- p.1 / Chapter 1.2 --- Introduction to Telomere / Chapter 1.2.1 --- General function and structure of telomere --- p.2 / Chapter 1.2.2 --- Role of shelterin complex in telomere maintenance / Chapter 1.2.2.1 --- TRF1 and TRF2 --- p.4 / Chapter 1.2.2.2 --- Pot1 --- p.5 / Chapter 1.2.2.3 --- TPP1 --- p.5 / Chapter 1.2.2.4 --- TIN2 --- p.6 / Chapter 1.2.2.5 --- RAP1 --- p.6 / Chapter 1.2.3 --- Telomere shortening and replicative senescence in human cell / Chapter 1.2.3.1 --- End replication problem of DNA polymerase --- p.6 / Chapter 1.2.3.2 --- Replicative senescence pathway --- p.7 / Chapter 1.2.4 --- Telomere shortening and cancer formation --- p.7 / Chapter 1.3 --- Introduction to Telomerase / Chapter 1.3.1 --- Function and organization of telomerase --- p.9 / Chapter 1.3.2 --- Telomerase expression in normal cells --- p.9 / Chapter 1.3.3 --- Role of telomerase in cancer cells --- p.10 / Chapter 1.3.4 --- Other roles of telomerase in cells --- p.12 / Chapter 1.3.5 --- Regulation and Recruitment of telomerase / Chapter 1.3.5.1 --- Protein counting model on telomerase regulation --- p.12 / Chapter 1.3.5.2 --- Evidences of telomerase activation on short telomere --- p.13 / Chapter 1.3.5.3 --- Telomerase regulation by shelterin and its associate factors --- p.14 / Chapter 1.3.5.4 --- Cell cycle dependent trafficking of telomerase --- p.15 / Chapter 1.4 --- Introduction to PinX1 / Chapter 1.4.1 --- Discovery of PinX1 as telomerase inhibitor --- p.16 / Chapter 1.4.2 --- Role of PinX1 in telomerase and telomere regulation / Chapter 1.4.2.1 --- Interaction between PinX1 and telomerase --- p.17 / Chapter 1.4.2.2 --- PinX1 mediates nucleolar localization of hTERT --- p.17 / Chapter 1.4.2.3 --- Interaction between PinX1 and TRF1 --- p.17 / Chapter 1.4.2.4 --- Dual role of PinX1 in telomere maintenance --- p.18 / Chapter 1.4.3 --- PinX1 expression in Cancer cells / Chapter 1.4.3.1 --- Genetic analysis of PinX1 in cancers --- p.19 / Chapter 1.4.3.2 --- Treating of cancer through PinX1 manipulation --- p.19 / Chapter 1.5 --- Introduction to Nucleophosmin / Chapter 1.5.1 --- Nucleophosmin (NPM) as a multi-functional protein / Chapter 1.5.1.1 --- NPM is a molecular chaperone --- p.21 / Chapter 1.5.1.2 --- Involvement of NPM in ribosome biogenesis --- p.21 / Chapter 1.5.1.3 --- NPM maintains genomic stability --- p.22 / Chapter 1.5.2 --- Role of Nucleophosmin in Cancer cell / Chapter 1.5.2.1 --- NPM as an oncogene? --- p.22 / Chapter 1.5.2.2 --- NPM as a tumor-suppressor gene? --- p.23 / Chapter 1.6 --- Long term impact and objectives of the study --- p.25 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Plasmids --- p.26 / Chapter 2.1.2 --- Bacterial Cells --- p.26 / Chapter 2.1.3 --- Mammalian Cells --- p.26 / Chapter 2.1.4 --- Serum and Antibodies --- p.27 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Molecular cloning / Chapter 2.2.1.1 --- Basic scheme of cloning --- p.28 / Chapter 2.2.1.2 --- Cloning of PinX1 constructs --- p.29 / Chapter 2.2.1.3 --- Cloning of Nucleophosmin (NPM) constructs --- p.29 / Chapter 2.2.1.4 --- Cloning of hTERT constructs --- p.29 / Chapter 2.2.2 --- Preparation of the competent cells --- p.30 / Chapter 2.2.3 --- Chemical Transformation of competent cells --- p.30 / Chapter 2.2.4 --- Expression of recombinant protein in bacteria --- p.31 / Chapter 2.2.5 --- Purification of GST-PinX1 and GST-PinX1-N --- p.31 / Chapter 2.2.6 --- Purification of PinX1-M and PinX1-C --- p.32 / Chapter 2.2.7 --- Purification of Sumo-NPM and its truncations --- p.33 / Chapter 2.2.8 --- In vitro Pull Down Assay of PinX1-C against HepG2 Lysate / Chapter 2.2.8.1 --- Immobilization of PinX1-C to NHS-column --- p.33 / Chapter 2.2.8.2 --- Preparation of nuclear fraction of HepG2 Lysate --- p.33 / Chapter 2.2.8.3 --- In vitro Pull Down Assay by NHS-column --- p.34 / Chapter 2.2.9 --- 2D-gel electrophoresis --- p.35 / Chapter 2.2.10 --- Mass Spectrommetry --- p.35 / Chapter 2.2.11 --- In-vitro pull down assay --- p.36 / Chapter 2.2.12 --- Plasmid Transfection into mammalian cells --- p.36 / Chapter 2.2.13 --- Co-immunoprecipitation --- p.37 / Chapter 2.2.14 --- Immunofluorescence / Chapter 2.2.14.1 --- Immunostaining of PinX1 and NPM --- p.37 / Chapter 2.2.14.2 --- Immunostaining of hTERT and TRF2 --- p.38 / Chapter 2.2.15 --- TRAP Assay / Chapter 2.2.15.1 --- Basic Scheme of TRAP Assay --- p.39 / Chapter 2.2.15.2 --- TRAP Assay with exogenous purified proteins --- p.40 / Chapter 2.2.16 --- Immunoprecipitation-TRAP Assay --- p.41 / Chapter 2.2.17 --- Transient knock-down of PinX1 or NPM by siRNA --- p.42 / Chapter 2.2.18 --- Synchronization of HeLa cells --- p.42 / Chapter 2.2.19 --- Cell cycle analysis of HeLa cells by flow cytometry --- p.42 / Chapter Chapter 3 --- Identification of PinX1-interacting partners / Chapter 3.1 --- Introduction --- p.50 / Chapter 3.2 --- Results / Chapter 3.2.1 --- Purification of PinX1 constructs --- p.52 / Chapter 3.2.2 --- Identification of PinX1 interacting partners by Pull Down assay --- p.55 / Chapter 3.2.3 --- Mass spectrometry analysis of potential PinX1 partners --- p.55 / Chapter 3.3 --- Discussion --- p.64 / Chapter Chapter 4 --- Role of PinX1/NPM interaction on telomerase regulation / Chapter 4.1 --- Introduction --- p.68 / Chapter 4.2 --- Results / Chapter 4.2.1 --- Confirmation of PinX1/NPM interaction / Chapter 4.2.1.1 --- Association of PinX1 and NPM inside the cell --- p.70 / Chapter 4.2.1.2 --- Direct interaction between PinX1 and NPM in vitro --- p.70 / Chapter 4.2.1.3 --- Co-localization of NPM and PinX1 within the nucleus --- p.73 / Chapter 4.2.2 --- PinX1/NPM/hTERT associated as a complex inside the cell --- p.73 / Chapter 4.2.3 --- Characterization of PinX1/NPM/hTERT interaction / Chapter 4.2.3.1 --- Nucleophosmin interacts with the C-terminal region of PinX1 --- p.76 / Chapter 4.2.3.2 --- PinX1 interacts with the N-terminal region of Nucleophosmin and E56, E61 and E63 of Nucleophosmin are critical for the interaction --- p.78 / Chapter 4.2.3.3 --- Nucleophosmin associates with hTERT through the interaction with PinX1 --- p.83 / Chapter 4.2.4 --- PinX1 recruits NPM to telomerase and attenuates the PinX1 inhibition on telomerase activity --- p.89 / Chapter 4.2.5 --- Nucleophosmin co-localize with hTERT in nucleolus during early S-phase of cell-cycle --- p.91 / Chapter 4.3 --- Discussion --- p.97 / Chapter Chapter 5 --- Importance of PinX1 in telomerase recruitment / Chapter 5.1 --- Introduction --- p.101 / Chapter 5.2 --- Results and Discussion / Chapter 5.2.1 --- Synchronization and silencing of PinX1 in HeLa cells --- p.103 / Chapter 5.2.2 --- Reduced telomerase localization to telomere in PinX1 down-regulated HeLa cells --- p.103 / Chapter 5.3 --- Discussion --- p.110 / Chapter Chapter 6 --- Discussion / Chapter 6.1 --- Concluding Remarks --- p.113 / Chapter 6.2 --- PinX1/NPM interaction as a potential target for cancer treatment --- p.115 / Chapter 6.3 --- Future Prospects / Chapter 6.3.1 --- Studies on other potential PinX1 interacting partners --- p.116 / Chapter 6.3.2 --- Cell-cycle dependent interaction between PinX1, NPM and hTERT --- p.116 / Chapter 6.3.3 --- Designation of inhibitory peptide to disrupt PinX1/NPM interaction --- p.117 / Chapter 6.3.4 --- Importance of PinX1/NPM interaction on tumor growth --- p.117 / Chapter 6.3.5 --- Interaction between NPM and other shelterin proteins --- p.118 / Literature Cited --- p.119

Acquired Immunodeficiency Syndromes (AIDS), a disease caused by the infection of human immunodeficiency virus (HIV), is still incurable to date. Various types of anti-viral drugs have been developed and most of these drugs are targeted on HIV reverse transcriptase and protease. Highly active antiretroviral therapy (HAART) has been used on AIDS treatment recently. However, new drugs are required to delay the resistance onset and to maximize the effectiveness of combination therapy by inhibiting a variety of targets simultaneously. / In the second part, the possibility of using the vector-based approach of RNA interference (RNAi) to reduce the expression of HIV-1 integrase and HIV replication in mammalian cells was examined. RNAi suppressed protein synthesis through the induction of sequence-specific gene silencing of 21-25 nucleotides (nt) double stranded RNA fragments, termed small interfering RNA (siRNA). pSilencer series vectors with different promoters (p Silencer 1.0-U6, pSilencer 2.0-U6, pSilencer 3.0-H1) were used on shRNA expression inside HeLa cells. Four different hairpin constructs containing the 19-nt corresponding to the nucleotide sequence of HIV integrase at positions 19-27, 79-96, 158-176 and 495-513 were generated for RNAi study. (Abstract shortened by UMI.) / Integrase is one of the important enzymes on HIV infection. It acts by integrating viral RNA to host DNA and this is one of the ideal targets for therapeutic intervention. Previous results in our laboratory demonstrated that luffin, a type-I ribosome inactivating protein (RIP), had high potency on integrase inhibition. In the first part of this thesis, alpha-luffin cDNA was cloned from the seed of Luffa cylindrica. Three different sets of expression vectors were used to produce recombinant luffin. Different deletion mutants of luffin were also generated for structural analysis on integrase inhibition. Recombinant alpha-luffin and its various deletion mutants were expressed exclusively in the form of inclusion bodies despite different expression conditions had been attempted. Various refolding strategies and conditions were carried out but the problem of insolubility was consistently found after removal of the denaturing reagents. The problem of insolubility was improved by using the maltose binding protein (MBP) luffin fusion construct. However, there is evidence that this soluble MBP-luffin formed a multimeric fusion protein complex rather than monomer and removal of MBP tag resulted in the precipitation of luffin. / Lau Tat San. / "August 2005." / Adviser: C. C. Wan. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3594. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 202-225). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

No description available.

Thesis (M.Sc. Earth and Ocean Sciences)--University of Waikato, 2007. / Title from PDF cover (viewed March 19, 2008) Includes bibliographical references (p. 97-107)

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 154-169).

Thesis (Ph.D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Pharmacology and Toxicology. Title from title-page of electronic thesis. Bibliography: leaves 98-107.