by Li Tin Yau. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 56-60). / Acknowledgment --- p.i / Table of Contents --- p.ii / Abstract --- p.iii / Abbreviation --- p.iv / Chapter I --- Introduction --- p.1 / Chapter I-1 --- General Background --- p.1 / Chapter I-2 --- Mechanistic Aspect of Glycosidase Inhibition --- p.4 / Chapter I-3 --- Previous Syntheses of Valienamine and its Diastereomers --- p.7 / Chapter II --- Results and Discussion --- p.16 / Chapter II-1 --- General Strategy --- p.16 / Chapter II-2 --- Synthesis of Tribenzyl ether69 --- p.18 / Chapter II-3 --- Synthesis of Diol67 --- p.20 / Chapter II-4 --- "Synthesis of N,O,O, O, O-Pentaacetylvalienamine30 and its 2-Epimer62" --- p.22 / Chapter II-5 --- Synthesis of Diacetate66 --- p.26 / Chapter II-6 --- Synthesis of the Valienamine Derivatives 63 and64 --- p.30 / Chapter III --- Conclusion --- p.34 / Chapter IV --- Experimental --- p.36 / Chapter V --- Reference --- p.56

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

Lectins and hemagglutinins are carbohydrate binding proteins present in a diversity of organisms including humans, vertebrate and invertebrate animals, plants, fungi, and bacteria. They are usually the abundant storage proteins in leguminous plants. They display a host of biological activities such as antitumor, antifungal, antiviral, insecticidal, and antibacterial activities. / The biological properties of isolated proteins, including hemagglutinating, antifungal, anti-tumor and HIV-1 reverse transcriptase inhibitory activities, were examined. Their biochemical and biological properties were compared with other purified proteins. / The seeds contain an abundance of proteins, some of which are storage proteins but may play a role of protection from pathogenic microbes and phytophagous insects. Antifungal peptides/proteins, antiviral proteins, ribosome-inactivating proteins, proteinase inhibitors, chitinases, proteinases, and defensins, are some examples of the myriad of seed proteins. The aforementioned proteins are collectively called plant defense proteins in view of their antipathogenic activities. These antifungal proteins exhibit a wide range of molecular masses and amino acid sequences. / Two lectins with potentially exploitable activities were purified from Capparis spinosa seeds and Hibiscus mutabilis seeds, respectively. A hemagglutinin was isolated from Phaselous vulgaris , cultivar "French bean 35", and detailed apoptotic pathway in breast cancer cells, MCF-7 cells, was investigated. A novel dimeric beta-lactoglobulin-like antifungal protein and an antifungal amidase were purified from Passiflora edilus seeds and Peltophorum pterocarpum, respectively. / Lam, Sze Kwan. / Adviser: Tsi Bun Ng. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 188-204). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

The altered protein expression and activity of receptor tyrosine kinases (TK) are implicated in the progression of various types of cancers. One such dysfunction is the overexpression of the epidermal growth factor receptor (EGFR) that correlates with aggressive tumor progression and poor prognosis. On the other hand, c-Src non-receptor tyrosine kinase is overexpressed and activated in a large number of human malignancies and has been strongly linked to progression to distant metastases. c-Src-induced phosphorylation of EGFR is required for EGF-mediated mitogenesis, tumorigenesis and tumour invasiveness. Thus we surmised that molecules termed "combi-molecules" designed to block both EGFR and c-Src should not only possess significant growth inhibitory potency but also strong anti-invasive properties. In this thesis, we utilized molecular modeling to design molecules containing two moieties: one that straddles the structure of the known Src inhibitor PP2 and the other that mimics the backbone of Iressa, a potent EGFR inhibitor. Of all the molecules synthesized, only SB163 containing the longest spacer between the two moieties was capable of inducing a dose dependent inhibition of both Src and EGFR. More importantly, SB163 blocked cell motility in the wound healing assay and showed significantly greater anti-invasive activity than a PP2+Iressa combination. The observation that SB163 was a less potent EGFR or Src inhibitor than Iressa and PP2 suggests that its superior potency when compared with the PP2+ Iressa combination may be at least partially attributed to mechanisms other than EGFR or Src blockade. This was also corroborated by the fact that SB163, despite its significant bulkiness (>700) could induce dose dependent inhibition of other kinase such PDFGR and Abl. The results in toto suggest that conferring multiple kinase targeting properties to single molecules can lead to highly anti-proliferative and anti-invasive agents. Traditionally, multi-kinase targeted molecules were discovered serendipitously through multi-kinase testing. Here we initiated a more rational approach to the design of single multi-targeted molecules. Cancer being a complex disease driven by tumours characterized by multiple disordered signaling pathways, this approach may well represent a novel avenue in the therapy of refractory malignancies.

No description available.

by Chung Wilson. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 126-149). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract (English) --- p.ii / Abstract (Chinese) --- p.v / Content --- p.vii / Abbreviations --- p.xii / List of Figures --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Alzheimer's disease --- p.1 / Chapter 1.1.1 --- History of Alzheimer's disease --- p.1 / Chapter 1.1.2 --- Definition of Alzheimer's disease --- p.2 / Chapter 1.1.3 --- Symptoms of Alzheimer's disease --- p.6 / Chapter 1.1.3.1 --- Memory deficit --- p.6 / Chapter 1.1.3.2 --- Difficulty in learning --- p.6 / Chapter 1.1.3.3 --- Language difficulties --- p.7 / Chapter 1.1.3.4 --- Decline in ability to perform routine tasks --- p.7 / Chapter 1.1.4 --- Prevalence of Alzheimer's disease --- p.8 / Chapter 1.2 --- Present treatment of Alzheimer's disease --- p.9 / Chapter 1.2.1 --- Acetylcholine and dementia --- p.9 / Chapter 1.2.2 --- Tacrine as first drug approved by US Food and Drug Administration --- p.9 / Chapter 1.3 --- Proposed theory of Alzheimer's disease formation --- p.10 / Chapter 1.3.1 --- The amyloid cascade hypothesis --- p.10 / Chapter 1.3.1.1 --- The amyloid precursor protein --- p.10 / Chapter 1.3.1.2 --- The processing of amyloid precursor protein --- p.12 / Chapter 1.3.1.3 --- Neurotoxic effect of amyloid plaque --- p.15 / Chapter 1.3.1.4 --- Genetic factors --- p.15 / Chapter 1.3.1.4.1 --- The amyloid precursor protein --- p.15 / Chapter 1.3.1.4.2 --- Apolipoprotein E (ApoE) --- p.16 / Chapter 1.3.1.4.3 --- Presenilin genes --- p.17 / Chapter 1.3.2 --- Tau and tangle hypothesis --- p.19 / Chapter 1.3.2.1 --- Tau protein --- p.19 / Chapter 1.3.2.2 --- Paired helical filaments (PHF) --- p.20 / Chapter 1.3.2.3 --- Tau protein kinase --- p.20 / Chapter 1.3.2.3.1 --- Glycogen synthase kinase-3 (GSK-3) --- p.21 / Chapter 1.3.2.3.2 --- Cyclin-dependent kinase 5 (CDK5) --- p.21 / Chapter 1.3.2.4 --- Tangle leads to dementia --- p.22 / Chapter 1.4 --- Cross-talk between the two hypotheses --- p.24 / Chapter 1.5 --- β -secretase (BACE) --- p.24 / Chapter 1.5.1 --- Discovery of β-secretase (BACE) --- p.24 / Chapter 1.5.2 --- Detailed structure of BACE --- p.25 / Chapter 1.5.3 --- Comparsion of human and mouse BACE --- p.27 / Chapter 1.5.4 --- Comparsion of BACE-1 with BACE-2 --- p.27 / Chapter 1.5.5 --- Properties of BACE-1 --- p.28 / Chapter 1.5.6 --- Expression of BACE in E.coli --- p.29 / Chapter 1.5.7 --- Expression of BACE in mammalian cells --- p.30 / Chapter 1.6 --- Objectives of the present study --- p.32 / Chapter Chapter 2 --- Materials and Methods --- p.34 / Chapter 2.1 --- Recombinant DNA techniques --- p.34 / Chapter 2.1.1 --- Amplification of genes by PCR techniques --- p.34 / Chapter 2.1.2 --- Agarose gel electrophoresis --- p.34 / Chapter 2.1.3 --- Extraction of DNA from agarose gel --- p.35 / Chapter 2.1.4 --- Digestion of various vectors and inserts --- p.36 / Chapter 2.1.5 --- Ligation of DNA fragments --- p.36 / Chapter 2.1.6 --- Preparation of Escherichia coli competent cells --- p.37 / Chapter 2.1.7 --- Bacterial transformation --- p.38 / Chapter 2.1.8 --- Minipreparation of plasmid DNA --- p.38 / Chapter 2.1.9 --- Large scale preparation of plasmid DNA --- p.39 / Chapter 2.1.10 --- Strain storage and revival --- p.40 / Chapter 2.1.11 --- Plasma DNA purification by High Pure plasmid isolation kit --- p.41 / Chapter 2.1.12 --- DNA sequencing --- p.42 / Chapter 2.1.13 --- Quantitation of DNA by spectrophotometric method --- p.43 / Chapter 2.2 --- Prokaryotic protein expression --- p.43 / Chapter 2.2.1 --- Selection of appropriate clones for recombinant protein expression using conventional method --- p.43 / Chapter 2.2.2 --- Selection of appropriate clones for recombinant protein expression using modified method --- p.44 / Chapter 2.2.3 --- Large -scale expression of recombinant human BACE protein using modified method --- p.45 / Chapter 2.2.4 --- Preparation of inclusion body from the bacterial expression culture --- p.46 / Chapter 2.2.5 --- Refolding of human BACE --- p.47 / Chapter 2.2.6 --- Purification of recombinant human BACE by immobilized metal ion affinity chromatography (IMAC) --- p.47 / Chapter 2.2.7 --- Protein concentration determination --- p.48 / Chapter 2.2.8 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.48 / Chapter 2.2.9 --- Western blotting --- p.50 / Chapter 2.2.10 --- Plasmid stability test --- p.50 / Chapter 2.3 --- Mammalian cell expression --- p.51 / Chapter 2.3.1 --- Transient transfection --- p.51 / Chapter 2.3.2 --- Measuring transfection efficiency --- p.52 / Chapter 2.3.3 --- Stable transfection --- p.52 / Chapter 2.3.4 --- Preparation of membrane extracts from CHO cells --- p.53 / Chapter 2.4 --- HPLC analysis --- p.53 / Chapter 2.4.1 --- Preparation of samples --- p.53 / Chapter 2.4.2 --- Reverse phase HPLC --- p.54 / Chapter 2.5 --- Fluorometric assay --- p.54 / Chapter 2.6 --- Immunohistochemistry --- p.55 / Chapter 2.7 --- Reagents and buffers --- p.55 / Chapter 2.7.1 --- Medium for bacterial culture --- p.56 / Chapter 2.7.2 --- Reagents for preparation of plasmid DNA --- p.56 / Chapter 2.7.3 --- Buffers for agarose gel electrophoresis --- p.57 / Chapter 2.7.4 --- Buffers for SDS-PAGE --- p.58 / Chapter 2.7.5 --- Buffer for purification of protein --- p.60 / Chapter 2.7.6 --- Buffer for Western Blotting --- p.61 / Chapter 2.7.7 --- Culturing medium of CHO cells --- p.62 / Chapter 2.7.8 --- Solutions for estimating transfection efficiency --- p.63 / Chapter 2.7.9 --- Reagents for HPLC --- p.64 / Chapter 2.7.10 --- Reagents for fluorometric assays --- p.65 / Chapter 2.7.11 --- Reagents for Immunohistochemistry --- p.66 / Chapter Chapter 3 --- Results --- p.67 / Chapter 3.1 --- Expression of BACE in E. coli --- p.67 / Chapter 3.1.1 --- Cloning of truncated human and mouse BACE into pRSET --- p.67 / Chapter 3.1.2 --- Expression of BACE in BL21(DE3)LysS cells --- p.70 / Chapter 3.1.2.1 --- Expression of truncated mouse and human BACEin BL21(DE3)LysS cells using conventional method --- p.70 / Chapter 3.1.2.2 --- Expression of truncated mouse and human BACEin BL21(DE3)LysS cells using modified method --- p.72 / Chapter 3.1.3 --- Analysis of BACE activity of purified recombinant proteins --- p.76 / Chapter 3.1.3.1 --- Fluorometric analysis --- p.76 / Chapter 3.2 --- Expression of BACE in mammalian cells --- p.81 / Chapter 3.2.1 --- "Cloning of full length mouse and human BACE into pCDNA3, pCDNA4HisMax" --- p.81 / Chapter 3.2.2 --- Transient transfection --- p.84 / Chapter 3.2.2.1 --- Western blot analysis --- p.86 / Chapter 3.2.2.2 --- Fluorometric analysis --- p.88 / Chapter 3.2.2.3 --- HPLC --- p.91 / Chapter 3.2.3 --- Stable transfection --- p.100 / Chapter 3.2.3.1 --- Western blot analysis --- p.101 / Chapter 3.2.3.2 --- Fluorometric analysis --- p.103 / Chapter 3.2.3.3 --- HPLC --- p.105 / Chapter 3.2.3.4 --- Immunohistochemistry --- p.112 / Chapter Chapter 4 --- Discussion --- p.115 / References --- p.126 / Appendix --- p.i / Chapter A1 --- Vector circle map --- p.i / Chapter A1-1 --- Vector circle map of pBluescript II- --- p.i / Chapter A1-2 --- Vector circle map of pCDNA3 --- p.ii / Chapter A1-3 --- Vector circle map of pCDNA4HisMax --- p.iii / Chapter A1-4 --- Vector circle map of pRSET --- p.iv / Chapter A2 --- Primer lists --- p.v / Chapter A3 --- Chemical structure of fluorophore and quench used in fluorometric assay --- p.vi

by Yuen-Kam Tsoi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 160-173). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iv / List of figures --- p.vii / List of tables --- p.x / Abbreviations --- p.xi / Chapter Chapter 1 --- Purification and characterization of the chymotrypsin inhibitor (MCCI) from the seeds of Momordica cochinchinensis / Chapter 1.1 --- Introduction l / Chapter 1.1.1 --- Classification of protease inhibitor --- p.2 / Chapter 1.1.2 --- Therapeutic potential of protease inhibitors --- p.4 / Chapter 1.2 --- Rationale of the present study --- p.6 / Chapter 1.3 --- Materials and methods / Chapter 1.3.1 --- Materials --- p.9 / Chapter 1.3.2 --- Preparation of chymotrypsin-Sepharose 4B affinity column --- p.10 / Chapter 1.3.3 --- Protein extraction --- p.11 / Chapter 1.3.4 --- Chymotrypsin-Sepharose 4B affinity chromatography --- p.12 / Chapter 1.3.5 --- Reversed phase high pressure liquid chromatography --- p.12 / Chapter 1.3.6 --- Assays for protease inhibitory activities --- p.14 / Chapter 1.3.6.1 --- Assay for chymotrypsin activity --- p.15 / Chapter 1.3.6.2 --- Assay for trypsin activity --- p.15 / Chapter 1.3.6.3 --- Assay for elastase activity --- p.16 / Chapter 1.3.6.4 --- Assay for subtilisin activity --- p.16 / Chapter 1.3.7 --- Determination of protein concentration --- p.17 / Chapter 1.3.8 --- Titration of chymotrypsin --- p.17 / Chapter 1.3.9 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis --- p.18 / Chapter 1.3.10 --- Determination of molecular weight by mass spectrometry --- p.19 / Chapter 1.3.11 --- Partial amino acid sequencing --- p.20 / Chapter 1.3.12 --- Effects of chymotrypsin on MCCI --- p.20 / Chapter 1.3.13 --- Stability assay --- p.21 / Chapter 1.4 --- Results --- p.21 / Chapter 1.4.1 --- Isolation of MCCI from the seeds of Momordica cochinchinensis --- p.21 / Chapter 1.4.2 --- N-terminal amino acid sequencing --- p.27 / Chapter 1.4.3 --- Determination of molecular weight --- p.31 / Chapter 1.4.4 --- Inhibitory activity of MCCI towards different proteases --- p.33 / Chapter 1.4.5 --- Effects of chymotrypsin on MCCI --- p.37 / Chapter 1.4.6 --- Stability of MCCI on heating and at different pH --- p.37 / Chapter 1.5 --- Discussion --- p.42 / Chapter Chapter 2 --- Immunomodulatory effect of MCCI / Chapter 2.1 --- Introduction of the immune system and protease inhibitors --- p.51 / Chapter 2.2 --- Rationale of the present study --- p.55 / Chapter 2.3 --- Materials and methods --- p.56 / Chapter 2.3.1 --- Materials --- p.56 / Chapter 2.3.2 --- Isolation of different types of immune cells --- p.57 / Chapter 2.3.3 --- Determination of cell proliferation --- p.60 / Chapter 2.3.4 --- Determination of H2O2 formation --- p.61 / Chapter 2.3.5 --- Assay of interleukin-2 --- p.61 / Chapter 2.3.6 --- Determination of cell viability --- p.62 / Chapter 2.4 --- Results --- p.63 / Chapter 2.4.1 --- Murine splenocytes --- p.63 / Chapter 2.4.1.1 --- In vitro effect of MCCI on the proliferation of murine splenocytes --- p.63 / Chapter 2.4.1.2 --- Effect of MCCI on cytokine production --- p.63 / Chapter 2.4.2 --- Murine lymphocytes --- p.66 / Chapter 2.4.2.1 --- In vitro effect of MCCI on the proliferation of lymphocytes --- p.66 / Chapter 2.4.2.2 --- Effect of MCCI on cytokine production --- p.66 / Chapter 2.4.3 --- Murine bone marrow cells --- p.69 / Chapter 2.4.3.1 --- Effect of MCCI on the growth of murine bone marrow cells --- p.69 / Chapter 2.4.4 --- Murine neutrophills --- p.69 / Chapter 2.4.4.1 --- Effect of MCCI on H2O2 formation --- p.69 / Chapter 2.4.5 --- Murine macrophages --- p.71 / Chapter 2.4.5.1 --- Effect of MCCI on the growth of macrophages --- p.71 / Chapter 2.4.5.2 --- Effect of external ATP on the growth of macrophages --- p.71 / Chapter 2.4.5.3 --- Effect of ATP on the growth of macrophages pre-treated with MCCI --- p.76 / Chapter 2.4.5.4 --- Effect of MCCI on the growth of macrophages pre-treated with ATP --- p.76 / Chapter 2.4.5.5 --- Effect of MCCI on H201production --- p.79 / Chapter 2.5 --- Discussion --- p.82 / Chapter Chapter 3 --- Anti-oxidative effect of MCCI in primary rat hepatocytes culture / Chapter 3.1 --- Introduction --- p.91 / Chapter 3.1.1 --- Liver disease and protease inhibitors --- p.91 / Chapter 3.1.2 --- Primary rat hepatocyte as a pharmacological model --- p.93 / Chapter 3.1.3 --- tert-Butyl hydroperoxide as an oxidative stress inducer --- p.94 / Chapter 3.1.4 --- Endogenous antioxidant enzymes against ROS --- p.96 / Chapter 3.2 --- Rationale of the present study --- p.99 / Chapter 3.3 --- Materials and methods --- p.101 / Chapter 3.3.1 --- Materials --- p.101 / Chapter 3.3.2 --- Isolation of primary rat hepatocytes --- p.101 / Chapter 3.3.2.1 --- Liver perfusion --- p.101 / Chapter 3.3.2.2 --- Preparation of collagen pre-coated culture plates --- p.103 / Chapter 3.3.2.3 --- Hepatocytes culturing --- p.103 / Chapter 3.3.3 --- Drug treatment and oxidative stress induction --- p.104 / Chapter 3.3.4 --- Cytotoxicity assessment --- p.105 / Chapter 3.3.5 --- Cellular GSH content determination --- p.105 / Chapter 3.3.6 --- Protein determination by Lowry's method --- p.106 / Chapter 3.3.7 --- Medium MDA determination --- p.106 / Chapter 3.3.8 --- Medium GSSG determination --- p.107 / Chapter 3.3.9 --- Antioxidant enzymes measurement --- p.108 / Chapter 3.3.9.1 --- Catalase measurement --- p.108 / Chapter 3.3.9.2 --- SOD measurement --- p.109 / Chapter 3.3.9.3 --- GST measurement --- p.109 / Chapter 3.3.9.4 --- GR measurement --- p.110 / Chapter 3.3.10 --- Statistical analysis --- p.110 / Chapter 3.4 --- Results --- p.111 / Chapter 3.4.1 --- Cytotoxicity of MCCI on rat hepatocytes --- p.111 / Chapter 3.4.2 --- Effect of tBHP and MCCI on hepatocytes viability --- p.111 / Chapter 3.4.3 --- Effects of tBHP and MCCI on hepatocytes GSH and GSSG content --- p.117 / Chapter 3.4.4 --- Effect of MCCI on lipid peroxidation of hepatocytes --- p.121 / Chapter 3.4.5 --- Effect of MCCI on antioxidant enzymes activities --- p.121 / Chapter 3.4.6 --- Comparison with typical antioxidants --- p.125 / Chapter 3.5 --- Discussion --- p.127 / Chapter Chapter 4 --- Cytotoxicity of MCCI on tumor cell lines / Chapter 4.1 --- Introduction --- p.134 / Chapter 4.1.1 --- Relationship between protease inhibitors and cancer --- p.134 / Chapter 4.1.2 --- Cell cycle and apoptosis --- p.137 / Chapter 4.2 --- Rationale of the present study --- p.140 / Chapter 4.3 --- Materials and methods --- p.141 / Chapter 4.3.1 --- Materials --- p.141 / Chapter 4.3.2 --- Cell culture --- p.141 / Chapter 4.3.3 --- MTT assay --- p.142 / Chapter 4.3.4 --- Cell cycle analysis --- p.142 / Chapter 4.3.5 --- DNA fragmentation --- p.143 / Chapter 4.4 --- Results --- p.136 / Chapter 4.4.1 --- Cytotoxicity of MCCI --- p.144 / Chapter 4.4.2 --- Cell cycle and apoptosis analysis --- p.147 / Chapter 4.5 --- Discussion --- p.152 / Conclusion and future perspectives --- p.157 / References --- p.160

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

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