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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

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

The influenza polymerase is a complex of three subunits, polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1) and polymerase acidic protein (PA). It associates with the viral RNA segment and nucleoprotein (NP) to form a viral ribonucleoprotein (vRNP) complex which is important for transcription and replication of the viral genome. Concurrently, the previous three influenza pandemics viruses contain reassorted vRNP of different origins. This leads to the aim of study to investigate the role of polymerase in the pandemic viruses. By reconstitution of vRNPs in human cells, it was demonstrated that vRNPs of H2N2 and H3N2 pandemic viruses had higher polymerase activity than the H2N2 seasonal viruses in-between them. The recombinant virus with H2N2 pandemic vRNP also showed faster growth kinetics in the early stage of viral replication and better adaptability to the selective environment with neuraminidase inhibitor than the recombinant virus with H2N2 seasonal vRNP, which had a lower polymerase activity. Reconstitution of chimeric vRNPs of H2N2 pandemic and seasonal viruses revealed that PB2, PB1 and PA were responsible for the difference in polymerase activity between them. Five residues, one in PB2, three in PB1 and one in PA were identified to be significant for the polymerase activity change. These polymerase subunits and residues may act as part of the determining factors for the H2N2 pandemic virus. Furthermore, PB2-627 has been shown to have stringent host specificity and affect polymerase activity and viral replication. Recombinant viruses in mammalian and avian cells with random mutation were generated at this position. It showed that the amino acids at this position are not restricted to those appear in the nature for generating viable viruses. It was also observed that the avian-derived viruses generally had lower polymerase activity and reduced growth kinetics in mammalian cells, while part of the mammalian-derived viruses had lower polymerase activity and reduced growth kinetics in avian cells. This consolidated the role of PB2-627 on host specificity and demonstrated the possibility of some novel amino acids for this position, which may play a role in the future influenza pandemic. The 2009 H1N1 pandemic virus contains a reassorted vRNP with subunits of avian, human and swine origins. This prompts me to compare the polymerase activity of all the 81 possible combinations of chimeric vRNPs of three different origins. The results were statistically analyzed and several single subunit factors and interactions between vRNP subunits were identified to significantly affect the polymerase activity. In order to reduce the effort and resources required, a fractional factorial design of 27 experimental runs was developed to substitute the 81-combination full factorial design for identifying the significant single subunit factors that affect the polymerase activity. Overall, this study identified some factors that may contribute to a pandemic virus and allows us to have better understanding of the role of polymerase in a pandemic virus. These findings may contribute to evaluating the pandemic potential of the novel virus that emerges or may emerge in the nature and enhances the preparedness towards the next pandemic influenza. / published_or_final_version / Public Health / Doctoral / Doctor of Philosophy

There is no abstract available for this thesis. / Department of Biology

Thesis (Ph. D.)--Ohio State University, 2007.

Thesis (M.Sc.)--York University, 2009. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 108-139). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR51542

Bunyamwera virus (BUNV) is the prototype of the family Bunyaviridae. It has a tripartite genome consisting of negative sense RNA segments called large (L), medium (M) and small (S). The S segment encodes the nucleocapsid protein (N) of 233 amino acids. The N protein encapsidates all three segments to form transcriptionally active ribonucleoproteins (RNPs). The aim of this project was to determine the domain map of BUNV N protein. To investigate residues in BUNV N crucial for its functionality, random and site- specific mutagenesis were performed on a cDNA clone encoding the BUNV N protein. In total, 102 single amino acid substitutions were generated in the BUNV N protein sequence. All mutant N proteins were used in a BUNV minigenome system to compare their activity to wt BUNV N. The mutant proteins displayed a wide-range of activity, from parental-like to essentially inactive. The most disruptive mutations were R94A, I118N, W134A, Y141C, L177A, K179I and W193A. Sixty-four clones carrying single substitutions in the BUNV N protein were used in the BUNV rescue system in an attempt to recover viable mutant viruses. Fifty recombinant mutant viruses were rescued and 14 N genes were nonrescuable. The 50 mutant viruses were characterized by: titration, protein labelling, western blotting, temperature sensitivity and host-restriction. Mutant viruses displayed a wide range of titers between 10³ -10⁸ pfu/ml, and three different plaque sizes large, medium and small. Protein labelling and western blotting showed that mutations in the N gene did not affect expression of the other viral genes as much as affecting N protein expression. It was demonstrated that single amino acid substitutions could alter N protein electrophoretic mobility in SDS- PAGE (e.g. P19Q and L53F). Temperature sensitivity tests showed that recombinant viruses N74S, S96S, K228T and G230R were ts, growing at 33˚C but not at 37˚C or 38˚C, while the parental virus grew at all temperatures. Using the northern blotting technique, mutant viruses N74S and S96G were shown to have a ts defect in genome-synthesis (late replication step), while mutant viruses K228T and G230R had a ts defect in antigenome- synthesis (early replication step). Host-restriction experiments were performed using 5 different cell lines (Vero-E6, BHK-21, 2FTGH-V, A549-V and 293-V). Overall, the parental virus grew similarly in all cell lines. Likewise, the majority of mutant viruses follow this pattern except mutant virus Y23A. It showed a 100-fold reduction in titer in 2FTGH-V cells. Comparing the ratios of intracellular and extracellular particles revealed that only 15% of the total virus particles of mutant Y23A was released as extracellular particles compared to 30% of the parental virus. Fourteen N genes were nonrescuable. They were characterized by (i) their activity in the BUNV minigenome system, (ii) their activity in BUNV packaging assay, (iii) their ability to form multimers, (iv) their ability to interact with L protein, and (v) their impact on RNA synthesis. In summary, BUNV N protein was shown to be multi-functional and involved in the regulation of virus transcription and replication, RNA synthesis and assembly, via interactions with the viral L polymerase, RNA backbone, itself or the viral glycoproteins.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy