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

Lau, Yuen Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 156-163). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.iii / Acknowledgements --- p.iv / Table of content --- p.V / List of figures --- p.ix / List of tables --- p.xiii / List of abbreviations --- p.xiv / Chapter 1 --- INTRODUCTION --- p.Page / Chapter 1.1 --- Embryonic stem cells (ESCs) --- p.1 / Chapter 1.1.1 --- What are ESCs and the characteristics of ESCs --- p.1 / Chapter 1.1.2 --- Promising use of ESCs in drug development and regenerative medicine --- p.1 / Chapter 1.1.3 --- Maintenance of self-renewal and pluripotent properties of ESCs --- p.3 / Chapter 1.2 --- Cell cycle in ESCs --- p.5 / Chapter 1.2.1 --- Cell cycle --- p.5 / Chapter 1.2.2 --- Characteristics of cell cycle of ESCs --- p.6 / Chapter 1.3 --- Telomere --- p.8 / Chapter 1.3.1 --- Telomere structure and the telomeric proteins --- p.8 / Chapter 1.3.2 --- End replication problem --- p.10 / Chapter 1.3.3 --- Telomere dysfunction in cancer and cellular aging --- p.11 / Chapter 1.4 --- Telomerase --- p.12 / Chapter 1.4.1 --- Telomerase and stem cell characteristics --- p.13 / Chapter 1.4.1.1 --- Telomerase and cell proliferation --- p.13 / Chapter 1.4.1.2 --- Telomerase and stem cell differentiation --- p.14 / Chapter 1.4.2 --- Regulation of telomerase expression/ activity --- p.15 / Chapter 1.4.2.1 --- Regulation of telomerase at different levels --- p.15 / Chapter 1.4.2.2 --- Regulation of telomerase activity by cellular components in ESCs --- p.16 / Chapter 1.5 --- PinXl --- p.18 / Chapter 1.5.1 --- Expression of PinXl --- p.18 / Chapter 1.5.2 --- Effects of PinXl on the activities and the sub-cellular localization of telomerase --- p.19 / Chapter 1.5.3 --- Structure-function relationship of PinXl --- p.19 / Chapter 1.5.4 --- Effect of PinXl on the growth rate of normal and cancer cells --- p.21 / Chapter 1.5.5 --- Other functions of PinX 1 V --- p.22 / Chapter 1.5.6 --- Mouse homolog of PinXl and its function in mESCs --- p.23 / Chapter 1.6 --- Aims of this study --- p.24 / Chapter 2 --- METERIALS AND METHODS --- p.Page / Chapter 2.1 --- mESC culture and differentiation --- p.25 / Chapter 2.1.1 --- Cell line --- p.25 / Chapter 2.1.2 --- Irradiation of MEF --- p.25 / Chapter 2.1.3 --- mESC culture --- p.26 / Chapter 2.1.4 --- Differentiation of mESCs --- p.26 / Chapter 2.1.5 --- Establishment and' culture of feeder-free mESCs --- p.28 / Chapter 2.1.6 --- Culture of feeder-free mESCs --- p.28 / Chapter 2.2 --- Trypan Blue Exclusion Assay --- p.29 / Chapter 2.3 --- Sub-cloning --- p.29 / Chapter 2.3.1 --- Amplification of the insert gene by PCR --- p.29 / Chapter 2.3.2 --- Purification of PCR products --- p.31 / Chapter 2.3.3 --- Restriction enzyme digestion --- p.32 / Chapter 2.3.4 --- Ligation of digested insert and vector --- p.33 / Chapter 2.3.5 --- Transformation of ligation product into competent cells --- p.34 / Chapter 2.3.6 --- Confirmation of positive clone by colony PCR --- p.34 / Chapter 2.3.7 --- Small scale preparation of the recombinant plasmid DNA --- p.35 / Chapter 2.3.8 --- Confirmation of positive clone by restriction digestion --- p.36 / Chapter 2.3.9 --- DNA sequencing of the recombinant plasmid DNA --- p.36 / Chapter 2.3.10 --- Large scale preparation of the recombinant plasmid DNA --- p.37 / Chapter 2.4 --- Design of siRNA targeting mPinXl and mPinXlt --- p.38 / Chapter 2.5 --- Transient transfection --- p.38 / Chapter 2.6 --- Cloning of siRNA into shRNA insert in Lentiviral Vector pLVTHM --- p.39 / Chapter 2.7 --- Lentiviral vector-mediated gene transfer to mESCs --- p.42 / Chapter 2.7.1 --- Lentivirus packaging --- p.42 / Chapter 2.7.2 --- Checking of successful transduction by lentivirus in HEK cells --- p.43 / Chapter 2.7.3 --- Multiple transductions to mESCs --- p.43 / Chapter 2.7.4 --- Selection of positive clones --- p.44 / Chapter 2.7.5 --- Monoclonal establishment --- p.44 / Chapter 2.8 --- "Total RNA preparation, Reverse Transcription (RT) and Quantitative Polymerase Chain Reaction (qPCR)" --- p.45 / Chapter 2.9 --- Immunocytochemistry --- p.46 / Chapter 2.10 --- Western Blotting --- p.48 / Chapter 2.10.1 --- Total Protein Extraction vi --- p.48 / Chapter 2.10.2 --- Measurement of Protein Concentration --- p.48 / Chapter 2.10.3 --- SDS-PAGE and chemiluminescent detection --- p.49 / Chapter 2.11 --- Co-immunoprecipitation --- p.51 / Chapter 2.12 --- Telomere Repeat Amplification Protocol (TRAP) Assay --- p.52 / Chapter 2.13 --- Cell cycle analysis --- p.54 / Chapter 2.14 --- MTT assay --- p.54 / Chapter 2.15 --- Statistical analysis --- p.55 / Chapter 3 --- RESULTS --- p.Page / Chapter 3.1 --- mPinXlt was discovered in mESCs --- p.56 / Chapter 3.2 --- mPinXl and mPinXlt were expressed at transcriptional level in the inspected mouse tissues --- p.61 / Chapter 3.3 --- Expression of mPinXl and mPinXlt changed upon differentiation --- p.64 / Chapter 3.4 --- mPinXl and mPinXlt were both located in the nucleolus and the nucleoplasm in undifferentiated mESCs --- p.69 / Chapter 3.5 --- Co-immunoprecipitation (Co-IP) of mPinXl and mPinXlt with mTERT --- p.73 / Chapter 3.6 --- Transient knockdown of mPinXl in mESCs --- p.78 / Chapter 3.6.1 --- Knockdown of mPinXl decreased proliferation but did not change cell viability --- p.79 / Chapter 3.6.2 --- Knockdown of mPinXl decreased telomerase activity --- p.79 / Chapter 3.6.3 --- Knockdown of mPinXl did not change pluripotency --- p.80 / Chapter 3.6.4 --- Knockdown of mPinXl did not affect cell cycle progression --- p.80 / Chapter 3.7 --- Transient knockdown of mPinXlt using siRNA against mPinXlt in mESCs --- p.88 / Chapter 3.8 --- Transient over-expression of mPinXl and mPinXlt in mESCs --- p.90 / Chapter 3.8.1 --- Over-expression of mPinXl and mPinXlt decreased cell proliferation but didn't affect cell viability --- p.91 / Chapter 3.8.2 --- Over-expression of mPinXl increased telomerase activity --- p.92 / Chapter 3.8.3 --- Over-expression of mPinXl and mPinXlt did not affect pluripotency --- p.93 / Chapter 3.8.4 --- Over-expression of mPinXl and mPinXlt did not affect cell cycle progression --- p.93 / Chapter 3.9 --- Stable over-expression and knockdown of mPinXl and mPinXlt in mESCs --- p.103 / Chapter 3.9.1 --- Expression of mPinXl and mPinXlt at mRNA and protein levels in all over-expression stable cell lines --- p.108 / Chapter 3.9.2 --- Expression of mPinXl and mPinXlt at mRNA and protein levels in mPinXl knockdown stable cell lines --- p.113 / Chapter 3.9.3 --- Proliferation of all stable cell lines --- p.116 / Chapter 3.9.4 --- Telomerase activity of all stable cell lines --- p.121 / Chapter 3.9.5 --- Cell cycle distribution of all stable cell lines --- p.123 / Chapter 3.9.6 --- Pluripotency of all stable cell lines --- p.127 / Chapter 3.9.7 --- Differentiation of the stable cell lines --- p.130 / Chapter 3.9.7.1 --- Size of EBs formed from stable cell lines at Day 7 --- p.130 / Chapter 3.9.7.2 --- Beating curves of the stable cell lines derived EBs --- p.130 / Chapter 4 --- DISCUSSIONS --- p.Page / Chapter 4.1 --- mPinXlt gene was detected in mESCs --- p.137 / Chapter 4.2 --- "Presence of mPinXl and mPinXlt in mouse tissues, mESCs and their differentiation derivatives" --- p.138 / Chapter 4.3 --- Differences in expressions of mPinXl and mPinXlt in undifferentiated mESCs and their differentiation derivatives --- p.139 / Chapter 4.4 --- mPinXl and mPinXlt are pre-dominantly localized in the nucleolus --- p.141 / Chapter 4.5 --- mPinXl and mPinXlt interacted with mTERT --- p.143 / Chapter 4.6 --- "Transient knockdown of mPinXl slightly inhibited, while over-expression of mPinXl slightly promoted telomerase activity" --- p.143 / Chapter 4.7 --- Both transient knockdown and over-expression of mPinXl inhibited the growth of mESCs --- p.146 / Chapter 4.8 --- Both stable knockdown and over-expression of mPinXl did not affect cell proliferation and telomerase activity of mESCs --- p.148 / Chapter 4.9 --- Involvement of mPinXl and mPinXlt in the differentiation process of mESCs --- p.149 / Chapter 4.10 --- Regulation of mPinXl gene expression by mPinXlt --- p.151 / Chapter 4.11 --- Future perspectives --- p.152 / Chapter 5 --- CONCLUSION --- p.154 / Chapter 6 --- REFERENCES --- p.156

O estresse é muito presente na vida das pessoas, e grande parte deste é de origem social. O estresse contínuo induz a uma série de efeitos nocivos no organismo. Estes efeitos também podem ser observados no cérebro, principalmente no hipocampo, onde promove alterações na sua morfologia e função. Recentemente, demonstrou-se uma redução do tamanho dos telômeros em leucócitos de mulheres com alto grau de estresse. Os telômeros são seqüências repetitivas dos nucleotídeos TTAGGG, responsáveis por proteger as extremidades dos cromossomos, e sua manutenção e alongamento é mediado, principalmente, pela enzima telomerase. Telômeros e telomerase têm sido alvos de muitos estudos envolvendo o envelhecimento e morte celular. Assim, o presente trabalho estudou os efeitos do estresse psicossocial prolongado em um modelo animal de subjugação/submissão sobre a biologia telomérica do hipocampo. O modelo de subjugação consistiu em um pareamento dos animais experimentais com um camundongo agressivo por 30 minutos. Camundongos machos jovens adultos da linhagem C57BL/6J passaram por este modelo de estresse diariamente por 21 dias. Observou-se um aumento na temperatura corpórea basal em repouso no 21º dia, bem como diminuição na hipertermia reativa a um estresse agudo de manipulação, demonstrando a efetividade do modelo em indução de estresse. Os tamanhos dos telômeros, a expressão gênica da enzima telomerase e sua atividade protéica foram investigados no hipocampo dos camundongos que sofreram o estresse crônico e comparados com animais do grupo controle. Observou-se, após o período de indução do estresse, uma diminuição em aproximadamente 58% no tamanho relativo médio dos telômeros das células hipocampais nos camundongos submetidos ao estresse psicossocial, quando comparados ao grupo controle. A quantidade de RNAm relativo ao gene da telomerase, bem como sua atividade enzimática, mostraram-se similares entre os grupos controle e experimental. Estes resultados sugerem que o estresse psicossocial prolongado reduz o tamanho dos telômeros do hipocampo de camundongos e que esta redução não parece ser devido a alterações gênicas ou enzimáticas da telomerase. Com este estudo procuramos entender melhor os mecanismos celulares e genômicos cerebrais modulados pelo estresse psicológico prolongado, de natureza social. Uma vez que muitos distúrbios psiquiátricos e neurodegenerativos são provenientes do estresse crônico, nossos resultados fornecem mais subsídios para se evidenciar a importância do ambiente social na saúde mental dos indivíduos. / Stressor stimuli are part of the daily life and the major part is derived from the social context. Long term stress can be very harmful, and induce several noxious events in the organism. These effects can also be perceived in the central nervous system, particularly in the hippocampus, in which morphological and functional damages may be observed. Recently a correlation of perceived stress and telomere length was demonstrated in leukocytes of stressed women. Telomeres, which are in tanden repeats of the nucleotides TTAGGG, are responsible to protect the extremities of the chromosomes, and it´s maintenance and elongation is mainly mediated by the enzyme telomerase. Telomeres and telomerase have been the targets of many studies in the last years, concerning aging and cell death. In this regard, the present study has investigated the effects of prolonged psychosocial defeat stress model in the telomeric biology of the hippocampus. Male C57Bl/6J young adult mice were submitted to a 21 days of psychosocial stress. We observed a raise in body temperature, as well as a decrease in reactive hyperthermia to the handling stress, demonstrating the effectiveness of this stress model. The telomere length, the transcript levels of telomerase mRNA, and the activity of the enzyme telomerase were also investigated in the hippocampus of psychosocially stressed mouse. A decrease of 58% in average telomere length was observed in the hippocampal cells of stressed mice when compared to control group. Levels of telomerase mRNA and telomerase activity were similar between control and defeat groups. In this study, we intend to better understand the cellular and genomic mechanisms promoted by long term psychological stress, with social nature, in brain. Since many psychiatric and neurodegenerative disorders have chronic stress underlying them, our results reinforce the importance of the social environment on individual mental health.

O estresse é muito presente na vida das pessoas, e grande parte deste é de origem social. O estresse contínuo induz a uma série de efeitos nocivos no organismo. Estes efeitos também podem ser observados no cérebro, principalmente no hipocampo, onde promove alterações na sua morfologia e função. Recentemente, demonstrou-se uma redução do tamanho dos telômeros em leucócitos de mulheres com alto grau de estresse. Os telômeros são seqüências repetitivas dos nucleotídeos TTAGGG, responsáveis por proteger as extremidades dos cromossomos, e sua manutenção e alongamento é mediado, principalmente, pela enzima telomerase. Telômeros e telomerase têm sido alvos de muitos estudos envolvendo o envelhecimento e morte celular. Assim, o presente trabalho estudou os efeitos do estresse psicossocial prolongado em um modelo animal de subjugação/submissão sobre a biologia telomérica do hipocampo. O modelo de subjugação consistiu em um pareamento dos animais experimentais com um camundongo agressivo por 30 minutos. Camundongos machos jovens adultos da linhagem C57BL/6J passaram por este modelo de estresse diariamente por 21 dias. Observou-se um aumento na temperatura corpórea basal em repouso no 21º dia, bem como diminuição na hipertermia reativa a um estresse agudo de manipulação, demonstrando a efetividade do modelo em indução de estresse. Os tamanhos dos telômeros, a expressão gênica da enzima telomerase e sua atividade protéica foram investigados no hipocampo dos camundongos que sofreram o estresse crônico e comparados com animais do grupo controle. Observou-se, após o período de indução do estresse, uma diminuição em aproximadamente 58% no tamanho relativo médio dos telômeros das células hipocampais nos camundongos submetidos ao estresse psicossocial, quando comparados ao grupo controle. A quantidade de RNAm relativo ao gene da telomerase, bem como sua atividade enzimática, mostraram-se similares entre os grupos controle e experimental. Estes resultados sugerem que o estresse psicossocial prolongado reduz o tamanho dos telômeros do hipocampo de camundongos e que esta redução não parece ser devido a alterações gênicas ou enzimáticas da telomerase. Com este estudo procuramos entender melhor os mecanismos celulares e genômicos cerebrais modulados pelo estresse psicológico prolongado, de natureza social. Uma vez que muitos distúrbios psiquiátricos e neurodegenerativos são provenientes do estresse crônico, nossos resultados fornecem mais subsídios para se evidenciar a importância do ambiente social na saúde mental dos indivíduos. / Stressor stimuli are part of the daily life and the major part is derived from the social context. Long term stress can be very harmful, and induce several noxious events in the organism. These effects can also be perceived in the central nervous system, particularly in the hippocampus, in which morphological and functional damages may be observed. Recently a correlation of perceived stress and telomere length was demonstrated in leukocytes of stressed women. Telomeres, which are in tanden repeats of the nucleotides TTAGGG, are responsible to protect the extremities of the chromosomes, and it´s maintenance and elongation is mainly mediated by the enzyme telomerase. Telomeres and telomerase have been the targets of many studies in the last years, concerning aging and cell death. In this regard, the present study has investigated the effects of prolonged psychosocial defeat stress model in the telomeric biology of the hippocampus. Male C57Bl/6J young adult mice were submitted to a 21 days of psychosocial stress. We observed a raise in body temperature, as well as a decrease in reactive hyperthermia to the handling stress, demonstrating the effectiveness of this stress model. The telomere length, the transcript levels of telomerase mRNA, and the activity of the enzyme telomerase were also investigated in the hippocampus of psychosocially stressed mouse. A decrease of 58% in average telomere length was observed in the hippocampal cells of stressed mice when compared to control group. Levels of telomerase mRNA and telomerase activity were similar between control and defeat groups. In this study, we intend to better understand the cellular and genomic mechanisms promoted by long term psychological stress, with social nature, in brain. Since many psychiatric and neurodegenerative disorders have chronic stress underlying them, our results reinforce the importance of the social environment on individual mental health.

abstract: The highly specialized telomerase ribonucleoprotein enzyme is composed minimally of telomerase reverse transcriptase (TERT) and telomerase RNA (TR) for catalytic activity. Telomerase is an RNA-dependent DNA polymerase that syntheizes DNA repeats at chromosome ends to maintain genome stability. While TERT is highly conserved among various groups of species, the TR subunit exhibits remarkable divergence in primary sequence, length, secondary structure and biogenesis, making TR identification extremely challenging even among closely related groups of organisms. A unique computational approach combined with in vitro telomerase activity reconstitution studies was used to identify 83 novel TRs from 10 animal kingdom phyla spanning 18 diverse classes from the most basal sponges to the late evolving vertebrates. This revealed that three structural domains, pseudoknot, a distal stem-loop moiety and box H/ACA, are conserved within TRs from basal groups to vertebrates, while group-specific elements emerge or disappear during animal TR evolution along different lineages. Next the corn-smut fungus Ustilago maydis TR was identified using an RNA-immunoprecipitation and next-generation sequencing approach followed by computational identification of TRs from 19 additional class Ustilaginomycetes fungi, leveraging conserved gene synteny among TR genes. Phylogenetic comparative analysis, in vitro telomerase activity and TR mutagenesis studies reveal a secondary structure of TRs from higher fungi, which is also conserved with vertebrates and filamentous fungi, providing a crucial link in TR evolution within the opisthokonta super-kingdom. Lastly, work by collabarotors from Texas A&M university and others identified the first bona fide TR from the model plant Arabidopsis thaliana. Computational analysis was performed to identify 85 novel AtTR orthologs from three major plant clades: angiosperms, gymnosperms and lycophytes, which facilitated phylogenetic comparative analysis to infer the first plant TR secondary structural model. This model was confirmed using site-specific mutagenesis and telomerase activity assays of in vitro reconstituted enzyme. The structures of plant TRs are conserved across land plants providing an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2019

No description available.

No description available.

(Uncorrected OCR) Abstract of thesis entitled Telomerase activation and telomeric repeats alteration in sex hormone-induced prostatic carcinogenesis in the Noble rat submitted by Chan Ching for the Degree of Master of Philosophy at the University of Hong Kong In November, 1998 Despite its distinction as the most frequently diagnosed cancer and the�second leading cause of cancer deaths in men, little is known about the causes and mechanisms of prostatic carcinogenesis. The animal model, based on the existing sex hormone-induced prostate carcinogenesis in the Noble rat, by substantially increasing the dosage of testosterone while keeping the level of estrogen unchanged has been reported. With this modified protocol, we have successfully induced high incidence of prostate carcinoma in Noble rats. The earliest time of development of dysplasia was two months, carcinoma in situ at 4 months and fully developed carcinoma at six months. The tumor incidence was 92% after 12 months hormonal implantation. This animal model is very useful in the investigation of prostate cancer as its multi -step nature mimics the human situation and its high incidence in relative short time. Telomerase activation is a characteristic of immortalized tumor cells and is thought to contribute to the mechanism by which these cells abort the normal process of senescence. Telomerase activity has been detected in various human cancers and there are reports showing that telomeric repeat fragment (TRF) length may be correlated to the staging of tumors. These findings suggested that telomerase activation and TRF length alteration might be useful in prognosis of different cancers. Study of prostate cancer on animal model can compensate for the difficulties in human cancer research, as stage-by-stage investigations are available. In this study, we proposed that telomerase would be activated in sex hormone-induced prostatic carcinogenesis and TRF length alterations might be correlated to the different stages of prostatic carcinogenesis. We have examined the telomerase activation as well as telomeric repeat fragment content alteration in the hormonal induced prostatic carcinogenesis in Noble rat. Telomerase activation is common in prostate carcinoma and also can be detected in 12% of non-malignant tissues. For the non-prostatic tissues tested, all the testis tissues (n=5) were strongly positive for telomerase activity and only one liver tissue (n=5) showed weak. telomerase activity. The high frequency of telomerase activation in prostatic carcinoma specimens suggested that it might be a useful malignancy marker for prognosis evaluation in prostatic carcinogenesis. There were alterations of TRF content in dorsal lateral prostate. The ventral prostate tissues have the similar results as the dorsal lateral prostate. It seems that the TRF content in normal tissues is less than that of hyperplasia, dysplasia and carcinoma tissues. However, no obvious relationships between the TRF content and clinicopathological properties of prostatic carcinogenesis were observed. Also, there was no significant relation between the telomerase activation and TRF content in this study. Hence, the TRF content appear to have no significant correlation with prostatic carcinogenesis. / abstract / toc / Anatomy / Master / Master of Philosophy

Thesis (Ph. D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Human Genetics. Title from resource description page. Includes bibliographical references.

Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2005. / "A dissertation in pharmaceutical sciences and chemistry." Advisor: Simon H. Friedman. Typescript. Vita. Description based on contents viewed June 23, 2006; title from "catalog record" of the print edition. Includes bibliographical references (leaves 327-353). Online version of the print edition.