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

The role of male germ cell death in spermatogenesis is an important one as it removes dysfunctional or genetically damaged germ cells and is necessary to maintain an optimal germ cell to Sertoli cell ratio. The formation of the bloodtestis barrier requires the elimination of excessive germ cells and a surge of germ cell apoptosis occurs prior to puberty regulating the ratio of germ cells to Sertoli cells. The aim of this study was to evaluate the presence of four apoptotic markers on sperm from patients with various grades of fertility using flow cytometry. Furthermore, any correlations between the apoptotic marker assays and the standard semen analysis results were identified. This study compares early and late parameters of apoptosis with morphological features in spermatozoa in the same samples. The three sample groups were identified as: teratozoospermic [G-pattern] (n=26), teratozoospermic [P-pattern] (n=98) and oligoteratozoospermic [Ppattern] (n=36). Standard semen analysis was conducted on the semen samples according to the WHO guidelines. Four apoptotic marker assays using flow cytometry was applied in this study to examine the apoptotic alterations in ejaculate sperm. These assays included the Annexin-V staining for the determination of phosphatidylserine exposure, APO-Direct to identify DNA fragmentation, caspase-3 to detect expression of this active protease during early apoptosis and Fas expression. For the Annexin-V and caspase-3 assays, statistically significant differences (P&lt / 0.05) were evident between the three groups. No significant differences (P&gt / 0.05) were found between the groups with respect to the APO-Direct assay. A significant difference (P&lt / 0.05) was found when comparing the teratozoospermic [G-pattern] group and the oligoteratozoospermic [P-pattern] group for the Fas assay. A strong positive correlation was evident between the Fas and the caspase-3 assays in the teratozoospermic [G-pattern] group. For the teratozoospermic [P-pattern group] the following positive correlations existed between the APO-Direct and the Fas assays, APO-Direct and caspase-3 assays and between caspase-3 and Fas assays. The only strong positive correlation was between the caspase-3 and APO-Direct assays in the oligoteratozoospermic [P-pattern] group. The presence of spermatozoa showing microscopic features resembling apoptosis has been identified in ten human ejaculate samples per sample group. Electron microscopy was used to identify morphological features of apoptosis in these human sperm samples. Classical apoptosis as observed in diploid cells could be identified in sperm and these included: loose fibrillarmicrogranular chromatin network, presence of vacuoles in the nuclear chromatin, membranous bodies within the vacuoles of the chromatin, partially disrupted nuclear membranes, plasma membrane protuberances and apoptotic bodies containing cytoplasmic vacuoles and dense masses. This study has confirmed that semen samples with abnormal semen parameters exhibit the presence of apoptotic markers in sperm. The identification of apoptotic markers on the sperm suggests that abnormalities occur during their developmental process, however, the exact mechanism thereof remains unclear. These findings may suggest that certain apoptotic markers may be an indicator of abnormal sperm function and possibly indicative of male infertility.

by Tsang Chi Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 137-155). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.iii / Abstract --- p.vi / 撮要 --- p.ix / Publications --- p.xi / Table of contents --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Human mast cells and their pathological roles in inflammation --- p.1 / Chapter 1.1.1 --- Morphology of mast cells --- p.1 / Chapter 1.1.2 --- Mediators of mast cells --- p.1 / Chapter 1.1.3 --- Migration and activation --- p.3 / Chapter 1.1.4 --- Pathological roles of mast cells --- p.3 / Chapter 1.1.5 --- Human mast cell-1 (HMC-1) --- p.5 / Chapter 1.2 --- Cytokines as stimulator of mast cells in inflammation --- p.7 / Chapter 1.2.1 --- SCF --- p.7 / Chapter 1.2.2 --- TNF-α --- p.8 / Chapter 1.2.3 --- IL-13 --- p.8 / Chapter 1.2.4 --- IL-18 --- p.9 / Chapter 1.2.5 --- IL-25 --- p.9 / Chapter 1.3 --- Interaction of mast cells with inflammatory cells through adhesion molecules and chemokines --- p.11 / Chapter 1.3.1 --- Adhesion molecules on mast cells --- p.11 / Chapter 1.3.2 --- Chemokines released by mast cells --- p.12 / Chapter 1.4 --- Intracellular signaling pathways in mast cells --- p.16 / Chapter 1.4.1 --- p38-MAPK pathway --- p.16 / Chapter 1.4.2 --- ERK pathway --- p.17 / Chapter 1.4.3 --- NF-kB Pathway --- p.18 / Chapter 1.4.3 --- Cross-talking of pathways --- p.18 / Chapter 1.5 --- Signal transduction pathways and pharmacological intervention --- p.23 / Chapter 1.6 --- Traditional Chinese Medicine and pharmacological intervention --- p.25 / Chapter 1.6.1 --- Anti-allergic effects of traditional Chinese Medicine --- p.25 / Chapter 1.6.2 --- Anti-asthmatic effects of a newly developed Wheeze-Relief Formula --- p.26 / Chapter 1.7 --- Aims and scope of the study --- p.30 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- HMC-1 cell Line --- p.32 / Chapter 2.1.2 --- Media and reagents for cell culture --- p.32 / Chapter 2.1.3 --- Recombinant human cytokines --- p.33 / Chapter 2.1.4 --- "Signal transduction pathway inhibitors: PD98035, SB203580 and BAY 117082" --- p.34 / Chapter 2.1.5 --- Monoclonal antibodies and reagents for immunofluorescent staining --- p.34 / Chapter 2.1.6 --- Reagents and buffers for chemokine detection --- p.35 / Chapter 2.1.7 --- Reagents and buffers for total RNA extraction --- p.36 / Chapter 2.1.8 --- Reagents and buffers for reverse transcription 一 polymerase chain reaction (RT-PCR) --- p.37 / Chapter 2.1.9 --- Reagents and buffers for protein extraction --- p.40 / Chapter 2.1.10 --- Reagents and buffers for detection of activated signaling pathways --- p.41 / Chapter 2.1.11 --- Reagents and buffers for agarose gel electrophoresis --- p.42 / Chapter 2.1.12 --- Reagents and buffers for SDS-polyacrylamide gel electrophoresis (PAGE) --- p.43 / Chapter 2.1.13 --- Reagents and buffers for Western blot analysis --- p.45 / Chapter 2.1.14 --- Reagents and buffers for cDNA expression array analysis --- p.47 / Chapter 2.1.15 --- Reagents and buffers for cell viability and proliferation assay --- p.48 / Chapter 2.1.16 --- Reagent kit for endotoxin level assay --- p.49 / Chapter 2.2 --- Methods --- p.49 / Chapter 2.2.1 --- HMC-1 cell cultures --- p.49 / Chapter 2.2.2 --- Flow cytometry of cell surface expression of ICAM-1 and ICAM-3 --- p.50 / Chapter 2.2.3 --- Total cellular RNA extraction --- p.50 / Chapter 2.2.4 --- Reverse Transcription - Polymerase Chain Reaction (RT-PCR) --- p.51 / Chapter 2.2.5 --- Agarose gel electrophoresis --- p.51 / Chapter 2.2.6 --- "Quantitative analysis of IL-8, IP-10,MCP-1 and RANTES" --- p.52 / Chapter 2.2.7 --- Quantitative analysis of 1-309 and MIP-1β --- p.52 / Chapter 2.2.8 --- Detection of phosphorylated-ERX and phosphorylated-p38 MAPK --- p.53 / Chapter 2.2.9 --- Detection of NF-kB activity --- p.53 / Chapter 2.2.10 --- Detection of phosphorylated-ATF-2 --- p.53 / Chapter 2.2.11 --- Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) --- p.54 / Chapter 2.2.12 --- Western blot analysis --- p.54 / Chapter 2.2.13 --- MTT assay --- p.55 / Chapter 2.2.14 --- Cell proliferation assay --- p.55 / Chapter 2.2.15 --- Hot water extraction of TCM --- p.56 / Chapter 2.2.16 --- Endotoxin level assay --- p.56 / Chapter 2.2.17 --- cDNA expression array analysis --- p.57 / Chapter 2.2.18 --- Statistical analysis --- p.57 / Chapter Chapter 3 --- Results / Chapter 3.1 --- The effects of cytokines on the expression of ICAM-1 and ICAM-3 on HMC-1 --- p.59 / Chapter 3.1.1. --- "SCF, TNF-α and IL-13 up-regulated ICAM-1 but not ICAM-3 expression on HMC-1 cells" --- p.59 / Chapter 3.1.2. --- "SCF, TNF-α and IL-13 up-regulated the mRNA expression of ICAM-1" --- p.59 / Chapter 3.1.3 --- "The combined treatment of SCF and TNF-α, and SCF and IL-13 showed synergistic and additive effect on ICAM-1 expression respectively" --- p.60 / Chapter 3.1.4 --- Synergistic up-regulation of ICAM-1 expression in combined treatment of SCF and TNF-α was dose-dependently enhanced by SCF --- p.60 / Chapter 3.2 --- "The effects of cytokines on the release of IL-8, IP-10, MCP-1, RANTES, 1-309 and MIP-1β from HMC-1 cells" --- p.66 / Chapter 3.2.1 --- "SCF induced the release of IL-8, MCP-1, RANTES, 1-309 and MIP-1β" --- p.66 / Chapter 3.2.2 --- "TNF-a induced the release of IL-8, IP-10, MCP-1, RANTES and 1-309" --- p.66 / Chapter 3.2.3 --- SCF and TNF-α did not enhance the proliferation rate of HMC-1 --- p.66 / Chapter 3.3 --- "The effect of SCF and TNF-α on the activation of ERK, p38 MAPK and NK-kB" --- p.71 / Chapter 3.3.1 --- SCF activated ERK but not p38 MAPK and NF-kB --- p.71 / Chapter 3.3.2 --- TNF-α activated p38 MAPK and NF-kB but not ERK --- p.71 / Chapter 3.4 --- The effect of inhibitors on the SCF and TNF-a-induced release of chemokines --- p.76 / Chapter 3.4.1 --- "The optimal dose of PD98059, SB203580 and BAY117082" --- p.76 / Chapter 3.4.2 --- "PD98059 suppressed the SCF induced IL-8, MCP-1, RANTES, 1-309 and MIP-1β release from HMC-1 cells" --- p.76 / Chapter 3.4.3 --- SB203580 and BAY117082 differentially suppressed the TNF-α induced chemokine release from HMC-1 cells --- p.77 / Chapter 3.5 --- The effect of inhibitors on the SCF and TNF-a-induced upregulation of ICAM-1 --- p.83 / Chapter 3.5.1 --- BAY117082 but not SB203580 suppressed the TNF-α-induced ICAM-1 expression --- p.83 / Chapter 3.5.2 --- PD98059 and BAY 117082 suppressed the combined treatment of SCF and TNF-α induced ICAM-1 expression --- p.83 / Chapter 3.6 --- "Effect of inhibitors on TNF-α and SCF-induced ERK, p38 MAPK and NF-kB activities in HMC-1 cells." --- p.85 / Chapter 3.6.1 --- PD98059 suppressed the SCF-induced activity of ERK --- p.85 / Chapter 3.6.2 --- SB203580 and BAY117082 suppressed the TNF-α induced p38 MAPKand NF-kB activity respectively --- p.85 / Chapter 3.6.3 --- PD98059 suppressed the enhanced NF-kB activity after the combined treatment of SCF and TNF-α for 18 hours --- p.86 / Chapter 3.7 --- Effect of TNF-α and SCF on the gene expression profile of inflammatory cytokines and receptors of HMC-1 cells. --- p.90 / Chapter 3.8 --- The effects of TCM on the SCF-induced 1-309 and MCP-1 from HMC-1 cells --- p.95 / Chapter 3.8.1 --- "Endotoxin level of Radix astragali, Radix Scutellariae, Radix stemonae, Bulbus Fritillariae cirrhosae and Cordyceps sinensis" --- p.95 / Chapter 3.8.2 --- The effects of TCM on the proliferation rate of HMC-1 cells --- p.95 / Chapter 3.9.3 --- The effects of TCM on the SCF-induced release of 1-309 from HMC-1 cells --- p.96 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Involvement of adhesion molecules and chemokines in mast cell-mediated immunological events --- p.107 / Chapter 4.2 --- HMC-1 as the in vitro mast cell model adapted in my project --- p.108 / Chapter 4.3 --- The effect of cytokines on the expression of ICAM-1 and ICAM-3 in HMC-1 cells --- p.109 / Chapter 4.4 --- The effect of cytokines on the release of chemokines in HMC-1 cells --- p.111 / Chapter 4.5 --- "The regulation of ICAM-1, IL-8, IP-10, MCP-1, RANTES, 1-309 and MIP-1β through p-38 MAPK, ERK and NF-kB signaling pathways in HMC-1 cells" --- p.115 / Chapter 4.6 --- Further characterization of HMC-1 cells using cDNA array --- p.119 / Chapter 4.7 --- Investigating the in vitro anti-allergic activities of a newly developed Wheeze-relief formula using cytokine-activated HMC-1 cells --- p.128 / Chapter 4.8 --- Concluding remarks and future prospective --- p.132 / References --- p.137 / Appendix --- p.156

Heart failure, as a result of myocardial infarction, is a major cause of mortality in human. The main cause of heart failure is that when adult cardiomyocytes die in the infarct site they do not regenerate. Instead the infract site is replaced by fibroblasts and collagen scar. It is generally believed that cardiomyocytes have terminally differentiated and can not divide to replace cardiomyocytes that have lost following injury. However, recently published data have provided new evidence that there is a small but continuously turnover of cardiomyocytes in the adult heart. These new findings provide a new theory that the heart does possess a limited ability to regenerate. / I also examined the regenerative ability of cardiomyocyte in adult heart. MRL mice were used because previously it has been reported that the cardiomyocyte could proliferate in response to injury. To understand how the cardiomyocytes in the MRL mouse heart, I used a cryo-injury approach. I discovered that the cardiomyoctyes in MRL mouse hearts were capable of dividing shortly after cryo-injury. These MRL hearts healed without scarring in contrast to C57BL/6 control mice. It was discovered that BMP-2, GATA4 and Nkx2.5 were involved in the healing process. The activation of these genes induced the cardiomyocyte to re-enter the cell cycle so that new cardiomyocytes could replace the cell that have been lost in the infarct site. I also discovered that stem cells may also play a minor role in the healing process. / In summary, my research findings revealed that cardiomyocytes regeneration in the heart is a very complex process that involves the participation of many cells and signalling pathway. There findings raise many intriguing and important questions and are worthy of being addressed in the future. / Stem cell therapy has been proposed as a potential treatment for various myocardial diseases. Chen et al. (2004) found small chemical called reversine that could dedifferentiate C2C12 cells to become stem-like cells. In this study, I demonstrated that reversine could inhibit the growth of C2C12 cell. The presence of reversine in cell culture could significantly inhibit muscle-specific genes MyoD, Myogenin and Myf5 expression. These 3 muscle specific transcriptional genes are essential for maintaining muscle differentiation. The down regulation of these gene showed that reversine could dedifferentiate C2C12 cells. We also discovered that reversine-treated C2C12 cells could differentiate into cardiomyocytes when they were cocultured with cardiomyocytes or when transplanted into the infarct site of a cryo-injured heart. / To investigate the regenerative potential of cardiomyoctyes in adult heart, we tried first to uncover the signals that direct post-natal cardiomyocytes to enter into growth arrest and differentiation. In the first part of my study, I established that the cardiomycytes divided extensively in 2 day-old post-natal hearts and that the majority of these cells entered into growth arrest and terminal differentiation at day 13. Comparative proteomic techniques were used in order to identify proteins that might be associated with cardiomyocytes proliferation during terminal differentiation the mouse heart. Several proteins were found to be differently expressed and amongst them was cyclin I protein. Cyclin I was found strongly expressed in 13 day old hearts. The protein is involved in signaling growth arrested in cells. / Liu, Ye. / "November 2006." / Adviser: Lee Ka Ho. / Source: Dissertation Abstracts International, Volume: 68-09, Section: B, page: 5658. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 142-172). / 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. / Abstracts in English and Chinese. / School code: 1307.

The cystic fibrosis transmembrane conductance regulator (CFTR) was identified to be involved in acidosis-induced ATP release from skeletal myocytes in vitro and from contracting muscle in vivo. My PhD studies aimed to investigate the underlying mechanism and identify the pathway for ATP release in acidosis-induced CFTR-regulated ATP release. Lactic acid (10 mM) decreased the intracellular pH of L6 skeletal myocytes to 6.87 ± 0.12 after 3 hours, and the lowered pH resulted in the elevation of ATP release from skeletal myocytes. The acidosis-induced ATP release was totally abolished by GlyH-101 (40 μM), an open-channel CFTR blocker, suggesting that CFTR was involved. The cAMP/PKA signaling pathway was involved in the CFTR-regulated ATP release from skeletal myocytes: 1). Forskolin increased the extracellular ATP and the phosphorylation of CFTR; IBMX, a phosphodiesterase inhibitor, further enhanced the forskolin-induced extracellular ATP and phosphorylation of CFTR; 2). Inhibition of PKA by its selective inhibitor KT-5720 abolished the acidosis-induced ATP release and the forskolin-induced phosphorylation of CFTR. In addition, the inhibition of Na+/H+ exchanger (NHE) by amiloride, or inhibition of Na+/Ca2+ exchanger (NCX) by its specific inhibitors SN-6 and KB-R7943 abolished the lactic-acid-induced ATP release from skeletal myocytes, indicating that NHE and NCX might be involved. Previous studies demonstrated that Connexin hemichannels and Pannexin channels were able to conduct ATP in response to stimuli. This study found that connexin 43 (Cx43) was strongly expressed on skeletal myocytes, while Pannexin 1 (Panx1) showed a strong expression in gastrocnemius muscle. Investigation of the role that Cx43 may play in acidosis-induced cAMP/PKA-activated CFTR-regulated ATP release from myocytes showed that: 1). Cx43 was immunoprecipitated with CFTR suggesting a physical interaction; 2). The opening of Cx hemichannels was increased by lactic acid and this lactic-acid-induced opening was inhibited by CFTRinh-172, suggesting the mediation of CFTR; 3). Inhibition of Cxs and Panxs with carbenoxolone abolished the acidosis-induced ATP release; moreover, specific silencing of the Cx43 gene using siRNA decreased both basal and acidosis-induced ATP release, suggesting that Cx43 was involved; 4). Overexpression of CFTR alone did not elevate the acidosis-induced ATP release, while overexpression of Cx43 alone doubled the acidosis-induced ATP, and co-overexpression of CFTR and Cx43 further elevated the acidosis-induced ATP release, supporting the concept that Cx43 functionally interacted with CFTR to induce the acidosis-induced ATP release. Panx1 was studied in native skeletal muscle, and found to be coimmunoprecipitated with CFTR. Inhibition of Panxs with gadolinium or probenecid abolished the muscle-contraction-induced ATP release, while inhibition with carbenoxolone or quinine reduced it to less than 10% of control, suggesting that Panx1 may be involved in the acidosis-induced ATP release during muscle contraction. All the in vitro and in vivo studies suggested that Cxs and Panx were involved in the acidosis-induced CFTR-regulated ATP release from skeletal myocytes and skeletal muscle. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Breast cancer is a very heterogeneous disease. This heterogeneity can be observed at many levels, including gene expression, chromosomal aberrations, and disease pathology. A clear understanding of these differences is important since they impact upon treatment efficacy and clinical outcome. Recent studies have demonstrated that the tumour microenvironment also plays a critical role in cancer initiation and progression. Genomic technologies have been used to gain a better understanding of the impact of gene expression heterogeneity on breast cancer, and have identified gene expression signatures associated with clinical outcome, histopathological breast cancer subtypes, and a variety of cancer-related pathways and processes. However, little work has been done in this context to examine the role of the tumour microenvironment in determining breast cancer outcome, or in defining breast cancer heterogeneity. Additionally, little is known about gene expression in histologically normal tissue adjacent to breast tumour, if this is influenced by the tumour, and how this compares with non-tumour-bearing breast tissue. By applying laser--capture microdissection and gene expression profiling to clinical breast cancer specimens the research presented in this thesis addresses these questions. / We have generated gene expression profiles of morphologically normal epithelial and stromal tissue, isolated using laser capture microdissection, from patients with breast cancer or undergoing breast reduction mammoplasty. We determined that morphologically normal epithelium and stroma exhibited distinct expression profiles, but molecular signatures that distinguished breast reduction tissue from tumour-adjacent normal tissue were absent. Stroma isolated from morphologically normal ducts adjacent to tumour tissue contained two distinct expression profiles that correlated with stromal cellularity, and shared similarities with soft tissue tumors with favourable outcome. Adjacent normal epithelium and stroma from breast cancer patients showed no significant association between expression profiles and standard clinical characteristics, but did cluster ER/PR/HER2-negative breast cancers with basal-like subtype expression profiles with poor prognosis. Our data reveal that morphologically normal tissue adjacent to breast carcinomas has not undergone significant gene expression changes when compared to breast reduction tissue, and provide an important gene expression data set for comparative studies of tumour expression profiles. / We compared gene expression profiles of tumour stroma from primary breast tumors and derived signatures strongly associated with clinical outcome. We present a new stroma-derived prognostic predictor (SDPP) that stratifies disease outcome independently of standard clinical prognostic factors and published expression-based predictors. The SDPP predicts outcome in several published whole tumour--derived expression data sets, identifies poor-outcome individuals from multiple clinical subtypes, including lymph node--negative tumors, and shows increased accuracy with respect to previously published predictors, especially for HER2-positive tumors. Prognostic power increases substantially when the predictor is combined with existing outcome predictors. Genes represented in the SDPP reveal the strong prognostic capacity of differential immune responses as well as angiogenic and hypoxic responses, highlighting the importance of stromal biology in tumour progression. / We show that gene expression in the breast tumour microenvironment is highly heterogeneous, identifying at least six different classes of tumour stroma with distinct expression patterns and distinct biological processes. Two of these classes recapitulate the processes identified in the stroma-derived prognostic predictor, while the others are new classes of stroma associated with distinct clinical outcomes. One of these is associated with matrix remodelling and is strongly associated with the basal molecular subtype of breast cancer. The remainder are independent of the previously published molecular subtypes of breast cancer. Additionally, based on independent data from over 800 tumors, the combinations of stroma classes and breast cancer subtypes identify new subgroups of breast tumors that show better discrimination between good and poor outcome individuals than the molecular breast cancer subtypes or the stroma classes alone, suggesting a novel classification scheme for breast cancer. This research demonstrates an important role for the tumour microenvironment in defining breast cancer heterogeneity, with a consequent impact upon clinical outcome. Novel therapies could be targeted at the processes that define the stroma classes suggesting new avenues for individualized treatment.

Blockade of the renin-angiotensin system lowers the rate of cardiovascular events in patients at risk for vascular disease and also improves endothelial function but the mechanism remains unclear. HUVECs were stimulated with Ang II (100 nM). Ang II produced a 2-fold increase in O2- production, which was measured by lucigenin-enhanced chemiluminescence. This increase was blocked by NAD(P)H oxidase inhibitor DPI, but not by eNOS inhibitor L-NAME. Ang II increased monocyte adhesion to ECs by 4.5-fold, and this increase was blocked by candesartan (AT1 receptor antagonist), DPI, L-NAME, wortmannin (PI3K inhibitor), dominant negative-AKT, and p22phox siRNA. Dominant active-AKT increased adhesion by 1.5-fold. Our findings indicate that the simultaneous activation by Ang II of eNOS and NAD(P)H oxidase leads to endothelial activation. This process can partially explain the therapeutic benefits of reducing the action of Ang II.

Insulin-like growth factor-1 (IGF-1) is essential for oligodendrocyte (OL) development, promoting their survival, proliferation and differentiation. Furthermore, IGF-1 null mutant mice have a decrease in CNS myelination and in the number of OL progenitors (OLPs). IGF-1 interacts with the Type I IGF receptor to activate two main downstream signalling pathways, the PI3K/Akt and the Ras-Raf-MEK/ERK cascades, which mediate survival or proliferation of OLPs. The objective of this study is to elucidate the transduction pathways involved in IGF-I-stimulated protein synthesis, important for growth and differentiation of OLs. In other cellular systems, the PI3K/Akt pathway is involved in protein translation. mTOR and the p70 S6 kinase are downstream effectors that phosphorylate translation initiation factors (e.g. eIF-4E) and their regulators (e.g. 4E-BP1). OLPs were obtained from primary cultures and were treated with IGF-1 with or without inhibitors LY294002 or wortmannin (PI3K), rapamycin (mTOR), Akt III or IV, an adenovirus with a dominant negative form of Akt or PD98059 (ERK). Protein synthesis was assessed by metabolic labeling with [35S]-methionine, and protein phosphorylation by Western blotting. Results from the former showed that IGF-1 stimulates protein synthesis in a dose-dependent manner. Moreover, IGF-1 increases protein synthesis in OLPs through PI3K, mTOR, Akt and ERK activation. Concordantly, Western blot analysis reveals that IGF-1 stimulates phosphorylation of Akt, mTOR, ERK, S6 and 4E-BP 1. Activation of S6 and inactivation of 4E-BP1 occur through phosphorylation and are required for protein synthesis to take place. These events are dependent on the upstream activation of PI3K, Akt and mTOR.

The study of diabetes mellitus is vital in this day and age because its incidence is increasing at an alarming rate. Diabetes results in the loss of function of beta-cells within the pancreas. Insulin resistance contributes to diabetes but the human body can compensate in various ways such as increasing the islet cell mass, glucose disposal and insulin secretion, in order to prevent the onset of diabetes. Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are two integral hormones important in both glucose homeostasis and islet cell growth. Early studies using cultured islet cells have demonstrated positive regulation of beta-cell growth by both GH and IGF-I. To evaluate their relevance on normal beta-cell growth, compensatory growth, as well as in insulin responsiveness, we have used two mouse models that represent opposite manipulations of the GH/IGF-I axis. Specifically, the growth hormone receptor gene deficient (GHR-/-) and the IGF-I overexpression (MT-IGF) mice, to help understand the role of glucose homeostasis and islet cell growth in the GH/IGF-I axis. GH is essential for somatic growth and development as well as maintaining metabolic homeostasis. It is known that GH stimulates normal islet cell growth. Moreover, GH may also participate in islet cell overgrowth and compensate for insulin resistance induced by obesity. To determine whether the islet cell overgrowth is dependent on GH signaling, we studied the response of GHR-/- mice to high-fat diet (HFD)-induced obesity. We also studied the insulin responsiveness in GHR-/- mice. On the other hand, IGF-I promotes embryonic development, postnatal growth and the maturation of various organ systems. The notion that IGF-I stimulates islet cell growth has been challenged in recent years by results from IGF-I and receptor gene targeted models. We have characterized MT-IGF mice which overexpress the IGF-I gene. / The results of our studies indicate that (1) GH is essential for normal islet cell growth, but not required for compensatory overgrowth of the islets in response to obesity, (2) GHR gene deficiency caused delayed insulin responsiveness in skeletal muscle; in contrast to elevated insulin sensitivity in the liver; (3) although overexpression does not stimulate islet cell growth, a chronic IGF-I elevation caused significant hypoglycemia, hypoinsulinemia, and improved glucose tolerance, (4) finally IGF-I overexpression mice are resistant to experimental diabetes.

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 5 uppsatser.