Sox2, a HMG box transcription factor, is well known for its role in stem cell maintenance, iPS (induced pluripotent stem cell) induction, and development of neural tissues such as central nervous system and sensory organs. Sox2 has been demonstrated to be essential for the development of inner ear sensory patches. It has been shown that Sox2 is under the regulation of multiple regulatory elements to obtain a tissue specific manner. Two allelic mouse mutants, yellow submarine (Ysb) and Light coat and circling (Lcc) show hearing and balance impairments with different severity. They were made by random insertions of a transgene (pAA2) and X-ray irradiation respectively. Ysb and Lcc are both localized to chromosome 3 and involve complex chromosomal rearrangements. The Sox2 coding region is intact in the mutants, while the Sox2 expression in the otocyst is greatly reduced in Ysb and totally lost in Lcc, which indicates the tissue specific reduction of Sox2 may be due to the rearrangement of Sox2 regulatory element(s). Since Sox2 null mutants die before implantation, the two Sox2 inner ear mutants are valuable models for studying Sox2 knock down (Ysb) and Sox2 knock out (Lcc) condition in the inner ear. To understand the molecular basis behind Sox2 regulation in the inner ear, this project aims to identify the Sox2 otic regulatory elements, and potential Sox2 downstream targets involved in the development of inner ear. Previous work has indicated that Nop1 and Nop2 are the otic specific regulatory elements of Sox2 in chicken ear. In this project, transgenic mice were generated using Nop1-Nop2, and the result showed Nop1-Nop2 could drive Sox2 expression to the dorsal side of the otiv vesicle, which is different from the endogenous Sox2 expression pattern. Therefore, Nop1 and Nop2 may require other regulatory element(s) to gain a correct regulatory pattern. BAC(RP23-335P23), which contained the DNA sequences close to Ysb integration site 1 was also been tested in transgenic mice. Interestingly, the result showed that BAC(RP23-335P23) could drive Sox2 expression to the ventral side of the otic vesicle, indicating that this BAC may contain the Sox2 otic regulatory element(s). In this project, the binding relationship between Sox2 protein and Math1 enhancer has also been identified using chromatin immunoprecipitation (Ch-IP). Results showed that Sox2 could bind to Math1 enhancer A in the inner ear cochlea. So Sox2 may regulate Math1 through binding to Math1 enhancer A in inner ear development. Using a bioinformatics approach, potential Sox2 target genes in inner ear development have been identified from public microarray data on E9 to E15 inner ear tissue by the presence of conserved Sox2 binding sites. Among these potential targets, 4 genes (Itga6, Erbb3, Sox10 and Mycn) have been selected based on their known functions. Their expression patterns in the cochlea of wild type, Ysb and Lcc were verified. The identification of Sox2 downstream target genes using a bioinformatics approach will help us to understand the molecular basis of Sox2 regulation, and also understand the role of Sox2 in the inner ear development. / published_or_final_version / Biochemistry / Master / Master of Philosophy

Type-2 diabetes is one of the most prevalent non-communicable diseases globally. The prevalence has increased rapidly in both developed and developing countries. Type-2 diabetes has become epidemic in China in the past three decades. With a relatively low obesity prevalence compared to western countries, such as the United States, the question has arisen as to why people in China are so susceptible to diabetes, but few studies have answered this question definitively. Asians have lower muscle mass than Caucasians which could be one possible explanation. This study aims to test the hypothesis that pubertal muscle mass acquisition, under the influence of testosterone, may be a risk factor for diabetes. This study recruited from the population-representative Chinese “Children of 1997” birth cohort with 8327 participants (88% of all infants born in April and May 1997) in Hong Kong. Participants were aged 15 years at the time of this study. The main objective was to examine the association of pubertal muscle mass with diabetes risk factors, and to examine determinants of pubertal muscle mass in this non-western setting. In June to August 2012, 502 participants from “Children of 1997” were recruited to complete a health assessment follow up at the Active Health Clinic in University of Hong Kong, including a questionnaire, blood tests and a physical examination. Multivariable linear regression was used to assess the associations of: (1) testosterone and pubertal muscle mass with diabetes risk factors, including fasting glucose, fasting insulin and homeostasis model assessment -insulin resistance (HOMA-IR), and any mediation of the association of testosterone with diabetes risk factors by pubertal muscle mass, and (2) environmental influences, including intergenerational influences, proxied by mother’s place of birth, and current life style influences (meat consumption and physical activity), with pubertal muscle mass. Higher pubertal testosterone was associated with lower fasting glucose (-0.008, 95% confidence interval (CI) -0.015 to -0.002), fasting insulin (-0.44, 95% CI -0.57 to -0.31) and HOMA-IR (-0.090, 95% CI -0.12 to -0.063) after adjusting for potential confounders, i.e, sex, birth weight, highest parental education, mother’s place of birth and physical activity. The association was partially mediated by skeletal muscle mass and body fat percentage. Skeletal muscle mass was negatively associated with fasting glucose (-0.017, 95% CI -0.025 to -0.009), insulin (-0.876, 95% CI -1.033 to -0.719), and HOMA-IR (-0.180, 95% CI -0.214 to -0.147) after adjusted for potential confounders. High meat consumption (0.176, 95% CI 0.000 to 0.351) and physical activity (0.157, 95% CI 0.059 to 0.254) were both associated with higher skeletal muscle mass in adolescents; however birth weight and mother’s place of birth had no influence on pubertal skeletal muscle composition. Higher pubertal muscle mass and testosterone are association with better glucose metabolism in adolescence. Food intake and physical activity may also influence the development of skeletal muscle, thus adolescence may be a sensitive period for the development of diabetes where interventions to increase muscle mass could have long-term protective effects. / published_or_final_version / Public Health / Master / Master of Philosophy

Human embryonic stem cells (hESCs) are characterized by unlimited proliferation (self-renewal), capability to differentiate into derivatives of all three germ layers (pluripotency), and abbreviated cell cycle structure. Despite tremendous efforts in identification of important regulators, the complicated molecular mechanisms and essential effectors underlying the distinctive features of hESCs have not yet been fully elucidated. Forkhead box transcription factor M1 (FOXM1) has been demonstrated to be critical for the maintenance of pluripotency in mouse embryonic stem cells (mESCs) and mouse embryonal carcinoma cells (mECCs). The present study hypothesized that FOXM1 is important to the self-renewing capacity and pluripotency of hESCs. The objectives of this study were to characterize FOXM1 expression in undifferentiated and differentiating hESCs, and to study the effect of perturbing FOXM1 expression on pluripotency and proliferation. Undifferentiated VAL3 analyzed by bivariate flow cytometric analysis revealed that FOXM1 expression was regulated in a cell cycle phase-dependent manner, with expression level increased from G1 through S phase, and eventually reached peak levels in G2/M phase. To study the subcellular localization of FOXM1 with respect to cell cycle progression, VAL3 cells were synchronized by nocodazole-mediated cell cycle block, followed by immunocytochemical analysis. The result indicated that FOXM1 underwent nuclear translocation at late-S and early-G2 phase of the cell cycle. When VAL3 spontaneously differentiated as embryoid bodies (EBs), the mRNA expression of FOXM1 displayed profound fluctuation over the differentiation process. Retinoic acid (RA) treatment induced rapid differentiation of VAL3, yet differential expression pattern of FOXM1 was observed for cells grown in different culture media. FOXM1 mRNA expression persisted in differentiating VAL3 cultured in mTeSR. By contrast, RA-driven differentiation of VAL3 cultured in conditioned medium was accompanied by transient depletion and resurgence of FOXM1 protein expression. Differentiation of VAL3 driven by Definitive Endoderm kit did not alter FOXM1 expression, whereas induced differentiation by Bone Morphogenic Protein 4 (BMP4) led to repression of FOXM1. The functional role of FOXM1 in hESCs was investigated with the use of siRNA. Transient knockdown of FOXM1in VAL3 did not induce substantial repression of pluripotent marker (OCT4, SOX2, NANOG) expression nor significant morphological change of colonies, despite upregulation of early differentiation marker SSEA-1. Intriguingly, FOXM1 depletion led to altered cell cycle progression and delay in G2 phase progression, possibly attributed to the downregulation of Cyclin B1 and Cdc25B. Also FOXM1 knockdown impaired VAL3 proliferation, yet no prominent defect in mitosis was observed. In conclusion, the present study reported for the first time the expression and functions of FOXM1 in undifferentiated hESCs. Upon differentiation, FOXM1 expression varied in cells committing to different lineages. Depletion of FOXM1 did not interfere with hESCs pluripotency, but hindered cell cycle progression and cell proliferation, suggesting that FOXM1 is mainly involved in promoting rapid proliferation of hESCs. The functional role and regulatory mechanics of FOXM1 in hESCs cell cycle control and differentiation warrant further investigation. / published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / abstract / toc / Psychiatry / Master / Master of Philosophy

published_or_final_version / abstract / toc / Dentistry / Doctoral / Doctor of Philosophy

published_or_final_version / Molecular Biology / Doctoral / Doctor of Philosophy

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