Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2009. / Includes bibliographical references (p. 140-165).

Thesis (Ph. D.)--University of North Texas, Dec., 2008. / Title from title page display. Includes bibliographical references.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 96-103). Also available in electronic version.

Thesis (Ph. D.)--University of Virginia, 2006. / Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 194-218). Also available in electronic version.

Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Environmental Health Science. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 128-138).

Mesoderm is one of the three primary germ layers from which the cardiovascular system, muscle and bone originate and derivatives of the mesoderm lineage are affected in a number of pathologies. Therefore, understanding the mechanisms regulating formation of mesoderm is interesting for a diversity of diseases and clinical application. In vivo study of human development beyond gastrulation is technically challenging and the mechanisms controlling mesoderm specification are difficult to study since the maximum number of days allowed to grow human embryos is 14 days. Thus, in this dissertation I use human pluripotent stem cells (hPSCs) as a simplified model of human development. Studies have shown that the cell cycle machinery plays a direct role in the differentiation of endoderm and ectoderm lineages but its role in guiding mesoderm subtype formation remains elusive. In this dissertation, I provide new insights of the importance of the cell cycle regulators in mesoderm specification. I first developed tools such as the FUCCI2A reporter line to isolate cells in the different cell cycle phases and to investigate propensity of mesoderm differentiation. I have shown that the propensity of differentiation into the three mesoderm subtypes lateral plate mesoderm, cardiac mesoderm and presomitic mesoderm varies during the cell cycle phases, with differentiation being more efficient in the G1 and to a lesser extend in G2/M phase. Furthermore, I developed a protocol where cells can be efficiently synchronised in the different cell cycle phases using the G2/M inhibitor nocodazole. Using this tool, I showed that developmental signalling pathways such as BMP and WNT are active in all cell cycle phases indicating that alternative mechanisms are involved in the differentiation process. In order to further explore these mechanisms, I investigated the role of cell cycle regulators controlling the G1 and G2 checkpoint. I have shown that the cell cycle regulators CDK4/6, CDK2, Retinoblastoma phosphorylation and CDK1 are essential for mesoderm subtype formation. Furthermore, I have shown that CDK1 regulates the activity of ERK1/2 signalling, an important pathway for the differentiation process confirming the existence of complex interplays between cell cycle machinery, signalling pathways and transcription factors in mesoderm subtype formation. This knowledge will be useful to further improve protocols for generating mesoderm subtypes from hPSCs for clinical applications such as drug screening, disease modelling and cell based therapy.

Coronary artery disease (CAD), characterized by the narrowing of coronary arteries through the complex manifestation and development of atherosclerosis, is a complex disease and one of the leading causes of death worldwide. Both genetic and environmental factors are believed to contribute equally to the risk of CAD. Recently, a study identified a non-synonymous coding variant, rs11556924, (MAF, 0.38) in ZC3HC1 associated with protection against CAD (p= 9.8x10-18; OR= 0.90). NIPA, encoded by ZC3HC1, is a characterized F-Box protein and regulator of cell cycle. Since the amino acid change (Arg363His) is in a conserved region of NIPA and is predicted to have functional effects (Polyphen-2), this study aimed at understanding the functional implications of this amino acid change on NIPA and cell cycle regulation. Here we are able to effectively show a) allele specific differences in mRNA expression in whole blood, b) a slight structural difference between NIPA363Arg and NIPA363His variants, c) proliferation rates of NIPA363Arg expressing cells were significantly increased, and d) phosphorylation of a critical serine residue in close proximity to aa.363 is not statistically different between the two variants. These results suggest that rs11556924 plays a direct role in development of CAD through its disruption of cell cycle regulation and NIPA mRNA availability. This study is the first to identify a molecular basis for the association of rs11556924 to CAD development.

In this study, DNA methylation was examined during the eukaryotic cell cycle, and shown to occur throughout the S phase as well as during the "early" G₂ phase. However, DNA synthesis and methylation of newly synthesized DNA did not occur simultaneously, but the latter lagged behind DNA synthesis by about two hours. Once added during the S phase, the methyl groups were stably maintained in the DNA. Various compounds which are known to affect DNA synthesis in tissue cultured cells, were tested for their ability to alter the methylation status of DNA. The effects of three DNA synthesis inhibitors, viz. hydroxyurea (HU), 1-S-D-arabinofuranosyl cytosine (ara-C) and aphidicolin were examined on a normal embryonic lung fibroblast cell line (WI-38) and its two transformed counterparts, a simian virus 40 (SV 40) transformed line (SVWI-38) and a y-irradiation transformed cell line (CT-1). HU was shown to enhance hypermethylation of pre-existing DNA strands in the normal cells, while ara-C and aphidicolin caused hypermethylation of newly synthesized DNA strands. The effects of various concentrations of a known inducer of gene expression, sodium butyrate, were examined on these three cell lines as well. During a 16-20 hour treatment period, at butyrate concentrations of between 5 and 20 mM, no adverse effect on cell morphology was observed. Cell growth, in the presence of butyrate for 14 hours, showed that butyrate was more toxic on the transformed cells than on the normal cells. However, at 5 mM butyrate, DNA synthesis was inhibited by 75% in the normal cells, and was unaffected in the transformed lines. RNA synthesis was not affected in the transformed cells, whilst in the normal cell line, RNA synthesis was decreased to 76% of the control value, at sodium butyrate concentrations as low as 5 mM. Protein synthesis also was unaffected in the transformed cells and only slightly (+ 10%) inhibited in the normal cells at 20 mM butyrate. SDS polyacrylamide gel electrophoresis of proteins synthesized in the presence of 10 mM sodium butyrate, showed that most proteins were unaffected. Two high molecular weight proteins in the WI-38 cells appeared to be modified during butyrate. treatment, while one protein was induced by butyrate treatment in the CT-1 cells. More importantly though, butyrate treatment also resulted in hypermethylation of DNA, as shown by MSP 1 and Hpa II restriction endonuclease digestion and high-pressure liquid chromatography analysis. Butyrate appeared to specifically cause hypermethylation of pre-existing DNA strands in the WI-38 cells, while the SVWI-38 and CT-1 cells showed preferential hypermethylation of newly synthesized DNA strands. However, the hyper-methylated state was only heritable if the methylation event occurred in newly synthesized DNA. Hypermethylation on pre-existing DNA was rapidly lost in the subsequent generation. It would therefore appear that methylcytosines are only maintained in the DNA if they are generated on newly synthesized DNA. This study has clearly shown that the heritability of DNA methylation patterns is closely linked to DNA replication.