published_or_final_version / Medicine / Doctoral / Doctor 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

Both cDNA tag-based and DNA chip hybridization assays have revealed widespread transcriptional activity across mammalian genomes, providing a rich source of novel protein-coding and non-coding transcripts. Annotation and functional evaluation of this undefined transcriptome space represents a major step towards the comprehensive definition of biomolecules regulating the properties of living cells, including embryonic stem cells (ESCs) and their derivatives. In this study I analysed 87 rare mRNA transcripts from human ESCs that mapped uniquely to the human genome, in regions lacking evidence for known genes or transcripts. In addition, the transcripts appeared enriched in the hESC transcriptome as enumerated by serial analysis of gene expression (SAGE). Full-length transcripts corresponding to twelve novel LongSAGE tags were recovered and evaluated with respect to gene structure, protein-coding potential, and gene regulatory features. In addition, transcript abundance was compared between RNA isolated from undifferentiated hESCs and differentiated cells. Analysis of full-length transcripts revealed that the novel ORFs did not exceed a size of 129 amino acids and no matches were observed to well characterized protein domains. Interesting protein level predictions included small disulfide-bonded proteins, known members of which are important in a variety of biological processes. Transcripts evaluated for differential expression by real-time RT-qPCR (Reverse Transcription followed by real-time quantitative Polymerase Chain Reaction) were found to be variably expressed (0.2- to 4.5-fold) in Day-2 or Day-4 retinoic acid-induced differentiation cultures compared to undifferentiated hESCs. Relative quantitation using a universal reference RNA (derived from pooled adult tissues) showed large differences in novel transcript levels (0.002- to 35-fold) compared to hESCs. Collectively, these results provide a detailed analysis of a set of novel hESC transcripts and their abundance in early and adult differentiated cell types, both of which may advance our understanding of the transcriptional events governing stem cell behavior.

No description available.

Both cDNA tag-based and DNA chip hybridization assays have revealed widespread transcriptional activity across mammalian genomes, providing a rich source of novel protein-coding and non-coding transcripts. Annotation and functional evaluation of this undefined transcriptome space represents a major step towards the comprehensive definition of biomolecules regulating the properties of living cells, including embryonic stem cells (ESCs) and their derivatives. In this study I analysed 87 rare mRNA transcripts from human ESCs that mapped uniquely to the human genome, in regions lacking evidence for known genes or transcripts. In addition, the transcripts appeared enriched in the hESC transcriptome as enumerated by serial analysis of gene expression (SAGE). Full-length transcripts corresponding to twelve novel LongSAGE tags were recovered and evaluated with respect to gene structure, protein-coding potential, and gene regulatory features. In addition, transcript abundance was compared between RNA isolated from undifferentiated hESCs and differentiated cells. Analysis of full-length transcripts revealed that the novel ORFs did not exceed a size of 129 amino acids and no matches were observed to well characterized protein domains. Interesting protein level predictions included small disulfide-bonded proteins, known members of which are important in a variety of biological processes. Transcripts evaluated for differential expression by real-time RT-qPCR (Reverse Transcription followed by real-time quantitative Polymerase Chain Reaction) were found to be variably expressed (0.2- to 4.5-fold) in Day-2 or Day-4 retinoic acid-induced differentiation cultures compared to undifferentiated hESCs. Relative quantitation using a universal reference RNA (derived from pooled adult tissues) showed large differences in novel transcript levels (0.002- to 35-fold) compared to hESCs. Collectively, these results provide a detailed analysis of a set of novel hESC transcripts and their abundance in early and adult differentiated cell types, both of which may advance our understanding of the transcriptional events governing stem cell behavior.

Thesis (M. S.)--Electrical and Computer Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Robert Butera; Committee Member: Pamela Bhatti; Committee Member: Todd McDevitt

Thesis (M. S.)--Computer Science, Georgia Institute of Technology, 2009. / Committee Chair: Dr. Samuel Dudley; Committee Member: Dr. Micheal Davis; Committee Member: Dr. Robert Butera

Thesis (Ph. D.)--Universität Mannheim, 2004/2005. / Includes bibliographical references (p. [313]-344).

Thesis (Ph.D.)--University of Missouri-Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 153-173). Also available on the Internet.

Thesis (Ph.D.)--University of Missouri-Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 153-173). Also available on the Internet.