Thesis (Ph. D.)--George Mason University, 2007. / Title from PDF t.p. (viewed Jan. 17, 2008). Thesis director: Richard E. Rubenstein. Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Conflict Analysis and Resolution. Vita: p. 228. Includes bibliographical references (p. 222-227). Also available in print.

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Embryonic stem (ES) cells have the remarkable ability to differentiate into all cells comprising the three germ layers of the developing embryo. It is this pluripotency that makes them attractive for use in regenerative medicine. However, in order to harness this potential, we must understand the molecular mechanisms regulating the ability of ES cells to self-renew and thereby generate identical pluripotent daughter ES cells. The Welham laboratory has previously described a requirement for PI3K signalling in maintaining self-renewal of murine ES (mES) (Paling et al., 2004; Storm et al., 2007). To identify the molecular mechanisms involved in regulating mES cell self-renewal downstream of PI3K signalling, an Affymetrix microarray screen was carried out prior to the start of this PhD. For the screen, mES cells were grown in the presence of LIF and treated with the reversible PI3K inhibitor LY294002 (LY) or a DMSO control for 24, 48 and 72 hours. A total of 646 statistically significant transcriptional changes were detected and subsequently divided into 12 clusters using k-means clustering. Experiments using pharmacological inhibitors suggest that genes within the same cluster are regulated by common mechanisms. To identify potential candidates involved in regulation of mES cell pluripotency, further analyses concentrated on transcription factors and genes with unknown functions. In our microarray data Zscan4c, a member of a SCAN-domain containing Zinc finger protein family, is one of the earliest down-regulated probe-sets. Loss-of-function experiments using siRNA approaches highlight a role for Zscan4 downstream of PI3Ks in regulation of ES cell self-renewal. Immunohistochemical staining of cells overexpressing Zscan4c showed nuclear accumulation of the protein. This, together with the fact that Zscan4c was mainly detectable in the nuclear protein fraction, strengthens a role of Zscan4c in transcriptional regulation. Potential Zscan4c protein interaction partners were identified by applying a combined immunoprecipitation (IP) - mass spectrometry strategy. Interestingly, the majority of potential Zscan4c interacting proteins identified are associated with functions related to transcriptional regulation and DNA damage response, all characteristics linked with Zscan4. Furthermore, the Class IA PI3K catalytic isoforms were genetically activated in mES cells, and liberation of the requirement for LIF was found upon over-expression of an activated p110 catalytic subunit.

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Includes bibliographies. Aims to identify homeobox genes which may have a developmental role during early embryogenesis by the characterization of homeobox gene expression in undifferentiated ES cells, and in a range of differentiated ES cell derivatives.

A Disintegrin-like And Metalloproteinase with Thrombospondin (TSP)-Type I sequence motifs 9 (ADAMTS9) is widely expressed in mouse and human fetal tissues. ADAMTS9 null mice cannot survive beyond E7.5 and its haploinsufficiency is associated with cardiac and aortic anomalies. This project hypothesized that ADAMTS-9 plays an important role during early embryogenesis. By using human embryonic stem cells (hESCs) as a model, the objectives of this study were to study the expression of ADAMTS9 and to determine the effects of ADAMTS9 perturbation on hESC differentiations. The expression of ADAMTS9 was compared between undifferentiated and differentiated hESCs. Its mRNA was maintained at similar levels in different passages of normal hESC lines. Interestingly, significantly lower expression was detected in karyotypically abnormal VAL4A when compared to the normal cells (H9 and VAL3). ADAMTS9 immunoreactivity was detected in cells located at the boundary of hESC colonies. The expression of ADAMTS9 was then studied during differentiation of hESC. ADAMTS9 mRNA and protein expression increased time-dependently during the first 24 days? of embryoid body (EB) formation. The expression pattern was similar to that of mesoderm and endoderm markers. Upon more specific lineage differentiation induced by retinoic acid and bone morphogenesis protein 4, ADAMTS9 mRNA expression was significantly increased. The positive correlation of ADAMTS9 with ESC differentiation was also found in mouse system, in which ADAMTS9 was increased time-dependently during mouse EB formation and down-regulated during reprogramming from somatic cells into induced pluripotent cells. Previous studies have shown that down-regulation of ADAMTS9 in several tumor tissues was attributed to ADAMTS9 hypomethylation. However, the present study demonstrated that the expression of ADAMTS9 was reduced in hESC after treatments with inhibitors of DNA methylation (5-aza-2?deoxycytidine) and histone deacetylase (VPA). The cellular localization of ADAMTS9 during hESC differentiation was further studied by co-localization of ADAMTS9 with several lineage specific markers. It was found that ADAMTS9 co-localized with mesoderm and endoderm markers. The functional role of ADAMTS9 in hESCs was then studied by transient ADAMTS9 knockdown. ADAMTS9 siRNA significantly decreased the expression level of mesoderm marker, REN. Thus, the role of ADAMTS9 during mesoderm differentiation was followed. ADAMTS9 was found to be dramatically increased after mesoderm differentiation of hESCs. In mesodermal cells, ADAMTS9 was co-expressed with vascular endothelial markers, VEGF and CD31, but not with pericyte markers, alpha muscle actin. Lentiviral vector encoding ADMATS9 shRNA was used for long term knockdown of ADAMTS9. ADAMTS9 down-regulation had no effect on the proliferation of hESCs. In agreement with the siRNA study, ADAMTS9 shRNA also significantly reduced the expression of REN. Upon mesoderm differentiation, ADAMTS9 knockdown resulted in a decreasing trend of mesoderm marker, CD34. In conclusion, the present study demonstrated a positive association of ADAMTS9 expression with hESC differentiation. ADAMTS9 was dramatically induced during mesoderm differentiation and its knockdown led to down-regulation of mesoderm markers. Together with the fact that ADAMTS9 expression was associated with endothelial cell markers suggested its possible role during endothelial cells formation. The roles of ADAMTS9 during hESC differentiation and early embryo development warrant further investigation. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

Includes bibliographies. / xi, 127, [90] leaves, [31] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to identify homeobox genes which may have a developmental role during early embryogenesis by the characterization of homeobox gene expression in undifferentiated ES cells, and in a range of differentiated ES cell derivatives. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1996

Thesis (Ph. D.)--University of Adelaide, Dept. of Biochemistry, 1996. / Includes bibliographical references.

Dissertation (DPhil)--University of Stellenbosch, 2007. / Bibliography. Also available via the Internet.