Modulation of the actin-based cytoskeleton and transcription factor regulation are merely two essential functions in a wide array of cellular activities that the Rho family of small GTPases is responsible for mediating. Aberrations in, or loss of, Rho GTPase signaling has been found to lead to multiple pathologies, including both metastatic progression and lenticular differentiation leading to cataractogenesis. This study has examined the transcriptional regulation of the metastasis suppressor, KiSS-1. Although the mechanism by which KiSS-1 modulates an anti-metastatic effect is not entirely known, it is known that KiSS-1 mediates stress fiber formation, increased adhesion and reduced migratory and invasive properties through modulation of the Rho family of small GTPases. The loss of KiSS-1 that commonly occurs during metastatic progression, leads to a loss of proper Rho GTPase regulation. This study has examined how KiSS-1 is regulated in two tissue types, breast and skin, and how the loss of AP-2(alpha) and DRIP-130, respectively, leads to the progression of breast cancer and melanoma. In addition, this study has also looked at the importance of Rac1 expression and function in the lens epithelium. Activation of Rac1 and its downstream effector, SRF, have been shown to be key regulators in lens cell differentiation, possibly leading to lens opacification via its transcriptional control of the structural crystallins within the lens. The results of this dissertation research have made significant strides in understanding the nature of the anti-metastatic effects registered by the novel KiSS-1 peptide and its cognate GPCR. Additionally, it has shed light on the Rho family regulation of lens epithelial cell differentiation, indicating the elaborate involvement of Rac1 in mediating lens fiber development. In all, this research has determined previously unknown roles of small molecule GTPases in both the progression of metastasis, as well as in normal and abnormal lens cell differentiation.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 196-221).

Thesis (Ph. D.)--University of Kentucky, 2002. / Title from document title page. Document formatted into pages; contains vii, 83 p. : ill. Includes abstract. Includes bibliographical references (p. 73-82).

The retina is an internal photosensitive neural tunic which absorbs light and prevents it from reflecting back. The light receptors and neurons of the retina are initial processor of visual information. Various anomalies of the retina such as retinitis pigmentosa, cone-rod dystrophy to retinal degenerative diseases cause severe loss of vision since they affect photoreceptors directly or indirectly. Conventional therapies have never been fully successful in restoring vision in such diseases. However current research in stem cell therapies has shown remarkable potential. In this project, induced pluripotent stem cells from mouse were coxed into photoreceptor fate in presence and absence of Dorsomorphin using specific media in a stepwise differentiation process. Dorsomorphin is an inhibitor of Bone Morphogenic Protein (BMP) whose suppression may influence neural differentiation. Studies were done using conventional inverted microscopy and fluorescent microscopy on mouse induced pluripotent stem cells (miPS cells). Immunolabelling techniques involving Pax6, Crx, RPE65, Rhodopsin and Opsin were used to evaluate the advantage of these as markers for stem cells differentiation. Reverse Transcriptase PCR was done to confirm the gene expression on the differentiated cells. Human iPS derived Mesenchymal stem cells were cultured and the effect of different concentrations of Retinoic Acid such as 0mM, 0.1mM and 0.5mM on cell proliferation was tested in both presence and absence of Dorsomorphin. The results revealed both control and Dorsomorphin treated miPS cells successfully differentiated into photoreceptors-like cells as detected by positive staining of Rhodopsin and Opsin. The cells were however negative for Pax6, and very weak staining for RPE and Crx. The presence or absence of Dorsomorphin did not make any difference on miPS differentiation. The same observation was made on differentiating human iPS-MSC where Dorsomorphin did not reveal much effect. However highest cell count of proliferating cells was observed in the subgroups containing 0.1mM Retinoic Acid on Day7 groups as control had an average of 590 ±±317.23 and treatment 1206 .33 ±±114.99 cells, with statistical significance of P<0.05. It appears that the presence of Retinoic Acid facilitated the proliferation of human iPS-MSC. In conclusion, the study reveals that iPS can be another potential stem cell source for therapies of retinal diseases involving photoreceptors where the question of ethical issue is not a problem unlike embryonic stem cells. Also it reveals the concentration of Retinoic Acid most suited for human iPS-MSC cell proliferation. Dorsomorphin did not seem to have much effect on either type of stem cells in terms of promoting photoreceptor differentiation. / published_or_final_version / Medicine / Master / Master of Medical Sciences

abstract / Biochemistry / Doctoral / Doctor of Philosophy

Cell proliferation and differentiation are coordinated by synchronized patterns of gene expression. The regulation of these patterns is achieved, in part, through epigenetic mechanisms that affect the nature of DNA packaging into chromatin. Specifically, post-translational modifications to histone tails impact the structural dynamics of nucleosomes, thereby affecting DNA accessibility to transcriptional complexes. Accumulating evidence suggests that transcriptional regulators facilitate these alterations, resulting in altered local gene transcription. Thus, the structural interpretations of histone modifications are responsible for the establishment and maintenance of discrete programs of gene expression that ultimately correspond with distinct biological outcomes. Most histone lysine methyltransferases catalyze methyl transfer by way of the SET domain, a module encoded within many proteins that regulate diverse processes, including some critical for development and proper progression of the cell cycle. One such group of proteins, the SET and MYND domain (Smyd) family have been demonstrated to be direct regulators of tumorigenesis and essential developmental processes. Presented here is a functional characterization of two members of that family, Smyd2 and Smyd3. Smyd2 is identified as a member of the Smyd family and reported here to possess SET-dependent histone H3, lysine 36-specific methyltransferase activity. Smyd2 specifically associates with the Sin3A histone deacetylase complex, suggesting a link between two independent chromatin modification activities. Finally, over-expression of Smyd2 in fibroblasts is shown to significantly suppress their rate of growth. It is therefore proposed that Smyd2-mediated chromatin modifications regulate specific gene expression, thereby having important implications for normal and neoplastic cell proliferation. Aberrant expression of the histone H3-lysine 4-specific methyltransferase, Smyd3, has been implicated in colorectal, hepatocellular, and breast cell carcinogenesis. Here, Smyd3 is also shown to target histone H4, lysine 20 (H4K20). However, over-expression of Smyd3 in fibroblasts results in global reduction of trimethylation at H4K20 and this is accompanied by a striking increase in cell proliferation. As the methylation of H3K4 and H4K20 are normally associated with conflicting biological functions, I predict that these differential activities of Smyd3 are manifest under spatially and/or temporally distinct conditions, in the presence of different associating complexes, thereby resulting in effects that may be antagonistic of one another. / text

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

No description available.

Staufen1 is a double-stranded RNA-binding protein known to be involved in the transport, localization, decay and increased translation of some mRNAs. The goal of the present study is to determine the role of Staufen1 during myogenic differentiation by characterizing the effects of Staufen1 over-expression in C2C12 cells. Immunofluorescence experiments revealed that Staufen1 over-expression causes a decrease in the fusion and differentiation indices and leads to the formation of myotubes with significantly fewer nuclei. We show, by western blot and qRT-PCR, that the protein expression of MyoD, myogenin and MyHC and the mRNA expression of MyoD, myogenin, Mef2A, Mef2C and p35 are significantly decreased during differentiation when Staufen1 is over-expressed. We then found that c-myc protein expression was increased during proliferation but that its mRNA expression remained unchanged. In this study we propose that Staufen1 negatively regulates skeletal muscle differentiation through the posttranscriptional regulation of c-myc, Mef2A, Mef2C and p35 transcripts.

No description available.