Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [75]-90).

Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xix, 201 p.; also includes graphics. Includes bibliographical references (p. 187-201).

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Vita. Includes bibliographical references.

Thesis (Ph.D.) - University of Queensland, 2003. / Includes bibliography.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 155-165).

Thesis (Ph. D.)--University of Chicago, Committee on Developmental Biology, March 2001. / Includes bibliographical references. Also available on the Internet.

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Cells have evolved the ability to change continuously and adapt to their environment. An important way in which this dynamic modulation is achieved is by reversible phosphorylation, mediated by protein kinases and phosphatases. This thesis focuses on the temporal variations in expression of the proteins protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A) and p53 tumour suppressor protein in proliferating and hexamethylene bisacetamide (HMBA) induced differentiating murine erythroleukaemic (MEL) cells. The study included analysis of variations of p53 mRNA in these cells. Protein variations were analysed in cell extracts using western immunoblotting. The p53 variations were evaluated further using enzyme-linked immunosorbent assay (ELISA); p53 mRNA was determined by reverse transcriptase polymerase chain reaction (RT-PCR). Dynamic variations in protein expression and mRNA were detected in both the untreated and HMBA treated MEL cell preparations. For PP1, an immunospecific band of molecular mass 36 kDa, corresponding to the catalytic subunit, was detected, while for PP2A, two immunospecific bands of 32 and 36 kDa were observed. For the PP2A, the 36 kDa band corresponded to the catalytic subunit of this protein and the 32 kDa band was believed to be a proteolytically cleaved form of the catalytic subunit. The mean values of results showed little significant difference between proliferating and differentiating MEL cells, emphasising that single time-point studies give incomplete and probably misleading information. Multiple time analysis for expression clearly showed evidence of oscillatory behaviour and modulation by the differentiating agent. The influence of HMBA on PP1, PP2A and p53 expression was variable for the different experiments and affected both the frequency and phasing of rhythms. The results add support to the view that dynamic oscillatory control processes play an important role in regulating cellular behaviour. Modulation of the dynamics of key proteins in the cell, such as PP1, PP2A and p53, may be an important mechanism of controlling cellular function and reversing neoplastic transformation.

The ability of a cell to respond precisely to environmental stress depends on the expression of a large number of genes in a finely coordinated manner. One of such genes is CHOP that encodes the CCAAT/Enhancer-Binding Protein Homologous Protein. CHOP is usually expressed to mediate apoptosis under the condition of excessive stress. The expression of CHOP therefore has to be stringently regulated as its expression will determine the fate of a cell under stress. The expression of many genes is regulated at the posttranscriptional level through the metabolism of their mRNA, such as maturation, transport, storage, and degradation of mRNA. Many metabolic processes of mRNA are known to be mediated by RNA-binding proteins that specifically interact with the mRNA. RNA-binding proteins that interact with the CHOP mRNA have until present not been identified. The aim of this study is to investigate what proteins may bind specifically to CHOP mRNA. The study will enable further understanding regarding how the expression of CHOP is regulated in cellular stress response. Proteins extracted from HeLa cells were incubated with a 335bp [3H]-labelled CHOP RNA probe that spans over a part of the coding region and the 3’UTR of CHOP mRNA. Sucrose density gradient ultracentrifugation revealed that after incubation with proteins extracted from HeLa cells, the sedimentation rate of the [3H]-CHOP RNA probe was significantly higher than that of the free [3H]-RNA probe. The formation of heavy molecular complexes involving the [3H]-CHOP RNA probe was therefore suggested. However, no increase in sedimentation rate of the [3H]-CHOP RNA probe was observed in the presence of an excess of unlabelled CHOP RNA probe. Similar observations were made when the experiments were performed using proteins isolated from cells treated with As2O3. Two putative sequence elements, the Adenylate-Uridylate-Rich Element (ARE) and the Putative Regulatory Element (PRE) located respectively in the 3’UTR and coding region of the CHOP mRNA were then examined for their involvement in RNA-protein interaction. The deletion of ARE and/or PRE, from the [3H]-CHOP RNA probe had little effect on the binding of the RNA probe to the HeLa cell proteins. Consistently, unlabelled CHOP RNA probes with the same deletions were only slightly weaker in competing with the intact [3H]-CHOP RNA probe to bind to HeLa cell proteins. Human Antigen R (HuR) was identified by Western blot analysis to be present in the proteins that were obtained by pull-down assays using biotinylated CHOP RNA as a probe. The deletion of ARE and/or PRE resulted in a slight reduction of HuR obtained by pull down assays. This study provides the first evidence that physical binding interaction occurs between intracellular RNA-binding proteins and CHOP mRNA. More importantly, one such protein is HuR. Data suggest that HuR binding to the CHOP mRNA is mediated by sequences in the CHOP mRNA other than ARE and PRE. / published_or_final_version / Biochemistry / Master / Master of Philosophy