Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 73-81). Also available in print.

Thesis (M.S.)--The University of North Carolina at Greensboro, 2009. / Directed by Paul Steimle; submitted to the Dept. of Biology. Title from PDF t.p. (viewed May 11, 2010). Includes bibliographical references (p. 37-39).

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 May 10

Thesis (Ph.D.)--Kent State University, 2006. / Title from PDF t.p. (viewed Jan. 11, 2007 ) Advisor: J Gary Meszaros. Keywords: cardiac fibroblast, angiotensin II, fibrosis, MAPK Includes bibliographical references (p. 150-168).

Thesis (M.Phil.) - University of Queensland, 2006. / Includes bibliography.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

Thesis (Ph. D. in Molecular Physiology and Biophysics)--Vanderbilt University, Aug. 2007. / Title from title screen. Includes bibliographical references.

Abstract Cardiac hypertrophy and remodelling are mechanisms of adaptation to increased workload and acute injuries of the heart. In the long-term, these initially beneficial mechanisms become detrimental and ultimately lead to the development of heart failure. The molecular determinant of myocardial remodelling and heart failure is altered intracellular signal transduction and a modified gene expression pattern in the individual cardiomyocyte. This study was aimed at characterising the changes in mitogen-activated protein kinases (MAPKs) and their nuclear effector, GATA-4, and their functional significance and interaction in experimental models of increased cardiac workload and remodelling. To study the effects of increased cardiac workload on MAPKs and GATA-4, isolated perfused rat hearts were subjected to increased left ventricular wall stress and their activities were determined using western blot and gel mobility shift assays. Left ventricular wall stress rapidly activated the DNA binding of GATA-4, and this activation was abolished in the presence of endothelin-1 (ET-1) and angiotensin II receptor antagonists. Furthermore, the activation of GATA-4 DNA binding was significantly attenuated by p38 MAPK and extracellular signal regulated kinase (ERK) inhibition. To gain further insights into the role of p38 MAPK as a regulator of cardiac transcription factors, gene expression and remodelling, a gene transfer protocol of increased p38 MAPK activity was established. Direct adenovirus-mediated gene transfer of wild-type p38α and constitutively active upstream kinase mitogen-activated kinase kinase 3b (MKK3b) selectively increased p38 MAPK activity in the left ventricle, which was followed by up-regulation of cardiac gene expression, myocardial inflammation and fibrosis. Using a DNA microarray approach, the cardiac target genes of p38 MAPK were identified, including several cell division, inflammation and signal transduction-associated genes. Furthermore, p38 MAPK over-expression was found to increase the DNA binding activities of several transcription factors, including GATA-4. Finally, the functional role of p38 MAPK was determined using adenovirus-mediated gene transfer in an experimental model of myocardial infarction. Post-infarction remodelling was characterised by a sustained down-regulation of p38 MAPK, while rescue of p38 MAPK activity attenuated post-infarction remodelling through anti-apoptotic and angiogenic mechanisms. These results indicate that p38 MAPK is a key regulator of GATA-4 transcription factor and cardiac gene expression during left ventricular wall stress and remodelling. They demonstrate that p38 MAPK, being cardioprotective in the infarcted heart but promoting inflammation and fibrosis in the normal heart, has a unique dual role in the myocardium.

The mechanism by which insulin and other polypeptide growth factors alter cellular metabolism is not fully understood. In the case of insulin, it is thought that phosphorylation/dephosphorylation mechanisms may play a central role in the signalling pathway. This is based on evidence which includes demonstration that the receptor for insulin is a tyrosine-specific protein kinase which is activated in response to insulin binding. Ultimately, insulin binding to its receptor on the surface of intact fat cells leads to altered levels of serine phosphorylation of several soluble proteins, including the phosphorylation of ATP-citrate lyase and acetyi-CoA carboxylase. Recently, studies involving site-specific mutagenesis have shown that the tyrosine kinase function of the insulin receptor is essential for insulin signalling. The studies described in this thesis have addressed the problem of how activation of the insulin receptor/tyrosine kinase results in the altered serine phosphorylation observed in intact cells in response to insulin. To gain further understanding of the cellular components required for insulin signalling, reconstitution experiments have been carried out mixing isolated cellular fractions with preparations of insulin receptors. The effects of insulin on altering protein-serine and protein-tyrosine phosphorylation have been determined in this reconstituted system. Results show that in a high-speed (100,000 x g) supernatant fraction prepared from rat adipose tissue endogenous protein-serine kinases are sensitive to conditions which are commonly employed for assaying insulin receptor/kinase activity. This includes inhibition by micromolar concentrations of MnCI₂, by 40 mM NaF, and by low reaction temperature (0°C). When the insulin receptor, present in a WGA-Sepharose-purified preparation of detergent-solublized rat liver membranes, was assayed in the complete absence of both MnCI₂ and NaF, receptor/tyrosine kinase activity was only slightly reduced with little or no decrease in the responsiveness to insulin. Furthermore, when the WGA-Sepharose-purified membrane fraction was incubated at 37°C in the presence of [ɣ -³²P]ATP several endogenous proteins were observed to be phosphorylated in addition to the β-subunit of the insulin receptor. These membrane proteins appear to be phosphorylated on tyrosine as indicated by their resistance to alkali hydrolysis. Upon reconstitution of the adipose tissue high-speed supernatant fraction with the WGA-Sepharose-purified preparation of insulin receptors the most striking effects observed were the phosphorylation of a 40 kd protein subunit (pp40) and the dephosphorylation of a 25 kd protein subunit (pp25) present in adipose tissue. The phosphorylation of pp40 occurs on tyrosine and is insulin-responsive, whereas the dephosphorylation of pp25 occurs following reconstitution with either untreated control, or insulin-activated insulin receptors. To assess the effect that reconstituted insulin receptors may have on the phosphorylation of endogenous ATP-citrate lyase in adipose tissue high-speed supernatant, it was found that a more pure preparation of insulin receptors was required. Further purification of the insulin receptor to homogeneity was therefore attempted using insulin-agarose affinity chromatography. However, difficulties including low yield and instability of the receptor through purification have prevented progress with these studies at present. In a separate study, highly purified acetyl-CoA carboxylase was reconstituted with a crude fraction consisting of total Triton-solublized membrane proteins. In this reconstituted system phosphorylation of acetyl-CoA carboxylase was enhanced to an extent greater than 6-fold after incubation with [ɣ -³²P]ATP. Following chromatography of the crude Triton-solublized extract over WGA-Sepharose this acetyl-CoA carboxylase kinase activity was found to be present in the flow-through void fraction and not in the N-acetylglucosamine eluted fraction. The acetyl-CoA carboxylase kinase, at present, does not appear to be insulin-responsive, but further studies are needed to confirm this observation. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate