Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 119-134). Also available in electronic version.

Thesis (Ph. D.)--University of Chicago, Dept. of Biochemistry and Molecular Biology, June 1999. / Includes bibliographical references. Also available on the Internet.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 99-117). Also available in print.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

My PhD project investigated the regulation of human cardiac transient outward potassium current (Ito) by protein tyrosine kinases (PTKs) and the functional expression of transient receptor potential (TRP) channels in human atrial myocytes to make an advanced understanding of human cardiac electrophysiology and pathophysiology. The modulation of human cardiac Itoby PTKs was studied in human atrial myocytes and HEK 293 cells expressing hKv4.3 (coding human cardiac Ito). We found that the broad-spectrum PTK inhibitor genistein, the selective EGFR kinase inhibitor AG556, and the Src-family kinases inhibitor PP2 inhibited human atrial Itoand the inhibitory effect was countered by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. Similar results were observed in hKv4.3-HEK cells. Interestingly, tyrosine phosphorylation of hKv4.3channels was reduced by genistein, AG556, and PP2,and the reduction was antagonized by orthovanadate. The mutant Y136F of hKv4.3 lost the inhibitory response to AG556, whileY108F lost the response to PP2.The double mutant Y108F-Y136F hKv4.3 failed to respond to both AG556 and PP2, and exhibited a dramatic reduction of tyrosine phosphorylation. These results indicate that native cardiac Itois regulated by both EGFR and Src family kinases. In the second part, we studied whether TRPC channels would mediate the nonselective cation current described previously in human atrial myocytes. It was found that TRPC1 channel activator thapsigargin activated the current, and the effect was suppressed by La3+or prevented by intracellular anti-TRPC1 antibody. Endothelin-1 and angiotensin II stimulated the current, andthe effect was inhibited by La3+and/or 2-APB. RT-PCR and Western blot analysis revealed that in addition to the TRPC1 channels mediating the nonselective cation current, the components of store-operated Ca2+channels (SOCs), STIM1 and Orai1 were abundantly expressed in human atria. The interaction of TRPC1, STIM1, and Orai1 was confirmed by co-immunoprecipitation. Interestingly, we found that protein expression of TRPC1 and STIM1, but not Orai1, was up-regulated in human atria with atrial fibrillation. The third part of the project determined whether TRPM7 channels were expressed in human atrial myocytes, since this channel was reported in human atrial fibroblasts, conferring atrial fibrosis in human atria with atrial fibrillation. We found a TRPM7 -like current which was potentiated by acidic pH, and inhibited by La3+and 2-APB, and a Ca2+-activated TRPM4 current. RT-PCR and Western blot analysis confirmed the expression of TRPM7 and TRPM4 channels in human atria. Moreover, we found TRPM7 protein, but not TRPM4 protein was significantly up-regulated in human atria with atrial fibrillation, suggesting the potential participation of TRPM7 channels in atrial remodeling of human atria with atrial fibrillation. Collectively, this PhD thesis project has demonstrated for the first time that human cardiac Itois modulated by EGFR kinase and Src kinases via phosphorylating Y136and Y108, respectively. TRPC1 channels mediate the nonselective cation current and SOCs.TRPM7 channels are expressed in human atrial myocytes. The up-regulation of TRPC1, STIM1, and TRPM7 channels in human atria with atrial fibrillation suggest that they are likely involved in atrial electrical and/or structure remodeling in patients with atrial fibrillation. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

 In rat testes, at stage VIII of the epithelial cycle of spermatogenesis, two cellular events, namely blood-testis barrier (BTB) restructuring and spermiation, take place simultaneously but at the opposite ends of the seminiferous epithelium. BTB is constituted by tight junctions (TJs), basal ectoplasmic specializations (ES), gap junctions and desmosomes, which must disassemble intermittently at stage VIII to facilitate preleptotene spermatocyte migration across the barrier. Synchronously, spermiation occurs at the luminal edge of the tubule lumen, involving the disruption of the apical ES, the only anchoring device there, and the release of sperm. The mechanism coordinating these events is not well understood. In this dissertation, I provide evidence that intercellular adhesion molecule (ICAM)-1 and -2, are working in concert with sarcoma (Src) family kinases to regulate these events. ICAMs comprise an immunoglobulin subfamily of cell adhesion proteins expressed by hematopoietic, endothelial and epithelial cells. They are known to function in the transendothelial migration of leukocytes. In the rat testis, ICAM-1 was shown to localize to both BTB and apical ES stage-specifically, with its immunoreactivity highest at stage VIII at the BTB. Besides co-immunoprecipitation and co-localization with BTB proteins, such as occludin and N-cadherin, ICAM-1 was found to promote BTB integrity in that its over-expression (O-E) in Sertoli cells in vitro increased transepithelial electrical resistance (TER). However, O-E of a truncated form of ICAM-1 (sICAM-1) that only consisted of the extracellular domain resulted in decreased TER and down-regulation of several BTB constituent proteins, possibly via the Src/Pyk2 signaling pathway. O-E of sICAM-1 in vivo also compromised the BTB integrity. These findings illustrate that ICAM-1 is an important regulator of the BTB. On the other hand, the localization of ICAM-2 was restricted to the Sertoli-germ cell interface and absent from the BTB, and associated with β1-integrin, nectin-3 and F-actin at the apical ES. Further, ICAM-2 was shown to interact with Src and Pyk2, as well as annexin II, a phospholipid-binding protein. Intriguingly, ICAM-2, Src and annexin II were specifically up-regulated during CdCl2-induced germ cell loss. These results reveal that ICAM-2 actively participates in the restructuring of apical ES based on studies using the cadmium model. The function of c-Yes, a member of the Src family, was also investigated. It was found to be stage-specifically expressed at the BTB and the apical ES, and it structurally associated with BTB components (e.g., occludin and N-cadherin) and with the apical ES proteins (e.g., β1-integrin, laminin β3 and γ3). In the study, the knockdown of c-Yes by RNAi in vitro and in vivo affected BTB and apical ES function, causing changes in the distribution/localization of adhesion proteins at the BTB and the apical ES, inducing germ cell loss from the seminiferous epithelium, possibly via an interference with the F-actin network. These findings implicate that ICAMs and c-Yes are regulatory molecules of cell adhesion at the BTB and the apical ES, and are biomarkers for male contraceptive development. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Paediatrics / Master / Master of Philosophy

FES protein-tyrosine kinase (PTK) activation downstream of the KIT receptor in mast cells (MC) promotes cell polarization and migration towards the KIT ligand Stem cell factor (SCF). A variety of tumours secrete SCF to promote MC recruitment and release of mediators that enhance tumour vascularization and growth. This study investigates whether FES promotes MC migration via regulation of microtubules (MTs), and if FES is required for MC recruitment to the tumour microenvironment. MT binding assays showed that FES has at least two MT binding sites, which likely contribute to the partial co-localization of FES with MTs in polarized bone marrow-derived mast cells (BMMCs). Live cell imaging revealed a significant defect in chemotaxis of FES-deficient BMMCs towards SCF embedded within an agarose drop, which correlated with less MT organization compared to control cells. To extend these results to a tumour model, mouse mammary carcinoma AC2M2 cells were engrafted under the skin and into the mammary fat pads of immune compromised control (nu/nu) or FES-deficient (nu/nu:fes-/-) mice. A drastic reduction in tumour-associated MCs was observed in FES-deficient mice compared to control in both mammary and skin tissue sections. This correlated with a trend towards reduced tumour volumes in FES-deficient mice. These results implicate FES signaling downstream of KIT, in promoting MT reorganization during cell polarization and for chemotaxis of MCs towards tumour-derived SCF. Thus, FES is a potential therapeutic target to limit recruitment of stromal mast cells or macrophages to solid tumours that enhance tumour progression. / Thesis (Master, Biochemistry) -- Queen's University, 2011-09-14 11:49:32.871

Agrin triggers the subsynaptic aggregation of acetylcholine receptor (AChR) via activation of the receptor tyrosine kinase MuSK (muscle-specific kinase). At present, the intracellular mechanisms utilized by MuSK to initiate such a complex process remain unknown. In the present study, I first tested if H-ras was involved in the process of synaptogenesis induced by agrin. The data presented suggest that ras could have a role in this process because a dominant inhibitory ras mutant (ras-N17) partially blocked the inductive effects of agrin while two activated ras mutants (ras-V12 and ras-V12-D38) induced agrin-independent AChR clusters. These effects were not due to major alterations in the levels of AChR, though more experiments are required to confirm these preliminary findings. / Second, I investigated whether the adaptor protein Shc was a downstream effector of activated MuSK. MuSK and Shc could be co-immunoprecipitated, but this association was not consistently observed nor was it modulated by agrin at all times. Generally, no alteration in Shc phosphotyrosine content was observed in response to agrin, and when an increase was detected, it was modest. Finally, agrin did not modulate the interaction between Shc and Grb2. Based on these results, I conclude that Shc interaction with MuSK is not regulated by agrin.

No description available.