published_or_final_version / Surgery / Master / Master of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

Tight junctions, as zones of close contact between epithelial and endothelial cells, form a physical barrier as one of the first host defense strategies that prevent the intrusion of pathogens across epithelia and endothelia. Recently, an interaction between the Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) envelope protein (E) and PALS1, a member of the CRB tight junction complex, was identified in the Virus-Host Interaction group at HKU-Pasteur Research Centre (Teoh et al, 2010). In this report, I present in vitro data which helps to better understand how this protein-protein interaction could interfere with the formation and maintenance of tight junctions at the apical domain of epithelial cells. In previous research, the interaction between E and PALS1 was identified through a yeast two-hybrid screen and confirmed in vitro. A PDZ-binding motif (PBM) was identified at the C-terminal end of E, which interacts with the PDZ domain of PALS1. The objective of my research was to further enhance the knowledge of this interaction by studying the effect of E expression on PALS1 localization and tight junction structure in epithelial cells. I have shown that expression of E is associated with a partial relocalization of PALS1 to the Golgi compartment. Also, I discovered that when wild-type E, E(wt), was expressed in the MDCKII cell model, the time required for tight junction formation was extended to 6-8 hours, while normal cells only required two hours. Interestingly, expression of the E protein with a deletion of the PBM, E(ΔPBM) did not affect the timing of tight junction formation. This finding indicates that the PBM plays a critical role in the process of alteration of tight junctions mediated by E, most likely through its interaction with PALS1. Furthermore, the localization pattern of E was altered when its PBM was deleted. In the MDCKII model, E(wt) located, as expected, at membranes of the Golgi compartment, whereas E(ΔPBM) had a diffused distribution in the cytosol. This observation suggests that the PBM acts as a localization signal for the E protein to the Golgi region, which is the assembly site of the virus. Finally, to examine the role of the PBM in the context of the whole virus, I participated in the production of SARS-CoV recombinant viruses, with mutations in the PBM of E. Though this work is still in progress, the use of these viruses should help to delineate the role of E PBM in SARS-CoV induced pathogenesis in vitro and ultimately in vivo. / published_or_final_version / Pathology / Master / Master of Philosophy

Two of the most challenging goals of evolutionary biology are to reconstruct the evolutionary relationships among all extant species and to understand the process by which new species form. Accomplishing these goals will require accurate computational methods for reconstructing phylogenetic trees, general analytic models of speciation, and powerful statistical tools for studying the process of speciation in natural systems. In the first chapter, I study the effects of improper model assumption on estimates of phylogeny. Using DNA sequence data simulated under a variety of models of sequence evolution, I demonstrate that use of oversimplified models can result in erroneous phylogeny estimates. This result suggests that if the models currently utilized are oversimplified then current estimates of phylogeny may be inaccurate and more complex models need to be developed and employed. In the second and third chapters, I study one process thought to be important in completing the final stages of speciation: reinforcement. Using simulations of a hybrid zone, I show that the process of reinforcement can result in patterns other than reproductive character displacement. I also show that speciation by reinforcement is more likely when the genes involved in reproductive isolation are sex-linked. In the fourth chapter, I develop a statistical method of quantifying the degree of isolation between species undergoing divergence. Using genotype data obtained from natural hybrid zones, this novel method can be used to estimate the fitness of hybrids during different stages of their life cycle. This approach offers a new approach to empirical biologists studying extrinsic postzygotic isolation in natural systems.

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

Dimorphic Leishmania donovani parasites exist as promastigotes in the sandfly vector and differentiate into amastigotes once injected into the skin of human hosts during a blood meal. The mechanisms and signals that are involved in triggering differentiation are not well understood in Leishmania. We have investigated whether tyrosine phosphorylation is a possible signalling component. Differential levels of tyrosine-phosphorylated proteins were observed in extracts from in vitro promastigote and amastigote cultures, with an overall reduction in the latter stage. Following this observation, the inhibition of tyrosine phosphorylation was examined in promastigotes using Tyrphostin AG1433, a broad-spectrum tyrosine phosphorylation inhibitor. AG1433 treated in vitro promastigote cultures differentiate into amastigote-like morphology, have reduced tyrosine phosphorylation level, and express the amastigote-specific marker A2 proteins. Our studies demonstrate that signal transduction mechanisms involving tyrosine phosphorylation/dephosphorylation events are involved in controlling L. donovani promastigote differentiation into amastigote forms.