published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves.

published_or_final_version / Zoology / Master / Master of Philosophy

Soluble form of Junctional adhesion molecule C (sJAM-C) has been identified to cause angiogenesis as well as chemotaxis in endothelial cells. However, the role of sJAM-C in the context of cancer has not been elucidated. Our atomic force microscopy (AFM) stiffness measurements of normal mammary epithelial cells (MCF 10A) have shown a two-fold decrease in cell's stiffness in response to sJAM-C. Changes in cell stiffness are indicative of modulations in a cell's mechanical properties. Our results indicated that sJAM-C increased the MCF 10A cell migration about two-fold and also promoted a three-fold increase in chemotaxis. Additionally, sJAM-C treatment resulted in considerable filamentous-actin loss and peripheral actin ring breakage. We also found activation of Rho signaling pathway to be the main mechanism behind sJAM-C mediated alterations in MCF 10A cell cytoskeleton and motility. Our data present for the first time that sJAM-C is a pro metastatic mediator for normal mammary epithelial cells. / by Anila Qureshi. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

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

Indiana University-Purdue University Indianapolis (IUPUI) / Carbon nanotubes (CNTs) are one of the most common nanoparticles (NP) found in workplace air. Therefore, there is a strong chance that these NP will enter the human body. They have similar physical properties to asbestos, a known toxic material, yet there is limited evidence showing that CNTs may be hazardous to human barrier epithelia. In previous studies done in our laboratory, the effects of CNTs on the barrier function in the human airway epithelial cell line (Calu-3) were measured. Measurements were done using electrophysiology, a technique which measures both transepithelial electrical resistance (TEER), a measure of monolayer integrity, and short circuit current (SCC) which is a measure of vectorial ion transport across the cell monolayer. The research findings showed that select physiologically relevant concentrations of long single-wall (SW) and multi-wall (MW) CNTs significantly decreased the stimulated SCC of the Calu-3 cells compared to untreated cultures. Calu-3 cells showed decreases in TEER when incubated for 48 hours (h) with concentrations of MWCNT ranging from 4µg/cm2 to 0.4ng/cm2 and SWCNT ranging from 4µg/cm2 to 0.04ng/cm2. The impaired cellular function, despite sustained cell viability, led us to investigate the mechanism by which the CNTs were affecting the cell membrane. We investigated the interaction of short MWCNTs with model lipid membranes using an ion channel amplifier, Planar Bilayer Workstation. Membranes were synthesized using neutral diphytanoylphosphatidylcholine (DPhPC) and negatively charged diphytanoylphosphatidylserine (DPhPS) lipids. Gramicidin A (GA), an ion channel reporter protein, was used to measure changes in ion channel conductance due to CNT exposures. Synthetic membranes exposed to CNTs allowed bursts of currents to cross the membrane when they were added to the membrane buffer system. When added to the membrane in the presence of GA, they distorted channel formation and reduced membrane stability.