Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Osmostasis relies on the efficient and selective transport of molecules across barriers, often in a directional manner and this, in turn, is dependent on the asymmetric arrangement of channels within the cells comprising the organs that regulate these processes. The primary aim of this thesis is the identification of the extracellular matrix-receptor interactions important in the generation of polarized channel distribution in two such organs, the kidney and the blood-brain barrier. In the first study, we investigated the involvement of fibronectin and laminin in the basolateral localization of Kir4.1, an inwardly-rectifying potassium channel, and aquaporin-4 (AQP4), a water-permeable channel, in polarized Madin-Darby canine kidney (MDCK) cells. We determined using a variety of approaches that laminin-1 and fibronectin, when present in the culture substrate, significantly stablize both channel types at the basolateral surfaces of these cells and accordingly induce their increased expression within this membrane domain without requiring a concomitant upregulation of de novo channel synthesis. We also show that the coexpressed laminin receptor dystroglycan (DG) is important for cell surface expression of Kir4.1 but not AQP4, and demonstrate via the use of disintegrin peptides and function-blocking antibodies that their cell-surface expression and stability is also partly reliant on integrin receptors, with αvβ3 being particularly important in the case of the latter. In the second study, we examined the possibility that laminin-dystroglycan binding is involved in the regulation of AQP4 turnover in astrocytes. We first determined that laminin, when applied to primary astrocytes in culture, causes AQP4 amounts at the plasma membrane to increase in a DG-dependent manner while depleting the channel from intracellular sites, brought about by the suppression of channel endocytosis. We then showed that DG binds to inactive dynamin, and that the latter, when freed from this inhibitory influence, functions in cooperation with clathrin to mediate the rapid internalization of AQP4. Finally, we demonstrated that laminin selectively upregulates the cell-surface expression of the M23 isoform of AQP4 only. Our findings therefore reveal that, through their roles in establishing the microscopic architecture of these systems, the extracellular matrix and cell-surface receptors are critical determinants in osmoregulation
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Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Astrocytes play an important role in a wide variety of physiological processes and indisease states such as ischemia. Ischemic damage in the brain involves apoptotic cell death ofneurons as well as astrocytes and it has been suggested that reactive oxygen species (ROS)generated as a consequence of ischemia are a major factor in triggering cell death. The waterpermeablechannel, aquaporin 4 (AQP4), which is expressed at high concentrations in astrocytes,is an important determinant of mortality and morbidity in mice subjected to ischemia, howeverthe effects of ROS on AQP4 expression and the underlying mechanisms are still obscure. In thepresent study, we used primary astrocyte cultures to examine the expression of AQP4 underoxidative stress using hydrogen peroxide. First, we showed that H₂O₂ induces a significantincrease of both AQP4 mRNA and protein levels and that this effect is inhibited by the antioxidantN-acetylcysteine. Second, we demonstrated using cell surface biotinylation that H₂O₂increases AQP4 plasma membrane expression independently of the newly synthesized pool ofAQP4. In parallel, we found that caveolin-1 undergoes a dose- and time-dependentphosphorylation in astrocytes treated with H₂O₂ and that this effect is inhibited by the src kinaseinhibitor PP2. More importantly, PP2 inhibits the H₂O₂-induced increase in AQP4 cell surfaceexpression, suggesting that the phosphorylation of caveolin-1 and possibly other proteins mayplay a role in this process. To investigate this further, we used MDA-435 cells expressing Y14Fand Y14D caveolin-1 mutants transfected with AQP4 and found that these cells exhibit adecrease and an increase in AQP4 membrane expression, respectively. Furthermore, caveolin-1knock down in astrocytes inhibits H₂O₂-induced increase in AQP4 cell surface expression.Together these findings show that the phosphorylation of caveolin-1 Y14 is a key regulator ofAQP4 cell surface expression in oxidative stress possibly by altering AQP4 internalization andtrafficking resulting in its redistribution within different compartments of the cell.
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