Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.692270
Title: Establishing a biological role for class II phosphoinositide 3-kinase (PI3K) enzyme PI3K-C2??
Author: Balakrishnan, Sanjeevi
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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Abstract:
Phosphoinositide 3-Kinase (PI3K) enzymes control a variety of cellular processes and their deregulation has been extensively investigated in disease conditions including renal disease, autoimmunity and chronic inflammation. Whilst PI3K enzymes have been the subject of intense research activity, the precise biological role of PI3K-C2?? enzyme remains unclear. PI3K-C2??-/- mice developed focal segmental glomerulosclerosis, damaging podocyte morphology and affecting function. My thesis explored the contribution of the PI3K-C2?? enzyme in the pathology of chronic diseases. PI3K-C2??flx/+ mice were mated with ??-actin cre mice, and the resulting F1 progeny, PI3K-C2??flx/- mice were backcrossed to produce PI3K-C2??-/- mice. Analysis of PI3K-C2??-/- mice revealed slightly elevated proteinuria and mild hyperglycaemia. Histological examination of their kidneys showed an increase in glomerular volume, intraglomerular nuclei, mesangial matrix expansion and an infiltration of T cell subsets in the glomeruli compared to control mice. In vitro, mesangial cells derived from PI3K-C2??-/- mice proliferated twice as fast as mesangial cells from control animals, without any stimulus. PI3K-C2??-/- mice exhibited an increase in the levels of leukocytes and displayed augmented proliferation, in presence of mitogenic stimuli. Wild type (WT) mice mesangial cells cultured in PI3K-C2??-/- mice splenocyte conditioned medium, exhibited a dose dependent increase in their proliferation. Induction of glomerulonephritis (GN) using a sub-nephritogenic dose of nephrotoxic serum (NTS) in PI3K-C2??-/- mice, resulted in impaired renal function and aggravated renal tissue damage. PI3K-C2??-/- mice had a greater influx of T cells and macrophages in the diseased glomeruli, but also of activated macrophage functions compared to WT mice. During GN, PI3K-C2??-/- mice developed splenomegaly leading to extramedullary haematopoiesis and increased splenocyte proliferation. PI3K-C2??-/- mice may mediate GN by polarising their cytokine effects via a Th1 pathway. PI3K-C2??-/- mice exhibit a delay in the repair of dermal injury during cutaneous wound healing. PI3K-C2??-/- mice recruit fewer macrophages to the wound site which affects re-epithelialisation, myofibroblast differentiation, angiogenesis and fibroblast mediated remodelling. Delayed wound healing in PI3K-C2??-/- mice might be a consequence of the failure to upregulate pro-inflammatory cytokines at the wound site, which are potential modulators for the recruitment of various cell types to the wound site to accelerate the healing process. To complement these in vivo studies, biochemical analysis on the putative Ras binding domain present in PI3K-C2?? and PI3K-C2?? enzymes was performed by expressing as recombinant proteins. The binding ability of the recombinant proteins to Ras family GTPases was tested. Although this approach initially proved unsuccessful, two putative binding partners, eEF1?? and eEF1?? were identified and have been shown to immunoprecipitate with full length enzymes. My data is the first study to demonstrate a role for the intracellular signalling enzyme, PI3K-C2?? in pathological conditions involving chronic inflammation. Agonist mediated activation of PI3K-C2?? enzyme might serve as a potential therapeutic target to treat chronic diseases.
Supervisor: Domin, Jan ; Pusey, Charles Sponsor: Imperial College London
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.692270  DOI: Not available
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