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Title: Intracellular signalling mechanisms of the unicellular parasite, T.b. brucei
Author: Cameron, A. L.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1998
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The primary aim of this work was to investigate phospholipid metabolism in trypanosomes, including a phosphoinositide signalling, and the metabolism of the VSG and its GPI anchor. The phosphoinositides of T.b brucei were labelled using 32P (from [32P)Pi or [γ32P]ATP) and [3H)inositol through a variety of methods. Most work was carried out with phospholipids labelled using [γ32P]ATP as the donor, with cells permeabilised by being placed in a buffer of low osmotic strength (swell dialysis). Species labelled with 32P were identified as PI 4-P and PI 4,5-P2 through HPLC analysis. There was no evidence of 3-phosphorylated phosphoinositides under the conditions used. Results showed that T.b brucei has GTP- and Ca2+-regulated phosphoinositide metabolism, similar to that seen in vertebrate cells. Additionally, an unidentified factor in rabbit serum caused a 2-fold increase in the phosphorylation of the polyphosphoinositides. Although Ca2+ is known to cause the release of VSG through the action of GPI-PLC, it is not clear whether this enzyme plays an important role in membrane-form (mf) VSG metabolism in intact cells. The relationship between VSG release and subsequent GPI anchor metabolism was examined using a GPI-PLC inhibitor (pCMPSA) and conditions known to cause the release of soluble form (s) VSG (Ca2+). Metabolism of the mfVSG GPI anchor was taken to correspond with [32P]PA generation in cells incubated in the presence of [γ32P)ATP. Anti-VSG antibodies were used to detect VSG in supernatant samples of T.b. brucei. A great deal of phospholipid metabolism sensitive to pCMPSA, so possibly related to GPI-PLC activity, was shown to occur in the absence of sVSG release. The primary role of GPI-PLC in viable trypanosomes may therefore be in the regulation of processes other than mfVSG metabolism. T.b. brucei was incubated with anti-VSG IgGs to observe whether interaction with anti-VSG antibodies caused the destruction of mfVSG and concomitant increase in PA labelling. Anti-VSG IgGs had no effect on PA metabolism. mfVSG endocytosed in bloodstream from T. brucei may therefore be returned intact to the cell surface.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available