Thèses (M.Sc.)--Université de Sherbrooke (Canada), 1999. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

Thèses (M.Sc.)--Université de Sherbrooke (Canada), 2001. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

A new procedure has been developed to concentrate the phosphofructokinase from muscle of Ascaris suum with minimum loss of activity. By utilizing this method, 50 ml fraction was concentrated to a final volume of 3 ml in about 1.5 h without loss in enzyme activity. The concentrated enzyme had a specific activity of 64 units per mg. Ascaris muscle-cuticle was incubated in 50 1M solutions of either acetylcholine, serotonin, y-aminobutyric acid, levamisole, or saline alone. Phosphate analysis of the isolated phosphofructokinase from each incubation revealed that the enzyme contained the following moles of phosphate per subunit: 2.9 (acetylcholine), 2.2 (serotonin), 2.0 (y-aminobutyric acid), 1.5 (levamisole), and 3.4 (salne alone). The present study did not establish a direct correlation between degree of phosphorylation and phosphofructokinase activity. Phosphofructokinase from muscle of Ascaris suum appears to contain several phosphorylation sites, and one of these sites is required to be phosphorylated in order for the enzyme to exhibit maximum activity under physiological conditions.

The phosphoinositide 3-kinase (PI3K) enzymes are well known for their regulation of pro-survival signalling cascades that result in increased cell survival and proliferation. However, most of what we understand is based on Class I PI3K enzymes and much less is understood about the Class II enzymes. Loss of PI3K C2α in mice results in embryonic lethality, or severe glomerular injury with increased morbidity. In contrast, PI3K C2β deficient mice display no apparent phenotype and are healthy and viable. Previous work in our laboratory revealed that administration of a sub-nephritogenic dose of nephrotoxic serum led to an augmented immune response resulting in glomerular damage and impaired renal function, which was associated with T-cell infiltration. Elucidating the immunological basis of this sensitivity was the basis of my project. In response to a subcutaneous injection of sheep IgG in complete Freund’s adjuvant, the spleens of PI3K C2β-/- mice showed prominent germinal centre associated cell proliferation that was absent in the controls. Analysis of splenocyte populations revealed that PI3K C2β-/- mice had an increased population of CD4+ T-cells and when cultured in vitro within a total splenocyte population, the increased CD4+ T-cell population was maintained. However, this effect was lost when T-cells were purified and maintained ex vivo. These data suggest that the increased PI3K C2β-/- CD4+ proliferation may be due to additional factors within the total splenocyte population. B-cell populations from the spleens of PI3K C2β-/- mice had higher CD19 expression compared to B-cells from control mice. Elevated levels of CD19 are associated with a reduced activation threshold. In response to stimulation with a sub-optimal dose of LPS and IL-4 PI3K C2β-/- B-cells underwent increased class switch recombination, displayed increased metabolic activity and remained viable for longer than B-cells from control mice. B-cell lysates from PI3K C2β-/- mice also revealed increased levels of phosphorylated MEK1/2. These data indicate that PI3K C2β may serve as a negative regulator of B-cell function and that loss of this PI3K enzyme isoform activity produces a heightened immune response which may lead to a predisposition to associated pathologies.