Thesis (Ph.D.)--University of Nebraska-Lincoln, 2007. / Title from title screen (site viewed July 10, 2007). PDF text: 115 p. : ill. UMI publication number: AAT 3252836. Includes bibliographical references. Also available in microfilm and microfiche formats.

Aerolysin is a channel-forming protein toxin secreted by virulent Aeromonas species. The toxin binds to receptors on cells, is proteolytically activated, and then assembles into a heptameric oligomer, which inserts into the plasma membrane forming a functional channel, resulting in cell death. To further characterize these steps receptor identification, the effect of membrane domains on channel formation and the mode of cell death were investigated on T lymphomas. Screening of cell lysates for proaerolysin-binding proteins N-glycosidase and phosphatidylinositol specific phospholipase C treatment and/or purification of these proteins resulted in the identification of a group of glycosylphosphatidylinositol (GPI)-anchored proteins, which included contactin, Thy-1, and placental alkaline phosphatase. Liposomes were used to show that these proteins were receptors for aerolysin as those containing Thy1 or placental alkaline phosphatase in their membranes were at least 100-fold more sensitive to aerolysin than those without protein. Similarly, cells expressing GPI-anchored proteins were 10⁴-fold more sensitive to aerolysin than cells lacking them. This is likely the result of these proteins concentrating aerolysin on the cell surface and thus promoting oligomerization. The fact that these proteins can be localized to membrane domains known as rafts, which are enriched in sphingomyelin and cholesterol has the potential to affect oligomerization. To investigate this possibility erythrocytes and T lymphomas were treated with methyl-β-cyclodextrin, which destroys rafts by sequestering cholesterol. Raft disruption did not decrease the sensitivity of these cells to aerolysin. Similarly, aerolysin was no more active against liposomes containing placental alkaline phosphatase in raft domains than those in which the receptor was in non-raft domains. Thus raft domains do not promote channel formation by aerolysin. The mechanism of cell death was next investigated. At high toxin concentrations cell death was shown to proceed by necrosis, whereas at subnanomolar concentrations aerolysin triggers apoptosis. Using inactive aerolysin variants it was determined that apoptosis was not a result of binding to GPI-anchored proteins nor was it triggered by receptor clustering induced by oligomerization. Instead the formation of a small number of channels was shown to trigger apoptosis. Taken together these studies have helped to clarify the mode of action of aerolysin. / Graduate

Magister Scientiae - MSc / Many genes are activated to influence the self-destruction programme of the cell. This programme entails synchronised instigation and implementation of numerous subprograms. The arrival of gene targeting aided in the determining of the functions of novel genes. Such genes may have been sequenced, but not functionally characterised. The fulfillment of this requirement through gene targeting technology has swiftly developed. The mode by which DWNN operate in organisms in which it is thought to be covalently linked to some other proteins, which have a definite role in apoptosis, is not yet unraveled. This study attempted the functional characterisation of DWNN in light to the hypothesis that it may be involved in Cytotoxic T lymphocyte killing and apoptosis. / South Africa

Philosophiae Doctor - PhD / The process of cytotoxic T lymphocyte (CTL) killing involves the recognition and destruction of foreign antigens by cytotoxic T cells and is of crucial importance to the defence of the organism against viral infections. Defects in this process can lead to various autoimmune diseases and cancer. The aim of this study was to identify more genes involved in the cell death pathway and to link CTL killing, apoptosis and cancer. / South Africa

No description available.

No description available.

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

No description available.

No description available.

No description available.