Date of Award

Winter 2007

Project Type

Thesis

Program or Major

Chemistry

Degree Name

Master of Science

First Advisor

Steven Levery

Abstract

Glycosphingolipids (GSLs) are ubiquitously distributed among all eukaryotes. In fungi, GSLs have also been implicated in a variety of cellular processes, including growth, cell signaling, differentiation and morphogenesis. Detailed knowledge of fungal GSL structure and biosynthesis is therefore expected to yield valuable insights into both their functions within the organism and their interactions with other organisms, such as with the immune system, of an infected host as well as to provide targets for diagnostic and therapeutic agents.

Research described in this thesis focused on fungal GSL structure and biosynthesis. Two projects are described. The first was aimed at elucidating the functions of GSLs in Neurospora crassa. The isolation and structural analysis of glycolinositolphosphoryl ceramides (GIPCs) from N. crassa wild type (ATCC 18889 and 24698) and a defensin-resistant mutant strain (SP 10) were attempted, using NMR, MS and other techniques. Although no GIPCs were ultimately isolated in this work, phosphatidylinositol, the donor substrate for inositolphosphorylceramide IPC synthase, was detected and characterized. In addition, a preliminary study of the effects of an inhibitor of GSL biosynthesis, 4'-ethylenedioxy-1-phenyl-2-palmitoyl-3-pyrrolidinopropanol (EDO-P4), was performed. The results of this experiment were consistent with inhibition of N. crassa glucosylceramide synthase (GCS) by EDO-P4.

In the second project, studies of the structures and biosynthesis of glycosphingolipids of the pathogenic model fungus, Cryptococcus neoformans were performed on both the wild type (JEC 21) and a mutant, termed cxt1cxt2Delta, in which the genes for C. neoformans beta1,2-xylosyltransferases, Cxt1 and Cxt2 had been disrupted. Our results demonstrated an altered expression in GIPC structures in the absence of the two xylosyltransferases, characterized by the expression of normal C. neoformans GIPC core structure, but without addition of characteristic Xylbeta1,2 residue. We detected no residual xylose-containing GIPCs in the cxt1cxt2Delta mutant sample, confirming that no other enzyme performs the function of xylose addition during GIPC synthesis.

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