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published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Bibliography: leaves 186-193 / vi, 193 leaves ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Organic Chemistry, 1991

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Results of previous research on whole-cell cultures of Penicillium chrysogenum have suggested that acetyl CoA, without being converted to malonyl CoA, supplies the two carbon units for the elongation of palmitic acid. The purpose of this study was to determine the mode of elongation of 1-t4C palmityl CoA by a 20,000 x g mitochondrial pellet from P. chrysogenum.Acetyl CoA or malonyl CoA was incubated with radioactivelylabeled palmityl CoA for 20 minutes. Avidin was added to some experimental reaction mixtures. The resulting fatty acids were saponified, extracted with hexane, methylated with diazomethane, and purified by thin layer chromatography. The methyl esters were separated and identified by gas-liquid chromatography. The radioactivity of each methyl ester was determined by liquid scintillation spectrometry.Elongation of palmityl CoA was observed in the presence of acetyl CoA, but not in the presence of malonyl CoA. The addition avidin produced a greater proportion of short-chained fatty acidsthe expense of palmitic acid, but did not decrease the percentage of long-chained fatty acids produced.A high proportion of label was recovered in the C18:3 fatty acid, linolenic acid. This suggested that two pathways of linolenic acid synthesis may be operating in this organism.Methods for detection and control of cancer encompass a large area of today's research. Recent use of granulomas as a model for such detection and control may be a promising field, especially for monitoring tumor antigens and immune responses. These granuloma systems are increasingly becoming vehicles in the study of tumor immunology. Although granulomas may be induced naturally by means of foreign bodies i.e. viral, fungal, or bacterial agents, new methods are being established to produce artificial granuloma systems. These systems include chemical or foreign body implantations followed by tumor vaccine challenges. The research presented here involved the use of a golf ball-induced granuloma for the purpose of establishment of a detection system for immune responses. The use of a golf ball-induced granuloma provided a closed system for monitoring cell-mediated and humoral responses to tumor antigens. Immune responses were monitored by means of hematocrits (packed blood cell counts), white blood cell differential counts, and electrophoretic results.Hematocrit results indicated no great immune response to the closed vaccine injected granulomasystems. Observations made on differential white blood cell counts indicated decreasing neutrophil/lymphocyte ratios for cellular immune responses. Electrophoretic results for granuloma fluids indicated decreases in albumin levels concurrent with increases in peak two,and complete loss of peak three following vaccination. Responses to tumor specific antigens in the form of cell-mediated immune responses are indicated by the results presented in this research. Utilization of the golf ball-induced granuloma system provided a means of separating the cell-mediated and humoral immune responses.Tumor specific antigens elicited various immune responses and provide hope for future identification of tumors by this method. Future development and utilization of the golf ball-induced granuloma system may be potential means of monitoring cell-mediated immune responses to tumor malignancies.

Similar to many cancers, pancreatic cancer (PDA) assumes an altered metabolic state that is designed to support the manufacture of macromolecules necessary for cell division. For example, membrane synthesis from lipid precursors is dependent on de novo fatty acid synthesis (dnFAS), a pathway that is upregulated in many cancers. The rate-limiting enzyme of dnFAS is fatty acid synthase (FASN), an enzyme that is overexpressed in cancer but found at very low levels in normal tissues. Here we demonstrate that fatty acid synthase (FASN) inhibition using novel small molecule inhibitor IPI-9119 induces apoptosis in FASN overexpressing epithelial cells of a pre-clinical pancreatic cancer mouse model (KPC). We also provide evidence that FASN inhibition insensitivity in PDA cells is due to flux through scavenging pathways, particularly macropinocytosis. We initially hypothesized that FASN overexpression is a survival strategy for PDA epithelial cells through which they acclimate to a low nutrient microenvironment. Indeed, K-ras driven metabolic reprograming has been shown to increase flux through the dnFAS pathway indicating that PDA cells should rely heavily on FASN activity. Surprisingly we found that PDA cell lines are minimally affected by FASN inhibition and rely chiefly on scavenging pathways. In vitro cells displayed a dependency on lysosomal function, determined using lysosomal inhibitor chloroquine (CQ). Dual therapy with FASN inhibitor, IPI-9119, and lysosomal inhibitor, CQ, increased apoptosis and decreased cell viability in human PDA cell lines and in bulk KPfl/flC PDA tumors. Our findings suggest that mutated epithelial tumor cells overexpress FASN and that these oncogenic cells can be specifically targeted using FASN inhibition. Our findings in regards to scavenging pathways demonstrate that PDA is reliant on lysosomal activity and may utilize both autophagy and macropinocytosis to maintain lipid levels in addition to dnFAS. Dual therapy with a FASN inhibitor and a lysosomal inhibitor induces apoptosis in PDA cell lines regardless of their sensitivity to FASN inhibition alone indicating that this therapeutic strategy, dual treatment with lysosomal inhibitors and FASN inhibitors, should be further developed. Our major conclusions from this work are: (1) that PDA is not solely dependent on FASN activity for lipid maintenance but short-term inhibition results in targeted apoptosis in FASN expressing cells, in vivo. (2) Lipid scavenging pathway macropinocytosis accounts for primary resistance to FASN inhibition in vitro, although autophagy may also contribute. (3) Dual inhibition of FASN and lysosomal function induces apoptosis and decreases proliferation in PDA cell lines and tumors.

Graduation date: 1997

Graduation date: 1995