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.

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"September 2002" Bibliography: leaves 207-230. Ch. 1. Literature review -- Ch. 2. General methods -- Ch. 3. Dietary n-3 PUFAs and reperfusion injury in isolated cardiomyocytes -- Ch. 4. The effect of dietary n-3 PUFAs on cardiomyocyte membrane fluidity, intracellular ROS and Ca 2+ levels during oxidative stress -- Ch. 5. The effect of dietary fish oil supplementation on antioxidant enzyme gene expression in rat myocardium -- Ch. 6. The effect of dietary lipids on ischaemia-reperfusion injury in rat myocardium -- Ch. 7. General discussion -- Ch. 8. Appendices. The broad aims of this thesis were to develop a cellular model for studying reperfusion injury, in order to investigate the reported protective effects of n-3 PUFAs, and to examine the underlying mechanisms associated with such protection.

The physicochemical characteristics of seven cold-pressed flaxseed oils sold in New Zealand were investigated for their fatty acid composition, tocopherol composition, moisture and volatile matter, free fatty acids, chlorophyll pigments, unsaponifiable matter, total phenolic acids and flavanoids, and colour. The seven cold-pressed flaxseed oils exhibited significant variations in their physicochemical characteristics. Quality of the oils in terms of oxidative stability was also investigated. Four oils were found to be within the limit of good stability oil indices, measured in terms of peroxide value, p-anisidine value, conjugated dienoic acids, specific extinction in ultraviolet spectrum, acid value and food oil sensor readings (to indicate total polar compounds). The role of minor constituents in the oxidative stability of two selected oils with different levels of fatty acid composition and minor constituents was investigated. Pan heating at 150�C caused loss of tocopherols, plastochromanol-8, phenolic acids, chlorophyll pigments, β-carotene and lutein and changes in the fatty acid composition. The pan-heated oils exceeded the limit of good stability oil indices using the measurement mentioned above except for acid value. The addition of α-tocopherol to the oils did not provide enhanced protection to the oils in accelerated aging of oil tests at 60�C. It was most likely that phenolic acids present in the oils played a dominant role in the oxidative stability of the oils. Lipase-catalyzed transesterification of triolein with cinnamic and ferulic acids using a commercially available immobilized lipase B from Candida antarctica (Novozym 435) was conducted to evaluate the antioxidant activity of the lipophilized products as model systems for enhanced protection of unsaturated oil. The lipophilized products were identified using Electrospray Ionization-Mass Spectroscopy (ESI-MS). Separation and isolation of two classes of lipophilized products was also achieved using a solid phase extraction method developed in this study for further investigation into the structure-free radical scavenging activity. Free radical scavenging activity was determined using the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) method. The polarity of the solvents proved important in determining the free radical scavenging activity of the substrates. Ferulic acid showed much higher free radical scavenging activity than cinnamic acid, which had limited activity. The esterification of cinnamic acid and ferulic acid with triolein resulted in significant increase and decrease in the free radical scavenging activity, respectively. These opposite effects were due to the effect of addition of electron-donating alkyl groups on the predominant mechanism of reaction (hydrogen atom transfer or electron transfer) of a species with DPPH. The effect of esterification of cinnamic acid was confirmed using ethyl cinnamate which greatly enhances the free radical scavenging activity. Although, compared with the lipophilized cinnamic acid product, the activity was lower. The free radical scavenging activity of the main component isolated from lipophilized cinnamic acid product using solid phase extraction, monocinnamoyldioleoylglycerol, was as good as the unseparated mixture of lipophilized product. Based on the ratio of a substrate to DPPH concentration, lipophilized ferulic acid was a much more efficient free radical scavenger than lipophilized cinnamic acid. Lipase-catalyzed transesterification of flaxseed oil with cinnamic and ferulic acids using Novozym 435 was conducted to evaluate whether the lipophilized products provided enhanced antioxidant activity in the oil. The lipophilized products were identified using ESI-MS and were examined for their free radical scavenging activity toward DPPH in ethanol and ethyl acetate. Ferulic acid showed the highest free radical scavenging activity among all substrates tested while cinnamic acid had negligible activity. The effect of esterification of cinnamic acid and ferulic acid with flaxseed oil was similar to that with triolein. Lipophilized ferulic acid was a better free radical scavenger as compared with lipophilized cinnamic acid and extended the naturally-occuring antioxidant capacity of the flaxseed oil. Lipophilized cinnamic acid did not provide much enhanced radical scavenging activity in the flaxseed oil as the presence of natural hydrophilic antioxidants in the oil had much greater radical scavenging activity. It may still be useful for unsaturated oils with a small amount of natural antioxidants in them. Lipophilized cinnamic and ferulic acids showed higher free radical scavenging activity when tested in a less polar solvent (ethyl acetate) whereas ferulic acid showed better activity in a more polar solvent (ethanol). These results indicate that the choice of solvent for the DPPH assay is critical in evaluating the free radical scavenging activity of substrates of differing polarity, and support previous observations by other authors that the solubility of an antioxidant in relation to the site of oxidation is an important factor for consideration in the use of antioxidants.

"September 2002" / Bibliography: leaves 207-230. / x, 230 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The broad aims of this thesis were to develop a cellular model for studying reperfusion injury, in order to investigate the reported protective effects of n-3 PUFAs, and to examine the underlying mechanisms associated with such protection. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 2002

Graduation date: 1997

Graduation date: 1995

To examine how dietary n-3 polyunsaturated fatty acids affect Th2 cell development, female C57BL/6 mice were fed a washout corn oil (CO) diet for 1 wk followed by 2 wk of either the same CO diet or a fish oil (FO) diet. CD4+ T cells were isolated from spleens and cultured under both neutral (anti-CD3 and phorbol myristate acetate (PMA)) and Th2 polarizing conditions (anti-CD3 and PMA, in presence of rIL-4, rIL-2, and anti-IFN-γ) in the presence of homologous mouse serum (HMS) or fetal bovine serum (FBS) for 2 d. Dietary n-3 PUFA significantly enhanced Th2 cell development and suppressed Th1 development under neutral conditions as assessed by intracellular cytokine staining for IL-4 and IFN-γ as the two prototypic Th2 and Th1 cytokines, respectively. However, under Th2 polarizing conditions, while the suppression of Th1 cells was maintained in FO-fed mice, no dietary effect was observed in Th2 cells. Dietary FO increased the Th2/Th1 ratio under both neutral and Th2 polarizing conditions with HMS in the cultures. To examine the effect of dietary n-3 PUFA on Th1 development, DO11.10 Rag2-/- mice expressing transgenic T cell receptor specific for ovalbumin (OVA) peptide were used. CD4+ T cells were isolated from spleens and lymph nodes and stimulated with ovalbumin (OVA) peptide and irradiated BALB/c splenocytes in the presence of rIL-12, anti-IL-4, and rIL-2 in HMS for 2d. Cells were expanded for another 3 d in the presence of rIL-2 and rIL-12. Dietary n-3 PUFA did not affect Th1 differentiation as assessed by the proportion of IFN-γ+, IL-4- T cells in the cultures, but suppressed rIL-2 induced expansion. The suppressed expansion was due to suppressed proliferation (p<0.05). In vivo expansion of antigen-specific T cells was visualized by flow cytometric analysis of CFSE-positive transgenic T cells. Dietary n-3 PUFA did not appear to affect antigen-induced CD4+ T cell cycle progression in vivo. Overall, these results suggest dietary n-3 PUFA have no direct effect on Th2 cell development but do directly suppress Th1 cell development following both mitogenic and antigenic stimulation in vitro.

El objetivo principal de esta tesis ha sido el desarrollo de nuevos poliuretanos a partir de ácidos grasos como fuentes renovables, utilizando química “click” en la síntesis de los dioles y polioles correspondientes. La primera parte del trabajo describe la preparación de los monómeros “vía” acoplamiento tiol-eno ó tiol-ino, su caracterización y las propiedades de los poliuretanos sintetizados. Como los polímeros a partir de ácidos grasos provienen de macromoléculas biológicas y pueden ser potencialmente biocompatibles, la segunda parte del trabajo está dirigida a incrementar la biocompatibilidad de los poliuretanos a través de diversas estrategias de funcionalización con moléculas bioactivas, para su implementación en ingeniería de tejidos. Tres metodologías fueron investigadas: mezclas con gelatina; inmovilización covalente de colágeno sobre la superficie mediante plasma; e inmovilización iónica de sulfato de condroitina sobre la superficie mediante aminólisis. / The main objective of this thesis has been the development of new polyurethanes from renewable sources such as fatty acids, using "click" chemistry in the synthesis of the corresponding diols and polyols. The first part of this study describes the preparation of monomers "via" thiol-ene or thiol-yne coupling, characterization and properties of the synthesized polyurethanes. As the polymers from fatty acids derived from biological macromolecules and can be potentially biocompatible, the second part of this work is focused on the enhancement of polyurethanes biocompatibility through bioactive molecules functionalization strategies, for tissue engineering purposes. Three approaches have been investigated: blends of polyurethanes and gelatin; covalent surface modification of polyurethanes with collagen by plasma treatment; and ionic surface modification of polyurethanes with chondroitin sulfate by aminolysis treatment.