published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Not available / text

published_or_final_version / Zoology / Master / Master of Philosophy

No description available.

No description available.

No description available.

The independent effect of a high polyunsaturated:saturated fat (P:S) diet on glycemic control in humans has been poorly studied. We propose that a P:S $>$ 1.0 vs P:S 1.0 vs. P:S 1.0. HDL and IGFl were significantly lower with the P:S $>$ 1.0. Si was unaffected by the P:S difference, however, trends towards decreased Sg and increased insulin secretion were seen with P:S $>$ 1.0. The small sample size limits the making of firm conclusions, however, it suggests that glycemic control may be improved through increased insulin secretion a result of an increase in P:S.

Amyloid fibrils are a type of protein nanofibres that form when a normally soluble protein aggregates in a regular fashion via self-association. Their organised and repetitive β-sheet structure is thought to be a generic property of all proteins, depending on the environmental conditions. The nanometre size and high stability of these protein nanofibres are attractive features to exploit in bionanomaterials. This thesis aimed to manipulate insulin amyloid fibrils, as a model protein nanofibre system, through investigating the effect of chemical modification on insulin fibril formation in heterogeneous mixtures. Using acetylation, reduction carboxymethylation, reduction pyridylethylation, trypsin digestion and chymotrypsin digestion, it was shown that nanofibres can form in heterogeneous mixtures of modified insulin at variable rates to produce fibrils of distinct morphologies. Distinctively well defined, long, unbranched nanofibres were observed in the crude reduced carboxymethylated insulin mixture after incubation at 60°C (pH 7.4), which formed at a faster rate than native insulin. The crude reduced pyridylethylated insulin revealed the formation of “wavy” fibrils when exposed to 60°C and pH 1.6, compared to the straight native insulin amyloid fibrils. Although, the trypsin digestion inhibited nanofibre formation at 60°C and pH 1.6, chymotrypsin digestion of insulin produced a mixture of long and short nanofibres under the same conditons. Thus chemical modification provides a simple means of manipulating protein nanofibre assembly for use in bionanotechnology. Protein nanofibres were incorporated into a model polymer polyvinylalcohol (PVOH) film in order to assess the impact on material properties. A systematic study involving both insulin and a crude source of crystallin proteins derived from bovine eye lens was undertaken. A protein nanofibre-PVOH nanocomposite was successfully fabricated by a procedure of solution mixing and casting. Dynamic mechanical analysis showed that the addition of insulin fibrils did not change the stiffness of the PVOH. However, an increase in the stiffness of the PVOH-crude bovine eye lens composites was found. Both insulin and bovine eye lens nanofibres reduced the damping properties of the polymer, which suggested a reduction in molecular mobility/slipping. The results revealed that protein nanofibre formation can be controlled through the modification of the protein and that nanofibres may alter polymer properties in a protein specific manner. Employing these findings in the development of novel bionanomaterials that use the protein nanofibres as a form of natural scaffolding offers a fruitful avenue of future research.

Pax4 is a paired-domain-containing transcription factor that plays a crucial role in the development of pancreatic β- and δ-cells. In the absence of Pax4, no β- or δ-cells develop, but an increase in the number of α-cells is observed. To gain insight into Pax4 function, a rat insulinoma cDNA library was screened and two Pax4-related clones were isolated. One clone encoded a 349 amino acid protein with a molecular weight of 38K that corresponded to the full-length sequence of Pax4. The second cDNA, termed Pax4c, was identical to Pax4 but lacked the sequences encoding 117 amino acids at the COOH-terminus. Intracellular localisation studies indicated that Pax4 was sequestered specifically in the cytoplasm of β-cells. To determine the effect of Pax4 on islet cell gene expression, Pax4 was co-transfected with a series of human insulin and islet amyloid polypeptide (IAPP) promoter constructs into the β-cell line MIN6, and transcriptional activity was measured by reporter gene assay. Pax4 was found to have an inhibitory effect on the human insulin gene promoter, which was mapped, to the region -229 to -258. Electrophoretic mobility shift assay was used to show that Pax4 could bind to the C2 element located at -253 to -244 within this region. Pax4 was also found to have an inhibitory effect on the IAPP promoter, which was mapped to a sequence downstream of -138. Using a GAL4 reporter system, the repressive properties of Pax4 was further mapped to separate regions of the promoter between amino acids 2-230 and 231-349.