Angiogenesis, the formation of new vessels from a preexisting vasculature, is critical for supplying a healing myocardium with oxygen and nutrients to sustain metabolism post myocardial infarction (MI). Interleukin-1β (IL-1β), a proinflammatory cytokine increased in the heart post-MI, is considered essential for angiogenesis in tumor growth and metastasis, arthritis, endometriosis, and wound healing. Matrix metalloproteinases (MMPs) are implicated in angiogenesis because of their ability to selectively degrade components of the extracellular matrix. Vascular endothelial growth factors (VEGFs) play a vital role in angiogenesis because of their involvement in the recruitment and proliferation of endothelial cells. The current study explores IL-1β-stimulated regulation of angiogenic genes in cardiac microvascular endothelial cells (CMECs), the signaling mechanisms involved, and the implications in the processes of angiogenesis. DNA microarray analysis indicated IL-1β modulates the expression of numerous angiogenesis-related genes, notably upregulating MMP-2 and downregulating VEGF-D expression. RT-PCR and Western blot analyses confirmed the differential expression in response to IL-1β. In-gel zymographic analysis demonstrated IL-1β-stimulated increase in MMP-2 activity. IL-1β activated ERK1/2 and JNKs, not p38 kinase, and activated PKCα/β1 independent of MAPKs. IL-1β inactivated GSK3β via ERK1/2. Pharmacological inhibition of these signaling cascades indicated IL-1β-stimulated regulation of MMP-2 and VEGF-D occurs via ERK1/2, JNKs, and PKCα/β1-dependent mechanisms. In addition, inactivation of GSK3β inhibited basal VEGF-D expression. H2O2 significantly increased MMP-2 protein levels while IL-1β-induced VEGF-D downregulation was further potentiated by ROS scavenging compounds and inhibition of NF-κB. Phalloidin-FITC stain indicated a sharp reduction in fibrillar actin in the cytoskeleton of IL-1β-stimulated cells. Wounding assays revealed that IL-1β induced CMEC migration but prevented cell-to-cell contact and restoration of the monolayer. Flow cytometric analysis revealed a G0/G1 phase cell cycle arrest in IL-1β-stimulated cells, indicative of decreased proliferation. IL-1β inhibited three-dimensional in vitro tube formation by CMECs. Lastly, IL-1β inhibited microvessel sprouting from aortic rings, an assay examining the collective response of multiple cell types. Collectively, the data presented in this study provide evidence that IL-1β differentially regulates important angiogenesis-related genes in CMECs. This differential regulation may lead to interruptions in the processes of angiogenesis, ultimately creating a dysfunctional phenotype for myocardial vessel formation.

Lactic acid bacteria are essential as flavor producers of cheese and fermented products. They are capable of catabolizing aromatic, branched chain, and sulfur amino acids to flavor compounds. During cheese ripening the numbers of lactococcal colonies decrease, but lactococci survive without replication in culture. This prompted an investigation into possible mechanisms of catabolism of branched chain amino acids into branched chain fatty acids and the physiological relevance of amino acid catabolism to the bacteria. We hypothesized that lactococci catabolize branched chain amino acids to branched chain fatty acids during nonculturability. Lactococci, lactobacilli, and brevibacteria catabolized both branched chain amino acids and keto acids into branched chain fatty acids. Lactococci survived carbohydrate-limited conditions for over 4 yrs. Their survival was represented by maintaining intracellular ATP, enzyme activity, membrane integrity, capability of ATP- and PMF-dependent substrate transport, transcription, and catabolism of amino acids to fatty acids. Assays conducted with NMR spectroscopy coupled with in silico analysis showed that branched chain substrates are catabolized via keto acids, HMG-CoA, and acetyl-CoA to branched chain fatty acids. A short list of candidate genes was identified for the pathway by gene expression analysis coupled to NMR analysis. The expression of these genes and the presence of the related catabolites were identified in long-term starved cultures of nonculturable lactococci. This verified that catabolism of branched chain amino acids to branched chain fatty acids occurred during the nonculturable state only and in conditions of carbohydrate deprivation. The pathway also facilitated fixation of carbon by lactococci, revealing the mechanism of survival of lactococci over 4 yrs in culture without the addition of external carbon sources. Between strains the availability of carbohydrate and acid stress played significant roles in modulating their ability to produce branched chain catabolites. The ability of lactococci to catabolize branched chain amino acids during sugar starvation represents a shift in carbon catabolic routes. The identified pathway also represented a balance between catabolism and anabolism, suggesting that the bacteria were in a homeostatic state during nonculturability. We accepted the hypothesis that nonculturable lactococci catabolized branched chain amino acids to branched chain fatty acids during starvation./p>

Abstract not available

Abstract not available

A number of methods for the analysis of free amino acids in melon fruit have been evaluated experimentally. Analysis of their tBDMS derivatives by GC (gas chromatography) was found to be the most suitable for the mix of free amino acid found in the melon matrix. It affords good yields of amino acid derivatives with excellent gas chromatographic properties and characteristic mass spectra. The single-step derivatization procedure is highly reproducible and allows simultaneous analysis of asparagine and glutamine together with their corresponding acids. Changes in amino acids, sugars, the principal acids, volatiles and minerals in the free form were studied in the fruit mesocarp during development, ripening and storage of the fruits. Sucrose was the principal sugar, absent in young fruit but showing a dramatic increase during ripening while the levels of fructose and glucose remained constant during the whole course of fruit growth or slightly decreased during ripening and storage. The quantitative determination of 22 free amino acids was achieved by GC analysis using the method developed. Total aroma volatiles were determined using a headspace-gas chromatographic technique. They increased and reached a maximum value just before fruit full slip. Most of the esters characteristic of melon aroma were absent in young fruit but developed at the ripening stage. Changes in the quantities of mineral nutrients present in the fruits were determined by ICP-AES analysis. The concentrations of most elements increased thoughout the fruit development except for Ca which decreased markedly and Cu which decreased during early growth then fluctuated around lower values later in the development stage. All of the above changes can be related to the metabolic activity during fruit growth and maturation. Statistical analysis showed changes in TV, TSS, TS, pH, some free amino acids and some minerals were strongly correlated. / Master of Science (Hons)

Limited published data exists on the Australian duck industry, particularly in relation to the nutritional requirements of the commercial duck (Anas Platyrhynchos). A series of seven experiments was designed to determine whether current nutritional recommendations for energy, lysine, methionine, threonine and tryptophan were sufficient to optimise growth, feed efficiency, and carcass characteristics of the duck. The ability of the duck to perform diet self selection was then examined for its potential use in the Australian industry. The outcome of the diet self selection experiments showed that ducks sometimes self select diets when offered choices from four diets differing in nutrient density. A key outcome of this research was the development of a revised set of nutrient specifications designed to maximise the performance of the Australian commercially grown duck. These proposed specifications could be economically beneficial to the expanding Australian duck industry / Doctor of Philosophy (PhD)

Selected food gums (hydrocolloids) were tested for their abilities to encapsulate enzymes for acclerating cheese ripening. The effect of pH and acidity on gel strength of the gums was determined. Enzyme was entrapped in k-carrageenan, gellan gum and milk fat and incorporated into cheese milk prior to cheese making. The cheese produced was tested for protein breakdown and amino acid production during ripening and textural and sensory properties of the ripened cheese were also evaluated. The findings and significance of this study and the literature review are presented. Gels were produced from alginate. Most gels showed reduced strength after treatment in solutions of either modified acidities or pH. There was, however, no significant change in gel strength between the different treatments for most of the gums. The activity of encapsulated enzyme were also investigated. Enzymes encapsulated in k-carrageenan, gellan and alginate gums retained higher activities than the other gums studied. Enzymes entrapped in agar gels had the least retention of activity. The retention of enzyme capsules produced from gellan, k-carrageenan and milk fat in cheese curd was investigated. Loss of encapsulated enzymes in cheese whey was also determined. Enzyme loss in the whey ranged from 5.6 to 17.9% with the highest losses observed with milk fat and the least with gellan gum capsules. Most of the cheeses treated with enzyme capsules showed higher levels of amino acid within two weeks than control cheese. After two weeks, all experimental cheeses showed higher production of amino acids than the control cheese. The addition of enzyme capsule to cheese did lead to a higher growth level of microorganisms. The experimental cheese exhibited lower score than the control cheese for most textural properties. The experimental cheeses were not significantly different in flavour and aroma from the control cheese. K-carrageenan treated cheeses were recorded as having the highest score for bitter after taste. Except for those cheeses treated with k-carrageenan capsules, the overall acceptability for the trial cheeses were not significantly different from that of 6 month old untreated cheese. / Master of Science (Hons)

Title from title screen. Interactive three dimensional molecular data and multiple colour images. Text presented in Hypertext Markup Language (.htm); images in standard formats (.jpg, .gif); molecules presented mostly as Cambridge Protein Data Bank format (.pdb); some molecules presented in alternative X.Mol cartesian co-ordinates format (.xyz); search facility in PERL script. Includes bibliographical references. Text, numeric and representational data System requirements: for text, any standard web browser on any platform, Netscape 2.x or higher, Internet Explorer 3.x or higher; for molecular structures, viewer such as Rasmol or preferably MDL's Chemscape Chime; for search facility , an appropriately configured web server. Links to all required software for browsing on various platforms are included in the software directory in the thesis. Mode of access: World Wide Web.

<p>The study and understanding of Alzheimer’s disease on protein level is fundamentally important in the search for its treatment and there is a demand for proteins that can be used together with candidate drugs in crystallography trials. The refolding time reaching up to three weeks for beta-site APP cleaving enzyme 1 (BACE1), the proposed disease-generating protein, is presently not optimal and new protein constructs are needed. In attempts to shorten the refolding time the six cysteins in BACE1 were substituted to hydrophobic valine or alanine residues. The proteins, both wild type and mutant BACE1, were expressed in <em>Escherichia coli</em>, refolded for one week and purified by ion exchange chromatography and gel filtration. The final products were characterised by measuring stability, homogeneity and enzyme activity. There was significantly lower protein yield for the mutants compared to the wild type BACE1, indicating that generation of the disulphide bonds are important for correctly folded and stable BACE1. Also, it was found that the three different disulphide bonds are not equally important during refolding, with Cys₂₇₈-Cys₄₄₃being the most important and Cys₂₁₆-Cys₄₂₀ and Cys₃₃₀-Cys₃₈₀ being of less importance. The present work shows that one week of refolding is enough for a sufficient protein yield of wt BACE1 and that the current refolding time for wt BACE1 can be shortened. Furthermore the disulphide bridges in BACE1 are important for forming an active protein with correct fold.</p>

Graduation date: 1998