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The aim of this study was to evaluate the factors, which affect insulin release and stability in simulated gastrointestinal fluid that ultimately will reduce the bioavailability of insulin in a nanoparticulate oral delivery system. The focus was on nanoparticulate carriers developed by C. Reis, which have the highest level of bioavailability reported in the literature thus far. Particles observed by TEM were spherical and particle analyzer data showed that the peak of the distribution was 10 nm. Entrapment efficiency of insulin was 85%. Insulin retention/release was evaluated in both enzymatic and enzyme-free simulated digestive fluids. HPLC measurement showed that insulin was stable in acid condition in presence and absence of pepsin. By changing the pH to 6.8 in an intestinal simulation, the amount of insulin decreased such that at the end of the 5 h simulation, 71% of the insulin was measureable in simulated GI fluid in presence of enzymes. Insulin release profile from nanoparticles was low in gastric condition. After changing the pH to 6.8, an initial release of insulin occurred in the first 1h followed by plateau state in the remaining 2h. After a total of 5h in acidic followed by neutral pH medium, the formulation retained 48% of the insulin in the particles in simulated GI fluid in presence of enzyme. As confirmation of the amount of retained insulin, after 5h, particles were dissolved and the formulation was shown to fully retain up to 45% of the insulin in extended simulated gastrointestinal condition in the presence of enzymes. Insulin release behavior was investigated in different simulated small intestinal media by incorporating phosphate buffer or bicarbonate buffer and physiological electrolytes. The release rate from particles in the phosphate buffer was faster compared to bicarbonate buffers. KBB-C showed a release profile that was very different from other media, with about 10% released in the first 30 min and 70% of the insulin remaining entrapped within the particles at the end of the experiment. Release in this buffer was reduced due to the decreased sodium to calcium ratio compared to the other KBB media. / Thesis (Master, Chemical Engineering) -- Queen's University, 2013-08-26 13:02:50.837

The dairy cow experiences a period of immunosuppression around the time of calving that contributes to the increased incidence and severity of infectious diseases observed during this period. This reduction in immune capacity is due in part to the impairment of neutrophil function, a key component of the innate immune system. In fact, the success of the host defense mechanisms against infection depends on the ability of neutrophils to reach the site of the infection, recognize, engulf and ultimately destroy the pathogen using several mechanisms such as the generation and release of reactive oxygen species (ROS) and the recently described neutrophil extracellular traps or NETs. The alteration in some of these functions and the overall killing ability of neutrophils during the periparturient period has been widely described. However, the physiological mechanisms underlying the period of immunosuppression are not completely elucidated. Interestingly, the impairment of these immune defense mechanisms coincides with the profound metabolic changes associated with parturition and lactogenesis. Changes in several hormones and metabolites have been proposed to be the cause of the reduction in neutrophil function, but the effect of insulin on the functional capacity of these cells has not been investigated. Not only does the concentration of plasma insulin fall as parturition approaches, but also the animal experiences a period of impaired insulin action, termed insulin resistance, during this same time-frame. Therefore, we isolated circulating neutrophils from periparturient and midlactating cows and incubated them with insulin alone or in combination with the insulin-sensitizing agent 2,4- thiazolidinedione (TZD). Subsequently, we measured the total, extracellular, and intracellular generation of ROS, NETs release, phagocytic and killing ability. Insulin did not improve any of the parameters used to assess neutrophil function. In contrast, TZD had a potent inhibitory effect on the total ROS generation, despite an increase in extracellular superoxide anion production. Surprisingly, TZD did not alter the ability of neutrophils to phagocytose and/or kill Staphylococcus aureus during an in vitro coculture. Results suggest that TZD can reduce the oxidative stress that neutrophils experience during their respiratory burst and diminish the damage that ROS cause to the surrounding tissue without compromising the capacity of neutrophils to eliminate the invading pathogen.

78 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, Dept. of Child Health, 1971

Beef carcass value is influenced by the quantity and distribution of adipose tissue. Elucidation of metabolic controls of caloric partitioning between adipose depots could lead to development of production solutions that enhance beef carcass value. Historical trends in Choice and Select beef supply and short-term demand structures for Choice and Select boxed beef were explored. Recent stabilization in slaughter mix may suggest an optimum is being approached. Evaluation of short-run demand supports this premise, and suggests that Choice and Select products may not be strong substitutes. Growth-based prediction equations relating carcass traits to growth traits using ultrasound measurements as the basis of projections under different growing systems were explored. Accuracy of carcass fat predictions from growth-based equations is influenced by weight gain between ultrasound and endpoint, breed, and gender; scans out to 120 d pre-harvest may be accurate. Angus steers were used to test effects of dietary energy source on muscle and adipose tissue metabolism and insulin sensitivity. Results suggest that feeding hay limited both glucose supply and tissue capacity to increase glucose utilization in response to insulin without altering acetate conversion to fatty acids. Because subcutaneous (s.c.) adipose tissue consistently utilized more acetate and oxidized more glucose than intramuscular (i.m.), these results suggest that hay-based diets may alter i.m. adipose tissue metabolism with less impact on s.c. adipose tissue. Additionally, s.c. adipose tissue may become resistant to insulin in steers fed to an excessive s.c. fat thickness. A final experiment was designed to test the effects of dietary energy source during backgrounding and compositional endpoint on adipose tissue metabolism and insulin sensitivity. Feeding hay during backgrounding may have differential effects on tissue lipogenesis. Feeding hay increased both glucose oxidation and incorporation of acetate into fatty acids; in i.m. insulin failed to stimulate glucose conversion to lipid. As physiological maturity increases, glucose conversion to CO2 and lactate increased, but the ability of insulin to stimulate lipid synthesis from glucose may be reduced. These data provide foundation for a hypothesis regarding diet-mediated regulation of differential adipose tissue metabolism. Validation of these hypotheses could generate nutritional strategies that alter the rate and site of adipose deposition.

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Title page, summary and table of contents only. The complete thesis in print form is available from the University of Adelaide Library. / The insulin and insulin-like growth factor receptor (IR and IGF-lR respectively) networks are ancient and fundamental systems that control growth and metabolism in multicellular organisms. This thesis has examined several aspects of this field focusing on mammalian receptor biology and a comparison of the similarities and differences between the insulin and IGF receptor signalling systems. The insulin receptor family of proteins consist of eleven structural domains, of which the extracellular domains contain all the ligand binding and specificity determinants. The insert domain, within the extracellular region is the least understood of all the domains, and it has no similarity to any other protein sequence. It does however contain the cleavage site which separates the receptor into two subunits and also a small stretch of residues shown to directly contact bound ligand and which is absolutely required for ligand binding in short recombinant forms of the receptor. In addition, the human insulin receptor, expressed as one of two isoforms, A and B, results in the exclusion or inclusion of 12 amino acids directly adjacent to the ligand contacting amino acids in the insert domain. The A isoform lacking exon11 is expressed ubiquitously and the B isoform containing exon11 is co-expressed mainly in the traditional insulin responsive tissues of liver, muscle, adipocytes and kidney, where it is the dominant isoform. In this thesis recombinant insert domain was expressed in a bacterial system in an attempt to purify folded protein suitable for NMR structural analysis. The results of the expression studies indicated that the insert domain was unstructured in isolation and was unable to be adequately refolded by all conditions tried, although hydrophobic conditions appeared to partially stabilize the structure. The overall conclusions of this project were that the Insert domain is likely to have limited structure, and probably buried within the receptor, and therefore requires the presence of the rest of the extracellular domains to adopt its correct structure. A comparison of the ligand binding and phsophorylation potential between the two human isoforms of the insulin receptor was made. A competition binding assay using europium labelled insulin was developed, that found that both IGF-l and IGF-2 had an increased affinity for the hIR-A, but insulin had a slightly reduced affinity. These results differ from the established literature in the raw values, however the relative ratios of binding strength are consistent. The most likely reason for this is that the europium labelled insulin has a different mode of binding the receptors due to the location of the europium chelate. Interestingly, using europium labelled IGF-l produced results nearly identical to those of conventional competition assays. Phosphorylation assays indicated that the hIR-B isoform was more responsive than hIR-A. Even though IGF-2 and IGF-l had improved affinity for hIR-A, the level of phosphorylation was not as high. The ability of each growth factor to promote cellular proliferation correlated well with the relative strength of binding and activation of the receptor. The regions of the IR and IGF-1R involved in binding substrates and regulators are predominantly found in the juxtamembrane domain and the C-terminal domain, which contain several potential tyrosine and serine phosphorylation target sequences. In this study the effect of mutations in unique tyrosine residues and other residues in the C-terminal domain of the hIGF-lR was investigated. Results of time-course phosphorylation assays showed that mutation of Tyrosine¹²⁵¹ to phenylalanine caused hyperphosphorylation of the receptor and increased proliferation, which was caused by deregulation of a tyrosine phosphatase. A Tyrosine¹²⁵⁰ to phenylalanine mutation had altered kinetics of phosphorylation, displaying an unchanging rate of phosphorylation over time after ligand stimulation. However, proliferation was unaltered, indicating that even under extended exposure to ligand, the initial strength of receptor activation is more critical to affecting the biological response. The Caenorhabditis elegans insulin-like peptide family is a very large family consisting of possibly 38 peptides likely to be both agonists and antagonists of Daf-2 Receptor (IR homologue) signalling. Comparative modelling of all 38 peptides was performed based on the known structures of mammalian peptides. The overall results indicated that good quality models of ins peptides could be made despite the low sequence similarity with the templates. This suggested that it is the conformational shape of the molecule allowable by the individual residues that is most important when modelling and not having a perfect sequence match. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1200443 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2005