Thesis (M.S.)--University of Hawaii at Manoa, 2006. / Includes bibliographical references (leaves 41-47). / vii, 77 leaves, bound 29 cm

Thesis (M.S.)--University of Hawaii at Manoa, 2006. / Includes bibliographical references (leaves 101-105). / x, 105 leaves, bound ill. 29 cm

Thesis (M.S.)--University of Hawaii at Manoa, 2006. / Includes bibliographical references (leaves 26-28). / ix, 77 leaves, bound ill. 29 cm

Includes bibliographical references. / 151 leaves : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The venom from the Australian brown snakes (Pseudonaja spp.) contains a strong procoagulant component that produces bleeding due to consumption of clotting factors in bite victims. Investigation of the role of phospholipid and calcium found neither was essential for activity, but calcium could shorten the clotting time. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 2000

Includes copies of articles co-authored by the author during preparation of this thesis. / Includes bibliographical references (leaves 185-191). / v, 192 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Sixteen aurein peptides are present in the host defence secretion from the granular dorsal glands of the Green and Golden Bell Frog (Litoria aurea) and seventeen from those of the related Southern Bell Frog (Litoria raniformis). All peptides have been sequenced using a combination of electrospray mass spectrometry and Lys-C digestion, with each sequence confirmed by automated Edman sequencing. Ten of these peptides are common to both species of frog. Thirteen of the aurein peptides show wide-spectrum antibiotic and anticancer activity. / Thesis (Ph.D.)--Adelaide University, Dept. of Chemistry, 2001

[Truncated abstract. Please see the pdf format for the complete text.] Interest in the role of peroxynitrite in the pathogenesis of atherosclerosis has increased due to many in vitro studies which have demonstrated its potent oxidising and nitrating capability and immunohistochemical staining studies which demonstrate nitration of tyrosine in vivo. It is frequently suggested that the production of nitric oxide and superoxide at sites of inflammation implicates peroxynitrite as the major damaging reactive nitrogen species in vivo. Evidence for a role for peroxynitrite is often demonstrated by measurement of 3-nitrotyrosine yet even this cannot distinguish peroxynitrite from other nitrating species. Clearly, however, if peroxynitrite is important in atherogenesis, then identification of mechanisms for its detoxification could provide a means of preventing such effects. Therefore, this Thesis has sought to determine whether phenolic compounds of dietary origin can be preferentially nitrated by reactive nitrogen species thereby protecting endogenous structures, such as low density lipoproteins, from atherogenic modifications. This Thesis focuses upon phenolic acids as they have received relatively less attention than other classes of phenolic compounds, such as flavonoids, yet they are quite abundant in socially important beverages such as red wine. In order to complete the required analyses, the development of methods to detect phenolic acids and their nitration products together with 3-nitrotyrosine, dityrosine and 5-nitro-γ-tocopherol was necessary. The initial in vitro experiments described herein sought to determine the products of reaction of peroxynitrite with phenolic acids of the 4-hydroxy and 3,4-dihydroxy type and then to examine whether these products could account for a protective effect upon tyrosine, lipids and endogenous anti-oxidants, if any was observed, when isolated LDL was treated with SIN-1, which releases peroxynitrite through the simultaneous generation of nitric oxide and superoxide. A concurrent minor focus was to examine the relationship between structure and activity of these phenolic acids under various regimes of oxidative insult. These experiments indicate that, at least in this in vitro model, oxidation is a dominant mechanism over nitration. Peroxynitrite was shown to nitrate coumaric acid in moderate yields but exclusive oxidation of caffeic acid appeared to occur. Although a potential role for γ-tocopherol as an anti-nitration agent was inferred, all types of chemical treatment of LDL in the presence of phenolic acids yielded oxidation as the primary end point. In fact, nitration of tyrosine was not detected and nitration of coumaric acid was at the limit of detection. Since nitration of tyrosine is generally regarded as important in many disease states, a more physiological nitrating mechanism involving artificially stimulated neutrophils was used. This system demonstrated that although physiologically relevant reactive nitrogen species can result in nitration of phenolic compounds, in a complex system including biological structures (LDL) and phenolic compounds, oxidation but not nitration of all species appears to occur. As a consequence of the results above, an examination of carotid plaque was undertaken to determine to what extent nitration occurred relative to oxidation in atherosclerotic tissue. These studies applied methods developed herein to detect 3-nitrotyrosine and dityrosine in complex biological matrices as markers of nitration and oxidation respectively. The data obtained demonstrated that nitration was a minor modification of protein (0.01%) compared to oxidation (0.3%) even in a highly diseased tissue such as carotid artery plaque. A secondary study examining plasma revealed that dityrosine, which has been implicated in irreversible albumin aggregation in chronic renal failure and more recently in heart disease, is elevated in chronic renal failure subjects compared to well matched controls. A separate examination of plasma from healthy subjects revealed that in both the fasting and post prandial state 3-nitrotyrosine could not be detected and, in fact, interfering species could be problematic in the GC-MS analysis of 3-nitrotyrosine. The lack of nitration of any substrate observed in vitro using reactive nitrogen species generated in the aqueous phase, the relative lack of nitration of tyrosine in plaque proteins and the lipophilicity of nitric oxide, the precursor of all reactive nitrogen species, suggested that nitration could be more closely associated with lipid structures. The known ability of γ-tocopherol to form 5-nitro-γ-tocopherol was used to probe this concept. The 5-nitro-γ-tocopherol content of lipid extracts obtained from carotid artery plaques was very high (30%). This indicated that nitration is predominantly a lipid phase phenomenon and that nitrating species are present in much greater abundance than oxidising species in vivo.

Includes bibliographical references. The venom from the Australian brown snakes (Pseudonaja spp.) contains a strong procoagulant component that produces bleeding due to consumption of clotting factors in bite victims. Investigation of the role of phospholipid and calcium found neither was essential for activity, but calcium could shorten the clotting time.

This thesis describes a long-term study in which the genetic health of a population accidentally exposed to ionizing radiation of cesium-137. The Goiânia (Brazil) radiological accident of September 1987 involved 249 individuals exposed to doses up to 7 Gy, and included four fatalities. We have investigated the genetic effects of radiation exposure in this population using both cytogenetic and molecular endpoints in T-lymphocytes. The micronucleus assay differentiated between groups exposed to different levels of ionizing radiation. At the molecular level two methods were employed: (1) the hprt clonal assay; and (2) the determination of microsatellite instability. The hprt assay involves in vitro culturing of T-cells and the selection of 6-thioguanine-resistant hprt mutant clones which were then characterized at the molecular level using both RT-PCR and genomic analysis. Exposure to ionizing radiation initially elevated the mutation frequency but this effect gradually diminished, so that 4.5 years no significant increase was observed. This limitation makes the hprt T-cell assay unsuitable for the study of long term past exposure. Analysis of the spectrum of hprt mutations recovered from 10 individuals exposed to relatively high doses of ionizing radiation revealed a significant increase (3.8-fold) in the frequency of A:T → G:C mutations in the exposed group. This increase in A:T → G:C transitions is consistent with the effects of ionizing radiation in prokaryotes and lower eukaryotes and likely reflects the mispairing of radiation-induced thymine, glycol with guanine. In addition, a two-fold increase in the frequency of deletions not readily explained by slippage events and hence which may reflect ionizing radiation-induced DNA strand breakage was also observed. Microsatellite instability was also investigated. Fluorescent PCR and automated DNA sequencer analysis, using genomic DNA from mononuclear cells, were used to investigate the frequency of microsatellite alterations in exposed and non-exposed populations. We examined a total of 200 and 190 alleles respectively and found that the microsatellite instability distribution in the two groups were not different. Our assay lacked sufficient sensitivity to discriminate between spontaneous and induced microsatellite instability and it is, therefore, not suitable for population monitoring. Finally, despite the minimal database, we used the micronucleus and hprt mutant frequency data to estimate the risk associated with radiation exposure for the Goiânia population. The estimated genetic risk for the exposed group was approximately a 24-fold increase in dominant disorders in the first post-exposure generation. Moreover, the risk of carcinogenesis in this population was estimated to be increased by a factor in the range of 1.4 to 1.5 compared to the population at large. / Graduate

A recently discovered peptide hormone, pituitary adenylate cyclase-activating polypeptide (PACAP) regulates several endocrine systems affecting essential physiological processes such as metabolism, growth, reproduction, and the stress response. PACAP acts as a hypophysiotropic factor, is a potent secretogogue of insulin, regulates production and release of catecholamines from the adrenal medulla and acts as a neuromodulator in the sympathetic and parasympathetic nervous system. The primary structure of PACAP has been highly conserved during the evolution of chordates suggesting it plays an important physiological role. The objective of my thesis was to identify PACAP’s primary physiological function and to determine if it is essential for survival by generating a mouse line deficient in PACAP through targeted disruption of the PACAP gene locus. Postnatal PACAP expression was examined to determine sites of peripheral PACAP production. In addition, several splice variants of the PACAP gene with alternate 5’untranslated regions were identified suggesting a complex system for regulating expression of the mouse PACAP gene. A targeting vector that allows tissue specific or developmental stage specific knockout of the PACAP gene was constructed in the event that PACAP gene deletion resulted in embryonic lethality. PACAP null mice were generated from homologously recombined embryonic stem cells. Initial characterization of the PACAP null mice determined that in the absence of PACAP, mice died within the first two postnatal weeks with abnormal lipid metabolism. Lipid accumulation was present in liver, heart and skeletal muscle and serum lipids were high. Mitochondrial dysfunction in the liver was not the cause of the lipid accumulation, as P-oxidative function was normal. I conclude that PACAP null mice are unable to regulate lipid release from white adipose tissue stores, resulting in a flood of lipids to non-adipose tissues. The abnormal distribution of lipids observed in the PACAP null mice is characteristic of diabetes type 2, yet classical insulin resistance is not observed. Thus, elevated insulin levels were accompanied by low blood glucose levels and the response to a glucose challenge was normal. The uncontrolled release of free fatty acids may result if glucose that is taken up by cells can not be utilized and an alternate energy source is required or if white adipocytes only are insulin resistant. The PACAP null mice were temperature sensitive, in that when raised at 21“C they exhibited metabolic dysfunction and died by two weeks of age. At 24°C most (85%) of the mice survived to adulthood with no obvious signs of metabolic dysfunction. We have determined that the inability of the PACAP null pups to thermoregulate normally when exposed to a lower environmental temperature may be associated with decreased norepinephrine levels to the brown adipose tissue. PACAP may be important for the production and release of catecholamines in the adrenal gland or within the sympathetic nervous system in times of prolonged stress. A mechanistic connection between the lipid abnormalities and the temperature sensitivity in the PACAP null pups has yet to be made. Catecholamines affect a wide range of tissues and the problems associated with insulin regulation within the PACAP null mice may be due to the imbalance in catecholamine production. As one of two main stress response systems, the sympathetic nervous system elicits a vital coping mechanism in times of stress and PACAP’s ability to regulate this system may explain why the primary structure of PACAP has remained so highly conserved. PACAP is a wide acting hormone and therefore the metabolic problems seen in the PACAP null mice may result from altered regulation of several endocrine systems at once. Targeted disruption of the PACAP gene in mouse has revealed a role for PACAP in the regulation of lipid metabolism and in the sympathetic control of thermoregulation. / Graduate

The amino acid, tryptophan, obtained through dietary means, is metabolised by the enzymes tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase (IDO) and tryptophan hydroxylase. All the enzymes have an effect on circulating tryptophan levels, especially TDO, since it is the major site of tryptophan catabolism in the liver and results in the production of kynurenine metabolites, viz. kynurenine, kynurenic acid, 3-hydroxyanthranilic acid and quinolinic acid. Extrahepatically, IDO is responsible for the synthesis of the kynurenine metabolites. Tryptophan 2,3-dioxygenase and IDO activity is increased by hormones or substrates such as tryptophan, and inflammation, in the case of IDO. Tryptophan availability for serotonin (5-HT) synthesis by the enzyme tryptophan hydroxylase is primarily dependent on TDO activity. A study was attempted in order to ascertain whether any of the endogenous metabolites of the kynurenine and serotonergic pathways would be able to inhibit TDO activity. Results showed that although the kynurenines had no effect, the indoleamines, except for the indoleacetic acids, were able to reduce TDO activity. 6-Methoxy-2-benzoxazolinone (6-MBOA), a structural analogue to melatonin, was the most potent inhibitor with a reduction in activity of 55 % compared with the control. The pineal gland in the rat brain has been shown to have the highest IDO activity. With induction, the kynurenine metabolite concentrations of kynurenic acid and quinolinic acid are increased. The effects of both compounds were determined on the serotonergic pathway. Although kynurenic acid produced no significant effect, quinolinic acid significantly reduced N-acetylserotonin and melatonin synthesis at concentrations of lOJLM and 100 JLM respectively. Many authors have implicated oxygen derived species as causative agents in the important neurodegenerative disorders such as Parkinson's and Huntington's disease. Increased radical generation and lipid peroxidation have been suggested to be responsible for the toxic destruction of neurons, especially in the brain because of its high lipid content and oxygen demand. The brain is therefore vulnerable to oxidative attack. During inflammatory diseases, IDO is induced with a resultant increase in kynurenines. This study was also an attempt at determining the effect of kynurenines on lipid peroxidation. All metabolites of the kynurenine pathway were able to induce lipid peroxidation significantly. The antioxidative potential of various tryptophan analogues, viz. serotonin, melatonin and 6-methoxy-2-benzoxazolinone, was determined using quinolinic acid-induced lipid peroxidation. Serotonin, melatonin and 6-MBOA were able to significantly reduce quinolinic acid-induced lipid peroxidation.