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Typescript (photocopy) / viii, 197 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Pharmacology, 1982

The severe hepatotoxicity of the antiepileptic drug valproic acid (VPA) is believed to be mediated through chemically reactive metabolites. The monounsaturated metabolite 4-ene VPA is steatogenic in the rat, and in a similar fashion to the hepatotoxin 4-pentenoic acid, is thought to be oxidized by mitochondria to a highly reactive α,β-unsaturated ketone, 3-keto-4-ene VPA. The tripeptide thiol, glutathione (GSH), is known to react with a variety of electrophilic compounds that have the potential to interact with cellular macromolecules. The identification and structural characterization of GSH conjugates provides a means of identifying short-lived unstable electrophiles and thus an insight into the mechanisms of toxicity. This thesis describes the synthesis and characterization of thiol conjugates of reactive metabolites derived from the in vivo metabolism of VPA, 4-ene VPA, (E)-2,4-diene VPA, and 4-pentenoic acid. A negative ion chemical ionization gas chromatographic/mass spectrometric (NICI/GC/MS) method for the determination of VPA and 14 of its metabolites in a single chromatographic run was developed. A combination of pentafluorobenzyl and trimethylsilyl derivatization resulted in the [M-181]̄̄̄ ̄anion as the base peak for all the metabolites measured. When these ions were monitored sensitivities in the low picogram range were achieved. The VPA metabolite profile was determined in pediatric patients on VPA monotherapy and on combined therapy with either carbamazepine or clobazam. 4-Ene- and (E)-2,4-diene-VPA were found to be minor metabolites with serum levels below 1% that of VPA. In patients on combined therapy with carbamazepine, the ω and ω-l pathways of VPA metabolism were induced, while products of β-oxidation were significantly decreased. Polytherapy had no significant effect on the serum levels of 4-ene- or (E)-2,4-diene-VPA. Rats were dosed intraperitoneally with 100 mg/kg of the sodium salts of VPA, 4-ene-, (E)-2,4-diene-VPA, 4-pentenoic or (E)-2,4-pentadienoic acids. Methylated bile extracts were analyzed by high pressure liquid chromatography and liquid chromatography/tandem mass spectrometry (LC/MS/MS) for GSH conjugates while urine samples were analyzed by GC/MS and LC/MS for N-acetylcysteine (NAC) conjugates and other metabolites. The GSH conjugate of (E)-2,4-diene VPA was detected in the bile of rats treated with 4-ene- and (E)-2,4-diene-VPA. The NAC conjugate was a major urinary metabolite of rats given (E)-2,4-diene VPA and was a prominent urinary metabolite of those animals given 4-ene VPA. The structures of these metabolites were confirmed by comparing GC/MS or LC/MS properties of the isolated metabolites to those of synthetic standards. The GSH and NAC conjugates of (E)-2,4-diene VPA were chemically synthesized and their structures established to be (E)-5-(glutathion-S-yl)-3-ene VPA and (E)-5-(N-acetylcystein-S-yl)-3-ene VPA by nuclear magnetic resonance spectroscopy and mass spectrometry. In contrast to the very slow reaction of the free acid of (E)-2,4-diene VPA with GSH, the methyl ester reacted rapidly with GSH to yield the adduct. In vivo it appears the diene forms an intermediate with enhanced electrophilic reactivity to GSH as indicated by the facile reaction of the diene with GSH in vivo (about 40% of the (E)-2,4-diene VPA administered to rats was excreted as the NAC conjugate in 24 hr). In rats treated with 4-pentenoic and/or (E)-2,4-pentadienoic acids the following conjugates were identified and characterized by synthesis: GSH and cysteine conjugates of 3-oxo-4-pentenoic acid, GSH and NAC conjugates of (E)-2,4-pentadienoic acid, and the NAC conjugate of acrylic acid. The results thus provided the first direct biochemical evidence for the in vivo formation of the metabolite of 4-pentenoic acid considered responsible for the irreversible inhibition of fatty acid metabolism. The results also revealed basic differences between the mitochondrial metabolism of 4-ene VPA and 4-pentenoic acid. The 3-keto-4-ene VPA and its GSH and NAC conjugates were synthesized in order to facilitate the in vivo identification of these compounds following the administration of VPA, 4-ene-, or (E)-2,4-diene-VPA to rats. However, neither the 3-keto-4-ene VPA nor its thiol derivatives were evident in any of the treatments. The NAC conjugate of (E)-2,4-diene VPA was also found to be a metabolite of VPA in patients. The level of the conjugate appeared to be higher in two patients who recovered from VPA-induced liver toxicity. The characterization of GSH and NAC (in humans and rats) conjugates of (E)-2,4-diene VPA suggests that VPA is metabolized to a chemically reactive intermediate that may contribute to the hepatotoxicity of the drug. / Pharmaceutical Sciences, Faculty of / Graduate

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Despite extensive research into the effects of alcohol consumption, there is no clear understanding into the mechanisms underlying human information processing impairment. The acute consumption of alcohol was investigated to determine the implications for human information processing capabilities, and to identify the extent to which these implications were stage-specific. Further aims included the investigation and quantification of caffeine-induced antagonism of alcohol impairment. Moreover, the aforementioned relationships were investigated in morning versus evening conditions. A test battery of six resource-specific tasks was utilised to measure visual perceptual, cognitive and sensory-motor performance, fashioned to return both simple and complex measures of each task. The tasks implemented were: visual perceptual performance (accommodation, visual detection, visual pattern recognition); cognition (memory recall- digit span); and motor output (modified Fitts‟ and a driving simulated line-tracking). Performance measures were recorded by the respective computer based tasks. Physiological variables measured included heart rate frequency, heart rate variability (RMSSD, High and Low Frequency Power) and body temperature. Saccade speed, saccade amplitude, pupil size and fixation duration were the oculomotor parameters measured. Three groups of participants (alcohol, caffeine+alcohol and control) n=36 were studied, split evenly between sexes in a mixed repeated/non-repeated measures design. The control group performed all test batteries under no influence. The alcohol group performed test batteries one and two sober, and three and four under the influence of a 0.4 g/kg dose of alcohol. Group caffeine+alcohol conducted test battery one sober, two under the effect of caffeine only (4 mg/kg), and three and four under the influence of both caffeine and alcohol (0.4 g/kg). The third test battery demonstrated the effects of alcohol during the inclining phase of the blood alcohol curve, and the fourth represented the declining phase. Morning experimentation occurred between 10:00 - 12: 45 and 10:30 -13:15 with evening experimentation between 19:00 - 21:30 and 19:30 - 22:00. Acute alcohol consumption at a dose of approximately 0.4 g/kg body weight effected an average peak breath alcohol concentration of 0.062 % and 0.059 % for the alcohol and caffeine+alcohol groups respectively. Task-related visual perceptual performance demonstrated significant decrements for simple reaction time, choice reaction time and error rate. Cognitive performance demonstrated no significant performance decrements, while motor performance indicated significant decrements in target accuracy only. Physiological parameters in response to alcohol consumption showed significantly decreased heart rate variability (RMSSD) in the modified Fitts‟ task only. A significant decrease in saccade amplitude in the memory task was the only change in oculomotor parameters. Prior caffeine consumption demonstrated limited antagonism to task-related alcohol impairment, significantly improving performance only in reduced error rate while reading. Caffeine consumption showed stimulating effects on physiological parameters, significantly increasing heart rate and heart rate variability when compared to alcohol alone. The design of the tasks allows for comparison between complex and simple task performance, indicating resource utilisation and depletion. Complex tasks demonstrated higher resource utilisation, however with no statistical performance differences to simple tasks. Physiological parameters showed greater change in response to alcohol consumption, than did the performance measures. Alcohol consumption imposed significant changes in physiological and oculomotor parameters for cognitive tasks only, significantly increasing heart rate frequency and decreasing heart rate variability, skin temperature and saccade amplitude. Caffeine consumption showed no antagonism of alcohol-induced performance measures. Physiological measures showed that caffeine consumption imposed stimulating effects in only the neural reflex and memory tasks, significantly increasing heart rate frequency and heart rate variability. Prior caffeine consumption significantly decreased fixation duration in the memory task only. The time of day at which alcohol was consumed demonstrated significant performance and physiological implications. Results indicated that morning consumption of alcohol imposes greater decrements in performance and larger fluctuations in physiological parameters than the decrements in evening experimental sessions. It can be concluded that alcohol consumption at a dose of 0.4 g/kg affects all stages in the information processing chain. Task performance indicates that alcohol has a greater severity on the early stages of information processing. Conversely, under the influence of alcohol an increased task complexity induces greater effects on central stage information processing. In addition, caffeine consumption at a dose of 4 mg/kg prior to alcohol does not antagonise the alcohol-induced performance decrements.

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published_or_final_version / Chinese Medicine / Master / Master of Philosophy

Tetracycline is a front line antibiotic for the treatment of chlamydial infections in both humans and animals, and the emergence of tetracycline resistant (tet[superscript R]) Chlamydia is of significant clinical importance. Recently, several tet[superscript R] chlamydial strains have been isolated from swine (Sus scrofa) raised in production facilities in Nebraska. Here, the intracellular development of two C. suis tet[superscript R] strains, R19 and R27, is characterized through the use of tissue culture and immunofluorescence. The strains grow in tetracycline up to 4 ��g/ml, while a tetracycline sensitive (tet[superscript S]) C. suis swine strain (S45) and a C. trachomatis strain of the human serovar L2 (LGV-434) grow in tetracycline up to 0.1 ��g/ml. Although inclusions form in the presence of tetracycline, many contain large aberrant RBs that do not differentiate into infectious EBs. The percentage of inclusions containing typical developmental forms decreases with increasing tetracycline concentrations, and at 3 ��g/ml of tetracycline, 100% of inclusions contain aberrant RBs. However, upon removal of the tetracycline, the aberrant RBs revert to typical RBs and a productive developmental cycle resumes. In addition, inclusions were found that contained both C. suis R19 and C. trachomatis L2 after sequential infection, demonstrating that two biologically distinct chlamydial strains could both develop within a single inclusion. / Graduation date: 2002

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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