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Summary Puerperal sepsis is a rare but serious and potentially lethal syndrome. It is imperative that severe postpartum malaise is taken seriously; early initiation of antibiotic therapy before sepsis becomes manifest can save lives.

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Cyanobacterial secondary metabolites, often identified as toxins such as microcystin, have also demonstrated biological functions including inhibition of bacterial and viral growth. In this study, 10 cyanobacterial strains were isolated from field sites around Adelaide and laboratory cultures and assessed for bioactivity against bacterial, viral and fungal pathogens. A comprehensive literature search identified a number of screening assays employed by research groups to identify cyanobacterial strains with biological activity. Within the review, methods to optimise extraction of the compounds were also noted. Combinations of extraction methods, solvents and assay procedures were investigated to optimise the success of this phase of the study. Bioactivity was confirmed by development of agar disc diffusion and microtitre plate assays to analyse cyanobacterial biomass extracts. Result of the assays indicated a methanolic extract of one species, Microcystis flos-aquae (Wittr Elenkin), inhibited growth of bacterial cells and viral infectivity and was selected for further analysis. The bioactive compound was isolated by HPLC and mass spectrometric analysis. Separation of the bioactive extract into component peaks indicated only one that was likely to represent the metabolite of interest, at a retention time of approximately 18 min. A second profile was constructed of a methanolic extract of the same species in a later growth stage that did not inhibit growth of either the bacterial or viral test organisms. Comparison of the profiles exposed the absence of the peak at 18 min retention time in the second profile. Accumulation of the fraction was conducted using a semi-preparative HPLC column for analysis by mass spectrometry. A sample of the isolated peptide was submitted to Proteomics International, a subsidiary of Murdoch University, WA, for identification and structural characterisation. Proteomics International analysed the data by electronspray ionisation time of flight mass spectrometry (LC/MS/TOF) followed by LC. De novo sequence analysis of the data was carried out using Analyst QS software; however, PI was unable to provide a readily interpretable, continuous amino acid sequence, despite their admission that some gaps in the fragmentation ladder corresponded to known amino acids. Interpretation of the data generated by Proteomics International by a research chemist within the University of Adelaide proposed the following amino acid sequence and subsequent structure for the compound: [Figures omitted] Proposed (a) amino acid sequence and (b) structure for the bioactive compound isolated from non-toxic M. flos-aquae. Comparison of the proposed sequence with those contained in peptide databases was unable to classify the compound (B Neilan, personal communication, April 2008), suggesting the bioactive metabolite is perhaps previously undetected and therefore may be considered a novel compound, or has undergone a modification and is thus a variant of a known compound. Taxonomic classification of the strain used during this study was completed by PCR amplification of 16S ribosomal RNA, using primers from alternative cyanobacterial sources. The sequence was classified in the following taxonomic hierarchy (with 100% assignment detail, for a confidence threshold of 95%): Domain: Bacteria Phylum Cyanobacteria Class Cyanobacteria Family Family 1.1 Genus Microcystis This classification confirms that the species investigated during this research is of the genus Microcystis. Synthesis of cyanobacterial metabolites is generally accepted to be a result of nonribosomal synthetic pathways. The presence of non-ribosomal peptide synthetase and polyketide synthetase genes in Microcystis flos-aquae was confirmed by PCR amplification using degenerate primers from other cyanobacterial sources. Analysis of sequence data identified the presence of an NRPS gene demonstrating significant similarity (98%) to the NRPS cyanopeptolin gene of Microcystis sp. However, the PKS (polyketide) gene identified verified only a 63% similarity to a known sequence, that of the PKS (mcyG) gene of M. aeruginosa PCC 7806 (Koch). Results of the molecular investigation imply this compound may belong within the cyanopeptolin family. Researchers have speculated that the majority of cyanobacteria possess genes for production of toxins, though in many instances the gene cluster may be incomplete or one or more genes may be absent or mutated. The presence of microcystin genes was confirmed by PCR amplification using primers from previously characterised cyanobacterial genes. Analysis of the sequence data identified the presence of several mcy genes generally found in toxic strains of cyanobacteria noted for synthesis of the toxin microcystin. The DNA sequences show significant similarity to the mcyA, mcyC, mcyD and mcyE genes described for Microcystis sp. and Microcystis aeruginosa PCC 7806. However, analysis of the sequence data for the mcyB gene revealed that this gene was not present. Further PCR amplification of the region between mcyA and mcyC using the reverse complements of the original primers indicated that a sequence was present that may have been a truncated variant of mcyB or another gene entirely. Time constraints prohibited submission of this region for sequence analysis. The primary objective of this research project was to screen a field strain of cyanobacteria for synthesis of biologically active secondary metabolites, and to isolate those compounds using a combination of analytical chemistry and molecular biotechnology. This study forms part of a collaborative project between the University of Adelaide, South Australian Research and Development Institute (Aquatic Sciences) and the Environmental Biotechnology Cooperative Research Centre, entitled “P6: Commercial scale integrated biosystems for organic waste and wastewater treatment for the livestock and food processing industries”. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1352988 / Thesis (Ph.D.) -- University of Adelaide, School of Chemical Engineering, 2009

The Wet Air Oxidation process has considerable attractions for the disposal of toxic organic wastes. In this thesis, a fundamental study is made of the mechanism of oxidation under wet air and related conditions of a series of well-defined substances known to occur as components of industrial wastes, and which are known to present difficulties in the Wet Air Oxidation process. In the initial stages, the oxidation of a series of simple alkylpyridines, namely 2-, 3-, and 4-methylpyridines, has been studied under simulated Wet Air Oxidation plant conditions in a laboratory autoclave operating at 250°C and 250 atmospheres. The progress of the oxidation was followed by withdrawing samples at intervals and subjecting these to chromatographic analysis, using Gas Chromatography-Mass Spectrometry and High Performance Liquid Chromatography, so as to establish the nature of the oxidation products. In the autoclave oxidation of 2-, and 4-methylpyridine, a wide range of oxidation products was detected, including a number of compounds which appeared to be derived from the reactions of pyridylalkyl radicals formed from the parent substance, implying that a free radical mechanism was occurring under Wet Air Oxidation conditions. Under these conditions, 3-methylpyridine appeared to be more resistant to oxidation, the only significant oxidation product being the related aldehyde. The literature suggests that the formation of the hydroxyl radical (OH) under Wet Air Oxidation conditions may be responsible for the initiation of the above reactions, and thus the possibility of catalysis of the above systems by reagents known to generate hydroxyl radicals has been explored. The literature suggests that Fenton's reagent, which is a mixture of iron(ll) and hydrogen peroxide, provides a source of hydroxyl radicals. Thus, the oxidation of the methylpyridines using Fenton's reagent at ambient temperature and atmospheric pressure was carried out and it was also used as a catalyst in the autoclave oxidation reactions. The effectiveness of other metal ion/hydrogen peroxide mixtures was explored, e.g. involving iron(lll), copper(ll), copper(l), titanium(lll), and vanadium(IV), as there is considerable evidence from the literature of their involvement in oxidation chemistry. In all of the oxidation reactions investigated, both under autoclave conditions, and at room temperature, evidence of destructive oxidation of the heteroaromatic ring has been gained for all three methylpyridines. However, in addition to ring destruction products, a range of intermediate oxidation products was observed and similarities were found between those products formed in the autoclave and those reactions carried out in the laboratory. However, recent literature has questioned the formation of hydroxyl radicals by Fenton and related reagents, and so the Fenton catalysed oxidation of each of the methylpyridines was explored further. This was done by the incorporation of appropriate radical trapping agents and complexing agents such as 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and ethylenediaminetetraacetic acid (EDTA) respectively. In each of the oxidation reactions studied, attempts have been made to identify as many as possible of the products observed by comparison with known substances. However, it has been necessary to develop procedures for the preparation of some of these compounds, notably a range of dimeric structures derived from the simple alkylpyridines, e.g. dipyridylethenes, dipyridylethanes, and dipyridylmethanes.

The aim of this dissertation was to study the absorption, and subsequent toxic side effects of transition metal based pharmaceuticals following oral administration to rats. Administration of cisplatin (30mg/kg), carboplatin (37mg/kg) and iproplatin (42mg/kg) by oral gavage resulted in their rapid absorption so that respective peak blood levels of 2.63mug platinum/ml, 1.48mug platinum/ml and 3.13mug platinum/ml were achieved within 2-4 hours. Approximately 3-4% of the dose was excreted in the urine, but the major route of elimination was the faeces (>75%). This indicated that although rapid, absorption was relatively poor. Absorption was enhanced by employing a period of starvation prior to administration. A series of novel platinum (IV) mixed amines were absorbed to a greater extent than cisplatin and its congeners, carboplatin and iproplatin. However, absorption was somewhat slower with peak blood levels being attained some 24 hours after administration. Of the other transition metal complexes studied, auranofin and ruthenium acetylacetonate were particularly well absorbed so that peak blood levels of 12.53mug gold/ml and 6.4mug ruthenium/ml were achieved respectively. Urinary clearance of the ruthenium complex was especially significant with up to 45% of the administered dose being eliminated by this pathway within 48 hours. In vitro everted gut sac and in situ perfusion techniques confirmed the in vivo finding that cisplatin is absorbed from the small intestine more readily than carboplatin. No evidence for active or carrier-mediated transport was found and kinetic studies confirmed that absorption was by passive diffusion. Toxicology studies after oral administration of cisplatin (57 or 30mg/kg) or carboplatin (282mg/kg) indicated that the toxicities associated with the perenteral use of the complexes would also apply to the oral route. This was exemplified by the fact that oral cisplatin was profoundly nephrotoxic, whereas carboplatin was not. In addition, gastro-intestinal toxicity manifested as acute necrotizing enteritis and ulcerogenicity of the stomach was potentiated by the oral route. Studies in the ferret indicated that cisplatin is significantly more emetogenic than carboplatin. Examination of liver morphology indicated changes, such as mitochondrial swelling and vesiculation of the endoplasmic reticulum, that might indicate a higher incidence of hepatotoxic responses associated with administration of platinum complexes by the oral route. Both cisplatin and carboplatin induced a degree of myelosuppression but the most pronounced haematological lesion associated with oral administration was severe erythrocytosis which occurred as a result of a dehydration related decrease in plasma volume. Electrothermal atomic absorption analysis of tissues excised from cisplatin and carboplatin rats indicated that platinum deposition was highest in the kidney. In an attempt to explain the prolonged retention of cisplatin in this organ, the intracellular location of the compound was studied using electron microprobe analysis and subcellular fractionation. The various lysosome populations of the proximal tubule were identified as sites of concentration of platinum and it was hypothesised that sequestration within these organelles might be an important mechanism of detoxification.

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