A model of growth and plasmid transfer between strains of Escherichia coli and Salmonella typhimurium was developed with reference to the literature. This was the organising principle for the collection of a complete set of in vitro life history parameters of one S. typhimurium and one E. coli strain. In the course of estimating these parameters two results of note were obtained. Fits of the Lotka-Volterra competition model were obtained for data on S. typhimuiurm growing in competition with E. coli. The first noteworthy discovery was the failure of this model to account for several characteristics of growth of these strains under competition. The growth rates of plasmid-bearing and plasmid-free strains were obtained. The second main result came from examination of the results of the growth rate data, which revealed that the cost to S. typhimuiurm 576 of bearing the resistance plasmid was low (4%). The model was also used to simulate the effect of antibiotic dose on the density of the donor, recipient and transconjugant populations over time. These simulations predicted that there would be a convex relationship between antibiotic dose and transconjugant density (i.e. that the density would first rise, then fall, with increasing dose). Following from this result, laboratory experiments and in vivo experiments in chickens were directed towards obtaining information on the relationship between these two variables. This convex relationship was not demonstrated within a single experiment, although some experimental environments produced an increase in transconjugant density with dose, and others, a decrease. Few transconjugants were formed in vivo. In order to investigate the low cost of resistance and low rate of in vivo transconjugant production, cost of resistance and plasmid transfer rate of this plasmid in several strain combinations of E. coli and S. typhimuiurm was evaluated.

Sustainable parasite control in livestock depends on anthelmintic drugs. The nematode Haemonchus contortus, the most important intestinal parasite of sheep and goats has developed resistance to all classes of anthelmintics including moxidectin, the most potent of the macrocyclic lactones. Pyrosequencing was used to screen H. contortus laboratory and field strains for single nucleotide polymorphisms (SNPs) associated with resistance in three genes, and determine their involvement in field resistance to macrocyclic lactones. Specific SNPs increased in frequency in ivermectin/moxidectin laboratory selected strains for all three genes. These did not protect a resistant field strain from a field dose of ivermectin and were not the major mechanism of resistance in the field strain. A gamma-aminobutyric acid chloride receptor SNP may be a potential marker for moxidectin resistance in the field. This study indicates results obtained from laboratory strains selected with sub-therapeutic doses of drug may not reflect the situation in the field.

Anthelmintic resistance is a major problem in livestock, and while it has been slower to emerge in cattle, there are reports of its occurrence. Three broad-spectrum anthelmintics are available for use, and one mechanism of resistance that is common to all is target site alteration. Glutamate-gated chloride channels (GluCls) are an important target for macrocyclic lactone anthelmintics (MLs), while beta-tubulin represents the benzimidazole (BZ) target. The objectives of this thesis were to determine whether GluCls are involved in ML resistance in the cattle parasite Cooperia oncophora , and whether beta-tubulin is involved in BZ and ML resistance. Two isolates of C. oncophora were used. In a fecal egg-count reduction test, ivermectin was found to be 100% effective against one isolate (IVS), and only 77.8% effective against the second isolate (IVR). Two full-length GluCl cDNAs, encoding GluClalpha3 and beta subunits, were cloned. These subunits share high sequence identity with similar GluCl subunits from Haemonchus contortus and Caenorhabditis elegans. Genetic variability analysis of the two genes showed significant differences in allele frequencies between IVS and IVR worms at the GluClalpha3 gene, but not the GluClbeta gene, suggesting that the GluClalpha3 gene is involved in ivermectin resistance. Sequencing of full-length GluCl subunit cDNAs from IVS and IVR worms revealed the presence of mutations in the N-terminal domains. Mutations in the GluClalpha3 caused modest but significant reductions in glutamate, ivermectin and moxidectin sensitivity, while mutations in the GluClbeta abolished glutamate sensitivity. Of the three mutations identified in the IVR GluClalpha3, the L256F mutation accounted for the difference in glutamate and ivermectin response between IVS and IVR GluClalpha3 channels. Two beta-tubulin isotypes cloned from C. oncophora were found to share a high homology with beta-tubulin isotypes from other trichostrongylids. Gen

The past few decades have witnessed an array of advances in DNA science including the introduction of genomics and bioinformatics. The quest for complete genome sequences has driven the development of microarray and massively parallel sequencing technologies at a rapid pace, yielding numerous scientific discoveries. My thesis applies several of these genome-scale technologies to understand genomic response to perturbation as well as chromatin structure, and it is divided into three major studies. The first study describes a method I developed to identify drug targets by overexpressing human genes in yeast. This chemical genomic assay makes use of the human ORFeome collection and oligonucleotide microarrays to identify potential novel human drug targets. My second study applies genome resequencing of yeast that have evolved resistance to antifungal drug combinations. Using massively parallel genomic sequencing, I identified novel genomic variations that were responsible for this resistance and it was confirmed in vivo. Lastly, I report the characterization of chromatin structure in a non-eukaryotic species, an archaeon. The conservation of the nucleosomal landscape in archaea suggests that chromatin is not solely a hallmark of eukaryotes, and that its role in transcriptional regulation is ancient. Together, these 3 studies illustrate how maturation of genomic technology for research applications has great utility for the identification of potential human and antifungal drug targets and offers an encompassing glance at the structure of genomes.

The glutamate-gated chloride (GluCl) channel family of receptors are members of the ligand-gated ion channel (LGIC) family. In addition to being essential for physiological and behavioural aspects of an organism, they are also exploited as a target for the drug ivermectin. A novel GluCl subunit in C. elegans, GLC-4, was characterized in order to further understand the diversity of GluCls and their implications in biological and behavioural processes. Sequencing of cDNAs generated by RT-PCR indicate that GLC-4 possesses the typical features of a GluCl subunit. In addition, they also suggest possible alternative splicing of glc-4 resulting in a slightly truncated transcript. A glc-4 mutant, glc-4(ok212), was used to investigate the role of GLC-4 in C. elegans ivermectin sensitivity and behaviour. Glc-4(ok212) worms were found to be hypersensitive to low concentrations of ivermectin and experienced a slight hyperreversal behaviour. Genetic, molecular, and electrophysiological evidence is also provided suggesting an interaction between GLC-4 and AVR-14, another GluCl subunit in C. elegans. We hypothesize a direct association between GLC-4 and AVR-14 which together form a heteromeric channel in vivo .

Appendix included in back. / Errata posted on back end cover. / Bibliography: leaves 236-268. / xxii, 268, [7] leaves, [41] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / A contribution to the knowledge of multidrug resistance and its role in acute leukaemia. / Thesis (Ph.D.)--University of Adelaide, Dept. of Cytogenetics and molecular genetics, 1997

Doctor of Philosophy (PhD) / The H69CIS200 cisplatin-resistant and H69OX400 oxaliplatin-resistant cell lines developed as part of this study, are novel models of low-level platinum resistance. These resistant cell lines do not have common mechanisms of platinum resistance such as increased expression of glutathione or decreased platinum accumulation. Rather, these cell lines have alterations in their cell cycle allowing them to proliferate rapidly post drug treatment in a process known as ‘regrowth resistance’. This alteration in cell cycle control has come at the expense of DNA repair capacity. The resistant cell lines show a decrease in nucleotide excision repair and homologous recombination repair, the reverse of what is normally associated with platinum resistance. The alterations in these DNA repair pathways help signal the G1/S checkpoint to allow the cell cycle to progress despite the presence of DNA damage. The decrease in DNA repair capacity has also contributed to the development of chromosomal alterations in the resistant cell lines. Similarities in chromosomal change between the two platinum resistant cell lines have been attributed to inherent vulnerabilities in the parental H69 cells rather than part of the mechanism of resistance. The H69CIS200 and H69OX400 resistant cells are cross-resistant to both cisplatin and oxaliplatin. This demonstrates that oxaliplatin does not have increased activity in low-level cisplatin-resistant cancer. Oxaliplatin resistance also developed more rapidly than cisplatin resistance suggesting that oxaliplatin may be less effective than cisplatin in the treatment of SCLC. The resistant cell lines have also become hypersensitive to taxol but show no alterations in the expression, polymerisation or morphology of tubulin. Rather, the PI3K/Akt/mTOR pathway is involved in both platinum resistance and taxol sensitivity as both are reversed with rapamycin treatment. mTOR is also phosphorylated in the resistant cell lines indicating that platinum resistance is associated with an increase in activity of this pathway. The mechanism of regrowth resistance in the platinum-resistant H69CIS200 and H69OX400 cells is a combination of activation of PI3K/Akt/mTOR signalling and alterations in control of the G1/S cell cycle checkpoint. However, more work remains to determine which factors in these pathways are governing this novel mechanism of platinum resistance.

Thesis (PhD)--University of Stellenbosch, 2005. / Bibliography. Also available via the Internet.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.