published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Molecular Biology / Doctoral / Doctor of Philosophy

published_or_final_version / Pharmacology / Master / Master of Philosophy

Anxiety is a common disorder which affects about 25% of the population and whose pathophysiology is still poorly understood. Animal models of disease have been widely used to investigate the molecular basis of human disorders, including psychiatric illnesses. This thesis is about the study of the genetic basis of a mouse model of anxiety. I have carried out a QTL mapping study of behavioural measures thought to model anxiety. I report results from 1,636 mice, assessed for a large number of phenotypes in five ethological tests. Mice belonged to two F2 intercrosses originated by four lines generated in a replicate selection experiment. By comparing mapping results between the two crosses, I have demonstrated that selection operated on the same relatively small number of loci in the four selected lines. Analysis of genetic effect of QTL across phenotypes has allowed me to identify loci with specific roles on different dimensions of anxious behaviour, therefore enhancing our understanding of the anxiety phenotype in mice. For some of these QTL I have also accomplished fine mapping experiments: a locus on chromosome 15 is now contained in an interval of only 3 centimorgans. This work is the basis for further molecular dissection of the genetic loci that underlie anxiety and provides a starting point for the discovery of genes involved in a common psychiatric condition.

Neutrophils accumulate around Trichinella spiralis larvae encysted in skeletal muscle cells of the host. The magnitude of the neutrophil infiltration follows a pattern relating to the stage of infection of T.spiralis. This study was performed to determine if there are factors chemotactic for neutrophils present in the sera of mice infected with T.spiralis, and if present, to compare the chemotactic potential of sera from several time points during the infection. Female MRL++ mice hosted the T.spiralis infection and provided neutrophils for all experiments. The chemotactic potentials of sera were tested by placing 150 ul of serum collected at zero, four, 11, or 28 days following initial infection of the mouse with T.spiralis, into the bottom well of a modified Boyden chamber called a deep well chemotaxis chamber. Next a PVP-free polycarbonate micropore membrane with a pore size of 5 um was placed over the bottom well. The top chamber was then fastened to the bottom well and filled with a neutrophil suspension which was obtained by gradient layer centrifugation using the NIM • 2 step gradient reagent system. A cover glass was placed over the opening of the top well and the entire apparatus was incubated at 37°C under 5% C02 in a humidified incubator for four hours. Following the incubation cells which had migrated through to the bottom well were counted using a hemocytometer and the membranes were stained with Diff Quick. The results demonstrate that there are factors present in the sera of mice infected with T.spiralis that elicit the chemotactic response of neutrophils in vivo. This work also demonstrates that sera from mice infected 11 and 28 days are significantly more chemotactic for neutrophils than are sera from uninfected mice and mice infected four days. These findings correlated with the histologic appearance of the infected skeletal muscle at four, 11 and 28 days post infection as determined by other members of our laboratory. The chemokines IL-8 and KC, and other chemotactic factors such as C5a are discussed as potential mediators of the neutrophil chemotaxis found in this experiment.

Helicobacter pylori is a unique bacterial pathogen which colonises the human stomach. Infection with H. pylori has been linked to several disease outcomes including gastric and duodenal ulcer, gastric cancer and MALT lymphoma. Considering the harsh environment in which it resides and the lack of competition from other bacteria, this host/pathogen relationship is particularly interesting. Microarray analysis is a new and powerful technique which can be used to investigate various aspects of these complex interactions. Expression profiling of bacteria using microarrays remains in its infancy and thus appropriate methods were developed herein for investigating the transcriptional responses of H. pylori to various environments in vitro. Studies showed the tight relationship between growth phase dependent expression of iron homeostasis, motility and virulence genes in H. pylori for the first time. Consequently, the late exponential phase of growth was implicated as the most virulent growth phase of this bacterium in vitro. In response to mammalian cell co-culture, induced expression of H. pylori metabolism/respiration genes, genes of unknown function and genes encoding the 2-component regulators, HP1021 and HP0166, were detected. These represent a set of genes likely to be important specifically in the context of infection. To investigate the host response to infection a new mouse colonising strain of H. pylori, the Sydney Strain 2000 (SS2000), was isolated for use in comparative studies with the established strain, Sydney Strain 1 (SS1). Both host and strain specific effects were studied in a 15 month colonisation experiment using C57BL/6 and BALB/c mice. Genomic typing was used to investigate dynamic changes that occurred in the mouse-adapted strains during colonisation. In these animals reponses relating to the severity of inflammation and to the infecting H. pylori isolate were revealed by gene expression profiling. Previously unrealised cellular responses were uncovered. These included the significant down-regulation of both ferritin and haemoglobin expression. This perhaps suggests a mechanism for H. pylori induced iron deficiency anaemia. Physiological connections between colonisation, acid secretion and expression of the endocrine hormones were also implicated. These experiments have shown the utility of microarray analysis in the investigation of pathogenesis and have highlighted many directions for further investigation.

published_or_final_version / abstract / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Pharmacology / Master / Master of Philosophy

published_or_final_version / Paediatrics / Master / Master of Philosophy