PhD by publication.

Includes bibliographical references (leaves 218-249) / xviii, 249 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The preferred theory to explain the aetiology of emphysema points to an imbalance in the protease-antiprotease systems within the lung with human leukocyte elastase and [alpha]1-protease inhibiter being the main candidates. Examines some aspects of this theory. / Thesis (Ph.D.)--University of Adelaide, Dept. of Pathology, 2001

Le lien entre la prolactine (PRL) et la tumorigenèse mammaire est soutenu par de nombreux arguments expérimentaux, cliniques et épidémiologiques. Cependant, aucune mutation des gènes de la PRL ou de son récepteur (PRLR) n'a jamais été identifiée dans le contexte du cancer du sein. En 2008, notre Laboratoire a identifié le premier polymorphisme gain-de-fonction du PRLR chez des patients présentant une forme rare de tumeur mammaire bénigne (polyadénomatose mammaire). En effet, la seule substitution de l'isoleucine 146 par une leucine dans le domaine extracellulaire du PRLR lui confère une activité de signalisation basale plus élevée que le PRLR sauvage. Dans le cadre de ma thèse, nous avons essayé de déterminer expérimentalement quelles pouvaient être les conséquences de l'expression d'un PRLR constitutivement activé dans une cellule mammaire saine ou tumorale. Pour ce faire, nous avons utilisé deux approches complémentaires. D'une part, nous avons généré deux modèles de souris knock-in ayant intégré une seule copie du PRLR humain, muté (I146L) ou sauvage (en contrôle). Nous avons ensuite analysé les phénotypes mammaires de ces souris à différents âges. Nous n'avons observé aucun développement tumoral, indiquant que le PRLR-I146L n'est pas un oncogène. Néanmoins, les glandes mammaires des souris âgées présentent certaines anomalies histologiques suggérant une interférence possible du PRLR muté avec la différenciation mammaire. L'analyse des ces phénotypes doit être poursuivie pour en déterminer l'ampleur exacte et les mécanismes potentiels. D'autre part, nous avons réalisé une étude in vitro en utilisant des cellules tumorales mammaires humaines. Une étude parallèle au Laboratoire ayant permis de montrer que la substitution I146D conduisait à une activité constitutive plus forte que le polymorphisme naturel I146L, nous avons généré des clones stables de deux lignées tumorales mammaires humaines exprimant le PRLR-I146D. Malgré une signalisation constitutive démontrée, l'expression de ce PRLR muté ne procure aucun avantage sélectif aux cellules tumorales en termes de prolifération, ni ne modifie leur phénotype histologique (Zhang, Cherifi et al, en révision). En conclusion, notre travail a permis de montrer que la seule expression d'un variant gain-de-fonction du PRLR est insuffisante pour transformer une cellule mammaire saine, ou pour favoriser la prolifération de cellules mammaires tumorales. / The link between prolactin (PRL) and mammary tumorigenesis is supported by many experimental, clinical and epidemiologic data. However, mutations on the gene coding for PRL or its receptor (PRLR) have never been identified in the context of breast cancer. In 2008, our Laboratory identified the first gain-of-function polymorphism of the PRLR in patients presenting with a rare form of benign mammary tumors (mammary polyadenomatosis). Indeed, the one-residue substitution of isoleucine 146 by a leucine in the extracellular domain of the PRLR conferred a higher basal activity than that of the wild-type PRLR. Within the framework of my thesis, we have tried to determine the consequences of expressing a constitutively active PRLR in a healthy or cancerous mammary cell. With this aim, we have used two complementary approaches. On one hand, we generated two models of knock-in mice carrying only one copy of the mutated (I146L) or wild-type (as control) human PRLR. We then analyzed the mammary phenotypes of these mice at various ages. We did not observe any tumor development, indicating that the PRLR-I146L is not an oncogene. Nevertheless, the mammary glands of old mice presented certain histological anomalies suggesting a possible interference of the mutated PRLR with normal mammary differentiation. The analysis of these phenotypes must continue to determine their extent and the potential underlying mechanisms. On the other hand, we carried out an in vitro study using human breast cancer cell lines. A parallel study in the Laboratory had showed that the I146D substitution led to a stronger constitutive activity of the PRLR than the natural I146L polymorphism. Thus, we generated stable clones of two human breast cancer cell lines expressing the PRLR-I146D. In spite of its constitutive activity, the expression of PRLR-I146D did not result in any selective advantage for tumor cells in terms of proliferation, nor did it modify their histological phenotype (Zhang, Cherifi et al., in revision). In conclusion, our work has shown that the expression of a gain-of-function mutation in the PRLR is insufficient to transform a healthy mammary cell, or to enhance the proliferation of mammary cancer cells.

This text is a compilation of lecture notes from pathophysiology courses I have taught over the last ten years. The goal of the text is to equip future advanced practice nurses with knowledge of pathophysiology for common diseases and disorders they may encounter in the primary care setting. / https://dc.etsu.edu/etsu-oer/1002/thumbnail.jpg

This work contains a summary of the current genetic, epigenetic and metagenomic knowledge of obesity, as well as an extensive overview of current genetic approaches in mouse models of obesity. Lastly, this work presents a candidate gene approach based on mouse models, which finds new variants associated with multiple obesity phenotypes in a multi-ethnic cohort. / The prevalence of obesity has been mainly be attributed to the rise in an obesogenic environment, in which individuals are more prone to high-dense energy foods and live a sedimentary lifestyle. Familial aggregation of obesity however, has led to numerous studies focused on understanding the genetic basis of this complex disorder. To this effect, this thesis summarizes the current knowledge of obesity genetics, including the monogenic, polygenic and epigenetics field. Given the tremendous contribution of animal models, especially mouse models, to our current knowledge of obesity genetics, this thesis summarizes the methodology of genetic studies in mice, and focuses on how the synergy between human and mouse studies has led to not only the discovery of obesity causal genes, but also their biological contribution to obesity. Lastly, this thesis summarizes a candidate gene approach based on the information from mouse models that have led to identification of a novel variant associated with body mass index (BMI), hip circumference and body adiposity index (BAI) in a multi-ethnic cohort. / Thesis / Master of Science (MSc) / Obesity is a heritable disease, and the genetic basis of obesity could result in better prevention and treatment options for this disorder. Obesity patterns are not uniform worldwide, and some ethnic groups are more prone to obesity than others, therefore having a multi-ethnic approach in studying obesity could yield to causal variants that could be generalized to all.

"December 2004" / Includes bibliographical references (leaves 342-379) / xiii, 379 leaves : ill. (col.) ; 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 Medicine, 2005

Reperfusion injury may mediate renal dysfunction following ischaemia. A murine model was developed to investigate the role of the tissue factor-thrombin-protease activated receptor pathway in renal ischaemia reperfusion injury (IRI). In this model, mice received 25 minutes of ischaemia and subsequent periods of reperfusion. C57BL6, protease activated receptor-1 (PAR-1) knockout mice, and tissue factor (TF) deficient mice were used. Following 24 hours IRI, PAR-1 deficiency resulted in protection against severe renal failure compared to the C57BL6 mice (creatinine, 118.2 ?? 6.3 vs 203 ?? 12 ??mol/l, p&lt0.001). This was confirmed by lesser tubular injury. By 48 hours IRI, this resulted in a survival benefit (survival, 87.5% vs 0%, p&lt0.001). Treatment of C57BL6 mice with hirudin, a specific thrombin inhibitor, offered renoprotection at 24 hours IRI (creatinine, 107 ?? 10 ??mol/l, p&lt0.001), leading to a 60% survival rate at 48 hours IRI (p&lt0.001). TF deficient mice expressing less than 1% of C57BL6 mouse TF were also protected (creatinine, 113.6 ?? 7 ??mol/l, p&lt0.001), with a survival benefit of 75% (p&lt0.001). The PAR-1 knockout, hirudin treated C57BL6 and TF deficient mice had reduced myeloperoxidase activity and tissue neutrophil counts compared to the C57BL6 mice, along with reduced KC and MIP-2 chemokine mRNA and protein expression. Hirudin treatment of PAR-1 knockout mice had no additional benefit over PAR-1 absence alone, suggesting no further contribution by activation of other protease activated receptors (creatinine at 24 hours IRI, 106.5 ?? 10.5 ??mol/l, p&gt0.05). Furthermore, immunofluoresence staining for fibrin(ogen) showed no difference between C57BL6 and PAR-1 knockout mice, suggesting no major contribution by fibrin in this model. Renal IRI resulted in increased levels of TF mRNA expression in the C57BL6, PAR-1 knockout, and hirudin treated C57BL6 mice compared to normal controls, suggesting that TF mRNA expression was upregulated in this model. This resulted in increased TF functional activity in the C57BL6 and PAR-1 knockout mice, but TF activity was negligible in hirudin treated C57BL6 and TF deficient mice. The data therefore suggests that the TF-thrombin cascade contributes to renal IRI by signalling via PAR-1 that then regulates chemokine gene expression and subsequent neutrophil recruitment.

Reperfusion injury may mediate renal dysfunction following ischaemia. A murine model was developed to investigate the role of the tissue factor-thrombin-protease activated receptor pathway in renal ischaemia reperfusion injury (IRI). In this model, mice received 25 minutes of ischaemia and subsequent periods of reperfusion. C57BL6, protease activated receptor-1 (PAR-1) knockout mice, and tissue factor (TF) deficient mice were used. Following 24 hours IRI, PAR-1 deficiency resulted in protection against severe renal failure compared to the C57BL6 mice (creatinine, 118.2 ?? 6.3 vs 203 ?? 12 ??mol/l, p&lt0.001). This was confirmed by lesser tubular injury. By 48 hours IRI, this resulted in a survival benefit (survival, 87.5% vs 0%, p&lt0.001). Treatment of C57BL6 mice with hirudin, a specific thrombin inhibitor, offered renoprotection at 24 hours IRI (creatinine, 107 ?? 10 ??mol/l, p&lt0.001), leading to a 60% survival rate at 48 hours IRI (p&lt0.001). TF deficient mice expressing less than 1% of C57BL6 mouse TF were also protected (creatinine, 113.6 ?? 7 ??mol/l, p&lt0.001), with a survival benefit of 75% (p&lt0.001). The PAR-1 knockout, hirudin treated C57BL6 and TF deficient mice had reduced myeloperoxidase activity and tissue neutrophil counts compared to the C57BL6 mice, along with reduced KC and MIP-2 chemokine mRNA and protein expression. Hirudin treatment of PAR-1 knockout mice had no additional benefit over PAR-1 absence alone, suggesting no further contribution by activation of other protease activated receptors (creatinine at 24 hours IRI, 106.5 ?? 10.5 ??mol/l, p&gt0.05). Furthermore, immunofluoresence staining for fibrin(ogen) showed no difference between C57BL6 and PAR-1 knockout mice, suggesting no major contribution by fibrin in this model. Renal IRI resulted in increased levels of TF mRNA expression in the C57BL6, PAR-1 knockout, and hirudin treated C57BL6 mice compared to normal controls, suggesting that TF mRNA expression was upregulated in this model. This resulted in increased TF functional activity in the C57BL6 and PAR-1 knockout mice, but TF activity was negligible in hirudin treated C57BL6 and TF deficient mice. The data therefore suggests that the TF-thrombin cascade contributes to renal IRI by signalling via PAR-1 that then regulates chemokine gene expression and subsequent neutrophil recruitment.

Elevated levels of homocysteine (HCy) are a known risk factor in several disease states (1). HCy has several other metabolites, homocysteine thiolactone (HCy-T) and homocysteic acid (HCA). Whilst HCy-mediated neurotoxicity has been extensively studied, the underlying mechanisms of HCy-T and HCA mediated neuronal damage remain largely unknown. This thesis aims to explore the underlying mechanisms, triggered by HCy and metabolites which result in neuronal cell death, and may be appropriate targets for future research on disease-modifying interventions in neurodegenerative disorders. As ageing is the greatest risk factor for neurodegeneration, a novel model of human neuronal ageing was established, permitting investigation of the pathways triggered by HCy in ageing. Using SH-SY5Y cells, a novel differentiation protocol was established and categorised, once fully differentiated, these cells were shown to be fully functional neurons and could be maintained for a month in culture. Using a range of concentrations of HCy and HCy-T, the concentration cell death occurs at was determined using crystal violet and lactate dehydrogenase assays. Mechanisms of toxicity were determined using pharmacological intervention at the NMDA receptor, nitric oxide scavengers and antioxidants. Using a combination of immunocytochemistry, live cell imaging and ELISA, alterations in markers of cell damage could be examined. The results showed HCy and HCy-T have distinct mechanisms of toxicity. Whilst both are neurotoxic, HCy directly acts via the NMDA receptor, however HCy-T appears to be less potent. Additionally, HCy-T caused a greater increase in reactive oxygen species generation than HCy, and each metabolite also displayed distinct mitochondrial network abnormalities. Finally, using the long-term culture methods, the chronic effects of HCy, HCy-T and HCA were examined. However, extensive cell death was apparent at low doses in all metabolites therefore no definitive mechanisms could be determined. This culture method was deemed not appropriate for toxicity experiments.

Psychiatric disorders and cognitive impairment are increasingly being described in patients with vestibular pathology. Yet frameworks that describe the link between emotion, memory and the vestibular system have yet to reach maturity, partly because studies have not yet provided detailed accounts of behavioral changes in experimental animals, or in man. One of the goals of this thesis was to use experimental psychology to define changes in memory and emotional behaviour in rats given bilateral vestibular deafferentation (BVD, n=18) or sham surgery (Sham, n=17). In an elevated-plus maze task, BVD rats made up to 166% greater open arm entries and spent up to 42% more time in the open arms compared to Sham rats. In an elevated-T maze task, BVD rats failed to develop a normal learned inhibition response to open space. In an open field maze BVD rats consistently showed 50-60% greater movement velocity, spent on average 35% more time in the inner most aversive part of the arena, and failed to show the normal boundary-seeking behaviour (thigmotaxis) typical of untreated or Sham rats. In a social interaction test BVD rats spent up to 34% less time engaged in social contact compared to Sham rats. In a hyponeophagia test, BVD rats� latency to eat was 70% greater than Sham rats at 3-weeks post-op., however this difference disappeared at 3- and 5-months. These findings suggest that BVD treatment may in some cases disrupt normal behavioral inhibition. Memory performance was also affected. In a T-maze task BVD rats achieved 40-60% correct arm entries, compared to 90-100% for Sham controls. In a foraging task carried out in darkness, BVD rats� initial homing angle was random, homing paths were ~70% longer, and reference memory errors were ~56% greater compared to Sham rats. To elucidate possible neurochemical substrates for these behavioral changes, western blot assays on monoamine proteins were carried out on tissue from a naïve set of rats (BVD n=6; Sham n=6). In BVD rats, serotonin transporter protein expression was 39% lower in CA1 hippocampus and 27% lower in the forebrain region, despite forebrain tryptophan hydroxylase expression being 34% upregulated. Tyrosine hydroxylase expression in the forebrain region was 27% lower in BVD rats. Proteins related to synaptogenesis were also investigated. In the dentate gyrus SNAP-25 was 37% upregulated in BVD rats, while in area CA2/3 of the hippocampus neurofilament-L was 13% upregulated. Forebrain and entorhinal cortex drebrin expression was 28% and 38% downregulated in BVD rats. Neurofilament-L was also 31% downregulated in the forebrain region of BVD rats. To test whether any of these behavioral or biochemical changes may have been attributable to chronic physiological stress, a corticosterone assay was carried out at the conclusion of behavioral testing; however, the no significant between treatment differences were found. In conclusion, vestibular information appears to be needed for the acquisition of spatial and reference memory as well as the normal expression of emotional behaviour. The neurochemical changes described herein point toward possible substrates for these behaviors, however their full significance has yet to be determined.