School of Biomedical Sciences - Theses
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ItemCharacterization of cyclic nucleotide-mediated signal transduction pathways in Toxoplasma gondii( 2017)T. gondii is amongst the most common human pathogens, chronically infecting more than 30% of the human population. T. gondii has a complex life cycle, often spanning multiple hosts. T. gondii parasites invade target host cells to establish infection. Residing inside host cells, parasites are immotile and active replicate. After multiple rounds of replication, T. gondii activates their motility and egress from host cells. Invasion and egress relies on parasites motility, which is strictly controlled by a number of second messenger-mediated signalling transduction pathways. The signalling pathways that controls parasite motility have become a focus of investigations in the past two decades due to its potential as a therapeutic target. Previous studies showed that activation of Ca2+-mediated signalling pathways and cGMP-mediated signalling pathways drives parasites microneme secretion and motility. Conversely, cAMP-mediated signalling pathways may work as suppressors on parasites motility. In Chapter 3, I evaluated the function of a putative guanylyl cyclase in T. gondii asexual growth. TgGC accumulates at apical tip of the parasites and alters its localization during intracellular growth and extracellular motility. TgGC is critical for T. gondii growth and it plays an important role in parasites microneme secretion, invasion and egress, highly suggestive of its function in cGMP generation. We showed that cGMP-mediated signalling pathways localize upstream of cytosolic Ca2+ rise and could be occurring by activation of phosphatidylinositol-specific phospholipase C. In Chapter 4, I investigated the regulation of cAMP signalling. I identified the cAMP-dependent protein kinase A regulatory domain TgPKAr that regulates activity of catalytic domain TgPKAc1, which has been suggested as a negative regulator of motility and Ca2+ signalling. TgPKAr and TgPKAc1 localize at the parasite periphery, which occurs likely through palmitoylation and myristoylation on the N-terminal TgPKAr sequence. Overall, my work highlights the pivotal role signal transduction pathways play in T. gondii parasites and sheds light on the complex hierarchy and interplay between second messengers-mediated signalling cascades that regulate parasites motility and infectivity.
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ItemDefining the roles of essential genes in the malaria parasite life cycle( 2017)The combination of drug resistance, lack of an effective vaccine and ongoing conflict and poverty mean that malaria remains a major global health crisis. Understanding metabolic pathways at all parasite life stages is important in prioritising and targeting novel anti-parasitic compounds. To overcome limitations of existing genetic tools to investigate all the parasite life stages, new approaches are vital. This project aimed to develop a novel genetic approach using post meiotic segregation to separate genes and bridge parasites through crucial life stages. The unusual heme synthesis pathway of the rodent malaria parasite, Plasmodium berghei, requires eight enzymes distributed across the mitochondrion, apicoplast and cytoplasm. Deletion of the ferrochelatase (FC) gene, the final enzyme in the pathway, confirms that heme synthesis is not essential in the red blood cell stages of the life cycle but is required to complete oocyst development in mosquitoes. The lethality of FC deletions in the mosquito stage makes it difficult to study the impact of these mutations in the subsequent liver stage. To overcome this, I combined locus-specific fluorophore expression with a genetic complementation approach to generate viable, heterozygous oocysts able to produce a mix of FC expressing and FC deficient sporozoites. In the liver stage, FC deficient parasites can be distinguished by fluorescence and phenotyped. Parasites lacking FC exhibited a severe growth defect from early to mid-stages of liver development in-vitro and could not infect naïve mice, confirming liver stage arrest. These results validate the heme pathway as a potential target for prophylactic drugs targeting liver stage parasites. Energy metabolism in malaria parasites varies remarkably over the parasite life cycle. Parasites depend solely on anaerobic glycolysis at blood stage but need Krebs cycle, the electron transport chain, and mitochondrial ATP synthase during mosquito stage development. Again, reverse genetic approaches to study the hepatic stage of Plasmodium have been thwarted because parasites with defects in energy pathways are unable to complete the mosquito stage. I used the genetic complementation approach established to study heme biosynthesis to bridge parasites lacking the β subunit of mitochondrial ATP synthase through mosquito stage and studied their development in the liver stage. ATPase knockouts were indistinguishable from wildtype in in-vitro liver stage assays of size, nuclear content, and merosome production. Robust progression to blood stage confirmed the dispensability of mitochondrial ATP synthesis in liver stages. I extended this approach to explore the essentiality of upstream mitochondrial electron transport and Krebs cycle during the liver stage. I speculate that energy metabolism in the liver stage resembles that in the blood stage, relying predominantly on glycolysis for ATP production. There are numerous genetic tools to manipulate the blood stage malaria parasite genome in general, but existing genetic tools to generate viable parasites with defects in blood stage essential genes are limited. To overcome this limitation, I have developed a novel strategy in which I first insert a complementary copy of the essential gene-of-interest, and then delete the endogenous gene, and then take advantage of meiosis and segregation during the mosquito stage to create haploid knockout sporozoites. I genotype the parasites along the way by fluorescence microscopy. As proof of principle, I created complemented knockouts of the blood stage essential 1-deoxy-D-xylulose-5- phosphate reductoisomerase (DXR) gene, crossed these with wildtype parasites, and then tracked the progeny through in-vitro and in-vivo liver development. Precomplementation proved difficult, perhaps due to inappropriate expression of important metabolic genes. Additionally, problems with apparent silencing of the fluorophore tags compromised my ability to genotype cross progeny preventing any firm conclusion on the function of isoprenoid precursor pathway of liver stage parasites. Nevertheless, my success in generating a blood stage essential gene knockout via precomplementation provides encouragement that this novel reverse genetic strategy can be implemented to investigate the role of blood stage-essential genes in sporozoite and liver stages of malaria parasites.
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ItemPathology of glycogen excess in diabetic cardiomyopathy( 2017)Background: Diabetic cardiomyopathy is a distinct cardiac pathology and the underlying mechanisms are unknown. Elevated glycogen content has been observed in the diabetic human myocardium, first recorded 80 years ago, suggesting that despite impaired glucose uptake cardiomyocytes accumulate glycogen. Anecdotal evidence of glycogen accumulation in the diabetic myocardium has since been recorded in the literature but a systematic investigation of this paradoxical phenomenon has not been conducted. Glycogen storage diseases demonstrate that increased cardiac glycogen is associated with severe functional deficits, and therefore the observed glycogen ‘excess’ in diabetic hearts may be an important and novel agent of pathology in diabetic cardiomyopathy. Aim: This body of work aimed to systematically investigate the role myocardial glycogen accumulation in diabetic cardiomyopathy, with a focus on glycophagy, a glycogen-specific autophagy process. Key metabolic signaling pathways (insulin, AMPK, β-adrenergic) were interrogated to investigate their therapeutic potential. The four experimental questions addressed in this thesis are: 1. Does myocardial glycogen accumulation contribute to functional deficits in the diabetic heart? (Chapter 2) 2. What glycogen processing mechanisms are disrupted and may be associated with glycogen accumulation in the diabetic myocardium? (Chapter 2) 3. Do simulated hyperglycemic and hyperinsulinemic conditions mediate cardiomyocyte glycogen accumulation? (Chapter 3) 4. Can key metabolic signaling pathways (AMPK, β-adrenergic signaling) be exploited to degrade excess cardiomyocyte glycogen? (Chapter 4) Methods: Type 1 diabetes (T1D) was induced in male Sprague-Dawley rats using Streptozotocin. C57Bl/6 mice were fed a high fat diet to induce obesity and insulin resistance – a state of early type 2 diabetes (T2D). Human atrial tissue from diabetic patients were examined for glycogen content. Echocardiography was conducted to assess functional outcomes in diabetic animals. Neonatal rat ventricular cardiomyocytes were cultured in extracellular high glucose (30mM) and insulin (1nM) and/or had suppressed GABARAPL1 expression (siRNA, siGABARAPL1). Influence of β-adrenergic or AMPK activation was assessed using isoproterenol (100µM, 1 hour) or AICAR (30µM, 30 minutes), respectively. Glycogen content in cardiac tissue homogenates and cell lysates was measured via enzymatic assay. Molecular markers of key signaling pathways were investigated using immunoblotting, immunohistochemistry and qPCR. Results: Some of the overall findings of this investigation are that: 1. Myocardial glycogen accumulation in in vivo models of insulin resistance and progressed T1D is associated with diastolic and systolic dysfunction. 2. Myocardial glycogen accumulation is associated with decreased GABARAPL1 lipidation, suggesting a disruption in glycophagosome scaffold processing in the insulin resistant mouse and diabetic human myocardium. This finding was also established in vitro where a suppression of GABARAPL1 mRNA induced cardiomyocyte glycogen excess. 3. High extracellular glucose (simulated hyperglycemia) only increases cardiomyocyte glycogen content in the presence of insulin and is associated with increased expression levels of the glycophagy adapter protein STBD1 in vitro. 4. In vitro activation of β-adrenergic signaling mediates a reduction in cardiomyocyte glycogen via activation of glycogen phosphorylase when glycophagy is disrupted (siGABARAPL1). In vitro activation of AMPK signaling decreases cardiomyocyte glycogen induced by disrupted glycophagy (siGABARAPL1), but is not effective in modulating glycogen loading induced by high extracellular glucose (simulated hyperglycemia). This study identifies glycogen accumulation as a novel agent of pathology in the development of diabetic cardiomyopathy, associated with a disruption in glycophagy. It is the first to show that cardiac dysfunction is linked with myocardial glycogen accumulation. In a glycophagy compromised setting, AMPK and β-adrenergic signaling may provide potential therapeutic targets to rescue cardiac glycogen excess. An increased understanding of the complex signaling pathways mediating glycogen synthesis and storage in early diabetes may provide a platform for the development of cardiac specific, targeted therapeutic interventions in diabetes.
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ItemExploring adaptation in a key Australian genus Brachyscome through experimentation( 2017)This thesis focuses on species adaptation in a key Australian genus Brachyscome, using three central themes in ecological theory; plants perform better where they naturally occur (local adaptation), widespread species outperform narrow endemics (niche breadth—range size hypothesis), and warming responses and range size (related species show ecological similarity). This study incorporates a broad ecological and experimental approach to test these three hypotheses. This approach involves combining reciprocal field based designs with a common garden comparison, and a multispecies common garden with a warming treatment. Firstly, under a combined reciprocal and common garden approach, and using different populations (at the seedling stage) of broadly distributed B. decipiens I questioned whether there was a ‘home site advantage’ with the hypothesis of populations showing greater fitness at their sites of origin. I had undertaken prior survey work which indicated significant morphological variation between different populations. When tested in a reciprocal design this was not the case, and no local adaptation was detected, and further to this, no variation between populations was detected in the common garden comparison. Keeping the same reciprocal and common garden approach and increasing the study species so that I now had a related group, I explored the niche breadth— range size hypothesis at the seed and seedling stage of plant development. I predicted species that naturally occurred over a greater area were more likely to show a higher performance in terms of survival and growth than species with a restricted range in novel environments, and further restricted species would show higher performance at their site of origin due to their specialised habitat preferences. I found performance came at a cost to survival in three of the widespread species, and the opposite pattern in two of the narrow endemic species. A trade off may exist. The niche breadth—range size hypothesis was partially supported, given one species with the most restricted range showed a similar response to the widespread species. So, although we found a pattern, an exception to this also exists. Moving beyond the alpine environment, I used a common garden approach with a treatment to explore species responses to warming. I extended my study species to nineteen Brachyscome taxa under the following criteria; 1) only occur in an alpine zone, 2) occur in and beyond an alpine zone and 3) do not occur in an alpine zone. I predicted species would show a phylogenetic signature for warm temperature responses, and that alpine species would be particularly susceptible to the treatment. Findings suggest species which were endemic to alpine areas were less likely to benefit from warming than widespread species. I found evolutionary history did not have a detectable effect on warming responses. While there was a moderate phylogenetic signal for plant growth in the absence of warming, there was no signal for growth changes in response to warming, despite variability among species that ranged from positive to negative growth responses to warming. There was also no strong effect of ecological context, as species that showed a positive response to warming did not necessarily originate from hotter environments. In fact, several species originating from hot environments grew relatively poorly when exposed to higher soil temperature. We found a strong phylogenetic signal suggesting that closely related species tend to occur in areas with similar annual variability in precipitation. As Brachyscome is an iconic Australian genus of ~80 species of daisies with very few in cultivation, I included one widely cultivated species Brachyscome multifida (because it had already demonstrated horticultural potential) to explore the horticulture potential of species which responded positively to warming. Brachyscome stuartii, and B. rigidula, both compact plants with attractive foliage, showed horticultural potential under warmer soil conditions, as did B multifida, supporting its popularity as an ornamental cultivar. Brachyscome is an intriguing group of daisies that can be found across a myriad of habitats in the Australian landscape. Tapping into this complex group by taking representatives from across diverse clades to address the three ecological concepts, this thesis helps build our understanding of local adaptation and variation in alpine systems, and helps in the identification of correlates of species performance under warming within the context of the niche—breadth and range size hypotheses. It highlights how plants might be selected that respond positively to our changing climate.
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ItemInvestigating the role of NK cells in allogeneic haematopoietic stem cell transplantation( 2017)Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is used to treat a range of haematological malignancies. However, the use of allo-HSCT is limited due to significant toxicity related to conditioning intensity, opportunistic infection, graft failure, and graftversus host disease (GVHD). Standard conditioning regimen including myeloablative total body irradiation (TBI) is considered to be the trigger for pro-inflammatory cytokine release and further induce acute GVHD (aGVHD). To decrease the risk of aGVHD, a reduced intensity conditioning (RIC) regimen was introduced to clinical practice because of its lower toxicity. However, the insufficient suppression of host immune system caused by RIC might lead to higher chances of graft failure. In this case, to identify the risk factors to graft rejection after RIC regimen was crucial. And by targeting the major contributor to graft failure post-transplantation, RIC can be utilised to prevent aGVHD while high donor engraftment can still be achieved. Recipient natural killer (NK) cells were found to be significantly more radio-resistant than other cytotoxic lymphocytes following TBI and thus could be a considerable contributor to acute allograft rejection. MHC-mismatched transplantation allo-BMT mouse models were utilised in this project to determine the extent by which radio-resistant recipient NKs are involved in causing acute allograft rejection. By using wild-type (WT) and the NK cell deficient (Bcl2fl/fl Ncr1-iCre) mice as recipients, donor cells were rapidly rejected in WT recipients with RIC-allo-BMT, whereas Bcl2fl/fl Ncr1-iCre recipients that were irradiated with the RIC 2×400 rad TBI achieved long-term engraftment and lower aGVHD clinical scores. This positive outcome with reduced TBI dose was attributed to lesser donor T cell expansion, lower pro-inflammatory cytokine levels and higher myeloid cell reconstitution in the NK cell deficient recipients, compared to recipients that had undergone transplantation with myeloablative TBI (2×600 rad). These findings clearly indicate that recipient radio-resistant NK cells are the main cause of graft failure and can be targeted to lower conditioning intensity and promote engraftment. Besides the Bcl2fl/fl Ncr1-iCre mouse models, other NK cell deficient or aberrant models were also studied in order to understand further how NK cells can regulate aGVHD and donor engraftment. Using an alternative approach with greater clinical relevance, WT mice administered with the BCL2-inhibitor S63845 and/or the MCL1-inhibitor ABT-199 exhibited similar outcomes as the BCL2- and MCL1-deficient mouse models. Inhibitor treatment prior to allogeneic transplantation with RIC regimen reduced the number of host residual NK cells. And in this case, mice that had the inhibitor treatment and RIC-allo-BMT had reduced risk of graft failure and could also be free from aGVHD. Another key finding of this study, which has significant potential for clinical translation was validation of the therapeutic graft-versus-leukaemia (GVL) effect in the RIC-allo-BMT models. Either genetically modified NK cell deficient mouse RIC-allo-HSCT models or inhibitor treatment mouse RIC-allo-HSCT models were proven to have intact GVL effect. Therefore, the less sufficient anti-neoplastic efficacy of RIC regimen could be compensated by the intact GVL effect. And most importantly, the overall mortality related to leukaemia relapse of the NK cell deficient RIC-allo-BMT models was significantly lower than the WT RIC-allo-BMT models.
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ItemDefining and developing novel host targeted therapies to eliminate Mycobacterium tuberculosis infections( 2017)Tuberculosis, caused by Mycobacterium tuberculosis (M.tb), is a massive global health problem with one third of the world’s population infected. Aproximately two billion people have been killed by tuberculosis over the last two centuries. With the growth in our understanding of tuberculosis disease and production of the Bacillus Calmette–Guérin (BCG) vaccine, the tuberculosis incidence and mortality have been dramatically reduced over the past century. However, the number of newly diagnosed tuberculosis cases per year is still very high in some parts of world. Complex issues confound our ability to tackle this disease including problems with detecting and differentiating infection and disease, inadequate sanitation, low BCG vaccination rates, poor vaccine efficacy and the emergence of drug resistant strains. These issues increase the pressure, need and urgency of developing better preventive management techniques to stop the spread of new infections and better agents to treat infection, particularly resistant infections. A characteristic of intracellular M.tb infection is its ability to cause or promote lytic forms of host cell death (e.g. necrosis). The major shortcoming of necrosis is that organisms are not killed and they escape the intracellular environment enabling them to infect more host cells. One mechanism that assists in the killing of intracellular M.tb is the activation of host cell inducible nitric oxide synthase (iNOS). Additionally, recent work in our laboratory has shown that apoptosis can be promoted during M.tb infection to help kill intracellular M.tb and clear infection. This was done using a drug that antagonises the inhibitor of apoptosis proteins (IAPs). IAPs activate a cell survival pathway downstream of TNF signalling. If IAPs are antagonised with small molecule inhibitors, TNF no longer activates a host cell survival signalling pathway but rather it activates an apoptotic signalling cascade. In my studies, I combined the IAP antagonists’ with bosentan, a clinical stage drug that has been developed to promote iNOS activity. I found that bosentan was not efficacious in reducing M.tb burdens in lung and this may be due the drugs ability to increase oxygenation in the lung. Perhaps the increased oxygenation promoted the ability of aerobic M.tb to proliferate. Interestingly, IAP antagonists reversed the negative effects of bosentan presumably by promoting apoptosis of infected cells. As well as killing intracellular M.tb, apoptosis is also thought to promote antigen presentation and hence immunity. If we are able to promote apoptotic cell death during BCG vaccination we may be able to promote more robust immunity to M.tb. So I examined the ability of IAPs antagonists to promote BCG efficacy and I found that these drugs, when administered at the correct time, did promote BCG vaccine efficacy. I additionally sought to examine the role of a subset of T cells called mucosal associated invariant T cells (MAIT cells) that have been implicated in facilitating the acquisition of adaptive immunity. MAIT cells are found in the mucosa and several organs including lung. These cells become activated when they encounter host cells presenting bacterially derived vitamin B metabolites in association with MHC class I-like molecule (MR1). I activated MAIT cells with bacterial vitamin B metabolites in both wild type and MR1 gene knock out mice and then immunized animals with BCG vaccine prior to M.tb challenge. I found that promoting MAIT cell activation had no significant effect on promoting BCG vaccine efficacy.
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ItemUnderstanding the factors that drive the emergence of dominant gene constellations during influenza A virus reassortment( 2017)The segmented nature of the influenza genome allows the virus to undergo reassortment (gene swapping) upon co-infection of a single cell. This process is a major contributing factor to the emergence of novel pandemic strains and in a practical context, reassortment can be utilised to produce viruses carrying the gene constellations that make them suitable for vaccine production. Despite its significance, the factors that govern the emergence of dominant gene constellations during reassortment are not well understood. Studies in this thesis have extensively examined influenza virus reassortment in the context of vaccine seed production. Using a methodology equivalent to that used for seasonal influenza vaccine seed production, eggs have been co-infected with the A/Udorn/307/72 (Udorn) virus as a model seasonal strain and the egg-adapted, high-yielding A/Puerto Rico/8/34 (PR8) virus. Tracking of the reassortant genotypes generated throughout the reassortment process performed under the selective pressure of antibody to PR8 surface glycoproteins, identified a large variety of reassortant viruses isolated in the initial stages. However, with subsequent rounds of replication and selection, specific gene constellations began to dominate. Enhanced replicative fitness was shown to explain the emergence of certain gene constellations; however, a few had reduced replicative abilities and we demonstrated, using competitive plasmid transfection experiments, that gene co-selection can explain the dominance of these viruses. We therefore postulate that preferential co-packaging of gene segments into progeny virions is an important driver for the emergence of certain gene constellations after reassortment. As well as confirming co-selection relationship between NA and PB1 from both Udorn and PR8 viruses, a novel putative co-selection relationship was identified between the PB1 and NP genes of both viruses and an additional putative co-selection relationship between the NA, PB2 and PB1 genes of the Udorn virus. Overall, the PB1 and NP genes of the Udorn virus and the PA and M genes of the PR8 virus were found to prevail in the final viral progeny. Seed viruses produced through classical reassortment are selected to have the seasonal haemagglutinin (HA) and neuraminidase (NA) and a high HA protein yield. From the final dominant gene constellations isolated through cloning via limiting dilution, four potential vaccine seed candidates were selected. Comparisons of viral fitness for the reassortants identified an incompatibility between the Udorn PB1 and PR8 NP that resulted in reduced replicative fitness. This incompatibility was further exacerbated by the presence of the Udorn HA. Analysis of the four vaccine seed candidates also revealed that either an increase in the HA protein density on the virion or an increase in the production of non-infectious particles were the main mechanisms contributing to the observed high HA yields. This study of the dynamics of reassortment show it to be a largely random process initially, but selective pressures such as replicative fitness, gene co-selection and antibody pressures restrict the final viruses that dominate. Influenza virus reassortment is a complicated process and understanding the factors that govern reassortment may aid in the prediction of future outbreak isolates.
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ItemExploring the role of early life respiratory infection in asthma( 2017)The underlying cause of asthma is yet to be determined. Asthma is presently the most common chronic disease affecting children, and it is becoming clear that disease originates in early life and as a result of complex synergistic interactions of various environmental exposures. There has been increasing interest in the role of neonatal infections; with evidence emerging that asymptomatic pneumococcal colonisation is a strong predictor of future asthma, which can be exacerbated by viral infection. It is therefore paramount that appropriate models of disease are developed in order to elucidate these complex immunological interactions that have profound pathogenic potential during this window of lung development. Importantly, small airways dysfunction is present in the majority of asthmatics, and remains relatively undertreated by existing therapy, contributing considerably to the severity of disease. The lung slice technique is a powerful in vitro tool that can be used to explore airway and vascular pharmacology, offering a unique experimental link between cell-based assays and in vivo experimentation. Methodological studies compared airway reactivity in lung slices in varying experimental conditions, and application of the lung slice technique to characterize reactivity following acute respiratory infection in vivo, permitting application of this technique within the rest of this thesis. Utilising novel mouse models of asthma, parameters of bronchial reactivity, lung immunity and structural changes were assessed in adulthood following exposures in infancy, elucidating key mechanisms driving the deleterious effects caused by early life exposures. Using a mouse model of neonatal respiratory co-infection, the effect of early life co-infection with Streptococcus pneumoniae (SP) and influenza A virus (IAV) on mouse lung health, immunity and structure in adulthood was investigated. Co-infection caused a significant increase in central airway resistance that was not associated with conventional airway remodelling such as mucus overproduction, smooth muscle thickening or epithelial leakage. Airways hyperresponsiveness (AHR) was not maintained in lung slices in vitro. An increase in hysteresivity was observed in both IAV and co-infected mice. Hence, it is possible that co-infection in infancy is causing distinct structural changes that contribute to ventilation heterogeneity, which persists into adulthood. A novel model of early life co-infection combined with house dust mite (HDM) aeroallergen challenge was developed and characterised. Neonatal co-infection in the background of HDM sensitisation resulted in chronic lung colonisation, exacerbated neutrophilic inflammation, increased mucin production and increased AHR. Hence, pneumococcal colonisation does not protect against allergic airways disease which is predominately driven by mechanisms that are independent of TH2 immunity. This study identified SAA, IL-17A and G-CSF as molecular markers for this phenotype, which parallels features of more severe asthma. Taken together, the strong neutrophilic/TH17/SAA signal, accompanied by AHR and mucus hyper-secretion generated in the present study in early life co-infection plus HDM group suggests this model may be capturing key features of severe, steroid insensitive asthma, providing novel insight into clinically relevant pathology. Ultimately, this model presents a unique opportunity to develop new treatment strategies to circumvent a highly relevant cause of childhood asthma.
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ItemDiabetic cardiomyopathy: sex-specific aspects of functional, structural and molecular remodelling( 2017)Background: Clinical studies have revealed increased cardiovascular risk in diabetic patients, which is substantially elevated in women. Perplexingly, while there has been extensive experimental effort in characterising cardiac dysfunction in the progression of diabetic cardiomyopathy, studies investigating sex differences are limited. A mechanistic understanding of sexual dimorphism in diabetic cardiomyopathy remains to be achieved. Aim: The aim of this Thesis was to examine female susceptibility to cardiac pathology in type 1 diabetes (T1D). In particular, this thesis focussed on examining cardiac responses to diabetes (functional and molecular) under basal conditions, during ischemia and with increased cardiac renin angiotensin system (RAS) signalling. The four experimental questions addressed in this thesis are: 1. Are systemic and cardiac T1D phenotypes different between males and females? [Chapter 3] 2. Is there an accentuated female vulnerability to ischemia reperfusion injury in T1D? [Chapter 4] 3. Are there sex-specific changes in cell death, autophagy and metabolism associated with diabetes? [Chapter 5] 4. Does cardiac RAS upregulation interact with sex-specific cardiac responses in T1D? [Chapter 6] Methods: A wide range of in vivo, ex vivo and molecular strategies were employed to characterise the role of sex differences in a streptozotocin (STZ)-induced T1D mouse model. Echocardiographic assessment was performed to examine T1D-induced functional and structural deficits in vivo. Ex vivo isolated heart perfusion analysis was used to characterise the role of sex differences in ischemia-reperfusion injury and recovery in T1D. The mechanistic basis of T1D-induced cardiac pathology was evaluated with various histological, biochemical and molecular techniques. Molecular findings from T1D models were also compared against changes from type 2 diabetic (T2D) mouse models (lean and obese). Finally, the role of RAS in exacerbating the T1D phenotype was assessed using a cardiac-specific angiotensinogen overexpressing mouse model. Results: The overall findings of this thesis are: 1. Although the extent of hyperglycaemia and increase in glycated haemoglobin (HbA1c) was less marked in female T1D in comparison to male T1D, diastolic dysfunction was evident in female T1D, but not in male T1D mice. 2. In males, diabetic hearts showed greater reperfusion recovery associated with reduced cardiac glycogen levels post-ischemia, suggesting better glycogen utilisation during ischemia, compared to male controls. In contrast, despite an earlier onset of ischemic contracture, the reperfusion recovery and glycogen levels were unchanged in female T1D hearts, compared to female control hearts. 3. GABARAPL1, a gene responsible for lysosomal breakdown of glycogen, was upregulated in T1D male hearts, whereas genes from conventional glycogen breakdown pathways (glycogen phosphorylase and glycogen debranching enzyme) were increased in female T1D. In addition, a pronounced increase in expression of genes from macro-autophagy pathway (protein bulk degradation) and apoptotic cell death pathway genes were observed in female T1D but not male T1D hearts. Interestingly, in lean and obese T2D mice, contrasting cardiac gene expression responses were observed in glycogen metabolic and macro-autophagy pathways. 4. With elevated cardiac AngII, T1D-induced cardiac functional and structural changes were exacerbated in males, but these changes were not apparent in females. Conclusion: Collectively, the novel findings in this thesis have contributed new knowledge to the literature on sex-specific attributes of diabetic cardiomyopathy. This study is the first demonstration that a less pronounced hyperglycaemic response in T1D female mice is associated with more marked functional cardiac pathology. This female vulnerability may be partially attributed to a preferential slower/inefficient processing of glycogen and heightened cell death pathology, evidenced from pronounced autophagic drive in female T1D mice. A sex-specific role for cardiac RAS in exacerbating the T1D phenotype has also been identified. The findings in this thesis support a case for sex-specific progression of diabetic cardiac pathology.