Heart failure is a complex clinical syndrome, associated with a significant burden of morbidity and mortality, including a high rate of rehospitalisation. Reducing the burden of rehospitalisation among patients with heart failure is a recognised priority for healthcare systems. However, in order to optimize outcomes, contemporary studies have emphasized the need for better understanding of the clinical heterogeneity of heart failure populations. Extending beyond the understanding of pathophysiological heterogeneity, the elucidation of sociodemographic factors may also reveal greater opportunities for targeted optimisation. Moreover, utilising new methods by which rehospitalisation is assessed may lead greater insights than would otherwise be captured by conventional means. This project aims to expand the understanding of rehospitalisation burden in evolving heart failure populations. It evaluates the adverse impact of cultural and linguistic diversity on rehospitalisation outcomes in patients with heart failure. A discussion of important methodological concepts into researching culturally and linguistically diverse (CALD) patient groups is carried out. In doing so, a novel operational approach to defining CALD patients is presented. This thesis also explores the measures of which rehospitalisation is evaluated in heart failure patients. It described the added value of analysing all recurrent hospital admissions (events), an approach which is very seldom performed in heart failure research. An assessment of several modelling techniques for evaluating rehospitalisation burden is performed, specifically in relation to heart failure type (i.e. heart failure with preserved vs reduced ejection fraction). In doing so, demonstrate the analysis of recurrent hospitalisations, compared to traditionally utilised first-event statistical approaches, may be a more informative and clinically relevant measure when evaluating the burden of heart failure rehospitalisation. As the prevalence of heart failure is rising and patients are becoming more diverse and complex, there is an increasing need to better characterise and understand these evolving heart failure populations. Further elucidating the heterogeneity of heart failure populations will help guide improvements to existing management approaches, as well as the direct the development of new targeted approaches.

Atherosclerotic coronary artery disease (CAD) is a major health burden worldwide and percutaneous coronary intervention (PCI) is an established treatment for this condition. Both PCI and invasive imaging techniques have evolved tremendously over the past few decades. Limitations of angiography were largely overcome, first by intra-vascular ultrasound, and then, optical coherence tomography (OCT), which is now recognized as the most sensitive and validated tool to examine the vessel lumen, plaque composition and stent-vessel wall interface. This thesis centers on randomized, OCT trials of coronary atherosclerosis and stents. In-vivo, comparative studies of drug eluting stents were conducted to directly observe their mechanical and healing characteristics. Results of these trials subsequently laid foundation for computational fluid dynamics (CFD) experiments and some illuminating observations were made around the effects of stent malapposition on intra coronary flow dynamics. It is the information of this kind that guides scientists to refine stent designs and clinicians, to improve procedural outcomes.

Background: Cardiometabolic disorders including atherosclerosis, diabetes, and metabolic syndrome (MetS) are major causes of mortality and morbidity. Nutrition is a lifestyle factor that plays an important role in the development of cardiometabolic disorders. Objective: The research project aimed to examine the association of omega-3 fatty acid intake and dietary quality with abdominal aortic calcification (AAC) among community-dwelling adults from a subset of the Melbourne Collaborative Cohort Study (MCCS) and whether protein intakes from different sources are differently associated with the incident diabetes and MetS in the whole population of the MCCS. Methods: Analyses regarding association of omega-3 fatty acid intakes and dietary quality with AAC were based on a subset (n=312) of the MCCS with diet assessed at baseline (1990-1994) and follow-up (2010-2011) and AAC measured using radiography and dual-energy x-ray absorptiometry (DXA) at follow-up (2010-2011). Analyses regarding protein intakes, diabetes and MetS were conducted using data from the MCCS of 41,514 participants. Dietary intakes, diabetes, and MetS were assessed at both baseline (1990-1994) and follow-up (2003-2007). A meta-analysis of the association between protein intakes and incident diabetes was also conducted. Results: Baseline energy-adjusted alpha linolenic acid (ALA) intake showed inverse associations with AAC severity by both radiography (odds ratio (OR) (95% CI) for tertiles 3 vs 1: 0.49 (0.23, 1.02)) and DXA (0.37 (0.16, 0.83)) in women. Women in the third tertile of total omega-3 fatty acid intake had lower AAC severity by radiography with OR (95% CI) 0.33 (0.16, 0.71) and DXA with OR (95% CI) 0.27 (0.12, 0.62) compared with those in the first tertile. Omega-3 fatty acid intake was not associated with AAC severity in men. Higher baseline dietary quality assessed by Alternate Healthy Eating Index-2010 was associated with lower AAC severity by both radiography (OR (95% CI) for tertiles 3 vs 1: 0.53 (0.29, 0.99)) and DXA (0.38 (0.20, 0.70)). Multivariate-adjusted ORs (95% CIs) of incident MetS for the highest compared with lowest quartile of percentage energy intake from total, animal and plant protein were 1.46 (1.01, 2.10), 1.67 (1.13, 2.48) and 0.60 (0.37, 0.97), respectively. The multivariate-adjusted OR (95% CI) for incident type 2 diabetes in the highest compared with the lowest quintile of animal protein intake as % energy was 1.29 (0.99, 1.67). In the meta-analysis of 11 prospective cohort studies, the pooled relative risks for type 2 diabetes comparing the highest with the lowest category of total, animal, and plant protein intakes were 1.09 (1.06, 1.13), 1.19 (1.11, 1.28), and 0.95 (0.89, 1.02), respectively. Conclusion: Higher intakes of ALA and total omega-3 fatty acids and high quality diets were associated with lower risk of AAC in adults. Higher plant protein and lower animal protein consumption, and substitution of animal protein, were associated with lower incidence of MetS and diabetes. This research provides novel evidence regarding the potential benefits of adherence to dietary guidelines especially high intakes of plant foods and low intakes animal foods in the prevention of cardiometabolic disorders.

The concept of screening entails detecting subclinical disease in the hope of altering the natural progression and thereby reducing long term mortality and morbidity. For a screening program to be effective it must be targeted at a disorder that is prevalent with accepted treatments and diagnostic tools. Coronary heart disease (CHD) is ideally positioned in this respect. There are multiple strategies available to diagnose asymptomatic CHD but the emerging field of coronary computer tomography angiography (CCTA) appears to have many of the required qualities. It is non-invasive, safe and has high sensitivity to diagnose CHD. The radiation dose has markedly decreased with evolving technology and the cost is comparable to current non-invasive cardiac investigation. An effective screening strategy for CHD maybe beneficial at all ages but applying it to the young may have a greater impact on society. The aim of this thesis was to highlight the burden of CHD in the young and to asses potential screening strategies to detect asymptomatic disease. In particular the potential role of CCTA as a screening tool has been investigated. Chapters 3 and 5 discusses the results of reviews looking at the literature focusing on myocardial infarction (MI) in the young and screening for asymptomatic CHD in the young respectively. Chapter 4 discusses the findings of a study that demonstrated the prevalence of MI in the young (defined as ≤55 years) in a low socioeconomic Australian urban setting was approximately 32%. The younger patients were more likely to be male, have a family history of premature CHD and be current smokers compared to the older cohort. In general this prevalence is higher than what is reported in the literature. Chapter 6 highlights the findings of a study that used CCTA to demonstrate the prevalence of asymptomatic CHD in the siblings of young MI patients. The participants of the study were aged 30-55 and 30-60 years if males and females respectively. Obstructive CHD (defined as ≥50% stenosis of at least one epicardial coronary artery) was demonstrated in 18% of participants. All of those with obstructive CHD were either current or ex-smokers. No statistically significant association was found between traditional cardiovascular risk scores and obstructive CHD by CCTA. A similar study conducted for this research project at another healthcare centre found the prevalence of asymptomatic CHD via screening CCTA in an all comer population was 18%. Males and those over the age of 55 years were more likely to have obstructive CHD. These results are discussed in chapter 7. Compared to the published literature the prevalence of obstructive CHD by CCTA demonstrated in these two studies is higher. Screening strategies have been demonstrated to alter modifiable risks in those who participate. Chapter 8 discusses the findings of a study that demonstrated 60% of the participants of a screening program for CHD using CCTA either stopped or reduced smoking one month after undergoing CCTA. This change was largely sustained at 12 months with 55% either stopping or reducing smoking. This rate appears to be high compared to more traditional methods such as nicotine replacement therapy. For CCTA to be accepted as an effective screening modality for CHD the risks of radiation exposure need to be as low as possible. Chapter 9 discusses the results of a study comparing the radiation doses associated with three new generation CT scanners when used to perform CCTA; the Siemens Somatom Definition Flash CT scanner, the GE Revolution CT scanner and the Toshiba Aquilion ONE ViSION CT scanner. The study found all three scanners exposed subjects to below a median dose of 5 mSv, fulfilling the Australian National Health and Medical Research Council guidelines of <5 mSv for research purpose. In addition it found the Siemens Somatom Definition Flash CT scanner exposed subjects to the lowest median radiation of 1.76 mSv. This appears to suggest the new generation CT scanners expose subjects to low enough radiation for CCTA to be a viable screening modality. In summary this thesis demonstrates a high prevalence of both symptomatic CHD (in the form of MI) and asymptomatic CHD in the young. It highlights the importance of family history of premature CHD and smoking as important risk factors for the presence of asymptomatic CHD in the young as detected by CCTA. In addition asymptomatic CHD as detected by CCTA has been shown to be more prevalent in males and those over the age of 55 years. Participation in a screening program for CHD is also shown to be an effective and sustained means of modifying smoking habits. Finally the very low radiation exposure that can be achieved with the use of the latest generation of CT scanners is illustrated. In combination these findings support further research investigating the use of CCTA in a screening strategy for CHD.

Thyroid-associated orbitopathy (TO) is an autoimmune-mediated orbital inflammation that affects 25% of patients with Graves’ disease. The close temporal relationship between onset of Graves’ disease and TO suggests they share a common aetiology. While the complex inheritance of Graves’ disease is better characterized, little is known of the genetic susceptibility in TO. Multiple environmental factors such as smoking, male, older age are known risk factors for development of TO, however the extent of gene-environmental interaction remains largely unknown. The molecular mechanisms driving the development of TO is incompletely understood, hence targeted treatment options for TO remained limited. This thesis is undertaken to test the hypothesis that there is genetic susceptibility that predispose to development of TO. The research project initially examined exogenous risk factors associated with TO in a large Australian cohort with Graves’ disease, in order to identify environmental factors important for subsequent covariates adjustment when analyzing genetic findings. The risk factors association study found smoking, older age and longer duration of Graves’ disease correlated positively with TO, and secondarily there was relative selenium deficiency in TO cases compared to Graves’ disease patients without eye involvement. A genome-wide association study using deoxyribonucleic acid pooling approach and high-throughout array platform were used to discover gene variants associated with thyroid-associated orbitopathy in a case-control study design. The genetic findings were followed by a second stage individual genotyping targeting fewer markers to validate the genetics variants identified through genome-wide association study in the discovery cohort and independent replication study cohort. MACROD2, a novel gene that encodes an eraser of mono-ADP-ribosylation, possibly has a role in nuclear factor κβ signaling, showed evidence of association with TO in genome-wide association study and also in validation genotyping. A secondary aim of the thesis is to determine differentially expressed genes a priori in active TO orbital adipose tissue using microarray to explore molecular mechanisms of TO and to correlate gene expression findings with the genetic study. The study found TIMD4, DEFA1, DEFA1B, and DEFA 3 were over-expressed in active TO compared with inactive TO suggesting a pathogenic role of the innate immune response in TO. Active TO was marked by up-regulation of multiple genes involved in cell-mediated, innate and inflammatory responses with concurrent enhancement of orbital adipogenesis. For the first time, epigenetic factors was implicated in the pathogenesis of TO. However MACROD2 were not differentially expressed in active TO when compared with either inactive TO or normal control. Overall the findings from this thesis further our understanding on the genetic and environmental risk factors involved in thyroid-associated orbitopathy and give new insights into the underlying complex molecular mechanisms. The novel insights into candidate molecules and pathways can be explored to develop alternative treatment strategies for TO.

Colorectal cancer (CRC) is a global health problem. It is the second leading cause of cancer related death worldwide and in Australia. Around 25% of CRC will experience recurrences postoperatively in the absence of lymph node metastasis and liver metastasis. We need to develop better diagnostic tools and more effective therapeutics. This will come through a thorough understanding of the molecular events in CRC progression. Erythropoietin producing hepatoma (Eph) receptors are the largest family of the receptor tyrosine kinases (RTK). They are divided into two subfamilies, A and B, Together with their cognate ligands, they play integral role in different body organs during embryo development and in adulthood. EphB receptors are considered as key coordinator of proliferation and migration of the intestinal stem cells (ISCs). They are Wnt signalling target genes. They are distributed in a complimentary pattern with their ligands, ephrinB1 and ephrinB2, along the intestinal crypt axis. Adenomatous polyposis coli gene mutation is considered as the initiating step in CRC development. It leads to Wnt signalling mutation and adenoma-carcinoma progression. Despite activation of Wnt signalling pathway, EphB receptors are inactivated during adenoma-carcinoma progression EphB1 and EphB2 receptors function as tumour suppressors in CRC, whereas for the EphB4 some studies found that EphB4 receptor is underexpressed whilst others found it overexpressed with the progression of the disease. It has also been suggested EphB4 expression is up-regulated as EphB2 expression is extinguished. Using immunohistochemistry we investigated pattern of expression of the three receptors together, EphB1, EphB2 and EphB4, across three stages of CRC, SII, SIII, SIV, in lymph node metastasis, and in liver metastasis, in a total population of 148 CRC patients. We found that: The three receptors are co-ordinately under-expressed in the primary CRC. The receptors are further under-expressed in the liver metastasis. The EphB1 receptor is under-expressed in most undifferentiated CRC and in liver metastasis. The EphB4 receptors follow the same down-regulation pattern as the other two in both primary tumours and metastases. There is no evidence that EphB2 –negative tumours express EphB4. The importance of this study of EphB receptors expression pattern in CRC and in liver metastasis is that it gives a general picture about the status of the three receptors together across different stages of CRC, in lymph node metastasis, and in liver metastasis of the same patients. Understanding the pattern of expression of three receptors together in CRC, highlights the significance of these receptors in CRC, and in long term revolutionizes current anticancer drug treatment of the disease.

Introduction: Uncomplicated pregnancies represent a hypercoaguable state. However, placental thrombosis is rare in these pregnancies which suggests that thrombin generation must be tightly regulated. In contrast, pregnancy disorders such as pre-eclampsia not only demonstrate an exaggerated increase in procoagulant activity which may contribute to the thrombotic lesions observed in the uteroplacental circulation of these pregnancies, but there is evidence of substantial cell growth, differentiation and angiogenic functional defects. Proteoglycans are abundantly expressed within the placenta compared to other human tissues. Particularly of interest are heparan sulphate proteoglycans which have important anticoagulant, anti-inflammatory and angiogenic properties. Hypothesis: Altered abundance, structure or function of placental heparan sulphate proteoglycans contributes to the development of pre-eclampsia by: increasing placental thrombin generation, interfering with cellular growth, differentiation, disrupting normal angiogenesis and altering growth factor interactions. Aims : 1) To determine the mRNA expression, protein abundance and cellular localisation of placental heparan sulphate proteoglycans from pregnancies complicated by pre-eclampsia and compare these to gestation matched controls. 2) To investigate the functional consequence of reduced HSPGs in placental cells 3) To determine the differences in the abundance and structure of heparan sulphate proteoglycans and heparan sulphate GAGs from placentae obtained from pregnancies complicated by pre-eclampsia and compare these to gestation matched controls. Methods: 1) The mRNA, protein abundance and cellular localisation of placental heparan sulphate proteoglycans was determined using real-time PCR, western immunoblotting and immunofluorescence, respectively. 2) To investigate the functions of reduced heparan sulphate proteoglycans, a cell culture model using short interference RNA was used. Cellular growth will be assessed using the xCELLigence system, thrombin generation using the calibrated automated thrombogram system and angiogenesis will be determined using a matrigel based assay. Cellular differentiation and apoptosis will be determined using real-time PCR. Growth factor signalling will be determined using a real-time PCR growth factor array. 3) To isolate proteoglycans from placenta, anion exchange chromatography will be utilised. Enzymatic digestion will be used to isolate the glycosaminoglycans, and enzyme-linked immunosorbant assays will be undertaken to determine the abundance of PGs and GAGs. Results: The mRNA expression of heparan sulphate proteoglycans, Syndecan 1, Syndecan 2, Glypican 1 and Glypican 3 are significantly reduced in the placentae of women whose pregnancies are complicated by pre-eclampsia. A cell culture model was utilised to determine the functions of heparan sulphate proteoglycans in the placenta. Successful downregulation of heparan sulphate proteoglycans was achieved in the cell lines using short interference RNA treatment, and a number of functions were significantly altered as a result. The downregulation of Syndecan 1, Glypican 1 and Glypican 3 resulted in significant alterations in the downstream growth factor targets. Reduced Syndecan 2 expression resulted in a significant reduction in the thrombin generation potential of endothelial cells. Investigation into the abundance and structure of glycosaminoglycans within the placenta, demonstrated heparan sulphate glycosaminoglycans to be significantly reduced in pregnancies complicated with pre-eclampsia compared to gestation matched controls. Conclusion: The reduction in heparan sulphate proteoglycans expression observed in pregnancies complicated with pre-eclampsia may be responsible for the altered growth factor interaction commonly observed in preeclamptic pregnancies. This study has provided us with a greater understanding of the biological role of heparan sulphate proteoglycans within the human placenta and its potential implications in the pathogenesis of pre-eclampsia.

Background: Vitamin D deficiency is a widespread global problem of particular importance for people with HIV. Vitamin D is an important hormone for endocrine and extra-endocrine functions, including modulating the immune system. Vitamin D deficiency is associated with reduced bone mineral density and infectious diseases, both common in people with HIV. Maintaining optimal vitamin D levels may help to reduce the risk of developing these diseases. The overall research questions for this thesis are: Firstly, which factors influence vitamin D metabolites, and secondly, what effect does vitamin D status have on bone and immunological outcomes, in people living with HIV infection? Method: To answer these questions, five studies were performed: 1. Cross-sectional comparison of 25(OH)D levels in HIV-infected and uninfected individuals in southern Australia. 2. Cross-sectional analysis of the determinants of 25(OH)D in people with HIV in Queensland and Melbourne, Australia. 3. Cross-sectional analysis of parathyroid hormone levels in antiretroviral-treated individuals. 4. Cross-sectional analysis of the effect of 25(OH)D on bone mineral density in antiretroviral-treated individuals. 5. Longitudinal analysis of CD4 cell count trajectory in antiretroviral-untreated individuals. Results: 1. People with HIV were more likely to be vitamin D deficient when compared with people without HIV in southern Australia. 2. Determinants of 25(OH)D levels: a. UV index and location are important determinants of 25(OH)D levels in people with HIV in Australia. b. Antiretroviral therapy impacts 25(OH)D levels: i. Efavirenz is associated with lower 25(OH)D levels, particularly in those with dyslipidaemia. ii. Protease inhibitors are associated with higher 25(OH)D levels. 3. The effect of tenofovir on parathyroid hormone levels depends on sex and ethnicity; parathyroid hormone levels were higher in non-white males using tenofovir. 4. Influence of ART and 25(OH)D on 1,25(OH)2D levels and bone mineral density: a. People using tenofovir have higher 1,25(OH)2D levels and people using protease inhibitors have lower 1,25(OH)2D levels. b. There is an interaction between tenofovir and 25(OH)D status on 1,25(OH)2D levels and bone mineral density. Vitamin D deficiency increases the odds of low bone mineral density only in those not using tenofovir. 5. Vitamin D deficiency may reduce the time to CD4 decline to <350 cells/μL in people living with HIV and untreated with antiretroviral therapy. Conclusions: Traditional risk factors for vitamin D deficiency and bone mineral density appear to be more important than HIV-related factors in people with HIV. In spite of this, antiretroviral therapy clearly alters vitamin D and bone metabolism. Understanding the mechanisms behind these alterations may assist in modifying the negative effects of vitamin D deficiency.

Background: Lower serum 25-hydroxyvitamin D (25OHD) levels have been consistently associated with increased type 2 diabetes mellitus (T2DM) prevalence and incidence in systematic reviews and meta-analyses of observational studies, however this association has not consistently been replicated in vitamin D supplementation trials. This disparity may be due to a number of different factors: lack of power in the trials due to small sample size, insufficient duration of dosing, baseline vitamin D or glycaemic status differing between studies, low supplementation compliance or insufficient vitamin D dose. Alternatively, lower 25OHD levels may be a product, rather than a cause of ill-health, or they may share pathology earlier in life or in disease progression so that supplementing with vitamin D in adulthood has no effect on disease outcome. This body of work presents another explanation: given that sun exposure is the most influential contributor to serum 25OHD levels, observational studies may be reporting an effect of sun exposure, rather than vitamin D, on T2DM. Therefore vitamin D supplementation trials may be failing to capture any additional benefits of sun exposure through non-vitamin D pathways. The aim of this body of work was to investigate the possible association between sun exposure and T2DM endpoints reported in scientific literature as well as in an original analysis. A major objective of the original analysis was to determine whether or not any association found between sun exposure and T2DM incidence was through non-vitamin D pathways. Methods: Following a literature review, a systematic review of observational studies reporting on associations between sun exposure variables and T2DM-related endpoints was conducted. The potential of an association between sun exposure- measured using ambient ultraviolet radiation (UVR), and five-year cumulative incidence of T2DM was explored using a prospective, national diabetes cohort (AusDiab). A causal mediation analysis was undertaken to explore whether or not there were effects of ambient UVR on cumulative T2DM incidence, via non-vitamin D pathways. Results: The systematic review revealed that high-level evidence for an association between sun exposure and T2DM-related outcomes was lacking. There was moderate-level evidence for greater sun exposure reducing T2DM incidence. The opposite was found in the original analysis using the AusDiab cohort: ambient UVR was associated with increased T2DM incidence (OR=1.17, 95% CI: 1.01-1.36, p=0.04). This association was independent of an effect of age, sex, body mass index, physical activity, ethnicity, smoking status and serum 25OHD levels, but was likely to be confounded by area-level determinants of health due to the nature of the exposure variable. The major limitations of this work were that the sun exposure measures were suboptimal. Time-of-year measures were the most common sun exposure variables contained in the systematic review, and ambient UVR at the site of participant recruitment was the proxy for sun exposure in the original analysis. Conclusion: There is likely to be a complex relationship between sun exposure and T2DM. The direction of the association between sun exposure and T2DM incidence, as well as delineation of the mechanistic pathways through which this association may exist, are yet to be confirmed. Future studies are encouraged to use person-level sun exposure measurements, and findings from such studies may influence sun protection policy.

Background: Prosthetic joint infection (PJI) is a serious infection that is difficult to cure and is associated with significant morbidity. The pathogenesis of PJI involves bacteria growing in biofilm adherent to the prosthesis surface, making them resistant to eradication with standard antibiotics. Recent evidence demonstrates successful treatment of early PJI with surgical debridement and retention of the prosthesis (DAR) and the biofilm-active antibiotic combination of rifampicin and a fluoroquinolone for staphylococcal infections. However, there are few studies investigating appropriate antibiotics to use in combination with rifampicin for PJI caused by staphylococci resistant to fluoroquinolones or which antibiotics to use for organisms other than staphylococci. Little is known about functional outcomes, quality of life (QOL) or complications after treatment of PJI. The aim of this thesis is to provide further evidence to help guide management in these areas. Methods: This thesis synthesizes three of my recent studies published in peer-reviewed journals and one study presented at a national scientific meeting. In the first study, outcomes were analysed for consecutive patients with staphylococcal PJI treated with DAR and a combination of rifampicin and fusidic acid. The second examined consecutive patients with a Gram-negative bacillus PJI treated with DAR and ciprofloxacin-based regimens. In the third study, consecutive patients treated for hip PJI with DAR and biofilm-active antibiotics were matched to controls that had hip arthroplasty with no infection, and their function, QOL and complications compared. In the fourth study, a large prospective hip and knee arthroplasty cohort was analysed to determine if PJI treated with DAR and biofilm-active antibiotics was predictive of adverse QOL outcomes. Results: Of 20 patients with staphylococcal PJI, treatment failure occurred in two patients. The cumulative risk of treatment failure after 1 year was 11.76% (95% CI 3.08–39.40%). Ten of 11 patients with infections involving methicillin-resistant Staphylococcus aureus had successful outcomes. Of 17 patients with Gram-negative bacillus PJI, treatment failure occurred in two patients. In only one patient was a relapsed Gram-negative infection responsible for the failure and this patient had not been treated with ciprofloxacin. The 2-year survival rate free of treatment failure was 94% (95% CI, 63–99%). In 19 hip PJI cases there was significant improvement 12-months post-arthroplasty in function according to Harris Hip Score and QOL according to the 12-item Short Form Health Survey Physical Component Summary. There was no significant difference in the improvement between 76 controls and the 19 cases. Medical complications occurred more frequently in cases than controls but the rate of surgical complications was the same. Of 2134 patients in a hip and knee arthroplasty cohort there were 37 patients with PJI treated with DAR and biofilm-active antibiotics. On multivariate logistic analysis, PJI treated this way was not predictive of adverse QOL outcomes according to SF-12 scores, however pre-arthroplasty SF-12, female gender, knee arthroplasty and a comorbidity index were. Conclusions: DAR in combination with rifampicin and fusidic acid is effective and should be considered for patients with early staphylococcal PJI, including those with infections involving fluoroquinolone-resistant organisms. DAR in combination with ciprofloxacin is effective for patients with early Gram-negative bacillus prosthetic joint infection. Treatment of PJI with DAR and biofilm-active antibiotics was well tolerated and results in good improvements in function and QOL.

TCD8+ of the adaptive immune system play critical roles in the host defence against viruses and other pathogens through elimination of infected host cells. After infection, TCD8+ proliferate and adopt effector functions following recognition of specific antigenic‐peptides in the groove of a MHC class I molecule expressed by antigen presenting cells (APC). Professional APCs such as dendritic cells (DCs) are specialized for the priming and activation of naïve TCD8+. DCs are the key cell type responsible for T cell priming. They can either be directly infected and present viral antigens (direct‐priming) or phagocytose infected cell debris and process and present phagocytosed antigens to the specific TCD8+ (cross‐priming). Little is known about the actual contribution of direct versus cross‐priming during an immune response against viral infections. Understanding how DCs regulate TCD8+ responses is central for our ability to favourably manipulate the immune system as well as develop effective targeting strategies for optimal anti‐viral and antitumoral vaccination. To determine the contributions of direct versus cross‐priming to the clearance of an in vivo viral infection, we generated a Cre‐indicator transgenic mouse model utilising cutaneous HSV‐Cre infection to conditionally trigger model antigen expression in infected cells. This was expected to enable us to discriminate between virally infected and hence direct‐presenting DCs from uninfected and cross‐presenting ones in vivo for the first time. Unexpectedly, all generated transgenic mouse lines showed various levels of tolerance towards our model antigen due to undesired protein leakiness at steady state. However, TCD8+ in these neo‐transgene expresser transgenic mice possessed antigen ignorant properties and were non‐specifically activated following acute viral infection and the introduction of other innate immune cell ligands. Therefore, although these generated transgenic mice could not be used for their initial study purpose, their varying expression levels of model antigen make them an excellent model for studying peripheral tolerance induction and its maintenance. Cross‐priming is especially important for anti‐tumour immunity as tumour cells, although carrying tumour associated antigens, do not activate naïve TCD8+ efficiently due to an absence of co‐stimulatory molecules. Remarkably, our group has recently shown that influenza A virus (IAV) infection of allogeneic cells led to tumour protection due to enhanced cross‐priming of TCD8+ specific to cellular antigen. We have previously demonstrated that this enhancement was partially mediated through TLR7 sensing and entirely dependent on MyD88 and IFN signalling pathways, yet independent of the IL‐1β‐inflammasome. To further increase our understanding of cross‐priming enhancement mechanisms found in our system, we have additionally investigated the involvement of other immunological mechanisms in this thesis. Here, we show that IAV enhanced crosspriming is independent from the IL‐18‐inflammasome signalling pathway but that TCD4+ helper play a surprisingly important role for optimal enhancement. Also, through investigations using Batf3‐/‐ mice, we not only confirm the specificity of CD8α+ and CD103+ DC subsets for cross‐presenting, but also demonstrated that there are two types of cross‐priming outcomes: a baseline cross‐priming that is innate signalling independent and an innate immune signal enhanced crosspriming pathway. Interestingly, both types of cross‐priming events were abolished in Batf3‐/‐ mice. This knowledge will be useful to aid future efforts to develop more robust cancer vaccines. Finally, efficient antigen processing and presentation of the TCD8+ epitope is a central factor that determines the extent of specific T‐cell responses. Our group has identified the most immunodominant T‐cell response in the Balb/c mice after IAV infection. Interestingly, this novel epitope is encoded by the intronic region of the non‐structural protein mRNA. To dissect the mechanism of such epitope translation, as being either through spliced mRNA translation or generation using an alternative open reading frame (AltORF), we disabled splicing mechanisms and discovered that generation of the novel epitope occurred through AltORF translation. We will focus on the identification of the exact mechanism(s) underlying the efficient generation of this novel epitope as such mechanisms may provide a great opportunity for developing more efficient IAV T‐cell based vaccine strategies.

High calcium intake can prevent age-related bone loss; however, its efficacy in fracture prevention is not clear. Some recent studies have associated calcium supplementation with adverse cardiovascular events (CVE). Scientific evidence on the long term effects of dietary calcium intake (DCI) and fracture or CVD risk lacks consistency.