Medicine (St Vincent's) - Theses
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ItemActivation of islet inflammation by cytokine signalling in pancreatic beta cells: understanding the role of protein tyrosine phosphatases( 2018)Type 1 diabetes is characterised by the autoimmune destruction of insulin producing beta-cells in the islets of Langerhans of the pancreas. Immune cells release pro-inflammatory cytokines such as interferon-g (IFN-g), tumour necrosis factor-a (TNF-a) and interleukin- 1b (IL-1b) into the islet microenvironment which activate phosphorylation cascades and gene expression in beta-cells that increase their susceptibility to autoimmune attack and destruction. Protein tyrosine phosphatases (PTPs) regulate phosphorylation based signalling pathways and have previously been shown to negatively regulate IFN-g induced cell death of beta-cells in vitro. We previously showed that during immune infiltration to the islet PTPs, including PTPN1 and PTPN6, are rendered catalytically inactive through oxidation resulting in loss of signal regulation. The overall aim of this thesis is to observe if antioxidant treatment can reduce autoimmune development in the NOD/Lt mouse through reduction of oxidised PTPs and dissect the role of PTPN1 and PTPN6 in the regulation of cytotoxic signalling events in the NIT-1 beta-cell line and isolated NODPI islets in vitro. Chapter 3 studies the effect of the mitochondrial targeted antioxidant mito-TEMPO on insulitis and diabetes development in the NOD/Lt mouse. Delivery of mito-TEMPO through drinking water reduced levels of oxidised PTPs in the pancreas of NOD/Lt mice but had no effect on the development of insulitis, activity or number of CD8+ and CD4+ T- cells in the periphery in NOD/Lt mice or diabetes development in a diabetes transfer model. Chapter 4 describes the regulation of cytokine signalling in NIT-1 cells and isolated NODPI islets by PTPN1. Inhibition of PTPN1 activity reduced IFN-g, TNF-a and IL-1b induced death of NIT-1 cells in vitro. Activation of the IFN-g, TNF-a and IL-1b signalling pathways and downstream transcription of pro-inflammatory gene signatures associated with autoimmune diabetes were also reduced with PTPN1 inactivation. Furthermore, PTPN1 inhibition reduced IFN-g induced MHC-I expression on the surface of NODPI beta-cells and reduced the ability of autoreactive NOD8.3 CD8+ T-cells to destroy isolated NOD/Lt islets. These studies showed that PTPN1 is a positive regulator of cytotoxic signalling in NIT-1 cells and NODPI islets and promotes immune cell mediated death, suggesting it may be a potential therapeutic target for type 1 diabetes. Chapters 5&6 study the role of PTPN6 in cytokine signalling regulation in NIT-1 cells. PTPN6 inhibition was found to enhance TNF-a induced NIT-1 cell death independent of IFN-g and IL-1b in vitro. TNF-a induced JNK signalling was enhanced with PTPN6 inhibition which resulted in reduced anti-apoptotic BCL-2 protein expression and enhanced caspase-3 cleavage. Pan-caspase inhibition prevented TNF-a induced cell death suggesting that cells were dying through apoptosis. TNF-a induced cell death was also prevented with RIPK1 inhibition which prevented enhanced caspase-8 cleavage in PTPN6 deficient cells. Collectively these studies showed that PTPN6 negatively regulates TNF-a induced intrinsic and extrinsic apoptosis of beta-cells in vitro. Overall, the data indicate that PTPs play a nonredundant role in the regulation of cytotoxic signalling in NIT-1 cells and NODPI islets. This finding is consistent with results in other disease pathologies. The results provide a mechanistic insight into how PTPN1 and PTPN6 have opposite roles in regulating cytokine signalling, highlighting how PTPN1 antagonism and PTPN6 agonism may prove beneficial in reducing beta-cell death in vitro. Whether these results are directly translatable into in vivo models of autoimmune diabetes remains undetermined. The use of PTPN1 inhibitors or PTPN6 agonists currently under development would allow direct translation of these results.
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ItemPredicting response to anti-TNF therapy in Crohn's disease: clinical and metabolic studies( 2018)Crohn’s disease is a chronic, disabling inflammatory condition that affects the gastrointestinal tract that is associated with significant morbidity. Up to 80 percent of patients require surgery at some point in their lives. A third of patients require surgery within five year of diagnosis even in the era of biologic therapy. Ant- Tumour Necrosis Factor alpha (Anti-TNF) therapies form the backbone of drug therapy in patients with moderate to severe Crohn’s disease and remain the most effective biologic drugs available. There are currently three classes of biological drugs available on the Pharmaceutical Benefits Scheme (PBS) for Australians with many more under development. Up to thirty percent of patients fail to have an adequate response to anti-TNF therapy and loss of response occurs at a rate of ten percent per year. There are at present no highly sensitive biomarkers of response to anti-TNF therapies. Without biomarkers to assist with selecting the most effective biologic for a particular patient, there is a risk of exposing patients to ineffective drug therapies with unnecessary side-effects and costs. This thesis comprises a range of clinical and scientific studies that seek to identify predictors of loss of response to anti-TNF therapies in Crohn’s disease. Two types of loss of response have been identified: Primary nonresponse (PNR), is defined as a lack of response to the initial drug therapy as determined at 12 weeks post induction, and Secondary Loss of Response (SLOR) is defined as the loss of response after the patient has had an initial response to the drug. Based on the clinical observation that patients with altered body composition and those with strictures have a variable to response to anti-TNF therapy, we sought to identify whether there were specific clinical, endoscopic, histologic and biochemical parameters that correlated with response to anti-TNF therapy amongst patients from whom longitudinal body composition and endoscopic data had been collected. A prospective cohort study of patients who had been newly started on anti-TNF therapies was also undertaken. In this cohort samples of urine, blood and faeces were taken prior to anti-TNF therapy delivery (baseline) and then at 3 monthly intervals thereafter. A combination of bioinformatic analyses and novel techniques evaluating the metabolome were applied to the cohort, in order to find clinical factors and biomarkers that might correlate with therapeutic response to anti-TNF therapy. This series of studies presented in this thesis on clinical and metabolic predictors of outcomes in patients receiving anti-TNF therapy for Crohn’s disease reveals new insights into the pathophysiology of Crohn’s disease and has identified biomarkers for the prediction of therapeutic response – thereby helping to come a step closer towards the ultimate goal of precision medicine.
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ItemInvestigating the natural history, serologic and genetic associations with disease outcomes, disability and cost of care in inflammatory bowel disease( 2018)Inflammatory bowel disease (IBD) is an emerging global health condition. In the second half of the 20th century, the incidence of Crohn’s disease (CD) and ulcerative colitis (UC) increased dramatically in the Western world. In the last two decades a corresponding rise has been observed in South East Asia(1) and Australia has one of the highest incidences of IBD in the world.(2) At this point in time there is no cure for IBD, and individuals with the condition usually receive lifelong treatment and a significant proportion will need intestinal surgery.(3) Early aggressive treatment with immunomodulator (IM) and biologic therapy can improve steroid free remission rates, reduce the need for surgery and hospitalisation, and reduce the rate of progression to a complex disease phenotype. (4-9) Medicare-subsidised biologic therapy for CD was first made available in Australia in late 2007, while infliximab was not available for use in UC in Australia until 2014. Although the rate of biologic prescription is steadily rising, the impact of increasing biologic use on the natural history and cost of care in the real-world is not clear. Furthermore, it is difficult to identify patients who would benefit from early biologic therapy, and it is uncertain if advancements in treatment are translating into improved patient function and reduced disability. Previous Australian researchers established a prospective community-based IBD inception registry in Barwon, Victoria in 2007 and collected cases to June 2013.(2, 10, 11) The initial aim of the registry was to establish the incidence and prevalence of IBD in Australia, to identify environmental exposures associated with disease aetiology, and to assess the course of disease and healthcare costs in the first 12 months from diagnosis. The aims of the work presented in this thesis were to use the community-based cohort from Barwon, in addition to a newly constructed tertiary IBD center hospital-cohort to determine the natural history of IBD in the first five years of disease, to identify clinical, serologic and genetic associations with disease course and outcomes in IBD, to determine the health care cost of IBD within the first four years of diagnosis from a health care system perspective and identify early clinical predictors of high cost of care, and to describe the burden of disability and identify determinants of disability in IBD. Incident cases diagnosed from July 2013 to April 2016 were identified through a multifaceted approach from Barwon and St Vincent’s Hospital Melbourne to ensure complete capture. Cases were enrolled into the IBD registry that was used as a basis to collect outcome data on disease course, healthcare costs, and disability. Serological analysis was performed for anti-Saccharomyces cerevisiae (ASCA) IgG and IgA antibodies in CD patients, as well as perinuclear anti-Neutrophil Cytoplasmic antibodies (p-ANCA) in CD and UC patients. DNA analysis was performed using 26 single nucleotide polymorphisms (SNPs) that were identified from the literature as potentially having a prognostic role. A total of 408 patients [243(59.6%) CD and 165(40.4%) UC] were included in the analysis of natural history, as well as the cost analysis. 56% of CD and 62% of UC patients were from the Community cohort. 20.8% of CD patients needed intestinal surgery at 5 years which is lower than older population-based studies from the pre-biologic era. 28.1% of CD patients progressed to complex disease phenotype and 8.9% of UC patients underwent colectomy by 5 years which is similar to population-based studies from the pre-biologic era. There was no significant difference in disease outcomes in CD or UC between the hospital and community cohorts. 38% of CD patients and 15% of UC patients were exposed to biologic therapy. Anti-Saccharomyces cerevisiae positivity was associated with progression to complex disease and surgery in CD. Anti-Neutrophil Cytoplasmic Antibody positivity was associated with the need for biologic therapy in UC. There were no significant genetic associations with disease outcomes in CD or UC. In both CD and UC, medication costs accounted for over 50% of total cost, of which biologics accounted for the majority of medication cost. In CD, the mean medication cost per patient per year increased by $3,144 between years 1 to 4 (<0.01), which was offset by a fall in medical and surgical hospitalisation costs of $1,547 (p <0.01) and $3,321 (p <0.01) respectively leading to a reduction in total cost of care over 4 years. There were no significant changes in cost over time in UC. In both CD and UC, surgery, biologic use, and hospitalisation use were associated with higher cost of care. Disability was measured using the newly developed and validated IBD Disability Index (IBD-DI). 334 patients completed the IBD-DI. 56% of all patients had no or only mild disability while 32% of patients in remission had moderate to severe disability. Patients who had previous surgery or current biologic therapy but were currently in remission were more likely to be disabled. Factors associated with greater disability on multivariate regression analysis were active disease, female gender, socioeconomic disadvantage, and biologic use. In summary, this thesis has shown a numerically lower rate of intestinal surgery in CD compared with studies from the true pre-biologic era. Colectomy rates in UC were stable compared with historical controls however biologic exposure rates in UC were modest. We have demonstrated for the first time the nature of changes in IBD-associated healthcare costs over 4 years. After the first year of diagnosis, the use of IM and biologics rose significantly in CD, while surgery and hospitalisation rates declined. Over this time, healthcare cost profiles shifted in a similar fashion, in that as biologic usage rates increased, so did the cost of medications. As hospitalisation and surgical rates fell, so too did their corresponding costs. Disability in IBD is prevalent, and although active disease is an important contributor to disability, a significant proportion of patients were disabled despite being in remission. This novel finding indicates that other factors such as mental health issues may be contributing to disability in IBD.
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ItemIntegration of advanced echocardiographic metrics and biomarkers to the fields of heart failure and exercise physiology in the era of precision medicine( 2018)Exercise testing is widely used for risk stratification of ischaemic heart disease, however, its application in other cardiovascular conditions, specifically heart failure and other disease states is less defined. Heart failure is a leading cause of mortality and morbidity. Despite advances in medical therapy, its prevalence is expected to increase. Recent developments in cardiac echocardiography, exercise testing and reducing costs of biomarker assays provides the opportunity to improve identification and prognostication of the heart failure syndrome. These principles may also prove useful in the discrimination of other disease states, in which a diagnostic biomarker is lacking. The principle aim of this thesis was to identify novel echocardiographic and non-invasive cardiopulmonary parameters during exercise that provide incremental prognostic value to established risk markers in heart failure. An exploratory sub aim was to investigate the ability of a personalised exercise test to discriminated disease states outside the cardiovascular system. Cohorts of heart failure patients undergoing assessment with echocardiography and cardiopulmonary exercise testing were selected to assess the incremental value of novel parameters. Recently, deformation imaging, also known as strain and quantitative measures of the right heart have shown prognostic value in patients with heart failure. These measures have rarely been analysed with cardiopulmonary exercise testing, therefore, their prognostic value in outcome models and contemporary risk scores were assessed. Dynamic changes in selected parameters were evaluated with regard to risk stratification potential. Finally, as biomarker assays are becoming increasingly used to identify the heart failure syndrome, their contribution to discrimination of disease and risk modeling when performed in conjunction with an exercise test was evaluated. In summary the main findings of this thesis were: 1. Echocardiographic contractile reserve and cardiopulmonary exercise testing parameters provide complementary information, therefore, in combination provide an opportunity to improve prognostication in heart failure. 2. Right heart metrics specifically, right atrial volume and exercise performance add value to previously validated heart failure risk scores in dilated cardiomyopathy. 3. The combination of exercise performance, left ventricular strain and left atrial volume presents a simple model for predicting heart failure outcomes in hypertrophic cardiomyopathy. 4. Percent predicted values of maximal oxygen consumption derived from the Fitness Registry and the Importance of Exercise National Database (FRIEND) registry equation appear to accurately predict heart failure outcomes. 5. Cytokine profiling post exercise appears to have greater discriminatory power than at rest when used to identify patients with myalgic encephalomyelitis / chronic fatigue syndrome; Echocardiographic parameters, have limited value. 6. The integration of cardiac biomarkers, cytokine profiling, exercise performance and cardiac imaging in a personalised exercise test is achievable and has the potential to improve risk profiling. In conclusion, this thesis demonstrated the opportunity to further refine risk stratification in heart failure using novel images techniques, specifically deformation imaging, right heart metrics and exercise performance. The use of serum samples post a bout of exercise may provide an opportunity to further refine diagnostics in the field. Future studies should address biological variability of serum biomarkers and investigate their value when integrated with established markers of risk.
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ItemInflammation in atherosclerosis: exploring novel risk factors, prognostic markers and role of colchicine as an anti-inflammatory therapy in acute coronary syndromes( 2018)Inflammation plays a crucial role in the pathophysiology of atherosclerosis and clinical manifestation of acute coronary syndromes (ACS). Despite optimal medical therapy and contemporary percutaneous coronary intervention (PCI) technology, ACS patients remain at heightened risk for recurrent events and adverse clinical outcomes. It is known that inflammation is a global phenomenon and inflammatory milieu is intensified during ACS. In addition, inflammation, microvascular dysfunction (MVD) and myocardial injury/oedema are independently associated with adverse cardiovascular outcomes in patients with ischaemic heart disease despite there been scarce evidence to suggest the potential links between them. Diagnosis of MVD and myocardial oedema involves cumbersome invasive catheterisation procedures and radiology scans, which may not be necessarily accessible to all patients. Therefore, there is a need to explore potential surrogate markers that are simple, readily available, reproducible and inexpensive. Furthermore, colchicine has recently gained attention as a potential anti-inflammatory therapy in cardiovascular disease. However, the safety and feasibility of long-term colchicine administration in ACS population has not been examined. The principle aims of this thesis were to delineate the associations between inflammation, microvascular injury and myocardial injury, to explore the utility and prognostic values of various biomarkers, and to examine the potential of colchicine as a novel anti-inflammatory therapy in ACS management. A number of study protocols were conducted to achieve the aims. These include assessing coronary microvascular function using pressure/temperature sensor guidewire in catheterisation laboratory, evaluating for presence of myocardial oedema on cardiac magnetic resonance (CMR) imaging, capturing long-term cardiovascular events in a cohort of ACS patients via telephone follow-up, and lastly conducting a double-blind, placebo-controlled randomised trial involving colchicine in ACS population. In summary, the main findings of this thesis are: (a) Inflammation is associated with MVD, and C-reactive protein (CRP) is a significant predictor of MVD. (b) Resting coronary microcirculatory status is related to degree of myocardial injury in patients with ischaemic heart disease. (c) An ischaemic ECG, peak serum troponin and CRP levels are significant predictors of myocardial oedema (d) Leukocyte parameters such as platelet-to-lymphocyte ratio (PLR) and neutrophil-to-lymphocyte ratio (NLR) are associated with long-term all-cause mortality in ACS patients. (e) Colchicine in addition to standard secondary prevention therapy can be safely administered in ACS population. (f) Long-term adherence to ACS guideline-recommenced medical therapy remains unsatisfactory. The findings of this PhD are novel and have potential implications for future risk-stratification and management strategies in patients presenting with ACS. Larger studies are warranted to validate the prognostic value and clinical utility of these biomarkers before they could be incorporated into current risk prediction models or utilised in routine clinical practice. Lastly, the colchicine feasibility study has paved the way for a large randomised controlled trial which is currently underway to assess the efficacy of colchicine in improving long-term cardiovascular outcomes in ACS patients.
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ItemNew approaches to induce tolerance in autoantigen-specific memory T cells in type 1 diabetes( 2018)Autoimmune disorders like type 1 diabetes (T1D) result from the failure of immune tolerance mechanisms. Antigen-specific therapy constitutes an attractive approach to re-establish a tolerant state but has so far not been successful in clinical settings. Importantly, treatment after the onset of autoimmunity is likely to be less effective due to established autoimmune responses and active inflammation in the islets. Moreover, a major hurdle might be the persistence of antigen-experienced islet-reactive T cells and the induction of tolerance in these memory cells is challenging. Hence, therapies intended to induce antigen-specific tolerance may need additional immunomodulatory treatments to be effective in individuals with established autoimmunity. Here, we aimed to identify a combination of interventions that induces tolerance in antigen-specific memory T cells. For this study, we generated non-obese diabetic (NOD) mice with tetracycline-regulated expression of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) in antigen presenting cells (TII mice) and tracked IGRP-specific CD8+ T cells using tetramer enrichment. Notably, while naïve IGRP-specific T cells are eliminated upon antigen expression, memory T cells are refractory to cell depletion. Interestingly, these memory T cells up-regulated several exhaustion markers in response to antigen exposure but showed no functional impairment. We hypothesized that combining short-term treatment with non-Fc-binding anti-CD3 F(ab’)2 and the co-stimulation blocker abatacept together with antigen expression might induce effective tolerance in TII mice. However, after initial depletion of antigen-experienced IGRP-specific cells by anti-CD3 F(ab’)2, these cells re-expanded and were still functional. Thus, our combination approach has only limited efficacy in established autoimmunity. Combination therapies of antigen-specific therapies with agents that support the induction of complete T cell exhaustion might be more successful and should be tested in future studies.
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ItemEpileptic seizures: mechanisms and forecasting( 2018)Seizure forecasting, like weather forecasting, was once considered the domain of charlatans and purveyors of science fiction. However, neuroscience has now advanced to the point of translating seizure forecasting research into widely available clinical applications. Just like weather apps that report the probability of rain on a given day, it is now conceivable that devices will inform people with epilepsy about their current likelihood of having a seizure. This information could be life-changing: restoring a sense of control and the ability to participate in everyday activities. Over 65 million people around the world have epilepsy; one third cannot control their seizures with medication. The unpredictability of seizures can be devastating, leading to persistent anxiety, exclusion from day-to-day life, serious injury or death. The aim of this thesis is to develop a clinically useful framework for forecasting seizures. The presented research addresses several key questions towards this goal: What drives seizure transitions? Are there underlying rhythms governing seizure onset? If underlying rhythms exist, how can they be integrated into a single determination of an individual's seizure likelihood? By presenting answers to these questions this thesis aims to form the basis for an innovative approach to seizure forecasting.
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ItemRole of purinergic signalling in the pathophysiology of antiphospholipid antibody induced miscarriages( 2017)Antiphospholipid syndrome (APS) is an autoimmune disorder in antibodies are generated against cell membrane and self-antigens (aPL-ab) hence the name Antiphospholipid disorder. This autoimmune disorder characterised by the presence of antibodies against β2Glycoprotein-1 and cardiolipin. Pathophysiology includes thrombosis ('blood clotting') and devastating pregnancy complications such as recurrent morbidity (miscarriage), low birth weight and preeclmapsia. Antiphospholipid antibodies bind to endothelial cells and platelets causing vascular complications including thrombosis ultimately leading to clot formation at the site of the injury. The key components implicated in the parthenogenesis of APS are complement cascade, Tissue factor expression, inflammation and coagulation. Increasing evidence shows extensive cross talk between inflammation and coagulation, wherein inflammation leads to activation of coagulation and coagulation considerably enhances inflammatory activity. Purinergic signalling involving catabolism of ATP “danger signal” to adenosine by orchestrated action of cell surface enzymes CD39 and CD73 has been shown to have anti-inflammatory and anti-thrombotic effects. In this project, we aimed to ascertain whether activities of CD39 and CD73are important in developing aPL-ab induced miscarraiges. We utilised mouse model of misccaraiges by passive transfusion of purified human aPL-ab to pregnant wildtype and mice that are modified for CD39 and CD73 activity. We were able to show that aPL-ab infusion in pregnant CD39 and CD73 knock out mice trigger an increase in the rate of miscarriages associated wth increased expression of tissue factor, complement deposition and elevated oxidative stress. There is also an increase in the pro-inflammatory TNF-α and IL-10 expression within the placental vasculature. In contrast to these observations, we also observed that mice over expressing CD39 were protected against aPL-ab induced miscarriages. These mice had reduced TF expression in the decidua, along with reduction in the complement C3D component deposition. Diminished lipid peroxidation and reduction in the proinflammatory TNF-αexpression was also observed in these mice. Taken together, our results provide a rationale for both perturbations in the purinergic pathway to explain disease associated with aPL-ab and for the development of endothelial cell targeted soluble CD39 as a novel therapeutic for management of APS.
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ItemPhysiological role of osteocytic parathyroid hormone-related protein (PTHrP)( 2018)Parathyroid hormone-related protein (PTHrP) is an autocrine/paracrine regulator in many tissues, including bone and mammary glands. Mice with conditional deletion of PTHrP in the osteoblast lineage showed an osteopenic phenotype, and low bone formation levels by the age of 6 weeks, suggesting a paracrine role for PTHrP in promoting bone formation. Since PTHrP is also expressed in osteocytes, I sought to determine the role of osteocytic PTHrP in bone. For this purpose, I have developed an in vitro model of PTHrP (gene: Pthlh) knockdown by using shRNA in a new osteocytic cell line, Ocy454 cells. Pthlh mRNA levels were knocked down by 80% (compared to luciferase) on 3D scaffolds. Pthlh knockdown increased osteocyte markers, Sost (>2-fold greater than luciferase control at day 7), Mepe and Dmp1 (3-4-fold higher than luciferase control at day 14), and reduced osteoblast markers Alpl and Bglap. Undifferentiated Pthlh knockdown cells also had lower levels of cAMP compared to vector control cells. These results showed that PTHrP acts as an autocrine/paracrine factor in osteocytes to regulate mineralisation and bone formation. Next, I generated 10kb-Dmp1Cre.Pthlhf/f mice, which had conditional deletion of PTHrP in osteocytes, from heterozygous breeders. My results demonstrating that Dmp1Cre.Pthlhf/f mice showed no significant differences in cortical or trabecular bone structure compared to Dmp1Cre.Pthlhw/w littermates at 6 weeks of age, indicate that osteocytic PTHrP is not essential for skeletal development in growing mice. However, trabecular bone mass was lower in male and female Dmp1Cre.Pthlhf/f mice compared to controls at 12 weeks of age (the peak of trabecular bone mass in adults). Dmp1Cre.Pthlhf/f mice had lower bone formation, with no difference in bone resorption, compared to controls. Although there was no significant alteration in cortical dimensions, three-point bending tests revealed that Dmp1Cre.Pthlhf/f femora had significantly lower fracture tolerance, with lower ultimate force and deformation, and yield point and fracture occurred at a lower strain compared to controls. These findings indicate that osteocyte-derived PTHrP is required to maintain trabecular bone mass and material strength of adult bones. Dmp1Cre.Pthlhf/f mice from homozygous breeders (Dmp1Cre.Pthlhf/f(hom)) showed a phenotype different from the above heterozygous-bred mice. To our surprise, Dmp1Cre.Pthlhf/f(hom) mice showed higher trabecular bone mass and wider long bones compared to Dmp1Cre.Pthlhw/w(hom) controls. These changes were initially observed in adult male mice, however, I confirmed that this wider cortical bone was detected at 12 days of age in both male and female Dmp1Cre.Pthlhf/f(hom) mice, and retained throughout life in male Dmp1Cre.Pthlhf/f(hom) mice. PTHrP is produced by mammary glands and is secreted into milk. Our results showed that Pthlh DNA recombination directed by Dmp1Cre occurred not only in bones, but also in mammary tissues. To determine the cause of this phenotype I assessed milk content during lactation and fetal bone development during embryogenesis. My results showed recombination of Pthlh in mammary tissues, however there was no difference in milk PTHrP content of Dmp1Cre.Pthlhf/f mothers, compared to controls. Studying the embryonic skeletons at E18.5 showed that Dmp1Cre.Pthlhf/f(hom) fetuses have wider femora, indicating accelerated fetal skeletal development. Finally, I studied the effect of osteocytic PTHrP on the anabolic action of parathyroid hormone (PTH), the only currently approved anabolic therapy for osteoporosis. These studies were initially carried out on homozygous-bred mice. Since these mice exhibited a different basal phenotype to heterozygous-bred mice, we repeated the experiments on the latter to exclude any effect of parental genotype. These two sets of experiment showed that deletion of endogenous osteocytic PTHrP (in both heterozygous- and homozygous-bred Dmp1Cre.Pthlhf/f mice) had no impact on PTH-induced bone formation. I conclude that PTHrP has both local and systemic functions driven by cells that express Dmp1Cre, that influence the skeleton. Osteocytic PTHrP, acting in an autocrine/paracrine manner, is required for normal gene expression by osteocytes and maintains trabecular bone mass and strength at the peak of bone mass in adults. Maternal PTHrP limits fetal skeletal development and radial growth; this effect of maternal PTHrP is important, not only in neonatal mice, but also influences the bone mass of adult male mice.
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ItemTowards the integration of palliative care in advanced cancer: an exploration of patient, family and community perceptions of palliative care and views of communication( 2018)Since its inception, palliative care practice has evolved to respond to the emerging evidence base and the needs of the population it serves. This has resulted in the increasing health system focus on models of early integration of palliative care, enabling improved quality of life, prevention of suffering, and the achievement of goals considered important by people with serious illness and their families. Yet, population data from international jurisdictions including those such as Australia with “advanced” systems of integrated care suggest a picture of missed opportunities, with many patients accessing palliative care late in the illness course. This thesis explored patient, carer, and community perceptions of palliative care, and their views of communication about this concept – including at the hospital for those who are experiencing serious illness and in the community more broadly. This was explored with the broader aim to improve engagement with palliative care, and ultimately facilitate the provision of quality care for those with serious illness. Consistent with the Medical Research Council Framework for the development of complex interventions, empirical work was undertaken using mixed methodology over two phases. Exploratory qualitative interviews were undertaken with patients with advanced cancer and their carers. These data informed the development of a public health intervention to communicate information about palliative care to the broader community, which was tested in an online randomised pilot trial. Patients and carers reported inherently negative perceptions of palliative care, understood as inpatient nursing care, relevant only when no other anti-cancer treatment options remained. Patients revealed the difficulties faced in speaking about death other than in implicit terms, with this topic perceived to be outside the realms of medical consultations. Instead, patients perceived health professionals used the term ‘palliative care’ as a tool to talk about dying. ‘Palliative care’ was understood as a euphemism for death, and personified to mean my death, in turn also becoming ‘unspeakable’. Carers reported their needs for communication about palliative care to be ideally staged overtime, providing education about the tasks of palliative care separate from referral. Once death was imminent, carers wanted open communication with their health professionals, including spoken acknowledgement that death was close, using simple, direct language, and including the words ‘death’ and ‘dying’. A narrative communication intervention was demonstrated to be an acceptable approach to provide education about palliative care to community members. Participants reported significant improvement in attitudes to palliative care post intervention compared to baseline. An evidence-based narrative spoken by a health professional was found to hold the most promise for further investigation in a definitive trial. The results of thesis have significant implications for achieving the routine integration of palliative care in clinical practice. They highlight key opportunities to target education and improve communication about palliative care, and can inform future public health efforts to develop a systematic, evidence-based approach to community engagement. As such, this work provides the next steps to begin to transform experiences of end of life care within health systems.