Medicine (RMH) - Theses
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ItemThe role of genetic factors in the aetiology of focal epilepsy and in the outcomes of epilepsy treatment( 2017)Epilepsy is one of the most common neurological disorders, affecting people of all ages, races, social classes, and nationalities. It is characterised by an enduring predisposition to generate epileptic seizures, and by the neurobiological, cognitive, psychological and social consequences of this condition. The aetiology of epilepsy has long been regarded as ‘unknown’ in about two-thirds of cases. However, evidence mainly from the past two decades has allowed to establish that many epilepsies initially considered to be of unknown cause have in fact a genetic basis. The importance of genetics in epilepsy is not limited to aetiology, and growing interest exists on the genetic contributions to epilepsy treatment outcomes. The overarching aim of this thesis was to investigate and characterise the role of genetic factors in determining the aetiology of focal epilepsies and influencing outcomes of epilepsy treatment. Firstly, I ascertained the extent to which newly diagnosed non-lesional mesial temporal lobe epilepsy (MTLE) actually represents familial MTLE (FMTLE). By directly assessing relatives of patients presenting to a First Seizure Clinic with non-lesional MTLE, the familial syndrome of FMTLE was found to account for about one-fifth of new diagnoses of non-lesional MTLE. Secondly, I examined the diagnostic yield and management implications of genetic testing in the routine clinical care of focal epilepsies. By applying whole exome sequencing (WES) with targeted gene analysis on patients with non-lesional focal epilepsy in whom a genetic aetiology was suspected, pathogenic or likely pathogenic variants were detected in one-eighth of cases. In an individual with drug-resistant epilepsy, the genetic finding altered clinical management, resulting in complete seizure control. Thirdly, I evaluated whether genetic factors influence seizure outcome after surgery for drug-resistant MTLE. Using a multicentre case-control WES study design, no definitive evidence for genetic influences on postsurgical outcome was found. However, a signal emerged for surgical failures, as a group, being enriched for individuals with deleterious variants in established epilepsy genes. Lastly, I investigated the genetic underpinnings of neural tube defects (NTDs) resulting from prenatal exposure to valproic acid (VPA). Adopting a family-based WES study design, two rare variants possibly conferring a substantial risk of VPA-associated NTDs were identified in the planar cell polarity genes CELSR1 and SCRIB. However, this interpretation remains speculative due to sample size limitations. Clearly, genetics has an important role in the aetiology of focal epilepsy. The results of this thesis also substantiate the excitement around the potential for genetics to improve outcomes of epilepsy treatment.
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ItemShear wave elastography in the assessment of liver fibrosis( 2017)The accurate quantification of liver fibrosis is essential to the prognostication and clinical management of patients with chronic liver disease (CLD). Whilst liver biopsy remains the gold standard for fibrosis assessment, it has a number of limitations which have seen its use become increasingly substituted by non-invasive techniques. Ultrasound shear wave elastography (SWE) includes some of the most widely used non-invasive technologies in clinical practice. This work evaluates two ultrasound SWE devices which are in differing stages of clinical development and use; the first being a well-validated point SWE technique from Siemens called Acoustic Radiation Force Impulse elastography or ‘ARFI’ and the second a new 2D-SWE platform by Toshiba. The differing study aims for the two technologies were assessed in separate patient cohorts. Hence the thesis is divided in two. ARFI (Siemens): Background: Acoustic Radiation Force Impulse elastography or ‘ARFI’ is a point shear wave elastography (SWE) technique that is in broad clinical use for the quantification of liver fibrosis. Whilst well validated, questions remain for a number of areas of ARFI performance. This includes the magnitude and likely mechanism of obesity’s impact on ARFI performance, the impact of hepatosteatosis on ARFI reliability and whether ARFI performance is dependent on operator experience. There is also conflicting information as to whether ARFI liver stiffness measurements (LSMs) correlate with cirrhosis severity and the presence of cirrhotic complications. Finally, clinicians have limited facility to gauge the validity of obtained ARFI measurements beyond the IQR/Median criteria. An additional study aim was therefore to develop new strategies to aid ARFI reliability assessment; specifically whether inter-operator disagreement predicts the presence of unreliable ARFI measurements. Method: ARFI performance was assessed amongst a cohort of 943 patients with diffuse CLD of mixed aetiology, who had ARFI LSMs taken as part of clinical fibrosis assessment. Patients were scanned independently by either two or three operators, with ARFI results analysed in the context of patient demographic and CLD information obtained from medical records. Anthropometric measures including body mass index (BMI) was recorded at the time of scanning, and the distance from the skin surface to liver capsule (SLD) was measured from ARFI screenshots as a marker of central adiposity. The cumulative number of scans completed by individual operators and the institution overall was recorded. Assessed performance measures included IQR/Median and inter-operator agreement. ARFI accuracy was also assessed amongst a subcohort of 55 patients who had undergone a liver biopsy within 6 months of ARFI. The performance of ARFI in assessing cirrhosis severity was assessed amongst a further subcohort of 186 patients with clinically diagnosed cirrhosis. The presence of cirrhotic complications was determined retrospectively from medical records and endoscopy reports. Prognostic indices including Child Pugh and Model for End stage Liver Disease (MELD) scores were calculated using bloods tests where available. Results: ARFI showed modest accuracy in assessing liver fibrosis, demonstrating an AUROC of 0.67, 0.76 and 0.70 at discriminating the F01/ F2, F2/ F3 and F3/F4 cut-offs, respectively. ARFI showed good sensitivity (80.0 – 88.9%) and NPV (70.6 – 95.3%), but relatively poor specificity (42.9 – 66.3%) and PPV (27.9 – 56.2%) at the three cut-offs. Body habitus, particularly skin-to-liver capsule distance or ’SLD’, was found to be the primary determinant of ARFI performance in multi-regression analyses. SLD had the strongest relationship with ARFI accuracy (R2 = 0.543) followed by necroinflammatory change (R2 = 0.167), whilst all other patient factors, including hepatosteatosis, failed to show an independent association. Patients with a SLD >2.5cm (indicating significant central adiposity) showed particularly poor ARFI performance and was associated with higher IQR/Median ratios (median = 0.363 vs. 0.187, p<0.001), greater deviation between operators (29.8% vs. 15.9%, p<0.001) and poorer correlation with biopsy (rho = -0.242 vs. 0.493) than those with a SLD ≤2.5cm. Individual operator experience showed a weak relationship with ARFI performance, with operators of <25 scans experience having similar median IQR/Median ratios (0.170 vs. 0.165, p=0.13), slightly greater deviation between operators (14.3% vs. 11.06%, p=0.014) and greater deviation from the biopsy reference range (mean deviation = 0.588 vs. 0.279m/s, p=0.004) than more experienced colleagues. There also appeared to be a similarly weak association between overall institutional experience and ARFI performance, with reliability being slightly reduced amongst the first 150 scans performed in the institution. In patients in whom both operators had concordant F score results, ARFI LSM showed greater correlation with biopsy (rho = 0.392) than in cases of inter-operator disagreement (rho = 0.010). When scanned by three operators, patients with three-way operator agreement showed even stronger correlation with histopathology (rho = 0.571). Amongst cirrhotic patients, ARFI showed a moderately strong correlation with prognostic scores of liver function, including both MELD score (rho = 0.342, p<0.001) and Child- Pugh Score (rho = 0.363, p<0.001). ARFI LSMs showed modest accuracy in predicting the presence of ascites (AUROC = 0.58), encephalopathy (AUROC = 0.60) and oesophageal varices (AUROC = 0.69). Conclusion: ARFI showed moderate performance in quantifying liver fibrosis in a clinical Australian setting. The technology’s strength appears to be in the exclusion of liver fibrosis, however the tool is prone to false positive results. Body habitus was found to be the primary determinant of ARFI performance, with necroinflammatory change and operator experience showing a weaker impact on scan reliability. Central adiposity, as indicated by SLD, showed a particularly strong relationship with ARFI performance and the routine measurement and reporting of SLD should be considered to help clinicians gauge the reliability of ARFI results. Scanning patients with multiple independent operators also showed value as a reliability indicator, with inter-operator discordance being a predictor of poor ARFI performance. 2D-SWE (Toshiba): Background: The second technology assessed is a new 2D-SWE platform from Toshiba, which has a number of technical innovations and theoretical advantages over Siemens’ ARFI system. The technology is in the early clinical phases of testing and therefore data on this new technique remains limited. Our study aim was therefore to evaluate specific technical parameters to help assist in the formation of acquisition guidelines. This included assessing the measurement variability of Toshiba 2D-SWE (i.e. IQR/Median), the number of measurements required per patient to yield a precise LSM estimate and whether the uniformity of shear wave velocities within the measurement ROI (i.e. ROI SD/Speed ratio) could be used to assess the reliability of individual 2D-SWE measurements. Method: 2D-SWE was assessed amongst fifty-five patients with mixed aetiology CLD using the Toshiba Aplio 500 ultrasound system. Ten measurements were obtained per patient by an operator blinded to all preceding readings. Measurement variability (i.e. IQR/Median) and the number of measurements required per patient to achieve a LSM estimate within 5% of the existing method using 10 samples was assessed. Results were analysed against scan and clinical information including CLD aetiology, BMI, SLD, presence and severity of hepatosteatosis and measurement depth within the liver. The ratio of the standard deviation of shear wave velocities within the measurement ROI to overall shear wave velocity (i.e. ROI SD/Speed) was calculated for each individual measurement, and its relationship with measurement consistency (i.e. deviation of the measurement from the set’s median) was assessed. Results: The median IQR/Median ratio for 2D-SWE was 0.131 (q1-q3: 0.089–0.174). Five readings provided an approximation within 0.11m/s or 4.2% of the median velocity of ten measurements. Factors associated with increased measurement variability included increasing BMI (rho=0.388, p=0.003), SLD (rho=0.426, p=0.002) and measurements taken within 1.5cm of the liver capsule (p<0.001). Measurements with heterogeneous shear wave profiles (indicated by a ROI SD/Speed >0.15) showed greater deviation from the set’s median velocity than those with a ROI SD/Speed ≤0.15 (0.421 vs. 0.219 m/s, p=0.0001). Conclusion: 2D-SWE showed low overall measurement variability, with a minimum of five readings providing equivalent precision to the existing method using 10 samples. Obesity (i.e. BMI>30kg/m2), increasing abdominal wall thickness (i.e. SLD), sub-capsular measurements and a ROI SD/Speed >0.15 were all associated with increased measurement variability. ROI SD/Speed warrants further evaluation as a quality assessment metric, as it may allow objective operator assessment of individual 2D-SWE measurement reliability in real-time.
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ItemFalling down the cascade: the gaps in care delivery for people living with chronic hepatitis B in Australia( 2017)In Australia, an estimated 239,000 people were living with chronic hepatitis B (CHB) in 2016. Most of the affected community were born overseas and acquired their infection early in life, in countries with high and intermediate prevalence of hepatitis B. The mortality attributable to CHB is from both liver cancer (hepatocellular carcinoma) and cirrhosis. Hepatocellular carcinoma has poor five-year survival, increasing incidence globally, and was projected to become the sixth most common cause of cancer death in Australia in 2016. The first “Global Hepatitis Health Sector Strategy” was signed in 2016 by all the member states of the World Health Assembly including Australia, with the aim of eliminating viral hepatitis, including hepatitis B, as a public health concern by 2030. The five studies presented in this thesis use the cascade of care framework to approach different aspects of the health system response to chronic hepatitis B in Australia. The studies have used different data sources and methodologies to measure the cascade and explore factors associated with the delivery of care. The first study presents an analysis of national data from 2012. It proposed, for the first time, a cascade of care for chronic hepatitis B in Australia that included a novel “enrolled in care” indicator. The second study presents findings from a multicentre retrospective study of adherence to antiviral therapy for chronic hepatitis B from 2010-2013. The study measured the proportion of people adherent to treatment in tertiary settings and analysed the demographic and health system factors associated with poor adherence. The third study analysed the association of a pharmacy-based adherence measure (the medication possession ratio) with viral outcomes using a time-to-event analysis for favourable and unfavourable viral outcomes. The fourth study presents findings from a retrospective analysis of primary care data in a community health centre that received external support from a tertiary service to improve the delivery of guideline-based care for chronic hepatitis B, including surveillance for hepatocellular carcinoma. This study evaluated four and a half years of data focusing on hepatocellular carcinoma surveillance participation and adherence. The fifth study presents findings from a qualitative study: semi-structured interviews of African-Australians living with chronic hepatitis B. This study explored participants’ understanding of health risks associated with hepatitis B, including their perceptions of their risk of developing hepatocellular carcinoma. Findings from this thesis have shown that few people living with chronic hepatitis B in Australia were enrolled in care. It provided the first multicentre estimates of the adherence of people on antiviral therapy for chronic hepatitis B in our health system (using medication possession ratio as the measure of adherence) and that factors associated with poor adherence were younger age and poor continuity of clinician. In a further study, the association between medication possession ratio and unfavourable viral outcomes was demonstrated for the first time. This analysis found that there was no true cut-off point or threshold to define adherence and the risk of poor outcomes. Rather, there was an increasing hazard ratio for unfavourable events with decreasing medication possession ratio. The findings also include results from a study that demonstrated hepatocellular carcinoma surveillance in a tertiary-supported general practice – with both participation and adherence to six-monthly scans with supported recall and reminder systems – is hard to achieve. Finally, the fifth study presented as part of this thesis found that African-Australians living with chronic hepatitis B perceived and experienced significant risks to social and emotional wellbeing from the shock of diagnosis, fear of infectiousness, and discrimination from telling others about their illness, as well as physical or liver-related problems. The results from this thesis have informed the development of the current Australian cascade of care for chronic hepatitis B and provided insights into the challenges of delivering health services to people living with chronic hepatitis B. These findings have led to recommendations for further development of the cascade at a national and regional level, and the need for further research and evaluation of the health system response to chronic hepatitis B. The work presented demonstrates that Australia has failed to meet the targets of the 2014-2017 National Strategy and needs to rapidly improve essential elements of the cascade, including increasing the proportion diagnosed and enrolled in care, to reach the targets of elimination of chronic hepatitis B as a public health concern by 2030.
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ItemThe neuropsychiatric disorders of focal epilepsy( 2017)Neuropsychiatric disorders commonly co-exist with focal epilepsy. Despite intense investigation there remains significant limitations to our current understanding of the aetiological relationship between these conditions, as well as the clinical and radiological factors that are associated with the development of mental illness in epileptic patients. To address these questions I assembled a large cohort of consecutive focal epilepsy patients reviewed at a large tertiary referral centre over an 11-year period, and analysed relevant clinical, radiological and demographic findings using both cross-sectional (baseline evaluation) and longitudinal (serial assessments) study designs. For cross-sectional analyses, psychiatric and epilepsy comorbidity were comprehensively identified and compared to baseline MRI-determined Deep Brain Structure (DSB) volumes. In the prospective study, standardised psychiatric and quality of life assessments were obtained in a subset of patients and the development of mental illness compared to interval changes in DSB volumes. I focus on depression and psychosis, as they represent the two most prevalent psychiatric disorders identified, and classify focal epilepsy patients according to the site of seizure origin and presence of a lesion. Contrary to the common medical belief, I found that the rates of neuropsychiatric disorders in temporal lobe epilepsy were equivalent to those in extratemporal lobe epilepsy. There were no differences between those with and without psychiatric disorder by age, gender, and laterality of seizure, epilepsy severity or duration. Patients with non-lesional epilepsy, both temporal and extratemporal, have double the rate of depression compared to those with lesional focal epilepsy. There were high rates of quality of life difficulties in people with epilepsy and comorbid psychiatric disorders but the pattern of subjective concerns does not match objective clinician ratings. The hippocampal and amygdalae volumes of epilepsy patients were reduced compared to normal controls. People with co-existing epilepsy and depression had a trend towards smaller reductions in temporal lobe structures, which may represent differential expressions of progression through inflammation, trauma or emotional processing needs with either relative sparing or volumetric increases. People with psychosis and epilepsy have bilaterally reduced hippocampi compared to those with epilepsy, where reductions are predominantly ipsilateral to seizure focus. There is less evidence for amygdala change in psychosis, but a small relative increased volume was observed compared to those with epilepsy alone. There was no evidence of progression over time at a population level over 3.9 years, although there was greater variability in size in all epilepsy subjects compared to normal controls. These results convincingly argue against assumptions about the primary role of temporal lobe foci in the pathogenesis of psychiatric comorbidities in patients with epilepsy, whilst allowing for the possibility that disruption to frontotemporal networks may be a component in the development of psychiatric disorders. Progression is not an inevitable part of the natural history, at least over a 4 year period but it is possible that individuals may exhibit marked changes. They highlight the as yet unexplored possibility that the absence of a lesion as an epileptic site may be associated with greater risks of neuropsychiatric illness and allow for speculation that this may be related to inhibitory surround impacts, more extensive underlying diffuse abnormalities and disruption to frontotemporal connectivity.
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ItemVitamin D status and related health in young women: the Safe-D study( 2017)Background: Vitamin D deficiency is emerging as a global public health issue in all age groups and public interest in vitamin D has increased significantly over the past decade. Deficiency is associated with increased risk of a number of adverse health outcomes, including poor musculoskeletal health, cardiovascular disease, autoimmune diseases, infectious disease, mental ill-health and several cancers. In Australia, vitamin D deficiency affects one in three adults and up to 50% of young women. Despite this, few studies have examined vitamin D status in late adolescent and young adult women. Aims: The Safe-D study was a cross-sectional study examining the association between vitamin D status and related health outcomes and aimed to define the prevalence of vitamin D deficiency in young women. The objectives of this doctoral research included examining predictors and correlates of vitamin D status, exploring potential associations between vitamin D status, mental and musculoskeletal health in young women, defining reference data for bone turnover markers (BTMs) in young women and examining determinants of bone health, including BTMs. Exploratory aims included examining potential interrelationships between these health domains. Methods: Females aged 16-25 years, living in Victoria, Australia were recruited into the Safe-D study, via Facebook advertisements. Participants were required to complete a comprehensive, online questionnaire, wear an ultraviolet dosimeter for two weeks to measure personal sun exposure and attend a site visit. Data collected through the study questionnaire included: participant demographics, medical history, sun-related behaviours, mental health, physical activity, nutrition, behavioural risk factors and lifestyle choices. Site visit measures included: serum 25-hydroxyvitamin D (25OHD) assayed by liquid-chromatography tandem-mass spectrometry, various serum biochemistry measures (including BTMs), areal bone mineral density and body composition by dual-energy X-ray absorptiometry, volumetric bone mineral density and measures of bone quality by peripheral quantitative computed tomography and muscle function by Leonardo mechanography®. Results: A total of 557 young women were recruited into the Safe-D study, of whom 410 completed site visits and 407 had serum 25OHD results. The prevalence of vitamin D deficiency (serum 25OHD <50 nmol/L) was 26%. Mean (± standard deviation) serum 25OHD was 68 ± 27 nmol/L. Factors associated with serum 25OHD, such as sex hormone-binding globulin and iron levels, sun exposure, season, multivitamin use and anthropometry, were able to explain 56% of the variation in 25OHD levels. Reference intervals for CTX and P1NP were calculated as 230–1000 ng/L and 27–131 μg/L, respectively, and both markers were found to be strongly associated with age and contraceptive use. No association was found between vitamin D status and health domains highly-relevant to women, mental and musculoskeletal health. Conclusion: Although vitamin D deficiency was found to be common in young Victorian women, it did not appear to be a major health risk for the majority in the immediate period. Further investigation of temporal relationships between vitamin D status and relevant health outcomes using longitudinal data, in addition to exploring the effects of improving vitamin D status in young women is warranted.
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ItemInsights into the mechanisms and effects of right ventricular septal pacing( 2017)Cardiac pacemakers have markedly improved the morbidity and survival of patients with bradyarrhythmias. However, major clinical trials have demonstrated that the traditional pacing site, the right ventricular apex (RVA), is associated with increased heart failure, atrial fibrillation and mortality. This has led to the exploration of alternative right ventricular (RV) pacing sites such as the RV septum to decrease the risk of pacemaker-induced heart failure. However, despite a multitude of experimental animal and clinical human studies, definitive demonstration of a benefit of RV septal over RVA pacing remains elusive. This thesis (1) systematically reviews pacemaker-induced cardiomyopathy and the clinical trials comparing RV apical to RV “septal” pacing sites, (2) expands the current fluoroscopic definition of RV “septal” pacing and categorises RV lead position instead into both the long and short axis using the gold standard imaging modality: cardiac computer tomography (CT) and describes novel fluoroscopic and echocardiographic schemas for accurate intraoperative RV lead placement, (3) describes the utility of cardiac CT in the assessment of pacing lead perforation and proximity to coronary arteries and (4) characterises LV electrical activation patterns of different RV “septal” and apical pacing sites and their implications for cardiac resynchronisation therapy.
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ItemAtrial fibrillation and systolic heart failure: the role of myocardial fibrosis and catheter ablation( 2017)Atrial fibrillation and systolic dysfunction are both emerging epidemics in the developed world and both frequently co-exist. Each condition, both individually and in combination, are associated with significantly worsened morbidity and mortality. Both share pathophysiological mechanisms and may promote the progression of each other. Traditional pharmacological therapies for AF have limited efficacy, which is also the case in patients with concurrent systolic dysfunction. Catheter ablation has emerged as an effective treatment for AF with superior outcomes compared to pharmacological rhythm control, the current standard of care. An increasing body of evidence has shown that catheter ablation is feasible and effective in patients with systolic dysfunction. Nonetheless, identifying those patients with systolic impairment likely obtain the greatest benefit from catheter ablation remains a evolving challenge. Additionally, the electrophysiological and structural changes associated with the co-morbid AF and systolic dysfunction is yet to be fully elucidated. The central aim of this thesis is to comprehensively evaluate the role of catheter ablation as a treatment for systolic dysfunction. Following a comprehensive review of the relevant existing literature in this area in Chapter 1, Chapters 2, 3 and 4, of this thesis seek to clinically evaluate the effectiveness of catheter ablation in selected patients with systolic impairment and AF, with a particular focus on utilising advanced imaging techniques such as cardiac magnetic resonance imaging (CMR), as a tool to optimise patent selection, and evaluate treatment outcomes. Secondly in Chapters 5 to 8, this thesis seeks to characterise the electrophysiological and structural characteristics of patients with AF in the setting of systolic impairment and additionally to highlight the limitations and challenges of catheter ablation in persistent AF, with a focus on pulmonary vein electrical activity and the role of intra-procedural adenosine.
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ItemTowards the further understanding of prognosis and outcomes for patients with high grade gliomas: A randomised phase II study of Carboplatin and Bevacizumab in recurrent glioblastoma( 2017)Glioblastoma (GBM) is the most common, and the most aggressive, adult brain tumour, affecting over one thousand Australians per year. It carries a high mortality burden and a high social burden to both the cancer sufferer and their family. GBM recurs in nearly all patients within months of treatment, and median overall survival from diagnosis remains less than 18 months. At recurrence, no chemotherapy drug has been associated with reliable and meaningful gains in survival, and no new drugs have been added to the Australian Pharmaceutical Benefits Scheme for GBM since temozolomide over a decade ago. We need better treatments for this devastating disease. Multiple trials have tested the role of targeted therapies – either alone or in addition to chemotherapy – in both the newly diagnosed and recurrent disease settings. Bevacizumab, a vascular endothelial growth factor inhibitor monoclonal antibody, showed promise in early and small clinical trials in recurrent GBM. We developed and conducted a multi-centre, stratified randomized phase II trial comparing bevacizumab monotherapy with bevacizumab + carboplatin chemotherapy in patients with recurrent GBM - the CABARET (Carboplatin And BevAcizumab in REcurrenT glioblastoma) clinical trial. CABARET represents a substantial achievement in Australian medical research. This is the largest investigator-initiated brain tumour clinical trial in Australia to date. It was conceptualized, developed and conducted entirely within Australia. The primary objective of CABARET was to determine the effect of bevacizumab plus carboplatin versus bevacizumab monotherapy on progression-free survival in patients with recurrent GBM. The trial found that adding carboplatin to bevacizumab resulted in more toxicity without additional clinical benefit: no significant differences in response rate, progression-free or overall survival. Several secondary and exploratory endpoints have been investigated. This was the first prospective study to assess the role of continuing bevacizumab beyond progression. Albeit with a limited sample size, no differences in survival outcome were noted after patients with progression on study underwent a second randomization to either continue or cease bevacizumab. We measured time to deterioration in health-related quality of life, finding no difference between the two arms but noting that up to 50% of patients gained some improvements in relevant quality of life parameters while on treatment, suggesting benefit from treatment. We also assessed the role of early MRI after four weeks on study, finding this to be a robust indicator for overall survival in this patient cohort. Finally, we found that using centralized radiology review resulted in significantly earlier time to progression, but the absolute difference for the cohort of 0.3 months was not felt to be clinically relevant. The study results have significantly improved knowledge regarding the use of bevacizumab in the setting of recurrent GBM. We successfully challenged and refuted the dogma, previously established from either retrospective studies or single arm early phase trials, that bevacizumab should be continued after progression or combined with chemotherapy in GBM. This will have significant impact on both future clinical trial design, and also managing patients with this disease, as we have further insight into anticipated effects and survival in the Australian context.
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ItemAssessing prion acute synaptotoxicity using an electrophysiology study paradigm( 2017)Prion diseases comprise a group of rare neurodegenerative disorders that affect both humans and animals. Regardless of the host species, misfolding of normal prion protein (PrPC) into abnormal conformers (PrPSc) with the subsequent aggregation and accumulation of PrPSc appears critical to disease pathogenesis although the precise neurotoxic species and how such species provoke neuronal dysfunction and loss leading to the onset of clinical illness remain unresolved. This project assessed the acute synaptotoxicity of ex vivo PrPSc prepared from brains of prion-infected mice using an electrophysiology study paradigm. The initial stages of this project were to develop and validate the electrophysiology model to assess prion acute synaptotoxicity. Electrophysiology studies employed multi-electrode arrays to stimulate and record CA1 region synaptic functions in wild type (WT) mouse hippocampal slices following brief exposure to ex vivo PrPSc preparations prepared from brains of prion infected mice at the terminal stage of disease (TSD). The principal synaptic functions assessed included long-term potentiation (LTP) and post-tetanic potentiation (PTP). Exposure of WT hippocampal slices to crude brain homogenate derived PrPSc from two different mouse-adapted prion strains (cM1000 and cMU02) as well as cell lysate derived PrPSc from chronically M1000-infected MoRK13 cells (MoRK13-Inf), caused significant LTP dysfunction. Evidence for PrPSc as the synaptotoxic agent was provided through the rescue of LTP following selective immuno-depletion of total prion proteins from cM1000, through modestly proteinase K (PK)-treated cM1000 retaining full synaptotoxicity, and through restoration of the LTP impairment when employing reconstituted, PK-eluted, immuno-precipitated M1000. These experiments validated the electrophysiology model as a bona fide model of prion acute synaptotoxicity. Additional model characterisation was achieved by further electrophysiological analyses, which demonstrated heightened pre-synaptic vulnerability to the acute synaptotoxicity, exemplified by impairment of PTP due to cumulative insufficiency of replenishment of the readily releasable pool of vesicles during repeated high-frequency stimulation utilised for induction of LTP. Further model characterisation was achieved by biochemical analyses of hippocampal slices manifesting LTP dysfunction revealing reduced expression of multiple key synaptic proteins, while such changes were absent in hippocampi demonstrating rescued LTP. PTP and LTP were impaired in hipocampal slices prepared from Prn-p gene ablated (PrPC knockout; PrPo/o) mice after exposure to ex vivo PrPSc, indicating that acute prion acute synaptotoxicity is largely independent of PrPC expression and PrPSc propagation. Further electrophysiological and biochemical analyses of PrPo/o slices manifesting synaptic dysfunction however, revealed that some effects of the prion acute synaptotoxicity were dependent on PrPC expression. The electrophysiology model was used to assess the presence of acutely synaptotoxic species by utilising brains of M1000-infected mice at 30%, 50%, 70%, and 100% of the TSD to determine the propagation kinetics of neurotoxic species during disease evolution. The results demonstrated that the neurotoxic species are propagated from early in the disease progression, which appears to overlap the propagation of transmitting species. Finally, the electrophysiology model was also utilized to assess the acute synaptotoxicity of oligomeric PrPSc obtained after size fractionation chromatography of cM1000 from these designated time points of the disease. The results demonstrated a clear correlation between the propagation of oligomeric PrPSc and acutely synaptotoxic species in prion disease natural evolution although the synaptotoxic species appear not to acquire PK resistance until relatively late in disease progression. Overall, the main findings in my thesis demonstrated that acutely synaptotoxic species in M1000 prion disease are PrPSc correlating with the presence of oligomeric PrPSc, propagated from relatively early in disease evolution. A number of future studies are proposed to further confirm and elaborate these principal findings.
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ItemCognition, NMDA receptors, and gamma oscillations: implications for schizophrenia( 2017)Cognitive deficits in schizophrenia represent the most disabling symptom category of the disorder for which there is currently a lack of consistently effective treatment. This may be attributable to our limited understanding of the causal mechanisms associated with these symptoms. Neural oscillations in the gamma frequency range (i.e 30-80Hz) have been associated with higher order cognitive function and the regulation of these high frequency neural oscillations is impaired in schizophrenia patients. Therefore, it is theorised that gamma oscillatory dysfunction may underpin cognitive deficits in schizophrenia patients. NMDA receptor (NMDAr) hypofunction is a potential molecular mechanism involved in the pathophysiology of schizophrenia. The NMDAr hypofunction hypothesis of schizophrenia suggests that aberrant signalling through these receptors may underlie symptoms of the disorder. This dysfunction is commonly modelled through administration of NMDAr antagonists to rodents, which also induces behavioural endophenotypes related to schizophrenia. In addition, administration of NMDAr antagonists can induce aberrant gamma oscillations, as well as disrupt high frequency oscillations (HFOs; 130-180Hz). Therefore, the research in this thesis aimed to investigate the relationship between gamma oscillations and cognition, particularly working memory and attention, and elucidate whether cognitive deficits induced by NMDAr antagonism are also associated with disruptions to gamma oscillations. Given that NMDAr hypofunction also influences HFOs, the role of HFOs in cognition was also examined. Systemic administration of NMDAr antagonist, MK801, to rodents can induce several endophenotypes of schizophrenia, including hyperlocomotion and cognitive deficits. The hyperlocomotive endophenotype can produce motor confounds during cognitive testing. Thus, we firstly assessed whether the effects of localised NMDAr antagonism would induce the same behavioural disturbances seen with systemic administration (Chapter 3). We studied the effect of localised NMDAr hypofunction on the generation of neural oscillations in local and distant regions, as well as behavioural psychosis-related endophenotypes of schizophrenia. Local infusion of MK801 into the prefrontal cortex (PFC), dorsal hippocampus (dHIPP), or nucleus accumbens (NAcc) of male Wistar rats all elevated gamma oscillations in both local regions and regions distant from the drug infusion site, and also induced hyperlocomotion, stereotypy and ataxic behaviours. This study demonstrated that local disruption of NMDAr function can disrupt global networks and psychosis-related behaviours, therefore, for the remaining research in this thesis, motor and motivational factors were assessed alongside cognition in order to ensure that external factors did not confound task performance. Working memory and attention were assessed in the rodent touchscreen chambers (Chapters 4-6). Administration of MK801 to rats disrupted working memory performance in the trial unique nonmatching-to-location (TUNL) task but attentional performance in the 5-choice serial reaction time task (5CSRTT) was preserved. Working memory was found to be associated with increased high gamma (65-80Hz) coherence between the PFC and dHIPP during the information maintenance stage of the task. MK801 disrupted gamma oscillations accompanied with working memory deficits, suggesting that working memory processing is associated with gamma activity and is NMDAr-dependent. Attentional processing, on the other hand, was not associated with gamma activity. However, gamma oscillations were still increased with MK801 administration without impairing attentional performance. This suggests that attentional processing is not NMDAr-dependent and not associated with gamma oscillations. Aberrant gamma oscillations induced by NMDAr antagonism are also not indicative of dysfunction in attentional processing. HFO activity was found to not be related to cognitive function in both domains. The results of this thesis suggest that working memory and attention are differentially influenced by NMDAr antagonism and are differentially related to gamma oscillations. NMDAr hypofunction and gamma oscillations may be related to some but not all cognitive symptoms relevant to schizophrenia. These findings aid understanding of mechanisms associated with cognitive deficits of schizophrenia and this data will guide future studies in finding biomarkers and effective treatment options for schizophrenia patients.