Medicine (RMH) - Theses
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Novel Aspects in the pathogenesis of Chronic Kidney Disease-Mineral Bone Disorder (CKD-MBD)
Chronic Kidney Disease – Mineral and Bone Disorder (CKD-MBD) is a clinical entity, broadly defined by (a) disturbances in mineral metabolism, (b) abnormal bone remodeling and (c) accelerated vascular calcification. CKD-MBD is ubiquitous as kidney function deteriorates and contributes significantly to increased fracture risk and excess cardiovascular morbidity and mortality. Due to the complex and multifactorial nature of this condition, there remains many unanswered questions on how to best investigate and manage all aspects of CKD-MBD. The general aim of this thesis was to investigate the pathophysiology, clinical outcomes and novel assessment strategies of the three domains of CKD-MBD. More specifically, this thesis aimed to 1) evaluate the outcomes of calciphylaxis in Australian patients, 2) develop a greater understanding of changes in skin and subcutaneous tissue in patients with kidney disease, 3) demonstrate the consequences of severe secondary hyperparathyroidism (SHPT) in the absence of calcimimetic treatment, 4) understand the temporal relationship between calciprotein particles and biochemical markers of CKD-MBD and 5) evaluate bone microarchitecture in patients with severe SHPT using a novel imaging modality. Although calciphylaxis is a rare disease, it can be considered a form of accelerated vascular calcification. It predominantly affects patients with chronic kidney disease (CKD) and is associated with significant morbidity and mortality due to progressive cutaneous calcification, necrotic ulceration and infection. Clinical registries have been established to better understand risk factors, optimal treatments and disease outcomes of calciphylaxis. To better understand outcomes of Australian patients with calciphylaxis, Chapter 2 investigated the five-year outcomes from the internet-based Australian Calciphylaxis Registry. Data was recorded on patient characteristics, biochemical parameters, treatments and disease outcomes. The Australian Calciphylaxis Registry highlights risk factors for calciphylaxis including diabetes, obesity and vitamin K antagonist use, whilst significantly elevated parathyroid hormone (PTH) levels were not a consistent finding at the time of diagnosis or in the preceding 12 months in this cohort. Unfortunately, resolution of calciphylaxis remains uncommon despite multimodal therapy and mortality from calciphylaxis in the first year following diagnosis is high. Vascular calcification is well described in large- and medium-sized vessels in patients with CKD, especially in those with end-stage kidney disease (ESKD) on dialysis. Medial calcification is particularly prevalent in this population and contributes to arterial stiffness and increased cardiovascular mortality and morbidity. Apart from in the setting of calciphylaxis, few studies have assessed skin and subcutaneous calcification and associations with abnormalities of bone and mineral metabolism in patients with CKD. In Chapter 3, patients with varying stages of CKD undergoing elective surgery underwent intraoperative incisional skin biopsy to evaluate for the histological presence of vascular calcification and upregulation of pro-calcific gene transcripts. This study reports the novel finding of dermal and subcutaneous small vessel calcification in multiple anatomical locations in 38% of patients with advanced CKD or ESKD undergoing elective surgery but free from calciphylaxis. Expression of pro-calcific gene transcripts, specifically TNAP or RUNX2, was not increased in samples from patients with CKD or those with histological evidence of vascular calcification. SHPT in patients with CKD is associated with cardiovascular and bone pathology. Measures to achieve target PTH values and control biochemical abnormalities associated with SHPT require complex therapies, and severe SHPT often requires parathyroidectomy or the calcimimetic cinacalcet. In Australia, cinacalcet prescription, in dialysis patients was reimbursed by the government from 2009 to 2015. However, after this time funding was withdrawn following publication of the EVOLVE study, which resulted in most patients on cinacalcet ceasing therapy. Changes to reimbursement of cinacalcet in Australia provided an opportunity to assess effects of medication cessation on biochemical and clinical outcomes in dialysis patients, including changes to novel biomarkers such as calciprotein particles (CPP). CPP are nanoparticles of mineral and protein in the circulation associated with increased vascular calcification in patients with CKD. Chapter 4 focused on changes to novel biochemical markers following cinacalcet withdrawal in dialysis patients with SHPT from a single centre with CPP levels assessed at four different time points over a 12-month period. Outcomes were compared with an age- and gender-matched cohort of cinacalcet-naive dialysis patients. Cinacalcet withdrawal in the real-world setting demonstrated increases in PTH, serum calcium and the novel prognostic marker CPP over a 12-month period. Following on from work in Chapter 4, Chapter 5 involved a multi-centre retrospective study of dialysis patients who ceased cinacalcet after August 2015 at 11 Australian institutions. Clinical outcomes and changes in biochemical parameters were assessed over a 12- and 24-month period from drug cessation. Significant increases in serum PTH, calcium and alkaline phosphatase occurred over a 12-month period following withdrawal of cinacalcet, without an associated increase in cardiovascular mortality and morbidity, fractures or calciphylaxis. SHPT in patients with CKD leads to complex bone disease, affecting both trabecular and cortical bone with resulting increased fracture risk. Optimal assessment of bone in patients with CKD is yet to be determined. High-resolution magnetic resonance imaging (MRI) can provide three-dimensional assessment of bone microarchitecture, as well as determination of mechanical strength with finite element analysis (FEA). Chapter 6 evaluated bone microarchitecture in patients with severe SHPT undergoing parathyroidectomy. Correlation of MRI findings of bone microarchitecture were made with biochemical markers. MRI in this patient population demonstrated evidence of significant changes in bone microarchitecture with trabecular deterioration, low trabecular and cortical bone volume, and reduced mechanical competence of bone with overall poor bone mechanical strength. In summary, this thesis presents insights into the vascular, biochemical and bone domains of CKD-MBD with both clinical and basic science research focusing on pathophysiology, novel biochemical markers and imaging techniques. The novel findings obtained in this thesis have broadened the understanding of all three domains of CKD-MBD, particularly with regards to CPP as a potential novel biomarker to evaluate biochemical derangement and vascular risk, and MRI as a novel imaging modality to gain a better understanding of bone microarchitecture. Further clinical and basic science studies are required to validate findings and to explore concepts established in this thesis, however, data presented here establishes important concepts for future research in this field.
Cather ablation techniques and insights into the relationship between AF and Heart Failure
Atrial fibrillation (AF) is a common cardiovascular condition due to increasing life expectancy, increased comorbidities and increased risk factors such as obesity and sedentary lifestyle. AF is associated with an increase in healthcare utilisation, mortality, medication use and decrease in quality of life. AF is also associated with a wide range of other comorbidities such as heart failure which worsens the prognosis further. More recently, we have come to recognize the importance of maintaining sinus rhythm especially in patients with comorbidities. Catheter ablation (CA) is a relatively new and novel invasive strategy used in the management of AF which has become increasingly accepted as the ideal option for managing patients with symptomatic AF. CA decreases the need for long term anti arrhythmic therapy which has been associated with poorer outcomes. Recent studies have highlighted the role of AF in the pathogenesis of HF with reduced ejection fraction (HFrEF) and established the role CA in this population. However, the role of AF in HF with preserved ejection fraction (HFpEF) remains unknown. The aim of this thesis is to explore the impact of CA in people with AF and HFrEF or HFpEF, focussing specifically on gender differences in outcomes and describe the structural, electrical and functional changes that result from restoring and maintaining sinus rhythm using CA. Chapter 1 describes and explores the relationship between arrhythmias and cardiomyopathies in general focussing on the paradigm shift in our understanding of the how arrhythmia leads to HFrEF. We then explore the evidence for link between AF and HF (both HFrEF and HFpEF) with some novel concepts in pathophysiology of HFpEF in people with AF. Chapter 2: The CAMERA-MRI study demonstrated that CA and maintenance of sinus rhythm in people with persistent AF (PsAF, AF lasting >7 days) and HFrEF resulted in normalisation of left ventricular ejection. However, the results were reported with only 6 months of follow up. We report the long-term efficacy and clinical impact of CA in the AF population with otherwise unexplained HFrEF. We conclude that CA is highly successful in restoring and maintaining sinus rhythm in people with AF and otherwise unexplained HFrEF. The improvements in left ventricular ejection fraction (LVEF) persisted at long term follow up. Patient selection is key to improved outcomes. Chapter 3: The AF outcomes of the CAMERA-MRI study was significantly superior to other reported series. All patients in the CAMERA-MRI study underwent posterior wall isolation (PWI) in addition to the standard pulmonary vein isolation (PVI). There is adequate evidence to suggest that PWI could improve arrhythmia outcomes in patients with PsAF. Hence, this is a technical description of a novel ablation technique to achieve PWI which can be used in conjunction with routine CA to decrease AF recurrence after CA. Chapter 4: The hypothesis behind the improved arrhythmic outcomes of the CAMERA-MRI study population was explored further with a detailed electro anatomical assessment using invasive and accurate assessment of the right atrium using special contact force capable catheters. This special equipment can accurately assess the electrical signal of the heart to determine if maintaining sinus rhythm and improvement in LVEF can lead to improvement in the substrate that facilitates AF. All participants had a baseline study at the time of their ablation returned for a follow up study after having maintained sinus rhythm following CA. We conclude that maintenance of sinus rhythm with improvement in LVEF is associated with reverse atrial remodelling that might partly explain the improved arrhythmia free survival in this patient population. Chapter 5 explores the negative prognostic markers for AF recurrence after CA in people with PsAF. Women are reported to have sub-optimal clinical outcomes after CA. We performed a large multi-centre retrospective analysis of arrhythmia recurrence in people who underwent more than one ablation procedure and reported on the gender differences in arrhythmia outcomes and discuss the potential basis for these differences. We conclude that women have a higher risk of arrhythmia recurrence compared to men which may be explained by higher incidence of non-pulmonary vein trigger for AF and higher prevalence of HFpEF. Chapter 6: The relationship between AF and HFpEF and importantly the role of AF in the pathophysiology of HFpEF has never been studied in detail previously. We performed a prospective analysis to determine the true prevalence of HFpEF in people considered for AF ablation using gold-standard invasive assessment of individuals pulmonary capillary wedge pressure (PCWP). We then reassessed these patients with the same gold standard invasive test to determine the impact of maintaining sinus rhythm on their exercise capacity and HFpEF haemodynamic parameters. We conclude that there is a high prevalence of undiagnosed HFpEF in people considered for CA for AF and that restoration and maintenance of sinus rhythm can reverse HFpEF in selected patient population. Chapter 7: AVNRT is the most common form of regular tachycardia in the world. It can coexist with AF and in some cases can lead to heart failure. CA is considered the best treatment for people with AVNRT. At the time of ablation, there is a cardiac rhythm response to ablation called junctional rhythm (JR) which is highly sensitive but not specific for successful ablation. It is not known if characteristics of the JR can guide ablation and predict success. We performed a prospective multicentre observational study to explore the link between JR and procedural outcome. We conclude that even short ablations at the effective site can be successful and that junctional rhythm does not help determine success during ablation.
Multimodal Ultra-High-Field MRI Biomarkers of Epilepsy
The Default Mode Network (DMN), whose major hub is the PCC/precuneus, is one of the key resting-state networks in the brain. Many research groups have investigated its role in a range of neurological and psychiatric conditions. In particular, resting-state fMRI has demonstrated DMN alterations in various forms of epilepsy. However, most of these studies recruited patients with either focal or generalised epilepsy, and few comparisons were made between the fMRI characteristics of these conditions. Magnetic resonance spectroscopy (MRS) was employed in several studies of patients with epilepsy. The concentrations of metabolites such as glutamate and gamma-amino butyric acid (GABA), the major excitatory and inhibitory neurotransmitters in the CNS, and glutathione, the major free radical scavenging compound in the brain, could not be accurately determined in magnetic fields up to 4T due to overlapping resonances from other molecules using conventional vendor supplied sequences. The advent of 7T MRI may help improve quantification of these metabolites. In this study we developed a multimodal protocol consisting of resting-state fMRI and MRS of a cubic 8 mL voxel located in the PCC/precuneus using the stimulated echo acquisition mode (STEAM) sequence with ultrashort TE at 7T. Resting-state fMRI was acquired from 12 right-handed healthy volunteers, and reproducibility of quantitative MRS of glutamate, glutathione and GABA was assessed in 10 of these volunteers. We later recruited 35 right-handed patients with epilepsy (19 with temporal lobe epilepsy and 16 with idiopathic generalised epilepsy) who underwent 7T MRI scans using a protocol comprising resting-state fMRI and a single MRS sequence identical to the ones used in the healthy controls. We discovered a linear correlation between the ratio of glutamate to GABA concentration in the PCC/precuneus of healthy controls and a similar linear correlation with age of the functional connectivity of the PCC/precuneus to other major DMN hubs. However, no independent correlation was seen between PCC/precuneus functional connectivity and glutamate, GABA, or the ratio of their concentrations, thus lending further support to the use of both MRS and fMRI as complementary methods in a multimodal pipeline exploring the effects of epilepsy in the patient groups. Analysis of MRS results demonstrated an overall increased concentration of glutathione in patients with idiopathic generalised epilepsy as compared to healthy controls. Analysis of fMRI data led to the identification of several cortical clusters, whose functional connectivity, and in some cases also pattern of activation, is different between patients with idiopathic generalised epilepsy and temporal lobe epilepsy. Subgroup analysis related to seizure focus laterality and short-term seizure-freedom was also performed. No significant correlation was found between the glutamate/GABA ratio or the mean functional connectivity of the PCC/precuneus with age or duration of epilepsy in the patient groups. In conclusion, multimodal MRI sequences have demonstrated distinct patterns of resting-state connectivity and metabolic profiles that are different between patients and controls and between patient subgroups. If corroborated in larger cohorts, such findings may be useful in the diagnosis and management of patients with epilepsy, and especially in expediting the diagnosis of the important subgroup of patients with drug-resistant epilepsy.
The role of neutrophils in immune responses in malaria infection
Abstract Malaria remains a major health challenge worldwide with an estimated incidence of more than 200 million cases per year, and is one of the leading causes of morbidity and mortality in tropical and subtropical regions. Plasmodium falciparum malaria causes severe morbidity and mortality, especially in young children and pregnant women. Clinical malaria is caused by blood-stage parasites. The mature stage of P. falciparum parasites produces the clonally variant adhesion protein called P. falciparum erythrocyte membrane protein 1 (PfEMP1) which is expressed on the surface of infected erythrocytes (IE) and mediates attachment to endothelial surface receptors. If malaria is caused during pregnancy, P. falciparum parasites produce PfEMP1 VAR2CSA protein on the surface of IEs and mediate the binding to placental receptor, chondroitin sulfate A. Repeatedly malaria-exposed individuals develop immunity and become protected from severe clinical malaria. Naturally acquired antibodies against VAR2CSA are produced during successive pregnancies, and VAR2CSA is also an important vaccine candidate. Parasite sequestration and inflammation are assumed to be the two main mechanisms of malaria pathogenesis and complications. Phagocytic cells play an important role in innate immunity to malaria. Neutrophils are the most abundant innate immune cells, and play a crucial role in pathogen clearance. However, neutrophil responses to human malaria are understudied. This dissertation aims to investigate the role of neutrophils in the immune response in malaria, including in phagocytosis of malaria-infected cells, and formation of neutrophil extracellular traps, or NETs in human tissues and in vitro. In chapter four, we evaluate functional antibody responses to pregnancy-specific IEs and VAR2CSA DBL antigens in association with protection against placental malaria. We used plasma samples from PNG pregnant women collected upon enrollment into a randomized controlled trial intermittent preventive treatment in pregnancy. Women were followed up until delivery and were grouped into placental malaria (PM) and infected but no placental malaria (no PM) and noninfected at delivery, based on light microscopy of Giemsa stained placental sections and peripheral blood smear together with PCR of peripheral blood. To measure ADNP activity in test plasma, neutrophils were isolated from whole blood collected from healthy donors by negative selection and were incubated with placental binding IEs or beads coated with VAR2CSA DBL antigens, and phagocytosis was measured by flow cytometry. Functional antibodies induce neutrophil phagocytosis of IEs expressing placental binding parasites and antibody-dependent neutrophil phagocytosis (ADNP) for IE and DBL2 coated beads, expressed as the average of 3 neutrophil donors, differed between pregnant women with malaria infection but no placental malaria and women with placental malaria. However, we observed no evidence of a difference between no PM and PM groups for DBL3 and DBL 5 coated beads, suggesting that DBL2 domain of VAR2CSA is an important target of protective immunity. In chapter five, we investigated the ability of IE to induce NET formation in vitro. Human neutrophils were cocultured with purified IEs or merozoites. SYTOX green was used to assess DNA release from neutrophils; confocal microscopy to assess changes in neutrophil morphology; and ELISA was used to quantify myeloperoxidase (MPO) release as a measure of neutrophil degranulation. Incubation of P. falciparum IEs with neutrophils did not cause significant DNA release. By confocal microscopy, neutrophils stimulated with IEs displayed normal lobulated nuclei and showed only very limited NET formation. The ability of IE to inhibit NET formation was investigated. For this purpose, neutrophils and IEs or purified merozoites were cocultured, with or without potent NETs stimulators phorbol 12-myristate 13-acetate (PMA), or lipopolysaccharide (LPS). The formation of NETs increased substantially upon stimulation with PMA or LPS, but IEs were effective in inhibiting PMA-induced, but not LPS-induced, NET formation. IEs also inhibited neutrophil degranulation, decreasing PMA-induced MPO release. By contrast, P. falciparum merozoites did not inhibit NET formation or neutrophil degranulation. We report, for the first time, that IEs inhibit NET formation and myeloperoxidase (MPO) release as a means of neutrophil degranulation. Inhibition of NET formation might help the malaria parasite escape host immune responses, contributing to parasite survival. Identification of the mechanism by which P. falciparum inhibits NET formation could contribute to the development of new treatment strategies for malaria. In chapter six, we hypothesized that neutrophil infiltration to the placenta is increased during malaria in pregnancy and leads to the formation of NETs in the IVS of the placenta. Sections of paraffin-embedded placental tissues were evaluated following staining with Giemsa and neutrophil specific markers (immunohistochemistry and immunofluorescence). Our findings showed that more neutrophils migrate into the IVS of placentas infected with malaria compared to uninfected controls. The presence of NETs was significantly higher in malaria infected placentas compared to controls. Overall, this Ph.D. thesis provides understanding of the essential role of neutrophils in the immune response to malaria including in opsonic phagocytosis of malaria-infected cells, and formation of NETs in response to malaria parasites in human tissues and in vitro.
Protein and energy delivery in critically ill adult patients and the impact on nutritional and patient-centred outcomes
Adult patients who survive critical illness are known to be at risk of several adverse health consequences, including substantial muscle loss, impaired muscle function and reduced quality of life. While nutrition interventions for critically ill patients may attenuate muscle loss, this therapeutic approach remains unproven. Evidence published by others over the period of this PhD program challenged the existing orthodoxy that greater overall calorie provision during the acute phase of critical illness improves overall patient outcomes. Accordingly, the PhD candidate specifically investigated the relationship between protein provision in critical illness and patient-centred outcomes. This thesis comprises several discrete but aligned studies, including a systematic and narrative review of the literature, three observational studies, and a pilot parallel group randomised clinical trial (RCT). The systematic review and meta-analysis established that current practice guidelines recommending the amount of enteral protein are not based on strong evidence. Moreover, there is a lack of high-quality data about the impact of protein provision on functional outcomes that are important to patients. The narrative review evaluated the methodologies and outcomes used to quantify the effect of nutrition therapy in the critically ill. While patient centred and less direct surrogate clinical outcomes are increasingly being used in the nutrition literature, there remains substantial variation in the tools used and the outcomes selected. In a prospective observational study of patients surviving an intensive care unit (ICU) admission following serious trauma, it was identified that many of the outcomes that would be desirable to measure were not feasible after ICU discharge in this patient cohort. In contrast, it was feasible to implement these methodologies in ICU in a separate cohort of critically ill patients, and cumulative nutrition deficits in the latter study were associated with inferior nutritional and patient-centred outcomes, including the presence of malnutrition and ICU-acquired weakness and greater loss of fat-free mass. To evaluate whether it was feasible to increase the enteral protein received by at-risk ICU patients to the doses recommended in practice guidelines, and establish point estimates for the impact of this strategy on patient-centred outcomes, a parallel group RCT was conducted. This single-centre single-blinded pilot trial compared an intervention of a ‘volume-based’ enteral feeding protocol with protein supplementation to usual care. The intervention delivered greater amounts of protein and energy than received by the usual care group, and this was associated with attenuation of muscle loss and reduced prevalence of malnutrition in survivors at ICU discharge. These data are novel, describing the first successful delivery of enteral protein within current guideline recommendations and a consequent favourable effect on muscle mass. Based on this program of work, a multi-centre randomised clinical trial is required to explore the signals observed in these studies, and to generate robust evidence regarding optimal provision of enteral protein during critical illness and the impacts on functional recovery and quality of life in those who survive.
Liver-bone relationships: integrative pathophysiology, diagnostic, prognostic, preventive and therapeutic considerations
Over the last 10 years we examined complex multidirectional interactions between the digestive system and osteoporotic fractures focusing on pathophysiological and clinical issues of liver-bone relationships. These included: vitamin D, vitamin K and parathyroid hormone in chronic liver and pancreatic diseases (2 papers: [1, 2]); associations between liver function, bone-mineral biomarkers, indices of iron metabolism and adipokines (leptin, adiponectin, resistin) in orthogeriatric patients (3 papers: [3-5]); diagnostic and prognostic indicators of osteoporosis, fractures, and in- hospital outcomes (5 papers: [6-10]). A shortened overview of our main findings is presented.
Investigating the role of macrophages in gastric cancer
Gastric cancer (GC) is lethal, with standard treatments offering little benefit. The immune component of the tumour microenvironment has been shown to highly influence GC progression and response to immune checkpoint therapies (ICTs). Tumour-associated macrophages (TAMs), one of the most abundant immune infiltrates in GC, are known for their plasticity and multiplicity of function. An abundance of TAMs is commonly associated with poor survival of cancer patients. However, their influences on survival in the GC setting are inconsistent between studies possibly due to a range of activation states and phenotypes and require further investigation. In this study, a human GC cohort was interrogated by multiplex immunohistochemistry and analysed using comprehensive bioinformatic spatial and transcriptomic techniques to investigate the spectrum of TAMs in GC. Their spatial distribution within the tumour and association with tumour-infiltrating T lymphocytes (TILs), GC microenvironment and clinical outcomes were determined. In silico and in vivo models were used to validate these findings. Seven predominant TAM populations were identified in a complexed multi-dimensional spectrum model. Each TAM population exhibited a unique combination of cellular markers and correlated with a specific environmental signature resembling published characteristics of in vitro-polarised macrophages. The spatial distribution pattern of TAMs was highly-related to their distance to tumour cells but was independent of their overall densities. This distribution pattern was described as an overall decrease in CD206, and increases in IRF8, CD68 and CD163 as TAMs near the tumour cells. The CD163+ TAMs expressing concurrent high CD68 (CD68++CD163+) were associated with improved patient prognosis. These TAMs co-existed with the CD3+CD8+ TILs within the same tumour. NOX2 and PD1 were identified as functional markers for these immune populations, respectively, and an upregulation of these markers on corresponding cells was observed when the cells were in proximity with each other. Inclusion of these functional markers improved the survival predictive value of these immune populations compared to using overall cell density, and suggests that these immune cells may be functioning through the NOX2/PD1 axis. This may reflect the antigen-presentation ability and activation statues of the TAMs and TILs, respectively. TAMs is commonly correlated with high programmed death ligand 1 (PDL1) expression, a current ICT target, in the literature. In this study, the CD68-only and CD206+ TAMs were identified as specific TAM populations that exhibit high PDL1. This indicates that these TAMs may be particularly associated with immunosuppression and may serve as a complementary biomarker for ICTs in addition to PDL1. An in vivo macrophage depletion protocol was established in an immune-incompetent mouse model to further test these hypotheses on macrophage subtypes. Concentrations of different clodronate-liposome constructs were optimised for investigating tumour invasion and progression in the absence of overall and CD206+ macrophages. In conclusion, this Thesis (i) establishes methods which allow investigation and reveal the spatial heterogeneity of TAMs in human GC, and (ii) identifies a specific TAM/TIL pair and PDL1+ TAM populations that may be utilised as potential therapeutic targets and biomarkers of patient survival and response to ICT.
Investigating the Role of Tau and TDP-43 Pathologies in Traumatic Brain Injury
Traumatic brain injury (TBI) is a leading cause of death and morbidity worldwide, and is also associated with the later onset of other neurodegenerative conditions. However, the pathogenesis of TBI is poorly understood, and there is currently no treatment known to improve long-term outcomes. Hyperphosphorylated tau has been implicated in the pathogenesis of TBI, where the accumulation of hyperphosphorylated tau has been associated with long term neurological deficits. The presence of protein inclusions consisting of TAR DNA-binding protein 43kd (TDP-43) are a pathological hallmark in motor neuron diseases (MND). Notably, TBI is a risk factor in the development of MND, and motor neuron loss, corticospinal tract degeneration and TDP-43 abnormalities have also been observed in individuals with a history of TBI. While preliminary studies suggest that each of these proteinopathies may occur in the aftermath of TBI, the role of tau and TDP-43 remain poorly understood in the TBI context. In this thesis, I examined the role of tau and TDP-43 using wild type and transgenic animal models in the context of TBI and repeated mild TBI. It was found that TBI resulted in abnormalities in both tau and TDP-43, and this was associated with functional consequences. It was also found that that treatment with sodium selenate reduced hyperphosphorylated tau and improved pathological and functional outcomes in a model of repeated mild TBI. Taken together, tau and TDP-43 pathologies appear to have an important role in preclinical TBI and represent potential pharmaceutical targets to improve outcomes.
The role of 18FDG-PET in predicting seizure outcomes and memory deficits following an anterior temporal lobe resection for drug resistant epilepsy
Successful epilepsy surgery transforms the lives of patients with drug-resistant epilepsy. Yet, the seizure recurrence seen in a significant proportion of patients calls for improved selection of surgical candidates. The findings of this study, focusing on the role of 18FDG-PET as a metabolic predictor of memory deficits and seizure outcomes in patients with drug-resistant mesial temporal lobe epilepsy (MTLE) following an anterior temporal lobe resection, demonstrated the laterality-specific differentiality of metabolic patterns, with significantly higher rates of bitemporal hypometabolism observed in right MTLE patients heralding a 5-fold increased risk of postoperative seizure recurrence, whereas left MTLE patients exhibited more extensive ipsitemporal hypometabolism. While the real-world clinical utility of 18FDG-PET in predicting memory deficits in patients with MTLE is limited, the study findings suggest the need for further research into the role of impaired arbitrary learning as a potential biomarker of bitemporal epileptic network involvement in patients with right MTLE as well as the need for using task-specific neurocognitive markers in the evaluation of epilepsy surgery candidates. Lastly, while the extent of the postoperative gliosis was not associated with late seizure recurrence, time-related postoperative changes could offer further insights into the role of glial astrocytes in epileptogenesis and neuroprotection. The study findings enhance our understanding of laterality specific epileptic networks and have the distinct potential to advance patient care by influencing the selection of surgical candidates and assisting with preoperative patient counselling.
Evaluation of renal bone disease: advancing our understanding of associations and diagnostic challenges
In patients with chronic kidney disease (CKD), disordered mineral metabolism, deterioration in bone quality and extra-skeletal calcification constitute the clinical syndrome known as chronic kidney disease – mineral and bone disorder (CKD–MBD). Renal osteodystrophy (ROD) is the skeletal component of CKD-MBD, and refers to abnormalities of bone turnover, mineralisation, volume, linear growth or strength. The erosion of bone quality in CKD confers an excess fracture risk through stages of CKD, is not attenuated by kidney transplantation and can be associated with persistent hyperparathyroidism in kidney transplant recipients (KTRs). The currently available tools in clinical practice have limited utility in evaluating ROD and add to diagnostic and therapeutic uncertainty in this area. The mechanisms of bone loss after transplantation and their relationship with changes in bone structure and markers of mineral metabolism are poorly understood. Tackling these challenges requires an enhanced understanding of the pathophysiology of ROD and development of tools for accurate evaluation of renal bone disease. The advent of high-resolution (HR) imaging and newer biomarkers has provided greater insight and generated research interest in CKD-MBD. HR imaging studies of bone microarchitecture have highlighted the contribution of cortical bone structure to overall bone strength and its association with hyperparathyroidism and bone fragility in KTRs and patients with CKD. HR magnetic resonance imaging (MRI) using customised processing techniques has proven to be an excellent non-invasive tool for studying bone microarchitecture. My research explored prevalent bone and mineral metabolism abnormalities and the role of various contemporary imaging modalities, in particular MRI, in evaluation of ROD. Chapter 2, a cross-sectional study of histological abnormalities of bone in a relatively young cohort of potential KTRs, highlights severe deterioration of cortical microarchitecture but predominantly normal trabecular parameters, reinforcing the importance of comprehensive cortical bone evaluation in patients with CKD. Chapter 3 describes a proof-of-concept evaluation of bone microarchitecture in patients with CKD using MRI with findings correlated to those obtained by bone biopsy and other bone imaging techniques (Dual-energy X-ray absorptiometry, DXA; peripheral quantitative computed tomography, pQCT; trabecular bone score, TBS), to highlight advantages of MRI as a diagnostic tool in ROD. MRI demonstrated significant and relevant associations with important bone biopsy and DXA parameters but could not establish a reliable relationship with bone turnover. No associations were observed with bone turnover markers. This study also underlined the inherent heterogeneity of bone microarchitecture at differing skeletal sites and the pitfalls of assessing these and comparing imaging modalities with vastly different resolutions and protocols. The study described in Chapter 4 monitored changes in bone density and microarchitecture in KTRs over one year after transplantation using MRI, DXA, pQCT and TBS and biomarkers. Trabecular bone structure and connectivity deteriorated independent of changes in BMD but associated with PTH levels, complemented by significant reduction in TBS. Unlike in other contemporary studies, there was no progressive deterioration in cortical bone. Chapter 5 describes an exploration of mineral metabolism and clinical outcomes after transplantation in a large cohort of KTRs, focusing on the impact of discontinuation of cinacalcet on these parameters. Persistent post-transplantation hyperparathyroidism and hypercalcaemia, consistent with available data, was demonstrated, but the clinical significance of these mineral metabolism markers was unclear. Historically, aluminium deposition in bone was recognised as a major cause of adynamic bone disease but is now less relevant due to increasingly rare use and phasing out of aluminium-containing phosphate binders in clinical practice. However, many centres continue to routinely screen dialysis patients for aluminium levels. The study in Chapter 6 examined the utility of routine testing of serum aluminium levels in the haemodialysis population in our centre via a retrospective audit of tests performed on 755 patients over four years. Only 0.5% of 2058 test results matched criteria for toxic aluminium levels, with no evidence of clinical toxicity and progressive reduction in the proportion of elevated aluminium levels over the years. This led to cessation of routine testing of aluminium levels in our dialysis unit. In conclusion, clinical studies presented in this thesis add to the increasing body of literature about the bone abnormalities prevalent in CKD and highlight the novel use of MRI in assessing bone quality in CKD and in KTRs. Overall, non-invasive HR imaging, particularly MRI, has the potential to close the vast gap between screening of BMD using DXA and comprehensive diagnosis by bone biopsy. This needs validation in large trials with hard clinical endpoints and should be viewed in the context of a plethora of technical caveats and issues of high cost and availability. The utility of MRI (and other HR imaging) would be enhanced by more accurate and reliable non-invasive markers of bone turnover and mineralisation. These challenges are significant, but not insurmountable. A coordinated, multidisciplinary, patient-focused effort is needed from the nephrology research community to address the unmet needs of patients with ROD and CKD-MBD.
Characterisation of bromodomain proteins in the malaria parasite Plasmodium falciparum
Plasmodium falciparum is a protozoan parasite that is responsible for the most severe and fatal form of human malaria. In addition to the canonical histones, the parasite possesses divergent and Apicomplexa-specific histone variants. The N-terminal tails of histones in P. falciparum are extensively acetylated and carry unique acetylation marks. P. falciparum relies on this network of histone post-translational modifications and interactive, chromatin-associated proteins to modulate its gene expression. Integral to this process are bromodomain (BRD) histone reader proteins which interact with acetylated lysine. The rapid emergence and development of human BRD inhibitors for the treatment of many diseases prompted our project to study P. falciparum BRD proteins (PfBDPs) and assess their value as potential, novel, antimalarial therapies. There are eight bromodomain proteins in P. falciparum. P. falciparum bromodomain protein 1 (PfBDP1) is critical for the coordinated regulation of invasion genes and is essential for parasite survival. In addition, a piggyBac transposon mutagenesis forward-genetic screen in P. falciparum showed that PfBDP2, PfGCN5 and PfTAF2 are essential for the parasite during the blood, asexual life cycle. Recent studies showed that PfBDPs can interact with histone acetylations and transcription factors as well as other chromatin-associated proteins to form a network of histone reader complexes. This thesis investigates the functions of PfBDP4 and PfBDP3 and their importance to parasite survival. Conditional disruption of PfBDP4 caused a growth defect from which an essential role in the blood stage, asexual life cycle was inferred. Chromatin immunoprecipitation sequencing (ChIP-seq) indicated that PfBDP4 was enriched upstream of highly expressed genes, especially genes that are important for parasite invasion. In addition, PfBDP4 shares genomic localisation with an Apicomplexa-specific transcription factor AP2-I and the BRD protein PfBDP1, confirming their interaction in a complex as observed in previous studies. PfBDP4 was also enriched upstream of genes that are involved in metabolic pathways such as proteolysis and phosphorylation which demonstrates that PfBDP4 likely regulates multiple critical pathways of the parasite. In contrast, parasite growth was not inhibited by knockdown of PfBDP3, which is consistent with previous studies. In trophozoites, PfBDP3 was enriched in heterochromatin or compact chromatin, where genes are generally silenced. As PfBDP3 has two BRDs, PfBDP3 may act as a scaffold for proteins that are associated with heterochromatin. Finally, hit compounds from collaborators biochemical screens of recombinant PfBDP BRDs were tested for P. falciparum in vitro growth inhibition. Potent hit compounds were identified. These confirmed hits should now be tested for specificity and selectivity to confirm that they are acting on-target and do not cause cytotoxic effects in human cells. Overall, this study showed that PfBDP4 is associated with active promoters of select processes and its BRD is essential for the parasite survival while PfBDP3 is associated with heterochromatin and is non-essential during the asexual life cycle. This work also highlighted PfBDPs as novel drug targets in P. falciparum and demonstrated the feasibility of identifying potent PfBDP inhibitors as candidates for future investigation.
Peripheral quantitative computed tomography (pQCT)-based finite element modelling and its application in different populations
Background: Dual energy X-ray absorptiometry (DXA), as the most established tool for the assessment of bone fragility, has limitations in identifying individuals with increased fracture risk. Peripheral quantitative computed tomography (pQCT) can provide additional information about bone fragility compared with DXA alone. The three-dimensional data acquired by peripheral quantitative computed tomography are a potential source to generate finite element analysis (FEA) models to further estimate bone mechanical properties. Aims: This doctoral project aimed to establish a FEA model based on pQCT data (pQCT-FEA) and to investigate its role in the assessment of bone fragility in three populations: older patients with low-trauma fracture, women following surgical menopause and young women with type 1 diabetes mellitus (T1DM). Methods: This project consisted of four parts. In the first study, FEA models were established using distal radius pQCT cross-sections and were validated against forearm failure load obtained from mechanical testing in laboratory. In the second study, variables of DXA, standard pQCT and pQCT-FEA were compared between older patients with low-trauma fracture and controls. Their ability to classify the two groups were also examined. In the third study, bone loss was examined between women who received and who did not receive hormone therapy following risk reducing bilateral salpingo-oophorectomy (RRBSO) and controls. In the fourth study, comparison was made in DXA, standard pQCT and pQCT-FEA variables between young women with T1DM and age-, height- and weight-matched controls. Results: For Study 1, high coefficient of determination (highest r2 = 0.86) was observed between pQCT-FEA stiffness variables and forearm failure load. For Study 2, there were significant difference in pQCT-FEA stiffness variables between fracture patients and controls. Enhanced area under the receiver operating characteristic curve (AUROC) was found in pQCT-FEA stiffness variables in both females (0.83 for shearing stiffness) and males (0.81 for bending stiffness) compared with DXA variables (0.71 and 0.62, respectively, for total hip areal bone density). For Study 3, an average 14.1% decrease was observed in bending stiffness by pQCT-FEA in women who did not receive hormone therapy following surgical menopause. Although DXA can also detect bone loss over the 24 months follow up, the change was smaller (up to 6.0% at the femoral neck). At the 24 months visit, pQCT-FEA variables differ between women following surgical menopause and controls. For Study 4, no difference was observed in DXA measurements between young women with T1DM (p >= 0.08) although there was a trend towards lower aBMD at the lumbar spine in T1DM subjects than in controls after adjustment for confounders (p = 0.053). Significantly lower stiffness by pQCT-FEA was detected in young women with T1DM. Conclusions: pQCT-FEA has good ability to predict bone failure load obtained from mechanical testing, which facilitates its application in clinical settings. It can provide additional information about bone strength deficits in patients with fragility fracture, women following surgical menopause and young women with T1DM. These findings warrant future study using pQCT-FEA for the assessment of bone fragility in other populations.