Medicine & Health

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Now showing 1 - 9 of 9
  • (2022) Cao, Jun
    This thesis focuses on the development and applications of magnetic resonance electrical properties tomography (MREPT), which is an emerging imaging modality to noninvasively obtain the electrical properties of tissues, such as conductivity and permittivity. Chapter 2 describes the general information about human research ethics, MRI scanner, MR sequence and the method of phase-based MREPT implemented in this thesis. Chapter 3 examines the repeatability of phase-based MREPT in the brain conductivity measurement using balanced fast field echo (bFFE) and turbo spin echo (TSE) sequences, and investigate the effects of compressed SENSE, whole-head B_1 shimming and video watching during scan on the measurement precision. Chapter 4 investigates the conductivity signal in response to short-duration visual stimulus, compares the signal and functional activation pathway with that of BOLD, and tests the consistency of functional conductivity imaging (funCI) with visual stimulation across participants. Chapter 5 extends the use of functional conductivity imaging to somatosensory stimulation and trigeminal nerve stimulation to evaluate the consistency of functional conductivity activation across different types of stimuli. In addition, visual adaptation experiment is performed to test if the repetition suppression effect can be observed using funCI. Chapter 6 explores if resting state conductivity networks can be reliably constructed using resting state funCI, evaluates the consistency of persistent homology architectures, and compares the links between nodes in the whole brain. Chapter 7 investigates the feasibility of prostate conductivity imaging using MREPT, and distinctive features in the conductivity distribution between healthy participants and participants with suspected abnormalities.

  • (2022) Aishah, Atqiya
    Obstructive sleep apnoea (OSA) pathogenesis is multifactorial with contributions from anatomical and non-anatomical endotypes. Current anatomical-orientated therapies are often inadequate or poorly tolerated with no pharmacotherapies available for OSA. Recent research shows that a combination of noradrenergic and anti-muscarinic agents increases upper-airway muscle activity (key non-anatomical endotype) and reduces OSA severity. Thus, my thesis aimed to investigate alternate therapies for OSA including novel pharmacotherapies targeted towards non-anatomical OSA endotypes as well as combining with existing anatomical approaches based on OSA endotype characterisation. Study 1 investigated the effects of the noradrenergic agent atomoxetine combined with 2 different anti-muscarinics (solifenacin or biperiden) with different receptor-selectivity profiles. Previous studies combined atomoxetine with the antimuscarinic oxybutynin which has broad receptor-selectivity. The goal was to gain mechanistic insight into specific antimuscarinic receptor subtypes for OSA pharmacotherapy which may also have a better side-effect profile versus oxybutynin. The different anti-muscarinics plus atomoxetine improved upper airway function and perceived next-day sleepiness in people with OSA albeit to a lesser extent compared to oxybutynin. This suggests broad or at least M2 muscarinic receptor selectivity may be important in mediating the efficacy of this drug combination for OSA pharmacotherapy. Previous studies with noradrenergic and antimuscarinic agents have been short term (≤1 week) and have not included different doses. Accordingly, in study 2, I investigated longer term (1-month) safety, tolerability, and efficacy of different doses of atomoxetine plus oxybutynin (ato-oxy) versus placebo. 1-month of ato-oxy was generally well-tolerated with a side effect profile consistent with the known profile of each agent alone. An 80/5 mg dosage combination of ato-oxy reduced key OSA severity metrics by ~50%. In study 3 I aimed to investigate if OSA endotype characterisation can be used to inform targeted therapy to resolve OSA in the clinically relevant group of patients who have an incomplete therapeutic response to oral appliance alone (~50% of patients). In these individuals, I systematically added existing anatomical therapies and emerging non-anatomical therapies (i.e., ato-oxy) according to OSA endotype characterisation. OSA was controlled in 50% of participants with addition of other existing anatomical interventions. Almost all the remaining participants were fully treated with the addition of non-anatomical pharmacotherapies. These novel findings provide important insight for the development of novel pharmacotherapy and combination therapy approaches informed by underlying physiological mechanisms for future treatment and management of OSA.

  • (2022) Ye, Pengpeng
    Introduction With rapid ageing of the population, falls have become a significant public health issue in China. Although falls prevention activities have been mandated in the National Essential Public Health Service Package since 2009, recommendations remain only as general advice and are not evidence-based. This thesis aims to generate an evidence base to support the integration of falls prevention programs for older people into the National Essential Public Health Service Package within the Chinese primary health care system. Methods This thesis contains five interrelated studies: an epidemiological study to understand the burden of falls among older people at the national and subnational level in mainland China from 1990 to 2019 using data from the Global Burden of Diseases, Injuries, and Risk Factors Study 2019; a scoping review to characterise existing evidence for fall-prevention interventions in community-dwelling older people in mainland China from 1990 to 2022; a policy review to document national healthy ageing-related policies from a health system perspective in mainland China from 2016 to 2020; a qualitative study to identify the perceptions of facilitators and barriers to implementation of falls prevention programs in primary health care settings in China; and a participatory design study to develop an implementation framework to support the integration of evidence-based falls prevention programs for older people into the National Essential Public Health Service Package. Results In mainland China, the incidence rate of falls in older adults increased substantially over the last three decades. Very few high-quality studies were identified to provide evidence on the effectiveness of existing interventions in China. The national policies on healthy ageing, including falls prevention, were fragmented and inadequate. At the primary health care level, major barriers, including the lack of confidence in delivering interventions, fragmentation in service delivery and limited multi-sectoral collaboration, posed challenges to the implementation of falls prevention in the National Essential Public Health Service Package. An implementation framework consisting of data, workforce, organisation, service and policy themes was jointly developed to support the integration of falls prevention into the National Essential Public Health Service Package. Conclusions In mainland China, falls remain as an ongoing health burden for older people. The challenges to preventing falls for older people primarily arise from low-quality evidence for intervention effectiveness, fragmented policy support and multiple barriers to implementation at the primary health care level. Establishing data-driven surveillance, generating high-quality evidence for the effectiveness of interventions, and creating a supportive environment are three key strategies to address these challenges and have the potential to integrate falls prevention into the National Essential Public Health Service Package within the Chinese primary health care system.

  • (2022) Subramanian, Shruthi
    Regulation of gene expression is crucial in establishing cell identity and function. Chromatin accessibility and binding of transcription factors to target sites in DNA and the assembly of protein complexes that regulate gene transcription is one of many levels of control. The protein products of such transcription may be components of a regulatory network that in turn influences the transcriptional output of itself and other genes. These gene regulatory networks (GRNs) establish and maintain cell-type-specific gene expression while their dynamic remodelling contributes to cell trajectories that direct differentiation of stem cells to more mature cell states. Human blood stem cells residing in the bone marrow continuously repopulate billions of mature circulating blood cells including red blood cells, white blood cells and platelets, each day throughout postnatal life. These haematopoietic stem cells (HSC) undergo a series of differentiating events to give rise to more mature and less stem-cell-like “progenitor” populations enroute to the production of mature circulating cells. Corruption of GRNs may lead to aberrant proliferation and differentiation of these progenitors resulting in bone marrow failure and/or leukaemia. As such, studying these networks in stem and progenitor populations in the bone marrow could provide vital information regarding normal blood cell development in humans. My study explores the interplay of a heptad of transcription factors- FLI1, ERG, GATA2, RUNX1, TAL1, LYL1 and LMO2, that play important roles during blood development. To map the binding patterns of these factors and construct gene regulatory networks in various primary stem and progenitor populations, I fractionated cells including HSCs, common myeloid progenitors (CMP), granulocyte monocyte progenitors (GMP) and megakaryocyte erythroid progenitors (MEP) using fluorescence-activated cell sorting and extracted chromatin from these populations for downstream assays. These included a) chromatin immunoprecipitation (ChIP) and ChIPmentation for genome-wide identification of active (H3K27ac, H3K4me3) and inactive (H3K27me3) histone marks, and transcription factor and co-factor (including CTCF and PU.1) binding. b) HiC to broadly classify the higher order 3D genome structures including compartments and topologically associated domains and c) H3K27Ac HiChIP to identify active looping structures between regulatory and promoter regions. I have shown that the heptad transcription factors exhibit shared and distinct patterns of binding across the stem and progenitor populations, with a preference for binding to regulator-like regions. These transcription factors exhibit combinatorial binding, with the binding of all seven factors showing highest significance. Interestingly, across lineage determining genes such as GATA1 and MPO, I noticed a dynamic accumulation of the heptad transcription factors at gene promoters as cells became more differentiated. Higher order genome architectures were conserved across the four cell types, while chromatin loops between gene promoters and distal regulatory regions showed cell type specificity. On resolving the regulatory network of the seven individual transcription factor genes, I found an interconnected network displaying combinatorial binding that was asymmetric across the four stem and progenitor populations. I was able to connect candidate distal gene regulatory regions with specific gene promoters and relate differential transcription factor binding to differential gene expression in relevant cell populations. Furthermore, I noticed patterns of binding that changed along the differentiation arc across the regulatory regions of genes expressed in mature cells. For example, at gene loci expressed in monocytes/granulocytes, there was an increase in GATA2, ERG, LYL1 and LMO2 and a decrease in TAL1 binding in GMP with respect to other populations. In contrast, at regulatory regions of genes expressed in erythroblasts/megakaryocytes, I noticed an increase in TAL1, GATA2, LYL1 and LMO2 and a concomitant decrease in ERG binding in MEP. Finally, by combining datasets generated in this study, I clustered 85,100 accessible regions present in HSPCs based on their regulatory potential and used transcription factor binding in stem and progenitor populations as a scaffold to map usage of candidate gene regulatory regions during hematopoietic stem cell differentiation. Taken together, my study provides a comprehensive characterisation of the genome wide gene regulatory landscape in rare human blood stem and progenitor cells. The results of this study constitute an important framework for accurate analysis of aberrant regulatory networks in leukemic cells and assist in devising better therapeutic strategies.

  • (2022) Truong, Tuan Kiet Peter
    Azacitidine (AZA) and Decitabine (DAC) are frontline hypomethylating agents (HMAs) effective at altering the natural course of Myelodysplastic Syndromes (MDS). Unfortunately, treatment resistance and failure are hallmarks of HMA therapy. Developing effective HMA combinatorial therapies is challenging as the underlying mechanisms governing AZA response remain uncertain. To address this, I leveraged genome-wide CRISPR-Cas9 dropout screening in a human MDS cell line to identify novel synthetic lethal gene-AZA relationships. First, I looked to ascertain a clinically relevant treatment dose of AZA in the MDS-L cell line through using a combination of cell viability, mass spectrometry, and transcriptomic approaches. I identified a concentration of AZA that exerted more anti-proliferative rather than direct cytotoxic effects, triggered global hypomethylation, and induced clinically relevant transcriptomic signatures. Using the defined dosage, I performed a genome-wide CRISPR-Cas9 screen in the MDS-L cell line where I identified and validated TOPORS as a bonafide target that confers hypersensitivity to AZA therapy. Given AZA is also for Acute Myeloid Leukemia (AML), I determined whether targeting TOPORS could be generalized. Targeting TOPORS significantly sensitized a panel of AML cell lines to AZA. Importantly this combinatorial approach did not functionally impair healthy CD34+ cells and was relatively less toxic compared to MDS cells. As TOPORS is implicated in regulating the DNA damage response through SUMOylation-dependent mechanisms and AZA is known to induce genome instability, I hypothesized that TOPORS contributed to genome stability in response to AZA. TOPORS-edited MDS-L were primed for apoptosis as they accumulated extensive DNA damage and arrested at a late S- G2/M checkpoint in response to AZA. Concordantly, transcriptomics and nuclear proteomics revealed a disruption in the DNA damage response. As there are currently no pharmacological inhibitors of TOPORS, to enable clinical translation I sought to target its key biological processes. Here I piloted the combination of TAK-981, a novel inhibitor of SUMOylation, with AZA or DAC. Combining TAK-981 and AZA was found to be additive in MDS-L and synergistic in MOLM-13, while TAK-981 synergized with DAC in both MDS-L and MOLM-13. Collectively, my data provides a framework for the translation of TAK-981 in combination with HMA as a potential therapeutic strategy for patients with MDS or AML.

  • (2022) Kelly, Shane
    Autoimmune diseases represent a significant cause of morbidity and mortality, for which there is no cure. The pathogenesis of autoimmunity is unknown. Identifying and profiling antigen-specific lymphocytes has the potential to advance our knowledge of disease pathogenesis, and potentially offer more targeted therapy. The overall aim of this thesis was to identify antigen-specific cells in a range of autoimmune diseases and characterise them proteomically, transcriptomically and genomically using novel sequencing technologies. The experimental design comprised identifying pathogenic B cells from peripheral blood by flow cytometry, followed by single cell sorting and sequencing by G&T seq. Those cells confirmed as antigen-specific then underwent in depth genomic, transcriptomic and proteomic analysis. For the cryoglobulinaemia project I succesfully charcaterised antigen-specific B cells in a pateint who had a rare IgA RF-producing clone. It harboured nonsense mutations in TNFAIP3 and in BCL-10, both of which it is anticipated lead to increased NFKB signalling. For the MAG neuropathy project I successfully characterised antigen-specific B cells in all three patients. All had multiple IgM kappa clonal populations, although for two I was able to show that they arose from a common progenitor, despite being heavily mutated. Essentially all clones, irrespective of MAG specificity, harboured a MYD88p.L265P mutation. I also identified a number of other somatic mutations, including in CXCR4 in one patient, and in SIGLEC11 which was present in only one clonal branch of another patient. For the polyclonal models of autoimmunity project (ie PR3-AAV, MPO-AAV and Ro60+SjS) I was successful for two of the three diseases, although the cell frequency was exceedingly rare. In the one patient (who had PR3-AAV) where I isolated enough antigen-specific B cells to perform a meaningful analysis it was notable that they were enriched for IgG4, lambda, J6 gene usage, IGHE expression, and demonstrated low levels of somatic hypermutation. In summary in this thesis I was successful in most of my aims in identifying and multiomic profiling of antigen-specific B cells in a range of autoimmune diseases including cryoglobulinaemic vasculitis, MAG neuropathy and PR3-AAV. However given the significant challenges in isolating such cells in polyclonal models of autoimmunity, consideration needs to be given in sourcing alternative samples to peripheral blood and/or modifying the current experimental strategy.

  • (2022) Kaur, Jagjit
    Secreted by pancreatic β-cells, insulin is the major anabolic hormone, regulating the metabolism of fats, proteins, and carbohydrates. Defects in insulin production or action can lead to diabetes characterized by derangements in glucose handling and metabolic disease. Diabetes affects 420 million people worldwide, increasing morbidity, mortality and placing a burden on healthcare of nations. There is a need for rapid and accurate monitoring of insulin levels to optimize diabetes management and facilitate early diagnosis of insulin related chronic diseases. Conventional strategies such as HPLC, MALDI-TOF, ELISA, etc. used for insulin detection are not suitable for point-of-care testing (POCT) as they are expensive, and require sample preparation, sophisticated instruments, and skilled personnel. Our goal was to develop techniques to allow POCT for insulin in real time. In this study we developed two lateral flow assays (LFAs) based POCT platforms using aptamers as the biorecognition molecules for colorimetric and fluorescent detection of insulin. A range of conditions were tested such as concentrations of aptamers, reporter molecules used, volume of sample required, and assay time to obtain quantify insulin levels using a standard LFA reader. The colorimetric LFAs had linear detection range of 0.01-1 ng.mL-1 and LOD of 0.01 ng.mL-1. The fluorescent LFAs exhibited a linear detection range of 0-4 ng.mL-1 and 0.1 ng.mL-1 LOD. Various signal amplification strategies were incorporated, ie., gold-silver amplification technique and rolling circular amplification (RCA) to further enhance the signal. The developed colorimetric LFAs were successfully used for insulin quantification in rat blood, human blood, and human saliva samples. Although insulin levels were quantified within 12 min, some issues arose such as coagulation, need for dilution, and non-uniform flow through the test strips. Further work is required to optimize blood handling to progress an insulin POCT in real time. Future work could develop a multiplexed strip for detection of different analytes such as HbA1c, glucose, and C-peptide for better management of diabetes, along with a smartphone reader App. This research goes some way to addressing the challenge of providing a reliable and rapid approach for highly sensitive and specific detection of insulin for POCT applications.

  • (2022) Sanchez Miranda, Marta
    The aim of this thesis is the development of microscopy-compatible electronic devices to advance the field of biosensing in two main directions. First, the development of a platform that allows electrical and optical studies of membrane proteins at the single molecule level. We have fabricated InAs nanowire field-effect transistors and PEDOT:PSS organic electrochemical-transistors on 170 μm thick glass slides for their integration with fluorescence microscopes. Each electronic device was fabricated at the bottom of a circular well sealed with a lipid bilayer. A wide range of membrane proteins can be inserted into this lipid bilayer, most of which work as pores or active pumps for the transport of ions across the membrane. This platform allows simultaneous electrical and optical studies of such proteins, achieving single-molecule resolution when a single protein is inserted in the bilayer. We present the fabrication procedures for these devices and the creation of a lipid membrane over them, as well as electrical characterization and ion sensitivity measurements. The development of novel hardware to achieve electrical contact with the devices while performing fluorescence microscopy is also presented. We demonstrate the viability of our platform via the correlation of electrical and optical signals in response to ion concentration and lipid bilayer rupture. We also present a computational model that provides insight into this system and propose future optimization steps towards the incorporation of membrane proteins into the system. Second, the creation of a sensor that allows electrical detection of a moving target at the single molecule level. In this thesis, we present a theoretical model to determine the feasibility of detecting an actin filament or microtubule passing in close proximity to a carbon nanotube field-effect transistor. This platform would allow the detection of moving biomolecules without the need to physically attach the molecules to the electronic devices. Our results showed that electrical detection is possible given the right experimental conditions. This would enable tracking of large numbers of molecules at once, an important advance for applications where the detection of biological agents is relevant, such as biocomputation.

  • (2022) Bello, Idris
    The inflammatory artery diseases atherosclerosis and abdominal aortic aneurysm (AAA) are major causes of morbidity and mortality and there is significant attention towards identifying and targeting prominent inflammatory mediators underpinning these cardiovascular diseases. In the first chapter, the role of the pro-inflammatory and pro-oxidant enzyme myeloperoxidase (MPO) in inflammatory artery disease was studied. A clinical study showed that while circulating plasma MPO levels were not different in AAA patients versus healthy controls, immunohistochemistry showed that the MPO protein was prevalent in human AAA tissue. In the angiotensin II (AngII)-infusion model of AAA and atherosclerosis in apolipoprotein-E gene-deficient (ApoE–/–) mice, administration of 2-thioxanthines (2-TX), a clinically-trialled MPO inhibitor, significantly inhibited AAA but not atherosclerosis. Paradoxically, MPO gene-deficiency did not affect AngII-induced AAA but attenuated atherosclerosis. Notably, 2-TX significantly inhibited AAA in ApoE–/–MPO–/– mice, indicating 2-TX protects against aortic disease in the absence of MPO. The role of MPO in the diabetes-accelerated atherosclerosis in ApoE–/– mice was also examined. While MPO gene-deficiency did not impact on the degree of diabetes it significantly reduced diabetes-accelerated atherosclerosis at the brachiocephalic artery and aortic sinus, but not aortic arch, indicating that MPO exhibits site-specific effect on atherosclerosis. A second chapter focused on semicarbizide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1), a pro-inflammatory enzyme that facilitates the vascular recruitment of activated leukocytes. Treatment of AngII-infused ApoE–/– mice with a clinically-viable SSAO/VAP-1 inhibitor significantly protected against AAA and atherosclerosis, independent of alterations to plasma lipid levels. A third chapter tested the therapeutic efficacy of apolipoprotein A-I (ApoA-I), the major cardioprotective protein in high-density lipoproteins, and a class of immunomodulatory nanoparticles (INPs), which selectively target and disable a pro-inflammatory monocyte subset. Although ApoA-I treatment did not impact on the development of arterial disease in AngII-infused, aged ApoE–/– mice, INPs provided significant protection against AAA and atherosclerosis in these mice. This novel research provides new insights on the roles of MPO and SSAO in AAA and atherosclerosis and identified clinically-viable inhibitors of MPO and SSAO and a class of biodegradable immunomodulatory nanoparticles as potential new therapeutics for treating inflammatory artery disease.