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(2022)ThesisThe homeostatic regulation of amino acid concentrations is crucial for optimal brain function and development. Different amino acid transporters at cell membranes work together to facilitate the movement of amino acids into and out of the brain. Despite countless in vitro and in vivo research on these amino acids' activities, many fundamental concerns about their metabolic function in different brain areas and pathophysiological conditions remain unanswered. In the framework of this thesis, the effects of exogenous administration of several non-essential amino acids and the participation of their specific transporters in brain metabolism were investigated in Guinea pig cortical brain slices and mouse brain tissues using a targeted neuropharmacological and metabolomic strategy. Alterations in brain metabolism were analyzed using 1H and 13C nuclear magnetic resonance spectroscopy to evaluate changes in metabolite pools and 13C-enriched substrates. All the amino acid transporters mentioned in this study were addressed by the existing solute carrier (SLC) gene nomenclature system for amino acid transporters. The effect of exogenous L-aspartate, L-ornithine, and their salt, L-aspartate-L-ornithine (LOLA), on brain metabolism was investigated with or without an intact blood-brain barrier (BBB). The results indicated that neither L-aspartate, L-ornithine, nor LOLA, affected brain metabolism with an intact BBB. In cortical tissue slices L-aspartate increased brain metabolism concentration-dependently, L-ornithine significantly slowed it at higher concentrations (100 μmol/L), and the effects of LOLA was largely dependent on the balance of its two constituent amino acids. D-aspartate, another isoform of aspartate, produced a range of metabolic impacts, particularly on glutamatergic and GABAergic systems, with varying concentrations. In principal component analysis, the effects of D-aspartate were clearly distinguished from those of L-aspartate, indicating a metabolic pattern distinct from that of excitatory mechanisms. L-Proline administration significantly inhibited brain metabolism in Guinea pig cortical tissue slices, indicating a GABA-like effect; however, it was not a significant metabolic substrate. While it was actively taken up by cells in a concentration-dependent manner but was not completely metabolized. The metabolic pattern revealed that L-proline's effects clustered with 3-aminopropyl(methyl)phosphinic acid (SKF 97541), GABA, 1,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) and (5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]thieno[2,3-f][1,4]diazepine-3-carboxylic acid) 1,1-dimethylethyl ester (RO194603) at lower concentrations (10 μmol/L) and with vigabatrin and RO194603 at higher concentrations (100 μmol/L); indicating that proline may act as a GABAB receptor agonist or GABAArho antagonist. Deletion of SLC6A17/NTT4 (neurotransmitter transporter 4) gene significantly impaired glutamate-glutamine cycle, reduced incorporation of 13C into Krebs cycle intermediates, and increased incorporation into lactate in the brain of mice lacking the gene. NTT4 knockout also altered several important metabolites in glutamatergic neurones, implying that it is a crucial transporter for maintaining brain amino acid homeostasis. Investigation of glutamine transport in cerebellum demonstrated that system A dominates glutamine transport in the cerebellum, with contributions from system N, which is inhibited by histidine and 2-(Methylamino)-2-methylpropionic acid (MeAIB) exerting the most metabolic influence. Inhibition of systems A and L by L-γ-Glutamyl-p-nitroanilide (GPNA) and 2-amino-4-bis(aryloxybenzyl)aminobutanoic acid (AABA) did not influence glutamine transport due to their low affinity for the transporters. Inhibition of systems L and B0 by 2-Aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) showed little effect on fluxes from [1-13C]D-glucose but increased the flux of [1,2-13C]acetate into Glu C4,5 and Gln C4,5. Effects of cycloleucine were comparable to BCH but less powerful. This study provided new insight into the role of several non-essential amino acids in brain metabolism and also showed how brain metabolism is regulated in different brain regions.
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(2022)ThesisThis 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.
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(2022)ThesisObstructive 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.
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(2022)ThesisThe emergence of single cell RNA sequencing technologies has opened a vast number of strategies to look at various aspects of immunity to infectious diseases, especially into the adaptive immune system. Here, we applied scRNA-seq to study rare antigen-specific memory B cells in Hepatitis C and COVID-19 disease. Firstly, a tool for the reconstruction of single T cell and B cell receptor (TCR and BCR) sequencing method was developed that can obtain full-length repertoire from short read sequencing of T and B cell at single cell resolution. This tool, VDJPuzzle was able to report the detection of V(D)J genes, isotypes, somatic hypermutations, membrane vs secreted exon isoforms. VDJPuzzle showed highly accurate detection of V(D)J when benchmarked against available tools together. The utility of this tool to explore infection dynamics associated between BCR and disease outcome was then explored for two diseases: Hepatitis C (HCV) and COVID-19. In the HCV study, properties associated with rare HCV-specific memory B cells and varied HCV infection outcome in primary infection and reinfection were examined. In the primary infection cohort, a higher maturation of memory B cells and a reduced level of aromatic residues was observed in individuals that developed chronic infection. In the reinfection cohort, no association of BCR recall was observed, however transcriptomic differences were observed prior to re-infection across clearers and chronics where, clearers had a pre-existing enrichment of genes associated with the TNF-α signaling via NF-kB pathway. There was also a population of memory B cells with increased CD95 surface expression that showed migration of these cells towards effector long-lived plasma cells showing higher expression of ZBTB32, TBX21, FAS, ITGAX, etc. In the COVID-19 study, we successfully extracted rare SARS-CoV-2 specific memory B cells from mild/moderate and severely infected subjects, at 1 month and 4 months convalescence. Interestingly, the VDJPuzzle tool observed a higher percentage of dual kappa-lambda chains in SARS-CoV-2-specific B cells. The presence of unique phenotypes was also identified in SARS-CoV-2-specific memory B cells from subjects with severe disease. In particular, there was an increase in two populations defined as ‘actBC1’ (CD80hiTNFAIP3hi) and ‘actBC2’ (CD11chiCD95hi) which we hypothesized might be associated with increased longevity of the memory B cell response. The severe patients also maintained a higher level of activation genes associated with TNF-α signaling via NF-kB pathway, which may relate to the ‘cytokine storm’ that is often observed in severe SARS-CoV-2 infected individuals. Whereas, in mild/moderate infected individuals a decline in activated genes was observed over time. In summary, this thesis took advantage of unique sets of scRNA-seq techniques to identify the causes associated with HCV-specific memory B cells towards the clearance and chronic infection outcome in primary and reinfection cohort. Furthermore, we looked at the potential mechanistic insights associated with SARS-CoV-2-specific memory B cell response towards how transcriptome contributes towards the long-term immunity in a mild/moderate or severe infected individuals up to 4 months at convalescence.
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(2022)ThesisIntroduction 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.
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(2022)ThesisRegulation 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.
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(2023)ThesisNormally, the neural control of the brain’s respiratory centre (ventral respiratory column – VRC) alters to accommodate ventilatory requirements in response to respiratory demand. Disruption of the tonic drive to the VRC has been implicated in several respiratory diseases, including chronic obstructive pulmonary disease. However, the pathophysiology of central respiratory circuit dysfunction and subsequent disruption of autonomic respiratory regulating mechanisms under these conditions is unknown. The altered brainstem peptidergic circuits could contribute to this disruption. Moreover, persistent inflammation is involved in the pathogenesis of chronic respiratory diseases, which are exacerbated by chronic hypercapnia and cigarette smoking. Therefore, we investigated the peptidergic alterations and inflammatory responses in mouse brain respiratory regions following hypercapnia and smoking. In Chapters 3 and 4, mice were exposed to hypercapnia or room air for 6 hours, 10 days, or 14 days. In Chapter 3, immunohistochemistry data revealed that galanin immunoreactivity levels remained unchanged in respiratory regions following 10 or 14 days of hypercapnia. Galanin terminals were closely apposite to neurons expressing excitatory Somatostatin (SST) or inhibitory Parvalbumin (PV) in the VRC. In Chapter 4, stress (CORT) and inflammatory responses (interleukin (IL) -6 and IL-1β) in the circulation and brain IL-1β were measured using ELISA. Plasma CORT levels were increased after 10 days but remained unchanged after 6 hours or 14 days of hypercapnia. Cytokine levels were unchanged in circulation but IL-1β levels were higher in the cerebellum following 6 hours and, in the hypothalamus, and VRC following 10 days of hypercapnia. In Chapter 5, dams and their adult offspring were exposed to 12 cigarettes per day or room air (dams for 6 weeks before mating until the offspring were weaned, and the adult offspring for 1-month) prior to culling. In the VRC, IL-1β levels were decreased in dams and increased in offspring following cigarette smoke. Overall, the close apposition of galanin terminals to PV and SST neurons in the VRC indicates that multiple mechanisms are used to generate respiratory rhythm. Hypercapnia or cigarette smoke induces inflammation in the respiratory neural circuits, demonstrating how these two factors may contribute to respiratory irregularities in patients with chronic respiratory diseases.
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Identifying novel genetic targets that regulate azacitidine sensitivity in myelodysplastic syndromes(2022)ThesisAzacitidine (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.
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(2023)ThesisThis doctoral research focuses on expanding medical students’ knowledge and understanding of a non-core curricular topic not normally offered in the medical curriculum. The critical juncture explores how to introduce a less prioritised subject into medical education that complements without competing for space/time in medical students’ core studies. This research focuses on answering the question, “what is important to medical students’ online learning in medical education using dental trauma as the subject for research?” This research is significant because it addresses how online medical education introduces the learning of important extra-curricular topics. Using dental trauma as an example, this research identified evidence to support the opportunity for medical students to learn about managing dental injuries and to raise their awareness of the need for accurate diagnosis and treatment of traumatic dental injuries (TDIs) before they graduate as medical doctors. The originality of the research is demonstrated with the introduction of a new non-core curricular topic related to the medical profession (i.e., dental trauma) into the medical curriculum. The newly developed (online dental trauma) course will be introduced into a new environment (the medical school). The contribution of this research is demonstrated by showing how the online dental trauma course provides a path for cross-disciplinary learning in medical education without the need for a specialist (e.g., dental) educator being present at the medical school. This research also contributes the development of a framework that can be applied for other disciplines (e.g., physiotherapy, optometry, etc.) and provides an online educational pathway to expand new knowledge into medical education. This research identified gaps in knowledge regarding medical doctors’ management of TDIs. Ten important features were identified for medical educators to consider when they develop an online course to introduce a non-curricular core topic. These features are 1) prioritising information to TDI, 2) provide specific facts and information to TDI, 3) information must be easily retrieved, 4) provide career-related information, 5) promote self-confidence, 6) promote learning of new knowledge, 7) easy to understand content materials, 8) establish a logical sequence of learning, 9) visual illustrations to complement written text, and 10) promote self-directed learning. In conclusion, this doctoral research has justified that online medical education enhances extra-curricular learning of a less traditional but important health-related topics considered important to medical professionals. A new topic not normally offered in the medical curriculum is made available through the medical school as an online learning course. Online learning provides an educational pathway for cross-disciplinary learning without any change to the existing curriculum. This research identified important key features that should be included in the design of an online course to provide a positive online learning experience for medical students.
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(2023)ThesisThe incorrect use of child car restraint systems is a longstanding and widespread problem. While child restraints offer good protection, incorrect use increases the risk of death and injury to child occupants in a crash. Strategies to address incorrect use continue to be developed and implemented. However, no strategy has successfully addressed the complexity of interactions between the users i.e., the adult, the child, and the restraint. There is a need to engineer design-based solutions that target the ergonomics of child restraints from a user-centred approach. This thesis presents four interrelated studies evaluating the potential of targeted restraint design to reduce incorrect use. A multimethod, user-centred approach was used to identify restraint design features or clusters of design features with the lowest propensity for misuse by both adult and child users. A diverse range of methods were used to capture the breadth of interactions that occur between the child, the adult and the restraint. Methods include in-depth observation studies, a laboratory trial, a driving trial and the retrospective analysis of naturalistic driving data. Descriptive and complex multilevel statistical analysis techniques were used, including those that allow for the control of potential confounders and clustering of data, as well as content analysis for qualitative data. Results from this body of work demonstrate the substantial scope for reducing incorrect use of child restraints through attention to their design. To realise this reduction, there is a need to shift to user-centred design that recognises the usability of a restraint as a precedent to its utility. Design that incorporates features with a low propensity for misuse by both the adult and child users will ensure a system offers the level of crash protection for which it was designed. There is a need to further investigate different restraint systems, with different features, across all tasks of restraint use from a user-centred approach.