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(2012)ThesisCell immunoisolation systems are fast becoming a favourable approach to cure various challenging diseases and disorders such as type I diabetes. Although the addition of biological molecules to cell immunoisolation devices can significantly enhance their performance by supporting cell viability and function, little is known about their effects on the immunoisolating membrane properties especially its permselectivity. Therefore, this research focused on examining the effect of combining biological molecules with a synthetic polymer on the permeability of hydrogels, with a specific emphasis on encapsulation of insulin producing cells for treatment of diabetes. The research aimed at achieving an optimum balance between a controlled permselectivity and cell survival support. It was hypothesised that covalent incorporation of small amounts of model extracellular matrix (ECM) molecules, heparin and gelatin, would support cell viability without compromising the controlled permselectivity and physico-mechanical properties of the base PVA network. Varying the number of functional groups per PVA backbone successfully controlled the PVA permeability and physico-mechanical properties. A suitable degree of permselectivity was achieved by the highly crosslinked hydrogels. Covalent incorporation of heparin and gelatin at low percentage was successfully achieved without interfering with either their biofunctionalities or the base PVA properties, including its permselectivity. Moreover, the incorporated ECM analogues supported the viability and metabolic activity of pancreatic β-cell lines encapsulated for two weeks. Consequently, biosynthetic hydrogels composed of permselective PVA base material and a small amount of biological molecules show promise as immunoisolating materials for cell-based therapy.
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(2019)ThesisA weave of collagen and elastin fibers supports every tissue in the body, with tissue-, age- and health status-specific spatial and temporal fibre distributions. The newly presented concept of biotextilogy refers to the creation of textiles that emulate nature's own. Microscopy Aided Design and ManufacturE (MADAME) describes an advanced manufacturing method by which spatial distributions of tissue structures such as porosity, structural protein fibers, and permeability characteristics can be engineered and manufactured using biotextilogy alone or in combination with additive manufacturing. Periosteum, a hyperelastic soft tissue sheath covering every bone in the body, exhibits stimuli-responsive or smart properties that would confer great benefit if integrated into medical textiles. These benefits include harnessing movement or displacements to deliver pressure gradients, e.g. to oedematous limbs (actuator function) or changing of its own form when strained by neighboring tissues (sensor function). This thesis also applies biotextilogy and MADAME to test textiles created to mimic the natural patterns of collagen and elastin in periosteum. First the mechanical and strain properties of compression sleeves were characterized. The mechanical testing results from this study showed a lack of gradients in textile samples taken along the length of the sleeve, providing a first step to develop more efficacious compression sleeves. MADAME was then applied, first using microscopy to study elastin and collagen in periosteum, then implementing a recursive approach to better incorporate gradients into textile design using elastic and stiffer fibers mimicking elastin and collagen. Textiles demonstrated spatially tuneable mechanical gradients and strain distributions, validating the concept. Once feasibility had been shown, a set of prototype textiles was constructed using different compositions and combinations of sutures as elastin and collagen analogues. Textiles were tested for biocompatibility by seeding with mammalian cells, and observing cell viability and proliferation over 15 days. Sterilization showed significant effects on material stiffness, though results vary across material and sterilisation procedure. Biotextilogy and MADAME provide a platform for a new class of smart materials and products that exhibit advantageous properties in bending, tension and compression, as well as the capacity to harness forces associated with physiological activity to activate the material’s smart properties.
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(2018)ThesisA patient’s own T cells can be genetically modified and amplified in the laboratory to target antigens expressed on tumour cells through the introduction of chimeric antigen receptor (CAR) genes. Despite considerable advances in the treatment of B cell leukaemia using chimeric antigen receptor T (CART) cells targeting the CD19 antigen, some patients have not responded well, particularly those with solid tumours. Understanding the biology of CART cell effector function may explain treatment failure, and lead to more effective cell therapy products. Population-based assays such as flow cytometry give a snapshot of these complex cellular systems, but do not allow one to study the fate of individual cells over time. Therefore, the aim of this thesis was to apply flow cytometry, time-lapse imaging and singlecell tracking to characterise the dynamics of CART and tumour cell interactions in vitro. The utility of time-lapse imaging and single-cell tracking was demonstrated by quantifying the cytotoxicity of CART cells targeting CD19+ leukaemia cell lines. This thesis also addresses the problem of minimal CART cell effector function against solid tumours by studying the potency of anti-GD2 re-directed Natural Killer (GD2NK) and T cells (GD2T) targeting neuroblastoma spheroids with time-lapse imaging. Flow cytometry studies show that at least 90% of CD19+ leukaemia cells were killed at high effector to target ratios (E:T=10:1), however only 10% killing was achieved at lower ratios (E:T=1:1). Cooperative killing by CART cells was observed by time-lapse imaging without serial killing. In the clinical context, localisation of effector cells through chemotaxis and proliferation at tumour sites may be required for tumour elimination. NK and GD2NK cell penetration of solid neuroblastoma spheroids was superior to T or CART cells. NK but not T cells were able to destroy tumour spheroids. However, coculture of neuroblastoma spheroids with NK and T cells resulted in loss of NK-mediated tumour killing. In conclusion, this thesis provides methodological advances to the study of anti-tumour response generated by innate and CAR-enhanced effector cells against haematological malignancy and solid tumours by application of time-lapse imaging. This thesis also provides insights into the detailed mechanism of effector cell killing by direct observation of the dynamics of effector cell migration, infiltration, conjugate formation and cytotoxicity using single-cell tracking.
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(2018)ThesisAs robotic applications move towards unstructured environments, there is a need for grippers that can manipulate objects securely (preventing slip) and efficiently (applying minimal grip forces). One approach for achieving this involves measuring the coefficient of static friction (COE) at the gripper-object interface. In the absence of torque, the minimum grip force to prevent slip can be estimated from COE and the contact forces . However, torque at the gripper-object interface is generated when the lifting force does not align with the weight vector of the object being lifted. Current robotic gripping systems ignore the need to measure COE, and there is little literature on the grip force required to prevent slip in the presence of a tangential torque (T) at the gripping interface. In this thesis, a grip force control system was developed, using the measurement of COE at the first contact between the gripper and the object, and the continuous measurement of contact forces and torque to approach the minimal grip force required to hold the object stably. The target grip force is determined in real-time during object manipulation. To demonstrate the importance of COE when no torque is present, objects were gripped with the target grip force dependent only on the measured loads and COE. Furthermore, a model was developed in which the minimum grip force preventing slip can be estimated based on COE, load force, and T. This friction model is first validated with respect to the grip force at which slip is predicted to occur, for varying COE, load force, and T. Objects were then gripped (varying load, COE, and T) with the target grip force calculated by the friction model. The results demonstrate that COE-dependent grip force control is superior to gripping without knowledge of COE, irrespective of T at the contact interface, with respect to the system’s ability to prevent slip and simultaneously minimise the grip force applied. The minimum grip force estimation model and grip force controller developed and validated in this thesis have highlighted the value of measuring COE and the necessity of countering torque to ensure a secure and efficient grip.
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(2019)ThesisRecent advancements in cardiac computational modelling allow for ready simulation of bileaflet mitral valve (MV) motion in a contracting left ventricle (LV), demonstrating the capability of computational modelling to simulate the MV diseased state and treatment strategies. Furthermore, recent advancements in image-based modelling can be used for pre-procedural planning of mitral prosthetic valve placement and analysis of intraventricular blood flow. This thesis aims to develop a set of computational models that can simulate normal MV function, MV disorders and treatments to help in the understanding of MV movement and its interaction with blood. In addition, a moving-wall LV computational framework was developed to provide pre-surgical guidance for determining optimal orientation of mitral prostheses. In this thesis, the structural boundaries of a 2D model of the left heart were represented as a spring-like elastic structure. A 3D LV model was subsequently developed, consisting of ideal geometric-shaped MV leaflets and the LV wall. An experimentally-based hyperelastic material formulation was used to model mechanical behaviour of the MV leaflets. For both 2D and 3D ideal models, the MV chordae tendineae and papillary muscle were incorporated. Finally, for the image-based models based on wide-volume full cycle cardiovascular CT images prior to transcatheter MV implantation (TMVI) were developed and analysed (n = 6 patients). Patient-specific computational fluid dynamics simulations of TMVI at various implant insertion angles were performed (n = 30). The 2D and 3D ideal models were successful in simulating the normal and prolapsed states of the MV. Additionally, both models were able to simulate blood movement after the MV prosthetic with and without left ventricular outflow tract (LVOT) flow obstruction. In the image-based models, computed pressure gradients following artificial valve placement compared well with clinical measurements and accurately predicted clinical LVOT obstruction. The simulations demonstrated that LVOT obstruction can be mitigated by adjusting the valve insertion angle, with the extent of residual obstruction contingent on the aorto-mitral-annular angle and LV anatomy.
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(2019)ThesisVisual neuroprostheses aim to restore vision to patients suffering from degenerative retinal diseases such as retinitis pigmentosa and age-related macular degeneration. Development of visual implants faces a great number of challenges in both device design and stimulation strategy. Computational modelling is a powerful tool for exploring and testing new visual prostheses design and stimulation strategies. In this thesis, we have proposed and validated a new version of the classical cable equation valid for any fibre morphology, electrode configuration, or non-uniformity in ion channel expression, implemented using a finite element approach. Moreover, we developed the first continuum multi-domain model of retinal electrical stimulation to represent all main retinal ganglion cell (RGC) compartments. The continuum model was validated against discrete morphologically-realistic OFF and ON RGC models as well as RGC excitation thresholds reported in recently published in vitro experimental studies using intra- and extra-cellular electrical stimulation. The continuum model reproduced the same results as that of the discrete model and in vitro experimental studies. Furthermore, the first degenerate model of retinal electrical stimulation accounting for observed changes occurring in the whole retina was developed, using a detailed model of electrical stimulation of OFF and ON RGCs. Interestingly, the model predicted that suprachoroidal stimulation of the degenerate retina exhibited increased current thresholds, mainly due to the presence of the glial scar layer. In contrast, epiretinal stimulation thresholds were almost similar for both healthy and degenerate models, implying epiretinal prostheses can bypass the influence of the glial scar layer. Various stimulation strategies were examined for both healthy and degenerate retinal models. No significant difference among the three return electrode configurations (monopolar, quasi-monopolar and hexapolar) was found when the distance between electrodes and RGCs was less than the electrode diameter. Electrode spacing was the significant factor underlying increased current thresholds, where electrode size had a marginal impact among all three return electrode configurations. Stimulus pulse polarities and durations were found to have a significant impact on the localisation of evoked phosphenes. Moreover, virtual electrodes could be elicited by using an appropriate time shift between two stimulus waveforms applied to the active electrodes.
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(2010)ThesisThis study investigated retinal ganglion cell (RGC) responses to epiretinal electrical stimulation delivered by hexagonally-arranged bipolar (Hex) electrodes. In vitro experiments were performed using rabbit retinal preparations and a computational model was developed. Electrical stimulation was delivered using 50 and 125 IJm diameter platinum Hex electrodes and single-unit RGC activities were recorded differentially with extracellular tungsten microelectrodes. The majority of the responses exhibited short latencies (<5 ms) and were time-locked to the stimulus pulse. With 100 IJS/phase anodic-first biphasic pulses, threshold charge densities were 24.0 ± 11.2 and 7.7 ± 3.2 1JC/cm2 for 50 1-1m and 125 IJm diameter electrodes, respectively. The estimated chronaxie from the strength-duration relationship was 214 IJS. The short-latency responses of most cells (7/11) persisted at 200 Hz stimulus frequencies. Cell responses (7/7) were not diminished in the presence of cadmium chloride synaptic blocker. RGC responses persisted as the stimulating electrodes moved along the axonal path. Threshold profiles and response characteristics strongly suggested that RGC axons were the neural activation site. Both the model and in vitro data indicated that the lowest threshold point occurred beneath the centre active electrode and thresholds increased rapidly with lateral electrode distance across the retina, suggesting that localised tissue stimulation is achieved with Hex electrodes. Threshold profiles were dependant on the orientation of the electrodes relative to the axons. Thresholds increased up to 20 times when the electrodes were lifted 100 1Jm above the retinal surface. Low thresholds for axonal activation as well as the threshold increase with electrode displacement above the retinal surface are major concerns for epiretinal implant design. Overall, the threshold profiles predicted by the model accurately matched the experimental data obtained in this study.
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(2017)ThesisAngiogenesis, the process through which new blood vessels are formed, relies on coordinated endothelial cell behaviours, regulated by key signalling pathways such as vascular endothelial growth factor (VEGF) and integrin pathways. In vitro study of endothelial cell guidance at multiple scales is vital to understand how different environmental cues are integrated at the subcellular level. The aim of this thesis was to develop high content imaging methods to capture temporal information at the cellular, subcellular and molecular scale in controlled microenvironments. To examine the hypothesis that persistent endothelial cell migration is directed by soluble and surface-bound gradients, a method to create steady soluble or immobilised gradients on radiofrequency-plasma-polymerised surfaces to support endothelial cell attachment and migration was developed. A novel method of imaging single molecule protein adsorption by Total Internal Reflection Fluorescence Microscopy (TIRF-M) was developed. This new single molecule counting method may have future applications, particularly the study of how proteins and cells interact with surfaces at the molecular scale. Endothelial cells should not be considered as homogeneous cell populations as they take on different roles during new vessel growth. Single cell live-cell imaging was developed to capture the heterogeneous nature of endothelial cell migration directed by a soluble gradient. Statistical methods for analysing directed cell motion were evaluated. Both circular and Hotelling’s T² statistics provided robust statistics for evaluating the effect of a chemical gradient on endothelial cell migration. It was surprising to discover that a soluble VEGF165 gradient on its own was not sufficient to direct endothelial cell migration, requiring synergy with a sphingosine-1-phosphate gradient to elicit optimal responses. The Rho GTPases are thought to coordinate cell surface signalling with remodelling of cytoarchitecture for directed endothelial cell motion. A Förster resonance energy transfer (FRET) probe (Raichu) was used to visualise Rho GTPase activity in live endothelial cells at subcellular scales in real time using fluorescence lifetime imaging and intensity-based measurements. A fluorescent protein toolbox was developed to quantify spectral bleed-through. A new quantification method has extended the resolution of live cell FRET measurements by statistical modelling of spectral bleed-through and biological noise.
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(2017)ThesisFalls are the number one cause of injury in the elderly. A fall event can cause adverse physical consequences and psychological and social impacts. More seriously, a long-lie after a fall can exacerbate these adverse consequences. Thus, it is necessary to provide a timely medical rescue for those fallers who live alone in either the community or nursing homes. A fall detector can automatically detect a fall event and alert a caregiver of the occurrence of this event. In this way, health complications related with falls and long-lies are minimised by reducing the time between a fall and the arrival of medical assistance. A wearable fall detector is usually powered by a battery with limited capacity. However, a wearable fall detector needs to continuously implement power-hungry data acquisition and computation tasks. As a result, such devices face the challenge of limited battery life. Furthermore, a short battery life will decrease user compliance. To overcome this impediment to the successful deployment of the device, this thesis presents two generations of the low-power fall detector based on triaxial acceleration and barometric pressure sensing. The proposed fall detectors (generation one and two) were developed to classify between a fall and an activity of daily living (ADL) effectively. More importantly, the devices reduce their power consumption and prolong their battery lives using various low-power technologies in the designs of hardware, firmware, and fall detection algorithm. The devices were evaluated based on data collected in several human trials (simulated falls, simulated ADLs, and free-living trials). Finally, the battery life of each fall detector design was estimated based on power measurements using benchtop electrical test and data from the free-living trial. As a result, the first generation of the fall detector achieves a sensitivity of 93.0% and a specificity of 87.3%, a false alarm rate of 0.022 alarms per hour, and a battery life of 685.1 days. The second generation of the fall detector achieves a sensitivity of 89.5% and a specificity of 96.1%, a false alarm rate of 0.012 alarms per hour, and a battery life of 1,186.4 days.
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(2018)ThesisThe mammalian adult heart is comprised of multiple cell types which interact with each other to maintain structure and function in homeostasis and disease. Extensive characterisation of such populations is therefore crucial to understanding how these cells can be manipulated for better injury resolution and repair. It is hypothesised that a subset of cardiac cells labelled as SCA1+PDGFR?+CD31- cells (S+P+) which reside in the cardiac interstitium are dedicated to cardiac homeostasis and regeneration. They may also support cardiomyocytes and vascular tissue through dedicated paracrine functions. To investigate the activation of these cells during initial stages of inflammation after myocardial infarction (MI), the gene expression (GE) profiles of more than 30,000 cells were analysed following induction of MI by coronary artery ligation. High throughput single cell RNA-seq (scRNAseq) was performed using GFP+CD31- and total interstitial cells (TIP) isolated from a mouse with a H2B-eGFP fusion gene knocked-in to the PDGFR? locus. In-depth bioinformatic analysis including clustering and diffusion mapping analysis revealed known and novel cell states and the presence of cell subsets with potential progenitor cell activity. While the major differentiation pathway directs S+P+ cells towards myofibroblasts differentiation, a novel minority progenitor population was characterised by expression of Wif1, an inhibitor of Wnt signalling. The growth factors PDGF and FGF may recruit S+P+ cells to form immature myofibroblast in vitro with similar GE signature to myofibroblasts isolated from mice following MI in vivo. Both Fluidigm and Chromium 10X scRNAseq technologies had complementary roles: While Fluidigm had much greater depth; there were too few cells for detailed cluster analysis, though cluster identity could be assigned to Fluidigm sequenced cells using a Random Forests classifier trained on the Chromium 10X data-set. High content, single cell GE analysis provides an extraordinary resolution of the cellular repair process. Our findings highlight the role of resident S+P+ as cardiac progenitor cells in the healthy and diseased heart. S+P+ cells are activated following MI, providing a source of differentiated cell types and secretory functions required for cardiac repair. These molecular findings may identify new therapeutic approaches for improving patient survival following MI.