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Mitochondria play a central role in oxidative stress-induced cell death. By increasing the production of reactive oxygen species, such as H2O2, oxidative stress causes mitochondrial fragmentation and apoptosis. It was hypothesised that Transient Receptor Potential Melastatin 2 (TRPM2) channels play a role in mitochondrial fragmentation and cell death. The rationale behind this hypothesis was the published evidence that oxidative stress stimulates TRPM2 channels, resulting in an increase in the cytosolic levels of Ca2+ and Zn2+, and that both these ions are detrimental to mitochondrial health and cell survival. To test the hypothesis, human umbilical vein endothelial cells (HUVECs) and endothelial cells isolated from wild-type and TRPM2 knock-out mice were used. TRPM2 actions were suppressed using pharmacological agents and small interfering RNA (siRNA). Fluorescent reporters were used to examine changes in intracellular ion distribution and organelle morphology. Molecular biology, biochemical and imaging techniques were used to examine the dynamics of ions and organelles. Exposure of HUVECs to H2O2 or high glucose stress led to TRPM2 activation, resulting in extracellular Ca2+ entry, lysosomal membrane permeability (LMP) and the release of lysosomal free Zn2+. Unexpectedly, this was accompanied by the accumulation of Zn2+ in the mitochondria. The rise in mitochondrial Zn2+ led to extensive mitochondrial fragmentation, mitochondrial outer membrane permeabilisation (MMP) and cell death. Silencing of TRPM2 channels with siRNA prevented intracellular Zn2+ redistribution, mitochondrial fragmentation and cell death. Endothelial cells derived from TRPM2 knock-out mice were resistant to oxidative stress-induced mitochondrial fragmentation. Biochemical and immunostaining experiments revealed an unexpected presence of TRPM2 channels in mitochondria, where they mediated mitochondrial Zn2+ uptake. Accumulation of Zn2+ in the mitochondria led to mitochondrial fragmentation by promoting the recruitment of cytoplasmic Drp1, an enzyme responsible for mitochondrial fission. Taken together, the results of this thesis revealed a novel mechanism for how oxidative stress can cause excessive mitochondrial fragmentation and cell death: the mechanism involves activation of TRPM2 channels leading to increased Ca2+ entry, LMP and release of lysosomal Zn2+; Zn2+ thus released is taken up by the mitochondria, leading to Drp1 recruitment, mitochondrial fragmentation and finally cell death. Since mitochondrial fragmentation is associated with several age-related chronic illnesses, including neuronal (Alzheimer’s, Parkinson’s), cardiovascular (atherosclerosis, myocardial infarction) and metabolic/inflammatory (diabetes) disorders, these results suggest that the TRPM2 channel is a novel target that could be explored for therapeutic intervention of age-related illnesses.

Electronic patient-reported outcome measures (e-PROMs) have been introduced to improve the collection of patient feedback and to facilitate data linkage with research databases. However, before implementing e-PROMs, it is important to understand patient’s feelings about and acceptance of these technologies. Until today, there has been no adequate questionnaire to understand patient acceptance of e-PROMs. So, this study aimed to study patient acceptance of e-PROMs through developing and validating a new questionnaire based on the Theory of Planned Behaviour (TPB) and additional factors including computer anxiety and patient characteristic factors. Not only did this study apply a quantitative method to understand the factors behind patient acceptance, the development and the psychometric testing of the new questionnaire was conducted using a variety of methodological approaches. This includes: (1) developing the initial version of the questionnaire based on the available literature, (2) an expert panel review (n=5) and cognitive interviews (n=10) to measure face and content validity, and (3) conducting field-testing (n=231) to measure construct validity and internal consistency reliability. The field-testing included testing the conceptual model with cancer survivors at an outpatient oncology clinic in Leeds Teaching Hospitals NHS Trust. Based on these study findings, the developed questionnaire shows good validity and reliability. Moreover, the conceptual model results show that patient attitudes (a TPB construct), computer anxiety and gender were significantly (P < 0.05) associated with behavioural intention to use e-PROMs. The most influential factor is patients’ attitude to computers, followed by computer anxiety then male gender. Overall, these model constructs explained around 87% of the variance in acceptance. The findings of this study strongly suggest that clinicians need to encourage their male patients to use e-PROMs and help them to reduce their computer anxiety.

Silk fibroin (SF) from the silkworm, Bombyx mori, is a natural fibrous protein with unique mechanical properties, biocompatibility and biodegradability. It has great potential in biomaterial applications for tissue engineering, drug delivery and biomedical devises. Most of the SF based biomaterials (e.g. films, scaffolds, hydrogels and electrospun fibres) are cast from regenerated silk fibroin (RSF) solution. Hence, it is important to acquire a comprehensive and deep understanding of the fibroin solution. This research applied a number of biophysical approaches, aiming to investigate the solution aggregation and interfacial adsorption behaviour of the SF polypeptides in aqueous solution. The methods for fabricating nanometre scale SF films are also explored carefully because well-controlled films and their surfaces enable direct characterisation of their interaction with other molecules and cells. Using the dynamic light scattering (DLS) technique, it was found that the particle size of newly made RSF peptides in solution was around 2.3~6.5 nm and they could remain stable for at least 10 weeks at 4 °C. Factors such as temperature, fibroin concentration, pH, alcohol and metallic ions can directly affect the assembly and aggregation of fibroin polypeptides as well as their solubility and stability in solution. The formation of large aggregation under certain conditions was possibly related to the conformational transition of SF from random coil/α-helix (Silk I) to β-sheets (Silk II). The interfacial adsorption of RSF solution at the SiO2/water interface was monitored by spectroscopic ellipsometry (SE), dual polarisation interferometry (DPI) and neutron reflection (NR). It was revealed that surface excess and thickness increased with concentration and decreased with rising pH and ionic strength. NR measurements revealed that the adsorbed polypeptide layers are characterised by a thin and dense inner region and a thick and diffuse outer region, a feature similar to the adsorbed layers from other polypeptides. The results from SE, DPI and NR are in good agreement. Multilayer ultrathin SF films were fabricated using the layer-by-layer spin coating method and were found to be stable in physiological conditions. The thickness and surface excess of the SF films were tuned by varying the concentrations while coating. Surface biocompatibility as demonstrated by MTT assays varied with the film thickness or the number of layers coated. With the aid of the cationic copolymer MPC30-DEA70, SF films successfully immobilised plasmid DNA, which demonstrates the potential of these multilayer SF films to be used in a drug delivery system. The ultrathin SF films were modified with gelatin (G). Preliminary cell culture experiments with 3T3 fibroblasts demonstrated that SF/G films with 1.2% ~ 20% (w/w) G content promoted cell attachment and proliferation compared with pure SF films. When films contain 10% ~ 20% (w/w) of G, they showed biocompatibility even superior to the pure G films. These enhanced cellular responses must result from improved film stability arising from SF and improved cytocompatibility arising from G.

This thesis presents the research-driven design and development of Stromohab: A motion-capture virtual-reality locomotion simulator for the research and rehabilitation of gait disorders following stroke. Software and hardware components are designed, developed and tested to facilitate and motivate patients in rehabilitative interactive avatar-based locomotor tasks. The system is then used to investigate systematically on healthy volunteers the known problem of distance underestimation in virtual environments by testing and analysing all combinations of cross-planar translation of leg movement to avatar actuated movement in a virtual environment. Specific performance deficits in the sagittal plane are confirmed and compared to those from coronal and transverse motion. Potential improvements of adding in isolation monocular cues for perspective, illumination, or size, and binocular cues from 3D stereo anaglyphs, are investigated, leading to a proposed movement model and scaling solution that both explains and resolves the observed deficit empirically in a practical locomotor task. Overall, the findings demonstrate the importance for the design and application of virtual environment interfaces of quantifying the underlying mechanisms in order to ensure accurate and controlled reproduction of a user’s movement. These would be of particular significance in medical rehabilitation for neurological patients, for whom consideration of cognitive load and the potential for improper re-adaptation when returning to real world environments can be critical. It is envisaged that this study will be useful to technologists, clinicians and other professionals who apply the rapidly developing, increasingly accessible and beneficial motion capture and virtual reality technologies to medicine and related applications.

Molecular profiling data based patient stratification plays a key role in clinical decision making, such as identification of disease subgroups and prediction of treatment responses of individual subjects. Many existing knowledge management systems like tranSMART enable scientists to do such analysis. But in the big data era, molecular profiling data size increases sharply due to new biological techniques, such as next generation sequencing. None of the existing storage systems work well while considering the three 'V' features of big data (Volume, Variety, and Velocity). New Key Value data stores like Apache HBase and Google Bigtable can provide high speed queries by the Key. These databases can be modeled as Distributed Ordered Table (DOT), which horizontally partitions a table into regions and distributes regions to region servers by the Key. However, none of existing data models work well for DOT. A Collaborative Genomic Data Model (CGDM) has been designed to solve all these is- sues. CGDM creates three Collaborative Global Clustering Index Tables to improve the data query velocity. Microarray implementation of CGDM on HBase performed up to 246, 7 and 20 times faster than the relational data model on HBase, MySQL Cluster and MongoDB. Single nucleotide polymorphism implementation of CGDM on HBase outperformed the relational model on HBase and MySQL Cluster by up to 351 and 9 times. Raw sequence implementation of CGDM on HBase gains up to 440-fold and 22-fold speedup, compared to the sequence alignment map format implemented in HBase and a binary alignment map server. The integration into tranSMART shows up to 7-fold speedup in the data export function. In addition, a popular hierarchical clustering algorithm in tranSMART has been used as an application to indicate how CGDM can influence the velocity of the algorithm. The optimized method using CGDM performs more than 7 times faster than the same method using the relational model implemented in MySQL Cluster.

This thesis addressed the sensory functions of the gastrointestinal (GI) tract with an emphasis on the interaction between afferents nerves and other cell types such as enterochromaffin (EC) cells and mast cells, and also investigated the influence of ageing on these interactions. Studies on mice focused on TRPA1, a central molecule in nociception and inflammation. TRPA1 signaling induced by allyl isothiocyanate (AITC) comprised direct activation of TRPA1 on extrinsic afferents, indirect activation of 5-HT3 receptors on extrinsic afferents following 5-HT release form EC cells and indirect signals evoked by contractions. TRPA1 signaling was attenuated with advanced age. This could be attributed to reduced primary afferent innervation and decreased gene expression of 5-HT3 in the gut wall. Sensory functions of human GI tract were investigated morphologically and functionally. Some changes were identified in aged human bowel, including increased EC cells, mast cells, decreased sensory innervation and enhanced mast cell-afferent associations. Afferent activities were recorded from isolated human bowel using a novel in vitro model, allowing direct characterization of the mechanical and chemical sensitivities of human gastrointestinal afferents.

Mutations in parkin cause autosomal recessive Parkinsonism and mitochondrial defects. A recent drug screen identified a steroid like class of hydrophobic compounds able rescue mitochondrial function in mutant parkin fibroblasts. These included Ursolic Acid, Ursocholanic Acid, and Ursodeoxycholic Acid. pH-sensitive polymersomes, nanoparticles composed of amphiphilic block co- polymer, have been shown to encapsulate hydrophobic cargoes, enter cells without detriment to viability and release their cargoes therein. PMPC25−PDPA65 nanoparticles successfully encapsulating drugs were made by thin film rehydration, and purified by hollow fibre filtration. High encapsulation efficiencies were revealed by HPLC. Particle characterisation by and Transmission Electron Microscopy revealed a spectrum of morphologies, including spherical particles, branched tubular assemblies, and large high genus lyotropic structures. Morphological fractionation was achieved through stepwise centrifugation, and mass quantification showed drug encapsulation increased the relative proportion of tubular assemblies. Polymersomes were found to enter into parkin mutant fibroblasts without cytotoxic induction, or detriment to mitochondrial function as assessed by LDH release, mitochondrial membrane potential and cellular ATP levels. Drug loaded polymersomes of both spherical and tubular morphology increased cellular ATP levels of parkin mutant fibroblasts, and were found to deliver a fluorescent steroid at least as effectively as DMSO. The results presented here suggest PMPC-PDPA nanoparticles are suitable for use as a therapeutic vector in parkin mutant cells. The production of spherical and tubular nanoparticle morphologies would be of interest to in vivo applications, each being known to show distinct properties affecting nanoparticle distribution within the body.

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Enteral nutrients, especially lipid, have been well recognized as signalling molecules which control various cellular processes and play an important role in food intake, metabolisms, inflammation and pain. Loss of appetite and altered functional property of TRPV1 ion channel have been found ageing. However, the transduction mechanism of extrinsic intestinal afferent nerves in lipid sensing from the gut remains poorly understood. Thus, the primary objective of this thesis was to investigate effect of lipid-containing nutrients on different subpopulations of jejunal afferents using an in vitro nerve-gut preparation from adult and aged mice using a sophisticated and comprehensive single unit analysis. The contribution of TRPV1 in aged related changes was also examined. In adult mice, both lipid-low and lipid-rich containing nutrients were found to increase intraluminal distension-induced afferent response in three functional different subpopulations; Low threshold (LT), wide dynamic range (WDR) and high threshold (HT) units. The effects were lipid concentration dependent. Only the response of LT unit to lipids was significantly attenuated by CCK-1 antagonist devazepide which also effectively inhibited exogenous applied CCK, strongly suggesting that lipids-induced enhanced mechanosensitivity was predominately mediated by CCK-1 vagal nerve pathway. Lipid rich-induced mechanosensitivity was further examined in aged mice. Response of both LT and HT was gradually reduced with ageing compared with adult. The reduced mechanosensitivity was restored in 12 months TRPV1-knockout mice, suggesting down-regulation role of TRPV1 in gut ageing. Finally, the role of lipid in modulating jejunal afferent mechanosensitivity, its changing and function of TRPV1 as a sensor during ageing process was discussed. Thus this study has advanced our understanding of biological mechanisms underlying gut afferent signalling in nutrient sensing at single nerve fibre level.