Antigen-specific immunotherapy (ASI) involves induction of tolerance to autoantigens. An important protein in the development of type 1 diabetes (T1D) is the autoantigen, proinsulin (PI), the precursor of insulin. Microneedles (MNs) are micron-sized needles that penetrate into the upper skin layers. MNs provide advantages for autoantigen delivery including targeted delivery to the skin's dendritic cells (DCs), with minimal inflammation. The aim of this Thesis was to develop a PI-coated solid MN system and investigate the potential of this system to induce peripheral tolerance in the non-obese diabetic (NOD) mouse model of T1D. A highly concentrated PI MN coating formulation was developed containing the PI, diluent and a surfactant. The formulation enabled uniform and reproducible coating of the PI on to MNs. Delivery of PI from the MN system was investigated in mouse skin. MN application method and duration were optimised and resulted in skin puncture and reproducible delivery of PI to the skin. In vitro studies identified the insulin-reactive G9 CD8+ T cell as an appropriate biological readout for PI delivery. In vivo delivery studies indicated that MNdelivered PI was delivered to the skin and subsequently processed by DCs into PI peptides, which were cross-presented in the skin draining lymph nodes to adoptively transferred G9 CD8+ T cells. This demonstrated that the PI-coated MN system has potential for inducing peripheral tolerance in the NOD mouse. T1D development was significantly delayed in NOD SCID mice that received cells from PI-treated NOD mice and cells from diabetic NOD mice (experimental group). However, no statistically significant difference in time to T1D development was observed between the experimental group and the control NOD SCID mice that received cells from untreated NOD mice and diabetic NOD mice. Further investigation of the dosage and dosing frequency of PI using the coated MN system is, therefore, warranted.

Site-directed mutagenesis and fluorescent-protein based techniques were used to evaluate the role of the C-terminal tail of human V\(_1\)\(_a\) vasopressin receptor (V\(_1\)\(_a\)R). Mutants engineered with the C-tail truncations were characterised with respect to cell-surface expression by enzyme linked immunosorbent assay (ELISA), agonist binding by competition radioligand binding assay and signalling capability by inositol phosphates (InsP-InsP\(_3\)) accumulation assay. A series of Ser/Thr mutations disrupted phosphorylation sites and identified a putative G-protein-coupled receptor kinase 2 (GRK2) consensus site contributing to V\(_1\)\(_a\)R internalisation rate and desensitisation in response to vasopressin. GRK2 and GRK2 constructs mutated at functional domains were used to identify the role of GRK2 in V\(_1\)\(_a\)R internalisation. A V\(_1\)\(_a\)R-mCherry fusion, V\(_1\)\(_a\)R-vYC and A2AR-vYC were generated and likewise functionally characterised. Addition of mCherry or vYC at the C-terminus of V\(_1\)\(_a\)R did not affect the binding affinity of V\(_1\)\(_a\)R, cell-surface expression and ability to signal via Gq/11 coupling. Similarly, A\(_2\)\(_A\)R-vYC displayed WT like binding profile and receptor internalisation. Total internal reflection fluorescence microscopy (TIRF-M) and confocal imaging were performed to monitor V\(_1\)\(_a\)R-mCherry internalisation upon agonist stimulation. Overall, results presented in this thesis provide insight into the role of the C-terminal tail domain in V\(_1\)\(_a\)R internalisation and desensitisation and identify functionally important residues including a putative GRK2 regulatory site.

Alloy compositions near to stoichiometric Nd2Fe14B produced under normal casting conditions, with subsequent isothermal heat treatments and with sintering have been characterised. The possible mechanisms for removing the soft magnetic phase, free α-Fe, via additions of 0.6 at% of Zr or 1.0 at% of ZrB2 have been investigated by EPMA (Electron Probe Microanalysis) and TEM (Transmission Electron Microscopy). 1.0 at% of ZrB2 has been found to be very effective in suppressing the formation of free α-Fe dendrites during solidification, but the excessive ZrB2-type needles formed in the NdFeB alloy may have a deleterious effect on the magnetic properties. However, the coercivity of the NdFeB sintered magnets can be improved to some extent by addition of Zr or ZrB2 to the composition Nd12.68Fe77.91-80.91B6.41-8.41Zr0-1 (at%) due to the smaller and more uniform grains of the Nd2Fe14B phase (~6.5 μm) in the sintered magnets. Both SEM (equipped with WDX) and TEM (equipped with EDX) have been employed to investigate ZrB2 needles in Nd12.68Fe77.91-80.91B6.41-8.41Zr0-1 (at%) alloys. It is found that suppression of free α-Fe dendrites in both Zr-containing NdFeB alloys resulted from different solidification routes. Since the sequences of formation of the ZrB2-type needles in Zr-containing alloys are different, the Nd2Fe14B grain growth is inhibited via different mechanisms. Meanwhile, evidence for the high temperature solubility of Fe in ZrB2 needles is found for both Zr-containing NdFeB alloys. Chemical analysis shows that the ZrB2-type needles in the NdFeB alloys are composed mainly of B (~70 at%) and Zr (~20 at%) with a small but significant amount of Fe (~7 at%), a formula which can be expressed as (Zr1-x, Fex)B2 (x~0.3).

In the 21st century - the era of big data science - chemical risk assessment procedures remain woefully dependent upon a suite of basic toxicological assays that offer little, if any, biochemical information pertaining to the underlying mechanism of toxicity. Metabolomics, defined as the holistic study of all naturally occurring, low molecular weight metabolites present within a biological system, holds huge potential as a tool to fill this knowledge gap, and thereby, to revolutionise the chemical risk assessment process through provision of rich molecular information . Owing to on-going challenges in the area of metabolite identification, however, which ultimately serves to impede derivation of biological knowledge from metabolomics data sets, the full potential of the metabolomics platform has yet to be realised in the context of (eco-)toxicological research. In this thesis, I present the experiments undertaken in establishing a bespoke bioanalytical workflow specifically designed and optimised to resolve this bottleneck. Ultimately, I demonstrate application of select components of this workflow in the characterisation of the metabolome of D. magna, a model organism for eco-toxicological research.

Plasmid DNA partitioning is a crucial process for the transfer of at least a single copy of plasmid to the daughter cells during bacterial cell division. Partitioning for various low-copy number plasmids involves a DNA-binding protein (ParB), a centromere-like DNA site ( parS) and a ParA-family protein. Interestingly, the RK2 plasmid encodes two ParA proteins of different lengths. The longer protein is IncC1 (364 a.a), while IncC2 lacks a N- terminal domain of 105 amino acids (IncC NTD). The secondary structure of IncC NTD by NMR spectroscopy and other biophysical methods has been determined as random coil. It appears to bind DNA weakly and non-specifically. The expression and purification of IncC1 and IncC2 proteins was optimized. The two proteins and IncC NTD were characterized using various biophysical methods including Circular Dichroism, Analytical Ultracentrifugation, Small Angle X-ray Scattering, Size Exclusion Chromatography-Multi Angle Light Scattering, and EMSAs. Bacterial two hybrid assays and chemical crosslinking showed the two IncC proteins form homo- and hetero-dimers and interact with KorB protein. IncC1 and IncC2 proteins bind to DNA, non-specifically. IncC1 binds DNA weakly in the absence of nucleotides but IncC2 protein was found to bind DNA only in the presence of nucleotides (ADP, ATP).

This thesis presents a body of work which advances the use of single crystal hydrogen terminated diamond as a semiconducting material. Surface transfer doping of intrinsic diamond is investigated, examining the current state of this technology and its limitations. New techniques for producing robust, thermally stable surface transfer doped diamond were achieved through use of transition metal oxides such as MoO3 and V2O5, as demonstrated experimentally by way of Hall measurement. Through use of these materials, thermal stability was greatly increased up to temperatures of at least 300oC. To achieve this higher temperature operation, encapsulation of MoO3 and V2O5 was found to be necessary in maintaining conductivity of the diamond surface due to suspected thermally-induced loss of hydrogen termination. Similarly, long term atmospheric stability is shown to necessitate annealing of the diamond surface prior to oxide deposition and for thinner layers of oxide, down to 10 nm, encapsulation of the oxide to isolate from atmosphere is shown to be required for increased stability. As well as the improvements in stability offered by these transition metal oxides, sheet resistance of the hydrogen terminated diamond surface was also greatly reduced. Carrier densities as high as ~7.5 ×1013 cm-2 were observed for MoO3-induced surface transfer doping, resulting in a low sheet resistance of ~ 3 kΩ/□. In parallel to the development of oxide acceptor materials, conditioning of the diamond surface was explored using Atomic Force Microscopy (AFM). Techniques for smoothing the surface after mechanical polishing were developed by way of RIE and ICP etching using both chlorine and oxygen mixtures. Surface roughness down to 2 angstroms was demonstrated, showing a significant improvement in roughness over mechanical polishing alone. Similarly, observed defects produced by polishing induced damage were removed through use of this etching strategy. The effects of varied plasma density during hydrogen termination was explored on etched surfaces, which produced higher quality hydrogen-terminated surfaces as verified by surface conductivity and AFM measurements. Finally, incorporation of MoO3 into a preliminary Field Effect Transistor (FET) device on diamond was attempted. Fabrication techniques to produce a FET device on hydrogen-terminated diamond is shown with preliminary results of MoO3 encapsulated devices. Insights into the fabrication of ohmic and gate contacts, incorporating MoO3, is also discussed.

The nuclear energy industry produces radioactive waste at various stages of the fuel cycle. In the United Kingdom, spent fuel is reprocessed at the Sellafield facility (formerly known as Windscale), in Cumbria on the north-west coast of England. Waste generated at the site comprises a wide range of radionuclides, including radiocarbon (14C) which, due to its long half-life (5730 years), environmental mobility and high bio-availability, is the largest contributor to collective dose commitment from the entire nuclear industry. 14C is disposed of in various forms, one of which is as highly soluble inorganic carbon within the low-level liquid radioactive effluent, via pipelines, into the Irish Sea. The discharged 14C is then rapidly incorporated into the dissolved inorganic carbon (DIC) pool. This project aimed to better understand the fate of Sellafield discharges of 14C to the marine environment. Investigations of intertidal sites in the Irish Sea and West of Scotland found 14C enrichment above ambient background levels in shell material, including at the most northerly site, located 265 km north of Sellafield. Dissolved inorganic 14C is readily utilised during shell formation by calcifying organisms and mussel shell 14C activities at sites closer to Sellafield appear to be varying in response to the total Sellafield 14C discharge activity over the preceding 5 years. Due to subsequent erosion of this material, 14C is transferred to finer fractions of intertidal sediments where it is accumulating. During photosynthesis, primary producing organisms also utilise carbon derived from the DIC pool. This uptake and the trophic-level transfer of 14C within the Irish Sea and West of Scotland marine environments were examined. The 14C activities of Irish Sea DIC and biota in the east and west basins were enriched and highly variable. A general decrease in 14C activity with distance from Sellafield was observed, although, enriched activities were also found in the West of Scotland where the activities were more homogenous. Organic sediments were significantly less enhanced than associated benthic organisms. This could be due to rapid scavenging of labile, 14C-enriched organic material by organisms and mixing to depth with older, 14C depleted material. Commercially important species were 14C enriched; however, the radiation dose from their consumption is extremely low and radiologically insignificant. To evaluate the transfer of 14C to top predators in the UK marine environment, 14C activities were examined in stranded marine mammals. All Irish Sea samples were enriched, as were most from the West of Scotland, although the 14C activities were lower. In demonstrating transfer of enriched 14C to apex predators for the first time, this study also showed that marine mammal activities correlated significantly with distance from Sellafield and Sellafield 14C discharge activities for 24 months prior to stranding. These measurements also provided some insight into harbour porpoise (Phocoena phocoena) ecology, indicating high foraging fidelity and suggesting the animals stranded on the West of Scotland did not forage in the Irish Sea. The studies in this thesis examined the dispersion of Sellafield-derived 14C to both near- and far-field sites and the subsequent ecosystem uptake and trophic transfer at these locations. However, it is important to attempt to understand the fate of 14C discharges beyond the limitations and scope of analytical investigations. To do so, the first spatial-temporal ecosystem model to predict the ecological fate of Sellafield-derived 14C was developed. The observed trends in 14C activities between different species were predicted by the model which illustrated the integration of 14C in species at higher trophic levels through time.

The beta 2 adrenergic receptor (β2AR) is a GPCR that is susceptible to multiple post-translational modifications (PTMs) including phosphorylation, ubiquitination, palmitoylation and glycosylation, which can alter how the β2AR orchestrates downstream intracellular signals. Following the discovery of SUMOylation of the cardiac signalling protein SERCA2a, we hypothesised that the β2AR, which is also involved in cardiac signalling, may be a substrate for SUMOylation. This notion was supported by previous findings that five different GPCRs have been identified as susceptible to SUMOylation, including metabotropic glutamate receptors (mGluR), a cannabinoid receptor, a serotonin receptor and a receptor involved in basal cell carcinoma known as smo. For the first time, we confirm the susceptibility of the β2AR to SUMOylation by identifying SUMO accepting lysines, interaction sites between the β2AR and the enzymes of the SUMOylation cascade, and the traditional “ghost” band, which is indicative of protein SUMOylation. SUMOylation has been shown to influence receptor signalling, and we now uncover a possible role for SUMOylation in β2AR signalling. I report that SUMOylation of the β2AR (mediated via overexpression of the E3 ligase PIASγ) reduces β2AR phosphorylation by PKA altering the receptor driven phospho-ERK response, inhibits β2AR ubiquitination and degradation, and delays β2AR internalisation. These changes could be associated with steric effects of the bulky SUMO modification and ubiquitin-SUMOylation competition for available surface associated lysines. With the importance of SUMOylation in multiple disease states (including cardiovascular disease, neurodegenerative disease and cancer), we worked in conjunction with the antibody production company Badrilla ® to produce a SUMO-substrate specific antibody for a site within the third intracellular loop of the β2AR. The antibody we have produced is successful in recognising the SUMOylated form of the receptor in both cell and tissue lysate, making it the first antibody of its kind to be generated. In conjunction with the Hajjar group at Mount Sinai Cardiovascular Research Centre (New York, USA) we used the SUMO-β2AR specific antibody to assess the influence of the SUMO modification in two animal models of heart failure (HF); transverse aortic constriction (TAC) pressure overload HF model in mice and the left anterior descending (LAD) artery balloon occlusion ischemic HF model in pigs. Prior work within the Hajjar group has revealed that SUMOylation of SERCA2a is reduced in HF, and restoring this modification to SERCA2a via SUMO-1 adeno-associated viral-mediated gene delivery is beneficial in restoring cardiac function. We hypothesised that SUMOylation of the β2AR would also be reduced in HF, however unexpectedly; the SUMOylated form of the β2AR was increased in the LAD artery balloon occlusion ischemic HF model in pigs. The role of the β2AR in HF is unclear. There are studies which both report a cardio-protective and a cardio-toxic role of the receptor. To ascertain the role SUMOylation of the β2AR plays, in vivo studies promoting SUMOylation of the β2AR in the HF models described above will help to determine if enhanced SUMOylation of the receptor worsens the HF phenotype or prevents its development. To conclude, we present the first evidence that the β2AR can be modified by SUMOylation, which acts to influence the receptors downstream signalling, desensitisation and degradation. We have designed a first-in-class SUMO-β2AR antibody – which paves the way for a panel of SUMO specific antibodies – and utilised it to assess SUMOylation of the β2AR in model cellular systems and animal models of HF. Similarly, to SERCA2a, SUMOylation does influence β2AR in the HF phenotype, although not a decrease in SUMOylation as was expected, but an increase. Future work in animal models promoting SUMOylation of the receptor is vital to assess the role of this highly novel post-translational modification of the β2AR.

Infectious diseases remain some of the most serious health threats facing the world. The immune system is equipped to initiate a rapid and specific response to foreign invaders of the body, with its ultimate aim being to protect an organism from injury and disease. Eicosanoids, including prostaglandins and leukotrienes, are a family of lipids that play key roles in inflammation including helping leukocytes fight infection. Cells of the innate immune system including tissue macrophages, neutrophils and sentinel dendritic cells are major contributors of local eicosanoids. In mammals an inflammatory insult will result in a cytokine cascade whereby tumour necrosis factor α (TNF-α) is released, followed by interleukin-1β (IL-1β) and then IL-6. Downstream of these cytokines, others are released that serve as potent chemoattractants to induce migration of neutrophils and macrophages to the site of infection. It is known that exposure to varying bacterial components results in a different profile of lipids and cytokines, and by characterising mediator signals it may be possible to define biomarker fingerprints predictive for early bacterial infections. To analyse this, a combination of a targeted lipidomic approach and cytokine immunoassays were employed to identify neutrophil and macrophage responses to individual bacterial components and the whole organism. Work in this thesis has identified potential markers of bacterial infection, such as 12- HETE, 14-HDOHE and TNF-α, which, along with future advances, could be used to develop novel strategies for clinicians, nurses and primary care staff to analyse patients suspected of bacterial infection at the bedside. Work here provides an insight into how the eicosanoid and cytokine storms are generated alongside each other to accompany classic inflammation during specific bacterial infection. The ability to distinguish between species of bacteria causing infection could prove invaluable, reducing the time taken to establish the cause of infection, ultimately leading to better patient outcomes.

This thesis shows how microwaves and nanoparticles can be used to enhance the disruption of spores in the context of a novel microwave-based bacterial detector. Infections linked to Clostridium difficile are a significant cause of suffering. In hospitals, the organism is primarily acquired through the faecal-oral route as spores excreted by infected patients contaminate the healthcare environment. Microwave-based spore disruption is the focus of this project offers a potential rapid diagnostic method to detect spores, including C. difficile spores by making them release DNA which is then detected. One of the limiting factors of this method was the power required and it was hypothesised that by using a new microwave cavity and adding nanoparticles, the DNA release from spores can be achieved with less power. First, the spore surface properties of the different isolates of C. difficile were compared. A significant variation in both spore morphology and spore hydrophobicity of clinical isolates of C. difficile was observed. In particular, the "pineapple-like" shape of strains was associated with higher hydrophobicity in spores, while the loose outer exosporium layer was associated with lower hydrophobicity. The isolates were then tested for DNA release in response to microwaving. Spores were shown to release single-stranded, but no double stranded DNA. The spores were also not visibly changed by microwave exposure, suggesting a non-destructive mechanism of disruption. To study this mechanism further, the microwave system was updated to remove overall sample heating and tested under electric fields, magnetic fields and a combination of the two. The electric field was shown to be causing the disruption of spores, field showed a positive correlation with increasing disruption. Finally, the spores with tested with microwaves and nanoparticles, where nanoparticles showed a significant improvement in two of the four tested isolates. A computer model of the spore and nanoparticle interactions was made, which offered a plausible mechanism for the nature of microwave mediated disruption and the improvement in disruption caused by nanoparticles.