Duchenne muscular dystrophy (DMD) is a muscle-wasting disease caused by the loss of sarcolemmal protein dystrophin. In DMD and the mouse model of the disease mdx, there is an increase in an associated protein, caveolin-3. In this study, mdx mice with deficiencies in caveolin-3 were generated to allow a distinction to be made between the pathology caused by the loss of dystrophin and that caused by an excess of caveolin-3. It was found that in late gestation embryos, there were perturbations in skeletal muscle stem cell populations and depletion of respiratory muscles in mdx and mdx/cav3\(^{+/-}\), both of which were more severe in mdx/cav3\(^{+/-}\) embryos. In post natal skeletal muscles, there was a trend in that the level of regeneration, believed to be indicative of previous degeneration, was consistently greater in mdx than mdx/cav3\(^{+/-}\). Taken together it would appear whereas increased caveolin-3 may compensate for the lack of dystrophin in embryonic mdx muscle; post natally, it may contribute to the muscle regeneration observed in mdx. The data presented in this thesis should help towards clarifying the contribution of caveolin-3 in the pathogenesis of DMD and in doing so expand on the understanding of the molecular aetiology of the disease.

Early synaptic degeneration in prion disease has developed into a subject of interest, because it is thought that it may allow therapeutic intervention to prevent the neuronal death which is often observed at the late stage of the disease. However, the events behind the synaptic degeneration in prion disease, that may ultimately lead to neuronal death, are still unclear. Studying the morphology of neuronal components, namely synaptic boutons, axons, spines, dendrites and cell bodies, of the population of origin may help in understanding the neuropathology of prion disease. Intrahippocampal injection of murine modified scrapie (ME7 homogenate) provides a model of prion disease in vivo. Animals injected with ME7 were compared to control animals (injected with normal brain homogenate, NBH) and both groups were killed at 13, 16 and 19 weeks. At each time point, Biotinylated Dextran Amine (BDA) tracer was injected into the CA3 area of the hippocampus to reveal the morphology of neurons and their components during the disease progression, using both light and electron microscopy. The results showed that at 13 weeks, the number of synaptic boutons, which are distributed along the axons in the CA1 stratum radiatum, was significantly reduced, while most of the remainder were hypertrophied. This correlated with an increase in both the synaptic spacing along the axonal segments and the presence of abnormal swellings on the axons throughout the stratum radiatum. At 13 weeks, electron microscopic studies revealed vacuole-like structures in the synaptic boutons, which differed from actual autophagic or spongiform vacuoles. The results also showed a significant reduction in the number of dendritic spines on CA3 neurons, associated with a reduction in dendritic arborizations and length. Abnormal swellings were also seen in dendrites and occasionally in the cell bodies. Changes in CA3 cell body size were not observed until 16 weeks, when the soma area had reduced. The results indicate that changes in the morphology of synaptic boutons and dendritic spines were observed at an early time point, 13 weeks the fist observation time, and progressed with time. These results showed for the first time that there is a correlation between both synaptic bouton and spine loss in the neuron of origin, which may suggest that there is continual, simultaneous degeneration between the efferent and afferent components of neuron, leading to an early impairment of the neuronal communication within the brain. This suggests the notion that the loss of communication of the neuron at the early stage may lead to neuronal dysfunction and degeneration at the late stage of the disease. The results also suggest that the synaptic vacuoles may play a crucial role in the hypertrophy or degeneration of the remaining synapses. Additionally, it has been shown for the first time that astrocytes in ME7-animals express features of pathology and degeneration, suggesting that the astrocytes may be another target of PrPSc in prion disease. The combination of these findings has opened new avenues in the field of prion studies. The possible mechanisms behind these results are discussed.

Creutzfeldt-Jakob disease (CJD) can be iatrogenically transmitted during transplants, grafts and transfusions from CJD infected donors and also contaminated surgical instruments. A variety of methods to amplify and detect the presence of infectious prions as disease markers are available. However, these techniques do not measure the infectivity potentially associated with these markers. Currently, animal-bioassays are used to detect infectivity; however, they have limitations in detectable prion strains, cost, ethical considerations and assay length. Novel cell-based infectivity assays offer the potential to overcome these limitations, lower the requirements for animal use and the application of different cell lines could detect a wider range of prion strains. This project utilised murine neuroblastoma, N2a #58 cells infected with 22L-murine scrapie to develop a highly sensitive assay for the in situ detection of amyloid-rich prion (PrPSc) accumulation as an indication of prion infectivity. The autofluorescence quenching properties of Sudan black (SB) were incorporated into a novel Thioflavin T (ThT) based protocol for amyloid staining with improved specificity and sensitivity. Cell passages were incorporated into the assay to increase incubation time, improve cell viability and subsequently improve assay sensitivity; thus, demonstrating the detection of infectivity from a final 10-10 dilution of 22L-infected brain homogenate. Introduction of 22L-inoculated, surgical grade stainless steel wires to the N2a #58 cells demonstrated the SB/ThT detection of prion infectivity pre and post decontamination, which was comparable to animal bioassay data. Furthermore, preliminary work on the incorporation of the SB/ThT detection of prion infectivity within neural stem cells (NSC’s), for prion propagation within a cell line that did not require genetic manipulation for increased prion susceptibility, highlighted problems with unspecific fluorescence of dead cells during NSC differentiation. Improvements in culture conditions of the NSC’s regarding atmospheric conditions and trophic support were addressed in preparation for their use in future prion infectivity assays.

Dialysis Related Amyloidosis (DRA) is a serious complication of long term haemodialysis. Amyloid deposits accumulate in the joints causing great pain & restricting mobility of sufferers. The main constituent of these amyloid deposits is fibrillar β2-microglobulin (β2m), although additional components are found which are thought to affect the formation and stability of the β2m fibrils. β2m fibrils formed in vitro under acidic conditions appear to have the same morphology as fibrils formed in vivo under pathological conditions when studied using electron microscopy and atomic force microscopy. However, the in vitro formed fibrils are not stable at neutral pH and quickly dissociate into monomeric and low oligomeric species. This raises the question as to why fibrils do not dissociate in vivo at physiological pH. In vivo serum amyloid P component (SAP) is always found associated with β2m fibrils and thought to stabilise the fibrils by preventing dissociation. Here we present evidence from pull-down assays that SAP binds tightly to acid produced β2m fibrils. The behaviour of the acid produced fibrils with and in the absence of SAP at neutral pH has being characterised using Thioflavin T fluorescence studies and has revealed that SAP does have a small stabilising effect on acid produced fibrils at the concentrations tested. The studies also imply that ionic strength as well as free protein concentration are important determining factors into the longevity of the fibrils at neutral pH. Studies of β2m in inclusion bodies prior to refolding demonstrate that they are not identical to β2m fibrils but that NMR studies do show areas of structural homogeneity with fibrils suggesting that the inclusion bodies may have structure and are not amorphous aggregate as previously thought. Soluble β2m has been assigned using solution-state NMR to identify regions of structural transition between soluble and fibrillar forms of β2m. Solid-state NMR spectra of acid produced fibrils have been acquired at both acidic and neutral pH and reveal that at a molecular level the fibrils are structurally homogenous, giving rise to spectra with site specific resolution. Sequential assignment of fibrillar β2m has therefore been possible using specific labelling techniques to overcome spectral crowding. To identify the interaction interface between β2m fibrils and SAP we have undertaken solid-state NMR studies of β2m with and without SAP bound. Comparing the chemical shifts from these studies has allowed us to identify that SAP is interacting with the side chain carboxylates of fibril aspartates and glutamates. Subsequent chemical modification of these carboxylates to remove their charge resulted in complete inhibition of SAP binding; confirming that they are essential for SAP binding to occur. However there is no strong interaction between monomeric β2m and SAP occurring demonstrating that a collective action of these acidic side chains is needed for binding to occur. Fibrils provide this in the form of acidic strips along the fibril axis brought about by the parallel and anti-parallel beta-strand structure of fibrils.

The aim of this study was to investigate the potential use of ELISA and LC-MS-MS in combination and as individual techniques, for the detection of drugs of abuse in biological matrices. Overall the LC-MS-MS method showed good correlation results for opiates compared to the GC-MS method. 6-MAM was however detected in more root segments and segments excluding roots by LC-MS-MS. Morphine was detected in a greater number of root segments by LC-MS-MS compared to GC-MS. However, morphine was detected in a greater number of segments excluding roots by GC-MS. Codeine and dihydrocodeine were also detected in a greater number of root segments and segments excluding roots by GC-MS. The cocaine results showed excellent qualitative correlation between the LC-MS-MS and GC-MS methods for cocaine and benzoylecgonine. The GC-MS method did not however extract greater concentrations of cocaine and its metabolites compared to LC-MS-MS due to the higher recovery of the drug group specific GC-MS method. Cocaethylene and EME were detected in some samples by LC-MS-MS method for opiates and cocaine and its metabolites compared to the GC-MS method; there may be some cases where the GC-MS method would detect the analytes where the LC-MS-MS method would not. This has been demonstrated in 3 samples for morphine and in 6 samples for codeine. The LC-MS-MS method analysed for and detected amphetamines in samples that were not tested for amphetamines by GC-MS. In one sample that was tested by both methods, amphetamine was detected in the root sample by LC-MS-MS where GC-MS failed to detect it. Also a greater concentration of amphetamine was extracted using the LC-MS-MS method in the segment without roots. The LC-MS-MS method was useful for the analysis of 17 drugs of abuse in post-mortem hair samples in forensic toxicology cases. Using this method, it is possible to obtain maximum information from one hair sample which is extremely useful when the sample weight is limited. The ability of the LC-MS-MS method to extract and analyse a greater number of drug groups from one hair sample highlights the advantages of using this method over GC-MS which targets individual drug groups and requires splitting of the sample. This method is particularly applicable for implementation in the forensic toxicology laboratory at the University of Glasgow where currently GC-MS methods that target individual drug groups are used for routine hair screening and confirmation.

The potential for nitric oxide (NO) metabolites (e.g. inorganic nitrite) to act as stable stores of “Transported Nitric Oxide” has excited huge interest due to the substantial potential therapeutic avenues. The prospect developing of a “silver bullet” that could target areas most in need of vasodilatation, by releasing NO in areas of hypoxia and ischaemia, could prove a massive advance in the treatment of vascular disease. In this thesis I examine the effects of nitrite infusion in both hypoxia and normoxia. I examine the effects both in health and heart failure, and investigate the potential roles of Nitric Oxide Synthase (NOS) and Xanthine Oxidase (XO) in mediating the reduction of nitrite. We found, and were the first to report in man, that intra-arterial infusions of nitrite had little effect upon the vasculature in high oxygen tension environments but led to significant vasodilatation during hypoxaemia. We found that patients suffering with Chronic Heart Failure responded differently to nitrite infusion to healthy controls, possibly as a result of differences in redox-stress. In healthy volunteers, at rest, neither NOS nor XO appeared to play a significant role in nitrite induced vasodilatation in normoxia and mild hypoxia. We found that vascular myoglobin contributes to the reduction of nitrite to nitric oxide and may play a role in prolonging the vasodilatation induced by nitrite infusion.