Amyotrophic Lateral Sclerosis (ALS) is a progressive fatal neurodegenerative disease characterised by motor neuron degeneration and muscle denervation, atrophy and eventual paralysis. Approximately 10% of ALS cases are familial, caused by mutations in a range of genes including superoxide dismutase 1 (SOD1), TDP-43 (TARDBP) and fused-in-sarcoma (FUS), although the most common genetic cause of ALS is now thought to be a hexanucleotide repeat in C9ORF72. Mouse models of ALS, most commonly created by overexpressing wildtype or disease-causing mutant human proteins, have been critical for our understanding of ALS pathogenesis. In this Thesis I investigated a number of new mouse models that express ALS-causing mutations including mutant mice created through N-ethyl-N-nitrosourea (ENU) mutagenesis, which induces random point mutations in the mouse genome, resulting in expression of the mutant proteins at physiological levels, as well as a new transgenic mouse which over-expresses wildtype human FUS. In the first experiments, a mutant mouse carrying a D83G mutation in mouse Sod1, analogous to a pathogenic human mutation, was examined. Sod1D83G/D83G mice show motor neuron degeneration and axonopathy. Examination of cultured primary embryonic motor neurons revealed that Sod1D83G/D83G motor neurons have a reduced viability, a reduction in neurite outgrowth and significant deficits in mitochondrial function. In the second set of experiments, four different mice with ENU-induced mutations in TDP-43 were examined. These mice did not show any signs of neuromuscular dysfunction in vivo, or signs of pathology in vitro, assessed by neurite outgrowth and stress granule formation. Furthermore, crossing TDP-43 mutant mice with SOD1G93A transgenic mice did not modify disease progression. In the final set of experiments, transgenic mice overexpressing wildtype human FUS were investigated. In vivo assessment of neuromuscular function in homozygote mice at 10 weeks of age revealed significant pathological changes in muscle function and motor unit survival. These results characterise a novel ENU-induced mouse model of mutant SOD1-induced ALS and a new transgenic mouse model in which overexpression of wildtype human FUS causes a severe motor phenotype. However, ENU-induced mutations in mouse TDP-43 do not result in any overt motor phenotype.

Cell encapsulation in collagen hydrogels affords the ability to rapidly create living yet mechanically weak tissue equivalents. A previously developed compression technique that removes the interstitial fluid from these hydrogels allows for a rapid improvement of the mechanical strength with adequate cell survival. However, although the UTS of the resulting compressed collagen-rich tissue sheets approach those found in vivo, the break strength, due to the small size of these sheets, does not allow for appropriate handling properties such as suturing. In the present study it was found that both the thickness and the break strength of the compressed sheets could be improved by increasing the (pre-compression) collagen hydrogel volume. It was found here, however, that the previously described anisotropic increase of the collagen density in the hydrogel at the surface through which the interstitial fluid content is removed forms a mass transfer dependent resistance to the outflow of fluid through this surface which indicates that there is a limit to the size and strength of living tissue that can be achieved with this technique. It was found here that the suturability of the compressed sheets could also be enhanced by the hybridisation with a synthetic degradable mesh without compromising cell survival or the speed of construction. Fibroblast seeded hybrid constructs featuring a near confluent epithelial monolayer and with a stratified epithelium, created within 5 hours and 2 weeks respectively, were tested in a lapine model of an acute full thickness skin defect to test for their safety and efficacy in wound healing. Only constructs with a stratified epidermis showed a significant increase in wound closure and neo-vascularisation of the wound bed compared to untreated wounds. Comparison of the present findings with currently available living biological dressings for the treatment of chronic wounds suggests the utility and possible cost-effectiveness of this rapid tissue engineering technique.

ldiopathic scoliosis is a tri-dimensional deformity of the spine surgically corrected by means of instrumentation such as the Harrington rod. This correction can be of two types: vertebral fusion and fusionless correction. During vertebral fusion, the spinal deformity is corrected, a metal implant fixed to several vertebrae and morsellised bone used to promote fusion. However, if performed in children and adolescents, it can hinder their trunk height affecting the growth of the ribcage and internal organs. Fusionless correction addresses this by performing a staged correction of the spine allowing for vertical growth, but it increases the risk of deep infection and trauma due to repeated surgeries. A non-invasive system based on a growing endoprosthesis, which permits staged correction and minimizes infection risk, was designed and its systems tested. This device provides a distraction force to overcome the natural soft tissue response which amounts to a maximum of 6OON. The drive unit providing this extension is comprised of a commercially available micro-motor, gear-head and encoder combination, attached to a specially designed gearbox which transforms the rotation into vertical extension via a threaded stainless steel rod attached to the uppermost vertebra of the deformity. The implant is sealed within a stainless steel chamber and controlled with an inductive link. The inductive link has been designed and tested for operation at increasing distance between its coils. This system provides the necessary power for the implant and transmits data from the drive unit which is used to perform automatic control with a closed-loop system. The control system maintains the power of the motor so that the extension is stable and comfortable to the patient. This thesis presents the design and testing of the inductive link, measurement of spinal forces, testing of the miniature motor and gearbox and mechanical implant fatigue testing.

Chordoma is a rare malignant bone tumour, showing notochordal differentiation, which occurs in the axial skeleton. Brachyury, a molecule involved in notochordal development, is a highly specific and sensitive marker for chordoma. It is hypothesised that brachyury or genes involved in its activation are implicated in the pathogenesis of chordoma. As there is currently no effective drug therapy for chordoma the aim of this study was to identify genetic events involved in chordoma pathogenesis with a view to identifying potential therapeutic targets. One hundred chordomas (50 skull-based, 50 non-skull based) were studied. Immunohistochemistry showed that the PI3K/AKT/TSC/mTOR pathway was activated in 65% of chordomas, thereby providing a rationale for testing mTOR inhibitors for the treatment of selected cases. DNA sequencing revealed no mutations in PI3KCA or RAS homologue enriched in brain (Rheb) in 23 tumours. Immunohistochemistry and Western blotting showed activation of the fibroblastic growth factor receptor (FGFR)/RAS/RAF/MEK/ERK/ETS2/brachyury pathway in more than 90% of cases, but no mutations were found in the genes analysed (FGFRs, KRAS, BRAF and brachyury) in 23 tumours. Three percent of cases revealed brachyury amplification but nearly half of the cases showed chromosomal abnormalities involving the brachyury locus. Knockdown of brachyury was achieved in the U-CH1 chordoma cell line using shRNA and resulted in premature cell senescence. These findings demonstrate that brachyury plays an important role in chordoma pathology. FISH analysis showed EGFR copy number gain in 45% of chordomas, including 6% with amplification and 39% with high level polysomy. The EGFR inhibitor, tyrphostin (AG1478) significantly inhibited growth of the chordoma cell line, and Western blotting showed this was associated with reduced phosphorylation of EGFR in a dose dependent manner. This study provides evidence for the first time that selected chordomas may be susceptible to treatment with EGFR inhibitors.

The thesis addresses questions of diagnosis and prognosis in connective tissue disease associated pulmonary hypertension, as addressed in analyses of clinical data from our patients over the last 15 years. Chapter 1 explores use of pulmonary function tests to improve the detection of patients at risk of pulmonary hypertension. A novel formula is derived and validated. Chapter 2 evaluates whether the formula described in the previous chapter can be combined with echocardiography and other measures in order to further improve the selection of patients for right heart catheterisation. In chapter 3 we turn to the prognostic value of ‘borderline pulmonary hypertenion’, exploring indicators of future progression to pulmonary hypertension with important implications for patient care. In chapter 4 we scrutinise our data on body size and inspected the relationship between obesity and pulmonary hypertension, finding intriguing associations which have not been reported before in this population. Chapter 5 addresses the very rare diagnosis of lupus associated pulmonary hypertension and compare this small group of patients to scleroderma patients with pulmonary hypertension in order to consider whether their response to treatment and prognosis are distinct. For chapter 6 we review the relationship between cross-sectional imaging and pulmonary haemodynamics, and specifically address CT findings suggestive of the rare pulmonary veno-occlusive disease. In chapter 7 we turn our attention to the unfortunate group of patients with systemic sclerosis who develop interstitial lung disease and pulmonary hypertension. We compare their survival to those patients who have pulmonary hypertension without interstitial lung disease and find an important predictor of survival. In chapter 8 we review our cohort of patients with systemic sclerosis and pulmonary hypertension and consider whether pulmonary function tests can be combined with pulmonary haemodynamic measures to improve prognostication.

A study was carried out in order to compare the effects of a cognitive-behavioural intervention, with a postal education package, on coping with osteoporosis. Thirty osteoporosis sufferers were divided into three groups. One group received an intervention programme consisting of education, relaxation techniques, and instruction in the use of coping strategies for pain and stress. A comparison group received an education booklet on osteoporosis sent to them through the postal system. The study also included a non-treatment control group. Measures of locus of control, depression, anxiety, pain, and disease knowledge, were taken pre-and post-intervention. lt was predicted that the intervention group would report a significant decrease in external locus of control (subdivided into chance happenings, and powerful doctor control beliefs), and an increase in internal locus of control, and in disease knowledge compared to the other two groups. A significant decrease in depression, anxiety, and pain ratings was also predicted for the intervention group compared to the comparison group and the control group. It was further predicted that their would be no relationship between the age of participants, and scores on any of the outcome measures. Analyses of variance carried out on the data revealed significant differences between the groups from baseline to follow-up on some of the measures. Depression and pain ratings decreased significantly for the intervention group compared to the other two groups. Although anxiety also showed a trend to decrease for the intervention group, this result was not significant. Both internal locus of control and disease knowledge increased significantly for the intervention group compared to the other two groups. No significant differences were found post-intervention between the groups for external locus of control. Correlational analysis revealed no significant relationship between the age of participants and any of the dependent measures. These results suggest that education delivered on an individual basis as part of a cognitive behavioural-intervention is more effective in increasing disease knowledge than an education package sent to osteoporosis sufferers through the postal system. The results further suggest that adaptation to osteoporosis can be facilitated by equipping sufferers with a repertoire of cognitive-behavioural coping strategies, and that older adults are just as likely to benefit from such an approach as their younger counterparts. The results are discussed in relation to previous research findings, and suggestions for future research are put forward.

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