Study 1: Study design: A non –randomised continuous retrospective cross sectional and observational study Objective 1) To evaluate the results of nonoperative treatment of symptomatic lumbar pars stress injuries or spondylolysis in sporting as well as non sporting individuals 2) To determine the factors responsible for non-operative method of managing symptomatic lumbar spondylolysis in young population 3) To evaluate the outcome in different types of sports 4) To establish the role of compulsory non-operative treatment for symptomatic lumbar spondylolysis in sporting individuals Summary of Background Data The treatment and management of symptomatic spondylolysis in sporting populations is mainly based on observation rather than experimental study. Conservative treatment in the form of bracing and avoidance of sports for at least three to six months has been recommended. Excellent or good results following bracing and physical therapy have been observed in 80% patients. Criteria for return to sport are dominated by symptom led decisions. Methods The research was carried out as a qualitative, descriptive and analytic study with a non-randomised cohort of patients investigated for spondylolysis in a single centre. A total number of 123 patients treated conservatively following confirmation by imaging studies (SPECT,CT or MRI scans) as having stress fractures of the lumbar pars interarticularis (PI) ranging in age from 8 to 35 years have been selected for the study. All patients attending the Back pain clinic has to follow a protocol of filling up the VAS, ODI and SF-36 questionnaires as a part of their assessment. At the time of the study these questionnaires along with the Back Pain & Sports Questionnaire (BPSQ) were sent to all but only 123 patients responded who were included in the study 1. The background data contains gender, age, date of onset of symptoms with current limitation in sport, pain in flexion or extension, type of sport, level of sport and length of treatment. The data also contains each subject with level, number, laterality and distribution of lumbar spondylolysis, investigations, outcome with VAS, ODI, SF-36 and Back pain and sports questionnaire (BPSQ) and return to sports. We classified the individual sports into seven types depending on the major movements of the body. Descriptive and analytical statistics was performed along with correlation testing between the outcome measures and predictive factors. Results The mean age of onset of back pain was 21.7 years (range 8-35 years). Most patients were between the ages of 15&19 years (43) followed by 20&24 years (32). The Male: Female ratio was 74:49. There were 98/123 (76.9%) sporting individuals. 35/98 (35%) were professional players, 29/98 (29.5%) were semi professional and 34/98 (34.6%) were amateur sportsmen and women. Cricket (22) followed by Football (22) were the most common type of sports played. Trunk twisting movement was the common denominator in most of the patients with pars defect. The cricketers (13) with unilateral pars defect had more commonly left sided pars defect than the right (10 left vs 3 right). Right sided pars defect was more commonly observed in soccer players (7:1). Most incomplete fractures were observed at L4 in the cricketers. The non sporting group had consulted with a delay of more than six months since the onset of pain. 60% pars lesion was observed at L5 followed by L4 (11.3%), L3 (9.7%) and L2 (2.4%). At L5 most were bilateral lesions (81%). Spina bifida was recorded in 16% patients. The mean pre and post treatment VAS score was 4.5 and 0.65 respectively (SD- 0.8,p<0.01). The mean pre and post treatment ODI was 35.5 (SD-7.8) and 6.9 (SD- 7.6) respectively (p<0.01). In the SF-36 scores, the mean score for the physical component of health improved from 34.9 (SD – 5.3) to 49.3 (SD -6.6) (p< 0.001). The mean score for the mental component of health improved from 40.2 (SD -5.2) to 52.0 (SD-6.0) (p<0.001). The mean BPSQ score was 52.5 (range 0-90). The mean pretreatment and post-treatment VAS and ODI scores were slightly better in males as compared to females. In the unilateral group, 28/36 (77%) patients had complete relief of pain by a mean time of 4.2 months (range 3-7 months). In the bilateral group, 47/59 (79%) patients had complete pain relief at a mean time of 6.5 months (3-12 months). In the unilateral pars defect group, 32/36 sporting individuals returned to active sports. In the bilateral pars defect group, 49/59 sporting individuals returned to active sports. There was significant difference between the sporting and the non-sporting group in their age (mean 20.7 vs 25.4 years, p <0.001). There was significant difference between the two groups in all pre and post treatment outcome scores. The pre treatment VAS score had most significant correlation with post treatment ODI ( =0.634, p <0.01) and post treatment VAS scores ( =0.626, p<0.01). Conclusion A treatment protocol of rest for 4-6 weeks followed by the functional restorative program has excellent or good outcome in 85% sporting individuals with symptomatic pars defect. Male sporting individuals have better outcome than females. Unilateral pars lesions have a better outcome than bilateral pars lesions. Bracing may not be required in most patients if the pain subsides on restriction of activity. Full functional recovery to previous level of activity is possible with the help of dynamic spinal stabilization exercises and physical therapy. The individuals involved in trunk twisting sports should be evaluated carefully for muscle imbalance in the lumbar spine and they should have altered techniques of sporting activity without compromising the performance in the rehabilitation phase. Study 2: Study Design: A non –randomised continuous retrospective observational study Objective 1) To identify the most significant determinant of surgical intervention in lumbar pars defect 2) To identify the independent factors that predict a successful outcome following surgery for lumbar pars defect in young sporting individuals 3) Can we establish an outcome predictive model based on these significant factors responsible for a successful outcome? Summary of Background Data Most athletes or young active professional sportsmen or women would like to return to their previous level of sports since they may be earning their livelihood through the sport. Early onset of symptoms and conservative treatment in these patients may lead to a good clinical outcome but it is difficult to predict which group or which individuals will require surgical repair of the defect. Young athletes to have returned to competitive sports after surgery have been reported only in few previous papers. The first cohort from this series was published in 2003. ODI (Oswestry Disability Index) and SF-36 (Short form) scores were used to evaluate the final outcome for the first time in lumbar spondyloysis for outcome analysis. Methods A total number of 55 patients treated operatively following confirmation by imaging studies (SPECT,CT or MRI scans) as having stress fractures of the lumbar pars interarticularis (PI) ranging in age from 8 to 35 years have been selected for the study. All patients attending the Back pain clinic has to follow a protocol of filling up the VAS, ODI and SF-36 questionnaires as a part of their assessment. At the time of the study these questionnaires along with the Back Pain & Sports Questionnaire (BPSQ) were sent to all but only 50/55 patients responded. The background data contains gender, age, date of onset of symptoms with current limitation in sport, pain in flexion or extension, type of sport, level of sport and length of treatment. The data also contains each subject with level, number, laterality and distribution of lumbar spondylolysis, investigations, outcome with VAS, ODI, SF-36 and Back pain & sports questionnaire (BPSQ) and return to sports.

Aims: The aims of this study were to determine the usefulness of a functional knee brace (FKB) or a spiral method of taping in modifying the impaired biomechanics of the ACL-deficient knees towards a safe and more normal pattern, and to assess any compensatory changes at the ankle and hip joints following knee bracing or taping. The study also aimed to compare the difference in gait patterns during simple· level walking and treadmill activities for ACL-deficient subjects. Methods: A prospective experimental study was carried out on 15 ACL-deficient and 15 carefully matched amateur athletes as controls. A comprehensive gait analysis study was designed using a high frequency CODA-mpx30 gait analysis system, force platform and electromyography (EMG) system. The study was carried out during simple level walking, treadmill walking (3.6 Km/hr) and treadmill running (10 Km/hr) which we describe as low and high level physical activities. Treatments investigated included a functional knee brace (FKB) or a special spiral taping method that was applied to the deficient knees. The temporospatial parameters, total range of motion (ROM), joint position, kinetics and EMG parameters were recorded in the knee, ankle and hip joints in different trials with different supports and the results were compared with the baseline data of both the patients and the data derived from the control subjects. Main Results: The FKB significantly reduced total ROM in the ACL-deficient subjects for all levels of walking trials (P<O.05). The FKB significantly reduced peak knee flexion during swing while walking on level ground, but increased maximum knee flexion in swing during walking on the treadmill (P<O.05). Taping significantly increased mean knee angle in stance in both walking modes (P<O.05). Neither FKBs nor taping showed any angulatory kinematic effects on the knee joint during running on the treadmill. The FKBs could significantly reduce the antero-posterior (A-P) displacement of the tibia relative to the femur during level walking mostly in the swing phase. Wearing a brace did not reduce the knee extensor moments, but significantly reduced the hip flexor moments. Taping, however, had no significant effects on knee moment, but increased the generation and absorption of ankle power and decreased hip generation power. Bracing reduced the "support moment" and "support power" in the lower limb, but taping did not change them. No quadriceps avoidance gait pattern was found in this study and the patients showed an extensor knee moment throughout the stance phase. The gastrocnemius muscle was found to have a principal role in the ACL-deficient subjects and wearing a FKB could significantly activate the gastrocnemius muscles earlier in the ACL-deficient subjects, although no effects on peak activity of the muscle were demonstrated. Conclusion: It can be concluded that the functional knee brace used in this study did not show any harmful effects in ACL-deficient knees. It was helpful particularly for low force activities such as level walking. The brace was as effective for walking on the treadmill as walking on level ground although some kinematic changes exist between these two different activities. Taping, however, is not recommended for ACL-deficient knees. Since the ACL-deficient subjects showed good knee control in most running trials, there would appear to be a need for more strenuous activities and these are strongly recommended.

Background: Low back pain is a main cause of sickness absence and work disability in the UK. The economic impact of this is considerable and there is a growing urgency to address the occupational management of low back pain through a vocational focus on rehabilitation. However there is a lack of evidence as to how the needs of this client group can best be met. Objective: The aim of this study was to test the feasibility of delivering an NHS vocational intervention to this client group, assess how acceptable the intervention was to the participants, and examine the costs involved. Methods: The study followed an iterative process of development, evaluation and implementation. The study used survey and interview methods to investigate current NHS provision of work-related advice and support to this client group, and determine how the effectiveness of vocational interventions might be measured. The findings were used to inform the design and test the feasibility of an individually targeted vocational intervention and economic evaluation with patients concerned about their ability to work due to low back pain. Results: The findings of this research demonstrated that there is limited advice and support available to people who are concerned about their ability to work due to low back pain, either from clinicians or in the workplace. Although routine multidisciplinary group rehabilitation reduced patients' concerns, its impact depended on the ability of the patient to apply condition management tools and techniques to the workplace. A total of 51 patients were recruited over a six month period to a feasibility randomised controlled trial with concurrent economic evaluation. Eighty-seven individual work support sessions were delivered. Outcome data was obtained for 38 participants at six month follow-up. Post-trial interviews were conducted with 22 of the trial participants. The intervention and the trial were acceptable to many of the participants, although not all were willing for the researcher to involve the workplace and some did not engage. For some, the demands of work itself were an obstacle to accessing treatment. Conclusions: This study showed that it is feasible to deliver an individually targeted NHS vocational intervention to this client group, that the protocol was acceptable to many of the participants, and that an economic evaluation could be conducted. However, the current design cannot be recommended for a definitive randomised controlled trial. Considerable methodological changes are needed to address the method of recruiting participants, the delivery of the intervention and the measurement tools used. Furthermore, routine rehabilitation may not be sufficiently reliable as a control. Finally, the impact of vocational interventions is likely to be limited unless partnership working between clinicians and employers becomes customary practice.

Mutations affecting the p62 signalling adapter protein are commonly found in patients with the skeletal disorder Paget‟s disease of bone (PDB). We have extended previous in vitro functional analyses of PDB-mutant p62 proteins (Cavey et al., 2006) to study the effects of several uncharacterised PDB-associated mutations on the ubiquitin-binding properties of p62. These include mutations which affect regions of p62 outside of the ubiquitin-binding UBA domain (A381V, D335E and a mutant equivalent to a predicted product of the G1205C splice-site mutation which lacks amino acids 351-388), as well as a double mutation involving the P392L and S399P changes on the same allele. In accordance with previous findings, both of the non-UBA domain mutations (A381V, ∆351-388) showed deleterious effects on ubiquitin-binding by p62 in pull-down assays, further emphasising the important role of non-UBA domain sequences in mediating ubiquitin-recognition, as well as in PDB aetiology. The D335E mutant retained its ubiquitin-binding function in vitro. The P392L/S399P double mutant showed a more severe effect on ubiquitin-binding than either of the single P392L or S399P missense mutations alone; as this double mutation is associated with a particularly severe phenotype, our findings are supportive of the proposal that disease severity in PDB with p62 mutations may be directly related to the effects of the mutations on the ubiquitin-binding function of the p62 protein. Since the in vitro pull-down assays are semi-quantitative at best, we sought to investigate if a more quantitative biophysical approach, two dimensional Heteronuclear Single Quantum Coherence (2D-HSQC) protein NMR, might be applied to investigate the effects of PDB-associated mutations on protein (ubiquitin-binding) function. Our results showed that protein NMR was not optimal to quantitatively assess the effects of the mutations on the interaction between p62 and ubiquitin in vitro. Using confocal microscopy, co-transfection of U20S cells showed that the selected PDB-associated p62 mutants (A381V, P392L, G425R) co-localised with ubiquitin with a cellular phenotype indistinguishable from wild type, as each PDB mutant formed cytoplasmic bodies with an area ranging from the detection limit of the microscope to 40μm2 or higher; in contrast the E396X truncating mutant did not form cytoplasmic bodies nor co-localise with ubiquitin. In addition to interacting with ubiquitin, p62 also interacts with the LC3 (an autophagic marker) through its LC3 interacting region (LIR) to mediate the formation of autophagosomes. By co-transfecting p62 constructs with LC3 We found that some of the p62-positive cytoplasmic bodies were autophagosomes, and that the D335E mutation of p62 (which lies within the LIR) did not appear to affect the formation of autophagosomes. The effects of the wild type and PDB-mutant p62 proteins on NF-κB signalling were assessed in HEK293 cells co-transfected with an NF-κB luciferase reporter construct. A381V mutant p62 produced a level of activation of NF-κB signalling greater than wildtype and similar to that of UBA domain mutants, indicating that non-UBA and UBA domain mutations may exert their effects through a common mechanism involving dysregulated NF-κB signalling. To further examine the function of p62 in the regulation of NF-κB signalling, we went on to determine possible effects of PDB-associated mutations on p62-CYLD (a DUB enzyme) interactions. Unexpectedly we found that CYLD expression appears to abrogate the formation of the p62 cytoplasmic bodies previously shown to be ubiquitin-positive. Finally, we went on to study the interaction of p62 (and its PDB mutants) with another important regulator of NF-κB signalling, IKKγ/NEMO. We concluded that wild type and PDB-mutant p62 proteins are capable of recruiting NEMO to cytoplasmic bodies which may represent autophagosomes, but do not appear to accelerate its degradation.

Background: Osteoarthritis (OA), one of the commonest joint diseases of unknown aetiology is a major source of pain and disability in the ageing population. Current therapeutic agents for OA focus on symptomatic relief. Patients often present to clinics with long established disease when the boundaries between ageing and pathology are indistinct. A greater understanding of early OA is thus required and may help achieve the goal of disease modification. OA is associated with chondropathy, synovitis, subchondral bone remodelling and osteophyte formation. Angiogenesis, the growth of new blood vessels from pre-existing ones may contribute to each of these features. Inflammation is increasingly recognised as an important feature of OA. Synovitis is detectable within the osteoarthritic joint both radiologically and histologically, evidenced by symptoms such as stiffness or pain, signs such as effusion and use of anti-inflammatory drugs for treatment of OA. Pain is the predominant symptom of OA, but little is known about the mechanism by which this pain arises. Objectives: This thesis describes studies examining the contributions of angiogenesis to inflammation, structural damage and pain in OA. Hypothesis: Inflammation and angiogenesis are co-dependent processes that can exacerbate and mediate structural damage and pain in OA. Methods: In-vivo, in animal models and ex-vivo in human meniscal tissue, using immunohistochemistry, joint histology and pain behaviour testing, the effects of enhancing synovitis on angiogenesis, structural damage and pain in OA, and whether inhibiting either synovitis or angiogenesis could reduce this structural damage and pain were investigated. Results: It is shown for the first time that blood vessel growth at the onset of resolving synovitis leads to its subsequent persistence. Exacerbating synovitis in the rat meniscal transection (MNX) model of OA enhanced synovial angiogenesis, total joint damage and pain behaviour. Following treatment with the anti-angiogenic compound, PPI-2458 [(1R)-1-carbamoyl-2-methyl]-carbamic acid-(3R, 3S, 5S, 6R)-5-methoxy-4-[(2R, 3R)-2-methyl-3-(3-methyl-but-2-enyl) oxiranyl]-1-oxaspiro (2*5) oct-6-yl ester], synovial angiogenesis, vascularisation of channels penetrating into the articular cartilage (osteochondral angiogenesis), synovitis, joint damage (mainly attenuation of osteophyte growth) and pain behaviour were all reduced. Anti-inflammatory drugs (dexamethasone and indomethacin) reduced synovitis, synovial angiogenesis and pain behaviour in the rat MNX model of OA. Treatment with dexamethasone reduced joint damage score by decreasing cartilage damage. Indomethacin however did not affect joint structure. Human meniscal tissue from knees with high chondropathy displayed increased degeneration of collagen bundles, increased vascular densities both in the synovium and at the fibrocartilage junction with a greater density of perivascular sensory nerve profiles in the outer region. Increased penetration of the synovial tissue towards the tip of the meniscus was noted in menisci from high chondropathy group compared to those from the low chondropathy group. Conclusion: Data from animal studies indicates that conversion of acute inflammation to chronic inflammation may be due to the stimulation of angiogenesis. Furthermore, these data provide evidence that synovitis contributes to joint pathology and pain behaviour in the rat MNX model of OA and this may be partly due to the angiogenesis in the synovium and at the osteochondral junction. Data from human studies highlights that tibiofemoral chondropathy is associated with altered matrix structure, increased vascular penetration and increased sensory nerve densities in the medial meniscus. Angiogenesis and associated sensory nerves in the meniscus may therefore contribute to pain in knee OA. Summary: These findings support the hypothesis that inflammation and angiogenesis are indeed co-dependent processes, exacerbating and mediating structural damage and pain in OA. Angiogenesis inhibition has the potential to reduce synovitis, joint damage and pain in OA.

Osteoarthritis and degenerative cartilage diseases affect millions of people. Therefore, there is huge interest in developing new therapies to repair, replace and/or regenerate cartilage. This necessitates advances in techniques which make earlier non-invasive diagnosis and objective quantitative evaluations of new therapies possible. Most previous research has focused on the knee and neglected the ankle joint. Hence, the aims of this thesis are to describe and quantify the geometric properties of ankle cartilage, to evaluate joint contact characteristics and develop techniques which allow quantitative measurements to be made in vivo. Chapters 3 and 6 describe the application of a high resolution stereophotography system for making highly accurate 3-D geometric models from which quantitative measurements of cartilage parameters and joint area contact can be made. Chapters 4 and 5 report the testing of image analysis algorithms designed to segment cartilage sensitive MR images. Work focused on initially on a semi-automated 2-D segmentation approach and subsequently on a pilot study of 3-D automated segmentation algorithm. The stereophotographic studies were highly accurately and demonstrated that ankle cartilage thickness is greater than previously reported with the thickest cartilage occurring where cartilage injuries are most commonly seen. Furthermore, joint contact area is larger than previously believed and corresponds to the regions of the thickest cartilage over the talar shoulders. The image analysis studies show that it is possible to accurately and reproducibly segment the thin cartilage layers of the ankle joint using a semi-automated approach. The feasibility of a fully automated 3D method for future clinical use is also shown. In conclusion this thesis presents novel methods for examining ankle articular cartilage in vitro and in vivo, showing that the thickest cartilage occurs in highly curved regions over the shoulders of the talus which correspond to regions of greatest contact. Importantly, the image analysis techniques may be used for future clinical monitoring of patients sustaining cartilage injuries or undergoing cartilage repair therapies.

Background. Knee osteoarthritis (OA) can result in considerable pain and disability for some people. Inflammation within the joint may be partly responsible for the pain associated with OA and a link between inflammation and disease progression has been suggested. Ultrasound (US) imaging has been successfully employed in the evaluation of knee joint effusion, synovial hypertrophy and power doppler signal (PDS) which are said to represent joint inflammation. The associations between US features of inflammation, knee pain and radiographic OA have yet to be firmly established. Objectives. The objectives of this thesis were to compare the frequency of US features of inflammation in 4 groups from a community sample, [1] those with normal knees (controls) [2] knee pain - without radiographic OA (KP) [3] radiographic OA (without pain) (ROA) and [4] symptomatic OA (SOA). Associations between US features, knee pain, radiographic change and clinical signs of inflammation could then be explored. Secondary objectives were to determine if US features change in tandem with fluctuations in knee pain (1) over time and (2) with improved pain following a therapeutic intervention in people with SOA. Methods. In a cross-sectional multiple group comparison study, 243 participants were divided into 4 groups based on the presence of absence of knee pain and ROA. All underwent an US examination for effusion, synovial hypertrophy, peri-articular cysts and PDS. The presence or absence of features, absolute measures (millimetres) and grade of PDS (0-3) was recorded for both knees. Radiographs and clinical evaluation of knee pain, biomechanical stiffness and function were also undertaken. Follow-up examination of control and SOA groups was undertaken at 3 months. Participants with SOA were then invited to take part in a randomised placebo-controlled study of intra-articular (IA) cortico-steroid and a saline placebo. Results. The frequency of US features in the control group (effusions (29%) synovial hypertrophy (8%), popliteal cysts (12%) and PD signal (2%)) was not significantly different from those in the KP group. US features were more common in ROA and higher again in SOA (effusion 81% and 92% respectively, synovial hypertrophy 41% and 82%, popliteal cysts 22% and 39%). PDS was not significantly different between ROA (6.3%) and SOA (16%). Synovial hypertrophy was the only US feature independently associated with knee pain after adjusting for ROA (aOR 6.6; 95% CI 2.85, 15.11). All grey-scale features were strongly associated with ROA and remained so after adjusting for pain (effusion aOR 13.39, 95%CI 6.14, 29.02; synovial hypertrophy aOR 14.39, 95%CI 6.28, 32.94; popliteal cysts aOR 2.82, 95%CI 0.76, 10.43). PDS was not association with either knee pain or radiographic OA. Change in pain severity was not found to correlate with and change in US measures among the participants followed up at 3 months or following improved pain among participants in the intervention study. Conclusion. These findings show that US features suggestive of inflammation are higher in participants with SOA but was only significant for synovial hypertrophy. Synovial hypertrophy was confirmed as an independent risk factor for knee pain but was not found to be responsive to temporal changes in pain or improved pain following an IA cortico-steroid or placebo injection. Further studies to understand the contribution of US features of inflammation to pain in knee OA are warranted.

Weakness and fatigue in the human triceps surae have been assessed objectively by the measurement of absolute force evoked using supramaximal stimulation. The effects of disuse, age and exercise were systematically investigated. Under control conditions the triceps surae of young men were found to generate high maximal tetanic forces, have a mean twitch time to peak tension of 107 msec and did not fatigue readily. This was indicative of a large muscle mass with a predominance of type I (slow twitch) fibres. Muscle temperature manipulation over the range 29.5 to 39.1°C did not affect maximal force generation but had a profound effect on the force and time course of twitch and unfused tetanic responses. The triceps surae of 70 year old men were found to be slower contracting weaker and yet, paradoxically more fatiguable than, those of young men. These changes may be explained by a slowing of the Ca 2+ kinetics in the remaining muscle fibres of the elderly and restricted blood supply during intermittent exercise. Long term immobilisation due to injury caused a substantial reduction in the force generating capacity of the triceps surae and a change in twitch time course which could be explained by selective type I fibre atropy. In contrast voluntary immobilisation for 2 weeks caused a reduction of maximal voluntary force and a prolongation of the twitch response which could not be accounted for by loss of contractile machinery. Voluntary dynamic exercise involving concentric contraction of the triceps surae produced small short lasting force decrements. Eccentric contractions caused large long lasting decreases in force particularly at low stimulus frequencies, which were explained by uncoupling of excitation and contraction. Responses to submaximal stimulation were found to be voltage dependent and did not accurately reflect the response of the whole muscle. The need for supramaximal stimulation in the assessment of weakness and fatigue in the human triceps surae was highlighted.

Sarcopenia or the involuntary age associated muscle wasting starts in the fourth decade of life and accelerates markedly from the fifth decade. This gradual loss of muscle mass eventually results in an inability of older people to carry out simple daily tasks, instability, is associated with an increased risk of falls and fractures, loss of independence, and reduced quality of life. As the number of older people is growing steadily in our society, this in turn places an increasing burden on health care resources, making the topic of sarcopenia and its consequences an important area for research. Resistance exercise and protein enriched feeding are potent stimulators of MPS and act synergistically to increase the MPS; however, the muscle protein synthetic responses to amino acids are blunted in the elderly in the resting state. Leucine has been shown to be the most potent branched-chain amino acid acting as a signal for accelerating MPS in the resting state. How intensity and duration of resistance exercise can affect MPS and anabolic signalling in the elderly is less well understood. Can leucine enriched protein supplementation coupled with resistance exercise rejuvenate the MPS responses in the elderly? We aimed to answer these questions. The results revealed a sigmoidal dose-response relationship between exercise intensity and the stimulation of MPS in the post absorptive state, with little increase from 20-40% 1RM, then a bigger rise at 60 % of 1 RM with no significant further increase up to 90% 1RM in both the young and the elderly. Both groups showed quantitatively similar increases in phosphorylation of both p70s6K and 4E-BP1, which were maximal for exercise at 60-90% 1 RM at 1 h post exercise, i.e. just before the maximal increase in MPS. However, older men demonstrated a blunted rise in MPS and anabolic signalling activity after exercise, suggesting a general pattern of a reduced protein synthetic response to exercise in the elderly. This may explain, in part the mechanisms through which muscle is lost gradually with ageing. Increasing exercise volume from 3 to 6 sets at 40% and 75% 1RM produced no additional MPS responses in post absorptive young men; however, in older men, it resulted in enhanced MPS and p70S6K responses at both intensities, suggesting that the muscle of older men requires a greater volume of exercise to activate the protein synthetic machinery sufficiently to achieve synthetic responses comparable to those seen in younger men. Exercise, irrespective of intensity and volume caused only short term stimulation in MPS (returned to basal level at 4h post exercise) in the post absorptive state. Leucine supplementation to protein feeding after resistance exercise appeared to overcome age-related anabolic blunting of responses of myofibrillar protein synthesis and p70S6K phosphorylation in skeletal muscle of older men by rejuvenating their synthetic responses. In summary, the results gave a clear indication as to the likely optimal exercise intensity and volume of acute resistance exercise (6 sets of 8-10 reps at 75% 1RM) coupled with optimal amino acid supplementation (leucine supplemented drink containing about 20 g of protein) required to effectively stimulate MPS and anabolic signalling in the elderly for maintenance of muscle mass. This work helps shed light on the pathophysiology of sarcopenia and suggests strategies that could be used to develop effective countermeasures to counteract sarcopenia.

Myotonic dystrophy (DM) is an autosomal dominant inherited multisystemic neuromuscular disease. The molecular mechanism for DM is mediated by toxic RNAs containing expanded repeat units. DMI is caused by a CTG repeat expansion in 3'-UTR of the DMPK gene while DM2 is caused by CCTG repeat in intronl of the ZNF9 gene. The molecular features of DM are formation of RNA foci, co-localisation of MBNL proteins with ribonuclear foci, splicing defects of a subset of pre-mRNAs with elevation ofCUGBPI in DMl. In order to develop therapy for DM, assays were designed based on the molecular characteristics of the disease to screen compounds. Two primary assays were based on disruption of nuclear foci and on correction of misregulated splicing involving intron2 CLCNI. The first part of this report deals with the development of a nuclear foci assay and splicing construct assay. Both assays were optimised in HTS and utilized in screens for molecules that clear nuclear foci from DM cells and correct misregulated splicing in intron2 of CLCNI respectively. High throughput screens of kinase and phosphatase inhibitor libraries using the nuclear foci assay and CLCNI splicing construct assay yielded two positive hits: protein kinase C inhibitors designated in the compound library as D8 (hypericin) and D9 (Ro-31-8220). The second part of this thesis deals with the confirmation of compound hits obtained from the primary screen. I examined two aspects: mutant DMPKtranscript entrapment in nucleus and splicing defects associated with the disease. A BpmJ restriction assay was used to test the effect of compounds on mutant DMPK transcripts showed that both D8 and D9 were unable to release the mutant transcript into the cytoplasm. D8 demonstrated efficacy in reversing spliceopathy in alternative splicing assays of JR, SERCAI MBNLI and MBNL2 while D9 did not have this effect except on MBNLI which showed a very minor efficacy.