Evolution has led to specialised shapes for each individual joint which are created during prenatal development through joint morphogenesis. Alterations or abnormalities in joint shape development can severely compromise joint function and have long-term health implications. Mechanical stimulation, due to fetal movements, plays an important role in the creation of joint shapes and a reduction in fetal movements has been highlighted as a risk factor for bone and joint shape malformation, which can lead to physical disability. This doctorate explored the relationship between mechanical stimulation and joint morphogenesis. A novel culture system was created in which embryonic chick hindlimb explants were subjected to controlled and quantified magnitudes of movements. The morphology of the knee joint from explants cultured under different mechanical conditions were compared to reveal how the magnitude of applied movement affects key aspects of the developing joint. A complex relationship was found between the magnitude of movement and the progression of joint shape development. This study provided evidence, for the first time, that the amount of movement experienced during development is a significant variable that impacts of the fate of prenatal joint development. Whilst dynamic movements were applied to developing joints, potential mechanotransduction pathways were tested for their involvement in modulating joint morphogenesis. Blocking the action of both stretch activated ion channels and voltage gated calcium channels produced joints shapes similar to those that had experienced no movement, suggesting the stimulating effects of dynamic movement were not acted upon. This research has provided a novel insight into how mechanical stimulation influences joint morphogenesis which is extremely valuable for understanding of the etiology of congenital joint conditions. Knowledge of how joints are first created is also valuable to the field of tissue engineering for developing treatments to regenerate degenerated joints.

Duchenne muscular dystrophy (DMD) is the most common inherited muscle disease in men and currently there is no effective treatment for this debilitating and lethal disorder. Although the absence of dystrophin is identified as the main cause of DMD, multiple secondary changes have been found to result from the dystrophin deficiency both in muscle and in non-muscle tissues. Among these abnormalities, our laboratory and others have demonstrated a dramatic increase in the expression of the P2RX7 receptor in cells and tissues from DMD patients and the mdx mouse model of DMD. The aim of this study was to determine the effects of P2RX7 ablation on biological functions in mdx muscle in situ and to identify suitable diagnostic and prognostic biomarkers for use in studies on the pharmacological inhibition of P2RX7. RNA transcription was profiled in muscle from wild-type and mdx mice, and also from double knock-out mdx/P2rx7^-/- mice which lack both functional Dmd and P2rx7 genes, and the effects of P2RX7 antagonists were assessed on pathological markers at the acute disease stage of disease in mdx mice, which resembles the human pathology. RNA sequencing was performed using the Illumina HiSeq 2000 platform to characterise the differential gene expression in tibialis anterior (TA) muscles from four week old wild type, mdx and mdx/P2rx7^-/- mice. The biological functions and molecules that are most affected in mdx and corrected in mdx/P2rx7^-/- tissues are those of the immune response, including the innate immune response, cytokine regulatory genes and the NF-кB pathway followed by fibrosis, telomerase regulatory genes, atherosclerosis signalling and the LXR/RXR activation pathway. Moreover, activation of the cell cycle, mitochondrial dysfunction, apoptosis and the adherens junction genes were found to be altered in mdx compared to wild type but not normalised in mdx/P2rx7^-/- muscles. This analysis also demonstrated that the mdx mutation disrupts the non-sense-mediated RNA decay and splicing mechanisms, which leads to an increase in out-of-frame transcripts that may have unexpected cellular impact. One of these altered transcripts, Bmp7, was significantly down-regulated in the mdx myoblasts, myofibres and in TA muscles and restored to the normal level in mdx/P2rx7^-/- muscle. Different analyses were used to map this alteration to the 5'exons of Bmp7 but the identification of this abnormal transcript was not successful. Four P2RX7 antagonists (oxidised ATP, A438079, AFC-5128 and azidothymidine (AZT)) were administered to male mdx mice and their effects on the pathology were analysed using different methods and biomarkers. All of the antagonists inhibited P2RX7 receptor-mediated responses in mdx mice without any detectable side effects. A438079 and AZT were found as the most effective in attenuating the wide range of the pathological features. The reduction in dystrophic features as results of ablation and antagonism the P2RX7 confirms the involvement of this purinoreceptor in the DMD pathology and making it an attractive target for a pharmacological treatment of this lethal disease. Additionally, as AZT is already in clinical use for other diseases, also in children, this drug could be relatively easily re-purposed and trialled for the treatment of DMD.

Musculoskeletal models allow non-invasive predictions of non-directly measurable forces exchanged within the human body in motion. Despite this information has plenty of potential applications, actual adoption of current models is impeded by limitations related to the insufficient number of validation studies or the drastic modelling assumptions often made. This thesis aims to address these limitations developing an innovative approach to the mechanical modelling of the tibiofemoral joint. To achieve this, three main sections are presented: Effects of the soft tissue artefact on current musculoskeletal models – this study used a statistical approach to develop a realistic distribution of soft tissue artefact, which was used to assess the sensitivity of the estimates of three publicly-available musculoskeletal models. Results showed joint-dependent variations, decreasing from hip to ankle, providing awareness for the research community on the investigated models and indications to better interpret simulation outcomes. Modelling the mechanical behaviour of the tibiofemoral joint using compliance matrices – this part of the thesis proposed a method to characterise the tibiofemoral joint mechanical behaviour using a discrete set of compliance matrices. Model calibration and validation was performed using data from ex vivo testing. Accurate results were found in close proximity to where the model was calibrated, opening the way to a more biofidelic joint representations. The developed model was included in the calculation pipeline to estimate joint kinematics using penalty-based method. For this inclusion, validation using in vivo data for these estimates was promising, providing remarkable alternatives to traditional methods. A force-based approach to personalised tibiofemoral models – this section attested on an ex vivo dataset that the model based on compliance matrices can be personalised using data from clinical tests. Since the latter are usually performed in vivo, this opens the way to future exciting applications.

Introduction: Rheumatoid Arthritis (RA) is a chronic inflammatory disorder that typically affects joints of the body such as knees, hands and feet. The prevalence of RA is around 1% in the general population and the incidence rate is about 41 per 100,000 persons per year. The use of adaptive designs in drug development is of increasing interests to medical researchers since adaptive design potentially delivers faster and better decisions compared to the traditional fixed sample-size designs. In RA clinical trials, after treatment starts, follow-up and outcome assessments usually take a number of months, with some outcomes collected up to two or more years after the start of study treatment. For this reason, adaptive designs are rarely undertaken in RA clinical trials as the timing of the endpoints make it difficult to make decisions from the interim analyses. Aims: The overall aim of this thesis is to assess whether or not we can apply an adaptive dose finding design, using Bayesian methods, to a Phase 2a Proof of concept (PoC) trials for the development of a new drug or pharmaceutical treatments for patients with RA disease when the dose response is likely to be non-monotonic. Data: The primary data was a PoC study which was re-analysed retrospectively using an adaptive design procedure to investigate the aspects of the study design. Method: In this dissertation, a Bayesian dose response model was applied to the adaptive design of a Phase 2a PoC clinical trial in RA patients. The first part of the design was to “learn” the dose response in multiple dose cohorts and the second part was to “confirm” the selected dose in a group sequential design with an O’Brien Fleming alpha spending correction. A delayed response predictive model using outcomes collected short-term (Day 14 post-randomisation) for the prediction of longer term outcomes (Day 56 post-randomisation time points) is proposed, based on retrospective analysis and a literature based meta-analysis which was undertaken to investigate the use of early time points to predict Day 56 responses with the intent of reducing the time taken to make decisions at interim analyses of efficient design. Simulations are used to evaluate the models in the desired settings. Results: It was shown that the two-part adaptive design with “learn” and “confirm” could be implemented in the Phase 2a PoC study with two potential improvements: 1) The delayed response predictive model can be used to predict Disease Activity Score (DAS28) Day 56 outcome based on Day 14 outcome to expedite the time to interim decisions in the context of a Phase 2a PoC design; 2) The Bayesian Normal Dynamic Linear Model (NDLM) can be used in the dose response analysis to handle both monotonic and non-monotonic dose responses without sacrificing statistical power or design performance. Conclusion: This thesis demonstrates that it is possible to apply an adaptive design to a PoC study in the treatment of RA. It is recommended that the dose response design with Bayesian NDLM model using predicted DAS28 outcomes at Day 56 based on Day 14 data can expedite the interim decision making. In most cases the Bayesian Emax model works effectively and efficiently, with low bias and a good probability of success in the case of a monotonic dose response. However, if there is a belief that the dose response could be non-monotonic based on prior knowledge then the NDLM is the superior model to assess the dose response. Based on the trial design proposed if the predictive model can be applied to a future adaptive trial, there is potential for a significant time-saving in Phase 2a study.

A search has been made for the presence of an initiating antigen in rheumatoid arthritis to which rheumatoids would specifically respond in vitro tests of delayed hypersensitivity. This has involved the testing of 25 synovial specimens prepared in a variety of ways. Crude synovial homogenates containing soluble and insoluble material have been tested in the leucocyte migration test (LMT) and the lymphocyte transformation test (LTT). Protein eluates and detergent solubilized protein have also been tested. Results have been presented indicating that rheumatoid arthritis patients and age and sex-matched controls will respond to the same extent to an ubiquitous test antigen, streptokinase and to a non-specific cell membrane antigen from homogenized erythrocytes. However RA's do respond specifically to IgG in both native and aggregated forms to which controls do not respond in the LMT. A protein has been eluted from inflammatory synovium which induces equal inhibition of migration in the LMT of both RA and control leucocytes and stimulation of RA and control lymphocytes in the LTT. The mechanism of action of this protein in the LMT and LTT has been discussed in the light of reports in the literature. A role for this protein as an initiating factor in rheumatoid arthritis has not been established. A speculative role in the perpetuation of this chronic rheumatoid response has been presented in terms of the aetiology and pathogenesis of this disease.

The knee joint is the most common joint affected by osteoarthritis (OA). The medial compartment of the knee is more commonly afflicted with OA than the lateral compartment. Lateral wedge insoles (LWI), a common treatment approach for the conservative management of medial compartment knee OA, have previously been considered effective in reducing external knee adduction moment (EKAM). LWI aim to shift a proportion of knee load from the medial compartment to the lateral compartment of the knee joint, providing some symptomatic relief. Foot posture may influence the efficacy of LWI in reducing the EKAM due to its effect on the dynamic function and mechanical alignment of the lower limbs. This thesis investigated the role of foot posture on the magnitude of EKAM and impact on the effectiveness of LWI for the treatment of medial compartment knee OA. Firstly, a repeatability trial was conducted to ensure investigator competency, and reliability of the methods and outcome measures utilised. Secondly, a trial using healthy subjects assessed rearfoot posture using the Foot Posture Index (FPI) to determine if a relationship existed between static foot posture and biomechanical rearfoot motion and their effects on EKAM when wearing LWI, to determine whether clinical foot parameters have a role in the magnitude of EKAM. The role of foot posture in response to LWI, the effects of foot posture on the efficacy of LWI, and effects and impact on EKAM in patients with medial compartment knee OA were assessed. No relationship was identified between clinical static foot posture, biomechanical rearfoot motion, and EKAM. However, a relationship existed between rearfoot motion and EKAM. Rearfoot range of motion can therefore predict the response to LWI. The thesis then examined research questions in regards to the collection of rearfoot motion using different methods (a heel cup cluster and a heel pin cluster) demonstrating that heel pin cluster marker sets are an acceptable method of determining rearfoot motion in barefoot and shod walking. Due to changes in walking speed with lateral wedge insoles the role of increased walking speed on the magnitude of EKAM when wearing LWI was assessed. Increasing walking speed with LWI reduced biomechanical response. The thesis has demonstrated a further understanding of foot posture and ankle motion in healthy subjects and in individuals with medial compartment knee OA. Further clinical studies investigating the role of rearfoot motion and biomechanical response to lateral wedge insoles are indicated.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with an unknown aetiology. Strong evidence exists which suggests that the disease is initiated through a combination of genetic susceptibility and exposure to an environmental factor. One potential environmental factor is mercury (Hg) exposure, which is typically encountered in the form of inorganic mercury (iHg) and methylmercury (MeHg), primarily through dental amalgams and fish consumption, respectively. The effect of mercury exposure in existing autoimmune disease is unknown. The aim of this research was to investigate the role of iHg and MeHg exposure on disease activity and disease associated damage in SLE patients whilst controlling for confounding factors such as n-3 long chain polyunsaturated fatty acids (LCPUFA) and the genotype of genes associated with Hg metabolism (glutathione). This aim was achieved through the completion of observational studies in SLE cohorts based in Northern Ireland. Patients were assessed for Hg concentrations in their hair and urine and also underwent clinical assessment for three disease activity indices and one disease associated damage index. The first observational study (n=52) found a relationship between hair Hg and disease activity and disease associated damage. This finding was repeated in a second observational study (n=98); however, this relationship no longer existed when controlling for n-3 LCPUFA status, indicating that the inverse relationship between hair Hg and disease activity may be due to the concomitant consumption of n-3 LCPUFA present in fish. There was no relationship between urinary Hg or dental amalgams and disease activity or disease associated damage in either study. Neither of the single nucleotide polymorphisms (SNPs) of glutathione related genes investigated were predictors of hair Hg, urinary Hg, disease activity or disease associated damage. An ex vivo study was undertaken to investigate the differences in peripheral blood mononuclear cell (PBMCs) MeHg-induced inflammation between SLE patients and healthy controls and also to determine the impact of n-3 LCPUFA on the MeHg-induced inflammatory response. This study identified a significantly greater pro-inflammatory response from PBMCs of SLE patients (n=12) compared to that of age and sex matched controls (n=12) following ex vivo MeHg exposure. Furthermore, n-3 LCPUFA was shown to ameliorate MeHg induced cytokine release; however, this reduction was less pronounced in SLE patients. The results from this study suggest that SLE patients are more sensitive to MeHg-induced inflammatory response, and that the simultaneous exposure to n-3 LCPUFA is likely to mitigate the adverse effects of MeHg. Taken together, these data further support the evidence that suggests low level MeHg and iHg exposure appears to elicit no adverse effects in SLE patients and furthermore, the work described in this thesis suggests that n-3 LCPUFA is beneficial to SLE patients.

Scoliosis is a spinal deformity characterised by lateral curvatures of the spine. It typically occurs during adolescence, but in rare incidences can occur in young children. In adolescents, if severe or progressive, surgical treatment in the form of spinal fusion using screw and rod constructs may take place. However, in younger patients the spine and lungs are still growing, therefore, fusion is not recommended before the age of eight years. For early onset scoliosis fusionless techniques are used, with the current gold standard being traditional growing rods, incorporating pedicle screws and rods, which are periodically lengthened through frequent surgical interventions. Due to the number surgeries throughout the treatment period, this treatment suffers from high complication rates. As this treatment is far from ideal, alternative treatments are being explored to reduce these issues. This thesis describes the initial development of an alternative implant for the treatment of early onset scoliosis to alleviate the issues of traditional growing rods. The implant was based on a pedicle screw and rod system, but incorporated a bearing component to allow free extension of the construct without the need for further surgery. The development of the implant design was achieved through finite element analysis of individual components and of constructs using contact conditions. It was initially intended to use a UHMWPE bearing component incorporating a crosslink and Ti-6Al-4V sliding rod, with a commercially available pedicle screw. However, issues of the construct size lead to the development of a novel screw design incorporating an alumina bearing, a decoupled crosslink and CoCrMo sliding rod. The results of the finite element analysis show that an implant incorporating an alumina bearing component is a promising area for development, however, additional investigations of the construct are still required.

A method has been obtained for the analysis of hexosamines and neutral sugars by gas-liquid chromatography. Hexuronic acids were found not to be resolved by the standard method and needed to be reduced under acid conditions. As this procedure was shown to be unreliable, polysaccharides containing both hexuronic acids and hexosamines were subjected to such modification reactions as methylation, but with no results. Hydrolytic procedures were applied to glycosaminoglycans to obtain the maximal yields of hexosamine and hexuronic acid. The same procedures were applied to samples of costal cartilage powder and, in addition, conditions for the maximum release of hydroxyproline, sialic acid, calcium and magnesium were determined. The normal range for each of the cartilage constituents was determined and the hydrolysis conditions applied to known cases of metabolic bone disease. In each case an abnormality in at least two constituents was observed. These abnormalities suggest areas for more intensive research and this simple approach to cartilage chemistry is regarded as a 'screening' procedure. Colourimetric hexuronic acid analysis of cartilage was non-reproducible and a new method involving modification of the acid moieties in glycosaminoglycans is presented.

The incidence, nature and significance of activated lymphocytes has been investigated in P.A. and healthy subjects. The incidence of these cells using discontinuous Ficoll gradient analysis was studied in R.A. patients with different disease activity and in those undergoing gold therapy. The density gradient also provided a method for separating lymphocyte subpopulations for the further study of surface markers. These included the possession of surface Ig and receptors for sheep erythrocytes and IgG (Fc). R.A. patients, particularly those with clinically active disease, had more circulatory activated lymphocytes (immunoblasts) and low density lymphocytes than healthy subjects. This increase in low density cells was not seen in patients receiving gold therapy. Surface marker analysis showed that the population of low density (activated) lymphocytes in R.A. were enriched in surface Ig bearing cells and depleted of cells with sheep erythrocyte receptors, in comparison with the high density (small) lymphocytes. However, by comparison, high avidity Fc receptors were found predominantly on the dense lymphocytes whereas low avidity Fc receptors were more common on the low density cells. The role of the activated cells ir R.A. is considered with particular reference to the increased B cell activation in peripheral blood. The propagation of R.A. by activated lymphocytes is discussed in relation to their abnormal migratory patterns to inflammatory sites.