Mechanical loading is an important regulatory factor in bone homeostasis, and plays an essential role in maintaining the structure and mass of bone throughout a lifetime. Although the exact mechanism is unknown the data presented in this thesis supports the concept that substrate signals influence MSC growth and differentiation. A better understanding of the cellular and molecular responses of bone cells to mechanical stimuli is the key to further improvements to therapeutic approaches in orthopaedics, orthodontics, periodontics, bone repair, bone regeneration, implantology and tissue engineering. However, the mechanisms by which cells transduce mechanical signals are poorly understood. There has also been an increased awareness of the need for improvement and development of 3-D in vitro models of mechanotransduction to mimic the 3-D environment, as found in intact bone tissue and to validate 2-D in vitro results. The aims of the project were (i) to optimize a model system by which bone cells can survive in 3-D static culture and their responses to mechanical stimuli can be examined in vitro, (ii) to test the effects of intermittent mechanical compressive loading on cell growth, matrix maturation and mineralization by osteoblastic cells, (iii) to examine the role of the primary cilia, (iv) to assess the effect of dynamic compressive loading on human mesenchymal stem cells in the 3-D environment. The optimized model system has the potential to be used in in vitro studies of bone in 3-D environments including a better understanding of the mechanically controlled tissue differentiation process and matrix maturation in the engineered bone constructs. It has less complicated equipment and techniques compared to dynamic seeding and culture systems making it easy to use in the laboratory. In addition, cells are not pre stimulated by any mechanical stimuli during seeding and culture which enables the researcher to study selected mechanical stimuli and mechanotransduction in bone tissue constructs. The model can mimic the bone environment providing a better physiological model than cells cultured in 2-D monolayer. Using our 3-D system, several loading regimens were compared and it was shown that intermittent short periods of compressive loading can improve cell growth and/or matrix production by MLO-A5 osteoblastic cells during 3-D static culture. This VI suggests that the cells are responding to the mechanical compression stimulus either by directly sensing the substrate strain or the fluid shear stress induced by flow through the porous scaffold. We also demonstrated that our mechanical loading system has the potential to induce osteogenic differentiation and bone matrix production by human MSCs in the same way as treatment with dexamethasone. Although the exact mechanism is unknown the data presented supports the concept that the dynamic compressive loading influence MSC growth, differentiation and production. In further experiments, we used the optimized 3-D model system to study the effects of mechanical loading on primary cilia, which have recently been shown to be potential mechanosensors in bone. We demonstrated that mature cells lacking a cilium were less responsive, less able to upregulate matrix protein gene expression and did not increase matrix production in response to mechanical stimulation suggesting that the primary cilia are sensors for mechanical forces such as fluid flow and/or strain induced shear stress.

The number of people with spinal cord injury (SCI) is increasing every year and walking has been found to be the most exciting and important prospect to these patients to improve their quality of life. Many individuals with incomplete SCI have the potential to walk and everyone of them wants to try. Unfortunately up to now, there is less than one third of patients could walk again after SCI. Residual function, the orthotic support, energy expenditure, patient motivation and control technique are some of the factors that influence the walking outcome of spinal cord injured people. In this thesis, a series of studies are carried out to investigate the possibility of enhancing the performance of the functional electrical stimulation (PES) assisted paraplegic walking with wheel walker through the development and implementation of intelligent control technique and spring brake orthosis (SBO) with full utilization of the voluntary upper body effort. The main aim of this thesis is to enable individuals with complete paraplegia to walk again with maximum performance and the simplest approach as possible. Firstly, before simulation of the system can be made, it is important to select the right model to represent the actual plant. In this thesis, the development of a humanoid and wheel walker models are carried out using MSC.visualNastran4D (vN4D) software and this is integrated with Matlab Simulink® for simulation. The newly developed quadriceps and hamstrings muscle models from the series of experiments are used to represent subject muscles after comparison and validation with other two well-known muscle models are performed. Several experiments are conducted to investigate the effect of stimulation frequency and pulse-width in intermittent stimulation with isometric measurement from paraplegic subjects. The results from this work can serve as a guidance to determine the optimum stimulation parameters such as frequency and pulse-width to reduce muscle fatigue during PES application. The ability test is introduced to determine the maximum leg force that can be applied to the specific paraplegic subject during FES functional task with minimum chance of spasm and leg injury. Investigations are carried out on the control techniques implemented for FES walking with wheel walker. PID control and fuzzy logic control (FLC) are used to regulate the electrical stimulation required by the quadriceps and hamstrings muscles in order to perform the FES walking manoeuvre according to predefined walking trajectory. The body weight transfer is introduced to increase the efficiency of FES walking performance. The effectiveness of body weight transfer and control strategy to enhance the performance of FES walking and reduce stimulation pulses required is examined. Investigations are carried out on the effectiveness of spring brake orthosis (SBO) for FES assisted paraplegic walking with wheel walker. A new concept in hybrid orthotics provides solutions to the problems that affect current 'hybrid orthosis, including knee and hip flexion without relying on the withdrawal reflex or a powered actuator and foot-ground clearance without extra upper body effort. The use of SBO can also eliminate electrical stimulation pulses required by the hamstrings muscle for the same FES walking system. Further improvement of the FES walking system is achieved by introducing finite state control (FSC) to control the switching time between springs, brakes and electrical stimulation during FES assisted walking with wheel walker with the combInation of FLC to regulate the electrical stimulation required for the knee extension. The results show that FSC can be used to accurately control the switching time and improve the system robustness and stability.

Objectives: To evaluate comprehensively the psychometric properties of the Functional Independence Measure (FIM) and the Functional Independence Measure + Functional Assessment Measure (FIM+FAM), and to compare their performance in stroke and multiple sclerosis (MS) patients and with the Barthel Index. To evaluate the conceptual models of both instruments using item analysis, and determine the feasibility of developing a short-form measure. To compare five methods of evaluating responsiveness. Design: Psychometric study. Subjects: 209 inpatients with a variety of neurological disorders recruited from three neurorehabilitation units in Southeast England. Method: Standard methods were used to evaluate the acceptability, reliability, validity, and responsiveness of the FIM and FIM+FAM. Detailed item analyses were performed including internal consistency, intercorrelations between scales and subscales, item convergent and discriminant validity, and principal components analysis. Item reduction techniques were used to develop a short-form FIM. Five methods were used to evaluate responsiveness: t - statistics, relative efficiency, effect size, standardised response mean, and the responsiveness index. Results: The FIM and FIM+FAM are acceptable, reliable, valid, and responsive measures of disability in neurorehabilitation. However, they demonstrate no psychometric advantage over the Barthel Index, show item redundancy, limited item discriminant validity, and inadequate support for hypothesised subscales. An 8-item short-form FIM is developed that shows similar psychometric performance to the 18-item FIM and 30-item FIM+FAM. Five methods of evaluating responsiveness rank order scales similarly, but generate numerical estimates of different magnitude. Conclusions: Results demonstrate the need for a more systematic and rigorous approach to the development and psychometric evaluation of instruments before their introduction into practice to ensure the accurate measurement of patient-oriented outcomes in health care. This approach includes the development of appropriate conceptual and measurement models, the application of standard item analysis and item reduction techniques during questionnaire development, and comprehensive evaluation of the recommended full range of psychometric properties.

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No description available.

The work presented in this thesis involves new methods of using nuclear magnetic resonance spectroscopy (NMR) to study ion transport <i>in vivo</i> in hypertension. A new NMR technique was developed which allowed measurement of absolute concentrations of rubidium (potassium) <i>in vivo</i>. This method was used to study rubidium (potassium) kinetics in spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats and showed evidence for increased potassium efflux and increased Na<SUP>+</SUP>/K<SUP>+</SUP>-ATPase activity in SHR skeletal muscle <i>in vivo</i>. NMR was also used to study <i>in vivo</i> Na<SUP>+</SUP>/H<SUP>+</SUP> antiporteractivity in the skeletal muscle of the rat and it was found that Na<SUP>+</SUP> /H^+ antiporter activity is increased by β2-adrenoceptor stimulation and by increasing the activity of the sympathetic nervous system. This stimulation of Na^+/H^+ antiporter activity in skeletal muscle by the sympathetic nervous system may be part of the well known but poorly understood 'fright and flight' mechanism. Using these new NMR techniques it was found that Na^+/H^+ antiporter activity is increased in SHR skeletal muscle <i>in vivo</i>. In addition, this difference in antiport activity can be removed by blocking L-type calcium channels. These differences in ion transport in SHRs occurred in association with increased skeletal muscle relaxation following contraction which is similar to the finding in vascular smooth muscle in essential hypertension. Despite differences in <i>in vivo</i> Na^+/H^+ antiporteractivity and <i>in vivo</i> potassium fluxes in SHRs no difference was found in either intracellular steady-state concentrations of potassium ions, hydrogen ions or bicarbonate. In addition, there was no difference in intrinsic cell buffering in SHRs <i>in vivo</i> compared with WKY rats. On the basis of these findings a new hypothesis is proposed which links ion transport abnormalities found in hypertension with cell volume and pH regulation.

No description available.

Arthritis is a painful and degenerative condition that affects synovial joints within the human body. Total knee replacement can reduce the pain and increase mobility. Metal on Ultra-high molecular weight polyethylene (UHMWPE) implants have been used successfully for the last 40 years. However, the increasing incidence of implantation in younger patients and the resultant increase in activity has led to higher mechanical demands on the implants. The work in this thesis aims to improve the understanding of UHMWPE with the clinical goal of improved resistance to wear by reducing type 2 fusion defects through improving processing methods and post-processing treatments. The wear properties of four variants of direct compression moulded (DCM) UHMWPE plates were assessed using statically loaded, multidirectional, pin-on-plate machines. Moulding temperatures and durations were varied between 145°C - 195°C, and 15 min - 30 min, respectively. Mean wear factors for the specific processing conditions were as follows 145°C 30 min - 4.362±0.535, 155°C 30 min - 2.903±0.443, 165°C 15 min - 3.026±0.379, 195°C 15 min - 2.631±0.295 (All wear factors x10^(-6)mm^(3)N^(-1)m^(-1)). Wear properties improved with increased temperature. For cases where moulding temperatures were within 10°C, an increase in moulding duration had a positive effect on wear properties. Surface analysis revealed a link between increased number of type 2 fusion defects and an increase in wear factor. Tests using roughened pins increased wear factors, but the differences between processing conditions became smaller. Molecular weight was also found to affect the wear factor with best performance exhibited by that with a molecular weight of 5,000,000 g/mol, compared to 600,000 g/mol and 9,000,000 g/mol specimens. A state-of-the-art Prosim knee simulator was used to assess the wear properties of both the current `gold standard' ArCom DCM UHMWPE and a new generation of vitamin E infused, highly cross-linked E1 UHMWPE inserts. Inserts were of geometrically identical designs and were assessed using a high kinematic test profile. A new load soak control (LSC) station was developed and incorporated into the simulator. Volumetric wear results were 6.46±1.80 mm^(3)/million cycles and 0.853±0.131 mm^(3)/million cycles for the ArCom and E1 inserts respectively. Radiation cross-linking was found to reduce wear by 87%. Both wear rates compared favourably to those obtained in similar tests at other research centres, however care must be taken when comparing results as material, experimental, and geometric design differences may all contribute to variations in wear rates. Identical testing using extremely high femoral surface roughness (Ra=0.295µm) increased wear rates to 100±38.5 mm^(3)/million cycles and 50.7±17.3 mm^(3)/million cycles for the ArCom and E1 inserts respectively, suggesting that with highly roughened counterfaces, some of the cross-linking advantage is lost. Tests on E1 inserts using smooth femoral components but increased axial force (4.0kN) revealed no detrimental effects on wear. Accelerated ageing and subsequent wear testing of the E1 inserts showed no change in wear rate, indicating excellent resistance to oxidative degradation afforded by the infused Vitamin E. Temperature monitoring revealed 6.5°C rises in test cell temperature due to frictional heating between the bearing surfaces. Lubricant uptake for both types of insert was found to increase under dynamic loading, indicating the importance of a LSC. In conclusion: it is possible to improve the wear properties of DCM UHMWPE with small changes in processing conditions. Cross-linking resulted in substantial decreases in wear, however this was less evident when tested using femoral components with increased surface roughness. Vitamin E proved successful in preventing oxidation of the E1 inserts.

No description available.

2A sequences are short 20 ~30 amino acid peptides initially characterised from foot and mouth disease virus (FMDV), but also encoded in a range of Picornaviruses and other viruses as well as non-LTR retrotransposons in a broad range of organisms. They direct a translational recoding event in which ribosomes that have reached the final codon of the 2A sequence pause and terminate translation in the absence of a stop codon and then restart translation. The effect is to separate a single ORF into 2 polypeptides, between the final 2 amino acids (glycine and proline) of 2A, ‘skipping’ a peptide bond. 2A sequences comprise 2 parts, a conserved GDV/IEXNPGP C-terminal motif, and a highly variable, but necessary, N-terminal portion. In the work described in this thesis, extensive mutagenesis studies were carried out across both the conserved and non-conserved portions of the FMDV 2A peptide, including alanine, glycine and proline scanning mutagenesis, site-directed mutagenesis screens. These data indicate that while the amino acid present at the first 2-3 positions of the peptide is not critical for activity, the residue at most other positions is important, including several positions that vary considerably between different 2A peptides. Intriguingly, results of alanine, glycine and proline scanning mutagenesis were very similar, suggesting that features of side chains, and not just the secondary structure propensity of the peptide, plays a critical role. Despite their great variability, each 2A peptide must provide a set of interactions between side chains within the peptide and with the ribosomal exit tunnel to allow the 2A reaction to proceed. Finally, the work also verified a previous finding that insertion of a stop codon in place of the final proline codon of 2A leads ribosomes to stall, unable to terminate translation. Efforts were made to establish a system for purification of such stalled ribosome-nascent chain complexes towards the eventual aim of structural characterisation. The data strongly support a model in which the interaction of the 2A peptide with the ribosome distorts ribosomal conformation.