This thesis is concerned with the study of biophysicochemical processes using electrochemistry and related techniques. The first part of the thesis discusses the electrochemical detection of biological species, and characterisation of the electrode materials employed. A comparison of two novel forms of carbon electrode, namely carbon nanotubes and polycrystalline boron doped diamond (pBDD), with more conventional carbon electrode materials reveals their enhanced characteristics for bioelectrochemistry, with improved sensitivity and resistance to fouling. These materials are further characterised using novel high-resolution electrochemical imaging methods, to determine heterogeneous electron transfer rates for a number of different redox species. The kinetic rate constants are determined from measured electrochemical currents using finite element method (FEM) modelling, which proves to be a powerful technique for the quantitative analysis of intrinsic system parameters that cannot be studied directly. The electrochemical response of isolated regions of pristine SWNTs is investigated using scanning electrochemical cell microscopy, demonstrating high electrochemical activity at the nanotube sidewalls. A similar analysis of the different facets of pBDD is performed using intermittent contact scanning electrochemical microscopy coupled with FEM simulations, revealing that the electroactivity is strongly in uenced by the local density of states of the material. New techniques are also presented for the investigation of transport processes at membrane interfaces. A new method of bilayer formation is developed, which overcomes many of the limitations of current techniques, and is used to investigate the permeation rates of a series of aliphatic carboxylic acids. Using confocal laser scanning microscopy (CLSM) with a pH-sensitive uorophore, the pH change as a weak acid permeates across the bilayer can be visualised, and the permeation coefficient determined by comparison with FEM simulations. This reveals a trend of increasing permeability with lipophilicity. Finally, CLSM is used to study the lateral diffusion of protons at lipid bilayers and other surfaces. Protons are generated galvanostatically by a UME positioned close to the substrate, altering the local pH which can be visualised by means of a pH-sensitive uorophore. The uorescence profile is again compared to FEM simulations, allowing the lateral diffusion coefficient to be determined.

Mesenchymal stem cells (MSC) are a tissue-resident stromal cell population that are able to regulate immune responses, in particular the capacity for endothelial cells (EC) to support leukocyte recruitment. In this thesis we examined the ability of MSC from different sources (bone marrow, Wharton’s jelly and trabecular bone) to regulate neutrophil recruitment to inflamed EC and how these responses are altered upon adipogenic differentiation of MSC. Using two flow based adhesion models with varying degrees of proximity between MSC and EC, we observed that all MSC populations suppressed neutrophil recruitment. IL-6 and TGFβ were identified as common bioactive agents found in all co-cultures. Upon differentiation, MSC exhibited a diminished capacity to suppress neutrophil, but not peripheral blood lymphocyte, recruitment. Loss of suppression by MSC-derived adipocytes was reversed by neutralising IL-6. Adipose tissue-derived mature adipocytes and culture differentiated pre-adipocytes did not recapitulate the effects of MSC-derived adipocytes. These data suggest that crosstalk between tissue-resident MSC and EC, dampens the endothelial response to cytokines and limits the aberrant infiltration of circulating leukocytes during inflammation. Upon adipogenic differentiation, MSC lose this regulatory capacity. This could impact on the beneficial effects of MSC in chronically inflamed sites where aberrant infiltration of leukocyte is a main driver of the disease.

Osteoarthritis is the most widespread musculoskeletal disease worldwide among the elderly. It causes joint pain that can affect locomotion and reduce mobility. For osteoarthritis patients, maintaining walking ability is considered the most beneficial way to preserve their quality of life. Walking sticks are widely used by elderly adults and have been shown to have a supportive role on locomotion. In this thesis I carried out four experiments: The first study investigated how footwear affects the locomotion of elderly patients suffering from this disease. In the second chapter the gait of elderly walking stick users was analysed in conjunction with their responses to a questionnaire with a view to understanding the causes and context of walking stick use in their everyday environments. My findings demonstrated that the majority of participants experienced greater pain after prolonged use of their walking stick. In the last two experiments I investigated how the use of a walking stick combined with aspects of the individual’s locomotor environment (e.g. indoor and outdoor, level and sloped surfaces) to influence gait. Overall, osteoarthritis, advanced age and challenging locomotor environments can influence their quality of life and the risk of falling.

The secretory pathway Ca\(^{2+}\)-ATPase (SPCA) provides the Golgi apparatus with a luminal Ca\(^{2+}\) store, which is used to modulate the activity of Ca\(^{2+}\)-dependent enzymes involved in controlling the secretory pathway and post-translational modification of proteins. This Ca\(^{2+}\) store controlled by SPCA is also believed to be agonist-releasable. Regucalcin (RGN), (also known as senescence marker protein 30 (SMP30)) is believed to be a Ca\(^{2+}\)-binding protein expressed in an age-dependent manner, whereby its protein levels decrease in a number of organs as aging progresses. It has been suggested to be able to affect the activities of the sarco/endo-plasmic reticulum Ca\(^{2+}\)-ATPase (SERCA), as well as other Ca\(^{2+}\)-dependent enzymes. On the other hand, RGN’s ability to bind Ca\(^{2+}\) has been argued against and this protein has been shown to modulate the activities of enzymes not involved in Ca\(^{2+}\) homeostasis, as well as have intrinsic enzymatic activity in itself as a gluconolactonase. It was of interest in the present studies to examine how SPCA can be regulated and how RGN can contribute to the regulation of Ca\(^{2+}\) homeostasis within intact cells. As a result, the following novel attributes of SPCA and RGN were shown: (1) SPCA expression and activity are sensitive to glucose homeostasis in rat vascular smooth muscle cells (VSMCs); (2) hSPCA1d activity can be inhibited without altering hSERCA2b activity by using bis-phenol and 2-APB; (3) TFP and TBBPA affect both hSPCA1d and hSERCA2b activities to the same degrees, while cADPR and NAADP have no effect on hSPCA1d (and possibly hSERCA2b in the case of cADPR) activity; (4) RGN mRNA expression can be found in some specialised cell types of the male rat reproductive system; and (5) human RGN over-expression can influence Ca\(^{2+}\) mobilisation stimulated by 1\(\mu\)M thapsigargin and 10\(\mu\)M histamine, possibly via alteration of SERCA expression. The present study has also made available a custom-made recombinant form of rat RGN and anti-RGN polyclonal antibodies. It can be strongly suggested that SPCA plays a role in diabetes because of its sensitivity to glucose concentrations, coupled with its involvement in the secretory pathway and ability to influence vasopressin response in VSMCs. Bis-phenol has now been quantitatively shown to be a potent and specific inhibitor of SPCA, giving it great potential to be used as a pharmacological tool for future research on this Ca\(^{2+}\)-ATPase. For RGN, it appears promising that this cytosolic protein has a role to play in male fertility, while its role in Ca\(^{2+}\) homeostasis is likely to involve modulating ER Ca\(^{2+}\) storage capacity and strength of Ca\(^{2+}\)-mediated hormone response.

Background: The non-invasive assessment of volume status in left ventricular systolic dysfunction (LVSD) is challenging. The main thesis objective was to establish the feasibility and potential clinical utility of repeated measures assessment of non-invasive biomarkers in defining changes in volume status within individual volunteers. Methods: Differential volume manipulation protocols were achieved in a three-staged plan of investigation, firstly, in patients with decompensated heart failure receiving intravenous furosemide, secondly, in normal volunteers receiving acute loads of oral water or intravenous saline, and thirdly, in stable LVSD patients on chronic furosemide dosing undergoing staged diuretic withdrawal and resumption. Repeated measures of biomarkers relevant to volume status including blood and urine biomarkers, echocardiographic and bioimepdance measures were performed to assess their sensitivity to the induced volume changes. Results Summary: I demonstrated the smallest variance for bioimpedance measures, and the largest variance for urine biomarkers. In the patients with decompensated heart failure, none of the biomarkers studied showed potential clinical utility at tracking acute volume response to intravenous furosemide. In the normal volunteers, a significant change in estimated blood volume was observed following intravenous saline, but not with acute oral water ingestion. Only whole-body and trunk bioimpedance measures, and to a lesser extent, mitral valve early peak velocity with and without the Valsalva manoeuvre, appeared sensitive enough to map these changes in volume status. In the stable LVSD patients, statistically significant increases in B-type natriuretic peptide, urinary creatinine, urinary kidney injury molecule 1, and in bioimpedance-estimated body water composition were observed with diuretic withdrawal, with levels of these markers reducing following diuretic resumption. However, the amplitude of the changes observed was either lower than the respective within-subject variance or too small to be potentially useful as a marker of volume change. This would thus limit the clinical utility of these biomarkers in the routine monitoring of volume status in LVSD. Conclusion: The repeated measures of biomarkers studied in response to different volume manipulations were interpreted in the context of their within-subject normal variance. None of the biomarkers studied appeared to have the ideal characteristics clinically for the monitoring of subclinical changes in volume status in stable LVSD or in response to acute diuresis in decompensated heart failure. The significant increases in urine biomarkers following diuretic withdrawal in stable LVSD suggested potentially beneficial renal effects of furosemide in stable LVSD.

Taking part in regular physical activity leads to adaptive response that enables the body’s antioxidant defence to be better equipped to fight against oxidative stress. Exercise intensity seems to be one of key factors that determines the effectiveness of exercise. The work presented in this thesis used novel approaches, through the application of emerging technologies, to study physical activity and its effects. This thesis contributes to the existing literature by being the first to investigate the effect of exercise on a marker of oxidative stress in the brain, an organ that becomes impaired (including oxidative damage) with ageing and diseases associated with ageing. The finding from this thesis suggests that brain glutathione (GSH) of young sedentary men as measured by magnetic resonance spectroscopy (MRS) was altered in response to acute exercise, in an exercise intensity dependent manner. Observed changes in peripheral markers of oxidative stress were also exercise intensity dependent. The brain seems to be protected against hyperperfusion injury during high intensity phase of high intensity interval exercise. Objectively measured physical activity levels were not significantly increased by an unsupervised home-based exercise intervention in older adults, potentially due to a lack of progressive goals based on adherence to physical activity.

This thesis aims to identify physiological and biochemical variables, comparing sex, training status, age and maturity in sub-elite, endurance trained adolescents. Maximal lactate steady state was investigated and the effects of endurance training programmes measured.T he first study assessedth e reliability of absolute running speed, V02, and HR that correspond to the fixed blood lactate reference values of 2.0 and 2.5 mmo1. L"1 and the lactate threshold (LT) and found these measures to be reliable after endurance-trained adolescent runners completed two identical incremental treadmill tests within a 7-10 d period The second study was designed to determine the relationship between physiological variables and endurance running performance in this age group. Track-based, running performance times were available for 18 boys and 14 girls for the 800 m, and 16 boys and 13 girls for the 1500 m. The participants were tested using a step-wise incremental treadmill test and a Wingate anaerobic power test (WAnT) on separate occasions. The results from this study found that for the 1500m, running speeds corresponding to the fixed [BLa ] were a useful measure for assessing performance in endurance trained boys and girls. Unlike previous studies, peak V02 was not a significant physiological predictor of 1500m performance in either boys or girls. For the 1500 m performance in girls the anaerobic measure was no longer significant once variations in size or age had been taken into consideration. Whereas V VO2 peak and running economy may prove to be of some value when considering the 800m for boys, the running speed corresponding to a [BLa ] of 2.5 mmol-L-1 was the only meaningful physiological predictor variable for girls once differences in age and body size had been accounted for. The third study had three main objectives: (1) to identify the exercise intensity that corresponds to the (MLaSS) in adolescent, endurance trained runners, (2) to examine possible between sex differences, and (3) to compare the MLaSS with commonly cited fixed blood lactate reference variables. The participants were first tested using a step-wise incremental treadmill test to establish the blood lactate profile and peak VO 2. The running speed and % peak VO 2 at the MLaSS were not significantly different to those corresponding to the fixed [BL& ] of 2.0 and 2.5 mmol-L-1 (P>0.05). The % HR max at 2.5 mmol-L-1 was also not different to that at the MLaSS, whereas at 2.0 mmol-L-1 it was slightly lower (P<0.05). The running speed, % peak VO 2, and % HR max at the fixed [BLa] of 4.0 mmol-L-1 were significantly higher than those at the MLaSS (P<0.05). In conclusion, it is clear that the MLaSS corresponded to the relatively high exercise intensity in this sample of athletes. It would appear that the running speed, % peak VO 2, and % HR max at the MLaSS lies somewhere between the fixed [BLa ] of 2.0 and 2.5 mmol"L-1. These results confirm earlier work that has suggested a fixed [BLa ] of 2.5 mmol-L-1 may be used with young people' to assess and monitor endurance running performance in place of the more commonly used 4.0 mmol-L-1 that has received so much attention in adult-based studies. The fourth study examined the effect of exercise training on endurance performance, blood lactate profile in relation to running speed (RV) and cardio respiratory function (peak V02) in adolescent runners. This study demonstrated that resting HR, LT and 1 Use of the expression young people is increasingly common since the publication of the text, Young People and Physical Activity by Armstrong and Weisman in 1997. It is used within this document to generically represent the 6 to 18 year age group. 11 RV, HR, V02 and peak V02 at LT were significantly influenced by endurance training. When running time, running velocity and run performance time pre and postintervention were included in the analysis, the intervention did not have a significant effect on peak VO2. When percentage body fat was included as a covariate, there was a positive association with pre and post-training for all groups. The conclusion from these data is that maturity and training both have an effect, especially at supra suggested training levels. The results of the four inter-linked studies support an age-related increase in endurance in aerobic and anaerobic performance and indicated significant differences between boys and girls. From a coaching viewpoint the results reveal that, from the age of 14 to 18 years, runners should be introduced to high intensity training and that changes to the format of middle distance running performance in adolescent competition are recommended.

This thesis describes the development of an anatomically meaningful musculo-skeletal model of the human arm, incorporating two modified Hill muscle models representing the elbow flexor and extensor muscles. In vivo experimental methods to determine parameter values are presented. The stimulus for this work was to enable the prediction of movement, to support development of prostheses and orthoses such as Functional Electrical Stimulation (FES). A key problem in model based movement studies is that the passive parameter values in the Hill muscle models and the joint had not been experimentally determined in vivo. The result has been an inability for predictive models to generate realistic predictions of human movement dynamics. In the model, movement dynamics of the forearm was described using the Newton-Euler method, which was validated from analysis of physical pendulum. Structural identifiability analyses of the muscle models ensured that values for the model parameters could be uniquely determined from perfect noise free data. A novel experimental procedure termed the passive movement method is described, which exclusively parameterised the model’s passive components. Simulated model dynamics were fitted to measured movements of the freely swinging forearm under gravity. Model values were obtained on an individual subject basis. The average muscle model spring and damping constants for four healthy subjects were 143N/m and 1.73Ns/m respectively. Separately, the force/length characteristics of the muscles’ active component, the contractile element (CE), were obtained from measurements of isometric maximum voluntary contraction (MVC) at different elbow angles. The results for the five healthy subjects showed good agreement with results reported in the literature. A preliminary experiment was performed to predict elbow flexion movement under FES. An electrical stimulus that generated a specified isometric elbow flexion moment (10% of MVC) was applied to generate elbow flexion movement. Simulated FES arm movement was compared with the measured results. The simulated change in elbow angle did not agree with the measured data. A major cause for this was believed to be skin movement causing a change in the current path across the muscle fibres, thus affecting the force generated. The passive movement method described in this thesis filled an important chapter to fully parameterise musculo-skeletal models in vivo. Although in the FES movement experiment, simulated change in elbow angle generated by FES did not agree with measured data, the shape of the dynamic response in the fitted simulated movement showed good agreement with the measured FES movement.

Aims: To fully characterise the RGC-5 cell line and determine whether it would be a suitable substitute for primary RGC cell culture. To optimise a CNTF Nogo-P4 inhibitory assay and establish whether (a) CNTF alone is capable of stimulating RGC neurite outgrowth and survival, or (b) an increase in intracellular cAMP is required for CNTF to be effective. To determine whether recombinant AAV2 viral constructs were capable of producing detectable levels of CNTF in HEK-293 transfected conditioned media. To optimise AAV2-eGFP delivery and establish whether AAV2-CNTF-hrGFP and AAV2-CNTF-shRhoA-hrGFP could promote RGC survival and regeneration after optic nerve crush surgery. Methods: The RGC-5 cell line was characterised using semi-quantitative PCR, sequencing and immunocytochemistry. RGC-5 cells were screened for a selection of neuronal, glial, progenitor, oligodendroglial lineages and cone photoreceptor cell markers to identify the cells origin. Retinal cultures were treated with recombinant CNTF and/or Forskolin to promote RGC neurite outgrowth and survival - this was quantified after 3 d. Retinal wholemounts were prepared to assess GFP transduction and survival after intravitreal delivery of AAV2-eGFP. Axonal regeneration and RGC survival were assessed through histological examination of optic nerves and retinal sections. Results: The RGC-5 cell line predominantly expressed oligodendroglial lineage markers and only weakly expressed \(\beta\)III-Tubulin mRNA. RGC-5 cells did not express mRNA for many of the phenotypic markers of RGC. CNTF was effective at stimulating RGC neurite outgrowth without the need for cAMP elevation - furthermore recombinant CNTF could disinhibit Nogo-P4 treated RGC in vitro. GFP transduction was low when injected alone, however, when administered with Pronase-E there was a significant increase in GFP expression. AAV2-CNTF-hrGFP and AAV2-CNTF-shRhoA-hrGFP did not promote RGC survival or regeneration 23 d post optic nerve crush. Conclusions: RGC-5 cells are not an appropriate substitute for primary retinal cell culture in vitro as they express many of the same markers as oligodendrocyte progenitors. CNTF is capable of stimulating RGC neurite outgrowth without an additional elevation of cAMP. AAV2-mediated GFP expression could be enhanced through the partial digestion of the inner limiting membrane - this seems to be the major obstacle in achieving optimal AAV2 transduction.

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