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The potential for nitric oxide (NO) metabolites (e.g. inorganic nitrite) to act as stable stores of “Transported Nitric Oxide” has excited huge interest due to the substantial potential therapeutic avenues. The prospect developing of a “silver bullet” that could target areas most in need of vasodilatation, by releasing NO in areas of hypoxia and ischaemia, could prove a massive advance in the treatment of vascular disease. In this thesis I examine the effects of nitrite infusion in both hypoxia and normoxia. I examine the effects both in health and heart failure, and investigate the potential roles of Nitric Oxide Synthase (NOS) and Xanthine Oxidase (XO) in mediating the reduction of nitrite. We found, and were the first to report in man, that intra-arterial infusions of nitrite had little effect upon the vasculature in high oxygen tension environments but led to significant vasodilatation during hypoxaemia. We found that patients suffering with Chronic Heart Failure responded differently to nitrite infusion to healthy controls, possibly as a result of differences in redox-stress. In healthy volunteers, at rest, neither NOS nor XO appeared to play a significant role in nitrite induced vasodilatation in normoxia and mild hypoxia. We found that vascular myoglobin contributes to the reduction of nitrite to nitric oxide and may play a role in prolonging the vasodilatation induced by nitrite infusion.

Inorganic carbon is fundamental to the physiology of all organisms, however elevated CO2 is generally detrimental. Numerous class III adenylyl cyclases (AC) from both prokaryotic and mammalian organisms have been shown to respond to inorganic carbon in vitro, however, at present there is limited evidence in vivo. This thesis demonstrates in cellulo evidence that hypercapnia CO2 blunts agonist induced cAMP signalling. The eect of CO2 is independent of changes in intracellular and extracellular pH, independent of the mechanism used to activate the cAMP signalling pathway, and is independent of the cell line employed. Through a combination of pharmacological and genetic tools this eect of elevated CO2 on cAMP signalling is demonstrated to require Ca2+ ion release from IP3 receptors in the endoplasmic reticulum. Consistent with these ndings, CO2 caused an increase in cytoplasmic Ca2+ concentrations which require the presence of active IP3 receptors and is absent under comparable acidotic conditions. Physiological relevance for this signalling mechanism is demonstrated through activity of the sodium dependant proton exchanger NHE3. This transporter exhibits well-characterised inhibition by cAMP dependant protein kinase PKA to increase bicarbonaturia in vivo. Overall these results provide conclusive evidence of potentially profound eects of inorganic carbon on intracellular cell signalling, which could lead to signicant insight into the pathophysiology and treatment of numerous disorders including metabolic acidosis, reperfusion injuries, acute lung injury and obesity.

The main aim of my research, presented here, is to develop proteomic research techniques, for their use in biomarker discovery and identification. This is broken down into three main chapters: • Biomarker Identification in Stroke Brain guided by MALDI-imaging. • Evaluation of the Effectiveness of Heat Treatment for Prevention of Proteomic Sample Degradation using Label Free Relative Quantitation. • Discovery and Identification of Biomarkers for Hypertension Within these chapters special attention is paid to sample preparation, development and assessment of new methods for biomarkers discovery and identification, the importance of experimental design and the application of relevant and useful statistical methods to enable the mining of useful information from rich datasets. The biological changes in stroke induced mouse brain tissue are studied, the prevention of degradation to tissue samples by a novel heat treatment method and changes to plasma samples from hypertensive, wild type and a congenic strain of rat are also studied. The outcomes of this work are multiple, namely: • The identification of a possible marker for stroke from mouse brain tissue • The effect of a new heat treatment device on proteomic data obtained from mouse brain tissue • The novel application of a statistical analysis to a new type of dataset (LC-MS and label free quantitation of biological samples) • The identification of possible biomarkers for hypertension in rat plasma using this method

The development of complex metazoans depends on the integration of a handful of signalling pathways that eventually modulate precise patterns of gene expression. The fact that just a few pathways are involved in the generation of such complexity in different organisms, suggests that these are highly regulated and conserved processes. The accurate spatio-temporal coordination of the signalling pathways controls the assignation of different cell fates and their patterning into tissues and organs. The source of diversity relies on the different possible interactions between signalling pathways, such as, the combination of signals and the order in which they are received by the cell or crosstalk. Due to their importance in development, abnormal signalling through these pathways has been strongly associated with developmental disorders, cancers and other diseases. The Notch and Wnt signalling pathways are key components of the intricate network that controls gene expression during development, and genetic analysis in Drosophila has highlighted that interactions between these two signalling pathways are important during this process.This thesis investigates the cross-regulatory interactions between Notch and Wnt signalling pathways in mammals. Using transcriptional reporter assays and biochemical analysis, I have found two molecular mechanisms underlying the inhibitory crosstalk between Notch and β-catenin, the effector of Wnt signalling pathway, in mammalian cells. At the membrane Notch inhibits β-catenin transcriptional activity through Deltex mediated endocytosis of Notch and a component required for β-catenin activation. This is similar to results observed in Drosophila. In the nucleus, I have identified a novel mechanism by which NICD-dependent transcription of Hes/Hey family of transcription factors prevents the activation of Wnt signalling pathway. This mechanism involves the formation of a physical complex between Hey1 and β-catenin/TCF, which allows Hey to block Wnt transcriptional activation. Additionally, I have found that these two mechanisms are conserved across vertebrates.Together the findings of this thesis improve our understanding of the molecular mechanism underlying the Notch/Wnt crosstalk. In turn, this will give an insight into unravelling how a handful of signalling pathways can generate sufficient diversity in signalling output to specify the hundreds of different cell fates generated to make a mammal. Elucidating these signalling networks will also contribute to our understanding of diseases, both their aetiology, by knowing how changes in one signal can influence another, and their treatment as mimicking points of crosstalk is likely to generate very specific therapeutic agents.

Water movement in the body is determined by the osmotic forces acting on the cell membrane. Ingestion of a highly-hypertonic glycerol solution resulting in high extracellular osmolality could drive water out of the intracellular space into the vascular space resulting in reduced muscle hydration and increased blood volume. The aim of this thesis was to study the effects of altered body water distribution during exercise. In chapter 3, ingestion of a 400 ml glycerol solution (1 g·kg-1 body mass) increased serum osmolality (309 mosmol·kg-1) which was associated with a 4.0% increase in blood volume due to a 7.2% increase in plasma volume, attributed to a shift of water from the intracellular space, resulting presumably in tissue dehydration. Glycerol ingestion was then used as a means of altering body water distribution in the other studies described in this thesis. Altered body water distribution had no acute effect on force production during quadriceps muscle isometric exercise (chapter 4), or handgrip strength (chapter 5). Regarding chronic effects (chapter 5), two groups of participants exercised handgrip and initiated recovery after ingestion of either a glycerol solution or placebo over a period of 8 weeks. Maximum handgrip strength increased in both groups and there was no statistically significant difference between the two groups. In chapters 6 and 7, the subjects performed a cycling exercise protocol to fatigue. In the glycerol trial, time to fatigue decreased compared with the iso-osmotic trial during an incremental VO2max test (chapter 6) and during cycling against a constant load at 100% VO2max intensity (chapter 7). In the glycerol trial, there was an accelerated increase in blood lactate and an accelerated increase in serum potassium (chapter 7), indicating altered muscle metabolism which may have contributed to the early development of fatigue.

INTRODUCTION: The magnetic resonance (MR) relaxation parameter T2* is used for non-invasive assessment of cardiac iron, with low cardiac T2* being associated with left ventricular (LV) impairment and the development of heart failure. However, there is very little data calibrating cardiac T2* and other MR relaxation parameters to human cardiac iron concentration, and the effects of cardiac iron on the right ventricle (RV) are little known. METHODS AND RESULTS: We studied the distribution of cardiac iron in a series of post-mortem hearts from transfusion-dependent patients using mass spectrometry derived iron concentration, and compared the results against the MR relaxation parameters T1, T2 and T2*. We found variable iron loading between cardiac tissues, but a mainly homogeneous deposition of iron in the LV myocardium, apart from a transmural gradient. We established a calibration equation for cardiac iron using T2*. Septal iron and T2* were very representative of whole-heart values. Cardiac iron was also measurable using T2, but T1 measurement was unreliable. Clinical studies of the RV showed a progressive fall in ejection fraction (EF) as iron loading increased and we established normal ranges for RV volumes and EF in patients with beta thalassaemia major (TM). Finally, a worldwide survey of the clinical use of T2* in over 3000 TM patients showed a high prevalence of cardiac iron loading with large regional variation, and confirmed that low T2* values are associated with heart failure and death. CONCLUSION: In transfusion dependent patients, cardiac iron is deposited variably in the cardiac tissues, but is mainly homogenous in the myocardium and can be calculated from cardiac T2* using the obtained calibration equation. There is similarity in myocardial response to iron loading between the RV and LV. Worldwide data shows a high prevalence of cardiac iron loading, but significant variation suggests that undetermined factors may influence the loading, of which genetic modulation is a prime candidate.

Biochemical sensors that are low cost, small in size and compatible with integrated circuit technology play an essential part in the drive towards personalised healthcare and the research described in this thesis is concerned with this area of medical instrumentation. A new biochemical measurement system able to sense key properties of biochemical fluids is presented. This new integrated circuit biochemical sensor, called the Chemical Current-Conveyor, uses the ion sensitive field effect transistor as the input sensor combined with the current-conveyor, an analog building-block, to produce a range of measurement systems. The concept of the Chemical Current-Conveyor is presented together with the design and subsequent fabrication of a demonstrator integrated circuit built on conventional 0.35μm CMOS silicon technology. The silicon area of the Chemical Current-Conveyor is (92μm x 172μm) for the N-channel version and (99μm x 165μm) for the P-channel version. Power consumption for the N-channel version is 30μW and 43μW for the P-channel version with a full load of 1MΩ. The maximum sensitivity achieved for pH measurement was 46mV per pH. The potential of the Chemical Current Conveyor as a versatile biochemical integrated circuit, able to produce output information in an appropriate form for direct clinical use has been confirmed by applications including measurement of (i) pH, (ii) buffer index ( ), (iii) urea, (iv) creatinine and (v) urea:creatinine ratio. In all five cases the device has been demonstrated successfully, confirming the validity of the original aim of this research project, namely to produce a versatile and flexible analog circuit for many biochemical measurement applications. Finally, the thesis closes with discussion of another potential application area for the Chemical Current Conveyor and the main contributions can be summarised by the design and development of the first: ISFET based current-conveyor biochemical sensor, called 'Chemical Current Conveyor, CCCII+' has been designed and developed. It is a general purpose biochemical analog building-block for several biochemical measurements. Real-time buffer capacity measurement system, based on the CCCII+, which exploits the imbedded analog computation capability of the CCCII+. Real-time enzyme based CCCII+ namely, Creatinine-CCCII+ and Urea-CCCII+ for real-time monitoring system of renal system. The system can provide outputs of 3 important parameters of the renal system, namely (i) urea concentration, (ii) creatinine concentration, and (ii) urea to creatinine ratio.

This thesis investigated whether highly concentrated sodium solutions ([HS] 126-164 mmol.L-1 NaCl) could provide viable strategies before during and after exercise in the heat to improve cardiovascular and thermoregulatory functioning and exercise performance. To do this it also examined the gustatory responses to HS drinks before, during and after exercise. All studies compared HS with a low sodium control ([LS] 10-27 mmol.L-1). Chapter 4 found that during 3 h recovery from dehydration, ingestion of 120 % body mass losses of HS restored fluid balance to a greater extent (121 vs. 84 %) than LS. Chapter 7 was the first to investigate the effects of ingesting HS during exercise in the heat and in an untrained population. HS attenuated the decline in stroke volume [SVDrift] and increase in heart rate [HRDrift], but did not affect rectal temperature [TRec], cardiac output, or oxygen uptake during the second of two consecutive 45 min bouts at 55% . In Chapters 8 and 9 untrained participants ingested either HS or LS during 30-45 min pre-exercise rest. HS reduced HRDrift and SVDrift but did not affect TRec during 45-60 min exercise at 10% of the difference between and gas exchange threshold [∆]. HS also increased both time to exhaustion and exercise toleranceduring subsequent exercise bouts at 60-70% ∆. Chapters 5 and 6 found that taste perceptions act as physiological regulators, in this case, one reflecting the priority to restore hyperosmolality over hypovolemia. Exercise-induced dehydration increased the palatability of water, and decreased the palatability HS, when measured before, immediately after and during 3 h recovery. The changes were highly correlated with physiological indicators of fluid balance. The ingestion of highly concentrated sodium solutions can be both an efficient and acceptable means to improve hydration, reduce cardiovascular stress, and improve exercise performance in the heat. Whilst highly effective, caution should apply since the unpleasant taste evoked by these solutions persists for at least three hours post exercise.

During exercise water and electrolytes are lost in sweat. There is a large variation in both sweat rate and sweat composition and as a consequence sweat electrolyte loss can be large, especially for sodium, the primary cation in sweat. The loss of large amounts of sodium in sweat has been linked with hyponatraemia and muscle cramps. Sodium intake is encouraged in some athletes and in some exercise situations, which is in direct contrast to guidelines aimed at the general population aimed at reducing average sodium intakes to 2.4g of sodium per day (6g salt/day). Dietary sodium intakes have been determined by numerous methods, including weighed dietary records and 24h urine collections. As dietary sodium intake in excess of basal requirement is primarily excreted in the urine in non-sweating individuals, and the basal requirement for sodium is small, 24h urine collections can provide an accurate estimate of dietary sodium intake. In Chapter 3, 24h urinary sodium excretion was measured in eighteen subjects on 4 separate occasions. Subjects consumed their normal diet with the exception of a 5g creatine supplement and 500ml of water, which was part of a separate investigation. The relationship between urine sodium excretion in each 24h collection period was weak, but on average males excreted 200 ± 48mmol of sodium per day and females excreted 157 ± 33mmol of sodium per day, which is equivalent to 4.6g and 3.6g of sodium, respectively. This is in excess of the current recommended intake. In chapter 4, the variation in sodium excretion was determined in eight subjects who consumed the same diet for 5 consecutive days. Despite the similar intake of sodium each day, a day to day variation in sodium excretion of 13% was still observed. This was not related to either sodium intake or potassium intake. In chapter 5, nine subjects consumed their normal diet for 5 consecutive days but weighed and recorded all food and drink consumed. During this period, 24h urine samples were also collected. No strenuous exercise was permitted apart from an exercise task on day 4. This involved intermittent cycling in the heat until 2% body mass (BM) was lost. Sweat was collected from four absorbent patches placed on the back, chest, forearm and thigh. Sweat sodium concentration was adjusted to account for the 35% over-estimation using this regional collection method. Subjects lost 1.51 ± 0.19L of sweat and 66 ± 16mmol (range 32 86mmol) of sodium. There was no difference in sodium balance between each 24h period due to a significant decrease in urine sodium excretion on the day of exercise (day 4). In chapter 6, the effect of prior exercise on sweat composition during a second exercise bout completed later that same day was determined. Eight healthy males cycled for 40 minutes in the heat on one or two occasions. A period of 5h elapsed between exercise bouts when two exercise sessions were performed. Sweat was collected using a whole body washdown method and by 4 absorbent patches placed on the back, chest, forearm and thigh. The main finding was that prior exercise did not affect sweat rate or sweat sodium, potassium and chloride concentrations in the second exercise bout when using the whole body washdown method. Chapter 7 determined the effects of two exercise sessions completed on the same day on electrolyte balance. Nine subjects followed their normal dietary behaviour but weighed and recorded all food and drink consumed during 5 consecutive days. During this period 24h urine samples were also collected. No strenuous exercise was permitted during this period apart from two exercise tasks on day 4. During exercise sweat was collected using a whole body washdown technique. Sweat rate and sweat sodium, potassium and chloride concentrations during the second exercise bout were found to be similar to the first exercise bout. Subjects lost 2.64L (range 1.80 3.48L) of sweat and 138 ± 106mmol of sodium (range 32 287mmol). Sodium balance was not significantly affected on the day of exercise, but urine sodium was lower than dietary sodium intake on the day of exercise (Day 4) and the day following exercise (day 5), indicating significant sodium conservation by the kidney. In contrast, no change in sodium intake was observed. In chapter 8, the effect of skimmed milk and a sports drink in restoring fluid balance was examined following exercise-induced dehydration. Seven physically active males cycled intermittently in the heat until 2% BM was lost. During a 1h rehydration period a sports drink (23mmol Na+/L) or skimmed milk (32mmol Na+/L) was consumed in a volume equivalent to 150% of BM loss. Fluid balance at the end of the 3h recovery period tended to be more positive when milk was consumed. Despite this, no difference in exercise capacity in the heat was observed. This thesis shows that exercise did not increase sodium intake, but this may be due to the already high dietary sodium intake of individuals. Sodium balance was maintained in the majority of individuals due to a significant conservation of sodium by the kidneys. When sweat sodium losses are large, urine sodium conservation may not be sufficient to prevent a negative sodium balance. When no food is consumed in the acute period post-exercise, the higher sodium content of skimmed milk than a sports drink may be partly responsible for the increased retention of the ingested fluid. But this did not enhance subsequent performance in the heat.

Epithelial sodium channels (ENaC) are of immense importance, controlling Na+ transport across epithelia and thus playing a critical role in all aspects of fluid clearance as well as numerous other functions. Although extensive studies have been carried out to invistegate the regulation mechanism of ENaC, many questions still remain unclear. Therefore, we employed various techniques including electrophysiology and molecular biology approaches to investigate the mechanisms underlying the regulation of ENaCs by lipid metabolites, oxygen and mechanical stress. We have identified profound regulation mechanisms of ENaC in distal renal epithelial cells and vascular endothelial cells by lipid metabolites, heme and mechanical forces. Our results revealed a novel O\(_2\) sensitive regulation pathway of ENaC channels, in which hemeoxygenase acts as the O\(_2\) sensor and the substrate and product of which either inhibits or stimulates ENaC activity. This finding may eventually lead to novel clinic strategies in dealing with diseases e.g. renal failure, kidney reperfusion injury, hypertension, pulmonary edema and pre-clampsia. In addition, we, at the first time, have revealed that ENaC is functionally expressed in a variety of endothelial cells and is able to serve as mechano-transducing sensors in the vascular endothelial cells which play important roles in vascular physiology and pathology progresses.

Breath is a mixture of nitrogen, oxygen, carbon dioxide, water vapour, inert gases, and a small fraction of trace volatile organic compounds (VOCs) in the parts per million by volume to parts per trillion by volume range. VOCs can be produced anywhere in the body as a result of physiological and pathophysiological processes, and are transported via the bloodstream to the lungs where they are exhaled in breath. On the basis that VOCs in breath are representative of VOCs in the blood and therefore biochemical processes occurring in the body, the analysis of exhaled breath may become a noninvasive tool for use in clinical practice. This thesis documents the development of the analytical technique of chemical ionisation reaction time-of-flight mass spectrometry (CIR-TOF-MS) for the analysis of VOCs in exhaled breath, exploring the challenges associated with breath sampling to its application in clinical studies. Initial work focused on the design of a suitable breath sampling device that coupled directly to the CIR-TOF-MS instrument to allow the on-line, real-time analysis of breath. The analysis of exhaled breath from healthy individuals allowed a common group of breath VOCs to be identified and quantified. The CIR-TOF-MS system was applied to a number of clinical trials examining the breath of individuals with cystic fibrosis (9 cystic fibrosis children, 4 healthy children), asthma (35 asthmatic subjects, 5 COPD, 28 healthy controls) and cancer (4 female cancer subjects, 10 healthy female controls), for which the latter study required the investigation of off-line breath collection. The analysis of VOCs emitted from bacterial and fungal cultures in vitro was also explored, as a means to support the hypothesis that the measurement of VOCs in exhaled breath could be used to identify infection status in vivo. Within these applications, CIR-TOF-MS was able to demonstrate that the chemical profile of breath has the potential to identify the presence of infection or disease.

Regular physical activity can lead to improvements in the immune system and so help to prevent many diseases such as cardiovascular disease (CVD), Type2 diabetes, and obesity. The objectives of this study were to assess the antioxidant and trace element status in serum from adolescent, female gymnasts and pubertal controls of the same age, using ICP-MS, AAS to measure trace elements (selenium, copper, and zinc) and (magnesium), respectively. Enzymatic assay were also used to measure inflammation, auto-immunity and endothelial activation including (GPx, SOD, CRP, ICAM-1, HSP27 antibodies and antigens IgG, and IgM). In addition, anthropometric, physical activity and dietary intake were estimated, as previously described (Nurmi-Lawton et al., 2004). Dual energy x-ray absorptiometry (DXA, Lunar DPX) was used to assess bone mineral content and density. The subjects of the current study, comprising 38 competitive gymnasts and 40 healthy, sedentary adolescent females, were part of a three-year long investigation into exercise and peak bone mass (PBM). Most previous studies have not taken into account the possibility of confounding factors that include anthropometric differences and dietary intake, which are also likely to vary between active and less active subjects. In this present study, it was our objective to take these factors into account. Our findings show that young, female gymnasts have an altered antioxidant enzyme profile, serum inflammatory markers and endothelial activation, compared to their less physically active peers. The current study also found a significant, positive association between levels of serum Zn and indices of bone mass in the active group. Further research should focus on examining the effect of vitamin D insufficiency on the above relationships and investigate further the interaction with and effect of hormonal changes and body weight.

Oxidative stress has been associated with a wide range of diseases, including cardiovascular diseases, Alzheimer's disease, atherosclerosis, Parkinson's disease and cancer. It also plays a role in the ageing process. Hyperhomocysteimia is commonly found to be associated with these diseases. The hyperhomocysteimia is a result of a deficiency in both folate and cobalamin Folate is known to reduce Hey and protect cells from apoptosis, but there are no studies investigating the impact of cobalamin on apoptosis induced by oxidative stress or the mechanism(s) of the protection. The aims of the research are to investigate the protective role of cobalamin and the possible mechanism(s) for this protection. It also examines the protective role of novel cobalamin and investigates their superior protection. The methods used in this research for apoptosis detection we used caspase-3 and the annexin-V, while for necrosis we used PI staining, where cell viability were detected using MTS assay. We also measured the generation of superoxide by Lucigenin-enhanced chemiluminescence and reactive oxygene species by using the redox active prob DCFH-DA. Moreover, the intracellular proteins were measured via staining with specific fluorescent-conjugated antibodies were detected using flowcytometry. Our result demonstrated that 25|iM of cobalamin protects cells from apoptosis. The protection by cobalamin was associated with induction of iHsp72 and iHO-1, and these are shown to be essential for the protection. Furthermore, our research demonstrated a novel mechanism of cobalamin-apoptosis protection involving induction of NfkB, ERK1/2 and AKT signal transduction pathways. In order to protect cells from apoptosis induced by oxidative stress, cobalamin induces the pNfkB which in turn regulate the iNOS and HO-1 induction. Cobalamin also induces thepERK1/2 which regulates the induction of Hps72 and Nrf2. And finally, pAKT induced by cobalamin which regulate the Nrf2 and HO-1 induction. The inhibition of any of theses pathways leads to loss the protection. The GSCbl and NACCbl provide a superior protection against oxidative stress, this protection involved induction of the signal transduction pathways and Hsps. To conclude; cobalamin provides protection against cells death induced by oxidative stress. Cobalamin achieves this by multiple pathways which include direct antioxidant stimulation and induction of signal transduction pathways. Different cobalamin derivatives have superior protections. These finding are a useful pharmaceutical tool in the treatment of the oxidative stress related diseases.

A reduced blood lactate concentration ([lac-]B) is commonly observed during whole-body exercise following inspiratory muscle training (IMT). However, whether the inspiratory muscles are, in part, the source of these reductions remains unknown. Accordingly, this thesis investigated: (I) the contribution of the respiratory muscles to the systemic [lac-]B and (II) the effects of IMT upon inspiratory muscle lactate exchange and clearance. In addition, the thesis also evaluated the determinants of inspiratory muscle strength (maximal inspiratory mouth pressure; MIP). All subjects were healthy, active and free of pulmonary and respiratory muscle disease. Under resting conditions, 10 min intense volitional hyperpnoea at 85% of maximal exercise minute ventilation (VE max) increased [lac-]B by 0.96 mmol.L-1. This was attenuated by 25% following 6 wks IMT. 8 min volitional hyperpnoea at 90% VE max imposed upon exercise at the maximal lactate steady state (MLSS) increased [lac-]B by 0.99 mmol.L-1. Following 6 wk IMT, the steady state and hyperpnoea-mediated increase in [lac-]B were lower by 8 and 26%, respectively. Relative to pre-IMT, loading the trained inspiratory muscles using a low-intensity pressure threshold resistance (15 cmH2O) immediately following maximal exercise accelerated both lactate exchange and clearance capacities by ~70%. Collectively these findings support the notion that the respiratory muscles are capable of net lactate production and are the first to suggest that IMT increases their capacity for lactate clearance. This thesis also demonstrates that the respiratory muscles are responsible, in part, for the reductions observed in [lac-]B during whole-body exercise following IMT. Finally, baseline MIP was positively correlated with the strength of the chest wall inspiratory muscles. The IMT-mediated increase in MIP was negatively correlated with the relative increase in chest wall muscle strength. Therefore, these findings are the first to demonstrate that the lower the initial strength of the chest wall inspiratory muscles, the lower the MIP and the greater the improvement in global inspiratory muscle strength following IMT.

The proton transfer reaction mass spectrometer (PTR-MS) was used to analyse volatile organic compounds (VOCs) in human breath and the headspace above microbial cultures. Real-time expirograms of acetone, isoprene and methanol show that end-expired VOC concentration depends on duration of exhalation so a robust method of breath sampling based on cyclic rebreathing was developed. Concentrations of VOCs in arterial (CAB) and venous blood (CVB) and breath (CBr), were measured in 10 healthy volunteers. Mean (range) CBr, CAB and CVB are 1090 (515-2335) ppbv, 26 (10-73) μmol/1 and 18 (9-39) μmol /1 respectively for acetone. For isoprene, the corresponding values are 465 (308-702) ppbv, 6.8 (3.7-11) nmol/1 and 14 (5.5-30) nmol/l. In-vivo blood/breath ratios were calculated and were 580 (280 -1060) for acetone and 0.47 (0.22 - 0.77) for isoprene. The limits of agreement between blood concentrations of acetone and isoprene in blood and breath were determined. A significant inter-individual correlation was found between breath and arterial blood for acetone but not for isoprene. Headspace above three types of nutrient broths inoculated with Staphylococcus aureus was compared with uninoculated broths. VOC emissions from the cultures were measured over 6 hours. The same VOCs were observed for each cultured broth but the time evolution was different

This thesis reports the effects in vivo of the sympathetic nervous system (SNS) in human subcutaneous abdominal white adipose tissue (WAT) and other tissues involved in energy storage and utilisation. Cannulation of superficial veins draining skin, abdominal subcutaneous WAT and deep forearm muscle combined with isotope turnover methodology and tissue blood flow estimation was used to investigate the behaviour of these tissues under varying experimental conditions. Glucose infusion study: This examined differential substrate uptake and utilisation in the three tissues. WAT was responsible for only a small amount of glucose disposal and deep forearm muscle took up but did not release NEFA. Skin was a net exporter of lactate. Results confirm the relative purity of the venous effluent from these tissues. Sympathetic Nervous System study: This examined whole body, WAT and forearm muscle SNS activity in lean and obese individuals under fasting and postprandial conditions. Whereas whole body SNS activity was increased in the obese, regional heterogeneity of SNS activity was evidenced by reduced SNS activity in WAT. Adipose tissue blood flow was significantly reduced in the obese. This may underlie abnormal lipolysis and/or blood flow regulation in obesity. 7 Pulsatility Studies: These studies examined whether lipolysis and leptin production in human WAT is uniform or pulsatile. Novel control datasets were used to test the robustness of a widely used pulse detection algorithm. Whereas NEFA release appeared truly pulsatile, apparent leptin ‘pulses’ occurred with similar frequency in the control datasets and appear likely simply to reflect variability. Anatomical studies: Confocal immuno-fluorescence microscopy was used to demonstrate innervation of WAT in man for the first time. Such innervation appears confined to the microvasculature and suggests that the defective SNS activity within WAT is likely to affect adipose tissue biology primarily through defective regulation of adipose tissue blood flow.

Prolonged and intense exercise is known to modulate and suppress certain aspects of the immunoendocrine system. Such effects are thought to be largely mediated by the release of stress hormones and regulatory cytokines which originate from a variety of stress related paradigms in sport. These include acute physical exertion, chronic and repetitive exercise as well as other psychological and psychosocial aspects of training and competing in an elite environment. It may be of particular interest to study the effects of regular competition and training on immunoendocrine markers in rugby union players. At the professional level, rugby is an intense and physically demanding game where a significant amount of tissue trauma occurs as a result of the many game collisions. The aims of the studies outlined in this thesis were to determine the effects of acute, repeated and chronic exercise exposure on immunoendocrine markers and illness incidence in professional rugby union. Additional case studies were also undertaken to supplement main study findings. The first part of the thesis documented the effects of acute and repeated exercise on immunoendocrine markers in a cohort of international rugby union players. Data in study 1 showed that large disturbances in immunoendocine and hormone levels occur in players (n = 10) following game play. The magnitude of this response appeared dependent on game physicality (number of rucks/mauls, tackles) and the number of collisions players received during match play. Findings also showed suppression in host immunity, and in particular, innate immune function (neutrophil degranulation) which was not resolved 38 h (-29%) into the recovery period. In study 2, bloods were taken from players (n = 8) across a 21-day international rugby series. Data revealed that players entered the international camp with residual muscle damage (creatine kinase; CK) and inflammation (hs-CRP) following previous club involvement in European cup rugby. Further increases in stress related markers (cortisol, IL-6, CK, CRP) were not evident throughout the players time at the international training base. Conversely, a progressive increase in anabolic-catabolic balance (T/C ratio) was observed across time. In comparison to values on camp-entry (day 1), increases in T/C ratio were evident on day-5 (9.8%), day-7 (13%), day-19 (35%) and day-21 (45%) (P < 0.05). This data is suggestive of improved physiological recovery and was commensurate with team fitness goals (reduced volume + maintenance of training intensity) for that time. Findings suggest that monitoring of player club activities and training load preceding international duty is pertinent if they are required to represent their country inside 7 days. The second part of the thesis evaluated the stress induced effects of chronic rugby exposure in professional club players. Questionnaire data analysed from study 3 showed that players (n = 65) perceived current season length as being ‘too long’ (55%), ‘poorly structured’ (56%) and that game demands are increasing with time (52%). Furthermore, the majority of players (80%) felt that time ‘away’ from rugby was not sufficiently long enough and were in favour of a mid-season break (2 wks in duration). Investigation into the effects of chronic exercise on illness incidence, immunological and psychological state was carried out in a squad of club players (n = 30) over a competitive season (n = 48 wks) in studies 4-6. Findings revealed that specific periods in a rugby season resulted in disturbances to hormonal and immune status. These periods occurred following breaks in club game fixtures [November international and Six-nations period: February/early March], times of increased training intensity and increased ratio of conditioning/rugby activity. Peaks in number of upper respiratory illnesses (URIs) and disturbances in psychometric variables also occurred during these time periods. In 23% of all URIs, players reported that the presence of the illness either reduced activity (14.4%) or felt the need to go to bed (8.6%). Positional differences in variables were also observed. A higher incidence of URIs (3.4 vs 4.3) and lower concentrations of resting immune markers [salivary lysozyme: s-Lys (-31%); immunoglobulin A: s-IgA (-27%)] was observed in ‘backs’ (vs forwards) over the season. Higher mid-season cortisol levels was also observed in backs (P < 0.05) while conversely, greater concentrations of plasma CK and CRP were found in forwards throughout the season. These findings indicate positional specific differences in response to exercise load and point to the role of group specific recovery at certain times during the season. Data from study 6 showed that the number of training related complaints decreased across the season, findings which closely resembled corresponding decreases in plasma CRP values. This data is suggestive of a ‘repeated-bout’ effect or ‘contact adaptation’ in rugby union. Finally, comparison of methods used in the recording of illness data revealed that players were more honest when disclosing the existence of banal infections to a web-based training diary and under-reported infections to medical staff.

The addition of silicon ions (10-500 μM) to the culture medium of MG63 osteoblast-like cells showed no changes in cell viability, metabolic activity, proliferation or morphology. Silicon ions resulted in a concentration-dependent increase in the expression of vascular endothelial growth factor (VEGF) by the MG63 cells. Addition of magnesium ions (1-50 mM) to the culture medium of MG63 cells caused a dose-dependent decrease in cell viability, metabolic activity and proliferation at each time point. In general, silicon and magnesium ions had no effect on the viability of a human endothelial cell line (HUVEC). A slight decreasing trend to the metabolic activity of the HUVECs was observed with increasing concentrations of silicon ions at all time points, but this decreasing trend was more pronounced with the addition of magnesium ions. The highest magnesium ion concentration studied (50 mM) caused a change in HUVEC morphology from a typical cobblestone appearance to a fibroblast-like shape. Lastly, the effect of silicon ions on angiogenesis in vitro was studied using two different in vitro assays. The first, using Matrigel as an extracellular matrix coating for the guidance of endothelial cells to form tube-like structures (an indicator of angiogenesis), proved unreliable for studying the promotion of angiogenesis. Additionally, tube-like structures were also observed with osteoblasts cells, raising questions about the efficiency of this assay. The second assay, AngioKit, was a suitable model for studying stimulation and inhibition of tube-like formation. Results obtained using this assay showed that silicon ions alone (500 μM) did not stimulate tube-like formation, but a significant increase in tube-like formation was observed with MG63 cell-conditioned media, with (500 μM) and without silicon ions, when compared to the control medium.

In humans, sweating is one of the most variable routes of water loss. This thesis investigates sweat volumes and sweat composition during single training sessions. Chapter 4 investigates the variability of sweat losses and sweat composition between four training sessions. Forty-five able-bodied individuals (twenty males and twenty-five females) volunteered for the study and ranged in age from 18–65 years. Data was collected during the participants normal training sessions, consisting of various recreational pursuits. Sweat losses were determined by changes in body mass over the training session, and corrected for any fluid intake and urine losses during training. A sample of participants' sweat was obtained via adhesive gauze patches and analysed for sodium, potassium and chloride concentrations. Sweat samples were obtained from the shoulder, chest, forearm and thigh. The 95% confidence intervals determined from this study demonstrated the reliability of a single training session to estimate an individual's future hydration to within 2% of initial body mass.

The zinc metalloprotease angiotensin-converting enzyme 2 (ACE2) and its homologue, ACE, are the central enzymes of the renin-angiotensin system (RAS), which regulates blood pressure, electrolyte balance and fluid volume homeostasis. ACE2 is thought to counterbalance ACE-mediated angiotensin (Ang) II formation by converting this peptide into the vasodilator Ang-(l-7). Both enzymes are implicated in cardiac fimction, renal disease, diabetes, atherosclerosis and acute lung injury, with ACE2 also being the functional receptor for the coronavirus that is linked to severe acute respiratory syndrome (SARS).

Triacylglycerols (TAG)-rich lipoproteins (TGRLPs), i.e. very-low-density lipoprotein (VLDL) and chylomicrons (CM), are believed to be important substrates for heart to produce ATP and to incorporate constituents of cardiomyocyte membranes. However, cardiac utilisation of TGRLPs has not been fully understood. Therefore, the aims of the study were to investigate metabolism of TGRLPs by the isolated 'working' rat heart under physiological and diabetic conditions. Radiolabelled VLDL and CM particles were prepared by an extended liver perfusion and a thoracic duct cannulation technique respectively. Streptozotocin (STZ)-treated rats and Zucker Diabetic Fatty (ZDF) rats were used as type 1 and 2 diabetic animal models. Under physiologically moderate workload conditions, both VLDL and CM could maintain the isolated heart in good 'working' performance. Lipoprotein lipase (LPL)mediated hydrolysis was the principal mechanism for heart to take up VLDL and CM. Receptor-mediated endocytosis, although less quantitatively important regarding the uptake of TGRLPs, was a mechanism which preferentially 'channelled' TAG assimilated towards oxidation rather than esterification. CM was a preferable substrate for heart to use, mainly as an energy source. VLDL, however, may have a different role in cardiac lipoprotein metabolism. Only about half of VLDL-TAG assimilated was oxidised and the oxidation rate of VLDL-TAG was markedly lower than that ofCM-TAG. In diabetic hearts, heparin-releasable LPL activity was significantly increased. But surprisingly, no significant change in TAG uptake was found in diabetes, although a slight increased trend was shown in ZDF rats. Significantly higher TAG oxidation rates and lower glucose oxidation rates were demonstrated in diabetic hearts, supporting the shift of cardiac substrate utilisation from glucose to lipids (not only non-esterified fatty acid but also TAG) in diabetes. The proportion of VLDL-TAG oxidised in diabetes was much higher than that in control, suggesting that VLDL could act as an extracellular energy store for hearts. The total tissue lipid esterification was unchanged but an enhanced TAG accumulation was found in STZtreated hearts, although cardiac performance was maintained. The effects of diabetic lipoproteins on metabolism were also demonstrated, possibly due to changes in lipoprotein particle composition, which was found to be disturbed in diabetes. These results suggest that VLDL and CM have a different metabolic role in the heart. The importance of the utilisation of these lipids was also confirmed by the findings in diabetes. Therefore, manipulation of lipoproteins and their utilisation may be important therapeutically.

The tetraazatriphenylene chromophore, dpqC, efficiently sensitises europium(III) and terbiiim(lll) emission. It has been incorporated into a series of ligands, based on the :yclen macrocycle, which saturate the coordination sphere of the metal ion and lead to complexes that exhibit high luminescent quantum yields in aerated aqueous media of the order of 15 - 50 %, with long emission hfetimes of the order of milliseconds.The complexes have been shown to be taken up by a number of eukaryotic cell lines and are amenable to time resolved fluorescence microscopy. By relating the structure of the complexes to their uptake and locahsation profiles, it should be possible to design complexes for specific intracellular applications, for example, as probes or components in biological assays.

Historically nitric oxide (NO) was viewed as playing a purely paracrine role in the vasculature, acting within the vicinity of its release. Today an endocrine role of NO metabolites is widely accepted, although the species accounting for the vascular regulatory function still remains in contention. The original aim of this thesis was to analyse the two main NO metabolite hypotheses (i.e., SNO-Hb and nitrite hypotheses) to provide a more comprehensive understanding of their potential significance in the human circulation. Initially this involved developing methods capable of measuring baseline levels of NO metabolites in human blood. During this work a major confounding factor was identified influencing the measurement of NO attached to red blood cells/haemoglobin. A means was developed to overcome this via the modification an assay reagent, which was used along with several other methods to analyse NO metabolites in human blood. Ultimately however, levels of SNO-Hb at baseline were on the border of methodological sensitivity, although haemoglobin-bound NO was distinguishable. Consequently my work focused on the potential of nitrite versus haemoglobin-bound NO per se to act as endocrine NO metabolites in the human vasculature. Nitrite metabolism in human whole blood was analysed whilst the vasodilatory properties of nitrite were investigated in relation to the relaxation response observed from native red blood cells under hypoxic conditions. Finally for the first time ever NO metabolites were measured across the coronary and pulmonary vascular beds in the human circulation to address their potential to regulate vascular tone at baseline and under conditions of increased oxygen demand.

The aim of this thesis was to compare the behavioural effects of selective serotonergic lesions of the PFC using 5,7-dihydroxytryptamine, in a New World primate, the common marmoset (Callithrix jacchus), on two tests of prefrontal cortical function designed to measure the flexible control of inhibitory responding. First, the role of 5-HT in orbitofrontal cortex function was assessed using a serial discrimination reversal task in which monkeys have to repeatedly shift their responding between one of two visual stimuli. In contrast to controls, 5-HT-lesioned monkeys were impaired in their ability to reverse stimulus-reward associations, due to perseveration at the previously-correct stimulus. Second, the role of 5-HT in lateral PFC function was assessed using an attentional set-shifting task in which monkeys have to shift their attention between two perceptual dimensions of a compound visual stimulus. Despite lesioned monkeys showing perseverative responding when reversing a stimulus-reward association, the same monkeys were unimpaired in their ability to shift between higher-order dimensions or rules. Finally, two studies were undertaken to characterize the perseverative deficit both neurochemically and psychologically. Although selective orbitofrontal cortex dopamine lesions were without effect, selective 5-HT lesions of the orbitofrontal and lateral PFC induced perseveration, which was not due to learned avoidance or proactive interference. These findings suggest that PFC 5-HT (but not dopamine) is critical for flexible responding at the level of changing stimulus-reward contingencies, but is not essential for the higher-order shifting of attentional sets, thus highlighting the differential sensitivity of distinct prefrontally-mediated psychological functions to serotonergic modulation. Further characterization of the 5-HT systems involved in this deficit may reveal drug targets for the treatment of psychiatric disorders in which these abilities are impaired.

Mutation of the N-terminal IGF-binding site of IGFBP-5 and its transfection into myoblasts revealed that the functions of IGFBP-5 in myogenesis can be divided in to IGF-dependent inhibition of myogenesis, and an IGF-independent anti-apoptotic role, assessed by decreased caspase-3 and PARP cleavage, caspase-3 and -9 activities, and decreased annexin V staining of cell populations overexpressing wild type (wtIGFBP-5) and non-IGF binding IGFBP-5 (mutIGFBP-5). Further analysis revealed the decreased apoptosis stimulated by wt and mutIGFBP-5 correlates with increased Bcl-XL protein, and Bad phosphorylation levels. Transfection of myoblasts with wtIGFBP-5 resulted in decreased Akt phosphorylation, increased p38 activation and slightly increased p21 protein levels. Closer examination of cells overexpressing mutIGFBP-5 revealed enhanced myogenesis, increased p38 activation, and slightly, but consistently, increased p21 levels. Intriguingly, increased Akt phosphorylation was also observed in mutIGFBP-5 overexpressing cells which occurred independently of type I receptor activation. This suggests that mutIGFBP-5 was activating an alternative pathway to enhance the myogenic programme. To study the influence of the secretory pathway on the functions of IGFBP-5, the signal peptide was removed (nsIGFBP-5). The overexpression of nsIGFBP-5 in C2 cells had little effect on the rate or extent of myogenesis, Akt phosphorylation or p38 activation when compared with control cells, but increased p21 levels to a greater extent than did wtIGFBP-5. However, more dead cells were visible. When apoptosis was examined in these cell populations, nsIGFBP-5 no longer had a protective effect, as assessed by annexin V staining and caspase-3. Indeed, cells overexpressing nsIGFBP-5 appeared to have slightly elevated apoptosis, and an intriguing 4-fold increase in caspase-9 activation, which was not translated fully in to an increase in caspase-3 activity. The slightly elevated caspase-3 activity interestingly appeared to correlate with an approximate 50% reduction in Bcl-2 protein levels.

This study involved the isolation and purification of collagen peptides and a characterisation of the effects of these peptides on cell behaviour. In vitro experiments were carried out on CNBr-derived peptides from collagens I and III, and these were shown to stimulate fibroblast growth and chemotaxis. Peptides CB3 and CB8 from collagen a1(I) and peptides CB4 and CB8 from collagen aI(III) showed the most potent stimulation. A chemotactic peptide, 22 amino acids in length, obtained by sequential enzymatic digestion of collagen I with bacterial collagenase and chymotrypsin was isolated and this peptide was also found to be a potent chemoattractant for fibroblasts. A tetra-peptide with the amino acid sequence Gly-Pro-Ala-Gly was identified as one of the most chemotactically active regions in the collagen molecule. The collagen peptide (22 amino acid sequence) was also tested in vivo for its ability to stimulate cellular infiltration and granulation tissue formation using the PVA (polyvinyl alcohol) model system in rats. The peptide was injected into PCVA sponges which had been implanted into rats. After 7 or 10 days the sponges were removed and analysed both histologically and biochemically. The levels of collagen, DNA and protein in the PVA sponges injected with the peptide increased significantly compared to control sponges, after 10 days. This effect was concentration dependent, with the optimum tested concentration being 10mg/ml. Experiments were also carried out on cell surface interactions. Binding of the peptide to fibroblasts was not inhibited by antibodies to the integrins a1b1, a2b1, a3b1, suggesting that collagen-receptor interaction in this case may be non-integrin mediated. From this work, it was shown that peptides isolated from collagens I and III were chemotactic for fibroblasts both in vitro and in vivo.

The thesis examines the regulation of neuronal nitric oxide synthase (nNOS) mRNA expression in magnocellular neurosecretory neurones under physiological circumstances including pregnancy, hyperosmotic challenges and prolonged stimulation of the axons of the magnocellular neurones, and the functional role of nitric oxide (NO) in regulating oxytocin and vasopressin neuronal activities, and the release of oxytocin. The studies have included measurement of mRNA expression by in situ hybridization, in vivo electrophysiological studies combined with intrasupraoptic nucleus retrodialysis, Fos immunocytochemistry studies, and functional studies involving measurement of oxytocin secretion. Expression of nNOS mRNA in the magnocellular neurones in the supraoptic (SON) and paraventricular nuclei (PVN) has been confirmed to be regulated in response to chronic osmotic stimuli, suggesting an important involvement of NO in this situation. It has been demonstrated that an induction of this gene is not a rapid response since an increase of NOS gene expression in the magnocellular neurones in the SON and PVN has been found at 6 h after acute hypertonic saline intraperitoneal injection, but not at 4 h after injection. Neuronal activity of oxytocin and continuous firing vasopressin neurones in the SON was inhibited by local application of NO donor, and was increased by local administration of NOS inhibitor. Nitric oxide donor inhibited neuronal activity of phasic vasopressin neurones, while NOS inhibitor did not alter neuronal activities of these neurones. Blocking the central production of NOS by NOS inhibitor enhanced the expression of Fos in the magnocellular neurones in the SON and PVN following high doses, but not low doses of hypertonic saline. In addition, inhibition of NOS potentiated the release of oxytocin evoked by electrical stimulation of the axons of the magnocellular neurones. Furthermore, prolonged electrical stimulation of the axons of the magnocellular neurones for 2 h produced a down-regulation nNOS mRNA expression in the magnocellular neurones in the SON. Thus NO, generated in an activity-dependent manner, appears to act as a feedback inhibitor of oxytocin release at the cell bodies and the terminals of oxytocin neurones. However, although NOS expression is up-regulated in conditions of chronic demand of oxytocin, the present experiments indicate that this does not reflect a coupling of spike activity to increased NOS mRNA expression. In experiments in virgin rats, central administration of NO donor attenuated the release of oxytocin in response to acute intraperitoneal hypertonic saline and systemic administration of NOS inhibitor potentiated oxytocin release induced by acute intraperitoneal hypertonic saline but not cholecystokinin.

Long-term exposure of Jurkat, L929 and J774 cells to exogenous NO inhibits cellular respiration at the level of complex IV that is reversed by oxyHb and at the level of complex I that is not reversed by oxyHb. Long-term exposure of NO to the cells causes a decrease in reduced glutathione concentration that precedes the inhibition at the level of complex I. This inhibition is reversed by addition of reduced glutathione or cold light, suggesting that S- nitrosylation of thiols is necessary for the activity of the enzyme. Furthermore, long-term exposure of the cells to NO decreases oxygen utilisation due to two distinct actions, one being the inhibition of cellular oxygen consumption and the other, the generation of a hypoxic microenvironment due to the oxygen consumption by the extracellular medium. Inhibition of basal NO, with a NO synthase inhibitor, increases cellular respiration in iNOS mutant, iNOS and eNOS wild type, but not in eNOS mutant mice. Administration of a low dose of endotoxin to the mice leads to a short-term generation of NO associated with a short-term inhibition of mitochondrial respiration resulting in non-lethal shock. Whereas administration of a high dose of endotoxin leads to a sustained generation of NO accompanied by a sustained inhibition of mitochondrial respiration, at the level (at least in part) of complex I, resulting in lethal shock. These results suggest that NO can be considered as a physiological regulator and a pathological inhibitor of tissue respiration. The basal release of small amounts of NO acts as a regulating mechanism, whereby cells respond to changes in their environment and regulate ATP production. By contrast the generation of high concentrations of NO over a sustained period decreases reduced glutathione concentration and pathologically inhibits cellular respiration leading to multiple organ failure and ultimately death.