Pulmonary arterial hypertension (PAH) is a progressive disease which results from increases in mean pulmonary artery pressure and pulmonary vascular resistance. If untreated it leads to right ventricular failure and death. 5-Hydroxytryptamine (5-HT) has been implicated in the disease process and is thought to promote both vasoconstriction and remodelling of the pulmonary vasculature. The activity of potassium ion (K+) channels plays a major role in influencing pulmonary artery tone by regulating resting membrane potential, intracellular Ca2+ concentration and contraction of vascular smooth muscle. This study aimed to investigate possible interactions between 5-HT and K+ channels in the development of PAH in the mouse. The actions of 5-HT and a range of K+ channel blockers were investigated on isolated intralobar pulmonary arteries (IPA) from wild type (WT) mice and mice over-expressing the serotonin transporter (5HTT), which spontaneously develop PAH. Both 5-HT and linopirdine, a KCNQ K+ channel inhibitor, were found to induce contraction of IPA, but were more potent in IPA from WT mice than 5-HTT+ mice. The 5-HT induced vasoconstriction was found to involve influx of Ca2+ from the extracellular space, Ca2+ release from the sacroplasmic reticulum and a rho kinase–dependent increase in the sensitivity of the contractile machinery of pulmonary artery smooth muscle cells (PASMC) to intracellular Ca2+. Ca2+ entered the cell via both voltage operated calcium channels (VOCC), activated by membrane depolarisation, and a separate Ca2+ entry pathway, the latter appearing to contribute more in 5-HTT+ mice. The effects of linopirdine were shown to be due entirely to the entry of Ca2+ through VOCC in both WT and 5-HTT+ mice IPA. The difference in vasoconstrictor potency between WT and 5HTT+ mice was not seen with any other K+ channel blocker, suggesting a selective loss of KCNQ channels and/or VOCC in PAH resulting from 5HTT over expression. KCNQ channel activity was further investigated using the KCNQ channel openers, flupirtine and retigabine. These agents were more potent in dilating IPA from WT mice compared to 5-HTT+ mice, consistent with the loss of expression or activity of KCNQ channels in 5-HTT+ mice. Despite this, orally administered flupirtine was shown to reverse two indices of established PAH in the 5HTT+ mice; right ventricular pressure and right ventricular hypertrophy. This action of flupirtine was also seen in chronic hypoxic mice, where it prevented the development of PAH. In conclusion, this study provides evidence of an interaction between KCNQ channels and the 5-HT system in the development of PAH. By showing that a KCNQ channel opener can attenuate PAH, both in the developing and established disease situation, this study proposes a new potential therapeutic target in the treatment of PAH.

Despite the abundance of the templates, both human and rodent SINEs are normally expressed at a very low level. DNA methylation-mediated silencing has been proposed as a possible cause of their transcriptional repression. The effect of DNA methylation and the effect of DNA methylation-dependent methyl-CpG-binding domain proteins (MBD proteins) on SINE transcription were studied here. It was shown that both human and rodent SINEs are bound by MeCP2, MBD1 and MBD2. Both human and rodent SINEs were also shown to be occupied by HDAC1, HDAC2 and a component of SWI/SNF complex, Brahma. Human Alus were also found to be occupied by components of two corepressor complexes, SIN3 and NuRD. Whether MBD proteins repress SINE transcription in a DNA methylation-dependent manner was further investigated using systems with low or near absent DNA methylation and, in the case of MeCP2 protein, by its direct removal. MeCP2 was found to have no repressive effect on B1 and B2 expression. RT-PCR analysis showed no increase in B1 and B2 RNA levels in MeCP2 null mice kidneys. ChIP analysis of Dnmt1n/n p53-/- embryonic fibroblasts, which have less than 5% of the normal DNA methylation level, showed significant reduction in MeCP2 and MBD2 binding, confirming that their presence is DNA methylation-dependant. RT-PCR comparison of Dnmt1+/+ p53-/- and Dnmt1n/n p53-/- cells, however, detected no increase in B1 or B2 RNA levels. This was consistent with results obtained from MeCP2 null mice, where lack of MeCP2 did not result in increased B1 and B2 expression and with a previous study involving human Alus (Yu et al., 2001). MBD2 also does not seem to repress SINE activity, as its release following loss of DNA methylation did not result in increased SINE RNA levels. Strikingly, all human and rodent SINEs studied here were found to be bound by transcription factors TFIIIB and TFIIIC at comparable levels with actively transcribed genes. Some RNA polymerase III was also detected, but at levels significantly lower than on active genes, suggesting a defect in RNA polymerase III loading onto SINEs. This occupancy of the transcriptional complex was comparable in cells with normal levels of DNA methylation and in cells with significantly reduced levels of DNA methylation, suggesting that the occupancy is not affected by methylated DNA or DNA methylation-dependent components of chromatin. Indeed, removal of 50% of histone H1 did not result in increased B1 or B2 expression in this study. The fact that all tested SINEs are occupied by TFIIIB and TFIIIC also brings an unprecedented insight into the number of these transcription factors present in the cell.

Chronic myeloid leukaemia (CML) is a stem cell (SC) disorder initiated by the reciprocal translocation between chromosome 9 and 22, giving rise to the Philadelphia (Ph) chromosome and the resulting expression of the oncogenic fusion protein BCR-ABL. The current first line of treatment is imatinib mesylate (IM), a tyrosine kinase inhibitor (TKI) that competes with ATP to block ABL kinase activity, which in turn prevents tyrosine phosphorylation of downstream molecules and selectively induces apoptosis of BCR-ABL cells. However, despite excellent cytogenetic responses, only a minority of patients achieve complete molecular response (CMR). We have previously identified a population of quiescent (q) Ph+ SC found in chronic phase (CP) CML that are relatively insensitive to IM and other TKIs and which may be responsible for the molecular persistence of this disease. This population may be insensitive because TKIs do not reach therapeutic concentrations within the cell. Such resistance to classical chemotherapeutic drugs, the phenomenon of multidrug resistance (MDR), is mediated by ABC transporters. In this study we have investigated whether CML SC express the clinically relevant ABC transporters and determine their interaction with TKIs. In addition, we determined whether the inhibition of these transporters increased the efficacy of TKI against CML SC. Using CML CD34+ cells isolated from newly diagnosed patients, normal CD34+ cells and cell lines transduced with specific transporters as controls, the relative expression of drug transporters were determined in CML CD34+ cells and intracellular staining confirmed protein expression. The interaction of drug transporters with TKIs was assessed using a combination of substrate displacement assays and radiolabelled assays. The effect of transporter inhibitors 3 with TKIs on the growth and differentiation of q34+ and more mature CD34+ cells from CML patients in CP were assessed with regard to cell division, apoptosis and BCR-ABL kinase activity. When compared to normal CD34+ cells, CML CD34+ cells over-expressed ABCG2 mRNA. In contrast MDR1 expression was reduced in CML CD34+ cells and MRP1 was detected at similar expression levels in both populations. All three drug transporters were expressed at the protein levels in CML CD34+ cells. It was determined that at therapeutic concentrations (5μM) IM and nilotinib both inhibited ABCG2 and MDR1 and nilotinib also inhibited MRP1. Neither drug was a substrate for any of the transporters. In contrast, dasatinib was shown to be a substrate for ABCG2 and MRP1, but had no effect on MDR1. Therefore activity and concentration of dasatinib but not IM or nilotinib may be altered by the activity of these proteins. In keeping with their inhibitory activity, neither IM nor nilotinib demonstrated significantly increased efficacy when combined with specific ABC transporter inhibitors (FTC or PSC 833). Surprisingly, although dasatinib was a substrate for ABCG2 and MRP1, dasatinib did not further increase apoptosis, or reduce the qSC population. Therefore, although MDR1, MRP1 and ABCG2 were found to be expressed and functional in CML CD34+ cells and to interact with TKI, the co-treatment of TKIs with drug transporter inhibitors did not further increase apoptosis, reduce BCRABL kinase activity or reduce the qSC population. Therefore, modulation of individual transporter activity is unlikely to reverse the resistance of this population of cells to TKI and will not improve the clinical response to these drugs.

Since the emergence of Kenyan endurance runners on the world stage at the 1968 Mexico Olympics, where they won 8 medals ranging from 400m relay to the 10 000m, Kenyan success has grown year on year. The staggering success of a country that compromises just 0.5 % of the world population has triggered a number of explanations. Heavily cited explanations are genetic superiority and environmental factors. Despite a number of investigations, genetic superiority remains to be determined, what is clear though is that the environmental factors that interact with each genetic element leading to world-class performance are particularly important. Aims and objectives: Given the importance environmental factors may have on the process leading to world class performance, the main aims of the following research were: 1) to determine the composition of elite Kenyan endurance runners diet and assess their energy balance status prior to major competition using "gold standard" methods; 2) to establish lifestyle practices of elite Kenyan endurance runners prior to major competition that will allow an insight in to the preparation of some of the best athletes in the world; 3) to ascertain the hydration status of elite Kenyan endurance runners during an important training period and directly compare these results to traditional paradigms and current thinking on optimal fluid intake for superior endurance running performance; 4) to investigate the training process leading to world class performance by quantifying training load in the lead up to major competition; 5) to determine the fluid intake behaviours of the world s best marathon runners during racing. This will allow an insight into current practices of elite runners that will act as a benchmark and comparison of current fluid intake guidelines; and 6) to validate and combine existing technologies of heart rate and accelerometry for quantifying energy expenditure during free living conditions. Methods: Chapters 2 and 3 detail extensively the diet, hydration, lifestyle and training practices of a group of highly successful elite Kenyan endurance runners during important training periods based at a high altitude camp in Kenya. Chapter 4 explores the significance of the hydration practices reported in Chapters 2 and 3 (i.e., ad libitum fluid intake) have on elite marathon running performance and the wider implications for fluid intake recommendations for elite marathon running. Chapter 5 investigates novel technology (i.e., the combined use of accelerometry and heart rate) that may further enhance our understanding of the physical activity patterns and training practices of elite Kenyan endurance runners on a day-to-day basis. Results and discussion: Chapter 2 reported elite Kenyan endurance runners are in negative energy balance prior to major competition as assessed by the gold standard doubly labeled water method (Energy intake: 13.2 +/- 1.3 MJ/d: vs. Energy expenditure: 14.6 +/- 1.0 MJ/d; p < 0.005). Considering the relatively high carbohydrate content of their diet (e.g., 67.3 +/- 7.8 %, 9.8 g/kg/bm) it is hypothesised the caloric deficit may not have a direct impact on their training performance. In fact the performance implications of reducing body mass as a result of energy deficiency is that the athletes will be lighter for competition and may thus be at an advantage as the energy cost per unit distance increases in direct proportion to the added load expressed as a percentage of body mass. Measured physical activity patterns (i.e., Physical Activity Ratio (PAR) and accelerometry) of elite Kenyan endurance runners strongly suggest rest between running training sessions is an important lifestyle factor as it was found time spent relaxing, in light activity, slow running (8.0-13.6 km/h), moderate running (13.7-17.3 km/h), and fast running (> 17.4 km/h) as estimated using the PAR method was 82 +/- 6 %, 8 +/- 6 %, 3 +/- 1 %, 5 +/- 1 %, 2 +/- 1 %, respectively. The reported time spent in light, moderate, hard and very hard activity as determined by accelerometry was 82 +/- 3 %, 11 +/- 2 %, 6 +/- 3 %, and 1 +/- 1 % respectively. A further striking finding in Chapter 2 was the relatively low daily fluid intake that consisted of primarily water (0.9 +/- 0.5 L/d) and milky tea (0.9 +/- 0.3 L/d). Chapter 3 found athletes remained hydrated day-to-day drinking ad libitum despite this relatively low daily fluid intake that corroborated prevailing fluid intake recommendations. This was evidenced by mean total body water and pre training body mass being maintained day-to-day throughout the recording period (p = 0.194 and p = 0.302, respectively). Furthermore, there was no significant difference between the osmolality of the morning urine sample and the evening sample (p = 0.685). It was also found that athletes remained in electrolyte balance (Na+ intake: 3245 +/- 901 vs. Na+ loss: 3254 +/- 1070 mg/d; p = 0.975) day-to-day thus negating the need for further supplementation.

SUMMARY: Every day we have to make choices, to solve small and big life problems, and we behave accordingly. The experience becomes crucial and determinant in sport situation. The research intends to propose an analysis, at a first level, of the main factors involved in choice and action. The direction and the conditions proposed by the study could be a framework for future research and for further developments in sport. Selection among options, choice, focal action and the time of reaction are the centre of the investigation. The main parameters are choice and reaction time. Choice describes the process of weighting and picking the more suitable alternative. Reaction time is the time of preparation and execution, the time during selection, choice, and the performing of the focal action, which ends at the recording of the data. Reaction time period and choice events were analysed separately. Choice, in particular, was related to a positive prediction, that is to a correct answer. Among the variables involved in the experimental situation, choice, reward, delay and cues were chosen. The main aim was to find the change in reaction time, when a correct response could be predicted. A special Reaction Time Device was built up, tailored on the specific needs of the experiments. The period between a visual “go” signal and a target touching by an arm, the focal action, was the time of reaction. The feedback given by the Device informed the subject on the correct or not correct choice. A basic protocol was defined, and the experimental plan, built up by 4 steps, related to specific aims, was fixed. The first step had an introductory role. It tested simple and choice conditions. Random sequences and random delays were presented. Choice reaction time was examined when dominant, non-dominant and either arm were performing. The final evaluations allow detecting correct and incorrect answer factors, and the effect of different delays, as first reference of these components. The data register no difference in reaction time between dominant and non dominant arm performed during choice. The second step involved reward factor. The aim was to compare reaction time without and with a possibility of reward. The results showed that at those conditions, no significant reaction time difference was registered. The third step examined the delay factor and tested the reaction time when a variable or a fixed foreperiod was presented, before the “go” signal. It was concluded that there is no significant difference in time of reaction between a constant and a changeable delay. The contractions of the subject’s focal and postural adjustment representative muscles were simultaneously recorded during the tests through EMG, to check the preparation phase and, eventually, a possible change in muscle contractions during the selection of the alternatives. The results confirmed that choice is a high level process and that, at least at those conditions, muscles are involved only at the last, final stage, after selection. The former 3-step experiments had the role of premise to test the weight of some relevant variables. The centre of decision-making process is guessing, is anticipating the event. It implies the presence of clues, which can be identified, recognised and can lead the person to predict the next answer. This was the main motive of the fourth step. Three of the four sequences had a pattern, made up by 3 numbers, presented 4 times in the same sequence. The reaction time results included very short and very long values, far from the normal distribution. They were transformed in Log (- 350), to get coefficients suitable to be processed through parametric tests. The numbers suggested that after the first experiences of the pattern, some subjects, having the feedback at each trial, identified and recognised the regularity and tended to be more correct at the last presentation. Some of them took a longer time to come to a choice, some were quicker. Two among 3 patterns were detected easier. In one of the 3 special sequences, the percentage of pattern correct answers was 48%, beyond the probability limit. It means that nearly half of the answers were positive and nearly half of the people guessed the pattern. There was a clear difference in percentage of correct answers between the random sequence, which remained within the probability percentage, 33%, and 2 of the 3 sequences. In addition the percentage of patterned correct answer was higher than the total percentage of positive responses, inside the same sequence. Nevertheless in statistical terms the p values were not significant. The 2 factors tested during the investigation, reaction time and choice, showed that, at the specific conditions of the experiments, there is no clear reciprocal correspondence. Unlike the studies in the field, correct answers were not directly related to lower reaction times, as expected, in patterned trials. Sometimes the subjects took more time to make their choice. Short and long reactions, within the same subject and among quick and slow volunteers, balanced the data. The results were not significant, nevertheless the differences were evident and in the right direction. The conclusion was that in a choice situation, when guessing was encouraged by cues to get correct response, the number of patterned correct trials tended to increase, in particular at the last repetition of the pattern. The analysis on reaction time could not confirm the expected relationship between pattern correct trials, a sign of guessed cue, and a decreasing time of reaction.

The oxidative stress response to exercise is a well-established phenomenon; however, the time course of this response has not been well characterised. There is little information in the literature regarding the oxidative stress response during exercise; most authors have measured oxidative stress solely during the recovery period from exercise. There are several different invasive methods available for assessment of oxidative stress, although there is no “gold standard” technique. A novel non-invasive technique utilising laser spectroscopy to quantify expired ethane concentration has become available, but has not yet been tested in relation to exercise. The first study described here aimed to use the laser spectroscopy technique for the first time to assess exercise-induced oxidative stress in three species: humans, horses and dogs; and to determine the utility of carbon monoxide monitoring as a means of assessment of oxidative stress. A further objective was to better characterise the oxidative stress response by the collection of data at frequent intervals during exercise and during recovery. Eight endurance-trained males performed incremental treadmill exercise to volitional exhaustion. Twelve racehorses and twelve racing greyhounds performed maximal exercise on a race track. Expired ethane concentration was measured throughout exercise in humans, and pre- and post-exercise in horses and dogs. Carbon monoxide concentration was assessed pre- and post-exercise in all species. Results indicated that the technique of laser spectroscopy was viable for use in relation to exercise in all three species. Oxidative stress was shown to increase significantly following exercise in all three species, thus supporting previous literature, and extending this finding to a trained human population for the first time. The pattern of response during incremental treadmill exercise was characterised for the first time and indicated a non-significant increase in oxidative stress in humans within 2 minutes of the onset of exercise, with the response progressively increasing alongside increases in work rate until exercise was terminated at exhaustion. The response returned close to the resting value by 20 minutes into the recovery period. Low subject number may have contributed to the lack of significant findings during exercise. Carbon monoxide was not a useful indicator of oxidative stress in any species. Increased functionality of the laser spectroscopy technique was investigated by pilot work in which real-time monitoring of expired ethane was attempted for the first time in relation to exercise. This allowed the observation of the oxidative stress response on a breath by breath basis. Initial tests, in which two healthy males performed incremental cycle ergometer exercise to exhaustion whilst breathing through a valve connected directly to the spectrometer, indicated that a useful output could be recorded during a prolonged period of exercise. However, the measurement of ambient ethane concentration, essential for accuracy, was not undertaken in the initial tests. Thus, further pilot work was successfully carried out in three healthy males to replicate the initial tests with concurrent ambient ethane monitoring. This pilot testing allowed development of data editing techniques. The oxidative stress response profile for incremental exercise in real-time was similar to that reported in the previous chapter. Additional tests were undertaken which illustrated that the rise in ethane output observed during incremental exercise was not simply a manifestation of the ventilatory response to exercise, rather than an indication of exercise-induced oxidative stress. This was accomplished by forcing an increase in ventilation, by imposition of an additional dead space volume during normal breathing in two individuals. This technique shows promise for more detailed characterisation of the time course of the oxidative stress response in future exercise studies via the capability for extremely high density data collection. The main aims of the second study were to investigate the oxidative stress response throughout the entire work rate range from rest to volitional exhaustion, rather than just the higher end of the work rate range as observed in study one; and to examine the magnitude and time course of the oxidative stress response to constant load exercise performed below and above the lactate threshold. Six healthy males performed incremental cycle ergometer exercise to exhaustion during which blood samples were collected regularly for later analysis for the presence of F2-isoprostanes. Results of the analysis were disappointing, with a high proportion of samples displaying a concentration outwith the range of the assay. However, preliminary malondialdehyde analysis suggested that the oxidative stress response may increase progressively alongside work rate throughout the entire work rate range. However, this observation is far from conclusive as it is based on data from a single subject only. The final study was intended to investigate the effect of contraction intensity on the oxidative stress response to isometric handgrip exercise sustained to exhaustion, and to clarify the time course of the oxidative stress response during the recovery period. Due to logistical limitations, it was possible to study one contraction intensity only. Initially, pilot work was undertaken to determine the suitability of the novel non-invasive technique for ethane assessment in relation to isometric exercise, since this assessment method had not been used previously with this exercise mode. Then, six healthy males performed sustained isometric exercise at 60 % of maximal voluntary contraction until fatigue. Oxidative stress was assessed during a 30 minute recovery period via expired ethane and also via F2-isoprostanes concentration in blood collected from both the exercised arm and the non-exercised arm. This was intended to allow comparison of blood sampling site, and of the systemic oxidative stress response measured both invasively and non-invasively; however this was not possible due to poor assay results. The previous finding of a peak oxidative stress response following isometric exercise within the first 5 minutes of the recovery period was supported. Oxidative stress was assessed by ethane output for the first time in relation to isometric exercise and was found to be a viable technique; however, its use remains to be validated against more traditional plasma markers. The potential value of non-invasive assessment was underlined by F2-isoprostanes analysis issues. In conclusion, the use of laser spectroscopy, including the use of real-time monitoring, appears to be a viable technique for the non-invasive assessment of exercise-induced oxidative stress, and may enhance our ability to characterise this response in future studies.

There were two purposes to the work of this thesis (a) to identify the role of isometric training intensity in the training-induced reductions in resting blood pressure, and (b) to identify whether the mechanism for the reduced resting blood pressure is best reflected in what can be broadly termed cardiovascular, neuromuscular or metabolic markers of that training. Firstly, in a cross-sectional study, the only strong correlation was found between heart rate variability (a cardiovascular marker) and resting blood pressure. Secondly, this cardiovascular marker was also significantly affected by a single session of isometric exercise, an effect that persisted for at least 4 hours after exercise. However, thirdly, this marker and other cardiovascular markers (such as cardiac output and stroke volume) did not correlate with reductions in blood pressure seen after 4 weeks of isometric training. Instead, the training-induced reductions in blood pressure correlated strongly with neuromuscular and metabolic markers of isometric training. The extent to which local muscle fatigue was induced during isometric training correlated with the reductions in resting blood pressure. Therefore (a) isometric training intensity appears to be of utmost importance in the reductions in resting blood pressure (when bilateral-leg exercise is performed in 2 minute bouts), and (b) the mechanism whereby the adaptations in resting blood pressure occur is best reflected in neuromuscular and metabolic markers of local muscle fatigue during that training. These findings are discussed with a particular focus on the possible role of muscle metaboreceptor stimulation, during isometric training in the mechanism of training-induced reduction in resting blood pressure.

1. The pressure-perfusion myograph. permeabilisation techniques and also intracellular membrane potential recordings were used to examine the regulation of myogenic tone in cerebral and mesenteric resistance arteries by metabolic agents and second messenger systems. 2. After pressurisation to 60 mmHg, rat isolated mesenteric and cerebral resistance arteries developed spontaneous myogenic tone, resulting in a 26 ± 1% (n = 42) and 30 ± 2% (n = 14) reduction in diameter respectively. 3. The metabolic vasodilator adenosine and the KATI' channel opener cromakalim each produced a dose-dependent dilatation of pressurised mesenteric arteries. The cromakalim-evoked dilatation was inhibited by glibenc1amide (l J.lM), demonstrating the presence of the KATI' channel in the mesenteric artery and their activation as the mechanism for cromakalim-evoked dilatation. In contrast neither adenosine nor cromakalim produced a dilatation of pressurised cerebral arteries. 4. Adenosine-evoked dilatation of mesenteric arteries was unaffected by the nitric oxide synthase inhibitor L-NAME (100 IlM). antagonists of the KATI' channel (gJibenclamide; 1 J.lM), the small conductance Ca2' activated K+ channel (apamin; 0.3 J.lM) and the large conductance, Ca2! activated K+ channel (TEA; 1 mM). Further to this, cromakalim (10 IlM) but not adenosine (100 J.lM) produced a hyperpolarisation of the pressurised mesenteric artery. This suggests that neither nitric oxide synthesis nor K+ channel activation contributed to the adenosine-evoked dilatation. 5. Adenosine evoked adose-dependent dilatation of p-escin permeabilised mesenteric arteries; where the intracellular Ca" concentration was clamped to ~600 nM. The mechanism of adenosine-evoked dilatation may involve a decreased myofilament Ca2+ sensitivity. 6. An increase in extracellular potassium ion concentration ([K+lo) may link increased neuronal activity and regional cerebral blood flow. Elevation of [K+Jofrom 4.7 to 10 mM evoked a sustained dilatation of isolated pressurised thalamo-perforating cerebral arterioles. 7. The K+-evoked dilatation was inhibited by the inward rectifier K+ channel (K1R) inhibitor Ba2+ (50J.lM), and the K+ channel inhibitor cesium (20mM) but was not blocked by inhibitors of the ATP-sensitive (KATP)and the Ca2 + -activated K+ channel (KcJ, glibenclamide (l J.lM) and TEA (lmM) respectively. Nor was the dilatation altered with the neurotoxin tetrodotoxin (TTX, 0.3 J.lM). The K+--evoked dilatation was associated with a membrane hyperpolarisation to -58 ± I mV (n = 5), from a control value of -42 ± 1 mV (n = 10). 8. It is proposed that increased [K+Jo evokes a dilatation of thalamoperforating cerebral arteries via an activation of KIR channels and smooth muscle cell hyperpolarisation. 9. An increase in [Ca2+]o to approximately 700 nM evoked a 30 ± 3 % (n = 28) constriction of isolated ~-escin permeabilised cerebral resistance arteries. 10. Under [Ca2+1 clamped conditions the putative PKC activator indolactam evoked a 20 ± 2% constriction of the artery. The PKC inhibitor (PKC(19_ 36); I IlM) produced a near maximal (85 ± 4 %) reversal of the indolactam-evoked constriction of the artery, while PKC(19_36) (1 IlM) produced only a minor (12 ± 3 %) reversal of the Ca2+-induced constriction, thus confirming that the indolactam-evoked constriction was due to an activation ofPKC. 11. The MLCK antagonist SM-l (100 JlM) reversed both the Ca2+_ and the indolactam-evoked constriction of the artery. The calmodulin antagonist RS-20 (0.1 - 100 JlM) dose-dependently reversed the Ca2 + -evoked constriction but, even up to a concentration of 300 JlM, did not reverse the indolactam evoked-constriction of the artery. 12. It is proposed that MLCK but not calmodulin plays a role in the PKCevoked smooth muscle contraction.

The primary objective of these series of experiments was to develop an optimal hyperhydration strategy for use during conditions of restricted water access or exercise-induced heat stress. This strategy was composed of two compounds, namely Cr and Gly which each targeted specific body water compartments in order to maximise the volume of retained water. Endurance-trained subjects were recruited to participate in the current series of three experiments, and following Cr/Gly supplementation, body water was estimated by multifrequency bioimpedance and the physiological responses to exercise in the heat (30°C, 70% relative humidity) recorded and compared to pre-supplementation values. The aim of the first study presented in this thesis (Chapter 3(a)) was to examine the effects of combined Cr and Gly supplementation on fluid retention and subsequently the effects on cardiovascular, thermoregulatory and metabolic responses and performance during exercise in the heat. The aim of Chapter 3 (b) was to examine the effects of a novel method of Cr and Gly delivery and ingestion on fluid retention and distribution. Chapter 4 (a) aimed to assess the effects of Cr and Gly supplementation ingested according to the loading protocol described in the previous chapter (6 days of Cr and Gly ingestion, with the final supplement consumed 3 hours prior to measurement) on cardiovascular, thermoregulatory and metabolic responses and performance during exercise in the heat. The aim of the study in Chapter 4 (b) was to examine the effects of extending the period of time between ingestion of the final Cr/Gly supplement on the retention and distribution of fluid. The experiment in chapter 5 compared the effects of the novel Cr and Gly loading protocol established in Chapter 4 (b) on cardiovascular, thermoregulatory and metabolic responses and performance during exercise in the heat. The aim of Chapter 6 was to compare Tc measurements obtained using an ingestible telemetry pill and a tympanic membrane thermometer with those from a rectal thermistor during rest and high intensity exercise conducted in a hot and humid environment (30°C and 70% relative humidity) intended to raise Tc above 39°C.

This project was designed to determine the effect of a wide range of grape and tea based extracts and green tea catechins on vascular tension in vitro, further determine the relationship between their vasodilatory and antioxidant activities and identify the major polyphenols present in green and black tea. Grape and tea based extracts were examined for their vasodilator activity in vitro using standard organ bath pharmacology. All extracts evoked biphasic concentration-dependent relaxation in rabbit aortic vessels. Grape based extracts were significantly better vasodilators than tea based extracts. Previous work by others has shown that grape based products induce vascular relaxation via nitric oxide (NO) and endothelium-dependent mechanisms. This study demonstrated that a range of grape based extracts induced vasorelaxation responses via complex mechanisms including endothelium-dependent and independent mechanisms via vasodilating prostaglandins by way of prostacyclin and endothelial NO production. Tea based extracts on the other hand, induced vasorelaxation via the combined interactions of vasodilating endothelium-dependent prostaglandins. The relationship between the vasodilation capacity, antioxidant activity, based on the reduction of the free Fremy's radical and ferric reducing power, and total phenolic content of each extract was also determined. In general, a significant inverse correlation was identified between the vasodilator abilities of the grape and tea extracts in vitro and their antioxidants activities. This study demonstrates that grape and tea based extracts induce vasorelaxation in isolated rabbit aortic vessels and are effective antioxidants in vitro, within a concentration range that may be reached in vivo by moderate wine and tea consumption. The results presented here also indicate that consumption of green tea may have greater benefits than consumption of black tea in terms of their vasodilatory activity and antioxidant capacity in vitro.