Exaggerated metabolic perturbations during the postprandial period are likely to play a role in the development of vascular and metabolic diseases. Elevated levels of postprandial triglycerides (TG) are associated with increased risk for atherosclerosis independently of other cardiovascular risk factors, and exaggerated postprandial insulin excursions are known to contribute to lipid dysmetabolism and chronic insulin resistance. This, together with the fact that free-living humans spend most of their time in the postprandial state, suggests that interventions focusing on the improvement of postprandial metabolism could play a role in the prevention and management of cardiovascular and metabolic diseases. Exercise has a potent role in improving postprandial metabolism, by effectively attenuating postprandial lipaemia and insulinemia, as well as increasing fat oxidation, all of which providing positive outcomes for the prevention and treatment of metabolic disorders. It is however unclear the extent to which these beneficial effects of exercise persist when food is consumed ad libitum. In addition, the effects of exercise on appetite regulation and food intake require further elucidation. It is possible that exercise may provoke compensatory adaptations in food intake in an effort to restore energy balance, through physiological and/or behavioural responses. This has implications for the efficacy of exercise in the regulation of a healthy body weight. Therefore, the overall aim of this thesis is to describe the effects of exercise on postprandial metabolism, appetite responses and feeding behaviour in overweight/obese men. The first two experimental chapters of this thesis (Chapters 3 and 4) aimed to investigate the effects of single vs. repeated exercise sessions (~700 kcal per session) on postprandial metabolism, energy intake, appetite and gut peptide responses in response to ad libitum feeding. Ten sedentary, overweight/obese men underwent: i) no-exercise control; ii) one exercise session (Day 3); and iii) three exercise sessions over three consecutive days (Days 1-3); prior to a 7-h metabolic assessment day (Day 4). Energy substrate utilisation, postprandial TG, insulin, acylated ghrelin, PYY3-36 as well as appetite responses and ad-libitum energy intake (breakfast, lunch, dinner) were determined. The findings of this study showed that the beneficial effects of a single exercise session on postprandial metabolism on postprandial metabolic responses persisted when meals were consumed ad libitum, but were not augmented by inducing a larger energy deficit by exercising on consecutive days. Furthermore, while a single exercise session did not elicit compensatory responses in appetite and energy intake, exercising on consecutive days led to a partial compensation (~24%) in energy intake as well as increased hunger sensation. Gut peptide responses were unaltered by exercise. The next chapter (Chapter 5) aimed to determine the effects of exercise timing relative to meal ingestion on postprandial metabolism, appetite responses, and ad libitum energy intake. Ten, sedentary overweight men exercised for an hour (~400 kcal) before or after consuming a standardised breakfast meal, followed by an 8.5 h metabolic assessment period. Energy substrate utilisation, postprandial TG, insulin, as well as appetite responses and ad-libitum energy intake (lunch, dinner) were determined. The findings indicated that exercise performed prior to a breakfast meal and exercise performed after a breakfast meal waas similarly beneficial in improving postprandial metabolism. Exercise timing relative to meal ingestion also did not influence appetite responses and ad libitum energy intake. In the final experimental chapter (Chapter 6), a pilot study was designed to examine the effects of acute exercise on non-metabolic factors related to appetite using a computer-based assessment. Twenty-seven men and women walked for an hour on the treadmill or rested on a control day. Appetite-related measures were assessed before and immediately after exercise, and hourly for 2 hours post exercise. The findings showed that an acute bout of moderate intensity exercise had an anorexigenic effect; characterised by diminished hunger and lower prospective food intake (ideal portion size) compared to no exercise. Although not a primary aim, this study discovered a novel association between loss aversion and prospective food intake and food liking. The collective findings of this thesis suggest that exercise attenuates postprandial TG and enhances fat oxidation in response to ad libitum feeding, indicating that exercise’s benefits can be extended into the ‘real world’ setting. The beneficial effects of exercise on postprandial metabolism are also independent of its timing relative to meal ingestion. In line with evidence in the literature, an acute bout of aerobic exercise does not induce compensatory responses in terms of energy intake and increased appetite, supporting the role of exercise in weight management. Other than physiological factors, the behavioural and cognitive aspects related to feeding can play a role in mediating compensatory responses to exercise and this requires further investigation.

Taekwondo has evolved from a traditional martial art into a modern-day Olympic combat sport. Despite this transition, knowledge of the physiological demands of this combat sport is in its infancy. This thesis investigates the physiological demands of competitive Taekwondo using experienced male international Taekwondo competitors. Physiological measures and activity profile information were initially collected in championship Taekwondo competition to determine the fundamental physiological demands of this combat sport. The activity profile of championship Taekwondo combat elicited near-maximal heart rate (HR) responses and high blood lactate concentrations. The activity levels and physiological responses (e.g. HR and blood lactate) increased significantly between round 1 and 3 of combat. These data collectively suggest that the activity pattern of Taekwondo combat imposes high aerobic and anaerobic demands on the competitors, and these energetic requirements are increased as the rounds progress. The activity profile in championship combat was also modulated by a competitor’s weight division. Most notably, the data highlighted a predominance of fighting activity for heavy weights, and longer preparatory actions and less frequent fighting exchanges for feather weights. A Taekwondo competition simulation was devised and implemented to examine the physiological and hormonal responses to Taekwondo combat in simulated and championship settings. The championship Taekwondo combats elevated the physiological (e.g. HR, plasma lactate, glucose and glycerol) and hormonal responses (e.g. plasma adrenaline and noradrenaline) in comparison to simulated combats performed in a controlled setting. These divergent responses were evident even though both combat settings exhibited comparable activity profiles. This suggests that the contrasting physiological and hormonal responses were mediated by the stress responses to fighting in championship events. The physiological and hormonal responses to performing successive Taekwondo combats were examined during a simulated championship event. Performing four combats in an ecologically valid competition time-structure modulated the physiological and hormonal responses to combat and perturbed homeostasis between the combats. Most notably, the successive combats resulted in reduced plasma noradrenaline and lactate responses to combat and increased HR responses earlier in combat. These responses may reflect a change in the activity of the competitors’ and/or altered metabolic function in favour of an increased reliance on aerobic metabolism and diminished anaerobic energy yield as the combats are repeated. Importantly, the HR and plasma concentrations II of glycerol, NEFA and lactate remained elevated above baseline levels between a number of the repeated combats. This suggests that the recovery processes were often incomplete between the combats. The collective findings of these investigations demonstrate that Taekwondo is an intermittent combat sport that elicits high demands upon both aerobic and anaerobic metabolism. The physiological requirements of Taekwondo combat may be regulated by a multitude of competition factors including a competitor’s weight division, the round of combat and performing successive combats with different recovery intervals. Taekwondo combat also activates the sympathetic-adrenal-medulla promoting the release of stress hormones (catecholamines) into the circulation. The stress-hormonal responses are mediated by the specific combat environment and the requirement to perform repeated combats within a single day. These original findings may serve as a valuable ergonomic framework to prepare competitors’ for the specific requirements of Taekwondo competition.

The primary objective of this series of experiments was to explore some of the reasons which lead to equivocal outcomes in the literature on dietary recommendations and to demonstrate the need for evidence-based data on well-trained and elite athletes. Therefore, the previous mentioned groups of athletes were recruited to participate in four research studies, each entailing a series of experimental trials. The data obtained were compared to previous research and/or to the established dietary recommendations. The aim of the first research study presented in this thesis (Chapter 2) was to examine the effects of Glycine-arginine-α-ketoisocaproic acid (GAKIC) supplementation on fatigue during high intensity, repeated cycle sprints in trained cyclists. It should be noted that despite the fact that studies on GAKIC supplementation involving well-trained subjects are lacking, athletes regularly use this commercially available supplement. This is is also the case for a vast range of other supplements whose suitability has been assessed in normal healthy subjects or recreationally active individuals. In the study presented in Chapter 2, 10 well-trained male cyclists completed two supra-maximal sprint tests each involving 10 sprints of 10 s separated by 50 s rest intervals on an electrically braked cycle ergometer. Participants ingested 11.2 g of GAKIC (according to protocols previously established in the literature) or Placebo (Pl) during a period of 45 min prior to the experimental trials. Peak power, mean power, fatigue index as well as heart rate (HR) and ratings of perceived exertion did not differ between conditions (GAKIC vs. Pl). Peak power declined from the 1st sprint (mean  SD) (Pl: 1332  307 W, GAKIC: 1367  342 W) to the 10th sprint (Pl: 1091  229 W, GAKIC: 1061  272 W) and did not differ between conditions (P = 0.88). Mean power declined from the 1st sprint (Pl: 892  151 W, GAKIC: 892  153 W) to the 10th sprint (Pl: 766  120 W, GAKIC: 752  138 W) and did not differ between conditions (P = 0.96). Fatigue index remained at ~38% throughout the series of sprints and did not differ between conditions (P = 0.99). HR and ratings of perceived exertion increased from the 1st sprint to the 10th sprint and did not differ between conditions (P = 0.11 and P = 0.83, respectively). The data reported, suggest that GAKIC has no ergogenic effect on repeated bouts of high intensity exercise in trained individuals. The reported data further contradicts previous performance studies where GAKIC was found to attenuate the decline in power output, improve muscle performance and delay muscle fatigue resulting in the improvement of total work during high intensity exercise. Notably, none of the previous studies involving GAKIC supplementation seem to control for a number of possible confounding factors that could have adversely affected the results. For instance, utilization of untrained individuals and failure to include baseline trials to establish the repeatability of performance trials leaves the reliability of the data open to question. Furthermore, the results in Chapter 2 highlight the importance of extrapolating decisions concerning the effectiveness of a marketed nutritional supplement from the best available research conducted on well-trained subjects. The aim of Chapter 3 was to assess the food and macronutrient intake of elite Ethiopian distance runners; a group of athletes that dominates endurance running. The results of the research allowed further examination and direct comparison of the nutrient intake to the established guidelines and previous studies conducted on African runners. The dietary intake of 10 highly-trained Ethiopian long distance runners, living and training at high altitude was assessed during a 7-day period of intense training prior to competition using the standard weighed intake method. Training was also assessed using an activity/training diary. Body mass (BM) was stable (i.e., was well maintained) over the assessment period (pre: 56.7 ± 4.3 kg vs. post: 56.6 ± 4.2 kg, P = 0.54). The diet comprised of 13375 ± 1378 kJ and was high in carbohydrate (64.3 ± 2.6% of total energy intake (TEI), 545 ± 49 g, 9.7 ± 0.9 g.kg-1). Fat and protein intake was 23.3 ± 2.1% TEI (83 ± 14 g) and 12.4 ± 0.6% TEI (99 ± 13 g, 1.8 ± 0.2 g.kg-1), respectively. Fluid intake comprised mainly of water (1751 ± 583 mL.day-1), while no fluids were consumed before or during training with only modest amounts being consumed following training. It was concluded that, as found in previous studies on elite Kenyan distance runners, the diet of these elite Ethiopian distance runners met most recommendations for endurance athletes in regard to macronutrient intake but not in regard to fluid intake. Nevertheless, it remains unclear in what way these differences in fluid consumption, before major competitions, have an impact on their performance. Therefore, Chapter 3 highlights the fact that more studies involving truly world-class athletes are required in order to assess and possibly improve the applicability of current recommendations to elite athletes. Chapter 3 also highlights the potential role of the commercial industry in the application of the science of exercise nutrition. Chapter 4 aimed to describe the drinking behaviours of elite male marathon runners during major city marathons. Retrospective video analysis of 10 male marathon runners during 13 major city marathons was undertaken. Total drinking durations during the marathons were determined by estimating the time spent ingesting fluid at each drinking station from video images. The ambient conditions during the 13 studied marathon races were 15.3 ± 8.6 oC (ambient temperature) and 59 ± 17% relative humidity; average marathon competition time was 02:06:31 ± 00:01:08 (h:min:s). Total drinking duration during these races was 25.5 ± 15.0 s (range: 1.6 - 50.7 s) equating to an extrapolated fluid intake rate of 0.55 ± 0.34 L.h-1 (range: 0.03 - 1.09 L.h-1). No significant correlations were found between total drink duration, fluid intake (rate and total), running speed and ambient temperature. Estimated BM loss based on calculated sweat rates and rates of fluid ingestion was 8.8 ± 2.1% (range: 6.6 - 11.7%). Measurements of the winner in the 2009 Dubai marathon revealed a BM loss of -9.8%. It was concluded that the most successful runners during major city marathons, drink fluids ad libitum (i.e., at one's pleasure) for less than ~60 s at an extrapolated fluid ingestion rate and is in accordance with the current recommendations by the American College of Sports Medicine of 0.4 - 0.8 L.h-1. Nevertheless, these elite runners do not seem to maintain their BM within the current recommended ranges of 2 – 3%. On the other hand, this apparently widely adopted ad libitum strategy during marathon racing seems to produce optimal/winning performances. This evidence and the finding that the runner, who set the previous world record (2008), finished a competitive race (Dubai, 2009) with a BM loss of 9.8%, suggest that a tolerable range for dehydration may exist. It is possible, that this tolerable limit of dehydration may not have a negative impact on running performance in elite runners and may even confer an advantage by preventing a significant increase in BM due to “over - consumption” of large volumes of fluid. Given the data extrapolated from “real world” studies (Chapters 3 and 4) and the established guidelines for fluid ingestion, the investigation in Chapter 5 aimed to examine a possible method that could bring together the established guidelines and the data extracted from “real world” studies. Therefore, the effects of a hyper-hydration method combining creatine (Cr) and glycerol (Gly) supplementation on thermoregulatory responses and running economy (RE) in hot and cool conditions were investigated. Cr•H2O (11.4 g), Gly (1 g•kg-1 BM) and glucose polymer (75 g) were administered twice daily to 15 male endurance runners during a 7-day period. Exercise trials were conducted pre- and post-supplementation at 10 and 35 °C and 70% relative humidity. Combined Cr and Gly supplementation increased BM and total body water by 0.90 kg and 0.71 L, respectively following supplementation. Despite the significant increase in BM, supplementation had no effect on oxygen uptake (V̇O2) and thus RE. Both HR and core temperature were attenuated significantly after supplementation. Combining Cr and Gly is effective in reducing thermal and cardiovascular strain during exercise in the heat without negatively impacting RE. The potential influence of the commercial industry on scientific objectivity, as well as the lack of properly evaluated, controlled and randomized studies are the two main weaknesses that prevent the establishment of well accepted guidelines for food and fluid intake of well-trained and elite athletes. The development of novel guidelines needs to be solely evidence-based. Therefore, in order to reach conclusions regarding specific categories of athletes, research should be conducted on homogeneous groups (i.e., either well-trained, or elite, or world-class). Furthermore, research must be conducted under environmental and other conditions that are equivalent to those met during ‘‘outdoor’’ exercise, in order to evaluate and even improve the prevailing recommendations.

Pulmonary arterial hypertension (PAH) is a devastating and progressive vasculopathy of the pulmonary arteries for which there is no cure. There is an urgent need for more effective therapies. PAH is characterised by elevated pulmonary arterial pressures and obstructive vascular lesions in the distal vasculature by excessive cellular proliferation. As a result, the right ventricle is placed under excessive strain resulting in adaptive hypertrophy which progresses to maladaptive hypertrophy and failure. PAH is more common in women than in men suggesting that estrogens may be integral to disease pathogenesis. Understanding the biological basis for this sex difference would offer a new treatment paradigm in this devastating cardiovascular disease. Here, we challenged the concept that the estrogen metabolic axis is dysregulated in PAH New insights have revealed a potential contribution of the estrogen metabolizing enzyme, cytochrome P450 1B1 (CYP1B1) in the development of PAH. 17β-estradiol (17β-E2) and estrone (E1) are metabolized by the activity of CYP1B1 to the 2-, 4- and 16-hydroxylated estrogens. Here, we defined the role of CYP1B1 in the pathogenesis of PAH. CYP1B1 expression was increased in both experimental (hypoxia and SU5416+hypoxia) and in heritable and idiopathic PAH (HPAH and IPAH, respectively). Both male and female CYP1B1 knockout mice (CYP1B1-/-) were challenged with chronic hypoxia to induce PAH as assessed by right ventricular systolic pressures (RVSP), right ventricular hypertrophy (RVH) and pulmonary vascular remodeling. CYP1B1-/- mice were protected against hypoxia-induced pulmonary hypertension (PH). CYP1B1 inhibition with the highly potent and selective inhibitor 2,3',4,5'-tetramethoxystilbene (TMS; 3 mg/kg/day by intra-peritoneal injection) attenuated the development of hypoxia-induced PH. Only moderate effects were observed with CYP1B1 inhibition in monocrotaline-induced PH, despite improving survival rates. Female mice that over-express the human serotonin transporter gene (SERT+ mice) develop a spontaneous PAH phenotype at 5 months of age which is dependent on circulating levels of 17β-E2. Here, we provide evidence that the estrogen metabolic axis is dysregulated in these mice and this may underlie their PAH phenotype. The estrogen synthesizing enzyme aromatase and CYP1B1 was increased in whole lung homogenates of female SERT+ mice compared to wild-type mice. Despite increased expression of aromatase, 17β-E2 concentrations were unchanged. CYP1B1 inhibition with TMS (1.5mg/kg/day by intra-peritoneal injection) attenuated the PAH phenotype in female SERT+ mice as assessed by RVSP and pulmonary vascular remodeling Other studies have identified that the 16-hydroxylated metabolites of estrogens (17β-E2 and E1) are the only CYP1B1 metabolites to induce cellular proliferation, with the most profound effects observed with 16α-hydroxyestrone (16α-OHE1). In mice exposed to chronic hypoxia, urinary concentrations of 16α-OHE1 were increased. Chronic dosing of 16α-OHE1 in mice (1.5mg/kg/day by intra-peritoneal injection for 28 days) resulted in the development of a PAH phenotype in female mice only. 16α-OHE1 induced cellular proliferation in human pulmonary arterial smooth muscle cells (hPASMCs) and this was inhibited by a scavenger of reactive oxygen species (ROS) and an inhibitor of extracellular regulated kinase 1/2 (ERK 1/2). 4-hydroxylation is the predominant metabolic pathway activated by CYP1B1 activity and we therefore investigated the effects of the 4-hydroxylated metabolite of 17β-E2 in vivo. 4-hydroxyestradiol (4-OHE2) had no effects on PAH parameters in mice (1.5mg/kg/day by intra-peritoneal injection for 28 days). However, serotonin-induced vasoconstriction of the intra-pulmonary arteries was dramatically reduced in arteries harvested from mice dosed with 4-OHE2. More recent studies have identified that 4-hydroxyestrone (4-OHE1) is the predominant CYP1B1 metabolite in the lungs of mice. Interestingly, despite evidence for a pathogenic function of CYP1B1 activity in vivo, 4-OHE1 inhibited cellular proliferation in hPASMCs as assessed by thymidine incorporation whilst no effects were reported on cell viability. We provide evidence for an altered estrogen metabolic axis in PAH, by in part, overexpression of the putatively pathological CYP1B1. Yet, the dynamic estrogen metabolic profile in pulmonary vascular cells remains undetermined. To address this, we developed a high fidelity HPLC method to quantitatively fate map estrogen metabolism in hPASMCs to determine the dynamic regulation of estrogen metabolism in PAH. We provide the first direct evidence that hPASMCs metabolize 17β-E2 and that estrogen metabolism is pathologically altered in PAH. Our metabolic screen revealed a prominent role for 17β-hydroxysteroid dehydrogenase enzymes in hPASMCs by rapid formation of E1 in all groups studied, increasing with time, with the highest activity in male control hPASMCs and the lowest activity in female control hPASMCs. In female control hPASMCs there was no evidence of CYP activity, whilst numerous metabolites were formed in the other groups studied. The formation of the pathogenic 16α-hydroxylated estrogens was only evident in PASMCs from both male and female PAH patients at 24 and 48 hours. Globally, this study introduces a platform to elucidate effects of PAH insults and potential therapies on the estrogen-metabolic profile in pulmonary vascular cells. Overall, we provide eminent evidence that the estrogen metabolic axis is pathologically altered in PAH and is influenced by gender. This provides a strong rationale for the application of estrogen-sensitive therapies in the management of this highly female discriminating disease.

Acute coronary artery occlusion is the most common cause of sudden cardiac death. In some cases an acute myocardial infarction (MI) can immediately lead to lethal arrhythmias, but the factors that determine whether an MI precipitates arrhythmias are uncertain. In this thesis, I compare and contrast the detailed electrophysiology of hearts that develop arrhythmias post MI compared to those that do not using voltage sensitive fluorescent dyes in isolated rabbit hearts. In an attempt to improve the information from voltage mapping studies, initial work involved attempts to use ratiometric imaging of the fluorescence from the dye RH237. These identified optimal filter settings to collect voltage data at two distinct wavebands that would eliminate movement artefact and permit absolute voltage measurements. But routine implementation of this technique was prevented by additional technical issues related to uneven illumination levels and alignment of the two cameras. In initial studies the drug E-4031, a selective blocker of the delayed rectifier potassium current (IKr), was used to assess the contribution of this channel to repolarisation in rabbit ventricle, both in the steady state at a range of physiological and sub-physiological frequencies and in the transition between step frequency changes. The data suggests that IKr has a small but significant contribution to repolarisation at normal heart rates, 300ms pacing cycle length; a close to maximal concentration of E-4031 (0.03M) increased action potential duration (APD90) by 8.5 ± 1.7ms (P<0.01). This contribution is considerably larger at lower stimulation frequencies; at 1Hz E-4031 increased APD by 73.7 ± 13.7ms (P<0.05). The EC50 for E-4031 in this study was 0.01M which is similar to that reported in the literature. The recovery of the channel from inactivation appeared an important determinant of the rate of adaptation of the action potential duration. In the main experimental section, a novel snare technique was used to produce the acute coronary artery occlusion in the apical region of the left ventricle (LV) free wall. From control experiments (n=21), 47.6% of the hearts develop ventricular fibrillation (VF) within 30 minutes of coronary artery occlusion. On average, hearts with intrinsically longer epicardial action potential duration prior to ischaemia (mean APD50 168.8 ± 5.5ms) did not develop VF, and those with shorter APD (mean APD50 141.5 ± 3.5ms) during pre-occlusion period were more prone to VF (P<0.001). However, artificially prolonging the APD with the drug E-4031 (0.03 concentration) prior to coronary artery occlusion did not significantly change the incidence of arrhythmia. Brief and transient exposure to isoprenaline (0.3Mconcentration) before the occlusion shortened the average APD prior to occlusion but still did not increase the likelihood of VF. Therefore, I concluded that shorter epicardial APD values prior to ischaemia are associated with a higher incidence of arrhythmia but are not the cause. To investigate this further, a panoramic optical mapping technique was used to look at the electrophysiological properties across the entire ventricular surface of the hearts. The panoramic optical mapping study confirmed the correlation between shorter APD pre-occlusion and the incidence of VF during occlusion and indicated that the region of the LV exhibiting a shorter APD is confined to the apical half of the LV, and does not include basal LV or RV electrophysiology. Panoramic imaging also revealed a delayed activation time predominantly in the basal aspects of the LV. Both of these events – shorter APD in the apex and longer activation time in the base – were a feature of hearts that developed VF on ligation of the coronary artery. Future work will investigate the cellular/molecular basis for these differences in ventricular electrophysiology.

Respiration is a complex rhythmic motor behaviour that metabolically supports all physiological processes in the body and is continuous throughout the life of mammals. A failure to generate a respiratory rhythm can be fatal. Understanding how the respiratory rhythm is generated by the brainstem presents a substantial challenge within the field of respiratory neurobiology. Studies utilising in vitro and in vivo rodent models have provided compelling evidence that a small bilateral region of the ventrolateral medulla, known as the preBötzinger complex (preBötC), is the site for respiratory rhythmogenesis. There is also evidence to suggest a second distinct neuronal group, the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG), plays a specialised role in respiratory rhythm generation in the neonatal rodent. During early life in rodents and humans, the respiratory system is immature and an irregular breathing pattern is generated, making this period of life potentially vulnerable to external perturbations. However a step in maturity occurs early in life after which breathing becomes regular. Currently, the underlying mechanisms involved in respiratory rhythm generation during early life are not fully understood. It is hypothesised that the RTN/pFRG functions as the dominant respiratory rhythm generating oscillator during early life when the respiratory system is immature, after which the preBötC becomes the dominant rhythm generator. However, how the preBötC and the RTN/pFRG interact in vivo to produce rhythmic breathing during postnatal development remains elusive. The first aim of this thesis was to assess postnatal maturation of breathing patterns in the mouse using non-invasive whole body plethysmography. Between postnatal day (P) 2 and P3, a critical maturation step occurred, whereby breathing transitioned from an unstable and dysrhythmic pattern to a regular and robust pattern. The second aim of the thesis was to investigate the influence of this postnatal maturation on central respiratory control. Mu (μ) opioid receptor agonists are known respiratory depressants. The activity of the preBötC is depressed by μ opioids in vitro. Furthermore, fentanyl, a potent μ opioid receptor agonist, evokes respiratory frequency depression in vivo by exclusively targeting and depressing preBötC neurons. Conversely, the RTN/pFRG is insensitive to μ opioids. Accordingly, fentanyl was utilised as a pharmacological tool to selectively perturb the preBötC in vivo throughout postnatal development and through to early adulthood. The acute respiratory depressive effects of fentanyl were measured in order to investigate the level of involvement of the preBötC in respiratory rhythm generation throughout this critical developmental time period. Based on the general hypothesis that the preBötC functions as the dominant respiratory rhythm generator when the respiratory system has matured, it was hypothesised that mice would be more susceptible to the respiratory depressive effects of fentanyl after the maturation step has occurred i.e. the respiratory sensitivity to fentanyl would be age-dependent. Initially, mice were repeatedly exposed to fentanyl throughout postnatal development. However, fentanyl failed to induce a respiratory depression at all postnatal ages, suggesting repeated exposure had induced a rapid desensitisation to fentanyl’s respiratory effects. The study design was consequently altered to allow the hypothesis to be sufficiently tested, whereby different mice were studied on each postnatal day i.e. each mouse was only exposed to fentanyl once. This study revealed a trend towards an age-dependent increase in respiratory sensitivity to fentanyl, where mice displayed a heightened respiratory frequency depression in response to fentanyl after the maturation step had occurred from P3 onwards. This data therefore lends support to the hypothesis that the preBötC functions as the dominant respiratory rhythm generator post-maturation. In the clinical setting fentanyl is widely utilised for treating chronic and acute pain. However, despite the potent respiratory depressive actions of fentanyl, the long-term respiratory consequences of repeated exposure remain unexplored both clinically and pre-clinically. Owing to the immaturity of the respiratory system and the corresponding fragile nature of breathing patterns during neonatal life in mammals, a further aim of the thesis was to determine the long-term effects of fentanyl exposure during this vulnerable respiratory time period in the mouse. To establish if the postnatal age of fentanyl-exposure influences long-term respiratory effects, fentanyl exposure during juvenile life, which is regarded as being post-respiratory maturation, was also assessed. Neonatal mice were exposed to fentanyl (0.04 mg/kg daily) from P1-P5 and juvenile mice were exposed from P9-P13. When mice reached adulthood, baseline respiratory activity and the respiratory response to a subsequent fentanyl challenge were assessed during wakefulness and under anaesthesia. When awake, neonatal-exposed mice exhibited a reduced baseline respiratory frequency and an attenuated respiratory sensitivity to fentanyl. Under anaesthesia, neonatal-exposed mice displayed a depressed baseline minute ventilation and a high frequency of spontaneous augmented breaths. In direct contrast to the wakeful state, when anaesthetised, neonatal-exposed mice exhibited a striking hypersensitivity to the acute respiratory depressive actions of fentanyl. In all neonatal-exposed mice, fentanyl evoked a respiratory failure. In juvenile-exposed mice, baseline respiratory activity remained unaltered in the wakeful state and fentanyl also failed to induce a respiratory depression. When anaesthetised, baseline minute ventilation remained unchanged and the high occurrence of augmented breaths exhibited by the neonatal-exposed mice was not observed. Unlike the wakeful state, fentanyl evoked a depression of respiratory activity in the juvenile-exposed mice when anaesthetised, however the augmented sensitivity to fentanyl and consequential respiratory arrest displayed by the neonatal-exposed was not observed. This data indicates that the anaesthetised state is more susceptible to respiratory depression. Furthermore, the data suggests that neonatal life represents a time period that is particularly vulnerable to the respiratory effects of opioid depression. The final aim of the thesis was to determine the long-term effects of neonatal fentanyl exposure on neurokinin-1 (NK1R) and μ opioid receptor expression within the ventral respiratory column (VRC), a region of the ventrolateral medulla comprising the preBötC. Neonatal-exposed mice exhibited significantly less NK1R and μ opioid receptor expressing cells in the region of the preBötC. This data suggests that repeated fentanyl exposure in neonatal life induces a long-term downregulation of these receptors. In conclusion, fentanyl’s acute respiratory effects were age-dependent, which lends supports to the hypothesis that the preBötC functions as the dominant rhythm generator post-maturation. Furthermore, this thesis highlights the vulnerabilities of neonatal life to the lasting effects of opioid respiratory depression, whilst also providing invaluable insight into state-dependent respiratory modulation and depression.

The complex interplay between regulatory factors of kidney mineral ion homeostasis are difficult to investigate in vivo. In addition, preclinical drug safety testing is limited by lack of translational tools for screening novel drugs for nephrotoxicity. At Cardiff University, my PhD project aimed to characterise tools to address these needs. At AstraZeneca, the use of tissue biomarkers as tools for assessing kidney injury in retrospective studies where urine biomarker samples are not available, was evaluated. To enable studies of mineral ion homeostasis, an in vitro human primary renal cell model was characterised. Cells expressed phenotypic marker proteins (E-, N-Cadherin) as well as proteins involved in mineral ion homeostasis (FGFR1-4, Klotho, NaPi IIa, NCX1, PMCA1). They also exhibited morphological features and functional characteristics of renal cells in vivo. Data were presented as an oral abstract communication at the American Society of Nephology Meeting, 2013. To enable studies of drug-induced nephrotoxicity, in vitro primary mouse and human renal cell models were characterised. A concentration-dependent increase in apoptosis was observed in mouse cells following 24-hour treatment with medium containing cisplatin, cyclosporin A and BEA. Following 24-hour treatment, the same nephrotoxins increased expression of the kidney injury biomarkers kidney injury molecule-1 (KIM-1) and osteopontin. Human cells responded to 24-hour treatment with medium containing cisplatin with expression of KIM-1, osteopontin and clusterin. Parallel use of such rodent and human models would revolutionise preclinical translational drug safety assessment. Cisplatin-treated rats were used to evaluate the use of tissue biomarkers as tools in studies where urine biomarker samples are not available, such as retrospective studies in drug safety testing. Of the biomarkers investigated, KIM-1 and osteopontin showed greatest correlation with their corresponding urinary biomarkers. As such, they offer greatest utility as tissue biomarkers in retrospective studies. Data were published in Toxicologic Pathology (Wadey et al. 2013).

The gut-derived bacteria, endotoxin (lipopolysaccharide), have been observed to be raised in patients with type 2 diabetes mellitus (T2DM) and cardiovascular diseases (CVD) which appears to represent a source of diet induced inflammation exacerbating metabolic disease. To further the current studies on endotoxin induced inflammation investigations examined a cohort of adult Saudi Arabian women with obesity/weight gain and or T2DM to ascertain (1) the impact of a post-prandial high SFA rich meal on systemic inflammation; (2) the direct effect of a 3 month diet intervention on cardiometabolic health (3) and assess how subtle changes in dietary interventions can impact on metabolic risk in different patient groups and what dietary components appear important. A total of 92 Saudi adult women with varying metabolic states [18 nondiabetic (ND) control subjects (Age 24.4±7.9 years; BMI 22.2±2.2 Kg/m2), 24 overweight-plus obese (overweight+) subjects (Age 32.0±7.8 years; BMI 28.5±1.5 Kg/m2) and 50 T2DM patients (Age 41.5±6.2 years; BMI 35.2±7.7 Kg/m2)] were recruited for this 3-month intervention study. Anthropometric data and fasting blood samples were taken at pre- and 3 months post-intervention with glucose, insulin, HOMA-IR, lipid profile and endotoxin measured. To establish whether a high-fat meal alters circulating endotoxin in different metabolic disease states, all subjects were given a high fat standardized meal (75g fat, 5g carbohydrate, 6g protein) after an overnight fast of 12–14 h. Blood samples were drawn via cannula at baseline (0 hour) and post-prandially (1, 2, 3, and 4 hours). For the dietary intervention, participants were prescribed a 500Kcal deficit energy diet less than their daily recommended dietary allowances. Targeted macronutrient composition was 20%-30% fat, <10% of saturated fatty acids, 50%-60% carbohydrates, 15%-20% protein and at least 15g of fibre per 1000 kcal. At baseline and with the exception of HDL-cholesterol, all anthropometric, glycemic parameters, lipid profile and endotoxin were significantly higher in the T2DM group. For the high fat challenge, the most notable changes were the postprandial increases in the triglycerides, insulin, HOMA-IR and endotoxin levels, and subsequent significant decrease in HDL-cholesterol in all groups (p<0.05). These same patterns of changes were observed after 3 months in the overweight+ and T2DM group. Endotoxin was found to be significantly and positively associated with total and LDL-cholesterol (p<0.05), modestly with triglycerides and inversely with HDL-cholesterol (p=NS). For the dietary intervention, significant improvements were noted in all anthropometric measures in the T2DM group and BMI in the overweight+ group (p<0.01). The noted weight loss was secondary to the significant decrease in carbohydrates, fats and total caloric intakes (p<0.05) which translated to a better cardiometabolic health in both groups noted clearly through lipid profile changes. Endotoxin was found to be inversely associated with fiber intake (p<0.05). Fiber intake was found to be positively associated with HDL-cholesterol (p<0.05), which appeared to be an important dietary component to be associated with health improvements. This current thesis expanded our knowledge and understanding on how a high fat oral challenge exacerbates cardiometabolic and inflammatory conditions (including endotoxemia) in Saudi Arabian women with different metabolic states, and how a 3-month caloric restriction may induce weight loss that leads to improved cardiometabolic health. Observations from the present thesis highlight strategies that may potentially be of clinical use in future dietary intervention studies in patients with T2DM and obesity in the Middle Eastern region.

Communication between neuronal and glial cells is important for many brain functions. Astrocytes can modulate synaptic strength by releasing gliotransmitters. The mechanisms underlying release of gliotransmitters remain uncertain with exocytosis being the most intriguing and debated pathway. Furthermore, the contribution of gliotransmitter release to synaptic transmission and plasticity is also debated. Therefore, the purpose of this project was to investigate whether astrocytes can release gliotransmitters via vesicular exocytosis and to investigate the role of this release in synaptic transmission and plasticity in the somatosensory cortex. To investigate this, the transgenic mice line called dnSNARE, where vesicular exocytosis of gliotransmitters is impaired specifically in astrocytes, were used in combination with electrophysiological and pharmacological approaches. It was shown that cortical astrocytes can release ATP via calcium-dependent vesicular exocytosis. The released astrocytic ATP can bind to neuronal P2X receptors. This results in inhibition of GABAergic transmission. This in turn leads to increase in NMDAR activity during synaptic transmission and plasticity. It was shown that NMDAR-dependent LTP is modulated via vesicular exocytosis of ATP from astrocytes. It was also shown that astrocytes can release glutamate and D-serine via vesicular exocytosis. However, release of D-serine was not required for LTP induction in the somatosensory cortex. Release of glutamate from astrocytes activated GluN2B-containing NMDARs on cortical neurones. These GluN2B-containing NMDARs contributed to LTP induction in the cortex. The role of endocannabinoid signalling in synaptic transmission and plasticity was also investigated. It was shown that application of anandamide could result in calcium elevation in astrocytes by acting on CB1 receptors on these cells. This lead to vesicular exocytosis of ATP from astrocytes onto P2X receptors on neurones. This vesicular release of ATP from astrocytes contributed to LTP induction in the somatosensory cortex.

Approximately one third of genes in the human genome (1) encode transmembrane (TM) proteins and form more than half of all drug targets (2). However, our understanding of how these proteins fold into their functional form, as well as how they may misfold into a disease-associated form, remains a difficult area of study. By observing the effects of single point mutations in the context of a native sequence, in addition to adding and mutating interhelical interaction motifs on a low complexity sequence background, we aim to elicit ‘rules’ of TM protein domain association. For the single point mutation in the context of a native sequence, the TM domain of the sequence Neu, along with its oncogenic substitution V664E form Neu*, were selected. Using molecular dynamics (MD) a united atom model of each dimer in a model bilayer system was subjected to umbrella sampling along an interhelical reaction coordinate to yield a free energy profile of self-association. The lipid order, bilayer thickness, and peptide tilt angle were calculated from trajectories taken from three points along the reaction coordinate. Helical composition, solvent accessible surface area, and hydrogen bond analysis (for the V664E substitution) were performed at the free energy minimum. Low complexity sequences of polyleucine and polyleucine-alanine heptad repeat sequences, with and without interaction motifs similar to those present in the Neu model, were ligated into PBLM100 plasmids. Transformed E. coli cells were subjected to semi-quantitative homo-interaction analysis using the GALLEX assay. The same TM sequences were modelled using a coarse grained (CG) forcefield. Umbrella sampling along an interhelical reaction coordinate was performed to yield a free energy profile of self-association. Single-linkage cluster analysis of peptides was performed at the global free energy minimum. A representative structure from each set was compared to an averaged structure from the clusters of an atomistic conformational search. The results presented in this study, could contribute to what in theory would be a large database of motif-driven rules for TM helical domain oligomerisation. This may encourage further investigation into TM protein design for novel application.