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This study was conducted to test the silver spoon hypothesis that earlylife nutritional conditions impact development, performance and fitness of the birdsgreat tit (Parus major) nestlings. We investigated whether fatty acid affects immunityand metabolism during the altricial period by examining the expressions of geneTLR4 (immunity related) and COX 2 (metabolism related) against a dietarymanipulation on great tit nestlings in urban vs. rural environments. The resultssuggested that arachidonic acid had no significant effect on TLR4 expression, but atendency to induce immune response, regardless of urban or rural conditions. Thestrength of immune response was however negatively correlated with laying date. Theurban great tit nestlings had a higher COX 2 gene expression than rural ones, andarachidonic acid suppressed COX 2.Thus no strong support to the hypothesis was found for the studied great titpopulations. It showed, however, i) there is a tendency of increasing immune responsewith extra fatty acid in the diet, and ii) arachidonic acid suppress metabolism. Fattyacid involved in a multiple physiological processes and this complex need to beelaborated in future studies.

It is known that during fluid restriction, obligatory water losses continue and hypohydration develops and that restricted energy intake leads to a concomitant restriction of all other dietary components, as well as hypohydration, but the specific effects of periods of fluid and/ or energy restriction on fluid balance, electrolyte balance and exercise performance have not been systematically described in the scientific literature. There were two main aims of this thesis. Firstly, to describe the effects of periods of severe fluid and/ or energy restriction on fluid and electrolyte balance; secondly, to determine the effect of electrolyte supplementation during and after energy restriction on fluid and electrolyte balance as well as energy exercise performance. The severe restriction of fluid and/ or energy intake over a 24 h period all resulted in body mass loss (BML) and hypohydration, but whilst serum osmolality increases during fluid restriction (hypertonic hypohydration), serum osmolality does not change during energy restriction (isotonic hypohydration), despite similar reductions in plasma volume (Chapter 3). These differences in the tonicity of the hypohydration developed are most likely explainable by differences in electrolyte balance, with fluid restriction resulting in no change in electrolyte balance over 24 h (Chapter 3) and energy restriction (with or without fluid restriction) producing significant reductions in electrolyte balance by 24 h (Chapter 3; Chapter 4; Chapter 5; Chapter 6; Chapter 7). Twenty four hour combined fluid and energy restriction results in large negative balances of both sodium and potassium, and whilst the addition of sodium chloride to a rehydration solution ingested after fluid and energy restriction increases drink retention, the addition of potassium chloride to a rehydration solution does not (Chapter 4). Supplementation of sodium chloride and potassium chloride during periods of severe energy restriction reduces the BML observed during energy restriction and maintains plasma volume at pre-energy restriction levels (Chapter 5; Chapter 6; Chapter 7). iv These responses to electrolyte supplementation during energy restriction appear to be related to better maintenance of serum osmolality and electrolyte concentrations and a consequential reduction in urine output (Chapter 5; Chapter 6; Chapter 7). Additionally, 48 h energy restriction resulted in a reduction in exercise capacity in a hot environment and an increase in heart rate and core temperature during exercise, compared to a control trial providing adequate energy intake. Whilst electrolyte supplementation during the same 48 h period of energy restriction prevented these increases in heart rate and core temperature and exercise capacity was not different from the control trial Chapter 8). In conclusion, 24-48 h energy restriction results in large losses of sodium, potassium and chloride in urine and a large reduction in body mass and plasma volume and supplementation of these electrolytes during energy restriction reduces urine output, attenuates the reduction in body mass and maintains plasma volume and exercise capacity.

It is well established that improved nutritional strategies can enhance both health and exercise performance. Scientific developments in recent years have furthered our understanding of cellular metabolism, which in turn, has provided an additional platform to investigate the impact of diet on health and adaptation. The overall aim of this research programme was to build on the current understanding of dietary intake in athletes and the impact dietary manipulation has on cellular and metabolic adaptation at rest and in combination with endurance training. It is postulated that nutrition is the most controllable risk factor impacting long-term health and chronic disease (World-Health-Organization, 2003), and enhanced knowledge of nutrition has been associated with improved dietary choices. A number of nutrition knowledge questionnaires have been developed to assess this; however the validity of each tool is reduced if implemented outside the target population. A valid and reliable general and sport nutrition knowledge questionnaire had not yet been developed. Using a parallel groups repeated measures study design (N = 101) the aim of the first experimental Chapter (Chapter 4) was to develop a new tool to measure general and sport nutrition knowledge in UK track and field athletes. Following the questionnaire design 53 nutrition educated and 48 non-nutrition educated participants completed the questionnaire on two occasions separated by three weeks. The results of the process demonstrated face and construct validity from the development of the question pool, content validity (the nutrition educated group scored > 30% higher that the non-nutrition educated group), reliability (test - retest correlation of 0.98, p < 0.05) and internal consistency (Chronbach's alpha value > 0.7) as such establishing a new tool (Nutrition knowledge Questionnaire for Athletes (NKQA)) for the assessment of general and sport nutrition knowledge in track and field athletes. Athletes' diets are commonly reported as inadequate and previous work has demonstrated a weak positive relationship between diet quality and nutrition knowledge. Additionally a commercially available tool, the metabolic typing questionnaire, claims to identify individual metabolic function and subsequently prescribe a personalised diet to optimise health. Thus the aim of the second experimental Chapter (Chapter 5) was to quantify nutrition knowledge (using the questionnaire developed in Chapter 4), measure diet intake and quality and investigate the efficacy of the metabolic typing questionnaire in UK track and field athletes. Using a parallel groups repeated measures design participants (UK track and field athletes n = 59, and non-athletic control group n = 29) completed a food diary, the NKQA and the metabolic typing questionnaire at two time points through the year (October and April) to investigate seasonal change. The results of the metabolic typing questionnaire concluded that 94.3% of the participants were the same dietary type and would subsequently have been prescribed the same diet. Athletes possess greater general and sport nutrition knowledge the non-athletes (60.4 ± 2.0 % vs. 48.6 ± 1.5 %) and also had better diet quality (76.8 ± 10.5 % vs. 67.6 ± 2.6 %). However no relationship was observed between individual nutrition knowledge score and diet quality (r2 = 0.003, p = 0.63). No difference in dietary intake was observed between power and endurance athletes; average diet intake consisted of 57.0% carbohydrate, 17.1% protein and 25.9% fat. The metabolic typing diet is based around three different diets: high carbohydrate, high protein and mixed diet. The results from Chapter 5 identified that the metabolic typing questionnaire was not able to differentiate between metabolic function in healthy individuals. Additionally all athletes, independent of event (power vs. endurance), consumed similar diets. With such similarities a clearer understanding of the impact such diets have at a cellular level is required. Therefore for the remainder of the thesis it was decided to investigate the impact of dietary manipulation utilising more robust measures. Mitochondria are responsible for energy production; their quantity and density have been associated with improved health and endurance performance. External stressors such as energy reduction, carbohydrate restriction and exercise are potent stimulators of transcription markers of mitochondrial biogenesis. Thus manipulating carbohydrate and energy availability in vivo may enhance cellular adaptation and limited literature exists on the impact increased protein intake has on this. The aim of Chapter 6 was to investigate the impact of acute (7-day) continuous dietary manipulation on metabolic markers, body composition and resting metabolic rate (RMR). Using a repeated measures parallel group (N = 45) design, participants were randomly assigned one of four diets: high protein hypocaloric, high carbohydrate hypocaloric, high protein eucaloric or high carbohydrate eucaloric. The macronutrient ratio of the high protein diets was 40% protein, 30 % carbohydrate and 30% fat, the high carbohydrate diets were 10% protein, 60% carbohydrate and 30% fat. Energy intake in the hypocaloric diets was matched to resting metabolic rate (RMR). Participants consumed habitual diet for 7-days then baseline measures were collected (skeletal muscle biopsy, dual energy X-ray absorptiometry scan (DXA) and RMR, habitual diet was consumed for a further 7-days and repeat testing was completed (these time points were used as a control), the intervention diet was then consumed for 7-days and post measures were collected. The results of the skeletal muscle biopsy demonstrated no group x time interaction in any marker, however a pre-post time difference subsequent to the high protein hypocaloric diet (the diet which induced the greatest metabolic stress) was observed in four transcriptional markers of mitochondrial biogenesis (pre-post intervention fold increase: PCG1-α 1.27, AMPK 2.09, SIRT1 1.5, SIRT3 1.19, p < 0.05). The results of the DXA scan demonstrated that the high protein hypocaloric group lost significantly more fat mass than the high carbohydrate eucaloric group (-0.99 kg vs. -0.50 kg, p < 0.015). Irrespective of macronutrient ratio, no energy-matched between group difference was observed in lean mass (LM) loss. However when matched for macronutrient ratio the high protein diet attenuated LM loss to a greater extent that the high carbohydrate diet, suggesting an important role of increased protein intake in the maintenance of lean mass. No time point or group difference in RMR was observed. This data suggests that a high protein low carbohydrate hypocaloric diet may provide a stimulus to promote skeletal muscle metabolic adaptation. The aim of the final experimental Chapter (Chapter 7) in this thesis was to explore the impact exercise in combination with a high protein diet on metabolic adaptation, substrate utilisation and exercise performance in well trained runners. Using a parallel groups repeated measures study design the participants (well-trained endurance runners, N = 16) consumed normal habitual diet for 7-days, then 7-days intervention diet (high protein eucaloric or high carbohydrate eucaloric, same dietary ratios as Chapter 6) and finally returned to habitual diet for 7-days, training was consistent throughout. A pre exercise muscle biopsy was taken subsequent to each diet and immediately followed by a 10 km sub-maximal run and a time to exhaustion run (TTE) at 95% of velocity at maximal aerobic capacity (vV̇O2max). Post intervention the high protein group presented significant changes in sub-maximal substrate utilisation with 101% increase in fat oxidation (0.59 g·min-1, p = 0.0001). No changes were observed in substrate utilisation in the high carbohydrate group. A trend towards a reduction in average weekly running speed was observed in the PRO group (-0.9 km·h-1), the high carbohydrate group maintained the same training speed. TTE was decreased (-23.3%, p = 0.0003) in the high protein group subsequent to the intervention, no change was observed in subsequent to the high carbohydrate diet. / The high carbohydrate group demonstrated preferential increases in markers of metabolic adaptations (fold increase: AMPK = 1.44 and PPAR = 1.32, p < 0.05) suggesting that training intensity, rather than carbohydrate restriction, may be a more profound driver of metabolic adaptation. All performance measures, in both groups, returned to pre intervention levels once habitual diet was returned; however the increased gene expression observed in the high carbohydrate group remained elevated 7-days post intervention. The increased metabolic stress imposed by reducing carbohydrate intake did not increase transcriptional markers of mitochondrial biogenesis. For continuous endurance training and high intensity endurance performance a high carbohydrate diet is preferential to a high protein diet.