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Enduro mountain bike racing (enduro) consists of timed downhill race stages linked by non-competitive transition stages and general classification is determined by accumulated race stage time. Limited research is available on the physiological requirements of enduro despite a large population of professional elite riders. For this thesis, nine elite enduro athletes (n=8 male, n=1 female; top 100 world ranking) were recruited. Measures of daily training load (TL) and resting heart rate variability (HRV) were collected between three laboratory based tests throughout a season of training and racing. The demands of an international race event were assessed by heart rate, terrain induced accelerations and vibration exposure. Leukocyte subset (Neutrophils, CD4+ T-cell, CD8+ T-cell, and NK cell) redistribution, cortisol and IL-6 concentration were assessed at each laboratory test (pre, post, 1h-post) and the race event (pre, 1h-post, 19h-post). Main findings were that successful performance in enduro requires a large aerobic capacity (VO₂peak = 61.1 ± 5.2 ml.kg.min⁻¹, power VO₂peak = 410.9 ± 18.2W) coupled with adequate skill, technique and muscle mass to ensure high velocities can be sustained over differing types of terrain. Elevated TL appears to be a key component of training habit and the upper limit of training volume before negative adaptation was identified (>800 A.U. LuTRIMP). No relationship was found between TL and HRV. No significant changes were observed in leukocyte subset redistribution between laboratory tests. The race event induced significantly larger changes in circulating numbers of certain leukocyte subsets when compared to the laboratory test and the magnitude of redistribution of CD4⁺ senescent T-cells was partially explained by vibration exposure (ΔR² = -0.673, F₍₃,₁₎ = 12.12, p = 0.04). A subsequent novel assessment of vibration loading in mountain biking revealed potentially damaging levels of vibration exposure that could be associated with long term health implications in enduro athletes (Ai(8) range 5.47 to 6.61ms⁻²). It was concluded that vibration exposure needs to be considered in future models of physiological loading in this discipline.

Reproduction is the only certain constant within animals and maintaining the integrity of this process is key to ensuring survival of the species. However, male fertility rates in the Western world are in decline and the use of assisted reproductive techniques rising (Makarow and Hojgaard, 2010). With emerging technologies such as nanotechnologies presenting both enormous benefits alongside new risks, there is a clear need to understand and manage these potential novel threats to male reproductive health. However, the animal burden associated with such research is extreme, therefore consideration of how to reduce, refine or replace animal use in the associated developmental and reproductive toxicity (DART) testing is paramount. This research aimed to assess the toxicity of engineered nanomaterials (ENM) in the male reproductive system and develop an improved testing strategy for hazard assessment of DART. Using a panel of highly representative ENM, assays to screen for toxicity in male testicular cell lines were established and optimised in vitro. Findings were then validated for reliability and reproducibility against those generated using similar test systems with cells from alternative organs of the body. Assessment of cell and endocrine function also provided a deeper understanding of cellular responses following acute sub-lethal ENM exposure. Comparison of outcomes in vitro to in vivo was enabled by appraisal of tissues from animals exposed orally to the same ENM. Through this, a new method by which to stage tubules for histopathological analysis was developed, and for the first time a truly thorough morphological and stereological examination of tissues for markers of effect provided. Assimilation of key findings made it possible to conclude that silver ENM are reproductive toxicants and potential endocrine disruptors. The accumulated results were also used to guide development of a novel Integrated Approach to Testing and Assessment (IATA) for male reproductive toxicity from ENM.

The circadian cycle, although seasonally adjusted, consists of two phases: a nocturnal period of darkness and a diurnal period of light. Many parameters of human physiology display either a nocturnal or diurnal peak as seen in athletic performance, immune, endocrine, physiology and cognitive function, respectively. The studies included in this thesis focused on the diurnal (morning or AM versus evening or PM) effect of high-intensity self-paced exercise on physiological and immunological measures and the influence of individual preference for activity or chronotype on these responses. Self-paced time-trials were completed in an environmental chamber (6°C) in the AM and PM and displayed a similar effect on physiological and biological parameters. Performance times were non-statistically quicker (P > 0.05) at PM. Physiological parameters heart rate, rate of perceived exertion, lung function and self-paced treadmill speed were not found to be statistically different at AM or PM trial (p > 0.05). Core body temperature (CBT), was significantly higher (p0.05). CD8+ T-lymphocyte cell-surface markers for naïve/senescence and differentiation (CD27CD45RA and CD27CD28) reported no diurnal difference between AM and PM exercise in experienced and experienced/recreational distance runners respectively (P > 0.05). However, a higher percentage of highly differentiated phenotypes were found in the recreationally active population. Neutrophils displayed a pronounced elevation in response to PM exercise with the mechanism driving this response unclear at this time. Cortisol concentration displayed less inflammatory responses in the morning compared to the evening with higher values pre, post and one-hour post AM trials. Chronotype showed no effect on physiology or biology at rest or in response to exercise. Chapter 4 presents data from a study that investigated diurnal physiology and immune response to high-intensity exercise in highly-trained men. Recreational and experienced endurance male runners at differing diurnal time-points were investigated in Chapter 5, while individual chronotype differences and circadian phase responses were explored in Chapter 6.In summary, it is concluded from this work that there was a lack of evidence showing a diurnal effect on running performance and subsequent immune response. Elevated circulating immune counts prior to exercise, irrespective of diurnal phase, appear to govern exercise-induced responses. The effect of high intensity exercise is subject to three distinct variables: the fitness status and experience of the individual completing the exercise, the time of day at which the exercise is undertaken, and the phase response of exercise at that point of the circadian cycle. No diurnal phase mediated a divergent effect on variables examined was observed at 09.00hrs and 17.00hrs. These time-points should be considered not sufficiently dissimilar to elucidate diurnal variation in trained and healthy males.

The nematode Caenorhabditis elegans is an alternative model used in biomedical research for the investigation of descriptive and mechanistic toxicity assessment of chemicals. There are considerable differences in published data, especially in terms of reproducibility and validation of toxicity endpoints, and the techniques used in the investigation of these endpoints. This thesis describes the evaluation of toxicological endpoints following the exposure of C. elegans to chemicals which include; zinc oxide nanoparticles (ZnONP), Diethylstilbestrol (DES) and derivatized target-specific anthelmintics. The results suggest that ZnONP prepared in anionic and cationic dispersants (AZNP and CZNP respectively) were the most toxic against the nematode resulting in the ‘bag of worms' (BOW) phenotype which can be exploited as a marker for reproductive toxicity. Also, worms treated with ZnONP prepared in 0.1% FBS (FZNP), molecular grade water (WZNP) or E. coli OP50 supernatant (SZNP) presented three-fold embryo elongation showing fully differentiated tissues encapsulated within the eggshell and still within the hermaphrodite gravid adult. The phenotype has been named accelerated embryonic development (AED) and could be used as a developmental toxicity endpoint. The results suggest that the AED endpoint is the most sensitive while lethality endpoint appears to be the least sensitive despite its extensive use in the literature. Also, microRNA microarray expression appears to be the most sensitive molecular endpoint while behavioural endpoints such as speed should be interpreted with caution, especially when performed manually. Importantly, good C. elegans culture practice (GCeCP) is required for reproducible chemical toxicity assessment and different endpoints may be required for different types of toxicity assessment. Additionally, the thesis describes a second but related study which explores a potential for enhanced anthelmintic targeting. Novel fluorophore-based asparagine-containing oligopeptide substrate probes were used to target the helminth protease, legumain. These probes were selectively cleaved by legumain in C. elegans, Haemonchus contortus and Teladorsagia circumcincta. The protease-specific probes could potentially be exploited to achieve protease-mediated prodrug activation and drug delivery.