Maximising strength and neurological adaptations to resistance training has long been sought to improve athletic performance and enhance clinical rehabilitation functional outcomes. In recent years, transcranial magnetic stimulation (TMS) and peripheral nerve stimulation (PNS) have been applied to investigate changes in the central nervous system (CNS). Conventional resistance training programmes consist of shortening and lengthening muscle contractions and have been shown to have uniquely different motor control strategies; how this neurological control is modified during specific muscle contraction resistance training is unknown. Additionally, understanding the detraining process will assist in designing tapers for elite athletes and improve our knowledge of detraining and inactivity in other populations. The overall aim of the thesis was to determine the TMS and PNS responses to, and following, shortening and lengthening resistance exercise and subsequent detraining.

Cardiovascular disease is the primary cause of global mortality (Naghavi, 2015). Given the global health issues associated with poor cardiovascular function, interventions that help reduce the severity and prevalence of these diseases would not only have economic implications, but would also improve health, wellbeing and quality of life. Epidemiological studies have suggested that polyphenol-rich foods can exert positive cardiovascular health benefits and as a result could reduce the severity of the primary pathology and increase the capacity to stay physically and mentally active (Joshipura et al., 1999; Bazzano et al., 2002; Hung et al., 2004). One of most studied polyphenol-rich, functional foods in recent years, in both the clinical and exercise domains, has been tart cherries. Tart cherries and their derivatives are high in numerous polyphenols (Wang et al., 1999; Seeram et al., 2001; Seymour et al., 2014; Bell et al., 2014) that include the flavonoids isorhamnetin, kaempferol, quercetin, catechin, epicatechin, procyanidins, and anthocyanins (Kim et al., 2005; Kirakosyan et al., 2009). Indeed, there has been an enormous research effort over the past decade to delineate the physiological and biochemical effects that tart cherries (and its constituents) might afford, and how these effects could be exploited to improve health outcomes. There is now strong evidence that tart cherries attenuate inflammation (Wang et al., 1999), oxidative stress (Howatson et al., 2010; Bell et al., 2014) and accelerate exercise recovery (Howatson et al., 2010; Bowtell et al., 2011; Bell et al., 2014; 2016). Furthermore, cherry extracts have been shown, in cell and animal models, to exert a range of cardioprotective effects that include increasing nitric oxide production and antioxidant (AOX) status, reducing lipid oxidation and inhibiting inflammatory pathways (Wang et al., 1999; Seeram et al., 2001). However, data from human trials are not always consistent. Furthermore, it is yet to be explored whether MC concentrate can be used for performance enhancement. Thus, the overarching aim of this thesis was to elucidate the effects of Montmorency, a specific cultivar of tart cherry, (MC) supplementation on vascular function and exercise performance in humans. The series of investigations that set out to address this aim have led to many novel and interesting findings. To begin, study 1 was the first to show that protocatechuic and vanillic acid were identified in the plasma post MC consumption. Furthermore, a combination of PCA and VA increased cell migration, but had no effect on the proliferation of vascular smooth muscle cells. Secondly, and perhaps the most novel finding of this thesis was that MC supplementation showed promise as an effective adjuvant in the management of hypertension. This was a consistent finding throughout the thesis; in all instances MC supplementation was able to significantly reduce systolic blood pressure. Another important finding was that MC supplementation resulted in an acute modulation of cerebral blood flow parameters in the front cortex during task performance with no changes in cognition or mood. Finally, the final study of this thesis demonstrated that MC supplementation can improve aspects of exercise performance with no changes in V̇O2 kinetics, NO2- concentrations or muscle oxygenation. All of these findings suggest that circulating phenolic metabolites derived from MC juice are at least partly responsible for these effects. Collectively, findings of this thesis provide novel information to literature surrounding the application of MC in health maintenance and exercise performance. In addition to identifying and quantifying some of the primary downstream metabolites of the principal anthocyanins contained in the concentrate, this research is the first to provide efficacy for the use of MC supplementation to acutely modulate various aspects of vascular function including systolic blood pressure, total and oxy-Hb during a cognitively challenging task. The underlying mechanisms that govern these effects remain elusive, although data from study 1, 2 and 4 would suggest that it is not likely to be attributed to NO, at least systemically. A more likely idea by which MC supplementation improves factors associated with CVD is based on the uptake of polyphenols that possess cardio-protective properties. To conclude, this thesis highlights the ability of MC to improve aspects of cardiovascular function and exercise performance. Circulating phenolic metabolites derived from MC are at least partly responsible for these acute improvements. Further work is required to; fully elucidate the mechanisms by which MC exerts its protective effects, determine whether the effects reported would be amplified using chronic supplementation and demonstrate effects of MC supplementation within habitual dietary practices.

Introduction: Tobacco smoking has been shown to have a detrimental impact on fracture healing and is often implicated in the non- and delayed-union of bone. Whilst numerous studies have concentrated on the demographic and clinical manifestations of fracture healing and smoking, very little analyses have been undertaken at the biochemical level. Aims: This research project will assess the impact of smoking on the cellular and molecular mechanisms of bone healing by analysing human tibial fracture haematomas using a variety of laboratory techniques. Materials and methods: Cell culture: Fracture haematomas (~2 mL) were collected from anaesthetised patients (n=48), who had sustained a fracture of the tibia. The semisolid material was explanted into 25 cm2 non-coated tissue culture flasks and allowed to clot. Complete culture media was prepared and pipetted into the vessels, which were then placed in an incubator (37˚C; humidified 5% CO2). After characterisation via immunocytochemistry (using antigen-specific markers to CD29, CD44, CD73, CD105, CD166 and CD34), isolated cells were counted using flow cytometry and proliferation rates were compared between non-smokers and smokers. Cigarette smoke extract (CSE) was manufactured based on the methods of Bernhard et al. (2004), in order to synthesise an in vitro smoking environment. Cells harvested from non-smokers (n=10) were cultured and infused with the CSE (2.5%) and proliferation and cell recovery rates were compared between the treated and untreated groups. Cellular nitric oxide levels and necrosis rates (using propidium iodide for the latter) were also compared in a similar way. An MTT assay was conducted to assess response to various concentrations of BMP-2 (10, 100 and 500 ng mL-1) infusion in cell xvi cultures. Molecular assays: Serum was extracted from the haematomas and resolved on 16% acrylamide gels before staining with either Coomassie blue or silver nitrate. The resolved gels were photographed using a Kodak gel imaging system and unknown bands were identified using MALDI-TOF. Intracellular TGF- and IL-6 were probed using antibodies, which were then analysed via flow cytometry in order to confirm the presence of these acute-phase proteins. In additional experiments, the ELISA was undertaken to quantify and compare the amounts of VEGF and IL-6 in haematoma serum and lysed fracture cells respectively. Biochemical testing on serum for b-ALP, albumin and total protein amounts in fracture serum was carried out, comparing smoker (n=10) and non-smoker (n=10) levels. Results: Cell culture: Spindle-shaped, fibroblast-like cells were visible in the primary culture, which were expanded through at least 10 further passages. The immunostaining of cells suggested a mesenchymal stem cell (MSC) lineage (CD29+, CD44+, CD73+, CD105+, CD166+, CD34-). Absolute cell counting using flow cytometry revealed marked proliferation of cells after 3 passages of culture. There was a reduction in the rate of proliferation of MSCs in smokers over 2 passages compared with non-smokers (-20%). A 5-day proliferation study in the CSE-treated vs. untreated cells showed a reduction in the rate of doubling in the treated group (-40% p<0.05). Cells showed a recovery response after CSE was withdrawn from culture, but was not significant. Cell necrosis analysis of CSE-treated vs. untreated cells showed that the CSE-treated cultures had a higher rate of necrosis than the untreated cells (CSE-treated 14.37% vs. untreated 7.98% p<0.05). Nitric oxide levels were lower in the CSE-treated cells (CSE-treated 3.0 M vs. untreated 3.6 M; p<0.05). MTT assay; BMP-2 infusions all improved cell viability compared to the non-infused cells, xvii with a BMP-2 concentration of 10 ng mL-1 increasing cell viability the most, though not significantly (+23%; p>0.05). Molecular assay: After staining, SDS-PAGE gels showed numerous bands of serum proteins and the haematoma serum lanes displayed unknown proteins which were later found to be haptoglobin (~20 kDa) and haemoglobin (~14 kDa) chains via mass spectrometry (MALDI-TOF). Cells were strongly positive (>96%) for the intracellular protein markers TGF- and IL-6. The VEGF-A (serum) and IL-6 (cells) preliminary data from the ELISA revealed a reduction of these acute-phase proteins in patients who were smokers (VEGF-A -10%; IL-6 -15%). In the biochemical assays, albumin was reduced in smokers’ serum (-13%, p<0.05), whereas b-ALP was raised (+20%; p>0.05 n.s.) and total protein lowered (-12%; p<0.01). Conclusions: The haematoma cultures produced colonies of adherent fibroblast-like cells that were of a MSC lineage. With the exception of a residual haemolysis-derived haptoglobin band, SDS-PAGE did not appear to show an over-expression of acute phase proteins, although it was useful for the characterisation of the fracture milieu, in that it was suggestive of a consistently haemolysed haematoma. The effect of smoking on bone fracture healing, therefore, appears to contribute to the inhibition of MSC proliferation, angiogenesis and the acute phase stress response. Cigarette smoke was also shown to cause excessive necrosis and reduce the amount of NO in those MSC cultures treated with CSE, which may indicate reduced vasodilatation to the fracture site. Bone alkaline phosphatase (b-ALP) was raised in the fracture serum of smokers, suggesting abnormally-high bone turnover, whilst the reduction of total protein and serum albumin likely indicates a lowered capacity for acute-phase protein synthesis and calcium transport in these patients. Exogenous BMP may be indicated for rectification of malunion in smokers.

Soccer matches have a typical duration of 90 min. However, when matches are drawn in some knockout cup scenarios and an outright winner is required, an additional 30 min period, termed extra-time (ET), is played. The performance and physiological responses to 90 min of soccer-specific exercise have been extensively investigated, however; there is a paucity of research investigating the demands of 120 min of soccer-specific exercise (i.e., matches requiring ET). Accordingly, the aims of this thesis were 1) to elucidate professional practitioner perceptions of ET, 2) to investigate the performance and physiological responses during prolonged actual and simulated match-play, and 3) to examine the influence of a nutritional intervention on performance during ET. To actuate aim one, a qualitative approach (i.e., an online survey) was used to assess practitioner perceptions of ET and their current applied practices. To accomplish aim two, quantitative research projects utilising performance analysis techniques and an analogue of match-play (simulated soccer match) were used. To actuate aim three, the same analogue of match-play was used to investigate the effect of carbohydrate-electrolyte gels ingested prior to ET on performance and physiology. Practitioners generally account for ET when preparing and recovering players and the majority (91%) of practitioners want research to be conducted on ET. Using notational analysis, reductions in technical performance (i.e., passing and dribbling) were observed during ET. Furthermore, when using a simulated match protocol, perturbations in both performance and physiology compared to the previous 90 min of exercise occur. Specifically reductions in both physical (i.e., sprint speeds) and technical (i.e., shooting speed) parameters, taxing of endogenous fuel sources, dehydration, and shifts in substrate utilisation (i.e., a move towards fat oxidation as a fuel source) were observed. The ingestion of carbohydrate-electrolyte gels prior to ET improved dribbling performance, however; this intervention was unable to attenuate decrements in physical performance and hydration status. In conclusion, ET influences both soccer-specific performance and physiological responses. In agreement with practitioners working in professional soccer, more research is required to investigate the efficacy of interventions (particularly hydro-nutritional interventions) that improve performance and ameliorate perturbations in physiology and metabolism.

This study used a qualitative method to focus on the perspectives, beliefs and expectations of low back pain sufferers. The research was undertaken within a hospital based pain clinic. In recent years low back pain research has proliferated, and the epidemiological evidence suggests that back pain is an increasing problem. Much attention has been paid to the impact of low back pain on the population, and to the increasing cost in economic and health terms. Biomedical and psychological evidence abounds to shape acute and chronic management of low back pain, but there is a dearth of information about the viewpoint of those suffering pain. This study attempted to bring the understanding of the back pain sufferer to the fore. Issues of quality of life, functional ability and the impact of back pain on their lifestyle were explored, along with the influence of contextual factors in relation to how back pain sufferers perceived themselves and how others perceived them. A narrative method was utilized to illuminate the journey with pain. Nine interviews were conducted, and the interpretation and presentation of the narratives generated was influenced by Ricoeur’s interpretative theory. Thematic analysis revealed that doctorability, agency, control, separation or acceptance of the pain and the concept of future life were key features within the narratives. The analysis highlighted that for the majority in this study pain arrived uninvited following a traumatic accident or incident, and back pain became a chronic condition. It was always unwanted and initially it was unexpected as the usual script for pain is one of a transient incapacity followed by recovery. It was precisely this deviation from the norm that resulted in difficulties for the people suffering the pain. Biographical differences did not appear to be identifiable in the themes discerned in the stories, nor in the overall structure.

Skeletal muscles are composed of thousands of muscle fibres (muscle cells), densely packed together in parallel and surrounded by connective tissue sheaths. These fibres are multinuclear in nature, which allows for the control and regulation of the highly organised, protein rich cellular interior. The primary function of skeletal muscle is to produce force, which allows for movement to occur or posture to be maintained, and the regulation of this function is in turn reliant on the expression of specific genes and proteins. Skeletal muscle exhibits a high degree of plasticity, and can adapt in response to stimuli such as increased/decreased use, metabolic perturbations or changes in the systemic environment which often occur as a result of exercise, ageing, disuse or disease. Examining responses and adaptations in skeletal muscle in vivo are challenging due to experimental restrictions, and studies are limited by ethical issues surrounding experimentation on human beings and indeed on animals following the principals of refinement, reduction and replacement. Thus in vitro studies are often conducted in order to further understand mechanisms involved in adaptation. However, the environment to which skeletal muscle cells are exposed to in vitro is far removed from that in the body, and the resulting cellular architecture is often abnormal in morphology. Tissue engineered skeletal muscle has shown much promise in rectifying these issues, as cells can be grown on/within a matrix which is biologically relevant and engineered to grow in a uniaxial manner in parallel to one another. However, this field is in its relative infancy, and to date little data exists with regards the behaviour and characteristics of human muscle derived cells (MDCs) in tissue engineered constructs. In this thesis, human skeletal MDCs were obtained, characterised and subsequently cultured in a suitable model for tissue engineering purposes. MDCs were seeded on to a fibrin based hydrogel, which self-assembled over time to form a cylindrically shaped construct held in place between two anchor points. In ii this model, the cells were shown to align uniaxially and in parallel to one another in a fascicular like structure. The model was improved in terms of biomimicity and maturation by both increasing the seeding density of the MDCs, and by increasing the ratio of myogenic to non-myogenic cells. These models appear to promote the development of a slow muscle, as evidenced by the favourably high levels of MYH7 transcription in comparison to other isoforms, and showed suggestions of sarcomeric organisation as indicated by the classically striated pattern of protein organisation when myosin heavy chain immunostaining was conducted. The work conducted in the final chapter of this thesis focussed on developing a system capable of assessing and quantifying the force produced by these tissue engineered human skeletal muscle constructs when electrically stimulated. Further work in this area should aim to determine these functional characteristics and thereafter use the model for physiological, cellular and molecular studies in exercise science.

Gait problems in Parkinson's disease (PD) are complex and not adequately addressed by current medical and surgical options. The focus of this thesis was a desire to optimise the effectiveness of cues after experience of delivering cueing therapy in the context of a multi-centre RCT. Cues provide information on how to adapt the stepping pattern either through external prompts or internally through focussing attention. Cues are known to improve gait in PD but there is a compromise between strategies which have large effects but limited practical application and those which are easily applied in complex situations but have more modest effects. A laboratory study explored the feasibility of a cueing strategy combining an external rhythmical cue with a focussed instruction to increase step size, targeting both temporal and spatial parameters. A group of 15 PD and 12 age and sex matched controls were tested and gait was measured with an instrumented walkway which uses pressure activated sensors. The combination cue was compared with two single parameter cueing strategies, a rhythmical auditory cue and an attentional strategy asking subjects to walk with large steps. Gait was assessed under single and dual tasks to establish the attentional demands of the different cues. Walking speed and step amplitude significantly increased with the attentional strategy and combination cue in single and dual tasks in PD and controls (see chapter 3). The combination cue had an additional benefit in significantly reducing stride time and double limb support time variability in PD subjects, whilst variability increased in controls (see chapter 4). The effects of cues on and off medication was tested in the home in a group of 50 PD subjects using the same dual task paradigm to explore the mechanisms underlying cueing compared to dopamine on gait control. Gait was measured using an in-shoe footswitch system allowing reliable gait data to be collected in the home. Walking speed and stride amplitude significantly improved with all cues in the single and dual tasks on medication and with the attentional strategy and combination cue off medication suggesting that cues have a different mechanism to dopamine. The greatest improvements were seen with the combination of cues and medication. Gait variability responded differently to cues on and off medication. The combination cue reduced variability on and off medication for single and dual tasks, the auditory cue reduced variability in all conditions except for single task on mediation and the attentional strategy increased variability in the single task on mediation and had no effect in other conditions (see chapter 5). Cues which are delivered externally result in different mechanisms of gait control than those generated internally. Measures of gait variability reflect the attentional cost of movement and underlying neural control but there is limited knowledge on their validity. The final stage of the research examined the clinical characteristics associated with increased gait variability to increase understanding of these variables. Non-cued gait variability was strongly associated with disease severity, but cued gait variability was not adequately explained suggesting involvement of more diverse parameters (see chapter 6). These findings provide new knowledge on the mechanism underlying cued gait, the involvement of dopaminergic pathways and the attentional cost of different cues. Focussed instruction can alter the response to an external cue in the form of a rhythmical auditory tone, targeting both temporal and spatial gait parameters and reducing the attentional cost of walking.