Дисертації з теми "Mitochondrial movement"

This thesis explores two distinct parts of mitochondrial physiology: the role of mitochondria in generation of reactive oxygen species (ROS) and mitochondrial morphology and dynamics within cells. The first area of research is covered in Chapters 1-8. Mitochondrial biofunctionality and ROS production are discussed in Chapter 1, followed by the strategy of targeting bioactive compounds to mitochondria by linking them to lipophilic triphenylphosphonium cations (TPP) (Chapter 2). ROS sensors relevant to the research are reviewed in Chapter 3. Chapter 4 presents design and synthesis of novel probes for superoxide detection in mitochondria (MitoNeo-D), cytosol (Neo-D) and extracellular environment (ExCellNeo-D). The results of biological validation of MitoNeo-D and Neo-D performed in the MRC MBU in Cambridge are presented in Chapter 5. A dicationic hydrogen peroxide sensor that utilizes in situ click chemistry is discussed in Chapter 6. Preliminary work on the synthesis of mitochondria-targeted superoxide generators, which led to the development of mitochondria-targeted analogue of paraquat, MitoPQ, is presented in Chapter 7. A set of bifunctional probes (BCN-Mal, BCN-E-BCN and Mito-iTag) for assessing the redox states of protein thiols is discussed in Chapter 8 along with their biological validation. The second part of the thesis is aimed at the study of mitochondrial morphology and dynamics and is presented in Chapters 9-11. Chapter 9 provides background on the classes of fluorophores relevant to the research, the phenomenon of fluorescence quenching and the principle of photoactivation with examples of photoactivatable fluorophores. Next, the background on mitochondrial morphology and heterogeneity is presented in Chapter 10, followed by the ways of imaging and tracking mitochondria within cells by conventional fluorophores and by photoactivatable fluorophores exploiting super-resolution microscopy. Chapter 11 presents the design and synthesis of four photoactivatable fluorophores for mitochondrial tracking, MitoPhotoRhod110, MitoPhotoNIR, Photo-E+, MitoPhoto-E+, along with results of biological validation of MitoPhotoNIR. The results and discussion concludes with Chapter 12, which is a summary and suggestions for future work, followed by the chemistry experimental procedures (Chapter 13), materials and methods for biological experiments (Chapter 14) and references.

Les mitochondries assurent des fonctions essentielles dans les cellules via la production d'énergie sous forme d'ATP, la captation de calcium et la régulation de la mort cellulaire par apoptose. Les dysfonctions des mitochondries apparaissent à des stades précoces de la maladie d'Alzheimer (MA), et ont été particulièrement associées au peptide toxique Aβ. Ce dernier est issu du clivage séquentiel de la protéine précurseur de l'amyloïde (APP) par la β- et la γ-sécrétase. Plusieurs traitements ciblant l'Aβ se sont avérés inefficaces contre la progression de la MA, orientant les recherches sur le potentiel toxique d'autres fragments issus du clivage de l'APP. L'hypothèse de mon projet porte sur la toxicité spécifique des fragments APP C-terminaux (APP-CTFs : C83 et C99 (précurseur direct de l'Aβ)) en se focalisant sur l'étude de la structure, la fonction des mitochondries et la mitophagie. J'ai également étudié l'impact de l'APP et de ses fragments sur la machinerie de transport des mitochondries, un mécanisme clé de leur renouvellement, en particulier dans les neurones.Premier axe : Impact de l'accumulation des APP-CTFs sur la structure, la fonction des mitochondries et sur la mitophagie. Nous décrivons une accumulation des APP-CTFs dans la fraction mitochondriale de modèles mimant les formes familiales de la MA in vitro (cellules de neuroblastome humains exprimant l'APP portant la double mutation suédoise (SH-SY5Y-APPswe), ou le fragment C99 (SH-SY5Y-C99)) et in vivo (souris transgéniques 3xTgAD exprimant les mutations APPswe, TauP301L, PS1 (M146V) ou bien le fragment C99 après infection virale). Par le biais d'une approche pharmacologique bloquant l'activité de la γ-sécrétase, nous démontrons in vitro et in vivo que l'accumulation des APP-CTFs, indépendamment de l'Aβ, est associée à l'agglomération de mitochondries structurellement altérées et surproduisant des espèces oxygénées réactives toxiques, conjointement à un blocage de la mitophagie. Nous avons conclu notre étude par la démonstration de l'altération de la mitophagie dans les cerveaux de patients atteints de la MA sporadique, corrélant avec les niveaux d'APP-CTFs (1, 2).Second axe : Etude des effets de l'APP, des APP-CTFS et de l'Aβ sur les protéines de transport mitochondrial. J'ai d'abord démontré l'impact de l'APP endogène et de la surexpression de l'APPswe sur les niveaux des protéines de la machinerie de transport mitochondrial in vitro (fibroblastes de souris KO pour l'APP ainsi que dans cellules SH-SY5Y-APPswe). J'ai discriminé le rôle de l'accumulation des APP-CTFs de celui de l'Aβ sur l'expression de ces protéines en traitant les cellules APPswe avec l'inhibiteur de la γ-sécrétase. J'ai validé ces observations en utilisant des fibroblastes de souris déplétés des présénilines (composants catalytiques de la γ-sécrétase) mimant une accumulation des APP-CTFs. J'ai par ailleurs démontré une implication des APP-CTFs et de l'Aβ dans le défaut de recrutement des mitochondries à la machinerie de transport dans les cellules SHSY-5Y différentiées. En analysant des cerveaux de souris 3xTgAD et WT à différent âges et des échantillons de cerveaux de patients Alzheimer sporadiques à différents stades de la maladie, nous rapportons que le développement de la MA et l'âge en lui-même ont un impact différentiel sur l'expression de certaines protéines de transport mitochondrial (3).Ces études démontrent de nouveaux mécanismes moléculaires impactant l'homéostasie mitochondriale au cours du développement de la MA. Ces découvertes permettront la mise en place de nouvelles pistes thérapeutiques ralentissant les dysfonctions des mitochondries et, ou favorisant leur renouvellement dans le contexte de la MA.(1). Vaillant-Beuchot L.*, Mary A.* et al. Acta Neuropathologica 2020.(2). Mary A.*, Vaillant-Beuchot L.* et al. Médecine/sciences 2021.(3). Vaillant-Beuchot et al. En cours de soumission
Mitochondria are essential organelles in cells, ensuring energy production with ATP synthesis, calcium buffering, apoptosis regulation. These functions are altered at early stages of Alzheimer's disease (AD) and are essentially induced by the Amyloid (Aβ), produced after the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretase. Aβ is a major actor of AD development but all the treatments targeting this peptide remain ineffective. C-terminal APP fragments (APP-CTFs: C83 and C99 (Aβ precursor) are other fragments presenting specific toxicity in AD and new potential therapeutic targets. My project is focus on the study of APP-CTFs toxicity, independently of Aβ, on the structure, function of mitochondria, their degradation by mitophagy and on mitochondrial transport proteins. They constitute the complex allowing mitochondrial transport in cells, especially in neurons, closely linked to mitochondrial renewal, particularly in neurons.First axe: APP-CTFs impact on mitochondrial structure, function and mitophagy. We described APP-CTFs accumulation in mitochondrial fraction in vitro (human neuroblastoma cells expressing APP Swedish double mutation (SH-SY5Y-APPswe) or C99 fragment (SH-SY5Y-C99)) and in vivo (3xTgAD mice expressing APPswe, TauP301L, PS1 (M146V) or C99 fragment after viral injection). We inhibit the cleavage of APP-CTFs and the production of Aβ by pharmacological approaches, to abolish γ-secretase activity. Ours results show for the first time in vitro and in vivo, that high concentration of APP-CTFs independently of Aβ, impact mitochondrial structure, function and alter mitophagy process, resulting in an accumulation of altered mitochondria producing high levels of toxic reactive oxygen species. In addition, our results in patient brains of sporadic AD (SAD) patients show altered mitophagic protein levels correlating with APP-CTFs accumulation (1-2).Second axe: study of the effects of APP, APP-CTFs and Aβ peptide on mitochondrial transport machinery. I reported the specific regulation of mitochondrial transport protein by endogenous APP (Mice fibroblasts APP WT and KO) and the overexpression of APPswe (and in SH-SY-5Y-APPswe cells). APP-CTFs and Aβ differentially regulate mitochondrial transport protein levels in treated SH-SY-5Y-APPswe cells with γ-secretase inhibitor. These results were validated in mice fibroblasts KO for presenilins (catalytic compounds of γ-secretase) avoiding APP-CTFs degradation. APP-CTFs and Aβ impair the recruitment of mitochondria to its transport machinery in differentiated SHSY-5Y. The progression of the disease deregulates the levels of mitochondrial transport protein in vivo (3xTgAD and WT mice brains, C99 injected mice brains) and in SAD patients brains. The analyses of young and old mice brains and of SAD patients samples at different stages of the disease, allowed us to demonstrate an impact of aging in the regulation of mitochondrial transport protein levels. This phenomenon occurs also in addition with AD progression (3).These studies highlight new molecular mechanisms impacting mitochondrial homeostasis during AD progression. Our findings will bring new therapeutic research to slow down mitochondrial dysfunctions and/or to stimulate their renewal in AD context.(1). Vaillant-Beuchot L.*, Mary A.* et al. Acta Neuropathologica 2020.(2). Mary A.*, Vaillant-Beuchot L.* et al. Médecine/sciences 2021.(3). Vaillant-Beuchot et al. En cours de soumission

The desire to age well is a common goal among the human population. How to do so is therefore, a popular question. One theory of ageing involves the accumulation of damage to mitochondrial protein and the subsequent loss of function the damage causes. Increasing the rate of mitochondrial protein synthesis, a variable that declines with advancing age, is one way to improve quality of life in the twilight years. A review of literature lead to a multi-level approach, with measurements of protein synthesis made at the whole body, muscle, and molecular levels. An acute bout of aerobic exercise, followed by feeding, two factors which have a positive effect on the rate of mitochondrial protein synthesis, was used. Adaptations to a period of exercise training are mediated by the accumulation of proteins due to each acute exercise bout, and so an acute intervention was postulated to be indicative of changes expected over the long term. A stable isotope infusion combined with sampling of breath, blood, and muscle was used to determine the rate of whole body protein synthesis in 12 older adults. Intracellular signalling for mitochondrial and whole body protein synthesis was examined using RT-quantitative PCR and Western blotting in eleven young adults. The rate of post-exercise whole body protein synthesis was 19% greater over the first four hours of post-exercise recovery, in subjects receiving a protein-plus-carbohydrate drink immediately after a bout of cycling than in those receiving a carbohydrate-only drink (p = 0.001). The same trend was revealed in signalling for whole body protein synthesis and the abundance of cytochrome c, a mitochondrial protein, although these results were not statistically significant (p = 0.2). In contrast there was a strong, albeit also statistically insignificant, tendency for signalling for mitochondrial protein synthesis to be higher in the skeletal muscle of subjects receiving a carbohydrate-only drink after a bout of cycling (p = 0.06). The exercise and feeding intervention described in this thesis may provide a means to enhance the rate of mitochondrial protein synthesis in older individuals and, in so doing, improve the quality of their old age.

Mitochondria are key components of skeletal muscles as they provide the energy required for almost all cellular activities, and play an important role in ageing and cell pathology. Different forms of exercise training have been associated with mitochondrial adaptations, such as increased mitochondrial content and function, and enhanced mitochondrial biogenesis, as well as improved endurance performance. However, the role of training intensity and training volume, in determining these changes remains elusive. Therefore, the aim of this thesis was to investigate the role of training intensity and volume on changes in mitochondrial content and function (as measured by mitochondrial respiration in permeabilised muscle fibres), in the skeletal muscle of healthy humans, and to study the molecular mechanisms underlying these changes. It was demonstrated that training intensity is a key factor regulating changes in mitochondrial respiration, but not mitochondrial content, and that an apparent dissociation exists between changes in these two parameters. Training consisting of repeated 30-s “all-out” sprints lead to improved mitochondrial (mt)-specific respiration (indicative of improved mitochondrial quality). Conversely, training volume was shown to be a key factor regulating mitochondrial content, with the associated increase in mitochondrial respiration being likely driven by the increase in mitochondrial content (i.e., unchanged mt-specific respiration). A training volume reduction resulted in a rapid decrease in most mitochondrial parameters, underlining the importance of maintaining the training stimulus to preserve training-induced mitochondrial adaptations. The protein content of PGC-1α, p53 and PHF20 was shown to be regulated in a training intensity-dependent manner, and was more strongly associated with changes in mitochondrial respiration rather than content, whereas changes in the protein content of TFAM were primarily associated with changes in mitochondrial content. Moreover, it was demonstrated that exercise intensity induced an increase in nuclear PGC-1α protein content and nuclear p53 phosphorylation, two events that may represent the initial phase of different pathways of the exercise-induced adaptive response. Collectively, this research provides novel information regarding mitochondrial adaptations to different training stimuli, and could have important implications for the design of exercise programs in conditions of compromised mitochondrial function.

Lung transplant (LTx) recipients have poor exercise tolerance, which persists in spite of the restoration of near normal lung function. This suggests that the exercise limitation is related to defects in skeletal muscle. Firstly, mitochondrial function and metabolism in resting skeletal muscle were examined for 7 LTx recipients, 3-24 months post operation. Secondly, exercise performance for patients with lung transplantation was investigated. Resistance training is an effective exercise mode for improving muscle bulk and strength in sports and medical rehabilitation. Recently, resistance training has become a popular exercise mode to improve muscular function and enhance exercise performance in patients with cardiopulmonary diseases. In a further study sixteen male volunteers participated in a study on the effects of resistance training on mitochondrial oxidative capacity in skeletal muscle.

Mitochondrial adaptation in skeletal muscle is promoted by a diverse array of stimuli, and changes in mitochondrial plasticity have been noted as a result of a many exercise modalities. High-intensity interval training is one such modality that promotes mitochondrial adaptation in response to repeated short-duration bouts of intense effort. Another result of intense muscular effort is a decrease in muscle pH, resulting in intracellular acidosis. The effect of this acidosis on oxygen consumption in muscle has received attention previously, with mixed findings. An aspect of skeletal muscle mitochondrial function that has received limited attention is the production of reactive oxygen species. To date a small number of studies have also provided evidence that attenuating the development of intracellular acidosis may have beneficial effects for mitochondrial adaptation. This thesis aimed to further investigate the effect of acidosis on mitochondrial function, and on intracellular signalling for mitochondrial biogenesis.

While seemingly simple, the underlying exercise prescription to bring about the desired adaptations to exercise training is as complicated as that of any drug. Prescribing the frequency, duration, or volume of training is relatively simple as these factors can be altered by manipulating the number of exercise sessions per week, the duration of each session, or the total work performed in a given time frame (e.g., per week). However, prescribing exercise intensity is more complex and there is controversy regarding the reliability and validity of the many methods used to determine and prescribe intensity. Despite their common use, it is apparent V̇ O2 and HR based exercise prescription has no merit to elicit a homogeneous and explicit homeostatic response. Alternatively, the use of submaximal anchors has been employed and perceived to represent shifts in the metabolic state of the working muscle and represent a demarcating point to define training zones. Whereas, the domains of exercise are independent of these anchors and defined by their distinct homeostatic responses, and offer a valid concept for normalising exercise intensity (Chapter 1; Review 1). Furthermore, the relationship between graded exercise test (GXT) derived anchors and constant work load derived anchors is at this point non sequitur and we discourage using submaximal anchors interchangeably (Chapter 2; Study 1). Mitochondria are organelles found inside skeletal muscle cells and their main role is the production of adenosine triphosphate (ATP) which is necessary for skeletal muscle contractions. The bioenergetics demands associated with aerobic exercise lead to a homeostatic perturbation, activating sensor proteins that initiates gene expression through transcriptional and translational processes leading to the development of mitochondrial proteins. The source of ATP production modulates the homeostatic perturbations that activate the sensor proteins which include: an increase in the redox state of the cell (NAD+/NADH), an increase in ATP turnover (measured via AMP/ATP), increased calcium flux and mechanical stress and these are largely influenced by the source of ATP production. These perturbations act as signals to activate sensor proteins that ultimately modulate transcriptional coactivators associated with mitochondrial biogenesis (Chapter 3; Review 2). Despite the relationship between exercise intensity and mitochondrial biogenesis, submaximal exercise intensity is almost exclusively prescribed relative to maximal oxygen uptake or maximal work rate. The well-established limitation of these methods is the inability to normalise exercise intensity; specifically, to elicit a homogenous homeostatic perturbations. Thus, employing methodology that normalises exercise intensity based on homeostatic perturbations may modulate the activation of signalling kinases and the extent of gene expression (Chapter 4; Study 2).

It is well documented that regular endurance exercise induces skeletal muscle mitochondrial biogenesis. However, the optimal training stimulus and nutritional intervention for which to maximize mitochondrial adaptations to endurance exercise is not well known. Developments in molecular techniques now permit the examination of the cell signalling responses to acute exercise therefore increasing our understanding of how manipulation of the training protocol and nutrient availability may enhance the training stimulus to a given bout of exercise. The primary aim of this thesis is to therefore characterise the skeletal muscle cell signalling responses thought to regulate mitochondrial biogenesis following an acute bout of high-intensity interval exercise and moderate- intensity continuous exercise.

Chronic heart failure (CHF) is characterised by an inability of the heart to pump enough blood to meet the body’s metabolic needs, resulting in exercise intolerance. A reduction in nitric oxide (NO) bioavailability has been implicated as an initiator and/or contributor to many of the peripheral skeletal tissue dysfunctions that contribute to the exercise intolerance in patients with CHF. Inorganic nitrate supplementation has been identified as an important mediator of exercise tolerance via increasing NO bioavailability, but the potential efficacy of this on patients with heart failure with reduced ejection fraction (HFrEF) as well as the effect on vascular function is not well understood and was the focus of Study 1. Additionally, to our knowledge, no previous study has examined the potential impact of nitrate supplementation on cardiac performance during submaximal exercise and mitochondrial respiration in individuals with HFrEF. These were the foci of Studies 2 and 3 respectively. Study 1: The effect of dietary inorganic nitrate supplementation on exercise tolerance and vascular function in patients with HFrEF. The primary aim of this study was to determine the effect of chronic inorganic nitrate supplementation on exercise tolerance, as measured by peak aerobic capacity (VO2peak) and time to exhaustion (TTE), during treadmill exercise in patients with HFrEF. A secondary aim was to determine the effect of chronic supplementation on vascular function (endothelial function) in these patients. Methods: Sixteen patients with HFrEF (15 men and 1 woman, 63 ± 4 y, BMI: 31.8 ±2.1 kg∙m-2) completed the primary outcome of this study (exercise tolerance), and 12 completed the vascular function component. Participants were randomly allocated, in a double-blind, crossover design, to consume either a nitrate rich beetroot juice (16mmol nitrate/day), or a nitrate-depleted placebo for five days prior to the first testing visit. Participants then continued daily dosing until they completed a cardiopulmonary exercise test (CPX) and a battery of vascular function assessments (peripheral and central blood pressure (BP) as well as aortic stiffness and brachial artery flow mediated dilation (BAFMD)). Results: There were significant increases in both plasma nitrate (p<0.001) and nitrite (p<0.05) following nitrate supplementation. No significant differences were observed in either VO2peak (nitrate 18.5 ± 5.7 ml∙kg-1∙min-1, placebo: 19.3 ± 1.4 ml∙kg-1∙min-1; p=0.13) or TTE (nitrate: 1165 ± 92 sec, placebo: 1207 ± 96 sec, p=0.16) between the two interventions. Similarly, there were no significant (p>0.05) changes in peripheral tissue oxygenation during exercise, as measured non-invasively with near-infrared spectroscopy (NIRS). There were no differences in the brachial blood pressure measurements including systolic blood pressure (SBP) (nitrate: 130 ± 4 mmHg, placebo: 132 ± 5 mmHg, p=0.58), diastolic blood pressure (DBP) (nitrate: 80 ± 3 mmHg, placebo: 81 ± 3 mmHg, p= .74) and mean arterial pressure (MAP) (nitrate: 96 ± 3 mmHg, placebo: 98 ± 4 mmHg, p=0.67). There were also no significant differences in aortic pressure or stiffness. BAFMD reactive hyperaemic percent change tended to improve (nitrate: 5.7% ± 1.1, placebo: 4.1% ± 0.7, (p=0.06), and this change had a moderate effect size (ES) (Cohen’s d 0.607). Conclusions: Results from this study indicate the nitrate appears ineffective at improving exercise tolerance and vascular function in HFrEF. Future studies should explore alternative interventions to improve peripheral muscle tissue function in HFrEF. Study 2: The effect of dietary nitrate supplementation on cardiac output and stroke volume during submaximal exercise in men with HFrEF: a pilot study. The primary aim of this exploratory study was to determine the effect of chronic inorganic nitrate supplementation on cardiac performance during three submaximal exercise bouts. Methods: Five male patients with HFrEF (61 ± 3y) completed this pilot study. Participants consumed either the nitrate-rich beetroot juice (16 mmol nitrate) or the placebo an average of 13 ± 2 days prior to the testing visit. They completed a three-stage (15-25 watts, 25-40 watts and 35-60 watts) discontinuous exercise protocol on an echo-compatible recumbent cycle ergometer with simultaneous Doppler echocardiography. Cardiac output (Q̇) and stroke volume (SV) were derived using the Doppler velocity time integral via the Huntsman method. Results: There were significant increases in both plasma nitrate (p=0.004, ES=3.54) and nitrite (p=0.01, ES=0.82) following nitrate supplementation. Although not statistically significant (all p>0.27), the differences in Q̇during stage two and stage three had medium to large ES (stage two: nitrate: 6.4 ± .4 L∙min-1, placebo: 5.3 ±. 2 L∙min-1, ES=1.51; stage three: nitrate: 7.5 ± 0.6 L∙min-1, placebo: 6.4 ± 0.7 L∙min-1, ES=0.50) exercise. Changes in Q̇ were accompanied by medium to large ES changes in SV (stage two: ES=0.97 and stage three: ES=0.57) and medium to large increases in heart rate (HR) at rest and all exercise stages. These differences were likely mediated by a reduction in total peripheral resistance (TPR) at stage two (ES=-1.62) and stage three (ES=-0.81). Conclusions: We report potentially clinically important improvements in measures of cardiac performance during submaximal exercise following nitrate supplementation in patients with HFrEF. The initial findings from this pilot study warrant further investigation in larger and more diverse samples in order to determine the efficacy of this intervention. Study 3: The effect of dietary nitrate supplementation on mitochondrial respiration in men with HFrEF. The primary aim of this exploratory study was to determine the effect of chronic inorganic nitrate supplementation on parameters of mitochondrial respiration in patients with HFrEF. Methods: Seven male participants (62 ± 2y) completed this invasive study. Participants consumed the nitrate rich beetroot juice (16mmol nitrate/day) or a placebo for an average of 15 ± 2 days prior to their muscle biopsy. Muscle samples were taken from the vastus lateralis. Mitochondrial respiration was assessed using high resolution respirometry. Western blot analysis was used to assess the protein content of mechanistic target of rapamycin complex 1 (mTORC1), p38 mitogen activated protein kinase (p38MAPK), protein kinase B (Akt), and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α). Results: Plasma nitrate increased (831%, p<0.001) following supplementation. Plasma nitrite also increased (100%) but this was not statistically significant (p=0.22). There were no differences in skeletal muscle maximal oxidative phosphorylation capacity as assessed as either mass-specific (p=0.93) or mitochondrial-specific (p=0.68) respiratory function of (CI+CII)p, nor were there any significant differences in other parameters of mitochondrial respiration (all p>0.05). Similarly, there were no differences in mitochondrial content, as assessed by citrate synthase activity (p=0.73) and no differences were noted in total and phosphorylated forms of mTORC1, p38MAPK, Akt, or PGC-1α (all p>0.10). Conclusions: Short-term nitrate supplementation, as a standalone treatment, may not be an effective way to improve mitochondrial function in patients with HFrEF and, as such, it may be clinically important to combine nitrate supplementation with other interventions known to affect mitochondrial function, such as exercise training. General Conclusions. Short-term inorganic nitrate supplementation had no effect on exercise tolerance (Study 1-Chapter 4), peripheral tissue oxygenation (Study 1- Chapter 4), or mitochondrial respiration (Study 3- Chapter 6) in patients with HFrEF. However, it may have a meaningful clinical effect on Q̇and SV during submaximal exercise (Study 2- Chapter 5). It may also improve vascular function (Chapter 4), reduce TPR (Chapter 5) and reduce DBP and MAP during submaximal exercise (Chapter 5) in these patients. Overall the data suggest that nitrate supplementation may be used in conjunction with other pharmacological and non-pharmacological (exercise training) interventions to improve clinical outcomes in this population. This hypothesis should be explored in the future by conducting a large-scale clinical trial.

The aim of this thesis was to investigate the acute and chronic effects of high-intensity intermittent exercise, in the form of repeated-sprint exercise (RSE) and indoor football (futsal), on performance responses and skeletal muscle molecular adaptations in young, healthy and middle-aged, sedentary individuals.

The increased time-demands imposed on modern society means that many people don’t meet nightly sleep recommendations. However, despite the obvious importance of sleep for the maintenance of good health, the effects of sleep loss are remarkably understudied. The consequences of sleep loss on aspects of metabolic health are becoming more apparent, with detrimental changes to glucose tolerance, insulin sensitivity, and an increased risk of numerous metabolic conditions being reported. Comparatively, the underlying mechanisms that lead to these changes are not well characterised, but may include circadian misalignment, changes in mitochondrial function, and inhibition of the molecular signalling pathways that govern protein synthesis. Due to the emergence of these detrimental metabolic changes, interventions that are capable of mitigating these effects (which remain following bouts of ‘recovery sleep’) should be investigated. Exercise improves glucose tolerance, improves mitochondrial function and is also thought to be able to shift circadian rhythms, making it an ideal candidate to mitigate some of the detrimental effects of sleep loss. Accordingly, the overall aim of this thesis was to investigate the metabolic consequences of sleep loss, and the underlying cellular mechanisms, and to determine the effectiveness of exercise on aspects of metabolic health during a period of sleep loss.

ACTN3 has been labelled as the ‘gene for speed’ due to the increased frequency of the R allele encoding the α-actinin-3 protein in elite sprint athletes compared to the general population. The results of the first study of this thesis demonstrate that elite athletes who express α-actinin-3 (ACTN3 RR genotype) have faster sprint times compared to those who do not express α-actinin-3 (ACTN3 XX genotype). Further analysis indicates that the ACTN3 genotype accounts for 0.92% in sprint speed amongst elite 200-m athletes. In study two, the same quantitative genetic epidemiological design applied to elite endurance athletes, showed no evidence that a trade-off existed. The endurance athletes with the ACTN3 XX genotype were no faster than those who express the α-actinin-3 protein. These results added to literature that it is unlikely the ACTN3 XX genotype to offer an advantage for endurance performance. While ACTN3 genotype does not appear to influence endurance performance in athletes, studies in mice that completely lack the α-actinin-3 protein suggest the ACTN3 genotype influences the adaptive response to endurance exercise. Based on these findings, the aim of study 3 was to investigate if ACTN3 genotype influences exercise-induced changes in the content of genes and proteins associated with mitochondrial biogenesis. At baseline, there was a compensatory greater α-actinin-2 protein content in ACTN3 XX vs ACTN3 RR participants (p=0.018) but there were no differences in the endurance-related phenotypes measured. There was a main effect of genotype (p=0.006), without a significant interaction effect, for RCAN1-4 or significant exercise-induced expression of genes associated with mitochondrial biogenesis. Together, these results suggest that ACTN3 genotype has a small but significant influence on sprint speed amongst elite sprint athletes. However, loss of α-actinin-3 protein is not associated with higher values for endurance-related phenotypes, endurance performance, or a greater adaptive response to a single session of high-intensity endurance exercise.

It is well established that endurance exercise training leads to cardiovascular, skeletal muscle, and metabolic adaptations, with important implications for both athletic performance and health. While many studies have addressed the effects of endurance exercise training on such adaptations, very few have examined the role of post-exercise nutritional supplementation in facilitating or increasing the magnitude of the adaptive response. The beneficial effects of post exercise nutrition, in the form of carbohydrate and protein, following an acute bout of exercise has been the focus of many investigations. The results however remain equivocal due to methodological differences, such as the training status of participants, the treatment groups not being isocaloric, differing CHO contents, different protein sources, exercise modes and protocols, and outcome measurements all being different. The quality of the protein source used in post exercise supplementation may also affect training adaptations. Animal-source of proteins, such as milk and the constituent proteins of milk, casein and whey, are classified as being of high biological availability and quality. The types of proteins that are best for achieving muscle recovery and adaptations after endurance exercise are not defined. The significant aim of this PhD candidature was to determine the role of protein supplementation, when included in the training diet over an extended period, on endurance training adaptations. Along with investigating how different proteins affect the signalling pathways that regulate endurance training adaptations. As such the research presented in this thesis examines 8 weeks of supplementation, with and without an endurance training program, with micellar caseins or caseinates, whey protein isolates or a carbohydrate matched and isocaloric group in animal and human models. The first study demonstrated that after 8 weeks of supplementing with whey protein isolates, this group had lower body fat compared to the carbohydrate group at week 4 (P < 0.05) and week 8 (P < 0.05). The micellar caseins group had lower body fat compared to carbohydrate group only after 8 weeks (P < 0.05). A key finding in the second study was that despite matching all rodents across groups according to exercise performance, the carbohydrate supplemented animals were unable to perform for as long in the time to exhaustion test compared to both whey protein isolates and micellar caseins groups (P < 0.05); 14:19 ± 4 min, 29:81 ± 11 min and 25:51 ± 6 min.. There was no significant difference between protein groups in several measures including; enzyme activity of citrate synthase and β-hydroxyacyl-CoA dehydrogenase (βHAD), mitochondrial respiration or lean mass. The third study examined post-exercise supplementation with calcium caseinates compared to whey protein isolates in trained cyclists in a double-blind manner. Participants were provided all meals and snacks for the duration of the study. Endurance exercise performance, body composition and mitochondrial respiration, along with proteins involved in mitochondrial biogenesis showed no difference between protein groups after 8 weeks of supplementation and endurance training. This research has established that micellar caseins and whey protein isolates may have beneficial effects on body composition and mitochondrial function without exercise, however following exercise training these mitochondrial function differences are diminished. Despite this, animals supplemented with the different proteins and undertook an endurance training protocol for 8 weeks performed significantly better in the time to exhaustion test. Thus, it appears that this improved time to exhaustion is not related to improved mitochondrial function, but by some other, yet to be determined factor.

Objective: The purpose of this study was to investigate the effects of regular postexercise hot water immersion (HWI) on selected physiological adaptations and on exercise performance in a temperate environment in trained road cyclists. Methods: Fourteen male cyclists were assigned to either an HWI (n = 7) group or a control (CON, n = 7) group. Both groups completed 9 high-intensity interval training sessions (over 3 weeks), with each training session followed by sitting in a water tub for 30 min. Participants from the HWI group were immersed in 42°C water, whereas a thermoneutral water temperature of 34°C was used for the CON group. Core and intramuscular temperature were continuously recorded during the first water immersion session and for 30 min post-session. Before and after the intervention, the cyclists performed a 20-km time trial test and an incremental test to exhaustion to determine lactate turn point, maximal oxygen consumption and peak power output. Venous blood at rest was sampled pre- and post-intervention to assess changes in plasma volume. Muscles biopsies were obtained from the vastus lateralis pre- and post-intervention to assess changes in mitochondrial function. Variables were analysed using t-test and two-way repeated measures analysis of variance. Results: Intramuscular temperature was significantly higher in the HWI than in the CON group at the end of the water immersion treatment (37.8 ± 0.4 vs 36.2 ± 0.5 °C, p=0.001) and 30 min post-immersion (36.7 ± 0.4 vs 35.1 ± 1.2 °C, p=0.01). In addition, HWI group had significantly higher core temperature immediately post-immersion than the CON group (37.8 ± 0.4 vs 37.1 ± 0.2 °C, p=0.01). However, all other measures were not significantly different between the groups. Nevertheless, there was a significant improvement in 20-km time trial performance in both the HWI (2009.8 ± 147.3 vs 1977.5 ± 134.5 seconds, p=0.01) and the CON group (2010.4 ± 182.3 vs 1974.2 ± 185.7 seconds, p=0.04). Conclusion: Three weeks of high intensity interval training led to an improved 20-km time trial performance, but the post-exercise HWI protocol used in this study did not provide additional performance benefits.

Although life expectancy is increasing, this often comes at the cost of declining health through an increased incidence of cancer, cardiovascular disease and arthritis in older age. In addition, a decline in muscular performance is commonly observed with increasing age, combining a loss of skeletal muscle (‘sarcopenia’), a decrease in muscle oxidative capacity and a reduction in muscle strength. Research has shown that it is possible to arrest, or even reverse, the changes in muscle mass and oxidative capacity that occur with age. Two of the most successful strategies identified to date in this regard are exercise, in particular resistance-based training, and protein supplementation. We devised a series of four related studies to investigate and refine strategies for the prevention or mitigation of sarcopenia among the elderly.