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PurposeIn this study we investigate the disease etiology in 12 patients with de novo variants in FAR1 all resulting in an amino acid change at position 480 (p.Arg480Cys/His/Leu).MethodsFollowing next-generation sequencing and clinical phenotyping, functional characterization was performed in patients' fibroblasts using FAR1 enzyme analysis, FAR1 immunoblotting/immunofluorescence, and lipidomics.ResultsAll patients had spastic paraparesis and bilateral congenital/juvenile cataracts, in most combined with speech and gross motor developmental delay and truncal hypotonia. FAR1 deficiency caused by biallelic variants results in defective ether lipid synthesis and plasmalogen deficiency. In contrast, patients' fibroblasts with the de novo FAR1 variants showed elevated plasmalogen levels. Further functional studies in fibroblasts showed that these variants cause a disruption of the plasmalogen-dependent feedback regulation of FAR1 protein levels leading to uncontrolled ether lipid production.ConclusionHeterozygous de novo variants affecting the Arg480 residue of FAR1 lead to an autosomal dominant disorder with a different disease mechanism than that of recessive FAR1 deficiency and a diametrically opposed biochemical phenotype. Our findings show that for patients with spastic paraparesis and bilateral cataracts, FAR1 should be considered as a candidate gene and added to gene panels for hereditary spastic paraplegia, cerebral palsy, and juvenile cataracts.

As the knowledge base and importance of mitochondrial physiology to human health expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow guidelines of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of databases of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery.

Background: Mitochondrial diseases, a group of multi-systemic disorders often characterized by tissue-specific phenotypes, are usually progressive and fatal disorders resulting from defects in oxidative phosphorylation. MTO1 (Mitochondrial tRNA Translation Optimization 1), an evolutionarily conserved protein expressed in high-energy demand tissues has been linked to human early-onset combined oxidative phosphorylation deficiency associated with hypertrophic cardiomyopathy, often referred to as combined oxidative phosphorylation deficiency -10 (COXPD10).

Background Mitochondrial diseases, a group of multi-systemic disorders often characterized by tissue-specific phenotypes, are usually progressive and fatal disorders resulting from defects in oxidative phosphorylation. MTO1 (Mitochondrial tRNA Translation Optimization 1), an evolutionarily conserved protein expressed in high-energy demand tissues has been linked to human early-onset combined oxidative phosphorylation deficiency associated with hypertrophic cardiomyopathy, often referred to as combined oxidative phosphorylation deficiency-10 (COXPD10). Material and methods Thirty five cases of MTO1 deficiency were identified and reviewed through international collaboration. The cases of two female siblings, who presented at 1 and 2 years of life with seizures, global developmental delay, hypotonia, elevated lactate and complex I and IV deficiency on muscle biopsy but without cardiomyopathy, are presented in detail. Results For the description of phenotypic features, the denominator varies as the literature was insufficient to allow for complete ascertainment of all data for the 35 cases. An extensive review of all known MTO1 deficiency cases revealed the most common features at presentation to be lactic acidosis (LA) (21/34; 62% cases) and hypertrophic cardiomyopathy (15/34; 44% cases). Eventually lactic acidosis and hypertrophic cardiomyopathy are described in 35/35 (100%) and 27/34 (79%) of patients with MTO1 deficiency, respectively; with global developmental delay/intellectual disability present in 28/29 (97%), feeding difficulties in 17/35 (49%), failure to thrive in 12/35 (34%), seizures in 12/35 (34%), optic atrophy in 11/21 (52%) and ataxia in 7/34 (21%). There are 19 different pathogenic MTO1 variants identified in these 35 cases: one splice-site, 3 frameshift and 15 missense variants. None have bi-allelic variants that completely inactivate MTO1; however, patients where one variant is truncating (i.e. frameshift) while the second one is a missense appear to have a more severe, even fatal, phenotype. These data suggest that complete loss of MTO1 is not viable. A ketogenic diet may have exerted a favourable effect on seizures in 2/5 patients. Conclusion MTO1 deficiency is lethal in some but not all cases, and a genotype-phenotype relation is suggested. Aside from lactic acidosis and cardiomyopathy, developmental delay and other phenotypic features affecting multiple organ systems are often present in these patients, suggesting a broader spectrum than hitherto reported. The diagnosis should be suspected on clinical features and the presence of markers of mitochondrial dysfunction in body fluids, especially low residual complex I, III and IV activity in muscle. Molecular confirmation is required and targeted genomic testing may be the most efficient approach. Although subjective clinical improvement was observed in a small number of patients on therapies such as ketogenic diet and dichloroacetate, no evidence-based effective therapy exists., Highlights • The phenotypic and genotypic spectrum of MTO1 deficiency is more variable than previously reported • Hallmark features: cardiomyopathy, lactic acidosis, developmental delay, failure to thrive, seizures, optic atrophy, ataxia • 19 pathogenic MTO1 mutations (splice-site, frameshift, missense) were identified with a geno-phenotype relation • Suspect MTO1 deficiency based on clinical features, mitochondrial markers in body fluids, low complex I III IV activity • Although ketogenic diet exerted subjective improvement in some cases, there is exists no evidence-based effective therapy

OBJECTIVE:To assess impact of a 52-week elosulfase alfa enzyme replacement therapy (ERT) on exercise capacity in Morquio A patients and analyze cardiorespiratory and metabolic function during exercise to uncover exercise limitations beyond skeletal abnormalities. METHODS:Morquio A patients aged ≥7years, able to walk >200m in the 6-minute walk test (6MWT), received elosulfase alfa 2.0mg/kg/week (N=15) or 4.0mg/kg/week (N=10) for 52weeks in the randomized, double-blind MOR-008 study ( ClinicalTrials.gov NCT01609062) and its extension. Exercise capacity was assessed by 6MWT, 3-minute stair climb test (3MSCT), and cardiopulmonary exercise test (CPET; N=15 dosage groups combined). RESULTS:Changes over 52weeks in 6MWT and 3MSCT were minimal. Baseline CPET results showed impaired weight-adjusted peak oxygen uptake (VO2), partly attributable to inability to increase tidal volume during exercise. CPET measures of exercise function showed significant improvement at 25 and/or 52weeks in exercise duration, peak workload, O2 pulse, and peak tidal volume (% increases in duration, 16.9 (P=0.0045) and 9.4 (P=0.0807); peak workload, 26.5 (P=0.0026) and 21.2 (P=0.0132); O2 pulse, 10.7 (P=0.0187) and 2.3 (P=0.643); peak tidal volume, 11.7 (P=0.1117) and 29.1 (P=0.0142)). In addition, decreased VO2/work ratio was noted (% decrease -7.6 [-11.9, 1.3] and -9.2 [-25.7, 5.1]), indicating performance of work at reduced oxygen cost. CONCLUSIONS:CPET uncovers limitation in exercise capacity in Morquio A related to reduced lung function. ERT improves exercise capacity and efficiency of oxygen utilization, not attributable to changes in cardiac or pulmonary function. Further study of the long-term impact of ERT on exercise capacity and the clinical relevance of the observed changes is warranted.

International audience; OBJECTIVE: Differential diagnosis of autosomal recessive cerebellar ataxias can be challenging. A ranking algorithm named RADIAL that predicts the molecular diagnosis based on the clinical phenotype of a patient has been developed to guide genetic testing and to align genetic findings with the clinical context. METHODS: An algorithm that follows clinical practice, including patient history, clinical, magnetic resonance imaging, electromyography, and biomarker features, was developed following a review of the literature on 67 autosomal recessive cerebellar ataxias and personal clinical experience. Frequency and specificity of each feature were defined for each autosomal recessive cerebellar ataxia, and corresponding prediction scores were assigned. Clinical and paraclinical features of patients are entered into the algorithm, and a patient's total score for each autosomal recessive cerebellar ataxia is calculated, producing a ranking of possible diagnoses. Sensitivity and specificity of the algorithm were assessed by blinded analysis of a multinational cohort of 834 patients with molecularly confirmed autosomal recessive cerebellar ataxia. The performance of the algorithm was assessed versus a blinded panel of autosomal recessive cerebellar ataxia experts. RESULTS: The correct diagnosis was ranked within the top 3 highest-scoring diagnoses at a sensitivity and specificity of \textgreater90% for 84% and 91% of the evaluated genes, respectively. Mean sensitivity and specificity of the top 3 highest-scoring diagnoses were 92% and 95%, respectively. The algorithm outperformed the panel of ataxia experts (p = 0.001). INTERPRETATION: Our algorithm is highly sensitive and specific, accurately predicting the underlying molecular diagnoses of autosomal recessive cerebellar ataxias, thereby guiding targeted sequencing or facilitating interpretation of next-generation sequencing data. Ann Neurol 2017;82:892-899.

Endurance exercise confers complete phenotype protection, suppresses early mortality, mitigates mitochondrial ROS-mediated oxidative damage, increases cellular antioxidant capacity, and 4 prevents cellular senescencmutator mice. Figure S2. Endurance exercise prevents dysregulated mitochondrial-induced apoptosis and reduces nuclear p53-mediated repression of PGC-1Îą and promotes mitochondrial biogenesis in mutator mice. Figure S3. Endurance exercise promotes systemic mitochondrial biogenesis in mtDNA mutator mice. Figure S4. Magnitude of mitochondrial ROS (physiological vs. pathological) regulates p53 subcellular localization. Figure S5. Pre-treatment with exogenous antioxidant preferentially shuttles p53 to mitochondria in response to stress. Figure S6. Endurance exercise-mediated attenuation of sarcopenia, increase in endurance capacity, skeletal muscle mitochondrial biogenesis, and repair of muscle mtDNA mutations is p53 dependent. Table S1. WT, PolG-SED, and PolG-END Skeletal Muscle Microarray IPA-GO Analysis. Table S2. Real-time PCR primer sequences. (PDF 1601Â kb)

The primary treatment outcomes of a phase 2, randomized, double-blind, pilot study evaluating safety, physiological, and pharmacological effects of elosulfase alfa in patients with Morquio A syndrome are herewith presented. Patients aged ≥7 years and able to walk ≥200 m in the 6-min walk test (6MWT) were randomized to elosulfase alfa 2.0 or 4.0 mg/kg/week for 27 weeks. The primary objective was to evaluate the safety of both doses. Secondary objectives were to evaluate effects on endurance (6MWT and 3-min stair climb test [3MSCT]), exercise capacity (cardio-pulmonary exercise test [CPET]), respiratory function, muscle strength, cardiac function, pain, and urine keratan sulfate (uKS) levels, and to determine pharmacokinetic parameters. Twenty-five patients were enrolled (15 randomized to 2.0 mg/kg/week and 10 to 4.0 mg/kg/week). No new or unexpected safety signals were observed. After 24 weeks, there were no improvements versus baseline in the 6MWT, yet numerical improvements were seen in the 3MSCT with 4.0 mg/kg/week. uKS and pharmacokinetic data suggested no linear relationship over the 2.0-4.0 mg/kg dose range. Overall, an abnormal exercise capacity (evaluated in 10 and 5 patients in the 2.0 and 4.0 mg/kg/week groups, respectively), impaired muscle strength, and considerable pain were observed at baseline, and there were trends towards improvements in all domains after treatment. In conclusion, preliminary data of this small study in a Morquio A population with relatively good endurance confirmed the acceptable safety profile of elosulfase alfa and showed a trend of increased exercise capacity and muscle strength and decreased pain.

Previous work has demonstrated that resistance exercise may increase dietary protein (PRO) requirements to a varying degree. Much of these discrepancies probably arose from differences in training intensity, classification of exercise, habituation to a training stimulus, and methodological considerations such as: 1. inadequate dietary adaptation periods; 2. failure to measure routes of nitrogen (N) loss (i.e., sweat); 3. failure to measure the dietary PRO (nitrogen (N)) content; 4. collection periods too short to measure an impact of exercise (i.e., delay in N excretion due to dehydration); and 5. the use of measurements insensitive to short-term alterations in PRO metabolism (i.e., lean body mass estimate from skinfolds to assess changes in muscle mass over a week of treatment). In addition, there is a paucity of information quantitating the influence of acute and chronic resistance exercise on PRO turnover. The overall purpose of this thesis was to examine N metabolism and the PRO requirements of resistance athletes, under well controlled conditions, with three discrete, but related studies. The first investigation was a randomized, cross-over study designed to determine the dietary PRO requirements of young men performing intensive body building resistance exercise during the early stages of training (novice) and to determine whether the consumption of excessive amounts of PRO had an ergogenic (work enhancing) effect upon muscle mass/strength gains. Twelve inexperienced, young, male volunteers received either a PRO supplement (total PROIN=2.62 g·kg⁻¹·d⁻¹) or a carbohydrate (CHO) supplement (total PROIN=1.35 g·kg⁻¹·d⁻¹) each for a period of 4 weeks during intensive (1.5 h·d⁻¹, 6 d·wk⁻¹) circuit weight training. Nitrogen balance (NBAL) measurements taken over the last 3 days on each diet were used to determine that the PRO intake (PROIN) for zero NBAL was 1.43 g·kg⁻¹·d⁻¹ (+1SD=1.62 g·kg⁻¹·d⁻¹ (recommended intake)). Pre- and post-training measurements of strength and estimates of muscle mass (density, creatinine excretion, and biceps muscle N content) were not different between diet treatments in spite of a significant training effect (increased strength and lean body mass) due to the exercise programme. It was recommended that young males performing body building-type resistance exercise require a dietary PROIN of 1.62 g·kg⁻¹·d⁻¹ during the first 2 months of training and that there are no greater increases in strength or muscle mass by consuming PRO in excess of this amount. The acute effects of resistance exercise upon leucine oxidation and whole body PRO synthesis (WBPS) were studied using stable isotope methodology in the second study. L-[1-¹³C] leucine was used as a tracer to calculate these variables in 6 healthy, fed, male athletes in response to a 1 h bout of circuit-set resistance exercise. The measurements were performed prior to, during and for 2 h after exercise and corrections were made for the background ¹³CO₂/¹²CO₂ breath enrichment and bicarbonate retention factor (c). Results demonstrated significant increases in the background ¹³CO₂/¹²CO₂ breath enrichment at 1 and 2 h after exercise and in c during exercise. At 15 min after exercise c was significantly lower than at rest. There were no effects of exercise on leucine oxidation, WBPS, nor the rate of appearance of endogenous leucine or total leucine flux. We concluded that circuit-set resistance exercise did not affect the measured variables of leucine metabolism. In addition, large errors in calculating leucine oxidation and WBPS during resistance exercise can occur if background ¹³CO₂/¹²CO₂ breath enrichment and c are not accounted for. In the final study, leucine kinetics and NBAL were used to determine the dietary PRO requirements of sedentary (S) and resistance trained (BB) subjects. Each subject was randomly assigned to each of 3 dietary PROIN (LP=0.86; MP=1.40; HP=2.40 g PRO·kg⁻¹·d⁻¹) for a total period of 13 days. Over the last 3 d, NBAL measurements were completed and on the last day WBPS and leucine oxidation were determined from L-[1-¹³C] leucine turnover. Regression analysis of the NBAL data was used to determine the PROIN for zero NBAL for S=0.69 g·kg⁻¹·d⁻¹ and BB=1.41 g·kg⁻¹·d⁻¹ and a recommended intake (ZERO intake +1 SD) for S=0.89 g·kg⁻¹·d⁻¹ and BB=1.76 g·kg⁻¹·d⁻¹. For BB the LP diet did not provide adequate PRO and resulted in an accommodated state (↓ WBPS vs MP and HP), the MP diet resulted in a state of adaptation as evident by the increase in WBPS (vs LP) and no increase in leucine oxidation (vs LP), while the HP diet did not result in increased WBPS compared to the MP diet but leucine oxidation did increase significantly indicating a nutrient overload. For S the LP diet provided adequate PRO and increasing PROIN did not increase WBPS. Leucine oxidation increased for S on HP diet. Taken together, these results indicated that the MP and HP diets were nutrient overloads for S. There were no effects of dietary treatment on indices of lean body mass (creatinine excretion, body density) for either group. Overall, this research provided evidence that the PRO requirements of resistance athletes are greater than for sedentary individuals and are above current Canadian and U.S. recommended daily PROIN for young, healthy males. The second study demonstrated that acute resistance exercise did not affect whole body leucine turnover and, in contrast to endurance exercise, there was no significant increase in leucine oxidation during exercise. In addition, this study demonstrated that changes occur in c and background breath enrichment during and after resistance exercise that can have an impact on leucine oxidation measurements. The final study also demonstrated that the positive NBAL that occurs at high PROIN does not appear to represent a physiological entity (i.e., no increase in WBPS or lean mass) but is probably an error in the NBAL method. Doctor of Philosophy (PhD)

Table S1. SCA29 Questionnaire. Table S2. Assessment of pathogenicity for ITPR1 variants identified in this study. (DOCX 1606 kb)

Table S1. SCA29 Questionnaire. Table S2. Assessment of pathogenicity for ITPR1 variants identified in this study. (DOCX 1606 kb)

BACKGROUND: GNE myopathy is a rare, autosomal recessive, muscle disease caused by mutations in GNE and is characterized by rimmed vacuoles on muscle biopsy and progressive distal to proximal muscle weakness. OBJECTIVE: Investigate the clinical presentation and progression of GNE myopathy. METHODS: The GNE Myopathy Disease Monitoring Program was an international, prospective, observational study in subjects with GNE myopathy. Muscle strength was assessed with hand-held dynamometry (HHD), with upper extremity (UE) and lower extremity (LE) composite scores reflecting upper and lower extremity muscle groups, respectively. The GNE myopathy-Functional Activity Scale (GNEM-FAS) was used to further assess impairment in mobility, upper extremity function, and self-care. RESULTS: Eighty-seven of 101 enrolled subjects completed the trial until study closure by the sponsor; 60 completed 36 months. Mean (SD) HHD UE composite score decreased from 34.3 kg (32.0) at baseline to 29.4 kg (32.6) kg at month 36 (LS mean change [95%CI]: -3.8 kg [-5.9, -1.7]; P = 0.0005). Mean (SD) HHD LE composite score decreased from 32.0 kg (34.1) at baseline to 25.5 kg (31.2) at month 36 (LS mean change [95%CI]: -4.9 [-7.7, -2.2]; P = 0.0005). GNEM-FAS scores were more severe at baseline in subjects who walked

BACKGROUND: Mitochondrial diseases, a group of multi-systemic disorders often characterized by tissue-specific phenotypes, are usually progressive and fatal disorders resulting from defects in oxidative phosphorylation. MTO1 (Mitochondrial tRNA Translation Optimization 1), an evolutionarily conserved protein expressed in high-energy demand tissues has been linked to human early-onset combined oxidative phosphorylation deficiency associated with hypertrophic cardiomyopathy, often referred to as combined oxidative phosphorylation deficiency-10 (COXPD10). MATERIAL AND METHODS: Thirty five cases of MTO1 deficiency were identified and reviewed through international collaboration. The cases of two female siblings, who presented at 1 and 2years of life with seizures, global developmental delay, hypotonia, elevated lactate and complex I and IV deficiency on muscle biopsy but without cardiomyopathy, are presented in detail. RESULTS: For the description of phenotypic features, the denominator varies as the literature was insufficient to allow for complete ascertainment of all data for the 35 cases. An extensive review of all known MTO1 deficiency cases revealed the most common features at presentation to be lactic acidosis (LA) (21/34; 62% cases) and hypertrophic cardiomyopathy (15/34; 44% cases). Eventually lactic acidosis and hypertrophic cardiomyopathy are described in 35/35 (100%) and 27/34 (79%) of patients with MTO1 deficiency, respectively; with global developmental delay/intellectual disability present in 28/29 (97%), feeding difficulties in 17/35 (49%), failure to thrive in 12/35 (34%), seizures in 12/35 (34%), optic atrophy in 11/21 (52%) and ataxia in 7/34 (21%). There are 19 different pathogenic MTO1 variants identified in these 35 cases: one splice-site, 3 frameshift and 15 missense variants. None have bi-allelic variants that completely inactivate MTO1; however, patients where one variant is truncating (i.e. frameshift) while the second one is a missense appear to have a more severe, even fatal, phenotype. These data suggest that complete loss of MTO1 is not viable. A ketogenic diet may have exerted a favourable effect on seizures in 2/5 patients. CONCLUSION: MTO1 deficiency is lethal in some but not all cases, and a genotype-phenotype relation is suggested. Aside from lactic acidosis and cardiomyopathy, developmental delay and other phenotypic features affecting multiple organ systems are often present in these patients, suggesting a broader spectrum than hitherto reported. The diagnosis should be suspected on clinical features and the presence of markers of mitochondrial dysfunction in body fluids, especially low residual complex I, III and IV activity in muscle. Molecular confirmation is required and targeted genomic testing may be the most efficient approach. Although subjective clinical improvement was observed in a small number of patients on therapies such as ketogenic diet and dichloroacetate, no evidence-based effective therapy exists.

Funder: Howard Hughes Medical Institute, Missense variants in RNA-binding proteins (RBPs) underlie a spectrum of disease phenotypes, including amyotrophic lateral sclerosis, frontotemporal dementia, and inclusion body myopathy. Here, we present ten independent families with a severe, progressive muscular dystrophy, reminiscent of oculopharyngeal muscular dystrophy (OPMD) but of much earlier onset, caused by heterozygous frameshift variants in the RBP hnRNPA2/B1. All disease-causing frameshift mutations abolish the native stop codon and extend the reading frame, creating novel transcripts that escape nonsense-mediated decay and are translated to produce hnRNPA2/B1 protein with the same neomorphic C-terminal sequence. In contrast to previously reported disease-causing missense variants in HNRNPA2B1, these frameshift variants do not increase the propensity of hnRNPA2 protein to fibrillize. Rather, the frameshift variants have reduced affinity for the nuclear import receptor karyopherin β2, resulting in cytoplasmic accumulation of hnRNPA2 protein in cells and in animal models that recapitulate the human pathology. Thus, we expand the phenotypes associated with HNRNPA2B1 to include an early-onset form of OPMD caused by frameshift variants that alter its nucleocytoplasmic transport dynamics.

Contains fulltext : 174061.pdf (Publisher’s version ) (Closed access) OBJECTIVES: The common data elements (CDE) project was developed by the National Institute of Neurological Disorders and Stroke (NINDS) to provide clinical researchers with tools to improve data quality and allow for harmonization of data collected in different research studies. CDEs have been created for several neurological diseases; the aim of this project was to develop CDEs specifically curated for mitochondrial disease (Mito) to enhance clinical research. METHODS: Nine working groups (WGs), composed of international mitochondrial disease experts, provided recommendations for Mito clinical research. They initially reviewed existing NINDS CDEs and instruments, and developed new data elements or instruments when needed. Recommendations were organized, internally reviewed by the Mito WGs, and posted online for external public comment for a period of eight weeks. The final version was again reviewed by all WGs and the NINDS CDE team prior to posting for public use. RESULTS: The NINDS Mito CDEs and supporting documents are publicly available on the NINDS CDE website ( https://commondataelements.ninds.nih.gov/ ), organized into domain categories such as Participant/Subject Characteristics, Assessments, and Examinations. CONCLUSION: We developed a comprehensive set of CDE recommendations, data definitions, case report forms (CRFs), and guidelines for use in Mito clinical research. The widespread use of CDEs is intended to enhance Mito clinical research endeavors, including natural history studies, clinical trial design, and data sharing. Ongoing international collaboration will facilitate regular review, updates and online publication of Mito CDEs, and support improved consistency of data collection and reporting.

Mitochondrial dysfunction has been linked to a number of age-associated disorders and recent evidence has shown that mitochondrial function is necessary for osteoblast differentiation and osteoclast survival. Although bone tissue was once thought to be an ‘inert’ tissue, research from the past decade in bone metabolism has demonstrated pathways linking bone and whole body energy metabolism. However, with mitochondria being the central energy provider in cells there is a paucity of research exploring the impact of mitochondrial function on bone tissue, with the bulk of work focused in cell culture. This thesis aimed to explore mitochondrial content and function after endurance training and an exhaustive bout of running in two different animal models. The first study explored the effect of progressive mitochondrial dysfunction on mitochondrial gene expression and bone strength in the polymerase gamma mouse model of mitochondrial dysfunction. The therapeutic efficacy of endurance training on mitochondrial parameters and bone strength were evaluated as well. The second study utilized the Koch-Britton rat model of low capacity and high capacity runners. This model has demonstrated that selection for low and high running capacity has led to a significant divergence in mitochondrial content and function between these two groups. Mechanical strain in the form of exercise has been established as playing a key role in regulating bone health however the underlying mechanisms are still being described. The untrained status and inherent differences in aerobic capacity allow for the elucidations of bone adaptations that may be a result of aerobic capacity. The effect of an exhaustive bout of exercise on mitochondrial gene expression and enzyme activity in LCR and HCR rats was studied. Taken together these studies demonstrate that exercise is beneficial for promoting bone health and may do so by altering mitochondrial content and stress resistance through the FoxO family of transcription factors. Doctor of Philosophy (Medical Science)

This thesis investigated the acute and chronic responses of human skeletal muscle to high-intensity exercise, with a particular focus on markers of mitochondrial content, and the potential for nutrition to manipulate the adaptive response in recreationally active individuals. The acute response was primarily assessed via measurement of signalling proteins and mRNA species linked to exercise-induced mitochondrial biogenesis. The chronic response was determined via changes in the protein content or maximal activities of mitochondrial enzymes after training. Study 1 examined whether the manner in which a given amount of high-intensity cycling work was performed (i.e., in an intermittent or continuous fashion) altered the acute metabolic response to exercise, and whether the acute response was indicative of longer-term adaptations. Despite the similar acute activation of signalling proteins after the intermittent and continuous matched-work exercise protocols, 6 wk of training with the continuous protocol did not increase mitochondrial content, contrary to what we have previously shown after 6 wk training with the intermittent protocol. This suggests that the intermittent application of a low-volume, high-intensity stimulus is important to elicit training-induced increases in mitochondrial content. Furthermore, Study 1 showed that acute changes in specific signalling proteins did not necessarily predict chronic adaptations. Studies 2 and 3 examined whether specific nutritional interventions, previously shown to modulate acute exercise capacity or metabolic response, altered the mitochondrial adaptive response to several weeks of HIT. Neither manipulating carbohydrate availability between twice daily training sessions, or chronic ingestion of β-alanine, augmented skeletal muscle adaptations in response to 2-6 wk of HIT. It is possible that small influences of nutrition were overwhelmed by the potency of HIT, which stimulated marked increases in mitochondrial content in this population. Overall this thesis advances our basic understanding of the skeletal muscle adaptive response to HIT and the influence of nutrition. Doctor of Philosophy (PhD)

Introduction: Mitochondrial dysfunction and oxidative stress increase with aging and may contribute to age-associated muscle atrophy (sarcopenia). Resistance exercise (RE) can promote the accretion of muscle mass, increase strength, and ultimately improve function in the elderly. Such beneficial effects are thought to be mitigated solely by increased muscle mass and strength; however, the contribution of the mitochondria to the beneficial effects of RE in aging have not been thoroughly characterized. While mitochondrial benefits have been established separately in both young and aged adults following chronic RE, the acute effects have not been well characterized. Methods: Sedentary young and aged adult males completed either an acute bout of fatiguing RE or endurance exercise (EE), and muscle biopsies were obtained at 3, 24 and 48 h post- exercise depending on the study. Results: Despite equivalent lean-body mass, increased age was associated with elevated mtDNA deletions, indicating potential for mitochondrial dysfunction. RE was associated with reduced mitochondrial content (transcripts, protein, and mtDNA copy number) at 48 h post-exercise, a response that did not differ with increasing age. Paradoxically, reduced mitochondrial content occurred alongside elevated total peroxisome proliferator-activated receptor γ coactivator one α (PGC-1α) mRNA; however, RE altered only the PGC-1α4 isoform post-exercise, a transcript that regulates myostatin and insulin-like growth factor one (IGF1) signalling and ultimately muscle hypertrophy and not mitochondrial adaptations. In addition, PGC-1α modulates the unfolded protein response (UPR), and RE was subsequently shown to elevate endoplasmic reticulum stress and elicit the UPR. Conclusion: PGC-1α mRNA increases regardless of exercise mode; however, differential expression or regulation of alternate PGC-1α isoforms or transcriptional binding partners co-activated by PGC-1α may dictate the specific phenotypic adaptations that occur following divergent modes of exercise. Furthermore, RE acutely decreases mitochondrial content despite elevated PGC-1α mRNA, and this response is not influenced by age. Doctor of Philosophy (Medical Science)

Introduction: Mitochondria are important organelles for skeletal muscle function. Mitochondria are susceptible to many forms of stress that alters their morphology, energy generation and reactive oxygen species (ROS) production, which collectively promote degeneration and dysfunction in skeletal muscle. These processes are implicated in many health disorders, particularly in the aging process itself. Exercise is well established to increase muscle mitochondrial content and thus may attenuate several aspects of mitochondrial deterioration. Methods: Both human and animal models of mitochondrial stress (aging, ROS) were utilized in order to determine their effects on mitochondrial and muscle function. Additionally, exercise training was used in order to assess its therapeutic potential in ameliorating defects in oxidative capacity, muscle atrophy and metabolic adaptation in skeletal muscle. Results: Aging resulted in reduced strength, aerobic capacity, larger intramyocelluar lipid droplets and fewer mitochondria in skeletal muscle. These changes were related to suppressed lipid metabolism, mitochondrial dynamics and organelle turnover. Habitual aerobic exercise partially attenuated the age-related loss of muscle strength and aerobic capacity, presumably due in part to improved mitochondrial function. Persistent mitochondrial oxidative stress prevented mitochondrial adaptations to exercise training in mice, a phenomenon that may explain why exercise cannot fully counteract the effects of aging in skeletal muscle. Conclusions and significance: This work furthers our knowledge of the mitochondrial consequences of aging and the therapeutic potential of aerobic exercise within skeletal muscle. These results can be applied to other differentiated tissues that are severely affected by aging (brain, heart) and the effects described here are likely relevant to other conditions that result in muscle atrophy and energetic insufficiency. Doctor of Philosophy (PhD)

This thesis examined the biochemical role of skeletal muscle mitochondria and metabolic consequences of mitochondrial adaptations to exercise in individuals with poor glycemic control. Mitochondrial dysfunction and/or ectopic lipid accumulation has been implicated in the pathogenesis of metabolic-related diseases such as obesity and type 2 diabetes (T2D). However, whether mitochondrial dysfunction is the cause of insulin resistance and T2D or is a consequence of this disorder remains controversial. Alternatively, pro-inflammatory stress signals initiated through altered secretion of adipocytokines and oxidative stress may be a unifying mechanism underlying insulin resistance and T2D. Furthermore, the impact of exercise on muscle adaptation in insulin-resistant states is not well defined. At rest and prior to exercise training, no evidence of mitochondrial dysfunction or disproportionate intramyocellular lipid (IMCL) accretion was detected in obese, insulin-resistant skeletal muscle biopsy samples vs. healthy, lean age-, and fitness-matched men. In response to exercise training (12 weeks, consisting of 32 sessions of 30-60 min @ 50-70% maximal oxygen uptake [VO2peak]), there was an increase in mitochondrial oxidative phosphorylation (OXPHOS) capacity, mitochondrial content, and IMCL deposition with sub-cellular specificity. Exercise training also reduced both skeletal muscle and systemic oxidative damage, already elevated in the obese. The improved adipocytokine profile associated with obesity after training also coincided with improvements in glycemic regulation. Patients with genetic mitochondrial mutations, resulting in skeletal muscle mitochondrial dysfunction have an increase prevalence of dysglycemia/T2D. However, when evaluated against age- and activity-matched normoglycemic myopathy controls, no differences in mitochondrial electron transport chain protein subunits, mitochondrial or IMCL density, or level of whole-body insulin resistance was detected. In fact, dysglycemic mitochondrial myopathy patients demonstrated higher skeletal muscle OXPHOS capacity and Akt activation, a key step in insulin-stimulated glucose transport activity as compared with normoglycemic mitochondrial myopathy patients. Interestingly, a significant impairment in β-cell function (defective insulin secretion), in the dysglycemic patients was observed coincident with elevated glucose levels during the oral glucose tolerance test (OGTT). These findings indicate that insulin resistance does not cause skeletal muscle mitochondrial dysfunction/IMCL accumulation or vice versa and provides evidence against a direct link between mitochondrial dysfunction and the development of insulin resistance/T2D. Perhaps, oxidative stress/inflammation and pancreatic β-cell erosion mediate the observed obesity-induced insulin resistance and mitochondrial myopathy-associated T2D, respectively? Twelve weeks of moderate endurance exercise is an effective strategy to improve mitochondrial capacity, oxidative damage, inflammation, and glycemic regulation in insulin-resistant, obese individuals, but an improvement in muscle insulin sensitivity did not appear to be required. Doctor of Philosophy (Medical Science)

Skeletal muscle exhibits a remarkable capacity for growth and regeneration in response to physiological stimuli. This extensive plasticity is, in part, due to a tissue-resident stem cell called the satellite cell. Satellite cells respond to myotrauma by upregulating a class of transcriptional networks which orchestrate myogenic specification. This process is controlled by four main transcription factors known as the myogenic regulatory factors: Myf5, MyoD, MRF4 and Myogenin. Satellite cells respond to molecular cues released from the muscle fiber or inflammatory cells in response to muscle damage. Although several regulators have been implicated in the control of the satellite cell response to exercise or damage, very few of these have been examined in humans. Insulin-like growth factor-1 (IGF-1) and Interleukin-6 (IL-6) have been demonstrated to enhance satellite cell proliferation in animal and cell culture models. IGF-1 has also been shown to induce myogenic differentiation, however little is known about IGF-1 and IL-6 in humans, in response to physiological levels of muscle damage. Myostatin has been identified as a negative regulator of muscle growth and an inhibitor of satellite cells in mice. To date no data exists regarding the relation of myostatin to the satellite cell response to exercise and in the context of aging. The work outlined in this thesis provides support for the proposed divergent effects of the IGF-1 splice variants on satellite cell function. IGF-1 appears to be preferentially spliced as IGF-1Ec during the proliferative phase of the myogenic program while IGF-1Ea and Eb appear as the predominant splice variants during the initiation of differentiation based on the expression of the MRFs. Furthermore, the localization of IGF-1 with Pax7 in muscle-cross sections in the post-exercise time-course lends support to the importance of IGF-1 in the myogenic response to myotrauma. This thesis also provides novel evidence to support the role of IL-6 in the regulation of satellite cell proliferation in response to acute muscle damage in humans. These data confirm that IL-6 imparts its action on the satellite cell via the JAK2/STAT3 pathway. In addition, for the first time, myostatin is demonstrated to be altered by acute exercise in both young and older adults and this effect is most notable in the satellite cell compartment. In addition, these data implicate myostatin as a contributing factor to age-related satellite cell dysfunction in response to exercise (or myotrauma). Doctor of Philosophy (PhD)

The purpose of this investigation was to examine the physiological effects of massage on indicators of skeletal muscle function following exhaustive endurance exercise. Specifically, we examined the effects of unilateral leg massage therapy on pain, muscle lactate, glycogen and damage, before (BASELINE) and at an early (EARLY, +30 min), and later (LATE, +2.5h) time points, following a single bout of exhaustive endurance exercise. Thirteen young, recreationally active males who exercise 2-3 times per week (age: 22.0 ± 2.7 y; weight: 75.2 ± 10.6 kg (mean ± SD)) were recruited to participate in the study. Participants underwent a baseline muscle biopsy of the vastus lateralis of one randomly assigned leg and performed a maximal oxygen uptake test (VO2peak) using an incremental cycle ergometry test to voluntary exhaustion (mean VO2peak = 45.9 ± 7.5 mL/kg/min, peak power = 270 ± 47 W). Two weeks later, the participants returned to the laboratory and completed an exhaustive endurance exercise protocol. Following the cycling protocol, participants were given 10 min of recovery, and were asked to report the pain levels of each of their quadriceps muscles using a visual analog scale (0 = no pain; 10 = worst pain imaginable) at two min post-exercise (post-exercise and pre-intervention (PRE)). A single Registered Massage Therapist randomly selected one leg for each participant to be massaged, while the other leg did not receive any treatment modality. Pain scores were again reported two min post-massage (post- intervention (POST)). Two muscle biopsies were taken from each leg (MAS and CON leg) on the second visit following completion of the endurance exercise protocol at +30 min and +2.5 h. There was no difference in postexercise/pre-massage pain scores between the legs (CON = 7.4 ± 2.0; MAS = 7.4 ± 2.0); however, post-exercise/post-massage pain scores were lower in the massaged leg (MAS = 2.3 ± 1.8; CON = 3.9 ± 2.3, P < 0.001). Massage had no effect on muscle lactate concentration, damage, or glycogen content, at any time point. In conclusion, massage therapy treatment following exhaustive endurance exercise was shown to lower pain scores but had no effect on muscle lactate, damage or glycogen content. These findings support the concept that post-endurance exercise massage therapy lowers pain perception but not through alterations in muscle lactate concentration, damage or glycogen content; consequently, the reduction in pain is more likely due to neurological and/or psychological effects associated with massage. Master of Science (MS)

Introduction: A higher rate of fat and lower rates of carbohydrate and protein oxidation are often observed during endurance exercise in women vs. men at the same relative workload. 17~-estradiol (E2) supplementation increases the oxidation of lipid, and reduces the oxidation of carbohydrate and amino acids in men. Women have a higher area percentage (%) of type I skeletal muscle fibers as compared with men. Consequently, we hypothesized that (i) sex, menstrual cycle phase and E2 would influence basal and exercise induced mRNA content for genes involved in substrate metabolism in human skeletal muscle; and (ii) sex would influence the basal mRNA content for genes involved in the determination of muscle fiber type. Methods: For studies concerning substrate selective utilization, 12 men and 12 women had muscle biopsies taken from their vastus lateralis before and after a 90 min bout of cycle exercise at 65 % V02peak. In a repeated measures design, men were randomly allocated to both E2 and placebo. Women were studied in the mid-follicular (FOL) and mid-luteal (LUT) phases ('" 2 wk apart). For studies concerning muscle fiber determination, a total of 24 women and 22 men had muscle biopsies taken from their vastus lateralis at rest. Twelve of the women were studied in mid-LUT, and 12 were studied in both the mid-FOL and mid-LUT, phases of the menstrual cycle. mRNA content was determined using TaqMan® real-time RT-PCR. Muscle fiber composition in 12 men and 12 women was determined from ATPase staining. Results: Exercise increased (P < 0.05) the mRNA content of peroxisome proliferator activated receptor gamma coactivator-1 alpha (PGC- 1a) (men, LUT) , cytosol fatty acid binding protein (FABPc) (men, FOL, LUT), carntine palmitoyltransferase I (CPTI) (men, LUT), long chain acyl-CoA dehydrogenase (LCAD) (LUT), hexokinase II (HKIl), asparatate aminotransferase (AST) and glucose transporter 4 (GLUT4) (men and women). Women had a higher (P ~ 0.05) mRNA content of membrane bound fatty acid transport protein 1 (F ATm), F ABPc, sterol regulatory element - binding protein 1 c (SREBP-l c) and mitochondrial glycerol phosphate acyltransferase (mtGPAT) (FOL, LUT), peroxisome proliferator activated receptor alpha (PPARa) (FOL), CPTI (FOL), LCAD (FOL), trifunctional protein alpha subunit (TFPa) (LUT), HKIl (FOL) and branched-chain 2-oxo acid dehydrogenase kinas (BCOADK) (FOL, LUT) pre- and post- exercise, peroxisome proliferator activated receptor delta (PPAR8) (FOL) and GLUT4 (FOL) at rest, and PPARa ( LUT) and LCAD (LUT) after exercise, and lower mRNA content for phosphofructokinase (PFK) (LUT) pre- and postexercise, than men. FOL women had a higher (P < 0.05) PPAR8, LeAD and glycogen phosphorylase and lower glycogenin mRNA content pre- and post- exercise, and tended to have higher GLUT4 mRNA content (P = 0.056) at rest, vs. LUT women. Postexercise FABPc mRNA content increased to a greater extent in LUT vs. FOL phase (P = 0.0001). E2 increased mRNA content of PPARa, CPTI, SREBP-lc, mtGPAT, LCAD, TFP-a, GLUT4 and GS-l (P < 0.05) in men at rest and during exercise, and PPAR8 at rest (P < 0.05). E2 attenuated the exercise induced increase in PGC-la mRNA content (P < 0.05). Women had lower mean area of individual fibers for type Ila and type Ilx, and a higher area % of type I fibers and lower area % of type II fibers vs. men (P < 0.05). Women had higher mRNA content for myosin heavy chain I (MHCI) (FOL, LUT, P ~ 0.05) and PPAR8 (FOL, P < 0.05), and a similar mRNA content of myosin heavy chain Ila (MHCIla), myosin heavy chain Ilx (MHCIlx), peroxisome proliferator activated receptor gamma coactivator-l alpha (PGC-l a), myostatin and PP AR8 (LUT), vs. men at rest. FOL women had a higher mRNA content of PPAR8 than LUT women (P = 0.0007). Menstrual cycle phase did not significantly alter the mRNA content of MHCI, PGC-l a or myostatin. Conclusions and Significance: We have comprehensively evaluated the effect of sex, acute endurance exercise, menstrual cycle and E2 on the content of mRNA species involved in substrate turnover in human skeletal muscle and found these changes directionally support a higher fat oxidation and lower protein oxidation in women vs. men. We have also evaluated the sex influences on the mRNA content of the genes involved in muscle fiber determination and found the change in PPAR8 mRNA content supports a greater area % of type I fibers in women than men. Doctor of Philosophy (PhD)

Gender differences in substrate utilization have been observed with females having higher lipid and less carbohydrate oxidation during endurance exercise. This thesis consists of a series of three studies conducted with the overall objective of advancing our understanding of gender differences in substrate metabolism and the mechanism behind these differences. The first and second studies investigated the effect of endurance training on whole-body substrate, glucose and glycerol utilization muscle enzyme adaptations in males and females. Carbohydrate utilization was lower following endurance training when tested at the same absolute exercise intensity. Glucose rate of appearance (Ra) and metabolic clearance rate (MCR) were lower following training. Females had a lower glucose MCR later in exercise as compared to males. Regardless of training status, females had a higher glycerol Ra as compared to males suggesting a greater whole-body lipolysis. Gender differences in lipid utilization were not accompanied by gender differences in maximal enzyme activities, which increased following endurance training for both genders. The third study investigated the effect of 17-β-estradiol on whole-body substrate, glucose and glycerol utilization during endurance exercise in males. The administration of 17-β-estradiol resulted in a lower MCR during exercise as compared to placebo. 17-β-estradiol also resulted in a higher plasma glucose concentration during exercise, but had no effect on substrate oxidation, glycerol turnover or plasma glycerol and FFA concentrations at rest or during endurance exercise. It can be concluded that females have a higher whole-body lipolysis and oxidize a greater proportion of fat. The glucose kinetic data from the training study together with the data from the 17-β-estradiol study suggested that females used less hepatic glucose during endurance exercise. Although alterations in glucose kinetics were found following the administration of 17-β-estradiol to males further research is required to unravel the mechanism(s) behind the gender difference in substrate metabolism. Doctor of Philosophy (PhD)