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Muscle aging is associated with a decrease in the number of satellite cells and their progeny, muscle progenitor cells (MPCs) that are available for muscle repair and regeneration. However, there is an increase in non-immuno-hematopoietic cells (CD45 negative) in regenerating muscle from aged mice characterized by high stem cell antigen -1(Sca-1) expression. In aged regenerating muscle, 14.2% of cells are CD45neg Sca-1pos while 7.2% of cells are CD45neg Sca-1pos in young adult muscle. In vitro, CD45neg Sca-1pos cells over express genes associated with fibrosis, potentially controlled by Wnt2. These cells are proliferative, non-myogenic and non-adipogenic, and arise in clonally-derived MPCs cultures from aged mice. Both in vitro and in vivo studies suggest that CD45neg Sca-1pos cells from aged muscle are more susceptible to apoptosis than their MPCs, which may contribute to depletion of the satellite cell pool. Therefore, with age, a subset of MPCs takes on an altered phenotype, which is marked by high Sca-1 expression. This altered phenotype prevents these cells from participating in muscle regeneration or replenishing the satellite cell pool, and instead may contribute to fibrosis in aged muscle.

Duchenne Muscular Dystrophy (DMD) is an x-linked neuromuscular disease that is caused by an absence of dystrophin protein, rendering skeletal muscle more susceptible to contraction-induced damage. One therapeutic strategy focuses on increasing the expression of endogenous utrophin A, a dystrophin homologue. Interestingly, slow muscle is more resistant to the dystrophic pathology and has increased utrophin A expression (Webster 1998; Gramolini 2001b). These observations led researchers to explore the therapeutic potential of stimulating the slow, oxidative myogenic program (SOMP) in the mdx context. Beneficial adaptations were seen with pharmacological activation of PPARδ and AMPK. We treated mdx mice with resveratrol (~100mg/kg/day), a putative SIRT1 activator, for 6-7 weeks and evaluated the activity of phenotypic modifiers that are known to influence the SOMP. SIRT1 activity and protein levels increased significantly, as well as downstream PGC-1α activity. There was evidence of a fibre type conversion as the treated mice had a higher proportion of the slow myosin heavy chain isoforms in both the EDL and Soleus skeletal muscles. Utrophin A protein levels showed modest, but consistent increases with resveratrol treatment. Finally, histological analysis revealed improvements in central nucleation and fibre size variability. These findings were promising, but raised the question of whether modifying the treatment regimen may result in greater therapeutic benefits. Surprisingly, we discovered that an elevated dose of 500mg/kg/day was ineffective in its promotion of the SOMP. SIRT1 was not activated and there was no change in utrophin A levels with resveratrol treatment. Taken together, this study demonstrates that resveratrol has the ability to promote the SOMP through SIRT1 and PGC-1α activation. It also highlights the importance of selecting an appropriate dose of resveratrol to maximize its effectiveness.

Patients with Peripheral Arterial Disease have a reduced capacity for exercise, the exact causes of which are poorly understood. This thesis investigated alternative testing procedures that aim to provide a more complete and precise description of the exercise capacities of these patients. Furthermore, the potential roles of gastrocnemius muscle fibre morphometry, capillary supply and glycogen stores in the exercise tolerance of PAD patients were studied. Study one aimed to determine the effect of test repetition on maximal exercise performance and test-to-test variability in PAD patients using an incremental treadmill walking test (T) (n=5), an incremental cycle test (C) (n=5), and incremental endurance (PF-endurance) and maximal strength (PF-strength) plantar flexion tests (n=5). Tests were conducted once per week for eight weeks. Performance was stable on the T (~530 s) and C (~500 s) tests across the eight weeks. Test-to-test variance on T decreased from 16%CV (CV: coefficient of variation) to 6%CV (p=.21,NS), and from ~8%CV to 2%CV on C (p<.05) over the eight week period. Variance of peak gas exchange variables tended to decrease with performance variance on both tests; however, other physiological variables, and the associated variance levels, were stable throughout the study. PF strength (635-712N) gradually increased over the initial 2-3 weeks (p<.05) which was accompanied by a reduction in variance from ~8%CV to ~3%CV (p<.05). Similarly, PF endurance increased over the first two weeks (~32,000 to 41500 N.s-1) while variance of this measure fell from ~21%CV to ~10%CV (p<.05) over the study duration. It is concluded that the implementation of familiarisation sessions leads to a reduction in whole body and local calf muscular performance variance in patients with PAD. Using a randomised crossover design, study two aimed to compare performance and the physiological and symptomatic responses between a T test and a C test in 16 patients with PAD. Peak exercise time on C (690 s) was greater than that on T (495 s); however the two were significantly correlated (n=16, r=.69, p<.05). Peak HR (120 bpm), VO2 (~1.22 l.min-1) and rate pressure product (~20') did not differ between the two tests, nor did the post exercise ankle pressure (T: 56; C: 61 mmHg). In two subjects with lower back pain during C, the ankle pressure of their "worst" limbs failed to fall by >10mmHg. Performance on both the T and C tests was closely related to the onset of leg symptoms; however the site of pain during C was much more variable than during T. Incremental cycle testing would overcome some of the limitations of treadmill testing (e.g. measurement of mechanical work), and it appears to be an acceptable alternative for measuring the exercise capacity and physiological exercise responses in known claudicants. Use of cycle ergometry for the diagnosis of PAD requires testing in the general population. Study three aimed to compare whole body (T test and C test) and local calf muscular (PF strength and endurance) exercise performance between 16 PAD patients (age: 63 ± 2; BMI: 25.9 ± 1.1) and 13 healthy, sedentary control (CON) subjects (age: 62 ± 1; BMI: 25.9 ± 0.4), and to describe relationships between the whole body and local calf muscular exercise capacities within the two groups. Furthermore, this study aimed to compare several histochemical characteristics of the medial gastrocnemius muscle fibres between PAD and CON, and to establish whether these factors were related to the exercise capacities of both groups. Maximal performance on T was 59% lower in the PAD group compared with the CON group, as was performance on C (50%), PF strength (25%), and PF endurance (58%). Compared with CON, PAD patients had a lower estimated calf muscle mass and a slight reduction (10%) in muscle fibre size (p=.14, NS). They also had a lower proportion of type I fibres (PAD: 49%; CON: 62%) that was offset by a greater proportion of type IIA fibres (PAD: 27%; CON: 16%), and a reduction in the capillary contacts per muscle fibre (PAD: 1.63; CON: 2.12) compared with CON. When expressed relative to fibre area there were no differences in capillarisation between PAD and CON; however this index was significantly related to resting and post exercise ABI in the PAD patients. There were no differences in the mixed muscle [glycogen], nor the optical density of glycogen in the individual fibres, between the two groups. PF endurance was poorly predictive of walking performance, and did not correlate with any of the morphological variables in both groups. Calf muscle mass correlated with PF strength (r=.59 - .62), and strength was correlated with T performance (r= .61 - .63) in both groups. In the PAD patients, T performance was correlated with the cross sectional area (n=12, r=.72, p<.05), capillary contacts (n=10, r=.81, p<.05) and glycogen density (n=9, r=.81, p<.05) of type I fibres. This study confirms that a reduction in calf strength, which appears to be mediated through muscle atrophy, plays some role in the reduced exercise capacity of claudicants. While both fibre area and capillary supply seem to be of relevance to the exercise capacity of PAD patients, these two factors are closely linked and further research is required to establish the determinants, and relative importance of both. An important, and possibly limiting role of carbohydrate oxidisation in PAD patients is supported by the strong relationship between type I glycogen stores and whole body exercise capacity.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2002. / Härtill 4 uppsatser.

Submitted by MARIANA LOPES BENITES null (mariana_benites@hotmail.com) on 2017-12-20T14:12:16Z No. of bitstreams: 1 repositório Mariana Benites.pdf: 2711972 bytes, checksum: 006ba62977278dd8c58404bd766be56d (MD5) / Submitted by MARIANA LOPES BENITES null (mariana_benites@hotmail.com) on 2017-12-21T12:31:27Z No. of bitstreams: 1 repositório Mariana Benites.pdf: 2711972 bytes, checksum: 006ba62977278dd8c58404bd766be56d (MD5) / Approved for entry into archive by Ana Paula Rimoli de Oliveira null (anapaula@foa.unesp.br) on 2017-12-22T13:19:15Z (GMT) No. of bitstreams: 1 benites_ml_dr_araca_int.pdf: 2616549 bytes, checksum: 09a481f5e45b2906fb14687e39c41b08 (MD5) / Made available in DSpace on 2017-12-22T13:19:15Z (GMT). No. of bitstreams: 1 benites_ml_dr_araca_int.pdf: 2616549 bytes, checksum: 09a481f5e45b2906fb14687e39c41b08 (MD5) Previous issue date: 2017-12-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Produzida pela glândula pineal exclusivamente em período noturno e na escuridão, a melatonina apresenta papel fundamental na sincronização dos ritmos circadianos com o ciclo claro-escuro do meio ambiente. A exposição à luz suprime a síntese e secreção de melatonina pela glândula pineal, podendo prejudicar a ação insulínica em células do músculo esquelético. O transporte ineficiente de glicose no músculo esquelético pode promover atrofia, geralmente acompanhada por perda de massa óssea. Resistência à insulina, atrofia muscular esquelética e perda de massa óssea são efeitos patológicos que podem ser prevenidos ou atenuados pela prática regular de exercícios físicos. O objetivo do presente estudo foi avaliar o efeito do exercício resistido (ER) sobre a sensibilidade à insulina e manutenção da musculatura esquelética e tecido ósseo em ratos pinealectomizados. 40 ratos Wistar machos foram distribuídos, aleatória e igualmente, em 4 grupos: 1) controle (CNS); 2) exercitado (CNEX); 3) pinealectomizado (PNX) e 4) pinealectomizado exercitado (PNXEX). O ER foi realizado em escada, durante 8 semanas, 3 sessões semanais (em dias não consecutivos), 9 repetições por sessão e intervalo de tempo de 2 minutos entre as repetições. O teste de força máxima foi realizado a cada 2 semanas e a intensidade selecionada foi 60% de 1 repetição máxima (1RM). Após o período de treinamento, os animais foram submetidos ao teste de tolerância à insulina. Após o sacrifício, amostras de sangue foram coletadas para fosforilação da glicemia e insulinemia. A partir destes valores, a resistência à insulina foi calculada pelo índice HOMA-IR. O cálculo da área de secção transversa do extensor digitorum longus (EDL) foi realizado a partir da análise histológica, utilizando o software IMAGEJ. Também foram avaliadas a expressão gênica e fosforilação de proteínas presentes nas vias de manutenção do músculo esquelético: GLUT4, TNF-α, atrogin-1, MuRF1, IGF-1, Akt1, miostatina e SMAD2/3. O cálculo da área do trabeculado ósseo esponjoso inferiormente à linha epifisial da tíbia foi realizado a partir da análise histológica em cortes longitudinais, utilizando o software IMAGEJ. Os parâmetros morfológicos do tecido ósseo foram avaliados por micro-CT; a análise mineral óssea foi avaliada pelo Dual-energy X-ray absorptiometry – DEXA e análise mecânica foi avaliada pelo teste de três pontos. As análises estatísticas foram realizadas por análise de variância com 2 fatores - ANOVA two way, seguida pelo teste de Bonferroni, ao nível de significância de 5%. A pinealectomia promoveu resistência à insulina e aumento na expressão de TNF-α em animais PNX. A prática de exercícios resistidos atuou na prevenção da resistência à insulina pelo aumento na expressão de GLUT4 em animais PNXEX. A pinealectomia também promoveu atrofia muscular esquelética nos grupos PNX e PNXEX. A prática de exercícios resistidos, realizada pelo período de tempo, frequência e intensidade selecionados, não foi suficiente para promover hipertrofia muscular no grupo CNEX ou prevenir a atrofia muscular no grupo PNXEX. Os grupos PNX e PNXEX apresentaram aumento da expressão gênica de miostatina e fosforilação proteica de SMAD2/3 em relação aos grupos CNS e CNEX. Não houve diferença na expressão gênica e fosforilação das demais proteínas analisadas entre os grupos estudados. Os resultados sugerem que a atrofia muscular esquelética, observada nos grupos PNX e PNXEX, está relacionada ao aumento na ativação da via da miostatina e, quem sabe, ao aumento de TNF-α em músculo esquelético. O aumento da DMO em animais PNX foi tomado como um mecanismo protetivo do osso. O exercício resistido promoveu melhora da qualidade óssea em animais pinealectomizados ou não. O presente estudo demonstrou que a prática regular de exercícios resistidos (60% de 1 RM) preveniu a resistência à insulina em animais pinealectomizados e melhorou a qualidade óssea em animais com e sem pineal. A partir desses resultados podemos aventar que o exercício físico, em trabalhadores noturnos, pode melhorar a sensibilidade à insulina e auxiliar na manutenção do tecido ósseo. / 99999.006989/2015-02

This thesis is concerned with a very common disorder of iron homeostasis: iron deficiency. The specific focus is the manner in which iron deficiency influences physiological responses to hypoxia in humans. This work is predicated on observations made over many decades in vitro and in vivo, suggesting that variations in the bioavailability of iron have important consequences for certain biological processes known to depend on oxygen availability. Three separate but related studies together form the basis for this thesis. The first two, Study A and Study B, adopt a similar approach in recruiting healthy volunteers who differ according to iron status, yielding iron-deficient and iron-replete groups in both cases. In Study A, the behaviour of the pulmonary circulation is investigated during a sustained hypoxic exposure, before and after an intravenous infusion of iron. In Study B, skeletal muscle metabolism is explored, both at the level of high-energy phosphate metabolism and the integrated physiological responses to exercise on a cycle ergometer. In the third study, Study C, a different approach is taken, recruiting patients with chronic obstructive pulmonary disease (COPD), and exploring the prevalence and associations of iron deficiency in this condition. Chapters 2 and 3 describe experiments using sustained hypoxia in a normobaric chamber, during which the pulmonary circulation is assessed non-invasively using Doppler echocardiography. These reveal augmented hypoxic pulmonary vasoconstriction (HPV) in iron-deficient individuals, who also exhibit greater sensitivity to the effects of an infusion of intravenous iron. Additionally, the way in which certain circulating mediators important for iron haemostasis change over the course of these hypoxic exposures, and how iron status influences these responses, is explored. Chapter 4 reports the findings of experiments using 31P-magnetic resonance spectroscopy and cardiopulmonary exercise testing, which demonstrate abnormal whole-body metabolism in iron-deficient individuals during large muscle-mass exercise, despite the absence of a clear defect in mitochondrial oxidative phosphorylation. Intravenous iron is found to have significant effects to alter the lactate threshold in healthy individuals, but the effects are more striking in iron-deficient individuals. Collectively, these experiments imply that iron deficiency promotes a more glycolytic phenotype. Chapter 5 explores iron deficiency in COPD, a condition in which pulmonary vascular disease, hypoxia and skeletal muscle dysfunction coexist, and examines some of the difficulties in assessing iron status in the setting of a chronic inflammatory disorder. Iron deficiency is found to be common, and unexpectedly associated with significantly more severe hypoxaemia, in patients with COPD. Possible reasons for these findings, and their clinical implications, are considered. Chapter 6 provides a summary of the main conclusions to be drawn from the studies presented in this thesis.

Le contrôle de la masse musculaire est sous la dépendance d'un équilibre entre les processus de synthèse et de dégradation. Sur le plan cellulaire, deux voies signalétiques majeures sont impliquées : la voie des facteurs de transcription de la famille FoxO qui contrôle l'expression des gènes impliqués dans les systèmes de dégradation (système ubiquitine-protéasome et autophagie), et la voie IGF-1/Akt/mTORC1 qui représente la voie majeure de la synthèse protéique. Nos travaux mettent en évidence, sur des cellules musculaires le rôle de la protéine kinase AMP-dépendante (AMPK) qui inhibe l'activité de la voie mTOR et régule les systèmes ubiquitine-protéasome et autophagiques de manière FoxO3 dépendante. Une nouvelle cible de l'AMPK a également été identifiée : la protéine Ulk1 qui possède une fonction clé dans l'activation de l'autophagie. Par ailleurs, nous avons montré le rôle centraldu facteur d'initiation à la traduction eIF3f dans l'induction de l'hypertrophie, et dans l'augmentation de l'activité de la voie mTORC1 associée. De plus, nous montrons que la surexpression d'un mutant d'eIF3f résistant à la dégradation est associée à une protection effective contre l'atrophie. / Skeletal muscle mass is depending upon a dynamic balance between anabolic and catabolic processes. At a cellular level, two major signaling pathways are involved: the transcription factors FoxO related pathway, implicated in the control of protein breakdown systems(ubiquitin-proteasome system and autophagy), and the IGF-1/Akt/mTORC1 pathway associated with the canonic pathway of protein synthesis. We show in muscle cells that theAMP-activated protein kinase (AMPK) decreases the mTORC1 pathway activity and simulate subiquitin-proteasome and autophagy systems in a FoxO3-dependant manner. Furthermore,we identify Ulk1 as a new interacting partner of AMPK, which plays a major role in the autophagy induction. Moreover, we demonstrate the key role of the eukaryotic translation initiation factor eIF3f in hypertrophy induction and in the associated increase of the mTORC1activity. In addition, we show that the overexpression of an eIF3f mutant resistant to the degradation is associated with a protection against muscle atrophy.

Highly-trained athletes are accustomed to varied and high-volume based exercise stimuli and eliciting adaptation in individuals already possessing the necessary physiology to compete at the highest level is difficult. Therefore, identifying novel, potent and time efficient methods of achieving cumulative training stress is a continual quest for coaches and exercise scientists. This thesis examined the acute and chronic effects of manipulating exercise and recovery during brief all-out sprint cycling on adaptive responses favouring enhanced endurance capacity. Chapter 3 highlighted that low-volume non-work matched all-out sprint cycling, whether it be interval- (4 x 30 s bouts) or continuous based (1 x 2 min bout) provides a similarly potent stimulus for the acute induction of cell signalling pathways and key growth factors associated with mitochondrial biogenesis and angiogenesis in trained individuals. In line with manipulating recovery and in attempting to identify a novel and potent exercise intervention capable of giving athletes more return on their training investment, Chapters 4-6 investigated the efficacy of combining sprint interval training with post-exercise blood flow restriction (BFR). Firstly, it was demonstrated that BFR potentiates HIF-1α mRNA expression in response to SIT, tentatively suggesting an enhanced stimulus for hypoxia- and/or metabolic-mediated cell signalling associated with mitochondrial biogenesis and angiogenesis over SIT alone. Secondly, four weeks of SIT combined with post-exercise BFR provides a greater training stimulus over SIT alone in trained individuals to enhance VO2max (4.7 v 1.1 % change) and MAP (3.8 v 0.2 % change), but not 15-km TT performance. Finally, in response to four weeks of SIT combined with post-exercise BFR, an international female track sprint cyclist increased her CP and W by 7 and 2 % and VO2max and absolute MAP by 3 and 4 %, respectively. Through a combination of an acute in vivo molecular experiment, a training study and an athlete case study, this thesis has introduced a potentially potent and novel training concept that appears capable of augmenting aerobic capacity.