Дисертації з теми "Muscle ageing"

An age-related loss of muscle mass is associated with increased frailty in the elderly. The effect is felt at both a national scale, with an increased budgetary demand for health services directed towards the ageing population, and by the individual where reduced mobility significantly reduces their quality of life. It is unclear whether all skeletal muscle types are affected in the same manner. This thesis considered how thiol signalling, facilitated through reactive thiol groups on cysteine amino acids, may affect skeletal muscle ageing as it is crucial for normal intracellular function. Several studies have identified reactive oxygen species (ROS) as crucial signalling molecules in healthy muscle and various proteins can detect and respond to changes in their concentration. The cysteines are evolutionarily conserved in functionally important locations and have a direct impact on protein function, affecting either its active site or conformation. In healthy muscle, proteins can quickly and efficiently respond to changes in ROS concentrations via this mechanism whereas in aged muscle these responses appear to be impaired. The quadriceps and soleus muscles were selected because of their differing primary metabolic pathways and physiology, reflecting fast and slow twitch muscle respectively. This enabled determination of age related changes to the redox proteome between two different skeletal muscles. They are hypothesised to age differently and to determine this, adult (12 months) and old (24 months) tissue were subjected to a deep proteomics investigation, elucidating changes to the global proteome of ageing mouse muscle as well as using differential labelling of reduced and reversibly oxidised cysteine residues to identify redox-susceptible locations on individual proteins. Prior to this a proteomics study had not analysed changes to the redox proteome between two skeletal muscle tissues before. Analysis of the quadriceps label free results identified changes to redox protein abundance such as a significant increase in Protein Disulphide Isomerase, crucial to disulphide bond formation and breakage. HSC70, important for protein folding, was significantly decreased with age. Differential labelling of specific cysteine residues demonstrated Cys46 increased in its reduced form with age in PARK7. Furthermore, many changes observed in the label free analysis highlighted cytoskeletal proteins as those primarily affected. The soleus label free results demonstrated significant decreases in abundance of a number of mitochondrial proteins involved in the electron transport chain such as NAD(P)H dehydrogenase and ATP Synthase. One example of differential labelling highlighted ATP Synthase Cys101 as becoming increasingly reduced with age. This increase in a reduced redox state of cysteines was observed across a range of other mitochondrial proteins, possibly indicating a negative impact on energy metabolism in the soleus with age. A successful preliminary study considered the effect of stretching C2C12 mouse skeletal muscle cells in vitro. A protocol for testing the effect of mechanical stretching on C2C12 cells was optimised with a future goal of producing replicable in vitro proteomics data and thereby reducing the requirement for animal tissue. The studies in this thesis identified various redox proteome changes in quadriceps and soleus muscle with age. This data will provide a basis for a targeted analysis of musculoskeletal proteins with a view to a better understanding of musculoskeletal ageing and its impact via the proteome.

In aged muscle, from humans and mice, the ryanodine receptor (RyR1) is leaky, leading to increased levels of resting Ca2+ in the myoplasm. This is also a feature of skeletal muscle disorders caused by variants in RyR1 such as malignant hyperthermia (MH), central core disease (CCD), exertional heat illness (EHI) and late-onset axial myopathy (LOAM). Elevated Ca2+ is damaging to mitochondria, leading to production of reactive oxygen and nitrogen species associated with MH susceptibility to inhalational anaesthetics. Mice with Ryr1 variants show premature muscle ageing and highlight the cycle of inefficient calcium handling and oxidative damage to mitochondria that impairs skeletal muscle energetics. Caenorhabditis elegans models of MH CCD EHI and LOAM variants, both homozygous and heterozygous forms, showed increased sensitivity to halothane. Altered caffeine sensitivity was evident in MH and CCD models, and at very high concentrations in EHI models. Strains with RyR1 variants exhibit age-related accelerated myosin disorganisation. Whole genome Affymetrix arrays revealed genes and pathways correlated with skeletal muscle ageing and MH. Of additional genes of interest investigated UNC13, CASQ1, ORAI1, MCU and MICU1 showed altered expression with age. Array data from blood has been used to identify a signature for MH susceptibility. There is loss of mitochondrial membrane integrity and alteration in mitochondrial number in MH. New apparatus, capable of quantifying heat produced during muscle contraction, has enabled calculation of skeletal muscle efficiency. Preliminary data indicates that there was loss of skeletal muscle efficiency in aged muscle from wild type mice. This work provides new information on the role of RYR1 variants in skeletal muscle ageing and the importance of calcium handling in muscle energetics.

The skeletal muscle fibre is a syncitium where each myonucleus regulates the gene products in a finite volume of cytoplasm i.e., the myonuclear domain (MND). A novel image analysis algorithm applied to confocal images, analyzing MND size and myonuclear spatial distribution in 3-dimensions in single skeletal muscle fibres has been used in this project. The goal was to explore the modulation of myonuclei count and MND size in response to muscle adaptation processes. The effects of ageing, gender, hormones, muscle hypertrophy and body size were investigated on MND size. A strong linear relationship was found between MND size and body size in the muscle fibres from mammals representing a 100,000-fold difference in body size. Independent of species, MND size was highly dependent on MyHC isoform type and mitochondrial contents of skeletal muscle fibres. In hypertrophic mice, a significant effect of MND size on specific force and myosin content was observed. This effect was muscle fibre type-specific and shows that the bigger MNDs in fast-twitch EDL muscle fibres are optimally tuned for force production while smaller MNDs in slow-twitch soleus muscle fibres have a much more dynamic range of hypertrophy without functional compromise. This indicates a critical volume individual myonuclei can support efficiently for a proportional gain in muscle fibre force and size. In human muscle fibres, spatial organization of myonuclei was affected by both ageing and MyHC isoform expression. In fibres expressing type I MyHC isoform, an increased MND size variability and myonuclear aggregates were observed in old age although average MND size was unchanged. In contrast, in type IIa fibres, the average MND size was smaller reflecting smaller size of muscle fibres. Those changes may influence the transcriptional activity per myonucleus and/or local cooperatively of myonuclei in a gender and muscle fibre-type specific manner. Finally, hormone replacement therapy was shown to negate menopause-related functional impairment in skeletal muscle fibres. The positive effect on force was due to quantitative effect in fibres expressing fast myosin isoform while the effect was both quantitative and qualitative in fibres expressing slow myosin isoform. The effect on MND size was fibre type dependent and was achieved by significantly reducing domain size in slow- but not the fast-twitch muscle fibres. Together, our data suggest that modulation of myonuclei count and MND size is a mechanism contributing to remodelling of skeletal muscle in muscle adaptation process. These findings should be considered when developing therapeutic approaches towards restoring muscle mass and strength in muscle wasting conditions.

The aims of this study were to investigate the mechanisms underlying (1) the ageing-related motor handicap at the whole muscle, cellular, contractile protein and myonuclear levels; and (2) ageing-related differences in muscle adaptability.

In vivo muscles function was studied in the knee extensors. Decreases were observed in isokinetic and isometric torque outputs in old age in the sedentary men and women and elite master sprinters. A 20-week long specific sprint and resistance training successfully improved the maximal isometric force and rate of force development in a subgroup of master sprinters.

In vitro measurements were performed in muscle biopsies from the vastus lateralis muscle. Immunocytochemical and contractile measurements in single membrane permeabilized muscle fibres demonstrated ageing- and gender-related changes at the myofibrillar level. In sedentary subjects, data showed a preferential decrease in the size of muscle fibres expressing type IIa MyHC in men, lower force generating capacity in muscle fibres expressing the type I MyHC isoform in both men and women and lower maximum velocity of unloaded shortening (V₀) in fibres expressing types I and IIa MyHC isoforms in both men and women. The master sprinters also experienced the typical ageing-related reduction in the size of fast-twitch fibres, a shift toward a slower MyHC isoform profile and a lower V₀ of type I MyHC fibres, which played a role in the decline in explosive force production capacity. The fast-twitch fibre area increased after the resistance training period. A model combining single muscle fibre confocal microscopy with a novel algorithm for 3D imaging of myonuclei in single muscle fibre segments was introduced to study the spatial organisation of myonuclei and the size of individual myonuclear domains (MNDs). Significant changes in the MND size variability and myonuclear organization were observed in old age, irrespective gender and fibre type. Those changes may influence the local quantity of specific proteins per muscle fibre volume by decreased and/or local cooperativity of myonuclei in a gender and muscle fibre specific manner.

In conclusion, the ageing-related impairments in in vivo muscle function were related to significant changes in morphology, contractile protein expression and regulation at the muscle fibre level. It is suggested that the altered myonuclear organisation observed in old age impacts on muscle fibre protein synthesis and degradation with consequences for the ageing-related changes in skeletal muscle structure and function. However, the improved muscle function in response to a 20-week intense physical training regime in highly motivated physically active old subjects demonstrates that all ageing-related in muscle function are not immutable.

Mechanical vibrations are common in today’s world. Studies have claimed that vibration training can enhance skeletal muscle performance and might be a useful therapy for sarcopenia, the age-related degeneration of muscle function and mass. Others, however, report debilitating consequences following excessive vibration exposure, such as Hand-Arm Vibration Syndrome. Further to this ambiguity, few studies have explored the function of skeletal muscles during vibration. Animal models are integral to furthering our understanding of the effects of vibration, and while efforts to reduce the use of vertebrate models are increasing, invertebrates may prove to be valuable alternatives. The contractile dynamics of the locust’s metathoracic extensor tibiae muscle were compared to the soleus and extensor digitorum longus muscles of C57 mice and then subjected to acute vibration, during which their contraction dynamics were analysed. The influence of sensory feedback, stimulus phase, vibration frequency and amplitude on the locust extensor tibiae muscle’s function were also analysed. Additionally, the effects of locust age on its behaviour, extensor tibiae muscle function and response to vibration were studied to determine whether it may provide insights into sarcopenia. The locust’s extensor tibiae muscle demonstrated higher peak forces than both murine muscles, but its force-frequency relationship showed a greater resemblance to that of the soleus muscle. The function of the extensor tibiae and soleus muscles were dependent on stimulus phase, frequency and amplitude of vibration treatments, and sensory feedback was shown to reduce the impact of vibration on the locust muscle. No behavioural changes or decreases in the extensor tibiae muscle’s mass were found in ageing locusts, but age-dependent variations in its contractile function and the neuromuscular response to vibration were measured. This work suggests that both vertebrate and invertebrate muscle function are compromised during vibration, and additional work utilising the locust as a model may further our understanding of the effects of vibration and ageing on the neuromuscular system.

Sarcopenia is the age-related loss of skeletal muscle mass and function; inflammation is thought to be one aetiological factor in its development. Adipose tissue accumulates with advancing age and adipose-derived cytokines (adipokines) contribute to inflammaging. Skeletal muscle myogenesis is one adaptaive mechanism by which skeletal muscle mass is sustained throughout the human lifespan. The effect of the adipose inflammatory milieu on such myogenesis is unknown, as is the relative importance of its constituent adipokines to myogenesis. This work demonstrates that conditioned medium generated from obese subcutaneuous adipose tissue has a detrimental effect on in vitro primary human myogenesis. Resistin is shown to be – in part – responsible for this phenomenon and is demonstrated to inhibit myogenesis by activating the classical NFκB pathway. Resistin is further shown to be a metabolic stressor of primary human myotubes, promoting increased oxygen consumption, fatty acid oxidation and lipid accumulation. It is important to identify more avenues for the development of pharmacological interventions in sarcopenia. To that end, this thesis also demonstrates for the first time that the myokine IL-15: 1) is pro-myogenic in primary human cultures; 2) can mitigate the detrimental effects of an inflammatory environment on myogenesis; and 3) supports myogenesis at autocrine concentrations.

The loss of muscle strength in the elderly is greater than the loss of muscle mass, termed specific force (SF) loss, and indicates that a decrease in muscle quality contributes to age-related muscular weakness. The present PhD thesis has studied age-related SF loss in human skeletal muscle using a skinned single muscle fibre model. A large variation in published skinned fibre SF measurements was found to exist in the literature. Therefore, a systematic review and meta-analysis was performed to identify factors causing this variability. The majority of publications were objectively divided into four research groups based on shared authorship. Methodological differences between research groups contributed to ~30% of the variance in the literature, suggesting that they are an important contributor to the variance in published SF values. Different research groups use different activating solutions to study skinned fibres, and were assessed experimentally. Skinned fibres were exposed to different, but commonly used activating solutions (termed A and B). A significantly higher SF and a shorter time to half peak tension (t50) was measured from the same fibres in solution B compared with solution A. The use of TES in solution B instead of Imidazole as a pH buffer largely caused the SF difference, and a lower Cl- concentration and the use of Glutathione in solution B partly caused the faster t50. These findings indicate that the use of different activating solutions likely affects the variance of published SF values. The final study in this thesis compared SF between skinned fibres from physically active and comparatively frail elderly cohorts to a young, healthy group. MHC I fibre SF was significantly higher in solution B than A within all groups. No significant differences in SF, myosin content (SDS PAGE) or order (X-ray diffraction) were observed between groups. These findings suggest that physical activity does not affect age-related skinned fibre SF loss and that SF is related to skinned fibre myosin content.

Older people experience skeletal muscle wasting, in part due to impaired proliferative capacity of quiescent skeletal muscle satellite cells and. The work presented in this thesis set out to examine the hypothesis that microenvironment of skeletal muscles can be influenced by immune cell secretions, which affect satellite cell proliferation, and that beneficial immune-muscle interactions in young people are blunted in elderly. The aim of this research was to investigate the role of ageing human lymphocytes on skeletal muscle cell behaviour. For this purpose, lymphocytes were isolated from whole blood of young (aged 18-25 years) and older (aged 78-85 years) healthy volunteers and older healthy volunteers. All the participants were healthy, with no history of muscle disease and not on immunosuppressant or corticosteroids treatment that affect immune function. Lymphocytes were cultured with, or without, anti-CD3/CD28 activators for 4 days to induce release of cytokines, interleukins and growth factors into the media. The secreted proteins were used to prepare conditioned media that were used to culture C2C12 myoblasts. Secretomes were analysed and fifteen secreted Th1/Th2 cytokines and IGF-I were quantified by multiplex immunoassay. The gene expression and protein concentrations of amphiregulin were determined from T-lymphocytes lysates by real-time PCR and ELISA respectively. The levels of CD25 and FoxP3 expression in lymphocytes were examined using flow cytometry. The expression of muscle transcription factors, MyoD and Myogenin were determined by real- time PCR. Activated Mek1/Erk1/2 and Akt/mTOR were measured by multiplex immunoassay. Our results demonstrate for the first time that a decrease in the levels of amphiregulin and CD25 coincides with the increase in FoxP3 with ageing, which may be involved in suppression of lymphocytes. Seven cytokines were differentially secreted by the young- compared with the old-activated lymphocytes. The secretome from young-activated lymphocytes had 30% (P < 0.005) higher IGF-I concentrations compared with old and control treatments. The conditioned media from young -activated lymphocytes increased the rate of proliferation of myoblasts by ~3-fold (P<0.005) and caused an approximate 4-fold (P < 0.005) increase in migration compared with non-activated lymphocyte control media. These responses were characterised the extended proliferation of young -treated myoblasts was also associated with a decrease in MyoD and Myogenin and an increase in mediators of proliferation Mek1/Erk1/2and a decrease in the key proteins for differentiation, Akt/mTOR. In contrast, myoblasts treated with conditioned media from old-activated lymphocytes exhibited a high degree of differentiation.

The aim of this thesis was to address the impact of the skeletal muscle adaptations that occur in Chronic Obstructive Pulmonary Disease (COPD) and subsequently to identify tools that may be relevant in the management of patients with structural and functional muscle abnormalities. To establish whether the type I to II fibre shift that occurs in COPD is associated with mortality, a retrospective multicentre analysis of 392 stable COPD outpatients was performed. Across the whole cohort, fibre shift was an independent predictor of mortality in a model also including age and FEV1%predicted. In patients with GOLD stage III or IV disease, quadriceps strength, but not fibre shift had an independent association with an increased risk of mortality. Therapies targeting quadriceps fibre shift and weakness may be of therapeutic value; practical tools that identify relevant patients may have clinical utility. Since the pathophysiological adaptations that occur may be considered to be a manifestation of accelerated biological ageing, potentially relevant physical and biological markers of ageing were identified following a review of the literature. The Short Physical Performance Battery (SPPB) is used in gerontology to assess lower limb function, but its determinants have not been previously evaluated in COPD. SPPB score was measured in 109 stable COPD outpatients; 31 also had a quadriceps biopsy. Quadriceps strength and exercise capacity were the only independent predictors of SPPB score. Stratification by SPPB score identified patients with locomotor muscle atrophy and impairment in strength, exercise capacity and daily physical activity. Patients with a reduced SPPB score had a higher proportion of type II fibres. Subsequently, the SPPB potentially has practical utility in the assessment of COPD patients. Systemic Klotho and GDF-15 levels are associated with mortality in non-selected populations; these proteins were evaluated initially in the serum and then the skeletal muscle of healthy controls, smokers and COPD patients. Circulating Klotho levels were reduced in COPD and smokers, related to quadriceps strength and increased after successful smoking cessation. Serum GDF-15 levels were elevated in COPD, and related to a marker of systemic oxidative damage and locomotor muscle atrophy. Klotho and GDF-15 were expressed in skeletal muscle. Quadriceps GDF-15 expression was elevated in COPD patients as compared to controls and diaphragm expression. Klotho levels were reduced in the locomotor muscle of human smokers and smoke-exposed mice. Humans had relatively higher Klotho levels in respiratory muscle. Quadriceps Klotho levels positively correlated with local protein carbonylation and were also unexpectedly elevated in patients with established skeletal muscle dysfunction; immunohistochemistry confirmed that Klotho was associated with regenerating muscle fibres. Modulation of Klotho and GDF-15 signalling, and potentially other age-related molecules, may provide therapeutic options to COPD patients.

Vascular smooth muscle cell (VSMC) phenotypic switching, from a contractile to a migratory phenotype, is essential for vascular repair and is compromised in ageing. Phenotypic transition involves dramatic actin reorganisation which is regulated by the linker of the nucleoskeleton and cytoskeleton (LINC) complex that spans the nuclear envelope (NE). The LINC complex physically anchors cytoskeletal filaments to the nucleoplasm via nesprin-SUN-nuclear lamina connections which enable rapid biophysical signalling between the nuclear interior and exterior. The nuclear lamina consists of A and B-type lamins that maintain nuclear architecture, however, the lamin A precursor protein, prelamin A, is a biomarker of VSMC ageing and this project investigates the impact of prelamin A upon LINC complex function. During VSMC ageing in-vitro, prelamin A accumulates at the presenescent growth phase which is associated with cellular elongation and focal adhesion reorganisation. Interference reflection imaging (IRM) and time-lapse microscopy revealed that presenescent VSMCs exhibit increased focal adhesion turnover with enhanced migratory speed and persistence. Importantly, prelamin A accumulation induced by siRNA-mediated knockdown of its processing enzyme, FACE-1, reiterates these morphological changes and enhances migratory persistence. Moreover, RhoA and Rac1 are well-established regulators of cell motility and their expression and activity diminishes in presenescent and FACE-1 depleted VSMCs. Fluorescence recovery after photobleaching (FRAP) also revealed that nuclear lamina disruption increases nesprin-2 dynamics at the nuclear exterior. Thus, we suggest that prelamin A impacts on actin-regulated processes including cell shape and motility via the LINC complex. We utilised an siRNA-mediated approach to investigate the importance of other LINC complex components in regulating cell morphology and migration. Interestingly, SUN2 levels decrease during in-vitro VSMC ageing and SUN2 knockdown enhances the migratory speed of VSMCs and fibroblasts similarly to presenescent VSMCs. The role of nesprins in regulating cell phenotype varies between different cells, highlighting that LINC complex organisation and function is flexible and cell-type specific. Together, our data reveal that the LINC complex is a versatile structure that is specialised to cell function and is an important regulator of cellular morphology, focal adhesion organisation, Rho GTPase activity and migration. VSMC ageing is associated with prelamin A accumulation and loss of SUN2 expression which consequently deregulates LINC complex organisation and functioning. Therefore, LINC complex disruption gives rise to an aged VSMC phenotype which we predict may underlie cardiovascular diseases such as atherosclerosis. In addition, IRM captured the release of adhesion-like structures, termed cell traces, from the rear of migrating VSMCs that outline their migratory path. Cell traces form physical tracks that enhance the speed and migrational directionality of neighbouring VSMCs. Therefore, we predict that cell traces support VSMC migration to injury sites and are important for vessel repair.

Thesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
In the last thirty years of the HIV epidemic, HIV has transitioned from a deadly to a chronic, manageable disease wherein infected individuals are living longer. However, despite the efficacy of antiretroviral therapy in suppressing viral burden, the quality of life is still impacted. HIV-infected individuals display symptoms associated with the elderly including a frailty related phenotype, declines in muscle and bone mass, and notably, a host response resulting in gradual increases in systemic inflammation. This physiological dysregulation leads to increased morbidity and mortality in the HIV positive population. Anabolic therapies targeting muscle loss have demonstrated the efficacy of androgen supplementation, particularly testosterone, in increasing muscle mass in both younger and older individuals, as well as in HIV positive patients. In this thesis, we drew from previous studies on aging and muscle biology to understand HIV associated decreases in muscle function. We evaluated changes in serum biomarkers between older and younger men at baseline and in response to testosterone to identify possible pathways for age related declines in muscle and testosterone response. We then evaluated genomic data from muscle biopsy tissues from HIV infected individuals, older men and younger men to identify pathways that are common to infection and aging and that may underlie declines in muscle mass and function. Finally, we characterized the identified human aging pathways in two animal models of HIV infection, the HIV transgenic rat and the SIV infected rhesus macaque. Here, we provided data on nine independent serum biomarkers related to aging and observed a subset of those that change with age to be affected by testosterone treatment. Furthermore, we found a ten gene signature in muscle that differed between young and old and showed premature expression of this signature in HIV infected people. Because the signature and biomarkers implicated TGFβ associated senescence and fibrotic pathways, we explored these pathways and phenotypes in models of HIV infection, and found suggestions of accelerated aging in muscle of HIV infected individuals. Overall, this thesis provides insights into natural aging pathways co-opted by HIV and how these pathways might play a role in declines in muscle mass and function.

La sarcopénie est une maladie dégénérative liée à l’âge qui se caractérise par une perte progressive et involontaire de la masse et de la force musculaire. Elle s’accompagne d’une atteinte de la régénération musculaire et d’une accumulation des espèces réactives de l’oxygène. Les cavéoles sont des invaginations de la membrane plasmique. Dans le muscle, elles jouent un rôle dans la différenciation des cellules satellites et dans le maintien de l’unité contractile dans le muscle différencié. Certaines formes de myopathies sont consécutives à l’absence de cavéoles dans le muscle. Elles sont également impliquées dans la médiation de signaux liés à la régulation du stress oxydant. Afin de mieux comprendre les mécanismes régulant la mise en place de la sarcopénie, nous avons étudié ici les relations existant entre le stress oxydant et les cavéoles. Des cellules musculaires de souris ont été traitées par l’H2O2 et une diminution du taux des cavéolines-1et -3 a été mise en évidence dans des myoblastes et les myotubes, respectivement. Il apparaît donc que les protéines constitutives des cavéoles sont effectivement sensibles au stress oxydant dans les cellules musculaires. En présence d’H2O2, la fonction des cavéoles (endocytose et résistance au stress mécanique) était également significativement altérée dans des myoblastes. L’ensemble des résultats obtenus suggère que le stress oxydant aurait un effet sur les cavéoles, ce qui pourrait entraîner des conséquences sur la régénération et le maintien de l’intégrité musculaire au cours du vieillissement
Sarcopenia is an age-related degenerative disease which is characterized by a progressive and involuntary loss of muscle mass and strength. It is accompanied by an impairment of muscle regeneration and accumulation of reactive oxygen species. Caveolae are invaginations of the plasma membrane. In muscle, they play a role in the differentiation of satellite cells and in maintaining the contractile unit of the differentiated skeletal muscle. Some myopathies are resulting from the absence of caveolae in muscle. Caveolae are also involved in mediating signals related to the regulation of oxidative stress. To better understand the mechanisms involved in the development of sarcopenia, we investigated here the relationship between oxidative stress and caveolae. Mouse muscle cells were treated with H2O2 and decreased levels of caveolin-1 and -3 were demonstrated in myoblasts and myotubes, respectively. It therefore appears that caveolae constituent proteins are actually sensitive to oxidative stress in muscle cells. In the presence of H2O2, caveolae functions (endocytosis and resistance to mechanical stress) were also significantly degraded in myoblasts. Altogether, these data suggest that oxidative stress would affect caveolae, which could have consequences on regeneration and maintenance of muscle integrity during aging

Autophagy is an ubiquitous degradation system, that is conserved through species. Cells activate autophagy to degrade long-lived proteins, damaged organelles or portions of cytoplasm, that are engulfed in double-membrane vesicles called autophagosomes, that ultimately fuse to lysosomes, where the cargo is degraded and breakdown products are recycled to sustain cellular energetic demands. Skeletal muscle is the most abundant tissue in mammals and controls 80% of the blood glucose. We have recently shown that an efficient autophagy is required for muscle mass maintenance (Masiero et al., 2009). During ageing, muscles inevitably undergo atrophy, a process named sarcopenia (Rossi et al. 2008). Moreover, it has been reported that autophagy declines with age (Tan et al., 2013). Since the mechanisms involved in age-related muscle loss remain obscure, we investigated whether autophagy impairment contributes to sarcopenia. In this work, the muscle-specific autophagy knockout (Atg7-/-MLC), that were recently generated in our laboratory, were characterized during ageing (Masiero et al., 2009). Aged Atg7-/- mice have reduced lifespan and exacerbated atrophic and myopathic phenotype. In vivo force measurements showed that they are weaker compared to age-matched control mice. Alteration of mitochondrial morphology is a typical feature of Atg7-/- muscles. Therefore, we studied mitochondrial function in adult mice. Mitochondria of Atg7-/- mice were dysfunctional, in fact they did not retain membrane potential upon inhibition of ATP synthase. This mitochondrial alteration induced an increase of oxidative stress. A proteomic approach on oxidized protein, in collaboration with Prof. Friguet at the University of Paris, revealed that contractile proteins, such as actin and myosin, were significantly more carbonylated when autophagy was blocked. Functional assays of force measurements on single isolated fibers and sliding properties of purified actin/myosin, performed in collaboration with Prof. Bottinelli at the University of Pavia, showed an impairment of these contractile proteins in Atg7-/- mice. Atg7-/- mice also undergo spontaneous denervation, as confirmed by upregulation of denervation markers, such as Muscle Specific Kinase (MuSK), Acetylcholine Receptor gamma subunit (AchR-gamma) and Neural Cell Adhesion Molecule (NCAM). Moreover, in collaboration with Dr. Rudolf at Karlsruhe Institute of Technology (KIT), in Karlsrhue, we performed in vivo imaging of neuromuscular junction (NMJ), that revealed NMJ fragmentation and instability in autophagy-deficient mice. These findings suggest that inhibition of autophagy specifically in muscle generates a series of events that affect NMJ and causes a precocious denervation, contributing to sarcopenia. Since oxidative stress is an important feature of Atg7-/- mice and is believed to contribute to ageing, we treated adult mice with an antioxidant vitamin E analogue (Trolox), for 30 days, and we monitored the effects on the phenotype of Atg7-/- muscles. Trolox treatment reduced the level of protein carbonylation, restored the sliding properties of actin and myosin and brought back the force to normal level. Mitochondria function was also ameliorated but we did not find any benefit on atrophy and NMJ morphology. However, there was a small amelioration on NMJ stability. These data showed that oxidative stress contributes only to some aspects of ageing features present in Atg7-/- mice. Therefore, other mechanisms are involved for the atrophy and the denervation aspects. We then hypothesized that muscles release neurotrophic factors that are critical for muscle-nerve interaction and stability. Initially, we tought for neurotrophic factors that were down-regulated in autophagy-deficient muscle both in adult and old mice. qRT-PCR identified FGF binding protein 1 (FGFBP1) to be the one that was always suppressed in Atg7-/- mice. FGFBP1 is protein involved in the bio-activation of FGF proteins, that are important pre-synaptic organizers. In order to investigate the role of FGFBP1 in NMJ instability we used loss and gain of function approaches. Down-regulation of FGFBP1 in control mice induced instability and fragmentation of NMJ. On the contrary FGFBP1 over-expression in Atg7-/- muscles reduced the number of denervated fibers and restored NMJ stability. Then we investigated the connection between autophagy impairment and FGFBP1 down-regulation, by analyzing MuSK activity, a kinase that is essential for NMJ maintenance. We observed an alterated MuSK clustering in NMJ of Atg7-/- mice. Moreover MuSK down-regulation in vivo leads to FGFBP1 suppression. These results suggest that NMJ requires the secretion of FGFBP1 neurotrophic factor that is under MuSK regulation and that autophagy is critical for a normal MuSK localization and activity. It has been consistently demonstrated that two lifestyle adaptations, namely caloric restriction and exercise, are able to extend lifespan and, in parallel, to mitigate age-related alterations in NMJ (Melov et al., 2007; Fontana et al., 2010; Sandri et al., 2013; Schiaffino et al., 2013; Coen et al., 2013; Toledo et al., 2013; Guarente, 2013). Moreover, both these conditions promote autophagy activation in skeletal muscles and in other tissues. It has also been reported that autophagy is required for exercise itself and for training-induced adaptations in glucose homeostasis (He et al., 2012). These findings remain controversial as skeletal muscle–specific autophagy-knockout mice show the opposite phenotype (Kim et al., 2013). In this scenario, it is still unknown whether it is whole body or muscle specific autophagy that is required to sustain contraction, maintain glucose homeostasis, and trigger exercise-induced benefits. For this reason, we used Tamoxifen-inducible, muscle-specific, Atg7 knockout mice (Atg7-/-HSA), that we have recently generated (Masiero et al., 2009), to investigate the role of autophagy in physical exercise. This inducible muscle-specific genetic model allows to minimize the chance of any adaptations and compensations that usually occur with constitutive deletion of genes. In order to investigate whether acute block of autophagy in muscle affects exercise performance, controls and autophagy-deficient mice were exercised on a treadmill. We used a concentric exercise protocol while monitoring the maximum distance ran to exhaustion. Surprisingly, we did not find any significant differences in running capacity between controls and inducible Atg7-/-. Thus, autophagy is not required to sustain muscle contraction during concentric physical activity. We hypothesized whether a damaging eccentric-type muscle contraction might unravel a novel role for autophagy during muscle repair after exercise. So we performed repeated bouts of eccentric exercise to exhaustion for three consecutive days to induce damaging eccentric contraction in controls and inducible Atg7-/- animals, and found out that in these conditions, autophagy-deficient mice ran significantly less than controls. Morphological analyses did not show any sign of inflammation or myofibre degeneration, thus suggesting that impaired performance of Atg7-/- muscles was not due to major structural alterations. We also looked for possible energetic imbalance upon exercise, by monitoring the activity of P-AMPK, one of the major sensor of energetic stress, and by checking glucose and lactate levels in the blood. However, no significant differences were observed, thus suggesting that autophagy is not required for metabolic regulation of skeletal muscle during exercise. Since autophagy is important for organelle quality control, we tested whether mitochondrial homeostasis was affected after exercise. Interestingly, isolated muscle fibers from inducible Atg7-/- animals contained dysfunctional mitochondria that well correlated with their impaired performance. Being mitochondria the main source of ROS in the cell, it was feasible to hypothesize that oxidative stress may play a role in this condition. To address that, we measured total protein carbonylation and ROS production in exercised muscles that indeed was higher in Atg7-/- muscles. All together these data showed that acute inhibition of autophagy led to accumulation of dysfunctional mitochondria, increased oxidative stress and reduced physical performance during eccentric contraction. Excessive oxidative stress impairs muscle function, thus potentially explaining the reduced physical performance of Atg7-/- mice. We therefore treated controls and inducible Atg7-/- mice with the anti-oxidant N-Acetyl Cysteine (NAC) for 6 weeks, and then exercised them eccentrically. Surprisingly, NAC treatment severely impaired performance of controls but did not elicit any benefit in inducible Atg7-/- animals. Moreover it impaired mitochondrial function of controls. This data were confirmed after treatment with another anti-oxidant (Mito-TEMPO), that was specific for mitochondria. It has been reported that anti-oxidant treatment reduces activation of autophagy in control animals and that ROS are important for signalling pathways in the cell (Underwood et al., 2010; Owusu-Ansah et al., 2013). Our findings support these evidences, suggesting that physiological levels of ROS are important for the correct basal and stimulus-induced autophagy activation. Our results highlight the role of autophagy in the maintenance of mitochondrial function but not in AMPK activation and exercise dependent glucose homeostasis, suggesting that autophagy is an adaptive response to exercise that ensures mitochondria-quality control during damaging contractions.
Il sistema autofagico-lisosomiale è un sistema di degradazione ubiquitario e conservato tra le diverse specie. Esso viene attivato dalla cellula per degradare proteine con lunga emivita, organelli danneggiati e porzioni citoplasmatiche, che vengono sequestrate da un network di vescicole a doppia membrana, dette autofagosomi. Gli autofagosomi che contengono il materiale da degradare fondono con i lisosomi, dove il loro contenuto viene degradato e i prodotti riciclati per soddisfare la richiesta energetica cellulare. Il muscolo scheletrico è il tessuto più abbondante nei mammiferi e utilizza l’80% del glucosio presente nel corpo. Un efficiente sistema autofagico è necessario per il mantenimento della massa muscolare (Masiero et al., 2009). Durante l’invecchiamento, il tessuto muscolare subisce un inevitabile processo di atrofia, detto sarcopenia, che è indipendente dall’attività del soggetto ma si aggrava in condizioni di disuso (Rossi et al., 2008). I meccanismi coinvolti nella perdita di massa muscolare non sono ancora stati individuati con chiarezza. Poiché l’autofagia diminuisce con l’età (Tan et al., 2013), abbiamo studiato il ruolo dell’autofagia durante l’invecchiamento del tessuto muscolare. In questo lavoro sono stati quindi caratterizzati topi knockout condizionali per il gene Atg7 (Atg7-/-), gene che codifica per un enzima critico per la formazione degli autofagosomi (Masiero et al., 2009). In questo modo è possibile ottenere il blocco del processo autofagico in modo specifico nel muscolo scheletrico. Questi animali e i rispettivi controlli sono stati analizzati durante l’invecchiamento. I topi Atg7-/- muoiono prima dei controlli e, da vecchi, presentano un fenotipo miopatico, in cui le condizioni di atrofia sono esacerbate rispetto agli animali Atg7-/- adulti. Misure di forza in vivo di questi animali hanno mostrato come gli animali Atg7-/- risultino più deboli dei controlli; inoltre, gli animali Atg7-/- adulti presentano la stessa forza dei controlli vecchi, suggerendo uno stato di indebolimento precoce. Poiché il sistema autofagico è importante per la rimozione degli organelli danneggiati, abbiamo studiato i mitocondri. Durante l’invecchiamento, i mitocondri dei muscoli Atg7-/- si accumulano e presentano un’alterata morfologia alla microscopia elettronica. Abbiamo quindi analizzato la loro funzionalità misurando la capacità di mantenere il potenziale di membrana mitocondriale dopo l’aggiunta di un inibitore dell’ATP sintasi. I mitocondri degli Atg7-/- sono risultati incapaci di mantenere il potenziale, al contrario dei controlli. L’alterata funzionalità mitocondriale induce un aumento della produzione di ROS con conseguente stress ossidativo. Mediante un approccio di proteomica in collaborazione con il Prof. Friguet dell’Univeristà di Parigi, abbiamo caratterizzato le proteine ossidate e abbiamo trovato che le proteine contrattili, actina e miosina, erano le proteine maggiormente carbonilate nei topi vecchi knockout rispetto ai controlli della stessa età. Per capire se questa alterazione contribuisse alla debolezza muscolare di questi animali abbiamo eseguito saggi funzionali in collaborazione con il gruppo del Prof. Bottinelli dell’Università di Pavia. Misurazioni della forza sulle singole fibre e della velocità di scorrimento dei filamenti di actina/miosina hanno mostrato che gli Atg7-/- hanno capacità contrattili minori e alterazioni nell’interazione actina/miosina. Sebbene la presenza di fibre denervate sia fisiologica durante l’invecchiamento, gli animali adulti Atg7-/- presentano segni di denervazione precoce, indicata dall’aumento di espressione di markers specifici come Muscle Specific Kinase (MuSK), Acetylcholine Receptor gamma subunit (AchR-gamma) e Neural Cell Adhesion Molecule (NCAM); inoltre la loro espressione aumenta ulteriormente con l’età. Abbiamo quindi deciso di analizzare in dettaglio la giunzione neuromuscolare in collaborazione con il gruppo del Dr. Rudolf presso Karlsruhe Institute of Technology (KIT) a Karlsrhue. Esperimenti di in vivo imaging hanno mostrato che le giunzioni degli Atg7-/- sono instabili e frammentate. Tali alterazioni sono già ben evidenti in animali adulti Atg7-/- suggerendo nuovamente un processo di invecchiamento precoce dovuto al blocco autofagico. Ci siamo poi focalizzati sul potenziale ruolo dello stress ossidativo nel generare e contribuire al fenotipo di questi animali. Abbiamo trattato gli animali per 30 giorni con un anti-ossidante (Trolox), analogo della vitamina E. Dopo il trattamento, le capacità contrattili di actina/miosina e di funzionalità mitocondriale sono tornate al livello dei controlli, mentre abbiamo osservato solo effetti minori sulla giunzione neuromuscolare e nessun miglioramento sull’ atrofia. Questi risultati indicano che lo stress ossidativo ha sicuramente un ruolo sulla funzionalità di proteine contrattili e dei mitocondri, ma che altri fattori sono implicati nel mantenimento della giunzione neuro-muscolare e nell’atrofia. Ci siamo quindi focalizzati su fattori neurotrofici secreti dal muscolo, che fossero alterati nei topi knockout, sia negli adulti che nei vecchi. Dopo uno screening mediante qRT-PCR abbiamo individuato FGF-binding protein 1 (FGFBP1) come l’unico fattore che risultava soppressonei topi Atg7-/- ad entrambe le età. FGFB1 è un importante attivatore di proteine FGFs coinvolte nell’organizzazione pre-sinaptica. A questo punto per capire il ruolo di FGFBP1, abbiamo effettuato esperimenti di silenziamento e di sovra-espressione in vivo. Inizialmenete abbiamo ridotto l’espressione di FGFBP1 in animali di controllo per mimare il fenotipo dei topi Atg7-/-. Due settimane di silenziamento sono state sufficienti per provocare instabilità e frammentazione della giunzione neuromuscolare. Successivamente abbiamo over-espresso FGFBP1 negli animali Atg7-/- per ristabilirne l’espressione ed abbiamo osservato un drastico miglioramento della stabilità della giunzione neuromuscolare. In ultimo, per far luce sul meccanismo che lega l’assenza di autofagia all’alterazione di FGFBP1, ci siamo concentrati su MuSK, una chinasi essenziale per la regolazione della maggior parte dei segnali implicati nello sviluppo e mantenimento della giunzione neuromuscolare. La localizzazione di MuSK risulta alterata negli animali Atg7-/- e il silenziamento di MuSK in vivo in animali di controllo porta all’abbattimento dell’espressione di FGFBP1. Questi risultati suggeriscono che il mantenimento della giunzione neuromuscolare richiede la secrezione di FGFBP1 da parte del muscolo e che l’autofagia è un processo critico per la giusta localizzazione e quindi attività di MuSK. Diversi lavori hanno dimostrato come la restrizione calorica e l’esercizio fisico migliorino la qualità della vita, siano in grado di ritardare l’insorgenza di caratteristiche proprie dell’invecchiamento ed avere effetti benefici sul mantenimento della giunzione neuromuscolare (Melov et al., 2007; Fontana et al., 2010; Sandri et al., 2013; Schiaffino et al., 2013; Coen et al., 2013; Toledo et al., 2013; Guarente, 2013). In letteratura sono presenti lavori che hanno analizzato il ruolo dell’autofagia nell’esercizio (He et al., 2012; Kim et al., 2013), essi però presentano risultati contrastanti. He et al. sostengono che l’autofagia sia richiesta per l’esercizio fisico e la regolazione dell’omeostasi del glucosio (He et al., 2009), al contario altri gruppi osservano un fenotipo opposto in animali in cui l’autofagia è assente costitutivamente nel muscolo scheletrico (Kim et al., 2013). In questo scenario, quindi, non è ancora chiaro il ruolo dell’autofagia durante l’esercizio e se gli effetti benefici dello stesso sono mediati da essa. Per investigare questo aspetto, abbiamo utilizzato animali in cui la delezione del gene Atg7, viene indotta specificamente nel muscolo scheletrico dopo somministrazione di Tamoxifen (Masiero et al., 2009). In questo modo è possibile escludere meccanismi di compensazione e adattamento presenti in modelli in cui le delezioni sono costitutive. Abbiamo deleto acutamente il gene Atg7 in animali adulti e, insieme ai rispettivi controlli, li abbiamo sottoposti ad un protocollo di esercizio concentrico su treadmill. Tuttavia non abbiamo osservato differenze nelle distanze percorse tra i due genotipi. Questo indica che l’autofagia non è richiesta per sostenere attività contrattile durante un normale esercizio concentrico. Abbiamo, poi, sottoposto gli animali ad un protocollo di tre giorni di esercizio eccentrico, per valutare se l’autofagia fosse invece richiesta per il mantenimento del tessuto muscolare in seguito a contrazioni che inducono danno. In questo caso abbiamo osservato che gli animali Atg7-/- corrono di meno rispetto ai controlli e, in particolare, questa differenza risulta significativa nelle femmine. Per investigare il motivo della ridotta performance abbiamo inizialmente analizzato la morfologia, senza però osservare segni di alterazione o infiammazione. Successivamente, abbiamo valutato aspetti metabolici, ma né i livelli di glicemia e di lattacidemia, né la fosforilazione della chinasi attivata da AMP (AMPK), uno dei maggiori indicatori di stress energetico, risultano differenti tra Atg7-/- e controlli dopo l’esercizio. Dato che l’autofagia è richiesta per il mantenimento del pool mitocondriale, abbiamo analizzato se la funzionalità dei mitocondri fosse alterata dopo l’esercizio. In questo caso abbiamo confermato che la delezione acuta di Atg7 causa l’accumulo di mitocondri disfunzionanti, e che la loro percentuale aumentava dopo l’esercizio. La presenza di mitocondri anomali causa un aumento dello stress ossidativo. Infatti abbiamo potuto dimostrare una maggiore carbonilazione delle proteine e aumentati livelli di produzione di ROS dopo l’esercizio, nei topi Atg7-/- rispetto ai controlli. Per valutare gli effetti dello stress ossidativo abbiamo trattato gli animali per sei settimane con un anti-ossidante generico N-acetil-cisteina (NAC). Sorprendentemente, il trattamento si è rivelato dannoso per la performance degli animali di controllo e in più non è stato in grado di migliorare l’attività dei topi Atg7-/-. L’antiossidante ha causato, inoltre, l’accumulo di mitocondri disfunzionanti nei topi di controllo. Questi risultati sono stati confermati anche dopo un trattamento con un diverso anti-ossidante (Mito-TEMPO), ad azione specifica sui mitocondri. E’ riportato in letteratura che il trattamento con anti-ossidanti riduce i livelli di autofagia in animali di controllo e che livelli fisiologici di ROS svolgono funzioni critiche nel signalling cellulare (Underwood et al., 2010; Owusu-Ansah et al., 2013). Negli animali di controllo trattati con anti-ossidante sono state confermate queste evidenze, ed infatti l’autofagia era bloccata. Questa inibizione potrebbe essere la causa dell’accumulo di mitocondri disfunzionanati e della loro performance. Questi risultati sottolineano il ruolo dell’autofagia nel mantenimento della funzionalità mitocondriale durante contrazioni eccentriche. Inoltre definiscono che l’autofagia non è richiesta per il supporto energetico durante le normali contrazioni e che AMPK e i livelli ematici di glucosio non dipendono dall’ attività del sistema autofagico.

O controle postural de idosos tem sido estudado com o objetivo de melhor entender os fatores que levam esta população a sofrer mais quedas que os indivíduos jovens. A redução da força e da potência muscular está entre os aspectos que contribuem para o declínio do controle postural. Entretanto, faltam esclarecimentos sobre a capacidade de produzir força e potência muscular em idosos de diferentes idades. Adicionalmente, faltam informações sobre a relação entre força e potência muscular com o desempenho do controle postural em idosas de diferentes faixas etárias. Sendo assim, o objetivo do presente estudo foi avaliar a força e a potência muscular e sua relação com o desempenho do sistema de controle postural de mulheres idosas de diferentes faixas etárias. Participaram do estudo 80 mulheres que foram divididas em 4 grupos de acordo com a idade: Grupo Jovens (n=20) com idade entre 18 e 30 anos; Grupo 60-64 anos (n=20) com idade entre 60 e 64 anos; Grupo 65-69 anos (n=20) com idade entre 65 e 69 anos; Grupo 70-74 anos (n=20) com idade entre 70 e 74 anos. A força (uma repetição máxima 1RM) e a potência dos músculos extensores e flexores de joelho produzida nas intensidades de 40%, 70% e 90% de 1-RM foram avaliadas através de uma cadeira extensora/flexora. A oscilação do centro de pressão (plataforma de força) e a atividade muscular (Vasto Lateral, Tibial Anterior, Bíceps Femoral e Gastrocnêmio Lateral) foram avaliadas durante a manutenção do controle postural em situação estática, dinâmica e com perturbação da base de suporte. Os resultados revelaram que idosas nas faixas etárias 60-64, 65-69 e 70-74 anos apresentam força e potência muscular semelhantes, porém inferiores em comparação com as jovens. Além disso, mostraram que o desempenho do controle postural de idosas nestas faixas etárias é semelhante, mas inferior em relação às jovens. Não houve diferença entre os grupos para a ativação muscular. Análises de regressão linear múltipla apontaram que a força e a potência muscular estiveram fracamente relacionadas com o desempenho do controle postural. Portanto, estes resultados indicam que, para mulheres com idade entre 60 e 74 anos, a força e a potência muscular são semelhantes e exercem pouca influência no desempenho do controle postural que também é similar nesta faixa etária.
The postural control of elderly has been studied with the aim of better understanding the factors that lead these women to have more falls than young individuals. The reduction in the muscle strength and power are among the aspects that contribute to the impairment of postural control. However, it is still unclear if the capacity of elderly to produce strength and muscle power is distinctive when comparing different ages. In addition, there is little information about the relationship between muscle strength and power with the performance of postural control in older women of different ages. Thus, the purpose of this study was to evaluate the muscular strength and power and its relation with the performance of postural control in elderly of different ages. Eighty women were divided into four groups according to their ages: Group Young (n=20) aged between 18 and 30 years; Group 60-64 (n = 20) aged between 60 and 64 years, Group 65-69 (n = 20) aged between 65 and 69 years and Group 70-74 (n = 20) aged between 70 and 74 years. The subjects performed tests for maximal strength (1-RM) and muscle power produced by knee extensor and flexor muscles in the intensities 40%, 70% and 90% of 1-RM using an extensor/flexor equipment. The sway of center of pressure (force plate) and the muscle activity (Vastus lateralis, Tibialis Anterior, Biceps Femoris and Gastrocnemius Lateralis) were analyzed during the maintenance of postural control in static, dynamics and with disturbance of the support base situations. The results revealed that women from three elderly groups (60-64, 65-69 and 70-74 years) have similar muscle strength and power, but lower compared to young group. Moreover, the results showed that women from three elderly groups are also similar with respect to the performance of postural control, but worse than young. There was no difference between groups for muscle activity. Multiple linear regression analyzes indicated that the strength and muscle power were weakly related to the performance of postural control. Therefore, these results indicate that for women aged between 60 and 74 years, muscular strength and power are similar and have little influence on the performance of postural control that is also similar in this age group.

This research has advanced our understanding of how the brain controls muscle activity during walking, in healthy adults of different ages and people with Parkinson's disease. It has shown that there is direct brain control of leg muscles during the double support phase of gait in these populations. Moreover, this brain control is reduced in people with Parkinson's compared to healthy individuals, and standard anti-Parkinsonian medication does not counteract this deficiency.

The understanding of the ageing process and of ageing-associated diseases represents a significant challenge for the scientific community, governments and society at large. I identified in skeletal muscle of murine models by microarray an increase in PPAR-β/δ expression during acute phase of hindlimb suspension (accelerated ageing), with a possible compensatory role, and an increase in expression levels of NR4A family of nuclear receptors in the skeletal muscle of caloric restricted rats (decelerated ageing). Adipose tissue has an endocrine role being actively involved in cross-talk with peripheral organs such as skeletal muscle. Visfatin is a recently discovered adipokine with pleiotropic functions. Unlike in other types of cells, in differentiated C2C12 myoblasts exogenous added visfatin (eNampt) did not act as an insulin-mimetic factor as shown by western blot and fluorescent assays. Visfatin treatment of differentiated C2C12 myotubes generated nevertheless an increase in the levels of reactive oxygen species as shown by fluorescent microscopy that was dependent on de novo transcription and translation of a new set of genes as revealed by RT-PCR. This increase in oxidative stress was independent of activation of the stress-activated protein kinases (MAPKs) such as ERK and p38, but dependent on NFkB activation as proved by western blot.

Ageing is accompanied by a progressive decline in skeletal muscle mass and strength which may lead to impaired ability to perform activities of daily living in older adults. Although the exact cause of the gradual decline in muscle mass is unknown, identifying efficient strategies aiming to prevent age-related loss of muscle mass and strength is important in order to promote healthy ageing. The overall aim of this thesis was to explore the effects of resistance training alone or combined with a healthy diet on skeletal muscle mass and function of healthy recreationally active older women and to determine mechanisms by which elevated systemic inflammation may contribute to the age-related decline of muscle mass in older adults. The combination of resistance training and a healthy diet induced gains in leg lean mass as well as greater gains in dynamic explosive force than resistance training alone in healthy recreationally active older women. The observed gains in leg lean mass were accompanied by increases in the size of type IIA muscle fibres together with down-regulation in gene expression of a pro-inflammatory factor (IL-1β) and upregulation in gene expression of a regulator of cellular growth (mTOR) in skeletal muscle of older women. Additionally, reduced muscle protein synthesis and size of muscle cells may mediate the detrimental effects of elevated circulating markers of inflammation on muscle mass in older adults. In conclusion, the present thesis depicts mechanistic links between elevated systemic marker of inflammation and muscle mass and provides new information on the effects of combined resistance training and healthy diet on muscle mass and strength in a group of healthy recreationally active older women. This knowledge is instrumental for development of strategies aiming to prevent age-related loss of muscle mass and function.

Le vieillissement s'accompagne d'un déclin des propriétés régénératrices de la plupart des tissus, et l’accumulation de cellules sénescentes au cours de l’âge est associée à ce déclin. La sénescence est une réponse cellulaire dû au raccourcissement des télomères ou à l’exposition aux stress provoquant une accumulation de dommages à l’ADN et / ou un stress oxydant. Cette réponse cellulaire est caractérisée par un arrêt irréversible du cycle cellulaire, une augmentation de l’activité β-galactosidase associée à la sénescence ainsi que la sécrétion du Senescence-Associated Secretory Phenotype ou SASP composé de cytokines, chimiokines, facteurs de croissance et protéases. La composition de ce SASP et par conséquent son rôle, dépendent du type cellulaire et de la nature du stress inducteur de sénescence et contribue aux effets délétères des cellules sénescentes. Parmi les tissus affectés, les muscles du squelette représentent un paradigme pour explorer les stratégies régénératives. Grâce à une population de cellules souches musculaires qui peuvent s'activer, proliférer et se différencier pour former de nouvelles myofibres, le muscle possède une remarquable capacité de régénération après blessure. Au cours du vieillissement et dans certaines dystrophies musculaires, ce potentiel s’épuise en raison de l'entrée progressive des cellules souches musculaires en sénescence. Le développement d’une sénotherapie basée sur l’utilisation de composés sénolytiques, capables d'éliminer les cellules sénescentes constitue donc une stratégie prometteuse. Cependant, les composés actuellement disponibles se caractérisent par un manque de spécificité. Ce travail de thèse a consisté à concevoir, synthétiser et évaluer pour la première fois deux nouveaux types de composés bifonctionnels innovants avec une sélectivité optimisée en ciblant la DPP4, protéase membranaire surexprimée sur la surface des cellules sénescentes. Le premier composé est la conjugaison entre le sénolytique puissant et un ligand de haute affinité de la DPP4 permettant un adressage moléculaire. La seconde est caractérisée, en plus, par un linker immolable, sensible à l’activité β-galactosidase associée à la sénescence permettant la libération spécifique du sénolytique au sein des cellules sénescentes. Les évaluations de ces composés ont été réalisé sur différentes lignées rendues sénescentes par différents stress et comparés à des sénolytiques et sénormorphiques de référence. En conclusion, les molécules bifonctionnelles développées au cours de cette thèse possèdent un pouvoir sénolytique similaire à la Piperlongumine, agent sénolytique utilisé pour la conception avec une sélectivité améliorée vis-à-vis des cellules non-sénescentes comparative aux sénolytiques de référence, Quercetin et Dasatinib par exemple. L’étude du mode d’action de la Piperlongumine a également été étudié, en particulier de l’état métabolique quelques cibles intracellulaires. Nos données constituent ainsi une excellente base pour développer un nouveau format de sénomodulateurs avec une sélectivité améliorée à des fins de régénération musculaire
Ageing is accompanied by a decline in the regenerative properties of most tissues, and the accumulation of senescent cells as we age is associated with this decline. Senescence is a cellular response due to telomere shortening or exposure to stresses causing an accumulation of DNA damage and/or oxidative stress. This cellular response is characterized by an irreversible cell cycle arrest, an increase in the β-galactosidase activity associated with senescence and the secretion of the Senescence-Associated Secretory Phenotype or SASP composed of cytokines, chemokines, growth factors and proteases. The composition of this SASP, and therefore its role, depends on the cell type and the nature of the senescence-inducing stress, and contributes to the deleterious effects of senescent cells. Among the tissues affected, skeletal muscle represents a paradigm for exploring regenerative strategies. Thanks to a population of muscle stem cells that can activate, proliferate and differentiate to form new myofibers, muscle has remarkable capacity for regeneration after injury. During the ageing process and muscular dystrophies, this potential is depleted as muscle stem cells gradually enter senescence. The development of senotherapy based on the use of senolytic compounds capable of eliminating senescent cells is therefore a promising strategy. However, the compounds currently available lack specificity. This thesis involved designing, synthesizing and evaluating for the first time two new types of innovative bifunctional compounds with optimized selectivity targeting DPP4, a membrane protease overexpressed on the surface of senescent cells. The first compound is characterized by a conjugation between a potent senolytic and a high-affinity ligand for DPP4, enabling molecular addressing. The second is additionally characterized by an immolative linker, sensitive to the senescence associated β-galactosidase activity, enabling specific release of the senolytic within senescent cells. These constructs were evaluated on different cell lines rendered senescent by various stresses and compared with reference senolytics and senormorphics. In conclusion, the bifunctional molecules developed during this thesis have a senolytic power similar to that of Piperlongumine, the parent senolytic agent used for design purposes, with improved selectivity towards non-senescent cells compared with reference senolytics such as Quercetin and Dasatinib. The mode of action of Piperlongumine was also studied in particular metabolism disruption, and intracellular targets. Hence, our data constitute an excellent basis to develop a new format of senomodulators with improved selectivity for muscle regeneration strategy purposes

Le métabolisme lipidique est stimulé lors de l'exercice musculaire. La contribution énergétique des lipides s'accentue pendant l'exercice d'endurance d'intensité modérée de longue durée (40% à 60% de VO2max). Outre les acides gras circulants, les réserves de lipides intramyocellulaires (IMCL) sont sensées être utilisées pendant des performances dépassant 4 heures. Devant le manque de preuves expérimentales jusqu'à ce jour, une 1ere étude a été entreprise sur 10 sportifs (40 ± 6 ans) lors d'une course de 24h. Les résultats obtenus sur le muscle vaste externe ont montré une baisse significative d'IMCL de 56% et 45% dans les fibres de type I et IIA respectivement, alors que le glycogène n'a diminué que dans les fibres I. Ces données indiquent un catabolisme d'IMCL plus efficace que celui du glycogène dans les fibres rapides lors de l'exercice d'ultra endurance, dont le mécanisme reste à déterminer. IMCL s'accumule lors du vieillissement ou de l'obésité et peut constituer un risque de résistance à l'insuline (RI). Un entraînement combiné en endurance (EE) et en résistance (ER) de 14 semaines a été mené sur des sujets âgés (73 ± 4 ans) et d'autres en surpoids (58 ± 5 ans). Dans les deux groupes IMCL a augmenté (p<0.05) dans le muscle vaste externe (après EE) mais est resté stable dans le muscle deltoïde (après ER) et s'est accompagné de l'augmentation (p<0.05) de la capacité enzymatique de la β-oxydation après EE. Les céramides musculaires, une classe de lipides impliquée dans RI, ont été diminués (p=0.052) par EE et non par ER. Ces résultats confirment que l'augmentation d'IMCL n'est pas un facteur de risque métabolique et que EE se traduit par une diminution des céramides et de RI
Lipid metabolism is involved during muscle exercise. Energetic contribution of lipids increases during long lasting endurance exercise of moderate intensity (40% à 60% of VO2max). As well as circulating free fatty acids, intramyocellular lipid storages (IMCL) are postulated to be used during performances longer than 4 hours. Due the the lack experimental evidences untill today, a first study was undertaken on 10 athletes (40 ± 6 yrs) during a 24h running. Results obtained on vastus lateralis muscle showed a significant 56% and 45% decrease of IMCL in type I and IIA fibres respectively while glycogen decreased only in type I fibres. These data indicate a more efficient catabolism of IMCL than those of glycogen in fast twitch fibres during ultra endurance exercise, of which mechanism remains to be explored. IMCL accumulates during ageing or overweighting and may constitute a risk of insulin resistance (IR). A combined 14 weeks endurance (ET) and resistance (RT) training was followed by older (73 ± 6 yrs) and overweighted (58 ± 5 yrs) subjects. In the two groups IMCL increased (p<0.05) in vastus lateralis muscle (after ET) but remained stable in deltoidus muscle (after RT) and was linked to an increase (p<0.05) of β-oxydation enzymatic capacity after ET. Muscle ceramides, a category of lipids implicated in IR, decreased (p=0.052) after ET and not after RT. These results confirm that increase in IMCL is not a metabolic risk factor and that ET induces a decrease of both ceramides and IR

The prevalence of muscle dysfunction in elderly populations represents a significant burden on healthcare due to the increased risk of injury, and difficulty in maintaining activities of daily living. This thesis describes the application of advanced computational techniques designed to “learn” the structure of molecular networks to understanding human skeletal muscle ageing. Using this approach we have been able to discover a link between protein translation and age-dependent metabolic reprogramming. Experimental validation using the haploinsufficient eukaryotic initiation factor 6 (eIF6) mouse confirmed this important hypothesis and revealed a substantial molecular reprogramming. The role of eIF6 in skeletal muscle and myoblasts was further investigated, revealing potential up and down-stream signalling mechanisms. The process of angiogenesis is an important step in morphogenesis including systems as diverse as muscle regeneration and tumour growth. This thesis presents the first temporal model of transcriptional alterations in tumour and the surrounding stroma during vascularisation. The application of reverse engineering approaches that are able to integrate perturbation data lead to the hypothesis that modulation of the pro-inflammatory cytokine IL1α may be an important upstream event in angiogenesis.

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.

The present habilitation thesis in theoretical biological physics addresses two central dynamical processes in cells and organisms: (i) active motility and motility control and (ii) self-organized pattern formation. The unifying theme is the nonlinear dynamics of biological function and its robustness in the presence of strong fluctuations, structural variations, and external perturbations. We theoretically investigate motility control at the cellular scale, using cilia and flagella as ideal model system. Cilia and flagella are highly conserved slender cell appendages that exhibit spontaneous bending waves. This flagellar beat represents a prime example of a chemo-mechanical oscillator, which is driven by the collective dynamics of molecular motors inside the flagellar axoneme. We study the nonlinear dynamics of flagellar swimming, steering, and synchronization, which encompasses shape control of the flagellar beat by chemical signals and mechanical forces. Mechanical forces can synchronize collections of flagella to beat at a common frequency, despite active motor noise that tends to randomize flagellar synchrony. In Chapter 2, we present a new physical mechanism for flagellar synchronization by mechanical self-stabilization that applies to free-swimming flagellated cells. This new mechanism is independent of direct hydrodynamic interactions between flagella. Comparison with experimental data provided by experimental collaboration partners in the laboratory of J. Howard (Yale, New Haven) confirmed our new mechanism in the model organism of the unicellular green alga Chlamydomonas. Further, we characterize the beating flagellum as a noisy oscillator. Using a minimal model of collective motor dynamics, we argue that measured non-equilibrium fluctuations of the flagellar beat result from stochastic motor dynamics at the molecular scale. Noise and mechanical coupling are antagonists for flagellar synchronization. In addition to the control of the flagellar beat by mechanical forces, we study the control of the flagellar beat by chemical signals in the context of sperm chemotaxis. We characterize a fundamental paradigm for navigation in external concentration gradients that relies on active swimming along helical paths. In this helical chemotaxis, the direction of a spatial concentration gradient becomes encoded in the phase of an oscillatory chemical signal. Helical chemotaxis represents a distinct gradient-sensing strategy, which is different from bacterial chemotaxis. Helical chemotaxis is employed, for example, by sperm cells from marine invertebrates with external fertilization. We present a theory of sensorimotor control, which combines hydrodynamic simulations of chiral flagellar swimming with a dynamic regulation of flagellar beat shape in response to chemical signals perceived by the cell. Our theory is compared to three-dimensional tracking experiments of sperm chemotaxis performed by the laboratory of U. B. Kaupp (CAESAR, Bonn). In addition to motility control, we investigate in Chapter 3 self-organized pattern formation in two selected biological systems at the cell and organism scale, respectively. On the cellular scale, we present a minimal physical mechanism for the spontaneous self-assembly of periodic cytoskeletal patterns, as observed in myofibrils in striated muscle cells. This minimal mechanism relies on the interplay of a passive coarsening process of crosslinked actin clusters and active cytoskeletal forces. This mechanism of cytoskeletal pattern formation exemplifies how local interactions can generate large-scale spatial order in active systems. On the organism scale, we present an extension of Turing’s framework for self-organized pattern formation that is capable of a proportionate scaling of steady-state patterns with system size. This new mechanism does not require any pre-pattering clues and can restore proportional patterns in regeneration scenarios. We analytically derive the hierarchy of steady-state patterns and analyze their stability and basins of attraction. We demonstrate that this scaling mechanism is structurally robust. Applications to the growth and regeneration dynamics in flatworms are discussed (experiments by J. Rink, MPI CBG, Dresden)
Das Thema der vorliegenden Habilitationsschrift in Theoretischer Biologischer Physik ist die nichtlineare Dynamik funktionaler biologischer Systeme und deren Robustheit gegenüber Fluktuationen und äußeren Störungen. Wir entwickeln hierzu theoretische Beschreibungen für zwei grundlegende biologische Prozesse: (i) die zell-autonome Kontrolle aktiver Bewegung, sowie (ii) selbstorganisierte Musterbildung in Zellen und Organismen. In Kapitel 2, untersuchen wir Bewegungskontrolle auf zellulärer Ebene am Modelsystem von Zilien und Geißeln. Spontane Biegewellen dieser dünnen Zellfortsätze ermöglichen es eukaryotischen Zellen, in einer Flüssigkeit zu schwimmen. Wir beschreiben einen neuen physikalischen Mechanismus für die Synchronisation zweier schlagender Geißeln, unabhängig von direkten hydrodynamischen Wechselwirkungen. Der Vergleich mit experimentellen Daten, zur Verfügung gestellt von unseren experimentellen Kooperationspartnern im Labor von J. Howard (Yale, New Haven), bestätigt diesen neuen Mechanismus im Modellorganismus der einzelligen Grünalge Chlamydomonas. Der Gegenspieler dieser Synchronisation durch mechanische Kopplung sind Fluktuationen. Wir bestimmen erstmals Nichtgleichgewichts-Fluktuationen des Geißel-Schlags direkt, wofür wir eine neue Analyse-Methode der Grenzzykel-Rekonstruktion entwickeln. Die von uns gemessenen Fluktuationen entstehen mutmaßlich durch die stochastische Dynamik molekularen Motoren im Innern der Geißeln, welche auch den Geißelschlag antreiben. Um die statistische Physik dieser Nichtgleichgewichts-Fluktuationen zu verstehen, entwickeln wir eine analytische Theorie der Fluktuationen in einem minimalen Modell kollektiver Motor-Dynamik. Zusätzlich zur Regulation des Geißelschlags durch mechanische Kräfte untersuchen wir dessen Regulation durch chemische Signale am Modell der Chemotaxis von Spermien-Zellen. Dabei charakterisieren wir einen grundlegenden Mechanismus für die Navigation in externen Konzentrationsgradienten. Dieser Mechanismus beruht auf dem aktiven Schwimmen entlang von Spiralbahnen, wodurch ein räumlicher Konzentrationsgradient in der Phase eines oszillierenden chemischen Signals kodiert wird. Dieser Chemotaxis-Mechanismus unterscheidet sich grundlegend vom bekannten Chemotaxis-Mechanismus von Bakterien. Wir entwickeln eine Theorie der senso-motorischen Steuerung des Geißelschlags während der Spermien-Chemotaxis. Vorhersagen dieser Theorie werden durch Experimente der Gruppe von U.B. Kaupp (CAESAR, Bonn) quantitativ bestätigt. In Kapitel 3, untersuchen wir selbstorganisierte Strukturbildung in zwei ausgewählten biologischen Systemen. Auf zellulärer Ebene schlagen wir einen einfachen physikalischen Mechanismus vor für die spontane Selbstorganisation von periodischen Zellskelett-Strukturen, wie sie sich z.B. in den Myofibrillen gestreifter Muskelzellen finden. Dieser Mechanismus zeigt exemplarisch auf, wie allein durch lokale Wechselwirkungen räumliche Ordnung auf größeren Längenskalen in einem Nichtgleichgewichtssystem entstehen kann. Auf der Ebene des Organismus stellen wir eine Erweiterung der Turingschen Theorie für selbstorganisierte Musterbildung vor. Wir beschreiben eine neue Klasse von Musterbildungssystemen, welche selbst-organisierte Muster erzeugt, die mit der Systemgröße skalieren. Dieser neue Mechanismus erfordert weder eine vorgegebene Kompartimentalisierung des Systems noch spezielle Randbedingungen. Insbesondere kann dieser Mechanismus proportionale Muster wiederherstellen, wenn Teile des Systems amputiert werden. Wir bestimmen analytisch die Hierarchie aller stationären Muster und analysieren deren Stabilität und Einzugsgebiete. Damit können wir zeigen, dass dieser Skalierungs-Mechanismus strukturell robust ist bezüglich Variationen von Parametern und sogar funktionalen Beziehungen zwischen dynamischen Variablen. Zusammen mit Kollaborationspartnern im Labor von J. Rink (MPI CBG, Dresden) diskutieren wir Anwendungen auf das Wachstum von Plattwürmern und deren Regeneration in Amputations-Experimenten

Yes
Background: Skeletal muscles undergo changes with ageing which can cause sarcopenia that can result in frailty. Quantitative MRI may detect the muscle-deficit component of frailty which could help improve the understanding of ageing muscles. Aims: To investigate whether quantitative MRI measures of T2, fat fraction (FF), diffusion tensor imaging and muscle volume can detect differences within the muscles between three age groups, and to assess how these measures compare with frailty index, gait speed and muscle power. Methods: 18 ‘young’ (18–30 years), 18 ‘middle-aged’ (31–68 years) and 18 ‘older’ (> 69 years) healthy participants were recruited. Participants had an MRI of their dominant thigh. Knee extension and flexion power and handgrip strength were measured. Frailty (English Longitudinal Study of Ageing frailty index) and gait speed were measured in the older participants. Results: Young participants had a lower muscle MRI T2, FF and mean diffusivity than middle-aged and older participants; middle-aged participants had lower values than older participants. Young participants had greater muscle flexion and extension power, muscle volume and stronger hand grip than middle-aged and older participants; middle-aged participants had greater values than the older participants. Quantitative MRI measurements correlated with frailty index, gait speed, grip strength and muscle power. Discussion: Quantitative MRI and strength measurements can detect muscle differences due to ageing. Older participants had raised T2, FF and mean diffusivity and lower muscle volume, grip strength and muscle power. Conclusions: Quantitative MRI measurements correlate with frailty and muscle function and could be used for identifying differences across age groups within muscle.
JDB is funded by a National Institute for Health Research (NIHR) (and Health Education England) Clinical Lectureship. This paper presents independent research funded/supported by the National Institute for Health Research (NIHR) Leeds Biomedical Research Centre (BRC). AC and LB are funded as part of the NIHR Collaboration for Leadership in Applied Health Research and Care, Yorkshire and Humber (NIHR CLAHRC YH).

Yes
Telomerase reverse transcriptase (TERT) is a key component of the telomerase complex. By lengthening telomeres in DNA strands, TERT increases senescent cell lifespan. Mice that lack TERT age much faster and exhibit age-related conditions such as osteoporosis, diabetes and neurodegeneration. Accelerated telomere shortening in both human and animal models has been documented in conditions associated with insulin resistance, including T2DM. We investigated the role of TERT, in regulating cellular glucose utilisation by using the myoblastoma cell line C2C12, as well as primary mouse and human skeletal muscle cells. Inhibition of TERT expression or activity by using siRNA (100nM) or specific inhibitors (100nM) reduced basal 2-deoxyglucose uptake by ~50%, in all cell types, without altering insulin responsiveness. In contrast, TERT over-expression increased glucose uptake by 3.25-fold. In C2C12 cells TERT protein was mostly localised intracellularly and stimulation of cells with insulin induced translocation to the plasma membrane. Furthermore, co-immunoprecipitation experiments in C2C12 cells showed that TERT was constitutively associated with glucose transporters (GLUTs) 1, 4 and 12 via an insulin insensitive interaction that also did not require intact PI3-K and mTOR pathways. Collectively, these findings identified a novel extra-nuclear function of TERT that regulates an insulin-insensitive pathway involved in glucose uptake in human and mouse skeletal muscle cells.

The Na+,K+-ATPase (NKA) is a key protein involved in the maintenance of skeletal muscle excitability and comprises 2 subunits (α and β), each of which express multiple isoforms at a protein level in skeletal muscle (α1-3 and β1-3). The fibre-specific expression, adaptability and roles of each isoform in human skeletal muscle are explored in this thesis. Research utilising muscle biopsies typically uses samples obtained from the vastus lateralis muscle, which in healthy young people comprises similar proportions of type I and II fibres. Analyses using whole muscle pieces don’t allow the detection of fibre-type specific differences and changes occurring at a cellular level. Hence analysis of skeletal muscle samples at the single fibre level offers important advantages in understanding NKA regulation. This thesis therefore investigated the isoform abundance of the NKA in human skeletal muscle single fibres and their adaptability following intense repeated-sprint exercise (RSE) training in healthy young adults (Study 1); with ageing (Study 2) and after high-intensity interval training (HIT) in the elderly (Study 3) and after voluntary inactivity and resistance training in healthy young adults (Study 4).

Guo, Anyun.
Thesis Ph.D. Chinese University of Hong Kong 2014.
Includes bibliographical references (leaves 153-164).
Abstracts also in Chinese.
Title from PDF title page (viewed on 21, December, 2016).

Background and objectives: Current research on respiratory diseases is mainly focused on the effects of drug treatments, however much less work has looked into the use of manual treatments of patients with symptoms of respiratory dysfunction. Studies currently being conducted through Victoria University are attempting to investigate whether manual treatments, such as HVLA and Lymphatic Pump techniques have an effect on lung function. There has however been no research into the effect of Muscle Energy Technique (MET) on measurable respiratory function even though it is such a widely used technique. This study aims to identify whether MET leads to a measurable change in ventilatory function in asymptomatic individuals. Conclusion: Thoracic MET performed on asymptomatic volunteers had no significant effect on FVC or FEV measurements immediately post, 10 minutes post or 20 minutes post-intervention. This minor thesis was written by post-graduate students as part of the requirements of the Master of Health Science (Osteopathy) program.

Oxidative stress has been implicated in various progressive degenerative conditions, such as skeletal muscle wasting and therefore this study sought to determine a role for the superoxide generating NADPH oxidase and antioxidant enzyme systems in conditions of skeletal muscle wasting. The results of these studies indicated changes in the gene expression of important components of NADPH oxidase in animal models of age-associated sarcopenia, cancer-induced cachexia and a model of antioxidant superoxide dismutase overexpression. Also observed were changes in superoxide dismutase that appeared to contribute significantly to alterations in cellular reactive oxygen species and contribute to skeletal muscle wasting in these conditions. While these oxidative and antioxidative systems demonstrated complex changes in these models, NADPH oxidase is indeed altered in response to aging, cancer and superoxide dismutase overexpression, which appear to be involved in complex redox-sensitive signaling that essentially regulates skeletal muscle atrophy and hypertrophy pathways.

Background and objectives: Current research on respiratory diseases is mainly focused on the effects of drug treatments, however much less work has looked into the use of manual treatments of patients with symptoms of respiratory dysfunction. Studies currently being conducted through Victoria University are attempting to investigate whether manual treatments, such as HVLA and Lymphatic Pump techniques have an effect on lung function. There has however been no research into the effect of Muscle Energy Technique (MET) on measurable respiratory function even though it is such a widely used technique. This study aims to identify whether MET leads to a measurable change in ventilatory function in asymptomatic individuals. Conclusion: Thoracic MET performed on asymptomatic volunteers had no significant effect on FVC or FEV measurements immediately post, 10 minutes post or 20 minutes post-intervention. This minor thesis was written by post-graduate students as part of the requirements of the Master of Health Science (Osteopathy) program.

Wang, Hui Chuan.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2013.
Includes bibliographical references (leaves 184-202).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

Falls and related injuries are the leading cause of mortality and morbidity in people aged 65 years and older. Despite all the many health benefits already reported from regular exercise participation including reduction of falls, this population group is still reported to be inadequately physically active. Therefore, it is critical to develop and validate new options that can enhance exercise uptake, sustain participation of this group and reduce their risk of having falls.

碩士
國立屏東科技大學
食品科學系所
101
The thermal gelation of cross-linked milkfish muscle proteins with 1-ethyl-3-（3-dimethylamino-propyl）carbodiimide（EDC）was studied by means of jelly-strength , ATP sensitivity , the activities of ATPases , SDS electrophoresis and chymotryptic digestibility. The treatment with EDC（0.1mg EDC/mg protein）gave higher jelly-strength values to the actomyosin for all the tested temperatures. Gel-disintegration for actomyosin and myosin was completely depressed by EDC. By treating the actomyosin with EDC（0.05 mg EDC / mg protein）, the activation of Mg-ATPase and inactivation of EDTA-ATPase occurred simultaneously while Ca-ATPase was unchanged. In EDC-treated actomyosin the decrease of myosin heavy chain and the formation of polymers were observed form SDS electrophoretograms. The EDC-treatment for actomyosins resulted in the decrease in the relative viscosity and the disappearance of ATP sensitivity.The desalting aggregation ability of actomyosins disappeared with EDC-treatment. The cross-linked myosin with EDC formed a polymerized complex. The thermal gelation of myosin by heating at 30-50℃ were enhanced in the presence of up to 25 mol EDC/105g protein and the EDC more than 25 mol effectively depressed gel-disintegration. The chymotryptic digestion of EDC-myosin suggested that there are two cross-linking sites with EDC above or below 1mM.

\({Background}\) The muscles of the trunk are essential for an individual’s ability to perform normal functional activities such as standing or walking and are involved in the control of balance and posture. Decreased trunk proprioception, muscle imbalance and functional decline of these muscles all have the potential to alter posture and balance, leading to an increase in the risk of falls and deterioration in physical function and quality of life. The trunk muscles investigated in this study are the rectus abdominis, transversus abdominis, internal oblique, external oblique and the lumbar multifidus (MF) muscles. The external and internal abdominal oblique muscles are torque producers of the trunk, while the transversus abdominis and MF muscles are tonically active during weight-bearing activities. While peripheral skeletal muscle mass and strength deteriorate with age (sarcopenia), the effect of ageing or pathology on muscles of the trunk, and the potential effects these changes have on physical function and quality of life are not fully understood. \({Aims}\) This thesis aimed to: 1. Summarise the evidence in older adults for: i. changes in function, composition and morphology of the abdominal and MF muscles and the effects of these changes on physical function. ii. validity and reliability of electromyographic and imaging measurements of abdominal and multifidus muscles among adults aged 50 years and older. 2. Determine the test-retest reliability of ultrasound imaging (USI) for the assessment of abdominal and MF muscle thickness and CSA at the L2 - L5 vertebral levels in older adults. These results will determine the appropriateness of using these measures in cross-sectional and longitudinal studies (aims 3 and 4), particularly as outcomes for randomised controlled trials (RCT) 3. Determine the effect of 12 months of vitamin D supplementation, on morphology and function of the abdominal and MF muscles of adults aged 50 to 79 years with low serum 25(OH)D levels. 4. Determine the associations between abdominal and MF muscle size and function and measures of physical activity, physical function and quality of life among older adults. \({Methods}\) The first part of this thesis provides a comprehensive and systematic assessment of the current literature investigating abdominal and multifidus muscle in older adults (Study 1). This study helped to inform the studies in the second part of this thesis (Studies 2 – 4) that aimed to fill some of the gaps in the literature identified in the systematic review. Studies 2 - 4 used data from an ultrasound imaging sub-study (n=217) of the Vitamin D Effect on Osteoarthritis (VIDEO) clinical trial, conducted between June 2010 and December 2013. The participants were community-dwelling adults aged 50-79 years with ongoing symptoms of knee osteoarthritis and serum 25(OH)D levels between 12.5 and 60 nmol/L. \({Key}\) \({findings}\) The key findings and evidence gaps identified by the systematic review (Study 1) were: • Research on abdominal and MF muscles in older adults was limited •There was limited evidence that imaging modalities were reliable tools for the assessment of abdominal and MF muscles of older adults • There was no evidence for the reliability of test-retest measures of abdominal or MF muscles using ultrasound imaging in older adults • There was limited evidence for an age-related decrease in abdominal and MF muscle size and an increase in intramuscular fat infiltrations that had the potential to affect physical function of older adults • There was limited, but consistent evidence of detrimental effects on abdominal and MF muscles by conditions that affect physical function such as various spinal conditions and low back pain (LBP) • There was limited or no evidence on the effect of other conditions such as stroke or vitamin D deficiency on abdominal or MF muscles • There were no studies in healthy, older adults investigating associations between trunk muscle measures and any aspect of physical activity or physical function nor identifying modifiable factors that could mitigate age-related changes in abdominal or MF muscles Study 2 determined the test-retest reliability for measurements of abdominal and MF muscle thickness and CSA. The estimates of reliability were as follows: • Substantial (ICC 0.87-0.98) for all measurements of abdominal muscles, except for the right internal oblique muscle in the contracted state which was moderate (ICC 0.75) • Substantial (ICC 0.84-0.91) for all measurements of MF muscle CSA • Fair to moderate (ICC 0.55-0.74) for all measurements of MF muscle thickness Establishing the test-retest reliability of USI for assessing abdominal and lumbar multifidus muscles confirmed the appropriateness of using these measures in longitudinal studies and particularly as outcomes for randomised controlled trials (RCT) We used this technique to assess muscle outcomes in the VIDEO RCT. Twelve months of vitamin D supplementation in vitamin D deficient older adults with knee osteoarthritis alone was not an effective means to improve or maintain abdominal or MF muscle size of active, community dwelling adults aged 50-79 years (Study 3). Given the lack of effect of vitamin D on muscle size, we proceeded to investigate another important gap in the literature described in section 1.4 of the introduction of this thesis and also identified the systematic review, namely a paucity of studies investigating associations between trunk muscle measures and any aspect of physical function in healthy older adults. This cross-sectional analysis revealed no cross-sectional correlations between abdominal or MF muscle size or function (assessed by changes in muscle thickness on contraction) and measures of physical activity (International Physical Activity Questionnaire and pedometer), physical function ( functional deficit subscale of the Western Ontario and McMaster Universities Arthritis Index and quality of life (Assessment of Quality of Life instrument) in healthy, active, community-dwelling adults with knee osteoarthritis (Study 4). However, such effects cannot be ruled out due to the limitations in the measures used to assess muscle function, physical activity and physical function available in VIDEO. \({Conclusion}\) Age and various spinal conditions have detrimental effects on abdominal and MF muscle size, strength, activation and muscle quality, reflected in an increase in intramuscular fat infiltrations. However, how these detrimental factors affect physical function and healthy ageing are not clearly understood. The findings in this thesis have contributed to fill some of the identified gaps in the literature. Particularly important are the finding of the RCT on the effect of vitamin D supplementation on abdominal and MF muscles suggesting that vitamin D supplementation alone is not an effective therapy to improve or reserve the size or ability to contract of postural muscle of the trunk. A question that was not investigated in this study and remains unanswered is the effect of vitamin D supplementation on other aspects of trunk muscle function such as strength, power or physical function. Study 4 contributed to a limited body of evidence on the associations between abdominal and MF muscle size and measures of physical function. Although our negative results are consistent with findings in the systematic review published in 2017 and some of the studies in the systematic review update in this thesis, the evidence is not robust enough to rule out associations between abdominal and MF muscle size and measures of physical function in older adults. Given that the evidence base remains limited, further research is definitely required, to further address important persisting gaps in the literature. These include: • what other factors affect trunk muscles of older adults? • what are the long-term effects of declines in muscle size on physical activity, physical function and quality of life of older adults? • what strategies can be implemented to mitigate the effect of age-related changes on these muscles? Finally, the literature in the systematic review update suggest that abdominal and MF muscle strength and muscle quality are important determinants of physical function in older adults. Therefore, these two areas of research would also appear to be promising for future research investigating the effect of age-related changes in abdominal and MF muscles on physical function and quality of life of older adults.

碩士
東海大學
環境科學與工程學系
97
The levels of beryllium ( Be ) in muscles of poultry and live-stock maybe very low and have not been reported in muscles of poultry and live-stock certified reference materials ( e.g. BCR CRM-384 ). The purpose of this thesis is to develop a method for the determination of Be in muscles of poultry and live-stock using microwave digestion, chelating with acetylacetone（acac）, pre-concentrating by solid-phase extraction, eluting with methanol and then measured by GFAAS. Trace amounts of Be (0.004 - 0.56 ng) in a dried pork muscle sample (20 mg) could be accurately determined by graphite-furnace atomic absorption spectrophtometry (GFAAS) after treating with microwave digestion (HNO3/H2O2) at 85C for 10 min and using acetylacetone as a chelating agent in the presence of an acetate buffer (pH 6.0). The method detection limit (MDL, 3) for Be was found to be 0.19 ng/g; the calibration graph was linear up to 28 ng/g. The Be contents measured in four muscle samples (BCR CRM - 384 pork muscle, and three samples collected in Kaohsiung, Taiwan, R. O. C.) were between 2.5 and 3.0 ng/g. Good spiked recoveries (95.7 – 103.2%) were obtained for these four samples with a relative standard deviation (RSD, n = 3) ≤ 2.1%. The proposed method could be applied to measurements of Be in muscles of poultry and live-stock.