THE THESIS EXPLAINED

Cachexia is a multifactorial and complex syndrome associated with chronic diseases such as cancer, which leads to an involuntary and progressive loss of body weight. Cachexia is characterized by an increased level of pro-inflammatory cytokines and other humoral factors, which induce autophagy and proteolysis mediating muscle fiber atrophy. Muscle wasting interferes with the therapy of the primary disease and is a negative prognostic factor for cancer patients. Physical activity has been proposed to counteract cachexia since it improves muscle metabolism and decreases systemic inflammation by regulating the balance between anti-and proinflammatory cytokines. Physical activity is thought to promote the release of beneficial myokines from the exercised muscle. Exercise is associated with an improvement in the quality of life and survival in cancer patients. However, the mechanisms underlying the response to physical exercise in cancer cachexia are not known. We hypothesized that the effects of exercise are mediated by a mechano-transduction response in muscle, involving Serum Response Factor (SRF), which is a gene transcription factor induced by mechanical cues mediated by actin polymerization. Since the broad involvement of SRF in muscle homeostasis includes a pivotal role in satellite cell proliferation and recruitment to myotubes, we also hypothesized that SRF beneficial effects are extended to the satellite cell niche.

To verify our hypothesis, we used an in vivo model consisting of mice injected or not with C26 tumor cells, to induce cancer cachexia. The animals were further divided into two groups, which performed or not voluntary exercise. In this way, we had four experimental groups allowing us to test any interaction between exercise and cancer cachexia. To specifically address the SRF role in the rescue of muscle mass by physical exercise in cancer cachexia, we used an SRF loss of function models, consisting of inducible, musclespecific SRF KO mice with the Cre loxP system activated by tamoxifen injections For these mice, a different tumor, i.e., the Lewis Lung Carcinoma (LLC) was used to induce cachexia, due to immune incompatibility resulting in tumor rejection in these nonimmunoexpressed mouse lines.

Our results showed that the C26 tumor induces loss of body weight, muscle wasting, protein degradation, including a decrease of SRF protein, which resulted from the downregulation of its transcriptional activity. We observed that the effects of C26 tumors were counteracted by physical activity, suggesting that voluntary exercise is an effective means to counteract cachexia; we also observed that the beneficial exercise effects are linearly correlated to the SRF protein levels in muscle, suggesting a pivotal role for SRF as a mediator of the response to exercise. To demonstrate that SRF is necessary to transduce exercise effects, we applied the same exercise protocol as above to LLC tumor-bearing SRF KO mice and observed that exercise could no longer rescue cachexia, thereby indicating that SRF is essential for the organismal response to exercise. Additionally, we observed opposite effects of the tumor and exercise on Pax7+ cells, i.e., the satellite cells which are responsible for muscle regeneration and homeostasis. Since we also observed the recruitment of nuclei within the muscle fibers in response to exercise, we concluded that the myogenic program is activated by exercise, resulting in stem cell recruitment to muscle fibers, which could contribute to muscle homeostasis. Overall, our data demonstrate that the positive effects of exercise on cancer cachexia are due to an SRF-mediated mechanical response of muscle fibers, resulting in transcriptional changes activating promyogenic genes and in the recruitment of muscle stem cells which ultimately result in the rescue of muscle mass.

INTRODUCTION

3.1 The adult skeletal muscle

The muscular system of human beings comprehends almost 670 skeletal muscles represents 40% of body weight in the adult. Contractility, excitability, elasticity, stretchable, and plasticity are among the most prominent features of the musculature. Its functions are locomotion, posture and body temperature regulation.

The adult skeletal muscle tissue contains multinuclear, cylindrical cells called muscle myofibers, which are organized in parallel bundles by a connective tissue network that peripherally continues with the tendons to transfer the contractile force from muscle fibers to the bone. The perimysium is conjunctive tissue which bring together between 20 and 80 myofibers, the whole being called a muscle fascicle. Within each fascicle, individual muscle fibers are separated one from the other by the endomysium. A specialized extracellular matrix (ECM) called basement membrane separates the muscle and the connective tissue [START_REF] Mouw | Extracellular matrix assembly: a multiscale deconstruction[END_REF].

Between the of the myofiber sarcolemma and the basement membrane, lies the another major cell population of the muscle tissue, the satellite cells, which are the stem cells of this tissue (Mauro1961).

Epimysium surrounds the whole muscle. Thanks to the connective tissue, the striated skeletal muscle is innervated and vascularized on a single-fiber basis. The area between the motoneuron and the myofiber is the neuromuscular junction; a single neuron is responsible for multiple neuromuscular junctions, thus innervating more than one fiber, constituting a motor unit [START_REF] Schwartsburd | Catabolic and anabolic faces of insulin resistance and their disorders: a new insight into circadian control of metabolic disorders leading to diabetes[END_REF].

The "sarcomere" is a muscle-specific structural and functional unit allowing contraction. Single sarcomeres are limited by Z lines and are approximately two micrometers long; however, hundreds of thousands of sarcomeres form a myofibril, the myofilaments arrangement forms a sequence of alternating light (I band) and dark bands (A band), reflecting the existence of two kinds of filaments partially intermingled. The major proteins of the myofilaments are myofilaments are Myosin, which forms thick filaments, and Actin, which forms thin filaments (with other proteins) (« Actin, Myosin, and Cell Movement -The Cell -NCBI Bookshelf » s. d.). Since a muscle cell is highly enriched in protein myofilaments and the muscle tissue accounts for a significant amount of body mass, the skeletal muscle is an important protein reserve for organisms; the status of the muscle is associated with the life span, quality of life and anabolism/catabolic balance [START_REF] Leal | Physical Exercise-Induced Myokines and Muscle-Adipose Tissue Crosstalk: A Review of Current Knowledge and the Implications for Health and Metabolic Diseases[END_REF]. Furthermore, muscle has been identified as an endocrine organ that synthesizes cytokines and other peptides and factors, collectively named myokines [START_REF] Pedersen | Voluntary Running Suppresses Tumor Growth through Epinephrine-and IL-6-Dependent NK Cell Mobilization and Redistribution[END_REF], representing the molecular basis for the crosstalk between skeletal muscle and other organs [START_REF] So | Exerciseinduced myokines in health and metabolic diseases[END_REF].

Skeletal Muscle homeostasis

Skeletal muscle is a highly plastic tissue that adapts to demands, which vary along the life span of an individual, by shifting its content proteins. Indeed, it is established that atrophy appears as a decrease of cytoplasm volume and myofibril content in muscle fibers, following a decrease of mechanical stimulation or nutriments [START_REF] Haddad | Atrophy responses to muscle inactivity. II. Molecular markers of protein deficits[END_REF][START_REF] Giger | Rapid muscle atrophy response to unloading: pretranslational processes involving MHC and actin[END_REF]). On the contrary, after an increase of mechanical stress or upon hypertrophying hormonal signals the cytoplasmic volume of myofibers can increase, thanks to an increase of the protein synthesis, on the inhibition of proteolysis signaling pathways, or both (true hypertrophy); also, incorporation of myoblasts has been associated with muscle growth, especially in the young [START_REF] Egan | Exercise Metabolism and the Molecular Regulation of Skeletal Muscle Adaptation[END_REF][START_REF] Bell | The beneficial role of proteolysis in skeletal muscle growth and stress adaptation[END_REF]. Taken together, the muscle is managed by a protein balance which requires e specific transcription program. The degradation of damaged protein has been highlighted by muscle atrophy studies, and shown to require a common set of genes: the atrogens [START_REF] Foletta | The Role and Regulation of MAFbx/Atrogin-1 and MuRF1 in Skeletal Muscle Atrophy[END_REF]). On the contrary, the specific activation of genes associated with the hypertrophic program permits the protein synthesis, a finding which represents an important stake in the treatments against sarcopenia and several pathologies associated with muscle atrophy [START_REF] Schwartsburd | Catabolic and anabolic faces of insulin resistance and their disorders: a new insight into circadian control of metabolic disorders leading to diabetes[END_REF]. Finally, thanks to the abundance of satellite cells, the muscle can fully regenerate in a few days after a muscular injury [START_REF] Bintliff | Radioautographic Study of Skeletal Muscle Regeneration[END_REF][START_REF] Capers | Multinucleation of Skeletal Muscle in vitro[END_REF].

Contribution of muscles stems cells in muscular hypertrophy

In 1961, using electron microscopy, Mauro first described the satellite cells (SCs) as mononuclear cells located between the basal lamina and the sarcolemma [START_REF] Mauro | SATELLITE CELL OF SKELETAL MUSCLE FIBERS[END_REF]. Satellite cells SCs are multipotent, quiescent mononuclear cells which represent 2-7% of the cells related to muscle fibers [START_REF] Snijders | Satellite cells in human skeletal muscle plasticity[END_REF][START_REF] Hawke | Myogenic satellite cells: physiology to molecular biology[END_REF]. Furthermore, they can redesign and regenerate the myofibers during postnatal growth and in response to overload and injury. The fact that SCs reside in a niche in proximity to the capillaries [START_REF] Nederveen | Skeletal muscle satellite cells are located at a closer proximity to capillaries in healthy young compared with older men[END_REF]) allows the interaction between SCs, myofibers and endothelial cells on each side of the basement membrane (Yin, Price, et Rudnicki 2013a). On a genetic point of view, SCs are characterized by the expression of transcription factors such as Pax3 or Pax7 until their activation by muscle damage [START_REF] Günther | Myf5-Positive Satellite Cells Contribute to Pax7-Dependent Long-Term Maintenance of Adult Muscle Stem Cells[END_REF].

Once the SCs activate, they re-enter the cell cycle and co-express Pax7 and Myf5, being then considered as myoblasts. Their divisions can be symmetric, leading to two activated cells (Pax7+/Myf5+) or asymmetric, leading to one activated cell (Pax7+/Myf5+) and a quiescent cell (Pax7+/ Myf5-) a process which reforms the SC pool, and is a typical stemness feature [START_REF] Gurevich | Asymmetric Division of Clonal Muscle Stem Cells Coordinates Muscle Regeneration in Vivo[END_REF].

Transcription factors such as Myf5, MyoD, myogenin, and MRF4 promote myogenic differentiation and their expression is inhibited by Pax7, which rather characterizes undifferentiated cells. The activated SCs fuse to existing damaged muscle fibers or together to form nascent myofibers, or myotubes, expressing MRF4 and the typical genes of terminal differentiation, such the heavy chain of myosin (MHC) (Yin, Price, et Rudnicki 2013a). Interestingly, the skeletal muscle possesses several stem cell types which may participate in skeletal muscle regeneration [START_REF] Sirabella | Sources for Skeletal Muscle Repair: From Satellite Cells to Reprogramming[END_REF]. The cells with myogenic potential which are addressing high interest for therapeutic applications are: endothelial progenitor cells, mesoangioblasts (MABs), CD133+ cells, muscle interstitial cells (PICs), side population stem cells, hematopoietic stem cells, mesenchymal stem cells, multipotent adult progenitor cells, and adipose derived stem cells [START_REF] Sirabella | Sources for Skeletal Muscle Repair: From Satellite Cells to Reprogramming[END_REF]. Synthesis and degradation of proteins are a dynamic equilibrium, which, depending on circumstances, can promote muscle gain or loss [START_REF] Egerman | Signaling Pathways Controlling Skeletal Muscle Mass[END_REF]. They are controlled by pathways influenced by mechanical stress, physical exercise, accessibility of nutrients and growth factors.

Insulin-like Growth Factor 1 (IGF-1) controls protein synthesis and degradation through the regulation of the protein kinase B (AKT) targets [START_REF] Sandri | Signaling in Muscle Atrophy and Hypertrophy[END_REF]. AKT activates protein synthesis by the phosphorylation of mTOR protein, which stimulates protein synthesis via phospho-S6 kinase and EIF4EBP1 and inhibits protein catabolic pathways by phosphorylation of transcriptional factors FoxO which excludes this latter from nuclei. To highlight its role in muscle hypertrophy, it has been shown in a gain of function model for AKT, a muscle hypertrophy and an increase in lean muscle mass [START_REF] Sandri | Foxo Transcription Factors Induce the Atrophy-Related Ubiquitin Ligase Atrogin-1 and Cause Skeletal Muscle Atrophy[END_REF][START_REF] Lai | Conditional Activation of Akt in Adult Skeletal Muscle Induces Rapid Hypertrophy[END_REF]). Worth noting, IGF-1 is known under multiple isoforms and the muscle secretes one specific, IGF-1E which is local with hypertrophic effect [START_REF] Musarò | Counteracting sarcopenia: the role of IGF-1 isoforms[END_REF]. Follistatin has an indirect hypertrophic effect, being an inhibitor of myostatin and activin A. The latter are members of TGF family (Transforming Growth Factor) which inhibits muscle hypertrophy via Smad2 and 3 [START_REF] Elkina | The Role of Myostatin in Muscle Wasting: An Overview[END_REF]. Follistatin binds myostatin and inhibits its activity by stopping its binding to the receptor. It was demonstrated that the overexpression of follistatin in vivo leads to muscle hypertrophy like that observed in Myostatin null mice [START_REF] Elkina | The Role of Myostatin in Muscle Wasting: An Overview[END_REF]. In contrary, in the follistatin null mice showed a decrease in muscle mass which suggests its significant role in the regulation of muscle homeostasis [START_REF] Hill | Regulation of Myostatin in Vivo by Growth and Differentiation Factor-Associated Serum Protein-1: A Novel Protein with Protease Inhibitor and Follistatin Domains[END_REF]). Among effector proteins, the MAPK family (Mitogen-Activated Protein Kinases) is involved in muscle hypertrophy. MAPK p38 allows the protein synthesis with activation of eIF4E and improves the myogenic differentiation with the MyoD phosphorylation and the activation of myogenin and p21 [START_REF] Lluís | E47 Phosphorylation by P38 MAPK Promotes MyoD/E47 Association and Muscle-Specific Gene Transcription[END_REF]).

Factors promoting muscle atrophy

Aging, fasting, malnutrition and immobilization all can induce muscular atrophy [START_REF] Gao | Muscle Atrophy Induced by Mechanical Unloading: Mechanisms and Potential Countermeasures[END_REF][START_REF] Ryan | Cancer-Associated Malnutrition, Cachexia and Sarcopenia: The Skeleton in the Hospital Closet 40 Years Later[END_REF]. Atrophy determines a decrease of muscle functional capacity, noticeably muscle strength [START_REF] Schwartsburd | Catabolic and anabolic faces of insulin resistance and their disorders: a new insight into circadian control of metabolic disorders leading to diabetes[END_REF]. Atrophy is often the consequence of a combination of anabolism inhibition and catabolism activation which leads to protein degradation via several systems: autophagy, ubiquitin-proteasome system, and proteases (calpains and caspases) [START_REF] Fanzani | Molecular and cellular mechanisms of skeletal muscle atrophy: an update[END_REF].

Autophagy is involved in protein and aged-organelle degradation [START_REF] Chun | Autophagy: An Essential Degradation Program for Cellular Homeostasis and Life[END_REF]. Three autophagy mechanisms have been studied: macro-autophagy, micro-autophagy and autophagy associated chaperons 'proteins. During macro-autophagy, the proteins are taken in phagosomes then degraded by lysosomal proteases. During this process, ATG7 and LC3 participated in the auto-phagosome formation and the substrates involve damaged organelles and cytosolic proteins [START_REF] Mizushima | The role of mammalian autophagy in protein metabolism[END_REF]. Microautophagy is a non-selective lysosomal degradative way, involving the engulfment direct of cytoplasmic content at a frontier membrane [START_REF] Li | Microautophagy: Lesser-Known Self-Eating[END_REF]. Chaperone-mediated autophagy implies one of the intracellular proteolytic pathway for the selective degradation of cytosolic proteins within lysosomes. The characteristics include absence of vesicle formation or membrane invagination in the procedure of protein supply to lysosomes [START_REF] Zhang | Chaperone-Mediated Autophagy Upregulation Rescues Megalin Expression and Localization in Cystinotic Proximal Tubule Cells[END_REF].

The ubiquitin-proteasome system is also involved in atrophy phenomena. Like autophagy, the role of the ubiquitin-proteasome system is to degrade the nonessential proteins [START_REF] Lilienbaum | Relationship between the proteasomal system and autophagy[END_REF].

To be degraded, the protein is associated with a ubiquitin by ubiquitin's ligases E3 which are recognized by the proteasome 26S.

The ubiquitin ligases MuRF-1 (Muscle Ring Finger-1) and MAFbx/atrogin-1 (Muscle Atrophy F-box) are muscle-specific Ubligases associated with several conditions of muscle atrophy, and their transcription is controlled by FoxO (Forkhead Box (Fox) O family). Consistent with their function, they are over expressed during the atrophy associated with cachexia, freeze injury, denervation or immobilization [START_REF] Foletta | The Role and Regulation of MAFbx/Atrogin-1 and MuRF1 in Skeletal Muscle Atrophy[END_REF].

The activation of calpains and caspase-3 promotes the proteolytic processes of protein muscles [START_REF] Nelson | Cross-talk between the calpain and caspase-3 proteolytic systems in the diaphragm during prolonged mechanical ventilation[END_REF]. The calpains are the main actors of calcium-depended proteolysis [START_REF] Storr | The Calpain System and Cancer[END_REF].

They can degrade the kinases, phosphatases and proteins like Actin and the Myosin [START_REF] Kuchay | Calpain-mediated regulation of platelet signaling pathways[END_REF]. The caspases are a class of enzymes triggering apoptosis. However, they also participate proteolysis. Following an activation cascade of different caspases, caspase-3 is activated which grants the beginning of the acto-Myosin complex dismantling in muscle fibers, then followed by the intervention of the ubiquitin-proteasome system [START_REF] Nelson | Cross-talk between the calpain and caspase-3 proteolytic systems in the diaphragm during prolonged mechanical ventilation[END_REF]). At humoral level, Myostatin is a protein associated with the control of muscle growth. It has been identified as a myokine that circulates in an inactive form but once cleaved, it is activated. It acts by binding to its receptor, Activin Receptor-IIB (ACTRIIB) Activins receptors ( [START_REF] Caestecker | The transforming growth factor-β superfamily of receptors[END_REF] and triggers the SMAD family pathway in the skeletal muscle. The overexpression of Myostatin led to the decrease of muscle mass suggesting myostatin is a negative regulator of skeletal muscle growth [START_REF] Amirouche | Down-Regulation of Akt/Mammalian Target of Rapamycin Signaling Pathway in Response to Myostatin Overexpression in Skeletal Muscle[END_REF]).

Interleukin-6 (IL-6) is a interleukin which is up regulated after exercise and plays a role in the inflammation [START_REF] Muñoz-Cánoves | Interleukin-6 myokine signaling in skeletal muscle: a double-edged sword?[END_REF]. A chronic IL-6 up-regulation characterizes various conditions such as heart failure, sepsis, burns acute respiratory distress syndrome, pneumonia and exacerbations of chronic obstructive pulmonary disease in the serum which is associated to atrophy induce by chronic inflammation [START_REF] Gabay | Interleukin-6 and chronic inflammation[END_REF]).

Cachexia

Cachexia derives from the Greek word κακός kakos, bad, and ἕξις hexis, condition. Cachexia is induced by several chronic or acute disease states, including cancer. Cachexia is a multifactorial syndrome that leads to severe body weight loss due to both fat and lean tissues wasting [START_REF] Donohoe | Cancer Cachexia: Mechanisms and Clinical Implications[END_REF][START_REF] Penna | Autophagy Exacerbates Muscle Wasting in Cancer Cachexia and Impairs Mitochondrial Function[END_REF]. Cachectic patients experience severe body weight loss, skeletal muscle wasting, asthenia, anemia, and anorexia. However, nutritional monitoring is not enough to counteract the severe body weight and muscle loss experienced in cachexia [START_REF] Gullett | Nutritional Interventions for Cancer-induced Cachexia[END_REF].

The progressive loss of skeletal muscle alters the whole metabolic balance of the body and physical functions, which ultimately compromise patients' life span and quality of life. Furthermore, cancer-cachexia interferes with the treatments against cancer (surgery, chemotherapy, radiotherapy) by lowering the functional status of organs and tissues and by debilitating the patient v [START_REF] Aoyagi | Cancer cachexia, mechanism and treatment[END_REF][START_REF] Vaughan | Cancer cachexia: impact, mechanisms and emerging treatments[END_REF]. It has been estimated that half of cancer patients eventually present with cachexia and the latter is responsible for 25% of cancer patient death [START_REF] Aoyagi | Cancer cachexia, mechanism and treatment[END_REF][START_REF] Skipworth | Pathophysiology of cancer cachexia: Much more than host-tumour interaction?[END_REF]). Cancer-cachexia has a progressive development and depends on the stages of the associated disease. The symptoms above can be associated with others sign less important such as taste alteration, early satiety [START_REF] Ohnuma | Manifestations of Cachexia[END_REF].

Regarding the patient's behavior, several characteristics are observed such as a decrease of physical activity, fatigue associated with a down energy. In parallel, mental capacities such as the ability to focus on a task are also decreased [START_REF] Hopkinson | Psychosocial Impact of Cancer Cachexia[END_REF].

Experiences with parabiotic transfers between healthy controls and tumors bearing animals have shown the occurrence of cachexia in control animals, indicating that it was triggered by the presence of circulating factors, likely cytokines [START_REF] Norton | Parabiotic Transfer of Cancer Anorexia/Cachexia in Male Rats[END_REF]. The latter is a heterogeneous group of low-molecular weight soluble polypeptides or glycoprotein, secreted mainly by immune cells. The so-called pro-inflammatory cytokines induce important metabolic perturbations inducing systemic inflammation [START_REF] Norton | Parabiotic Transfer of Cancer Anorexia/Cachexia in Male Rats[END_REF][START_REF] Argiles | Counteracting Inflammation: A Promising Therapy in Cachexia ». Critical Reviews&trade[END_REF]. In addition, cytokines directly target muscle cells, their effects not being limited to immune cells. Several studies have shown direct effects of TNF-α on the skeletal muscle of cancer patients, associating the high TNF-α plasmatic level with muscle atrophy, lipolysis and anorexia. In rodents an acute treatment by TNF-α injection induces an increase of catabolism and a decrease of anabolism in skeletal muscle, associated with neuroinflammation [START_REF] Reid | Tumor necrosis factor-α and muscle wasting: a cellular perspective[END_REF][START_REF] Biesmans | Peripheral Administration of Tumor Necrosis Factor-Alpha Induces Neuroinflammation and Sickness but Not Depressive-Like Behavior in Mice[END_REF]. Others have shown that TNF-α activates the NFκB nuclear translocation in skeletal muscle, which, in turn, activates several catabolic pathways including protein degradation [START_REF] Baltgalvis | Interleukin-6 and Cachexia in ApcMin/+ Mice[END_REF]. While the inhibition of TNF-α has been led to positive effects in some cancer models [START_REF] López-Soriano | Lipid Metabolism in Tumour-Bearing Mice: Studies with Knockout Mice for Tumour Necrosis Factor Receptor 1 Protein[END_REF], nowadays, no significant results have been obtained with this approach in clinical trials involving cancer patients [START_REF] Jatoi | A Placebo-Controlled Double Blind Trial of Etanercept for the Cancer Anorexia/Weight Loss Syndrome[END_REF]). IL-6 is a pleiotropic cytokine important in cell differentiation or in the inflammatory and exercise response. Rising of circulating IL-6 induces liver growth, insulin resistance, adipose tissue lipolysis and disrupts muscle protein turnover [START_REF] Gabay | Interleukin-6 and chronic inflammation[END_REF]). Furthermore, IL-6 has been associated with cachexia development though several studies in mice models which have shown a negative correlation between IL-6 plasma levels and the survival of metastatic lung cancer patients [START_REF] White | IL-6, Cancer and Cachexia: Metabolic Dysfunction Creates the Perfect Storm[END_REF][START_REF] Ando | Tocilizumab, a Proposed Therapy for the Cachexia of Interleukin6-Expressing Lung Cancer[END_REF]. Worth noting, the treatment with an anti-IL-6 receptor neutralizing antibody reduced the progression of cachexia in lung cancer patients [START_REF] Ando | Possible Role for Tocilizumab, an Anti-Interleukin-6 Receptor Antibody, in Treating Cancer Cachexia[END_REF].

Cytokines of host origin are not the only pro-cachectic factors. Proteolysis-inducing factor (PIF) and lipase maturation factor (LMF) are responsible for proteolysis in skeletal muscle and lipolysis in fat tissue, respectively [START_REF] Argilés | Inter-Tissue Communication in Cancer Cachexia[END_REF]. Both factors are of tumor origin and their serum levels increase in cachexia. IFN-γ can inhibit lipoprotein lipase activity, thereby increasing lipolysis. Several studies have shown that IFN-γ has a major role in cancer-cachexia. The administration of monoclonal antibodies against IFN counteracts cachexia in LLC bearing mice [START_REF] Miller | The Immunological Regulation of Cancer Cachexia and Its Therapeutic Implications[END_REF].

Even though skeletal muscle is a primary, direct target of cytokines, cancer-cachexia is a systemic phenomenon affecting various organs and systems.

Effects on skeletal muscle. Cancer-cachexia is defined by skeletal muscle wasting and muscle fiber atrophy, a loss in the amount of muscle resulting in weakness and fatigue. The metabolic dysfunctions are responsible for the loss of muscle mass observed in patients with cachexia [START_REF] Argilés | Inter-Tissue Communication in Cancer Cachexia[END_REF]. Studies have shown alterations in protein synthesis, protein degradation and amino acid uptake in the cachectic patients, pointing to altered protein metabolism as a key factor leading to sarcomere dismantling and muscle mass loss (Sandri 2016). Besides, an increase in apoptosis associated with an impaired capacity for regeneration contributes to muscle wasting [START_REF] Bossola | Skeletal Muscle Regeneration in Cancer Cachexia[END_REF][START_REF] Pigna | Aerobic Exercise and Pharmacological Treatments Counteract Cachexia by Modulating Autophagy in Colon Cancer[END_REF]. All these alterations lead to the negative nitrogen balance observed in the cachectic patients. Numerous studies agree that the main catabolic pathway involved in muscle protein degradation in cachexia is the ubiquitin-proteasome pathway (Sandri 2016). In healthy condition, this pathway controls the degradation of damaged proteins, however, in cachectic conditions, even intact myofibrillar proteins are degraded [START_REF] Cohen | Ubiquitylation by Trim32 Causes Coupled Loss of Desmin, Z-Bands, and Thin Filaments in Muscle Atrophy[END_REF]. The main consequences of muscle wasting are the reduction of the respiratory function and locomotion.

Effects on cardiac muscle. In parallel to the muscle wasting, cancer cachexia relates to significant wasting of the heart muscle, which leads to a cardiac dysfunction and remodeling [START_REF] Murphy | The pathogenesis and treatment of cardiac atrophy in cancer cachexia[END_REF]. The mortality of cancer patients induced by cardiac failure results from the association between depletion of heart mass and function and cardio toxicity of the cancer treatment [START_REF] Ewer | Erratum: Cardiotoxicity of Anticancer Treatments[END_REF]. Consistent with this, numerous studies in tumors bearing mice model, have found that the observed cardiac atrophy is associated with a heart dysfunction [START_REF] Sweeney | Cardiac Atrophy and Heart Failure In Cancer[END_REF]. Cancercachexia in mice leads to cardiac fibrosis, deregulation of the myocardial structure, and increasing the expression of fetal gene expression program [START_REF] Tian | Cardiac Alterations in Cancer-Induced Cachexia in Mice[END_REF].

Effects on adipose tissue. Despite muscle wasting has been the most studied target of cachexia research, adipose tissue wasting represents an important study field in the understanding of cancercachexia. The importance of adipocyte tissue is highlighted by its loss of precedes muscle wasting in cancer-cachexia [START_REF] Vaitkus | The role of adipose tissue in cancer-associated cachexia[END_REF]. In addition, its dysfunction in cachexia can have supplementary systemic effects. The depletion of lipid storage leads lipotoxicity, which increases the lipid accumulation of heart, muscle, and liver [START_REF] Dahlman | Adipose Tissue Pathways Involved in Weight Loss of Cancer Cachexia[END_REF]. This consequence is associated with altered secretion of lipokines and adipokines which participate in the metabolic dysfunction and systemic inflammation. Cancer cachexia has also been associated with inflammation and changes in function of multiple other organs as liver, brain, bone, blood, pancreas or gut tissues [START_REF] Porporato | Understanding cachexia as a cancer metabolism syndrome[END_REF].

Physical Activity

Physical activity is defined as "any bodily movement produced by skeletal muscles that requires energy expenditure [START_REF] Caspersen | Physical activity, exercise, and physical fitness: definitions and distinctions for health-related research[END_REF]. Indeed, physical activity affects muscle homeostasis and induces protein synthesis in skeletal muscle, decreases insulin resistance, and diminishes the catabolic effects of cachexia and other disease states [START_REF] Gould | Cancer cachexia prevention via physical exercise: molecular mechanisms[END_REF]). The latter effect highlights the relevance of physical activity not only in muscle physiology but also as a therapeutic intervention against disease. The effects of training on muscle physiology and metabolism depend on several factors such as the frequency, the intensity, the exercise charge, and the timing.

Resistance training (RT).

There is not a full consensus on defining this type of exercise, but we can describe RT as an exercise leading to muscle contraction against an external resistance. RT induces important adaptations in the musculoskeletal apparatus, improving strength, fiber size, by inducing structural and functional adaptations of the skeletal muscle [START_REF] Krüger | Functional and Muscular Adaptations in an Experimental Model for Isometric Strength Training in Mice[END_REF].

Aerobic training (AT).

We can describe AT as a physical exercise of low to high intensity that depends primarily on the aerobic energy-generating process (MacInnis et Gibala 2017). It induces important adaptations in the cardiovascular, musculoskeletal and hematological systems. It can increase the capillary networks around myofibers, which is involved in the improvement of muscle oxygenation [START_REF] Hellsten | Cardiovascular Adaptations to Exercise Training[END_REF]. Furthermore, AT increases mitochondria number, size, and biochemical activity, ultimately leading to increased ATP production and aerobic capacity [START_REF] Montero | Haematological Rather than Skeletal Muscle Adaptations Contribute to the Increase in Peak Oxygen Uptake Induced by Moderate Endurance Training[END_REF]. The biochemical adaptations to endurance exercise include an increase of the respiratory capacity, a slower utilization of muscle glycogen and blood glucose, a greater reliance on fat oxidation, and a lower lactate production [START_REF] Alves | Aerobic exercise training as therapy for cardiac and cancer cachexia[END_REF].

Both types of exercise have pleiotropic effects and present numerous advantages, possibly exploitable against the development of chronic diseases.

Exercise influences the transcriptional activity of the myofibers, modifying their cytoskeleton, metabolism and promoting the secretion of growth factors which can induce the activation of satellite cells (Magaudda et al, 2004). Exercise also increases energy expenditure and reduces the risk of developing obesity and excessive adiposity [START_REF] Norman | Influence of Excess Adiposity on Exercise Fitness and Performance in Overweight Children and Adolescents[END_REF]. Behind the musculoskeletal system, physical exercise affects the immunological response by reducing inflammation, by means of three mechanisms: the reduction of visceral fat mass (fat plays a role in maintaining inflammation), the increase of anti-inflammatory cytokines produced by the skeletal muscle and reduced expression of Toll-like receptors on monocytes and macrophages. [START_REF] Petersen | The Anti-Inflammatory Effect of Exercise[END_REF].

Numerous studies have also demonstrated that physical exercise affects cognition, brain plasticity, and well-being. The positive biological and psychological effects are possible by the potent neuroplastic phenomena and promotion of genes and protein expression [START_REF] Mandolesi | Effects of Physical Exercise on Cognitive Functioning and Wellbeing: Biological and Psychological Benefits[END_REF]) Worth noting, cytokines pass the brain-blood barrier. They can, therefore, affect both the central and the peripheral nervous system (Pongratz et Straub 2014.)

Based on the all above, it appears easy to understand that physical activity and fitness reduce the risk of stroke, colon and breast cancer, chronic heart failure, chronic obstructive pulmonary disease, depression, osteoporosis, coronary heart disease or type 2 diabetes mellitus [START_REF] Petersen | The Anti-Inflammatory Effect of Exercise[END_REF].

Exercise and Cachexia

Physical training is a non-pharmacological treatment which improves muscle strength and metabolic function in healthy and pathological conditions [START_REF] Zinna | Exercise Treatment to Counteract Protein Wasting of Chronic Diseases[END_REF]. Therefore, it appeared reasonable to exploit exercise as a therapeutic approach to counteract cachexia. Worth noting, muscle wasting is also intensified by the decrease or even lack of activity, which characterizes cachectic patients, therefore even the goal of maintaining acceptable levels of activity appears to be important in this pathological condition [START_REF] Murphy | Importance of functional and metabolic impairments in the characterization of the C-26 murine model of cancer cachexia[END_REF]. It has in fact demonstrated that cancer patients experience fatigue therefore having a reduced physical activity [START_REF] Narayanan | Fatigue in Cancer: A Review of Literature[END_REF]. Today, exercise protocols have been set to ameliorate clinical status of cachectic patients. Indeed, exercise training improves the quality of life through changes on body composition, metabolism and chronic inflammation [START_REF] Lira | The Therapeutic Potential of Exercise to Treat Cachexia[END_REF]. For example, a walking exercise protocol with has shown positive results on the body weight of cachectic patients (Unpublish data-Seelaender). Yoga is other type of possible intervention which showed also promising results.

Adapting yoga intervention showed to be efficiency to decrease the fatigue, psychological morbidity, distressful symptoms, improving the quality of life [START_REF] Vadiraja | Effects of Yoga in Managing Fatigue in Breast Cancer Patients: A Randomized Controlled Trial[END_REF]. Therefore, it is necessary to adapt different protocols to effort intensity, volume and session of exercise training protocol in function of patients.

Studies with AT protocols in cachectic patients showed the muscle wasting is hampered thanks to the inhibition of MurF-1 expression [START_REF] Bowen | Skeletal Muscle Wasting in Cachexia and Sarcopenia: Molecular Pathophysiology and Impact of Exercise Training[END_REF]. Furthermore, AT increases the muscular anabolism via IGF-1-PI3K-AKt pathway, promoting protein synthesis. AT, such as the treadmill running, can be combined with nutritional approaches and lead to a reduction of tumor growth and improvement of muscle mass [START_REF] Deuster | Endurance Exercise Modifies Cachexia of Tumor Growth in Rats[END_REF]. In another model, it has been shown the overexpression of PGC-1α, a gene which is physiologically induced by exercise and promotes muscle endurance, is sufficient to counteract muscle wasting via suppression of FoxO3 and atrogens [START_REF] Geng | PGC-1α Promotes Nitric Oxide Antioxidant Defenses and Inhibits FOXO Signaling Against Cardiac Cachexia in Mice[END_REF].

Given the pro-anabolic effects of RT, early studies on animal models showed inhibition of muscle wasting by this type of exercise, either performed before or after the tumor inoculation [START_REF] Al-Majid | Resistance Exercise Training Attenuates Wasting of the Extensor Digitorum Longus Muscle in Mice Bearing the Colon-26 Adenocarcinoma[END_REF]. Interestingly, these findings were soon validated in humans, showing that resistance exercise combined with protein ingestion increases muscle protein synthesis in cancer patients [START_REF] Petersen | The Anti-Inflammatory Effect of Exercise[END_REF]. In addition the effect of protein synthesis, the degradation of damaged proteins is stimulated by both acute endurance and resistance exercise and so it accelerates protein turnover [START_REF] Wolfe | Skeletal Muscle Protein Metabolism and Resistance Exercise[END_REF][START_REF] Pigna | Aerobic Exercise and Pharmacological Treatments Counteract Cachexia by Modulating Autophagy in Colon Cancer[END_REF]. Recent studies are focused on the anti-inflammatory effects of exercises which have been described in healthy adults and chronic disease patients. Physical exercise stimulates anti-inflammatory cytokines, which are produced from skeletal muscle [START_REF] Gleeson | The Anti-Inflammatory Effects of Exercise: Mechanisms and Implications for the Prevention and Treatment of Disease[END_REF][START_REF] Pigna | Aerobic Exercise and Pharmacological Treatments Counteract Cachexia by Modulating Autophagy in Colon Cancer[END_REF]. In particular, exercise induces IL-6 secretion which, in turn, stimulates the production of the anti-inflammatory cytokines such as IL-1ra or IL-10 and inhibits the production of the proinflammatory cytokines, such as TNF-α [START_REF] Steensberg | IL-6 enhances plasma IL-1ra, IL-10, and cortisol in humans[END_REF][START_REF] Mizuhara | T Cell Activation-Associated Hepatic Injury: Mediation by Tumor Necrosis Factors and Protection by Interleukin 6[END_REF]. AT has a stronger anti-inflammatory effect with respect to RT.

Serum Response Factor

The transcriptional factor Serum response factor (SRF) has been identified for the first time in 1987 [START_REF] Treisman | Identification and purification of a polypeptide that binds to the c-fos serum response element[END_REF], and is produced in large amounts of all skeletal muscle tissues. Serum Response Factor SRF is a member of the MADS transcription factors and binds the core sequence of CArG boxes [CC (A/T) 6 GG] as a homodimer [START_REF] Posern | Actin' together: serum response factor, its cofactors and the link to signal transduction[END_REF]. SRF is conserved from drosophila to humans; it is coded by one gene located on chromosome 6p2.1 in humans and chromosome 17 in mice. SRF gene is composed of 7 exons separated by 6 introns and possess 10607 pair of bases. [START_REF] Chai | Serum Response Factor: Discovery, Biochemistry, Biological Roles and Implications for Tissue Injury Healing[END_REF][START_REF] Coletti | Serum Response Factor in Muscle Tissues: From Development to Ageing[END_REF]). Four isoforms have been identified in the mouse: SRF-L, SRF-M, SRF-S et SRF-I. SRF-M, SRF-S and SRF-I are devoid of a part of the domain of transactivation by deletion of the exon 5 for SRF-M, exons 4 and 5 for SRF-S and exons 3, 4 and 5 for SRF-I [START_REF] Kemp | Four isoforms of serum response factor that increase or inhibit smooth-muscle-specific promoter activity[END_REF]. In its major form, SRF-L protein has 508 aminoacids and a molecular weight of 67kDa. The N-terminal region includes a nuclear localization domain, while the medium region presents by the MADS domain, which contains a dimerization domain and a DNA fixation domain [START_REF] Chai | Serum Response Factor: Discovery, Biochemistry, Biological Roles and Implications for Tissue Injury Healing[END_REF]; in the C-terminal region SRF, has a domain responsible for its transcriptional activity. SRF works in a homodimer form and binds the core sequence of CC(A/T)6GG (CArG boxes). The target genes of SRF are characterized in the promotor region by the presence of at least one CArG box which permits the binding between DNA and SRF. Its ability to regulate different sets of downstream target genes may depend on the promoter context, but also on SRF association with different cofactors. Interestingly, SRF has two CArG functional boxes in its promoter, which makes it is own target gene. Among the other SRF target genes, we distinguish several groups: immediate early genes (IEG) like c-fos, junB, erg-1 and erg-2; their expression is quickly activated after a given stimulus without the activation of protein synthesis; they control genes which are involved in the cell cycling or coding for growth factors [START_REF] Chai | Serum Response Factor: Discovery, Biochemistry, Biological Roles and Implications for Tissue Injury Healing[END_REF]. On the other hand, SRF also controls several targets genes which are involved in adhesion, cytoskeleton, contractile or signaling function such as vinculin, tropomyosin, actinin 1, integrin, MyoD, myosin heavy chain, myosin light chain [START_REF] Chai | Serum Response Factor: Discovery, Biochemistry, Biological Roles and Implications for Tissue Injury Healing[END_REF]. [START_REF] Guerci | Srf: a key factor controlling skeletal muscle hypertrophy by enhancing the recruitment of muscle stem cells[END_REF][START_REF] Coletti | Serum Response Factor in Muscle Tissues: From Development to Ageing[END_REF]. Importantly, SRF KO mice also show an alteration of the regenerative response suggesting that SRF play a crucial role in the activation of satellite cells. Indeed, SRF has been shown to induce the secretion of both IL-6 and IL-4, which induce satellite cell proliferation and recruitment to nascent myotubes, respectively, in the same studies, they have shown that absence of adaptive response after the overload protocol in mutant mice [START_REF] Guerci | Srf-Dependent Paracrine Signals Produced by Myofibers Control Satellite Cell-Mediated Skeletal Muscle Hypertrophy[END_REF]

SRF and exercise

Physical exercise induces the transduction of the extracellular stress signals to intracellular response, gene transcription and protein translation [START_REF] Russell | Molecular Regulation of Skeletal Muscle Mass[END_REF], which can stimulate the STARS/RhoA/MRTF pathways [START_REF] Mahadeva | Ms1, a Novel Stress-Responsive, Muscle-Specific Gene That Is up-Regulated in the Early Stages of Pressure Overload-Induced Left Ventricular Hypertrophy[END_REF][START_REF] Wallace | The regulation and function of the striated muscle activator of rho signaling (STARS) protein[END_REF]. In fact, it has been demonstrated in the polymerization of actin by RhoA and STARS is increased after mechanical stress, therefore releasing MRTF, the co-activator of SRF, which increases its nuclear translocation and the transcriptional activity of SRF (Arai, Spencer, et Olson 2002b).

Others have demonstrated an increase in SRF protein level after 8 days of mechanical overloading in rat plantaris muscle [START_REF] Gordon | Selected Contribution: Skeletal muscle focal adhesion kinase, paxillin, and serum response factor are loading dependent[END_REF]. Inversely, SRF mRNA and protein levels return to their respective basal after a detraining period [START_REF] Lamon | The STARS Signaling Pathway: A Key Regulator of Skeletal Muscle Function[END_REF].

SRF and Disease

Numerous studies have studied the regulation of SRF in pathological conditions. Interestingly, in the aging skeletal muscle, SRF is down regulated, which suggests that, it could mediate sarcopenia. In addition, deregulation of SRF is associated with different metabolic and muscle diseases: for instance, in chronic obstructive pulmonary disease, SRF expression is decreased in the skeletal muscle, suggesting that the alteration of SRF signaling may contribute to muscle dysfunction [START_REF] Lahoute | Premature Aging in Skeletal Muscle Lacking Serum Response Factor[END_REF][START_REF] Lewis | Downregulation of the Serum Response Factor/MiR-1 Axis in the Quadriceps of Patients with COPD[END_REF]).

AIM

Exercise increases the well-being and the life span in cancer patients by improving muscle homeostasis and function. Similar findings in animal models of cancer cachexia have allowed the understanding of some of the signaling pathways induced by exercise in skeletal muscle tissue. However, the response to exercise in terms of transcription activity and gene expression in cachexia is greatly unknown. Acquiring additional information on exercise targets in the musculature would allow the understanding of the adaptative muscle responses to exercise, especially in pathological conditions. In turn, this knowledge would improve exercise-based protocols against cancer cachexia and help us to develop novel pharmacological or non-pharmacological interventions against this devastating syndrome. SRF is a crucial factor in mediating cell responses to mechanical stimuli affecting actin dynamics. SRF is highly expressed in muscle tissues and plays a pivotal role in skeletal muscle development and homeostasis. The objective of this thesis is to characterize SRF regulation in cancer cachexia and SRF role in mediating the response to exercise, i.e., increased muscle activity, in this pathological condition.

RESULTS AND DISCUSSION

Voluntary wheel running counteracts cancer cachexia

To study exercise effects on cachexia, we used control (CTRL) and C26-tumor-bearing (C26) mice, which were allowed to perform voluntary wheel-running (WR) or not, as previously described [START_REF] Pigna | Aerobic Exercise and Pharmacological Treatments Counteract Cachexia by Modulating Autophagy in Colon Cancer[END_REF]. Cancer-cachexia is characterized by systemic inflammation, an involuntary loss of lean body mass and negative protein and energy balance [START_REF] Aoyagi | Cancer cachexia, mechanism and treatment[END_REF]. Therefore, we measured body weight before and after the treatments and noticed that body weight was significantly decreased in the C26 mice with respect to the control, a phenomenon which was not observed in the C26+WR group, while WR per se had no effect on healthy mice (Figure 7a). Since skeletal muscle mass accounts for the major part of body weight, we measured the effects of exercise on muscle mass and histology by analyzing the tibialis anterior (TA) as a representative muscle, which will be therefore used throughout this study. We observed that WR did not change significantly the TA weight, while we observed a significant decrease in TA weight of the C26 group, which confirm the result above; WR counteracts the loss of TA mass in tumor-bearing-mice (Figure 7b). To observe the effects of C26 and WR on fiber atrophy, we performed a morphometric analysis of the muscle fiber cross-sectional area (CSA), following the immunofluorescence for dystrophin, which highlights the sarcolemma of the muscle fibers (Figure 7c). As expected, we observed a significant decrease of CSA in C26 compared with CTRL group, a difference which was no longer observed in C26+WR, nor it was in the CTRL+WR, as expected (Figure 7d). Therefore, muscle changes are actually due to changes in muscle fiber size and the atrophy observed in C26 mice is efficiently counteracted by voluntary exercise.

Muscle fiber atrophy is characterized by the up-regulation of the muscle-specific E3-ubiquitin ligases, Atrogin-1 and MuRF1 [START_REF] Coletti | Spontaneous Physical Activity Downregulates Pax7 in Cancer Cachexia[END_REF]. Therefore, we analyzed the effects of the C26 tumor and WR on the expression of Atrogin-1 and MuRF-1 in muscle extracts from the groups above. Our results showed that both Atrogin-1 and MuRF-1 expression was significantly upregulated in the muscle from the C26 group when compared with the CTRL group or any other condition; therefore, WR prevented the up-regulation of Ubiquitin-ligases and likely the proteasomemediated protein degradation which accounts for muscle wasting in cachexia (Figure 7e).

Physical exercise reported to be an effective therapeutic strategy against muscle wasting induced in cancer-cachexia [START_REF] Alves | Aerobic exercise training as therapy for cardiac and cancer cachexia[END_REF][START_REF] Coletti | Spontaneous Physical Activity Downregulates Pax7 in Cancer Cachexia[END_REF][START_REF] Pigna | Aerobic Exercise and Pharmacological Treatments Counteract Cachexia by Modulating Autophagy in Colon Cancer[END_REF]. We showed that the WR counteracts the loss of body weight, skeletal muscle mass and fiber size in tumor-bearing mice. Furthermore, it reduces Atrogin-1 and MuRF1 gene expression, implying that aerobic exercise stopped the muscle atrophy by targeting the catabolic pathways as efficiently as resistance training [START_REF] Zanchi | Chronic Resistance Training Decreases MuRF-1 and Atrogin-1 Gene Expression but Does Not Modify Akt, GSK-3beta and P70S6K Levels in Rats[END_REF]). The data, normalized by GAPDH expression, are expressed over the CTRL and are shown as mean+/-SEM; n=5. Two-way ANOVA revealed an interaction between the presence of the tumor and the running activity (for Atrogin-1: F=73.02 df=1; p<0,01 for tumor; F=68.91 df=1; p<0,01 for wheel running condition; F=78. 24 df=1; p<0,01 For interaction; for MuRF-1: F=73.02 df=1; p<0,01 for tumor; F=68.91 df=1; p<0,01 for wheel running condition; F=78.24 df=1; p<0,01For interaction); #= p<0.01 by Tukey's HSD test.

C26 tumor and exercise independently affect SRF protein level in muscle

SRF is a transcriptional factor essential in the muscle plasticity and plays a pivotal role in muscle physiology, pathology and homeostasis, especially by promoting myoblast recruitment and the myotubes and by preventing sarcopenia [START_REF] Coletti | Serum Response Factor in Muscle Tissues: From Development to Ageing[END_REF][START_REF] Guerci | Srf: a key factor controlling skeletal muscle hypertrophy by enhancing the recruitment of muscle stem cells[END_REF]). In addition SRF plays a role in the muscle homeostasis in presence of mechanical stimulation [START_REF] Chai | Serum Response Factor: Discovery, Biochemistry, Biological Roles and Implications for Tissue Injury Healing[END_REF]. To examine the contribution of SRF in the rescue by voluntary exercise in cancercachexia, we analyzed the SRF mRNA and protein levels in CTRL and C26 in presence or not of WR. After 21 days of treatment, the expression of SRF was analyzed by real-time PCR and we did not observe any significant effect of either WR or the tumor on SRF transcription (Figure 8a). However, striking differences were observed when we analyzed SRF at the protein level by western blot: by two-way ANOVA we observed that both WR and C26 affect SRF, and that the two treatments negatively interact (Figure 8c). In particular, SRF was downregulated in the C26 group, which no linger held true in the C26+WR, showing a rescue of SRF protein level similar to the CTRL group (Figure 8b).

Based on these observations, we associated the observed downregulation of SRF level to muscle atrophy in cachexia. Indeed, previous studies have shown that the SRF expression was significantly decreased in denervation and aging models [START_REF] Lamon | The STARS Signaling Pathway: A Key Regulator of Skeletal Muscle Function[END_REF][START_REF] Lahoute | Premature Aging in Skeletal Muscle Lacking Serum Response Factor[END_REF], while other studies showed in COPD decrease of SRF translocation in the nuclei associated with decreasing of transcriptional activity [START_REF] Ma | Reduced Nuclear Translocation of Serum Response Factor Is Associated with Skeletal Muscle Atrophy in a Cigarette Smoke-Induced Mouse Model of Chronic Obstructive Pulmonary Disease[END_REF]. Our data not only show for the first time a decrease of the SRF protein in cancer-cachexia, confirming that SRF deregulation may play a major role in muscle atrophy, but also show that the rescue of SRF level is associated with a rescue in muscle mass. The negative regulation of SRF expression observed in the C26 group could be explained by the up regulation of ubiquitin ligase observed in the C26 group (Figure 7e). Indeed, it has been shown in cardiomyocyte culture, the association between the ubiquitin ligase MURF1, 2 or 3 with SRF leads to reduce nuclear SRF [START_REF] Perera | Developmental regulation of MURF ubiquitin ligases and autophagy proteins nbr1, p62/SQSTM1 and LC3 during cardiac myofibril assembly and turnover[END_REF].

We can suppose the same mechanism exist in skeletal muscle and the reduction of this ubiquitin ligase has a positive effect on the SRF expression. 5.3 The amount of SRF protein in the skeletal muscle correlates with body weight changes

We noticed higher levels of SRF in the musculature of mice that were gaining weight during the experiments, and the reverse in cachectic animals; in addition, we noticed a certain degree of heterogeneity in the C26+WR group for what concerns SRF levels (see the quadruplicate for C26+WR in Figure 8c). Whilst still considering the average SRF value as representative of the population, we decided to do a more detailed analysis of the distribution of SRF protein levels in relation to the displayed changes in body weight between the beginning and the end of the experiments (negative delta body weight corresponding to cachectic mice). The statistical analysis of our result showed a significant correlation between body weight changes and SRF protein level. In particular, there was a linear correlation between body weight gain or loss and high or low levels of SRF expression, respectively (Figure 9).

These novel findings strongly suggested that SRF played a major role in mediating exercise beneficial effects and prompted us to further investigate SRF transcriptional activity in the different experimental conditions.

C26 tumor and exercise independently affect SRF transcriptional activity

We have shown that SRF is downregulated in cancer cachexia and rescued by voluntary exercise and that its levels correlate with body weight gain, suggesting that SRF transcriptional activity plays a role in mediating the beneficial effects of exercise on muscle homeostasis. For this reason, we focused on SRF transcriptional activity: Dystrophin, α Sk-Actin, Vinculin, MHC2a, IGF-1, BCL-2 are all known as being SRF target genes. Globally, our analysis showed that WR increases the SRF transcriptional activity, independently of the presence of the tumor, suggesting the importance of mechanical stimulation in mediating SRF activity and the activity of its targets (Table 1). In particular: 1) Dystrophin is a protein that links intracellular the cytoskeleton network to transmembrane components, the deregulation of its expression has several.

The analyses of Dystrophin mRNA expression in the C26 group showed a down-regulation which is counteracted by WR; this result is in agreement with the reported membrane damage in cancer cachexia [START_REF] Acharyya | Dystrophin glycoprotein complex dysfunction: A regulatory link between muscular dystrophy and cancer cachexia[END_REF]) and could provide a mechanistic explanation for this phenomenon.

2) Skeletal actin (Sk-Actin) is the principal actin isoform in the sarcomeric thin filaments of adult skeletal muscle; our analysis showed two opposite effects exerted by WR and by C26 consistent with a dismantling of sarcomeres in the presence of the tumor and with an increase in sarcomere-genesis upon physical activity.

3) Vinculin is a cytoskeleton protein showing the same regulation as skeletal actin, consistent with the strengthening of structural elements in the presence of mechanical stimulation, which is needed by the musculature to perform exercise. 4) Myosin (MHC2A) is a member of the motor protein superfamily known for its role in muscle contraction, constituting the thick filaments in sarcomeres. Myosin are the primary target of protein degradation in cachexia, as confirmed by our data, and MHC2A expression is rescued in the C26+WR group, similarly to the other SRF target genes above. 5) IGF-1 is the principal muscle growth factor, which stimulates muscle anabolism. Our analyses showed a WR effect on IGF-1 expression, indicating that running has a pro-hypertrophic effect. 6) BCL-2 is an apoptosis inhibitor, which is included in the established SRF target genes. BCL-2 maintains mitochondrial membrane integrity through the inhibition of Bax, preventing apoptosis. Even though we did not further investigate apoptosis in cachexia, we noted that WR and the tumor have two opposites' effects on BCL-2 expression indicating that the presence of the tumor could favor apoptosis by the BCL-2 down-regulation in skeletal muscle while WR has the opposite effect.

In addition to all the above, two major targets of SRF are MyoD and integrin beta. Given the relevance of their role in muscle cell differentiation and homeostasis, we dedicated a specific analysis to these two factors and validated the Q-PCR findings at protein level.

Table1. Exercise counteracts the C26-induced effects on SRF transcriptional activity. Real-Time PCR analysis for different SRF target genes: Dystrophin, α Sk-Actin, Vinculin, MHC2a, IGF-1, and BCL-2 in the TA muscle. Control (CTRL) and C26 tumor-bearing (C26) mice, which were hosted in individual cages, in the absence or presence (WR) of a wheel, which allows running voluntary exercise. GAPDH has been used as a normalizer, the data are expressed over the CTRL and are shown as mean+/-SEM; n=6. Two-way ANOVA revealed an effect of the running activity (for Dystrophin: F=3.78 df=1; p=0,0613 for tumor; F=4.93 df=1; p<0,05 for wheel running condition; F=5.35 df=1 ; p<0,05 for interaction; for α Sk-Actin; F=8.15 df=1; p<0,01 for wheel running condition; for Vinculin: F=5.17 df=1; p<0,038 for wheel running condition; no interaction; for MHC2a: F=11.5 df=1; p<0,01 for tumor; F=8.96 df=1; p<0,01 for wheel running condition; F=5.59 df=1; p<0,05 for interaction; for IGF-1: F=3.04 df=1; p<0,09 for tumor; F=5.17 df=1; p<0,05 for wheel running condition; no interaction; for BCL-2 ; F=5.55 df=1; p<0,05 for wheel running condition; F=4.33 df=1; p= 0,05 for interaction).

MyoD expression in the context of cachexia and exercise

MyoD is major regulator of skeletal muscle differentiation in the embryo and of satellite cell activation as an early response to muscle injury [START_REF] Yamamoto | Loss of MyoD and Myf5 in Skeletal Muscle Stem Cells Results in Altered Myogenic Programming and Failed Regeneration[END_REF]). Also, it is well established that MyoD is a main SRF target gene, consistent with SRF role in muscle hypertrophy as a consequence of satellite cells fusion [START_REF] Randrianarison-Huetz | Srf Controls Satellite Cell Fusion through the Maintenance of Actin Architecture[END_REF]. MyoD expression, analyzed by real-time PCR, showed a significant up-regulation by WR in both the CTRL and C26 groups, irrespectively of the tumor (Figure 10a). At protein level, by western-blot analysis, we observed that SRF was increased by WR, while C26 had an opposite effect; WR negatively interfered with the tumor effect and the MyoD down regulation in respect to the CTRL was no longer significant in the WR+C26 group (Figure 10b andc).

In conclusion, the MyoD down regulation induced by cachexia is consistent with studies showing that the myogenic program and regeneration capacity are altered in cachexia (Coletti, 2005). In addition, cytokine-dependent NF-κP activation leads to MyoD degradation in skeletal muscle [START_REF] Guttridge | NF-KappaB-Induced Loss of MyoD Messenger RNA: Possible Role in Muscle Decay and Cachexia[END_REF] and to the up regulation of Pax7, which is inhibitory for myogenic differentiation of satellite cells [START_REF] He | NF-κB-mediated Pax7 dysregulation in the muscle microenvironment promotes cancer cachexia[END_REF][START_REF] Coletti | Spontaneous Physical Activity Downregulates Pax7 in Cancer Cachexia[END_REF]. Even though MyoD remains significantly lower in the C26+WR group in respect to control, it is clear that MyoD regulation depends on several transcription factors and the full mechanisms of its regulation upon exercise and in cachexia are not fully understood, yet. 5.6 Integrin-β1 expression is down-regulated in cachexia, an effect which is blunted by exercise Integrin-β1 is essential for the interaction between cells and ECM, for the cytoskeleton organization of the post-synaptic apparatus and is known to be SRF target gene. In our experimental settings, Integrin-β1 showed a significant down regulation in C26 group compared to CTRL, which was no longer observed in the C26+WR group (Figure 11a), due to a statistically significant, negative interaction between the two treatments, i.e., tumor and exercise. This result was confirmed at protein level (Figure 11b andc). The downregulation of integrin-β1 has often been associated with the upregulation of atrogens in cancer-cachexia [START_REF] Judge | Genome-Wide Identification of FoxO-Dependent Gene Networks in Skeletal Muscle during C26 Cancer Cachexia[END_REF], and we provide a mechanistic explanation for the reported integrin-β1 down-regulation, which we propose to be due to the decrease of SRF transcriptional activity. [START_REF] Wallace | The regulation and function of the striated muscle activator of rho signaling (STARS) protein[END_REF]. STARS activation permits the nuclear localization of the cofactor of SRF, MRTF-A (Arai, Spencer, et Olson 2002a). We performed a preliminary analysis on these pathways by looking at STARS and RhoA mRNA expression. The latter were significantly decreased in the C26 group compared to the matching exercised groups, consistent with ANOVA which showed a negative interaction between exercise and tumor for both factors (Figure 12a andb); in addition, STARS appeared to be more significantly modulated than RhoA, for instance in the presence of C26 tumor. The diminution of the expression of STARS/RhoA in the presence of the tumor could be explained by a decrease in spontaneous mice activity. Indeed, it has been demonstrated, the inactivity-induced muscle unloading decreases STARS and RhoA mRNA expression [START_REF] Wallace | The regulation and function of the striated muscle activator of rho signaling (STARS) protein[END_REF]. The perturbation of STARS/RhoA pathway is expected to result in, and indeed is associated with, down regulation of SRF expression as shown above (Figure 8). Furthermore, two major transcriptional targets downstream of STARS/RhoA are MyoD and SRF [START_REF] Wallace | The regulation and function of the striated muscle activator of rho signaling (STARS) protein[END_REF], which could explain their down regulation in cachectic conditions. Inversely, in the presence of voluntary exercise, STARS and RhoA mRNAs are significantly up regulated compared the C26 group. This response can be explained by the increasing of contraction-induced mechanical stress, having as a consequence actin polymerization and MRTF-A and SRF activation. Indeed, an up regulation of STARS and RhoA expression was reported following both resistance and aerobic exercise [START_REF] Wallace | Overexpression of Striated Muscle Activator of Rho Signaling (STARS) Increases C2C12 Skeletal Muscle Cell Differentiation[END_REF]), and we can suppose that the muscle response was he same in our experimental settings. The dysregulation of the STARS/RhoA axis is usually associated with myopathies or musclewasting conditions [START_REF] Lamon | The STARS Signaling Pathway: A Key Regulator of Skeletal Muscle Function[END_REF]. Furthermore, in loss of function experiments, the RhoA inhibition leads to the decrease of transcriptional activity of SRF (Arai, Spencer, et Olson 2002a). 5.8 SRF is associated to a positive body weight balance and depends on a threshold running activity

The western blot analyses on SRF level, as exemplified by figure 8, highlighted two types of results within the C26+WR. On average, we observed a rescue of the SRF protein amount in C26+WR mice, as presented in figure 8b. However, we also noted in some samples a down-regulation of SRF expression in the C26+WR group. Since our experimental model is based on voluntary wheel running, an activity which is heterogeneous by definition, we correlated on individual basis for some mice in the C26+WR group: the amount of SRF protein in the musculature, the amount of exercise in terms of Km run per day, and the changes in body weight -a negative delta body weight meaning a negative body weight balance at the end of the experiment (Supplemental data 2A and B).

Figures Suppl 2A and 2B represent two independent experiments in duplicate, with the whole data set reported for each mouse. We focused our attention on mice # 4 and 8 in the C26+WR group, which had a very different response in terms of SRF levels (Supplemental data 2Aa) and noted that the two mice had an opposite response in terms of body weight loss (Supplemental data 2Ab), associating a body weight gain with high SRF levels (mouse # 4) and vice versa (mouse # 8). The two mice also differed for the amount of exercise, monitored in the last five days before the sacrifice (Supplemental data 2Ac): the mouse #4, which displayed high SRF levels and a body weight increased consistently run more than 2 KM/day, while the other did not. When we focused our attention on mice # 2 and 3 in the C26+WR group, which behaved identically and did not show the expected rescue of SRF levels, we noted that both were cachectic and both performed less than 2 Km/day of wheel running (Supplemental data 2Ba, b and c).

Taken together, these novel observations suggest the importance of a daily threshold in physical activity to maintain the SRF protein level at high levels even in the presence of a tumor and, ultimately, to rescue cachexia. In addition, our results reinforce the notion that SRF is a mechano-transductor and suggest that it mediates the beneficial effects of exercise in the presence of mechanical stimulation. 5.9 Characterization of the conditional, muscle specific SRF KO mice: genetic studies

To study the role of SRF in skeletal muscle with a genetic approach, in the absence of the lethally associated with a global deletion of SRF in mice, a murine line with a muscle-specific deletion of SRF was generated. Tissue specify is guaranteed by the use of the Crelox system to express the enzyme responsible for excision of exon 2 in SRF gene resulting in an out-frame mutation [START_REF] Charvet | New Role for Serum Response Factor in Postnatal Skeletal Muscle Growth and Regeneration via the Interleukin 4 and Insulin-like Growth Factor 1 Pathways[END_REF].

Cre being under the control of Human Skeletal Actin (HSA promotor, its expression is restricted in muscular fibers; it has been shown that this HSA-Cre begins to be expressed at the embryonic stage E9, and its maximum level of expression is observed during the perinatal period. By crossing the Cre mice above with floxed-SRF mice, we obtained double transgenic mice, which became muscle specific SRF KO mice upon tamoxifen injections (for details, see M&M). We systematically validated the knockout of SRF expression by both RT-PCR and WB analyses. In particular, SRF RNA was decreased by about 80% in muscle lysates from SRF KO mice (as expected, since the musculature contains contaminants, WT tissues other than the muscle-specific KO tissue) (Figure 13a). Figure 13b shows a representative experiment in duplicate with muscle extracts from the four experimental groups, consisting of WT and KO mice, which performed or not physical activity by WR: as expected, SRF is not detectable in the musculature of KO mice.

5.10

Characterization of the conditional, muscle specific SRF KO mice: physiological studies Wheel running is a form of aerobic exercise that induces muscle adaptations leading to increase of the physical performance [START_REF] Alves | Aerobic exercise training as therapy for cardiac and cancer cachexia[END_REF]. Since we planned to perform experiments on wheel running with SRF KO mice, we wanted to know in preliminary experiments whether the deletion of SRF in the musculature did not perturb muscle adaptation to exercise, given the pivotal role played by SRF in muscle tissue. We hypothesized that WR would have been performed similarly between WT and KO mice, which would have been compatible with the planned experiments without the risk of bias due to the genotype. To confirm this hypothesis, we performed an incremental test and analyzed the effects of maximal running speed and V02 peak.

Our results showed that voluntary exercise increased the maximal running speed of mice in both WT+WR and KO+WR groups (Figure 14a). The exercised WT and SRF KO mice showed an improvement in the maximal speed at 21 days (T3) compared to non-exercised WT and KO mice, demonstrating in both groups: 1) the adaptation to voluntary exercise; 2) similar outcomes in terms of performance.

In addition, we analyzed and compared the maximum oxygen uptake capacity, which is a marker of the aerobic capacity of an animal. Our results showed a significant increase in the V ̇O2 peak between to beginning of experience (T1) and 21 days (T3) in both groups of exercised-mice (KO+WR and WT+WR) compared to non-exercised-mice, highlighting the independence of this adaptation of the genotype (Figure 14b). The results presented above suggest that the effects induced by voluntary exercise in mice are similar to the two groups and independent from the SRF expression. Therefore, the muscle deletion of SRF had not impacted the performance, the muscle adaptation or the general behavior of mice having the possibility to perform voluntary exercise. The fulfillment of this condition was essential to perform experiments involving exercise with SRF KO mice.

5.11

Detailed analysis of the exercisedependent metabolic and strength adaptations in WT and SRF KO mice Following 21 days of wheel running, we analyzed the body weight in WT and SRF KO mice in the absence or presence of WR, showing no significant differences between the groups. A more detailed analysis of the body composition in terms of lean and fat mass showed a significant difference in the exercised WT and KO mice compared to the matching non-exercised groups (Table 2a). Since our exercise protocol was based on voluntary exercise, we analyzed the running distance over the 3-week protocol period noticing no differences between groups and a regular running time within a given group (Table 2b). Due to the observed increase of lean mass in exercised mice, we decided to investigate muscle function by using repeatable grip test studies. In particular, we analyzed the mean and the maximal strength. In non-exercised WT and KO mice, the results showed a significant increase in the mean strength of the WT compared to KO, suggesting that muscle performance could be negatively affected by the lack of SRF. However, they exercised, SRF KO mice showed a significant improvement of the Fmax, which even was higher than WT+WR, as compared to the respective control (Table 2c). Therefore, we concluded that SRF KO mice do not have a diminished capacity to develop a force. The respiratory exchange ratio (RER) is the ratio between the quantity of carbon dioxide (CO2) produced in metabolism and oxygen (O2) consumed. We analyzed and compared RER rest between WT and KO mice in the presence or not of WR training and we observed no difference between the groups (Table 2c). However, the RER peak analyses showed a significant decrease in the WT+WR group compared to others.

5.12

SRF is necessary for the beneficial effects of exercise in tumor-bearing mice Once we demonstrated the feasibility of experiments involving exercise and SRF KO mice, we could perform experiments to demonstrate whether SRF is necessary for the beneficial effects of exercise in cachexia. To do so, we exploited WT and SRF KO mice, in the absence or presence of WR; in all these 4 groups we injected cells of Lewis Lung Carcinoma (LLC) and used the same exercise protocol presented above: for this reason, it is worth noting that here the control groups represents WT, tumor-bearing mice, which are indeed cachectic, and not WT, healthy mice as in the previous part of the study. Indeed, the analysis of the final body weight showed a significant increase of body weight in WT+WR group compared to WT, like expected if WT mice were indeed WT tumor-bearing mice, losing weight in the course of the experiment due to the tumor burden. When we compare the effect of WR on SRF KO mice, we didn't observe any difference between the two groups, indicative of the fact that both exercised (WR) and not exercised mice were losing weight in the presence of LLC tumor. In other words, voluntary exercise rescued body weight in the WT group but no in the KO group (Figure 15a).

To strengthen the previous observation, we analyzed the mass of the tibialis anterior (TA) muscle. We observed that WR increased the TA mass in WT+WR compared to WT, which no longer held in the KO+WR compared to the KO group (Figure 15b). To measure muscle fiber atrophy, we performed histological and morphometric analyses on TA cross-sections. We specifically measured the crosssectional area (CSA) of the glycolytic muscle fibers, identified by using NADH staining by histochemistry, which highlights Type I (dark) and Type II (light) myofibers, as well as intermediate ones (Figure 15c). The result showed a significantly increase of the CSA of glycolytic fibers (the most sensitive to fiber size changes in cachexia) in the WT+WR group as compared to the WT group. This difference was no longer observed in the KO+WR as compared to the KO group (Figure 15d). As show in figure 7, in cachexia an up-regulation of the musclespecific E3-ubiquitin ligases, MuRF1 and Atrogin-1 occurs (Figure 7e). We analyzed the effects of LLC and WR on these ubiquitin ligase expression in SRF KO and WT muscles. In both cases, our results showed a significant Ub-ligase down-regulation in the muscles from the WT+WR group when compared with the WT group confirming our previous results obtained in a different mouse strain bearing a different tumor (Figure 15e andf). When we analyzed the Ub-ligases in KO mice, we did not see a difference between the KO and the KO+WR. These results indicate that SRF plays a necessary role in the rescue of muscle mass in cancer-cachexia by exercise. The cross-sectional area of glycolytic muscle fibers was measured using Image J program and showed an exercise-mediated rescue of glycolytic fiber size exclusively in WT mice in the presence of LLC tumor. Data are shown as mean+/-SEM; n=4. Student's t-test revealed a significant difference between WT and WT+WR; #=p<0.05. (e) Real-Time PCR analysis for muscle ubiquitin ligases MuRF-1 and Atrogin-1 of the same groups as above. The data, normalized by GAPDH expression, are expressed over the WT and are shown as mean+/-SEM; n=3. Two-way ANOVA revealed an interaction between the presence of the tumor and the running activity (for MuRF-1: F=25.52 df=1; p<0,01 for tumor; F=9.56 df=1; p<0,05 for wheel running condition; F=26.02 df=1; p<0,01 for interaction; for Atrogin-1: F= 11.14 df=1; p<0,05 for tumor; F= 11 df=1; p<0,05 for wheel running condition; F= 7.43; df=1; p<0,05 for interaction) #= p<0.05 by Tukey's HSD test.

5.13

Exercise and the tumor exert opposite effects on the number of Pax7-expressing cells In normal muscle regeneration, satellite cells are activated, proliferate, and ultimately committed to myogenic differentiation to give rise to newly formed myofibers. Regulation of Pax7 is important for muscle homeostasis: Pax7 is expressed by quiescent satellite cells and its progressively down-regulated upon Sc activation, since it represents an inhibitory signal for the expression of MRFs, such as MyoD (Yin, Price, et Rudnicki 2013b). Since we and others have observed an up-regulation of Pax7 in cachectic muscle [START_REF] He | NF-κB-mediated Pax7 dysregulation in the muscle microenvironment promotes cancer cachexia[END_REF][START_REF] Coletti | Spontaneous Physical Activity Downregulates Pax7 in Cancer Cachexia[END_REF], we expected to observe an increase of Pax7 positive cells in cachexia and we wanted to verify whether exercise would rescue Pax7 levels to control levels. Indeed, exercise diminishes the number of Pax7+nuclei increased by the presence of a tumor, restoring the expression observed to control levels (Figure 16a andb). As cited above, the abnormal increase of Pax7 expression keeps satellite cells in a nondifferentiate and proliferating status, possibly sustained by tumorderived factors, such as pro-inflammatory cytokines [START_REF] Coletti | Spontaneous Physical Activity Downregulates Pax7 in Cancer Cachexia[END_REF][START_REF] He | NF-κB-mediated Pax7 dysregulation in the muscle microenvironment promotes cancer cachexia[END_REF]. Interestingly, the C26+WR group was not showing abnormal levels of Pax7+ cells any longer (Figure 16). Some studies have shown that the muscles of mutant mice are not able to shift to a compensatory hypertrophy of muscle in experimental conditions [START_REF] Guerci | Srf: a key factor controlling skeletal muscle hypertrophy by enhancing the recruitment of muscle stem cells[END_REF]. The myofibers' diameters and muscle mass did not increase in the mutant muscles compared to CTRL mice. The same team has presented the crucial role of SRF in recruitment and fusion of satellite cells. (b) Quantitative analysis of the Pax7 positive nucleus number, normalized by fiber number, in the same groups as above. Data are shown as mean+/-SEM; n=6 . Two-way ANOVA revealed an interaction between the CTRL and C26 muscles treated or not with WR (F=9,9 df=1 p<0,05 for the tumor; F=5,8 df=1; p<0,05 For interaction); p<0.05 by Tukey's HSD test.

5.14

Exercise and the tumor exert opposite effects on the number of nuclei incorporated in skeletal muscle fibers Since the observed decrease in the number of Pax7 expressing cells in exercised muscles suggested that these cells were activated, differentiated into myoblasts and ultimately fused to participate to muscle fibers, we hypothesized that these proliferating, i.e., activated, cells could incorporate fluorescent EDU. EDU incorporation in the cell DNA makes this labeling stable and, therefore, a reliable cell tracker [START_REF] Pereira | Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescenceintensity analysis[END_REF]). Since we quantified the number of EDU+ nuclei within the muscle fibers (for these experiments dystrophin was used, which is underneath the sarcolemma, and only the EDU+ nuclei within the dystrophin boundary were considered) and since myonuclei are not divided by definition, we assumed that an EDU+ nucleus could only derive from that of a satellite cell that proliferated and was ultimately incorporated within a myofiber upon myogenic differentiation (for details on EDU injections, see M&M). As expected, we did not observe a difference between the CTRL and the CTRL+WR groups, while a significant decrease was noted in the C26 group, indicative that the presence of the tumor is inhibitory for the incorporation of nuclei into the myofibers (Figure 17). On the contrary, a significant increase of EDU + nuclei inside the myofibers in the C26+WR group was observed. Taken together, our data suggest that the tumor maintains Pax7 expression elevated in the satellite cells, which represents an inhibitory signal for their incorporation into the myofibers. On the other hand, voluntary exercise promotes the incorporation of nuclei, likely deriving from satellite cells, into the myofibers. This phenomenon could participate in the rescue by voluntary exercise of myofiber size upon cachexia [START_REF] Bazgir | Satellite Cells Contribution to Exercise Mediated Muscle Hypertrophy and Repair[END_REF][START_REF] Biressi | Heterogeneity in the muscle satellite cell population[END_REF]. Muscle dystrophin (red), nuclei (blue), and EDU (green) show the presence of EDU-labeled nuclei inside (with arrow) or outside the muscle fibers. Dystrophin highlights the myofiber plasma membrane and envelops exclusively myonuclei; since EDU is incorporated into the nuclei of proliferating cells (for details on EDU treatments, see M&M), EDU+ myonuclei derives from mononuclear, proliferating cells that have been incorporated into the myofibers. Scale bar= 50 micron. (b) Analyses of the number of EdU positive nuclei in muscle sections, normalized by the total number of EDU+ nuclei per section, from the wild type, BALB/c mice, divided in four experimental groups: Control (CTRL) and C26 tumor-bearing (C26) mice, which were hosted in individual cages, in the absence or presence (WR) of a wheel, which allows running voluntary exercise. Data are shown as mean+/-SEM; n=4. Two-way ANOVA revealed an interaction between the CTRL and C26 muscles treated or not with WR; (F=9.53 df=1; p<0,05 for wheel running condition; F=17.48 df=1; p<0,01 for interaction); p<0.05 by Tukey's HSD test.

6.

MATERIALS AND METHODS To analyze the effects of voluntary wheels running (WR) on mice, they were individually hosted in custom-made, wheel equipped. All wheels were supplied with a tachometer to record physical activity data, including the average speed, the total distance covered, and the average daily distance [START_REF] Pigna | Aerobic Exercise and Pharmacological Treatments Counteract Cachexia by Modulating Autophagy in Colon Cancer[END_REF]. Mice of the exercised groups were hosted with wheels equipped cages from the first day of tumor injection until the sacrifice. The group without wheels was considered as non-exercised group. Each day, the weight, the data on running activity have been taken.

Body composition

The body weight of the mice was registered daily, five times per week. Their body composition, such as lean body mass and body fat, were measured at T1, T2 and T3 by a nuclear magnetic resonance system (LF90II, Bruker, Germany).

Evaluation of gripping strength

To evaluate the grip strength, we used the Strength Meter Grip (BIO-GS3, Bioseb, France). The force measurements were performed weekly at T1, T2 and T3. The test was repeated 5 times in a row with a recovery time of 30 seconds between each test. The average value (Mean) as well as the maximum value (Fmax) of the 5 tests were recorded as the gripping force of the forelimb and hind limb for each mouse.

Incremental exercise on a treadmill

For the evaluation of the physical performance we used a singletrack unidirectional treadmill with a speed adjustable from 0 to 1.07 m.s-1, equipped with a calorimetric cage (Phenomaster, TSE, Germany). The mice had an acclimation period of 10 minutes at a speed of 0.05 m.s-1, 24 hours before the beginning of the protocol. The running speed had not been increased further, to avoid a training effect. The mice were acclimated on the treadmill 3 minutes just before evaluation at low rates of 0.01 to 0.05 m.s-1. In order to evaluate the physical performance of the mice, the mice were exercised at an incremental speed, which was increased by 1 cm.s-1 every 15 seconds. Oxygen consumption (V ̇O2), carbon dioxide production (V ̇CO2) and respiratory exchange ratio (RER = V ̇CO2 / V ̇O2) were recorded every second. The peak oxygen consumption (V ̇O2 peak in ml.h-1) and RER peak was defined as the highest values achieved during exercise. Stopping the exercise corresponds to the maximum speed (Smax in m.s -1 ) reached by each mouse.

Indirect calorimetry at rest

The resting V ̇O2 and RER were measured for each mouse in the stationary treadmill equipped with the calorimetric cage. The V ̇O2 rest and RER rest correspond to the lowest values over a period of 1 hour.

Histology and Histochemistry

The TA muscle was dissected, covered by tissue freezing medium (Leica), and frozen in liquid nitrogen-cooled isopentane. Muscle were stored at -80 °C until needed, then the cryosections of 8 𝜇m thickness were obtained using a Leica cryostat (Leica Biosystems).

NADH staining

The muscle cryosections were fixed by 4% paraformaldehyde for 10 minutes at RT, then incubated with NADH staining solution (0.4 mg/ml NADH + 0.8 mg/ml NBT in 0.1 M Tris / HCl) for 60 min at 37 °C. After several washings with dd H2O, the sections are dehydrated in 75% Ethanol for 1 min, 95% Ethanol for 1 min and 100% Ethanol for 5 min successively. At last, ethanol was removed with Toluene (AKA Toluolo) for 5 min and the slide are mounted in Eukitt medium.

Immunofluorescence

The muscle sections were fixed at 4% paraformaldehyde for 10 minutes at RT. After saturation with 5% bovine serum albumin (BSA) for 60 minutes, the sections were incubated with a polyclonal anti-laminin Ab (Sigma) (1 : 500 to 4% BSA) and polyclonal antidystrophin (Sigma) (1 : 500 to 4% BSA), followed by the incubation of the anti-mouse Alexafluor 555 (1 : 500; Molecular probes; Invitrogen), or anti-rabbit Alexafluor 488 (1 : 500; Molecular probes; Invitrogen) for 35 minutes while Hoechst 33342 (Sigma) is used for nuclear staining (1: 10000). For Pax7, the sections were fixed at 4% paraformaldehyde for 10 minutes at RT, then permeabilized in 0.2% Triton for 20 minutes at RT. After incubation with 5 % Igg-free-BSA for 120 minutes, the sections were saturated with Affini Pure Fab Fragment Goat antimouse IgG (1: 100, Jackson ImmunoResearch) for 20 minutes. Primary mouse anti-Pax7 antibody (1: 50; Santa Cruz Biotechnology) and anti-laminin Ab (1: 500; Sigma) were incubated. After three washing, the slides were incubated the antimouse Alexafluor 555 (1: 500; Molecular probes; Invitrogen) and the anti-rabbit Alexafluor 488 (1: 500; Molecular probes; Invitrogen) for 35 minutes at RT. Nucleus staining was performed using HOESCHT. Muscle sections were then mounted in Glycerol Mounting Medium and kept at 4°C until image acquisition.

Nuclear labeling by EDU and detection

The mice received 25 µg/g 5-ethynyl-2'-deoxyuridine (EdU) (Life Technologies) at 12-day, 13-day and 14-day after the inoculation of C26 tumors. EdU detection was performed using a Click-it-EdU Alexa Fluor 647 kit, according to the manufacturer's instructions (Life Technologies).

Western Blot

Samples were treated with lysis buffer which contains 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% TRITON, and a protease inhibitor cocktail. Proteins (30-50 micrograms) were separated by means of SDS-PAGE with varying % of acrylamide, depending on the protein molecular weight to be analyzed, and transferred to Nitrocellulose Blotting membrane 0.45µm (Amersham Protran; GE Healthcare). The running period was 60 minutes at 100V with the Running buffer, and the transfer period was 60 minutes at 100 in Transfer buffers. Membrane of nitrocellulose was incubated with blocking buffer TBS-Tween (20 mM Tris, 137 mM NaCl, 0.1% Tween 20) with 5% not fat milk for 1 hour at room temperature; then the membranes were incubated at 4 °C, overnight with Ab solutions. The primary antibodies were: anti-MyoD (1: 1000; Sigma), anti-integrin β1 (1:1000; AbCam), anti-SRF (1:1000; Santa Cruz), anti-GAPDH (1:30000; Sigma); they were incubated in the presence of TBS-Tween 5% of milk. The secondary antibodies were HRP-conjugated-anti-mouse or antirabbit (Biorad; 1:10000) in TBST, and signals were detected using ECL reagent (CYANAGEN; 1: 1). Intensity of Western blot signals was quantified by using ImageJ software or Image Lab.

6.8 RNA extraction, cDNA synthesis and Q -PCR Total RNA was isolated from TA muscles, with Trizol reagent (Invitrogen) following the manufacturer's protocol. RNA concentration was determined by measuring the absorbance in 260 nm/280 nm in a NanoDrop spectrophotometer from 1000 ng of extracted RNA, first-strand cDNA was then synthesized using the high capacity applied PrimeScript RT reagent Kit (TaKaRa).

LightCycler® 480 was used to detected SYBR Green (Applied Biosystem). Data were collected and analyzed using the LightCycler® 480 software, release version 1.5.0.

Primers list:

6.9 Statistical analysis VassarStat a statistical computation website available at http://vassarstats.net/ was used for statistical analyses. Statistical significance was determined by sing two tailed Student t -test when two conditions were compared, or with two-way analysis of variance (ANOVA) followed by Tukey's HSD as a post-hoc test, to compare the treatments. All values were expressed as mean +/-SEM. The value of p < 0.05 was considered to indicate a statistically significant difference.

5. 4

 4 C26 tumor and exercise independently affect SRF transcriptional activity ............................................................... 5.5 MyoD expression in the context of cachexia and exercise ………………………………………………………….42 5.6 Integrin-β1expression is down-regulated in cachexia, an effect which is blunted by exercise ........................................... 5.7 The voluntary exercise promotes the STARS/RhoA expression .................................. ………………………………46 5.8 SRF is associated to a positive body weight balance and depends on a threshold running activity .................................... 5.9 Characterization of the conditional, muscle-specific SRF KO mice: genetic studies ........................................................... 5.10 Characterization of the conditional, muscle-specific SRF KO mice: physiological studies ................................................. 5.11 Detailed analysis of the exercise-dependent metabolic and strength adaptations in WT and SRF KO mice ......................... 5.12 SRF is necessary for the beneficial effects of exercise in tumor-bearing mice ................................................................... 5.13 Exercise and the tumor exert opposite effects on the number of Pax7-expressing cells ............................................... 5.14 Exercise and the tumor exert opposite effects on the number of nuclei incorporated in skeletal muscle fibers ........... 6. Materials and Methods .......................................................... 6.1 Mice, wheel running and tumors transplantation ........... 6.2 SRF KO mice ................................................................. 6.3 Exercise protocols .......................................................... 6.4 NADH staining ............................................................... 6.5 Immunofluorescence ...................................................... 6.6 Nuclear labeling by EDU and detection ......................... 6.7 Western Blot ................................................................... 6.8 RNA extraction, c DNA synthesis and Q -PCR ............. 6.9 Statistical analysis ........................................................... 7. REFERENCES ....................................................................... 8. LIST OF PUBLICATIONS ................................................... muscle activator of Rho signaling STAT: Signal Transducers and Activators of Transcription TA: Tibialis Anterior TCF: Ternary complex factor TGF-β: Tumor Growth Factor β TNF α: Tumor Necrosis Factor α

Figure 1 :

 1 Figure 1: The structure of the skeletal muscle and of the myofibril, showing the cross-sectional and longitudinal organization from "A chemo-mechanical model for the single myofibril in striated muscle contraction" Naldi, G. Meccanica (2017) 52: 3357

Figure 2 :

 2 Figure 2: Alterations in myonuclear number during skeletal muscle hypertrophy and atrophy from "Oxidative stress and disuse muscle atrophy" Scott K. Powers, Scott K. Powers.

Figure 3 :

 3 Figure 3: Symmetric and Asymmetric Division of Muscle Satellite Cells from "Oriented Cell Divisions and Muscle Satellite Cell Heterogeneity" Cossu and Tajbakhsh, Cell; Volume 129, Issue 5, 1 June 2007, Pages 859-861

Figure 4 :

 4 Figure 4: The simplified scheme shows the mainly organs affected during cachexia progression from "Understanding cachexia as a cancer metabolism syndrome". P. E. Porporato, Oncogenesis; (2016).

Figure 5 :

 5 Figure 5: Roles of serum Response Factor adapted Chai J, 2002Despite the low intrinsic transactivating capacity, SRF is a potent regulator of gene transcription. Indeed, the SRF activity is controlled by the actin dynamics through the transcription co-factor Myocardin-related transcription factors (MRTFs, including two members: MRTF-A and MRTF-B)[START_REF] Guerci | Srf-Dependent Paracrine Signals Produced by Myofibers Control Satellite Cell-Mediated Skeletal Muscle Hypertrophy[END_REF]. The latter are ubiquitous, and their localization is regulated by the actin dynamics, ultimately being the primary responsible for SRF transcriptional activity. As shown in Figure7, actin polymerization favors MRTF nuclear import, and therefore the activation of SRF transcriptional activity by diminishing the cytoplasmic concentration of G-actin[START_REF] Posern | Actin' together: serum response factor, its cofactors and the link to signal transduction[END_REF]. The Rho GTPases are responsible for the increasing of actin polymerization, which, in turn, regulates SRF and its co activator MRTF. Study realized on fibroblasts in culture showed the MRTF-localization is mainly cytoplasmic and the stimulation with serum leads to its nuclear accumulation. It has been also shown the activation of Rho/actin signal pathways can induce the entry of MRTF-A in the nuclei and that the mechanism involved in MRTF-A inhibition is the binding between monomeric actin and MRTF-A[START_REF] Esnault | Rho-actin signaling to the MRTF coactivators dominates the immediate transcriptional response to serum in fibroblasts[END_REF]. Furthermore, the regulation of MRTF-A localization is dynamic and subjected to import/export systems: in the absence of serum, the concentration of monomeric actin increases, also leading to an increased MRTF-A export[START_REF] Weissbach | Myocardin-Related Transcription Factor A Activation by Competition with WH2 Domain Proteins for Actin Binding[END_REF]).

Figure 6 :

 6 Figure 6: Regulation of actin by RhoA/STARS pathway from Muscle-Specific Signaling Mechanism that Links Actin Dynamics to Serum Response Factor. Koichiro Kuwahara, Tomasa Barrientos, G.C.Teg Pipes, Shijie Li, Eric N. Olson ; Molecular and Cellular Biology Mar 2005, 25 (8)

Figure 7 .

 7 Figure 7. Voluntary exercise counteracts cancer-cachexia (a) Analysis of the final body weight (for tumor-bearing mice, which is the net weight obtained by subtracting the tumor weight from the total weight of the animal at 21 days following tumor cell injection). Control (CTRL) and C26 tumor-bearing (C26) mice were hosted in individual cages, in the absence or presence (WR) of a wheel, which allows voluntary exercise. Data are shown as mean+/-SEM; CTRL n=32; CTRL+WR n=22; C26 n=42; C26+WR n=24. Two-way ANOVA revealed an interaction between the presence of the tumor and the running activity on body weight (F=39,24 df=1; p<0,01 for tumor; F=15,9 df=1; p<0,01 for wheel running condition; F=7,95 df=1 ; p<0,05 For interaction); # = p<0.05 by Tukey's HSD test. (b) Analysis of the Tibialis Anterior (TA) muscle weight in the same animal as (a): control (CTRL) and C26 tumor-bearing (C26) mice, in the absence of

Figure 8 .

 8 Figure 8. Voluntary exercise counteracts the tumor-dependent decrease in SRF protein levels (a) Real-Time PCR analysis for SRF in the TA muscle. Control (CTRL) and C26 tumor-bearing (C26) mice were hosted in individual cages, in the absence or presence (WR) of a wheel, which allows voluntary exercise. The data, normalized by GAPDH expression, are expressed over the CTRL and are shown as mean+/-SEM; n=6 for each group. Two-way ANOVA revealed an effect of the running activity: (F=1.26 df=1; p<0,05 for wheel running condition). (b) Quantification of the Western blot analysis (see below) for SRF protein levels in the TA muscle in the same conditions as above.GAPDH is used as a loading control, the data are expressed over the CTRL and are shown as mean+/-SEM; results from WB membranes from three independent experiments performed at least in triplicate have been used for the quantitative analysis (n=9 for CTRL and CTRL+WR and n=10 for C26 and C26+WR). Two-way ANOVA revealed an interaction between the presence of the tumor and the running activity (F=10.17 df=1; p<0,01 for tumor; F=14.7 df=1; p<0,01 for wheel running condition; F=5.22 df=1 ; p<0,01 for interaction); p<0.05 by Tukey's HSD test. (c) Representative blot showing the three SRF bands and the GAPDH band in the same conditions as above. The three SRF bands represent the SRF isoforms, the weight of active form is 67kd. This specific membrane CTRL and CTRL+WR have been loaded in triplicate, while C26 and C26+WR in quadruplicates, along with the molecular markers (M) in the same gel.

Figure 9 .

 9 Figure 9. SRF protein levels directly correlate with the change in body weight Regression analysis of the changes in body weight between day 0 and day 21 (delta body weight) from the control and the C26 tumor-bearing mice, in the absence or presence of voluntary exercise, as a function of the SRF protein levels measured by WB in the TA muscle extracts and normalized by GAPDH. The calculated equation shows a statistically significant correlation between the amount of SRF protein and a positive body weight change (y = 21,109 x -12,853; r=0.6756; p< <0.001); n=38.

Figure 10 .

 10 Figure 10. Voluntary exercise counteracts the tumor-dependent decrease in MyoD protein levels Real-Time PCR analysis for MyoD in the TA muscle. Control (CTRL) and C26 tumor-bearing (C26) mice, which were hosted in individual cages, in the absence or presence (WR) of a wheel, which allows running voluntary exercise. The data, normalized by GAPDH expression, are expressed over the CTRL and are shown as mean+/-SEM; n=6 for each group. Two-way ANOVA revealed an interaction between the presence of the tumor and the running activity: (F=46.34 df=1; p<0,05 for wheel running condition; F=4.59; df=1; p=0.07 quite significant for interaction). (b) Quantification of the Western blot analysis (see below) for MyoD protein levels in the TA muscle in the same conditions as above. GAPDH is used as a loading control, the data are expressed over the CTRL and are shown as mean+/-SEM; WB experiments performed in triplicate or quadruplicate, n=3 for CTRL and CTRL+ WR; n=4 for C26 and C26+WR have been used for this analysis. Two-way ANOVA revealed an interaction between the presence of the tumor and the running activity (F= 17.31 df=1; p< 0.01 for tumor; F= 11.42 df=1; p< 0.01 for wheel running condition; F= 6.83 df=1 ; p< 0.05 f or interaction) ; p<0.05 by Tukey's HSD test. (c) Representative blot showing the MyoD band and the GAPDH band in the same conditions as above. For this specific membrane CTRL and CTRL+WR have been loaded in triplicate, while C26 and C26+WR in quadruplicates, along with the molecular markers (M) were loaded in the same gel.

Figure 11 .

 11 Figure 11. Voluntary exercise counteracts the tumor-dependent decrease in Integrin-β1 protein levels (a) Real-Time PCR analysis for Integrin-β1 in the TA muscle. Control (CTRL) and C26 tumor-bearing (C26) mice, which were hosted in individual cages, in the absence or presence (WR) of a wheel, which allows running voluntary exercise. GAPDH has been used as a normalizer, the data is expressed over the CTRL and are shown as mean+/-SEM; n=6. Two-way ANOVA revealed an interaction between the presence of the tumor and the running activity (F=3,6 df=1; p<0,08 for tumor; F=4,89 df=1; p<0,05 interaction). (b) Quantification of the Western blot analysis (c) for Integrin-β1 protein levels in the TA muscle in the same conditions as above. GAPDH is used as a loading control, the data are expressed over the CTRL and are shown as mean+/-SEM; WB membranes from three independent experiments performed in triplicate or quadruplicate, n= 7 for CTRL and CTRL+ WR; n=8 for C26 and C26+WR, have been used for this analysis. Two-way ANOVA revealed an interaction between the presence of the tumor and the running activity (F=4,29 df=1; p<0,05 interaction); p<0.05 by Tukey's HSD test. (c) Representative blot showing the three Integrin-β1 bands and the GAPDH band in the same conditions as above. CTRL and CTRL+WR triplicate, and C26 and C26+WR quadruplicates, along with the molecular markers (M) were loaded in the same gel.

Figure 12 .

 12 Figure 12. The voluntary exercise counteracts and activates the STARS/RhoA/MRTFs pathway Real-Time PCR analysis for RhoA and (b) STARS in the TA muscle. Control (CTRL) and C26 tumorbearing (C26) mice were hosted in individual cages, in the absence or presence (WR) of a wheel, which allows voluntary running exercise. The data, normalized by GAPDH expression, are expressed over the CTRL and are shown as mean+/-SEM; n=6. Two-way ANOVA revealed an effect of the running activity (for STARS: F=3.07 df=1; p=0,09 for tumor; F=13.52 df=1; p<0,05 for wheel running condition; F=7.69 df=1 ; p<0,05 for interaction; for RhoA F=7.69 df=1; p<0,05 for wheel running condition; F=6.29 df=1 ; p<0,05 for interaction).

  Supplemental data 2A. Correlative analysis of SRF protein level, changes in body weight and running distance of individual mice. (a) SRF protein levels in the TA of Control (CTRL) and C26 tumorbearing (C26) mice which were hosted in individual cages, in the absence or presence (WR) of a wheel, allowing voluntary running exercise. GAPDH is used as a loading control. A representative WB membrane, loaded in duplicate, and including the molecular weight marker (M) is shown. The table presents the murine final body weight (BW) at day 21 and the body weight changes (delta BW) for individual mice. The red squares and circles highlight data from mouse #4 and #8 in the C26+WR group, showing two different responses to exercise for SRF expression. (b) Analysis of the changes in body weight between day 0 and day 21 (delta body weight) in the control and the C26 tumor-bearing mice, in the absence or presence of voluntary exercise, as a function of the SRF protein levels measured by WB in the TA muscle extracts and individually normalized by GAPDH. (c) Individual, daily measure of running distance in the last five days of the experiment (D17-D21) and total running distance Correlative analysis of SRF protein level, changes in body weight and running distance of individual mice. (a) SRF protein levels in the TA of Control (CTRL) and C26 tumor-bearing (C26) mice which were hosted in individual cages, in the absence or presence (WR) of a wheel, allowing voluntary running exercise. GAPDH is used as a loading control. A representative WB membrane, loaded in duplicate, and including the molecular weight marker (M) is shown. The table presents the murine final body weight (BW) on day 21 and the body weight changes (delta BW) for individual mice. The red squares and circles highlight data from mouse #2 and #3 in the C26+WR group, showing two different responses to exercise for SRF expression. (b) Analysis of the changes in body weight between day 0 and day 21 (delta body weight) in the control and the C26 tumor-bearing mice, in the absence or presence of voluntary exercise, as a function of the SRF protein levels measured by WB in the TA muscle extracts and individually normalized by GAPDH. (c) Individual, daily measure of running distance in the last five days of the experiment (D17 to D21) total running distance (e) for the mouse #2 and #3.

Figure 13 .

 13 Figure 13. SRF mRNA and protein levels decrease in KO mice Real-Time PCR analysis for SRF in the TA muscle of wild Type (WT) and SRF knockout (KO) mice, 21 days following the last tamoxifen injection. The data, normalized by GAPDH expression, are expressed over the CTRL and are shown as mean+/-SEM; n=3. Student's t-test revealed a significant difference between WT and WT+WR; # = p<0.05. (b) Quantification of the Western blot analysis (see below) for SRF protein levels in the TA muscle. WT and KO mice were hosted in individual cages, in the absence or presence (WR) of a wheel, which allows running voluntary exercise. The data, normalized by GAPDH expression, are expressed over the WT and are shown as mean+/-SEM; n=4. Two-way ANOVA revealed an interaction between the presence of the tumor and the running activity; # =p<0.05 by Tukey's HSD test. (c) Representative blot showing for SRF and GAPDH in WT, WT+WR, KO and KO+WR duplicates, along with the molecular markers (M), loaded in the same gel.

Figure 14 .

 14 Figure 14. WT and SRF KO mice show any difference in the running performance and endurance (a) Measure of oxygen consumption (V ̇O2) at three-time points: 1 day before the beginning of the experiment (T1), at 2 weeks (T2) and at 3 weeks (T3) of training. V ̇O2 peak was measured during an incremental speed test on individual treadmill equipped with a calorimetric system. Wild Type (WT) and SRF knockout (KO) mice were hosted in individual cages, in the absence or presence (WR) of a wheel, which allows running voluntary exercise. At 3 weeks, both WT+WR and KO+WR mice showed significantly increased V ̇O2 peak compared to WT and KO. Data are shown as mean+/-SEM;

Table 2 :

 2 Exercise-dependent metabolic and strength adaptations in WT and Srf KO Wild-type (WT) and SRF KO mice were analyzed 1 day before the experiment (T1) and 3 weeks after (T3) the beginning of the training. (a) Body composition was measured by a nuclear magnetic resonance system. Wild Type (WT) and SRF knockout (KO) mice were hosted in individual cages, in the absence or presence (WR) of a wheel, which allows running voluntary exercise, and both showed an increase in lean body mass upon exercise. Data are shown as mean+/-SEM; n=4. T0 vs. T3: * p <0.05, ** p <0.01. (b) Running distance and time wheel training have been measured on the last day experiment, i.e., at 21 days of training. Data are shown as mean+/-SEM; n=4. Student's t test showed no difference in the running distance between WT and KO. (c) F max (maximal Force) and F mean (mean Force) have been measured by the grip test, while RER peak (peak of respiratory exchange ratio = V ̇ CO2/V ̇ O2) and RER rest (the lowest value of respiratory exchange ratio) have been measured by indirect calorimetry test. For n-both cases, mice were analyzed 1 day before the experiment (T1) and 3 weeks (T3) after the beginning of the training. Data are shown as mean+/-SEM; n=4; T0 vs. T3: * p <0.05, ** p <0.01.

Figure 17 .

 17 Figure 17. SRF is necessary to mediate exercise beneficial effect against cachexia Analysis of the final body weight (for tumor-bearing mice, that is the net weight obtained by subtracting the tumor weight from the total weight of the animal at 30 day following tumor cells injection). Wild Type (WT) and SRF knockout (KO) mice were hosted in individual cages, in the absence or presence (WR) of a wheel, which allows running voluntary exercise. All mice are LLC tumor-bearing mice (+LLC), therefore the WT displays cachexia. For technical reasons runners (+WR) were compared to the respective control in two different experiments: one for the WT mice and one for the KO mice. Data are shown as mean+/-SEM; n=5. Student's t-test revealed a significant difference between WT and WT+WR; #= p<0.05. (b) Analysis of the Tibialis Anterior (TA) muscle from the same experimental groups as (a). Data are shown as mean+/-SEM; n=4. Student's t-test

Fig. 16 .

 16 Fig.16. Voluntary exercise counteracts the tumor-dependent increase of Pax7 positive nuclei (a) Immunofluorescence analysis of transverse cryosections of the TA muscle, from wild type, BALB/c mice, divided in four experimental groups: Control (CTRL) and C26 tumor-bearing (C26) mice, which were hosted in individual cages, in the absence or presence (WR) of a wheel, which allows running voluntary exercise. Muscle laminin (green), nuclei (blue), and Pax7 (red) were immunestained, while nuclei were counter-stained in blue. The presence of Pax7-expressing, satellite cells underneath the basement membrane is highlighted by arrows. Scale bar= 50 micron.

Fig. 17 .

 17 Fig.17. Percentage of positive nuclei of EDU in the skeletal muscle fibers increases in exercised mice (a) Immunofluorescence analysis of transverse cryosections of the TA of the same groups as above. Muscle dystrophin (red), nuclei (blue), and EDU (green) show the presence of EDU-labeled nuclei

Supplemental data1 .

 data1 Tumor-bearing mice perform endurance exercise to an extent comparable to healthy mice. (a) Gross appearance of BALB/c and C57BL/6 control (CTRL) compared with C26 and LLC tumor-bearing mice, respectively (right) at 3 weeks following the tumor implant.(b) Quantification of total running distance between CTRL (CTRL+WR) and C26 tumor-bearing Balb/c mice (C26+WR). (c) Quantification of total running distance between WT (WT+WR) and LLC tumor-bearing in c57bl/6 mice (LLC+WR) 6.3.2. Evaluation of physical performance KO and WT mice were divided into four different groups: WT, WT+WR, KO, KO+WR. The exercised-mice were installed in individual cages equipped with a wheel (BIO-ACTIVE -Bioseb, France) connected to a tachometer to record physical activity data. Data were recorded each day for a period of 21 days. The evaluation of the physical performance of the mice took place at 3 moments: just before the beginning of the protocol T1 and at 14 days and 21 days after the beginning of the training protocol (T2, and T3 respectively).

  The generation of SRF KO mice, via the tamoxifen activated Cre-recombinase technique, has allowed the study of SRF role in skeletal muscle in perinatal life. For three days after the birth, the newborns are normal, but later they present a delayed muscle growth compared to WT mice. Half of KO mice survive after few weeks, the other show a severe growth problem, scoliosis and a 75% lower muscle mass compared to health mice; in particular, the musculature shows muscle atrophy and sarcomere disorganization[START_REF] Parlakian | Targeted Inactivation of Serum Response Factor in the Developing Heart Results in Myocardial Defects and Embryonic Lethality[END_REF]). Adult stage. Numerous studies agreed on the implication of SRF in the muscle plasticity in adult life. During aging, SRF is downregulated in human and mice skeletal muscle which could contribute to the sarcopenia commonly observed in the elderly. The loss of SRF function in the adult skeletal muscle by the Crerecombinase induced by tamoxifen injection showed an overt phenotype only several months following the tamoxifen injection. The phenotype of the prematurely aging muscle in the SRF KO mice displayed atrophy, fibrosis, lipid accumulation and a perturbed regeneration

3.10

SRF Knock-out in skeletal muscle cells Embryonic stage. The invalidation of SRF in skeletal muscle during the embryonic stage leads to severe muscular hypoplasia and the death of perinatal stage

[START_REF] Coletti | Serum Response Factor in Muscle Tissues: From Development to Ageing[END_REF]

.

Postnatal stage.

ACKNOWLEDGMENT

First, I would like to thank Dario Coletti for the daily support, his patience and gave me the opportunity to lead this project. I thank Sergio Adamo and Zhigang Xue my two co-directors for their scientific knowledges and their support. I thank Zhelin Li, Denise Paulin, Ara Parlakian, Jean-Francois Decaux, Jocelyne Blanc, Jacqueline Gao-Li and Alexandra Benoni for their scientific skills and their help during these three years. I thank also Viviana Moresi, Alexandra Renzini and Carla Ramina for kindness and scientific skills. I thank Athanassia Sotiropoulos from Cochin Institut for the SRF KO mice. Finally, I thank my family to give me the chance to follow my dreams.

Conference participation, oral and poster presentation: