Objective: Cervical vestibular evoked myogenic potentials (cVEMPs) are short-latency electromyogram (EMG) evoked by high-level acoustic stimuli recorded from the activated sternocleidomastoid muscle and used to evaluate otolith organ function. The purpose of this study was to investigate the effects of aging on the cVEMP and on the sternocleidomastoid muscle EMG level. Design: A cross-sectional observational study was used to investigate differences in cVEMP and sternocleidomastoid muscle EMG level in a group of 24 younger and 24 older individuals. cVEMPs were recorded during activation of the sternocleidomastoid muscle at target EMG levels ranging from 0 to 90 μV and during maximum voluntary contraction of the sternocleidomastoid muscle. Results: The sternocleidomastoid muscle EMG amplitude increased as a function of target EMG level for both age groups; however, the mean EMG amplitude was greater for the younger group than the older group, and the variability of EMG amplitude was greater for the older group. The EMG amplitude at maximum voluntary contraction ranged from 88 to 279 μV for the younger subjects and from 32 to 230 μV for the older subjects, and the mean EMG amplitude at maximum voluntary contraction was significantly greater for the younger group than the older group. The cVEMP amplitude increased as a function of EMG target level for each age group. Although cVEMP amplitude increased as a function of target EMG level for both groups, the older group exhibited smaller cVEMP amplitudes, overall, compared with the younger group. To separate the influence of EMG level from aging on cVEMP amplitude, only the responses obtained at the 30 μV target EMG level were considered for the statistical analysis because there was no significant difference in EMG level between groups at the 30 μV target level. The mean cVEMP amplitudes at the 30 μV target level were 101 and 51 μV for the younger and older groups, respectively, and a statistical analysis indicated that cVEMP amplitude for the younger group was significantly greater than the older group. Conclusions: The findings suggest that the decrement in cVEMP amplitude is related to both age-related changes in the vestibular system and age-related changes in the sternocleidomastoid muscle.

Au cours de la myogenèse post-natale, la voie de signalisation de Notch participe au développement et à la régénération du muscle squelettique chez les mammifères. Elle permet le maintien de l'état prolifératif des myoblastes, contrôle la quiescence des cellules satellites in vivo et préserve une sous-population de cellules de réserve indifférenciées in vitro. L' activation de la voie et l'interaction du récepteur Notch avec ses ligands est dépendante de leur entité glucidique, notamment de leurs O-fucosylglycannes. La synthèse de ces derniers est initiée par la protéine O-fucosyltransférase 1 (Pofut1) qui greffe un O-fucose sur des domaines peptidiques particuliers appelés EGF-like. Bien que les acteurs moléculaires de la différenciation myogénique aient été largement étudiés par la communauté scientifique, la contribution de la glycosylation des protéines dans ce processus reste peu documentée. Une approche expérimentale in vitro basée sur l'utilisation de la lignée myoblastique murine C2C12 nous a permis d'identifier une expression importante de Pofut1 dans les cellules de réserve tandis qu' elle est restreinte dans les myotubes durant la différenciation myogénique. Plusieurs lignées de cellules C2C12 ont été générées pour qu' elles expriment de manière stable et différentielle Pofut1. Elles permettent ainsi d' évaluer l' importance du niveau d' expression de Pofut1 sur la différenciation myogénique.La sous-expression de Pofut1 réduit l' activation de la voie de signalisation de Notch conduisant à une entrée précoce des myoblastes dans le programme myogénique. Ceci a pour conséquence la dépletion des cellules de réserve Pax7+/MyoD- au profit d' une augmentation du nombre de myotubes. Des études morphométriques ont révélé un défaut d' accrétion nucléaire dans les myotubes sous-exprimant Pofut1, caractéristique d' une altération de la fusion secondaire. Ces observations sont accompagnées d' une diminution significative de l' expression du récepteur à l' interleukine 4 dans les cellules de reserve sous-exprimant Pofut1. Les lignées cellulaires ré-exprimant Pofut1 présentent une activation de la voie de signalisation de Notch et un processus de fusion myoblastique correctement restaurés.Ces travaux de thèse ont mis en exergue pour la première fois le rôle essentiel de Pofut1 dans le devenir cellulaire et la fusion des myoblastes au cours de la différenciation myogénique. / During post-natal myogenesis, Notch signaling pathway is involved in the development and regeneration of skeletal muscle in mammals. It maintains progenitor cell properties during the development of the myogenic lineage and controls the transition of satellite cells from a quiescent to an active state and preserves a subpopulation of reserve cells, in cell culture, in an undifferentiated state. The interaction between Notch and its ligands and the activation of this signaling is mainly controlled by the activity of protein O-fucosyltranferase 1 (Pofut1) and thus by the O-fucosylation state of the EGF-like repeats.Although the molecular players in myogenic differentiation have been extensively studied by the scientific community, the contribution of glycosylated proteins in this process remains poorly documented. An experimental in vitro study based on the C2C12 mouse myoblast cell line allowed us to identify a high expression of Pofut1 in reserve cells while a low expression was found in myotubes during myogenic differentiation. Several C2C12 cell lines were generated to express Pofut1 at different levels. They were used to evaluate the contribution of Pofut1 expression to the myogenic differentiation.The knockdown of Pofut1 repressed Notch signaling pathway activation leading to an earlier entrance of myoblasts in myogenic program. This resulted in the depletion of reserve cells Pax7+/MyoD- and an increase in the number of myotubes. Morphometric analysis revealed a nuclear accretion defect in Pofut1 knockdown myotubes. A significant decrease in the expression of the interleukin-4 receptor in Pofut1 knockdown reserve cells was also observed. Cell lines re-expressing correctly Pofut1 restored Notch signaling pathway and subsequently myoblast fusion process.This thesis work highlights, for the first time, the crucial role of Pofut1 in the cell fate decision and the fusion of myoblasts during myogenic differentiation.

Orientador: Maeli Dal Pai Silva / Tese ( doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T12:24:51Z (GMT). No. of bitstreams: 1 Carani_FernandaRegina_D.pdf: 24872848 bytes, checksum: e4b7c794fa866be614e932ec5a33eaf6 (MD5) Previous issue date: 2011 / Resumo: O pirarucu (Arapaima gigas) caracteriza-se como uma espécie promissora para a Aqüicultura, devido principalmente às suas características de rápido crescimento e rusticidade. Sua criação em regime intensivo tem obtido enorme sucesso, podendo alcançar até 10 quilos de peso corporal em apenas um ano de criação. O pirarucu é considerado hoje uma das mais importantes espécies de peixes da bacia Amazônica e, por esta razão, é primordial que se investigue os mecanismos celulares e moleculares que controlam o rápido crescimento muscular, contribuindo com novas estratégias de criação e com a manutenção da espécie. O crescimento do músculo estriado esquelético nos peixes pode ocorrer por dois mecanismos: hipertrofia e/ou hiperplasia das fibras, a partir de células satélites ou mioblastos. Esse processo é controlado por Fatores Reguladores Miogênicos (MRFs) e pelo fator de crescimento Miostatina. O objetivo do presente estudo foi avaliar as características morfológicas e o crescimento muscular hipertrófico e hiperplásico, bem como analisar a expressão gênica e protéica da MyoD, da Miogenina e da Miostatina na musculatura esquelética do pirarucu (A. gigas), em diferentes fases de crescimento. Os animais utilizados no presente estudo foram provenientes de duas pisciculturas: na primeira, foram obtidos os "alevinos" (pós-larvas; 48 g); na segunda, os animais em diferentes estágios de crescimento, divididos em quatro grupos de acordo com o peso corporal. Grupo A (50 gramas, n=7), grupo B (420 gramas, n=7), grupo C (5,5 quilogramas, n=7) e grupo D (9,1 quilogramas, n=7). As amostras musculares foram coletadas, congeladas e submetidas à coloração HE, para avaliação do padrão morfológico das fibras, e à reação para a enzima NADH-TR, para avaliar o metabolismo oxidativo das fibras. Para avaliar o padrão de crescimento hiperplásico e hipertrófico da musculatura branca, foi calculado o menor diâmetro de uma população de fibras por animal, e estas foram distribuídas em classes, na dependência do seu diâmetro. A análise da expressão gênica de MyoD, miogenina e miostatina foi feita por Reação em Cadeia da Polimerase após Transcrição Reversa (RTqPCR); para análise da expressão protéica, foi utilizado o Western Blot. A distribuição das fibras em classes de diâmetro exibiu o seguinte padrão: o grupo A mostrou a maior parte das fibras na classe 20, o grupo B, na classe 50, o grupo C, nas classes 50 e 80, e o grupo D, na classe 80. Isso indica uma alta taxa de hiperplasia das fibras nos grupos menores (A e B) e alta hipertrofia das fibras nos grupos maiores (C e D). Para a análise da expressão gênica de MyoD e miogenina no músculo vermelho e branco dos "alevinos", não houve diferença estatística; para a miostatina, houve expressão diferencial, com os maiores níveis encontrados no músculo branco em comparação com o músculo vermelho. Na avaliação da expressão de MyoD e miogenina, tanto a expressão gênica como a expressão protéica não mostraram diferença significativa. Por outro lado, a expressão gênica da miostatina foi menor no grupo A e maior nos demais, e a expressão da proteína miostatina foi maior no grupo A, diminuindo nos demais grupos. Estes resultados refletem as características de crescimento muscular da espécie e sugerem que a expressão dos MRFs e da miostatina são responsáveis pelo balanço entre a hiperplasia e a hipertrofia das fibras, contribuindo para o rápido crescimento da espécie e a manutenção das características do filé / Abstract: Pirarucu (Arapaima gigas) is a promising fish species for Aquaculture programs mainly by the fast growing feature and rusticity. Their rearing under intensive conditions generated much successful results, as they reach up to 10 kilograms in just one year. Considered one of the most important fish species from Amazon basin, it is of primary interest to investigate the cellular and molecular mechanisms that control the fast muscle growth in pirarucu, providing information for new rearing strategies and species conservation. In most fish, skeletal muscle growth occurs by two mechanisms: hypertrophy and hyperplasia, from satellite cells or myoblasts. These process are under control by Myogenic Regulatory Factors (MRFs) and by the growth factor Myostatin. The animals were obtained from two pisciculture, where we got the alevin pirarucu (n=7; 48 grams weight), and the specimens at different growth stages, divided into groups according body weight. Group A (50 grams, n=7), group B (420 grams, n=7), group C (5,5 kilograms, n=7) and group D (9,1 kilograms, n=7). Muscle samples were collected, frozen and stained with HE for morphological analysis, and submitted to NADH-TR enzyme reaction for oxidative methabolism analysis. To evaluate hyperplasic and hypertrophic muscle growth, it was measured the smallest diameter from a set of fibers, which were grouped into diameter classes. Gene expression analysis of MyoD, Myogenin and Myostatin were performed by Quantitative Polimerase Chain Reaction after Reverse Transcription (RT-qPCR); protein content analysis was by Western Blot technique. Muscle fibers distribution in classes showed the following pattern: group A showed most fibers in class 20, group B, in class 50, group C, in classes 50 and 80, and group D, in class 80. This is an indicative of high fiber hiperplasia rate in groups A and B, and high hypertrophy in groups C and D. There was no statistical difference in MyoD and Myogenin genes expression in red and white muscles of pirarucu; however, Myostatin expression showed high levels in white muscle compared to red muscle. Evaluating MyoD and Myogenin expression in white muscle of pirarucu at different growth stages, both gene and protein levels were similar. Myostatin gene expression was low in group A and high in the other groups; on the other hand, Myostatin protein was high in group A and low in the other groups. These results reflect the muscle growth characteristics in pirarucu and suggest that the MRFs and Myostatin expression are controlling the balance between hyperplasic and hypertrophic mechanisms, promoting the fast rate feature of pirarucu and their fillet quality / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

La protéine O-fucosyltransférase 1 (POFUT1) réticulaire, dont le gène est localisé dans la région chromosomique 20q11.21 chez l’Homme, catalyse le transfert d’un fucose qui sera O-lié sur la sérine ou la thréonine présente dans la séquence consensus (C2X4S/TC3), portée par un domaine EGF-like d’une glycoprotéine membranaire ou sécrétée. Le knockdown de Pofut1 (Po -) dans la lignée myoblastique murines C2C12 conduit à la formation de myotubes allongés et minces, à faible nombre de noyaux ainsi qu’à une sous-expression du marqueur myogénique tardif Myf6, suggérant des défauts significatifs dans la fusion secondaire. La signalisation NFATc2/IL-4 est décrite comme la voie principale associée à cette étape. Nous montrons que la moindre expression de Nfatc2 dans les myotubes Po - est corrélée à une baisse de l'IL-4 sécrétée et à une faible quantité de son récepteur (IL-4Rα) présent chez les cellules de réserve qui doivent participer à la fusion avec les myotubes naissants. La neutralisation de l’IL-4Rα sur les C2C12 sauvages provoque des défauts d'accrétion myonucléaire, semblables à ceux observés pour les Po -. Ainsi, POFUT1 pourrait être un nouveau médiateur de la croissance des myotubes au cours du processus myogénique, notamment par la signalisation NFATc2/IL-4. La glycoprotéine WIF1, cible potentielle de POFUT1, est un antagoniste de la signalisation WNT via sa fixation aux protéines WNT. Cette voie est connue pour être impliquée dans la prolifération et la différenciation des myoblastes. Néanmoins, aucune donnée ne concerne le rôle de WIF1 dans le processus myogénique. Par un apport exogène de WIF1, nous avons montré l’augmentation de la prolifération et l’altération de la différenciation myoblastique des C2C12. Lors de la prolifération, une augmentation de l’expression de Myf5 et une diminution de MyoG sont observées. A 7 jours de différenciation, les myotubes Po - ont un diamètre plus petit que les myotubes sauvages et ils sont plus nombreux à avoir un faible nombre de noyaux, traduisant des défauts de fusion. Nous démontrons pour la première fois, l’implication de la protéine WIF1 dans le processus myogénique. Récemment, POFUT1 a aussi été proposé comme nouveau biomarqueur pour certains cancers, mais pas évalué dans le cancer colorectal (CCR). Nous avons donc collecté des données issues de 626 tumeurs et 51 tissus adjacents non tumoraux disponibles dans FireBrowse, celles de lignées cellulaires cancéreuses colorectales et de prélèvements tumoraux provenant du Centre de Ressources Biologiques du CHU de Limoges. Une surexpression de POFUT1 est observée dès le stade I, majoritairement due à une amplification de la région 20q11.21. Elle est significativement associée aux adénocarcinomes non mucineux et à une localisation rectale. De plus, l’expression de POFUT1 est corrélée à celles des récepteurs NOTCH ainsi qu’au processus métastatique, probablement par activation de la voie NOTCH. A ce titre, POFUT1 pourrait être considéré comme un nouveau biomarqueur pour le diagnostic du CCR. / The ER protein O-fucosyltransferase 1 (POFUT1), whose gene is located at the 20q11.21 chromosomic region in humans, catalyzes O-linked fucose addition to serine or threonine present in the consensus sequence (C2X4S/TC3) carried by EGF-like domain of membrane or secreted glycoprotein. Pofut1 knockdown (Po -) in murine myoblast C2C12 cell line leads to formation of elongated and thin myotubes, with a low number of nuclei and to downexpression of the late myogenic marker Myf6, suggesting significant defects in secondary fusion. NFATc2/IL-4 signaling is described as the main pathway associated to this step. We showed that the slightest expression of Nfatc2 in Po - myotubes is correlated with a decrease in IL-4 secretion and a lower quantity of IL 4Rα in reserve cells, which had to fuse with nascent myotubes. IL-4Rα neutralization on wild-type C2C12 causes myonuclear accretion defects, similar to those observed in Po -. Then, POFUT1 could be a new mediator of myotube growth during myogenic process, particularly through NFATc2/IL-4 signaling. The glycoprotein WIF1, potential POFUT1 target, is an antagonist of WNT signaling via its binding to WNT proteins. This pathway is involved in proliferation and differentiation of myoblasts. However, no data are available on WIF1 role in the myogenic process. Through exogenous WIF1 treatment, we showed a proliferation increase and a myoblast differentiation impairment in C2C12. During proliferation, increase in Myf5 and decrease in MyoG expressions are observed. At 7 days of differentiation, Po - myotubes have a smaller diameter than wild-type ones and are more numerous to have a small number of nuclei, reflecting fusion defects. For the first time, we demonstrate the involvement of WIF1 in the myogenic process. Recently, POFUT1 was proposed to be a new biomarker for several cancers, but not evaluated in colorectal cancer (CRC). We used data from 626 tumors and 51 adjacent non-tumor tissues available at FireBrowse, colorectal cancer cell lines and tumor samples from the Biological Resource Centre of Limoges hospital. A POFUT1 overexpression is observed from stage I, mainly due to amplification of 20q11.21 region. It is significantly associated to non-mucinous adenocarcinoma and to rectum location. Moreover, POFUT1 expression is correlated with those of NOTCH receptors as well as the metastatic process, probably by activation of the NOTCH pathway. POFUT1 could therefore be considered as a new biomarker for CRC diagnosis.

Ce travail a pour but de décrire les relations réciproques entre prise de perspective et informations vestibulaires. Une étude chez des patients avec un déficit vestibulaire bilatéral ancien et des sujets contrôles a montré que l’ancrage du soi sur le corps et la simulation implicite de la perspective visuo-spatiale d’autrui étaient similaires chez les deux groupes. Ainsi, une perte vestibulaire ancienne n’entraînerait pas de conflits multisensoriels, connus pour évoquer un sentiment de perspective désincarnée chez des patients avec des déficits vestibulaires aigus. Une étude chez des volontaires sains a combiné des stimulations vestibulaires naturelles sur fauteuil rotatoire à des tâches de prise de perspective dans un environnement virtuel embarqué. Les temps de prise de perspective étaient modulés en fonction de la direction de la rotation. Cette influence n’était pas présente pour la rotation mentale d’objets 3D. La contribution vestibulaire canalaire modulerait donc spécifiquement les rotations mentales du point de vue. Enfin, les modulations cognitives du traitement des informations vestibulaires ont été analysées par l’enregistrement de potentiels évoqués myogéniques vestibulaires sur les muscles sternocléidomastoïdiens et trapèzes. L’amplitude des potentiels évoqués était significativement modulée par l’angle séparant le point de vue du participant et celui d’un avatar distant. Nos travaux théoriques et les résultats de cette série d’expériences démontrent la contribution des informations vestibulaires à la prise de perspective visuo-spatiale. / This thesis aims at describing the reciprocal relations between perspective taking and the vestibular system. A study in patients with bilateral vestibular deficits and controls showed that the anchoring of the self to the body and implicit visuo-spatial perspective taking were similar in both groups. Our negative findings offer insight into the multisensory mechanisms of embodiment: only acute peripheral vestibular disorders and neurological disorders in vestibular brain areas may evoke disembodied experiences. A second study, combined natural vestibular stimulation on a rotatory chair with virtual reality to test how vestibular signals are processed to simulate the view point of a distant avatar. While they were rotated, participants tossed a ball to a virtual character from the view point of a distant avatar. Our results showed that participants were faster when their physical body rotated in the same direction as the mental rotation needed to take the avatar's viewpoint. Altogether, these data indicate that vestibular signals have a direction-specific influence on visuo-spatial perspective taking, but not a general effect on mental imagery. Finally, cognitive modulations of vestibular information processing were analyzed by recording vestibular-evoked myogenic potentials on the sternocleidomastoid and trapeze muscles. The amplitude of evoked potentials was significantly modulated by the angle separating the participant’s viewpoint to that of a distant avatar. To conclude, our theoretical work, together with results from this series of experiments, demonstrate the contribution of vestibular information to visuo-spatial perspective taking.

Chronic kidney disease (CKD) is defined as glomerular filtration rate (GFR) less than 60 mL/min/1.73 m2 for 3 months and is characterized by progressive loss of renal function. The second leading cause of CKD is hypertension. More than half of CKD patients also suffer from hypertension. Arteries and arterioles adjust to the fluctuations in the systematic blood pressure through a mechanism called autoregulation. In the kidneys, autoregulation protects the delicate glomeruli capillaries from high blood pressure and occurs through myogenic constriction (MC). MC refers to contraction of arterioles in response to an increase in the blood pressure. Chronically hypertensive individuals and animal models have an enhanced MC, leading to minimal renal injury despite their elevated blood pressure. Experimental and clinical evidence point to a role for the MC in the pathogenesis of the CKD, however, the mechanism through which preglomerular arterial MC contributes to CKD has not been fully elucidated. This thesis showed that augmented MC in chronically hypertensive animal models was due to increased thromboxane A2 prostaglandin that was not released from the endothelium (Chapter 2). Nevertheless, inhibiting MC while also reducing the blood pressure prevented salt-induced renal injury even though the blood pressure was still not normalized compared to the normotensive controls (Chapter 3). The resulting improvement in renal structure and function could be attributed to the reduction in the blood pressure, albumin, and uromodulin (UMOD) excretion (Chapter 3). UMOD is a kidney-specific glycoprotein that, based on a genome-wide association study have the strongest association to CKD (Chapter 3). Comparing two CKD hypertensive animal models further revealed that CKD progression was independent of the blood pressure and strongly associated with UMOD excretion levels (Chapter 4). Collectively, the data discussed in this thesis demonstrates potential therapeutic targets in CKD hypertensive animal models. / Dissertation / Doctor of Philosophy (PhD)

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L’instabilité posturale est fréquente chez les séniors et peut entrainer la chute. La chute chez les séniors est un problème majeur de santé publique. Les chiffres épidémiologiques sont éloquents : une personne sur trois âgées de plus de 70 ans fera une chute dans l’année. Les causes sont multifactorielles : ostéo-articulaire, visuelle, cognitive, vestibulaire…. Dans cette étude, nous nous sommes intéressés à l’évolution de la fonction des récepteurs vestibulaires périphériques avec l’âge et à la perception de rotation à partir des entrées canalaires horizontales (système vestibulaire central et projections vestibulaires corticales). Notre but est d’essayer de comprendre l’implication du vieillissement du système vestibulaire dans l’instabilité posturale des séniors. Au niveau périphérique, nous avons quantifié la fonction des canaux semi-circulaires horizontaux par le test calorique et le vidéo-head impulse test. La fonction des récepteurs otolithiques (utriculaire et sacculaire) a été évaluée par les potentiels évoqués myogéniques recueillis au niveau cervical (voies sacculo-spinales) et oculaire (voies utriculo-oculaires). Au niveau central, la perception de l’entrée vestibulaire canalaire horizontale a été appréciée après irrigation à l’eau chaude du conduit auditif externe en appliquant un score de perception (présence ou absence de sensation rotatoire). Finalement, l’équilibre a été quantifié grâce au test d’organisation sensorielle sur l’Equitest et grâce à un système que nous avons récemment mis au point en collaboration avec le Professeur Curthoys à Sydney, comprenant une Wii Balance Board, un tapis mousse et un masque de réalité virtuelle (Oculus Rift). Les résultats ont montré une diminution des réponses oculaires au test calorique après 70 ans mais une absence de baisse du gain du réflexe vestibulo-oculaire horizontal au vidéo-head impulse test. La fonction otolithique, sacculaire et utriculaire, est altérée avec l’âge quelle que soit la stimulation utilisée (aérienne ou osseuse). La perception de l’entrée vestibulaire canalaire horizontale induite par une stimulation calorique nous a permis de montrer pour la première fois que certains séniors ne percevaient pas la sensation de rotation malgré une réponse oculaire normale (vitesse maximale de la phase lente du nystagmus oculaire supérieure à 15°/s). Dans notre population, nous avons pu ainsi définir deux types de séniors : un groupe présentant une perception de vertige rotatoire et un groupe « négligeant » ne pouvant pas reconstruire une sensation rotatoire à partir des entrées vestibulaires canalaires horizontales. La comparaison de ces deux groupes de séniors appariés sur l’âge ne montre aucune différence de la fonction canalaire horizontale ni de la fonction otolithique sacculaire et utriculaire. Néanmoins, les séniors négligents présentent en majorité des performances anormales (chute ou score diminué) à l’Equitest notamment en conditions 5 et 6. De plus, leur score au DHI est plus élevé relevant ainsi le handicape ressenti par ces séniors à cause de leur instabilité. En conclusion, les troubles de l’équilibre chez certains seniors pourraient résulter en partie d’une dysfonction vestibulaire centrale. Des études ultérieures permettront de déterminer si l’augmentation du seuil de perception rotatoire est un bon facteur prédictif du risque de chute. / Postural instability is common in seniors and can lead to falls which seniors are a major problem for Public Health. Epidemiological studies clearly show the magnitude of this problem: one in three people aged than more 70 years will fall in a year. This is caused by multiple factors including: musculoskeletal, visual, cognition, vestibular… The present study concerns the effect of age on the vestibular peripheral receptors function and on the perception of rotation from horizontal canal inputs (central vestibular processing and vestibular cortical projection). The aim is to try to understand the vestibular mechanisms involved in postural instability and mobility with age. At the peripheral level, the horizontal canal function was assessed using caloric test and video-Head Impulse Test. Otolith function (saccular and utricular) was assessed using vestibular evoked myogenic potentials recorded at cervical level (sacculo-spinal pathways) and at ocular level (utriculo-ocular pathways). At the central level, perception of motion from vestibular horizontal canal inputs was studied after caloric stimulation with warm water using a subjective perceptual score (presence or absence of rotatory vertigo). Finally, postural equilibrium was assessed with the Sensory Organization Test on the Equitest machine and also with a new system developed in collaboration with Prof. Curthoys (Sydney) using a Wii Balance Board, a foam rubber pad and a virtual reality headset (Oculus Rift DK2). Results showed decreased ocular responses induced by caloric stimulation after 70 years of age but healthy horizontal gain of the vestibulo-ocular reflex assessed by video-head impulse testing. The otolithic (saccular and utricular) function is impaired with age for all the stimuli used (air or bone conducted). Perception of motion induced by caloric stimulation (vestibular horizontal canal inputs) allowed us to show for the first time that some seniors are unable to feel the induced rotatory vertigo even with normal ocular responses (peak of the slow phase eye velocity higher than 15°/s). We defined two types of seniors: one senior group having a normal feeling of vertigo and one senior ‘neglect’ group who did not feel any sensation of rotation from horizontal canal inputs. The comparison of these two age-matched groups showed no difference in horizontal canal function, or otolithic function. The majority of the ‘neglect’ seniors with an absence of perception exhibited falls or a decreased score in conditions 5 and 6 during the Equitest. Moreover, their DHI scores were higher, showing the handicap induced by postural instability in these seniors. In conclusion, postural instability and falls in seniors may result from central vestibular impairment (inadequate central processing). A prospective study is needed to determine whether the increase perceptual threshold of rotation could be a good predictor of fall risk in seniors.

Skeletal muscle is a complicated and heterogeneous striated muscle tissue that serves critical mechanical and metabolic functions in the organism. The process of generating skeletal muscle, myogenesis, is elaborately coordinated by members of the protein kinase family, which transmit diverse signals initiated by extracellular stimuli to myogenic transcriptional hierarchy in muscle cells. Mitogen-activated protein kinases (MAPKs) including p38 MAPK, c-Jun N terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) are components of serine/threonine protein kinase cascades that play important roles in skeletal muscle differentiation. The exploration of MAPK upstream kinases identified mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4), a serine/threonine protein kinase that modulates p38 MAPK, JNK and ERK activities in multiple cell lines. Our lab further discovered that Map4k4 regulates peroxisome proliferator-activated receptor γ (PPARγ) translation in cultured adipocytes through inactivating mammalian target of rapamycin (mTOR), which controls skeletal muscle differentiation and hypotrophy in kinase-dependent and -independent manners. These findings suggest potential involvement of Map4k4 in skeletal myogenesis. Therefore, for the first part of my thesis, I characterize the role of Map4k4 in skeletal muscle differentiation in cultured muscle cells. Here I show that Map4k4 functions as a myogenic suppressor mainly at the early stage of skeletal myogenesis with a moderate effect on myoblast fusion during late-stage muscle differentiation. In agreement, Map4k4 expression and protein kinase activity are declined with myogenic differentiation. The inhibitory effect of Map4k4 on skeletal myogenesis requires its kinase activity. Surprisingly, none of the identified Map4k4 downstream effectors including p38 MAPK, JNK and ERK is involved in the Map4k4-mediated myogenic differentiation. Instead, expression of myogenic regulatory factor Myf5, a positive mediator of skeletal muscle differentiation is transiently regulated by Map4k4 to partially control skeletal myogenesis. Mechanisms by which Map4k4 modulates Myf5 amount have yet to be determined. In the second part of my thesis, I assess the relationship between Map4k4 and IGF-mediated signaling pathways. Although siRNA-mediated silencing of Map4k4 results in markedly enhanced myotube formation that is identical to the IGF-induced muscle hypertrophic phenotype, and Map4k4 regulates IGF/Akt signaling downstream effector mTOR in cultured adipocytes, Map4k4 appears not to be involved in the IGF-mediated ERK1/2 signaling axis and the IGF-mediated Akt signaling axis in C2C12 myoblasts. Furthermore, Map4k4 does not affect endogenous Akt signaling or mTOR activity during C2C12 myogenic differentiation. The results presented here not only identify Map4k4 as a novel suppressor of skeletal muscle differentiation, but also add to our knowledge of Map4k4 action on multiple signaling pathways in muscle cells during skeletal myogenesis. The effects that Map4k4 exerts on myoblast differentiation, fusion and Myf5 expression implicate Map4k4 as a potential drug target for muscle mass growth, skeletal muscle regeneration and muscular dystrophy.