Conséquences fonctionnelles de mutations affectant le récepteur de la vasopressine de type 2 et implications thérapeutiques

Le récepteur de la vasopressine de type 2 (V2R) joue un rôle crucial dans l’homéostasie hydrique. Exprimé principalement au niveau du rein, son activation par l’hormone antidiurétique arginine-vasopressine (AVP) favorise la réabsorption d’eau, participant ainsi à diminuer la diurèse. Plus de 200 mutations dans le gène du V2R ont été associées au diabète néphrogénique insipide congénital (DINc), une maladie causée par une perte de fonction du récepteur. À l’opposé, trois mutations découvertes récemment induisent un gain de fonction du V2R, et sont la cause du syndrome néphrogénique de l’anti-diurèse inappropriée (NSIAD). Les travaux de cette thèse visent à mieux comprendre les bases moléculaires responsables de la perte ou du gain de fonction des récepteurs mutants associés à ces deux maladies. Dans plus de 50% des cas, les mutations faux-sens affectent négativement l’adoption d’une conformation native par le V2R, provoquant la reconnaissance et la rétention intracellulaire des mutants par le système de contrôle de qualité du réticulum endoplasmique. Nos résultats ont démontré que l’interaction entre les récepteurs mutants et le chaperon moléculaire calnexine est dépendante de N-glycosylation et que sa durée varie en fonction de la mutation. De plus, l’importance de cette modification co-traductionnelle et des interactions lectines-sucres dans le processus de maturation d’un mutant donné s’est avérée une caractéristique intrinsèque, puisque l’absence de N-glycosylation n’a pas affecté le mutant Y128S (phénotype léger) tandis que la maturation du mutant W164S (phénotype sévère) a été totalement abolie. Nos résultats suggèrent aussi que l’action des chaperons pharmacologiques (CP), molécules favorisant la maturation des mutants du V2R, peut survenir à différentes étapes au cours du processus de maturation, selon le mutant réchappé. Ces différences entre muta nts suggèrent des processus biosynthétiques ‘personnalisés’ dictés par la nature de la mutation impliquée et pourraient expliquer la différence de sévérité des manifestations cliniques chez les patients porteurs de ces mutations. Bien qu’une récupération de fonction ait été obtenue pour les mutants Y128S et W164S par un traitement au CP, il n’en est pas de même pour toutes les mutations occasionnant un défaut conformationnel. C’est ce que nous avons démontré pour le mutant V88M, affligé de deux défauts, soit une faible efficacité de maturation combinée à une basse affinité pour l’AVP. Dans ce cas, et malgré une augmentation du nombre de récepteurs mutants la surface cellulaire, la diminution de l’affinité apparente du récepteur mutant pour l’AVP a été exacerbée par la présence résiduelle de CP à son site de liaison, rendant impossible l’activation du récepteur aux concentrations physiologiques d’AVP. Les mutants R137C et R137L ont une activité constitutive élevée et mènent au NSIAD tandis que la substitution de cette même arginine par une histidine (R137H) mène au DINc. Ces trois mutants se sont avéré partager plusieurs caractéristiques, dont une efficacité de maturation réduite et une désensibilisation spontanée élevée. La seule différence iden tifiée entre ces mutants est leur niveau d’activité constitutive. Le CP utilisé dans nos études possède aussi la propriété d’agoniste inverse, mais n’a pourtant pas diminué l’activité constitutive des mutants R137C/L, suggérant une conformation active ‘figée’. Seul l’effet chaperon a été observé, entraînant la hausse de récepteurs à la surface cellulaire, qui se traduit par une augmentation de la production de second messager. Nous avons par contre suggéré l’utilisation d’AVP puisqu’il favorise l’endocytose des récepteurs R137/L sans promouvoir leur activation, diminuant ainsi le nombre de récepteurs actifs à la surface cellulaire. Nous avons identifié la première mutation occasionnant un gain de fonction du V2R qui n’implique pas l’arginine 137. Le mutant F229V a une activité constitutive élevée et, contrairement aux R137C et R137L, il n’est pas sujet à une désensibilisation spontanée accrue. L’observation que des agonistes inverses sont aptes à inhiber l’activité constitutive de ce nouveau mutant est une découverte importante puisque l’insuccès obtenu avec les mutations précédentes suggérait que ces molécules n’étaient pas utiles pour le traitement du NSIAD. Considérés globalement, ces travaux illustrent le caractère particulier des formes mutantes du V2R et l’importance de bien cerner les conséquences fonctionnelles des mutations afin d’apporter aux patients atteints de DINc ou NSIAD une thérapie personnalisée, et de développer de nouveaux agents thérapeutiques adaptés aux besoins.The vasopressin type 2 receptor (V2R) plays an important role in water homeostasis. Mainly expressed in the collecting ducts of the kidney, V2R activation by the antidiuretic hormone arginine-vasopressin (AVP) leads to water reabsorption, resulting in a decrease urine output. More than 200 mutations in the V2R gene have been link to the aetiology of the congenital form of nephrogenic diabetes insipidus (cNDI), resulting from a receptor loss-of-function. In contrast, three recently identified mutations have been shown to cause a gain-of-function of the V2R leading to the nephrogenic syndrome of inappropriate antidiuresis (NSIAD). The work presented herein is focussed on a better understanding of the molecular determinants leading to the loss- or gain -of-function of V2R mutants. More than 50% of missense mutations affecting the V2R were shown to hamper the receptor’s ability to adopt its native conformation and to cause its intracellular retention by the endoplasmic reticulum quality control system. We thus looked at the role of N-glycosylation and calnexin (Cnx) in the maturation process of mutant V2R, and their importance for receptor rescue by pharmacological chaperones (PC). Our results have shown that N-glycosylation is required for Cnx binding to the receptors and that the duration of this interaction is correlated to the severity of the misfolded state of the mutant. The importance of N-glycosylation and to sugar-mediated interactions in the maturation process of a given V2R mutant was found to be an intrinsic property, as it had no significant repercussion on the mild phenotype-associated Y128S mutant, while it completely abolished maturation of the W164S mutant, associated with a severe phenotype. Moreover, we have shown that pharmacological chaperoning can occur at different steps during the maturation process, according to the mutant studied. These mutant-specific differences indicate that the biosynthetic processing of mutant V2R is highly influenced by the nature of the mutation itself and could partially explain the variations in the clinical outcome severity among NDI-causing mutant V2Rs. Although a functionality rescue of W164S and Y128S mutants was obtained upon exposure to PC, it is not the case for all V2R mutants with a maturation defect. The V88M-V2R was found affected both in its maturation and its affinity toward AVP. In this case, and despite a significant increase in maturation and cell surface expression, the PC treatment led to a further loss in the receptor’s affinity for AVP, preventing its activation at physiological AVP concentrations. The R137C and R137L mutants are endowed with a high constitutive activity leading to NSIAD. Stunningly, substitution of this arginine by histidine (R137H) was associated with cNDI. These three mutant V2R were found to share many characteristics, of which a compromised maturation and elevated spontaneous desensitization. The only difference between these mutants relies on their constitutive activity levels. The PC used in our studies is also an inverse agonist, but failed to reduce the constitutive activity of the R137C/L mutants, entailing a ‘locked’ active conformation. Instead, the chaperoning property of the compound led to an increase in the number of constitutively active receptor at the cell surface. We have thus proposed the use of AVP as a treatment, as it was shown to cause receptor’s endocytosis without promoting their activation, leading to a reduced active receptor number at the cell surface. We have identified a new gain-of-function mutation affecting the V2R, the first not involving arginine 137. The F229V substitution was shown to confer high constitutive activity to the receptor, but unlike the two other NSIAD-causing mutants, it does not undergo elevated spontaneous desensitization. The observation that inverse agonists are efficient at inhibiting the constitutive activity of the F229V mutant is an important discovery since the unfruitful attempts obtained with the other constitutively active mutants led some investigators to the erroneous conclusion that inverse agonists were not useful for the treatment of NSIAD. Taken together, these findings underline the ‘individuality’ of V2R mutants and the importance of their functional characterization in order to bring personalized therapeutic strategies for patients with cNDI or NSIAD, and to develop new therapeutics adapted to the patients’ needs

Séparation d'un mélange sous-déterminé de sources par simulation stochastique

Nous proposons dans cet article un algorithme de Gibbs pour l'identification, la séparation et la déconvolution d'un mélange mono-capteur de sources i.i.d. mutuellement indépendantes régies par une loi de Bernoulli-Gauss, et filtrées linéairement. Il s'agit d'une étude préparatoire, l'objectif à moyen-terme étant l'analyse des signaux électromyographiques pour le diagnostic des pathologies neuro-musculaires. L'extension au cas multi-capteur (mélange convolutif) est immédiate

estimation du couple articulaire pendant un mouvement de squat

International audienceA repetitive movement over a long period of time, habituation to poor posture, handling heavy load can lead to musculoskeletal disorders. The most important thing is to prevent MSDs by automating repetitive tasks. But it is also necessary to help a patient with rehabilitation when an MDS has appeared. Regarding the human musculoskeletal system of movement analyses, joint torques developed by muscles are useful to prevent or treat MSDs. The knowledge of these torques is important to evaluate the possibilities of assisting a joint with an orthosis for instance. In this study, we analyze a specific half squat motion considered as a planar movement. Joint torques are estimated with two models -a 3D Opensim and a sagittal Matlab model. Torque variations from the two models are consistent even if the amplitudes differ. This result is attributable to the different anthropometric tables which the two models are based on. Another strategy developed here consists in estimating joint torques without the measured ground reaction forces. In that case, global vertical reaction force is well estimated. The use of the sagittal Matlab model is a simple and efficient way to preliminarily analyze squat trajectories.Un mouvement répété sur une longue période, l'accoutumance à une mauvaise posture, la manipulation de charges lourdes peuvent entraîner des troubles musculosquelettiques (TMS). Il est important de prévenir les TMS en automatisant les tâches répétitives. Mais il est également nécessaire d'aider un patient en phase de rééducation lorsqu'une TMS apparait. En ce qui concerne le système musculosquelettique humain, les couples articulaires développés par les muscles sont utiles pour prévenir ou traiter les TMS. La connaissance de ces couples est importante pour évaluer les possibilités d'assister une articulation avec une orthèse par exemple. Dans cette étude, nous analysons un mouvement de demi-squat spécifique considéré comme un mouvement planaire. Les couples articulaires sont estimés à l'aide de deux modèles: un modèle 3D sur Opensim et un modèle sagittal sur Matlab. Les variations de couple issues des deux modèles sont cohérentes même si les amplitudes peuvent différer. Ce résultat s'explique par le fait que les modèles se basent sur des tables anthropométriques différentes. Une autre stratégie développée ici consiste à estimer les couples articulaires sans utiliser la mesure des forces de réaction du sol sur les pieds. Dans ce cas, la force de réaction verticale totale est bien estimée. L'utilisation du modèle sagittal Matlab est un moyen simple et efficace d'analyser préalablement les trajectoires de squat

Link travel time estimation in urban areas by detectors and probe vehicles fusion

International audienceThis paper presents an approach to estimate link travel time in urban areas. This approach consists of a data fusion from underground loop detectors and probe vehicles equipped with global positioning system (GPS). This method is expected to be more accurate, reliable and robust than using either of these data sources alone. In this approach, an algorithm is developed. This algorithm is based on the unscented Kalman filter using vehicle counts and flows from loop detectors located at the end of every link, and travel time from probe vehicles. From these counts the average travel time is calculated using the "cumulative plot" method. Furthermore, in order to incorporate the GPS data, a map-matching method is used to associate a travel time to the appropriate link

Practical identification of a glucose-insulin dynamics model

Glycemia regulation algorithms which are designed to be implemented in several artificial pancreas projects are often model based control algorithms. However, actual diabetes monitoring is based throughout the world on the so-called Flexible Insulin Therapy (FIT) which does not always cope with current mathematical models. In this paper, we initiate an identification methodology of those FIT parameters from some standard ambulatory clinical data. This issue has an interest per se, or for a further use in any closed-loop regulation system

Feeding ecology of Liza spp. in a tidal flat: Evidence of the importance of primary production (biofilm) and associated meiofauna

International audienceGrey mullets are unique among temperate-region fish species in their ability to feed on mudflat biofilm. In this study, we examined mullet feeding strategies on biofilm and associated meiofauna by using a diet study and stable isotope analysis to explore functional interactions between mullets and tidal flats. A stomach vacuity investigation showed that mullets did not import any materials from subtidal areas into the mudflat but exported mud, biofilm, and associated meiofauna. The results of mullet stomach content and fecal analyses, when compared to the availability of tidal flat resources, showed evidence of mullets' ability to ingest and assimilate biofilm and to concentrate major meiofauna grazers such as nematodes, copepods and, secondarily, foraminifers and ostracods. Isotopic ratios confirmed diet investigations, and as recently shown in salt marsh habitats, mullets exhibited an intermediate trophic position, supporting the hypothesis that they can assimilate both biofilm and major meiofauna grazers. The function of the tidal flat as a feeding habitat for gray mullets and the role of mullets as the main export pathway of biofilm from tidal flat ecosystems are discussed

Reconstruction du spectre directionnel de la houle

- Cette étude consiste en l'élaboration d'une procédure d'estimation du spectre directionnel de la houle, à partir des mesures de mouvement (tangage, roulis, lacet, pilonnement, embardée, cavalement) d'un navire se déplaçant en translation rectiligne uniforme. La connaissance de cet état de mer absolu doit permettre d'élaborer des lois de commande adaptatives pour la tranquillisation du navire, ainsi que d'utiliser celui-ci comme bouée de mesures océanographiques. Cependant, cette étude est menée dans le contexte de la boucle ouverte, c'est-à-dire que l'information obtenue sur l'état de la mer n'est pas utilisée en vue de la tranquillisation du navire. On propose une modélisation du système à l'aide d'une représentation dans l'espace d'état, suivie d'une procédure d'identification des paramètres de spectres directionnels paramétriques

A comprehensive protocol for chemical analysis of flame combustion emissions by secondary ion mass spectrometry

International audienceRATIONALE. Time of flight secondary ion mass spectrometry (ToF-SIMS) is used to provide detailed information on the surface chemical composition of soot. An analytical protocol is proposed and tested on a laboratory flame, and the results are compared with our previous measurements provided by two-step laser mass spectrometry (L2MS). METHODS. This work details: (1) the development of a dedicated apparatus to sample combustion products from atmospheric flames and deposit them on substrates suitable for ToF-SIMS analysis; (2) the choice of the deposition substrate and the material of the sampling line, and their effect on the mass spectra; (3) a method to separate the contributions of soot and condensable gas based on impact deposition, and finally (4) the post-acquisition data processing. RESULTS. Compounds produced during flame combustion are detected on the surface of different deposition substrates and attributed a molecular formula based on mass defect analysis. Silicon and titanium wafers perform similarly, while the surface roughness of glass microfiber filters results in a reduced mass resolution. The mass spectra obtained from the analysis of different locations of the deposits obtained by impaction show characteristic patterns that are attributed to soot/condensable gas. CONCLUSIONS. A working method for the analysis of soot samples and the extraction of useful data from mass spectra is proposed. This protocol should help avoiding common experimental issues like sample contamination, while optimizing the setup performance by maximizing the achievable mass resolution

Identification and Characterization of an Activating F229V Substitution in the V2 Vasopressin Receptor in an Infant with NSIAD

Gain-of-function mutations in the gene encoding the V2 vasopressin receptor (V2R) cause nephrogenic syndrome of inappropriate antidiuresis. To date, reported mutations lead to the substitution of arginine 137 by either a cysteine or leucine (R137C/L). Here, we describe a 3-month-old hyponatremic infant found to have a phenylalanine 229 to valine (F229V) substitution in V2R. Characterization of this substitution in vitro revealed that it leads to high constitutive activity of the receptor, compatible with spontaneous antidiuresis. In contrast to R137C/L mutant receptors, F229V receptors do not undergo spontaneous desensitization, which results in sustained, high basal activity. Notably, the V2R-selective inverse agonists tolvaptan and satavaptan completely silenced the constitutive signaling activity of the F229V mutant receptor, indicating that this substitution does not lock the receptor in an irreversible active state. Thus, inverse agonists might prove to be effective therapies for treating patients with this or other spontaneously activating mutations that do not lock the V2R in its active state. These results emphasize the importance of genetic testing and the functional characterization of mutant receptors for patients with nephrogenic syndrome of inappropriate antidiuresis because the results might inform treatment decisions

The use of DNA barcoding to monitor the marine mammal biodiversity along the French Atlantic coast

International audienceIn the last ten years, 14 species of cetaceans and five species of pinnipeds stranded along the Atlantic coast of Brittany in the North West of France. All species included, an average of 150 animals strand each year in this area. Based on reports from the stranding network operating along this coast, the most common stranding events comprise six cetacean species (Delphinus delphis, Tursiops truncatus, Stenella coeruleoalba, Globicephala melas, Grampus griseus, Phocoena phocoena) and one pinniped species (Halichoerus grypus). Rare stranding events include deep-diving or exotic species, such as arctic seals. In this study, our aim was to determine the potential contribution of DNA barcoding to the monitoring of marine mammal biodiversity as performed by the stranding network. We sequenced more than 500 bp of the 5' end of the mitochondrial cox1 gene of 89 animals of 15 different species (12 cetaceans, and three pinnipeds). Except for members of the Delphininae, all species were unambiguously discriminated on the basis of their cox1 sequences. We then applied DNA barcoding to identify some "undetermined" samples. With again the exception of the Delphininae, this was successful using the BOLD identification engine. For samples of the Delphininae, we sequenced a portion of the mitochondrial control region (MCR), and using a non-metric multidimentional scaling plot and posterior probability calculations we were able to determine putatively each species. We then showed, in the case of the harbour porpoise, that cox1 polymorphisms, although being lower than MCR ones, could also be used to assess intraspecific variability. All these results show that the use of DNA barcoding in conjunction with a stranding network could clearly increase the accuracy of the monitoring of marine mammal biodiversity