Characterization of the molecular mechanisms of insulin exocytosis and of their adaptation to physiopathological conditions

Résumé large public Le glucose est une source d'énergie essentielle pour notre organisme, indispensable pour le bon fonctionnement des cellules de notre corps. Les cellules β du pancréas sont chargées de réguler l'utilisation du glucose et de maintenir la glycémie (taux de glucose dans le sang) à un niveau constant. Lorsque la glycémie augmente, ces dernières sécrètent l'insuline, une hormone favorisant l'absorption, l'utilisation et le stockage du glucose. Une sécrétion insuffisante d'insuline provoque une élévation anormale du taux de glucose dans le sang (hyperglycémie) et peut mener au développement du diabète sucré. L'insuline est sécrétée dans le sang par un mécanisme particulier appelé exocytose. Une meilleure compréhension de ce mécanisme est nécessaire dans l'espoir de trouver des nouvelles thérapies pour traiter les 170 millions de personnes atteintes de diabète sucré à travers le monde. L'implication de diverses protéines, comme les SNAREs ou Rabs a déjà été démontrée. Cependant leurs mécanismes d'action restent, à ce jour, peu compris. De plus, l'adaptation de la machinerie d'exocytose à des conditions physiopathologiques, comme l'hyperglycémie, est encore à élucider. Le but de mon travail de thèse a été de clarifier le rôle de deux protéines, Noc2 et Tomosyn, dans l'exocytose ; puis de déterminer les effets d'une exposition prolongée à un taux élevé de glucose sur l'ensemble des protéines de la machinerie d'exocytose. Noc2 est un partenaire potentiel de deux Rabs connues pour leur implication dans les dernières étapes de l'exocytose, Rab3 et Rab27. Grâce à l'étude de différents mutants de Noc2, j'ai montré que l'interaction avec Rab27 permet à la protéine de s'associer avec les organelles de la cellule β contenant l'insuline. De plus, en diminuant sélectivement l'expression de Noc2, j'ai déterminé l'importance de cette protéine pour le bon fonctionnement du processus d'exocytose et le relâchement de l'insuline. Quant à Tomosyn, une protéine interagissant avec les protéines SNAREs, j'ai démontré son importance dans la sécrétion d'insuline en diminuant de manière sélective son expression dans les cellules β. Ensuite, grâce à une combinaison d'approches moléculaires et de microscopie, j'ai mis en évidence le rôle de Tomosyn dans les dernières étapes de l'exocytose. Enfin, puisque la sécrétion d'insuline est diminuée lors d'une hyperglycémie prolongée, j'ai analysé l'adaptation de la machinerie d'exocytose à ces conditions. Ceci m'a permis de découvrir que l'expression de quatre protéines essentielles pour le processus d'exocytose, Noc2, Rab3, Rab27 et Granuphilin, est fortement diminuée lors d'une hyperglycémie chronique. L'ensemble de ces données met en évidence l'importance de Noc2 et Tomosyn dans la sécrétion d'insuline. L'inhibition, par un taux élevé de glucose, de l'expression de Noc2 et d'autres protéines indispensables pour l'exocytose suggère que ce phénomène pourrait contribuer au développement du diabète sucré. Résumé L'exocytose d'insuline, en réponse au glucose circulant dans le sang, est la fonction principale de la cellule β. Celle-ci permet de stabiliser le taux de glucose sanguin (glycémie). Le diabète de type 2 est caractérisé par une glycémie élevée due, principalement, à un défaut de sécrétion d'insuline en réponse au glucose. La compréhension des mécanismes qui contrôlent l'exocytose d'insuline est essentielle pour clarifier les causes du diabète sucré. Plusieurs composants impliqués dans ce processus ont été identifiés. Ceux-ci incluent les SNAREs Syntaxin-1, VAMP2 et SNAP25 et les GTPases Rab3 et Rab27 qui jouent un rôle dans les dernières étapes de l'exocytose. Pendant mon travail de thèse, j'ai étudié le rôle de Noc2, un des partenaires de Rab3 et Rab27, dans l'exocytose d'insuline. Nous avons déterminé que Noc2 s'associe aux granules de sécrétion d'insuline grâce à son interaction avec Rab27. La diminution de l'expression de Noc2 dans la lignée cellulaire β INS-1E, par ARN interférence, influence négativement la sécrétion d'insuline stimulée par différents sécrétagogues et prouve que cette protéine Noc2 est essentielle pour l'exocytose d'insuline. L'interaction avec Munc13, une protéine impliquée dans l'arrimage des vésicules, suggère que Noc2 participe au recrutement des granules d'insuline à la membrane plasmique. Ensuite, j'ai analysé l'adaptation de la machinerie d'exocytose à des concentrations supraphysiologiques de glucose. Le niveau d'expression de Rab3 et Rab27 et de leurs effecteurs Granuphilin/S1p4 et Noc2 est fortement diminué par une exposition prolongée des cellules β à haut glucose. L'effet observé est en relation avec l'induction de l'expression de ICER, un facteur de transcription surexprimé dans des conditions d'hyperglycémie et également dans des modèles génétiques de diabète de type 2. La surexpression de ICER dans des cellules INS-1E diminue l'expression de Rab3, Rab27, Granuphilin/Slp4 et Noc2 et par conséquent l'exocytose d'insuline. Ainsi, l'induction de ICER, après une exposition prolongée à haut glucose, régule négativement l'expression de protéines essentielles pour l'exocytose et altère la sécrétion d'insuline. Ce mécanisme pourrait contribuer au dysfonctionnement de l'exocytose d'insuline dans le diabète de type 2. Dans la dernière partie de ma thèse, j'ai investigué le rôle de la protéine Tomosyn-1 dans la formation du complexe SNARE. Cette protéine a une forte affinité pour Syntaxin-1 et contient un domaine SNARE. Tomosyn-1 est concentrée dans les régions cellulaires enrichies en granules de sécrétion. La diminution sélective de l'expression de Tomosyn-1 induit une réduction de l'exocytose stimulée par différents sécrétagogues. Cet effet est dû à un défaut de fusion des granules avec la membrane plasmique. Ceci nous indique que Tomosyn-1 intervient dans une phase importante de la préparation des vésicules à la fusion, qui est nécessaire à l'exocytose. Abstract: Insulin exocytosis from pancreatic β-cells plays a central role in blood glucose homeostasis. Diabetes mellitus is a complex metabolic disorder characterized by secretory dysfunctions in pancreatic β-cells and release of amounts of insulin that are inappropriate to maintain blood glucose concentration within normal physiological ranges. To define the causes of β-cell failure a basic understanding of the molecular mechanisms that control insulin exocytosis is essential. Some of the molecular components involved in this process have been identified, including the SNARE proteins VAMP2, Syntaxin-1 and SNAP25 and the two GTPases, Rab3 and Rab27, that regulate the final steps of insulin secretion. I first investigated the role of Noc2, a potential Rab3 and Rab27 partner, in insulin secretion. I found that Noc2 associates with Rab27 and is recruited by this GTPase on insulin- containing granules. Silencing of the Noc2 gene by RNA interference led to a strong impairment in the capacity of the β-cell line INS-1E to respond to secretagogues, indicating that appropriate levels of the protein are essential for insulin exocytosis. I also showed that Noc2 interacts with Munc13, a protein that controls vesicle priming, suggesting a possible involvement of Noc2 in the recruitment of secretory granules at the plasma membrane. In the second part of my thesis, I investigated the adaptation of the molecular machinery of exocytosis to physiopathological conditions. I found that the expression of Rab3, Rab27 and of their effectors Granuphilin/Slp4 and Noc2 is dramatically decreased by chronic exposure of β-ce1ls to supraphysiological glucose levels. The observed glucotoxic effect is a consequence of the induction of ICER, a transcriptional repressor that is increased by prolonged hyperglycemia and in genetic models of type 2 diabetes. Overexpression of ICER reduced Granuphilin, Noc2, Rab3 and Rab27 levels and inhibited exocytosis. These results suggest that the presence of inappropriate levels of ICER diminishes the expression of a group of proteins essential for exocytosis and contributes to defective insulin release in type 2 diabetes. In the last part of my thesis, I focused my attention on the role of Tomosyn-1, a Syntaxin-1 binding protein possessing a SNARE-like motif, in the control of SNARE complex assembly. I found that Tomosyn-1 is concentrated in cellular compartments enriched in insulin-containing secretory granules. Silencing of Tomosyn-1 did not affect the number of secretory granules docked at the plasma membrane but decreased their release probability, resulting in a reduction in stimulus-induced insulin exocytosis. These findings suggest that Tomosyn-1 is involved in a post-docking event that prepares secretory granules for fusion and is necessary to sustain exocytosis in response to insulin secretagogues.

Generative FDG-PET and MRI model of aging and disease progression in Alzheimer's disease.

The failure of current strategies to provide an explanation for controversial findings on the pattern of pathophysiological changes in Alzheimer's Disease (AD) motivates the necessity to develop new integrative approaches based on multi-modal neuroimaging data that captures various aspects of disease pathology. Previous studies using [18F]fluorodeoxyglucose positron emission tomography (FDG-PET) and structural magnetic resonance imaging (sMRI) report controversial results about time-line, spatial extent and magnitude of glucose hypometabolism and atrophy in AD that depend on clinical and demographic characteristics of the studied populations. Here, we provide and validate at a group level a generative anatomical model of glucose hypo-metabolism and atrophy progression in AD based on FDG-PET and sMRI data of 80 patients and 79 healthy controls to describe expected age and symptom severity related changes in AD relative to a baseline provided by healthy aging. We demonstrate a high level of anatomical accuracy for both modalities yielding strongly age- and symptom-severity- dependant glucose hypometabolism in temporal, parietal and precuneal regions and a more extensive network of atrophy in hippocampal, temporal, parietal, occipital and posterior caudate regions. The model suggests greater and more consistent changes in FDG-PET compared to sMRI at earlier and the inversion of this pattern at more advanced AD stages. Our model describes, integrates and predicts characteristic patterns of AD related pathology, uncontaminated by normal age effects, derived from multi-modal data. It further provides an integrative explanation for findings suggesting a dissociation between early- and late-onset AD. The generative model offers a basis for further development of individualized biomarkers allowing accurate early diagnosis and treatment evaluation.

A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast.

Cell-cell fusion is essential for fertilization. For fusion of walled cells, the cell wall must be degraded at a precise location but maintained in surrounding regions to protect against lysis. In fission yeast cells, the formin Fus1, which nucleates linear actin filaments, is essential for this process. In this paper, we show that this formin organizes a specific actin structure-the actin fusion focus. Structured illumination microscopy and live-cell imaging of Fus1, actin, and type V myosins revealed an aster of actin filaments whose barbed ends are focalized near the plasma membrane. Focalization requires Fus1 and type V myosins and happens asynchronously always in the M cell first. Type V myosins are essential for fusion and concentrate cell wall hydrolases, but not cell wall synthases, at the fusion focus. Thus, the fusion focus focalizes cell wall dissolution within a broader cell wall synthesis zone to shift from cell growth to cell fusion.

Recurrent loss of heterozygosity in 1p36 associated with TNFRSF14 mutations in IRF4 translocation negative pediatric follicular lymphomas.

Pediatric follicular lymphoma is a rare disease that differs genetically and clinically from its adult counterpart. With the exception of pediatric follicular lymphoma with IRF4-translocation, the genetic events associated with these lymphomas have not yet been defined. We applied array-comparative genomic hybridization and molecular inversion probe assay analyses to formalin-fixed paraffin-embedded tissues from 18 patients aged 18 years and under with IRF4 translocation negative follicular lymphoma. All evaluable cases lacked t(14;18). Only 6 of 16 evaluable cases displayed chromosomal imbalances with gains or amplifications of 6pter-p24.3 (including IRF4) and deletion and copy number neutral-loss of heterozygosity in 1p36 (including TNFRSF14) being most frequent. Sequencing of TNFRSF14 located in the minimal region of loss in 1p36.32 showed nine mutations in 7 cases from our series. Two subsets of pediatric follicular lymphoma were delineated according to the presence of molecular alterations, one with genomic aberrations associated with higher grade and/or diffuse large B-cell lymphoma component and more widespread disease, and another one lacking genetic alterations associated with more limited disease.

Novel approaches in anti-arenaviral drug development.

Hemorrhagic fevers caused by arenaviruses are among the most devastating emerging human diseases. Considering the number of individuals affected, the current lack of a licensed vaccine, and the limited therapeutic options, arenaviruses are arguably among the most neglected tropical pathogens and the development of efficacious anti-arenaviral drugs is of high priority. Over the past years significant efforts have been undertaken to identify novel potent inhibitors of arenavirus infection. High throughput screening of small molecule libraries employing pseudotype platforms led to the discovery of several potent and broadly active inhibitors of arenavirus cell entry that are effective against the major hemorrhagic arenaviruses. Mechanistic studies revealed that these novel entry inhibitors block arenavirus membrane fusion and provided novel insights into the unusual mechanism of this process. The success of these approaches highlights the power of small molecule screens in antiviral drug discovery and establishes arenavirus membrane fusion as a robust drug target. These broad screenings have been complemented by strategies targeting cellular factors involved in productive arenavirus infection. Approaches targeting the cellular protease implicated in maturation of the fusion-active viral envelope glycoprotein identified the proteolytic processing of the arenavirus glycoprotein precursor as a novel and promising target for anti-arenaviral strategies.

Neonatal dexamethasone induces premature microvascular maturation of the alveolar capillary network.

Postnatal glucocorticoid treatment of preterm infants was mimicked by treating newborn rats with dexamethasone (0.1-0.01 microg/g, days 1-4). This regimen has been shown to cause delayed alveolarization. Knowing that microvascular maturation (transformation of double- to single-layered capillary networks in alveolar septa) and septal thinning prevent further alveolarization, we measured septal maturation on electron photomicrographs in treated and control animals. In treated rats and before day 10, we observed a premature nonreversing microvascular maturation and a transient septal thinning, which both appeared focally. In vascular casts of both groups, we observed contacts between the two capillary layers of immature alveolar septa, which were predictive for capillary fusions. Studying serial electron microscopic sections of human lungs, we were able to confirm the postulated fusion process for the first time. We conclude that alveolar microvascular maturation indeed occurs by capillary fusion and that the dexamethasone-induced impairment of alveolarization is associated with focal premature capillary fusion.

Cytogenetic characterisation of childhood acute lymphoblastic leukemia in Nicaragua

BACKGROUND: Within the frame of a twinning programme with Nicaragua, The La Mascota project, we evaluated in our study the contribution of cytogenetic characterization of acute lymphoblastic leukemia (ALL) as prognostic factor compared to clinical, morphological, and immunohistochemical parameters. METHODS: All patients with ALL treated at the only cancer pediatric hospital in Nicaragua during 2006 were studied prospectively. Diagnostic immunophenotyping was performed locally and bone marrow or blood samples were sent to the cytogenetic laboratory of Zurich for fluorescence in situ hybridization (FISH) analysis and G-banding. RESULTS: Sixty-six patients with ALL were evaluated. Their mean age at diagnosis was 7.3 years, 31.8% were >or=10 years. Thirty-four patients (51.5%) presented with hyperleucocytosis >or=50 x 10(9)/L, 45 (68.2%) had hepatosplenomegaly. Immunophenotypically 63/66 patients (95%) had a B-precursor, 2 (3%) a T- and 1 (1.5%) a B-mature ALL. FISH analysis demonstrated a TEL/AML1 fusion in 9/66 (14%), BCR/ABL fusion in 1 (1.5%), MLL rearrangement in 2 (3.1%), iAMP21 in 2 (3.1%), MYC rearrangement in 1 (1.5%), and high-hyperdiploidy in 16 (24%). All patients but two with TEL/AML1 fusion and high-hyperdiploidy were clinically and hematologically in the standard risk group whereas those with poor cytogenetic factors had clinical high-risk features and were treated intensively. CONCLUSIONS: Compared to Europe, the ALL population in Nicaragua is older, has a higher proportion of poor prognostic clinical and hematological features and receives more intensive treatment, while patients with TEL/AML1 translocations and high-hyperdiploidy are clinically in the standard risk group. Cytogenetics did not contribute as an additional prognostic factor in this setting.

Self-potential investigations of a gravel bar in a restored river corridor

Self-potentials (SP) are sensitive to water fluxes and concentration gradients in both saturated and unsaturated geological media, but quantitative interpretations of SP field data may often be hindered by the superposition of different source contributions and time-varying electrode potentials. Self-potential mapping and close to two months of SP monitoring on a gravel bar were performed to investigate the origins of SP signals at a restored river section of the Thur River in northeastern Switzerland. The SP mapping and subsequent inversion of the data indicate that the SP sources are mainly located in the upper few meters in regions of soil cover rather than bare gravel. Wavelet analyses of the time-series indicate a strong, but non-linear influence of water table and water content variations, as well as rainfall intensity on the recorded SP signals. Modeling of the SP response with respect to an increase in the water table elevation and precipitation indicate that the distribution of soil properties in the vadose zone has a very strong influence. We conclude that the observed SP responses on the gravel bar are more complicated than previously proposed semi-empiric relationships between SP signals and hydraulic head or the thickness of the vadose zone. We suggest that future SP monitoring in restored river corridors should either focus on quantifying vadose zone processes by installing vertical profiles of closely spaced SP electrodes or by installing the electrodes within the river to avoid signals arising from vadose zone processes and time-varying electrochemical conditions in the vicinity of the electrodes.

Méthodes sismiques actives adaptées à l'étude de milieux hétérogènes : application aux glissements de terrain

Résumé Les glissements de terrain représentent un des principaux risques naturels dans les régions montagneuses. En Suisse, chaque année les glissements de terrains causent des dégâts qui affectent les infrastructures et ont des coûts financiers importants. Une bonne compréhension des mécanismes des glissements peut permettre d'atténuer leur impact. Celle-ci passe notamment par la connaissance de la structure interne du glissement, la détermination de son volume et de son ou ses plans de glissement. Dans un glissement de terrain, la désorganisation et la présence de fractures dans le matériel déplacé engendre un changement des paramètres physiques et en particulier une diminution des vitesses de propagation des ondes sismiques ainsi que de la densité du matériel. Les méthodes sismiques sont de ce fait bien adaptées à l'étude des glissements de terrain. Parmi les méthodes sismiques, l'analyse de la dispersion des ondes de surface est une méthode simple à mettre en oeuvre. Elle présente l'avantage d'estimer les variations des vitesses de cisaillement avec la profondeur sans avoir spécifiquement recours à l'utilisation d'une source d'onde S et de géophones horizontaux. Sa mise en oeuvre en trois étapes implique la mesure de la dispersion des ondes de surface sur des réseaux étendus, la détermination des courbes de dispersion pour finir par l'inversion de ces courbes. Les modèles de vitesse obtenus à partir de cette procédure ne sont valides que lorsque les milieux explorés ne présentent pas de variations latérales. En pratique cette hypothèse est rarement vérifiée, notamment pour un glissement de terrain dans lequel les couches remaniées sont susceptibles de présenter de fortes hétérogénéités latérales. Pour évaluer la possibilité de déterminer des courbes de dispersion à partir de réseaux de faible extension des mesures testes ont été effectuées sur un site (Arnex, VD) équipé d'un forage. Un profil sismique de 190 m de long a été implanté dans une vallée creusée dans du calcaire et remplie par des dépôts glacio-lacustres d'une trentaine de mètres d'épaisseur. Les données acquises le long de ce profil ont confirmé que la présence de variations latérales sous le réseau de géophones affecte l'allure des courbes de dispersion jusqu'à parfois empêcher leur détermination. Pour utiliser l'analyse de la dispersion des ondes de surface sur des sites présentant des variations latérales, notre approche consiste à déterminer les courbes de dispersions pour une série de réseaux de faible extension, à inverser chacune des courbes et à interpoler les différents modèles de vitesse obtenus. Le choix de la position ainsi que de l'extension des différents réseaux de géophones est important. Il tient compte de la localisation des hétérogénéités détectées à partir de l'analyse de sismique réfraction, mais également d'anomalies d'amplitudes observées sur des cartes qui représentent dans le domaine position de tir - position du récepteur, l'amplitude mesurée pour différentes fréquences. La procédure proposée par Lin et Lin (2007) s'est avérée être une méthode efficace permettant de déterminer des courbes de dispersion à partir de réseaux de faible extension. Elle consiste à construire à partir d'un réseau de géophones et de plusieurs positions de tir un enregistrement temps-déports qui tient compte d'une large gamme de distances source-récepteur. Au moment d'assembler les différentes données une correction de phase est appliquée pour tenir compte des hétérogénéités situées entre les différents points de tir. Pour évaluer cette correction nous suggérons de calculer pour deux tir successif la densité spectrale croisée des traces de même offset: Sur le site d'Arnex, 22 courbes de dispersions ont été déterminées pour de réseaux de géophones de 10 m d'extension. Nous avons également profité du forage pour acquérir un profil de sismique verticale en ondes S. Le modèle de vitesse S déduit de l'interprétation du profil de sismique verticale est utilisé comme information à priori lors l'inversion des différentes courbes de dispersion. Finalement, le modèle en deux dimension qui a été établi grâce à l'analyse de la dispersion des ondes de surface met en évidence une structure tabulaire à trois couches dont les limites coïncident bien avec les limites lithologiques observées dans le forage. Dans celui-ci des argiles limoneuses associées à une vitesse de propagation des ondes S de l'ordre de 175 m/s surmontent vers 9 m de profondeur des dépôts de moraine argilo-sableuse caractérisés par des vitesses de propagation des ondes S de l'ordre de 300 m/s jusqu'à 14 m de profondeur et supérieur ou égal à 400 m/s entre 14 et 20 m de profondeur. Le glissement de la Grande Combe (Ballaigues, VD) se produit à l'intérieur du remplissage quaternaire d'une combe creusée dans des calcaires Portlandien. Comme dans le cas du site d'Arnex les dépôts quaternaires correspondent à des dépôts glacio-lacustres. Dans la partie supérieure la surface de glissement a été localisée à une vingtaine de mètres de profondeur au niveau de l'interface qui sépare des dépôts de moraine jurassienne et des dépôts glacio-lacustres. Au pied du glissement 14 courbes de dispersions ont été déterminées sur des réseaux de 10 m d'extension le long d'un profil de 144 m. Les courbes obtenues sont discontinues et définies pour un domaine de fréquence de 7 à 35 Hz. Grâce à l'utilisation de distances source-récepteur entre 8 et 72 m, 2 à 4 modes de propagation ont été identifiés pour chacune des courbes. Lors de l'inversion des courbes de dispersion la prise en compte des différents modes de propagation a permis d'étendre la profondeur d'investigation jusqu'à une vingtaine de mètres de profondeur. Le modèle en deux dimensions permet de distinguer 4 couches (Vs1 < 175 m/s, 175 m/s < Vs2 < 225 m/s, 225 m/s < Vs3 < 400 m/s et Vs4 >.400 m/s) qui présentent des variations d'épaisseur. Des profils de sismiques réflexion en ondes S acquis avec une source construite dans le cadre de ce travail, complètent et corroborent le modèle établi à partir de l'analyse de la dispersion des ondes de surface. Un réflecteur localisé entre 5 et 10 m de profondeur et associé à une vitesse de sommation de 180 m/s souligne notamment la géométrie de l'interface qui sépare la deuxième de la troisième couche du modèle établi à partir de l'analyse de la dispersion des ondes de surface. Abstract Landslides are one of the main natural hazards in mountainous regions. In Switzerland, landslides cause damages every year that impact infrastructures and have important financial costs. In depth understanding of sliding mechanisms may help limiting their impact. In particular, this can be achieved through a better knowledge of the internal structure of the landslide, the determination of its volume and its sliding surface or surfaces In a landslide, the disorganization and the presence of fractures in the displaced material generate a change of the physical parameters and in particular a decrease of the seismic velocities and of the material density. Therefoe, seismic methods are well adapted to the study of landslides. Among seismic methods, surface-wave dispersion analysis is a easy to implement. Through it, shearwave velocity variations with depth can be estimated without having to resort to an S-wave source and to horizontal geophones. Its 3-step implementation implies measurement of surface-wave dispersion with long arrays, determination of the dispersion curves and finally inversion of these curves. Velocity models obtained through this approach are only valid when the investigated medium does not include lateral variations. In practice, this assumption is seldom correct, in particular for landslides in which reshaped layers likely include strong lateral heterogeneities. To assess the possibility of determining dispersion curves from short array lengths we carried out tests measurements on a site (Arnex, VD) that includes a borehole. A 190 m long seismic profile was acquired in a valley carved into limestone and filled with 30 m of glacio-lacustrine sediments. The data acquired along this profile confirmed that the presence of lateral variations under the geophone array influences the dispersion-curve shape so much that it sometimes preventes the dispersion curves determination. Our approach to use the analysis of surface-wave dispersion on sites that include lateral variations consists in obtaining dispersion curves for a series of short length arrays; inverting each so obtained curve and interpolating the different obtained velocity model. The choice of the location as well as the geophone array length is important. It takes into account the location of the heterogeneities that are revealed by the seismic refraction interpretation of the data but also, the location of signal amplitude anomalies observed on maps that represent, for a given frequency, the measured amplitude in the shot position - receiver position domain. The procedure proposed by Lin and Lin (2007) turned out to be an efficient one to determine dispersion curves using short extension arrays. It consists in building a time-offset from an array of geophones with a wide offset range by gathering seismograms acquired with different source-to-receiver offsets. When assembling the different data, a phase correction is applied in order to reduce static phase error induced by lateral variation. To evaluate this correction, we suggest to calculate, for two successive shots, the cross power spectral density of common offset traces. On the Arnex site, 22 curves were determined with 10m in length geophone-arrays. We also took advantage of the borehole to acquire a S-wave vertical seismic profile. The S-wave velocity depth model derived from the vertical seismic profile interpretation is used as prior information in the inversion of the dispersion-curves. Finally a 2D velocity model was established from the analysis of the different dispersion curves. It reveals a 3-layer structure in good agreement with the observed lithologies in the borehole. In it a clay layer with a shear-wave of 175 m/s shear-wave velocity overlies a clayey-sandy till layer at 9 m depth that is characterized down to 14 m by a 300 m/s S-wave velocity; these deposits have a S-wave velocity of 400 m/s between depths of 14 to 20 m. The La Grand Combe landslide (Ballaigues, VD) occurs inside the Quaternary filling of a valley carved into Portlandien limestone. As at the Arnex site, the Quaternary deposits correspond to glaciolacustrine sediments. In the upper part of the landslide, the sliding surface is located at a depth of about 20 m that coincides with the discontinuity between Jurassian till and glacio-lacustrine deposits. At the toe of the landslide, we defined 14 dispersion curves along a 144 m long profile using 10 m long geophone arrays. The obtained curves are discontinuous and defined within a frequency range of 7 to 35 Hz. The use of a wide range of offsets (from 8 to 72 m) enabled us to determine 2 to 4 mode of propagation for each dispersion curve. Taking these higher modes into consideration for dispersion curve inversion allowed us to reach an investigation depth of about 20 m. A four layer 2D model was derived (Vs1< 175 m/s, 175 m/s <Vs2< 225 m/s, 225 m/s < Vs3 < 400 m/s, Vs4> 400 m/s) with variable layer thicknesses. S-wave seismic reflection profiles acquired with a source built as part of this work complete and the velocity model revealed by surface-wave analysis. In particular, reflector at a depth of 5 to 10 m associated with a 180 m/s stacking velocity image the geometry of the discontinuity between the second and third layer of the model derived from the surface-wave dispersion analysis.

Development of appropriateness criteria for the surgical treatment of symptomatic lumbar degenerative spondylolisthesis (LDS).

PURPOSE: Spine surgery rates are increasing worldwide. Treatment failures are often attributed to poor patient selection and inappropriate treatment, but for many spinal disorders there is little consensus on the precise indications for surgery. With an aging population, more patients with lumbar degenerative spondylolisthesis (LDS) will present for surgery. The aim of this study was to develop criteria for the appropriateness of surgery in symptomatic LDS. METHODS: A systematic review was carried out to summarize the current level of evidence for the treatment of LDS. Clinical scenarios were generated comprising combinations of signs and symptoms in LDS and other relevant variables. Based on the systematic review and their own clinical experience, twelve multidisciplinary international experts rated each scenario on a 9-point scale (1 highly inappropriate, 9 highly appropriate) with respect to performing decompression only, fusion, and instrumented fusion. Surgery for each theoretical scenario was classified as appropriate, inappropriate, or uncertain based on the median ratings and disagreement in the ratings. RESULTS: 744 hypothetical scenarios were generated; overall, surgery (of some type) was rated appropriate in 27 %, uncertain in 41 % and inappropriate in 31 %. Frank panel disagreement was low (7 % scenarios). Face validity was shown by the logical relationship between each variable's subcategories and the appropriateness ratings, e.g., no/mild disability had a mean appropriateness rating of 2.3 ± 1.5, whereas the rating for moderate disability was 5.0 ± 1.6 and for severe disability, 6.6 ± 1.6. Similarly, the average rating for no/minimal neurological abnormality was 2.3 ± 1.5, increasing to 4.3 ± 2.4 for moderate and 5.9 ± 1.7 for severe abnormality. The three variables most likely (p < 0.0001) to be components of scenarios rated "appropriate" were: severe disability, no yellow flags, and severe neurological deficit. CONCLUSION: This is the first study to report criteria for determining candidacy for surgery in LDS developed by a multidisciplinary international panel using a validated method (RAM). The panel ratings followed logical clinical rationale, indicating good face validity. The work refines clinical classification and the phenotype of degenerative spondylolisthesis. The predictive validity of the criteria should be evaluated prospectively to examine whether patients treated "appropriately" have better clinical outcomes.

Regulation of the expression of Fit insect toxin locus genes in the root-associated biocontrol pseudomonad CHA0

The root-colonizing Pseudomonas fluorescens strain CHA0 is a biocontrol agent of soil-borne plant diseases caused by fungal and oomycete pathogens. Remarkably, this plant-beneficial pseudomonad is also endowed with potent insecticidal activity that depends on the production of a large protein toxin termed Fit (for P. fluorescens insecticidal toxin). In our present work, the genomic locus encoding the P. fluorescens insect toxin is subjected to a detailed molecular analysis. The Fit toxin gene fitD is flanked upstream by the fitABC genes and downstream by the fitE gene that encode the ABC transporter, membrane fusion, and outer membrane efflux components of a type I protein secretion system predicted to function in toxin export. The fitF, fitG, and fitH genes located downstream of fitE code for regulatory proteins having domain structures typical of signal transduction histidine kinases, LysR-type transcriptional regulators, and response regulators, respectively. The role of these insect toxin locus-associated control elements is being investigated with mutants defective for the regulatory genes and with GFP-based reporter fusions to putative promoter regions upstream of the transporter genes fitA and fitE, the toxin gene fitD, and the regulatory genes fitF and fitH. Our preliminary findings suggest that the three regulators interact with known global regulators of biocontrol factor expression to control Fit toxin expression and secretion.

Tomosyn-1 is involved in a post-docking event required for pancreatic beta-cell exocytosis.

Although the assembly of a ternary complex between the SNARE proteins syntaxin-1, SNAP25 and VAMP2 is known to be crucial for insulin exocytosis, the mechanisms controlling this key event are poorly understood. We found that pancreatic beta-cells express different isoforms of tomosyn-1, a syntaxin-1-binding protein possessing a SNARE-like motif. Using atomic force microscopy we show that the SNARE-like domain of tomosyn-1 can form a complex with syntaxin-1 and SNAP25 but displays binding forces that are weaker than those observed for VAMP2 (237+/-13 versus 279+/-3 pN). In pancreatic beta-cells tomosyn-1 was found to be concentrated in cellular compartments enriched in insulin-containing secretory granules. Silencing of tomosyn-1 in the rat beta-cell line INS-1E by RNA interference did not affect the number of secretory granules docked at the plasma membrane but led to a reduction in stimulus-induced exocytosis. Replacement of endogenous tomosyn-1 with mouse tomosyn-1, which differs in the nucleotide sequence from its rat homologue and escapes silencing, restored a normal secretory rate. Taken together, our data suggest that tomosyn-1 is involved in a post-docking event that prepares secretory granules for fusion and is necessary to sustain exocytosis of pancreatic beta-cells in response to insulin secretagogues.

Cytogenetic characterization of childhood acute lymphoblastic leukemia in Nicaragua.

BACKGROUND: Within the frame of a twinning programme with Nicaragua, The La Mascota project, we evaluated in our study the contribution of cytogenetic characterization of acute lymphoblastic leukemia (ALL) as prognostic factor compared to clinical, morphological, and immunohistochemical parameters. METHODS: All patients with ALL treated at the only cancer pediatric hospital in Nicaragua during 2006 were studied prospectively. Diagnostic immunophenotyping was performed locally and bone marrow or blood samples were sent to the cytogenetic laboratory of Zurich for fluorescence in situ hybridization (FISH) analysis and G-banding. RESULTS: Sixty-six patients with ALL were evaluated. Their mean age at diagnosis was 7.3 years, 31.8% were >or=10 years. Thirty-four patients (51.5%) presented with hyperleucocytosis >or=50 x 10(9)/L, 45 (68.2%) had hepatosplenomegaly. Immunophenotypically 63/66 patients (95%) had a B-precursor, 2 (3%) a T- and 1 (1.5%) a B-mature ALL. FISH analysis demonstrated a TEL/AML1 fusion in 9/66 (14%), BCR/ABL fusion in 1 (1.5%), MLL rearrangement in 2 (3.1%), iAMP21 in 2 (3.1%), MYC rearrangement in 1 (1.5%), and high-hyperdiploidy in 16 (24%). All patients but two with TEL/AML1 fusion and high-hyperdiploidy were clinically and hematologically in the standard risk group whereas those with poor cytogenetic factors had clinical high-risk features and were treated intensively. CONCLUSIONS: Compared to Europe, the ALL population in Nicaragua is older, has a higher proportion of poor prognostic clinical and hematological features and receives more intensive treatment, while patients with TEL/AML1 translocations and high-hyperdiploidy are clinically in the standard risk group. Cytogenetics did not contribute as an additional prognostic factor in this setting.

Identification of Genes Affecting Vacuole Membrane Fragmentation in Saccharomyces cerevisiae.

The equilibrium of membrane fusion and fission influences the volume and copy number of organelles. Fusion of yeast vacuoles has been well characterized but their fission and the mechanisms determining vacuole size and abundance remain poorly understood. We therefore attempted to systematically characterize factors necessary for vacuole fission. Here, we present results of an in vivo screening for deficiencies in vacuolar fragmentation activity of an ordered collection deletion mutants, representing 4881 non-essential genes of the yeast Saccharomyces cerevisiae. The screen identified 133 mutants with strong defects in vacuole fragmentation. These comprise numerous known fragmentation factors, such as the Fab1p complex, Tor1p, Sit4p and the V-ATPase, thus validating the approach. The screen identified many novel factors promoting vacuole fragmentation. Among those are 22 open reading frames of unknown function and three conspicuous clusters of proteins with known function. The clusters concern the ESCRT machinery, adaptins, and lipases, which influence the production of diacylglycerol and phosphatidic acid. A common feature of these factors of known function is their capacity to change membrane curvature, suggesting that they might promote vacuole fragmentation via this property.

Zonation for 3D aquifer characterization based on joint inversions of multimethod crosshole geophysical data

Predictive groundwater modeling requires accurate information about aquifer characteristics. Geophysical imaging is a powerful tool for delineating aquifer properties at an appropriate scale and resolution, but it suffers from problems of ambiguity. One way to overcome such limitations is to adopt a simultaneous multitechnique inversion strategy. We have developed a methodology for aquifer characterization based on structural joint inversion of multiple geophysical data sets followed by clustering to form zones and subsequent inversion for zonal parameters. Joint inversions based on cross-gradient structural constraints require less restrictive assumptions than, say, applying predefined petro-physical relationships and generally yield superior results. This approach has, for the first time, been applied to three geophysical data types in three dimensions. A classification scheme using maximum likelihood estimation is used to determine the parameters of a Gaussian mixture model that defines zonal geometries from joint-inversion tomograms. The resulting zones are used to estimate representative geophysical parameters of each zone, which are then used for field-scale petrophysical analysis. A synthetic study demonstrated how joint inversion of seismic and radar traveltimes and electrical resistance tomography (ERT) data greatly reduces misclassification of zones (down from 21.3% to 3.7%) and improves the accuracy of retrieved zonal parameters (from 1.8% to 0.3%) compared to individual inversions. We applied our scheme to a data set collected in northeastern Switzerland to delineate lithologic subunits within a gravel aquifer. The inversion models resolve three principal subhorizontal units along with some important 3D heterogeneity. Petro-physical analysis of the zonal parameters indicated approximately 30% variation in porosity within the gravel aquifer and an increasing fraction of finer sediments with depth.