The role of proteolytic processing and the stable signal peptide in expression of the Old World arenavirus envelope glycoprotein ectodomain.

Maturation of the arenavirus GP precursor (GPC) involves proteolytic processing by cellular signal peptidase and the proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P), yielding a tripartite complex comprised of a stable signal peptide (SSP), the receptor-binding GP1, and the fusion-active transmembrane GP2. Here we investigated the roles of SKI-1/S1P processing and SSP in the biosynthesis of the recombinant GP ectodomains of lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV). When expressed in mammalian cells, the LCMV and LASV GP ectodomains underwent processing by SKI-1/S1P, followed by dissociation of GP1 from GP2. The GP2 ectodomain spontaneously formed trimers as revealed by chemical cross-linking. The endogenous SSP, known to be crucial for maturation and transport of full-length arenavirus GPC was dispensable for processing and secretion of the soluble GP ectodomain, suggesting a specific role of SSP in the stable prefusion conformation and transport of full-length GPC.

Assessment of the in vitro synergy of daptomycin plus linezolid against multidrug-resistant enterococci

The widespread incidence of enterococci resistant to ampicillin, vancomycin and aminoglycosides, the first-line anti-enterococcal antibiotics, has made the treatment of severe enterococcal infections difficult and alternatives should be explored. We investigated the activity of daptomycin combined with linezolid against three Enterococcus faecalis and four Enterococcus faecium strains resistant to standard drugs used for therapy. Minimum inhibitory concentrations (MICs) were determined by the broth dilution method. Drug interactions were assessed by the checkerboard and time-kill methods. Synergy was defined by a fractional inhibitory concentration index (FICI) of ≤0.5 or a ≥2 log10 CFU/mL killing at 24 h with the combination in comparison with killing by the most active single agent. Indifference was defined by a FICI > 0.5-4.0 or a 1-2 log10 CFU/mL killing compared with the most active single agent. MICs of daptomycin were 2-4 μg/mL for E. faecalis and 2-8 μg/mL for E. faecium. MICs of linezolid were 1-2 μg/mL for all bacteria. In the checkerboard assay, five isolates showed synergism (FICI < 0.5) and two showed indifference (FICIs of 0.53 and 2). Killing studies revealed synergy of daptomycin plus linezolid against four isolates (2.2-3.7 log10 CFU/mL kill) and indifference (1.1-1.6 log10 CFU/mL kill) for the other three strains. Antagonism was not observed. In conclusion, the combination of daptomycin and linezolid had a synergistic or indifferent effect against multidrug-resistant enterococci. Additional studies are needed to explore the potential of this combination for severe enterococcal infections when first-line antibiotic combinations cannot be used.

Synapses on motoneuron dendrites in the brachial section of the frog spinal cord: a computer-aided electron microscopic study of cobalt-filled cells.

Cobalt-labelled motoneuron dendrites of the frog spinal cord at the level of the second spinal nerve were photographed in the electron microscope from long series of ultrathin sections. Three-dimensional computer reconstructions of 120 dendrite segments were analysed. The samples were taken from two locations: proximal to cell body and distal, as defined in a transverse plane of the spinal cord. The dendrites showed highly irregular outlines with many 1-2 microns-long 'thorns' (on average 8.5 thorns per 100 microns 2 of dendritic area). Taken together, the reconstructed dendrite segments from the proximal sites had a total length of about 250 microns; those from the distal locations, 180 microns. On all segments together there were 699 synapses. Nine percent of the synapses were on thorns, and many more close to their base on the dendritic shaft. The synapses were classified in four groups. One third of the synapses were asymmetric with spherical vesicles; one half were symmetric with spherical vesicles; and one tenth were symmetric with flattened vesicles. A fourth, small class of asymmetric synapses had dense-core vesicles. The area of the active zones was large for the asymmetric synapses (median value 0.20 microns 2), and small for the symmetric ones (median value 0.10 microns 2), and the difference was significant. On average, the areas of the active zones of the synapses on thin dendrites were larger than those of synapses on large calibre dendrites. About every 4 microns 2 of dendritic area received one contact. There was a significant difference between the areas of the active zones of the synapses at the two locations. Moreover, the number per unit dendritic length was correlated with dendrite calibre. On average, the active zones covered more than 4% of the dendritic area; this value for thin dendrites was about twice as large as that of large calibre dendrites. We suggest that the larger active zones and the larger synaptic coverage of the thin dendrites compensate for the longer electrotonic distance of these synapses from the soma.

Atypical EPO profiles in anti-doping analyses

Résumé : Erythropoietin (EPO) is a glycoprotein hormone endogenously produced by the kidney, whose main physiological role is the stimulation of erythropoiesis. Since the beginning of the nineties, recombinant human EPO (rhEPO), a potent anti-anaemia treatment drug, has been manufactured by pharmaceutical industries. However, the erythropoiesis stimulating power of rhEPO was rapidly misused by unscrupulous athletes in order to improve their performances in endurance sports. Endogenous EPO has the same amino-acid backbone as most of recombinant forms; the molecules however differ through their respective glycosylation patterns. This difference constitutes the basis of the usual EPO screening test (IEF) developed in 2000 and still currently used in all anti-doping laboratories of the world. Nowadays, 3 EPO generations have been commercialized. The fight against EPO abuse is a continuous challenge for anti-doping laboratories. The diversity of recombinant EPO forms and the continuous development of new ones considerably confuse the identification of EPO doping. Several facets of this fight were investigated in this work. One of the limiting aspects of doping agents screening is the availability of positive samples. Therefore, 2nd and 3rd generation EPOS, namely NESP and C.E.R.A., were injected to healthy subjects in the frame of pilot clinical studies. These latter allowed to review the current EPO identification criteria defined by the World Anti-Doping Agency (WADA) in the case of NESP and to validate and implement a new assay targeting C.E.R.A. in human serum. Both studies resulted in the determination of the respective detection windows of NESP and C.E.R.A. in biological fluids. Following that, Dynepo, a 1st generation EPO presenting similarities with the endogenous form, was also in the centre of a similar clinical study. Our work aimed to overcome the actual identification criteria, which are not adapted to Dynpeo, and to propose an alternative pattern classification method based on the discriminant analysis of IEF EPO profiles. This method might be validated for other EPO forms in the future. The detection window of this molecule was also determined. Under particular conditions, confounding effects can complicate the identification of EPO in biological matrices. For example, athletes having performed a strenuous physical effort can excrete modified isoforms of endogenous EPO, making it very similar to some recombinant forms. Such phenomena, called effort urines, were reproduced under controlled conditions and, after characterization of effort EPO, an urinary biochemical marker was proposed to unequivocally identify effort urines. It also happens that EPO analyses fail to detect endogenous levels of EPO. Such profiles were thoroughly investigated and potential causes identified. Natural reasons relying on urine properties and test specificity were underlined, but the possible addition of adulterant agents in urine samples was also considered. Therefore, a simple biochemical assay targeting the suspected substances was set up. Our work was based on the characterization of atypical EPO profiles from different origins. Therefore, 3 EPO molecules representing the 3 generations of the drug and 2 confounding effects confusing the results interpretation were studied. These studies resulted in tangible applications for the laboratory, the best example of which being the C.E.R.A. assay, but also in scientific findings allowing to improve our comprehension of EPO doping in sport.

Constitutively active G protein-coupled receptors: potential mechanisms of receptor activation.

Mutations of G protein-coupled receptors can increase their constitutive (agonist-independent) activity. Some of these mutations have been artificially introduced by site-directed mutagenesis, others occur spontaneously in human diseases. The analysis of the constitutively active G protein-coupled receptors has provided important informations about the molecular mechanisms underlying receptor activation and drug action.

Active learning methods for remote sensing image classification

In this paper, we propose two active learning algorithms for semiautomatic definition of training samples in remote sensing image classification. Based on predefined heuristics, the classifier ranks the unlabeled pixels and automatically chooses those that are considered the most valuable for its improvement. Once the pixels have been selected, the analyst labels them manually and the process is iterated. Starting with a small and nonoptimal training set, the model itself builds the optimal set of samples which minimizes the classification error. We have applied the proposed algorithms to a variety of remote sensing data, including very high resolution and hyperspectral images, using support vector machines. Experimental results confirm the consistency of the methods. The required number of training samples can be reduced to 10% using the methods proposed, reaching the same level of accuracy as larger data sets. A comparison with a state-of-the-art active learning method, margin sampling, is provided, highlighting advantages of the methods proposed. The effect of spatial resolution and separability of the classes on the quality of the selection of pixels is also discussed.

Contribution to the geology of Southern Turkey: new insights from the Mersin mélanges and from the Lycian and Antalya nappes

Abstract The study of fossil Tethyan continental margins implies the consideration of the oceanic domains to which they were connected. The advent of plate tectonics confirmed the importance of the detection of accretion-related mélanges. Ophiolitic mélanges are derived from both an upper ophiolitic obducting plate and a lower oceanic plate. Besides ophiolitic elements, the mélanges may incorporate parts of a magmatic arc and dismembered fragments of a passive continental margin. As the lower plate usually totally disappears during the obduction process, it can only be reconstructed from its elements found in the mélanges. Because of their key location at active margin boundaries, preserved accretion-related mélanges provide strong constraints on the geological evolution of former oceanic domains and their adjacent margins. The identification of Palaeotethyan remnants as accretionary series or reworked during the Late Triassic Eo-Cimmerian event, as well as the recognition of HugluPindos marginal sequences in southern Turkey and in the external Hellenides represent the main achievements of this work, making possible to establish new palaeogeographical correlations. The Mersin mélanges (Turkey), together with the Antalya and Mamonia (Cyprus) domains, are characterized by a series of exotic units found now south of the main Taurus range and compose the South-Taurides Exotic Units. The Mersin mélanges are subdivided in a Triassic and a Late Cretaceous unit. These units consist of the remnants of three major Tethyan oceans, the Palaeotethys, the Neotethys and the Huglu-Pindos. The definition and inventory of the Upper Antalya Nappes (Turkey) are still a matter of controversies and often conflicting interpretations. The recognition of Campanian radiolarians on top of the Kerner Gorge unit directly overlain by the Ordovician Seydi§ehir Fm. of the Tahtah Dag Nappe outlines a tectonic contact and demonstrates that the Upper Antalya Nappes system is composed of three different nappes, the Kerner Gorge, Bakirli and the Tahtah Dag nappes. Additionally, a limestone block in a doubtful tectonic position at the base of the Upper Antalya Nappes yielded for the first time two middle Viséan associations of foraminifers and problematic algae. The Tavas Nappe in the Lycian Nappes (Turkey) is classically divided into the Karadag, Teke Dere, Köycegiz and Haticeana units. As for the Mersin mélanges, the Tavas Nappe is highly composite and includes dismembered units belonging to the Palaeotethyan, Neotethyan and HugluPindos realms. The Karadag unit consists of a Gondwana-type platform succession ranging from the Late Devonian to the Late Triassic. It belongs to the Cimmerian Taurus terrane and was part of the northern passive margin of the Neotethys. The Teke Dere unit is composed of different parts of the Palaeotethyan succession including Late Carboniferous OIB-type basalts, Carboniferous MORB-type basalts, an Early Carboniferous siliciclastic series and a Middle Permian arc sequence. The microfauna and microflora identified in different horizons within the Teke Dere unit share strong biogeographical affinities with the northern Palaeotethyan borders. Kubergandian limestones in primary contact above the Early Carboniferous siliciclastics yielded a rich and diverse microfauna and microflora also identified in reworked cobbles within the Late Triassic Gevne Fm. of the Aladag unit (Turkey). The sedimentological evolution of the Köycegiz and Haticeana series is in many points similar to classical Pindos sequences. These series originated in the Huglu-Pindos Ocean along the northern passive margin of the Anatolian (Turkish transect) and Sitia-Pindos (Greek transect) terranes. Conglomerates at the base of the Lentas Unit in southern Crete (Greece) yielded a microfauna and microflora presenting also strong affinities with the northern borders of the Palaeotethys. This type of reworked sediments at the base of Pindos-like series would suggest a derivation from the Palaeotethyan active margin. -Résumé (French abstract) L'étude des marges continentales fossiles de l'espace téthysien implique d'étudier les domaines océaniques qui y étaient rattachés. Les progrès de la tectonique des plaques ont confirmé l'importance de la reconnaissance des mélanges d'accrétion. Les mélanges ophiolitiques dérivent d'une plaque supérieure ophiolitique qui obducte, et d'une plaque inférieure océanique. En plus d'éléments ophiolitiques, les mélanges peuvent aussi incorporer des parties d'un arc magmatique, ou des fragments d'une marge continentale passive. Comme la plaque inférieure disparaît généralement complètement durant le processus d'obduction, elle ne peut être reconstruite qu'au travers de ses éléments trouvés dans les mélanges. A cause de leur situation aux limites de marges actives, les mélanges d'accrétion bien préservés permettent de contraindre l'évolution géologique d'anciens océans et de leurs marges. L'identification de vestiges de la Paléotéthys en série d'accrétion ou remaniés lors de l'orogenèse éo-cimmérienne au Trias supérieur, ainsi que l'observation de séquences marginales de Huglu-Pinde en Turquie du sud et dans les Hellénides externes représentent les principaux résultats de ce travail, permettant d'établir de nouvelles corrélations paléogéographiques. Les mélanges de Mersin (Turquie), avec les domaines d'Antalya et de Mamonia (Chypre), sont caractérisés par des unités exotiques se trouvant au sud de la chaîne taurique, et forment les Unités Exotiques Sud-Tauriques. Les mélanges de Mersin sont subdivisés en une unité triasique, et une autre du Crétacé supérieur. Ces unités comprennent les reliques de trois principaux océans téthysiens, la Paléotéthys, la Néotéthys et Huglu-Pinde. L'inventaire et la définition des nappes supérieures d'Antalya (Turquie) sont encore matière à controverse et donne lieu à des interprétations conflictuelles. La découverte de radiolaires campaniens au sommet de l'unité de la Gorge de Kemer, directement recouverts par la formation ordovicienne de Seydisehir de la nappe du Tahtali Dag met en évidence un contact tectonique et démontre que les nappes supérieures sont composées de trois différentes nappes, celle de la Gorge de Kemer, celle du Bakirli et celle Tahtali Dag. De plus, un bloc de calcaire dont la position tectonique demeure incertaine à la base des nappes supérieures a fourni pour la première fois deux associations viséennes de foraminifères et d'algues problématiques. La nappe de Tavas dans les nappes lyciennes (Turquie) est séparée en unités du Karadag, du Teke Dere, de Köycegiz et d'Haticeana. Comme pour les mélanges de Mersin, la nappe de Tavas est composite et inclut des unités appartenant à la Paléotéthys, à la Néotéthys et à Huglu-Pinde. L'unité du Karadag est une plateforme carbonatée de type Gondwana se développant du Dévonien supérieur au Trias supérieur. Elle appartient au domaine cimmérien du Taurus et formait la marge nord de la Néotéthys. L'unité du Teke Dere est composée de différentes écailles paléotéthysiennes et inclut des basaltes d'île océanique du Carbonifère supérieur, des basaltes de ride océanique du Carbonifère, une série siliciclastique du Carbonifère supérieur et un arc du Permien moyen. Les microfaunes et -flores trouvées à différents niveaux de la série du Teke Dere partagent de fortes affinités paléogéographiques avec les marges nord de la Paléotéthys. Des calcaires du Kubergandien en contact primaire au-dessus de la série siliciclastique a donné de riches microfaunes et -flores, également identifiées dans des galets remaniés dans la formation de Gevne du Trias supérieur de l'Aladag. L'évolution sédimentologique des séries de Köycegiz et d'Haticeana sont très similaires aux séries classiques du Pinde. Ces séquences prennent leur racine dans l'océan de Huglu-Pinde, le long de la marge passive nord anatolienne (profil turc) et de la marge de Sitia-Pinde (profil grec). Des conglomérats à la base de l'unité de Lentas au sud de la Crète (Grèce) ont donné des microfaunes et flores partageant également de fortes similitudes avec les bordures nord de la Paléotéthys. Le type de sédiments remaniés à la base d'unités de type Pinde suggère une dérivation depuis la marge active de la Paléotéthys. -Résumé grand public (non-specialized abstract) Au début du 20ème siècle, Alfred Wegener bouleverse les croyances géologiques de l'époque et publie plusieurs articles sur la dérive ou la translation des continents. En utilisant des arguments géographiques (similarités des lignes de côte), paléontologiques (faunes et flores similaires) et climatiques (dépôts tropicaux et glaciaires), Wegener explique qu'il y a plusieurs millions d'années, les terres émergées actuelles ne devaient former qu'un seul et grand continent. La fin du 20ème siècle verra l'avènement de la théorie de la tectonique des plaques suite à la reconnaissance du cycle de Wilson, des rides médio-océaniques, des anomalies magnétiques dans les océans et des sutures océaniques qui représentent les reliques d'océans disparus. Le Cycle de Wilson se caractérise par une suite d'évènements géologiques majeurs pouvant se résumer de la manière suivante : (1) séparation d'un craton continental en deux parties, créant une limite de plaque divergente. C'est ce que l'on appelle un rift; (2) développement et croissance d'un océan entre ces deux blocs. Des roches magmatiques remontent à la surface de la terre et forment une chaîne de montagne sous-marine que l'on appelle ride médio-océanique ou dorsale. L'océan continue de se développer, et des sédiments se déposent à sa surface formant la suite ophiolitique ou trinité de Steinmann; (3) après une phase d'expansion plus ou moins longue, les conditions imposées aux limites des plaques à la surface de la terre changent, et l'océan se met à se refermer par disparition progressive (subduction) de sa croûte océanique sous une croûte continentale par exemple. Ceci crée une nouvelle limite de plaque, convergente cette fois; (4) la subduction de la plaque océanique sous la plaque continentale provoque une remontée de magma formant des chaînes volcaniques à la surface de la Terre ; (5) une fois que la plaque océanique a complètement disparu, les deux blocs préalablement séparés par l'océan font collision, formant ainsi une chaîne de montagne. Les chaînes de montagnes sont de manière générale formées par un empilement plus ou moins complexe de nappes. C'est au coeur de certaines de ces nappes que se trouvent les vestiges de l'océan disparu. Un des objectifs de ce travail était la recherche de ces vestiges dans le domaine téthysien de la Méditerranée orientale. Pour ce faire, nous avons parcourus une grande partie du sud de la Turquie, nous sommes allés à Chypre, dans le Sultanat d'Oman, en Iran, en Crète, et nous avons visités quelques îles grecques du Dodécanèse. La région de la Méditerranée orientale est une zone qui a été tectoniquement très active, et qui continue de l'être de nos jours par des phénomènes de subduction (ex. les volcans de Santorin), et par des mouvements coulissants entre des plaques continentales (ex. la faille nord-anatolienne) qui donnent régulièrement lieu à des tremblements de terre. Pour le géologue, la complexité de ces zones d'étude réside dans le fait que les chaînes de montagne actuelles ne contiennent en général pas seulement les restes d'un océan, mais bien de plusieurs bassins océaniques qui se sont succédés dans l'espace et dans le temps. Les nappes qui se trouvent au sud de la Turquie et dans le Dodécanèse forment un important jalon dans la chaîne alpine qui s'étend depuis les Alpes jusque dans l'Himalaya. L'idée d'un continuum au coeur de ce système se basait principalement sur l'âge des océans et sur la reconnaissance de similarités dans l'évolution des séries sédimentaires. La localisation des vestiges de la Paléotéthys ainsi que l'identification des séries sédimentaires ayant appartenu à l'océan de HugluPinde repris sous forme de nappes en Turquie et en Grèce sont cruciales pour permettre de bonnes corrélations locales et régionales. La reconnaissance, la compréhension et l'interprétation de ces séries sédimentaires permettront d'élaborer un modèle d'évolution géodynamique régional, s'appuyant sur des faits de terrains indiscutables, et prenant en compte les contraintes globales que ce genre d'exercice implique.

The application of trend surface models to the analysis of time factors in Swiss cancer mortality.

To study different temporal components on cancer mortality (age, period and cohort) methods of graphic representation were applied to Swiss mortality data from 1950 to 1984. Maps using continuous slopes ("contour maps") and based on eight tones of grey according to the absolute distribution of rates were used to represent the surfaces defined by the matrix of various age-specific rates. Further, progressively more complex regression surface equations were defined, on the basis of two independent variables (age/cohort) and a dependent one (each age-specific mortality rate). General patterns of trends in cancer mortality were thus identified, permitting definition of important cohort (e.g., upwards for lung and other tobacco-related neoplasms, or downwards for stomach) or period (e.g., downwards for intestines or thyroid cancers) effects, besides the major underlying age component. For most cancer sites, even the lower order (1st to 3rd) models utilised provided excellent fitting, allowing immediate identification of the residuals (e.g., high or low mortality points) as well as estimates of first-order interactions between the three factors, although the parameters of the main effects remained still undetermined. Thus, the method should be essentially used as summary guide to illustrate and understand the general patterns of age, period and cohort effects in (cancer) mortality, although they cannot conceptually solve the inherent problem of identifiability of the three components.

Mechanisms of antifungal resistance in the opportunistic fungus Aspergillus fumigatus

ABSTRACT Aspergillus fumigatus is one of the most prevalent airbone fungal pathogen and can cause severe fatal invasive aspergillosis in immunocompromised patients. Several antifungal agents are available to treat these infections but with limited success. These agents include polyenes (amphotericin B), echinocandins (caspofungin) and azoles, which constitute the most important class with itraconazole (ITC) and voriconazole as major active compounds. Azole-derived antifungal agents target the ergosterol biosynthesis pathway via the inhibition of the lanosterol 14α-demethylase (cyp51/ERG1 1), a cytochrome P450 responsible for the conversion of lanosterol to ergosterol, which is the main component of cell membrane in fungi. A. fumigatus is also found in the environment as a contaminant of rotting plant or present in composting of organic waste. Among antifungal agents used in the environment for crop protection, the class of azoles is also widely used with propiconazole or prochloraz as examples. However, other agents such as dicarboximide (iprodione), phenylamide (benalaxyl) or strobilurin (azoxystrobin) are also used. Emergence of clinical azole-resistant isolates has been described in several European countries. However the incidence of antifungal resistance has not been yet reported in details in Switzerland. In this study, the status of antifungal resistance was investigated on A. fumigatus isolates collected from Swiss hospitals and from different environmental sites and. tested for their susceptibility to several currently used antifungal agents. The data showed a low incidence of resistance for all tested agents among clinical and environmental isolates. Only two azole-resistant environmental isolates were detected and none among the clinical tested isolates. In general, A. fumigatus was susceptible to all antifungals tested in our study, except to azoxystrobin which was the less active agent against all isolates. Since mechanisms of antifungal resistance have been poorly investigated until now in A. fumigatus, this work was aimed 1) to identify A. fumigatus genes involved in antifungal resistance and 2) to test their involvement in the development of resistance in sampled isolates. Therefore, this work proposed to isolate A. fumigatus genes conferring resistance to a drug-hypersusceptible Saccharomyces cerevisiae strain due to a lack of multidrug transporter genes. Several genes were recovered including three distinct efflux transporters (atrF, atrH and mdrA) and a bZip transcription factor, yapA. The inactivation of each transporter in A. fumigatus indicated that the transporters were involved in the basal level of azole susceptibility. The inactivation of YapA led to a hypersusceptibility to H2O2, thus confirming the involvement of this gene in the oxidative stress response of A. fumigatus. The involvement of the abovementioned transporters genes and of other transporters genes identified by genome analysis in azole resistance was tested by probing their expression in some ITC-resistant isolates. Even if upregulation of some transporters genes was observed in some investigated isolates, the correlation between azole resistance and expression levels of all these transporters genes could not be clearly established for all tested isolates. Given these results, the present work addressed 1) alteration in the expression of cyp51A encoding for the azole target enzyme, and 2) mutation(s) in the cyp51A sequence as potential mechanisms of azote resistance in A. . However, overexpression of cyp51A in the investigated isolates was not linked with azote resistance. Since it was reported that mutation(s) in cyp51A were participating in azote resistance in A. fumigatus, a functional complementation of cyp51A cDNAs from ITC-resistant A. fumigatus strains in S. cerevisiae ergl 1 Δ mutant strain was attempted. Expression in S. cerevisiae allowed the testing of these cDNAs with regards to their functionality and involvement in resistance to specific azote compounds. We could demonstrate that Cyp51A protein with a G54E or M220K mutations conferred resistance to specific azoles in S. cerevisiae, therefore suggesting that these mutations were important for the development of azote resistance in A. fumigatus. In conclusion, this work showed a correlation between ITC resistance and mechanisms involving overexpression of transporters and cyp51A mutations in A. fumigatus isolates. However, azole resistance of some isolates has not been solved and thus it will be necessary to approach the study of resistance mechanisms in this fungal species using alternative methodologies. RESUME Aspergillus fumigatus est un champignon opportuniste répandu et est la cause d'aspergilloses invasives le plus souvent fatales chez des patients immunodéprimés. Plusieurs antifongiques sont disponibles afin de traiter ces infections, cependant avec un succès limité. Ces agents incluent les polyènes (amphotericin B), les échinocandines (caspofungin) et les azoles, qui représentent la plus importante classe d'antifongiques avec l'itraconazole (ITC) et le voriconazole comme principaux agents actifs. Les dérivés azolés ciblent la voie de biosynthèse de l'ergostérol via l'inhibition de la lanostérol 14α-demethylase (cyp51/ERG11), un cytochrome P450 impliqué dans la conversion du lanostérol en ergostérol, qui est un composant important de la membrane chez les champignons. A. fumigatus est également répandu dans l'environnement. Parmi les antifongiques employés en agriculture afin de protéger les cultures, les azoles sont aussi largement utilisés. Cependant, d'autres agents tels que les dicarboximides (iprodione), les phenylamides (benalaxyl) et les strobilurines (azoxystrobin) peuvent être également utilisés. L'émergence de souches cliniques résistantes aux azoles a été décrite dans différents pays européens. Cependant, l'incidence d'une telle résistance aux azoles n'a pas encore été reportée en détails en Suisse. Dans ce travail, l'émergence de la résistance aux antifongiques a été étudiée par analyse de souches d'A. fumigatus provenant de milieux hospitaliers en Suisse et de différents sites et leur susceptibilité testée envers plusieurs antifongiques couramment utilisés. Les données obtenues ont montré une faible incidence de la résistance parmi les souches cliniques et environnementales pour les agents testés. Seulement deux souches environnementales résistantes aux azoles ont été détectées et aucune parmi les souches cliniques. Les mécanismes de résistance aux antifongiques ayant été très peu étudiés jusqu'à présent chez A. fumigatus , ce travail a eu aussi pour but 1) d'identifier les gènes d' A. fumigatus impliqués dans la résistance aux antifongiques et 2) de tester leur implication dans la résistance de certaines souches. Ainsi, il a été proposé d'isoler les gènes d' A. fumigatus pouvant conférer une résistance aux antifongiques à une souche de Saccharomyces cerevisiae hypersensible aux antifongiques. Trois transporteurs à efflux (atrF, atrH et mdrA) et un facteur de transcription appartenant à la famille des bZip (YapA) ont ainsi été isolés. L'inactivation, dans une souche d'A. fumigatus, de chacun des ces transporteurs a permis de mettre en évidence leur implication dans la susceptibilité d'A. fumigatus aux antifongiques. L'inactivation de YapA a engendré une hypersusceptibilité à l' H2O2, confirmant ainsi le rôle de ce gène dans la réponse au stress oxydatif chez A . fumigatus. La participation dans la résistance aux antifongiques des gènes codant pour des transporteurs ainsi que d'autres gènes identifiés par analyse du génome a été déterminée en testant leur niveau d'expression dans des souches résistantes à l'ITC. Bien qu'une surexpression de transporteurs ait été observée dans certaines souches, une corrélation entre la résistance à l'ITC et les niveaux d'expression de ces transporteurs n'a pu être clairement établie. Ce présent travail s'est donc porté sur l'étude de 2 autres mécanismes potentiellement impliqués dans la résistance aux azoles : 1) la surexpression de cyp51A codant pour l'enzyme cible et 2) des mutations dans cyp51A. Cependant, la surexpression de cyp51A dans les souches étudiées n'a pas été constatée. L'effet des mutations de cyp51A dans la résistance aux azoles a été testée par complémentation fonctionnelle d'une souche S. cerevisiae déletée dans son gène ERG11. L'expression de ces gènes chez S. cerevisiae a permis de démontrer que les protéines Cyp51Ap contenant une mutation G54E ou M220K pouvaient conférer une résistance spécifique à certains azoles, ainsi suggérant que ces mutations pourraient être importantes dans le développement d'une résistance aux azoles chez A. fumigatus. En conclusion, ce travail a permis de mettre en évidence, dans des souches d'A. fumigatus , une corrélation entre leur résistance à l' ITC et les mécanismes impliquant une surexpression de transporteurs et des mutations dans cyp51A. Cependant, ces mécanismes n'ont pu expliquer la résistance aux azoles de certaines souches et c'est pourquoi de nouvelles approches doivent être envisagées afin d'étudier ces mécanismes.

Activation mechanisms of the protease MALT1 and cleavage of one of its targets - the ribonuclease Regnase-1- in lymphocytes and lymphoma cell lines

Persistent infection induces an adaptive immune response that is mediated by T and B lymphocytes. Upon triggering with an antigen, these cells become activated and turn into fast expanding cells able to efficiently defend the host. Lymphocyte activation is controlled by a complex composed of CARMA1, BCL10 and MALT1 which regulates the NF-KB signaling pathway upon antigen triggering. Abnormally high expression or activity of either one of these three proteins can favor the development of lymphomas, while genetic defects in the pathway are associated with immunodeficiency. MALT1 was identified as a paracaspase sharing homology with other cysteine proteases, namely caspases and metacaspases. In order to be active, caspases need to dimerize. Based on their sequence similarity with MALT1, we hypothesized that dimerization might also be a mechanism of activation employed by MALT1. To address this assumption, we performed a bioinformatics modelling based on the crystal structures of several caspases. Our model suggested that the MALT1 caspase-like domain can indeed form dimers. This finding was later confirmed by several published crystal structures of MALT1. In the dimer interface of our model, we noticed the presence of charged amino acids that could potentially form salt bridges and thereby hold both monomers together. Mutation of one of these residues, E549, into alanine completely blocked the catalytic activity of MALT1. Additionally, we provided evidence for a role of E549 in promoting the MALTl-dependent growth of cells derived from diffuse large B cell lymphoma (DLBCL) of the aggressive B cell-like type (ABC). To our initial surprise, the E549A mutation showed only a partial defect in dimerization, indicating that additional residues are essential to form a stable dimer. The MALT1 crystal structures revealed a key function for E549 in stabilizing the catalytic site of the protease via its interaction with an arginine which is located next to the catalytic active cysteine. In an additional study, we discovered that MALT1 monoubiquitination is required for the catalytic activity of the protease. Interestingly, we found that the MALT1 dimer interface mutant E549A could not be monoubiquitinated. Based on these findings, we suggest that correct formation of the dimer interface is a prerequisite for monoubiquitination. In a second project, we discovered a novel target of the protease MALT1, the ribonuclease Regnase¬la It was described that the RNase activity of Regnase-1 negatively regulates immune responses. We could show that in ABC DLBCL cell lines, Regnase-1 is not only cleaved by MALT1 but also phosphorylated, at least in part, by the inhibitor of KB kinase (IKK). Both regulations appear to restrain the RNase function of Regnase-1 and thereby allow the production of pro-survival proteins. In conclusion, our studies further highlight and explain the importance of the catalytic activity of MALT1 for the activation of lymphocytes and provide additional knowledge for the development of specific drugs targeting the catalytic activity of MALT1 for immunomodulation and treatment of lymphomas.  SUMMARY IN FRENCH PhD Thesis Katrin Cabalzar 2 SUMMARY IN FRENCH Une infection persistante induit une réponse immunitaire adaptative par l'intermédiaire des lymphocytes T et B. Quand elles reconnaissent l'antigène, ces cellules sont activées et se multiplient très rapidement pour défendre efficacement l'hôte. L'activation des lymphocytes est transmise par un complexe composé de trois protéines, CARMA1, BCL10 et MALT1, qui régule la voie de signalisation NF-KB lorsque l'antigène est reconnu. L'expression ou l'activité anormalement élevée de l'une de ces trois protéines peut favoriser le développement de lymphomes, tandis que des défauts génétiques de cette voie de signalisation sont associés à l'immunodéficience. MALT1 a été identifiée comme étant une paracaspase qui partage des séquences homologues avec d'autres protéases à cystéine, comme les caspases et les métacaspases. Pour être actives, les caspases ont besoin de dimériser. Etant donné leur similarité de séquence avec MALT1, nous avons supposé que la dimérisation pouvait aussi être un mécanisme d'activation utilisé par MALT1. Pour vérifier cette hypothèse, nous avons conçu un modèle bioinformatique à partir des structures cristallographiques de plusieurs caspases. Et notre modèle a suggéré que le domaine catalytique de MALT1 était effectivement capable de former des dimères. Cette découverte a été confirmée plus tard par des publications qui montrent des structures cristallographiques dimériques de MALT1. Dans l'interface du dimère de notre modèle, nous avons remarqué la présence d'acides aminés chargés qui pouvaient former des liaisons ioniques et ainsi réunir les deux monomères. La mutation de l'un de ces résidus, E549, pour une alanine, a complètement inhibé l'activité catalytique de MALT1. De plus, nous avons mis en évidence un rôle d'E549 dans la croissance dépendante de MALT1, des cellules dérivées de lymphomes B diffus à grandes cellules (DLBCL) de sous-type cellules B actives (ABC). Dans un premier temps nous avons été surpris de constater que cette mutation révélait seulement un défaut partiel de dimérisation, ce qui indique que des acides aminés supplémentaires sont indispensables pour former un dimère stable. Les structures cristallographiques de MALT1 ont révélé un rôle primordial d'E549 dans la stabilisation du site catalytique de la protéase via son interaction avec une arginine qui se trouve à côté de la cystéine du site actif. Dans une autre étude, nous avons découvert que la monoubiquitination de MALT1 est requise pour l'activité catalytique de la protéase. A remarquer que nous avons trouvé que le mutant E549A de l'interface dimère de MALT1 n'a pas pu être monoubiquitiné. Sur la base de ces résultats, nous suggérons que la formation correcte de l'interface du dimère est une condition préalable pour la monoubiquitination. Dans un second projet, nous avons découvert une nouvelle cible de la protéase MALT1, la ribonucléase Regnase-1. Il a été décrit que l'activité RNase de Regnase-1 régulait négativement les réponses immunitaires. Nous avons pu montrer que dans les lignées cellulaires ABC DLBCL, la Regnase-1 n'était pas seulement clivée par MALT1 mais également phosphorylée, au moins en partie, par la kinase de l'inhibiteur de KB (IKK). Les deux régulations semblent supprimer la fonction RNase de Regnase-1 et permettre ainsi la stabilisation de certains ARN messagers et la production de protéines favorisant la survie. En conclusion, nos études mettent en évidence le rôle-clé de la dimérisation de MALT1 et expliquent l'importance de l'activité catalytique de MALT1 pour l'activation des lymphocytes. Ainsi, nos résultats apportent des connaissances supplémentaires pour le développement de médicaments spécifiques ciblant l'activité catalytique de MALT1, qui pourraient être utiles pour modifier les réponses immunitaires et traiter des lymphomes.

Constitutive activity and inverse agonism at the alpha1adrenoceptors.

Mutations of G protein-coupled receptors (GPCR) can increase their constitutive (agonist-independent) activity. Some of these mutations have been artificially introduced by site-directed mutagenesis, others occur spontaneously in human diseases. The alpha(1B)adrenoceptor was the first GPCR in which point mutations were shown to trigger receptor activation. This article briefly summarizes some of the findings reported in the last several years on constitutive activity of the alpha(1)adrenoceptor subtypes, the location where mutations have been found in the receptors, the spontaneous activity of native receptors in recombinant as well as physiological systems. In addition, it will highlight how the analysis of the pharmacological and molecular properties of the constitutively active adrenoceptor mutants provided an important contribution to our understanding of the molecular mechanisms underlying the mechanism of receptor activation and inverse agonism.

Transcriptional control of the Notch1 tumour suppressor gene

Abstract : The Notch pathway is an important regulator of differentiation and carcinogenesis. In keratinocytes and possibly other specific epithelial cell types, it acts as tumour suppressor. Expression of endogenous Notch1 gene is markedly reduced in keratinocyte-derived squamous cell carcinoma (SCC) and cervical cancer cells, as well as in prostate cancer cell lines, and this difference is, at least in part, at the transcriptional level. Little is known on transcriptional control of the Notch1 gene with the exception that it is a p53-target. Our work focused on the mechanisms involved in the different transcription level of the Notch1 gene in normal versus cancer cells. We show that the fully active minimal Notch1 promoter is differentially controlled in normal versus cancer cells. It consists of two distinct regions, one downstream of the transcription start site, which is likely to bind the basic transcription apparatus, and one upstream region characterized by highly GC-rich sequence. This latter region binds Sp/KLF family members, specifically Spa and KLF4, which is upregulated in cancer cells. This is functionally significant as KLF4 overexpression is sufficient to downmodulate Notchl gene transcription, while KLF4 knockdown, in combination with Spa, results in Notch1 upregulation. Control of Notch1 by KLF4/Sp3 is independent of p53. Biochemically, KLF4/Sp3 seem to affect preferentially the initiation step of Notch1 gene transcription, while p53 controls both initiation and elongation steps. Thus, the Notch1 gene is a negative Sp3/KLF4-target and this mechanism contributes, in parallel with p53, to Notch1 downregulation in cancer. Résumé : La voie de signalisation induite par Notch est considérablement impliquée dans la différenciation des cellules et dans la carcinogénèse. Dans les kératinocytes ainsi que dans d'autres types cellulaires de l'épithelium, il agit comme suppresseur de tumeur. L'expression endogène de Notch1 est remarquablement réduite dans les cellules du carcinome spino-cellulaire et du cancer du col de l'utérus ou dans les lignées cellulaires du cancer de la prostate. Cette différence s'explique, du moins en partie, par le niveau de transcription. Peu de choses sont connues sur le contrôle transcriptionnel de Notch1 à l'exception du fait qu'il soit une cible de p53. Notre travail s'est concentré sur les mécanismes impliqués dans la transcription de Notch1, mécanismes qui diffèrent entre les cellules normales et les cellules cancéreuses. Nous avons trouvé la plus petite région du promoteur de Notch1 qui est suffisante pour induire un haut niveau transcriptionnel et qui est contrôlée différemment dans les cellules normales et les cellules cancéreuses. Elle est constituée de deux régions distinctes: une en aval du site de départ de la transcription, qui lie probablement le complexe de base pour la transcription, et une en amont caractérisée par une séquence riche en GC. Cette région lie les membres de la famille Sp/KLF, spécifiquement Sp3 et KLF4, qui sont surexprimés dans les cellules cancéreuses. Ceci est fonctionnellement significatif car la surexpression de KLF4 dans les kératinocytes est suffisante pour diminuer la transcription de Notch1, alors que l'inhibition de KLF4 et de Spa, résulte en une augmentation de Notch1. En outre, le contrôle de Notch1 par KLF4 et Spa est indépendant de p53. Biochimiquement, KLF4 et Spa semblent plutôt affecter l'initiation de la transcription de Notch1 alors que p53 contrôle aussi bien l'initiation que l'élongation. En conclusion, le gène Notch1 est inhibé par Spa et KLF4: ce mécanisme contribue, en parallèle à p53, à diminuer l'expression de Notch1 dans les cellules cancéreuses.

Fast Geodesic Active Fields for Image Registration Based on Splitting and Augmented Lagrangian Approaches.

In this paper, we present an efficient numerical scheme for the recently introduced geodesic active fields (GAF) framework for geometric image registration. This framework considers the registration task as a weighted minimal surface problem. Hence, the data-term and the regularization-term are combined through multiplication in a single, parametrization invariant and geometric cost functional. The multiplicative coupling provides an intrinsic, spatially varying and data-dependent tuning of the regularization strength, and the parametrization invariance allows working with images of nonflat geometry, generally defined on any smoothly parametrizable manifold. The resulting energy-minimizing flow, however, has poor numerical properties. Here, we provide an efficient numerical scheme that uses a splitting approach; data and regularity terms are optimized over two distinct deformation fields that are constrained to be equal via an augmented Lagrangian approach. Our approach is more flexible than standard Gaussian regularization, since one can interpolate freely between isotropic Gaussian and anisotropic TV-like smoothing. In this paper, we compare the geodesic active fields method with the popular Demons method and three more recent state-of-the-art algorithms: NL-optical flow, MRF image registration, and landmark-enhanced large displacement optical flow. Thus, we can show the advantages of the proposed FastGAF method. It compares favorably against Demons, both in terms of registration speed and quality. Over the range of example applications, it also consistently produces results not far from more dedicated state-of-the-art methods, illustrating the flexibility of the proposed framework.

Appropriateness of initial treatment for severe Crohn's disease: application of EPACT criteria to the EC-IBD European cohort

Background: The appropriateness of use of therapy for severe active luminal Crohn's disease (CD) cases has never been formally assessed. The European panel on the appropriateness of Crohn's disease therapy [EPACT (http://www.epact.ch)] developed appropriateness criteria. We have applied these criteria to the EC-IBD prospectively assembled, uniformly diagnosed European population-based inception cohort of Inflammatory Bowel Disease (IBD) patients diagnosed between 1991 and 1993. Methods: 426 CD patients from 13 European participating centers (10 countries) were included at the time of diagnosis (first flare, naive patients, no maintenance treatment, no steroids). We used the EPACT definition of the severe active luminal CD, agreed upon by the panel experts (acute flare, hospitalized patient, without documented fistula or stenosis and who did not undergo surgery for abscess drainage or a fistulectomy). The various treatments were analyzed to determine the appropriateness of the medical decision, according to the EPACT criteria. Results: 84 (20%) patients met the inclusion criteria. Considering at least one appropriate (A) treatment as appropriate: 60 patients (71%) received an appropriate treatment, 24 patients (29%) an inappropriate treatment (I). Furthermore, in 87% of the cases with one appropriate treatment an additional mostly inappropriate treatment was added or continued. Detailed results are indicated in the table below. Conclusion: In the EC-IBD cohort, the treatment for severe active luminal CD was appropriate for more than 70% of the patients, but frequently an inappropriate treatment was continued or added, thus increasing the risk of adverse reactions, drugs interactions and costs.

A new classification of the Turkish terranes and sutures and its implication for the paleotectonic history of the region

The Turkish part of the Tethyan realm is represented by a series of terranes juxtaposed through Alpine convergent movements and separated by complex suture zones. Different terranes can be defined and characterized by their dominant geological background. The Pontides domain represents a segment of the former active margin of Eurasia, where back-arc basins opened in the Triassic and separated the Sakarya terrane from neighbouring regions. Sakarya was re-accreted to Laurasia through the Balkanic mid-Cretaceous orogenic event that also affected the Rhodope and Strandja zones. The whole region from the Balkans to the Caucasus was then affected by a reversal of subduction and creation of a Late Cretaceous arc before collision with the Anatolian domain in the Eocene. If the Anatolian terrane underwent an evolution similar to Sakarya during the Late Paleozoic and Early Triassic times, both terranes had a diverging history during and after the Eo-Cimmerian collision. North of Sakarya, the Küre back-arc was closed during the Jurassic, whereas north of the Anatolian domain, the back-arc type oceans did not close before the Late Cretaceous. During the Cretaceous, both domains were affected by ophiolite obduction, but in very different ways: north directed diachronous Middle to Late Cretaceous mélange obduction on the Jurassic Sakarya passive margin; Senonian synchronous southward obduction on the Triassic passive margin of Anatolia. From this, it appears that the Izmir-Ankara suture, currently separating both terranes, is composite, and that the passive margin of Sakarya is not the conjugate margin of Anatolia. To the south, the Cimmerian Taurus domain together with the Beydağları domain (part of the larger Greater Apulian terrane), were detached from north Gondwana in the Permian during the opening of the Neotethys (East-Mediterranean basin). The drifting Cimmerian blocks entered into a soft collision with the Anatolian and related terranes in the Eo-Cimmerian orogenic phase (Late Triassic), thus suturing the Paleotethys. At that time, the Taurus plate developed foreland-type basins, filled with flysch-molasse deposits that locally overstepped the lower plate Taurus terrane and were deposited in the opening Neotethys to the south. These olistostromal deposits are characterized by pelagic Carboniferous and Permian material from the Paleotethys suture zone found in the Mersin mélange. The latter, as well as the Antalya and Mamonia domains are represented by a series of exotic units now found south of the main Taurus range. Part of the Mersin exotic material was clearly derived from the former north Anatolian passive margin (Huğlu-type series) and re-displaced during the Paleogene. This led us to propose a plate tectonic model where the Anatolian ophiolitic front is linked up with the Samail/Baër-Bassit obduction front found along the Arabian margin. The obduction front was indented by the Anatolian promontory whose eastern end was partially subducted. Continued slab roll-back of the Neotethys allowed Anatolian exotics to continue their course southwestward until their emplacement along the Taurus southern margin (Mersin) and up to the Beydağları promontory (Antaya-Mamonia) in the latest Cretaceous-Paleocene. The supra-subduction ocean opening at the back of the obduction front (Troodos-type Ocean) was finally closed by Eocene north-south shortening between Africa and Eurasia. This brought close to each other Cretaceous ophiolites derived from the north of Anatolia and those obducted on the Arabian promontory. The latter were sealed by a Maastrichtian platform, and locally never affected by Alpine tectonism, whereas those located on the eastern Anatolian plate are strongly deformed and metamorphosed, and affected by Eocene arc magmatism. These observations help to reconstruct the larger frame of the central Tethyan realm geodynamic evolution.