NS4B Self-Interaction through Conserved C-Terminal Elements Is Required for the Establishment of Functional Hepatitis C Virus Replication Complexes.

Hepatitis C virus (HCV) is an important human pathogen, persistently infecting more than 170 million individuals worldwide. Studies of the HCV life cycle have become possible with the development of cell culture systems supporting the replication of viral RNA and the production of infectious virus. However, the exact functions of individual proteins, especially of nonstructural protein 4B (NS4B), remain poorly understood. NS4B triggers the formation of specific, vesicular membrane rearrangements, referred to as membranous webs, which have been reported to represent sites of HCV RNA replication. However, the mechanism of vesicle induction is not known. In this study, a panel of 15 mutants carrying substitutions in the highly conserved NS4B C-terminal domain was generated. Five mutations had only a minor effect on replication, but two of them enhanced assembly and release of infectious virus. Ten mutants were replication defective and used for selection of pseudoreversions. Most of the pseudoreversions also localized to the highly conserved NS4B C-terminal domain and were found to restore replication competence upon insertion into the corresponding primary mutant. Importantly, pseudoreversions restoring replication competence also restored heterotypic NS4B self-interaction, which was disrupted by the primary mutation. Finally, electron microscopy analyses of membrane alterations induced by NS4B mutants revealed striking morphological abnormalities, which were restored to wild-type morphology by the corresponding pseudoreversion. These findings demonstrate the important role of the C-terminal domain in NS4B self-interaction and the formation of functional HCV replication complexes.

Molecular modeling of peptide-MHC class I complexes : applications to peptide based immunotherapy

Summary The specific CD8+ T cell immune response against tumors relies on the recognition by the T cell receptor (TCR) on cytotoxic T lymphocytes (CTL) of antigenic peptides bound to the class I major histocompatibility complex (MHC) molecule. Such tumor associated antigenic peptides are the focus of tumor immunotherapy with peptide vaccines. The strategy for obtaining an improved immune response often involves the design of modified tumor associated antigenic peptides. Such modifications aim at creating higher affinity and/or degradation resistant peptides and require precise structures of the peptide-MHC class I complex. In addition, the modified peptide must be cross-recognized by CTLs specific for the parental peptide, i.e. preserve the structure of the epitope. Detailed structural information on the modified peptide in complex with MHC is necessary for such predictions. In this thesis, the main focus is the development of theoretical in silico methods for prediction of both structure and cross-reactivity of peptide-MHC class I complexes. Applications of these methods in the context of immunotherapy are also presented. First, a theoretical method for structure prediction of peptide-MHC class I complexes is developed and validated. The approach is based on a molecular dynamics protocol to sample the conformational space of the peptide in its MHC environment. The sampled conformers are evaluated using conformational free energy calculations. The method, which is evaluated for its ability to reproduce 41 X-ray crystallographic structures of different peptide-MHC class I complexes, shows an overall prediction success of 83%. Importantly, in the clinically highly relevant subset of peptide-HLAA*0201 complexes, the prediction success is 100%. Based on these structure predictions, a theoretical approach for prediction of cross-reactivity is developed and validated. This method involves the generation of quantitative structure-activity relationships using three-dimensional molecular descriptors and a genetic neural network. The generated relationships are highly predictive as proved by high cross-validated correlation coefficients (0.78-0.79). Together, the here developed theoretical methods open the door for efficient rational design of improved peptides to be used in immunotherapy. Résumé La réponse immunitaire spécifique contre des tumeurs dépend de la reconnaissance par les récepteurs des cellules T CD8+ de peptides antigéniques présentés par les complexes majeurs d'histocompatibilité (CMH) de classe I. Ces peptides sont utilisés comme cible dans l'immunothérapie par vaccins peptidiques. Afin d'augmenter la réponse immunitaire, les peptides sont modifiés de façon à améliorer l'affinité et/ou la résistance à la dégradation. Ceci nécessite de connaître la structure tridimensionnelle des complexes peptide-CMH. De plus, les peptides modifiés doivent être reconnus par des cellules T spécifiques du peptide natif. La structure de l'épitope doit donc être préservée et des structures détaillées des complexes peptide-CMH sont nécessaires. Dans cette thèse, le thème central est le développement des méthodes computationnelles de prédiction des structures des complexes peptide-CMH classe I et de la reconnaissance croisée. Des applications de ces méthodes de prédiction à l'immunothérapie sont également présentées. Premièrement, une méthode théorique de prédiction des structures des complexes peptide-CMH classe I est développée et validée. Cette méthode est basée sur un échantillonnage de l'espace conformationnel du peptide dans le contexte du récepteur CMH classe I par dynamique moléculaire. Les conformations sont évaluées par leurs énergies libres conformationnelles. La méthode est validée par sa capacité à reproduire 41 structures des complexes peptide-CMH classe I obtenues par cristallographie aux rayons X. Le succès prédictif général est de 83%. Pour le sous-groupe HLA-A*0201 de complexes de grande importance pour l'immunothérapie, ce succès est de 100%. Deuxièmement, à partir de ces structures prédites in silico, une méthode théorique de prédiction de la reconnaissance croisée est développée et validée. Celle-ci consiste à générer des relations structure-activité quantitatives en utilisant des descripteurs moléculaires tridimensionnels et un réseau de neurones couplé à un algorithme génétique. Les relations générées montrent une capacité de prédiction remarquable avec des valeurs de coefficients de corrélation de validation croisée élevées (0.78-0.79). Les méthodes théoriques développées dans le cadre de cette thèse ouvrent la voie du design de vaccins peptidiques améliorés.

Metamorphism on Chromite Ores from the Dobromirtsi ultramafic massif, Rhodope Mountains (SE Bulgaria)

Podiform chromitite bodies occur in highly serpentinized peridotites at Dobromirtsi Ultramafic Massif (Rhodope Mountains, southeastern Bulgaria). The ultramafic body is believed to represent a fragment of Palaeozoic ophiolite mantle. The ophiolite sequence is associated with greenschist - lower-temperature amphibolite facies metamorphosed rocks (biotitic gneisses hosting amphibolite). This association suggests that peridotites, chromitites and metamorphic rocks underwent a common metamorphic evolution. Chromitites at Dobromirtsi have been strongly altered. Their degree of alteration depends on the chromite/silicate ratio and to a lesser extent, on the size of chromitite bodies. Alteration is recorded in individual chromite grains in the form of optical and chemical zoning. Core to rim chemical trends are expressed by MgO- and Al2O3- impoverishment, mainly compensated by FeO and/or Fe2O3 increases. Such chemical variations correspond with three main alteration events. The first one was associated with ocean-floor metamorphism and was characterized by a lizardite replacement of olivine and the absence of chromite alteration. The second event took place during greenchist facies metamorphism. During this event, MgO- and SiO2-rich fluids (derived from low temperature serpentinization of olivine and pyroxenes) reacted with chromite to form chlorite; as a consequence, chromite became altered to a FeO- and Cr2O3-rich, Al2O3-poor chromite. The third event, mainly developed during lower temperature amphibolite facies metamorphism, caused the replacement of the primary and previously altered chromite by Fe2O3-rich chromite (ferritchromite).

Stress reaction of kidney epithelial cells to inorganic solid-core nanoparticles.

A route of accumulation and elimination of therapeutic engineered nanoparticles (NPs) may be the kidney. Therefore, the interactions of different solid-core inorganic NPs (titanium-, silica-, and iron oxide-based NPs) were studied in vitro with the MDCK and LLC-PK epithelial cells as representative cells of the renal epithelia. Following cell exposure to the NPs, observations include cytotoxicity for oleic acid-coated iron oxide NPs, the production of reactive oxygen species for titanium dioxide NPs, and cell depletion of thiols for uncoated iron oxide NPs, whereas for silica NPs an apparent rapid and short-lived increase of thiol levels in both cell lines was observed. Following cell exposure to metallic NPs, the expression of the tranferrin receptor/CD71 was decreased in both cells by iron oxide NPs, but only in MDCK cells by titanium dioxide NPs. The tight association, then subsequent release of NPs by MDCK and LLC-PK kidney epithelial cells, showed that following exposure to the NPs, only MDCK cells could release iron oxide NPs, whereas both cells released titanium dioxide NPs. No transfer of any solid-core NPs across the cell layers was observed.

Characterization of the role of Rho activating signalling complexes in G protein-coupled receptor induced cardiac fibrosis

Dans certaines conditions pathologiques, telles que l'hypertension artérielle ou l'infarctus du myocarde, le coeur répond à une augmentation de la post-charge par des processus de remodelage aboutissant à une hypertrophie du ventricule gauche. L'hypertrophie cardiaque est caractérisée par une croissance hypertrophique des cardiomyocytes, ainsi que par une différenciation des fibroblastes en un phenotype présentant une capacité accrue de synthèse protéiques, nommés myofibroblastes. Ceci résulte en une accumulation excessive des constituants de la matrice extracellulaire, ou autrement dit fibrose. En raison de son effet délétère sur la contractilité du coeur, menant sur le long terme à une insuffisance cardiaque, de nombreux efforts ont été déployés, afin de définir les mécanismes moléculaires impliqués dans la réponse profibrotique. A ce jour, de nombreuses études indiquent que la petite GTPase RhoA pourrait être un médiateur important de la réponse profibrotique du myocarde. Cependant, les facteurs d'échanges impliqués dans la transduction de signaux profibrotiques, via la régulation de son activité au niveau des fibroblastes cardiaques, n'ont pas encore été identifiés. De précédentes études menées dans le laboratoire, ont identifiées une nouvelle protein d'ancrage de la PKA, exprimée majoritairement dans le coeur, nommée AKAP-Lbc. Il a été montré que cette protéine, en plus de sa fonction de protein d'ancrage, possédait une activité de facteur d'échange de nucléotide guanine (GEF) pour la petite GTPase RhoA. Au niveau des cardiomyocytes, il a été montré que l'AKAP-Lbc participe à une voie de signalisation pro-hypertrophique, incluant la sous-unité alpha de la protéine G hétérotrimerique G12 et RhoA. Chose intéressante, des observations antérieures à cette étude, indiquent que dans le coeur, l'AKAP-Lbc est également exprimée dans les fibroblastes. Cependant aucunes études n'a encore reporté de fonction pour ce facteur d'échange dans les fibroblastes cardiaques. Dans ce travail, les résultats obtenus indiquent que dans les fibroblastes cardiaques, I'activation de RhoA par l'AKAP-Lbc est impliquée dans la transmission de signaux profibrotiques, en aval des récépteurs à l'angiotensine II. En particulier, nous avons observé que la suppression de l'expression de l'AKAP-Lbc dans les fibroblastes ventriculaires de rat adultes, réduisait fortement Γ activation de Rho induite par l'angiotensine II, la déposition de collagène, la capacité migratoire des fibroblastes ainsi que leur différenciation en myofibroblastes. A notre connaissance, l'AKAP-Lbc est le premier RhoGEF identifié comme médiateur de la réponse profibrotique dans les fibroblastes cardiaques. - In pathological conditions such as chronic hypertension or myocardial infarction, the myocardium is subjected to various biomechanical and biochemical stresses, and undergoes an adverse ventricular remodelling process associated with cardiomyocytes hypertrophy and excess deposition of extracellular matrix proteins resulting in fibrosis. During the fibrotic response, cardiac fibroblasts differentiate into a more mobile and contractile phenotype termed myofibroblasts. These cells, possess a greater synthetic ability to produce ECM proteins and have been implicated in diseases with increased ECM deposition including cardiac fibrosis. Because fibrosis impairs myocardial contractility and is associated with the progression to heart failure, a major cause of lethality worldwide, many efforts have been made to define the molecular players involved in this process. During these last years, increasing evidence suggests a role for the small GTPase RhoA in mediating the fibrotic response in CFbs. However the identity of the exchange factors that modulate its activity and transduce fibrotic signals in CFbs is still unknown. Earlier work in our laboratory identified a novel PKA anchoring protein expressed in the heart termed AKAP-Lbc that has been shown to function as anchoring protein as well as a guanine nucleotide exchange factor (GEF) for the small GTPase RhoA. In response to several hypertrophic stimuli we have shown that RhoGEF activity of AKAP-Lbc mediated by Gan promotes the activation of a signaling pathway including RhoA, leading to cardiomyocytes hypertrophy. Within the heart, previous observations made in the laboratory indicated that AKAP-Lbc was also expressed in fibroblasts. However its role in cardiac fibroblasts remained to be determined. In the present study, we show that AKAP-Lbc is critical for activating RhoA and transducing profibrotic signals downstream of angiotensin II receptors in cardiac fibroblasts. In particular, our results indicate that suppression of AKAP-Lbc expression by infecting adult rat ventricular fibroblasts with lentiviruses encoding AKAP-Lbc specific short hairpin RNAs strongly reduces angiotensin II-induced RhoA activation, collagen deposition as well as cell migration and differentiation. These findings identify AKAP-Lbc as the first Rho-guanine nucleotide exchange factor involved in a profibrotic signalling pathway at the level of cardiac fibroblasts.

Scientific value of systematic reviews: survey of editors of core clinical journals.

BACKGROUND: Synthesizing research evidence using systematic and rigorous methods has become a key feature of evidence-based medicine and knowledge translation. Systematic reviews (SRs) may or may not include a meta-analysis depending on the suitability of available data. They are often being criticised as 'secondary research' and denied the status of original research. Scientific journals play an important role in the publication process. How they appraise a given type of research influences the status of that research in the scientific community. We investigated the attitudes of editors of core clinical journals towards SRs and their value for publication.¦METHODS: We identified the 118 journals labelled as "core clinical journals" by the National Library of Medicine, USA in April 2009. The journals' editors were surveyed by email in 2009 and asked whether they considered SRs as original research projects; whether they published SRs; and for which section of the journal they would consider a SR manuscript.¦RESULTS: The editors of 65 journals (55%) responded. Most respondents considered SRs to be original research (71%) and almost all journals (93%) published SRs. Several editors regarded the use of Cochrane methodology or a meta-analysis as quality criteria; for some respondents these criteria were premises for the consideration of SRs as original research. Journals placed SRs in various sections such as "Review" or "Feature article". Characterization of non-responding journals showed that about two thirds do publish systematic reviews.¦DISCUSSION: Currently, the editors of most core clinical journals consider SRs original research. Our findings are limited by a non-responder rate of 45%. Individual comments suggest that this is a grey area and attitudes differ widely. A debate about the definition of 'original research' in the context of SRs is warranted.

Two-stage prograde and retrograde Variscan metamorphism of glaucophane-eclogites, blueschists and greenschists from Ile de Groix (Brittany, France)

On Ile de Groix, Variscan metamorphic former tholeiitic and alkaline basalts occur as glaucophane-eclogites, blueschists and greenschists in isolated lenses and layers within metapelites. Whole-rock delta O-18(SMOW) values of the metabasites show limited variations (10.4-12.0 parts per thousand) and no systematic differences among rock types and metamorphic grades. This provides no argument for large-scale blueschist-to-greenschist transformation driven by infiltration of externally derived fluids. Metamorphic mineralogical changes should have been triggered by internal fluids. Element variations in interlayered blue- and greenschists can be attributed to magmatic fractionation. Assemblages with garnet, clinopyroxene and glaucophane of a high-pressure/low-temperature (HP-LT) metamorphism M1, and NaCa-amphiboles (barroisite, magnesiohornblende, actinolite) of a medium-pressure/medium-temperature metamorphism M2 crystallized during deformation Dl. Detailed core-rim zonation profiles display increasing and then decreasing Al-IV in glaucophane of M1. NaCa-amphiboles of M2, mantling glaucophane and crystallized in porphyroblasts, show first increasing, then decreasing, Al-IV and Al-IV. Empirically calibrated thermobarometers allowed P-T path reconstructions. In glaucophane-eclogites of a metamorphic zone I, a prograde evolution to M1 peak conditions at 400-500 degreesC/10-12 kbar was followed by a retrograde P-T path within the glaucophane stability field. The subsequent M2 evolution was again prograde up to > 600 degreesC at 8 kbar and then retrograde. Similarly, in metamorphic zones II and III, prograde and retrograde paths of MI and M2 at lower maximal temperatures and pressures exist. The almost complete metamorphic cycle during M2 signalizes that the HP-LT rocks escaped from an early erosion by a moderate second burial event and explains the longlasting slow uplift with low average cooling rates.

On the monotonic core

The monotonic core of a cooperative game with transferable utility (T.U.-game) is the set formed by all its Population Monotonic Allocation Schemes. In this paper we show that this set always coincides with the core of a certain game associated to the initial game.

Impact of precipitation on the water table level of different ombrotrophic raised bog complexes, central Estonia

Sadannan vaikutus vedenpinnan tasoon kohosuolla

On the dimension of the core of the assignment game

The set of optimal matchings in the assignment matrix allows to define a reflexive and symmetric binary relation on each side of the market, the equal-partner binary relation. The number of equivalence classes of the transitive closure of the equal-partner binary relation determines the dimension of the core of the assignment game. This result provides an easy procedure to determine the dimension of the core directly from the entries of the assignment matrix and shows that the dimension of the core is not as much determined by the number of optimal matchings as by their relative position in the assignment matrix.

Multi-sided Böhm-Bawerk assignment markets: the nucleolus and the core-center

En aquest treball mostrem que, a diferència del cas bilateral, per als mercats multilaterals d'assignació coneguts amb el nom de Böhm-Bawerk assignment games, el nucleolus i el core-center, i. e. el centre de masses del core, no coincideixen en general. Per demostrar-ho provem que donant un m-sided Böhm-Bawerk assignment game les dues solucions anteriors poden obtenir-se respectivament del nucleolus i el core-center d'un joc convex definit en el conjunt format pels m sectors. Encara més, provem que per calcular el nucleolus d'aquest últim joc només les coalicions formades per un jugador o m-1 jugadors són importants. Aquests resultats simplifiquen el càlcul del nucleolus d'un multi-sided ¿¿ohm-Bawerk assignment market amb un número molt elevat d'agents.

The Lorenz-maximal core allocations and the kernel in some classes of assignment games

En aquest treball demostrem que en la classe de jocs d'assignació amb diagonal dominant (Solymosi i Raghavan, 2001), el repartiment de Thompson (que coincideix amb el valor tau) és l'únic punt del core que és maximal respecte de la relació de dominància de Lorenz, i a més coincideix amb la solucié de Dutta i Ray (1989), també coneguda com solució igualitària. En segon lloc, mitjançant una condició més forta que la de diagonal dominant, introduïm una nova classe de jocs d'assignació on cada agent obté amb la seva parella òptima almenys el doble que amb qualsevol altra parella. Per aquests jocs d'assignació amb diagonal 2-dominant, el repartiment de Thompson és l'únic punt del kernel, i per tant el nucleolo.

Regular population monotonic allocation schemes and the core

A subclass of games with population monotonic allocation schemes is studied, namelygames with regular population monotonic allocation schemes (rpmas). We focus on theproperties of these games and we prove the coincidence between the core and both theDavis-Maschler bargaining set and the Mas-Colell bargaining set

Ubiquitin-specific protease 2-45 (Usp2-45) binds to epithelial Na+ channel (ENaC)-ubiquitylating enzyme Nedd4-2.

Regulation of the epithelial Na(+) channel (ENaC) by ubiquitylation is controlled by the activity of two counteracting enzymes, the E3 ubiquitin-protein ligase Nedd4-2 (mouse ortholog of human Nedd4L) and the ubiquitin-specific protease Usp2-45. Previously, Usp2-45 was shown to decrease ubiquitylation and to increase surface function of ENaC in Xenopus laevis oocytes, whereas the splice variant Usp2-69, which has a different N-terminal domain, was inactive toward ENaC. It is shown here that the catalytic core of Usp2 lacking the N-terminal domain has a reduced ability relative to Usp2-45 to enhance ENaC activity in Xenopus oocytes. In contrast, its catalytic activity toward the artificial substrate ubiquitin-AMC is fully maintained. The interaction of Usp2-45 with ENaC exogenously expressed in HEK293 cells was tested by coimmunoprecipitation. The data indicate that different combinations of ENaC subunits, as well as the α-ENaC cytoplasmic N-terminal but not C-terminal domain, coprecipitate with Usp2-45. This interaction is decreased but not abolished when the cytoplasmic ubiquitylation sites of ENaC are mutated. Importantly, coimmunoprecipitation in HEK293 cells and GST pull-down of purified recombinant proteins show that both the catalytic domain and the N-terminal tail of Usp2-45 physically interact with the HECT domain of Nedd4-2. Taken together, the data support the conclusion that Usp2-45 action on ENaC is promoted by various interactions, including through binding to Nedd4-2 that is suggested to position Usp2-45 favorably for ENaC deubiquitylation.

The assignment game: core bounds for mixed-pair coalitions

In the assignment game framework, we try to identify those assignment matrices in which no entry can be increased without changing the coreof the game. These games will be called buyer¿seller exact games and satisfy the condition that each mixed¿pair coalition attains the corresponding matrix entry in the core of the game. For a given assignment game, a unique buyerseller exact assignment game with the same core is proved to exist. In order to identify this matrix and to provide a characterization of those assignment games which are buyer¿seller exact in terms of the assignment matrix, attainable upper and lower core bounds for the mixed¿pair coalitions are found. As a consequence, an open question posed in Quint (1991) regarding a canonical representation of a ¿45o¿lattice¿ by means of the core of an assignment game can now be answered