On the transcriptional role of NLRC5 and the function of NLRC5-driven MHC class I expression in the immune system

Major histocompatibility complex (MHC) molecules are of crucial importance for the immune system to recognize and defend the body against external attacks. Foreign antigens are presented by specialized cells, called antigen presenting cells, to T lymphocytes in the context of MHC molecules, thereby inducing T cell activation. In addition, MHC molecules are essential for Natural Killer (NK) cell biology, playing a role in NK cell education and activation. Recently, the NOD-like receptor (NLR) family member NLRC5 (NLR caspase recruitment domain containing protein 5) was found to act as transcriptional regulator of MHC class I, in particular in T and NK cells. Its role in MHC class I expression is however minor in dendritic cells (DCs). This raised the question of whether inflammatory conditions, which augment the levels of NLRC5 in DCs, could increase its contribution to MHC class I expression. Our work shows that MHC class I transcript and intracellular levels depend on NLRC5, while its role in MHC class I surface expression is instead negligible. We describe however a general salvage mechanism that enables cells with low intracellular MHC class I levels to nevertheless maintain relatively high MHC class I on the cell surface. In addition, we lack a thorough understanding of NLRC5 target gene specificity and mechanism of action. Our work delineates the unique consensus sequence in MHC class I promoters required for NLRC5 recruitment and pinpoints conserved features conferring its specificity. Furthermore, through genome-wide analyses, we confirm that NLRC5 regulates classical MHC class I genes and identify novel target genes all encoding non-classical MHC class I molecules exerting an array of functions in immunity and tolerance. We finally asked why a dedicated factor co-regulates MHC class I expression specifically in T and NK lymphocytes. We show that deregulated NLRC5 expression affects the education of NK cells and alters the crosstalk between T and NK cells, leading to NK cell-mediated killing of T lymphocytes. Altogether this thesis work brings insights into molecular and physiological aspects of NLRC5 function, which might help understand certain aspects of immune responses and disorders. -- Les molécules du complexe majeur d'histocompatibilité (CMH) sont essentielles au système immunitaire pour l'initiation de la réponse immunitaire. En effet, l'activation des lymphocytes T nécessite la reconnaissance d'un antigène étranger présenté par les cellules présentatrices d'antigènes sur une molécule du CMH. Les molécules du CMH ont également un rôle fondamental pour la fonction des cellules Natural Killer (NK) puisqu'elles sont nécessaires à leur processus d'éducation et d'activation. Récemment, NLRC5 (NLR caspase recruitment domain containing protein 5), un membre de la famille des récepteurs de type NOD (NLRs), a été décrit comme un facteur de transactivation de l'expression des gènes du CMH de classe I. A l'état basai, cette fonction transcriptionnelle est essentielle dans les lymphocytes T et NK, alors que ce rôle reste mineur pour l'expression des molécules du CMH de classe I dans les cellules dendritiques (DCs). Dans des conditions inflammatoires, l'expression de NLRC5 augmente dans les DCs. Notre travail démontre que, dans ces conditions, les transcrits et les niveaux intracellulaires des molécules du CMH de classe I augmentent aussi d'une façon dépendante de NLRC5. A contrario, le rôle de NLRC5 sur les niveaux de molécules de surface reste minoritaire. Cette observation nous a conduits à l'identification d'un mécanisme général de compensation qui permet aux cellules de maintenir des niveaux relativement élevés de molécules de CMH de class I à leur surface malgré de faibles niveaux intracellulaires. De plus, il semblait nécessaire de s'orienter vers une approche plus globale afin de déterminer l'étendue de la fonction transcriptionnelle de NLRC5. Par une approche du génome entier, nous avons pu décrire une séquence consensus conservée présente dans les promoteurs des gènes du CMH de classe I, sur laquelle NLRC5 est spécifiquement recruté. Nous avons pu également identifier de nouveaux gènes cibles codant pour des molécules de CMH de classe I non classiques impliqués dans l'immunité et la tolérance. Finalement, nous nous sommes demandé quel est l'intérêt d'avoir un facteur transcriptionnel, en l'occurrence NLRC5, qui orchestre l'expression du CMH de classe I dans les lymphocytes T et NK. Nous montrons que la dérégulation de l'expression de NLRC5 affecte l'éducation des cellules NK et conduit à la mort cellulaire des lymphocytes T médiée par les cellules NK. Dans l'ensemble ce travail de thèse contribue à la caractérisation du rôle de NLRC5, tant au niveau moléculaire que physiologique, ce qui présente un intérêt dans le cadre de la compréhension de certains aspects physiopathologique de la réponse immunitaire.

Molecular mechanism of a green-shifted, pH-dependent red fluorescent protein mKate variant.

Fluorescent proteins that can switch between distinct colors have contributed significantly to modern biomedical imaging technologies and molecular cell biology. Here we report the identification and biochemical analysis of a green-shifted red fluorescent protein variant GmKate, produced by the introduction of two mutations into mKate. Although the mutations decrease the overall brightness of the protein, GmKate is subject to pH-dependent, reversible green-to-red color conversion. At physiological pH, GmKate absorbs blue light (445 nm) and emits green fluorescence (525 nm). At pH above 9.0, GmKate absorbs 598 nm light and emits 646 nm, far-red fluorescence, similar to its sequence homolog mNeptune. Based on optical spectra and crystal structures of GmKate in its green and red states, the reversible color transition is attributed to the different protonation states of the cis-chromophore, an interpretation that was confirmed by quantum chemical calculations. Crystal structures reveal potential hydrogen bond networks around the chromophore that may facilitate the protonation switch, and indicate a molecular basis for the unusual bathochromic shift observed at high pH. This study provides mechanistic insights into the color tuning of mKate variants, which may aid the development of green-to-red color-convertible fluorescent sensors, and suggests GmKate as a prototype of genetically encoded pH sensors for biological studies.

Investigation of classical epidemiological links between patients harbouring identical, non-predominant meticillin-resistant Staphylococcus aureus genotypes and lessons for epidemiological tracking.

According to molecular epidemiology theory, two isolates belong to the same chain of transmission if they are similar according to a highly discriminatory molecular typing method. This has been demonstrated in outbreaks, but is rarely studied in endemic situations. Person-to-person transmission cannot be established when isolates of meticillin-resistant Staphylococcus aureus (MRSA) belong to endemically predominant genotypes. By contrast, isolates of infrequent genotypes might be more suitable for epidemiological tracking. The objective of the present study was to determine, in newly identified patients harbouring non-predominant MRSA genotypes, whether putative epidemiological links inferred from molecular typing could replace classical epidemiology in the context of a regional surveillance programme. MRSA genotypes were defined using double-locus sequence typing (DLST) combining clfB and spa genes. A total of 1,268 non-repetitive MRSA isolates recovered between 2005 and 2006 in Western Switzerland were typed: 897 isolates (71%) belonged to four predominant genotypes, 231 (18%) to 55 non-predominant genotypes, and 140 (11%) were unique. Obvious epidemiological links were found in only 106/231 (46%) patients carrying isolates with non-predominant genotypes suggesting that molecular surveillance identified twice as many clusters as those that may have been suspected with classical epidemiological links. However, not all of these molecular clusters represented person-to-person transmission. Thus, molecular typing cannot replace classical epidemiology but is complementary. A prospective surveillance of MRSA genotypes could help to target epidemiological tracking in order to recognise new risk factors in hospital and community settings, or emergence of new epidemic clones.

Population genetics of Capercaillie (Tetrao urogallus) in the Jura and the Pyrenees: a non-invasive approach to avian conservation genetics

RÉSUMÉ Le Grand tétras est un galliforme de montagne apparenté au faisan et au tétras lyre. Il est distribué de manière continue à travers la toundra et les montagnes de moyenne altitude en Europe de l'ouest. Toutefois, les populations d'Europe de l'ouest ont subi un déclin constant au cours des derniers siècles. Les causes de ce déclin sont probablement liées à l'activité humaine, telle .que l'élevage ou le tourisme, qui ont engendré une modification et une fragmentation de l'habitat de l'espèce. Malheureusement, les populations soumises à de forts déclins démographiques peuvent subir des effets génétiques (augmentation de la consanguinité et perte de diversité génétique) pouvant diminuer leur potentiel de reproduction et conduire irrémédiablement à l'extinction. Cette thèse présente les analyses conduites dans le but d'estimer l'impact du déclin démographique des populations de Grand tétras sur l'étendue et la distribution de leur variabilité génétique dans le Jura et dans les Pyrénées. Du fait de la législation locale protégeant les tétraonidés en général, mais également en raison de la biologie très cryptique du Grand tétras, l'ensemble des analyses de cette étude a été réalisé à partir de matériel génétique extrait des fientes (ou échantillonnage génétique non invasif). Dans la première partie de l'étude, je détaille les protocoles d'extraction. d'ADN et d'amplification par PCR modifiés à partir des protocoles classiques utilisant des échantillons conventionnels, riches en ADN. L'utilisation d'ADN fécal impose des contraintes dues à la mauvaise qualité et à la faible quantité du matériel génétique à disposition dans les fientes. Ces contraintes ont pu être partiellement contournées en réalisant des répétitions multiples du génotypage afin d'obtenir un degré de fiabilité suffisante. J'ai également analysé les causes de la dégradation de l'ADN dans les excréments. Parmi les causes les plus communes, telles que l'activité bactérienne, l'hydrolyse spontanée et la dégradation enzymatique par les DNases libres, c'est ce dernier facteur qui apparaît comme étant la cause majeure et la plus rapide responsable de la dégradation de la qualité des échantillons. La rapidité de l'action enzymatique suggère que les plans d'échantillonnages de excréments sur le terrain pourraient être optimisés en les réalisant dans des conditions climatiques froides et sèches, favorisant ainsi l'inhibition des DNases. La seconde partie de la thèse est une étude par simulation visant à déterminer la capacité du logiciel Structure à identifier les structures génétiques complexes et hiérarchiques fréquemment rencontrées dans les populations naturelles, et ce en utilisant différents types de marqueurs génétiques. Les troisième et quatrième parties de cette thèse décrivent le statut génétique des populations résiduelles du Jura et des Pyrénées à partir de l'analyse de 11 loci microsatellites. Nous n'avons pas pu mettre en évidence dans les deux populations des effets liés à la consanguinité ou à la réduction de la diversité génétique. De plus, la différenciation génétique entre les patches d'habitats favorables reste modérée et corrélée à la distance géographique, ce qui suggère que la dispersion d'individus entre les patches a été importante au moins pendant ces dernières générations. La comparaison des paramètres de la diversité génétique avec ceux d'autres populations de Grand tétras, ou d'autres espèces proches, indique que la population du Jura a retenu une proportion importante de sa diversité originelle. Ces résultats suggèrent que le déclin récent des populations a jusqu'ici eu un impact modéré sur les facteurs génétiques et que ces populations semblent avoir conservé le potentiel génétique nécessaire à leur survie à long terme. Finalement, en cinquième partie, l'analyse de l'apparentement entre les mâles qui participent à la parade sur les places de chant (leks) indique que ces derniers sont distribués en agrégats de manière non aléatoire, préférentiellement entre individus apparentés. De plus, la corrélation entre les distances génétique et géographique entre les leks est en accord avec les motifs d'isolement par la distance mis en évidence à d'autres niveaux hiérarchiques (entre patches d'habitat et populations), ainsi qu'avec les études menées sur d'autres espèces ayant choisi ce même système de reproduction. En conclusion, cette première étude basée uniquement sur de l'ADN nucléaire aviaire extrait à partir de fèces a fourni des informations nouvelles qui n'auraient pas pu être obtenues par une méthode d'observation sur le terrain ou d'échantillonnage génétique classique. Aucun oiseau n'a été dérangé ou capturé, et les résultats sont comparables à d'autres études concernant des espèces proches. Néanmoins, la taille de ces populations approche des niveaux au-dessous desquels la survie à long terme est fortement incertaine. La persistance de la diversité génétique pour les prochaines générations reste en conséquence liée à la survie des adultes et à une reprise du succès de la reproduction. ABSTRACT Capercaillie (Tetrao urogallus) is a large grouse that is continuously distributed across the tundra and the mid-high mountains of Western Europe. However, the populations in Western Europe have been showing a constant decline during the last decades. The causes for this decline are possibly related to human activities, such as cattle breeding and tourism that have both led to habitat modification and fragmentation. Unfortunately, populations that have undergone drastic demographic bottlenecks often go through genetic processes of inbreeding and loss of diversity that decrease their fitness and eventually lead to extinction. This thesis presents the investigations conducted to estimate the impact of the demographic decline of capercaillie populations on the extent and distribution of their genetic variability in the Jura and in the Pyrenees mountains. Because grouse are protected by wildlife legislation, and also because of the cryptic behaviour of capercaillie, all DNA material used in this study was extracted from faeces (non-invasive genetic sampling). In the first part of my thesis, I detail the protocols of DNA extraction and PCR amplification adapted from classical methods using conventional DNA-rich samples. The use of faecal DNA imposes specific constraints due to the low quantity and the highly degraded genetic material available. These constraints are partially overcome by performing multiple genotyping repetitions to obtain sufficient reliability. I also investigate the causes of DNA degradation in faeces. Among the main degraders, namely bacterial activity, spontaneous hydrolysis, and free-¬DNase activities, the latter was pointed out as the most important according to our experiments. These enzymes degrade DNA very rapidly, and, as a consequence, faeces sampling schemes must be planned preferably in cold and dry weather conditions, allowing for enzyme activity inhibition. The second part of the thesis is a simulation study aiming to assess the capacity of the software Structure to detect population structure in hierarchical models relevant to situations encountered in wild populations, using several genetic markers. The methods implemented in Structure appear efficient in detecting the highest hierarchical structure. The third and fourth parts of the thesis describe the population genetics status of the remaining Jura and Pyrenees populations using 11 microsatellite loci. In either of these populations, no inbreeding nor reduced genetic diversity was detected. Furthermore, the genetic differentiation between patches defined by habitat suitability remains moderate and correlated with geographical distance, suggesting that significant dispersion between patches was at work at least until the last generations. The comparison of diversity indicators with other species or other populations of capercaillie indicate that population in the Jura has retained a large part of its original genetic diversity. These results suggest that the recent decline has had so forth a moderate impact on• genetic factors and that these populations might have retained the potential for long term survival, if the decline is stopped. Finally, in the fifth part, the analysis of relatedness between males participating in the reproduction parade, or lek, indicate that capercaillie males, like has been shown for some other grouse species, gather on leks• among individuals that are more related than the average of the population. This pattern appears to be due to both population structure and kin-association. As a conclusion, this first study relying exclusively on nuclear DNA extracted from faeces has provided novel information that was not available through field observation or classical genetic sampling. No bird has been captured or disturbed, and the results are consistent with other studies of closely related species. However, the size of these populations is approaching thresholds below which long-term survival is unlikely. The persistence of genetic diversity for the forthcoming generations remains therefore bond to adult survival and to the increase of reproduction success.

NS2 protein of hepatitis C virus interacts with structural and non-structural proteins towards virus assembly.

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.

The philosophical foundations of classical "rDzogs chen" in Tibet : investigating the distinction between dualistic mind (sems) and primordial knowing (ye shes)

While the syncretistic Tibetan tradition known as rDzogs chen ("Great Perfection") has attracted considerable attention over the past few decades, its philosophical foundations remain largely unknown to those unacquainted with its primary sources. This thesis looks at the essentials of rDzogs chen philosophy through the lens of two principal distinctions that the tradition has considered indispensable for understanding its distinctive views and practices: dualistic mind (sems) versus primordial knowing (ye shes) and dharmakâya versus the 'ground of all' (kun gzhi) conditioned experience. Arguing that the distinctions provided classical rDzogs chen scholars with a crucial framework for (a) articulating the necessary conditions of nondual primordial knowing, the conditio sine qua non of rNying ma soteriology, and (b) schematizing the relationship between the exoteric and esoteric vehicles of Indian Buddhism within a unifying conception of the Buddhist path as the progressive disclosure of primordial knowing, the thesis shows how the rDzogs chen philosophy of mind has been integral to the tradition's complex soteriology. The thesis consists of two parts: (1) a detailed philosophical investigation of the distinctions and (2) an anthology of previously untranslated Tibetan materials on the distinctions accompanied by critical editions and introductions. The first part systematically invesigates the nature and scope of the distinctions and traces their evolution and complex relationships with Indian Buddhist Cittamâtra, Madhyamaka, Pramàriavàda, and Vajrayâna views. It concludes with an exploration of some soteriological implications of the mind/primordial knowing distinction that became central to rDzogs chen path hermeneutics in the classical period as authors of rDzogs chen path summaries used this distinction to reconcile progressivist sutric and non-progressivist tantric models of the Buddhist path. The translations and texts included in part two of the thesis consist of (a) a short treatise from Klong chen pa's Miscellaneous Writings entitled Sems dang ye shes kyi dris lan (Reply to Questions Concerning Mind and Primordial Knowing), (b) selected passages on the distinctions from this author's monumental summary of the rDzogs chen snying thig system, the Theg mchog mdzod (Treasury of the Supreme Vehicle), and (c) an excerpt on rDzogs chen distinctions taken from 'Jigs med gling pa's (1729-1798) 18th century Klong chen sNying thig path summary entitled Treasury of Qualities (Yon tan mdzod) along with a word-by- word commentary by Yon tan rgya mtsho (b. 19th c.).

Insulin-like growth factor-I and C-reactive protein during pregnancy and maternal risk of non-epithelial ovarian cancer: a nested case-control study.

BACKGROUND: Insulin-like growth factor-I (IGF-I) and C-reactive protein (CRP) may be positively associated with the risk of epithelial ovarian cancer (EOC) but no previous studies have investigated their associations with non-epithelial ovarian cancers (NEOC). METHODS: A case-control study was nested within the Finnish Maternity Cohort. Case subjects were 58 women diagnosed with sex cord-stromal tumors (SCST) and 30 with germ cell tumors (GCT) after recruitment. Control subjects (144 for SCST and 74 for GCT) were matched for age, parity, and date of blood donation of the index case. RESULTS: Doubling of IGF-I concentration was not related to maternal risk of either SCST (OR 0.97, 95% CI 0.58-1.62) or GCT (OR 1.13, 95% CI 0.51-2.51). Similarly, doubling of CRP concentrations was not related to maternal risk of either SCST (OR 1.10, 95% CI 0.85-1.43) or GCT (OR 0.93, 95% CI 0.68-1.28). CONCLUSIONS: Pre-diagnostic IGF-I and CRP concentrations during the first trimester of pregnancy were not associated with increased risk of NEOC in the mother. Risk factors for NEOC may differ from those of EOC.

Differential migration of in vivo primed B and T lymphocytes to lymphoid and non-lymphoid organs.

Our study describes tissue-specific migration of T and B cells during a localized anti-viral immune response. After mouse mammary tumor virus (MMTV) injection, B lymphocytes of the draining lymph node become infected and present a retroviral superantigen to CD4(+) T lymphocytes. Infected B cells receive superantigen-mediated help in a fashion comparable to classical immune responses. To investigate the fate of T and B lymphocytes that had interacted via cognate help in the same peripheral lymph node microenvironment we adoptively transferred them into naive recipients. Here we show that MMTV-infected B cells and superantigen-stimulated T cells were programmed to migrate to distinct sites of the body. Plasmablasts but not T cells migrated to the mammary gland and activated alpha4beta1 integrins were found to have a crucial role in the migration to the mammary gland. In contrast, T cells had a much higher affinity for secondary lymphoid organs and large intestine. This demonstrates that upon antigen-driven B and T lymphocyte interaction in the local draining lymph node a subset-specific homing program for B and T lymphocytes is induced.

IL28B alleles associated with poor hepatitis C virus (HCV) clearance protect against inflammation and fibrosis in patients infected with non-1 HCV genotypes.

Genetic polymorphisms near IL28B are associated with spontaneous and treatment-induced clearance of hepatitis C virus (HCV), two processes that require the appropriate activation of the host immune responses. Intrahepatic inflammation is believed to mirror such activation, but its relationship with IL28B polymorphisms has yet to be fully appreciated. We analyzed the association of IL28B polymorphisms with histological and follow-up features in 2335 chronically HCV-infected Caucasian patients. Assessable phenotypes before any antiviral treatment included necroinflammatory activity (n = 1,098), fibrosis (n = 1,527), fibrosis progression rate (n = 1,312), and hepatocellular carcinoma development (n = 1,915). Associations of alleles with the phenotypes were evaluated by univariate analysis and multivariate logistic regression, accounting for all relevant covariates. The rare G allele at IL28B marker rs8099917-previously shown to be at risk of treatment failure-was associated with lower activity (P = 0.04), lower fibrosis (P = 0.02) with a trend toward lower fibrosis progression rate (P = 0.06). When stratified according to HCV genotype, most significant associations were observed in patients infected with non-1 genotypes (P = 0.003 for activity, P = 0.001 for fibrosis, and P = 0.02 for fibrosis progression rate), where the odds ratio of having necroinflammation or rapid fibrosis progression for patients with IL28B genotypes TG or GG versus TT were 0.48 (95% confidence intervals 0.30-0.78) and 0.56 (0.35-0.92), respectively. IL28B polymorphisms were not predictive of the development of hepatocellular carcinoma. CONCLUSION: In chronic hepatitis C, IL28B variants associated with poor response to interferon therapy may predict slower fibrosis progression, especially in patients infected with non-1 HCV genotypes.

Postoperative subconjunctival mitomycin-C injection after non-penetrating glaucoma surgery.

PURPOSE: To evaluate subconjunctival mitomycin C (MMC) injection efficacy and safety in patients with failing glaucoma non-penetrating filtering blebs. METHODS: Twenty-eight eyes were consecutively recruited for this study. Only one eye for each patient was randomly selected. All the recruited patients had glaucoma and uncontrolled intraocular pressure after a non-penetrating filtering glaucoma surgery and/or a pathological aspect of the filtering bleb (i.e., vascularized and/or encysted). One or more MMC injections were performed under the conjunctiva closed to the bleb to improve filtration. Local effects and complications of subconjunctival MMC injections were analyzed. RESULTS: Out of the 28 patients, 21 (75%) had MMC also applied intraoperatively. The mean postoperative IOP before MMC injections was 17 +/- 6.6 mmHg. The final IOP after MMC injections was 13.9 +/- 2.9 mmHg after a mean follow-up of 6 months. A total of 67 subconjunctival MMC injections were performed with a mean of 2.9 (ranging from 1 to 5) injections per patient. The only complication found to be possibly related to MMC injections was two cases of corneal Dellen. CONCLUSION: From these preliminary results, subconjunctival MMC injections in selected cases appear to be not only safe but also effective in promoting further the postoperative IOP drop.

A non-interventional phase IV Belgian survey to assess the antiviral effectiveness of pegylated interferon-alpha-2b and ribavirin treatment according to the stage of liver fibrosis in previously untreated patients with genotype 1/4/5/6 chronic hepatitis C (PRACTICE).

BACKGROUND AND STUDY AIMS: This was an observational, non-interventional, multicenter, phase IV study, in patients with genotype 1/4/5/6 chronic hepatitis C (CHC). The primary objectives were to evaluate SVR in patients with no or minimal fibrosis (METAVIR F0-F1) versus well established fibrosis (F2-F4), and to estimate response on Weeks 12, 24 and 48 on treatment in previously untreated patients with genotypes 1/4/5/6 CHC. PATIENTS AND METHODS: 538 patients treated with pegylated interferon alfa 2b 1.5 mcg/kg in combination with ribavirin 800-1200 mg/day were enrolled in 55 sites in Belgium and Luxembourg, 505 being considered for the analysis. 40% of the patients were female and 60% male, the average age was 47.5 years, 10.5% were 65 or older. RESULTS: SVR was observed in 35% of the patients, EVR in 68%, of which pEVR in 33% and cEVR in 35%. SVR was observed in 43% of the low fibrosis group (F0, F1) and 30% of the high fibrosis group (F2, F3, F4) (p = 0.005). SVR rates were 34% for genotype 1, 37% for genotype 4, and 47% for genotype 5 (NS). Multivariate analysis showed that EVR and baseline METAVIR score are independent prognostic factors for SVR. CONCLUSIONS: This trial confirms that fibrosis stage and early viral response are the most important key-factors to predict sustained response, suggesting that the earlier patients are treated, the better the outcome. Non-invasive techniques enable us to closely monitor progression of fibrosis, allowing a better selection of patients for antiviral treatment in the DAA-era.

Hydrogen isotope fractionations between amphiboles, micas, and fluids in alkaline igneous intrusions

RÉSUMÉ DE LA THÈSE Les teneurs des amphiboles en éléments majeurs et en isotopes stables ont été analysées dans plusieurs complexes ignés alcalins et hyperalcalins, dans le but de déterminer l'importance des variations de composition des minéraux pour le fractionnement isotopique de l'hydrogène dans un système naturel minéral-magma-fluide. Cette étude se concentre principalement sur les syénites néphéliniques de complexes intrusifs alcalins bien connus mais à chimie variable, dont les amphiboles, ainsi que d'autres silicates hydratés tels que micas et eudialytes, lorsque cela était possible, ont été séparés. L'intérêt principal s'est porté sur le complexe alcalin d'Ilímaussaq de la Province du Gardar, au Sud du Groenland. Dans une optique de comparaison, nous avons collecté et analysé d'autres échantillons provenant du complexe de Tugtutôq (Sud Groenland), des complexes de Khibina et Lovozero (Péninsule de Kola, Russie), du Mont St-Hilaire et du Mont Royal (Canada) et de 6 autres du nord-ouest de la Namibie (Cape Cross, Okenyenya, Messum, Etaneno, Kalkfeld,et Okorusu). Les compositions isotopiques de l'hydrogène des amphiboles des ces différentes zones présentent de grandes variations (-227 à -700/00), ce qui est atypique pour des magmas d'origine mantellique. Les valeurs comprises entre -80 et -400/00 indiquent une provenance du manteau. Ces larges variations de compositions ainsi que l'extrême appauvrissement en isotope lourd de l'hydrogène (D), en comparaison avec d'autres roches ignées, semblent être propres.aux roches alcalines et hyperalcalines de ce type, ce qui indiquerait un processus commun. Les différents complexes alcalins choisis présentent un large intervalle de composition chimique des amphiboles. La caractérisation des amphiboles par microscopie électronique et par spectroscopie Mössbauer contribuent à observer le contrôle du Fe sur le fractionnement des isotopes de l'hydrogène. En effet, cela a mis en évidence un contrôle du Fe sur le fractionnement et même, dans le cas du complexe hyperalcalin d'Ilímaussaq, une relation entre le rapport Fei3+/FeT et les variations du rapport D/H. Les complexes étudiés diffèrent de par leur index agpaïtique (Na+K/Al) et également de par leur contenu en fer. Les plus hautes valeurs en Fe (27-35 wt%) et en éléments alcalins dans les amphiboles, ainsi que les teneurs de D/H les plus basses et leur grande variation, sont celles du complexe d'Ilímaussaq. Les amphiboles de la Péninsule de Kola et du Canada sont similaires, mais toutefois moins appauvries en D. En ce qui concerne les amphiboles des complexes du NO de la Namibie, elles présentent des compositions isotopiques de l'hydrogène magmatiques normales (-73 à -100 0/00), contiennent moins de Fe (15-17 wt%) et sont fortement enrichies en Ca et moins en Na. Dans ce cas, l'alcalinité est moins importante en comparaison des autres complexes étudiés. En dehors des teneurs en éléments alcalins des amphiboles, l'alcalinité des fluides s'avère également un facteur important, ce qui est cohérent avec certaines suggestions à partir de systèmes expérimentaux. Afin de mieux contraindre ce facteur, des expériences d'échanges hydrothermaux entre les amphiboles et les fluides de salinité différente ont été effectuées en simulant des conditions naturelles. L'approximation d'amphiboles naturelles de complexes ignés alcalins, couplée aux expériences d'échange, aide à préciser les facteurs contrôlant le fractionnement des isotopes de l'hydrogène dans les roches alcalines. Les valeurs extrêmement basses de 3D des amphiboles de ces complexes alcalins peuvent être dues à une combinaison de différents facteurs, telles qu'une haute alcalinité, une haute teneur en Fe et une faible profondeur d'intrusion. Les grandes variations ainsi que les faibles valeurs de SD des amphiboles étudiées peuvent résulter d'un processus magmatique interne et il est peu probable que de l'eau météorique soit impliquée et/ou que le dégazage magmatique ait joué un rôle. THESIS ABSTRACT Major element and stable isotope compositions of amphiboles were analyzed from a number of alkaline and peralkaline igneous complexes in order to determine the importance of compositional variations in minerals to hydrogen isotope fractionations in natural mineral-melt-fluid systems. The thesis mainly focuses on nepheline syenites of well-studied, but chemically variable alkaline intrusive rocks, from which amphiboles and, if possible, other hydrous silicates such as micas and eudialytes were separated. The system of primary interest was the alkaline Ilímaussaq Complex of the Gardar Province of South Greenland. For the purpose of comparison additional samples were collected and examined from the Tugtutôq Complex (South Greenland), the Khibina and Lovozero Complexes (Kola Peninsular, Russia), Mount St-Hilaire and Mount Royal (Canada) and six further complexes from NW Namibia (Cape Cross, Okenyenya, Messum, Etaneno, Kalkfeld, and Okorusu). The hydrogen isotope compositions of amphiboles from the localities studied differ greatly, which is atypical for amphiboles from mantle, range between - 227 and - 700/00 (latter compatible with a simple mantle origin). As this wide range in compositions and the extreme depletion in the heavy hydrogen isotope (D) content relative to other igneous rocks appear to be unique to alkaline to peralkaline rocks of this type, a common process is indicated. The different alkaline complexes chosen cover a wide range of amphibole chemical compositions. Detailed chemical characterization of amphiboles by electron microprobe and Mössbauer spectroscopy analyses helped to constrain the control of Fe on the H-isotope fractionations. Complete characterization of the chemical compositions of the amphiboles support Fe-control on fractionations and at least for the peralkaline Ilímaussaq complex a relationship between Fe3+/FeT ratios and variations in D/H. The studied complexes differ in their agpaitic index (Na+K/Al) and also in their Fe-content. The most iron (27-35 wt. %) and alkaline element rich amphiboles, with the lowermost D/H ratio, as well with very wide range, are the ones from Ilímaussaq complex. Similar, but less D depleted amphiboles are from the Kola Peninsula and the Canadian localities. The complexes described from NW Namibia have amphiboles with normal magmatic hydrogen isotope composition (-730/00 to -1000/00), and have less Fe-content (15-17 wt. %), and are more Ca-and less Na-rich. In this case alkalinity is not that important in comparison to the other studied complexes. Beside the alkaline element contents in the amphiboles, the alkalinity of the fluids has been found to be an important factor, in conjunction with earlier suggestions from experimental systems. To further constrain this factor, hydrothermal exchange experiments between amphiboles and fluids of different salinity simulating natural conditions were performed. The approach of examining natural amphiboles from alkaline igneous complexes in parallel to performing exchange experiments - helped to further constrain the factors controlling the H-isotope fractionations in alkaline rocks. The observed changes between the hydrogen and oxygen isotope compositions of amphiboles and fluids before and after the experiments suggest that another phase was produced during the experiments, which influenced the final hydrogen isotope composition of the system. This presumably hydrous phase has also influenced the Fe3 +/Fe2+ ratio of the amphiboles, which became more oxidized. The extremely low SD values of amphiboles in these alkaline complexes may be due to a combination of different factors such as high alkalinity, high Fe-content, and shallow intrusion depths. This wide range and the low SD values of the amphiboles studied might be a result of internal, magmatic processes and it is unlikely that meteoric water was involved and/or magmatic degassing played an important role. RÉSUMÉ DE LA THÈSE (pour le grand public) Fractionnement isotopique de l'hydrogène entre amphiboles, micas et fluides dans des intrusions alcalines Zsófia Wáczek Directeur de thèse, Prof. Torsten W. Vennemann Institut de Minéralogie et Géochimie, Université de Lausanne Les roches alcalines et celles qui leurs sont associées sont des sources importantes de nombreux minéraux et minerais, tels l'apatite, le niobium, le diamant et autres pierres précieuses. Cette étude se concentre sur des complexes alcalins localisés dans le sud du Groenland, au Canada, dans la péninsule de Kola en Russie et au nord-ouest de la Namibie. Ces complexes sont composés de roches ayant cristallisé à partir de magmas et de fluides très enrichis en alcalins. Cet enrichissement permet la précipitation de minéraux inhabituels riches en potassium et/ou sodium, telles les amphiboles sodiques, également enrichies en fer. Les amphiboles étudiées ont des compositions calciques, sodi-calciques et sodiques, qui reflètent leurs différents environnements de formation. Des études précédentes ont révélé une large gamme de rapports isotopiques de l'hydrogène dans les amphiboles de roches hyperalcalines, dont certains extrêmement bas. Cette variation importante est très intrigante, sachant que des valeurs entre -40 et -800/00 correspondent à des silicates ignés hydratés et non altérés, alors que des valeurs descendant jusqu'a -1500/00 nécessiteraient une altération par de l'eau météorique et/ou une contamination par les roches environnantes ou des sédiments riches en matière organique. Dans lé cas précis du complexe d'Ilímaussaq (sud du Groenland), aucune de ces explications n'a pu être démontrée et des valeurs encore plus faibles ont été trouvées. Le complexe d'Ilímaussaq présente des valeurs de rapport isotopique de l'hydrogène entre -227 et -500/00 dans les amphiboles. Une origine mantellique permet d'expliquer les valeurs élevées, mais d'autres processus doivent entrer en jeu pour engendrer les valeurs les plus négatives. C'est à l'identification de ces processus que nous nous sommes attachés dans ce travail. Les grandes variations observées dans les teneurs en fer et dans le rapport Fe3+/FeT des roches et des minéraux de ces complexes sont corrélées avec d'autres paramètres chimiques, tels que la composition isotopique de l'hydrogène dans les amphiboles. Nous avons dès lors abordé les questions suivantes: quelle est la relation entre la teneur en fer des amphiboles et leur composition isotopique? Que nous apprennent les changements de la teneur en fer et les changements dans le rapport Fe3+/FeT sur les processus pétrologiques dans ces roches? Pour répondre à ces questions, nous avons analysé les compositions isotopiques de l'oxygène et de l'hydrogène dans les amphiboles et d'autres silicates hydratés. La composition chimique et le rapport Fe3+/FeT des amphiboles ont également été déterminés. Des expériences hydrothermales simulant des conditions naturelles ont été entreprises afin de mieux comprendre les processus de fractionnement isotopiques dans ces systèmes très alcalins. Nos conclusions sont les suivantes: (1) Les valeurs extrêmement faibles ainsi que les larges variations des rapports isotopiques de l'hydrogène des amphiboles de ces complexes alcalins sont dues à une combinaison de facteurs tels que la forte alcalinité, la haute teneur en fer et la profondeur très faible de l'intrusion. (2) Ces valeurs sont probablement le résultat de processus magmatiques internes. (3) Il est peu probable que les eaux météoriques et/ou le dégazage magmatique aient joué un rôle lors de la formation de ces amphiboles. (4) Certaines corrélations, en accord avec les études précédentes, ont pu être trouvées au niveau des concentrations en fer. (5) Dans le cas du complexe d'Ilímaussaq exclusivement, une relation a été trouvée entre le rapport Fe3+/FeT et la composition isotopique de l'hydrogène des amphiboles.

Inhibition of angiogenesis by non-steroidal anti-inflammatory drugs: from the bench to the bedside and back.

The formation of new blood vessels, a process globally referred to as angiogenesis, occurs in a number of pathological conditions, such as cancer and chronic inflammation. Recent findings indicate that cyclooxygenase-2 (COX-2), the inducible form of the cyclooxygenase (COX) isoenzymes, acts as a potent inducer of angiogenesis. Non-steroidal anti-inflammatory drugs (NSAIDs) are classical inhibitors of COX enzymes, which are widely prescribed for the treatment of inflammation, pain and fever. Selective COX-2 inhibitors (COXIBs) have been subsequently developed with the purpose to improve the safety profile of this class of therapeutics. More recently, substantial preclinical evidence demonstrated that NSAIDS and COXIBs have anti-angiogenic properties. This newly recognized activity opens the possibility of using these drugs for the treatment of angiogenesis-dependent diseases. In this article we review the most recent advances in understanding the mechanisms by which NSAIDs and COXIBs suppress angiogenesis, and we discuss their potential clinical use as anti-angiogenic drugs.

Correlation between microtensile bond strength data and clinical outcome of Class V restorations.

OBJECTIVE: To determine if the results of resin-dentin microtensile bond strength (µTBS) is correlated with the outcome parameters of clinical studies on non-retentive Class V restorations. METHODS: Resin-dentin µTBS data were obtained from one test center; the in vitro tests were all performed by the same operator. The µTBS testing was performed 8h after bonding and after 6 months of storing the specimens in water. Pre-test failures (PTFs) of specimens were included in the analysis, attributing them a value of 1MPa. Prospective clinical studies on cervical restorations (Class V) with an observation period of at least 18 months were searched in the literature. The clinical outcome variables were retention loss, marginal discoloration and marginal integrity. Furthermore, an index was formulated to be better able to compare the laboratory and clinical results. Estimates of adhesive effects in a linear mixed model were used to summarize the clinical performance of each adhesive between 12 and 36 months. Spearman correlations between these clinical performances and the µTBS values were calculated subsequently. RESULTS: Thirty-six clinical studies with 15 adhesive/restorative systems for which µTBS data were also available were included in the statistical analysis. In general 3-step and 2-step etch-and-rinse systems showed higher bond strength values than the 2-step/3-step self-etching systems, which, however, produced higher values than the 1-step self-etching and the resin modified glass ionomer systems. Prolonged water storage of specimens resulted in a significant decrease of the mean bond strength values in 5 adhesive systems (Wilcoxon, p<0.05). There was a significant correlation between µTBS values both after 8h and 6 months of storage and marginal discoloration (r=0.54 and r=0.67, respectively). However, the same correlation was not found between µTBS values and the retention rate, clinical index or marginal integrity. SIGNIFICANCE: As µTBS data of adhesive systems, especially after water storage for 6 months, showed a good correlation with marginal discoloration in short-term clinical Class V restorations, longitudinal clinical trials should explore whether early marginal staining is predictive for future retention loss in non-carious cervical restorations.

Extension of Murray's law using a non-Newtonian model of blood flow.

BACKGROUND: So far, none of the existing methods on Murray's law deal with the non-Newtonian behavior of blood flow although the non-Newtonian approach for blood flow modelling looks more accurate. MODELING: In the present paper, Murray's law which is applicable to an arterial bifurcation, is generalized to a non-Newtonian blood flow model (power-law model). When the vessel size reaches the capillary limitation, blood can be modeled using a non-Newtonian constitutive equation. It is assumed two different constraints in addition to the pumping power: the volume constraint or the surface constraint (related to the internal surface of the vessel). For a seek of generality, the relationships are given for an arbitrary number of daughter vessels. It is shown that for a cost function including the volume constraint, classical Murray's law remains valid (i.e. SigmaR(c) = cste with c = 3 is verified and is independent of n, the dimensionless index in the viscosity equation; R being the radius of the vessel). On the contrary, for a cost function including the surface constraint, different values of c may be calculated depending on the value of n. RESULTS: We find that c varies for blood from 2.42 to 3 depending on the constraint and the fluid properties. For the Newtonian model, the surface constraint leads to c = 2.5. The cost function (based on the surface constraint) can be related to entropy generation, by dividing it by the temperature. CONCLUSION: It is demonstrated that the entropy generated in all the daughter vessels is greater than the entropy generated in the parent vessel. Furthermore, it is shown that the difference of entropy generation between the parent and daughter vessels is smaller for a non-Newtonian fluid than for a Newtonian fluid.