Molecular and isotopic characterization of biomarkers in the Frick Swiss Jura sediments: A palaeoenvironmental reconstruction on the northern Tethys margin

Molecular and stable carbon isotope compositions of source-specific hydrocarbons have been used to reconstruct palaeoenvironmental conditions during deposition of the Middle Hettangian to Upper Sinemurian sediments on the northern epicontinental Tethys margin, Frick Swiss Jura. Increasing algal, cyanobacterial and phytoplanktonic (i.e., dinoflagellate) contributions associated with the C-13-enrichment of cyanobacteria derivatives (i.e., hopanes and monomethylalkanes) suggest enhanced primary productivity upsection. This is related to the C-13-enrichment of dissolved CO2 in the upper layers and the progressive increase of depth and oxygenation of the water column. In the Middle Hettangian shallow-water environments (lagoon), the occurrence of green sulfur bacteria (Chlorobiaceae) derivatives indicates that the lower part of the water column was strictly anoxic and rich in H2S. Since these bacteria require very low light intensity to grow, these euxinic conditions may be extended up to the photic zone, allowing for anaerobic photosynthesis. Light penetration depth is most likely reduced by high productivity and/or turbidity in the photic zone. In these sediments, C-13-depleted hopanoids (-39.5 parts per thousand) are most likely associated with phototrophic purple sulfur bacteria utilizing isotopically light organic carbon at the base of the aerobic zone. These purple sulfur bacteria may have consumed the H2S used by Chlorobiaceae in the deeper layers and thus, sustained the algae and cyanobacteria productivity in the upper layers. The C-13-depleted carbonate (-13.3 parts per thousand) may be partially related to the anaerobic oxidation of the organic matter during bacterial sulfate-reduction. (c) 2006 Elsevier Ltd. All rights reserved.

Identification and functional characterization of a monofunctional peroxisomal enoyl-CoA hydratase 2 that participates in the degradation of even cis-unsaturated fatty acids in Arabidopsis thaliana.

A gene, named AtECH2, has been identified in Arabidopsis thaliana to encode a monofunctional peroxisomal enoyl-CoA hydratase 2. Homologues of AtECH2 are present in several angiosperms belonging to the Monocotyledon and Dicotyledon classes, as well as in a gymnosperm. In vitro enzyme assays demonstrated that AtECH2 catalyzed the reversible conversion of 2E-enoyl-CoA to 3R-hydroxyacyl-CoA. AtECH2 was also demonstrated to have enoyl-CoA hydratase 2 activity in an in vivo assay relying on the synthesis of polyhydroxyalkanoate from the polymerization of 3R-hydroxyacyl-CoA in the peroxisomes of Saccharomyces cerevisiae. AtECH2 contained a peroxisome targeting signal at the C-terminal end, was addressed to the peroxisome in S. cerevisiae, and a fusion protein between AtECH2 and a fluorescent protein was targeted to peroxisomes in onion cells. AtECH2 gene expression was strongest in tissues with high beta-oxidation activity, such as germinating seedlings and senescing leaves. The contribution of AtECH2 to the degradation of unsaturated fatty acids was assessed by analyzing the carbon flux through the beta-oxidation cycle in plants that synthesize peroxisomal polyhydroxyalkanoate and that were over- or underexpressing the AtECH2 gene. These studies revealed that AtECH2 participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2E-enoyl-CoA for further degradation through the core beta-oxidation cycle.

Methods for the ex vivo characterization of human CD8+ T subsets based on gene expression and replicative history analysis.

The generation of an antigen-specific T-lymphocyte response is a complex multi-step process. Upon T-cell receptor-mediated recognition of antigen presented by activated dendritic cells, naive T-lymphocytes enter a program of proliferation and differentiation, during the course of which they acquire effector functions and may ultimately become memory T-cells. A major goal of modern immunology is to precisely identify and characterize effector and memory T-cell subpopulations that may be most efficient in disease protection. Sensitive methods are required to address these questions in exceedingly low numbers of antigen-specific lymphocytes recovered from clinical samples, and not manipulated in vitro. We have developed new techniques to dissect immune responses against viral or tumor antigens. These allow the isolation of various subsets of antigen-specific T-cells (with major histocompatibility complex [MHC]-peptide multimers and five-color FACS sorting) and the monitoring of gene expression in individual cells (by five-cell reverse transcription-polymerase chain reaction [RT-PCR]). We can also follow their proliferative life history by flow-fluorescence in situ hybridization (FISH) analysis of average telomere length. Recently, using these tools, we have identified subpopulations of CD8+ T-lymphocytes with distinct proliferative history and partial effector-like properties. Our data suggest that these subsets descend from recently activated T-cells and are committed to become differentiated effector T-lymphocytes.

Characterization of Adult Stem/Progenitor Cell Populations From Bone Marrow in a Three-Dimensional Collagen Gel Culture System.

Stem cell transplantation therapy using mesenchymal stem cells (MSCs) is considered a useful strategy. Although MSCs are commonly isolated by exploiting their plastic adherence, several studies have suggested that there are other populations of stem and/or osteoprogenitor cells which are removed from primary culture during media replacement. Therefore, we developed a three-dimensional (3D) culture system in which adherent and non-adherent stem cells are selected and expanded. Here, we described the characterization of 3D culture-derived cell populations in vitro and the capacity of these cells to differentiate into bone and/or cartilage tissue when placed inside of demineralized bone matrix (DBM) cylinders, implanted subcutaneously into the backs of rat for 2, 4 and 8 weeks. Our results demonstrates that 3D culture cells were a heterogeneous population of uncommitted cells that express pluripotent, hematopoietic, mesenchymal and endothelial specific markers in vitro and can undergo osteogenic differentiation in vivo.

Identification and characterization of a caspase-2 activating complex

Résumé Les caspases sont des protéases essentielles lors de l'induction de l'apoptose ou pour la maturation de certaines cytokines. Elles peuvent être divisées en deux groupes: les caspases initiatrices, qui sont les premières activées lors d'un signal pro-apoptotique, et les caspases effectrices, qui sont activées par les caspases initiatrices et sont responsables du clivage et de la dégradation des substrats cellulaires. Les caspases initiatrices sont activées dans des complexes de haut poids moléculaire: l'apoptosome pour la caspase-9 et le DISC pour la caspase-8. La caspase-2 est également une caspase initiatrice qui contient un domaine CARD. Cependant son mécanisme d'activation n'est pas encore connu. Lors de cette étude, nous avons découvert et caractérisé le complexe qui permet l'activation de la caspase-2. Ce complexe, appelé le PIDDosome, est composé de PIDD/LRDD, de la protéine adaptatrice RAIDD et de la protéase caspase-2. L'expression forcée de PIDD induit l'activation constitutive de la caspase-2. Cela entraîne la mort ou la sensibilisation à la mort des cellules selon la lignée étudiée. Cet effet est expliqué par une perte du potentiel de membrane de la mitochondrie, certainement dû à un effet direct de la caspase-2. Peu de choses sont connues sur PIDD: c'est une protéine contenant un domaine DD qui peut être induite par p53. Nous avons caractérisé PIDD et montré qu'elle est exprimée de façon ubiquitaire. PIDD est constitutivement auto-clivée environ au milieu de la protéine, ce qui génère deux fragments qui restent liés l'un à l'autre. Le fragment N-terminal a une activité régulatrice et le C-terminal une activité effectrice. De plus, PIDD peut se déplacer entre le cytoplasme et le noyau. Enfin, nous avons découvert que PIDD est également impliquée dans l'induction de NF¬ -κB en réponse à des dommages à l'ADN. PIDD est responsable de la modification par sumo de NEMO, étape nécessaire à l'induction de NF-κB après des dommages à l'ADN. Ainsi PIDD semble être à l'intersection de la décision que prend la cellule entre survivre et réparer les dommages, ou entrer en apoptose. Summary Caspases are a family of proteases that fulfill varied and often critical roles in mammalian apoptosis or proteolytic activation of cytokines. Caspases can be divided into two sub-groups: initiator caspases, which are the first activated after a pro-apoptotic signal, and effector caspases, which are activated by initiator caspases and that are responsible for the cleavage and degradation of cellular components. Initiator caspases are activated in high molecular weight platforms such as the apoptosome for caspase-9 or the DISC for caspase-8. Caspase-2 is a CARD-containing initiator caspase whose mechanism of activation was not yet known. In this study we have identified an activating platform for caspase-2. This high molecular weight complex, called the PIDDosome, is composed of PIDD/LRDD, the adaptor protein RAIDD and caspase-2. Constitutive expression of PIDD led to constitutive activation of caspase-2, which in some cell lines was sufficient to induce cell death while in others it merely sensitizes. Active caspase-2 was found to disturb directly the mitochondria by inducing a partial loss of the transmembrane potential. Very little was known on PIDD. It can be induce by p53 and inhibition of its expression by antisense oligonucleotides diminishes p53-dependent apoptosis. We decided to further characterize PIDD function and expression. PIDD possesses seven LRR, two Zu5 domains and one DD. It is ubiquitously expressed and appears to be constitutively cleaved by auto- processing into two main fragments equal in size. The two fragments remain bound to one another and constitute a regulatory N-terminal fragment and an active C-terminal fragment. In addition, PIDD can shuttle between the cytoplasm and the nucleus. Finally, investigating the possible relevance of new interaction partners, we found that PIDD is implicated in DNA damage-induced NF- κB. PIDD binds to RIP1 and to NEMO. In response to DNA damage, PIDD translocates to the nucleus and mediates sumo- modification of NEMO, a necessary step in DNA damage-induced NF-κB. All together these results raise the possibility that PIDD acts as a molecular switch between proliferation and repair, and apoptosis following DNA damage.

Characterization of a cAMP-regulated enhancer-binding protein.

The transcription regulation of many hormone genes is modulated by intracellular second messengers such as cAMP. The cAMP response element binding protein, CREB, binds to the 8 base pair CRE enhancer, TGACGTCA, that is found in the 5'-flank of certain genes including those for somatostatin and the alpha-subunit of human chorionic gonadotropin. The recent characterization of CREB and CREB-related cDNA clones, combined with Southwesterns and Northern blot analyses, reveals a family of transcription factors that dimerize via a leucine zipper motif and bind to the CRE through positively charged basic regions. The CREB cDNA encoding a 327 residue protein is transcriptionally activated via phosphorylation by protein kinases, including the cAMP-dependent protein kinase-A.

Biochemical characterization of recombinant human telomerase

Résumé Les télomères sont les structures ADN-protéines des extrémités des chromosomes des eucaryotes. L'ADN télomérique est constitué de courtes séquences répétitives. L'intégrité des télomères est essentielle pour protéger les extrémités des chromosomes contre les systèmes de dégradations et pour les distinguer des cassures de l'ADN double brin. Parce que la machinerie de la réplication de l'ADN n'est pas capable de répliquer l'extrémité des chromosomes, les télomères raccourcissent au fur et à mesure des cycles de réplication. Dès que les télomères atteignent une longueur critique, leur structure protectrice est perdue. Cela induit un signal de dommage de l'ADN et l'arrêt du cycle cellulaire. Pour contrebalancer le raccourcissement des télomères, les cellules qui s'auto régénèrent, dont les cellules de la moelle osseuse, les lymphocytes activés et 80-90% des cellules cancéreuses, expriment la télomérase. C'est une ribonucléoprotéine qui a la capacité de synthétiser des séquences télomériques par transcription inverse d'une courte séquence contenue dans sa propre sous-unité ARN avec laquelle elle est associée. La télomérase humaine est une enzyme processive au niveau de l'addition des nucléotides et aussi des répétitions télomériques. La télomérase de levure et la télomérase humaine sont toutes deux dimériques et il a été montré que la télomérase humaine recombinante contient deux ARN qui coopèrent pour fonctionner ainsi que deux sous-unités catalytiques. Cependant, il n'a pas encore été montré quel est le rôle de la dimérisation dans l'activité de la télomérase. Afin d'élucider ce rôle, nous avons exprimé, reconstitué et purifié la télomérase humaine dimérique recombinante. Et pour étudier l'effet d'ARN mutants sur l'activité de la télomérase, nous avons développé une méthode pour reconstituer et enrichir en hétérodimères de télomérase. Les hétérodimères contiennent une sous-unité ARN sauvage et une sous-unité ARN mutée au niveau de la séquence de la matrice. Sur l'ARN muté nous avons introduit une étiquette aptamer ARN-S1 puis nous avons purifié la télomérase via l'etiquette Si. Nous avons montré que la dimérisation est essentielle pour l'activité de la télomérase. Nos données indiquent que chaque télomérase du dimère allonge leur substrat, l'ADN télomérique, indépendamment l'une de l'autre à chaque cycle d'élongation mais que l'addition itérative de répétitions télomériques nécessite une coopération entre les deux télomérases du dimère. Nous proposons donc un modèle dans lequel les deux télomérases du dimères se lient et allongent deux substrats télomères et que pendant l'élongation processive les deux enzymes subissent un changement de conformation de manière coordonnée, ce changement va permettre le repositionnement des substrats pour d'autres cycles d'additions de répétitions télomériques. Dyskeratosis congenita est une maladie mortelle due majoritairement au disfonctionnement de la moelle osseuse. Dans la forme autosomale de la maladie, l'ARN de la télomérase contient des mutations. En utilisant notre système de reconstitution, nous avons montré que ces ARN mutés, qui ont perdu leur activité enzymatique dans le cas d'un homodimère de mutants, sont dominant négatifs quand ils sont présents dans les hétérodimères sauvage/mutant. Cet effet trans-dominant négatif pourrait contribuer à la progression de la maladie. Abstract Telomeres are protein-DNA structures at the ends of linear eukaryotic chromosomes. The telomeric DNA consists of tandemly repeated sequences. Telomeric integrity is essential to protect chromosomal ends from nucleolytic degradation and to prevent their recognition as DNA double strand breaks. Due to the inability of the conventional DNA replication machinery to replicate terminal DNA stretches, telomeres shorten with continuous rounds of DNA replication. As soon as telomeres reach a critical length, their protective structure is lost and the deprotected telomeres will induce a DNA damage response leading to cell cycle arrest. To counteract telomere shortening, self-renewing cells, including bone marrow cells, activated lymphocytes and 80-90% of cancer cells express the cellular reverse transcriptase telomerase, which has the capacity to synthesize telomeric repeats by reverse transcription of a short template sequence encoded by its stably associated RNA subunit. Human telomerase is a processive enzyme for nucleotide as well as repeat addition. Both yeast and human telomerase are dimeric enzymes and recombinant human telomerase has been shown to contain two functionally cooperating RNAs and most probably also two protein subunits. However, it has remained unclear how dimerization may contribute to telomerase activity. To study the role of dimerization, we expressed, reconstituted and purified recombinant human telomerase. We also developed a new method to reconstitute and enrich for telomerase heterodimers containing wild-type (wt) and mutant telomerase RNA subunits. To this end we introduced an S1-RNA-aptamer tag into telomerase RNA and purified telomerase reconstituted with a mixture of untagged and tagged RNA via the S1-tag. Using this experimental system, we introduced template mutations in the tagged RNA subunit and examined the effect of mutant RNAs on wt telomerase activity in wt/mutant heterodimers. We obtained evidence that dimerization is essential for telomerase activity. Our data indicate that the two subunits elongate telomere substrates independently of each other during single rounds of elongation, but that iterative addition of telomeric repeats requires cooperation between the two subunits. We suggest a model, in which dimeric telomerases bind and elongate two telomere substrates and that the two subunits undergo coordinated conformational changes during processive elongation that enable repositioning the substrates for subsequent rounds of repeat addition. Dyskeratosis congenita is a multisystemic disease with bone marrow failure as the major cause of death. The autosomal form of this disease was found to harbor mutations in the telomerase RNA. Using our reconstitution system, we tested whether mutant dyskeratosis telomerase RNAs behaved in a dominant negative manner. We observed that dyskeratosis telomerase RNA mutants, which lacked enzymatic activity were dominant negative, when present in wt/ mutant heterodimers. The transdominant negative effect of these mutants may contribute to disease progression.

Molecular characterization of signalling complexes involved in G-protein coupled receptor-induced cardiac hypertrophy

In response to pathological stresses, the heart undergoes a remodelling process associated with cardiac hypertrophy. Since sustained hypertrophy can progress to heart failure, there is an intense investigation about the intracellular signalling pathways that control cardiomyocyte growth. Accumulating evidence has demonstrated that most stimuli known to initiate pathological changes associated with the development of cardiac hypertrophy activate G protein-coupled receptors (GPCRs) including the αl-adrenergic- (αl-AR), Angiotensin II- (AT-R) and endothelin-1- (ET-R) receptors. In this context, we have previously identified a cardiac scaffolding protein, called AKAP-Lbc (Α-kinase anchoring protein), with an intrinsic Rho specific guanine nucleotide exchange factor activity, that plays a key role in integrating and transducing hypertrophic signals initiated by these GPCRs (Appert-Collin, Cotecchia et al. 2007). Activated RhoA controls the transcriptional activation of genes involved in cardiomyocyte hypertrophy through signalling pathways that remain to be characterized. Here, we identified the nuclear factor-Kappa Β (NF-κΒ) activating kinase ΙΚΚβ as a novel AKAP-Lbc interacting protein. This raises the hypothesis that AKAP-Lbc might promote cardiomyocyte growth by maintaining a signalling complex that promotes the activation of the pro-hypertrophic transcription factor NF-κΒ. In fact, the activation of NF- κΒ-dependent transcription has been detected in numerous disease contexts, including hypertrophy, ischemia/reperfusion injury, myocardial infarction, allograft rejection, myocarditis, apoptosis, and more (Hall, Hasday et al. 2006). While it is known by more than a decade that NF-κΒ is a critical mediator of cardiac hypertrophy, it is currently poorly understood how pro-hypertrophic signals controlling NF-κΒ transcriptional activity are integrated and coordinated within cardiomyocytes. In this study, we show that AKAP-Lbc and ΙΚΚβ form a transduction complex in cardiomyocytes that couples activation of αl-ARs to NF-κB-mediated transcriptional reprogramming events associated with cardiomyocyte hypertrophy. In particular, we can show that activation of ΙΚΚβ within the AKAP-Lbc complex promotes NF-κB-dependent production of interleukine-6 (IL-6), which, in turn, enhances foetal gene expression. These findings indicate that the AKAP-Lbc/ΙΚΚβ complex is critical for selectively directing catecholamine signals to the induction of cardiomyocyte hypertrophy.

Characterization of the role of AKAP-Lbc/p38 complex in the hypertrophic response of the heart

In response to chronic stress the heart undergoes an adverse remodeling process associated with cardiomyocyte hypertrophy, increased cellular apoptosis and fibrosis, which ultimately causes cardiac dysfunction and heart failure. Increasing evidence suggest the role of scaffolding and anchoring proteins in coordinating different signaling pathways that mediate the hypertrophic response of the heart. In this context, the family of Α-kinase anchoring proteins (AKAPs) emerged as important regulators of the cardiac function. During my thesis work I have conducted two independent projects, both of them aiming at elucidating the role of AKAPs in the heart. It has been shown that AKAP-Lbc, an anchoring protein that possesses an intrinsic Rho- specific exchange factor activity, organizes a signaling complex that links AKAP-Lbc- dependent activation of RhoA with the mitogen activated protein kinase (MAPK) p38. The first aim of my thesis was to study the role of this novel transduction pathway in the context of cardiac hypertrophy. Here we show that transgenic mice overexpressing in cardiomyocytes a competitor fragment of AKAP-Lbc, which specifically disrupts endogenous AKAP-Lbc / p38 complexes, developed early dilated cardiomyopathy in response to two weeks of transverse aortic constriction (TAC) as compared to controls. Interestingly, inhibition of the AKAP-Lbc / p38 transduction pathway significantly reduced the hypertrophic growth of single cardiomyocytes induced by pressure overload. Therefore, it appears that the AKAP- Lbc / p38 complex is crucially involved in the regulation of stress-induced cardiomyocyte hypertrophy and that disruption of this signaling pathway is detrimental for the heart under conditions of sustained hemodynamic stress. Secondly, in order to identify new AKAPs involved in the regulation of cardiac function, we followed a proteomic approach which allowed us to characterize AKAP2 as a major AKAP in the heart. Importantly, here we show that AKAP2 interacts with several proteins known to be involved in the control of gene transcription, such as the nuclear receptor coactivator 3 (NCoA3) or the ATP-dependent SWI/SNF chromatin remodeling complex. Thus, we propose AKAP2 as a novel mediator of cardiac gene expression through its interaction with these transcriptional regulators.

Modulation of the airway allergic response : characterization of the cellular and molecular mechanisms

ABSTRACT Allergic asthma is a major complication of atopy. Its severity correlates with the presence of activated T lymphocytes and eosinophils in the bronchoalveolar lavage fluid (BALF). Mechanisms that protect against asthma are poorly understood. Based on oral models of mucosal tolerance induction, models using the nasal route showed that uptake of important amounts of antigen can induce tolerance and reverse the allergic phenotype. 1L-10 producing regulatory T cells were proposed as key players in tolerance induction, but other players, e.g. dendritic cells (DC), B cells and epithelial cells may have to be taken into consideration. The objective of the present study is to characterize the effects of a therapeutic intranasal treatment (INT) in a murine model of asthma and to determine, in this model, the cellular and molecular mechanisms leading to protection against asthma. First, we established an asthma model by sensitizing the BALB/c mouse to ovalbumin (OVA) by two intraperitoneal injections of alum-adsorbed OVA and three inhalations of aerosolized OVA. Then OVA was applied to the nasal mucosa of OVA- sensitized mice. Mice were later re-exposed to OVA aerosols to assess the protection induced by OVA INT. OVA sensitization induced strong eosinophil recruitment, OVA-specific T cell proliferation and IgE production. Three intranasal treatments at 24-hour intervals with 1.5 mg OVA drastically reduced inflammatory cell recruitment into the BALF and inhibited OVA-specific IgE production upon allergen re-exposure. T cell proliferation in ex vivo bronchial lymph node (BLN) cells was inhibited, as well as TH2 cytokine production. Protection against OVA-induced bronchial inflammation was effective for an extended period of time and treated mice resisted a second re-exposure. Transfer of CD4+ cells from BLN and lungs of OVA-treated mice protected asthmatic recipient mice from subsequent aerosol challenge indicating an involvement of CD4+ T regulatory cells in this protection. RESUME L'asthme allergique est une manifestation clinique majeure de l'atopie. La sévérité de l'asthme est liée à la présence de lymphocytes T activés ainsi que d'éosinophiles dans le lavage broncho-alvéolaire (LBA). Les mécanismes permettant de se prémunir contre l'asthme sont mal connus. Basés sur des modèles muqueux d'induction de tolérance par la voie orale, des modèles utilisant la voie nasale ont montré que d'importantes quantités d'antigène peuvent induire une tolérance et ainsi reverser le phénotype allergique. Des cellules régulatrices produisant de l'IL-10 pourraient jouer un rôle clé dans l'induction de la tolérance mais d'autres acteurs tels que les cellules dendritiques, les cellules B et les cellules épithéliales doivent aussi être prises en compte. L'objectif de la présente étude est de caractériser les effets d'un traitement intranasal thérapeutique dans un modèle murin d'asthme et de déterminer dans ce modèle les mécanismes cellulaires et moléculaires conférant une protection contre l'asthme. En premier lieu, un modèle d'asthme allergique a été établi en sensibilisant des souris BALB/c à l'ovalbumine (OVA) par deux injections intraperitonéales d'OVA adsorbé sur de l'alum et trois séances d'OVA en aérosol. Dans un second temps, de l'OVA a été administrée sur la muqueuse nasale des souris sensibilisées à l'OVA. Les souris furent ensuite challengées par des aérosols d'OVA afin d'évaluer la protection conférée par le traitement intranasal à l'OVA. La sensibilisation à l'OVA a induit un fort recrutement d'éosinophiles, une réponse proliférative des cellules T à l'OVA ainsi qu'une production d'lgE spécifiques. Trois traitements intranasaux à 24 heures d'intervalle avec 1.5 mg d'OVA ont permis de réduire drastiquement le recrutement des cellules inflammatoires dans le LBA ainsi que d'inhiber la production d'lgE spécifiques à l'OVA produits lors d'une ré-exposition à l'OVA. La prolifération en réponse à l'OVA de cellules extraites ex vivo de ganglions bronchiques a, elle aussi, été inhibée de même que la production de cytokines TH2. La protection contre l'inflammation provoquée par l'aérosol est efficace pour une longue période et les souris traitées résistent à une seconde ré- exposition. Le transfert de cellules CD4+ issues de ganglions bronchiques et de poumons de souris traitées à l'OVA protège les souris asthmatiques receveuses contre les effets inflammatoires d'un aérosol, indiquant que des cellules T CD4+ régulatrices pourraient être impliquées dans cette protection. RESUME DESTINE A UN LARGE PUBLIC L'asthme est une affection des voies respiratoires qui se caractérise par une contraction de la musculature des voies aériennes, une production de mucus et d'anticorps de l'allergie (IgE). On parle d'asthme allergique lorsque les facteurs déclenchant l'asthme sont des allergènes inhalés tels que acariens, pollens ou poils d'animaux. Le système immunitaire des patients asthmatiques a un défaut de programmation qui le rend réactif à des substances qui sont normalement inoffensives. Le traitement actuel de l'asthme repose sur le soulagement des symptômes grâce à des produits à base de stéroïdes. Les techniques permettant de reprogrammer le système immunitaire (immunothérapie) ne sont pas efficaces pour tous les antigènes et prennent beaucoup de temps. En conséquence, il est nécessaire de mieux comprendre les mécanismes sous-tendant une telle reprogrammation afin d'en améliorer le rendement et l'efficacité. Dans ce but, des modèles d'immunothérapie ont été mis au point chez la souris. Ils permettent une plus grande liberté d'investigation. Dans cette étude, un modèle d'asthme allergique dans la souris a été établi par une sensibilisation à un antigène particulier : l'ovalbumine (OVA). Ce modèle présente les caractéristiques principales de l'asthme humain : recrutement de cellules inflammatoires dans les poumons, augmentation de la production d'anticorps et de la résistance des bronches aux flux respiratoires. Cette souris asthmatique a ensuite été traitée par application nasale d'OVA. Comparées aux souris non traitées, les souris traitées à l'OVA ont moins de cellules inflammatoires dans leurs poumons et produisent moins d'anticorps IgE. D'autres marqueurs inflammatoires sont aussi fortement diminués. Des cellules de poumons ou de ganglions bronchiques prélevées sur des souris traitées injectées dans des souris asthmatiques améliorent les symptômes de l'asthme. Ces cellules pourraient donc avoir un rôle régulateur dans l'asthme. Les caractériser et les étudier afin d'être capable de les générer est crucial pour les futures thérapies de l'asthme.

Molecular and isotopic characterization of organic samples from the wreck of the Saint-Etienne merchant ship (XVIIIth century): Identification of pitch, fat, hair and sulfur

The wreck U Pezzo, excavated within the Saint Florent Gulf in northern Corsica was identified as the pink, Saint Etienne, a merchant ship which sank on January 31, 1769. In order to determine the composition of organic materials used to coat the hull or to waterproof different parts of the pink, a study of several samples, using molecular biomarker and carbon isotopic analysis, was initiated. The results revealed that the remarkable yellow coat, covering the outside planks of the ship's bottom under the water line, is composed of sulfur, tallow (of ox and not of cetacean origin) and black pitch which corresponds to a mixture called ``couroi'' or ``stuff'. Onboard ropes had been submitted to a tarring treatment with pitch. Hairs mixed with pitch were identified in samples collected between the two layers of the hull or under the sheathing planking. The study also provides a key model for weathering of pitch, as different degrees of degradation were found between the surface and the heart of several samples. Accordingly, molecular parameters for alteration were proposed. Furthermore novel mixed esters between terpenic and diterpenic alcohols and the free major fatty acids (C(14:0), C(16:0), C(18:0)) were detected in the yellow coat. (C) 2009 Elsevier Ltd. All rights reserved.

Redox index of soil carbon stability

As an alternative to the relatively complex and expensive spectroscopic methods, the redox properties of humic acids, determined by potentiometric titrations, have been used to evaluate the stability of soil organic C. The objective of the present study was to establish a Redox Index of C Stability (RICS) and to correlate it with some properties of the humic acids extracted from different modal soils in Brazil (distinct weathering stages or management) to facilitate system comparison. The RICS was efficient for soil comparison and variations were comparable to those of the chemical and spectroscopic methods used for humic acid characterization. The values of soil pH, point of zero salt effect, sum of bases, exchangeable Ca content, weathering index, as well as the humic acid O/C ratio, quinone and semiquinone free radical contents, aromatic C and fluorescence intensity were closely related with the RICS. The RICS was higher in less weathered soils, with more active clays and higher fertility. The RICS values of soils under long-term sugarcane management were ranked in decreasing order: unburned, burned with vinasse, burned without vinasse.

CHARACTERIZATION OF NrsZ, A NOVEL SMALL REGULATORY NON-CODING RNA INVOLVED IN THE ADAPTATION OF PSEUDOMONAS AERUGINOSA PAO1

The opportunistic ubiquitous pathogen Pseudomonas aeruginosa strain PAOl is a versatile Gram-negative bacterium that has the extraordinary capacity to colonize a wide diversity of ecological niches and to cause severe and persistent infections in humans. To ensure an optimal coordination of the genes involved in nutrient utilization, this bacterium uses the NtrB/C and/or the CbrA/B two-component systems, to sense nutrients availability and to regulate in consequence the expression of genes involved in their uptake and catabolism. NtrB/C is specialized in nitrogen utilization, while the CbrA/B system is involved in both carbon and nitrogen utilization and both systems activate their target genes expression in concert with the alternative sigma factor RpoN. Moreover, the NtrB/C and CbrA/B two- component systems regulate the secondary metabolism of the bacterium, such as the production of virulence factors. In addition to the fine-tuning transcriptional regulation, P. aeruginosa can rapidly modulate its metabolism using small non-coding regulatory RNAs (sRNAs), which regulate gene expression at the post-transcriptional level by diverse and sophisticated mechanisms and contribute to the fast physiological adaptability of this bacterium. In our search for novel RpoN-dependent sRNAs modulating the nutritional adaptation of P. aeruginosa PAOl, we discovered NrsZ (Nitrogen regulated sRNA), a novel RpoN-dependent sRNA that is induced under nitrogen starvation by the NtrB/C two-component system. NrsZ has a unique architecture, formed of three similar stem-loop structures (SL I, II and II) separated by variant spacer sequences. Moreover, this sRNA is processed in short individual stem-loop molecules, by internal cleavage involving the endoribonuclease RNAse E. Concerning NrsZ functions in P. aeruginosa PAOl, this sRNA was shown to trigger the swarming motility and the rhamnolipid biosurfactants production. This regulation is due to the NrsZ-mediated activation of rhlA expression, a gene encoding for an enzyme essential for swarming motility and rhamnolipids production. Interestingly, the SL I structure of NrsZ ensures its regulatory function on rhlA expression, suggesting that the similar SLs are the functional units of this modular sRNA. However, the regulatory mechanism of action of NrsZ on rhlA expression activation remains unclear and is currently being investigated. Additionally, the NrsZ regulatory network was investigated by a transcriptome analysis, suggesting that numerous genes involved in both primary and secondary metabolism are regulated by this sRNA. To emphasize the importance of NrsZ, we investigated its conservation in other Pseudomonas species and demonstrated that NrsZ is conserved and expressed under nitrogen limitation in Pseudomonas protegens Pf-5, Pseudomonas putida KT2442, Pseudomonas entomophila L48 and Pseudomonas syringae pv. tomato DC3000, strains having different ecological features, suggesting an important role of NrsZ in the adaptation of Pseudomonads to nitrogen starvation. Interestingly the architecture of the different NrsZ homologs is similarly composed by SL structures and variant spacer sequences. However, the number of SL repetitions is not identical, and one to six SLs were predicted on the different NrsZ homologs. Moreover, NrsZ is processed in short molecules in all the strains, similarly to what was previously observed in P. aeruginosa PAOl, and the heterologous expression of the NrsZ homologs restored rhlA expression, swarming motility and rhamnolipids production in the P. aeruginosa NrsZ mutant. In many aspects, NrsZ is an atypical sRNA in the bacterial panorama. To our knowledge, NrsZ is the first described sRNA induced by the NtrB/C. Moreover, its unique modular architecture and its processing in similar short SL molecules suggest that NrsZ belongs to a novel family of bacterial sRNAs. -- L'agent pathogène opportuniste et ubiquitaire Pseudomonas aeruginosa souche PAOl est une bactérie Gram négative versatile ayant l'extraordinaire capacité de coloniser différentes niches écologiques et de causer des infections sévères et persistantes chez l'être humain. Afin d'assurer une coordination optimale des gènes impliqués dans l'utilisation de différents nutriments, cette bactérie se sert de systèmes à deux composants tel que NtrB/C et CbrA/B afin de détecter la disponibilité des ressources nutritives, puis de réguler en conséquence l'expression des gènes impliqués dans leur importation et leur catabolisme. Le système NtrB/C régule l'utilisation des sources d'azote alors que le système CbrA/B est impliqué à la fois dans l'utilisation des sources de carbone et d'azote. Ces deux systèmes activent l'expression de leurs gènes-cibles de concert avec le facteur sigma alternatif RpoN. En outre, NtrB/C et CbrA/B régulent aussi le métabolisme secondaire, contrôlant notamment la production d'importants facteurs de virulence. En plus de toutes ces régulations génétiques fines ayant lieu au niveau transcriptionnel, P. aeruginosa est aussi capable de moduler son métabolisme en se servant de petits ARNs régulateurs non-codants (ARNncs), qui régulent l'expression génétique à un niveau post- transcriptionnel par divers mécanismes sophistiqués et contribuent à rendre particulièrement rapide l'adaptation physiologique de cette bactérie. Au cours de nos recherches sur de nouveaux ARNncs dépendant du facteur sigma RpoN et impliqués dans l'adaptation nutritionnelle de P. aeruginosa PAOl, nous avons découvert NrsZ (Nitrogen regulated sRNA), un ARNnc induit par la cascade NtrB/C-RpoN en condition de carence en azote. NrsZ a une architecture unique, composée de trois structures en tige- boucle (TB I, II et III) hautement similaires et séparées par des « espaceurs » ayant des séquences variables. De plus, cet ARNnc est clivé en petits fragments correspondant au trois molécules en tige-boucle, par un processus de clivage interne impliquant l'endoribonucléase RNase E. Concernant les fonctions de NrsZ chez P. aeruginosa PAOl, cet ARNnc est capable d'induire la motilité de type « swarming » et la production de biosurfactants, nommés rhamnolipides. Cette régulation est due à l'activation par NrsZ de l'expression de rhlA, un gène essentiel pour la motilité de type swarming et pour la production de rhamnolipides. Étonnamment, la structure TB I est capable d'assurer à elle seule la fonction régulatrice de NrsZ sur l'expression de rhlA, suggérant que ces molécules TBs sont les unités fonctionnelles de cet ARNnc modulaire. Cependant, le mécanisme moléculaire par lequel NrsZ active l'expression de rhlA demeure à ce jour incertain et est actuellement à l'étude. En plus, le réseau de régulations médiées par NrsZ a été étudié par une analyse de transcriptome qui a indiqué que de nombreux gènes impliqués dans le métabolisme primaire ou secondaire seraient régulés par NrsZ. Pour accentuer l'importance de NrsZ, nous avons étudié sa conservation dans d'autres espèces de Pseudomonas. Ainsi, nous avons démontré que NrsZ est conservé et exprimé en situation de carence d'azote par les souches Pseudomonas protegens Pf-5, Pseudomonas putida KT2442, Pseudomonas entomophila L48, Pseudomonas syringae pv. tomato DC3000, quatre espèces ayant des caractéristiques écologiques très différentes, suggérant que NrsZ joue un rôle important dans l'adaptation du genre Pseudomonas envers la carence en azote. Chez toutes les souches étudiées, les différents homologues de NrsZ présentent une architecture similaire faite de TBs conservées et d'espaceurs. Cependant, le nombre de TBs n'est pas identique et peut varier de une à six copies selon la souche. Les différentes versions de NrsZ sont clivées en petites molécules dans ces quatre souches, comme il a été observé chez P. aeruginosa PAOl. De plus, l'expression hétérologue des différentes variantes de NrsZ est capable de restaurer l'expression de rhlA, la motilité swarming et la production de rhamnolipides dans une souche de P. aeruginosa dont nrsZ a été inactivé. Par bien des aspects, NrsZ est un ARNnc atypique dans le monde bactérien. À notre connaissance, NrsZ est le premier ARNnc décrit comme étant régulé par le système NtrB/C. De plus, son unique architecture modulaire et son clivage en petites molécules similaires suggèrent que NrsZ appartient à une nouvelle famille d'ARNncs bactériens.

Characterization of sustained BOLD activation in the rat barrel cortex and neurochemical consequences.

To date, only a couple of functional MR spectroscopy (fMRS) studies were conducted in rats. Due to the low temporal resolution of (1)H MRS techniques, prolonged stimulation paradigms are necessary for investigating the metabolic outcome in the rat brain during functional challenge. However, sustained activation of cortical areas is usually difficult to obtain due to neural adaptation. Anesthesia, habituation, high variability of the basal state metabolite concentrations as well as low concentrations of the metabolites of interest such as lactate (Lac), glucose (Glc) or γ-aminobutyric acid (GABA) and small expected changes of metabolite concentrations need to be addressed. In the present study, the rat barrel cortex was reliably and reproducibly activated through sustained trigeminal nerve (TGN) stimulation. In addition, TGN stimulation induced significant positive changes in lactate (+1.01μmol/g, p<0.008) and glutamate (+0.92μmol/g, p<0.02) and significant negative aspartate changes (-0.63μmol/g, p<0.004) using functional (1)H MRS at 9.4T in agreement with previous changes observed in human fMRS studies. Finally, for the first time, the dynamics of lactate, glucose, aspartate and glutamate concentrations during sustained somatosensory activation in rats using fMRS were assessed. These results allow demonstrating the feasibility of fMRS measurements during prolonged barrel cortex activation in rats.

CHARACTERIZATION OF HEADWATERS PEATS OF THE RIO ARAÇUAÍ, MINAS GERAIS STATE, BRAZIL

Peatlands are soil environments that accumulate water and organic carbon and function as records of paleo-environmental changes. The variability in the composition of organic matter is reflected in their morphological, physical, and chemical properties. The aim of this study was to characterize these properties in peatlands from the headwaters of the Rio Araçuaí (Araçuaí River) in different stages of preservation. Two cores from peatlands with different vegetation types (moist grassland and semideciduous seasonal forest) from the Rio Preto [Preto River] headwaters (conservation area) and the Córrego Cachoeira dos Borges [Cachoeira dos Borges stream] (disturbed area) were sampled. Both are tributaries of the Rio Araçuaí. Samples were taken from layers of 15 cm, and morphological, physical, and chemical analyses were performed. The 14C age and δ13C values were determined in three samples from each core and the vertical growth and organic carbon accumulation rates were estimated. Dendrograms were constructed for each peatland by hierarchical clustering of similar layers with data from 34 parameters. The headwater peatlands of the Rio Araçuaí have a predominance of organic material in an advanced stage of decomposition and their soils are classified as Typic Haplosaprists. The organic matter in the Histosols of the peatlands of the headwaters of the Rio Araçuaí shows marked differences with respect to its morphological, physical, and chemical composition, as it is influenced by the type of vegetation that colonizes it. The peat from the headwaters of the Córrego Cachoeira dos Borges is in a more advanced stage of degradation than the peat from the Rio Preto, which highlights the urgent need for protection of these ecosystems/soil environments.