Analysis of Centrosome duplication in "Caenorhabditis elegans" : the role of "sas" genes

Abstract: The centrosome is the major microtubule organizing center (MTOC) of most animal cells. As such, it is essential for a number of processes, including polarized secretion or bipolar spindle assembly. Hence, centrosome number needs to be controlled precisely in coordination with DNA replication. Cells early in the cell cycle contain one centrosome that duplicates during S-phase to give rise to two centrosomes that organize a bipolar spindle during mitosis. A failure in this process is likely to engage the spindle assembly checkpoint and threaten genome stability. Despite its importance for normal and uncontrolled proliferation the mechanisms underlying centrosome duplication are still unclear. The Caenorhabditis elegans embryo is well suited to study the mechanisms of centrosome duplication. It allows for the analysis of cellular processes with high temporal and spatial resolution. Gene identification and inactivation techniques are very powerful and a wide set of mutant and transgenic strains facilitates analysis. My thesis project consisted of characterizing three sas-genes: sas-4, sas-5 and sas-¬6. Embryos lacking these genes fail to form a bipolar spindle, hence their name (spindle assembly). I established that sas-4(RNAi) and sas-6(RNAi) embryos do not form daughter centrioles and thus do not duplicate their centrosomes. Furthermore, I showed that both proteins localize to the cytoplasm and are strikingly enriched at centrioles throughout the cell cycle. By performing fluorescent recovery after photobleaching (FRAP) experiments and differentially labeling centrioles, I established that both proteins are recruited to centrioles once per cell cycle when daughter centrioles form. In contrast, SAS-5, PLK-1 and SPD-2 shuttle permanently between the cytoplasm and centrioles. By showing that SAS-5 and SAS-6 interact in vivo, I established a functional relationship between the proteins. Testing the putative human homologue of SAS-6 (HsSAS-6) and a distant relative of SAS-4 (CPAP), I was able to show that these proteins are required for centrosome duplication in human cells. In addition I found that overexpression of GFP¬HsSAS-6 leads to formation of extra centrosomes. In conclusion, we identified and gained important insights into proteins required for centrosome duplication in C. elegans and in human cells. Thus, our work contributes to further elucidate an important step of cell division in normal and malignant tissues. Eventually, this may allow for the development of novel diagnostic or therapeutic reagents to treat cancer patients. Résumé: Le centrosome est le principal centre organisateur des microtubules dans les cellules animales. De ce fait, il est essentiel pour un certain nombre de processus, comme l'adressage polarisé ou la mise en place d'un fuseau bipolaire. Le nombre de centrosome doit être contrôlé de façon précise et en coordination avec la réplication de l'ADN. Au début du cycle cellulaire, les cellules n'ont qu'un seul centrosome qui se duplique au cours de la phase S pour donner naissance à deux centrosomes qui forment le fuseau bipolaire pendant la mitose. Des défauts dans ce processus déclencheront probablement le "checkpoint" d'assemblage du fuseau et menaceront la stabilité du génome. Malgré leurs importances pour la prolifération normale ou incontrôlée des cellules, les mécanismes gouvernant la duplication des centrosomes restent obscures. L'embryon de Caenorhabditis elegans est bien adapté pour étudier les mécanismes de duplication des centrosomes. Il permet l'analyse des processus cellulaires avec une haute résolution spatiale et temporelle. L'identification des gènes et les techniques d'inactivation sont très puissantes et de larges collections de mutants et de lignées transgéniques facilitent les analyses. Mon projet de thèse a consisté à caractérisé trois gènes: sas-4, sas-5 et sas-6. Les embryons ne possédant pas ces gènes ne forment pas de fuseaux bipolaires, d'où leur nom (spindle assembly). J'ai établi que les embryons sas-4(RNAi) et sas-6(RNAi) ne forment pas de centrioles fils, et donc ne dupliquent pas leur centrosome. De plus, j'ai montré que les deux protéines sont localisées dans le cytoplasme et sont étonnamment enrichies aux centrioles tout le long du cycle cellulaire. En réalisant des expériences de FRAP (fluorscence recovery after photobleaching) et en marquant différentiellement les centrioles, j'ai établi que ces deux protéines sont recrutées une fois par cycle cellulaire aux centrioles, au moment de la duplication. Au contraire, SAS-5, PLK-1 et SPD-2 oscillent en permanence entre le cytoplasme et les centrioles. En montrant que SAS-5 et SAS-6 interagissent in vivo, j'ai établi une relation fonctionnelle entre les deux protéines. En testant les homologues humains putatifs de SAS-6 (HsSAS-6) et de SAS-4 (CPAP), j'ai été capable de montrer que ces protéines étaient aussi requises pour la duplication des centrosomes dans les cellules humaines. De plus, j'ai montré que la surexpression de GFP-HsSAS-6 entrainait la formation de centrosomes surnuméraires. En conclusion, nous avons identifié et progressé dans la compréhension de protéines requises pour la duplication des centrosomes chez C. elegans et dans les cellules humaines. Ainsi, notre travail contribue à mieux élucider une étape importante du la division cellulaire dans les cellules normales et malignes. A terme, ceci devrait aider au développement de nouveaux diagnostics ou de traitements thérapeuthiques pour soigner les malades du cancer.

Phase I dose-finding study of cabazitaxel administered weekly in patients with advanced solid tumours.

BACKGROUND Cabazitaxel is approved in patients with metastatic hormone-refractory prostate cancer previously treated with a docetaxel-containing regimen. This study evaluated a weekly cabazitaxel dosing regimen. Primary objectives were to report dose-limiting toxicities (DLTs) and to determine the maximum tolerated dose (MTD). Efficacy, safety and pharmacokinetics were secondary objectives. METHODS Cabazitaxel was administered weekly (1-hour intravenous infusion at 1.5-12 mg/m2 doses) for the first 4 weeks of a 5-week cycle in patients with solid tumours. Monitoring of DLTs was used to determine the MTD and the recommended weekly dose. RESULTS Thirty-one patients were enrolled. Two of six patients experienced DLTs at 12 mg/m2, which was declared the MTD. Gastrointestinal disorders were the most common adverse event. Eight patients developed neutropenia (three ≥ Grade 3); one occurrence of febrile neutropenia was reported. There were two partial responses (in breast cancer) and 13 patients had stable disease (median duration of 3.3 months). Increases in Cmax and AUC0-t were dose proportional for the 6-12 mg/m2 doses. CONCLUSION The MTD of weekly cabazitaxel was 12 mg/m2 and the recommended weekly dose was 10 mg/m2. The observed safety profile and antitumour activity of cabazitaxel were consistent with those observed with other taxanes in similar dosing regimens. TRIAL REGISTRATION The study was registered with ClinicalTrials.gov as NCT01755390.

Analysis and quantification of vitamin D metabolites in serum by ultra-performance liquid chromatography coupled to tandem mass spectrometry and high-resolution mass spectrometry: a method comparison and validation.

RATIONALE: The aim of the work was to develop and validate a method for the quantification of vitamin D metabolites in serum using ultra-high-pressure liquid chromatography coupled to mass spectrometry (LC/MS), and to validate a high-resolution mass spectrometry (LC/HRMS) approach against a tandem mass spectrometry (LC/MS/MS) approach using a large clinical sample set. METHODS: A fast, accurate and reliable method for the quantification of the vitamin D metabolites, 25-hydroxyvitamin D2 (25OH-D2) and 25-hydroxyvitamin D3 (25OH-D3), in human serum was developed and validated. The C3 epimer of 25OH-D3 (3-epi-25OH-D3) was also separated from 25OH-D3. The samples were rapidly prepared via a protein precipitation step followed by solid-phase extraction (SPE) using an HLB μelution plate. Quantification was performed using both LC/MS/MS and LC/HRMS systems. RESULTS: Recovery, matrix effect, inter- and intra-day reproducibility were assessed. Lower limits of quantification (LLOQs) were determined for both 25OH-D2 and 25OH-D3 for the LC/MS/MS approach (6.2 and 3.4 µg/L, respectively) and the LC/HRMS approach (2.1 and 1.7 µg/L, respectively). A Passing & Bablok fit was determined between both approaches for 25OH-D3 on 662 clinical samples (1.11 + 1.06x). It was also shown that results can be affected by the inclusion of the isomer 3-epi-25OH-D3. CONCLUSIONS: Quantification of the relevant vitamin D metabolites was successfully developed and validated here. It was shown that LC/HRMS is an accurate, powerful and easy to use approach for quantification within clinical laboratories. Finally, the results here suggest that it is important to separate 3-epi-25OH-D3 from 25OH-D3. Copyright © 2012 John Wiley & Sons, Ltd.

Daptomycin plus fosfomycin versus daptomycin monotherapy in treating MRSA: protocol of a multicentre, randomised, phase III trial.

INTRODUCTION Despite the availability of new antibiotics such as daptomycin, methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia continues to be associated with high clinical failure rates. Combination therapy has been proposed as an alternative to improve outcomes but there is a lack of clinical studies. The study aims to demonstrate that combination of daptomycin plus fosfomycin achieves higher clinical success rates in the treatment of MRSA bacteraemia than daptomycin alone. METHODS AND ANALYSIS A multicentre open-label, randomised phase III study. Adult patients hospitalised with MRSA bacteraemia will be randomly assigned (1:1) to group 1: daptomycin 10 mg/kg/24 h intravenous; or group 2: daptomycin 10 mg/kg/24 h intravenous plus fosfomycin 2 gr/6 g intravenous. The main outcome will be treatment response at week 6 after stopping therapy (test-of-cure (TOC) visit). This is a composite variable with two values: Treatment success: resolution of clinical signs and symptoms (clinical success) and negative blood cultures (microbiological success) at the TOC visit. Treatment failure: if any of the following conditions apply: (1) lack of clinical improvement at 72 h or more after starting therapy; (2) persistent bacteraemia (positive blood cultures on day 7); (3) therapy is discontinued early due to adverse effects or for some other reason based on clinical judgement; (4) relapse of MRSA bacteraemia before the TOC visit; (5) death for any reason before the TOC visit. Assuming a 60% cure rate with daptomycin and a 20% difference in cure rates between the two groups, 103 patients will be needed for each group (α:0.05, ß: 0.2). Statistical analysis will be based on intention to treat, as well as per protocol and safety analysis. ETHICS AND DISSEMINATION The protocol was approved by the Spanish Medicines and Healthcare Products Regulatory Agency (AEMPS). The sponsor commits itself to publishing the data in first quartile peer-review journals within 12 months of the completion of the study. TRIAL REGISTRATION NUMBER NCT01898338.

Tonotopic gradients in human primary auditory cortex: concurring evidence from high-resolution 7 t and 3 t FMRI.

The tonotopic representations within the primary auditory cortex (PAC) have been successfully mapped with ultra-high field fMRI. Here, we compared the reliability of this tonotopic mapping paradigm at 7 T with 1.5 mm spatial resolution with maps acquired at 3 T with the same stimulation paradigm, but with spatial resolutions of 1.8 and 2.4 mm. For all subjects, the mirror-symmetric gradients within PAC were highly similar at 7 T and 3 T and across renderings at different spatial resolutions; albeit with lower percent signal changes at 3 T. In contrast, the frequency maps outside PAC tended to suffer from a reduced BOLD contrast-to-noise ratio at 3 T for a 1.8 mm voxel size, while robust at 2.4 mm and at 1.5 mm at 7 T. Overall, our results showed the robustness of the phase-encoding paradigm used here to map tonotopic representations across scanners.

Conflict resolution and evolution of social structures in insect societies

In colonies of social Hymenoptera (which include all ants, as well as some wasp and bee species), only queens reproduce whereas workers generally perform other tasks. The evolution of worker's reproductive altruism can be explained by kin selection, which states that workers can indirectly transmit copies of their genes by helping the reproduction of relatives. The relatedness between queens and workers may however be low, particularly when there are multiple queens per colony, which limits the transmission of copies of workers genes and increases potential conflicts between colony members. In this thesis, we investigated the link between social structure variations and conflicts, and explored the mechanisms involved in variation of colony queen number in ants. According to kin selection, workers should rear the brood they are most related to. In social Hymenoptera, males are haploid whereas females (workers and queens) are diploid. As a result, workers can be up to three times more related to females than males in some colonies, where they should consequently favour the production of females. In contrast, queens are equally related to daughters and sons in all types of colonies and therefore should favour a balanced sex ratio. In a meta-analysis across all studies of social Hymenoptera, we showed that colony sex ratio is generally largely influenced by workers. Hence, the evolution of social structures where queens and workers are equally related to males and females may contribute to decrease the conflict between the two castes over colony sex ratio. Another conflict between queens and workers can occur over male production. Many species contain workers that still have the ability to lay haploid eggs. In some social structures, workers are on average more related to sons of queens than to sons of other workers. As a result, workers should eliminate worker-laid eggs to favour queen-laid eggs. We showed that in the ant Formica selysi, workers eliminate more worker-laid than queen-laid eggs, independently of colony social structure. These results therefore suggest that worker policing can evolve independently from relatedness, potentially because of costs of worker reproduction at the colony-level. Colony queen number is a key parameter that influences relatedness between group members. Queen body size is generally linked to the success of independent colony foundation by single queens and may influence the number of queens in the new colony. In the ant F. selysi, single-queen colonies produce larger queens than multiple-queen colonies. We showed that this association results from genes or maternal effects transmitted to the eggs. However, we also found that queens produced in colonies of the two social forms did not differ in their general ability to found new colonies independently. Queen body size may also influence queen dispersal ability and constrain small queens to be re-adopted in their original nest after mating at proximity. We tested the acceptance of new queens in another ant species, Formica paralugubris, which has numerous queens per colony. Our results show that workers do not discriminate between nestmate and foreign queens, and more generally accept new queens at a limited rate. To conclude, this thesis shows that mechanisms influencing variation in colony queen number and the influence of these changes on conflict resolution are complex. Data gathered in this thesis therefore constitute a solid background for further research on the evolution and the maintenance of complex organisations in insect societies.

Implications of the serpentine phase transition on the behaviour of beryllium and lithium-boron of subducted ultramafic rocks

The Totalp-Platta-Malenco ophiolites in the Eastern Central Alps offer a unique opportunity to study the behaviour of Li, Be and B in ultramafic rocks in response to serpentinization and to progressive Alpine metamorphism. These units represent the remnants of a former ocean-continent transition that was intensely serpentinized during exposure on the Jurassic seafloor of the Ligurian Tethys. From north to the south, three isograd reactions (lizardite double right arrow antigorite + brucite; lizardite + talc double right arrow antigorite; lizardite + tremolite double right arrow antigorite + diopside) have been used to quantify the evolution of the light element content of metamorphic minerals. We determined the Li, Be and B concentrations in major silicate minerals from the ultramafic bodies of Totalp, Platta and Malenco by secondary ion mass spectrometry. Mantle minerals have Be concentrations (e.g. <0.001-0.009 mu g/g in olivine) similar to the metamorphic minerals that replace them (e.g. <0.001-0.016 mu g/g in serpentine). The mantle signature of Be is thus neither erased during seafloor alteration nor by progressive metamorphism from prehnite-pumpellyite to epidote-amphibolite facies. In contrast, the Li and B inventories of metamorphic minerals are related to the lizardite-to-antigorite transition. Both elements display higher concentrations in the low-temperature serpentine polymorph lizardite (max. 156 mu/g Li, max. 318 mu g/g B) than in antigorite (max. 0.11 mu g/g Li, max. 12 mu g/g B). Calculated average B/Li ratios for lizardite (similar to 1395) and antigorite (similar to 115) indicate that Li fractionates from B during the lizardite-to-antigorite transition during prograde metamorphism in ultramafic rocks. In subduction zones, this signature is likely to be recorded in the B-rich nature of forearc fluids. Relative to oceanic mantle the Be content of mantle clinopyroxene is much higher, but similar to Be values from mantle xenoliths and subduction-related peridotite massifs. These data support previous hypothesis that the mantle rocks from the Eastern Central Alps have a subcontinental origin. We conclude that Be behaves conservatively during subduction metamorphism of ultramafic rocks, at least at low-temperature, and thus retains the fingerprint of ancient subduction-related igneous events in mantle peridotites. (C) 2010 Elsevier Ltd. All rights reserved.

The role of Toll-like Receptor (TLR) 9 in the development of a T-helper (Th)1/Th2 response in the murine model of infection with " Leishmania major "

1.1 SUMMARY The role of the non-specific innate immune system is as important as the elaboration of the adaptive immune system in the initiation of an immune response to pathogens. The role of the Toll-like receptors (TLRs) in the innate immune response to virus and bacterial pathogens is widely recognised, however, little is known about the role of TLRs in host defence against eukaryotic pathogens. Immunologic investigations on the marine model of infection with Leishmania major (L. major) have correlated the outcome of the disease with expansion of different subsets of CD4+ cells, designated Th1 and Th2. The resistance of C57BL/6, CBA and C3H/He mice is linked with an IL-12 driven Th1 response. In BALB/c mice the susceptibility correlates with an IL-4 driven Th2 response. The initial event promoting the development of a Th1 or Th2 response still remains elusive. Recently, the contribution of the TLR signalling pathway in the innate and acquired immune response to infection with the intracellular protozoan parasite L. major has been demonstrated. Thus, the purpose of this study is to determine whether TLRs may play a role in influencing the outcome of the infection by directing the development of a Th1 or a Th2 response during infection with L, major parasites, in resistant C57BL/6 and susceptible BALB/c mice, respectively. We demonstrated that MyD88, the major TLR adaptor molecule is necessary for C57BL/6 to develop a resistant Th1 response following L. major infection. Our data show the essential role of MyD88 in the establishment of a protective Th1 response. We subsequently aimed to determine which TLRs may be involved in the protective response. Since TLR2 and TLR4 have shown to have a potential role for Leishmania recognition, we analysed the course of infection in TLR2 and TLR4 deficient mice on a C57BL/6 resistant background following L. major infection. Our results clearly demonstrate that TLR2 or TLR4 aze dispensable to control the outcome of the disease as the TLR2 and TLR4 knockout mice developed a protective Th1 response. With the aim of determining a potential TLR candidate important in the initiation of the Thl response, we assessed the mRNA expression of different TLRs (TLR1 to TLR9) using quantitative real-time RT-PCR at different time points during the first week of infection. The results clearly showed an upregulation of TLR7 and TLR9 mRNA expression during the early phase of infection in resistant C57BL/6 mice but not in susceptible BALB/c mice. To provide in vivo evidence for the role for, these TLRs in the outcome of cutaneous leishmaniasis, studies using TLR7 and TLR9 deficient mice on a resistant C57BL/6 background were performed. The TLR7 deficient mice developed a resistance phenotype that was comparable with C57BL/6 wild type mice. Thus, the presence of TLR7 is not indispensable for the development of a Th1 response and resistance to infection. On the contrary, TLR9 deficient mice on the C57BL/6 resistant background showed high variability in the outcome of the disease. Although some mice behave as resistant C57BL/6 mice, half of them developed high lesion following infection and showed a decrease in IFN-γ production and an increase in IL-4 as compared to wild type mice. These results suggest that TLR9 may be involved in the control of infection. To test the hypothesis that regulatory T cells (Treg) are playing a role in the high variability in the disease outcome in TLR9 deficient mice, depletion of CD4+CD25+ T cells with a specific antibody three days before infection with L. major were performed Interestingly, these treated mice developed large lesions, low IL-4 and decreased IFN-γ producion when compared to untreated mice. A better understanding of the mechanism by which Treg cells influence the outcome of the disease in TLR9 deficient mice following L. major infection is currently under investigation. Altogether, this study demonstrates the importance of TLR9 in the induction of a protective T'h1 response, a process that is involved in the resolution of the lesion induced by L. major infection. 1.2 RÉSUMÉ Le rôle de la réponse immunitaire innée a longtemps été négligé quant à l'impact qu'elle pourrait avoir dans l'initiation d'une réponse immune adaptative efficace dirigée contre un pathogène. Si l'importance des récepteurs Toll-like (TLR) du système inné dans la reconnaissance des virus et bactéries a été démontrée, son rôle dans la défense contre les pathogènes eucaryotes reste encore très élusif. Récemment, il a été montré que les voies de signalisation provenant de l'activation des TLRs pouvaient initier la réponse immunitaire innée et adaptative après une infection avec le parasite protozoaire Leishmania major (L. major). Dans un modèle marin d'infection avec L. major alors que la plupart des souches de souris telles que C57BL/6 sont résistantes à l'infection et développent une réponse immunitaire de type T helper 1 (Th1) induite par IL-12, peu de souches dont les BALB/c sont sensibles et développent une réponse Th2 induite par IL-4. La différentiation Th1/Th2 est un événement qui prend place de manière définitive lors de la première semaine après infection. Les événements précoces promouvant le développement d'une réponse Th1 ou Th2 n'étant pas connus, l'objectif de ce travail a été de démontrer un rôle des TLRs dans l'initiation d'une réponse immune innée et adaptative suite à l'infection par L. major. Nous avons démontré que MyD88, une molécule importante dans le processus de signalisation des TLRs, est nécessaire pour que les souris résistantes C57BL/6 développent une réponse Th1 protectrice. L'importance du rôle de TLR2 et TLR4 dans la reconnaissance du parasite Leishmania ayant été démontrée, nous avons privilégié l'analyse de la réponse immunitaire suite à une infection in vivo de souris déficiente en TLR2 ou TLR4 sur un fond génétique résistant. Les résultats obtenus montrent que la présence de ces récepteurs n'est pas indispensable pour le contrôle de l'infection et la polarisation d'une réponse Th1 caractéristique de la résistance à L. major. Cependant d'autres TLRs peuvent aussi activer la voie de signalisation MyD88 dépendante. L'expression de l'ARNm des différents TLRs dans les ganglions drainant de souris sensibles et résistantes pendant la première semaine d'infection a été déterminée par PCR quantitative en temps réel. Les résultats obtenus montrent que l'ARNm de TLR7 et TLR9 était régulé positivement suite à l'infection par L. major chez les souris résistantes C57BL/6 alors qu'aucune modulation n'était détectable chez les souris sensibles BALB/c. Le rôle des récepteurs TLR7 et TLR9 a donc été évalué par l'infection par L. major des souris déficientes en TLR7 et TLR9 sur fond génétique C57BL/6. Nos résultats ont clairement démontré que les souris déficientes en TLR7 montrent une réponse immunitaire identique à celle des souris résistantes C57BL/6, signifiant que TLR7 n'est pas indispensable au développement d'une Th1 ainsi qu'au contrôle de la parasitémie. Paz contre, les souris déficientes en TLR9 sur un fond génétique résistant ont montré une grande variabilité dans la réponse à l'infection. En effet, la moitié des souris deviennent sensibles à l'infection, ceci étant associé à une diminution dans la production d'IFN-γ et à une augmentation de la production d'IL-4. Ces résultats suggèrent que TLR9 est impliqué dans le contrôle de la lésion et de la réponse immunitaire suite à l'infection avec L. major. Cependant les résultats avec les souris déficientes en TLR9 montrant une grande hétérogénéité et une balance Th1/Th2 instable, nous avons émis l'hypothèse que les cellules T régulatrices pouvaient être impliquées dans ce phénomène. Nous avons effectivement constaté qu'après déplétion des cellules CD4+CD25+, les souris déficientes en TLR9 développent des lésions aussi grandes que les souris BALB/c après infection par L. major. Cependant le nombre de parasites reste le même que chez les souris C57BL/6. De plus la production d'IL-4 ainsi que celle d'IFN-γ reste extrêment bas. Les mécanismes régulateurs impliqués dans ce processus sont en cours d'analyse. Ce travail met en évidence l'importance du TLR9 dans le développement d'une réponse Th1 lors d'une infection avec L. major, un processus nécessaire pour la résistance à l'infection. 1.3 RESUME POUR UN LARGE PUBLIC La leishmaniose est une maladie parasitaire répandue dans le monde entier et touchant plus de 88 pays. L'incidence mondiale de la leishmaniose cutanée et de 1 à 1,5 million de nouveaux cas par année. Plus de 12 millions de personnes sont affectées par la maladie et 350 millions de personnes sont une population à risque. Un modèle marin d'infection avec Leishmania major (L. major) a été établi qui reproduit plusieurs tableaux cliniques observés dans le cas de la leishmaniose cutanée chez l'homme. L'analyse de la réponse immunitaire dans les souris infectées par L. major a permis de distinguer deux groupes : les souris de la plupart des souches telles que C57BL/6 sont résistantes à l'infection et développent une réponse immunitaire de type T helper 1 (Th1), alors que quelques souches dont les BALB/c sont sensibles et développent une réponse de type Th2. La réponse immune adaptative dans le modèle d'infection avec L. major à été largement étudiée. Cependant, les événements précoces déterminants pour le développement d'une réponse Th1 ou Th2 restent encore très flous. Récemment, plusieurs publications ont montré que les récepteurs Toll-like (TLR) peuvent contribuer à l'initiation de la réponse immunitaire lors d'une infection avec le parasite intracellulaire L. major. Dans ce travail de thèse, nous avons étudié le rôle de MyD88, une molécule importante dans le processus de signalisation des TLRs, dans la réponse immune suite à une infection avec L. major. En l'absence de MyD88, les souris normalement résistantes à l'infection avec L. major deviennent sensibles et développent des lésions importantes. Ces souris ne sont plus capables de développer une réponse Thl, normalement caractéristique de leur phénotype résistant. Nous avons ensuite tenté de comprendre quels TLRs, plus précisément, pouvait être impliqué dans ce processus. Malgré quelques évidences démontrant que TLR2 et TLR4 pouvaient avoir un rôle important dans l'initiation d'une réponse immunitaire adaptative à Leishmania, nous avons montré que, in vivo après infection avec L. major, la déficience d'un de ces récepteurs n'était pas suffisante à faire basculer la réponse immunitaire. Les souris C57BL/6 déficient en TLR2 ou TLR4 peuvent parfaitement contrôler l'évolution de la maladie. De plus, ces souris, malgré l'absence de TLR2 ou TLR4, sont capables de monter une parfaite réponse Thl. Etant donné que TLR2 et TLR4 n'étaient pas essentiels pour la résistance à la maladie, nous avons analysé les TLRs, parmi les 12 décrits qui pouvaient être indispensables au développement d'une réponse de type Th1 associée à la résistance à l'infection par Leishmania. Nos expériences ont montré que l'expression de l'ARN messager (ARNm) de TLR7 et TLR9 était modulée suite à l'infection par L. major chez la souris résistante C57BL/6 alors qu'aucune modulation n'était visible chez les souris sensible BALB/c. Pensant que ces TLRs pourraient jouer un rôle dans la réponse immunitaire au parasite, nous avons étudié l'évolution de l'infection dans les souris déficientes en TLR7 et TLR9. Nos résultats ont clairement démontré que TLR7 n'était pas indispensable à la résistance au parasite alors que l'absence de TLR9 avait des conséquences radicales sur le contrôle de la lésion et de la réponse immunitaire suite à l'infection avec L. major. Ce travail révèle ainsi l'importance du TLR9 dans le développement d'une réponse Th1 lors d'une infection avec L. major, un processus nécessaire pour la résistance à l'infection. Il est a noté que nos résultats sont en accord avec le fait que les motifs CpG, qui sont des immunostimulateurs interagissant avec le TLR9, ont une activité adjuvante importante dans la préparation de vaccins contre la leishmaniose. Une meilleure compréhension des mécanismes immunologiques impliquant le TLR9 dans la reconnaissance du parasite est alors indispensable pour le développement de vaccins thérapeutiques efficaces.

Bitumens in the late Variscan hydrothermal vein-type uranium deposit of Pribram, Czech Republic: Sources, radiation-induced alteration, and relation to mineralization

The late Variscan (275-278 Ma) Pribram uranium deposit is one of the largest known accumulations of uraniferous bitumens in hydrothermal veins. The deposit extends along the northwestern boundary of the Central Bohemian pluton (345-335 Ma) with low-grade metamorphosed Late Proterozoic and unmetamorphosed Cambrian rocks. From a net uranium production of 41,742 metric tons (t), more than 6,000 t were extracted from bitumen-uraninite ores during 43 years of exploration and mining. Three morphological varieties of solid bitumen are recognized: globular, asphaltlike, and cokelike. While the globular bitumen is uranium free, the other two types are uraniferous. The amount of bitumen in ore veins gradually decreases toward the contact with the plutonic body and increases with depth. Two types of bitumen microtextures are recognized using high-resolution transmission electron microscopy: amorphous and microporous, the former being less common in uraniferous samples. A lower Raman peak area ratio (1,360/1,575 cm(-1)) in mineralized bitumens (0.9) compared with uranium-free samples (2.0) indicates a lower degree of microtextural organization in the latter The H/C and O/C atomic ratios in uranium-free bitumens (0.9-1.1 and 0.09, respectively) are higher than those in mineralized samples (H/C = 0.3-0.8, O/C = 0.03-0.09). The chloroform extractable matter yield is Very low in uranium-free bitumens (0.30-0.35% of the total organic carbon,TOC) and decreases with uranium content increase. The extracted solid uraniferous bitumen infrared spectra show depletion in aliphatic CH2 and CH3 groups compared to uranium-free samples. The concentration of oxygen-bearing functional groups relative to aromatic bonds in the IR spectra of uranium-free and mineralized bitumen, however, do not differ significantly. C-13 NMR confirmed than the aromaticity of a uraniferous sample is higher (F-ar = 0.61) than in the uranium-free bitumen (F-ar = 0.51). Pyrolysates from uraniferous and nonuraniferous bitumens do not differ significantly, being predominantly cresol, alkylphenols, alkylbenzenes, and alkylnaphthalenes. The liquid pyrolysate yield decreases significantly with increasing uranium content. The delta(13)C Values of bulk uranium-free bitumens and low-grade uraniferous, asphaltlike bitumens range from -43.6 to 52.3 per mil. High-grade, cokelike, uraniferous bitumens are more C-13 depleted (54.5 to -58.4 parts per thousand). In contrast to the very light isotopic ratios of the high-grade uraniferous cokelike bitumen bulk carbon, the individual n-alkanes and isoprenoids (pristane and phytane) extracted from the same sample are significantly C-13 enriched. The isotopic composition of the C13-24 n-alkanes extracted from the high-grade uraniferous sample (delta(13)C = -28.0 to 32.6 parts per thousand) are heavier compared with the same compounds in a uranium-free sample (delta(13)C = 31.9 to 33.8 parts per thousand). It is proposed that the bitumen source was the isotopically light (delta(13)C = 35.8 to 30.2 parts per thousand) organic matter of the Upper Proterozoic host rocks that were pyrolyzed during intrusion of the Central Bohemian pluton. The C-13- depleted pyrolysates were mobilized from the innermost part of the contact-metamorphic aureole, accumulated in structural traps in less thermally influenced parts of the sedimentary complex and were later extracted by hydrothermal fluids. Bitumens at the Pribram deposit are younger than the main part of the uranium mineralization and were formed through water-washing and radiation-induced polymerization of both the gaseous and liquid pyrolysates. Direct evidence for pyrolysate reduction of uranium in the hydrothermal system is difficult to obtain as the chemical composition of the original organic fluid phase was modified during water-washing and radiolytic alteration. However, indirect evidence-e.g., higher O/C atomic ratios in uranium-free bitumens (0.1) relative to the Upper Proterozoic source rocks (0.02-0.05), isotopically very light carbon in associated whewellite (delta(13)C = 31.7 to -28.4 parts per thousand), and the striking absence of bitumens in the pre-uranium, hematite stage of the mineralization-indicates that oxidation of organic fluids may have contributed to lowering of aO(2) and uraninite precipitation.

Use of an in-house approach to study the three-dimensional structures of various outer membrane proteins: structure of the alcaligin outer membrane transporter FauA from Bordetella pertussis.

Bordetella pertussis is the bacterial agent of whooping cough in humans. Under iron-limiting conditions, it produces the siderophore alcaligin. Released to the extracellular environment, alcaligin chelates iron, which is then taken up as a ferric alcaligin complex via the FauA outer membrane transporter. FauA belongs to a family of TonB-dependent outer membrane transporters that function using energy derived from the proton motive force. Using an in-house protocol for membrane-protein expression, purification and crystallization, FauA was crystallized in its apo form together with three other TonB-dependent transporters from different organisms. Here, the protocol used to study FauA is described and its three-dimensional structure determined at 2.3 A resolution is discussed.

Kinetics of crystal growth and equilibrium domains in eclogite of the Sesia Zone, Western Alps

Kinetics of crystal growth and equilibrium domains in eclogite of the Sesia Zone, Western Alps Darbellay Bastien Institut de Minéralogie et Géochimie Résumé grand public Comme toute matière, la roche est sensible à son environnement et cherche à s'adapter pour acquérir un état stable (état d'équilibre). Les changements des conditions physiques (température et pression) vont ainsi impliquer des modifications dans la roche. Le métamorphisme est l'étude de ces changements. Les minéraux qui constituent la roche peuvent modifier, leur structure, leur chimie ou être remplacer par d'autres minéraux plus stables. Il est ainsi crucial de déterminer les processus responsables et limitant de la croissance minérale. Trois processus permettent la croissance ; (1) la dissolution des éléments du réactant, (2) le transport de ces éléments vers le site de croissance, (3) l'incorporation de ces éléments dans la nouvelle structure. Cette thèse se focalise sur les structures des minéraux de haute pression (forme, zonation chimique, structure interne) pour essayer de déterminer les facteurs importants à l'origine de leur état final. Les zones d'étude se situent dans la zone de Sésia. La première partie traite de la problématique liée à l'incorporation d'un élément dans une structure minérale. A l'image de la croissance humaine, les irrégularités minéralogiques permettent de mettre en lumière un dysfonctionnement de la croissance due à un excès ou à une carence d'un élément. Bien dosé, cet élément est cependant essentiel à la croissance. Les zoisites (épidotes) des métabasites de la région de Cima di Bonze montrent une zonation chimique en sablier. Dans cette zonation la teneur en fer excède la capacité maximum que peut contenir la structure orthorhombique de la zoisite. Des défauts de structure permettent l'accommodation de cet excès. La zoisite peut ainsi adapter sa structure pour permettre l'incorporation d'une relativement grande quantité de fer. Les études précédentes montraient, pour des conditions similaires, la formation de deux épidotes distinctes. La deuxième partie se penche sur la compétition entre le minéral qui fait sa croissance et les minéraux (réactants) qui l'entourent. Les métapélites de la région du Monte Mucrone contiennent des grenats atollaires. Des études détaillées de la texture et de la zonation chimique du grenat ainsi qu'une modélisation thermodynamique ont permis de mieux cerner les facteurs importants responsables de la forme atollaire. Cette structure est obtenue par un changement du comportement de la croissance du grenat le long d'un chemin P-T hercynien. Dans un premier stade, le grenat croît rapidement et consume peu le quartz de la matrice. La croissance se fait ainsi le long des jointures des grains de quartz. Dans un second temps, les changements de conditions PT donnent une croissance lente du grenat et une forte consommation du quartz. Le grenat peut ainsi développer sa forme dodécaédrale classique. La troisième partie s'intéresse aux distances de transport par diffusion d'un élément (ici l'argon) durant la haute pression. Pour ce faire, un profile d'âges 40Ar/39Ar sur biotite a été mesuré depuis un veine de haute pression riche en argon jusque dans son encaissant (granitoïd du Monte Mucrone). Le profile montre une répartition des âges suivant une courbe de diffusion. Le transport se fait sur une longueur de deux centimètre avec l'aide d'un fluide. Il est réduit à une échelle millimétrique quand la phase fluide disparaît. Cette étude montre ainsi les difficultés de transport des éléments durant la haute pression ne permettant pas un rééquilibrage de la roche à grande échelle. Kinetics of crystal growth and equilibrium domains in eclogite of the Sesia Zone, Western Alps Darbellay Bastien Institut de Minéralogie et Géochimie Résumé de thèse Les processus de croissance (diffusion des éléments et les réactions d'interface) et les conditions dans lesquelles les minéraux grandissent (température, pression, fluide, composition chimique de la roche), déterminent la texture ainsi que la zonation des minéraux. Cette thèse se focalise, par le biais de textures peu communes, sur trois différents processus impliqués dans la croissance minérale à haute pression (Zone de Sésia, Alpes de l'Ouest, Italie). L'incorporation d'un élément dans une structure minérale ne peut se faire que dans des sites en accord avec la taille et la charge ionique de l'élément. De plus, la balance de charge doit être maintenue dans le minéral. La régularité de la structure cristalline fixe ainsi une limite maximum de concentration d'un élément donné. Les zoisites provenant des métabasites de la région de Cima di Bonze montrent des zonations en sablier caractérisées par une concentration anormale en fer. La zonation se marque par une différente teinte de biréfringence et par un plus grand angle d'extinction que le reste de la zoisite. Une inter-croissance de clinozoisite à l'intérieur de la structure orthorhombique de la zoisite peut ainsi être suspectée. Les analyses XRD (diffraction des rayons x) ainsi que les analyses Raman ne confirment pas cette suspicion. Seules les analyses TEM (microscope à électrons transmis) montrent des défauts de structure pouvant être interprétés comme des modules de clinozoisite. Ils ne peuvent cependant pas être considérés comme une phase thermodynamique. Un nouveau trou d'immiscibilité entre deux zoisite (X ep= 0.1 and Xep = 0.15) a ainsi pu être établi. Dans les métapélites la région du Monte Mucrone, des grenats fortement zonés montrent une évolution texturale singulière. Ils présentent une forme initiale de `champignon' qui se développe pour former une structure atollaire finale. L'étude conjuguée de la structure 3D et des zonations, ainsi que l'établissement d'un model thermodynamique, indiquent que ces structures proviennent de deux épisodes de croissances : (1) La croissance du grenat durant un chemin prograde hercynien (de 525 °C et 6.2 kbar à 640 °C et 9 kbar) permet la formation des textures atollaires. Elles sont le résultat d'une croissance poecilitique initiale suivie d'une croissance idiomorphique du grenat. (2) La structure est rendue plus complexe par la cristallisation d'un grenat homogène tout autour ainsi qu'à l'intérieur du grenat hercynien durant la haute pression alpine (550 °C and 20 kbar). L'arrivée de l'eau durant la haute pression facilite le transport d'éléments et permet une cristallisation rapide du grenat. La diffusion peut être un facteur limitant de la croissance minéralogique. Elle a aussi une grande importance pour la géochronologie. Une veine de haute pression à l'intérieur du granitoïde du Monte Mucrone a été étudiée dans le but de déterminer la distance de diffusion de l'argon. Le profile d'âges 40Ar/39Ar sur biotites, établi de la veine vers le métagranitoïde, suit une courbe de diffusion. Les âges sont élevés proche de la veine (800 Ma) puis décroissent jusqu'à des âges homogènes (170-150 Ma) à deux centimètres de la veine. La présence de fluide, marqué par de hautes concentrations en chlore, permet une diffusion centimétrique. Cependant, la distance est réduite à une échelle millimétrique quand le fluide est absent. Les très faibles distances de diffusion préservent les âges pré-alpins et impliquent un événement géologique pour les âges de 170-150 Ma. Kinetics of crystal growth and equilibrium domains in eclogite of the Sesia Zone, Western Alps Darbellay Bastien Institut de Minéralogie et Géochimie Thesis abstract Rock textures and zonings are the consequence of growth processes (element diffusion and interface reaction) steered by the environment in which they grew (pressure, differential stress, temperature, fluid and rock composition). The thesis presented here focuses on three different topics, each of it dealing with aspects of mineral growth processes during subduction, in a high-pressure environment. All studies were conducted in the Sesia Zone of the Western European Alps, Italy. The first study addresses the crystallography and geochemistry of element incorporation in zoisite, one of the major hydrous minerals found in subduction zone rocks. Elements can be incorporated into a mineral structure only on crystallographic sites that offer enough space for the ion and the overall charge balance has to be maintained. Element concentrations are hence limited. Incorporation of some elements produces complex zoning, including hourglass like patterns, which are the focus of the first contribution. Zoisites from Cima di Bonze (Sesia Zone) show spectacular hourglass zoning defined by Fe-content variations. The hourglass zones have a distinct birefringence and a different extinction angle than the regular part of the zoisite. We show by detailed XRD (X-ray diffraction) and confocal Raman analyses that the high Fe-zones are nevertheless zoisite, and not clinozoisite as one might expect. High resolution TEM (transmission electron microscopy) analyses show planar defects on (100) that can be interpreted as small-scale clinozoisite modules. However, these clinozoisites cannot be interpreted as a distinctive thermodynamic phase and the entire mineral has to be considered as zoisite. The miscibility gap between two zoisites (Xep = 0.1 and Xep = 0.15) can be then definite at 550 ± 50°C and 14 to 20 Kbar. Strongly zoned garnets in quartz rich metapelite from the Monte Mucrone area (Sesia Zone) show evolution form 3D mushroom to atoll structure. The second contribution presents textural investigations, garnet zoning and thermodynamic modeling that demonstrate that atoll garnets are the result of two distinctive growth events. (1) Garnet atoll structure is already formed during a prograde Hercynian path from 525 °C and 6.2 kbar to 640 °C and 9 kbar. It results in an initial poikilitic growth followed by a final idiomorphic growth event. (2) Alpine HP garnet are homogenous (550 °C and 20 kbar) and grew around and also inside the Hercynian garnet. Lack of prograde Alpine garnet and fast growth of the HP garnet is explained by the absence of water during much of the prograde path. Water saturation was only observed towards the end, close towards the peak metamorphic conditions. Diffusion could be a limiting factor for crystal growth. It has also a great importance in geochronology. HP vein inside the metagranitoide of the Monte Mucrone (~300 Ma) was investigated to determine argon diffusion scales during high-pressure metamorphism. 40Ar/39Ar biotite ages profile from the vein toward the metagranodiorite show a diffusion curve: old ages (800 Ma) located close to the vein decrease until homogenous 170-150 Ma ages are obtained, two centimeter away from the vein. Centimeter-scale diffusion occurs with help of a fluid phase marked by high chlorine concentrations. Argon diffusion is reduced to a millimeter scale when free fluid is absent. Very short diffusion distance permits to preserve pre-Alpine ages. The 170-150 Ma ages are considered to be geologic meaningful, probably resulting from the extensional tectonics linked to opening of the Tethian ocean.

Investigation into Improved Pavement Curing Materials and Techniques: Part 2 - Phase III, March 2003

Appropriate curing is important for concrete to obtain the designed properties. This research was conducted to evaluate the curing effects of different curing materials and methods on pavement properties. At present the sprayed curing compound is a common used method for pavement and other concrete structure construction. Three curing compounds were selected for testing. Two different application rates were employed for the white-pigmented liquid curing compounds. The concrete properties of temperature, moisture content, conductivity, and permeability were examined at several test locations. It was found, in this project, that the concrete properties varied with the depth. Of the tests conducted (maturity, sorptivity, permeability, and conductivity), conductivity appears to be the best method to evaluate the curing effects in the field and bears potential for field application. The results indicated that currently approved curing materials in Iowa, when spread uniformly in a single or double application, provide adequate curing protection and meet the goals of the Iowa Department of Transportation. Experimental curing methods can be compared to this method through the use of conductivity testing to determine their application in the field.

Neuroprotective gene therapy for Huntington's disease, using polymer-encapsulated cells engineered to secrete human ciliary neurotrophic factor: results of a phase I study.

Huntington's disease (HD) is a monogenic neurodegenerative disease that affects the efferent neurons of the striatum. The protracted evolution of the pathology over 15 to 20 years, after clinical onset in adulthood, underscores the potential of therapeutic tools that would aim at protecting striatal neurons. Proteins with neuroprotective effects in the adult brain have been identified, among them ciliary neurotrophic factor (CNTF), which protected striatal neurons in animal models of HD. Accordingly, we have carried out a phase I study evaluating the safety of intracerebral administration of this protein in subjects with HD, using a device formed by a semipermeable membrane encapsulating a BHK cell line engineered to synthesize CNTF. Six subjects with stage 1 or 2 HD had one capsule implanted into the right lateral ventricle; the capsule was retrieved and exchanged for a new one every 6 months, over a total period of 2 years. No sign of CNTF-induced toxicity was observed; however, depression occurred in three subjects after removal of the last capsule, which may have correlated with the lack of any future therapeutic option. All retrieved capsules were intact but contained variable numbers of surviving cells, and CNTF release was low in 13 of 24 cases. Improvements in electrophysiological results were observed, and were correlated with capsules releasing the largest amount of CNTF. This phase I study shows the safety, feasibility, and tolerability of this gene therapy procedure. Heterogeneous cell survival, however, stresses the need for improving the technique.

New validated prognostic models and prognostic calculators in patients with low-grade gliomas diagnosed by central pathology review: a pooled analysis of EORTC/RTOG/NCCTG phase III clinical trials.

Background In a previous study, the European Organisation for Research and Treatment of Cancer (EORTC) reported a scoring system to predict survival of patients with low-grade gliomas (LGGs). A major issue in the diagnosis of brain tumors is the lack of agreement among pathologists. New models in patients with LGGs diagnosed by central pathology review are needed. Methods Data from 339 EORTC patients with LGGs diagnosed by central pathology review were used to develop new prognostic models for progression-free survival (PFS) and overall survival (OS). Data from 450 patients with centrally diagnosed LGGs recruited into 2 large studies conducted by North American cooperative groups were used to validate the models. Results Both PFS and OS were negatively influenced by the presence of baseline neurological deficits, a shorter time since first symptoms (<30 wk), an astrocytic tumor type, and tumors larger than 5 cm in diameter. Early irradiation improved PFS but not OS. Three risk groups have been identified (low, intermediate, and high) and validated. Conclusions We have developed new prognostic models in a more homogeneous LGG population diagnosed by central pathology review. This population better fits with modern practice, where patients are enrolled in clinical trials based on central or panel pathology review. We could validate the models in a large, external, and independent dataset. The models can divide LGG patients into 3 risk groups and provide reliable individual survival predictions. Inclusion of other clinical and molecular factors might still improve models' predictions.

Contrast agent-enhanced, free-breathing, three-dimensional coronary magnetic resonance angiography.

For free-breathing, high-resolution, three-dimensional coronary magnetic resonance angiography (MRA), the use of intravascular contrast agents may be helpful for contrast enhancement between coronary blood and myocardium. In six patients, 0.1 mmol/kg of the intravascular contrast agent MS-325/AngioMARK was given intravenously followed by double-oblique, free-breathing, three-dimensional inversion-recovery coronary MRA with real-time navigator gating and motion correction. Contrast-enhanced, three-dimensional coronary MRA images were compared with images obtained with a T2 prepulse (T2Prep) without exogenous contrast. The contrast-enhanced images demonstrated a 69% improvement in the contrast-to-noise ratio (6.6 +/- 1.1 vs. 11.1 +/- 2.5; P < 0.01) compared with the T2Prep approach. By using the intravascular agent, extensive portions (> 80 mm) of the native left and right coronary system could be displayed consistently with sub-millimeter in-plane resolution. The intravascular contrast agent, MS-325/AngioMARK, leads to a considerable enhancement of the blood/muscle contrast for coronary MRA compared with T2Prep techniques. The clinical value of the agent remains to be defined in a larger patient series. J. Magn. Reson. Imaging 1999;10:790-799.