Sputum containing zinc enhances carbapenem resistance, biofilm formation and virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa chronic lung infections are the leading cause of mortality in cystic fibrosis patients, a serious problem which is notably due to the numerous P. aeruginosa virulence factors, to its ability to form biofilms and to resist the effects of most antibiotics. Production of virulence factors and biofilm formation by P. aeruginosa is highly coordinated through complex regulatory systems. We recently found that CzcRS, the zinc and cadmium-specific two-component system is not only involved in metal resistance, but also in virulence and carbapenem antibiotic resistance in P. aeruginosa. Interestingly, zinc has been shown to be enriched in the lung secretions of cystic fibrosis patients. In this study, we investigated whether zinc might favor P. aeruginosa pathogenicity using an artificial sputum medium to mimic the cystic fibrosis lung environment. Our results show that zinc supplementation triggers a dual P. aeruginosa response: (i) it exacerbates pathogenicity by a CzcRS two-component system-dependent mechanism and (ii) it stimulates biofilm formation by a CzcRS-independent mechanism. Furthermore, P. aeruginosa cells embedded in these biofilms exhibited increased resistance to carbapenems. We identified a novel Zn-sensitive regulatory circuit controlling the expression of the OprD porin and modifying the carbapenem resistance profile. Altogether our data demonstrated that zinc levels in the sputum of cystic fibrosis patients might aggravate P. aeruginosa infection. Targeting zinc levels in sputum would be a valuable strategy to curb the increasing burden of P. aeruginosa infections in cystic fibrosis patients.

Multimodal MRI analysis of structural and functional networks in the human brain : application to two clinical studies : driving abilities under THC intoxication and early white matter changes in FXTAS

Les approches multimodales dans l'imagerie cérébrale non invasive sont de plus en plus considérées comme un outil indispensable pour la compréhension des différents aspects de la structure et de la fonction cérébrale. Grâce aux progrès des techniques d'acquisition des images de Resonance Magnetique et aux nouveaux outils pour le traitement des données, il est désormais possible de mesurer plusieurs paramètres sensibles aux différentes caractéristiques des tissues cérébraux. Ces progrès permettent, par exemple, d'étudier les substrats anatomiques qui sont à la base des processus cognitifs ou de discerner au niveau purement structurel les phénomènes dégénératifs et développementaux. Cette thèse met en évidence l'importance de l'utilisation d'une approche multimodale pour étudier les différents aspects de la dynamique cérébrale grâce à l'application de cette approche à deux études cliniques: l'évaluation structurelle et fonctionnelle des effets aigus du cannabis fumé chez des consommateurs réguliers et occasionnels, et l'évaluation de l'intégrité de la substance grise et blanche chez des jeunes porteurs de la prémutations du gène FMR1 à risque de développer le FXTAS (Fragile-X Tremor Ataxia Syndrome). Nous avons montré que chez les fumeurs occasionnels de cannabis, même à faible concentration du principal composant psychoactif (THC) dans le sang, la performance lors d'une tâche visuo-motrice est fortement diminuée, et qu'il y a des changements dans l'activité des trois réseaux cérébraux impliqués dans les processus cognitifs: le réseau de saillance, le réseau du contrôle exécutif, et le réseau actif par défaut (Default Mode). Les sujets ne sont pas en mesure de saisir les saillances dans l'environnement et de focaliser leur attention sur la tâche. L'augmentation de la réponse hémodynamique dans le cortex cingulaire antérieur suggère une augmentation de l'activité introspective. Une investigation des ef¬fets au niveau cérébral d'une exposition prolongée au cannabis, montre des changements persistants de la substance grise dans les régions associées à la mémoire et au traitement des émotions. Le niveau d'atrophie dans ces structures corrèle avec la consommation de cannabis au cours des trois mois précédant l'étude. Dans la deuxième étude, nous démontrons des altérations structurelles des décennies avant l'apparition du syndrome FXTAS chez des sujets jeunes, asymptomatiques, et porteurs de la prémutation du gène FMR1. Les modifications trouvées peuvent être liées à deux mécanismes différents. Les altérations dans le réseau moteur du cervelet et dans la fimbria de l'hippocampe, suggèrent un effet développemental de la prémutation. Elles incluent aussi une atrophie de la substance grise du lobule VI du cervelet et l'altération des propriétés tissulaires de la substance blanche des projections afférentes correspondantes aux pédoncules cérébelleux moyens. Les lésions diffuses de la substance blanche cérébrale peu¬vent être un marquer précoce du développement de la maladie, car elles sont liées à un phénomène dégénératif qui précède l'apparition des symptômes du FXTAS. - Multimodal brain imaging is becoming a leading tool for understanding different aspects of brain structure and function. Thanks to the advances in Magnetic Resonance imaging (MRI) acquisition schemes and data processing techniques, it is now possible to measure different parameters sensitive to different tissue characteristics. This allows for example to investigate anatomical substrates underlying cognitive processing, or to disentangle, at a pure structural level degeneration and developmental processes. This thesis highlights the importance of using a multimodal approach for investigating different aspects of brain dynamics by applying this approach to two clinical studies: functional and structural assessment of the acute effects of cannabis smoking in regular and occasional users, and grey and white matter assessment in young FMR1 premutation carriers at risk of developing FXTAS. We demonstrate that in occasional smokers cannabis smoking, even at low concentration of the main psychoactive component (THC) in the blood, strongly decrease subjects' performance on a visuo-motor tracking task, and globally alters the activity of the three brain networks involved in cognitive processing: the Salience, the Control Executive, and the Default Mode networks. Subjects are unable to capture saliences in the environment and to orient attention to the task; the increase in Hemodynamic Response in the Anterior Cingulate Cortex suggests an increase in self-oriented mental activity. A further investigation on long term exposure to cannabis, shows a persistent grey matter modification in brain regions associated with memory and affective processing. The degree of atrophy in these structures also correlates with the estimation of drug use in the three months prior the participation to the study. In the second study we demonstrate structural changes in young asymptomatic premutation carriers decades before the onset of FXTAS that might be related to two different mechanisms. Alteration of the cerebellar motor network and of the hippocampal fimbria/ fornix, may reflect a potential neurodevelopmental effect of the premutation. These include grey matter atrophy in lobule VI and modification of white matter tissue property in the corresponding afferent projections through the Middle Cerebellar Peduncles. Diffuse hemispheric white matter lesions that seem to appear closer to the onset of FXTAS and be related to a neurodegenerative phenomenon may mark the imminent onset of FXTAS.

Bond strength tests of dental adhesive systems and their correlation with clinical results : a meta-analysis

OBJECTIVE: To evaluate the variability of bond strength test results of adhesive systems (AS) and to correlate the results with clinical parameters of clinical studies investigating cervical restorations. MATERIALS AND METHODS: Regarding the clinical studies, the internal database which had previously been used for a meta-analysis on cervical restorations was updated with clinical studies published between 2008 and 2012 by searching the PubMed and SCOPUS databases. PubMed and the International Association for Dental Research abstracts online were searched for laboratory studies on microtensile, macrotensile and macroshear bond strength tests. The inclusion criteria were (1) dentin, (2) testing of at least four adhesive systems, (3) same diameter of composite and (4) 24h of water storage prior to testing. The clinical outcome variables were retention loss, marginal discoloration, detectable margins, and a clinical index comprising the three parameters by weighing them. Linear mixed models which included a random study effect were calculated for both, the laboratory and the clinical studies. The variability was assessed by calculating a ratio of variances, dividing the variance among the estimated bonding effects obtained in the linear mixed models by the sum of all variance components estimated in these models. RESULTS: Thirty-two laboratory studies fulfilled the inclusion criteria comprising 183 experiments. Of those, 86 used the microtensile test evaluating 22 adhesive systems (AS). Twenty-seven used the macrotensile test with 17 AS, and 70 used the macroshear test with 24 AS. For 28 AS the results from clinical studies were available. Microtensile and macrotensile (Spearman rho=0.66, p=0.007) were moderately correlated and also microtensile and macroshear (Spearman rho=0.51, p=0.03) but not macroshear and macrotensile (Spearman rho=0.34, p=0.22). The effect of the adhesive system was significant for microtensile and macroshear (p<0.001) but not for macrotensile. The effect of the adhesive system could explain 36% of the variability of the microtensile test, 27% of the macrotensile and 33% of the macroshear test. For the clinical trials, about 49% of the variability of retained restorations could be explained by the adhesive system. With respect to the correlation between bond strength tests and clinical parameters, only a moderate correlation between micro- and macrotensile test results and marginal discoloration was demonstrated. However, no correlation between these tests and a retention loss or marginal integrity was shown. The correlation improved when more studies were included compared to assessing only one study. SIGNIFICANCE: The high variability of bond strength test results highlights the need to establish individual acceptance levels for a given test institute. The weak correlation of bond-strength test results with clinical parameters leads to the conclusion that one should not rely solely on bond strength tests to predict the clinical performance of an adhesive system but one should conduct other laboratory tests like tests on the marginal adaptation of fillings in extracted teeth and the retention loss of restorations in non-retentive cavities after artificial aging.

Targeted inactivation of hypocretin receptors in dopaminergic and noradrenergic systems alters brain activity in wakefulness

Since the discovery of hypocretins/orexins (Hcrt/Ox) in 1998, several narcoleptic mouse models, such as Hcrt-KO, Hcrtrl-KO, Hcrtr2-KO and double receptors KO mice, and orexin-ataxin transgenic mice were generated. The available Hcrt mouse models do not allow the dissection of the specific role of Hcrt in each target region. Dr. Anne Vassalli generated loxP-flanked alleles for each Hcrt receptor, which are manipulated by Cre recombinase to generate mouse lines with disrupted Hcrtrl or Hcrtr2 (or both) in cell type-specific manner. The role of noradrenaline (NA) and dopamine (OA) in ttie regulation of vigilance states is well documented. The purpose of this thesis is to explore the role of the Hcrt input into these two monoaminergic systems. Chronic loss of Hcrtrl in NA neurons consolidated paradoxical sleep (PS), and altered wakefulness brain activity in baseline, during the sleep deprivation (SD), and when mice were challenged by a novel environment, or exposed to nest-building material. The analysis of alterations in the sleep EEG delta power showed a consistent correlation with the changes in the preceding waking quality in these mice. Targeted inactivation of Hcrt input into DA neurons showed that Hcrtr2 inactivation present the strongest phenotype. The loss of Hcrtr2 in DA neurons caused modified brain activities in spontaneous wakefulness, during SD, and in novel environmental conditions. In addition to alteration of wakefulness quality and quantity, conditional inactivation of Hcrtr2 in DA neurons caused an increased in time spent in PS in baseline and a delayed and less complete PS recovery after SD. In the first 30 min of sleep recovery, single (i.e. for Hcrtrl or Hcrtr2) conditional knockout receptor mice had opposite changes in delta activity, including an increased power density in the fast delta range with specific inactivation of Hcrtr2, but a decreased power density in the same range with specific inactivation of Hcrtrl in DA cells. These studies demonstrate a complex impact of Hcrt receptors signaling in both NA and DA system, not only on quantity and quality of wakefulness, but also on PS amount regulation as well as on SWS delta power expression. -- Depuis la découverte des hypocrétines/orexines (Hcrt/Ox) en 1998, plusieurs modèles de souris, narcoleptiques telles que Hcrt-KO, Hcrtr2-KO et récepteurs doubles KO et les souris transgéniques orexine-ataxine ont été générés. Les modèles de souris Hcrt disponibles ne permettaient pas la dissection du rôle spécifique de l'Hcrt dans chaque noyau neuronal cible. Notre laboratoire a généré des allèles loxP pour chacun des 2 gènes codant pour les récepteurs Hcrtr, qui sont manipulés par recombinase Cre pour générer des lignées de souris avec Hcrtrl inactivé, ou Hcrtr2 inactivé, (ou les deux), spécifiquement dans un type cellulaire particulier. Le rôle de la noradrénaline (NA) et la dopamine (DA) dans la régulation des états de vigilance est bien documentée. Le but de cette thèse est d'étudier le rôle de l'afférence Hcrt dans ces deux systèmes monoaminergiques au niveau de l'activité cérébrale telle qu'elle apparaît dans l'électroencéphalogramme (EEG). Mon travail montre que la perte chronique de Hcrtrl dans les neurones NA consolide le sommeil paradoxal (PS), et l'activité cérébrale de l'éveil est modifiée en condition spontanée, au cours d'une experience de privation de sommeil (SD), et lorsque les souris sont présentées à un nouvel environnement, ou exposées à des matériaux de construction du nid. Ces modifications de l'éveil sont corrélées à des modifications de puissance de l'activité delta du sommeil lent qui le suit. L'inactivation ciblée des Hcrtrs dans les neurones DA a montré que l'inactivation Hcrtr2 conduit au phénotype le plus marqué. La perte de Hcrtr2 dans les neurones DA mène à des modification d'activité cérébrale en éveil spontané, pendant SD, ainsi que dans des conditions environnementales nouvelles. En plus de l'altération de la qualité de l'éveil et de la quantité, l'inactivation conditionnelle de Hcrtr2 dans les neurones DA a provoqué une augmentation du temps passé en sommeil paradoxal (PS) en condition de base, et une reprise retardée et moins complète du PS après SD. Dans les 30 premières minutes de la récupération de sommeil, les modèles inactivés pour un seul des récepteurs (ie pour Hcrtrl ou Hcrtr2 seulement) montrent des changements opposés en activité delta, en particulier une densité de puissance accrue dans le delta rapide avec l'inactivation spécifique de Hcrtr2, mais une densité de puissance diminuée dans cette même gamme chez les souris inactivées spécifiquement en Hcrtrl dans les neurones DA. Ces études démontrent un impact complexe de l'inactivation de la neurotransmission au niveau des récepteurs d'Hcrt dans les deux compartiments NA et DA, non seulement sur la quantité et la qualité de l'éveil, mais aussi sur la régulation de quantité de sommeil paradoxal, ainsi que sur l'expression de la puissance delta pendant le sommeil lent.

Dustiness and deagglomeration testing: interlaboratory comparison of systems for nanoparticle powders

Different types of aerosolization and deagglomeration testing systems exist for studying the properties of nanomaterial powders and their aerosols. However, results are dependent on the specific methods used. In order to have well-characterized aerosols, we require a better understanding of how system parameters and testing conditions influence the properties of the aerosols generated. In the present study, four experimental setups delivering different aerosolization energies were used to test the resultant aerosols of two distinct nanomaterials (hydrophobic and hydrophilic TiO2). The reproducibility of results within each system was good. However, the number concentrations and size distributions of the aerosols created varied across the four systems; for number concentrations, e.g., from 10(3) to 10(6) #/cm(3). Moreover, distinct differences were also observed between the two materials with different surface coatings. The article discusses how system characteristics and other pertinent conditions modify the test results. We propose using air velocity as a suitable proxy for estimating energy input levels in aerosolization systems. The information derived from this work will be especially useful for establishing standard operating procedures for testing nanopowders, as well as for estimating their release rates under different energy input conditions, which is relevant for occupational exposure.

Coevolution of restriction factors and retroviruses

Les virus exploitent la machinerie cellulaire de l'hôte pour se répliquer. Ils doivent s'adapter pour infecter la cellule hôte de manière optimale tout en échappant à la vigilance du système de défense de l'hôte. Ainsi l'hôte et les virus se livrent à de constantes batailles évolutives. Mon travail de thèse a porté sur l'étude des signatures évolutives de facteurs de l'hôte agissant comme des 'facteurs de restriction' en bloquant la réplication rétrovirale chez les primates. Plus spécifiquement, mon travail a visé à utiliser des données évolutives pour renseigner les analyses fonctionnelles et la biologie. Nous avons étudié le facteur anti-VIH-1 nommé TRIM5a (i) chez les prosimiens pour mieux comprendre son rôle dans le contrôle d'un lentivirus endogène, (ii) dans son activité contre d'autres anciennes infections représentées par des rétrovirus endogènes humains et (iii) en tant que protéine capable de générer des mutants de la capside. Premièrement nous nous sommes intéressés à TRIM5a chez deux espèces de lémuriens dont Microcebus murinus qui porte le lentivirus endogène PSIV dans son génome depuis plusieurs millions d'années,. Nous avons observé que TRIM5a chez M. murinus a un spectre d'activité antivirale réduit à l'opposé de TRIM5a chez le Lemur catta - non porteur du PSIV endogène - qui bloque une large variété de rétrovirus dont le PSIV. De ce fait TRIM5a aurait pu contribuer à protéger certaines espèces de lémuriens vis-à-vis d'anciennes infections par le PSIV. A l'inverse du PSIV, des virus dérivés des rétrovirus endogènes humains HERV-K and HERV-H se sont révélés largement résistants à l'inhibition par TRIM5a. Ces données illustrent une absence de protection par TRIM5a face à d'autres anciennes infections rétrovirales. Puis, pour évaluer l'impact de la protéine TRIM5a humaine sur le VIH-1, nous avons testé l'effet de mutations des résidues sous sélection positive dans la capside du VIH-1 sur l'inhibition par TRIM5a. Nos résultats montrent que TRIM5a ne jouerait pas un rôle significatif dans l'évolution de la capside du VIH-1. Enfin notre travail a porté sur le facteur anti-VIH-1 SAMHD1 récemment découvert, que nous avons séquencé chez 25 espèces de primates. L'analyse évolutive des sites sous sélection positive et des expériences fonctionnelles ont permis d'identifier le domaine de SAMHD1 interagissant avec la protéine lentivirale Vpx. De même que d'autres protéines virales contrecarrent les facteurs de restriction en les menant à la dégradation, nous avons observé que Vpx induit la dégradation de SAMHD1 de manière spécifique à l'espèce. Ces découvertes contribuent à comprendre comment les facteurs de restriction et les virus co-évoluent pour se neutraliser l'un l'autre. - Viruses hijack the host cellular machinery to replicate. They adapt to infect optimally host cells while escaping host defense systems. Viruses and the host coevolve in an evolutionary struggle. My thesis work has been devoted to study the evolutionary signatures of host factors acting as restriction factors that block retroviral replication in primates. Specifically, my work aimed at using evolutionary data to inform functional analyses and biology. We studied the anti-HIV-1 factor TRIM5a (i) in prosimians to better understand its possible role in the control of an endogenous lentivirus, (ii) in its activity against other ancient infections - as represented by HERVs, and (iii) as a protein capable of generating escape mutants in the viral capsid. First, my work focused on two lemur species, one of which was the gray mouse lemur that carries the endogenous lentivirus PSIV integrated in its genome for several million years. TRIM5a from gray mouse lemur exhibited a limited antiviral spectrum as opposed to TRIM5a from ring-tailed lemur - not a host of PSIV - that is able to block diverse retroviruses notably PSIV. These results support the possible contribution of TRIM5a in protecting lemur species from ancient infection by PSIV. In contrast, chimeric viruses derived from two human endogenous retroviruses were broadly resistant to TRIM5a-mediated restriction, suggesting TRIM5a lack of activity against other types of ancient infections. To evaluate the recent impact of human TRIM5a on HIV-1 evolution, we tested whether variants at positively selected sites in the HIV-1 capsid affected the ability of human TRIM5a alleles to restrict HIV-1. Our results indicate that TRIM5a does not play a significant role in the evolution of HIV1 capsid. At last, our work concentrated on the newly discovered anti-HIV-1 restriction factor SAMHD1. We determined its coding sequence in a panel of 25 species of primates. Evolutionary analyses of positively selected sites in SAMHD1 domains and functional assays identified the domain of SAMHD1 interacting with the lentiviral protein Vpx. Similar to other viral countermeasures targeting cellular restriction factors, Vpx was responsible of the degradation of SAMHD1 orthologs in a species-specific manner. These findings contributed to understanding how restriction factors and viruses evolve to counteract each other.

The application of chemometrics on Infrared and Raman spectra as a tool for the forensic analysis of paints

The aim of this work is to evaluate the capabilities and limitations of chemometric methods and other mathematical treatments applied on spectroscopic data and more specifically on paint samples. The uniqueness of the spectroscopic data comes from the fact that they are multivariate - a few thousands variables - and highly correlated. Statistical methods are used to study and discriminate samples. A collection of 34 red paint samples was measured by Infrared and Raman spectroscopy. Data pretreatment and variable selection demonstrated that the use of Standard Normal Variate (SNV), together with removal of the noisy variables by a selection of the wavelengths from 650 to 1830 cm−1 and 2730-3600 cm−1, provided the optimal results for infrared analysis. Principal component analysis (PCA) and hierarchical clusters analysis (HCA) were then used as exploratory techniques to provide evidence of structure in the data, cluster, or detect outliers. With the FTIR spectra, the Principal Components (PCs) correspond to binder types and the presence/absence of calcium carbonate. 83% of the total variance is explained by the four first PCs. As for the Raman spectra, we observe six different clusters corresponding to the different pigment compositions when plotting the first two PCs, which account for 37% and 20% respectively of the total variance. In conclusion, the use of chemometrics for the forensic analysis of paints provides a valuable tool for objective decision-making, a reduction of the possible classification errors, and a better efficiency, having robust results with time saving data treatments.

Role of the stress sigma factor RpoS in GacA/RsmA-controlled secondary metabolism and resistance to oxidative stress in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens biocontrol strain CHA0, the two-component system GacS/GacA positively controls the synthesis of extracellular products such as hydrogen cyanide, protease, and 2,4-diacetylphloroglucinol, by upregulating the transcription of small regulatory RNAs which relieve RsmA-mediated translational repression of target genes. The expression of the stress sigma factor sigmaS (RpoS) was controlled positively by GacA and negatively by RsmA. By comparison with the wild-type CHA0, both a gacS and an rpoS null mutant were more sensitive to H2O2 in stationary phase. Overexpression of rpoS or of rsmZ, encoding a small RNA antagonistic to RsmA, restored peroxide resistance to a gacS mutant. By contrast, the rpoS mutant showed a slight increase in the expression of the hcnA (HCN synthase subunit) gene and of the aprA (major exoprotease) gene, whereas overexpression of sigmaS strongly reduced the expression of these genes. These results suggest that in strain CHA0, regulation of exoproduct synthesis does not involve sigmaS as an intermediate in the Gac/Rsm signal transduction pathway whereas sigmaS participates in Gac/Rsm-mediated resistance to oxidative stress.

Dimensionality of drinking consequences - cross-cultural comparability and stability over time

Despite the long tradition for asking about the negative social and health consequences of alcohol consumption in surveys, little is known about the dimensionality of these consequences. Analysing cross-sectional and longitudinal data from the Nordic Taxation Study collected for Sweden, Finland, and Denmark in two waves in 2003 and 2004 by means of an explorative principal component analysis for categorical data (CATPCA), it is tested whether consequences have a single underlying dimension across cultures. It further tests the reliability, replicability, concurrent and predictive validity of the consequence scales. A one-dimensional solution was commonly preferable. Whereas the two-dimensional solution was unable to distinguish clearly between different concepts of consequences, the one-dimensional solution resulted in interpretable, generally very stable scales within countries across different samples and time.

Mendacastrum n. gen. and Domuzdagia n. gen., two Jurassic spherical Spumellaria (Radiolaria) with hagiastrid medullary shell

Two new spumellarian radiolarian genera, Mendacastrum and Domuzdagia, are described from the lower Tithonian and lower Pliensbachian respectively. Both have a spherical cortical shell of actinommid type and a spherical or subspherical double medullary shell with the inner medullary shell of hagiastrid s.l. type. The inner medullary shell of Mendacastrum is of dactyliosphaerid or higumastrid s. sit. type, whereas that of Domuzdagia is of angulobracchiid type. Since they cannot be assigned to any described Mesozoic pyloniacean families, they are considered as type genera of two new families: Mendacastridae and Domuzdagiidae respectively.

Cell Therapy Assistance in Reconstructive Surgery for Musculoskeletal Tissues Following Burn and Trauma: Swiss Cellular Transplantation Platform

The sterol compositions of three oceanic jellyfish have been determined using gas chromatographic mass spectrometric techniques involving the use of two separate gas chromatographic column systems. The components in overlapping peaks have been identified by comparison of the mass spectra of peaks in the two column systems using subtractive techniques. A mid-water animal, Periphylla periphylla, was found to contain a very complex and unusual sterol profile including rare 5alpha-stanols, whereas two other oceanic jellyfish Pelagia noctiluca and Atolla wyvillei contained similar mixtures of delta5 sterols to those previously isolated from coastal species.

Controlling cytokinesis in the fission yeast Schizosaccharomyces pombe : the role of dma1; and ubc8

ABSTRACT The fission yeast Schizosaccharomyces pombe is a single celled eukaryote that has proved to be an excellent model system for the study of cell cycle control. S. pombe cells are rod shaped and grow mainly by elongation at their tips. They divide by formation of medially-placed cell wall, or septum, which cleaves the cell in two. Once the cell commits itself to mitosis the site of division is determined by formation of an acto-myosin based contractile ring at the cell cortex. The ring is assembled in stages throughout mitosis and contracts at the end of anaphase, coincident with spindle disassembly. The contraction, but not the assembly, of the ring requires the signal transduction network called the septation initiation network or SIN. The core components of the SIN are three protein kinases (cdc7p, sidl p and sid2p) and their regulatory subunits (spg1 p, cdcl4p and moblp, respectively). Signalling is dependent upon the nucleotide status of the GTPase spgl p, which is regulated by a two-component GAP protein, cdc16p-byr4p. Signalling is thought to emanate from the spindle pole body, where core SIN components are anchored to a scaffold comprised of sid4p and cdc11p. Activation of the SIN requires the protein kinase plolp, which also has additional roles in mitosis. SIN signalling is tightly regulated to assure the proper co-ordination of mitosis and cytokinesis. Ectopic activation of the SIN in interphase can uncouple septum formation from mitosis, while deregulated SIN signalling leads to formation of cells with multiple septa that do not cleave. Regulators of SIN activity are therefore of considerable interest. This study has concentrated upon two of these, dma1 and ubc8. I have demonstrated that dmal becomes essential when SIN signalling is activated. This leads me to propose a tripartite model for regulation of the SIN during the mitotic cell cycle. Increased expression of dma1 inhibits SIN signalling and prevents cell division. To identify potential targets and mediators of this, multicopy suppressors of dma1 toxicity were identified. One of these, ubc8, is the subject of this thesis. Genetic and molecular analyses are consistent with the view that ubc8p acts as an inhibitor of the SIN Localisation of ubc8p indicates that it is a nuclear protein. The ubc8 gene is not essential, but in its absence cells are unable to prevent septum formation if progression through mitosis is impaired. These data suggest that it may be an effector of the spindle assembly checkpoint. Together, these data shed new light upon the mechanisms by which cytokinesis is regulated in S. pombe. RESUME La levure Schizosaccharomyces pombe est un eucaryote unicellulaire qui est un bon système d'étude du cycle cellulaire. Les cellules de S. pombe sont en forme de bâtonnets et poussent par allongement aux deux bouts. Elles se divisent en formant une paroi au milieu de la cellule, qui s'appelle un septum et qui sépare la cellule en deux. Une fois que la cellule est engagée dans la mitose, le site de clivage est déterminé par la formation d'un anneau contractile d'acto-myosine au niveau du cortex cellulaire. Cet anneau est séquentiellement assemblé au cours de la mitose et se contacte à la fin de l'anaphase, au moment où le fuseau mitotique et désassemblé. La contraction, mais non pas l'assemblage, de l'anneau dépend d'un réseau de signalisation appelé septation initiation netvvork' ou SIN. Les composants centraux du SIN sont trois kinases (cdc7, sidi et sid2) ainsi que leurs sous-unités régulatrices (spgl, cdc14 et mob1, respectivement). La signalisation dépend du nucléotide rattaché à la GTPase spgl qui est régulée par une GAP comprenant deux sous-unités cdc16 et byr4. La signalisation est présumée provenir du pôle du fuseau où les composants centraux du SIN sont ancrés grâce à un échafaudage comprenant sid4 et cdcl 1. La signalisation est étroitement régulée pour assurer une bonne coordination entre mitose et cytokinèse. Une activation ectopique du SIN en interphase peut découpler la formation du septum de la mitose, engendrant des cellules à multiples septa qui ne sont pas clivés. C'est pourquoi les régulateurs du SIN sont d'un intérêt considérable. Cette étude se concentre autour de deux ces régulateurs, dma1 et ubc8. J'ai montré que dma1 devient essentiel quand la signalisation du SIN est activée. Ceci m'amène à proposer un modèle en trois parties pour la régulation du SIN durant la mitose. Une expression élevée de dma1 inhibe la signalisation du SIN et empêche la division cellulaire. Afin d'identifier des substrats ou médiateurs potentiels de la toxicité de dma1, des supresseurs en copies multiples ont été identifiés. Un de ces supresseurs, ubc8, constitue le deuxième sujet de cette thèse. Les études génétiques et moléculaires suggèrent un rôle inhibiteur du SIN par ubc8. Ubc8p est une protéine nucléaire, non essentielle, mais en son absence les cellules ne peuvent pas restreindre la fomation du septum, lorsque la progression de la mitose est perturbée. Les données suggèrent que ubc8 pourrait être un effecteur de point de contrôle de l'assemblage du fuseau mitotique. Prises dans leur ensemble, ces données apportent un nouvel éclairage sur les mécanismes de régulation de la cytokinèse dans S. pombe.

Validation study of the French version of the observer-rater scales measuring coping and cognitive errors (CE-CAP) on a non-clinical population

This study examined the validity and reliability of the French version of two observer-rated measures developed to assess cognitive errors (cognitive errors rating system [CERS]) [6] and coping action patterns (coping action patterns rating system [CAPRS]) [22,24]. The CE measures 14 cognitive errors, broken down according to their valence positive or negative (see the definitions by A.T. Beck), and the CAP measures 12 coping categories, based on an comprehensive review literature, each broken down into three levels of action (affective, behavioural, cognitive). Thirty (N = 30) subjects recruited in a community sample participated in the study. They were interviewed according to a standardized clinical protocol: these interviews were transcribed and analysed with both observer-rated systems. Results showed that the inter-rater reliability of the two measures is good and that their internal validity is satisfactory, due to a non-significant canonical correlation between CAP and CE. With regard to discriminant validity, we found a non-significant canonical correlation between CAPRS and CISS, one of most widely used self-report questionnaire measuring coping. The same can be said for the correlation with a self-report questionnaire measuring symptoms (SCL-90-R). These results confirm the absence of confounds in the assessment of cognitive errors and of coping as assessed by these observer-rated scales and add an argument in favour of the French validation of the CE-CAP rating scales. (C) 2010 Elsevier Masson SAS. All rights reserved.

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites.

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA'-'lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

Testing the use of two types of nest box by the common dormouse Muscardinus avellanarius

British mammalogists have used two different systems for surveying the common dormouse Muscardinus avellanarius: a modified bird nest box with the entrance facing the tree trunk, and a smaller, cheaper model called a "nest tube". However, only few data comparing different nest box systems are currently available. To determine which system is more efficient, we compared the use of the large (GB-type) and small nest boxes (DE-type, a commercial wooden mouse trap without a door) in three Swiss forest. The presence of Muscardinus, potential competitors, and any evidence of occupation were examined in 60 pairs of nest boxes based on 2,280 nest box checks conducted over 5 years. Mean annual occupation and cumulative numbers of Muscardinus present were both significantly higher for the DE than for the GB boxes (64.6% versus 32.1%, and 149 versus 67 dormice, respectively). In contrast, the annual occupation by competitors including Glis glis, Apodemus spp. and hole-nesting birds was significantly higher in the GB than in the DE boxes in all forest (19-68% versus 0-16%, depending on the species and forest). These results suggest that smaller nest boxes are preferred by the common dormouse and are rarely occupied by competitors. These boxes hence appear to be preferable for studying Muscardinus populations.