Colour, form, and movement are not perceived simultaneously

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Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors.

BACKGROUND: Mitogen activated protein kinases (MAPK) play an essential role in integrating extra-cellular signals and intra-cellular cues to allow cells to grow, adapt to stresses, or undergo apoptosis. Budding yeast serves as a powerful system to understand the fundamental regulatory mechanisms that allow these pathways to combine multiple signals and deliver an appropriate response. To fully comprehend the variability and dynamics of these signaling cascades, dynamic and quantitative single cell measurements are required. Microscopy is an ideal technique to obtain these data; however, novel assays have to be developed to measure the activity of these cascades. RESULTS: We have generated fluorescent biosensors that allow the real-time measurement of kinase activity at the single cell level. Here, synthetic MAPK substrates were engineered to undergo nuclear-to-cytoplasmic relocation upon phosphorylation of a nuclear localization sequence. Combination of fluorescence microscopy and automated image analysis allows the quantification of the dynamics of kinase activity in hundreds of single cells. A large heterogeneity in the dynamics of MAPK activity between individual cells was measured. The variability in the mating pathway can be accounted for by differences in cell cycle stage, while, in the cell wall integrity pathway, the response to cell wall stress is independent of cell cycle stage. CONCLUSIONS: These synthetic kinase activity relocation sensors allow the quantification of kinase activity in live single cells. The modularity of the architecture of these reporters will allow their application in many other signaling cascades. These measurements will allow to uncover new dynamic behaviour that previously could not be observed in population level measurements

L’histoire du freeski

Ce travail expose de quelle manière le freeski s’est construit pour devenir une discipline à part entière. Il permet de proposer une première définition historique de mouvement qui sera indispensable pour un travail plus approfondi sur l’univers du freeski et sur certaines de ses particularités. Dans un premier temps, nous exposerons la méthodologie suivie, pour fixer les repères bibliographiques, de l’histoire du freeski, puis les sources traitant du freeski, et enfin nous étudierons l’histoire du freeski

Size distribution of EC and OC in the aerosol of Alpine valleys during summer and winter

International audienceCollections of samples were conducted for the determination of the size distributions of EC and OC during the intensive sampling campaigns of the POVA program, in two Alpine valleys of the French Alps, in summer and in winter. The comparison of concentrations obtained for samples collected in parallel with impactor- and filter-based methods indicates that the correction of pyrolysis seems to work for the impactor samples despite non even deposits. The size distributions of the concentrations of EC and OC present large evolutions between winter and summer, and between a suburban and a rural site. In winter, an overwhelming proportion of the mass fraction of both species is found in the droplet and accumulation modes, often (but not always) in association with sulfate and other chemical species resulting from secondary formation processes. Some indications of gas/particles exchanges can be found for the other parts of the size spectrum (the Aitken and super micron modes) in the case of the rural site. In summer, the changes are more drastic with, according to the case, a dominant droplet or accumulation mode. Particularly at the rural site, the large extent of processing of the aerosol due to gas/particles exchanges is evident for the Aitken and super micron modes, with increasing of the OC mass fractions in these size ranges. All of these observations give indications on the degree of internal vs. external mixing of the species investigated in the different modes

Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters.

Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise

Regulation of transcription dynamics in single cells

In mammalian cells, the MAPK pathways regulate vital processes such as differentiation, growth or death in response to a wide array of stimuli, like hormones or stresses. To achieve this, they interpret extracellular cues and reprogram the cell transiently or on longterm, through the transcriptional regulation of target genes. Understand the regulation of the MAPK signaling and resulting transcription is of high importance because of the involvement of mis-regulation of these pathways in many diseases (cancer, diabetes, cardiovascular diseases). As these signaling cascades are conserved among various organisms, we study them in the budding yeast Saccharomyces cerevisiae. This widely used model organism possesses its own MAPK network, and is easy to genetically modify. The transcription is a crucial process in cellular life, as it drives the production of proteins involved in all possible aspects of life. As such, it is highly regulated through complex combination of factors. Moreover, the transcription is a dynamic process that can occur either continuously or in a bursty manner. These bursts can be variable in their frequencies and duration, leading to various mRNA productions within cells from a clonal population. Hence, the transcription regulation needs to be studied at the single cell level and in real-time. To this purpose, we developed a new kind of gene expression reporters able to quantify in real-time and at the single cell level the expression arising from a promoter, that we named dPSTR. Using this system, we measured the extremely fast and transient gene expression resulting from hyper osmotic stress, and observed a difference on the sub-minute timescale in gene induction. Then, we used the dPSTR to quantify gene expression triggered by the mating signaling. We found that despite a signaling activity occurring minutes after addition of exogenous pheromone, resulting gene expression occurs on different timescales. Finally, by means of genetic analysis and modification of endogenous promoters, we defined some rules governing the expression kinetics of mating-induced genes

Evolution de la surveillance des PM10 en France : épisodes de pollution par les particules au printemps 2007

National audienceSince the 1st January 2007, PM10 monitoring network in France has evolved, in order to account for volatile fraction of PM10. This evolution permitted the observation of high peaks of PM10 during spring 2007. Concentrations during these peaks would have been largely underestimated with measuring techniques used before 2007. A study, coupling chemical and modelling approach of the phenomenon has been launched by LCSQA (Laboratoire Central de Surveillance de la Qualité de l'Air), in collaboration with several AASQA (Association Agrée de Surveillance de la Qualité de l'Air, air monitoring network). Chemical analysis of PM10 show that ammonium nitrate is the component at the origin of the observed peaks. The French forecasting system for air quality Prév'Air (www.prevair.org) largely underestimated the peaks, and especially ammonium nitrate concentrations. A sensibility study shows that emissions due to agricultural practices seem to be at the origin of the peaks. The necessity of reconsidering these emissions as a function of meteorological conditions (especially the temperature) is highlightedDepuis le 1er janvier 2007, le dispositif de surveillance des PM10 en France a évolué, de façon à prendre en compte la fraction volatile des particules. Une conséquence de cette évolution est l'observation, au printemps 2007, d'importants pics de particules : ces pics auraient été largement sous-évalués avec les techniques précédemment utilisées. Le LCSQA (Laboratoire Central de Surveillance de la Qualité de l'Air), en collaboration avec plusieurs AASQA (Association Agrée de Surveillance de la Qualité de l'Air) a entrepris un travail de compréhension de l'origine des pics, couplant mesures de terrain et modélisation Les résultats expérimentaux montrent que, pour la plupart des pics étudiés, la fraction volatile est généralement supérieure à la fraction non-volatile. Des analyses chimiques ont permis de mettre en évidence la présence prépondérante du nitrate d'ammonium dans la plupart des cas, expliquant ponctuellement les larges fractions volatiles mesurées. Sur le plan de la modélisation, le modèle CHIMERE utilisé dans le système Prév'Air (www.prevair.org) a sous-estimé ces fortes concentrations, et en particulier les concentrations en nitrate d'ammonium. Les émissions d'espèces azotées lors des épandages massifs d'engrais à cette période de l'année, mal prises en compte, pourraient en grande partie expliquer les résultats. La nécessité de reconsidérer dans le modèle la dépendance de ces émissions aux conditions météorologiques (notamment les températures élevées qui favorisent l'évaporation) a été mise en évidenc

Size distribution of EC and OC in the aerosol of Alpine valleys during summer and winter

Collections of samples were conducted for the determination of the size distributions of EC and OC during the intensive sampling campaigns of the POVA program, in two Alpine valleys of the French Alps, in summer and in winter. The comparison of concentrations obtained for samples collected in parallel with impactor- and filter-based methods is rather positive with slopes of 0.95 and 0.76 for OC and EC, respectively and correlations close to 1 (0.92 and 0.90 for OC and EC, respectively, n=26). This is an indication that the correction of pyrolysis seems to work for the impactor samples despite non even deposits. The size distributions of the concentrations of EC and OC present large evolutions between winter and summer, and between a suburban and a rural site. In winter, an overwhelming proportion of the mass fraction of both species is found in the droplet and accumulation modes, often (but not always) in association with sulfate and other chemical species resulting from secondary formation processes. Some indications of gas/particles exchanges can be found for the other parts of the size spectrum (the Aitken and super micron modes) in the case of the rural site. In summer, the changes are more drastic with, according to the case, a dominant droplet or accumulation mode. Particularly at the rural site, the large extent of processing of the aerosol due to gas/particles exchanges is evident for the Aitken and super micron modes, with increasing of the OC mass fractions in these size ranges. All of these observations give indications on the degree of internal vs. external mixing of the species investigated in the different modes

New families of single integration vectors and gene tagging plasmids for genetic manipulations in budding yeast.

The tractability of the budding yeast genome has provided many insights into the fundamental mechanisms regulating cellular life. With the advent of synthetic biology and single-cell measurements, novel tools are required to manipulate the yeast genome in a more controlled manner. We present, here, a new family of yeast shuttle vectors called single integration vectors (pSIV). Upon transformation in yeast, these plasmids replace the entire deficient auxotrophy marker locus by a cassette containing an exogenous marker. As shown using flow cytometry, this complete replacement results in a unique integration of the desired DNA fragment at the marker locus. In addition, a second transcriptional unit can be inserted to achieve the simultaneous integration of two constructs. The selection marker cassettes, present in the pSIV, were also used to generate a complete set of gene tagging plasmids (pGT) encompassing a large palette of fluorescent proteins, from a cyan fluorescent protein to a near-infrared tandem dimer red fluorescent protein. These tagging cassettes are orthogonal to each other thanks to the use of different TEF promoter and terminator couples, thereby avoiding marker cassette switching and favoring integration in the desired locus. In summary, we have created two sets of robust molecular tools for the precise genetic manipulation of the budding yeast

Polycyclic aromatic hydrocarbons (PAHs) in the atmospheres of two French alpine valleys: sources and temporal patterns

International audienceAlpine valleys represent some of the most important crossroads for international heavy-duty traffic in Europe, but the full impact of this traffic on air quality is not known due to a lack of data concerning these complex systems. As part of the program "Pollution des Vallées Alpines" (POVA), we performed two sampling surveys of polycyclic aromatic hydrocarbons (PAHs) in two sensitive valleys: the Chamonix and Maurienne Valleys, between France and Italy. Sampling campaigns were performed during the summer of 2000 and the winter of 2001, with both periods taking place during the closure of the "Tunnel du Mont-Blanc". The first objective of this paper is to describe the relations between PAH concentrations, external parameters (sampling site localization, meteorological parameters, sources), and aerosol characteristics, including its carbonaceous fraction (OC and EC). The second objective is to study the capacity of PAH profiles to accurately distinguish the different emission sources. Temporal evolution of the relative concentration of an individual PAH (CHR) and the PAH groups BghiP+COR and BbF+BkF is studied in order to differentiate wood combustion, gasoline, and diesel emissions, respectively. The results show that the total particulate PAH concentrations were higher in the Chamonix valley during both seasons, despite the cessation of international traffic. Seasonal cycles, with higher concentrations in winter, are also stronger in this valley. During winter, particulate PAH concentration can reach very high levels (up to 155 ng.m-3) in this valley during cold anticyclonic periods. The examination of sources shows the impact during summer of heavy-duty traffic in the Maurienne valley and of gasoline vehicles in the Chamonix valley. During winter, Chamonix is characterized by the strong influence of wood combustion in residential fireplaces, even if the temporal evolution of specific PAH ratios are difficult to interpret. Information on sources given by PAH profiles can only be considered in qualitative terms