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Deciphering mechanisms of oocyte development in female fishes still remains challenging and a comprehensive overview of this process at the level of the organ is still needed. The recent development optical tissue clearing methods have tremendously boosted the 3D imaging of large size biological samples that are naturally opaque. However, no attempt of clearing on fish ovary that accumulates extremely high concentration of lipids within oocytes has been reported to date. To face with this ovarian-specific challenge, we combined two existing clearing methods, the non-toxic solvent-based Eci method for efficient clearing and the CUBIC method to enhance lipid removal and reduce non-specific staining. The methyl green fluorescent dye was used to stain nuclei and delineate follicles. Using this procedure (named C-Eci), ovaries of both medaka and trout could be imaged in 3D and all follicles analyzed. To our knowledge this is the first procedure elaborated for clearing and imaging fish ovary in 3D. The C-Eci methods thus provides an interesting tool for getting precise quantitative data on follicular content in fish ovary and promises to be useful for further morphological studies.

Conjugation of fluorescent dyes to proteins - a prerequisite for the study of conformational dynamics by single molecule Förster resonance energy transfer (smFRET) - can lead to substantial changes of the dye’s photophysical properties, ultimately biasing the quantitative determination of inter-dye distances. In particular the popular cyanine dyes and their derivatives, which are by far the most used dyes in smFRET experiments, exhibit such behavior. To overcome this, a general strategy to site-specifically equip proteins with FRET pairs by chemo-selective reactions using two distinct non-canonical amino acids simultaneously incorporated through genetic code expansion inEscherichia coliwas developed. Applied to human NADPH- cytochrome P450 reductase (CPR), the importance of homogenously labeled samples for accurate determination of FRET efficiencies was demonstrated. Furthermore, the effect of NADP+ on the ionic strength dependent modulation of the conformational equilibrium of CPR was unveiled. Given its generality and accuracy, the presented methodology establishes a new benchmark to decipher complex molecular dynamics on single molecules.

The performance of buffer zones for removing pesticides from runoff water varies greatly according to landscape settings, hydraulic regime, and system design. Evaluating the performance of buffers for a range of pesticides and environmental conditions can be very expensive. Recent studies suggested that the fluorescent dyes uranine and sulforhodamine B could be used as cost-effective surrogates of herbicides to evaluate buffer performance. However, while transformation mechanisms in buffers have been extensively documented, sorption processes of both dyes have rarely been investigated. In this study, we measured the adsorption, desorption, and kinetic sorption coefficients of uranine and sulforhodamine B for a diverse range of buffer zone materials (soils, litters, plants) and compared the adsorption coefficients (Kd) to those of selected herbicides. We also compared the global sorption capacity of 6 ditches, characterized by varying proportions of the aforementioned materials, between both dyes and a set of four herbicides using the sorption-induced pesticide retention indicator (SPRI). We found that both the individual Kd of uranine for the diverse buffer materials and the global sorption capacity of the ditches are equivalent to those of the herbicides diuron, isoproturon, and metolachlor. The Kd of sulforhodamine B on plants and soils are equivalent to those of glyphosate, and the global sorption capacities of the ditches are equivalent for both molecules. Hence, we demonstrate for the first time that uranine can be used as a proxy of moderately hydrophobic herbicides to evaluate the performance of buffer systems, whereas sulforhodamine B can serve as a proxy for more strongly sorbing herbicides.

En lien avec la notice : http://prodinra.inra.fr/?locale=fr#!ConsultNotice:355849; National audience

National audience; Dans le cadre de recherches génériques sur les déterminants génétiques, moléculaires et biochimiques de la qualité du bois, il a été mis en évidence d’intéressantes propriétés biologiques et colorantes au niveau de deux types d’extraits de bois de robinier (Robinia pseudoacacia). Bien représentée en métropole et en région Centre, cette essence fixe naturellement l’azote atmosphérique, s’accommode de sols peu fertiles et est tolérante au stress hydrique. Elle représente de ce fait une espèce de choix pour la production de biomasse et la séquestration de carbone sur des friches non-agricoles qui ne peuvent être valorisées par d’autres espèces pérennes généralement plantées dans ces buts comme le peuplier ou le saule. Vu la proportion importante de bois de coeur présente chez le robinier, la mise en place, d’unités de production d’extraits ou de molécules de haute valeur ajoutée à partir de cette ressource locale semble judicieuse en complément des filières d’utilisation classiques de ce bois (bois-énergie, fabrication de piquets et de menuiseries extérieures). En relation avec des partenaires régionaux fortement impliqués dans les filières cosmétiques et phytosanitaires potentiellement utilisatrices de ces nouveaux produits issus du bois, le projet ValRob vise à confirmer la nature et l’activité des deux molécules en jeu, à estimer la variabilité génétique de leurs teneurs au sein des ressources génétiques françaises de robinier et finalement, à concrétiser leur production et leur valorisation au niveau industriel en tenant compte des données socio-économiques de la filière bois-forêt bien implantée au niveau régional.

International audience; Fluorescent proteins (FPs) of the green fluorescent protein family blink and bleach like all fluorophores. However, contrary to organic dyes, the mechanisms by which transient losses of fluorescence occur in FPs have received little attention. Here, we focus on the photoactivatable IrisFP, for which a transient non-fluorescent chromophoric state with distorted geometry was recently reported (Adam, V.; et al. J. Am. Chem. Soc. 2009, 131, 18063). We investigated the chemical nature of this blinked state by employing quantum chemical/molecular mechanical calculations. Our findings suggest two previously unidentified dark states that display similar distorted chromophores with a transiently ruptured pi-electron system. Both are protonated at atom C(alpha) of the chromophore methylene bridge. Transient protonation may occur via proton transfer from the nearby Arg66 either in the triplet state T(1) after intersystem crossing or in an anionic radical (doublet) ground state. As Arg66 is conserved in green-to-red photoconvertible FPs, these dark states are predicted to be common to all these proteins. We also suggest that C(alpha) protonated dark states may accelerate photobleaching by favoring decarboxylation of the fully conserved Glu212.

L’artiste moderne cherche continuellement de nouvelles techniques. Des nouveaux matériaux tels que l’éponge, le polyester, le sable, etc. sont devenus courants. Ces développements amènent de nouveaux problèmes dans le domaine de la conservation (préventive) des matériaux en question. Chaque matériau doit être analysé individuellement afin de préserver le mieux possible l’intention de l’artiste à long terme. La présente recherche concerne des matériaux très récents: les pigments et les couleurs fluorescentes. Ces derniers n’ont fait leur apparition qu’au vingtième siècle. Pour cette raison les données concernant leur dégradation et leur conservation sont peu nombreuses. La majorité des tableaux fluorescents sont conçus pour être montrés sous éclairage UV. En cas de dommage, si l’œuvre a besoin d’être retouchée, ceci pose un grand problème esthétique. La retouche devient visible sous les lampes UV. La première partie de la recherche concerne la composition des différentes sortes de pigments et de peintures. Par la suite, les différentes causes de dégradation- lumière UV, chaleur, etc.- sont analysées en détail. Pour conclure, un possible remède est proposé.In modern art we can see that artists are breaking with traditional techniques. New materials like sponge, polyester, sand, etc. are being used. This causes a lot of new problems in the (preventive) conservation of works containing these materials. Every material needs to be researched individually so the exact intention of the artist can be preserved for a (relatively) long period. My research is about very recent materials: fluorescent pigments and paints. These only started to be used by artists from the 60’s. That's why there is not much information about their aging and ways to (preventively) conserve them. A lot of fluorescent paintings are meant to be shown under UV-light. If a fluorescent painting has damage and needs to be retouched there is a big esthetical problem. The retouched damage is visible under UV-light. The title of this thesis is born out of this last problem: Conservation problems with paintings containing fluorescent layers of paint. The first topic of research for this thesis concerns the composition of these different sorts of paint and pigments. Afterwards, the causes of damage resulting from UV-light –and other factors- were thoroughly analyzed. Finally, a possible remedy is being proposed.