[en] We focused first on americium(III) and uranium(VI), that can have a simpler redox chemistry, as compared to plutonium, in the conditions of fungi culture. The high specific activity of americium limits its manipulation, which is why europium(III) was used as a chemical analogue. The accumulation of the actinides was followed in a filamentous model fungus, Podospora anserina. This fungus is easily cultivated under laboratory conditions and grows rapidly with a complete life cycle of 7 days in standard conditions. The culture medium used is a synthetic one whose composition is perfectly known. The medium has been adapted (absence of phosphates) in order to work with actinides without leading directly to the precipitation of the latter. First, the influence of actinide concentration on the fungus development was evaluated to identify the concentration range that the fungi can cope with. In order to better understand the mechanisms involved, the actinide accumulation was studied during the different development stages of the fungus (mycelium growth and sexual reproduction). Once actinides have been accumulated in fungi, they were localized in the different fungus parts (mycelia and perithecia, the site of the sexual reproduction) using ICP-MS and confocal fluorescence microscopy. The fungi part responsible of the actinides accumulation was then pinpointed by comparing the initial speciation of actinide in the culture medium with the one in fungi, thanks to speciation modelling combined to spectroscopic techniques like x-ray absorption spectroscopy. For instance, the presence of europium in the culture medium, at concentrations higher than 10^-4 M, has a strong impact on both mycelial growth and sexual reproduction, inhibiting the growth and leading to the formation of non-mature perithecia. Moreover, the accumulation mechanisms at europium concentrations 10^-5 M and 5x10^-4 M seems to be different. These distinct behaviors could be explained by a difference in speciation of europium in the culture medium. At 10^-5 M, europium is mainly present as citrate complexes, whereas at higher europium concentration, sulfate species and non-complexed species prevailed. However, in each case, europium(III) accumulation was observed, reaching concentrations from 180 to 4500 ppm in the mycelia and from 36 and 1900 ppm in the perithecia after 14 days of doping at 10^-5 M and 5x10^-4 M respectively. The accumulated europium could be localized as homogeneously distributed in the mycelium. The use of concanavalin A, a fluorescent lectin displaying high affinity for glycoproteins present at the cellular membranes, could pinpoint the absence of europium on the membranes of the mycelia. Within the perithecia, the accumulation of europium occurs mainly during the differentiation of the asci and then is concentrated in the ascospores during the meiotic division. Europium fluorescence is not observed in non mature asci and therefore does not appear to be accumulated at this time of development. First results on uranium accumulation in P. anserina will be also presented

[en] The threat of a dirty bomb which could cause internal contamination has been of major concern for the past decades. Because of their high chemical toxicity and their presence in the nuclear fuel cycle, uranium and neptunium are two actinides of high interest. Calmodulin (CaM) which is a ubiquitous protein present in all eukaryotic cells and is involved in calcium-dependent signaling pathways has a known affinity for uranyl and neptunyl ions. The impact of the complexation of these actinides on the physiological response of the protein remains, however, largely unknown. An isothermal titration calorimetry (ITC) was developed to monitor in vitro the enzymatic activity of the phosphodiesterase enzyme which is known to be activated by CaM and calcium. This approach showed that addition of actinyl ions (AnO₂ⁿ⁺), uranyl (UO₂²⁺) and neptunyl (NpO₂⁺), resulted in a decrease of the enzymatic activity, due to the formation of CaM-actinide complexes, which inhibit the enzyme and alter its interaction with the substrate by direct interaction. Results from dynamic light scattering rationalized this result by showing that the CaM-actinyl complexes adopted a specific conformation different from that of the CaM-Ca²⁺ complex. The effect of actinides could be reversed using a hydroxypyridonate actinide decorporation agent (5-LIO(Me-3,2-HOPO)) in the experimental medium demonstrating its capacity to efficiently bind the actinides and restore the calcium-dependent enzyme activation. (authors)

[en] Herein, we describe the structural investigation of one possible uranyl binding site inside a non structured protein. This approach couples spectroscopy, thermodynamics, and theoretical calculations (DFT) and studies the interaction of uranyl ions with a phospho-peptide, thus mimicking a possible osteopontin (OPN) hydroxyapatite growth-inhibition site. Although thermodynamical aspects were investigated by using time-resolved laser fluorescence spectroscopy (TRLFS) and isothermal titration calorimetry (ITC), structural characterization was performed by extended X-ray absorption fine structure (EXAFS) at the U L(III)-edge combined with attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. From the vibrational and fluorescence spectra, several structural models of a UO_2"2"+/peptide complex were developed and subsequently refined by using theoretical calculations to fit the experimental EXAFS obtained. The structural effect of the pH value was also considered under acidic to moderately acidic conditions (pH 1.5-5.5). Most importantly, the uranyl/peptide coordination environment was similar to that of the native protein. (authors)