Neutral Products Desorption from DNA Thin Films Induced by Low-Energy Electrons (0.5-20 eV)

International audienceLow-energy electrons (LEEs) are produced in great amount in the biological medium, when submitted to high-energy radiations. They have the ability to induce strand breaks in the DNA duplex, as proven by electrophoresis analysis of irradiated dry deposits. LEE interactions with target molecules induce the formation of different species such as anions, cations, radicals and neutrals. The desorption of anionic species from oligonucleotides and DNA under LEEs irradiation has been intensively explored. The involved mechanisms and sites were successfully identified, including the resonant formation of transient negative ions (TNI) below 15 eV. However, the desorption of neutral products was less explored [4], due to their difficult detection. Exploring this aspect will provide additional information and complete the picture of the dissociating pathways followed by TNIs

La construction du site pédagogique numérique CHIMACTIV : analyse d'une coopération réussie entre enseignants

International audienceUn collectif d'enseignants inter-établissements s'est organisé pour concevoir et co-construire un site pédagogique numérique. L'équipe de conception initiale a fortement coopéré (au sein de chaque établissement et entre établissements) et interagi étroitement avec différents acteurs (cellules TICE, étudiants, prestataires externes) pour aboutir à une version bilingue du site. La volonté d'élargir et de diversifier le champ des utilisateurs (enseignants et étudiants) a conduit à ouvrir ce collectif à de nouveaux enseignants, afin de faire évoluer le site et compléter son contenu. Après une analyse de l'organisation mise en place, nous discuterons des obstacles à surmonter, des facteurs de réussite et du ressenti des enseignants ayant vécu cette coopération, avant de conclure sur ce qu'apporte l'aspect « numérique » des ressources développées dans la coopération entre enseignants sur la base de notre expérience

Gold Nanoparticle Uptake in Tumor Cells: Quantification and Size Distribution by sp-ICPMS

Gold nanoparticles (AuNPs) are increasingly studied for cancer treatment purposes, as they can potentially improve both control and efficiency of the treatment. Intensive research is conducted in vitro on rodent and human cell lines to objectify the gain of combining AuNPs with cancer treatment and to understand their mechanisms of action. However, using nanoparticles in such studies requires thorough knowledge of their cellular uptake. In this study, we optimized single particle ICPMS (sp-ICPMS) analysis to qualify and quantify intracellular AuNP content after exposure of in vitro human breast cancer cell lines. To this aim, cells were treated with an alkaline digestion method with 5% TMAH, allowing the detection of gold with a yield of 97% on average. Results showed that under our experimental conditions, the AuNP size distribution appeared to be unchanged after internalization and that the uptake of particles depended on the cell line and on the exposure duration. Finally, the comparison of the particle numbers per cell with the estimates based on the gold masses showed excellent agreement, confirming the validity of the sp-ICPMS particle measurements in such complex samples

Vers la compréhension de l effet radiosensibilisateur de nanoparticules d or soumises à un rayonnement X

La radiothérapie est l une des stratégies thérapeutiques parmi les plus répandues dans la lutte contre le cancer mais son efficacité est limitée par la tolérance des tissus sains. De fait, son association avec des agents radiosensibilisants vise à accroître son efficacité en augmentant spécifiquement les dommages aux cellules tumorales. C est dans ce cadre que nous avons étudié la radiosensibilisation par des nanoparticules d or (NPo) associées à un rayonnement X in vitro. Ce travail de thèse a connu trois temps. Le premier a consisté en la mise en place des outils nécessaires à cette étude, à savoir la synthèse et la caractérisation de NPo ainsi que des différentes sources de rayonnement utilisées. Puis nous avons démontré la radiosensibilisation par les NPo combinées aux rayons X de trois cibles cellulaires potentielles : la membrane, les protéines et l ADN. Nous avons alors montré que la concentration des NPo, leur taille et l énergie des rayons X incidents étaient trois paramètres gouvernant cet effet radiosensibilisateur. Enfin, nous nous sommes intéressés aux mécanismes à l origine de cet effet : une surproduction de radicaux hydroxyle a été observée en présence de NPo et l effet direct des électrons émis par l or semble perdurer en solution. Nous avons également évalué les conditions de transposition de cette radiosensibilisation du in vitro au in vivo.Radiotherapy is one of the most widespread cancer treatments but it suffers from its lack of selectivity. That s why associated with a radiosensitizing agent that specifically enhances the radiation effect in the tumour, it could be more efficient. In this context, we studied the radiosensitization by gold nanoparticles (NPo) submitted to X-rays in vitro. Three steps could be distinguished in this work. The first one consisted in implementing the mandatory tools, namely NPo synthesis and characterization and calibration of the different irradiation sources. Then we demonstrated the radiosensitization by NPo combined to X-rays of three potential cellular targets: membrane, protein and DNA. We showed that NPo concentration, size and X-ray energy were three parameters governing this effect. Finally, we got interested in the underlying mechanisms: hydroxyle radicals overproduction was observed in the presence of NPo and direct effect from electrons emitted by gold seems to still occur in solution. Besides, we assessed the conditions required for the translation of this fundamental research topic to clinical applications.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Damage induced to DNA by low-energy (0-30 eV) electrons under vacuum and atmospheric conditions.

International audienceIn this study, we show that it is possible to obtain data on DNA damage induced by low-energy (0-30 eV) electrons under atmospheric conditions. Five monolayer films of plasmid DNA (3197 base pairs) deposited on glass and gold substrates are irradiated with 1.5 keV X-rays in ultrahigh vacuum and under atmospheric conditions. The total damage is analyzed by agarose gel electrophoresis. The damage produced on the glass substrate is attributed to energy absorption from X-rays, whereas that produced on the gold substrate arises from energy absorption from both the X-ray beam and secondary electrons emitted from the gold surface. By analysis of the energy of these secondary electrons, 96% are found to have energies below 30 eV with a distribution peaking at 1.4 eV. The differences in damage yields recorded with the gold and glass substrates is therefore essentially attributed to the interaction of low-energy electrons with DNA under vacuum and hydrated conditions. From these results, the G values for low-energy electrons are determined to be four and six strand breaks per 100 eV, respectively

A novel cryo-reduction method to investigate the molecular mechanism of nitric oxide synthases

International audienceNitric oxide synthases (NOSs) are hemoproteins responsible for the biosynthesis of NO in mammals. They catalyze two successive oxidation reactions. The mechanism of oxygen activation is based on the transfer of two electrons and two protons. Despite structural analogies with cytochromes P450, the molecular mechanism of NOS remains yet to be elucidated. Because of extremely high reaction rates, conventional kinetics methods failed to trap and characterize the major reaction intermediates. Cryo-reduction methods offer a possibility to circumvent this technological lock, by triggering oxygen activation at cryogenic temperatures by using water radiolysis. However, this method is not adapted to the NOS mechanism because of the high instability of the initial Fe(II)O2 complex (extremely fast autoxidation and/or reaction with the cofactor H4B). This imposed a protocol with a stable Fe(II)O2 complex (observed only for one NOS-like protein) and that excludes any redox role for H4B. A relevant approach to the NOS mechanism would use H4B to provide the (second) electron involved in oxygen activation; water radiolysis would thus provide the first electron (heme reduction). In this context, we report here an investigation of the first electron transfer by this alternative approach, i.e., the reduction of native NOS by water radiolysis. We combined EPR and resonance Raman spectroscopies to analyze NOS reduction for a combination of different substrates, cofactor, and oxygen concentrations, and for different NOS isoforms. Our results show that cryo-reduction of native NOS is achieved for all conditions that are relevant to the investigation of the NOS mechanism

Radiolysis of proteins in the solid state: an approach by EPR and product analysis.

Radio-induced modifications in proteins have been studied using several techniques. Electron paramagnetic resonance (EPR) was used to characterize free radicals, and analysis methods (high-performance liquid chromatography, capillary electrophoresis) were employed to visualize final degraded forms. Whereas EPR indicates that perthiyl radicals are formed, analysis does not detect any compound in which such bonds would be broken. Since EPR signals decay with time, it is concluded that rearrangements occur at subsequent steps, in which the solvent used during the analysis might play a role

Hydrogen plasma treated nanodiamonds lead to an overproduction of hydroxyl radicals and solvated electrons in solution under ionizing radiation

International audienceIn numerous fields of application (environmental remediation, catalysis, nanomedicine), production ofhydroxyl radicals and solvated electrons by nanomaterials is a cornerstone. Through a very sensitive,nanoparticle-compatible, coumarin-based protocol, we quantified hydroxyl radicals in solution whenhydrogenated (H-ND) and oxidized (Ox-ND) detonation nanodiamonds were irradiated by MeV photons.We highlighted a blatant difference between the two surface chemistries as only H-ND led to 50% moreradicals, for irradiation doses and ND concentrations relevant in nanomedicine. For the first time, we alsoquantified solvated electrons after keV irradiation of both suspensions and showed that in the presenceof H-ND, hydroxyl radicals and solvated electrons were available in solution in equivalent and higheramounts than in water only. This asks the question of the mechanisms at stage and beside the negative/positive electron affinity hypothesis usually mentioned, we proposed, as for other nanomaterials, thatinterfacial water could play an essential role in radicals’ production in solution when detonation H-NDare irradiate

Milled nanodiamonds overproduce solvated electrons while scavenging hydroxyl radicals under gamma irradiation

International audienceAmong the different nanodiamonds, milled nanodiamonds (MNDs) from high pressure high temperature (HPHT) synthesis exhibit a crystalline quality close to the bulk diamond, making it a serious candidate for quantum or energy-related fields. In this study, MNDs aqueous suspensions with oxidized (MND-Ox) and hydrogenated (MND-H) surface chemistries were irradiated with gamma-rays. Using optimized nanoparticle-compatible methods, we revealed that MNDs in suspension enhance the production of solvated electrons under irradiation, with a higher yield for MND-H. Also, for the first time, a surface chemistry-dependent scavenging effect of hydroxyl radicals was highlighted for this type of nanodiamonds

Selective Cold Welding of Colloidal Gold Nanorods

International audienceA new cold nanowelding technique for cetyltrimethylammonium bromide (CTAB)-stabilized colloidal nanorods in water is reported. This technique leads to the formation of micrometric nanowires by end-to-end self-organization and welding of nanorods. This cold welding strategy could be generally applicable to colloidal nanoparticles and opens new outlooks for the bottom-up fabrication of nanodevices using colloidal building blocks