Search

This paper describes in detail the implementation of Geant4 Livermore electromagnetic physics models based on the EPICS2017 database for the low energy transport of photons. These models describe four photon processes: gamma conversion, Compton scattering, photoelectric effect and Rayleigh scattering. New parameterizations based on EPICS2017 were performed for scattering functions of Compton effect, subshell cross-sections of the photoelectric effect and form factors of Rayleigh scattering, in order to improve the precision of fitted values compared to tabulated values. Comparisons between new and old parameterizations were also carried out to evaluate the precision of the new parameterizations. The models were tested through a comparative study, in which the mass attenuation coefficient was calculated for both total photon interaction and each process using Geant4 simulations based on EPICS2017 and EPDL97 respectively. The results obtained from the simulations were found in good agreement with the XCOM reference data.

Proton computed microtomography is a technique that reveals the inner content of microscopic samples. The density distribution of the material (in g·cmIn this study, we describe how the 3D variations in the mass density were taken into account in the reconstruction code, in order to quantify the correction according to the position of the proton beam and the position and aperture angle of the X-ray detector. Moreover, we assess the accuracy of the reconstructed densities using Geant4 simulations on numerical phantoms, used as references.The correction process was successfully applied and led, for the largest regions of interest (little affected by partial volume effects), to an accuracy ≤ 4% for phosphorus (compared to about 40% discrepancy without correction).This study demonstrates the accuracy of the correction method implemented in the tomographic reconstruction code for thick samples. It also points out some advantages offered by Geant4 simulations: i) they produce projection data that are totally independent of the inversion method used for the image reconstruction; ii) one or more physical processes (X-ray absorption, proton energy loss) can be artificially turned off, in order to precisely quantify the effect of the different phenomena involved in the attenuation of X-ray spectra.

A compact low-energy and high-intensity X-ray source for radiation biology applications is presented. A laser-induced plasma moves inside a 30 kV diode and produces a beam of 10$^{14}$ electrons at the anode location. An aluminum foil converts a part of the energy of these electrons into X-ray photons which are characterized using filtered imaging plates. The dose that would be deposited by these X-ray photons in C. elegans larvae is calculated from Geant4 simulations. It can be set to a value ranging between 10 $\mu$Gy and 10 mGy per laser shot by simply changing the aluminum foil thickness and the diode voltage. Therefore, this versatile and compact X-ray source opens a new path to explore the radiation effects induced by dose rates varying over several orders of magnitude., Comment: 5 pages, 6 figures

International audience; Proton imaging can be carried out on microscopic samples by focusing the beam to a diameter ranging from a few micrometers down to a few tens of nanometers, depending on the required beam intensity and spatial resolution. Three-dimensional (3D) imaging by tomography is obtained from proton transmission (STIM: Scanning Transmission Ion Microscopy) and/or X-ray emission (PIXE: Particle Induced X-ray Emission). In these experiments, the samples are dehydrated for under vacuum analysis. In situ quantification of nanoparticles has been carried out at CENBG in the frame of nanotoxicology studies, on cells and small organisms used as biological models, especially on Caenorhabditis elegans (C. elegans) nematodes. Tomography experiments reveal the distribution of mass density and chemical content (in g.cm−3) within the analyzed volume. These density values are obtained using an inversion algorithm. To investigate the effect of this data reduction process, we defined different numerical phantoms, including a (dehydrated) C. elegans phantom whose geometry and density were derived from experimental data. A Monte Carlo simulation based on the Geant4 toolkit was developed. Using different simulation and reconstruction conditions, we compared the resulting tomographic images to the initial numerical reference phantom. A study of the relative error between the reconstructed and the reference images lead to the result that 20 protons per shot can be considered as an optimal number for 3D STIM imaging. Preliminary results for PIXE tomography are also presented, showing the interest of such numerical phantoms to produce reference data for future studies on X-ray signal attenuation in thick samples.

Charged-particle microbeams (CPMs) provide a unique opportunity to investigate the effects of ionizing radiation on living biological specimens with a precise control of the delivered dose, i.e. the number of particles per cell. We describe a methodology to manipulate and micro-irradiate early stage C. elegans embryos at a specific phase of the cell division and with a controlled dose using a CPM. To validate this approach, we observe the radiation-induced damage, such as reduced cell mobility, incomplete cell division and the appearance of chromatin bridges during embryo development, in different strains expressing GFP-tagged proteins in situ after irradiation. In addition, as the dosimetry of such experiments cannot be extrapolated from random irradiations of cell populations, realistic three-dimensional models of 2 cell-stage embryo were imported into the Geant4 Monte-Carlo simulation toolkit. Using this method, we investigate the energy deposit in various chromatin condensation states during the cell division phases. The experimental approach coupled to Monte-Carlo simulations provides a way to selectively irradiate a single cell in a rapidly dividing multicellular model with a reproducible dose. This method opens the way to dose-effect investigations following targeted irradiation.

International audience; We report the development of thin single crystal diamond membranes suitable for dose control in targeted cell irradiation experiments with a proton microbeam. A specific design was achieved to deliver single protons with a hit detection efficiency approaching 100%. The membranes have thicknesses between 1.8 and 3 pm and arc used as vacuum windows on the microbeam line. The impact of these transmission detectors on the microbeam spot size is estimated by Monte-Carlo simulations, indicating that a beam lateral resolution below 2 µm is achieved. This is confirmed by experiments showing the accumulation online of X-ray Repair Cross-Complementing protein 1 (XRCC1)-Green Fluorescent Protein (GFP) at DNA damaged sites in living cells.

The reliance of all cell types on the mitochondrial function for survival makes mitochondria an interesting target when trying to understand their role in the cellular response to ionizing radiation. By harnessing highly focused carbon ions and protons using microbeams, we have performed in situ live cell imaging of the targeted irradiation of individual mitochondria stained with Tetramethyl rhodamine ethyl ester (TMRE), a cationic fluorophore which accumulates electrophoretically in polarized mitochondria. Targeted irradiation with both carbon ions and protons down to beam spots of in situ after targeted microbeam irradiation. The methods developed and the results obtained have the ability to shed new light on not just mitochondria’s response to radiation but to further elucidate a putative mechanism of radiation induced depolarization and mitochondrial response.

The successful development of nanomaterials illustrates the considerable interest in the development of new molecular probes for medical diagnosis and imaging. Substantial progress was made in the synthesis protocol and characterization of these materials, whereas toxicological issues are sometimes incomplete. Nanoparticle-based contrast agents (CAs) tend to become efficient tools for enhancing medical diagnostics and surgery for a wide range of imaging modalities. The multimodal nanoparticles (NPs) are much more efficient than the conventional molecular-scale CAs. They provide new abilities for in vivo detection and enhanced targeting efficiencies through longer circulation times, designed clearance pathways, and multiple binding capacities. Properly protected, they can safely be used for the fabrication of various functional systems with targeting properties, reduced toxicity, and proper removal from the body. This review mainly describes the advances in the development of mono- to multimodal NPs and their in vitro and in vivo relevant biomedical applications ranging from imaging and tracking to cancer treatment. Besides the specific applications for classical imaging (magnetic resonance imaging, positron emission tomography, computed tomography, ultrasound, and photoacoustic imaging), the less common imaging techniques such as terahertz molecular imaging (THMI) or ion beam analysis (IBA) are mentioned. The perspectives on the multimodal theranostic NPs and their potential for clinical advances are also mentioned.

International audience; At very low radiation dose rates, the effects of energy depositions in cells by ionizing radiation is best understood stochastically, as ionizing particles deposit energy along tracks separated by distances often much larger than the size of cells. We present a thorough analysis of the stochastic impact of the natural radiative background on cells, focusing our attention on E. coli grown as part of a long term evolution experiment in both underground and surface laboratories. The chance per day that a particle track interacts with a cell in the surface laboratory was found to be 6 × 10−5 day−1, 100 times less than the expected daily mutation rate for E. coli under our experimental conditions. In order for the chance cells are hit to approach the mutation rate, a gamma background dose rate of 20 μGy hr−1 is predicted to be required.

Microbeam facilities provide a unique opportunity to investigate the effects of ionising radiation on living biological cells with a precise control of the delivered dose. This paper describes dosimetry calculations performed at the single-cell level in the microbeam irradiation facility available at the Centre d'Etudes Nucleaires de Bordeaux-Gradignan in France, using the object-oriented Geant4 Monte Carlo simulation toolkit. The cell geometry model is based on high-resolution three-dimensional voxelised phantoms of a human keratinocyte (HaCaT) cell line. Such phantoms are built from confocal microscopy imaging and from ion beam chemical elemental analysis. Results are presented for single-cell irradiation with 3 MeV incident alpha particles.

International audience; Correlative microscopy is the application of two or more distinct microscopy techniques to the same region of a sample, generating complementary morphological and structural information that exceeds what is possible with any single technique to answer a biological question. We propose an approach based on a multimodal correlative microscopy, via two imaging and analytical techniques: fluorescence microscopy (FM) and ion beam analysis (IBA) to investigate in vitro nanoparticles (NPs) interactions. Indeed, the explosive growth in Nanotechnology has led to their utilization in a wide range of applications from therapeutics to multimodal imaging labeling. However, the risks for adverse health effects have not been clearly established. Detecting and tracking nanomaterials in biological systems are thus challenging and essential to understand the possible NPs-induced adverse effects. Indeed, assessing in situ NPs internalization at the single cell level is a difficult but critical task due to their potential use in nanomedicine. One of the main actual challenges is to control the number of NPs internalized per cell. The data obtained by both FM and IBA were strongly correlated in terms of detection, tracking, and colocalization of fluorescence and metal detection. IBA provides the in situ quantification not only of exogenous elements in a single cell but also of all the other endogenous elements and the subsequent variation in their cellular homeostasis. This unique property gives access to dose-dependent response analyses and therefore new perspectives for a better insight on the effect of metal oxide NPs on cellular homeostasis.

International audience; Charged-particle microbeams (CPMs) allow the targeting of sub-cellular compartments with a counted number of energetic ions. While initially developed in the late 1990s to overcome the statistical fluctuation on the number of traversals per cell inevitably associated with broad beam irradiations, CPMs have generated a growing interest and are now used in a wide range of radiation biology studies. Besides the study of the low-dose cellular response that has prevailed in the applications of these facilities for many years, several new topics have appeared recently. By combining their ability to generate highly clustered damages in a micrometric volume with immunostaining or live-cell GFP labelling, a huge potential for monitoring radiation-induced DNA damage and repair has been introduced. This type of studies has pushed end-stations towards advanced fluorescence microscopy techniques, and several microbeam lines are currently equipped with the state-of-the-art time-lapse fluorescence imaging microscopes. In addition, CPMs are nowadays also used to irradiate multicellular models in a highly controlled way. This review presents the latest developments and applications of charged-particle microbeams to radiation biology

International audience; The Centre d'Etudes Nucléaires de Bordeaux-Gradignan (CENBG) will soon be equipped with a new state of the art accelerator facility including a single-ended HVEE® 3.5 MV Singletron. Its performance in terms of brightness and energy stability will provide the unique opportunity to develop a high spatial resolution nanobeam line. The actual microbeam line dedicated to ion beam analysis (scanning transmission ion microscopy, particle induced X-ray emission and Rutherford backscattering spectrometry) and to cellular irradiation will be reinstalled on the new machine. In this paper, expected performances of the two beam lines are presented from ray tracing simulations using the Geant4 toolkit, capabilities of which at the sub-micron scale have been investigated and validated previously.

Friedreich ataxia (FRDA), the most common recessive ataxia, is characterized by degeneration of the large sensory neurons of the spinal cord and cardiomyopathy. It is caused by severely reduced levels of frataxin, a mitochondrial protein involved in iron-sulfur cluster (ISC) biosynthesis. Through a spatiotemporally controlled conditional gene-targeting approach, we have generated two mouse models for FRDA that specifically develop progressive mixed cerebellar and sensory ataxia, the most prominent neurological features of FRDA. Histological studies showed both spinal cord and dorsal root ganglia (DRG) anomalies with absence of motor neuropathy, a hallmark of the human disease. In addition, one line revealed a cerebellar granule cell loss, whereas both lines had Purkinje cell arborization defects. These lines represent the first FRDA models with a slowly progressive neurological degeneration. We identified an autophagic process as the causative pathological mechanism in the DRG, leading to removal of mitochondrial debris and apparition of lipofuscin deposits. These mice therefore represent excellent models for FRDA to unravel the pathological cascade and to test compounds that interfere with the degenerative process.

International audience; Assessing in situ nanoparticles (NPs) internalization at the level of a single cell is a difficult but critical task due to their potential use in nanomedicine. One of the main actual challenges is to control the number of internalized NPs per cell. To in situ detect, track, and above all quantify NPs in a single cell, we propose an approach based on a multimodal correlative microscopy (MCM), via the complementarity of three imaging techniques: fluorescence microscopy (FM), scanning electron microscopy (SEM), and ion beam analysis (IBA). This MCM was performed on single targeted individual primary human foreskin keratinocytes (PHFK) cells cultured and maintained on a specifically designed sample holder, to probe either dye-modified or bare NPs. The data obtained by both FM and IBA on dye-modified NPs were strongly correlated in terms of detection, tracking, and colocalization of fluorescence and metal detection. IBA techniques should therefore open a new field concerning specific studies on bare NPs and their toxicological impact on cells. Complementarity of SEM and IBA analyses provides surface (SEM) and in depth (IBA) information on the cell morphology as well as on the exact localization of the NPs. Finally, IBA not only provides in a single cell the in situ quantification of exogenous elements (NPs) but also that all the other endogenous elements and the subsequent variation of their homeostasis. This unique feature opens further insights in dose-dependent response analyses and adds the perspective of a better understanding of NPs behavior in biological specimens for toxicology or nanomedicine purposes.

International audience; Detecting and tracking nanomaterials in biological systems is challenging and essential to understand the possible interactions with the living. In this context, in situ analyses were conducted on human skin cells and a multicellular organism (Caenorhabditiselegans) exposed to titanium dioxide nanoparticles (TiO2 NPs) using nuclear microprobe. Coupled to conventional methods, nuclear microprobe was found to be suitable for accurate description of chemical structure of biological systems and also for detection of native TiO2 NPs. The method presented herein opens the field to NPs exposure effects analyses and more generally to toxicological analyses assisted by nuclear microprobe. This method will show applications in key research areas where in situ imaging of chemical elements is essential.

The autosomal dominant mutation causing myotonic dystrophy (DM1) is a CTG repeat expansion in the 3'-UTR of the DM protein kinase (DMPK) gene. This multisystemic disorder includes myotonia, progressive weakness and wasting of skeletal muscle and extramuscular symptoms such as cataracts, testicular atrophy, endocrine and cognitive dysfunction. The mechanisms underlying its pathogenesis are complex. Recent reports have revealed that DMPK gene haploinsufficiency may account for cardiac conduction defects whereas cataracts may be due to haploinsufficiency of the neighboring gene, the DM-associated homeobox protein (DMAHP or SIX5) gene. Furthermore, mice expressing the CUG expansion in an unrelated mRNA develop myotonia and myopathy, consistent with an RNA gain of function. We demonstrated that transgenic mice carrying the CTG expansion in its human DM1 context (>45 kb) and producing abnormal DMPK mRNA with at least 300 CUG repeats, displayed clinical, histological, molecular and electrophysiological abnormalities in skeletal muscle consistent with those observed in DM1 patients. Like DM1 patients, these transgenic mice show abnormal tau expression in the brain. These results provide further evidence for the RNA trans-dominant effect of the CUG expansion, not only in muscle, but also in brain.

The energy and specific energy absorbed in the main cell compartments (nucleus and cytoplasm) in typical radiobiology experiments are usually estimated by calculations as they are not accessible for a direct measurement. In most of the work, the cell geometry is modelled using the combination of simple mathematical volumes. We propose a method based on high resolution confocal imaging and ion beam analysis (IBA) in order to import realistic cell nuclei geometries in Monte-Carlo simulations and thus take into account the variety of different geometries encountered in a typical cell population. Seventy-six cell nuclei have been imaged using confocal microscopy and their chemical composition has been measured using IBA. A cellular phantom was created from these data using the ImageJ image analysis software and imported in the Geant4 Monte-Carlo simulation toolkit. Total energy and specific energy distributions in the 76 cell nuclei have been calculated for two types of irradiation protocols: a 3 MeV alpha particle microbeam used for targeted irradiation and a 239Pu alpha source used for large angle random irradiation. Qualitative images of the energy deposited along the particle tracks have been produced and show good agreement with images of DNA double strand break signalling proteins obtained experimentally. The methodology presented in this paper provides microdosimetric quantities calculated from realistic cellular volumes. It is based on open-source oriented software that is publicly available.

A high resolution focused beam line has been recently installed on the AIFIRA (“Applications Interdisciplinaires des Faisceaux d’Ions en Region Aquitaine”) facility at CENBG. This nanobeam line, based on a doublet–triplet configuration of Oxford Microbeam Ltd. OM-50™ quadrupoles, offers the opportunity to focus protons, deuterons and alpha particles in the MeV energy range to a sub-micrometer beam spot. The beam optics design has been studied in detail and optimized using detailed ray-tracing simulations and the full mechanical design of the beam line was reported in the Debrecen ICNMTA conference in 2008. During the last two years, the lenses have been carefully aligned and the target chamber has been fully equipped with particle and X-ray detectors, microscopes and precise positioning stages. The beam line is now operational and has been used for its first applications to ion beam analysis. Interestingly, this set-up turned out to be a very versatile tool for a wide range of applications. Indeed, even if it was not intended during the design phase, the ion optics configuration offers the opportunity to work either with a high current microbeam (using the triplet only) or with a lower current beam presenting a sub-micrometer resolution (using the doublet–triplet configuration). The performances of the CENBG nanobeam line are presented for both configurations. Quantitative data concerning the beam lateral resolutions at different beam currents are provided. Finally, the first results obtained for different types of application are shown, including nuclear reaction analysis at the micrometer scale and the first results on biological samples.

The use of ion microbeams as probes for computed tomography has proven to be a powerful tool for the three-dimensional characterization of specimens a few tens of micrometers in size. Compared to other types of probes, the main advantage is that quantitative information about mass density and composition can be obtained directly, using specific reconstruction codes. At the Centre d’Etudes Nucleaires de Bordeaux Gradignan (CENBG), this technique was initially developed for applications in cellular biology. However, the observation of the cell ultrastructure requires a sub-micron resolution. The construction of the nanobeam line at the Applications Interdisciplinaires des Faisceaux d’Ions en Region Aquitaine (AIFIRA) irradiation facility has opened new perspectives for such applications. The implementation of computed tomography on the nanobeam line of CENBG has required a careful design of the analysis chamber, especially microscopes for precise sample visualization, and detectors for scanning transmission ion microscopy (STIM) and for particle induced X-ray emission (PIXE). The sample can be precisely positioned in the three directions X , Y , Z and a stepper motor coupled to a goniometer ensures the rotational motion. First images of 3D tomography were obtained on a reference sample containing microspheres of certified diameter, showing the good stability of the beam and the sample stage, and the precision of the motion.

International audience; Abstract Deciphering the molecular basis of toxicology mechanism induced by nanoparticles (NPs) remains an essential challenge. Ion Beam Analysis (IBA) was applied in combination with Transmission Electron Microscopy and Confocal Microscopy to analyze human keratinocytes exposed to TiO(2)-NPs. Investigating chemical elemental distributions using IBA gives rise to a fine quantification of the TiO(2)-NPs uptake within a cell and to the determination of the intracellular chemical modifications after TiO(2)-NPs internalization. In addition, fluorescent dye-modified TiO(2)-NPs have been synthesized to allow their detection, precise quantification and tracking in vitro. The internalization of these TiO(2)-NPs altered the calcium homeostasis and induced a decrease in cell proliferation associated with an early keratinocyte differentiation, without any indication of cell death. Additionally, the relation between the surface chemistry of the TiO(2)-NPs and their in vitro toxicity is clearly established and emphasizes the importance of the calcium homeostasis alteration in response to the presence of TiO(2)-NPs.

Background Pharmacological high-throughput screening (HTS) represents a powerful strategy for drug discovery in genetic diseases, particularly when the full spectrum of pathological dysfunctions remains unclear, such as in Friedreich ataxia (FRDA). FRDA, the most common recessive ataxia, results from a generalized deficiency of mitochondrial and cytosolic iron-sulfur cluster (ISC) proteins activity, due to a partial loss of frataxin function, a mitochondrial protein proposed to function as an iron-chaperone for ISC biosynthesis. In the absence of measurable catalytic function for frataxin, a cell-based assay is required for HTS assay. Methods Using a targeted ribozyme strategy in murine fibroblasts, we have developed a cellular model with strongly reduced levels of frataxin. We have used this model to screen the Prestwick Chemical Library, a collection of one thousand off-patent drugs, for potential molecules for FRDA. Results The frataxin deficient cell lines exhibit a proliferation defect, associated with an ISC enzyme deficit. Using the growth defect as end-point criteria, we screened the Prestwick Chemical Library. However no molecule presented a significant and reproducible effect on the proliferation rate of frataxin deficient cells. Moreover over numerous passages, the antisense ribozyme fibroblast cell lines revealed an increase in frataxin residual level associated with the normalization of ISC enzyme activities. However, the ribozyme cell lines and FRDA patient cells presented an increase in Mthfd2 transcript, a mitochondrial enzyme that was previously shown to be upregulated at very early stages of the pathogenesis in the cardiac mouse model. Conclusion Although no active hit has been identified, the present study demonstrates the feasibility of using a cell-based approach to HTS for FRDA. Furthermore, it highlights the difficulty in the development of a stable frataxin-deficient cell model, an essential condition for productive HTS in the future.

Cobalt is known to be toxic at high concentration, to induce contact dermatosis, and occupational radiation skin damage because of its use in nuclear industry. We investigated the intracellular distribution of cobalt in HaCaT human keratinocytes as a model of skin cells, and its interaction with endogenous trace elements. Direct micro-chemical imaging based on ion beam techniques was applied to determine the quantitative distribution of cobalt in HaCaT cells. In addition, synchrotron radiation X-ray fluorescence microanalysis in tomography mode was performed, for the first time on a single cell, to determine the 3D intracellular distribution of cobalt. Results obtained with these micro-chemical techniques were compared to a more classical method based on cellular fractionation followed by inductively coupled plasma atomic emission spectrometry (ICP-AES) measurements. Cobalt was found to accumulate in the cell nucleus and in perinuclear structures indicating the possible direct interaction with genomic DNA, and nuclear proteins. The perinuclear accumulation in the cytosol suggests that cobalt could be stored in the endoplasmic reticulum or the Golgi apparatus. The multi-elemental analysis revealed that cobalt exposure significantly decreased magnesium and zinc content, with a likely competition of cobalt for magnesium and zinc binding sites in proteins. Overall, these data suggest a multiform toxicity of cobalt related to interactions with genomic DNA and nuclear proteins, and to the alteration of zinc and magnesium homeostasis.

A single-ended HVEE® 3.5 MV Singletron electrostatic accelerator has been installed since October 2005 at the Centre d’Etudes Nucleaires de Bordeaux-Gradignan (CENBG) in France. This facility is equipped with a microbeam line dedicated to ion beam analysis (scanning transmission ion microscopy – STIM, particle induced X-ray emission – PIXE, Rutherford back scattering) and cellular irradiation in single event mode. A high demagnification nanobeam line will be installed on the same facility in the near future. This paper focuses on the simulation of the microbeam and nanobeam lines performances using the Geant4 Monte Carlo simulation toolkit. Comparisons with experimental data collected on the microbeam line are presented.

Micros 2005 Special Issue - Venise; International audience; A comparison of three cellular irradiation techniques using the Monte Carlo simulation toolkit Geant4 is presented in this paper. They involve electrodeposited source of alpha particle-emitting radionuclides, random classical alpha beam irradiation and single cell targeted irradiation using a focused alpha microbeam line. The simulation allows the calculation of hit distributions among the cellular population as well as the absorbed dose for two typical cellular geometries.

International audience; Friedreich ataxia (FRDA) results from a generalized deficiency of mitochondrial and cytosolic iron-sulfur protein activity initially ascribed to mitochondrial iron overload. Recent in vitro data suggest that frataxin is necessary for iron incorporation in Fe-S cluster (ISC) and heme biosynthesis. In addition, several reports suggest that continuous oxidative damage resulting from hampered superoxide dismutases (SODs) signaling participates in the mitochondrial deficiency and ultimately the neuronal and cardiac cell death. This has led to the use of antioxidants such as idebenone for FRDA therapy. To further discern the role of oxidative stress in FRDA pathophysiology, we have tested the potential effect of increased antioxidant defense using an MnSOD mimetic (MnTBAP) and Cu,ZnSOD overexpression on the murine FRDA cardiomyopathy. Surprisingly, no positive effect was observed, suggesting that increased superoxide production could not explain by itself the FRDA cardiac pathophysiology. Moreover, we demonstrate that complete frataxin-deficiency neither induces oxidative stress in neuronal tissues nor alters the MnSOD expression and induction in the early step of the pathology (neuronal and cardiac) as previously suggested. We show that cytosolic ISC aconitase activity of iron regulatory protein-1 progressively decreases, whereas its apo-RNA binding form increases despite the absence of oxidative stress, suggesting that in a mammalian system the mitochondrial ISC assembly machinery is essential for cytosolic ISC biogenesis. In conclusion, our data demonstrate that in FRDA, mitochondrial iron accumulation does not induce oxidative stress and we propose that, contrary to the general assumption, FRDA is a neurodegenerative disease not associated with oxidative damage.

Friedreich ataxia (FRDA), a progressive neurodegenerative disorder associated with cardiomyopathy, is caused by severely reduced frataxin, a mitochondrial protein involved in Fe-S cluster assembly. We have recently generated mouse models that reproduce important progressive pathological and biochemical features of the human disease. Our frataxin-deficient mouse models initially demonstrate time-dependent intramitochondrial iron accumulation, which occurs after onset of the pathology and after inactivation of the Fe-S dependent enzymes. Here, we report a more detailed pathophysiological characterization of our mouse model with isolated cardiac disease by echocardiographic, biochemical and histological studies and its use for placebo-controlled therapeutic trial with Idebenone. The Fe-S enzyme deficiency occurs at 4 weeks of age, prior to cardiac dilatation and concomitant development of left ventricular hypertrophy, while the mitochondrial iron accumulation occurs at a terminal stage. From 7 weeks onward, Fe-S enzyme activities are strongly decreased and are associated with lower levels of oxidative stress markers, as a consequence of reduced respiratory chain activity. Furthermore, we demonstrate that the antioxidant Idebenone delays the cardiac disease onset, progression and death of frataxin deficient animals by 1 week, but does not correct the Fe-S enzyme deficiency. Our results support the view that frataxin is a necessary, albeit non-essential, component of the Fe-S cluster biogenesis, and indicate that Idebenone acts downstream of the primary Fe-S enzyme deficit. Furthermore, our results demonstrate that Idebenone is cardioprotective even in the context of a complete lack of frataxin, which further supports its utilization for the treatment of FRDA.

Myotonic dystrophy (DM) is caused by a CTG repeat expansion in the 3'UTR of the DM protein kinase (DMPK) gene. A very high level of instability is observed through successive generations and the size of the repeat is generally correlated with the severity of the disease and with age at onset. Furthermore, tissues from DM patients exhibit somatic mosaicism that increases with age. We generated transgenic mice carrying large human genomic sequences with 20, 55 or >300 CTG, cloned from patients from the same affected DM family. Using large human flanking sequences and a large amplification, we demonstrate that the intergenerational CTG repeat instability is reproduced in mice, with a strong bias towards expansions and with the same sex- and size-dependent characteristics as in humans. Moreover, a high level of instability, increasing with age, can be observed in tissues and in sperm. Although we did not observe dramatic expansions (or 'big jumps' over several hundred CTG repeats) as in congenital forms of DM, our model carrying >300 CTG is the first to show instability so close to the human DM situation. Our three models carrying different sizes of CTG repeat provide insight on the different factors modulating the CTG repeat instability.

A (CTG) n expansion in the 34-untranslated region (UTR) of the DM protein kinase gene (DMPK) is responsible for causing myotonic dystrophy (DM). Major instability, with very large expansions between generations and high levels of somatic mosaicism, is observed in patients. There is a good correlation between repeat size (at least in leucocytes), clinical severity and age of onset. The trinucleotide repeat instability mechanisms involved in DM and other human genetic diseases are unknown. We studied somatic instability by measuring the CTG repeat length at several ages in various tissues of transgenic mice carrying a (CTG) 55 expansion surrounded by 45 kb of the human DM region, using small-pool PCR. These mice have been shown to reproduce the intergenerational and somatic instability of the 55 CTG repeat suggesting that surrounding sequences and the chromatin environment are involved in instability mechanisms. As observed in some of the tissues of DM patients, there is a tendency for repeat length and somatic mosaicism to increase with the age of the mouse. Furthermore, we observed no correlation between the somatic mutation rate and tissue proliferation capacity. The somatic mutation rates in different tissues were also not correlated to the relative inter-tissue difference in transcriptional levels of the three genes (DMAHP, DMPK and 59) surrounding the repeat.

Human body is perpetually subjected to environmental exposures as sun, natural, urban or industrial pollutions and societal exposures. Skin is the main physiological barrier facing to these different stresses; however its response mechanisms are still not well understood. Skin models are thus necessary to understand skin physiology and behavior in normal conditions or under stress. Because of the large natural interindividual variability and of some difficulties in obtaining human skin biopsies, we have been developing and characterizing different skin models such as reconstructed epidermis in vitro, native pigskin and human keratinocyte cells. These models allow us to study the mechanisms involved in ion homeostasis in correlation with structural organization and biological responses. In the present study, we have characterized the pig ear skin model using micro-particle induced x-ray emission (PIXE) analysis with the aim of evaluating the distribution of inorganic ions under normal or stress conditions together with the transepidermal penetration of external agents, such as aluminum oxides. The nuclear microprobe analysis allowed us to validate the culture conditions of the tissue and we have shown an alteration of the inorganic ion patterns after exposure to a chemical compound (sodium dodecyl sulfate) known for its deleterious effects on the barrier function. Finally, we have shown no transepidermal penetration of aluminum oxides after different exposures to cosmetic formulations. Copyright (C) 2009 John Wiley & Sons, Ltd.

International audience; Although titanium dioxide nanoparticles (TiO2 NPs) have been extensively studied, their possible impact on health due to their specific properties supported by their size and geometry, remains to be fully characterized to support risk assessment. To further document NPs biological effects, we investigated the impact of TiO2 NPs morphology on biological outcomes. To this end, TiO2 NPs were synthesized as nanoneedles (NNs), titanate scrolled nanosheets (TNs), gel-sol-based isotropic nanoparticles (INPs) and tested for perturbation of cellular homeostasis (cellular ion content, cell proliferation, stress pathways) in three cell types and compared to the P25. We showed that TiO2 NPs were internalized at various degrees and their toxicity depended on both titanium content and NPs shape, which impacted on intracellular calcium homeostasis thereby leading to endoplasmic reticulum stress. Finally, we showed that a minimal intracellular content of TiO2 NPs was mandatory to induce toxicity enlightening once more the crucial notion of internalized dose threshold beside the well-recognized dose of exposure.

Objectifs: to define place and impact of nanoparticles in radio-oncology. To study high Z nanoparticles radiopotentialization with a literature review and experimentation by Titane Dioxyde nanoparticles (TiO2NPs). Materials and Methods: IB115 cell line (liposarcoma) were exposed to TiO2Nps, at 0.2 et 4 µg.cm-2, and were irradiated at doses of 0, 1, 2 and 6 Gy by 6MeV photons on a medical LINAC facility. Multi-elementay chemical composition were analysed by AIFAIRA facility after cryofixation. Stress pathways genes induction (NFKB, NRF2, p53, ATF4, ATF6, ERSE) were analysed by BMYScreen facility. Mitochondrial function and apoptosis were analysed by immunodetection (Cytochrome C, clived Caspase 3). Endoplasmic Reticulum (ER) and oxidative stress pathways were analysed by RTqPCR. Results: TiO2NPs internalization is exclusively intracytoplasmic and perinuclear. It induce a Cytochrome C cytoplasmic relocalization and a Clived Caspase 3 activation by a mitochondrial action. TiO2Nps induce an activation of ER stress pathway and an intracellular Calcium (CAic) increase, without oxidative stress. Ionizing radiation exposition leads to a CAic increase and a ER stress pathway activation. NPs-radiotherapy combination potentialize the CAic increase and ER stress pathway activation. Conclusions: TiO2NPs are not biologically inert. Combined with radiotherapy, they imply the ER stress pathway, a mitochondrial dysfunction, an intrinsic apoptotic pathway activation and a calcic homeostasis alteration. This combination’s mode of action is therefor a radiosensibilization, and not only a radiopotentialization., Objectifs : effectuer une revue de la littérature pour définir la place dans la stratégie thérapeutique, l’impact actuel et le possible impact futur des nanoparticules (NPs) en onco-radiothérapie. Étudier le potentiel radio-potentialisant des NPs de numéro atomique élevé à travers une revue de la littérature et une partie expérimentale avec des NPs de dioxyde de Titane (TiO2 NPs). Matériels et Méthodes : la lignée cellulaire IB115 (liposarcome para-testiculaire) a été exposée à des TiO2 NPs (nanofeuillets, longueur moyenne 178 nm) natifs ou fonctionnalisées au TRITC, à 0, 2 et 4 μg.cm-2, et a été irradiée à des doses de 0, 1, 2 et 6 Gy sur un accélérateur linéaire médical de type CLINAC (Varian Medical System) par des photons de 6 MeV. La composition chimique multi-élémentaire a été analysée sur la plateforme AIFIRA après cryofixation. Une analyse par gènes rapporteurs de voies de stress cellulaire (NFKB, NRF2, p53, ATF4, ATF6, ERSE) a été effectuée sur la plateforme BMYScreen. La fonction mitochondriale et l’activation de l’apoptose ont été analysées par immunodétection (Cytochrome C, Caspase 3 clivée). L’activation des voies du stress du réticulum endoplasmique (RE) et du stress oxydant ont été analysées par RTqPCR. Résultats : l’internalisation des TiO2NPs est exclusivement cytoplasmique et péri-nucléaire. Elle induit une relocalisation cytoplasmique du Cytochrome c et une activation de la Caspase 3 clivée, signant une action médiée par la mitochondrie. L’exposition aux TiO2 NPs induit l’activation des gènes de la voie du stress du RE et une augmentation du calcium intracellulaire, sans activation de la voie du stress oxydant. L’exposition aux radiations ionisantes induit une augmentation du calcium intracellulaire et une activation de la voie du stress du RE. La combinaison TiO2NPs-radiothérapie potentialise l’augmentation du calcium intracellulaire et l’activation de la voie du stress du RE. Conclusions : les NPs de dioxyde de Titane ne sont pas biologiquement inertes. Leur action en combinaison avec la radiothérapie implique la voie du stress du RE, une dysfonction mitochondriale, une activation de la voie apoptotique intrinsèque et une altération de l’homéostasie calcique. Le mode d’action de la combinaison TiO2NPs-radiothérapie se définit par une radio-sensibilisation et non une seule radio-potentialisation.

Few years ago, the paradigm of radiation biology was that the biological effects of ionizing radiations occurred only if cell nuclei were hit, and that cell death/dysfunction was strictly due to unrepaired/misrepaired DNA. Now, next this “DNA-centric” view several results have shown the importance of “non-DNA centered” effects. Both non-targeted effects and DNA-targeted effects induced by ionizing radiations need to be clarified for the evaluation of the associated radiation resistance phenomena and cancer risks. A complete overview on radiation induced effects requires the study of several points: (i) analyzing the contribution of different signaling and repair pathways activated in response to radiation-induced injuries; (ii) elucidating non-targeted effects to explain cellular mechanisms induced in cellular compartments different from DNA; and (iii) improving the knowledge of sensitivity/resistance molecular mechanisms to adapt, improve and optimize the radiation treatment protocols combining ionizing radiations and nanoparticles. Charged particle microbeams provide unique features to answer these challenge questions by (i) studying in vitro both targeted and non-targeted radiation responses at the cellular scale, (ii) performing dose-controlled irradiations on a cellular populations and (iii) quantifying the chemical element distribution in single cells after exposure to ionizing radiations or nanoparticles. By using this tool, I had the opportunity to (i) use an original micro-irradiation setup based on charged particles microbeam (AIFIRA) with which the delivered particles are controlled in time, amount and space to validate in vitro methodological approaches for assessing the radiation sensitivity of different biological compartments (DNA and cytoplasm); (ii) assess the radiation sensitivity of a collection of cancerous cell lines derived from patients in the context of radiation therapy; (iii) study metal oxide nanoparticles effects in cells in order to understand the potential of nanoparticles in emerging cancer therapeutic approaches., Ces dernières années, le paradigme de la radiobiologie selon lequel les effets biologiques des rayonnements ionisants ne concernent strictement que les dommages à l'ADN et les conséquences liées à leur non réparation ou à leur réparation défectueuse, a été remis en question. Ainsi, plusieurs études suggèrent que des mécanismes «non centrés » sur l'ADN ont une importance significative dans les réponses radio-induites. Ces effets doivent donc être identifiés et caractérisés afin d’évaluer leurs contributions respectives dans des phénomènes tels que la radiorésistance, les risques associés au développement de cancers radio-induits, les conséquences des expositions aux faibles doses. Pour ce faire, il est nécessaire : (i) d'analyser la contribution de ces différentes voies de signalisation et réparation induites en fonction de la dose et de la zone d’irradiation; (ii) d’’étudier les réponses radio-induites suite à l’irradiation exclusive de compartiments subcellulaires spécifiques (exclure les dommages spécifiques à l'ADN nucléaire); (iii) d’améliorer la connaissance des mécanismes moléculaires impliqués dans les phénomènes de radiosensibilité/radiorésistance dans la perspective d’optimiser les protocoles de radiothérapie et d’évaluer in vitro de nouvelles thérapies associant par exemple les effets des rayonnements ionisants et de nanoparticules d’oxydes métalliques. Les microfaisceaux de particules chargées offrent des caractéristiques uniques pour répondre à ces questions en permettant (i) des irradiations sélectives et en dose contrôlée de populations cellulaires et donc l’étude in vitro des effets « ciblés » et « non ciblés » à l'échelle cellulaire et subcellulaire, (ii) de caractériser l’homéostasie de cultures cellulaires en réponses à des expositions aux rayonnements ionisants et/ou aux nanoparticules d’oxydes métalliques (micro-analyse chimique multi-élémentaire). Ainsi, au cours de ma thèse, j'ai validé et exploité des méthodes d’évaluation qualitatives et quantitatives (i) in cellulo et en temps réel de la réponse radio-induite de compartiments biologiques spécifiques (ADN, mitochondrie, …) ; (ii) in vitro de la radiosensibilité de lignées sarcomateuses issues de patients; et (iii) in vitro des effets induits par des expositions à des nanoparticules d'oxydes métalliques afin d’évaluer leur potentiel thérapeutique et anti-cancéreux.

To understand the mechanisms of interaction of ionizing radiation with living tissues exposed to low and protracted doses remains a major issue for risk evaluation. The response cannot be found in epidemiological studies because the only available data concern accidental exposures to high doses of radiation. In addition, this kind of exposure is very difficult to reproduce in vitro by irradiating cell lines. The method per preference is based on random irradiation of cell populations. The mean number of particles interacting with cells is calculated on the basis of Poisson's statistics: inevitable multiple impacts, numerous potential intracellular targets (nuclei, cytoplasms...), indirect effects induced by the impact of particles on neighbouring cells or simply the extra-cellular targets, constitute phenomena that make more complex the interpretation of experimental data.A charged particle microbeam was developed at CENBG to perform the targeted irradiation of individual cells with a few microns precision, to deliver a counted number of particles down to the ultimate dose (one alpha per cell), to target predetermined cells and observe the neighbouring cells response. This facility has been validated during this work on human keratinocyte cells expressing a recombinant fluorescent histone (H2B-GFP). Confocal microscopy and quantitative analysis allowed DNA double strand breaks measurement via histone H2A.X phosphorylation in single cells, DNA repair mechanism and apoptosis induction. Data obtained during this thesis validate the methodology we developed by demonstrating targeting reproducibility and dose control showed dose-effect relationship as a function of time., Comprendre les modes d'interaction des rayonnements ionisants avec la matière vivante, notamment lors de l'exposition à de faibles doses telles que celles que l'on peut trouver dans un environnement industriel ou dans la nature, reste un enjeu majeur pour l'évaluation du risque associé. Il s'agit d'un problème de société qui n'a pu malheureusement trouver de réponse dans les études épidémiologiques classiques dans la mesure où les quelques données fiables concernent plutôt des expositions accidentelles à des doses beaucoup plus élevées. L'exposition naturelle représente pourtant la première source dans la vie courante juste devant les sources d'origines médicales (radiologie, radiothérapie). Ce type d'exposition est très difficile à reproduire en laboratoire sur des lignées cellulaires. La méthode principalement utilisée, basée sur l'irradiation aléatoire de populations cellulaires, consiste à calculer le nombre moyen de particules ayant interagi par cellule et repose ainsi sur des lois de distribution statistique (loi de Poisson). En plus des inévitables impacts multiples, la variété des cibles intracellulaires touchées (noyau, cytoplasme), les effets indirects induits par les impacts sur les cellules voisines ou simplement extracellulaires sont autant de phénomènes qui compliquent alors sérieusement l'interprétation des données.Dans ce contexte, un microfaisceau de particules  a été développé au CENBG pour réaliser des irradiations ciblées à l'échelle sub-cellulaire avec une précision de quelques micromètres. Il est ainsi possible de contrôler le nombre exact de particules délivrées par cellule (jusqu'à la dose ultime d'un ion par cellule), de prédéterminer avec précision le point d'impact et d'irradier certaines cellules tout en vérifiant la réponse de cellules voisines.La validation de ce dispositif a été réalisée au cours de ce travail de thèse, sur des kératinocytes humains exprimant une protéine recombinante nucléaire fluorescente (histone H2B-GFP) en mettant en évidence des dommages nucléaires radio-induits spécifiques et dose-dépendant. La combinaison de techniques telles que le microfaisceau d'ions, la microscopie confocale et l'analyse quantitative numérique a permis de mesurer, in situ et à l'échelle de la cellule unique, la cinétique de phosphorylation de la protéine histone H2A.X et d'aborder ainsi l'étude des processus de réparation de l'ADN et d'induction de l'apoptose. Les résultats expérimentaux ont validé la méthodologie développée en démontrant la reproductibilité du tir et le contrôle de la dose grâce à la mise en évidence d'une relation dose-effet qui a été également étudiée en fonction du temps.

Titanium dioxide nanoparticles are nowadays used in numerous domains. Theyenter in particular in the constitution of every day’s products such as paints or cosmetics(sun screen). The nanometer scale gives them a high reactivity which raises worries abouttheir possible toxicity. Nanoparticles with controlled morphologies were synthesized andcharacterized. They were then introduced in the medium of human cells in cultivation inorder to observe in vitro interactions and identify the reasons of this toxicity. The study wasthen extended to in vivo with a multi-cellular specimen: the nematode Caenorhabditiselegans (C. elegans).Researches have been focalized on detection, tracking and quantification of nanoparticles atthe scale of single-cells and then in nematodes. The different kinds of synthesizednanoparticles allowed identifying the physico-chemical properties involved in their toxicity.Ion beam analyses were conducted in order to visualize and in some case quantify thisnano-objects at the single cell-scale., Les nanoparticules de dioxyde de titane sont aujourd’hui utilisées dans denombreux domaines. Elles entrent notamment dans la constitution de produits d’usagecourant comme les peintures ou certains produits cosmétiques (crèmes solaires). La taillenanométrique leur confère une réactivité accrue ce qui soulève des inquiétudes sur leurpossible toxicité. Des nanoparticules de morphologies contrôlées ont été synthétisées etcaractérisées. Elles ont ensuite été introduites dans le milieu de cellules humaines enculture afin d’observer les interactions in vitro et identifier les raisons de cette toxicité.L’étude a ensuite été étendue à l’in vivo avec un organisme pluricellulaire : le nématodeCaenorhabditis elegans (C. elegans).Les recherches ont été focalisées sur la détection, la localisation et la quantification desnanoparticules à l’échelle de la cellule unique puis dans les nématodes. Les différentesnanoparticules synthétisées ont permis d’identifier les propriétés physico-chimiquessusceptibles d’influer sur la toxicité. Les techniques d’analyse par faisceau d’ions ont permisde visualiser voire même de quantifier ces nano-objets à l’échelle de la cellule unique.