Jean-Pierre Pouget

M Guy

Infine Stefano

Keywords: Targeted irradiation, DNA damage, low/high LET irradiations, Radiation sensitivity, Nanoparticles irradiation induces depolarization without persistent membrane alteration . . Particle-Induced X-Rays Emission RBS: Rutherford Backscattered Spectrometry STIM: Scanning Transmission Ion Microscopy

Je tiens ici à remercier toutes celles et ceux qui ont contribué de près ou de loin à ce projet et qui m'ont permis de le mener à la fin. Que ceux que je pourrais oublier acceptent déjà mes excuses et soient assurés de toute ma reconnaissance. Firstly, I would like to thank all members of the SPRITE project. They contributed to open my mind to other different fields. All along these three years, during numerous training courses shared together, I learnt a lot of things (scientific and not) from all of them. Je remercie Philippe Moretto comme Directeur du laboratoire de m'avoir accueillie au CENBG et aussi comme membre du jury avec Mme Claire Lafrasse-Rodriguez, M

Etude des effets biologiques radio-induits et microirradiation par particules chargées. Des mécanismes moléculaires aux thérapies émergeantes anti-cancéreuses

Résumé

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 telle 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 impliquées 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.

Mots-clés: Micro-irradiation ciblé, Dommages ADN radio-induits, Irradiations bas/haut TEL, Radiosensibilité, Nanoparticules

Résumé substantiel

La radiobiologie est le domaine des sciences médicales qui étudie l'influence des rayonnements ionisants (RI) sur les cellules, les tissus biologiques et les organismes vivants. Depuis de nombreuses années, le « dogme classique » de la radiobiologie considère que l'ADN est la cible « critique » et que les conséquences radio-induites ne se produisent que si les noyaux cellulaires sont effectivement touchés et que la mort/dysfonction cellulaire est strictement liée à un défaut ou à une absence de réparation de l'ADN. Aujourd'hui, cette vision « centrée » sur l'ADN (« targeted » effect) est remise en question et de nombreuses études suggèrent l'importance des effets « non centrés » sur l'ADN (« non targeted effect », NTE). Ainsi, il apparaît que des facteurs tels que le microenvironnement cellulaire/tumoral, la génétique, l'adaptation et la signalisation impliquées dans les réponses aux RI ont un rôle fondamental. De fait, ces mécanismes radio-induits doivent être identifiés afin de permettre une meilleure évaluation du risque associé à l'exposition aux RI (cancer) et ce, aussi bien dans le cadre des fortes et faibles doses d'exposition.

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 impliquées 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 RI et de nanoparticules d'oxydes métalliques. Les nanoparticules sont en effets des agents très intéressants permettant d'augmenter localement la dose délivrée dans la tumeur lors de traitements en radiothérapie externe. Cependant, l'exploitation de leurs propriétés de « dose-enhancers » nécessite le développement de protocoles destinés à quantifier l'internalisation et la toxicité des nanoparticules dans les cellules. L'amélioration des connaissances des mécanismes fondamentaux des réponses radio-induites en fonction de la dose d'exposition nécessite une étude profonde de tous les domaines mentionnés cidessus. Dans ce but, il faut répondre à plusieurs questions: (i) quels mécanismes moléculaires doivent être pris en considération pour une évaluation des effets radio-induits à court et long terme ? (ii) quels sont les effets radio-induits suite à une irradiation non ciblée sur l'ADN ? (iii) comment définir la notion de «dose» moyenne dans le cadre d'exposition in vitro ou l'organisme biologique est une population cellulaire et non un organe ? (iv) comment exploiter des données expérimentales in vitro afin de valider de nouveaux protocoles thérapeutiques ? etc, … Cette thèse s'est donc concentrée sur la caractérisation moléculaire et cellulaire des effets biologiques radio-induits et sur l'exploitation potentielle de ces mécanismes dans le cadre de nouvelle thérapie anti-cancéreuse. Plusieurs thèmes sont abordés dans ce travail de thèse et il s'organise en trois parties. La première partie concerne la mise en oeuvre des approches méthodologiques in vitro pour évaluer la radiosensibilité de différents compartiments cellulaires (ADN et cytoplasme) suite à des expositions contrôlée en dose, dans le temps et à l'échelle cellulaire à l'aide de l'utilisation d'un microfaisceau de particules chargées (AIFIRA). La seconde partie concerne l'évaluation in vitro de la radiosensibilité d'une collection de lignées cellulaires cancéreuses provenant de patients (lignées sarcomateuses). Enfin, la troisième et dernière partie porte sur l'étude des effets biologiques induits en réponse à des expositions à des nanoparticules d'oxydes métalliques afin de caractériser leur potentiel thérapeutique (thérapie anti-cancéreuse).

L'objectif de la première partie de cette étude concerne la caractérisation des effets biologiques radio-induits en fonction de la dose délivrée et de la cible irradiée (ADN, mitochondries). Dans cette étude, les réponses biologiques ont également été évaluées en fonction du transfert d'énergie linéique des particules considérées : proton et particule .

Cette étude a ainsi permis de caractérises la dynamique et la cinétique de réponse de protéines impliquées dans les mécanismes de reconnaissance et de réparation des cassures ADN induites par des particules chargées de basse énergie (3 MeV protons -particules ). Dans ce sens, une collection de lignées cellulaires exprimant des protéines fluorescentes recombinées avec la GFP (green fluorescent protein) a été établie. J'ai ainsi étudié les réponses radio-induites des protéines GFP-XRCC1 et GFP-RNF8 en fonction (i) de l'énergie déposée et (ii) du TEL (Transfert d'Energie Linéique) des particules. Pour ce faire, j'ai contribué à la validation d'un système de détection original qui permet d'irradier de manière sélective et contrôlée des cellules avec la dose « ultime » d'une seule particule  par noyau. Ce dispositif permet de détecter des particules  individuelles sans interférer avec l'énergie et la taille du microfaisceau. En utilisant notre système d'irradiation, j'ai mesuré que la protéine GFP-RNF8 s'accumule continuellement sur des traces de particules  pendant les 30 premières minutes après l'irradiation (1). J'ai ensuite pu évaluer que la protéine GFP-RNF8 est recrutée sur les sites endommagés d'une manière dépendante du TEL et que sa vitesse de recrutement est 10 fois inférieure à celle observée pour la protéine GFP-XRCC1 (2).

Ensuite, pour étudier les « effets non ciblés », j'ai collaboré avec l'Université de Bundeswehr de Munich et la plateforme d'irradiation SNAKE (consortium Marie-Curie, SPRITE) dans un projet visant à cibler les mitochondries et à évaluer leur réponse suite à une irradiation locale, ciblée et contrôlée en dose. La difficulté de ce projet résidait dans la capacité à (i) micro-irradier sélectivement des mitochondries, (ii) à réaliser des expériences similaires entre 2 facilités d'irradiation en terme de dépôt d'énergie et de modalité d'irradiation. Les résultats, obtenus lors de ces expériences, ont montré une dépolarisation locale des mitochondries en réponse à des micro-irradiations ciblées à l'aide de carbone (55 MeV, SNAKE) et de protons (3 MeV, AIFIRA). Les mécanismes induisant la dépolarisation des mitochondries restent encore inconnus mais nous avons observé que cette dépolarisation n'était pas due à la rupture de la membrane mitochondriale. En fait, afin de valider le maintien de l'intégrité de la membrane, j'ai établi des cellules transfectées exprimant une protéine de la matrice mitochondriale appelée Matrix-roGFP2. Suite aux irradiations réalisées au SNAKE et à AIFIRA, aucune modification n'a pu être observée (pas de modification de la localisation et de l'intensité de la fluorescence de la matrice mitochondriale) (3).

Enfin, la mobilisation et les interactions des molécules impliquées dans la réponse cellulaire aux RI peut être aussi mesurée avec des expériences de FRAP pour « Fluorescence Recovery after Photobleaching ». Pour réaliser ces expériences, le système d'irradiation de la ligne de microfaisceau a été complété avec une source laser équipée d'un système de balayage permettant de réaliser le photoblanchiment de marqueurs fluorescents. J'ai été donc impliqué dans la validation de ce dispositif expérimental en développant de lignées cellulaires d'intérêts telle que GFP-H2B et GFP-Nop52 afin de permettre l'étalonnage de ce nouveau système en termes d'intensité de « bleaching », de résolution et de ciblage.

Les objectifs de la deuxième partie de ma thèse ont été de développer des protocoles in vitro, en utilisant un faisceau d'ions caractérisé et avec d'études dosimétriques pour mieux comprendre les mécanismes biologiques induits par les électrons, les photons (couramment utilisés en radiothérapie conventionnelle) et les protons mais également l'impact de différentes modalités d'irradiation en terme de distribution spatiale en énergie (à l'échelle cellulaire) sur les lignées cellulaires cancéreuses issues des patients (sarcome). Deux lignées cellulaires, issues d'une collection unique établie par F.

Chibon, ont été sélectionnées pour leur histologie et leurs caractéristiques génétiques afin d'en étudier l'influence sur la réponse intrinsèque aux rayonnements. En collaboration avec le service de radiothérapie de l'Institut Bergonié, j'ai développé et adapté des protocoles d'irradiation in vitro pour estimer l'efficacité d'un faisceau médical de haute énergie (électrons de 9 MeV et photons de 6 MV) et l'efficacité de un faisceau de protons de basse énergie (3 MeV). Les irradiations d'électrons et de photons ont été réalisées avec un accélérateur linéaire médical. Les irradiations de protons ont été réalisées avec un microfaisceau, ce qui offre la possibilité d'étudier les effets de deux distributions d'énergie où l'énergie peut être déposée de manière très focalisée à l'échelle cellulaire ou bien de manière plus homogène sur la totalité de la surface cellulaire (faisceau focalisé versus faisceau large).

La dosimétrie assure que la même dose a été administrée par cellule au cours de ces différentes modalités d'irradiation. Les paramètres biologiques, tels que la quantification et la persistance dans le temps des dommages à l'ADN, la prolifération et la survie clonogénique, ont été analysées pour l'évaluation de la radiosensibilité de ces lignées cellulaires. Pour réduire les erreurs expérimentales, j'ai validé deux méthodes de comptage automatique développées avec le logiciel ImageJ qui permettent à la fois la quantification des dommages radio-induits (foci H2AX et P-ATM) et le comptage des colonies et du nombre de cellules par colonie. En utilisant ces approches, j'ai observé que les protons induisent des effets plus délétères que les électrons, indépendamment de la distribution d'énergie à l'échelle cellulaire et qu'une lignée cellulaire est plus radiosensible pour les paramètres biologiques analysés et cette radiosensibilité semble être en relation avec les caractéristiques génomique (4).

Dans une prochaine étape, ces établis protocoles in vitro peuvent être adaptés pour évaluer la potentialité de nouvelles approches thérapeutiques combinant RI et nanoparticules d'oxydes métalliques. Comme déjà mentionné, les nanotechnologies sont des domaines émergents étudiés afin d'augmenter localement la dose délivrée et ensuite, améliorer l'efficacité thérapeutique de la radiothérapie. L'un des points les plus importants qui contribue à disséquer les effets biologiques radio-induits en présence des nanoparticules est d'évaluer la concentration internalisée et la localisation des nanoparticules à l'échelle de la cellule unique.

Dans ce contexte, la troisième partie de mon manuscrit a été consacrée au développement de nouveaux protocoles qui combinent l'analyse par faisceau d'ions et les techniques de microscopie en fluorescence pour quantifier la distribution des éléments chimiques à l'échelle de la cellule unique.

Une microsonde nucléaire présente au CENBG permet de détecter, quantifier et localiser la présence d'éléments chimiques dans différents tissus et dans des populations cellulaires à l'échelle de la cellule unique. Dans ce contexte particulier, j'ai participé au développement d'une méthodologie d'imagerie originale qui combine l'analyse par faisceau d'ions et la microscopie de fluorescence et utilisant cette méthodologie, nous avons montré que les nanoparticules de dioxyde de titane (TiO 2 ) s'accumulent dans le cytoplasme tout autour du noyau et sont exclues du noyau et des mitochondries. Nous avons également observé que le contenu intracellulaire des nanoparticules peut être très hétérogène avec des variations très importantes de l'ordre de 10 fois entre différentes cellules d'une même population exposée. De plus, nous avons observé une altération de l'homéostasie cellulaire avec une augmentation d'ion intracellulaire, tel que le calcium, directement corrélée à la teneur de nanoparticules de TiO 2 (5).

Ces données suggèrent une altération de l'homéostasie cellulaire induite par la présence de nanoparticules de TiO 2 . Donc, pour définir les mécanismes moléculaires et cellulaires impliqués dans la toxicité des nanoparticules dans les cellules eucaryotes, j'ai participé au projet de Marina Simon qui vise à étudier les réponses de l'homéostasie cellulaire induite par différentes nanoparticules de TiO 2 (en termes de forme, de taille et de réactivité de surface) dans différentes populations de cellules humaines primitives et immortalisées. Nous avons observé qu'une concentration intracellulaire minimale de nanoparticules de TiO 2 (seuil minimal) est nécessaire pour (i) induire une altération de l'homéostasie cellulaire à travers l'altération du calcium et (ii) induire une voie de stress spécifique comme celle du réticulum endoplasmique associée à une dysfonction mitochondriale. De plus, nous avons observé que la fonction cellulaire (endothéliale versus épidermique), le type cellulaire (primaire versus immortalisé/cancéreuse) et l'hétérogénéité de la distribution intracellulaire de nanoparticules de TiO 2 dans une population modifient profondément la réponse cellulaire (6).

En résumé, j'ai (i) validé de nouveaux systèmes et protocoles pour étudier les effets radio-induits sur l'ADN et les mitochondries, (ii) exploré les mécanismes spatio-temporels activés dans les cellules en réponse aux IR, (iii) réalisé des analyses multiparamétriques pour extraire des informations biologiques sur la réponse intrinsèque des lignées cellulaires cancéreuses aux RI, et (iv) en collaboration avec mes collègues, j'ai analysé les altérations des éléments chimiques induites par les nanoparticules et/ou les RI.

Table of Contents

INTROD UCT ION

The path to the discovery of ionizing radiations is paved by important physics discoveries. In 1895, the German physicist Röntgen discovered a "new kind of ray", that he called temporarily X-Rays as a designation of something "unknown", and with which he took the first picture of his wife's hand and skeletal structure. In 1896, Becquerel discovered that uranium compounds naturally emitted similar rays, and the adverse health effects due to exposure to X-Rays, were soon reported. Inadvertently, he left a radium container in his vest pocket and he described the skin erythema appeared 2 weeks later. Two years later, Pierre and Marie Curie named this phenomenon radioactivity. They isolated the radioactive polonium and radium that, within few years, was used for the treatment of cancer.

Marie Curie died in 1934 of aplastic anemia probably developed from extended exposure to various radioactive materials.

These are few examples of the applications or effects that ionizing radiations (IR) could cause in cells, organs and tissues of the human body. It is clear that there are health risks for humans exposed to radiations, despite the considerable benefits obtained from the use of radiations in medicine 1 .

Everyday a person, group of people or even an entire population might be chronically or acutely exposed to ionizing radiations and suffer from acquired damages. These IR come from nuclear accidents, natural sources or produced for medical purposes, energy production and other industrial uses. Nuclear bombings of Hiroshima and Nagasaki by USA during World War II in 1945 are examples for nuclear warfare. Nuclear reactor accidents, such as Chernobyl in 1986, affected staff, clean-up workers, and the population of the region. There are also natural sources of IR, such a cosmic rays and radiation from elements present in the earth's crust (for example radon). Other examples of expositions to IR are in medicine for both imaging/nuclear medicine and for cancer treatment (radiotherapy) where damaged is caused not only in cancerous cells but also in healthy tissues.

For all these and many other reasons, the knowledge of interaction between IR and living tissues has an important role in many fields, such as health risks associated to low dose exposure from natural or working environment, the appearance of radiation-induced tumors, and the new cancer therapeutic approaches.

IR are electromagnetic waves or massive particles that carry enough energy to ionize (i.e. removes an electron from) an atom or a molecule of the medium through which it propagates. These changes can induce a variety of biological effects depending on the physical nature, duration, dose and dose-rate of exposure. Low doses of IR can be found in natural (cosmic gamma rays, ingestion of potassium-40, radon exposure) and industrial environment (nuclear power plants, waste processing plants, medical environment). Thus, we are increasingly exposed to radiation during the course of INTRODUCTION 20 our lives from routine medical usage, such as diagnostic X-Rays and computed tomography (CT) scanning, from the increased frequency of flying, and from radiotherapy treatments 2,3 . The elevated usage of radiation for medical purposes and nuclear power for energy enhances the risk of accidental exposure from industrial accidents and for workers in the radiation industry 4 .

Worldwide, the average human exposure to radiation from natural sources is 2.4 mSv per year, about half of which is due to the effects of radon progeny (daughters) (radioactive elements produced by the decay of 222 Ra). Diagnostic medical exposures add about 0.4 mSv per year, atmospheric nuclear testing about 0.005 mSv per year, the Chernobyl accident fallout 0.002 mSv per year, and nuclear power production about 0.0002 mSv per year 5,6 . However, the magnitude of health risks at low doses and dose-rates (below 100 mSv and 0.1 mSv.min -1 , respectively) remains controversial due to the difficulty of direct data collection 7 . To date, this issue cannot be addressed by epidemiological studies because it is difficult to accurately assess the risk of a population exposed to various carcinogens: it is well established that low-dose effects of IR are more difficult to measure than high-dose effects. The most important of the epidemiological studies for risk assessment is the Life Span Study of the survivors of the atomic bombings of Hiroshima and Nagasaki 8,9 . These epidemiological studies of the survivors provide strong evidence of increased risk of developing both solid cancers and leukemia from high doses and high dose-rates of radiation. However, significantly increased cancer risk is also observed in atomic bomb survivors exposed to lower doses of radiations 1,10,11 . Another convincing evidence of low-dose effects comes from studies of in utero exposure, which suggest that there are elevated risks of leukemia and most other cancer types following 10-20 mGy diagnostic exposure 12,13 . The radiation risks (per unit dose) implied by these studies are similar in magnitude to those following much higher dose exposure in early life in the Japanese atomic bomb survivors 14 .

Although the link between the initial damage and cancer remains elusive, the target theory is the basis for the model describing risk of cancer and heritable effects, and it has been used to establish international rules and standards of radiation protection. These assumptions are described collectively as the linear no-threshold (LNT) model, where the risk of low doses is extrapolated from the risk assessed at high doses. The LNT relationship implies a proportionality between the dose and the cancer risk 7 . The validity of using this dose-response model is controversial because accumulated evidences indicated that living organisms respond differently to low dose radiation than they do to high dose radiation 15 . The range in sensitivity between individuals is, perhaps, best illustrated by the response to radiotherapy where 1-5% of treated patients have a more dramatic response compared INTRODUCTION 21 to the average individual. This represents the response to high-dose exposure but current evidence suggests that there will be individuals with enhanced sensitivity to low-dose exposure. Therefore, while some progress has been made in understanding these phenomena, the knowledge has yet to be assembled into a coherent body of understanding that can be readily applied to the assessment of low-dose risk. However, it is recognized that epidemiological studies can be limited in statistical power to detect excess risk under these conditions. This is because the population size required to detect added risk becomes enormous when very small increases in risk are being investigated. The advances in radiation biology during the past two decades, the understanding of carcinogenesis, and the discovery of defenses against carcinogenesis challenge the LNT model, which appears nowadays outdated 16,17 .

Quite a lot is currently known about the general mechanisms of action of radiation. However, much remain to be learnt about the specific mechanisms by which radiations exert their effects, in particular the effects of low doses over long periods of time. Improved knowledge of genetic and epigenetic mechanisms involved in radiation sensitivity will allow a better definition of radiation protection rules and the development of more effective treatment plans in case of deleterious exposure.

The absorption of IR by living cells can directly disrupt atomic structures, producing physical, chemical and biological changes (direct effects). It can also act indirectly through radiolysis of water, thus generating reactive chemical species that may damage nucleic acids, proteins and lipids (the socalled indirect effects) 18 . Since the discovery of the DNA (in 1953, by Watson and Crick) and the discovery that radiations could introduce strand breaks into DNA double helix (in 1961, by Lett and colleagues), the DNA was considered as the main target of IR 19 . It was assumed that the amount of DNA damages is proportional to the radiation dose received, and there is a clear relationship between DNA damages, mutations and cancer development, which drive to a DNA-centered paradigm, known as 'the target theory' 20 . Indeed, upon DNA damages induction by IR, there is an extensive response in the chromatin surrounding the break. Hundreds of molecules and DNA damage response proteins accumulate at DNA damaged sites, forming large nuclear aggregates, that appear as ionizing radiation-induced nuclear foci (IRIF) 21 . Of particular importance is to analyze the contribution of these different signaling and repair pathways activated in response to radiationinduced injuries that can affect human health and promote cancer development 6,17 .

Alongside the damages induced directly to DNA, it has been put-forward that the radiation-induced effects can be mediated by 'Non-Target Effects' (NTE). NTE occur when IR energy has not been INTRODUCTION 22 deposited in the DNA or the DNA has not been targeted. In 1990's the 'membrane theory', where membranes are considered as a second major target of radiation effects, challenges the 'DNAcentered theory'. Furthermore, early effects of lipid soluble vitamins under radiation response were observed 22 . In those years, it raised the idea that the signaling of damage from irradiated cells could be induced in non-hit cells, calling this phenomena 'bystander effects'. Nowadays, the distinct classes of NTE are identified in genomic instability, bystander effect, adaptive response, and low dose radiation-induced hypersensitivity.

Elucidating the mechanisms of NTE calls for further researches; moving away from the conventional DNA targeted framework, it is necessary to develop new experimental strategies to evaluate the impact of IR on specific targets different from DNA.

As well as the effects of IR at low doses need to be clarified for the evaluation of the associated cancer risks 23 , understanding how high doses of IR interacts with molecular mechanisms responsible for the radiation resistance phenomena (that could be innate or derived from a previous radiation therapy treatment) remains a challenge. Improving the knowledge of sensitivity/resistance molecular mechanisms is useful to adapt, improve and optimize the radiation treatment protocols.

Indeed, radiation therapy exploits the capability of IR to kill cells. The effectiveness of radiation therapy depends not only on the type of radiation (X-Rays or protons) but also, on the nature of cancer, on the individual patient, and on the combination of radiations with other treatments.

The evolution of radiotherapy has been strongly correlated with the development of imaging techniques, which allow radiologists to determine the location of both internal organs and tumor 24 .

However, when using conventional external-beam radiotherapy (photons and electrons), healthy tissues are exposed to radiations. Several strategies investigate the use of heavy charged particles (hadrons) 25 , and the addition of radiation sensitizing agents to increase the radiation-induced effects in the tumor volume, and to spare the healthy tissues surrounding tumor 26,27 . Charged particles, indeed, deposit little energy at the entrance to the body, and most at the end of their range in the tissue delivering conformal dose distribution in the tumor and minimizing the normal tissue exposed to radiation 28 .

Recently, nanotechnologies have paved the way to new approaches in local cancer therapy.

Nanoparticles, with high electron density, offer the possibility to deposit high amounts of energy within the cancer cells, when activated by IR. To exploit the capability to increase the deposit of energy via secondary electron emission (photoelectric and/or Compton Effect) and atomic deexcitation processes (Auger electrons, X-Rays), the uptake of nanoparticles by cells is a very It is necessary to improve the knowledge about the fundamental mechanisms of cellular response to IR at both low and high doses. To this aim, different questions need to be answered: (i) which biomolecular mechanisms induced in cellulo by IR have to be considered for a correct evaluation of radiation-induced effects? (ii) what are the effects of IR when the target is not the DNA? (iii) what is the mean 'dose' when radiobiological in vitro studies are conducted on cells and not on whole organs? (iv) which predictive methodologies can allow us to translate the results obtained in vitro to medical applications? (v) where nanoparticles accumulate in cells and how quantify the intracellular nanoparticles content? etc, …

The aim of my PhD thesis is to decipher the biological effects of ionizing radiations from the molecular mechanisms to their applications in radiation therapy. Deciphering the radiation-induced effects, from molecular mechanisms in single cells to the behavior of a cell population, requires the development of original approaches using in vitro models and highly-controlled experimental conditions. Multidisciplinary knowledge is required, from physics to fundamental and medical biology to: (i) develop accurate irradiation setups where the dose is controlled in time, amount and space, (ii) study complex molecular and cellular regulation pathways activated after DNA damage at both low and high doses, (iii) explore the intricate radiobiological responses to IR and (iv) investigate the interaction of nanoparticles with cells.

Charged particle microbeams provide unique features to study in vitro the targeted and non-targeted radiation responses and have been shown to be powerful tools to localize and quantify chemical elements in cells. The "Centre d'Etudes Nucléaires de Bordeaux-Gradignan" (CENBG) is equipped with a Singletron™ particle accelerator that produces focalized charged particle microbeams. In particular during my work, I made use of two microbeam lines. One microbeam line is addressed to perform micro-irradiations of living cells and small organisms (C. elegans) where dose, target and time are highly controlled. Indeed, this set-up allows targeting energetic protons and helium ions in living biological specimens with micrometer precision. It is coupled with live cell imaging tools such as an epifluorescence microscopy for real time cell visualization and a laser photobleaching setup.

The second beam line exploits the interactions of charged particles with matter for chemical element imaging and analytical techniques such as µ-PIXE, µ-RBS and µ-STIM permit to obtain the spatial distribution and quantification of these chemical elements at the single cell level.

INTRODUCTION 24

This manuscript is structured in three main parts.

FIRST PART: BIOLOGICAL EFFECTS INDUCED BY IONIZING RADIATIONS. STUDY OF DOSE-EFFECT

PROTEINS KINETICS AND NON-TARGETED EFFECTS

After an introduction devoted to a general overview of IR, their effects on biological specimens and the description of the AIFIRA irradiation microbeam line, the experimental results obtained are summarized in three articles. These three manuscripts are then followed by a section where outcomes and impacts of the individual projects are discussed.

 The first result shows the development of a thin membrane for detection of single -particles minimizing beam scattering. These single particles were used to irradiate living cells and to study their impact on GFP-proteins accumulation at DNA damaged sites.

 The second result identifies a correlation between the number of delivered particles (starting from the "ultimate dose" of one single particle per cell), the Linear Energy Transfer of particles and the kinetics profile of proteins involved in the DNA damage signaling and repair. In particular, we chose to describe the kinetics profiles of different stable GFP-transfected cell lines, which are involved in the DNA signaling and repair mechanisms, such as XRCC1 (Single Strand Breaks and BER/NER pathways) and RNF8 (Double Strand Breaks ligase protein). These proteins accumulate at DNA damaged sites forming large nuclear aggregates that appear as ionizing radiation-induced nuclear foci (IRIF) 21 . DNA damage-induced foci are highly dynamic structures, subject to precise spatio-temporal regulation and the precise order and timing of recruitment is thought to provide the kinetics of how this lesion is processed and resolved after irradiations 29 .

 The third result, obtained thanks to the collaboration with my colleague Dietrich Walsh and the SNAKE microbeam facility (Bundeswehr University Munich), shows the radiation-induced effects after different doses of 3 MeV protons and 55 MeV carbon ions on mitochondria. The cytoplasm, which is the environment where the majority of processes involved in the maintenance of cellular integrity take place, has rarely been taken into account as a target of ionizing radiation.

Mitochondria constitute a large volume of the cytoplasm in all cell types and they have been selected in this study to highlight the effect of targeted irradiation. We showed that targeted irradiations with both carbon ions and protons induce instant mitochondrial depolarization without changes the mitochondrial matrix.

 The fourth result describes the implementation of the microbeam line end-station with a 488 nm laser system. This intense light source can be focused into the object plane of our online fluorescence microscope and the focal point can be moved to selected positions to permanently bleach GFP molecules. These experiments enable us to measure the exchange and binding INTRODUCTION 25 behavior of repair proteins after bleaching and this validated system can now be coupled with targeted irradiations.

SECOND PART: IN VITRO EVALUATION OF RADIATION SENSITIVITY OF SARCOMA CELL LINES DERIVED FROM PATIENTS

The second part of this PhD thesis starts with a general overview of sarcomas that are particularly radiation resistant types of tumor for which radiation therapy has to be improved.

The objective of this part is the development of protocols, ranging from dosimetry to biological assays, to describe the radiation sensitivity of patient's derived cell lines. Two cells lines, from a wide collection established by F. Chibon, are selected for their histology and genetic characteristics to study the influence of IR on the intrinsic radiation response. Working together with the Radiation Therapy Department of the Institut Bergonié, we developed and adapted an irradiation protocol to estimate the effectiveness of medical beams (9 MeV electrons and 6 MV photons), usually used in external radiotherapy, and the effectiveness of low energy protons (3 MeV). Electron and photon irradiations were carried out with a medical linear accelerator, and proton irradiations were carried out with the AIFIRA microbeam, which offers the possibility to study the effects of two different deposited energy distributions at the cellular level, such as the effects of focalized versus broad beam. The results obtained are described in the fourth manuscript that is followed by a discussion and perspectives opened by this work.

THIRD PART: IN SITU AND IN CELLULO DETECTION AND QUANTIFICATION OF METAL OXIDE

NANOPARTICLES. TOWARD THE ELUCIDATION OF NANOTOXICITY MECHANISMS AND PERSPECTIVES FOR CANCER THERAPY

Finally, the third part of this manuscript is devoted to the development of new protocols to combine nuclear microprobe analysis and microscopy techniques for quantification of chemical element distribution in single cells. After a general introduction of the microprobe present at the AIFIRA platform, its applications are presented. In particular, the quantification of nanoparticles at the single cell level is needed to understand the mechanisms involved in new emerging concepts of radiation therapy proposing to combine IR and nanoparticles to enhance the local dose deposition inside the tumors. We developed a procedure for the in situ detection and in vitro quantification of chemical elements present in human cells, as well as metal oxide nanoparticles and by using these methods we observed the effects of different nanoparticles in different cell types. These results are presented in two manuscripts which are followed by a discussion and perspectives for future works.

PART I Biological effects induced by ionizing radiations. Study of dose-effects proteins kinetics and non-targeted effects P A R T I

When the integrity of the genetic material of cells, the DNA, is compromised, swift and efficient measures must be taken to restore it. Failures can lead to the occurrence and propagation of mutations and even cell death. An intricate cellular machinery has evolved to prevent the deleterious consequences of DNA damage. If successful repair cannot be achieved, the apoptotic suicide pathway may eliminate cells with compromised DNA to protect the organism against potential tumourigenesis. Collectively, the network of pathways that ensure the above objectives is called the DNA damage response. This network also affects cellular processes like transcription and replication of DNA and constitutes an important barrier against cancer development. The tight correlation between the DNA damage response and cancer development has solicited a massive effort to map the underlying molecular pathways and to understand their roles in human pathogenesis. Our understanding of the DNA damage response is already quite considerable and most of the central proteins and their functions have been described in details. Nevertheless, one key issue that remains somewhat under-represented in the field is how these important reactions are organized in time and space. The spatio-temporal aspects of the DNA damage response have been intensively conducted using a variety of tools including femto-second lasers and particle micorbeams. However, due to the nature of lasers, the energy deposited with these systems is not quantifiable. Charged particles micro-irradiation enables a quantification of deposited energy in a defined nuclear area by delivering a precise number of particles.

The aim of the first part of this project is to further develop our capabilities in detecting and irradiating cells with a highly controlled number of particles, and apply these techniques to investigate the spatio-temporal properties of the DNA damage response. In addition, the focusing capabilities of microbeams allow us to target single mitochondria and in collaboration with the University of Bunderswehr and the facility installed at SNAKE in Munich we studied the effects of charged particles on mitochondrial membrane.

The main body of this first part is divided into three sections: Background, Experimental Results and Discussion. The Background section deals with the micro-irradiation techniques, and outlines the theory and current status of the DNA damage response and DNA repair processes induced by IR. In Experimental Results section, I present three manuscripts produced during my PhD studies on which I appear as an author. Then, I discuss outcomes and impacts of these projects in the light of subsequent findings in this expanding fields.

Background

Chapter 1

Ionizing radiations and matter interactions

Radiation can be defined as the propagation of energy through matter or space. We define a nonionizing radiation, a radiation whose associated energy can excite an atom (raise an electron to a higher energy level) but not remove an electron from it. If the energy is sufficient to remove electrons from their orbits from atoms or molecules, then the radiation is called ionizing radiation (Figure 1). to block these types of radiations (Figure 2). (iv) Neutrons are high speed nuclear particles that have an exceptional ability to travel great distances in air and require very thick hydrogen-containing material to block them 1 . Linear Energy Transfer. When IR traverse matter, they deposit energy along their tracks. Such energy is typically measured in electron volt (eV). The energy deposit along the track is strictly dependent on the type of incident radiation and it is described by the Linear Energy Transfer (LET). Indeed, the LET is the amount of energy transferred to matter per unit length of the track. The unit usually used for this quantity in radiation biology is kiloelectron volt per micrometer (keV.m -1 ) per density unit. For a given charged particle, the LET is influenced by its energy and its charge. On the basis of the LET, IR can be divided into sparsely ionizing radiations (X-Rays, -Rays and electrons with low LET) and densely ionizing radiations (particle radiations with high LET). 10 keV.µm -1 is generally accepted as the threshold between low and high LET radiations. For example, -particles of few MeV are classified as high LET radiations because they depose their energy on a short linear range (20 to 100 µm), producing a dense ionization along their tracks. The energy lost by these particles during the interaction with matter rapidly increases after a small distance, generating the so-called Bragg peak, and particles are then stopped. While the energy deposition of protons and heavier charged particles is characterized by the presence of the Bragg peak, this is not the case for photons 1,2 .

Absorbed Dose. Some radiation damage causes permanent chemical change that can lead to harmful biological effects. Biological effects are found to be directly related to the amount of energy that is deposited by the radiation per unit of mass of tissue. The amount or quantity of absorbed radiation is expressed in terms of the absorbed dose, measured in Gray (Gy). One Gy corresponds to an energy deposition of 1 Joule per kilogram of irradiated matter. This unit is especially used when an organ is irradiated in a homogeneous manner, but this concept loses of meaning when the dose is delivered in a small area such as cells. When single cells are irradiated is more appropriate to speak about "deposited energy" instead of "dose" 3 .

Relative Biological Effectiveness. Equal doses of different types of radiation do not produce equal biological effects. The effectiveness of different type of IR is evaluated by the relative biological effectiveness (RBE). This parameter is obtained by comparing the dose of one type of radiation needed to cause a specific effect with the dose of another type of radiation needed to obtain an equal biological effect. The typical standard to define RBE of different radiations is X-rays. The RBE values for low LET radiations are close to 1 and increase as LET increases. The RBE generally increases as the dose is decreased, and it varies greatly according to the tissue or cell line studied 4 .

Chapter 2

An original tool to observe ionizing radiation effects:

Charged Particle Microbeam

To study the effects of IR on living cells, methods and models which spanning from physics to biology are necessary. Synergy of various fields gives the possibility to explore livings and to understand the fundamental physiologic and pathologic mechanisms from original and complementary points of view.

From the end of 1990s, charged particle microbeams were developed as a specific tools to investigate the effects of IR on living samples 5 . The rational for developing such devices was initially motived by the necessity to study the cellular response to low doses. Using broad beam or radioactive sources, the delivering of an average of one particle per cells leads to a Poisson distribution of the number of particle traversals. This means that 37% of the cells receive no particle at all, 37% receive one particle and 26% receive more than one particle 6 . For this reason there was an increasing interest in the use of microbeam systems designed to deliver single particles with a position resolution of a few micrometers in biological targets. This allows the study of biological responses to charged particles at both single cell (nucleus) and subcellular (nucleolus or cytoplasm) levels. In addition, a large quantity of cells can be simultaneously irradiated within a relatively short time by moving the beam from cell to cell using fast electromagnetic scanning systems, which permit to study the effects on a cell population. Also, the dose that is delivered to cells can be accurately measured and controlled.

Nowadays, there are 11 microbeam facilities fully-operational or under-development worldwide and all of them contribute to the fundamental knowledge of cellular response to IR 7 . The feasible applications of microbeams are described below and schematized in Figure 3 8,9 :

(i) Study of biological effects of single charged particles in single cell. The main advance of these facilities is to deliver single ion in single cell, and to measure the effect of a single particle track on mutagenic and oncogenic transformation 10,11 , cellular toxicity, micronuclei formation and genomic instability [12][13][14] .

(ii) Study of DNA damage response. By their ability to target the cell nucleus in a highlycontrolled way, microbeams are used to study DNA damage and repair machinery 15 .

Indeed, DNA damage signaling and repair factors can accumulate around radiation induced lesions in microscopically discernible structures known as ionizing radiation-induced foci (IRIF). The protein accumulation can be visualized using immunofluorescence techniques or live cell imaging. For live cell imaging, the protein of interest need to be fluorescently tagged, e.g. by fusion to GFP. In this way the formation of IRIF can be followed before, during, and after irradiation by using microbeam lines equipped with advanced fluorescence microscopy end-stations [16][17][18][19][20] .

(iii) Cellular compartment targeting. The micrometric resolution gives the possibility to target sub-nuclear compartments and allows studying the impact of IR on these structures.

Initially, the sub-micrometer size of the beam was achieved at GSI Helmholtz Centre for Heavy Ion Research and nowadays this resolution permits to study the DNA repair response in heterochromatin centers 21,22 . Recently, at SNAKE (Superconducting Nanoscope for Applied nuclear physics Experiments) facility the ability to target larger structures such as nucleoli was shown 23 .

(iv) Study of non-targeted effects. The possibility to irradiate the cytoplasm, without irradiate the nucleus gives the opportunity to study non-targeted effects, that also include effects on non-irradiated cells that respond to their neighbors irradiated cells 5,24 . Several reports even point to the involvement of mitochondria in the signaling pathways of the bystander response, induced by both nuclear and cytoplasmic irradiations [25][26][27][28] .

(v) Development of approaches for tumor therapy. A series of reports have been addressed to study the effects of specially modified proton microbeams to irradiate cell systems and tissue models. As example, with microbeams is possible to study the dose-rate effects where similar doses can be delivered with different fluences (continuous or pulsed microbeams) [29][30][31] . Similar doses can also differently distributed on cell populations or on human skin models, as recently demonstrated at the SNAKE facility. These studies offer the possibility to analyze the effects of dose distributions in cancerous and healthy tissues 32,33 .

Figure 3. Microbeam irradiation applications. Using a microbeam it is possible to study the radiation-induced biological responses at the single cell level after targeted irradiations in the nucleus, in the cytoplasm or in subcellular compartments. In addition, thanks to the focalization and scanning system, is possible to study the effects of two dose distributions on a cell monolayer.

The major limitation of these devices is their restricted availability within the scientific community. This is due to the size requirement of these facilities and the costs of building and maintaining them in operation. Additionally, each charged particle microbeam facility is limited in the particle spectra and energies provided, and numerous technical problems had to be solved to perform radiobiological experiments 9 .

Besides these disadvantages, with respect to classical irradiation systems (random sources, X-Rays, -Rays and UV irradiations), microbeam target irradiation stands out for different characteristics:

-the spatial resolution, that determines the precision of the sample targeting -the temporal resolution, which allows for fast samples irradiation within few seconds -the dose resolution, that permits to deliver a controlled and precise number of particles at the cellular level.

A microbeam line with these characteristics is installed at the AIFIRA platform (Applications

Interdisciplinaires des Faisceaux d'Ions en Région Aquitaine) situated in the CENBG (Centre d'Études

Nucléaires de Bordeaux-Gradignan). This equipment, described in Bourret et al. 20 , represents a major resource to study the biological responses to protons and -particles in both single cell (at subcellular level, nucleus and cytoplasm) and cell population.

Briefly, the beam line is constituted by an accelerator (Singletron TM , High Voltage Engineering Europa, The Netherlands) that delivers protons and Helium ions with energies up to 3 MeV. To target single living cells, the beam is strongly collimated to reduce the particles flux to a few thousand ions per second on target and focused using a triplet of magnetic quadrupoles to achieve a sub-micron resolution under vacuum. The ion beam is extracted in air through a 200 nm thick Si Recently, to complete the imaging capabilities present on the beam line, we installed a commercially available laser diode (Roper Scientific, iLas2 TM ) coupled to a galvo-scanned mirror on our microscope.

This set-up allows performing fluorescence recovery after photobleaching (FRAP) measurements in combination with charged particle irradiations (Figure 4). Some preliminary results obtained using this system are discussed in the Experimental results section. 34 show that 3 MeV -particles pass through 18 µm and 3 MeV protons pass through 148 µm of water before to lose all their energy. The inset shows the position and thickness of polypropylene foil (grey square) and of cell (green square) with respect to the particle deposited energy.

It is important to mention that biological responses not only depend on the localization of irradiation, but also on the type of particle and on the deposited energy. Monte Carlo simulations allows the quantification of dose distributions at the microscopic level in well-defined conditions (beam focalization, extraction window, and cellular phantom). Modeling radiation-induced damages is today an active and intense field of research and more and more refined simulations are currently developed 35 . The Monte Carlo Geant4 toolkit has been widely adopted in the radiobiology community since it can reproduce the stochastic nature of interactions between elementary particles and matter 36 . Numerous specialized Monte Carlo codes, usually called "track structure codes", have been developed for microdosimetry simulations and are able to simulate precisely particle-matter interactions, with (i) the "physics" stage, (ii) the "physico-chemical" and (iii) the "chemical" stages allowing in particular the simulation of oxidative radical species. To better understand the biological effects of IR at the cellular scale, it is crucial to model the radiation energy deposit in cells and cellular responses.

The iRIBio (ionizing Radiation interactions and Biology) group has initiated a simulation activity based on the Geant4 simulation toolkit (an open-source and publicly available simulation platform) in order to develop models of track structure caused by IR traversals through living biological specimens and to compare predictions with experimental data 37 . The Geant4 tracking capabilities at the submicrometer scale were first extended and validated by the group, allowing to propose a complete simulation platform for the design of specific micron and sub-micron beam irradiation. These stateof-the-art setups allow for example a precise control of delivered ionizing doses to living organisms.

Geant4 is further extended for the development of high-resolution 3D cellular "phantom" models obtained from confocal microscopy imaging and from ion beam analysis techniques available on the microbeam line facility. These "phantoms" allow high-resolution modeling of cell geometries and a more realistic estimation of deposited doses 37,38 . In this way the total energy distribution is calculated and early biological damage induced by IR at the DNA scale can be estimated. (All simulations presented in this work were carried out with the help and courtesy of Dr. P. Barberet).

Chapter 3 Ionizing radiations induce cluster of DNA lesions

When IR interact with biological material, they cause a sequence of events. Complex events that accompany the absorption of IR can be divided into four consecutive, temporal stages (Figure 6).

During the first or ''physical'' stage, the energy deposition is caused by the incident radiation and secondary electrons are generated. The resulting species are extremely unstable and undergo fast reorganization in the second or ''physicochemical'' stage. These processes produce radical and molecular products of radiolysis that are distributed in a highly non-homogeneous track structure.

The initial (10 -15 s) spatial distribution of reactants is then directly used as the starting point for the so-called third stage of ''non-homogeneous chemistry''. During this stage, the various chemically reactive species diffuse and react with one another or with the environment, until all intra-track reactions are complete (10 -6 s). Finally, in a physiological system, there is a ''biological'' stage in which cells respond to the damage resulting from the products formed in the previous stages. During this stage (10 -3 s and longer), the biological responses affecting the long-term consequences of radiation exposure are induced [39][40][41][42][43][44] . IR produces through direct and indirect effects a variety of DNA lesions, such as single strand breaks (SSBs), double strand breaks (DSBs), abasic sites (either apurinic or apyrimidinic), a variety of base modifications, sugar modifications, and DNA-DNA and DNA-protein cross-links 45,46 . The damage spectrum is influenced by dose, dose-rate and type of radiation exposure. DSBs are the most lethal lesions since, if unrepaired, they can result in cell death and, if misrepaired, they can cause chromosomal translocation: an early step in the etiology of carcinogenesis 47 . Also, complex damages, defined by the proximity of DSB to other lesions such as DSB or SSB, could be induced by the high concentration of ionizing events along the particle track. Indeed, by using Monte Carlo track structure simulations, the increase of DSB complexity as a function of the LET has been shown (Figure 7) [48][49][50][51] . To protect their genome from the constant and severe assault from external and internal DNA damaging agents, cells have evolved elaborate defensive strategies, collectively termed the DNA damage response 52 . The choice of repair complex from the large panel of repair mechanisms depends on the type of damage occurred.

Mispaired DNA bases are replaced with correct bases by mismatch repair (MMR), and small chemical alterations of DNA bases are repaired by base excision repair (BER) through excision of the damaged base. More complex lesions, such as pyrimidine dimers and intra-strand crosslink, are corrected by nucleotide excision repair (NER), through the removal of an oligonucleotide of approximately 30 bp containing the damaged bases [53][START_REF] Okano | Spatial and temporal cellular responses to single-strand breaks in human cells[END_REF][START_REF] Jiricny | The multifaceted mismatch-repair system[END_REF][START_REF] Okano | Cellular responses and repair of single-strand breaks introduced by UV damage endonuclease in mammalian cells[END_REF][START_REF] Friedberg | The role of endogenous and exogenous DNA damage and mutagenesis[END_REF][START_REF] Bauer | The current state of eukaryotic DNA base damage and repair[END_REF] . SSBs are repaired by single-strand break repair (SSBR), whereas DSBs are processed either by non-homologous end joining (NHEJ) or homologous recombination (HR) [START_REF] West | Molecular views of recombination proteins and their control[END_REF][START_REF] Caldecott | Single-strand break repair and genetic disease[END_REF] .

The various DNA repair pathways sometimes compete with each other for processing the same lesion, and each step in a multistep repair pathway creates an intermediate that constitutes another lesion, which may be susceptible to intervention by enzymes from another pathway. There are increasing evidences that the various DNA repair pathways are not separated, but well interlinked 53 .

The DNA damage response (DDR) is a signal transduction pathway that senses DNA damage and replication stress and sets in motion a choreographed response to protect the cell and ameliorate the threat to the organism 52,[START_REF] Schaue | Opportunities and challenges of radiotherapy for treating cancer[END_REF] .

Sometimes the first protein to access the lesion may be a transcription factor or another protein that is not directly involved in DNA repair. Also, the response to damage may require a threshold level of damage so that very low levels of lesions might be overlooked, whereas substantial amounts of damage or particular type of lesions may induce a robust response [START_REF] Hanawalt | Historical perspective on the DNA damage response[END_REF] .

Base Excision and Single Strand Break Repair. The base excision repair is designed to correct oxidized bases, abasic sites and SSBs. The sensory component of the repair system is made up by a large family of DNA glycosylases that continuously scan the genome for base modifications. Once bound to its target, the glycosylase separates the modified base from the sugar-phosphate backbone by enzymatic cleavage, leaving an abasic site [START_REF] Fortini | The base excision repair: mechanisms and its relevance for cancer susceptibility[END_REF][START_REF] Barnes | Repair and genetic consequences of endogenous DNA base damage in mammalian cells[END_REF] . Subsequently, other sensor proteins such as ATR, phosphorylate mediator proteins, which can amplify the DNA damage response by recruiting ATR substrates. The activation of poly(ADP-ribose) polymerase-1 (PARP-1), which binds the strand break site and reorganizes the chromatin, is one of the first event induced to repair SSBs. PARP-1 recruits DNA repair and chromatin modifying complexes at the DNA damage sites. In a PARP-dependent manner, XRCC1 is recruited and promotes SSB repair following DNA end-processing by XRCC1 interacting proteins, such as DNA polymerase , polynucleotide kinase (PNK), and the nuclease APE1 [START_REF] Caldecott | Single-strand break repair and genetic disease[END_REF][START_REF] Campalans | Distinct spatiotemporal patterns and PARP dependence of XRCC1 recruitment to single-strand break and base excision repair[END_REF][START_REF] Polo | Regulation of DNA-damage responses and cell-cycle progression by the chromatin remodelling factor CHD4[END_REF][START_REF] Chou | A chromatin localization screen reveals poly (ADP ribose)-regulated recruitment of the repressive polycomb and NuRD complexes to sites of DNA damage[END_REF] . The reparation finished when ligase 3 (Lig III) ligates the broken DNA back together resolving the SSB with the correct insert. When damage is repaired all proteins dissociate from the SSB [START_REF] El-Khamisy | A requirement for PARP-1 for the assembly or stability of XRCC1 nuclear foci at sites of oxidative DNA damage[END_REF] (Figure 8). The observation of the fast and slow components of DSB repair by DNA fragmentation analysis, has suggested the role played by the chromatin compactness, in different cell types after irradiation to low and high LET radiations 51,[START_REF] Radulescu | Chromatin organization contributes to nonrandomly distributed double-strand breaks after exposure to high-LET radiation[END_REF][START_REF] Elmroth | Influence of chromatin structure on induction of double-strand breaks in mammalian cells irradiated with DNA-incorporated 125I[END_REF] . The DNA is packaged in a three-dimensional structure and chromatin is in a high-compacted state (heterochromatin), except when it is relaxed during active transcription (euchromatin). In response to DNA damage, chromatin undergoes to rapid local and global decondensation, a process that has been proposed to facilitate genome surveillance by enhancing access of DDR proteins to damaged sites [START_REF] Beucher | ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2[END_REF][START_REF] Woodbine | Endogenously induced DNA double strand breaks arise in heterochromatic DNA regions and require ataxia telangiectasia mutated and Artemis for their repair[END_REF][START_REF] Soutoglou | The Emerging role of nuclear architecture in DNA repair and genome maintenance[END_REF] .

The ATM kinase plays a central role in chromatin relaxation, and it is responsible for recognition of DSBs, and the recruitment of repair factors to the break [START_REF] Stiff | ATM and DNA-PK Function Redundantly to Phosphorylate H2AX after Exposure to Ionizing Radiation[END_REF][START_REF] Shanbhag | ATM-Dependent chromatin changes silence transcription in cis to dna double-strand breaks[END_REF] . ATM phosphorylates itself and a specialized histone H2AX on Ser139 (named H2AX) and initiates a cascade of factor assembly [START_REF] Paull | A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage[END_REF] . ATM also phosphorylates MDC1 that, through its fork head-associated (FHA) domain, further propagates H2AX spreading. The formation of extensive H2AX regions is important for sustaining the DNA damage response that is achieved through the recruitment of an intricate network of chromatinmodifying enzymes regulating ubiquitination, sumoylation, acetylation and methylation. It is worth noticing that the ubiquitin ligase RNF8 has emerged as key regulator of this molecular pathway with critical roles in early formation of DNA repair foci complex [START_REF] Bartocci | Put a RING on it: regulation and inhibition of RNF8 and RNF168 RING finger E3 ligases at DNA damage sites[END_REF] . The ubiquitin ligase RNF8 protein belongs to the RING (Really Interesting New Gene) domain containing E3 ubiquitin ligase family, and thus, it is able to attach ubiquitin molecules to target proteins for propagation of molecular signal.

Together with two more ubiquitin ligases, RNF168 and HERC2, RNF8 promotes the recruitment of the BRC1-A complex, a ligase that amplifies the cascade signal 52,[START_REF] Mok | The ubiquitin ligases RNF8 and RNF168 display rapid but distinct dynamics at DNA repair foci in living cells[END_REF][START_REF] Dantuma | Spatiotemporal regulation of posttranslational modifications in the DNA damage response[END_REF] . Faced with a double strand break in the DNA, cells can employ two different pathways for its repair:

Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) (Figure 9). Although NHEJ factors are recruited to DSB more rapidly than HR factors, and NHEJ and HR factors are independently recruited to DSB, there is a period of time when both sets of factors are present at the PART I -Background 47 damage sites [START_REF] Kim | Independent and sequential recruitment of NHEJ and HR factors to DNA damage sites in mammalian cells[END_REF] . This is consistent with the idea that the pathway choice may be regulated by one or more proteins acting in both pathways [START_REF] Shrivastav | Regulation of DNA double-strand break repair pathway choice[END_REF] .

NHEJ is fast and the simplest repair pathway, that is active during the whole cell cycle [START_REF] Rothkamm | Pathways of DNA Double-Strand Break Repair during the Mammalian Cell Cycle[END_REF] . This DSB repair pathway, which binds DNA double strand breaks in juxtaposition and even incompatible ends, is mostly error prone. The NHEJ repair is executed by two core protein complexes: the DNAdependent protein kinase (DNA-PKcs) complex, composed of the Ku70/Ku80 heterodimer (Ku) and catalytic subunit of the DNA-PKcs, and a second complex of ligase IV with its co-factors XRCC4 and XFL (also known as Cernunnos). The mechanism starts with the Ku complex activation that recognizes and binds the DSB ends of the damaged site [START_REF] Mahaney | Repair of ionizing radiation-induced DNA double strand breaks by non-homologous end-joing[END_REF][START_REF] Lieber | The Mechanism of Double-Strand DNA Break Repair by the Nonhomologous DNA End-Joining Pathway[END_REF] ; then the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is recruited and it stabilizes the DSB ends, promoting the phosphorylation of several substrates including p53, Ku complex, DNA Ligase IV/XRCC4, all of which may facilitate end processing reactions [START_REF] Roberts | Ku is a 5'-dRP/AP lyase that excises nucleotide damage near broken ends[END_REF] . End processing involves the removal of damaged or mismatched nucleotides by nucleases, such as PNK and Artemis, and/or synthesis of single strand DNA (ssDNA) by DNA polymerases [START_REF] Mansour | The alternative end-joining pathway for repair of DNA double-strand breaks requires PARP1 but is not dependent upon microhomologies[END_REF] . For heterochromatic DSBs, ATM phosphorylation of Kap1 allows the localized chromatin relaxation, facilitating the repair by NHEJ. This process requires Artemis and mediator proteins in addition to ATM [START_REF] Jeggo | Cellular radiosensitivity: how much better do we understand it?[END_REF] . HR is active during the S-and G2-phase of the cell cycle, when sister chromatids are available. This DSB-repair pathway is regarded as error-free, since HR uses an undamaged homologous sister chromatid as a template. During the first step of HR, the broken ends are resected to generate long stretches of single stranded DNA with 3' ends. The preparation of these single strands is mediated by the MRE11-RAD50-NBS1 (MRN) complex, which detects the lesion and promotes the activation of the key DDR signaling kinase ATM. Then, phosphorylation of H2AX, recruitment of MDC1, and association of 53BP1 and BRCA1 generate ssDNA overhangs around DSB [START_REF] Williams | Mre11-Rad50-Nbs1 is a keystone complex connecting DNA repair machinery, double-strand break signaling, and the chromatin templateThis paper is one of a selection of papers published in this Special Issue, entitled 28th International West Coast Chromatin a[END_REF] . The resulting ssDNA is recognized and coated by replication protein A (RPA) that protects and prevents the formation of higher order structures. At this point, RAD51, RAD52 and RAD54, after scanned the surroundings for a homologous sequence, promote the invasion into the homologous template provided by the sister chromatid [START_REF] Beucher | ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2[END_REF][START_REF] Soutoglou | The Emerging role of nuclear architecture in DNA repair and genome maintenance[END_REF][START_REF] Jeggo | The role of homologous recombination in radiationinduced double-strand break repair[END_REF][START_REF] Pellegrini | Insights into DNA recombination from the structure of a RAD51-BRCA2 complex[END_REF] .

Chapter 4 Ionizing radiation and non-targeted effects

Accumulating evidences have shown that an energy deposition in the nuclear DNA is not only event which can trigger to cellular damage. There is an increasing evidence from a number of laboratories indicating that extra-nuclear target and or extra-cellular events may also play an important role in determining the biological responses to ionizing radiation [START_REF] Hei | Radiation induced non-targeted response: mechanism and potential clinical implications[END_REF][START_REF] Zhou | Artesunate induces apoptosis via a Bak-mediated caspase-independent intrinsic pathway in human lung adenocarcinoma cells[END_REF] . A cellular reaction to radiations could derive from molecules other than DNA, such as lipids, proteins, and cytoplasm. These phenomena are termed as "non-targeted effects" (NTE) and include the responses to radiation exposure of nontargeted molecules or of molecules which have not directly interacted with radiation 25,[START_REF] Waldren | Classical radiation biology dogma, bystander effects and paradigm shifts[END_REF] . These effects include radiation-induced bystander effects, genomic instability, adaptive response, low dose hyperadiosensitivity (HRS) and radiation induction of genes expression/modulation 25 . Consideration of NTE is important because probably there is not a direct correlation between the number of cells exposed to radiation and the number of cells that are at risk of showing effects such as mutation, chromosomal damage or apoptosis 41,[START_REF] Kadhim | Non-targeted effects of ionising radiation-Implications for low dose risk[END_REF] . NTE imply that radiation may affect targets other that directly irradiated cellular nuclear DNA, such as proteins, cell membranes, and sub-cellular compartments 24,[START_REF] Hong | Mechanism of genotoxicity induced by targeted cytoplasmic irradiation[END_REF][START_REF] Wu | Targeted cytoplasmic irradiation with alpha particles induces mutations in mammalian cells[END_REF][START_REF] Hu | Intrachromosomal changes and genomic instability in site-specific microbeamirradiated and bystander human-hamster hybrid cells[END_REF] . Mitochondria, as well as nucleoli, could be interesting targets and the understanding of their role in the cellular response to IR is nowadays a challenge.

Mitochondria may account for up to 30% of the total cell volume and they are the only sites where extra-nuclear DNA resides. Therefore, mitochondria are likely to be a major target of IR together with the cell nucleus 26 . Mitochondria dysfunction can lead to an increased release of reactive oxygen species (ROS), produced during normal oxidative respiration, which have been associated DNA lesions, increased levels of cell death, and cancer [START_REF] Waypa | Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells[END_REF][START_REF] Sedelnikova | Role of oxidatively induced DNA lesions in human pathogenesis[END_REF] . Today, more and more studies aim at studying the effect of mitochondria irradiations. Zhou et al. reported an increase mitochondrial mass 4 hours after X-Rays exposure and, more importantly, they showed a decrease in the mitochondrial membrane potential 100 . The mitochondrial polarization state is directly linked to the mitochondria function. When the membrane potential is maintained, mitochondria are considered polarized and fully functional. A loss of potential across the membrane is accompanied by a variety of cellular responses, such as apoptotic cell death 101,102 . Wu et al. found an increased level of cell mutations after cytoplasmic irradiation using -particle microbeam [START_REF] Wu | Targeted cytoplasmic irradiation with alpha particles induces mutations in mammalian cells[END_REF] . Moreover, several groups have reported an involvement of mitochondria in the signaling pathway involved in both cytoplasm irradiated and

Questions addressed in this work

The knowledge of interaction between IR and living tissues has an important role in many fields, such as health risks associated to low dose exposure from natural or working environment, the appearance of radiation-induced tumors, and the new cancer therapeutic approaches.

Conventionally, the effects of IR have been explained using the "Target Theory". Therefore, deleterious effects of IR, such as mutagenesis and carcinogenesis, can be attributed to the damage of a cellular target, usually identified as nuclear DNA via direct absorption of radiation energy.

Therefore, numerous studies show the involvement of other cellular structure in response to ionizing radiations. The observation of early cellular responses activated to ionizing radiations requires highly controlled irradiation conditions. The AIFIRA charged particle microbeam allows us (i) to control the dose by delivering a precise number of particles, (ii) to irradiate a specific target with a micrometer precision, (iii) to visualize in real time living cells.

In this context, during the PhD my efforts were dedicated to:

 establish stable transfected cell lines expressing GFP-tagged proteins involved in different DNA damage signaling and repair pathways. In addition, stable transfected cell lines expressing GFPtagged proteins involved in the structure of different cellular compartments permit to irradiate other targets than DNA.

 irradiate cells expressing GFP-proteins in highly controlled conditions and to visualize DNA damages induced by one -particle. To achieve this point a thin membrane which detects single -particles without interfering with beam lateral resolution and trajectory was developed.

 correlate protein responses with delivered doses and particles Linear Energy Transfer (LET). The mean recruitment time of proteins to the DNA damages gives the spatio-temporal organization of DNA damage response machinery.  develop a system that allow the measurement of protein dynamics and binding ability in the damaged site. A 488 nm laser able to bleach GFP signal is installed and validated in order to perform FRAP experiments.

 participate in a project conducted by the Munich University where single mitochondria are irradiated with different particles.

Experimental Results

The micro-irradiation beam line installed at AIFIRA routinely functions for micro-irradiation experiments. Micro-irradiation is a promising approach to provide more localized induction of DNA damage in highly controlled conditions. These highly controlled conditions consist in irradiating living cells with a precise number of particles in a precise area. The area can be chosen between nucleus and cytoplasm and successive analysis of cell responses can be performed. The microbeam line need continue technical improvements that I contribute to develop. Figure 10 shows in a simplified manner the main results that I obtained during my PhD. These results are discussed with more details in the following sections and in three manuscripts.

Stable transfections of GFP-tagged proteins for DNA and sub-cellular structures visualization

Since its discovery, the use of GFP-expressing cells is hugely increased to monitor genes expression and proteins localization in living cells 103 . One of my principal efforts was to develop, establish and characterize stable transfected cell lines which enable to visualize, target and study different proteins Second, I defined stable transfected cell lines to visualize specific subcellular compartments, such as the nuclei (GFP-H2B) 108 , nucleoli (GFP-Nop52) 109 and mitochondria (Matrix-roGFP2) [START_REF] Waypa | Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells[END_REF] . GFP-H2B and GFP-Nop52 were used to validate the 488 nm laser installed on the microbeam line end-station with which we are able to perform FRAP experiments. Matrix-roGFP2 triggers a good collaboration with the Bundeswehr University Munich which leads to mitochondria irradiations by ensuring the membrane integrity.

Third, I established stable transfected cells expressing GFP-polyQ peptides (23 and 74 repeats) 110 to test the radiation-induced effects on the proteostasis. Indeed, when exposed to different stressors, polyQ peptides could agglomerate and precipitate in cells and could be detected and quantified in situ and in cellulo.

The constructs containing GFP-tagged proteins have been recovered from different sources which are listed in Table

Validation of a thin membrane for single -particles detection and irradiation of GFP-RNF8 transfected cells

A significant part of the natural background radiation exposure of humans is caused by -particles from the inhalation of radon gas 3 . In addition, -particles are increasingly considered in medical applications, such as targeted radiation therapy, where -emitting radionuclides are specifically localised to deliver a cytotoxic radiation dose to cancerous tissues, while sparing surrounding healthy tissues [111][112][113] . Therefore, potential health effects resulting from -particles exposure continue to be the focus of numerous studies 10,11,[114][115][116][117][118] . Understanding cellular responses to complex DNA damages specifically induced by -particles is of particular importance and requires specific tools that allow

GFP-RNF8 and GFP-XRCC1 proteins show different spatiotemporal kinetics after proton and -particle microirradiations

The AIFIRA microbeam therefore permits highly controlled irradiations with 3 MeV -particles and

55

wavelength, energy, exposure time, and on the setup itself 9 . With respect to laser micro-irradiation systems, charged particle-induced DNA damage is better defined and protein kinetics can be correlated with a measurable deposited energy 19,124 . Charged particle microbeams permit to vary particles and number of delivered particles per nucleus adjusting the deposited energy per cell and allowing the study of both particles LET and dose. Monte Carlo simulations and its extensions Geant4 and the Geant4-DNA 125-127 allow to measure the deposited energy per nucleus and particle track structures. By using the combination of charged particle microbeam and Monte Carlo simulations, it

is possible to answer to challenging questions such as how fast is the recognition of various types of damaged DNA? Which proteins arrive first to the sites of DNA damage? What is the affinity of different repair proteins for clustered DNA lesions? How do distinct proteins recognize clustered DNA lesions? 128 In particular, we studied the recruitment time to radiation-induced DNA damages of GFP-XRCC1 and GFP-RNF8 proteins, known to be involved in the recognition, signaling and repairing of DNA damages, as a function of the deposited energy and the particle LET which give the spatial distribution of ionizations. XRCC1 is an essential scaffold protein required for the coordination of different repair pathways and associated to Base Excision Repair (BER) and SSB Repair (SSBR) pathways [START_REF] Campalans | Distinct spatiotemporal patterns and PARP dependence of XRCC1 recruitment to single-strand break and base excision repair[END_REF]119,129 . RNF8 is an ubiquitin ligase that promotes DSB-associated chromatin ubiquitination, and it interacts directly with MDC1 which accumulates at DSB sites among the first proteins 106,130-132 .

Performing targeted irradiation with increasing number of 3 MeV protons and -particles, for the first time, we showed the evidence that GFP-RNF8 recruitment time is 10 times slower with respect to GFP-XRCC1 recruitment time for the same deposited energy. In addition, the GFP-RNF8 recruitment time is primarily impacted by particle LET with respect to XRCC1 which recruitment time is mainly influenced by the deposited energy. Indeed, the recruitment of RNF8 takes place 4 times at higher speed after -particles (high LET irradiation) than after protons (lower LET). The recruitment time of GFP-XRCC1 does not depend on LET but mainly on the deposited energy. These results are detailed in the Manuscript 2.

PART I -Experimental Results

56

This work opens other questions not only about the investigation of the recruitment time to the DNA damaged sites of other proteins but also on the time lapse image acquisition in case of an instantaneous protein recruitment. The first protein, GFP-H2B, was selected because associated with the chromatin and then, fundamentally immobile 134 . The control software MetaMorph® was used for bleaching selected patterns (for example point, line, circle) with defined location, intensity and timing. GFP-H2B is an ideally target to calibrate the laser system because of its immobility. Figure 12a shows the first measurements of fluorescence intensity after photobleaching experiments of GFP-H2B protein. No recovery of fluorescence is observed during 3 min after bleach and the protein immobile fraction is calculated corresponding to 98%.

The second protein, GFP-Nop52, was selected because it represents a tiny target in the cell nucleus and allowed for the validation of targeting small areas 135 . Preliminary evaluation of the time-lapse images following methods reported in other studies 134,136 permits to obtain FRAP times of t 1/2 = 43 s (N = 18 cells). The immobile fraction of nucleolus expressing GFP-Nop52 calculated is 33% (Figure 12b).

The validation of this system allow us to move toward experiments combining micro-irradiation and photobleaching to study the proteins bond in the site of damage. Nowadays, micro-irradiation and laser diode for photobleaching experiments were available only at the GSI 137 .

Targeted irradiation of mitochondria with two microbeam facilities: the AIFIRA microbeam in Bordeaux and the SNAKE microbeam in Munich

Finally, establishing Mito-roGFP2 cell line, I actively participated in a work realized in collaboration with the Bundeswehr University Munich and in particular with my colleague Dietrich Walsh. The main purpose of his project is to understand the role of cytoplasm and in particular the role of mitochondria in response to ionizing radiations. The prevailing dogma in radiation biology is that a high dose of energy deposited to the nucleus will lead to the destruction of cells [START_REF] Waldren | Classical radiation biology dogma, bystander effects and paradigm shifts[END_REF] . However, the cytoplasm which is the cellular environment in which the majority of processes involved in the maintenance of cellular integrity take place has been rarely been taken into account. Despite cytoplasmic irradiation has been shown to be involved in inducing bystander effects and mutation induction [START_REF] Wu | Targeted cytoplasmic irradiation with alpha particles induces mutations in mammalian cells[END_REF]138 In addition, roGFP protein was created to be a reduction-oxidation-sensitive GFP protein whit two fluorescence excitation maxima at about 400 and 490 nm. In response to changes in redox conditions, roGFP exhibits reciprocal changes in intensity at the two excitation maxima, and its ratiometric characteristics make it sensitive to expression levels 139,140 . These characteristics could be used in the future to measure the mitochondrial redox state in irradiated cells.

Discussion and perspectives

The effects of ionizing radiations on the human body have been linked to the formation of DNA lesions, and have been classified as a risk factor for development of cancer. The "classical dogma" in radiation biology is that a high enough deposited energy in a cell nucleus will lead to the destruction of that cell. Numerous evidences showed that cytoplasm, in addition to DNA, can be also involved in bystander effects and mutations induction.

The observation of early cellular responses at charged particles damaged sites (at both nuclear and cytoplasmic levels) was achieved by using a microbeam set-up, which provides controlled irradiations of single cells with a determined number of charged particles.

The first development here presented demonstrates the ability to deliver single MeV -particles to the cell nuclei with a micrometric precision. Subsequently, the formation of ionizing radiationinduced foci of GFP-RNF8 protein was followed over time. Post-irradiation analysis shows that GFP-RNF8 accumulates at single -particle tracks during the first 30 minutes after irradiation. The intensity and the recruitment time varies from one IRIF to another reflecting most probably the chromatin heterogeneity inside the nucleus.

To investigate the influence of damage density and complexity on recruitment kinetics of GFP-RNF8

and GFP-XRCC1 proteins, we irradiated cells with 3 MeV protons and 3 MeV -particles. Delivering a precise number of particles having a LET well-identified (12 and 148 keV.µm -1 , respectively) provides crucial insight into how DNA is damaged. In our study, we showed that the recruitment kinetics of GFP-RNF8 is dependent on the particles LET, indeed for the same deposited energy, GFP-RNF8 is recruited 5 times faster after -particles (higher LET) than after protons (lower LET). Then, we found that the GFP-XRCC1 recruitment time is dependent on the deposited energy for both -particles and protons and is not dependent on the LET. These different responses may be correlate with the molecular role of these proteins. RNF8 is recruited in a more specific manner to complex DNA damages (faster recruitment after high-LET particles) compare to XRCC1 that is a loading platform for other proteins and does not depend on damage complexity (recruitment faster when the deposited energy is increased independently on the particles LET). These data raise important questions about the mechanisms of DNA signaling and repair machinery and deserve more detailed studies.

As mentioned before, the DNA is not the only target of IR. Mitochondria constituting a large volume of the cytoplasm in all cell types are interesting targets to investigate. In the third presented study, mitochondria have been selected as the target of micro-irradiations. We showed that highly-localized

PART II

In vitro evaluation of radiation sensitivity of sarcoma cell lines derived from patients

PART II 121

PART II

In 1896 X-Rays were used for medical purpose to locate a piece of knife in the backbone of a drunken sailor, who was paralyzed until the fragment was removed by tracking its location. This new technology spread rapidly through Europe and United States, and the field of diagnostic radiology was born. Therapeutically, the surgeon Freund demonstrated the disappearance of a hairy mole following treatment with X-Rays. Medical uses of machine-produced radiations and radionuclides have also developed, and play a significant role in medical diagnosis and treatment. Controlled amounts of radiations in the form of X-Rays have been used for a century as aid in the diagnosis and treatment of diseases in humans and animals. Despite these recognized benefits, radiation has several well-established effects on human health, and cancer is considered the major long-term contributor to health risk. Radiation therapy, with surgery and chemotherapy, is one of the main modality involved in cancer treatments. Radiation therapy is based on the use of high-energy ionizing radiation to kill malignant cells or to control their proliferation. However, several associated side effects such as the damage inflicted to the surrounding healthy tissues and the appearance of the radiation resistance phenomena, are some of the main factors that influence the efficiency of this treatment.

To optimize and adapt the therapeutic benefit of radiation therapy, a lot of efforts are invested to improve the beam ballistics and to understand the mechanisms which cause the radiation resistance phenomena (that could be innate or consecutive to a radiation treatment).

The main current developments in radiation therapy are based on the final aim to increase the radiation-induced effects in the tumor volume, reduce secondary radiation induced-effects in healthy tissues and define sub-group of tumors that could be treated in the same manner. For these purpose, several strategies are investigated such as the use of heavy charged particles, the use of new techniques of irradiation, the addition of radiation sensitizing agents inside the tumor volume, and the analysis of the tumor genetics characteristics to develop new personalized therapies.

A better understanding of in vitro interaction of IR with biological systems is mandatory and must combine a realistic dosimetry in cells exposed to different IR, the ability to irradiate using different modalities, and the knowledge of the genetic characteristics that influence biological responses.

The aim of the second part of this project is to further develop our capabilities to evaluate the biological responses of cell lines exposed to different radiations. To achieve these objectives we developed irradiation protocols, based on realistic dosimetry obtained with Monte Carlo simulations, to evaluate the radiation sensitivity/resistance of two patient's derived sarcoma cell lines whose genetics is characterized. We defined the biological endpoints in order to compare the biological PART II 122 effects induced by several doses of electrons (conventional particles used in radiation therapy), protons, and to compare two spatial energy distributions (broad beam versus focused microbeam).

The main body of this second part is divided in three parts: Background that deals with the overview of sarcomas, the radiation therapy techniques, the intrinsic mechanisms of cellular radiation sensitivity, and the use of microbeam in radiation biological studies; Experimental results that summarizes the results obtained; and Discussion and Perspectives that details the impact and the further possibilities opened by this work.

PART II -Background 123

Background

Today, one the major health problems for humanity is cancer. The World Health Organization estimated 14.1 million new cases of cancer occurred worldwide, and 8.2 million cancer-related deaths in 2012 1 . The universal demand in oncology is early cancer diagnosis, and to remove cancer or precancerous growths. The armamentarium of cancer treatments includes surgery, radiotherapy and chemotherapy. Among them, treating cancer with a high-energy photon beam is a well-established therapeutic technique. Indeed, in developed countries, over 50% of cancer patients will undergo traditional X-Rays radiation therapy during their disease. Despite the benefits in destroying cancer IR therapy has also side effects as the damage can occur in healthy tissues surrounding the tumor. The process of ionization in living material necessarily changes atoms and molecules, at least transiently, and may thus damage cells. If cellular damage occurs and is not adequately repaired, the cell may not survive, reproduce or perform its normal function. Indeed, DNA damage from radiation is cumulative and can result in carcinogenesis or other adverse cellular events, months or years after exposure 2 .

Further studies are needed because the carcinogenic potential of low and high dose of IR is not completely understood, and this uncertainty is much higher for cancer induced by charged particles 3 .

PART II -Background 124 Chapter 1

Sarcomas: particular types of cancer

Cancer is a group of diseases caused by normal cells changing so that they grow in an uncontrolled way. It is possible to discriminate more than 100 different types of cancer. Sarcomas represent a heterogeneous group of rare tumors, accounting for approximately 1% of adult cancers with an estimated incidence averaging 5/100 000/year in Europe, and for nearly 21% of all pediatric solid malignant cancers 4,5 . Currently, more than 50 histological subtypes have been identified 6 . They are derived from mesenchymal tissue including bones (osteosarcoma), muscles, cartilages and other connective tissues (soft tissue sarcomas).

In summarizing the current knowledge on histology, clinical features, and specific molecular events that define tumor subtype, we can recognize four groups of sarcomas. Sarcomas of the first group have nonpleomorphic histology and known pathognomonic molecular events (e.g. GIST with activating KIT mutations). In the second group, we find sarcomas which affect younger patients and generally have nonpleomorphic histology and karyotypes of limited complexity, however pathognomonic molecular events have yet to be identified. The third group includes sarcomas which occur in the adult population, and show pleomorphic histology, but on a background of complex changes, it includes consistently identified molecular events (e.g. dedifferentiated liposarcoma with CDK4/MDM2 amplifications). Finally, the fourth group, the most common in adult population, have complex karyotypes, pleomorphic histology, and lack of identifiable molecular events 7 .

The sarcoma etiology is unknown, but external radiation therapy is a well-established risk factor for soft tissue sarcoma 5,8 . Indeed, sarcomas are a type of cancer that can be radiation-induced and they present a higher resistance to conventional radiation therapy. Studies devoted to deeper understand the susceptibility of radiation therapy-related cancers could be important in identifying high-risk individuals and may lead to clinical benefits if radiation exposure could be reduced in these patients 9 .

When radiations are the etiologic factor, post-irradiation sarcomas are also called radiation-induced sarcoma or radiation-associated sarcomas. Evidences are given, for instance, by the mutation of TP53 gene in 88% of post-irradiation sarcomas with respect to the 20% frequency of TP53 mutations in sporadic soft-tissue sarcomas 10 .

PART II -Background 125 Despite the sarcoma variety, the clinical behavior of most soft tissue sarcomas correlates with anatomic location, histological grade, and tumor size 11 . However, the variability in patients' clinical outcome underestimates the heterogeneity of the biological aggressiveness of these tumors, and the therapeutic management remains one of the most difficult tasks for oncologists. Contemporary management of sarcomas often includes a multidisciplinary approach involving a combination of surgery, radiation therapy, and chemotherapy specific for tumor type, histological grade, and stage of disease.

Surgery is the standard primary approach for all patients with adult-type, localized sarcoma. It must be performed by a surgeon specifically trained in the treatment of this disease. Chemotherapy is preferably given before surgery, in order to assess tumor response and thus modulate the length of treatment, and it is an option in non-resectable tumors. In this case, doxorubicin chemotherapy is the single most active broadly applicable anti-sarcoma chemotherapeutic but only has an approximate 30% overall response rate with additional breakthrough tumor progression and recurrence after initial chemo-responsiveness and further problematic features in sarcomas management. There clearly still remains a desperate need for new systemic therapies.

Radiation therapy is added for those patients with residual tumors following surgery, in the case of low-grade or superficial sarcomas, and or as neoadjuvant therapy. The optimal mode (external beam or brachytherapy), and timing (preoperative, intraoperative, or postoperative) of radiation therapy have yet to be defined 12 . But, in generally it should be administered postoperatively, with the best technique available, at a dose of 50-60 Gy, with fractions of 1.8-2 Gy, depending on presentation and quality of surgery 4 . Sarcoma radio-responsiveness is very variable and depends on the histology, etiology, and kind of radiation used for treatment 13 . Bone and soft-tissue tumors including several histological subtypes are generally very radiation resistant 14 . As these tumors are highly radiation resistant, sarcomas are ideal candidates for therapy with high-LET radiations. Nowadays, the proton beam therapy is employed for sarcomas treatment. It reduces healthy tissue radiation dose by up to 50-60% with respect to photon therapy and is increasingly the preferred treatment modality for pediatric sarcoma patients, where a higher risk for late radiation-associated malignancy is expected 15,16 .

PART II -Background 126 Chapter 2

Radiation biology for studying in vitro radiation resistance and radiation sensitivity

Cancer is treated with both invasive and non-invasive treatment modalities, such as surgery, radiation therapy, chemotherapy, as well as other therapeutic modalities (e.g. immunotherapy, hormone therapy, photodynamic therapy), and a combination of these. Today, radiation therapy is one of the most common non-invasive way to treat cancer, and one million of patients each year undergo radiation therapy, which is a clinical modality dealing with the use of IR 17 . The fundamental challenge in radiation therapy is to deliver a precisely measured dose to a well-defined tumor volume with as minimal damage as possible to surrounding healthy tissue 18 . Considerable efforts are being devoted to improve radiation therapy. There are different ways in which such an improvement in radiation therapy might be obtained: (i) by focusing the delivered dose on the tumor; (ii) by raising the efficiency in cell killing; and (iii) by minimizing the energy deposition in healthy tissues.

Today, most radiation treatments are based on photon irradiations, but there is an increasing interest in proton and carbon ions radiation therapies because these particles have an impact on the above mentioned developments.

Radiobiological research includes investigation of the factors, which cause the different severity of damages induced by the same dose of IR and offers a fundamental basis in the understanding of how radiation therapy works. This knowledge is important in both planning radiation therapy and radiation protection.

The same dose of IR causes differential effects determined by the genetic and functional characteristics of the "targeted" type of cells, tissues, organs or organisms. Radiation therapy simplified this problem where the total dose is adjusted to the most sensitive individuals so that not more than 20% of the patients will have an adverse healthy tissue effects. For the same therapy treatment approximately 5% could experience some severe or even fatal effects from standard radiation therapy. As a consequence, the majority of the patients receive a suboptimal dose, as they would have tolerated a higher total dose and gained a better tumor control, without getting severe side effects 19,20 . Indeed, depending on the time of occurrence, an organ or tissue expresses response PART II -Background 127 to radiation damage either as an acute effect or as a late (chronic) reaction. Acute reaction induces endothelial cell dysfunction, which is characterized by increased permeability, detachment from the underlying basement membrane and apoptosis. Endothelial cell dysfunction and apoptosis contribute to post-irradiation inflammation and fibrosis. Early biological events are usually transient and normally resolved within 3 months of completing treatment. Late reactions occur after 3 months up to many years and may be caused by the absorption of radiation directly in the target tissue or consequential to damage in overlying tissue such as mucosa or epidermis. Furthermore, studies imply that patients with no acute tissue reactions to therapy have a higher incidence of local recurrence, indicating that all cancerous cells were not eradicated. And more, early and late responses of normal tissues to irradiation are dose-limiting factors in radiotherapy, affecting therapeutic efficacy as well as the quality of life of patients and these events also result in secondary malignancy 21,22 . Thus, if tumor therapy could be individualized based on radiation sensitivity, more patients would be cured and the most severe adverse reactions could be avoided.

Radiation sensitivity is the relative susceptibility of cells, tissues, organs, or organisms to the harmful effect of IR. It was suggested in one study by Safwat et al. that 80-90% of variability in late normal tissue radiation sensitivity was due to patient-related factors 23 . These uncertainties are considered as a problem in radiation protection practices where the dose limit applied aims to provide a robust protection for the whole population.

That variability in cellular radiation sensitivity may result from differences in efficiency and/or accuracy of DNA repair. DSB is the lesion most likely to be the cause of the lethal effects on cellular level. If left unrepaired, DSBs can result in permanent cell cycle arrest, induction of apoptosis, or mitotic cell death caused by loss of genomic material; if repaired incorrectly, they can lead to carcinogenesis through translocation, inversions, or deletions 24,25 . Also micronuclei and chromosomal rearrangements can result from non-repaired or misrepaired DNA damage induced by IR. Depending on the damage extent and the ability of damaged cell to cope with, damage may lead to defect/loss of cellular function, senescence, cessation of proliferation, cellular death, and mutagenesis, which may cause genomic instability 26,27 . In vitro assays normally measure only one particular cell function and multiple tests have been used and described to assess individual radiation sensitivity, which have a practical importance in the radiotherapy field 28 . The existing experimental end-points allow measurements of (i) induction of cellular death (loss of metabolic activity); (ii) apoptosis (programmed cell death); (iii) clonogenic survival; (iv) DNA repair capacity (pulsed filed electrophoresis, comet assay, measurement of H2AX phosphorylation); (v) cytogenetic effects, micronuclei, chromosomal aberrations and chromosomal instability.

A general conclusion based on these studies is that individual sensitivity to radiation is likely to be multifactorial in its origin and several biomarkers/bioassays will be needed to gain the predictability necessary for implementation in the clinic.

Chapter 3 Microbeam for radiation biological approaches

The immense progress in the advancement of cellular and molecular biology techniques has raised the possibility to better understand the molecular consequences of particle irradiation in cells and

(artificial) tissues. In vitro studies and the application of physics accelerators for radiation biological studies are necessary to improve the understanding of IR exposure. The long term aim of this thesis was to develop tools to assess the individual radiation sensitivity of sarcoma patient's cell lines and to better understand the mechanisms behind the radiation sensitivity and radiation effects after IR exposures.

Micro-irradiation devices are able to control the target area and to apply an established number of ions in such determined area. These characteristics permit different applications [29][30][31][32][33] , such as (i) the study of low-doses, where each targeted cells can be irradiated with a precise number of ions 34,35 ; (ii) the analysis of molecular process at sites of DNA damage (this applications is the object of the first part of this PhD thesis); and (iii) study new approaches for tumor therapy extending studies from cellculture models to more complex tissue models and in vivo systems 30,36 .

A series of reports have been addressed to evaluate new approaches for tumor therapy by irradiating cell systems and tissue models with a specially modified proton microbeam. Indeed microbeam offers the possibility to study the influence of the dose-rate on RBE. Schmid and colleagues administrated on a 3D human reconstructed skin model the same dose of 20 MeV protons either as a continuous or nanosecond pulsed proton beam. They analyzed several endpoints and concluded that there were no significant differences in RBE of continuous or pulsed applied protons, for the endpoints that were investigated, including clonogenic cell survival [37][38][39] .

Another example can be illustrated by the study of effects induced during microbeam radiation therapy (MRT). In general, the MRT is a novel radiotherapy method invented by Slatkin and coworkers in 1992 40 and it is based on a spatial fractionation of synchrotron-generated X-Ray beams where collimated, planar, quasi-parallel microbeams are delivered to the tumor. This technic leads to the radiation resistance of the skin and the radiation sensitization of the tumor tissue induced by

mechanisms not yet completely understood 41,42 . Microbeam offers the opportunity to be focused to micrometer scale or defocused in order to study these mechanisms and at the SNAKE facility PART II -Background 130 different studies started with the scope of elucidating the dosimetry 43 and biological effects of different proton dose distributions 44 .

PART II -Questions addressed in this work 131

Questions addressed in this work

The development of biomedical applications of ion beams is one objective of particular interest of the training program in which I participated during my PhD (SPRITE project). Transfer of knowledge from fundamental research to clinical applications is the final aim of this program. One fundamental issue that can be faced using standard research protocols is the evaluation in vitro of the radiation sensitivity or resistance in different biological samples. To achieve this purpose it is necessary to understand the biological mechanisms induced by electrons (commonly used in conventional radiation therapy), protons and different spatial energy distributions on patient's cell lines.

Developing protocols able to assess in vitro mechanisms that lead to radiation sensitivity or resistance of cancerous cells requires:

 to define biological samples which permit a correlation of radiation sensitivity of patient's derived sarcoma cell lines whit their genetic characteristics. Two sarcoma cell lines were selected for their different genetics: a known amplicon based genetics and a complex genetics.

 the ability to compare different particles effects. Cells were irradiated with a medical linear accelerator (9 MeV electrons) and with the AIFIRA microbeam at CENBG (3 MeV protons). To ensure that the mean delivered dose by these two irradiation systems was equivalent, we performed the dosimetry at the cellular scale using Monte Carlo techniques.

 to evaluate the biological effects of two deposited energy distributions. For that, cells were irradiated with the same dose of protons but the energy at the cellular scale was deposited with different patterns. Indeed, the microbeam line allows us to focus the beam to the minimum beam spot size (2 µm) (microbeam) or to defocus it to obtain a homogeneous proton distribution (broad beam).

 to develop and validate protocols for deciphering in vitro radiation sensitivity. Several biological endpoints such as cell proliferation, surviving fraction, immunodetection of DNA damage signaling proteins (such as the phosphorylated proteins ATM and H2AX) were analyzed. For evaluating these end-points we developed two ImageJ routines: one is conceived for quantifying the radiation-induced foci per nucleus and the other one for counting the number of cells constituting a colony and the number of colonies after irradiation.

Experimental results

To achieve the defined objectives, it is necessary to develop a multidisciplinary approach where biology, genetics, physics and medical physics are combined for a better understanding of radiationinduced effects. So that, I integrated diverse teams working on these different fields. dosimetry was needed to develop irradiation protocols appropriate to irradiate the selected biological samples with controlled doses of electrons or photons; (iii) Taking advantage of the expertise of the iRIBio team, I adapted all protocols developed for a medical facility to proton microbeam irradiations in order to compare the responses of sarcoma cells exposed to these ionizing radiations. Protons are, in effect, particles used today in hadron therapy and analyze their effects on cancerous cells is essential. Despite the fact that the energy of our proton beam is not in the medical range (AIFIRA is able to deliver 3 MeV protons), the advantage of microbeam lies in its ability to control the dose, the time and the space of irradiation allowing to elucidate the impact of two energy distributions at the cellular level (proton broad beam versus proton microbeam). As mentioned in Background section, the use of microbeams and different spatial energy distributions are increasing for medical applications for which more knowledge is worth considering. The study of biological responses was matched with the micro-dosimetry at the cellular scale that displays the total deposited energy per nucleus and the deposited energy per spot.

PART II -Experimental results

133

The objective of this work was to combines all expertise above mentioned and to develop a methodology to assess radiation sensitivity of patient's derived cells. The methodology developed is schematized in Figure 13. To develop this methodology a multi-step work was necessary. The first approach tested in order to assess the radiation sensitivity of selected cells was the cytogenetic approach. Indeed, cytogenetic approaches are one of the ways that have enormously contributed to the understanding of radiation sensitivity. It enables to (i) precisely label the chromosomal location of any gene using different colored dots and (ii) identify cells that have lost or gained a specific chromosome, undergone a translocation event involving a specific set of chromosomes. To identify specific translocations radiation-induced, I performed a cytogenetic method called in multicolor fluorescence in situ hybridization (mFISH). This technique utilizes 24-color probes sets to label each human chromosome with a distinct color. Because each chromosome has its own color, chromosomal translocation are easily detected allowing researchers to detect small chromosomal rearrangements or chromosomal aberrations 45 . To estimate the chromosomal rearrangements, I started to perform mFISH assays in one cell type exposed to electron irradiations following the protocol described by Geigl et al. 46 . Figure 14 shows three karyotypes after exposure to 0, 2 and 6 Gy of electrons. From the performed analysis on these samples and from previous performed analysis on the same cell type after -particle irradiations (Vincent Atallah's Master thesis), we confirmed that the high number of chromosome rearrangements already present in control cells makes impossible the identification of rearrangements due to irradiations. Then, another technique to perform genome-wide scans to identify chromosomal regions associated with loss or gain of genetic information is called microarray-based comparative genome hybridization (CGH). This method uses arrays containing thousands of base-pair fragments of the human genome adhered to a microchip. Each individual DNA fragment, which is located in a specific position on the chip, corresponds to a known DNA sequence that has been mapped to a specific chromosomal region. The same color-coded probes (green for the control group, and red for the experimental group) are then used in hybridization experiments with the CGH microarray platform, which can be scanned using an automated approach. Unaltered chromosome regions show equal binding of the green and red probes and a resulting orange/yellow color, whereas amplified and deleted chromosomal regions in the experimental group appear red and green, respectively 47 . By using this techniques, it is possible determine the chromosomal regions and genes that are amplified or missed. In this study and in previous studies performed at passages 2 and 5 after irradiation (Vincent Atallah's Master thesis) significant quantitative changes of the genomic profile with respect to control cells are not showed.

These two cytogenetic tests, mFISH and CGH, are not conclusive in our case due to the high complex genetic of samples which do not permit to correlate genetic/genomic alterations with radiationinduced effects.

For these reasons, I developed a multi-parametric methodology based on the more common assays aimed to evaluate the in vitro radiation sensitivity 48 . This methodology is well-detailed in the Manuscript 4 and evaluates three biological endpoints, such as quantification and persistence over time of DNA damage, proliferation and surviving fraction. These end points are analyzed for two types of ionizing radiations (electrons versus protons), two different energy distributions at the cellular scale (focused proton beam versus proton broad beam) and for two different sarcoma cell lines. This developed multi-parametric analysis required (i) the quantification over time of radiationinduced foci in a consistent number of cells, (ii) the quantification of colonies present in a cell dish and (ii) the quantification of cells belonging to each colony. To figure out with these difficulties, I validated two automatized routines ImageJ which allow the quantification of foci radiation-induced, the counting of colonies and the counting of cells in each colony by reducing the experimenter errors.

Using this approach, I observed that (i) radiation-induced effects and biological responses correlate with the delivered absorbed dose; (ii) protons induce more deleterious effects compared to electrons, independently from the dose distribution at the cellular scale; and (iii) one cell line is more sensitive than the other one for the endpoints analyzed. At first sight, this different radiation sensitivity can be associated to the genetics of samples; indeed, the higher radiation resistance is observed for the cell line with a complex genomic profile.

Discussion and Perspectives

Despite the ability to build machine that deliver radiation with ever great accuracy and conformability progress, radiotherapy will continue to fall short of its promise. The next leap in progress will require radiation oncologists to immerse themselves in the biological basis of cancer and its response to radiation. The hallmarks of cancer represent potential routes to clinically useful biomarkers and targeted agents that will allow flexibility and undisputed therapeutic impact of radiation to find its full expression. No general biomarker of cellular radiation sensitivity has been identified yet. One probable reason for this is that the genetics behind radiation sensitivity is complex. It is also probable that the biology behind radiation sensitivity is diverse and caused by many different factors. A combination of biomarkers will most certainly improve the diagnostic and treatment power. However, many factors need to be carefully considered to achieve an individualized treatment for patients, such as acute radiation sensitivity, late radiation sensitivity, the risk of secondary cancers and the risk for local recurrences 49,50 . Developing a 'system biology' approach to the radiation response, in which multidisciplinary expertise is used to analyze vast data sets, may identify the molecular targets that would probably yield tangible clinical benefits 51 . In this work, we developed protocols to evaluate two cell lines radiation sensitivity which genetics is known and seems to correlate with biological responses.

Indeed, IB115 cells (cell line with a known amplicon-based genetics) show lower proliferation, higher number and lower rate of disappearance of DNA damages, revealing a higher radiation sensitivity with respect to IB106 (cell line with complex genetics). In addition, we show a considerably higher efficacy of protons in comparison to electrons on these sarcoma cells. More difficult was to define if differences occurred after proton broad beam and proton microbeam for the endpoints analyzed.

The main difficult of data analysis in this work is created by the use of patient's cell lines whose genomic profiles are complex. This complexity could be derived from the original biopsy and/or from the different nature of the cell lines analyzed. For example, IB106 cell line is constituted from cancerous and peritumoral cells and IB115 cell line shows the presence of more clones with similar but not identical genomic profiles. These particular characteristics could explain the impossibility to highlight specific radiation-induced chromosome rearrangements or genomic profile alterations.

A next step that could be considered to better understand the radiation-induced effects is the study of transcriptomics, also referred as expression profiling, which examines the expression level of mRNAs in a given cell population, often using high-throughput techniques based on DNA microarray PART II -Discussion and Perspectives 137 technology such as RNA-Seq 52,53 . The expression profile can reveal specific pathways radiationinduced.

These developed protocols can be improved by the addition of other end points but lay the groundwork for analyzing the radiation sensitivity of other patient's cell lines to verify first difference in response to ionizing radiations. In addition, the development of these well-defined protocols, allowed us to begin parallel works for the purpose of evaluating the effects of metal and metal oxide nanoparticles which use is a new increasing interesting approach in the battle against cancer. Metal and metal oxide nanoparticles have been proposed as potential contrast agents and represent an important improvement of the therapeutic ratio by increasing the local control (increasing dose to the target), or by decreasing the normal tissue complications. The amplification of radiation effects induced by the presence of intra-cellular high-Z nanoparticles are explained in terms of early stage physical processes that take place in the cells in the surrounding of nanoparticles. Nevertheless, the relation between biological effects and the radiation dose is not fully understood at present. These new approaches need to develop calculation methods with experimental validation to precisely predict dose-effect correlation in the context of IR and nanoparticles interaction in cancerous cell lines. In addition, is necessary to determine the physicochemical and bio-molecular mechanism induced in cellulo by IR and metal nanoparticles interaction. Tests of feasibility using different nanoparticles are in progress and are the objects of future studies. Strategy to optimize radiation therapy relies on increasing the dose ratio between the tumor and the surrounding tissues to kill cancerous cells while minimizing the risk of secondary radiation-induced effects in healthy cells. Thus, the use of radiation enhancement with metal oxide nanoparticles (NPs) is a promising strategy to both improve cancer treatment and reduce associated radiation side effects. Increased radiation effects induced by the presence of intracellular high atomic number (Z)

NPs is due to early stage physical processes that take place in cells around NPs. Nevertheless, the relation between the biological effects and the delivered dose is not fully understood at present. In addition, nanomaterials are used in diverse fields including food, cosmetic, and medical industries.

However, the risks for their adverse health effects have not been clearly established. A better understanding of the in vitro interactions of NPs with biological systems is mandatory and requires a multisciplinary approach covering chemical, physical, and biological aspects.

The AIFIRA platform at the CENBG allows not only the selective irradiation of a target with controlled doses of ionizing radiations (microbeam) but also allows in vitro quantification and localization of subcellular chemical elements at the single cell level (microprobe). This equipment represents a major resource to localize and quantify nanoparticles in both single cells and cell population. In particular, the iRIBio team has gained long-standing experience about titanium dioxide nanoparticles (TiO 2 NPs). These NPs are widely used for a wide variety of applications and are produced in diverse shapes, but it remains largely unknown how their modifications may alter bioavailability, biological effects and in vitro and in vivo toxicity.

There is a real need for the development of analytical methods able to in situ detect and quantify NPs, whatever their size, nature, and surface reactivity and whether they are native or functionalized. For this purpose, diverse TiO 2 NPs are synthesized, an approach based on correlative microscopies (fluorescence microscopy and ion beam analysis) is developed, and their effects on the cellular homeostasis responses are investigated.

This third part of this PhD thesis is divided in three sections: Background, which firstly describes the use of nanoparticles in nanomedicine with a particular regard to TiO 2 NPs and secondly the microprobe line of CENBG which allows the detection of NPs in biological systems; Experimental Results are summarized in two articles which present the protocols employed and NPs exposure consequences in cells. Finally, Discussion and Perspectives illustrate the impact of these researches and the future directions associated to these works.

Background

Chapter 1

Nanoparticles in nanomedicine

The use of nanoparticles for biomedical applications is prevalent in the literature and continues to be a rapidly growing research field, with heavy emphasis on imaging and drug delivery. Nanomaterials and nanoparticles (NPs) are generally defined as any particulate material for which at least one dimension lies in the range of 1-100 nm 1 . NPs can exist in various shapes and sizes. The various chemical process that guide the synthesis of materials at the nanometer scale can be defined as nanochemistry, which play a critical role in tailoring the physical and chemical proprieties of NPs. For their unique mechanical, thermal, electrical, magnetic and optical properties, NPs are suitable for a wide range of applications, ranging from electronics, to energy storage, to communications, to biology and to medicine 2 .

Nanomedicine is the application of NPs in medicine and NPs intended for medical use have drawn inspiration from the various 'natural' nanoparticles discovered in the body. These include various nanosized vesicles, lipids, proteins, and complex biomacromolecules that regulate the natural functioning in the body, and may act as carries of active molecules. Alongside, an increasing awareness about novel medical applications of smaller, inorganic-based NPs, processing unique properties at the nanoscale, has led to a burst of research activities in the development of 'nanoprobes' for diagnostic medicine and agents for novel, externally activated therapies 3,4 .

Nanocarries are the most common application because they have several key advantages over conventional molecular agents in medicine. They enable stable aqueous dispersions of active, but poorly water soluble molecular agents, for delivery in the biological medium. Their composition, size, shape and surface proprieties can protect the encapsulated agents from degradation by various endogenous defense mechanisms. Control of their structure allows them to be targeted to not only specifics organs/tissues in the body, but even with cellular and subcellular specificity 3,5,6 .

NPs are also commonly used to perform biomedical imaging. A nanoparticle platform provides optical, magnetic, and radioactive functionalities to enhance contrast in various imaging methods for early detection, screening, diagnosis, and image-guided treatment of life-threatening diseases and cancer 3,5 .

Nanoparticles in radiation therapy. Recent advances in nanochemistry have paved the way for new strategies for the development of efficient sensitizers to enhance the biological effects of Xirradiations. It is known that X-Rays and photons deposit most of their energy through secondary electrons that are generated by photoelectric or Compton effects. In principle, loading the tumor with high-Z materials could result in a greater photoelectric absorption or Compton effect, when keV or MeV energy photons are delivered, respectively, within the tumor itself and in the surrounding tissues, and thereby enhance the dose delivered to a tumor during radiation therapy 7,8 . The nanoparticles enhanced-dose effect need to be estimated via dosimetry systems, such as Monte Carlo simulations and electron spin resonance 9,10 . In pioneering studies, several groups demonstrated that high atomic number materials, such as Iodine, have radiosensitizing effects on cultured cells 11,12 .

Then, several studies -including in vitro and in vivo experiments -investigated gold nanoparticles (AuNPs) 8,13,14 , as well as hafnium (HfNPs) 15,16 and titanium (TiNPs) 17 nanoparticles effects, to improve the radiation therapy effectiveness. Dose enhancement obtained by combining ionizing radiations and high-Z NPs has been simulated and showed by numerous in silico and in vitro studies 9,18,19 . These studies concluded that the increased density of ionization is significantly localized and occurred in short distances from the nanoparticles [7][8][9][10]19,16,20 . So an important factor, affecting the sensitizing proprieties, is the localization of nanoparticles, because the energy deposition occurs very close to the irradiated nanoparticles. It has generally been believed that localization in the nucleus gives a higher sensitization, however different studies show a localization of the nanoparticles only in the cytoplasm not in the cell nucleus [21][22][23] . Although nanoparticles may not be incorporated into cell nuclei, they could contribute to the cell killing by attacking cytoplasmic targets. Quantifying and localizing the amount of nanoparticles at the single cell level is a challenge that is being pursued in various laboratories worldwide.

While the promises of nanomaterials in medicine are numerous, it is also worthwhile to consider possible risks associated with their interactions with biological systems. Ideally, once delivered in the body for a particular medical intent, NPs should perform their desired function and exit from body without causing any deleterious effect. In reality, a number of investigations, ranging from simple in vitro analyses to clinical observations, have revealed that many NPs exhibit minor to major hazardous/toxicological effects 24 . Thus, it is essential to comprehensively analyze various nanoparticles from their toxicological perspective.

The team iRIBio, in collaboration with the chemist Marie-Hélène Delville, achieved a strong expertise in designing, synthetizing and characterizing TiO 2 NPs of different shapes and sizes. Using ion beam techniques, the group developed a methodology to obtain an accurate quantification of exogenous and endogenous chemical element distribution in single cells.

TiO 2 Nanoparticles. The high photocatalytic and super-hydrophilic properties of titanium dioxide nanoparticles (TiO 2 NPs) have made them popular for a wide variety of applications. Titanium dioxide pigment (TiO 2 ) is a white powder with high opacity, brilliant whiteness, excellent covering power, and resistance to color change 25 . These properties have made it a valuable pigment for a broad range of applications in white paints, household products, plastic goods, medications (pills), foods, orthopedic implants, paper and toothpaste. A very common application of TiO 2 NPs is as an additive in sunscreen cosmetics as UV-attenuating filter agents. In recent years, many studies have focused on the biomedical applications of TiO 2 NPs in areas such as cancer therapy, drug delivery systems, cell imaging, genetic engineering, biosensors, and biomedical experiments 26,17,27 .

TiO 2 is a natural mineral found primarily in rutile, anatase, and brookite forms. TiO 2 was previously classified as biologically inert but after such widespread use, its potential to penetrate skin (dermal penetration), gastrointestinal tract (ingestion), respiratory tract (inhalation) and blood circulation (injection), and its pathogenic role was re-examined.

TiO 2 NPs are produced in "spherical" shape (such as AEROXIDE P25) but could be engineered in terms of shapes and sizes. The reduction of the particle size leads to higher specific surface area. Tailoring sphere-shaped to fiber-shaped NPs such as nanowires, nanobelts and nanotubes is very attractive, because these nanomaterials have advantage in the application of photocatalysis, charge transfer and sensing due to its unique structures. For example, titanate scrolled nanosheets have been recently developed for their exceptional electronic, optical and photocatalytic performance [26][27][28][29] .

Despite promising new applications in industry and in nanomedicine, TiO 2 NPs may engender environmental and health risks due to their specific properties supported by their size and geometry what is attracting great concerns. Many studies have reported that TiO 2 NPs elicit a toxic response in different biological systems including animals, mammalian cells, model organisms, and bacteria [30][31][32][33][34][35][36][37][38][39] .

Although there are many observations of these TiO 2 NPs toxicity, the detailed molecular mechanisms induced are not so clear. Recent studies report endoplasmic reticulum stress (ER stress) as a common mechanism in TiO 2 NPs related toxicity. The endoplasmic reticulum (ER) is an important organelle and functions in folding and assembling of cellular proteins, in synthesis of lipids and sterols, and in regulating the maintenance of the calcium ion (intracellular calcium homeostasis), processes which are all dependent on the ER internal homeostasis. ER stress is usually a short-term response, which induces a series of transcriptional activities for cell survival; in a prolonged stress situation, ER stress activates apoptotic cell death pathways [40][41][42][43] . Finally, whereas there are evidences of TiO 2 NPs toxicity, the involved mechanisms are not fully elucidated.

To date, only a few studies have investigated the relationship between NPs morphology and their toxicity without providing any obvious correlations 24,[44][45][46] . Their toxic potential remains largely unclear and it is a real challenge to detect, track and quantify the NPs in living and biological samples and to correlate this with toxicity effects. Particle-Induced X-Rays Emission (PIXE). PIXE is a spectrometry technique, which can be used for nondestructive, simultaneous elemental analysis. It is based on X-Rays emission after collision of incident protons with the inner shell electrons of the target atoms. The incident proton may remove an electron, creating a vacancy. The energies of the X-Rays, which are emitted when higher shell electrons fill the created vacancies, are characteristic of the element from which they originate. The number of X-Rays emitted is proportional to the mass of the element in the analyzed sample.

Therefore, this technique provides not only the chemical composition of the sample, but also its twodimensional elemental mapping of a specific region of interest (ROI) in defined cellular compartments. The distribution of trace elements, from sodium to heavy metals, is acquired simultaneously with a subcellular spatial resolution and high sensitivity 23 .

Rutherford Backscattered Spectrometry (RBS). This method is based on the energy measurement of backscattered protons after elastic collision with sample atom nuclei. The ion beam is directed onto a solid sample, it enters the sample, where it loses energy by inelastic collisions on electrons, it scatters on atomic nuclei and it travels back out with a characteristic energy distribution that can be detected. From it, it is possible to determine the dry mass of organic samples. Carried out simultaneously with PIXE, RBS allows identifying low-Z elements, such as carbon, nitrogen and oxygen.

PART III -Background 178 Scanning Transmission Ion Microscopy (STIM). In this technique, the particles are transmitted through the sample and the energy loss is measured. The magnitude of the energy loss depends on the thickness and density of the sample. Thereby the local change in cell density and cell structures such as the nucleus, nucleolus and cytoplasm can be deduced 22 . Thus, STIM can be used both as an imaging technique and as a thickness measurement. The transmitted particles are detected in the beam direction, allowing the use of very small beam currents, which makes it possible to focus the beam to about 300 nm.

The microprobe present at CENBG is used to quantify, localize and study the presence of inorganic elements in different tissues and at the single cell level. In this particular context, the capabilities of the microprobe are coupled to the classical imaging techniques, such as optical and fluorescence microscopy, to quantify and localize nanoparticles within cells. The importance of nanoparticles quantification is stressed not only in the study of their effects on cells homeostasis but also in their use in radiation therapy as a dose-enhancer agent.

Questions addressed in this work

The work package 1 of the SPRITE project has been ideated to develop biomedical applications of ion beams and, in this context, the iRIBio expertise can be exploited to localize and quantify the intracellular content of nanoparticles and reveal chemical alterations due to nanoparticle and/or ionizing radiation exposure. Recently, the combined use on NPs and ionizing radiations have been extensively investigated for biomedical application, such as in radiation therapy. Indeed, different studies showed an increased density of ionization significantly localized in short distances from nanoparticles integrated in cells. Despite the use of nanoparticles are investigated for these reasons and also used in diverse field including food, cosmetic, and medical industries, the risks for their adverse health effects have not been clearly established. Their use in biological system still raises challenging questions about the internalization mechanisms, the localization and quantification in single cells.

Another important parameter that merits attention is to define how modifications of NPs morphology may alter their bioavailability and then consequent effects on biological systems.

To answer these problematic issues, the goals of this work are to:

 develop an original imaging methodology that allows in vitro studies for the detection, tracking and quantification of NPs in biological systems.

 explore the toxicity of different forms of NPs and to define precisely the molecular and cellular mechanisms involved in the toxicity of NPs in eukaryotic cells.

Experimental results

To optimize the therapeutic benefit of radiation therapy, a lot efforts are invested to improve the

Discussion and perspectives

Nanotechnology is a fast-expanding area of science. This area of research is anticipated to lead to the development of novel, sophisticated, multifunctional applications which can recognize cancer cells, deliver drugs to target tissue, aid in reporting outcome of therapy, provide real-time assessment of therapeutic and surgical efficacy, and most importantly, monitor intracellular changes to help prevent precancerous cells from becoming malignant. On-going efforts by scientists, researchers, and medical personnel can sincerely ensure to 'do big thing using the very small'. The main challenge when working with very small particles is to provide a comprehensive study to fully explore the toxicity of these nanomaterials. A better knowledge of cellular mechanisms nano-induced may help to better understand their deleterious health effects and create environmentally friendly and biologically relevant nanoparticles.

The behavior of NPs inside living cells is still an enigma, and no metabolic responses induced by these NPs are understood so far. We took up this challenge to decipher the molecular events that regulate bioaccumulation, biopersistence, and toxicity of metal oxide NPs. In particular, we validate and integrated a methodology involving chemistry, physics and biology to predict potential effects of NPs.

We investigated the toxicity of different TiO 2 NPs in terms of morphologies and the relation between intracellular titanium and calcium contents and a specific metabolic pathway: Endoplasmic Reticulum Stress. Our results show that TiO 2 NPs exerts different toxicity levels according to their morphologies (size, shape, surface reactivity), to their related behavior in biological media, and to the considered cell type (in vitro). Indeed, the morphology and the behavior of TiO 2 NPs in biological media will determine their capability to interact in vitro with the cell and then, to be internalized. According to this, it will favor heterogeneous distribution of the TiO 2 NPs within the cell population and an important variability in terms of titanium content at the single level. As we demonstrated that a minimal intracellular content of titanium (TiO 2 NPs) is mandatory to induce marked change of the cellular homeostasis, it is essential to consider the intracellular content (and not only the exposure concentration) of the considered NPs.

The fundamental and basic knowledge acquired will provide a unique opportunity to develop freeenvironmental risk nanomaterials and also offer new perspectives in cancer therapy. The future progresses of this project will address fundamental question in the emerging and promising fields of

CONCLUSI ONS A ND PERSPE CTIVES

Since the discovery of X-Ray in 1895 by Rontgen, ionizing radiation (IR) has been employed as a powerful tool for imaging and radiotherapy. In the early days after the discovery of radiation, scientists were interested in understanding the physics of different types of IR as well as their applications. It did not take a long time to discover the hazards of radiation. Skin erythema due to high doses of radiation was among the first symptoms observed. Eventually it was noticed that radiation is a potential risk for cancer induction by observing the cancer incidence of exposure to radiations, among them Marie Curie and her daughter. IR is known as a double edged sword that could cause or be used to eradicate cancer. IR has been studied mainly by its effects, however all mechanisms of action are still not fully understood.

The European project SPRITE, in which I was recruited, aimed to exploit the ion beam techniques and their applications. All along my PhD, I utilized ion microbeam abilities to expand the fundamental knowledge of IR effects on biological systems (microbeam for micro-irradiation) and to exploit the interactions between IR and matter to obtain information on the chemical elements distribution in cells (microprobe for multi-elements chemical analysis). All together these techniques aimed to decipher IR-induced mechanisms at the single cell level or in cell population which would be exploited for improving cancer management or treatment.

In response to IR, cells activate DNA repair and cell signaling processes to protect their genetic pool either by repairing or by causing cell death in order to avoid adverse effects, such as mutation, chromosome aberration, and cancer. DNA repair plays the central role in the cell response to radiation. Intensive laboratory research is evolving in DNA repair and cell signaling process, however the link from DNA damage to mutation, cancer and cell death is not easily formed. The advances in understanding the mechanisms of DNA repair and cell signaling pathways and human genome research have opened up opportunities to develop new approaches. These approaches are aimed at linking induced DNA damage through cellular DNA repair processes with the dose of IR, the type of IR and with the potential adverse health consequences (cancer and hereditary effects) that may ensue.

The first part of this work focused on the study of induced DNA damages, the repair processes and the responses activate after exposure to IR. We developed a system able to control the dose of different IR, in space and time, and deliver them on living cells. The AIFIRA microbeam, indeed, permits to correlate charged particles-induced responses and dose-response at the cellular level.

-particles that are increasingly studied because constituting significant part of the radioactive natural background and increasingly considered in medical applications. Up to now, the knowledge about -particles effects on living cells is acquired by using radioactive sources with which we cannot precisely control the number of traversals at the single cell scale and it is difficult to correlate the biological responses with the traversal. Microbeam can control the number and the position of delivered particles. However, two major issues needed to be solved due to the low energy of - For a complete characterization of the radiation sensitivity or resistance of cancerous cells to examine in depth others biological markers such as the cellular death, the cell cycle arrest, changes of genomic profiles can improve the radiation therapy protocols and estimate genetic and carcinogenic risks to human populations exposed to IR. In addition, several stress pathways could be modified after IR exposure or nanoparticles exposure, such as the ER stress induced by certain nanoparticles. To characterize different stress pathways activated after IR and/or nanoparticles exposure, a collaboration with the platform BMYScreen has been established. This platform develops approaches consisting in screening molecular stress pathways such as ER stress, inflammatory response and oxidative stress (Figure 16). Deciphering the biological effects of ionizing radiations using charged particle microbeam: from molecular mechanisms to perspectives in emerging cancer therapies

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.

Keywords: Targeted irradiation, DNA damage, low/high LET irradiations, Radiation sensitivity, Nanoparticles

  INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PART I: BIOLOGICAL EFFECTS INDUCED BY IONIZING RADIATIONS. STUDY OF DOSE-EFFECT PROTEINS KINETICS AND NON-TARGETED EFFECTS . . . . . . . BACKGROUND . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 1: Ionizing radiations and matter interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Linear Energy Transfer Absorbed Dose Relative Biological Effectiveness Chapter 2: An original tool to observe ionizing radiation effects: Charged Particle Microbeam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Study of biological effects of single charged particles in single cells Study of DNA damage response Cellular compartment targeting Study of non-targeted effects Development of approaches for tumor therapy Chapter 3: Ionizing radiations induce cluster of DNA lesions . . . . . . . . . . . . . . . . . . . . . . . . . Base Excision and Single Strand Break Repair Double Strand Break Repair Chapter 4: Ionizing radiation and non-targeted effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . QUESTIONS ADDRESSED IN THIS WORK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EXPERIMENTAL RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DISCUSSION AND PERSPECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ANNEX INTRODUCTION INTRODUCTION 19

  Developments of protocols addressed to quantify and localize these nanoparticles are needed.

Figure 1 .

 1 Figure1. Ionizing radiations and non-ionizing radiations in the electromagnetic spectrum. When the energy of radiations is higher than 13.6 eV, it is considered as ionizing radiations. Adapted from www.nasa.gov

Figure 2 .

 2 Figure 2. Penetration power of ionizing radiations in different materials.

  3 N 4 window (Silson Ltd., Northampton, England) and enters the sample through a 4-m thick polypropylene foil (Goodfellow) used as a cells support. The ion beam is positioned on target by means of electrostatic scanning plates situated downstream of the last quadrupole. In case of protons, the mean number of particles (N) hitting cells is linearly related to the opening time and the relative statistical fluctuation in the number of traversals delivered decreases as N increases. The exposure of targeted cells to charged particles is controlled using a fast electrostatic beam deflector allowing to open and close the beam within 1 s (DEI PVM-4210). In case of -particles, each particle is detected upstream with a BNCD (Boron-doped Nano-Crystalline Diamond) from which secondary electrons emitted are collected using a channeltron electron multiplier (These new achievements are obtained during my PhD and are presented in Manuscript 1).The irradiation end-station is constituted of a motorized inverted fluorescence microscope (AxioObserver Z1, Carl Zeiss Micro-Imaging GmbH) equipped with a 14 bits Rolera EM-C 2TM Camera (QImaging) which is positioned horizontally at the end of the beam line. It can be equipped with up to 6 objectives and fluorescence filter sets. A good compromise between high numerical aperture (NA) and long working distance is obtained with a 63x objective (LD Plan-Neofluar 63x/0.75, Optical resolution of about 400 nm, Carl Zeiss MicroImaging GmbH). Fluorescence light is provided by Light Emitting Diode (LED) illuminating system (Colibri2 TM , Zeiss) with negligible heat production.

Figure 4 .

 4 Figure 4. Scheme of the micro-irradiation set-up and imaging system. Charged particles are focused in a micrometer spot, using a triplet of magnetic lenses, and driven to the target cell under vacuum. The beam is extracted in air through a 200 nm thick extract window. A fluorescence microscope (Zeiss AxioObserver Z1) equipped with a 14-bits Rolera Camera is placed at the end of the beam line to visualize cells and perform online fast time-lapse imaging. A 488 nm Laser diode (iLas2 TM , Roper Scientific) coupled to a galvo-scanned mirror is installed on the microscope. The whole experiment is controlled by custom-made irradiation and acquisition software.

Figure 5 .

 5 Figure 5. Simulations of 3 MeV protons and -particles in water. SRIM 2013 simulations34 show that 3 MeV -particles pass through 18 µm and 3 MeV protons pass through 148 µm of water before to lose all their energy. The inset shows the position and thickness of polypropylene foil (grey square) and of cell (green square) with respect to the particle deposited energy.

Figure 6 .

 6 Figure 6. Representation of ionizing radiation effects. High and lose doses of ionizing radiation can damage the DNA and other target in direct and indirect way. Cell response mechanisms act in a time scale from second to few minutes by signaling the damage and repairing it if possible. If misrepaired, the cell damage can lead to biological effects more or less important for the human life.

Figure 7 .

 7 Figure 7. Radiation-induced spectra of DNA damages. The DNA damage can be of different entities, from a modification of bases to double strand breaks. Ionizing radiations produce clusters of DNA damage combining different types of damage depending on the particle LET used. Each dot represents event of ionization or excitation.

Figure 8 .

 8 Figure 8. Simplified representation of SSB repair pathways and the actors involved in this repair mechanism.

Figure 9 .

 9 Figure 9. Schematization of NHEJ and HR mechanisms involved in DSBs repair.

Figure 10 .

 10 Figure 10. Main results obtained by using the micro-irradiation beam line.

  involved or in the DNA repair pathways or in the structure of subcellular compartments. All along my PhD, I validated different stable transfected cell lines (HTB96-U2OS cells) and I established numerous transfected clones expressing several GFP-tagged proteins. The principal reason that lead to the choice of proteins involved in DNA repair pathways was the fact that despite the biochemical mechanisms of eukaryotic DNA damage repair have been largely studied, much remains to be elucidated regard the regulation of the DNA repair pathways and the connections between them. Another critical question that is worthwhile to answer is on roles played by subcellular organelles in response to ionizing radiations. First, I focused on the generation of cell lines expressing GFP-tagged proteins involved in DNA repair pathways: GFP-XRCC1 (BER/NER pathways) 104 , GFP-OGG1 (BER, 8-oxo-guaunine) 105 , GFP-RNF8 (Homologous recombination) 106 , GFP-Ku70 and GFP-Ku80 (Non-homologous End-joining) 107 . These cell lines are established to study the dynamics and kinetics induced by 3 MeV charged particles (protons and -particles) as a function of the delivered dose and the particle LET.

  the selective irradiation of single cells and follow-up observations of induced damage via dedicated biological markers (DNA damage signalling, DNA repair protein). By using a 239 Pu -particles emitting source, I irradiated GFP-RNF8 and GFP-XRCC1 cells. Using this irradiation method and combining it with H2AX in situ immunodetection assay, I observed that GFP-RNF8 relocalized to the DNA damage induced by one traversal and it persisted within the damaged site 30 min after irradiation. XRCC1 was difficult to visualize a posteriori because this protein dissociate from damaged site within 15 min 119 .Despite this source has extensive and of easier access with respect to the micro-irradiation beam line, the fluence of delivered particles is very low (30 min are necessary to obtain a mean number of hits per nucleus of 7.6 ± 3.3), and the irradiation time and position are unknown37 . Alternatively, PART I -Experimental Results 54 charged-particle microbeams can target living cells with single charged particles and can be used extensively to study various biological endpoints. Modern end-stations, equipped with fluorescence time-lapse imaging, provide the opportunity to visualize and quantify in real-time the early radiationinduced cellular response. However, due to the limited range of -particles in matter (a few tens of micrometres), it is difficult to detect single -particle without significantly altering the microbeam energy and size. For this reason was necessary to develop a system able to detect single -particles without interfering with the particle energy and trajectory and visualize the DNA effects in real time.Thanks to the collaboration with Michal Pomorski (CEA) and Philippe Barberet, we developed a Boron-doped Nano-Crystalline Diamond (BNCD) membrane that allows reliable single -particles detection and single cell irradiation with negligible beam scattering. The BNCD membranes give reproducible detection efficiency and are very homogeneous on millimetre surfaces. In addition to the detection capability, they are transparent and non-fluorescent, making them compatible with bright field and fluorescence imaging. The detection of single -particle traversals allows controlled irradiation of living cells. Post-irradiation analyses showed that GFP-RNF8 accumulates continuously at single -particle track during the first 30 minutes after irradiation. We observed that the intensity of irradiation-induced foci varies from one to another reflecting most probably the chromatin heterogeneity inside the nucleus. These achievements are detailed in the Article 1 published in Scientific Reports.

  photons) generate different types of DNA damages within the nucleus in a manner dependent on

Figure 11 . 4 .

 114 Figure 11. Visualization and targeting of cell nucleus containing the GFP-Ku80 protein before, during and after irradiation (left). Relocalization of the GFP-Ku80 protein in the irradiated area (right). The nucleus is irradiated in a micrometer spot (red cross) with 10 5 3 MeV protons 20 s after the images acquisition starts. Each image is taken every 100 ms with a new high-sensitivity camera (fast imaging). Scale bar: 10 m. Red: Measure of fluorescence intensity in the irradiated area; Cyan: fluorescence in the rest of nucleus; Blue: control nucleus used for normalization.In particular, GFP-Ku80 occurred in very few seconds, as shown in Figure11, when 10 5 3 MeV protons are delivered in a 2 m spot and this very rapid protein recruitment need to be monitored with a very fast image acquisition. To solve this issue and to perform fast imaging a new highsensitivity camera was installed on the microbeam microscope. This new Rolera Camera EM-C 2TM is able to take images every 100 ms allowing a very fast imaging. Further studies are now necessary to study the response of this protein when different amount of protons or -particles are delivered.

Figure 12 .

 12 Figure 12. FRAP experiment on cells (a) GFP-H2B and (b) GFP-Nop52 (nucleolus protein). Left: Time-lapse

  the question about the contribution of cytoplasmic components have in response to ionizing radiation remains open. Mitochondria constitute a large volume of cytoplasm and have been selected in this work to highlight the effect of targeted irradiation. The experiments of micro-PART I -Experimental Results 59 irradiation were carried out using the SNAKE facility in Munich and the AIFIRA facility in Bordeaux. These two facilities permitted mitochondria irradiations with both 55 MeV carbon ions and 3 MeV protons in a variety of patterns which allowed for irradiations with the same total deposited energy per unit area. In particular, I contributed to adapt the irradiation protocols between SNAKE and AIFIRA facilities. The first result observed is the local depolarization of irradiated mitochondria, assessed by the loss of the accumulated tetramethyl rhodamine ester (TMRE), without effect on the rest of not-irradiated mitochondria of the targeted cell. These findings are confirmed repeating the experiments with different numbers of particles 5 times at SNAKE and then 2 times at AIFIRA. To test if the targeted irradiation causes mitochondrial membrane rupture, membrane integrity was tested. My contribution was to provide Mito-roGFP2 transfected cells to verify the integrity of membranes. The fluorescence signal of roGFP2 in the mitochondrial matrix stayed constant after irradiation excluding alterations of the mitochondrial matrix composition. Results obtained are detailed in the Manuscript 3 which is submitted to Scientific Reports.

PART I -Discussion and perspectives 61 targeted mitochondrial irradiations using 55 1 "

 61551 MeV carbon ions and 3 MeV protons induce mitochondrial depolarization. Mitochondrial depolarization, as confirmed by the relocalization of TMRE from the mitochondria to the cytoplasm and then to the extracellular space, indicates a distinct change in mitochondrial membrane potential. These studies underlined the importance of elucidating the biological mechanisms and responses activated by exposure to ionizing radiations in order to improve our knowledge and consequently the associated cancer risks. Further studies and several approaches are needed to completely understand the cellular mechanisms. The wide range of transfected cell lines developed during my PhD permits to study different DNA repair pathways (SSB and DSB Repair, 8-oxo guanine, BER, etc…) as a function of type of particle, deposited energy and track structure for which the Monte Carlo toolkit represents a fundamental tool. In addition modifications of chromatin and proteostasis induced by ionizing radiations and the epigenetic mechanisms can be a subject of future studies. The investigation of radiation-induced effects on other cellular compartments different from DNA such as mitochondria and nucleoli is a point to clarify. Mitochondria depolarization were investigated during this work and more studies are necessary to explain the depolarization mechanisms that are not based on the membranes rupture but probably due to changes in the membrane structure and permeability. The nucleolus, another interesting target, is a prominent non-membrane-bound nuclear substructure that organizes around chromosome segments containing nucleolar-organizing regions (NORs). It is the center of the rRNA transcription and ribosome biogenesis. Also additional functions, such as the cell cycle regulation, the telomerase activity, and the p53 metabolism have been attributed to the nucleolus itself 135,141,142 . In 1960 Montgomery et al. showed the first results of continuous ultraviolet microbeam irradiations of living cells nucleoli 143 . In 1971 Berns and colleagues irradiated with a laser microbeam the chromosome constriction and found a modified nucleolar organization 144 . Recently, Sorokin et al. investigated the movement and the morphology of nucleolar protein after gammaradiations 145 . From these studies an increasing interest in target nucleoli and in understanding their role in the radiation responses rose. Article Single -particle irradiation permits real time visualisation of RNF8 accumulation at DNA damaged sites"

  (i) The team of Frederic Chibon works on the action of oncogenesis and implication of genetics and biology mechanisms in sarcomas at the Institut Bergonié (Inserm Unit 1218). By means of this collaboration, I got access to the wide collection of patient's sarcoma cell lines established and characterized from the genetic point of view by Chibon's group. Genetics is a relevant factor that influences cellular responses but more studies and more biomarkers identification will certainly improve the knowledge for patient's individualized treatments. Characterizing the response of different patient's cell lines is of main importance because could relate the cellular radiation sensitivity with that of patients. From this collection, two cell lines with different genetic (known amplicon-based genetics profile versus complex genetics) were selected to evaluate their radiation sensitivity under different irradiation modalities: dose, nature of IR and irradiation procedure (focalized versus broad beams); (ii) I worked with the Department of Radiation therapy of the Institut Bergonié and in particular with the radiation therapist Guy Kantor and the medical physicist Mikael Antoine. This partnership gave me access to a medical facility (Clinac) used at the Institut Bergonié for radiation therapy patient's treatment. A

Figure 13 .

 13 Figure 13. Schematized procedures to study and characterize patient's cell lines radiation sensitivity.

Figure 14 .

 14 Figure 14. mFISH after electron irradiations. Chromosomes are stained with 5 fluorochromes where unique label combinations identify individual chromosome. Translocations result in chromosome composed of fragments with different color combinations. No differences are observed between (a) control, (b) 2 and (c) 6 Gy irradiated cells due to the high number of translocations already present before irradiation in control cells.

Manuscri pt 4 Manuscript 4 "

 44 Multiparametric analysis of radiation sensitivity of two sarcoma cell lines derived from patients after both electron and proton irradiations"Muggiolu G, Simon M, Devès G, Antoine M, Kantor G, Chibon F, Barberet P and Seznec H

Figure 15 .

 15 Figure 15. Ion Beam analysis principle and system for biological samples. STIM is used to map the cell density area by energy loss measurement. PIXE and RBS analyses provide the spatial distribution and quantification of chemical elements at the single cell level.

  beam ballistic and to increase the Relative Biological Effectiveness (RBE) of the radiation inside the tumor. The main current developments in radiation therapy are based on add radiation sensitizing agents with the final aim to enhance the dose in the tumor volume and to reduce risks to healthy tissues. High-Z metal or metal oxide nanoparticles have been proposed as good candidates. Starting from the collaboration with the Institut Bergonié, I learnt of a clinical assay leaded from a private company (NANOBIOTIX) consisting in the injection within the tumor of Hafnium oxide NPs to increase the radiation effects. By working together with Marina Simon and based on the results previously obtained, we started to adjust these in vitro protocols for evaluating the potentiality of new emerging therapeutic agents. As mentioned before, nanotechnologies are emerging areas studied for increasing the dose-rate effects, and to enhance the therapeutic effectiveness of radiation therapy.Within this context, we collaborated with the private company NANOBIOTIX to study the effects of the combination of metal oxide nanoparticles (HfO 2 , TiO 2 ) with IR. Preliminary confidential results are not discussed in this thesis but they originated important points to be clarified, such as the internalization, quantification and localization of nanoparticles in single cells. Indeed, besides the numerous physical and chemical advantages exhibited by NPs for medical applications, their use in biological system still raises challenging questions: (i) How and where accumulated NPs in cells? (ii) How quantify in situ the intracellular NPs content of exposed cells? (iii) How clarify the interactions between NPs and biological system? (iv) How NPs interact with ionizing radiations?To answer these questions, I contributed in collaboration with Marina Simon to quantify, localize and decipher the molecular mechanisms induced by NPs in cells. First, the research program tried to answer the following questions about TiO 2 NPs: (i) What are the parameters influencing the TiO 2 NPs bio-availability and interaction? (ii) What are the TiO 2 NPs physicochemical properties that determine their bio-distribution, bioaccumulation and bio-persistence? (iii) What are the molecular and cellular mechanisms involved? (iv) What are the relations between bio-distribution and toxicity? Second, the goal was to apply the methodology develop for TiO 2 NPs analysis to other metal or metal oxide nanoparticles.I participated to the development of a procedure suitable for in situ detection and in vitro quantification of chemical elements present in human cells. This method is well suited to any cell type and it is particularly useful for quantitative chemical analysis in single cells following in vitro PART III -Experimental results 181 metal oxide nanoparticles exposure. By using this methodology, we showed that TiO 2 nanoparticles accumulate in the cytoplasm all around the nucleus and are excluded from nucleus and mitochondria. Interestingly, we also observed that the nanoparticles concentration can be 10 fold higher in one cell compared to another of the same exposed population. In addition, we noticed an increase of intracellular ions, such as potassium and calcium associated to the highest uptake of TiO 2 nanoparticles. The protocol describing all steps is detailed in the Manuscript 5 and it is accompanied by a movie describing procedures. This work was invited for a publication in Journal of Visualized Experiments (JoVE).The development of this original imaging and analytical methodology (multimodal and correlative microscopies) allowed me to participate in another in vitro study combining the detection, tracking, and quantification of TiO 2 NPs. The main goal was to define precisely the molecular and cellular mechanisms involved in the toxicity of TiO 2 NPs in eukaryotic cells. The main results obtained are summarized in the Article 6. Briefly, the study investigated the cellular homeostasis responses induced by different TiO 2 NPs (in terms of shape, size and surface reactivity) in different in vitro primary and immortalized human cell populations (human umbilical vein endothelial cells -HUVEC, human epidermal primary new-born keratinocytes -HEKn and, immortalized cancerous cell line -Hela). A careful attention was paid to the quantification of NPs internalization in order to correlate it with the intracellular ion homeostasis, the ER stress response and the cell fate (differentiation, proliferation, death). We showed that (i) the presence of intracellular TiO 2 NPs is dependent on both their morphology and their bioavailability in biological media. In addition, we have established that (ii) a minimal intracellular content of TiO 2 NPs (minimal threshold) is needed to induce the positively correlated alteration of the intracellular calcium homeostasis (without any dysregulation of the calcium metabolism) and (iii) the induction of one specific ER stress-pathways associated with mitochondrial dysfunction that are strictly related to the nanotoxic responses. We also observed that (iv) the cellular function (endothelial versus epidermal), the cell type (primary versus immortalized/cancerous) and the heterogeneity of the intracellular distribution/content of TiO 2 NPs in a cell population/type deeply modify the cellular response.

  nanomedicine and will help us to (i) determine the physicochemical and bio-molecular mechanisms induced by ionizing radiation interactions and metal/metal oxide NPs in cellulo (with in vitro distribution and quantification data), (ii) develop and validate predictive methodologies from chemistry, physics and biology (Monte Carlo simulation), (iii) develop calculation methods with experimental validation to precisely predict dose-effect correlation in the context of ionizing radiation and NPs interactions, (iv) identify adverse side effects due to potential NPs toxicity in relation to their physicochemical characteristics and the targeted cell type (cancerous versus normal cells), (v) define in vitro and preclinical assays to validate potential new radiation therapy protocol combining NPs and ionizing radiation. The physical and modeling aspect of the project, beam characterization and cell dosimetry in realistic conditions are some aspects of a PhD thesis in medical physics following my PhD work.

  particles: the -particles detection without stopping them and the observation of induced damages in real time. During my PhD, the collaboration with Michal Pomorski leaded to the development of a thin Boron-doped Nano-Crystalline Diamond membranes able to detect single -particles without modifying their trajectory and energy. In addition, the biological marker GFP-RNF8 protein permited to visualize and follow the DNA damages induced by single -particles in real time. These technical developments allowed us, for the first time, to irradiate and visualize in real time the GFP-RNF8 protein that accumulates at DNA damage sites induced by one -particle. These results open a series of studies devoted to elucidate the effects of low doses in living.Then, our facility was used to investigate repair processes activated after different types of damages induced by increasing numbers of charged particles with different LET. Increasing number (from 1 to 1000) of protons (LET = 12 keV.µm -1 ) or -particles (LET = 148 keV.µm -1 ) were delivered on living cells to create cluster of DNA damages with different complexities. The responses of GFP-RNF8 and GFP-XRCC1, involved in two DNA repair mechanisms, were followed after irradiations. The correlations between their recruitment time to damages and both deposited energies and particle structures were done. With this study, I showed that two proteins acting in two DNA repair mechanisms react in two different manners. In response to IR, GFP-RNF8 exhibits a kinetics significantly dependent on the spatial distribution of damages, while the kinetics of GFP-XRCC1 is mainly dependent on the deposited energy. These results indicate a coherence between the protein function and the biological response to IR and open new challenging questions for understanding how the repair machinery acts in response to different types of damages, particles and doses.In addition during my PhD, I developed not only transfected cell lines to support the DNA irradiation but also stable transfected cell lines that allow the visualization and targeting of others sub-cellular compartments such as Matrix-roGFP2 and GFP-Nop52 to visualize mitochondria and nucleoli, respectively. We made these cell lines available to other research teams which triggers a collaboration with the Munich University. By establishing the cell line expressing Matrix-roGFP2 which is a sensor of mitochondrial matrix, I had the opportunity to participate in the PhD project of Dieter Walsh. The goals of Dieter's project are to target and irradiate mitochondria for evaluating their role in response to IR. Irradiations were performed at both facilities SNAKE (in Munich) and AIFIRA (in Bordeaux) with carbon ions and protons, respectively. Mitochondrial membrane depolarization without disrupting membrane integrity was shown after charged particle irradiations.Despite the mechanisms of mitochondrial depolarization remain unclear and further studies are necessary to explain such behavior, we demonstrated that this depolarization was not due to the membrane rupture ensured by the constant presence of Matrix-roGFP2 in the mitochondrial matrix.Recently, the microbeam line end-station has been implemented with a 488 nm laser able to bleach the GFP which allowed us to perform Fluorescence Recovery after Photobleaching experiments. I was involved in the calibration and validation of this new system that was achieved partly thanks to the development of stable GFP-H2B and GFP-Nop52 cell lines. Now this system can be combined with micro-irradiation experiments to measure the exchange and binding behavior of repair proteins recruited at the DNA damaged sites.The repair processes activated after IR-induced damages if not well-completed could lead to cellular modifications and then trigger mechanisms for cancer genesis and/or radiation resistance. In collaboration with the Insitut Bergonié, I studied the responses of sarcoma cell lines exposed to particles and doses commonly used in medical conditions and particles produced by a fundamental research accelerator. Two patients-derived sarcoma cell lines were selected for their genetic characteristics and were irradiated with 9 MeV electrons and 3 MeV protons. Additionally, protons were delivered by following two energy depositions at the cellular scale: homogeneous versus focused beams. Protocols to elucidate the radiation resistance after irradiation were developed and validated. In particular, automated ImageJ routines for DNA damage quantification and colonies counting were improved. Three biological end-points (radiation-induced foci, surviving fraction and proliferation) allowed us to show that (i) the biological responses are related to the genetic characteristics, (ii) the protons have more pronounced effects than electrons, and (iii) no differences are noticed between two energy distributions.Implementation of protocols to assess radiation sensitivity or resistance in patient cell lines drives to study the characterization of biological effects induced by IR in the presence of metal nanoparticles.The use of nanoparticles is an emerging field in radiation therapy with the objective to improve the deposit of IR in the tumor sparing the healthy tissues. As nanoparticles are relatively new technologies, it is necessary to elucidate the effects that nanoparticles could induce in biological systems. It is worth to know the cellular localization and concentration after exposure to define the effects of these agents. To this purpose, protocols to localize, quantify and study the effects of nanoparticles in cells were improved. Nuclear microprobe analysis are exemplary techniques allowing the investigation of nanoparticles bioaccumulation and consequent effects. By participating in the project of Marina Simon, I could observe the effects induced by different nanoparticle shapes and sizes in different cell types. We showed that nanoparticles induced biological effects positively correlated to ion homeostasis balance disequilibrium (calcium) and to the induction of metabolic stress pathways (Endoplasmic Reticulum stress). These published data validated an original methodology (multimodal and correlative microscopies) that allows in vitro detection, tracking, and quantification of TiO 2 NPs in parallel to the use of biological indicators for ion homeostasis, cell metabolism and cell fate.All these data, obtained thanks to the multidisciplinary expertise of the team I worked with, contributed to improve the knowledge about the interactions of IR and living. By combining DNA repair targeting and new radiation therapy approaches the radiation therapeutic benefits could be enhanced and uprising of radiation resistant cancers could be reduced. The deeper understanding of these mechanisms (IR or/and NPs induced effects) gives new insights to fundamental and therapeutic approaches.Numerous works are started and need to be continued at CENBG thanks to the wide versatility of the microbeam lines.Other biological markers are under development and investigation, such as GFP-Ku70 and GFP-Ku80 for elucidating NHEJ pathway, GFP-OGG1 for studying the ROS effects; beyond DNA, other substructures as nucleoli and cytoplasmic changes deserve attention and for these reasons stable transfected cell lines are under development such as GFP-FBL and GFP-PolyQ mutants. The use of these different constructs permits the characterization of different mechanisms induced by IR. The validation of the laser system permits now to move towards studies direct to combine microirradiation and FRAP experiments. These performances can elucidate, in addition to the kinetics, the dynamics of investigated proteins present at the damaged sites. In addition, PCR-based approaches constitute a suitable technique for DNA-damage quantification. LORD-Q PCR allows to accurate quantification of DNA damage in both nuclear DNA and mitochondrial DNA.

Figure 16 .

 16 Figure 16. The influence of TiO 2 nanoparticles or/and IR can be investigated at the single cell level. The NPs internalization as well as IR alter the intracellular ion homeostasis, leading to endoplasmic reticulum stress and can activate other different stress pathways.

  Rays are used in medical applications to treat cancer. Similarly, X-Rays are typically used to provide static images of body parts. Several meters of concrete, or a few meters of dense material, are able

	PART I -Background	33
	emitted during the decay of heavy radionuclides. -particles strongly interact with matter, but they
	have a very limited ability to penetrate. These particles can be blocked by a sheet of paper, skin, or
	even a few centimeters of air. Nonetheless, materials that emit -particles are potentially dangerous
	if they are inhaled or swallowed. (ii) Beta particles or electrons are light charged particles and are
	emitted from naturally occurring materials, such as strontium-90. In general beta particles have a
	great ability to penetrate few meters in the air, and can penetrate skin. Nonetheless, a thin sheet of
	metal, or plastic, or a block of wood can stop them. (iii) -Rays and X-Rays are two types of
	electromagnetic radiations with short wavelengths and high energies. X-Rays are produced by
	electrical devices accelerating electrons to high energy and stopping them in a target. -Rays are
	emitted by radioactive isotopes and are very similar to X-Rays in their effects on living organisms. -

Table 1 .

 1 List of constructs used to produce stable transfected cell lines containing selected plasmids where the gene of interest is coupled with the GFP gene.

	1.

Microdosimetric simulations razionalize in cellulo radiation-induced spatiotemporal dynamics of GFP-tagged DNA repair proteins at the cellular scale"

	Manuscript 2 Article 3
	time visualization of RNF8 accumulation at DNA damaged Seznec H and Barberet P Muggiolu, G. et al. Single α-particle irradiation permits real "Muggiolu G, Torfeh E, Simon M, Devès G, Incerti S, Lampe N, "
	sites. Sci. Rep. 7, 41764; doi: 10.1038/srep41764 (2017)

Live cell imaging of mitochondria following targeted irradiation in situ reveals rapid and highly localized loss of membrane potential"

	Walsh, D.W.M. et al. Live cell imaging of mitochondria
	following targeted irradiation in situ reveals rapid and highly
	localized loss of membrane potential. Sci. Rep. 7, 46648;
	doi:10.1038/srep46684 (2017).

  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 telle 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 impliquées 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.

Mots clés : Micro-irradiation ciblé, Dommages ADN radio-induits, Irradiations bas/haut TEL, Radiosensibilité, Nanoparticules

bystander cells24,26 . Understanding the impact of IR on mitochondrial functions (a major intracellular source of reactive oxygen) and the handling of free radicals is therefore likely to be useful.

Manuscript 2PART I -Manuscript 2

Scientific Reports (submitted)

Manuscript 4

Manuscript 5PART III -Manuscript 5

PART III -Article 6

Article 6

Acknowledgements

Chapter 2 Nuclear Microprobe techniques for chemical multielements micro-analysis

One of the main applications of microbeams is the ability to perform the micro-analysis which measures the physical/chemical characteristics of a sample.

Since the 1960s, charged-particle microbeams have been used for quantitative elemental analysis of geological, historical and biological samples. Two-dimensional elemental maps are obtained by scanning a small ion beam across a sample and monitoring the X-Rays produced by sample elements.

The interaction of charged particles and matter allows one to obtain quantitative information about the chemical composition of matter. The beam focalization and scanning on the sample give the spatial distribution of these chemical elements. These characteristics make the ion beam analysis a multi-elemental, quantitative, sensitive (down to few parts per million), and non-destructive powerful tool to investigate the composition of biological specimens 23 . It allows the measurement of trace elements at the single cell level (Fe, Cu, Zn, Se, S, …), the study the intracellular homeostasis (Na, Mg, Cl, K, Ca) and the investigation of the presence of metal nanoparticles (Ti, Al, Hf, Pt).

Various techniques are available for ion beam analysis, but just some of them are used for biological samples. Principally, these methods are: (i) PIXE -Particle-Induced X-Ray Emission, (ii) RBS -Rutherford Backscattering Spectrometry, and (iii) STIM -Scanning Transmission Ion Microscopy (Figure 15). These techniques rely on the detection and energy analysis of characteristic radiations emitted by atoms and nuclei of the target when bombarded by MeV ion beams, because the energy of the emitted radiation is typical of the targeted material 47 .