Abstract : To understand the mechanisms of interaction of ionizing radiation with living tissues exposed to low and protracted doses remains a major issue for risk evaluation. The response cannot be found in epidemiological studies because the only available data concern accidental exposures to high doses of radiation. In addition, this kind of exposure is very difficult to reproduce in vitro by irradiating cell lines. The method per preference is based on random irradiation of cell populations. The mean number of particles interacting with cells is calculated on the basis of Poisson's statistics: inevitable multiple impacts, numerous potential intracellular targets (nuclei, cytoplasms...), indirect effects induced by the impact of particles on neighbouring cells or simply the extra-cellular targets, constitute phenomena that make more complex the interpretation of experimental data.
A charged particle microbeam was developed at CENBG to perform the targeted irradiation of individual cells with a few microns precision, to deliver a counted number of particles down to the ultimate dose (one alpha per cell), to target predetermined cells and observe the neighbouring cells response. This facility has been validated during this work on human keratinocyte cells expressing a recombinant fluorescent histone (H2B-GFP). Confocal microscopy and quantitative analysis allowed DNA double strand breaks measurement via histone H2A.X phosphorylation in single cells, DNA repair mechanism and apoptosis induction. Data obtained during this thesis validate the methodology we developed by demonstrating targeting reproducibility and dose control showed dose-effect relationship as a function of time.