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CEMOVIS, développements méthodologiques et étude ultrastructurale de la cellule HT29 : De la cellule aux nucléosomes

Abstract : The ultrastructure of HT29 cells (human epithelial adenocarcinoma cell line) was studied by CEMOVIS (Cryo-Electron Microscopy of Vitreous Sections) with a special emphasis on chromatin organization in the cell nucleus. We proposed methodological improvements for this technique:- We first developed a grid holding micromanipulator to facilitate both cryosections collect and deposition on carbon-coated TEM grids.- We also developed new metallic thin films (to replace carbon-base supports) to enhance the adhesion of cryosections on their support. The TiO2 thin films that we produced and analysed by electron microscopy (high resolution imaging, EELS and chemical mapping) seem to be an interesting alternative to carbon films for the deposition of cryosections. Their adhesive properties could be due to Titanium high electric conductance at low temperature (although this relation has not been clearly demonstrated yet).In HT 29 cells, we indentified cell organites (nucleus; cytoskeleton filament bundles, multilamellar bodies) in situ. Selected imaging conditions provide for a high enough resolution to visualise the two membrane leaflets. In the cell nucleus, we observed striated patterns separated from 2.7 to 3.5 nm that we assume to be DNA molecule turns wrapped around the histone protein core. Compared with the dense phases formed in vitro by nucleosome core particle in solution, our images suggest that nucleosomes are locally ordered in chromatin. This observation is discussed regarding the chromatin polymeric models.
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Submitted on : Tuesday, April 10, 2012 - 2:15:08 PM
Last modification on : Saturday, June 25, 2022 - 10:06:41 PM
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Nicolas Lemercier. CEMOVIS, développements méthodologiques et étude ultrastructurale de la cellule HT29 : De la cellule aux nucléosomes. Sciences agricoles. Université Paris Sud - Paris XI, 2012. Français. ⟨NNT : 2012PA114812⟩. ⟨tel-00685425⟩

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