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Translocation d'acides nucleiques au travers d'une bicouche lipidique : du nanopore au bacteriophage

Abstract : This work highlights two nucleic acid translocation mechanisms through a lipid membrane : first the electrophoretically driven translocation of oligomers through alpha-hemolysin nanopore, then the passive translocation of genomic DNA outside T5 bacteriophage capsid. The first part of this thesis focuses on double-stranded DNA molecules unzipping through the alpha-hemolysin nanopore. The translocation time of single molecules through the pore are experimentally measured as a function of DNA sequence, DNA length and the applied force. Distributions obtained are compared with a model describing DNA translocation as a diffusion of an opening fork in a 1D energy landscape determined by the sequence of the molecule. The second part deals with an in vitro system reconstituting the initial steps of bacteriophage T5 infection. The interaction of T5 with its detergent purified membrane receptor FhuA generates a sequence of events leading to the release of the viral genome out of the capsid : (i) receptor binding, (ii) activation leading to the opening of a DNA channel, (iii) DNA release. The dynamics of the three steps is measured using both bulk and single virus assays. The DNA release step is compared to a physical model which exhibits a largely out of equilibrium dynamics. Finally, FhuA is reconstituted into giant lipid vesicles to monitor the ejection through a lipid membrane using fluorescence microscopy and electrophysiology.
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Submitted on : Thursday, December 9, 2010 - 3:37:35 PM
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Nicolas Chiaruttini. Translocation d'acides nucleiques au travers d'une bicouche lipidique : du nanopore au bacteriophage. Biophysique [physics.bio-ph]. Université Paris-Diderot - Paris VII, 2010. Français. ⟨pastel-00545086⟩

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