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Développement d’un criblage automatisé de l’activité kinase d’un biosenseur Aurora A par FLIM

Abstract : Overexpression of Aurora A is a major marker of some epithelial cancers. This gene encodes the multifunctional Aurora A kinase and its activation is required for entry and progression to mitosis. So far, no inhibitor of this oncogene has been approved by the FDA and it is therefore essential to identify new molecules. Our team developed a Forster Resonance Energy Transfer (FRET) biosensor for Aurora A kinase activity, consisting of the entire kinase flanked by two fluorophores, a GFP and a mCherry. The conformational change of Aurora A when it is activated brings the fluorophores closer and increases FRET efficiency. It is thus possible to follow the activation of Aurora A in living cells expressing the biosensor at endogenous levels. We can measure FRET using FLIM (Fluorescence Lifetime Imaging Microscopy) technique using a microscope developed in the team called fastFLIM. My thesis work consisted of developing a robust and automated screening strategy by combining the capabilities of fastFLIM and the Aurora A activity biosensor. This strategy based on automation of acquisitions and data analysis allowed to screen a library of 96-well plate molecules for potential inhibitors of Aurora A kinase activity. In addition, I participated in the validation of the biosensor for kinase activity monitoring in living cells, showing that the FRET variations measured correspond to the phosphorylation state of Aurora A on the Threonine 288 residue, a marker of its activation. Finally, I participated in the development of new microscopy techniques to monitor the activity of the biosensor. For that, I used a homoFRET biosensor with the challenge of being able to use several biosensors in a multiplex context. I also used the 2c-FCCS (2-color Fluorescence Cross Correlation Spectroscopy) technique on the Aurora A biosensor to measure FRET in regions where it is weakly expressing and whose lifetime measurement of Fluorescence is not possible by FLIM. Thus, my thesis work is part of the trend to develop a quantitative and autonomous microscopy with the challenge of providing a large number of phenotypic data.
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Submitted on : Wednesday, March 4, 2020 - 3:33:45 PM
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  • HAL Id : tel-02498589, version 1


Florian Sizaire. Développement d’un criblage automatisé de l’activité kinase d’un biosenseur Aurora A par FLIM. Biologie du développement. Université Rennes 1, 2019. Français. ⟨NNT : 2019REN1B033⟩. ⟨tel-02498589⟩



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