Mesure par microscopie confocale du métabolisme mitochondrial et du niveau énergétique cellulaire au cours d’épisodes de carences en substrats et/ou en oxygène

Abstract : Mitochondria form an information hub at the center of the cellular metabolism because of its physiological role consisting in the porduction of ATP from the degradation of porducts stemming from our food through the OXPHOS process. However, changes in the functionnig of the mitochondria can be responsible for numerous diseases. Among the different foms of metabolic stress leading to mitchondrial dysfunctions, ischemia-reperfusion can be found in numerous pathological situations. This work aims at developing a methodological approach based on confocal microscopy and image analysis to dissect –at cell level- the consequences of metabolic stress induced by episodes of deprivation in substrata associated or not with hypoxia or anoxia. Having developed the program of image analysis based on the « tophat » method, two approaches were designed to vizualize and quantify the mitochondrial function. The first one, combining TMRM labelling with NADH fluorescence made it possible to highlight some differences in the response to the stress caused by ischemia-reperfusion at the level of the respiratory chain or concerning the PTP opening in the four cellular types that were tested : HMEC-1, INS1, RT112 or pirmary heaptocyes. The second approach consisted in testing the use of biosensors designed to follow the variations of ATP concentration (ATeam) or the activation of AMPK (AMPKAR). The experimental conditions established in this work did not allow us to validate their use.
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Cécile Cottet‐rousselle. Mesure par microscopie confocale du métabolisme mitochondrial et du niveau énergétique cellulaire au cours d’épisodes de carences en substrats et/ou en oxygène. Biologie cellulaire. École pratique des hautes études - EPHE PARIS, 2016. Français. ⟨NNT : 2016EPHE3096⟩. ⟨tel-02105228⟩

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