PhD defense of Mr. Alan Vandenberghe

VANDENBERGHE Alan, PhLAM Laboratory - UMR8523 - Team DYSCO

Title: Experimental study of the coupling between circadian clock and metabolism

Jury: M. LEFRANC (PhLAM, supervisor), A. FURLAN (UMR 9020, supervisor), F.-Y. BOUGET (Sorbonne Université), F. DELAUNAY (Université Côte d'Azur), E. Werkmeister (Université de Lille), A. Ballesta (Université Paris Sciences & Lettres)

Abstract:

The circadian clock is a biological function that regulates various physiological processes throughout the day, allowing anticipation of daily changes resulting from the day/night cycle. Proper functioning of this clock requires constant synchronization with light/darkness and feeding/fasting cycles, which is ensured by signals related to ambient light and various metabolic factors. However, the mechanisms involved in clock synchronization through food intake remain poorly understood. To address this question, the laboratory has developed a mathematical model integrating metabolic factors into the molecular network of the circadian clock to explain synchronization by cell energy sensors. This model suggests that AMPK and SIRT1, cell energy sensors, control this action on the clock. As part of this thesis, I verified this implication of AMPK in circadian clock synchronization. To address this issue, we adopted a quantitative approach of single-cell measurements based on fluorescence microscopy, and we worked on the establishment of fluorescent reporters of the circadian cycle. Unfortunately, this approach has not yet yielded results, but it has allowed me to set up a videomicroscopy system coupled with a microfluidic station, as well as analysis and tracking tools through which I assessed the level of AMPK activity depending on the dose of AICAR administered to the cells, using a FRET biosensor. To understand the importance of AMPK in regulating the circadian cycle, I used a more established model, namely U2OS pBmal1-luc cells expressing luciferase under the control of the circadian promoter pBmal1. By modulating AMPK activity through AICAR administration, an AMP analogue, I demonstrated a dose-dependent positive impact of AICAR on Bmal1 promoter activity. In order to quantify the observed changes induced by AICAR, I implemented a circadian signal processing procedure, allowing the instantaneous phase and period of circadian signals to be obtained using the Hilbert transform. I was thus able to show that the addition of AICAR caused an increase in the period of the circadian cycle in human U2OS cells and that it temporally shifted the clock by an amount dependent on the administration time. Interestingly, these experimental results are similar to those generated in simulations conducted with the laboratory model. This consistency thus strengthens the initial hypothesis regarding the integration of metabolic factors relayed by AMPK in the synchronization of the circadian clock via the regulation of the Bmal1 promoter.