Defense of thesis Alan Vanderberghe

Defense of thesis Alan Vanderberghe - laboratory PhLAM

Abstract :

The circadian clock is a biological function that provides our organisms with an internal measure of time throughout the day. It orchestrates various physiological processes appropriately, anticipating daily changes resulting from the day/night cycle. The proper functioning of this clock requires constant synchronization to light/dark and feeding/fasting cycles, which is ensured by signals related to ambient light and various metabolic factors. However, the mechanisms involved in the synchronization of the clock through food intake remain poorly understood. To address this question, a mathematical model was previously developed in the laboratory, integrating the metabolic sensors AMPK and SIRT1 into the molecular network of the circadian clock.
In this thesis, I focused on experimentally verifying the involvement of AMPK in the synchronization of the circadian clock. I used a well-established cellular model, namely stable U2OS B6 cells transfected with a circadian reporter pBmal1-luc expressing a luciferase under the control of the clock gene Bmal1 promoter. By modulating AMPK activity through the administration of AICAR, an AMP analogue, I demonstrated that it significantly and dose-dependently increased the activity of the Bmal1 promoter. In order to quantify the effects of AICAR on the instantaneous phase and period of the clock, I implemented a circadian signal processing procedure using the Hilbert transform to robustly obtain these metrics. I was able to show that the administration of AICAR caused an elongation of the circadian cycle period, and this effect on the clock phase depends on the timing of administration. Remarkably, these different effects depend on NAD+, as we demonstrated through the administration of FK866, an inhibitor of the enzyme NAMPT. These experimental results are consistent with the predictions of the mathematical model, reinforcing the initial hypothesis regarding the role of an AMPK-Bmal1 axis in the synchronization of the circadian clock.
We also established a single-cell measurement approach coupling a video microscopy system with a microfluidic device. While the development of fluorescent circadian reporters has not yet been successful, the use of a FRET biosensor allowed us to precisely characterize the response of AMPK activity to AICAR and confirm that this activation was significant at the doses used in our studies.

Keywords : Circadian Clock,Metabolism,AMPK