FOURNIER Marie: New measurement tools for the study of molecular dynamics applied to the regulation of transcription in response to stress.

PhD thesis: The three-dimensional architecture of the cell nucleus, and more precisely chromatin, plays an important role in the dynamics of transcription factors (FTs) and in the regulation of gene expression. However, at present, the dynamics of transcription factors within the nucleus remains poorly understood. The aim of this thesis is to develop new tools for measuring molecular dynamics combining 1) a light film microscopy device, 2) photoconvertible proteins, 3) fluorescence correlation spectroscopy (Fluorescence correlation spectroscopy). Light-sheet illumination allows unmatched optical isolation of the cell nucleus in other optical microscopy methods, and allows, among other things, to achieve confocal-like resolutions in a more rapid manner. In addition, this technique rejects out-of-focus contributions and allows for a higher signal-to-noise ratio, which is essential when the sample is as complex and congested as the cell nucleus. The use of photoconvertible proteins makes it possible to address photo-activated localization microscopy protocols by single-particle-tracking Photo-Activated Localization Microscopy (spt-PALM) which is a super-resolution technique (price Nobel of chemistry 2014) to determine the molecular dynamics over times ranging from ten ms to ten minutes. The use of FCS makes it possible to measure intra-nuclear diffusion over very short times (μs-ms). The challenge of data mining is to understand the spatio-temporal dynamics of transcription factors seen by two measurement methods at different time ranges. The interest is to supplement the information derived from the FCS which is the representation of an average behavior of the molecules of interest by more precise spatio-temporal data resulting from spt-PALM realized on a smaller number of molecules but which allow to observe subpopulations with different behaviors (confinement, alternation of fast and slow diffusion ...).

From a biological point of view, the study will focus on the PTEFb complex, an activating transcription factor for transcriptional elongation and its interaction with Pol II RNA. This research will have implications for the understanding of uncontrolled activation of the stress pathway in various pathologies such as cancer, or in the recruitment of PTEFb in retrotranscription mechanisms following retrovirus infection such as eg HIV

FOURNIER Marie - marie.fournier[chez]univ-lille[point].fr 

Thesis director: HELIOT Laurent