ph.D thesis of Eliana Akoury (2024-2027)

The thesis is part of the FluoPath project funded by the French National Research Agency (ANR) over the period April 2024-March 2028. This project brings together 7 partners, including 4 academic research laboratories (UMR PAM, UMR SECALIM, UMR SQPOV and LUBEM), 2 Agro-Industrial Technical Institutes (AERIAL and ADRIA) and the dairy interprofession (CNIEL).
The primary aim of the FluoPath project is to determine new biomarkers (promoters inducing the expression of genes of interest) coupled with a fluorescent biosensor, enabling new insights to be gained into the physiological state of two pathogens (Listeria monocytogenes and Bacillus cereus) in dairy environments (milk, diluted model cheese and, if possible, solid model cheese) in relation to the impact of technological disturbances. Ultimately, this knowledge, combined with that available in the scientific literature, will be used to improve predictive models for microbiological risk in dairy products.
The thesis will focus on the pathogen L. monocytogenes, and will be carried out in the UMR PAM (Dijon) and UMR SECALIM (Nantes) academic laboratories, according to a schedule that will not require the student to travel regularly between the 2 cities, due to their geographical distance (travel expenses will be borne by the host laboratories). The 3-year thesis will start in October 2024 and end in September 2027.

The thesis will be structured around 4 axes
Axis 1: Selection of two strains out of six of L. monocytogenes and two relevant stress conditions
The main objective of this axis will be to evaluate the resistance of six L. monocytogenes strains to two stress conditions encountered in the dairy industry's unit operation chains. Statistical analysis of these phenotypes of interest will enable us to select, from these six strains, two with different levels of resistance. These strains will be used throughout the thesis.
Axis 2: Identification of mRNAs specific to each phenotype of interest
The aim of this axis is to identify virulence or stress genes that are over-expressed when the two stress conditions are applied to the two strains selected in Axis 1. The level of expression of the genes of interest, identified by a literature review, will be measured by RT-qPCR. In order to generate new fundamental knowledge, an unbiased transcriptomic analysis will be carried out using RNAseq, combined with a bioinformatics analysis (ontology, identification of promoter sequences specifically involved in each phenotype, etc.).
Axis 3: Development of a fluorescent mutant bank by transcriptional (chromosomal) fusion
The genes of interest identified in Axis 2 will be considered as candidates for building a mutant bank from the two strains previously selected (Axis 1). These mutants, which fluoresce due to the insertion of a gene coding for a fluorescent protein (GFP, RFP, CFP or YFP), will be used to generate new knowledge relating to the impact of stress conditions on the resistance and virulence of L. monocytogenes.
Axis 4: Quantification of mutant fluorescence under different stress conditions
In this area, we will evaluate the fluorescence emitted by mutants placed under previously established stress conditions. In particular, the experimental conditions will be optimized to avoid artifacts such as autofluorescence of the milk matrix.