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Outils microfluidiques pour le tri et la caractérisation biophysiques des spores fongiques

Year: 2025
Type: dissertation
Abstract: B. cinerea is a necrotrophic plant pathogen that can infect over 200 plant species worldwide and is considered the second most significant fungal pathogen. Genetically modified strains are often produced by biologists in order to study the influence of specific genes on fungal pathogenesis. However, because B. cinerea’s spores are multinucleated, obtaining a purified population in which all nuclei contain the desired mutation is a long and tedious process that can take up to several months. One strategy to speed up the purification process is to isolate mononucleated spores, hence ensuring homogenous DNA. To do so, we first examined a possible correlation between spore size and nuclei number, thus demonstrating that spores with a single nucleus tend to be smaller than with multiple nuclei. Hence, we propose a microfluidic tool implementing a size-based sorting approach to enrich a spore suspension with mononucleated spores, using deterministic lateral displacement (DLD). We first characterize the DLD chip using a commercial bead suspension, in order to optimize the device design, flow parameters, and suspending media composition. We then quantify its sorting efficiency with the same bead suspension. Finally, we test the chip with B. cinerea spores and observe its ability to enrich a suspension initially containing a mix of mono- and multinucleated spores. After processing, the sample is enriched with mononucleated spores, increasing the likelihood of obtaining homogeneous DNA and demonstrating the chip's potential to accelerate fungal genetic purification. In addition, biophysical signatures of the fungal cell wall (FCW) were investigated to provide insights into its mechanical and electrical properties, using Atomic Force Microscopy (AFM) and Electrical Impedance Spectroscopy (EIS), respectively. For the AFM experiments, we employed a tipless cantilever to compare the indentation response of the wild-type strain and the mutant strain ΔAGS; the latter impacting the FCW. This allowed us to assess differences in mechanical stiffness between the two strains. For the impedance measurements, we fabricated a microfluidic device for single spore trapping over integrated pairs of gold electrodes. Preliminary tests were performed using polystyrene beads to validate the setup. To our knowledge, this has not been previously addressed on this strain of fungal spores and our goal is to get knowledge of how the fungal wall can be affected by mutations disrupting genes involved in its synthesis as well as finding a signature correlated to the number of nuclei.
Language: English
Open Access status: green
OpenAlex ID: W7131333036
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