| Doctorant : |
Nizar AYADI
|
| Directeur de thèse : |
Fabrice FLEURY ,
Professeur Université |
| Encadrant : |
Houda BENHELLI ,
Maître de conférences Université |
| Financement : |
ANR (projet Freebiowave) |
| Date de la soutenance : |
jeudi 12 juin 2025, 14h30 |
| Modalité : |
- Lieu : Amphithéâtre Pasteur, bâtiment 2, campus Lombarderie
|
| Jury : |
- Rapporteur : Benoît Chénais, Professeur, BIOSSE, Université du Mans
- Rapporteur : Masayuki Takahashi, Directeur de recherche, Institut des Sciences, Tokyo
- Examinateur : Ibtissam Talhaoui, Chargée de recherche, Centre de Biophysique Moléculaire, Orléans
- Examinateur : Alena Sukhanova, Ingénieure de recherche, BioSpecT-EA7506, Université de Reims
- Directeur de thèse :
Fabrice FLEURY ,
Professeur Université
- Encadrant :
Houda BENHELLI ,
Maître de conférences Université
|
Actuellement, pour surveiller la malignité d’une tumeur, des biopsies sont nécessaires, mais cela représente un risque pour le patient car c’est invasif. De nouvelles techniques non invasives sont toujours en cours de développement afin d’assurer la surveillance en continue de la tumeur. Ainsi, les puces à protéines sont apparues comme essentielles dans le diagnostic, la découverte de médicaments et dans la protéomique. Dans ce contexte, on a développé un prototype d’imagerie multiparamétrique basé sur les avantages des cristaux photoniques qui est une alternative à la SPRi. Les données de cette étude ont démontré premièrement, qu’il était possible de détecter en temps réel et sans marquage, plusieurs interactions en simultanée. Dans un second temps, la technologie a été utilisée dans le suivi en temps réel du recrutement de la protéine RAD51, protéine centrale de la recombinaison homologue, sur l’ADNsb. Ainsi les résultats montrent une forte sensibilité de détection des évènements d’association-dissociation, permettant de calculer les paramètres cinétiques et ouvrent la voie pour un criblage à haut débit de nouveaux inhibiteurs de RAD51 impliqué dans la chimiorésistance. Dans ce même contexte, j’ai mené une étude dans la compréhension des mécanismes de régulation de RAD51, par la phosphorylation, ou encore par des cofacteurs tels que l’ATP et le calcium. Les résultats obtenus montrent une double régulation par le calcium, une fixation au niveau de la poche ATPase et pour la première fois, une fixation du calcium au niveau la région N-terminale de la protéine sur les résidus E16, E17 et E18.
En ce qui concerne la régulation de RAD51 par les MPTs, on a découvert une nouvelle phosphorylation sur la Ser97 du motif SRM. Ici on a démontré pour la première fois, que la PhosphoS97 de RAD51 augmente l’activité de la D-loop, la fixation et l’hydrolyse de l’ATP ainsi que l’affinité de RAD51 pour l’ADNsb.
Publications
2023
Nifontova, Galina; Petrova, Irina; Gerasimovich, Evgeniia; Konopsky, Valery N.; Ayadi, Nizar; Charlier, Cathy; Fleury, Fabrice; Karaulov, Alexander; Sukhanova, Alyona; Nabiev, Igor
Label-Free Multiplexed Microfluidic Analysis of Protein Interactions Based on Photonic Crystal Surface Mode Imaging Article de journal
Dans: International Journal of Molecular Sciences, vol. 24, no. 5, 2023, ISSN: 1422-0067.
@article{ijms24054347b,
title = {Label-Free Multiplexed Microfluidic Analysis of Protein Interactions Based on Photonic Crystal Surface Mode Imaging},
author = {Galina Nifontova and Irina Petrova and Evgeniia Gerasimovich and Valery N. Konopsky and Nizar Ayadi and Cathy Charlier and Fabrice Fleury and Alexander Karaulov and Alyona Sukhanova and Igor Nabiev},
url = {https://www.mdpi.com/1422-0067/24/5/4347},
doi = {10.3390/ijms24054347},
issn = {1422-0067},
year = {2023},
date = {2023-02-22},
urldate = {2023-02-22},
journal = {International Journal of Molecular Sciences},
volume = {24},
number = {5},
abstract = {High-throughput protein assays are crucial for modern diagnostics, drug discovery, proteomics, and other fields of biology and medicine. It allows simultaneous detection of hundreds of analytes and miniaturization of both fabrication and analytical procedures. Photonic crystal surface mode (PC SM) imaging is an effective alternative to surface plasmon resonance (SPR) imaging used in conventional gold-coated, label-free biosensors. PC SM imaging is advantageous as a quick, label-free, and reproducible technique for multiplexed analysis of biomolecular interactions. PC SM sensors are characterized by a longer signal propagation at the cost of a lower spatial resolution, which makes them more sensitive than classical SPR imaging sensors. We describe an approach for designing label-free protein biosensing assays employing PC SM imaging in the microfluidic mode. Label-free, real-time detection of PC SM imaging biosensors using two-dimensional imaging of binding events has been designed to study arrays of model proteins (antibodies, immunoglobulin G-binding proteins, serum proteins, and DNA repair proteins) at 96 points prepared by automated spotting. The data prove feasibility of simultaneous PC SM imaging of multiple protein interactions. The results pave the way to further develop PC SM imaging as an advanced label-free microfluidic assay for the multiplexed detection of protein interactions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
High-throughput protein assays are crucial for modern diagnostics, drug discovery, proteomics, and other fields of biology and medicine. It allows simultaneous detection of hundreds of analytes and miniaturization of both fabrication and analytical procedures. Photonic crystal surface mode (PC SM) imaging is an effective alternative to surface plasmon resonance (SPR) imaging used in conventional gold-coated, label-free biosensors. PC SM imaging is advantageous as a quick, label-free, and reproducible technique for multiplexed analysis of biomolecular interactions. PC SM sensors are characterized by a longer signal propagation at the cost of a lower spatial resolution, which makes them more sensitive than classical SPR imaging sensors. We describe an approach for designing label-free protein biosensing assays employing PC SM imaging in the microfluidic mode. Label-free, real-time detection of PC SM imaging biosensors using two-dimensional imaging of binding events has been designed to study arrays of model proteins (antibodies, immunoglobulin G-binding proteins, serum proteins, and DNA repair proteins) at 96 points prepared by automated spotting. The data prove feasibility of simultaneous PC SM imaging of multiple protein interactions. The results pave the way to further develop PC SM imaging as an advanced label-free microfluidic assay for the multiplexed detection of protein interactions.
2020
Ayadi, Nizar; Lafont, Florian; Charlier, Cathy; Benhelli-Mokrani, Houda; Sokolov, Pavel; Sukhanova, Alyona; Fleury, Fabrice; Nabiev, Igor
Comparative Advantages and Limitations of Quantum Dots in Protein Array Applications Chapitre d'ouvrage
Dans: Quantum Dots, vol. 2135, p. 259–273, Springer, New York, NY, Humana, 2020.
@inbook{cEQ3:ayadi_FLEURY:2020,
title = {Comparative Advantages and Limitations of Quantum Dots in Protein Array Applications},
author = {Nizar Ayadi and Florian Lafont and Cathy Charlier and Houda Benhelli-Mokrani and Pavel Sokolov and Alyona Sukhanova and Fabrice Fleury and Igor Nabiev},
year = {2020},
date = {2020-04-01},
booktitle = {Quantum Dots},
volume = {2135},
pages = {259--273},
publisher = {Springer},
address = {New York, NY},
edition = {Humana},
series = {Methods in Molecular Biology},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}
2018
Lafont, Florian; Ayadi, Nizar; Charlier, Cathy; Weigel, Pierre; Nabiev, Igor; Benhelli-Mokrani, Houda; Fleury, Fabrice
Assessment of DNA-PKcs kinase activity by quantum dot–based microarray Article de journal
Dans: Scientific Reports, vol. 8, no. 1, p. 1–12, 2018, ISSN: 20452322.
@article{Lafont2018,
title = {Assessment of DNA-PKcs kinase activity by quantum dot–based microarray},
author = {Florian Lafont and Nizar Ayadi and Cathy Charlier and Pierre Weigel and Igor Nabiev and Houda Benhelli-Mokrani and Fabrice Fleury},
doi = {10.1038/s41598-018-29256-2},
issn = {20452322},
year = {2018},
date = {2018-01-01},
journal = {Scientific Reports},
volume = {8},
number = {1},
pages = {1--12},
abstract = {Therapeutic efficacy against cancer is often based on a variety of DNA lesions, including DNA double-strand breaks (DSBs) which are repaired by homologous recombination and non-homologous end joining (NHEJ) pathways. In the past decade, the functions of the DNA repair proteins have been described as a potential mechanism of resistance in tumor cells. Therefore, the DNA repair proteins have become targets to improve the efficacy of anticancer therapy. Given the central role of DNA-PKcs in NHEJ, the therapeutic efficacy of targeting DNA-PKcs is frequently described as a strategy to prevent repair of treatment-induced DNA damage in cancer cells. The screening of a new inhibitor acting as a sensitizer requires the development of a high-throughput tool in order to identify and assess the most effective molecule. Here, we describe the elaboration of an antibody microarray dedicated to the NHEJ pathway that we used to evaluate the DNA-PKcs kinase activity in response to DNA damage. By combining a protein microarray with Quantum-Dot detection, we show that it is possible to follow the modification of phosphoproteomic cellular profiles induced by inhibitors during the response to DNA damage. Finally, we discuss the promising tool for screening kinase inhibitors and targeting DSB repair to improve cancer treatment.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Therapeutic efficacy against cancer is often based on a variety of DNA lesions, including DNA double-strand breaks (DSBs) which are repaired by homologous recombination and non-homologous end joining (NHEJ) pathways. In the past decade, the functions of the DNA repair proteins have been described as a potential mechanism of resistance in tumor cells. Therefore, the DNA repair proteins have become targets to improve the efficacy of anticancer therapy. Given the central role of DNA-PKcs in NHEJ, the therapeutic efficacy of targeting DNA-PKcs is frequently described as a strategy to prevent repair of treatment-induced DNA damage in cancer cells. The screening of a new inhibitor acting as a sensitizer requires the development of a high-throughput tool in order to identify and assess the most effective molecule. Here, we describe the elaboration of an antibody microarray dedicated to the NHEJ pathway that we used to evaluate the DNA-PKcs kinase activity in response to DNA damage. By combining a protein microarray with Quantum-Dot detection, we show that it is possible to follow the modification of phosphoproteomic cellular profiles induced by inhibitors during the response to DNA damage. Finally, we discuss the promising tool for screening kinase inhibitors and targeting DSB repair to improve cancer treatment.
Lien
