Characterization and development of a supramolecular hydrogel matrix for 3D cellualized culture : towards a bio-indicator of the effect of manufactured nanoparticles

Le projet de cette thèse s’inscrit dans la démarche de développement de méthodes alternatives aux cultures cellulaires conventionnelles dites bi-dimensionnelles (2D), limitées en termes de représentativité du comportement cellulaire natif, et aux modèles animaux qui se heurtent à des questions socio-scientifiques poussant à réduire leurs recours. Les présents travaux portent sur une série d’études impliquant l’application d’une nouvelle matrice à base d’hydrogels supramoléculaires pour la culture cellulaire dite tri-dimensionnelle (3D).Le consortium de chercheurs impliqués dans ce projet s’intéresse aux gélifiants supramoléculaires basés sur la chimie des acides nucléiques. Dans un premier temps, certaines molécules candidates ont donc été étudiées sur le plan fondamental afin d’approfondir notre compréhension des mécanismes thermodynamiques et cinétiques sous-jacents du processus de gélification supramoléculaire.Les matrices candidates ont ensuite été caractérisées dans l’objectif d’aboutir à un support biocompatible aux propriétés physico-chimiques optimales, répondant notamment aux spécificités de la culture 3D des cellules du glioblastome. En parallèle de cette étude, le modèle cellulaire a été affiné dans le but d’améliorer la reproductibilité des résultats par le développement d’une méthode de tri cellulaire, permettant de sélectionner une sous-population homogène de cellules souches parmi la population totale de cellules cancéreuses étudiée.Une fois validée, cette nouvelle méthode de culture cellulaire 3D a été mise à contribution pour l’étude des effets de nanoparticules manufacturées d’argent. Dans cette optique, il est nécessaire de pouvoir relier les caractéristiques des nanoparticules à leur impact sur le sphéroïde tumoral. La stratégie pour appréhender cette question se divise en plusieurs étapes : 1) la détermination de l’état des nanoparticules dans le milieu de culture ; 2) l’étude de la diffusion des nanoparticules au sein de la matrice de culture ; 3) la mise en place des méthodes de suivi de l’évolution du système biologique en présence des nanoparticules.Ces travaux pluridisciplinaires dans les domaines de la chimie supramoléculaire, de la culture cellulaire et de la chimie analytique ont été réalisés au sein d’un consortium de partenaires des universités de Bordeaux, Limoges et Pau. Ils ouvrent la voie à des études ultérieures plus poussées sur l’effet de ces nanoparticules par le biais d’un nouveau bio-indicateur.The project of this thesis involves the development of alternative methods to conventional cell cultures called two-dimensional (2D), which are limited in terms of representativeness of the native cell behavior, and to animal models which are facing socio-scientific issues leading to reduce their use. The present work focuses on a series of studies implying the application of a new matrix based on supramolecular hydrogels for three-dimensional (3D) cell cultures. The consortium of researchers involved in this project is interested in supramolecular gelling agents based on nucleic acid chemistry. In a first step, some potential molecules have been studied at the fundamental level in order to deepen our understanding of the thermodynamic and kinetic mechanisms underlying the supramolecular gelation process.The candidate matrices were then characterized in order to obtain a biocompatible support with optimal physico-chemical properties, particularly in response to the specificities of the 3D culture of glioblastoma cells. In parallel to this study, the cell model was refined in order to improve the reproducibility of the results by developing a cell sorting method, allowing the selection of a homogeneous subpopulation of stem cells among the total population of cancer cells studied.Once validated, this new 3D cell culture method was used to study the effects of manufactured silver nanoparticles. In this perspective, it is necessary to be able to link the characteristics of the nanoparticles to their impact on the tumor spheroid. The strategy to address this issue is divided into several steps: 1) determination of the state of the nanoparticles in the culture medium; 2) study of the diffusion of the nanoparticles within the culture matrix; 3) implementation of methods to monitor the evolution of the biological system in the presence of nanoparticles.This multidisciplinary work in the fields of supramolecular chemistry, cell culture and analytical chemistry was carried out within a consortium of partners from the universities of Bordeaux, Limoges and Pau. They open the way to further studies on the effect of these nanoparticles through a new bio-indicator

Evaluation of Amyloid Inhibitor Efficiency to Block Bacterial Survival

info:eu-repo/semantics/publishe

Biomaterials for Three-Dimensional Cell Culture: From Applications in Oncology to Nanotechnology

Three-dimensional cell culture has revolutionized cellular biology research and opened the door to novel discoveries in terms of cellular behavior and response to microenvironment stimuli. Different types of 3D culture exist today, including hydrogel scaffold-based models, which possess a complex structure mimicking the extracellular matrix. These hydrogels can be made of polymers (natural or synthetic) or low-molecular weight gelators that, via the supramolecular assembly of molecules, allow the production of a reproducible hydrogel with tunable mechanical properties. When cancer cells are grown in this type of hydrogel, they develop into multicellular tumor spheroids (MCTS). Three-dimensional (3D) cancer culture combined with a complex microenvironment that consists of a platform to study tumor development and also to assess the toxicity of physico-chemical entities such as ions, molecules or particles. With the emergence of nanoparticles of different origins and natures, implementing a reproducible in vitro model that consists of a bio-indicator for nano-toxicity assays is inevitable. However, the maneuver process of such a bio-indicator requires the implementation of a repeatable system that undergoes an exhaustive follow-up. Hence, the biggest challenge in this matter is the reproducibility of the MCTS and the associated full-scale characterization of this system’s components

Self-Assembly of Nucleoside-Derived Low-Molecular-Weight Gelators: A Thermodynamics and Kinetics Study on Different Length Scales

International audienceBiocompatible materials are of paramount importance in numerous fields. Unlike chemically bridge polymer-based hydrogels, low-molecular-weight gelators can form a reversible hydrogel as their structures rely on noncovalent interaction. Although many applications with this type of hydrogel can be envisioned, we still lack their understanding due to the complexity of their self-assembly process and the difficulty in predicting their behaviors (transition temperature, gelation kinetics, the impact of solvent, etc.). In this study, we extend the investigations of a series of nucleosidederived gelators, which only differ by subtle chemical modifications. Using a multitechnique approach, we determined their thermodynamic and kinetic features on various scale (molecular to macro) in different conditions. Monitored at the supramolecular level by circular dichroism as well as macroscopic scales by rheology and turbidimetry, we found out that the sol-gel and gel-sol transitions are greatly dependent on the concentration and on the mechanisms that are probed. Self-assembly kinetics depends on hydrogel molecules and is modulated by temperature and solvent. This fundamental study provides insight on the impact of some parameters on the gelation process, such as concentration, cooling rate, and the nature of the solvent

Epigallocatechin Gallate Remodelling of Hfq Amyloid-Like Region Affects Escherichia coli Survival

Hfq is a pleiotropic regulator that has key roles in the control of genetic expression. The protein noticeably regulates translation efficiency and RNA decay in Gram-negative bacteria, due to the Hfq-mediated interaction between small regulatory noncoding RNA and mRNA. This property is of primary importance for bacterial adaptation and virulence. We have previously shown that the Hfq E. coli protein, and more precisely its C-terminal region (CTR), self-assembles into an amyloid-like structure. In the present work, we demonstrate that epigallocatechin gallate (EGCG), a major green tea polyphenol compound, targets the Hfq amyloid region and can be used as a potential antibacterial agent. We analysed the effect of this compound on Hfq amyloid fibril stability and show that EGCG both disrupts Hfq-CTR fibrils and inhibits their formation. We show that, even if EGCG affects other bacterial amyloids, it also specifically targets Hfq-CTR in vivo. Our results provide an alternative approach for the utilisation of EGCG that may be used synergistically with conventional antibiotics to block bacterial adaptation and treat infections

Interactions between DNA and the Hfq Amyloid-like Region Trigger a Viscoelastic Response

10.1021/acs.biomac.0c00747BIOMACROMOLECULES2193668-367

Revised role for Hfq bacterial regulator on DNA topology

Abstract Hfq is a pleiotropic regulator that mediates several aspects of bacterial RNA metabolism. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, usually via its interaction with small regulatory RNA. Besides these RNA-related functions, Hfq has also been described as one of the nucleoid associated proteins shaping the bacterial chromosome. Therefore, Hfq appears as a versatile nucleic acid-binding protein, which functions are probably even more numerous than those initially suggested. For instance, E. coli Hfq, and more precisely its C-terminal region (CTR), has been shown to induce DNA compaction into a condensed form. In this paper, we establish that DNA induces Hfq-CTR amyloidogenesis, resulting in a change of DNA local conformation. Furthermore, we clarify the effect of Hfq on DNA topology. Our results evidence that, even if the protein has a strong propensity to compact DNA thanks to its amyloid region, it does not affect overall DNA topology. We confirm however that hfq gene disruption influences plasmid supercoiling in vivo, indicating that the effect on DNA topology in former reports was indirect. Most likely, this effect is related to small regulatory sRNA-Hfq-based regulation of another protein that influences DNA supercoiling, possibly a nucleoid associated protein such as H-NS or Dps. Finally, we hypothesise that this indirect effect on DNA topology explains, at least partially, the previously reported effect of Hfq on plasmid replication efficiency

Isolation of glioblastoma cancer stem cells by sedimentation field flow fractionation for the development of 3D models in supramolecular hydrogels

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Interactions between DNA and the Hfq Amyloid-like Region Trigger a Viscoelastic Response

International audienceMolecular transport of biomolecules plays a pivotal role in the machinery of life. Yet, this role is poorly understood due the lack of quantitative information. Here, the role and properties of the C-terminal region of Escherichia coli Hfq is reported, involved in controlling the flow of a DNA solution. A combination of experimental methodologies has been used to probe the interaction of Hfq with DNA and to measure the rheological properties of the complex. A physical gel with a temperature reversible elasticity modulus is formed due to formation of non-covalent crosslinks. The mechanical response of the complexes shows that they are inhomogeneous soft solids. Our experiments indicate that Hfq C-terminal region could contribute to genome's mechanical response. The reported viscoelasticity of the DNA-protein complex might have implications for cellular processes involving molecular transport of DNA or segments thereof