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Effets des champs électriques pulsés milli et nanosecondes sur cellules et tissus

Louise Chopinet-Mayeux 1
1 LAAS-NBS - Équipe NanoBioSystèmes
LAAS - Laboratoire d'analyse et d'architecture des systèmes
Abstract : Electropermeabilization is a physical technique first developed to transfer cytotoxic drugs in tumor. It consists in the transient permeabilization of the plasma membrane following electric field application. In specific electric conditions using long pulses of several milliseconds, the membrane destabilization can allow transferring plasmid DNA into the cell, thus allowing the development of gene therapy. For now, one clinical trial has been published using gene eletro-transfer and several others are ongoing. However the efficiency of the technique remains low compared to other transfer methods. This thesis gets interested in how pulsed electric fields affect cell membranes, in the concrete situation of gene transfer by electroporation. The comprehension of electro-gene transfer process need to be well understood in order to optimize it. In this context, we focus on the 3 barriers that DNA is confronted to during its transfer: first at the cell level: plasma membrane and nuclear envelope, second at the tissue level: the complexity of a multicellular environment. i) We first studied the efficiency of gene transfer on multicellular spheroid model. This work allowed the validation of this model for electro-transfection study, and the further optimization of the technique by raising some of the failures encountered in gene transfer in tissue. ii) The second part of the work has been dedicated to study plasma membrane destabilization due to electroporation by Atomic Force Microscopy. We used both innovating imaging modes and spectroscopy modes to analyze the effects on living cells, which resulted in the measurement of a decrease in elasticity, linked to side effects of electric fields on actin cytoskeleton destabilization. iii) The last part has been dedicated to the effects of nanopulses (nsEP) on both plasma membrane and the second barrier encountered by gene during its transfer, namely nuclear envelope. The effects of these very short (ns) and intense (several kV/cm) pulses have been indeed shown to affect both cell membrane and internal envelope (organelles ones). We first study their effect on membrane using patch-clamp to discriminate in the implication of actin cytoskeleton in nanopores formation. We secondly aimed to study how these nanopulses affect the special structure that is nuclear envelope during gene transfer, for validating their potential use on humans, and their possible role in optimization for gene transfer. P-clamp study revealed that actin is not involved in nanopores formation, and gene transfer one that nsEP do not affect positively transfection efficiency. Altogether this thesis brings new insights in electropermeabilization mecanisms understanding and barriers for gene transfer in tissue. Methods, models and tools have been set and validated. They are now usable for investigating electric field effect on living organisms.
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Submitted on : Wednesday, October 1, 2014 - 7:00:06 AM
Last modification on : Thursday, June 10, 2021 - 3:01:28 AM
Long-term archiving on: : Friday, January 2, 2015 - 10:10:23 AM

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  • HAL Id : tel-00874040, version 1

Citation

Louise Chopinet-Mayeux. Effets des champs électriques pulsés milli et nanosecondes sur cellules et tissus. Micro et nanotechnologies/Microélectronique. Université Paul Sabatier - Toulouse III, 2013. Français. ⟨tel-00874040⟩

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