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Reference : UMR5221-PASETI-001
Workplace : MONTPELLIER
Date of publication : Wednesday, June 24, 2020
Scientific Responsible name : Pascal ETIENNE
Type of Contract : PhD Student contract / Thesis offer
Contract Period : 36 months
Start date of the thesis : 1 October 2020
Proportion of work : Full time
Remuneration : 2 135,00 € gross monthly
Description of the thesis topic
In humans, at the muscular level, there is great interindividual variability in response to the same mechanical stress. This phenotypic variability is multifactorial, influenced by environmental factors as well as by multiple genetic variants. Thus, for the same level of muscle injury, two subjects of the same anthropometric characteristic, of the same age, with the same medical history and the same level of physical activity, will show a very variable level of muscle regeneration underpinned by biological function. stem cells and their microenvironment. Faced with the same training, certain athletes can develop a muscular pathology with iterative lesions and deficits of repair more debilitating than others without the substratum of this greater susceptibility being known.
Several recent works have highlighted the presence of genome modifications linked to the environment and which could be transmitted intergenerationally. This result may explain the interindividual variability with regard to the biological function of muscle stem cells involved in the regulation of muscle mass and its function [Papadimitriou ID & al., BMC Genomics (2016) 17: 285], [Petrella JK , J Appl Physiol (2008) 104: 1736]. Thus, these epigenetic events which seem to regulate the adaptation of the muscle to mechanical stress, are still poorly understood, due to the small number of studies on the subject. Indeed, these studies require a multifactorial analysis on muscle biopsies from cohorts which must be as homogeneous as possible, well characterized clinically with regard to the adaptation of the muscle to mechanical stress. This in vivo characterization remains complex due to its lack of reproducibility and can be disabling in athletes. The possibility, from a simple muscle micro biopsy, to isolate the satellite cells in order to reconstitute a muscle on a longitudinal axis will allow us to standardize the evaluation of the biological function of the satellite cells which will represent a precise measurement for each sportsman of its muscle regeneration capacity.
Thus, the strategy in the medical accompaniment of each high level sportsman and the re-athletization could be individualized with his capacity of muscular regeneration evaluated on muscular organoid allowing to model on a lesion stress.
Scientific objectives / hypotheses / problems:
The first objective of this thesis is to propose a new method for precise and reproducible evaluation of the muscle regeneration capacity involved in the recovery of a muscle injury. The second objective is to assess the impact of training on this capacity for muscle regeneration. For this we propose a new approach by modeling mechanical stress on a muscular organoid created from stem cells of the sportsman.
The work of the doctoral student will firstly, from stem cells, validate a complete protocol for manufacturing the organoid representative in vitro of its regeneration capacity. In a second step, the doctoral student will have to propose, from the organoid, a reproducible method of evaluation of the biological function of stem cells, guaranteeing the capacity of muscular regeneration. This method will focus on the application of an eccentric mechanical stress, on the organoid, allowing to model in vitro a muscular lesion within the framework of an intensive effort. It will make it possible to monitor and evaluate in the laboratory, the cellular mechanisms put in place by the organoid to ensure the regeneration of its muscle fibers.
Scientific obstacles to overcome / challenges / challenges:
This project should follow on from a very positive working basis carried out using diaphragmatic muscle stem cells which have made it possible to model in vivo diaphragmatic dysfunction induced by mechanical ventilation (DDIV) in intensive care patients. The use of biocompatible and microstructured surfaces in the form of grooves to differentiate and linearly grow stem cells has shown excellent results (Figure 1). The first challenge is to optimize growth in order to successfully produce a true three-dimensional muscular organoid, representative of the in-vivo model. Several microstructuring models will be tested. A second challenge lies in the ability to implement mechanical stress in the context of the application of eccentric stress. As direct traction of the organoid is not an option, we propose to multiply and differentiate muscle stem cells on a flexible silicone-type support which can be stretched mechanically once the organoid is obtained. The success of such an experiment requires a strong link between the organoid and the membrane for the transfer of mechanical effort. The classic use of a porous and poorly adherent Matrigel® has been tested but does not appear to be relevant. We propose a better strategy which consists in grafting peptide ligands which have the advantage of being biomimetic but above all of being covalently attached to silicone. The last challenge for this thesis will be, with regard to the microgrooves, support of the muscular fibers of our organoid, to insert stimulation electrodes. These will allow us, during mechanical stretching, to induce a contraction of the organoid, making it possible to model an eccentric stress at the base of a muscle injury.
Outline of the methodology to be implemented:
In terms of sports seasonality, under the technical coordination and supervision of CREPS de Montpellier and the Fédération Française d'Athlétisme with the help of sports structures Occitanie Ligue du sport universitaire and Montpellier Athletic Méditerranée Métropole, we will define a cohort of 20 athletes young adults and 20 senior athletes (on a gender basis) who will undergo a defined training program with weekly strength exercises to increase muscle mass. Calibrated physical tests will regularly validate the muscular evolution of the subjects on the basis of common and identical activities. A muscular micro biopsy of the quadriceps and a blood sample will be taken at the start and end of the training program. The blood sample is part of a perspective of future work that will arise from this thesis. It will later assess whether an epigenetic signature predicting the muscular response to a lesional phenomenon could also be found in blood cells. Indeed, this study, complementary to this thesis subject, brings into play the hypothesis of an epigenetic origin on the resistance and the muscular regeneration capacity of the athlete. It should be led by the Genome and Cellular Plasticity in Development and Aging team, led by Jean-Marc Lemaître (IRBM).
The stem cells from these athletes will form the basis for building the organoid. We plan to cultivate and differentiate satellite cells on a support along a single longitudinal axis similar to an in vivo environment. To do this, we will use a rapid prototyping method to print guide grooves with a biocompatible hybrid material on a silicone support. The grooves will force the alignment of muscle fibers along a single axis thanks to the contact principle, reproducing the physiological organization of a muscle. In addition, to induce an eccentric contraction, the organoid will be built on a flexible stretchy silicone membrane in which, opposite each microgroove, a stimulation microelectrode will have been integrated. Although biocompatible, the silicone support is bio-inert. To guarantee the adhesion of the muscular fibers of the organoid which will have to resist the mechanical stresses induced by the eccentric contraction, the silicone will be functionalized. The strategy proposed by the peptides and proteins team of Gilles Subra (IBMM) will consist in covalently grafting onto the silicone surface relevant peptide ligands derived from adhesion proteins of extracellular matrices [Pinese C. & al., Materials Today Chemistry (2017) 4:73]. Then, the OptoMicrofluidic Platform of Montpellier (POMM) and the team of Pascal Etienne (L2C) will bring their expertise on the organic-inorganic photostructurable hybrid materials (MHOIP) already used for applications in the fields of integrated optics and microfluidics [Yaacoub S. & al., J. of sol-gel sci. & Tech. (2013) 67 (2) 384]. MHOIPs have adjustable properties by modifying the chemical composition and molecular structure. The microstructures are obtained by deposition of MHOIP followed by the sequential action of localized UV irradiation using a laser writing machine and then wet etching of exposed or unexposed areas depending on the type of MHOIP.
The satellite cells will then be cultured on biocompatible support microstructures to develop a “muscle-on-a-chip” device so that they can proliferate on a single longitudinal axis, in the form of myoblasts and myotubes according to an experimental protocol described and validated. by the unit of Jacques Mercier (PhyMedExp). Once the organoid has been obtained, the challenge will be to impose eccentric stress on it. For this, the organoid will be, thanks to a Flexcell® type device, stretched mechanically and electrically stimulated to induce a synchronized muscle contraction.
We will test different eccentric contraction protocols, which will allow to induce in a reproducible and standardized way lesional phenomena stimulating muscle regeneration. Thus the objective will be to be able to determine the power of organogenesis after muscle damage of the satellite cells constituting the organoid in connection with its microenvironment. This assessment will include direct measurement of oxidative stress induced by eccentric stress, activation of homeostatic signaling pathways of proteins, the ability of myoblasts to proliferate, dimensions of regenerated myotubes and fusion abnormalities.
Expected results :
An athlete who has a low capacity for muscle regeneration will enter a cycle of iterative muscle damage at a minimum, limiting his sports performance. Thus in the work of this thesis we expect to find interindividual variability in the muscular response to the same eccentric stress. We also expect that the parameters of evaluation of muscle recovery evaluated in vitro can be correlated with the performance of the athlete.
The perspectives for this thesis work are numerous. They will allow, for future work, to assess human interindividual variability in the muscular response to the same mechanical stress and to study its link with dynamic epigenetic changes specific to each sportsman. Thus these first works will allow in the near future to assess whether an epigenetic signature predicting the muscular response to a lesional phenomenon could also be found in blood cells. This perspective will pave the way for blood tests that make it possible to predictively differentiate athletes who are likely to develop more than others iterative muscle lesions requiring modalities and an adaptation of their training intensity. Finally, this tool developed during this thesis will make it possible to test in vitro in humans, biomolecules active on the biological function of stem cells and on muscle regeneration.
The PhD student will be enrolled in the Information, Structures and Systems doctoral school (I2S, www.edi2s.univ-montp2.fr).
He will be assigned to the Charles Coulomb Laboratory (www.coulomb.umontpellier.fr) within the "Hybrid Materials and Nanostructures" Team and will carry out part of his work within POMM, the OptoMicrofluidic platform in Montpellier (www.pomm. cnrs.fr). He will manufacture all the microstructures supporting the growth of organoids. The doctoral student will also study and design the experimental tools put in place as part of mechanical measurements on organoids.
Due to the transdisciplinary aspect of the project, the doctoral student will have to interact with several other teams from the MUSE site.
In particular, the doctoral student will collaborate with the Aminoacids and Peptides team of the Max Mousseron Institute of Biomolecules in order to achieve the functionalization of the organoid growth supports, allowing better anchoring of the differentiated muscle fibers.
On the other hand, to test the capacity of the supports in the genesis of organoids from a stem cell culture, the doctoral student will be welcomed in the PhyMedExp unit (UMR CNRS 9214 - Inserm U1046, CHU Montpellier) of University of Montpellier (www.u1046.edu.umontpellier.fr). He will have access to the Culture Room (L2) of the Unit to set up the muscular cell model from human myotubes, these myotubes themselves being derived from stem cells purified from biopsies of human skeletal muscles. The biopsies necessary for carrying out this research project will be carried out in the thoracic surgery department of the Arnaud de Villeneuve hospital, Montpellier University Hospital. This protocol was already validated in July 2016 by the various competent bodies (CPP Sud Méditerranée I: RO – 2016/30).
Finally, the doctoral student will also have to maintain a close and permanent relationship with the partners of the sports movement (MA2M and FFSU Occitanie) responsible for initiating and leading cohorts under the additional supervision of the scientific referent of the Department of Sports Performance of CREPS of Montpellier. This relationship will be administrative and technical (calendar of operations, constitution of cohorts, establishment and implementation of training protocols, biological samples and feedback on experiences)
The student will have access to all the instrumental resources available within these structures and through the various platforms of the University of Montpellier.
Constraints and risks
The PhD student will be the link between the many partners. He must therefore have a great sense of organization and good autonomy to manage interactions as well as possible and demonstrate maturity in planning his work in interface with the various laboratories involved.
From an experimental point of view, the doctoral student will handle chemical reagents for his syntheses and biological material for the preparation of organoids. He will therefore be required to work in rooms with controlled access and requiring the wearing of specific safety equipment.
Although all studies must be done in Montpellier, the doctoral student may be required, if necessary, to develop collaborations with other French or European partners. Travel is therefore to be expected. The doctoral student must also present his work at national or international congresses at least once a year and ensure good dissemination of his work within the local scientific community.
The required profile is primarily a biochemist-type experimenter with a curriculum showing an openness to the science of materials. A biologist profile is also possible on the condition that he shows a motivation towards chemistry and materials science.
Knowledge of cell culture and muscle physiology through either teaching or internships.
Very strong autonomy and organizational skills
Mastery of project management
Good experimental skills (demonstrated during internships)
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