Consequences of heavy ion exposure on spermatogonial stem cells of adult mice

Heavy ions appear to be the more toxic component of galactic cosmic radiation. There is no evaluation of the effects of low-dose heavy ions on male fertility, which is necessary to assess the hereditary risk associated with a stay in deep space and to improve healthcare and countermeasures.
We are investigating the effects of a single 56Fe ion exposure (10 mGy, 100 mGy or 1 Gy) on adult mouse spermatogonial stem cells. Tissue lesions will be examined by immunolabeling and the irradiated undifferentiated spermatogonia population will be analysed. Transcriptional regulatory pathways will be studied by scRNA seq in irradiated spermatogonial enriched populations to identify a cell specific signature and biomarkers, and to define the fate of spermatogonial stem cells. The altered molecular pathways will be used as a reference in future studies of the effects of chronic very low dose, or mixed ion exposures that are closer to galactic cosmic radiation.

Microbiota-microglia interactions in human brain development: role on cortical circuit formation and childhood epilepsy.

The PhD student will join a project investigating microbiota-microglia interactions in the development of cortical circuits and epilepsy onset in the human pediatric brain. Human brain development progresses through complex stages that establish cortical connectivity, where disruptions can lead to cortical malformations and epilepsy. Gut microbiota mediators can penetrate in the brain and significantly influence microglia, the brain's immune cells. Microglia play an active role in circuit development and changes in their properties may contribute to malformations and epilepsy onset in pediatric patients. To validate these hypotheses, the candidate will collect fresh brain cortical biopsies from young patients with malformations (Necker Hospital, Paris) and learn to perform live imaging of microglia in human acute ex vivo cortical slices, using two-photon microscopy. Paired with electrophysiological patch-clamp recordings, these techniques will clarify microglia’s role in human cortical circuits in pathology. Additionally, the student will test whether gut microbiota metabolites modulate microglial activity using organotypic human cortical slices as a human brain long-term ex vivo model. We expect these findings to clarify the microbiota’s impact on microglial activity and cortical development, potentially guiding personalized therapeutic strategies to support brain health in children with developmental defects.

Trans-splicing gene therapy for Stargardt disease: construction of molecular and cellular tools to target ABCA4 gene mutations

This project aims to develop an innovative therapeutic approach for Stargardt disease, a macular degeneration caused by mutations in the ABCA4 gene. The strategy is based on SMaRT (Spliceosome-Mediated RNA Trans-splicing) technology, which enables mutation correction at the transcriptome level by replacing mutated exons of endogenous mRNA by trans-splicing with an exogenous RNA (PTM). Since the PTM contains only a part of the mRNA to be corrected, this approach can overcome the obstacle of the large size of the ABCA4 cDNA, which exceeds the carrying capacity of AAV vectors. The project will consist of several phases using molecular and cell biology techniques: construction of viral vectors for the expression of PTMs, production of cell lines to test the efficacy of binding domains (BD) to induce trans-splicing, and screening of BDs to optimize PTMs. Selected PTMs will then be tested in retinal organoids and animal models to demonstrate their therapeutic potential for the treatment of this genetic disease. As AAV is currently the most effective vector for retinal transduction, this project could open new therapeutic perspectives for Stargardt disease.

Towards a better understanding of membrane proteins through AI

Despite the remarkable advances in artificial intelligence (AI), particularly with tools like AlphaFold, the prediction of membrane protein structures remains a major challenge in structural biology. These proteins, which represent 30% of the proteome and 60% of therapeutic targets, are still significantly underrepresented in the Protein Data Bank (PDB), with only 3% of their structures resolved. This rarity is due to the difficulty in maintaining their native state in an amphiphilic environment, which complicates their study, especially with classical structural techniques.

This PhD project aims to overcome these challenges by combining the predictive capabilities of AlphaFold with experimental small-angle scattering (SAXS/SANS) data obtained under physiological conditions. The study will focus on the translocator protein TSPO, a key marker in neuroimaging of several serious pathologies (cancers, neurodegenerative diseases) due to its strong affinity for various pharmacological ligands.

The work will involve predicting the structure of TSPO, both in the presence and absence of ligands, acquiring SAXS/SANS data of the TSPO/amphiphile complex, and refining the models using advanced modeling tools (MolPlay, Chai-1) and molecular dynamics simulations. By deepening the understanding of TSPO’s structure and function, this project could contribute to the design of new ligands for diagnostic and therapeutic purposes.

Hyperpolarized Xenon NMR to probe the functionality of biological barriers

Optical pumping of xenon, giving rise to an intense NMR signal, is a specialty of the LSDRM team. Xenon, which is soluble in biological media, has a wide range of chemical shifts, which we use here to study the properties of cell barriers. Numerous pathologies stem from an alteration of these barriers.

In this thesis, we aim to develop a specific methodology using hyperpolarized xenon to study the functionality (integrity, permeability, selectivity) of biological barriers, using in vitro and in vivo spectroscopy and imaging. The thesis will be divided into two parts: in vitro, the aim will be to develop a device and NMR protocols for studying model cell assemblies; in vivo, studies on rodents will enable us to assess xenon's ability to reach organs more or less close to the lungs while maintaining its polarization, and to measure kinetics across barriers. This topic will enable major instrumental and methodological advances, as well as a deepening of our knowledge of selective transport processes at different biological barriers.

Complex brain organoid model reproducing the glioblastoma tumor niche and its immune component for the development of personalized medicine

Glioblastoma, responsible for 3,500 annual deaths in France, is an extremely aggressive brain tumor that is resistant to current treatments. Clinical trials in immunotherapy have shown only transient effects, underscoring the importance of understanding resistance mechanisms and developing more targeted therapeutic strategies.

We have developed an innovative model of glioma stem cell invasion in immunocompetent and vascularized brain organoids derived from induced pluripotent stem cells (iPSCs) (Raguin et al., submitted). This model faithfully reproduces the glioblastoma tumor niche, including vascular co-option, reprogramming of microglia into tumor-associated macrophages, and tumor recurrence following radiotherapy.

The aim of this PhD project is to derive a universal brain organoid model for the transfer of glioma cells from patients and lymphocytes to optimize the immunotherapy approach (CAR-T cells).

The project involves creating a universal model of human brain organoids that are immunologically "silent" by suppressing the expression of the HLA class I/II system in iPSCs (CRISPR/CAS9 for the ß2M and CIITA genes). Additionally, it aims to elucidate the mechanisms of immunosuppression induced by irradiation, such as the reprogramming of microglial/macrophage cells and the involvement of senescence. Various approaches to make the tumor microenvironment more conducive to immunotherapy will be explored, including activating the type I interferon pathway through genetic modification or with cGAS/STING pathway agonists. Subsequently, the use of CAR-T cells targeting an antigen overexpressed by glioblastoma cells (CD276/B7-H3) will be studied. This model could be used in personalized medicine by co-cultivating patients' tumor cells, monocytes, and CAR-T cells.

This project offers innovative perspectives for the personalized treatment of glioblastoma via immunotherapy and could represent a major advancement in this therapeutic approach.

Crosstalk between adipocytes and T lymphocytes, key players in immunity in adipose tissue

The metabolic and endocrine role of adipose tissue (AT) is established. The AT is composed mainly of adipocytes but also of immune cells, best known for controlling the metabolic homeostasis of the tissue. The immune activity of TA is associated with the secretion of cytokines and metabolites that modulate its immune function. Obesity, characterized by an accumulation of AT at the subcutaneous or visceral level, is associated with the local inflammation of AT. However, the AT can be infected by different pathogens and it constitutes a site of accumulation of CD8 T lymphocytes (TL) specific for them, which protect against reinfection. These data therefore encourage us to investigate the interactions between adipocytes and CD8-TL in the AT.
The project will be carried out in the CoVir team, which is developing various projects aimed at deciphering the anti-infectious properties of adipose tissue. It is part of a consortium established for an ANR project (INSERM, CNRS, Institut Pasteur de Lille). The objective of the team's project is to study the local contribution of adipocytes and CD8-TL residing in the TA during influenza virus infection in a non-human primate (NHP) model. The NHP presents metabolic and immune responses similar to humans. In addition to in-vivo approaches in NHP, we will develop a 3D co-culture model to target the interaction between adipocytes and CD8-TL, without interference from inflammatory and metabolic signals external to TA. This project benefits from the historical expertise developed in the institute (IMVA-HB UMR 1184/I IDMIT, directed by R. Le Grand) concerning the study of viral infections in the preclinical model of PNH. IDMIT platforms provide equipment and expertise in histology, cytometry (LFC), cytokine/chemokine detection (L2I) and animal welfare (ASW).
The thesis project will focus on in vitro approaches. We will compare the interactions between adipocytes and LT, by studying the metabolic and immune response of each of these fractions. Indeed, adipocyte cells exhibit strong metabolic activity but exert their own immune activity (through the production of microbicidal peptides) and immunomodulatory activity. Concerning immune cells, their functional activity is dependent on their metabolic functions and it is crucial to evaluate the immune-metabolic modifications of immune cells in the presence of adipocytes. The organoid model will allow us to evaluate : (i) the impact of the context of obesity to those in a context of metabolic normality, (ii) the impact of viral pathogens on each of these two fractions. In the medium term, this model will make it possible to test strategies for modulating TA functions during metabolic pathologies or viral infections.

Biogas upgrading with an advanced Biorefinery for CO2 conversion

The use of renewable energy sources is a major challenge for the coming decades. One way of meeting the growing demand for energy is to valorize waste. Among the various strategies currently developed, the recovery of biogas from anaerobic digestion plants appears to be a promising approach. Biogas is composed mainly of methane, but also of unused CO2 (around 40%). The project proposed here is to reform biogas using a renewable biohydrogen source to convert the remaining CO2 into pure CH4. We propose to set up a stand-alone advanced biorefinery that will combine photoproduction of hydrogen from waste (e.g.: lactoserum) by the bacterium Rhodobacter capsulatus combined with the CO2 present in the biogas in a biomethanation unit containing a culture of Methanococcus maripaludis, a methanogenic archaea able to produce CH4 from CO2 and H2 only (according to the Sabatier reaction). The aim is to produce CH4 in an energy-efficient and environmentally-friendly way.

Multiplexed whole-body in vivo imaging monitoring of pathogen dissemination and immune responses dynamics in tuberculosis

This thesis is dedicated to set up a multiplexed medical imaging monitoring of pathogen colonization and associated immune responses dynamics at the whole body scale for various infectious diseases. This could provide an innovative and non-invasive tool to better understand dynamics links between immune responses and pathogen distribution throughout the body and potentially provide new biomarkers associated to several diseases. To tackle this issue this thesis would implement such strategy in tuberculosis disease. The main aim is to determine the relationship between Mycobacterium tuberculosis dissemination and associated immune responses across the whole body during the course of tuberculosis infection from early infection to latent or active tuberculosis thanks to innovative multiplexed imaging protocols. The goal of this study is to provide correlations in time and space between local bacterial burden and several immune cell infiltrations (activated macrophages and T lymphocytes subsets) occurring following infection and detected over time by imaging. These findings could then provide, with minimal invasiveness, predictive biomarkers on disease or local granuloma progression and may provide also valuable insight on potential immune targets for future preventive or curative strategies based on modulation of the immune system. To do so, this thesis would take advantage of the preclinical Non-human primate model of tuberculosis developed in France and on our in vivo imaging of pathogens and immune cells expertise in NHPs. Of note, deeper immune cell profiling in samples of interest (imaging guided) will be assessed by spatial or single-cell transcripomic technologies in tissue samples to provide additional readouts on TB pathophysiology and potential treatment efficacy.

Unraveling the mechanism of enzymatic carbon fixation

The Synchrotron Group at the Institut de Biologie Structurale in Grenoble is currently developing an innovative method called TR-FOX (Time-Resolved Functional Oscillation Crystallography). This technique aims to elucidate, firstly, the global dynamics of biological macromolecules in action and, secondly, their fine catalytic mechanism. It relies on the use of an injector capable of depositing onto the crystal, during the course of the X-ray diffraction data collection, a nanoliter droplet containing the substrate and cofactor of the studied reaction. This triggers the enzymatic reaction within the crystal. The approach can be combined with UV-Visible absorption spectroscopy to characterize the reaction kinetics more precisely. The goal is to obtain a series of structures during the catalytic cycle in order to make a molecular movie depicting the functioning of the enzyme. This thesis has two objectives: (i) improve and validate the TR-FOX method and, (ii) study the catalytic mechanism of two enzymes involved in carbon fixation either by capture or conversion of CO2.

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