Study of the links between the dysregulations of metabolism and epigenetics marks in Huntington’s disease
We want to focus on epigenetic dysregulation in Huntington’s Disease (HD), a pathogenic mechanism implicated in accelerated aging of striatal neurons. Specifically, we will investigate the interplay between altered energy metabolism and epigenetic impairment in HD striatal neurons to identify new targets/pathways for disease-modifying intervention. We aim to obtain detailed maps of histone post-translational modifications (PTMs), especially of methylations, acetylation and the recently described lactylation, which might be critical in the HD brain. Indeed, these PTMs are tightly regulated by the metabolic status of the cells. We will use proteomics which is the best suited approach to identify and quantify multiple protein PTMs. We consider working on the striatum of WT, R6/1 transgenic mice and the more progressive Q140 knock in model at various stages of disease, to assess evolution of histone PTMs and metabolism with aging. Additionally, to get a dynamic view of the links between metabolic and epigenetic imbalance in HD, we will inject intraperitoneally HD mice and controls with 13C-glucose and analyze over a time course the incorporation of 13C into histone PTMs. Finally, acetyl-CoA, the precursor for histone lysine acetylation, has been shown to be locally produced in the nucleus, by either acetyl-CoA synthetase 2 (ACSS2), ATP-citrate lyase (ACLY) or the pyruvate dehydrogenase complex. Regarding lactylation, it is currently unknown where, and by which enzymes, the pool of lactate used for modifying histone lysines by lactylation is produced. ACSS2 is a very good candidate, as it can catalyze the production of acyl-CoA molecules from the corresponding short chain fatty acids (SCFA). To address the question of the production of metabolites in the vicinity of chromatin in striatal cells, we will use epigenomics (ChIPseq or CUT&tag) to get the genomic distribution of ACSS2 and ACLY and compare it to distributions of acetyl and lactyl histone marks.
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.
Condensates and Chromatin: How Phase Separation Shapes Plant Temperature Responses
Plants must adapt their development to environmental conditions, including rising temperatures due to climate change. Heat stress significantly impacts plant physiology, and to mitigate these effects, plants have evolved heat shock responses (HSR), with Heat Shock Factor A1a (HSFA1a) serving as a master regulator in Arabidopsis thaliana. Under nonstress conditions, HSFA1a remains cytosolic and inactive, bound to heat shock proteins (HSPs). Heat stress triggers HSP dissociation, enabling HSFA1a nuclear translocation, trimerization, chromatin binding, and activation of stress-responsive genes. Recent studies reveal that HSFA1a might act as a pioneer transcription factor to access closed chromatin regions and initiate HSR. Additionally, preliminary findings also suggest that HSFA1a undergoes liquid-liquid phase separation (LLPS) to form nuclear condensates that regulate gene expression. This project aims to 1) explore how temperature affects HSFA1a structure and oligomerization, 2) investigate LLPS of HSFA1a with and without DNA, 3) characterize HSFA1a pioneer activity, and 4) determine the physiological importance of LLPS in HSR.
Characterization of the molecular mechanism involved in the detection of rare earth elements in Pseudomonas putida and associated biosensors development.
Rare earths (REE) are widely used in high technology, and demand for REE is set to double over the next 30 years. The selective extraction and recycling of REE has a triple challenge: economic, technological and ecological. Currently, less than 1% of REEs are recycled. What's more, extraction methods are tedious and polluting. They require several stages with acids or solvents. The discovery in 2011 of enzymes that naturally use light REE has opened up new prospects. The development of biosourced methods could be a key element in unlocking current selectivity and extraction barriers. This thesis is part of the biotechnologies of tomorrow theme. The aim of this thesis is to acquire fundamental data on the molecular mechanism of a biological system for the selective perception of REE through a robust screening, in order to take advantage of it for the development of biosensors responding to certain specific REE. Cell biology, biochemistry and in silico analysis techniques based on artificial intelligence will be used to accomplish this project. The results obtained will enable us to identify: 1) the molecular mechanism of REE detection and the factors influencing its selectivity, 2) the binding sites of the regulator and the genes involved in this response, and 3) the development from 1) and 2) of robust and selective biosensors.
New rapid diagnostic tool for sepsis: microfluidic biochip for multi-target detection by isothermal amplification
Sepsis is among the main cause of death across the world, and is caused by severe bacterial infection but can also originate from viruses, fungi or even parasites. In order to drastically increase survival rates, a rapid diagnostic and appropriate treatment is of paramount importance. The commercially available tools for nucleic acid detection by qPCR are able to sense multiple targets. However, these multiplexed analyses arise from the accumulation of analysis channels or reaction chambers where only one target can be detected. The original sample has to be divided, resulting in a loss of sensibility since a smaller amount of targets is available in channels or chambers.
In order to tackle the question of “How to detect multiple targets without a loss in sensibility?”, the PhD candidate will have to develop a multiplexed detection in a single reaction chamber by localized immobilization of LAMP primers (Loop-mediated isothermal amplification) on a solid substrate like COC or glass.
The expected outcome is a biochip allowing for real-time and fast (minutes) detection of several molecular DNA targets including: primers design and selection, primers immobilization on surface, integration of the biochip into a microfluidic cartridge and data collection and management for fluorescence detection of targets.
This innovative work will provide the PhD candidate with strong skills in diverse scientific domains such as molecular biology, surface functionalization, modelling and simulation, in a very multidisciplinary working environment.
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.
Towards a detailed understanding of the regulation of gene expression by acetylation and lactylation of histone proteins
In eukaryotic cells, DNA is wrapped around histone proteins to form chromatin. Dynamic modification of histones by various chemical structures enables fine regulation of gene expression. Alterations in these complex regulatory mechanisms are at the root of many diseases. Histone lysine acetylation is known to induce gene expression. Other structures can be added to histones, whose effects on transcription remain largely to be elucidated. Most of them, like lactylation discovered in 2019, depend on cellular metabolism. We have begun to study lactylation in the context of murine spermatogenesis. This process of cellular differentiation is a model of choice for studying the regulation of transcription, due to the dramatic changes in chromatin composition and the gene expression program. We have generated novel epigenetic profiles consisting of the genome-wide distribution of acetylated and lactylated marks on three histone H3 lysines. The aim of this thesis is to contribute to the deciphering of the “histone code”, firstly by studying the role of lactylations on the transcriptional program. Secondly, the prediction of chromatin states will be refined by integrating our new data with existing epigenomic data at the two studied cellular stages, within neural network models.
ROLE OF UNFOLDED PROTEIN RESPONSE IN MAINTAINING THE SPERMATOGONIAL STEM CELL POOL IN THE ADULT MOUSE
Adverse conditions (oxidative stress, imbalanced lipid, glucose or calcium levels, or inflammation) induce the accumulation of abnormal proteins resulting in ER stress. The Unfolded Stress Response (UPR) is activated to restore cellular homeostasis, but severe or chronic stress results in apoptotic cell death. Uncontrolled UPR signaling promotes many human diseases (diabetes, Parkinson's, Alzheimer's, liver disease, cancer...), but nothing is known about its implication in adult male sterility. Spermatozoa production relies on Spermatogonial Stem Cells (SSC) which are maintained by self-renewal throughout life. We have shown that the clonogenic activity of SSC is drastically impaired after ER stress through differentiation entry. An HTS screen has highlighted 2 of the 3 UPR branches as being involved in the clonogenic activity of SSC in vitro. The role of these 2 UPR pathways will be further investigated in SSC cultures of mice to determine whether they are involved in the induction of cell death or in the balance between self renewal and differentiation. In treated SSC cultures, cell death, cell cycle, induction of differentiation and synergy between UPR pathways will be analyzed. As the effect of each pathway is mediated by transcriptional factors, the target genes will be characterized by RNAseq in order to identify the gene networks controlled by UPR effectors and involved in the fate of SSC. For the most relevant pathway, an in vivo study will confirm the role of the UPR effector in CSS property.
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.
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.