Involvement of Rad51 paralogs in Rad51 filament formation in DNA repair

Homologous recombination (HR) is a major repair mechanism for DNA double-strand breaks induced by ionizing radiation. A key step in HR is the formation of Rad51 nucleoprotein filaments on the single-stranded DNA generated from these breaks. We have shown that strict control of these filaments is essential, so that HR does not itself induce chromosomal rearrangements (eLife 2018, Cells 2021). In humans, functional homologs of control proteins are tumor suppressors. Thus, the control of HR appears to be as important as the HR mechanism itself. Our project involves the use of new molecular tools enabling a real breakthrough in the study of these controls. We will be using a functional fluorescent version of the Rad51 protein developed for the first time by our collaborators A. Taddei (Institut Curie), R. Guérois and F. Ochsenbein (I2BC, Joliot, CEA). This major advance will enable us to observe the influence of control proteins on DNA repair by microscopy in living cells. We have also developed highly accurate structural models of control protein megacomplexes in association with Rad51 filaments. This study also led to the identification of specific domains for each paralog protein, outside the structurally conserved Rad51-like core, that might define the specificity of each paralog proteins. We will use a multidisciplinary approach based on genetic, molecular biology, biochemistry, protein structure and live microscopy methods and yeast as model organism to study the consequences of the ablation of these specific domains. We will also search for proteins specifically binding these domains. Their identification would be crucial to understand the function of Rad51 paralog complexes and help to develop new therapeutic approaches.

Prion-like proteins in marine plankton: a quest towards new molecular factors of thermal adaptation

Climate change is reshaping the distribution of species on the planet and mechanisms for adaptation to thermal stress are then called upon. Recently, in terrestrial plants the role of prion-like proteins has been highlighted in flowering and vernalization mechanisms. However, these atypical proteins have not been characterized in the marine world where plankton plays an essential role in the biological carbon pump and the marine food web. To explore the world of prion-like proteins and their role in thermal adaptation of marine plankton species, we propose a three years PhD program in the computational biology team of the CEA-SEPIA in the François Jacob Institute of Biology located in Fontenay-aux-Roses, France. The first objective of the thesis is to identify and characterize the function of marine prion-like proteins and their biogeography in the world oceans. The student will also reconstruct the molecular evolution of these proteins across a wide spectrum of marine plankton species through gain/loss and adaptation signal analyses. The research approach will be based on comparative genomics and phylogeny on Tara Oceans metagenomic and metatranscriptomic data. Additionally, the student will identify prion-like proteins involved in the adaptation to temperature by integrating spatial and environmental data collected by the Tara Oceans expeditions. In a context of climate change, this research fits into the understanding of the molecular evolution of prion-like proteins, shedding light on their role in the thermal adaptation of species playing a key role in the marine food chain and geochemical cycles.

PPARy, a major player in bone marrow stromal homeostasis and a therapeutic target for myelofibrosis?

Myelofibrosis (MF) is the most severe of the Philadelphia-negative myeloproliferative neoplasias (MPNs), with a median survival of 5-6 years. Whether diagnosed de novo (Primary Myelofibrosis, PMF) or secondary to another MPN, the features of MF are similar. A subpopulation of haematopoietic cells derived from the pathological clone releases pro-inflammatory cytokines and growth factors into the bone marrow microenvironment. In response, the bone marrow microenvironment undergoes remodelling, resulting in osteosclerosis and fibrosis of the mesenchymal stromal cells (MSCs) associated with loss of haematopoietic support. The 2016 WHO classification includes a premyelofibrosis state to facilitate early diagnosis of patients at increased risk of progression. However, although major progress has been made in understanding the pathogenesis of the disease, notably with the description of the so-called "driver" mutations responsible for myeloproliferation (JAK2, CALR and MPL), apart from haematopoietic stem cell allotransplantation, which only concerns a minority of patients, current treatments are mainly symptomatic and have little influence on the natural history of MF.
Recently, we demonstrated that activation of the nuclear receptor PPARy (Peroxisome Proliferator-Activated Receptor-gamma) by its pharmacological ligands (Actos®) or (Pentaza®) reduced the development of osteosclerosis and reticulin fibrosis of the bone marrow (BM) and prevented anaemia resulting from bone marrow remodelling in three preclinical mouse models of MF (Lambert, Saliba et al. 2021). These results position PPARy agonists as interesting therapeutic candidates. However, before considering their therapeutic repositioning in the treatment of MF, it is imperative to characterise the status and function of PPARy within medullary MSCs both at the physiological stage and during the development of NMPs.
In this project, our initial results show that PPARy expression is decreased in murine and human MSCs at the MF stage. In contrast, no change in PPARy expression was observed in MSCs derived from other MPNs. Transcriptomic analyses also demonstrated that TGF-B, a major cytokine in the development of MF, is capable of negatively regulating PPARy expression in MSCs. In order to mimic this expression defect, we invalidated PPAR-y (KO) in two bone marrow MSC lines, the first murine (MS5), the second human (HS5, under characterisation). Under these conditions, basal expression of a panel of genes associated with MF is increased in MSC-KO to the level of wild-type lines stimulated by TGF-B. Expression of this panel was further increased in MSC-KO in the presence of TGF-B, indicating potentiation of the TGF-B-mediated signal in the absence of PPARy. This transcriptomic signature associated with KO-MSCs is found in murine MSCs from the thrombopoietin (TPOhigh) induced MF model as well as in human MSCs from patients with PMF. However, this expression profile was not found in MSCs from patients with another MPN, indicating that it is indeed a sign of a stage of MF.
Invalidation of PPARy does not affect the phenotypic signature of bone marrow MSCs, but their multipotent character is altered with a loss of adipocyte differentiation capacity associated with an increase in osteo-chondrocyte differentiation potential. These histological observations are corroborated by the decrease in the production of adipocyte factors by MSC-KO and an increase in the expression of the osteoblastic factor Runx-2. In addition, the supernatant of the KO line showed a marked increase in osteoprotegerin (OPG), a soluble molecule produced by osteoblasts that leads to apoptosis of osteoclasts. This deregulation of the osteoblast/osteoclast balance in KO conditions could explain the osteosclerosis observed in patients with MF. In addition, the production of CXCL12 (CXC motif Chemokine Ligand 12) and the bone marrow growth factor SCF (c-kit ligand) are greatly reduced in MSC-KO conditions, at both transcriptomic and protein levels. These data recapitulate the results described during transcriptomic analyses of MSC from patients with fibrosis. At the same time, the capacity of MSC-KO to support haematopoiesis, in both the short and long term, is significantly reduced, reflecting the cytopenias associated with MF.
In silico, RNA-Seq analyses were carried out on the MS5-WT and MS5-KO lines. Initial gene set enrichment analyses (GSEA) show that the pathways most significantly affected are inflammation, myogenesis (MSC to myofibroblast transition) and the cell cycle. Comprehensive analyses are now required to identify new therapeutic candidate genes and gain a better understanding of the development of bone marrow fibrosis.
These initial in vitro results support the key role of the PPARy receptor in the homeostasis of the bone marrow microenvironment and in the genesis of its remodelling during the development of myelofibrosis. However, in vitro approaches alone are unable to capture the full complexity of a disease involving multiple players including haematopoietic cells, immunological cells and all the cell types making up the bone marrow microenvironment. To integrate all these parameters, we have established a mouse model in which PPARy expression is reduced (haploinsufficiency) or invalidated (KO) in the medullary MSCs of animals. It is the study of this model that will form the core of the project. Initially, in vivo, it will be used to:
1) Characterise the role of PPARy in the homeostasis of the bone marrow microenvironment.
2) Assess the impact of reduced expression on the development of bone marrow fibrosis.
3) To validate the positioning of PPARy as a therapeutic target in the management of bone marrow fibrosis and to consider the repositioning of its pharmacological agonists (Actos®; Pentaza®) in this pathology.

The presence of medullary pre-fibrosis/fibrosis is a poor prognostic factor in MPN or acute myeloid leukaemia (AML). However, it is difficult to determine whether this condition is simply an indicator or whether it plays an active role in the development of haemopathies. The use of these models (Haplo-insufficient or KO for PPARy in MSCs) in association with preclinical mouse models of MPN (CML (BCR-ABL); PV (JAK2 V617F), ET (CALRDel52)) will allow, in a second phase, to determine whether:
1) The presence of a predisposition to bone marrow fibrosis influences the natural history of haemopathies.
2) In these diseases, which are purely haematopoietic in origin (mutation of the haematopoietic stem cell), it is appropriate to combine treatment targeting the malignant clone with treatment aimed at preventing the development of bone marrow fibrosis (activation of the PPARy receptor by its ligands in the haploinsufficiency condition).

This entire project is part of the Tomorrow's Biotechnologies (F) initiative, which aims to improve patient care through the development of personalised medicine.

Imaging disease associated astrogliosis by Positron Emission Tomography in Alzheimer’s disease

This PhD fellowship focuses on the assessment of astrocytic reactivity induced by tauopathy, associated with Alzheimer's disease, using PET imaging or 3D-autoradiography using the radioligand [11C]BU99008 or [3H]BU99008 specific for I2-BS receptors, target of astrocytic reactivity. The main objectives of this research project are: 1- to validate the specificity of the [11C]/[3H]BU99008 ligand binding in different models of tauopathy; 2- to characterise the signature of reactive astrocytes detected by BU99008; and 3- to assess the specific binding of [11C]/[3H]BU99008 as well as gene expression following a therapeutic strategy targeting I2-BS receptors in one of the tauopathy models. Our methodological approach adopts a multiscale perspective, combining PET imaging with microscopic and transcriptomic analyses, thus enabling a multiscale analysis ranging from macro (in vivo) to micro (in vitro). These studies not only provide a fundamental understanding of astrocytic reactivity, but also open important perspectives for the development of therapeutic strategies targeting astrocytic reactivity in the context of neurodegenerative diseases.

Effects of ionizing radiation and radiosensitizing molecules in a relevant murine model of breast cancer

The project aims at evaluating the efficacy of molecules combined with radiotherapy, in in vitro and in vivo models of breast cancer.
On the one hand, the student will evaluate the radioenhancer effect of bimetallic nanoparticles designed in the laboratory, on a murine model mainly. A clinical, histological, and immune monitoring will confirm the added value of such molecules for combination with radiotherapy. In addition, those innovative nanoparticles have been designed as biodosimeters, using unique physical properties of metallic nanoparticles. Therefore the project includes an evalution of the biodosimetry potential, in collaboration with physicists from CEA, who developed detection tools.
On the other hand, specific inhibitors for DNA repair will be used to block radiation-triggered repair. Thus, damaged cancer cells will be oriented towrads cell death, even in the case of radioresistant cells. The objective of the PhD program is to evaluate these molecules effect on in vitro cellular models, as well as on murine models of breast cancer.
Overall, the research project will benefit from the laboratorys’ collaborations with physicists and chemists, as well as the platforms of IRCM (irradiation, animal experimentation, microscopy, cytometry, etc...)

Intra-tumor cytotoxic bystander T lymphocytes with anti-tumor potential are inhibited by the HLA-G/ILT2 checkpoint: study of a new paradigm with therapeutic perspectives.

Objective of the thesis: to participate in the research theme summarized below and to take charge of part of it. This project is financed by a PRT-K grant and is a collaborative work Saclay/Paris/Grenoble
Knowledge of immunology and basic immunology techniques. Valued multidisciplinarity, in particular in relation to bioinformatics/biostatistics.
Summary of the research theme 2023-2025
T cells specific for tumor antigens play a central role in anti-tumor immunity and immunotherapy. However, recent data show that they constitute only a fraction of tumor-infiltrating T cells (TILs), and that many TILs (called 'bystanders') recognize non-tumor antigens.
Immunotherapeutic strategies using these bystander T cells are at the preclinical stage. Our work on the HLA-G/ILT2 inhibitory checkpoint in patients with urological cancers shows that alongside PD1+ TILs known to be antitumor and specific for tumor antigens, ILT2+ TILs constitute a significant reservoir of highly cytotoxic lymphocytes, sensitive to inhibition by HLA-G but not by PDL1, and therefore insensitive to anti-PD1/-PDL1 immunotherapies. These ILT2+ TILs are anti-viral, and their activation may be of the innate type, independent of the TCR. ILT2+ T lymphocytes are therefore potentially anti-tumor bystander TILs. This situation is specifically human and cannot be observed in animal models in which the HLA-G/ILT2 checkpoint does not exist. The transition from preclinical to clinical therefore requires studies in humans.
Our goal is to demonstrate in urological cancers that
(i) when designing new immunotherapies based on bystander TILs, HLA-G, their main inhibitory checkpoint must absolutely be taken into account,
(ii) a therapeutic strategy can take advantage of the large population of intratumoral ILT2+ bystanders and their large reservoir of peripheral precursors, to redirect their functions specifically towards tumor cells while blocking their main inhibitory checkpoint HLA-G.
To achieve this objective, we brought together scientists from the CEA in onco-immunology (Paris) and bioinformatics (Grenoble), and oncologists, urologists and pathologists from Saint-Louis Hospital (Paris)

Monkeypox Sexual Transmission: Understanding Pathogenesis, Immunity and Vaccine Development in a non-human primate model (MONKIVAX)

In 2022-2023, an unprecedented global outbreak of Monkeypox virus (MPXV), a double-stranded DNA virus related to smallpox, emerged in non-endemic areas, causing over 87,000 reported cases in 170 countries. This prompted the WHO to classify it as a Public Health Emergency of International Concern (PHEIC). France alone documented 5,014 cases, with 83% confirmed biologically.
The outbreak strain, responsible for the 2022-2023 epidemic, was identified as the West African B.1 lineage Clade 2b, known as "mpox”.This outbreak was characterized by its rapid global spread, unique clinical symptoms, and high prevalence among men who have sex with men (MSM). Transmission of mpox can occur through various forms of contact, but sexual contact plays a significant role, with over 91% of cases linked to it. Lesions in genital, perianal, and oral areas suggest sexual transmission, and MPXV DNA has been found in the semen of infected patients.
No specific mpox vaccines exist, but previous evidence indicates that smallpox vaccines can provide some protection due to cross-reaction. JYNNEOSTM, a live, non-replicating Modified Vaccinia Ankara (MVA)-based vaccine, was recently approved for mpox prevention but showed varying efficacy against the 2022 outbreak, highlighting the need for more effective vaccines.
Our understanding of mpox pathogenesis, immunity, and transmission is limited, especially during the recent outbreak. To address these questions, a study using cynomolgus macaques as a model to simulate mucosal MPXV infection with the current outbreak strain is proposed.
The project has two main aims:
1) Elucidate the mechanisms of mpox pathogenesis and the immune response to natural infection using a non-human primate (NHP) model of sexual transmission.
2) Assess the immune response elicited by the currently available MVA vaccine, and identify novel mechanisms that future vaccine candidates may need to activate in order to enhance vaccine effectiveness and improve the immune response to vaccination.
The PhD candidate will receive comprehensive training in theoretical and practical aspects of immunology, virology, and microbiology. This includes experimental techniques for studying virus transmission and the host's immune response to natural infection and vaccination. In addition, the student will gain proficiency in a wide range of techniques, such as cell/tissue cultures, flow cytometry, histology, transcriptomic, and more, using both in vitro and in vivo approaches with an NHP model.
Overall, this project aims to deepen our understanding of mpox pathogenesis and immunity, guide vaccine development, and contribute to public health efforts in combating this emerging infectious disease.

Aptamer-based molecular fingerprinting for the diagnosis of neurodegenerative diseases.

The PhD project consists of developing a novel diagnostic capable of detecting the signature of a pathological forms of protein intimately associated to neurodegenerative diseases. The aim is to improve the diagnosis of patients suffering from neurodegenerative proteinopathies due to the aggregation of the proteins alpha-synuclein and tau, e.g. Parkinson’s and Alzheimer’s diseases. This project builds on our team's expertise in aptamer technology (nucleic acid-based ligands) and the production of structurally distinct aggregates of alpha-synuclein and tau that we demonstrated to trigger distinct synucleinopathies and tauopathies. During this work, different aptamer libraries will be evaluated against different polymorphs of protein fibers found in different diseases. These aptamers will then be used to design a diagnostic test using a recently patented method (AptaFOOT-Seq). The student should have a strong interest in biomedical research, particularly the molecular aspects of biology. This thesis will provide in-depth training in RNA and protein synthesis and purification, directed molecular evolution, quantitative PCR (qPCR and droplet PCR), high-throughput sequencing, bioinformatics analysis and structural biochemistry. The aim of the thesis is to obtain results that can be exploited in terms of intellectual property and to enable the student to envisage a career in an academic or industrial environment.

Response of Spermatogonial Stem Cells to heavy ion irradiation: functional assessment and transcriptome profiling in adult mice.

In deep space, astronauts will be exposed to galactic cosmic rays, whose high-energy heavy ions - minority elements - are highly toxic to cells. The consequences for the body of this chronic, low-dose exposure are still poorly understood, due to a lack of human data. In order to assess the impact of a prolonged stay in space on male fertility, this project proposes to study the effects of irradiation by a 56Fe ion beam on mouse spermatogonial stem cells (SSCs). In adults, the continuous production of spermatozoa relies on a stock of SSCs maintained by self-renewal. The integrity of the activity of irradiated CSS will be tested in vivo using transplantation tests. Various parameters of irradiated CSS will be analysed (DNA lesions, mortality, cell cycle, etc.). A transcriptional signature and markers of exposure to heavy ions will be sought in undifferentiated spermatogonia (single cell - RNA seq), and the gene networks involved in stress responses will be studied in particular. All of this data could serve as a basis for studying the hereditary and epigenetic risk associated with space flights, as well as for improving protective measures.