Abstracts

Here you can find all of the abstracts of the 2nd years Phd students.

Guilhen PRUNIER (INFINITy - Rle LD)
Remodeling of the CD8+ T cell immune synapse by co-stimulatory receptors

Cytotoxic CD8+ T cells (CTL) eliminate infected or tumor cells through an immunological synapse (IS) based on the activation of surface receptors, mainly the T Cell Receptor (TCR). In addition, many co-stimulatory receptors that modulate CTL activity are engaged at the IS. While these receptors were initially characterized as TCR signal regulators, their abilities to modulate IS dynamics are ill defined. These receptors display distinct intracytoplasmic domains, recruit different molecular adapters and activate different signaling pathways. Our working hypothesis is that each co-stimulatory receptors may differentially regulate IS dynamics. Therefore, we are comparing the effects of the engagement of co-stimulatory receptors of clinical interest ICOS, CD2, CD226, CD27, OX40 and GITR in the IS architecture, IS related events and IS dependent functional properties of primary human CTL. Using a high content confocal microscopy approach we have shown that the engagement of co-stimulatory receptors by antibodies or coated ligands promotes T cells adhesion by affecting IS morphology to varying degrees. Engagement of CD226, ICOS and CD2 had the most prominent effect on IS spreading and associated actin-rich protrusions. Strikingly, CD226 and ICOS promoted formation of the peripheral ring of LFA-1 integrin, a structure playing a role in the adhesion and killing of CTL. Consistently our preliminary data indicate that CD226 engagement potentiates adherence to target cell and enhances their elimination. In parallel, we employed phosphoproteomics to dissect the signaling downstream of receptor engagement. CD2 and CD226 engagement alone induced the phosphorylation of hundreds of phosphoproteins. As expected, the majority of these events belonged to signaling pathways associated with TCR activation. Further analysis indicated that CD2 and CD226 differentially phosphorylated modulators of the actin cytoskeleton, possibly explaining the effects of these co-receptors on IS dynamics.

Eloïse DUFOURD (INFINITy - Team 4 AS/RL)
Study of antigen specific interactions between endothelial cells of the BBB and naive CD8 T cells

Endothelial cells of the blood brain barrier (BBB-EC) are able to present self-antigens but also to cross-present antigens from the periphery or from the brain parenchyma. While antigen specific interactions between BBB-EC and activated CD8 T cells, leading to brain inflammation have been described in various pathologies, little is known about antigen specific interactions between BBB-EC and naive CD8 T cells, in non-inflammatory condition. Using an original mouse model, we show that BBB-EC expressing hemagglutinin (HA) are able to activate HA-specific naive CD8 T cells both in vivo and in vitro. This antigen specific interaction in vivo leads to an accumulation of HA-specific CD8 T cells in the brain parenchyma. These infiltrating CD8 T cells are persisting in the brain up to 30 days after injection and they seem to acquire a tissue resident memory phenotype in the brain. The consequences of these interactions and the persistence of a CD8 T cell population in the brain will be assessed, notably on further brain inflammation and on aging and cognitive abilities.

Romain PFEIFER (CRCT - Team 7)
Identification of long non-coding RNAs involved in Acute Myeloid Leukemia chemoresistance

Acute myeloid leukemia (AML) is caused by the acquisition of mutations in hematopoietic stem cells and progenitors resulting in an accumulation of immature cells in the bone marrow. AML patients are treated with a chemotherapy combining cytarabine and anthracycline. However, resistance occurs in 50% of cases. Hence, we have to better understand the mechanisms contributing to chemoresistance. So far, studies have focused on protein-coding genes but a major part of the genome is transcribed into non-coding RNAs. Among them, long non-coding RNAs (lncRNAs) are now recognized as key regulators of all biological processes. However, only few articles report the involvement of lncRNAs in AML chemoresistance. In order to identify lncRNAs involved in AML chemoresistance, we quantified the expression of all lncRNAs listed in the LNCipedia database from public RNAseq data including 364 AML patients (BeatAML cohort). Then, using the available clinical data, we identified 697 lncRNAs whose high expression is associated with a poor prognosis. A more stringent analysis allowed us to select the 14 most interesting candidates. In order to determine if these lncRNAs are involved in chemoresistance mechanisms, we modulated individually their expression in AML cell lines. We induced the endogenous expression of our candidates using the CRISPR-SAM system which allowed us to identify HCP5, a lncRNA able to block apoptosis in cytarabine-treated OCI-AML3 cells. Furthermore, by transient transfection of gapmeR in these cells, we identified 3 lncRNAs (SENCR, LINC00899 and LINC02916) whose inactivation sensitizes cells to cytarabine. In conclusion, we identified 4 lncRNAs which seem to be involved in AML chemoresistance. We have to validate these results in other AML cell lines and in primary leukemic cells. Then, we will decipher the molecular mechanisms at the origin of these resistances. This study will allow us to better adapt therapies for patients and to identify new therapeutic targets.

Aymeline DEBONLIER (INFINITy - Team 4 Inflammatory Diseases of the Central Nervous System - Mechanisms and Therapies)
Sex difference in a human regulatory T cell pathway important for MS

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) characterized by demyelination and axonal and neuronal degeneration. Women are three times more susceptible to develop inflammatory autoimmune diseases such as multiple sclerosis than men. The involvement of sex hormones in this difference has already been demonstrated, but the results remain controversial. The costimulatory protein CD46 induces T lymphocyte activation leading to the conversion of Th1 cells into Tr1 (type I regulatory T cells). This pathway has been shown to be defective in MS patients, through reduced IL-10 production. Unpublished data from our team show the existence of a crosstalk between CD46 and another costimulatory molecule, CD226 promoting the expansion of Tr1 and the production of IL-10. However, the existence of a potential sex bias in this activation pathway has not been apprehended. We have examined the existence of male/female differences in this activation pathway in CD4+ T cells concomitantly costimulated with anti-CD3, anti-CD46 and anti-CD226 Abs compared with simple costimulation. We observed male/female differences in IL-10 production by CD4+ T cells and by Tr1. Women seem to be less activated by these activation pathways compared to men. Moreover, there seems to be a gender bias in response to activation by CD46 and CD226 molecules simultaneously. An increase in the proportion of Tr1 by this activation pathway is observed in both sexes, however the Tr1 from women have a reduced secretion of IL-10 compared to men. Preliminary data suggest a potential involvement of estrogens in these differences.

Amalia MARTINEZ (CERPOP - EQUITY)
The influence of sex and/or gender on the frequency of occurrence of colorectal cancer in the general population in developed countries: a scoping review

Background: Sex differences in the incidence of colorectal cancer are visible and have recently been explored through the prism of gender. Gender refers to socially prescribed roles that impose different behaviours, interests, expectations and divisions of labour for girls and boys and then for women and men. Failure to take gender into account when studying incident cancer cases may underestimate the consequences of risk factor exposures and mask gender inequalities.

Objective: Our aim is to conduct a scoping review to select articles that address the role of sex as a determinant of CRC risk, and then to differentiate between those that consider this sex difference as gender or not.

Methods: We conducted a scoping review of the literature using two databases (PubMed and Google Scholar) up to August 2022. We grouped studies according to how sex and/or gender variables were used, quantified the type and frequency of sex and/or gender variables. We also identified other social exposure variables mentioned in the articles.

Results: We reviewed 99 studies and 7 articles were included in the analysis. Only 1 article defined its exposure variable "sex" as gender. Of the 7 articles all analysed the incidence of colorectal cancer by gender, but only 3 used the term "gender" to define exposure. Individual social variables were more often associated with sex and/or gender, particularly race/ethnicity and age, and cancer subsite.

Discussion: The effect of gender in colorectal cancer disease history research should be considered more systematically and better defined, given its role in the distribution of incident cases. This could help to understand how gender-related social mechanisms interact to produce a health state with the aim of mitigating social inequalities in health and preventing the emergence of new ones through more accurate, rigorous and valid results.

Sofia GALANOU (CRCT - Oncosarc)
Response to chemotherapy in osteosarcoma patients

Osteosarcoma is a rare bone tumor associated with a poor prognosis occurring mostly in teenagers. Neoadjuvant chemotherapy followed by surgical resection is the current treatment for this type of cancer. The 5-year overall survival is around 66% and drops under 20% for metastatic patients. Furthermore, there has not been any improvement in the last 40 years. The driving mechanisms of poor response to chemotherapy have not yet been identified due to its intratumoral heterogeneity and no molecular targeted therapy has been developed. This heterogeneity results in an irregular distribution of cell populations across the tumor and may trigger resistance to treatment. The goal of this project is to better understand the response mechanisms to chemotherapy by identifying the resistant tumor cell subpopulations and their interaction with the tumor microenvironment. The cell transcriptome has been analysed by bulk RNA-seq. Furthermore, we will focus on a single nuclei level performing snRNA-seq and spatial transcriptomic in territories of good and poor response in a unique collection of surgical resections and compare them with their coupled biopsy at diagnosis. Our cohort consists of 10 patients with osteosarcoma from 7-31 y.o (average of 16 y.o) having received methotrexate as neoadjuvant chemotherapy. A total of 35 RNA extractions using those tumor samples were performed for bulk sequencing and the results are being analyzed. From the same tumor region, a second specimen will be used for the snRNA seq. We are currently optimizing the protocol for nuclei isolation on bone tissue that we will use later in our cohort. We are focusing on snRNA-seq because of our frozen collection of surgical resections and the lack of fresh tumor samples.

Chaïma AZOUZI (MCD - Organisation et Dynamique Nucléaire)
Characterization of the mode of action of the antitumoral molecule BMH-21, which targets RNA Polymerase I transcription

Among the three nuclear RNA polymerases (RNA Pol) in eukaryotic cells, Pol I is the most active and specialized since it produces only one RNA specie: the pre-ribosomal RNA (rRNA) which represents 60% of total cellular RNAs. Despite being the most active polymerase in the cell, the mechanistic regulating Pol I elongation process, known to be discontinuous with high occurrence of pauses, has to be clarified. Our project aims at deciphering Pol I elongation complex features using a Pol I mutant called “SuperPol” which displays increased transcriptional activity in vivo compared to WT Pol I. Our results revealed that WT Pol I is prone to frequent premature termination of transcription with production of abortive transcripts in vivo. These events are strongly reduced in the SuperPol mutant. Furthermore, investigations on Pol I elongation dynamic through the use of simplified in vitro system revealed that SuperPol cleaves better and restart elongation more efficiently after a pause compared to WT Pol I: in other words, SuperPol mutant is more processive than WT Pol I. Finally, we discovered that the modified elongation features of SuperPol allows resistance to BMH-21, a promising antitumoral drug targeting Pol I activity, whose mode of action need to be elucidated. Thus, SuperPol mutant allows, in one hand, the opportunity to understand the mechanistic of WT Pol I elongation regulation, and in other hand, to understand and put in evidence the target of BMH-21 mode of action. The understanding of the precise mechanisms of Pol I transcription and the implication of each inherent elongation feature opens wide prospects on health-related areas of research, such as a large number of genetic diseases collectively called ribosomopathies and cancer therapy aiming at inhibiting Pol I activity.

Alexia DE CARO (IPBS - Cellular Biophysic (Rols))
Efficiency of new high frequency electrical protocols for in vitro electrochemotherapy with cisplatin, bleomycin and calcium

For more than 20 years, electroporation has been developing in the field of cancer and is becoming increasingly common in the treatment of skin cancers. By increasing the cytotoxic effect of anti-tumor drugs (bleomycin, cisplatin), electrochemotherapy has already proven its effectiveness on tumors in human medicine but also in veterinary practice. However, this treatment requires loco-regional or even general anesthesia, as electrical pulses can be painful and cause muscle contractions. Several publications proved that application of high frequency pulses (5,000 Hz) resulted in much less discomfort to the patient than the 1 Hz protocol used in the clinical practice in the 90’s and 2000’s.

In order to maintain the effectiveness of the treatment and reduce the pain associated with contractions, we are developing new protocols using a high-frequency generator, and new multipolar electrodes. Our ongoing results obtained on a colorectal cancer cells line (HCT-116) cultured both 2D and 3D (spheroids) showed a clear effect on cell death. Using our new protocols, the viability rate on cell suspension were around 10 % forty-eight hours after treatment and less than 5 % ten days after. Results on spheroid models show a significant decrease of spheroid size, which allow to conclude on growth impairment. These results were comparable to ESOPE (European standard operating procedures for electrochemotherapy) protocol currently use in clinic. Therefore, our high frequency electroporation protocols appear promising for an effective cancer treatment with similar temperature increase than ESOPE protocol and without noticeable muscular contraction.

Maria CHAOUKI (CRCT - SigDYN Intégration des signaux cellulaires et PI3K de classe I II III)
Treating B cell lymphoma patiens with inhibitors OFPI3Kδ(phosphoinositide 3-kinases) : how to increase the efficacy ?

PI3K delta (PI3Kδ) is a protein that is overexpressed in many B-cell malignancies and plays a role in the survival, proliferation and migration of these cancer cells. Its targeting is very effective in treating certain patients with chronic lymphocytic leukemia (CLL). The PI3Kδ inhibitor, Idelalisib (IDL), is thus available for the treatment of chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL), but induces numerous toxicities at the recommended doses. My research team aims to optimize how PI3Kδ inhibitors are used to reduce toxicity while maintaining maximum efficacy. To provide answers, two avenues will be explored :1.As new PI3Kδ inhibitors have been shown to be less toxic in treated patients, my project aims to compare their cellular and molecular effects on cells from lymphoma patients to better understand their action and reduced toxicity. In particular, I will focus on a novel PI3K inhibitor that inhibits PI3Kδ differently than the reference inhibitor Idelalisib. 2.Since a clinical trial conducted by my team at IUCT-O shows that lower doses are effective and reduce toxicity, I would like to study the molecular effects of these low doses. In conclusion, by demonstrating that each PI3Kδ inhibitor induces different molecular effects or that the same PI3Kδ inhibitor at lower doses does not induce the molecular changes associated with toxicity, my project will provide evidence that these inhibitors can be effective without being toxic and guide future clinical trials with PI3Kδ inhibitors under development.

Charlène MARTIN (INFINITy)
Impact of hCMV infection on the miRNA composition of small placental extracellular vesicles and consequences on the biology of fetal neural stem cells

Human Cytomegalovirus (hCMV) is the most commonly transmitted virus in utero. Congenital hCMV infection is the most frequent cause of cerebral malformations and deafness of infectious origin, without any reliable prognosis methods today. Although the virus has a direct effect when infecting neural progenitors of the fetus, the response of the placenta could also contribute to disturbances in fetal brain development, in particular via the secretion of small extracellular vesicles (sEV). Indeed, the protein composition of these placental membranous nanovesicles is modified during hCMV infection. Using first-trimester human cytotrophoblasts, we examined the impact of hCMV infection on the miRNA content of sEV released into the culture supernatant. We observed an enrichment of sEV in viral miRNAs in the infected condition, as well as a deregulation of cellular miRNAs highlighting a decrease in the expression of miRNAs involved in the development of the central nervous system. As this difference in composition may have consequences on fetal brain development, we then evaluated the impact of sEV from cytotrophoblasts - infected or not - on the biological properties of human neural stem cells (NSC). The growth, survival and proliferation of the latter are not impacted by sEV from cytotrophoblasts, whether infected or not, however a significant difference was observed concerning the differentiation of NSCs into neurons. These results support the hypothesis of a positive influence of placental sEVs on fetal brain development, which would be altered during hCMV infection. In conclusion, hCMV could therefore act indirectly on the fetal cerebral compartment by modifying the composition of placental sEVs, which would impact the biology of neuronal progenitors in a paracrine manner.

Emilie GUEMAS (INFINITy - Team 6 EIPIC & LAAS - M3)
Study of the I431V mutation of DHPS in Plasmodium falciparum

Malaria remains a major public health problem. In addition to protective measures against the mosquito vector, the sulfadoxine-pyrimethamine (SP) combination is used throughout Africa for the intermittent preventive treatment of malaria in pregnancy (IPTp) and the seasonal malaria chemoprevention (SMC) to prevent malaria in children under 5 years. However, the efficacy of this chemoprophylaxis is threatened by the emergence of mutations in the Plasmodium falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes, which code for the enzymes targeted by pyrimethamine and sulfadoxine, respectively. A new mutation in PfDHPS, I431V, was identified in Nigeria in 20071 and in Cameroon in 2010. This mutation was mainly found in association with 4 other PfDHPS mutations forming a vagKgs quintuple mutant. The mechanisms of resistance to sulfadoxine are very poorly studied. We obtained a complete PfDHPS WT structure from a PfDHPS crystal structure (PDB ID: 6JWQ) by filling the missing parts. Then, 5 PfDHPS mutants of interest, with various combinations of mutations were generated. First, molecular dynamics (MD) simulations were performed with AMBER software, for a period of 200 ns. This allow us to study the conformational properties and dynamic behaviour of the enzyme-substrate complexes with natural substrate (pABA) and inhibitor (sulfadoxine). These molecular dynamics trajectories were analysed to compare the substrate binding free energies, which suggest that the vagKgs quintuple mutant has an increased affinity for pABA. During MD simulations, sulfadoxine was released from the active site in all 6 systems. Based on these structures obtained from the MD simulations, we will perform hybrid quantum mechanics/molecular mechanics (QM/MM) calculations to refine the understanding of the effects of mutation on active site structure and stability, solvent accessibility and charge distribution.

Asja GARLING (IRSD - Team 2 PATHOGENIE ET COMMENSALISME DES ENTEROBACTERIES)
Development of a vaccine platform against pathogenic Escherichia coli

Confidential abstract ! 

Audrey GOMAN (IRSD, TEAM 2 PATHOGENIE ET COMMENSALISME DES ENTEROBACTERIES)
A short-chain dehydrogenase/reductase encoded by the core genome of Pseudomonas aeruginosa induced the production of deleterious Outer Membrane Vesicles

Confidential abstract !

Chen HAOXIANG  (LMGM - Group IEVA)
Interactome of bacterial envelope-assembly machineries: biogenesis and remodelling programs in bacterial pathogens

Insertion of outer membrane protein (OMP) into the outer membrane (OM) of Gram-negative bacteria is -barrel assembly machinery (or BAM complex). The BAM assembles MltA-interacting protein (MipA) to form a -barrel structure. Here we report the discovery of MipA. We verified the interaction between BAM and MipA and analysis MipA quaternary architecture. We also found that MipA interacts with OmpC, LptD, LamB, SurA, Skp and DolP, the most interesting of which is that MipA only interacts with unfolded LamB.

Grégoire MONGIN (IPBS)
Uncovering the mechanisms that increased in vivo persistence and antibiotic tolerance of tuberculosis bacilli through experimental evolution

Persistence and efficient transmission in humans are the hallmark of Mycobacterium tuberculosis, the most frequent tuberculosis bacilli (TB). These features determine the TB pandemics. Pathogenomic evidence suggests that M. tuberculosis evolved from an environmental ancestor similar to Mycobacterium canettii, a rare human pathogen, which displays reduced transmission in humans and reduced capacity to persist in the mouse model. However, the genetic and phenotypic adaptations responsible for the higher epidemic capacity of M. tuberculosis vs M. canettii remains poorly characterised. Eight M. canettii populations from two distinct genetic backgrounds were evolved experimentally by infecting mice to select mutants with enhanced persistence in vivo when compared to parental strains. Genome sequencing of 140 M. canettii mutants, isolated at various steps during the process, revealed parallel and convergent evolution of the different populations. Mutations fixed in these populations identified a limited number of targeted pathways corresponding to cAMP metabolism and import/utilization of nutrients. Most of the tested mutants were more resistant than their parental strains to several stress encountered in vivo, such as nitric oxide, an important effector of immunity against M. tuberculosis infection. Interestingly, results suggests that theses mutants also display increased tolerance toward common antimycobacterial drugs. Hence, the aim of my project is to continue the phenotypical characterization of those mutants and most importantly, to understand how the mutations endow M. canettii with an increased capacity to withstand different stresses from the host, or the environment. Those results will help us find out how as the ancestor of M. tuberculosis was able to adapt to those stresses, which eventually led to his emergence and spread across the human population to become the pathogen we know today.

Lucie GELON (IPBS - Endothelial cells in immunity, inflammation and cancer)
Lung thrombopoiesis : regulation and functions during lung inflammation

"My thesis team has recently demonstrated the existence of pulmonary thrombopoiesis but its specificity, the mechanisms that regulate it and its functions remain unknown. Our preliminary data suggest that pulmonary megakaryocytes have a specific role in inflammation and that platelet activation and production are modified during lung injury by local inflammatory mediators, including IL-33, a danger signal involved in allergic inflammation and the development of asthma. The main goal of my thesis project is to provide a better fundamental understanding of the regulation of pulmonary thrombopoiesis and its specificities, in particular by assessing the importance of IL33 in platelet production and specialization during allergic inflammation. To do so, we expose mice to recombinant IL-33 or to a fungus (alternaria alternata), causing IL-33-dependent pulmonary inflammation. The differentiation of megakaryocytes as well as the production, activation and specialization of platelets in wild type and IL-33 deficient mice is followed by flow cytometry, proteomics and intravital microscopy. Thus, this project aims to improve the understanding of the regulation and role of platelets in the development of pulmonary inflammatory diseases such as asthma and to identify new therapeutic targets."

Rémi MONTANE (RESTORE - Team 3 Metabolink)
Role of brown and beige adipose tissues in lactate systemic fluxes

Energy homeostasis is based on metabolic cooperation between several organs such as liver, muscle and adipose tissues. However, these dialogues are altered during aging and this contributes to metabolic disorders. Among the various metabolites that support inter-organs metabolic communication, lactate plays an important role. This metabolite, which is produced when the glycolytic flux exceeds mitochondrial oxidative capacities, is involved in redox homeostasis, feeds several biosynthetic pathways as well as mitochondrial oxidative metabolism, and can regulate gene expression through epigenetic mechanisms. The team demonstrated that lactate is a powerful activator of the beiging process, which corresponds to the appearance of brown-like adipocytes into white adipose tissues. The canonical function of brown and beige adipose tissues - that strongly express the mitochondrial uncoupling protein UCP1 - is the regulation of body temperature through non-shivering thermogenesis. They play a critical role in glucose and lipids levels, their presence confers protection against cardio-metabolic diseases. The activation of these tissues by lactate allowed us to propose a new function from brown and beige adipose tissues in the regulation of systemic lactate levels thanks to their strong oxidative capacities. We hypothesize that the decrease in brown and beige adipose tissues with age would contribute to the alteration of systemic lactate fluxes and the onset of metabolic diseases. We performed lactate tolerance tests and in vivo 13C lactate isotope tracing on different mice models such as aged mice, mice with genetic invalidation of UCP1 or exposed to different temperatures. Label incorporation into different metabolites was analysed by mass spectrometry in different organs. This work reveals the role of brown and beige adipose tissues in lactate systemic fluxes through inter-organ dialogues and how their dysregulation change lactate metabolic fate at the systemic level.

Céline MARTIN (INRAe - Toxalim - Integrative toxicology and metabolism (TIM))
Pre-clinical study of the effects of thermoneutrality on non-alcoholic fatty liver disease

"Non-alcoholic fatty liver disease (NAFLD) including steatosis and its more advanced stage non-alcoholic steato-hepatitis (NASH) are pathologies whose prevalence is increasing in direct relation to that of obesity and obesity and type 2 diabetes. In the absence of available treatments, research into preclinical models is necessary in order to study the inflammatory component and the progression to the irreversible stages of the disease (NASH). In this work, we evaluated the effects of thermoneutrality on obesity, glucose homeostasis and NAFLD. In this work, we evaluated the effects of thermoneutrality on obesity, glucose homeostasis and NAFLD.

Male mice were fed a control (Chow) or obesogenic (Western Diet, WD) diet for 13 weeks and housed at either thermoneutrality (TN; 30°C) or room temperature (RT; 30°C). WD mice show significantly higher obesity, fasting blood glucose and HOMA-IR at thermoneutrality than at room temperature.

In addition, the WD induced steatosis associated with inflammation and fibrosis at thermoneutrality than at room temperature.

Hepatic genome expression analysis revealed that the number of genes significantly modulated by the WD is more than 2-fold higher in TN (WD vs. Chow 705 genes, induction factor >2, Pcorrected<0.01) than in RT (WD vs. Chow 323 genes) with an overexpression of genes involved in inflammation and a decrease in the expression of genes involved in lipid metabolism regardless of the housing temperature.

These results demonstrate that thermoneutral housing exacerbates the metabolic and hepatic disturbances induced by a WD. This pre-clinical model thus allows us to recapitulate in rodents the different stages of the transition from steatosis to NASH in rodents and is of medical interest for the study of for the study of new pharmacological molecules for example."

Maxime BORRET (I2MC - Team 7 LIPSIPLAT)
Platelets role on early phases liver metabolic disorder

Non-Alcoholic Fatty Liver Diseases (NAFLD) represent a major public health problem worldwide. The WHO categorizes them as the number one cause of liver disease in Western countries with hundreds of thousands of people affected in France. They are metabolic disorders of the liver that begin with a strong accumulation of lipids, called hepatic steatosis, and which evolve for ~30% of patients, to an inflammatory and fibrotic state called steatohepatitis (or NASH, Non-Alcoholic Steato-Hepatitis). These two stages, which are still reversible, may remain asymptomatic or progress to cirrhosis and liver cancer (or hepatocellular carcinoma) with a survival rate of 10% at 5 years. Because of their multifactorial origin, the etiology of NAFLD is not the same in all patients and remains largely unresolved, which complicates the prevention and management of these diseases. From a therapeutic point of view, the only current recommendation to prevent or limit the evolution of these diseases is to take change lifestyle by practicing a regular physical activity with a balanced diet. Liver transplantation can also be an invasive alternative to the treatment of NAFLD. Beyond their primary role in hemostasis and thrombosis for the arrest of bleeding during vascular injury, blood platelets are involved in many pathophysiological processes, due to the molecules they release and their increased adhesive capacity with the multitude of receptors expressed on their surface. Platelets have also been show to be involved in the regulation of liver regeneration and fibrosis mechanisms. About platelets role in NAFLD, only one recent study shows a detrimental role of platelets in the late stages of NASH and hepatocarcinoma. This effect is mediated by platelet secretion and platelet interaction with endothelial cells and macrophages via the platelet receptor GPIbα. The role of platelets in the early and still reversible phases of NAFLD, hepatic steatosis and early phases of NASH, remains unknown.

Elene Maria FISTES (EVOLSAN)
The quest for truth in science, and in particular in oral surgery

"Our scientific knowledge is based on studies and experience. In oral surgery treatments and management guidelines taught at university but also recommended by groups of authority are supposed to be based on Evidence Based Medecine and Evidence Based Dentistry. However, some of our treatments are dictated by experience and are not necessarily based on high-level-of-proof studies. Some of the treatments we provide are not validated by a meta-analysis but are based on our observation of the use of this therapy by our peers with favorable results over several years.

What is considered high level of proof in medicine? We will go through the history of the quest of truth in science and attempt to define high level of proof in medicine, and detail the tools we have today to limit error and bias in medical research.

Is the knowledge that dictates our treatments and clinical management in oral surgery of a high level of proof?

What are the ethical implications of the level of proof of our present knowledge?"

Saha NASERI (CERPOP - EQUITY)
Antenatal care service delivery for Afghan women: From Afghanistan to France

"Afghanistan has been internationally recognized for the significant health gains made over the past two decades, mainly in maternal health outcomes, as a result of implementation of many innovative interventions. However, the collapse of the government by Taliban, in August 2021, which was followed by the halt of international aids to Afghanistan, posed significant challenges the young and donor-dependent health system, which raised many questions about the resilience and sustainability of these interventions. In the first phase of this PhD thesis, it is aimed to review and analyze the implemented antenatal care interventions in Afghanistan between 2002 and 2022, in order to identify and explain the mechanisms of success and failure of these interventions, using a Rapid Realist Review (RRR) methodology. In parallel, as a result of the government collapse, hundreds of thousands of people have migrated to other countries. By the end of 2020, there were 455,295 refugees from different countries registered in France, with Afghanistan featuring at the top of the list. Between August 16 and 27, 2021, around 2600 Afghan refugees threatened by the Taliban also evacuated to France. The unmet need of refugees for health services has been observed in France, notably on access of women to healthcare and prevention services. It is documented in some developed countries, on the other hand,  that Afghan nationals have higher unmet to health services need compared to other refugee nationalities, due to their complex, forced, and long-duration migration experience and other unknown reasons. The experience of Afghan refugees in France is overlooked. In the second phase of this PhD thesis, it is aimed to assess the access of Afghan women refugees to antenatal care services, and accordingly to develop an intervention prototype to improve the access of Afghan women refugees to antenatal care services in France. This thesis will have a community-based participatory approach."

Garance CASTINO (RESTORE - Team 3 Metabolink)
Low-responders to exercise among aged individuals: understanding mechanisms

"Physical activity is widely acknowledged as a beneficial practice for both the body and mind. In terms of muscle benefits, engaging in physical activity can help maintain and sometimes even increase muscle mass and strength, regardless of age. This is especially important for elderly individuals, as aging can have a negative impact on locomotor function, which can lead to a loss of autonomy and a decrease in overall quality of life. Therefore, exercise is one of the main strategies recommended to prevent the decline of muscle strength and mass.

However, some studies have shown that certain individuals may not experience significant muscular benefits after training, and are thus referred to as ""low-responders"" to exercise. Since muscle benefits are particularly important for elderly individuals, it is crucial to focus on aged low-responders. To study this group more precisely, we aim to first mimic the heterogeneity of response to exercise in an animal model. We will then use an index based on the progress of each animal in different functional tests to identify the low-responders. Finally, we will try to identify the cells and mechanisms responsible for the low-response to exercise, in order to propose strategies to improve the responsiveness to exercise for aged individuals."

Jason SHOURICK (CERPOP - MAINTAIN)
Weather and Seasonality Impact on Physical and Cognitive Measures

Summary
Although an effect of high temperature on cognitive and physical performance has been shown in experimental conditions, weather is not taken into account in the standardization of measurements taken during clinical trials and therefore some measures may not always reflect the participant’s actual performance. 

Methods
We re-analyzed 1350 participants aged over 70 years from MAPT, a multicenter randomized controlled prevention trial which evaluated non-drug multidomain interventions​ versus placebo over 3 years. This trial did not demonstrate a beneficial effect of the interventions on its main outcome, cognition measured using a composite cognitive Z-score of 4 cognitive tests. Physical performance was evaluated using gait speed, the Short Physical Performance Battery (SPPB) and hand grip strength.

We analyzed participants with a meteorological station less than 10 km from their study center. We used ground observed data from Meteo France, the French national meteorology institution. We modeled participants’ trajectories for cognitive and physical performance using linear mixed models and considered observations lower than expected by at least the MCID (Minimal Clinical Important Difference) as abnormally low. We then used logistic regression with abnormally low measures as the outcome and weather parameter aggregated per day (minimum, mean and maximum temperature), as well as season, as explanatory variables.

Results
None of the weather and seasonality parameters were associated with abnormally low cognitive performance. Higher temperature was linked with abnormally low gait speed or SPPB, but there was no effect of temperature on hand grip strength.

Regarding seasons, Spring and Winter were associated with a lower risk of abnormally low total SPPB and hand strength, and Summer with a higher risk of abnormally low gait speed.

Conclusion
We showed that weather had an impact on the measure of gait speed and SPPB but not on cognition.

Emma GRANDGIRARD (RESTORE - Team 1 STROMAGICS & IRIT - ADRIA/DEEL)
Breaking the 2D barrier: developing an image simulation tool for 3D nuclei segmentation in adipose tissue

Artificial intelligence methods, particularly deep learning algorithms, are widely used to perform various processing tasks on microscopic images to investigate complex biological issues in different contexts (biological tissues, whole organisms, or organoids, in physiological or pathological states). Despite their increasing popularity, two major challenges remain. First, these algorithms require a substantial amount of data to be properly trained. However, the time, material resources, and expertise required to conduct biological experiments make it challenging to collect a sufficiently large dataset for deep learning. The second challenge lies in dimensionality, as complete and accurate analyses can’t be performed without considering the 3D organization of the tissues. I will we present a data simulation pipeline which uses existing 2D microscopic images to create new images in 2D and 3D. The specific aim is to improve nuclei segmentation in 3D, but this approach could be extended to other 3D image processing tasks.

Ronan BOUZIGNAC (CBI & IGMM)
Nuclear response to physical forces during EMT

"Cells are constantly confronted to physical forces, either internal forces, generated by the cytoskeleton or external forces coming from a challenging environment. In response to these mechanical stimuli, the cells are able to produce a biochemical response. This is called mechanotransduction. Several in vitro studies have shown that the nucleus can resist to forces to protect the genome, but can also be an actor in mechanotransduction. However, due to the intrinsic complexity of tissues, the role of the nucleus in the mechanotransduction remains poorly understood in vivo.

To overcome this lack of knowledge, I will address the role of mechanotransduction in vivo, in cells generating forces and use the epithelial to mesenchymal transition (EMT) as an integrated model. Indeed, Suzanne's team has shown that in Drosophila, at the initiation of the EMT process, cells generate mechanical forces.

Thus, the aim of my project will be to decrypt the impact of these forces on the nucleus in terms of nuclear envelope dynamics and transcriptional dynamics,  using the mesoderm invagination of the Drosophila embryo (a classic EMT model).

I first analyzed the dynamics of the nuclear envelope during the mesoderm invagination. I observed  stereotyped deformations of the apical domain of nuclei in response to mechanical forces, that coincide with the reorganization of some nuclear components specifically in the deformed region. In order to characterize the transcriptional dynamics, I am currently focusing on identifying potential mechanosensitive genes through different approaches: A global approach via a nascent RNAseq and a candidate approach focused on mesodermic genes. The mechanical response of potential candidates will then be analyzed using micromanipulation approaches (compression and optical tweezers) and following transcription in live.

Overall, my project will contribute to a better understanding of the impact of mechanical forces on the nucleus in the EMT process, in vivo."

Henri MBOUMBA (MCD & CBI)
Study of the molecular bases of Wapl counteracting cohesion-dependent loops 

"Cohesin is a  protein complex known to maintain sister chromatid cohesion. At its core, cohesin is composed of two rode-shape subunits,  Smc1and Smc3, along with a  kleisin subunit, Scc1. When assembled, each subunits interact simultaneously with the other two, therefore giving to the complex the form of a tripartite ring. In order to establish sister chromatid cohesion, the cohesin complex encircles one chromatid. Subsequently, after chromatid duplication, the sister chromatid end up traped inside the cohesin ring . Moreover, the encirclement of chromatid, in the ring, is regulated by cohesin partners. Indeed, the cohesin entrapment of chromatin is counteracted by a protein named Wapl. Wapl causes chromatid to escape from cohesin entrapement by an opening of at least one of cohesin three interfaces (Smc3-Scc1, Scc1-Smc1 or Smc1-Smc3).  

Besides maintaining sister chromatid cohesion, cohesin is also known to organize chromatin into loops, probably, via a process called “loop extrusion” (LE). During LE, cohesin captures a small chromatin loop which is then enlarged by cohesin translocation along chromatin. Although the mechanisms of loop establishment are not completely understood, there is evidence that Wapl inhibits loop enlargement. Perhaps, Wapl causes an opening of the ring, leading to cohesin to fall off the extruded loop. If this is the case, artificially closing the interface(s) opened by Wapl could be a way to counteract Wapl inhibiting effect on loop enlargement. Therefore during my PhD, I mainly intend to identify which cohesin interface(s) is open by Wapl during loop enlargement. For that purpose, I intend to artificially close each one of cohesin interfaces using a crosslink reagent, and by this strategy, indentify the interface(s) opened by Wapl during loop enlargement."

Zoltan UDVARDY (IPBS - Proteomics and Mass Spectrometry of Biomolecules)
Development of Intelligent Mass Spectrometry Acquisition Methods Using Real-Time Control of Instruments 

Mass Spectrometry (MS) based proteomics is an important tool in molecular, cellular and systems biology. One of the key components of MS workflows are acquisition methods that define the operation of mass spectrometer instruments during acquisitions. Currently used approaches such as Data Dependent Acquisition (DDA), Data Independent Acquisition (DIA) and Parallel Reaction Monitoring (PRM) have their advantages but they also come with certain limitations. DDA and DIA are high-throughput, but low abundant peptides are often undetected, while PRM has high specificity but low-throughput. The analysis of proteomics samples would benefit from using sophisticated, intelligent and customised MS acquisition techniques. The ability to control MS instruments in real-time was proven to be pivotal in creating such methods. Interfacing with MS instruments requires in-depth knowledge and considerable effort, but there are no open-source solutions aiding this process. In this project an open-source software framework is created that can control instruments in real-time and facilitate the development of new acquisition methods. Using this framework, novel intelligent acquisition techniques are designed to overcome some limitations of current approaches. The developed framework called MSReact can control different instrument families. It is equipped with tools for easy method prototyping and provides integration with popular scientific and machine learning Python libraries. With the aid of MSReact an intelligent PRM method is being implemented that applies a novel real-time retention time (RT) calibration approach. This technique enables peptide monitoring using narrow time windows, which is key to deliver high-throughput PRM. Preliminary results show that a large majority of targeted peptides could be identified and quantified. As a next step MSReact will be made publicly available and the intelligent PRM method will be further improved and benchmarked.

Sudip KARKI (CERPOP - SPHERE)
Medications for chronic pathologies and pregnancy: Pharmacoepidemiological Studies within the framework of European Project “ConcePTION”

"It is becoming more common for women who have long-term health conditions to become pregnant. Several new medications are marketed for these chronic diseases however data regarding their safety during pregnancy remain scarce. 

In this thesis I will first define the chronic exposure to medicine or chronic prescription in health data sources either in terms of number of days per year or number of prescriptions per year and apply this definition in a cohort of pregnant women to study its impact on health of the mother as well as pregnancy outcome. 

So far, a literature review has been conducted to define the chronic exposure to medicine in administrative health data sources

Several pathologies for which there is a chronic exposure to medications will be investigated:

First, a Population based cohort study was carried out on the risk of hypertensive disorder in pregnancy. The association between exposure to serotonin norepinephrine reuptake inhibitor and the occurrence of gestational hypertension was assessed.

Second, I started to work on chronic diseases within the European Innovative Medicine Initiative project (IMI) “ConcePTION” that is a European ecosystem that plan to efficiently, systematically, and in an ethically responsible manner, generate and disseminate reliable evidence-based information regarding effects of medications used during pregnancy and breastfeeding to women and their healthcare providers. Health conditions that will be studied will be systemic lupus erythematosus (SLE), multiple sclerosis (MS) and neuropathic pain. A case-malformed control study is being carried out to explore the evidence of risk of congenital anomaly associated with disease modifying therapy used during first trimester of pregnancy in women with MS."

Quan LI (Musée de Tautavel)
Comparison of the occupation levels between the Early Paleolithic (bifacial) sites in Caunedel'Arago (Tautavel, France) and China (Bose Basin)

"The Caune de l'Arago (Tautavel, Pyrénées Orientales) is an emblematic archaeological site in the South-East of France. 690,000 to 92,000 years ago, human populations followed one another on this site that recorded one of the most complete archaeological sequences in Europe. This sequence provides us with essential information, not only on the biological evolution of human populations, but also on the evolution of their behaviors. The relationships between these technical behaviors and biological evolution are at the center of this thesis topic. This research is also part of an international collaboration. Indeed, the observations carried out on the Caune de l'Arago will be compared to those obtained on many archaeological sites in southern China, dating back about 800,000 years (Han River basin, Bose basin).

This thesis research will collect unpublished information, of great archaeological value, in several Paleolithic sites in southern China. The differences between the archaeological characteristics of southern China and the Caune de l'Arago in the early Paleolithic will then be analyzed. Thus, for the first time, it will be possible to highlight possible convergences or, on the contrary, relations between these two biological and cultural areas."

Emilie POISSON-BEUVART (EVOLSAN - Team 2 Médecine orale et traitements hybrides)
Chronic pain and Anthropology of Health : care structures and  care pathway in occluso-posturology

Orofacial pain affects one in five people with a prevalence of acute form that is easy to treat by the dentist. Unfortunately, the diagnosis of the chronic form is more difficult. Indeed, the average time to relieve is about 5 years. This anthropological study aims to understand the links and the organization of the care/professional structures having taken care of chronic pain patients presenting with occluso-postural disorders. The objective is to reduce the waiting time for treatment and to optimize therapeutic efficacy. The study includes field observation as well as interviews with professionals and patients in order to better understand the care pathway and the associated obstacles. The first results show difficulties of orientation of the professionals, a therapeutic wandering and an important suffering of these patients.

Raphaël MARTIN-ROY (Centre for Anthropobiology and Genomics of Toulouse - AGES)
Genomic and Morphological Evolution of Mytilus mussels

The relationship between phenotypic and genomic adaptation to environmental conditions is an important question in Evolution, especially under the rapid changes caused by human activities during the last 200 years. Related changes in marine environments (e.g., increased seawater temperature) are stressors hindering the capacity of shell-producing organisms to calcify. This is the case for mussels of the genus Mytilus, which are of great ecological and economic importance, and offer a unique opportunity to study adaptation to environmental changes. Mytilus mussels are indeed composed of seven interbreeding species found in a large variety of habitats and along environmental gradients subjected to rapid changes. Here, we first describe the genetic and shell phenotypic variations in respectively 63 and 194 mussel individuals from 18 sites along the environmental and genomic (M. edulis x M. trossulus hybrid zones) gradients of the Baltic Sea and Greenland. To do so, we apply a multidisciplinary approach combining 3D morphometrics (diffeomorphisms) and genomics. Preliminary results show 3D morphological variations along the Baltic Sea possibly linked to a combination of genetic and environmental factors. In Greenland, mussels show morphological similarities to Baltic individuals despite varying environmental conditions, possibly indicating an effect of genetic background on mussel morphology. Generalized mixed models will later be developed to more precisely describe and quantify the interactions between phenotype, genotype and environment helping to better understand how mussels are impacted by environmental changes. This work will finally be broadened by studying the adaptation of Mytilus mussels to environmental changes through time by applying the same multidisciplinary approach to a collection of Mytilus mussels dating back to the last 7000 years.

Pauline CLARAZ (CRCT)
Patients with swallowing disorders: galenic and pharmacokinetic implication in the modification of oral drug forms

In several situations, physiological (children under 6) or pathological (glioblastoma, throat adenocarcinoma), cancer patients may not be able to swallow their medication safely. In these cases, in order to provide the best care, the hospital pharmacist can formulate medication adapted to the clinical presentation of the patient. The gold standard for such disorders are oral liquids (oral solution, oral suspension). Due to the lipophilic nature of the most widely used oral anticancer drugs, the latter is mainly developed. With these modes of administration, there are two major issues. The first is the physico-chemical and microbiological instability in relation to the intrinsic properties of the drinkable suspension (solid phase dispersed in a non-solvent liquid) which can lead to inhomogeneity of the mixture and then to an inappropriate delivered dose. The second issue is pharmacokinetic modification regarding the galenic modification performed as it may lead to ineffectiveness or undesirable increase in toxicity. In this thesis, we propose to work on these two major axes. Regarding the galenic question, this work will focus on the use of a new suspending agent: gellan gum to fight against sedimentation. In this context, a new oral liquid galenic base is formulated to be compatible with administration to the youngest and oldest patients. The rheological behaviour of this vehicle is compared to existing products. Then, the stability of the formulated suspension is investigated. The chemical stability is determined by a validated liquid chromatography (LC-UV and LC-MS/MS) stability indicating method to highlight degradation products. On the second time, concerning pharmacokinetics issues, thanks to the results of the therapeutic drug monitoring of the patient who benefits from the oral suspension, a population pharmacokinetic approach will make it possible to evaluate the impact of the galenic modification on pharmacokinetic parameters such as apparent clearance.

Hugo BARRET (AMIS)
What methods should be used to measure glenoid bone loss?  A software study using a healthy glenoid

Introduction
There is no consensus on how to measure this glenoid bone loss (GBL) and on the threshold for choosing the type of surgery on anterior instability. The objective is to evaluate the accuracy and reproducibility of 7 methods of measuring GBL.

Methods
From a database of 174 CT scans, the inclusion criteria allowing to have only ""healthy"" glenoid were: (1) a version between 7 degrees of anteversion and 7 degrees of retroversion and (2) a superior inclination between 0 and 11 degrees then a review by a surgeon who validated glenoid without lesion. Once the ""healthy glenoid"" CTs were selected (n=14), 5 steps were necessary to reach the result of the GBL assessment.). Seven calculation methods were compared (step 5): a height/width ratio (M1), a missing surface calculation (M2), a circle method (M3), a radius ratio (M4), a linear measurement percentage (M5), a defect width ratio (M6), and a volume ratio (M7). We evaluated 2D and 3D measurement, accuracy and reliability of measurements for each method with each variation: bone loss, different circles, tilt.

Results
When assessing AI bone loss in 3D, methods 2, 3, and 7 were closest to constant for all levels of bone loss. For anteroinferior bone loss, the difference between virtually created and measured bone loss was less than 5% at all bone loss levels for M2, M3, M7. For anterior bone loss, the difference at all levels was less than 5% only for M2 and M3. For the other methods, the error was larger which shows the accuracy of M2 and M3. The variation of the circle showed a linear trend for M2 and M3 

Discussion
Anteroinferior glenoid bone loss is better assessed than anterior bone loss, which is closer to clinical practice. The area-based methods seem to be the most reliable and accurate. They are 3D and standardized. It should be commonly used by surgeons to define a threshold value and guide the most appropriate therapeutic decision.

Antoine DUBUC (CERPOP - SPHERE)
Improvement of Mucosal Lesion Diagnosis with Machine Learning Based on Medical and Semiological Data: An Observational Study

Despite artificial intelligence used in skin dermatology diagnosis is booming, application in oral pathology remains to be developed. Early diagnosis and therefore early management, remain key points in the successful management of oral mucosa cancers. The objective was to develop and evaluate a machine learning algorithm that allows the prediction of oral mucosa lesions diagnosis. This cohort study included patients followed between January 2015 and December 2020 in the oral mucosal pathology consultation of the Toulouse University Hospital. Photographs and demographic and medical data were collected from each patient to constitute clinical cases. A machine learning model was then developed and optimized and compared to 5 models classically used in the field. A total of 299 patients representing 1242 records of oral mucosa lesions were used to train and evaluate machine learning models. Our model reached a mean accuracy of 0.84 for diagnostic prediction. The specificity and sensitivity range from 0.89 to 1.00 and 0.72 to 0.92, respectively. The other models were proven to be less efficient in performing this task. These results suggest the utility of machine learning-based tools in diagnosing oral mucosal lesions with high accuracy. Moreover, the results of this study confirm that the consideration of clinical data and medical history, in addition to the lesion itself, appears to play an important role.

Honorat AGBECI (CERPOP)
Household expenditures in primary health care centers for children under 5 in Burkina Faso, Guinea, Mali and Niger, 2021/2022

Despite payment exemption policies (PEP) in Africa, the persistence of out-of-pocket (OOP) payments exposes households to catastrophic health care expenditures, exacerbating social inequalities. We measure household expenditures in West African primary health centers (PHCs) for the management of children under 5 years. The UNITAID-funded AIRE project is evaluating the implementation of the pulse oximeter integrated with IMCI in PHCs in Burkina Faso, Guinea, Mali, and Niger. PEP is full in Burkina Faso and Niger, and partial elsewhere. Children <5 years seen in IMCI consultation, classified as simple respiratory, moderate, or severe cases were included with parental consent in four PHCs per country. Each month, 5 simple/moderate, and 5 severe cases were randomly selected per PHC for the cost study. Direct medical costs (DMC) of cases fully managed at the PHC level and not referred were measured from the household perspective. From June 2021 to June 2022, 15,962 children were included in the AIRE project. Among 1,548 children selected for the cost study, 1,320 children were fully managed at the PHC level. The median (IQR) age ranged from 15 months (8-30) in Niger to 20 months (9-36) in Mali. Overall, the median (IQR) DMC at PHC level in USD was 0 (0-1.6) and 3.6 (1.8-5.7) in Burkina Faso and Niger; and 5.2 (3.1-7.9) and 7.9 (5.1-10.7) in Mali and Guinea. Drug costs accounted for more than 72% of DMC. The DMC of severe cases ranged from 0.7 (0-3.6) to 4.9 (2.3-7.6) in Burkina Faso and Niger and from 5.3 (0.9-9.5) to 8.5 (0-12.9) in Mali and Guinea. In West Africa, OOP payments persist for children <5 at PHC level, regardless of PEP, and the main expenditure being on drugs. With the exception of Burkina Faso, OOP payments remain a burden. Health policies should be improved to ensure financial protection for households.

Ariane WEYL (UMR1297 - ESTER)
Endometriosis and estrogen receptor: the alpha versus beta battle

"Endometriosis is a chronic, estrogen-dependent inflammatory disease that has a serious impact on quality of life. In this pathology, endometrial cells implant ectopically outside the uterine cavity. Various theories have been put forward to explain the implantation and persistence of these endometrial cells, which thus regain autonomy in parallel with the « eutopic » endometrium. Several forms of endometriosis have been described according to their location: in particular the superficial peritoneal form, and the deep peritoneal form, in which invasion of the neighbouring organs (digestive tract, bladder, uterine ligaments) can be observed. As for uterine physiology, estrogens, and mainly 17β-estradiol (E2), are key hormones for the growth and persistence of endometriotic tissue as well as for the inflammation and pain associated with it, but our understanding of the roles of estrogens and especially of their receptors (ERα and ERβ) in the pathogenesis of endometriosis need to be clarified.

In collaboration with the Gynecological Surgery Department of the Toulouse University Hospital, we wanted to better understand the role of ER alpha and ER beta respetively in the growth and persistence of endometriosis. For this purpose, we collected endometriotic lesions from patients treated surgically for this pathology, without hormonal treatment and with knowledge of the cycle phase.

The objective was to characterize the expression level of Erα and ERβ in endometriosis lesions using antibodies validated in the recognition of ERα (SP1 antibody) and/or ERβ (PPZ0506 antibody,) in a western blot, RT-PCR and immunohistochemistry study on human tissues. Because of the constitution of endometriosis lesions, in which the endometrial glands represent only a small area of the injured tissue, the gold technique is immunohistochemistry. We were thus able to challenge the existing paradigm, by demonstrating the absence of Erβ in endometriosis lesions and the predominant role of Erα."

Pierre VANDE PERRE (CRCT - Team 14 DIAD (Individualisation des Doses de Médicaments Anticancéreux))
Environment and eXome to Profile Early-onset Colon Tumor risk uSing MachIne LEarning – EXPECT SMILE

Incidence of early-onset colorectal cancer (EOCRC, diagnosis ≤ 40) increase by 2% annually since two decades. In France, having EOCRC is an oncogenetic indication for germline analysis on a consensus 14 genes “DIGE” panel: a genetic predisposition is found in only 25% of EOCRC. To highlight new variants in unexplained EOCRC cases, firstly we analyzed germline 584 genes panel variants in a cohort of 88 EOCRC patients without mutation in DIGE panel (DIGE-). 18 pathogenic/likely pathogenic variants (PV/LPV) in 16 genes were found to be statistically overrepresented in EOCRC group compared to exome (WES) variants of 51,377 control non-cancer European subjects from the GnomAD database These 16 genes were tested in a 2nd cohort of 82 Late-onset colorectal cancer (LOCRC, diagnosis ≥50) : 5 PV/LPV in 4 genes were found. Thus, whereas the involvement of these 4 genes in colorectal risk cannot be excluded, they are not specifically associated with early disease onset. By inference, we identified 14 germline candidate variants (12 distinct genes) statistically associated with EOCRC, of which 35% (5/14) were in genes involved in DNA repair (Vande Perre et al., in preparation). Secondly, we aim to identify EOCRC multifactorial predisposing profile (EOCRC-PP) by combining patients’ environment and germline WES, using machine learning approaches. Two populations will be explored: i) an 130 EOCRC local cohort (DIGE-) whose WES and environment data will be prospectively collected by sequencing and telephone interview and ii) subjects from the UK Biobank (45,000 volunteers, with data on lifestyle, social environment, health, and WES). Cohorts will be combined into two dataset: a Training and a Validation one. EOCRC-PP will allow the identification, among individuals exposed to define environmental and lifestyle factors, those who have a genetic profile at high risk of developing a EOCRC and who should therefore benefit from a tailored screening and prevention program.

Sebastien BOURDON (CRCT  - RNAReg)
Epitranscriptomic regulation of RNA G-Quadruplexes and impact on cancer genes expression

RNA G-quadruplexes (RG4s) are four-stranded structures known to control post-transcriptional gene expression. Their impact on RNA biology allows these structures to shape cellular processes relevant to cancer development, progression and treatment, making their targeting for therapeutic purposes an attractive option. My host team has largely contributed to the characterization of these structures, their binding partners (RNA-binding proteins, or RBPs), their function in cancer (Decorsiere et al, 2011, Cammas et al, 2015, Cammas et al, 2016), as well as their therapeutic potential (Herviou et al, 2020). In a recent review (Dumas et al, 2021) they brought to the forefront under-explored issues linking RG4s to cancer that constitute major challenges in the field for future research. Emerging evidence supports a link between RG4s and epitranscriptomics, specifically with N6-methyladenosine (m6A), the most prevalent internal RNA modification in mRNAs. m6A are emerging as critical regulator of post-transcriptional steps impacting cancer development and treatment, thus raising a growing interest for their regulation in cancer cells. One major challenge in the field lies in understanding the mechanisms controlling selectivity in m6A deposition, reading and erasing of modifications. Among these, the interplay with RNA structure is emerging as a critical, yet unexplored, mechanism to regulate m6A dynamics and function. The importance of RG4s and m6As in cancer, as well as the results obtained during my Master 2 internship supporting the possibility that they interact to modulate post-transcriptional gene expression, provide a strong rationale for studying the interplay between RG4s and m6As in cancer cells in order to understand the regulatory mechanisms and their impact on cellular processes relevant to cancer progression and treatment. 

Nikolaos KONTOPOULOS (CRCT  - Team 10 Therapeutic Innovation In Pancreatic Cancer)
Characterization of the mode of entry of oncolytic virus in pancreatic cancer cells

Oncolytic viruses (OV) are a novel bioactive class of cancer therapy that replicate in and specifically kill cancer cells. We recently demonstrated that SG33 strain which derives from Myxoma virus is oncolytic in PDAC, but still not all primary PDAC cultures are equal to oncolytic infection. Thus, there is a need to better understand the molecular basis that drive SG33 infection of PDAC cells. Host cell entry is one the first challenges for viruses to infect cells to exert their oncolytic capacities. Macropinocytosis (MPC) is a regulated endocytic mechanism that is elevated in PDAC as it supports the metabolic needs of the cancer cells. MPC is also utilized by large particles including viruses to penetrate into the cytoplasm of mammalian cells. Thus we make the hypothesis that SG33 entry and oncolytic activity in PDAC cells rely to some extent to MPC. In this work, we used pancreatic cancer cell lines and primary pancreatic cancer cultures. MPC was quantified by measuring 70kDa fluorescent dextran uptake into cells using microscopy. Following serum starvation alone or together with glutamine starvation or EGF treatment, a well-known inducer of MPC, the cells were classified into three groups, depending on the dextran signal. We next addressed whether blocking constitutive MPC with EIPA, which is an inhibitor of NA+/H+ exchange, may alter SG33 infectivity. We found that EIPA treatment inhibits by 64.1+/-8.4 % the replication of SG33 in primary cells as compared to control. Remarkably, we found that SG33 infection also promotes MPC as dextran uptake in AsPC-1 increases by nearly three-fold (3.4+/- 1.6) in the presence of virus. In these preliminary studies, we show that SG33 replication is significantly inhibited using blockers of MPC. In addition, we found that SG33 infection significantly induces MPC. This may call for future studies as SG33 may improve the delivery of clinically-relevant drugs, such as nab paclitaxel, that rely on MPC to enter cells. 

Elodie MITRI (MCD & CBI)
Mechanisms of cytokinesis regulation and importance in polyploidy and genetic instability in tumors 

"Cytokinesis is the final step of cell division during which daughter cells physically separate in a process called abscission. It is a complex and finely regulated process whose perturbation can result in the formation of polyploid cells. Polyploidy is a source of aneuploidy and chromosomal and genomic instability, which are well-established causes of cancer development and progression. Despite tremendous progress in our understanding of cytokinesis in recent years, the roles of many actors of this process remain unclear. Citron-Kinase (CitK) is a critical component of the cytokinesis machinery. Although CitK plays an important role in cytokinesis in all cells, it is essential in neural progenitors and CitK mutations in human and mice cause microcephaly due to neuronal death caused by cytokinesis failure. CitK is a large multi-domain protein composed of an N-terminal Ser/Thr kinase domain followed by several protein-protein interaction domains. How CitK is regulated and its exact functions remain largely unknown.

The objective of this project is to investigate the mechanisms that control cytokinesis at the molecular and cellular levels, focusing on the regulation and functions of CitK."

Lisa MARY (CRCT - Melasphinx & IQAC - Rubam)
Multi-OMICS to unveil the role of lipid metabolism in the control of cellular senescence and phenotypic plasticity in melanoma

Confidential abstract !

Sanzhar AITBAY (CRCT)
Bio-mechanical stress & signal integration by PI3Ks for the control of tumour growth

A new approach for treating cancer involves manipulating the mechanical properties of tumors in order to have an impact on their overall characteristics. This strategy focuses on changing the physical properties of tumors to potentially disrupt their growth and progression. Although it is well-established that class I PI3K signaling is elevated in over half of both solid tumors (including pancreatic cancer) and liquid cancer patients, the mechanism by which PI3K integrates mechanical signals is currently unclear. Additionally, it is not known whether modifying the mechanical properties of cancers could affect the effectiveness of PI3K inhibitors in treating cancer. In the project we work on deciphering early molecular events triggered by application of compressive mechanical stress and understanding later adaptation of cells to this mechanical stress in pancreatic cancer settings. Further, in this project we are going to specifically investigate the axis of PI3K signaling in pancreatic cancer cells’ response to mechanical environment by: i) mapping how PI3K activity influences response to mechanical compressive stress and find the mechanosensitive determinants that can prevent cell growth & migration in 2D, ii) establish an in vitro 3D multicellular spheroid/organoid model that will mimic some aspects of mechanical compressive stress found in pancreatic cancer, iii) validate the most efficient approach for preclinical settings in vivo in mouse pancreatic cancer model. 

Elise PONTHIER (CBI & LAAS)
Development of 3D microphysiological systems to study intestinal stem cell fate and model early steps of tumorigenesis

With 1 million deaths pear year, colorectal cancer is the world’s third deadliest cancer. Over-activation of the Wnt signaling pathway (Wnt/Apc/β-Catenin axis) that initiates hyperproliferation of crypts, which contain intestinal stem cells, appears to be the primary driver of this cancer. Conventional therapeutic approaches frequently fail due to tumor heterogeneity and resistance of cancer stem cells to treatments, causing relapse of the disease. Moreover, animal testing is not reliable and over 90% of translation from animal models to clinical trials fail, mainly because of the biological differences between species. Therefore, there is a need for new models that recapitulate human intestinal physiology and complexity more closely. Thus, different in vitro models have been developed, some reproducing mechanical cues such as peristalsis-like movements, others recapitulating the villi and/or crypt topography. However, none of the models have combined both aspects. My project aims at reproducing an intestinal epithelium in microphysiological systems (MPS) by using 3D printing and microfluidic devices. The first part of my project, ongoing, is to develop a photosensitive biomaterial allowing the adhesion and proliferation of primary intestinal cells derived from organoids, that is compatible for 3D printing and deformable to peristaltic movements. The second step will be to integrate these scaffolds into microfluidic devices for the mechanical cues. Thus, the MPS will reproduce different aspects of in vivo organ such as topography, peristaltic movements and microenvironmental parameters. Another goal is to reproduce the early steps of tumorigenic transformation in the MPS using cells carrying inducible mutants in the Wnt/Apc pathways. Ultimately, we aim to show that these devices could provide standardized and reproducible in vitro models for the study of stem cell fate and cancer etiology and to perform preclinical drug testing. 

Esmaa SELLAM (CRCT - IGAALD)
Impact of the germline mutation GATA2 R396Q in leukemic initiation and study of its cooperation with ASXL1 somatic mutations in leukemic transformation

Hematological malignancies are caused by genetic alterations in the hematopoietic stem and progenitors cells (HSPC) of the bone marrow. The majority of these hemopathies are de novo, but many studies have identified germline mutations in transcription factors essential for hematopoiesis such as GATA2. In 2011 we identified the germline mutation GATA2R396Q in a family with a high incidence of AML. We modelled this mutation in mice to explore its impact on hematopoiesis. Gata2R396Q/+ mice show altered distribution of HSPC subpopulations and functional defects. However, they do not develop hematological malignancies at long-term, suggesting as in patients, the need for additional alterations to induce leukemic transformation. Our team sequenced 72 DNA samples from the French-English cohort of leukemic or myelodysplastic patients with germline mutations in GATA2. This study has revealed recurrent somatic mutations affecting the ASXL1 gene.The GATA2R396Q missense mutation is located on the second zinc finger, involved in interactions with DNA. Therefore the mutation could modify the interactions of GATA2 with DNA but also with members of the heptameric complex of which GATA2 is a part. The first aim of my work is to determine the impact of this mutation on DNA binding and complex formation. To this end, we are studying the DNA binding of GATA2R396Q using the Cut&Run technique, and we are setting up the interactome of the wild type and mutated form of GATA2 using immunoprecipitation and Bio-ID.The second axis of my thesis focuses on cooperative events acquired during the evolution of malignant transformation, particularly ASXL1 loss-of-function mutations. To mimic these heterozygous mutations, we decreased the expression of Asxl1 in wild type or Gata2 mutated HSPCs using shRNAs. We then transplanted these cells into recipient mice which will serve as the basis to understand the cooperation between these two mutations, using several complementary in and in vivo approaches.

Léa MAGNE (IRSD - Team 6 Environnement et épithélium intestinal)
Modeling tissue architecture in colorectal physio(patho)logy by image analysis

Understanding the interplay between biology and mechanics involved in tissue architecture is challenging, especially for the 3D organization of tissue. It requires both an appropriate biological model, allowing multi-scale imaging observations, and pertinent approaches allowing accessing the morphological alteration undergone by the tissue concomitantly to the alteration of the 3D supporting matrix. Recently, using human colon epithelium organoids as biological model, and combining vertex and Finite Element Method (FEM), we generated a comprehensive elastic finite element model of the human colon organoid and demonstrated its flexibility. This FEM model provides a basis for relating cell shape, tissue deformation, and strain at the cellular level due to imposed stresses. In order to understand the mechanical interplays between the organoid and its matrix, we now aim to implement this model into its 3D environment. Our hypothesis is that extracting information on local mechanical properties of the matrix during tissue architecture modification helps to implement our numerical model. This could allow a better understanding of the cellular and molecular events involved in the modification of the tissue architecture. We thus worked on 3D human colon epithelium organoids seeded in Matrigel®. In order to have mechanical information about this matrix, we implemented the addition of fluorescent silica beads into the Matrigel. We combined this biological model with a timelapse confocal microscopy approach to follow the evolution of organoids morphology and spatial displacements of the matrix. These displacements are studied by Digital Image Correlation, with a specialized software (VicVolume). The global aim of this work is to create a FEM model of the matrix and its mechanical properties, and to combine the two numerical models (one for the tissue, another one for the matrix) to understand the interplays between epithelium architecture and matrix modification.

Camille GUYON (CRCT - SigDYN - Intégration des signaux cellulaires et PI3K de classe I, II, III)
Vps34 role in pancreatic ductal adenocarcinoma carcinogenesis

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with poor prognosis. Although the importance of the activating mutation of KRAS in initiation is recognized, most of the cellular and molecular mechanisms downstream of KRAS controlling the aggressiveness and heterogeneity of this cancer remain unknown. We are particularly interested in the possible involvement of the class III PI3-Kinase, VPS34. To mimic low VPS34 protein activity, we developed a mouse model combining the Kras G12D mutation with heterozygous inactivation of VPS34 protein in the exocrine pancreas. The pancreatic pathology of KrasG12D mutated and Vps34 inactivated mice (named KCVps34) is compared to that of KrasG12D mutated mice (named KC). Experimentally, the KCVps34 mouse cohort shows a drastic decrease in prognosis compared to the KC cohort; the first clinical signs of metastatic spread (cachexia) are observed in mice as young as 4 months of age. From a histological point of view, in the KCVps34 model, we observe an earlier appearance of high grade lesions compared to KC mice, and this in the majority of this cohort, whereas the pancreas of KC mice presents essentially precancerous lesions. Moreover, these high grade lesions (adenocarcinoma) are more aggressive with the presence of metastasis. Associated with these cancerous lesions, immune infiltrates are observed and the adjacent pancreatic parenchyma shows an accumulation of lipids within the acinar cells. Additional data are needed to understand the signaling pathways involved in VPS34 inactivation that would explain this poor prognosis; in particular, the impact on the metabolic niche of tumor cells in the tumor and remotely will be studied. We want to understand how VPS34 activity is altered by studying a possible correlation between pesticide exposure and modulations of autophagy by VPS34. Understanding the pathology of PDAC and the molecules involved in its poor prognosis is essential to improve the care of this pathology.

Lara FERNANDEZ MARTINEZ (CRCT - SIGNATHER)
Role of transcription in the secondary resistance to EGFR-TKIs in lung cancer

EGFR tyrosine kinase inhibitors (EGFR-TKIs) are effective targeted therapies for lung cancer with EGFR mutations. However, they are limited by the acquisition of secondary resistances. Few tumour cells with no pre-existing resistance mutations can survive the treatment and become non/slow-proliferating drug-tolerant cells (DTCs). Over time, some of them acquire resistance mutations, re-proliferate in the presence of the drug and restore the tumour. To date, the mechanisms by which non-replicating DTCs acquire secondary resistance mutations are unclear. Transcription can be a threat to genome integrity in non-dividing cells, primarily through R-loops, nucleic acid structures consisting of an RNA/DNA hybrid and a displaced single-stranded DNA (ssDNA). Preliminary results show that EGFR-TKI treatment induces R-loop increase in EGFR-mutated lung cancer cell lines. Furthermore, modulation of the R-loop level affects the emergence of resistant cells after prolonged exposure to EGFR-TKIs. Therefore, our preliminary data support a potential role of R-loops as promoters of secondary resistance and our aim is to investigate by which mechanisms. 

Célia RAMADE (CRCT - T2I : Anti-Tumor Immunity And Immunotherapy)
Analysis of T-cell responses using omics approaches to understand mechanisms of resistance to immunotherapy in bronchial and urothelial cancers

Adaptive immunity plays a central role in tumor control, yet the attainment of its full tumor rejection potential is hindered by regulatory mechanisms, including expression of inhibitory immune checkpoints by T cells. Although immunotherapy by immune checkpoint blockade (ICB) has transformed cancer care, not all patients experience clinical benefit. Studies aimed at identifying predictive biomarkers of response to ICB showed that infiltration of tumors by CD8 T cells before treatment favors clinical responses, indicating that ICB could release effector functions of pre-existing tumor-specific T cells. Thus, clinical responses to ICB provide an opportunity to study T-cell responses and the tumor antigens (Ags) for which they are specific, allowing to identify those that have the potential to mediate tumor rejection. In addition, the association of high tumor mutational burden (TMB) with treatment efficacy suggested that unleashing T cells targeting neoepitopes encoded by somatic mutations could underlie ICB-mediated tumor control or regression. Nevertheless, TMB and neoantigenic loads are not consistently related to clinical response to ICB, potentially because most studies have addressed the influence of TMB and neoantigenic load by in silico prediction without systematic analysis of actual neoAg-specific T-cells. Here, using WES and RNA-seq in a cohort of 58 NSCLC and bladder cancer patients receiving anti-PD-1/PD-L1, we assessed the antigenome, defined as the largest set of tumor Ags expressed in a tumor, including potential neoAgs as well as cancer testis Ags (CTAs). In our cohort, high TMB or neoantigenic loads were not predictive of responses to ICB, supporting our hypothesis that actual tumor Ag-specific T-cell responses are responsible for patients response to ICB. Thus, we developed a workflow allowing for the assessment of CD8 and CD4 T-cell responses to CTAs and more than 400 prioritized neoAgs in responder and non-responder patients to ICB.

Alexia RIVERO (CBI-MCD)
SmORF peptides : potential new markers for cancer related disorders

More than half of cancer patients suffer from systemic metabolic dysfunction, known as cancer cachexia, which is manifested by adipose and muscle wasting. Better management of patients requires the discovery of new markers and regulators of oncogenesis and induced cachexia. Due to the high gene conservation between humans and Drosophila, we are using this integrated model to identify the mechanisms underlying tumorigenesis and cachexia. In particular, we are targeting the novel smORF (100AA) family of peptides, translated from small ORFs present in coding and non-coding RNAs. Due to their small size, these peptides have long been ignored, unexploited and could act locally or systemically. Our objectives are 1) to identify by bioinformatics and then biochemically novel smORF peptides deregulated in intestinal cancer and in cachexia target organs, and 2) to test the function of these signature smORF peptides in tumor development and cachexia establishment. The conservation of oncogene se regulatory mechanisms between humans and Drosophila supports that the identified functional smORF peptides could be a source of therapeutic innovation; due to their small size, they can be easily synthesized or targeted.

Gonçalo POMBA DE ALMEIDA (RESTORE - Team 3 Metabolink & CRCT)
Systemic redistribution of metabolic fluxes upon chemotherapy in AML

Acute Myeloid Leukemia (AML) is a hematological malignancy characterized by an abnormal and uncontrolled proliferation of progenitor cells, blocked at an early stage of their differentiation. The overall prognosis of patients with acute myeloid leukemia (AML) remains poor and relapse rate remains high, mainly due to the presence of chemoresistant leukemia clones (RLCs). Results obtained previously by our team show that RLCs have a high oxidative and mitochondrial metabolism. In addition, some tumors have the ability to divert the metabolism of the body to their benefit. This behavior has been demonstrated recently in the case of leukemia. However, the impact of chemotherapy on systemic metabolism is unknown to date. It was recently shown that, besides metabolic interactions with their microenvironment, leukemia cells can also take benefit from the organismal systemic metabolism. This observation is raising a number of questions regarding how host metabolism hijacking can favor tumor growth and chemoresistance. Our project intends to demonstrate the impact of leukemia cells and specific treatment on the systemic metabolism of the immunodeficient mouse NSG type and that of the most common mutations for TP53 using an in vivo metabolic tracing approach with infusion of 13C substrates and analysis of metabolic fluxes by NMR and LC-MS. Mutations in the tumor suppressor gene TP53 encoding the p53 protein appear to have the worse prognosis in AML. These mutations are present in 10% of AML patients and are responsible for limited post-chemotherapy survival. We will therefore as a first step select the most discriminant mutants amongst the most common mutations of TP53 in vitro. The completion of this project will deepen our understanding of the mechanisms underlying AML  chemoresistance, specifically in the case of TP53-mutated AML and the identification and functional validation of novel metabolic targets for AML treatment.

Marlène LAC (I2MC - MetaDiab)
Heart-brown adipose tissue dialogue: Role of natriuretic peptides in the pathogenesis of type 2 diabetes

"Several cohort studies show that high prevalence/activity of brown adipose tissue (BAT) on the one hand, and circulating levels of natriuretic peptides on the other hand, are associated with a significant reduction in the risk of T2DM in humans. The pathophysiological and molecular links between these two observations remain unresolved.

We hypothesized that a defect in ANP signaling in the BAT could contribute to the development of T2DM. We generated a mouse model of conditional inducible deletion of the GCA ANP receptor specifically in the BAT. Wild-type (WT) and knockout (KO) male and female animals were reared on standard or hyperlipidic diets and housed under standard animal house conditions (21°C) or thermoneutrality (30°C).

Basal and acute cold stress regulation of body temperature in WT and KO mice was equivalent. The absence of GCA in the BAT results in glucose intolerance and in response to gluconeogenic substrates such as pyruvate and glycerol in KO mice housed at 21°C. This phenotype is exacerbated in KO mice housed at 30°C with an additional increase in basal blood glucose and a defect in glucose transport in the BAT. Collectively, these preliminary data indicate that a deficiency of the GCA natriuretic peptide receptor in the BAT may contribute to the development of pre-diabetes in a standard nutritional setting, independent of gender and BAT thermogenic activity. Further work is aimed at elucidating the molecular mechanisms involved."

Laura BOTTIN (I2MC - MetaDiab)
Enzymatic and non-enzymatic roles of a key protein in the lipid droplet of adipocytes: the hormone-sensitive lipase 

"Lipid droplets (LDs) of white adipocytes are unique in size and metabolism. The surface of LDs consists of a monolayer of lipids with specific proteins. The interior of LD is mainly composed of triglycerides (TG). Certain proteins associated with adipocyte LD are essential for the formation, maintenance and degradation of LD. One of them, called hormone-sensitive lipase (HSL), is involved in the hydrolysis of TG to fatty acids, a catabolic process called adipose tissue lipolysis. HSL is suspected to have non-enzymatic actions. For example, independently of its catalytic activity, HSL shows a physical interaction and modulates the activity of the glucose-sensitive transcription factor ChREBP. Furthermore, it appears that HSL navigates between 3 intracellular compartments, the cytosol, the LD and the nucleus (unpublished data).

To dissect the enzymatic and non-enzymatic roles of HSL, new human adipocyte models are generated to invalidate or replace the endogenous gene with mutant versions. These models will be used to study protein-protein interactions and adipocyte metabolism. In vivo, transgenic mouse models will be analyzed."

Camille FOURNES-FRARESSO (I2MC)
Control of insulin secretion by adipocyte lipolysis: an inter-organ dialogue ?

"Insulin secretion from the beta-pancreatic islet cells is essential for the maintenance of carbohydrate homeostasis in the body. Its stimulation in response to glucose is well documented and can be modulated by other stimuli such as hormones (GLP1, somatostatin...), amino acids and fatty acids. The latter can indeed potentiate insulin secretion in response to glucose, suggesting a potential dialogue between adipose tissue and islets. However, the mechanisms directly involved in this dialogue are not well known. 

In order to study this inter-organ dialogue, we developed a triple transgenic ATGLfl/fl HSLfl/fl adipoQ-CreERT2+ mouse model invalidated for the two neutral lipases ATGL and HSL in adipocytes: DaKO (for Double adipoKO).

As expected, this model without adipocyte lipolysis shows a decrease in plasma fatty acids. Surprisingly, analysis of insulin secretion in these mice revealed a drastic reduction in plasma insulin levels in response to different stimuli (glucose, arginine, beta-adrenergic). However, the islets of DaKO mice do not show any defect compared to the islets of control mice and are functional ex vivo.  Experiments with conditioned media of control or DaKO adipocytes on isolated islets confirm our in vivo results and will help to determine the molecular mechanisms involved. Lipidomic analyses are in progress to identify the lipids involved. Interestingly, DaKO mice also appear to have a defect in glucagon secretion, suggesting the involvement of a potential paracrine mechanism within the islet.

Our results reveal the importance of the inter-organ dialogue between the adipose tissue and the endocrine pancreas through the control of insulin secretion by adipocyte lipolysis. Further work is needed to determine whether this mechanism is altered during obesity and diabetes."

Vishnu KRISHNAKUMAR (MCD-CBI)
Mechanical regulation of epithelial tissue integrity in the mammalian gut

The gut epithelium is the fastest renewing epithelium in the body, and is unique for withstanding continuous mechanical, chemical and biological insults while maintaining renewal and barrier function throughout adult life. The main goal of my project is to understand the regulatory principles of epithelial tissue renewal in the mammalian gut — how this tissue with its astonishing dynamics is maintained in adult organ homeostasis, using the mouse gut as a model. During epithelial renewal in the gut, the critical processes of cell proliferation, differentiation, migration and extrusion are tightly coordinated to preserve the tissue size, function and integrity. It is unclear how these processes are regulated, especially in the context of mechanical forces that are present in the adult organ physiology.

To understand how mechanical forces are transmitted and sensed throughout the tissue and their potential role in regulation of tissue renewal and cell differentiation, I will employ state-of-the-art imaging, ex vivo tissue culture and organoid models, combined with pharmacological and genetic perturbations. By unravelling the role of force-responsive elements and mechanosensors in the gut epithelium, we will uncover new mechanoregulatory pathways and explain how the gut is performing its physiological barrier function amidst the continuous tissue turnover and mechanical insults.

Honora LABRANA (Metabolic and Cardiovascular Research Institute -  CREATED)
Hyperglycemia worsen smooth muscle foam cells formation through PI3Kγ-dependent defective autophagy

Rational : Diabetic patients are particularly susceptible to complications following atherosclerosis treatment by angioplasty such as restenosis and neo-atherosclerosis. Hyperglycemia may contribute to these complications as elevated level of glycated hemoglobin level has been linked to the development of neo-atherosclerosis. To investigate the underlying mechanism, we investigated in vivo and in vitro the impact of hyperglycemia on smooth muscle cell foam cell formations. 

Methods and results : Using a model of atherosclerosis induced by arterial injury in hyperglycemic mice, we showed that hyperglycemia increased lipid accumulation in smooth muscle cell after arterial injury suggesting that high glucose increased smooth muscle foam cell formation. In vitro, we confirmed that high glucose concentration (HG) increases SMC foam cells formation in presence of oxidized LDL compared to low glucose concentration (LG). Moreover, we found that autophagic processes were highly decreased in HG-cultured SMC compared to LG-cultured SMC and that autophagy reactivation prevents lipid droplet accumulation and induces free cholesterol efflux. Furthermore, blocking PI3kγ/Akt/mTOR pathway, over-induced under HG condition, prevents foam SMC formation by restoring the autophagic flux, suggesting a major role of this pathway in this process. 

Conclusion : Our study revealed that hyperglycemia worsen foam SMC formation through defective autophagy and reveal an important role of PI3Kγ in these processes. This SMC homeostasis disruption could in part explain complications of atherosclerosis treatment in diabetic patients and, suggest novel approach involving PI3Kγ inhibitors for the treatment of neo-atherosclerosis in these high-risk populations. 

Maxime BERGER (IRSD - Team 4 Métabolisme du fer : de la régulation à la thérapie)
Investigate the hepatic role of Matriptase 2 in iron homeostasis

"Matriptase 2 (MT2) is a transmembrane serine protease encoded by TMPRSS6 gene and is predominantly expressed in the liver. In hepatocytes, MT2 inhibits the production of hepcidin, a secreted peptide responsible for the regulation of iron absorption. In humans, TMPRSS6 mutations cause iron-refractory iron deficiency anaemia (IRIDA). 

Matriptase 2 is also expressed in other tissues such as spleen, kidney, lung, brain etc.  However, its role in iron homeostasis from these tissues has still not been elucidated and needs to be clarified. To investigate this, we generated a tissue-specific knockout mouse model in which Tmprss6 is specifically deleted in hepatocytes, by breeding Tmprss6 LoxP/LoxP mice with Alb-Cre expressing mice. 

Our results showed that Tmprss6 Alb/Alb mice display increased hepcidin expression in the liver, associated with microcytic anaemia, demonstrated by reduced red blood cells count, mean cell volume and haemoglobin concentration. Hence, hepatic MT2 suppression recapitulates the phenotype of total knockout mice, confirming the central role of the hepatocytic MT2. Next, we challenged Tmprss6 Alb/Alb mice with erythropoietin  injections in order to induce an erythroid stress. Indeed, we demonstrated before that full deletion of MT2 prevents the inhibition of hepcidin expression in response to erythroid stress. And interestingly, we observed that deletion of matriptase-2 in hepatocytes also prevented EPO-mediated hepcidin suppression. 

Overall, our data suggests that MT2 regulates hepcidin expression through its hepatocytic location only. Further studies are required to understand the role of extra-hepatic Matriptase-2."

Mélanie GADELORGE (RESTORE - Team 2 Flames)
Emergency myelopoiesis in skin wound healing :  Development of in vitro complex human study models

Defects in skin wound healing affect millions of people with various pathologies. These defects are caused in part by the mysregulation of emergency myelopoiesis (EM), involving a mysregulated production by the bone marrow of circulating myeloid cells then infiltrating the skin wound. EM and bone marrow-skin crosstalk are complex processes not completely elucidated yet, due essentially to limits of existing in vitro culture models and imperfect animal models. Here, we developed fully human bone marrow and 3D skin models to aid further investigation of normal and pathological EM after skin injury. At first, we developed a protocol to produce hematopoietic cells in vitro. In a 2-step culture process, we generated a comprehensive medullary stroma from human primary cells from femoral head containing all main cellular non-hematopoietic components of bone marrow microenvironment: osteoblasts, adipocytes and organized endothelial cells. Then, we performed coculture experiments with CD34+ cells sorted from umbilical cord blood. After 14, 21 and 28 days, hematopoietic cells were recovered and analysed by flow cytometry. Results show that stroma was capable to drive hematopoietic differentiation and to produce myeloid cells. In parallel, we developed a 3D human skin model composed of epidermal and dermal parts with a high cellular complexity. Our model is based on the self-organization of primary cells from skin biopsies. The formed spheroids were futher assembled and matured for 3 weeks. The constructs were characterized by histological and immunofluorescence analysis. Results show that in addition to the organization of keratinocytes and cutaneous fibroblasts, main skin cell populations, our model is composed of melanocytes, immune cells and a network of endothelial cells. To conclude, we generated 2 complex human models: bone marrow, myeloid cell producing tissue, and skin, recipient tissue, which could enable the study of EM in a skin wound healing context.

Chloé BRODEAU (RESTORE - Team 3 Metabolink)
Human skin fibroblast features as biomarkers of frailty syndrome

The issue of unhealthy aging has become a matter of public health concern due to the increase of life expectancy. The WHO recommend monitoring 5 essential functions (locomotion, cognition, vitality, sensory and psychosocial). The decline of those functions leads to frailty syndrome which is a clinical  indicator of unhealthy aging. However, there are no predictive biomarkers or biological models to understand frailty syndrome. The clinical state of an individual is the reflect of his tissular and cellular homeostasis. Loss of homeostasis can induce cellular dysfunctions and impairment in organs functions. Fibroblasts play a central role in repair mechanism and restoration of organs functions ability after experiencing stress (inflammatory, metabolic). Recent studies have shown that stress resistance of isolated fibroblasts could be correlated to life expectancy in many species. It also has been shown that human skin fibroblasts could be the mirror of age. In addition, the bioenergetic profile of human skin fibroblasts can be linked to the onset and evolution of functional decline in some diseases. Our hypothesis is that human skin fibroblasts can serve as an indicator of frailty syndrome. To test this hypothesis, we use human skin fibroblasts from 133 donors from the INSPIRE-T cohort from 20 to 100 years, with different frailty statuses. Our first objective is to evaluate fibroblasts stress response using several challenges that mimic life stresses. Then we plan to predict frailty syndrome by developing a classification algorithm using clinical data and our generated experimental data. We will use Shapley Additive exPlanations method to explain this model to determine the importance of each features on the prediction and potential biomarkers. This project will allow stratification of individuals to develop personalized medical care. These biomarkers may lead to new physiopathological hypothesis and models to achieve a better understanding in frailty mechanism.

Pinar ALTINER PINAR (IPBS - Proteomics and Mass Spectrometry of Biomolecules)
Development of New Signal Processing Algorithms of Ion Mobility MS Data to Improve the Quantification of Phospho-proteomics Samples

In mass spectrometry based-phospho-proteomic studies, determining the precise localization of its phosphorylation sites might be ambiguous. Various localization algorithms exist to define the specific sites where phosphorylation occurs in a protein. These algorithms assign a significance score to these sites by considering each spectrum at a time. To improve the accuracy of phospho-localization, we developed peptide-centric approach, which gathers site-determining ions information from all MS/MS spectra related to a given phospho-peptide. This algorithm summarizes fragment ion intensities for alternative phosphate positions, and analyzes them to cluster phospho-sites having similar fragment ion signatures. Using a public synthetic dataset, we generated heatmaps of the obtained signatures for a given peptide ion, enabling the quick visualization of phosphorylation patterns. In parallel, we also started a benchmarking study to compare the accuracy and precision of our method to existing algorithms. We analyzed 180 synthetic phospho-peptides in pure sample preparations and also at different concentrations in presence of an E. coli background. We also purified phospho-peptides from mouse T-cells and spiked them into a similar E. coli background, to generate a more complex phospho-dataset. More than 450 sample injections were performed using different mass spectrometers and tested with different acquisition methods, both with and without the ion mobility dimension. According to initial results, determining the correct phospho-site by clustering fragment ion signatures of several MS/MS spectra seems possible, but requires more development for a direct comparison with existing algorithms. To achieve this, a scoring function and a target-decoy method will be implemented, enabling automatic determination of phospho-sites position for a given FDR. 

Marie SORBARA (CRCT - ImPact-Therapeutic innovation in pancreatic cancer)
Targeting cytidine deaminase with intracellular antibodies to defeat resistance to treatment in pancreatic cancer

Cytidine deaminase, or CDA, is an enzyme that deaminates cytidine into uridine to maintain the pyrimidine pool of cells. In several tumors including pancreatic cancer (PDAC), CDA participates to therapeutic resistance by deaminating cytidine analogues, such as gemcitabine. Catalytic inhibitors exist but they present off target effects and were inefficient in clinical trial so far. Therefore, developing alternative strategies to inhibit CDA and defeat resistance to treatment in PDAC is necessary. We developed intracellular antibodies able to degrade CDA through the proteasome (i.e. antibody-based degraders). We identified CDA degraders via a cell-based screening, characterized them and showed their potency to deplete endogenous CDA protein in several PDAC cell lines. CDA degraders decrease gemcitabine IC50 in several PDAC cell lines and inhibit cell proliferation via an increased induction of apoptosis. Finally, in vivo data of cell derived xenograft show that the combined treatment CDA degrader and gemcitabine strongly impedes tumour growth. We will next address the delivery of these CDA degraders in experimental PDAC models, both in vitro and in vivo.

Jerko LJUBETIC (IPBS & CRCT)
High Endothelial Venules (HEVs) and T cell trafficking in agressive B cell lymphomas

"High endothelial venules (HEVs) are specialized post-capilary venules found in lymph nodes (LNs) and most other secondary lymphoid organs which mediate lymphocytes extravasation from the blood. By being key players in lymphocyte homing , HEVs play an essential role in lymphocyte recirculation and immune surveillance. To fufill this role, HEV cells (HECs) are equipped with peripheral node adressin (PNAd) which bind to lymphocytes expressing L-selectin. PNAd is specifically recognized by the HEV-specific antibody MECA-79 an important tool for immunofluorescence or flowcytometry studies. Thus, immunofluorescence revealed that HECs have a plump morphology, very different from other endothelial cells (Blanchard and Girard).

In the past few years HEV-like blood vessels have been found in solid tumors and appears to be a very good prognosis factor for survival and response to immunotherapy (Asrir et al.).

Despite an increased density of blood vessels in LNs during B Cell Lymphoma (BCL), HEVs seem to be found in much lower proportions, with a flat morphology, offering a perfect niche for lymphoma to develop.

With the arrival of immunotherapies such as αPD-1 or chimeric antigen engineered T (CAR-T) cells dependent on T cell trafficking, it appears relevant to interrogate the mechanisms leading to HEVs loss of function and to determine wether HEVs can be modulated or reinduced to fight lymphoma development.

The first line treatment for agressive B cell lymphoma is R-CHOP, a combination of a depleting antibody targeting B cells, with chemotherapy. In order to mimic the effects of this treatment we use a murine model that develop B cell lymphoma to study how R-CHOP affects T cell trafficking and HEVs, and whether those changes could be exploit to improve the treatment of patients."

Léa RIMAILHO (CRCT - Team 9 - NoLymIT)
Identification of CD39 and CD73 as new therapeutic targets in non-Hodgkin's lymphomas

Follicular lymphoma (FL) and diffuse large cell B-cell lymphoma (DLBCL) are the two most common subtypes of non-Hodgkin's lymphomas (NHL) whose treatment has benefited from the introduction of the anti-CD20 monoclonal antibody Rituximab (RTX) used in combination with chemotherapy. However, a significant fraction of patients does not respond and all patients relapse. The identification of mechanisms of resistance to therapies represents a major challenge. In this context, my team characterized a specific immuno-escape signature (IE) of NHL involving the expression of 54 genes among them, CD39 and CD73, two ectonucleotidases playing a key role in the adenosine pathway. In the literature, it has been shown that CD39 and CD73 are highly expressed in FL and DLBCL biopsies and that the level of tumor infiltrating lymphocytes (TILs) expressing CD73 correlates with a worse prognosis in DLBCL. Moreover, CD39+ expressing TILs in FL appear more abundant compared to reactive lymph nodes and reflect their hyporesponsiveness. Finally, it has been shown that TCR stimulation induced CD39 expression in normal gdT lymphocytes, and that tumors expressing CD39 are more resistant in vivo to RTX. Thus, we hypothesize that TIL expressing CD39/CD73 in FL and DLBCL could correspond to gdT cells and that their targeting may provide therapeutical benefit by improving the efficacy of anti-lymphomatous treatment. We are currently exploring the role of CD39 based on in vitro (NHL cell lines co-cultured withT cells) and ex vivo (patient-derived lymphoma spheroids) experiments and evaluate if CD39 targeting by a pharmacological inhibitor improves antibody-dependent cell cytotoxicity in presence of RTX. Moreover, by using public transcriptomics analysis we are currently exploring the influence of ENTPD1 (CD39 encoding gene) on NHL survival. Altogether, we expect to identify CD39 and/or CD73 as potential new therapeutical target in FL/DLBCL, diseases that are still considered as incurable. 

Tom MAILLET (CRCT - RADOPT (Radiotherapy Optimization))
Interrelationships between tumor metabolic heterogeneity and metabolic response of Glioblastoma stem cells to irradiation : impact on radioresistance

Glioblastoma (GB) is the most frequent and aggressive primary brain tumor. Standard therapeutic management consists in a surgical resection combined with radio/chemotherapy. Despite this treatment, median survival doesn’t exceed 15 months, due to the highly invasive nature of these tumors, linked with chemo/radioresistance capacities, leading to systematic relapse. This recurrence process is partly explained by the presence, within the tumor, of a cellular subpopulation with stem-like cells features (Glioblastoma Stem Cell, GSC). Multipotent, tumorigenic and highly invasive, GSC would facilitate relapse by resistance to conventional treatments. The use of Magnetic Resonance Spectroscopic Imaging (MRSI) in the clinical field allows to highlight the metabolic heterogeneity in GB notably through the Choline/N-AcetylAspartate (NAA) Index (CNI) measurement. An increased production of Choline is associated with a high cellular proliferation, but a decreased production of NAA is linked to a loss of neuronal function. MRSI analysis identifies two different subtypes of tumor metabolic areas: CNI+ (CNI>2, metabolically active) and CNI- (CNI<2, metabolically inactive). Linked to preoperative MRSI analysis in GB, our previous results demonstrated that CNI+ areas are predictive of post-radiation relapse sites. Moreover, our previous work showed in GB patients a significant GSC enrichment in CNI+ areas compared to CNI-. Given the major involvement of GSC in resistance and relapse, and the metabolic adaptation processes considered as a hallmark of cancer, my thesis subject focuses on the metabolic characterization of five pairs of GSC from CNI+ and CNI- areas, cultured from patients biopsies. This project will highlight differences in the GSC energetic metabolism from CNI+/- areas, as well as ionizing radiations (IR) impact on the GSC metabolism and resistance capacities, in order to better understand and target the recurrence mechanisms in these metabolically active tumor areas.

Léo ROLLAND (CBI)
Role of p27 in governing autophagy, metabolism and growth, and potential for targeting cancer cells

"p27Kip1 is a cell cycle inhibitor via its ability to bind to and inhibit cyclin-CDK activity. Due to this function, nuclear p27 acts as a tumor suppressor. However, p27 also has cyclin-CDK independent functions and is involved in regulating other cellular processes, like autophagy. Autophagy is a catabolic self-digestion process allowing cells to adapt their metabolism to environmental conditions. Recent studies showed that metabolic stress affects p27 activity. Under glucose starvation, the nutrient sensing kinases LKB1 and AMPK adapt p27 activity to promote autophagy via the regulation of autophagosomes positioning and fusion with lysosomes, thereby promoting cell survival. Under amino acid restriction, p27 promotes autophagy by binding to Ragulator on the lysosomal surface and inhibiting MTORC1 signaling, the master regulator of cellular growth. MTORC1 inhibition allows the nuclear translocation of transcription factor EB, triggering pro-autophagic genes expression.

Thus, p27 status affects cell metabolism and survival by distinct mechanisms in function of environmental conditions.

Here, I propose to: 

(1) Investigate the mechanisms by which p27 controls autophagy in response to glucose or amino acid starvation, especially how p27 is targeted to lysosomes and how p27 participates in MTORC1 inhibition on the lysosomal surface.                                                                                                                              

(2) Determine how p27 may contribute to metabolic reprogramming and controlling cell growth via its action on MTORC1 signaling.

(3) Determine if p27 status could be used to promote apoptosis in cancer cells when targeting specific metabolic pathways.

This project will give novel information about the role of p27 in autophagy and metabolic control, and if p27 status can be exploited by targeting specific metabolic or signaling pathways to induce cancer cell apoptosis and prevent treatment resistance."

Marine HERNANDEZ (IPBS - Microenvironnement, cancer et adipocytes)
A metabolic crosstalk occurs between human bone marrow adipocytes and prostate cancer cells

Prostate cancer (PCa) -related deaths are mainly caused by bone metastasis. Bone marrow adipocytes (BMAds) are the major component of bone microenvironment. My team has shown that periprostatic adipocytes increase tumor progression in localized PCa by fueling cancer cells with fatty acid (FA) released after lipolysis activation, a key adipocytes function (PMID: 30606769). However, there is no evidence of this crosstalk at metastatic site where PCa cells are in contact with BMAds. To investigate this, we establish a collaboration with orthopedic surgeons to obtain human bone marrow adipose tissue (BMAT) during hip replacement surgery. Two types of BMAds exist: red BMAds (rBMAds) in rBMAT and yellow BMAds (yBMAds) in yBMAT. Since PCa metastatic sites are frequently found near rBMAds, we set up a 3D culture system of these cells in fibrin matrix to preserve their viability until 5 days. Coculture of PCa cells with rBMAds resulted in an increase in neutral lipid content stored in lipid droplets, mainly composed of triglycerides (TG) as shown by lipidomic approaches. Using rBMAds loaded with fluorescent FA, we directly demonstrated that FA released by rBMAds are taken up by cancer cells and re-esterified into TG. The mechanism contributing to FA release is under investigation since these rBMAds are devoid of lipolysis as we previously demonstrated in yBMAds (PMID: 31995765). Coculture of different models of PCa cells increase migratory activity of tumor cells, a key process for cancer cells to propagate to multiple bone metastatic sites. The metabolic and transcriptional remodeling induced by rBMAd in PCa cells are being studied.  Overall, we showed for the first time that a metabolic crosstalk occurs between human mature rBMAds and PCa cells affecting cancer aggressiveness whose mechanistic nature remains to be determined. Deciphering this crosstalk could lead to pharmacological targets for bone metastases treatment for which therapeutic options remain very limited.

Claire LAC (CRCT - Team 6 MICROPANC & LAAS)
Do mechanical forces induce a protumoral dialogue  between the tumor and the adjacent healthy tissue ?

"Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant stroma composed predominantly of activated fibroblasts (CAFs) and dense extracellular matrix (ECM). Tumor cell growth and ECM modifications generate forces named “solid stress” documented to promote tumor progression. While it is recognized that these forces also result in compression of surrounding healthy tissue, their impact on the biology of adjacent normal cells, especially normal fibroblasts (Pancreatic Stellate Cells, PSCs) remain poorly described. 

In order to mimic the mechanical force transmitted by the tumor to the surrounding healthy tissue, we added an agarose cushion on a monolayer of PSCs. We show that a low pressure (85 Pa) is sufficient to induce the durable expression of 3 CAF activation markers associated with drastic changes in cell morphology, mitochondrial network rearrangement, and the activation of the mecanosensor FAK. The consequence of this mechano-induced fibroblast activation is an important increase of their ECM production and secretion; ECM that will in turn enhance tumor cell migration.

We are currently developing a 3D device to analyse this phenomenon in a model more relevant of the pathology. Our device will allow the application of a quantifiable and homogeneous pressure on cells cultured in 3D in a hydrogel and is compatible with a wide range of techniques. 

In conclusion, we obtained in 2D the proof of concept that solid stress activates PSCs in a durable manner. These mechanically activated fibroblasts acquire protumoral properties such as matrix secretion leading, in fine, to enhanced tumor cell migration."

Mehdi LIAUZUN (CRCT - Team 6 MICROPANC)
Tumor/stroma symbiosis in a new subtype of pancreatic ductal adenocarcinoma auxotrophic for serine

Pancreatic ductal adenocarcinoma (PDAC), the 4th leading cause of death in Europe, presents various alterations in mRNA translation in cancer cells and their microenvironment, supporting tumor growth. Current classification of PDAC patients, based on transcriptomic analyses, correlates with patient survival but is unsuitable for personalized treatment. Thus, we aimed to characterize PDAC tumor cell translated mRNA profile (translatome) using a collection of twenty-seven pancreatic Patient-Derived Xenografts (PDX), a model where tumor and stromal reads can be separated bioinformatically due to the replacement of the human stroma by murine cells. Through the application of blind source separation of the tumor translation efficacy, we identified a new tumor subtype, named “ISR-activated”. These tumors are characterized by a global downregulation of protein synthesis associated with an enhanced translation of mRNAs implicated in the integrated stress response (ISR). Using a collection of tumor cell lines and cancer cells directly derived from the PDX collection, we characterized this "ISR-activated" subtype and demonstrated an increased resistance to chemotherapies and apoptosis with metabolic dependence on exogenous serine. The absence of phosphoglycerate dehydrogenase (PHGDH), in the serine synthesis pathway and cystathionine B synthase (CBS) were found as key markers of deficiencies in serine metabolism of "ISR-activated" cells. Here, we aim to determine the tumor microenvironment that sustains the progression of these tumors. Immunohistochemistry on “ISR-activated” xenografts and co-culture assays drew a communication network with cytokines and metabolites exchanges between tumor cells, the immune system and specific cancer-associated fibroblasts (CAFs) that can provide serine. Based on these results, we are developing an orthotopic grafting approach in an immunocompetent model to better understand the exchanges between the immune system, CAFs and tumor cells.

Koffi Bertin VIANOU (PHARMADEV - CHIPS & IRCB-Bénin)
Elevated plasma interleukin-8 as a risk factor for mortality in children presenting with cerebral malaria

Malaria remains a major public health problem despite major scientific advances. WHO reports over 400,000 deaths due to malaria each year, mainly among children in Sub-Saharan Africa. Cerebral malaria (CM) is one of the most severe forms of malaria and remains fatal in 15 to 40% of cases. Despite administration of elective anti-malarial drugs, it unfortunately leads to very high mortality rates. Although the late management of these cases contributes to the disease severity, it seems that the disruption of the cellular immune response also contributes to the fatal outcome due to blood-brain barrier damage. Cytokines, chemokines and lipid mediators are key molecules in the intercellular dialogue. Therefore, our study aimed to study such plasma and urine markers in CM patients and identify markers of death. The study was conducted in Beninese children presenting with CM. Plasma and urine samples were collected at inclusion and following clinical management at Day 3 and Day 30. Using Luminex technology, levels of 17 markers of pro-and anti-inflammatory responses were simultaneously measured in plasma. For urine markers, levels were measured using commercial enzyme immunoassays kits. Socio-demographic, clinical and biological data were analyzed by univariate analysis and logistic regression. A total 50 children who survived and 20 who died were included. Multivariate linear regression analysis revealed a strong correlation between increased plasma IL-8 and death. Plasma IL-8 concentration  57.5 pg/ml increased dramatically the risk of mortality during CM [aOR = 15.3, 95% CI 2.75-85.4, P = 0.002]. By contrast, the other plasma and urine markers measured were not associated to death outcome. Interestingly, plasma TNF-α, IL-6, IL-10, CXCL10 and ICAM-1 decreased gradually from Day 0 to Day 30, whereas CXCL5, CCL17 and CCL22, suggesting deleterious and protective roles, respectively. This study highlighted plasma IL-8 as a marker of death in childhood cerebral malaria. 

Sonia BELKAI (IPBS - Immunomodulation By Mycobacterial Lipids and Glycoconjugates)
Identifying actors involved in Mycobacterium tuberculosis lipid antigen presentation by CD1b 

"Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, remains a major health problem. As the BCG vaccine shows flawed efficacy and antibiotic-resistant strains are emerging, it is necessary to developp new vaccine strategies. 

One of these strategies relies on lipid antigen recognition by the immune system. 

A subset of T cells are able to recognize antigenic lipids, when they are presented by the Human Cluster of Differenciation 1 (CD1) proteins. Five CD1 protein isoforms exist in human : CD1a, b, c, d and CD1e. These proteins present amphipathic lipids at the surface of the expressing cells. Although CD1a, c and d are also able to present Mtb glycolipids to specific T cells, CD1b presents the most. CD1b possesses a large hydrophobic groove that can contain an acyl chain up to 90 carbon long, with the example of the Mycolic Acid (MA). 

The loading of these Mtb lipids on CD1b takes place in the late endosomes and lysosomes, where the low pH enables a defolding of the protein as well as because many luminal hydrolases are present. Indeed, some lipids that are known to be presented by CD1b appear to have a wide structure and are most certainly processed by lysosomal enzymes in order to fit in the T cell receptor (TCR) that binds the hydrophilic part of the glycolipids. Some factors involved in the processing of Mtb lipids have already been discovered. For example, the hexamannosylated phosphatidyl-myo-inositol (PIM6), has to undergo a mannosidase processing that requires CD1e. It also undergoes a lipase processing in order to go from tetra to di-acylated form. Some other lysosomal proteins known as Lipid Transfer Proteins (LTPs) appear to be important to lead the membrane-embedded lipid to the CD1 groove. Namely, Saposins (in particular SAP-B and SAP-C) are a family of LTPs involved in the loading of CD1 proteins. 

Our work aims at identifying key actors of lipid antigen processing and loading, focusing on antigens presented by CD1b isoform."

Jeanne MANARANCHE (LCC - Team V Nouvelles molécules antipaludiques et approches pharmacologiques & PHARMADEV)
Study of the mechanisms of regulation of the redox system, involved in the resistance of Plasmodium falciparum to artemisinins

Current treatments for malaria are based on the use of artemisinin derivatives. Unfortunately, the parasite Plasmodium falciparum has developed a form of resistance to these molecules, based on its ability to enter a quiescent state, stopping its multiplication. The parasite is thus insensitive to treatments during this dormant phase. At the end of the treatment, the parasite can resume its proliferation, leading to a therapeutic failure. The triggering of the quiescence process and the survival of the resistant parasites cannot be dissociated from the mode of action of artemisinins, in particular the oxidative damages linked to the production of reactive oxygen species (ROS). The capacity of Plasmodium falciparum to manage oxidative stress appears to be a key element of the parasite resistance to artemisinins. The objective of this project is to understand the involvement of reactive oxygen species in the entry, maintenance and exit of quiescence. This is achieved by sorting quiescent parasites, and comparing the capacity of entry and exit of quiescence of different strains that are either sensitive or resistant to artemisinins. The effect of artemisinins on redox levels in sensitive and resistant parasites will also be studied. 

Marion LAURENT (LCC - Nouvelles molécules antipaludiques et approches pharmacologiques)
Prediction of the acquisition of artemisinin resistance by malaria parasites

Since 2008, the fight against malaria in Asia has been confronted with the resistance of the Plasmodium falciparum parasite to artemisinins. Artemisinins are the core component of artemisinin-based combination therapies (ACTs), which combine an artemisinin derivative with another antimalarial molecule and are the first-line treatment against malaria. In 2020, artemisinin-resistant parasites were reported in South America and Africa, where, by 2040, artemisinin resistance will lead to an increase in incidence of several million cases. To date, this resistance can only be monitored in the field once it has emerged. The objective of this thesis project is to explore the biology of the Plasmodium falciparum parasite (in particular African field isolates), subjected to artemisinin pressure, in order to identify molecular or biochemical markers predictive of the acquisition of artemisinin resistance in this parasite. The challenge of this project is therefore to identify tools that could allow a better understanding of the mechanisms of this resistance and to detect its acquisition at an early stage. This should allow to improve epidemiological monitoring and to adapt therapeutic policies in the field as quickly as possible, by anticipating the risk of emergence of resistant parasites in endemic areas, especially in Africa.

Romane DUSFOUR-CASTAN (LMGM)
Horizontal gene transfer between bacteria in a model microbiota

"Conjugative plasmids are mobile genetic elements (MGEs) that are major contributors to the propagation of antibiotic resistance genes especially since they play a central role in the horizontal transfer of other MGEs (such as transposable elements).

While the molecular bases of the mechanisms of MGEs propagation are widely described, there is still a significant lack of knowledge about these processes and the factors that influence them in situ, within natural complex bacterial communities such as microbiota. The only available information essentially comes from numerous metagenomic analyses, which do not provide the spatio-temporal dynamics of these processes.

Thus, this project aims to study the spatio-temporal dynamics of MGEs exchanges in the model intestinal microbiota of the nematode Caenorhabditis elegans. By combining fluorescent microscopy, bio-informatics and genetic techniques, its aim is to analyze and compare the fate of conjugative plasmids and transposons inside this model microbiota. 

The data obtained could provide a better understanding of how MGEs propagate in complex ecosystems such as microbiota and thus contribute to the search for ways to limit the emergence of multi-resistant bacteria, a serious threat currently weighing on global health.

Prior this work, the first step of this project was to focus on the establishment of the microbiota of C.elegans in its intestine (where in the intestine the different species of the microbiota are located, their proportion, the stability of the microbiota along time,…); as few data are available on this subject and would be needed for the work on the dynamic of MGEs exchanges. 

Thus, this presentation will mainly focus on the data obtained about the establishment of the microbiota of C. elegans and the beginning of the data obtained about MGEs exchanges."

Yassin ABUTA'A (LMGM - Regulation and transport of proteins across cell membranes)
Maintenance of the Enterobacterial Envelope Permeability Barrier to Antibiotics

Gram negative bacteria such as Enterobacteria are particularly able to resist antibiotics and deleterious conditions. This protection relies on their peculiar multilayered envelope composed of an inner membrane (IM) and an outer membrane (OM), separated by an aqueous space called the periplasm. The OM represents the first line of interaction with the extracellular environment and forms a semipermeable barrier as it ensures import of many nutrients while effectively protecting the cell from deleterious environmental conditions. Such function requires different OM proteins, such as integral OM proteins (OMPs) and OM-lipoproteins which must be efficiently transported and assembled by dedicated machineries: the BAM complex and the LOL pathway. The proper functioning of these assembly machines is essential to preserve the homeostasis of the OM and thus, their activities are carefully monitored by envelope stress response systems that help the cell to cope with possible dysfunctions. For instance, defects in OMPs assembly by the BAM complex cause the accumulation of unfolded OMPs in the periplasm, which triggers the σE response. On the other hand, the Cpx two components system (TCS) can be activated by different signals, some of which are sensed by NlpE, an OM-lipoprotein. The global coordination of these processes to maintain the envelope homeostasis remains today only marginally understood. My work identifies NlpE as an interactor of the BAM complex, highlighting a point of convergence between the Cpx-TCS and OMP assembly by BAM. My current and future work aims at addressing the mechanism by which NlpE interacts with the BAM complex and the physiological conditions that are monitored by this interaction in the enterobacterial model organisms E.coli. More globally, this project will help to decipher how the interplay between the BAM complex and a stress-surveillance system contributes to preserve OM integrity and promote bacteria resistance to antibiotics.

Sarah MAUREL (IRSD - Team 3)
Rôle des lipopeptides bactériens dans la douleur viscérale associée au syndrome de l’intestin irritable

"The first 1000 days of life, from in utero to two years of age, is a critical developmental period. Stress during pregnancy is one of the disturbances that can impact this window and lead to the development of pathologies such as irritable bowel syndrome later in life. The lack of knowledge on the mechanisms at the origin of the pathology and of effective treatments makes it a major public health issue. 

Stress during pregnancy was modeled in a mouse model and its impact evaluated in male and female offspring. Visceral hypersensitivity, reflecting the visceral pain experienced by IBS patients, correlated with a decrease in a strain of Lactobacillus, as well as a decrease in a lipopeptide it produces, were demonstrated. Treatment of these mice with the decreased lipopeptide restored visceral normo-sensitivity. The same type of compound was found to be decreased in the stool of IBS patients, correlated with clinical pain scores. 

The identification of bacterial lipid metabolites with therapeutic potential is a major advance in the treatment of IBS pain and the understanding of the effects of the microbiota on the physiology of its host."

Renzo GUTIERREZ LOLI (EPIIC)
How does Toxoplasma gondii modulate neuronal responses and antigen presentation to evade immune recognition during latent infection ? 

"IFN-γ-induced mechanisms are instrumental for parasite control and host protection during acute T. gondii (Tg) infection. Upon brain invasion by Tg, neurons upregulate surface MHC class I molecules, allowing them to present tachyzoite antigens to CD8+ T cells, which is key to avoid excessive parasite burden. Neurons also restrict parasite growth in response to IFN-γ, although this ability seems less efficient than other cells. Tg eventually persists as bradyzoites within intra-neuronal cysts. The mechanisms by which Tg bradyzoites evade immune control, in particular CD8+ T cell recognition, remain unclear. 

Here we address how Tg effectors that hijack IFN-γ responses in various cell types, modulate the responses of infected neurons. Using primary cultures of hippocampal neurons stimulated or not with IFN-γ, we assessed the nuclear translocation of IRF1 transcription factor and MHC I surface expression in neurons containing mostly tachyzoites (24 hpi) or bradyzoites (72 hpi). As expected, IRF1 was translocated in the host cell nucleus and surface expression of MHC I was upregulated after IFN-γ stimulation. Neurons infected by WT tachyzoites and bradyzoites failed to accumulate IRF1 in their nuclei. Conversely, neurons infected with TgIST-deficient parasites showed higher nuclear IRF1 intensity. Strikingly, neurons infected by tachyzoites had similar levels of MHC I as uninfected ones, but neurons infected by bradyzoites showed a clear TgIST-dependent decrease of MHC I.

These results suggest that the TgIST-mediated subversion of neuronal MHC I is delayed compared to the subversion of IRF1, therefore occurring after or upon bradyzoite differentiation.  Experiments are underway to explore the impact of TgIST and other effectors on the transcriptional responses of neurons to IFN-γ, and on the ability of CD8+ T cells to control the parasite in vivo."

Louise BATTUT (IRSD - Team 3 POGBA)
Effects of repeated interleukin-33 stimulations on mast cells response

"Active players in the innate immune system, mast cells (MC) are long-lived cells that monitor tissues integrity. Following their activation by various stimuli from their environment (microbes, danger signals), MC release mediators (cytokines, chemokines, histamine, proteases...) ensuring the recruitment and regulation of other immune cells. 

Inflammatory bowel diseases or IBD (Crohn's disease, ulcerative colitis) are chronic disabling and incurable pathologies characterized by intestinal lesions associated with inflammatory outbreaks. The damaged epithelial cells repeatedly release danger signals such as interleukin-33 (IL-33), which activates MC in particular. IL-33 promotes the production of inflammatory mediators by MC. My previous work shows that IL-33 promotes the ability of MCs to activate other immune cells (helper T cells and eosinophils) (L. Battut*, E. Leveque* et al., in preparation). All these results were obtained after a single stimulation of IL-33. However, during IBD, the immune cells are repeatedly solicited so MC could ‘memorize’ previous stimulations and adapt their functions. To date, no study has analyzed the effects of repeated IL-33 stimulations on MC response. My thesis work aims (1) to study these effects in vitro and to identify the associated molecular mechanisms and (2) to know their consequences in vivo during colon inflammation (colitis). By combining multidisciplinary approaches, and through the development of different techniques, my first results indicate that repeated stimulations of IL-33 reprograms MC in vitro and that the IL-33 / MC axis appears to be deleterious during colitis."

Maryam SALIM (IPBS - New antimalarial molecules pharmacological Approaches & LCC)
Innovative Plasmodium G-quadruplexes ligands as new potential antimalarial drugs  

"Plasmodium parasites are the causative agents of human malaria, a disease responsible for widespread morbidity and about more than million deaths each year. Most malaria deaths are caused by the species Plasmodium falciparum. Unfortunately, Plasmodium resistance to current antimalarial drugs is widespread in malaria endemic areas. Guanin-rich DNA and RNA sequences can fold into four–stranded non-canonical secondary structures called G-quadruplexes(G4). The presence of G4 structures in Plasmodium has been confirmed by the use of a specific G4-detecting antibody."

Cédric GARCIA (I2MC - Team 7 & Hematologie CHU Toulouse)
Ultrastructural Changes in Platelets and Viral Xenophagy Pathway in Severely Affected COVID-19 Patients with Evolving SARS-CoV-2 Variants

Thirty-seven severe patients with COVID-19 were recruited in this study after their hospitalization in the intensive care unit at Toulouse university Hospital. Twenty-seven patients were infected with the Delta variant, and ten with the Omicron variant. Using transmission electron microscopy (TEM), we observed the presence of viral-like particles in 25% of the platelet population regardless of the variant involved. SARS-CoV-2 is known to manipulate the autophagy system in infected cells. We observed several active autophagic structures in approximately 20% of platelets for the Delta variant, as well as significant colocalization of the spike protein with LC3B and partial colocalization with RAB7, suggesting the presence of SARS-CoV-2 material in autophagosomes and late endosomes. However, by observing these types of structures in patients infected with the Omicron variant, we observed a different ultrastructure and altered localization of the spike protein in their platelets. Overall, our results showed the presence of the virus in platelets associated with activities of SARS-CoV-2 xenophagy regardless of the variant involved. However, However, mutations acquired in the Omicron variant appear to impair the entry and degradation pathways in platelets that are involved in the viral clearance mechanism.

Tamara MIčKOVá (IPBS - NIGOU)
Structural and functional characterisation of new immunomodulators of Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, has evolved a plethora of different strategies to evade and undermine host immunity, with lipids being one of the mayor players in modulation of immune response. However, several evidences support that the current knowledge of Mtb lipidome’s composition and immunomodulatory properties are incomplete. Based on previous studies together with our preliminary data, we believe, there is number of yet uncharacterized mycobacterial lipid species that are produced in regular conditions as well as in a response to stress. Therefore, we are analysing mycobacterial lipidomes in different study models including in vitro broth cultures of bacteria, mycobacteria-infected macrophages and in vitro broth cultures of bacteria under stress conditions that are encountered during infection. To highlight new lipids in these different models, we rely on a lipidomic approach (set up at the Toulouse MetaToul platform) that has been developed for the global and unbiased analysis of complex lipid extracts such as Mtb lipidome. The method consists of a Supercritical Fluid Chromatography-coupled to a (Electrospray Ionisation)-Quadrupole-Time-Of-Flight-Mass Spectrometer (SFC-(ESI)-QTOF-MS). The diol chromatography (direct phase) allows prior separation of the lipid families based on their polar heads before their detection by the mass spectrometer. Subsequently, the generated datasets are processed and analysed thanks to bioinformatic tools.

Martin CAMPOY (IPBS - "Structural Biophysics" and "Mycobacterial Envelopes and Therapeutic Targets")
Structural and biochemical characterization of prodrugs-activating enzymes from Mycobacterium tuberculosis

Tuberculosis (TB), caused by the infectious agent Mycobacterium tuberculosis (Mtb), is a major global health threat with 1.5 million deaths and half a million people infected with drug-resistant Mtb strains in 2020 (WHO). Treatment of drug-resistant strains requires the use of second-lines drugs that are much more toxic and expensive than first-lines drugs, which highlights the urgent need to fill up the anti-TB drug pipeline. As many antibiotics against Mtb are prodrugs that need to be converted to active compounds by specific mycobacterial enzymes, an innovative approach consists of solving prodrug-activating enzymes structure for rational drug design. Here, we investigate structural and enzymatic properties of 2 key Baeyer-Viliger MonoOxygenases (BVMO) from Mtb, EthA and MymA, responsible for the activation of second-line drugs such as the commonly used ethionamide. 

Chloé RIVIERE (IPBS - Immunomodulation par les lipides et glycoconjugués mycobactériens)
Structural and functional characterization of the Protein O-MannosylTransferase of M. tuberculosis (MtPMT) : a potential target of innovative therapeutic inhibitors 

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis, which remains one of the most deadly infectious diseases in the world, with approximately 1.8 million victims per year (OMS). A resurgence of infections has been observed over the last twenty years, due in particular to the emergence of multi or totally antibiotic-resistant mycobacterial strains. The identification of new molecular targets likely to limit the emergence of resistance is issential for the development of new therapeutic strategies to fight tuberculosis. In this respect, a promising new concept of targeting so-called « non-essential » bacterial virulence factors is of attracting growing interest. In this context, Mtb Protein O-MannosylTransferase (MtPMT), a member of the C-type glycosyltransferase family, has been identified as a virulence factor. Inhibition of MtPMT therefore appears to be an original potential therapeutic approach targeting a non-essential bacterial virulence factor. To search for selective inhibitors of MtPMT, we have chosen to develop new semi-rational screening approaches based on (1) the characterizarion by point mutation of specific potentially druggable domains of MtPMT; (2) on the search for targetable interactions of MtPMT with functional partner proteins. Results: (1) By functional mutational screening of the elementary GT-C scaffold of MtPMT, we characterize a new essential specific domain probably involved in the recognition of substrate proteins; (2) By interactomic analysis based on an original IP-MS (Immunoprecipitation-Mass spectrometry) approach, we identified several functional partners of MtPMT involved in the essential biosynthetic pathway of bacterial mannoproteins and post-modification processes. These results open the door to the search for selective pharmacological inhibitors of bacterial PMT of great potential therapeutic value.

Olivia PARONETTO (INFINITy - Infections virales)
Study of the mechanisms enhancing the replicative capacity of virus host recombinant variants of hepatitis E virus 

Hepatitis E virus (HEV) is the leading cause of viral hepatitis in the world. HEV can lead to chronic infection in immunocompromised patients, in whom recombinant viruses that have integrated human gene fragments into the proline-rich region (PPR) of their genome were identified. These human fragments increase HEV replication in vitro but the mechanisms involved are unknown. Recently, 7  human gene fragments were identified by our group but their impact has not been studied in vitro. All the recombination events occurred in the region encoding PPR. The PPR protein could be an intrinsically disordered region, making possible interactions with many cellular partners. We hypothesize that the human fragments into the PPR could inhibit factors limiting HEV replication. The impact of the 7 human fragments on the replicative capacity of HEV was studied by inserting them into the plasmid encoding the Kernow virus. The strain without insertion was used as a control. After infection of the HepG2/C3A cell line, the viral RNA was quantified by RT-qPCR in the culture supernatant and cell lysate. The infectious foci were counted by immunofluorescence. To study the interactome, the region encoding PPR protein with and without insertion were cloned into an expression vector with an HA Tag. These plasmids were individually transfected into the HepG2C3A. The PPR protein was co-immunoprecipitated and interacting partners were identified by mass spectrometry. We found higher RNA concentrations in the culture supernatant and in the cell lysate, and more foci for 5 recombinant viruses compared to the strain without insertion. We identified 44 interacting proteins for the recombinant PPR compared to the wild type PPR. The partners will be confirmed by repeating experiments. Our work showed that insertions do not always improve the replicative capacity. Mechanisms conferring such replicative advantage are still under investigations.

Rébecca BARASCUD (INFINITy - Team 4)
Impact of maternal prenatal stress on offspring's susceptibility to CNS inflammation

The prenatal period is a delicate time during which intrauterine exposures to environmental factors may modulate the course of development of both immune system and central nervous system (CNS) and confer a lasting effect on offspring that later in life impact their susceptibility to brain associated diseases. There is compelling evidence that maternal prenatal stress (PS) is linked to cognitive, behavioral, physical, and emotional problems such as autism and attention-deficit hyperactivity disorder in children, as well as depression and schizophrenia in adulthood (Graignic-Phillippe et al,. 2014). Of note, prenatal maternal stress in utero impact also the child’s adaptive immune system by exaggerating the type 2 response skewing and reducing type 1 cytokine response to specific antigens (Entringer et al,. 2008). Although these studies established the relationship between PS and immune response of the offspring, the impact of PS on susceptibility to inflammatory diseases of the CNS such as MS remains unknow.

Lucie FABRIZI (CRCT - Team 5)
New function(s) of CHK1 kinase in the autophagy process

"DNA damage response (DDR) is a complex signalling network activated upon different kinds of genotoxic events. Activation of this pathway, which implicates the phosphorylation and activation of the CHK1 kinase by the ATR kinase, leads to cell cycle arrest and DNA repair to preserve genomic integrity. Autophagy is a biological process activated in response to different types of metabolic stress, including DNA damage. It is a highly conserved self-eating process during which cells digest some of their structural components to maintain cell homeostasis and metabolic equilibrium.

Both autophagy and DDR are therefore essential for survival and maintenance of cellular homeostasis in response to different categories of stress. Several studies report that autophagy can be activated by different types of DNA damage, and that it is involved in cellular responses such as DNA repair, senescence or cell death. Although functional links exist between DDR and autophagy, few studies have reported the implication of CHK1 in this cross-talk.

The hypothesis of this project is that CHK1 is by itself an actor of autophagy. The aim of my project is to demonstrate the existence of this new CHK1 function, and to decipher the molecular elements of this pathway. Our first results suggest that CHK1 is involved into autophagy induction in response to different types of metabolic stress independently of DNA damage. Also, we observe a modification of the CHK1 phosphorylation status (Serine 280) in response to different types of stress. In addition, our preliminary data suggest that the role of CHK1 in the survival of leukemic cells during metabolic stress is dependent on an “autophagic” function of the kinase.

Altogether, we propose a new function of the CHK1 kinase as an autophagy actor. In the future, CHK1 inhibition in combination with autophagy inhibition, or autophagy inhibition as an alternative to CHK1 inhibition, could be proposed as new therapeutic strategies."

Hussein HAMZE (MCD - Eukaryotic Ribosome Biogenesis team (Eukaribio))
Early 60S ribosomal subunit maturation steps require direct interactions between the Npa1 complex and the box C/D snoRNP chaperone snR190

The early steps of large ribosomal subunit assembly feature among the least understood steps of ribosome synthesis in eukaryotes. We and others previously showed that the Npa1 complex, formed by association between the α-solenoid scaffold proteins Npa1 and Npa2, the Dbp6 DEAD box helicase, the Nop8 RNA binding protein and Rsa3, is required for the production or stability of the first independent precursor particle to the large ribosomal subunit in S. cerevisiae. Our UV cross-linking (CRAC) analyses demonstrate that Npa1 complex members interact with the 5’ domain I and 3’ domains V and VI of 25S rRNA, suggesting that the Npa1 complex is involved in the folding and/or clustering of these domains, a crucial early event during 60S ribosomal subunit production. In addition, our CRAC analyses show that Npa1 complex members interact predominantly with the box C/D snoRNA snR190. This snoRNA does not guide 2’O-methylation of rRNAs. Instead, by establishing base-pairing interactions with 5’ and 3’ regions of the 25S rRNA, it is likely involved in early 25S rRNA folding events. We show that the Npa1 complex and snR190 interact outside pre-ribosomal particles and are likely incorporated in these particles as a preformed module. Moreover, we provide evidence that the Npa1 complex and snR190 mutually stabilize their integration within pre-ribosomal particles. snR190 features a central stem-loop structure not found in canonical box C/D snoRNAs. This extension is required for the interaction between snR190 and the Npa1 complex. Moreover, deleting this extension impairs snR190 activity in early pre-rRNA processing events. We conclude that a direct interaction between snR190 internal stem loop and the Npa1 complex is important for snR190 chaperone activity during early 25S rRNA folding events. 

Nesrine HIFDI (I2MC)
Regulation of lysosome homeostasis by phosphoinositides and role of ubiquitin ligase FBXW7 in myogenesis: molecular mechanisms and implications in centronuclear myopathy

"Homeostasis of long living cells such as neurones and muscles depend on a tight control of protein synthesis, folding and degradation, a process known as proteostasis. The evolutionary conserved nutrient sensing signalling pathway activated by Ser/Thr protein kinase mammalian target of rapamycin complex 1 (mTORC1) plays a central role in maintaining proteostasis. Nutrients promote mTORC1 translocation and activity at late endosome/lysosome (LE/Lys) surface (localization of active mTORC1) from where it triggers multiple pro-anabolic processes including protein synthesis. On a contrary, lack of nutrients inhibits mTORC1 signalling and enhances proteolysis. Yet, the picture is far from being complete. Surprisingly, we have recently discovered that unexpected signals from proteostasis pathways control mTORC1. Indeed, the Ubiquitin-Proteosome System (UPS)-mediated degradation of mTOR protein emerged as an important terminator of mTORC1 signalling. Mechanistically, we have demonstrated that mTOR is a substrate of a specific E3 ubiquitine ligase complex (Fbw7 E3 ligase in Skp1-Cullin 1-F-box protein/SCF complex).

Notably, Fbw7 protein has a sensing role in this E3-ligase complex as it facilitates recruitment of ubiquitination substrates. It does so through WD40-repeat domains that also bind the lipid second messenger phosphoinositides (PIs), particularly PI3P and PI(3,5)P2. Altogether, these recent observations prompted us to ask a fundamental question: how phosphoinositide signalling and Fbw7-mediated proteosomal degradation of mTORC1 sensor are integrated in nutrient sensing ?"

Inès SELMI (MCD)
Regulation of wing size by the histone demethylase dLsd1

"Organismal development and cell differentiation are critically dependent on chromatin state transitions. Chromatin refers to a mixture of DNA and histone proteins, its architecture islargely controlled by histone proteins. Histones are subject to posttranslational modifications which impact their interactions with DNA. One of these modifications is methylation, a reversible modification that is regulated by two different enzymes: the Histone methyl transferase and the Histone demethylase. The first histone demethylase to be discovered was the lysine specific demethylase 1 (LSD1).

Multiple studies have implicated LSD1 in the control of cell proliferation, cell differentiation and cell death. However, the underlying mechanisms by which LSD1 controls  tissue homeostasis in vivo remain to be fully elucidated. In this project, we are interested in studying the role of LSD1 in regulating wing size in drosophila. 

Here, we show that loss of function mutation of the histone demethylase dLsd1 results in a significant reduction of Drosophila wing size due to a decreased cell number. Our work shows that dLsd1 plays an important role in organ size control and that its depletion causes cell cycle arrest and increased DNA damage. These phenotypic effects correlate with misregulation of the expression of specific gene networks and with de-repression of transposons. Finally, our results suggest that increased TE mobility due to dLsd1 loss affects organ size. 

These results raise a series of new questions and open a new avenue of research devoted to better understand LSD1 role in the silencing of TEs.  As future goals of this project, we will take advantage of the possibility to spatiotemporally control dLsd1 activity in vivo through the use of an auxin-inducible degron (AID) system to address the following question : What are the primary mechanisms by which dLsd1 controls TEs silencing?"

Tomas CAETANO (LMGM - Genomics of integrated systems)
RNA sequencing, how quantifying the in vivo 5’-ends helps understanding RNA metabolism

"RNA-seq has revolutionized the study of gene expression, providing major insights into the transcriptome.

One aspect of RNA-Seq is the identification of transcription start sites (TSS) and their corresponding 5’-end sequences obtained via RNA-Seq protocols called 5’-end RNA-Seq (CAGE, TSS-EMOTE, PARE-Seq, etc.).

Over the last few years, some methods have been developed to analyze such data but they are either tailored to eukaryotes (CAGEfightR) or they provide only a specific type of analysis (identification of TSS, differential expression, detection of alternative promoters, etc.).

Here, we present a novel computational pipeline that provides a comprehensive and general analysis of bacterial 5’-end RNA-Seq data. Our methods enable multiple analysis options including identification of TSS, study of ribosomes and exonucleases dynamics, differential analysis. Additionally, our pipeline includes quality control and normalization steps that ensure reliable and reproducible results across multiple samples and experimental conditions. 

To demonstrate the utility of our pipeline, we applied it to two major biological applications: 

(1) Quantification of 5’-end phosphorylation state (proportion of 5’PPP vs. 5’P) in order to identify ribopyrophosphohydrolases and their RNA targets in Staphylococcus aureus.

(2) Study of ribosome and RNase J dynamics based upon the observed co translational mRNA degradation.

Overall, our methods are integrated in an R library for the analysis of 5’-end RNA-Seq data, with various potential applications."

Haibin MU (CBI)
Investigating the Role of dLSD1/CoREST in the Maintenance of Genome Integrity through Transposon Silencing during Drosophila Oogenesis

"Transposable elements, also known as transposons, are DNA sequences capable of moving within and between genomes, potentially causing gene mutations and disruptions in gene function. In Drosophila oogenesis, transposon silencing mechanisms are crucial for maintaining genome integrity and proper development. Previous research from our team suggests that dLSD1 and its cofactor play a significant role in regulating transposon silencing in Drosophila. However, the specific mechanisms and impacts of this complex on chromatin remodeling and transposon silencing are not fully understood.

To address this question, we investigated the role of CoREST130, a critical dLSD1 cofactor for transposon silencing in Drosophila oogenesis. We used knockdown and overexpression experiments to analyze the impact of CoREST130 on progeny production, transposon expression, histone modifications, and chromatin accessibility. Our findings suggest that the dLSD1/CoREST complex is a critical epigenetic regulator of transposon silencing in Drosophila oogenesis, as knockdown of CoREST130 resulted in increased transposon expression and altered chromatin modifications. We also found that overexpressing CoREST130 fully restored fertility and repressed active transposons in the fly line with depleted CoREST130. Additionally, our study showed that inhibiting retrotransposon mobilization partially restored ovary defects caused by CoREST130 loss. We performed ChIP-qPCR to investigate whether the loss of CoREST130 would reduce demethylase accessibility on target transposon genome loci.

Overall, our study provides important insights into the mechanisms underlying transposon silencing in Drosophila oogenesis and highlights the critical role of epigenetic regulators in maintaining genome integrity during development."

Léa FERRAYE (INFINITy)
Causal link between bi-allelic expression of TLR7 and the development of pathogenic autoantibodies in systemic lupus

The incidence of autoimmune diseases, like Systemic Lupus Erythematosus (SLE) is markedly increased in women and in 47 XXY Klinefelter syndrome men, as compared to 46 XY men, possibly in relationship with differences in X chromosome dosage. While in women, one of the two X chromosomes is inactivated (XCI), some genes can escape XCI, leading to increased expression of some X-linked genes. Tlr7 gene localized on the X chromosome encodes to an endosomal receptor and recognizes ssRNA and can also be engaged by endogenous ligands, potentially leading to inflammation or autoimmunity. Indeed, expressed at double dose, Tlr7 is sufficient to trigger autoimmunity in mice. We have recently demonstrated that TLR7 evades X silencing in an important proportion of immune cells from healthy women, resulting in enhanced immune responses. This observation supports the notion that TLR7 overexpression through biallelism is a candidate contributor to the higher risk of SLE, in women and KS men. Our working hypothesis is that the X-linked genetic risk to autoimmunity is an evolutionary consequence of positive selection for favorable immune response to infection. My thesis objective is to address the causal relationship between Tlr7 expression from the inactive X chromosome and the development of dysregulated immune responses involved in disease pathogenesis in SLE. We generated an original dual reporter/deleter mouse model in order to track and eliminate Tlr7 bi allelic cells to force monoallelic expression of Tlr7. This model will be use to evaluate the susceptibility to developing the disease in spontaneous SLE in the congenic B6.Sle123 and in a model of pristane-induced lupus. I will have a particular focus on the newly identified “age(autoimmunity)-associated B cell” (ABC) subset, emerging as key cellular source of autoantibodies in SLE. The results will provide knowledge on the nature of the mechanism underlying the enhanced female susceptibility to autoimmune diseases.

Arnaud DEBERNARDI (MCD)
Investigating LSM-8-/- mutant phenotypes with the Auxin Inducible Degradation (AID) systems

In eukaryotic genomes, heterochromatin correlates with silencing of genes. Regulation of gene expression and more particularly of gene silencing through heterochromatin is critical for the maintenance of developmental pathways and for ensuring appropriate responses to environmental stimuli. For years, heterochromatin was considered as an inert and transcriptionally inactive structure. Recently however, our lab identified the LSM2-8 complex as a key player in a pathway of post-transcriptional silencing of heterochromatin. We have found that the LSM2-8 complex promotes the selective degradation of transcripts arising from heterochromatic loci bearing H3K27me3, through the exoribonuclease XRN-2. This LSM2-8-mediated silencing of H3K27me3-bound regions defines a new mechanism for selective RNA degradation, and a previously unknown level of regulation for facultative heterochromatin in animals. Here we show that this post-transcriptional silencing pathway is essential for proper development and mutant worms lacking a functional LSM2-8 complex display several phenotypes such as sterility, developmental delay, and a protruding vulva among other phenotypes. To better characterize these phenotypes, we took advantage of the Auxin Inducible Degradation (AID) system to conditionally deplete the LSM-8 protein in a temporal and cell-type specific manner. To overcome some limitations inherent to the AID system, we also investigated these phenotypes with the improved AID (AID2) system. Overall, we were able to recapitulate phenotypes seen in LSM-8 knock out mutants by conditionally depleting the LSM-8 protein with the AID and AID2 systems and get previously inaccessible temporal & tissue specific information, such as the requirement of LSM-8 in somatic but not in germline cells for fertility. 

Haomiao WANG (RESTORE - Team 4 GOT-IT)
Explanations as a new metric for feature selection: a systematic approach

With the extensive use of Machine Learning (ML) in the biomedical field, there was an increasing need for Explainable Artificial Intelligence (XAI) to improve transparency and user trust, while meeting regulatory requirements. Feature Selection (FS) is widely used as a part of a ML pipeline to address the curse of dimensionality while preserving as much information as possible. However, the choice of FS methods affects the entire pipeline including the final prediction explanations, whereas very few works investigate on the relationships between FS and model explanations. In this work, a systematic workflow including the calculation of multiple metrics on 145 datasets was proposed to investigate the impact of the combination of several FS/ML models on the final explanations obtained. Since explanations contain both the influence of features and their importance, the present work demonstrated the complementarity of two metrics based on ranking and influence changes, to provide a comprehensive view of explanation differences. While retention rate and accuracy are fairly well-established metrics for FS method evaluation, new metrics based on explanations, and in particular measuring how much explanations differ with/without FS are particularly promising for FS methods recommendation. Since no single FS method can satisfy all needs, positioning FS methods in a tridimensional space, integrating explanations-based metrics, accuracy and retention rate, would allow the user to choose the priorities to be given on each of the dimensions.

Divyendu Kishore VALAPPIL (RESTORE - Team 4 GOT-IT & ONERA)
Multiscale methods for imaging and optical diagnostics of complex media using light scattering: from aerosols to biological tissues

"3D optical imaging and study of mechanical properties of living organisms is of considerable interest. Brillouin spectroscopy (BS) is an emerging tool for biology that allows a non-invasive assessment of viscoelastic properties of materials using the concept of Brillouin light scattering (BLS). BLS detects a narrow range of frequencies (0.1 – 20 GHz) with picometric shifts in the frequency. To resolve the extremely low signal to noise ratio (SNR), we use a VIPA (virtually imaged phase array), a high resolution spectrometer that increases the detection efficiency of BS. In this project, we combine the concept of both a digital laser scanned scattering microscope (DLSSM) and a BM. This system with digital line-scanning

decreases the acquisition time drastically allowing 3D optical imaging of living organisms as well as other optically dense media in a non-destructive manner. Overall, this poster provides a comprehensive overview of the state-of-the-art in multiscale methods for imaging and optical diagnostics of complex media using light scattering, with a particular emphasis on Brillouin microscopy."

Sébastien AUBER (MCD - Team Legube)
DSB SNPs

"Single Nucleotid Polymorphism are short mutations of one base. When a SNP occurs in a gene or in a regulatory region of a gene of a protein involved in Double Strand Breaks (DSB) repair it can lead to hereditary diseases (eg: a SNP in ATM  can result in ataxia telengecstasia). However, GWAS studies show SNPs that are not involved in DNA Damaged Repair (DDR) genes. To explain those we propose here the concept of DSB SNPs, which are SNPs that can occur in regulatory region of genes,  altering DNA repair protein binding. We uncovered

novel non-coding SNPs that could contribute to genome instability, and therefore, act as genetic drivers

of hereditary diseases. Our first results suggests that those DSB SNP could be associated with diverse diseases such as cancers, Central Nervous system diseases, and autoimmune diseases."

Kilian MAIRE (INFINITy)
Confidential title

Confidential abstract !