Laboratoire Biosciences et bioingénierie pour la Santé

Multiorgan-on-a-chip (multi-OoC) platforms have great potential to redefine the way in which human health research is conducted. After briefly reviewing the need for comprehensive multiorgan models with a systemic dimension, we highlight scenarios in which multiorgan models are advantageous. We next overview existing multi-OoC platforms, including integrated body-on-a-chip devices and modular approaches involving interconnected organ-specific modules. We highlight how multi-OoC models can provide unique information that is not accessible using single-OoC models. Finally, we discuss remaining challenges for the realization of multi-OoC platforms and their worldwide adoption. We anticipate that multi-OoC technology will metamorphose research in biology and medicine by providing holistic and personalized models for understanding and treating multisystem diseases.

Onset of myelination in Schwann cells is governed by several transcription factors, including Krox20/Egr2, and mutations affecting Krox20 result in various human hereditary peripheral neuropathies, including congenital hypomyelinating neuropathy (CHN) and Charcot-Marie-Tooth disease (CMT). Similar molecular information is not available on the process of myelin maintenance. We have generated conditional Krox20 mutations in the mouse that allowed us to develop models for CHN and CMT. In the latter case, specific inactivation of Krox20 in adult Schwann cells results in severe demyelination, involving rapid Schwann cell dedifferentiation and increased proliferation, followed by an attempt to remyelinate and a block at the promyelinating stage. These data establish that Krox20 is not only required for the onset of myelination but that it is also crucial for the maintenance of the myelinating state. Furthermore, myelin maintenance appears as a very dynamic process in which Krox20 may constitute a molecular switch between Schwann cell myelination and demyelination programs.

Abstract Sexual reproduction of Toxoplasma gondii , confined to the felid gut, remains largely uncharted owing to ethical concerns regarding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described 1,2 . Here we found that the transcription factors AP2XII-1 and AP2XI-2 operate during the tachyzoite stage, a hallmark of acute toxoplasmosis, to silence genes necessary for merozoites, a developmental stage critical for subsequent sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a marked change in the transcriptional program, promoting a full transition from tachyzoites to merozoites. These in vitro-cultured pre-gametes have unique protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit MORC and HDAC3 (ref. 1 ), thereby limiting chromatin accessibility and transcription. Consequently, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. Successful production of merozoites in vitro paves the way for future studies on Toxoplasma sexual development without the need for cat infections and holds promise for the development of therapies to prevent parasite transmission.

In the injured adult central nervous system (CNS), activation of pro-growth molecular pathways in neurons leads to long-distance regeneration. However, most regenerative fibers display guidance defects, which prevent reinnervation and functional recovery. Therefore, the molecular characterization of the proper target regions of regenerative axons is essential to uncover the modalities of adult reinnervation. In this study, we use mass spectrometry (MS)-based quantitative proteomics to address the proteomes of major nuclei of the adult visual system. These analyses reveal that guidance-associated molecules are expressed in adult visual targets. Moreover, we show that bilateral optic nerve injury modulates the expression of specific proteins. In contrast, the expression of guidance molecules remains steady. Finally, we show that regenerative axons are able to respond to guidance cues ex vivo, suggesting that these molecules possibly interfere with brain target reinnervation in adult. Using a long-distance regeneration model, we further demonstrate that the silencing of specific guidance signaling leads to rerouting of regenerative axons in vivo. Altogether, our results suggest ways to modulate axon guidance of regenerative neurons to achieve circuit repair in adult.

Biofilms represent a major concern in the food industry and healthcare. The use of probiotic bacteria and their derivatives as an alternative to conventional treatments to fight biofilm development is a promising option that has provided convincing results in the last decades. Recently, membrane vesicles (MVs) produced by probiotics have generated considerable interest due to the diversity of roles they have been associated with. However, the antimicrobial activity of probiotic MVs remains to be studied. In this work, we showed that membrane vesicles produced by Lacticaseibacillus casei BL23 (LC-MVs) exhibited strong antibiofilm activity against Salmonella enterica serovar Enteritidis (S. Enteritidis) without affecting bacterial growth. Furthermore, we found that LC-MVs affected the early stages of S. Enteritidis biofilm development and prevented attachment of bacteria to polystyrene surfaces. Importantly, LC-MVs did not impact the biomass of already established biofilms. We also demonstrated that the antibiofilm activity depended on the proteins associated with the LC-MV fraction. Finally, two peptidoglycan hydrolases (PGHs) were found to be associated with the antibiofilm activity of LC-MVs. Overall, this work allowed to identify the antibiofilm properties of LC-MVs and paved the way for the use of probiotic MVs against the development of negative biofilms.

Successful subversion of translation initiation factors eIF4E determines the infection success of potyviruses, the largest group of viruses affecting plants. In the natural variability of many plant species, resistance to potyvirus infection is provided by polymorphisms at eIF4E that renders them inadequate for virus hijacking but still functional in translation initiation. In crops where such natural resistance alleles are limited, the genetic inactivation of eIF4E has been proposed for the engineering of potyvirus resistance. However, recent findings indicate that knockout eIF4E alleles may be deleterious for plant health and could jeopardize resistance efficiency in comparison to functional resistance proteins. Here, we explored the cause of these adverse effects by studying the role of the Arabidopsis eIF4E1, whose inactivation was previously reported as conferring resistance to the potyvirus clover yellow vein virus (ClYVV) while also promoting susceptibility to another potyvirus turnip mosaic virus (TuMV). We report that eIF4E1 is required to maintain global plant translation and to restrict TuMV accumulation during infection, and its absence is associated with a favoured virus multiplication over host translation. Furthermore, our findings show that, in the absence of eIF4E1, infection with TuMV results in the production of a truncated eIFiso4G1 protein. Finally, we demonstrate a role for eIFiso4G1 in TuMV accumulation and in supporting plant fitness during infection. These findings suggest that eIF4E1 counteracts the hijacking of the plant translational apparatus during TuMV infection and underscore the importance of preserving the functionality of translation initiation factors eIF4E when implementing potyvirus resistance strategies.

Although the involvement of protein kinase CK2 in cancer is well-documented, there is a need for selective CK2 inhibitors suitable for investigating CK2 specific roles in cancer-related biological pathways and further exploring its therapeutic potential. Here, we report the discovery of AB668, an outstanding selective inhibitor that binds CK2 through a bivalent mode, interacting both at the ATP site and an allosteric αD pocket unique to CK2. Using caspase activation assay, live-cell imaging, and transcriptomic analysis, we have compared the effects of this bivalent inhibitor to representative ATP-competitive inhibitors, CX-4945, and SGC-CK2-1. Our results show that in contrast to CX-4945 or SGC-CK2-1, AB668, by targeting the CK2 αD pocket, has a distinct mechanism of action regarding its anti-cancer activity, inducing apoptotic cell death in several cancer cell lines and stimulating distinct biological pathways in renal cell carcinoma.

In a scenario where the discovery of new molecules to fight antibiotic resistance is a public health concern, ribosomally synthesized and post-translationally modified peptides constitute a promising alternative. In this context, the Gram-positive human gut symbiont Ruminococcus gnavus E1 produces five sactipeptides, Ruminococcins C1 to C5 (RumC1–C5), co-expressed with two radical SAM maturases. RumC1 has been shown to be effective against various multidrug resistant Gram-positives clinical isolates. Here, after adapting the biosynthesis protocol to obtain the four mature RumC2-5 we then evaluate their antibacterial activities. Establishing first that both maturases exhibit substrate tolerance, we then observed a variation in the antibacterial efficacy between the five isoforms. We established that all RumCs are safe for humans with interesting multifunctionalities. While no synergies where observed for the five RumCs, we found a synergistic action with conventional antibiotics targeting the cell wall. Finally, we identified crucial residues for antibacterial activity of RumC isoforms.

Microtubules are dynamic polymers that interconvert between phases of growth and shrinkage, yet they provide structural stability to cells. Growth involves hydrolysis of GTP-tubulin to GDP-tubulin, which releases energy that is stored within the microtubule lattice and destabilizes it; a GTP cap at microtubule ends is thought to prevent GDP subunits from rapidly dissociating and causing catastrophe. Here, using in vitro reconstitution assays, we show that GDP-tubulin, usually considered inactive, can itself assemble into microtubules, preferentially at the minus end, and promote persistent growth. GDP-tubulin-assembled microtubules are highly stable, displaying no detectable spontaneous shrinkage. Strikingly, islands of GDP-tubulin within dynamic microtubules stop shrinkage events and promote rescues. Microtubules thus possess an intrinsic capacity for stability, independent of accessory proteins. This finding provides novel mechanisms to explain microtubule dynamics.

Mass measurements in the mega-to giga-Dalton range are essential for the characterization of natural and synthetic nanoparticles, but very challenging to perform using conventional mass spectrometers. Nano-electro-mechanical system (NEMS) based MS has demonstrated unique capabilities for the analysis of ultra-high mass analytes. Yet, system designs to date included constraints transferred from conventional MS instruments, such as ion guides and high vacuum requirements. Encouraged by other reports, we investigated the influence of pressure on the performances of the NEMS sensor and the aerodynamic focusing lens that equipped our first-generation instrument. We thus realized that the NEMS spectrometer could operate at significantly higher pressures than anticipated without compromising particle focusing nor mass measurement quality. Based on these observations, we designed and constructed a new NEMS-MS prototype considerably more compact than our original system, and which features an improved aerodynamic lens alignment concept, yielding superior particle focusing. We evaluated this new prototype by performing nanoparticle deposition to characterize aerodynamic focusing, and mass measurements of calibrated gold nanoparticles samples. The particle capture efficiency showed nearly two orders of magnitude improvement compared to our previous prototype, while operating at two orders of magnitude greater pressure, and without compromising mass resolution.

Abstract Slow acquisition time and instrument stability are the two major limitations for the application of confocal Brillouin microscopy to biological materials. Although overlooked, coupling the microscope to the spectrometer with a multimode fiber (MMF) is a simple yet viable solution to increase both the detection efficiency and the stability of the classical single-mode fiber-coupled virtually imaged phase array (VIPA) instruments. Here we implement the first successful MMF-coupled VIPA spectrometer for confocal Brillouin applications and present a dimensioning strategy to optimize its collected power. The use of an MMF brings a tremendous improvement on the stability of the spectrometer that allows performing experiments over several weeks without realignment of the device. For instance, we map the Brillouin shift and linewidth in growing ductal and acinar organoids with a spatial resolution of <?CDATA $1 \times 1 \times 6\,\mu\textrm{m}^3$?> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" overflow="scroll"> <mml:mrow> <mml:mn>1</mml:mn> <mml:mo>×</mml:mo> <mml:mn>1</mml:mn> <mml:mo>×</mml:mo> <mml:mn>6</mml:mn> <mml:mstyle scriptlevel="0" /> <mml:mi>μ</mml:mi> <mml:msup> <mml:mtext>m</mml:mtext> <mml:mn>3</mml:mn> </mml:msup> </mml:mrow> </mml:math> and <?CDATA $50\,\textrm{ms}$?> <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" overflow="scroll"> <mml:mrow> <mml:mn>50</mml:mn> <mml:mstyle scriptlevel="0" /> <mml:mtext>ms</mml:mtext> </mml:mrow> </mml:math> dwell time. Our results clearly reveal the formation of a lumen in these organoids. Careful examination of the data also suggests an increase in the viscosity of the cells of the assembly.

Immunotherapies have recently emerged as a standard of care for advanced cancers, offering remarkable improvements in patient prognosis. However, only a small subset of patients benefit, and robust molecular predictors remain elusive. We present a computational framework leveraging sample-specific gene co-expression networks to identify features predictive of immunotherapy response in kidney cancer. Our results reveal that patients with similar clinical outcomes exhibit comparable gene co-expression patterns. Notably, increased gene connectivity and stronger negative gene-gene associations are hallmarks of poor responders. We further developed sample-specific pathway-level network scores to detect dysregulated biological pathways linked to treatment outcomes. Finally, incorporating these sample-level network features improves the predictive performance of gene expression-based machine learning models. This work highlights the value of personalized gene network features for stratifying patients with cancer and optimizing immunotherapy strategies.

Xeroderma Pigmentosum C is a dermal hereditary disease caused by a mutation in the DNA damage recognition protein XPC that belongs to the Nucleotide excision repair pathway. XPC patients display heightened sensitivity to light and an inability to mend DNA damage caused by UV radiation, resulting in the accumulation of lesions that can transform into mutations and eventually lead to cancer. To address this issue, we conducted a screening of siRNAs targeting human kinases, given their involvement in various DNA repair pathways, aiming to restore normal cellular behavior. We introduced this siRNA library into both normal and XPC patient-derived fibroblasts, followed by UVB exposure to induce DNA damage. We assessed the reversal of the XPC phenotype by measuring reduced photosensitivity and enhanced DNA repair. Among the 1292 kinase-targeting siRNAs screened, twenty-eight showed significant improvement in cellular survival compared to cells transfected with non-targeting siRNA after UV exposure in XPC cells. From these candidates, PIK3C3 and LATS1 were identified as particularly effective, promoting over 20% repair of 6-4 photoproduct (6-4PP) DNA lesions. Specifically targeting the autophagy-related protein PIK3C3 alone demonstrated remarkable photoprotective effects in XPC-affected cells, which were validated in primary XPC patient fibroblasts and CRISPR-Cas9 engineered XPC knockout keratinocytes. PIK3C3 knock down in XP-C cells ameliorated in UVB dose response analysis, decreased apoptosis with no effect on proliferation. More importantly, PIK3C3 knock down was found to induce an increase in UVRAG expression, a previously reported cDNA conveying lower photosensitivity in XP-C cells. Thus, attempts to improve the XPC photosensitive and deficient repair phenotype using PIK3C3 inhibitors could pave a way for new therapeutic approaches delaying or preventing tumor initiation.

T cells have the potential to maintain immunological memory and self-tolerance by recognizing antigens from pathogens or tumors. In pathological situations, failure to generate de novo T cells causes immunodeficiency resulting in acute infections and complications. Hematopoietic stem cells (HSC) transplantation constitutes a valuable option to restore proper immune function. However, delayed T cell reconstitution is observed compared to other lineages. To overcome this difficulty, we developed a new approach to identify populations with efficient lymphoid reconstitution properties. To this end, we use a DNA barcoding strategy based on the insertion into a cell chromosome of a lentivirus (LV) carrying a non-coding DNA fragment named barcode (BC). These will segregate through cell divisions and be present in cells' progeny. The remarkable characteristic of the method is that different cell types can be tracked simultaneously in the same mouse. Thus, we in vivo barcoded LMPP and CLP progenitors to test their ability to reconstitute the lymphoid lineage. Barcoded progenitors were co-grafted in immuno-compromised mice and their fate analyzed by evaluating the BC composition in transplanted mice. The results highlight the predominant role of LMPP progenitors for lymphoid generation and reveal valuable novel insights to be reconsidered in clinical transplantation assays.

The prediction of pathological changes on single cell behaviour is a challenging task for deep learning models. Indeed, in self-supervised learning methods, no prior labels are used for the training and all of the information for event predictions are extracted from the data themselves. We present here a novel self-supervised learning model for the detection of anomalies in a given cell population, StArDusTS. Cells are monitored over time, and analysed to extract time-series of dry mass values. We assessed its performances on different cell lines, showing a precision of 96% in the automatic detection of anomalies. Additionally, anomaly detection was also associated with cell measurement errors inherent to the acquisition or analysis pipelines, leading to an improvement of the upstream methods for feature extraction. Our results pave the way to novel architectures for the continuous monitoring of cell cultures in applied research or bioproduction applications, and for the prediction of pathological cellular changes.

Abstract Immune checkpoint blockade (ICB) therapies have improved the overall survival (OS) of many patients with advanced cancers. However, the response rate to ICB varies widely among patients, exposing non-responders to potentially severe immune-related adverse events. The discovery of new biomarkers to identify patients responding to ICB is now a critical need in the clinic. We therefore investigated the tumor microenvironment (TME) of advanced clear cell renal cell carcinoma (ccRCC) samples from primary and metastatic sites to identify molecular and cellular markers of response to ICB. We revealed a significant discrepancy in treatment response between subgroups based on cell fractions inferred from metastatic sites. One of the subgroups was enriched in non-responders and harbored a lower fraction of CD8+ T cells and plasma cells, as well as a decreased expression of immunoglobulin genes. In addition, we developed the Tumor-Immunity Differential (TID) score which combines features from tumor cells and the TME to accurately predict response to anti-PD-1 immunotherapy (AUC-ROC=0.88, log-rank tests for PFS P &lt; 0.0001, OS P = 0.01). Finally, we also defined TID-related genes ( YWHAE , CXCR6 and BTF3 ), among which YWHAE was validated as a robust predictive marker of ICB response in independent cohorts of pre- or on-treatment biopsies of melanoma and lung cancers. Overall, these results provide a rationale to further explore variations in the cell composition of metastatic sites, and underlying gene signatures, to predict patient response to ICB treatments. One Sentence Summary Tumor microenvironment balance of metastasis and associated genes are key predictors of immunotherapy patient response in kidney cancer.

International audience

Le carcinome canalaire pancréatique (PDAC) est une maladie dévastatrice avec un taux de survie global à 5 ans de seulement 11 %. Sa létalité est due à un diagnostic tardif et à une progression rapide vers les métastases. Diverses études génomiques à grande échelle ont identifié plusieurs mutations potentiellement responsables du PDAC, avec les gènes KRAS (K), TP53 (T), CDKN2A (C) et SMAD4 (S) montrant les fréquences de mutation les plus élevées. Notamment, les mutations du gène KRAS sont présentes dans plus de 90 % des tumeurs du PDAC.Dans notre projet de recherche, nous avons cherché à développer un modèle précancéreux qui reproduit le développement du PDAC en introduisant les quatre mutations clés dans des cellules épithéliales pancréatiques. Nous avons caractérisé ce modèle de manière exhaustive en cultures 2D et 3D et évalué son adéquation pour les tests de médicaments.Pour générer le modèle cellulaire, nous avons utilisé la technologie de modification génétique CRISPR/Cas9 pour cibler les cellules épithéliales canalaires pancréatiques humaines immortalisées (H6c7) afin de neutraliser trois gènes suppresseurs de tumeurs (C-T-S). De plus, nous avons conçu des vecteurs plasmidiques codant pour KRAS pour une expression continue et conditionnelle (en utilisant des systèmes TetR) du KRAS mutant. Pour améliorer l'efficacité de l'intégration du KRAS mutant, nous avons employé la transposase PiggyBac. Simultanément, nous avons neutralisé le gène KRAS pour explorer les voies cellulaires influencées par la perte de KRAS et identifier des cibles potentielles complémentaires à KRAS. Suite à chaque modification génétique, une sélection clonale a été effectuée, et les mutations génétiques ont été confirmées par des essais biologiques.Pour les gènes suppresseurs de tumeurs C-T-S, nous avons réussi à générer des lignées cellulaires avec des mutations simples et triples. Notamment, les cellules avec mutation triple ont montré une expression accrue de KRAS, entraînant une augmentation de la prolifération, de l'apoptose et des dommages à l'ADN. Cependant, lorsque nous avons tenté une expression continue des mutants de KRAS en utilisant des vecteurs plasmidiques, nous avons observé une induction de la sénescence dans les cellules de type sauvage et les cellules mutantes triples, empêchant l'établissement de clones stables. Pour surmonter ce défi, nous avons utilisé des vecteurs PiggyBac (PB) pour une expression conditionnelle de KRAS, validés par des essais de transfection. Par la suite, des lignées cellulaires stables PB ont été établies et caractérisées. Nous avons en outre évalué l'utilité de ces lignées cellulaires pour le dépistage de médicaments, en particulier en nous concentrant sur les dégradateurs et les inhibiteurs de KRAS mutant. De plus, nous avons exploré le potentiel de ces lignées cellulaires pour étudier l'initiation de la carcinogenèse en cultures 2D et 3D. Dans nos expériences de neutralisation de KRAS, nous avons réussi à obtenir des clones sur des bases de type sauvage et de mutation triple C-T-S. Nous avons observé que la perte de KRAS affectait la morphologie et la prolifération cellulaires.En conclusion, bien que p53 et p16 (produit du gène CDKN2A) soient traditionnellement reconnus comme des facteurs clés responsables de la sénescence induite par l'oncogène KRAS, nos premières constatations avec les mutants triples C-T-S indiquent que la délétion de ces gènes seuls n'est pas suffisante pour initier la carcinogenèse ou pour contrer la sénescence induite par KRAS. Cela suggère donc l'implication d'autres facteurs encore non identifiés, essentiels pour la carcinogenèse induite par KRAS. Nos données impliquant des cellules surexprimant KRAS et des cellules avec KRAS neutralisé soutiennent cette notion, indiquant que KRAS influence de multiples voies cellulaires, y compris celles régissant la taille, la morphologie, la dynamique et la régulation du cycle cellulaire.

Le diabète sucré est une maladie chronique qui touche 61 millions de personnes vivant en Europe, soit 1 adulte sur 11. Dans le diabète de type 1, une destruction auto-immune des cellules bêta productrices d’insuline conduit à une hyperglycémie. Le seul traitement des patients consiste en des injections pluri quotidiennes d’insuline afin de contrôler leur taux de glucose dans le sang, sans les guérir. Ces patients ont désespérément besoin d'un traitement durable, qui leur permettent d’améliorer leur qualité de vie. Une possibilité concerne la transplantation d'îlots pancréatiques provenant d’un donneur en état de mort cérébrale, permettant ainsi de rétablir le contrôle de la glycémie normale. Il existe plusieurs limites à cette thérapie cellulaire de remplacement, dont le nombre de patients diabétiques qui est bien supérieur au nombre de donneurs. Ainsi, une nouvelle source de cellules bêta est nécessaire pour que la transplantation puisse être disponible pour tous les patients. Les cellules souches ont le potentiel de fournir une source illimitée de nouvelles cellules productrices d'insuline. Plusieurs équipes ont obtenus ces cellules avec succès dans une boîte de Pétri en exposant les cellules souches à une succession de combinaisons de molécules qui imitent le développement du pancréas humain. Cependant, comme ces cellules sont cultivées dans une boîte de Pétri, elles sont dépourvues du micro environnement pancréatique qui joue un rôle important pendant leur développement. L'objectif de ma thèse est de produire des cellules bêta dérivées de cellules souches pluripotentes induites (CSPi) humaines et de leur fournir un micro environnement et une vascularisation humains. Pour y parvenir, nous avons d'abord utilisé un modèle d'organoïdes-sur-puce qui imite les caractéristiques clés du micro environnement du pancréas natif. Dans une puce microfluidique perfusable, les îlots seront vascularisés via des cellules endothéliales et perfusés assurant ainsi un meilleur apport en oxygène et en nutriments. Nous avons démontré la biocompatibilité de notre plateforme microfluidique qui a permis de vasculariser des organoïdes de cellules bêta dérivées de CSPi avec une perfusion continue du milieu à un débit contrôlé et un micro environnement avec un hydrogel de soutien enrichi de fibroblastes et de cellules endothéliales. Dans la seconde stratégie, nous avons fusionné et vascularisé des organoïdes d'îlots et de vaisseaux sanguins dérivés de CSPi pour améliorer la réponse à l'insuline au fil du temps. Nous avons montré que la co-culture d'organoïdes d'îlots et de vaisseaux sanguins a un effet positif sur la fonctionnalité des îlots et maintient la réactivité au glucose des cellules bêta pendant une culture prolongée. Dans l'ensemble, cette étude explore de nouvelles stratégies in vitro pour la vascularisation des îlots en utilisant les technologies des organoïdes-sur-puce et des cellules souches pluripotentes induites humaines, ouvrant la voie à leur utilisation future dans la médecine personnalisée et régénérative.

La régulation de l’expression des gènes est assurée par la dynamique des modifications post-traductionnelles des histones. Pendant ces 15 dernières années, un vaste panel de nouvelles structures a été découvert, collectivement appelées acylations. Ces structures permettent de réguler finement la transcription par l’état métabolique de la cellule. Toutefois, leurs rôles dans la régulation de l'expression génique restent peu caractérisés. Mon projet de thèse s’est centré sur cette problématique, dans le contexte de la spermatogenèse murine.Dans la première partie de ce projet, j’ai exploré par spectrométrie de masse et par immunoprécipitation couplée à la spectrométrie de masse les marques crotonyl, butyryl et bêta-hydroxybutyryl pour essayer de valider leur présence dans les histones extraites du testicule de souris. Les expériences autour de ces marques, principalement sur la lysine 27 de l’histone H3 (H3K27), ainsi que sur d’autres résidus, n’ont pas abouti à leur identification. En complément, j’ai contribué à une étude sur l’existence de variants souris-spécifiques. En particulier, j’ai interprété des données middle-down qui ont permis de valider avec confiance la présence de l’histone canonique H3.1 et des variants H3.3 et Testis-Specific H3 dans les testicules de souris, mais pas la présence des variants spécifiques de la souris H3mm7 et H3mm13.La deuxième partie de ce projet réside dans l’identification et la caractérisation de la lactylation, récemment découverte. Ce travail constitue l’aspect novateur et le plus abouti de mon projet de thèse. En effet, j’ai établi une cartographie de la lactylation et de l'acétylation sur de nombreuses lysines des histones H3 et H4, par analyse protéomique exploratoire (DDA). La quantification quasi impossible de la lactylation par DDA en raison d'événements d'oxydation nous a incitées à développer une méthode d’analyse protéomique ciblée, en utilisant des peptides synthétiques marqués par des isotopes stables et modifiés avec les énantiomères L-lactyl et D-lactyl. Les résultats ont montré que les peptides portant du L-lactyl ou du D-lactyl étaient d’abondances similaires sur la plupart des lysines de H3 et H4. La stœchiométrie de la lactylation a finalement été estimée : cette modification est très peu abondante et stable (0.01-0.5 %) sur toutes les lysines étudiées.Enfin, une dernière partie a porté sur la caractérisation d’un anticorps dirigé contre la marque H3K18(L-lactyl). Par Résonance Plasmonique de Surface (SPR) et par immunoprécipitation couplée à la spectrométrie de masse, j’ai montré que cet anticorps n’est pas parfaitement spécifique de sa cible. Il présente aussi une forte affinité pour la marque lactyl sur la lysine H3K23. De plus, il reconnait les modifications structurellement proches de lactyl telles que l’hydroxy-iso-butyrylation, et dans une moindre mesure l’acétylation, qui est d’abondance relative importante sur ce résidu. Les résultats de spectrométrie de masse ont montré un net enrichissement des marques lactylées sur H3K18 ou H3K23, mais dans une certaine mesure aussi de marque acétylée sur H3K18 et de la forme di-acétylée sur H3K18/K23, que cela soit sur H3.1 et sur le variant TSH3. De manière intéressante, cet anticorps montre une reconnaissance similaire des deux énantiomères L-lactyl et D-lactyl.Mes travaux de thèse illustrent la complexité de l’étude des nouvelles acylations sur les histones. Mon projet sur la lactylation a mis en lumière la présence des deux énantiomères L-lactyl et D-lactyl sur un grand nombre de lysines des histones H3 et H4, à l’échelle du testicule de souris. L’identification et la quantification fiables de peptides d’histones L- et D-lactylés résultent d’un travail pointilleux. L’étude de la lactylation est en pleine expansion depuis sa découverte en 2019, mais la caractérisation des deux marques L-lactyl et D-lactyl simultanément est très peu explorée. Mon travail s’inscrit ainsi dans une démarche originale et novatrice.