Auswahl der wissenschaftlichen Literatur zum Thema „Profilage ARN cellule unique“

Background:Janus kinase (JAK) inhibitors (JAKinibs) show similar efficacy in rheumatoid arthritis (RA). However, in vitro studies have shown differences in JAK selectivity profiles for baricitinib (BARI), tofacitinib (TOFA), upadacitinib (UPA) and filgotinib (FIL).1,2 These lead to distinct pharmacologic profiles in cellular signaling assays that may impact clinical efficacy or safety1. NK cells are innate lymphocytes important in anti-pathogen responses and immune surveillance, which function via production of cytokines and cell killing3. NK cell proliferation and IFNγ production are JAK-dependent pathways and may be modulated by JAKinibs. Clinical findings show transient decreases in NK cell numbers in patients treated with JAKinibs, but the link to safety is unclear4Objectives:To extend upon findings in proximal cell signaling assays, we compared the selectivity and potency of clinical JAKinibs on NK cell function by assessing proliferation mediated by IL-15 (JAK1/3) and IFN-γ production driven by IL-12 (JAK2/TYK2)+IL-18.Methods:NK cells were isolated from healthy donor PBMC, incubated in vitro with 8 concentrations of each evaluated JAKinib (TOFA, BARI, FIL, FIL metabolite, UPA) and stimulated with IL-15 for proliferation or IL-12/18 for IFNγ production. Proliferation was assessed by Cell Trace dye dilution after 6 days and IFNγ production by intracellular flow cytometry 4hrs post-stimulation. Half maximal inhibitory concentration (IC50) values were calculated for CD56bright, CD56dim, and total NK cells. Steady-state pharmacologic profile over a clinical dosing interval was modeled using concentration-time profiles from JAKinib population pharmacokinetic data in RA subjects under the therapeutic dose5-7. For each JAKinib, the time above IC50 and average daily inhibition of IFNγ or proliferation were calculated for each NK cell population in each donor.Results:Cellular assays in purified NK cells showed dose-dependent inhibition of IL-15-induced proliferation by all JAKinibs with TOFA showing the highest average inhibition and time above IC50 (35-60% inhibition for 8-15 hrs; TOFA>UPA>BARI≈FIL). The differences between JAKinibs are in line with differences in pSTAT inhibition downstream of IL-151. Interestingly, IL-12/18-induced production of IFNγ, which is mediated via JAK2/TYK2 (IL-12) and non-JAK dependent pathways (IL-18), showed weaker inhibition for all compounds. Moreover, all JAKinibs showed <25% average inhibition of IFNγ production over 24hrs and did not show any time above IC50 for IFNγ production or pSTAT4 inhibition at clinical doses. CD56dim and CD56bright sub-populations of NK cells are proposed to have distinct functions and unique expression of surface receptors. Analysis of the IC50 for pSTAT4 and IFNγ production showed ~2-10-fold weaker inhibition by JAKinibs in CD56bright NK cells, suggesting less dependence on JAK-dependent signals in CD56bright NK cells than CD56dim NK cells.Conclusion:NK cell proliferation depends on JAK1 and JAK3-mediated signaling and is differentially inhibited at clinical doses of distinct JAKinibs. In contrast, functional responses downstream of JAK2/TYK2-dependent IL-12/18 were not substantially inhibited by any of the JAKinibs studied. Inhibition of functional and proliferative responses in purified NK cells aligned well with proximal pSTAT inhibition. JAKinib modulation of NK cell proliferation, but not response to IL-12, reflects unique pharmacologic profiles of the drugs studied and could be one component underlying clinical safety observations, including increased risk of viral infections or malignancy4.References:[1]Traves PG et al. Ann Rheum Dis 2021 (in press)[2]McInnes IB, et al. Arthritis Res Ther 2019;21:183.[3]Cooper MA, Fehniger TA, Caligiuri MA. Trends Immunol 2001 Nov;22(11):633-40.[4]Winthrop KL. Nat Rev Rheumatol 2017; 13(4):234-243[5]Zhang X, et al. CPT Pharmacometrics Syst Pharmacol 2017;6(12):804-13.[6]CDER. Application Number: 203214Orig1s000. NDA 203214: Tofacitinib.[7]Klunder B et al. Clin Pharmacokinet 2019;58(8):1045-58.Disclosure of Interests:Paqui Gonzalez-Traves Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Laura Simpson Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Bernard Murray Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Amy Meng Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Julie A. Di Paolo Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Ethan Grant Shareholder of: Gilead Sciences, Employee of: Gilead Sciences, Gundula Min-Oo Shareholder of: Gilead Sciences, Employee of: Gilead Sciences

Abstract STUDY QUESTION Which transcriptional program triggers sex differentiation in bipotential gonads and downstream cellular events governing fetal testis and ovary development in humans? SUMMARY ANSWER The characterization of a dynamically regulated protein-coding and non-coding transcriptional landscape in developing human gonads of both sexes highlights a large number of potential key regulators that show an early sexually dimorphic expression pattern. WHAT IS KNOWN ALREADY Gonadal sex differentiation is orchestrated by a sexually dimorphic gene expression program in XX and XY developing fetal gonads. A comprehensive characterization of its non-coding counterpart offers promising perspectives for deciphering the molecular events underpinning gonad development and for a complete understanding of the etiology of disorders of sex development in humans. STUDY DESIGN, SIZE, DURATION To further investigate the protein-coding and non-coding transcriptional landscape during gonad differentiation, we used RNA-sequencing (RNA-seq) and characterized the RNA content of human fetal testis (N = 24) and ovaries (N = 24) from 6 to 17 postconceptional week (PCW), a key period in sex determination and gonad development. PARTICIPANTS/MATERIALS, SETTING, METHODS First trimester fetuses (6–12 PCW) and second trimester fetuses (13–14 and 17 PCW) were obtained from legally induced normally progressing terminations of pregnancy. Total RNA was extracted from whole human fetal gonads and sequenced as paired-end 2 × 50 base reads. Resulting sequences were mapped to the human genome, allowing for the assembly and quantification of corresponding transcripts. MAIN RESULTS AND THE ROLE OF CHANCE This RNA-seq analysis of human fetal testes and ovaries at seven key developmental stages led to the reconstruction of 22 080 transcripts differentially expressed during testicular and/or ovarian development. In addition to 8935 transcripts displaying sex-independent differential expression during gonad development, the comparison of testes and ovaries enabled the discrimination of 13 145 transcripts that show a sexually dimorphic expression profile. The latter include 1479 transcripts differentially expressed as early as 6 PCW, including 39 transcription factors, 40 long non-coding RNAs and 20 novel genes. Despite the use of stringent filtration criteria (expression cut-off of at least 1 fragment per kilobase of exon model per million reads mapped, fold change of at least 2 and false discovery rate adjusted P values of less than &lt;1%), the possibility of assembly artifacts and of false-positive differentially expressed transcripts cannot be fully ruled out. LARGE-SCALE DATA Raw data files (fastq) and a searchable table (.xlss) containing information on genomic features and expression data for all refined transcripts have been submitted to the NCBI GEO under accession number GSE116278. LIMITATIONS, REASONS FOR CAUTION The intrinsic nature of this bulk analysis, i.e. the sequencing of transcripts from whole gonads, does not allow direct identification of the cellular origin(s) of the transcripts characterized. Potential cellular dilution effects (e.g. as a result of distinct proliferation rates in XX and XY gonads) may account for a few of the expression profiles identified as being sexually dimorphic. Finally, transcriptome alterations that would result from exposure to pre-abortive drugs cannot be completely excluded. Although we demonstrated the high quality of the sorted cell populations used for experimental validations using quantitative RT-PCR, it cannot be totally excluded that some germline expression may correspond to cell contamination by, for example, macrophages. WIDER IMPLICATIONS OF THE FINDINGS For the first time, this study has led to the identification of 1000 protein-coding and non-coding candidate genes showing an early, sexually dimorphic, expression pattern that have not previously been associated with sex differentiation. Collectively, these results increase our understanding of gonad development in humans, and contribute significantly to the identification of new candidate genes involved in fetal gonad differentiation. The results also provide a unique resource that may improve our understanding of the fetal origin of testicular and ovarian dysgenesis syndromes, including cryptorchidism and testicular cancers. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the French National Institute of Health and Medical Research (Inserm), the University of Rennes 1, the French School of Public Health (EHESP), the Swiss National Science Foundation [SNF n° CRS115_171007 to B.J.], the French National Research Agency [ANR n° 16-CE14-0017-02 and n° 18-CE14-0038-02 to F.C.], the Medical Research Council [MR/L010011/1 to P.A.F.] and the European Community’s Seventh Framework Programme (FP7/2007-2013) [under grant agreement no 212885 to P.A.F.] and from the European Union’s Horizon 2020 Research and Innovation Programme [under grant agreement no 825100 to P.A.F. and S.M.G.]. There are no competing interests related to this study.

AbstractThe Intergovernmental Panel on Climate Change (IPCC) 6th Assessment Report (AR6) forecasts a sea level rise (SLR) of up to 2 m by 2100, which poses significant risks to regional geomorphology. As a country with a rapidly developing economy and substantial population, Bangladesh confronts unique challenges due to its extensive floodplains and 720 km-long Bay of Bengal coastline. This study uses nighttime light data to investigate the demographic repercussions and potential disruptions to economic clusters arising from land inundation attributable to SLR in the Bay of Bengal. By using geographical information system (GIS)-based bathtub modeling, this research scrutinizes potential risk zones under three selected shared socioeconomic pathway (SSP) scenarios. The analysis anticipates that between 0.8 and 2.8 thousand km2 of land may be inundated according to the present elevation profile, affecting 0.5–2.8 million people in Bangladesh by 2150. Moreover, artificial neural network (ANN)-based cellular automata modeling is used to determine economic clusters at risk from SLR impacts. These findings emphasize the urgency for land planners to incorporate modeling and sea inundation projections to tackle the inherent uncertainty in SLR estimations and devise effective coastal flooding mitigation strategies. This study provides valuable insights for policy development and long-term planning in coastal regions, especially for areas with a limited availability of relevant data.

Introduction: Disease states are being characterized at finer and finer levels of resolution via biomarker or gene expression profiles, while at the same time. Machine learning (ML) is increasingly used to analyze and potentially classify or predict the behavior of biological systems based on such characterization. As ML applications are extremely data-intensive, given the relative sparsity of biomedical data sets ML training of artificial neural networks (ANNs) often require the use of synthetic training data. Agent-based models (ABMs) that incorporate known biological mechanisms and their associated stochastic properties are a potential means of generating synthetic data. Herein we present an example of ML used to train an artificial neural network (ANN) as a surrogate system used to predict the time evolution of an ABM focusing on the clinical condition of sepsis.Methods: The disease trajectories for clinical sepsis, in terms of temporal cytokine and phenotypic dynamics, can be interpreted as a random dynamical system. The Innate Immune Response Agent-based Model (IIRABM) is a well-established model that utilizes known cellular and molecular rules to simulate disease trajectories corresponding to clinical sepsis. We have utilized two distinct neural network architectures, Long Short-Term Memory and Multi-Layer Perceptron, to take a time sequence of five measurements of eleven IIRABM simulated serum cytokine concentrations as input and to return both the future cytokine trajectories as well as an aggregate metric representing the patient’s state of health.Results: The ANNs predicted model trajectories with the expected amount of error, due to stochasticity in the simulation, and recognizing that the mapping from a specific cytokine profile to a state-of-health is not unique. The Multi-Layer Perceptron neural network, generated predictions with a more accurate forecasted trajectory cone.Discussion: This work serves as a proof-of-concept for the use of ANNs to predict disease progression in sepsis as represented by an ABM. The findings demonstrate that multicellular systems with intrinsic stochasticity can be approximated with an ANN, but that forecasting a specific trajectory of the system requires sequential updating of the system state to provide a rolling forecast horizon.

Les cellules souches hématopoïétiques (CSH) possèdent un potentiel de régénération et de différentiation leur permettant de générer toutes les cellules hématopoiétiques (CH) au cours de la vie d’un individu. Chez les vertébrés, les CSH sont générées au cours du développement embryonnaire via un processus nommé la Transition-Endothélio-Hématopoïétique (TEH). Au cours de la TEH, contrôlée notamment par Runx1, une sous-population de cellules endothéliales aortiques —dites hémogéniques— est extrudée du plancher de l’aorte. En utilisant l‘embryon de poisson zèbre comme modèle, mon laboratoire a précédemment montré que cette extrusion repose sur l’invagination de la membrane luminale de la cellule émergente, la contraction d’une ceinture apicale d’acto-myosine, et est caractérisée par une organisation anisotropique des composantes jonctionnelles. Suite à leur émergence, les précurseurs de CSH migrent pour coloniser les organes hématopoiétiques embryonnaires, qui, chez le poisson-zèbre, sont le Tissu Caudal Hématopoïétique, le thymus et le pronephros. Nos data non publiées montrent que, de manière surprenante et contrairement à ce qui est connu dans d’autres processus d’extrusion, les cellules en TEH maintiennent et renforcent une polarité apico-basale tout au long de leur émergence. De plus, nous avons décrit l’existence d’un second type de TEH jusque là non-identifié, qui est caractérisé par une absence d’invagination de sa membrane luminale et de polarité apico-basale. Les deux objectifs de ma thèse étaient les suivants: 1° caractériser certaines étapes clés de l’interaction dynamique entre les cellules hémogéniques/en TEH et leurs voisines endothéliales, et 2° questionner le destin des cellules descendant des deux types d’émergence, en terme de potentiel de migration, différentiation et prolifération et de potentiel souche. Pour mon premier axe, je me suis intéressée à des régulateurs potentiels de la TEH, qui lieraient la régulation de la contraction d’acto-myosine et la polarité apico-basale, en particulier la protéine Rho GEF ArhGEF11. J’ai utilisé des approches interférentes couplées à de l’analyse phénotypique à haute résolution grâce à de la microscopie live, ainsi qu’à des analyses quantitatives de dynamique jonctionnelle par FRAP. Nous avons découvert que l’interférence avec Runx1 impacte l’expression et l’épissage alternatif de l’ARNm d’ArhGEF11. Interférer avec l’épissage alternatif d’ArhGEF11 modifie la dynamique de renouvellement des composantes jonctionnelles entre cellules endothéliales et hémogéniques, résultant en un défaut dans l’intercalation des cellules endothéliales autour des cellules hémogéniques. Cette absence d’intercalation, normalement nécessaire à l’extrusion, conduit in fine à l’accumulation de cellules hémogéniques dans l’aorte. Pour mon deuxième axe de recherche, j’ai développé un protocole de suivi à l’échelle de la cellule unique qui me permet de photoconvertir une cellule au moment de son émergence et de suivre sa descendance trois jours après. J’ai associé cette approche à de la microscopie ainsi qu’à du tri cellulaire suivi d’analyse transcriptomique (MARS-seq) pour étudier en détail les destins respectifs des descendantes des deux types de TEH. Bien qu’elles donnent toutes deux naissances à des cellules multipotentes, j’ai observé une différence dans leur capacité à nicher dans le thymus, ainsi que dans leur capacité à générer diverses cellules de la lignée lymphoïde. De plus, contrairement à de précédentes études qui montrent que les CH entrent directement dans la circulation après leur émergence, nous avons observé que certaines restent dans l’espace sous-aortique (nommé AGM) pour une longue période (jusqu’à 3 jours). L’analyse transcriptomique de ces CH de l’AGM montre la présence de toutes les lignées hématopoiétiques ainsi que de différentes sous populations de CSH, suggérant que cette région pourrait être une niche qui contribuerait à la spécification des CSH
Long-term hematopoietic stem cells (LT-HSC) are cells with long-term regenerative potential that give rise to cells of all hematopoietic lineages. In vertebrates, they are generated during a short window of the embryonic development via a developmental process called the Endothelial-to-Hematopoietic Transition (EHT). During this transition, placed under the control of the transcription factor Runx1, a subpopulation of aortic cells — the hemogenic endothelium — extrudes from the floor of the aorta. My lab previously reported, using the zebrafish embryo model, that this extrusion involves the invagination of the cell’s luminal membrane, the contraction of an apical acto-myosin belt, and is characterized by the anisotropic organization of junction components. After emergence, newly generated HSC precursors (pre-HSC) migrate to seed embryonic hematopoietic organs (namely the Caudal Hematopoietic Tissue (CHT), thymus and pronephros in the zebrafish). Unpublished data from my lab showed that, surprisingly and contrary to what is observed in many cell extrusion processes, EHT-undergoing cells maintain/reinforce an apico-basal polarity throughout their emergence. Moreover, we reported the existence of a hitherto undescribed second type of EHT. This second type of EHT is characterised by an absence of luminal invagination, an absence of apico-basal polarity, and an apparent crawling of neighboring endothelial cells on its luminal membrane. In this context, the two goals of my PhD were: first to characterize some critical steps of the dynamic interplay between emerging Hematopoietic Stem Cells precursors and endothelial neighbours before and during the EHT, and second to investigate whether the two types of EHT give rise to progenies with (dis)similar migration, proliferation, differentiation and stemness potential. In the first line of results, I investigated the role of potential regulators of EHT, linking contractile actomyosin to apico-basal polarity, i.e. Rho GEF proteins recruited at apical junctions via a PDZ domain. I used interfering approaches coupled to phenotypic analysis using high-resolution live imaging as well as quantitative analysis of junction components renewal by FRAP. We uncovered the sensitivity of ArhGEF11/PDZ-RhoGEF mRNA expression and alternative splicing to Runx1 interference. Interfering with ArhGEF11 alternative splicing alters the dynamics of junction components around hemogenic cells, thus hindering the intercalation of endothelial cells around hemogenic cells, a necessary step for extrusion completion. This modification of the interplay between endothelial and hemogenic cells leads to an accumulation of hemogenic cells in the aorta. For the second line of results, I developed a single cell tracing protocol that allowed me to photoconvert single cells during their emergence and follow their progeny three days after. I coupled this approach to in vivo tracing using confocal microscopy and single cell sorting followed by transcriptomic analysis (MARS-seq). This strategy allowed me to study in depth the respective fates of both EHT cell types, spatially, temporally and transcriptionally. I show that both types of EHT give rise to multipotent hematopoietic cells and are able to generate cells of all hematopoietic lineages (erythroid/megakaryocyte, myeloid and lymphoid). However, they display a discrepancy in their ability to niche in the thymus and bias in their ability to generate different cell types of the lymphoid lineage. Moreover, contrary to previous investigations suggesting that hematopoietic cells directly enter the circulation after their emergence, we observed that some cells stay in the sub-artic region for long periods of time (up to 3days). Transcriptomic analysis of those cells shows the presence of all hematopoietic lineages as well as multiple HSC subpopulations. This suggests that the sub-aortic space might be an overlooked niche that could contribute to the specification of HSC

Le niveau d’expression des 21 000 gènes que comporte une cellule humaine doit être très précisément régulé en fonction de nombreux signaux extra- et intracellulaires. A ce titre, des défauts dans le contrôle de l’expression génique sont souvent impliqués dans des maladies comme les cancers. Le choix des gènes, ainsi que leurs niveaux d’expression, sont le résultat de la régulation de l’ARN polymérase II par une combinaison de facteurs de transcription. Généralement, ces évènements sont étudiés sur de larges populations cellulaires, masquant d’éventuelles variations entre cellules d’une même population. Au cours de ma thèse, je me suis particulièrement intéressé à comprendre la régulation transcriptionnelle du proto-oncogène c-Fos à l’échelle de la cellule unique. Pour cela, nous avons développé un outil permettant de compter les ARNm unique et les ARN naissant sur les sites de transcription à l’aide d’expériences d’Hybridation in situ en Fluorescence. A l’aide de ce programme, nous avons découvert un mode de régulation de c-Fos particulièrement simple et efficace. Le gène est transcrit durant une brève période appelé burst transcriptionnel. Nous avons montré que l’amplitude des bursts n’est pas régulée alors que leur fréquence est modulée par le niveau d’activation des voies de signalisation intracellulaire. Nous avons également observé des agrégations dynamiques d’ARN polymérase II sur les gènes. Ces agrégations pourraient fournir une explication à l’origine moléculaire de ces bursts transcriptionnels tout en apportant un cadre pour déchiffrer leurs régulations
The expression level of the 21,000 genes present in a human cell must be precisely controlled according to several extra- and intracellular signals. Failures in the control of gene expression are often involved in diseases such as cancer. The choice of genes, as well as their expression level, are the result of the regulation of RNA polymerase II by a combination of transcription factors. Usually, these events are studied over large cell populations, thus masking variations between cells of the same population. In my work, I particularly focused on the transcriptional regulation of the c-Fos proto-oncogene at the single cell level. To this end, we developed a tool for quantifying single mRNA and nascent RNA on transcription site from Fluorescence in situ Hybridization data. With this program, we discovered a remarkably simple regulation of c-Fos transcription. Multiple transcripts are produced during short and infrequent transcriptional bursts. We have shown that while the burst size is not regulated, their frequency is modulated by the level of activation of intracellular signaling pathways. We also observed a dynamics clustering of RNA polymerase II on genes. This clustering may provide an explanation for the molecular origin of these transcriptional bursts as well as providing a framework to decipher their regulation

Les technologies d'analyse à l'échelle de la cellule unique ont vu le jour il y a quelques années et sont depuis en constante évolution. Ces technologies permettent de mieux comprendre le fonctionnement d'ensemble de cellules très hétérogènes. Elles permettent par exemple de découvrir et suivre des sous types cellulaires, avec des applications en cancérologie ou encore en neurobiologie. Nous avons développé une technologie pour étudier le profil d'expression de gènes d'intérêt au niveau d'une cellule unique, en utilisant la microfluidique en gouttes. En limitant le nombre de gènes étudiés comparé aux technologies commerciales de transcriptome entier, l’approche ciblée a plusieurs avantages potentiels : gagner en profondeur de séquençage, augmenter le nombre de cellules étudiées, optimiser la détection pour les bas niveaux d’expression, tout en réduisant la complexité des données et des coûts. Le ciblage est parfois indispensable, notamment lorsque les ARNs ne portent pas de séquence d’amorce générique, comme dans le cas des ARNs viraux. Deux applications sont présentées : l'analyse de l'inflammation des cellules immunitaires du cerveau aux premières étapes du développement, ainsi que l'étude de la recombinaison génétique chez le virus
Single cells technologies were introduced a few years ago and have been dramatically evolving ever since. These technologies have revolutionized biology, making it possible to better understand how heterogeneous cell systems works. For example, they permit to discover and follow cell subtypes, with applications in oncology or neurobiology. We have developed a technology to study the expression profile of genes of interest at the level of a single cell, using droplet-based microfluidics. By limiting the number of genes studied compared to commercial whole-transcriptome technologies, the targeted approach has several potential benefits: gaining deeper sequencing, increasing the number of cells studied, optimizing detection for low levels of expression, while reducing the complexity of data and costs. Targeting is sometimes essential, especially when the RNAs do not carry a generic primer sequence, as in the case of viral RNAs. Two applications are presented: the analysis of inflammation of the immune cells of the brain in the early stages of development, as well as the study of genetic recombination in the virus

Le carcinome hépatocellulaire (CHC) est parmi les principales causes de mortalité dans le monde et les traitements disponibles sont insuffisants. Ceci est dû à la connaissance limitée de la complexité biologique et du microenvironnement hépatiques en situation normale et pathologique. Pour répondre à ces besoins, nous avons développé un protocole de séquençage d’ARN sur cellule unique (scRNA-seq) à partir de tissus primaires de foie humain. Nous avons assemblé un atlas de cellules du foie humain et comparé le profil scRNA-seq du foie normal au profil du CHC. L’atlas a révélé l’hétérogénéité au sein des principales populations de cellules hépatiques, la zonation transcriptomique des cellules endothéliales et l'existence de progéniteurs épithéliaux dans le foie adulte capable de se différencier à la fois en cholangiocytes et en hépatocytes. L'analyse par scRNA-seq du CHC a dévoilé l'hétérogénéité marquée de cette tumeur, les modifications de son microenvironnement cellule par cellule et les interactions entre les cellules tumorales et le virus de l’hépatite B en découvrant des voies et des facteurs moteurs de la cancérogenèse hépatique jusque-là inconnus
Hepatocellular carcinoma (HCC) is a leading cause of death worldwide and the current treatments are unsatisfactory. One reason is the limited knowledge on the complexity and microenvironment of healthy and diseased liver. To address this gap, we have developed a single cell RNA sequencing (scRNA-seq) pipeline for primary human liver tissues. We have assembled an atlas of human liver cells and compared the scRNA-seq profile of normal liver and HCC. The atlas revealed an unknown heterogeneity within the main populations of liver cells, the transcriptomic zonation of endothelial cells and the existence of an epithelial progenitor in the adult liver capable of differentiating into both cholangiocytes and hepatocytes. ScRNA-seq analysis uncovered the marked cell heterogeneity of HCC, its microenvironment changes at single-cell level and the interactions between tumor cells and hepatitis B virus discovering previously unknown pathways and drivers of hepatocarcinogenesis

Les communications cellulaires sont indispensables au bon fonctionnement des organismes multicellulaires, notamment pour s’adapter à un environnement changeant en permanence. Les cellules du système immunitaire n’échappent pas à cette règle mais les interactions entre cellules immunitaires restent peu connues et compliquée à étudier. La récente apparition des technologies de séquençage dites ‘cellules uniques’ représente une opportunité unique pour étudier ces communications. Dans cette thèse, différentes approches expérimentales et analytiques ont été développées pour étudier ces communications à une échelle de cellules uniques. Ces stratégies ont ensuite été appliquées à différents contextes pathologiques, incluant le COVID-19, la maladie d’Alzheimer ou une immunisation par des pathogènes inactivés, et ont permis d’identifier des voies de communications cellulaires jusqu’ici inconnues ou mal comprises. Néanmoins, l’efficacité de ces approches est limitée par l’absence d’informations sur la localisation des cellules et des travaux supplémentaires intégrant ce genre de données est essentiel pour aller plus loin dans la dissection des communications entre cellules immunitaires
Cellular communications are essential to the proper functioning of multi-cellular organisms, particularly in order to adapt to a constantly changing environment. The cells of the immune system are no exception to this rule, but the interactions between immune cells remain little known and complicated to study. The recent emergence of 'single cell' sequencing technologies represents a unique opportunity to study these communications. In this thesis, different experimental and analytical approaches have been developed to study these communications on a single cell scale. These strategies were then applied to different disease contexts, including COVID-19, Alzheimer's disease or immunisation with inactivated pathogens, and identified previously unknown or poorly understood cellular communication pathways. However, the effectiveness of these approaches is limited by the lack of information on cell location and further work integrating such data will be essential to go further in the dissection of immune cell communications

La nature segmentée du génome des virus de la grippe A (IAV) permet une évolution rapide par réassortiment génétique. Bien que le nombre théorique de génotypes issus d'un réassortiment entre deux virus soit de 256 (28), la panoplie complète des différents génotypes n'a jamais été observée et certains gènes ont tendance à co-ségréger, suggérant que le réassortiment génétique est biaisé. Cependant, à ce jour, les contraintes qui façonnent le réassortiment génétique restent largement méconnues. L'objectif de mon projet est de progresser dans la compréhension des règles sous-jacentes au réassortiment génétique afin d'améliorer notre capacité à prédire le réassortiment entre les IAV circulant dans la nature.Nous avons dans un premier temps étudié l’incompatibilité entre sous-unités hétérologues de la polymérase virale (FluPol) réunies suite à un réassortiment génétique. En effet, nous avons observé qu'un virus réassortant dont le segment PB2 dérive du virus A/WSN/33 (WSN) dans un fond génétique A/PR/8/34 (PR8) était atténué, malgré un degré d’identité de 97% entre les protéines PR8-PB2 et WSN-PB2. Des passages en série indépendants ont conduit à la sélection de révertants phénotypiques portant des mutations secondaires distinctes sur les sous-unités PA, PB1 et PB2. L’impact de ces mutations a été étudié par génétique inverse et à l’aide de tests d’activité sur les polymérase virales. Pour chaque virus révertant, au moins une mutation a été localisée à l'interface de dimérisation de FluPol et s'est avérée réguler son taux de dimérisation. La mutation PA-E349K en particulier joue un rôle majeur dans la correction d'un défaut initial de réplication virale (ARNc -> ARNv). Nos résultats montrent que les sous-unités de la FluPol co-évoluent non seulement pour assurer des interactions optimales entre sous-unités, mais également des niveaux appropriés de dimérisation, indispensables à une réplication efficace. Ainsi, la dimérisation de la FluPol pourrait être l’un des facteurs limitant l’issue du réassortiment génétique.Parallèlement, afin d’étudier le réassortiment génétique de manière exhaustive et avec une puissance statistique suffisante, nous avons cherché à adapter un système déjà éprouvé de microfluidique en goutte pour un séquençage ciblé, à haut débit et massivement parallélisé, de > 105 IAV issus d’un réassortiment entre deux IAVs. Pour établir la faisabilité du système deux souches virales saisonnières circulantes ont été choisies et des amorces ciblant les huit segments d’ARNv de chaque virus ont été conçues, testées et optimisées. Une expérience contrôle préliminaire réalisée sur des cellules uniques infectées individuellement, montre que les informations sont correctement préservées au niveau de la cellule unique mais que la détection des segments et des souches était déséquilibrée. De nouvelles amorces ont été conçues et des stratégies d'amplification alternatives mises en œuvre et optimisées. Après analyse du réassortiment entre les deux souches saisonnières et validation des données par comparaison avec les données de surveillance, notre système sera appliqué au réassortiment génétique entre les virus saisonniers humains et les virus animaux d’intérêt zoonotique.À long terme, les données générées via notre plateforme devraient aider à la compréhension des mécanismes de réassortiment génétique entre virus influenza. Notre plateforme pourrait également devenir un outil prédictif s’ajoutant aux outils d'évaluation du risque de pandémie grippale ainsi qu’un outil de surveillance
The segmented nature of the genome of influenza A viruses (IAVs) allows rapid evolution by genetic reassortment. Although the theoretical number of genotypes that can emerge from reassortment between two viruses is 256 (28), the full panel of different genotypes was never observed and certain genes tend to co-segregate, suggesting that genetic reassortment is biased. However, to date, the constraints that shape genetic reassortment remain largely unknown. The objective of my project is to make progress in understanding the rules underlying genetic reassortment in order to improve our capacity to predict reassortment among co-circulating IAVs.First, we investigated the incompatibility between non-cognate subunits of the influenza polymerase complex (FluPol) brought together by genetic reassortment. Indeed, we observed that a 7:1 reassortant virus whose PB2 segment derives from the A/WSN/33 (WSN) virus in an otherwise A/PR/8/34 (PR8) backbone was attenuated, despite a 97% identity between the PR8- and WSN-PB2 proteins. Independent serial passages led to the selection of phenotypic revertants bearing distinct second-site mutations on PA, PB1 and PB2. The constellation of mutations present on the revertant viruses was studied using reverse genetics and cell-based reconstitution of the viral polymerase. For each revertant virus, at least one mutation was located at the FluPol dimerization interface and was found to regulate the levels of FluPol dimer. For one of them, PA-E349K, a major role in correcting an initial defect in viral replication (cRNA -> vRNA) was demonstrated. Hence, our results show that the FluPol subunits co-evolve not only to ensure optimal inter-subunit interactions but also proper levels of dimerization of the heterotrimer, essential for efficient viral RNA replication. Thus, we suggest that FluPol dimerization is one of the factors that can restrict the outcome of genetic reassortment.In parallel, in order to study the outcome of genetic reassortment comprehensively and achieve adequate statistical power, we aimed at adapting a proven droplet-based microfluidic single-cell RNA-seq system for customized high-throughput massively parallelized targeted sequencing of > 105 reassortant IAVs. For a proof-of-concept, two circulating seasonal viral strains were chosen and gene specific primers targeting their eight segments were designed, tested and optimized. From a preliminary compartimentalized control experiment, we found that single cell information was well preserved but that segment and strain detection were imbalanced. New primers were designed and alternative amplification strategies were implemented and optimized. A new control experiment will be performed prior to analysis of reassortment between the two seasonal strains and validation of the data by comparison with surveillance data. Once validated, our system will be applied to genetic reassortment between human seasonal viruses and animal viruses of zoonotic interest. In the long term, the data generated through our platform should help understanding the mechanism of IAV genetic reassortment and become a valuable predictive tool added to the Pandemic Influenza Risk Assessment Tools for pandemic preparedness

Telomeres are nucleoprotein structures that cap the physical ends of eukaryotic chromosomes. They consist of repetitive DNA sequences 5’-TTAGGG-3’ assembled with proteins which form the shelterin complex. This complex protects the ends of chromosomes by inhibiting DNA repair pathways at telomeres and avoid their recognition as double-strand breaks. Telomeres have been identified as a transcriptionally silent zone until 2007 when a noncoding RNA called TERRA (TElomeric Repeat containing RNA) transcribed from telomeres was discovered. This RNA gave rise to many questions: How is TERRA regulated? How is TERRA expressed? Does TERRA interact with proteins, DNA or RNA? After several studies, we know that TERRA is frequently expressed in cancer cells and it interacts with a large proteome. Nevertheless, its specific function remains unknown. In this thesis, we studied this RNA in human cancer cells using live-cell microscopy which allowed us to get information on TERRA’s dynamics, localization and its interactome. Moreover, we used single-molecule imaging on TERRA 15q labeled by the MS2-GFP system, which allowed the visualization of TERRA transcripts. This study resulted in the discovery of two types of TERRA population from telomere 15q: one of the population is characterized by the formation of clusters and a second population is constituted of unique molecules more dynamic in the nucleus. Finally, in order to better understand TERRA’s functions, we developed a new approach which consists on immunoprecipitating TERRA using the MS2 stem-loops as a tag to identify TERRA-interacting proteins such as the telomeric factor TRF2 or RNA-binding proteins like hnRNP -A1 or FUS.
Les télomères forment les extrémités des chromosomes chez les eucaryotes. Ces séquences répétées en tandem 5’-TTAGGG-3’ font partie d’un complexe nucléoprotéique appelé shelterin. En effet, cet assemblage de protéines télomériques permet la protection des extrémités des chromosomes, permettant à celles-ci de ne pas être reconnues comme des cassures dans l’ADN et d’activer les voies de réparation de l’ADN. Les télomères ont longtemps été reconnus comme étant des zones de transcription inactives, ce jusqu’en 2007 lorsqu’une équipe de recherche découvrit un ARN non codant appelé TERRA (Telomeric Repeat containing RNA). Ce dernier a suscité de nombreuses questions : quel est le rôle de cet ARN? Comment est-il exprimé et régulé? Interagit-il avec d’autres facteurs cellulaires? Les différentes recherches menées sur cet ARN ont permis de conclure que celui-ci était fréquemment induit dans les cellules de cancer, que ses partenaires d’interactions sont nombreux, mais que ses fonctions restent encore mal définies. Par ailleurs, ces différentes études ont toujours été ou presque réalisées sur des cellules fixées, sur une population totale d’ARN télomérique TERRA. Afin d’apporter de nouvelles réponses et de mieux caractériser cet ARN, nous avons étudié ce transcrit dans des cellules de cancer humain en utilisant la technique de microscopie en temps réel, qui permet de récolter des données sur la dynamique, la localisation de cet ARN et ses éventuels partenaires. De plus, nous nous sommes intéressés à des molécules uniques de TERRA issues du télomère 15q en exploitant la technique de marquage avec des tiges-boucles MS2 (MS2-GFP). Cette étude de microscopie a permis de découvrir deux types de population de l’ARN TERRA 15q : une population caractérisée par des assemblages d’ARN dit clusters (agrégats d’ARN) et une population plus singulière qui semble avoir une diffusion plus importante dans le noyau de la cellule. Par ailleurs, l’expression de l’ARN TERRA semble être différente d’un type cellulaire à un autre et nous avons donc cherché à connaître le niveau d’expression de cet ARN au sein de la lignée étudiée au cours de ce projet de recherche. Enfin, afin de découvrir de nouveaux rôles pour cet ARN, nous avons développé une approche de co-immunoprécipitation de l’ARN TERRA pour identifier des interactions avec des protéines du complexe shelterin comme TRF2, ou des protéines liant l’ARN comme hnRNP-A1 ou encore FUS.

DNA polymorphisms are variations in the genetic sequence that occur within a population. These polymorphisms can be used as genetic markers to identify individuals, determine familial relationships, and study population genetics. Genetic fingerprinting is a widely used method for identifying individuals based on DNA polymorphisms. Genetic fingerprinting involves the analysis of DNA polymorphisms at multiple loci to generate a unique genetic profile for an individual. The most common types of DNA polymorphisms used for genetic fingerprinting are short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). STRs are short, repeated sequences of DNA that vary in length between individuals. They are highly polymorphic and generate DNA profiles by analyzing the number of repeats at each locus. SNP markers, on the other hand, are single nucleotide variations that occur at specific positions within the genome. They are less polymorphic than STRs, but genetic mapping and association studies are widely used. Genetic fingerprinting has many applications, including forensic science, paternity testing, and conservation biology. However, the use of genetic data also raises ethical concerns regarding privacy and discrimination.

Antibacterial and antifungal agents aim to kill pathogens, or at the very least incapacitate them. To achieve this aim these agents must have a reasonable degree of toxicity at the cellular level. If this toxicity was equally manifest against all cell types then the drugs would be unusable in patients as the side effect profile would be unacceptably severe. Selective toxicity, whereby the agents are orders of magnitude more toxic to bacteria or fungi than human cells, allows for the safe and effective administration of these agents to patients. There are a number of different mechanisms by which an antimicrobial agent can yield selective toxicity: ● Target a cellular structure that exists only in bacteria/fungi—e.g. the cell wall; ● Target a cellular structure that has a significantly different structure in bacteria/ fungi— e.g. the ribosome; the fungal cell membrane; ● Target cellular enzymes that are significantly different in bacteria/fungi e.g. topoisomerase; ● Target a synthetic pathway that exists only in bacteria e.g. folate synthesis. Broadly, antibacterial drugs can be divided into the following categories: ● Agents that target the cell wall; ● Agents that target the cell membrane; ● Agents that inhibit protein synthesis; ● Agents that inhibit DNA replication/ transcription of RNA; ● Agents that target folate synthesis; ● Agents that directly damage intracellular structures. The cell wall is unique to bacteria, and therefore an ideal target. Disrupting the complex cross-linking process required to produce the cell wall leads to loss of bacterial cell integrity and therefore to cell death. The following classes of antibiotics target the cell wall: The first class to be discovered, and still in many cases the most effective, incorporates the four-membered beta-lactam ring—its homology to d-alanyl-d-alanine allows beta-lactam-containing compounds to bind to cell wall peptidoglycans and act as chain terminators. The beta-lactam ring is fused to a five-membered sulphur-containing ring. Variations in side chains account for the differing pharmacokinetics and spectra of action of the different compounds—for example, the addition of an amino group to benzylpenicillin produces ampicillin.

Timely estrus detection is one of the critical factors for increasing reproductive efficiency in animals. Estrus physiology is under the influence of the endocrine signals that include a network of miRNAs. EV miRNAs are more stable than the other cell free miRNAs as they are doubly protected from endogenous RNase activity by means of cellular packing within the membrane-enclosed structures. Review of literature indicated the differential expression of miRNA at the estrus stage and other stages of the estrous cycle in various biological fluids, the role of miRNAs in oviductal function as well as their relation to the dynamics of preovulatory sex-steroid concentration or vice-versa by influencing the genes of miRNA biogenesis pathway. Interestingly, overlapping expression of miRNAs between tissues and EVs released from tissue fluids, as well as unique and differential expression of miRNA between bodily fluids and EV fractions of biological fluids has been identified. Studies focusing on the miRNA secreted in easily accessible urinary extracellular vesicles during the estrus stage in relation to the endocrine profile may pay the way for the identification of biomarkers for detecting estrus.

The acute phase response—trauma, tissue necrosis, infection, inflammation, and malignant neoplasia induce a complex series of nonspecific systemic, physiological, and metabolic responses including fever, leucocytosis, catabolism of muscle proteins, greatly increased de novo synthesis and secretion of a number of ‘acute phase’ plasma proteins, and decreased synthesis of albumin, transthyretin, and high- and low-density lipoproteins. The altered plasma protein concentration profile is called the acute phase response. Acute phase proteins—these are mostly synthesized by hepatocytes, in which transcription is controlled by cytokines including interleukin 1, interleukin 6, and tumour necrosis factor. The circulating concentrations of complement proteins and clotting factors increase by up to 50 to 100%; some of the proteinase inhibitors and α‎₁-acid glycoprotein can increase three- to fivefold; but C-reactive protein (CRP) and serum amyloid A protein (an apolipoprotein of high-density lipoprotein particles) are unique in that their concentrations can change by more than 1000-fold. C-reactive protein—this consists of five identical, nonglycosylated, noncovalently associated polypeptide subunits. It binds to autologous and extrinsic materials which contain phosphocholine, including bacteria and their products. Ligand-bound CRP activates the classical complement pathway and triggers the inflammatory and opsonizing activities of the complement system, thereby contributing to innate host resistance to pneumococci and probably to recognition and safe ‘scavenging’ of cellular debris. Clinical features—(1) determination of CRP in serum or plasma is the most useful marker of the acute phase response in most inflammatory and tissue damaging conditions. (2) Acute phase proteins may be harmful in some circumstances. Sustained increased production of serum amyloid A protein can lead to the deposition of AA-type, reactive systemic amyloid.

The interaction between tires and soft soils is a complex process that has not yet been fully understood. Attempts to create analytical models which realistically simulate these interactions have proven to be exceedingly time consuming for each tire model and have achieved only limited success. Thus, the exploration and evaluation of traction concepts by analytical means is impractical. This paper posits that significantly more reliable, rapid, and cost effective development is achieved through the prototyping and experimental evaluation of traction concepts. Here, three traction prototypes are developed and evaluated by undergraduate teams in the course of an academic semester. These concepts explore the performance of grousers, inverted geometry (dimples), and cellular materials respectively using a wheel endurance and soft-soil traction testing system at Clemson University. Each concept is tested at different acceleration rates from 0–10km/h and at different loads while measuring the slip between the tire and soil surfaces. The results of experimental evaluation indicate that cellular materials present a unique slip profile which is superior to that of the two other purely geometric concepts studied. The worst performance was seen on the inverse geometry concept which presented a behavior of steadily increasing slip with respect to wheel velocity regardless of acceleration. Grousers also presented this behavior but only at higher accelerations. This suggests that not only that prototyping is preferable, but that traditional soft-soil traction approaches may be in error.