Glioblastoma is one of the most devastating forms of primary brain tumor, with a survival of 14.6 months from the diagnosis. Despite the aggressive treatments used in the clinic, consisting of surgical resection followed by radiotherapy and concurrent chemotherapy using temozolomide, the absence of novel treatments and the development of resistance to standard-of-care therapies continue to position glioblastoma as the most challenging brain tumor in adults. The main factor contributing to the incurability of glioblastoma is the extensive heterogeneity. This heterogeneity, which is evident both at intratumoral and inter-patient levels, represents a substantial obstacle to the achievement of effective treatments. In this context, the use of single-cell resolution appears one of the most powerful means for understanding the intricacies and unravelling the heterogeneity of glioblastoma. Although single-cell RNA sequencing studies have provided and still provide valuable insights, they lack the essential spatial context that is critical for unravelling the heterogeneity of glioblastoma. This limitation impedes the understanding of interactions among distinct subpopulations and their intricate relationships with the neuronal microenvironment. To gain insights into the heterogeneity of glioblastoma, we used the spatially resolved RNA sequencing technology to analyse glioblastoma samples deriving from 3 different patients. For each patient, we focused on four distinct tumor regions, i. e. the proliferating tumor area, the necrotic core, the infiltrating area, and a distal healthy area. Cancer cells identified in the infiltrating regions exhibited a unique pattern of cell subpopulations, with the oligodendrocytes as the most represented in this area. In addition, we managed to generate patient-derived glioblastoma organoids from nearly all areas, with the tumor regions displaying the highest growth rates. These patient-derived organoids, which represent fundamental models useful to faithfully replicate the disease in vitro, may be employed in future analyses. Finally, we also derived glioblastoma stem cell cultures from the different tumor regions, with the proliferating tumor area showing the highest rate of success. In the second part, we aimed to gain a deeper understanding of the molecular mechanisms that underlie the development of glioblastoma, which is crucial for developing effective treatments, and characterise ELAVL2 role, highlighting its potential role as a potential tumor suppressor in glioblastoma. Collectively, our findings suggest that ELAVL2 promotes the exit of glioblastoma stem cells from quiescence, boosting their self-renewal capacity while also facilitating neuronal differentiation. The in vivo validation of our results using an orthotopic human glioblastoma stem cell xenograft model and a D. melanogaster genetic model strongly supports our findings and points to deletion of ELAVL2 as a factor that increases aggressiveness in glioblastoma stem cells and in vivo.

Doctor of Philosophy / Department of Entomology / Yoonseong Park / Complex life cycles (CLCs), developmental programs in which life-history stages are distinct in morphology, behavior, and physiology, are common throughout the biosphere. However, it is still unclear why and how CLCs evolve. The adaptive decoupling hypothesis (ADH) postulates that CLCs evolve to decouple the developmental processes that underlie traits across ontogeny to allow for independent, stage-specific responses to selection. This ultimately could lead to alternate life-history stages adapting to unique environments, thus optimizing fitness across development. However, few empirical tests of the ADH are available. Detecting genetic and transcriptomic decoupling of thermal hardiness using robust techniques in a model system, D. melanogaster, was the goal of this dissertation. Furthermore, this work illustrates that different life-history stages have the potential to become adapted to unique ecological niches. I performed three primary studies to test the ADH: 1.) estimation of the genetic correlation for cold hardiness between larvae and adults using isogenic lines of D. melanogaster to determine if unique genetic architectures underlie variation in cold stress response using standard quantitative genetic and Genome-Wide Association (GWA) methods, 2.) testing whether developmental acclimation is genetically correlated across stages, and whether acclimation alters cross-stage correlations in cold hardiness, and 3.) analysis of the transcriptional responses of both larvae and adults to extreme cold to determine if stage-specific stress response mechanisms exist across development.

No description available.

Il existe un certain nombre de situations au cours desquelles l’intégrité du génome cellulaire est mise en danger. Ceci se produit notamment lors de mouvements de gènes (translocation, éléments mobiles), lors d’infections virales parfois intégratives (AAV, HBV, HPV) ou lors d’infections rétrovirales. En effet, le cycle de réplication des rétrovirus nécessite une étape d’intégration du génome viral dans l’ADN génomique de la cellule infectée. Malgré les nombreuses études menées sur les infections par le VIH, les cancers viro-induits ou non et les thérapies géniques basées sur les rétrovirus, aucune donnée n’est actuellement disponible sur les modifications cellulaires induites par de telles perturbations chromosomiques. L’objectif du projet était d’identifier des mécanismes cellulaires induits par des atteintes à l’intégrité du génome, plus précisément par l’insertion de molécules d’ADN non cellulaire dans les chromosomes. L’intégration de matériel génétique additionnel a été induite par des vecteurs lentiviraux dérivés du VIH-1. Les modifications cellulaires uniquement dues à l’intégration ont été isolées par comparaison de vecteurs intégratif et non intégratif. Des cellules primaires du derme humain ont été sélectionnées pour l’étude. Les temps post infection et les doses virales les plus adéquates ont été sélectionnés grâce à des expériences de cinétiques d’intégration couplées à une quantification des ADN viraux intégrés. L’analyse des modifications cellulaires induites par l’intégration de l’ADN étranger a portée sur l’ensemble du transcriptome et sur l’ensemble du protéome cellulaire. Dans le but d’effectuer une analyse transcriptomique, des puces à ADN, représentant le génome humain complet, ont été utilisées. Cette étude a démontré une forte répression transcriptionnelle induite par l’intégration. De plus, toutes les fonctions cellulaires sont perturbées par le processus. Finalement, une classification par interactions et fonctions biologiques a mis en évidence cinq processus cellulaires majoritairement affectés par l’intégration de l’ADN étranger, qui correspondent au cycle et à la mort cellulaire, au remodelage et à la réparation de la chromatine et à la réponse immunitaire ou au stress. Dans le but de compléter cette analyse transcriptomique, une étude protéomique a été réalisée. Les protéines cellulaires ont été séparées sur des gels bi-dimensionnels. Parmi les neuf protéines identifiées en spectrométrie de masse, certaines appartiennent au cytosquelette et d’autres aux mécanismes de réponse au stress. L’intégration d’un ADN étranger au sein du génome provoque donc bien des perturbations cellulaires. L’intégration de matériel génétique additionnel ne concernant pas seulement les rétrovirus, les données obtenues lors de cette étude pourront permettre (i) de développer des stratégies de défenses contre les rétrovirus ou contre les autres maladies caractérisées par des atteintes à l’intégrité du génome, (ii) d’évaluer les risques encourus par l’intégration d’un vecteur thérapeutique dans le génome cellulaire lors des thérapies géniques ou des expériences de transfert de gènes / In numerous situations, cell genome integrity could be in danger. This is the case during gene movements (translocations, mobiles elements), during viral infections that could be sometimes integrative (AAV, HBV, HPV) or during retroviral infections. Indeed, the replication cycle of retroviruses requires a viral genome integration step. In spite of the studies on HIV infections, viral or non viral cancers and retrovirus-based gene therapy, no data are available concerning the cellular modifications induced by such chromosomal disruptions. The aim of work was to identify cellular mechanisms induced by the insertion of a non cellular DNA into the chromosomes. The integration of additional DNA was provoked by HIV-1-based lentiviral vectors. Cellular modifications only due to the integration step were isolated by comparison of integrative and non integrative vectors. Primary human dermal fibroblasts cells were selected for the study. Optimal times post infection and viral quantities were defined using kinetic integration experiments and integrated viral DNA quantifications. The study of cellular modifications induced by the integration of the foreign DNA was applied on the cellular global transcriptome and proteome. In order to perform a transcriptomic analyses, DNA microarray corresponding to the whole human genome, were used. This study revealed a strong transcriptional repression induced by the integration. Moreover, every cellular function are disturbed by the process. Finally, a network based on molecular interactions and biological functions underlined five cellular processes mostly affected by the foreign DNA integration and corresponding to the cell cycle and death, the remodelling and repair of chromatin and the immunity or stress responses. To complete this transcriptomic analyses, a proteomic study was realized. Cellular proteins were separated on 2D gels. Among the nine proteins identified by mass spectrometry, some are linked to the cytoskeleton and other to the cellular stress. Thus, integration of a foreign DNA into the genome provoked cellular perturbations. As additional DNA integration do not only concern retroviruses, data obtained during this study could allow (i) the development of defensive strategies against retroviruses or other diseases implicating the genome integrity and (ii) the evaluation of risks linked to the integration of a therapeutic vector into the genome during gene therapy and gene transfer experiments

This thesis describes the use of in vivo and in vitro models to better understand the molecular correlates of sleep and sleep deprivation. Unlike previous studies, we utilise a timecourse based experimental design throughout, which has the advantage of identifying how the abundance of molecules return to baseline following sleep deprivation. Chapter 3 outlines the transcriptome of mouse cortex collected over 54 hours from mice subjected to varied durations of sleep deprivation. The timecourse experimental design aids in the identification of genes that are induced during both spontaneous and enforced wakefulness, and facilitates the dissociation of genes whose expression is tightly linked to the current wake state of the animal from those whose expression is linked to the total amount of wakefulness recently experienced by the animal. Like previous studies, we identify several genes involved in the unfolded protein response and synaptic function that are upregulated by sleep deprivation. We also find that increasing durations of sleep deprivation progressively reduces the total number of rhythmically expressed genes in mouse cortex, with only a handful of transcripts identified as diurnal following 12 hour sleep deprivation. Chapter 4 outlines the proteomic and metabolomic effects of 12 hour sleep deprivation. Proteomic analyses indicate that the abundance of ribosomal and nucleosomal proteins is suppressed for at least 24 hours following sleep deprivation, whilst the abundance of several phosphodiesterases are acutely increased following sleep deprivation. Metabolomic analyses of sleep deprived mouse cortex identified 3 molecular species whose abundance profile implicate them as sleep homeostats. Finally, we also set out to develop an in vitro model of sleep deprivation based on the optogenetic activation of a neuroblastoma cell line, which is outlined in Chapter 5. Following several rounds of optimisation, the stable expression of an opsin was found to induce intracellular calcium spikes and immediate early gene expression during illumination. Transcriptomic profiling of illuminated SH-SY5Y cells induced large scale transcriptomic changes, and modulated the expression of genes involved in synapses, cholesterol synthesis, the molecular clock and the unfolded protein response. Although these functional classes are reminiscent of those modulated by in vivo sleep deprivation, there was only a slight enrichment of individual genes modulated by in vivo sleep deprivation amongst the blue light sensitive genes, indicating further work is required to more closely model in vivo sleep deprivation.

O comportamento social pode ser descrito como qualquer atividade de interação intraespecífica incluindo a escolha entre parceiros reprodutivos, reconhecimento da espécie, comportamento altruísta e organização da sociedade animal. Entre as espécies de animais mais sintonizadas com seu ambiente social estão os insetos que, como por exemplo nas espécies de abelhas das tribos Apini e Meliponini, apresentam um padrão complexo de socialidade conhecido como comportamento altamente eussocial. As abelhas constituem um grupo ideal para o estudo das bases da evolução deste comportamento, pois apresentam uma grande diversidade de organização social, desde espécies solitárias até altamente eussociais. Embora a evolução da eussocialidade tenha sido motivo de muitos estudos, as mudanças genéticas envolvidas nesse processo não são completamente conhecidas. Dados da literatura fornecem um ponto de partida para o entendimento da relação entre alterações gênicas específicas e a eussocialidade, mas questões fundamentais na evolução do comportamento social ainda precisam ser respondidas. Recentemente, novas tecnologias de sequenciamento têm permitido o estudo de organismos modelo e não modelo de forma mais detalhada e não direcional. Análises deste tipo são promissoras para o estudo evolutivo de características complexas como o comportamento. Neste contexto, realizamos um amplo estudo sobre as bases moleculares envolvidas em diferentes características comportamentais relacionadas à evolução da socialidade em abelhas. Para tanto, o padrão global de expressão de genes, em espécies e fases do desenvolvimento distintas, foram analisados comparativamente através de múltiplas abordagens. No Capítulo 1, utilizamos contaminantes do transcriptoma da abelha solitária Tetrapedia diversipes para analisar os recursos florais utilizados por esta espécie em suas duas gerações reprodutivas. Neste estudo concluímos que a riqueza de espécies visitadas durante a primeira geração é muito maior do que durante a segunda geração, o que está provavelmente relacionado à floração de primavera durante o primeiro período reprodutivo. No Capítulo 2, verificamos que o padrão de expressão dos genes das fêmeas fundadoras possivelmente afeta o desenvolvimento larval em T. diversipes. O padrão bivoltino de reprodução desta espécie, com diapausa em uma das gerações, pode ser importante para a evolução do comportamento social. Além disso, entre os genes possivelmente envolvidos nessa característica, podemos encontrar genes mitocondriais e lncRNAs. Os resultados obtidos no Capítulo 3 sugerem que a especialização em subcastas de operárias ocorreu posteriormente nas diferentes linhagens de abelhas, envolvendo genes específicos. No entanto, esses genes afetam processos biológicos comuns nas diferentes espécies. Por sua vez, o Capítulo 4 apresenta um método promissor para a identificação de genes comportamentais em diferentes espécies de abelhas, através de uma análise de expressão comparativa. Com base nessas análises, 787 genes comportamentais, que possivelmente fazem parte de um toolkit eussocial em abelhas, foram encontrados. O padrão de metilação desses genes, em espécies com diferentes níveis sociais, indicou ainda que o contexto genômico da metilação pode ser relevante para eussocialidade. Os resultados obtidos nesses estudos apresentam novas perspectivas metodológicas e evolutivas para o estudo da evolução do comportamento social em abelhas / The social behaviour can be widely described as any intraspecific interaction in the animal life, including but not restricted to, female choice, species recognition, altruistic behaviour and the organization of animal society. Among the animal species most attuned to their social environment are the insects that, for example, in the Apini and Meliponini tribes, present a complex behaviour known as highly eusocial. Bees are an ideal group to study the evolution of the social behaviour because they have a great diversity of social life styles that evolved independently. The tribes Apini and Meliponini comprise only highly eusocial species whereas various levels of sociality can be detected in other tribes, being most bees indeed solitary. Although the evolution of eusociality has been the subject of many studies, the genetic changes involved in the process have not been completely understood. Results from studies conducted so far provide a starting point for the connection between specific genetic alterations and the evolution of eusocial behaviour. However fundamental questions about this process are still open. Recently, new sequencing technologies have allowed genetic studies of model and non-model organisms in a deep and non-directional way, which is promising for the study of complex characteristics. Herein, we present a broad analysis of the molecular bases of different behavioural characteristics related to the evolution of sociality in bees. To that end, the global expression pattern of genes involved in different behavioural features, in a number of bee species and distinct developmental stages, was comparatively studied using multiple approaches. Through these approaches different results were obtained. In Chapter 1, we used contaminant transcripts from the solitary bee Tetrapedia diversipes to identify the plants visited by this bee, during its two reproductive generations. These contaminant transcripts revealed that the richness of plant species visited during the first reproductive generation was considerably greater than during the second generation. Which is probably related to the floral boom occurring in spring during the first reproductive period. In Chapter 2, data suggests that the expression pattern in foundresses affect larval development in T. diversipes. The bivoltinism presented by this species, with diapause in one generation, might be an important feature for the evolution of sociality. Our results suggest that mitochondrial genes and lncRNAs are involved in this reproductive pattern. Results described in Chapter 3 indicate that specialization in worker subcastes occurred posteriorly in distinct bee lineages, driven by specific genes. However, these genes affected common biological processes in the different species. In Chapter 4 is described a promising analyses method to identify, comparatively, genes involved in bee social behaviour. Using this approach, we identified 787 behavioural genes that might be involved in social behaviour of different species. The methylation pattern of these genes suggests that the DNA context in which methylation marks occur, might be especially relevant to bee sociality. Results obtained here presents new methodological and evolutionary approaches to the study of social behaviour in bees

Neste estudo foi implementado um pipeline bioinformático para processar e analisar dados de RNA-Seq total e fita-específico gerados em nosso laboratório a partir de amostras pareadas de tumor e tecido adjacente não tumoral de 14 pacientes com o objetivo de catalogar com alta-resolução a composição e alterações no transcritoma no PDAC incluindo genes codificadores e não codificadores de proteína. / In the present work, we applied a bioinformatic pipeline to process and analyse data from total RNA-seq strand-oriented generated in our laboratory from matched samples of tumor and non-tumor adjacent pancreatic tissue from 14 patients with the goal of generate a high resolution catalog of the composition and the alterations in the transcriptome of PDAC, including protein coding and non coding genes.

Le combat contre les infections bactériennes reste un enjeu majeur de santé publique notamment avec la dissémination des Entérobactéries productrices de carbapénèmases, capables d’hydrolyser l’ensemble des β-lactamines. On assiste à l’émergence de certains clones épidémiques, se distinguant par leur distribution mondiale, leur forte transmissibilité et leur capacité à persister chez les patients. L’exemple le plus parlant est le cas du clone de Klebsiella pneumoniae appartenant au « sequence type » (ST) 258 et responsable de la diffusion mondiale de la carbapénèmase KPC (Klebsiella Pneumoniae Carbapenemase) portée majoritairement par des plasmides de la famille InFIIk. Les raisons du succès de ce clone et de l’association KPC/IncFIIk/ST258 ne sont pas encore totalement élucidées. Par ailleurs, il n’existe pas de corrélation entre le niveau d’expression d’une carbapénèmase, la sensibilité in vitro de la souche vis à vis des carbapénèmes et l’efficacité clinique d’un traitement par ces molécules. Les phénomènes d’héterorésistance aux carbapénèmes sont fréquents chez les souches produisant KPC, mais l’impact clinique est inconnu. Les mécanismes de régulation de l’expression des carbapénèmases ne sont pas élucidés.Les objectifs de cette thèse résident dans l’analyse des facteurs génétiques associés à la diffusion et la persistance de clones multi-résistants ainsi que l’analyse de l’expression des β-lactamases associées.La première partie de ce travail porte sur l’analyse de l’évolution in vivo d’une souche de K. pneumoniae ST258 produisant KPC ayant persisté chez un patient pendant près de 5 ans. L’analyse comparative des génomes provenant de 17 isolats a permis de mettre en évidence une diversification génétique importante ainsi que la sélection de mutations modifiant la virulence de la souche et sa sensibilité aux antibiotiques. Afin de caractériser les raisons du succès de certains plasmides portant KPC, une analyse transcriptomique d’une souche de Escherichia coli TOP10 transformée par un plasmide multi-réplicon IncFIIk-IncFIB exprimant KPC-2, a été réalisée en présence ou non d’imipénème. Les gènes les plus exprimés dans ces conditions sont les gènes de résistance aux antibiotiques et certains gènes essentiels à la réplication du plasmide. La présence d’imipénème affecte peu la transcription des gènes plasmidiques mais induit un stress oxydatif important dans l’ensemble de la souche. Par ailleurs, l’analyse de l’expression du gène blaKPC-2 dans différentes espèces par 5’-RACE a permis de révéler que ce gène de résistance est sous la dépendance de plusieurs promoteurs, dont la force diffère selon le fond génétique. Cette caractéristique pourrait expliquer le succès de certains isoformes du transposon Tn4401 permettant une meilleure expression du gène blaKPC-2, dans certaines espèces. Les outils développés dans cette thèse ont également été appliqués à l’analyse d’un clone d’Enterobacter kobei ST125 dont la céphalosporinase naturelle ACT-28 possède une activité d’hydrolyse accrue vis à vis de l’imipénème. Enfin, l’analyse du génome de la première souche Shewanella sp. produisant une BLSE de type CTX-M-15 a permis de révéler la présence d’un nouveau variant oxacillinase chromosomique avec activité carbapénèmase, appelé OXA-535. OXA-535 est proche d’OXA-436, un autre variant carbapénèmase porté par un plasmide ayant déjà disséminé chez les Entérobactéries. L’analyse de l’environnement génétique des gènes blaOXA-535 et blaOXA-436 confirme le rôle du genre Shewanella comme progéniteur des carbapénèmases de classe D. Ce travail contribue à une meilleure compréhension de la diffusion de certains clones multi-résistants et des mécanismes contrôlant l’expression des gènes de résistance aux β-lactamines. / Multidrug resistant bacteria and in particular carbapenemase-producing Enterobacteriaceae remain a major health public challenge. Some successful clones are emerging globally, due to their high transmissibility and their ability to colonize and persist in patients over time. Genomic analyses revealed that the dissemination of KPC carbapenemase is closely related to the spread of Klebsiella pneumoniae of the sequence-type (ST) 258 and to few successful plasmids linked to IncFIIk family. However, the reasons of the association between K. pneumoniae ST258, IncFIIk plasmids and KPC that led to the rapid spread of this clone are currently unknown.Furthermore, there is no correlation between expression level of a carbapenemase-encoding gene, in vitro susceptibility to carbapenems and efficiency of a carbapenem-based treatment. Most of the time, KPC-producing K. pneumoniae exhibit a heteroresistant phenotype with carbapenems, but its clinical impact remains unknown. The mechanisms underlying the regulation of carbapenemases expression remain to be explored.The objectives of the thesis are to obtain deeper insights into genomic plasticity of carbapenemase–producing clones, and into the expression of their β-lactamases.The first part of this work was dedicated to the in vivo evolution of a single strain of KPC-producing K. pneumoniae ST258 that colonized a patient for almost 5 years. Genomic comparison of 17 isolates revealed a remarkable diversification with occurrence of several mutations with impact on bacterial virulence and susceptibility to antibiotics.Several studies extensively described the genetic structures of blaKPC-carrying plasmids, but information regarding gene expression at the whole plasmid level are lacking. Accordingly, we performed RNA-seq on Escherichia coli TOP10 transformed with an IncFIIk-IncFI blaKPC-2-carrying plasmid, with or without imipenem exposure. In both conditions, plasmid-encoded genes related to antimicrobial resistance and involved in plasmid replication were the most expressed. Imipenem exposure led to a more general response with overexpression of E. coli numerous chromosome-encoded genes involved in oxidative stress response. In addition, analysis of blaKPC-2 gene expression in several species using 5’RACE revealed the presence of several promoters whose strength depends on the bacterial genetic background. This could promote higher expression of blaKPC-2 gene and explain the association of some isoforms of Tn4401 in different species. The tools developed in the frame of this work were also applied to study a single Enterobacter kobei ST125 clone whose natural cephalosporinase (ACT-28) has increased hydrolytic activity towards imipenem. Finally, genomic analysis of the first ESBL-producing Shewanella sp. was performed. It revealed the presence of blaCTX-M-15 and blaSHV-2 genes carried on an IncA/C plasmid and a new chromosomally-encoded oxacillinase variant of OXA-48 with carbapenemase activity, called OXA-535. OXA-535 was found to be closely related to OXA-436, another carbapenemase which has recently spread in Enterobacteriaceae. Analysis of the genetic environment of both blaOXA-48-like genes confirmed the role of Shewanella spp. as progenitors of class D carbapenemases.Overall, this work contributes to a better comprehension of the diffusion of multi-drug resistant clones and of the mechanisms implicated in β-lactamase expression.

La reprogrammation, qui est la réversion d’un noyau d’un état somatique vers un état moins différencié, constitue un enjeu majeur pour la thérapie cellulaire. Cependant, les mécanismes initiaux qui président à la reprogrammation restent mal connus. Le transfert nucléaire (clonage) met à profit les propriétés de reprogrammation uniques du cytoplasme ovocytaire, et constitue une approche expérimentale intéressante pour analyser ces processus. Le but de cette thèse est d’étudier la différence de capacité de cellules fibroblastiques à être reprogrammées efficacement, en tirant partie d’une situation-modèle d'efficacité différentielle de reprogrammation après clonage chez le bovin. Ce modèle est constitué de deux lots de fibroblastes donneurs de noyaux, qui forment des embryons clonés dont la différence d’efficacité de développement à terme varie d’un facteur 8. L’analyse des cellules donneuses a montré une augmentation des anomalies de ploïdie dans les cellules à faible potentiel, et la similitude transcriptomique entre les cellules donneuses, alors que la comparaison des transcriptomes des embryonsclonés a montré des différences de reprogrammation de l’expression génique dès le stade suivant l’activation du génome embryonnaire. Des différences de méthylation de l’ADN entre cellules donneuses ont été observées dans les promoteurs de gènes candidats différentiellement reprogrammés, ainsi que dans une analyse plus globale par RRBS. Nous avons enfin étudié la distribution des cellules filles des deux premiers blastomères au stade blastocyste, la distribution « orthogonale » et l’aptitude au développement à terme des embryons de souris clonés étant liées (Liu et al., 2012). Nous avons montré l’existence de trois distributions dans les embryons fécondés mais n’avons pas observé de différence de proportions de celles-ci entre embryons bovins clonés. En conclusion, dans notre modèle, la distribution des cellules filles des deux premiers blastomères au stade blastocyste ne semble pas associée à l’efficacité de reprogrammation dans les embryons bovins clonés, contrairement aux différences épigénétiques entre cellules donneuses. / Reprogramming, which is the return of a nucleus from a somatic state to a less differentiated state, is a major issue for cell therapy. However, the initial mechanisms governing the reprogramming remain poorly understood. Nuclear transfer (cloning) takes advantage of the unique reprogramming properties of the oocyte cytoplasm, and therefore is an interesting experimental approach to analyze these processes. The aim of this thesis is to study the difference in fibroblasts’ ability to be reprogrammed by taking advantage of a model-situation of differential reprogramming efficiency after cloning in cattle. This model consists of two batches of donor fibroblasts, which form cloned embryos having an 8 fold difference in development to term efficiency. Analysis of donor cells has shown increase ploidy abnormalities in cells of low potential, and transcriptomic similarity between the donor cells, whereas comparison ofcloned embryos transcriptomes showed gene expression reprogramming differences just after embryonic genome activation. Differences in DNA methylation between donor cells were observed in the promoters of candidate genes differentially reprogrammed and in a more comprehensive analysis by RRBS. Finally we studied the distribution of the first two blastomeres’ daughter cells at the blastocyst stage, as an "orthogonal" distribution and development to term of mice cloned embryos are linked (Liu et al., 2012). We have shown the existence of three distributions in the fertilized embryos but haven’t seen any difference of proportions between bovine cloned embryos. In conclusion, in our model, the distribution of the first two blastomeres’ daughter cells at the blastocyst stage does not seem related to the reprogramming efficiency in bovine cloned embryos, unlike epigenetic differences between donor cells.

abstract: Spatial resolved detection and quantification of ribonucleic acid (RNA) molecules in single cell is crucial for the understanding of inherent biological issues, like mechanism of gene regulation or the development and maintenance of cell fate. Conventional methods for single cell RNA profiling, like single-cell RNA sequencing (scRNA-seq) or single-molecule fluorescent in situ hybridization (smFISH), suffer either from the loss of spatial information or the low detection throughput. In order to advance single-cell analysis, new approaches need to be developed with the ability to perform high-throughput detection while preserving spatial information of the subcellular location of target RNA molecules. Novel approaches for highly multiplexed single cell in situ transcriptomic analysis were developed by our group to enable single-cell comprehensive RNA profiling in their native spatial contexts. Reiterative FISH was demonstrated to be able to detect >100 RNA species in single cell in situ, while more sophisticated approaches, consecutive FISH (C-FISH) and switchable fluorescent oligonucleotide based FISH (SFO-FISH), have the potential for whole transcriptome profiling at the single molecule sensitivity. The introduction of a cleavable fluorescent tyramide even enables sensitive RNA profiling in intact tissues with high throughput. These approaches will have wide applications in studies of systems biology, molecular diagnosis and targeted therapies. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2019