The recent advent of High-Throughput Sequencing (HTS) methods has triggered a revolution in gene regulation studies. Demand has never been higher to process the immense amount of emerging data to gain insight into the regulatory mechanisms of the cell. We address this issue by describing methods to analyze, integrate and interpret HTS data from different sources. In particular, we developed and benchmarked Pyicos, a powerful toolkit that offers flexibility, versatility and efficient memory usage. We applied it to data from ChIP-Seq on progesterone receptor in breast cancer cells to gain insight into regulatory mechanisms of hormones. Moreover, we embedded Pyicos into a pipeline to integrate HTS data from different sources. In order to do so, we used data sets from ENCODE to systematically calculate signal changes between two cell lines. We thus created a model that accurately predicts the regulatory outcome of gene expression, based on epigenetic changes in a gene locus. Finally, we provide the processed data in a Biomart database to the scientific community. / La llegada reciente de nuevos métodos de High-Throughput Sequencing (HTS) ha provocado una revolución en el estudio de la regulación génica. La necesidad de procesar la inmensa cantidad de datos generados, con el objectivo de estudiar los mecanismos regulatorios en la celula, nunca ha sido mayor. En esta tesis abordamos este tema presentando métodos para analizar, integrar e interpretar datos HTS de diferentes fuentes. En particular, hemos desarollado Pyicos, un potente conjunto de herramientas que ofrece flexibilidad, versatilidad y un uso eficiente de la memoria. Lo hemos aplicado a datos de ChIP-Seq del receptor de progesterona en células de cáncer de mama con el fin de investigar los mecanismos de la regulación por hormonas. Además, hemos incorporado Pyicos en una pipeline para integrar los datos HTS de diferentes fuentes. Hemos usado los conjuntos de datos de ENCODE para calcular de forma sistemática los cambios de señal entre dos líneas celulares. De esta manera hemos logrado crear un modelo que predice con bastante precisión los cambios de la expresión génica, basándose en los cambios epigenéticos en el locus de un gen. Por último, hemos puesto los datos procesados a disposición de la comunidad científica en una base de datos Biomart.

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O trigo (Triticum aestivum L.) é uma importante cultura no Brasil. Poucas cultivares são recomendadas para produção do tipo sequeiro no Bioma Cerrado onde a escassez de água limita o rendimento de grãos. Aqui reportamos uma análise de transcriptoma do MGS1 Aliança (cultivar de trigo adaptada ao Cerrado) sob estresse de seca. Um grupo de 4.422 transcritos diferencialmente expressos foi encontrado em raízes e folhas. O número de transcritos reprimidos em raiz (1.102) foi menor que os transcritos induzidos (1.706), enquanto o oposto ocorreu em folhas (1,017 induzidos e 647 reprimidos). O número de transcritos comuns entre ambos órgaõs foi 1.249, enquanto 2.124 foram específicos para raíz e 1.049 específicos para folhas. Análises de RT-qPCR de 35 transcritos selecionados ao acaso revelou uma correlação de 0,78 com os dados de transcriptoma. Os transcritos diferencialmente expressos foram distribuídos por todos os cromossomos e componentes do genoma. O número de transcritos no genoma B foi maior do que nos genomas A e D. Ainda, um grande número de transcritos relacionados à seca foi mapeado nos cromossomos 3B, 5B e 2B. Quando consideramos ambos órgãos, 116 diferentes rotas metabólicas foram alteradas. Uma rota em comum, entre as três mais alteradas em ambos órgãos, foi o metabolismo do amido e da sacarose. A comparação de transcritos derivados de raiz e de folha permite a identificação de transcritos importantes relacionados à respota ao estresse de seca em cada um destes órgãos. Os dados obtidos, também, abrem caminho para o desenvolvimento de futuros marcadores e seleção de genes candidatos ligados à característica. Estes resultados são úteis para o entendimento de rotas metabólicas envolvidas na tolerância à seca em trigo. A informação gerada será usada, a mais longo prazo, para propósitos de transgenia. Para isto, a metodologia de duplo-haploides é desejável e uma primeira investigação sobre a eficiência de protocolo se mostrou necessária. Micrósporos são células gaméticas com capacidade de dar origem a uma nova planta via embriogênese in vitro. Plantas duplo-haploides geradas pela cultura de micrósporos isolados são completamente homozigotas e representam uma importante ferramenta para estudos genéticos e melhoramento de plantas O processo androgenético é desencadeado por diferentes pré-tratamentos de estresse, os quais são empregados para mudar os micrósporos da rota gametofítica para a rota esporofítica. Embora a cultura de micrósporos isolados tenha inúmeras vantagens, importantes limitações tem impedido sua apliação em larga escala. Diferenças genotípicas na resposta androgenética e na formação de plantas albinas ainda constituem desafios. Embora o albinismo seja principalmente uma característica genética, pré-tratamentos e meios de cultura apropriados podem evitar este fenômeno até certo ponto. A resposta androgenética de cinco genótipos de trigo brasileiro foi avaliada no presente estudo. Dois pré-tratamentos foram testados: frio (4°C) e ácido 2-hidroxinicotinico (100 mg/L). O frio foi melhor que o pré-tratamento químico, produzindo mais plantas verdes em quatro de cinco genótipos. Somente dois genótipos brasileiros tratados com ácido 2-hidroxinicotinico produziram plantas, e um deles apenas uma única planta albina. Nossos reultados mostram, também, que o meio semilíquido (contendo 10% de Ficoll) promoveu uma maior resposta androgenética que o meio líquido, aumentando o número de embriões e plantas regeneradas. / Wheat (Triticum aestivum L.) is an important crop cultivated in Brazil. Few cultivars are recommended for rainfed production in the Cerrado Biome where water scarcity limits grain yield. Here we report a transcriptome analysis of MGS1 Aliança (a wheat cultivar adapted to the Cerrado) under drought stress. A set of 4,422 differentially expressed transcripts was found in roots and leaves. The number of down-regulated transcripts in roots (1,102) was lower than the up-regulated transcripts (1,706), while the opposite occurred in leaves (1,017 induced and 647 repressed). The number of common transcripts between the two tissues was 1,249, while 2,124 were specific to roots and 1,049 specific to leaves. Quantitative RT-PCR analysis of 35 randomly selected transcripts revealed a 0.78 correlation with the transcriptome data. The differentially expressed transcripts were distributed across all chromosomes and component genomes. The number of transcripts on the B genome was greater than on the A and D genomes. Additionally, a greater number of drought related transcripts was mapped on chromosomes 3B, 5B and 5D. When considering both tissues, 116 different metabolic pathways were changed. One common pathway, among the top three changed pathways in both tissues, was starch and sucrose metabolism. The comparison of root- and leaf-derived transcripts allows the identification of important transcripts related to water stress response in each of these tissues. It also paves the way for future marker development and selection of candidate genes linked to that trait. These results are useful for understanding the metabolic pathways involved in wheat drought response. The information generated will be used for transgenic wheat purposes. For this the doubled-haploid method is desirable and an investigation about the protocol eficiency is needed. Microspores are gametic cells with capacity to give rise to a new plant via in vitro embryogenesis. Doubled haploid plants generated by isolated microspore culture are completely homozygous and represent an important tool for plant genetics and breeding research. This process is triggered by different stress pretreatments, which are employed to switch microspores from gametophytic to a sporophytic pathway. Although isolated microspore culture has innumerous advantages, important limitations have prevented its application on a large scale. Genotypic differences in androgenic response and the formation of albino plants remain great challenges. Although albinism is a major genetic characteristic, appropriated pretreatments and culture medium can avoid this phenomenon to some extent. The androgenic response of five Brazilian wheat genotypes was evaluated in the present study. Two pretreatments were tested: cold (4°C) and 2-hydroxynicotinic acid (100 mg/L). Cold was better than chemical pretreatment, producing more green plants in four out of five genotypes. Only two Brazilian genotypes treated with 2-hydroxynicotinic acid produced plants, and one of them produced a single albino plant. Our results also show that semi-liquid medium (containing 10% Ficoll) promoted a higher androgenic response than did liquid medium, increasing the number of embryos and regenerated plants.

O trigo (Triticum aestivum L.) é uma importante cultura no Brasil. Poucas cultivares são recomendadas para produção do tipo sequeiro no Bioma Cerrado onde a escassez de água limita o rendimento de grãos. Aqui reportamos uma análise de transcriptoma do MGS1 Aliança (cultivar de trigo adaptada ao Cerrado) sob estresse de seca. Um grupo de 4.422 transcritos diferencialmente expressos foi encontrado em raízes e folhas. O número de transcritos reprimidos em raiz (1.102) foi menor que os transcritos induzidos (1.706), enquanto o oposto ocorreu em folhas (1,017 induzidos e 647 reprimidos). O número de transcritos comuns entre ambos órgaõs foi 1.249, enquanto 2.124 foram específicos para raíz e 1.049 específicos para folhas. Análises de RT-qPCR de 35 transcritos selecionados ao acaso revelou uma correlação de 0,78 com os dados de transcriptoma. Os transcritos diferencialmente expressos foram distribuídos por todos os cromossomos e componentes do genoma. O número de transcritos no genoma B foi maior do que nos genomas A e D. Ainda, um grande número de transcritos relacionados à seca foi mapeado nos cromossomos 3B, 5B e 2B. Quando consideramos ambos órgãos, 116 diferentes rotas metabólicas foram alteradas. Uma rota em comum, entre as três mais alteradas em ambos órgãos, foi o metabolismo do amido e da sacarose. A comparação de transcritos derivados de raiz e de folha permite a identificação de transcritos importantes relacionados à respota ao estresse de seca em cada um destes órgãos. Os dados obtidos, também, abrem caminho para o desenvolvimento de futuros marcadores e seleção de genes candidatos ligados à característica. Estes resultados são úteis para o entendimento de rotas metabólicas envolvidas na tolerância à seca em trigo. A informação gerada será usada, a mais longo prazo, para propósitos de transgenia. Para isto, a metodologia de duplo-haploides é desejável e uma primeira investigação sobre a eficiência de protocolo se mostrou necessária. Micrósporos são células gaméticas com capacidade de dar origem a uma nova planta via embriogênese in vitro. Plantas duplo-haploides geradas pela cultura de micrósporos isolados são completamente homozigotas e representam uma importante ferramenta para estudos genéticos e melhoramento de plantas O processo androgenético é desencadeado por diferentes pré-tratamentos de estresse, os quais são empregados para mudar os micrósporos da rota gametofítica para a rota esporofítica. Embora a cultura de micrósporos isolados tenha inúmeras vantagens, importantes limitações tem impedido sua apliação em larga escala. Diferenças genotípicas na resposta androgenética e na formação de plantas albinas ainda constituem desafios. Embora o albinismo seja principalmente uma característica genética, pré-tratamentos e meios de cultura apropriados podem evitar este fenômeno até certo ponto. A resposta androgenética de cinco genótipos de trigo brasileiro foi avaliada no presente estudo. Dois pré-tratamentos foram testados: frio (4°C) e ácido 2-hidroxinicotinico (100 mg/L). O frio foi melhor que o pré-tratamento químico, produzindo mais plantas verdes em quatro de cinco genótipos. Somente dois genótipos brasileiros tratados com ácido 2-hidroxinicotinico produziram plantas, e um deles apenas uma única planta albina. Nossos reultados mostram, também, que o meio semilíquido (contendo 10% de Ficoll) promoveu uma maior resposta androgenética que o meio líquido, aumentando o número de embriões e plantas regeneradas. / Wheat (Triticum aestivum L.) is an important crop cultivated in Brazil. Few cultivars are recommended for rainfed production in the Cerrado Biome where water scarcity limits grain yield. Here we report a transcriptome analysis of MGS1 Aliança (a wheat cultivar adapted to the Cerrado) under drought stress. A set of 4,422 differentially expressed transcripts was found in roots and leaves. The number of down-regulated transcripts in roots (1,102) was lower than the up-regulated transcripts (1,706), while the opposite occurred in leaves (1,017 induced and 647 repressed). The number of common transcripts between the two tissues was 1,249, while 2,124 were specific to roots and 1,049 specific to leaves. Quantitative RT-PCR analysis of 35 randomly selected transcripts revealed a 0.78 correlation with the transcriptome data. The differentially expressed transcripts were distributed across all chromosomes and component genomes. The number of transcripts on the B genome was greater than on the A and D genomes. Additionally, a greater number of drought related transcripts was mapped on chromosomes 3B, 5B and 5D. When considering both tissues, 116 different metabolic pathways were changed. One common pathway, among the top three changed pathways in both tissues, was starch and sucrose metabolism. The comparison of root- and leaf-derived transcripts allows the identification of important transcripts related to water stress response in each of these tissues. It also paves the way for future marker development and selection of candidate genes linked to that trait. These results are useful for understanding the metabolic pathways involved in wheat drought response. The information generated will be used for transgenic wheat purposes. For this the doubled-haploid method is desirable and an investigation about the protocol eficiency is needed. Microspores are gametic cells with capacity to give rise to a new plant via in vitro embryogenesis. Doubled haploid plants generated by isolated microspore culture are completely homozygous and represent an important tool for plant genetics and breeding research. This process is triggered by different stress pretreatments, which are employed to switch microspores from gametophytic to a sporophytic pathway. Although isolated microspore culture has innumerous advantages, important limitations have prevented its application on a large scale. Genotypic differences in androgenic response and the formation of albino plants remain great challenges. Although albinism is a major genetic characteristic, appropriated pretreatments and culture medium can avoid this phenomenon to some extent. The androgenic response of five Brazilian wheat genotypes was evaluated in the present study. Two pretreatments were tested: cold (4°C) and 2-hydroxynicotinic acid (100 mg/L). Cold was better than chemical pretreatment, producing more green plants in four out of five genotypes. Only two Brazilian genotypes treated with 2-hydroxynicotinic acid produced plants, and one of them produced a single albino plant. Our results also show that semi-liquid medium (containing 10% Ficoll) promoted a higher androgenic response than did liquid medium, increasing the number of embryos and regenerated plants.

Life history traits affect the timing and pattern of maturation, reproduction, and survival during an organism’s lifecycle and are the major components influencing Darwinian fitness. Co-evolved patterns of these traits are known as life history strategies (LHS) and variation occurs between individuals, populations, and species. The polymorphisms underlying LHS are important targets of natural selection, yet the underlying genes and physiological mechanisms remain largely unknown. Mapping the genetic basis of a LHS and subsequently unraveling the associated physiological mechanisms is a challenging task, as complex phenotypes are often polygenic. However, in several systems discrete LHS are maintained within the population and are inherited as a single locus with pleiotropic effects. These systems provide a promising starting point for investigation into LHS mechanisms and this thesis focuses on one such strategy - the Alba polymorphism in Colias butterflies. Alba is inherited as a single autosomal locus, expressed only in females, and simultaneously affects development rate, reproductive potential, and wing color. Alba females are white, while the alternative morph is yellow/orange. About 28 of 90 species exhibit polymorphic females, though whether the Alba mechanism and associated tradeoffs are conserved across the genus remains to be determined. In this thesis I primarily focus on the species Colias croceus and integrate results from lipidomics, transcriptomics, microscopy, and genomics to gain insights to the proximate mechanisms underlying Alba and Alba’s evolution within the genus. Lipidomics confirm that, consistent with findings in New World species, C. croceus Alba females have larger abdominal lipid stores than orange, an advantage which is temperature dependent and arises primarily due to mobilized lipids. Gene expression data suggests differences in resource allocation, with Alba females investing in reproduction rather than wing color, consistent with previous findings in other Colias species. Additionally, I identify a morphological basis for Alba’s white wing color. Alba females from C. croceus, an Old World species, and Colias eurytheme, a New World species both exhibit a significant reduction in pigment granules, the structures within the wing scale that contain pigment. This is a trait that seems to be unique to Colias as other white Pierid butterflies have an abundance of pigment granules, similar to orange females. I also map the genetic basis of Alba to a single genomic region containing an Alba specific, Jockey-like transposable element insertion. Interestingly this transposable element​ is located downstream of BarH-1, a gene known to affect pigment granule formation in Drosophila. Finally, I construct a phylogeny using a global distribution of 20 Colias species to facilitate investigations of Alba’s evolution within the genus. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript. Paper 4: Manuscript.</p>

Hypoxia pathways are associated with the pathogenesis of both ischaemic and neoplastic diseases. In response to hypoxia the transcription factor hypoxia‐inducible factor (HIF) induces the expression of hundreds of genes with diverse functions. These enable cells to adapt to low oxygen availability. To date, pan-genomic analyses of these transcriptional responses have focussed on protein-coding genes and microRNAs. However, the role of other classes of non-coding RNAs, in particular lncRNAs, in the hypoxia response is largely uncharacterised. My thesis aimed at improving understanding of the transcriptional regulation of the non-coding transcriptome in hypoxia. I performed an integrated genomic analysis of both non-coding and coding transcripts by massively parallel sequencing. This was interfaced with pan-genomic analyses of DNAse hypersensitivity and HIF, H3k4me3 and RNApol2 binding in hypoxic cells. These analyses have revealed that hypoxia profoundly regulated all RNA classes. snRNAs and tRNAs are globally downregulated in hypoxia, whilst miRNAs, mRNAs and lncRNAs are both up- and downregulated with an overall trend towards slight upregulation. In addition, a significant number of previously non-annotated (and largely hypoxia upregulated) transcripts were identified, including novel intergenic transcripts and natural antisense transcripts. HIF bound close to genes for mRNAs, miRNAs and lncRNAs that were upregulated by hypoxia, but was excluded from binding at genes for RNA classes that showed global downregulation. This suggests that HIF acts as a transcriptional activator (but not repressor), of lncRNAs as well as mRNAs and miRNAs. Consistent with direct regulation by HIF, many of these hypoxia-inducible, HIF-binding lncRNAs were downregulated following HIF knockdown. Analysis of RNApol2 binding and DNAse HSS signals at HIF transcriptional target genes indicated that HIF-dependent transcriptional activation occurs through release of RNApol2 that is pre-bound to open promoters of lncRNAs as well as mRNAs. In these datasets, NEAT1 was the most hypoxia-upregulated, HIF-targeted lncRNA in MCF-7 cells and, despite binding of both HIF-1 and HIF-2 isoforms at its promoter, was selectively regulated by HIF-2 alone. Furthermore, NEAT1 was induced by hypoxia in a wide range of breast cancer cell lines and in hypoxic xenograft models. Functionally, NEAT1 is required for the assembly of nuclear paraspeckle structures. Increased nuclear paraspeckle formation was observed in hypoxia and was dependent on both NEAT1 and HIF-2. Knockdown of hypoxia-induced NEAT1 significantly reduced cell proliferation and survival and induced apoptosis. Finally, high expression of NEAT1 correlated with poor clinical outcome in a large cohort of breast cancer patients. These findings extend the role of the hypoxic transcriptional response in cancer into the spectrum of non-coding transcripts and provide new insights into molecular roles of hypoxia-regulated lncRNAs, which may provide the basis for novel therapeutic targets in the future.

Tendons are able to undergo repeated cyclic loading in vivo without permanent deformation or mechanical failure. However, diseased, traumatised and decellularised tendons gradually lose the ability to resist load and fail because of creep deformation. The molecular basis of the mechanical properties of tendon and how cells establish and maintain these properties is poorly understood. New knowledge in this area is required to develop novel medical strategies to improve tendon repair and regeneration. Recent advances in tissue bioengineering have led to the formation of fibrin-based tendon-like tissue (‘tendon constructs’) that display the mechanical properties and ultrastructure of embryonic tendon. This thesis presents the characterisation of the tendon constructs derived from primary fibroblasts to understand the relationship between the cells and matrix during tissue development, and to establish the standard of in vitro engineered tendons. These findings facilitated protocol development to engineer human tendon-like tissue derived from stem cells. Novel findings of constructs formed from differentiated human pluripotent stem cells in feeder and feeder-free systems are presented. Fibrin gels were seeded with human dermal fibroblasts (HDF), chick tendon fibroblasts (CTF), MAN5 (Manchester, embryonic stem) cells, human embryonic stem cells (HuES7) and induced pluripotent stem cells (iPS). The gels were cultured until isometric tendon-like constructs were formed (T0) or continued for four or ten days post-formation. The mechanical properties, histology and gene expression of the constructs were analysed and compared between the constructs seeded with the aforementioned cell types. Varying the initial cell number (tested in CTF-seeded fibrin and collagen based constructs) significantly affected the final cell count and the mechanical properties of the constructs differentially at T0 and T10. A non-linear relationship exists between the initial and final cell number, and, between the initial cell number and mechanical properties. However, the results showed that cell number impacted cell-matrix stabilisation as strength per se was strongly dependent on initial cell number. Collagen-based constructs showed a significantly lower stiffness compared with fibrin-based constructs at T0 and T10. The stem cells and primary cells reproducibly underwent morphogenesis to form a 3D tissue similar to embryonic tendon in vivo expressing ECM markers such as collagens type I and III. The tissue also exhibited the ultrastructural characteristics and biomechanical profile of immature tendons. RNA seq and qPCR results demonstrated the upregulation of tendon-specific genes. Tendon-like tissue generated from human stem cells and HDFs in vitro has the potential to replace functional tissue lost through disease and to advance the understanding of the molecular basis of human tenogenesis.

This thesis is devoted to designing and applying advanced algorithmical and statistical tools for analysis of NGS data related to cancer and infection diseases. NGS data under investigation are obtained either from host samples or viral variants. Recently, random peptide phage display libraries (RPPDL) were applied to studies of host's antibody response to different diseases. We study human antibody response to breast cancer and mouse antibody response to Lyme disease by sequencing of the whole antibody repertoire profiles which are represented by RPPDL. Alternatively, instead of sequencing immune response NGS can be applied directly to a viral population within an infected host. Specifically, we analyze the following RNA viruses: the human immunodeficiency virus (HIV) and the infectious bronchitis virus (IBV). Sequencing of RNA viruses is challenging because there are many variants inside population due to high mutation rate. Our results show that NGS helps to understand RNA viruses and explore their interaction with infected hosts. NGS also helps to analyze immune response to different diseases, trace changing of immune response at different disease stages.

Cardiovascular disease is a major cause of mortality in the world, and is rapidly increasing. To understand the inflammatory response of various diseases, we have made tremendous advances in molecular and cellular research to show clear evidence that macrophages and dendritic cells play central roles in cardiovascular diseases, such as atherosclerosis and myocardial infarction. We have identified the two conventional DCs subsets (cDC1 and cDC2) in heart of healthy mouse. As expected, we found that the IRF8-expressing cDC1, but not the IRF4-expressing cDC2, was dependent on Flt3 for its development. Myocardial infarction significantly increased the infiltration of CD45+ leukocytes in the infarcted heart, including macrophages, neutrophils and DCs. Among cDC, the most significantly increased subset was the cDC2. The diphtheria toxin receptor (DTR) mediated depletion of Zbtb46-expressing DCs in myocardial infarcted mice led to a significant decrease of IL-1β expression together with an increased recruitment of leukocytes. This dramatically reduced the infarcted size and improved cardiac function, suggesting that cardiac DCs play an important role in immune cell infiltration following myocardial infarction. In another study, we developed, for the first time, a lipid probe-based flow cytometry method to analyze foam cells in atherosclerotic aorta. Our method enables to isolate foamy and non-foamy macrophages in order to perform the transcriptomic analysis of these cells. In addition, this technique allows to assess the severity of atherosclerosis and the characteristics of foam cells. RNA-seq analysis showed that lipid-laden foamy macrophages in atherosclerotic lesions clearly expressed different transcripts compared to non-foamy macrophages. Surprisingly, we found that non-foamy macrophages showed a pro-inflammatory signature reflected by the up-regulation of cytokines, such as Il1β, Tnf and Nlrp3. However, foamy macrophages up-regulated genes related to the transport and uptake of lipids (cholesterol and fatty acid). Collectively, we specifically identified the function of dendritic cells and macrophages in the pathogenesis of cardiovascular diseases. Understanding the precise role of these cells will help to develop appropriate immune-therapeutic strategy to attenuate cardiovascular disease. / Les maladies cardiovasculaires représentent un risque majeur de mortalité dans le monde et la prévalence de ces pathologies ne cesse d’augmenter. Pour comprendre la réponse inflammatoire de ces maladies, nous avons fait d’énormes progrès dans la recherche moléculaire et cellulaire afin de démontrer clairement que les macrophages et les cellules dendritiques jouent un rôle central dans l’athérosclérose et l’infarctus du myocarde, deux maladies cardiovasculaires mortelles. Nous avons trouvé que, à l’état normal, le coeur de souris abrite les deux types de cellules dendritiques classiques (cDC1 et cDC2). Conformément à ce qui est connue sur ces les cDC, nous avons constaté que les cDC1, qui expriment IRF8, dépendent du Flt3, un facteur de croissance important pour leur développement alors que les cDC2, qui expriment IRF4, sont Flt3 indépendantes. Nous avons trouvé que le nombre total leucocytes CD45+ augment dans le coeur des souris ayant subi l'infarctus du myocarde. Ces leucocytes étaient majoritairement des macrophages, des neutrophiles et des cDCs. Parmi ces dernières, les cDC2 ont connu une plus forte augmentation. Dans un premier projet, nous avons vérifié le rôle de ces cDCs dans cette pathologie. Pour ce faire, nous avons procédé à la déplétion de ces cellules en utilisant un modèle de souris exprimant le récepteur de la toxine diphtérique (DTR) au même temps que Zbtb46. L’expression spécifique de ce dernier dans les cDCs induit l’expression de la DRT et l’injection ultérieure de la toxine diphtérique provoque la déplétion des cDCs chez ces souris transgéniques. Nos travaux ont montré que la déplétion des cDCs diminue le recrutement des leucocytes et l'expression de la cytokine inflammatoire l'IL-1β dans le coeur des souris ayant subi l’infarctus du myocarde. Cela réduit considérablement l’ampleur de l'infarctus et améliore la fonction cardiaque, suggérant que les cDCs jouent un rôle important dans l'infiltration de cellules immunitaires après infarctus du myocarde. Dans une autre étude, nous avons mis au point, pour la première fois, une méthode basée sur l’utilisation de sonde lipidique pour analyser les cellules spumeuses de l'aorte par cytométrie en flux. Cette méthode permet de cibler et isoler les macrophages spumeux et non-spumeux afin d’analyser leur profile transcriptomique. En plus de caractériser ces cellules, cette technique permet d’évaluer la taille des plaques d’athérosclérose. Le séquençage des ARN (ARN-seq) a montré que les macrophages spumeux chargés de lipides dans les lésions athérosclérotiques expriment clairement différents transcrits par rapport aux macrophages non-spumeux. De manière surprenante, nous avons constaté que les macrophages nonspumeux présentaient une signature pro-inflammatoire reflétée par une régulation à la hausse de l’Il1β, le Tnf et Nlrp3. Par contre, les macrophages spumeux présentait un profile non inflammatoire caractérisé une augmentation de l’expression des gènes liés au transport et à l'absorption de lipides (cholestérol et acides gras). Contrairement à ce qui est connue jusqu’à maintenant sur le rôle pathogénique des macrophages spumeux, nos résultats montrent que ces cellules jouent un rôle plutôt protecteur dans l’athérosclérose. Collectivement, nous avons spécifiquement identifié la fonction des cDC et des macrophages dans la pathogenèse des maladies cardiovasculaires. Comprendre le rôle précis de ces cellules aidera à développer une stratégie immunothérapeutique appropriée pour atténuer les maladies cardiovasculaires.

Depuis plusieurs années, la sénescence cellulaire a été majoritairement étudiée comme un processus causé par le vieillissement des cellules et un mécanisme pour limiter la propagation des cellules précancéreuses. Cette perspective a changé suite aux publications des groupes Serrano et Keyes, qui ont démontré la présence des cellules sénescentes très tôt durant le développement embryonnaire de la souris. Dernièrement, le laboratoire de Dr Roy a identifié le pronéphros, un organe transitoire qui est remplacé par le mésonéphros (homologue du rein chez les humains) à la maturité, les fascicules du nerf olfactif ainsi que les gencives comme des zones riches en cellules sénescentes durant le développement de l’axolotl. Ces évidences suggèrent que la sénescence est une réponse biologique survenant non seulement lors du vieillissement, mais est également une réponse physiologique pouvant être activée par différents signaux présents dans son environnement. À ce jour, bien que plusieurs chercheurs ciblent l’identification des points de contrôle de la sénescence cellulaire, on connaît peu de chose sur le rôle des cellules sénescentes durant le développement embryonnaire et la manière dont celui-ci pourrait influencer la croissance et la morphogenèse des organismes. Dans la présente étude, on cherche à identifier les gènes qui sont importants dans la sénescence développementale. Les résultats de séquençage d’ARN dans le pronéphros de l’axolotl ont démontré un enrichissement significatif du gène PEBP1, qui code pour une protéine surtout connue pour son rôle inhibiteur sur la kinase Raf. De plus, nos résultats semblent démontrer une diminution de l’activité bêta galactosidase dans le pronéphros de l’axolotl en développement lorsque celui-ci est traité à la Locostatin, un inhibiteur pharmacologique qui bloque l’interaction de PEBP1 avec RAF. Nous suggérons que PEBP1 pourrait être important pour soit l’activation ou le maintien du caractère sénescent dans les organes en développement de l’axolotl. Un phénomène qui semble être important pour conserver la fonctionnalité de l’organe en transition. / For several years, cellular senescence has been mainly studied as a process caused by the aging of cells and a mechanism to limit the propagation of precancerous cells. This perspective changed following the publications of Serrano and Keyes, who demonstrated the presence of senescent cells very early in the embryonic development of mice. Recently, the laboratory of Dr. Roy identified the pronephros, a transient organ which is replaced by the mesonephros (kidney counterpart in humans) at maturity, the olfactory nerve fascicles as well as the gums as areas rich in senescent cells during the development of axolotl. The evidence suggests that senescence is not only related to the aging process, but rather a physiological response which is activated by various signals present in its environment. To date, although several researchers are targeting the identification of control points for the activation of senescence, little is known about the role of senescent cells during embryonic development and how it could influence growth and morphogenesis. In the present study, we seek to identify the genes that are important in developmental senescence. The results of RNA sequencing in the axolotl pronephros have, among other things, demonstrated a significant enrichment of the PEBP1 gene which codes for a protein that acts as an inhibitor of the Raf kinase. Our findings support the idea that cellular senescence that occurs during the embryonic development of the axolotl is dependent on PEBP1 since the pharmacological inhibitor of PEBP1 (Locostatin), which blocks the interaction of PEBP1 with RAF, seems to affect the activity of senescent beta galactosidase. We suggest that PEBP1 is necessary for either the activation or the maintenance of senescence in the pronephros during embryogenesis. We further suggest that embryonic senescent is crucial for the morphogenesis of the developing organ perhaps by keeping the organ functional during the transition.