L'expression du gène Igf2, qui est soumis à l'empreinte génomique parentale chez les mammifères, est hautement régulée au cours du développement embryonnaire et de la période périnatale grâce à divers mécanismes transcriptionnels et post-transcriptionnels. Ces mécanismes mettent à contribution de longs ARN non codants produits au sein même du locus, dont le plus connu est l'ARN H19. En utilisant une approche de complémentation génétique par un transgène H19 dans myoblastes H19 KO de souris, nous démontrons l'existence de plusieurs nouveaux promoteurs d'Igf2. L'un de ces promoteurs, qui est conservé chez l'homme, peut être activé par un ARN ectopique antisens d'H19 (lncARN 91H) en dépit d'une méthylation complète de la région de contrôle empreinte située en cis sur le même allèle. Nous montrons également que les lncARN 91H présentent une certaine spécificité tissulaire et que leur transcription peut être initiée à partir des séquences conservées CS4, CS5 et CS9 situées en aval du gène H19. Quant à l'ARN H19, qui est l'ARN non codant majeur du locus, il semble pouvoir réguler ses transcrits antisens dans les myoblastes H19 KO complémentés par le transgène H19, mais surtout il participe activement à la régulation post-transcriptionnelle du gène Igf2 chez la souris. Nous observons en effet qu'il favorise la coupure endoribonucléolytique de l'ARN Igf2 par un mécanisme qui reste à découvrir. Enfin, nous mettons en évidence l'existence d'un l'arrêt de l'élongation de la transcription du gène d'Igf2, pour lequel nous proposons un modèle de régulation faisant intervenir un autre long ARN non codant du locus: le lncARN PIHit. Au-delà des mécanismes qui restent à explorer, nos résultats renforcent l'idée que la structure tridimensionnelle de la chromatine participe à la régulation de l'expression des gènes chez les mammifères. / In mammals, the expression of the Igf2 gene, which is subject to parental genomic imprinting, is tightly regulated during embryonic development and the perinatal period through several transcriptional and post-transcriptional mechanisms. These mechanisms are involving long non-coding RNAs (lncRNAs) produced within the locus; among them the best known is probably the H19 RNA. Using a genetic complementation assay consisting in transfections of an H19 transgene into H19 KO myoblasts, we discovered several novel Igf2 promoters in the mouse. One of these promoters, that is conserved in the human, can be activated by ectopic H19 antisens RNAs (91H lncRNAs) despite a complete methylation of the Imprinting-Control Region located in cis on the same allele. We also show that the 91H lncRNAs possess some tissue-specific features and that their transcription can be initiated from the CS4, CS5 and CS9 conserved sequences located downstream of the H19 gene. On the other hand, the H19 RNA, that is the major lncRNA of the locus, appears to regulate its antisense transcripts in H19 KO myoblasts complemented with the H19 transgene, but its major function seems to be in regulating post-transcriptionally the Igf2 gene expression. Indeed, we have observed that it favours the endoribonucleolytic cleavage of the Igf2 messenger RNAs through a mechanism that remains to be elucidated. Finally, we reveal the existence of a premature transcriptional elongation stop of the Igf2 gene, for which we propose a regulation model involving another lncRNA of the locus: the PIHit lncRNA. Beyond the mechanisms that remain to be explored, our results strengthen the idea that, in mammals, the three-dimensional organization of the chromatin is involved in regulating gene expression.

RNAs não codificantes (ncRNAs) têm ganho crescente prestígio nos últimos anos devido a recentes e contínuas descobertas revelando sua diversidade e importância. Porém, a identificação dessas moléculas ainda é um problema em aberto. Em particular, Plasmodium falciparum é um desafio para a pesquisa de ncRNAs, onde poucos foram identificados até o momento. P. falciparum é o parasita que causa uma malária humana letal. A descoberta de novos ncRNAs neste organismo pode auxiliar no desenvolvimento de novos tratamentos. Este trabalho faz um estudo sobre técnicas computacionais para a predição de ncRNAs e, utilizando como objeto de estudo P. falciparum, propõe uma metodologia de predição que seja aplicável inclusive a genomas com viés composicional. A ênfase deste estudo foi a predição de ncRNAs família-específicos, utilizando o componente RNA da telomerase como objeto de estudo. Este é um importante RNA que, devido à sua alta taxa de mutação, é de difícil identificação. Este RNA ainda não foi identificado em P. falciparum. No entanto, evidências biológicas indicam que este RNA é presente, funcional e deve ser essencial ao parasita, caracterizando-se como um alvo de drogas. Além disso, foi realizado um trabalho preliminar sobre a predição de ncRNAs em geral em P. falciparum utilizando uma abordagem comparativa. / Noncoding RNAs (ncRNAs) have been receiving increasing prestige in the last years due to recent and continuous discoveries revealing their diversity and importance. However, the identification of these molecules is still an open problem. In particular, Plasmodium falciparum is a challenge for the ncRNA research, in which few ncRNAs have been identified. P. falciparum is the parasite that causes a lethal human malaria. The discovery of new ncRNAs in this organism may help in the development of new treatments. This work does a research of computational techniques for the ncRNA prediction and, by using P. falciparum as target, proposes a prediction methodology which is also applicable to compositionally biased genomes. The emphasis of this study was the prediction of family-specific ncRNAs, by using the telomerase RNA component as target. This is an important RNA that has a high mutation rate, being difficult to predict. This RNA has not been identified in P. falciparum, yet. However, biological evidences indicate this RNA is present, functional and might be essential for the parasite, being a drug target. In addition, this work presents preliminary results about the prediction of general ncRNAs in P. falciparum by using a comparative approach.

A dengue é uma infecção viral sistêmica que pode se manifestar clinicamente de diversas formas, desde febres leves a hemorragia e síndrome do choque, condições potencialmente fatais. Diversos estudos já foram publicados investigando as mudanças globais de expressão que ocorrem durante a evolução da doença nesses diferentes quadros clínicos. Porém, nenhum desses estudos analisou o papel dos RNAs não codificadores longos (lncRNAs) na progressão da doença. Neste projeto, foi realizada uma metanálise dos dados de expressão provenientes desses estudos de dengue focando na expressão de lncRNAs e seus possíveis mecanismos de regulação gênica. Foram identificados dezenas de lncRNAs cuja expressão aumenta ou diminui em pacientes infectados com dengue comparado com pessoas saudáveis. Através de análise de \"guilty-by-association\", procurou-se identificar genes codificadores de proteína possivelmente regulados por esses lncRNAs ou genes que os regulem. Nossos resultados fornecem evidência de novos mecanismos de regulação entre lncRNAs e mRNAs. / Dengue fever is a systemic viral infection that can manifest clinically in a variety of ways, from mild fever to potentially fatal conditions such as hemorrhage and shock syndrome. Several studies have already been published investigating the global changes in expression that occur during the evolution of the disease in these different clinical settings. However, none of these studies analyzed the role of long non-coding RNAs (lncRNAs) in disease progression. In this project, we performed a meta-analysis of transcriptome data obtained from these dengue studies and focused on the expression of lncRNAs and their possible mechanisms of gene regulation. Dozens of lncRNAs have been identified whose expression increases or decreases in patients infected with dengue compared to healthy individuals. Through guilty-by-association analysis, we identified several lncRNAs that possibly regulate protein coding genes. Our results provide evidence of novel regulatory mechanisms between lncRNAs and mRNAs.

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Les organismes bactériens sont en contact direct avec leur environnement et doivent donc constamment s’acclimater aux variations de celui-ci. Pour cela, plusieurs leviers de régulations peuvent être actionnés. Récemment, la régulation post-transcriptionnelle par les ARN régulateurs a été proposée comme un mécanisme de régulation rapide et peu coûteux pour la cellule. Chez le phytopathogène Dickeya dadantii, la régulation de la virulence a quasi exclusivement été étudiée au niveau transcriptionnel et l’implication des ARN régulateurs dans la virulence reste très peu connue. Pour cela, nous avons tout d’abord étudié le rôle des chaperons à ARN dans la pathogénie de D. dadantii et mis en évidence leur implication dans de nombreux facteurs de virulence comme la production d’enzyme de dégradation de la paroi végétale. Puis, nous avons développé une nouvelle méthode d’identification d’ARN à partir de données RNA-seq. Cette méthode a été développée pour tirer profit des séquençages réalisés en paired-end, permettant de séquencer les deux extrémités d’un transcrit. Son évaluation dans sa capacité à détecter de manière précise des ARN connus a montré une performance supérieure aux méthodes de détection existantes. Enfin, cette nouvelle méthode a été appliquée sur des données de séquençage de petits transcrits. Cette analyse nous a permis d’identifier plus d’un millier d’ARN régulateurs potentiels, dont plusieurs pourraient être impliqués dans la régulation de la virulence. Ces travaux ont donc permis de mettre en lumière l’existence d’une régulation post-transcriptionnelle chez D. dadantii et de proposer des pistes concernant les acteurs et mécanismes concernés / Bacterial organisms are directly exposed to environmental conditions and have to respond to environmental stress. To do so, several regulation network are known. Recently, post transcriptional regulation with small RNAs was suggested to be a fast and cheap in energy regulation mechanism. In the phytopathogen Dickeya dadantii, investigations on pathogenic process mostly focused on its control by transcriptional regulators. Knowledge of post-transcriptional regulation of the virulence factors is still in its infancy.To this end, we first studied the impact of RNA chaperones in the virulence of D. dadantii and showed that they were involved in the regulation of several virulence factors, like production of cell wall degrading enzyme. Then, we developed a new method to detect sRNAs from paired-end bacterial RNA-seq data. This method take paired end sequencing into account, which allow the sequencing of the both ends of each fragment. A comparative assessment showed that this method outperforms all the existing methods in terms of sRNA detection and boundary precision. Finally, this method was applied to sequencing data. With this analysis, more than one thousand sRNAs has been detected, with the identification of several candidates potentially involved in virulence.Thereby, this work highlight the existence of post-transcriptionnal regulation in D. dadantii and suggest candidates and mechanisms involved in this regulation

A dengue é uma infecção viral sistêmica que pode se manifestar clinicamente de diversas formas, desde febres leves a hemorragia e síndrome do choque, condições potencialmente fatais. Diversos estudos já foram publicados investigando as mudanças globais de expressão que ocorrem durante a evolução da doença nesses diferentes quadros clínicos. Porém, nenhum desses estudos analisou o papel dos RNAs não codificadores longos (lncRNAs) na progressão da doença. Neste projeto, foi realizada uma metanálise dos dados de expressão provenientes desses estudos de dengue focando na expressão de lncRNAs e seus possíveis mecanismos de regulação gênica. Foram identificados dezenas de lncRNAs cuja expressão aumenta ou diminui em pacientes infectados com dengue comparado com pessoas saudáveis. Através de análise de \"guilty-by-association\", procurou-se identificar genes codificadores de proteína possivelmente regulados por esses lncRNAs ou genes que os regulem. Nossos resultados fornecem evidência de novos mecanismos de regulação entre lncRNAs e mRNAs. / Dengue fever is a systemic viral infection that can manifest clinically in a variety of ways, from mild fever to potentially fatal conditions such as hemorrhage and shock syndrome. Several studies have already been published investigating the global changes in expression that occur during the evolution of the disease in these different clinical settings. However, none of these studies analyzed the role of long non-coding RNAs (lncRNAs) in disease progression. In this project, we performed a meta-analysis of transcriptome data obtained from these dengue studies and focused on the expression of lncRNAs and their possible mechanisms of gene regulation. Dozens of lncRNAs have been identified whose expression increases or decreases in patients infected with dengue compared to healthy individuals. Through guilty-by-association analysis, we identified several lncRNAs that possibly regulate protein coding genes. Our results provide evidence of novel regulatory mechanisms between lncRNAs and mRNAs.

Application of cytotoxic chemotherapy still remains the essential treatment strategy in advanced colorectal cancer. The intrinsic and acquired drug resistance represents one of the reasons that may even lead to failure of cancer therapy. The DNA damage response pathways have been shown to play an important role in the development of chemoresistance. There is sufficient evidence showing the high-frequency deregulated expression of many DNA repair genes across multiple cancer types. An example of such gene in colorectal cancer is MRE11, which encodes protein known as a sensor of DNA double-strand breaks. In year 2016, there was a substantial study published by our group at The Department of Molecular Biology of Cancer (IEM CAS, Prague), the study analysed the association of polymorphisms in predicted microRNA target sites of double-strand breaks (DSBs) repair genes, including MRE11, and clinical outcome and efficacy of chemotherapy in colorectal cancer. Our hypothesis, based on the mentioned study, is that specifically and exactly defined microRNAs with ability to regulate certain DNA repair proteins may not only affect the survival of colorectal cancer cells, but also the sensitivity to chemotherapy. In practical part of the submitted thesis we have identified miR-140 as a potential regulator of...

Chez tous les eucaryotes, la régulation de l’expression génique est primordiale pour le contrôle du cycle cellulaire. Un large éventail de gènes, incluant des régulateurs essentiels du cycle, mais aussi d’autre gènes impliqués dans la transduction du signal, la régulation hormonale et le métabolisme sont ainsi exprimé à certaines phases du cycle. Ces changements sont contrôlés à de multiples niveaux, notamment de façon transcriptionnelle et post-traductionnelle. Chez les mammifères, il est aujourd’hui évident que les micro ARNs contribuent à cette régulation en ciblant spécifiquement les transcrits d’un grand nombre de gènes régulés au cours du cycle. Cependant, nous n’avons que très peu d’informations à ce jour concernant le rôle des petits ARNs sur le contrôle de la prolifération cellulaire chez les plantes. Mes travaux de thèse ont permis de démontrer que la perte d’AGO1 affecte la prolifération cellulaire et l’activité du méristème racinaire. Nous avons également séquencé les transcrits, les petits ARNs et le dégradome à partir de cellules BY-2 synchronisées afin de déterminer le répertoire et la fonction des petit ARNs au cours du cycle cellulaire. / In all eukaryotes, regulated gene expression is key to orchestrate cell cycle progression. Not only genes encoding important core cell cycle regulators, but also genes of a variety of other factors involved in signal transduction, hormonal regulation and metabolic control are expressed at specific time points of the cell cycle. These changes in gene expression are controlled at multiple levels, including transcriptional and post-translational controls. In mammals, it became evident that microRNAs contribute to this regulation by targeting the transcripts of numerous cell cycle-regulated genes. However, in plants we still know little about the regulatory roles of small RNAs in the control of cell proliferation. During my thesis, I showed that depletion of Arabidopsis AGO1 impairs cell proliferation and root meristem activity. To further determine the repertoire and role of sRNAs in cell cycle regulation, we thus sequenced total RNAs and small RNAs, AGO1-associated small RNAs and the RNA degradome of synchronized BY2 cells at S-, G2-, M- and G1-phases of the cell cycle.

Comme tous les ciliés, P. tetraurelia réarrange son génome à chaque génération sexuelle pendant le développement de son macronoyau somatique ¿ partir du micronoyau germinal. Les réarrangements incluent l’excision précise de courtes séquences dérivant de transposons et appelés IES (Internal Eliminated Sequences) dont la majorité sont intragéniques. L’excision d’une fraction d’entre elles dépend de petits ARN maternels (appelés scnARN) qui sont produits à partir de tout le génome germinal pendant la méiose. Ce mécanisme pose un problème lors d’une conjugaison entre deux souches présentant des polymorphismes d’insertion : une cellule sera théoriquement incapable d’exciser une IES portée par l’allèle paternel reçu si cette IES est absente de l’allèle maternel ou si la séquence est trop divergente. Mes résultats montrent cependant que les allèles paternels divergents sont correctement excisés en utilisant les scnARN produit par la cellule paternelle. Dans le cas d’un polymorphisme absence/présence, l’IES que j’ai étudié est excisée chez 70 % des hétérozygotes F1, également via les scnARN paternels. Nous avons exploré deux hypothèses pour expliquer comment ils pouvaient agir. Il pourrait s’agir d’une programmation précoce des noyaux gamétiques ou alors d’un échange cytoplasmique des scnARN. Finalement, j’ai montré qu’un défaut de scnARN maternels n’est pas une cause possible de dysgénésie hybride. Cependant, 30 % des hétérozygotes F1 présentent une rétention variable de l’IES étudié via un mécanisme inconnu. Si cela est généralisable à toutes les IES homozygotes, alors ce mécanisme aurait un effet délétère sérieux sur les F1 et pourrait contribuer à l’isolement reproductif. / Like all ciliates, P. tetraurelia entirely rearranges its genome during development of the somatic macronucleus from the germline micronucleus, in each sexual generation. Rearrangements include the precise excision of IESs (Internal Eliminated Sequences), single-copy intervening sequences likely derived from transposon insertions. At least for a fraction of IESs, correct excision, which is required to reconstitute functional genes in the macronucleus, is thought to depend on their recognition by Piwi-bound small RNAs (called scnRNAs) produced from the maternal germline genome during meiosis. This raises a problem during conjugation between strains presenting insertion polymorphisms: a cell will be theoretically unable to excise an IES from the incoming (paternal) allele if that IES is absent from the maternal allele, or if its sequence is too divergent. Our results, however, indicate that divergent paternal alleles are correctly rearranged, using scnRNAs produced by the paternal cell. In the case of an absence/presence polymorphism, the IES we studied is excised in 70% of heterozygotes, also using paternal scnRNAs. We explored two hypotheses to explain how they can act. It could be either an early programming of the gametic nuclei or through cytoplasmic exchange of scnRNAs. My results seem to favor the latter. Overall, I showed that the lack of maternal scnRNAs is not a possible cause of hybrid dysgenesis. However, 30% of heterozygous F1 display a variable retention of the IES through an unknown mechanism. If this is true for all hemizygous IESs then it will have a strong deleterious effect on hybrid F1s and may contribute to reproductive isolation.