Дисертації з теми "Edition génétique"
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Castanon, velasco Oscar. "Targeting the transposable elements of the genome to enable large-scale genome editing and bio-containment technologies." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLX006.
Повний текст джерелаProgrammable and site-specific nucleases such as CRISPR-Cas9 have started a genome editing revolution, holding hopes to transform human health. Multiplexing or the ability to simultaneously introduce many distinct modifications in the genome will be required for basic and applied research. It will help to probe the physio-pathological functions of complex genetic circuits and to develop improved cell therapies or anti-viral treatments. By pushing the boundaries of genome engineering, we may reach a point where writing whole mammalian genomes will be possible. Such a feat may lead to the generation of virus-, cancer- or aging- free cell lines, universal donor cell therapies or may even open the way to de-extinction. In this doctoral research project, I outline the current state-of-the-art of multiplexed genome editing, the current limits and where such technologies could be headed in the future. We leveraged this knowledge as well as the abundant transposable elements present in our DNA to build an optimization pipeline and develop a new set of tools that enable large-scale genome editing. We achieved a high level of genome modifications up to three orders of magnitude greater than previously recorded, therefore paving the way to mammalian genome writing. In addition, through the observation of the cytotoxicity generated by multiple double-strand breaks within the genome, we developed a bio-safety switch that could potentially prevent the adverse effects of current and future cell therapies. Finally, I lay out the potential concerns and threats that such an advance in genome editing technology may be bringing and point out possible solutions to mitigate the risks
Shirazi, Parsa Hadi. "Engineering of eIF4E gene to resistance against potyviridae viruses in muskmelon using genome editing." Electronic Thesis or Diss., université Paris-Saclay, 2022. http://www.theses.fr/2022UPASB002.
Повний текст джерелаMelon (Cucumis melo L.) is a diploid plant of the Cucurbitaceae family. Since the 17th century, melon has been the object of an active varietal selection using hybridization techniques. In order to accelerate the development of varieties adapted to climatic changes and integrating new characteristics of interest, it is important to adapt to melon, the new methods of selection and genome edition. Melon is a recalcitrant species to genetic transformation. Thus, the development of a protocol for genetic transformation and seedling regeneration is a first step towards the use of the latest genome editing technologies. In the first section of this thesis, different factors affecting the efficiency of the transformation were evaluated. First, we showed that the optimal co-culture time between the explant and the inoculation medium was 20 minutes. The transformation efficiency in an agrobacteria culture at an optical density (OD600) of 0.8 was 11% higher than that of a culture with OD600 of 0.4. In a second step, other factors such as filter paper, concentration of culture medium (10 mM MES) and temperature (24 °C) had a positive effect on the transformation efficiency. The use of filter paper instead of agar to solidify the co-culture medium strongly improved the transformation efficiency. Finally, the effect of ethylene, known to inhibit genetic transformation, was evaluated by adding AVG, AgNO₃ and KMnO₄ to plant tissue culture medium. KMnO₄ was found to be the most effective product increasing the transformation efficiency by more than 50%.Once the transformation and regeneration protocol was set up, the first transgenesis experiments showed a transformation efficiency of 4.72%. 90% of the transformed plants were diploid. In order to develop potyvirus resistant melons, we initiated the editing of target amino acids in the translation initiation factor eIF4E. Targeted genomic editing was performed using the CRIPR-Cas-9 system and guide RNAs designed to target specific amino acids of eIF4E. The analysis of 2500 explants, allowed us to identify 59 transformed lines for an overall efficiency of 2.4 %. After amplification and sequencing of the eIF4E gene in these lines, we identified 17 lines presenting sequence modifications within the eIF4E gene. In T1 lines, nine alleles of eIF4E were identified. Eight alleles were predicted to be deleterious to eIF4E function. These edited lines will be evaluated for their resistance to ZYMV, WMV, CMV, PRSV
Kadner, Maxime. "Développements méthodologiques et applications en biologie translationnelle de techniques d'édition du génome chez le blé tendre." Electronic Thesis or Diss., Université Clermont Auvergne (2021-...), 2023. http://www.theses.fr/2023UCFA0029.
Повний текст джерелаA major challenge for agriculture will be its capacity to feed the world in the 21st century in the face of climate change and population growth, while meeting the need for agro-ecological transition. Bread wheat (Triticum aestivum) will be a key issue, and biotechnologies a key tool to be mobilised. Three lines of research involving the CRISPR-Cas tool were addressed in this PhD. First, we evaluated the use of alternative enzymes to Cas9, in particular for their ability to perform multiple mutations simultaneously. We tested the Cas12a enzyme, recognising a different PAM from Cas9 and generating a cohesive cleavage site, as well as the CAS9-NG enzyme, recognising a shorter PAM than Cas9. We obtained very interesting results since we were able to induce up to 16 mutations simultaneously in the same plant, which is particularly encouraging for the future use of this tool in varietal selection programmes. Secondly, we sought to carry out targeted insertion of stop codon cassettes in order to induce more precise gene silencing than is currently possible. Preliminary results obtained by transient expression and analysed by nested PCR have shown that numerous targeted insertion events can be demonstrated, that undesirable degradation events occur preferentially at the ends of the cassette compared to the ends of the genomic DNA generated by the CRISPR-Cas cut, as well as an asymmetry in the occurrence of stochastic DNA insertion events at the ends of the cassette. However, we did not succeed in regenerating a plant with a stable insertion, which implies that a joint improvement of transformation and integration efficiencies will have to be achieved to reach this goal. The results obtained have been the subject of a patent application by the industrial partner, and a publication has been written for submission after the eventual acceptance of the patent. The third axis of our thesis had an applicative vocation of the tools developed in the first two axes on the theme of disease resistance. Indeed, as the agro-ecological transition induces a drastic reduction of synthetic pesticides, a special effort must be made to improve genetic control of diseases, which implies major research efforts to understand plant-pathogen interactions. In this context, in collaboration with the MDC laboratory of the GDEC, we focused on 20 candidate genes for Fusarium head blight (FHB) susceptibility in wheat. The results obtained show that it is possible to extinguish three genes simultaneously, which was previously impossible using conventional mutagenesis techniques. The resulting plant will be phenotyped for its pathogen resistance profile. Taken together, the results of the thesis show that the CRISPR systems are capable of reaching a large number of loci simultaneously in common wheat, and that a targeted insertion of a small DNA fragment is possible but with a very low frequency in stable transformation in the current state of the technology. These results could be applied for the first time to solve the problem of phenotyping QTLs associated with weak effects. They show the need to master and develop these new technologies to answer research questions. These results are also a step towards using editing techniques to support varietal selection, for example by deleting previously identified susceptibility genes in elite varieties. Implications for breeding programmes and possible developments in the light of EU regulatory guidelines are discussed
Soury, Thomas. "Les Fêtes de l'Hymen et de l'Amour de Jean-Philippe Rameau : étude historique, génétique et critique." Thesis, Tours, 2013. http://www.theses.fr/2013TOUR2013/document.
Повний текст джерелаThis thesis focuses on Les Fêtes de l’Hymen et de l’Amour, an heroic ballet written by Jean-Philippe Rameau with a libretto by Louis de Cahusac, created on March 15th, 1747 in Versailles for the Dauphin’s second wedding. The study is about the history of the piece, exploring its various representations, its successive changes and the influence of this opera on the lyric show of the xviiith century. It is also interested in the figure of Louis de Cahusac, his theories on opera and ballet, the inspirations of his libretto drawing Egyptology and Freemasonry. It also addresses the composer’s musical treatment. Lastly, this study offers an edition of the libretto and the score accompanied by a critical apparatus and a catalog’s sources of the opera
Hébert, Sophie Clémentine. "Pour une poétique du carnet dans la littérature française du XXe siècle." Thesis, Grenoble, 2014. http://www.theses.fr/2014GRENL018.
Повний текст джерелаOriginally a medium of genesis, both textual and intellectual, an archetypal memory space, the notebook also develops, thanks to the specific writing conditions it favorises, the poetics of attention and regard from which « incident » (Barthes) style and seizure of the moment style emanate directly. The formal discontinuity that these styles create, World writing rather than writings of Self, seems, on the other hand, to encourage the poetics of the anti-journal – perceptible from both the metatextual and formal points of view. Moreover, in the notebook, specific and complex etha unfold, based upon subtle dialectics situated between the « in » and the « out ». Finally, the question of transforming a notebook into a book has to be asked, from the angle of publication as well as in it's reception : which editorial protocol should be priviledged ? What type of reader for a writer's notebook ?
Frésard, Laure. "Recherche de phénomènes singuliers impliqués dans la régulation fine de l'expression du génome chez la poule via l'utilisation de données issues du séquençage haut débit." Toulouse 3, 2014. http://thesesups.ups-tlse.fr/2472/.
Повний текст джерелаSince the beginning of domestication crosses have been used in farm animal selection in order to use genetic variability at its best to obtain phenotypes better adapted to our expectations. The main goal is to produce more, ie select animals with good reproduction capacities, growing fast and with good production performances (muscle mass, milk production, egg production. . . ). But another objective is to raise non aggressive and unstressed animals, handling environmental conditions changes without damaging the environment. Most frequently, numerous genes are implicated in phenotypic variability. One big issue is to identify factors causing genetic variability and when possible, to understand the underlying mechanisms, their application being a possible lever to optimize production. Accumulating knowledge on a subject allows defining dogmas, which constitute the bases of our "scientific beliefs". For example, it is known that in a diploid organism, in classical expression conditions, both paternal and maternal transcripts faithfully reproduce coding parts of parental DNA from which they come, in particular concerning genetic variants. However, two exceptions do exist for this rule: parental genomic imprinting and RNA editing. Genomic imprinting is an epigenetic phenomenon leading to a parent-of-origin dependent expression of some genes and the existence of such event in Birds is undergoing a strong debate in literature. RNA editing corresponds to post-transcriptional modifications on RNA without modification of DNA sequence from which it is transcribed. This thesis participates in characterizing these singular phenomena taking part in genome functioning in Chicken, through high-throughput sequencing data analyses. Two chicken lines as inbred and genetically distant as possible were chosen in order to detect numerous polymorphisms and to identify the parental origin of the allele in offspring resulting from their cross. Two reciprocal crosses were made between these lines; genome and transcripts from generated embryos were then sequenced. The analysis of these sequences, which constitutes the major part of this work, allowed us to conclude to the absence of genomic imprinting in Birds, at least in whole embryo at 4. 5 days. These results constitute the first transcriptome-wide approach to seek for genomic imprinting in Chicken. This thesis was also the occasion to characterize RNA editing at genome scale in this species. Results tend to prove that this event is not frequent in comparison to Primates, even if, notably, some edited sites are conserved. Moreover, most modifications correspond to canonical A-to-G substitutions. Finally, we confirm the tissue and stage specificity of this phenomenon
Cullot, Grégoire. "Génotoxicité des systèmes CRISPR-Cas9." Thesis, Bordeaux, 2019. http://www.theses.fr/2019BORD0344.
Повний текст джерелаGene therapy is a promising therapeutic strategy for the monogenic diseases treatment. If the first approaches, called additive, have relied on the use of viral vectors, a growing share is now turning to gene editing. Less than a decade after its characterization, the CRISPR-Cas9 system has moved gene editing to a clinical stage. However, in the same period of time, several questions have been raised regarding the genotoxicity that can be induced by Cas9. An emerging literature points to the risk of genotoxicity at the targeted site. The thesis work presented here is part of this theme. The first part of the study aimed to describe the genotoxicity induced by a single double-stranded break made by Cas9. Characterization of the effects was done both at the nucleotide level, by monitoring the HDR / InDels balance, but also at the chromosome scale. The monitoring of chromosomal integrity has brought to light a new risk of genotoxicity that was not characterized. A sensitive and specific detection system for this risk has been developed to further characterize it. The second objective was to address the limitations of unwanted genotoxicity by developing a safer and more efficient gene editing method through the use of a single single-stranded breakage by Cas9D10A-nickase
Dubruque, Julien. "Edition critique, histoire, genèse et esthétique des deux versions du Temple de la Gloire de Voltaire et Rameau." Thesis, Tours, 2014. http://www.theses.fr/2014TOUR2024/document.
Повний текст джерелаThe court commissioned Voltaire and Rameau to write Le Temple de la Gloire in celebration of the king’s victorious return from Fontenoy. Premiered in Versailles in 1745, restaged in Paris in December, the opera closed down after its critical and box-office failure. It was considerably reworked, restaged in Paris in April 1746, and then forgotten, although many of its pieces were reused in other works. It is the only Voltaire opera staged at the Royal Academy of Music. This study consists of a double critical edition of the libretto and music of the two versions, from 1745 and 1746. It traces the history of the work from 1745 to the present day (Chapter 1), discusses it genetically (Chapter 2), and analyzes its esthetic reach (Chapter 3), which is considerable. Voltaire is not a courtly flatterer here ; he does not present a model of the conquering king, but rather one of a king who makes the people happy. Thus, in 1745, Voltaire extends his dramatic reforms to opera (less love, more entertainment, Metastasian gravitas). But in the end, in 1746, he gives into Rameau, to the Royal Academy of music’s protocol, and to his audience
Bourara, Khaoula. "Mise en évidence d'un processus d'édition des ARNm rétroviraux au cours de l'expression du virus de l'immunodéficience humaine de type 1." Bordeaux 2, 2000. http://www.theses.fr/2000BOR28751.
Повний текст джерелаDahman, Yacine. "Caractérisation de la RNase P nucléaire de Candida glabrata et amélioration des outils d’édition de son génome." Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAJ053/document.
Повний текст джерелаCandida glabrata is an opportunistic pathogenic yeast, and is today the second causative agent of candidemia in Europe and North America. This yeast has many genomic peculiarities such as the presence of new structural domains within ubiquitous non-coding RNAs. The first aspect of this thesis was the study of the atypical RNA subunit of the nuclear Ribonuclease P of C. glabrata. This RNA contains three large additional domains giving the transcript an overall size more than three times larger than the average eukaryotic RNase P RNA subunits. The experiments performed led to a better understanding of the role of these additional domains and demonstrated for the first time the presence of the Rcl1 protein within the RNase P complex. Secondly, this thesis work also contributed to the improvement of existing genome editing tools in C. glabrata. New small and positively selectable integrative cassettes have been developed. These elements exhibited all the required characteristics for their use in wild-type strains and clinical isolates of C. glabrata
Castandet, Benoît. "Mécanisme et origine de l’édition des ARN messagers des mitochondries de plante." Thesis, Bordeaux 2, 2010. http://www.theses.fr/2010BOR21789/document.
Повний текст джерелаRNA editing is an exception to the central dogma of molecular biology which states that the information encoded by the gene is faithfully transmitted to the protein. The plant mitochondrial transcriptome undergoes hundreds of specific C-to-U changes by RNA editing, mainly in mRNAs. To understand the mechanism used by the plant to select the C targets on the transcript, we studied the role of the neighbors -1 and +1 nucleotides in wheat cox2 editing sites. Under this scheme, four different recognition patterns can be distinguished: (a) +1 dependency (b) -1 dependency (c) +1/-1 dependency and (d) no dependency on nearest neighbor residues. An important observation was that distal elements can influence the editing efficiency, indicating that some sites are not autonomous for the reaction. We propose that these results could be a consequence of the fate of transcripts during the different maturation steps. To test this hypothesis, we constructed intronless cox2 and rps10 genes. RNA editing was strongly reduced in these constructs, suggesting that efficient RNA processing may require a close interaction of factors engaged in different maturation processes. Our results on editing events in non coding region, particularly in introns, indicate that editing is essential for splicing by remodeling the secondary structure required to excise the intron. To gain insight into the splicing mechanism for scattered mitochondrial genes, we have settled an in organello trans-splicing assay. By this way, it should be possible to decipher the molecular determinants of the reaction and the eventual role of RNA editing in this process. Finally, we proposed a new hypothesis explaining the origin and evolution of RNA editing in plant mitochondria. We assume that the nucleo-cytoplasmic conflict was the driving force allowing the settlement of T-to-C mutations in the mitochondrial genome. The nuclear response was the correction of these mutations on the RNA, i.e. RNA editing
Jouffroy, Olivier. ""El Maquiavelismo degollado" (1636-37) de Claude Clément, édition et étude : l'évolution d'une pensée politique entre mondes ancien et moderne." Thesis, Bourgogne Franche-Comté, 2017. http://www.theses.fr/2017UBFCC023/document.
Повний текст джерелаEl maquiavelismo degollado sounds as a challenge to all statesmen who could be tempted to follow the pragmatic way shown by Machiavelli rather than by the Roman Catholic Church. Then, Claude Clément was considered as a champion of the radical line in the anti-Machiavellian school of thought; however, the book has never been republished and, therefor, is not very well known. El maquiavelismo degollado is not a unique work knowing that three different books have been published, using two different languages and that the Spanish version really seems to be a re-written text rather than a simple translation. Scattered with reproductions of foreign documents, enriched with many allusions, quotations, some of them hidden away, this work seems to be a miscellany of political theories of its time. Using digital databases, this study attempts first to establish the text of El maquiavelismo degollado respecting its complexity, then to explain the main mechanism of its evolution from one version to the other and finally to determine how other works could have influenced it
Deuring, Silvia. "Rechtliche Herausforderungen moderner Verfahren der Intervention in die menschliche Keimbahn : ein deutsch-französischer Rechtsvergleich zum Einsatz von CRISPR/Cas9 und hiPS-Zellen sowie zum Mitochondrientransfer." Thesis, Paris 1, 2019. http://www.theses.fr/2019PA01D012.
Повний текст джерелаThe discovery of new biotechnological processes calls into question the ability of the law to provide sufficient protection for human beings from the beginning of their lite. These new methods, such as the CRISPR/Cas9 method -also known as "genome editing" -mitochondrial donation, and the creation of human induced pluripotent stem cells (hiPS cells), make it possible to manipulate and influence in a fundamental way the genetic make-up of one's offspring and future generations. This thesis aims to prepare a draft law addressed to the German legislature. ln so doing, it takes into account two aspects : on the one hand, it aims to optimise, on the basis of a comparative analysis of German and French law, current German legislation by identifying possible advantages of the regulatory approach in France. On the other hand – assuming that the techniques in question can one day be applied with controllable risks – it examines, on the basis of an analysis of German constitutional law, whether such a future application could, in principle, be justified and implements these considerations by drafting a legislative proposal
Dos, Santos Thierry Proença. "De Ilhéus a Canga, de Horácio Bento de Gouveia : a narrativa e as suas (re)escritas (com uma proposta de edição crítico-genética e com uma tradução parcial do romance para francês)." Paris 3, 2007. http://www.theses.fr/2007PA030121.
Повний текст джерелаThe work of Horácio Bento de Gouveia is a cornerstone in the process of the statement of a Maderian identity and a segment of Portuguese Literature yet to be fully explored. His first novel was chosen because of its fruitful subject. It was initially entitled Ilhéus (1949, 1960) and later, after significant remodelling, called Canga (1975). In truth, besides its literary and linguistic potential which is revealing of Bento de Gouveia’s poetics, there is some rather interesting material surrounding this novel in what relates to its textual variation, its editorial history and its transversality into other artistic domains. Based on the established corpus, on the fixation and interpretation of the text of the novel and on the translation, the guideline of this research was set on the reflections about the problematics of rewriting, of literary reformulation and of transcodification into other artistic language. This work resulted in an essay where the basis for a rewriting theory was emphasized. The reformulation of the narrative mode stems, on the other hand, from the demands of the adopted medium. In volume 2, the first part entitled “Projectos” includes the proposal of a critical-genetic edition of Ilhéus I Canga, with an introduction, annotations to the novel and a glossary, a French translation of the first part of the novel and some material pertaining to the adaptations. The second part, which is entitled “Dossier”, includes a chronological synopsis of the life and works of Bento de Gouveia and various documents such as the chronicles blended into the novel, the correspondence from the editor to the author, newspaper clippings about the colonia issue and photographs
Limones, Méndez Mariana Cecilia. "Développement d’outils moléculaires et cellulaires pour générer des variétés de Pomelo « Star Ruby » ne produisant pas de Furocoumarines." Electronic Thesis or Diss., Université de Lorraine, 2019. http://www.theses.fr/2019LORR0045.
Повний текст джерелаFuranocoumarins are phenolic compounds involved in defense against herbivores. These molecules are mainly described in four botanical families. Rutaceae, one of those families, includes Citrus species. Furanocoumarins are phototoxic compounds, which can be problematic for their use in cosmetics or in phytotherapy. Furanocoumarin ingestion via citrus juice consumption, may inhibit human enzymes of detoxification, such as human CYP3A4. This can lead to drug overdoses known as the “Grapefruit Juice Effect”. This work consisted in the development of tools that will allow to generate new varieties of pomelo that no longer produce furanocoumarins by targeted genome edition. We have covered the essential steps for the implementation of a global strategy: i) reproducible methods have been developed for the production of protoplasts and cell cultures of Star Ruby grapefruit; ii) conditions for protoplast transformation by electroporation have also been developed; iii) finally, to specifically inhibit the furanocoumarin biosynthetic pathway, we chose to implement a genome editing approach using a CRISPR / Cas9 methodology. The development of the method was carried out with a gene encoding umbelliferon 6-dimethylallyltransferase. The results obtained indicate that the strategy is feasible. To strengthen the CRISPR / Cas9 strategy, we implemented a method to identify additional target genes. Using a data mining approach of available genomic and transcriptomic databases we identified 18 candidate sequences potentially involved in the furanocoumarin biosynthetic pathway. Heterologous expression of the corresponding proteins and their functional characterization made it possible to show that CYP706J12 is able to metabolize herniarin (a coumarin). This result provides elements to hypothesize about the convergent evolution of coumarin and furanocoumarin synthesis in higher plants
Youssef, Divana. "Recherches de méthodes innovantes issues des biotechnologies pour l'amélioration génétique du blé tendre (Triticum aestivum L.)." Thesis, Université Clermont Auvergne (2017-2020), 2017. http://www.theses.fr/2017CLFAC054.
Повний текст джерелаThe genetic improvement of common wheat (Triticum aestivum L.), one of the three most cultivated cereals, is of strategic interest to the food security of the world's population. This genetic improvement will require a better understanding of the molecular and physiological mechanisms involved, and will also require increased efficiency in our ability to modify finely the genome. In recent years, the major advances in biotechnology have made it possible to envisage new fields of action for a deeply understanding of agronomic traits of wheat as well as for genetic improvement, and also provide new tools for innovate in the field of genome editing. In this PhD manuscript, we sought to develop innovations for wheat improvement using three new biotechnology tools. We first demonstrated that the extinction of the pds gene by a strategy of artificial micro RNA succeeded in the obtaining of the expected phenotype and that the expression of the artificial RNA was related to this phenotype. We have begun to explore the possibility of using wheat microRNAs to achieve the same extinction, with no results at this time. We have then shown that specific cuts of a given sequence can be obtained in vivo in wheat using a meganuclease, and that when the cleavage sites frame a given sequence a deletion of the framed fragment may be obtained. We finally carried out the first tests of the CRISPR-Cas9 system in the laboratory and generated a line expressing the Cas9 transgene constitutively. Unexpected results obtained during these experiments have also made it possible to improve the process of genetic transformation of soft wheat used in the laboratory. The applications of our results can be used for gene validation experiments and a better understanding of the molecular mechanisms involved, but also in the future for wheat genome editing. Strategic choices in terms of technological development and innovation in the field of biotechnology and within the framework of the objectives of a public laboratory are discussed
Blanc, Valérie. "Editing des ARNs mitochondriaux de plantes. Approche biochimique du processus de conversion C-U. Mise en évidence d'une réaction spécifique de désamination." Bordeaux 2, 1996. http://www.theses.fr/1996BOR28421.
Повний текст джерелаLimones, Méndez Mariana Cecilia. "Développement d’outils moléculaires et cellulaires pour générer des variétés de Pomelo « Star Ruby » ne produisant pas de Furocoumarines." Thesis, Université de Lorraine, 2019. http://www.theses.fr/2019LORR0045.
Повний текст джерелаFuranocoumarins are phenolic compounds involved in defense against herbivores. These molecules are mainly described in four botanical families. Rutaceae, one of those families, includes Citrus species. Furanocoumarins are phototoxic compounds, which can be problematic for their use in cosmetics or in phytotherapy. Furanocoumarin ingestion via citrus juice consumption, may inhibit human enzymes of detoxification, such as human CYP3A4. This can lead to drug overdoses known as the “Grapefruit Juice Effect”. This work consisted in the development of tools that will allow to generate new varieties of pomelo that no longer produce furanocoumarins by targeted genome edition. We have covered the essential steps for the implementation of a global strategy: i) reproducible methods have been developed for the production of protoplasts and cell cultures of Star Ruby grapefruit; ii) conditions for protoplast transformation by electroporation have also been developed; iii) finally, to specifically inhibit the furanocoumarin biosynthetic pathway, we chose to implement a genome editing approach using a CRISPR / Cas9 methodology. The development of the method was carried out with a gene encoding umbelliferon 6-dimethylallyltransferase. The results obtained indicate that the strategy is feasible. To strengthen the CRISPR / Cas9 strategy, we implemented a method to identify additional target genes. Using a data mining approach of available genomic and transcriptomic databases we identified 18 candidate sequences potentially involved in the furanocoumarin biosynthetic pathway. Heterologous expression of the corresponding proteins and their functional characterization made it possible to show that CYP706J12 is able to metabolize herniarin (a coumarin). This result provides elements to hypothesize about the convergent evolution of coumarin and furanocoumarin synthesis in higher plants
Antoniani, Chiara. "A genome editing approach to induce fetal hemoglobin expression for the treatment of β-hemoglobinopathies". Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB077.
Повний текст джерелаΒ-hemoglobinopathies (β-thalassemias and sickle cell disease) are genetic anemias affecting thousands of newborns annually worldwide. β-thalassemias and sickle cell disease (SCD) are caused by mutations affecting the adult hemoglobin expression and are currently treated by red blood cell transfusion and iron chelation regiments. For patients affected by severe β-hemoglobinopathies, allogenic hematopoietic stem cell (HSCs) transplantation is the only definitive therapy. However, transplantation of autologous, genetically corrected HSCs represents an alternative therapy for patients lacking a suitable HSC donor. Naturally occurring large deletions encompassing β- and δ-globin genes in the β-globin gene cluster, defined as Hereditary Persistence of Fetal Hemoglobin (HPFH) traits, lead to increased fetal hemoglobin (HbF) expression ameliorating both thalassemic and SCD clinical phenotypes. In this study, we integrated transcription factor binding site analysis and HPFH genetic data to identify potential HbF silencers in the β-globin locus. Based on this analysis, we designed a CRISPR/Cas9 strategy disrupting: (i) a putative δγ-intergenic HbF silencer targeted by the HbF repressor BCL11A in adult erythroblasts; (ii) the shortest deletion associated with elevated HbF levels (“Corfu” deletion) in β-thalassemic patients, encompassing the putative δγ-intergenic HbF silencer; (iii) a 13.6-kb genomic region including the δ- and β-globin genes and the putative intergenic HbF silencer. Targeting the 13.6-kb region, but not the Corfu and the putative δγ-intergenic regions, caused a robust HbF re-activation and a concomitant reduction in β-globin expression in an adult erythroid cell line and in healthy donor hematopoietic stem/progenitor cells (HSPC)-derived erythroblasts. We provided a proof of principle of this potential therapeutic strategy: disruption of the 13.6-kb region in HSPCs from SCD donors favored the β-to-γ globin switching in a significant proportion of HSPC-derived erythroblasts, leading to the amelioration of the SCD cell phenotype. Finally, we dissected the mechanisms leading to HbF de-repression demonstrating changes in the chromatin conformation and epigenetic modifications within the β-globin locus upon deletion or inversion of the 13.6-kb region. Overall, this study contributes to the knowledge of the mechanisms underlying fetal to adult hemoglobin switching, and provides clues for a genome editing approach to the treatment of SCD and β-thalassemia
Figueroa, Thomas. "Régulation et rôle d'ADAR1 dans l'hyper-édition phase-dépendante des transcrits ERL du GaHV-2 : un ARNInc antisens des pri-miARN des régions Rl." Thesis, Tours, 2016. http://www.theses.fr/2016TOUR4020/document.
Повний текст джерелаMarek’s disease virus (GaHV-2) is an "-herpesvirus that induces T-cell lymphoma in chickens. In this study, we have shown that some pri-miRNAs, which are specific of miR-M4, -M11, M31 et -M1, initiated at dispersed transcription start sites. They are located in internal positions from previously described pri-miRNAs but upstream sequences lack promoter activity, indicated a potential regulation by the upstream prmiR-M9M4, characterised during this study, or the prMeq. The 7.5 kbp gene of the ERL (Edited Repeat Long) lncRNA, which is alternatively spliced et antisense of these pri-miRNAs was defined. An extensive A-to-G hyperediting of it sequence was observed strongly linked to the lytic phase, indicated a functional repression during this phase. We showed that, like the human one, the chicken ADAR1 expression was positively controlled by the IFN response pathway et negatively by the suppressor of cytokine signaling 1 (SOCS1). Like the human et murine miR-155-5p, the chicken gga-miR-155-5p et the GaHV-2 analog mdv1-miR-M4-5p deregulate this pathway by targeting et repressing expression of SOCS1, leading to the upregulation of ADAR1
Hocq, Rémi. "Clostridium beijerinckii DSM 6423, une souche plateforme émergente pour la bioproduction de solvants." Thesis, Paris, Institut agronomique, vétérinaire et forestier de France, 2019. http://www.theses.fr/2019IAVF0026.
Повний текст джерелаClostridium beijerinckii DSM 6423 is an atypical bacterial strain which can ferment a wide array of substrates and naturally produce a solvent mixture composed of isopropanol, butanol and ethanol (IBE). Despite its potential, the prospect of using this microorganism for a biobased isopropanol synthesis process remains limited. In particular, its poor selectivity to this alcohol and its weak tolerance to the solvent mixture reduce isopropanol recoverability, although these drawbacks might be alleviated by targeted genome editing approaches. For this matter, developing genetic tools, that were until this work inexistent for this bacterium, and increasing our knowledge of its metabolism are important steps. In this context, a first IFPEN thesis did the ground work by drawing its genetic map and isolating isopropanol-tolerant mutants.Based on this context, my thesis first aimed at developing efficient targeted genetic tools for this relevant strain. Use of Dam- Dcm- plasmids permitted for the first time the successful transformation of this microorganism, enabling the development of a reusable, scarless, genome editing tool based on the CRISPR-Cas9 technology. As a proof of concept, the 2 genes CIBE_0562 (upp) and CIBE_3859 (catB) were inactivated. This second modification made the strain sensitive to thiamphenicol and allowed the use of a dual-plasmid CRISPR system that had previously been engineered in IFPEN to modify the genome of a reference solventogenic species: Clostridium acetobutylicum. After the deletion of the pNF2 endogenous plasmid, transformation efficiencies were drastically increased, resulting in a ∆catB ∆pNF2 platform strain that may be used in the future to construct an efficient strain for isopropanol production.In addition to the development of these genetic tools, my thesis also aimed at characterizing the microorganism by different approaches. Firstly, we studied the impact of several model or industrial carbon sources on the strain performances and on its metabolism. Focusing on three substrates (glucose, sucrose, beetroot molasse), a continuous fermentation process was next designed to isolate the two IBE-characteristic physiological states and coupled to a multi-omic analysis. Proteomics preliminary results highlight the severe impacts of industrial substrates on growth.Secondly, the Capp-Switch sequencing transcriptomic approach was used to map transcription start sites (TSS) at the genome scale for the DSM 6423 strain and two other reference solventogenic strains. A comparative TSS analysis notably revealed a differential regulation of the butyryl-CoA synthesis operon between the C. beijerinckii and C. acetobutylicum species.Although initially not planned at the beginning of this work, the sequencing of the AA strain, one of the mutants that were obtained in the first thesis and that is unable to produce isopropanol, as well as a targeted genome approach reproducing its phenotype in a C. beijerinckii model strain, also revealed the crucial role of σ54 in controlling solvent synthesis and alternative sugar uptake pathways.To conclude, by deepening our knowledge on C. beijerinckii DSM 6423 and by allowing targeted genome editing approaches, my thesis is involved in a series of work conducted at IFPEN that aim at making this strain an IBE fermentation model organism, in order to use it for the bioproduction of isopropanol at the industrial scale
Orsini, Sarah. "Les "Carmina" de Pascoli : édition traduite et commentée d'une sélection de poèmes latins et édition numérique d'une sélection de brouillons." Thesis, Lyon, 2019. http://www.theses.fr/2019LYSE2090.
Повний текст джерелаGiovanni Pascoli (1855-1912) is a classic of Italian literature. A bilingual poet writing in Italian and Latin. He has distinguished himself in the field of Neo-Latin literature by composing poems awarded in the certamen poeticum hoeufftianum. His Latin work, the Carminas, is almost unknown in France. The first objective of this doctoral research is to propose a bilingual edition, translated and commented, of a selection of Pascoli's Latin poems, accessible to a French audience not specialised in Latin or Italian literature. We have tried to select poems of various forms (narrative poems, epigrams, dedications odes) in order to give the reader an idea of what a complete edition of the Carmina could be like. In the introduction, we presented the biographical elements and the various aesthetic and linguistic aspects of Pascolian poetry that could facilitate the understanding of the texts. We also offer an analysis of the practice consisting in writing in Latin as a poetic experiment, and tried to situated this practice in relation to his contemporaries. We also tried as much as possible to study Pascoli's Latin poetry in relation to his Italian poetry. The second aspect of this thesis concerns the posthumous nature of this work, edited by the poet's sister, Maria Pascoli, with the help of philologist Ermenegildo Pistelli. Since the poet did not have time to propose a structure of his work in a collection, the Latin poems are organized according to thematic sections that are not always satisfactory (for example, the epigrams are published without organizational logic in a section containing all the short poems or the poems that could not be classified in the thematic sections). The digitization of all of G. Pascoli's drafts enabled us to explore the drafts of the author's plans, and we propose two possible collections in the introduction to this thesis: the first organized according to the chronology of the writing, the second organized according to the chronology of the facts represented. Our commented edition of the Carmina is presented according to the first organization, in order to offer an overview of Pascoli's writing as a process that has evolved over time. This study of the Pascholian writing process was also carried out at the poem level, in the detail of the composition. We made an XML-TEI genetic edition of a draft folder, that of the poem Crepereia Tryphaena. In this edition, we have described with great precision the writing gestures visible on the page (add, delete, substitute, move, rewrite a new version) and we have tried, when possible, to analyze the mental operations that caused these gestures. We have also tried to reconstruct the chronology of the writing of each draft, in order to create an editing prototype that allows us to read the drafts of poetry by displaying the text one writing campaign after the other and to visualize the modifications made by the author up to the scale of the letter. Thanks to this edition, we were able to analyse in detail the composition steps, the methods used by the author and the gestures made to carry out his project, and we deliver the results of this analysis at the end of the introduction of this thesis
Giovanni Pascoli (1855-1912) fa parte dei classici della letteratura italiana. Poeta bilingue scrivendo in italiano e in latino, è diventato famoso nell’ambito della letteratura neolatina per avere composto poemi premiati o lodati quasi ogni anno dal 1892 al certamenpoeticum hoeufftianum. La sua opera latina, i Carmina, è sconosciuta in Francia.Il primo obbiettivo di questa ricerca dottorale è di proporre un’edizione bilingue tradotta e commentata di una selezione dei poemi latini del Pascoli e di rendere quest’edizione accessibile a un lettorato francese che non sia specialista né di letteratura latina né di letteratura italiana. Abbiamo provato a selezionare poemi di forme varie (poemi narrativi, epigrammi, odi dedicatorie) per poter dare al lettore una visione d’insieme di quello che potrebbe essere un’edizione integrale dei Carmina. Abbiamo presentato in introduzioneelementi biografici e vari aspetti estectici e linguistici della poesia pascoliana suscettibili di agevolare la comprensione dei testi. Ci presentiamo anche un’analysi delle modalità della scrittura in latino come esperienza poetica, ambientando questa pratica nel contesto culturale in cui scriveva il Pascoli. Abbiamo inoltre provato a studiare la poesia latina del Pascoi al contatto di quella italiana.Il secondo aspetto di questa tesi riguarda il carattere postumo dei Carmina, la cui edizione princeps è stata realizzata da Maria Pascoli e Ermenegildo Pistelli. Il poeta non ha avuto il tempo di raggruppare i suoi poemi in una raccolta strutturata, quindi i Carmina sono stati organizzati in sezioni tematiche che non sono sempre soddisfacenti (per esempio, gli epigrammi sono pubblicati senza logica di organizzazione in una sezione ragruppando poemi di forma breve e poemi che non entravano nei temi delle sezioni). Grazie alla digitalizzazione dell’insieme delle carte di G. Pascoli, abbiamo potuto esplorare i tentativi di strutturazionedell’opera realizzati dal poeta, e proponiamo nell’introduzione di questa testi due strutture possibili : la prima secondo la cronologia della scrittura, e l’altra secondo la cronologia dei fatti rappresentati. Abbiamo scelto per presentare l’edizione commentata dei Carmina la prima struttura, per permettere di visualizzare la scrittura dei Carmina come un processo che ha evoluto nel tempo.Questo studio del processo della scrittura pascoliana, l’abbiamo anche portato al livello del poema, nel dettaglio della composizione. Abbiamo costituito un’edizione genetica XMLTEI di una cartella di carte, quella del poema Crepereia Tryphaena. In questa edizione, abbiamo descritto con una massima precisione possibile i gesti di scrittura visibili sulla pagina (aggiungere, cancellare, sostituire, spostare, riscrivere una nuova versione), e abbiamo tentato, quando era possibile, di analizzare le operazioni mentali che hanno provocato questi gesti. Inoltre, abbiamo provato a ricostituire la cronologia della scrittura di ogni carta, percreare un prototipo di edizione che permetta di leggere le carte di una poesia facendo apparire una stesura dopo l’altra, e permettendo di visualizzare le modifiche fino al livello della lettera. Grazie a quest’edizione abbiamo potuto analizzare precisamente le varie tappe della composizione di poesia, i metodi usati dall’autore et i gesti effettuati per condurre il suo progetto creativo. Presentiamo i risultati di quest’analisi alla fine dell’introduzione della tesi
Maroc, Laetitia. "Etude sur le changement de type sexuel et les cassures chromosomiques chez Candida glabrata A single Ho-induced doublestrand break at the MAT locus is lethal in Candida glabrata A new inducible CRISPR-Cas9 system useful for genome editing and study of double-strand break repair in Candida glabrata." Thesis, université Paris-Saclay, 2021. http://www.theses.fr/2021UPASL008.
Повний текст джерелаMating-type switching is one of the strategies developed by fungi to promote sexual reproduction and propagation. This mechanism enables one haploid cell to give rise to a cell of the opposite mating-type so that they can mate. It has been extensively studied in the sexual yeast Saccharomyces cerevisiae but little is known about why the mating-type switching components have been conserved in species like Candida glabrata, in which neither sexual reproduction nor mating-type switching is observed. We have previously shown that mating-type switching can be triggered, in C. glabrata, by expression of the endonuclease responsible of this mechanism in S. cerevisiae, but this leads to massive cell death. In this work, we studied the link existing between mating-type switching and cell death in C. glabrata
Joffredo, Thierry. "Approches biographiques de l'"Introduction à l'analyse des lignes courbes algébriques" de Gabriel Cramer." Thesis, Université de Lorraine, 2017. http://www.theses.fr/2017LORR0255/document.
Повний текст джерелаThe publication in 1750 of the Introduction à l'analyse des lignes courbes algébriques, the only published work by Gabriel Cramer, professor of mathematics at the Geneva Academy, is an important milestone in the history of geometry of curves and algebra. Almost ten years passed between the time when the Genevan wrote the first lines of his treatise on curves in the autumn of 1740 and its actual publication, making it a lifetime achievement.This book has a history, both intellectual and material, which takes place in the scientific, professional, academic and social contexts in which evolve its author and its readers. From the genesis of a handwritten text as a work in progress of which we will follow the evolutions during the process of writing and according to the events of its author's life, to the various mathematicians and historians' readings of the published text which are made in the two centuries following its publication, I would like to write here, insofar as this expression makes sense - which I shall endeavour to demonstrate - a « biography » of Gabriel Cramer's Introduction
Peyny, Maud. "L'expression du gène BCAR4 (Breast Cancer anti-estrogen Resistance 4) et son rôle dans la reproduction chez la lapine." Thesis, Tours, 2019. http://www.theses.fr/2019TOUR4031.
Повний текст джерелаThe BCAR4 gene (Breast Cancer anti-estrogen Resistance 4) has been previously characterized in cattle as a gene expressed preferentially in the oocyte and early embryo, whose inhibition alters embryonic development in vitro. However, its role in oogenesis, folliculogenesis and overall in fertility in vivo remains unknown. Since this gene is conserved in various mammals but not in rodents, the rabbit has been chosen to investigate its expression and function in vivo. By reverse transcription coupled to PCR, BCAR4 transcript is detected in the ovary when primordial follicles are formed, and in ovarian follicles at the preantral and antral stages, as well as in ovulated oocytes. Its abundance decreases after fertilization and throughout preimplantation development to disappear in the blastocyst, a typical profile for a maternal transcript.In order to elucidate the role of BCAR4 in vivo in female reproduction, rabbits carrying an altered BCAR4 gene were created and a line was generated. Both homozygous and heterozygous carriers of the genetic alteration are viable and appear healthy. The genetic alteration abolishes BCAR4 expression in ovarian follicles of homozygous animals, as the transcript abundance is down thirty-fold as compared to the wild-type phenotype. Females were phenotyped on several parameters related to reproduction. The genotype did not have a significant impact on follicular development or ovarian activity, as estimated by follicular count onto ovarian sections, anti-mullerian hormone concentration in plasma, and the response to ovarian stimulation. To evaluate their fertility and prolificacy, females were inseminated three times every six-weeks. Homozygous females had a significantly lower farrowing rate than heterozygous females, 22±7% vs 71±11% (mean±sem), while prolificacy was 1.5±0.7 vs 5.8±1.5 pups per insemination. In conclusion, BCAR4 is not essential for follicular development but the gene contributes to optimal fertility of female rabbits
Roumani, Marwa. "Les phénolamides de la tomate : développement d’une approche d’ingénierie métabolique pour l’étude de leurs fonctions in planta, et évaluation de leurs activités biologiques." Electronic Thesis or Diss., Université de Lorraine, 2021. http://www.theses.fr/2021LORR0091.
Повний текст джерелаPhenolamides are a family of specialized metabolites that accumulate in a wide variety of plant species. They have functions in flower initiation, are involved in plant responses to stress, and they are part of the composition of the walls of pollen grains. In addition, their therapeutic bioactivity has been described as an antioxidant, anti-inflammatory, anti-microbial or anti-cancer molecule. Recent laboratory work has shown that the tomato produces phenolamides, especially when it is attacked by the pest Tuta absoluta. Their role in the defense of plants remains unclear. In this context, a first objective of my thesis was to modulate the composition of phenolamides in tomatoes in order to assess the consequences of this modulation on the functioning of the plant and its ability to defend itself. A second objective was to determine the therapeutic potential of these phenolamides identified in tomatoes, by screening tests for activity. A prerequisite for the generation of plants genetically modified for their capacity to synthesize phenolamides, has been to identify new genes controlling the accumulation of phenolamides in the plant. We have exploited our RNA Seq tomato bank and work in other Solanaceae. We have identified ten candidate genes, two of which code for transcription factors and eight for acyltransferases. The functional characterization of these candidates has so far not led to the identification of any new enzymatic function associated with the phenolamide pathway. This work should be continued by broadening the spectrum of substrate tested on the identified candidates and by targeting other candidate genes. To generate the genetically modified plants, I carried out a methodological work, in the laboratory, to develop transformation and regeneration protocols on the tomato species, and the gene editing technique, (CRISPR / cas9). The techniques developed lead to a high efficiency of transformation, whether in overexpression or in gene editing. We thus generated 94 lines of tomatoes in which the expression of the putrescine hydroxycinnamoyl transferase (PHT) genes responsible for the accumulation of caffeoylputrescine was modified upward or downward, as was the expression of the transcription factor solyc06g083900 .2 likely to be involved in the accumulation of a wider set of phenolamides. The plants obtained are now available for characterization work on their metabolic content and the physiological consequences of the modifications generated. We evaluated the therapeutic potential of some tomato phenolamides induced by the herbivory of T. absoluta. Obtaining pure molecules required the help of chemists. Among the molecules evaluated, caffeoylputrescine and kukoamine A exhibit moderate antibacterial activities against Staphylococcus aureus. The tests revealed the anti-inflammatory property of caffeoylputrescine on macrophage cells. This work confirms the therapeutic potential of phenolamides. They will be prosecuted for the evaluation of their anti-cancer properties
Weber, Leslie. "New therapeutic strategies for the treatment of β-hemoglobinopathies". Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC272.
Повний текст джерелаHighly efficient curative therapeutic strategies are in great demand for patients affected by β-hemoglobinopathies, namely sickle cell disease (SCD) and β-thalassemia. Indeed, the poor access to compatible donors restrains the application of the only approved definitive therapy, the allogeneic hematopoietic stem cell (HSC) transplantation. In the first part of this thesis, we aimed at optimizing an established therapeutic alternative consisting in the autologous transplantation of lentivirus (LV)-corrected hematopoietic HSCs. The development of β-globin expressing LVs and the improvement of HSC transduction conditions have led to a clear clinical benefit for SCD and β-thalassemia patients treated with this approach in the frame of recent clinical trials. Despite these significant progresses, there is room for further improvement. Indeed, the correction of severe transfusion-dependent B-thalassemia and SCD patients requires high levels of transgene expression. The goal of this project was to select a high-titer LV able to transduce efficiently HSCs and to drive high levels of transgene expression in HSC-derived RBCs. To this purpose, we compared different combinations of regulatory elements, in order to define the minimal regulatory cassette needed for achieving high levels of globin expression in the frame of LVs. We constructed 2 mini-LCRs containing either HS2 and HS3 (total size 2.6 kb) or HS2, HS3 and HS4 (total size 3.7 kb) derived from the 16-kb Locus Control Region. These cassettes were inserted in the β-AS3 and β-AS3 HS4 LVs, respectively, driving the expression of an anti-sickling βAS3-globin transgene. First, we aimed at comparatively evaluate the transduction efficiency of β-AS3 and β-AS3 HS4 in SCD hematopoietic stem progenitor cells (HSPCs) and long term-repopulating HSCs. The second aim of the study was to assess β-AS3 and β-AS3 HS4 derived transgene expression in RBCs produced from SCD HSPCs, and to evaluate the efficacy of the best-performing LV in rescuing the SCD phenotype. The second part of this thesis aimed to develop a novel genome editing-based strategy to restore fetal γ-globin genes expression. This therapeutic approach stems from the observation that the clinical course of β-thalassemia and SCD is improved in the presence of elevated HbF levels. By using the innovative CRISPR/Cas9 technology, we aimed at disrupting repressors binding sites in the γ-globin promoters to reactivate HbF expression in SCD HSPCs-derived RBCs. Reactivating fetal γ-globin genes at their endogenous genomic locus can circumvent the difficulties associated with the relatively low LV-derived transgene expression per vector copy, likely because the low LV vector capacity allows the usage only of short DNA stretches from the LCR, arranged in a non-physiological manner. In addition, this strategy offers a potentially safer targeted approach compared to the LV-based gene addition. In SCD, this therapeutic approach can favor the anti-sickling γ-globin expression, at the expense of the mutated βS-globin, given the competition between the fetal and the adult genes for the interaction with the LCR. In a comparative approach, we intended to evaluate novel and known therapeutic targets in the γ-globin promoters. To this purpose, several gRNAs have been designed to target 3 regions of the γ-globin promoters, where variants associated with elevated HbF levels and/or binding sites for HbF repressors have been described. We aimed to screen these gRNAs in an adult erythroid cell line (HUDEP-2) and SCD HSPCs-derived RBCs in terms of HbF reactivation and correction of the patient phenotype, to select the best therapeutic target for an efficient and safe therapeutic approach for β-hemoglobinopathies
Cattenoz, Pierre. "Caractérisation de l'expression des éléments Alu et du phénomène d'édition de l'ARN chez l'humain et la souris." Phd thesis, Université de Strasbourg, 2012. http://tel.archives-ouvertes.fr/tel-00715812.
Повний текст джерелаMaikova, Anna. "The CRISPR-Cas system of human pathogen Clostridium difficile : function and regulation." Thesis, Université de Paris (2019-....), 2019. http://www.theses.fr/2019UNIP7091.
Повний текст джерелаClostridium difficile (the novel name – Clostridioides difficile) is a Gram-positive, strictly anaerobic spore forming bacterium, found in soil and aquatic environments as well as in mammalian intestinal tracts. C. difficile is one of the major pathogenic clostridia. This bacterium has become a key public health issue associated with antibiotic therapy in industrialized countries. C. difficile-associated diarrhoea is currently the most frequently occurring nosocomial diarrhoea in Europe and worldwide. Since the last decade the number of severe infection forms has been rising due to emergence of the hypervirulent and epidemic strains as ribotype 027 R20291 strain. C. difficile infection causes diarrhoea, colitis and even death. Many aspects of C. difficile pathogenesis remain poorly understood. Particularly, the molecular mechanisms of its adaptation to changing conditions inside the host are to be scrutinized. During the infection cycle C. difficile survives in bacteriophage-rich gut communities possibly by relying on some special systems that control the genetic exchanges favored within these complex environments. During the last decade, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems of adaptive prokaryotic immunity against exogenic genetic elements has become the center of interest among various anti-invader bacterial defense systems.Previous studies revealed the presence of abundant and diverse CRISPR RNAs in C. difficile. C. difficile has an original CRISPR system, which is characterized by the presence of an unusually large set of CRISPR arrays (12 arrays in the laboratory 630 strain and 9 ones in the hypervirulent R20291 strain), of two or three sets of cas genes conserved in the majority of sequenced C. difficile genomes and the prophage location of several CRISPR arrays. However, the role CRISPR-Cas plays in the physiology and infectious cycle of this important pathogen remains obscure.The general aims of this work run as follows: 1) to investigate the role and the functionality of C. difficile CRISPR-Cas system in the interactions with foreign DNA elements (such as plasmids), 2) to reveal the way C. difficile CRISPR-Cas system expression is regulated and functions in different states of bacterial culture, including its response to stresses. In the present PhD thesis the functionality of C. difficile CRISPR-Cas system was investigated (Chapter 2). Through conjugation efficiency assays defensive function (in interference) of C. difficile CRISPR-Cas system was demonstrated. The correlation between the previously known levels of expression of CRISPR RNAs and the observed levels of interference has also been shown. Moreover, through the series of interference experiments the functionality of PAMs (protospacer adjacent motifs) was confirmed, which have already been predicted in silico. Additionally, the general functional PAM consensus was determined using PAM libraries experiments. Furthermore, an adaptive function of C. difficile CRISPR-Cas system was shown for laboratory strain. The role of multiple cas operons in C. difficile CRISPR functionality is also demonstrated in this Chapter.In Chapter 3 the link between C. difficile CRISPR-Cas system and a new type I toxin-antitoxin system is demonstrated, as well as a possible co-regulation under biofilm and stress conditions of CRISPR-Cas system and these toxin-antitoxin modules. This Chapter also defines a possible role of c-di-GMP in regulation of C. difficile CRISPR-Cas system. Additionally, Chapter 4 describes the utilization of endogenous C. difficile CRISPR-Cas system as a novel tool for genome editing in C. difficile. Altogether, the obtained data highlight the original features of active C. difficile CRISPR-Cas system and demonstrate its biotechnological potential
NOWACKI, Kacper Wiktor. "La Dynamique de l'érotisme : étude comparative des Romans : La Marge d'André Pieyre de Mandiargues et la La Pornographie de Witold Gombrowicz." Doctoral thesis, Università degli studi di Bergamo, 2015. http://hdl.handle.net/10446/32799.
Повний текст джерелаLa recherche se focalise sur l’étude comparative de l’érotisme dans deux romans : La Marge d’André Pieyre de Mandiargues et La Pornographie de Witold Gombrowicz. Conformément à la nouvelle approche comparative (Apter, Casanova, Moretti) et dans une perspective culturelle et littéraire, le projet explore la façon dont l’érotisme peut être entendu (ou malentendu) dans l’histoire des idées, dans la critique littéraire et dans les œuvres littéraires. À partir d’une enquête épistémologique, de l’histoire contrastive du contexte littéraire franco-polonais et des enjeux critiques développés par Bataille, Foucault, Barthes et Deleuze, le projet montre les différences culturelles dans la représentation de l’érotisme littéraire. En outre, il compare la façon dont Mandiargues et Gombrowicz défendent la nécessité et le danger de l’érotisme dans la littérature, à travers leur écriture critique. Enfin, grâce à l’analyse des deux romans, l’étude tend à expliquer la dynamique de l’érotisme littéraire compris comme un thème tantôt descriptif tantôt narratif. Les deux romans montrent comment le rêve érotique peut être exploré à travers la temporalité narrative ou à travers l’espace et, par conséquent, comment ils peuvent conduire à des interprétations photographiques ou cinématographiques. Cette recherche vise à mettre en évidence le rôle de ces écrivains dans une discussion sur l’ars erotica contemporain dans la littérature mondiale et cherche à encourager l’étude de l’érotisme dans la littérature comparée.
Lessard, Samuel. "Deciphering causal genetic determinants of red blood cell traits." Thèse, 2017. http://hdl.handle.net/1866/19329.
Повний текст джерелаGenome-wide association studies (GWAS) have revealed several genetic variants associated with complex phenotypes. This is the case for red blood cell (RBC) traits, which are particularly amenable to GWAS as they are routinely and accurately measured. Understanding RBC trait variation is important given their significance as clinical markers and modifiers of disease severity. Notably, increased fetal hemoglobin (HbF) production in sickle cell disease (SCD) patients is associated with a higher life expectancy and decreased morbidity. Nonetheless, most variants identified through GWAS fall in non-coding regions of the human genome, increasing the difficulty of identifying causal links. The main goal of this project was to identify and characterize genes influencing complex traits, and in particular RBC phenotypes. First, I developed an approach to identify and test potential gene knockouts affecting anthropometric traits in a large sample from the general population, which did not yield significant associations. Then, I characterized the DNA methylome and transcriptome of erythroblasts differentiated ex vivo from hematopoietic progenitor stem cells (HPSC), and identified several genes potentially implicated in fetal and adult-stage erythroid programs. I also identified microRNAs (miRNA) that show specific developmental expression patterns and that are enriched in inversely expressed targets. Finally, I mapped expression quantitative trait loci (eQTL) in erythroblasts, and identify erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of RBCs. These genetic variants are associated with RBC traits and malaria susceptibly, and overlap an erythroid-specific enhancer of ATP2B4. Deletion of this regulatory element using CRISPR/Cas9 experiments in human erythroid cells minimized ATP2B4 expression and increased intracellular calcium levels. In conclusion, large and comprehensive genotyping datasets will be necessary to test the role of rare gene knockouts on complex phenotypes. The transcriptomes and DNA methylomes of erythroblasts show substantial differences correlating with their developmental stages and that may be implicated in HbF production. These results also suggest a strong implication of erythroid enhancers and miRNAs in developmental stage specificity. Finally, characterizing the erythroid-specific enhancer of ATP2B4 suggest that integrating epigenomic, transcriptomic and gene editing experiments can be a powerful approach to characterize non-coding genetic variants. These results implicate ATP2B4 in erythroid cell hydration, which is associated with malaria susceptibility and SCD severity, suggesting that therapies targeting this gene could impact diseases affecting millions of individuals worldwide.