In eukaryotes, DNA is wrapped around a group of proteins termed histones that are required to precisely control gene expression during development. The amino acids of both the globular domains and unstructured tails of these histones can be modified by chemical moieties, such as methylation, acetylation and ubiquitination. The ‘histone code’ hypothesis proposes that specific combinations of these and other histone modifications contain transcriptional information, which guides the cell machinery to activate or repress gene expression in individual cell types. Chromatin immunoprecipitation (ChIP) experiments using undifferentiated stem cell populations have identified the genomic co-localisation of histone modifications reported to have opposing effects on transcription, which is known as bivalency. The human α-globin promoter, a well-established model for the study of transcriptional regulation, is bivalent in embryonic stem (ES) cells and this bivalency is resolved once the ES cells terminally differentiate (i.e. only activating or repressing marks remain). In a humanised mouse model, the deletion of a bone fide enhancer within the human α-globin locus results in heterogeneous expression patterns in primary erythroid cells. Notably, this correlates with an unresolved bivalent state at this promoter in terminally differentiated cells. Using this mouse model it is not feasible to ascertain whether the transcriptional heterogeneity observed in the cells lacking an α-globin enhancer is reflective of epigenetic heterogeneity (i.e. a mixed population of cells) rather than co-localisation of bivalent histone modifications within the same cells. Furthermore, the functional contribution of bivalency to development has yet to be described. To address these difficulties, I aimed to generate a fluorescent reporter system for human α-globin to facilitate the separation of transcriptionally heterogeneous erythroid cells. This model will provide material for ChIP studies on transcriptionally active and inactive populations to determine whether the epigenetic bivalency is reflective of a mixed cell population or true bivalency. In addition, I aimed to produce epigenetic editing tools to target bivalent promoters, which in combination with in vitro differentiation assays would provide an interesting framework to test the function of bivalency during development. In this study, I extensively tested gene-editing strategies for generating a fluorescent reporter knock-in in humanised mouse ES cells. I validated the suitability of humanised mouse ES cell lines for gene targeting studies and optimised a robust in vitro differentiation protocol for studying erythropoiesis. I utilised both recombineering and CRISPR/Cas9 gene editing tools in tandem with PiggyBac transposon technology, to knock-in the reporter gene. I made significant steps in gene targeting and successfully inserted the reporter downstream of the α-globin gene. I also generated a cloning system to express site-specific DNA-binding domains (TALEs) fused to epigenetic regulators with the aim to resolve bivalent histone modifications in vitro. From preliminary tests using these fusion proteins targeting Nrp1, a bivalent promoter in mES cells, I observed mild but significant changes in gene expression although histone modifications were unchanged. The various tools generated and tested in this study provide a solid foundation for future development of genetic and epigenetic editing at the human α-globin and other bivalent loci.

Magnetic resonance spectroscopy (MRS) allows the non-invasive measurement of selected biological compounds in vivo. Despite MRS proven potential it is not yet a routine clinical tool operated by clinicians. This is mainly due to the complex procedure of MRS acquisition, lack of standardisation in both acquisition and analysis protocols along with lack of a standard quality control. This thesis intended to address these issues with the focus on four metabolites glutathione, glutamate, glutamine and GABA using MEGA-PRESS pulse sequence. Recommendations on acquisition and spectra analysis is made for the MRS protocol MEGA-PRESS aiming to detect glutathione in vivo. This is based on an investigation of glutathione acquisition in vivo and in vitro and was aimed to answer the question: can glutathione be measured reliably using conventional pulse sequence PRESS or does it require editing? The results showed strong evidence of using editing in order to have a reliable glutathione concentration measurement. An analysis along with a quality control method is also presented to enable the extraction of glutamate and glutamine from a GABA-optimised MEGA-PRESS pulse sequence. This enables simultaneous measurements of GABA, glutamate and glutamine in a single acquisition. A criterion of NAA linewidth < 8 Hz and Glx CRLB < 16% were defined as optimum features in the GABA-edited spectrum for a reliable glutamate and glutamine quantification. Finally, due to the increasing interest in functional MRS of GABA using MEGAPRESS an investigation on the feasibility of measuring GABA in a functional-MRS setting was performed with recommendations on study designs and subject size. Power calculations suggest that detecting a 40% change in GABA using a 4'30" acquisition requires 9-93 subjects per group in a between-group study design and 13- 68 participants in a within-session design, depending on the region of interest. This thesis is set out in the Journal format thesis. Three introductory chapters, with each experimental study presented as a chapter and a final chapter that summarizes and discusses the work. Results in this thesis provide a basis for a standard and reliable MRS pipeline to reliably measure glutathione, glutamate, glutamine and GABA using MEGA-PRESS pulse sequence at 3 Tesla.

This thesis describes the dynamics of mitochondrial RNA-binding protein 1 and 2 (MRP1/2) complex in different cell lines of Trypanosoma brucei under an optimized immobilized condition. This study reveals the influence of RNA on the complex's dynamics. Furthermore, the function of RNA-binding complex 1 (MRB1) core has been studied via reverse genetic, biochemical and molecular techniques, with its role in RNA editing being proposed.

This thesis characterizes RNA footprints of several RNA-binding proteins (RBPs) thatare involved in U-insertion/deletion, A-to-I, and C-to-U RNA editing in Trypanosoma brucei. Relying on iCLIP data and biochemical methods it shows that two paralogs proteins from the MRB1 complex regulate distinct editing fates of the mitochondrial transcripts. Further, this thesis provides evidence where the combinatorial interplay of RBPs might fine-tune the levels of edited mRNA. Finally, the presented thesis adds to the growing evidence of the importance of RBPs in post-transcriptional gene regulation.

The function of two subunits of the putative mitochondrial RNA binding complex (MRB1) associated with RNA editing in parasitic protist Trypanosoma brucei was studied using various in vivo and in vitro methods of molecular biology.

Le cancer du sein est le cancer le plus fréquent chez la femme et représente la principale cause de mortalité liée au cancer. Le décés est habituellement causé par le développement de résistance aux traitements et la propagation métastatique de la maladie. Malgré la pertinence clinique, la complexité moléculaire de la maladie et sa dynamique restent à ce jour peu connues.Depuis longtemps, l’hétérogénéité du cancer du sein a été observée au niveau histologique et du profil évolutif clinique, et ces différences ont servi de base pour la classification de la maladie. Avec le développement des technologies à haut débit, telles que les puces à damier (microarrays) et le séquençage à haut débit, cette classification a été affinée et une complexité génétique jusqu'alors inconnue a été révélée.Des études utilisant ces techniques ont montré que des différences moléculaires existent non seulement entre les différentes patientes atteintes d’un cancer du sein (hétérogénéité inter-tumorale), mais aussi chez la même patiente (hétérogénéité intra-tumorale). En outre, l'hétérogénéité intra-tumorale peut exister non seulement entre les différentes parties d'une tumeur (hétérogénéité intra-tumorale spatiale) mais elle peut aussi résulter de l’évolution moléculaire d'une tumeur au cours du temps (hétérogénéité intra-tumorale temporelle). Cette complexité pourrait avoir un impact important sur la façon dont les patientes atteintes d’un cancer du sein sont prises en charge et traitées.La recherche que j’ai menée dans le Breast Cancer Translational Research Laboratory sous la direction du Professeur Christos Sotiriou avait deux objectifs principaux. Le premier était de déterminer l'ampleur et les implications cliniques de l'hétérogénéité intra-tumorale dans deux scénarios cliniques courants, à savoir: les cancers du sein multifocaux (MFBCs) et les cancers du sein métastatiques ER positif / HER2 négatif. Le deuxième était d'étudier l'impact de l'édition de l'ARN dans la détermination de l'hétérogénéité inter-tumorale, phénomène encore peu caractérisé. Notre recherche a notamment montré que:1) Les lésions de tous les MFBCs que l’on a étudiés partagent une origine commune. Malgré cela, et malgré des caractéristiques pathologiques similaires, chez un tiers des patientes, les lésions multifocales d’une même patiente ne partageaient aucune substitution et aucune insertion/déletion. De plus, l’hétérogénéité inter-lésion a été observée pour des mutations oncogéniques dans des gènes tels que PIK3CA, TP53, GATA3 et PTEN;2) En se concentrant sur un nombre défini de gènes associés au cancer, une concordance substantielle des mutations et du nombre de copies des gènes a été observée entre les lésions primaires et métastatiques appariées de cancers du sein ER positif / HER2 négatif. Des différences entre les lésions appariées ont cependant été trouvées pour les niveaux d’expression de certains gènes. Dans les lésions primaires, seuls les niveaux d’expression de quelques gènes et un niveau élevé d'amplification de FGFR1 ont été associés à la survie;3) L'édition de l’ARN est une source généralisée de variation du transcriptome dans le cancer du sein. Dans ce cancer, et potentiellement dans tous les cancers, l'édition de l’ARN est principalement contrôlée par deux facteurs, à savoir l'amplification de 1q et l'inflammation, qui sont toutes deux très répandues parmi les cancers humains. La magnitude de l'édition de l’ARN, en combinaison avec la conservation des sites d'édition détectés dans les tissus et les patientes, suggère qu'il pourrait y avoir des implications cliniques et thérapeutiques pour un large éventail de patientes atteintes d’un cancer.Nos résultats suggèrent qu'une caractérisation moléculaire approfondie des cancers du sein multifocaux et métastatiques est importante pour apprécier leur complexité, et que dans la recherche sur le cancer du sein, plus d’importance devrait être accordée à l'édition de l'ARN, un phénomène encore peu étudié qui pourrait influencer notre connaissance sur le développement et l'évolution de la maladie. / Breast cancer still represents the most frequently diagnosed cancer and the leading cause of cancer-related mortality in women. Death is usually caused by the development of resistance to treatments and the resulting metastatic spread of the disease. Despite the clinical relevance, little is known about the molecular complexity of the disease and its dynamics.Breast tumor heterogeneity has been observed at the level of the histology and the natural history of the disease for a long time, and these differences have served as the basis for disease classification. With the advent of high-throughput technologies, such as gene expression microarrays and massively parallel sequencing, this classification has been refined and a previously unknown genetic complexity has been revealed. Studies implementing these technologies have shown that molecular dif¬ferences exist not only between different breast cancer patients (inter-tumor heterogeneity), but also within the same patient (intra-tumor heterogeneity). Furthermore, intra-tumor heterogeneity could occur either between different regions of a tumor (spatial intra-tumor heterogeneity), or as the result of the molecular evolu¬tion of a tumor over time (temporal intra-tumor heterogeneity). This complexity might have a profound impact on the way breast cancer patients are managed and treated. The research work that I carried out in the Breast Cancer Translational Research Laboratory under the direction of Prof Christos Sotiriou had two main aims. The first was to determine the extent and the clinical implications of intra-tumor heterogeneity in two common clinical scenarios, namely: multifocal breast cancers (MFBCs) and metastatic ER positive/HER2 negative breast cancers. The second was to investigate the potential impact of yet poorly characterized phenomenon, such as RNA editing, in determining inter-tumor heterogeneity. For this purpose, I have conducted three main projects, which resulted in three manuscripts.We showed that:1) The lesions of all the investigated MFBCs shared a common origin. Despite this, and despite having similar pathological features, in up to a third of the patients the lesions of the same MFBC didn’t share any substitution/indels, and inter-lesion heterogeneity was observed for oncogenic mutation(s) in genes such as PIK3CA, TP53, GATA3, and PTEN; 2) When focusing on a defined number of cancer-associated genes, a substantial concordance for mutations and copy number aberrations could be found between primary and matched metastatic lesions of ER positive/HER2 negative breast cancers. Differences between matched pairs could however be found for the level of expressions of few genes. In primary lesions, only the expression levels of few genes and high FGFR1 amplification levels were associated with OS;3) A-to-I RNA editing is a pervasive source of transcriptome variation in breast cancer. In breast and potentially all cancers, A-to-I editing is mainly controlled by two factors, namely 1q amplification and inflammation, both of which are highly prevalent among human cancers. The wide-spread editing observed, in combination with the conservation of editing sites detected across tissues and patients, suggests that there might be clinical and therapeutic implications for a wide range of cancer patients.Our results suggest both that a thorough molecular characterization of multifocal and metastatic breast cancers is important to appreciate their genomic complexity, and that in breast cancer research more relevance should be given to RNA editing, a yet poorly investigated phenomenon that has the potential to impact the development and the evolution of the disease. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

Cette thèse propose d’étudier l’oeuvre tant cinématographique que télévisuelle d’un cinéaste singulier, Jacques Rozier, au regard des différents dialogues qu’elle engage entre l’art et la vie afin de questionner ce couple conceptuel. Auteur d’une oeuvre prolifique et figure emblématique de la Nouvelle vague avec Adieu Philippine (1963), Rozier constitue un modèle de cinéaste atypique tant par ses méthodes de création par la forme que prend son parcours du milieu des années 1950 à aujourd’hui. Son rapport à l’écriture cinématographique se caractérise notamment par une conception de la création comme aventure justifiant l’invention de conditions de tournage susceptibles de faire advenir l’imprévisible. Ces méthodes engagent ainsi un type de relation au réel, à la technique, aux acteurs, au tournage, soit plus généralement à la mise en scène, qu’il s’agira de qualifier. Naviguant au gré du vent, il ne cesse d’effectuer des va-etvient entre long et court métrage, fiction et documentaire, cinéma et télévision, culture savante et culture populaire. En s’intéressant à l’ensemble de ces objets autant du point de vue de l’étude esthétique que de l’analyse génétique en passant par la compréhension des modalités de fabrication ou de réception par le spectateur, il s’agit de définir une stylistique et d’interroger le fonctionnement des différents mécanismes d’interactions, de circulations et de débordements en jeu dans ce travail. / This doctoral dissertation aims to investigate the cinematographic as well as televisual work of a singular filmmaker, Jacques Rozier, in the light of the different dialogues it engages between art and life in order to interrogate this conceptual couple. Author of a prolific work and emblematic figure of the French New wave with Adieu Philippine (1963), Rozier represents an atypical template of filmmaker, both because of the ways he used to create and of the shape taken by its career from the mid-1950s until today. His relationship with cinematographic writing is notably characterised by the conception of creation as an adventure, justifying the invention of a shooting environment allowing the unpredictable to happen. Such methods thus engage a kind of association with reality, with technique, with performers, with shooting, infact more generally with mise-en-scène, which is going to be characterised here. Sailing with the wind, he is continually going back and forth between long and short films, between fiction and documentary, between cinema and television, between intellectual and popular culture. By focusing on all these items in terms of aesthetic study as well as genetic analysis, and through an understanding of the terms of creation or reception by the viewer, it is about defining stylistics and interrogating the operations of the different mechanisms of interactions, of motions and of overflows involved in this work.

My aim for That Dark Remembered Day was to create a work of fiction with a strong sense of the literary, one whose themes of violence, landscape and survivor guilt shone a light on the human condition, specifically the effects of post-war trauma on one family. The novel’s structure would be crucial in achieving its emotional heft, as each of the central characters is allocated at least one section, the reader experiencing events from disparate and increasingly illuminating perspectives. Fragmenting the book’s chronology (both throughout, but particularly in Part 3) by employing a technique of temporal blurring, helped to recreate a sense of disorientation in the reader, a key symptom of post-traumatic stress disorder. And despite an at times lyrical style, readability was important to me, in that I wanted the novel to retain its pace as a psychological thriller, albeit one where the reader is told what happens at the outset, the remainder of the book tasked with explaining how and why. Crucial to this thesis is the relationship I developed with my appointed editor, specifically how conflict emerged and was for the most part resolved during revision of the work. Significantly, I argue that these negotiations, together with my exploration of the author/editor relationship, contributed to my development as an author, and to the realisation of the novel. Research was conducted into the Falklands conflict by studying first-hand accounts of those who served, allowing me to blur fact with fiction, a process that delivered its own ethical challenges.

Poet/ Editor/ Publisher is an annotated edition of the selected correspondence of Sylvia Beach, publisher and owner of the Shakespeare and Company bookstore, the writer and editor Bryher (Annie Winifred Ellerman), and her partner, the poet H.D. (Hilda Doolittle). The years covered by this selection, 1918 to 1931, are some of the most prolific for these women and for modernism. Beach published James Joyce’s Ulysses, H.D. wrote several books of poetry and prose, Bryher established POOL Productions and Close Up, the first magazine devoted to film criticism, and much more. The relationships fostered among H.D., Bryher, and Beach express an unconventional model for creative production—one more concerned with helping each other than making a profit. This model is expressed not only in Bryher’s publishing endeavors and financial support of Shakespeare and Company and other artists in her sphere, but also in the well-documented sacrifices Beach made to bring out Ulysses. Chatty and endearing, the letters demonstrate the way these relationships passed seamlessly from social to professional and back again. They are full of gossip, but also valuable professional advice and encouragement. For Bryher and H.D., who lived in Territet, Switzerland, Beach provided an essential connection not only to a major center of avant-garde art, but also, and more practically, to the mechanisms of distributing modernist writing: publishers, editors, literary journals, and printers. This dissertation joins a recovery of the work of women in the early twentieth century as well as a reconsideration of the roles each woman played in developing the modernist canon. These letters offer evidence of the influence of each woman’s efforts on an international network of artists and insight into the labor behind the great works of modernism.

Pluripotente Stammzellen haben das Potential, in unterschiedliche Zelltypen zu differenzieren und können genutzt werden, um organähnliche Mikrostrukturen zu generieren. Somit können molekulare Unterschiede verschiedenster künstlich differenzierter Gewebe, etwa zwischen Mensch und Schimpanse, anhand von pluripotenten Ausgangszellen untersucht werden. Da die Genome unserer nächsten ausgestorbenen Verwandten Neandertaler und Denisovaner aus konservierter DNA in alten Knochen sequenziert wurden, könnten ebenso Unterschiede zwischen Mensch und diesen Spezies oder dem letzten gemeinsamen Vorfahren untersucht werden. Dies erfordert jedoch die Generierung neandertalisierter Stammzellen durch künstliche Integration von Neandertalerallelen in humane Stammzellen, etwa durch die CRISPR Genomeditierungstechnik. Durch CRISPR kann ein DNA-Doppelstrangbruch an einer gewünschten Stelle im Genom eingefügt werden. Die zelluläre Reparatur des Doppelstrangbruchs ermöglicht dann die Editierung des Genoms. Basierend auf einer DNA-Matrize, die die gewünschte Modifikation trägt, kann das Genom an dieser Stelle präzise editiert werden. Die Effizienz präziser Editierung ist jedoch sehr niedrig im Vergleich zu unpräziser Reparatur. Um möglichst effizient neandertalisierte Stammzellen generieren zu können, wurden im Zuge dieser Doktorarbeit Methoden entwickelt, welche die präzise Genomeditierungseffizienz drastisch steigern. Zum einen wurde aus mehreren niedermolekularen Substanzen, welche mit Proteinen der DNA-Reparaturen interagieren, ein optimierter Mix entwickelt. Weiterhin konnte durch eine Mutation in einem zentralen Reparaturprotein die Effizienz für die Editierung eines einzelnen Gens auf 87% erhöht werden. Diese hohe Effizienz ermöglicht erstmals die präzise homozygote Editierung von vier Genen auf einmal in ein und derselben Zelle