Esta dissertação de mestrado propõe-se a apresentar um estudo sobre a obra ficcional de Seamus Deane Reading in the Dark à luz das recentes idéias sobre a redefinição do conceito de identidade norte-irlandesa. No pano de fundo deste romance autobiográfico, identificamos a presença de episódios históricos envolvendo o choque entre unionistas pró-britânicos e Nacionalistas irlandeses, que levou ao conflito conhecido como "The Troubles". Esses episódios, e suas conseqüências, são apresentados através da perspectiva de um protagonista autodiegético, que relata três décadas, de 1940 a 1960. Enquanto o personagem cresce, sua percepção obviamente vai-se alterando. O efeito final da minha leitura do romance - que foi escrito na década de 1990 - é a abertura de uma nova perspectiva, relacionada com a necessidade de redefinir questões da nacionalidade irlandesa. Reading in the Dark é um romance sobre contradições entre duas culturas que não conseguem - mas necessitam - co-existir, vistas através da perspectiva de um adolescente inteligente e bem intencionado. Este texto literário oferece uma formulação sobre os novos avanços a em relação às questões de identidade e tolerância, as quais podem ser abordadas de três formas: o conflito pode ser analisado internamente, através da oposição entre as comunidades Católicas e Protestantes, ou externamente, considerando os interesses da ilha da Irlanda, em oposição aos oitocentos anos de dominação inglesa. A terceira solução propõe uma redefinição de todos os conceitos implicados. Como conseqüência dessa crise, o romance denuncia e redefine os sistemas políticos usados como instrumentos de dominação e de manutenção e validação do choque entre as duas ideologias existentes que levaram ao sectarismo no território da Irlanda do Norte. A discussão levada a cabo nesta dissertação está baseada nos presentes debates sobre estudos culturais, especialmente como propostos por Terry Eagleton e por outros membros do "Field Day Theatre Company", que analisam as questões relativas à identidade. Esses intelectuais escolheram reavaliar as narrativas dominantes sobre a Irlanda, incluindo a formação dos mitos que motivou o acirramento dessa hostilidade contra a parte oposta. Esta dissertação está estruturada em três capítulos principais. Dois deles contextualizam o plano de fundo da narrativa e da agenda política crítica do "Field Day Theatre Company". O capítulo de análise é centrado em treze cenas fortes selecionadas do romance, as quais são comentadas a partir de considerações tecidas nos limites dos capítulos anteriores. No final do trabalho, eu espero validar a importância do romance autobiográfico de Seamus Deane Reading in the Dark no processo de reexame dos discursos que levaram à falta de comunicação entre duas comunidades que vivem em um mesmo território. / This thesis consists of a study of Seamus Deane's Reading in the Dark in the light of recent ideas regarding the redefinition of the concept of Northern Irish identity. In the background of this auto-biographical novel we identify the presence of historical episodes involving the clash between British Unionists and Irish Nationalists, which led to the conflicts known as "The Troubles." These episodes, and their consequences, are presented through the filter of an autodiegetic protagonist/narrator, through a time-span of three decades, from the 1940s to the 1960s. As the character grows, perception is obviously altered. The final effect of my reading of this novel - which was written in the 1990's - is the opening a new perspective, related to the need of redefining issues of national identity. Reading in the Dark is a novel about the contradictions between two cultures which cannot - but must - co-exist, as seen through the eyes of one growing perceptive, well-meaning intelligent young man. This literary text offers a statement about a new advance towards the issues of identity and toleration, which can be approached in three ways: the conflict can be analyzed internally, through the opposition between the Catholic and the Protestant parts of the community; or externally, considering the interests of the island of Ireland, as opposed to eight-hundred years of English domination. The third solution proposes a redefinition of all concepts implied. As a consequence of this crisis, the novel simultaneously denounces and redefines the political systems used as instruments of domination, and the maintenance and validation of the clash between the two existing ideologies that led to sectarianism within the northern territory. The discussion held in this thesis is based on the present state of the debate regarding Cultural Studies, especially as proposed by Terry Eagleton and by other members of the Field Day Theatre Company, who analyze the questions concerning identity. These intellectuals choose to revaluate the dominant narratives about Ireland, including the formation and the use made of myths that have heightened the sense of hostility against the opposite part. This thesis is structured in three main chapters. Two of them contextualize the background of the narrative and present the critical-political agenda of the Field Day Theatre Company. The chapter of analysis centers on thirteen strong scenes selected from the novel, which are woven within the framing previous chapters. At the end of the work, I hope to validate my belief in the social function of literature, by stressing the importance of Seamus Deane's Reading in the Dark in this process of re-examination of old discourses that led to the failure of communication between the two communities living in the same territory.

Orientador: Mónica Graciela Zoppi Fontana / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Estudos da Linguagem / Made available in DSpace on 2018-08-26T03:51:09Z (GMT). No. of bitstreams: 1 Favari_OdairAparecidoLourenco_M.pdf: 8308819 bytes, checksum: 784c42eb41e57e634392ae1146a213cf (MD5) Previous issue date: 2014 / Resumo: Este trabalho de mestrado tem por finalidade mostrar as diferenças encontradas na formação discursiva ¿ por meio da análise de discurso ¿ da produção textual do jornalista e escritor Fausto Wolff durante a sua colaboração no jornal O Pasquim 21. Dividida em dois momentos, a análise terá início com as crônicas produzidas no ano de 2002 e, em seguida, com as de 2003 e 2004 ¿ divididas igualmente em um montante de 13 textos para cada período. As crônicas serão analisadas de acordo com suas condições de produção levando em conta os preceitos da Análise de Discurso Francesa. Por se tratar de um momento histórico, devido a uma sucessão presidencial disputada por dois grandes partidos (PT e PSDB) que se opunham e tentavam demarcar um lugar nas urnas, o que se pretende com os textos analisados, além de definir o lugar da fala do autor e caracterizar a posição de seu discurso, é revelar através das imagens construídas no discurso de FW o político ideal / Abstract: This paper of Master¿s Degree aims to show the differences found at the discursive formation ¿ through the analysis of discourse ¿ of the textual production by the journalist and writer Fausto Wolff during his collaboration on the O Pasquim 21 newspaper. Split in two moments, the analysis will begin with the chronicles produced in 2002 and then with those from 2003 and 2004 ¿ equally split in an amount of 13 texts for each period. The chronicles will be analyzed according to their conditions of production taking into consideration the precepts of the French Analysis of Discourse. As it is a historical moment, due to a presidential succession disputed by two major parties (PT and PSDB) that were antagonists and tried to take their place at the ballot box, the analyzed texts intend, besides defining the place of the author¿s speech and characterizing the position of his discourse, to reveal the ideal politician through the images built in Fausto Wolff¿s discourse / Mestrado / Divulgação Científica e Cultural / Mestre em Divulgação Científica e Cultural

Dès le déclenchement de la mobilisation générale, le 2 septembre 1939, des centaines de détachements prévôtaux sont affectés aux unités françaises et aux armées alliées. Ils sont chargés, pendant les dix mois de la campagne de 1939-1940, du maintien de l’ordre sur les deux fronts métropolitains de la zone des armées : le Nord-Est et le front des Alpes. Incluse dans le système de contrainte qui pèse sur les cinq millions de mobilisés français, la gendarmerie prévôtale est appelée à remplir plusieurs missions. Elle traque les réfractaires à la mobilisation en lutant contre la désertion, les absences illégales et les violences des militaires. Elle assure également la gestion des prisons militaires tout en effectuant les transfèrements de détenus. Comme auxiliaire du commandement militaire, la prévôté veille à l’application des mesures organisant la nation en temps de guerre. Elle régule et discipline ainsi la circulation des militaires, surveille et restreint celle des civils afin de permettre la fluidité sur les routes empruntées par les troupes. La crainte d’une action subversive aux armées, amène aussi les prévôtés à prévenir et à contrecarrer les menées défaitistes. Le déclenchement des opérations actives, le 10 mai 1940, met la gendarmerie prévôtale à l’épreuve de la guerre de mouvement. Après le choc des Ardennes et la déroute des armées du Nord, elle se réorganise pour répondre aux tâches les plus urgentes : aiguiller la retraite des troupes, enrayer la panique et lutter contre les pillage. L’objectif est double : remplir une mission « ingrate » et mettre fin à la « légende noire » des prévôtés, héritée de la Première Guerre mondiale. / From the launching of general mobilization, on september 2nd 1939, hundreds of provost detachments are appointed to french units and to allied armies. They are loaded, during the ten months of the campaign of 1939-1940, the maintaining of order in the two fronts of the zone of armies: the North-East and the Alps. Included in the system of constraint which weighs on the five millions French mobilized, the provost gendarmerie is called to complete several missions. It tracks down recalcitrant to the mobilization and it struggles against desertion, irregular absences and soldiers violence. She also assures the management of the military prisons and prisoners' transferences. As assistant helper of the military command, the provost gendarmerie watches in the application of measurements organizing the nation in times of war. It regulates and disciplines the circulation of the soldiers, watches and restrains that of the civilians to allow fluidity on roads borrowed by troops. The fear of a subversive action in armies, also leads provost gendarmerie to warn and to prevent led defeatists. The launching of active operations, on may 10th, 1940, puts the provost gendarmerie in the test of the war of movement. After the shock of Ardennes and the crushing defeat of the armies of the North, it reorganizes itself to answer the most urgent tasks: direct the retirement of troops, check panic and struggle against depredation. Objective is double: commit a "ungrateful" mission and put an end to the «black legend » of the provost gendarmes, inherited from the First World war.

'Direct experience of God' is a term frequently used by theologians without adequate clarification regarding its meaning. The understanding thereof has become increasingly complicated by the process of secularization. In the 1960's, it was repeatedly asserted that modern people could not have direct experiences of God, albeit that one could still live by faith and by commitment to the way of Jesus in a world in which, it was asserted, "God is dead". This claim, although long predominant, has been challenged by the upsurge of interest in mysticism, both Eastern and Western, and the burgeoning of Pentecostalism and the Charismatic movement, in which circles direct experience of God was frequently claimed. If direct experience of God is something natural to humanity, interpretation of it will vary in exactly the same way as interpretation of all other human experiences. This could be a possible reason for it being so very poorly integrated into everyday life, resulting in the loss of meaning and value.

Orientador: Fabio Akcelrud Durão / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Estudos da Linguagem / Made available in DSpace on 2018-08-18T14:56:13Z (GMT). No. of bitstreams: 1 Tinti_TauanFernandes_M.pdf: 690660 bytes, checksum: f383a642a724f05ed1c7d2d14b1d9166 (MD5) Previous issue date: 2011 / Resumo: Este trabalho consiste em uma leitura de Fim de Partida, de Samuel Beckett, construída a partir da hipótese de que esta peça, com o que pode ser definido como uma recusa sistemática a tudo o que lhe venha de fora, é capaz de integrar à sua própria estrutura formal os impasses gerados pelas tentativas de interpretá-la, em um movimento que paradoxalmente fortalece cada vez mais sua lógica interna à medida que a interpretação é negada. Ao longo de três capítulos, busca-se investigar as diferentes ramificações dessa ideia como forma de esboçar a posição-limite na qual a peça se encontra: no primeiro capítulo, a hipótese em questão é desenvolvida a partir da leitura de alguns objetos de Fim de Partida, e a partir disso se argumenta que os personagens se aproximam mais de seus objetos fraturados e ausentes do que de pessoas; no segundo, a condição desses personagens é desenvolvida no sentido de um confronto entre duas formas de temporalidade, a progressão e a circularidade, submetidas ao mesmo princípio de escassez que atravessa outros níveis da peça; no terceiro capítulo, a hipótese central é a de que em Endgame são colocados em questão diversos procedimentos do humor de forma altamente destrutiva, e a significação retroativa que seus destroços passam a ter adquirido nesse processo podem ser extrapolados de modo a produzir um pequeno vislumbre, de dentro da própria peça, tanto de seu estatuto de obra de arte, quanto de sua relação complexa com a tradição / Abstract: This work consists in a reading of Samuel Beckett's Endgame built upon the hypothesis that the play, through what can be defined as a deliberate exclusion of everything external to it, is capable of integrating the resulting deadlocks from the attempts of its interpretation to its own formal structure, in a paradoxical movement that strengthens the play's internal logic through the denial of interpretation. Throughout three chapters, this work aims to explore different ramifications of that idea in order to outline the boundary represented by the play: in the first chapter, the interpretative hypothesis is developed upon a reading of the objects of Endgame, culminating in the idea that its characters are closer to the absent and fractured objects that they demand than to proper persons; in the second chapter, their condition is developed in the sense of a confrontation between two forms of temporality, progression and circularity, which are submitted to the same principle of scarcity that penetrates other levels of the play; in the third chapter, the central hypothesis is that Endgame puts into question some procedures of humor in a highly destructive manner, and that the retroactive meaning acquired by the resulting ruins of this process may be extrapolated into a glimpse, from within the play itself, both of its status as a work of art and of its complex relationship with tradition / Mestrado / Teoria e Critica Literaria / Mestre em Teoria e História Literária

Orientador: José Alves de Freitas Neto / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Filosofia e Ciências Humanas / Made available in DSpace on 2018-08-27T07:58:10Z (GMT). No. of bitstreams: 1 Silva_CaioPedrosada_D.pdf: 7283480 bytes, checksum: 6bac580ba2d433d2dfdfef8b0eebc488 (MD5) Previous issue date: 2015 / Resumo: Entre as décadas de 1910-1940, diversos sacerdotes católicos foram fuzilados por tropas revolucionárias mexicanas. Alguns desses personagens foram constantemente lembrados nas décadas posteriores como mártires da "perseguição religiosa". O mais conhecido dos mártires foi o sacerdote jesuíta Miguel Agustín Pro (padre Pro), que terminou fuzilado em 1927 na capital mexicana. A história do padre Pro foi escrita em diferentes contextos como forma de afirmar o lugar do catolicismo na nação mexicana, porém esse lugar não era, de forma alguma, ponto pacífico entre aqueles que se definiam como católicos. O presente trabalho analisa a história dos textos sobre os mártires católicos ¿ em especial o padre Pro ¿ pensando na maneira como eles forneciam uma visão católica para o período revolucionário que contrastava com as construções narrativas que enalteciam a revolução. A elaboração de uma narrativa da Igreja como mártir para o período revolucionário mexicano, realizada entre 1927 e 1960, serviu como antídoto para as narrativas pátrias produzidas por liberais e revolucionários que marginalizavam a importância da Igreja católica na formação nacional, ou mesmo apresentavam-se como abertamente anticlericais / Abstract: Between the decades of 1910-1940, a number of Catholic priests were executed by Mexican revolutionary troops. Quite often, these characters were reminded in the following decades as martyrs of the "religious persecution". The best known of this martyrs was the Jesuit priest Miguel Agustín Pro (padre Pro), killed in front of a firing squad in Mexico City in 1927. Catholics wrote the history/story of padre Pro in different contexts as a way of defining the place of Catholicism in the formation of Mexico as a country. However, this place was not taken for granted among those who defined themselves as Catholics. This dissertation examines the history of the texts about the Catholic martyrs - especially padre Pro - aiming to discuss how they provided a Catholic vision for the revolutionary period that contrasted to the narrative built to praise the revolution. The development, between 1927 and 1960, of a narrative of the Church as a martyr in the Mexican revolutionary period served as an antidote to the narrative produced by liberal and revolutionary authors that marginalized the importance of the Catholic Church in the national formation, or that even presented themselves as openly anti-clerical / Doutorado / Politica, Memoria e Cidade / Doutor em História

En mai 1940, la population ardennaise fuit devant l’arrivée des troupes allemandes. Les ressources économiques et agricoles du département, qui faisaient pourtant l’objet de plans d’évacuation préparés dès les années 1930, sont abandonnées à l’occupant. Quelques semaines plus tard, une vaste zone du nord-est de la France, dont les Ardennes font partie, est déclarée « zone interdite ». Les terres cultivables sont confisquées à leurs propriétaires et prises en charge au profit du Reich par une entreprise appelée Ostland, qui a déjà orchestré un semblable mouvement de spoliation en Pologne depuis son invasion. L’une de ses filiales régionales, la WOL III, met en place dans les Ardennes un vaste projet d’implantation des méthodes agricoles nationales-socialistes qui nécessite une abondante main-d’œuvre. Des agriculteurs allemands, appelés « chefs de culture » sont diligentés sur place et gèrent de grandes exploitations dans lesquelles travaillent plusieurs milliers de prisonniers français et coloniaux ainsi que 5 000 agriculteurs ardennais contraints à se mettre à leur service. Des ouvriers juifs sont également recrutés et des milliers de Polonais, expulsés de leurs villages, sont déportés pour travailler dans ces fermes qui exercent une agriculture intensive. Cette situation engendre des tensions sociales qui s’expriment particulièrement lors de la Libération et lors de procès d’épuration qui visent certains employés de l’Ostland. Les autorités françaises tentent de gérer au mieux la liquidation de l’entreprise allemande et l’organisation du rapatriement des Polonais dans leur pays, deux opérations difficiles qui nécessitent de longs mois. La reconnaissance des victimes de l’Ostland est inégale et tardive puisqu’elle n’intervient qu’à partir des années 1990. Des mémoires distinctes et spécifiques aux différents groupes de travailleurs émergent aussi à cette époque et s’expriment lors de commémorations. / In May 1940, the population of the Ardennes fled from the arrival of the German troops. The economic and agricultural ressources of the department, which yet had been subject to evacuation plans since the thirties, were given up to the occupying forces. A few weeks later, a large area of the North-East of France including the Ardennes was declared « forbidden zone ». The cultivable land was confiscated from its owners and taken over for the benefit of the Reich by a company named Ostland, which had already orchestrated a similar spoliation movement in Poland since its invasion. One of its local subsidiaries, WOL III , set up in the Ardennes a vast project to implement the National Socialist agricultural methods which required an abundant workforce. Some German farmers, called crop managers, were sent out there to run large farms on which several thousands of French and colonial prisoners as well as 5000 Ardennes farmers were working under duress. Jewish labourers were also recruited and thousands of Poles, expelled from their villages, were deported to work on these farms with intensive agriculture. This situation caused social tensions that were particuliarly evident during the Liberation and during the « purification » trials involving some WOL employees. French authorities tried to manage the liquidation of the German company and the organisation of the repatriation of the Poles, two difficult operations that took many months to complete. Recognition of Ostland victims was uneven and late since it occurred only from the 1990s onwards. Distinct memories specific to the different groups of workers also emerged at that time and were expressed during commemorations.

Genomweite Bemühungen haben eine große Anzahl langer nichtkodierender RNAs (lncRNAs) identifiziert, obwohl ihre möglichen Funktionen weitgehend rätselhaft bleiben. Hier verwendeten wir ein System zur synchronisierten Blüteninduktion in Arabidopsis, um 4106 blütenbezogene lange intergene RNAs (lincRNAs) zu identifizieren. Blütenbezogene lincRNAs sind typischerweise mit funktionellen Enhancern assoziiert, die bidirektional transkribiert werden und mit verschiedenen funktionellen Genmodulen assoziiert sind, die mit der Entwicklung von Blütenorganen zusammenhängen, die durch Koexpressionsnetzwerkanalyse aufgedeckt wurden. Die Master-regulatorischen Transkriptionsfaktoren (TFs) APETALA1 (AP1) und SEPALLATA3 (SEP3) binden an lincRNA-assoziierte Enhancer. Die Bindung dieser TFs korreliert mit der Zunahme der lincRNA-Transkription und fördert möglicherweise die Zugänglichkeit von Chromatin an Enhancern, gefolgt von der Aktivierung einer Untergruppe von Zielgenen. Darüber hinaus ist die Evolutionsdynamik von lincRNAs in Pflanzen, einschließlich nicht blühender Pflanzen, noch nicht bekannt, und das Expressionsmuster in verschiedenen Pflanzenarten war ziemlich unbekannt. Hier identifizierten wir Tausende von lincRNAs in 26 Pflanzenarten, einschließlich nicht blühender Pflanzen. Ein direkter Vergleich von lincRNAs zeigt, dass die meisten lincRNAs speziesspezifisch sind und das Expressionsmuster von lincRNAs einen hohen Transkriptionsumsatz nahe legt. Darüber hinaus zeigen konservierte lincRNAs eine aktive Regulation durch Transkriptionsfaktoren wie AP1 und SEP3. Konservierte lincRNAs zeigen eine konservierte blütenbezogene Funktionalität sowohl in der Brassicaceae- als auch in der Grasfamilie. Die Evolutionslandschaft von lincRNAs in Pflanzen liefert wichtige Einblicke in die Erhaltung und Funktionalität von lincRNAs. / Genome-wide efforts have identified a large number of long non-coding RNAs (lncRNAs), although their potential functions remain largely enigmatic. Here, we used a system for synchronized floral induction in Arabidopsis to identify 4106 flower-related long intergenic RNAs (lincRNAs). Flower-related lincRNAs are typically associated with functional enhancers which are bi-directionally transcribed and are associated with diverse functional gene modules related to floral organ development revealed by co-expression network analysis. The master regulatory transcription factors (TFs) APETALA1 (AP1) and SEPALLATA3 (SEP3) bind to lincRNA-associated enhancers. The binding of these TFs is correlated with the increase in lincRNA transcription and potentially promotes chromatin accessibility at enhancers, followed by activation of a subset of target genes. Furthermore, the evolutionary dynamics of lincRNAs in plants including non-flowering plants still remain to be elusive and the expression pattern in different plant species was quite unknown. Here, we identified thousands of lincRNAs in 26 plant species including non-flowering plants, and allow us to infer sequence conserved and synteny based homolog lincRNAs, and explore conserved characteristics of lincRNAs during plants evolution. Direct comparison of lincRNAs reveals most lincRNAs are species-specific and the expression pattern of lincRNAs suggests their high evolutionary gain and loss. Moreover, conserved lincRNAs show active regulation by transcriptional factors such as AP1 and SEP3. Conserved lincRNAs demonstrate conserved flower related functionality in both the Brassicaceae and grass family. The evolutionary landscape of lincRNAs in plants provide important insights into the conservation and functionality of lincRNAs.

Der Gen-Knockdown mittels RNAi hat sich als essentiell erwiesen, um Inhibitoren der induzierten Transdifferenzierung in C. elegans zu identifizieren (Tursun et al., 2011). Bakterienstämme, die dsRNA exprimieren, das die Expression spezifischer Gene mindert, können dem Wurm direkt zugefüttert werden, um einen genomweiten RNAi-screen der insgesamt 20.000 Gene in C. elegans durchzuführen. Allerdings werden die meisten biologischen Prozese durch mehr als ein Gen reguliert, was den Bedarf nach einer Methode generiert, die es erlaubt, zwei oder mehr Gene gleichzeitig herunter zu regulieren, um die Steuerung biologischer Prozesse studieren zu können. Die derzeitig vorhandenen Methoden liefern entweder nicht reproduzierbare Ergebnisse oder sind nicht skalierbar. Wir nutzen baktierelle Konjugation, die es durch ein konjugatives Plasmid ermöglicht Bakterienzellen zu generieren, die zwei verschiedene RNAi-Plasmide enthalten. Das Ziel war es, modifizierte RNAi-Donor-Plasmide mittels bakterieller Konjugation an eine Vielzahl anderer Bakterienzellen zu übertragen, die bereits ein anderes RNAi-Plasmid enthalten und dies dann im Hochdurchsatzverfahren durchführen zu können. Um Enhancer induzierter Expression von unc-25::gfp in der Keimbahn, ermöglicht durch den Knockdown des Histonchaperons LIN-53 (RbAp46/48 in Menschen), zu finden, wurden RNAi-Klone generiert, die gleichzeitig lin-53 als auch eines von insgesamt 800 verschiedenen Chromatin-bezogenen Gene herunter regulieren. Dabei identifizierten wir RBBP-5, Mitglied des Set1/ MLL-Methyltransferase-Komplexes, als neuen Barrierefaktor der induzierten Transdifferenzierung. RBBP-5 agiert dabei mutmaßlich parallel zu LIN-53. Doppelte RNAi, ermöglicht durch bakterielle Konjugation, erlaubt den simultanen Knockdown zweier oder mehr Gene, um genetische Interaktionen studieren zu können und erweitert damit die Einsatzmöglichkeiten von RNAi-Screens, um untereinander verbundene biologische Prozesse zu studieren. / The knock down of genes by RNAi has been fundamental to identify inhibitors of induced cell transdifferentiation in C. elegans (Tursun et al., 2011). Bacteria strains expressing dsRNA that target specific genes can be fed to the worm allowing straightforward whole-genome RNAi screens of the 20,000 genes in theC. elegans genome. However, many biological processes are regulated by more than one gene raising the need for simultaneous knock down of two or more genes to more fully interrogate the regulation of complex biological processes. Two approaches are currently available for double RNAi knockdown, − two bacteria strains expressing specific dsRNA can be mixed and grown together and fed simultaneously, which gives highly variable results. Alternatively, a new bacterial clone can be generated carrying a plasmid on which two RNAi targets of interest are 'stitched' together, which is not scalable. To address this challenge, we have developed a protocol using bacterial conjugation mediated by the 'Fertility Factor' (F) Episome in order to combine two different RNAi plasmids in a single bacterium. The objective was to be able to transfer a single RNAi plasmid to a large number of bacterial cells carrying different RNAi clones in one step in a high-throughput manner for large scale 'double' or even 'triple' RNAi screens. To find enhancers of induced unc-25::gfp expression in the germ line enabled by the depletion of histone chaperone LIN-53 (RbAp46/48 in humans), double RNAi clones targeting lin-53 and a total of 800 chromatin-related genes were generated and screened. We identified the Set1/MLL methyltransferase complex member RBBP-5 as a novel reprogramming barrier that putatively acts in a parallel pathway to LIN-53. Double RNAi by conjugation permits to reliably knock down two genes simultaneously in order to study genetic interactions at a genome-wide level, thus further increasing the versatility of RNAi screens to investigate interconnected biological processes.

Cis-regulatorische Elemente wie Promotoren und Enhancer, die die Regulation der Transkription von Genen steuern, befinden sich in Regionen des dekondensierten Chromatins. DNase-seq und ATAC-seq sind weit verbreitete Verfahren, um solche offenen Chromatinregionen genomweit zu untersuchen. Die einzel-Nukleotid-Auflösung von DNase-seq wurde des Weiteren genutzt, um Transkriptionsfaktor-Bindungsstellen (TFBS) in regulatorischen Regionen durch TF-Footprinting zu bestimmen. Kürzlich durchgeführte Studien haben jedoch gezeigt, dass DNase I einen Sequenzbias aufweist, welcher nachteilige Auswirkungen auf die Footprinting-Effizienz hat. Auch wurden das Footprinting und die Auswirkungen des Sequenzbias auf ATAC-seq noch nicht umfassend untersucht. In dieser Arbeit nehme ich einen systematischen Vergleich der beiden Methoden vor und zeige, dass die beiden Methoden unterschiedliche Sequenzbiases haben und korrigiere diese protokollspezifischen Biases beim Footprinting. Der Einfluss von Bias-Korrekturen der Footprinting Ergebnisse ist für DNase-seq größer als für ATAC-seq, und Footprinting mit DNase-seq führt zu besseren Ergebnissen in unserer Datensätze. Trotz dieser Unterschiede zeige ich, dass die Integration replizierter Experimente die Ableitung von qualitativ hochwertigen Footprints ermöglicht, wobei die beiden Techniken weitgehend übereinstimmen. Diese Techniken werden ferner eingesetzt, um die cis-regulatorischen Elemente zu charakterisieren, die die Embryogenese der Fruchtfliege Drosophila melanogaster bestimmen. Durch die Verwendung von Embryonen die sich im richtigen Entwicklungsstadium befinden, sowie gewebespezifischer Kernsortierung mit offenem Chromatin-Profiling können zeitlich und gewebespezifisch aufgelöste vermeintliche cis-regulatorische Elemente definiert werden. Zusammengenommen demonstrieren diese Analysen die Fähigkeit der offenen Chromatin-Profilierung und der Computeranalyse zur Aufklärung der Mechanismen der Genregulation. / Cis-regulatory elements such as promoters and enhancers, that govern transcriptional gene regulation, reside in regions of open chromatin. DNase-seq and ATAC-seq are broadly used methods to assay open chromatin regions genome-wide. The single nucleotide resolution of DNase-seq has been further exploited to infer transcription factor binding sites (TFBS) in regulatory regions through TF footprinting. However, recent studies have demonstrated the sequence bias of DNase I and its adverse effects on footprinting efficiency. Furthermore, footprinting and the impact of sequence bias have not been extensively studied for ATAC-seq. In this thesis, I undertake a systematic comparison of the two methods and demonstrate that the two methods have distinct sequence biases and correct for these protocol-specific biases when performing footprinting. The impact of bias correction on footprinting performance is greater for DNase-seq than for ATAC-seq, and footprinting with DNase-seq leads to better performance in our datasets. Despite these differences, I show that integrating replicate experiments allows the inference of high-quality footprints, with substantial agreement between the two techniques. These techniques are further employed to characterize the cis-regulatory elements governing the embryogenesis of a complex organism, the fruit fly Drosophila melanogaster. Combining tight staging of embryos and tissue-specific nuclear sorting with open chromatin profiling, enables the definition of temporally and tissue-specifically resolved putative cis-regulatory elements. Taken together, these analyses demonstrate the power of open chromatin profiling and computational analysis in elucidating the mechanisms of transcriptional gene regulation.