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Face à la rapidité des changements climatiques, les arbres doivent faire preuve de plasticité phénotypique. Les mécanismes épigénétiques font partie des pistes de recherche actuelles pour expliquer la plasticité phénotypique. Cette thèse visait à évaluer le rôle de la méthylation de l’ADN dans la plasticité phénotypique d’un organisme pérenne séquencé, le peuplier, en réponse à des variations de disponibilité en eau du sol. Les travaux, combinant écophysiologie et épigénomique, se sont focalisés sur le méristème apical caulinaire, centre de la morphogenèse de la tige feuillée. Trois résultats majeurs sont issus de cette thèse : i) Chaque état hydrique est associé à un méthylome et un transcriptome spécifiques, ii) Certaines régions différentiellement méthylées sont conservées dans le temps et entre contextes environnementaux, iii) Des lignées RNAi hypométhylées soumises à différents contextes hydriques présentent une réponse modifiée. Les résultats acquis lors de cette thèse appuient une contribution de la méthylation de l’ADN à la plasticité phénotypique et suggèrent un rôle des mécanismes épigénétiques dans la mémoire d’un stress chez les arbres. / Due to rapid climate changes, trees must exhibit phenotypic plasticity. Epigenetic mechanisms are part of current research to explain phenotypic plasticity. This thesis aimed to evaluate the role of DNA methylation in phenotypic plasticity of a perennial sequenced organism, poplar, in response to variations in soil water availability. The work, combining ecophysiology and epigenomics, focused on the shoot apical meristem, the center of morphogenesis of the leafy stem. Three major results emerge from this thesis: (i) Each hydric state is associated with a specific methylome and transcriptome, (ii) Some differentially methylated regions are conserved in time and between environmental contexts, (iii) Hypomethylated RNAi lines subjected to different contexts show a modified response. The results obtained during this thesis support a contribution of DNA methylation to phenotypic plasticity and suggest a role of epigenetic mechanisms in stress memory in trees.

Transkriptionelle Netzwerke (Transkriptionsfaktoren, epigenetische Modulatoren und spezifische Zielgene) stellen die unterste Ebene der zellinternen Signalübertragung dar. Eingebettet in verschiedene stimulusabhängige Signalwege bedienen sich ihre Komponenten genetischer und epigenetischer Mechanismen, um Zielgene transkrip-tionell zu regulieren und Veränderungen der Chromatinstruktur hervorzurufen. In der vorliegenden Arbeit wurde die hierarchische Organisation und Zusam-mensetzung des MEK-ERK-abhängig gesteuerten transkriptionellen Netzwerks und seine Veränderung im Zuge der HRAS-vermittelten onkogenen Transformation von HA1-Zellen untersucht. Viele Arbeiten haben sich bereits eingehend mit der Charak-terisierung einzelner Komponenten und Zielgene beschäftigt (Wagner et al. 2005, Reddy et al. 2002, Sun et al. 2006, Kapitel 1). Im Unterschied zu den zitierten Studien wurde in der vorliegenden Arbeit ein umfassendes Protokoll zur genomweiten De-chiffrierung transkriptioneller Netzwerke unter Kombination von experimentellen und bioinformatischen Methoden entwickelt und durchgeführt. Die Analyse ge-nomweiter Expressionsprofile un- und U0126-behandelter immortaler und HRASV12-transformierter humaner Nierenepithelzellen (HA1EB, HA1ER) erlaubte die Identifi-zierung von 138 auf- und 103 abregulierten genspezifischen IDs der RAS-ERK-abhängig gesteuerten Signalkaskade. Regulierte Transkriptionsfaktoren wurden i-dentifiziert und im Westernblot, sowie zum Teil mittels Durchflusszytometrie und RT-PCR validiert und nachfolgend transienten siRNA-Experimenten unterzogen. Für die Transkriptionsfaktoren ELK3, SRF und den hierarisch darunter liegenden Faktor FRA1 wurden Expressionsprofile der spezifischen siRNA-vermittelten Hemmung in beiden Zelllinien erstell und mit bioinformatischen Methoden (TRAP, GSEA-GO) a-nalysiert um direkte und indirekte sowie gemeinsame Zielgene zu ermitteln. Zusätz-lich wurde der Effekt auf phänotypischer Ebene (Softagar, MTT) überprüft. In der vorliegenden Arbeit ließ sich keine direkte Hierarchie der drei Transkrip-tionsfaktoren SRF, FRA1 und ELK3 bestätigen. Allerdings konnte zum ersten Mal eine gemeinsam regulierte Gruppe von Genen identifiziert werden, die darauf schließen lässt, dass die drei Transkriptionsfaktoren sowohl in HA1EB, als auch in HA1ER Teile eines gemeinsam regulierenden Netzwerks sind. Aus den Proliferationsexperimenten wurde zudem bestätigt, dass jeder Transkriptionsfaktor individuell eine essentielle Rolle bei der Promotion maligner Eigenschaften spielt. Für alle drei Transkriptionsfak-toren konnte eine RAS-abhängige starke Verschiebung der spezifisch angesteuerten Gene nachgewiesen werden. Diese Verschiebung wurde mittels TRAP und GSEA auch für alternative Regulatoren der spezifischen Zielgene festgestellt. Die nähere Analyse der FRA1-abhängigen Zielgene führte zu neuen Erkenntnis-sen zur Umordnung des Transkriptoms im Zuge der onkogenen Transformation. Die FRA1-spezifischen Zielgene in HA1EB und HA1ER weisen unterschiedliche Funktio-nalitäten auf. So wurden in HA1EB viele Gene identifiziert, die im Rahmen der Im-munantwort eine Rolle spielen und in HA1ER nicht reguliert werden. In den RAS-transformierten HA1ER konnten dagegen Gene identifiziert werden, die in der Tu-morprogression eine Rolle spielen (FRA1, STAT3, MTA1, TCFL5). Die Verifizierungen mittels qPCR und ChIP bestätigten 5 der 38 möglichen FRA1-Zielgene. Von diesen, FRA1, AEBP1, FRA1, TCFL5, NPAS2 und YWHAZ ist lediglich FRA1 bereits als FRA1-Zielgen beschrieben. Die Funktionen der neu identifizierten RAS-abhängigen FRA1-Zielgene untermauerten bereits bekannte Funktionen der FRA1-vermittelten Transkription (Differenzierung, Proliferation, zirkadiane Rhythmen, Apoptose) und erweitern sie um verschiedene Aspekte wie Metabolismus und Rückkopplungen in die Signaltransduktion, die noch nicht für die RAS-abhängige FRA1-vermittelte Transktiption beschrieben worden sind. Dazu gehören unter anderem Interaktionen mit TGFbeta, WNT, JAK/STAT und JNK. Daneben sind in den HA1ER eine Vielzahl von Regulatoren des RHO-Signalwegs identifiziert worden, was für FRA1 auf bisher unbekannte Interaktionen mit RAC/RHO-Signalwegen schließen lässt. / Transcriptional networks represent the final level of internal signal transmission. They are embedded in different signalling pathways and use genetic as well as epi-genetic mechanisms to regulate their according target genes. During oncogenic trans-formation they are undergoing massive rearrangements in composition, regulation and interaction. This leads to radical changes in the transcriptome and drives the on-cogenic phenotype of the according cells. My thesis employs the composition of the MEK-ERK-dependent transcriptional net-work and its alteration during the HRAS-oncogene-mediated transformation in HA1-cells. By commencing from already known components: SRF, Ternary Complex Fac-tors (TCF: SAP1, SAP2/ELK3, ELK1) and members of the AP1-complex (JUN, FOS-proteins) I analyzed the alteration in expression of secondary targets and their inter-action as well as their relation to the superior factors. Therefore I compared genome wide expression profiles (Affymetrix, HG-U133A) of immortal HA1EB and HRASV12-oncogene-transformed HA1ER-cells with and without U0126-induced MEK/ERK-inhibition and extracted several MEK/ERK-dependent transcription factors. Among them where FRA1 and ELK3, two transcription factors already known to be involved in oncogenesis and proliferation associated processes. ELK3 needs SRF as crucial binding partner to function. Therefor I also included SRF into the subsequent analysis. The three transcription factors function in different time-dependent hierarchy states so we supposed a putative hierachical network be-tween them. I established transient knockdown cells deriving from HA1EB and HA1ER for all three transcription factors and generated further expression profiles from them. Additionally I verified the importance of these transcription factors on survival and proliferation via MTT and Softagar experiments. Using different statis-tically and bioinformatical methods (GSEA, TRAP) in collaboration with the Max-Planck-Institute for molecular Genetics Berlin, several direct and indirect targets of these transcription factors were predicted. These were partially overlapping in all transcription factors. Also, in comparison of the immortal and the transformed cell line, a shift of functionalities and composition of the different target gene populations and collaborating factors could be detected for all three transcription factors. It was found that in HA1EB FRA1 seems more likely to regulate immunresponsive genes as well as genes associated with the cytoskeleton and nucleus organisation whereas in HA1ER FRA1 regulates a large group of transcription- and signalling-associated genes. Additionally it could be shown that in both cell lines FRA1 regulates genes in-volved in epigenetic processes as well as circadian rhythms which are known to be important aspects in oncogenic transformation. I verified 37 different putative target genes of FRA1 using qRT-PCR (Taqman) and partially also ChIP-analysis. Of these 37genes, 5 were fully validated as directly regu-lated targets of FRA1: FRA1, AEBP1, YWHAZ, NPAS2 and TCFL5. They imply functionalities connected to proliferation and differentiation (AEBP1, FRA1, TCFL5) as well as apoptosis (YWHAZ) cell cycle control and circadian rhythm (NPAS2, AEBP1), feedbacks into the signalling (YWHAZ, AEBP1) and metabolism (NPAS2, AEBP1). Summarised the work of this thesis contributes to the decipherment of the direct and indirect targets of the according transcription factors and strengthens the argument of a general and massive shift of the transcriptional network during oncogenic trans-formation of cells. The importance of all three transcription factors on the survival of genes could be proved via proliferation assays. Additionally the functionality of their according targets could be integrated into processes connected to oncogenic trans-formation.

Genetic control strategies are a novel method for reducing populations of pest insects such as the yellow fever mosquito Aedes aegypti, a major vector of several important arboviral diseases. This thesis describes efforts to develop new tools to engineer the Ae. aegypti genome and to better understand existing tools, and furthermore to use these to engineer a gene drive system in Ae. aegypti. The piggyBac transposon was found to be extremely stable in the germline of Ae. aegypti, and transposons engineered into the germline could not be remobilized with either an endogenous or exogenous source of piggyBac transposase. Conversely, somatic remobilization of piggyBac transposons was found to be readily detectable in the presence of a source of active transposase, the first report of such remobilization in Ae. aegypti. Toward new tools for genome engineering, the site-specific integrase from the phage φC31 was successfully used to promote exchange between a transgene cassette inserted into the genome of Ae. aegypti and a cassette in a plasmid vector, in the first demonstration of recombinase mediated cassette exchange technology in a pest insect species. The integrases from phages φRV1 and Bxb1 were not found to be active in the germline of the mosquito. Finally, development of a gene drive system in Ae. aegypti using an RNAi-mediated killer-rescue mechanism was attempted. Tissue-specific expression of tTAV-regulated-toxic effectors genes, using the promoter regions of the blood meal induced genes Carboxypeptidase A-1, 30Kb and Vitellogenin A, was possible, but sex-specificity was not achieved. A blood meal inducible lethal phenotype was not possible using the chosen promoters, with expression of the effectors either leading to death in early development or to a sublethal phenotype. RNAi against tTAV fused to the Mnp fragment of the dengue virus’ genome was tissue specific, but was found to be highly effective in the fat body suggesting that the Vitellogenin A was the best candidate for the engineering of killer-rescue systems in the mosquito.

RNA interference (RNAi) is a post transcriptional gene silencing mechanism that allows potent and specific silencing of cognate mRNA transcripts. Selective silencing can be used to dissect complex polygenic diseases, elucidate the function of known genes and provide a tool for genetic therapy. Its use in the case of dominant inherited disorders including disorders of the central nervous system, depends on its ability to confer single nucleotide discrimination between normal and mutant gene alleles. In this thesis the ability of RNAi effector molecules to provide single nucleotide specificity was examined by targeting two dominant inherited mutations of the acetylcholine receptor that cause slow-channel syndrome. Allele-specific silencing was achieved for one mutation. The other mutation was also silenced but not in an allele specific way despite employing known techniques for increasing single-nucleotide specificity. The model used in this thesis is the congenital myasthenia slow-channel syndrome. This is a dominant inherited disorder of the neuromuscular junction which is both well-characterised and more readily accessible compared to the central nervous system, thus provides a prototype for development of allele-specific RNAi therapeutics. Here we describe a new transgenic animal model of the slow-channel syndrome and show good representation of the human disorder. The need for defining the characteristics that determine the effectiveness and the specificity of RNAi effectors at single-nucleotide level, along with the future uses of the newly described animal model are discussed.

Le sarcome de Ewing est la seconde tumeur pédiatrique de l’os la plus fréquente. Elle est caractérisée par une translocation chromosomique résultant à la fusion de EWSR1 avec un membre de la famille ETS. Chez 85% des patients, cette fusion conduit à l’expression de la protéine chimérique EWS-FLI1 qui est l’oncogène majeur de ce sarcome. Ce dernier agit principalement par son action transcriptionelle sur des cibles qui lui sont propres. Au niveau thérapeutique, le sarcome d’Ewing est traité par chimiothérapie, chirurgie locale et par radiothérapie. La survie à long terme des patients est de l’ordre de 70%, mais beaucoup plus basse pour les patients métastatiques et quasi nulle lors d’une récidive. Parmi maintes caractéristiques, certains cancers présentent une dérégulation énergétique. L’influence d’EWS-FLI1 sur cet aspect n’a fait l’objet d’aucune étude dans le contexte du sarcome d’Ewing. Nous avons donc étudié par profilage métabolomique des cellules de sarcome d’Ewing en présence ou en absence d’EWS-FLI1. En comparant ces deux conditions, des modulations du profil énergétique relatif au cycle de Krebs, des précurseurs de le glycosylation ainsi que des métabolites de la voie de la méthionine et du tryptophane ont été observés. En parallèle, grâce à un crible de banque de shRNAs réalisé dans des conditions expérimentales similaires à l’étude métabolomique (lignée d’Ewing avec ou sans EWS-FLI1), nous avons pu identifier des gènes présentant des caractéristiques « synthétique létales », c'est-à-dire tuant uniquement les cellules du sarcome d’Ewing en présence de son oncogène. / Ewing sarcoma, the second most commonly occurring pediatric bone tumor, is most often characterized by a chromosomal translocation between EWSR1 and FLI1. The gene fusion EWS-FLI1 accounts for 85% of all Ewing sarcoma and is considered the major oncogene and master regulator of Ewing sarcoma. EWS-FLI1 is a transcriptional modulator of targets, both directly and indirectly. Ewing sarcoma is aggressively treated with chemotherapy, localized surgery and radiation and has an overall survival of about 70%, however, survival for metastasis or relapsed cases remains low. One of the cancer hallmarks, metabolic deregulation, is most likely partly dependent on EWS-FLI1 in Ewing sarcoma cells. In order to get a better understanding of Ewing sarcoma biology and oncogenesis, it might be of high interest to investigate the influence of EWS-FLI1 in Ewing sarcoma cells. We therefore performed a global metabolic profiling of Ewing sarcoma cells with or without inhibition of EWS-FLI1. Several changes in the energy metabolism were observed throughout this study; the observed changes were consistent with an energy profile that moved from a cancer cell energy metabolism towards the energy metabolism of a more normal cell upon EWS-FLI1 inhibition, primarily based on the TCA cycle. Levels of TCA intermediates, glycosylation precursors, methionine pathway metabolites and amino acids, especially changes in the tryptophan metabolic pathway, were altered upon EWS-FLI1 inhibition. Parallel to this study, we performed a high-throughput synthetic lethality screen, in order to not only identify essential genes for cell survival and proliferation, but also to identify new synthetic lethal targets that could specifically target Ewing sarcoma cells carrying the EWS-FLI1 fusion gene.

<p>Angiogenesis is defined as the formation of new capillary blood vessels from pre-existing ones. This process involves several steps including: migration, proliferation and differentiation of endothelial cells into blood vessels. Angiogenesis is initiated by binding of specific growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), to their cell surface receptors. Shb is a ubiquitously expressed adaptor protein with the ability to bind several tyrosine kinase receptors. My aim has been to identify the role of Shb in FGF- and VEGF-signalling in endothelial cells. Shb was found to be phosphorylated in a Src-dependent manner upon both FGF- and VEGF-stimulation. This was confirmed using fibroblasts overexpressing temperature sensitive v-Src. Furthermore, Shb-induced cell spreading on collagen of immortalised brain endothelial (IBE) cells was also Src-dependent. FGF stimulation led to a direct association between Shb and FAK, which was mediated by the phosphotyrosine binding domain of Shb. IBE cells overexpressing wild-type or R522K Shb (inactive SH2 domain) displayed increased FAK activation on collagen.</p><p>The SH2-domain of Shb was found to bind to tyrosine 1175 in the VEGFR-2 in a phosphotyrosine dependent manner using PAE cells expressing VEGFR-2. Furthermore, by use of siRNA, Shb knock-down experiments revealed that Shb regulates FAK activity, cellular migration and stress fiber formation in response to VEGF stimulation of VEGFR-2. In summary, Shb binds to both FGFR-1 and VEGFR-2 and regulates the activity of FAK and thereby stress fiber formation and cellular migration, which are necessary for formation of new blood vessels. IBE cells with an inactive SH2 domain of Shb displayed disorganised formation of tubular structures in the tube formation assay, while overexpression of wild-type Shb led to accelerated tubular morphogenesis.</p><p>Taken together, my data show that the adaptor protein Shb plays an important role in the process angiogenesis, in response to angiogenic tyrosine kinase receptors, by interacting with FAK and regulating spreading, stress fiber formation and cellular migration.</p>