Tout au long de la vie d’un individu, il existe un nombre optimal de cellules à produire et de progéniteurs à conserver en réserve. On parle de maintien de l’homéostasie tissulaire. De façon générale, l’organisme a cinq possibilités pour réguler l’homéostasie : l’autorenouvellement et la quiescence, souvent utilisés pour maintenir un ‘pool’ fonctionnel de progéniteurs, la différenciation qui permet de produire des cellules effectrices, l’apoptose et la sénescence, qui permettent de limiter la production de cellules ou encore d’en faire diminuer le nombre quand elles sont en excès. La régulation de ces quatre mécanismes peut se faire de façon extrinsèque en passant par différentes voies de signalisation combinées à l’action intrinsèque de facteurs de transcription comme Ikaros et GATA1. Le facteur de transcription Ikaros joue un rôle critique dans le devenir des cellules progénitrices et la différenciation des lignages hématopoïétiques. Cependant, il demeure surtout connu pour son influence sur la voie Notch dans les cellules lymphoïdes, notamment les lymphocytes T. Les cellules érythroïdes sont hautement sensibles à l’environnement et donc, particulièrement adaptées à l’étude des régulations de l’homéostasie. Les résultats de différentes études ont permis de démontrer qu’Ikaros et la voie Notch influencent l’érythropoïèse. Cependant le détail de leurs actions demeure en grande partie inconnu à ce jour. Au cours de notre étude nous avons voulu déterminer l’action d’Ikaros dans le maintien de l’homéostasie des cellules érythroïdes et si son rôle passe par un dialogue avec la voie Notch. Nous avons voulu décrypter les mécanismes de régulation transcriptionnelle utilisés par Ikaros et par Notch au cours de l’érythropoïèse et leurs effets. Notre étude montre qu’Ikaros réprime à l’aide de GATA1 le gène Hes1, une cible importante de la voie Notch, en recrutant un complexe de la famille Polycomb, le PRC2 ii (Polycomb Repressive Complex 2). Cette répression permet la promotion de la différenciation des cellules érythroïdes. Au niveau du maintien de l’homéostasie par régulation de l’apoptose, Ikaros est connu pour cibler l’anti-apoptotique Bcl2l1 dans les lymphocytes. Puisque Gata-1, partenaire préférentiel d’Ikaros cible Bcl2l1 dans les cellules érythroïdes, nous avons caractérisé leur effet sur l’expression de Bcl2l1. Nous avons découvert qu’Ikaros active de façon directe Bcl2l1 et qu’il recrute sur le gène deux complexes partenaires d’élongation : un de la famille SET1/MLL, et le complexe P-TEFb-NuRD. En l’absence d’Ikaros, le fragment intracellulaire de Notch (NICD) et son cofacteur RBP-J remplacent Ikaros et favorisent l’hyper-activation de l’expression de Bcl2l1. Ceci est associé à la modification du complexe d’élongation recruté, ainsi qu’à la mise en place de modifications épigénétiques distinctes de celles observées avec Ikaros ce qui modifie l’élongation transcriptionnelle du gène. Ikaros et Notch sont fréquemment mutés ou présentent des fonctions altérées dans les leucémies. Notre étude montre un dialogue Ikaros/Notch influençant aussi bien la différenciation que l’apoptose et met en évidence l’existence d’un circuit génétique dont le dérèglement pourrait favoriser l’apparition d’une hématopoïèse maligne. / Throughout the life of an individual, there is an optimal count of cells to produce and progenitors to conserve in stock. This is the tissue homeostasis maintenance. In a general fashion the organism has five means to regulate the homeostasis. Self-renewal and quiescence, often used in order to maintain a functional progenitors pool. Differentiation enhances effector cells production. Apoptosis and senescence can limit cell production and reduce cell number in case of excess. These regulation mechanisms can be performed in an extrinsic fashion using different signaling pathways combined with the action of transcription factors like Ikaros and GATA1. The transcription factor Ikaros is critical for progenitor cells fate and hematopoietic lineages differentiation. However, Ikaros is mostly known for its influence on Notch signaling in lymphoid cells, notably T lymphocytes. Erythroid cells are highly sensitive to the environment thus, particularly adapted to study homeostasis maintenance regulation. Results obtained in different studies showed Ikaros and Notch signaling influencing erythropoiesis. However, the detail of their effect remains mainly unknown to day. Our aim was to determine Ikaros effect on erythroid cells homeostasis maintenance and if its role involved a cross-talk with Notch signaling. We will decipher transcription regulation mechanisms used by Ikaros and Notch during erythropoiesis and their effects. We show Ikaros uses GATA1 to repress Hes1, a major Notch target by recruiting a Polycomb family complex, the PRC2 (Polycomb Repressive Complex 2). This repression promotes erythroid cells differentiation. At the apoptosis mediated control of homeostasis level, Ikaros is known to target Bcl2l1 in lymphocytes. As GATA1, Ikaros preferential partner, targets Bcl2l1 in erythroid cells, we assessed their effect on Bcl2l1 expression. We discovered Ikaros directly activates Bcl2l1 iv and recruits two elongation associated complexes: one from SET1/MLL complex family, and the P-TEFb-NuRD complex. In the absence of Ikaros, the intracellular fragment of Notch (NICD) and its cofactor RBP-J replace Ikaros and favors Bcl2l1 overactivation. This is associated with a switch of recruited elongation associated complex and the establishment of distinct epigenetic modifications from those observed with Ikaros, which modifies the gene transcriptional elongation. Ikaros and Notch are frequently mutated or present altered functions in leukemia. Our works present an Ikaros/Notch cross-talk influencing as well differentiation as apoptosis and reveal the existence of a genetic circuit for which a malfunction could favor hematologic disorders. Keywords : transcription, homeostasis, erythropoiesis

The sex determining region Y-box 2 (sox2) gene is one of the most important transcription factors during development, particularly the development of the central nervous system (CNS). It is expressed in embryonic stem cells and later in neural stem cells, where it modulates their maintenance and differentiation. In humans, heterozygous mutations are associated with eye malformations, including anophthalmia and severe microphthalmia. Also, a subset of patients has extra-ocular phenotypes, such as hearing loss, seizures and pituitary hypoplasia. Although the roles of sox2 in embryonic stem cells and eye development are well studied, the function of sox2 in brain development and disease is still elusive. The aim of this project was to characterize a novel role for sox2 in the development of zebrafish epithalamus, which was identified from an in silico screen previously performed in our laboratory. The zebrafish epithalamus, located in the dorsal diencephalon, consists of three main structures: the pineal gland, the parapineal organ and the habenular nuclei. The pineal gland, also known as epiphysis, is a photoreceptive (in zebrafish) and neuroendocrine organ that detects light and rhythmically produces melatonin in order to regulate the circadian rhythms. The parapineal organ is located to the left side of the pineal gland and is important for the elaboration of the asymmetries observed between the left and right habenular nuclei. Finally, the bilateral habenulae are part of the dorsal diencephalic conduction system that links the forebrain with the mid- and hindbrain. The left and right habenulae show both molecular and neuroanatomical asymmetries, including differences in neuropil organization, in levels of gene expression and in the morphology and connectivity of their neurons’ projections. The relatively simple architecture of the pineal gland and the asymmetric character of the habenulae provide a useful tool for studying cell-fate determination, cell migration and establishment of brain asymmetries. In this study, we used zebrafish as a model to dissect the novel functions of sox2 in the development of the epithalamus. We showed that sox2 works synergistically with Notch pathway to negatively regulate neurogenesis within the pineal gland. The pineal gland consists of only two cell types: the photoreceptors and the projection neurons. Previous studies showed that the Notch and BMP pathways are important for the proper specification of these cells. Here, we show that sox2 normally inhibits the photoreceptor cell fate, whereas it has no effect on the number of projection neurons. Therefore, sox2 complements Notch and BMP pathways in cell-fate determination within the pineal gland. In addition, downregulation of sox2 results in abnormal parapineal organ development and disruption of the asymmetric architecture of the habenulae. A subset of sox2 morphant embryos develops right-sided parapineal organs, which is consistent with abnormal bilateral expression of the Nodal gene, pitx2 (paired-like homeodomain transcription factor 2). Also, timelapse experiments showed that migration of the parapineal cells is defective, resulting in scattered cells. The aberrant parapineal development leads to disorganization of the habenular nuclei, as shown by the abnormal neuropil arrangement and the expression of the asymmetric marker kctd12.1 (potassium channel tetramerisation domain containing 12.1).

No description available.

This subject mainly uses Aluminum sheet metal material as the research object which is used in Volvo XC90 door, the elastic plasticity deformation within the different material angles and notch shape investigated with MTS tensile test equipment under uniaxial tension. The local strain curve studied by using DIC method to obtain the conventional physical quantity. Combining ABAQUS to analyze the distribution of stress in elastic and plastic regions, also use MATLAB to combine various parameters, and finally analyze the desired experimental results to find a relatively stable notch shape based on the yield curve and stress concentration factor.

[pt] O presente trabalho avalia a evolução da microestrutura e as propriedades mecânicas devido à influência do aporte de calor exercido por um procedimento de soldagem que utilizou sequencialmente dois processos de soldagem sobre um tubo de aço API 5L X80, fabricado pelo processo UOE, de um aço produzido por laminação controlada sem resfriamento acelerado. A soldagem foi realizada em um tubo de 20 de diâmetro nominal e 3/4 de espessura, fixado na posição horizontal simulando condições de campo, usando o processo MAG de curtocircuito de corrente controlada com gás de proteção CO2 (100%) para o passe de raiz e o processo por Arame Tubular com proteção gasosa Ar - CO2 (80% - 20%) para os demais passes. As propriedades mecânicas foram avaliadas segundo os ensaios mecânicos exigidos na norma API 1104, além dos ensaios de microdureza Vickers e de impacto Charpy V. As mudanças microestruturais na Zona Afetada Termicamente e Material de Solda foram avaliadas por microscopia eletrônica de varredura (MEV) e microscopia óptica. A avaliação mecânica segundo a norma API 1104 foi reprovada, onde os resultados dos ensaios de tração e Nick-Break foram aceitos e o ensaio de dobramento lateral um corpo-de-prova apresentou uma trinca superior ao comprimento máximo aceitável. Os resultados da microdureza foram aceitáveis e o resultado do impacto Charpy V, segundo a norma DNV-OS-F101, para a temperatura de 0 °C foi insatisfatório na região do metal de solda dos passes de acabamento. A região da ZTA apresentou maior energia de impacto quando comparado com o material de base à temperatura de 0 °C, embora com presença do microconstituinte A-M. / [en] The present work evaluates the changes in the microstructural and mechanical properties of an API 5L X80 steel tube due to the influence of heat input exerted during a welding procedure that used two sequential welding processes. The tubes were manufactured using the UOE process, from steel that was produced by controlled rolling without accelerated cooling. The welding was carried out on a 3/4 thick and 20 nominal diameter pipe, while it was held in a horizontal position in order to simulate field conditions, using a controlled short circuit GMAW process with CO2 (100%) gas shielding for the root pass and a flux cored arc welding process with Ar-CO2 (80% - 20%) gas shielding for the other passes. The evaluation of the mechanical properties was done by means of mechanical tests according to the API 1104 standard, in addition to the Vickers microhardness and Charpy V-notch tests. The changes in the microstructure of the Heat Affected Zone (HAZ) and the welded metal were evaluated by means of scanning electronic microscopy (SEM) and optical microscopy. The mechanical evaluation was unsatisfactory according to the API 1104 standard, while the tensile and Nick-Break test results were acceptable. The side bend test showed a crack in a specimen that exceeded the maximum acceptable value. The Vickers microhardness results were acceptable and the Charpy V-notch result, according to the DNV-OS-F101 standard, at a temperature of 0 °C, was unsatisfactory in the weld metal region of the over cap. The HAZ region showed greater energy of impact absorption compared to the base metal, at a temperature of 0 °C, even with existence of the microconstituent M-A.

A intensa exposição ao Sol sujeita o lábio, principalmente o inferior, aos danos provocados pela absorção de radiação ultravioleta (UV). O carcinoma epidermoide é a neoplasia maligna que se desenvolve nos lábios após exposição crônica prolongada aos raios UV e acredita-se que todos os casos sejam precedidos pela desordem potencialmente maligna denominada queilite actínica. Ambas as lesões são causados pelos efeitos nocivos da radiação UV agindo diretamente sobre o DNA, por meio do fenômeno da fotocarcinogênese. Nesse processo, a radiação provoca mutações que são capazes de causar a iniciação, progressão e a promoção de neoplasias. No entanto, é também importante considerar que outros eventos moleculares, além das mutações, estão envolvidos na iniciação e progressão do câncer. Alterações moleculares com ganho ou perda de função de componentes da via de sinalização Notch estão envolvidas em diferentes tipos de cânceres hematológicos e sólidos. Entretanto, a participação da sinalização Notch em câncer de lábio ainda é desconhecida. Assim, o objetivo desse trabalho foi investigar se a via Notch está relacionada às lesões de queilite actínica e de carcinoma epidermoide de lábio e sua participação na fotocarcinogênese bucal. Para isso, foram utilizados 45 casos de queilite actínica, 15 casos de carcinoma epidermoide de lábio e 05 casos de lábio com aspecto de normalidade, nos quais foi analisada a expressão de Notch1 e Jagged1 por meio da técnica de imuno-histoquímica. Os resultados demonstraram que houve perda da expressão de Notch1 em 40% dos carcinomas epidermoides de lábio, sugerindo que a expressão reduzida de Notch1 pode converter os queratinócitos a um estado ativado e imaturo. Observou-se ainda, diferença nos padrões de marcação de Nocth1 e Jagged1 nas células epiteliais sugerindo que o sinal da via Notch seja transmitido a partir de uma célula apical para uma célula basal devido a localização das células que expressam o receptor e das que expressam o ligante. Concluiu-se, assim, que os resultados imuno-histoquímicos não apontam a uma regulação diferencial da expressão da proteína Notch1 e Jagged1 em lesões UV induzidas. / The intense exposure to the sun subject the lips, particularly the lower, the damage caused by the absorption of ultraviolet (UV) radiation. The squamous cell carcinoma is a malignant tumor that develops on the lips after prolonged chronic exposure to UV rays and it is believed that all cases are preceded by potentially malignant disorder called actinic cheilitis. Both lesions are caused by the harmful effects of UV radiation acting directly on the DNA, through the phenomenon of photocarcinogenesis. In this process, the radiation causes mutations that are capable of causing the initiation, progression and promotion of cancer. However, it is also important to consider that other molecular events, apart from the mutations are involved in the initiation and progression of cancer. Molecular abnormalities with gain or loss of Notch pathway components function are involved in several types of hematological and solid cancer. However, the participation of Notch signaling in lip cancer is still unknown. The objective of this study was to investigate whether the Notch pathway is related to injuries actinic cheilitis and squamous cell carcinoma of the lip and participation in oral photocarcinogenesis. For this, were used 45 cases of actinic cheilitis, 15 cases of squamous cell carcinoma of the lip and lip 05 cases with normal aspect in which we analyzed the expression of Notch1 and Jagged1 by immunohistochemistry. The results showed loss of Notch1 expression in 40% of squamous cell carcinomas of the lip, suggesting that reduced expression of Notch1 can convert to an activated keratinocytes and immature state. There was also a difference in labeling patterns of Notch 1 and Jagged1 epithelial cells suggesting that the Notch pathway signal is transmitted from an apical cell to a basal cell due to localization of cells expressing the receptor and expressing the ligand. In summary, the immunohistochemical results do not show a differential regulation of Notch 1 and Jagged1 expression in UV induced lesions.

A intensa exposição ao Sol sujeita o lábio, principalmente o inferior, aos danos provocados pela absorção de radiação ultravioleta (UV). O carcinoma epidermoide é a neoplasia maligna que se desenvolve nos lábios após exposição crônica prolongada aos raios UV e acredita-se que todos os casos sejam precedidos pela desordem potencialmente maligna denominada queilite actínica. Ambas as lesões são causados pelos efeitos nocivos da radiação UV agindo diretamente sobre o DNA, por meio do fenômeno da fotocarcinogênese. Nesse processo, a radiação provoca mutações que são capazes de causar a iniciação, progressão e a promoção de neoplasias. No entanto, é também importante considerar que outros eventos moleculares, além das mutações, estão envolvidos na iniciação e progressão do câncer. Alterações moleculares com ganho ou perda de função de componentes da via de sinalização Notch estão envolvidas em diferentes tipos de cânceres hematológicos e sólidos. Entretanto, a participação da sinalização Notch em câncer de lábio ainda é desconhecida. Assim, o objetivo desse trabalho foi investigar se a via Notch está relacionada às lesões de queilite actínica e de carcinoma epidermoide de lábio e sua participação na fotocarcinogênese bucal. Para isso, foram utilizados 45 casos de queilite actínica, 15 casos de carcinoma epidermoide de lábio e 05 casos de lábio com aspecto de normalidade, nos quais foi analisada a expressão de Notch1 e Jagged1 por meio da técnica de imuno-histoquímica. Os resultados demonstraram que houve perda da expressão de Notch1 em 40% dos carcinomas epidermoides de lábio, sugerindo que a expressão reduzida de Notch1 pode converter os queratinócitos a um estado ativado e imaturo. Observou-se ainda, diferença nos padrões de marcação de Nocth1 e Jagged1 nas células epiteliais sugerindo que o sinal da via Notch seja transmitido a partir de uma célula apical para uma célula basal devido a localização das células que expressam o receptor e das que expressam o ligante. Concluiu-se, assim, que os resultados imuno-histoquímicos não apontam a uma regulação diferencial da expressão da proteína Notch1 e Jagged1 em lesões UV induzidas. / The intense exposure to the sun subject the lips, particularly the lower, the damage caused by the absorption of ultraviolet (UV) radiation. The squamous cell carcinoma is a malignant tumor that develops on the lips after prolonged chronic exposure to UV rays and it is believed that all cases are preceded by potentially malignant disorder called actinic cheilitis. Both lesions are caused by the harmful effects of UV radiation acting directly on the DNA, through the phenomenon of photocarcinogenesis. In this process, the radiation causes mutations that are capable of causing the initiation, progression and promotion of cancer. However, it is also important to consider that other molecular events, apart from the mutations are involved in the initiation and progression of cancer. Molecular abnormalities with gain or loss of Notch pathway components function are involved in several types of hematological and solid cancer. However, the participation of Notch signaling in lip cancer is still unknown. The objective of this study was to investigate whether the Notch pathway is related to injuries actinic cheilitis and squamous cell carcinoma of the lip and participation in oral photocarcinogenesis. For this, were used 45 cases of actinic cheilitis, 15 cases of squamous cell carcinoma of the lip and lip 05 cases with normal aspect in which we analyzed the expression of Notch1 and Jagged1 by immunohistochemistry. The results showed loss of Notch1 expression in 40% of squamous cell carcinomas of the lip, suggesting that reduced expression of Notch1 can convert to an activated keratinocytes and immature state. There was also a difference in labeling patterns of Notch 1 and Jagged1 epithelial cells suggesting that the Notch pathway signal is transmitted from an apical cell to a basal cell due to localization of cells expressing the receptor and expressing the ligand. In summary, the immunohistochemical results do not show a differential regulation of Notch 1 and Jagged1 expression in UV induced lesions.

La sensibilité des structures composites à la présence d'endommagements importants en zones singulières (trou, pointe d'entaille.. impose d'évaluer leur tolérance aux dommages. Dans un premier temps, un dialogue essai-calcul pour des sollicitations uniaxiales de traction simple sur composite stratifiés lisses, troués (plusieurs diamètres) et entaillés est mis en place. En utilisant différentes méthodes de suivi expérimental insitu (corrélation d'images, thermographie infrarouge) et post-mortem (micro-tomographie aux rayons-X), les scénarios de ruptures sont identifiés et comparés à ceux déterminés par simulation numérique « Discrete Ply Model ». Le modèle numérique est démontré valide pour simuler les cas étudiés (traction uniaxiale). Les influences de plusieurs paramètres du modèle sont étudiées dont la taille de maille et la présence de fissures discrètes. Dans un second temps, une étude expérimentale des stratifiés entaillés soumis à des sollicitations combinées à l'échelle supérieure (détail structural) est menée à l'aide du montage VERTEX conçu spécifiquement pour ces travaux. La modélisation de ce type d'essai est amorcée sur une plaque en aluminium pour valider la méthodologie de transfert de conditions limites obtenues par corrélation d'images. Cette stratégie est ensuite appliquée à un des drapages composite étudiés pour modéliser plusieurs types de sollicitations pour valider le « Discrete Ply Model » sur des cas de charge supplémentaires. / Composite structures sensitivity to substantial damage around notches leads us to assess their damage tolerance. First, both experiments and numerical simulations are being performed on small coupons under uniaxial tension for different configurations (plain, open-hole With different diameters and U-notch specimens). Using different methods of in situ experimental monitoring (image correlation, infrared thermography) and post-mortem micro-tomography (X-ray), failure scenarios are identified and compared to those determined by numerical simulations. The "Discrete Ply Model" is then proven valid to simulate the cases studied (uniaxial tension). The influences of several parameters such as mesh size and the presence of discrete cracks are investigated. Second, an experimental study of notched laminates subjected to complex loadings (structural detail scale) is conducted With the VERTEX rig, designed specifically for this work. Then, simulating this ype of tests is initiated on an aluminum plate to validate the methodology of boundary conditions (obtained by image correlation) transfer. This strategy is then applied to a notched laminated composite to validate the "Discrete Ply Model" on additional loading cases.

2.5 million people sustain a traumatic brain injury (TBI) annually in the United States. Although there is potential for functional recovery following TBI, there is no definitive treatment to improve recovery after TBI. Our lab has shown that TBI enhances an endogenous neurogenic response in the subventricular zone and hippocampus. TBI-induced neural stem cells (NSCs) can integrate into regions such as the hippocampus and olfactory bulb. Although the mechanism behind TBI-enhanced neurogenesis remains unknown, the Notch signaling pathway has been implicated as a regulator in the maintenance and survival of NSCs. This thesis explores the effects of Notch pathway manipulation on functional recovery following TBI. We hypothesize that Notch signaling plays a critical role in recovery after TBI. Activation of this pathway via a Notch agonist (Notch1) will facilitate post-injury recovery while inhibition of this pathway via a Notch antagonist (recombinant Jagged-1 Fc) will deter post-injury recovery. Functional recovery was assessed within 30 days or 60 days post-injury in two different cohorts of animals. The behavior assays conducted in this study included motor, cognitive, and olfactory assessment. In the 30-day phase, Notch pathway manipulation following TBI did not affect functional performance. In the 60-day study, significant group differences were found. While the FPI+Vehicle animals exhibited a functional recovery in Morris water maze, injured animals with Notch inhibition failed to show this cognitive recovery, indicating the involvement of the Notch pathway in cognitive recovery at the chronic stage following TBI. Motor and olfaction were not significantly affected by Notch pathway manipulation.

Hey1, Hey2 and HeyL are downstream effectors of the Notch signalling pathway. Hey genes play decisive roles during embryonic development for example in cardiovascular development. However, the precise transcriptional programmes and genes, which are affected by each single Hey gene, are still poorly understood. One drawback for the analysis of Hey1, Hey2 or HeyL single gene function is that these genes are co-expressed in many tissues and share a high degree of functional redundancy. Thus, it was necessary to establish a system, which is either devoid of Hey expression, or just comprises one single Hey gene family member. For this, Hey1(fl/fl)/Hey2(-/-)/HeyL(-/-)- as well as Hey-triple- knock out (KO)-ES cells (embryonic stem cells) were generated in this work, because ES cells and their differentiation as EBs (embryoid bodies) represent a valuable tool for the in vitro analysis of embryonic developmental processes. After the establishment of Hey1(fl/fl)/Hey2(-/-)/HeyL(-/-)- and Hey-triple- KO-ES cells, it could be seen by ALP staining and pluripotency marker expression that loss of Hey expression did not affect ES cell pluripotency features. Thus, these ES cells represent bona fide ES cells and could be further used for the differentiation as EBs. Here, differences in gene expression between Hey1(fl/fl)/Hey2(-/-)/HeyL(-/-)- and Hey-triple- KO-ES cells (after the loss of Hey1) could be observed in realtime-RT-PCR analysis for the endodermal marker AFP as well as for neural and myogenic markers in d10 EBs. However, the establishment of inducible Hey1, Hey2 or HeyL ES cell lines will be essential to confirm these findings and to search for novel Hey target genes. To get further insight into the mode of Hey action, the analysis of Hey interaction partners is necessary. One such binding partner, the Bre protein, has previously been found in a yeast-two-hybrid screen. Bre has been described to be a member of two distinct complexes (i.e. the nuclear BRCA1-A complex with a function in DNA damage response and the cytoplasmic BRISC complex), to directly interact with the TNF-receptor and Fas and to interfere with apoptotic signalling. The Hey-Bre interaction could be further corroborated in this work; yet, it was not possible to narrow down the interaction site of Bre with Hey1. It rather seems that non-overlapping parts of the Bre protein may bind to Hey. This interaction may be direct– pointing to more than one interaction site inside the Bre protein – or via a common binding partner such as the endogenous Bre protein itself. Besides the interaction studies, functional assays were performed for a more detailed characterisation of Hey1 and Bre interaction. Here, it could be shown that Hey1 over-expression did not have any influence on Bre sub-cellular localisation. Interestingly, it could be demonstrated that Bre positively interfered with Hey1 repressive function in luciferase assays at three of four promoters analysed. Moreover, interaction with Bre seems to lead to a stabilisation of Hey1. As Bre has been described to modulate the E3-ligase activity intrinsic to the BRCC complex it was analysed whether Bre over-expression results in an ubiquitination of Hey1. Yet, this could not be observed in the present work. Furthermore, an interaction of Bre with ubiquitinated proteins could not be demonstrated in an ubiquitin binding assay. To obtain a better insight into Bre function, Bre LacZ gene trap-ES cells and animals were generated. However, realtime-RT-analyses revealed that these cells and mice did not show a loss of Bre expression on mRNA level indicating that insertion mutagenesis did not occur as expected. However, embryos derived from these mice could nevertheless be used for the detection of tissues with Bre expression by β-galactosidase staining. Bre deficiency on mRNA levels was only achieved after the deletion of the floxed exon 3 resulting in the generation of Bre del-mice. Bre del-mice were fertile and without any obvious phenotype and they were used for the generation of Bre del- and wt-MEFs (murine embryonic fibroblasts). Characterisation of these cells showed that proliferation was not affected after loss of Bre (neither under normal nor under stress conditions). However, loss of Bre notably resulted in a reduction in the BRCA1 DNA damage response, in a slightly increased sensitivity towards apoptosis induction by FasL treatment and in an increase in the K63-poly-ubiquitin content in Bre del-cytoplasmic fractions, probably linked to a change in the BRISC de-ubiquitinase activity. Even though these results have the same tendencies as observed in former studies, the effects in the present work are less striking. Further studies as well as intercrossing of Bre del- to Hey KO-animals will be necessary to further understand the functional relevance of Hey and Bre interaction. / Hey1, Hey2 und HeyL sind Zielgene des Notch Signalwegs und spielen eine entscheidende Rolle während der Embryonalentwicklung, z. B. bei der Bildung des kardiovaskulären Systems. Die genauen Effekte eines jeden einzelnen Hey Gens auf Transkriptionsprogramme und einzelne Gene sind allerdings noch relativ unbekannt. Einer der Gründe hierfür liegt vermutlich in der Koexpression von Hey-Proteinen in vielen Geweben bzw. in der daraus resultierenden funktionellen Redundanz. Daher sollte in dieser Arbeit ein System entwickelt werden, in dem entweder keines oder jeweils nur eines der Hey-Gene intakt ist. Hierzu wurden Hey1fl/fl/Hey2-/-/HeyL-/- und Hey-triple-knock out (KO) ES-Zellen (embryonale Stammzellen) etabliert. ES-Zellen stellen ein hervorragendes Modellsystem für die Embryonalentwicklung dar, weil ihre in vitro Differenzierung als sog. „embryoid bodies“ (EBs) embryonale Entwicklungsprozesse widerspiegelt. Der Verlust der Hey-Genexpression hatte keinen Einfluss auf den Stammzellcharakter der etablierten Zellen, da sowohl die generierten Hey-triple-KO- als auch die Hey1fl/fl/Hey2-/-/HeyL-/--ES-Zellen eine positive ALP-Färbung sowie eine hohe Expression von Pluripotenzmarkern zeigten. Daher konnten die Zellen im Folgenden als EBs differenziert und auf Genexpressionsunterschiede während der Differenzierung untersucht werden. Zwischen Hey1fl/fl/Hey2-/-/HeyL-/-- (mit intakter Hey1-Expression) und Hey-triple- KO- ES Zellen konnten an EB Tag 10 mittels realtime-RT-PCR Unterschiede in der Genexpression für den endodermalen Marker AFP, sowie für neurale und myogene Marker festgestellt werden. Um diese Ergebnisse zu bestätigen, aber auch, um neue Hey Zielgene ausfindig machen zu können, ist jedoch die Etablierung induzierbarer ES-Zellen (für Hey1, Hey2 bzw. HeyL) notwendig. Um einen tieferen Einblick in die Funktionsweise der Hey-Gene gewinnen zu können ist die Untersuchung von Hey Interaktionspartnern wichtig. Das Bre-Protein ist ein solcher Bindepartner und wurde zuvor in einem Yeast-two-hybrid Assay gefunden. Bre ist in zwei verschiedenen Komplexen beschrieben worden: dem nukleären BRCA1-A-Komplex, der eine Rolle bei der Detektion von DNA-Schäden spielt und dem cytoplasmatischen BRISC-Komplex. Es ist außerdem bekannt, dass Bre direkt mit dem TNF-Rezeptor und mit Fas interagiert und die apoptotische Antwort in der Zelle beeinflusst. Die Interaktion zwischen Bre und Hey1 konnte in dieser Arbeit zunächst bestätigt werden; in weiteren Ko-immunpräzipitations-Experimenten war es aber nicht möglich, den Bereich des Bre-Proteins zu bestimmen, der die Interaktion mit Hey1 vermittelt, da verschiedene nicht überlappende Bereiche des Bre-Proteins eine Interaktion mit Hey1 zeigten. Ob es sich hierbei um direkte Interaktionen handelte und Bre somit mehrere Bindestellen für Hey1 aufweist oder ob die Interaktion indirekt über einen gemeinsamen Bindepartner wie z.B. das endogene Bre-Protein selbst vermittelt wird, ist noch nicht geklärt. Für eine weitere Charakterisierung der Interaktion zwischen den beiden Proteinen wurden funktionelle Versuche durchgeführt. Hierbei konnte gezeigt werden, dass die Überexpression von Hey1 keinen Einfluss auf die subzelluläre Lokalisation des Bre Proteins hat. Mit Hilfe von Luziferase Assays konnte aber interessanterweise nachgewiesen werden, dass Bre bei drei von vier untersuchten Promotern positiv auf die Repression durch Hey1 einwirkte. Außerdem scheint die Überexpression von Bre möglicherweise eine Stabilisierung des Hey1-Proteins zu bewirken. Da Bre eine Verstärkung der E3-Ligasefunktion des BRCC-Komplexes zugeschrieben wird, wurde außerdem untersucht, ob die Überexpression von Bre zu einer Ubiquitinylierung von Hey1 führt. Dies konnte allerdings nicht festgestellt werden. Desweiteren konnte in einem Ubiquitin-Bindeassay keine Interaktion von Bre mit anderen ubiquitinylierten Proteinen gezeigt werden. ...