Genome instability is a hallmark of human cancers. Patients with Bloom’s syndrome, a rare chromosome breakage syndrome caused by inactivation of the RecQ helicase BLM, result in phenotypes associated with accelerated aging and develop cancer at a very young age. Patients with Bloom’s syndrome exhibit hyper-recombination, but the role of BLM and increased genomic instability is not fully characterized. Sgs1, the only member of the RecQ family of DNA helicases in Saccharomyces cerevisiae, is known to act both in early and late stages of homology-dependent repair of DNA damage. Exo1, a 5′–3′ exonuclease, first discovered to play a role in mismatch repair has been shown to participate in parallel to Sgs1 in processing the ends of DNA double-strand breaks, an early step of homology-mediated repair. Here we have characterized the genetic interaction of SGS1 and EXO1 with other repair factors in homology-mediated repair as well as DNA damage checkpoints, and characterize the role of post-translational modifications, and protein-protein interactions in regulating their function in response to DNA damage. In S. cerevisiae cells lacking Sgs1, spontaneous translocations arise by homologous recombination in small regions of homology between three non-allelic, but related sequences in the genes CAN1, LYP1, and ALP1. We have found that these translocation events are inhibited if cells lack Mec1/ATR kinase while Tel1/ATM acts as a suppressor, and that they are dependent on Rad59, a protein known to function as one of two sub-pathways of Rad52 homology-directed repair. Through a candidate screen of other DNA metabolic factors, we identified Exo1 as a strong suppressor of chromosomal rearrangements in the sgs1∆ mutant. The Exo1 enzymatic domain is located in the N-terminus while the C-terminus harbors mismatch repair protein binding sites as well as phosphorylation sites known to modulate its enzymatic function at uncapped telomeres. We have determined that the C-terminus is dispensable for Exo1’s roles in resistance to DNA-damaging agents and suppressing mutations and chromosomal rearrangements. Exo1 has been identified as a component of the error-free DNA damage tolerance pathway of template switching. Exo1 promotes template switching by extending the single strand gap behind stalled replication forks. Here, we show that the dysregulation of the phosphorylation of the C-terminus of Exo1 is detrimental in cells under replication stress whereas loss of Exo1 suppresses under the same conditions, suggesting that Exo1 function is tightly regulated by both phosphorylation and dephosphorylation and is important in properly modulating the DNA damage response at stalled forks. It has previously been shown that the strand exchange factor Rad51 binds to the C-terminus of Sgs1 although the significance of this physical interaction has yet to be determined. To elucidate the function of the physical interaction of Sgs1 and Rad51, we have generated a separation of function allele of SGS1 with a single amino acid change (sgs1-FD) that ablates the physical interaction with Rad51. Alone, the loss of the interaction of Sgs1 and Rad51 in our sgs1-FD mutant did not cause any of the defects in response to DNA damaging agents or genome rearrangements that are observed in the sgs1 deletion mutant. However, when we assessed the sgs1-FD mutant in combination with the loss of Sae2, Mre11, Exo1, Srs2, Rrm3, and Pol32 we observed genetic interactions that distinguish the sgs1-FD mutant from the sgs1∆mutant. Negative and positive genetic interactions with SAE2, MRE11, EXO1, SRS2, RRM3, and POL32 suggest the role of the physical interaction of Sgs1 and Rad51 is in promoting homology-mediated repair possibly by competing with single-strand binding protein RPA for single-stranded DNA to promote Rad51 filament formation. Together, these studies characterize additional roles for domains of Sgs1 and Exo1 that are not entirely understood as well as their roles in combination with DNA damage checkpoints, and repair pathways that are necessary for maintaining genome stability.

This thesis explains the design, fabrication and characterization steps of a high dynamic range CMOS-MEMS capacitive accelerometer array and on-chip environmental sensors for bias drift compensation. Inertial navigation under harsh environments requires a high dynamic range accelerometer that can survive and provide continuous readout accuracy through shock events, while having a large dynamic range to capture fine-scale motions. The dynamic range target is set as 156 dB in accordance with navigation standard macro-electromechanical accelerometers, which corresponds to around 1 mG acceleration resolution in 50 kG input range. The small accelerometer cell design ensures shock survivability (e.g. up to 50 kG) by keeping the stress at the anchors below the fracture strength of thin-film oxide. Arraying multiple accelerometer cells in parallel lowers the fundamental thermomechanical noise limit set by the small mass of the individual accelerometer cells. Resonance frequency staggering between accelerometer cells suppresses ring-down oscillations. Parasitic capacitance of the high-impedance transduction signal is important to mitigate; undercut of the underlying silicon substrate and an aluminum etch of the top metal layer, incorporated in the CMOS-MEMS process flow, reduces the parasitic capacitance and improves sensitivity. PTAT temperature sensors, piezoresistive stress sensors and resonator-oscillators integrated across the accelerometer chip provide high-resolution environmental measurements for the compensation of long-term bias and scale factor drift. Simultaneous measurements from the accelerometer and environmental sensors demonstrate the correlation between environmental variations and long-term drift. Finite-element analysis shows that the scale factor stability of the accelerometer can be improved up to 1 ppm given the sensor array’s measurement resolution. The CMOS-MEMS accelerometer system-on-chip is fabricated in a TowerJazz 0.18 μm CMOS process. The post-CMOS MEMS processing steps are tuned to reduce the top metal milling and sidewall polymer deposition. A reactive ion etch recipe is developed for the removal of the top metal in order to reduce the parasitic capacitance and eliminate the risk of metal creep at spring beam anchors, thereby improve the bias stability. The PTAT temperature sensors have 3.1 mV/K measured sensitivity and 7.1 mK resolution with high repeatability. The compensation of the accelerometer readout for temperature variations down to 7.1 mK translates to 2.6 ppm scale factor stability for the accelerometer. The characterization of the stress sensors through the application of normal stress on the device package leads to an uncertainty in the amount of stress transferred to the stress sensors on the chip surface. The maximum measured stress sensitivity is 36.5 pV/Pa, which leads to 24.7 kPa stress resolution and translates to 1.7 ppm scale factor stability for the accelerometer without taking the stress attenuation into account. The measured sensitivity sets a lower bound on the sensitivity of the stress sensors implying that the stress resolution and the corresponding accelerometer scale factor stability is higher in practice. The measured frequency stability of the resonator-oscillator is 0.4 ppm, thereby the resonance frequency based variations of the accelerometer readout can be compensated to reach up to 0.8 ppm scale factor stability. However, the initial drift in the resonance frequency of the oscillators due to dielectric charging requires a long wait-time before these sensors can be used for accelerometer drift compensation. The accelerometer array is demonstrated to have 23.7 mG/√Hz noise floor and 70 mG bias stability. The maximum input acceleration applied on the device is limited to 4 kG by the split Hopkinson bar test setup. Improvement of the setup to transfer acceleration amplitudes up to 50 kG should validate the designed input range of the accelerometer array and lead to 117 dB dynamic range for the current design. The measurement bandwidth is fundamentally set by the 126 kHz resonance frequency of the accelerometer cells and can be further limited by filtering the readout signal to attenuate the transient oscillations faster. The nonlinearity of the accelerometer response is better than 1.2% in ±10 kG input range; however, it gets up to 19.0% in ±50 kG maximum input range. The long term bias drift of the accelerometer is shown to be correlated with the temperature and stress variations. Compensation of the accelerometer readout based on the stress and temperature sensor measurements leads to an observable improvement in the long term drift. However, the bias stability of the accelerometer is limited by excessive flicker noise in the system, which is believed to result from noise folding from higher frequencies. Suppression of the flicker noise in the system should allow for a more detailed study of the effect of environmental variations on the accelerometer readout and evaluation of more elaborate fitting algorithms for model based prediction and compensation of the bias drift to reach the target bias stability and dynamic range.

Mon travail de thèse s'intéresse au rôle du récepteur à l'immunité innée NLRP3, composant essentiel de l'inflammasome, dans le développement tumoral pulmonaire. Nos résultats révèlent que les cellules épithéliales pulmonaires immortalisées expriment un inflammasome NLRP3 fonctionnel. De façon inattendue, nous montrons que l'expression du récepteur NLRP3 est fortement diminuée, voire perdue des lignées tumorales de CBNPC et dans des tumeurs de patients, comparé au tissu sain adjacent. Nous montrons que NLRP3, de façon totalement indépendante de l'inflammasome, est impliquée dans la régulation transcriptionnelle de H2AFX, le gène codant pour le variant d'histone H2AX, élément clé de la signalisation des dommages à l'ADN. L'absence de NLRP3 dans les cellules HBEC altère l'amplification et la transmission du signal en réponse à des cassures double brin, résultant in fine à moins de réparation. Ce défaut de réparation des cassures se traduit par une instabilité génomique, qui est en effet plus forte dans les adénocarcinomes pulmonaires exprimant de faible niveau de NLRP3. Mon travail de thèse identifie donc le récepteur NLRP3 comme un facteur clé de la réponse aux dommages à l'ADN et du maintien de l'intégrité génomique en promouvant la transcription de H2AFX dans les cellules épithéliales pulmonaires. Ce nouveau rôle de NLRP3, associé à sa perte dans les tumeurs de CBPNC en font un potentiel suppresseur de tumeur / During my PhD, I have been interested in the role of the innate immune receptor NLRP3, a key component of the inflammasome, in lung cancer development. Our results show the presence of a functional NLRP3 inflammasome in normal human bronchial epithelial cells (HBEC). Surprisingly, NLRP3 expression is strongly down-regulated in a large panel of NSCLC cell lines and patient tumors compared to healthy tissue. Moreover, we unravel that NLRP3 contributes to the transcription of H2AFX, the coding gene for the histone variant H2AX, in an inflammasome independent-manner. The deletion of NLRP3 in HBEC impairs double strand break signal amplification and transduction, resulting in a decrease in DNA repair. This repair defect leads to genomic instability, which is increased in lung adenocarcinomas expressing low levels of NLRP3. My PhD work identifies NLRP3 as a key factor of the DNA damage response and genomic integrity maintenance by regulating the transcription of H2AFX. This new role for NLRP3, together with its loss in NSCLC, makes it as a potential tumor suppressor

L'hélium solide métastable est un candidat possible pour la supersolidité. Notre équipe a démontré en 2011 qu'on pouvait obtenir de l'hélium solide métastable à des pressions inférieures à la pression de fusion à l'aide d'une onde acoustique focalisée. Cependant, une instabilité inattendue apparaît lorsque la pression locale du crystal atteint 21 bar c'est à dire 4 bar sous la pression de fusion. J'ai donc commencé ma thèse en étudiant le temps d'apparition de l'instabilité, et j'ai confirmé qu'elle apparaît toujours dans des phases de décompression de l'onde sonore, c'est à dire à une pression inférieure à la pression de fusion. Ensuite, j'ai étudié la limite de cavitation de l'hélium superfluide à pression négative. En utilisant une méthode interférométrique développée par mon prédécesseur Fabien Souris, j'ai mesuré directement la densité de cavitation de l'hélium superfluide métastable. J'ai trouvé que, à 1 K, l'hélium superfluide cavite lorsque sa densité locale a diminué de 8.4%. En utilisant une équation d'état bien établie théoriquement, on peut convertir ce résultat en pression de cavitation pour le comparer avec ceux obtenus par d'autres groupes. À ma grande surprise, mon résultat n'est pas compatible avec ces derniers. Cette incompatibilité soulève des questions intéressantes quant à la possibilité de nucléation de la bulle sur des vortex quantifiés. Enfin, j'ai étudié la dynamique de la bulle d'hélium déclenchée par la cavitation. En analysant l'équation du mouvement de la bulle et le transfert de chaleur correspondant, j'ai expliqué avec succès pourquoi la durée de vie de la bulle a une transition dramatique quand l'hélium passe de liquide normal à superfluide. / Metastable solid helium is a possible candidate for supersolidity. In 2011, our group has demonstrated that we could obtain the metastable solid helium at pressures below the melting pressure using a focused acoustic wave. However, an unexpected instability occurs when the local pressure of the crystal reaches 21 bar which is 4 bar below the melting pressure. So I started my thesis by studying the appearance time of the instability, and I confirmed that it always appears at the low pressure swing of the acoustic wave. Then, I studied the cavitation limit of superfluid helium at negative pressure. Using an interferometric method developed by my predecessor Fabien Souris, I directly measured the cavitation density of metastable superfluid helium. I found that at 1 K, superfluid helium cavitates when its local density is lowered by 8.4%. Using a theoretically well-established equation of state, this result can be converted to a cavitation pressure in order to compare our results with those obtained by others groups. To my surprise, my result is not consistent with the others'. This incompatibility raises interesting questions about the possibility of nucleation of the bubble on quantified vortices. Finally, I studied the dynamics of the helium bubble triggered by cavitation. By analyzing the equation of motion of bubble and the corresponding heat transfer, I have successfully explained why the bubble's lifetime has a dramatic transition as the helium passes from normal liquid to superfluid.

[pt] O objetivo desta tese é estudar os mecanismos de escape em sistemas estruturais sensíveis a imperfeições quando submetidos a certas classes de carregamentos dinâmicos, identificar os parâmetros que controlam o escape e criar critérios capazes de prever a fronteira de escape e a perda de estabilidade da estrutura no espaço dos parâmetros de controle. Isto permitirá um melhor entendimento dos processos de perda de estabilidade e servirá de base para o cálculo e controle da integridade dessas estruturas. Após a descrição dos fenômenos que podem ocorrer na dinâmica dessa classe de estruturas, são testados e adaptados alguns critérios existentes na literatura, que verificam a estabilidade de uma estrutura a partir do conhecimento dos parâmetros de controle. Em seguida estuda-se a evolução da estabilidade global do conjunto das soluções medida pela área da bacia de atração, e pelas características de sua fronteira. Desenvolvem-se expressões gerais para o critério de Melnikov, e mostra-se, a partir de perturbações aleatórias nos parâmetros de controle e na força externa, que essas expressões podem ser tomadas como um limite inferior para o carregamento de escape e conseqüentemente como uma contribuição para o desenvolvimento de critérios de projeto. Verifica-se também que os valores obtidos pelos critérios de escape podem ser tomados como limites superiores para o valor da força de escape. / [en] The purpose of this thesis is to study the escape mechanisms in imperfection sensitive structural systems under certain dynamical loading conditions. Other objectives are to identify the parameters that control the escape phenomenon and to create some criteria capable of predicting the escape boundary and the structures stability in the control parameters space. This will allow a better understanding of the stability loss process and can serve as a basis to the integrity control and design of these structures. After a description of the phenomena that can occur in the dynamics of this class of structures, some predictive criteria, found in literature, that verify the structure stability based on the control parameters knowledge, are adapted and tested. Following is a study of the evolution of the global stability of the set of solutions measured by the basin of attraction area, and by the characteristics of its boundary. Some general expressions for the Melnikov criterion are developed, and it is shown by randomly perturbing the control parameters and the external force, that these expressions can be taken as a lower bound for the escape load, and consequently as a contribution to the development of design criteria. It is also observed that the values obtained by the escape criteria can be taken as an upper bound for the values of the escape force. / [es] EL objetivo de esta tesis es estudiar los mecanismos de escape en sistemas extructurales que son sensibles a imperfecciones cuando son sometidos a ciertas clases de cargas dinámicas. Outro objetivo es identificar los parámetros que controlan el escape y crear criterios capaces de preveer la frontera de escape y la pérdida de estabilidad de la extructura en el espacio de los parámetros de control. Esto permitirá una mejor comprensión de los procesos de pérdida de estabilidad y servirá de base para el cálculo y control de la integridad de esas extructuras. Después de describir los fenómenos que pueden ocurrir en la dinámica de esta clase de extructuras, se prueban y adaptan algunos criterios existentes en la literatura, que verifican la estabilidad de una extructura a partir del conocimiento de los parámetros de control. Seguidamente, se estudia la evolución de la estabilidad global del conjunto de las soluciones, se dearrollan expresiones generales para el criterio de Melnikov, y se muestra, a partir de perturbaciones aleatorias en los parámetros de control y en la fuerza externa, que esas expresiones pueden ser tomadas como límite inferior para la carga de escape y conseqüentemente como una contribución para el desarrollo de criterios de proyecto. Se verifica también que los valores obtenidos por los criterios de escape pueden ser tomados como límites superiores para el valor de la fuerza de escape.

[pt] Arco segmentado é uma estrutura composta de vários segmentos retilíneos,emendados em pontos pertencentes a uma curva em forma de arco que circunscreve a estrutura. A coincidência dos pontos das emendas (nós) com essa curva permite a redução dos momentos fletores nesses pontos. O arco segmentado assim idealizado apresenta algumas vantagens para a indústria de pré-moldados, quais sejam: permite a utilização das fôrmas empregadas na moldagem de outros tipos de peças retilíneas como pilares e vigas; permite a construção de arcos com qualquer relação entre a flecha e o vão, empregando-se as mesmas fôrmas, bastando para isso variar os ângulos entre os segmentos emendados. Neste trabalho é feito um estudo teórico sobre a estabilidade dos arcos segmentados,utilizando para isto o modelo computacional baseado no método nos elementos finitos. Dentro deste estudo são realizadas análises sobre a influência da relação altura/vão, da variação do número de segmentos, da presença de imperfeições iniciais e da ação do vento no comportamento global destes arcos. Também é desenvolvido um procedimento para o cálculo da carga crítica dos arcos segmentados bi e tri-articulados e que leva em consideração tanto as cargas caracterizadas por pontos limite como as por pontos de bifurcação. São apresentados os detalhes e resultados de um ensaio experimental de um arco segmentado de concreto armado, que teve como objetivo principal a verificação do comportamento global deste tipo de estrutura e da eficiência do modelo computacional aqui utilizado. / [en] Segmental arch is a structure made of several straight segments of precast concrete connected at points belonging to a curve in the form of an arch which circumscribes the structure. This type of arch has some advantages for the precasting industry: a) it allows the use of the same forms used to cast other types of straight pieces such as beams and columns; b) it allows the construction of arches with any height/span ratio, using the same forms, which is achieved just by varying the angle between the connected segments. In this work, a study of the stability of this type of arch is developed, with the use of a computer model based on the finite elements method. In side of this study, the effects of height/span ratio variation, segment number variation, initial imperfections presence and the wind action are investigated. In addition, a calculus procedure of the critical load to the segmental arches is developed. This procedure takes in consideration the loads characterized by limit points as much as that by bifurcation points. Is also presented an experimental test, of a segmental arch made of reinforced concrete, that was conducted with the purpose of studying the global behavior of this type of structure, and verify the efficiency of the computer model used. / [es] El arco segmentado es una extructura compuesta de varios segmentos rectilíneos,enmendados en puntos que pertenecen a una curva en forma de arco que circunscribe la extructura. La intercepción de los puntos de las enmiendas (nodos) con esa curva permite la reducción de los momentos flectores en esos puntos. El arco segmentado presenta algunas ventajas para la industria de premoldados como son: permite la reutilización de las formas para el moldeado de otros tipos de piezas rectilíneas como pilares y vigas; permite la construcción de arcos con cualquier relación entre la flecha y el vano, utilizando las mismas formas, tan solo variando los ángulos entre los segmentos enmendados. En este trabajo, se desarrolla un estudio teórico sobre la estabilidad de los arcos segmentados,utilizando el modelo computacional basado en el método en los elementos finitos. Dentro de este estudio se analiza la influencia de: la relación altura/vano, la variación del número de segmentos, la presencia de imperfecciones iniciales y la acción del viento en el comportamiento global de estes arcos. También se desarrolla un procedimiento para el cálculo de la carga crítica de los arcos segmentados bi y triarticulados que lleva en consideración tanto las cargas caracterizadas por puntos límites como por puntos de bifurcación. Se presentan detalles y resultados de un ensayo experimental de un arco segmentado de concreto armado, cuyo objetivo principal es verificar el comportamiento global de este tipo de extructura y la eficiencia del modelo computacional aqui utilizado.

[pt] Dutos são sistemas eficientes e seguros para o transporte de óleo e gás. O estudo da capacidade de carga de dutos em condições de serviço envolvendo o transporte de produtos agressivos e sujeitos a altas temperaturas e pressões ganha atualmente maior interesse dada a severidade das condições de operação e a crescente necessidade de manutenção e adaptação dos dutos existentes às condições atuais de serviço. No entanto,a presença de defeitos e imperfeições em segmentos de dutos pode ocasionar a redução na resistência do mesmo e eventualmente sua falha. Este trabalho tem como objetivo a previsão do comportamento de dutos sujeitos a cargas combinadas, tendo em vista os carregamentos que podem ocorrer em campo. São contemplados nestes carregamentos efeitos térmicos, movimentos geotécnicos,pressão de operação. No caso de assentamento de solo, o duto pode estar submetido a efeitos de flexão que resultam em curvaturas excessivas. Como consequência ocorre a flambagem local do duto e desenvolvem-se rugas. Nestas condições podem surgir deformações acima dos valores limites estabelecidos em normas.Tendo em vista a natureza tri-dimensional do problema e a investigação do fenômeno de flambagem local, faz-se necessário o entendimento do comportamento tridimensional de cascas em condições de grandes deslocamentos e grandes deformações em regime elasto- plástico.Neste trabalho desenvolvem-se alguns modelos para a previsão do comportamento de dutos submetidos a cargas combinadas. Procura-se reproduzir as condições de ensaio de laboratório de modo a se desenvolver um modelo de previsão confiável. Para tal foram tomados como referência alguns resultados de ensaios disponíveis na literatura. Aspetos de modelagem de condições de contorno e carregamento são enfocados. Dado o desenvolvimento de rugas e de grandes deformações estão presentes também outros aspectos importantes da modelagem como o aparecimento de regiões de auto-contato nas rugas.Alguns exemplos numéricos analisados com o programa comercial de elementos finitos ABAQUS são apresentados. / [en] Pipelines are efficient and safe systems for the transport of oil and gas. The study of the load capacity of these systems under functional conditions which include the transport of aggressive products under high temperatures and pressures has gained larger interest due to the severe operation conditions and growing needs for maintenance and adaptation of the existing pipelines to these conditions. However, the presence of defects and imperfections in pipe segments may lead to reduction of resistance and eventually to failure. The scope of this dissertation is the prediction of the behavior of pipelines subjected to combined load conditions and sequences till collapse, reproducing those which occur on field and in laboratory tests. Axial forces, internal pressure and bending are applied to the model in order to reproduce thermal effects, operating pressure and ground movements. In the case of soil settlement the pipeline may be subject to bending effects which result in excessive curvatures. As a consequence local buckling occurs with the development of wrinkles. Under these conditions strains above those recommended in the codes can take place.Due to the three dimensional nature of the problem the investigation of the local buckling phenomena, requires the understanding of the behavior shells under large displacement and large strain conditions in elasto- plastic regime.In the present work some models are developed for the prediction of the behavior of pipelines subjected to combined loading. Validation of these models allows us to accompany and establish experimental programs. Some reference laboratory results available in the literature are included. Aspects of the modeling such as boundary and loading conditions, material modeling and solution procedure are addressed. The development of wrinkles and large deformations brings into the model additional aspects such as the presence of self-contact areas. Numerical examples are analyzed using the commercial finite element program ABAQUS.

L’instabilité génomique est une caractéristique majeure des cellules cancéreuses. Dans les premières étapes du développement du cancer, l’activation des oncogènes induit du stress réplicatif à l’origine de cette instabilité. Le mécanisme par lequel la dérégulation des oncogènes induit un blocage des fourches de réplication et du gammaH2AX sur la chromatine reste peu compris. Ainsi déterminer l'origine de ce stress réplicatif dans les cellules précancéreuses est donc essentiel afin de mieux comprendre les premières étapes de la tumorigenèse. Il a été montré dans notre laboratoire que la déplétion dans des cellules humaines de la topoisomérase 1 ou du facteur d’épissage ASF/SF2 perturbe la progression des fourches de réplication, active le checkpoint de phase S et induit des cassures chromosomiques (Tuduri et al., 2009). Puisque les dommages à l’ADN et les défauts de réplication sont corrigés par la RNase H1, il est possible que ce stress réplicatif soit dû à la formation de R-loops. Ces hybrides ADN/ARN se forment au cours de la transcription lorsque l’ARN naissant revient s’hybrider avec sa matrice d’ADN laissant le brin non-transcrit sous forme simple brin. Les R-loops se formant dans des sites spécifiques du génome, nous avons alors cartographié leurs distributions et l’avons comparé à celle de marqueurs de stress réplicatif et de cassure double brin (CDB) de l’ADN dans nos cellules shASF et shTop1. Nous avons donc combiné différentes approches génomiques comme le DRIP-seq (R-loops), le ChIP-seq (pRPA et gammaH2AX) et le BLESS (CDB ; Crosetto et al., Nature Methods, 2013). Nos données montrent une corrélation importante entre les régions formant du stress réplicatif et la formation de R-loops appuyant l’idée que l’interférence entre réplication et transcription augmente l’instabilité génomique dans les cellules humaines. Toutefois puisque les R-loops ont de multiples rôles physiologiques, toutes régions qui en forment ne corrèlent pas avec l’induction de stress réplicatif. Ce projet devrait nous aider à déterminer dans quelles conditions les R-loops représentent une menace pour l’intégrité du génome. Par microscopie confocale à fluorescence, nous avons confirmé que les R-loops s’accumulaient dans nos lignées HeLa déplétées pour ASF et Top1. A noter que les R-loops s’accumulent également dans des fibroblastes immortalisées exprimant la forme oncogénique de Ras et dans des préplasmablastes, une étape du développement plasmocytaire particulièrement à risque pour le développement de myélome multiple. Ensemble, ces données indiquent que les R-loops pourraient être une source du stress réplicatif induit par les oncogènes. / Genome instability is a hallmark of cancer cells. It has been proposed that at early stages of the cancer process, genomic instability is caused by oncogene-induced replication stress, a poorly-understood process characterized with the accumulation of stalled replication forks and gammaH2AX on chromatin. Understanding the origin of chronic replication stress represents a major challenge in cancer biology. We have previously shown that depletion of DNA Topoisomerase 1 or the splicing factor ASF/SF2 in mammalian cells interferes with replication fork progression, activating the DNA damage response and inducing chromosome breaks (Tuduri et al., 2009). Since DNA damage and replication fork stalling are relieved by RNaseH1, an attractive hypothesis could be that replication stress is caused by R-loops. These RNA-DNA hybrid structures form when nascent RNA re-anneals to the template DNA strand, leaving the non-template strand unpaired. Using immunofluorescence confocal microscopy with the S9.6 antibody that recognizes RNA-DNA hybrids, we confirmed that R-loops accumulate in ASF/SF2 and Top1-depleted HeLa cells. Since R-loops are enriched at specific sites in the human genome, wecombined different genomic approaches, including DRIP-seq (R-loops), ChIP-seq (gammaH2AX, pRPA) and BLESS (DSBs; Crosetto et al., Nature Methods, 2013) to monitor their distribution relative to replication stress markers and DNA double-strand breaks (DSBs) in the absence of Top1 or ASF/SF2. . Our data reveal a significant correlation between replication stress and cotranscriptional R-loops, supporting the view that the interference between replication and transcription promotes genomic instability in human cells. However, not all R-loops forming regions colocalize with replication stress since these structures have multiple physiological roles. This approach allowed us to determine the conditions in which R-loops may represent a threat to genome integrity. Moreover, we also observed the accumulation of R-loops in immortalized fibroblasts expressing an oncogenic form of Ras and in preplasmablast during plasma cell differentiation, a crucial process during which multiple myeloma may evolve. In clonclusion, our data indicate that R-loops may represent an important source of oncogene-induced replication stress.

We augment a model of endogenous credit cycles by Matsuyama et al.(2016) with human capital to study the impact of human capital on the stability of central economic aggregates. Thus we offer a linkage between human capital formation and credit market instability on a macrolevel combined with an analysis of functional income distribution. Human capital is modelled as pure external effect of production following a learning-by-producing approach. Agents have access to two different investment projects, which differ substantially in their next generations spillover effects. Some generate pecuniary externalities and technological spillovers through human capital formation whereas others fail to do so and are subject to financial frictions. Due to this endogenous credit cycles occur and a pattern of boom and bust cycles can be observed. We explore the impact of human capital on the stability of the system by numerical simulations which indicate that human capital has an ambiguous effect on the evolution of the output. Depending on the strength of the financial friction and the output share of human capital it either amplifies or mitigates output fluctuations. This analysis shows that human capital is an essential factor for economic stability and sustainable growth as a high human capital share tends to make the system's stability robust against shocks. / Series: Department of Economics Working Paper Series

Le raccourcissement télomérique est la source majeure de l'instabilité chromosomique (CIN) au cours de la progression tumorale. Nous avons montré que les cellules humaines embryonnaires de rein (cellules HEK) ayant traversé une période de CIN subissent des vastes changements dans l'expression des microARNs, ce qui induit une transition épithélio-mésenchymateuse (TEM), un processus permettant aux cellules cancéreuses épithéliales migrer et envahir de nouveaux tissus et former des métastases. Notre travail a aussi suggéré que les cellules ayant subi une TEM étaient capables de former des tumeurs dans un microenvironnement sénescent. De surcroît, cette évolution dans la capacité tumorale était associée à une dérégulation supplémentaire des microARNs et à l'acquisition des propriétés des cellules souches. Afin d'étudier comment ce potentiel est mis en place au cours de l'instabilité chromosomique et au contact avec le microenvironnement sénescent, nous avons modulé les niveaux d'expression de miR-145 et avons démontré que la répression de miR-145 était nécessaire pour le développement des caractéristiques des cellules souches. Afin de mieux comprendre l'impact de CIN sur le programme génétique des cellules épithéliales, nous avons utilisé des approches de haut débit et avons caractérisé les changements des paysages chromatiniens et leur mise en place dans les cellules ayant traversé une période de CIN. Nos résultats révèlent pour la première fois que l'instabilité télomérique modifie profondément la distribution des marques d'histones en conduisant aux changements d'expression des gènes et au processus de transformation des cellules épithéliales pré-tumorales. / Telomere shortening is a major source of chromosome instability (CIN) at early stages during carcinogenesis. However, the mechanisms through which telomere-driven CIN (T-CIN) contributes to the acquisition of tumor phenotypes remain uncharacterized. We have shown that human epithelial kidney (HEK) cells undergo massive microRNA deregulation upon CIN, in particular a miR-200-dependent epithelial-mesenchymal transition (EMT), which is thought to enable epithelial cancer cells to migrate and invade other tissues to form metastases. Our work also indicated that CIN+ cells that underwent EMT were able to form tumors in a senescent microenvironment. Notably, this progression in tumor capacity was associated with further microRNA deregulation and the manifestation of enhanced stem-like properties. To investigate how stem-like properties are acquired in CIN+ cells in the contact with senescent microenvironment we adapted knockdown and overexpression approaches to modulate miR-145 expression, and demonstrated that enhanced stem-like properties depended on miR-145 repression. To fully apprehend the impact of CIN on the genetic program of epithelial cells, we used an unbiased approach to characterize the chromatin state of HEK CIN+ cells and uncover genome wide redistributions that were in direct correlation with gene expression changes. Our results reveal for the first time that T-CIN profoundly modifies the chromatin landscape genome-wide thereby fueling the transformation process of pre-tumor epithelial cells.