Common fragile sites are a set of genomic locations with a propensity to form lesions, breaks and gaps on mitotic chromosomes upon induction of replication stress. While the exact reasons for their fragility are unknown, CFS display instability in a cell-type specific manner, suggesting a substantial contribution from an epigenetic component. CFSs also overlap with sites of increased breakage and deletions in tumour cells, as well as evolutionary breakpoints, implying that their features shape genome stability in vivo. Previously, factors such as delays in replication timing, low origin density and transcription of long genes have been implicated in instability at CFS locations but comprehensive molecular studies are lacking. Chromatin structure, an important factor that fits the profile of cell-type specific contributor, has also not been investigated yet. Throughout their efforts to determine the factors that lead to the appearance of CFS lesions, investigators have focused on a single component at a time, potentially missing out complex interactions between cellular processes that could underlie fragility. Additional difficulties come from the cell-type specificity of CFS breakage: it indicates that only cell type-matched data would be informative, limiting the scope for studies using publicly available data. To perform a comprehensive study defining the role of different factors in determining CFS fragility, I explored replication timing, transcriptional landscapes and chromatin environment across a number of CFSs in two cell types exhibiting differential CFS breakage. Initially, I characterised the patterns of CFS fragility in the two cell types on both the cytogenetic and the molecular level. I then used a FISH-based technique to investigate the process of mitotic compaction at active CFS sites and found that the cytogenetically fragile core of these sites sits within larger regions which display a tendency to mis-fold in mitosis. The aberrant compaction of these regions could be observed on cytogenetically normal metaphase chromosomes, suggesting that finer scale abnormalities in chromosome structure underlie the cytogenetically visible breaks at fragile sites. I also investigated the links between transcription of long genes and CFS fragility using two approaches: I quantified levels of expression across all fragile sites using RNA-seq and modified transcription at a single active CFS using the CRISPR genome engineering methodology. My results indicate a complex interplay between transcription and CFS fragility: no simple linear correlation can be observed, but an increase of transcriptional levels at the active CFS led to a corresponding increase in fragility. To investigate the influence of the cell type specific replication programme and replication stress on CFS instability, I mapped replication timing genome-wide in unperturbed cells and under conditions of replication stress in both cell types. I found that replication stress induces bi-directional changes in replication timing throughout the genome as well as at CFS regions. Surprisingly, the genomic regions showing the most extreme replication timing alterations under replication stress do not overlap with CFS, implying that CFS instability is not fully explained by replication delays as previously suggested. Instead, I observed a range of replication-stress induced timing changes across CFS regions: while some CFSs appear under-replicated, others display switches to both earlier and later replication as well as differential recruitment of both early and late origins, implying that dis-regulation of replication timing and origin firing, rather than simply delays, underlie the sensitivity to CFS regions to replication stress. Finally, I investigated large-scale chromatin states at two active CFSs throughout S phase and into G2, the cell cycle stages most relevant stage for CFS breakage. I found that changes in large-scale chromatin architecture accompany the replication timing shifts triggered by replication stress, raising the possibility that such alterations contribute to instability. In conclusion, I assessed the influence of multiple relevant factors on CFS fragility. I found that bi-directional replication timing changes and alterations in interphase chromatin structure are likely to play a role, converging to promote mitotic folding problems which ultimately result in the well-described cytogenetic lesions on metaphase chromosomes and genomic instability.

Expansion of the CGG repeat region of the FMR1 gene from less than 45 repeats to between 55 and 200 repeats is known as the FMR1 premutation. Carriers of the FMR1 premutation may develop a neurodegenerative disease called fragile X-associated tremor/ataxia syndrome (FXTAS), which involves progressive symptoms of tremor, ataxia and cognitive decline. Evidence also suggests that premutation carriers experience other psychiatric difficulties throughout their lifespan. The present study aimed to investigate and delineate neurodegenerative and neurodevelopmental aspects of the premutation utilising primarily fMRI, clinical assessments and molecular measurements in 17 premutation carrier participants and 17 age-matched control participants, aged between 20 and 70 years. The functional imaging protocol included a motor task and an emotional processing task. A battery of clinical and neuropsychological tests outside of the scanner and blood-based measurements of FMR1 CGG repeat length, FMRP levels and FMR1 mRNA levels were also carried out. In the motor task, premutation carriers demonstrated significantly less cerebellar activation than controls during sequential versus random finger tapping (FWEcorr < 0.001). In addition, there was a significant age by group interaction in the hippocampus, inferior parietal cortex and temporal cortex originating from a more negative relationship between brain activation and age in the carrier group compared to the controls (FWEcorr < 0.001). Quantative real-time PCR analysis revealed that mean age-matched FMR1 mRNA levels display a trend towards being higher in carriers and clinical testing of motor skills additionally showed significantly worse tremor and co-ordination scores in non-FXTAS carriers. No significant associations were seen between these measurements and neuroimaging data. During the emotional processing task, carriers exhibited significantly lower activation compared to controls (FWEcorr < 0.001) at the bilateral superior parietal lobe, bilateral Brodmann Area (BA) 17 (V1), right intraparietal area and right BA18 (V2) when comparing high arousal and low arousal conditions. Group by age interaction analyses indicated no significant between group differences at a whole brain level. Clinical assessment revealed that carriers displayed significantly worse symptoms of obsessive-compulsiveness, anxiety, global severity of psychiatric symptoms, facial emotion recognition and autistic traits compared to controls and FMRP levels were comparable between groups. No significant associations were seen between these measurements and neuroimaging data. Here, we present for the first time functional imaging-based evidence for early movement-related neurodegeneration in Fragile X premutation carriers. These changes pre-exist the diagnosis of FXTAS and are greatest in older carriers suggesting that they may be indicative of FXTAS vulnerability. Additionally, we show significantly altered emotional processing at neuropsychological, clinical and functional neuroimaging levels in carriers compared to controls, which appear to display stability over age. Overall, we present new evidence in keeping with possible neurodegenerative and neurodevelopmental traits in FMR1 premutation carriers.

O processo de dano em materiais quasi-frágeis pode ser caracterizado pela perda de isotropia para certos níveis de carga. A localização de deformações, o efeito cooperativo entre regiões danificadas e a avalanche de rupturas são características particulares na medição do dano neste tipo de material. As características mencionadas criam diferentes formas de dissipação de energia, que não são fáceis de representar utilizando métodos baseados na hipótese dos meios contínuos. No presente trabalho uma versão do Método dos Elementos Discretos Formado por Barras é empregado. Neste método a massa do contínuo é concentrada nos nós, os quais são interconectados por barras sem massa. Essas barras possuem uma lei constitutiva bilinear, que é usada para simular a ruptura da estrutura em estudo. A distribuição dos nós permite formar uma treliça tridimensional regular, e a partir dessa discretização espacial é possível chegar a um sistema de equações de movimento, que é resolvido com um esquema explícito de integração numérica (diferenças finitas centrais). Neste método a fratura e a fragmentação são levadas em conta de forma natural, já que as barras que rompem durante o processo são desativadas, respeitando o balanço energético. É possível introduzir heterogeneidade no modelo considerando as propriedades do material como campos espaciais aleatórios com distribuição de probabilidades de Weibull e comprimento de correlação conhecido. Nessa dissertação, é analisado o processo de dano que aparece em estruturas de geometria simples quando solicitadas até o colapso. Diferentes índices são apresentados para realizar a medição do dano. O desempenho desses índices, e a maneira com que eles ajudam na interpretação da evolução do dano, são discutidos nesse trabalho. / The process of damage in quasi-fragile materials is characterized by loss of isotropy for certain load levels. The strain localization, the cooperative effect between damaged regions and the avalanche of ruptures are particular features in measuring the damage in this kind of material. The mentioned features create different forms of energy dissipation, which are not easy to represent with a continuous approach. In the present work a version of the Lattice Discrete Element Method is employed. In this method the mass of the solid is concentrated on node points, which are interconnected by uniaxial elements. These elements have a bilinear constitutive law, which is used to simulate the rupture of the structure under study. The node distribution allows the formation of a regular three-dimensional lattice, and from this spatial discretization it is possible to arrive at a system of equations of motion, which is solved by an explicit numerical integration scheme (central difference). In this method the fracture and fragmentation are taken into account in a natural manner, since the bars that reached their limit strength during the process are disabled of the system, respecting the energy balance. It is possible to introduce heterogeneity in the model considering the material properties as random fields with spatial Weibull probability distribution and known correlation length. In this dissertation, the damage process, which appears in structures of simple geometry, when they are loaded until collapse, is analysed. Different indexes are presented to perform the measurement of the damage. The performance of those indexes, and the way they help in the interpretation of the damage evolution, are discussed in this paper.

Made available in DSpace on 2014-06-11T19:23:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-12Bitstream added on 2014-06-13T20:29:49Z : No. of bitstreams: 1 curtis_kmc_me_araiq.pdf: 767278 bytes, checksum: b9eefeb12752b6e4c943bbfc29b1a46c (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A síndrome do cromossomo X frágil (SXF) é a forma mais comum de deficiência mental herdada. A doença ocorre pela expansão das repetições de trinucleotídeos na região 5’ não traduzida do gene FMR1 no cromossomo X. Dependendo do número de repetições CGG originam-se 4 tipos de alelos: normal (NL), pré-mutado (PM), gray zone (GZ) e mutação completa (FM). A instabilidade e expansão das repetições, aliado à metilação do DNA, causam a diminuição ou ausência na produção da proteína FMRP, a qual é essencial para a função cerebral. O diagnóstico da SXF tem sido realizado principalmente por análise molecular Southern blot. Porém, este método é trabalhoso, demorado e de custo elevado. Recentemente foi desenvolvido um novo método molecular para triagem da SXF por PCR, que segundo os autores, é rápido, de baixo custo, e eficiente na detecção das repetições CGG em homens e mulheres. No entanto, notou-se a ausência de informações importantes para reprodução do método. Os objetivos deste estudo foram: (i) padronizar a técnica de PCR proposta por Tassone et al., (2008), adaptando-a, devido a carência de informações metodológicas; (ii) comprovar a exatidão (acurácia), sensibilidade e especificidade do método, comparando-a ao Southern blot; (iii) avaliar a aplicação da técnica utilizando DNA extraído de diferentes materiais biológicos/métodos de extração; (iv) estimar o custo e o tempo de execução do método no mercado nacional. Os materiais biológicos utilizados foram: sangue coletado por sistema à vácuo e células da mucosa oral, que foram extraídos por solventes orgânicos e sangue coletado em cartões FTA, purificado pelo kit Whatman. Obtevese sucesso na reprodução do método da PCR em 75 indivíduos utilizando a enzima Expand Long Template PCR System (Roche Diagnostics). A exatidão (acurácia), sensibilidade e especificidade foram... / Fragile X Syndrome (FXS) is the most common form of inherited mental retardation. The disease occurs by the expansion of triplet nucleotide repeats in the 5' untranslated of the FMR1 gene on chromosome X. Depending on the number of CGG repeats four types of alleles originate from it: normal (NL), pre-mutated (PM), gray zone (GZ) and full mutation (FM). The instability and expansion of these repetitions, together with the methylation of DNA, cause a decrease or absence in the production of the protein FMRP, which is essential for the brain function. The diagnosis of FXS has been done mainly by molecular analysis Southern blot. However, this method is laborious, time consuming and expensive. Recently we have developed a new molecular method for FXS screening by PCR, which according to the authors, is rapid, inexpensive, and efficient in the detection of CGG repeats in male and female. However, we noted the absence of important information for breeding method. The objectives of this study were: (i) to standardize the PCR technique proposed by Tassone et al. (2008), adapting it, due to the lack of methodological information, (ii) verify the accuracy, sensitivity and specificity of the method, comparing it to the Southern blot, and (iii) to evaluate the technique using DNA extracted from different biological materials / extraction methods, and (iv) estimate the cost and time of the method execution in the domestic market. The biological materials used were: blood collected by vacuum system and oral mucosal cells, which were extracted by organic solvents and blood collected on FTA cards, purified by Whatman kit. Success was achieved in the reproduction of the PCR method in 75 individuals using the enzyme Expand Long Template PCR System (Roche Diagnostics). The accuracy, sensitivity and specificity were 100% when analyzing the total sample, indicating that the technique can detect the presence... (Complete abstract click electronic access below)

O processo de dano em materiais quasi-frágeis pode ser caracterizado pela perda de isotropia para certos níveis de carga. A localização de deformações, o efeito cooperativo entre regiões danificadas e a avalanche de rupturas são características particulares na medição do dano neste tipo de material. As características mencionadas criam diferentes formas de dissipação de energia, que não são fáceis de representar utilizando métodos baseados na hipótese dos meios contínuos. No presente trabalho uma versão do Método dos Elementos Discretos Formado por Barras é empregado. Neste método a massa do contínuo é concentrada nos nós, os quais são interconectados por barras sem massa. Essas barras possuem uma lei constitutiva bilinear, que é usada para simular a ruptura da estrutura em estudo. A distribuição dos nós permite formar uma treliça tridimensional regular, e a partir dessa discretização espacial é possível chegar a um sistema de equações de movimento, que é resolvido com um esquema explícito de integração numérica (diferenças finitas centrais). Neste método a fratura e a fragmentação são levadas em conta de forma natural, já que as barras que rompem durante o processo são desativadas, respeitando o balanço energético. É possível introduzir heterogeneidade no modelo considerando as propriedades do material como campos espaciais aleatórios com distribuição de probabilidades de Weibull e comprimento de correlação conhecido. Nessa dissertação, é analisado o processo de dano que aparece em estruturas de geometria simples quando solicitadas até o colapso. Diferentes índices são apresentados para realizar a medição do dano. O desempenho desses índices, e a maneira com que eles ajudam na interpretação da evolução do dano, são discutidos nesse trabalho. / The process of damage in quasi-fragile materials is characterized by loss of isotropy for certain load levels. The strain localization, the cooperative effect between damaged regions and the avalanche of ruptures are particular features in measuring the damage in this kind of material. The mentioned features create different forms of energy dissipation, which are not easy to represent with a continuous approach. In the present work a version of the Lattice Discrete Element Method is employed. In this method the mass of the solid is concentrated on node points, which are interconnected by uniaxial elements. These elements have a bilinear constitutive law, which is used to simulate the rupture of the structure under study. The node distribution allows the formation of a regular three-dimensional lattice, and from this spatial discretization it is possible to arrive at a system of equations of motion, which is solved by an explicit numerical integration scheme (central difference). In this method the fracture and fragmentation are taken into account in a natural manner, since the bars that reached their limit strength during the process are disabled of the system, respecting the energy balance. It is possible to introduce heterogeneity in the model considering the material properties as random fields with spatial Weibull probability distribution and known correlation length. In this dissertation, the damage process, which appears in structures of simple geometry, when they are loaded until collapse, is analysed. Different indexes are presented to perform the measurement of the damage. The performance of those indexes, and the way they help in the interpretation of the damage evolution, are discussed in this paper.

A critical exploration of seven peer-reviewed published papers supports the author’s contention that learning in Higher Education is a fragile system of conscious and unconscious transactions that serve to weaken a process that is already precarious. Over the course of this essay and the accompanying papers, the submission is that learning is brittle, and easily broken. The Fragile Learner is described as someone close to conceding defeat to circumstances that threaten his education. The Fragile Learner might be a student of a Higher Education Institution, but also might be an appointed educator. Alongside notions of barriers to learning, this submission explores identities and tensions. Although some of the ideas that make up my picture of Fragile Learning have been researched by other contributors (notably Meyer and Land; Britzman), my own contribution sees the complexities through various psychoanalytic lenses. Fundamentally, it is the addition of psychoanalysis that makes Fragile Learning original. It is argued that anxiety is an important part of adult learning. Fragile Learners might experience anxieties that are internal and complex but which appear to be attacks from other people. Alternatively, Fragile Learning might be a consequence of learners having suffered illness or indisposition. It is important that something can be blamed. The themes of fragility and anxiety – not to mention the difficulties that arise from distance learning – are present throughout.

O processo de dano em materiais quasi-frágeis pode ser caracterizado pela perda de isotropia para certos níveis de carga. A localização de deformações, o efeito cooperativo entre regiões danificadas e a avalanche de rupturas são características particulares na medição do dano neste tipo de material. As características mencionadas criam diferentes formas de dissipação de energia, que não são fáceis de representar utilizando métodos baseados na hipótese dos meios contínuos. No presente trabalho uma versão do Método dos Elementos Discretos Formado por Barras é empregado. Neste método a massa do contínuo é concentrada nos nós, os quais são interconectados por barras sem massa. Essas barras possuem uma lei constitutiva bilinear, que é usada para simular a ruptura da estrutura em estudo. A distribuição dos nós permite formar uma treliça tridimensional regular, e a partir dessa discretização espacial é possível chegar a um sistema de equações de movimento, que é resolvido com um esquema explícito de integração numérica (diferenças finitas centrais). Neste método a fratura e a fragmentação são levadas em conta de forma natural, já que as barras que rompem durante o processo são desativadas, respeitando o balanço energético. É possível introduzir heterogeneidade no modelo considerando as propriedades do material como campos espaciais aleatórios com distribuição de probabilidades de Weibull e comprimento de correlação conhecido. Nessa dissertação, é analisado o processo de dano que aparece em estruturas de geometria simples quando solicitadas até o colapso. Diferentes índices são apresentados para realizar a medição do dano. O desempenho desses índices, e a maneira com que eles ajudam na interpretação da evolução do dano, são discutidos nesse trabalho. / The process of damage in quasi-fragile materials is characterized by loss of isotropy for certain load levels. The strain localization, the cooperative effect between damaged regions and the avalanche of ruptures are particular features in measuring the damage in this kind of material. The mentioned features create different forms of energy dissipation, which are not easy to represent with a continuous approach. In the present work a version of the Lattice Discrete Element Method is employed. In this method the mass of the solid is concentrated on node points, which are interconnected by uniaxial elements. These elements have a bilinear constitutive law, which is used to simulate the rupture of the structure under study. The node distribution allows the formation of a regular three-dimensional lattice, and from this spatial discretization it is possible to arrive at a system of equations of motion, which is solved by an explicit numerical integration scheme (central difference). In this method the fracture and fragmentation are taken into account in a natural manner, since the bars that reached their limit strength during the process are disabled of the system, respecting the energy balance. It is possible to introduce heterogeneity in the model considering the material properties as random fields with spatial Weibull probability distribution and known correlation length. In this dissertation, the damage process, which appears in structures of simple geometry, when they are loaded until collapse, is analysed. Different indexes are presented to perform the measurement of the damage. The performance of those indexes, and the way they help in the interpretation of the damage evolution, are discussed in this paper.

Les aciers ferritiques pour structures industrielles présentent une transition fragile-ductile de ténacité et de résilience avec leur température. Leur résistance à la rupture fragile joue un rôle essentiel dans la certification de la sécurité des structures industrielles importantes. De nos jours, le souci de performance et de longévité sont des points clés pour des acteurs majeurs comme EDF.Dans ces études de transition ductile-fragile, de ténacité et de résilience, la ténacité est prédite à partir de la résilience. Plusieurs travaux antérieurs ont déjà montré que la probabilité de rupture par clivage peut être correctement décrite dans le palier fragile par une approche locale de la rupture. Mais ces études supposent que le matériau ne subit pas de vieillissement sous déformation, ce qui est en fait rarement pertinent pour les aciers bas carbone et peu calmés. Le travail a consisté d'une part à caractériser le comportement et d'autre part à en proposer une modélisation robuste et explicite des phénomènes observés. La caractérisation a consisté en la réalisation d'essais de traction entre -150°C et 20°C à plusieurs vitesses de déformation. Un modèle capable de simuler le vieillissement statique est identifié en mettant en place une stratégie adéquate et systématique. Des essais de résilience permettent de construire la courbe de transition ductile-fragile du matériau pour différentes conditions afin de comprendre et d'observer l'influence du vieillissement statique sur la rupture. Enfin, la modélisation de la rupture fragile a été décrite pour toutes les conditions expérimentales testées en utilisant le modèle de comportement développé et identifié dans la partie précédente afin de prédire numériquement la transition pour les différentes conditions du matériau. / Ferritic steels for industrial structures have a brittle-ductile transition toughness and impact energy with temperature. Their resistance to the brittle fracture plays an essential role in the safety certification of industrial structures. Nowadays, the performance and the durability are key issues for major players such as EDF. In these approaches ductile-to-brittle transition toughness and impact energy, toughness is predicted from resilience. Several previous studies have shown that the probability of cleavage fracture can be adequately described in brittle plateau by a local approach to fracture. However, these studies assume that the material does not undergo strain aging, which is rarely relevant for low carbon steels and low calmed down. The work consisted firstly to characterize the behavior and secondly to propose a robust and explicit modeling of the observed phenomena. Characterization consisted of performing tensile tests between -150degreC~and 20degreC for several strain rates. A model able to simulate the static aging is identified by implementing an appropriate and systematic strategy. Impact resistance test allows us to build the curve of ductile-to-brittle transition of the material for different conditions to understand and observe the influence of static strain aging on the failure. Finally, the modeling of the brittle fracture has been described for all experimental conditions tested using the model developed and identified in the previous section to predict the transition for different material conditions.

Les Sites Fragiles Communs (SFC) sont des loci incluant des grands gènes où des cassures chromosomiques apparaissent chez des cellules soumises à un stress réplicatif. Il est couramment admis que ces sites sont des régions dont la réplication n’est pas terminée lorsque les cellules entrent en mitose mais le mécanisme sous-jacent restait mal compris. Notre laboratoire a montré par la technique du peignage moléculaire que l’instabilité du site FRA3B chez les lymphocytes est due à la présence d’une grande région pauvre en origines de réplication actives (cœur) dont la réplication ne peut pas se terminer en cas de stress réplicatif. Dans le but de généraliser ces conclusions, j’ai étudié par la technique du « Répli-Seq », le timing de réplication à l’échelle du génome entier chez des cellules lymphoblastoïdes humaines traitées ou non à l’aphidicoline. Les résultats confirment clairement que les SFC connus dans ce type cellulaire correspondent à des régions incluant un grand gène et qui sont déficitaires en réplication sous un stress réplicatif. D’autre part, j’ai montré que retarder l’entrée des cellules en mitose par l’utilisation du RO-3306, un inhibiteur de CDK1, abolit complètement la fragilité des SFC induite par l’aphidicoline chez des lymphocytes et des fibroblastes humains. De plus, j’ai montré que cette suppression des cassures au niveau de FRA3B et FRA16D chez les lymphocytes et de FRA1L chez les fibroblastes ne s’accompagne pas d’une diminution de la transcription de FHIT, WWOX et NEGR1, les trois grands gènes contenus respectivement dans ces trois SFC. Par ailleurs, j’ai corrélé l’expression des gènes FHIT, WWOX, NEGR1 et LSAMP à la fragilité des sites FRA3B, FRA16D, FRA1L et FRA3L dans 7 lignées de fibroblastes et 7 lignées de lymphocytes. J’ai aussi mis en évidence une expression atypique de ces gènes dans certaines de ces lignées et montré que cette expression atypique s’accompagne d’une fragilité atypique du SFC correspondant. Cependant, j’ai montré que l’inhibition de la transcription par deux inhibiteurs différents, le triptolide et l’α-amanitine, ne diminue pas la fragilité induite par l’aphidicoline. Je propose un modèle pour expliquer ces résultats apparemment contradictoires. Enfin, j’ai corrélé la disparition des cassures au niveau de FRA3B dans les cellules traitées à l’aphidicoline et au RO-3306 à une augmentation des événements d’initiation dans la région cœur. J’ai montré que l’inhibition de CDK1 par le RO-3306 permet l’accumulation à la chromatine des facteurs CDC6, CDT1, ORC1 et MCM7 dans les cellules bloquées en phase G2 et que l’accumulation de CDT1 et de CDC6 est indispensable à la diminution de la fragilité sous RO-3306. Je propose un modèle permettant de rendre compte de ces observations qui postule l’existence d’origines de réplication latentes partiellement chargées en complexes de pré-réplication. / Common Fragile Sites (CFS) are loci harboring large genes where chromosome breaks occur in cells under replication stress. It is widely accepted that these sites are regions whose replication is incomplete when cells enter mitosis, but the underlying mechanism remains poorly understood. Our laboratory has shown by the technique of molecular combing, that the instability of FRA3B in lymphocytes is due to the presence of a large region poor in active replication origins (core) whose replication can not be completed under replicative stress. In order to generalize these findings, I studied by the technique of "Repli-Seq", the replication timing across the entire genome in human lymphoblastoid cells treated or not with aphidicolin. The results clearly confirm that CFS known in this cell type correspond to regions including large genes and which are underreplicated under replication stress conditions. Moreover, I have shown that delaying entry of cells into mitosis by using RO-3306, an inhibitor of CDK1, completely abolishes the fragility of SFC induced by aphidicolin in human lymphocytes and fibroblasts. I also have shown that abolition of the breaks at FRA3B and FRA16D in lymphocytes and at FRA1L in fibroblasts is not accompanied by a decreased transcription of FHIT, WWOX and NEGR1, respectively, the three large genes including these three SFC. Moreover, I have correlated the expression of FHIT, WWOX, NEGR1 and LSAMP genes to the fragility of FRA3B, FRA16D, FRA1L and FRA3L sites in 7 lines of fibroblasts and 7 lines of lymphocytes. I also highlighted an atypical expression of these genes in some of these lines and showed that this atypical expression is accompanied by an unusual fragility of the corresponding CFS. However, I have also shown that inhibition of transcription by two different inhibitors, triptolide and α-amanitine, does not diminish the fragility induced by aphidicolin. I propose a model to explain these apparently contradictory results.Finally, I have correlated the disappearance of breaks at FRA3B in cells treated with aphidicolin and RO-3306 with an increase in initiation events in the core region. I have shown that inhibition of CDK1 by the RO-3306 allows the accumulation of factors CDC6 CDT1, ORC1 and MCM7 on the chromatin in cells blocked in G2 phase, and that the accumulation of CDT1 and CDC6 is essential to reducing fragility under RO-3306. I propose a model to explain these observations that postulates the existence of latent replication origins partially loaded with the pre-replication complex.

This thesis examines and compares the humanitarian interventions in Kosovo, Libya and Somalia. The purpose of this study is to examine if the presence of strong tribal structures within a nation can increase the risk of terrorist activities, and subsequently contribute to a failed state following a humanitarian intervention. By applying a theory on tribes and critical terrorism studies, this thesis argues that policymakers might underestimate the significance of tribal structure within a state, before intervening. With Mills method of concomitant variation, this thesis has examined and compared the leadership, interventions, radical presence, and tribal structures of Kosovo, Libya and Somalia. This thesis has also examined if interventions can increase radicalism. The result from the analysis shows us that the presence of strong tribal structures can increase the terrorist activities and subsequently contribute to a failed state. Humanitarian intervention can further lengthen the weak state apparatus if the external actors neglect of the local structures of a state.