Endocytose joue un rôle dans l'activation du récepteur Notch. Des mutations dans le gène drosophilien lethal giant discs (lgd), provoque une prolifération cellulaire en perturbant l'endocytose de Notch. Les orthologues murins mlgd1 et 2 peuvent sauver ce phénotype, démontrant une fonction conservée. Cependant, des publications récentes suggèrent que les orthologs humains de lgd (hgd1/2) sont nucléaires. Dans cette étude, il est démontré que chez la Drosophile, le mutant dlgd(08) provoque l'accumulation de Notch dans des vésicules et une surprolifération de neuroblastes . Ceci suggère que Notch est activé a l'intérieur des endosomes dans les neuroblastes. L'immunohistochimie de cellules Hela indique que hlgd1 et 2 ne sont pas nucléaires, mais associés à des strctures endosomales. Enfin, la baisse d'expression par shRNA des gènes murins mlgd1 et mlgd2 provoque une différenciation accélérée des cellules souches hématopoïétiques dans la lignée lymphopoïèse T et bloque la transition DN3 / CD4+CD8+, suggérant une suractivation de Notch. / Endocytosis plays a role in the activation of the Notch receptor. Mutations in the Drosophila gene lethal giant discs (lgd), causes cellular overgrowth by perturbing Notch endocytosis. This Drosophila phenotype is rescued by the murine orthologs mlgd1 and 2, indicating conserved function. However, recent publications suggest that the human orthologs (hlgd1/2) are nuclear. This study demonstrates that the dlgd(08) mutant in Drosophila causes accumulation of Notch in vesicles and the overproliferation of neuroblasts. This suggests Notch is activated from within endosomes in neuroblasts. Immunohistochemistry of Hela cells indicates that hlgd1 is associated with early endosome while, hlgd2 with later endosome and lysosome, and not with the nucleus. Finally, down regulation of murine mlgd1 and mlgd2 by shRNA caused an accelerated differentiation of hematopoietic stem cell into the T lymphopoiesis lineage and blocked the DN3 to CD4+CD8+ transition, suggesting that Notch is overactivated in these cells.

A survey of relevant literature on the topic of wave propagation and scattering in pipes is given first. This review is followed by a theoretical framework which is pertinent to wave propagation in homogeneous, isotropic, pipes. Emphasis is placed on approximate solutions stemming from a computer based, Semi-Analytical Finite Element (SAFE) formulation. A modal analysis of the dynamic response of homogeneous, isotropic pipes, when subjected to a transient ultrasonic excitation, demonstrates that dominant features, i.e., singularities in an unblemished pipe’s displacement Frequency Response Function (FRF) coincide with its cutoff frequencies. This behaviour is confirmed experimentally. A novel technique is developed to deduce such a pipe’s wall thickness and elastic properties from three cutoff frequencies. The resulting procedure is simulated numerically and verified experimentally. Agreement between the new ultrasonic procedure and traditional destructive tests is within experimental uncertainty. Then a hybrid-SAFE technique is used to simulate waves scattered by various open rectangular notches. The simulations show, for the first time, that singularities distinct from the unblemished pipe’s cutoff frequencies arise in a displacement FRF when an axisymmetric notch is introduced. They also suggest that the new singularities depend on the properties of the parent pipe and the finite element region but effects are local to a notch. It is demonstrated further that the difference between the frequency at which a singularity introduced by a notch occurs and the nearest corresponding unblemished pipe’s cutoff frequency is a function of the notch’s dimensions. By plotting contours of constant frequency differences, it is shown that it is usually possible to characterize the notch’s dimensions by using two modes. However, the frequency difference for a third mode may be also needed occasionally. The more general case of nonaxisymmetric notches is shown to be a straightforward extension of the axisymmetric case.

The survival for high-grade glioma patients is poor and the treatment may cause severe side effects. A common obstacle in the treatment is chemoresistance. To improve the quality of life and prolong survival for these patients prognostic biomarkers and new approaches for chemotherapy are needed. To this end, a strategy to evade chemoresistance was evaluated by combining chemotherapeutic drugs with agents inhibiting resistance mechanisms identified by a bioinformatic analysis (paper I). The prognostic value of 13 different proteins was analyzed in this thesis (papers II-IV). Two of them, p38 mitogen-activated protein kinase (MAPK) and protein tyrosine phosphatase non-receptor type 6 (PTPN6, also known as SHP1) were analyzed for their potential as targets in combination chemotherapy (in paper III and IV, respectively).   We found that: PTPN6 expression and methylation status may be important for survival of anaplastic glioma patients, p38 MAPK phosphorylation may be a potential negative prognostic biomarker for high-grade glioma patients and FGF2 expression may be a potential negative prognostic biomarker for proneural glioma patients. PTPN6 may be a useful target for combination chemotherapy with cisplatin, melphalan or bortezomib in high-grade gliomas. The following drug combinations; camptothecin combined with an EGFR or RAC1 inhibitor, imatinib combined with a Notch or RAC1 inhibitor, temozolomide combined with an EGFR or FAK inhibitor and vandetanib combined with a p38 MAPK inhibitor may be useful combination chemotherapy for high-grade gliomas.

A survey of relevant literature on the topic of wave propagation and scattering in pipes is given first. This review is followed by a theoretical framework which is pertinent to wave propagation in homogeneous, isotropic, pipes. Emphasis is placed on approximate solutions stemming from a computer based, Semi-Analytical Finite Element (SAFE) formulation. A modal analysis of the dynamic response of homogeneous, isotropic pipes, when subjected to a transient ultrasonic excitation, demonstrates that dominant features, i.e., singularities in an unblemished pipe’s displacement Frequency Response Function (FRF) coincide with its cutoff frequencies. This behaviour is confirmed experimentally. A novel technique is developed to deduce such a pipe’s wall thickness and elastic properties from three cutoff frequencies. The resulting procedure is simulated numerically and verified experimentally. Agreement between the new ultrasonic procedure and traditional destructive tests is within experimental uncertainty. Then a hybrid-SAFE technique is used to simulate waves scattered by various open rectangular notches. The simulations show, for the first time, that singularities distinct from the unblemished pipe’s cutoff frequencies arise in a displacement FRF when an axisymmetric notch is introduced. They also suggest that the new singularities depend on the properties of the parent pipe and the finite element region but effects are local to a notch. It is demonstrated further that the difference between the frequency at which a singularity introduced by a notch occurs and the nearest corresponding unblemished pipe’s cutoff frequency is a function of the notch’s dimensions. By plotting contours of constant frequency differences, it is shown that it is usually possible to characterize the notch’s dimensions by using two modes. However, the frequency difference for a third mode may be also needed occasionally. The more general case of nonaxisymmetric notches is shown to be a straightforward extension of the axisymmetric case.

Comprendre comment les facteurs génétiques et environnementaux interagissent au cours du développement est une question fondamentale en biologie. Je me suis intéressée à cette question en utilisant le réseau de développement de la vulve du nématode C. elegans comme système modèle. L'objectif de mon projet était une étude quantitative de la modulation par l'environnement des voies de signalisation impliquées dans ce processus telles que, Ras, Delta-Notch et Wnt. J'ai tout d'abord analysé comment un facteur environnemental spécifique (la carence nutritionnelle) modifie les activités et les interactions entre les voies de signalisation sous-jacentes au développement vulvaire chez C. elegans. J'ai ainsi mis en évidence que l'augmentation de l'induction vulvaire par la carence passe par une augmentation de l'activité de la voie Ras et est indépendante de la voie Wnt. Cet effet de l'environnement est assuré par la détection de la diminution de l'apport en nutriments, probablement par l'action de la voie TOR, et affecte l'induction vulvaire en parallèle ou en amont du récepteur à l'EGF. J'ai ensuite examiné la sensibilité environnementale du système de développement de la vulve de Caenorhabditis dans une perspective évolutive et ce, grâce à l'analyse comparative de différents isolats. J'ai pu observer que l'exposition à des températures extrêmes induit des variants et des défauts de manière fortement dépendante de la souche et de l'espèce. L'occurrence de certains défauts développementaux induits par la température révèlent en outre que certaines cellules précurseurs de la vulve et les voies de signalisation associées présentent une sensibilité environnementale différente.

With the dawn of metazoans, the ability of cells to communicate with each other became of paramount importance in maintaining tissue homeostasis. The transforming growth factor β (TGF-β) signaling pathway, which plays important roles during embryogenesis and in the adult organism, signals via a heterodimeric receptor complex consisting of two type II and two type I receptors. After receptor activation through ligand binding, Smads mediate the signal from the receptor complex to the nucleus, where they orchestrate transcription. Depending on the context of activation, TGF-β can mediate a plethora of cellular responses, including proliferation, growth arrest, apoptosis and differentiation. In cancer, TGF-β can act as both as a tumor suppressor and promoter. During early stages of tumorigenesis, TGF-β prevents proliferation. However, TGF-β is also known to promote tumor progression during later stages of the disease, where it can induce differentiation of cancer cells towards a migratory phenotype. The aim of this thesis was to investigate how cells can differentiate their response upon TGF-β pathway activation. The first paper describes the role of Notch signaling in TGF-β induced growth arrest, demonstrating that TGF-β promotes Notch activity and that Notch signaling is required for prolonged TGF-β induced cell cycle arrest. In the second and third paper, we investigate the role of SIK, a member of the AMPK family of kinases, mediating signaling strength of TGF-β through degradation of the TGF-β type I receptor ALK5. While the second paper focuses on the effect of SIK on ALK5 stability and subsequent alterations in TGF-β signaling, the third paper emphasizes cooperation between SIK, Smad7 and the E3 ligase Smurf in degradation of ALK5. Finally, the fourth paper explores a novel role of SIK during TGF-β induced epithelial to mesenchymal transition (EMT). SIK binds to and degrades the polarity protein Par3, leading to enhanced EMT.

The most common cause for revision total hip replacement surgey is aseptic loosening of the acetabular component. This thesis explores the effect of three techniques to improve the depth and quality of cemented acetabular component fixation in primary total hip replacement. This may have beneficial effects on the longevity of cemented acetabular components and reduce the rate of revision surgery for aseptic loosening. Aims: 1. Determine the effect of the rim cutter on cement pressure during cup insertion. 2. Examine the effect of the rim cutter on cement penetration distance. 3. Evaluate the effect of bone grafting of the acetabular notch. 4. Determine the effect of iliac suction during cement pressurisation. 5. Compare the behaviour of bone cement with Play Dough. Materials and Methods: 1. Sawbones hemi pelvis models were fitted with pressure transducers at the rim and apex of the acetabulum. Peak pressure was measured upon insertion of cups with different flange sizes and when the acetabulum was prepared with the rim cutter. 2. Foam cavities were used to measure the depth of cement penetration when the same cups and rim cutter were used. 3. Hemi pelvis models were modified to simulate bone grafting of the acetabular notch. Again, pressure sensors were mounted at the apex and rim of the acetabulum. Intra-acetabular cement pressure was compared with native acetabulae. 4. A back bleeding model of the acetabulum was fitted with a suction catheter. The effect on cement penetration into cancellous bone was measured compared with no suction. 5. Play Dough pressurisation and penetration into hemi pelvises and foam was compared to bone cement. Results: 1. Significant increase in peak apex and rim pressures when flanged cup inserted into an acetabulum prepared with the rim cutter compared with both flanged and unflanged cups alone. 2. Significant increase in cement penetration at the rim of the acetabulum when rim cutter used and flanged cup inserted when compared with flanged and unflanged cups alone. 3. Significant increase in intra-acetabular pressure when cement pressurised in presence of simulated acetabular notch bone grafting compared with normal acetabulae. 4. Significant increase in cement penetration distance when suction used compared with no suction. 5. Significant differences in the flow characteristics between bone cement and Play Dough. Conclusion: The authors recommend preparation of the acetabular rim with the rim cutter and bone grafting of the acetabular notch to improve the depth and uniformity of the cement mantle in cemented primary THA. Play Dough at room temperature is not a suitable substitute for bone cement in in-vitro cementing studies.

The host immune response to parasitic helminths is usually characterized by a Th2 phenotype. As the Jagged/Notch pathway has been implicated in driving Th2 development, it was hypothesized that host macrophages and dendritic cells (DCs) could detect helminth products and mount an appropriate response via this pathway. Schistosoma mansoni soluble egg antigen (SEA) rapidly up-regulated expression of the Notch ligand, Jagged 1, in both mouse and human macrophages, as well as in conventional mouse DCs. Other factors associated with Th cell development, including the Th1-promoting factor IL-12 p40, as well as another potential Th2-promoting factor, interleukin (IL)-33, were not transcriptionally responsive to SEA in these same cell types, thus indicating the selectivity of the response. Inducible gene expression was modified by the presence of the macrophage growth factor colony-stimulating factor (CSF)-1, which inhibited Jagged 1 induction by SEA and lipopolysaccharide (LPS), but enhanced LPS-induced IL-12p40 expression. Despite the observation that SEA upregulated Jagged 1 in both macrophages and DCs, only SEA-pulsed DCs promoted IL-4 production upon T-cell activation, suggesting that Jagged 1 induction alone is insufficient for instructing Th2 development. A recombinant form of the extracellular region of Jagged 1 did, however, enhance IFN-γ production in splenocytes, thus implying that the rapid induction of Jagged 1 in macrophages and DCs can regulate T cell responses. A potential role for SEA-induced Jagged 1 in autocrine responses in macrophages was also investigated through studies with recombinant extracellular Jagged 1, as well as ectopic expression of Jagged 1 in macrophages. A comparison of the responses initiated in macrophages by SEA and the bacterial endotoxin lipopolysaccharide (LPS) revealed common activation of extracellular signal regulated kinase-1/2 (ERK-1/2) and p38 phosphorylation. However, only LPS triggered IκB degradation, phosphorylation of c-Jun N-terminal kinase (JNK) and phosphorylation of Tyr701 of signal transducer and activator of transcription 1 (STAT1). SEA robustly activated signalling in HEK293 cells expressing either Toll-like receptor 2 (TLR2) or TLR4/MD2, as well as variably in cells expressing TLR3. Jagged 1 upregulation by SEA was not abrogated in TLR4 knockout macrophages, in contrast to the LPS response. Pharmacological inhibition of the ERK-1/2 pathway impaired both SEA- and LPS-inducible Jagged 1 expression in macrophages. In conclusion, the data within this thesis suggests that Jagged 1 is an ERK-dependent target of TLR signalling that has a macrophage-specific function in the response to SEA.

[Truncated abstract] Human complement receptor 2 (CR2/CD21) is a cell surface glycoprotein detected on specific cells involved in immunity, which binds complement C3 cleavage fragments, cellular ligands IFN-? and CD23 as well as the EBV coat protein, gp350/220. During the early stages of B-cell development CR2/CD21 is silenced. Expression is initiated on immature B-cells escaping negative selection. During peripheral maturation CR2/CD21 is up-regulated with B-cell sub-populations showing distinctive surface levels (comparatively low, intermediate or high). CR2/CD21 is silenced upon terminal plasmacytic differentiation. Appropriate timing and expression level of CR2/CD21 is important for the development of a healthy B-cell repertoire. Previous studies have identified sequences within the proximal promoter and first intron of CR2/CD21 that cooperate within native chromatin to control cell-specific silencing. Further, analysis of cultured human cells has revealed chromatin structural variation causing DNase I hypersensitivity at these regulatory sites in a CR2/CD21-expressing mature B-cell line (Raji) which are absent in a non-lymphoid cell type (K562). The primary focus of the present study involved characterising chromatin structural variation over previously recognized DNase I hypersensitive regions at the CR2/CD21 locus in human cells to understand how chromatin structure might regulate developmental expression of CR2/CD21. ... These studies provide evidence that notch signaling influences CR2/CD21 expression in human cell lines. First, in vivo binding of CBF1 to CR2/CD21 sequences in the proximal promoter and CRS implies that CR2/CD21 is a direct target of notch activation. Second, the effect of exogenous notch signalling molecules on CR2/CD21 proximal promoter activity was modulated by factors binding tandem E-boxes near the transcriptional start site suggesting that the notch pathway may also influence CR2/CD21 expression via control of HLH molecules. Third, initiation of CR2/CD21 expression was observed in a nonexpressing pre-B cell line (Reh) by co-culture with stromal cells expressing a notch ligand (OP9-DL) but not control stroma (OP9-GFP). Together, these findings support a role for notch regulation of B-cell maturation and invite speculation that initiation of CR2/CD21 expression following negative selection of immature B-cells involves crosstalk between HLH transcriptional regulators and the notch pathway. Furthermore, the Reh/OP9-DL co-culture system may provide a model to directly study the relationship between cell signalling molecules, transcription factor regulation, chromatin structural variation and differentiation of B-cells.

A armadura de tração de dutos flexíveis é a camada responsável por suportar as cargas axiais destes equipamentos em campo. Com a tendência de aumento das profundidades de exploração e produção de petróleo offshore, principalmente dos campos do pré-sal brasileiro, faz-se necessário o uso de materiais com desempenho superior. Este trabalho buscou avaliar e comparar dois aços de alta resistência mecânica para tal aplicação, um predominantemente perlítico e outro martensítico. Durante a vida em serviço destes componentes, eles sofrem tensões axiais cíclicas associadas ao processo de corrosão, que gera pites como concentradores de tensões. Considerando estes aspectos, os materiais foram avaliados segundo seu comportamento à fadiga sem e com a presença de um pequeno defeito conhecido, que atua como um entalhe e busca simular um pite de corrosão. Além disso, esses materiais foram caracterizados metalúrgica e mecanicamente e sua tenacidade à fratura foi estimada via o método do CTOD. A partir dos dados experimentais, foi quantificada a sensibilidade ao entalhe, guiada essencialmente pelo processo de iniciação de trinca, ao passo que a tenacidade à fratura não foi decisiva. O material martensítico apresentou desempenho superior em fadiga e menor sensibilidade ao entalhe e mostrou-se promissor para a aplicação como arame da armadura de tração. / The tensile armor of flexible pipes is the layer responsible for support axial loads of this equipment in field. With the trend of deep increase in offshore oil exploration and production, mainly in pre-salt fields, the use of materials with high performance becomes necessary. The aim of this dissertation is evaluate and compare two high strength steels for such application, one with perlite and other with martensitic matrix. During service life of these components, they are damaged by cyclic loading associated with corrosion process which promote corrosion pits like stress concentrators. Considering these aspects, the materials were evaluated according their fatigue behavior with and without a small defect that act as a notch and simulates a corrosion pit. In addition, they were characterized metallurgical and mechanically and their fracture toughness was estimated through CTOD test. From experimental data, notch sensitivity was quantified. This was driven by initiation process, and fracture toughness was not decisive. The martensitic material showed higher fatigue performance and lower notch sensitivity and presented itself promising for tensile armor wire application.