Plankerbverzahnungen zählen zu den Vertretern der Kupplungsverzahnungen, mit deren Hilfe zwei Bauteile koaxial miteinander verbunden werden können. Neben einer hohen Winkelgenauigkeit zeichnen sie sich durch eine enorme Drehmomentkapazität bei gleichzeitig geringem Montageaufwand aus. Die zur Leistungsübertragung erforderliche Vorspannkraft kann über eine zentrale Spannschraube oder mehrere dezentral, auf einem gemeinsamen Teilkreis, angeordnete Spannschrauben erzeugt werden. Diese Arbeit liefert einen Beitrag zur beanspruchungsgerechten Dimensionierung von Plankerbverzahnungen mit zentraler und dezentraler Verschraubung. Es wird eine Berechnungsmethode zur Bestimmung der Tragfähigkeit des Zahnfußes, der Zahnflanke und der Spannschrauben vorgestellt und anhand von Beispielrechnungen erläutert. Neben den umfangreichen theoretischen Betrachtungen werden die Ergebnisse zahlreicher experimenteller Untersuchungen präsentiert. Diese dienen der Gewinnung von grundlegenden Aussagen zur Stützwirkung und zum Mittelspannungseinfluss an einfach und mehrfach gekerbten Proben sowie zur Verifizierung des Tragfähigkeitsnachweises.

The role of Notch in cell fate determination and lineage restriction in the bone marrow (BM) is controversial in the field. Recent studies have convincingly shown that Notch is dispensable for haematopoietic stem cell (HSC) regulation in adult haematopoiesis (Maillard et al., 2008). In contrast, Notch signaling has been proposed to be of importance in the regulation of BM megakaryocyte progenitor differentiation, based on dominant negative genetic approaches, identifying a potentially distinct role for Notch in adult BM haematopoiesis (Mercher et al., 2008). Here, I found that by selectively ablating the gene coding the transcription factor recombination signal-binding protein J kappa (RBP-Jk), to which all canonical Notch signaling converges, canonical Notch signaling does not mediate HSC maintenance, neither in steady state nor in conditions of stress. Furthermore, I propose, in contrast with previous studies (Mercher et al., 2008), that canonical Notch signaling plays no role in myeloerythropoiesis cell lineage commitment in the BM. My data also show that key Notch target genes are suppressed by RBP-Jk, as their expression is unaffected in Notch1-deficient BM progenitors, while target genes are upregulated in Rbp-Jk-deleted megakaryocyte and erythroid progenitors. This establishes for the first time in mammalian cells in vivo, that Notch target genes are kept in a suppressed state by RBP-Jk, potentially restricting T cell commitment to the thymus and not to the BM, at the expense of myeloerythropoiesis. Notch signaling and GATA3 are two master regulators in T cell commitment (Han et al., 2002; Ho et al., 2009; Pui et al., 1999; Radtke et al., 1999; Zhu et al., 2004). However, although very well established as being involved in the thymic stages of T cell restriction, there is little evidence of Notch and GATA3 being involved in the migration of a thymus settling progenitor (TSP) from the BM to the thymus or in the establishment of the earliest thymic progenitor (ETP) in the thymus. From this thesis work, I conclude that Notch signaling is essential for the emergence of ETPs in the thymus in a NOTCH1-independent manner. Moreover, I demonstrate, as supported by a very recent published study (Hosoya et al., 2009), that GATA3 is important for the development of the earliest T cell progenitor. GATA1 and GATA2 mediate haematopoietic stem cell maintenance in the BM. GATA1 is required for erythropoiesis, megakaryocytes and eosinophils while GATA2 is important for the proliferation and survival of HSCs. In contrast, a role for GATA3 in the BM has never been established. By using a Gata3-conditional knockout mouse model, I demonstrate that GATA3 is dispensable for HSC maintenance in steady state and following active haematopoietic regeneration as well as for HSC self-renewal in the BM.

Tumour tissue is characterised by fluctuating oxygen concentrations, decreased nutrient supply, and acidic pH. Angiogenic signalling pathways that drive a certain metabolic 'configuration' may give endothelial cells a selective advantage in the tumour environment. Previously it has been shown that glycolysis drives proliferation, migration and tip cell formation during sprouting of endothelial cells (De Bock, Georgiadou et al. 2013), and is increased by VEGFA. DLL4-NOTCH has been shown to limit angiogenesis and slow down proliferation of endothelial cells, and promote stalk cell formation during angiogenic sprouting, leading to sprout elongation. DLL4-NOTCH is implicated in tumour angiogenesis, and its overexpression is a potential mechanism of resistance to anti-VEGFA therapy (Li, Sainson et al. 2011). This thesis aimed at investigating the effect of the DLL4-NOTCH signalling pathway on endothelial metabolism and its implications in angiogenesis. Firstly, it was found that DLL4-NOTCH decreases the glycolytic rate and mitochondrial respiratory parameters in endothelial cells. When given exogenous fatty acids, DLL4-NOTCH activation caused increased fatty acid uptake, storage and oxidation. This shows that the induction of DLL4-NOTCH signalling results in increased fatty acid utilisation. Secondly, this research identified fatty acid oxidation as a target metabolism pathway for angiogenic therapy. More specifically, inhibition of fatty acid oxidation decreased proliferation of endothelial cells, decreased sprout elongation in the sprouting assay, and decreased sprouting from the axial vein in the zebrafish model. ATP production was not affected. Therefore, it was hypothesised that DLL4-NOTCH activation promotes and maintains the stalk cell phenotype through an increase of fatty acid oxidation, thereby promoting biomass production for endothelial cell proliferation and growth during angiogenic sprout elongation. Thirdly, a key fatty acid metabolism gene, fatty acid binding protein 4 (FABP4), was identified, that is positively regulated by NOTCH at its promoter region. FABP4 is a candidate for mediating increased fatty acid flux in endothelial cells in response to DLL4-NOTCH. This study shows that FABP4 is induced by VEGFA in a manner dependent on DLL4-NOTCH, and the insulin-responsive transcription factor FOXO1 was required for FABP4 expression in response to DLL4-NOTCH. FABP4 is pro-angiogenic and implicated in tumour angiogenesis in ovarian cancer omental metastasis. Taken together, this study shows for the first time that DLL4-NOTCH signalling increases FABP4 induction, contributing to a key pro-angiogenic pathway, and also fatty acid utilisation in endothelial cells, and thereby contributes to the formation of blood vessels.

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.

Although the physiological consequences of Notch signaling in hematopoiesis have been extensively studied, the differential effects of individual notch cleavage products remain to be elucidated. Given that a disintegrin and metalloproteinase 10 (ADAM10) is a critical regulator of Notch and that its deletion is embryonically lethal, we generated transgenic mice that overexpress ADAM10 at early stages of lymphoid and myeloid development (A10Tg). ADAM10 transgene expression alters hematopoiesis post-hematopoietic Lineage-Sca-1+c-kit+ (LSK) subset differentiation but prior to lineage commitment of progenitor populations. This results in delayed T cell development, abrogated B2 cell development, and dramatic expansion of functionally active myeloid derived suppressor cells (MDSCs) in A10Tg mice. Given ADAM10’s role in Notch signaling, we hypothesized that the observed hematopoietic alterations may be a consequence of perturbed Notch signaling. In fact, blockade of ADAM10 (S2) rescues B cell development and reduces myeloid cells in A10Tg LSKs. Inhibition of γ-secretase (S3) in wild type (WT) LSKs results in enhanced myelopoiesis, mimicking the phenotype of A10Tg mice. Collectively, these findings indicate that the differential cleavage of Notch into S2 and S3 products regulated by ADAM10 is critical for hematopoietic cell-fate determination. Albeit arising in a tumor-free host, A10Tg MDSCs are functionally and phenotypically analogous to tumor-derived MDSCs. A10Tg MDSCs inhibit T cell activation in vitro, and inhibit adoptive immunotherapy (AIT) of metastatic melanoma in vivo, which can be reversed with MDSC depletion. Intriguingly, A10Tg mice are resistant to parasitic infection upon inoculation of Nippostrongylus brasiliensis. However, depletion of MDSCs abrogates this response, while adoptive transfer (AT) of MDSCs into WT mice increases their resistance. This polarized activity of MDSCs is heavily dependent upon interaction with mast cells (MCs). In fact, B16 melanoma cells metastasize more rapidly in WT mice infused with MDSCs when compared to MC-deficient mice (Kit Wsh/Wsh), with or without MDSC AT. Parallel to B16 progression, the ability of MDSCs to promote anti-Nb immunity is significantly diminished in MC-deficient (Kit Wsh/Wsh) mice even with MDSC AT. This augmentation of MDSC activity in the presence of MCs is further corroborated by in vitro co-culture assays that demonstrate a synergistic increase in cytokine production. Furthermore, MDSCs preferentially migrate to the liver in a MC-dependent manner. This interaction is mediated by MC-released histamine. In fact, MDSCs express histamine receptors (HR) and histamine induces MDSC survival, proliferation, and activation. We demonstrate that MDSC activity is abrogated with histamine blockade. Moreover, in humans, allergic patients present with an increase in MDSC population, and MDSCs purified from a stage I breast cancer patient exhibit increased survival in the presence of histamine. Taken together, our studies indicate that MCs and MC-released histamine are critical for the observed functional duality of MDSCs, ranging from immunosuppressive to immunosupportive, depending on the disease state.

Engineering assemblies are very frequently subject to fretting fatigue, which is a damage process that results when very small slip displacements arise at nominally stationary frictional interfaces. Fretting accelerates the initiation and early propagation of fatigue cracks, thereby causing significant reductions in the fatigue performance of many critical engineering components. A majority of the previous research on fretting fatigue has focused on incomplete (i.e. smooth-edged) contacts, while complete (i.e. sharp-edged) contacts have received less attention. The aim of this thesis is to contribute to the theoretical understanding of complete contacts, especially when they are subject to fatigue conditions. This problem is addressed in two separate ways. First, because fretting failures almost invariably initiate from the edge of contact, a detailed understanding of the conditions in this region should enable more accurate assessments of fatigue performance to be made. Thus, an asymptotic analysis is presented, which provides an accurate description of the contact edge under many conditions. This is done by using the elasticity solution for a semi-infinite notch to represent the state of stress near the contact edge in an asymptotic sense. Attention is then placed on the fact that cyclically loaded frictional contacts tend toward a steady-state response in which less frictional slip (and energy dissipation) occurs than in the first few load cycles. To investigate this effect, a numerical sub-structuring procedure is described, which significantly reduces the number of degrees of freedom in finite element models of frictional contact. This reduced model is then used to calculate the shakedown limit, i.e. the amplitude of cyclic load above which frictional slip is guaranteed to persist in the steady state. The sensitivity of the steady-state solution to the initial residual displacement state is then investigated, and it is shown that initial conditions can have a large influence on the steady-state behaviour of complete contacts.

Mechanical components often possess notches. These notches give rise to stress concentrations, which in turn increases the likelihood that the material will undergo yielding. The finite element method (FEM) can be used to calculate transient stress and strain to be used in fatigue analyses. However, since yielding occurs, an elastic-plastic finite element analysis (FEA) must be performed. If the loading sequence to be analysed with respect to fatigue is long, the elastic-plastic FEA is often not a viable option because of its high computational requirements. In this thesis, a method that estimates the elastic-plastic stress and strain response as a result of input elastic stress and strain using plasticity modelling with the incremental Neuber rule has been derived and implemented. A numerical methodology to increase the accuracy when using the Neuber rule with cyclic loading has been proposed and validated for proportional loading. The results show fair albeit not ideal accuracy when compared to elastic-plastic finite element analysis. Different types of loading have been tested, including proportional and non-proportional as well as complex loadings with several load reversions. Based on the computed elastic-plastic stresses and strains, fatigue life is predicted by the critical plane method. Such a method has been reviewed, implemented and tested in this thesis. A comparison has been made between using a new damage parameter by Ince and an established damage parameter by Fatemi and Socie (FS). The implemented algorithm and damage parameters were evaluated by comparing the results of the program using either damage parameter to fatigue experiments of several different load cases, including non-proportional loading. The results are fairly accurate for both damage parameters, but the one by Ince tend to be slightly more accurate, if no fitted constant to use in the FS damage parameter can be obtained.

As ligas Ti-??constituem um grupo promissor de ligas de titânio em termos de processamento, propriedades e aplicações potenciais. Além do projeto da liga em termos de composição, a obtenção de microestruturas adequadas por meio de tratamentos térmicos é necessária para otimizar o balanço entre resistência mecânica e tenacidade. O presente trabalho teve por objetivo estudar o comportamento em fadiga de alto ciclo da liga Ti-13V-11Cr-3Al, uma liga de alta resistência mecânica destinada ao emprego na indústria aeroespacial, sob duas condições de tratamento. O material foi recebido na forma de barras com 7,6 mm de diâmetro, apresentando microestrutura composta por grãos alongados e propriedades em tração combinando alto limite de resistência (1.479 MPa) e razoável ductilidade (deformação verdadeira de fratura igual a 0,217). O material foi tratado termicamente em duas condições: recozimento a vácuo a 750?C por 1 h com resfriamento lento, e tratamento flash em banho de sal a 650?C por 6 min, com posterior têmpera em água. O tratamento flash resultou em melhor combinação de resistência mecânica e ductilidade, avaliadas pelo ensaio de tração. O estudo da resistência à fadiga baseou-se na obtenção de curvas ?/N por meio de ensaios de flexão rotativa (R = -1) empregando-se corpos de prova do tipo viga em balanço. Para cada condição microestrutural, foram obtidas duas curvas ?/N, uma com amostras polidas e outra empregando-se corpos de prova com concentradores de tensão geométricos (entalhes), visando avaliar a sensibilidade ao entalhe desta liga nas duas condições microestruturais. O material submetido ao tratamento flash apresentou maior dispersão e menor resistência à fadiga, comparado ao material recozido; no entanto apresentou também menor sensibilidade ao entalhe em vidas longas (106 ciclos). O trabalho foi complementado por exames fractográficos para a identificação dos pontos de iniciação da fratura. / The Ti-??alloys are a promising group of titanium alloys in terms of processing, properties and potential applications. In order to achieve the optimum balance between strength and toughness it is necessary, besides determining a proper composition in the alloy design, to obtain suitable microstructures by means of thermo-mechanical process and heat treatment. This study aimed to assess the high cycle fatigue behavior of Ti-13V-11Cr- 3Al alloy, a high-strength alloy intended for use in the aerospace industry. The material was received in the form of bars with 7.6 mm in diameter, whose microstructure comprises elongated grains and whose mechanical properties combines high tensile strength (1,479 MPa), and reasonable ductility (true strain fracture equal to 0.217). The material was heat treated in two conditions: vacuum annealing at 750?C for 1 h with slow cooling, and flash 650?C salt bath for 6 min with subsequent water quenching. The study was based on obtaining ??/ N curves through by rotary bending (R = -1) fatigue tests employing cantilever beam specimens. For each microstructural condition, two ??/ N curves were obtained, the former with smooth samples and the latter employing specimens with geometric stress concentrators (notches), to evaluate the notch sensitivity of this alloy in both microstructural conditions. The material subjected to flash treatment showed greater dispersion and lower fatigue strength compared to annealed; however also showed lower notch sensitivity at long life (106 cycles). The study was complemented by fractographics analysis to identify the fracture initiation points.

Ao longo deste trabalho foi proposta uma nova rota de tratamento térmico para o aço AISI 52100, principal material utilizado na fabricação de rolamentos. Esta nova rota visa refinar a microestrutura de carbonetos presentes no material, com o objetivo de otimizar as propriedades mecânicas. Os tratamentos térmicos realizados conduziram a uma diminuição no tamanho dos carbonetos quando comparados com a amostra tradicional. Para medir a tenacidade à fratura evitando a dificuldade experimental de se nuclear pré-trincas em materiais frágeis, foi utilizada a metodologia Chevron, como formalizada na ASTM E-1304(97). Algumas das amostras submetidas a nova rota apresentaram maior tenacidade à fratura quando comparadas a amostra tratada na rota convencional. O material tratado na nova rota de tratamento térmico foi avaliado também em ensaio de impacto, usando-se corpos de prova Charpy de secção reduzida, sem entalhe. Os resultados obtidos mostraram que o material com carbonetos mais finos apresentavam menor energia absorvida no impacto quando comparados com a amostra tratada na rota convencional. Este comportamento a princípio contraditório foi explicado pela maior presença de sítios de nucleação na amostra com carbonetos mais finos. A maior densidade de sítios nas condições do ensaio de impacto conduziu a uma intensificação da nucleação de trincas, levando a uma menor energia absorvida durante o ensaio de impacto; este efeito ocorre mesmo quando o mecanismo de nucleação de trincas envolve a nucleação e coalescimento de microcavidades. Procurou-se fazer uma correlação entre a morfologia das superfícies de fratura obtidas no ensaio Chevron, o tamanho do carboneto das amostras e o micromecanismo atuante. Para amostras com superfície de fratura com aspecto fibroso foi associado o micromecanismo de fratura controlado por deformação. Para amostras com superfície de fratura com aspecto intergranular foi associado o micromecanismo de fratura controlado por tensão. / This work proposes a new heat treatment route for AISI 52100 steel, the most used material for bearings. The new route is designed so as to refine the carbides distribution in material, with the aim of increasing the fracture toughness. In order to measure the fracture toughness a Chevron notch methodology was used, as described in ASTM E-1304(97). Obtaining a static pre-crack on brittle materials is difficult and expensive; the Chevron notch methodology allows avoiding this difficulty. The heat treatments reduced the carbide sizes as compared with conventionally heat-treated steels. Some samples treated by this new route presented an increase in Chevron notch fracture toughness. The materials treated by the new heat treatment route were also tested in impact using a subsize Charpy specimen, without notch. Materials with fine carbides presented less dissipated energy in impact test then the material conventionally treated. This behavior is associated with the increase in number of carbide particles, as second phase particles are known to behave as sites for microvoid initiation and growth. The higher density of such sites presented in the material with fine carbides enhance this mechanism, lowering the dissipated energy measured in impact test. A correlation between fracture morphology, carbide size and fracture mechanisms was attempted. Samples with fine carbides presents fibrous transgranular fracture morphologies with strain controlled fracture micromechanism while samples with coarse carbides presented intergranular type morphologies with stress controlled micro-mechanism.

Senescent cells interact with the surrounding microenvironment achieving both pro- oncogenic and tumour-suppressive outcomes. In addition to autocrine and paracrine signalling mediated by factors of the senescence-associated secretory phenotype (SASP), we have recently identified that NOTCH1 can drive a unique form of senescence in adjacent cells via juxtacrine signalling. Here, we show that NOTCH1 signalling confers a dramatic impact on chromatin structure during senescence. RAS-induced senescent (RIS) fibroblasts often develop chromatin structures called senescence-associated heterochromatic foci (SAHF). We find that NOTCH1 inhibits SAHF formation at least partially through transcriptional repression of a critical structural component, high-mobility group A (HMGA). Using ATAC-sequencing (assay for transposase accessible chromatin) we demonstrate that nucleosome positioning is substantially altered in RIS and that this re-distribution is also antagonised by NOTCH1, resulting in a distinct chromatin landscape. Importantly, normal or cancer cells that express the NOTCH ligand jagged-1 can drive similar chromatin structural changes in adjacent cells in a cell-cell contact dependent manner. In addition, using a highly optimised chromatin immunoprecipitation (ChIP-seq) protocol and the proximity ligation assay ‘Hi-C’, we demonstrate that HMGA proteins are directly involved in the formation of long-range interactions in RIS cells that may underpin SAHF formation. These ChIP-seq data have also allowed us to identify a unique HMGA1 binding profile, potentially suggesting a novel role for HMGA1 in gene regulation. Together, our data indicate that NOTCH signalling, both cell-autonomously and non-cell-autonomously, can repress HMGA1, a multi-faceted protein that regulates nucleosome positioning (1D structure), SAHF formation (3D structure) and potentially mRNA abundance.