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Είναι ευρέως αποδεκτό και καλά τεκμηριωμένο ότι ο υποδοχέας του επιδερμικού αυξητικού παράγοντα (epidermal growth factor receptor, EGFR) ελέγχει σημαντικές λειτουργίες των καρκινικών κυττάρων, όπως τον πολλαπλασιασμό και την απόπτωση, αλλά και διαδικασίες όπου συμμετέχουν περισσότεροι του ενός τύποι κυττάρων, όπως τη διήθηση και την αγγειογένεση. Μεταξύ των τύπων καρκίνου, στην ανάπτυξη των οποίων συμμετέχει ο EGFR, είναι και ο μη μικροκυτταρικός καρκίνος του πνεύμονα (ΜΜΚΠ). Πολύ πρόσφατα δεδομένα δείχνουν ότι ένα άλλο μόριο που εμπλέκεται στην ανάπτυξη του καρκίνου του πνεύμονα είναι το Notch. Ο ρόλος του είναι περίπλοκος και διττός: Έχει προταθεί ότι το Notch επάγει την ανάπτυξη του ΜΜΚΠ και αναστέλλει την ανάπτυξη του μικροκυτταρικού καρκίνου του πνεύμονα (ΜΚΠ). Επιπλέον, έχει βρεθεί ότι το μονοπάτι μεταγωγής σήματος του Notch επηρεάζει, αλλά και επηρεάζεται από άλλα μόρια. Στην παρούσα μεταπτυχιακή εργασία διερευνήθηκε ο ρόλος του EGFR και του Notch στην ανάπτυξη κυττάρων ΜΜΚΠ χρησιμοποιώντας τον προσδέτη του EGFR, EGF και τον αναστολέα της γ-σεκρετάσης DAPT. Για τη διεξαγωγή των πειραμάτων χρησιμοποιήθηκαν οι ανθρώπινες καρκινικές κυτταρικές σειρές ΜΜΚΠ Η23, Α549, Η661 και ΗCC827. Οι κυτταρικές σειρές Η23, Α549 και Η661 εκφράζουν τον αγρίου τύπου (wild type, wt) EGFR και η κυτταρική σειρά HCC827 εκφράζει EGFR που φέρει τη μετάλλαξη (mutation) (DE746- A750). Αρχικά με ανάλυση κατά western μελετήθηκε το προφίλ των κυττάρων ως προς τα επίπεδα έκφρασης του ενδοκυττάριου τμήματος του Notch (Notch Intracellular Domain, NICD). Βρέθηκε ότι τα κύτταρα Η23 εκφράζουν τα υψηλότερα επίπεδα Notch ICD, τα κύτταρα Η661 και HCC827 μέτρια επίπεδα και τα κύτταρα Α549 τα χαμηλότερα. Στη συνέχεια με τη μέθοδο του ΜΤΤ έγινε έλεγχος του DAPT στον πολλαπλασιασμό των κυττάρων και βρέθηκε ότι τα κύτταρα Η661 είχαν τη μεγαλύτερη αναστολή, παρόμοια συμπεριφορά έδειξαν και τα Α549. Τα κύτταρα Η23 εμφάνισαν μικρότερη ανταπόκριση σε σχέση με τα Η661 ενώ τα κύτταρα HCC827 εμφανίστηκαν ανθεκτικά στο DAPT. Η ανασταλτική δράση του DAPT στα κύτταρα Η661 συνοδεύτηκε με επαγωγή της απόπτωσης η οποία προσδιορίστηκε με τη μέθοδο αννεξίνης V καθώς και με επαγωγή της αυτοφαγίας η οποία ανιχνεύτηκε κάνοντας ανάλυση κατά western για τα πρωτεϊνικά επίπεδα της beclin-1. Περαιτέρω τα κύτταρα ενεργοποιήθηκαν με EGF και εν συνεχεία προστέθηκε DAPT. Παρατηρήθηκε ότι στα κύτταρα Η23 η προσθήκη του EGF δεν επέτρεψε να δράσει ανασταλτικά το DAPT ενώ στα Η661 εν μέρει ο EGF αντέστρεψε την ανασταλτική δράση του DAPT. Επιλέγοντας τις κυτταρικές σειρές Η23 και Η661, μελετήθηκε η δράση του DAPT και του EGF στα επίπεδα του Notch ICD. Παρατηρήθηκε ότι στα κύτταρα Η23, το DAPT μείωσε με χρονοεξαρτώμενο τρόπο τα πρωτεϊνικά επίπεδα του Notch ICD μέχρι και 6 ώρες μετά την προσθήκη του στα κύτταρα ενώ 24 ώρες μετά το φαινόμενο αντιστράφηκε. Η προσθήκη του EGF δεν επηρέασε τα επίπεδα του Notch ICD σε καμία από τις χρονικές στιγμές που μελετήθηκαν. Στα Η661 κύτταρα το DAPT προκάλεσε χρονοεξαρτώμενη μείωση των επιπέδων Notch ICD η οποία διήρκησε μέχρι και 24 ώρες μετά τη προσθήκη του DAPT. Ο EGF όπως και προηγουμένως δεν επηρέασε τα επίπεδα του Notch ICD. Παρατηρώντας ότι στα Η661 το DAPT ασκεί δράση με μεγαλύτερη διάρκεια σε σχέση με τα κύτταρα Η23, τα κύτταρα Η661 ενεργοποιήθηκαν με EGF και στη συνέχεια προστέθηκε το DAPT προκειμένου να δούμε τη δράση του συνδυασμού στα επίπεδα του Notch ICD. Βρέθηκε ότι ο EGF αντέστρεψε την μείωση των Notch ICD επιπέδων που προκαλεί μόνο του το DAPT. Τα αποτελέσματα αναδεικνύουν ότι τα μονοπάτια του EGFR και του Notch, συνηγορούν προς την ίδια κατεύθυνση για τη μείωση του όγκου και αυτό υποδηλώνει έναν ελκυστικό δρόμο συνδυαστικών προσεγγίσεων για τη θεραπεία του ΜΜΚΠ, που μπορεί να ενισχύσει τη δράση των ανασταλτικών παραγόντων του EGFR σε όγκους. Συμπερασματικά, θα μπορούσαμε να υποθέσουμε ότι στο ΜΜΚΠ: α) τα δύο μονοπάτια EGFR και Notch συνεπικουρούν για την ανάπτυξη του όγκου, β) η αναστολή του Notch είναι πιο αποτελεσματική σε κύτταρα με ενδιάμεσα επίπεδα ενεργού Notch 1, προκαλώντας τόσο απόπτωση όσο και αυτοφαγία, και γ) η μετάλλαξη του EGFR προσφέρει αντίσταση στη δράση αναστολέα της γ-σεκρετάσης. / It is widely accepted and well established that the epidermal growth factor receptor (EGFR) controls important processes of tumor cells, such as proliferation and apoptosis, but also processes involving more than one type of cells such as invasion and angiogenesis. It has been found that the EGFR has an important role in the development of several types of cancer including non-small cell lung cancer (NSCLC). Very recent data indicate that another molecule, which is involved in the development of lung cancer, is Notch. Its role is complicated and is under investigation. It is suspected that Notch has a growth promoting function in NSCLC, whereas exerts an inhibitory effect in small cell lung cancer (SCLC). Furthermore it has been found that the signaling pathway of Notch can affect/ can be affected by other molecules. This thesis investigated the role of EGFR and Notch in cell growth of NSCLC cells using the ligand of EGFR, EGF and gamma-secretase inhibitor, DAPT. To conduct the experiments the human NSCLC cell lines H23, A549, H661 and HCC827 were used. The cell lines H23, A549 and H661 express the wild type (wt) EGFR and the cell line HCC827 expresses EGFR bearing the mutation (mt) DE746-A750. Initially, we studied the profile of NSCLC cells regarding the protein levels of Notch intracellular domain (Notch ICD) using western blot analysis. It was found that H23 cells express the higher levels Notch ICD, H661 and HCC827 cells express intermediate levels and A549 cells express the lowest levels of Notch ICD. The next step was the evaluation of DAPT effect in cell proliferation using the MTT assay. We found that DAPT caused the greatest inhibition to H661 and A549 cells. DAPT was less effective to H23 cells while had no effect to HCC827 cells. The inhibitory effect of DAPT in H661 cells was in line with the induction of apoptosis and autophagy, as was detected using annexin V assay and western blot analysis for beclin-1, respectively. Furthermore, cells were stimulated with EGF and subsequently DAPT was added. We found that the stimulatory effect of EGF was not reversed by DAPT in H23 cells. However a partial reverse of EGF stimulation was observed in H661 cells. The next step was to study the effect of DAPT and EGF at Notch ICD protein levels, in H23 and H661 cells. We found that DAPT reduced the protein levels of Notch ICD in H23 cells, with a time-dependent manner, up to 6 hours after DAPT addition and this effect reversed 24 hour later. The addition of EGF did not affect the levels of Notch ICD at any time point tested. In H661 cells, DAPT caused a time-dependent reduction of Notch ICD protein levels up to 24 hours after DAPT addition to cells. EGF as previously, did not affect the levels of Notch ICD in these cells. Since DAPT was more effective to H661 cells, these cells stimulated with EGF and then DAPT was added in order to study the effect of the combination at the levels of Notch ICD. We found that EGF reversed the decrease of Notch ICD protein levels caused by DAPT alone. These results indicate that the pathways of EGFR and Notch might act with a synergistic fashion and this could be an attractive approach for the treatment of NSCLC. Summarizing our results, we might assume that in NSCLC: a) both pathways of EGFR and Notch exert a significant role in tumor growth, b) the inhibition of Notch is more effective in cells with intermediate levels of activated Notch 1, causing both apoptosis and autophagy, and c) the EGFR mutation confers resistance to the effect of γ- secretase inhibitor.

<p>Understanding signalling pathways that control pancreatic endocrine tumour (PET) development and proliferation may reveal novel targets for therapeutic intervention. The pathogenesis for sporadic and hereditary PETs, apart from mutations of the <i>MEN1</i> and <i>VHL</i> tumour suppressor genes, is still elusive. The protein product of the <i>MEN1</i> gene, menin, regulates many genes. The aim of this thesis was to identify genes involved in pancreatic endocrine tumourigenesis, with special reference to Notch signalling.</p><p>Messenger RNA and protein expression of NOTCH1, HES1, HEY1, ASCL1, NEUROG3, NEUROD1, DLK1, POU3F4, PDX1, RPL10, DKK1 and TPH1 were studied in human PETs, sporadic and MEN 1, as well as in tumours from heterozygous <i>Men1</i> mice. For comparison, normal and <i>MEN1</i> non-tumourous human and mouse pancreatic specimens were used. Nuclear expression of HES1 was consistently absent in PETs. In mouse tumours this coincided with loss of menin expression, and there was a correlation between <i>Men1</i> expression and several Notch signalling factors. A new phenotype consisting of numerous menin-expressing endocrine cell clusters, smaller than islets, was found in <i>Men1</i> mice. Expression of NEUROG3 and NEUROD1 was predominantly localised to the cytoplasm in PETs and islets from MEN 1 patients and <i>Men1</i> mice, whereas expression was solely nuclear in wt mice. Differences in expression levels of Pou3f4, Rpl10 and Dlk1 between islets of <i>Men1</i> and wt mice were observed.</p><p>In addition, combined RNA interference and microarray expression analysis in the pancreatic endocrine cell line BON1 identified 158 target genes of ASCL1. For two of these, DKK1 (a negative regulator of the WNT/β-catenin signalling pathway) and TPH1, immunohistochemistry was performed on PETs. In concordance with the microarray finding, DKK1 expression showed an inverse relation to ASCL1 expression.</p><p>Altered subcellular localisation of HES1, NEUROD1 and NEUROG3 and down-regulation of DKK1 may contribute to tumourigenesis.</p>

Au cours de l'ovogenèse, les stades vitellogéniques nécessitent une énergie considérable, et leur formation doit être ajustée en fonction d'autres besoins physiologiques. En utilisant la drosophile comme modèle, j'ai montré que la signalisation Insuline régule une transition du cycle cellulaire, mitose/ endocyle (M/E), une étape critique qui contrôle l'entrée des follicules en vitellogenèse. Mes travaux montrent que la transition M/E porte le rôle d'un point de contrôle nutritionnel. La carence protéique induit un blocage de cette transition au travers d'une interaction entre FoxO, Cut et Notch, empêchant une perte d'énergie. Ce blocage reste réversible, autorisant la reprise de l'ovogenèse sous retour à une alimentation normale. Ce travail montre qu'un point de contrôle nutritionnel au cours de l'ovogenèse permet de coupler des signaux métaboliques et développementaux pour protéger les tissus des dommages liés à la carence. D'autre part, j'ai montré que la signalisation Insuline contrôle la migration d'une cohorte de cellules d'origine épithéliale pour assurer la fertilité de l'ovocyte. L'insuline participe à la formation d'extensions cytoplasmiques riches en actine. Lors de ce processus, la signalisation Insuline contrôle notamment l'expression de chickadee, qui code pour la Profiline, une protéine nécessaire pour la polymérisation de l'actine qui permet la motilité des cellules. L'ensemble de ce travail montre que des tissus somatiques assurent l'homéostasie de l'ovogenèse malgré des conditions de nutritions fluctuantes. Ces travaux posent les bases de l'étude de nouveaux aspects de l'ovogenèse, potentiellement conservés chez les mammifères.

The unique thermo-mechanical behavior of Shape Memory Alloys (SMAs), such as their ability to recover the original shape upon heating or being able to tolerate large deformations without undergoing plastic transformations, makes them a good choice for actuators. This work studies their application in the aerospace and defense industries where SMA components can serve as release mechanisms for gates of enclosures that have to be deployed remotely. This work provides a novel approach in evaluating the stress and heat induced change of phase in a SMA, in terms of the transformation strain tensor. In particular, the FEA tool ANSYS has been used to perform a 2-D analysis of a Cu-Al-Zn-Mn SMA specimen undergoing a nontraditional loading path in two steps with stress and heating loads. In the first load step, tensile displacement is applied, followed by the second load step in which the specimen is heated while the end displacements are held constant. A number of geometric configurations are examined under the two step loading path. Strain results are used to calculate transformation strain which provides a quantitative measure of phase at a material point; when transformation strain is zero, the material point is either twinned martensite, or austenite depending on the temperature. Transformation strain value of unity corresponds to detwinned martensite. A value between zero and one indicates mixed phase. In this study, through two step loading in conjunction with transformation strain calculations, a method for mapping transient non-uniform distribution of phases in an SMA is introduced. Ability to obtain drastically different phase distributions under same loading path by modifying the geometry is demonstrated. The failure behavior of SMAs can be designed such that the load level the crack initiates and the path it propagates can be customized.

La voie de signalisation Notch est conservée au cours de l'évolution. Elle joue un rôle clé dans le développement, et elle est impliquée dans de nombreuses décisions de destin cellulaire, dans le maintien des cellules souches, et dans le contrôle de la prolifération et de la différenciation cellulaires. Une dérégulation de la signalisation Notch est impliquée dans diverses maladies et cancers, y compris les tumeurs solides, comme les cancers du sein et du col de l'utérus, et les leucémies, comme la Leucémie Aiguë Lymphoblastique des cellules T (LAL-T). Notch est un récepteur transmembranaire activé par des ligands transmembranaires de la famille DSL (Delta/Serrate/Lag-2). Bien que plusieurs mutations oncogéniques ont été identifiées au niveau du récepteur Notch, de nombreux cancers modulés par Notch demeurent ligand-dépendants. Étonnamment, les mécanismes moléculaires régulant l'activation du ligand sont encore relativement peu caractérisés par rapport à ceux qui régissent le récepteur Notch lui-même. Utilisant un essai de co-culture avec un rapporteur luciférase de Notch, nous avons effectué le premier crible d'ARNi pan-génomique visant spécifiquement à identifier des régulateurs des ligands de Notch dans la cellule émettrice du signal. Nous avons ainsi pu découvrir de nouvelles classes de régulateurs communs pour les ligands Delta-like1 et 4. Ces régulateurs comprennent des inhibiteurs de protéases, des facteurs de transcription, et des gènes divers à fonction inconnue, tels que Tmem128 « Transmembrane protein 128 », ou à fonction préalablement caractérisée tels que la co-chaperonne moléculaire Cdc37 « Cell division cycle 37 homolog ». Par la suite, nous avons développé des cribles secondaires fonctionnels où nous avons démontré l'importance de ces régulateurs pour des événements Notch-dépendants, comme la différenciation des cellules T normales, et la survie des cellules souches pré-leucémiques isolées à partir d'un modèle murin de LAL-T. En outre, nous avons prouvé que les régulateurs les plus forts du crible de survie sont également nécessaires pour l'activité d'auto-renouvellement des cellules souches pré-leucémiques. Finalement, nous avons entamé une caractérisation moléculaire préliminaire de deux régulateurs nouvellement identifiés; Tmem128 et Cdc37 afin d'étudier leur mécanisme d'action sur les ligands. En conclusion, cette étude nous a permis d'identifier de nouveaux régulateurs de la voie Notch qui pourraient servir de cibles thérapeutiques potentielles dans les cancers; tel qu'illustré par le modèle LAL-T. La compréhension des détails moléculaires sous-jacents aux fonctions de ces régulateurs sera essentielle afin de développer des inhibiteurs pharmacologiques pour bloquer leur action et entraver la signalisation Notch dans le cancer. / The Notch signalling pathway is evolutionarily conserved. It plays a key role in development and it is involved in multiple cell fate decisions, in the maintenance of stem cells, and in the regulation of cell proliferation and differentiation. Misregulation of Notch signalling is implicated in various diseases and cancers including solid tumours, such as breast and cervical cancers, and leukemias, such as T-cell Acute Lymphoblastic Leukemia (T-ALL). Notch is a transmembrane receptor activated by transmembrane ligands of the DSL family (Delta/Serrate/Lag-2). Whereas oncogenic mutations have been identified in the Notch receptor, many Notch-mediated cancers remain ligand-dependent. Strikingly, the molecular mechanisms that regulate ligand activation are still poorly characterized as compared to those regulating the Notch receptor itself. Using a co-culture assay with a luciferase Notch reporter, we performed the first genome-wide RNAi screen aiming specifically at identifying regulators of Notch ligands in the signal-sending cell. We thereby unraveled new classes of common regulators for both Delta-like1 and 4 ligands. These regulators include protease inhibitors, transcription factors and various genes of unknown function such as Tmem128 (Transmembrane protein 128), or of previously characterized function such as the molecular co-chaperone Cdc37 (Cell division cycle 37 homolog). We next developed functional secondary screens where we demonstrated that our hits are important for Notch-mediated events, such as normal T-cell differentiation, and survival of pre-leukemic stem cells (pre-LSCs) isolated from a mouse model of T-ALL. Moreover, we showed that top hits from the pre-LSC survival screen are also required for the self-renewal activity of pre-LSCs. Finally, we performed a preliminary molecular characterization of two newly identified regulators; Tmem128 and Cdc37 in order to investigate their mechanism of action on Delta-like ligands. Altogether, this study led to the identification of novel Notch pathway regulators that could serve as potential therapeutic targets in Notch cancers, as exemplified by the T-ALL model. Elucidating the finer details that underlie the molecular functions of these regulators will be critical to develop pharmacological inhibitors to counteract their action and impede Notch signalling in cancer.

As ligas de alumínio são aplicadas em cerca de 70% dos componentes estruturais dos aviões e o processo de fadiga e o modo de falha predominante em estruturas aeronáuticas, para a maioria das quais a presença de concentradores de tensão e inevitável. O comportamento em fadiga e as propriedades mecânicas das ligas de alumínio endurecíveis por precipitação são fortemente influenciadas por parâmetros como tamanho, espaçamento e densidade dos precipitados endurecedores. Neste contexto, pesquisas anteriores tem mostrado que o envelhecimento interrompido (T6I4) pode proporcionar melhores combinações de propriedades mecânicas para estas ligas. O presente trabalho tem como objetivo o estudo das propriedades mecânicas e do comportamento em fadiga de alto ciclo das ligas de alumínio AA6351 e AA7050 nas condições de tratamento térmico convencionais (T6 e T7451, respectivamente) e na condição T6I4, bem como da influência das características microestruturais e do tratamento térmico sobre estas propriedades. Dentro deste contexto, analises de Microscopia Eletrônica de Transmissão (MET) da liga AA6351 mostraram que a condição T6I4 resultou em uma maior densidade de precipitados endurecedores com tamanho heterogêneo, quando comparada com a condição T6. Para esta liga, a condição T6I4 também resultou em menores valores de tensão limite de escoamento, resistência a tração, resistência a fadiga e sensibilidade ao entalhe, com maior ductilidade e tenacidade. Para a liga AA7050, as análises de MET mostraram que a condição T6I4 resulta em uma maior densidade de precipitados endurecedores com menor tamanho, promovendo um melhor impedimento ao movimento de discordâncias durante a deformação por fadiga, quando comparada com a condição T7451. Esta alteração microestrutural proporcionou a condição T6I4 valores de resistência ao escoamento e resistência a tração similares a condição T7451, com maior ductilidade e tenacidade. A resistência a fadiga da condição T6I4 foi similar a condição T7451, entretanto o envelhecimento interrompido resultou em um melhor comportamento em sensibilidade ao entalhe. / Aluminum alloys are applied in approximately 70% of the aircraft structural components and the fatigue process is the dominant failure mode in aeronautical structures, for the most of which, the presence of stress concentrators is unavoidable. The fatigue behavior and the mechanical properties of the age hardenable aluminum alloys are strongly influenced by parameters including the size, spacing and density of strengthening precipitates. Within this context, previous researches have shown that the interrupted ageing (T6I4) could provide an improved combination of mechanical properties for these alloys. This work aims to study the mechanical properties and the high cycle fatigue behavior of AA6351 and AA7050 aluminum alloys in the conventional heat treatment conditions (T6 and T7451, respectively) and in the T6I4 condition, as well as the influence of the microstructural characteristics and of the heat treatment on these properties. Within this context, Transmission Electron Microscopy (TEM) analyzes of the AA6351 alloy showed that T6I4 condition resulted in higher density of hardening precipitates with heterogeneous size compared to T6 condition. For this alloy, the T6I4 condition resulted in lower values of yield stress, ultimate tensile strength, fatigue strength and notch sensitivity, with higher ductility and toughness. For the AA7050 alloy, TEM analyses showed that T6I4 condition presented a higher density of strengthening precipitates with smaller size promoting an improved dislocation pinning effect during the fatigue deformation compared to T7451 condition. This microstructural change provided to T6I4 condition yield stress and ultimate tensile strength similar to T7451, with higher ductility and toughness. The fatigue strength of T6I4 condition was also similar to T7451, however the interrupted ageing provided a better notch sensitivity behavior.

A indústria automotiva tem mostrado um crescente interesse pelas ligas de alumínio, em especial as ligas de alumínio da série 6xxx. Esta classe é amplamente empregada nas áreas de construção e de transporte, devido a sua boa resistência mecânica e excelente resistência à corrosão. No setor automobilístico sempre houve a necessidade de aprimoramento dos estudos do comportamento em fadiga, pois os componentes estruturais são submetidos a carregamentos vibratórios, esforços e tensões cíclicas e, como consequência, podem trincar e finalmente fraturar. A presença de um entalhe geralmente diminui a vida em fadiga, criando regiões de altas tensões triaxiais localizadas que restringem a deformação plástica, fragilizando o material. A resposta mecânica depende das solicitações, microestrutura, componentes da liga e propriedades do material. Neste trabalho foi realizado o estudo do comportamento em fadiga de alto ciclo e da sensibilidade ao entalhe de três ligas de alumínio da série 6xxx destinadas à fabricação de componentes de carroçarias para caminhões e ônibus: AA 6005, AA 6063 e AA 6351, todas na condição T6. As curvas S/N foram obtidas por meio de ensaios de fadiga em flexão rotativa (R = -1). Ensaios com peças entalhadas (Kt ? 3,0) permitiram determinar e comparar o fator de concentração de tensão em fadiga e a sensibilidade ao entalhe das ligas estudadas. Também foi estudada a influência da microestrutura e das partículas intermetálicas sobre as propriedades de fadiga. As superfícies das peças fraturadas foram observadas ao MEV e verificou-se que a maioria dos sítios de nucleação de trincas por fadiga ocorreram próximos a partículas de segunda fase que atuam como concentradores de tensão. / The automotive industry has shown a growing interest in aluminum alloys, particularly the AA 6xxx series. This class of alloys is widely used in construction and transportation because of their good mechanical strength, easy fabrication and excellent corrosion resistance. In the automotive sector there was always a need for improved studies of the fatigue behavior, because the structural components are subjected to vibratory loads and cyclic stresses and, as a consequence, may eventually crack and fracture. The presence of a notch generally decreases the fatigue life, creating regions of high triaxial stresses which restrain plastic deformation, weakening the material. The response depends on the mechanical solicitations, microstructure, the alloy components and the material properties. In this work it was conducted a study of the high cycle fatigue behavior and the notch sensitivity of three aluminum alloys used in components of truck and bus bodies: AA 6005, AA 6063 and AA 6351, all provided in T6 condition. The S/N curves were obtained by tests in rotating bending fatigue (R = -1). Tests with notched samples (Kt?3.0) allowed to determine and compare the fatigue concentration factor and the notch sensitivity of the studied alloys. It was also studied the influence of microstructure and the intermetallic particles on the fatigue properties. The surfaces of the fractured samples were observed via SEM and showed that most of the nucleation sites of fatigue cracking occurred near the second phase particles, which act as stress concentrators.

This thesis provides a structural and materials engineering explanation for many of the running fractures that occurred in steel structures during the destructive Kobe and Northridge earthquakes in the mid 1990s. A method is developed that allows the ductile endurance of structural steel members subjected to cyclic plastic deformation during earthquakes to be assessed and for pre-necking running fractures to be avoided. The study commenced following the 2000 World Earthquake Conference in Auckland. The conference brought together the findings of the huge research effort, in America, Japan, Europe and New Zealand, that followed the Kobe and Northridge earthquakes. The running fractures that had occurred in steel structures represented an unpredicted failure mode that structural engineers have not known how to predict or suppress through the engineering design process. A clear fundamental understanding of the causes and how to prevent the fractures did not arise from the conference. In fact apparently conflicting results were reported. Full scale cyclic tests in New Zealand on structural assemblies had not resulted in running fractures, whereas tests in American and Japan had. Structural engineers designing earthquake resistant structures rely on constructional steel to be materially homogeneous and nominally tri-linear in behaviour. Steel is expected to behave elastically under regular in-service loading, have a reliable and flat yield stress-strain characteristic, and under overload then develop predictable levels of strain-hardening in conjunction with significant plastic elongation up to its ultimate tensile strength. Steel is expected to eventually fracture after further plastic elongation and necking. Ductile design strategies and methods utilise the plastic elongation characteristics of steel to protect structures in earthquake. Plastic deformation is considered to beneficially dissipate energy generated in the structure by a severe earthquake and also dampen the structure’s response. The occurrence of running fracture without significant cyclic plastic deformation and before section necking in steelwork, therefore undermines the basis of the ductile seismic design approach. The initial part of the thesis is devoted to bringing together the fundamental aspects of materials engineering related to fracture of constructional steel. This is intended to provide a bridge of knowledge for structural engineering practitioners and researchers not fully conversant with materials engineering aspects of fracture. Fracture behaviour in steel is a broad and complex topic that developed rapidly in the twentieth century driven by the demands of technological growth. The unexpected fracture of welded liberty ships at sea in World War 2; the need for reliable long term containment for the nuclear reactors in the 1950s and 1960s; and prevention of fatigue failures in aircraft frames since the 1950s all drove engineering research into steel fracture behaviour. There are many subtle variations in definitions in the published literature on fracture that can be confusing. Therefore an attempt has been made to clarify terminology. The term brittle fracture in particular is only used in this thesis as applying to running fracture when the general or far field tensile stresses are below the yield stress of the steel. The term pre-necking or running fracture is preferred to describe the condition more broadly which may occur prior to and also after general yielding, but before section necking. Running fracture is a manifestation of pre-necking fracture in which insufficient plastic flow is available in the assembly to absorb the energy released upon fracture. The experimental studies investigated the behaviour of constructional steel commonly used in New Zealand, at various levels of plastic strain. This started with Charpy V-Notch (CVN) testing which revealed that a significant transition temperature shift and curve shape change occurs with increasing plastic strain and the associated strain-hardening. This showed that the ability of steel to avoid pre-necking or running fracture reduces as the level of plastic strain-hardening increases. Temperature controlled Crack Tip Opening Displacement (CTOD) testing was then undertaken. The setting of testing temperatures for the CTOD tests were guided by review of the CVN test results, using published CVN to fracture toughness correlation methods. However running cleavage fractures developed in the CTOD specimens at higher than predicted temperatures of 10 oC and 20 oC. These are typical service temperatures for structures in New Zealand and so are very likely to occur at the time of an earthquake. The implication from this is that there are levels of strain-hardening and conditions of material notching constraint that can lead to pre-necking and running fracture in New Zealand fabricated steel structures, under severe earthquake loading. Care was taken in the CTOD testing to monitor and maximise the capture of data electronically using a specially developed Direct Current Potential Drop method. This allowed the test results to be analysed and considered in varying ways, leading to a consistent assessment of the CTOD, crack growth, and the specific work of fracture in each test piece. While CTOD test results have sometimes been published by structural and welding engineering researchers in the wake of Kobe and Northridge, the results were typically of little use for this study as the CTOD initiation point was generally not identified effectively. The effect of remote plastic flow in the specimens was also not adequately accounted for. The CTOD test results were often simply used to help correlate other factors observed by the researchers. Side-grooving of specimens was not reported as having been used in any of the published results reviewed. When conducting CTOD test with highly ductile constructional steels it is very difficult to get useful CTOD results if the specimens are not side-grooved, as significant necking and tunnelling will otherwise occur and limit the usefulness of the results. Work by Knott and also by McRobie and Smith was seminal in terms of identifying some critical aspects of plane strain development in CTOD tests, and the links to non-metallic particle density with respect to fracture toughness and CTOD at initiation. Some of their findings with regards to the effect of pre-strain on CTOD initiation were subsequently found to confirm the experimental findings in this study. No effective methodology for prediction of pre-necking or running fracture in a structural member or assembly when subjected to gross plastic cyclic deformation was found to exist in the literature. It was concluded however that the principles of specific work of fracture, and monotonic and cyclic fracture similitude were particularly relevant. These were therefore utilised in the development of the design method proposed in this thesis. The CTOD test results were reviewed, isolating the remote plastic flow component, to determine the critical specific work of fracture property Rc of the steels tested. A meeting with Professor Kuwamura at the University of Tokyo was providential, allowing discussion of his similitude principle, and observations in person of some of the fractured specimens developed during his full scale test series’. Running fractures with cleavage were evident in the specimens, with their tell-tale chevron markings. He had predicted running fracture problems in structures in Japan ahead of the Kobe earthquake and been largely ignored. His insights were subsequently seriously considered in Japan after the earthquake. He and his colleagues developed the principle of structural similitude that relates monotonic fracture displacement ductility to cyclic fracture displacement ductility for a particular assembly. This arose from their observation that running fractures developed from ductile crack formation at blunt notches in structures. The similitude principle has echoes of the Coffin-Manson approach to ductile crack initiated low cycle fracture. The principle of similitude has a log–log relationship as does the Manson-Coffin relationship. So where notch plasticity controls the initiation of fracture in a structural assembly it is conceptually reasonable to expect that the number of cycles to initiation of fracture from a notch will have a log–log relationship to the amplitude of the cyclic strain developed in the notch. Kuwamura found that steel assemblies with lower CVN energy had reduced cyclic fracture endurance than the same assemblies made with steel with higher CVN impact energy. However no method of predicting performance of any particular assembly could be developed from his observations. The benefit of his method primarily relates to the minimising of testing necessary to assess the fracture limited cyclic displacement ductility of a structural assembly. However it doesn’t provide a means for designing a structural assembly to achieve specific levels of ductile endurance other than clearly identifying the need to use steel with good CVN characteristics. The most significant development arising from this thesis is therefore the development of a design method to assess cyclic ductile endurance. The method utilises the specific work of fracture properties obtained from CTOD specimens of the steel in conjunction with a relatively simple fracture mechanics assessment and an elasto-plastic finite element analysis (FEA). The FEA model is used to determine the displacement ductility of the assembly at the calculated onset of pre-necking fracture. The elasto-plastic stress–strain properties of the steel in various pre-strain states required for the FEA may be derived from tensile testing. Kuwamura’s similitude principle is then used to predict cyclic plastic endurance at various constant displacement ductility amplitudes. The method is extended using Miner’s rule to allow for the effects of increasing variable amplitude cyclic plastic loading. In summary the thesis explains why pre-necking and running fractures occur in steel members subjected to cyclic plastic deformation during a severe earthquake. In addition a method for consistently assessing the ability of structural steel assemblies to achieve a specified level of ductile endurance during earthquakes is proposed. The method is verified against published results for a cyclic test of a simple steel member with a crack at mid-span. / Whole document restricted, but available by request, use the feedback form to request access.

Understanding signalling pathways that control pancreatic endocrine tumour (PET) development and proliferation may reveal novel targets for therapeutic intervention. The pathogenesis for sporadic and hereditary PETs, apart from mutations of the MEN1 and VHL tumour suppressor genes, is still elusive. The protein product of the MEN1 gene, menin, regulates many genes. The aim of this thesis was to identify genes involved in pancreatic endocrine tumourigenesis, with special reference to Notch signalling. Messenger RNA and protein expression of NOTCH1, HES1, HEY1, ASCL1, NEUROG3, NEUROD1, DLK1, POU3F4, PDX1, RPL10, DKK1 and TPH1 were studied in human PETs, sporadic and MEN 1, as well as in tumours from heterozygous Men1 mice. For comparison, normal and MEN1 non-tumourous human and mouse pancreatic specimens were used. Nuclear expression of HES1 was consistently absent in PETs. In mouse tumours this coincided with loss of menin expression, and there was a correlation between Men1 expression and several Notch signalling factors. A new phenotype consisting of numerous menin-expressing endocrine cell clusters, smaller than islets, was found in Men1 mice. Expression of NEUROG3 and NEUROD1 was predominantly localised to the cytoplasm in PETs and islets from MEN 1 patients and Men1 mice, whereas expression was solely nuclear in wt mice. Differences in expression levels of Pou3f4, Rpl10 and Dlk1 between islets of Men1 and wt mice were observed. In addition, combined RNA interference and microarray expression analysis in the pancreatic endocrine cell line BON1 identified 158 target genes of ASCL1. For two of these, DKK1 (a negative regulator of the WNT/β-catenin signalling pathway) and TPH1, immunohistochemistry was performed on PETs. In concordance with the microarray finding, DKK1 expression showed an inverse relation to ASCL1 expression. Altered subcellular localisation of HES1, NEUROD1 and NEUROG3 and down-regulation of DKK1 may contribute to tumourigenesis.