Osteoarthritis (OA) is the leading cause of disability in North American and has major economic consequences for society. People with knee OA experience the worst quality of life, among musculoskeletal conditions, with function and mobility being influenced by symptoms such as pain and stiffness. However, the impact of OA symptoms varies due to intrinsic and extrinsic factors, leading many researchers to employ biopsychosocial and other population health frameworks to study the disease. These population health approaches have not been adopted when studying knee OA outcomes in bioarchaeology, where a limited biological lens prevails due to the sole reliance on skeletal remains. The purpose of this research was to explore methods for identifying the impairment potential of knee OA in archaeological populations using a clinical sample and population health approaches. Clinical studies have the advantage of assessing not only the biological implications of knee OA but also the functional outcomes. By creating a knee OA grading system applicable for both MRI and dry bone femora samples (Clinical Archaeological Osteoarthritis Score) a link between clinical and archaeological populations was proposed. Using this link to infer functional deficits onto archaeological populations using population health frameworks, a theoretical analysis was performed with two populations; the 17th century Huron and the 19th century Inuit from the Igloolik region of Nunavut. The results demonstrated the increased impairment potential of knee OA in the Inuit population versus the Huron population, produced by contrasting factors captured by the determinants of health, including social and physical environments.

Crumbs is a transmembrane protein and apical determinant in Drosophila epithelial cells. Its cytoplasmic tail contains a PDZ and a FERM domain-binding site through which Crumbs interacts with the FERM proteins Yurt, Moesin and Expanded. Recent evidence suggests that Crumbs can also interact with the uncharacterised FERM proteins CG34347 and Cdep. The main objective of my thesis was to generate mutations in CG34347 and Cdep to facilitate the functional analysis of these genes. I generated a mutation for Cdep that remains to be characterised and two mutant lines for CG34347; one lacking the first exon and one lacking the entire gene, using a FRT-based recombination strategy. Both CG34347 mutants cause severe ovarian defects. The most consistent defect is a multilayering of the interfollicular stalk. These defects are also observed when Notch, Hippo, Wingless and Hedgehog signalling pathways are overactive in ovaries suggesting that CG34347 participates in one of those pathways.

Crumbs is a transmembrane protein and apical determinant in Drosophila epithelial cells. Its cytoplasmic tail contains a PDZ and a FERM domain-binding site through which Crumbs interacts with the FERM proteins Yurt, Moesin and Expanded. Recent evidence suggests that Crumbs can also interact with the uncharacterised FERM proteins CG34347 and Cdep. The main objective of my thesis was to generate mutations in CG34347 and Cdep to facilitate the functional analysis of these genes. I generated a mutation for Cdep that remains to be characterised and two mutant lines for CG34347; one lacking the first exon and one lacking the entire gene, using a FRT-based recombination strategy. Both CG34347 mutants cause severe ovarian defects. The most consistent defect is a multilayering of the interfollicular stalk. These defects are also observed when Notch, Hippo, Wingless and Hedgehog signalling pathways are overactive in ovaries suggesting that CG34347 participates in one of those pathways.

En este trabajo se valida la TDC en dos materiales con comportamiento elástico–lineal en estado fisurado: el polímero PMMA y la aleación Al7075-T651. La validación se ha realizado sobre probetas de fractura y se han establecido rangos de validez de la TDC en régimen elástico-lineal. Igualmente, se demuestra la aplicabilidad de la TDC para la evaluación en rotura de componentes con cualquier concentrador de tensiones (entalla en U, entalla en V, taladro circular, etc) y para cargas de distinta naturaleza (tracción vs. Flexión). Además, se plantea y valida un modelo de evaluación de la integridad estructural de componentes con entallas que combina la TDC con los Diagramas de Fallo, extendiendo el rango de aplicación de la TDC a situaciones con comportamiento elastoplástico. Finalmente, el análisis se completa con un detallado estudio mediante microscopía electrónica de barrido de los micromecanismos de fractura de las probetas analizadas. / This study validates the TDC in two materials with linear elastic behavior in cracked conditions: polymer PMMA and alloy AL7075-T651. The validation has been performed on fracture specimens and validity ranges of the TDC in linear elastic regime have been established. Similarly, it is demonstrated the applicability of the TDC for evaluation of components containing any kind of stress riser (e.g., U-notch, V-notch, hole circular, etc.) and subjected to loads of different nature (tensile vs. bending). In addition, a model for the structural integrity assessment of notched components is presented and validated, combining the TDC with Failure Assessment Diagram. This extends the vailidity range of the TDC to situations with elastic-plastic behaviour. Finally, the analysis is completed with a detailed study by scanning electron microscopy of the fracture micromechanisms of the samples being analysed.

In modern broadband microwave applications, receivers are very sensitive to interference signals which can come from the system itself or from hostile emitters. Electronically tunable bandstop filters can be used to eliminate these interference signals with adaptation to changing frequency conditions. In this thesis, electronically tunable bandstop filter design techniques are investigated for microwave frequencies. The aim is to find filter topologies which allow narrowband bandstop or &lsquo / notch&rsquo / filter designs with low-Q resonators and with tuning capability. Tunability will be provided by the use of electronically tunable capacitors, specifically varactor diodes. For this purpose, firstly direct bandstop filter techniques are investigated and their performances are analyzed. Then phase cancellation approach, which enables high quality bandstop filter design with lossy circuit elements, is introduced and analyzed. Lastly, a novel notch filter design technique called as all-pass filter approach is introduced. This approach allows a systematic design method and enables to design very good tunable notch filter characteristics with low-Q resonators. Three filter topologies using this approach are given and their performances are analyzed. Also prototype tunable notch filters operating in X-Band are designed and implemented by using these three topologies.

This thesis is devoted to the study and designs of two important proportional weirs having the discharge-head characteristics of Q α H 2/3 In the first design a geometrically simple weir in the form of a rectangular weir over a inverted V-notch (Chimney weir) is presented. This weir gives for all flows above a threshold depth a discharge proportional to H 2/3 within a maximum percentage error of ±1.5, (measured above a reference plane) within certain limits of head. Second design is concerned with the self-basing weir in which a portion of the weir above the crest acts as a base. This design is achieved by using the complementary weir profile of a Quadratic weir above the parabolic base which has the significant property of fast convergence. This weir gives discharge for all flows above the threshold depth, proportional to (head)2/3 measured above a reference plane, with increasing accuracy as head increases. Experiments with these two weirs confirm the theory by giving a constant average Coefficient of Discharge (Cd) of 0.62. The importance of these weirs as a sensitive discharge measuring device in field and laboratory is highlighted.

Notch receptors and ligands are type I transmembrane proteins that regulate development and differentiation during cell-cell contact. There are four Notch receptor homologues and five notch ligands, identified in humans till date. Upon ligand activation, Notch1 intracellular domain (NIC-1) is released into the cytoplasm, which binds to several proteins as well as translocates into the nucleus to effect the Notch signaling. In the absence of the activated Notch signaling, the Notch target genes are kept repressed by the transcriptional repressor C protein binding factor 1 (CBF1) also known as RBPjk or CSL for CBF1/Su(H)/Lag1. RBPjk binds to the sequence “CGTGGGAA” and acts as a constitutive repressor. Upon ligand dependent activation, NIC-1 enters into the nucler and converts RBPjk from transcriptional repressor to an activator. Notch binding to CSL replaces the SMRT corepressor complex with a coactivator complex including SKIP, Mastermind like 1 (MAML1) (Mastermind in Drosophila), and histone acetyl transferases PCAF, GCN5 and p300 activating the transcription of target genes. Mastermind-like (MAML), a family of transcriptional activator proteins comprising of 3 members 1 to 3, has been shown to be required for Notch signaling. MAML forms a ternary complex with RBPjk-NIC by directly interacting with NIC. In turn, MAML recruits the histone acetyl transferase p300/CBP, which acetylates the histones, thereby altering the structure of chromatin amenable for transcription. Activation of Notch pathway induces oncogenesis, which can be divided into two categories including 1) Inhibition of Apoptosis and 2) Induction of proliferation. In T cells, activation of Notch1 protects cells from T cell receptor, dexamethasone and etoposide-mediated apoptosis, Fas receptor-mediated signaling by up regulating IAP (Inhibitor of Apoptosis) and Bcl-2 families, as well as FLIP (FLICE-like inhibitor protein). Notch signaling also promotes the survival of T cells through maintenance of cell size as well as through the promotion of glucose uptake and metabolism. Notch-1 has been shown to protect against anoikis (apoptosis induced by matrix withdrawal) or p53-mediated apoptosis in immortalized epithelial cells, T cell receptor-induced apoptosis in mature cells and dexamethasone-mediated apoptosis in thymocytes. This study was carried out to functionally characterize NIC-1 (human Notch1-intracellular domain) as an inhibitor of apoptosis and to evaluate the therapeutic potential of reversal of this apoptosis inhibition. The main objectives of this study are 1. Construction of recombinant adenovirus expressing human Notch1-intracellular domain (Ad-NIC-1) and characterization of NIC-1 as an inhibitor of chemotherapy and p53-induced cytotoxicity and apoptosis. 2. Role of PI3 kinase -Akt/PKB -mTOR pathway in NIC-1-mediated inhibition of p53-induced apoptosis. 3. Essential role of association between mTOR and NIC-1 and the dependent NIC-1 phosphorylation in Notch1-mediated transcription and survival signaling. 4. Identification of NIC-1 as an inhibitor of E1A-induced apoptosis and the role of mTOR in NIC-1-mediated inhibition of E1A-induced apoptosis. Activated Notch1 was first linked to tumorigenesis through identification of a recurrent t(7;9)(q34;q34.3) chromosomal translocation involving the human Notch1 gene that is found in a subset of human pre-T-cell acute lymphoblastic leukemia’s (T-ALL). Deregulated Notch signaling is oncogenic, inhibits apoptosis and promotes survival. In order to understand survival signaling induced by Notch1 and its possible role in chemoresistance, we have generated a replication deficient recombinant adenovirus expressing human Notch1-intracellular domain (Ad-NIC-1) and shown that it produces functional NIC-1 protein. Using this overexpression system, we characterized that activated Notch1-inhibits chemotherapy and in particular p53 induced apoptosis. Notch1-mediated inhibition of p53-induced apoptosis does not include coactivator squelching. p53 was inefficient in binding to its DNA in NIC-1 overexpressing cells. The levels of phosphorylation at Ser15, Ser20, and Ser392 of p53 expressed from Ad-p53 significantly reduced in NIC-1 preinfected cells. These results suggest that NIC-1-mediated inhibition of p53-mediated apoptosis involves reduced DNA binding, reduced nuclear localization and reduced post translational modifications and thus reduced transactivation of its target genes. Notch1-mediated inhibition of p53 was found to occur mainly through mammalian target of rapamycin (mTOR) using PI3 kinase-Akt/PKB pathway, as the mTOR inhibitor; rapamycin treatment was able to reverse Notch-1 mediated inhibition of p53 and chemoresistance. Consistent with this, rapamycin failed to reverse NIC-1 induced chemoresistance in cells expressing rapamycin resistant mTOR. Our results also suggest that the N-terminal HEAT repeat and the kinase function of mTOR are essential for Notch mediated inhibition of p53. Further, ectopic expression of eIF4E, a translational regulator that acts downstream of mTOR, inhibited p53-induced apoptosis and conferred protection against p53-mediated cytotoxicity to similar extent as that of NIC-1 overexpression, but was not reversed by rapamycin, which indicates that eIF4E is the major target of mTOR in Notch1-mediated survival signaling. Notch1-intracellular domain (NIC-1), following proteolytic cleavage, binds to RBPjk and regulates transcription. Active NIC-1 located in the nucleus is phosphorylated, which makes it more stable and bind better to RBPjk. NIC-1 was also shown to bind to Deltex1 in the cytoplasm. Next, we studied the requirement of components of Notch1 signaling pathway for this function. By using variety of approaches, we found that both RBPjk and Maml1 and hence transcription activation is required for NIC-1-mediated survival signaling and inhibition of p53 functions. Interestingly, while we found the other Notch1 effector, Deltex1 is also required for above functions, Notch1 failed to activate PI3 kinase -Akt/PKB -mTOR pathway in Deltex1, but not in RBPjk silenced cells. Our results suggest that Notch-Deltex1 pathway activates PI3 kinase. Previous studies show that NIC-1 interacts with Deltex1 and Grb2 interacts with PI3 kinase. Our data shows that Deltex1 interacts with SH3 domain of Grb2. Since Notch1-Deltex1 and PI3 kinase-Grb2 interactions are known, we conclude that Notch1 activation of PI3 kinase involves Deltex1 and Grb2. We found activated mTOR was able to binds to NIC-1 and regulates its phosphorylation. Inhibition of mTOR either by PI3 kinase inhibitors or mTOR inhibitor treatment or silencing of Akt/PKB or mTOR reduced the phosphorylation of NIC-1 with the concomitant reduction in NIC-1-mediated transcription. Further, endogenous Notch1 receptor activated by the DSL ligand failed to activate transcription efficiently in rapamycin treated cells, implying a positive role for mTOR in mammalian Notch signaling. These studies reveal that Notch1 activates PI3 kinase -Akt/PKB -mTOR signaling through Deltex1 and subsequently activated mTOR modulates Notch1 signaling by direct binding and possibly thorough phosphorylation of the intracellular domain of Notch. Adenoviral E1A, in the absence of cooperating oncogene, suppresses primary tumor growth and reverses the transformed phenotype of human tumor cells by inducing apoptosis. E1A requires p53 for efficient induction of apoptosis and was shown to induce apoptosis by down regulating Akt and the activation of pro apoptotic factor p38 MAP kinase. Since our results suggest Notch1 inhibits chemotherapy and p53-induced apoptosis, we analyzed the ability of Notch1 to protect cells from E1A-induced apoptosis. Here we show that NIC-1 suppresses the ability of E1A to induce apoptosis. NIC-1 requires mTOR-dependent signal to inhibit E1A-mediated apoptosis, as the rapamycin, an mTOR inhibitor was able to completely reverse the ability of Notch1 to protect cells against E1A-induced apoptosis. The role of mTOR in NIC-1-mediated survival signaling was further confirmed by using the cells stably expressing rapamycin resistant mTOR. Rapamycin was able to reverse Notch1-mediated protection in cells expressing wild type mTOR but not in rapamycin resistant mTOR expressing cells. We also found that E1A was able to induce apoptosis in cells silenced for the pro apoptotic factor p38 and NIC-1 continued to inhibit E1A-induced apoptosis in these cells. These results confirm that Notch1 requires the activation of mTOR signaling but not p38 MAP kinase for inhibition of E1A-induced apoptosis. These results also suggest that the combination therapy utilizing E1A-mediated gene delivery in combination with inhibition of mTOR pathway may prove successful in treating Notch overexpressing cancers. Chemotherapy remains a major treatment modality for human cancers. Chemoresistance is a clinical problem that severely limits treatment success. It can be divided into two forms: intrinsic and acquired. Intrinsic resistance is the essence of oncogenic transformation, resulting from activation of oncogenes and the loss of tumor suppressors, and manifests itself as alterations in cell cycle checkpoints and apoptotic pathways. It is now widely accepted that the apoptotic capacity of the cancer cell is crucial in determining the response to chemotherapeutic agents. Indeed, several gene products that regulate apoptosis, i.e., p53, Akt and PI3K are frequently altered in cancer cells. In this study, we identified that cells with aberrant Notch1 signaling are chemoresistant. Activated Notch1 overexpression makes cells resistant to chemotherapy in a wild type p53 dependent manner. Notch protected p53 wild type cells but not p53 mutated or p53 deleted cells against chemotherapy induced cytotoxicity. Further, inactivation of p53 by specific silencing abrogated the ability of NIC-1 to protect H460 cells against adriamycin induced cytotoxicity. Most importantly, NIC-1 mediated chemoresistance can be reversed by blocking PI3 kinase -Akt/PKB -mTOR pathway. Collectively, these results suggest that cancers with activated Notch1 signaling are chemoresistant and provide basis for the reversal of chemoresistance.

Prostate cancer is one of the leading causes of cancer-related deaths in men worldwide, with 10,000 new cases/year diagnosed in Sweden. In this context, there is an urgent need to identify new biomarkers to detect prostate cancer at an initial stage for earlier treatment intervention. Although how prostate cancer develops has not been fully established, the male sex hormone testosterone is a known prerequisite for prostate cancer development. High levels of transforming growth factor-β (TGFβ) are prognostically unfavorable in prostate cancer patients. TGFβ is a multifunctional cytokine that regulates a broad range of cellular responses. TGFβ signals through either the canonical Smad or the non-Smad signaling cascade. Cancerous cells develop different strategies to evade defense mechanisms and metastasize to different parts of the body. This thesis unveils one such novel mechanism related to TGFβ signaling. The first two articles provide evidence that TGFβ receptor type I (TβRI) is ubiquitinated by tumor necrosis factor receptor-associated factor 6 (TRAF6) and is cleaved at the ectodomain region by tumor necrosis factor alpha converting enzyme (TACE) in a protein kinase C zeta type-dependent manner. After TβRI is shed from the ectodomain, it undergoes a second cleavage by presenilin 1 (PS1), a γ-secretase catalytic subunit, which liberates the TβRI intracellular domain (TβRI-ICD) from the cell membrane. TRAF6 promotes TGFβ-dependent Lys63-linked polyubiquitination and recruitment of PS1 to the TβRI complex, and facilitates the cleavage of TβRI by PS1 to generate a TβRI-ICD. The TβRI-ICD then translocates to the nucleus, where it binds with the transcriptional co-activator p300 and regulates the transcription of pro-invasive target genes such as Snail1. Moreover, the nuclear translocated TβRI-ICD cooperates with the Notch intracellular domain (NICD), a core component in the Notch signaling pathway, to drive the expression of invasive genes. Interestingly, treatment with g-secretase inhibitors was able to inhibit cleavage of TβRI and inhibit the TGFβ-induced oncogenic pathway in an in vivo prostate cancer xenograft model. In the third article, we identified that Lysine 178 is the acceptor lysine in TβRI that is ubiquitinated by TRAF6. The TβRI K178R mutant was neither ubiquitinated nor translocated to the nucleus, and prevented transcriptional regulation of invasive genes in a dominant negative manner. In the fourth article, we show that TGFβ utilizes the E3-ligase TRAF6 and the p38 mitogen-activated protein kinase to phosphorylate c-Jun. In turn, the phosphorylated c-Jun activates p21 and Snail1 in a non-canonical Smad-independent pathway, and thereby promotes invasion in cancerous cells. In summary, we elucidate a new mechanism of TGFβ-induced oncogenic signal transduction in cancer cells in which TRAF6 plays a fundamental role. This opens a new avenue in the field of TGFβ signaling.

The mechanical behavior of a high pressure die cast AM60B Mg alloy is studied. Constant load amplitude fatigue tests were conducted at room, elevated and cold temperatures, with a stress ratio of R=0.1, and frequency of 30 Hz. The objective was to identify the possible effects of temperature on fatigue life cycle. In addition, fatigue crack propagation tests were conducted to ascertain the fatigue response of the alloy and determine its fatigue crack growth rate as a function of the applied stress ratio, experimentally, analytically and computationally, using Walker’s model. The results demonstrated that temperature had a significant influence on the fatigue life, and that the life increased at cold temperature but decreased at elevated temperature as compared to that evaluated at room temperature. In this study, the limit for applicability of LEFM was established for AM60B magnesium alloy. In addition, fatigue crack propagation test results were used to evaluate the coefficients of the Paris model.

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