River training structures are widely used to create and maintain navigable waterways, to restore rivers and channels in a more stable condition, to promote environmental benefits, and to protect people and infrastructure from damages or floods. Few historical datasets on the changes and impacts in secondary waterbodies resulting from notched river training structures are available for the Lower Mississippi River. Access to the notched training structures on the Lower Mississippi River remains difficult and inhibits data collection for monitoring efforts. This increases the need for alternative methods such as numerical models for assessing the performance of the notched training structures. A quasi-three-dimensional Adaptive Hydraulics model was assembled and used to provide a hydraulic assessment of seven notched river training structures in the Lower Mississippi River. The hydraulic assessment of the notches included assessing the impacts to navigation, the long-term trends, and the potential for aquatic wildlife habitat diversity.

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Glioblastoma is the most common malignant brain tumor, accounting for 17% of all primary brain tumors in the United States. Despite the available surgical, radiation, and chemical therapeutic options, the invasive and infiltrative nature of the tumor render current treatment options minimally effective. Recent reports have identified multiple regulators of glioblastoma progression and invasiveness. It has been demonstrated that ADAM12, A Disintegrin And Metalloproteinase encoded by ADAM12 gene, is over-expressed in glioblastoma and directly correlated with tumor proliferation. Additionally, dysregulation of the Notch signaling pathway has been implicated in the pathogenesis of many gliomas. Lastly, an evolving role of microRNAs, small noncoding RNAs, in carcinogenesis is progressively growing. A recent study has identified ADAM12 as a notch-related gene, and another demonstrated that inhibition of notch signaling decreased glioblastoma recurrence. However the mechanisms of regulation are still unknown. In this study, we hypothesize that direct downregulation of microRNA-29, downstream of over-expression of notch enhances glioblastoma malignancy through upregulation of ADAM12. Although our data demonstrate upregulation of Notch1, its downstream target HES1, and ADAM12 in U87MG glioblastoma cell line. Expression of the cleaved intracellular Notch1 was not detected. Furthermore, we were unable to demonstrate an inhibitory effect of ɣ-secretase inhibitor on Notch signaling, likely reflecting the requirement for modifying culturing conditions or detection in our assays. Furthermore, miR-29 was detected in glioblastoma cells. The expression of miR-29 was further elevated by ɣ-secretase inhibitor treatment, suggesting a role for Notch1 inhibition on miR-29 expression. Although no conclusive results are shown in our work, a role of Notch1 through miR-29 is implicated in the pathogenesis of glioblastoma pathogenesis warranting further investigation into the role downstream target genes in the Notch signaling pathway.

The Notch signalling pathway is an evolutionary conserved pathway, named after the Notch receptors, Notch1-4 in mammals, which upon cell-cell contact and ligand binding releases the intracellular domain (NICD). NICD translocates into the nucleus where it binds the transcriptional repressor RBP-Jk, which together with co-activators belonging to the Mastermind-like family of proteins form a transcriptional activation complex. This complex activates genes controlling cell fate decision, embryonic development, proliferation, differentiation, adult homeostasis and stem cell maintenance. On the other hand, disrupted Notch signalling may result in pathological conditions like cancer, although the mechanisms behind the disruption are often complex and in many cases largely unknown. Notch1 drives the lymphocyte differentiation towards a T-cell fate and activating mutations in the gene have been suggested to be involved in T-cell lymphoma. In paper I, genetic alterations in Notch1 and the Notch1 regulating gene CDC4 were investigated in tumours from murine T-cell lymphoma induced with phenolphthalein, 1,3-butadiene or 2’,3’-dideoxycytidine. We identified activating Notch1 mutations in 39% of the lymphomas, suggesting that Notch1 is an important target gene for mutations in chemically induced lymphomas. While it is known that constitutively activated Notch signalling has a clear oncogenic function in several solid malignancies as well, the molecular mechanisms are less known in this context. Unpublished data of our lab, together with other recent studies, suggest that mutations of Notch and Notch-related genes per se are uncommon in solid malignancies including colorectal cancer, while a growing body of evidence indicates that aberrant Wnt/b-catenin signalling may result in pro-tumoural Notch activation in these contexts. In paper II, we therefore investigated potential transcriptional interactions between the Notch and Wnt signalling pathways in colorectal cancer cell lines. The proximal Notch and Wnt pathway gene promoters were bioinformatically identified and screened for putative TCF/LEF1 and RBP-Jk sites. In canonical Wnt signalling, Apc negatively regulates b-catenin leading to repression of TCF/LEF1 target genes. Upon repression of the Wnt pathway we observed that several genes in the Notch pathway, including Notch2, were transcriptionally downregulated. We also confirmed binding of Lef1 to Notch2 as well as other Notch pathway gene promoters and luciferase assays showed an increased activity for at least one LEF1/TCF-site in the Notch2 promoter upon co-transfection of HT29 or HCT116 cells with mutated b-catenin. HT29 cell lines were also treated with the g-secretase inhibitor DAPT, leading to inactivation of the Notch pathway by preventing release of NICD. However, results showed no effects on Apc, b-catenin or their target cyclin D1. Taken together, these results indicate that the Wnt pathway may function as a regulator of the Notch pathway through the TCF/LEF1 target gene program in colon cancer cell lines. In summary, Notch pathway deregulation is of importance in both murine T-cell lymphoma and human colorectal cancer, although the mechanisms differ. The current results give new insights in Notch pathway alterations as well as the signalling networks in which the Notch pathway interacts, and thus increase the understanding of Notch’s involvement in malignant diseases. / Studies on molecular genetic alterations in colorectal cancer

The ophthalmic trigeminal placode (opV) exclusively gives rise to sensory neurons. A number of signaling pathways including Wnt, PDGF, FGF, and Notch are all involved in the progression of an undifferentiated cell in the opV placode to a proneural cell in the condensing opV ganglion. However, the regulatory relationships between these signal transduction pathways are still unknown. To determine if FGF activation acts to modulate Notch signaling in the sensory neurogenesis pathway, a novel multifactorial approach was employed: FGF signaling was inhibited in individual cells and globally with simultaneous inactivation of Notch signaling in chick embryos to investigate if FGF activation downregulates Notch thereby driving neurogenesis. These experiments resulted in few differentiating opV cells in the mesenchymal region of future ganglion formation suggesting an alternate regulatory relationship between FGF and Notch where either reduced Notch activity allows for FGFR4 expression (leading to FGF signaling and neurogenesis), or a parallel relationship where FGF and Notch act independently of one another to induce neurogenesis. To distinguish between these two possibilities Notch signaling was inhibited with DAPT, a gamma-secretase inhibitor, and assayed for FGFR4 mRNA expression. These results indicated FGFR4 is not upregulated by reduced Notch activity, suggesting that FGF and Notch act in parallel to promote neurogenesis. During these experiments it was observed that Notch inhibition resulted in an undefined ectoderm in the opV placode region. To investigate this, FGF and Notch were inhibited by SU5402, an FGF antagonist, and DAPT, and later sectioned and stained for Laminin. In DAPT treated embryos the basement membrane became highly fragmented, a remarkable observation not yet reported. From these data a proposed mechanism was established where activation of FGF with parallel downregulation of Notch leads to disruption of extracellular matrix proteins in the basement membrane resulting in fragmentation and subsequent delamination of differentiating opV placode cells.

The behavior of materials as they are subjected to combined thermal and mechanical fatigue loads is an area of research that carries great significance in a number of engineering applications. Power generation, petrochemical, and aerospace industries operate machinery with expensive components that undergo repeated applications of force while simultaneously being exposed to variable temperature working fluids. A case of considerable importance is found in steam turbines, which subject blades to cyclic loads from rotation as well as the passing of heated gases. The complex strain and temperature histories from this type of operation, combined with the geometric profile of the blades, make accurate prediction of service life for such components challenging. Development of a deterministic life prediction model backed by physical data would allow design and operation of turbines with higher efficiency and greater regard for reliability. The majority of thermomechanical fatigue (TMF) life prediction modeling research attempts to correlate basic material property data with simplistic strain and thermal histories. With the exception of very limited cases, these types of efforts have been insufficient and imprecise in their capabilities. Early researchers did not account for the multiple damage mechanisms that operate and interact within a material during TMF loads, and did not adequately address the extent of the relationship between smooth and notched parts. More recent research that adequately recognizes the multivariate nature of TMF develops models that handle life reduction through summation of constitutive damage terms. It is feasible that a modification to the damage-based approach can sufficiently include cases that involve complex geometry. The focus of this research is to construct an experimentally-backed extension of the damage-based approach that improves handling of geometric discontinuities. Smooth and notched specimens of Type 304 stainless steel were subjected to several types of idealized fatigue conditions to iv assemble a clear picture of the types of damage occurring in a steam turbine and similarly-loaded mechanical systems. These results were compared with a number of idealized TMF experiments, and supplemented by numerical simulation and microscopic observation. A nonuniform damage-summation constitutive model was developed primarily based on physical observations. An additional simplistic model was developed based on phenomenological effect. Findings from this study will be applicable to life prediction efforts in other similar material and load cases.

Increased production of amyloid beta (A-beta) peptide, via altered proteolytic cleavage of amyloid protein precursor (APP), and abnormalities in neuronal calcium homeostasis play central roles in the pathogenesis of Alzheimer's disease (AD). Notch1, a membrane receptor that controls cell fate decisions during development of the nervous system, has been linked to AD because it is a substrate for the gamma-secretase protein complex in which mutations cause early-onset inherited AD. Numb is an evolutionarily conserved endocytic adapter involved in the internalization of transmembrane receptors. Mammals produce four Numb isoforms that differ in two functional domains, a phosphotyrosine-binding domain (PTB) and a proline-rich region (PRR). Recent studies showed that the PTB domain of Numb interacts with the cytoplasmic tails of APP and Notch but the functional relevance of these interactions with respect to AD pathogenesis is not clear. In the current studies, we proposed to investigate the biological consequences of the interaction of the Numb proteins with APP and Notch in neural cells stably overexpressing each of the four human Numb proteins. In the first part of our studies, we found that expression of the Numb isoforms lacking the insert in the PTB (SPTB-Numb) caused the abnormal accumulation of cellular APP in the early endosomes, and increased the levels of C-terminal APP fragments and A-beta. By contrast, expression of the Numb isoforms with the insert in PTB (LPTB-Numb) leads to the depletion of cellular APP and coincides with significantly lower production of APP derivatives and A-beta. The contrasting effects of the Numb isoforms on APP metabolism were not attributed to differences in the expression of APP nor the activities of the various APP-processing secretases. In the second part of our studies, we found that expression of SPTB-Numb protein enhances neuronal vulnerability to serum deprivation-induced cell death by a mechanism involving the dysregulation of cellular calcium homeostasis. Neural cells expressing SPTB-Numb exhibited enhanced Notch activity, which markedly upregulated the expression of transient receptor potential canonical 6 (TRPC6) channels enhancing calcium entry in response to store depletion. We also found that serum deprivation increased TRPC6 expression, mediating the serum deprivation-induced death in neural cells. Interestingly, expression of LPTB-Numb protein suppressed serum deprivation-induced activation of Notch and the subsequent upregulation of TRPC6 and cell death. Finally, we showed that the Numb proteins differentially impact Notch activation by altering the endocytic trafficking and processing of Notch. Taken together, these studies demonstrate that aberrant expression of the Numb proteins may influence APP metabolism and Notch-mediated cellular responses to injury by altering their endocytic trafficking and processing.

Th17 cells are pro-inflammatory cells that are characterized by the production of their signature cytokine, IL-17. Although they are thought to have arisen to protect against extracellular bacteria and fungi they have been shown to mediate autoimmune diseases such as EAE and psoarisis. Notch protein is a cell-surface receptor that has been widely conserved among species. It plays an essential role in determining multiple cell fates. More recently, it has been implicated in regulating peripheral CD4+ T-cell responses. In these studies, we report that blockade of Notch signaling significantly down-regulates the production of IL-17 and associated cytokines in both mouse and human in-vitro polarized Th17 cells, suggesting an intrinsic requirement for Notch during Th17 differentiation in both species. We also present evidence, using promoter reporter assays, knockdown studies as well as chromatin immunoprecipitation, that IL-17 and RORt are direct transcriptional targets of Notch signaling in Th17 cells, with Notch 1 being the responsible Notch family member important in regulating the differentiation of human Th17 cells. In-vivo inhibition of Notch signaling reduced IL-17 production and Th17 mediated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. In addition, by using Notch1 and Notch3 knockout mice, we have shown that Notch 3 is the Notch family member that is essential for murine Th17 differentiation. We have also investigated noncanonical Notch signaling in Th17 cells by using CD4+ T-cells from CSL/RBP-Jk knockout mice. Based on data obtained, we have concluded that canonical Notch signaling is dispensable in Th17 responses. Thus, this study highlights the importance of different Notch family members in Th17 differentiation and indicates that selective targeted therapy against Notch may be an important tool to treat autoimmune disorders, including multiple sclerosis.

Over the past decades, information has accumulated concerning the mechanism how an exterior signal induced by ligand on neighboring cells is transmitted to the nucleus through the Notch receptor and the cellular effects of Notch signaling on the regulation of differentiation, proliferation and apoptosis in many cell types. However, the function and the mechanism of Notch signaling in peripheral T cells still remains to be addressed. Therefore, we asked whether Notch1 is involved in CD8+ cytolytic effector T cell (CTLs) maturation and effector functions and how Notch1 exerts its cellular function in the nucleus and in the cytoplasm. The maturation of naïve CD8+ T cells into CTLs is a critical feature of a functional adaptive immune system. Development of CTLs depends, in part, upon the expression of the transcriptional regulator, Eomesodermin (EOMES), which is thought to regulate the expression of two key effector molecules, perforin and granzyme B. In addition, the data from previous studies in our lab showed that Notch signaling results in the activation of NF-κB, IFN-γ secretion and cell proliferation both in CD4+ and CD8+ T cells. Therefore, we hypothesized that Notch1 may be involved in CD8+ T cell maturation and effector function. We observed that Notch1 regulates the expression of EOMES, perforin and granzyme B through direct binding to the promoters of these crucial effector molecules. By abrogating Notch signaling, both biochemically as well as genetically, we conclude that Notch activity mediates CTL development through direct regulation of EOMES, perforin and granzyme B. We further investigated the molecular steps leading to the formation of intracellular Notch1 (N1ICD)/CSL (also known as CBF1/RBP-Jκ in mammals; Suppressor of Hairless in Drosophila; and Lag-1 in C. elegans) with other co-factors in target promoters of Notch1 signaling. We proposed that the association of two nuclear complexes with N1ICD controls the transcription of genes, allowing the development of effector CTL in the immune system. Recent studies proposed a model where Notch1 colocalizes with CD4, a component of the immune synapse, upon T cell stimulation and directly associates with p56Lck and CD28, as well as PI3K. However, the link between Notch and the TCR signalosome needed further investigation. We found that Notch1 functions as a scaffold, associated with the cytosolic components, Carma1, Bcl10, PKCθ and the IKK complex upon TCR stimulation, leading to the activation of NF-κB and IL-2 production. We further showed that the N-terminal region of N1ICD is essential for interaction with Carma1 and that deficiency of Notch1 abolishes the nuclear binding of NF-κB on the il- 2 promoter, leading to reduced IL-2 production.

The application of uniformly distributed RC networks as feedback elements in the design of active circuits has been investigated. Distributed RC structures were fabricated using Mylar Film, Teledeltos resistance paper and metallic foil, and used to experimentally verify the predicted responses of particular active and passive configurations. By exploiting the frequency selective feedback provided by a distributed notch filter it was possible to construct an active band-pass filter operating at 1 MHz which achieved a Q of 50 without the use of inductance. For the design it was important that the notch parameter was deliberately chosen to be less than optimum (i.e., a< 17.78) so that the feedback circuit did not apply positive feedback in the region of the notch frequency. Application of positive feedback (a> 17.78) and sufficient amplifier gain would convert the active filter into a feedback oscillator. As predicted, the band-pass filter response was strongly influenced by the amplifier gain and phase characteristic, while amplifier impedance exerted only minor effects. / Thesis / Master of Engineering (ME)