The Notch signalling pathway is a highly conserved cell-to-cell signalling pathway involved in developmental cell fate determination in all metazons. When Notch is signalling, differentiation is inhibited and a progenitor-like state is favoured. This signalling pathway has been implicated in the developing retina, where the inhibition of Notch has been shown to skew the proportion of different retinal neuronal cell types. Although functional knockout studies have allowed us to characterize some of the roles of Notch in the retina, low protein levels have made it difficult to characterize the location of Notch receptors and ligands in neuronal tissue. Here we sought to characterize the localization of the Notch signalling pathway components in both the developing and the adult mouse retina. Using RT-PCR we were able to show the presence of mRNA for Notch receptors, ligands, and DNA binding cofactors for the Notch intracellular domain, CBF1, throughout postnatal development as well as in the adult retina. In situ hybridization confirmed the presence of Notch1, Notch2, and CBF1 mRNA in the embryonic (E14.5) and early postnatal (P1.5) retina similar to what has been reported in earlier studies, but in the adult retina (P40), levels were below detection. To further explore the role of Notch in the adult retina we used two transgenic mouse reporter models in which a Notch responsive element directs the expression of EGFP or Venus. In the adult retina of the NTR line (Tg(Cp-EGFP)25Gaia/J) reporter expression was detected in rod ON and cone type 2 OFF bipolar cells, as well as in a subset of both amacrine and ganglion cells. In the CBFRE:H2B-Venus line adult reporter expression was detected in photoreceptors, and a large proportion of both amacrine and ganglion cells. Together this data supports the conclusion that Notch is expressed and actively signalling in the retina throughout development and possibly in the adult retina, although below levels of in situ hybridization detection. These results represent the possibility of a previously unknown role for Notch in the adult retina. / Graduate / 0317

Ulm, Univ., Diss., 2008.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

The Notch receptor is a key signaling protein that also acts as a transcriptional co-activator in numerous cell fate decisions in animals, including Drosophila melanogaster. Like many other transcriptional activators interacting on the DNA, the Notch protein carries a polyglutamine tract encoded by the opa repeats of the Notch gene. Here, I show that considerable variation exists within this tract across populations from the United States and Malawi. This variation is distributed asymmetrically across the range of possible alleles, with a peak in each population at opa31 (typically encoding Q₁₃HQ₁₇) and/or opa32 (typically encoding Q₁₃HQ₁₈) and a tendency towards a large tail of longer alleles and few shorter alleles. Variation in this pattern between populations may be a result of certain tracts being less harmful in certain backgrounds, or it may be due to the ancestry of these populations. This variation has real effects, such that lines bearing alleles longer or shorter than the common 31- and 32-codon alleles exhibit abnormal phenotypes, gene expression disruption, and decreased viability, effects that persist even when the Notch gene is outcrossed or recombined into other backgrounds. I also describe lines bearing CRISPR-Cas9-edited opa repeats and highlight a potential interaction between Notch and the transcriptional activator Dorsal. Dorsal also exhibits variation in the length of its polyglutamine tract, and short tracts at Dorsal most frequently appear alongside short tracts at Notch, while long tracts of each also frequently co-occur.

Breast cancer is the most common cancer and the leading cause of cancer-related death among Canadian women. Despite improvements in treatment and early detection, there is still a need to develop novel therapies for breast cancer management. Aberrant Notch signaling is tumorigenic and is associated with poor clinical outcomes in breast cancer, as well as in several other types of cancer. Activation of Notch signaling requires -secretase-mediated Notch receptor cleavage. Thus, strategies to inhibit Notch signaling, including -secretase inhibition, are being evaluated for potential anti-tumor effects. The strongest justification for targeting Notch in breast cancer, and more specifically for using -secretase inhibitors, came from two studies that reported that the -secretase inhibitor (GSI) Z-LLNle-CHO inhibited the growth of breast cancer cells both in vitro and in vivo without causing significant side effects. In Chapter 2, we compared the enzymatic activities and cytotoxicity of Z-LLNle-CHO to those of two other specific GSIs and three proteasome inhibitors and demonstrated that the cytotoxicity of Z-LLNle-CHO in breast cancer cells is mediated by proteasome inhibition, not by -secretase inhibition. In Chapter 3, we characterized the protein complexes formed in breast cancer cells by the intracellular domains (NICD) of the four Notch paralogs. We found that the assembly of NICD protein complexes is dose-dependent and availability of MAML proteins becomes the limiting factor for continuous formation of NICD/RBPj/MAML transactivation complex. This suggests that the formation of some non-canonical NICD complex might occur preferentially at high levels of NICD, conditions under which aberrant Notch signaling induces tumorigenesis in breast cancer. Consequently, these non-canonical interactions might be good targets to specifically block oncogenic, but not physiological, Notch signaling. In addition, we found that the relative affinities of individual NICD paralogs to several known NICD-interacting proteins were different. This may account for the paralog-specific activities of Notch that have been previously reported. Together, these results may be of value for the development of new reagents to block Notch signaling for therapeutic benefit in breast cancer treatment. / Experimental Oncology

The Notch receptor and its ligands constitute a ubiquitous signaling pathway found throughout all multicellular animal life. In addition to its highly conserved function in development, a growing body of evidence suggests Notch signaling has important roles to play in adult processes, including long-term memory (LTM) formation. Building on previous work showing a specific requirement for the Notch1 receptor in spatial memory in mice, I show here a similar requirement for the Notch ligand Jag1. Mice with mutations to Dll1 (another Notch ligand) and Lfng (a Notch regulatory protein) do not display such phenotypes. I propose a model in which signaling between Notch pathway components found in the adult mouse hippocampus (such as Notch1 and Jag1) is required for LTM encoding, with no requirement for pathway components not expressed in this tissue (such as Dll1 and Lfng).

The Notch receptor and its ligands constitute a ubiquitous signaling pathway found throughout all multicellular animal life. In addition to its highly conserved function in development, a growing body of evidence suggests Notch signaling has important roles to play in adult processes, including long-term memory (LTM) formation. Building on previous work showing a specific requirement for the Notch1 receptor in spatial memory in mice, I show here a similar requirement for the Notch ligand Jag1. Mice with mutations to Dll1 (another Notch ligand) and Lfng (a Notch regulatory protein) do not display such phenotypes. I propose a model in which signaling between Notch pathway components found in the adult mouse hippocampus (such as Notch1 and Jag1) is required for LTM encoding, with no requirement for pathway components not expressed in this tissue (such as Dll1 and Lfng).

Characterization of dispersion curves in plate-like structures is possible with guided Lamb waves. In this research, experimental development of dispersion curves relies on the spectrogram, which suffers from the Heisenberg Uncertainty Principle. Reassignment is capable of localizing ill--defined dispersion curves. Unfortunately, reassignment also introduces spurious components, which reduce reassignment performance. This research develops an algorithm that provides both localization of dispersion curves and elimination of spurious components. To achieve this, an alternative formulation of reassignment called differential reassignment is modified and superimposed with nonlinear anisotropic diffusion. This study first examines reassignment and diffusion components individually. Three different versions of differential reassignment are considered, two of which are modifications explicitly derived in this research. The combined algorithm is then applied to reassign experimentally measured spectrograms, leading to a significant increase in clarity and notch detection performance.

Sprouting angiogenesis is a multi-step process consisting of basement membrane degradation, endothelial cell (EC) activation, proliferation, invasion, lumen formation, and stabilization. Such complexity reveals that the orchestration of individual genes and multiple signaling pathways are required. To better understand the mechanisms that direct the transformation of adherent ECs on the surface of collagen matrices to multicellular invading sprouts, we analyzed differential gene expression with time using an in vitro model of EC invasion driven by the combination of sphingosine-1-phosphate (S1P) and angiogenic growth factors. Gene expression changes were confirmed by real-time PCR and Western blot analyses. In addition, we have undertaken a proteomic screen to dissect downstream targets of the S1P receptors that possibly regulate EC invasion. Gene silencing or overexpression were used to examine the involvement and role of downstream targets of S1P in EC invasion into three-dimensional collagen matrices. We demonstrated that various cell adhesion molecule genes involved in adherens junction and cell-extracellular matrix (ECM) interactions were upregulated; whereas a set of genes associated with tight junctions were downregulated. Numerous genes encoding ECM proteins and proteases were induced, indicating that biosynthesis and remodeling of ECM is indispensable for sprouting angiogenesis. Knockdown of a highly upregulated gene, A Disintegrin and Metalloproteinase with Thrombospondin-type repeats-1 (ADAMTS1), decreased invasion responses, confirming a role for ADAMTS1 in mediating EC invasion. Furthermore, differential expression of multiple members of the Wnt (wingless) and Notch pathways were observed. Functional experiments indicated that inhibition and activation of the Notch signaling pathway stimulated and inhibited EC invasion responses, respectively. In addition, we identified annexin 2 as a regulator of endothelial morphogenesis. We observed that S1P triggered annexin 2 translocation from cytosol to plasma membrane and its association with vascular endothelial (VE)-cadherin. Moreover, annexin 2 depletion attenuated Akt activation, which was associated with increased phosphorylation of VE-cadherin and endothelial barrier leakage. Disrupting homotypic VE-cadherin interactions resulted in decreased Akt (but not Erk1/2) activation. Furthermore, expression of constitutively active Akt restored reduced EC invasion observed with annexin 2 and VE-cadherin knockdown. Collectively, we report that annexin 2 regulates endothelial morphogenesis through an adherens junction-mediated pathway upstream of Akt.

The purpose of thesis is aimed to analyze fracture and fatigue behaviors of notched composite laminates by numerical method. Types of notch are both central notch 4mm£rand double-edged crack. We employed finite element method analysis with ANSYS to perform the numerical analysis. We adopted Tsai-Wu failure criterion to approach failure of central notch laminates by a step increment method and considered the effect of failure area gradually expanded, thus we obtained the ultimate stress of a laminate. In the work of fatigue testing and evaluation, we used Miner¡¦s rule to predict the extension of fatigue damage zone, and compared it with experimental results. In the analysis of a double-edged crack laminate, it is first assumed mix mode failure. Then, we calculated the stress intensity factors of various crack length by extrapolation, and developed a function of configuration correction factor. Finally, this study can be concluded as follows. The accuracy of ultimate stress prediction with step-by-step increment analysis is satisfactorily well. The outcome failure area of a laminate is closely corresponding with the observation of experiment. In predicting the fatigue damage zone, the predicted initial damage zone is close to experimental observation, but it is inconsistent with the actual damage extension due to crack propagation. Stress intensity factors and configuration correction factors are directly in proportion to the square root of crack length. It is corresponding with fracture mechanics. However there are some errors in quasi-isotropic laminates due to the existing of ¡Ó45¢Xplies.