Browsing by Subject "Notch"
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Item Biomaterial-based ligand presentation to induce notch signaling in vitro(2013) Mehta, Naveen; Roy, KrishnenduT cell adoptive transfer is an important procedure in immunotherapy. In immunocompromised patients, T cells may need to be developed from stem cells, requiring the induction of Notch signaling events responsible for native T cell differentiation in the thymus. Current methods to develop T cells in vitro fail to mimic the 3D thymic niche and are not mechanically tunable. In this study, we improve on these approaches with a 2D polymer system with controllable mechanical properties, and with a 3D system that more accurately mimics the thymic niche. 3D inverse opal poly(ethylene glycol) (PEGDA) scaffolds were fabricated with defined pores using poly(methyl methacrylate) microspheres that are soluble in acetic acid, leaving negative space for cell growth. The PEGDA surface was made bioactive via biotinylation, followed by a streptavidin linker attaching biotinylated Notch ligand Delta-Like Ligand 4 (bDLL4). 2D polyacrylamide gels were prepared by sandwiching a pre-polymer droplet between two glass surfaces during redox initiation. Mechanical properties were modified by adjusting the concentrations of acrylamide and bisacrylamide. Gels were functionalized by immobilizing streptavidin via a SANPAH linker and binding bDLL4. After fabricating the systems and demonstrating their chemical and mechanical tunability, RT-PCR was used to detect Hes-1 expression, a downstream target of Notch, and flow cytometry was used to detect T cell differentiation levels following incubation of T cell progenitors with functionalized polymer systems. We show that a) the thymic niche can be effectively mimicked by synthetic systems, b) these systems effectively induce Notch signaling, and c) Notch signaling results in early T cell differentiation. These data are promising and suggest the potential to develop T cell banks from stem cells for the purposes of immunotherapy.Item Crack nucleation on V-notched PMMA and Polycarbonate(2022-08-10) Eom, Kyumin; Ravi-Chandar, KrishnaswamyThis study examined the criteria for a crack nucleation on a V-notched geometry for Polymethylmethacrylate (PMMA) and Polycarbonate (PC). V-notched geometries are known to have their specific notch stress intensity factor depending on the V-notch angle and the depth of the V-notch itself. It is crucial to understand the relationship between the stress concentration effect the notch geometry imposes and crack initiation. By investigating the relationship and quantifying driving factors, a reliable conclusion can be made on the criteria for crack nucleation on V-Notch. This study carried out symmetric three-point bend tests on PMMA and PC with varying V-notch angle and depth. The V-notch angles range from 60° to 120° while the depths range from 0.1 to 0.4 of the specimen height. The asymptotic analysis from Williams (1952) combined with a full 3-D finite element analysis were used to determine the V-notch stress intensities. Later, fractography was performed on the fractured specimens to determine the initial crack location and size to estimate the fracture toughness. PMMA showed exclusively brittle fracture while PC showed moderate to significant plastic deformation dominance depending on the V-notch angle and depth. Additionally, PMMA mostly showed a half-elliptical initial crack that nucleated and propagated slowly first, and then transitioned into an unstable, fast fracture. However, PC showed signs of a complex combination of nucleation of brittle fracture as well as significant plastic deformation depending on specimen geometry. A digital camera was used to capture the initial crack nucleation and the slow-crack growth before fast-fracture. This investigation showed that the theory of linear elastic fracture mechanics is effective in predicting crack initiation in V-notched geometries if proper account is taken of the initiation and growth of a sharp crack from the notch. Finally, as a conservative estimate, it is suggested that notches be considered to be cracks of corresponding depth, providing a lower bound for the allowable load in structures.Item Determining the role of a small GTPase, Ral, and an endocytic factor, epsin, in Drosophila Notch signaling(2011-12) Cho, Bomsoo; Fischer, Janice AnnCell-cell communication events are crucial to determine the fate of each cell during development. Notch signaling is involved in many different contexts in determining cell fate by mediating cell-cell communication. Furthermore, regulation of the Notch transduction pathway is critical for normal cellular function, which is implicated in various diseases, including cancers. At a certain developmental time point, intrinsic or extrinsic developmental cues induce biases in ligands and Notch receptors between neighboring cells. These initial biases are further amplified by various cellular factors which eventually dictate cell fates. In Drosophila, two Notch ligands, Delta and Serrate, trigger Notch receptor activation in nearby cells by virtue of numerous regulating factors. One important question in this area is how cells become Notch signal sending or receiving cells for cell fate decisions. I show evidence about a distinct mechanism for biasing the direction of Notch signaling that depends on a small GTPase, Ral, during Drosophila photoreceptor cell development. Investigations described here indicate that Fz signaling up-regulates Ral transcription in a signal sending fate cell, the R3 precursor, and Ral represses ligand-independent activation of Notch in the R3 precursor. This event ensures R3 to become a signaler and contributes to asymmetric Notch activation in the neighboring cell, R4. Ral is a small Ras-like GTPase that regulates membrane trafficking and signaling. Here, possible Ral effector pathways that are important for Notch regulation will be proposed. To trigger Notch activation in adjacent cells, Notch ligand endocytosis by the signaling cells is necessary. Recently, it was suggested that control of membrane trafficking is important not only for ligand signaling, but also for Notch receptor activation. Furthermore, Notch receptor trafficking regulates critical cellular functions, including proliferation, which is implicated in tumors. Therefore, another important question in Notch signaling is about the role of membrane trafficking in regulation of the Notch transduction pathway. Drosophila endocytic epsin, Liquid facets [Lqf], is a key component necessary for ligand endocytosis, thereby triggering Notch activation in adjacent cells. However, its function in signal receiving cells for Notch activation has not been studied. In this dissertation, I provide evidence that epsin is also required in signal receiving cells for Notch activation in developmental contexts. Furthermore, genetic and molecular evidence suggests that epsin regulates Notch receptor trafficking via Rab5-mediated endosomal sorting pathway for Notch activation. These studies support the idea that Notch activation at the plasma membrane is not the only way to transduce Notch signaling, but the Notch receptor must enter through an epsin-mediated endocytic pathway into subcellular compartments to be activated, at least in some contexts.Item Inhibitor of differentiation 2A influences growth and differentiation of the developing vertebrate retina upstream of the notch signaling pathway(2012-08) Uribe, Rosa Anna; Gross, Jeffrey Martin; Vokes, Steven A.; Eberhart, Johann; Stein, David; Agarwala, SeemaInhibitor of differentiation (Id) family helix-loop-helix proteins regulate the proliferation, survival and differentiation of numerous cell types during development, however their function during retinal development has not been analyzed. Using loss-of-function and overexpression assays in zebrafish, I demonstrate that Id2a levels modulate retinoblast cell cycle kinetics and thereby influence neuron and glia formation in the retina. Id2a-deficient retinas possess increased numbers of cells occupying S phase, at the expense of mitotic cells, and kinetic analyses demonstrate that Id2a is required for S-phase progression and/or the transition from S to M phase. Id2a-dependent defects in retinoblast proliferation lead to microphthalmia and to an absence of nearly all differentiated inner and outer nuclear layer cell types. Overexpression of id2a has the opposite effect on retinoblast cell cycle kinetics: id2a-overexpressing retinoblasts progress from S to M phase more rapidly and they undergo mitosis more frequently, which results in macrophthalmia. Mosaic analyses reveal that Id2a function in facilitating both cell cycle progression and neuronal differentiation in the retina is non-cell-autonomous, suggesting that Id2a functions upstream of the extrinsic pathways that regulate retinogenesis. In an effort to identify which extrinsic pathways function downstream of Id2a, I discovered that Id2a function is necessary and sufficient to limit Notch pathway activity during retinogenesis. Id2a-deficient retinae possess elevated levels of Notch pathway component gene expression, while retinae overexpressing id2a possess reduced expression of Notch pathway component genes. Attenuation of Notch signaling activity by DAPT or by morpholino knockdown of Notch1a is sufficient to rescue both the proliferative and differentiation defects in Id2a-deficient retinae. In addition to regulating Notch pathway activity, through an RNA-Seq and differential gene expression analysis of Id2a-deficient retinae, I identified a number of additional intrinsic and extrinsic regulatory pathway components whose expression is regulated by Id2a. These data highlight the integral role played by Id2a in the gene regulatory network governing the transition from retinoblast proliferation to terminal differentiation during vertebrate retinogenesis.Item JNK2 inhibits luminal cell commitment in normal mammary glands and tumors(2013-05) Cantrell, Michael Andrew; Van Den Berg, CarlaBreast cancer is a heterogeneous disease with vastly different tumor progression kinetics and survival outcomes depending upon the differentiation state and gene expression patterns of the tumor. Effective treatments exist for patients with endocrine therapy sensitive or HER2 overexpressing tumors, but targeted treatments are not available for other tumor types. The mechanisms governing mammary tumor phenotype generation could prove critical to finding treatments. The c-Jun N-terminal kinase (JNK) pathway has recently been implicated in the inhibition of breast tumor luminal differentiation (1, 2) and JNK2, in particular, is important in mammary tumorigenesis and tumor progression (3-8). Therefore, the involvement of JNK2 in inhibition of mammary luminal cell differentiation was investigated in normal glands and tumors. Studies found that JNK2 inhibits luminal cell populations in normal mammary ducts. Additionally, JNK2 suppresses Notch activity in stem cell niche of the developing mammary gland. In vitro assays show that control over differentiation by JNK2 is due to suppression of p53-dependent Notch1 expression. Inhibition of luminal cell populations by JNK2 is also apparent in tumor cell models regardless of p53 expression. In the p53-competent Polyoma Middle T-antigen model, Notch1 expression is suppressed by JNK2. In the absence of p53, JNK2 suppresses luminal populations independent of Notch1. In this model, decreased luminal marker expression is accompanied by increased epithelial to mesenchymal transition. It was also found that JNK2-dependent epithelial to mesenchymal transition inhibits luminal populations and is driven by JNK2-dependent suppression of Brca1. JNK2 also confers resistance to estrogen signaling inhibition, and increases the metastatic ability of tumor cells in vivo. These data establish the importance of JNK2 in mammary epithelial cell differentiation in normal glands and tumors. They also suggest that JNK2 may be an effective prognostic marker or treatment target.Item Ligand-immobilized biomaterial surfaces for Notch signaling and T cell differentiation(2012-12) Kim, Myunghee Michelle; Roy, Krishnendu; Maynard, Jennifer A; Schmidt, Christine E; Suggs, Laura J; Tucker, HaleyIn vitro T cell differentiation from hematopoietic progenitor cells is a potential alternative source of T cells for adoptive therapy in treatment of cancers as well as T cell deficiencies. Presentation of Notch ligands immobilized on a surface is necessary in designing a stroma-free in vitro T cell differentiation system. Current methods for in vitro T cell differentiation have advanced greatly in the recent years, allowing development of functional T cells in vitro. However, these are limited to 2D coculture with stromal cells or culture on hard plastic surfaces with immobilized ligands, and have yet to report quantitative effects of variables such as substrate stiffness. This dissertation discusses the fabrication of 2D and 3D systems of various properties for presentation of Notch ligands for development of an efficient culture system, at the same time offering insight into the science of cell signaling and cell-material interactions. Magnetic microbeads, liposomes, as well as 2D and 3D soft hydrogel surfaces were fabricated to present Notch ligands at varying ligand densities and to study their quantitative effect on Notch signaling and T cell differentiation. The x findings of this dissertation demonstrate that substrate material plays a role in Notch signaling in combination with ligand density, and may affect downstream events of T lineage commitment. Insights gained from this research provide a new direction in the importance of culture substrate in ligand-presenting systems and allow development of new systems to support efficient generation of T cells in vitro.Item The role of auxilin and endocytosis in delta signaling(2012-05) Banks, Susan Marie-Louise; Fischer, Janice Ann; Huibregtse, Jon; Macdonald, Paul; Morgan, Jennifer; O'Halloran, TheresaNotch signaling is important for cell-cell signaling during development. Notch signaling is highly conserved across all metazoans and failure in Notch signaling is causative in many human diseases. In the Drosophila eye, activation of the Notch pathway requires Lqf (Drosophila Epsin)-dependent and Clathrin-dependent internalization of the Notch receptor ligands, Delta or Serrate, by the signal-sending cells. However, it is unclear why ligand must be internalized into the signal-sending cells to activate Notch signaling in the signal-receiving cells. Evidence suggests that in addition to Clathrin and Epsin, Auxilin is essential for signaling and is indirectly required for internalization of the Notch receptor ligand Delta. Auxilin functions in uncoating Clathrin-coated vesicles to maintain a pool of free Clathrin and Epsin in the cell. auxilin mutants were used as an entryway to identify previously unknown components of the Notch signaling pathway. An F1, FLP/FRT, EMS screen was performed and enhancers of an auxilin mutant rough eye defect were isolated. The enhancers ultimately formed one complementation group on the 2nd chromosome and fourteen complementation groups on the 3rd chromosome. Three of the 3rd chromosome complementation groups were each identified as Delta, lqf, or hsc70. A single allele was identified as faf. Delta and Epsin have known roles in signaling cells to activate Notch as described above. Hsc70 is an ATPase that functions with Auxilin to uncoat Clathrin-coated vesicles and Faf is a deubiquitinating enzyme that maintains levels of active Epsin in the cell. These results suggest I have isolated mutations in genes closely tied to Notch signaling or functioning directly with Auxilin. Mutations in two genes previously undescribed in Notch signaling in the developing Drosophila eye were also isolated from the screen and identified. The second chromosome complementation group was identified as α-adaptin. α-Adaptin is a subunit of the heterotetrameric Clathrin adaptor protein AP-2. One of the third chromosome complementation groups was identified as crumbs. Crumbs is an integral membrane protein that functions at adherens junctions and in establishing apical/basal polarity in cells. Characterizing roles for α-Adaptin and Crumbs during Notch signaling may elucidate the purpose for Delta internalization to activate Notch signaling.