Cell-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. / text

Epsin is an endocytic protein that binds Clathrin, the plasma membrane, Ubiquitin, and also a variety of other endocytic proteins through well-characterized motifs. Although Epsin is a general endocytic factor, genetic analysis in Drosophila and mice revealed that Epsin is essential specifically for internalization of ubiquitinated transmembrane ligands of the Notch receptor, a process required for Notch activation. How Epsin promotes ligand endocytosis and thus Notch signaling is unclear. Here, by generating Drosophila lines containing transgenes that express a variety of different Epsin deletion and substitution variants, I tested each of the five protein or lipid interaction modules of Epsin for a role in Notch activation by each of the two Drosophila ligands, Serrate and Delta. here are five main results of this work that impact present thinking about endocytic machinery/Epsin, Epsin/ligand, or ligand/receptor interactions at the plasma membrane. First, I discovered that deletion or mutation of both UIMs destroys Epsin’s function in Notch signaling and has a greater negative effect on Epsin’s ability to function than removal of any other module type. Second, only one of the two UIMs of Epsin is essential. Third, the lipid-binding function of the ENTH domain is required for maximal Epsin activity. Fourth, although the C-terminal Epsin modules that interact with Clathrin, the adapter protein complex AP-2, or endocytic accessory proteins are necessary collectively for Epsin activity, their functions are highly redundant. Finally, I detected no ligand-specific requirements for Epsin modules. Most unexpected was the finding that Epsin’s Clathrin binding motifs were dispensable. All of these observations are consistent with a model where Epsin’s essential function in ligand cells is to link ubiquitinated Notch ligands to Clathrin-coated vesicles through other Clathrin adapter proteins. / text

Experiments conducted at the Texas A&M Turbomachinery Laboratory and field applications have shown that pocket damper seals (PDS) can be used to suppress vibrations in compressors. A mathematical model is presented for the notched PDS. The notch is a prominent feature in all the PDS manufactured in recent times. The notch is provided at the exit blades of the PDS to act as a diverging clearance, which is one of the conditions for the damper seal to perform satisfactorily The model to be presented has been adapted from a theory previously developed to predict the direct stiffness and damping coefficients. The flow equations are numerically solved and a computer program is developed correspondingly. The predictions from this notched model are compared with the existing model to highlight the effect of the notch in the analysis. These predictions correlate well with the experimental results from the notched PDS. Also experimental results from testing of a two bladed PDS are compared to the code predictions thus validating the notched model. The notched model performs satisfactorily to predict the direct damping coefficients. Coastdown tests are conducted on a four bladed eight pocket PDS with a partial arc notch of large radius across the exit blades. The PDS offers positive direct damping which increases with an increase in seal inlet pressure. The low stiffness of the test rig combined with the negative stiffness of the seal made it impracticable to conduct testing above inlet pressures of 64.7 psia (4.461 bar). The existing theoretical models are compared with the experimental data collected up to 64.7 psia (4.461 bar).

The Notch receptor is part of a highly conserved signaling pathway shared in Drosophila, C. elegans and mammals. Extensive studies of Notch signaling have revealed its participation in the development of diverse organ systems including brain, blood cells, blood vessels, gut, and skin. Many genetic modifiers of the Notch signaling pathway have been identified, including some which act at the membrane and others in the nucleus. One such member is Deltex, an E3 ubiquitin ligase, which was originally identified as a modifier of Notch in a Drosophila genetic screen. In early lymphoid development, Deltex has been demonstrated functionally to antagonize Notch signaling but the precise molecular mechanism for this functional antagonism between Notch and Deltex is not understood. However, in Drosophila, recent data supports the formation of a trimeric complex between Deltex, Kurtz and Notch that promotes Notch ubiquitin-mediated proteosomal degradation. Beta-arrestin1 is one of the closest mammalian homologues of Kurtz and functions as an adaptor protein in a variety of cellular processes such as endocytosis, ubiquitination and nuclear shuttling. We hypothesize that a similar interaction occurs in mammalian cells between Notch, beta-arrestin1 and Deltex to negatively modulate the Notch signaling pathway. Our data reveal a physical interaction between beta-arrestin1 and the Notch receptor. We could not, however, detect an interaction between Deltex and beta-arrestin1 by co-immunoprecipitation. We also demonstrate that Notch and beta-arrestin1 physically associate with both a membrane-bound form of activated Notch, as well as the intracellular form of Notch after membrane cleavage. Using RNA interference, as well as overexpression of beta-arrestin1, we demonstrate that beta-arrestin1 negatively regulates a Notch/CSL dependant reporter assay. We also show that the presence of Deltex enhances the negative modulation of the Notch signaling pathway mediated by beta-arrestin1. Therefore, we reveal a new Notch interacting protein and a novel role for beta-arrestin1 in the Notch signaling pathway.

Podocytes are terminally differentiated epithelial cells which regulate glomerular permselectivity by their cell-cell junctions, known as slit diaphragms (SD). Notch signaling regulates podocyte cell fate specification and downregulation of Notch targets occurs with terminal podocyte differentiation. The effects of constitutive Notch activation in developing podocytes on podocyte differentiation and function were determined using a podocyte-specific Cre-lox-p(Neph/Cre) approach. Proteinuria was noted shortly after birth denoting loss of glomerular permselectivity in transgenic mice (CRE;NIC mice). Histologic and molecular analyses of CRE;NIC-expressing mice at onset of proteinuria, show morphologic and cellular changes in podocytes including de-differentiation, proliferation and de novo expression of Pax2. Prior to onset of proteinuria, lower protein levels of key SD proteins are observed while SD mRNA expression is preserved in CRE;NIC mice. Consequently, constitutive Notch signaling in developing podocytes opposes terminal differentiation with deleterious consequences on SD assembly and thereafter, glomerular permselectivity.

No description available.

Podocytes are terminally differentiated epithelial cells which regulate glomerular permselectivity by their cell-cell junctions, known as slit diaphragms (SD). Notch signaling regulates podocyte cell fate specification and downregulation of Notch targets occurs with terminal podocyte differentiation. The effects of constitutive Notch activation in developing podocytes on podocyte differentiation and function were determined using a podocyte-specific Cre-lox-p(Neph/Cre) approach. Proteinuria was noted shortly after birth denoting loss of glomerular permselectivity in transgenic mice (CRE;NIC mice). Histologic and molecular analyses of CRE;NIC-expressing mice at onset of proteinuria, show morphologic and cellular changes in podocytes including de-differentiation, proliferation and de novo expression of Pax2. Prior to onset of proteinuria, lower protein levels of key SD proteins are observed while SD mRNA expression is preserved in CRE;NIC mice. Consequently, constitutive Notch signaling in developing podocytes opposes terminal differentiation with deleterious consequences on SD assembly and thereafter, glomerular permselectivity.

The Notch receptor is part of a highly conserved signaling pathway shared in Drosophila, C. elegans and mammals. Extensive studies of Notch signaling have revealed its participation in the development of diverse organ systems including brain, blood cells, blood vessels, gut, and skin. Many genetic modifiers of the Notch signaling pathway have been identified, including some which act at the membrane and others in the nucleus. One such member is Deltex, an E3 ubiquitin ligase, which was originally identified as a modifier of Notch in a Drosophila genetic screen. In early lymphoid development, Deltex has been demonstrated functionally to antagonize Notch signaling but the precise molecular mechanism for this functional antagonism between Notch and Deltex is not understood. However, in Drosophila, recent data supports the formation of a trimeric complex between Deltex, Kurtz and Notch that promotes Notch ubiquitin-mediated proteosomal degradation. Beta-arrestin1 is one of the closest mammalian homologues of Kurtz and functions as an adaptor protein in a variety of cellular processes such as endocytosis, ubiquitination and nuclear shuttling. We hypothesize that a similar interaction occurs in mammalian cells between Notch, beta-arrestin1 and Deltex to negatively modulate the Notch signaling pathway. Our data reveal a physical interaction between beta-arrestin1 and the Notch receptor. We could not, however, detect an interaction between Deltex and beta-arrestin1 by co-immunoprecipitation. We also demonstrate that Notch and beta-arrestin1 physically associate with both a membrane-bound form of activated Notch, as well as the intracellular form of Notch after membrane cleavage. Using RNA interference, as well as overexpression of beta-arrestin1, we demonstrate that beta-arrestin1 negatively regulates a Notch/CSL dependant reporter assay. We also show that the presence of Deltex enhances the negative modulation of the Notch signaling pathway mediated by beta-arrestin1. Therefore, we reveal a new Notch interacting protein and a novel role for beta-arrestin1 in the Notch signaling pathway.

Thesis (Ph.D.) in Biochemistry and Molecular Biology--University of Maine, 2007. / Includes vita. Includes bibliographical references (leaves 103-119).

Techn. University, Diss., 2005--München.