Automatic Train Control(ATC) of Signaling System for a Metro Systems comprise Automatic Train Supervision(ATS), Automatic Train Protection(ATP) and Automatic Train Operation(ATO). Based on theAutomatic Train Control(ATC) of Signaling System for a Metro Systems comprise Automatic Train Supervision(ATS), Automatic Train Protection(ATP) and Automatic Train Operation(ATO). Based on the Signaling Control System of Kaohsiung Mass Rapid Transit System(KMRT),the relationship of ATO propulsion power consumption and the run time between stations of train sets under the constraint of Automatic Train Protection(ATP) is investigated. By integrating the ATC function for the planning of timetable, the optimal timetable can be established. ATO solves the speed command for train sets operation by considering the propulsion system, track alignment and the operating timetable to achieve the efficiency of energy consumption by applying the operation modes of Cruising and Coasting to derive the speed profiles of train sets. Because of the correlationship of ATP, ATO and Operation Timetable of train sets, the ATP system is investigated first to ensure the safety of system operation. Based on the constraint of ATP, the energy conservation of train sets with ATO system is simulated. The optimal timetable is then derived by minimizing the objective function, which consists of the costs of train sets, driver¡¦s manpower and the simulated energy consumption. The Genetic Algorithm (GA) is applied to solve the optimal timetable by representing the Run Time Reserve and Loading Factor of train sets as the chromosomes. With the mutation of GA method, the global optimal solution can be obtained without falling into the local optimum. It is concluded that the optimal operation timetable solved by the proposed GA method can enhance the system performance of KMRT systems by reducing the operation cost of train sets.

Membrane-bound receptors transmit information from the cell exterior to the cell interior. Bacterial receptors capable of transmitting this information include sensor kinases, which control gene expression via response regulators, and methyl-accepting chemotaxis proteins (MCPs), which control rotation of the flagellar motor. These receptors, which have a similar general architecture and function, are predicted to share similar mechanisms of transmembrane signaling. The majority of such work has been conducted on MCPs. Our goal is to extend this work to the closely related sensor kinases by creating functional hybrid transducers. I show that a chimeric protein (Nart) that joins the periplasmic, ligandbinding domain of the sensor kinase NarX (nitrate/nitrite sensor) to the cytoplasmic signaling domain of the chemoreceptor Tar is capable of modulating flagellar rotation in response to both nitrate and nitrite. Consistent with the properties of NarX, our Nart elicits a stronger response to nitrate than to nitrite. Introduction of mutations into a highly conserved periplasmic region affects Nart signaling in a fashion that is consistent with the effects seen in NarX. I also present the first example of a substitution in a presumed ligand-binding domain that confers a reverse-signal phenotype for both nitrate and nitrite in Nart. These results support the hypothesis that the key aspects of transmembrane signaling are closely similar in homodimeric bacterial chemoreceptors and sensor kinases.

This thesis describes the design of a device for generating a reproduceable noise signals. The noise signal is generated by adding three periodic waveforms having non-multiple periods. Pulse techniques are used in the generation of the member functions so that the output may be reproduced exactly. Theoretical and experimental determinations of the amplitude probability distribution and of the autocorrelation function of the signal were made. On the basis of tests and observations made, it is concluded that the signal generated may be considered a noise signal having a near-Gaussian amplitude probability distribution, very little correlation for time-shifts greater than 30 seconds, and a bandwidth of about 60 cps. / Applied Science, Faculty of / Electrical and Computer Engineering, Department of / Graduate

Thesis PlanB (M.S.)--University of Wisconsin--Stout, 2009. / Includes bibliographical references.

Medulloblastoma (MB), a cancer of the cerebellum, is the most common solid tumor affecting children. In the cerebellum, Sonic Hedgehog (Shh) drives the proliferative expansion of granule neuron progenitors (GNP). These cells are located in the external granule layer (EGL) and are the cells of origin of Shh-MB. We recently identified Norrie Disease Pseudoglioma (Ndp) as a novel downstream target of Hh signaling in the developing retina. Ndp encodes an X-linked cysteine-rich secreted protein called Norrin, which is best known for its role in angiogenesis and blood brain barrier (BBB) maintenance in the developing retina and cerebellum, respectively. Norrin mediates this effect by binding to its receptor Frizzled4 (Fzd4) and co-receptors LRP5/6 and Tpsan12 to activate the canonical, β-catenin-dependent Wnt signaling pathway in endothelial cells (ECs). We detected the expression of Ndp and all required receptors in mouse GNPs and MB samples. To investigate a potential role for Ndp in Hh-driven MB, we genetically and pharmacologically inactivated Ndp/Fzd4 signaling in Ptch+/- mice (a mouse model for human Gorlin syndrome), which dramatically increased the incidence and reduced the latency of MB. This accelerated rate of tumorigenesis was caused by an increase in the number of preneoplastic lesions (PNLs), the precursor lesions to MB, and a faster conversion of these lesions to MB. We showed that Ndp mediates this increase in tumorigenesis by signaling through endothelial cell receptor Fzd4 to alter the GNP stroma, which is characterised by 5 major alterations: 1) activated angiogenic program, 2) open BBB, 3) aberrant deposition of extracellular matrix, 4) aberrant lymphocyte recruitment and 5) reduction in meningeal lymphatic vasculature. We propose that these stromal alterations are associated with a pro-tumor microenvironment that promotes DNA damage in GNPs and leads to enhanced lesion formation and progression towards MB. This research highlights 1) an unanticipated role for Ndp/Fzd4 signaling in Shh-MB initiation and progression, 2) a role for stromal signaling in the regulation of MB development and 3) a previously undescribed role for Ndp signaling in maintaining meningeal cerebellum lymphatic vessels.

No description available.

A delicate balance in the number, specific type and function of leukocytes is required for proper functionality of the mammalian immune system. Innate immunity, which quickly recognizes pathogens, represents the first line of defense. Later, a more specific response is generated via adaptive immunity. Deregulation of the immune system is manifested by the inability to control infection, development of allergic, autoimmune disorders or even cancer, and ultimately can lead to death. To fulfill their functions, cells develop an intricate network of intra- as well as extra-cellular molecules organized into signaling cascades, which allows them to communicate between each other. Better understanding of the molecular mechanisms of signaling pathways in leukocytes is critical for design of efficient therapies. In this thesis, leukocyte signaling was studied in several aspects. First, the role of adhesion molecules in pathogenesis of cervical cancer and the regulation of their expression was investigated. The second publication describes a new transmembrane adaptor protein (TRAP), called prolin rich 7 (PRR7), as a potentially interesting regulator of signaling and apoptosis in activated T cells. The final publication characterized the role of the Btk kinase downstream of the triggering receptor expressed...

<b>Background:</b> Sustained elevations in catecholaminergic signaling, mediated primarily through β-adrenergic receptor (β-AR) stimulation, are a hallmark neurohormonal alteration in heart failure (HF) that contribute to pathophysiologic cardiac remodeling. An important pathophysiological change during sustained β-AR stimulation is functional inhibition of the slow delayed rectifier potassium current, IKs, which has been demonstrated to prolong action potential duration (APD) and increase ventricular arrhythmogenesis in HF. Though functional inhibition of IKs has been consistently reproduced in cellular, animal, and limited human studies of HF, the mechanisms that mediate IKs inhibition during HF remain poorly understood.<div><br></div><div>In addition, HF results in aberrant calcium handling that is known to contribute to the disease. HF has been demonstrated to increase the expression and function of calcium/calmodulin-dependent protein kinase II (CaMKII), a key regulator of calcium homeostasis and excitation-contraction coupling in cardiomyocytes. Enhanced CaMKII signaling has been consistently demonstrated to contribute to increased arrhythmogenesis in a number of cardiac diseases, including HF. CaMKII is a known pathological regulator of many cardiac ion channels resulting in APD prolongation and the development of arrhythmias.<br></div><div><br></div><div><b>Objective:</b> This investigation aims to assesses the potential for CaMKII regulation of KCNQ1 (pore-forming subunit of IKs) during sustained β-AR stimulation and to characterize the potential functional implications on IKs. Furthermore, this investigation seeks to elucidate the mechanism underlying CaMKII-mediated IKs inhibition during sustained β-AR stimulation.<br></div><div><br></div><div><b>Methods: </b>Phosphorylation of KCNQ1 was assessed using a tandem liquid chromatography- mass spectrometry/ mass spectrometry (LCMS/MS) approach during sustained β-AR stimulation via treatment with 100 nM isoproterenol (ISO) for 4-24 hours and during co-expression with KCNE1. Whole-cell, voltage-clamp patch clamp electrophysiology experiments were performed in HEK 293 cells transiently co-expressing wild-type (WT) or mutant KCNQ1 (mutations conferring mimics of dephosphorylation and phosphorylation were introduced at phosphorylation sites identified by LCMS/MS) and KCNE1 (auxiliary subunit) during ISO treatment, treatment with CaMKII or protein kinase A (PKA) inhibitors, or during lentiviral δCaMKII overexpression. A robotic peptide synthesizer was used to create fifteen residue peptide fragments on a nitrocellulose membrane corresponding to KCNQ1 intracellular domains and the KCNQ1 residues identified via LCMS/MS; membranes were incubated with activated CaMKII or PKA in the presence of radiolabeled ATP to identify potential sites of phosphorylation. Bimolecular fluorescence complementation (BiFC) experiments were performed in HEK 293 cells to assess the impact of CaMKII-mediated KCNQ1 phosphorylation on the interaction of KCNQ1 and KCNE1 subunits. Protein immunoblot experiments were performed to (1) assess CaMKII activation during ISO treatment and (2) to assess plasma membrane expression of KCNQ1 and KCNE1 subunits with mimics of differential KCNQ1 phosphorylation following a membrane protein biotinylation procedure.<br></div><div><br></div><div><b>Results:</b> In Aim 1, we investigated the regulation of the KCNQ1 carboxyl terminus during sustained β-AR stimulation and assessed the associated functional implications on IKs. An LCMS/MS approach identified five novel KCNQ1 carboxyl terminal sites that demonstrated basal phosphorylation, with T482 and S484 having enhanced phosphorylation during treatment with 100 nM ISO for 24 hours (p<0.01 at both sites). Using patch clamp electrophysiology, we demonstrated that treatment with 100 nM ISO for 12-24 hours reduced IKs current density (p=0.01) and produced a depolarizing shift in the voltage dependence of activation (p<0.01) relative to vehicle. Mimics of phosphorylation (mutations to aspartic acid; Triple-D KCNQ1) at S457, T482, and S484 in combination, meanwhile, reduced IKs activation current density relative to dephosphorylation (mutations to alanine; Triple-A KCNQ1) mimics (p=0.02) but did not affect the voltage dependence of activation (p=0.66). Functional assessment of these sites individually revealed that phosphorylation mimics at S457 (p=0.02) and S484 (p=0.04), but not at T482 (p=0.53), reduced IKs current density relative to mimics of dephosphorylation. Similarly, the voltage dependence of activation was right-shifted with phosphorylation mimics at S457 (p=0.03) and S484 (p=0.02), but not at T482 (p=0.99), relative to mimics of dephosphorylation.<br></div><div><br></div><div>The focus of Aim 2 was to assess the potential for CaMKII signaling to regulate increased KCNQ1 phosphorylation and reduced IKs function during sustained β-AR stimulation. Peptide fragments corresponding to the KCNQ1 carboxyl terminal sites demonstrating basal phosphorylation via LCMS/MS analysis were synthesized on a nitrocellulose membrane and exposed to activated δCaMKII. Only peptide fragments corresponding to S484 demonstrated CaMKII phosphorylation. Patch clamp experiments demonstrated that CaMKII inhibition via the chemical inhibitor KN-93 (p=0.02) and the peptide inhibitor CN21 (p<0.01) reversed ISO-treatment associated inhibition of IKs activation current density relative to appropriate controls (KN-92 and CN21-Alanine, respectively). Inhibition with KN-93 and CN21 (p<0.01 for both) also reversed ISO-treatment associated right shifts in the voltage dependence of activation relative to appropriate controls. The ability of ISO treatment to activate CaMKII in HEK 293 cells was confirmed via protein immunoblot wherein T287 phosphorylation (CaMKII residue conferring constitutive activity) was increased during ISO treatment (p<0.05). Lentiviral overexpression of δCaMKII inhibited IKs activation current density with WT IKs (p=0.01) but not with Triple-A IKs (p=0.20) relative to lentiviral control. Inhibition of IKs activation current density during δCaMKII overexpression was attenuated with S484A IKs (p=0.04) but not with S457A (p=0.99) relative to WT IKs during δCaMKII overexpression. The voltage dependence of activation was also right-shifted during δCaMKII overexpression relative to lentiviral control (p=0.03). PKA inhibition with the peptide inhibitor PKI did not reverse ISO-treatment associated inhibition of IKs activation current density (p=0.51), and PKA did not phosphorylate peptide fragments corresponding to any of residues identified via LCMS/MS.<br></div><div><br></div><div>Aim 3 investigated the mechanism through which CaMKII-mediated phosphorylation at KCNQ1 S484 inhibits IKs function. To assess whether interaction with KCNE1 affects KCNQ1 phosphorylation, we performed LCMS/MS experiments during expression of KCNQ1 alone and during co-expression with KCNE1. Phosphorylation at S484 was reduced during co-expression with KCNE1 relative to expression of KCNQ1 alone (p<0.01). In addition, mimics of phosphorylation at S484 (S484D) did not affect activation current density (p=0.96) or the voltage dependence of activation (p=0.51) relative to dephosphorylation mimics (S484A). Based on these results, we hypothesized that S484 phosphorylation affected the interaction between KCNQ1 and KCNE1 subunits; accordingly, we assessed the KCNQ1-KCNE1 interaction using BiFC experiments in HEK 293 cells. In accordance with our hypothesis, Venus fluorescent intensity (corresponding to KCNQ1-KCNE1 interaction) was reduced during ISO treatment relative to vehicle (p<0.05) and with S484D KCNQ1 relative to S484A (p<0.01). The role of CaMKII in mediating this disruption of KCNQ1-KCNE1 interaction was demonstrated BiFC experiments that showed co-treatment with ISO and KN-93 attenuated reduced Venus intensity during co-treatment with ISO and KN-92 (p<0.01). These results were corroborated by BiFC experiments with Long QT Syndrome Phenotype 1 (LQT1) mutations that demonstrated that an LQT1 mutation predicted to disrupt CaMKII phosphorylation at S484 (R481I) attenuated reduced Venus intensity during ISO treatment relative to an LQT1 mutations predicted to not affect CaMKII regulation of S484 (S484T; p<0.01). The ability of S484 phosphorylation to affect KCNQ1 and/or KCNE1 trafficking was assessed via protein immunoblot experiments to detect KCNQ1 and KCNE1 following a biotinylation procedure to isolate plasma membrane-bound proteins. Biotinylation experiments demonstrated that KCNQ1 and KCNE1 plasma membrane expression were reduced by ~15% and ~33%, respectively, with S484D KCNQ1 relative to S484A (p<0.05 for both).<br></div><div><b><br></b></div><div><b>Conclusion: </b>CaMKII phosphorylates KCNQ1 S484 during sustained β-AR stimulation to inhibit IKs function. S484 phosphorylation inhibits IKs function by disrupting the interaction between KCNQ1 and KCNE1 subunits and by reducing the plasma membrane expression of KCNQ1 and KCNE1. Pathological regulation of KCNQ1 by CaMKII (and subsequent inhibition of IKs) during sustained β-AR stimulation may contribute to increased arrhythmogenesis during physiologic states of chronically increased catecholaminergic tone, such as during HF.<br></div>

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

We have previously reported that non-muscle myosin II (NMMII) and myosin light chain kinase (MLCK) are required for oncogenic Ras signaling to ERK in Ki-Ras transformed rat fibroblsasts (Helfman and Pawlak, J. Cell Biochem. 95(5), 1069-80, 2005). Here I examine if MLCK plays a role in ERK signaling in various tumor derived human epithelial cell lines. I also determined whether genetic inhibition of NMMII isoforms IIA and IIB, or MLCK also inhibits ERK activation in the MCF 10A human breast epithelial cell line expressing oncogenic H-Ras. Inhibition of MLCK by pharmacological inhibitors such as ML-7 and ML-9 was used to determine the role of MLCK in ERK signaling in an array of H/K-Ras transformed and tumor derived cell lines: T-24 bladder carcinoma, HCT 116 colon carcinoma, and MCF 10A Ras breast cancer cells. Genetic inhibition was carried out using specific siRNA targeted towards MLCK and NMMIIA or IIB. The knock down of NMM IIA and IIB did not inhibit active ERK, which suggested either a redundant function of NMM IIC or an alternate substrate for MLCK. Inhibition of MLCK by ML-7/ML-9 reduced activated ERK in all H/K-Ras transformed, or human tumor derived cell lines we tested. The possible mechanism of how MLCK could play a role in ERK signaling was tested by co-immunoprecipitation (co-IP) of MAPK scaffolding proteins with MLCK. That the ERK scaffold KSR1 regulates ERK signaling in MCF 10A Ras, was demonstrated through inhibition of KSR1 with siRNA. Moreover, KSR was shown to interact with MLCK because it was found to co-precipitate with MLCK.