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Strategies aimed at mimicking or enhancing the action of the incretin hormone glucagon-like peptide 1 (GLP-1) therapeutically improve glucose-stimulated insulin secretion (GSIS); however, it is not clear whether GLP-1 directly drives insulin secretion in pancreatic islets. Here, we examined the mechanisms by which GLP-1 stimulates insulin secretion in mouse and human islets. We found that GLP-1 enhances GSIS at a half-maximal effective concentration of 0.4 pM. Moreover, we determined that GLP-1 activates PLC, which increases submembrane diacylglycerol and thereby activates PKC, resulting in membrane depolarization and increased action potential firing and subsequent stimulation of insulin secretion. The depolarizing effect of GLP-1 on electrical activity was mimicked by the PKC activator PMA, occurred without activation of PKA, and persisted in the presence of PKA inhibitors, the KATP channel blocker tolbutamide, and the L-type Ca(2+) channel blocker isradipine; however, depolarization was abolished by lowering extracellular Na(+). The PKC-dependent effect of GLP-1 on membrane potential and electrical activity was mediated by activation of Na(+)-permeable TRPM4 and TRPM5 channels by mobilization of intracellular Ca(2+) from thapsigargin-sensitive Ca(2+) stores. Concordantly, GLP-1 effects were negligible in Trpm4 or Trpm5 KO islets. These data provide important insight into the therapeutic action of GLP-1 and suggest that circulating levels of this hormone directly stimulate insulin secretion by β cells.

The spatial and temporal control of polarity is fundamental to the survival of all organisms. Cells define their polarity using highly conserved mechanisms that frequently rely upon the action of small GTPases, such as Ras and Cdc42. Schizosaccharomyces pombe is an ideal system with which to study the control of cell polarity since it grows from defined tips using Cdc42-mediated actin remodeling. Here we have investigated the importance of Ras1-GTPase activity for the coordination of polarized cell growth during fission yeast mating. Following pheromone stimulation, Ras1 regulates both a MAPK cascade and the activity of Cdc42 to enable uni-directional cell growth towards a potential mating partner. Like all GTPases, when bound to GTP, Ras1 adopts an active conformation returning to an inactive state upon GTP-hydrolysis, a process accelerated through interaction with negative regulators such as GAPs. Here we show that, at low levels of pheromone stimulation, loss of negative regulation of Ras1 increases signal transduction via the MAPK cascade. However, at the higher concentrations observed during mating, hyperactive Ras1 mutations promote cell death. We demonstrate that these cells die due to their failure to coordinate active Cdc42 into a single growth zone resulting in disorganized actin deposition and unsustainable elongation from multiple tips. These results provide a striking demonstration that the deactivation stage of Ras signaling is fundamentally important in modulating cell polarity.

We present a novel parameter identification algorithm for the estimation of parameters in models of cell motility using imaging data of migrating cells. Two alternative formulations of the objective functional that measures the difference between the computed and observed data are proposed and the parameter identification problem is formulated as a minimisation problem of nonlinear least squares type. A Levenberg–Marquardt based optimisation method is applied to the solution of the minimisation problem and the details of the implementation are discussed. A number of numerical experiments are presented which illustrate the robustness of the algorithm to parameter identification in the presence of large deformations and noisy data and parameter identification in three dimensional models of cell motility. An application to experimental data is also presented in which we seek to identify parameters in a model for the monopolar growth of fission yeast cells using experimental imaging data. Our numerical tests allow us to compare the method with the two different formulations of the objective functional and we conclude that the results with both objective functionals seem to agree.

The spatial and temporal control of polarity is fundamental to the survival of all organisms. Cells define their polarity using highly conserved mechanisms that frequently rely upon the action of small GTPases, such as Ras and Cdc42. Schizosaccharomyces pombe is an ideal system with which to study the control of cell polarity since it grows from defined tips using Cdc42-mediated actin remodeling. Here we have investigated the importance of Ras1-GTPase activity for the coordination of polarized cell growth during fission yeast mating. Following pheromone stimulation, Ras1 regulates both a MAPK cascade and the activity of Cdc42 to enable uni-directional cell growth towards a potential mating partner. Like all GTPases, when bound to GTP, Ras1 adopts an active conformation returning to an inactive state upon GTP-hydrolysis, a process accelerated through interaction with negative regulators such as GAPs. Here we show that, at low levels of pheromone stimulation, loss of negative regulation of Ras1 increases signal transduction via the MAPK cascade. However, at the higher concentrations observed during mating, hyperactive Ras1 mutations promote cell death. We demonstrate that these cells die due to their failure to coordinate active Cdc42 into a single growth zone resulting in disorganized actin deposition and unsustainable elongation from multiple tips. These results provide a striking demonstration that the deactivation stage of Ras signaling is fundamentally important in modulating cell polarity.

A vast array of cellular processes are controlled by the highly conserved guanine nucleotide-regulated molecular switches, such as Ras. The spatial and temporal regulation of Ras signalling, both positively, through the action of guanine nucleotide exchange factors (GEFs) and negatively, via interaction with GTPase activating proteins (GAPs) is critical to maintain cell viability. Due to the presence of multiple Ras isoforms, regulatory proteins and effectors, the study of Ras signalling in higher eukaryotes is technically challenging. Fission yeast provides a simple system in which to study Ras signalling containing a single Ras homologue (Ras1) that modulates two distinct processes. Ras1 signalling is terminated through GTP-hydrolysis accelerated by the action of a GAP, Gap1. This thesis describes the development of a quantitative FRET assay to visualise the dynamics of Ras1 activity within single cells. Using this reporter, in combination with a variety of other quantitative techniques, the role of the negative regulator during signal transduction was explored. These results highlighted an important difference between the requirement for GTPhydrolysis during signal propagation. Finally, use of the FRET reporter revealed an alternative G protein state, within the Ras1 activation cycle. These data question the accepted models of G protein signal propagation and prompt further investigation into the roles of GTP-hydrolysis in signal transduction. Several positive regulators of Ras1 have previously been described in fission yeast. These proteins have been proposed to direct signalling via the different Ras1 effectors. Using quantitative image analysis and pheromoneresponsive reporter strains, this thesis presents data to suggest that it is not simply a one-activator-one-effector response. Further, the role of the previously uncharacterised, scaffold protein, Ral2 in mediating signal transduction is also investigated. Finally the possibility that the heterotrimeric G protein, Gpa1 can directly activate MAPK signalling to provide a pheromone-sensing mechanism within fission yeast is discussed.

Chemokine receptor antagonists appear to access two distinct binding sites on different members of this receptor family. One class of CCR4 antagonists has been suggested to bind to a site accessible from the cytoplasm while a second class did not bind to this site. In this report, we demonstrate that antagonists representing a variety of structural classes bind to two distinct allosteric sites on CCR4. The effects of pairs of low‐molecular weight and/or chemokine CCR4 antagonists were evaluated on CCL17‐ and CCL22‐induced responses of human CCR4+ T cells. This provided an initial grouping of the antagonists into sets which appeared to bind to distinct binding sites. Binding studies were then performed with radioligands from each set to confirm these groupings. Some novel receptor theory was developed to allow the interpretation of the effects of the antagonist combinations. The theory indicates that, generally, the concentration‐ratio of a pair of competing allosteric modulators is maximally the sum of their individual effects while that of two modulators acting at different sites is likely to be greater than their sum. The low‐molecular weight antagonists could be grouped into two sets on the basis of the functional and binding experiments. The antagonistic chemokines formed a third set whose behaviour was consistent with that of simple competitive antagonists. These studies indicate that there are two allosteric regulatory sites on CCR4. e00019

This study used the Adult Attitudes Toward Continuing Education Scale (AACES) to determine the relationship between attitudes and participation in continuing education activities. Along with the Behavior Index, the AACES instrument was utilized to identify differences between sub-groups of the sample population. The AACES survey, composed of items representing attitude-to-object and attitude-to-situation, was completed by a total of 1,782 full-time gainfully employed adults. The subjects worked in manufacturing industries in two rural and two urban counties in the South Carolina Midlands. Subjects also completed a sociodemographic survey, along with the Behavior Index, which requested information concerning their participation in current and past continuing education activities. Findings indicated that the workforce expressed generally favorable attitudes toward continuing education (p$<$ .0001). Relationships between attitude and income, race, gender, educational level, family income level, and rural or urban location were significant (p$<$ .05). Results also indicated that the variables of location, educational level, and gender may be significant predictors of attitudes (p$<$ .05) toward participation in adult continuing education programs. Behavior emerged as the single most important predictor of attitude (p$<$ .001). These findings suggest that studies need to be conducted to explore differences in attitude. Both workforce managers and practitioners may benefit from findings which lead to strategies for influencing adults to develop and maintain positive attitudes toward the concept of lifelong learning. Additional research may provide insight into the phenomenon of why adults choose, or do not choose, to participate in continuing education activities.

We present a novel parameter identification algorithm for the estimation of parameters in models of cell motility using imaging data of migrating cells. Two alternative formulations of the objective functional that measures the difference between the computed and observed data are proposed and the parameter identification problem is formulated as a minimisation problem of nonlinear least squares type. A Levenberg-Marquardt based optimisation method is applied to the solution of the minimisation problem and the details of the implementation are discussed. A number of numerical experiments are presented which illustrate the robustness of the algorithm to parameter identification in the presence of large deformations and noisy data and parameter identification in three dimensional models of cell motility. An application to experimental data is also presented in which we seek to identify parameters in a model for the monopolar growth of fission yeast cells using experimental imaging data.

The UK IBD Genetics Consortium and the Wellcome Trust Case Control Consortium 2 report results of a genome-wide association study of ulcerative colitis. They identify three new loci associated with the disease, including the HNF4A region on 20q13. Ulcerative colitis is a common form of inflammatory bowel disease with a complex etiology. As part of the Wellcome Trust Case Control Consortium 2, we performed a genome-wide association scan for ulcerative colitis in 2,361 cases and 5,417 controls. Loci showing evidence of association at P < 1 × 10 −5 were followed up by genotyping in an independent set of 2,321 cases and 4,818 controls. We find genome-wide significant evidence of association at three new loci, each containing at least one biologically relevant candidate gene, on chromosomes 20q13 ( HNF4A ; P = 3.2 × 10 −17 ), 16q22 ( CDH1 and CDH3 ; P = 2.8 × 10 −8 ) and 7q31 ( LAMB1 ; P = 3.0 × 10 −8 ). Of note, CDH1 has recently been associated with susceptibility to colorectal cancer, an established complication of longstanding ulcerative colitis. The new associations suggest that changes in the integrity of the intestinal epithelial barrier may contribute to the pathogenesis of ulcerative colitis.