Regulation of Epac by cAMP

The role of cAMP as a second messenger in cellular signalling is well established. In the eukaryotic cell the targets of cAMP are PKA, cyclic nucleotide gated ion channels and Epac. All these proteins have a cAMP-binding domain of same architecture in common. Its function is to bind cAMP and thereby sense the presence of cAMP. Binding of cAMP to this sensor domain is transformed into an altered property of the whole protein. In spite of the important role of cAMP in regulating cellular events, the knowledge on a molecular level of how cAMP binding is translated into protein action was limited so far. For a long time structural information was available only for cAMP-bound (i.e. active) cAMP-binding domains, but information of cAMP free (i.e. inactive) cAMP-binding domains was missing. We determined the cAMP free crystal structure of the regulatory region of Epac2 containing two cAMP-binding domains. The high conservation of both sequence and structure between Epac and PKA allows to compare these structures as representatives of the ligand bound and the ligand free state of cAMP-binding domains in general. From these analyses a general model was developed of how cAMP-binding is sensed by cAMP-binding domains. A relative small rearrangement of residues directly involved in cAMP binding is transduced to a reorientation of the C-terminal helical part of the cAMP-binding domain. The helices of the different classes of cAMP-regulated proteins have common features related to the rearrangements induced by cAMP and unique features to allow communication to the respective remote parts of the protein. By adaptation of the C-terminal helix to the individual requirements in the context of the regulated protein class, the cAMP-binding domain can be used as an universal switch unit. The critical importance of this helix for the regulation of PKA, CAP and cyclic nucleotide gated ion channels is described in the literature and was shown for Epac by various approaches in this thesis. In Epac the helix forms an interface interacting with the catalytic region. This interaction blocks the catalytic centre in the absence of cAMP. This binding and therefore the blockage is terminated after binding of cAMP. To complete the structural investigation, extensive biochemical analysis was undertaken. This confirmed already mentioned the importance of the C-terminal helix and corroborated the mechanism suggested for cAMP sensing. In addition, the requirements for productive cAMP-binding and activation were characterised. Thus, the action of Epac specific activators such as 8-pCPT-2-O-Me-cAMP can be explained. This analogue is able to activate Epac with higher efficiency than normal cAMP, but can neither activate nor inhibit PKA. These studies showed also that it is in principle possible to inhibit Epac with certain cAMP analogues. Based on these findings it should be possible to develop Epac specific inhibitors. This will allow the specific investigation of Epac mediated effects

The simulation of ionospheric conditions for space vehicles

Plasma wind tunnel to simulate ionospheric conditions for space vehicle

Rapid distortion theory for differential diffusion

Rapid distortion theory (RDT) is used to examine differential diffusion of active and passive scalars in unsheared, initially isotropic turbulence. RDT is well suited to study differential diffusion because it applies to strongly stratified flows with weak turbulence—that is, the conditions under which differential diffusion occurs. The theory reproduces several key features of the evolution of scalar fluxes and scalar flux spectra observed in direct numerical simulations (DNS). Predictions of the diffusivity ratio match laboratory results well when a parameter of the theory is related to a parameter of the experiments. RDT also allows parameters such as molecular diffusivities to be varied over a wider range than DNS can currently reach. RDT may prove to be a useful tool for computing mixing in weakly turbulent parts of the stratified ocean interior and possibly for parameterizing subgrid scale mixing in general circulation models

Modeling and Analysis of Power Processing Systems

The feasibility of formulating a methodology for the modeling and analysis of aerospace electrical power processing systems is investigated. It is shown that a digital computer may be used in an interactive mode for the design, modeling, analysis, and comparison of power processing systems

Multi-kilowatt modularized spacecraft power processing system development

A review of existing information pertaining to spacecraft power processing systems and equipment was accomplished with a view towards applicability to the modularization of multi-kilowatt power processors. Power requirements for future spacecraft were determined from the NASA mission model-shuttle systems payload data study which provided the limits for modular power equipment capabilities. Three power processing systems were compared to evaluation criteria to select the system best suited for modularity. The shunt regulated direct energy transfer system was selected by this analysis for a conceptual design effort which produced equipment specifications, schematics, envelope drawings, and power module configurations

Identification of a Novel, Small Molecule Partial Agonist for the Cyclic AMP Sensor, EPAC1

Screening of a carefully selected library of 5,195 small molecules identified 34 hit compounds that interact with the regulatory cyclic nucleotide-binding domain (CNB) of the cAMP sensor, EPAC1. Two of these hits (I942 and I178) were selected for their robust and reproducible inhibitory effects within the primary screening assay. Follow-up characterisation by ligand observed nuclear magnetic resonance (NMR) revealed direct interaction of I942 and I178 with EPAC1 and EPAC2-CNBs in vitro. Moreover, in vitro guanine nucleotide exchange factor (GEF) assays revealed that I942 and, to a lesser extent, I178 had partial agonist properties towards EPAC1, leading to activation of EPAC1, in the absence of cAMP, and inhibition of GEF activity in the presence of cAMP. In contrast, there was very little agonist action of I942 towards EPAC2 or protein kinase A (PKA). To our knowledge, this is the first observation of non-cyclic-nucleotide small molecules with agonist properties towards EPAC1. Furthermore, the isoform selective agonist nature of these compounds highlights the potential for the development of small molecule tools that selectively up-regulate EPAC1 activity

Buoyancy generated turbulence in stably stratified flow with shear

The energy evolution in buoyancy-generated turbulence subjected to shear depends on the gradient Richardson number Ri and the stratification number St, which is a ratio of the time scale of the initial buoyancy fluctuations to the time scale of the mean stratification. During an initial period, the flow state evolves as in the unsheared case. After this period, shear generates fluctuating velocity components for St=0.25, but it depletes the fluctuating vertical velocity component and temperature variance faster than in the unsheared case for St=4. Weak shear causes the kinetic and total energy to decrease faster than in the unsheared case, whereas strong shear adds more energy in comparison with the unsheared case. Energy increased with time in only one case considered (St=0.1 and Ri=0.04). When St\u3e1, the nonlinearity of the flow does not become significant even when Ri is small. Thus, results from rapid distortion theory and direct numerical simulation compare well. In particular, the theory reproduces trends in the energy evolution for St\u3e1

The Rap1 Guanine Nucleotide Exchange Factor C3G Is Required for Preservation of Larval Muscle Integrity in Drosophila melanogaster

C3G is a guanine nucleotide exchange factor (GEF) and modulator of small G-protein activity, which primarily acts on members of the Rap GTPase subfamily. Via promotion of the active GTP bound conformation of target GTPases, C3G has been implicated in the regulation of multiple cellular and developmental events including proliferation, differentiation and apoptosis. The Drosophila C3G orthologue exhibits a domain organization similar to that of vertebrate C3G. Through deletion of the C3G locus, we have observed that loss of C3G causes semi-lethality, and that escaping adult flies are characterized by a reduction in lifespan and general fitness. In situ hybridization reveals C3G expression in the developing embryonic somatic and visceral muscles, and indeed analysis of C3G mutants suggests essential functions of C3G for normal body wall muscle development during larval stages. C3G mutants display abnormal muscle morphology and attachment, as well as failure to properly localize βPS integrins to muscle attachment sites. Moreover, we show that C3G stimulates guanine nucleotide exchange on Drosophila Rap GTPases in vitro. Taken together, we conclude that Drosophila C3G is a Rap1-specific GEF with important functions in maintaining muscle integrity during larval stages

De Novo Heterozygous POLR2A Variants Cause a Neurodevelopmental Syndrome with Profound Infantile-Onset Hypotonia

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