Allosteric modulation by sodium ions and amilorides of G protein-coupled receptors : a closer look at the sodium ion site of the adenosine A2a receptor and development of a mass spectrometry ligand binding assay for adenosine A1 and A2a receptors

The main theme of this thesis, allosteric modulation effectuated through the sodium ion site of GPCRs, is inspired by the important role that this site appears to play in GPCR signaling. As sodium ions are abundant under physiological conditions they may affect GPCR signaling considerably. Receptor activation causes a substantial rearrangement of the sodium ion site, suggesting an important role in this process. Chapter 2 reviews the current knowledge on allosteric modulation of amiloride and its derivatives binding to the sodium ion site of Class A GPCRs. Chapters 3 to 5 follow-up on the recent crystal structure of the adenosine A2A receptor with a sodium ion bound. Chapters 3 and 4 complement the crystal structure with additional results from combined biochemistry, biophysical, molecular dynamics, and mutational studies. Chapter 5 describes the synthesis of novel amiloride derivatives that bind in the sodium ion site but also protrude into the orthosteric binding site. In Chapters 3 to 5, radio-labeled ligands were used to quantify ligand binding to the receptor, and Chapter 6 describes an alternative approach towards ligand binding assays. Instead of using a radio-label, mass spectrometry was used to quantify binding of an unlabeled ligand to adenosine A1 and A2A receptors.  Medicinal Chemistr

An Analysis Pathway for the Quantitative Evaluation of Public Transport Systems

We consider the problem of evaluating quantitative service-level agreements in public services such as transportation systems. We describe the integration of quantitative analysis tools for data fitting, model generation, simulation, and statistical model-checking, creating an analysis pathway leading from system measurement data to verification results. We apply our pathway to the problem of determining whether public bus systems are delivering an appropriate quality of service as required by regulators. We exercise the pathway on service data obtained from Lothian Buses about the arrival and departure times of their buses on key bus routes through the city of Edinburgh. Although we include only that example in the present paper, our methods are sufficiently general to apply to other transport systems and other cities

Mass spectrometry-based ligand binding assays on adenosine A(1) and A(2A) receptors

Conventional methods to measure ligand-receptor binding parameters typically require radiolabeled ligands as probes. Despite the robustness of radioligand binding assays, they carry inherent disadvantages in terms of safety precautions, expensive synthesis, special lab requirements, and waste disposal. Mass spectrometry (MS) is a method that can selectively detect ligands without the need of a label. The sensitivity of MS equipment increases progressively, and currently, it is possible to detect low ligand quantities that are usually found in ligand binding assays. We developed a label-free MS ligand binding (MS binding) assay on the adenosine A(1) and A(2A) receptors (A(1)AR and A(2A)AR), which are well-characterized members of the class A G protein-coupled receptor (GPCR) family. Radioligand binding assays for both receptors are well established, and ample data is available to compare and evaluate the performance of an MS binding assay. 1,3-Dipropyl-8-cyclopentyl-xanthine (DPCPX) and 4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol (ZM-241,385) are high-affinity ligands selective for the A(1)AR and A(2A)AR, respectively. To proof the feasibility of MS binding on the A(1)AR and A(2A)AR, we first developed an MS detection method for unlabeled DPCPX and ZM-241,385. To serve as internal standards, both compounds were also deuterium-labeled. Subsequently, we investigated whether the two unlabeled compounds could substitute for their radiolabeled counterparts as marker ligands in binding experiments, including saturation, displacement, dissociation, and competition association assays. Furthermore, we investigated the accuracy of these assays if the use of internal standards was excluded. The results demonstrate the feasibility of the MS binding assay, even in the absence of a deuterium-labeled internal standard, and provide great promise for the further development of label-free assays based on MS for other GPCRs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11302-015-9477-0) contains supplementary material, which is available to authorized users

Towards a logic for performance and mobility

Klaim is an experimental language designed for modeling and programming distributed systems composed of mobile components where distribution awareness and dynamic system architecture configuration are key issues. StocKlaim [R. De Nicola, D. Latella, and M. Massink. Formal modeling and quantitative analysis of KLAIM-based mobile systems. In ACM Symposium on Applied Computing (SAC). ACM Press, 2005. Also available as Technical Report 2004-TR-25; CNR/ISTI, 2004] is a Markovian extension of the core subset of Klaim which includes process distribution, process mobility, asynchronous communication, and site creation. In this paper, MoSL, a temporal logic for StocKlaim is proposed which addresses and integrates the issues of distribution awareness and mobility and those concerning stochastic behaviour of systems. The satisfiability relation is formally defined over labelled Markov chains. A large fragment of the proposed logic can be translated to action-based CSL for which efficient model-checkers exist. This way, such model-checkers can be used for the verification of StocKlaim models against MoSL properties. An example application is provided in the present paper

Allosteric modulation of G protein-coupled receptors by amiloride and its derivatives. Perspectives for drug discovery?

The function of G protein-coupled receptors (GPCRs) can be modulated by compounds that bind to other sites than the endogenous orthosteric binding site, so-called allosteric sites. Structure elucidation of a number of GPCRs has revealed the presence of a sodium ion bound in a conserved allosteric site. The small molecule amiloride and analogs thereof have been proposed to bind in this same sodium ion site. Hence, this review seeks to summarize and reflect on the current knowledge of allosteric effects by amiloride and its analogs on GPCRs. Amiloride is known to modulate adenosine, adrenergic, dopamine, chemokine, muscarinic, serotonin, gonadotropin-releasing hormone, GABA(B), and taste receptors. Amiloride analogs with lipophilic substituents tend to be more potent modulators than amiloride itself. Adenosine, alpha-adrenergic and dopamine receptors are most strongly modulated by amiloride analogs. In addition, for a few GPCRs, more than one binding site for amiloride has been postulated. Interestingly, the nature of the allosteric effect of amiloride and derivatives varies considerably between GPCRs, with both negative and positive allosteric modulation occurring. Since the sodium ion binding site is strongly conserved among class A GPCRs it is to be expected that amiloride also binds to class A GPCRs not evaluated yet. Investigating this typical amiloride-GPCR interaction further may yield general insight in the allosteric mechanisms of GPCR ligand binding and function, and possibly provide new opportunities for drug discovery.Medicinal Chemistr

Automated Verification of Quantum Protocols using MCMAS

We present a methodology for the automated verification of quantum protocols using MCMAS, a symbolic model checker for multi-agent systems The method is based on the logical framework developed by D'Hondt and Panangaden for investigating epistemic and temporal properties, built on the model for Distributed Measurement-based Quantum Computation (DMC), an extension of the Measurement Calculus to distributed quantum systems. We describe the translation map from DMC to interpreted systems, the typical formalism for reasoning about time and knowledge in multi-agent systems. Then, we introduce dmc2ispl, a compiler into the input language of the MCMAS model checker. We demonstrate the technique by verifying the Quantum Teleportation Protocol, and discuss the performance of the tool.Comment: In Proceedings QAPL 2012, arXiv:1207.055

A Process Algebraic Fluid Flow Model of Emergency Egress

Abstract-Pervasive environments offer an increasing number of services to a large number of people moving within these environments including timely information about where to go and when. People using these services interact with the system but they are also meeting other people and performing other activities as relevant opportunities arise. The design of such systems and the analysis of collective dynamic behaviour of people within them is a challenging problem. In previous work we have successfully explored a scalable analysis of stochastic process algebraic models of smart signage systems. In this paper we focus on the validation of a representative example of this class of models in the context of emergency egress. This context has the advantage that there is detailed data available from studies with alternative analysis methods. A second aim is to show how realistic human behaviour, often observed in emergency egress, can be embedded in the model and how the effect of this behaviour on building evacuation can be analysed in an efficient and scalable way

On-the-fly Uniformization of Time-Inhomogeneous Infinite Markov Population Models

This paper presents an on-the-fly uniformization technique for the analysis of time-inhomogeneous Markov population models. This technique is applicable to models with infinite state spaces and unbounded rates, which are, for instance, encountered in the realm of biochemical reaction networks. To deal with the infinite state space, we dynamically maintain a finite subset of the states where most of the probability mass is located. This approach yields an underapproximation of the original, infinite system. We present experimental results to show the applicability of our technique

QuantUM: Quantitative Safety Analysis of UML Models

When developing a safety-critical system it is essential to obtain an assessment of different design alternatives. In particular, an early safety assessment of the architectural design of a system is desirable. In spite of the plethora of available formal quantitative analysis methods it is still difficult for software and system architects to integrate these techniques into their every day work. This is mainly due to the lack of methods that can be directly applied to architecture level models, for instance given as UML diagrams. Also, it is necessary that the description methods used do not require a profound knowledge of formal methods. Our approach bridges this gap and improves the integration of quantitative safety analysis methods into the development process. All inputs of the analysis are specified at the level of a UML model. This model is then automatically translated into the analysis model, and the results of the analysis are consequently represented on the level of the UML model. Thus the analysis model and the formal methods used during the analysis are hidden from the user. We illustrate the usefulness of our approach using an industrial strength case study.Comment: In Proceedings QAPL 2011, arXiv:1107.074

Bisimulation of Labeled State-to-Function Transition Systems of Stochastic Process Languages

Labeled state-to-function transition systems, FuTS for short, admit multiple transition schemes from states to functions of finite support over general semirings. As such they constitute a convenient modeling instrument to deal with stochastic process languages. In this paper, the notion of bisimulation induced by a FuTS is proposed and a correspondence result is proven stating that FuTS-bisimulation coincides with the behavioral equivalence of the associated functor. As generic examples, the concrete existing equivalences for the core of the process algebras ACP, PEPA and IMC are related to the bisimulation of specific FuTS, providing via the correspondence result coalgebraic justification of the equivalences of these calculi.Comment: In Proceedings ACCAT 2012, arXiv:1208.430