Automatic Verification of Parametric Specifications with Complex Topologies

The focus of this paper is on reducing the complexity in verification by exploiting modularity at various levels: in specification, in verification, and structurally. \begin{itemize} \item For specifications, we use the modular language CSP-OZ-DC, which allows us to decouple verification tasks concerning data from those concerning durations. \item At the verification level, we exploit modularity in theorem proving for rich data structures and use this for invariant checking. \item At the structural level, we analyze possibilities for modular verification of systems consisting of various components which interact. \end{itemize} We illustrate these ideas by automatically verifying safety properties of a case study from the European Train Control System standard, which extends previous examples by comprising a complex track topology with lists of track segments and trains with different routes

Molecular studies on iron-sulfur proteins in Desulfovibrio

Desulfovibrio vulgaris (Hildenborough) . The organism described in this thesis, is an anaerobic gram-negative sulfate reducing bacterium (SRB). Its natural environments are the anaerobic sediments in lower levels of lakes and pools. This habitat is rich in sulfate that is used as terminal electron-acceptor by the organism and by performing this, D. vulgaris contributes to the important sulfur-cycle in nature. D. vulgaris can both utilize lactate (by anaerobic oxidation) and molecular hydrogen as energy source. The oxidation of lactate to acetate and C0 2 occurs in the cytoplasm or at the cytoplasmic membrane and results in the production of ATP and the release of protons and electrons. When D. vulgaris uses molecular hydrogen as substrate, the oxidation of the hydrogen occurs at the periplasmic side of the inner membrane. This creates a proton motive force that drives ATP synthesis.Periplasmic Fe-hydrogenase . Molecular hydrogen has been shown to play an important role in both energy-evolving systems described above. So far, three hydrogenases, catalyzing the reversible H 2 oxidation and reduction of protons have been identified in D. vulgaris (Hildenborough). The precise physiological function of each of these hydrogenases remains unclear. Two of these enzymes are localized in the cytoplasmic membrane and contain nickel in addition to iron-sulfur clusters as cofactor. The third enzyme contains only iron-sulfur clusters as cofactors and resides in the periplasmic space of the bacterium. This enzyme exhibits one of the highest catalytic activities ever described for hydrogenases. It occurs as a heterodimer that is composed of a large cc subunit (46 kDa) and a small 0 subunit (10 kDa). Only the small subunit is translated as a precursor (13 kDa) with a cleavable signal sequence for export that is probably involved in the export of both hydrogenase subunits across the cytoplasmic membrane. The catalytically active enzyme contains three iron-sulfur clusters as cofactors. Two of them are typical ferredoxin-like [4Fe-4S] clusters (F-clusters) involved in electron transport. The third (H-cluster) cluster contains six iron and sulfide ions coordinated in an unknown structure and is part of the catalytic center of the enzyme.Ile gene encoding the Fe-hydrogenase was the first hydrogenase gene that was isolated and expressed in E. coli . From these expression studies it became apparent that only very small amounts of αand βsubunits were assembled into an αβdimer and transported across the membrane. Also the iron-sulfur cluster incorporation was incomplete in the recombinant enzyme. The enzyme contained sub-stoichiometric amounts of F-clusters, while the H-cluster was not incorporated at all. These results indicated that the assembly and export of hydrogenase generating a catalytically active enzyme, are not spontaneously occurring processes, but involve specific helper components, as has been shown for other enzymes with redox-active metal clusters (reviewed in Chapter 1 ).Studies regarding the biosynthesis of Fe-hydrogenase: the hydC gene . As genes serving a single pathway are often clustered in the genome, the identification of genes encoding these additional activating components, was started by the isolation of large DNA fragments surrounding the structural hydrogenase genes. Surprisingly, one of the large isolated DNA fragments contained a gene, hydC ( Chapter 2 ) with homology in primary structure to the αand βsubunits of the Fe-hydrogenase.HydC has a high degree of similarity with both the αsubunit of the Fe-hydrogenase (in its central part) and with the βsubunit, minus the leader peptide (in its C-terminal part). Analogous to the FeMo co-factor insertion in nitrogenase component 1 which involves genes ( nifEN ) with high similarities to the structural subunits, it was speculated that the hydC gene might code for a helper protein that is involved in the processing of the hydrogenase. 'Me primary structure of hydC contains a N-terminus with no homology with one of the hydrogenase subunits. Subsequently, it was found that this N-terminal segment has homology with mitochondrial NADH-ubiquinone reductase, with subunits of a NAD+-reducing NiFe-hydrogenase from Alcaligenes eutrophus and with the Fe-hydrogenase I from Clostridium pasteurianum ( Chapter 3 ). On the basis of what is known about iron-sulfur cluster contents of these three enzymes and the conservation of cysteine motifs in these proteins, it was suggested that these motifs coordinate [2Fe-2S] clusters. Unfortunately, the HydC protein could not be purified from D. vulgaris, because no growth conditions were found resulting in a sufficient production of HydC protein. This hampered a furtherbiochemical and spectroscopical characterization of the protein. On the other hand, the high degree of homology with the C. pasteurianum Fe-hydrogenase, strongly suggests that HydC is a second alternative Fe-hydrogenase and not a helper protein involved in the processing of Fe-hydrogenase.Numerous attempts have been made to exchange the genes for the subunits of the Fe- hydrogenase and the hydC gene in the D. vulgaris genome with inactivated, interrupted copies of the genes. This type of marker exchange experiments would also be very useful for the identification of genes involved in biosynthesis of hydrogenase. One of the requirements for marker exchange is a system for the introduction of plasmids into Desulfovibrio. Such a plasmid transfer system has been developed, but subsequent experiments to apply it for marker exchange have been unsuccessful.The prismane protein . The inability to design a system for marker-exchange mutagenesis in Desulfovibrio blocked further study of the biosynthesis of the Fe-hydrogenase Therefore, investigations on another protein from D. vulgaris, the prismane protein, were started that are described in the second part of this thesis. As mentioned earlier, some indications were obtained that the H-cluster of Fe-hydrogenase is a [6Fe-6S] cluster.Stronger indications for the existence of such "supercluster" were obtained by Hagen and Pierik in our department for another iron-sulfur containing protein from D. vulgaris, the prismane protein. They isolated a protein containing six irons and sulfide ions coordinated in only one [6Fe-6S] cluster. The putative [6Fe-6S] prismane cluster occurs in four different redox- states: the three-electron reduced state [6Fe-6S] 3+(S=1/2), [6Fe-6S] 4+(S = even), [6Fe-6S] 5+(S = 1/2 and S = 9/2) and the fully oxidized [6Fe-6S] 6+(S=0) that shows no EPR spectrum. Chapter 4 and 5 describe the isolation of the genes for the prismane proteins from D. vulgaris (Hildenborough) and D. desulfuricans (ATCC 27774) and the determination of the amino acid sequence. Both proteins are highly conserved (66% identical residues), except for a 100 residues segment (residue 50-150). Besides this, both proteins contain typical cysteine motifs at the N-terminus. These motifs have also been found in the sequence of the a subunit of CO dehydrogenase from Methanothrix soehngenii and, in a slightly modified form, in that of CO dehydrogenase from Clostridium thermoaceticum. Also for the CO dehydrogenase from M. soehngenii a supercluster has been proposed. Therefore, it is tempting to speculate about the involvement of this motif in the ligation of the [6Fe-6S] prismane cluster.Prismane protein is produced only in small amounts in D. vulgaris. Since large amounts of purified prismane protein are required for X-ray crystallography and Mössbauer studies, efforts were made for overproduction of the protein ( Chapter 6 ). In a first attempt, the protein was overproduced in E. coli. In this host, a high production of prismane protein was obtained, but no iron-sulfur cluster was incorporated into the protein. The overproduced protein occurred as large insoluble protein-complexes. A second attempt for the overproduction of prismane protein was performed in D. vulgaris by using the aforementioned cloning system. A 25-fold overproduction of prismane protein was obtained by the introduction of extra copies of the gene encoding the prismane protein on a stable plasmid. Biochemical and spectroscopic properties of the protein overproduced in D. vulgaris were shown to be identical to wild-type prismane with one exception: in the as-isolated, oneelectron-reduced state the protein shows EPR signals belonging to a second (S=1/2), spin system that was not observed in the wild-type protein. These additional signals were also described for the wild-type prismane protein purified from D. desulfuricans by Moura and co-workers in Portugal. EPR signals belonging to this second (S=1/2), spin system disappear upon reduction/re-oxidation of the overproduced prismane protein, indicating that this spin system represents a different magnetic form of the [6Fe-6S] cluster. There are no indications for a second cluster as proposed by Moura et al. Determination of the three dimensional structure by X-ray crystallography and further Mössbauer spectroscopy of the overproduced prismane protein are subject for further study in our department and will ultimately lead to insight into the structure of this novel iron-sulfur cofactor

On Deciding Local Theory Extensions via E-matching

Satisfiability Modulo Theories (SMT) solvers incorporate decision procedures for theories of data types that commonly occur in software. This makes them important tools for automating verification problems. A limitation frequently encountered is that verification problems are often not fully expressible in the theories supported natively by the solvers. Many solvers allow the specification of application-specific theories as quantified axioms, but their handling is incomplete outside of narrow special cases. In this work, we show how SMT solvers can be used to obtain complete decision procedures for local theory extensions, an important class of theories that are decidable using finite instantiation of axioms. We present an algorithm that uses E-matching to generate instances incrementally during the search, significantly reducing the number of generated instances compared to eager instantiation strategies. We have used two SMT solvers to implement this algorithm and conducted an extensive experimental evaluation on benchmarks derived from verification conditions for heap-manipulating programs. We believe that our results are of interest to both the users of SMT solvers as well as their developers

Efficient Interpolation for the Theory of Arrays

Existing techniques for Craig interpolation for the quantifier-free fragment of the theory of arrays are inefficient for computing sequence and tree interpolants: the solver needs to run for every partitioning $(A, B)$ of the interpolation problem to avoid creating $AB$-mixed terms. We present a new approach using Proof Tree Preserving Interpolation and an array solver based on Weak Equivalence on Arrays. We give an interpolation algorithm for the lemmas produced by the array solver. The computed interpolants have worst-case exponential size for extensionality lemmas and worst-case quadratic size otherwise. We show that these bounds are strict in the sense that there are lemmas with no smaller interpolants. We implemented the algorithm and show that the produced interpolants are useful to prove memory safety for C programs.Comment: long version of the paper at IJCAR 201

The structures and biological activities of the lipo-oligosaccharide nodulation signals produced by type I and II strains of Bradyrhizobium japonicum

Bradyrhizobium japonicum produces lipo-oligosaccharide signal molecules that induce deformation of root hairs and meristematic activity on soybeans. B. japonicum USDA135 (a Type I strain) produces modified chitin pentasaccharide molecules with either a terminal N-C16:0- or N-C18:1-glucosamine with and without an O-acetyl group at C-6 and with 2-O-methylfucose linked to C-6 of the reducing N-acetylglucosamine. An additional molecule has N-C16:1-glucosamine and no O-acetyl group. All of these molecules cause root hair deformation on Vicia sativa and Glycine soja. The C18:1-containing molecules were tested and found to induce meristem formation on G. soja. USDA61 (a Type II strain) produces eight additional molecules. Five have a carbamoyl group on the terminal N-acylglucosamine. Six have chitin tetrasaccharide backbones. Three have a terminal N-acyl-N-methylglucosaminosyl residue. In four molecules, the reducing-end N-acetylglucosamine is glycosidically linked to glycerol and has a branching fucosyl, rather than a 2-O-methylfucosyl, residue. One molecule has a terminal N-acylglucosamine that has both acetyl and carbamoyl groups (one each).Plant science

Host Cell Egress and Invasion Induce Marked Relocations of Glycolytic Enzymes in Toxoplasma gondii Tachyzoites

Apicomplexan parasites are dependent on an F-actin and myosin-based motility system for their invasion into and escape from animal host cells, as well as for their general motility. In Toxoplasma gondii and Plasmodium species, the actin filaments and myosin motor required for this process are located in a narrow space between the parasite plasma membrane and the underlying inner membrane complex, a set of flattened cisternae that covers most the cytoplasmic face of the plasma membrane. Here we show that the energy required for Toxoplasma motility is derived mostly, if not entirely, from glycolysis and lactic acid production. We also demonstrate that the glycolytic enzymes of Toxoplasma tachyzoites undergo a striking relocation from the parasites' cytoplasm to their pellicles upon Toxoplasma egress from host cells. Specifically, it appears that the glycolytic enzymes are translocated to the cytoplasmic face of the inner membrane complex as well as to the space between the plasma membrane and inner membrane complex. The glycolytic enzymes remain pellicle-associated during extended incubations of parasites in the extracellular milieu and do not revert to a cytoplasmic location until well after parasites have completed invasion of new host cells. Translocation of glycolytic enzymes to and from the Toxoplasma pellicle appears to occur in response to changes in extracellular [K+] experienced during egress and invasion, a signal that requires changes of [Ca2+]c in the parasite during egress. Enzyme translocation is, however, not dependent on either F-actin or intact microtubules. Our observations indicate that Toxoplasma gondii is capable of relocating its main source of energy between its cytoplasm and pellicle in response to exit from or entry into host cells. We propose that this ability allows Toxoplasma to optimize ATP delivery to those cellular processes that are most critical for survival outside host cells and those required for growth and replication of intracellular parasites

A Novel Family of Toxoplasma IMC Proteins Displays a Hierarchical Organization and Functions in Coordinating Parasite Division

Apicomplexans employ a peripheral membrane system called the inner membrane complex (IMC) for critical processes such as host cell invasion and daughter cell formation. We have identified a family of proteins that define novel sub-compartments of the Toxoplasma gondii IMC. These IMC Sub-compartment Proteins, ISP1, 2 and 3, are conserved throughout the Apicomplexa, but do not appear to be present outside the phylum. ISP1 localizes to the apical cap portion of the IMC, while ISP2 localizes to a central IMC region and ISP3 localizes to a central plus basal region of the complex. Targeting of all three ISPs is dependent upon N-terminal residues predicted for coordinated myristoylation and palmitoylation. Surprisingly, we show that disruption of ISP1 results in a dramatic relocalization of ISP2 and ISP3 to the apical cap. Although the N-terminal region of ISP1 is necessary and sufficient for apical cap targeting, exclusion of other family members requires the remaining C-terminal region of the protein. This gate-keeping function of ISP1 reveals an unprecedented mechanism of interactive and hierarchical targeting of proteins to establish these unique sub-compartments in the Toxoplasma IMC. Finally, we show that loss of ISP2 results in severe defects in daughter cell formation during endodyogeny, indicating a role for the ISP proteins in coordinating this unique process of Toxoplasma replication

Big data in daily manufacturing operations

Best Paper Award MSAM 2014 (Modeling and Analysis of Semiconductor Manufacturing), part of Wintersim Conference. Abstract Big data analytics is at the brink of changing the landscape in NXP Semiconductors Back End manufacturing operations. Numerous IT tools, implemented over the last decade, collect gigabytes of data daily, though the potential value of this data still remains to be explored. In this paper, the software tool called Heads Up is presented. Heads Up intelligently scans, filters, and explores the data with use of simulation. The software provides real-time relevant information, which is of high value in daily, as well as long term, production management. The software tool has been introduced at the NXP high volume manufacturing plant GuangDong China, where it is about to shift the paradigm on manufacturing operations