E. coli and Salmonella enterica have multiple c-di-GMP cyclases and phosphodiesterases. Absence of a specific phosphodiesterase YhjH impairs motility in both bacteria. yhjH mutants have elevated c-di-GMP levels and require YcgR, a c-di- GMP-binding protein, for motility inhibition. This study demonstrates that YcgR interacts with the flagellar switch-complex proteins FliG and FliM, with the primary interaction site located within FliM. Interaction of YcgR with these proteins induces a CCW motor bias and reduces the efficiency of torque generation, thus inhibiting both chemotaxis and the speed of movement. In collaboration with David Blair’s group at the University of Utah, we propose a "backstop brake" model showing how both effects of YcgR on the motor can result from an initial disruption of the FliM/FliG interface, followed by destabilization and disorganization of the FliG C-terminal domain, which interacts with the stator protein MotA. Support for this order of events i.e. induction of a CCW bias followed by reduction of torque, is provided for S. enterica motors. Data from single motor analysis show that E. coli and S. enterica motors have inherently different properties, but that YcgR is solely responsible for disruption of motor function in both bacteria. This study also finds that E. coli and S. enterica employ c-di-GMP in additional and different pathways to impede motility. Inhibition of motility and chemotaxis may represent a bacterial strategy to prepare for sedentary existence by disfavoring migration away from a substrate on which a biofilm is to be formed. / text

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The quality of fish products is indispensably linked to the freshness of the raw material modulated by appropriate manipulation and storage conditions, specially the storage temperature after catch. The purpose of the research presented in this thesis, which was largely conducted in the context of a research project funded by Italian Ministry of Agricultural, Food and Forestry Policies (MIPAAF), concerned the evaluation of the freshness of farmed and wild fish species, in relation to different storage conditions, under ice (0°C) or at refrigeration temperature (4°C). Several specimens of different species, bogue (Boops boops), red mullet (Mullus barbatus), sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax), during storage, under the different temperature conditions adopted, have been examined. The assessed control parameters were physical (texture, through the use of a dynamometer; visual quality using a computer vision system (CVS)), chemical (through footprint metabolomics 1H-NMR) and sensory (Quality Index Method (QIM). Microbiological determinations were also carried out on the species of hake (Merluccius merluccius). In general obtained results confirmed that the temperature of manipulation/conservation is a key factor in maintaining fish freshness. NMR spectroscopy showed to be able to quantify and evaluate the kinetics for unselected compounds during fish degradation, even a posteriori. This can be suitable for the development of new parameters related to quality and freshness. The development of physical methods, particularly the image analysis performed by computer vision system (CVS), for the evaluation of fish degradation, is very promising. Among CVS parameters, skin colour, presence and distribution of gill mucus, and eye shape modification evidenced a high sensibility for the estimation of fish quality loss, as a function of the adopted storage conditions. Particularly the eye concavity index detected on fish eye showed a high positive correlation with total QIM score. / La qualità dei prodotti ittici è indispensabilmente legata alla freschezza della materia prima modulata, dalla manipolazione appropriata dalle condizioni di conservazione, specialmente la temperatura di stoccaggio dopo cattura. Lo scopo della ricerca presentata in questa tesi, che è stata condotta in gran parte nel contesto di un progetto di ricerca finanziato dal Ministero Italiano delle Politiche Agricole, Alimentari e Forestali (MIPAAF), riguarda la valutazione della freschezza di specie ittiche d'allevamento e selvatiche in relazione alle diverse condizioni di stoccaggio (campioni conservati sotto ghiaccio (0°C) o a temperatura di refrigerazione (4°C)). Vari esemplari di specie diverse, quali boga (Boops boops), triglia di fango (Mullus barbatus), orata (Sparus aurata) e spigola (Dicentrarchus labrax)), sono stati esaminati durante lo stoccaggio nelle condizioni adottate. Come parametri di controllo sono state effettuate valutazioni di tipo fisico (texture, attraverso l'uso di un dinamometro; qualità visiva utilizzando un sistema di visione del computer (CVS)), chimico (attraverso (1H-NMR)) e sensoriale (attraverso il quality index method (QIM)). Sulla specie nasello (Merluccius merluccius) sono state effettuate anche determinazioni microbiologiche. L’applicazione di queste tecniche è stata finalizzata alla valutazione delle modificazioni qualitative del prodotto. In generale, i risultati hanno mostrato che la temperatura di manipolazione/conservazione è un fattore chiave per il mantenimento della freschezza del pesce. Spettroscopia NMR ha mostrato di essere in grado di quantificare e valutare la cinetica di composti selezionati durante la degradazione pesce, anche a posteriori. Questo può essere adatto per lo sviluppo di nuovi parametri relativi alla qualità e freschezza. Dalla ricerca è emerso come lo sviluppo di metodi fisici per la valutazione della freschezza del pesce, presenti un elevato potenziale per lo sviluppo di un indice qualitativo strumentale correlato sia con gli indici chimici che sensoriali, al fine di integrare/sostituire i metodi sensoriali attualmente utilizzati.

Inorganic phosphate (Pi) plays a critical role in many biological structures and processes. However, Pi typically occurs at low concentrations, particularly in marine environments. In comparison, naturally occurring organophosphonates, which are characterized by a stable carbon-phosphorus (CP) bond, are frequently present at higher concentrations. Accordingly, bacteria have evolved different mechanisms for cleaving the CP bond of organophosphonates to liberate Pi for metabolic use. Two prominent enzyme pathways for catabolic cleavage of a CP bond are examined in this thesis. The first, called CP-lyase, is encoded by the phn operon that consists of 14 genes (phnCDEFGHIJKLMNOP). CP-lyase has long been of interest for its ability to degrade a wide array of organophosphonates through a homolytic CP bond cleaving reaction. A soluble protein complex consisting of PhnGHIJK was isolated from E. coli, suggesting that protein-protein interactions are important for CP bond cleavage. Intermediates of organophosphonate catabolism by E. coli CP-lyase were also detected and isolated, including -D-ribosyl-1,2-cyclic phosphate and N-acetylated aminoalkylphosphonates, 2-N-acetamidoethylphosphonate and 5’-phospho--D-ribosyl-1’-alkylphosphonates. The former compound was shown to be converted by the phosphodiesterase PhnP to -D-ribosyl-1-phosphate. It was also shown that PhnO is an aminoalkylphosphonate N-acetyl transferase and that N-acetylation by this enzyme is necessary for CP bond cleavage of 1-aminoalklyphosphonates. These results demonstrated that in addition to forming protein complexes, CP-lyase also comprises a catabolic pathway, with ribosylation of organophosphonates playing a key part in setting up the CP bond cleaving reaction. The second pathway examined in this thesis is comprised of marine bacterial enzymes PhnY and PhnZ and is specific for 2-aminoethylphosphonate. PhnY was shown to be an -ketoglutarate / Fe(II) dependent dioxygenase that hydroxylates the -carbon of 2-aminoethylphosphonate to form (R)-2-amino-1-hydroxyethylphosphonate. PhnZ was shown to be a novel Fe(II) dependent oxygenase that converts (R)-2-amino-1-hydroxyethylphosphonate to glycine and Pi. Site directed mutagenesis, kinetic analysis, reactions with substrate analogues, and X-ray crystallography examined the roles of active site residues and the di-iron active site. Additionally, a unique induced-fit mechanism was discovered which appears to synchronize substrate binding with activation of molecular oxygen. Overall these results show that PhnZ represents a new mechanism for catabolic cleavage of a CP bond. / Thesis (Ph.D, Chemistry) -- Queen's University, 2013-09-13 16:24:17.261

Interaction protocols establish how different computational entities can interact with each other. The interaction can be finalized to the exchange of data, as in 'communication protocols', or can be oriented to achieve some result, as in 'application protocols'. Moreover, with the increasing complexity of modern distributed systems, protocols are used also to control such a complexity, and to ensure that the system as a whole evolves with certain features. However, the extensive use of protocols has raised some issues, from the language for specifying them to the several verification aspects. Computational Logic provides models, languages and tools that can be effectively adopted to address such issues: its declarative nature can be exploited for a protocol specification language, while its operational counterpart can be used to reason upon such specifications. In this thesis we propose a proof-theoretic framework, called SCIFF, together with its extensions. SCIFF is based on Abductive Logic Programming, and provides a formal specification language with a clear declarative semantics (based on abduction). The operational counterpart is given by a proof procedure, that allows to reason upon the specifications and to test the conformance of given interactions w.r.t. a defined protocol. Moreover, by suitably adapting the SCIFF Framework, we propose solutions for addressing (1) the protocol properties verification (g-SCIFF Framework), and (2) the a-priori conformance verification of peers w.r.t. the given protocol (AlLoWS Framework). We introduce also an agent based architecture, the SCIFF Agent Platform, where the same protocol specification can be used to program and to ease the implementation task of the interacting peers.

Advances in wireless networking and content delivery systems are enabling new challenging provisioning scenarios where a growing number of users access multimedia services, e.g., audio/video streaming, while moving among different points of attachment to the Internet, possibly with different connectivity technologies, e.g., Wi-Fi, Bluetooth, and cellular 3G. That calls for novel middlewares capable of dynamically personalizing service provisioning to the characteristics of client environments, in particular to discontinuities in wireless resource availability due to handoffs. This dissertation proposes a novel middleware solution, called MUM, that performs effective and context-aware handoff management to transparently avoid service interruptions during both horizontal and vertical handoffs. To achieve the goal, MUM exploits the full visibility of wireless connections available in client localities and their handoff implementations (handoff awareness), of service quality requirements and handoff-related quality degradations (QoS awareness), and of network topology and resources available in current/future localities (location awareness). The design and implementation of the all main MUM components along with extensive on the field trials of the realized middleware architecture confirmed the validity of the proposed full context-aware handoff management approach. In particular, the reported experimental results demonstrate that MUM can effectively maintain service continuity for a wide range of different multimedia services by exploiting handoff prediction mechanisms, adaptive buffering and pre-fetching techniques, and proactive re-addressing/re-binding.