Thesis advisor: Jianmin Gao / Phosphatidylinositol-specific phospholipase C (PI-PLC) cleaves the substrate phosphatidylinositol through two steps: the first step occurs in the interface between lipid and solution, while the second step only takes place in water soluble environment. For interfacial catalysis, the enzyme should bind to the lipid surface first before engaging its substrate, therefore interfacial kinetics include both interfacial binding and an interfacial catalytic step. The Bacillus thuringiensis PI-PLC is activated by binding to zwitterionic surfaces; phosphatidylcholine (PC) and two tryptophan residues (Trp47 in the two-turn helix B and Trp242 in a disordered loop) at the rim of the barrel structure, in particular, are critical for this interaction. The helix B region in PI-PLC orients the side chains of Ile43 and Trp47 so that they form a hydrophobic protrusion from the protein surface that likely facilitates initial membrane binding. An earlier crystal structure of the dimeric W47A/W242A mutant, which is unable to bind to PC, showed that the helix B region was reorganized into an extended loop. Whether this conformational change occurred in the wild type PI-PLC was tested with a series of mutations targeting helix B residues and surrounding regions. Results strongly suggest that, while hydrophobic groups and presumably an intact helix B are critical for the initial binding of PI-PLC to membranes, disruption of helix B to allow enzyme dimerization is likely to play a role in the activated PI-PLC conformation. Besides the helix B residues, a number of hydrophobic residues along the rim of the <em>f</em>Ñ<em>f</em>Ò-barrel and close to both helix B and the active site were also altered to assess their contribution to membrane binding and kinetic activation. Results showed that Tyr86 and Tyr88, but not Tyr118, contribute to the protein binding to PC vesicles. These residues are capable of cation-<em>f</em>à interactions with the choline headgroup of the phospholipid PC. Although mammalian PLC<em>f</em>Ô1 is a complex multidomain protein, the catalytic domain resembles the bacterial PI-PLC enzymes. Little work has been done to characterize the extent to which this domain contributes to membrane binding. A mutated protein that removes the very anionic X/Y linker region that covers the active site was constructed. The interfacial binding and the corresponding enzyme activity of this mutant against WT were measured in both micelles and large unilamellar vesicles. The results showed at <em>f</em>ÝM protein concentration there was no large difference between the PLC<em>f</em>Ô1 and the deletion mutant in terms of vesicle binding. However, the deletion mutant showed much higher membrane binding affinity at nM concentrations. These results shed some light on the activation or inhibition role of the catalytic domain and pointed to a possible direction of future studies, for example examining specific mutant enzymes in the interfacial loop region. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Mary F. Roberts / Phosphatidylinositol-specific phospholipase C (PI-PLC) from B. thuringiensis is activated by phosphatidylcholine (PC) surfaces for both phosphatidylinositol (PI) cleavage to inositol 1,2-(cyclic)-phosphate (cIP) and subsequent hydrolysis of cIP to inositol-1-phosphate. These enzyme kinetics strongly suggest that this PI-PLC has two discrete binding sites for phospholipids - the active site binding PI (or substrate competitors) and an activator site specific for PC. However, it is difficult to determine the orientation and conformation of peripheral membrane proteins when docked to target membranes, let alone where sites for these might be on the protein. In this thesis, various biophysical techniques were applied to this bacterial PI-PLC to obtain structural information in the absence and presence of membranes to characterize specific conformational changes that occur when the protein binds to activating membranes. The crystal structures of an interfacially impaired double mutant of PI-PLC, W47A/W242A, was solved and showed the protein as a homodimer. The major interactions came from four clustered surface tyrosine residues from each monomer. This structure suggested the possibility of PI-PLC dimerization on membrane surfaces as part of the mechanism for interfacial activation. Mutations of these tyrosines showed a loss of activity and membrane binding. Crystal structures of these mutant proteins showed no significant change in the proteins, consistent with either disruption of a dimerization interface of a specific PC binding motif. FRET was used to try and monitor oligomerization of PI-PLC, derivatized on a cysteine introduced at residue 280 (W280C) with either a donor or acceptor fluorophore, on vesicle surfaces. The results suggested some specific aggregation could occur on very PC-rich surfaces but not on phospholipid vesicles with at least 50 mol% anionic phospholipids, strongly suggesting that a stable dimer was not forming when the enzyme was bound to vesicles mimicking conditions where enzyme specific activity is high. If dimerization occurs on surfaces, it must be transient. To examine which portions of the PI-PLC are interacting with membrane and to further explore if there is any evidence for PI-PLC dimerization on membrane surface, deuterium exchange coupled by mass spectrometry experiments were carried out with wild type PI-PLC, W47A/W242A and a covalent dimer formed from W242C that is more active than wild type enzyme. Results showed (i) a stable short helix B (containing an exposed tryptophan thought to insert into membranes) in wild type PI-PLC and its complete destabilization in W47A/W242C, (ii) a flexible surface loop (containing another tryptophan thought to partition into the membrane) that became protected when the protein was bound to vesicles, and (iii) reduced deuterium exchange for the peptide containing the tyrosines that either mediate transient dimerization or form a PC binding site.. These observations modify how we envision the protein anchoring to substrate-containing membranes. / Thesis (PhD) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Les systèmes ouverts reconfigurables sont aujourd'hui omniprésents dans le paysage informatique : réseaux mobiles, calculs et données dans “le nuage”, etc. Une particularité de ces systèmes est que leur topologie de communication évolue dynamiquement - nous parlerons de reconfiguration - en conséquence d'activités concurrentes internes ou externes. Les systèmes de transitions étiquetées pour les systèmes ouverts permettent de prendre en considération l'environnement extérieur de façon implicite.Les systèmes ouverts reconfigurables sont souvent modélisés par des formalismes inspirés ou dérivés du π-calcul. Le passage de nom permet de modéliser la dynamique des topologies de communication. Dans cette thèse, nous introduisons les π-graphs, une variante du π-calcul qui possède, entre autre, une interprétation graphique naturelle. De plus, le formalisme a été conçu pour servir de langage intermédiaire entre le π-calcul abstrait et des formalismes plus concrets, en particulier dans la famille des réseaux de Petri de haut-niveau.Nous proposons tout d'abord une traduction formelle et prouvée des π-graphes vers des réseaux de Petri de haut niveau supportés par des outils de modélisation et de vérification courants. Nous montrons que cette traduction peut-être élevée au rang d'isomorphisme entre les deux formalismes. Ainsi, les outils prototypes que nous avons développés dans le cadre des π-graphes peuvent travailler de concert avec des outils plus stables et plus généraux basés sur les réseaux de Petri.En se basant sur cette traduction bi-directionnelle, nous développons une extension de la logique temporelle linéaire (LTL) - la logique des systèmes ouverts reconfigurables - permettant de spécifier des propriétés portant sur la dynamique d'évolution de la topologie de communication dans le cadre d'environnements ouverts. Les propositions atomiques de cette logique caractérisent précisément les propriétés d'état des π-graphs.Un prototype d'outil a été développé dans le cadre de cette thèse pour valider expérimentalement l'approche proposée. Cet outil fournit un simulateur pour les modèles exprimés dans le formalisme des π-graphes. Ces modèles peuvent être compilés en réseaux de Petri de haut niveau et manipulés dans le cadre de l'outil SNAKES. Enfin, nous proposons une traduction de la logique des systèmes ouverts reconfigurables vers la logique de plus bas niveau supportée par le vérificateur de modèle NECO. Grâce à notre prévue constructive d'isomorphisme entre les π-graphes et leur traduction en réseaux de Petri, les contre-exemples générés pour les réseaux de Petri en cas d'invalidation de proposition par NECO peuvent être réinterprétées et expliquées dans les termes des π-graphes. / Today we witness the rapid spread of highly dynamic reconfigurable and distributed infrastructures that we group under the common name of open reconfigurable systems. The communication topology of those systems can dynamically change - or reconfigure - as a consequence of an internal or external concurrent activity. Labelled transition semantics for open systems allow to take into account the external activities in an implicit way.Open reconfigurable systems are commonly modeled using formalisms inspired by the π-calculus. Name passing makes it possible to model dynamic communication topologies. In this thesis, we introduce the π-graphs, a variant of the π-calculus that among other things enjoys a natural graphical interpretation. Moreover, the formalism has been designed to serve as an intermediate language between the abstract π-calculus and more concrete realizations, especially in the realm of high-level Petri nets.We first propose a faithful encoding of π-graphs into high-level Petri nets that are supported by common modelling and verification tools. We show that the translation can be lifted to an isomorphism between the two formalisms. Hence, the prototype tools that are developed specifically for π-graphs can be used in conjunction with more mature and general tools for Petri nets.Based on this bidirectional encoding, we develop a high-level variant of the linear temporal logic - namely the open system logic - to specify properties about the dynamic evolution of systems taking into account interactions within open environments. The atomic propositions of the logic precisely capture the state properties of π-graphs processes.The whole framework has been implemented during the course of this thesis. Our prototype tool provides a simulator for π-graphs models. These models can also be compiled into high-level Petri nets and then manipulated using the SNAKES framework. Finally, we provide an encoding of open system logic into linear temporal logic with state properties to be used with the NECO model checker for the automated verification of properties. Thanks to the bidirectional encoding from-and-to high-level Petri nets, counterexamples provided by the model checker for Petri nets can be easily reconstructed in terms of the original π-graphs.

Thesis--Bonn. / Vita. Includes bibliographical references (p. 213-228).

Thesis--Bonn. / Vita. Bibliography: p. 213-228.

The branching ratios of the radiative decays η(')→ π+π-π0γ are still unknown. Only an upper limit for η has been established and η' has not been observed. In this thesis the decays of η and η' into the final state π+π-π0γ are studied through the dominant contributions from bremsstrahlung and intermediate vector meson states respectively. Simulations have been done using ROOT, a framework based on C++ language, to determine if the decay to the final state can be observed. Based on theories describing the dominant contributions to the final state signal, event generators have been designed in this thesis to produce particle distributions for the mentioned decay branches. The process η' → ωγ, ω → π+π-π0 is 6.16±0.83 more likely than η'→ π+π-π0 decay. Probabilities for the η → π+π-π0γ contributions are presented as a function of different photon energies. / Den bakomliggande dynamiken hos de radiativa sönderfallen η(')→ π+π-π0γ är fortfarande okänd. I nuläget finns bara en övre gräns för η och η' har ännu inte fastställts. I detta projekt studeras sönderfallen av η och η' till sluttillståndet π+π-π0γ genom de dominanta bidragen från bromsstrålning av η och ett mellantillstånd där η' först sönderfaller till ωγ och ett ytterligare sönderfall av ω till π+π-π0. Simulationer har genomförts med hjalp av ROOT, som är ett program baserat pa C++, for att avgöra om sönderfallen kan observeras. Händelsegeneratorn baseras på tidigare arbeten som beskriver sönderfallsprocesserna och resultaten presenteras i from av Dalitz-grafer. Sannolikheten for processen η' → ωγ, ω → π+π-π0 är 6.16±0.83 gånger mer trolig än η'→ π+π-π0. Sannolikheten för η → π+π-π0γ presenteras i resultatdelen av rapporten för olika energier av fotonen som avges genom bromsstrålning.

Studying the annihilation of e+e- into hadrons is of great interest in the search for physics beyond the standard model. The cross sections of specific hadronic channels can be used in precise test of the Standard Model, or to estimate the anomalous muon magnetic moment, for which there are discrepancies between theory and experiment. This thesis focuses on one particular hadronic channel, e+e- → ηπ+π- with the final state π+π-γγ. As background, the reaction e+e- → π+π-π0 is chosen. By simulations using the Monte Carlo event generator PHOKHARA and the data analysis framework ROOT, the feasibility of detecting the aforementioned reaction with the detector KLOE at the electron-positron collider DAΦNE is studied. The detector acceptance and expected number of events is evaluated both for the signal and the background. An analysis program that can be used for feasibility studies of e+e- → ηπ+π- was written. The simulations indicate that the signal significance from an experiment at DAΦNE would be 2.4σ and thus lower than the confidence level required for detection. A definitive statement can however only be made after further investigations into minimizing the background and studies of additional final states with other decays of the η-meson and their corresponding background reactions. / I sökandet efter fysik bortom standardmodellen är studier av elektron-positron-annihilation till hadroner av stort intresse. Tvärsnitten för specifika hadroniska kanaler kan användas för precisa tester av standardmodellen, eller för att uppskatta myonens anomala magnetiska moment, vars teoretiska värde avviker från det experimentella. Detta examensarbete fokuserar på en specifik hadronisk kanal, e+e- → ηπ+π- med sluttillståndet π+π-γγ. Som bakgrund väljs reaktionen e+e- → π+π-π0. Med hjälp av simulationer byggda på den Monte Carlo-baserade händelsegeneratorn PHOKHARA, och ROOT, ett ramverk för dataanalys, studeras om det är genomförbart att detektera den ovan nämnda reaktionen med detektorn KLOE vid acceleratorn DA\(\Phi\)NE. Detektoracceptansen och det förväntade antalet händelser utvärderas både för signalen och bakgrunden. Ett analysprogram som skulle kunna användas för andra genomförbarhetsstudier för e+e- → ηπ+π- har skrivits. Simulationerna visar signifikansen för signal från experiment vid DAΦNE skulle vara 2.4σ, vilket är lägre än den konfidensnivå som krävs för att reaktionen med säkerhet ska kunna detekteras. Dock kan ett slutgiltigt besked ges först efter att möjligheten att ytterligare minska antalet bakgrundshändelser har undersökts och fler sluttillstånd med andra η-sönderfall och motsvarande bakgrundsreaktioner har studerats.

The space B(pi), defined by s.n. function (Phi)(pi)(x_{1},...), were discussed in details in [8], and it is shown that B(pi) is the dual space of B(Pi)^0, which is the closure of the polynomials in B(Pi), the space of analytic function defined by the s.n. function (Phi)(Pi)(x_{1},...), provided that {pi_{n}} satisfies some regularity condition. In this article, we will show that in fact we have the relation (B(Pi)^0)^* approx B(pi), B(pi)^* approx B(Pi). This is an interesting analogy to the classical duality between the operator ideal (S(Pi))^(0), S(pi) and S(Pi), or, the s.n. ideal defined by (Phi)(Pi) and (Phi)(pi).

I dagens IT-samhälle är säkerhet en viktig aspekt. Ett sätt att nå högre säkerhet är att bygga upp säkerheten i lager. I hemmanätverk är två vanliga lager antivirus och brandvägg. Den här kandidatuppsatsen undersöker om ett intrångsdetekteringsystem (IDS) är ett bra komplement till säkerheten i ett hemmanätverk.För att hålla systemet så attraktivt som möjligt för hemmanätverket fokuserar man på att hålla priset nere och konfigurationen enkel. Vi valde enkorts-datorn (Raspberry Pi) med programvaran IPFire, som är open-source, där IDS:en Snort ingår och IPFire har ett enkelt gränssnitt för konfiguration.För att mäta hur effektivt systemet fungerar, mäts det hur många hot Snort upptäcker. Mätningar gjordes också för att undersöka om systemet orsakade prestandaförluster i hemmanätverket.Av resultaten drogs slutsatsen att systemet är ett bra komplement till säkerheten i ett hemmanätverk. Det gick inte att säkerställa någon prestandaförlust på nätverket förens vid en uppkoppling på 100 mbit och däröver.

Despite an ever growing number of reports concerning the anion-π interaction, controversy surrounding the nature of these weak supramolecular interactions continues. In an effort to further explore the nature and properties of anion-π interactions, experimental and computational methods were employed to study their occurrence in inorganic, organic and biological systems. As part of ongoing research in the Dunbar group on the topic of anion-π based supramolecular interactions, the ligand 3,6-bis(2′-pyrimidyl)-1,2,4,5-tetrazine (bmtz) was synthesized and reacted with [Cu(NCMe)4][BF4] to form the octanuclear complex [Cu8(bmtz)6][BF6]6·6MeCN. Crystallographic evidence indicates an in situ reduction of two of the complexed bmtz ligands to radical anions. A second blue compound has also been observed in this reaction, and recent work has resulted in a direct synthesis of this compound. Preliminary results indicate that this second compound contains Cu(II) centers, as expected. Further work is necessary to identify the second blue compound. In an effort to explore the fundamental question of whether or not anion-π interactions can occur between complex anions and olefins a series of Density Functional Theory and ab initio computations have been preformed for the tetracyanoethylene, 7,7,8,8-tetracyanoquinodimethane, 7,7,8,8-tetracyano-1,2,4,5-tetrafluoroquinodimethane and octacyanoquinodimethane molecules with the anions tetrafluoroborate and hexaflurophosphate. These optimizations indicate favorable interactions for all cases and are further supported by critical point analysis, performed using the Atoms in Molecules theory, and Natural Bond Orbitals analysis. DFT and NBO computations were also employed to explore the simultaneous anion-π and π-type charge transfer interactions observed between 1,4,5,8,9,12-hexaazatriphenylene-hexacarbonitrile and the chloride, bromide and iodide anions, as observed previously by the Dunbar group. Computations involving chloride and bromide anions are in full agreement with the previously reported spectroscopic and crystallographic evidence. Finally, the question of whether or not anion-π interactions occur in proteins was investigated by searching the Protein Data Bank for interactions between chloride or iodide anion and the aromatic moieties of the phenylalanine, tyrosine and tryptophan residues. Computer scripts were specifically written for this search and revealed promising interactions between chloride anions and all three amino acids. Procedural and statistical considerations preclude these examples from being definitive.