<p>Fibronectin (FN) is a large extracellular matrix (ECM) protein that is made up of</p><p>type I (FNI), type II (FNII), & type III (FNIII) domains. It assembles into an insoluble</p><p>supra-­‐‑molecular structure: the fibrillar FN matrix. FN fibrillogenesis is a cell‐‑mediated process, which is initiated when FN binds to integrins on the cell surface. The FN matrix plays an important role in cell migration, proliferation, signaling & adhesion. Despite decades of research, the FN matrix is one of the least understood supra-­‐‑molecular protein assemblies. There have been several attempts to elucidate the exact mechanism of matrix assembly resulting in significant progress in the field but it is still unclear as to what are FN-­‐‑FN interactions, the nature of these interactions and the domains of FN that</p><p>are in contact with each other. FN matrix fibrils are elastic in nature. Two models have been proposed to explain the elasticity of the fibrils. The first model: the ‘domain unfolding’ model postulates that the unraveling of FNIII domains under tension explains fibril elasticity.</p><p>The second model relies on the conformational change of FN from compact to extended to explain fibril elasticity. FN contain 15 FNIII domains, each a 7-­‐‑strand beta sandwich. Earlier work from our lab used the technique of labeling a buried Cys to study the ‘domain unfolding’ model. They used mutant FNs containing a buried Cys in a single FNIII domain and found that 6 of the 15 FNIII domains label in matrix fibrils. Domain unfolding due to tension, matrix associated conformational changes or spontaneous folding and unfolding are all possible explanation for labeling of the buried Cys. The present study also uses the technique of labeling a buried Cys to address whether it is spontaneous folding and unfolding that labels FNIII domains in cell culture. We used thiol reactive DTNB to measure the kinetics of labeling of buried Cys in eleven FN III domains over a wide range of urea concentrations (0-­‐‑9M). The kinetics data were globally fit using Mathematica. The results are equivalent to those of H-­‐‑D exchange, and</p><p>provide a comprehensive analysis of stability and unfolding/folding kinetics of each</p><p>domain. For two of the six domains spontaneous folding and unfolding is possibly the reason for labeling in cell culture. For the rest of the four domains it is probably matrix associated conformational changes or tension induced unfolding.</p><p>A long-­‐‑standing debate in the protein-­‐‑folding field is whether unfolding rate</p><p>constants or folding rate constants correlate to the stability of a protein. FNIII domains all have the same ß sandwich structure but very different stabilities and amino acid sequences. Our study analyzed the kinetics of unfolding and folding and stabilities of eleven FNIII domains and our results show that folding rate constants for FNIII domains are relatively similar and the unfolding rates vary widely and correlate to stability. FN forms a fibrillar matrix and the FN-­‐‑FN interactions during matrix fibril formation are not known. FNI 1-­‐‑9 or the N-­‐‑ terminal region is indispensible for matrix formation and its major binding partner has been shown to be FNIII 2. Earlier work from our lab, using FRET analysis showed that the interaction of FNI 1-­‐‑9 with a destabilized FNIII 2 (missing the G strand, FNIII 2ΔG) reduces the FRET efficiency. This efficiency is restored in the presence of FUD (bacterial adhesion from S. pyogenes) that has been known to interact with FNI 1-­‐‑9 via a tandem ß zipper. In the present study we</p><p>use FRET analysis and a series of deletion mutants of FNIII 2ΔG to study the shortest fragment of FNIII 2ΔG that is required to bind FNI 1-­‐‑9. Our results presented here are qualitative and show that FNIII 2ΔC’EFG is the shortest fragment required to bind FNI 1-­‐‑9. Deletion of one more strand abolishes the interaction with FNI 1-­‐‑9.</p> / Dissertation

The aim of this work is the study of the existence and multiplicity of sign changing nonradial solutions to elliptic boundary value problems on annular domains.

The cardinality of the set of units, and of the set of equivalence classes of primes in non-trivial Euclidean domains is discussed with reference to the categories "finite" and "infinite." It is shown that no Euclidean domains exist for which both of these sets are finite. The other three combinations are possible and examples are given. For the more general Euclidean rings, the first combination is possible and examples are likewise given. Prime factorization is also discussed in both Euclidean rings and Euclidean domains. For Euclidean rings, an alternative definition of prime elements in terms of associates is compared and contrasted to the usual definitions.

This publication directly follows the bachelor thesis. It contains necessary theory of HTTP, SMTP and some other protocols and services. This knowledge is then used to draw a methodology to build additional tests to verify availability and functionality of basic Internet services of a domain name. This methodology is then implemented as an application that uses distributed processing to analyse a large number of domains. Obtained results are then compiled into statistical outputs. One chapter is also devoted to overview of the attacks on DNS and security options of DNS servers and domain records.

Protein interactions as mediated by catalytic or non-catalytic protein domains contribute to cellular signal transduction processes by covalent protein modification of or non-covalent binding to interaction partners. Ena/VASP homology 1 (EVH1) domains are found in different signal transduction proteins as N-terminal non-catalytic adaptor modules of ~ 115 amino acids sharing a common fold. By targeting their host proteins to subcellular sites of action they are involved in several signalling cascades which include protein phosphorylation and cytoskeletal reorganisation. In this study, protein interactions of the two EVH1 domain containing proteins VASP and Spred2 were studied according to their involvement in different and non-overlapping signal transduction pathways of the cell. EVH1 domains were first described in the Ena/VASP protein family with the Vasodilator-stimulated phosphoprotein VASP being its founding member. As a cytoskeleton-associated protein VASP not only interacts with different proteins of the actin network but it is also a substrate for cAMP- and cGMP-dependent protein kinases. However the full complement of protein kinases targeting VASP as their substrate is still unknown. Here we used mouse cardiac fibroblast (MCFB) cells in order to study the phosphorylation status of VASP and identify new candidate protein kinases involved after serum stimulation of these cells. Using phosphosite-specific antibodies we found that serum stimulation induces a phosphorylation of VASP at Ser-157 in a time-dependent manner reaching its maximum after 90 min of stimulation. We developed an interaction graph model of possible candidate protein kinases involved. Using a pharmacological perturbation analysis with different combinations of specific protein kinase inhibitors and activators we excluded any contribution of cGMP-dependent protein kinase and Rho kinases to this process and identified a combined action of classical isoforms of PKCs and PKA in serum-stimulated VASP phosphorylation at Ser-157 positioning PKC upstream of PKA in this signalling pathway. We hypothesise that PKC receives an external stimulatory signal upon serum stimulation of MCFB cells which is passed either directly or indirectly to PKA which finally phosphorylates VASP at Ser-157. A new EVH1 domain has been described recently in the Spred proteins (Sprouty related proteins containing an EVH1 domain) which are inhibitors of the Ras/Raf/MAP kinase pathway. Our laboratory has been involved in the elucidation of the atomic structure of the human Spred2 EVH1 domain by protein NMR spectroscopy (PDB 2JP2; 2007). A positively charged binding interface of this EVH1 domain suggests an interaction with negatively charged ligands; however no interaction partners of this domain have been described so far. In the second part of this study, we used different genetic and biochemical screening methods to search for ligands of the Spred2 EVH1 domain. A bacterial two-hybrid system was established using a physically well characterized interaction of the VASP EVH1 domain with a panel of its ActA binding peptides as positive controls to screen a human brain cDNA expression library at different stringencies for candidate Spred2 EVH1 interaction partners. However none of the clones isolated could be genetically and physically validated to support Spred2 EVH1 specific interactions. An in-vitro screening of a 9-mer phage display peptide library using purified GST-Spred2 EVH1 fusion protein was performed together with a Fyn-SH3 fusion protein as a positive control. In contrast to the Fyn-SH3 domain the majority of phages isolated with the Spred2 EVH1 domain either carried no inserts or inserts with stop codons suggesting a highly non-specific interaction of the phage coat protein with the latter domain but neither the Fyn-SH3 domain nor the GST moiety. Isolation of a 13-mer proline-rich sequence was particularly surprising in this context. In order to address possible interactions of the Spred2 EVH1 domain with non-peptidergic ligands protein-lipid interaction assays were performed. Quantitative binding studies to purified Spred2 EVH1 using a liposome sedimentation assay however excluded any interaction of candidate phospholipids of the phosphatidyl inositol phosphate class with the Spred2 EVH1 domain. A natively folded and thus binding-competent conformation of the purified proteins used was assessed independently by 1H protein NMR spectroscopy. In summary the cumulative evidence of our genetic and biochemical screening experiments suggests that the still elusive Spred2 EVH1 ligand(s) may be formed of hydrophobic peptide epitopes larger than nine amino acids in size and carrying negative charge(s). A phosphorylation of Spred2 EVH1 binding epitopes by a post-translational modification should be seriously considered in future experiments. / Proteininteraktionen, wie sie durch katalytisch oder nicht-katalytisch wirksame Proteindomänen vermittelt werden können, spielen eine wesentliche Rolle in zellulären Signaltransduktionsprozessen durch die kovalente Modifikation oder nicht-kovalente Bindung von Interaktionspartnern. Ena/VASP Homologie 1 (EVH1) Domänen finden sich als N-terminale, nicht-katalytische, etwa 115 Aminosäuren große und konserviert gefaltete Adaptormodule in vielen verschiedenen Signaltransduktionsproteinen. Indem sie ihre jeweiligen Wirtsproteine an deren subzellulärem Wirkort verankern helfen, sind sie an vielen verschiedenen Signalkaskaden wie z.B. Proteinphosphorylierungen oder Umbauprozessen des Zytoskeletts beteiligt. In dieser Arbeiten wurden Proteininteraktionen der beiden EVH1 domänen-haltigen Proteine VASP and Spred2 untersucht, die in nicht überlappenden Signaltransduktionswegen der Zelle vorkommen. EVH1 Domänen wurden zuerst innerhalb der Ena/VASP-Proteinfamilie beschrieben, deren Gründungsmitglied das Vasodilator-stimulierte Phosphoprotein VASP ist. Als zytoskelett-assoziiertes Protein wechselwirkt VASP nicht nur mit verschiedenen Aktin-bindenden Proteinen, sondern ist auch ein Substrat der cAMP- und cGMP-abhängigen Proteinkinasen. Der vollständige Satz jener Proteinkinasen, die VASP als eines ihrer Substrate aufweisen, ist immer noch unbekannt. Hier haben wir kardiale Mausfibroblasten (MCFB) Zellen verwendet, um nach Serum-Stimulation dieser Zellen den Phosphorylierungsstatus von VASP zu bestimmen und daran beteiligte, neue Kandidaten-Proteinkinasen zu identifizieren. Mit Hilfe von Phosphorylierungsstellen-spezifischen Antikörpern konnten wir zeigen, dass eine Serum-Stimulation eine zeitabhängige Phosphorylierung von VASP an Serin 157 induziert, die ein Maximum 90 min nach Stimulation erreicht. Wir entwickelten ein Interaktionsgraphen-Modell möglicher Kandidaten-Proteinkinasen, die an diesem Prozess beteiligt sein könnten. Mit Hilfe pharmakologischer Perturbationsexperimente auf der Grundlage spezifischer Proteinkinase-Inhibitoren und Aktivatoren konnten wir einerseits eine Beteiligung der löslichen cGMP-abhängigen Proteinkinase und von Rho-Kinasen an diesem Prozess ausschliessen und anderseits die gemeinsame Beteiligung der klassischen Proteinkinase C Isoform(en) und der cAMP-abhängigen Proteinkinase nachweisen. In diesem Signalweg liegt dabei die Proteinkinase C stromaufwärts vor letzterer. Nach unserer Interpretation der Daten wird die PKC nach Serum-Stimulation der MCFB-Zellen aktiviert und aktiviert ihrerseits direkt oder indirekt die cAMP-abhängige Proteinkinase, die schliesslich VASP als proximales Substrat am Serin 157 phosphoryliert. Eine neue EVH1 Domäne wurde kürzlich in den Spred Proteinen (Sprouty related proteins containing an EVH1 domain) beschrieben, die neue Inhibitoren im Ras/Raf/MAP-Kinase-Signalweg darstellen. Unser Labor war an der NMR-gestützten Aufklärung der atomaren Struktur der Spred2 EVH1 Domäne beteiligt (PDB 2JP2; 2007). Die positiv geladene Bindungsfurche dieser EVH1 Domäne legt eine Interaktion mit anionischen Liganden nahe. Interaktionspartner für diese Domäne sind bisher jedoch nicht beschrieben worden. Im zweiten Teil dieser Arbeit verwendeten wir verschiedene genetische und biochemische Suchverfahren zur Identifizierung möglicher Spred2 EVH1 Liganden. Ein bakterielles Two-Hybrid-System mit der Spred2 EVH1 Domäne als Köderprotein wurde dazu etabliert unter Verwendung der physikalisch gut charakterisierten Wechselwirkung der VASP EVH1 Domäne mit ihren ActA Bindungspeptiden als eines positiven Kontroll-Interaktionspaars und zum verschieden stringenten Durchmustern einer humanen cDNA Expressionsgenbank aus Gehirn eingesetzt. Keiner der isolierten Klone ließ sich jedoch genetisch oder nach Sequenzierung in Hinblick auf eine Spred2 EVH1 spezifische Wechselwirkung validieren. Mittels gereinigtem GST-Spred2 EVH1 Protein wurde daher eine 9-mer Peptid-Genbank im Phage-Display-Verfahren durchgemustert unter Verwendung eines Fyn-SH3 Fusionsproteins als positiver Kontrolle. Im Gegensatz zu den Ergebnissen mit letzterer trugen die mit der Spred2 EVH1 Domäne isolierten Phagen überwiegend keine Inserts oder solche mit Stop-Codons, was eine unspezifische Wechselwirkung mit den Phagen-Hüllenproteinen dieser Domäne nicht jedoch der Fyn-SH3 Domäne oder des GST-Partners nahelegt. Die Isolierung einer 13-mer großen prolin-reichen Bindesequenz war in diesem Zusammenhang besonders überraschend. Um eine mögliche Wechselwirkung von Spred2 EVH1 mit nicht-peptidergen Liganden zu untersuchen, wurden Protein-Phospholipid-Interaktionsassays durchgeführt. Mittels quantitativer Bindungsstudien unter Verwendung der isolierten Domäne konnte eine Interaktion mit Kandidaten-Phospholipiden aus der Klasse der Phosphatidylinositolphosphate in einem Liposomen-Sedimentationsassay ausgeschlossen werden. Eine native Faltung und damit prinzipiell bindungskompetente Konformation(en) der gereinigten Proteine konnten mittels 1H Protein-NMR-Spektroskopie sichergestellt werden. Zusammengenommen lassen unsere Experimente vermuten, dass es sich bei den noch immer nicht dingfest gemachten Spred2 EVH1 Liganden um hydrophobe, negative geladene, mehr als neun Aminosäuren umfassende Peptidepitope handeln könnte. Bei deren Identifizierung in zukünftigen Experimenten sollte mit ihrer Phosphorylierung durch post-translationale Modifikationen gerechnet werden.

A Dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Science. Johannesburg, 2013 / Many of the problems in mathematics have very elegant solutions. As complex, real–world geometries come into play, however, this elegance is often lost. This is particularly the case with meshes of physical, real–world problems. Domain mapping helps to move problems from some geometrically complex domain to a regular, easy to use domain. Shape transformation, specifically, allows one to do this in 2D domains where mesh construction can be difficult. Numerical methods usually work over some mesh on the target domain. The structure and detail of these meshes affect the overall computation and accuracy immensely. Unfortunately, building a good mesh is not always a straight forward task. Finite Element Analysis, for example, typically requires 4–10 times the number of tetrahedral elements to achieve the same accuracy as the corresponding hexahedral mesh. Constructing this hexahedral mesh, however, is a difficult task; so in practice many people use tetrahedral meshes instead. By mapping the geometrically complex domain to a regular domain, one can easily construct elegant meshes that bear useful properties. Once a domain has been mapped to a regular domain, the mesh can be constructed and calculations can be performed in the new domain. Later, results from these calculations can be transferred back to the original domain. Using harmonic functions, source domains can be parametrised to spaces with many different desired properties. This allows one to perform calculations that would be otherwise expensive or inaccurate. This research implements and extends the methods developed in Voruganti et al. [2006 2008] for domain mapping using harmonic functions. The method was extended to handle cases where there are voids in the source domain, allowing the user to map domains that are not topologically equivalent to the equivalent dimension hypersphere. This is accomplished through the use of various boundary conditions as the void is mapped to the target domains which allow the user to reshape and shrink the void in the target domain. The voids can now be reduced to arcs, radial lines and even shrunk to single points. The algorithms were implemented in two and three dimensions and ultimately parallelised to run on the Centre for High Performance Computing clusters. The parallel code also allows for arbitrary dimension genus-0 source domains. Finally, applications, such as remeshing and robot path planning were investigated and illustrated.

Thesis advisor: Liane Young / Thesis advisor: James Dungan / Moral psychologists disagree over whether descriptively different moral violations represent distinct cognitive domains or are in fact unified by common cognitive mechanisms. The Moral Foundations Theory (MFT; Haidt, 2007) offers five different domains of moral transgressions: Harm/Care, Fairness/Reciprocity, Ingroup/Loyalty, Authority/Respect, and Purity/Sanctity. Both intentionality and omission bias (e.g. omissions such as letting someone die being judged less harshly than actions such as killing someone) have been shown to impact moral judgments; however, it remains unclear how these rules modulate judgments across moral transgressions of various types. Here, we investigate the role of intentionality and omission bias across different moral violations to determine if the divide between moral domains represent true cognitive, (as opposed to descriptive), differences. We utilized a 2 x 2 x 5 design to create stories across the 5 domains posited by MFT that were intentional/accidental cases of actions/omissions. Importantly, this study also looks at four distinct moral judgments of wrongness, responsibility, blameworthiness, and punishment to assess the role of these rules across judgments. We found that intent and action play different roles across judgments, particularly when comparing wrongness and punishment. Intent seems to matter more for wrongness, whereas action matters more for punishment. Further, these rules also differ across domains. We found that intent matters more for the individualizing foundations of harm and fairness (versus the binding foundations of ingroup, authority, and purity) in judgments of wrongness and punishment. The difference between action and omission is also more important for the individualizing foundations for punishment. These data suggest intentionality and omission bias manifest themselves uniquely across moral judgments and domains and provide evidence that there are meaningful differences between domains. / Thesis (BS) — Boston College, 2014. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Psychology Honors Program. / Discipline: Psychology .

Abstract We study in this thesis functional spaces involved in crack problems in unbounded domains. These spaces are defined by closing spaces of Sobolev H1 regularity functions (or vector fields) of bounded support, by the L2 norm of the gradient. In the case of linear elasticity, the closure is done under the L2 norm of the symmetric gradient. Our main result states that smooth functions are in this closure if and only if their gradient, (respectively symmetric gradient for the elasticity case), is in L2. We provide examples of functions in these newly defined spaces that are not in L2. We show however that some limited growth in dimension 2, or some decay in dimension 3 must hold for functions in those spaces: this is due to Hardy's inequalities.

The increasing importance of the Internet as an integral part of contemporary society has stressed the need to formalize the process of response to security incidents that accompany it inseparably. Security teams of the CERT/CSIRT type are established at different levels for this purpose. These teams respond to reports from their constituency and cooperate with other teams. This thesis introduces the reader to the issues these teams deal with and analyzes their needs in resolving threats and problems related to DNS and its domains. Part of the work is an overview of the basic existing tools to support the work of CERT/CSIRT teams to solve problems with domains, the design of a tool Malicious Domain Manager and description of its implementation. The results of test run of this tool by CZ.NIC-CSIRT team show its contribution to security of DNS.

Understanding how changes in functional requirements of the cell select for changes in protein sequence and structure is a fundamental challenge in molecular evolution. This dissertation delineates some of the underlying evolutionary forces using as a model system, the Haloalkanoate Dehalogenase Superfamily (HADSF). HADSF members have unique cap-core architecture with the Rossmann-fold core domain accessorized by variable cap domain insertions (delineated by length, topology, and point of insertion). To identify the boundaries of variable domain insertions in protein sequences, I have developed a comprehensive computational strategy (CapPredictor or CP) using a novel sequence alignment algorithm in conjunction with a structure-guided sequence profile. Analysis of more than 40,000 HADSF sequences led to the following observations: (i) cap-type classes exhibit similar distributions across different phyla, indicating existence of all cap-types in the last universal common ancestor, and (ii) comparative analysis of the predicted cap-type and functional diversity indicated that cap-type does not dictate the divergence of substrate recognition and chemical pathway, and hence biological function. By analyzing a unique dataset of core- and cap-domain-only protein structures, I investigated the consequences of the accessory cap domain on the sequence-structure relationship of the core domain. The relationship between sequence and structure divergence in the core fold was shown to be monotonic and independent of the corresponding cap type. However, core domains with the same cap type bore a greater similarity than the core domains with different cap types, suggesting coevolution of the cap and core domains. Remarkably, a few degrees of freedom are needed to describe the structural diversity in the Rossmann fold accounting for the majority of the observed structural variance. Finally, I examined the location and role of conserved residue positions and co-evolving residue pairs in the core domain in the context of the cap domain. Positions critical for function were conserved while non-conserved positions mapped to highly mobile regions. Notably, we found exponential dependence of co-variance on inter-residue distance. Collectively, these novel algorithms and analyses contribute to an improved understanding of enzyme evolution, especially in the context of the use of domain insertions to expand substrate specificity and chemical mechanism.