Photoactive yellow protein: A structural prototype for the three-dimensional fold of the PAS domain superfamily

PAS domains are found in diverse proteins throughout all three kingdoms of life, where they apparently function in sensing and signal transduction. Although a wealth of useful sequence and functional information has become recently available, these data have not been integrated into a three-dimensional (3D) framework. The very early evolutionary development and diverse functions of PAS domains have made sequence analysis and modeling of this protein superfamily challenging. Limited sequence similarities between the ∼50-residue PAS repeats and one region of the bacterial blue-light photosensor photoactive yellow protein (PYP), for which ground-state and light-activated crystallographic structures have been determined to high resolution, originally were identified in sequence searches using consensus sequence probes from PAS-containing proteins. Here, we found that by changing a few residues particular to PYP function, the modified PYP sequence probe also could select PAS protein sequences. By mapping a typical ∼150-residue PAS domain sequence onto the entire crystallographic structure of PYP, we show that the PAS sequence similarities and differences are consistent with a shared 3D fold (the PAS/PYP module) with obvious potential for a ligand-binding cavity. Thus, PYP appears to prototypically exhibit all the major structural and functional features characteristic of the PAS domain superfamily: the shared PAS/PYP modular domain fold of ∼125–150 residues, a sensor function often linked to ligand or cofactor (chromophore) binding, and signal transduction capability governed by heterodimeric assembly (to the downstream partner of PYP). This 3D PAS/PYP module provides a structural model to guide experimental testing of hypotheses regarding ligand-binding, dimerization, and signal transduction.

The wing of the enhancer-binding domain of Mu phage transposase is flexible and is essential for efficient transposition.

A tetramer of the Mu transposase (MuA) pairs the recombination sites, cleaves the donor DNA, and joins these ends to a target DNA by strand transfer. Juxtaposition of the recombination sites is accomplished by the assembly of a stable synaptic complex of MuA protein and Mu DNA. This initial critical step is facilitated by the transient binding of the N-terminal domain of MuA to an enhancer DNA element within the Mu genome (called the internal activation sequence, IAS). Recently we solved the three-dimensional solution structure of the enhancer-binding domain of Mu phage transposase (residues 1-76, MuA76) and proposed a model for its interaction with the IAS element. Site-directed mutagenesis coupled with an in vitro transposition assay has been used to assess the validity of the model. We have identified five residues on the surface of MuA that are crucial for stable synaptic complex formation but dispensable for subsequent events in transposition. These mutations are located in the loop (wing) structure and recognition helix of the MuA76 domain of the transposase and do not seriously perturb the structure of the domain. Furthermore, in order to understand the dynamic behavior of the MuA76 domain prior to stable synaptic complex formation, we have measured heteronuclear 15N relaxation rates for the unbound MuA76 domain. In the DNA free state the backbone atoms of the helix-turn-helix motif are generally immobilized whereas the residues in the wing are highly flexible on the pico- to nanosecond time scale. Together these studies define the surface of MuA required for enhancement of transposition in vitro and suggest that a flexible loop in the MuA protein required for DNA recognition may become structurally ordered only upon DNA binding.

Report.

Mode of access: Internet.

Conservation C.O.N.G.R.E.S.S. : Department of Conservation analysis of recommendations.

Cover title.

Proceedings

Mode of access: Internet.

Mechanisms of negative regulation of inflammasome activation

Thesis (Ph.D.)--University of Washington, 2016-06

Towards the preparative and large-scale precision manafacture of virus-like particles

Virus-like particles (VLPs) are of interest in vaccination, gene therapy and drug delivery, but their potential has yet to be fully realized. This is because existing laboratory processes, when scaled, do not easily give a compositionally and architecturally consistent product. Research suggests that new process routes might ultimately be based on chemical processing by self-assembly, involving the precision manufacture of precursor capsomeres followed by in vitro VLP self-assembly and scale-up to required levels. A synergistic interaction of biomolecular design and bioprocess engineering (i.e. biomolecular engineering) is required if these alternative process routes and, thus, the promise of new VLP products, are to be realized.

Investigations into a dual band 2.4/5.2GHz antenna for WLAN applications

The design of a dual-band 2.45/5.2 GHz antenna for an access point of a wireless local area network (WLAN) is presented. The proposed antenna is formed by an assembly of a radial line slot array (RLSA) operating at 2.4 GHz and a microstrip patch working at 5.2 GHz. The design of this antenna system is accomplished using commercially available finite element software, high frequency structure simulator (HFSS), of Ansoft. The performance of the designed antenna is assessed in terms of return loss (RL), radiation pattern and polarization purity in the two investigated frequency bands.

Reformulation of a thermostable broadly protective recombinant vaccine against human papilloma virus

The causal relationship between Human Papilloma Virus (HPV) infection and cervical cancer has motivated the development, and further improvement, of prophylactic vaccines against this virus. 70% of cervical cancers, 80% of which in low-resources countries, are associated to HPV16 and HPV18 infection, with 13 additional HPV types, classified as high-risk, responsible for the remaining 30% of tumors. Current vaccines, Cervarix® (GlaxoSmithKline) and Gardasil®(Merk), are based on virus-like particles (VLP) obtained by self-assembly of the major capsid protein L1. Despite their undisputable immunogenicity and safety, the fact that protection afforded by these vaccines is largely limited to the cognate serotypes included in the vaccine (HPV 16 and 18, plus five additional viral types incorporated into a newly licensed nonavalent vaccine) along with high production costs and reduced thermal stability, are pushing the development of 2nd generation HPV vaccines based on minor capsid protein L2. The increase in protection broadness afforded by the use of L2 cross-neutralizing epitopes, plus a marked reduction of production costs due to bacterial expression of the antigens and a considerable increase in thermal stability could strongly enhance vaccine distribution and usage in low-resource countries. Previous studies from our group identified three tandem repeats of the L2 aa. 20-38 peptide as a strongly immunogenic epitope if exposed on the scaffold protein thioredoxin (Trx). The aim of this thesis work is the improvement of the Trx-L2 vaccine formulation with regard to cross-protection and thermostability, in order to identify an antigen suitable for a phase I clinical trial. By testing Trx from different microorganisms, we selected P. furiosus thioredoxin (PfTrx) as the optimal scaffold because of its sustained peptide epitope constraining capacity and striking thermal stability (24 hours at 100°C). Alternative production systems, such as secretory Trx-L2 expression in the yeast P. pastoris, have also been set-up and evaluated as possible means to further increase production yields, with a concomitant reduction of production costs. Limitations in immune-responsiveness caused by MHC class II polymorphisms –as observed, for example, in different mouse strains- have been overcome by introducing promiscuous T-helper (Th) epitopes, e.g., PADRE (Pan DR Epitope), at both ends of PfTrx. This allowed us to obtain fairly strong immune responses even in mice (C57BL/6) normally unresponsive to the basic Trx-L2 vaccine. Cross-protection was not increased, however. I thus designed, produced and tested a novel multi-epitope formulation consisting of 8 and 11 L2(20-38) epitopes derived from different HPV types, tandemly joined into a single thioredoxin molecule (“concatemers”). To try to further increase immunogenicity, I also fused our 8X and 11X PfTrx-L2 concatemers to the N-terminus of an engineered complement-binding protein (C4bp), capable to spontaneously assemble into ordered hepatmeric structures, previously validated as a molecular adjuvant. Fusion to C4bp indeed improved antigen presentation, with a fairly significant increase in both immunogenicity and cross-protection. Another important issue I addressed, is the reduction of vaccine doses/treatment, which can be achieved by increasing immunogenicity, while also allowing for a delayed release of the antigen. I obtained preliminary, yet quite encouraging results in this direction with the use of a novel, solid-phase vaccine formulation, consisting of the basic PfTrx-L2 vaccine and its C4bp fusion derivative adsorbed to mesoporus silica-rods (MSR).

ANÁLISE SISTÊMICA DAS RELAÇÕES DE PODER EM GRUPOS DE LIDERANÇA OFICIAL DE IGREJAS DE GOVERNO CONGREGACIONAL: Estudo de Caso em Igrejas Menonitas de Curitiba

A pesquisa a seguir tem o poder como objeto de estudos, especificamente na forma de relações de poder em conselhos diretores (pastoral e administrativo) de igrejas menonitas em Curitiba. A forma presumida de governo dessas igrejas é historicamente congregacional, isto é, as assembléias das respectivas igrejas são supremas e últimas nas deliberações. A metodologia de pesquisa é sociológica, indutiva, funcionalista, com tendências fenomenológicas, tendo como perspectiva principal a teoria sistêmica e seus recursos de pesquisa. A teoria geral dos sistemas orienta os fundamentos da pesquisa, a teoria dos sistemas sociais é o arcabouço da pesquisa. O método de pesquisa é a observação-participante com registros em vídeo e áudio, transcrição, análise e elaboração de conclusões. A técnica de pesquisa citada demonstrou-se eficaz e útil para o levantamento de dados em pesquisas de campo que tratam de grupos e suas funções diferenciadas em agremiações eclesiásticas. Os objetivos buscam identificar a dinâmica sistêmica nas reuniões dos conselhos observados, bem como identificar o processo das relações de poder nos mesmos. As hipóteses lançadas como ponto de partida afirmam que a teoria sistêmica em qualquer perspectiva é observável, bem como os elementos (conselheiros) das relações de poder se demonstram inconscientes de seus atos de poder. As hipóteses principais e secundárias foram confirmadas pela observação-participante, a saber: a pesquisa verifica a hipótese geral de que mesmo igrejas e seus sistemas sociais evidenciam princípios sistêmicos segundo a teoria geral dos sistemas e a teoria dos sistemas sociais de Niklas Luhmann. As hipóteses específicas verificam se os conselheiros dos grupos de liderança evidenciam consciência do poder exercido e do poder implícito em suas funções e papéis; se os grupos formais, eleitos pela assembléia exercem poder de fato ou se existe influência do poder informal; se existe uma correlação, entre o poder formal e as fronteiras rígidas , o poder informal e a fronteira difusa ; se é possível trabalhar preventivamente e interventivamente através do conceito de relações de poder e os princípios sociais sistêmicos. As igrejas menonitas de Curitiba de maneira geral preservam traços de governo congregacional, mas, a transição para estilos de governo pastorcêntricos e autocráticos a partir dos conselhos observados é um fato e parece irreversível. Aparentemente a causalidade desse movimento surge no contexto social metropolitano e suas implicações, mais do que numa mudança estrategicamente planejada pelas lideranças. Portanto, a transição parece ser cultural. Aliás, as transformações sociais das tradições menonitas são diretamente proporcionais à sua inclusão e inculturação no contexto social em que se situam, confirmando assim os princípios sistêmicos da sociedade em geral.(AU)

Measuring the arts: The case of Dade County nonprofits arts organizations

The theoretical analysis and research of cultural activities have been limited, for the most part, to the study of the role the public sector plays in the funding and support of nonprofit Arts organizations. The tools used to evaluate this intervention follow a macroeconomic perspective and fail to account for microeconomic principles and assumptions that affect the behavior of these organizations. This dissertation describes through conceptual models the behavior of the agents involved in the artistic process and the economic sectors affected by it. The first paper deals with issues related to economic impact studies and formulates a set of guidelines that should be followed when conducting this type of study. One of the ways to assess more accurately the impact culture has in a community is by assuming that artists can re-create the public space of a blight community and get it ready for a regeneration process. The second paper of this dissertation assumes just that and explains in detail all the cultural, political, economic and sociological interactions that are taking place in the Arts-led regeneration process in Miami Beach, Florida. The paper models the behavior of these agents by indicating what their goals and decision process mechanisms are. The results give support to the claim that the public space artists create in a city actually stimulate development. The third paper discusses the estimation of a demand function for artistic activities, specifically the New World Symphony (NWS) located in Miami Beach, Florida. The behavior of the consumers and producers of NWS' concerts is modeled. The results support the notion that consumers make their decisions based, among other things, on the perceived value these concerts have. Economists engage in the analysis of the effects of cultural activities in a community since many cities rely on them for their development. The history of many communities is not told by their assembly lines and machinery anymore but by their centers of entertainment, hotels and restaurants. Many cities in Europe and North America that have seen the manufacturing sector migrate to the South are trying to face the demands of the new economy by using the Arts as catalysts for development. ^

Diketonylpyridinium cations as a support of new ionic liquid crystals and ion-conductive materials: analysis of counter-ion effects

Ionic liquid crystals (ILCs) allow the combination of the high ionic conductivity of ionic liquids (ILs) with the supramolecular organization of liquid crystals (LCs). ILCs salts were obtained by the assembly of long-chained diketonylpyridinium cations of the type [HOO^(R(n)pyH)] + and BF_(4)^(-) , ReO_(4)^(-), NO_(3)^(-), CF_(3)SO_(3)^(-), CuCl_(4)^(2-) counter-ions. We have studied the thermal behavior of five series of compounds by differential scanning calorimetry (DSC) and hot stage polarized light optical microscopy (POM). All materials show thermotropic mesomorphism as well as crystalline polymorphism. X-ray diffraction of the [HOO^(R(12)pyH)][ReO_(4)] crystal reveals a layered structure with alternating polar and apolar sublayers. The mesophases also exhibit a lamellar arrangement detected by variable temperature powder X-ray diffraction. The CuCl_(4)^(2-) salts exhibit the best LC properties followed by the ReO_(4)^(-) ones due to low melting temperature and wide range of existence. The conductivity was probed for the mesophases in one species each from the ReO_(4)^(-) , and CuCl_(4)^(2-) families, and for the solid phase in one of the non-mesomorphic Cl^(-) salts. The highest ionic conductivity was found for the smectic mesophase of the ReO_(4)^(-) containing salt, whereas the solid phases of all salts were dominated by electronic contributions. The ionic conductivity may be favored by the mesophase lamellar structure.

Data analysis of collapse mechanisms of a 3D printed groin vault in shaking table testing

The aim of this novel experimental study is to investigate the behaviour of a 2m x 2m model of a masonry groin vault, which is built by the assembly of blocks made of a 3D-printed plastic skin filled with mortar. The choice of the groin vault is due to the large presence of this vulnerable roofing system in the historical heritage. Experimental tests on the shaking table are carried out to explore the vault response on two support boundary conditions, involving four lateral confinement modes. The data processing of markers displacement has allowed to examine the collapse mechanisms of the vault, based on the arches deformed shapes. There then follows a numerical evaluation, to provide the orders of magnitude of the displacements associated to the previous mechanisms. Given that these displacements are related to the arches shortening and elongation, the last objective is the definition of a critical elongation between two diagonal bricks and consequently of a diagonal portion. This study aims to continue the previous work and to take another step forward in the research of ground motion effects on masonry structures.

Modelling the influence of diradical character and aggregation on conjugated molecular materials

Molecular materials are made by the assembly of specifically designed molecules to obtain bulk structures with desired solid-state properties, enabling the development of materials with tunable chemical and physical properties. These properties result from the interplay of intra-molecular constituents and weak intermolecular interactions. Thus, small changes in individual molecular and electronic structure can substantially change the properties of the material in bulk. The purpose of this dissertation is, thus, to discuss and to contribute to the structure-property relationships governing the electronic, optical and charge transport properties of organic molecular materials through theoretical and computational studies. In particular, the main focus is on the interplay of intra-molecular properties and inter-molecular interactions in organic molecular materials. In my three-years of research activity, I have focused on three major areas: 1) the investigation of isolated-molecule properties for the class of conjugated chromophores displaying diradical character which are building blocks for promising functional materials; 2) the determination of intra- and intermolecular parameters governing charge transport in molecular materials and, 3) the development and application of diabatization procedures for the analysis of exciton states in molecular aggregates. The properties of diradicaloids are extensively studied both regarding their ground state (diradical character, aromatic vs quinoidal structures, spin dynamics, etc.) and the low-lying singlet excited states including the elusive double-exciton state. The efficiency of charge transport, for specific classes of organic semiconductors (including diradicaloids), is investigated by combining the effects of intra-molecular reorganization energy, inter-molecular electronic coupling and crystal packing. Finally, protocols aimed at unravelling the nature of exciton states are introduced and applied to different molecular aggregates. The role of intermolecular interactions and charge transfer contributions in determining the exciton state character and in modulating the H- to J- aggregation is also highlighted.

Innovative photoresponsive materials & supramolecular structures

This thesis focuses on two main topics: photoresponsive azobenzene-based polymers and supramolecular systems generated by the self-assembly of lipophilic guanosines. In the first chapters describe innovative photoresponsive devices and materials capable of performing multiple roles in the field of soft robotics and energy conversion. Chapter 2 describes a device obtained by coupling a photoresponsive liquid-crystalline network and a piezoelectric polymer to convert visible light into electricity. Chapter 3 deals with a material that can assume different shapes when triggered by three different stimuli in different environments. Chapter 4 reports a highly performing artificial muscle that contracts when irradiated. The last two chapters report on supramolecular structures generated from functionalized guanosines dissolved in organic solvents. Chapter 6 illustrates the self-assembly into G-quadruplexes of 8- and 5’-functionalized guanosines in the absence of templating ions. Chapter 7 describes the supramolecular structure generated by the assembly of a lipophilic guanosine in the presence of silver cations. Chapter 6 is reproduced from an already published paper, while the other chapters are going to be submitted to different journals in a couple of months.