Plants and animals share functionally common bacterial virulence factors

By exploiting the ability of Pseudomonas aeruginosa to infect a variety of vertebrate and nonvertebrate hosts, we have developed model systems that use plants and nematodes as adjuncts to mammalian models to help elucidate the molecular basis of P. aeruginosa pathogenesis. Our studies reveal a remarkable degree of conservation in the virulence mechanisms used by P. aeruginosa to infect hosts of divergent evolutionary origins.

National policies for technical change: Where are the increasing returns to economic research?

Improvements over the past 30 years in statistical data, analysis, and related theory have strengthened the basis for science and technology policy by confirming the importance of technical change in national economic performance. But two important features of scientific and technological activities in the Organization for Economic Cooperation and Development countries are still not addressed adequately in mainstream economics: (i) the justification of public funding for basic research and (ii) persistent international differences in investment in research and development and related activities. In addition, one major gap is now emerging in our systems of empirical measurement—the development of software technology, especially in the service sector. There are therefore dangers of diminishing returns to the usefulness of economic research, which continues to rely completely on established theory and established statistical sources. Alternative propositions that deserve serious consideration are: (i) the economic usefulness of basic research is in the provision of (mainly tacit) skills rather than codified and applicable information; (ii) in developing and exploiting technological opportunities, institutional competencies are just as important as the incentive structures that they face; and (iii) software technology developed in traditional service sectors may now be a more important locus of technical change than software technology developed in “high-tech” manufacturing.

Handoff from recombinase to replisome: Insights from transposition

Bacteriophage Mu replicates as a transposable element, exploiting host enzymes to promote initiation of DNA synthesis. The phage-encoded transposase MuA, assembled into an oligomeric transpososome, promotes transfer of Mu ends to target DNA, creating a fork at each end, and then remains tightly bound to both forks. In the transition to DNA synthesis, the molecular chaperone ClpX acts first to weaken the transpososome's interaction with DNA, apparently activating its function as a molecular matchmaker. This activated transpososome promotes formation of a new nucleoprotein complex (prereplisome) by yet unidentified host factors [Mu replication factors (MRFα2)], which displace the transpososome in an ATP-dependent reaction. Primosome assembly proteins PriA, PriB, DnaT, and the DnaB–DnaC complex then promote the binding of the replicative helicase DnaB on the lagging strand template of the Mu fork. PriA helicase plays an important role in opening the DNA duplex for DnaB binding, which leads to assembly of DNA polymerase III holoenzyme to form the replisome. The MRFα2 transition factors, assembled into a prereplisome, not only protect the fork from action by nonspecific host enzymes but also appear to aid in replisome assembly by helping to activate PriA's helicase activity. They consist of at least two separable components, one heat stable and the other heat labile. Although the MRFα2 components are apparently not encoded by currently known homologous recombination genes such as recA, recF, recO, and recR, they may fulfill an important function in assembling replisomes on arrested replication forks and products of homologous strand exchange.

Redirecting the specificity of ubiquitination by modifying ubiquitin-conjugating enzymes.

Depletion of specific cellular proteins is a powerful tool in biological research and has many medical and agricultural benefits. In contrast to genetic methods currently available to attenuate protein levels, we describe an alternative approach that redirects the ubiquitin-dependent proteolytic pathway to facilitate specific proteolytic removal. Degradation via the ubiquitin pathway requires the prior attachment of multiple ubiquitins to the target protein. This attachment is accomplished, in part, by a family of enzymes designated E2s (or ubiquitin-conjugating enzymes), some of which use domains near their C termini for target recognition. Here, we demonstrate that E2 target recognition can be redefined by engineering E2s to contain appropriate protein-binding peptides fused to their C termini. In five dissimilar examples, chimeric E2s were created that recognized and ubiquitinated their respective binding partners with high specificity. We also show that ubiquitination of one protein targeted by this method led to its ATP-dependent degradation in vitro. Thus, by exploiting interacting domains derived from natural and synthetic ligands, it may be possible to design E2s capable of directing the selective removal of many intracellular proteins.

New multivalent calixarene-based ligands for protein recognition and inhibition

One of the challenges that concerns chemistry is the design of molecules able to modulate protein-protein and protein-ligand interactions, since these are involved in many physiological and pathological processes. The interactions occurring between proteins and their natural counterparts can take place through reciprocal recognition of rather large surface areas, through recognition of single contact points and single residues, through inclusion of the substrates in specific, more or less deep binding sites. In many cases, the design of synthetic molecules able to interfere with the processes involving proteins can benefit from the possibility of exploiting the multivalent effect. Multivalency, widely spread in Nature, consists in the simultaneous formation between two entities (cell-cell, cell-protein, protein-protein) of multiple equivalent ligand-recognition site complexes. In this way the whole interaction results particularly strong and specific. Calixarenes furnish a very interesting scaffold for the preparation of multivalent ligands and in the last years calixarene-based ligands demonstrated their remarkable capability to recognize and inhibit or restore the activity of different proteins, with a high efficiency and selectivity in several recognition phenomena. The relevance and versatility of these ligands is due to the different exposition geometries of the binding units that can be explored exploiting the conformational properties of these macrocycles, the wide variety of functionalities that can be linked to their structure at different distances from the aromatic units and to their intrinsic multivalent nature. With the aim of creating new multivalent systems for protein targeting, the work reported in this thesis regards the synthesis and properties of glycocalix[n]arenes and guanidino calix[4]arenes for different purposes. Firstly, a new bolaamphiphile glycocalix[4]arene in 1,3-alternate geometry, bearing cellobiose, was synthesized for the preparation of targeted drug delivery systems based on liposomes. The formed stable mixed liposomes obtained by mixing the macrocycle with DOPC were shown to be able of exploiting the sugar units emerging from the lipid bilayer to agglutinate Concanavalin A, a lectin specific for glucose. Moreover, always thanks to the presence of the glycocalixarene in the layer, the same liposomes demonstrated through preliminary experiments to be uptaken by cancer cells overexpressing glucose receptors on their exterior surface more efficiently respect to simple DOPC liposomes lacking glucose units in their structure. Then a small library of glycocalix[n]arenes having different valency and geometry was prepared, for the creation of potentially active immunostimulants against Streptococcus pneumoniae, particularly the 19F serotype, one of the most virulent. These synthesized glycocalixarenes bearing β-N-acetylmannosamine as antigenic unit were compared with the natural polysaccharide on the binding to the specific anti-19F human polyclonal antibody, to verify their inhibition potency. Among all, the glycocalixarene based on the conformationally mobile calix[4]arene resulted the more efficient ligand, probably due its major possibility to explore the antibody surface and dispose the antigenic units in a proper arrangement for the interaction process. These results pointed out the importance of how the different multivalent presentation in space of the glycosyl units can influence the recognition phenomena. At last, NMR studies, using particularly 1H-15N HSQC experiments, were performed on selected glycocalix[6]arenes and guanidino calix[4]arenes blocked in the cone geometry, in order to better understand protein-ligand interactions. The glycosylated compounds were studied with Ralstonia solanacearum lectin, in order to better understand the nature of the carbohydrate‐lectin interactions in solution. The series of cationic calixarene was employed with three different acidic proteins: GB1, Fld and alpha synuclein. Particularly GB1 and Fld were observed to interact with all five cationic calix[4]arenes but showing different behaviours and affinities.

Desenvolvimento de microssensores eletroquímicos e aplicações no estudo de processos dinâmicos interfaciais utilizando microscopia eletroquímica de varredura

O trabalho descrito nesta tese mostra de forma detalhada a fabricação e caracterização de diferentes microssensores eletroquímicos os quais têm sido recentemente utilizados como sondas em grupo de técnicas conhecida como Scanning Electrochemical Probe Microscopy (SEPM). Desta forma, a caracterização de superfícies pode ser feita explorando diferentes fenômenos interfaciais relevantes à Ciência. Neste sentido, as interfaces de materiais cristalinos como hidroxiapatita (materiais dentários) e calcita foram o foco de estudo neste trabalho. Assim, diferentes técnicas SEPM foram exploradas no sentido de se obter informações relevantes relacionadas aos processos dentários, como a erosão ácida e hipersensibilidade. Inicialmente, microeletrodos de platina foram desenvolvidos empregando uma metodologia convencional na qual são utilizados microfibras encapsuladas em capilares de vidro. Scanning Electrochemical Microscopy (SECM) no modo amperométrico foi utilizada para obtenção de informações com relação às alterações de topografia do esmalte dentário causadas pelo contato com substâncias ácidas. Adicionalmente, SECM foi empregada no estudo do transporte de espécies eletroativas em amostras de dentina e investigações relacionadas ao bloqueio dos túbulos empregando-se cremes dentais comerciais foram realizadas. A permeação de peróxido de hidrogênio pela dentina também foi estudada. Os resultados de SECM foram comparados com imagens SEM obtidas nas mesmas condições experimentais. Microeletrodos de membrana ionófora íon-seletiva (Ion Selective Microelectrodes-ISMEs) sensíveis a íons cálcio também foram desenvolvidos e caracterizados, com subsequente aplicação em SECM no modo potenciométrico. A dissolução ácida de esmalte bovino (erosão dentária) foi investigada em diferentes valores de pH (2,5; 4,5; 6,8). Além disso, o transporte de íons cálcio através de membranas porosas sintéticas foi avaliado a uma distância tip/substrato de 300µm. Alterações no fluxo de íons cálcio foram correlacionadas em experimentos realizados na presença e ausência de campos magnéticos gerados por nanopartículas de magnetita incorporadas à membrana porosa. Microcristais de calcita facilmente sintetizados pelo método de precipitação foram utilizados como superfície modelo para investigações interfaciais, cujos resultados podem ser correlacionados aos materiais dentários. Desta forma, nanopipetas de vidro preenchidas com eletrólito suporte foram fabricadas e utilizadas como sonda em Scanning Ion Conductance Microscopy (SICM). O mapeamento topográfico de alta resolução espacial da superfície de um microcristal de calcita foi obtido utilizando o modo de varredura hopping mode. Adicionalmente, sondas multifuncionais ISME-SICM também foram desenvolvidas e caracterizadas para investigações simultâneas com relação às alterações topográficas e quantificação de íons cálcio sobre a superfície de um microcristal de calcita. A adição de reagentes ácidos no canal SICM promoveu a dissolução da superfície do microcristal, sendo obtidos dados cinéticos de dissolução. Investigações em meio neutro também foram realizadas utilizando a sonda ISME-SICM. Os resultados experimentais obtidos também foram comparados com aqueles oriundos de simulação computacional.

Cosmological perturbations in coherent oscillating scalar field models

The fact that fast oscillating homogeneous scalar fields behave as perfect fluids in average and their intrinsic isotropy have made these models very fruitful in cosmology. In this work we will analyse the perturbations dynamics in these theories assuming general power law potentials V(ϕ) = λ|ϕ|^n /n. At leading order in the wavenumber expansion, a simple expression for the effective sound speed of perturbations is obtained c_eff^ 2  = ω = (n − 2)/(n + 2) with ω the effective equation of state. We also obtain the first order correction in k^ 2/ω_eff^ 2 , when the wavenumber k of the perturbations is much smaller than the background oscillation frequency, ω_eff. For the standard massive case we have also analysed general anharmonic contributions to the effective sound speed. These results are reached through a perturbed version of the generalized virial theorem and also studying the exact system both in the super-Hubble limit, deriving the natural ansatz for δϕ; and for sub-Hubble modes, exploiting Floquet’s theorem.

Becoming a State: Zionist and Palestinian Movements for National Liberation

This study examines the road to statehood for the Zionist and Palestinian movements. There are three components which frame this investigation: 1. social movements and the practices in which they engage that are aimed at establishing statehood for a people; 2. distinctive configurations of the international system and the manner in which both the material and ideational foundations of that system pulls units towards conformity and predictable behavior; and finally, 3. the role of agency, that is, the way in which instrumentally rational individuals attempt to push the structure in which they are embedded towards a configuration that is better suited to their interests and objectives The most influential factor guiding these struggles for national liberation are those forces which emanate from the prevailing structure of the international system. Not only was it demonstrated that the established material and ideational preferences of existing states have strong bearing on a movement’s ideological orientation and by consequence its chosen course of struggle, but hegemonic order configurations also define political cleavages and in so doing present movement leaders with both tactical and strategic opportunities by harnessing or exploiting those cleavages. From the agency perspective, the cases showed that the leadership of each movement was highly influential in the determination of a movement’s success or failure.

Coordenação da entrega de ajuda a vítimas de desastre: uma abordagem multicritério com pensamento focado em valor.

Este trabalho trata da logística envolvida em operações de resposta a desastres, com foco na entrega final de suprimentos destinados a ajudar vítimas. Seu propósito é investigar os objetivos pertinentes ao planejamento do transporte da carga e encontrar uma metodologia para definir estratégia que sirva à tomada de decisão em campo. Para tanto, primeiramente identifica-se os objetivos adotados em modelos de Pesquisa Operacional para a tarefa em questão, através da análise de conteúdo das publicações pertinentes. Então, a abordagem do Pensamento Focado em Valores é utilizada para estruturar o problema. Finalmente, o método Simple Multi-Attribute Rating Technique Exploiting Ranks (SMARTER) é empregado na construção de um modelo de Análise da Decisão Multicritério (ADM), com consulta a um profissional experiente da área humanitária e aproveitando a análise da literatura previamente realizada. Neste processo, são elaboradas e avaliadas seis alternativas para a tomada de decisão condizentes com os valores da comunidade humanitária. Os resultados obtidos mostram que existe incompatibilidade entre os critérios de desempenho identificados nas publicações existentes e os objetivos perseguidos pelo Tomador da Decisão (TD) real. De acordo com o modelo construído, o atendimento de prioridades e a manutenção da sustentabilidade da operação são os objetivos que devem ser levados em conta para planejar a entrega de carga em pós-desastre, sendo que o custo e a equidade da distribuição não devem ser considerados. Conclui-se que o método adotado é útil à definição destes critérios e também ao desenvolvimento de estratégias que resultem em distribuições de ajuda melhores, aos olhos do próprio TD. Desta forma, ressalta-se que este trabalho contribui à área da Logística Humanitária com a investigação dos objetivos, assim como ao campo da ADM pela formalização dos processos de elaboração de alternativas, além da adição de mais uma aplicação possível ao repertório do método SMARTER.

Estudio de la fluencia de una calcarenita: la Piedra de San Julián (Alicante)

La fluencia de las rocas tiene gran importancia en la evaluación del comportamiento a largo plazo de elementos construidos con estos materiales. En este trabajo, se ha caracterizado física y mecánicamente una calcarenita porosa bien conocida localmente como Piedra de San Julián. Se han realizado ensayos de compresión uniaxial de 96 h. a carga constante. Se ha utilizado un modelo de fluencia bien conocido, el Código-modelo CEB-FIP 2010, usado para modelizar otro material pétreo (hormigón). Además, se ha propuesto un modelo reológico. El objetivo principal de este trabajo es investigar la posibilidad de aprovechar la gran experiencia acumulada en el estudio del hormigón, con el fin de obtener un enfoque para el comportamiento de la roca, para tiempos de prueba muy largos difíciles de implementar en laboratorio. Se propone una función de fluencia adaptada a la roca estudiada dependiente sólo de sus características elásticas y mecánicas.

Polarimetric Response of Rice Fields at C-Band: Analysis and Phenology Retrieval

A set of ten RADARSAT-2 images acquired in fully polarimetric mode over a test site with rice fields in Seville, Spain, has been analyzed to extract the main features of the C-band radar backscatter as a function of rice phenology. After observing the evolutions versus phenology of different polarimetric observables and explaining their behavior in terms of scattering mechanisms present in the scene, a simple retrieval approach has been proposed. This algorithm is based on three polarimetric observables and provides estimates from a set of four relevant intervals of phenological stages. The validation against ground data, carried out at parcel level for a set of six stands and up to nine dates per stand, provides a 96% rate of coincidence. Moreover, an equivalent compact-pol retrieval algorithm has been also proposed and validated, providing the same performance at parcel level. In all cases, the inversion is carried out by exploiting a single satellite acquisition, without any other auxiliary information.

Crop Phenology Estimation Using a Multitemporal Model and a Kalman Filtering Strategy

In this letter, a new approach for crop phenology estimation with remote sensing is presented. The proposed methodology is aimed to exploit tools from a dynamical system context. From a temporal sequence of images, a geometrical model is derived, which allows us to translate this temporal domain into the estimation problem. The evolution model in state space is obtained through dimensional reduction by a principal component analysis, defining the state variables, of the observations. Then, estimation is achieved by combining the generated model with actual samples in an optimal way using a Kalman filter. As a proof of concept, an example with results obtained with this approach over rice fields by exploiting stacks of TerraSAR-X dual polarization images is shown.

Calmness of the Feasible Set Mapping for Linear Inequality Systems

In this paper we deal with parameterized linear inequality systems in the n-dimensional Euclidean space, whose coefficients depend continuosly on an index ranging in a compact Hausdorff space. The paper is developed in two different parametric settings: the one of only right-hand-side perturbations of the linear system, and that in which both sides of the system can be perturbed. Appealing to the backgrounds on the calmness property, and exploiting the specifics of the current linear structure, we derive different characterizations of the calmness of the feasible set mapping, and provide an operative expresion for the calmness modulus when confined to finite systems. In the paper, the role played by the Abadie constraint qualification in relation to calmness is clarified, and illustrated by different examples. We point out that this approach has the virtue of tackling the calmness property exclusively in terms of the system’s data.

Dynamical Approach for Real-Time Monitoring of Agricultural Crops

In this paper, a novel approach for exploiting multitemporal remote sensing data focused on real-time monitoring of agricultural crops is presented. The methodology is defined in a dynamical system context using state-space techniques, which enables the possibility of merging past temporal information with an update for each new acquisition. The dynamic system context allows us to exploit classical tools in this domain to perform the estimation of relevant variables. A general methodology is proposed, and a particular instance is defined in this study based on polarimetric radar data to track the phenological stages of a set of crops. A model generation from empirical data through principal component analysis is presented, and an extended Kalman filter is adapted to perform phenological stage estimation. Results employing quad-pol Radarsat-2 data over three different cereals are analyzed. The potential of this methodology to retrieve vegetation variables in real time is shown.

The Goodness of Simultaneous Fits in ISIS

In a previous work, we introduced a tool for analyzing multiple datasets simultaneously, which has been implemented into ISIS. This tool was used to fit many spectra of X-ray binaries. However, the large number of degrees of freedom and individual datasets raise an issue about a good measure for a simultaneous fit quality. We present three ways to check the goodness of these fits: we investigate the goodness of each fit in all datasets, we define a combined goodness exploiting the logical structure of a simultaneous fit, and we stack the fit residuals of all datasets to detect weak features. These tools are applied to all RXTE-spectra from GRO 1008−57, revealing calibration features that are not detected significantly in any single spectrum. Stacking the residuals from the best-fit model for the Vela X-1 and XTE J1859+083 data evidences fluorescent emission lines that would have gone undetected otherwise.