User Experience in Human-Technology Interaction. Communication, context and evaluation methodology

En esta Tesis se presentan dos líneas de investigación relacionadas y que contribuyen a las áreas de Interacción Hombre-Tecnología (o Máquina; siglas en inglés: HTI o HMI), lingüística computacional y evaluación de la experiencia del usuario. Las dos líneas en cuestión son el diseño y la evaluación centrada en el usuario de sistemas de Interacción Hombre-Máquina avanzados. En la primera parte de la Tesis (Capítulos 2 a 4) se abordan cuestiones fundamentales del diseño de sistemas HMI avanzados. El Capítulo 2 presenta una panorámica del estado del arte de la investigación en el ámbito de los sistemas conversacionales multimodales, con la que se enmarca el trabajo de investigación presentado en el resto de la Tesis. Los Capítulos 3 y 4 se centran en dos grandes aspectos del diseño de sistemas HMI: un gestor del diálogo generalizado para tratar la Interacción Hombre-Máquina multimodal y sensible al contexto, y el uso de agentes animados personificados (ECAs) para mejorar la robustez del diálogo, respectivamente. El Capítulo 3, sobre gestión del diálogo, aborda el tratamiento de la heterogeneidad de la información proveniente de las modalidades comunicativas y de los sensores externos. En este capítulo se propone, en un nivel de abstracción alto, una arquitectura para la gestión del diálogo con influjos heterogéneos de información, apoyándose en el uso de State Chart XML. En el Capítulo 4 se presenta una contribución a la representación interna de intenciones comunicativas, y su traducción a secuencias de gestos a ejecutar por parte de un ECA, diseñados específicamente para mejorar la robustez en situaciones de diálogo críticas que pueden surgir, por ejemplo, cuando se producen errores de entendimiento en la comunicación entre el usuario humano y la máquina. Se propone, en estas páginas, una extensión del Functional Mark-up Language definido en el marco conceptual SAIBA. Esta extensión permite representar actos comunicativos que realizan intenciones del emisor (la máquina) que no se pretende sean captadas conscientemente por el receptor (el usuario humano), pero con las que se pretende influirle a éste e influir el curso del diálogo. Esto se consigue mediante un objeto llamado Base de Intenciones Comunicativas (en inglés, Communication Intention Base, o CIB). La representación en el CIB de intenciones “no claradas” además de las explícitas permite la construcción de actos comunicativos que realizan simultáneamente varias intenciones comunicativas. En el Capítulo 4 también se describe un sistema experimental para el control remoto (simulado) de un asistente domótico, con autenticación de locutor para dar acceso, y con un ECA en el interfaz de cada una de estas tareas. Se incluye una descripción de las secuencias de comportamiento verbal y no verbal de los ECAs, que fueron diseñados específicamente para determinadas situaciones con objeto de mejorar la robustez del diálogo. Los Capítulos 5 a 7 conforman la parte de la Tesis dedicada a la evaluación. El Capítulo 5 repasa antecedentes relevantes en la literatura de tecnologías de la información en general, y de sistemas de interacción hablada en particular. Los principales antecedentes en el ámbito de la evaluación de la interacción sobre los cuales se ha desarrollado el trabajo presentado en esta Tesis son el Technology Acceptance Model (TAM), la herramienta Subjective Assessment of Speech System Interfaces (SASSI), y la Recomendación P.851 de la ITU-T. En el Capítulo 6 se describen un marco y una metodología de evaluación aplicados a la experiencia del usuario con sistemas HMI multimodales. Se desarrolló con este propósito un novedoso marco de evaluación subjetiva de la calidad de la experiencia del usuario y su relación con la aceptación por parte del mismo de la tecnología HMI (el nombre dado en inglés a este marco es Subjective Quality Evaluation Framework). En este marco se articula una estructura de clases de factores subjetivos relacionados con la satisfacción y aceptación por parte del usuario de la tecnología HMI propuesta. Esta estructura, tal y como se propone en la presente tesis, tiene dos dimensiones ortogonales. Primero se identifican tres grandes clases de parámetros relacionados con la aceptación por parte del usuario: “agradabilidad ” (likeability: aquellos que tienen que ver con la experiencia de uso, sin entrar en valoraciones de utilidad), rechazo (los cuales sólo pueden tener una valencia negativa) y percepción de utilidad. En segundo lugar, este conjunto clases se reproduce para distintos “niveles, o focos, percepción del usuario”. Éstos incluyen, como mínimo, un nivel de valoración global del sistema, niveles correspondientes a las tareas a realizar y objetivos a alcanzar, y un nivel de interfaz (en los casos propuestos en esta tesis, el interfaz es un sistema de diálogo con o sin un ECA). En el Capítulo 7 se presenta una evaluación empírica del sistema descrito en el Capítulo 4. El estudio se apoya en los mencionados antecedentes en la literatura, ampliados con parámetros para el estudio específico de los agentes animados (los ECAs), la auto-evaluación de las emociones de los usuarios, así como determinados factores de rechazo (concretamente, la preocupación por la privacidad y la seguridad). También se evalúa el marco de evaluación subjetiva de la calidad propuesto en el capítulo anterior. Los análisis de factores efectuados revelan una estructura de parámetros muy cercana conceptualmente a la división de clases en utilidad-agradabilidad-rechazo propuesta en dicho marco, resultado que da cierta validez empírica al marco. Análisis basados en regresiones lineales revelan estructuras de dependencias e interrelación entre los parámetros subjetivos y objetivos considerados. El efecto central de mediación, descrito en el Technology Acceptance Model, de la utilidad percibida sobre la relación de dependencia entre la intención de uso y la facilidad de uso percibida, se confirma en el estudio presentado en la presente Tesis. Además, se ha encontrado que esta estructura de relaciones se fortalece, en el estudio concreto presentado en estas páginas, si las variables consideradas se generalizan para cubrir más ampliamente las categorías de agradabilidad y utilidad contempladas en el marco de evaluación subjetiva de calidad. Se ha observado, asimismo, que los factores de rechazo aparecen como un componente propio en los análisis de factores, y además se distinguen por su comportamiento: moderan la relación entre la intención de uso (que es el principal indicador de la aceptación del usuario) y su predictor más fuerte, la utilidad percibida. Se presentan también resultados de menor importancia referentes a los efectos de los ECAs sobre los interfaces de los sistemas de diálogo y sobre los parámetros de percepción y las valoraciones de los usuarios que juegan un papel en conformar su aceptación de la tecnología. A pesar de que se observa un rendimiento de la interacción dialogada ligeramente mejor con ECAs, las opiniones subjetivas son muy similares entre los dos grupos experimentales (uno interactuando con un sistema de diálogo con ECA, y el otro sin ECA). Entre las pequeñas diferencias encontradas entre los dos grupos destacan las siguientes: en el grupo experimental sin ECA (es decir, con interfaz sólo de voz) se observó un efecto más directo de los problemas de diálogo (por ejemplo, errores de reconocimiento) sobre la percepción de robustez, mientras que el grupo con ECA tuvo una respuesta emocional más positiva cuando se producían problemas. Los ECAs parecen generar inicialmente expectativas más elevadas en cuanto a las capacidades del sistema, y los usuarios de este grupo se declaran más seguros de sí mismos en su interacción. Por último, se observan algunos indicios de efectos sociales de los ECAs: la “amigabilidad ” percibida los ECAs estaba correlada con un incremento la preocupación por la seguridad. Asimismo, los usuarios del sistema con ECAs tendían más a culparse a sí mismos, en lugar de culpar al sistema, de los problemas de diálogo que pudieran surgir, mientras que se observó una ligera tendencia opuesta en el caso de los usuarios del sistema con interacción sólo de voz. ABSTRACT This Thesis presents two related lines of research work contributing to the general fields of Human-Technology (or Machine) Interaction (HTI, or HMI), computational linguistics, and user experience evaluation. These two lines are the design and user-focused evaluation of advanced Human-Machine (or Technology) Interaction systems. The first part of the Thesis (Chapters 2 to 4) is centred on advanced HMI system design. Chapter 2 provides a background overview of the state of research in multimodal conversational systems. This sets the stage for the research work presented in the rest of the Thesis. Chapers 3 and 4 focus on two major aspects of HMI design in detail: a generalised dialogue manager for context-aware multimodal HMI, and embodied conversational agents (ECAs, or animated agents) to improve dialogue robustness, respectively. Chapter 3, on dialogue management, deals with how to handle information heterogeneity, both from the communication modalities or from external sensors. A highly abstracted architectural contribution based on State Chart XML is proposed. Chapter 4 presents a contribution for the internal representation of communication intentions and their translation into gestural sequences for an ECA, especially designed to improve robustness in critical dialogue situations such as when miscommunication occurs. We propose an extension of the functionality of Functional Mark-up Language, as envisaged in much of the work in the SAIBA framework. Our extension allows the representation of communication acts that carry intentions that are not for the interlocutor to know of, but which are made to influence him or her as well as the flow of the dialogue itself. This is achieved through a design element we have called the Communication Intention Base. Such r pr s ntation of “non- clar ” int ntions allows th construction of communication acts that carry several communication intentions simultaneously. Also in Chapter 4, an experimental system is described which allows (simulated) remote control to a home automation assistant, with biometric (speaker) authentication to grant access, featuring embodied conversation agents for each of the tasks. The discussion includes a description of the behavioural sequences for the ECAs, which were designed for specific dialogue situations with particular attention given to the objective of improving dialogue robustness. Chapters 5 to 7 form the evaluation part of the Thesis. Chapter 5 reviews evaluation approaches in the literature for information technologies, as well as in particular for speech-based interaction systems, that are useful precedents to the contributions of the present Thesis. The main evaluation precedents on which the work in this Thesis has built are the Technology Acceptance Model (TAM), the Subjective Assessment of Speech System Interfaces (SASSI) tool, and ITU-T Recommendation P.851. Chapter 6 presents the author’s work in establishing an valuation framework and methodology applied to the users’ experience with multimodal HMI systems. A novel user-acceptance Subjective Quality Evaluation Framework was developed by the author specifically for this purpose. A class structure arises from two orthogonal sets of dimensions. First we identify three broad classes of parameters related with user acceptance: likeability factors (those that have to do with the experience of using the system), rejection factors (which can only have a negative valence) and perception of usefulness. Secondly, the class structure is further broken down into several “user perception levels”; at the very least: an overall system-assessment level, task and goal-related levels, and an interface level (e.g., a dialogue system with or without an ECA). An empirical evaluation of the system described in Chapter 4 is presented in Chapter 7. The study was based on the abovementioned precedents in the literature, expanded with categories covering the inclusion of an ECA, the users’ s lf-assessed emotions, and particular rejection factors (privacy and security concerns). The Subjective Quality Evaluation Framework proposed in the previous chapter was also scrutinised. Factor analyses revealed an item structure very much related conceptually to the usefulness-likeability-rejection class division introduced above, thus giving it some empirical weight. Regression-based analysis revealed structures of dependencies, paths of interrelations, between the subjective and objective parameters considered. The central mediation effect, in the Technology Acceptance Model, of perceived usefulness on the dependency relationship of intention-to-use with perceived ease of use was confirmed in this study. Furthermore, the pattern of relationships was stronger for variables covering more broadly the likeability and usefulness categories in the Subjective Quality Evaluation Framework. Rejection factors were found to have a distinct presence as components in factor analyses, as well as distinct behaviour: they were found to moderate the relationship between intention-to-use (the main measure of user acceptance) and its strongest predictor, perceived usefulness. Insights of secondary importance are also given regarding the effect of ECAs on the interface of spoken dialogue systems and the dimensions of user perception and judgement attitude that may have a role in determining user acceptance of the technology. Despite observing slightly better performance values in the case of the system with the ECA, subjective opinions regarding both systems were, overall, very similar. Minor differences between two experimental groups (one interacting with an ECA, the other only through speech) include a more direct effect of dialogue problems (e.g., non-understandings) on perceived dialogue robustness for the voice-only interface test group, and a more positive emotional response for the ECA test group. Our findings further suggest that the ECA generates higher initial expectations, and users seem slightly more confident in their interaction with the ECA than do those without it. Finally, mild evidence of social effects of ECAs was also found: the perceived friendliness of the ECA increased security concerns, and ECA users may tend to blame themselves rather than the system when dialogue problems are encountered, while the opposite may be true for voice-only users.

Analysis of the problems associated with dynamic interaction between train, track and structure at transition zones

El gran desarrollo experimentado por la alta velocidad en los principales países de la Unión Europea, en los últimos 30 años, hace que este campo haya sido y aún sea uno de los principales referentes en lo que a investigación se refiere. Por otra parte, la aparición del concepto super − alta velocidad hace que la investigación en el campo de la ingeniería ferroviaria siga adquiriendo importancia en los principales centros de investigación de los países en los que se desea implantar este modo de transporte, o en los que habiendo sido ya implantado, se pretenda mejorar. Las premisas de eficacia, eficiencia, seguridad y confort, que este medio de transporte tiene como razón de ser pueden verse comprometidas por diversos factores. Las zonas de transición, definidas en la ingeniería ferroviaria como aquellas secciones en las que se produce un cambio en las condiciones de soporte de la vía, pueden afectar al normal comportamiento para el que fue diseñada la infraestructura, comprometiendo seriamente los estándares de eficiencia en el tiempo de viaje, confort de los pasajeros y aumentando considerablemente los costes de mantenimiento de la vía, si no se toman las medidas oportunas. En esta tesis se realiza un estudio detallado de la zonas de transición, concretamente de aquellas en las que existe una cambio en la rigidez vertical de la vía debido a la presencia de un marco hidráulico. Para realizar dicho estudio se lleva a cabo un análisis numérico de interacción entre el vehículo y la estructura, con un modelo bidimensional de elemento finitos, calibrado experimentalmente, en estado de tensión plana. En este análisis se tiene en cuenta el efecto de las irregularidades de la vía y el comportamiento mecánico de la interfaz suelo-estructura, con el objetivo de reproducir de la forma más real posible el efecto de interacción entre el vehículo, la vía y la estructura. Otros efectos como la influencia de la velocidad del tren y los asientos diferenciales, debidos a deformaciones por consolidación de los terraplenes a ambos lados el marco hidráulico, son también analizados en este trabajo. En esta tesis, los cálculos de interacción se han llevado a cabo en dos fases diferentes. En la primera, se ha considerado una interacción sencilla debida al paso de un bogie de un tren Eurostar. Los cálculos derivados de esta fase se han denominado cálculos a corto plazo. En la segunda, se ha realizado un análisis considerando múltiples pasos de bogie del tren Eurostar, conformando un análisis de degradación en el que se tiene en cuenta, en cada ciclo, la deformación de la capa de balasto. Los cálculos derivados de esta fase, son denominados en el texto como cálculos a largo plazo. Los resultados analizados muestran que la utilización de los denominados elementos de contacto es fundamental cuando se desea estudiar la influencia de asientos diferenciales, especialmente en transiciones terraplén-estructura en las que la cuña de cimentación no llega hasta la base de cimentación de la estructura. Por otra parte, tener en cuenta los asientos del terraplén, es sumamente importante, cuando se desea realizar un análisis de degradación de la vía ya que su influencia en la interacción entre el vehículo y la vía es muy elevada, especialmente para valores altos de velocidad del tren. En cuanto a la influencia de las irregularidades de la vía, en los cálculos efectuados, se revela que su importancia es muy notable, siendo su influencia muy destacada cuanto mayor sea la velocidad del tren. En este punto cabe destacar la diferencia de resultados derivada de la consideración de perfiles de irregularidades de distinta naturaleza. Los resultados provenientes de considerar perfiles artificiales son en general muy elevados, siendo estos más apropiados para realizar estudios de otra índole, como por ejemplo de seguridad al descarrilamiento. Los resultados provenientes de perfiles reales, dados por diferentes Administradores ferroviarios, presentan resultados menos elevados y más propios del problema analizar. Su influencia en la interacción dinámica entre el vehículo y la vía es muy importante, especialmente para velocidades elevadas del tren. Además el fenómeno de degradación conocido como danza de traviesas, asociado a zonas de transición, es muy susceptible a la consideración de irregularidades de la vía, tal y como se desprende de los cálculos efectuados a largo plazo. The major development experienced by high speed in the main countries of the European Union, in the last 30 years, makes railway research one of the main references in the research field. It should also be mentioned that the emergence of the concept superhigh − speed makes research in the field of Railway Engineering continues to gain importance in major research centers in the countries in which this mode of transportation is already implemented or planned to be implemented. The characteristics that this transport has as rationale such as: effectiveness, efficiency, safety and comfort, may be compromised by several factors. The transition zones are defined in railway engineering as a region in which there is an abrupt change of track stiffness. This stiffness variation can affect the normal behavior for which the infrastructure has been designed, seriously compromising efficiency standards in the travel time, passenger comfort and significantly increasing the costs of track maintenance, if appropriate measures are not taken. In this thesis a detailed study of the transition zones has been performed, particularly of those in which there is a change in vertical stiffness of the track due to the presence of a reinforced concrete culvert. To perform such a study a numerical interaction analysis between the vehicle, the track and the structure has been developed. With this purpose a two-dimensional finite element model, experimentally calibrated, in a state of plane stress, has been used. The implemented numerical models have considered the effects of track irregularities and mechanical behavior of soil-structure interface, with the objective of reproducing as accurately as possible the dynamic interaction between the vehicle the track and the structure. Other effects such as the influence of train speed and differential settlement, due to secondary consolidation of the embankments on both sides of culvert, have also been analyzed. In this work, the interaction analysis has been carried out in two different phases. In the first part a simple interaction due to the passage of a bogie of a Eurostar train has been considered. Calculations derived from this phase have been named short-term analysis. In the second part, a multi-load assessment considering an Eurostar train bogie moving along the transition zone, has been performed. The objective here is to simulate a degradation process in which vertical deformation of the ballast layer was considered. Calculations derived from this phase have been named long-term analysis. The analyzed results show that the use of so-called contact elements is essential when one wants to analyze the influence of differential settlements, especially in embankment-structure transitions in which the wedge-shaped backfill does not reach the foundation base of the structure. Moreover, considering embankment settlement is extremely important when it is desired to perform an analysis of track degradation. In these cases the influence on the interaction behaviour between the vehicle and the track is very high, especially for higher values of speed train. Regarding the influence of the track irregularities, this study has proven that the track’s dynamic response is heavily influenced by the irregularity profile and that this influence is more important for higher train velocities. It should also be noted that the difference in results derived from consideration of irregularities profiles of different nature. The results coming from artificial profiles are generally very high, these might be more appropriate in order to study other effects, such as derailment safety. Results from real profiles, given by the monitoring works of different rail Managers, are softer and they fit better to the context of this thesis. The influence of irregularity profiles on the dynamic interaction between the train and the track is very important, especially for high-speeds of the train. Furthermore, the degradation phenomenon known as hanging sleepers, associated with transition zones, is very susceptible to the consideration of track irregularities, as it can be concluded from the long-term analysis.

Preferential interaction of the his pause RNA hairpin with RNA polymerase β subunit residues 904–950 correlates with strong transcriptional pausing

RNA secondary structures (hairpins) that form as the nascent RNA emerges from RNA polymerase are important components of many signals that regulate transcription, including some pause sites, all ρ-independent terminators, and some antiterminators. At the his leader pause site, a 5-bp-stem, 8-nt-loop pause RNA hairpin forms 11 nt from the RNA 3′ end and stabilizes a transcription complex conformation slow to react with NTP substrate. This stabilization appears to depend at least in part on an interaction with RNA polymerase. We tested for RNA hairpin interaction with the paused polymerase by crosslinking 5-iodoUMP positioned specifically in the hairpin loop. In the paused conformation, strong and unusual crosslinking of the pause hairpin to β904–950 replaced crosslinking to β′ and to other parts of β that occurred in nonpaused complexes prior to hairpin formation. These changes in nascent RNA interactions may inhibit reactive alignment of the RNA 3′ end in the paused complex and be related to events at ρ-independent terminators.

Interaction of pigeon cytochrome c-(43–58) peptide analogs with either T cell antigen receptor or I-Ab molecule

We determined that a pigeon cytochrome c-derived peptide, p43–58, possesses two anchor residues, 46 and 54, for binding with the I-Ab molecule that are compatible to the position 1 (P1) and position 9 (P9) of the core region in the major histocompatibility complex (MHC) class II binding peptides, respectively. In the present study to analyze each binding site between P1 and P9 of p43–58 to either I-Ab or T cell antigen receptor (TCR), we investigated T cell responses to a series of peptides (P2K, P3K, P4K, P5K, P6K, P7K, and P8E) that sequentially substituted charged amino acid residues for the residues at P2 to P8 of p43–58. T cells from C57BL/10 (I-Ab) mice immunized with P4K or P6K did not mount appreciable proliferative responses to the immunogens, but those primed with other peptides (P2K, P3K, P5K, P7K, and P8E) showed substantial responses in an immunogen-specific manner. It was demonstrated by binding studies that P1 and P9 functioned as main anchors and P4 and P6 functioned as secondary anchors to I-Ab. Analyses of Vβ usage of T cell lines specific for these analogs suggested that P8 interacts with the complementarity-determining region 1 (CDR1)/CDR2 of the TCR β chain. Furthermore, sequencing of the TCR on T cell hybridomas specific for these analogs indicated that P5 interacts with the CDR3 of the TCR β chain. The present findings are consistent with the three-dimensional structure of the trimolecular complex that has been reported for TCR/peptide/MHC class I molecules.

Comparative analyses of the secondary structures of synthetic and intracellular yeast MFA2 mRNAs

The overall folded (global) structure of mRNA may be critical to translation and turnover control mechanisms, but it has received little experimental attention. Presented here is a comparative analysis of the basic features of the global secondary structure of a synthetic mRNA and the same intracellular eukaryotic mRNA by dimethyl sulfate (DMS) structure probing. Synthetic MFA2 mRNA of Saccharomyces cerevisiae first was examined by using both enzymes and chemical reagents to determine single-stranded and hybridized regions; RNAs with and without a poly(A) tail were compared. A folding pattern was obtained with the aid of the mfold program package that identified the model that best satisfied the probing data. A long-range structural interaction involving the 5′ and 3′ untranslated regions and causing a juxtaposition of the ends of the RNA, was examined further by a useful technique involving oligo(dT)-cellulose chromatography and antisense oligonucleotides. DMS chemical probing of A and C nucleotides of intracellular MFA2 mRNA was then done. The modification data support a very similar intracellular structure. When low reactivity of A and C residues is found in the synthetic RNA, ≈70% of the same sites are relatively more resistant to DMS modification in vivo. A slightly higher sensitivity to DMS is found in vivo for some of the A and C nucleotides predicted to be hybridized from the synthetic structural model. With this small mRNA, the translation process and mRNA-binding proteins do not block DMS modifications, and all A and C nucleotides are modified the same or more strongly than with the synthetic RNA.

A transcriptional activating region with two contrasting modes of protein interaction

A C-terminal segment of the yeast activator Gal4 manifests two functions: When tethered to DNA, it elicits gene activation, and it binds the inhibitor Gal80. Here we examine the effects on these two functions of cysteine and proline substitutions. We find that, although certain cysteine substitutions diminish interaction with Gal80, those substitutions have little effect on the activating function in vivo and interaction with TATA box-binding protein (TBP) in vitro. Proline substitutions introduced near residues critical for Gal80 binding abolish that interaction but once again have no effect on the activating function. Crosslinking experiments show that a defined position in the activating peptide is in close proximity to TBP and Gal80 in the two separate reactions and show that binding of the inhibitor blocks binding to TBP. Thus, the same stretch of amino acids are involved in two quite different protein–protein interactions: binding to Gal80, which depends on a precise sequence and the formation of a defined secondary structure, or interactions with the transcriptional machinery in vivo, which are not impaired by perturbations of either sequence or structure.

DNA annealing by Rad52 Protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA

Homologous recombination in Saccharomyces cerevisiae depends critically on RAD52 function. In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects the annealing of RPA–ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA–Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA–ssDNA complex does not. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast.

Brownian flocculation of polymer colloids in the presence of a secondary minimum

Most analyses of Brownian flocculation apply to conditions where London–van der Waals attractive forces cause particles to be strongly bound in a deep interparticle potential well. In this paper, results are reported that show the interaction between primary- and secondary-minimum flocculation when the interparticle potential curve reflects both attractive and electrostatic repulsive forces. The process is highly time-dependent because of transfer of particles from secondary- to primary-minimum flocculation. Essential features of the analysis are corroborated by experiments with 0.80-μm polystyrene spheres suspended in aqueous solutions of NaCl over a range of ionic strengths. In all cases, experiments were restricted to the initial stage of coagulation, where singlets and doublets predominate.

Formation of a GNRA tetraloop in P5abc can disrupt an interdomain interaction in the Tetrahymena group I ribozyme

The secondary structure of a truncated P5abc subdomain (tP5abc, a 56-nucleotide RNA) of the Tetrahymena thermophila group I intron ribozyme changes when its tertiary structure forms. We have now used heteronuclear NMR spectroscopy to determine its conformation in solution. The tP5abc RNA that contains only secondary structure is extended compared with the tertiary folded form; both forms coexist in slow chemical exchange (the interconversion rate constant is slower than 1 s−1) in the presence of magnesium. Kinetic experiments have shown that tertiary folding of the P5abc subdomain is one of the earliest folding transitions in the group I intron ribozyme, and that it leads to a metastable misfolded intermediate. Previous mutagenesis studies suggest that formation of the extended P5abc structure described here destabilize a misfolded intermediate. This study shows that the P5abc RNA subdomain containing a GNRA tetraloop in P5c (in contrast to the five-nucleotide loop P5c in the tertiary folded ribozyme) can disrupt the base-paired interdomain (P14) interaction between P5c and P2.

The specificity of the secondary DNA binding site of RecA protein defines its role in DNA strand exchange.

The RecA protein-single-stranded DNA (ssDNA) filament can bind a second DNA molecule. Binding of ssDNA to this secondary site shows specificity, in that polypyrimidinic DNA binds to the RecA protein-ssDNA filament with higher affinity than polypurinic sequences. The affinity of ssDNA, which is identical in sequence to that bound in the primary site, is not always greater than that of nonhomologous DNA. Moreover, this specificity of DNA binding does not depend on the sequence of the DNA bound to the RecA protein primary site. We conclude that the specificity reflects an intrinsic property of the secondary site of RecA protein rather than an interaction between DNa molecules within nucleoprotein filament--i.e., self-recognition. The secondary DNA binding site displays a higher affinity for ssDNA than for double-stranded DNA, and the binding of ssDNA to the secondary site strongly inhibits DNA strand exchange. We suggest that the secondary binding site has a dual role in DNA strand exchange. During the homology search, it binds double-stranded DNA weakly; upon finding local homology, this site binds, with higher affinity, the ssDNA strand that is displaced during DNA strand exchange. These characteristics facilitate homologous pairing, promote stabilization of the newly formed heteroduplex DNA, and contribute to the directionality of DNA strand exchange.

Targeting nucleic acid secondary structures by antisense oligonucleotides designed through in vitro selection.

Using an in vitro selection approach, we have isolated oligonucleotides that can bind to a DNA hairpin structure. Complex formation of these oligonucleotides with the target hairpin involves some type of triple-stranded structure with noncanonical interaction, as indicated by bandshift assays and footprinting studies. The selected oligomers can block restriction endonuclease cleavage of the target hairpin in a sequence-specific manner. We demonstrate that in vitro selection can extend the antisense approach to functional targeting of secondary structure motifs. This could provide a basis for interfering with regulatory processes mediated by a variety of nucleic acid structures.

Genetic and comparative analyses reveal an alternative secondary structure in the region of nt 912 of Escherichia coli 16S rRNA.

Mutations at position 912 of Escherichia coli 16S rRNA result in two notable phenotypes. The C-->U transition confers resistance to streptomycin, a translational-error-inducing antibiotic, while a C-->G transversion causes marked retardation of cell growth rate. Starting with the slow-growing G912 mutant, random mutagenesis was used to isolate a second site mutation that restored growth nearly to the wild-type rate. The second site mutation was identified as a G-->C transversion at position 885 in 16S rRNA. Cells containing the G912 mutation had an increased doubling time, abnormal sucrose gradient ribosome/subunit profile, increased sensitivity to spectinomycin, dependence upon streptomycin for growth in the presence of spectinomycin, and slower translation rate, whereas cells with the G912/C885 double mutation were similar to wild type in these assays. Comparative analysis showed there was significant covariation between positions 912 and 885. Thus the second-site suppressor analysis, the functional assays, and the comparative data suggest that the interaction between nt 912 and nt 885 is conserved and necessary for normal ribosome function. Furthermore, the comparative data suggest that the interaction extends to include G885-G886-G887 pairing with C912-U911-C910. An alternative secondary structure element for the central domain of 16S rRNA is proposed.

Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces a spectrum of exoproducts many of which have been implicated in the pathogenesis of human infection. Expression of some of these factors requires cell-cell communication involving the interaction of a small diffusible molecule, an "autoinducer," with a positive transcriptional activator. In P. aeruginosa PAO1, LasI directs the synthesis of the autoinducer N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), which activates the positive transcriptional activator, LasR. Recently, we have discovered a second signaling molecule-based modulon in PAO1, termed vsm, which contains the genes vsmR and vsmI. Using HPLC, mass spectrometry, and NMR spectroscopy we now establish that in Escherichia coli, VsmI directs the synthesis of N-butanoyl-L-homoserine lactone (BHL) and N-hexanoyl-L-homoserine lactone (HHL). These compounds are present in the spent culture supernatants of P. aeruginosa in a molar ratio of approximately 15:1 and their structures were unequivocally confirmed by chemical synthesis. Addition of either BHL or HHL to PAN067, a pleiotropic P. aeruginosa mutant unable to synthesize either of these autoinducers, restored elastase, chitinase, and cyanide production. In E. coli carrying a vsmR/vsmI'::lux transcriptional fusion, BHL and HHL activated VsmR to a similar extent. Analogues of these N-acyl-L-homoserine lactones in which the N-acyl side chain has been extended and/or oxidized at the C-3 position exhibit substantially lower activity (e.g., OdDHL) or no activity (e.g., dDHL) in this lux reporter assay. These data indicate that multiple families of quorum sensing modulons interactively regulate gene expression in P. aeruginosa.

Pleasant sweeping grooming touch in rhesus monkey: effects on autonomic nervous system and its codification in posterior insular and secondary somatosensory cortex

Il tatto assume un'importanza fondamentale nella vita quotidiana, in quanto ci permette di discriminare le caratteristiche fisiche di un oggetto specifico, di identificarlo e di eventualmente integrare le suddette informazioni tattili con informazioni provenienti da altri canali sensoriali. Questa è la componente sensoriale-discriminativa del tatto. Tuttavia quotidianamente il tatto assume un ruolo fondamentale durante le diverse interazioni sociali, positive, come quando abbracciamo o accarezziamo una persona con cui abbiamo un rapporto affettivo e negative, per esempio quando allontaniamo una persona estranea dal nostro spazio peri-personale. Questa componente è la cosiddetta dimensione affettiva-motivazionale, la quale determina la codifica della valenza emotiva che l'interazione assume. Questa componente ci permette di creare, mantenere o distruggere i legami sociali in relazione al significato che il tocco assume durante l'interazione. Se per esempio riceviamo una carezza da un familiare, questa verrà percepita come piacevole e assumerà un significato affiliativo. Questo tipo di tocco è comunente definito come Tocco Sociale (Social Touch). Gli aspetti discriminativi del tatto sono stati ben caratterizzati, in quanto storicamente, il ruolo del tatto è stato considerato quello di discriminare le caratteristiche di ciò che viene toccato, mentre gli aspetti affettivi sono stati solo recentemente indagati considerando la loro importanza nelle interazioni sociali. Il tocco statico responsabile dell'aspetto discriminante attiva a livello della pelle le grandi fibre mieliniche (Aβ), modulando a livello del sistema nervoso centrale le cortecce sensoriali, sia primarie che secondarie. Questo permette la codifica a livello del sistema nervoso centrale delle caratteristiche fisiche oggettive degli oggetti toccati. Studi riguardanti le caratteristiche del tocco affiliativo sociale hanno messo in evidenza che suddetta stimolazione tattile 1) è un particolare tocco dinamico che avviene sul lato peloso delle pelle con una velocità di 1-10 cm/sec; 2) attiva le fibre amieliniche (fibre CT o C-LTMRs); 3) induce positivi effetti autonomici, ad esempio la diminuzione della frequenza cardiaca e l'aumento della variabilità della frequenza cardiaca; e 4) determina la modulazione di regioni cerebrali coinvolte nella codifica del significato affiliativo dello stimolo sensoriale periferico, in particolare la corteccia insulare. Il senso del tatto, con le sue due dimensioni discriminativa e affiliativa, è quotidianamente usato non solo negli esseri umani, ma anche tra i primati non umani. Infatti, tutti i primati non umani utilizzano la componente discriminativa del tatto per identificare gli oggetti e il cibo e l'aspetto emotivo durante le interazioni sociali, sia negative come durante un combattimento, che positive, come durante i comportamenti affiliativi tra cui il grooming. I meccanismi di codifica della componente discriminativa dei primati non umani sono simili a quelli umani. Tuttavia, si conosce ben poco dei meccanismi alla base della codifica del tocco piacevole affiliativo. Pur essendo ben noto che i meccanorecettori amilienici C-LTMRs sono presenti anche sul lato peloso della pelle dei primati non umani, attualmente non ci sono studi riguardanti la correlazione tra il tocco piacevole e la loro modulazione, come invece è stato ampiamente dimostrato nell'uomo. Recentemente è stato ipotizzato (Dunbar, 2010) il ruolo delle fibre C-LTMRs durante il grooming, in particolare durante il cosiddetto swepping. Il grooming è costituito da due azioni motorie, lo sweeping e il picking che vengono eseguite in modo ritmico. Durante lo sweeping la scimmia agente muove il pelo della scimmia ricevente con un movimento a mano aperta, per poter vedere il preciso punto della pelle dove eseguire il picking, ovvero dove prendere la pelle a livello della radice del pelo con le unghie dell'indice e del pollice e tirare per rimuovere parassiti o uova di parassiti e ciò che è rimasto incastrato nel pelo. Oltre il noto ruolo igenico, il grooming sembra avere anche una importante funzione sociale affiliativa. Come la carezza nella società umana, cosi il grooming tra i primati non umani è considerato un comportamento. Secondo l'ipotesi di Dunbar l'attivazione delle C-LTMRs avverrebbe durante lo sweeping e questo porta a supporre che lo sweeping, come la carezza umana, costituisca una componente affiliativa del grooming, determinando quindi a contribuire alla sua codifica come comportamento sociale. Fino ad ora non vi è però alcuna prova diretta a sostegno di questa ipotesi. In particolare, 1) la velocità cui viene eseguito lo sweeping è compatibile con la velocità di attivazione delle fibre CT nell'uomo e quindi con la velocità tipica della carezza piacevole di carattere sociale affiliativo (1-10 cm/sec)?; 2) lo sweeping induce la stessa modulazione del sistema nervoso autonomo in direzione della modulazione del sistema vagale, come il tocco piacevole nell'uomo, attraverso l'attivazione delle fibre CT?; 3) lo sweeping modula la corteccia insulare, cosi come il tocco piacevole viene codificato come affiliativo nell'uomo mediante le proiezioni delle fibre CT a livello dell'insula posteriore? Lo scopo del presente lavoro è quella di testare l'ipotesi di Dunbar sopra citata, cercando quindi di rispondere alle suddette domande. Le risposte potrebbero consentire di ipotizzare la somiglianza tra lo sweeping, caratteristico del comportamento affiliativo di grooming tra i primati non umani e la carezza. In particolare, abbiamo eseguito 4 studi pilota. Nello Studio 1 abbiamo valutato la velocità con cui viene eseguito lo sweeping tra scimmie Rhesus, mediante una analisi cinematica di video registrati tra un gruppo di scimmie Rhesus. Negli Studi 2 e 3 abbiamo valutato gli effetti sul sistema nervoso autonomo dello sweeping eseguito dallo sperimentatore su una scimmia Rhesus di sesso maschile in una tipica situazione sperimentale. La stimolazione tattile è stata eseguita a diverse velocità, in accordo con i risultati dello Studio 1 e degli studi umani che hanno dimostrato la velocità ottimale e non ottimale per l'attivazione delle C-LTMRs. In particolare, nello Studio 2 abbiamo misurato la frequenza cardiaca e la variabilità di questa, come indice della modulatione vagale, mentre nello Studio 3 abbiamo valutato gli effetti dello sweeping sul sistema nervoso autonomo in termini di variazioni di temperatura del corpo, nello specifico a livello del muso della scimmia. Infine, nello Studio 4 abbiamo studiato il ruolo della corteccia somatosensoriale secondaria e insulare nella codifica dello sweeping. A questo scopo abbiamo eseguito registrazioni di singoli neuroni mentre la medesima scimmia soggetto sperimentale dello Studio 2 e 3, riceveva lo sweeping a due velocità, una ottimale per l'attivazione delle C-LTMRs secondo gli studi umani e i risultati dei tre studi sopra citati, ed una non ottimale. I dati preliminari ottenuti, dimostrano che 1) (Studio 1) lo sweeping tra scimmie Rhesus viene eseguito con una velocità media di 9.31 cm/sec, all'interno dell'intervallo di attivazione delle fibre CT nell'uomo; 2) (Studio 2) lo sweeping eseguito dallo sperimentatore sulla schiena di una scimmia Rhesus di sesso maschile in una situazione sperimentale determina una diminuzione della frequenza cardiaca e l'aumento della variabilità della frequenza cardiaca se eseguito alla velocità di 5 e 10 cm/sec. Al contrario, lo sweeping eseguito ad una velocità minore di 1 cm/sec o maggiore di 10 cm/sec, determina l'aumento della frequenza cardiaca e la diminuzione della variabilità di questa, quindi il decremento dell'attivazione del sistema nervoso parasimpatico; 3) (Studio 3) lo sweeping eseguito dallo sperimentatore sulla schiena di una scimmia Rhesus di sesso maschile in una situazione sperimentale determina l'aumento della temperatura corporea a livello del muso della scimmia se eseguito alla velocità di 5-10 cm/sec. Al contrario, lo sweeping eseguito ad una velocità minore di 5 cm/sec o maggiore di 10 cm/sec, determina la diminuzione della temperatura del muso; 4) (Studio 4) la corteccia somatosensoriale secondaria e la corteccia insulare posteriore presentano neuroni selettivamente modulati durante lo sweeping eseguito ad una velocità di 5-13 cm/sec ma non neuroni selettivi per la codifica della velocità dello sweeping minore di 5 cm/sec. Questi risultati supportano l'ipotesi di Dunbar relativa al coinvolgimento delle fibre CT durante lo sweeping. Infatti i dati mettono in luce che lo sweeping viene eseguito con una velocità (9.31 cm/sec), simile a quella di attivazione delle fibre CT nell'uomo (1-10 cm/sec), determina gli stessi effetti fisiologici positivi in termini di frequenza cardiaca (diminuzione) e variabilità della frequenza cardiaca (incremento) e la modulazione delle medesime aree a livello del sistema nervoso centrale (in particolare la corteccia insulare). Inoltre, abbiamo dimostrato per la prima volta che suddetta stimolazione tattile determina l'aumento della temperatura del muso della scimmia. Il presente studio rappresenta la prima prova indiretta dell'ipotesi relativa alla modulazione del sistema delle fibre C-LTMRs durante lo sweeping e quindi della codifica della stimolazione tattile piacevole affiliativa a livello del sistema nervoso centrale ed autonomo, nei primati non umani. I dati preliminari qui presentati evidenziano la somiglianza tra il sistema delle fibre CT dell'uomo e del sistema C-LTMRs nei primati non umano, riguardanti il Social Touch. Nonostante ciò abbiamo riscontrato alcune discrepanze tra i risultati da noi ottenuti e quelli invece ottenuti dagli studi umani. La velocità media dello sweeping è di 9.31 cm / sec, rasente il limite superiore dell’intervallo di velocità che attiva le fibre CT nell'uomo. Inoltre, gli effetti autonomici positivi, in termini di battito cardiaco, variabilità della frequenza cardiaca e temperatura a livello del muso, sono stati evidenziati durante lo sweeping eseguito con una velocità di 5 e 10 cm/sec, quindi al limite superiore dell’intervallo ottimale che attiva le fibre CT nell’uomo. Al contrario, lo sweeping eseguito con una velocità inferiore a 5 cm/sec e superiore a 10 cm/sec determina effetti fisiologici negativo. Infine, la corteccia insula sembra essere selettivamente modulata dallo stimolazione eseguita alla velocità di 5-13 cm/sec, ma non 1-5 cm/sec. Quindi, gli studi sul sistema delle fibre CT nell’uomo hanno dimostrato che la velocità ottimale è 1-10 cm/sec, mentre dai nostri risultati la velocità ottimale sembra essere 5-13 cm / sec. Quindi, nonostante l'omologia tra il sistema delle fibre CT nell'umano deputato alla codifica del tocco piacevole affiliativo ed il sistema delle fibre C-LTMRs nei primati non umani, ulteriori studi saranno necessari per definire con maggiore precisione la velocità ottimale di attivazione delle fibre C-LTMR e per dimostrare direttamente la loro attivazione durante lo sweeping, mediante la misurazione diretta della loro modulazione. Studi in questa direzione potranno confermare l'omologia tra lo sweeping in qualità di tocco affiliativo piacevole tra i primati non umani e la carezza tra gli uomini. Infine, il presente studio potrebbe essere un importante punto di partenza per esplorare il meccanismo evolutivo dietro la trasformazione dello sweeping tra primati non umani, azione utilitaria eseguita durante il grooming, a carezza, gesto puramente affiliativo tra gli uomini.

Examining the Relationship Between Secondary Traumatic Stress and Sickness Absenteeism within 9-1-1 Emergency Call Centers

Thesis (Master's)--University of Washington, 2016-06