(Table 1) Isotopic and trace-element composition of ODP Leg 127/128 basalts

The western Pacific includes many volcanic island arc and backarc complexes, yet multi-isotopic studies of them are rare. Basement rocks of the Sea of Japan backarc basin were encountered at Sites 794,795, and 797, and consisted of basaltic sills and lava flows. These rocks exhibit a broad range in isotopic composition, broader than that seen in any other western Pacific arc or backarc system: 87Sr/86Sr = 0.70369 to 0.70499, 143Nd/144Nd = 0.51267 to 0.51317, 206Pb/204Pb = 17.64 to 18.36. The samples form highly correlated arrays between very depleted mid-ocean ridge basalt (MORB) and the Pacific pelagic sediment fields on Pb-Pb plots. Similarly, on plots of Sr-Pb and Nd-Pb, the Sea of Japan samples lie on mixing curves between depleted mantle and enriched mantle ("EM II"), which is interpreted to be of average crustal or pelagic sediment composition. The source of these backarc rocks appears to be a MORB-like mantle source, contaminated by pelagic sediments. Unlike the Mariana and Izu arc/backarc systems, Japanese arc and backarc rocks are indistinguishable from each other in a Sr-Nd isotope plot, and have similar trends in Pb-Pb plots. Thus, sediment contamination of the mantle wedge appears to control the isotopic compositions of both the arc and backarc magmas. Two-component mixing calculations suggest that the percentage of sediments in the magma source varies from 0.5% to 2.5%.

Pore water isotopic ratios of CO2 and SO4, and sediment CH4 content of ODP Leg 164 sites

A unique set of geochemical pore-water data, characterizing the sulfate reduction and uppermost methanogenic zones, has been collected at the Blake Ridge (offshore southeastern North America) from Ocean Drilling Program (ODP) Leg 164 cores and piston cores. The d13C values of dissolved CO2 (sum CO2) are as 13C-depleted as -37.7 per mil PDB (Site 995) at the sulfate-methane interface, reflecting a substantial contribution of isotopically light carbon from methane. Although the geochemical system is complex and difficult to fully quantify, we use two methods to constrain and illustrate the intensity of anaerobic methane oxidation in Blake Ridge sediments. An estimate using a two-component mixing model suggests that ~24% of the carbon residing in the sum CO2 pool is derived from biogenic methane. Independent diagenetic modeling of a methane concentration profile (Site 995) indicates that peak methane oxidation rates approach 0.005 µmol/cm**3/yr, and that anaerobic methane oxidation is responsible for consuming ~35% of the total sulfate flux into the sediments. Thus, anaerobic methane oxidation is a significant biogeochemical sink for sulfate, and must affect interstitial sulfate concentrations and sulfate gradients. Such high proportions of sulfate depletion because of anaerobic methane oxidation are largely undocumented in continental rise sediments with overlying oxic bottom waters. We infer that the additional amount of sulfate depleted through anaerobic methane oxidation, fueled by methane flux from below, causes steeper sulfate gradients above methane-rich sediments. Similar pore water chemistries should occur at other methane-rich, continental-rise settings associated with gas hydrates.

Approximate Relational Reasoning for Probabilistic Programs

La seguridad verificada es una metodología para demostrar propiedades de seguridad de los sistemas informáticos que se destaca por las altas garantías de corrección que provee. Los sistemas informáticos se modelan como programas probabilísticos y para probar que verifican una determinada propiedad de seguridad se utilizan técnicas rigurosas basadas en modelos matemáticos de los programas. En particular, la seguridad verificada promueve el uso de demostradores de teoremas interactivos o automáticos para construir demostraciones completamente formales cuya corrección es certificada mecánicamente (por ordenador). La seguridad verificada demostró ser una técnica muy efectiva para razonar sobre diversas nociones de seguridad en el área de criptografía. Sin embargo, no ha podido cubrir un importante conjunto de nociones de seguridad “aproximada”. La característica distintiva de estas nociones de seguridad es que se expresan como una condición de “similitud” entre las distribuciones de salida de dos programas probabilísticos y esta similitud se cuantifica usando alguna noción de distancia entre distribuciones de probabilidad. Este conjunto incluye destacadas nociones de seguridad de diversas áreas como la minería de datos privados, el análisis de flujo de información y la criptografía. Ejemplos representativos de estas nociones de seguridad son la indiferenciabilidad, que permite reemplazar un componente idealizado de un sistema por una implementación concreta (sin alterar significativamente sus propiedades de seguridad), o la privacidad diferencial, una noción de privacidad que ha recibido mucha atención en los últimos años y tiene como objetivo evitar la publicación datos confidenciales en la minería de datos. La falta de técnicas rigurosas que permitan verificar formalmente este tipo de propiedades constituye un notable problema abierto que tiene que ser abordado. En esta tesis introducimos varias lógicas de programa quantitativas para razonar sobre esta clase de propiedades de seguridad. Nuestra principal contribución teórica es una versión quantitativa de una lógica de Hoare relacional para programas probabilísticos. Las pruebas de correción de estas lógicas son completamente formalizadas en el asistente de pruebas Coq. Desarrollamos, además, una herramienta para razonar sobre propiedades de programas a través de estas lógicas extendiendo CertiCrypt, un framework para verificar pruebas de criptografía en Coq. Confirmamos la efectividad y aplicabilidad de nuestra metodología construyendo pruebas certificadas por ordendor de varios sistemas cuyo análisis estaba fuera del alcance de la seguridad verificada. Esto incluye, entre otros, una meta-construcción para diseñar funciones de hash “seguras” sobre curvas elípticas y algoritmos diferencialmente privados para varios problemas de optimización combinatoria de la literatura reciente. ABSTRACT The verified security methodology is an emerging approach to build high assurance proofs about security properties of computer systems. Computer systems are modeled as probabilistic programs and one relies on rigorous program semantics techniques to prove that they comply with a given security goal. In particular, it advocates the use of interactive theorem provers or automated provers to build fully formal machine-checked versions of these security proofs. The verified security methodology has proved successful in modeling and reasoning about several standard security notions in the area of cryptography. However, it has fallen short of covering an important class of approximate, quantitative security notions. The distinguishing characteristic of this class of security notions is that they are stated as a “similarity” condition between the output distributions of two probabilistic programs, and this similarity is quantified using some notion of distance between probability distributions. This class comprises prominent security notions from multiple areas such as private data analysis, information flow analysis and cryptography. These include, for instance, indifferentiability, which enables securely replacing an idealized component of system with a concrete implementation, and differential privacy, a notion of privacy-preserving data mining that has received a great deal of attention in the last few years. The lack of rigorous techniques for verifying these properties is thus an important problem that needs to be addressed. In this dissertation we introduce several quantitative program logics to reason about this class of security notions. Our main theoretical contribution is, in particular, a quantitative variant of a full-fledged relational Hoare logic for probabilistic programs. The soundness of these logics is fully formalized in the Coq proof-assistant and tool support is also available through an extension of CertiCrypt, a framework to verify cryptographic proofs in Coq. We validate the applicability of our approach by building fully machine-checked proofs for several systems that were out of the reach of the verified security methodology. These comprise, among others, a construction to build “safe” hash functions into elliptic curves and differentially private algorithms for several combinatorial optimization problems from the recent literature.

Optimization of Pumping System in Solar Pumped Laser.

The coupling between solar light radiation and laser rod medium in a solar pumped laser affects the efficiency of the laser. To optimize the pumping system, simulation of the two-stage pumping system with a Fresnel lens and conic pumping cavity is carried out with Tracepro software. According to the power density distribution along the axis at focal place of the Fresnel lens, the diameter and position of the pumping cavity window and the distance of the window from the Fresnel lens are optimized. The power density distributions along the laser rod axis of different cavity lengths and different cavity tapers are also analyzed. The optimal structure of taper cavity is obtained. The mirror relecting cavity and ceramic cavity are introduced in detail.

Monitorización de sistemas en alta disponibilidad

El objetivo del proyecto es implantar un sistema de monitorización, con la peculiaridad de encontrarse en alta disponibilidad, esto es, que el servicio (la monitorización de una infraestructura) se preste forma continua y no se vea interrumpido. Dado que el propósito del sistema es monitorizar activamente una infraestructura, ha sido necesario desplegar una infraestructura, además del sistema de monitorización. La infraestructura en cuestión está compuesta por un servidor de documentación, un servidor de base de datos, un servidor de aplicaciones y un servidor web. El sistema de monitorización se ha desplegado en la misma red de área local de esta infraestructura y monitoriza que los servicios prestados por los componentes de esta infraestructura se encuentren operativos y funcionando adecuadamente. Así pues, se tendría un sistema de monitorización local funcional. No obstante, el proyecto plantea un sistema escalable, que esté preparado para el crecimiento de la infraestructura y continúe siendo eficiente. Para ello, sistema de monitorización se encuentre dividido por dos componentes:  Sonda delegada: monitoriza localmente los activos de la infraestructura a monitorizar, es el escenario anteriormente descrito.  Sonda maestra: recibe los resultados de la monitorización realizada, este sistema puede estar desplegado en otra red distinta a la sonda delegada. Este enfoque no solo es escalable, sino también es fiel a la realidad, pues puede darse el caso de que las sondas pertenezcan a distintas infraestructuras e inclusive, distintas organizaciones, y se comuniquen a través de internet, mediante un mecanismo confiable a ser posible. El proyecto plantea que ambas sondas se encuentren en alta disponibilidad (en adelante HA, referente a high availability), y que cada sonda está compuesta por dos equipos (nodos, en adelante). Como se analizará en posteriores capítulos, existen diversas configuraciones que permiten implantar un sistema en HA, la configuración escogida para el proyecto es Activo – Pasivo(los detalles de esta configuración también se explican en posteriores capítulos). Para finalizar, se estudiara la posibilidad de ofrecer respuestas activas en ciertas situaciones y configuraciones adicionales sobre el sistema de monitorización base. Por otro lado, para la implantación del proyecto se ha usado software de código abierto para la virtualización de la infraestructura (Virtual Box y GNS3), los sistemas operativos base (Linux), el sistema de monitorización(Nagios Core) así como el software que implementa la HA (corosync y pacemaker).---ABSTRACT---The aim of the Project is to implement a monitoring system, with the peculiarity of being deployed in high availability, what it is that the service (monitoring infrastructure) is provided continuously and not interrupted. As the purpose of the system is monitoring infrastructure actively, an infrastructure has been deployed, and also the monitoring system. The infrastructure monitored is composed of a documentation server, a server database, an application server and a Web server. The monitoring system has been also deployed on the same LAN of this infrastructure and monitors the services provided by the components of this infrastructure are operational and working as expected. This is a local monitoring system functional. However, the project also proposes a scalable system that is ready for growth of infrastructure and efficient. This is the reason of divide the system in two components:  Slave Component: monitors locally the infrastructure assets to be monitored, this is the scenario described above.  Master Component: get the results from the monitoring, provided by the Slave Component. This system can be deployed in a different network than the slave component. This approach is not only scalable but also a real scenario, as may be the case that the Components belongs to different infrastructures and even, different organizations, also this components can communicate over the Internet, through a reliable mechanism if possible. The project proposes that both Components are deployed in high availability (HA onwards concerning high availability), each Component is composed of two servers (nodes, hereafter). As will be discussed in later chapters, there are several settings available to deploy a system in HA, the configuration chosen for the project is Active - Passive (details of this configuration are also explained in later chapters). Finally the possibility of offering active responses in certain situations and additional settings on the monitoring system will be discussed. On the other hand, for the implementation of the project, open source software has been used, for virtualization infrastructure (Virtual Box and GNS3), code-based operating systems (Linux), the monitoring system (Nagios core), as well as the software that implements the HA (corosync and pacemaker).

Estudio teórico-experimental para la mejora de la resistencia estructural de autocares basado en el diseño de uniones y asientos

La presente tesis analiza la mejora de la resistencia estructural ante vuelco de autocares enfocando dos vías de actuación: análisis y propuestas de requisitos reglamentarios a nivel europeo y la generación de herramientas que ayuden al diseño y a la verificación de estos requisitos. Los requisitos reglamentarios de resistencia estructural a vuelco contemplan la superestructura de los vehículos pero no para los asientos y sistemas de retención. La influencia de los pasajeros retenidos es superior a la incluida en reglamentación (Reg. 66.01) debiendo considerarse unida al vehículo un porcentaje de la masa de los pasajeros del 91% para cinturón de tres puntos y del 52% para cinturón subabdominal frente al 50% reglamentario para todos los casos. Se ha determinado la cinemática y dinámica del vuelco normativo en sus diferentes fases, formulando las energías en las fases iniciales (hasta el impacto contra el suelo) y determinando la fase final de deformación a través del análisis secuencial de ensayos de módulos reales. Se han determinado los esfuerzos para los asientos que se dividen en dos fases diferenciadas temporalmente: una primera debida a la deformación estructural y una segunda debida al esfuerzo del pasajero retenido que se produce en sentido opuesto (con una deceleración del pasajero en torno a 3.3 g). Se ha caracterizado a través de ensayos cuasi.estáticos el comportamiento de perfiles a flexión y de las uniones estructurales de las principales zonas del vehículo (piso, ventana y techo) verificándose la validez del comportamiento plástico teórico Kecman.García para perfiles de hasta 4 mm de espesor y caracterizando la resistencia y rigidez en la zona elástica de las uniones en función del tipo de refuerzo, materiales y perfiles (análisis de más de 180 probetas). Se ha definido un método de ensayo cuasi.estático para asientos ante esfuerzos de vuelco, ensayándose 19 butacas y determinándose que son resistentes (salvo las uniones a vehículo con pinzas), que son capaces de absorber hasta más de un 17% de la energía absorbida, aunque algunos necesitan optimización para llegar a contribuir en el mecanismo de deformación estructural. Se han generado modelos simplificados para introducir en los modelos barra.rótula plástica: un modelo combinado unión+rótula plástica (que incluye la zona de rigidez determinada en función del tipo de unión) para la superestructura y un modelo simplificado de muelles no.lineales para los asientos. Igualmente se ha generado la metodología de diseño a través de ensayos virtuales con modelos de detalle de elementos finitos tanto de las uniones como de los asientos. Se ha propuesto una metodología de diseño basada en obtener el “mecanismo óptimo de deformación estructural” (elevando la zona de deformación lateral a nivel de ventana y en pilar o en costilla en techo). Para ello se abren dos vías: diseño de la superestructura (selección de perfiles y generación de uniones resistentes) o combinación con asientos (que en lugar de solo resistir las cargas pueden llegar a modificar el mecanismo de deformación). Se ha propuesto una metodología de verificación alternativa al vuelco de vehículo completo que contempla el cálculo cuasi.estático con modelos simplificados barra.rótula plástica más el ensayo de una sección representativa con asientos y utillajes antropomórficos retenidos que permite validar el diseño de las uniones, determinar el porcentaje de energía que debe absorberse por deformación estructural (factor C) y verificar el propio asiento como sistema de retención. ABSTRACT This research analyzes the improvement of the structural strength of buses and coaches under rollover from two perspectives: regulatory requirements at European level and generation of tools that will help to the design and to the verification of requirements. European Regulations about rollover structural strength includes requirements for the superstructure of the vehicles but not about seats, anchorages and restraint systems. The influence of the retained passengers is higher than the one included currently in the Regulations (Reg. 66.01), being needed to consider a 91% of the passenger mass as rigidly joint to the vehicle (for 3 points’ belt, a 52% for 2 points’ belt) instead of the 50% included in the Regulation. Kinematic and dynamic of the normative rollover has been determined from testing of different sections, formulating the energies of the first phases (up to the first impact with the ground) and determining the last deformation phase through sequential analysis of movements and deformations. The efforts due to rollover over the seats have been established, being divided in two different temporal phases: a first one due to the structural deformation of the vehicle and a second one due to the effort of the restrained passenger being this second one in opposite sense (with a passenger deceleration around 3.3 g). From quasi.static testing, the behavior of the structural tubes under flexural loads, including the principal joints in the vehicle (floor, window and roof), the validity of the theoretical plastic behavior according Kecman.García theories have been verified up to 4 mm of thickness. Strength of the joints as well as the stiffness of the elastic zone has been determined in function of main parameters: type of reinforcement, materials and section of the tubes (more than 180 test specimens). It has been defined a quasi.static testing methodology to characterize the seats and restrain system behavior under rollover, testing 19 double seats and concluding that they are resistant (excepting clamping joints), that they can absorb more than a 17 of the absorbed energy, and that some of them need optimization to contribute in the structural deformation mechanism. It has been generated simplified MEF models, to analyze in a beam.plastic hinge model: a combined model joint+plastic hinge (including the stiffness depending on the type of joint) for the superstructure and a simplified model with non.lineal springs to represent the seats. It has been detailed methodologies for detailed design of joints and seats from virtual testing (MEF models). A design methodology based in the “optimized structural deformation mechanism” (increasing the height of deformation of the lateral up to window level) is proposed. Two possibilities are analyzed: design of the superstructure based on the selection of profiles and design of strength joints (were seats only resist the efforts and contribute in the energy absorption) or combination structure.seats, were seats contributes in the deformation mechanism. An alternative methodology to the rollover of a vehicle that includes the quasi.static calculation with simplified models “beam.joint+plastic hinge” plus the testing of a representative section of the vehicle including seats and anthropomorphic ballast restrained by the safety belts is presented. The test of the section allows validate the design of the joints, determine the percentage of energy to be absorbed by structural deformation (factor C) and verify the seat as a retention system.

A PII-like protein in Arabidopsis: Putative role in nitrogen sensing

PII is a protein allosteric effector in Escherichia coli and other bacteria that indirectly regulates glutamine synthetase at the transcriptional and post-translational levels in response to nitrogen availability. Data supporting the notion that plants have a nitrogen regulatory system(s) includes previous studies showing that the levels of mRNA for plant nitrogen assimilatory genes such as glutamine synthetase (GLN) and asparagine synthetase (ASN) are modulated by carbon and organic nitrogen metabolites. Here, we have characterized a PII homolog (GLB1) in two higher plants, Arabidopsis thaliana and Ricinus communis (Castor bean). Each plant PII-like protein has high overall identity to E. coli PII (50%). Western blot analyses reveal that the plant PII-like protein is a nuclear-encoded chloroplast protein. The PII-like protein of plants appears to be regulated at the transcriptional level in that levels of GLB1 mRNA are affected by light and metabolites. To initiate studies of the in vivo function of the Arabidopsis PII-like protein, we have constructed transgenic lines in which PII expression is uncoupled from its native regulation. Analyses of these transgenic plants support the notion that the plant PII-like protein may serve as part of a complex signal transduction network involved in perceiving the status of carbon and organic nitrogen. Thus, the PII protein found in archaea, bacteria, and now in higher eukaryotes (plants) is one of the most widespread regulatory proteins known, providing evidence for an ancestral metabolic regulatory mechanism that may have existed before the divergence of these three domains of life.

Proteolytic Processing and Ca2+-binding Activity of Dense-Core Vesicle Polypeptides in Tetrahymena

Formation and discharge of dense-core secretory vesicles depend on controlled rearrangement of the core proteins during their assembly and dispersal. The ciliate Tetrahymena thermophila offers a simple system in which the mechanisms may be studied. Here we show that most of the core consists of a set of polypeptides derived proteolytically from five precursors. These share little overall amino acid identity but are nonetheless predicted to have structural similarity. In addition, sites of proteolytic processing are notably conserved and suggest that specific endoproteases as well as carboxypeptidase are involved in core maturation. In vitro binding studies and sequence analysis suggest that the polypeptides bind calcium in vivo. Core assembly and postexocytic dispersal are compartment-specific events. Two likely regulatory factors are proteolytic processing and exposure to calcium. We asked whether these might directly influence the conformations of core proteins. Results using an in vitro chymotrypsin accessibility assay suggest that these factors can induce sequential structural rearrangements. Such progressive changes in polypeptide folding may underlie the mechanisms of assembly and of rapid postexocytic release. The parallels between dense-core vesicles in different systems suggest that similar mechanisms are widespread in this class of organelles.

The Gcd10p/Gcd14p complex is the essential two-subunit tRNA(1-methyladenosine) methyltransferase of Saccharomyces cerevisiae

The modified nucleoside 1-methyladenosine (m1A) is found at position 58 in the TΨC loop of many eukaryotic tRNAs. The absence of m1A from all tRNAs in Saccharomyces cerevisiae mutants lacking Gcd10p elicits severe defects in processing and stability of initiator methionine tRNA (tRNAiMet). Gcd10p is found in a complex with Gcd14p, which contains conserved motifs for binding S-adenosylmethionine (AdoMet). These facts, plus our demonstration that gcd14Δ cells lacked m1A, strongly suggested that Gcd10p/Gcd14p complex is the yeast tRNA(m1A)methyltransferase [(m1A)MTase]. Supporting this prediction, affinity-purified Gcd10p/Gcd14p complexes used AdoMet as a methyl donor to synthesize m1A in either total tRNA or purified tRNAiMet lacking only this modification. Kinetic analysis of the purified complex revealed KM values for AdoMet or tRNAiMet of 5.0 μM and 2.5 nM, respectively. Mutations in the predicted AdoMet-binding domain destroyed GCD14 function in vivo and (m1A)MTase activity in vitro. Purified Flag-tagged Gcd14p alone had no enzymatic activity and was severely impaired for tRNA-binding compared with the wild-type complex, suggesting that Gcd10p is required for tight binding of the tRNA substrate. Our results provide a demonstration of a two-component tRNA MTase and suggest that binding of AdoMet and tRNA substrates depends on different subunits of the complex.

Rational design of a global inhibitor of the virulence response in Staphylococcus aureus, based in part on localization of the site of inhibition to the receptor-histidine kinase, AgrC

Two-component signaling systems involving receptor-histidine kinases are ubiquitous in bacteria and have been found in yeast and plants. These systems provide the major means by which bacteria communicate with each other and the outside world. Remarkably, very little is known concerning the extracellular ligands that presumably bind to receptor-histidine kinases to initiate signaling. The two-component agr signaling circuit in Staphylococcus aureus is one system where the ligands are known in chemical detail, thus opening the door for detailed structure–activity relationship studies. These ligands are short (8- to 9-aa) peptides containing a thiolactone structure, in which the α-carboxyl group of the C-terminal amino acid is linked to the sulfhydryl group of a cysteine, which is always the fifth amino acid from the C terminus of the peptide. One unique aspect of the agr system is that peptides that activate virulence expression in one group of S. aureus strains also inhibit virulence expression in other groups of S. aureus strains. Herein, it is demonstrated by switching the receptor-histidine kinase, AgrC, between strains of different agr specificity types, that intragroup activation and intergroup inhibition are both mediated by the same group-specific receptors. These results have facilitated the development of a global inhibitor of virulence in S. aureus, which consists of a truncated version of one of the naturally occurring thiolactone peptides.

Complete genome sequence of Caulobacter crescentus

The complete genome sequence of Caulobacter crescentus was determined to be 4,016,942 base pairs in a single circular chromosome encoding 3,767 genes. This organism, which grows in a dilute aquatic environment, coordinates the cell division cycle and multiple cell differentiation events. With the annotated genome sequence, a full description of the genetic network that controls bacterial differentiation, cell growth, and cell cycle progression is within reach. Two-component signal transduction proteins are known to play a significant role in cell cycle progression. Genome analysis revealed that the C. crescentus genome encodes a significantly higher number of these signaling proteins (105) than any bacterial genome sequenced thus far. Another regulatory mechanism involved in cell cycle progression is DNA methylation. The occurrence of the recognition sequence for an essential DNA methylating enzyme that is required for cell cycle regulation is severely limited and shows a bias to intergenic regions. The genome contains multiple clusters of genes encoding proteins essential for survival in a nutrient poor habitat. Included are those involved in chemotaxis, outer membrane channel function, degradation of aromatic ring compounds, and the breakdown of plant-derived carbon sources, in addition to many extracytoplasmic function sigma factors, providing the organism with the ability to respond to a wide range of environmental fluctuations. C. crescentus is, to our knowledge, the first free-living α-class proteobacterium to be sequenced and will serve as a foundation for exploring the biology of this group of bacteria, which includes the obligate endosymbiont and human pathogen Rickettsia prowazekii, the plant pathogen Agrobacterium tumefaciens, and the bovine and human pathogen Brucella abortus.

Solution structure of DFF40 and DFF45 N-terminal domain complex and mutual chaperone activity of DFF40 and DFF45

Apoptotic DNA fragmentation is mediated by a caspase-activated DNA fragmentation factor (DFF)40. Expression and folding of DFF40 require the presence of DFF45, which also acts as a nuclease inhibitor before DFF40 activation by execution caspases. The N-terminal domains (NTDs) of both proteins are homologous, and their interaction plays a key role in the proper functioning of this two-component system. Here we report that the NTD of DFF45 alone is unstructured in solution, and its folding is induced upon binding to DFF40 NTD. Therefore, folding of both proteins regulates the formation of the DFF40/DFF45 complex. The solution structure of the heterodimeric complex between NTDs of DFF40 and DFF45 reported here shows that the mutual chaperoning includes the formation of an extensive network of intermolecular interactions that bury a hydrophobic cluster inside the interface, surrounded by intermolecular salt bridges.

Regulated transposition of a fish transposon in the mouse germ line

Tc1/mariner elements are able to transpose in species other than the host from which they were isolated. As potential vectors for insertional mutagenesis and transgenesis of the mouse, these cut-and-paste transposons were tested for their ability to transpose in the mouse germ line. First, the levels of activity of several Tc1/mariner elements in mammalian cells were compared; the reconstructed fish transposon Sleeping Beauty (SB) was found to be an order of magnitude more efficient than the other tested transposons. SB then was introduced into the mouse germ line as a two-component system: one transgene for the expression of the transposase in the male germ line and a second transgene carrying a modified transposon. In 20% of the progeny of double transgenic male mice the transposon had jumped from the original chromosomal position into another locus. Analysis of the integration sites shows that these jumps indeed occurred through the action of SB transposase, and that SB has a strong preference for intrachromosomal transposition. Analysis of the excision sites suggests that double-strand breaks in haploid spermatids are repaired via nonhomologous end joining. The SB system may be a powerful tool for transposon mutagenesis of the mouse germ line.

The alginate regulator AlgR and an associated sensor FimS are required for twitching motility in Pseudomonas aeruginosa.

Mucoid strains of Pseudomonas aeruginosa isolated from the lungs of cystic fibrosis patients produce large amounts of the exopolysaccharide alginate. AlgR has long been considered a key regulator of alginate production, but its cognate sensor has not been identified. Here we show that AlgR is required for twitching motility, which is a form of bacterial surface translocation mediated by type 4 fimbriae. Adjacent to algR we have identified a sensor gene (fimS), which is also required for twitching motility. However, FimS does not appear to be required for alginate production in mucoid strains. FimS and AlgR are representative of a new subclass of two-component transmitter-receiver regulatory systems. The alternative sigma factor AlgU also affects both alginate production and twitching motility. Therefore, these two virulence determinants appear to be closely associated and coordinately regulated.

Creation of a reversible on/off system for site-specific in vivo control of exogenous gene activity in the renal glomerulus.

Using genetically engineered glomerular mesangial cells, an in vivo gene transfer approach was developed that specifically targets the renal glomerulus. By combining this system with a tetracycline (Tc)-responsive promoter, the present study aimed to create a reversible on/off system for site-specific in vivo control of exogenous gene activity within the glomerulus. In the Tc regulatory system, a Tc-controlled transactivator (tTA) encoded by a regulator plasmid induces target gene transcription by binding to a tTA-responsive promoter located in a response plasmid. Tc inhibits this tTA-dependent transactivation via its affinity for tTA. In double-transfected cells, therefore, the activity of a transgene can be controlled by Tc. Cultured rat mesangial cells were cotransfected with a regulator plasmid and a response plasmid that introduces a beta-galactosidase gene. In vitro, stable double-transfectant MtTAG cells exhibited no beta-galactosidase activity in the presence of Tc. However, following withdrawal of Tc from culture media, expression of beta-galactosidase was induced within 24 h. When Tc was again added, the expression was rapidly resuppressed. Low concentrations of Tc were sufficient to maintain the silent state of tTA-dependent promoter. MtTAG cells were then transferred into the rat glomeruli via renal artery injection. In the isolated chimeric glomeruli, expression of beta-galactosidase was induced ex vivo in the absence of Tc, whereas it was repressed in its presence. When Tc-pretreated MtTAG cells were transferred into the glomeruli of untreated rats, beta-galactosidase expression was induced in vivo within 3 days. Oral administration of Tc dramatically suppressed this induction. These data demonstrate the feasibility of using mesangial cell vectors combined with the Tc regulatory system for site-specific in vivo control of exogenous gene expression in the glomerulus.