Mechanical behaviour of laminated functionally graded fibre-reinforced self-compacting cementitious composites

El hormigón autocompactante (HAC) es una nueva tipología de hormigón o material compuesto base cemento que se caracteriza por ser capaz de fluir en el interior del encofrado o molde, llenándolo de forma natural, pasando entre las barras de armadura y consolidándose únicamente bajo la acción de su peso propio, sin ayuda de medios de compactación externos, y sin que se produzca segregación de sus componentes. Debido a sus propiedades frescas (capacidad de relleno, capacidad de paso, y resistencia a la segregación), el HAC contribuye de forma significativa a mejorar la calidad de las estructuras así como a abrir nuevos campos de aplicación del hormigón. Por otra parte, la utilidad del hormigón reforzado con fibras de acero (HRFA) es hoy en día incuestionable debido a la mejora significativa de sus propiedades mecánicas tales como resistencia a tracción, tenacidad, resistencia al impacto o su capacidad para absorber energía. Comparado con el HRFA, el hormigón autocompactante reforzado con fibras de acero (HACRFA) presenta como ventaja una mayor fluidez y cohesión ofreciendo, además de unas buenas propiedades mecánicas, importantes ventajas en relación con su puesta en obra. El objetivo global de esta tesis doctoral es el desarrollo de nuevas soluciones estructurales utilizando materiales compuestos base cemento autocompactantes reforzados con fibras de acero. La tesis presenta una nueva forma de resolver el problema basándose en el concepto de los materiales gradiente funcionales (MGF) o materiales con función gradiente (MFG) con el fin de distribuir de forma eficiente las fibras en la sección estructural. Para ello, parte del HAC se sustituye por HACRFA formando capas que presentan una transición gradual entre las mismas con el fin de obtener secciones robustas y exentas de tensiones entre capas con el fin de aplicar el concepto “MGF-laminados” a elementos estructurales tales como vigas, columnas, losas, etc. El proceso incluye asimismo el propio método de fabricación que, basado en la tecnología HAC, permite el desarrollo de interfases delgadas y robustas entre capas (1-3 mm) gracias a las propiedades reológicas del material. Para alcanzar dichos objetivos se ha llevado a cabo un amplio programa experimental cuyas etapas principales son las siguientes: • Definir y desarrollar un método de diseño que permita caracterizar de forma adecuada las propiedades mecánicas de la “interfase”. Esta primera fase experimental incluye: o las consideraciones generales del propio método de fabricación basado en el concepto de fabricación de materiales gradiente funcionales denominado “reología y gravedad”, o las consideraciones específicas del método de caracterización, o la caracterización de la “interfase”. • Estudiar el comportamiento mecánico sobre elementos estructurales, utilizando distintas configuraciones de MGF-laminado frente a acciones tanto estáticas como dinámicas con el fin de comprobar la viabilidad del material para ser usado en elementos estructurales tales como vigas, placas, pilares, etc. Los resultados indican la viabilidad de la metodología de fabricación adoptada, así como, las ventajas tanto estructurales como en reducción de costes de las soluciones laminadas propuestas. Es importante destacar la mejora en términos de resistencia a flexión, compresión o impacto del hormigón autocompactante gradiente funcional en comparación con soluciones de HACRFA monolíticos inclusos con un volumen neto de fibras (Vf) doble o superior. Self-compacting concrete (SCC) is an important advance in the concrete technology in the last decades. It is a new type of high performance concrete with the ability of flowing under its own weight and without the need of vibrations. Due to its specific fresh or rheological properties, such as filling ability, passing ability and segregation resistance, SCC may contribute to a significant improvement of the quality of concrete structures and open up new field for the application of concrete. On the other hand, the usefulness of steel fibre-reinforced concrete (SFRC) in civil engineering applications is unquestionable. SFRC can improve significantly the hardened mechanical properties such as tensile strength, impact resistance, toughness and energy absorption capacity. Compared to SFRC, self-compacting steel fibre-reinforced concrete (SCSFRC) is a relatively new type of concrete with high flowability and good cohesiveness. SCSFRC offers very attractive economical and technical benefits thanks to SCC rheological properties, which can be further extended, when combined with SFRC for improving their mechanical characteristics. However, for the different concrete structural elements, a single concrete mix is selected without an attempt to adapt the diverse fibre-reinforced concretes to the stress-strain sectional properly. This thesis focused on the development of high performance cement-based structural composites made of SCC with and without steel fibres, and their applications for enhanced mechanical properties in front of different types of load and pattern configurations. It presents a new direction for tackling the mechanical problem. The approach adopted is based on the concept of functionally graded cementitious composite (FGCC) where part of the plain SCC is strategically replaced by SCSFRC in order to obtain laminated functionally graded self-compacting cementitious composites, laminated-FGSCC, in single structural elements as beams, columns, slabs, etc. The approach also involves a most suitable casting method, which uses SCC technology to eliminate the potential sharp interlayer while easily forming a robust and regular reproducible graded interlayer of 1-3 mm by controlling the rheology of the mixes and using gravity at the same time to encourage the use of the powerful concept for designing more performance suitable and cost-efficient structural systems. To reach the challenging aim, a wide experimental programme has been carried out involving two main steps: • The definition and development of a novel methodology designed for the characterization of the main parameter associated to the interface- or laminated-FGSCC solutions: the graded interlayer. Work of this first part includes: o the design considerations of the innovative (in the field of concrete) production method based on “rheology and gravity” for producing FG-SCSFRC or as named in the thesis FGSCC, casting process and elements, o the design of a specific testing methodology, o the characterization of the interface-FGSCC by using the so designed testing methodology. • The characterization of the different medium size FGSCC samples under different static and dynamic loads patterns for exploring their possibilities to be used for structural elements as beams, columns, slabs, etc. The results revealed the efficiency of the manufacturing methodology, which allow creating robust structural sections, as well as the feasibility and cost effectiveness of the proposed FGSCC solutions for different structural uses. It is noticeable to say the improvement in terms of flexural, compressive or impact loads’ responses of the different FGSCC in front of equal strength class SCSFRC bulk elements with at least the double of overall net fibre volume fraction (Vf).

Axisymmetric long liquid bridges in a time-dependent microgravity field

This paper deals with the dynamics of liquid bridges when subjected to an oscillatory microgravity field. The analysis has been performed by using a one-dimensional slice model, already used in liquid bridge problems, which allows to calculate not only the resonance frequencies of a wide range of such fluid configurations but also the dependence of the dynamic response of the liquid bridge on the frequency on the imposed perturbations. Theoretical results are compared with experimental ones obtained aboard Spacelab-Dl, the agreement between theoretical and experimental results being satisfactory

Integral energy behaviour of photovoltaic semi-transparent glazing elements for building integration

La hipótesis general que esta tesis quiere demostrar es que la integración arquitectónica de sistemas fotovoltaicos semitransparentes (STPV) puede contribuir a mejorar la eficiencia energética de los edificios. Por lo tanto, la investigación se centra en el desarrollo de una metodología capaz de cuantificar la reducción de la demanda energética del edificio proporcionada por estas novedosas soluciones constructivas. Al mismo tiempo, los parámetros de diseño de las soluciones STPV se han analizado para establecer cuales presentan el mayor impacto sobre el balance energético global del edificio y por lo tanto tienen que ser cuidadosamente definidos a la hora de optimizar el comportamiento energético del mismo. A la luz de estos objetivos, la metodología de estudio se ha centrado en tres puntos principales:  Caracterizar el comportamiento energético global de sistemas STPV en condiciones de operación realistas, similares a las que se darían en un sistema real;  Caracterizar el comportamiento energético global de sistemas STPV en condiciones controladas, con el objetivo de estudiar la variación del comportamiento del los elementos en función de parámetro de diseño y operación;  Evaluar el potencial de ahorro energético global de los sistemas STPV en comparación con soluciones acristaladas convencionales al variar de las condiciones de contorno constituidas por los parámetros de diseño (como el grado de transparencia), las características arquitectónicas (como el ratio entre superficie acristalada y superficie opaca en la fachada del edificio) y las condiciones climáticas (cubriendo en particular la climatología europea). En síntesis, este trabajo intenta contribuir a comprender la interacción que existe entre los sistemas STPV y el edificio, proporcionando tanto a los fabricantes de los componentes como a los profesionales de la construcción información valiosa sobre el potencial de ahorro energético asociado a estos nuevos sistemas constructivos. Asimismo el estudio define los parámetros de diseño adecuados para lograr soluciones eficientes tanto en proyectos nuevos como de rehabilitación. ABSTRACT The general hypothesis this work seeks to demonstrate is that the architectural integration of Semi-Transparent Photovoltaic (STPV) systems can contribute to improving the energy efficiency of buildings. Accordingly, the research has focused on developing a methodology able to quantify the building energy demand reduction provided by these novel constructive solutions. At the same time, the design parameters of the STPV solution have been analysed to establish which of them have the greatest impact on the global energy balance of the building, and therefore which have to be carefully defined in order to optimize the building operation. In the light of these goals, the study methodology has focused on three main points:  To characterise the global energy behaviour of STPV systems in realistic operating conditions, similar to those in which a real system will operate;  To characterise the global energy behaviour of STPV systems in controlled conditions in order to study how the performance varies depending on the design and operating parameters;  To assess the global energy saving potential of STPV systems in comparison with conventional glazing solutions by varying the boundary conditions, including design parameters (such as the degree of transparency), architectural characteristics (such as the Window to Wall Ratio) and climatic conditions (covering the European climatic conditions). In summary, this work has sought to contribute to the understanding of the interaction between STPV systems and the building, providing both components manufacturers and construction technicians, valuable information on the energy savings potential of these new construction systems and defining the appropriate design parameters to achieve efficient solutions in both new and retrofitting projects.

Why neutron guides may end up breaking down? Some results on the macroscopic behaviour of alkali-borosilicate glass support plates under neutron irradiation

In this paper we report on a first part of a study on the mechanisms leading to brittle fracture in neutron guides made of glass as structural element. Such devices are widely used to deliver thermal and cold neu tron beams to experimental lines in most large neutron research facilities. We present results on macroscopic properties of samples of guide glass substrates which are subjected to neutron irradiation at relatively large fluences. The results show a striking dependence of some of the macroscopic properties such as density, shape or surface curvature upon the specific chemical composition of a given glass. The relevance of the present findings for the installation of either replacement guides at the existing facilities or for the deployment of instruments for ongoing projects such as the European Spallation Source is briefly discussed.

The effects of tantalum addition on the microtexture and mechanical behaviour of tungsten for ITER applications

Tungsten (W) and its alloys are very promising materials for producing plasma-facing components (PFCs) in the fusion power reactors of the near future, even as a structural part in them. However, whereas the properties of pure tungsten are suitable for a PFC, its structural applications are still limited due to its low toughness, ductile to brittle transition temperature and recrystallization behaviour. Therefore, many efforts have been made to improve its performance by alloying tungsten with other elements. Hence, in this investigation, the thermo-mechanical performance of two new tungsten-tantalum materials has been evaluated. Materials with We5wt.%Ta and We15wt.%Ta were processed by mechanical alloying (MA) and later consolidation by hot isostatic pressing (HIP), with distinct settings for each composition. Thus, it was possible to determine the relationship between the microstructure and the addition of Ta with the macroscopic mechanical properties. These were measured by means of hardness, flexural strength and fracture toughness, in the temperature range of 300e1473 K. The microstructure and the fracture surfaces features of the tested materials were analysed by Field Emission Scanning Electron Microscopy (FESEM).

NF-κB-dependent inhibition of apoptosis is essential for host cell survival during Rickettsia rickettsii infection

The possibility that bacteria may have evolved strategies to overcome host cell apoptosis was explored by using Rickettsia rickettsii, an obligate intracellular Gram-negative bacteria that is the etiologic agent of Rocky Mountain spotted fever. The vascular endothelial cell, the primary target cell during in vivo infection, exhibits no evidence of apoptosis during natural infection and is maintained for a sufficient time to allow replication and cell-to-cell spread prior to eventual death due to necrotic damage. Prior work in our laboratory demonstrated that R. rickettsii infection activates the transcription factor NF-κB and alters expression of several genes under its control. However, when R. rickettsii-induced activation of NF-κB was inhibited, apoptosis of infected but not uninfected endothelial cells rapidly ensued. In addition, human embryonic fibroblasts stably transfected with a superrepressor mutant inhibitory subunit IκB that rendered NF-κB inactivatable also underwent apoptosis when infected, whereas infected wild-type human embryonic fibroblasts survived. R. rickettsii, therefore, appeared to inhibit host cell apoptosis via a mechanism dependent on NF-κB activation. Apoptotic nuclear changes correlated with presence of intracellular organisms and thus this previously unrecognized proapoptotic signal, masked by concomitant NF-κB activation, likely required intracellular infection. Our studies demonstrate that a bacterial organism can exert an antiapoptotic effect, thus modulating the host cell’s apoptotic response to its own advantage by potentially allowing the host cell to remain as a site of infection.

Region-dependent dynamics of cAMP response element-binding protein phosphorylation in the basal ganglia

The cAMP response element-binding protein (CREB) is an activity-dependent transcription factor that is involved in neural plasticity. The kinetics of CREB phosphorylation have been suggested to be important for gene activation, with sustained phosphorylation being associated with downstream gene expression. If so, the duration of CREB phosphorylation might serve as an indicator for time-sensitive plastic changes in neurons. To screen for regions potentially involved in dopamine-mediated plasticity in the basal ganglia, we used organotypic slice cultures to study the patterns of dopamine- and calcium-mediated CREB phosphorylation in the major subdivisions of the striatum. Different durations of CREB phosphorylation were evoked in the dorsal and ventral striatum by activation of dopamine D1-class receptors. The same D1 stimulus elicited (i) transient phosphorylation (≤15 min) in the matrix of the dorsal striatum; (ii) sustained phosphorylation (≤2 hr) in limbic-related structures including striosomes, the nucleus accumbens, the fundus striati, and the bed nucleus of the stria terminalis; and (iii) prolonged phosphorylation (up to 4 hr or more) in cellular islands in the olfactory tubercle. Elevation of Ca2+ influx by stimulation of L-type Ca2+ channels, NMDA, or KCl induced strong CREB phosphorylation in the dorsal striatum but not in the olfactory tubercle. These findings differentiate the response of CREB to dopamine and calcium signals in different striatal regions and suggest that dopamine-mediated CREB phosphorylation is persistent in limbic-related regions of the neonatal basal ganglia. The downstream effects activated by persistent CREB phosphorylation may include time-sensitive neuroplasticity modulated by dopamine.

Crossover isomer bias is the primary sequence-dependent property of immobilized Holliday junctions

Recombination of genes is essential to the evolution of genetic diversity, the segregation of chromosomes during cell division, and certain DNA repair processes. The Holliday junction, a four-arm, four-strand branched DNA crossover structure, is formed as a transient intermediate during genetic recombination and repair processes in the cell. The recognition and subsequent resolution of Holliday junctions into parental or recombined products appear to be critically dependent on their three-dimensional structure. Complementary NMR and time-resolved fluorescence resonance energy transfer experiments on immobilized four-arm DNA junctions reported here indicate that the Holliday junction cannot be viewed as a static structure but rather as an equilibrium mixture of two conformational isomers. Furthermore, the distribution between the two possible crossover isomers was found to depend on the sequence in a manner that was not anticipated on the basis of previous low-resolution experiments.

Rho-dependent Regulation of Cell Spreading by the Tetraspan Membrane Protein Gas3/PMP22

Gas3/PMP22 plays a crucial role in regulating myelin formation and maintenance, and different genetic alterations in gas3/PMP22 are responsible for a set of human peripheral neuropathies. We have previously demonstrated that Gas3/PMP22 could regulate susceptibility to apoptosis in NIH3T3 cells but not in REF 52 cells. In this report we demonstrate that when the apoptotic response triggered by gas3/PMP22 was counteracted by Bcl-2 coexpression, morphological changes were observed. Time-lapse analysis confirmed that Gas3/PMP22 can modulate cell spreading, and this effect was strengthened after inhibition of phosphoinositide 3-kinase. Using the active form of the small GTPase RhoA, we have been able to dissect the different Gas3/PMP22 biological activities. RhoA counteracted the Gas3/PMP22-dependent morphological response but was unable to neutralize the apoptotic response. Treatment of NIH3T3 cells with cytotoxic necrotizing factor 1, which activates endogenous Rho, also counteracted Gas3/PMP22-mediated cell shape and spreading changes. Treatment of REF 52 cells, which are unresponsive to Gas3/PMP22 overexpression, with the C3 exoenzyme, inhibiting Rho activity, renders REF 52 cells responsive to Gas3/PMP22 overexpression for cell shape and spreading changes. Finally, assembly of stress fibers and focal adhesions complexes, in response to lysophosphatidic acid–induced endogenous Rho activation, was impaired in Gas3/PMP22-overexpressing cells. We hypothesize that cell shape and spreading regulated by Gas3/PMP22 through the Rho GTPase might have an important role during Schwann cells differentiation and myelinization.

Microtubule-dependent Recruitment of Staufen-Green Fluorescent Protein into Large RNA-containing Granules and Subsequent Dendritic Transport in Living Hippocampal NeuronsV⃞

Dendritic mRNA transport and local translation at individual potentiated synapses may represent an elegant way to form synaptic memory. Recently, we characterized Staufen, a double-stranded RNA-binding protein, in rat hippocampal neurons and showed its presence in large RNA-containing granules, which colocalize with microtubules in dendrites. In this paper, we transiently transfect hippocampal neurons with human Staufen-green fluorescent protein (GFP) and find fluorescent granules in the somatodendritic domain of these cells. Human Stau-GFP granules show the same cellular distribution and size and also contain RNA, as already shown for the endogenous Stau particles. In time-lapse videomicroscopy, we show the bidirectional movement of these Staufen-GFP–labeled granules from the cell body into dendrites and vice versa. The average speed of these particles was 6.4 μm/min with a maximum velocity of 24.3 μm/min. Moreover, we demonstrate that the observed assembly into granules and their subsequent dendritic movement is microtubule dependent. Taken together, we have characterized a novel, nonvesicular, microtubule-dependent transport pathway involving RNA-containing granules with Staufen as a core component. This is the first demonstration in living neurons of movement of an essential protein constituent of the mRNA transport machinery.

Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human β-globin in engrafted mice

Transcriptional silencing of genes transferred into hematopoietic stem cells poses one of the most significant challenges to the success of gene therapy. If the transferred gene is not completely silenced, a progressive decline in gene expression as the mice age often is encountered. These phenomena were observed to various degrees in mouse transplant experiments using retroviral vectors containing a human β-globin gene, even when cis-linked to locus control region derivatives. Here, we have investigated whether ex vivo preselection of retrovirally transduced stem cells on the basis of expression of the green fluorescent protein driven by the CpG island phosphoglycerate kinase promoter can ensure subsequent long-term expression of a cis-linked β-globin gene in the erythroid lineage of transplanted mice. We observed that 100% of mice (n = 7) engrafted with preselected cells concurrently expressed human β-globin and the green fluorescent protein in 20–95% of their RBC for up to 9.5 mo posttransplantation, the longest time point assessed. This expression pattern was successfully transferred to secondary transplant recipients. In the presence of β-locus control region hypersensitive site 2 alone, human β-globin mRNA expression levels ranged from 0.15% to 20% with human β-globin chains detected by HPLC. Neither the proportion of positive blood cells nor the average expression levels declined with time in transplanted recipients. Although suboptimal expression levels and heterocellular position effects persisted, in vivo stem cell gene silencing and age-dependent extinction of expression were avoided. These findings support the further investigation of this type of vector for the gene therapy of human hemoglobinopathies.

Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody

The hyperpermeability of tumor vessels to macromolecules, compared with normal vessels, is presumably due to vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) released by neoplastic and/or host cells. In addition, VEGF/VPF is a potent angiogenic factor. Removal of this growth factor may reduce the permeability and inhibit tumor angiogenesis. To test these hypotheses, we transplanted a human glioblastoma (U87), a human colon adenocarcinoma (LS174T), and a human melanoma (P-MEL) into two locations in immunodeficient mice: the cranial window and the dorsal skinfold chamber. The mice bearing vascularized tumors were treated with a bolus (0.2 ml) of either a neutralizing antibody (A4.6.1) (492 μg/ml) against VEGF/VPF or PBS (control). We found that tumor vascular permeability to albumin in antibody-treated groups was lower than in the matched controls and that the effect of the antibody was time-dependent and influenced by the mode of injection. Tumor vascular permeability did not respond to i.p. injection of the antibody until 4 days posttreatment. However, the permeability was reduced within 6 h after i.v. injection of the same amount of antibody. In addition to the reduction in vascular permeability, the tumor vessels became smaller in diameter and less tortuous after antibody injections and eventually disappeared from the surface after four consecutive treatments in U87 tumors. These results demonstrate that tumor vascular permeability can be reduced by neutralization of endogenous VEGF/VPF and suggest that angiogenesis and the maintenance of integrity of tumor vessels require the presence of VEGF/VPF in the tissue microenvironment. The latter finding reveals a new mechanism of tumor vessel regression—i.e., blocking the interactions between VEGF/VPF and endothelial cells or inhibiting VEGF/VPF synthesis in solid tumors causes dramatic reduction in vessel diameter, which may block the passage of blood elements and thus lead to vascular regression.

Temperature, ordering, and equilibrium with time-dependent confining forces

The concepts of temperature and equilibrium are not well defined in systems of particles with time-varying external forces. An example is a radio frequency ion trap, with the ions laser cooled into an ordered solid, characteristic of sub-mK temperatures, whereas the kinetic energies associated with the fast coherent motion in the trap are up to 7 orders of magnitude higher. Simulations with 1,000 ions reach equilibrium between the degrees of freedom when only aperiodic displacements (secular motion) are considered. The coupling of the periodic driven motion associated with the confinement to the nonperiodic random motion of the ions is very small at low temperatures and increases quadratically with temperature.

Protective long-term antibody memory by antigen-driven and T help-dependent differentiation of long-lived memory B cells to short-lived plasma cells independent of secondary lymphoid organs

Memory is a hallmark of immunity. Memory carried by antibodies is largely responsible for protection against reinfection with most known acutely lethal infectious agents and is the basis for most clinically successful vaccines. However, the nature of long-term B cell and antibody memory is still unclear. B cell memory was studied here after infection of mice with the rabies-like cytopathic vesicular stomatitis virus, the noncytopathic lymphocytic choriomeningitis virus (Armstrong and WE), and after immunization with various inert viral antigens inducing naive B cells to differentiate either to plasma cells or memory B cells in germinal centers of secondary lymphoid organs. The results show that in contrast to very low background levels against internal viral antigens, no significant neutralizing antibody memory was observed in the absence of antigen and suggest that memory B cells (i) are long-lived in the absence of antigen, nondividing, and relatively resistant to irradiation, and (ii) must be stimulated by antigen to differentiate to short-lived antibody-secreting plasma cells, a process that is also efficient in the bone marrow and always depends on radiosensitive, specific T help. Therefore, for vaccines to induce long-term protective antibody titers, they need to repeatedly provide, or continuously maintain, antigen in minimal quantities over a prolonged time period in secondary lymphoid organs or the bone marrow for sufficient numbers of long-lived memory B cells to mature to short-lived plasma cells.

Redox-dependent activation of CO dehydrogenase from Rhodospirillum rubrum

Studies of initial activities of carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum show that CODH is mostly inactive at redox potentials higher than −300 mV. Initial activities measured at a wide range of redox potentials (0–500 mV) fit a function corresponding to the Nernst equation with a midpoint potential of −316 mV. Previously, extensive EPR studies of CODH have suggested that CODH has three distinct redox states: (i) a spin-coupled state at −60 to −300 mV that gives rise to an EPR signal termed Cred1; (ii) uncoupled states at <−320 mV in the absence of CO2 referred to as Cunc; and (iii) another spin-coupled state at <−320 mV in the presence of CO2 that gives rise to an EPR signal termed Cred2B. Because there is no initial CODH activity at potentials that give rise to Cred1, the state (Cred1) is not involved in the catalytic mechanism of this enzyme. At potentials more positive than −380 mV, CODH recovers its full activity over time when incubated with CO. This reductant-dependent conversion of CODH from an inactive to an active form is referred to hereafter as “autocatalysis.” Analyses of the autocatalytic activation process of CODH suggest that the autocatalysis is initiated by a small fraction of activated CODH; the small fraction of active CODH catalyzes CO oxidation and consequently lowers the redox potential of the assay system. This process is accelerated with time because of accumulation of the active enzyme.