Avaliação do estresse oxidativo induzido por superfícies de titânio

Commercially pure Titanium (cp Ti) is a material largely used in orthopedic and dental implants due to its biocompatibility properties. Changes in the surface of cp Ti can determine the functional response of the cells such as facilitating implant fixation and stabilization, and increased roughness of the surface has been shown to improve adhesion and cellular proliferation. Various surface modification methods have been developed to increase roughness, such as mechanical, chemical, electrochemical and plasma treatment. An argon plasma treatment generates a surface that has good mechanical proprieties without chemical composition modification. Besides the topography, biological responses to the implant contribute significantly to its success. Oxidative stress induced by the biomaterials is considered one of the major causes of implant failure. For this reason the oxidative potential of titanium surfaces subjected to plasma treatment was evaluated on this work. CHO-k1 cells were cultivated on smooth or roughed Ti disks, and after three days, the redox balance was investigated measuring reactive oxygen species (ROS) generation, total antioxidant capacity and biomarkers of ROS attack. The results showed cells grown on titanium surfaces are subjected to intracellular oxidative stress due to hydrogen peroxide generation. Titanium discs subjected to the plasma treatment induced less oxidative stress than the untreated ones, which resulted in improved cellular ability. Our data suggest that plasma treated titanium may be a more biocompatible biomaterial.

Modificações na produtividade e nas características físicas e químicas da madeira de Eucalyptus grandis causadas pela adubação com lodo de esgoto tratado

O Eucalyptus grandis é uma das espécies mais cultivadas no Brasil devido à sua produtividade e qualidade da madeira. Avaliaram-se o efeito da aplicação de lodo de esgoto tratado (0 a 40 t ha-1 base seca) e uma dose de adubo mineral nos atributos físicos e químicos da madeira de Eucalyptus grandis de árvores com cinco anos de idade, no Município de Itatinga, São Paulo, Brasil. O tipo de solo foi caracterizado como Latossolo Vermelho-Amarelo Distrófico (argila = 120 g kg-1 na camada de 0-20 cm) e o clima, como mesotérmico úmido (Cwa), segundo a classificação de Köeppen. O delineamento experimental foi o de blocos ao acaso, com seis tratamentos e quatro repetições. O diâmetro à altura do peito (DAP), a altura das árvores e o volume de madeira foram obtidos em todas as parcelas de oito árvores com DAP na classe de maior freqüência. As caracterizações físicas e químicas da madeira foram realizadas de acordo com as normas da ABTCP, TAPPI e ABNT. O lodo de esgoto diminuiu a densidade básica da madeira, mas não afetou os teores de celulose, lignina, extrativos e o poder calorífico da madeira. O decréscimo de densidade da madeira pela adubação com lodo de esgoto foi compensado pela maior produtividade de madeira.

Influência da lubrirrefrigeração na qualidade superficial de metais retificados

Na fabricação de componentes mecânicos precisos, que necessitam de alta resistência mecânica e ao desgaste, utiliza-se o processo de retificação, para conferir o acabamento final desejado e, também, para eliminar as deformações ocorridas durante a têmpera do aço. No entanto, as condições de retificação devem ser adequadas, para que não sejam introduzidas falhas na peça. Novos conceitos de lubrificação e refrigeração, para o processo de retificação, estão sendo pesquisados, de forma a diminuir os custos e os danos ambientais causados pelos fluidos de corte. Nesse trabalho, é analisada a influência das técnicas de mínima quantidade de lubrificante (MQL), refrigeração otimizada e refrigeração convencional, com diferentes vazões e velocidade de aplicação do fluido de corte, na qualidade das peças produzidas com aço ABNT 4340 endurecido, no processo de retificação cilíndrica externa de mergulho com a utilização de rebolos de CBN. O Aço ABNT 4340 apresenta várias aplicações industriais, sendo considerado de uso aeronáutico devido, sua alta resistência mecânica sem aumentar o peso dos componentes que o utilizam. A análise da qualidade das peças foi realizada com a verficação das rugosidades e com a análise de microscopias eletrônicas de varredura. Verificou-se, ainda, a força tangencial de corte. em relação às diferentes formas de aplicação do fluido de corte, notou-se o melhor desempenho da aplicação otimizada, para maiores velocidades, mostrando a eficiência do bocal utilizado. O processo otimizado e o processo MQL foram capazes de manter a integridade superficial das peças produzidas. Exceção somente para a condição MQL com vazão de fluido de corte de 40ml/h, que produziu trincas e queima superficial. Rebolos com baixa concentração de CBN, conseqüentemente mais baratos, proporcionaram bons resultados, quando associados com técnicas mais eficientes de aplicação de fluido de corte apresentando desgaste reduzido.

Editorial

Presented in this bulletin educational innovation that changes the traditional teaching of library and documentation with the inclusion of tools such as computer and support documents: the compact disc, floppy disks, optical media mail. It proposes a contemporary conception of storage and retrieval revolutionizing the traditional world of paper.

Phenolic compounds in Coleostephus myconis (L.) Rchb.f.: characterization by HPLC-DAD-ESI/MS and phenology effects

The Asteraceae family is spread worldwide. In Portugal, there are more than 300 species, standing out as one of the botanical families with largest representation in the Portuguese flora. Coleostephus myconis (L.) Rchb.f. is a scarcely studied Asteraceae species, characterized as having ruderal growth and persistence in abandoned soils (an expanding problem due to the desertification phenomena in rural areas). In this work, the flowers of C. myconis were collected in three different flowering stages (i: flower bud; ii: flower in anthesis; iii: senescent flower) from the Northwestern area of the Portuguese territory. Powdered samples (1 g) were extracted twice with ethanol:water 50:50 (v/v). After removing solvents, the combined extracts were re-dissolved, filtered through 0.22-μm disposable LC filter disks and analyzed by high performance liquid chromatography coupled to a diode array detector and electrospray ionization-mass spectrometry (HPLC-DAD/ESI-MS). The phenolic compounds were characterized according to their UV and mass spectra, and retention times. For the quantitative analysis, calibration curves of standard compounds were used. According to the UV spectra (λmax = 314-330 nm) and pseudomolecular ions ([M-H]-) at m/z 353 and 515, all producing an m/z 191 ion, four compounds derived from quinic acid were detected: 3-O-caffeoylquinic acid (Figure 1A), 5-O-caffeoylquinic acid (Figure 1B), 3,5-O-dicaffeoylquinic acid (Figure 1C) and 4,5-O-dicaffeoylquinic acid (Figure 1D), as also supported by the literature [1,2]. A fifth phenolic acid was identified as protocatechuic acid. The detected flavonoid were quercetin-O-glucuronide, quercetin-3-Oglucoside, myricetin-O-methyl-hexoside and a second glycosylated myricetin (not possible to identify completely). Some statistically significant changes were detected among the different assayed flowering stages; nevertheless, 3,5-O-dicaffeoylquinic acid was the major compound, independently of the phenologic stage. According to the previous results, C. myconis might be considered as a potential natural source of these valuable bioactive compounds, especially considering the high botanical representativeness of this plant and its inexpensiveness.

Análise e avaliação dos elementos de penetração no solo de equipamentos rodoviários após revestimento

The main objective is to analyze the abrasive wear resistance to the low stress of the elements that make up the organs of road machinery that are exposed directly to contact with abrasives. These samples were analyzed after these elements are coated superficially by the process of welding electrode coated with (SAER) and the manual process of coating type LVOF thermal spraying. As well, is to provide suggestions for a better recovery and return of these elements, which are reducing costs and avoiding downtime in the fronts of service. The samples were made from a substrate of carbon ABNT 1045 tempered steel, following the same specifications and composition of metals and alloys of constituents was followed the standard governing the dimensions of these samples and in accordance with the corresponding size. The results were evaluated by testing the hardness, abrasion resistance to wear by the low stress and the loss of volume involving the microstructure of coatings analyzed

Influência das espécies ativas na absorção de intersticiais durante a carbonitretação a plasma do TI

Physical-chemical properties of Ti are sensible to the presence of interstitial elements. In the case of thermochemical treatments plasma assisted, the influence of different active species is not still understood. In order to contribute for such knowledge, this work purposes a study of the role played by the active species atmosphere into the Ar N2 CH4 carbonitriding plasma. It was carried out a plasma diagnostic by OES (Optical Emission Spectroscopy) in the z Ar y N2 x CH4 plasma mixture, in which z, y and x indexes represent gas flow variable from 0 to 4 sccm (cm3/min). The diagnostic presents abrupt variations of emission intensities associated to the species in determined conditions. Therefore, they were selected in order to carry out the chemical treatment and then to investigate their influences. Commercial pure Ti disks were submitted to plasma carbonitriding process using pre-established conditions from the OES measurements while some parameters such as pressure and temperature were maintained constant. The concentration profiles of interstitial elements (C and N atoms) were determined by Resonant Nuclear Reaction Analysis (NRA) resulting in a depth profile plots. The reactions used were 15N(ρ,αγ)12C and 12C(α,α)12C. GIXRD (Grazing Incidence X-Ray Diffraction) analysis was used in order to identify the presence of phases on the surface. Micro-Raman spectroscopy was used in order to qualitatively study the carbon into the TiCxN1 structure. It has been verified which the density species effectively influences more the diffusion of particles into the Ti lattice and characteristics of the layer formed than the gas concentration. High intensity of N2 + (391,4 nm) and CH (387,1 nm) species promotes more diffusion of C and N. It was observed that Hα (656,3 nm) species acts like a catalyzer allowing a deeper diffusion of nitrogen and carbon into the titanium lattice.

Construção, caracterização e aplicação de eletrodos para descarga por arco de cátodo oco

The system built to characterize electrodes and, consequently, deposited fine films are constituted by a hollow cathode that works to discharges and low pressures (approximately 10-3 to 5 mbar), a source DC (0 to 1200 V), a cylindrical camera of closed borossilicato for flanges of stainless steel with an association of vacuum bombs mechanical and spread. In the upper flange it is connected the system of hollow cathode, which possesses an entrance of gas and two entrances for its refrigeration, the same is electrically isolated of the rest of the equipment and it is polarized negatively. In front of the system of hollow cathode there is a movable sample in stainless steel with possibility of moving in the horizontal and vertical. In the vertical, the sample can vary its distance between 0 and 70 mm and, in the horizontal, can leave completely from the front of the hollow cathode. The sample and also the cathode hollow are equipped with cromel-alumel termopares with simultaneous reading of the temperatures during the time of treatment. In this work copper electrodes, bronze, titanium, iron, stainless steel, powder of titanium, powder of titanium and silício, glass and ceramic were used. The electrodes were investigated relating their geometry change and behavior of the plasma of the cavity of hollow cathode and channel of the gas. As the cavity of hollow cathode, the analyzed aspects were the diameter and depth. With the channel of the gas, we verified the diameter. In the two situations, we investigated parameters as flow of the gas, pressure, current and applied tension in the electrode, temperature, loss of mass of the electrode with relationship at the time of use. The flow of gas investigated in the electrodes it was fastened in a work strip from 15 to 6 sccm, the constant pressure of work was among 2.7 to 8 x 10-2 mbar. The applied current was among a strip of work from 0,8 to 0,4 A, and their respective tensions were in a strip from 400 to 220 V. Fixing the value of the current, it was possible to lift the curve of the behavior of the tension with the time of use. That curves esteem in that time of use of the electrode to its efficiency is maximum. The temperatures of the electrodes were in the dependence of that curves showing a maximum temperature when the tension was maximum, yet the measured temperatures in the samples showed to be sensitive the variation of the temperature in the electrodes. An accompaniment of the loss of mass of the electrode relating to its time of use showed that the electrodes that appeared the spherical cavities lost more mass in comparison with the electrodes in that didn't appear. That phenomenon is only seen for pressures of 10-2 mbar, in these conditions a plasma column is formed inside of the channel of the gas and in certain points it is concentrated in form of spheres. Those spherical cavities develop inside of the channel of the gas spreading during the whole extension of the channel of the gas. The used electrodes were cut after they could not be more used, however among those electrodes, films that were deposited in alternate times and the electrodes that were used to deposit films in same times, those films were deposited in the glass substrata, alumina, stainless steel 420, stainless steel 316, silício and steel M2. As the eletros used to deposit films in alternate time as the ones that they were used to deposit in same times, the behavior of the thickness of the film obeyed the curve of the tension with relationship the time of use of the electrode, that is, when the tension was maximum, the thickness of the film was also maximum and when the tension was minimum, the thickness was minimum and in the case where the value of the tension was constant, the thickness of the film tends to be constant. The fine films that were produced they had applications with nano stick, bio-compatibility, cellular growth, inhibition of bacterias, cut tool, metallic leagues, brasagem, pineapple fiber and ornamental. In those films it was investigated the thickness, the adherence and the uniformity characterized by sweeping electronic microscopy. Another technique developed to assist the production and characterization of the films produced in that work was the caloteste. It uses a sphere and abrasive to mark the sample with a cap impression, with that cap form it is possible to calculate the thickness of the film. Through the time of life of the cathode, it was possible to evaluate the rate of waste of its material for the different work conditions. Values of waste rate up to 3,2 x 10-6 g/s were verified. For a distance of the substratum of 11 mm, the deposited film was limited to a circular area of 22 mm diameter mm for high pressures and a circular area of 75 mm for pressure strip. The obtained films presented thickness around 2,1 µm, showing that the discharge of arch of hollow cathode in argon obeys a curve characteristic of the tension with the time of life of the eletrodo. The deposition rate obtained in this system it is of approximately 0,18 µm/min

Tratamento térmico do titânio e suas consequências sobre as propriedades físico-químicas e de biocompatibilidade

The titanium and titanium alloys are widely used as biomaterial in biomedical device and so research have been developed aiming to improve and/or better to understand interaction biomaterial/biological environment. The process for manufacturing of this titanium implants usually involves a series of thermal and mechanical processes which have consequence on the final product. The heat treatments are usually used to obtain different properties for each application. In order to understand the influence of these treatments on the biological response of the surface, it was done, in this work, different heat treatments in titanium and analyzed their influence on the morphology, adhesion and proliferation of the pre-osteoblastic cells (MC3T3-E1). For such heat-treated titanium disks were characterized by optical microscopy, contact angle, surface energy, roughness, microhardness, X-ray diffraction and scanning through the techniques (BSE, EDS and EBSD). For the analysis of biological response were tested by MTT proliferation, adhesion by crystal violet and β1 integrin expression by flow cytometry. It was found that the presence of a microstructure very orderly, defined by a chemical attack, cells tend to stretch in the same direction of orientation of the material microstructure. When this order does not happen, the most important factor influencing cell proliferation is the residual stress, indicated by the hardness of the material. This way the disks with the highest level state of residual stress also showed increased cell proliferation

Détection et caractérisation de nouveaux disques circumstellaires autour d’étoiles de faibles masses et naines brunes jeunes.

La présence de disques circumstellaires signale la formation actuelle ou passée de systèmes planétaires, pour lesquels les processus de formation sont encore mal compris. Ce mémoire porte sur la détection et la caractérisation de disques circumstellaires autour d’étoiles de faibles masses (types spectraux > K5) et de naines brunes qui sont candidates ou membres d’associations cinématiques jeunes. Nous présentons ici les résultats de cette recherche ainsi que son implication pour la compréhension des processus de formation et d’évolution des systèmes planétaires. De l’échantillon initial composé de ∼ 1600 objets provenant des relevés BANYAN de Malo et al. ainsi que Gagné et al., dont seulement 600 satisfont nos critères de qualité sur les données, quatre nouveaux candidats de disque ont été découverts en détectant leur excès d’émission infrarouge dans les données d’archive de la mission WISE. Les données du relevé 2MASS ainsi que les spectres synthétiques BT-Settl ont été conjointement utilisés pour modéliser l’émission des étoiles. Les nouveaux candidats, dont les types spectraux sont tardifs (M4.5 à L0) et les masses se situent entre ∼ 13 et 120 M_Jup, ont des températures de disque de ∼ 135–520 K et des luminosités fractionnaires de 0,021–0,15. Pour deux des cibles, nous avons obtenu des spectres dans les longueurs d’onde visibles et infrarouges proches. Ces nouveaux spectres montrent respectivement des signes d’émission en Hα et Paβ, indiquant la présence d’accrétion, et ainsi de gaz, et renforçant l’hypothèse que ces objets sont réellement jeunes. Ces deux objets, vraisemblablement âgés de 40 Ma, pourraient représenter la première détection et caractérisation de disques porteurs de gaz plus vieux que 20 Ma.

Emergence and fate of siloxanes in waste streams: Release mechanisms, partitioning and persistence in three environmental compartments

Siloxanes are widely used in personal care and industrial products due to their low surface tension, thermal stability, antimicrobial and hydrophobic properties, among other characteristics. Volatile methyl siloxanes (VMS) have been detected both in landfill gas and biogas from anaerobic digesters at wastewater treatment plants. As a result, they are released to gas phase during waste decomposition and wastewater treatment. During transformation processes of digester or landfill gas to energy, siloxanes are converted to silicon oxides, leaving abrasive deposits on engine components. These deposits cause increased maintenance costs and in some cases complete engine overhauls become necessary. ^ The objectives of this study were to compare the VMS types and levels present in biogas generated in the anaerobic digesters and landfills and evaluate the energetics of siloxane transformations under anaerobic conditions. Siloxane emissions, resulting from disposal of silicone-based materials, are expected to increase by 29% within the next 10 years. Estimated concentrations and the risk factors of exposure to siloxanes were evaluated based on the initial concentrations, partitioning characteristics and persistence. It was determined that D4 has the highest risk factor associated to bioaccumulation in liquid and solid phase, whereas D5 was highest in gas phase. Additionally, as siloxanes are combusted, the particle size range causes them to be potentially hazardous to human health. When inhaled, they may affix onto the alveoli of the lungs and may lead to development of silicosis. Siloxane-based COD-loading was evaluated and determined to be an insignificant factor concerning COD limits in wastewater. ^ Removal of siloxane compounds is recommended prior to land application of biosolids or combustion of biogas. A comparison of estimated costs was made between maintenance practices for removal of siloxane deposits and installation/operation of fixed-bed carbon absorption systems. In the majority of cases, the installation of fixed-bed adsorption systems would not be a feasible option for the sole purpose of siloxane removal. However they may be utilized to remove additional compounds simultaneously.^

Détection et caractérisation de nouveaux disques circumstellaires autour d’étoiles de faibles masses et naines brunes jeunes

La présence de disques circumstellaires signale la formation actuelle ou passée de systèmes planétaires, pour lesquels les processus de formation sont encore mal compris. Ce mémoire porte sur la détection et la caractérisation de disques circumstellaires autour d’étoiles de faibles masses (types spectraux > K5) et de naines brunes qui sont candidates ou membres d’associations cinématiques jeunes. Nous présentons ici les résultats de cette recherche ainsi que son implication pour la compréhension des processus de formation et d’évolution des systèmes planétaires. De l’échantillon initial composé de ∼ 1600 objets provenant des relevés BANYAN de Malo et al. ainsi que Gagné et al., dont seulement 600 satisfont nos critères de qualité sur les données, quatre nouveaux candidats de disque ont été découverts en détectant leur excès d’émission infrarouge dans les données d’archive de la mission WISE. Les données du relevé 2MASS ainsi que les spectres synthétiques BT-Settl ont été conjointement utilisés pour modéliser l’émission des étoiles. Les nouveaux candidats, dont les types spectraux sont tardifs (M4.5 à L0) et les masses se situent entre ∼ 13 et 120 M_Jup, ont des températures de disque de ∼ 135–520 K et des luminosités fractionnaires de 0,021–0,15. Pour deux des cibles, nous avons obtenu des spectres dans les longueurs d’onde visibles et infrarouges proches. Ces nouveaux spectres montrent respectivement des signes d’émission en Hα et Paβ, indiquant la présence d’accrétion, et ainsi de gaz, et renforçant l’hypothèse que ces objets sont réellement jeunes. Ces deux objets, vraisemblablement âgés de 40 Ma, pourraient représenter la première détection et caractérisation de disques porteurs de gaz plus vieux que 20 Ma.

PROTEIN-TRIGGERED SUSTAINED DRUG RELEASE CONTROLLED BY APTAMERS BOUND TO OLIGOMER-CROSSLINKED ALGINATE

Sustained drug release systems provide many advantages over traditional delivery methods such as extending the time in which the drug is found to be within an effective concentration within the therapeutic window, which decreases the frequency of administration of the drug, and increases patient compliance. Research using polyacrylamide crosslinked by oligomers containing an aptamer sequence, has demonstrated a pulsatile release over 50 minutes triggered by a 2 mM target adenosine concentration. This thesis aims to build off this concept by designing a system that delivers in a sustained manner when triggered by micromolar target concentrations reflective of disease in vivo, using macromolecular targets. For example, the disease wet age related macular degeneration (wet AMD) is associated with increased concentrations of the protein vascular endothelial growth factor (VEGF-A) – a macromolecule. Patients with wet AMD would benefit from the implantation of devices or microspheres that release drugs in a sustained manner in response to local VEGF concentrations. In this thesis, we hypothesize that the protein lysozyme, used to demonstrate proof-of-concept, could trigger the increased release of drugs from oligomer-crosslinked alginate. The objectives are to (i) demonstrate sustained release from alginate, (ii) design oligomer crosslinked alginate that degrades in response to lysozyme, and then (iii) use these systems to control the release of FITC-dextran with and without lysozyme. A series of control experiments and analyses were used to optimize the crosslinking of alginate by annealed oligomers. The cumulative release of FITC-dextran (MW 20,000) from oligomer crosslinked alginate increased by 3.4 μg when lysozyme (3 μM) was introduced at 48 hours, as opposed to controls which released only 0.2 μg. FITC-loaded alginate microspheres coated by oligomer-crosslinked alginate released 15% more FITC-dextran over 120 hours when placed into 3 μM of lysozyme than without lysozyme. Controls of alginate crosslinked with PEG or control oligomers (without a lysozyme aptamer sequence) had no changes in release with lysozyme. The incorporation of a lysozyme aptamer onto oligomers used to crosslink alginate disks or alginate coatings on microspheres resulted in different diffusion and release of FITC-dextran into PBS with or without lysozyme. This approach could be adapted for the delivery of drugs to diseases with specific protein profiles such as wet AMD.

Effects of spin on high-energy radiation from accreting black holes

Observations of jets in X-ray binaries show a correlation between radio power and black hole spin. This correlation, if confirmed, points toward the idea that relativistic jets may be powered by the rotational energy of black holes. In order to examine this further, we perform general relativistic radiative transport calculations on magnetically arrested accretion flows, which are known to produce powerful jets via the Blandfordâ Znajek (BZ) mechanism. We find that the X-ray and γ-ray emission strongly depend on spin and inclination angle. Surprisingly, the high-energy power does not show the same dependence on spin as the BZ jet power, but instead can be understood as a redshift effect. In particular, photons observed perpendicular to the spin axis suffer little net redshift until originating from close to the horizon. Such observers see deeper into the hot, dense, highly magnetized inner disk region. This effect is largest for rapidly rotating black holes due to a combination of frame dragging and decreasing horizon radius. While the X-ray emission is dominated by the near horizon region, the near-infrared (NIR) radiation originates at larger radii. Therefore, the ratio of X-ray to NIR power is an observational signature of black hole spin.

Initiation and early crack growth in VHCF of stainless steels : Experimental and theoretical analysis

Mechanical fatigue is a failure phenomenon that occurs due to repeated application of mechanical loads. Very High Cycle Fatigue (VHCF) is considered as the domain of fatigue life greater than 10 million load cycles. Increasing numbers of structural components have service life in the VHCF regime, for instance in automotive and high speed train transportation, gas turbine disks, and components of paper production machinery. Safe and reliable operation of these components depends on the knowledge of their VHCF properties. In this thesis both experimental tools and theoretical modelling were utilized to develop better understanding of the VHCF phenomena. In the experimental part, ultrasonic fatigue testing at 20 kHz of cold rolled and hot rolled stainless steel grades was conducted and fatigue strengths in the VHCF regime were obtained. The mechanisms for fatigue crack initiation and short crack growth were investigated using electron microscopes. For the cold rolled stainless steels crack initiation and early growth occurred through the formation of the Fine Granular Area (FGA) observed on the fracture surface and in TEM observations of cross-sections. The crack growth in the FGA seems to control more than 90% of the total fatigue life. For the hot rolled duplex stainless steels fatigue crack initiation occurred due to accumulation of plastic fatigue damage at the external surface, and early crack growth proceeded through a crystallographic growth mechanism. Theoretical modelling of complex cracks involving kinks and branches in an elastic half-plane under static loading was carried out by using the Distributed Dislocation Dipole Technique (DDDT). The technique was implemented for 2D crack problems. Both fully open and partially closed crack cases were analyzed. The main aim of the development of the DDDT was to compute the stress intensity factors. Accuracy of 2% in the computations was attainable compared to the solutions obtained by the Finite Element Method.