Development of calix[4]arene-functionalized microcantilever array sensing system for the rapid, sensitive and simultaneous detection of metal ions in aqueous solutions

The work described in this thesis was conducted with the aim of: 1) investigating the binding capabilities of calix[4]arene-functionalized microcantilevers towards specific metal ions and 2) developing a new16-microcantilever array sensing system for the rapid, and simultaneous detection of metal ions in fresh water. Part I of this thesis reports on the use of three new bimodal calix[4]arenes (methoxy, ethoxy and crown) as potential host/guest sensing layers for detecting selected ions in dilute aqueous solutions using single microcantilever experimental system. In this work it was shown that modifying the upper rim of the calix[4]arenes with a thioacetate end group allow calix[4]arenes to self-assemble on Au(111) forming complete highly ordered monolayers. It was also found that incubating the microcantilevers coated with 5 nm of Inconel and 40 nm of Au for 1 h in a 1.0 M solution of calix[4]arene produced the highest sensitivity. Methoxy-functionalized microcantilevers showed a definite preference for Ca²⁺ ions over other cationic guests and were able to detect trace concentration as low as 10⁻¹² M in aqueous solutions. Microcantilevers modified with ethoxy calix[4]arene displayed their highest sensitivity towards Sr²⁺ and to a lesser extent Ca²⁺ ions. Crown calix[4]arene-modified microcantilevers were however found to bind selectively towards Cs⁺ ions. In addition, the counter anion was also found to contribute to the deflection. For example methoxy calix[4]arene-modified microcantilever was found to be more sensitive to CaCl₂ over other water-soluble calcium salts such as Ca(NO₃)₂ , CaBr₂ and CaI₂. These findings suggest that the response of calix[4]arene-modified microcantilevers should be attributed to the target ionic species as a whole instead of only considering the specific cation and/or anion. Part II presents the development of a 16-microcantilever sensor setup. The implementation of this system involved the creation of data analysis software that incorporates data from the motorized actuator and a two-axis photosensitive detector to obtain the deflection signal originating from each individual microcantilever in the array. The system was shown to be capable of simultaneous measurements of multiple microcantilevers with different coatings. A functionalization unit was also developed that allows four microcantilevers in the array to be coated with an individual sensing layer one at the time. Because of the variability of the spring constants of different cantilevers within the array, results presented were quoted in units of surface stress unit in order to compare values between the microcantilevers in the array.

Documentation of IPY Seamount

During Leg ANT-XXIII/9 on 2007-04-04 the German research vessel POLARSTERN mapped a significant bathymetric feature with its swath sonar system in the area of the Indian Ridge in the Southern Indian Ocean. The feature is a vulcano located 800 km northwest of Crozet Island. The vulcano with a crater has an absolute height of 1370 m, extending from 3100 m mean depth of the surrounding sea floor to a depth of 1730 m at the top of the crater rim. The crater has a depth of about 135 m. Due to the fact, that the feature was discovered just a month after the fourth International Polar Year (IPY) 2007/2009 has started, it was named "IPY Seamount". The undersea feature name proposal was submitted to the International Hydrographic Organisation (IHO) and the Intergovernmental Oceanographic Commission (IOC of UNESCO) on 2007-05-11. The name was officially accepted by the GEBCO Sub-Committee on Undersea Feature Names (SCUFN) at its 20th meeting in July and was added to the GEBCO Gazetteer of UFN.

Radiocarbon ages and pollen record of a sediment core from Lake Lyadhej-To

An 1180-cm long core recovered from Lake Lyadhej-To (68°15'N, 65°45'E, 150 m a.s.l.) at the NW rim of the Polar Urals Mountains reflects the Holocene environmental history from ca. 11,000 cal. yr BP. Pollen assemblages from the diamicton (ca. 11,000-10,700 cal. yr BP) are dominated by Pre-Quaternary spores and redeposited Pinaceae pollen, pointing to a high terrestrial input. Turbid and nutrient-poor conditions existed in the lake ca. 10,700-10,550 cal. yr BP. The chironomid-inferred reconstructions suggest that mean July temperature increased rapidly from 10.0 to 11.8 °C during this period. Sparse, treeless vegetation dominated on the disturbed and denuded soils in the catchment area. A distinct dominance of planktonic diatoms ca. 10,500-8800 cal. yr BP points to the lowest lake-ice coverage, the longest growing season and the highest bioproductivity during the lake history. Birch forest with some shrub alder grew around the lake reflecting the warmest climate conditions during the Holocene. Mean July temperature was likely 11-13 °C and annual precipitation-400-500 mm. The period ca. 8800-5500 cal. yr BP is characterized by a gradual deterioration of environmental conditions in the lake and lake catchment. The pollen- and chironomid-inferred temperatures reflect a warm period (ca. 6500-6000 cal. BP) with a mean July temperature at least 1-2 °C higher than today. Birch forests disappeared from the lake vicinity after 6000 cal. yr BP. The vegetation in the Lyadhej-To region became similar to the modern one. Shrub (Betula nana, Salix) and herb tundra have dominated the lake catchment since ca. 5500 cal. yr BP. All proxies suggest rather harsh environmental conditions. Diatom assemblages reflect relatively short growing seasons and a longer persistence of lake-ice ca. 5500-2500 cal. yr BP. Pollen-based climate reconstructions suggest significant cooling between ca. 5500 and 3500 cal. yr BP with a mean July temperature 8-10 °C and annual precipitation-300-400 mm. The bioproductivity in the lake remained low after 2500 cal. yr BP, but biogeochemical proxies reflect a higher terrestrial influx. Changes in the diatom content may indicate warmer water temperatures and a reduced ice cover on the lake. However, chironomid-based reconstructions reflect a period with minimal temperatures during the lake history.

Methane and sulphate consumption in different habitats of the Håkon Mosby Mud Volcano (HMMV)

The Hakon Mosby Mud Volcano is a highly active methane seep hosting different chemosynthetic communities such as thiotrophic bacterial mats and siboglinid tubeworm assemblages. This study focuses on in situ measurements of methane fluxes to and from these different habitats, in comparison to benthic methane and oxygen consumption rates. By quantifying in situ oxygen, methane, and sulfide fluxes in different habitats, a spatial budget covering areas of 10-1000 -m diameter was established. The range of dissolved methane efflux (770-2 mmol m-2 d-1) from the center to the outer rim was associated with a decrease in temperature gradients from 46°C to < 1°C m-1, indicating that spatial variations in fluid flow control the distribution of benthic habitats and activities. Accordingly, total oxygen uptake (TOU) varied between the different habitats by one order of magnitude from 15 mmol m-2 d-1 to 161 mmol m-2 d-1. High fluid flow rates appeared to suppress benthic activities by limiting the availability of electron acceptors. Accordingly, the highest TOU was associated with the lowest fluid flow and methane efflux. This was most likely due to the aerobic oxidation of methane, which may be more relevant as a sink for methane as previously considered in submarine ecosystems.

Prevalência da síndrome metabólica em receptores de transplante renal de acordo com o gênero e tempo pós-transplante /

Metabolic syndrome (MS) is defined as a set of cardiovascular risk factors including obesity, systemic high blood pressure (SHBP), changes in glucose metabolism and dyslipidemia. The prevalence of MS in renal transplant recipients (RTR) ranges from 15% to 65%, increasing the risk of cardiovascular disease (CVD) and reducing renal allograft survival in the long term. The objectives of this study were to determine the prevalence and frequency of MS in renal transplant patients according to gender and time of transplantation and to evaluate renal function in patients with and without MS. Patients and Methods: Crosssectional study conducted from August 2012 to September 2013 involving 153 renal transplant recipients. MS was defined according to the National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III). The sample was divided into two groups: patients with metabolic syndrome (WMS patients) and patients without metabolic syndrome (WoMS patients) and according to gender. The WMS patients were stratified into quartiles according to the renal transplantation period (RTP), and variables related to MS were analyzed for both sexes. Results: MS was diagnosed in 58.1% of the studied population, specifically in MS was found 58.4% of men and 41.6% of women (P ˂ 0.05). The male and female with MS were 48.8 ± 11.6 years old vs. 47.1 ± 12.7 years old and the time of post transplantation was 76.1 ± 76.5 months vs. 84.7 ± 65.4 months, respectively (P >0,05). When we compared the sexes in the WMS group, systolic blood pressure (SBP) was higher in men (137.0 ± 18.1 vs. 128.9 ± 13.6 mmHg, P= 0.029), while the other components of MS did not exhibit significant differences. With respect to renal function, when we compared the sexes in the WMS group, the serum creatinine (sCr) was higher in men (1.73 ± 0.69 vs. 1.31 ± 0.47 mg/dL, P= 0.0012), while the urinary protein/creatinine ratio was higher in women (0.48 ± 0.69 vs. 0.37 ± 0.48 mg/dL, P=0.0150). We found no significant difference in the estimated glomerular filtration rate (eGFR) between WMS and WoMS patients for women and men (50.6 ± 19.1 vs. 50.1 ± 18.3 mL/min/1.73 m², P=0.909). We found a significant positive association between eGFR and HDL-c levels (r=0.3371; P=0.0145) for WMS men. The MS components showed no significant differences in RTP for different interquartile ranges, except for diastolic blood pressure (DBP) in women, where there was a significant variation among the quartiles evaluated (P=0.0009). Conclusion: the prevalence of MS was similar in the different quartiles in both sexes, in relation to time post TX. There was no significant difference in eGFR in patients WMS and WoMS, in both sexes. Concluding that the MS did not vary in relation to time post transplant.

Pathogen-Specific Adaptations to Conserved Signaling Pathways in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that causes significant disease worldwide. Even though this fungus has not evolved specifically to cause human disease, it has a remarkable ability to adapt to many different environments within its infected host. C. neoformans adapts by utilizing conserved eukaryotic and fungal-specific signaling pathways to sense and respond to stresses within the host. Upon infection, two of the most significant environmental changes this organism experiences are elevated temperature and high pH.

Conserved Rho and Ras family GTPases are central regulators of thermotolerance in C. neoformans. Many GTPases require prenylation to associate with cellular membranes and function properly. Using molecular genetic techniques, microscopy, and infection models, I demonstrated that the prenyltransferase, geranylgeranyl transferase I (GGTase I) is required for thermotolerance and pathogenesis. Using fluorescence microscopy, I found that only a subset of conserved GGTase I substrates requires this enzyme for membrane localization. Therefore, the C. neoformans GGTase I may recognize its substrate in a slightly different manner than other eukaryotic organisms.

The alkaline response transcription factor, Rim101, is a central regulator of stress-response genes important for adapting to the host environment. In particular, Rim101 regulates cell surface alterations involved in immune avoidance. In other fungi, Rim101 is activated by alkaline pH through a conserved signaling pathway, but this pathway had yet been characterized in C. neoformans. Using molecular genetic techniques, I identified and analyzed the conserved members of the Rim pathway. I found that it was only partially conserved in C. neoformans, missing the components that sense pH and initiate pathway activation. Using a genetic screen, I identified a novel Rim pathway component named Rra1. Structural prediction and genetic epistasis experiments suggest that Rra1 may serve as the Rim pathway pH sensor in C. neoformans and other related basidiomycete fungi.

To explore the relevance of Rim pathway signaling in the interaction of C neoformans with its host, I characterized the Rim101-regulated cell wall changes that prevent immune detection. Using HPLC, enzymatic degradation, and cell wall stains, I found that the rim101Δ mutation resulted in increased cell wall chitin exposure. In vitro co-culture assays demonstrated that increased chitin exposure is associated with enhanced activation of macrophages and dendritic cells. To further test this association, I demonstrated that other mutant strains with increased chitin exposure induce macrophage and dendritic cell responses similar to rim101Δ. We used primary macrophages from mutant mouse lines to demonstrate that members of both the Toll-like receptor and C-type lectin receptor families are involved in detecting strains with increased chitin exposure. Finally, in vivo immunological experiments demonstrated that the rim101Δ strain induced a global inflammatory immune response in infected mouse lungs, expanding upon our previous in vivo rim101Δ studies. These results demonstrate that cell wall organization largely determines how fungal cells are detected by the immune system.

Baseline characteristics of climate, permafrost, and land cover from Samoylov Island, Lena River Delta, Siberia

Samoylov Island is centrally located within the Lena River Delta at 72° N, 126° E and lies within the Siberian zone of continuous permafrost. The landscape on Samoylov Island consists mainly of late Holocene river terraces with polygonal tundra, ponds and lakes, and an active floodplain. The island has been the focus of numerous multidisciplinary studies since 1993, which have focused on climate, land cover, ecology, hydrology, permafrost and limnology. This paper aims to provide a framework for future studies by describing the characteristics of the island's meteorological parameters (temperature, radiation and snow cover), soil temperature, and soil moisture. The land surface characteristics have been described using high resolution aerial images in combination with data from ground-based observations. Of note is that deeper permafrost temperatures have increased between 0.3 to 1.3 °C over the last five years. However, no clear warming of air and active layer temperatures is detected since 1998, though winter air temperatures during recent years have not been as cold as in earlier years.

Grain size distribution of the lagoonal deposits within the South Malé Atoll, Maldives, Indian Ocean

Seismic and multibeam data, as well as sediment samples were acquired in the South Malé Atoll in the Maldives archipelago in 2011 to unravel the stratigraphy and facies of the lagoonal deposits. Multichannel seismic lines show that the sedimentary succession locally reaches a maximum thickness of 15-20 m above an unconformity interpreted as the emersion surface which developed during the last glacial sea-level lowstand. Such depocenters are located in current-protected areas flanking the reef rim of the atoll or in infillings of karst dolinas. Much of the 50 m deep sea floor in the lagoon interior is current swept, and has no or very minor sediment cover. Erosive current moats line drowned patch reefs, whereas other areas are characterized by nondeposition. Karst sink holes, blue holes and karst valleys occur throughout the lagoon, from its rim to its center. Lagoonal sediments are mostly carbonate rubble and coarse-grained carbonate sands with frequent large benthic foraminifers, Halimeda flakes, red algal nodules, mollusks, bioclasts, and intraclasts, some of them glauconitic, as well as very minor ooids. Finer-grained deposits locally are deposited in current-protected areas behind elongated faros, i.e., small atolls which are part of the rim of South Malé Atoll. The South Malé Atoll is a current-flushed atoll, where water and sediment export with the open sea is facilitated by the multiple passes dissecting the atoll rim. With an elevated reef rim and tower-like reefs in the atoll interior it is an example of a leaky bucket atoll which shares characteristics of incipiently drowned carbonate banks or drowning sequences as known from the geological record.

(Table 1) Hydrocarbon gas composition of sediment and gas hydrate collected in video-guided grabs from Chapopote

In the Campeche Knolls, in the southern Gulf of Mexico, lava-like flows of solidified asphalt cover more than 1 square kilometer of the rim of a dissected salt dome at a depth of 3000 meters below sea level. Chemosynthetic tubeworms and bivalves colonize the sea floor near the asphalt, which chilled and contracted after discharge. The site also includes oil seeps, gas hydrate deposits, locally anoxic sediments, and slabs of authigenic carbonate. Asphalt volcanism creates a habitat for chemosynthetic life that may be widespread at great depth in the Gulf of Mexico.

Standing stocks and carbon isotopes of live benthic foraminifera from the South Atlantic

Live (Rose Bengal stained) and dead benthic foraminifera of surface and subsurface sediments from 25 stations in the eastern South Atlantic Ocean and the Atlantic sector of the Southern Ocean were analyzed to decipher a potential influence of seasonally and spatially varying high primary productivity on the stable carbon isotopic composition of foraminiferal tests. Therefore, stations were chosen so that productivity strongly varied, whereas conservative water mass properties changed only little. To define the stable carbon isotopic composition of dissolved inorganic carbon (d13CDIC) in ambient water masses, we compiled new and previously published d13CDIC data in a section running from Antarctica through Agulhas, Cape and Angola Basins, via the Guinea Abyssal Plain to the Equator. We found that intraspecific d13C variability of all species at a single site is constantly low throughout their distribution within the sediments, i.e. species specific and site dependent mean values calculated from all subbottom depths on average only varied by +/-0.09 per mil. This is important because it makes the stable carbon isotopic signal of species independent of the particular microhabitat of each single specimen measured and thus more constant and reliable than has been previously assumed. So-called vital and/or microhabitat effects were further quantified: (1) d13C values of endobenthic Globobulimina affinis, Fursenkoina mexicana, and Bulimina mexicana consistently are by between -1.5 and -1.0 per mil VPDB more depleted than d13C values of preferentially epibenthic Fontbotia wuellerstorfi, Cibicidoides pachyderma, and Lobatula lobatula. (2) In contrast to the Antarctic Polar Front region, at all stations except one on the African continental slope Fontbotia wuellerstorfi records bottom water d13CDIC values without significant offset, whereas L. lobatula and C. pachyderma values deviate from bottom water values by about -0.4 per mil and -0.6 per mil, respectively. This adds to the growing amount of data on contrasting cibicid d13C values which on the one hand support the original 1:1-calibration of F. wuellerstorfi and bottom water d13CDIC, and on the other hand document severe depletions of taxonomically close relatives such as L. lobatula and C. pachyderma. At one station close to Bouvet Island at the western rim of Agulhas Basin, we interpret the offset of -1.5 per mil between bottom water d13CDIC and d13C values of infaunal living Bulimina aculeata in contrast to about -0.6 +/- 0.1 per mil measured at eight stations close-by, as a direct reflection of locally increased organic matter fluxes and sedimentation rates. Alternatively, we speculate that methane locally released from gas vents and related to hydrothermal venting at the mid-ocean ridge might have caused this strong depletion of 13C in the benthic foraminiferal carbon isotopic composition. Along the African continental margin, offsets between deep infaunal Globobulimina affinis and epibenthic Fontbotia wuellerstorfi as well as between shallow infaunal Uvigerina peregrina and F. wuellerstorfi, d13C values tend to increase with generally increasing organic matter decomposition rates. Although clearly more data are needed, these offsets between species might be used for quantification of biogeochemical paleogradients within the sediment and thus paleocarbon flux estimates. Furthermore, our data suggest that in high-productivity areas where sedimentary carbonate contents are lower than 15 weight %, epibenthic and endobenthic foraminiferal d13C values are strongly influenced by 13C enrichment probably due to carbonate-ion undersaturation, whereas above this sedimentary carbonate threshold endobenthic d13C values reflect depleted pore water d13CDIC values.

(Table 1) Age control points and dating methods for sediment cores of the Sea of Okhotsk

Marine- and terrestrial-derived biomarkers (alkenones, brassicasterol, dinosterol, and long-chain n-alkanes), as well as carbonate, biogenic opal, and ice-rafted debris (IRD), were measured in two sediment cores in the Sea of Okhotsk, which is located in the northwestern Pacific rim and characterized by high primary productivity. Down-core profiles of phytoplankton markers suggest that primary productivity abruptly increased during the global Meltwater Pulse events 1A (about 14 ka) and 1B (about 11 ka) and stayed high in the Holocene. Spatial and temporal distributions of the phytoplankton productivity were found to be consistent with changes in the reconstructed sea ice distribution on the basis of the IRD. This demonstrates that the progress and retreat of sea ice regulated primary productivity in the Sea of Okhotsk with minimum productivity during the glacial period. The mass accumulation rates of alkenones, CaCO3, and biogenic opal indicate that the dominant phytoplankton species during deglaciation was the coccolithophorid, Emiliania huxleyi, which was replaced by diatoms in the late Holocene. Such a phytoplankton succession was probably caused by an increase in silicate supply to the euphotic layer, possibly associated with a change in surface hydrography and/or linked to enhanced upwelling of North Pacific Deep Water.

Physical oceanography from CTD during Maria S. Merian cruise MSM20/4 in spring 2012

With an extension of > 40 km**2 the recently discovered Campeche cold-water coral province located at the northeastern rim of the Campeche Bank in the southern Gulf of Mexico belongs to the largest coherent cold-water coral areas discovered so far. The Campeche province consists of numerous 20-40 m-high elongated coral mounds that are developed in intermediate water depths of 500 to 600 m. The mounds are colonized by a vivid cold-water coral ecosystem that covers the upper flanks and summits. The rich coral community is dominated by the framework-building Scleractinia Enallopsammia profunda and Lophelia pertusa, while the associated benthic megafauna shows a rather scarce occurrence. The recent environmental setting is characterized by a high surface water production caused by a local upwelling center and a dynamic bottom-water regime comprising vigorous bottom currents, obvious temporal variability, and strong density contrasts, which all together provide optimal conditions for the growth of cold-water corals. This setting - potentially supported by the diel vertical migration of zooplankton in the Campeche area - controls the delivering of food particles to the corals. The Campeche cold-water coral province is, thus, an excellent example highlighting the importance of the oceanographic setting in securing the food supply for the development of large and vivid cold-water coral ecosystems.

(Table S1) Age determination ofs ediment cores of the Azores Current System

In order to test the sensitivity of marine primary productivity in the midlatitude open ocean North Atlantic to changes in the Atlantic Meridional Overturning Circulation (AMOC), we investigated two spliced sediment cores from a site south of the Azores Islands at the northern rim of the North Atlantic subtropical gyre. For this purpose we analyzed coccolithophore assemblages, diatom abundances, alkenones and conducted X-ray fluorescence (XRF) core scanning. During times of reduced AMOC, especially during Heinrich event 1 (H1) and the Younger Dryas, we observe a strong increase in productivity as evidenced by high coccolith accumulation rates, high alkenone concentrations/accumulation rates, high Ba/Ti-ratios, high abundances of diatoms and low abundances ofF. profunda. The increased productivity is partly caused by a more southern position of the Azores Front (AzF), and hence by a less northward extension of the subtropical gyre, as deduced from high abundances of the temperate coccolithophore species G. muellerae and low abundances of subtropical species (Oolithotus spp., Umbellosphaera spp., Umbilicosphaeraspp.). However, to explain the full range of the observed productivity increase, other factors like increased westerly winds and advection of nutrient-rich surface waters have also to be considered. Because this pattern can also be observed in other sediment cores from the midlatitude North Atlantic, we propose that during times of reduced AMOC there has been a band of strongly increased productivity across the North Atlantic at the northern rim of the contracted subtropical gyre, which partly counteracts the decreased organic carbon pump in the high northern latitudes.

A Mineralogical Study of a Dravitic Tourmaline from the Grenville Province

Tourmaline from a gem-quality deposit in the Grenville province has been studied with X-ray diffraction, visible-near infrared spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscopy, electron microprobe and optical measurements. The tourmaline is found within tremolite-rich calc-silicate pods hosted in marble of the Central Metasedimentary Belt. The crystals are greenish-greyish-brown and have yielded facetable material up to 2.09 carats in size. Using the classification of Henry et al. 2011 the tourmaline is classified as a dravite, with a representative formula shown to be (Na0.73Ca0.2380.032)(Mg2+2.913Fe2+0.057Ti4+0.030) (Al3+5.787Fe3+0.017Mg2+0.14)(Si6.013O18)(BO3)3(OH)3((OH,O)0.907F0.093). Rietveld analysis of powder diffraction data gives a = 15.9436(8) Å, c = 7.2126(7) Å and a unit cell volume of 1587.8 Å3. A polished thin section was cut perpendicular to the c-axis of one tourmaline crystal, which showed zoning from a dark brown core into a lighter rim into a thin darker rim and back into lighter zonation. Through the geochemical data, three key stages of crystal growth can be seen within this thin section. The first is the core stage which occurs from the dark core to the first colourless zone; the second is from this colourless zone increasing in brown colour to the outer limit before a sudden absence of colour is noted; the third is a sharp change from the end of the second and is entirely colourless. These events are the result of metamorphism and hydrothermal fluids resulting from nearby felsic intrusive plutons. Scanning electron microscope, and electron microprobe traverses across this cross-section revealed that the green colour is the result of iron present throughout the system while the brown colour is correlated with titanium content. Crystal inclusions in the tourmaline of chlorapatite, and zircon were identified by petrographic analysis and confirmed using scanning electron microscope data and occur within the third stage of formation.

Female employment from the household. Towards a comprehensive analysis of work-life articulation

Research on women’s employment has proliferated over recent decades, often under a perspective that conceptualizes female labour market activity as independent of male presences and absences in the productive and reproductive spheres. In the face of these approaches, the article argues the need to focus on the couple as the unit of analysis of work-life articulation. After referring to the main theoretical arguments that, from a gender perspective within labour studies, have pointed out the relevance of placing the household as the central space for the analysis of the sexual division of labour, the article reviews different empirical contributions that have incorporated such perspective in the international literature. Next, the state of the art in the Spanish literature is presented, before arguing the desirability of applying such framework of analysis to the study of employment and care work in Spanish households, which are at present undergoing major transformations.