NMR methods have been designed and employed in studying ofcomplex liquids and solids formed by surfactants. PGSE NMRexperiments are easily biased by convection; this artifact canbe avoided by changing the sample holder and by usingconvection-compensated pulse sequences. The temperaturedistribution within samples was controlled using thetemperature dependent order parameter for CBr2H2dissolved in a thermotropic nematic solvent.Electronic ringing that often spoils accurate NMR experimentsfor broad lines was removed by the using composite pulses andquadrupole echo sequences with appropriate phase cycles. Field-dependent81Br and35Cl NMR relaxation studies in micellar solutions ofC16TAX surfactants showed that the structure ordynamics of the hydration shell is more influenced by thesurfactant cation for bromide than for chloride, in agreementwith their position in the Hoffmeister series. The presence ofa small but significant frequency-dependent relaxation showedthat the lateral self diffusion of the anions may be reduced ascompared to its bulk value in diluted solutions but only with afactor of 1.0 - 2.5. The ions are clearly not "bound" to thesurface. A field-dependent2H NMR relaxation study on the CTABr-α-d2and benzene-d6showed an initial one-dimensional micellargrowth followed by the appearance of microemulsion droplets onaddition of benzene. The local mobility of the benzene wasreduced when solubilized in small amounts, consistent with aninitial average location of benzene at the micellar interface.The surfactant diffusion coefficients fromconvection-compensated PGSE NMR experiments in the C12E8-D2O system showed monotonous growth of the micellesupon increasing temperature. Emulsion droplets in the C12E5-decane-D2O system where shown to coarsen according to theOstwald ripening theory after being brought out of equilibriumby a temperature drop. X-ray scattering and2H NMR line-shape and relaxation experimentssuggested that complex solids formed by a partly-sulfatedpolysaccharide and CnTAB exhibit regular ordering at both microscopicand mesoscopic length scales. <b>Keywords</b>: CTAB, CTAC, C12E8, C12E5, decane, benzene, CBr2H2, polysaccharide, micelle, microemulsion, emulsion,Ostwald ripening, NMR,81Br,35Cl,2H, field- dependent spin relaxation, PGSE, selfdiffusion, convection, ringing, thermometer, generalized Blochequations, EXORCYCLE, quadrupole echo, SAXS, WAXS, cryo-TEM.

<p>NMR methods have been designed and employed in studying ofcomplex liquids and solids formed by surfactants. PGSE NMRexperiments are easily biased by convection; this artifact canbe avoided by changing the sample holder and by usingconvection-compensated pulse sequences. The temperaturedistribution within samples was controlled using thetemperature dependent order parameter for CBr₂H₂dissolved in a thermotropic nematic solvent.Electronic ringing that often spoils accurate NMR experimentsfor broad lines was removed by the using composite pulses andquadrupole echo sequences with appropriate phase cycles.</p><p>Field-dependent⁸¹Br and³⁵Cl NMR relaxation studies in micellar solutions ofC₁₆TAX surfactants showed that the structure ordynamics of the hydration shell is more influenced by thesurfactant cation for bromide than for chloride, in agreementwith their position in the Hoffmeister series. The presence ofa small but significant frequency-dependent relaxation showedthat the lateral self diffusion of the anions may be reduced ascompared to its bulk value in diluted solutions but only with afactor of 1.0 - 2.5. The ions are clearly not "bound" to thesurface. A field-dependent²H NMR relaxation study on the CTABr-α-<i>d</i><i>2</i>and benzene-<i>d</i><i>6</i>showed an initial one-dimensional micellargrowth followed by the appearance of microemulsion droplets onaddition of benzene. The local mobility of the benzene wasreduced when solubilized in small amounts, consistent with aninitial average location of benzene at the micellar interface.The surfactant diffusion coefficients fromconvection-compensated PGSE NMR experiments in the C₁₂E₈-D₂O system showed monotonous growth of the micellesupon increasing temperature. Emulsion droplets in the C₁₂E₅-decane-D₂O system where shown to coarsen according to theOstwald ripening theory after being brought out of equilibriumby a temperature drop. X-ray scattering and²H NMR line-shape and relaxation experimentssuggested that complex solids formed by a partly-sulfatedpolysaccharide and C_nTAB exhibit regular ordering at both microscopicand mesoscopic length scales.</p><p><b>Keywords</b>: CTAB, CTAC, C₁₂E₈, C₁₂E₅, decane, benzene, CBr₂H₂, polysaccharide, micelle, microemulsion, emulsion,Ostwald ripening, NMR,⁸¹Br,³⁵Cl,²H, field- dependent spin relaxation, PGSE, selfdiffusion, convection, ringing, thermometer, generalized Blochequations, EXORCYCLE, quadrupole echo, SAXS, WAXS, cryo-TEM.</p>

Submitted by Eunice Novais (enovais@uepg.br) on 2018-06-15T18:10:58Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Kerolin Ap Alves Gomes.pdf: 2922365 bytes, checksum: ccbcc929da0afe5876dd9273d11519e6 (MD5) / Made available in DSpace on 2018-06-15T18:10:58Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Kerolin Ap Alves Gomes.pdf: 2922365 bytes, checksum: ccbcc929da0afe5876dd9273d11519e6 (MD5) Previous issue date: 2018-01-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A utilização de materiais ambientalmente corretos, de fonte renovável, assim como a celulose, tem se apresentado como assunto de interesse para muitos pesquisadores. Neste trabalho os nanocristais de celulose (NCC) foram extraídos a partir da celulose microcristalina comercial (CMC) com posterior ajuste de pH e modificação superficial com o surfactante catiônico brometo de cetil trimetil amônio (CTAB), objetivando-se realizar a secagem dos NCC e posterior redispersão em água. Para tanto, foram estudadas as influências dos procedimentos de diálise, da faixa de pH, da modificação superficial e também da técnica de secagem sobre a estabilidade coloidal e também sobre a distribuição de tamanhos de partículas. Três técnicas de secagem foram estudadas: (1) secagem em estufa, (2) secagem por liofilização e (3) secagem por spray drying. Na secagem em estufa houve a formação de aglomerados indissolúveis impedindo a redispersão dos NCC, nesta técnica foi avaliado principalmente o efeito do ajuste de pH, observando-se que para condições ácidas de pH ocorre a degradação dos NCC. Na secagem por liofilização foi possível redispersar os NCC em água, porém não foi obtido um material seco estável, pois ocorreu a dessorção da água. Observou-se também que para adições maiores do contra-íon +, obtiveram-se melhores resultados de redispersão quanto à distribuição de tamanho de partículas. Na técnica de secagem por spray drying foi testada a influência do surfactante sobre a redispersão. A adsorção do surfactante catiônico à superfície dos NCC foi confirmada pela técnica de espectroscopia no infravermelho por transformada de Fourier (FTIR). Ficou demonstrado que com a adição de 1% de surfactante às suspensões de NCC é possível redispersá-las obtendo características muito próximas as das suspensões antes do processo de secagem, no entanto, observou-se perda da estabilidade coloidal para algumas amostras após a redispersão. As técnicas de microscopia eletrônica de varredura (MEV), difração de raios X (DRX), análise termogravimétrica (ATG) e análise reológica foram usadas para caracterizar o material obtido na secagem por spray drying, que foi determinada como a melhor técnica de secagem. A morfologia das partículas apresentou-se no formato esférico, com a formação de aglomerados. Foram obtidos elevados índices de cristalinidade para as amostras com adsorção do surfactante CTAB, porém com reduzida estabilidade térmica. A avaliação da viscosidade cinemática indicou um comportamento pseudoplástico para as suspensões do NCC e um aumento na viscosidade das amostras após a secagem e redispersão indicou a formação de aglomerados / The use of environmentally friendly materials from renewable sources, as well as cellulose, has been presented as a subject of interest to many researchers. In this work the cellulose nanocrystals (CNC) were extracted from commercial microcrystalline cellulose (CMC) with subsequent pH adjustment and surface modification with the cationic surfactant cetyl trimethyl ammonium bromide (CTAB), aiming to dry the CNC and rehydrate them in water. The influence of dialysis procedure, pH range, surface modification and drying technique on colloidal stability and particle size distribution were studied. Three drying techniques were studied: (1) oven drying, (2) drying by lyophilization and (3) spray drying. The oven drying process showed the formation of indissoluble agglomerates preventing the redispersion of CNC, in this technique the effect of the pH adjustment was evaluated, and it was observed that for acidic pH conditions the degradation of CNC occurs. On drying by lyophilization it was possible to redisperse the CNC in water, but it was not possible to obtain a stable dry material, as water desorption occurred. It was also observed that for larger additions of the counterion +, better particle size distribution results were obtained for the redispersed suspensions. The influence of the surfactant on redispersion was tested in the spray drying drying technique. The adsorption of the cationic surfactant to the surface of the CNC was confirmed by the Fourier transform infrared spectroscopy (FTIR) technique. It has been shown that with the addition of 1% surfactant to the CNC suspensions it is possible to redisperse them obtaining characteristics very close to those of the suspensions prior to the drying process, however, loss of colloidal stability has been observed for some samples after redispersion. Scanning electron microscopy (SEM), X-ray diffraction (XRD), thermogravimetric analysis (TGA) and rheological analysis were used to characterize the material obtained by spray drying, which was determined as the most suitable drying technique. The particles were presented with a spherical morphology and with the formation of agglomerates. High crystallinity indexes were obtained for samples with adsorption of CTAB surfactant, but with low thermal stability. The kinematic viscosity assessment indicated a pseudoplastic behavior for the CNC suspensions and an increase in the viscosity of the samples after drying and redispersion indicated the formation of agglomerates.

L’introduction des nano-charges dans le milieu des polymères donna naissance aux nanocomposites et révolutionna l’univers des matériaux. Les nanocomposites sont à l’instar des composites traditionnels, un mélange de polymère et d’une charge avec au moins une dimension nanoscopique. Dans le cadre de notre projet, nous avons travaillé avec des nanocomposites à base des polymères biodégradables : polyacidelactique et polycaprolactone, combinés avec différentes nano-argiles «kaolin et bentonite» et avec différentes nano-organo-argiles. Ces nanocomposites ont été préparés par différentes méthodes (intercalation en solution, polymérisation in situ et la méthode de la voie fondue). L’avantage de ces polymères est dans leur dégradabilité ainsi que leur biocompatibilité. Une étude des relations entre l’élaboration, la microsctructure et les propriétés physico-chimiques des nanocomposites, des argiles modifiées par 3-aminopropyltriéthoxysilane (APTES) et Bromure d’Hexadecyltriméthy ammonium (CTAB) et des argiles non traitées ont été réalisés. La polymérisation in situ et le mélange à l’état fondu sont les méthodes les plus appropriées pour obtenir une meilleure dispersion nommée exfoliation. L’étude de la capacité d’adsorption du Co (II), Cd (II), Cu (II) et Ni (II) par ces deux adsorbants bruts (kaolin et bentonite) croit considérablement avec l’augmentation du pH. Cette capacité est 2 fois et demie plus importante dans le cas de la bentonite que dans celui du kaolin. L’étude comparative des cinétiques d’adsorption, nous a révélé une cinétique rapide de l’ordre de 6 min dans le cas du cobalt, 10 min dans le cas du nickel, 15 min dans le cas du cadmium et une cinétique moyenne de l’ordre de 120 minutes pour Cu (II) aussi bien pour la bentonite que pour le kaolin. L’étude de l’adsorption du Co (II), Cd (II), Cu(II) et Ni (II) par ces matériaux (argiles brutes, argiles organiquement modifiées et nanocomposites préparés par différentes méthodes) montre que la capacité d’adsorption de ces métaux lourds est importante dans le cas de l’argile modifiée par APTES / The introduction of nano-fillers into polymers field lead to the creation of the nanocomposites. This creation is starting up a new revolution into the world of materials. Nanocomposites are similar to traditional composite of a polymer blend and filler with at least one nano-scopic dimension. In our project, we worked with nanocomposites of biodegradable polymers: polyacidlactic and polycaprolactone, combined with different nanoclays "kaolin and bentonite" and with different nano-organo-clays. These nanocomposites have been prepared by different methods (solution intercalation, in situ polymerization and melt mixture methods). The advantage of these polymers is their degradability and biocompatibility. A study of the relationship between development, microsctructure and physico-chemical properties of nanocomposites, clays modified with 3-aminopropyltriethoxysilane (APTES) and Hexadecyltriméthy ammonium bromide (CTAB) and untreated clays were made. In situ polymerization and melt mixture methods are most suitable methods to get a better dispersion named exfoliation. The study of the adsorption capacity of Co (II), Cd (II), Cu (II) and Ni (II) by these two raw adsorbents (kaolin and bentonite) increases significantly with increasing pH. This capacity is two and half times higher in the case of bentonite than in kaolin. The comparative study of adsorption kinetics, we revealed rapid kinetics of the order of 6 minutes for the Co (II), 10 min for the Ni (II), 15 min for the Cd (II) and an average kinetic order of 120 minutes for Cu (II) both for the bentonite that for kaolin. The study of the adsorption of Co (II), Cd (II), Cu (II) and Ni (II) by these materials (raw clays, organically and nanocomposites prepared by different methods) shows that the adsorption capacity of these heavy metals is important in the case modified clay by APTES

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-01-29T19:55:46Z No. of bitstreams: 2 Dissertação - Fernanda Bueno Sampaio - 2013.pdf: 855102 bytes, checksum: 072ad37c90c900b69a5b7d188b872f7c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-01-29T19:56:16Z (GMT) No. of bitstreams: 2 Dissertação - Fernanda Bueno Sampaio - 2013.pdf: 855102 bytes, checksum: 072ad37c90c900b69a5b7d188b872f7c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-01-29T19:56:16Z (GMT). No. of bitstreams: 2 Dissertação - Fernanda Bueno Sampaio - 2013.pdf: 855102 bytes, checksum: 072ad37c90c900b69a5b7d188b872f7c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-11-21 / Citrus Black Spot (CBS) caused by Guignardia citricarpa is classified as quarantine disease, imposing restrictions to fresh fruits shipping to the European Union countries. In the state of Goiás, despite systematic phytosanitary surveys, its distribution and occurrence are unknown due to lack of available technologies for diagnosis. The occurrence of latent infections, the presence of an endophytic species morphologically similar to G. citricarpa, as well as time consuming for diagnosis through conventional methods require the validation of fast, efficient, reproducible, safe and sensitive methods of diagnosis to provide reliability of diagnosis and disease surveys for its detection and delimitation. Modifications of the “Cationic hexadecyl trimethyl ammonium bromide” method (CTAB) to extract DNA of G. citricarpafrom symptomatic tissues with validation of chemical purity, structural integrity, and absence of DNA inhibiting substances, for further use in diagnosis by PCR were tested. There was no difference in the average of DNA extracted for hygienized and non-hygienized tissues (328.62 ng µL -1 and 322.79 ng µL -1 , respectively), with low concentration of proteinsin the DNA solution. The extractor of DNA in amount (>300 ng µL -1 ) and quality (structural integrity) was sufficient to perform the conventional and real-time PCR analyses. The absence of inhibitors was demonstrated by real-time PCR, adding 0.1 % genetically modified standard corn DNA (event MON 810 standard ERM®-BF413b), with the amplification of the specific region of this event in all test samples. The modified CTAB method sowed repeatability and partial reproducibility among the limits acceptablein the methodology with coefficient of variability lower than 30 %. To comply with the ISO/TEC 17025:2005 norm, the method for the diagnosis of the fungus G. citricarpaby PCR conventional and real time PCR was validated with the specific evaluation of the limitof detection. Conventional and real time PCR methods were specificity and adequacy to detect G. citricarpa. Conventional PCR presented detecting limit of 10 ng µL -1 of the fungus DNA, with repeatability. Real time PCR presented higher sensitivity, having the detecting limit determined for the technique with repeatability, at the concentration of 10 fg of DNA of the fungus. In two farms 24 external asymptomatic leaves from orange trees variety Pera Rio were collected; eight from each third (lower, medium and upper); totaling twent plants. The modified CTAB method for DNA extraction was used. The presence of the fungus in very low concentrations was detected in the asymptomatic leaves, which made it impossible when we used the conventional PCR technique. In those conditions, the real-time PCR proved to be feasible, reproducible and highly sensitive for G. citricarpa detection, amplifying between 232 and 232 x 10 2 DNA copies of the fungus from asymptomatic leaf samples; being an excellent option for the diagnosis of thispathogen in asymptomatic orchards. / A pinta preta dos citros (PPC), causada pelo fungo Guignardia citricarpa, é considerada uma doença quarentenária, que impõe restrições ao transporte de frutas frescas para países da União Europeia. Em Goiás, apesar dos sistemáticos levantamentos fitossanitários, ainda não se conhece a real distribuição da ocorrência da PPC em função da tecnologia disponível para o diagnóstico. A ocorrência de infecção latente, a presença de uma espécie endofítica muito semelhante morfologicamente à G. citricarpae o tempo para o diagnóstico por métodos convencionais levam à necessidade de validar métodos moleculares rápidos, eficientes, reprodutíveis, seguros e sensíveis de diagnóstico, que garantem confiabilidade dos diagnósticos e dos levantamentos de detecção e delimitação da distribuição dessa doença. Foram testadas modificações do método “Cationic hexadecyl trimethyl ammonium bromide” (CTAB) para extração de DNA de G. citricarpaem tecidos de frutos cítricos sintomáticos, com a avaliação dapureza química, integridade estrutural e ausência de substâncias inibidoras na solução de DNA, para posterior uso em diagnóstico por reação em cadeia da polimerase (PCR). Não houve diferença significativa na quantidade média de DNA extraído para tecidos higienizados e não higienizados (328,62 ng µL -1 e 322,79 ng µL -1 , respectivamente), com baixa concentração de proteínas na suspensão de DNA. A obtenção de DNA em quantidade (>300 ng µL -1 ) e qualidade (integridade estrutural) foi suficiente para realização das análises de PCR convencional e PCR em tempo real. A ausência de compostos inibidores foi demostrada por PCR em tempo real pela adição de DNA padrão de milho, geneticamente modificado 0,1 % (evento MON 810 padrão ERM®-BF413b), com a amplificação da região específica desse evento em todas as amostras teste. O método CTAB modificado apresentou repetitividade e reprodutibilidade parcial dentro dos limites aceitáveis para a metodologia, apresentando coeficientes de variação inferiores a 30 %. Em atendimento à norma ISO/IEC 17025:2005, foi validado o método para diagnóstico do fungo G. citricarpa por PCR convencional e PCR em tempo real com a avaliação da especificidadee do limite de detecção. Os métodos de PCR convencional e PCR em tempo real demonstraram serem específicos e adequados para a detecção de G. citricarpa. A PCR convencional apresentou o limite de detecção de 10 ng µL -1 de DNA do fungo, com repetitividade. A PCR em tempo real apresentou maior sensibilidade, sendo o limite de detecção determinado para a técnica, com repetitividade, na concentração 10 fg de DNA do fungo. Em duas propriedades foram coletadas 24 folhas externas assintomáticas de laranja-pera rio, sendo oito em cada terço (inferior, médio e superior) da planta, em um total de vinte plantas. Para a extração do DNA, foi utilizado o método CTAB modificado. Em folhas assintomáticas, apresença do fungo foi detectada em baixíssimas concentrações, o que inviabiliza a utilização da técnica PCR convencional. Nessas condições, a PCR em tempo real demonstrou ser viável, reprodutível e altamente sensível para a detecção de G. citricarpa, sendo detectado na concentração de 232 a 232 x 10² números de cópia do DNA do fungo em amostras de folhas assintomáticas, constituindo uma excelente opção para o diagnóstico desse patógeno em pomares assintomáticos.

Orientadora: Ivanise Gaubeur / Tese (doutorado) - Universidade Federal do ABC. Programa de Pós-Graduação em Ciência e Tecnologia/Química, 2013

<p>The project aim is to determine if good quality DNA can be extracted from barley, malt and hop, ingredients used in beer brewing. Good quality DNA is important in DNA fingerprinting techniques which can be used for identification of ingredients. The 3 methods tested are the cetyltrimethylammonium bromide (CTAB) method, QIAGEN DNeasy Plant Mini Kit and Meyer’s method as published in 1996 with QIAGEN DNeasy Plant Mini Kit in combination. To evaluate the DNA quality after extraction we used 3 different techniques:</p><p>(i) spectrophotometry to estimate purity by using the ratio A260/A280; (ii) agarose gel electrophoresis after DNA extraction to determine the success of the extraction and evaluate the amount of high molecular weight DNA and degradation; and (iii) the polymerase chain reaction with 4 different primer pairs, together with agarose gel electrophoresis, to determine if the extracted DNA could be used in downstream applications, see the effect of inhibitors and estimate the fragmentisation of the DNA. The results achieved using the above mentioned methods were then used to evaluate the success of each of the extraction methods in their function of extracting high quality DNA from barley, malt and hop as well as determining whether the treatment of the ingredients has an effect on the DNA quality.</p>

The project aim is to determine if good quality DNA can be extracted from barley, malt and hop, ingredients used in beer brewing. Good quality DNA is important in DNA fingerprinting techniques which can be used for identification of ingredients. The 3 methods tested are the cetyltrimethylammonium bromide (CTAB) method, QIAGEN DNeasy Plant Mini Kit and Meyer’s method as published in 1996 with QIAGEN DNeasy Plant Mini Kit in combination. To evaluate the DNA quality after extraction we used 3 different techniques: (i) spectrophotometry to estimate purity by using the ratio A260/A280; (ii) agarose gel electrophoresis after DNA extraction to determine the success of the extraction and evaluate the amount of high molecular weight DNA and degradation; and (iii) the polymerase chain reaction with 4 different primer pairs, together with agarose gel electrophoresis, to determine if the extracted DNA could be used in downstream applications, see the effect of inhibitors and estimate the fragmentisation of the DNA. The results achieved using the above mentioned methods were then used to evaluate the success of each of the extraction methods in their function of extracting high quality DNA from barley, malt and hop as well as determining whether the treatment of the ingredients has an effect on the DNA quality.

abstract: Alkali treated aluminosilicate (geopolymer) was functionalized by surfactant to increase the hydrophobicity for making Pickering emulsion for the first part of this work. In the first part of this study, alkali treated metakaolin was functionalized with cetyltrimethylammonium bromide ((C16H33)N(CH3)3Br, CTAB). The electrostatic interaction between this quaternary ammonium and the surface of the aluminosilicate which has negative charge has taken place. The particles then were used to prepare Pickering emulsion. The resulting stable dispersions, obtained very fast at very simple conditions with low ratio of aluminosilicate to liquid phase. In the second part, the interaction between geopolymer and glycerol was studied to see the covalent grafting of the geopolymer for making geopolymer composite. The composite material would be the basis material to be used as support catalyst, thin coating reagent and flame retardant material and so on, Variety of techniques, Thermogravimetric (TGA), Particle-induced X-ray emission (PIXE), FTIR, Solid state NMR, Powder X-ray diffraction (PXRD), BET surface area, Elemental analysis (CHN), TEM, SEM and Optical microscopy were used to characterize the functionalized geopolymer. / Dissertation/Thesis / M.S. Chemistry 2012

In the food industry, it is very important to take care of the quality, safety and organoleptic properties of the products supplied. For this reason, food must be checked. However, not all information can be found using conventional techniques such as immunoassays, chromatographic techniques, etc. DNA-based techniques can be used for these cases where traditional procedures are insufficient. Among them, the best known technique is PCR. The aim of the thesis was to isolate DNA from vegetable samples (broccoli, beetroot, carrot and pepper). DNA was isolated using the magnetic particle method and the traditional CTAB method. Both methods were able to isolate the DNA from the vegetable samples in quality and at a concentration suitable for PCR, where the 35S rDNA gene region was amplified (more precisely about 700 bp of the 18S-ITS1-5,8S region). After amplification, the PCR products were subjected to restriction reactions and the results compared to bioinformatic analysis. These steps have succeeded in finding suitable enzymes for diferentiation of PCR products from the tested vegetable species.