High interstitial fluid pressure (IFP) in tumours is recognized as a barrier to drug delivery resulting in reduced efficacy. High IFP impedes the normal process of convective fluid flow (CFF) from blood vessels into the interstitium. The aim of this study was to develop an in vitro model that could be used to measure CFF and to study its effects on drug delivery. The model consists of a transwell cell culture insert which supports the growth of multicell layers (MCL) on collagen coated membranes. A graduated tube is inserted into the transwell and a pressure gradient is applied across the membrane by raising the volume of medium in the tube above that of the bottom chamber. CFF is determined by measuring the weight of medium in the bottom chamber as a function of time. CFF was inversely proportional to MCL thickness and 41.1±3.6µm thick MCL has completely stopped CFF. Using a physiologically relevant hydrostatic pressure of 28mmHg, a CFF of 21µL/min was recorded using a DLD-1 MCL that was 12.21±3.2µm thick. Under these conditions, the rates of penetration of doxorubicin, imatinib and gefitinib were respectively 42, 26 and 13 folds greater than when no CFF exists. Reversing the CFF so that it opposed the drug diffusion gradient significantly impairs drug penetration. In conclusion, a novel in vitro model for assessing the impact of CFF on drug delivery has been developed. This model could be used to evaluate strategies designed to increase drug delivery to solid tumours by modifying the CFF.

No description available.

No description available.

<p>Hydraulic systems are commonly used for actuation and manipulation of heavy loads. They are found in a variety of different industries, such as in automotive, manufacturing, robotics, construction, and aerospace. Conventional hydraulic systems use a centralized constant pressure supply system. Pressurized fluid is then channeled to actuators using servo-valves. The advantages of these systems are their high torque to mass ratio, and the ability to control speed and direction with relative precision. However, there are also disadvantages such as the requirement of a bulky centralized supply, leakage, noise, and reduced energy efficiency due to orifice flow and the requirement for maintaining a constant supply pressure.</p> <p>Electro-Hydrostatic Actuation systems (EHA) alleviate many of the above mentioned shortcomings of servo-valve controlled hydraulic systems. In the EHA position control is achieved by regulating the pumping action. Here, a fixed or a variable displacement pump can be used to move oil from one chamber of the actuator to the other. In these actuators, the presence of nonlinearities associated with pump/motor static friction and backlash, pressure drop in the piping system, and nonlinear friction at the load have a significant effect on the performance and positional precision of the system.</p> <p>This research will focus on developing a multiple inner-loop control strategy by implementing multiple inner-loops that utilize the differential pump/load position and velocity. The main goal will be to decrease the effect of the pump backlash as well as the nonlinear friction at the load; both of which negatively impact positional precision. Therefore, the main benefit of this method is an improvement in trajectory tracking precision, which is particularly important for high precision hydrostatic systems. Furthermore, a sliding mode control strategy will be incorporated into the design to suppress load oscillations reported in precision trajectory tracking applications. The research hypothesis states that sliding mode control in conjunction with multiple inner-loops, will improve the trajectory tracking performance of a hydrostatic actuation system by partially compensating the effects of static friction at the load. Theoretical analysis, simulation supported by experimental results are presented to demonstrate the effectiveness of the newly developed methods in suppressing the effects of nonlinearities on the EHA performance, with the downside of an increased complexity due to the increased number of controller parameters.</p> / Doctor of Philosophy (PhD)

According to the American Heart Association, cardiovascular disease (CVD) is the leading cause of death in the U.S., representing about 20-30% of all deaths every year in the U.S. Major risk factors for developing CVD include high blood lipid and LDL-cholesterol levels. A large number of heart attacks and strokes could be prevented by controlling these factors through lifestyle modifications and diet interventions. Epidemiological evidence shows that consumption of dry or common beans (Phaseolus vulgaris L.) has positive effects on reducing blood LDL-cholesterol and lipid levels. These health benefits are mainly attributed to the high content of dietary fiber (DF) of beans, including soluble and insoluble DF (SDF and IDF). Some proposed mechanisms to explain the cholesterol and lipid-lowering effects of DF are related to the physico-chemical properties (e.g. viscosity) of DF, and involve binding to bile salts (BS) in the small intestinal to prevent BS re-absorption which further promote cholesterol catabolism and delay lipid digestion. Nevertheless, the precise mechanisms are not fully understood yet. In addition, cooking and processing operations, and in particular high-hydrostatic pressure (HHP) processing, can modify the composition, structure and functional properties of foods; however, whether HHP affects the ability of beans to interfere with different aspects of lipid digestion remains unknown. The overall goal of this research is to understand how common beans and HHP processing impact the ability of beans to bind BS and influence lipid digestion in vitro. The specific objectives are 1) to evaluate the effect of HHP treatments (and compared it with conventional cooking (HT)) on the thermo-rheological and functional properties of dry beans; 2) to identify the impact of major bean components on the in vitro BS-binding ability of beans, the role played by the bean matrix and how this is affected by HHP processing; 3) to investigate how bean (micro)structure and fiber fractions, as well as HHP processing of dry beans, influence lipid digestion in vitro. Results showed that HT caused complete starch gelatinization and protein denaturation of beans, while HHP treatments induced partial or no starch gelatinization and a lower degree of protein denaturation, which resulted in enhanced protein solubility and emulsifying activity/stability. It was observed that, while HT treatment reduced the capacity of bean flours to retain BS because of severe disruption of the bean cell wall integrity, protein matrices, and starch granules, HHP treatments maintained or enhanced BS retention, possibly by promoting the formation of starch/protein/fiber networks able to entrap BS. Furthermore, by using an in vitro dialysis-based digestion model combined with viscosity measurements and thermal analysis, it was shown that the interaction between bean tissue materials and primary BS was not only related to viscosity but also involved hydrophobic linkages. The contribution of IDF and proteins (other than SDF) to retain BS was also significant. There was a different binding preference of beans to four primary BS with sodium glycochenodeoxycholate, the more hydrophobic BS, showing the largest retention levels while sodium taurocholate being the least effectively retained BS by beans. Diverse sequences of the same processing operations showed distinct impacts on BS-retention by dry beans. By means of an in vitro digestion model simulating conditions in the upper gastrointestinal tract, bean flours delayed the digestion of extrinsic lipids to a higher extent, compared to isolated IDF and SDF. Furthermore, HHP treatment and less severe mechanical disintegration maintained the ability of beans to modulate lipid digestion, which suggests the importance of bean structural integrity in reducing the lipolysis rate and extent by beans. Overall, this research work shows that HHP processing is a promising minimal processing technology to produce bean flours with improved functionality. It also highlights the importance of considering the structure of foods, and not just their nutrient content, when evaluating potential health impacts. This knowledge could be applied to develop a range of bean-based ingredients and formulations with desirable health benefits. This work can be extended to research the influence of beans on the gut microbiota and profile of secondary BS and short-chain fatty acids, which are also closely related to cholesterol and lipid metabolism. / Doctor of Philosophy / According to the American Heart Association, cardiovascular disease (CVD) is the leading cause of death in the U.S., representing about 20-30% of all deaths every year in the U.S. Around the world, millions of people are struggling to control the risk of CVD. Major risk factors for developing CVD include high blood lipid and LDL-cholesterol levels. A large number of heart attacks and strokes can be prevented by controlling the major risk factors through lifestyle modifications and diet interventions. Epidemiological evidence shows that consumption of dry beans (Phaseolus vulgaris L.) has positive effects on reducing blood LDL-cholesterol and lipid levels. These health benefits are mainly attributed to the high content of dietary fiber (DF) in beans. DF is carbohydrate polymers that are not hydrolyzed by the endogenous enzymes in humans. However, some of them (water-soluble DF) could increase viscosity and retain the absorption of bile salts (BS) in the small intestinal. The BS retention or the binding of BS could promote more cholesterol convert to BS (thus reduce cholesterol levels) and decrease lipid digestion. Therefore, due to the increased viscosity and BS retention ability of DF, dry beans could help to reduce the blood cholesterol and lipid levels and further help to prevent CVD. Moreover, different cooking and processing method could also affect the composition, microstructure and functional properties of foods. The purpose of this research was to determine how common beans and high hydrostatic pressure (HHP) (compared with hydrothermal (HT)) processing, a non-thermal processing, influence the ability of dry beans to retain bile salts and modulate lipid digestion in vitro. This study showed that severe HT treatment disrupted the bean cell wall integrity severally and reduced the BS retaining the efficiency of dry beans, while HHP treatment, produced minimally processed beans, improved the application properties of dry beans and maintained/enhanced BS-retention by dry beans. It also showed that the whole bean matrix (other than soluble DF) also contributes to retain BS and modulate lipid digestion, indicating the importance of retaining intact food structures. The integrity of bean structures through HHP treatment and less severe mechanical treatment could help to retain the ability of dry beans to reduce lipid digestion. These findings suggest that dry beans, with a high content of dietary fiber and resistant starch, have significant health benefits related to lowering cholesterol and lipid levels. Increasing the consumption of dry beans would definitely help to improve overall health. HHP, as a non-thermal processing technology, showed the potential to produce minimally processed bean products with enhanced health benefits and diverse application properties. This study could be extended through continuing research into the influence of beans on the gut microbiota, which are also closely related to the cholesterol and lipid metabolism regulation.

[ES] Actualmente, una de las principales preocupaciones de las autoridades sanitarias y de la industria alimentaria sigue siendo garantizar la inocuidad microbiológica en la cadena alimentaria ya que a pesar de los notables esfuerzos realizados, el número de brotes y casos de enfermedades de transmisión alimentaria causadas por microorganismos sigue elevándose. Por otro lado, otro de los problemas que debe afrontar la industria alimentaria es la implementación de nuevas alternativas de producción más sostenibles, manteniendo altos estándares de seguridad y garantizando la disponibilidad de alimentos. En este contexto se enmarca la presente tesis doctoral, cuyo principal objetivo es la evaluación in vivo e in vitro de la capacidad antimicrobiana de distintas sustancias naturales procedentes de fuentes alternativas, más sostenibles (extracto de coliflor, espirulina y soluciones de quitosano), frente a microorganismos patógenos de importancia en la industria de alimentos. Los estudios demuestran la capacidad antimicrobiana in vitro del quitosano de insecto y crustáceo frente a microorganismos patógenos como E. coli, L. monocytogenes y S. Typhimurium siendo dependiente del pH del medio estudiado. Además, cuando el quitosano se combina con un tratamiento suave de altas presiones hidrostáticas, se pone de manifiesto una relación sinérgica entre ambas tecnologías llegando a inactivar completamente la carga microbiana de S. Typhimurium. Los estudios in vivo llevados a cabo con Caenorhabditis elegans muestran que el nematodo aumenta su supervivencia en presencia de extractos de coliflor y espirulina. En cuanto a la capacidad antimicrobiana, los extractos de espirulina y de coliflor reducen la colonización intestinal por S. Typhimurium en el nema todo mostrando así su capacidad antimicrobiana in vivo. En la presente tesis doctoral, se hace un recorrido completo sobre la capacidad antimicrobiana de ciertos extractos vegetales o componentes procedentes de animales, fundamentalmente basados en estudios in vivo. Este es un paso importante en la materialización de estos compuestos como alternativas más naturales y sostenibles a otros compuestos sintéticos usados en conservación de alimentos. / [CA] Actualment, una de les principals preocupacions de les autoritats sanitàries i de la indústria alimentària continua sent garantir la innocuïtat microbiològica en la cadena alimentària ja que malgrat els notables esforços realitzats, el nombre de brots i casos de malalties de transmissió alimentària causades per microorganismes continua elevant-se. D'altra banda, un altre dels problemes que ha d'afrontar la indústria alimentària és la implementació de noves alternatives de producció més sostenibles, mantenint alts estàndards de seguretat i garantint la disponibilitat d'aliments. En aquest context s'emmarca la present tesi doctoral, el principal objectiu de la qual és l'avaluació in vivo i in vitro de la capacitat antimicrobiana de diferents substàncies naturals procedents de fonts alternatives, més sostenibles (extracte de coliflor, espirulina i solucions de quitosano), enfront de microorganismes patògens d'importància en la indústria d'aliments. Els estudis demostren la capacitat antimicrobiana in vitro del quitosano d'insecte i crustaci enfront de microorganismes patògens com a E. coli, L. monocytogenes i S. Typhimurium sent dependent del pH del mig estudiat. A més, quan el quitosano es combina amb un tractament suau d'altes pressions hidroestàtiques, es posa de manifest una relació sinèrgica entre totes dues tecnologies arribant a inactivar completament la càrrega microbiana de S. Typhimurium. Els estudis in vivo duts a terme amb Caenorhabditis elegans mostren que el nematode augmenta la seua supervivència en presència d'extractes de coliflor i espirulina. Quant a la capacitat antimicrobiana, els extractes d'espirulina i de coliflor redueixen la colonització intestinal per S. Typhimurium en el nematode mostrant així la seua capacitat antimicrobiana in vivo. En la present tesi doctoral, es fa un recorregut complet sobre la capacitat antimicrobiana d'uns certs extractes vegetals o components procedents d'animals, fonamentalment basats en estudis in vivo. Aquest és un pas important en la materialització d'aquests compostos com a alternatives més naturals i sostenibles a altres compostos sintètics usats en conservació d'aliments. / [EN] Currently, one of the main concerns of the health authorities and the food industry continues to be ensuring microbiological safety in the food chain, as despite considerable efforts, the number of outbreaks and cases of foodborne diseases caused by microorganisms continues to rise. On the other hand, another chalenge facing the food industry is the implementation of new, more sustainable production alternatives while maintaining high safety standards and ensuring food availability. It is in this context, the main objective of this PhD thesis is the in vivo and in vitro evaluation of the antimicrobial capacity of different natural substances from alternative, more sustainable sources (cauliflower extract, spirulina and chitosan solutions) against pathogenic microorganisms of importance in the food industry. Studies demonstrate the in vitro antimicrobial capacity of insect and crustacean chitosan against pathogenic microorganisms such as E. coli, L. monocytogenes and S. Typhimurium, being dependent on the pH of the medium studied. Moreover, when chitosan is combined with a mild treatment of high hydrostatic pressures, a synergistic relationship between both technologies is evident, reaching the point of completely inactivating the microbial load of S. Typhimurium. In vivo studies carried out with Caenorhabditis elegans show that the nematode increases its survival in the presence of cauliflower and spirulina extracts. In terms of antimicrobial capacity, spirulina and cauliflower extracts reduce intestinal colonization by S. Typhimurium in the nematode, thus showing their antimicrobial capacity in vivo. In this doctoral thesis, a complete overview of the antimicrobial capacity of certain plant extracts or animal-derived compounds is presented, mainly based on in vivo studies. This is an important step in the materialisation of these compounds as more natural and sustainable alternatives to other synthetic compounds used in food preservation. / La presente tesis doctoral se enmarca dentro del Programa de Doctorado Ciencia, Tecnología y Gestión Alimentaria de la Universidad Politécnica de Valencia. El trabajo experimental se realizó gracias a fondos del Ministerio de Ciencia e Innovación y del Fondo Europeo de Desarrollo Regional (FEDER), a través del proyecto: Validación de nuevas herramientas y procesos para el análisis y la mejora de la seguridad alimentaria microbiológica: aplicación a nuevas matrices alimentarias (AGL2017-86840-C2-2-R) del Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad. / Ibáñez Peinado, D. (2022). Estudio de la capacidad antimicrobiana de sustancias naturales in vivo e in vitro [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/184317

Electro Hydrostatic Actuators (EHA) are emerging as a viable option for industrial machine builders as the design combines the best of both electro-mechanical and electro-hydraulic technologies. The EHA is a highly integrated, compact alternative to traditional hydraulic solutions. Automation engineers moving toward electro-mechanical actuation in pursuit of energy efficiency and environmental cleanliness, will find an EHA an attractive option for high force density actuators. This paper will address the factors to consider when assessing an industrial machine’s application suitability for this latest innovation in actuation. It describes principal base circuits, a concept for EHA building blocks and a realized pilot application as well as challenges on actuator and components level.

In this study we investigate the interactions between two co-rotating vortices. These vortices are subject to rapid rotation and stable stratification such as are found in planetary atmospheres and oceans. By conducting a large number of simulations of vortex interactions, we intend to provide an overview of the interactions that could occur in geophysical turbulence. We consider a wide parameter space covering the vortices height-to-width aspect-ratios, their volume ratios and the vertical offset between them. The vortices are initially separated in the horizontal so that they reside at an estimated margin of stability. The vortices are then allowed to evolve for a period of approximately 20 vortex revolutions. We find that the most commonly observed interaction under the quasi-geostrophic (QG) regime is partial-merger, where only part of the smaller vortex is incorporated into the larger, stronger vortex. On the other hand, a large number of filamentary and small scale structures are generated during the interaction. We find that, despite the proliferation of small-scale structures, the self-induced vortex energy exhibits a mean `inverse-cascade' to larger scale structures. Interestingly we observe a range of intermediate-scale structures that are preferentially sheared out during the interactions, leaving two vortex populations, one of large-scale vortices and one of small-scale vortices. We take a subset of the parameter space used for the QG study and perform simulations using a non-hydrostatic model. This system, free of the layer-wise two-dimensional constraints and geostrophic balance of the QG model, allows for the generation of inertia-gravity waves and ageostrophic advection. The study of the interactions between two co-rotating, non-hydrostatic vortices is performed over four different Rossby numbers, two positive and two negative, allowing for the comparison of cyclonic and anti-cyclonic interactions. It is found that a greater amount of wave-like activity is generated during the interactions in anticyclonic situations. We also see distinct qualitative differences between the interactions for cyclonic and anti-cyclonic regimes.

Longitudinal growth of long bone occurs at growth plates by a process called endochondral ossification. Endochondral ossification is affected by both biological and mechanical factors. This thesis focuses on the mechanical modulation of femoral bone growth occurring at the proximal growth plate, using mechanobiological theories reported in the literature. Finite element analysis was used to simulate bone growth. The first study analyzed the effect of subject-specific growth plate geometry over simplified growth plate geometry in numerical prediction of bone growth tendency. Subject-specific femur finite element model was constructed from magnetic resonance images of one able- bodied child. Gait kinematics and kinetics were acquired from motion analysis and analyzed further in musculoskeletal modelling to determine muscle and joint contact forces. These were used to determine loading on the femur in finite element analysis. The growth rate was computed based on a mechanobiological theory proposed by Carter and Wong, and a growth model in the principal stress direction was introduced. Our findings support the use of subject- specific geometry and of the principal stress growth direction in prediction of bone growth. The second study aimed to illustrate how different muscle groups’ activation during gait affects proximal femoral growth tendency in able-bodied children. Subject-specific femur models were used. Gait kinematics and kinetics were acquired for 3 able-bodied children, and muscle and joint contact forces were determined, similar to the first study. The contribution of different muscle groups to hip contact force was also determined. Finite element analysis was performed to compute the specific growth rate and growth direction due to individual muscle groups. The simulated growth model indicated that gait loading tends to reduce neck shaft angle and femoral anteversion during growth. The muscle groups that contributes most and least to growth rate were hip abductors and hip adductors, respectively. All muscle groups’ activation tended to reduce the neck shaft and femoral anteversion angles, except hip extensors and adductors which showed a tendency to increase the femoral anteversion. The third study’s aim was to understand the influence of different physical activities on proximal femoral growth tendency. Hip contact force orientation was varied to represent reported forces from a number of physical activities. The findings of this study showed that all studied physical activities tend to reduce the neck shaft angle and anteversion, which corresponds to the femur’s natural course during normal growth. The aim of the fourth study was to study the hypothesis that loading in the absence of physical activity, i.e. static loading, can have an adverse effect on bone growth. A subject-specific model was used and growth plate was modeled as a poroelastic material in finite element analysis. Prendergast’s indicators for bone growth was used to analyse the bone growth behavior. The results showed that tendency of bone growth rate decreases over a long duration of static loading. The study also showed that static sitting is less detrimental than static standing for predicted cartilage-to-bone differentiation likelihood, due to the lower magnitude of hip contact force. The prediction of growth using finite element analysis on experimental gait data and person- specific femur geometry, based on mechanobiological theories of bone growth, offers a biomechanical foundation for better understanding and prediction of bone growth-related deformity problems in growing children. It can ultimately help in treatment planning or physical activity guidelines in children at risk at developing a femur or hip deformity. / <p>QC 20170616</p>

La supraconductivité non-conventionnelle a récemment été observée à proximité d'un point critique induit sous pression dans CePt$_2$In$_7$ et dans FeSe. Le premier est un fermion lourd tandis que le deuxième fait parti de la famille des supraconducteurs à base de Fer. Cette thèse a pour objectif de contribuer à la compréhension de ces systèmes à électrons fortement corrélés en étudiant les évolutions des structures cristallographiques et magnétiques, ainsi que les surfaces de Fermi sous conditions extrêmes.Tout d'abord, nous présentons une étude de diffraction de neutrons dans la phase magnétique de CePt$_2$In$_7$. Une seule structure magnétique, avec comme vecteur de propagation $textbf{Q} = (0.5,0.5,0.5)$ et $0.45~mu_B$ par atome de Cérium à 2 K, a été détectée en dessous de $T_N = 5.5$ K.Ensuite, des mesures de torque sous champ pulsé de CePt$_2$In$_7$ ne montrent aucun changement des surfaces de Fermi jusqu'à 70 T, bien au-dessus du point critique quantique induit sous champ, attendu à $55-60$ T selon la littérature. Cependant, ces mesures révèlent une claire anomalie métamagnétique à 47 T, très peu dépendante de la température et de l'orientation du champ ainsi qu'une chute des masses effectives vers 50 T. Nous suggérons que ces deux derniers éléments sont la manifestation d'un changement de valence des atomes de Ce de l'ordre de 0.06 électron par atomes de Cérium.L'étude des surfaces de Fermi de CePt$_2$In$_7$ sous pression a nécessité un développement instrumental à partir d'un circuit résonant à base d'une diode tunnel, combinée avec une cellule de pression de type Bridgman. Nous montrons qu'il est tout à fait possible de sonder les surfaces de Fermi à la fois sous champ magnétique et sous pression avec cette technique. Cependant, la fragilité du système résonnant nous amène à considérer des améliorations en vue de fiabiliser la mesure.Enfin, nous analysons une expérience de diffraction de rayons X sur un échantillon de FeSe sous pression hydrostatique. A 20 K, nous quantifions un durcissement de l'axe cristallographique $c$ qui s'opère à 1.9 GPa. A 50 K, outre le passage d'une maille orthorhombique à tétragonale à 1 GPa, nous mettons en évidence qu'une symétrie monoclinique s'installe à partir de 2 GPa, où, selon la littérature, une phase antiferromagnétique apparaît. / Unconventional superconductivity was recently observed in the vicinity of a pressure-induced quantum critical point in CePt$_2$In$_7$ and FeSe. The former is a heavy fermion compound, while the latter is an iron-based superconductor. This PhD thesis aims at improving our understanding of the physics of these newly discovered strongly correlated electron systems. This is achieved by experimental investigation of the evolution of crystal and magnetic structures, as well as of the Fermi surfaces under extreme conditions of high magnetic fields, high pressure, and low temperatures.We have investigated the magnetic structure of CePt$_2$In$_7$ by neutron diffraction. We observed only one magnetic propagation vector $textbf{Q} = (0.5, 0.5, 0.5)$ below $T_N = 5.5$ K. The magnetic moment is estimated at $0.45~mu_B$ per cerium atom at 2 K.Our torque measurements on CePt$_2$In$_7$ in pulsed fields suggest that the Fermi surfaces remain unchanged up to 70 T. This is well above the field-induced quantum critical point, which is expected to occur at $55-60$ T according to previous studies. However, a clear metamagnetic-like anomaly is found at 47 T. The anomaly is almost temperature and field-angle independent. Furthermore, a sudden drop of the effective mass is observed at about the same field. We suggest that the last two observations are most naturally accounted for by a valence crossover, where the cerium valence changes by about 0.06, the order of magnitude expected in Ce-based compounds.In order to study the Fermi surfaces of CePt$_2$In$_7$ under pressure, we developed a tunnel diode oscillator combined with a Bridgman-type pressure cell. We have demonstrated that this set-up is suitable for measuring quantum oscillations both at high magnetic fields and under high pressure. However, the working conditions of the oscillator have to be improved in order to obtain a more reliable system.Finally, we performed an X-ray diffraction experiment on FeSe under hydrostatic pressure. At 20 K, we found a change of the bulk modulus along the $c$ axis at 1.9 GPa. At 50 K, the orthorhombic to tetragonal crystallographic phase transition occurs at 1 GPa. We found evidences that this is followed by the emergence of a monoclinic symmetry above 2 GPa, where an antiferromagnetic phase was previously reported.