Genetic epidemiology of gallbladder disease in Mexican Americans and cholesterol 7$\alpha$-hydroxylase gene sequence variation

Among Mexican Americans, the second largest minority group in the United States, the prevalence of gallbladder disease is markedly elevated. Previous data from both genetic admixture and family studies indicate that there is a genetic component to the occurrence of gallbladder disease in Mexican Americans. However, prior to this thesis no formal genetic analysis of gallbladder disease had been carried out nor had any contributing genes been identified.^ The results of complex segregation analysis in a sample of 232 Mexican American pedigrees documented the existence of a major gene having two alleles with age- and gender-specific effects influencing the occurrence of gallbladder disease. The estimated frequency of the allele increasing susceptibility was 0.39. The lifetime probabilities that an individual will be affected by gallbladder disease were 1.0, 0.54, and 0.00 for females of genotypes "AA", "Aa", and "aa", respectively, and 0.68, 0.30, and 0.00 for males, respectively. This analysis provided the first conclusive evidence for the existence of a common single gene having a large effect on the occurrence of gallbladder disease.^ Human cholesterol 7$\alpha$-hydroxylase is the rate-limiting enzyme in bile acid synthesis. The results of an association study in both a random sample and a matched case/control sample showed that there is a significant association between cholesterol 7$\alpha$-hydroxylase gene variation and the occurrence of gallbladder disease in Mexican Americans males but not in females. These data have implicated a specific gene, 7$\alpha$-hydroxylase, in the etiology of gallbladder disease in this population.^ Finally, I asked whether the inferred major gene from complex segregation analysis is genetically linked to the cholesterol 7$\alpha$-hydroxylase gene. Three pedigrees predicted to be informative for linkage analysis by virtue of supporting the major gene hypothesis and having parents with informative genotypes and multiple offspring were selected for this linkage analysis. In each of these pedigrees, the recombination fractions maximized at 0 with a positive, albeit low, LOD score. The results of this linkage analysis provide preliminary and suggestive evidence that the cholesterol 7$\alpha$-hydroxylase gene and the inferred gallbladder disease susceptibility gene are genetically linked. ^

REGULATION OF TOXIN SYNTHESIS BY CLOSTRIDIUM DIFFICILE

Clostridium difficile is the leading definable cause of nosocomial diarrhea worldwide due to its virulence, multi-drug resistance, spore-forming ability, and environmental persistence. The incidence of C. difficile infection (CDI) has been increasing exponentially in the last decade. Virulent strains of C. difficile produce either toxin A and/or toxin B, which are essential for the pathogenesis of this bacterium. Current methods for diagnosing CDI are mostly qualitative tests that detect the bacterium, the toxins, or the toxin genes. These methods do not differentiate virulent C. difficile strains that produce active toxins from non-virulent strains that do not produce toxins or produce inactive toxins. Based on the knowledge that C. difficile toxins A and B cleave a substrate that is stereochemically similar to the native substrate of the toxins, uridine diphosphoglucose, a quantitative, cost-efficient assay, the Cdifftox activity assay, was developed to measure C. difficile toxin activity. The concept behind the activity assay was modified to develop a novel, rapid, sensitive, and specific assay for C. difficile toxins in the form of a selective and differential agar plate culture medium, the Cdifftox Plate assay (CDPA). This assay combines in a single step the specific identification of C. difficile strains and the detection of active toxin(s). The CDPA was determined to be extremely accurate (99.8% effective) at detecting toxin-producing strains based on the analysis of 528 C. difficile isolates selected from 50 tissue culture cytotoxicity assay-positive clinical stool samples. This new assay advances and improves the culture methodology in that only C. difficile strains will grow on this medium and virulent strains producing active toxins can be differentiated from non-virulent strains. This new method reduces the time and effort required to isolate and confirm toxin-producing C. difficile strains and provides a clinical isolate for antibiotic susceptibility testing and strain typing. The Cdifftox activity assay was used to screen for inhibitors of toxin activity. Physiological levels of the common human conjugated bile salt, taurocholate, was found to inhibit toxin A and B in vitro activities. When co-incubated ex vivo with purified toxin B, taurocholate protected Caco-2 colonic epithelial cells from the damaging effects of the toxin. Furthermore, using a caspase-3 detection assay, taurocholate reduced the extent of toxin B-induced Caco-2 cell apoptosis. These results suggest that bile salts can be effective in protecting the gut epithelium from C. difficile toxin damage, thus, the delivery of physiologic amounts of taurocholate to the colon, where it is normally in low concentration, could be useful in CDI treatment. These findings may help to explain why bile rich small intestine is spared damage in CDI, while the bile salt poor colon is vulnerable in CDI. Toxin synthesis in C. difficile occurs during the stationary phase, but little is known about the regulation of these toxins. It was hypothesized that C. difficile toxin synthesis is regulated by a quorum sensing mechanism. Two lines of evidence supported this hypothesis. First, a small (KDa), diffusible, heat-stable toxin-inducing activity accumulates in the medium of high-density C. difficile cells. This conditioned medium when incubated with low-density log-phase cells causes them to produce toxin early (2-4 hrs instead of 12-16 hrs) and at elevated levels when compared with cells grown in fresh medium. These data suggested that C. difficile cells extracellularly release an inducing molecule during growth that is able to activate toxin synthesis prematurely and demonstrates for the first time that toxin synthesis in C. difficile is regulated by quorum signaling. Second, this toxin-inducing activity was partially purified from high-density stationary-phase culture supernatant fluid by HPLC and confirmed to induce early toxin synthesis, even in C. difficile virulent strains that over-produce the toxins. Mass spectrometry analysis of the purified toxin-inducing fraction from HPLC revealed a cyclic compound with a mass of 655.8 Da. It is anticipated that identification of this toxin-inducing compound will advance our understanding of the mechanism involved in the quorum-dependent regulation of C. difficile toxin synthesis. This finding should lead to the development of even more sensitive tests to diagnose CDI and may lead to the discovery of promising novel therapeutic targets that could be harnessed for the treatment C. difficile infections.

Intercellular adhesion and membrane polarity in rat hepatocytes: Localization of cell -CAM 105 and other domain-specific membrane proteins {\it in situ\/} and {\it in vitro\/} by electron microscopy

Cell-CAM 105 has been identified as a cell adhesion molecule (CAM) based on the ability of monospecific and monovalent anti-cell-CAM 105 antibodies to inhibit the reaggregation of rat hepatocytes. Although one would expect to find CAMs concentrated in the lateral membrane domain where adhesive interactions predominate, immunofluorescence analysis of rat liver frozen sections revealed that cell-CAM 105 was present exclusively in the bile canalicular (BC) domain of the hepatocyte. To more precisely define the in situ localization of cell-CAM 105, immunoperoxidase and electron microscopy were used to analyze intact and mechanically dissociated fixed liver tissue. Results indicate that although cell-CAM 105 is apparently restricted to the BC domain in situ, it can be detected in the pericanalicular region of the lateral membranes when accessibility to lateral membranes is provided by mechanical dissociation. In contrast, when hepatocytes were labeled following incubation in vitro under conditions used during adhesion assays, cell-CAM 105 had redistributed to all areas of the plasma membrane. Immunofluorescence analysis of primary hepatocyte cultures revealed that cell-CAM 105 and two other BC proteins were localized in discrete domains reminscent of BC while cell-CAM 105 was also present in regions of intercellular contact. These results indicate that the distribution of cell-CAM 105 under the experimental conditions used for cell adhesion assays differs from that in situ and raises the possibility that its adhesive function may be modulated by its cell surface distribution. The implications of these and other findings are discussed with regard to a model for BC formation.^ Analysis of molecular events involved in BC formation would be accelerated if an in vitro model system were available. Although BC formation in culture has previously been observed, repolarization of cell-CAM 105 and two other domain-specific membrane proteins was incomplete. Since DMSO had been used by Isom et al. to maintain liver-specific gene expression in vitro, the effect of this differentiation system on the polarity of these membrane proteins was examined. Based on findings presented here, DMSO apparently prolongs the expression and facilitates polarization of hepatocyte membrane proteins in vitro. ^

Altered cholesterol metabolism in a hypercholesterolemia -resistant rabbit colony

A colony of rabbits has been developed at the University of Texas Medical School at Houston that is resistant to dietary-induced hypercholesterolemia. The liver of resistant rabbits had higher levels of ($\sp{125}$I) $\beta$-VLDL binding and 3-hydroxy-3-methylglutaryl (HMGCoA) reductase activity, but lower acyl coenzyme A:cholesterol acyltransferase (ACAT) activity than normal rabbits. Direct quantitation of intracellular cholesterol content of the liver revealed that the resistant rabbits had $<$10% of the intracellular free cholesterol present in normal rabbits. Fibroblasts isolated from normal and resistant rabbits exhibited differences in ($\sp{125}$I) LDL binding, HMGCoA reductase activity and ACAT activity that were similar to those found in the liver. No structural differences were found in the LDL receptor of normal and resistant fibroblasts that would account for the increased binding capacity of the resistant cells. The regulation of LDL receptor levels by exogenous oxygenated sterols was similar in normal and resistant fibroblasts. The regulation of LDL receptor binding capacity by LDL was attenuated in the resistant compared to normal fibroblasts, suggesting that the resistant fibroblasts have an alternate pathway for processing lipoprotein-derived cholesterol. Sterol-balance studies revealed that the cholesterol-fed resistant rabbits increased lithocholic acid excretion compared to the basal state, and had higher levels of deoxycholic acid excretion than cholesterol-fed normal rabbits. In addition, the specific activity and mRNA levels of cholesterol 7$\alpha$-hydroxylase (C7$\alpha$H) were higher in resistant rabbits than normal rabbits, suggesting that the increased bile acid excretion was due to an increase in bile acid synthesis. Increased clearance of cholesterol relieves the negative feedback inhibition cholesterol exerts on expression of the LDL receptor. The number of cell surface LDL receptors is then increased in resistant rabbits and allows rapid clearance of lipoproteins from the plasma compartment, thereby reducing plasma cholesterol levels. The low intracellular cholesterol level also relieves the negative feedback inhibition cholesterol exerts on HMGCoA reductase. Increased synthesis of cholesterol from acetate provides cells with cholesterol for bile acid synthesis and/or homeostasis. The activity of ACAT is then decreased due to the flux of cholesterol through the bile acid synthetic pathways. ^

Characterization of the cholesterol 7$\alpha$-hydroxylase promoter in hypercholesterolemia -resistant rabbits

Our laboratory has developed and partially characterized a strain of New Zealand white rabbits that are resistant to the hypercholesterolemia which typically occurs in normal rabbits when fed a cholesterol-enriched diet. This phenotype is most likely attributed to an increase in bile acid excretion by hypercholesterolemia-resistant (CRT) rabbits as a result of elevated enzyme activity of cholesterol 7$\alpha$-hydroxylase (C7$\alpha$H), the rate-limiting enzyme in bile acid synthesis. Northern analysis revealed that CRT rabbits, in comparison to normal rabbits, have a 7-fold greater steady-state C7$\alpha$H mRNA levels irrespective of dietary regimen. The C7$\alpha$H gene in both phenotypes was determined to be a single copy gene. The hypothesis was that the elevated C7$\alpha$H mRNA levels in CRT rabbits, in comparison to normal animals, was due to an increase in the transcription rate of the C7$\alpha$H gene as a result of a mutation in a cis-acting element and/or a trans-acting factor within the hepatocyte. To isolate the C7$\alpha$H gene from both normal and CRT rabbits, genomic libraries were prepared from both phenotypes into $\lambda$GEM12 vectors using conventional techniques. Three CRT and one normal phage clones that contained the C7$\alpha$H gene were identified by screening the library with a series of probes located within different exons of the C7$\alpha$H cDNA. Sequencing analysis confirmed that approximately 1100 bp of the C7$\alpha$H 5'-flanking region from both normal and CRT phenotypes was identical. The increase in C7$\alpha$H mRNA levels was not attributed to a cis-acting mutation within this region. Liver nuclear extracts were prepared from normal and CRT rabbits maintained either on a basal or 0.25% cholesterol-enriched diet and incubated with several radiolabeled DNA fragments from the C7$\alpha$H gene. A 37 basepair region, located between nucleotides $-$452 to $-$416 was identified that had altered binding patterns between normal and CRT rabbits as a function of diet. Two additional regions, $-$747 to $-$575 and $-$580 to $-$442, produced banding patterns which were identical, irrespective of phenotype or diet. In conclusion, these studies suggested that the increase in C7$\alpha$H mRNA in CRT rabbits was due to differences in binding of a cholesterol-responsive transcription factor to the C7$\alpha$H promoter. ^

(Table 3) Liver weight, blood liver parameters, and blood plasma PCB concentrations in 14 sledge dog bitches and their 14 pups in Aasiaat, west Greenland

We assessed the relationship between exposure to organohalogen polluted minke whale (Balaenoptera acutorostrata) blubber and liver morphology and function in a generational controlled study of 28 Greenland sledge dogs (Canis familiaris). The prevalence of portal fibrosis, mild bile duct hyperplasia, and vascular leukocyte infiltrations was significantly higher in the exposed group (all Chi-square: p<0.05). In case of granulomas, the frequency was significantly highest in the bitches (P generation) while the prevalence of portal fibrosis was highest in the F generation (pups) (both Chi-square: p<0.05). No significant difference between exposed and controls was found for bile acid, ALAT, and ALKP, while ASAT and LDH were significantly highest in the control group (both ANOVA: p<0.05). We therefore suggest that a daily intake of 50-200 g environmentally organohalogen polluted minke whale blubber can cause liver lesions in Greenland sledge dogs. It is reasonable to infer that other apex predators such as polar bears (Ursus maritimus) and humans may suffer from similar impacts.

Distribution of the coal flow in the mill-duct system of the As Pontes Power Plant using CFD modeling

The efficiency of a Power Plant is affected by the distribution of the pulverized coal within the furnace. The coal, which is pulverized in the mills, is transported and distributed by the primary gas through the mill-ducts to the interior of the furnace. This is done with a double function: dry and enter the coal by different levels for optimizing the combustion in the sense that a complete combustion occurs with homogeneous heat fluxes to the walls. The mill-duct systems of a real Power Plant are very complex and they are not yet well understood. In particular, experimental data concerning the mass flows of coal to the different levels are very difficult to measure. CFD modeling can help to determine them. An Eulerian/Lagrangian approach is used due to the low solid–gas volume ratio.

Túneles en macizos calcáreos karstificados: impacto en las obras subterráneas de los fenómenos kársticos, su evaluación y tratamiento

En esta Tesis Doctoral se aborda un tema poco estudiado en el ámbito de los túneles y cuya problemática está basada en los riesgos e incertidumbres que representa el diseño y ejecución de túneles en macizos calizos karstificados. Mediante un estudio profundo del comportamiento de distintos casos reales de túneles en macizos kársticos calizos, aportando la realización de modelos y la experiencia constructiva del autor, se pretende proponer un procedimiento que permita sistematizar las actuaciones, investigación, evaluación y tratamiento en los túneles en karst, como herramienta básica para la toma de decisiones primarias correctas. Además, se proponen herramientas que pueden ayudar a mejorar el diseño y a decidir las medidas más eficientes para afrontar los problemas que genera el karst en los túneles, minimizando los riesgos para todos los actores, su probabilidad e impacto. Se profundiza en tres fases principales (que son referidas en la tesis en cuatro Partes): La INVESTIGACIÓN del macizo rocoso: La investigación engloba todas las actuaciones observacionales encaminadas a obtener el IK (Índice de Karstificación), así como las investigaciones necesarias mediante recopilación de datos superficiales, hidrogeológicos, climáticos, topográficos, así como los datos de investigaciones geofísicas, fotointerpretación, sondeos y ensayos geotécnicos que sean posibles. Mediante la misma, se debe alcanzar un conocimiento suficiente para llevar a cabo la determinación de la Caracterización geomecánica básica del macizo rocoso a distintas profundidades, la determinación del Modelo o modo de karstificación del macizo rocoso respecto al túnel y la Zonificación del índice de karstificación en el ámbito de actuación en el que se implantará el túnel. En esta primera fase es necesaria la correcta Definición geométrica y trazado de la obra subterránea: En función de las necesidades que plantee el proyecto y de los condicionantes externos de la infraestructura, se deben establecer los requisitos mínimos de gálibo necesarios, así como las condiciones de máximas pendientes para el trazado en alzado y los radios mínimos de las curvas en planta, en función del procedimiento constructivo y motivación de construcción del túnel (ferrocarril, carretera o hidráulico, etc...). Estas son decisiones estratégicas o primerias para las que se ha de contar con un criterio y datos adecuados. En esta fase, son importantes las decisiones en cuanto a las monteras o profundidades relativas a la karstificación dominante e investigación de las tensiones naturales del macizo, tectónica, así como las dimensiones del túnel en función de las cavidades previstas, tratamientos, proceso de excavación y explotación. En esta decisión se debe definir, conocida ya de forma parcial la geotecnia básica, el procedimiento de excavación en función de las longitudes del túnel y la clasificación geomecánica del terreno, así como sus monteras mínimas, accesos y condicionantes medioambientales, pero también en función de la hidrogeología. Se establecerá la afección esperable en el túnel, identificando en la sectorización del túnel, la afección esperable de forma general para las secciones pésimas del túnel. Con todos estos datos, en esta primera aproximación, es posible realizar el inventario de casos posibles a lo largo del trazado, para poder, posteriormente, minimizar el número de casos con mayores riesgos (técnicos, temporales o económicos) que requieran de tratamiento. Para la fase de EVALUACIÓN de la matriz de casos posibles en función del trazado inicial escogido (que puede estar ya impuesto por el proyecto, si no se ha podido intervenir en dicha fase), es necesario valorar el comportamiento teórico del túnel en toda su longitud, estudiando las secciones concretas según el tipo y el modo de afección (CASOS) y todo ello en función de los resultados de los estudios realizados en otros túneles. Se debe evaluar el riesgo para cada uno de los casos en función de las longitudes de túnel que se esperan que sean afectadas y el proceso constructivo y de excavación que se vaya a adoptar, a veces varios. Es importante tener en cuenta la existencia o no de agua o relleno arcilloso o incluso heterogéneo en las cavidades, ya que los riesgos se multiplican, así mismo se tendrá en cuenta la estabilidad del frente y del perímetro del túnel. En esta segunda fase se concluirá una nueva matriz con los resultados de los riesgos para cada uno de los casos que se presentarán en el túnel estudiado. El TRATAMIENTO, que se debe proponer al mismo tiempo que una serie de actuaciones para cada uno de los casos (combinación de tipos y modos de afección), debiendo evaluar la eficacia y eficiencia, es decir la relevancia técnica y económica, y como se pueden optimizar los tratamientos. Si la tabla de riesgos que se debe generar de nuevo introduciendo los factores técnicos y económicos no resulta aceptable, será necesaria la reconsideración de los parámetros determinados en fases anteriores. Todo el desarrollo de estas tres etapas se ha recogido en 4 partes, en la primera parte se establece un método de estudio e interpretativo de las investigaciones superficiales y geotécnicas, denominado índice de karstificación. En la segunda parte, se estudia la afección a las obras subterráneas, modelos y tipos de afección, desde un punto de vista teórico. La tercera parte trata de una recopilación de casos reales y las consecuencias extraídas de ellos. Y finalmente, la cuarta parte establece los tratamientos y actuaciones para el diseño y ejecución de túneles en macizos kársticos calizos. Las novedades más importantes que presenta este trabajo son: El estudio de los casos de túneles realizados en karst calizo. Propuesta de los tratamientos más efectivos en casos generales. La evaluación de riesgos en función de las tipologías de túnel y afecciones en casos generales. La propuesta de investigación superficial mediante el índice de karstificación observacional. La evaluación mediante modelos del comportamiento teórico de los túneles en karst para casos muy generales de la influencia de la forma, profundidad y desarrollo de la karstificación. In this doctoral thesis is studied the recommended investigation, evaluation and treatment when a tunnel is planed and constructed in karstic calcareous rock masses. Calcareous rock masses were selected only because the slow disolution produces stable conduct and caves instead the problems of sudden disolutions. Karstification index (IK) that encompasses various aspects of research karstic rock mass is presented. The karst rock masses are all unique and there are no similarities between them in size or density cavities ducts, but both their formation mechanisms, like, geological and hydrogeological geomorphological evidence allow us, through a geomechanical survey and geological-photo interpretation in the surface, establish a karst evaluation index specific for tunnelling, which allows us to set a ranking of the intensity of karstification and the associated stadistic to find caves and its risk in tunnelling. This index is estimated and useful for decision-making and evaluation of measures to be considered in the design of a tunnel and is set in degrees from 0 to 100, with similar to the RMR degrees. The sectorization of the tunnel section and the types of condition proposed in this thesis, are estimated also useful to understand the different effects that interception of ducts and cavities may have in the tunnel during construction and service life. Simplified calculations using two-dimensional models contained in the thesis, have been done to establish the relationship between the position of the cavities relative to the section of the tunnel and its size in relation to the safety factors for each ground conditions, cover and natural stresses. Study of over 100 cases of tunnels that have intercepted cavities or karst conduits have been used in this thesis and the list of risks found in these tunnels have been related as well. The most common and effective treatments are listed and finally associated to the models and type of affection generated to give the proper tool to help in the critical decision when the tunnels intercept cavities. Some existing studies from Marinos have been considered for this document. The structure of the thesis is mainly divided 4 parts included in 3 steps: The investigation, the evaluation and the treatments, which match with the main steps needed for the critical decisions to be made during the design and construction of tunnels in karstic rockmasses, very important to get a successfully project done.

Algoritmos genéticos en control activo de ruido

Este proyecto se centra en la implementación de un sistema de control activo de ruido mediante algoritmos genéticos. Para ello, se ha tenido en cuenta el tipo de ruido que se quiere cancelar y el diseño del controlador, parte fundamental del sistema de control. El control activo de ruido sólo es eficaz a bajas frecuencias, hasta los 250 Hz, justo para las cuales los elementos pasivos pierden efectividad, y en zonas o recintos de pequeñas dimensiones y conductos. El controlador ha de ser capaz de seguir todas las posibles variaciones del campo acústico que puedan producirse (variaciones de fase, de frecuencia, de amplitud, de funciones de transferencia electro-acústicas, etc.). Su funcionamiento está basado en algoritmos FIR e IIR adaptativos. La elección de un tipo de filtro u otro depende de características tales como linealidad, causalidad y número de coeficientes. Para que la función de transferencia del controlador siga las variaciones que surgen en el entorno acústico de cancelación, tiene que ir variando el valor de los coeficientes del filtro mediante un algoritmo adaptativo. En este proyecto se emplea como algoritmo adaptativo un algoritmo genético, basado en la selección biológica, es decir, simulando el comportamiento evolutivo de los sistemas biológicos. Las simulaciones se han realizado con dos tipos de señales: ruido de carácter aleatorio (banda ancha) y ruido periódico (banda estrecha). En la parte final del proyecto se muestran los resultados obtenidos y las conclusiones al respecto. Summary. This project is focused on the implementation of an active noise control system using genetic algorithms. For that, it has been taken into account the noise type wanted to be canceled and the controller design, a key part of the control system. The active noise control is only effective at low frequencies, up to 250 Hz, for which the passive elements lose effectiveness, and in small areas or enclosures and ducts. The controller must be able to follow all the possible variations of the acoustic field that might be produced (phase, frequency, amplitude, electro-acoustic transfer functions, etc.). It is based on adaptive FIR and IIR algorithms. The choice of a kind of filter or another depends on characteristics like linearity, causality and number of coefficients. Moreover, the transfer function of the controller has to be changing filter coefficients value thought an adaptive algorithm. In this project a genetic algorithm is used as adaptive algorithm, based on biological selection, simulating the evolutionary behavior of biological systems. The simulations have been implemented with two signal types: random noise (broadband) and periodic noise (narrowband). In the final part of the project the results and conclusions are shown.

Measurement of velocity in rotational flows using ultrasonic anemometry: the flowmeter

In this paper a previously developed theoretical model of the measurement process performed by a transit-time ultrasonic anemometer is applied to a fluid flowing through a circular section pipe. This model considers the influence of the shift of the acoustic pulse trajectory from straight propagation due to the flow on the measured speed. The aim of this work is to estimate the errors induced in the measured velocity by the shift of the acoustic pulse trajectory. Using different duct’s flow models, laminar and turbulent regimes have been analyzed. The results show that neglecting the effect of shift of the acoustic pulse trajectory leads to flow rate measurement underestimation.

Definición e implantación de herramientas avanzadas 3D en el diseño integrado y eficiente de sistemas HVAC en buques, desde el proyecto conceptual hasta la producción

Uno de los aspectos más complicados del diseño de sistemas HVAC en buques es la correcta evaluación de las necesidades de aire fresco y el correcto dimensionado de los conductos que suministran dicho aire y evacuan el calor generado a bordo. Contrariamente a lo que sucede en los sistemas de tuberías, las características particulares del caudal de aire hacen que el dimensionado de los conductos sea muy sensible al trazado y geometría de los mismos, por lo que para obtener un buen diseño es necesaria una relación muy estrecha y una integración bidireccional entre los cálculos y el trazado de los propios conductos en el buque. Asumida la utilización de sistemas CAD/CAM para las tareas de diseño, históricamente, aquellos que permitían modelar conductos HVAC no incluían en su alcance de suministro los aspectos de cálculo, y como consecuencia de ello, el trazado de conductos se reducía a la inclusión en el modelo 3D de circuitos y sistemas previamente calculados y dimensionados, Así, servían únicamente para calcular interferencias con otros elementos del modelo 3D y para obtener posteriormente planos de fabricación y montaje. Esto, que por sí no es poco, dejaba el diseño de sistemas HVAC pendiente de una importante interacción manual y de habituales retrabajos, ya que cualquier modificación en el trazado de los conductos, consecuencia de otras necesidades del diseño, obligaba a los diseñadores a recalcular y redimensionar los conductos en un entorno diferente al del propio sistema CAD/CAM, y volver a realizar el modelado de los mismos, reduciendo significativamente las ventajas de la utilización de un modelo 3D. Partiendo de esta situación real, y con objeto de solucionar el problema que para el diseño y la propia producción del buque se creaba, se concibió una herramienta que permitiera la definición en el modelo 3D de diagramas de ventilación, el cálculo de pérdidas de presión, el dimensionado automático de los conductos, y que toda esta información pudiera estar disponible y reutilizarse en las etapas posteriores del diseño. Con ello, los diseñadores podrían realizar su trabajo en un entorno único, totalmente integrado con el resto de disciplinas. El objeto de esta Tesis Doctoral es analizar en detalle el problema y las ineficiencias actuales del diseño de HVAC, describir la innovadora herramienta concebida para paliar estas ineficiencias, detallando las bases sobre la que se construye, y destacar las ventajas que se obtienen de su uso. La herramienta en cuestión fue concebida como una funcionalidad adicional del sistema CAD/CAM naval FORAN, referente tecnológico en el mundo del diseño y la construcción navales, y como consecuencia de ellos se llevó a cabo el desarrollo correspondiente. En la actualidad, el sistema FORAN incluye en su alcance de suministro una primera versión de esta herramienta, cuya utilidad queda avalada por el uso que de la misma hacen astilleros y oficinas técnicas en todo el mundo. Esta Tesis Doctoral es eminentemente práctica. No es un estudio teórico de dudosa aplicación, sino que tiene por objeto aportar una solución eficiente a un problema real que muchos astilleros y oficinas técnicas, incluidas los más avanzados, padecen hoy en día. No tiene otra motivación que servir de ayuda para lograr diseñar y construir mejores barcos, en un plazo más corto, y a un coste menor. Nada más, pero nada menos. ABSTRACT One of the most complicated aspects of the design of HVAC systems in shipbuilding is the correct evaluation of the fresh air needs, the correct balancing of the ducts that supply this air and evacuate the existing heat on board. In opposition to piping systems, due to the particular characteristics of the air flow, the balancing of the ducts is very sensitive to the routing and the aspect of the ducts, so the correct design requires a close interconnectivity between calculations and routing. Already assumed the use of CAD/CAM systems for design tasks, historically, those CAD/CAM systems capable of modelling HVAC ducts did not cover calculation aspects, with the result that the routing of HVAC ducts was reduced solely to the input of previously balanced circuits into the 3D Product Model for the purpose of interference checking and generation of fabrication and assembly drawings. This situation, not negligible at all, put the design of HVAC ducts very dependent on manual operations and common rework task, as any modification in the routing of the HVAC ducts, derived from design needs, obliged engineers to re-balance the ducts and eventually to re-size them independently of the CAD-CAM environment, thus annulling the advantages of the 3D Product Model. With this situation in mind, and with the objective of filling the gap created in the design and construction of the ship, it was conceived a tool allowing the definition, within the 3D Product model, of HVAC diagrams, the calculation of pressure drops, the automatic dimensioning of ducts. With this, engineers could make the complete HVAC design in a single working environment, fully integrated with the rest of the disciplines. The present Ph. D. thesis analyses in deep the existing problem and the current lack of efficiency in HVAC design, describes the innovative tool conceived to minimize it, details the basis on which the tool is built, and highlights the advantages of its use. This tool was conceived as an additional functionality of the marine CAD/CAM system FORAN, a technological reference in the shipdesign and shipbuilding industry. As a consequence, it was developed, and nowadays FORAN System includes in its scope of supply a first version of the tool, with its usefulness endorsed by the fact that it is used by shipyards and shipdesign offices all over the world. This Ph. D. thesis is on top everything, of practical nature. It is not a theoretical study with doubtful application. On the contrary, its objective is to provide with an efficient solution for solving a real problem that many shipyards and shipdesign offices, including those more advanced, suffer nowadays. It has no other motivation that to help in the process of designing and building better and cheaper ships, within a shorter deliver time. Nothing more, but nothing less.

In vivo inhibition of rat stellate cell activation by soluble transforming growth factor β type II receptor: A potential new therapy for hepatic fibrosis

Transforming growth factor β (TGF-β) is a well characterized cytokine that appears to play a major role in directing the cellular response to injury, driving fibrogenesis, and, thus, potentially underlying the progression of chronic injury to fibrosis. In this study, we report the use of a novel TGF-β receptor antagonist to block fibrogenesis induced by ligation of the common bile duct in rats. The antagonist consisted of a chimeric IgG containing the extracellular portion of the TGF-β type II receptor. This “soluble receptor” was infused at the time of injury; in some experiments it was given at 4 days after injury, as a test of its ability to reverse fibrogenesis. The latter was assessed by expression of collagen, both as the mRNA in stellate cells isolated from control or injured liver and also by quantitative histochemistry of tissue sections. When the soluble receptor was administered at the time of injury, collagen I mRNA in stellate cells from the injured liver was 26% of that from animals receiving control IgG (P < 0.0002); when soluble receptor was given after injury induction, collagen I expression was 35% of that in control stellate cells (P < 0.0001). By quantitative histochemistry, hepatic fibrosis in treated animals was 55% of that in controls. We conclude that soluble TGF-β receptor is an effective inhibitor of experimental fibrogenesis in vivo and merits clinical evaluation as a novel agent for controlling hepatic fibrosis in chronic liver injury.

Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain

A novel multispecific organic anion transporting polypeptide (oatp2) has been isolated from rat brain. The cloned cDNA contains 3,640 bp. The coding region extends over 1,983 nucleotides, thus encoding a polypeptide of 661 amino acids. Oatp2 is homologous to other members of the oatp gene family of membrane transporters with 12 predicted transmembrane domains, five potential glycosylation, and six potential protein kinase C phosphorylation sites. In functional expression studies in Xenopus laevis oocytes, oatp2 mediated uptake of the bile acids taurocholate (Km ≈ 35 μM) and cholate (Km ≈ 46 μM), the estrogen conjugates 17β-estradiol-glucuronide (Km ≈ 3 μM) and estrone-3-sulfate (Km ≈ 11 μM), and the cardiac gylcosides ouabain (Km ≈ 470 μM) and digoxin (Km ≈ 0.24 μM). Although most of the tested compounds are common substrates of several oatp-related transporters, high-affinity uptake of digoxin is a unique feature of the newly cloned oatp2. On the basis of Northern blot analysis under high-stringency conditions, oatp2 is highly expressed in brain, liver, and kidney but not in heart, spleen, lung, skeletal muscle, and testes. These results provide further support for the overall significance of oatps as a new family of multispecific organic anion transporters. They indicate that oatp2 may play an especially important role in the brain accumulation and toxicity of digoxin and in the hepatobiliary and renal excretion of cardiac glycosides from the body.

Design of compounds that increase the absorption of polar molecules

Hydrophilic drugs are often poorly absorbed when administered orally. There has been considerable interest in the possibility of using absorption enhancers to promote absorption of polar molecules across membrane surfaces. The bile acids are one of the most widely investigated classes of absorption enhancers, but there is disagreement about what features of bile acid enhancers are responsible for their efficacy. We have designed a class of glycosylated bile acid derivatives to evaluate how increasing the hydrophilicity of the steroid nucleus affects the ability to transport polar molecules across membranes. Some of the glycosylated molecules are significantly more effective than taurocholate in promoting the intestinal absorption of a range of drugs, showing that hydrophobicity is not a critical parameter in transport efficacy, as previously suggested. Furthermore, the most effective glycosylated compound is also far less damaging to membranes than the best bile acid absorption promoters, presumably because it is more hydrophilic. The results reported here show that it is possible to decouple absorption-promoting activity from membrane damage, a finding that should spark interest in the design of new compounds to facilitate the delivery of polar drugs.

Disruption of the murine gene encoding phosphatidylethanolamine N-methyltransferase

All nucleated cells make phosphatidylcholine via the CDP-choline pathway. Liver has an alternative pathway in which phosphatidylcholine is made by methylation of phosphatidylethanolamine catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). We investigated the function of PEMT and its role in animal physiology by targeted disruption of its gene, Pempt2. A targeting vector that interrupts exon 2 was constructed and introduced into mice yielding three genotypes: normal (+/+), heterozygotes (+/−), and homozygotes (−/−) for the disrupted PEMT gene. Only a trace of PE methylation activity remained in Pempt2(−/−) mice. Antibody to one form of the enzyme, PEMT2, indicated complete loss of this protein from Pempt2(−/−) mice and a decrease in Pempt2(+/−) mice, compared with Pempt2(+/+) mice. The levels of hepatic phosphatidylethanolamine and phosphatidylcholine were minimally affected. The active form of CTP:phosphocholine cytidylyltransferase, the regulated enzyme in the CDP-choline pathway, was increased 60% in the PEMT-deficient mice. Injection of [l-methyl-3H]methionine demonstrated that the in vivo PEMT activity was eliminated in the Pempt2(−/−) mice and markedly decreased in the Pempt2(+/−) mice. This experiment also demonstrated that the choline moiety derived from PEMT in the liver can be distributed via the plasma throughout the mouse where it is found as phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin. Mice homozygous for the disrupted Pempt2 gene displayed no abnormal phenotype, normal hepatocyte morphology, normal plasma lipid levels and no differences in bile composition. This is the first application of the “knockout mouse” technique to a gene for phospholipid biosynthesis.