Objetivo: El principal objetivo de esta tesis fue estudiar la relación de los ácidos grasos y de las vitaminas liposolubles antioxidantes y carotenoides plasmáticos con la obesidad en la etapa gestacional y la adolescencia. Además, se planteó desarrollar un método analítico para analizar vitaminas y carotenoides en muestras de plasma. Metodología: Por una parte, se incluyeron 165 mujeres embarazadas (normopeso, sobrepeso y obesas) de 18 a 44 años de edad, y 162 recién nacidos. Se determinaron los ácidos grasos de los fosfolípidos plasmáticos (por cromatografía de gases) de las gestantes a las 24 y 34 semanas de gestación y en el parto, así como de los hijos al nacer. Por otra parte, se reclutaron 127 adolescentes con sobrepeso u obesidad, de 12 a 17 años de edad, los cuales fueron sometidos a un tratamiento integral de adelgazamiento. En éstos se analizaron las vitaminas A y E, β-caroteno y licopeno (por cromatografía líquida rápida de alta eficacia, UHPLC) y los ácidos grasos totales (por cromatografía de gases), tanto en condiciones basales como a los 2 y 6 meses de tratamiento. Asimismo, se estimó la actividad de algunas enzimas involucradas en el metabolismo de los ácidos grasos, empleando los siguientes índices: elongasa-6 de ácidos grasos=C18:0/C16:0, estearoil-coenzima A desaturasa-16=16:1n-7/16:0, estearoil-coenzima A desaturasa-18=C18:1n-9/C18:0, delta-5-desaturasa=C20:4n-6/C20:3n-6 y delta-6-desaturasa=C18:3n-6/C18:2n-6. Resultados: Las embarazadas obesas presentaron una menor proporción de los ácidos grasos C18:1 n-9, C17:0 y C18:3 n-3 que las gestantes con un estado nutricional normal, en los tres períodos de estudio. Los hijos de embarazadas obesas tuvieron una menor cantidad de C20:5 n-3 que el grupo control. La proporción de C14:0, C16:0, C18:0, C20:3 n-9, C22:5 n-3, C18:2 n-6 en plasma materno y el contenido plasmático de C20:2 n-6 y C22:5 n-3 de los hijos se relacionaron con los indicadores antropométricos de crecimiento fetal. Los recién nacidos macrosómicos presentaron una menor cantidad de C18:0 que los niños de peso normal. En los adolescentes, el aumento de las concentraciones de α-tocoferol, retinol y β-caroteno, ocurrido durante el tratamiento, se relacionó con la reducción de los indicadores antropométricos de adiposidad general, con una mayor pérdida de peso y con la mejora de los marcadores de riesgo cardiometabólico. El descenso plasmático de los ácidos C14:0 y C18:2 n-6, y el incremento del C18:1 n-9 y de la actividad enzimática estimada de estearoil-coenzima A desaturasa-18 también se asociaron con la reducción de los indicadores antropométricos de adiposidad general y con una mayor pérdida de peso. La disminución del C14:0 y de la actividad estimada de elongasa-6 de ácidos grasos y delta-6-desaturasa, y el aumento de delta-5-desaturasa se relacionaron con la mejora de los marcadores de riesgo cardiometabólico de los adolescentes. Conclusiones: se desarrolló y validó un método analítico rápido, preciso y sensible, con linealidad y exactitud óptimas, que permite, en sólo tres minutos, la determinación simultánea de α-tocoferol, retinol, β-caroteno y licopeno en plasma por cromatografía líquida rápida de alta eficacia. La obesidad materna se relacionó con la composición plasmática de ácidos grasos de las embarazadas y de sus hijos. Los ácidos grasos saturados y poliinsaturados de las madres, y los ácidos grasos poliinsaturados de cadena larga y el ácido esteárico de los hijos se relacionaron con el crecimiento fetal. Los cambios de la composición plasmática de ácidos grasos, de la actividad enzimática estimada y de las vitaminas liposolubles antioxidantes y carotenoides de los adolescentes se asociaron con el descenso de la adiposidad, con una mayor pérdida de peso y con la mejora del perfil cardiometabólico de los adolescentes. / Aim: The main objective of this thesis was to explore the relationships between fatty acids (FA) and antioxidant lipo-soluble vitamins and carotenoids plasma levels and the obesity during pregnancy and adolescence. Moreover, the improvement of the analytical methodology to analyze vitamins and carotenoids in plasma samples was addressed. Methods: Two different populations were studied. On the one hand, 165 pregnant women (obese, overweight and normal weight) and their newborns were included. FA of plasmatic phospholipids were determined in maternal plasma at 24 and 34 weeks of gestation and at delivery, as well as in cord blood by gas chromatography (GC). On the other hand, 127 adolescents (obese and overweight) involved in a weight-loss program were recruited. Samples of plasma were obtained during the intervention, at baseline, 2 and 6 months. Vitamins A and E, β-carotene and lycopene were analyzed by ultra-high performance liquid chromatography (UHPLC) and total plasmatic FA by GC. Moreover, the enzymatic activity of some enzymes involved in FA metabolism was estimated using the respective product:substrate ratio. Results: Obese pregnant women presented lower levels of C18:1 n-9, C17:0 and C18:3 n-3 than normal weight ones, and the newborns from obese women had lower levels of C20:5 n-3. Maternal plasmatic levels of C14:0, C16:0, C18:0, C20:3 n-9, C22:5 n-3, C18:2 n-6 and cord blood levels of C20:2 n-6 and C22:5 n-3 were related to anthropometric indicators of fetal growth. In adolescents, the observed increase of α-tocopherol, retinol and β-carotene during the treatment was related with the decrease in anthropometric indicators of adiposity, weight loss and with an improvement in the cardiometabolic risk markers. Additionally, plasmatic variations during treatment of C14:0, C18:2 n-6, C18:1 n-9 and some of the studied enzymatic activities were also related with anthropometric indicators of adiposity and cardiometabolic risk markers. Conclusions: A reliable, sensitive and low-time consuming method for the simultaneous determination of α-tocopherol, retinol, β-carotene and lycopene by UHPLC was developed. The maternal and umbilical cord plasma FA composition was associated with maternal obesity. Some maternal and cord blood FA were related to fetal growth. In obese adolescents, the changes in plasmatic FA and vitamins levels during the intervention were associated with weight loss and an improvement in adiposity and cardiometabolic profile.

El estudio de la relación entre la dieta o factores específicos de la dieta y el estado de salud requieren medidas exactas de la exposición dietética. Los análisis tradicionales de exposición alimentaria, como las encuestas dietéticas o los cuestionarios de frecuencia de consumo contienen una serie de errores sistemáticos, como la limitación en la lista de alimentos considerados o estimaciones erróneas en el tamaño y la frecuencia de la ingesta, que han provocado una cierta inconsistencia en el verdadero papel de la alimentación sobre la salud humana. Como resultado de este hecho, existe un intenso esfuerzo por parte de la comunidad científica hacia la identificación de biomarcadores nutricionales. El objetivo principal de esta tesis ha sido el estudio de los biomarcadores de consumo de vino tinto, incluyendo principalmente aquellos metabolitos originados por la acción de la microbiota intestinal, mediante la generación de la base de datos Phenol-Explorer y su aplicación en estudios clínicos con vino tinto. Para ello se ha desarrollado la primera base de datos que considera el metabolismo de los compuestos fenólicos, concretándose en la generación del módulo de metabolismo de la base de datos Phenol-Explorer y se ha aplicado al estudio del metabolismo del vino. El conocimiento generado de este trabajo se aplicó a un estudio clínico y a un subestudio de similares características, ambos aleatorizados, cruzados y controlados de intervención con vino tinto, vino tinto desalcoholizado y ginebra como control para la identificación de biomarcadores. Se describió el más amplio perfil metabólico de metabolitos urinarios fenólicos mayoritariamente aquellos originados por la microbiota intestinal (n=61) mediante UPLCMS/ MS tras el consumo de vino tinto desalcoholizado observando un aumento de metabolitos (n=49) respecto al momento basal, principalmente aquellos originados por el metabolismo microbiano de los antocianos y los flavan-3-oles. Cuando se comparó la ingesta de vino alcoholizado y desalcoholizado no se observaron diferencias de excreción de metabolitos entre los dos vinos, por lo que se evaluó la capacidad discriminatoria de dichos metabolitos como biomarcadores de su consumo. A través de una regresión logística se estableció un modelo que incluía derivados del ácido gálico y el etilgalato. Tras su evaluación, estos resultaron ser buenos predictores del consumo de vino tanto para plasma como para orina. Estos resultados demuestran que la combinación de diversos biomarcadores procedentes del consumo de vino mejora su capacidad discriminatoria y predictiva respecto a la consideración de biomarcadores individuales. La comparación entre ambas matrices estableció que la orina 24 horas proporciona un mayor poder discriminatorio entre consumidores o no consumidores de vino respecto a la de plasma, sin embargo, existe una correlación entre los grupos de metabolitos y el modelo generado. En el subestudio realizado con 10 participantes donde también se recogieron muestras de heces, el análisis de la flora fecal tras el consumo de vino permitió establecer el efecto prebiótico de los polifenoles del vino sobre la microbiota intestinal, aumentando significativamente Enterococcus, Bifidobacterium y Eggerthella lenta tras el consumo de polifenoles, independientemente de la presencia de alcohol. De manera adicional se observaron mejoras de parámetros bioquímicos como el colesterol o la proteína-C reactiva correlacionados con cambios en los géneros Bifidobacterium y Bacteroides. Finalmente, los cambios bacterianos se asociaron con los cambios en la excreción de metabolitos fenólicos observándose que los aumentos de las bifidobacterias se correlacionaban con aumentos de metabolitos microbianos como el siríngico y 4-hidroxibenzoico derivados de los antocianos del vino, y la existencia de una correlación inversa para el Enterococcus y la Eggerthella lenta, con el 3(4-hidroxi)fenilacético y los ácidos hidroxicinámicos respectivamente. / The study on the relationship between diet and health status require accurate measurements of dietary exposure. Traditional analysis of dietary exposure might lead to bias in the estimation of the role of food on human health. Therefore, there is an intense effort by the scientific community on the identification of biomarkers. The principal objective of the Thesis has been to contribute to the identification and evaluation of biomarkers of red wine consumption, focusing on the intestinal microbiota metabolites. To this purpose the Phenol-Explorer database metabolism module was developed and applied to study wine metabolism. The knowledge generated in this work was applied to a clinical study and a substudy of similar characteristics, both randomized, crossed and controlled intervention with red wine, dealcoholized red wine and gin as a control for biomarkers identification. Therefore, it has been described the broadest phenolic metabolic profile, mainly representing metabolites originated by microbiota (n=61), by UPLC-MS/MS in urine after consumption of dealcoholized red wine. Most of increased metabolites from baseline (n=49) were of anthocyanins and flavanols microbial metabolism origin. When comparing the intake of red wine and dealcoholized red wine no differences in metabolite excretion were observed, so both wines were used to evaluate the discriminatory power of these metabolites as biomarkers of wine intake. Through a logistic regression, a model including derivatives of gallic acid and ethylgallate was established. This model was found to be better predictor of wine consumption in plasma and urine than individual metabolites. These results demonstrated that the combination of different biomarkers could improve the discriminatory and predictive ability regarding the consideration of individual biomarkers. In the substudy conducted with 10 participants, fecal samples were also collected. Their analysis on microbiota content allowed establishing the prebiotic effect of polyphenols, where significant increases of Enterococcus, Bifidobacterium and Eggerthella lenta after both wines were detected and correlated to improvements in biochemical parameters such as cholesterol or C-reactive protein. Finally, bacterial changes were associated with changes in the excretion of phenolic metabolites, as bifidobacterias, which increase was correlated with anthocyanins derived microbial metabolites.

Actualment, s’estan invertint molts esforços en la detecció de components bioactius, com ara els polifenols, en fruites i hortalisses pel seu possible paper en la prevenció i el tractament de malalties cròniques. La quantitat de polifenols ingerits en la dieta és d’aproximadament 1 g/dia, quantitat més gran que la que prové de qualsevol altre antioxidant de la dieta. De fet, és aproximadament 10 vegades superior a la de vitamina C i 100 vegades superior a la de vitamina E i a la ingesta de carotens. D’altra banda, algunes dades epidemiològiques han mostrat una associació inversa entre el risc de mortalitat total o malalties cardiovasculars i el consum d’aliments rics en polifenols, com ara les fruites i hortalisses, el te, l’oli d’oliva verge i el vi. El tomàquet és l’hortalissa més consumida a Espanya (14.5 kg/any per persona) amb una important repercussió econòmica. En els últims 5 anys, el consum del tomàquet ha crescut un 2.8% anual. En l’àmbit de la fruita/hortalissa transformada, el tomàquet també ocupa el primer lloc en consum amb una ingesta de tomàquet fregit envasat de 4.1 kg/any per persona i de tomàquet natural envasat de 3 kg/any per persona. Els tomàquets espanyols destaquen pel seu elevat contingut en flavonoides, encara que no hi ha gaires estudis que s’hagin centrat en com afecta el processat i l’elaboració industrial de les salses als polifenols, així com tampoc l’evolució d’aquests compostos durant la vida útil d’aquests productes. El tomàquet i els seus derivats contenen altres fitoquímics valuosos com ara els carotens. El trans-licopè és el carotè més important dels tomàquets i el millor amortidor d’oxigen atòmic (captador de radicals lliures). A més del trans-licopè, els tomàquets també contenen altres carotens com els cis-isòmers del licopè i també α-, beta-carotè i luteïna. La importància dels isòmers cis recau en la seva elevada biodisponibilitat en l’organisme humà, per la qual cosa són necessaris mètodes per a la identificació d’aquests carotens. Alguns estudis han investigat l’efecte del tractament tèrmic en els carotens de salses de tomàquet, i s’hi ha observat un increment d’isòmers cis durant el processat. No obstant això, no hi ha estudis que valorin la influència de l’origen, el processat i l’emmagatzematge de tomàquets, salses, quètxups, gaspatxos i sucs de tomàquet quant als polifenols. / Currently many efforts are being invested in the detection of bioactive components of nutrients in fruits and vegetables, such as polyphenols, and their possible role in the prevention and treatment of chronic diseases. Total dietary polyphenol levels are approximately 1 g/day, a much larger amount than any other antioxidant that comes from the diet. In fact, it is about 10 times the dietary vitamin C and 100 times higher than vitamin E and carotene intake. Moreover, some epidemiological data have shown an inverse association between the risk of mortality or cardiovascular disease and consumption of foods rich in polyphenols, such as fruits and vegetables, tea, olive oil and wine. The tomato is the most consumed vegetable in Spain (14.5 kg/year per person) and makes a significant economic impact. In the last 5 years, the consumption of tomatoes has grown by 2.8% annually. In the field of fruit/vegetables, tomatoes also rank first with the consumption of fried tomatoes at an intake of 4.1 kg/year and that of raw tomatoes at 3 kg/year per person. Spanish tomatoes are known for their high content of flavonoids, although there is a lack of studies focused on how the industrial processing of sauces affects the content of polyphenols, and also the evolution of these compounds during their shelf life. The tomato and its derivatives contain other valuable phytochemicals such as carotenoids. trans-Lycopene is the most important carotene of tomato and it is an excellent free-radical scavenger; its capacity is more than twice that of beta-carotene. In addition to trans-lycopene, tomatoes also contain other carotenoids such as cis-isomers of lycopene and α- and beta-carotene and lutein. The importance of cis-isomers lies in their high bioavailability in the human body. Therefore, methods are needed to identify these carotenoids. Some studies have studied the effect of heat treatment on carotenoids in tomato sauces, and an increase has been observed during processing. However, no studies have been focused on evaluating the influence of the origin, processing and storage of tomatoes, sauces, ketchups, tomato juices and gazpachos on the level of polyphenols.

La consulta íntegra de la tesi, inclosos els articles no comunicats públicament per drets d'autor, es pot realitzar prèvia petició a l'Arxiu de la UPC / Vegetables and fruits are foods that have large number of compounds beneficial to health, as well as being essential in the diet. Its components include the phenolic substances. These compounds have a great antiradical activity and can be used to increase the stability of food, since they possess the ability to avoid the lipid peroxidation and can protect even against oxidation caused by metal cations. These cations favor the creation of reactive oxygen species, harmful to health and can participate directly or indirectly in the oxidation of lipids. The food industry produces large amounts of by-products and waste, which can be used for substances with high added value, such as natural antioxidants to replace the synthetic ones. In addition, this type of waste has a significant environmental impact due to high organic load and costs due to handling, transport and storage, among other processes. The present Doctoral thesis has obtained extracts rich in polyphenols, from three agro-industrial waste, natural: leaves of Borage, avocado seed and pericarp of pineapple. The conventional extraction by batch or continuous, has been applied to obtain polyphenols from these wastes. The conventional process has been optimized through the use of response surface methodology and equations of mass transfer: Fick law, theory of film, empirical, Peleg and speed law have been studied to establish a representative mathematical model. The diffusion coefficients were determined with the Fick law equation. Also have been applied new techniques, such as magnetic pulses and extraction assisted by ultrasound, to improve or accelerate the process of extraction, which has provided a 280% increase in the quantity of bioactive components obtained seed from avocado applying extraction assisted with ultrasound and a 520% of the leaves of Borage with extraction using magnetic pulses. In the kinetics of extraction, the studied equations had a adjustment, R2, between 0.99 and a 0.81. The extracts obtained have been evaluated in different models of food: emulsions, mincemeat and cupcakes, to determine if they increase the stability and prevent or retard the oxidation of fats and oils. There has been a decrease in the formation of oxidation products, both primary and secondary : from 80 to 90% (hydroperoxides) in emulsions, up to a 90% in products of secondary oxidation in the minced meat with extracts of Borage and avocado seed; and between 50 and 90% in the muffins with pineapple extract. Therefore it is shown that has been prolonged useful life time and increased stability against oxidation. Conclusion, in the study, was demonstrated that the use of by-products from agroindustry was useful to prepare natural extracts with antioxidants. These wastes represent a cost for industry and can be used to obtain compounds with high added value. Also, They slow down the oxidation of fats in different models of food. The by-products of the production industry of Borage (leaves), avocado (seeds) and pineapple (epicarp) contain large amounts of polyphenols and show a remarkable antioxidant capacity. In addition, these wastes may be material for the production of polyphenols such as acid rosmarinic and chlorogenic employing technologies appropriate for its refining. / Los vegetales y frutas son alimentos que poseen gran cantidad de compuestos beneficiosos para la salud, además de ser imprescindibles en la dieta. Entre sus componentes se encuentran las sustancias fenólicas. Estos compuestos poseen una gran actividad antiradicalaria y pueden ser utilizados para incrementar la estabilidad de los alimentos, ya que poseen la capacidad de evitar la peroxidación de los lípidos y pueden proteger incluso frente a la oxidación causada por cationes metálicos. Estos cationes favorecen la creación de especies reactivas de oxígeno, perjudiciales para la salud y pueden participar directa o indirectamente en la oxidación de los lípidos. La industria de los alimentos produce gran cantidad de subproductos y desperdicios, que se pueden utilizar para obtener sustancias de alto valor añadido, como antioxidantes naturales y sustitutos de los sintéticos. Además, este tipo de residuo posee un gran impacto ambiental debido a la gran carga orgánica y tiene un coste debido a manipulación, transporte y almacenamiento, entre otros procesos. En la presente Tesis Doctoral se ha estudiado la obtención de extractos naturales, ricos en polifenoles, a partir de tres desperdicios agroindustriales: Hojas de borraja, semilla de aguacate y epicarpio de piña. Se ha aplicado la extracción convencional, por lotes y en continuo, para obtener polifenoles a partir de estos residuos. Se han optimizado los procesos convencionales mediante el uso de la metodología de superficie de respuesta y se han aplicado las ecuaciones de transferencia de masa: Ley de Fick, teoría del film, empírica, Peleg y ley de velocidad, para obtener un modelo matemático representativo. La ecuación de la ley de Fick se ha usado para conseguir los coeficientes de difusión en las extracciones. Además se han aplicado técnicas novedosas, tales como, pulsos electromagnéticos y extracción asistida por ultrasonidos, para así mejorar y/o acelerar el proceso de extracción, lo que ha proporcionado un incremento de un 280% en la cantidad de componentes bioactivos obtenidos de la semilla de aguacate aplicando extracción asistida con ultrasonidos y un 520% de las hojas de borraja con la extracción mediante el uso de pulsos electromagnéticos. Con respecto a la cinética de extracción, el ajuste R2 de las ecuaciones estudiadas está entre un 0.81 y 0.99. Se han evaluado los extractos obtenidos en diferentes modelos de alimentos: emulsiones, carne picada y magdalenas, para determinar si aumentan la estabilidad y evitan o retardan la oxidación de las grasas y aceites. Se ha observado una disminución en la formación de los productos de la oxidación, tanto primaria como secundaria: entre un 80 y un 90% (hidroperóxidos) en las emulsiones, en un 90% los productos de oxidación secundaria en la carne picada con extractos de borraja y aguacate; y entre un 50 y un 90% en las madalenas con extracto de piña. Por lo tanto queda demostrado que se ha prolongado el tiempo de vida útil y ha aumentado la estabilidad frente a la oxidación. Como conclusión global, se ha demostrado que el uso de subproductos de la agroindustria es muy útil para la elaboración de extractos naturales que incorporan antioxidantes y es una materia prima importante que pasa de ser un residuo por el que se ha de pagar a ser un producto que permite obtener compuestos de alto valor añadido. Ralentizan la oxidación de las grasas en diversos modelos de alimentos. Los subproductos de la industria de la producción de borraja (hojas), aguacate (semillas) y piña (epicarpio) contienen gran cantidad de polifenoles y muestran una remarcable capacidad antioxidante. Además, estos desperdicios pueden ser material para la producción de polifenoles tales como el ácido rosmarínico y clorogénico empleando las tecnologías adecuadas para su refinación.

Tesi realitzada a l'Institut Hospital del Mar d'Investigacions Mèdiques (IMIM) / Cardiovascular diseases (CVD) are the principal cause of mortality worldwide. Lifestyle plays a crucial role in preventing the development of CVD, and one of its key elements is diet, which directly affects cardiometabolic health and cardiovascular risk. Given the complexity of diet and also of cardiovascular disease etiology, a lot of recent research into the association between diet and disease have focused on dietary patterns, as this is currently the most holistic way to study dietary habits in the population. In this work, we examined dietary patterns and their components that are associated with cardiometabolic health. Data were obtained from two population-based cross-sectional surveys conducted in Girona (Spain) in 2000 and 2005. The first survey included 3,058 randomly selected free-living men and women aged 25 to 74 years. The second survey included 6,352 men and women aged 35 to 80 years. These cohorts were re-examined in 2010 and 2012, with a follow up rate of 80.6% and 78.0%, respectively. At baseline and follow-up, we collected data on diet, using a validated food frequency questionnaire (FFQ), and on cardiovascular risk. We performed validation studies of a priori and a posteriori dietary patterns. In the a priori analysis, we found that a Diet Quality Index derived from a short Diet Quality Screener had good ability to predict future cardiovascular health complications. The a posteriori analysis showed good reproducibility and modest validity of dietary patterns defined using cluster analysis of the FFQ data. In a further step, we analysed the impact of energy under-reporting on dietary patterns and secular trends in dietary patterns. We concluded that energy under-reporting is a serious problem in nutritional epidemiology, especially when developing dietary guidelines for the general population, and more solid research is urgently needed in this area. To explore the association between diet and cardiovascular risk factors and CVD, we focused on abdominal obesity, a risk factor that had received increasing interest in recent years. Adherence to the Mediterranean diet and substitution of soft drinks with other caloric beverages, such as milk and juice, was associated with decreased abdominal adiposity. Finally, we studied the association between dairy products and cardiovascular events, and concluded that consumption of dairy products does not have an adverse impact on CVD incidence, and that frequent intake of yogurt was negatively associated with CVD incidence, presumably due to its prebiotic properties. In conclusion, this PhD research shows a smaller increase in waist circumference among individuals with high adherence to the Mediterranean diet, and the opposite for soft drink consumption. Furthermore, the results support the hypothesis that yogurt consumption is cardioprotective. Finally, it underlines the need to control for energy misreporting in epidemiological studies, and to validate dietary patterns. / En este trabajo, hemos examinado qué patrones dietéticos y cuáles de sus componentes están asociados con la salud cardiometabólica. Los datos se han obtenido a partir de dos estudios transversales de base poblacional realizados en España en 2000 y 2005. El primer estudio incluyó 3.058 y el segundo - 6.352 hombres y mujeres de 25 a 80 años de edad. Estas dos cohortes fueron re-examinadas en el año 2010 y 2012. Al inicio y al final del estudio se recogieron los datos dietéticos y los datos sobre los factores de riesgo cardiovascular. En el análisis a priori, encontramos que el Índice de Calidad Alimentaria derivado de un Cuestionario corto de Calidad de la Dieta tenía buena capacidad para predecir acontecimientos relacionados con la salud cardiovascular. El análisis a posteriori mostró una buena reproducibilidad y una validez modesta de los patrones dietéticos definidos mediante análisis de clústers de los datos de un cuestionario de frecuencia de alimentos. En el análisis del impacto de la declaración inferior de los niveles reales de energía consumida sobre los patrones dietéticos y las tendencias seculares en los mismos, llegamos a la conclusión de que la energía declarada por debajo de valores reales es un problema grave en la epidemiología nutricional, sobre todo en el desarrollo de guías alimentarias para la población en general. El seguimiento de una dieta mediterránea y la substitución de las bebidas gaseosas por otras bebidas calóricas, se asoció con una disminución de la adiposidad abdominal, un factor de riesgo de enfermedades cardiovasculares (ECV). Por último, llegamos a la conclusión de que el consumo frecuente de yogur se asoció negativamente con la incidencia de ECV. En conclusión, esta investigación doctoral muestra un menor aumento de la circunferencia de la cintura entre los individuos que siguen una dieta mediterránea, y lo contrario para el consumo de bebidas gaseosas. Además, los resultados apoyan la hipótesis de que el consumo de yogur actúa como cardioprotector. Por último, subraya la necesidad de controlar la declaración errónea del consumo de energía en los estudios epidemiológicos, y de validar los patrones dietéticos.

Encara que els gens de resistència a antibiòtics són una eina important per a la selecció i manteniment de plàsmids recombinants, les autoritats reguladores consideren el seu ús inacceptable en diverses àrees dins la indústria biotecnològica, sobretot quan el producte serà utilitzat amb finalitat terapèutica. Un sistema d’expressió basat en el vector pQE (Quiagen) amb un marcador de selecció plasmídic alternatiu (basat en l’auxotròfia de glicina) va ser desenvolupat prèviament (Vidal et al., 2008). El sistema d’expressió pQE es basa en el promotor T5, induïble per IPTG. La no-­‐ estabilitat del promotor en absència d’inductor pot provocar una inestabilitat estructural del vector, que portarà a nivells baixos d’expressió deguts, per exemple, a fenòmens de recombinació a la regió del promotor T5. El repressor lac, codificat pel gen lacI, s’uneix molt fortament al promotor, interferint en la transcripció del gen d’interès. Així doncs, els nivells transcripcionals del gen lacI tenen un paper crític en els sistemes d’expressió basats en el promotor T5, influenciant els nivells basals de transcripció i la concentració d’inductor. Encara que la sobreexpressió de la proteïna d’interès és un factor important en quant a càrrega metabòlica, també cal tenir en compte la contribució deguda a l’expressió d’altres gens plasmídics. En aquest sentit, per tal de millorar la robustesa del sistema i minimitzar la càrrega metabòlica relacionada amb els gens codificats als plàsmids, s’han eliminat tots els gens de resistència a antibiòtics i s’han afinat els nivells d’expressió del marcador auxotròfic (glyA) i del repressor lac (lacI). En aquest treball s’han construït uns cassettes d’expressió on els gens lacI i glyA estan sota el control d’un seguit de promotors constituents sintètics, per tal d’obtenir suficient inhibidor lacI per evitar la “no-­‐estabilitat del promotor” i assegurar el nivell mínim de transcripció de glyA necessaris tant per al manteniment del plàsmid com per al correcte creixement del cultiu en medi definit. A més, el gen de resistència a antibiòtic en el vector d’expressió va ser reemplaçat per la construcció glyA-­‐lacI, obtenint un sistema totalment lliure de gens per a la resistència a antibiòtic. Finalment, s’ha estudiat la capacitat per a la sobreexpressió de FucA recombinant a diferents escales (flascons agitats i bioreactor) i en diversos modes d’operació (discontinu i discontinu alimentat) per tal d’obtenir alts nivells d’expressió. Per últim, per tal d’analitzar la població bacteriana a nivell de cèl·∙lules individuals, els canvis morfològics (FSC i SSC) i les característiques físiques i bioquímiques de les diferents cèl·∙lules dins la població bacteriana, s’han realitzat anàlisis de citometria de flux. / While antibiotic resistance marker genes are a powerful system for selection and maintenance of recombinant plasmids in hosts such as E. coli, its use has been considered unacceptable in many areas of biotechnology by regulatory authorities, particularly when the recombinant product will be used in the therapeutic field. Previously, we have developed an expression system based on the pQE vector series (Qiagen) with an alternative plasmid selection marker based on glycine auxotrophy complementation (Vidal et al., 2008). The pQE expression system is based on the IPTG-­‐induced T5 promoter. Promoter leakiness in inducer absence may lead to structural instability of the expression vector, resulting in reduced expression levels, e.g. due to recombination events in the T5 promoter region. The lac repressor, encoded by the lacI gene, binds very tightly to the promoter, interfering with the transcription of the gene of interest. Therefore, lacI transcriptional level plays a key role in T5 promoter-­‐based expression systems, influencing the basal transcriptional levels and the concentration of inducer. Furthermore, although the overexpression of the plasmid-­‐encoded protein of interest is a major factor in the metabolic burden, expression of other plasmid-­‐genes may also contribute. Thus, in order to improve the system robustness, all the antibiotic resistance genes have been removed and the expression levels of the auxotrophic marker (glyA) and the lac repressor (lacI) genes have been fine-­‐tuned, in order to minimize the metabolic burden related to plasmid-­‐encoded genes. In this study, an expression cassette has been constructed where the lacI and glyA genes have been placed under the control of selected synthetic constitutive promoters in order to obtain the sufficient lacI inhibitor ensuring lack of “promoter leakiness” and the minimal glyA transcriptional levels needed for plasmid maintenance and optimal cell growth in defined media. Moreover, in the expression vector the antibiotic resistance gene was replaced by the lacI-­‐glyA cassette, thus yielding a completely antibiotic resistance gene free system. Finally, in order to obtain high expression levels of the protein of interest, the capacity for recombinant FucA overexpression has been investigated at different scales (shake flasks and bioreactors) and operation modes, batch and fed-­‐batch. Lastly, flow cytometry analyses were performed in order to analyze the bacterial populations at the single-­‐cell level: changes in morphology (FSC and SSC) and physical and biochemical characteristics of individual cells within a bacterial population.

La presente tesis realizó el análisis y evaluación de los métodos estadísticos para valorar la calidad del café. En este sentido, se dio un aporte para tratar de cuantificar y cualificar la calidad del café mediante las técnicas estadísticas abordados desde la estadística bayesiana, multivariada y del diseño experimental. Los métodos que se describen en cada uno de los estudios, muestran la relación y las diferencias de las diferentes técnicas empleadas. / This thesis was devoted to the analysis and evaluation of statistical methods to assess the quality of the coffee. In this sense, a contribution to try to quantify and qualify the coffee quality through statistical techniques addressed from Bayesian statistics, multivariate and experimental design were given. The methods described in each of the studies carried out, show the relationship and the differences of the different techniques used. / En aquesta tesi es va realitzar l’anàlisi i l’avaluació dels mètodes estadístics per a valorar la qualitat del cafè. En aquest sentit, es va tractar de quantificar i qualificar la qualitat del cafè mitjançant tècniques estadístiques abordades des de l’estadística Bayesiana multivariada i del disseny experimental. Els mètodes que es descriuen en cadascun dels estudis mostren la relació i les diferències de les distintes tècniques emprades.

Este trabalho foi realizado com o objetivo de caracterizar, em termos da constituição elementar, vinhos procedentes de diferentes regiões do estado do Rio Grande do Sul, Brasil, com ênfase na produção do Vale dos Vinhedos, estabelecendo se existem peculiaridades advindas da região em que o vinho é produzido, e que naturalmente estão relacionados ao ciclo de produção: solo, folha, uva e mosto. Além disto, estudar possíveis influências do processo de armazenamento do produto. Para tanto, foram utilizadas duas técnicas analíticas, PIXE e RBS. A análise por PIXE, realizada com o varietal Cabernet Sauvignon, mostrou que os vinhos, em geral, possuem elementos com número atômico entre 11 e 38, como Na, P, S, K, Ti, Mn, Fe, Cu, Zn, Br, Rb e Sr. Foram observadas algumas diferenças nas concentrações de alguns elementos, de acordo com a região de procedência, como, por exemplo, nas concentrações do Mn e do Rb. Rolhas também foram analisadas. Elementos como P, K, Ca, Ti, e Fe ficam retidos na parte da rolha que manteve contato com o vinho, sendo que este efeito é mais evidente para o Si, com relação aos demais elementos. Resultados referentes ao processo de cultivo e elaboração mostraram que o Cu é um elemento que apresenta grande variação de uma amostra para outra do ciclo de produção, com maior concentração na folha e no solo e menor concentração no vinho. Outros elementos como Ni e Rb também apresentaram variações no processo de vinificação. RBS foi usada na determinação da matriz do vinho e das rolhas, que é constituída, no caso do vinho, basicamente por C (68%) e O (32%). / In this work we studied the elemental concentration of wines from different regions of Rio Grande do Sul, Brazil, with principal focus in the production steming from Vale dos Vinhedos. Two analytical techniques were used, Particle- Induced X-Ray Emission (PIXE) and Rutherford Backscattering Spectrometry (RBS). The PIXE spectra of wine consisted typically of seventeen elements with atomic number between 11 and 38, as Na, P, S, K, Ti, Mn, Fe, Cu, Zn, Br, Rb and Sr , with some variations in the elemental concentrations between wines from different regions. We also analyzed the corks. The analysis of the internal side of the cork showed some elements stuck in the cork, as P, K, Ca, Ti and Fe. This result is more evident to Si element. Analysis of grape cultivation and wine processing samples showed Cu as an abundant element in the soil and leaves. Others elements showed variations in the vinification process, as Ni and Rb. The matrix composition of the wine was obtained by RBS. It revealed an amount of 32% of oxygen and 68% of carbon approximately.

O trabalho foi conduzido com uvas Cabernet Sauvignon do distrito de Pinto Bandeira, Bento Gonçalves, RS. Essa cultivar se destaca no Brasil pela produção de vinhos tintos de guarda, tendo-se adaptado relativamente bem na Serra Gaúcha. Como outras cultivares tintas, possue, em determinadas safras, dificuldades para obtenção de uma adequada maturação, dificultando a elaboração de vinhos com boa estrutura. Dentre as estratégias adotadas para minimizar esse problema, está a adoção de um adequado manejo do vinhedo e acompanhamento da maturação, aliados a operações enológicas, como controles na maceração e uso de insumos enológicos. Em função desse contexto objetivou-se estudar a aplicação de taninos enológicos e goma arábica para avaliar este uso na composição físico-química e qualidade sensorial do vinho Cabernet Sauvignon. Na safra 2004 foram usados taninos de quebracho e de castanheira, aplicados em três dosagens (5,0 g.hl-1 , 10,0 g.hL-1, 20,0 g.hl-1 ), e em três momentos de aplicação (maceração, 02 dias após o esmagamento; descuba, 08 dias após o esmagamento; após a fermentação malolática, 04 meses após o esmagamento). Na safra 2005 aplicaram-se os taninos de quebracho, de castanheira e de acácia negra, todos na dosagem de 10,0 g.hl-1 , aplicados na maceração. Ainda nessa safra, os vinhos foram tratados com quatro dosagens de goma arábica (0,0 g.L-1 , 1 ,O g.L-1 , 2,0 g.L-1 e 3,0 g.L-1 ) no ato do engarrafamento. Na safra 2006 aplicaram-se os três taninos testados na safra 2005, todos na dosagem de 20,0 g.hl-1 , durante a maceração, e mais um "pool" de quatro taninos (casca de uva, semente de uva, quebracho, carvalho), na dosagem de 10,0 IPT (Índice de Polifenóis Totais), durante a maceração e no vinho estabilizado. Ainda se testaram duas dosagens de goma arábica (0,0 g.L-1 e 3,0 g.L-1 ), aplicadas no momento do engarrafamento. Nos vinhos estabilizados, realizaram-se as análises físico-químicas clássicas, mais características cromáticas e polifenóis, além de uma análise sensorial detalhada. Em função das boas condições meteorológicas apresentadas nas safras estudadas, que levaram à obtenção de uvas de boa qualidade, no que conceme às características físico-químicas clássicas, de modo geral, se verificou que a adição dos taninos teve pouca influência na composição do vinho. Aquelas variáveis que seriam, hipoteticamente, mais influenciáveis, como antocianinas e índices de cor, mostraram, da mesma forma, poucas mudanças significativas. A goma arábica influenciou uma série de características, denotando que sua utilização afeta de maneira mais significativa a composição do vinho. Na análise sensorial, principalmente o perfil aromático dos vinhos foi influenciado pelo uso dos taninos enológicos, especialmente quando aplicados na maceração. Assim, parece ser prudente inferir que o uso associado de taninos de origens diversas, contribuiu de maneira mais efetiva para a qualidade final dos vinhos. A goma arábica, por sua vez, manteve a tendência de influir positivamente na qualidade gustativa dos vinhos, em especial no aumento da maciez e redução da adstringência. Mesmo que parcialmente, em função das diferentes safras, comprovou-se a expectativa que os taninos enológicos contribuem para aumentar a capacidade antioxidante dos vinhos. / The study was performed with Cabemet Sauvignon grapes from the Pinto Bandeira district, Bento Gonçalves, Rio Grande do Sul, Brazil, a cultivar known throughout the country for the production of cellaring red wine which adapted fairly well to the Serra Gaucha region. Like other red wine vines, there can be obstacles to proper maturation in certain harvests, which can curtail the production of well-structured wines. Among the strategies adopted to minimize the problem are using correct vineyard management and monitoring the maturation process, as well as enological operations such as contrais in maceration and the use of enological inputs. This situation has led to the study of the application of enological tannins and gum arabic to assess their use in the physicochemical composition and sensory properties of Cabernet Sauvignon wine. Quebracho and chestnut tannins were used in the 2004 harvest in three different dosages (5.0 g.hL-1, 10.0 g.hL-1, 20.0 g.hl-1 ) and at three different moments (maceration, 02 days after crushing; devatting, 08 days after crushing; after malolactic fermentation, 04 months after crushing). Quebracho, chestnut and black green-wattle acacia tannins were applied to the 2005 harvest, at 10.0 g.hL-1 dosages, ali during maceration . Wines from the same vintage were also treated with four dosages of gum arabic (0.0 g.L-1 , 1.0 g.L-1 , 2.0 g.L-1 and 3.0 g.L-1 ) at bottling. Three tannins tested on the 2005 harvest were applied to the 2006 one, ali at 20.0 g.hl-1 dosages during maceration, as well as a pool of four tannins (grape skins, grape pips, quebracho, oak) at dosages of 10.0 TPI (Total Polyphenollndex) during maceration and to the stabilized wine. Two gum arabic dosages were also tested (0.0 g.L-1 and 3.0 g.L-1 ), applied at bottling. The classical physicochemical analyses were performed on stabilized wines as well as chromatic characteristics, polyphenols and detailed sensory analysis. Due to the good weather conditions of the vintages studied, which led to the harvest of quality grapes regarding the classical physicochemical characteristics, tannin addition had little overall influence on wine composition. Those variables that would, hypothetically, be the most affected by their addition, such as anthocyanines and colar indexes, did not have significant changes either. Gum arabic influenced several characteristics, which shows that their use has more significant effects on wine composition. In sensory analysis, enological tannins primarily affected the aromatic profile of the wines, especially when applied in the maceration, so it seems safe to inter that the concomitant use of tannins from various origins contributed effectively to the final quality of the wines. Gum arabic, in turn, continued to have a positive effect on the palate of the wines, especially by increasing their softness and decreasing their astringency. The hypothesis that enological tannins contribute to the increase in antioxidant capacity in wines proved correct, even if only partially so due to the different harvests.

A qualidade e o desenvolvimento são duas questões complexas que se manifestam com veemência no Vale dos Vinhedos, principal área produtora de vinhos fmos no Brasil. A pesquisa, então, versa sobre a construção social da qualidade dos vinhos fmos e a dinâmica do desenvolvimento na microrregião, a qual foi demarcada para iniciar um processo de reconhecimento de indicações geográficas. Cada vez mais os mercados têm sido percebidos como construções sociais, em que os atores são capazes de criar produtos diferenciados. Nesse sentido, o objetivo do estudo centra-se na caracterização e na análise do Vale dos Vinhedos como um ambiente institucional e organizacional construido socialmente, com destaque a elementos da coordenação dos agentes. Dessa forma, a fundamentação do trabalho apoia-se na convergência e na complementaridade entre teorias referentes ao desenvolvimento, à qualidade e à construção social, em que a teoria das convenções denota o tema. A fim de que se cumpram os objetivos propostos, o método de pesquisa é do tipo exploratório com enfoque qualitativo, em que houve a seleção de uma amostra não probabilística composta por treze organizações e nove instituições relacionadas ao ambiente investigado. A apresentação dos dados e sua análise são realizadas com base nos modelos analítico e teórico que foram esquematizados. Assim, a atividade da vitivinicultura situa-se entre o homem e a natureza, presente em um contexto econômico, social, cultural, humano e ambiental. O V ale dos Vinhedos obteve prestígio ao ser pioneiro em conquistar a primeira indicação geográfica do Brasil. Nesse contexto, a microrregião mobiliza-se e vem experimentando conquistas, tais como melhora da qualidade dos vinhos, maior valor agregado aos produtos e ao território, e multiplicadores locais motivados pelo enoturismo. Dessa forma, a análise da coordenação dos atores permeia motivações, relevância, ações e cooperação, compondo competências, bem como possui valores de ordem conforme apregoa a teoria das convenções. Portanto, chega-se a conclusões de que agentes organizados, com destaque às instituições, compõem uma complexa rede de atores com capacidade de criar produtos mais sofisticados, à medida que ativam recursos que lhes são peculiares para qualificar os produtos e que dinamiza o desenvolvimento. Assim, a qualidade e o desenvolvimento revelam-se endogenamente, porém com motivação exógena, bem como a coordenação produz efeitos positivos e desafios. Por fim, a construção que promove acréscimo da qualidade e desenvolvimento, sobretudo assenta uma identidade por um saber-fazer local que também está sendo conhecido e afirmado. / The quality and development are two complex issues that present intensely in the V ale dos Vinhedos, to which is the main production area o f fine wines in Brazil. The focus of research is the social construction of quality fine wines and dynarnic development in the microregion that was demarcated for a process of recognition of geographical indications. Ever more markets are perceived as social constructions, in which actors are able to create differentiated products. Thus, the objective of this study is to characterize and analyze the Vale dos Vinhedos as an institutional and organizatioual environment that is socially constructed, based on elements of the coordination o f agents. So the research is based on the convergence and complementarity between theories of development, quality and social construction, principally by the theory of conventions. For the purpose o f reach the objectives, the method used is an exploratory and qualitative approach, in which there was the selection of a nonprobabilistic sample consists of thirteen organizations and nine institutions related to the environment investigated. The data presentation and analysis are performed on the basis on analytical and theoretical model defined. Therefore vitiviniculture is between man and nature, present in an economic, social, cultural, human and environmental. And the Vale dos Vinhedos eamed prestige for pioneering conquest of the frrst geographical indication of Brazil. In this context, microregion has experienced some successes which are improving quality o f wines, value-added products and the territory, also local multipliers, motivated by enotourism. The analysis of coordination of actors is engaged for motivations, relevance, actions and cooperation, forming abilities, but also has values of order, according to the theory o f conventions. Thus can be concluded that organized agents, with special emphasis on the role of institutions, form a complex network of actors with the ability to create more elaborate products when activate features that are peculiar them, to qualify products and it encourages development. Hence the quality and development are endogenously expressed, but the motivations are exogenous, yet the coordination cause positive effects and challenges. Fiually, the construction social that has promoted quality and development, particularly carries an identity by a local know-how which is also being known and affirmed.