Solving the mechanistic puzzle of gold-catalyzed cyclization of 1,6-enynes and beyond

La tesis se centra en el estudio del mecanismo de las reacciones de cicloisomerización de 1,6-eninos catalizada por complejos de oro(I). Evidencia experimental de la existencia de los intermedios propuestos se ha obtenido mediante la reacción de atrapamiento intermolecular de dichos intermedios, con alquenos, mediante la reacción de bisciclopropanación, y también por adición externa de nucleófilos. Por otra parte, la reacción de cicloadición [4 + 2] de aril eninos sustituidos fue estudiada. Esta reacción se ha utilizado como metodología clave para la síntesis de productos naturales como la familia de las picnanthuquinonas, También se ha estudiado el mecanismo de trasposición por rututa de enlace simple o doble y la formación de ciclobutenos en la reacción de 1,6-eninos aril substituidos.This Thesis is focused on the mechanism of the cycloisomerization reaction of 1,6-enyes catalyzed by gold(I) complexes. We obtained experimental evidence for the existence of the proposed intermediates by intermolecular trapping with alkenes, through a biscyclopropanation reaction and by reaction with external nucleophiles. On the other hand, the [4 + 2] cycloaddition reaction of substituted aryl enynes was studied. This reaction was used in an approach for the total synthesis of natural products such as the pycnanthuquinones. We have also studied the mechanism of the simple/double cleavage rearrangement and the formation of cyclobutenes in the reaction of aryl substituted 1,6-enynes. Moreover, new silver and copper complexes were prepared and structurally characterized. The new complexes were assayed in the cycloisomerization and cyclopropanation reactions. Metal-arene interactions were also studied

Mineral carbonation of ceramic brick at low pressure and room temperature. A simulation study for a superficial CO2 store using a common clay as sealing material

This research explores the possibilities of CO2 sequestration on ceramic bricks in a short time and at surface conditions. The experiment was carried out in a specially designed reaction chamber, filled with brick wastes and sealed with common clays. The brick used were composed of quartz, wollastonite, diopside, orthoclase and anhydrite, and the common clay was a marl composed of calcite, quartz, illite, smectite and kaolinite. Experimental condition in the reaction chamber were: reaction time 5 months, pressure of CO2 0.5 bar, 4:1 solid/water ratio. The experiment was followed by XRD, XRF, BET, physical sorption by N2 and CO2, Hg porosity, TG-DTA, SEM and ICP-EOS. After the CO2 treatment, wollastonite and anhydrite were practically destroyed and some diopside and orthoclase. Calcite precipitated as new phase (up to 48 wt%), and small amount of illite was the result of orthoclase alteration. Concerning the sealing clay, the CO2 produced an increment of calcite content (from 32 to 41 wt%) and a partial destruction of smectite, particularly close to the upper part of the brick layer. These results are hopeful in relation with the possible mineral carbonation of building ceramic waste in a short time at surface conditions, and open the opportunity to use those wastes for CO2 trapping in an appropriate system, as a quarry reclamation

Limitations of the Standard Procedure for the Evaluation of Marble and Limestone for Use in Construction: A Critical Analysis and Proposal for Modification

The selection of natural stone for each specific use must be based on determinations which assess their technical quality and ensure their suitability for the environment in which they will be utilized. Among the criteria to be considered, a petrographic characterization is basic to deduce the behaviour of the stone to external agents. Current European regulations for the valuation and use of a stone present serious constraints in connection with the petrographic characterization and with the definition of some of the technological tests, which can endanger the suitability of a stone for use in construction. Two commercial limestones and one marble, are studied following the existing European Standards, to explain reasonably the behavior of these materials and deduce the most appropriate uses. Finally the results lead to certain recommendations to modify existing regulations

On the Influence of Shade in Improving Thermal Comfort in Courtyards

This study analyzes the thermal performance of courtyards in traditional buildings in the city center of Córdoba (South of Spain), one of them displaying a shading component, to determine the influence of this precise element. The courtyards have been monitored simultaneously during a summer period when temperatures during the day reached over 45 °C. The obtained data was contrasted, and we confirmed that the shading element provided an improvement of the thermal performance of the courtyard which doubled the thermal leap between outdoor and inside the courtyard temperatures when the shading element was installed, in comparison to the courtyard without shade. Therefore, the tempering effect of courtyards can be significantly improved by means of using these simple elements

The Principles of Gold-Catalyzed Molecular Gymnastics

L'or(I) dirigeix el contorsionisme molecular mitjançant reaccions intramoleculars i intermoleculars d'enins per mitjà d'intermedis ciclopropil o carbens altament distorsionats. La síntesi de productes naturals com (+)-orienalol F i (-)-englerin A mostra l'estat d'art de la catàlisi de l'or(I) per a construir complexitat molecular.Gold(I) orchestrates molecular gymnastics by intraand intermolecular reaction of enymes via highly distorted cyclopropyl gold carbenes as intermediates. The synthesis of natural products such as (+)-orientalol F and (−)-englerin A illustrates the state of the art of gold (I) catalysis for the buildup of molecular complexity

Las tiendas de mercadería y pulperías en la ciudad de Santafé, Nuevo Reino de Granada, 1772-1810

Las tiendas de mercadería y pulperías fueron los espacios comerciales predilectos por los habitantes de la ciudad de Santafé de Bogotá (1772-1810) para abastecerse de productos de primera necesidad como de bienes de lujo. Por esto este artículo se propone explorar las mercancías que se podían encontrar enlos estantes y mostradores de estos dos tipos de tiendas, también con el propósito de fijar una posible diferenciación entre estos espacios. Asimismo, se caracterizan a los propietarios y la inversión que debían realizar para poder establecer o mantener sus tiendas abiertas al público. Por último, se establece una cartografía comercial de las tiendas de mercadería y pulperías al interior de la ciudad.Palabras clave: Tiendas de mercadería, pulperías, comercio menudo, cartografía comercial, Santafé, siglo XVIII.***********************************The merchandise stores and grocery stores in the city of Santafé, Nuevo Reino de Granada, 1772-1810.AbstractThe merchandise stores and pulperías were the favorite commercial spaces for the inhabitants of the city of Santafé de Bogotá (1772-1810) to stock up on basic necessities as well as luxury goods. For this reason, this article aims to explore the merchandise that could be found on the shelves and counters ofthese two types of stores, also with the purpose of establishing a possible differentiation between these spaces. Likewise, the owners and the investment they had to make in order to establish or keep their stores open to the public are characterized. Finally, a commercial cartography of the merchandise storesand pulperías in the interior of the city is established.Key words: Merchandise stores, pulperías, small commerce, commercial cartography, Santafé, XVIII century.***********************************As lojas de mercadorias e pulperías na cidade de Santafé, Nuevo Reino de Granada, 1772-1810.ResumoAs lojas de mercadorias e pulperías eram os espaços comerciais preferidos dos habitantes da cidade de Santafé de Bogotá (1772-1810) para estocar tanto mercadorias de primeira necessidade como de luxo. Por esta razão, este artigo se propõe a explorar a mercadoria que poderia ser encontrada nas prateleiras e balcões destes dois tipos de lojas, também com o objetivo de estabelecer uma possível diferenciação entre estes espaços. Também caracteriza os proprietários e o investimento que tiveram que fazer para estabelecer ou manter suas lojas abertas ao público. Finalmente, é estabelecida uma cartografia comercial das lojas de mercadorias e pulperías dentro da cidade.Palavras-chave: Merchandise shops, pulperías, pequeno comércio, cartografia comercial, Santafé, século XVIII

Development of a detection method for butyric spores based on Real-Time PCR and biomagnetic separation in milk

La hinchazón tardía (HT) se produce en quesos de pasta dura y semi-dura como consecuencia de la fermentación producida por bacterias butíricas, dando lugar a cambios en el sabor y aroma, y a la aparición de agujeros o roturas. Los quesos afectados por HT no pueden ser comercializados lo que produce perdidas económicas en el sector de la industria láctea. Normalmente, estos quesos se destinan a queso rallado. Las bacterias acido butíricas son Gram-positivas y esporuladas. Sus esporos, la forma de resistencia de estos microorganismos, son muy difíciles de eliminar completamente de la leche cruda porque pueden sobrevivir a una gran variedad de tratamientos.Actualmente, no existe un método rápido para detectar las bacterias butíricas en leche cruda, que permita decidir el destino final de la leche contaminada para elaborar diferentes productos lácteos, excepto para quesos de pasta dura y semi-dura en los que tiene lugar la HT. El método de rutina para detectar las bacterias butíricas es el Numero Mas Probable (NMP), en el cual los resultados se obtienen entre dos y siete días. Por esta razón, métodos mas rápidos y específicos alternativos al NMP, son necesarios para prevenir la HT y reducir las perdidas económicas en la industria láctea.Aunque diferentes bacterias butíricas pueden producir la HT, C. tyrobutyricum es considerado como el principal agente causante. El principal objetivo de esta tesis doctoral ha consistido en elaborar un método de detección para esporos de C. tyrobutyricum en leche cruda basado en PCR a tiempo real (qPCR) y bioseparación magnética.La primera parte de esta tesis doctoral ha consistido en el cribado de diferentes métodos de ruptura de esporos de C. tyrobutyricum para obtener ADN genómico puro. Disponer de un método efectivo resulta clave para obtener suficiente cantidad de ADN para su análisis mediante qPCR, considerando la resistencia de los esporos en comparación con las células vegetativas. Teniendo en cuenta los resultados obtenidos, se seleccionaron los tratamientos por microondas (MW) y bead beating (BB), seguidos de una purificación del ADN mediante columna de sílica. Para recuperar los esporos, laleche se trato con subtilisina y después de una centrifugación, se aplicaron los tratamientos de MW y BB al precipitado de esporos para su posterior análisis mediante qPCR. Finalmente, el tratamiento de MW fue seleccionado como el mas apto en leche UHT como paso previo a la detección de los esporos de C. tyrobutyricum mediante qPCR.Aunque el tratamiento de MW y la posterior purificación del ADN mediante columna, se selecciono como el mejor método a aplicar en tampón acuoso (PBS) y leche UHT; la aplicación de este método en leches crudas procedentes de laboratorios de control de calidad de la leche españoles, dio una baja precisión en el ensayo de qPCR.Por esta razón, se evaluó un tercer método basado en un kit comercial y que precisa de un sistema automatizado denominado King Fisher (KF) Duo Prime System.La curva de calibración realizada con el método KF permitió conseguir valores de Ct y un limite de detección (LD) mas bajo en el ensayo de qPCR que el tratamiento de MW, como paso previo a la purificación del ADN con columna. Después de esta verificación, 202 muestras de vaca, oveja y cabra, procedentes de tres zonas geográficas españolas diferentes, fueron analizadas para determinar la concentración de esporos de C. tyrobutyricum. Los valores de concentración de esporos se encontraron en el rango de 102-103 esporos/mL, aunque un importante numero de las muestras analizadas seencontraron por debajo del LD y no pudieron ser cuantificadas. Por esta razón, aunque la precisión de la qPCR mejoro claramente al aplicar el método de KF, este método se ha considerado como cualitativo.Para verificar la presencia de C. tyrobutyricum en las muestras de leche cruda, estas fueron cultivadas en un medio selectivo para bacterias butíricas que contiene Dcicloserina y rojo neutro. Las colonias fueron analizadas mediante PCR multiplex y secuenciación del 16S rADN. Los resultados mostraron la presencia de otros microorganismos, como Lactobacillus y Paenibacillus, que también podían crecer en este medio. C. tyrobutyricum solo fue aislado en leche de vaca, y otras especies como C. sporogenes y C. perfringens, fueron identificadas, sugiriendo que también podrían contribuir a la HT. Ademas, las especies de Clostridium identificadas, fueron diferentes dependiendo del tipo de leche analizada (vaca, oveja o cabra).La segunda parte de esta tesis se ha centrado en el desarrollo de un sistema de captura de esporos basado en partículas magnéticas funcionalizadas con ligandos afines.Los resultados del análisis mediante qPCR de las leches crudas, había revelado que muchas de las muestras se encontraban por debajo del LD. Por esta razón, un paso de captura de los esporos directamente en la leche, podría ser una buena estrategia para mejorar la detección. Con este objetivo, se evaluó el péptido pCZS1 obtenido mediante la técnica de Phage Display y afín por los esporos de C. tyrobutyricum. En primer lugar, la afinidad del péptido pCZS1 por los esporos butíricos, se analizo mediante Calorimetría de Titulación Isotérmica (CTI) y citometría de flujo. Después, se evaluó la capacidad de captura de esporos de C. tyrobutyricum en PBS y leche UHT por partículas magnéticas funcionalizadas con pCZS1. Los resultados de la captura fueron positivos en PBS y en leche UHT tratada con subtilisina. Sin embargo, el principal objetivo, consistía en aplicar la captura de los esporos directamente en leche, por lo que se evaluaron otros ligandos con el fin de conseguir este objetivo.La proteina G3P transmembrana del fago M13, se expreso unida al péptido pCZS1 en el extremo N-terminal de la proteína para incrementar la exposición del péptido a los esporos. Primero, se expreso solo el dominio soluble de la proteína G3P junto con pCZS1, denominándola como tG3P. Después, se diseño una segunda proteína formada por la G3P completa y el péptido pCZS1, denominándola cG3P. Ambas proteínas fueron expresadas en Escherichia coli y purificadas mediante cromatografía de afinidad aplicando un método para solubilizar los cuerpos de inclusión. La proteína tG3P tuvo mejor rendimiento que la cG3P, probablemente porque la parte transmembrana pudiera afectar al proceso de purificación. La proteína tG3P fueevaluada mediante CTI y citometría de flujo confirmando su afinidad por los esporos butíricos. La evaluación mediante CTI de la proteína cG3P, obtuvo un valor de Kd muy similar al obtenido para la proteína tG3P. Sin embargo, debido a los problemas de estabilidad de la proteína cG3P, las partículas magnéticas solo se funcionalizaron con la proteina tG3P. Los esporos de C. tyrobutyricum fueron capturados con las partículas magnéticas funcionalizadas con tG3P, pero se obtuvieron valores muy bajos de captura tanto en PBS como en leche UHT. Por esta razón, se concluyo que la proteína G3P no aportaba ninguna ventaja frente al péptido pCZS1.La ultima parte de esta tesis se ha centrado en la purificación de anticuerpos policlonales procedentes del suero de conejos inmunizados con esporos de C. tyrobutyricum. Después de la purificación, los anticuerpos se unieron a partículasmagnéticas con proteína A y G. La eficiencia de captura de los esporos con las partículas funcionalizadas con los anticuerpos se evaluó en PBS y leche UHT, obteniendo valores de recuperación cercanos al 90%. Como aplicación final, la inmunocaptura se realizo en leche cruda y después se realizo el ensayo de qPCR para detectar los esporos. La extracción del ADN de los esporos se realizo mediante tratamiento de MW y purificación en columna. Las partículas con proteína A, proporcionaron valores de Ct y porcentajes de amplificación mejores que los ensayos realizados con las partículas con proteína G.Estos resultados confirman que la immunocaptura de esporos de C. tyrobutyricum directamente en leche y el posterior análisis mediante qPCR son posibles.Los resultados obtenidos en esta tesis sientan las bases para el desarrollo de un método de detección de esporos butíricos en leche cruda mediante qPCR revelando los puntos críticos. Ademas, se propone un sistema basado en la inmunocaptura de esporos con partículas magnéticas funcionalizadas con anticuerpos para recuperar los esporos de C. tyrobutyricum de la leche cruda y para su posterior detección mediante qPCR.Late blowing defect (LBD) is produced in hard and semi-hard cheeses due to the fermentation produced by butyric bacteria, leading into changes on flavor, smell and also to the appearance of cracks. The cheeses with LBD cannot be commercialized, which produces important economic loses for dairy industry. Normally, these cheeses are destinated to melted cheese. Butyric bacteria are Gram positive and sporulated, and spores, the resistance form of these microorganisms, are very difficult to remove completely from raw milk because they can survive to several treatments. Nowadays, there is not a fast method to detect butyric bacteria in raw milk, which would allow to use contaminated raw milk in different dairy products except for hard or semi-hard cheeses in which LBD can take place. The routine microbiological method used to detect butyric bacteria in raw milk is the Most Probable Number (MPN), which provides results between two and seven days. For this reason, faster and more specific methods alternative to MPN method are needed to prevent LBD and reduce economic losses in dairy industry. Although different species of butyric bacteria can produce LBD, C. tyrobutyricum is considered the main causative agent. The main objective of this thesis has been to develop a detection method for C. tyrobutyricum spores in raw milk based on Real-Time PCR and biomagnetic separation. The first part of this thesis has consisted on the screening of different disruption methods to obtain pure genomic DNA from C. tyrobutyricum spores. An effective disruption is a key point to have enough DNA for qPCR analysis taking into account the resistance of spores in comparison with vegetative cells. From the results obtained, microwaves (MW) treatment and bead beating (BB) followed by column purification were selected. To facilitate the recovery of spores, milk was treated with subtilisin and after centrifugation, MW and BB treatment were applied to the precipitate of spores for further analysis by qPCR. Finally, MW treatment was found successful in UHT milk as a previous step to the qPCR analysis to detect C. tyrobutyricum spores. Although MW treatment followed by column purification of DNA was found as the best choice in an aqueous buffer and UHT milk, the application of this method in cow raw milk samples coming from Spanish laboratories of milk quality control, gave results with low precision by qPCR. For this reason, a third method was evaluated based on a commercial kit and automated system named King Fisher (KF) Duo Prime System. This method disrupts the spores based on BB and the released DNA is recovered with magnetic particles. The calibration curve made with the KF method allowed to achieve better LOD and lower Ct by qPCR analysis than MW and column purification. After this verification step, 202 samples of cow, ewe and goat from three different geographical regions of Spain were analyzed to determine C. tyrobutyricum spore concentration. The spore levels found were in the range 10^2-10^3 spores/mL, although an important number of samples were below the LOD and could not be quantified. For this reason, this method was considered as qualitative, though qPCR precision was clearly improved by applying the KF method. To verify C. tyrobutyricum presence in raw milk samples, they were cultured in a selective media for butyric bacteria composed of RCM with D-cycloserine and neutral red. The grown colonies were analyzed by multiplex PCR and 16S rDNA sequencing. The results obtained showed that other microorganisms, such as Lactobacillus and Paenibacillus can grow in the selective medium. C. tyrobutyricum was only isolated in cow milk and other species, such as C. sporogenes and C. perfringens, were identified suggesting that they could contribute to LBD in cheese. Moreover, the identified species of Clostridium were different depending on the milk type analyzed (cow, ewe or goat). The second part of this thesis was focused on the development of a capture system based on magnetic particles functionalized with affine ligands. The results from qPCR analysis revealed that most of the samples analyzed were below the LOD, for this reason a step of spore capture directly from milk could be a good strategy to improve their detection. With this aim, the pCZS1 peptide obtained by Phage Display technique as affine for C. tyrobutyricum spores was evaluated. First, the affinity of pCZS1 for butyric spores was checked by isothermal titration calorimetry (ITC) and flow cytometry. After that, particles were functionalized with pCZS1 and C. tyrobutyricum spore capture was done in PBS and UHT milk. Positive results were achieved only in PBS and UHT milk treated with subtilisin. However, as the main objective was to apply the spore capture directly in milk, other ligands were assayed to achieve this goal. The transmembrane G3P protein from M13 phage was expressed with pCZS1 attached to the N-terminal end to increase the exposition of the peptide to the spores. First, only the soluble domain of G3P, named as tG3P, together with pCZS1 were expressed. A second ligand was designed based on the complete structure of tG3P together with pCZS1, named as cG3P, and pCZS1. Both proteins were expressed in Escherichia coli cells and purified by affinity chromatography by applying a protocol to solubilize inclusion bodies. tG3P protein provided better yields than cG3P protein probably because the transmembrane domain affects the purification process. tG3P was evaluated by ITC and flow cytometry confirming its affinity for butyric spores. cG3P was only evaluated by ITC with a similar Kd value in comparison with tG3P but due to the stability problems of tG3P, only tG3P was bound to magnetic particles. C. tyrobutyricum spores were captured with particles functionalized with tG3P reporting low capture values in PBS and milk with no successful results. For this reason it was concluded that G3P protein did not provide any advantage over pCZS1. The last part of this thesis has focused on the purification of polyclonal antibodies from the serum of rabbits immunized with C. tyrobutyricum spores. After the purification, Protein A and Protein G magnetic particles were functionalized with the specific antibodies. The capture efficiency for the spores was evaluated in PBS and UHT milk with both particles providing rates close to 90%. As a final application, the immunocapture was done in raw milk followed by qPCR detection. The DNA extraction from spores was made by MW treatment followed by column purification. Protein A particles reported better values in terms of amplification rates and Ct values than Protein G particles, confirming that the immunocapture of C. tyrobutyricum spores directly from milk, followed by qPCR detection is possible. The results obtained in this thesis set up the basis to develop a detection method for C. tyrobutyricum spores in raw milk by qPCR revealing the critical points. Furthermore, an immunocapture system based on magnetic particles coated with antibodies is proposed to recover C. tyrobutyricum spores from raw milk for further qPCR detection. <br /

Thermodynamic performance enhancement of courtyards using a shading device

The design of more sustainable buildings is one of the main concerns of our society given the necessity to reduce energy consumption to reduce climate change. Incorporate passive strategies in buildings should be the first step because of its zero energy consumption. One of the most used passive strategies in the Mediterranean Climate is the use of inner courtyards as a tempering element of buildings. The performance of these courtyards has been traditionally improved by installing shading devices such as canvas. The aim of this study is to quantify the effect of the shading element installed in two courtyards with different geometries in the south of Spain by monitoring the temperature in the courtyards and the outdoor during warm and extremely hot day