Tesis por compendio / [EN] Dehydration is one of the most commonly used operations in the food industry, and although its aim is to extend the shelf life of foods by reducing their water activity, it could also involve quality degradation. Vacuum freeze-drying may be considered one of the best drying methods for the purposes of preserving the organoleptic and nutritional properties of the fresh product, but its high processing cost limits its use to high value-added products. Convective drying at low temperatures could be considered an alternative means of obtaining high quality products at lower cost. However, the low drying rates at low temperatures (T<20ºC) and atmospheric pressure makes its industrial application difficult. In this sense, high intensity ultrasound (US) has been used to intensify mass transfer phenomena in food processing. It could be of great interest to apply US in low temperature drying because the ultrasonic effects are mainly mechanical (non-thermal). In this context, the main aim of this thesis was to determine the feasibility of US application in low temperature drying, addressing the effect on both the drying kinetics and the quality of the obtained products. For this purpose, apple, eggplant and cod samples were dried at different temperatures (-10, -5, 0, 5 and 10ºC), air velocities (1, 2, 4 and 6 m/s) and applying different ultrasonic powers (0, 25, 50 and 75 W). Diffusion models were used to describe the drying kinetics and to quantify the influence of the process variables. Moreover, different quality parameters (rehydration capacity, texture, antioxidant capacity...) of the dried products were determined. The application of US significantly (p<0.05) shortened the drying time under every drying condition and with each product tested, reducing the drying time by up to 80, 87 and 60% in apple, eggplant and cod samples, respectively. Thus, the greater the ultrasonic power applied, the shorter the drying time. The drying temperature and air velocity influenced the US efficiency and the best performance was achieved at the lowest drying temperatures and air velocities. In general terms, the diffusion model adequately fitted the drying kinetics of the three products tested. Although, in the case of US assisted drying, a better fit of the experimental data was obtained when the external resistance to water transfer was considered. The URIF (Uniformly Retreating Ice Front) model successfully fitted the atmospheric freeze drying kinetics. This model was validated under different experimental conditions. As regards the effect of the process variables on the quality parameters, in overall terms, it was observed that neither the US application nor the air velocity greatly influenced the quality of the obtained products. However, the temperature affected some quality parameters, such as rehydration capacity and color, especially at temperatures below the samples' freezing point. Finally, as a technology employed for the purposes of obtaining porous food matrices to be used further in the development of functional foods, US-assisted low temperature drying could be considered of great potential. Thus, from dried apple samples impregnated with olive leaf extract, it was observed that US application during drying did not significantly (p<0.05) influence the infusion capacity but did increase the antioxidant capacity of the final product. Therefore, high intensity ultrasound could be considered an interesting technology with which to speed-up the low temperature drying processes without greatly affecting the quality of the dried product. / [ES] La deshidratación, una de las operaciones más utilizadas en la industria agroalimentaria, mejora la estabilidad de los alimentos al reducir su actividad de agua, aunque puede afectar a su calidad. Entre las diferentes técnicas de secado existentes, destaca la liofilización a vacío por ser una de las que mejor conservan las propiedades organolépticas y nutricionales de los productos. Sin embargo, esta operación resulta muy cara y sólo se utiliza en productos de alto valor añadido. El secado convectivo a baja temperatura (T<20ºC) representa una alternativa para obtener productos de alta calidad a menor coste aunque su baja velocidad de proceso dificulta su implementación a nivel industrial. En este sentido, los ultrasonidos de alta intensidad (US) se han aplicado para intensificar operaciones de transferencia de materia en diferentes procesos agroalimentarios. Sus efectos son principalmente mecánicos (no térmicos), por lo que su uso en el secado a baja temperatura resulta altamente interesante. En este contexto, el objetivo general de la presente tesis doctoral fue determinar la viabilidad de la aplicación de US en procesos de secado a baja temperatura, abordando tanto su efecto en la cinética como en la calidad de los productos obtenidos. Para ello, se deshidrataron muestras de manzana, berenjena y bacalao a diferentes temperaturas (-10, -5, 0, 5 y 10ºC) y velocidades de aire (1, 2, 4 y 6 m/s) y aplicando diferentes niveles de potencia acústica (0, 25, 50 y 75 W). Se utilizaron modelos difusivos para describir las cinéticas de secado y cuantificar la influencia de las variables de proceso. Además, se determinaron diferentes parámetros de calidad (capacidad de rehidratación, textura, capacidad antioxidante,¿) de los productos deshidratados. La aplicación de US permitió reducir significativamente (p<0.05) el tiempo de secado en todas las condiciones experimentales y productos analizados, obteniendo reducciones de tiempo de secado de hasta el 80, 87 y 60% en manzana, berenjena y bacalao, respectivamente. La reducción del tiempo de secado fue mayor cuanto mayor fue la potencia acústica aplicada. La temperatura y la velocidad del aire de secado influyeron en la efectividad de la aplicación de US, siendo mayor el efecto de los US a las temperaturas y velocidades más bajas. En general, la teoría difusional describió adecuadamente la cinética de secado de los tres productos estudiados. En las experiencias con aplicación de US se obtuvo un mejor ajuste a los datos experimentales cuando se consideró la resistencia externa en el modelo. Asimismo, en condiciones de liofilización a presión atmosférica, el modelo URIF (Uniformly Retreating Ice Front) se ajustó adecuadamente a los datos experimentales. Además, este modelo se validó en diferentes condiciones experimentales. Respecto al efecto de las variables de proceso en los parámetros de calidad, en general, se observó que ni la aplicación de US ni la velocidad de aire influyeron de manera importante en la calidad de los productos obtenidos. En cambio, la temperatura afectó de manera relevante a parámetros como la capacidad de rehidratación y el color, especialmente a temperaturas por debajo del punto de congelación de las muestras. Por otro lado, el secado a baja temperatura asistido con US tiene un alto potencial para la obtención de matrices porosas alimentarias para su posterior utilización en el desarrollo de alimentos funcionales. Así, en muestras de manzana deshidratada e impregnada con extracto de hoja de olivo, se observó que la aplicación de US durante el secado no afectó significativamente (p<0.05) a la capacidad de impregnación, pero sí incrementó la capacidad antioxidante del producto obtenido. Por lo tanto, los ultrasonidos de alta intensidad se pueden considerar como una tecnología interesante para acelerar los procesos de secado a baja temperatura sin afectar en gran medida a la calidad del producto obtenido. / [CA] La deshidratació, una de les operacions més utilitzades en la indústria agroalimentària, millora l'estabilitat dels aliments en reduir la seua activitat d'aigua, encara que pot afectar-ne la qualitat. Entre les diferents tècniques d'assecatge que hi ha, destaca la liofilització al buit per ser una de les que millor conserven les propietats organolèptiques i nutricionals dels productes, però resulta molt cara i només s'utilitza en productes d'alt valor afegit. L'assecatge convectiu a baixa temperatura (T<20ºC) representa una alternativa per a obtenir productes d'alta qualitat a menor cost. No obstant això, la baixa velocitat d'assecatge dificulta la seua implementació a nivell industrial. En aquest sentit, els ultrasons d'alta intensitat (US) s'han aplicat per a intensificar operacions de transferència de matèria en diferents processos agroalimentaris. El seu ús en l'assecatge a baixa temperatura resulta altament interessant pel fet que els seus efectes són principalment mecànics (no tèrmics). En aquest context, l'objectiu general de la present tesi doctoral va ser determinar la viabilitat de l'aplicació d'US en processos d'assecatge a baixa temperatura, abordant tant l'efecte en la cinètica del procés com en la qualitat dels productes obtinguts. Amb aquesta finalitat, es van deshidratar mostres de poma, albergínia i bacallà a diferents temperatures (-10, -5, 0, 5 i 10ºC) i velocitats d'aire (1, 2, 4 i 6 m/s) i aplicant diferents nivells de potència acústica (0, 25, 50 i 75 W). Es van utilitzar models difusius per a descriure les cinètiques de l'assecatge i quantificar la influència de les variables del procés. A més, es van determinar diferents paràmetres de qualitat (capacitat de rehidratació, textura, capacitat antioxidant...) dels productes deshidratats. L'aplicació d'US va permetre reduir significativament (p<0.05) el temps d'assecatge en totes les condicions experimentals i tots els productes analitzats; es van obtenir reduccions de temps de fins al 80%, 87% i 60% en pomes, albergínies i bacallà, respectivament. La reducció del temps d'assecatge va ser més alta com més alta va ser la potència acústica aplicada. La temperatura i la velocitat de l'aire de l'assecatge van influir en l'efectivitat de l'aplicació d'US, sent major l'efecte dels US a les temperatures i velocitats més baixes. En general, la teoria difusional va descriure adequadament la cinètica de l'assecatge dels tres productes estudiats. En les experiències amb aplicació d'US es va obtenir un millor ajust a les dades experimentals quan es va considerar la resistència externa en el model. Així mateix, en condicions de liofilització a pressió atmosfèrica, el model URIF (Uniformly Retreating Ice Front) es va ajustar correctament a les dades experimentals. A més, aquest model es va validar en diferents condicions experimentals. Respecte a l'efecte de les variables del procés en els paràmetres de qualitat, en general, es va observar que ni l'aplicació d'US ni la velocitat de l'aire van influir de manera important en la qualitat dels productes obtinguts. En canvi, la temperatura va afectar de manera rellevant a paràmetres com la capacitat de rehidratació i el color, especialment a temperatures per davall del punt de congelació de les mostres. D'altra banda, l'assecatge a baixa temperatura assistit amb US presenta un alt potencial per a obtenir matrius poroses alimentàries per a la posterior utilització en el desenvolupament d'aliments funcionals. Així, en mostres de poma deshidratada i impregnada amb extracte de fulles d'olivera, es va observar que l'aplicació d'US durant l'assecatge no va afectar significativament (p<0.05) la capacitat d'impregnació, però sí que va incrementar la capacitat antioxidant del producte obtingut. Per tant, els ultrasons d'alta intensitat es poden considerar com una tecnologia interessant per a accelerar els processos d'assecatge a baixa temperatura sense afectar de manera re / Santacatalina Bonet, JV. (2016). Contribución al estudio de la aplicación de ultrasonidos de alta intensidad en procesos de secado a baja temperatura [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61484 / Compendio

Living cells accumulate compensatory solutes for protection against the harmful effects of extreme environmental conditions such as high salinity, temperature and desiccation. Even at high concentrations these solutes do not disrupt the normal cellular functions and at times counteract by stabilizing the cellular components. These properties of compensatory solutes have been exploited for stabilizing proteins and cells in vitro. Betaines are widespread natural compensatory solutes that have also been used in other applications such as therapeutic agents and polymerase chain reaction (PCR) enhancers. Some biomedical applications of novel synthetic analogues of natural betaines were investigated. Natural compensatory solutes are either dipolar zwitterionic compounds or polyhydroxyl compounds, and the physical basis of compensation may differ between these, so one focus was on synthetic betaines with hydroxyl substituents. The majority of the synthetic solutes stabilized different model proteins against stress factors such as high and low temperatures. The presence of hydroxyl groups improved protection against desiccation. The observed stabilization effect is not just on the catalytic activity of the enzyme, but also on its structural conformation. Synthetic compensatory solutes have a potential application as protein stabilizers. Dimethylthetin was evaluated as a therapeutic agent and found to be harmful in a sheep model. However, from the study we were able to generate a large-animal continuous ambulatory peritoneal dialysis (CAPD) model and showed that glycine betaine could be added to the dialysis fluid in chronic renal failure. Some synthetic compensatory solutes reduce the melting temperatures of DNA better than most natural solutes. Synthetic solutes were identified that have potential to enhance PCR and could replace some reagents marketed by commercial suppliers. Density, viscosity and molecular model data on the solutes showed correlations with the biochemical effects of the solutes, but no physical measurements were found that reliably predicted their potential for biotechnological applications.

Cortical malformations as a result of altered development are a common cause of human epilepsy. The cellular mechanisms that render neurons of malformed cortex epileptogenic remain unclear. Using a rat model of the malformation of microgyria, a previous study showed an alteration in the number of immunocytochemically-identified parvalbumin cells, a GABAergic inhibitory interneurons subtype (Rosen et al., 1998). A second study showed no change in the total number of GABAergic neurons (Schwarz et al., 2000). Consequently, we hypothesize that interneuron subtypes are differentially affected by maldevelopment. The present study investigated (1) whether interneuron subtype identity is retained in malformed cortex, based on chemical content, and (2) whether the proportion of three chemical subtypes is altered in malformed cortex. Here we demonstrate that three non-overlapping subtype markers remain non-overlapping in malformed cortex, but show altered distributions. These findings suggest that an increase in one subpopulation of interneurons may compensate for a corresponding decrease in a second subset.

L’apport de sédiments fins au cours d’eau est un phénomène naturel mais qui sous les effets de l’anthropisation peut devenir excessif et conduire à perturber le fonctionnement de l’hydrosystème. Cet apport excessif provoque le colmatage du lit des rivières et peut perturber les échanges hydriques, les processus biogéochimiques et les communautés d’invertébrés benthiques et hyporhéiques. D’un point de vue méthodologique, la mesure de la conductivité hydraulique dans le substrat permet d’obtenir une image du niveau de colmatage de la zone hyporhéique et le pompage Bou Rouch permet d’étudier qualitativement la faune hyporhéique. D’un point de vue biologique, les effets du colmatage sont un peu plus importants pour l’hyporhéos que pour le benthos notamment au niveau de la richesse taxonomique, des assemblages faunistiques et des traits biologiques / Fine sediment input to the streambeds is a natural phenomenon but in addition to the effects of human impacts may become excessive and lead to disruption of the functioning of the hydrosystem. This excessive intake causes the clogging of river beds and can disrupt water exchange, biogeochemical processes and benthic and hyporheic invertebrate communities. From a methodological point of view, measurement of hydraulic conductivity in the substratum provides a realistic picture of the clogging level of the hyporheic zone and the Bou Rouch pump a good sampling for a qualitative study of the hyporheic fauna. From a biological point of view, the clogging effects on the hyporheos are higher compared to the benthos regarding taxonomic richness, changes in faunal assemblages and biological traits

Denna studie har undersökt om förvärmning av luft är en bra metod för att undvika frostbildning i motströmsplattvärmeväxlare i luftbehandlingsaggregat. Frostbildning i värmeväxlare för ventilation är ett problem i kalla klimat och sänker den återvunna energimängden när potentialen för energiåtervinning är som högst.   Tidigare forskning i området har utförts för att bland annat: 1) Definiera säkra förhållanden utan frostbildning i värmeväxlare med laminär luftströmning.  2) Jämföra olika frostkontrollstrategier. 3) Analysera frostbildningens egenskaper och effekt på värmeväxlare i ventilation. Att forskningen inte är entydig för vilken frostkontrollstrategi som är bäst belyser vikten av detta arbete.   I en fallstudie kvantifieras empiriska värmeöverföringskoefficienter som används i en analytisk modell som förutspår energianvändningen för luftvärmning under olika driftfall för ett luftbehandlingsaggregat. Värmeöverföringskoefficienterna tas fram genom mätningar i ett luftbehandlingsaggregat lokaliserat i ett flerbostadshus. Mätobjektet har ett installerat värmebatteri före värmeväxlaren vilket värmer inkommande uteluft till konstant lufttemperatur.  Tillverkaren proklamerar att det sker turbulent luftströmning i den studerade värmeväxlaren. Vid olika lufthastigheter ändras värmeöverföringskoefficienten mer vid turbulent strömning än vid laminär strömning. Att olika lufthastigheter har betydlig inverkan på värmeöverföringskoefficienten överensstämmer med resultatet från mätningarna. Effektiviteten av den aktuella styrningen av förvärmaren har granskats och brister har upptäckts. En ny optimerad styrning föreslås för frostfri drift och minimerad energianvändning. Den optimerade styrningen jämförs sedan med en vanlig avfrostningsmetod och utförs med hjälp av flödesbalansstatistik från ett aggregat med sektionsavfrostningsstyrning. Resultatet visar att förvärmning av luft för frostfri drift använder mindre energi än vid sektionsavfrostningsstyrning.  Den föreslagna styrningen innebär att bypass-spjället öppnas då förvärmaren går på full effekt. Detta för att förhindra påfrostning vid extrema utetemperaturer, då förvärmaren inte ger tillräcklig effekt för att undvika påfrostning. Den föreslagna styrningen regleras utifrån daggpunkten, temperaturen på tilluften innan värmeväxlaren och temperaturen på avluften. Den framtagna värmeöverföringskoefficienten i studien tar inte i beaktning ökad koefficient under fuktiga förhållanden då daggpunkten i frånluften är över fryspunkten och det sker kondens. En framtida studie kan hitta värmeöverföringskoefficienter som förutspår yttemperaturen under kondensutfällning då daggpunkten är över 0°C vilket ger medel för att ta fram en ännu mera energieffektiv styrning. Detta kan utföras genom mätning i ett luftbehandlingsaggregat med hög temperaturverkningsgrad placerat i en fuktig miljö. / This study has analysed if preheating of air is a good method to prevent frost formation in a counter flow plate heat exchanger used in Air Handling Units. Frosting in heat exchangers used in AHU-systems is a problem in cold climates and lowers the energy recovery when its potential is the highest.   Previous research has been done to 1) Define safe operating conditions without frost formation in heat exchangers with laminar flow. 2) Compare different frost control strategies. 3) Analyse frost formation properties and its effects on heat exchangers in AHU-systems. That the research is not unambiguous for which frost control method is the best highlights the importance of this work. Empirical heat transfer coefficients are quantified in this study and are used in the creation of an analytical model of a counter flow heat exchanger that predicts the energy use for heating the air at different operating modes. The heat transfer coefficients are produced by measurements in an AHU-systems located in an apartment building. The AHU has an installed air heater before the heat exchanger which heats the outdoor inlet air to constant temperature.   The manufacturer of the AHU-system proclaims turbulent flow in the heat exchanger. Change of airflow has greater impact on the heat transfer coefficients during turbulent flow compared to laminar flow conditions. This is also derived from the results of the measurements. The effectiveness of the current control system is analyzed, and flaws are discovered. A new control system is therefore proposed for frost free operation and higher energy efficiency. The proposed control system is compared to a frequently used frost control system which uses bypass-dampers to redirect the inlet air to the reheater, to let the warm air stream melt formed frost on the warm air side. This comparison is accomplished by analyzing the usage of the bypass-dampers during different outdoor temperatures in a comparable AHU-system that have the mentioned frost control method. The results show that preheating of inlet air for frost free operation uses less energy than usage of the bypass-dampers to melt formed frost on the warm air side. The proposed control system needs the bypass-dampers to be used when the preheater is on full operation to prevent frost formation at extreme outdoor temperatures when the preheating may not be enough. The proposed control system is regulated by the dewpoint and the temperatures of the exhaust air stream and the supply air stream. The derived heat transfer coefficient that is used to predict the plate-temperature take no account for condensation on the warm air side during humid conditions when the dew point is above the freezing point. Future studies can derive the heat transfer coefficient during condensation which will improve the prediction of the plate-temperature. This would make the system more energy efficient during humid air conditions. This can be done by measurements in an AHU-system with high temperature efficiency placed in a humid environment.

As L-asparaginases bacterianas (L-asparaginase amidohidrolase, E.C.3.S.1.1) são enzimas de alto valor terapêutico devido ao seu uso no tratamento de leucemia linfocítica aguda. A L-asparaginase da Escherichia coli por ser uma enzima periplásmica com alta afinidade, é particularmente efetiva em certas terapias de câncer infantil. Muitos agentes terapêuticos recentes são proteínas e peptídeos que surgiram do design molecular de drogas e tecnologia de DNA recombinante. Numerosos estudos têm demonstrado que aditivos preservam a estrutura e a atividade biológica de cada molécula destas proteínas. Entretanto, o mecanismo de proteção, pelo qual estes aditivos funcionam, não tem sido totalmente elucidado. O objetivo do presente trabalho é investigar detalhadamente o efeito da liofilização sobre a estrutura e a atividade enzimática da L-asparaginase, tanto em células íntegras de Escherichia coli, como na enzima purificada. Até recentemente a maneira para se avaliar o comportamento de um aditivo e o comportamento da água na estabilização de uma proteína durante a liofilização consistia na medida dos parâmetros de atividade após a reidratação, porém atualmente modernas técnicas de Ressonância Magnética Nuclear (RMN) de baixa resolução são utilizadas para se entender o comportamento da água nas interações com proteínas. E a Espectroscopia de Infravermelho por Transformada de Fourier apresenta grande potencial no estudo de estabilização de proteínas durante a liofilização. Utilizou-se o cálculo de porcentagem de retenção de atividade para expressar os valores de atividade enzimática estudados. Estes cálculos foram realizados para os sistemas congelados e para os sistemas congelados e liofilizados. Os sistemas foram tratados em velocidades de congelamento diferentes (20°C/min, SOC/min e 2°C/min), sendo em seguida liofilizados por 24 horas. São apresentados resultados sobre o efeito das diferentes velocidades de congelamento e o efeito da liofilização nos diferentes sistemas aditivo-enzima e aditivo-célula. Utilizaram-se os aditivos: sacarose, maltose, lactose, inositol, manitol e trealose testados em diferentes concentrações (30, 90 e 150mM). Para identificar quais as condições e os aditivos que apresentaram uma crioproteção satisfatória. Nos sistemas maltose-enzima e trealose-enzima observou-se um aumento da crioproteção com o aumento da concentração de aditivo. Para os sistemas maltose-enzima, congelados lentamente, os resultados de retenção de atividade foram: 8,67%, 14,02% e 30,80% respectivamente para 30, 90 e 150mM. O sistema enzima-maltose (150mM) congelado rapidamente e liofilizado apresentou a maior retenção de atividade (111,11 %) e também o maior valor de T2 (81&#181;s) nos resultados referentes a RMN. Nos sistemas trealose-enzima nas concentrações de 90 e 150mM apresentaram retenção de atividade 89,93% e 79,74%, respectivamente. / Bacterial L-asparaginase (L-asparaginase amidohydrolase, E.C. 3.5.1.1) are enzymes of high therapeutic value due to their use in the treatment of lymphocytic acute leukemia. Escherichia coli L-asparaginase is a periplasmic enzyme of high affinity, particularly effective in some kinds of childhood cancer therapies. Several studies have showed that there are specific stabilizing additives preserve the structure and the biological activity of protein molecules (lyoprotectant). However, the protection mechanism for these excipients has not been totally elucidated yet. The aim of this work was investigate the effect of freeze-drying on the enzymatic activity of L-asparaginase using both the purified enzyme as well as intact cells of Escherichia coli. Until recently the way to evaluate the behavior of an addictive one and the behavior of the water in the stabilization of a protein during the freeze-drying consisted of the measure of the activity parameters after the reidratação, even so now modem techniques of the Nuclear Magnetic Resonance of low resolution have been used to understand the behavior of the water in the interactions with proteins. It is Infrared Spectroscopy for Fourier Transformed it presents a great potential in the study of stabilization of proteins during the freeze-drying. The calculation of percentage of activity retention was used to express the studied values of enzymatic activity. These calculations were accomplished for the frozen systems and for the frozen systems and freeze-dryied. The systems were treated in three speeds of different freezing (20°C/min, 5°C/min and 2°C/min), being the freeze-drying for 24 hours. Results are presented on the effect of the freeze-drying in the different systems addictive-enzyme and addictive-cell. The addictive ones were used: sucrose, maltose, lactose, inositol, manitol and trehalose tested in different concentrations (30, 90 and 150mM), to identify which the conditions and the addictive ones that presented a satisfactory cryoprotection. For the systems enzyme-maltose, frozen slowly, the results of activity retention were: 8,67%, 14,02% e 30,80% to 30,90 e 150mM,respectively . The system enzyme-maltose (150mM) frozen quickly and freeze-dryied presented the largest activity retention (111,11 %) and also the largest value of T2 (81 &#181;s) in the referring results NMR. The systems enzyme-trealose in the concentrations of 90 and 150mM they presented retention of activity 89,93% and 79,74%, respectively.

Die Tight Junction (TJ) reguliert den parazellulären Transport von Ionen, Wasser und Soluten an Epithelien und Endothelien und ist von entscheidender Bedeutung für die Aufrechterhaltung der Funktion von Organen und Geweben. Obwohl Claudin-3 zu den zuerst identifizierten und ubiquitär exprimierten Komponenten der TJ gehört, konnte seine spezifische Funktion bislang nicht geklärt werden. Für die funktionelle Charakterisierung des humanen Claudin-3-Proteins wurden stabile Überexpressionsklone der lecken Nierenepithel-Zelllinie MDCK II generiert. Die Überexpression von Claudin-3 führte zu einer deutlichen Änderung des TJ-Strangmusters sowie zu einer starken Zunahme des transepithelialen Widerstandes und einer verminderten Permeabilität für Ionen und Moleküle der Größe 332 Da und 4000 Da. Der parazelluläre Durchtritt von Wasser war unverändert. Claudin-3 konnte eindeutig als abdichtende Komponente der TJ identifiziert werden. Anhand des endothelialen Zellkulturmodells HUVEC wurden Expression und Regulation von Claudin-3 und anderen TJ-Proteinen unter dem Einfluss mechanischer Strömungsverhältnisse und des Sauerstoffpartialdrucks analysiert. Die Behandlung mit fehlender Wandschubspannung führte zur Hochregulation der abdichtenden TJ-Proteine Occludin, Claudin-3, Claudin-5 und Claudin-11, nicht aber Claudin-23. Die Regulation der einzelnen TJ-Komponenten wurde durch unterschiedliche Signalwege vermittelt, wobei der verstärkten Proteinexpression jeweils eine Hochregulation auf mRNA-Ebene zugrunde lag. Die kombinierte Behandlung mit fehlender Wandschubspannung und Hypoxie resultierte in einer sehr stark erhöhten Expression von Claudin-3. Durch die Hochregulation abdichtender TJ-Komponenten unter Bedingungen fehlender Wandschubspannung und Hypoxie, wie sie in verschiedenen physiologischen und pathologischen Situationen auftreten, könnte einem unerwünschten Durchtritt von Substanzen aus dem Blut in das umliegende Gewebe vorgebeugt werden. / The tight junction (TJ) regulates the paracellular transport of ions, water and solutes in epithelia and endothelia and is of particular importance for a correct function of organs and tissues. Although claudin-3 is one of the first identified and ubiquitously expressed TJ components, its specific function was unsolved as yet. For functional characterization, human claudin-3 was stably overexpressed in the leaky epithelial cell line MDCK II. Overexpression of claudin-3 led to a marked alteration of TJ meshwork pattern, a strong increase in transepithelial resistance and a decrease in permeability for ions and paracellular tracers (332 or 4000 Da). Paracellular water transport was not affected. It was proved that claudin-3 acts as a „tightening“ TJ component. The endothelial cell culture model HUVEC was used for analysis of expression and regulation of claudin-3 and several other TJ proteins under different conditions of wall shear stress and oxygen saturation. Treatment with lacking wall shear stress led to an upregulation of the “tightening” TJ proteins occludin, claudin-3, claudin-5, and claudin-11, but not claudin-23. Upregulation of all proteins was due to increased mRNA levels. Apparently, different signaling pathways were involved in regulation of particular TJ components. Combined treatment with lacking shear stress and hypoxia resulted in drastically increased claudin-3 expression. Upregulation of tightening TJ components under lacking shear stress and hypoxic conditions as occuring in different physiological or pathological situations would limit the passage of solutes from the blood into the surrounding tissue.

Presently, over 300 proteins or peptide based therapeutic medicines have been approved by the FDA owing to advances in protein engineering and technology. However, majority of these protein-based medications are unstable or have limited shelf life when in aqueous form. During pre-formulation and manufacturing, various technological processes including mixing, dissolving, filling (through pipes) can produce strong mechanical stresses on proteins. These stresses may cause the protein molecule to unfold, denature or aggregate. To improve stability upon formulation, they may be manufactured as freeze dried cakes that requires reconstitution with a buffer or water prior to administration. Although it has been successful in improving the stability of protein-based formulations, the freeze drying process itself also contributes to protein aggregation. This process introduces other stresses such as freezing, thawing and drying. In addition to these stresses, the agitation processes used during reconstitution may also destabilize the protein’s native structure. Two key processes used in preparation of protein based formulations were studied in this work; mechanical agitation and freeze drying. The aim of this project was to explore the aggregation of proteins that occur due to the various technological processes typical in the production of protein based formulations. The project has two parts that relates to liquid and solid formulations. In the first part, the effect of different methods of mechanical agitations on BSA protein was investigated. In the second part, the focus was on the effect of formulation (i.e. the application of amino acids) on aggregation of protein (BSA) in freeze dried formulations. Arginine and lysine were added individually into protein-based freeze-dried formulation to study their potential of improving the stability of the proteins during manufacturing, storage and reconstitution. In the formulation development, additional excipients were added to prevent moisture uptake due to the hygroscopic properties of the amino acids and to provide lyo- and cryo- protection for the protein molecule during freeze drying. Without further purification, BSA solutions prepared by using sonication, low shear rotor mixer or high shear tube/pipe mixing were studied using dynamic light scattering (DLS). Thioflavin T assay and turbidimetry analysis were used as complementary studies. In protein-based freeze dried formulations, at accelerated storage conditions, the presence of aggregates were studied in samples containing arginine or lysine using ThT assay and turbidimetry analysis. Characterisation of the freeze dried cakes was performed relative to their moisture sorption, cake shrinkage, mechanical properties and morphology using various analytical techniques. iv In the BSA solution studies, particle size analysis indicated two distributions for non-agitated BSA solution that corresponds to the average particle sizes of BSA molecules and their aggregates. Under mechanical stresses (all types), the intensity of distribution centered ≈ 7.8 nm reduces and broadens as the agitation time increases, indicating a reduction in the amount of “free” BSA macromolecules. The second distribution, as a result of increasing agitation time or shear intensity, reveals a significant shift towards larger sizes, or even splits into two particle size populations. These particle size growths reflect the formation of aggregates due to intensive collisions and, as a result, partial unfolding followed by hydrophobic interactions of exposed non-polar amino acids. UV spectra showed that aggregation in both low shear and mechanical vibration agitations were lower compared to the high shear stress. When compared to non-agitated BSA solution, ThT assay recorded ≈15 times higher fluorescence emission from the high shear samples, ≈2 times fluorescence emission from low shear and ≈6 times fluorescence emission from mechanical vibrations. Thus all the three agitation methods showed a good correlation between the results. The second part of this project was performed in three stages. In the initial 2 stages, 2- and 3-excipients component system were investigated to develop an optimal preliminary formulations which will be used in the final protein based 4-components formulations. From the 1st stage (ArgHCl/LysHCl + sugar/polyol), among 4 tested excipients (polyol and sugar), mannitol was observed to have resisted moisture uptake by the highly hygroscopic ArgHCl/LysHCl amino acids. However, mannitol is considered a good cryoprotector but has poor lyoprotection properties. Therefore, in the following stage, a 3rd excipient (in a 3-excipients component system) sucrose or trehalose, was introduced into the formulation. The formulation was made up of 20% ArgHCl (LysHCl), and various ratios of mannitol and sugar were explored. The criteria for selecting the best systems were based on ideal physicochemical properties i.e. moisture uptake, shrinkage, mechanical properties, matrix structure and appearance, and thermal properties. The final stage was the formulation of a 4-components system comprising the three excipients and combinations selected from the stage 2 studies, and the addition of BSA as the model protein. To study aggregation in this system, a freeze dried 4-components excipient/protein system was reconstituted and incubated at accelerated storage conditions over time. Fluorescence spectroscopy and turbidimetry were used to study aggregation of proteins, moisture uptake kinetics with gravimetric balance, and thermal analytical techniques were used to characterise the freeze dried cakes with and without BSA protein. This study represented a systematic analysis of aggregation of proteins in both liquid and solid formulations. Some of the novel aspects of this study include: v 1. The new experimental results obtained for aggregation of proteins in solution subjected to mechanical agitations. The high shear stress created by syringe agitation, simulated the real situation in post manufacturing process during filling through narrow pipes, and has been shown here to strongly affect the aggregation of protein macromolecules. 2. The development of a methodical approach for optimization of multi component (up to 4 excipients) protein based formulations. 3. The unexpected non-linear behavior of the physicochemical properties of the 3-excipients component system as a function of composition. To the best of my knowledge, this novel aspect has not been previously reported in literature. 4. Application of amino acid in protein based formulations has shown the inhibition of aggregation of BSA, with the highest effect observed with ArgHCl. The results of this study coincide with the conclusions published previously for aggregation of proteins in solution.

As L-asparaginases bacterianas (L-asparaginase amidohidrolase, E.C.3.S.1.1) são enzimas de alto valor terapêutico devido ao seu uso no tratamento de leucemia linfocítica aguda. A L-asparaginase da Escherichia coli por ser uma enzima periplásmica com alta afinidade, é particularmente efetiva em certas terapias de câncer infantil. Muitos agentes terapêuticos recentes são proteínas e peptídeos que surgiram do design molecular de drogas e tecnologia de DNA recombinante. Numerosos estudos têm demonstrado que aditivos preservam a estrutura e a atividade biológica de cada molécula destas proteínas. Entretanto, o mecanismo de proteção, pelo qual estes aditivos funcionam, não tem sido totalmente elucidado. O objetivo do presente trabalho é investigar detalhadamente o efeito da liofilização sobre a estrutura e a atividade enzimática da L-asparaginase, tanto em células íntegras de Escherichia coli, como na enzima purificada. Até recentemente a maneira para se avaliar o comportamento de um aditivo e o comportamento da água na estabilização de uma proteína durante a liofilização consistia na medida dos parâmetros de atividade após a reidratação, porém atualmente modernas técnicas de Ressonância Magnética Nuclear (RMN) de baixa resolução são utilizadas para se entender o comportamento da água nas interações com proteínas. E a Espectroscopia de Infravermelho por Transformada de Fourier apresenta grande potencial no estudo de estabilização de proteínas durante a liofilização. Utilizou-se o cálculo de porcentagem de retenção de atividade para expressar os valores de atividade enzimática estudados. Estes cálculos foram realizados para os sistemas congelados e para os sistemas congelados e liofilizados. Os sistemas foram tratados em velocidades de congelamento diferentes (20°C/min, SOC/min e 2°C/min), sendo em seguida liofilizados por 24 horas. São apresentados resultados sobre o efeito das diferentes velocidades de congelamento e o efeito da liofilização nos diferentes sistemas aditivo-enzima e aditivo-célula. Utilizaram-se os aditivos: sacarose, maltose, lactose, inositol, manitol e trealose testados em diferentes concentrações (30, 90 e 150mM). Para identificar quais as condições e os aditivos que apresentaram uma crioproteção satisfatória. Nos sistemas maltose-enzima e trealose-enzima observou-se um aumento da crioproteção com o aumento da concentração de aditivo. Para os sistemas maltose-enzima, congelados lentamente, os resultados de retenção de atividade foram: 8,67%, 14,02% e 30,80% respectivamente para 30, 90 e 150mM. O sistema enzima-maltose (150mM) congelado rapidamente e liofilizado apresentou a maior retenção de atividade (111,11 %) e também o maior valor de T2 (81&#181;s) nos resultados referentes a RMN. Nos sistemas trealose-enzima nas concentrações de 90 e 150mM apresentaram retenção de atividade 89,93% e 79,74%, respectivamente. / Bacterial L-asparaginase (L-asparaginase amidohydrolase, E.C. 3.5.1.1) are enzymes of high therapeutic value due to their use in the treatment of lymphocytic acute leukemia. Escherichia coli L-asparaginase is a periplasmic enzyme of high affinity, particularly effective in some kinds of childhood cancer therapies. Several studies have showed that there are specific stabilizing additives preserve the structure and the biological activity of protein molecules (lyoprotectant). However, the protection mechanism for these excipients has not been totally elucidated yet. The aim of this work was investigate the effect of freeze-drying on the enzymatic activity of L-asparaginase using both the purified enzyme as well as intact cells of Escherichia coli. Until recently the way to evaluate the behavior of an addictive one and the behavior of the water in the stabilization of a protein during the freeze-drying consisted of the measure of the activity parameters after the reidratação, even so now modem techniques of the Nuclear Magnetic Resonance of low resolution have been used to understand the behavior of the water in the interactions with proteins. It is Infrared Spectroscopy for Fourier Transformed it presents a great potential in the study of stabilization of proteins during the freeze-drying. The calculation of percentage of activity retention was used to express the studied values of enzymatic activity. These calculations were accomplished for the frozen systems and for the frozen systems and freeze-dryied. The systems were treated in three speeds of different freezing (20°C/min, 5°C/min and 2°C/min), being the freeze-drying for 24 hours. Results are presented on the effect of the freeze-drying in the different systems addictive-enzyme and addictive-cell. The addictive ones were used: sucrose, maltose, lactose, inositol, manitol and trehalose tested in different concentrations (30, 90 and 150mM), to identify which the conditions and the addictive ones that presented a satisfactory cryoprotection. For the systems enzyme-maltose, frozen slowly, the results of activity retention were: 8,67%, 14,02% e 30,80% to 30,90 e 150mM,respectively . The system enzyme-maltose (150mM) frozen quickly and freeze-dryied presented the largest activity retention (111,11 %) and also the largest value of T2 (81 &#181;s) in the referring results NMR. The systems enzyme-trealose in the concentrations of 90 and 150mM they presented retention of activity 89,93% and 79,74%, respectively.

0 comportamento higroscópico do suco de laranja liofilizado, e aditivado com malto-dextrinas e lactose, e de misturas mecânicas do suco liofilizado com os aditivos secos, foi estudado através da cinética e das isotermas de sorção de água, em diferentes temperaturas. Avaliou-se as características espectrais no visível e ultravioleta, e o teor de vitamina C, de amostras de suco de laranja liofilizado expostas a diferentes umidades relativas e temperaturas. Estudou-se a influência do valor de pH e natureza do tampão sobre a retenção do limoneno emulsionado em soluções de lactose, liofilizadas. A liofilização praticamente não alterou o teor de vitamina C e as características espectrais do suco de laranja, mas aumentou o valor monomolecular da malto-dextrina (dextrose equivalente de 9 a 12%). A lactose liofilizada apresentou-se no estado amorfo. Os teores de umidade de equilíbrio das misturas aditivadas liofilizadas, foram menores que os das misturas mecânicas. Os aditivos reduziram a sorção de umidade em função do tempo. A retenção do limoneno foi influenciada pela sua concentração inicial, sendo que ocorreu a major perda, durante a liofilização na major concentração utilizada. / The higroscopic behavior of freeze-dried orange juice, with added maltodextrins and lactose and mechanical mixtures with this additives was studied through the kinetics and isoterms of water sorption in different temperatures. It was evaluated the spectral characteristics, in the visible and ultraviolet range, and the vitamin C content from samples of freeze-dried orange juice exposed at different relative humidities and temperatures. It was studied the influence of pH and buffer composition, on the limonene retention of freeze-dried emulsions. The spectral characteristics and vitamin C content showed no alterations after liofilization the maltodextrin (9-12 % dextrose equivalent monomolecular value increased. Freezedried lactose was amorphous. The equilibrium humidity content of the samples with additives were reduced when compared with mechanical mixtures. The additives also reduced the rate of water sorption. The limonene retention during freeze-drying was influenced by the initial concentration of the emulsion. The greater concentration studied showed the lower retention value.