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Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Artificial lipid bilayers formed on solid surface supports are widespread model systems to study physical, chemical, as well as biological aspects of cell membranes and fundamental interfacial interactions. The approach to use a thin polymer film representing a cushion for lipid bilayers prevents incorporated membrane proteins from pinning to the support and mimics the native environment of a lipid bilayer in certain aspects of the extracellular matrix and intracellular structures. A key component for cell anchorage to extracellular fibronectin is the transmembrane adhesion receptor alpha(5)beta(1) integrin. Its transport dynamics and clustering behavior plays a major role in the assembly of focal adhesions, which mediate mechanical forces and biochemical signals of cells with their surrounding. The system investigated herein is envisioned to use extrinsically controlled stimuli-responsive polymer cushions to tune the frictional drag between polymer cushion and mobile membranes with incorporated integrins to actively regulate lipid membrane characteristics. To attain this goal, a temperature- and pH-responsive polymer based on poly(N-isopropylacrylamide) copolymers containing varying amounts of carboxyl-group-terminated comonomers at different aliphatic spacer lengths (PNIPAAm-co-carboxyAAM) was surface-grafted to a poly(glycidyl methacrylate) anchorage layer. The swelling transitions were characterized using atomic force microscopy, ellipsometry and quartz crystal microbalance with dissipation monitoring (QCM-D) and found to be tunable over a wide range of temperature and pH. In agreement with the behavior of the polymers in solution, longer alkyl spacers decreased the phase transition temperature T(P) and higher contents of carboxylic acid terminated comonomers increased T(P) at alkaline conditions and decreased T(P) at acidic conditions. Remarkably, the point where the degree of carboxyl group deprotonation balances the T(P)-lowering effect of the alkyl spacer was distinctive for each alkyl spacer length. These findings illustrate how the local and global balance of hydrophilic and hydrophobic interactions along the copolymer chain allows to adjust the swelling transition to temperatures below, comparable, or above those observed for PNIPAAm homopolymers. Additionally, it could be shown that surface-grafting leads to a decrease in T(P) for PNIPAAm homopolymers (7°C) and copolymers (5°C - 10°C). The main reason is the increase in local polymer concentration of the swollen film constrained by dense surface anchorage in comparison to the behavior of dilute free chains in solution. In accordance with the Flory-Huggins theory, T(P) decreases with increasing concentration up to the critical concentration. Biological functionalization of the PNIPAAm-co-carboxyAAm thin films was demonstrated for the cell adhesion ligand peptide cRGD via carbodiimide chemistry to mimic extracellular binding sites for the cell adhesion receptors integrin. The outcome of QCM-D measurements of cRGD-functionalized surfaces showed a maintained stimuli-responsiveness with slight reduction in T(P). A drying/rehydration procedure of a 9:1 lipid mixture of the cationic lipid dioleoyl-trimethylammoniumpropane (DOTAP) and the zwitterionic dioleoyl-phosphatidylcholine (DOPC) was utilized to form lipid bilayer membranes on PNIPAAm-co-carboxyAAM cushions. Fluorescence recovery after photobleaching (FRAP) revealed that lipid mobility was distinctively higher (6.3 - 9.6) µm2 s-1 in comparison to solid glass support ((3.0 - 5.9) µm2 s-1). In contradiction to the initial expectations, modulation of temperature and pH led to poor variations in lipid mobility that did not correlate with the PNIPAAm cushion swelling state. The results suggested a weak coupling of the lipid bilayer with PNIPAAm polymer cushions that can be slightly tuned by electrostatic interactions. The transmembrane adhesion receptor alpha(5)beta(1) integrin was reconstituted into liposomes consisting of DOPC/sphingomyelin/cholesterol 2:2:1 for the formation of polymer cushioned bilayers. PNIPAAm- co-carboxyAAM and maleic acid (MA) copolymers were used as cushions, both with the option for cRGD functionalization. On the MA copolymer cushions, fusion of proteoliposomes resulted in supported bilayers with mobile lipids as confirmed by FRAP. However, incorporated integrins were immobile. In an attempt to explain this observation, the medium-sized cytoplasmic integrin domain was accounted to hamper the movement by steric interactions with the underlying polymer chains in conjunction with electrostatic interactions of the cationic cytoplasmic domain with the oppositely charged MA copolymer. On the PNIPAAm-co-carboxyAAM cushion only a drying/rehydration procedure lead to bilayer formation. However, again the integrins were immobile, presumably due to the harsh treatment during preparation. Nevertheless, the results of the investigated set of PNIPAAm copolymer films suggest their application as temperature- and pH-responsive switchable layers to control interfacial phenomena in bio-systems at different physiological conditions. The PNIPAAm-co-carboxyAAm cushioned bilayer system represents a promising step towards extrinsically controlled membrane – substrate interactions.

Orientador: Flavia Maria Netto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-19T21:19:52Z (GMT). No. of bitstreams: 1 Silva_FernandaGuimaraesDrummonde_M.pdf: 1363127 bytes, checksum: 68b1a97c95798f4425019ee900814160 (MD5) Previous issue date: 2012 / Resumo: Existem evidências numerosas sobre o papel dos radicais livres em uma série de condições patológicas, incluindo envelhecimento, câncer, esclerose múltipla, doenças cardiovasculares. Hidrolisados protéicos de diferentes fontes têm sido estudados por seu potencial antioxidante. A atuação antioxidante da proteína, na maioria das vezes, encontra-se limitada devido à conformação espacial, que concentra resíduos capazes de neutralizar radicais livres no interior da molécula, dificultando o acesso das espécies reativas aos centros nucleofílicos. A hidrólise da proteína contribui para aumentar a exposição desses resíduos de aminoácidos, aumentando sua atuação como antioxidante. Compostos fenólicos podem estar presentes em hidrolisados proteicos de origem vegetal, devido a sua associação com as proteínas. Métodos in vitro que simulam as condições do trato gastrointestinal permitem estudar como a digestão pode interferir na atividade antioxidante de peptídeos e compostos fenólicos. O presente trabalho tem como objetivos obter hidrolisados proteicos com capacidade antioxidante a partir da farinha de linhaça e avaliar o efeito da digestão in vitro pode interferir nessa atividade. A farinha de linhaça marrom foi desengordurada, obtendo-se a farinha de linhaça marrom desengordurada (FLMD). O concentrado proteico de linhaça (CPL) foi obtido a partir da FLMD por extração alcalina e precipitação no ponto isoelétrico seguida de neutralização. Para obtenção dos hidrolisados proteicos (HPL), a partir do CPL, com Alcalase, foi realizado um delineamento composto central rotacional (DCCR) 2². As variáveis independentes foram pH que variou entre 7,5 a 9,5 e relação enzima: substrato (E:S) que variou de 1:150 a 1:30. As variáveis dependentes foram grau de hidrólise (GH), teor de substâncias redutoras do reagente de Folin-Ciocalteau e atividade antioxidante, determinada por FRAP e ORAC. Teor de substâncias redutoras e atividade antioxidante foram avaliados a partir dos extratos aquosos e metanólico (metanol 70%). Os hidrolisados de maior atividade antioxidante, a FLMD e o CPL foram submetidos à digestão in vitro, simulando as condições da digestão gastrintestinal. As amostras antes e após a digestão in vitro foram caracterizadas por eletroforese em sistema SDS-PAGE Tricina e por cromatografia liquida de alta eficiência de fase reversa (HPLC- RP). O teor de substâncias redutoras e da atividade antioxidante das amostras FLMD, CPL e HPL foram avaliados antes e após a digestão in vitro. As condições ótimas para obtenção de HPL de maior GH (21,0%) são pH entre 7,5 e 8,0 e E:S entre 1:60 e 1:30, indicando que a faixa de pH ótimo da enzima e a alta E:S favorecem maior hidrólise do CPL. Para obtenção de HPL com maior teor de substâncias redutoras para os extratos aquoso (24 mg EAG/ g HPL) e metanólico (20 mg EAG/ g HPL) as condições ótimas são pH ~ 8,5 /E:S 1:30. Este resultado parece estar relacionado à liberação de compostos fenólicos ligados a proteína e também de peptídeos durante a hidrólise. Açúcares e aminoácidos aromáticos presentes no hidrolisado podem interferir na reação e superestimar o teor de fenóis dos HPL. A maior atividade antioxidante determinada pelo método de FRAP para o extrato aquoso (42 mg SF/ g HPL) se dá nas condições de pH ~ 9,5/E:S ~1:150 e para o extrato metanólico (40 mg SF/ g HPL) pH entre 8,5 e 9,0/E:S entre 1:90 a 1:150. Para o método de ORAC, as condições ótimas para maior atividade antioxidante no extrato aquoso (300 µmol TE/ g HPL) são pH entre 7,5 a 9,5/E:S ~ 1:30 ou ~1:150 e para o extrato metanólico (330 µmol TE/ g HPL) são pH ~ 8,5/E:S entre 1:150 e 1:30. Os hidrolisados de maior atividade antioxidante foram os obtidos em pH 8,5/E:S 1:90, e em pH 9,2/E:S 1:133 denominados HPL 0 e HPL 3, respectivamente. Para a FLMD, CPL e os hidrolisados, após a digestão in vitro, observou-se que o teor de substâncias redutoras totais aumentou (9 a 20 vezes) para todas as amostras. O teor de substâncias redutoras do CPL (~24 mg EAG/ g amostra), em ambos os extratos, após a digestão in vitro se igualou ao teor dos hidrolisados (~23 mg EAG/ g amostra). Este resultado sugere que tanto a hidrólise com Alcalase quanto o processo digestório liberam compostos redutores, dentre eles fenólicos da proteína de linhaça. A atividade antioxidante dos extratos de FLMD e CPL, determinada por FRAP, também aumentou (de 3 a 10 vezes) após a digestão, mas não se igualou à atividade antioxidante dos hidrolisados (48 mg SF/g amostra). No entanto, o CPL apresentou atividade antioxidante determinada por ORAC semelhante à dos hidrolisados no extrato aquoso (~420,24 µmol TE/ g amostra) e 10 % maior que o encontrado para os hidrolisados (~365 µmol TE/ g amostra) no extrato metanólico. Após a digestão in vitro, os hidrolisados apresentaram a maior atividade antioxidante medida por FRAP (50 mg SF/ g amostra), e o CPL, a maior atividade determinada pelo método de ORAC (~430 µmol TE/ g amostra). Estes resultados sugerem o processo digestório é tão ou mais eficiente que a Alcalase em liberar os compostos com atividade redutora no CPL. Uma vez que a metodologia de determinação da atividade antioxidante por ORAC tem maior proximidade com o mecanismo de oxirredução que ocorre in vivo, esses resultados sugerem o uso do CPL como melhor produto protéico da linhaça com maior potencial antioxidante para a formulação de nutracêuticos e alimentos funcionais / Abstract: There are several evidences which indicate the role of free radicals on a series of pathological conditions, including aging, cancer, multiple sclerosis and cardiovascular disease. Hydrolysates from different sources have been studied because of their antioxidant potential. The antioxidant activity of the protein, in most cases, is limited due to their conformation, which concentrates residues capable of neutralize free radicals in the molecule¿s core, hampering the access of the reactive species to nucleophilic sites. The protein hydrolysis contributes to increasing the exposure of these amino acid residues, increasing their role as antioxidants. Phenolic compounds may also be present in vegetable protein hydrolysates because of their association with proteins. In vitro methods that simulate the conditions of the gastrointestinal digestion are an important way to evaluate how the digestion affects the antioxidant activity of phenolic compounds and peptides. This study aims at obtaining hydrolysates with antioxidant capacity from defatted flaxseed flour and evaluate the effect of the in vitro digestion on this activity. The brown flaxseed flour was defatted, resulting in the brown defatted flaxseed meal (BDFM). The flaxseed protein concentrate (FPC) was obtained from the BDFM by alkaline extraction and precipitation at the isoelectric point followed by neutralization. To obtain the flaxseed protein hydrolysates (FPL), using FPC and Alcalase, a central composite rotational design (DCCR) was performed. The independent variables were pH ranging from 7.5 to 9.5 and enzyme: substrate ratio (E: S) that ranged from 1:150 to 1:30. The dependent variables were the degree of hydrolysis (DH), total phenolic content and antioxidant activity, determined by FRAP and ORAC. Phenolic and antioxidant activity were evaluated from the aqueous and methanol (70% methanol). The hydrolysates with the highest antioxidant activity, the CPL FLMD were submitted to the in vitro digestion. The samples obtained before and after the in vitro digestion were characterized by electrophoresis SDS-PAGE- tricine and HPLC. The total phenolic content and antioxidant activity of FLMD, CPL and HPL were evaluated before and after in vitro digestion. The optimum conditions to obtain HPL with the highest GDH (21.0%) are pH (7.5-8) and E:S ratio (1:60-1:30), which indicates that the Alcalase optimum pH and highest E:S ratio collaborates to highest hydrolysis of CPL. To obtain HPL with higher content of Folin-Ciocalteau reducing compounds content in aqueous (EAG 24 mg / g HPL) and methanol (20 mg EAG / g HPL) extracts, the optimum conditions were pH ~ 8.5 / E: S 1:30. This result seems to be related to the release of phenolic compounds bound to protein and also of peptides during hydrolysis. The highest antioxidant activity determined by the FRAP method in the aqueous extract (42 mg SF / g HPL) occurs under pH ~ 9.5 / E: S ~ 1:150 and the methanol extract (40 mg SF / g HPL) pH 8.5-9.0 / E: S 1:90-1:150. For the ORAC method, optimum conditions for increased antioxidant activity in aqueous extract (300 µmol TE / g HPL) are pH 7.5-9.5 / E: S ~ 1:30 or 1:150 and the methanol extract (330 µmol TE / g HPL) are pH ~ 8.5 / E: S 1:30-1:150. The hydrolysates with the highest antioxidant activities were obtained at pH 8.5 / E: S 1:90, and at pH 9.2 / E: S 1:133 denominated HPL ) and HPL 3, respectively. For FLMD, CPL and hydrolysates, after in vitro digestion, the content increased (9-20 times) for all samples. The Folin-Ciocalteau reducing capacity of the CPL (EAG ~ 24 mg / g sample) in both extracts after in vitro digestion equaled the content of hydrolysates (EAG ~ 23 mg / g sample). This result suggests that both hydrolysis with Alcalase and the digestion process are able to release phenolic compounds from the flaxseed products. The antioxidant activity of extracts of FLMD, CPL determined by FRAP, also increased (from 3 to 10 times) after digestion, but did not reached the antioxidant activity of hydrolysates (48 mg SF / g sample). However, when the activity was determined by ORAC, the FPC showed value similar to the hydrolysates, measured on the aqueous extract (~ 420.24 µmol TE / g sample) and 10% higher than on the methanol extract (~ 365 µmol TE / g sample). After in vitro digestion, hydrolysates showed the highest antioxidant activity measured by FRAP (SF 50 mg / g sample), and the FPC, the highest activity determined by ORAC method (~ 430 micromol TE / g sample). These results suggest that digestive process are equally or more effective than Alcalase in releasing peptides and phenolic compounds present in the FPC. Since the methodology for determining the antioxidant activity by ORAC utilizes a biologically relevant radical source, these results suggest the use of FPC as the best protein product of flaxseed with potential antioxidant in the formulation of nutraceuticals and functional foods / Mestrado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Mestre em Alimentos e Nutrição

Artificial lipid bilayers formed on solid surface supports are widespread model systems to study physical, chemical, as well as biological aspects of cell membranes and fundamental interfacial interactions. The approach to use a thin polymer film representing a cushion for lipid bilayers prevents incorporated membrane proteins from pinning to the support and mimics the native environment of a lipid bilayer in certain aspects of the extracellular matrix and intracellular structures. A key component for cell anchorage to extracellular fibronectin is the transmembrane adhesion receptor alpha(5)beta(1) integrin. Its transport dynamics and clustering behavior plays a major role in the assembly of focal adhesions, which mediate mechanical forces and biochemical signals of cells with their surrounding. The system investigated herein is envisioned to use extrinsically controlled stimuli-responsive polymer cushions to tune the frictional drag between polymer cushion and mobile membranes with incorporated integrins to actively regulate lipid membrane characteristics. To attain this goal, a temperature- and pH-responsive polymer based on poly(N-isopropylacrylamide) copolymers containing varying amounts of carboxyl-group-terminated comonomers at different aliphatic spacer lengths (PNIPAAm-co-carboxyAAM) was surface-grafted to a poly(glycidyl methacrylate) anchorage layer. The swelling transitions were characterized using atomic force microscopy, ellipsometry and quartz crystal microbalance with dissipation monitoring (QCM-D) and found to be tunable over a wide range of temperature and pH. In agreement with the behavior of the polymers in solution, longer alkyl spacers decreased the phase transition temperature T(P) and higher contents of carboxylic acid terminated comonomers increased T(P) at alkaline conditions and decreased T(P) at acidic conditions. Remarkably, the point where the degree of carboxyl group deprotonation balances the T(P)-lowering effect of the alkyl spacer was distinctive for each alkyl spacer length. These findings illustrate how the local and global balance of hydrophilic and hydrophobic interactions along the copolymer chain allows to adjust the swelling transition to temperatures below, comparable, or above those observed for PNIPAAm homopolymers. Additionally, it could be shown that surface-grafting leads to a decrease in T(P) for PNIPAAm homopolymers (7°C) and copolymers (5°C - 10°C). The main reason is the increase in local polymer concentration of the swollen film constrained by dense surface anchorage in comparison to the behavior of dilute free chains in solution. In accordance with the Flory-Huggins theory, T(P) decreases with increasing concentration up to the critical concentration. Biological functionalization of the PNIPAAm-co-carboxyAAm thin films was demonstrated for the cell adhesion ligand peptide cRGD via carbodiimide chemistry to mimic extracellular binding sites for the cell adhesion receptors integrin. The outcome of QCM-D measurements of cRGD-functionalized surfaces showed a maintained stimuli-responsiveness with slight reduction in T(P). A drying/rehydration procedure of a 9:1 lipid mixture of the cationic lipid dioleoyl-trimethylammoniumpropane (DOTAP) and the zwitterionic dioleoyl-phosphatidylcholine (DOPC) was utilized to form lipid bilayer membranes on PNIPAAm-co-carboxyAAM cushions. Fluorescence recovery after photobleaching (FRAP) revealed that lipid mobility was distinctively higher (6.3 - 9.6) µm2 s-1 in comparison to solid glass support ((3.0 - 5.9) µm2 s-1). In contradiction to the initial expectations, modulation of temperature and pH led to poor variations in lipid mobility that did not correlate with the PNIPAAm cushion swelling state. The results suggested a weak coupling of the lipid bilayer with PNIPAAm polymer cushions that can be slightly tuned by electrostatic interactions. The transmembrane adhesion receptor alpha(5)beta(1) integrin was reconstituted into liposomes consisting of DOPC/sphingomyelin/cholesterol 2:2:1 for the formation of polymer cushioned bilayers. PNIPAAm- co-carboxyAAM and maleic acid (MA) copolymers were used as cushions, both with the option for cRGD functionalization. On the MA copolymer cushions, fusion of proteoliposomes resulted in supported bilayers with mobile lipids as confirmed by FRAP. However, incorporated integrins were immobile. In an attempt to explain this observation, the medium-sized cytoplasmic integrin domain was accounted to hamper the movement by steric interactions with the underlying polymer chains in conjunction with electrostatic interactions of the cationic cytoplasmic domain with the oppositely charged MA copolymer. On the PNIPAAm-co-carboxyAAM cushion only a drying/rehydration procedure lead to bilayer formation. However, again the integrins were immobile, presumably due to the harsh treatment during preparation. Nevertheless, the results of the investigated set of PNIPAAm copolymer films suggest their application as temperature- and pH-responsive switchable layers to control interfacial phenomena in bio-systems at different physiological conditions. The PNIPAAm-co-carboxyAAm cushioned bilayer system represents a promising step towards extrinsically controlled membrane – substrate interactions.

Dans ce travail, nous présentons une étude de biogels à base de chaînes de chitosane (polyélectrolyte naturel de la famille des polysaccharides) réticulées chimiquement en solution aqueuse. Notre objectif est double : caractériser la structure de ces gels et comprendre comment s'y déroule la diffusion de masse. À partir de mesures de spectroscopie mécanique, nous avons montré que l'on pouvait moduler la cinétique de formation et la structure de ces hydrogels en fonction de la masse molaire et la concentration de chitosane, du pH et de la teneur en POE (polyoxyéthylène). Ces résultats ont été confirmés par la détermination de la longueur de corrélation entre points d'enchevêtrements des hydrogels à l'aide de mesures de diffusion de neutrons aux petits angles. Nous avons ensuite étudié la diffusion de macromolécules fluorescentes de dextran en fonction de leur masse molaire dans les réseaux de chitosane réticulé ou non-réticulé à l'aide de la technique de recouvrement de fluorescence après photolyse par réseau de franges (FRAP). L'évolution du coefficient de diffusion avec la masse molaire de dextran en loi de puissance avec un exposant -1 dans les solutions de chitosane a permis de proposer une diffusion de type Rouse. Dans les gels de chitosane, nous avons clairement mis en évidence le passage entre une diffusion semblable à celle dans les solutions et une dynamique bloquée à partir d'une masse molaire critique de dextran.

Les cellules épithéliales de l'intestin possèdent une membrane plasmique spécialisée, la bordure en brosse, qui est constituée de protubérances en forme de doigts appelées microvillosités. Chaque microvillosité est composée d'un faisceau de filaments d'actine et d'un réseau en hélice de molécules de myosine I de la bordure en brosse (BBMI) formant des liens entre la membrane plasmique et les filaments d'actine. Lors de l'assemblage de la bordure en brosse, il se produit des phénomènes hautement dynamiques. En particulier, le nombre et la taille des microvillosités augmentent durant la dernière étape de différenciation des entérocytes adultes, comme au cours de la phase finale de l'embryongenèse. Le renouvellement rapide des protéines membranaires et du cytosquelette dans les entérocytes matures suggère également que des processus dynamiques ont lieu pour permettre à ces cellules de conserver leur morphologie. La dynamique des microvillosités pourrait être due à la polymérisation de l'actine qui est suffisante pour assurer la propulsion des bactéries, par exemple. Alternativement, BBMI pourrait être responsable de cette dynamique, soit en générant une force comme proposé par Sheetz pour l'extension des cônes de croissance des filopodes, soit en acheminant des composants membranaires au pôle apical de la cellule. Afin d'élucider la dynamique de BBMI et de l'actine dans la bordure en brosse des entérocytes, nous avons mis au point un instrument permettant la mesure de la mobilité tridimensionnelle des protéines dans des cellules vivantes en combinant l'imagerie de cellules vivantes en microscopie à deux photons avec la technique de FRAP (Fluorescence Recovery After Photobleaching). Nos résultats montrent que BBMI et l'actine sont mobiles, mais avec une dynamique différente. De plus, nous avons montré, pour la première fois dans des cellules vivantes, que BBMI a une activité motrice dans les microvillosités.

abstract: Biological membranes are critical to cell sustainability by selectively permeating polar molecules into the intracellular space and providing protection to the interior organelles. Biomimetic membranes (model cell membranes) are often used to fundamentally study the lipid bilayer backbone structure of the biological membrane. Lipid bilayer membranes are often supported using inorganic materials in an effort to improve membrane stability and for application to novel biosensing platforms. Published literature has shown that a variety of dense inorganic materials with various surface properties have been investigated for the study of biomimetic membranes. However, literature does not adequately address the effect of porous materials or supports with varying macroscopic geometries on lipid bilayer membrane behavior. The objective of this dissertation is to present a fundamental study on the synthesis of lipid bilayer membranes supported by novel inorganic supports in an effort to expand the number of available supports for biosensing technology. There are two fundamental areas covered including: (1) synthesis of lipid bilayer membranes on porous inorganic materials and (2) synthesis and characterization of cylindrically supported lipid bilayer membranes. The lipid bilayer membrane formation behavior on various porous supports was studied via direct mass adsorption using a quartz crystal microbalance. Experimental results demonstrate significantly different membrane formation behaviors on the porous inorganic supports. A lipid bilayer membrane structure was formed only on SiO2 based surfaces (dense SiO2 and silicalite, basic conditions) and gamma-alumina (acidic conditions). Vesicle monolayer adsorption was observed on gamma-alumina (basic conditions), and yttria stabilized zirconia (YSZ) of varying roughness. Parameters such as buffer pH, surface chemistry and surface roughness were found to have a significant impact on the vesicle adsorption kinetics. Experimental and modeling work was conducted to study formation and characterization of cylindrically supported lipid bilayer membranes. A novel sensing technique (long-period fiber grating refractometry) was utilized to measure the formation mechanism of lipid bilayer membranes on an optical fiber. It was found that the membrane formation kinetics on the fiber was similar to its planar SiO2 counterpart. Fluorescence measurements verified membrane transport behavior and found that characterization artifacts affected the measured transport behavior. / Dissertation/Thesis / Ph.D. Chemical Engineering 2011

La prevalencia de los trastornos metabólicos está creciendo a nivel mundial. Botánicos, como el romero (Rosmarinus officinalis L.) y las semillas de fresno (Fraxinus excelsior L.), pueden ser una alternativa para mejorar estos trastornos. El objetivo de esta tesis doctoral fue generar extractos industriales de las partes comestibles de estas plantas, identificar sus compuestos por HPLC, HPLC-MS y RMN, y determinar su eficacia y seguridad. Se desarrollaron tres extractos de romero - estandarizados al 20% de ácido carnósico, 20% de ácido rosmarínico, y 40% de ácido ursólico. El extracto rico en ácido ursólico mostró la menor capacidad antioxidante en modelos ORAC y FRAP in vitro, mientras que los otros dos extractos tuvieron capacidades más elevadas. Sin embargo, el extracto rico en ácido carnósico (RE) fue superior en la inhibición de la oxidación de LDL ex vivo, y fue el único seleccionado para estudios posteriores. Se desarrolló un extracto de semilla de fresno (FE) de acuerdo a su uso tradicional en Marruecos, siendo identificados salidrosido y 9 glucósidos secoiridoides, dos de ellos fueron descubiertos por primera vez: Excelside A y Excelside B. En estudios in vitro, RE y FE activaron receptores nucleares que regulan la homeostasis de la glucosa y la energía, y actuaron sobre mecanismos contra la obesidad y la dislipidemia - RE activa PPAR? e inhibe la lipasa pancreática, y FE estimula PPAR? y evita la diferenciación de preadipocitos. La eficacia de ambos extractos para el control de trastornos metabólicos fue confirmada en ratones C57BL/6J administrados con una dieta baja en grasa, una dieta alta en grasa, o una dieta alta en grasa más 0.5% RE (HFD.RE) o 0.5% FE (FED) durante 16 semanas. HFD.RE limitó el aumento del peso corporal y grasa en el epidídimo en un 69% (P<0.01) y 79% (P<0.001), respectivamente. HFD.RE también redujo la glucemia (72%, P<0.01) y los niveles de colesterol (68%, P<0.001). FED redujo la glucemia (76.52%, P<0.001), insulinemia (53.43%, P< / Ibarra, SA. (2011). OBTENTION AND CHARACTERIZATION OF ROSEMARY AND ASH TREE SEED EXTRACTS AND STUDY OF THEIR PREVENTIVE EFFECTS ON METABOLIC DISORDERS [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/10795 / Palancia

<p>Dendritic spines are mushroom-shaped appendages on the dendritic branches of neurons. They are invaluable to the function of the brain as they form the major site for excitatory signal transmission in the mammalian brain. These ubiquitous structures have several invaluable and unique characteristics – namely that their morphological and functional characteristics are activity-dependent and undergo remodeling as the spine experiences stimulation. This activity-dependent regulation then in turn modulates the excitatory postsynaptic potential that propagates into the adjacent parent dendrite, and which ultimately reaches the somatic compartment. The mediation of this modulatory effect on the postsynaptic signal by dendritic spines renders them invaluable to the brain’s ability to change neuronal circuits as it learns. The relationship between the structural and functional change in dendritic spines as plasticity is induced remains poorly understood; while efforts have been made to examine the morphology of dendritic spines during plasticity as well as the change to receptor insertion on the postsynaptic density, a comprehensive methodology to interrogate the concomitant changes to several aspects of dendritic spine structure and function as plasticity occurs has not been established. In this study, such a methodology was developed in order to facilitate future study of how a dendritic spine’s diffusional neck resistance, head volume, calcium-sensitive channels (on the postsynaptic density), and excitatory postsynaptic potential amplitude change concurrently as the spine undergoes activity-dependent regulation. This activity-dependent regulation also occurs in groups of spines called “clusters” <em>in vivo</em>, and the structural and functional dynamics of spines as these groups are formed also remains unknown. In order to to facilitate future <em>in vivo</em> studies on how clustered dendritic spines may change dynamically in both structure and function, a methodology for surgically accessing and recording calcium-based activity from the primary auditory cortex was developed, as the frequency-specific tuning of dendritic spines in this cortical area forms a compelling environment in which to study the relationship between spine form and function. </p>