Research was conducted to investigate the effect of phenolic compounds derived from inherently rich antioxidant grape extracts (GE) on α-glucosidase inhibitory activity in vitro and in vivo blood glucose control, oxidative stress, and inflammation associated with obesity-induced type 2 diabetes. Because intestinal α-glucosidase plays a key role in the digestion and absorption of complex carbohydrates, the inhibition of this enzyme is a metabolic target for managing diabetes by improving post-prandial blood glucose control. Initially, red Norton wine grape (Vitis aestivalis) and pomace extracts were evaluated and determined to have notable phenolic content and antioxidant properties. Next, grape skin (GSE) and pomace extract (GPE) were tested and both had in vitro yeast and mammalian α-glucosidase inhibitory activity. The GSE was 32-times more potent at inhibiting yeast α-glucosidase than acarbose, a commercial oral hypoglycemic agent. From HPLC and LC-MS analysis, three phenolics from the GSE (resveratrol, ellagic acid, and catechin) were identified as active inhibitory compounds. The acute administration of GPE (400 mg/kg bw) to mice reduced postprandial blood glucose level by 35% following an oral glucose tolerance test compared to the control. The daily supplementation (250 mg/kg bw) of GSE and GPE for 12-weeks to mice affected fasting blood glucose levels, oxidative stress, and inflammatory biomarkers associated with obesity and type 2 diabetes. At the end of the study, the GSE group gained significantly (P < 0.05) more weight (24.6 g) than the control, high fat, or GPE groups (11.2, 20.2, 19.6 g, respectively). Both GSE and GPE groups had lower fasting blood glucose levels (119.3 and 134.2 mg/dL, respectively) compared to the high fat group (144.6 mg/dL). The 12-week supplementation of GSE was associated with a higher plasma oxygen radical absorbance capacity (ORAC), lower liver lipid peroxidation as measure by TBARS, and lower levels of inflammation as measured by plasma C-reactive protein compared to the high fat group. In conclusion, our collective observations from these studies provide insight into the potential effects of antioxidant rich grape extracts on diabetes-related biomarkers through a dual mechanism of antioxidant protection and specific inhibition of intestinal α-glucosidases. / Ph. D.

Chez les bactéries à Gram-négatif, deux systèmes très bien conservés et essentiels à la survie de la cellule bactérienne ont été identifiés : les systèmes Tol et TonB. Ces deux systèmes utilisent la force proton motrice, issue de la membrane interne et transfert l’énergie associée pour le transport actif de molécules (TonB) ou nécessaire au maintien de l’intégrité membranaire (Tol). Ces 2 systèmes ont été détournés de leurs fonctions initiales et parasités par les colicines, leur conférant un rôle primordial dans le mécanisme d’import de la colicine. Une colicine est une bactériocine (toxine) produite par Escherichia coli pour tuer des souches apparentées. Ce sont des toxines spécifiques et hautement actives. Cependant E.coli a développé des mécanismes de protection afin de résister à l’action cytotoxique des colicines. Ces mécanismes de résistance consistent essentiellement à produire des protéines d’immunité, qui vont pour la plupart se fixer sur le domaine catalytique de la colicine et l’empêcher d’exercer son action létale. La bactérie Yersinia pestis, agent de la peste, possède une colicin-like bactériocine, la pesticine, dont l’activité est de dégrader le peptidoglycane. L’action de la pesticine est inhibée par une protéine d’immunité, Pim, localisée dans le périplasme. Le principal objectif de ce projet est de comprendre les mécanismes d’inhibition de la pesticine par sa protéine d’immunité, grâce à des données biochimiques et structurales, mais aussi d’apporter des solutions pour contourner ce problème de résistance. La structure de la pesticine révèle des homologies structurales avec le T4 lysozyme du bactériophage T4. Pour contourner le problème de la résistance bactérienne liée à la protéine d’immunité, une solution a été de fusionner le domaine de réception/translocation de la pesticine avec le T4 lysozyme. Nous avons ainsi pu créer et résoudre la structure tridimensionnelle d’une protéine chimère fonctionnelle, capable de se fixer sur FyuA (récepteur de la pesticine) et tuer une souche exprimant ce récepteur et dont l’activité létale n’est pas inhibée par Pim. / In Gram-negative bacteria, two essential systems for cell survival have been characterized: the Tol and TonB system. Both Ton and Tol systems are very well conserved in Gram-negative bacteria and coupled to the proton motive force across the inner membrane, acting as energy transducers for active transport (Ton) or maintenance of outer envelope integrity (Tol). Both systems have been embezzled from their primary function and hijacked by colicins as part of the colicin killing pathway. Colicin is a bacteriocin (toxin) produced by and toxic to some strains of Escherichia coli. Colicins are highly effective toxins. However E.coli could develop protective mechanisms to resist to colicin cytotoxic effect. These mechanisms essentially consist to produce an immunity protein. These proteins bind to colicin catalytic domain and inhibit its lethal activity. Yersinia pestis, plague agent, possesses its own colicin-like bacteriocin, Pesticin, which degrades murein. Pesticin activity is inhibited by an immunity protein, Pim, localized in the periplasm. The main goal of this project is to understand inhibition mechanisms between Pim and Pesticin by biochemical and structural data and to provide solution to overcome the resistance issue, since Pesticin was thought to be used as antimicrobial agent. The Pesticin structure has revealed that Pesticin share structural homologies with the T4 lysozyme from the bacteriophage T4. To overcome the resistance issue due to the immunity protein, one solution has been to fuse the Pesticin binding/translocation domain with the T4 lysozyme. Thus, we could engineered and solved the three-dimensional structure of a chimera protein, able to bind FyuA (Pesticin physiological receptor) and kill a FyuA expressing strain, in which the lethal activity is not affected by Pim.

Foram examinadas as propriedades conformacionais de um peptídeo (YRWPFGNHL-NH2) presente na primeira alça extra-celular do receptor AT1 do hormônio angiotensina II. Espectros de dicroísmo circular (CD) e fluorescência foram obtidos em solução aquosa (em função do pH e da temperatura), na presença de um solvente indutor de estrutura secundária (trifluoroetanol, TFE), e na presença de micelas carregadas negativamente (dodecil sulfato de sódio, SDS) ou zwitteriônicas (N-hexadecil-N,N-dimetil-3-amônio-1-propano sulfonato, HPS e lisofosfatidilcolina, liso-PC), e de bicamadas contendo um fosfolipídio zwitteriônico (1-palmitoil-2-oleoil fosfatidilcolina, POPC) ou uma mistura de POPC e um fosfolipídio carregado negativamente (ácido l-palmitoil-2-oleoil fosfatídico, POPA). O estudo por calorimetria de titulação permitiu analisar a termodinâmica da ligação. Espectros de CD indicaram uma estrutura flexível em solução aquosa, modulada pelo pH e pela temperatura. Na presença de TFE, de micelas, e de vesículas de POPC:POPA o peptídeo adquire estrutura secundária, sugerindo a presença de uma dobra beta. Espectros de fluorescência intrínseca (W3) mostraram um deslocamento do comprimento de onda máximo de emissão (&#955;max) para o azul na presença de micelas e de vesículas de POPC:POPA. Observou-se também um aumento da intensidade de fluorescência, exceto no caso de SDS. Esses resultados indicaram que o peptídeo interagiu com os agregados. Não se observou alteração da fluorescência na presença de POPC. Medidas de anisotropia de fluorescência mostraram que, quando ligado, o peptídeo toma-se mais imobilizado. Estudos de supressão de fluorescência empregando agentes supressores aquossolúveis e de membrana sugeriram que o W3 localiza-se próximo à interface bicamada-água. Os resultados mostraram que o peptídeo se liga a micelas zwitteriônicas e negativas, enquanto que, no caso de bicamadas (mais empacotadas), a ligação depende da presença de cargas negativas. Experimentos de calorimetria mostraram que o &#916;H de ligação do peptídeo a vesículas é negativo, compatível com a ocorrência de interações eletrostáticas. Foram observadas diferenças qualitativas e quantitativas na ligação do peptídeo às micelas de HPS e liso-PC. A fim de examinar se essas diferenças eram devidas ao empacotamento molecular, foram obtidos espectros de ressonância paramagnética eletrônica de marcadores de spin intercalados nos dois sistemas. Diferenças foram observadas principalmente na região da cabeça polar, as quais poderiam ser responsáveis pela diferença de comportamento do peptídeo em ambos os agregados. Os resultados obtidos para a primeira alça extra-celular do receptor AT1 indicam que a sua conformação pode ser modulada pelo pH e pela polaridade do ambiente, que ela pode interagir com a membrana através de interações hidrofóbicas e eletrostáticas, e ainda que essa interação depende do grau de empacotamento da fase lipídica. Esses resultados estão de acordo com a visão de que domínios extra-membranares de GPCRs situam-se na interface membrana-água. As alterações conformacionais induzidas pelo meio, bem como pela interação entre domínios extra-membranares de GPCRs e a fase lipídica ou pela ligação do agonista, poderiam ter um papel no mecanismo molecular de transdução de sinal. / In this work the conformational properties of a peptide (YRWPFGNHL-NH2) whose sequence is present in the first extra-cellular loop of the angiotensin II AT1 receptor were examined. Circular dichroism (CD) and fluorescence spectra were obtained in aqueous solution (as a function of pH and temperature), in the presence of a secondary structure inducing solvent (trifluoroethanol, TFE), and in the presence of negatively charged (sodium dodecyl sulfate, SDS) or zwitterionic (N-hexadecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate, HPS and lysophosphatidylcholine, liso-PC) micelles, and of bilayers containing a zwitterionic phospholipid (l-palmitoyl-2-oleoyl phosphatidylcholine, POPC) or a mixture of POPC and a negatively charged phospholipid (l-palmitoyl-2-oleoyl phosphatidic acid, POPA). Studies of titration calorimetry allowed the analysis of the binding thermodynamics. CD spectra indicated a flexible structure in aqueous solution, which was modulated by pH and temperature. In the presence of TFE, micelles and of POPC:POPA vesicles, the peptide acquired secondary structure, which is suggestive of a &#946; turn. The intrinsic fluorescence spectra (W3) showed a blue-shift of the maximum emission wavelenght (&#955;max) in the presence of micelles and of POPC:POPA vesicles. An increase in fluorescence intensity was also observed, except in the case of SDS. These results indicated that the peptide interacted with the aggregates. No fluorescence changes were observed in the presence of POPC. Fluorescence anisotropy measurements showed that, when bound to the aggregates, the peptide becomes more immobilized. Fluorescence quenching studies using water soluble and membrane-bound quenchers suggested that W3 is located close to the water-bilayer interface. The results showed that the peptide binds to negativelly charged and zwitterionic micelles, while in the case of bilayers (which are more tightly packed) the binding depends on the presence of negative charges. Titration calorimetry showed that &#916;H of peptide binding to the vesicles is negative, which is compatible with the ocurrence of eletrostatic interactions. Qualitative and quantitative differences in binding of the peptide to HPS and liso-PC micelles were observed. In order to examine whether these differences were due to molecular packing, electron paramagnetic resonance spectra of spin labels intercalated in both systems were obtained. Differences were observed, mainly in the polar head group region, that could be responsible for the different behaviour displayed by the peptide in both aggregates. The results obtained for the first extra-cellular loop of the AT1 receptor indicated that its conformation can be modulated by pH and by the polarity of the medium, and that it can interact with the membrane through hydrophobic and electrostatic interactions, and also that this interaction depends on the molecular packing of the lipid phase. These results are in aggrement with the idea that the GPCRs extra-membrane domains are located at the water-membrane interface. Conformational changes induced by the medium, as well as by the interaction between extra-cellular segments and the membrane or by ligand binding, could play a role in the molecular mechanism of signal transduction.

Die vorliegende Arbeit präsentiert eine umfassende Untersuchung zu einem speziellen lichtempfindlichen Makromolekül: ein Azobenzol Photoschalter, eingebettet in den Hauptstrang eines synthetischen stäbchenförmigen Polymers. Dazu wurden die Polymere zunächst aus Lösung auf eine Einfachlage Octadecylamin (ODA) aufgebracht, welche auf einer Oberfläche von hochgeordnetem pyrolytischem Graphit (HOPG) lag. Die besondere Eigenschaft der amphiphilen ODAs, sich in Nanolamellen anzuordnen, diente nicht nur der Immobilisierung und Isolierung der stabförmigen Polymere, sondern auch deren Orientierung auf der Oberfläche. Diese Orientierung, insbesondere die Ausrichtung relativ zu den Lamellen, wurde mit einem Modell, basierend auf den hydrophoben W echselwirkungen zwischen den Seitenketten der Polymere und der amphiphilen Unterlage aus ODA, untersucht. Mittels SFM konnte die Kontraktion beziehungsweise Expansion bei Belichtung mit UV- oder sichtbarem Licht untersucht werden. In zeitaufgelösten Messungen wurden lichtinduzierte morphologische Änderungen in Abhängigkeit von der Belichtungszeit der Polymere gemessen. Dies verdeutlichte die lichtinduzierte Bewegung dieser Strukturen innerhalb der Makromoleküle und offenbarte eine kriechende Fortbewegung der Polymere über die Oberfläche. Polymere, die auf Oberflächendefekten nur schwach gebunden waren, änderten ihre Orientierung bei Belichtung zufällig. Daraus schlussfolgere ich, dass die wohldefinierte lokale Umgebung mit Korngrenzen oder Lamellen eine wichtige Rolle als Templat spielt und die Bewegungsrichtung maßgeblich bestimmt. Die entwickelte experimentelle Methode kann verwendet werden, um die Wechselwirkungen zwischen Oberfläche und Polymer zu optimieren, und die anschließende Untersuchung von lichtinduzierten Bewegung der Makromoleküle kann die Entwicklung neuartiger optomechanischer Nanosysteme ermöglichen. / This work presents a comprehensive investigation of one particular photoresponsive macromolecule: Azobenzene photoswitches incorporated into the backbone of synthetic rigid-rod polymers. Firstly, the polymers were deposited from solution onto a monolayer of octadecylamine (ODA) covering the basal plane of highly oriented pyrolytic graphite (HOPG). The unique ODA amphiphilic nanorails, self- assembled on HOPG, served not only to immobilize and isolate the polymers, but also to orient them on the surface. The orientations of rod- like polymers on an ODA surface, i.e., predominally perpendicular or parallel with respect to the underlying lamellar surface, were analyzed with a model based on the hydrophobic interaction of the side chains of the polymers with the amphiphilic nanorails of the ODA molecules. Upon the irradiations with UV and visible light, respectively, large light- induced contractions and extensions of the single macromolecules have been visualized by SFM. An SFM’s time-laps study of morphological changes of polymers at different irradiating times also detailed the light-induced movements within the macromolecules and a crawling movement across the surface. For weakly adsorbed polymers on surface defects, those surface-directed folding/unfolding (or contracting/extending) movements exhibited a random change in orientation. Thus, I conclude, that well-defined local environments, such as domain boundaries or lamellae within the ODA monolayer, play important roles in the template that directs the folding and unfolding movements of polymers during irradiation. The developed setup allows to promote the development of optomechanical nanosystems by optimizing the interaction between single macromolecules and ODA surfaces, followed by visualization of light- induced, on-surface motions of single macromolecules.

Although nonflexible, scaled molecular models like Pauling-Corey's and its descendants have made significant contributions in structural biology research and pedagogy, recent technical advances in 3D printing and electronics make it possible to go one step further in designing physical models of biomacromolecules: to make them conformationally dynamic. We report the design, construction, and validation of a flexible, scaled, physical model of the polypeptide chain, which accurately reproduces the bond rotational degrees-of-freedom in the peptide backbone. The coarse-grained backbone model consists of repeating amide and alpha-carbon units, connected by mechanical bonds (corresponding to phi and psi angles) that include realistic barriers to rotation that closely approximate those found at the molecular scale. Longer-range hydrogen-bonding interactions are also incorporated, allowing the chain to easily fold into stable secondary structures. This physical model can serve as the basis for linking tangible bio-macromolecular models directly to the vast array of existing computational tools to provide an enhanced and interactive human-computer interface. We have explored the boundaries of this direction at the interface of computational tools and physical models of biological macromolecules at the nano-scale. Using a CAD-biocomputational framework, we have provided a methodology to design and build physical protein models focusing on shape and dynamics. We have also developed a workflow and an interface implemented for such bio-modeling tools. This physical-digital interface paradigm, at the intersection of native state proteins (P), computational models (C) and physical models (P), provides new opportunities for building an interactive computational modeling tool for protein folding and drug design. Furthermore, this model is easily constructed with readily obtainable parts and promises to be a tremendous educational aid to the intuitive understanding of chain folding as the basis for macromolecular structure. / Ph. D.

Le présent travail est destiné à acquérir un ensemble de données expérimentales et quantitatives cohérentes sur le comportement physico-chimique d'un système constitué d'un polysaccharide linéaire, flexible et chimiquement fonctionnalisé en groupements carboxyliques, le carboxyméthyl-dextrane (CMD) et d'un milieu ionique comportant des cations d'affinité variée pour ces fonctions : Na+, Ca2+ et Cd2+. La densité de sites et leur constante de dissociation - complexation ont été déterminées par titrage potentiométrique avec des électrodes spécifiques (proton et cadmium). Les propriétés électro-hydrodynamiques et les transitions conformationnelles ont été étudiées en combinant la conductimétrie, l'électrophorèse, la diffusion dynamique de lumière et la viscosimétrie. Enfin, la stabilité colloïdale en relation avec les interactions intermoléculaires a été étudiée par turbidimétrie et diffusion dynamique de lumière. En présence d'ions monovalents, le comportement du CMD, typique d’une particule microgel molle, est déterminé par la force ionique et la concentration en polysaccharide. A basse force ionique, le CMD est en condition de bon solvant lorsqu'il est peu concentré tandis que le recouvrement des doubles-couches électriques autour des macromolécules détermine les propriétés électro-hydrodynamiques du CMD en régime concentré. A haute salinité, les interactions électrostatiques intramoléculaires et interparticulaires sont négligeables, et la macromolécule a un comportement caractéristique de polymère en mauvais solvant à haute fraction volumique. En présence de cations divalents, le calcium, et plus encore le cadmium, entrent en compétition avec le proton pour l’occupation des sites carboxyliques, ce qui s’accompagne par une réorganisation locale des chaînes polymères. A haute force ionique, la taille élevée des agrégats, la vitesse initiale d'agrégation élevée, ainsi que la persistance d'une forte turbidité au maximum d'effet, suggèrent que les agrégats sont formés en régime de type agrégation limitée par la diffusion des particules (DLA) / The present work focused on the acquisition of experimental and quantitative data on the physico-chemical properties of a linear, flexible and chemically functionalized polysaccharide by carboxymethyl grafting, yielding carboxymethyldextran macromolecules (CMD) and an aqueous electrolyte containing various ions Na+, Ca2+ et Cd2+ with different chemical affinity for these chemical functions. Charge density and complexation – dissociation constants have been evaluated using specific electrodes-based potentiometric titration. The electro-hydrodynamic properties and the conformational transitions were examined through electrical conductivity increment measurements, electrophoresis, dynamic light scattering and viscosimetry. Also, colloidal stability has been investigated by means of turbidimetry and dynamic light scattering. In the presence of monovalent ions, the behaviour of CMD, typically that of a soft microgel particle, is strongly depending on ionic strength and polysaccharide content. For low ionic strengths and in dilute regime, CMD is in situation of good solvent while in concentrated regime, overlapping double layers that develop around macromolecules governs the electro-hydrodynamic features of CMD. For sufficiently high ionic strengths, intermolecular and intramolecular electrostatic interactions are nearly suppressed, and CMD behaves as a polymer in bad solvent upon increase of its concentration in the medium. In the presence of divalent ions, calcium ions and cadmium ions are in competition with hydronium ions to occupy the carboxylic sites, and this situation is concomitantly accompanied by local reorganization of polymer chains. The large size of formed clusters, the high initial aggregation rate and the increased turbidity all suggest that aggregates are generated according to a diffusion limited aggregation (DLA) type of mechanism

Este trabalho é direcionado ao estudo dos efeitos da radiação UV sobre amostras de poliestireno (PS) e de copolímeros de estireno com proporções variáveis de 2-trimetilsililóxi-1,3-butadieno (TSB, I), 2-metóxi-1,3-butadieno (MEB, II) e 2-etóxi-1,3-butadieno (ETB, III), em solução aerada, visando principalmente à amplificação da degradação ambiental do PS quando exposto a condições de intemperismo natural. (Ver no arquivo em PDF). Os polímeros foram sintetizados via polimerização radicalar em solução, resultando em uma massa molar ponderai média (Mw) de cerca de 30,0 kg/mol para o homo e copolímeros. Estes produtos, após serem submetidos a um processo de hidrólise na presença de fluoreto de tetrabutilamônio (série TSB) ou HCI aquoso (séries MEB e ETB), resultaram em PS contendo grupos carbonila na cadeia principal sendo, a seguir, isolados e caracterizados por meio de técnicas analíticas como espectroscopia de infravermelho (IR), análise elementar, cromatografia de exclusão por tamanho (SEC), espalhamento de luz estático dependente do tempo (TDSLS), análise térmica (TG e DSC) e microscopia eletrônica de varredura (MEV). As soluções poliméricas foram irradiadas com lâmpada de mercúrio de baixa pressão (99,5% de emissão a 254 nm), na presença de ar e à temperatura ambiente, por um período de dez horas. As alterações sofridas pelos polímeros foram acompanhadas por meio de medidas de IR, TDSLS e SEC. Os espectros no infravermelho mostraram um aumento contínuo da intensidade de algumas bandas: na região entre 3800 - 3000 cm-1, houve um alargamento das bandas que pode ser atribuído à formação de grupos hidroxila e/ou hidroperóxido; na região de 1900 - 1500 cm-1, observou-se o aparecimento de um ombro em 1712 cm-1, que pode ser atribuído à formação de grupos cetona e aldeído como funções terminais na cadeia do polímero e, na região entre 1500 - 1000 cm-1, o alargamento das bandas foi atribuído à formação de grupos anidrido. Os estudos realizados por TDSLS revelaram claramente que os copolímeros degradam com mais rapidez que o PS puro, e as taxas de degradação aumentam proporcionalmente ao aumento no teor de carbonilas incorporadas ao polímero. Tal observação tem suporte nas massas molares ponderais médias obtidas via SEC (Mw) dos fragmentos resultantes da fotólise, com valores cada vez menores com o aumento dos efeitos da fotodegradação, iniciando com cerca de 34,0 kg/mol (TSB5), por exemplo, e decrescendo a valores de Mw tão baixos quanto 4,8 kg/mol (TSB15). Para finalizar, tentou-se verificar o comportamento dos polímeros quando expostos a condições reais de utilização, submetendo-se corpos de prova a um envelhecimento acelerado em câmara de intemperismo artificial e avaliando seu comportamento mecânico. Os resultados mostraram que, para homo e copolímeros, houve uma redução na resistência à tração com o aumento do tempo de exposição (0 a 600 horas), sendo este efeito muito mais acentuado para os copolímeros. / This work was directed to the study of the UV radiation effects upon samples of polystyrene (PS) and copolymers of styrene with different fractions of 2-trimethylsililoxy-1,3-butadiene (TSB, I), 2-methoxy-1 ,3-butadiene (MEB, II) and 2-ethoxy-1,3-butadiene (ETB, III), in normally aerated solution, aiming at the optimization of the PS degradation when exposed to weathering conditions. (See in the PDF file). The polymers were prepared by free-radical polymerization in solution, resulting in average molecular weight (Mw) of about 30.0 kg/mol for the homo and copolymers. The resulting polymers were submitted to hydrolysis in the presence of tetrabutylamonium fluoride (TSB series) or aqueous HCI (MEB and ETB series), resulting in PS containing carbonyls inserted in its backbone. The polymers were isolated and characterized by infrared spectroscopy (IR), elemental analysis, size exclusion chromatography (SEC), time dependent static light scattering (TDSLS), thermal analysis (TG and DSC) and scanning electron microscopy (SEM). The polymeric solutions were irradiated with a low pressure mercury lamp (99.5% emission at 254 nm), in the presence of air at room temperature, for a period of 10 hours. The polymer changes were followed by IR, TDSLS and SEC. The infrared spectra showed a continuous increase in the intensity of some bands: the broad band in the region of 3800 - 3000 cm-1 was assigned to the formation of hydroxides or hydroperoxides groups; the band at 1900 - 1500 cm-1 with a shoulder at 1712 cm-1 can be assigned to ketones or aldehydes, which appear as chain termination groups and the band at 1500 - 1000 cm-1 was attributed to the formation of anhydrides. The TDSLS studies clearly showed that the copolymers degradated much faster than the pure PS, and the degradation rates were directly related to the increase in the TSB, MEB and ETB contents of the polymer. This observation is supported by the decreasing molecular weight values obtained by SEC (Mw) for the resulting fragments from photolysis, when the carbonylic content of the polymer increased, starting at around 34.0 kg/mol (TSB5), for instance, decreasing to Mw values as low as 4.8 kg/mol (TSB15). Finally, we tried to found out the polymer behavior when exposed to the real outdoor conditions of use, by submitting samples to an accelerated aging process in a Weather-o-Meter chamber and evaluating its mechanical behavior afterwards. The results showed that for both, homo and copolymers, there was a reduction in the tensile strength with increasing in the exposition time (0 to 600 hours). This behavior is much stronger in the copolymers.

In this thesis, fluid characteristics and sampling efficiency of high molecular weight cut-off microdialysis are presented, with the aim of improving the understanding of microdialysis sampling mechanisms and its performance regarding extraction efficiency of biological fluid and biomarkers. Microdialysis is a well-established clinical sampling tool for monitoring small biomarkers such as lactate and glucose. In recent years, interest has raised in using high molecular weight cut-off microdialysis to sample macro molecules such as neuropeptides, cytokines and proteins. However, with the increase of the membrane pore size, high molecular weight cut-off microdialysis exhibits drawbacks such like unstable catheter performance, imbalanced fluid recovery, low and unstable molecule extraction efficiency, etc. But still, the fluid characteristics of high molecular weight cut-off microdialysis is rarely studied, and the clinical or in vitro molecule sampling efficiency from recent studies vary from each other and are difficult to compare.   Therefore, in this thesis three aspects of high molecular weight cut-off microdialysis have been explored. The first, the fluid characteristics of large pore microdialysis has been investigated, theoretically and experimentally. The results suggest that the experimental fluid recovery is in consistency with its theoretical formula. The second, the macromolecule transport behaviour has been visualized and semi-quantified, using an in vitro test system and fluorescence imaging. The third, two in vitro tests have been done to mimic in vivo cerebrospinal fluid sampling under pressurization, using native and differently surface modified catheters. As results, individual protein/peptide extraction efficiencies were achieved, using targeted mass spectrometry analysis. In summary, a theory system of the fluid characteristics of high molecular weight cut-off microdialysis has been built and testified; Macromolecular transport of microdialysis catheter has been visualized; In vivo biomolecules sampling has been simulated by well-defined in vitro studies; Individual biomolecular extraction efficiency has been shown; Different surface modifications of microdialysis catheter have been investigated. It was found that, improved sampling performance can be achieved, in terms of balanced fluid recovery and controlled protein extraction efficiency.

Este trabalho é direcionado ao estudo dos efeitos da radiação UV sobre amostras de poliestireno (PS) e de copolímeros de estireno com proporções variáveis de 2-trimetilsililóxi-1,3-butadieno (TSB, I), 2-metóxi-1,3-butadieno (MEB, II) e 2-etóxi-1,3-butadieno (ETB, III), em solução aerada, visando principalmente à amplificação da degradação ambiental do PS quando exposto a condições de intemperismo natural. (Ver no arquivo em PDF). Os polímeros foram sintetizados via polimerização radicalar em solução, resultando em uma massa molar ponderai média (Mw) de cerca de 30,0 kg/mol para o homo e copolímeros. Estes produtos, após serem submetidos a um processo de hidrólise na presença de fluoreto de tetrabutilamônio (série TSB) ou HCI aquoso (séries MEB e ETB), resultaram em PS contendo grupos carbonila na cadeia principal sendo, a seguir, isolados e caracterizados por meio de técnicas analíticas como espectroscopia de infravermelho (IR), análise elementar, cromatografia de exclusão por tamanho (SEC), espalhamento de luz estático dependente do tempo (TDSLS), análise térmica (TG e DSC) e microscopia eletrônica de varredura (MEV). As soluções poliméricas foram irradiadas com lâmpada de mercúrio de baixa pressão (99,5% de emissão a 254 nm), na presença de ar e à temperatura ambiente, por um período de dez horas. As alterações sofridas pelos polímeros foram acompanhadas por meio de medidas de IR, TDSLS e SEC. Os espectros no infravermelho mostraram um aumento contínuo da intensidade de algumas bandas: na região entre 3800 - 3000 cm-1, houve um alargamento das bandas que pode ser atribuído à formação de grupos hidroxila e/ou hidroperóxido; na região de 1900 - 1500 cm-1, observou-se o aparecimento de um ombro em 1712 cm-1, que pode ser atribuído à formação de grupos cetona e aldeído como funções terminais na cadeia do polímero e, na região entre 1500 - 1000 cm-1, o alargamento das bandas foi atribuído à formação de grupos anidrido. Os estudos realizados por TDSLS revelaram claramente que os copolímeros degradam com mais rapidez que o PS puro, e as taxas de degradação aumentam proporcionalmente ao aumento no teor de carbonilas incorporadas ao polímero. Tal observação tem suporte nas massas molares ponderais médias obtidas via SEC (Mw) dos fragmentos resultantes da fotólise, com valores cada vez menores com o aumento dos efeitos da fotodegradação, iniciando com cerca de 34,0 kg/mol (TSB5), por exemplo, e decrescendo a valores de Mw tão baixos quanto 4,8 kg/mol (TSB15). Para finalizar, tentou-se verificar o comportamento dos polímeros quando expostos a condições reais de utilização, submetendo-se corpos de prova a um envelhecimento acelerado em câmara de intemperismo artificial e avaliando seu comportamento mecânico. Os resultados mostraram que, para homo e copolímeros, houve uma redução na resistência à tração com o aumento do tempo de exposição (0 a 600 horas), sendo este efeito muito mais acentuado para os copolímeros. / This work was directed to the study of the UV radiation effects upon samples of polystyrene (PS) and copolymers of styrene with different fractions of 2-trimethylsililoxy-1,3-butadiene (TSB, I), 2-methoxy-1 ,3-butadiene (MEB, II) and 2-ethoxy-1,3-butadiene (ETB, III), in normally aerated solution, aiming at the optimization of the PS degradation when exposed to weathering conditions. (See in the PDF file). The polymers were prepared by free-radical polymerization in solution, resulting in average molecular weight (Mw) of about 30.0 kg/mol for the homo and copolymers. The resulting polymers were submitted to hydrolysis in the presence of tetrabutylamonium fluoride (TSB series) or aqueous HCI (MEB and ETB series), resulting in PS containing carbonyls inserted in its backbone. The polymers were isolated and characterized by infrared spectroscopy (IR), elemental analysis, size exclusion chromatography (SEC), time dependent static light scattering (TDSLS), thermal analysis (TG and DSC) and scanning electron microscopy (SEM). The polymeric solutions were irradiated with a low pressure mercury lamp (99.5% emission at 254 nm), in the presence of air at room temperature, for a period of 10 hours. The polymer changes were followed by IR, TDSLS and SEC. The infrared spectra showed a continuous increase in the intensity of some bands: the broad band in the region of 3800 - 3000 cm-1 was assigned to the formation of hydroxides or hydroperoxides groups; the band at 1900 - 1500 cm-1 with a shoulder at 1712 cm-1 can be assigned to ketones or aldehydes, which appear as chain termination groups and the band at 1500 - 1000 cm-1 was attributed to the formation of anhydrides. The TDSLS studies clearly showed that the copolymers degradated much faster than the pure PS, and the degradation rates were directly related to the increase in the TSB, MEB and ETB contents of the polymer. This observation is supported by the decreasing molecular weight values obtained by SEC (Mw) for the resulting fragments from photolysis, when the carbonylic content of the polymer increased, starting at around 34.0 kg/mol (TSB5), for instance, decreasing to Mw values as low as 4.8 kg/mol (TSB15). Finally, we tried to found out the polymer behavior when exposed to the real outdoor conditions of use, by submitting samples to an accelerated aging process in a Weather-o-Meter chamber and evaluating its mechanical behavior afterwards. The results showed that for both, homo and copolymers, there was a reduction in the tensile strength with increasing in the exposition time (0 to 600 hours). This behavior is much stronger in the copolymers.

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