L’ischémie reperfusion pulmonaire et sa traduction clinique la dysfonction primaire du greffon sont responsables d’une morbi-mortalité importante en transplantation pulmonaire aussi bien à court terme qu’à long terme. Nous avons voulu faire la démonstration de la pertinence des microparticules comme marqueur de l’ischémie reperfusion pulmonaire. Nous avons reproduit et validé la stabilité d’un modèle de perfusion ventilation ex vivo de poumon de rat aussi bien en conditions normales (pas d’ischémie pulmonaire avant reperfusion) qu’en conditions extrêmes (1 h d’ischémie chaude avant reperfusion pulmonaire). Nous avons étudié la génération de microparticules par des poumons soumis à des conditions variables d’ischémie froide et d’ischémie chaude. Les poumons soumis à de fortes conditions d’ischémie froide (20h) produisent un pic précoce de microparticules d’origine épithéliale alvéolaire, leucocytaire et endothéliale. Nous en concluons que le modèle de perfusion ventilation ex vivo de poumons de rats est un modèle pertinent pour l’étude des réactions d’ischémie reperfusion. Les microparticules apparaissent comme un marqueur précoce des lésions d’ischémie reperfusion pulmonaires dans ce modèle. / Lung ischemia reperfusion and its clinical expression as primary graft dysfunction are provider of immediate and long term morbidity and mortality for patients. We aimed at demonstrating the usefulness and relevance of microparticles as biomarkers for lung ischemia reperfusion injury. We first reproduced an ex vivo rat lung perfusion and ventilation experimental model. Stability of the model was validated for normal conditions (no ischemia before reperfusion) as well as for extreme conditions (1 hour warm ischemia before reperfusion). The generation of microparticles was studied in that model for variable conditions of cold ischemia and for warm ischemia. Lung submitted to strong ischemic injury (20hours cold ischemia) generate an early pike of microparticles originated from leukocyes, endothelial cells, and epithelial alveolar cells. We may conclude the ex vivo model of rat lung perfusion and ventilation is relevant for the study of lung ischemia reperfusion injury. Microparticles are relevant markers of rat lung ischemia reperfusion injury in our model.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-08-31T17:28:06Z No. of bitstreams: 1 Thassila Nogueira Pitanga Efeito das micropartículas....pdf: 1105021 bytes, checksum: 9c6f0280b9ffe2b38105f8844c3a2b0f (MD5) / Made available in DSpace on 2012-08-31T17:28:06Z (GMT). No. of bitstreams: 1 Thassila Nogueira Pitanga Efeito das micropartículas....pdf: 1105021 bytes, checksum: 9c6f0280b9ffe2b38105f8844c3a2b0f (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / Micropartículas (MPs) são vesículas liberadas da membrana plasmática após ativação celular ou fases iniciais de apoptose. MPs de neutrófilos (NMPs) apresentam em suas membranas diferentes proteínas com diferentes funções e diretamente relacionadas com seus efeitos biológicos, tais como mediadores inflamatórios (integrinas e L-selectina) e mieloperoxidase (MPO). A MPO é uma enzima catiônica, presente nos grânulos azurófilos de neutrófilos e monócitos, responsável pela formação de espécies reativas de oxigênio (ROS). Na presença de peróxido de hidrogênio (H2O2), a enzima catalisa a oxidação de íons cloro com a formação de ácido hipocloroso (HOCl). MPs de diversas origens parecem afetar a função vascular por mecanismos dependentes do estresse oxidativo. No presente trabalho investigamos o possível papel de NMPs na lesão de células endoteliais vasculares in vitro. NMPs foram produzidas por ativação de polimorfonucleares (PMNs) humanos com ionóforo de cálcio e caracterizadas por microscopia eletrônica de transmissão e varredura como estruturas delimitadas por bicamada lipídica e com diâmetro menor do que 1 m. A presença da MPO foi confirmada por citometria de fluxo, utilizando o anticorpo anti-MPO, e pela atividade enzimática detectada por quimioluminescência de HOCl, uma ROS catalisada unicamente pela MPO. A adição de azida, inibidor de peroxidases, ou de taurina, “scavanger” de HOCl, inibiu ou reduziu a atividade enzimática, respectivamente. Identificamos moléculas expressas na superfície das NMPs (CD62L, CD66b, CD45, CD95 e fosfatidilserina). A atividade de MPO foi observada após incubação das células endoteliais vasculares in vitro com NMPs, sugerindo que estas apresentam um potencial de carrear a MPO para a superfície endotelial vascular. A exposição de células endoteliais a NMPs induziu alterações morfológicas e perda da integridade da membrana celular, analisadas, respectivamente, por microscópio de contraste de fase e microscopia de fluorescência. Nossos resultados sugerem que NMPs atuam como carreadoras de MPO para a superfície de células endoteliais vasculares e induzem lesão destas células em um mecanismo dependente de MPO, sugerindo sua participação no desenvolvimento das complicações cardiovasculares. / Microparticles (MPs) are vesicles released from the plasma membrane after cell activation or early stages of apoptosis. In its membranes, neutrophil MPs (NMPs) present different proteins with different functions, directly associated with the cell’s biological activities, such as inflammatory mediation via integrins, L-selectin and myeloperoxidase (MPO). MPO is a cationic enzyme found in the azurophilic granules of neutrophils and monocytes that produces reactive oxygen species (ROS). In the presence of hydrogen peroxide (H2O2) it catalyzes the oxidation of chlorine ions with the formation of hypochlorous acid (HOCl). MPs from different sources seem to affect vascular function through oxidative stress dependent mechanisms. In this study we investigated the possible role of NMP in vascular endothelial cell injury in vitro. NMPs were produced by activation of human polymorphonuclear (PMN) cells with calcium ionophore and characterized as structures bounded by lipid bilayer and with less than 1 m diameter by transmission and scanning electron microscopy. MPO presence was confirmed by flow cytometry using anti-MPO antibody, and the enzyme activity detected by chemiluminescence of HOCl, a ROS catalyzed solely by the MPO. The addition of azide, peroxidase inhibitor, or taurine, HOCl scavanger, inhibited and reduced enzymatic activity, respectively. We identified molecules expressed on the NMPs surface (CD62L, CD66b, CD45, CD95 and phosphatidylserine). MPO activity was observed after incubation of vascular endothelial cells in vitro with NMPs, suggesting that they have the potential to carry MPO to the vascular endothelial surface. Exposure of endothelial cells to NMPs induced morphological changes and loss of cell membrane integrity, which were evaluated, respectively, by phase contrast microscopy and fluorescence microscopy. Our findings suggest that NMPs act as carriers of MPO to the surface of vascular endothelial cells and induce damage to these cells with an MPO-dependent mechanism, suggesting its participation in the development of cardiovascular complications.

Made available in DSpace on 2014-06-11T19:25:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-15Bitstream added on 2014-06-13T20:33:03Z : No. of bitstreams: 1 rossanezi_g_me_arafcf.pdf: 544804 bytes, checksum: 730652528635cea19a056b12c29e4b28 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / As doenças que afetam o globo ocular de maneira geral têm a terapêutica limitada pela grande dificuldade de se atingir e manter níveis efetivos de fármacos nessa região. Isso porque as formas farmacêuticas convencionais para as doenças oculares são destinadas a aplicação tópica, e não proporcionam níveis terapêuticos no corpo vítreo, retina e coróide. Dessa forma, os sistemas de liberação prolongada de fármacos para aplicação intraocular representam um interesse significativo para a terapêutica em oftalmologia. Sendo assim, o presente trabalho teve como objetivo produzir através da técnica de “Spray Drying” micropartículas biodegradáveis a partir do ácido poli-láctico-co-glicólico (PLGA) contendo cetorolaco de trometamina (CETO), um antiinflamatório não-esteróide que tem apresentado resultados relevantes no tratamento pós-operatório de cirurgias oftálmicas, proporcionando maior conforto e diminuindo os efeitos colaterais causados pelos antiinflamatórios esteróides. Utilizando o método proposto foram produzidas micropartículas com diferentes proporções de CETO:PLGA. As micropartículas foram visualizadas através da Microscopia Eletrônica de Varredura (MEV), apresentando formato esférico e uniforme, com superfícies lisas, e o tamanho médio e distribuição de tamanho das partículas foram determinados através do espalhamento de luz. Somente uma das amostras foi descartada, devido a não formação de microesferas. As propriedades físico-químicas de todos os sistemas foram estudadas utilizando espectroscopia de infravermelho (IR), calometria diferencial exploratória (DSC) e termogravimetria/termogravimetria derivada. Os resultados destes estudos mostraram que após a obtenção as estruturas químicas dos componentes foram preservadas... / The diseases that affect the ocular globe generally have limited treatment due their difficulty to achieve and sustain effective levels of drugs in this region. Because conventional pharmaceutical forms for ocular diseases are destined to topical application, and not provide therapeutic levels in vitreous, retina and choroid. In this way, the ocular drug delivery systems represent a strategy for therapy in ophthalmology. Thus, the present work had as aim to produce through Spray drying technique biodegradable microparticles of poly-lactic co-glycolic acid (PLGA) containing ketorolac tromethamine (CETO), a nonsteroidal anti-inflammatory that has presented relevant results in post-operative treating of ophthalmic surgery, providing greater comfort and reducing the adverse effects caused by steroidal anti-inflammatory. Using the considered method were produced microparticles with different ratios of CETO: PLGA. The microparticles were accessed through Scanning Electronic Microscopy (SEM), presenting itself spherical and uniform, smooth surface, and the particle size analysis and mean diameter were determined by Dynamic Light Scattering. Only one non spherical sample was dismissed. The physicochemical properties of all systems were studied using infrared spectroscopy (IR), differential scanning calorimetry (DSC) and thermogrametry/ derivative thermogravimetry (TG/DTG). The results of these studies showed that after produced the chemical structure of components were preserved, only having the necessaries interactions to promote prolonged released. The amounts of encapsulated CETO were determined by UV-Vis spectrophotometry... (Complete abstract click electronic access below)

Cell-derived microvesicles (MVs) are naturally released into the human circulation and an increase in the concentration of certain MV populations have been observed after exercise. However, the MV appearance dynamics, the exercise-related stimuli that induce their formation and physiological relevance are poorly understood. Hence, the overall objectives of this thesis were to: 1) characterise the circulating platelet (PMV) and endothelial-derived MVs (EMVs) responses during exercise and recovery, as well as their arteriovenous dynamics, 2) investigate the potential role of haemodynamic forces on MVs formation in vivo by vascular shear stress manipulations, and 3) explore the putative proliferative, chemotactic and angiogenic potential of exercise-derived MVs upon human vascular endothelial cells in vitro. Chapter 5 of this thesis describes the time-course of MV appearance in response to prolonged cycling, and demonstrates that intravascular [PMV] increases during and after exercise performed in the heavy intensity domain, whereas [EMV] remains unaltered. Moreover, [PMV] during exercise was related to estimates of vascular shear stress and plasma noradrenaline levels. Results from chapter 6 revealed that PMVs increased in the arterial circulation during passive heat stress, and in the arterial as well as venous circulation during short duration very heavy exercise engaging either a large or small muscle mass. The increases in [PMV] were not directly linked to local changes in vascular shear stress through heat stress and exercise, indicating a systemic PMV response. Finally, chapter 7 revealed that exercise-derived MVs supported endothelial proliferation and migration, while displaying pro-angiogenic potential in vitro. In conclusion, results of this thesis provide original information about MV dynamics, by demonstrating that PMV increase systemically in the circulation not only after but during exercise involving a small and large muscle mass. This MV response seems to be modulated by exercise intensity, and is only partially linked to levels of vascular shear stress. Moreover, circulating MVs produced during exercise present stimulatory angiogenic and mitogenic effects upon endothelial cells in vitro, suggesting a novel potential link between vascular adaptation and exercise training.

O presente trabalho teve como objetivo desenvolver e avaliar a eficácia de micro e nanopartículas lipídicas sólidas contendo óleo de café verde, cera de abelha e alfa-tocoferol. O óleo de café verde (OCV) foi quantificado por cromatografia gasosa, apresentando em sua composição ácido palmítico (28,74%), ácido linoléico (42,77%), ácido oleico (12,51%), ácido esteárico (10,62%), ácido araquídico (3,57%) entre outros ácidos graxos (1,79%), estes valores foram próximos àqueles descrito na literatura. As micropartículas foram preparadas pelo método de spray congealing e as nanopartículas pela técnica da microemulsão a quente. Os rendimentos de micropartículas variaram de 42 a 58% com tamanhos entre 63,3 a 101,2 ?m. Os rendimentos das nanopartículas variaram entre 96 a 97% e com tamanhos entre 249 a 766 nm. Posteriormente foram definidas as condições para o processo de produção das micro e nanopartículas, bem como a concentração de OCV, alfa tocoferol e cera de abelha (CA). Um estudo mais detalhado das proporções de OCV, cera de abelha e alfatocoferol foi realizado com o auxílio de um planejamento de misturas, avaliando a estabilidade oxidativa por testes Rancimat e Termogravimétricos. Porém, nas condições do presente estudo, esses resultados não foram estatisticamente diferentes, por isso escolheu-se a maior concentração de OCV proposta (50%). Após estas etapas, as partículas foram adicionadas em gel aristoflex®, bem como o OCV em sua forma líquida, formando três géis de mesma concentração para comparar e avaliar a viabilidade do preparo de micro e nanopartículas frente a forma convencional já comercializada. Estudos em Artemia salina foram realizados como teste preliminar para avaliar a citotoxicidade dos três géis, sendo que em concentrações de 500?g/mL os géis contendo nanopartículas apresentaram maior letalidade do que aqueles compostos por micropartículas ou OCV líquido. Testes de FPS in vitro apresentaram baixos índices de proteção solar, entre 0,19 e 0,28, porém o OCV apresenta proteção contra os raios UVB podendo ser utilizado como potencializador na ação fotoprotetora de filtros químicos. As análises de viabilidade celular mostraram que mesmo na forma de micro ou nanopartículas, o OCV e a cera de abelhas se mantiveram seguros para a utilização em formulações tópicas. Nos testes de estabilidade química, transcorrido o tempo de armazenamento, os géis contendo OCV em sua forma líquida apresentaram teores de ácido palmítico de 56 e 38%, nas temperaturas de 25oC e 40oC respectivamente. Enquanto que as micropartículas apresentaram teores de 69 e 53% e as nanopartículas 73 e 69%, nas referidas temperaturas. Nos testes clínicos, as micropartículas apresentaram um melhor desempenho para a melhora do conteúdo aquoso do estrato córneo depois de 2 horas de aplicação do produto. Já as nanopartículas apresentaram os melhores resultados para a perda de água transepidérmica, conferindo à pele uma menor perda de água. Nos testes de permeação cutânea, não houve quantificação pelo método utilizado em nenhum tempo da fase receptora, porém o presente estudos apresentou maiores concentrações do ácido palmítico na derme dos ensaios feitos para as nanopartículas e no estrato córneo daqueles feitos para as micropartículas. Portanto, diante dos resultados obtidos, a micro e nanoencapsulação do OCV é uma alternativa interessante para aumentar a estabilidade deste óleo, além de promover melhoras em sua eficácia. / This study aimed to develop and assess the effectiveness of solid lipid micro and nanoparticles containing green coffee oil, beeswax and alpha-tocopherol. Green coffee oil was quantified by gas chromatography, showing the composition in their palmitic acid (28.74%), linoleic acid (42.77%), oleic acid (12.51%), stearic acid (10.62%), arachidic acid (3.57%) and other fatty acids (1.79%), these values were similar to those described in the literature. The microparticles were prepared by spray congealing method, and the nanoparticles by the technique of hot microemulsion. Microparticles yields ranged 42-58% in size from 63.3 to 101.2 micrometers. Yields of nanoparticles ranged from 96-97% and ranging in size from 249-766 nm. Thereafter the conditions were set for the process of production of micro and nanoparticles, as well as the concentration of green coffee oil, alpha tocopherol and beeswax. A more detailed study of the proportions of GCO, beeswax and alpha-tocopherol was performed with the aid of a planning mixtures evaluating the oxidative stability by Rancimat and thermogravimetric tests. However, under the conditions of this study, these results were not statistically different, so it was chosen the highest concentration of green coffee oil proposal (50%). After these steps, the particles were added in aristoflex® gel, and the green coffee oil in liquid form, forming three gels of the same concentration to compare and assess the viability of micro and nanoparticles prepared against conventionally already commercialized. Studies were conducted on Artemia salina as a preliminary test to evaluate the cytotoxicity of the three gels, whereas at concentrations of 500?g/ml nanoparticles containing gels had a higher mortality than those composed of microparticles or liquid GCO. SPF vitro tests showed low levels of sunscreen, between 0.19 and 0.28, but GCO shows protection against UVB rays can be used as potentiating the action of chemical sunscreen filters. The cell viability tests have shown that even in the form of microparticles or nanoparticles, the GCO and beeswax kept safe for use in topical formulations. In the chemical stability test, the elapsed storage time, the gels containing GCO presented in liquid form palmitic acid levels of 56 and 38% at temperatures of 25°C and 40°C respectively. While the microparticles showed levels of 69 and 53% and the nanoparticles 73 and 69% in those temperatures. In clinical tests, the microparticles showed a better performance for the improvement of the water content of the stratum corneum after 2 hours of application of the product. Already nanoparticles showed the best results for the transepidermal water loss, giving the skin a smaller loss water. The skin permeation tests, no quantification by the method used at any time from the receiving phase, but this study showed higher concentrations of palmitic acid in the dermis of the tests made to the nanoparticles and the stratum corneum of those made to the microparticles. Therefore, opposite the results obtained, the micro and nanoencapsulção of green coffee oil is an interesting alternative to increase the stability of this oil, and promote improvements in their effectiveness

Les nanotubes de carbone (CNTs), intégrant à la fois la structure parfaite, la géométrie unique, et des propriétés exceptionnelles, sont d'une grande importance dans le domaine des nanotechnologies. Leur association avec d'autres matériaux produit de nouvelles propriétés remarquables, et étend par conséquent leurs domaines d'applications comme charges multifonctionnelles. Cette thèse vise à développer un nouveau matériau hybride avec une structure multi-échelle à base de CNTs et de particules micrométriques d'alumine (mAl2O3) par une méthode de dépôt chimique en phase vapeur (CVD). Nos études démontrent que les structures CNTs-mAl2O3 ont une propriété exceptionnelle en matière de transport thermique dans les composites polymères. Celle-ci nous a amenée à explorer plus en profondeur les mécanismes de l’organisation des CNTs sur mAl2O3, et d’étudier la cinétique de réactions chimiques dans l’espace gazeux du réacteur CVD. Dans le premier chapitre, nous faisons une revue de l'état de l'art sur la structure, les propriétés et les applications des CNTs, ainsi que les mécanismes de croissance de CNTs par CVD. Une attention particulière est également accordée aux structures hybrides nano-micrometriques qui sont synthétisées par greffage in-situ des CNTs sur des substrats micrométriques. Dans le deuxième chapitre, nous présentons trois types de structures hybrides, qui sont classifiées selon différents modes d'organisation des CNTs sur les microsphères d'alumine. L'évolution des structures hybrides est démontrée en faisant varier le diamètre, la longueur et la densité numérique des CNTs sur mAl2O3. L’organisation specifique et la dispersion homogène des CNTs permettent de diminuer considérablement leurs résistances de contacts thermiques lorsque les matériaux hybrides CNTs-mAl2O3 sont utilisés comme charges dans les composites polymères. Une amélioration importante de la conductivité thermique des composites Epoxy/CNTs-mAl2O3, par rapport à celle des composites constitués de CNTs et de résine époxy, est obtenue à une fraction massique ultra-faible en CNTs. Dans le troisième chapitre, nous avons étudié en détail les rôles joués par les paramètres CVD et les microparticules sphériques d’alumine dans la construction de structures hybrides multiformes. En particulier, les fortes corrélations entre la température, les sources de carbone et les ratios d'hydrogène ont été discutées. Le lien entre les CNTs et les microparticules est mis en évidence, ainsi que la dynamique de croissance des CNTs. L’auto-organisation des CNTs sur mAl2O3 est expliquée par les deux mécanismes suivants. Dans un premier temps, la structure hétérogène de la surface des particules entraîne une distribution différente des particules du catalyseur, et leur arrangement cristallin spécifique détermine potentiellement l’orientation des CNTs. Dans un deuxième temps, l'auto-assemblage des CNTs est dû à l’interaction des forces faibles de Van der Waals entre CNTs voisins. Le calcul basé sur le modèle du nano-cantilever montre que l’auto-assemblage des CNTs dépend fortement de leur diamètre, de leur longueur et de leur densité numérique sur mAl2O3. Dans le quatrième chapitre, la cinétique chimique des réactions dans l’espace gazeux du réacteur CVD est numériquement analysée. Le processus non-équilibré de CVD qui contient plusieurs phénomènes physico-chimiques est simulé avec succès en combinant la cinétique des réactions chimiques avec les phénomènes de transport physique. Les champs des concentrations de chaque espèce est révélée aux températures utilisées par simulation des réactions chimiques. Les sources effectives de carbone et de fer pour la croissance des CNTs ont été éclaircies en comparant les résultats de simulation avec les observations expérimentales, y compris les mesures de spectrométrie de masse. Ces analyses sont nécessaires pour améliorer la production des hybrides avec des structures homogènes. / Carbon nanotubes (CNTs), integrating perfect structure, unique geometry, and exceptional properties, are of great significance in nanotechnology. Their hybridization with a variety of other materials generates huge amounts of attractive properties, and thus expands largely their application fields as multifunctional fillers. This thesis aims to develop a novel multi-scale hybrid material based on carbon nanotubes and micrometer alumina particles (mAl2O3) by an in-situ floating chemical vapor deposition (CVD) method. Our studies demonstrate that the CNTs-mAl2O3 structures have outstanding thermal transport properties in polymer composites. This greatly motivates us to further explore the organization mechanisms of CNTs on microparticles, and to investigate gas phase chemical reaction kinetics in CVD reactor. In the first chapter, we review the state of the art of research in CNT structure, properties and applications, as well as CNT growth mechanisms in CVD. Special attention is also paid to the nano-micro hybrid structures which are synthesized by in-situ grafting CNTs on micrometer substrates. In the second chapter, we present three types of hybrid structures which are classified according to distinct CNT organization patterns on alumina microspheres. The evolution of the hybrid structures is demonstrated by varying CNT diameter, length and number density on mAl2O3. The specific organization and homogeneous dispersion of CNTs could significantly reduce their thermal contact resistances when the CNTs-mAl2O3 hybrid materials are used as fillers in polymer composites. Enhanced thermal conductivities of the Epoxy/CNTs-mAl2O3 composites are obtained at ultra-low CNT weight fractions compared with that of the composites constituted of pristine CNTs and epoxy. In the third chapter, we investigate in detail the roles played by CVD parameters and alumina spherical microparticles in the construction of multiform hybrid structures. In particular, the strong correlations among the temperature, carbon sources and hydrogen ratios are discussed. The connection between the CNTs and the microparticles are demonstrated, along with the CNT growth dynamics on mAl2O3. The self-organization behavior of CNTs on mAl2O3 is explained by the following two mechanisms: first, heterogeneous surface structures of mAl2O3 generate varied nucleation of catalyst particles, and their specific crystal arrangement potentially determines CNT growth orientations; second, the self assembly of CNTs is due to weak Van der Waals interaction forces between neighboring nanotubes. The calculation based on the nano-cantilever model shows that the CNT self assembly is greatly dependent on their diameter, length and number density on mAl2O3. In the forth chapter, gas phase chemical reaction kinetics in CVD reactor is numerically analyzed. The non-equilibrium CVD processes which involve multi physical-chemical phenomena are successfully simulated by combining the chemical reaction kinetics with the physical transport phenomena. The space-dependent concentration distribution of each species is revealed by simulating the reacting fluid at the used temperatures. The effective carbon and iron precursors for CNT growth are illuminated by comparing simulation results with experimental observations including mass spectrometry measurements. These analyses of chemical reactions in CVD system are helpful to improve the production of the hybrids with homogeneous structures.

Orientadores: Carlos Raimundo Ferreira Grosso / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-10T13:38:28Z (GMT). No. of bitstreams: 1 Leonel_AldaJusceline_M.pdf: 1114899 bytes, checksum: 0e2498fa7d8941ce552ac5eced6c86a2 (MD5) Previous issue date: 2008 / Resumo: Apesar de micropartículas lipídicas possuírem vantagens, como baixa toxicidade e não utilizarem solventes orgânicos em sua produção, elas apresentam alguns inconvenientes como a baixa capacidade de encapsulação do recheio e a possibilidade de expulsão do material de recheio encapsulado durante o armazenamento. Este trabalho teve como objetivo estudar a formação e a estabilidade de micropartículas de lipídios preparadas por spray cooling compostas por ácidos graxos (esteárico e oléico) e gordura vegetal hidrogenada como matriz, lecitina de soja como tensoativo e solução de glicose como material de recheio. Foram avaliados morfologia de superfície, tamanho de partícula, quantidade de recheio encapsulada e não encapsulada (superficial) e comportamento de liberação do recheio em solução aquosa. Para o estudo da estabilidade, medidas da retenção do recheio ao longo do tempo em temperatura controlada, medidas de difração de raios-X (0, 7, 14, 21 e 28 dias) e medidas calorimétricas (tempo zero) foram efetuadas. Valores de eficiência de encapsulação foram superiores a 70%, e as maiores quantidades de glicose não encapsulada foram encontradas nas formulações nas quais foram adicionadas maiores quantidades de recheio. A liberação do recheio também apresentou correlação com a quantidade de recheio utilizado, onde menores quantidades de recheio foram mais bem retidas. Os diâmetros médios das micropartículas situaram-se entre 25 e 32 µm, e as partículas apresentaram paredes rugosas porém contínuas. No estudo da estabilidade, as formulações com maiores quantidades de recheio (solução de glicose 80%) ou sem a adição de tensoativo (solução de glicose 40%, sem lecitina) apresentaram, na quantidade de glicose superficial, 18 e 56% de recheio não encapsulado no último dia de análise, respectivamente, sendo significativamente diferente das outras duas formulações, uma com solução de glicose 60% e outra com solução de glicose 40%, que apresentaram resultados de 5% e 2% no último dia de análise. Os resultados de difração de raios-X não apresentaram alterações ao longo dos 28 dias de estudo e os resultados de DSC foram semelhantes no tempo zero entre as diferentes formulações estudadas. A utilização da mesma matriz lipídica nas formulações parece responsável pelos resultados da difração de raios-X e calorimétricos obtidos / Abstract: Although lipid microparticles have advantages, just as low toxicity and no use of organic solvents in their production, they present some incovenients, as low capacity of encapsulation of core material and the expulsion of encapsulated core during its storage. The aim of this work was to study the formation and the stability of lipid microparticles prepared by spray cooling, composed by fatty acids (stearic and oleic) and hydrogenated vegetable fat as matrix, soya lecithin as surfactant and glucose solution as core material. For this propose, surface morphology, particle size, no encapsulated (surficial) core measure, total encapsulated core material and core release behavior in aqueous solution were evaluated. The stability study was evaluated with measures of core retention along of the time in controlled temperature, X-ray diffraction measurements (0, 7, 14, 21 and 28 days) and calorimetric measurements (zero time). The efficiency of encapsulation was above 70% and the biggest amounts of glucose no encapsulated were found in the formulations in which were added the biggest amount of core material. Results of release of core material also showed correlation with the amount of core material utilized, where low amount of core material showed better retention. The mean diameters of microparticles remained between 25 and 32 µm and they exhibited rough walls, but continuous. In the stability study, formulations with high amounts of core material (solution of glucose 80%) or without the addition of surfactant (solution of glucose 40%, without lecithin) showed 18 and 56% of no encapsulated core material in the last day of analysis, respectively. This result were significantly different compared with the other two formulations, one with solution of glucose 60% and the other with solution of glucose 40%, that showed results of 5% and 2% of superficial glucose in the last day of analysis. The X-ray diffraction results didn¿t show alterations after 28 days. The DSC results showed similar profile for the formulations studied at zero time. The use of the same lipid materials to produce the microparticles during the stability study may be responsible for X-ray and calorimetric results obtained. / Mestrado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Mestre em Alimentos e Nutrição

Diversos biomateriais podem ser aplicados na engenharia de tecidos, mas poucos são utilizados em contato direto com células-tronco na forma de suportes de micropartículas, devido à falta de adesão, espalhamento e toxicidade do material, de forma que os tornam inviáveis junto ao cultivo celular. Um biomaterial promissor para bioengenharia é a fibroína, proteína fibrosa presente no casulo do bicho da seda (Bombyx mori), devido à sua resistência mecânica, biocompatibilidade e mínima reação inflamatória, porém, suas caracteristicas são pouco conhecidas na literatura. O mesmo não ocorre com o colágeno que já é bastante estudado por pesquisadores e, assim como a fibroína, apresenta propriedades naturais que incluem baixa resposta imunológica, baixa toxicidade e habilidade de promover o crescimento celular, porém o uso do colágeno em sua maior parte é em forma de filmes, esponjas e membranas. Como existem poucos métodos relacionados para preparação e caracterização de micropartículas em formatos esféricos e porosos, este trabalho teve por objetivo desenvolver e caracterizar micropartículas à base de colágeno ou fibroína, tratadas ou não com glutaraldeído (GA), para ser utilizado como suporte para células-tronco mesenquimais e avaliar a citotoxicidade destes materiais em cultura celular.Nos resultados de Calorimetria Exploratória Diferencial para ambos os materiais, colágeno e fibroína quando submetidas a tratamento com GA, a temperatura de desnaturação e degradação aumenta, respectivamente. Na microscopia ótica, eletrônica de varredura e birrefringência, observa-se o aparecimento de rugosidade e poros e/ou bolhas de ar no interior das micropartículas em maior quantidade quando tratadas com GA, o que pode ser um fator positivo para aderência celular no suporte. A porcentagem de água absorvida é maior no colágeno devido às estruturas hidrofóbicas em maior quantidade na fibroína, porém, quando tratadas com GA, a absorção é estabilizada em um curto tempo em ambos os materiais. Os picos nos espectros de FTIR mostram as bandas amidas I, II e III dos materiais e as alterações sofridas quando em contato com GA e os testes de citotoxicidade que ambos materiais tratados ou não, são atóxicos, mas o desenvolvimento celular nas micropartículas de fibroína é mais lento e diminui quando tratados com GA, por possuir mais estruturas ordenadas na forma de -folha quando se necessita de um crescimento mais controlado das células nas micropartículas. / There are several biomaterials can that be used in tissue engineering, but few are used in direct contact with stem cells like scaffold in the microparticle form, because of the lack of adhesion, spreading and toxicity of the biomaterial, in order to make them nonviable in the cell culture. A promising biomaterial for bioengineering is fibroin, a fibrous protein present in the fibers of silkworm (Bombyx mori) cocoon, because of its mechanical strength, biocompatibility and minimal inflammatory reaction; however, little is still described in the literature. Not so with the collagen that is already well studied by researchers and as the fibroin, has natural properties that include low immune response, low toxicity and ability to promote cell growth, but the use of collagen is mostly in form of films, sponges and membranes. As there are few methods reported for preparation and characterization of microparticles in spherical shapes and porous, this study aimed to develop and characterize microparticles based on collagen or fibroin, treated or not with glutaraldehyde (GA), to be used as a support for cells mesenchymal stem cells and evaluating the cytotoxicity of these materials in cell culture.In the results of Differential Scanning Calorimetry (DSC) curves for both materials, collagen and fibroin when subjected to treatment with GA, the denaturation and degradation temperatures increases, respectively. In Optical Microscopy, MSCanning electronic Microscopy and Birefringence results, it is observed the onset of surface roughness and porosity and or air pockets within the microparticles in greater quantity when treated with GA, which may be a positive factor for cell attachment on the support. The percentage of water absorbed is greater in the collagen structures due to more hydrophobic structure than silk fibroin, but when in treated with GA, absorption is stabilized in a shorter time in both materials. The peaks in FTIR spectra show bands amide I, II and III of the materials and the changes suffered when in contact with GA and cytotoxicity tests are non-toxic to both biomaterials treated or not, however, in the growth of cells, the fibroin microparticles is slower and decreases when treated with GA, due to its more ordered structure in the form of -sheet and more spherical than collagen due to its more ordered -sheet structures, which may be very interesting when it needs a more controlled growth of cells on microspheres.

A Trombocitopenia imune (PTI) é uma doença imuno mediada adquirida de adultos e crianças caracterizada por plaquetopenia transitória ou persistente, onde o grau de plaquetopenia aumenta o risco de sangramento. Geralmente, os pacientes apresentam manifestações clínicas apenas em plaquetopenias abaixo de 50x103/mm3, e contagem de plaquetas entre 100 e 150 x103/mm3, se estável por mais de 6 meses, necessariamente não indica uma condição patológica. Tem sido sugerido diferentes processos fisiopatológicos relacionados às plaquetas de acordo com a intensidade da plaquetopenia, e que alterações na megacariocitopoiese e diminuição da sobrevida plaquetária são eventos determinantes na PTI. Contagem de plaquetas reticuladas em citometria de fluxo é um teste muito útil para avaliação da plaquetopenia, pois reflete a atividade megacariocitopoiética, destruição das plaquetas e a própria contagem de plaquetas. Tanto as plaquetas quanto os megacariócitos apresentam a via intrínseca da apoptose. A atividade dos principais mediadores da apoptose intrínseca, como Bax a Bak, é regulada por proteínas anti-apoptótica da família Bcl-2, tais como Bcl-xL. Um balanço entre Bcl-xL e Bax regula a sobrevivência plaquetária. Pacientes de PTI apresentam aumento da ativação plaquetária e da formação de micropartículas derivadas de plaquetas (MPP). Nosso objetivo foi avaliar a relação entre produção e apoptose plaquetária, e associá-la com a ativação plaquetária e a formação de MPP nos diferentes graus de intensidade da PTI. Os pacientes recrutados foram diagnosticados para trombocitopenia imune (PTI) primária, \"idiopática\", não esplenectomizados, acompanhados regularmente no ambulatório de Doenças Hemorrágicas e Trombóticas, do Serviço de Hematologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, e foram divididos grupos de acordo com contagem de plaquetas: < 50x103/mm3 (n=7); entre 50-100x103/mm3 (n=7); > 100x103/mm3 (n=7). Como grupo controle, foram avaliados 10 doadores de sangue da Fundação Pró-Sangue Hemocentro de São Paulo. A produção plaquetária foi estabelecida através da contagem de plaquetas reticuladas por citometria de fluxo com laranja de tiazol. Também por citometria de fluxo avaliamos a ativação plaquetária pela expressão de P-selectina (CD62P), e a formação de MPP através de beads de 1um e dupla positividade para CD41a (GPIIbIIIa) e CD31 (molécula de adesão PECAM-1). A apoptose foi avaliada pela relação entre as expressões das proteínas anti apoptótica, Bcl-xL, e pró apoptótica, Bax, (Bcl-xL/Bax) em Western Blotting. Evidenciamos que na PTI há um aumento do numero de plaquetas reticuladas, predomínio de Bax em relação à Bcl-xL, uma maior ativação plaquetária e formação de MPP, mas em níveis variados dependentes do grau de plaquetopenia instalado. Sugerimos que o nível de predomínio do sistema pró apoptose sobre o anti apoptótico estabelece a intensidade da plaquetopenia na PTI. E que os eventos ativação plaquetária e formação de MPP são regulados pelo balanço entre Bcl-xL/Bax / Immune thrombocytopenic (ITP) is an immune-mediated acquired disease of adults and children characterized by transient or persistent decrease of the platelet count and, depending upon the degree of thrombocytopenia, increased risk of bleeding. ITP usually presents with clinical manifestations only in platelet counts below 50x103/mm3, and counts between 100 and 150x103/mm3 if they have been stable for more than 6 months do not necessarily indicate a pathologic condition. It has been suggested the existence of different pathophysiological processes involving platelets according to the severity of thrombocytopenia, and that alterations in the megakaryopoiesis and reduced platelet lifespan play a key role in ITP. The flow cytometric analysis of reticulated platelets is useful for evaluating thrombocytopenia that reflect the activity megakaryopoiesis, platelets destruction, plataelet count, and age. Platelets and megakaryocytes contain intrinsic pathway of apoptosis. The activity of key mediators of intrinsic apoptosis, Bak and Bax, is tightly controlled by anti-apoptotic Bcl-2 family members, of which Bcl-xL, wich have been shown to coordinately regulate platelet survival. ITP patients have a larger amount of activated platelets that express P-selectin as well as an increase of platelets-derived microparticles (PMP). We suggest a relationship between platelet production and Bcl-xL and Bax expression in ITP patients from different degrees of thrombocytopenia, that has relate with platelet activation and PMP. Our aim was assessment of ratio between platelets production and apoptosis, and it associate with platelets activation and PMP in differents thrombocytopenia degree in ITP. Patients were considered eligible for the study if they were on regular follow-up at the clinic of haemorrhagic and thrombotic diseases of the Hematology Service of Clinics Hospital of University of São Paulo Medicine School diagnosed with primary immune thrombocytopenia, \"idiopathic\". Patients were divided into groups according to levels of thrombocytopenia: platelets count between 100 and 150x103/mm3 ( > 100x103/mm3); between 50 and 100x103/mm3 (50-100x103/mm3); and below 50x103/mm3 ( < 50x103/mm3). Healthy volunteers were blood donors in the Pró-Sangue Foundation of Blood Center of São Paulo, with platelets count between 150 and 450 x103/mm3. Platelets production was through reticulated platelets count by flow cytometry wiht thiazole orange. Also by flow cytometry evaluated platelet activation through expression of P-selectin, and PMP by beads of 1um and double positive for CD41a, and CD31. Apoptosis was evaluated by the relationship between anti apoptotic proteins Bcl- XL, and pro apoptotic, Bax (Bcl-XL / Bax) in Western blotting. We show that there higher reticulated platelets in ITP as well as Bax predominantly in relation to Bcl-xL, increased platelet activation and PMP, but at varying levels depending on the degree of thrombocytopenia. We suggest that the level of dominance of pro apoptotic system on the anti-apoptotic establishes the intensity of thrombocytopenia in ITP, and that platelet activation and formation of MPP events are regulated by the balance between Bcl-xL / Bax

Made available in DSpace on 2018-06-27T18:56:37Z (GMT). No. of bitstreams: 3 Flabio da Rosa Pons Junior.pdf: 1999620 bytes, checksum: e5045f0611ac73ef991604e04484fbb8 (MD5) Flabio da Rosa Pons Junior.pdf.txt: 174053 bytes, checksum: 8140591651031d4c7b893b3bc3ce8675 (MD5) Flabio da Rosa Pons Junior.pdf.jpg: 3814 bytes, checksum: 8bd8fcbd7817488781e11c3f2a9361f5 (MD5) Previous issue date: 2011-08-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The fungal infections in humans occur in several ways, can this is superficial and systemic mycoses mycoses. The use of new antifungal made important progress in the treatment of different types of fungal infections. Among the drugs used to treat, there is ketoconazole, azole derivative, a synthetic molecule that acts by inhibiting biosynthesis of ergosterol molecule, exerting fungicidal. Treatment with ketoconazole requires frequent applications of the product, which causes skin irritation, the adherence to treatment. Initial studies have shown that drugs have nanocarrier improvement in selectivity, reducing side effects by modifying the release and increasing the effectiveness of the asset. This study aimed to characterize, evaluate the physical and chemical stability and antimicrobial activity of systems composed of nanoparticles or microparticles incorporated into formulations containing ketoconazole shampoo. All studies were carried out comparing ketoconazole as nanostructured and microstructured with the free form. The samples were stored under conditions of 25 ± 2 ° C, 4 ± 2 ° C and 40 ± 2 ° C and 75 ± 5% RH and exposed to UV light for 90 days and analyzed at 0, 7, 15, 30, 60 and 90 days in relation to the determination of pH, particle diameter, polydispersity index, zeta potential, rate of association and the drug dosing, antimicrobial activity and viscosity for the samples of shampoo. The average particle size for the nanocapsules was less than 370 nm, for microparticles less than 52 micrometers. All samples consist of the nanocarrier showed negative zeta potential. The values for the rate of association were 92.10% for the nanocapsules and 97.69% for the microparticles. Regarding the content of the suspensions and tested for stability, the samples comprising nanocapsules showed the loss of a smaller amount of assets (17.9% and 19.7%) in conditions of 25 ± 2 ° C and ± 4 2 ° C respectively. For formulations of shampoo made by nanocarrier systems, the same place, but with losses (9.3% and 13.6%) for the same conditions of suspension. In the analysis of antimicrobial activity, suspensions of nanocapsules had power of 98.15% and 100.25% of microparticles. Samples of shampoo were analyzed by microbiological assay sensitivity, which analyzed both qualitatively and showed that the samples have antifungal activity. The validation of analytical methodologies for the suspensions showed satisfactory results for all parameters. The results obtained, it can be concluded that the suspensions have demonstrated good stability, even when incorporated into a topical pharmaceutical vehicle, thus representing a better technological feasibility for the pharmaceutical area. / As infecções fúngicas nos seres humanos ocorrem de formas variadas, podendo tratar-se de micoses superficiais ou micoses sistêmicas. O uso de novos antifúngicos permitiu importantes progressos na terapêutica dos diferentes tipos de infecções micóticas. Dentre os fármacos utilizados para o tratamento, destaca-se o cetoconazol, derivado dos azóis, uma molécula sintética que age inibindo na biossíntese da molécula do ergosterol, exercendo ação fungicida. O tratamento com cetoconazol exige aplicações freqüentes do produto, o que provoca irritação cutânea, dificultando a adesão ao tratamento. Estudos iniciais têm demonstrado que fármacos nanocarreados apresentam melhora na seletividade, reduzindo efeitos colaterais, modificando a liberação e aumentando a eficácia do ativo. Este trabalho teve como objetivo, caracterizar, avaliar a estabilidade físico química e a atividade antimicrobiana de sistemas compostos de nanopartículas ou micropartículas contendo cetoconazol incorporadas em formulações de xampu. Todos os estudos foram realizados comparando-se o cetoconazol na forma nanoestruturada e microestruturada com a forma livre. As amostras foram armazenadas nas condições de 25 ± 2 °C, 4 ± 2 °C e 40 ± 2 °C e 75 ± 5% UR e expostas à luz UV, durante 90 dias, sendo analisadas em períodos 0, 7, 15, 30, 60 e 90 dias, em relação à determinação do pH, diâmetro de partícula, índice de polidispersão, potencial zeta, taxa de associação e doseamento do fármaco, atividade antimicrobiana e viscosidade para as amostras de xampu. O tamanho médio das partículas para as nanocápsulas foi inferior a 370 nm, para as micropartículas inferior a 52 &#956;m. Todas as amostras compostas pelos nanocarreadores apresentaram potencial zeta negativo. Os valores para a taxa de associação foram de 92,10 % para as nanocápsulas e 97,69 % para as micropartículas. Em relação ao teor das suspensões submetidas aos testes de estabilidade, as amostras compostas por nanocápsulas foram as que apresentaram uma menor perda de quantidade do ativo (17,9 % e 19,7 %) nas condições de 25 ± 2 °C e 4 ± 2 °C respectivamente. Para as formulações de xampu compostas pelos sistemas de nanocarreadores, o mesmo ocorreu, porém com perdas de (9,3 % e 13,6 %) para as mesmas condições das suspensões. Na análise da atividade antimicrobiana, as suspensões de nanocapsulas apresentaram potência de 98,15 % e as de micropartículas 100,25 %. As amostras de xampu foram analisadas pelo ensaio microbiológico de sensibilidade, o qual analisado de forma qualitativa e demonstrou que as amostras possuem atividade antifúngica. As validações das metodologias analíticas para as suspensões apresentaram resultados satisfatórios para todos os parâmetros analisados. Através dos resultados obtidos, pode-se concluir que as suspensões demonstraram ter boa estabilidade, mesmo quando incorporadas em um veículo farmacêutico de uso tópico, representando assim, uma melhor viabilidade tecnológica para a área farmacêutica.