Made available in DSpace on 2016-08-17T18:39:47Z (GMT). No. of bitstreams: 1 5353.pdf: 2805182 bytes, checksum: dbd89699431c4bbf40d6959bb375df30 (MD5) Previous issue date: 2013-04-29 / Financiadora de Estudos e Projetos / The increasing interest in the employment of multipotent mesenchymal stromal cells (MSCs) to applications in tissue engineering and cell therapy is mainly attributed to their plasticity and regenerative capacity. The MSCs represent a revolution in the treatment of countless diseases and in the understanding of tissue repair mechanisms. Due to their low availability in the tissues (0.00001% to 0.0002%) as well as to the need of high doses for therapeutic applications (approximately 2 x 106 cells/kg patient), and to the inefficiency of traditional in vitro cultures (in monolayer) to meet the existing high demand to these applications, development of new technologies of ex vivo expansion on a large scale became indispensable. The use of spinner flasks-like bioreactors with microcarriers in suspension is an effective alternative system for this expansion. However, bibliographic report of cultures with this system have indicated the formation of large clusters of microcarriers with cells as a possible obstacle to obtain a greater cellular productivity. These clusters hamper the diffusion of nutrients, gases (especially oxigen), and reagents of indirect cellular quantification within them; and they harm the cell recovery at the end of culture. Alginate microspheres have become interesting to reduce the frequency of collisions between microcarriers, due to the fact that the microspheres do not adhere to cells nor to microcarries and can prevent the union of same. The dextran sulfate has been described in the literature as an inhibitor of cell-to-cell contact as well as of the encounter between microcarriers in culture, therefore, it too has become a promising option to avoid the formation of clusters of microcarriers. Thus, the aim of this work was to develop a methodology for the culture of MSCs in spinner flasks with microcarriers Cultispher-S for application in cell therapy and evaluate the influence of alginate microspheres and dextran sulfate on this culture. For this, the lineage hMSCTERT and 3 g/L Cultispher-S were held in 100 mL spinner containing α-MEM culture medium (supplemented with 15% of bovine fetal serum, glucose and glutamine) at 37ºC, with pH control. The results showed that the addition of dextran sulfate (at 0.5% and 1%) and of 100% of alginate microspheres (in relation to the number of Cultspher-S particles) to the culture caused considerable cell death; and the control (without additives) as well as the addition of 50% of alginate microspheres promoted cell growth. The formation of clusters was observed in cultures; however, it was delayed with the addition of 50% of alginate microspheres. This occurrence allowed a cellular expansion factor of 50.4 times (with dilution factor correction) and of 13.1 times (without correction). The recovered cells after expansion kept their immunophenotypic characteristics. / O crescente interesse na utilização de células mesenquimais estromais multipotentes (CMMs) para aplicações na engenharia de tecidos e na terapia celular é atribuído principalmente à sua plasticidade e capacidade regenerativa. As CMMs representam uma revolução no tratamento de inúmeras doenças e no entendimento dos mecanismos de reparo tecidual. Sua baixa disponibilidade nos tecidos (de 0,00001 à 0,0002%) associada à necessidade de elevadas doses para as aplicações terapêuticas (aproximadamente 2 x 106 células/kg paciente) e à ineficiência dos cultivos tradicionais (em monocamada) in vitro para atender à alta demanda existente para essas aplicações, tornaram indispensável o desenvolvimento de novas tecnologias de expansão ex vivo em larga escala. A utilização de biorreatores tipo frascos spinner com microcarregadores em suspensão é um sistema alternativo eficaz para esta expansão. O relato bibliográfico de cultivos com esse sistema, no entanto, tem indicado a formação de grandes aglomerados de microcarregadores com células como um possível obstáculo na obtenção de maior produtividade celular. Tais aglomerados dificultam a difusão de nutrientes, gases (principalmente o oxigênio) e reagentes de quantificação celular indireta em seu interior; e prejudicam a recuperação das células no final do cultivo. Microesferas de alginato tornaram-se interessantes para diminuir a frequência de colisões entre os microcarregadores, pois não se aderem às células nem aos microcarregadores podendo evitar a união dos mesmos. O sulfato de dextrana foi descrito na literatura tanto como inibidor do contato célula-célula, quanto do encontro entre microcarregadores em cultivo, por isso também passou a ser uma opção promissora para evitar a formação de aglomerados de microcarregadores. Assim, os objetivos deste trabalho foram desenvolver uma metodologia de cultivo de CMMs em frasco spinner com microcarregadores Cultispher-S para aplicação em terapia celular e avaliar a influência de microesferas de alginato e do sulfato de dextrana nesse cultivo. Para isso, a linhagem hMSC-TERT e 3 g/L Cultispher-S foram mantidos em spinner de 100 mL contendo meio de cultura α-MEM (suplementado com 15% de soro fetal bovino, glicose e glutamina), a 37ºC, com controle de pH. Os resultados mostraram que a adição de sulfato de dextrana (à 0,5% e 1%) e de 100% de microesferas de alginato (em relação ao número de partículas de Cultispher-S) ao cultivo provocaram morte celular considerável; e que o controle (sem aditivos), assim como a adição de 50% de microesferas de alginato promoveram crescimento celular. A formação de aglomerados foi observada nos cultivos, no entanto, ela foi atrasada com adição de 50% de microesferas de alginato. Esse fato permitiu a obtenção de um fator de expansão de 50,4 vezes (com a correção do fator de diluição) e de 13,1 vezes (sem a correção). As células recuperadas após a expansão mantiveram suas características imunofenotípicas.

Neste trabalho foi feito um estudo sobre a preparação e caracterização de microesferas poliméricas à base de poli(ácido metacrílico-co-divinilbenzeno) por polimerização por precipitação. As partículas foram sintetizadas e analisadas em diferentes condições de reação. Partículas esféricas políméricas foram sintetizadas na faixa de 1,66 - 8,41 m, assim como partículas no estado de microgel. As partículas foram caracterizadas pelas técnicas de espalhamento de luz dinâmica (DLS), análise termogravimétrica (TGA), espectroscopia na região do infravermelho (FTIR), adsorção de nitrogênio pelos métodos BET (Brunauer, Emmett e Teller) e BJH (Barret, Joyner e Halenda), microscopia ótica, microscopia eletrônica de varredura, e testes de razão de inchamento. A análise das partículas foi feita para verificar a influência da mudança na composição de comonômeros, grau de reticulação, relação de monômeros totais/diluentes em massa/volume (g/100 mL), e quanto à relação volumétrica de diluentes. Verificou-se que houve um aumento no tamanho das partículas e da resistência térmica com a diminuição da fração molar de MAA (ácido metacrílico). Na preparação de partículas com fração molar de 50% de MAA, e relação volumétrica acetonitrila/tolueno de 75/25, quanto maior a relação de monômeros totais/diluentes (g/100 mL), maior o tamanho e o rendimento das partículas. Com a mudança da relação volumétrica de diluentes, houve mudança nas características de porosidade, tamanho das partículas, e grau de inchamento das partículas, sendo que na relação volumétrica acetonitrila/tolueno de 50/50, houve formação de microgel / The preparation and characterization of polymeric microspheres based on poly(methacrylic acid-co-divinylbenzene) by precipitation polymerization was reported. The particles were synthesized and analyzed at different reaction conditions. Spherical polymer particles were synthesized in the range from 1,66 to 8,41 m, in addition to microgel particles. The particles were characterized by dynamic light scattering (DLS), thermogravimetric analysis (TGA), infrared spectroscopy (FTIR), nitrogen adsorption methods by BET (Brunauer, Emmett and Teller) and BJH (Barrett, Joyner and Halenda), optical microscopy, scanning electron microscopy, and swelling ratio tests. The particles analysis was made to determine the influence of the change in comonomer composition, degree of crosslinking, ratio of total monomers/diluents by weight / volume (g/100 mL), and the volume ratio of diluents. It was found that there was an increase in particle size and thermal resistance with decreasing molar fraction of MAA (methacrylic acid). In preparing particles with the mole fraction of 50% MAA, and volume ratio acetonitrile/toluene 75/25, the larger the ratio of total monomers/diluents (g/100 mL), the larger the particle size and yield. By changing the volume ratio of diluents, there was a change in the characteristics of porosity, particle size and degree of swelling of the particles, and in the volume ratio acetonitrile / toluene of 50/50, there was microgel formation

Diferentes floculantes ecológicos têm sido estudados na literatura para o tratamento de água. Entre tais substâncias, a quitosana e os taninos têm sido utilizados, regularmente, em diversos trabalhos relacionados ao tratamento de água. Neste contexto, o objetivo deste trabalho foi a obtenção de microesferas de quitosana comercial (QTSc) e tanino extraído da casca de Eucalyptus urograndis (TAN), para a utilização piloto no tratamento de água bruta com baixa turbidez (100-110 NTU). Primeiramente, avaliou-se o grau de desacetilação da quitosana comercial (QTSc) e da quitosana na microesfera (QTSm) para a avaliação da porcentagem de grupamentos aminas livres ou desacetiladas. Em seguida, a quitosana desacetilada foi utilizada como matriz na preparação de microesferas empregando duas concentrações de TAN (5,0 e 7,5 % v/v). O material preparado foi caracterizado por FTIR, DRX, MEV e TGA. Os espectros de FTIR apresentaram aumento da intensidade dos picos à medida que havia aumento de concentração de TAN nas microesferas formadas. Os espectros de DRX apresentaram um material amorfo. As micrografias por MEV mostraram que a interação da QTS com o TAN modifica as características superficiais da microesfera provocando mudanças na superfície. As análises de DTA/TGA mostraram que as microesferas preparadas com 0 e 5% apresentaram três estágios de degradação térmica e dois estágios para QTS/TAN 7,5% mostrando que o material com QTS/TAN 7,5% apresenta resistência térmica diferenciada quando comparado ao QTS/TAN 0 e 5%. As microesferas foram avaliadas em ensaios de coagulação/floculação por Jar test no tratamento de água bruta utilizando planejamento fatorial experimental (23) no qual fatores utilizados os paramentos de massa de coagulante de 10 mg e 25 mg, pH=7,0 e pH=8,0 e tempo de decantação de 15 min e 30 min. Os fatores foram avaliados quanto a porcentagem de remoção de turbidez e cor. Os resultados mostraram-se satisfatórios quanto a remoção de turbidez, com mínimo de 51,52 % e máximo de 76,67 % e remoção de cor com mínimo de 42,86 % e máximo de 66,00 %. Os melhores resultados de % remoção foram obtidos quando foram utilizadas as microesferas de QTS/TAN 5% em pH = 8,0 com tempo de decantação de 30 min e massa de coagulante de 25 mg. Os resultados permitiram avaliar as microesferas de QTS/TAN como materiais promissores no tratamento de água. / Different ecological flocculants have been studied in the literature for treatment of water and wastewater. Among such substances, chitosan and tannins have been used regularly in several works related to water treatment. In this context, the aim of this study was to obtain commercial chitosan microspheres (QTSc) and tannin extracted from barks of Eucalyptus urograndis (TAN) for pilot utilization in the treatment of raw water with low turbidity (100-110 NTU). At first degree of deacetylation of both, commercial chitosan (QTSc) and the chitosan microsphere (QTSm) was verified for assessment of degree of deacetylation of amines. Microspheres were prepared at different concentrations of TAN ranging from 0 - 7.5% v/v and then characterized by FTIR, XRD, SEM and DTA/TGA. FTIR showed an increase of peak intensity as increased the concentration of the TAN microspheres formed. XRD presented an amorphous material. SEM micrographs allowed verifying the interaction of chitosan and tannin in the modification of the surface. Analyses of DTA/TGA showed that chitosan microspheres CTS/TAN 0% and 5% exhibited three stages of thermal degradation and two stages were observed for 7.5% CTS/TAN, allowed to conclude that CTS/TAN 7.5% has lower heat resistance compared to CTS/TAN 0 and 5%. The microspheres were evaluated in terms of coagulation/ flocculation assays by Jar test using an experimental factorial design (23). The coagulant mass of 10 mg and 25 mg, pH = 7.0 and pH = 8.0 and settling time of 15 min and 30 min were used as main factors. The factors were assessed for percentage removal of turbidity and color, which gave satisfactory results in the removal of turbidity with a minimum at 51.52% and a maximum at 76.67% and color removal with a minimum at 42.86% and maximum at 66.00%. The best results of were obtained using microspheres CTS/TAN 5% at pH 8.0 and settling time of 30 min and 25 mg of coagulant. Microspheres of CTS/TAN are promising in the treatment of water, but additional studies are necessary for searching the better conditions/results for water treatment.

Les microrésonateurs à modes de galerie en matériaux amorphes sont faciles à produire par des techniques de fusion. Leur facteur de qualité est cependant limité à quelques 108 du fait du phénomène de contamination de surface ou d'une absorption résiduelle plus importante que celle des matériaux cristallins. Nous avons développé deux méthodes utilisant des micro-résonateurs actifs, fabriqués par fusion de poudres de verres ZBLALiP dopés Erbium, permettant de dépasser cette limite. Il est tout d'abord possible de compenser les pertes en introduisant du gain optique dans le micro-résonateur. Des facteurs de qualité chargés de 5×109 correspondant à un facteur de qualité intrinsèque de 1010 ont ainsi pu être mesurés. En plus d'une augmentation du facteur de qualité, le gain interne offre un paramètre supplémentaire de contrôle du couplage, cela nous a permis d'obtenir tous les régimes de couplage d'un résonateur à partir du sous couplage jusqu'au régime d'amplification sélective. Nous avons atteint en particulier le régime de transparence, un micro-résonateur dans un tel régime peut trouver des applications comme ligne à retard variable transparente ayant un retard de groupe qui peut être modifié en jouant sur le taux de couplage entre le résonateur et la ligne d'accès. La deuxième méthode consiste à augmenter très fortement la valeur de l'indice de groupe du milieu par des effets de lumière lente. Un indice de groupe d'environ 4×106 a été obtenu via le phénomène d'Oscillations Cohérentes de Populations (OCP) ce qui caractérise un très fort ralentissement de la lumière. Ceci nous a permis d'augmenter, dans une micro-cavité, le temps de stockage des photons de 200 ps à 2,5 ms ce qui correspond à un facteur Q de 3×1012. / Glass-based whispering gallery mode (WGM) microresonators are easy to produce by melting techniques. However, they suffer from surface contamination or residual absorption which limits their long term quality factor to only about 108. We show that an optical gain provided by erbium ions can compensate for residual losses. Moreover it is possible to control the coupling regime of an ultrahigh Q-factor microresonator from undercoupling to spectral selective amplification by changing the pumping rate. We demonstrate a critically coupled fluoride glass WGM microresonator with a diameter of 220 μm and a loaded Q-factor of 5,3×109. We also show that by introducing slow-light effects in a monolithic WGM microresonator it is possible to enhance the photon lifetime by several orders of magnitude and circumvent fabrication limitations. We experimentally demonstrate Erbium-doped fluoride glass microresonators with a photon lifetime up to 2,5 ms at room temperature, corresponding to a Q-factor of 3×1012 at 1530 nm, by combining WGM resonance effect and population oscillations.

In the present thesis we propose the development of hybrid polymer titanium dioxide (TiO2) nanoparticles for use in biomedical applications. TiO2 exhibits high biocompatibility in the dark however, upon illumination in aqueous media with near UV light it produces an array of reactive oxygen species (ROS) which have the capability to induce death in neighbouring cells. The process of inducing cell death using a photosensitive material which produces ROS is known as photodynamic therapy (PDT) and is used to treat a wide range of maladies from psoriasis to cancer.We have demonstrated the ability to produce anatase nanoparticles with high control over their resulting size through a novel water mediated sol-gel synthetic method in benzyl alcohol, using either Ti(OnPr)4, Ti(OnBu)4 or Ti(OiPr)4 as the metal precursor. Through dynamic light scattering (DLS) analysis we have shown that the mechanism of nanoparticle growth appears to proceed through the agglomeration of primary nanoparticles formed instantly upon adding the reagents together. After synthesis the nanoparticles could be easily redispersed in aqueous media at pH2 with any further agglomeration being controlled by the parent alkoxide.After synthesis the nanoparticles were coated with PEG, conjugated to either a catechol or phosphate as ligand, in order to stabilise the nanoparticles at neutral pH. Uncoated nanoparticles exhibited good photoactive capability in the photooxidation of methylene blue. However, on coating with catechols the photoactivity of the nanoparticles was abolished. Coating with phosph(on)ates on the other hand preserved or even enhanced the photoactivity which makes this system promising for in vivo applications.At the same time this thesis also reports preliminary investigations on the use of TiO2 embedded into the walls of model drug loaded poly electrolyte multilayer microspheres for UV triggered delivery applications.

Probiotic lactic acid bacteria and bifidobacteria are natural part of microflora of gastrointestinal tract. In the present, day they are grossly exploited in food processing industry. The aim of the work was molecular identification of bacteria of genus Lactobacillus and Bifidobacterium in complex matrices of two food additives. Total DNA was isolated from crude cell lysates by magnetic particles P(HEMA-co-GMA). Isolated DNA was amplified in genus-specific and species-specific PCRs. Amplicons were detected by agarose gel electrophoresis. Results were compared with declared specification given by producers in three different batches.

Probiotic lactic acid bacteria (LAB) are an important part of fermented dairy products, pharmaceuticals and food supplements. At present, rapid and accurate identification of bacteria is carried out using molecular biological methods based on DNA amplification. The aim of the thesis was to identify by non-cultivation bacteria of genus Lactobacillus and bacteria of species Lactobacillus acidophilus in complex matrices at total of seven different food supplements. Total DNA was isolated from crude cell lysates using magnetic carrier P(HEMA-co-GMA). Amplificability of DNA was verified by PCR using primers specific for the domain Bacteria. In next step isolated DNA was amplified using primers specific for the genus Lactobacillus and species Lactobacillus acidophilus to demonstrate the presence of this bacterial genus and species declared by the producers. The results of bacteria identification obtained by PCR were compared with declared specification given by the producers.

The work was focused on isolation of PCR-ready DNA and the identification of probiotic lactic acid bacteria that were isolated from five milk product for infant nutrition. DNA was isolated from crude cell-lysates of the products by magnetic P(HEMA-co-GMA) microspheres. DNAs isolated from crude cell lysates of control strains using phenol extraction method were used as positive controls. Using PCRs DNA of genera Bifidobacterium and species B. animalis, B. bifidum, B. breve, B. infantis, B. longum and Streptococcus thermophilus species were identified in products. The results obtained are consistent with the data declared by the manufacturers.

No description available.

Nanotechnology builds devices from the bottom up with atomic accuracy. Among the basic nano-components to fabricate such devices, semiconductor nanoparticle quantum dots (QDs), metal nanocrystals, proteins, and nucleic acids have attracted most interests due to their potential in optical, biomedical, and electronic areas. The major objective of this research was to prepare nano-components in order to fabricate functional nano-scale devices. This research consisted of three projects. In the first two projects, we incorporated two desirable characteristics of QDs, which are their abilities to serve as donors in fluorescence energy transfer (FRET) and surface energy transfer (SET) as well as to do multiplexing, to engineer QD-based nanoconjugates for optical and biomedical applications. Immobilizing luminescent semiconductor CdSe/ZnS QDs to a solid platform for QD-based biosensors offers advantages over traditional solution-based assays. In the first project, we designed highly sensitive CdSe/ZnS QD SET-based probes using gold nanoparticles (AuNPs) as FRET acceptors on polystyrene (PS) microsphere surfaces. The emission of PS-QD was significantly quenched and restored when the AuNPs were attached to and then removed from the surface. The probes were sensitive enough to analyze signals from a single bead and for use in optical applications. The new PS-QD-AuNP SET platform opens possibilities to carry out both SET and FRET assays in microparticle-based platforms and in microarrays. In the second project, we applied the QD-encoded microspheres in FRET-based analysis for bio-applications. QDs and Alexa Fluor 660 (A660) fluorophores are used as donors and acceptors respectively via a hairpin single stranded DNA. FRET between QD and A660 on the surface of polystyrene microspheres resulted in quenching of QD luminescence and increased A660 emission. QD emission on polystyrene x microspheres was restored when the targeted complementary DNA hybridized the hairpin strand and displaced A660 away from QDs. The third project involved fabrication of different nanoconjugates via self-assembly of template-based metal nanowires and metal nanoparticles using oligonucleotides as linkers. These nanoconjugates can serve as building blocks in nano-electronic circuits. The template method restricted the oligonucleotides attachment to the tip of the nanowires. Nanowires tagged with hybridizable DNA could connect to complementary DNA-modified metal crystals in a position-specific manner.