Made available in DSpace on 2014-10-09T12:34:40Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:00:26Z (GMT). No. of bitstreams: 1 17901.pdf: 1984096 bytes, checksum: e265a4443e82668567ad545f9d03f397 (MD5) / Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP; Faculdade de Odontologia, Universidade de São Paulo, São Paulo

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Made available in DSpace on 2014-10-09T12:52:35Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:02:35Z (GMT). No. of bitstreams: 0 / A papaína é uma enzima proteolítica extraída do látex das folhas e frutos do mamão verde adulto. Tem sido amplamente utilizada como agente debridante de escaras e cicatrizante de feridas. No entanto, apresenta baixa estabilidade, o que limita seu uso a formulações de manipulação extemporânea ou de curto prazo de validade. O objetivo deste trabalho foi incorporar a papaína em uma matriz polimérica de modo a obter um sistema de liberação controlada do fármaco. Polímeros de aplicação médica foram selecionados e inicialmente avaliados quanto à sua citotoxicidade. Os polímeros não-citotóxicos foram submetidos ao ensaio de irritação cutânea primária in vivo em animais, para avaliar sua capacidade de causar irritação na pele humana. Diversas membranas foram preparadas com os polímeros considerados adequados para aplicação biomédica para incorporação da papaína. As membranas preparadas com 2% de papaína foram selecionadas para serem submetidas ao ensaio de liberação com células de difusão de Franz. Parte dessas membranas foi irradiada com raios γ na dose de 25 kGy para esterilização do material. As membranas irradiadas e não-irradiadas foram testadas simultaneamente a fim de verificar se a radiação γ interferiria no perfil de liberação do fármaco. Os resultados do ensaio de liberação indicaram que o fármaco é liberado de maneira constante durante as 12 horas iniciais do experimento. A análise, por Microscopia Eletrônica de Varredura, das membranas irradiadas revelou que as membranas formadas são bastante densas e que seus poros são pequenos. / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:35:30Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:04:37Z (GMT). No. of bitstreams: 0 / Muitas das proteínas de valor biomédico relevante são encontradas em baixas concentrações em suas fontes nativas. O alto nível de expressão de proteínas recombinantes em E. coli, muitas vezes gera o acúmulo de proteínas como agregados insolúveis no citoplasma e/ou periplasma da bactéria, denominados de corpos de inclusão (CI). A alta pressão tem sido amplamente utilizada no estudo da conformação das proteínas,ela modula as interações proteína-proteína e proteína-solvente através de mudanças no volume das mesmas, promovendo a entrada de água nas cavidades não expostas da molécula e promovendo hidratação e solubilização dos agregados. O presente trabalho teve como objetivo a renaturação de proteínas recombinantes expressas como CI em Escherichia coli usando alta pressão hidrostática como condição branda de dissociação dos agregados. As tiorredoxinas TsnC e TrxA, a proteína YbbN e a proteína comigratória com bacterioferritina (Bcp), todas de Xylella fastidiosa, foram estudadas neste trabalho. As condições de renaturação foram otimizadas, utilizando-se diferentes proporções do par redox, concentrações de GdnHCl, presença de aditivos e esquemas de descompressão. Para a quantificação e análise da eficácia do processo de renaturação das proteínas sob pressão foram utilizadas as técnicas de microscopia eletrônica de varredura dos CI e de SDS-PAGE, e ensaios de atividade enzimática das proteínas. A TsnC foi renaturada em Tris HCl 50 mM com proporção de 10GSH:1GSSG em concentração final de 10 mM, 0,75 M GdnHCl, na presença de 0,5 M de Triton-X e a pressão utilizada foi de 2,4 kbar por 1 hora e 30 minutos seguida de descompressão direta e incubação por 16 horas em pressão atmosférica. O rendimento final de obtenção de TsnC solúvel foi altíssimo, de até 89,9%. A renaturação de proteína YbbN, nunca antes descrita, foi obtida em tampão de renaturação Tris HCl 50 mM, na presença de 0,5 M de L-Arginina e a pressão utilizada foi de 2,4 kbar por 1 hora e 30 minutos seguida de descompressão direta e incubação por 16 horas em pressão atmosférica. A proteína YbbN, que apresentou atividade de tioredoxina, foi renaturada com rendimento de até 98% a partir da proteína insolúvel nos CI. Foi possível a solubilização da tiorredoxina TrxA e Bcp sob alta pressão hidrostática em tampão de renaturação Tris HCl 50 mM, utilizando diferentes proporções do par redox na concentração final de 10 mM e 1,5 M de GdnHCl, porém não foi possível obter a atividade biológicas destas proteínas. Mostramos também que a L-Arginina apresenta efeito auxiliar na solubilização dos CI induzida pela alta pressão, e ao mesmo tempo se mostrou altamente protetora contra a inativação da atividade da YbbN promovida pela incubação em altas temperaturas, o que sugere que a presença deste aminoácido pode ter alta aplicabilidade juntamente com a aplicação de altas pressões para elevar os rendimentos de renaturação de proteínas recombinantes a partir de CI. / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

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Philosophiae Doctor - PhD / Transition metal oxides magneli phases present crystallographic shear structure which is of great interest in multiple applications because of their wide range of valence, which is exhibited by the transition metals. The latter affect chemical and physical properties of the oxides. Amongst them we have nanostructures VO2 system of V and O components which are studied including chemical and physical reactions based on non-equilibrium thermodynamics. Due to their structural classes of corundum, rocksalt, wurtzite, spinel, perovskite, rutile, and layer structure, these oxides are generally used as catalytic materials which are prepared by common methods under mild conditions presenting distortion or defects in the case of VO2. Existence of an intermediate phase is proved using an x-ray thermodiffraction experiment providing structural information as the nanoparticles are heated. Potential application as gas sensing device has been the first time obtained due to the high surface to volume ratio, and good crystallinity, purity of the material and presence of suitable nucleating defects sites due to its n-type semiconductor behavior. In addition, annealing effect on nanostructures VO2 nanobelts shows a preferential gas reductant of Ar comparing to the N2 gas. Also, the hysteresis loop shows that there is strong size dependence to annealing treatment on our samples. This is of great interest in the need of obtaining high stable and durable material for Mott insulator transistor and Gas sensor device at room temperature.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / The blood brain barrier (BBB) is a signaling interface between the blood and the central nervous system (CNS), which prohibits the entry of harmful blood-borne substances into the brain micro-environment, thus maintaining brain homeostasis. The crucial role of the BBB is protecting the CNS, which may adversely be affected by alcohol. The central component of the BBB, endothelial cells (ECs), regulates BBB transport by regulating the permeability both transcellularly and through their paracellular junctions, by structures called tight junctions (TJs) that are composed of proteins. The aim of this study was to investigate the in vitro effects of ethanol (EtOH) and fermented rooibos (Rf) on a monolayer of bEnd5 mouse brain ECs, by determining the effects of EtOH and Rf on bEnd5 (i) cell viability (ii) cell proliferation (iii) rate of cell division (iv) cell toxicity (v) claudin-5 transcription (vi) permeability across a monolayer of bEnd5 ECs and (vii) morphology, for a selected experimental timeline of 24, 48, 72, and 96hrs. We then investigated if the simultaneous exposure of Rf and EtOH could reverse or alleviate the EtOHinduced effects on the bEnd5 ECs. EtOH metabolism induces oxidative stress and results in a range of adverse physiological effects. Aspathalus linearis (rooibos) contains many phenolic compounds, of which the main antioxidant activity is attributed to aspalathin. Our underlining hypothesis is that the antioxidants in an aqueous rooibos extract may therefore protect against the potential oxidant damaging effects of alcohol on the BBB. Cells were exposed for 24hrs to selected concentrations of EtOH (25mM and 100mM), a concentration of Rf containing equivalent of 1.9nM aspalathin, and the combinations of EtOH and Rf. Cell viability and cell toxicity was determined, while cell proliferation and rate of cell division was estimated using the trypan blue exclusion assay. Real time quantitative PCR was implemented to quantify claudin-5 transcription, normalized against housekeeping genes, GAPDH and HPRT. Transepithelial electrical resistance (TEER) was measured using the Ohm Millicell-electrical resistance system, while bEnd5 monolayer morphology was analysed using the Zeiss scanning electron microscope. Both concentrations of EtOH led to an overall decrease in cell viability, and a decreased number of live cells across 72hrs. Consistent with this, EtOH resulted in increased cell toxicity across the 96hr experimental timeframe and a diminished rate of cell division. The transcription of claudin-5 in bEnd5 ECs exposed to 25mM and 100mM EtOH varied dramatically across the 96hr timeframe. While 25mM EtOH resulted in an overall decrease in TEER, cells exposed to 100mM EtOH only decreased TEER between 48 and 96hrs. Morphologically, both concentrations of EtOH led to compromised paracellular spaces as endorsed by high definition SEM analysis. The administration of Rf on its own resulted in an initial decrease in viability, followed by recovery between 72 and 96hrs. Exposure to Rf diminished live cell numbers at 72 and 96hrs, accompanied by a compromised rate of cell division and an overall increase in cell toxicity. In addition, Rf down-regulated claudin-5 transcription across the course of the experiment, particularly between 24 and 48hrs. In alignment with this, Rf also led to an increase in BBB permeability from 24 to 96hrs. However, SEM studies were not able to discriminate any differences between control and Rf treated cells. Our study showed that the BBB could be protected against the adverse effects of EtOH, and this at the plasma concentration induced by 500ml’s of Rooibos tea. The simultaneous exposure of Rf and EtOH was able to negate the effects of EtOH on cell viability, cell proliferation, and cell toxicity but exacerbated the effects of EtOH on claudin-5 transcription and paracellular permeability. Morphologically, co-exposure with Rf only reversed the effects of 25mM EtOH while exacerbating the effects of 100mM EtOH at 96hrs. In conclusion, EtOH was shown to be detrimental to the integrity of bEnd5 ECs, and the addition of a minuscule quantity of the Rf extract was able to partially alleviate excess ROS-induced effects.

M.Sc. / Despite several decades of extensive research and development in pharmaceutical chemistry, the poor solubility of lipophilic compounds in aqueous media remains a major barrier to their absorption, bioavailability and clinical efficacy. This poor solubility is also a problem in other areas such as the flavour and fragrance industry. In cosmetics, for example, poor aqueous solubility and instability of oily compounds causes problems in formulation and fragrance stability. One approach to overcome these difficulties is to encapsulate oily compounds in biocompatible materials. As a drug delivery system such an approach is attractive if the size of the capsule is reduced to the micrometer or nanometer scale. Naturally occurring polysaccharides like sodium alginate (NaALG) and chitosan (CS) are generally regarded as safe (GRAS) for use in human use and have therefore gained much attention recently. As a drug delivery system, this polymer matrix can be used to prevent drug degradation in the gastro intestinal tract (GIT) and often provides controlled release of the encapsulant. Cyclodextrins (CDs) on the other hand offer an alternative approach. These cyclic oligosaccharides have the ability to form non-covalent inclusion complexes with a range of organic compounds, and in so doing alter their physiochemical properties such as solubility. This study was aimed at exploring these concepts by using ALG and CS as an entrapment matrix for an essential oil, tagette oil (used as a model oily drug) that is insoluble in aqueous media. Alginate/chitosan (ALG/CS) nanoparticles were prepared in a 3-step procedure; emulsification of tagette oil in aqueous Na-ALG solution, followed by ionotropic pre-gelation of the ALG core with CaCl2 and further crosslinking with CS. Morphology and particle size measurements were performed by scanning and transmission electron microscopy (SEM and TEM), and Malvern Zetasizer.

>Magister Scientiae - MSc / Magnetic luminescent nanoparticles of an iron oxide (Fe₃O₄) superparamagnetic core and an indium phosphide/zinc selenide (InP/ZnSe) quantum dot shell are reported. The magnetic nanoparticles (MNP’s) and quantum dots (QD’s) were each synthesized separately before conjugation. The MNP’s were functionalized with a thiol-group allowing the QD shell to bind to the surface of the MNP by the formation of a thiol-metal bond. The nanocomposite was capped with 3-mercaptopropionic acid, 1-propanethiol, 2-methyl-1-propanethiol and their properties investigated using the characterization techniques: high- resolution transmission electron microscopy (HR-TEM), energy-dispersive spectroscopy (EDS), UV-vis, scanning electron microscopy (SEM), superconducting quantum interference device (SQUID), and photoluminescence. These techniques yielded significant information on particle size, morphology, dispersion, and chemical composition including luminescence and florescence.