Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-03-17T17:34:41Z No. of bitstreams: 1 Nicoletti, Caroline Deckmann [Dissertação, 2015].pdf: 2492191 bytes, checksum: 2cc1df517b27092f16baf567910a3c1a (MD5) / Made available in DSpace on 2017-03-17T17:34:41Z (GMT). No. of bitstreams: 1 Nicoletti, Caroline Deckmann [Dissertação, 2015].pdf: 2492191 bytes, checksum: 2cc1df517b27092f16baf567910a3c1a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A papaína é uma enzima proteolítica utilizada no tratamento de feridas. É parcialmente solúvel em água e tem baixa estabilidade em preparações farmacêuticas, dessa forma os géis com concentrações de papaína acima de 1% podem apresentar precipitados. O presente trabalho teve como objetivo o desenvolvimento de géis de papaína a 4% (p/p) usando tensoativos como agente solubilizante. Como fase inicial do estudo realizou-se a determinação da Concentração Micelar Crítica (CMC) por meio da medida de tensão superficial de geis de papaína a 4,0% (p/p) com e sem L-cisteína e diferentes concentrações de polissorbato 80. Novas preparações foram desenvolvidas usando-se concentrações de polissorbato e L-cisteína que resultavam na completa solubilização da papaína. O estudo de estabilidade dos géis de papaína a 4,0% (p/p) com polissorbato 80 foi planejado e executado a partir de desenho experimental fatorial 33 em amostras armazenadas sob refrigeração por 30 dias. As variáveis independentes estipuladas foram concentração de L-cisteína, concentração de polissorbato 80 e o tempo de armazenagem; a variável dependente avaliada foi a concentração de papaína ativa. Todas as amostras foram analisadas quanto à manutenção das características sensoriais e físico-químicas. Durante o período de avaliação as preparações conservaram-se como géis homogêneos, mantendo-se estáveis quanto aos valores de pH, de viscosidade e de estabilidade termodinâmica. Estas amostras não apresentaram mudanças no seu comportamento reológico. As preparações estudas apresentaram concentrações de papaína ativa entre 90 e 110% apos 24 horas de preparo, mas não foi possível a manutenção desta atividade durante o período de estudo. As avaliações estatísticas decorrentes do desenho experimental fatorial resultaram em gráficos de superfície de resposta e de contorno, que demonstraram que o tempo é o fator de maior influência sobre a perda da atividade da papaína nas preparações em gel. A L-cisteína interfere favoravelmente para a manutenção da atividade, mas sua ação isolada é incapaz de sobrepor-se ao efeito do tempo. A associação da L-cisteína e do polissorbato 80 também apresenta ação favorável a manutenção da atividade enzimática, que se torna evidente somente nas maiores concentrações estudadas para estes adjuvantes / Papain is a proteolytic enzyme used in the treatment of wounds. It is partially soluble in water and has low stability in pharmaceutical preparations thus gels with papain above 1% concentration may have precipitated. This study aimed to the development of papain gels at 4% (w / w) using surfactants as solubilizing agent. As the initial phase of study was carried out to determine the critical micellar concentration (CMC) by action of surface tension of papain gels at 4.0% (w / w) with and without L-cysteine and different concentrations of polysorbate 80. New preparations were developed using concentrations of polysorbate and Lcysteine resulted in complete solubilization of papain. The stability study of papain gels at 4.0% (w / w) polysorbate 80 was planned and carried out from 33 factorial experimental design for samples stored under refrigeration for 30 days. The independent variables were prescribed concentration of L-cysteine, concentration of polysorbate 80 and the storage time; the evaluated dependent variable was the concentration of active papain. All samples were analyzed for the maintenance of sensorial and physicochemical characteristics. During the evaluation period preparations presented as homogeneous gels, remaining stable as the pH, viscosity and thermodynamic stability. These samples showed no changes in their rheological behavior. The preparations studied showed ative papain concentrations between 90 and 110% after 24 hours of preparation, but it was not possible to maintain this activity over the study period. The statistical evaluations resulting from the factorial experimental design resulted in response surface graphs and contour, showing that time is the most influential factor on the loss of papain activity in preparations gel. L-cysteine favorably affects the maintenance of the activity, but its action alone is unable to override the effect of the time. The combination of Lcysteine and polysorbate 80 also presents favorable action to maintain the enzyme activity, which becomes evident only at the highest concentrations investigated for these adjuvants

The goal of this project was to develop a non-toxic amphiphilic diblock copolymer nanoparticulate drug delivery system that will solubilize paclitaxel (PTX) and retain the drug in plasma. Methoxy poly(ethylene glycol)-block-poly(ε-caprolactone) (MePEG-b-PCL) diblock copolymers loaded with PTX were characterized and their physicochemical properties were correlated with their performance as nanoparticulate drug delivery systems. A series of MePEG-b-PCL was synthesized with PCL blocks ranging from 2-104 repeat units and MePEG blocks of 17, 44 or 114 repeat units. All copolymers were water soluble and formed micelles except MePEG₁₁₄-b-PCL₁₀₄, which was water insoluble and formed nanospheres. Investigation of the effects of block length on the physicochemical properties of the nanoparticles was used to select appropriate copolymers for development as PTX nanoparticles. The critical micelle concentration, pyrene partition coefficient and diameter of nanoparticles were found to be dependent on the PCL block length. Copolymers based on a MePEG molecular weight of 750 g/mol were found to have temperature dependent phase behavior. Relationships between the concentration of micellized drug and the compatibility between the drug and core-forming block, as determined by the Flory-Huggins interaction parameter, and PCL block length were developed. Increases in the compatibility between PCL and the drug, as well as longer PCL block lengths resulted in increased drug solubilization. The physicochemical properties and drug delivery performance characteristics of MePEG₁₁₄-b-PCL₁₉ micelles and MePEG₁₁₄-b-PCL₁₀₄ nanospheres were compared. Nanospheres were larger, had a more viscous core, solubilized more PTX and released it slower, compared to micelles. No difference was seen in the hemocompatibility of the nanoparticles as assessed by plasma coagulation time and erythrocyte hemolysis. Micellar PTX had an in vitro plasma distribution similar to free drug. The majority of micellar PTX associated with the lipoprotein deficient plasma fraction (LPDP). In contrast, nanospheres were capable of retaining more of the encapsulated drug with significantly less PTX partitioning into the LPDP fraction. In conclusion, although both micelles and nanospheres were capable of solubilizing PTX and were hemocompatible, PTX nanospheres may offer the advantage of prolonged blood circulation, based on the in vitro plasma distribution data, which showed that nanospheres retained PTX more effectively. / Pharmaceutical Sciences, Faculty of / Graduate

The importance of interactions between transmembrane domains of integral membrane proteins has been well-established in a range of essential cellular functions. Most integral membrane proteins also possess regions that lie on the exterior of the membrane that may influence the ability of these transmembrane domains to interact. We sought to test this hypothesis by quantifying the energetics of transmembrane helix self-association in the absence and presence of an amphipathic helix that can bind to the membrane surface. The model chosen for this study was the major coat protein (MCP) of M13 bacteriophage, which has an N-terminal amphipathic helix linked to its single transmembrane segment via a flexible linker. Dimerization of both full-length MCP and a peptide containing only the transmembrane domain (MCPTM) was studied by solution NMR in SDS micelles. We found that there was an increase in the apparent dimerization affinity in the absence of the N-terminal helix. However, this increase in apparent affinity could be attributed to differences in detergent-binding properties of the two polypeptides in monomeric versus dimeric states when the empty micelle was considered to be a participant in the dimer dissociation. Preliminary results from the integral membrane protein, p7 of the hepatitis C virus are also presented in this thesis. It has been demonstrated that p7 enhances viral infectivity and accumulation, and that this function may require oligomerization in the membrane. While we encountered limitations due to challenges in the generation of sufficient quantities of pure p7 samples, we were able to perform circular dichroism spectroscopy under conditions that may favor different oligomeric states. These studies suggest that there is a change in the degree of helicity upon oligomerization, and suggest that SDS could be a suitable system to characterize the interactions of the p7 oligomer in the future.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23031号 / 理博第4708号 / 新制||理||1675(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 寺嶋 正秀, 教授 林 重彦, 教授 渡邊 一也 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23219号 / 工博第4863号 / 新制||工||1759(附属図書館) / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 田中 庸裕, 教授 近藤 輝幸, 准教授 菅瀬 謙治, 教授 佐藤 啓文 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Presented diploma thesis deals with drug encapsulation of Sulfathiazole (STA) in thermosensitive hydrogel composed of amphiphilic fibrous molecules of copolymer poly(D,L-lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(D,L-lactide-co-glycolide) (PLGA-PEG-PLGA). In the experimental part, the structure of the micelles which is dependent on the concentration of the default copolymer, was thoroughly described. The method of dynamic light scattering (DLS) was used to dimensionally characterize the micellar size in aqueous solution. At the same time, the Cryo-Transmission Electron Microscopy (Cryo-TEM) imaging technique confirmed the transition from spherical micelles to fibrous branched structures with a number of topological interactions. The change in viscoelastic properties of micellar hydrogel after addition of STA was studied by rheological analysis. The studied triblock copolymer appears to be a suitable tissue engineering material as a carrier in applications for targeted drug delivery and tissue regeneration.

The objective of this work is to enhance the understanding of the delay observed in the early stages of E-coat deposition. E-coat deposition has been widely used by industries such as the automotive industry to form the primary protective coating against corrosion. Currently, models that are used to find the best conditions under which the desired coating coverage for the entire auto body can be achieved do not accurately predict the coating coverage in recessed areas. The accuracy of large-scale models can be improved by enhancing our understanding of the mechanisms responsible for the observed delay. To accomplish this, experiments are performed to define the processes that control deposition initiation and then a model is developed to describe those processes. Simulation results are compared with experimental measurements for a range of conditions to assess the validity of the results. The delay before the onset of deposition is influenced by the type of substrate and properties of the E-coat solution. The impact of the substrate type on the onset of deposition was experimentally investigated. The results of experiments indicated that surface characteristics such as adhesion of bubbles to the surface and the formation of an initial coating increase the local current density on the surface. Investigations of the morphology of the initial coating on different types of substrates indicated that deposition began at areas where the local current density was higher. Increasing the local current density due to the adhesion of bubbles to the surface resulted in a 40% reduction in the time required for the onset of deposition on galvanized steel compared to bare steel. The processes in the solution adjacent to the surface were also investigated to understand the mechanisms responsible for the onset of deposition. Convection was used as a tool to determine the impact of the accumulation of hydroxide ions on the onset of deposition. The results of rotating disk electrode (RDE) experiments showed that the observed delay before deposition was not due to the time required for accumulation of hydroxide ions at the surface. The results of additional experiments showed that the accumulation of micelles was critical to the deposit initiation. The impact of micelle accumulation on the deposit initiation was further explored by developing a mathematical model of the physical processes in the solution adjacent to the surface. The model was evaluated at different conditions and was found to agree with experimental results at different current densities and bulk micelle concentrations. The model and the experimental results from this study help to explain the observed delay in the early stages of E-coat deposition and provide a basis for improving large-scale simulation of E-coat deposition.

Enzymic milk coagulation was monitored by measuring changes in curd firmness and apparent absorbance of undiluted milk. Detection of coagulation, visually or rheologically, occurred after the milk changes from a system of aggregating particles to an extended space network. This change was observed as a shoulder in apparent absorbance plots and coagulation time was defined as the critical point in the aggregation process analogously to non-linear condensation polymerization reactions. It corresponds to the inflexion point during the period when apparent absorbance was rapidly increasing and can be calculated by fitting curd firmness data to an exponential equation. Addition of calcium chloride to milk reduced coagulation time with a minimum occurring at .05M calcium. Also, curd firmness increased with a maximum at .02M calcium. It appears that calcium affects all stages of coagulation: proteolysis, micelle aggregation, and gelation. When bulk culture media was added to milk, the pH of the media had a greater effect on coagulation time than did presence of phosphate in the media. Non-specific proteolytic activity of milk coagulants affects the initial rate of curd firming but not the maximum firmness. The more proteolytic the enzyme the slower the curd firming rate. This can be used to rapidly assay for pepsin content of bovine rennets.

Tato práce je zaměřena na agregační chování nativního a hydrofobně modifikovaného hyaluronanu, v různých molekulových hmotnostech a stupních substituce, ve vodném prostředí. Pro studium bylo vybráno šest fluorescenčních sond s různými vlastnostmi (Pyren; Nilská červeň; Perylen; Akridinová oranž; 6-(p-Toluidino)-2-nafthalenesulfonová kyselina; PRODAN). a výsledky získané pomocí těchto sond byly porovnány s jednoduchým anionaktivním tenzidem (Dodecylsíran sodný). U všech použité sond byly testovány jejich spektrální vlastnosti v závislosti na polaritě okolí a/nebo na koncentraci. Pro stanovení vlastností nepolárního jádra hyaluronového agregátu byly vybrány dvě sondy (Pyren, Nilská červeň). U domén byly sledovány polarita a viskozita vnitřního prostředí a jejich závislost na iontové síle a teplotě. Pro modifikované hyaluronany bylo stanoveno, že jejich kritická agregační koncentrace klesá s rostoucí molekulovou hmotností a stupněm substituce. Pro vlastní doménu platí, že její kompaktnost roste s rostoucí iontovou silou, ale klesá s rostoucí teplotou.

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