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Strategies to improve the aging, barrier and mechanical properties of chitosan, whey and wheat gluten protein filmsOlabarrieta, Idoia January 2005 (has links)
Chitosan, Whey Protein Isolate (WPI) and vital wheat gluten (WG) are three biomaterials that have quite promising properties for packaging purposes. They have good film forming properties and good gas barrier properties in dry conditions. Moreover, because they are produced from industrial waste of food processing, they offer an ecological advantage over polymers made from petroleum. However, their physicochemical characteristics still must be improved for them to be of commercial interest for the food packaging industry. The purpose of this work was to study different strategies aiming to improve the water resistance and aging properties of these polymers, which are some of the key disadvantages of these materials. The produced solution cast chitosan and WPI films were characterised with scanning electron microscopy (SEM), density measurements and thermogravimetry. The water vapour transmission rate was determined at a relative humidity of 11%. In the first part, mechanical properties of solid films and seals were assessed by tensile testing. WG film’s tensile properties and oxygen and water vapour permeabilities were measured as a function of aging time. The changes in the protein structure were determined by infrared spectroscopy and size-exclusion high-performance liquid chromatography and the film structure was revealed by optical and scanning electron microscopy. Gluten-clay nanocomposites were characterised by tensile testing, X-ray diffraction and transmission electron microscopy. The incorporation of a hydrophobic biodegradable polymer, poly ( ε-caprolactone), PCL, in both chitosan and whey protein, yielded a significant decrease in water vapour transmission rate. It was observed that a certain amount of the PCL particles were ellipsoidal in chitosan and fibrous in WPI. The obtained data also indicated that the particle shape had an important influence in the water vapour transmission rate. In the second part, the aging properties of WG films, plasticized with glycerol and cast from water/ethanol solutions with pH=4 or pH=11 were investigated. WG films made from alkaline solutions were mechanically more time-stable than the acidic ones, the latter being initially very ductile but turning brittle towards the end of the aging period. The protein solubility measurements indicated that the protein structure of the acidic films was initially significantly less aggregated than the in basic films. During aging the acidic films lost more mass than the basic films through slow evaporation of volatiles (water/ethanol) and through migration of glycerol to the paper support. The oxygen permeability was also lower for the basic films. In the last part, the properties of new and aged glycerol-plasticized WG films at acidic and basic conditions containing ≤4.5 wt% natural or quaternary-ammonium-salt-modified montmorillonite were studied. Films of WG with montmorillonite were possible to produce by solution casting. The aging rate of acidic and basic films was unaffected by the incorporation of clay. However, the large reduction in water vapour permeability for most systems suggested that the clay sheets were evenly distributed within the films. The film prepared from basic solution and containing natural clay was almost completely exfoliated as revealed by transmission electron microscopy and X-ray diffraction. The best water vapour barrier properties were obtained by using modified clay. / QC 20101013
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Preparación y evaluación de formulaciones acrílicas autocurables de baja toxicidad modificadas con polímeros biodegradables para cirugía ortopédica y mínimamente invasivaFranco Marquès, Elena 20 July 2012 (has links)
In the present work, a study of the preparation of new formulations of acrylic bone cements (ABC) has been carried, to obtain materials with capacity for controlled drug delivery of medicaments useful in the therapeutic treatment of osteoporosis. The main research line has been focused on the partial modification of the solid phase of the conventional formulation of ABC, by substituting part of the PMMA beads with different microparticles of synthetic as well as natural biodegradable polymers. The incorporation of these polymers to the conventional formulation, with a structure based on PMMA, has been focused on two well defined points of view: as a structural component, and as a model component to study its delivery from the material to the external medium, to determine the capacity of the formulation for drug delivery. The work was finished with the evaluation of the capacity of controlled release of proteins and the determination of biocompatibility. / En este trabajo se ha llevado a cabo el estudio de nuevas formulaciones de cementos óseos acrílicos (COA) para su posterior evaluación como sistema de liberación controlada de medicamentos útiles en el tratamiento terapéutico de la osteoporosis. El trabajo se ha fundamentado en la modificación parcial de la fase sólida de los COA, mediante la substitución parcial de las microesferas de PMMA, por diferentes micropartículas de polímeros biodegradables, tanto de naturaleza sintética como natural. La incorporación de estos polímeros a la formulación del COA convencional, cuya estructura polimérica básica es el PMMA, ha sido enfocada desde dos puntos de vista bien diferenciados: como un componente estructural, que aporta resistencia mecánica al material, o como un componente que nos permita determinar la capacidad de liberación de un fármaco como el Ibandronato o de una proteína modelo como el colágeno hidrolizado. Finalmente se ha evaluado la biocompatibilidad de los COA modificados.
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Tailoring of Biomaterials using Ionic Interactions : Synthesis, Characterization and ApplicationAtthoff, Björn January 2006 (has links)
The interactions between polymers and components of biological systems are an important area of interest within the fields of tissue engineering, polymer chemistry, medicine and biomaterials. In order to create such a biomimetic material, it must show the inherent ability to reproduce or elicit a biological function. How do we design synthetic materials in order to direct their interactions with biological systems? This thesis contributes to this research with aspects of how polymers interact with biological materials with the help of ionic interactions. Polyesters, biodegradable or not, may after a hydrolytic cleavage interact ionically with protonated amines by the liberated carboxylate functions. Amines are found in proteins and this fact will help us to anchor proteins to polyester surfaces. Another type of interaction is to culture cells in polymeric materials, i.e. scaffolds. We have been working on compliant substrates, knitted structures, to allow cell culture in three dimensions. A problem that arises here is how to get a high cell seeding efficiency? By working on the interactions between polymers, proteins and finally cells, it is possible to create a polarized protein membrane that allows for very efficient cell seeding, and subsequent three dimensional cell cultures. Finally a synthetic route to taylor interaction was developed. Here a group of polymers known as ionomers were synthesized. In our case ionic end groups have been placed onto biodegradable polycarbonates, we have created amphiphilic telechelic ionomers. Functionalization, anionic or cationic, changes the properties of the material in many ways due to aggregation and surface enrichment of ionic groups. It is possible to add functional groups for a variety of different interactions, for example introducing ionic groups that interact and bind to the complementary charge of proteins or on the other hand one can chose groups to prevent protein interactions, like the phosphorylcholine zwitterionomers. Such interactions can be utilized to modulate the release of proteins from these materials when used in protein delivery applications. The swelling properties, Tg, degradation rate and mechanical properties are among other things that will easily be altered with the choice of functional groups or backbone polymer.
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Sequential Growth Factor Delivery From Polymeric Scaffolds For Bone Tissue EngineeringYilgor, Pinar 01 September 2009 (has links) (PDF)
Tissue engineering is a promising alternative strategy to produce artificial bone substitutes / however, the control of the cell organization and cell behavior to create fully functional 3-D constructs has not yet been achieved. To overcome these, activities have been concentrated on the development of multi-functional tissue engineering scaffolds capable of delivering the required bioactive agents to initiate and control cellular activities. The aim of this study was to prepare tissue engineered constructs composed of polymeric scaffolds seeded with mesenchymal stem cells (MSCs) carrying a nanoparticulate growth factor delivery system that would sequentially deliver the growth factors in order to mimic the natural bone healing process. To achieve this, BMP-2 and BMP-7, the osteogenic growth factors, were encapsulated in different polymeric nanocapsules (poly(lactic acid-co-glycolic acid) (PLGA) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)) with different properties (degradation rates, crystallinity) and, therefore, different release rates to achieve the early release of BMP-2 followed by the release of BMP-7, as it is in nature. Initially, these nanoparticulate delivery systems were characterized and then the effect of single, simultaneous and sequential delivery of BMP-2 and BMP-7 from these delivery systems was studied in vitro using rat bone marrow MSCs. The effect of using these two growth factors in a sequential manner by mimicking their natural bioavailability timing was shown with maximized osteogenic activity results. BMP-2 loaded PLGA nanocapsules were subcutaneously implanted into Wistar rats and according to initial results, their biocompatibility as well as the positive effect of BMP-2 release on the formation of osteoclast-like cells was shown. To complete the construction of the bioactive scaffold, this nanoparticulate sequential delivery system was incorporated into two different types of polymeric systems / natural (chitosan) and synthetic (poly(& / #949 / -caprolactone) (PCL)). 3-D fibrous scaffolds were produced using these materials by wet spinning and 3-D plotting. Incorporation of nanocapsules into 3-D chitosan scaffolds was studied by two different methods: incorporation within and onto chitosan fibers. Incorporation into 3-D PCL scaffolds was achieved by coating the nanocapsules onto the fibers of the scaffolds in an alginate layer. With both scaffold systems, incorporation of nanocapsule populations capable of delivering BMP-2 and BMP-7 in single, simultaneous and sequential fashion was achieved. As with free nanocapsules, the positive effect of sequential delivery on the osteogenic differentiation of MSCs was shown with both scaffold systems, creating multi-functional scaffolds capable of inducing bone healing.
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Strategies for building polymers from renewable sources : Using prepolymers from steam treatment of wood and monomers from fermentation of agricultural productsSöderqvist Lindblad, Margaretha January 2003 (has links)
<p>A strategic research area today is development of polymericproducts made from renewable sources. The ways of utilizingrenewable sources studied in this thesis are using 1)prepolymers obtained by steam treatment of wood and 2) monomersobtainable by fermentation of agricultural products.</p><p>Novel hemicellulose-based hydrogels were prepared by usingprepolymers obtained from steam treatment of spruce.Hemicellulose was first modified with well-defined amounts ofmethacrylic functions. Hydrogels were then prepared by radicalpolymerization with 2-hydroxyethyl methacrylate orpoly(ethylene glycol) dimethacrylate to form hydrogels. Theradical polymerization reaction was carried out in water usinga redox initiator system. The hydrogels were in generalelastic, soft and easily swollen in water. Frequency sweeptests indicated that the hydrogel system displayed prevailingsolid-like behavior. Comparison of the hemicellulose-basedhydrogels with pure poly(2-hydroxyethyl methacrylate)-basedhydrogels showed that it was possible to preparehemicellulose-based hydrogels with properties similar to thoseof pure poly(2-hydroxyethyl methacrylate)-based hydrogels.</p><p>Polyester-based materials were prepared by using themonomers 1,3- propanediol and succinic acid obtainable byfermentation. α,ω-Dihydroxyterminatedoligomeric polyesters produced by the thermal polycondensationof 1,3-propanediol and succinic acid were chain-extended toobtain sufficiently high molecular weight. Depending on thechain-extension technology adopted, poly(ester carbonate)s orpoly(ester urethane)s were obtained. In the case of poly(estercarbonate)s, the chain-extended products ofα,ω-dihydroxyterminated oligomeric copolyesters werealso produced using 1,3-propanediol/1,4-cyclohexanedimethanol/succinic acid mixtures toimprove thermal and mechanical properties. Segmented poly(esterether carbonate)s fromα,ω-dihydroxyterminated oligo(propylenesuccinate)s and poly(ethylene glycol) were also synthesized toincrease the hydrophilicity.</p><p>Molecular weights and polydispersity were analyzed by SECfor all materials. Their structures were also identified by NMRspectroscopy (1H NMR and 13C NMR). All characterizations werein agreement with the proposed structures. Thermal parameterswere characterized by DSC. Tensile testing anddynamic-mechanical tests were performed and in additionpreliminary processing trials were carried out in some cases.The results demonstrate the feasibility of using monomersderived from renewable sources to build up new polymericstructures endowed with a variety of physical and mechanicalproperties.</p>
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Etudes diélectriques des matériaux biodégradables et/ou bio-sourcés / Dielectric study of biodegradable and/or bio-based polymeric materialsHegde, Vikas 13 July 2017 (has links)
L'utilisation extensive des combustibles fossiles, l'émission de gaz à effet de serre dans l'atmosphère et les difficultés de recyclage des résidus de consommation incitent au recours accru aux polymères biodégradables. Dans le domaine du génie électrique, de nombreux polymères trouvent des applications dans les systèmes d'isolation des matériels électriques. Ces polymères sont issus du pétrole, peu respectueux de l'environnement et la plupart d'entre eux ne sont pas biodégradables. Dans le but de les remplacer, des polymères biodégradables ont été explorés.Une revue des travaux de recherche sur les polymères biodégradables dans le domaine du génie électrique a été réalisée. Les propriétés thermiques et électriques de polymères biodégradables et conventionnels sont rapportées et comparées.Les polymères biodégradables et / ou biosourcés sélectionnés et mis en œuvre sont le Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), le polycaprolactone (PCL) et l’acide polylactique PLA. Un matériau nanocomposite basé sur PLA a également été préparé. Les propriétés diélectriques pour une large gamme de température et de fréquence ont été mesurées par spectroscopie diélectrique et analysées en s’appuyant sur les propriétés thermiques obtenues par DSC et DMA. La résistivité volumique et la tenue diélectrique ont pu également être mesurées. Ces polyesters biodégradables sont comparés aux polymères conventionnels. / The declining resources of fossil fuels, increase in wide-spread pollution, emission of green-house gases and difficulties in recycling waste materials are pushing biodegradable polymers into prominence. In the domain of electrical engineering, many polymers find applications in various electrical insulation systems. These polymers are petro-based, not eco-friendly and most of them are not biodegradable. With an objective to replace conventional products, biodegradable polymers are explored for their dielectric properties.In this work, a detailed study on the present status in the research work on biodegradable polymers in the electrical engineering domain is presented. Thermal and electrical properties of both biodegradable and classical polymers are compiled and compared.The polymers processed and studied were Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), Polycaprolactone (PCL), Poly(lactic acid) (PLA) and PLA based nanocomposite. Dielectric properties for a wide range of temperature and frequency were measured by dielectric spectroscopy and analyzed with the help of DSC and DMA experiments. Volume resistivity and electrical breakdown were measured on few polymers. These biodegradable polyesters were compared with conventional polymers.
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Desenvolvimento e caracterização de filmes poliméricos biodegradáveis para aplicação em sacolas descartáveis e sua análise de ecoeficiênciaCarvalho, Jéssika Souza de January 2017 (has links)
Orientador: Prof. Dr. Derval dos Santos Rosa / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Ciência e Tecnologia Ambiental, 2017. / Nos dias de hoje, tem sido crescente a preocupação com os impactos ambientais provocados pelo descarte inadequado de sacolas plásticas após seu uso. Em busca de uma solução parcial para o descarte dessas, nos últimos anos vem aumentando o interesse em incrementar e desenvolver materiais biodegradáveis com características que permitam a sua utilização em embalagens e utensílios descartáveis e que contribuam com a sustentabilidade. Esse trabalho teve como objetivo desenvolver filmes poliméricos biodegradáveis, para uso em sacolas descartáveis de supermercado. Os filmes foram preparados utilizando PBAT, PLA e CaCO3, em diferentes composições. Os filmes das composições foram caracterizados por ensaios mecânicos e avaliação da biodegradação. Foram analisados também os desempenhos ambiental e econômico, ao longo do ciclo de vida dos filmes preparados, em comparação com sacolas usuais disponibilizadas nos supermercados (kraft e PEAD). Essa Análise de Ecoeficiência foi realizada segundo as normas ABNT NBR da série ISO 14040, com base em ferramentas de cálculo e bancos de dados de Avaliação de Ciclo de Vida internacionalmente reconhecidos. Os resultados do ensaio mecânico demonstraram que a incorporação de CaCO3 nos filmes diminuiu o alongamento na ruptura e a resistência máxima à tração e aumentou o módulo de Young dos materiais. Todas amostras apresentaram caráter hidrofílico, com ângulos de contato menores que 90º. As amostras com CaCO3 não apresentaram nítidas separação de fases, diferentemente da amostra sem carga, demonstrando que a incorporação do material favoreceu a dispersão e interação de PBAT/PLA. O CaCO3 reduziu e temperatura máxima de degradação das amostras. Com exceção da amostra de papel, todas apresentaram pequenos percentuais de massa sorvida. Após 360 dias do ensaio de biodegradação, os resultados mostraram que não houve uma perda de massa expressiva, no entanto, as amostras já apresentaram uma modificações superficiais, indicando ação de micro-organismos. Na Análise de Ecoeficiência foi possível observar que a incorporação de CaCO3 diminuiu significantemente os impactos econômicos das alternativas em estudo e favoreceu a ecoeficiência dos polímeros biodegradáveis. / Nowadays, there has been growing concern about the environmental impacts caused by the improper disposal of disposable plastic bags after use. In search of a partial solution for their disposal, the interest in improving and developing biodegradable materials of low cost with characteristics that allow its use in disposable containers and utensils that contribute to the sustainability have increased in recent years. This work aimed to develop polymeric biodegradable films for use in disposable supermarket bags. The films were prepared using PBAT, PLA and CaCO3, in different compositions. These films were characterized by mechanical tests and evaluation of biodegradation. There were also analyzed the environmental and economic performance over the life cycle of the films prepared in comparison with the usual alternatives available in supermarkets (paper and PEAD). This Eco-efficiency Analysis was performed according to NBR ISO 14040 standards series, based on calculation internationally recognized tools and databases of Life Cycle Assessment. The results of the mechanical tests demonstrated that the incorporation of CaCO3 in the films decreased the elongation at break and the maximum tensile strength at break of the materials, but increased the Young's modulus. All samples presented a hydrophilic character, with contact angles smaller than 90º. The samples with CaCO3 did not present a clear phase separation, unlike the sample with no load, demonstrating that the incorporation of the material favors the interaction of PBAT with PLA. The CaCO3 reduced the maximum degradation temperature of the samples. With the exception of the paper sample, all presented small percentage of sorbed mass. After 360 days of biodegradation test, the results showed that there was no significant mass loss, however, the samples already presented a superficial modifications, indicating action of microorganisms. In the Eco-efficiency Analysis it was possible to observe that the incorporation of CaCO3 significantly decreased the economic impacts of the alternatives under study and favored the eco-efficiency of the biodegradable polymers.
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Avaliação do potencial de Burkholderia sacchari produzir o copolimero biodegradável poli(3-hidroxibutirato-co-3-hidroxihexanoato) [P(3HB-co-3HHX)]. / Evaluating the potential of Burkholderia sacchari to produce the biodegradable copolymer poly (3-hydroxybutirate-co-3-hydroxyhexanoate).Thatiane Teixeira Mendonça 11 February 2010 (has links)
A capacidade de B. sacchari acumular poli-3-hidroxibutirato-co-3-hidroxihexanoato (P3HB-co-3HHx) foi confirmada, com até 2 mol% de 3HHx no PHA total (<10% do 3HHx máximo teórico a partir do ácido), indicando flexibilidade da PHA sintase por substratos, porém alta eficiência nas vias catabólicas do hexanoato. Análise da estabilidade térmica do PHA indicou uma temperatura de degradação reduzida, compatível com a presença de unidades 3HHx. Mutantes incapazes de crescer em ácido hexanóico foram obtidos com UV e transposon mini-Tn5, que ainda acumulavam 3HHx a partir de hexanoato mas com redução na capacidade do acúmulo de 3HB e 3HHx. Foram construídos recombinantes abrigando o gene phaB (codificador de 3-cetoacil-CoA redutase) de Ralstonia eutropha ou phaJ1 e phaJ2 (codificadores de enoil-CoA hidratases R-específicas) de Pseudomonas aeruginosa. A expressão de phaB ou phaJ1 aumentou a canalização de 3HB para a PHA sintase, apesar de não aumentar as frações de 3HHx. Monômeros de 3HHx e 3HO foram detectados a partir de ácidos butírico e octanóico, respectivamente. / The ability of B. sacchari to accumulate poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (P3HB-co-3HHx) from glucose and hexanoic acid was confirmed. 3HHx content was up to 2 mol% of PHA (<10% of the maximum theoretical 3HHx from the acid), indicating a substrate flexibility of B. sacchari PHA synthase, but high efficiency of hexanoate catabolic pathways. Thermal stability analysis of the copolymer indicated a reduced degradation temperature compatible with 3HHx units. Mutants unable to grow on hexanoic acid were obtained with UV and mini-Tn5 transposon. They still accumulated 3HHx from hexanoate, but the ability to accumulate 3HB and 3HHx was reduced. Recombinants harboring the Ralstonia eutropha phaB (encoding 3-ketoacyl-CoA reductase) and Pseudomonas aeruginosa phaJ1 and phaJ4 genes (encoding R-specific enoyl-CoA hydratases) were constructed. Expression of both phaB and phaJ1 increased the channeling of 3HB to the PHA synthase, despite no increase on 3HHx fraction was observed. 3HHx and 3HO monomers were detected from butyric and octanoic acids, respectively.
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Toxoide diftérico: nova roupagem para uma vacina tradicional / Diphtheric toxoid: new clothes for a traditional vaccineJocimara Ambrosio de Moraes Namur 27 November 2007 (has links)
O processo de micrencapsulação de proteínas em microesferas (MS) de PLGA [poli (ácido lactico-co-glicolico)] é fácil de fazer e é uma ferramenta útil para melhorar tanto uma formulação quanto para aumentar a atividade imunológica de vacinas de novas gerações. A MS-PLGA têm caráter adjuvante porque é um sistema particulado e, além disto, controla a liberação do antígeno. O escopo desta tese foi o de dar uma nova roupagem para um antígeno vacinal tradicional e muito bem estudado- o toxoide diftérico (Dtxd). Estudaram-se a produção de MS de tamanho desejado; os mecanismos que controlam danos nas proteínas durante o processo de micrencapsulação; a produção de microesferas com características de liberações em tempos distintos e ensaios biológicos. O tamanho de MS é um determinante fundamental para controlar a velocidade de liberação de um soluto. Para se produzir MS com tamanhos controlados usou-se um desenho fatorial experimental com três fatores distintos e três pontos centrais, para se determinar a influência das variáveis (concentração de poli álcool vinílico; velocidade de agitação e relação fase dispersa/fase contínua) na determinação do tamanho das MS. Foram obtidas MS esféricas e lisas de 4- 15 µm de diâmetro. Estes resultados abrem a possibilidade de se formular PLGA-MS com tamanhos planejados através de um mínimo de experimentos. O mecanismo de danos conformacionais nas proteínas nas várias fases do processo de produção de PLGA-MS é ainda uma questão em aberto. Usaram-se várias técnicas biofísicas (HPLC, espectroscopias no uv, fluorescência e CD) além de ELISA para se testar a interferência dos sais da série de Hofmeister sobre a solubilidade e estabilidade da proteína durante a emulsificação e do contacto com a interface água/cloreto de metileno (primeira etapa do processo de preparação de MS). Estudaram-se também a influência de oligômeros de PLGA e SDS sobre a estrutura da proteína no meio de liberação (etapa de liberação do soluto). A emulsificação de Dtxd na presença de Mg2+ induziu agregação protéica, com exposição de resíduos hidrofóbicos para o meio; variações no ângulo diédrico do S-S proteico e perda de identidade imunológica. Esta agregação foi quase abolida pelo caotrópico SCN- (toxicidade = 30 g/ homem adulto de 70 kg). A conformação \"nativa\" do Dtxd e sua atividade biológica foram protegidas pelo KSCN. Os oligômeros de PLGA e o SDS induziram uma conformação de Dtxd nova. A adição de KSCN na fase aquosa aumentou a eficiência de encapsulação de Dtxd pela PLGA-MS em 20 %. Esta foi a solução mais simples quando comparada com aquelas descritas na literatura. Produziram-se seis formulações diferentes (diferentes massas molares e carboximetilações do PLGA) com pelo menos três cinéticas de liberações distintas. Imunizaram-se camundongos com 5 µg de Dtxd encapsulado em MS-PLGA usando-se dois polímeros de 12 kDa (-COOH livre ou metilado) e um outro de 63 kDa (metilado). O padrão de resposta e a maturidade imunológicas foram medidos por titulações de IgG1 e IgG2a. Mantiveram-se os mesmos padrões de resposta humoral (desejável). Menores quantidades de antígenos foram necessárias para se obter os mesmos benefícios gerados pela vacina tradicional de Dtxd. Aumentaram-se a produção e a seletividade de anticorpos através de duas manipulações simples: a formulação e o tempo da aplicação da dose de reforço. Estes resultados colocam estas formulações na área de vacinas de sucesso uma vez que também foram obtidas memórias imunológicas. / The protein microencapsulation within microspheres (MS) of PLGA (Poly-lactide-co-glycolide) is easy to do and, it is a useful tool to enhance formulation and immunologic performances for new generation vaccines. MS-PLGA has adjuvant character because it is a particulate system and can control the antigen release. The question addressed in this thesis was to give this new dress for the traditional and well studied vaccine antigen - the diphtheria toxoid (Dtxd). The steps of MS control size production; mechanism to control protein damages; MS production with different polymers and biological assay were addressed here. MS size is a primary determinant of solute release velocity. A full factorial experimental design 23 with triplicate at the central point was used to determine the influence of variables (polyvinyl alcohol concentration, stirring velocity and the relationship between dispersed /continuous phase) on MS size. Uniformly spherical and smooth microspheres (4 - 15 µm of diameter) were obtained. These results open the possibility of formulating PLGA microspheres with custom sizes performing a minimum of experiments as required for specific applications. It stills an open question to detail the conformational mechanism of protein damages during the various steps of the PLGA microencapsulation process. Various techniques (HPLC gel filtration, ELISA, Fluorescence, UV and Circular dichroism spectroscopies) were tested on the interference of the Hofmeister ion series over protein solubility and stability during the emulsification and contact with the interface water/CH2Cl2 interface (First step on MS preparation). The interference of SDS and PLGA olygomers over protein structure in the liberation media was also studied (solute liberation step). The Dtxd emulsification in the presence of Mg2+ was followed by protein aggregation, with exposition of hydrophobic residues and changes on the dihedral S-S protein angle and loses on immunological identity. This aggregation is 95% avoided by the chaotropic and little toxic salt KSCN (30g/ adult human of 70 kg). All the \"native\" Dtxd conformation and biological properties were maintained by KSCN. MS with different liberation kinetics profile and different erosion characteristics were obtained by using six different polymers. The SDS and PLGA olygomers exerted a generation of new Dtxd molecular organization. The KSCN increased Dtxd encapsulation within PLGA-MS in more than 20 %. This was the simplest solution used to solve protein aggregation compared with others solutions used in the literature. The six different formulations produced (differing in molar mass and carboxymethylation) produced, at least, three different Dtxd liberation profiles. Mice were primed with 5 µg of Dtxd microencapsulated within MS prepared with 12 kDa (ended carboxymethylated or free PLGA) and with 63 kDa (methylated) PLGA. The response patterns and the immune maturity were measured by IgG1 and IgG2a titrations. The humoral pattern was maintained, but fewer antigens were needed to obtain the same traditional Dtxd vaccine benefits. The simple change on Dtxd-PLGA formulation and timing of the booster enhanced both, antibody production and selectivity. An immunological memory was also obtained, putting so, these formulations in the field of successful vaccine.
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Obtenção de mutantes deficientes no acúmulo de PHA e construção de linhagens recombinantes para o controle da composição monomérica / Mutants deficient on polyhydroxyalkanoate (PHA) accumulation and construction of recombinants to control monomer composition on PHARogério de Sousa Gomes 03 February 2010 (has links)
Pseudomonas putida produz PHA de cadeia média (PHAMCL) a partir de carboidratos e óleos vegetais. Genes de biossíntese de P3HB (poli-3-hidroxibutirato) de Ralstonia eutropha foram inseridos em P. putida IPT046 selvagem e mutantes PHA-. A expressão de phaC, codificador da PHA sintase, permitiu o acumulo de PHA com alto teor de 3HB, indicando que P. putida possui enzimas geradoras de 3HB. A expressão de phaB mostrou que seu produto canaliza 3HB e 3-hidroxihexanoato (3HHx) da <font face=\"Symbol\">β-oxidação para a PHA sintase. Ao expressar phaC em IPT461, afetado na PHA sintase, 30% de P3HB-co-3HHx-co-3HO foram produzidos a partir de carboidratos, mostrando que há vias metabólicas eficientes para suprir 3HAMCL, que são incorporados pela PHA sintase de R. eutropha. Usando transposon mini-Tn5, obtiveram-se mutantes deficientes na biossíntese de PHA a partir de glicose ou glicose e octanoato. Não se detectaram mutantes exclusivamente deficientes no acúmulo de PHA a partir de octanoato, indicando que diferentes produtos gênicos canalizam intermediários da <font face=\"Symbol\">β-oxidação para a síntese de PHA. / Pseudomonas putida produces medium-chain-length PHA (PHAMCL) from carbohydrates and plant oils. Recombinants harboring the P3HB (polyhydroxybutyrate) biosynthesis genes from Ralstonia eutropha were constructed on P. putida IPT046 wild type and PHA- mutants. Expression of phaC, encoding, PHA synthase, allowed the synthesis of PHA with high 3HB content, indicating that P. putida has enzymes to generate 3HB. Expression of phaB showed that its product channels 3HB and 3-hydroxyhexanoate (3HHx) from <font face=\"Symbol\">β-oxidation to the PHA synthase. When phaC was expressed on IPT461, affected in the PHA synthase, 30% P3HB-co-3HHx-co-3HO were accumulated from carbohydrates, showing that there are metabolic pathways efficient to supply 3HAMCL, which are incorporated by the R. eutropha PHA synthase. Using mini-Tn5 transposon, mutants deficient on PHA biosynthesis from glucose or glucose and octanoate were produced. No mutant deficient exclusively on PHA biosynthesis from octanoate was detected. Thus different gene products may channel intermediates from <font face=\"Symbol\">β-oxidation to PHA biosynthesis.
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