In this work, the synthesis and characterization of functionalized biosurfaces that can be used for bioseparations and bio-nanotechnology are reported. A novel protein purification technique that incorporates chelating ligands and polymers onto the same chromatographic matrix is explored. A polysaccharide based gel, agarose, was modified systematically with hybrid ligands of the chelator iminodiacetic acid (IDA) and the polymer polyethylene glycol (PEG). The PEG molecule acts as a blocking polymer that can allow only small proteins to permeate onto the matrix surfaces and form conventional immobilized metal ion affinity chromatographic (IMAC) interactions with the chelators. Kinetic studies of chelator and polymer attachment were performed in order to effectively control the chelator and polymer densities on the matrix. Studies with different PEG surface densities and their effects on the adsorption of several proteins (e.g. myoglobin, lysozyme and bovine serum albumin (BSA)) were evaluated to characterize these new hybrid size exclusion IMAC (SEIMAC) matrices. An exclusion effect was observed while adsorption as observed in IMAC systems took place.Functionalization schemes and procedures were extended in the activation and incorporation of affinity ligands on inorganic surfaces such as gold surfaces. Functional gold platforms were explored for development of nano-interconnects via functionalized self assembled monolayers (FSAMs) on gold to attach specific affinity ligands as linkers to immobilize biomolecules, such as microtubules (MTs). MTs eventually could be utilized as self assembling structures and templates for fabrication of nano-scale bio-interconnect arrays and networks. In this work, different organothiols were used to form FSAMs and anti-glutathione S-transferase was attached as a linker to utilize the attachment of MT cap proteins, gamma-tubulin. The gamma-tubulin could recognize specifically a heterodimer of the MTs and can provide a nucleation center for MT growth. Several methodologies were employed including photolithographic methods and the use of photoreactive compounds for proper micro/nano scale dual protein functionalization of surfaces with homogeneous affinity ligands and with heterogeneous ligands as well.

ROCHA, Thays Nogueira. Solubilização do fármaco Griseofulvina e da droga Oncocalixona A utilizando misturas dos copolímeros F127/P123/PEG35K como aditivo. 2015. 76 f. Dissertação (Mestrado em Química)-Universidade Federal do Ceará, Fortaleza, 2017. / Submitted by Celia Sena (celiasena@dqoi.ufc.br) on 2017-07-20T17:06:43Z No. of bitstreams: 1 2015_dis_tnrocha.pdf: 1233446 bytes, checksum: 2d595df0b79e6ef007f1edceda88db7f (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2017-11-06T22:27:16Z (GMT) No. of bitstreams: 1 2015_dis_tnrocha.pdf: 1233446 bytes, checksum: 2d595df0b79e6ef007f1edceda88db7f (MD5) / Made available in DSpace on 2017-11-06T22:27:16Z (GMT). No. of bitstreams: 1 2015_dis_tnrocha.pdf: 1233446 bytes, checksum: 2d595df0b79e6ef007f1edceda88db7f (MD5) Previous issue date: 2015 / A continuing challenge of the health sciences and pharmaceutical industry is to improve the performance of drugs in the human body, reducing administration doses, toxicity levels and side effects, improving their efficiency. Thus, the objective of this work was to investigate the solubilization of the micellar solutions composed of the copolymer E98P67E98 (F127)/P123, as well as their mixtures, using the polymer PEG 35K as additive against the aromatic drug griseofulvin and the bioactive oncocalixone A, combining characteristics of gelification of F127 and solubilization. The methodology used in the solubilization of the drug griseofulvin was the direct dissolution method and, due to the fact that the bioactive oncocalixone A presented instability in this resource, it was studied through the solvent evaporation (Film) method. For the characterization of the copolymers and their mixtures the rheology was used and for the study of the gelling properties, the technique of tube inversion. In order to characterize the systems that presented the best solubilization results for oncocalixone A, infrared spectroscopy (FT-IR), rheology and in vitro release experiments were performed. Solubilization was measured by UV-Vis spectrophotometry and particle size by dynamic light scattering (Zetasizer). The copolymers P123 and F127 as well as their mixtures in the PEG 35K additive showed promising vehicles in the administration of the hydrophobic drug griseofulvin, the concentration of surfactant being 1% which provided the best solubilization result. The bioactive oncocalixone A presented a high solubility value, both in aqueous medium and in the presence of PEG 35K, when compared to the drug griseofulvin, the PF 50 mixture being in aqueous medium and in the presence of the additive, which proposed better values in the solubilization for this drug. It is seen from the particle size data in the presence and absence of the oncocalixone A drug that the mixtures as well as the compositions composed of the copolymers isolated in PEG 35K presented sizes below 200 nm, avoiding that there is a prior uptake and elimination by the phagocytic mononuclear system. / Um desafio contínuo das ciências da saúde e da indústria farmacêutica é aperfeiçoar o desempenho dos fármacos no organismo humano, reduzindo as doses de administração, o nível de toxicidade e os efeitos colaterais, melhorando a sua eficiência. Dessa forma, o objetivo desse trabalho foi investigar a solubilização das soluções micelares compostas pelo copolímero E98P67E98 (F127) e P123, bem como suas misturas, utilizando o polímero PEG 35K como aditivo frente ao fármaco aromático griseofulvina e da droga oncocalixona A, combinando as características desejáveis de geleificação (F127) e solubilização. A metodologia utilizada na solubilização do fármaco griseofulvina foi o método da dissolução direta e, devido ao fato do bioativo oncocalixona A apresentar instabilidade nesse recurso, o mesmo foi estudado através do método da evaporação do solvente (Filme). Para a caracterização dos copolímeros e suas misturas fez-se o uso da reologia e para o estudo das propriedades geleificantes, a técnica de inversão de tubo. Visando ainda a caracterização dos sistemas que apresentaram os melhores resultados de solubilização para a oncocalixona A foi realizado o estudo desses pelas técnicas de espectroscopia na região do infravermelho (FT-IR), reologia e experimentos de liberação in vitro. A solubilização foi medida através da espectrofotometria UV-Vis e o tamanho de partícula por espalhamento de luz dinâmico (Zetasizer). Os copolímeros P123 e F127 bem como suas misturas no aditivo PEG 35K mostraram-se promissores veículos na administração do fármaco hidrofóbico griseofulvina, sendo a concentração do surfactante a 1 % a que proporcionou o melhor resultado de solubilização. O bioativo oncocalixona A apresentou, tanto em meio aquoso como na presença do PEG 35K, um elevado valor de solubilidade, quando comparado ao fármaco griseofulvina, sendo a mistura PF 50, em meio aquoso e na presença do aditivo, a que propôs melhor valores na solubilização para essa droga. Vê-se, a partir dos dados de tamanho de partícula, na presença e ausência da droga oncocalixona A, que as misturas bem como as composições compostas pelos copolímeros isolados em PEG 35K, apresentaram tamanhos abaixo de 200 nm, evitando com que haja uma prévia captação e eliminação pelo sistema mononuclear fagocitário.

Made available in DSpace on 2014-06-12T15:51:22Z (GMT). No. of bitstreams: 2 arquivo3089_1.pdf: 770384 bytes, checksum: 9c54cc6a2a126071254fd185f79c29bd (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / As colagenases são enzimas, obtidas a partir de procariotos e eucariotos, que clivam a cadeia protéica do colágeno em pH e temperatura fisiológicos. Estas enzimas são empregadas em diversas aplicações industriais com destaque para a indústria farmacêutica, onde são aplicadas no tratamento médico de feridas, cicatrizes e queimaduras. A proposta para a utilização do sistema de duas fases aquosas (SDFA) é devido à alta relação custo-benefício, baixa tensão interfacial, fácil escalonamento e a sua capacidade de purificar uma proteína em um ambiente rico em água (80-90%), o que favorece a estrutura protéica e retém assim a sua atividade biológica após purificação. Este trabalho visa à purificação de colagenase produzida por Penicillium aurantiogriseum (URM-4622) utilizando SDFA PEG/fosfato. O planejamento experimental (23) foi usado para selecionar as variáveis significativas no processo de purificação, e a massa molar do PEG (MMPEG), concentração do PEG (CPEG) e concentração do fosfato (CFOS) foram as variáveis estudadas. O sistema de duas fases aquosas foi composto de PEG 550, 1500 e 4000 g/mol nas concentrações de 15, 17,5 e 20% (m/m) e concentrações de fosfato de 12,5, 15 e 17,5% (m/m). As variáveis de resposta escolhidas foram: coeficiente de partição (K), rendimento de atividade (Y) e fator de purificação (PF). Os dados e gráficos obtidos passaram por análise estatística. Observou-se que PEG de massa molar 550 (g/mol) em concentração de 20% (p/p), concentração de fosfato 17,5% (p/p) e pH 6.0 foram as melhores condições para purificação de colagenase. Estas condições geraram um coeficiente de partição de 1,01, um rendimento de atividade de 242% e fator de purificação de 23,5. Os resultados mostraram que SDFA foi seletivo para colagenase e esta enzima particionou para a fase rica em polímero

This thesis provides a brief intro to the peg-and-hole problem and goes through two active and passive compliance strategies as well as the cases that the compliance center position is not ideal. A specific scenario of peg being gripped at an error angle due to large vision uncertainties is raised and studied. Such setup can lead to an off compliance center position relative to the peg and can happen in real life but has not been solved by previous approaches. A potential solution to it by combining active control and passive compliance is provided and analyzed. By using the force and torque feedback and the robot joint angle information, the compliance center for the above scenario could be estimated without vision feedback to by-pass the potential accuracy limitation of vision sensors. And a position control with reference determined in real-time by these sensors would be able to cancel out the majority of the effect caused by an off compliance center. Additional recommendations to future work on integration of active and passive compliance strategies and utilization of arbitrary compliance center positions are provided as well.

Combining enzymes with synthetic materials is the new frontier of biocatalysis, materials science, and protein engineering. Enzymes are biological macromolecule catalysts with incredible efficiency and specificity that are desirable for use in a variety of different fields. However, commercial applications have been limited by the stability and reusability of un-altered enzymes. An avenue for overcoming the challenges to harnessing enzyme power is to combine enzymes with materials to create an enzymatically-active material that has enhanced stability and activity. Unfortunately, the catalytic activity of the hybrid material is often lower than that of the enzyme alone. The activity of an enzyme is directly dependent on its structure and dynamics. Therefore, a deeper understanding of enzyme structure and dynamics upon incorporation into materials will provide the data necessary to rationally design enzymatically-active materials with the desired features. This dissertation explores the behavior of a model enzyme, T4 Lysozyme, with two different artificial material systems, metal-organic frameworks and polyethylene glycol. The underlying structural rationale for the behavior is probed using a variety of techniques, notably, Electron Spin Paramagnetic Resonance. Herein, the implications of structural alterations on activity and opportunities for exploitation are discussed. T4 Lysozyme is a perfect model for this study because it has a well characterized structure-activity relationship, thus providing a vast literature understanding which can be pulled from to verify and assist with interpretation of data. The structural basis of enzyme activity alteration in artificial materials can be used to rationally design systems with desired characteristics. After successfully demonstrating the tunability of proteins in artificial materials using T4L as a model, human Cu/Zn superoxide dismutase 1 was chosen for continuing studies due to its importance in diseased states. However, the superoxide dismutase mutant chosen is aggregation prone, which makes it difficult to express recombinantly in large amounts. Therefore, an efficient protocol for producing the superoxide dismutase protein was developed to set the stage for future studies.

Peg solitaire is a game in which pegs are placed in every hole but one and the player jumps over pegs along rows or columns to remove them. Usually, the goal is to have a single peg remaining. In a 2011 paper, this game is generalized to graphs. In this paper, we consider a variation in which each peg must be jumped twice in order to be removed. For this variation, we consider the solvability of several graph families. For our major results, we characterize solvable joins of graphs and show that the Cartesian product of solvable graphs is likewise solvable.

No description available.

The delivery of nucleic acids at the tissue and cellular levels remains one of the major hurdles in this scientific area. Since nucleic acids are bulky macromolecules and unstable in the presence of nucleases, vehicles are required to compact them into nanosized particles, offer protection from degradation in vivo, and release the therapeutic cargo at the desired location. Polycationic vehicles are good candidates for these purposes since they can be chemically modified to tune the desired properties in nanoparticle formulations. We designed a family of trehalose-oligoethyleneamine copolymers that showed promising plasmid DNA (pDNA) transfection results in the presence of serum proteins. A diazidotrehalose monomer was copolymerized with linear oligoethyleneamines of varying length and containing alkyne end-groups via step-growth Cu(I)-catalyzed azide-alkyne cycloaddition polymerization resulting in a series of trehalose copolymers with a range of secondary amines (from 4 to 6) within the polymer backbone. Upon electrostatic complexation of the polycations and pDNA in aqueous media, nanosized particles were formed, and their sizes and zeta-potentials were characterized via dynamic light scattering (DLS). The glycopolymers were tested for pDNA binding, toxicity, cellular uptake, and transfection efficiency in vitro. Characterization of these polymers revealed a significant influence of minor structural modifications on bioactivity. In general, all of the polymers efficiently bind pDNA at low nitrogen to phosphate (N/P) ratios forming nanoparticles below 100 nm in size and demonstrated cellular uptake and transfection. Polymers comprised of trehalose moieties and four secondary amines in the repeat unit showed the greatest promise in pDNA delivery in vitro. Because of its large hydration volume, we hypothesize that trehalose contributes to particle stabilization in serum. The trehalose-based polymers with four secondary amines (Tr4) were subsequently modified with PEG (5kDa). This modification lead to the development of well-defined polymeric structures with PEG moieties selectively incorporated at the ends of linear trehalose-oligoethyleneamine polycations. The study of the effect of this modification on bioactivity revealed that there were no significant difference in the toxicity profiles within this series of PEGylated and non-PEGylated materials; however, overall results suggest that both modified and unmodified trehalose-oligoethyleneamine copolymers have a great promise for stem cell-based and regenerative therapies. / Ph. D.

A viabilidade da produção em escala industrial de produtos biotecnológicos de interesse comercial e terapêutico, como os fármacos, depende significativamente das técnicas de separação e purificação utilizadas. A aplicação do sistema de duas fases aquosas (SDFA) é proposta como alternativa para a purificação, pois permite a separação e análise de biomoléculas, de modo que estas não percam sua atividade ou propriedades desejadas. Esta técnica é interessante para a purificação em larga escala, pois permite partição seletiva, com potencial de obtenção de altos rendimentos, além de apresentar boa relação custo-benefício. O presente trabalho estudou a purificação por extração líquido-líquido do ácido clavulânico em SDFA utilizando um novo sistema polimérico aquoso, formado pelos polímeros polietileno glicol (PEG) e ácido poliacrílico (APA). Foram estudadas diferentes composições do sistema polimérico aquoso PEG/APA, empregando diferentes massas molares e concentrações para o PEG e utilizando a massa molar 8000g/mol para o APA. Com base nas informações obtidas o melhor ponto de extração para o ácido clavulânico na presença de Na2SO4 foi definido como MPEG=400 g/mol, CPEG=17,5% (m/m) e CNaPA=22,5% (m/m) com K= 19,14, ηT=91,21%, BM=101,69 e R=0,45. Enquanto que na presença de NaCl, o melhor ponto encontrado foi: MPEG=400 g/mol, CPEG=35% (m/m) e CNaPA=10% (m/m) com K=11,96 ηT=80,04%, BM=90,18 e R=0,66. No trabalho será avaliada, também, a influência da temperatura, pH e força iônica nesse sistema. Estabeleceram-se os melhores parâmetros de separação do ácido clavulânico presente em meio fermentado produzido por Streptomyces clavuligerus utilizando a metodologia de fermentação extrativa com SDFA PEG/APA. O efeito do ácido clavulânico no diagrama de fases do sistema PEG-APA, bem como sua partição na forma pura e na presença de homogeneizado celular, foi estudado principalmente através da determinação do coeficiente de partição e recuperação do respectivo fármaco. / The viability of industrial scale production of commercial and therapeutical biotechnological products, such as drugs, is significantly dependent on the separation and purification techniques applied. The use of two-aqueous phase systems (ATPS) is proposed as an alternative to purification because it allows the separation and analysis of biomolecules, so that they do not lose their activities or desired properties. This technique is interesting for large scale purification because it allows selective partition with high potential yield and good cost/benefit ratio. The present work studied the purification of clavulanic acid (CA) by liquid-liquid extraction in ATPS applying a new aqueous polymeric system composed of two polymers, namely polyethylene-glicol (PEG) and sodium polyacrylate (NaPA). Different compositions of the aqueous polymeric system (PEG/PAA) were utilized, employing different PEG molar masses (MPEG) and concentrations (CPEG) and a molar mass of PAA of 8000 g/mol. In the light of the results obtained, the best conditions for clavulanic acid extraction, in the presence of Na2SO4, were MPEG = 400 g/mol, CPEG = 17.5% (m/m) and CNaPA = 22.5% (m/m), which allowed obtaining a partition coefficient (K) of 19.14, a yield in the top phase (ηT) of 91.21%, a mass balance (MB) of 101.69 and a volume ratio (R) of 0.45. On the other hand, in the presence of NaCl, the best results (K = 11.96, ηT = 80.04%, MB = 90.18 and R = 0.66) were found at: MPEG = 400 g/mol, CPEG = 35% m/m and CNaPA = 10% m/m. The effect of clavulanic acid in the PEG-PAA system phase diagram and its partition either in its pure form or in the cell homogenate were studied mainly through both the determination of the partition coefficient and the recovery of the drug selected for this study.

O Monometoxi-polietilenoglicol succinimidil ácido propiônico (mPEG-SPA 5 e 20 kDa) foi analisado como adjuvante e inibidor da atividade neurotóxica da toxina tetânica (TxT) adsorvida ou não em Al(OH)3, à qual o polímero foi conjugado. Avaliou-se a toxicidade das amostras por DL50, demonstrando que a atividade neurotóxica da TxT foi inibida. A via subcutânea foi mais efetiva na indução de resposta à TxT tratada pelo mPEG-SPA e o efeito adjuvante do Al(OH)3 se deu pela intramuscular. Trinta cavalos foram submetidos a esquema de imunização seletiva, dividindo-se os dezoito escolhidos em grupos para imunização com TxT conjugada ao mPEG-SPA 5.000 e 5.000(2X) e TxT adsorvida ou não. Os soros dos cavalos foram analisados por ToBI Teste, que avaliou a evolução da resposta imune. Os soros também foram analisados por imunodifusão, eletroforese e immunoblotting, tendo este indicado uma provável superioridade antigênica da TxT Fluida relativamente aos adjuvantes. A conjugação mPEG-SPA provou ser efetiva na produção do soro antitetânico terapêutico para uso humano. / Monometoxypolyethylene glycol succinimidyl propionic acid (SPA-mPEG 5 and 20 kDa) was analyzed as adjuvant and inhibitor of tetanus toxin neurotoxic activity (TxT) adsorbed or not by Al(OH)3, to which the polymer was conjugated. The samples toxicity was evaluated by DL50, disclosing that TxT neurotoxic activity was inhibited. The subcutaneous inoculation was more effective in induction of response to TxT treated with SPA-mPEG and the adjuvant effect of Al(OH)3 was evidenced by the intramuscular. Thirty horses were submitted to a selective scheme of immunization and eighteen were divided in groups to be immunized with TxT conjugated to SPA-mPEG 5,000 and 5,000(2X) and TxT adsorbed or not. The horses sera were analysed by ToBI Test, which evaluated the immune response development. The sera were also analysed through immunodifusion, electrophoresis and immunoblotting and the last one indicates a probable antigenic superiority of TxT fluid relatively to the adjuvants. The SPA-mPEG conjugation proved to be effective for anti-tetanus human therapeutic serum production.