In the cancer research, it is important to understand protein dynamics which are involved in cell signaling. Therefore, particular protein detection and analysis of target protein behavior are indispensable for current basic cancer research. However, it usually performed by conventional biochemical approaches, which require long process time and a large amount of samples. We have been developed the new applications based on microfluidics and Raster image Correlation spectroscopy (RICS) techniques. A simple microfluidic 3D hydrodynamic flow focusing device has been developed for quantitative determinations of target protein concentrations. The analyte stream was pinched not only horizontally, but also vertically by two sheath streams by introducing step depth cross junction structure. As a result, a triangular cross-sectional flow profile was formed and the laser was focused on the top of the triangular shaped analyte stream. Through this approach, the target protein concentration was successfully determined in cell lysate samples. The RICS technique has been applied to characterize the dynamics of protein 53 (p53) in living cells before and after the treatment with DNA damaging agents. P53 tagged with Green Fluores-cent Protein (GFP) were incubated with and without DNA damaging agents, cisplatin or eptoposide. Then, the diffusion coefficient of GFP-p53 was determined by RICS and it was significantly reduced after the drug treatment while that of the one without drug treatment was not. It is suggested that the drugs induced the interaction of p53 with either other proteins or DNA. This result demonstrates that RICS is able to detect protein-protein or protein-DNA interactions in living cells and it may be useful for the drug screening. As another application of microfluidics, an integrated microfluidic platform was developed for generating collagen microspheres with encapsulation of viable cells. The platform integrated four automated functions on a microfluidic chip, (1) collagen solution cooling system, (2) cell-in-collagen microdroplet generation, (3) collagen microdroplet polymerization, and (4) incubation and extraction of the microspheres. This platform provided a high throughput and easy way to generate uniform dimensions of collagen microspheres encapsulating viable cells that were able to proliferate for more than 1 week.

Hollow glass microspheres have been used extensively in the automotive and marine industries as an additive for reducing weight and saving material costs. They are also added to paints and other materials for their reflective properties. They have shown promise for weight critical applications, but have thus far resulted in materials with low fracture toughness and impact resistance when combined with thermosetting resins in syntactic foam. The advent of commercially available microspheres with a wide range of crushing strengths, densities and adhesive properties has given new impetus to research into syntactic foam with better fracture behaviour. Current research suggests that the beneficial effects on fracture and impact resistance gained by the addition of solid reinforcements such as rubber and ceramic particles are not seen with the addition of hollow glass microspheres. The research presented in this paper has examined the mechanisms for fracture resistance in glass microsphere filled epoxy (GMFE) syntactic foams, as well as determined the effect microsphere crushing strength and adhesion strength has on the material???s fracture toughness. The flexural properties of various GMFE have also been determined. GMFE were manufactured with varying microsphere volume fraction up to 50%, and with variances in microsphere crushing strength and adhesion. The specimens were tested for Mode I fracture toughness in a three point single edge notched bending setup as described in ASTM D5045 as well as a three point flexural setup as described in ASTM D790-3. Fracture surfaces were inspected using scanning electron microscope imaging to identify the fracture mechanisms in the presence of microspheres. Results indicate a positive effect on fracture toughness resulting from new fracture areas created as tails in the wake of the microspheres in the fracture plane. Results also indicate a negative effect on fracture toughness resulting from weak microspheres or from interfacial disbonding at the fracture plane. These two effects combine to show an increase in GMFE fracture toughness as the volume fraction of microspheres is increased to between 10 ??? 20% volume fraction (where the positive effect dominates), with a reduction in fracture toughness as microspheres are added further (where the negative effect dominates).

Thesis (Ph.D.) -- McMaster University, 2001. / Includes bibliographical references. Also available via World Wide Web.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

O objetivo do estudo foi desenvolver um cimento endodôntico com microesferas contendo amoxicilina e avaliar suas propriedades. As microesferas foram produzidas por secagem e caracterizadas por Microscopia eletrônica de varredura (MEV) e difração a laser. A formulação da resina base para um cimento endodôntico dual foi obtida pela mistura, em massa, de 70% de UDMA, 15% de GDMA e 15% de BISEMA. Como sistema iniciador/ativador, foram incorporados canforoquinona, DHEPT e peróxido de benzoíla, a 1% em mol e BHT em 0,01% em massa. Foram adicionados à resina base, em massa, 10 e 15% de microesferas de amoxicilina, além de um grupo sem microesferas (controle). Em todos os grupos foi adicionado trifluoreto de itérbio (10% em massa) como agente radiopacificante. Os cimentos foram avaliados quanto ao grau de conversão (GC) por Raman (n=3) imediatamente e após 24 horas de armazenamento, degradação em solvente (n=3) após 1 hora de imersão em álcool 70%, perfil de liberação do fármaco (n=3), atividade antimicrobiana contra Enterococcus Faecallis (n= 3), escoamento (n=3) e espessura de película (n=3)e citotoxicidade (n=3). Os dados foram analisados por ANOVA e Tukey com nível de significância de 5%. As microesferas apresentaram diâmetro médio de 2,664 μm. O grau de conversão imediato variou entre 51,73% e 55,13% e em 24h variou entre 60,79% e 73,80% sem apresentar diferença estatística entre os grupos. O percentual de degradação em solvente apresentou diferença significativa entre o grupo controle e o grupo com 15% de microesferas, variando entre 54,44% e 56,21% de redução. O perfil de liberação do fármaco mostrou que em 96h ocorreu uma liberação média de 73,76% do fármaco. A atividade antimicrobiana apresentou redução significativa dos grupos experimentais em 24 e 48h. Em 96h o grupo com 15% não apresentou diferença estatística quando comparado ao grupo controle (p>0,05). O escoamento apresentou uma redução significativa nos grupos experimentais comparados ao grupo controle (p<0,05). A espessura de película variou, mas não apresentou diferença estatística entre os grupos (p=0,63). A citotoxicidade apresentou alta viabilidade celular no tempos avaliados. Com base nesses resultados, pode-se concluir que a adição de até 10% de microesferas contendo amoxicilina apresentou característica antimicrobiana e não alterou as propriedades do cimento endodôntico experimental. / The objective of the study was to develop an endodontic sealer with amoxicillin-loaded microsphere and to evaluate its properties. The microspheres were produced by drying and characterized by Scanning Electron Microscopy (SEM) and laser diffraction. The formulation of the base resin dual cure endodontic cement was obtained by mixing, by weight, 70% UDMA, 15% GDMA and 15% BISEMA. As initiator / activator system, camphorquinone, DHEPT and benzoyl peroxide, 1 mol% and BHT were incorporated in 0.01% by weight. 10 and 15% of amoxicillin microspheres were added to the base resin, in addition to a group without microspheres (control). Ytterbium trifluoride (10% by weight) as radiopacifier was added in all groups. The cements were evaluated for Raman (n=3) conversion grade immediately after 24 hours storage, solvent degradation (n=3) after 1 hour immersion in 70% alcohol, drug release profile (n=3), antimicrobial activity against E. faecallis (n=3), flow (n=3) and film thickness (n=3) and cytotoxicity (n=3). Data were analyzed by ANOVA and Tukey with significance level of 5%. The microspheres had an average diameter of 2,664 μm. The degree of immediate conversion ranged from 51.73% to 55.13%, and in 24h conversion ranged from 60.79% to 73.80%, with no statistical difference between the groups. The percentage of degradation in solvent showed a significant difference between the control group and the group with 15% of microspheres, varying between 54.44% and 56.21% reduction. The drug release profile showed that a mean release of 73.76% of the drug occurred in 96h. The antimicrobial activity showed a significant reduction of the experimental groups in 24 and 48h. In 96h the group with 15% presented no statistical difference when compared to the control group (p> 0.05). The flow showed a significant reduction in the experimental groups compared to the control group (p <0.05). The film thickness varied, but did not present statistical difference between the groups (p = 0.63). Cytotoxicity showed high cellular viability at the determined times. Based on these results, it can be concluded that the addition of up to 10% of microspheres containing amoxicillin showed antimicrobial characteristics and did not alter the properties of the experimental endodontic cement.

O objetivo do estudo foi desenvolver um cimento endodôntico com microesferas contendo amoxicilina e avaliar suas propriedades. As microesferas foram produzidas por secagem e caracterizadas por Microscopia eletrônica de varredura (MEV) e difração a laser. A formulação da resina base para um cimento endodôntico dual foi obtida pela mistura, em massa, de 70% de UDMA, 15% de GDMA e 15% de BISEMA. Como sistema iniciador/ativador, foram incorporados canforoquinona, DHEPT e peróxido de benzoíla, a 1% em mol e BHT em 0,01% em massa. Foram adicionados à resina base, em massa, 10 e 15% de microesferas de amoxicilina, além de um grupo sem microesferas (controle). Em todos os grupos foi adicionado trifluoreto de itérbio (10% em massa) como agente radiopacificante. Os cimentos foram avaliados quanto ao grau de conversão (GC) por Raman (n=3) imediatamente e após 24 horas de armazenamento, degradação em solvente (n=3) após 1 hora de imersão em álcool 70%, perfil de liberação do fármaco (n=3), atividade antimicrobiana contra Enterococcus Faecallis (n= 3), escoamento (n=3) e espessura de película (n=3)e citotoxicidade (n=3). Os dados foram analisados por ANOVA e Tukey com nível de significância de 5%. As microesferas apresentaram diâmetro médio de 2,664 μm. O grau de conversão imediato variou entre 51,73% e 55,13% e em 24h variou entre 60,79% e 73,80% sem apresentar diferença estatística entre os grupos. O percentual de degradação em solvente apresentou diferença significativa entre o grupo controle e o grupo com 15% de microesferas, variando entre 54,44% e 56,21% de redução. O perfil de liberação do fármaco mostrou que em 96h ocorreu uma liberação média de 73,76% do fármaco. A atividade antimicrobiana apresentou redução significativa dos grupos experimentais em 24 e 48h. Em 96h o grupo com 15% não apresentou diferença estatística quando comparado ao grupo controle (p>0,05). O escoamento apresentou uma redução significativa nos grupos experimentais comparados ao grupo controle (p<0,05). A espessura de película variou, mas não apresentou diferença estatística entre os grupos (p=0,63). A citotoxicidade apresentou alta viabilidade celular no tempos avaliados. Com base nesses resultados, pode-se concluir que a adição de até 10% de microesferas contendo amoxicilina apresentou característica antimicrobiana e não alterou as propriedades do cimento endodôntico experimental. / The objective of the study was to develop an endodontic sealer with amoxicillin-loaded microsphere and to evaluate its properties. The microspheres were produced by drying and characterized by Scanning Electron Microscopy (SEM) and laser diffraction. The formulation of the base resin dual cure endodontic cement was obtained by mixing, by weight, 70% UDMA, 15% GDMA and 15% BISEMA. As initiator / activator system, camphorquinone, DHEPT and benzoyl peroxide, 1 mol% and BHT were incorporated in 0.01% by weight. 10 and 15% of amoxicillin microspheres were added to the base resin, in addition to a group without microspheres (control). Ytterbium trifluoride (10% by weight) as radiopacifier was added in all groups. The cements were evaluated for Raman (n=3) conversion grade immediately after 24 hours storage, solvent degradation (n=3) after 1 hour immersion in 70% alcohol, drug release profile (n=3), antimicrobial activity against E. faecallis (n=3), flow (n=3) and film thickness (n=3) and cytotoxicity (n=3). Data were analyzed by ANOVA and Tukey with significance level of 5%. The microspheres had an average diameter of 2,664 μm. The degree of immediate conversion ranged from 51.73% to 55.13%, and in 24h conversion ranged from 60.79% to 73.80%, with no statistical difference between the groups. The percentage of degradation in solvent showed a significant difference between the control group and the group with 15% of microspheres, varying between 54.44% and 56.21% reduction. The drug release profile showed that a mean release of 73.76% of the drug occurred in 96h. The antimicrobial activity showed a significant reduction of the experimental groups in 24 and 48h. In 96h the group with 15% presented no statistical difference when compared to the control group (p> 0.05). The flow showed a significant reduction in the experimental groups compared to the control group (p <0.05). The film thickness varied, but did not present statistical difference between the groups (p = 0.63). Cytotoxicity showed high cellular viability at the determined times. Based on these results, it can be concluded that the addition of up to 10% of microspheres containing amoxicillin showed antimicrobial characteristics and did not alter the properties of the experimental endodontic cement.

Esta tese envolveu o estudo de encapsulação de vacina bivalente contra difteria e tétano de uso adulto (dT) em microesferas de ácido poli(láctico-coglicólico) (PLGA), com o objetivo de desenvolver uma formulação que induzisse uma resposta protetora com um número reduzido de doses, ou preferencialmente de apenas uma dose. Para ter sucesso, uma formulação como tal deve ter um perfil de liberação de antígeno que simule as múltiplas doses que recebem o indivíduo. Assim, nesta tese são mostrados os resultados dos experimentos de caracterização bioquímica e imunológica da formulação citada, sem o uso de estabilizadores protéicos, a fim de reduzir a complexidade do sistema em estudo. Do ponto de vista das proteínas encapsuladas, tal caracterização envolveu estudos de fluorescência, de dicroísmo circular, de atividade antigênica e determinação por HPLC da degradação durante o processo de encapsulação. Do ponto de vista da formulação, foram realizados ensaios de degradação in vitro e de atividade imunogênica in vivo, em camundongos e cobaias. Os dados obtidos indicaram que a encapsulação em microesferas de PLGA de vacina dT, sem o uso de estabilizadores, permitiu a produção de uma formulação vacinal viável, capaz de estimular uma resposta imunológica protetora e de memória, abrindo caminho para o desenvolvimento da tecnologia de produção de vacinas polivalentes encapsuladas em microesferas de PLGA. / The study on encapsulating a bivalent single dose vaccine against diphtheria and tetanus for adults (dT) within PLGA microspheres is described in this thesis. A successful single dose vaccine must show an antigen release profile mimicking the several doses a person should receive during the life. Therefore, results from the immunological and biochemical characterization of the formulation, prepared without protein stabilizers, is showed here. From the encapsulated protein point of view the characterization involved fluorescence, circular dichroism, antigenicity and HPLC analysis. From the formulation point of view, there were performed in vitro release assays and immunogenicity on mice and on guinea-pigs. The results indicated that the dT microencapsulation within PLGA microspheres without protein stabilizers lead to the production of a viable vaccine formulation, able to elicit protective and memory immune response.

No Brasil, e em outros paises de clima tropical, os carrapatos se tornaram um enorme problema economico de^$de que a industria do gado se desenvolveu. O carrapato Boophilus microplus, um dos artropodes mais importantes na veterinaria, causa efeitos direto, como suc,cao de sangue, e indireto, como a transmissao de uma grande variedade de patogenos que normalmente resulta em infec,c~es letais. As vacinas genicas contendo o antigeno Bm86, uma proteina ligada a membrana do intestino do carrapato B. microplus, representam uma alternativa atrativa aos acaricidas para controlar as infesta~oes por carrapatos em contrapartida aos inconvenientes produtos quimicos. Devido sua administra,cao ser feita em 4 doses no primeiro ano, seguida de refor,cos a cada seis meses, estas formula,coes vacinais nao s3c adequadas para paises com cria,cao extensiva de gado, como no Brasil. Visando uma libera~ao sustentada do antigeno Bm86, neste trabalho desenvolveuse uma vacina de dose unica baseada em microesferas polimericas. Para obter o padrao de liberac,ao desejavel, diferentes formula,coes e parametros de processo foram variados, como a composi,cao do polimero, a taxa entre os monomeros ^Uacido latico:acido glicolico\" e o tamanho das microparticulas. As formula,coes foram preparadas pelo metodo de emulsao multipla e evapora,cao do solvente. A formula~ao que melhor se enquadrou nos objetivos da vacina de dose unica foi preparada com PLGA 75:25, solu,cao 3% de PVA como estabilizante, agita,cao de 11000 rpm para forma,cao da emulsao primaria e de 800 rpm para forma,cao da emulsao multipla e evapora,cao do solvente. As particulas assim obtidas apresentaram um tamanho medio de 25 ,um, uma taxa de encapsula,cao maior que 90% e aproximadamente 50% da proteina foi liberada in vitro em 60 dias. Analises por SDS-PAGE e Westem Bloning revelaram que a proteina se manteve integra apos encapsula,cao. Os resultados da avalia,cao da imunogenicidade em bovinos mostraram que a formula,cao baseada em microesferas polimericas biodegradaveis e habil a conseguir, com uma unica dose, uma resposta imune similar aquela conseguida com tres doses das formula,coes convencionais da vacina de Bm86. / In Brazil, and in others tropical countries, the ticks have become a huge economic problem since the industry of livestock has developed. Ticks and tick-borne diseases affect animal and human health and are the cause of significant economic losses. The cattle tick Boophilus microplus is one of the most important arthropods in veterinary. This tick species causes both direct effects, such as blood sucking, and indirect effects, such as transmission of a wide variety of pathogens, which usually result in lethal infections. The gene vaccines based on Bm86 antigen, a midgut membrane-bound protein of the cattle tick B. microplus, represent a good alternative to control tick infestations, compared to chemicals. However, due to these vaccine formulations need 4 doses over the first year with booster at each 6 months to be effective, they are not suitable for countries with extensive cattle raising, like Brazil. Aiming a sustained release of Bm86 antigen, in this work we developed a single shot vaccine based on Bm86 loaded polymeric microspheres. In order to obtain desired release patterns, different formulations and processing parameters were varied, for example, the composition of the polymer, the monomer ratio lactic acid:glycolic acid and the size of the microparticles. The formulations were prepared by solvent evaporation method based on double emulsion. The formulation that presented better result as single shot vaccine was prepared with PLGA 75:25, solution 3% of PVA as stabilizer, agitation of 11000 rpm to form the primary emulsion and 800 rpm to obtain the double emulsion and solvent evaporation. The particles thus obtained presented an average size of 25 m, encapsulation ratio greater than 90% and approximately 50% of the protein was released in vitro in 60 days. Analysis by SDSPAGE and Western Blot showed that the integrity of the protein remained after encapsulation. The immunogenic studies showed that the formulation based onbiodegradable polymeric microspheres is able to elicit, with a single dose, an immune response and protection similar to that attained with 3 doses of conventional Bm86 vaccine formulations.

Lactobacilli are dominant bacteria of the vaginal flora. Lactobacillus-containing probiotics products are used for the treatement and profylaxis of bacterial urogenital infections. This work is focused on DNA identification and species identification of probiotic bacteria in 5 different vaginal tablets using molecular-genetic methods. Total DNA isolated from complex matrix of vaginal tablets was used for amplification in polymerase chain reaction. DNA was isolated from crude cell lysates by magnetic particles P(HEMA-co-GMA) and by method of phenol extraction. Identification of species of probiotic bacteria was verified using genus-specific and species-specific PCRs. Results of bacterial identification obtained by PCR were compared with declared specification given by producers. Bacteria of genus Lactobacillus were proved in all tablets whereus species identification was in accordance with the stated composition in 1 tablet only.

Stroke is a medical condition in which poor blood flow to the brain results in cell death. The current treatment options are limited and only very few patients can benefit from these treatments. Stroke causes brain swelling and often a decompressive craniectomy is performed for some of the patients to release intracranial pressure to prevent further damage. As a result, a duraplasty is implanted to replace the surgically-damaged dura mater to protect the brain. In view of that, the purpose of this project was to develop double-walled microspheres (DWMS) which can be used as a drug delivery system when incorporated into duraplasty to promote endogenous stem cell therapy to treat stroke. The DWMS were composed of poly (l-lactic acid) (PLLA) and poly (lactic-co-glycolic acid) (PLGA) using a solvent evaporation method. Bovine serum albumin (BSA), as a model protein, was entrapped within these DWMS with different core-shell thicknesses and compositions to investigate the distribution of protein, encapsulation efficiency, and in vitro release. The fabrication process parameters of DWMS were also optimized to attain higher yields, and the phase separation and surface morphology were examined by differential scanning calorimetry and scanning electron microscopy.