Biological sample analysis is a costly and time-consuming process. It involves highly trained technicians operating large and expensive instruments in a temperature and dust controlled environment. In the world of rising healthcare cost, the drive towards a more cost-effective solution calls for a point-of-care device that performs accurate analyses of human blood samples. To achieve this goal, today's bulky laboratory instruments need to be scaled down and integrated on a single microchip of only a few square centimeters or millimeters in size. Dielectrophoresis (DEP), a phenomenon where small particles such as human blood cells are manipulated by non-uniform electric fields, stands to feature prominently in the point-of-care device. An original device that enhances DEP effect through novel geometry of the electrodes is presented. When activated with two inverting sinusoidal waveforms, the novel-shaped electrodes generate horizontal bands of increasing electric fields on the surface of the microchip. With these bands of electric fields, particles can be manipulated to form a straight horizontal line at a predictable location. Experimental results showing the collection, separation, and transportation of mammalian cells are presented. A strategy for simultaneous processing of two or more types of particles is also demonstrated. With capabilities for an accurate position control and an increased throughput by parallel processing, the novel microchip device delivers substantial improvements over the existing DEP designs. The research presented here explores the effects of novel electrode geometries in cell manipulations and contributes to the overall progress of an automated blood analysis system.

The work presented in this thesis aims to further the understanding and implementation of the Baculovirus Expression Vector System (BEVS) for varied uses such as protein production and viral vector production. To this end, three projects have been presented, two of which deal with methods to quantify baculovirus titres and the last deals with tracking baculovirus transcripts in infected insect cells. The first project examined assumption-free analysis as a method for data analysis of Real Time PCR data in order to enable direct comparison of baculovirus titres between samples, without the need for a traditional standard curve. It concluded that assumption-free analysis was well suited for this purpose and fold differences of baculovirus titres of different samples obtained using this method corresponded to real differences in sample titres. The second project aimed to develop a cheap and reliable method for sample preparation for Real Time PCR which would remove the need for the use of commercially available extraction kits. Samples were subjected to various combinations of Triton X-100 at different concentrations and different numbers of freeze/thaw cycles in order to determine the combination which would provide the best baculovirus genome exposure. One of these combinations was found to be at least as good as commercially available kits in reliably extracting baculovirus DNA and providing baculovirus titres that are at least as accurate. The third project was a preliminary study examining the effects of multiplicity of infection on the levels of baculovirus Gp-64 transcript in insect cell culture. The study concludes that at high multiplicities of infection, there seems to be no increase in baculovirus transcripts when the multiplicity of infection is further increased. This study served to allow for familiarization with tracking transcript levels, and the principles and techniques demonstrated here will form the basis for an exhaustive future study on the same subject.

The work presented in this thesis aims to further the understanding and implementation of the Baculovirus Expression Vector System (BEVS) for varied uses such as protein production and viral vector production. To this end, three projects have been presented, two of which deal with methods to quantify baculovirus titres and the last deals with tracking baculovirus transcripts in infected insect cells. The first project examined assumption-free analysis as a method for data analysis of Real Time PCR data in order to enable direct comparison of baculovirus titres between samples, without the need for a traditional standard curve. It concluded that assumption-free analysis was well suited for this purpose and fold differences of baculovirus titres of different samples obtained using this method corresponded to real differences in sample titres. The second project aimed to develop a cheap and reliable method for sample preparation for Real Time PCR which would remove the need for the use of commercially available extraction kits. Samples were subjected to various combinations of Triton X-100 at different concentrations and different numbers of freeze/thaw cycles in order to determine the combination which would provide the best baculovirus genome exposure. One of these combinations was found to be at least as good as commercially available kits in reliably extracting baculovirus DNA and providing baculovirus titres that are at least as accurate. The third project was a preliminary study examining the effects of multiplicity of infection on the levels of baculovirus Gp-64 transcript in insect cell culture. The study concludes that at high multiplicities of infection, there seems to be no increase in baculovirus transcripts when the multiplicity of infection is further increased. This study served to allow for familiarization with tracking transcript levels, and the principles and techniques demonstrated here will form the basis for an exhaustive future study on the same subject.

No description available.

Ongoing work in the laboratory of Professor Chris Backhouse is aimed at developing a portable and inexpensive lab on a chip instrument. A system capable of molecular biology protocols including sample preparation (SP), polymerase chain reaction (PCR), and melting curve analysis (MCA) would meet the requirements for point of care genetic analysis. The SP, PCR, and MCA modules were designed and tested on a standalone basis and then integrated for analysis of raw clinical samples. An automated XY stage was developed for magnetic bead-based DNA purification. In addition, a LED/CCD-based optical detection module was employed for real time PCR and MCA. Data analysis algorithms and protocols were implemented to remove noise and interpret data. This work culminated in proof of principle on-chip SP-PCR-MCA to detect ß2m DNA from human buccal cells in a modular and inexpensive system. / Biomedical Engineering

Thesis (MSc.(Chemical pathology))--University of Pretoria, 2009. / Includes bibliographical references.

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177f. / Submitted by Ana Hilda Fonseca (anahilda@ufba.br) on 2013-04-03T15:12:44Z No. of bitstreams: 1 Tese - José Tiago P. Barbosa - Versão Digital.pdf: 2697646 bytes, checksum: 8a6596b96acedc91d9eedf51d2ff97e2 (MD5) / Approved for entry into archive by Ana Hilda Fonseca(anahilda@ufba.br) on 2013-04-23T15:43:21Z (GMT) No. of bitstreams: 1 Tese - José Tiago P. Barbosa - Versão Digital.pdf: 2697646 bytes, checksum: 8a6596b96acedc91d9eedf51d2ff97e2 (MD5) / Made available in DSpace on 2013-04-23T15:43:22Z (GMT). No. of bitstreams: 1 Tese - José Tiago P. Barbosa - Versão Digital.pdf: 2697646 bytes, checksum: 8a6596b96acedc91d9eedf51d2ff97e2 (MD5) Previous issue date: 2011 / CAPES / A soja é uma leguminosa que possui notável relevância no cenário internacional essencialmente em função de suas características funcionais e nutracêuticas. O consumo da soja é considerado benéfico não somente pela potencialidade nutricional associada, como também pelo efeito preventivo contra inúmeras doenças crônicas principalmente em virtude da presença de isoflavonas em sua constituição. O objetivo do trabalho consistiu no desenvolvimento de estratégias para a determinação da composição multielementar e do teor das isoflavonas daidzeína e genisteína em amostras de soja em grãos e derivados comercializadas/cultivadas nas cidades de Salvador e Barreiras, Bahia e Uberlândia, Minas Gerais. Para a avaliação multielementar foram propostos dois procedimentos: decomposição por via úmida assistida por radiação micro-ondas empregando ácidos diluídos (PAD) e decomposição utilizando combustão induzida por micro-ondas (MIC), empregando técnicas espectrométricas (ICP OES e ICP-MS) para detecção dos analitos. O procedimento de análise cromatográfica foi desenvolvido para a determinação de daidzeína e genisteína nas amostras estudadas empregando a técnica HPLC-DAD. Para o procedimento PAD foram avaliadas diferentes concentrações de HNO3 (2,1 – 14,5 mol L-1) utilizando H2O2 como agente oxidante auxiliar para a decomposição de aproximadamente 250 mg de amostra. A eficiência de decomposição para as diferentes concentrações ácidas foi avaliada considerando os parâmetros acidez e teor de carbono residual e percentual de recuperação dos analitos, de modo que a concentração de 2,1 mol L-1 em HNO3 foi selecionada. O procedimento de decomposição empregando MIC permitiu a decomposição de uma massa de amostra de 400 mg, utilizando como solução absorvedora HNO3 a 6,0 mol L-1 para a determinação multielementar. Para a determinação dos halogênios Cl, Br e I, foi utilizada como solução absorvedora NH4OH a 100 mmol L-1. A exatidão dos procedimentos PAD e MIC foi avaliada com material de referência certificado (NIST-1568a) e o desempenho quanto à precisão e LOQ foi também avaliado. Estes procedimentos se mostraram adequados para a determinação multielementar nas amostras sendo considerados alternativas promissoras, devido à minimização do consumo de reagentes e de geração de efluentes, de acordo com os princípios da química verde. Na determinação das isoflavonas, foi proposto um procedimento de análise cromatográfica empregando-se extração acelerada (ASE) com solvente (metanol) e posterior separação e detecção destes analitos por HPLC-DAD utilizando os solventes acetonitrila e ácido acético em eluição isocrática. Para a validação do método foram considerados os parâmetros: seletividade, linearidade, precisão, limites de detecção e quantificação. Os resultados obtidos estiveram nas faixas: daidzeína (10,3 a 34,0 μg g-1) e genisteína (18,5 a 77,8 μg g-1). Este procedimento se mostrou adequado para a identificação e quantificação destas isoflavonas nas amostras. O presente trabalho contribuiu para traçar um perfil preliminar das amostras de soja em grãos e derivados em termos de micronutrientes, contaminantes e as isoflavonas daidzeína e genisteína. / Salvador

98f. / Submitted by Ana Hilda Fonseca (anahilda@ufba.br) on 2013-04-10T15:28:07Z No. of bitstreams: 1 Dissertação versão final. 2012.pdf: 2926493 bytes, checksum: 7c8eb202f8e1d49db66d28957c7f2ad6 (MD5) / Approved for entry into archive by Ana Hilda Fonseca(anahilda@ufba.br) on 2013-05-10T15:54:03Z (GMT) No. of bitstreams: 1 Dissertação versão final. 2012.pdf: 2926493 bytes, checksum: 7c8eb202f8e1d49db66d28957c7f2ad6 (MD5) / Made available in DSpace on 2013-05-10T15:54:03Z (GMT). No. of bitstreams: 1 Dissertação versão final. 2012.pdf: 2926493 bytes, checksum: 7c8eb202f8e1d49db66d28957c7f2ad6 (MD5) Previous issue date: 2011 / CAPES / Devido a aspectos nutricionais e socioeconômicos os crustáceos possuem grande importância no cenário nacional por serem alimentos usualmente consumidos na região costeira. O objetivo deste trabalho foi avaliar as concentrações de elementos essenciais e não essenciais em amostras de caranguejo, siri e aratu, na forma catada, comercializados em feiras livres na cidade de Salvador, Bahia, Brasil, de forma a traçar um perfil comparativo em termos de concentrações macro e microelementos. As amostras foram adquiridas nos principais pontos de comercialização de pescados da cidade Feira de São Joaquim, Mercado Popular, Feira dois de Julho e CEASA, sendo oriundas de diversas regiões da Baía de Todos os Santos. No pré-tratamento, as amostras foram secas em liofilizador, a moagem foi realizada com moinhos de bolas e o preparo das amostras foi realizado utilizando forno microondas com cavidade, usando uma mistura de ácido nítrico e peróxido de hidrogênio. Para a determinação dos elementos foi empregada a espectrometria de emissão óptica com plasma indutivamente acoplado (ICP OES). As concentrações encontradas nas amostras variaram expressivamente mesmo quando as mesmas foram indicadas ser de mesmo local de captura. Foram obtidos os seguintes teores médios de concentração, em μg.g-1: para aratu Cu (64,5 ± 1,5), Fe (65,7 ± 2,0), Mn (2,91 ± 0,25), Zn (163 ± 4); para as amostras de caranguejo Cu (53,3 ± 2,5), Fe (69,8 ± 6,0), Mn (3,69 ± 0,31), Zn (309 ± 6); e para as amostras de siri Cu (67,4 ± 3,3), Fe (31,5 ± 3,6), Mn (7,48 ± 2,51), Zn (184 ± 8). As concentrações de todos os elementos analisados mostraram contribuir para o teor de ingestão diária recomendada (IDR). No entanto, as concentrações de Cu e Zn para todas as amostras estão acima do limite máximo de tolerância estabelecido pela ANVISA. Estudos complementares estão sendo realizados visando contribuir para o desenvolvimento de medidas práticas de gerenciamento de modo a proteger a saúde da população. / Salvador

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