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In this project a method based on surface plasmon resonance has been developed for determining the concentration of several His-tagged proteins in complex solutions. It showed large dynamic range, no measureable non-specific binding and high sensitivity (with linear range around 0.1–10 μg/ml depending on the proteins). The method showed a low variation when checked on MBP-His during an extended time period. The concentrations of the His-tagged protein in the lysate has also been determined and compared with other alternative methods. This method will later be used to analyse protein concentrations during development and optimization of chromatographic purification process.

Although advances in microfluidic technology have enabled increasingly sophisticated biosensing and bioassay operations to be performed at the microscale, many of these applications employ such small amounts of charged biomolecules (DNA, proteins, peptides) that they must first be pre-concentrated to a detectable level. Efficient strategies for precisely handling minute quantities of biomolecules in microchannel geometries are critically needed, however it has proven challenging to achieve simultaneous concentration, focusing, and metering capabilities with currentgeneration sample injection technology. Using microfluidic chips incorporating arrays of individually addressable microfabricated electrodes, we demonstrate that DNA can be sequentially concentrated, focused into a narrow zone, metered, and injected into an analysis channel. The technique used in this research transports charged biomolecules between active electrodes upon application of a small potential difference (1 V), and is capable of achieving orders of magnitude concentration increases within a small device footprint. The collected samples are highly focused, with sample zone size and shape defined solely by electrode geometry. In addition to achieving the objectives of the research project, this setup was found to provide added functionality as a label-free biomolecule detection technique due to the formation of light scattering phases of charged biomolecules on top of the capture electrode.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-10-06T17:03:40Z No. of bitstreams: 1 texto completo.pdf: 247331 bytes, checksum: ae57be7c82f87c29d3a84555f4712a48 (MD5) / Made available in DSpace on 2016-10-06T17:03:40Z (GMT). No. of bitstreams: 1 texto completo.pdf: 247331 bytes, checksum: ae57be7c82f87c29d3a84555f4712a48 (MD5) Previous issue date: 2004-07-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O dobramento de proteínas secretórias no lúmen do retículo endoplasmático é altamente facilitado por chaperones moleculares. A proteína BiP é um dos chaperones mais bem caracterizados residentes do RE. BiP tem demonstrado ser um modulador multifuncional de vários processos que ocorrem no retículo endoplasmático. Essa proteína auxilia no dobramento e enovelamento das cadeias polipeptídicas nascentes e exerce papel no controle de qualidade do RE, reconhecendo e direcionando proteínas incorretamente dobradas para degradação. Também funciona como um sensor da via de resposta a proteínas incorretamente enovelada (UPR), regulando indiretamente a atividade da cinase eIF-2 e a sua própria expressão. As proteínas de reserva são sintetizadas em ribossomos associados à membrana do retículo endoplasmático, sendo co-traducionalmente translocadas para o lúmen do RE, onde podem permanecer associadas em corpos protéicos ou ser transportadas, via complexo de Golgi, para vacúolos de proteínas de reserva. Evidências na literatura indicam que BiP associa-se com proteínas de reserva in vitro, e seu acúmulo nas sementes está coordenado com a síntese de proteínas de reserva. Nesta investigação, foram analisados o nível de BiP nos diferentes estádios de desenvolvimento de sementes de soja e a correlação entre as concentrações de BiP e de proteínas de reserva de sementes. Para isso, utilizaram-se sementes de soja das variedades CAC-1 e CC3 com diferentes teores de proteína, determinados pelo método Kjeldahl. BiP acumulou-se predominantemente nos estádios iniciais de desenvolvimento das sementes, coincidindo com a síntese ativa de proteínas de reserva. O acúmulo temporal de BiP foi significativamente superior nas sementes CC3 que apresentam maior teor protéico. A capacidade de BiP associar-se a proteínas de reserva foi funcionalmente avaliada por meio do sistema duplo híbrido. Este ensaio demonstrou que BiP interage eficientemente com a subunidade de - conglicinina em leveduras. A partir desses resultados, surgiu a hipótese de que um aumento da expressão de BiP poderia resultar em elevação do teor de proteína das sementes. O efeito da superexpressão da proteína BiP no teor de proteína total foi diretamente avaliado em sementes de Nicotiana tabacum transgênicas (geração T4) superexpressando o gene BiP da soja. Embora o nível de BiP nas sementes transgênicas, detectado por immunoblotting e quantificado por densitometria, tenha sido maior nas linhagens transgênicas senso em relação às controle e anti-senso, a superprodução constitutiva de BiP não foi correlacionada diretamente com o aumento do teor protéico, determinado pelo método Kjeldahl. Esses resultados sugerem que a capacidade de processamento do retículo endoplasmático em tabaco durante o desenvolvimento da semente não constitui o fator limitante do processo de síntese e acúmulo de proteínas de reserva. / The folding of secretory proteins within the lumen of the endoplasmic reticulum (ER) is greatly facilitated by molecular chaperones. The binding protein BiP is one of the best characterized ER-resident molecular chaperones. In mammalian cells, BiP has been demonstrated to serve as a multifunctional modulator of various ER-supported processes including regulation of eIF-2 kinase and mRNA translation, regulation of BiP expression, and the catalysis of protein folding, as well as, potentially, the targeting of misfolded proteins for degradation. The storage proteins from soybean are synthesized in ER membrane-bound polyribosomes and through a Golgi-mediated translocation are deposited into specialized vacuoles, designated protein bodies. We have previously demonstrated that BiP associated detectably with storage proteins in vitro and the efficiency of BiP synthesis correlated with the accumulation of seed storage protein, such that soybean cultivars that exhibited a greater content of storage proteins also accumulated higher levels of BiP. In this investigation, we further analyzed the synthesis of soybean BiP during seed development and the naturally occurring correlation between the content of BiP and seed storage proteins. Immunoblottings of total protein extracts from CAC1 (normal protein content) and CC3 (high protein content) cultivars demonstrated that, during the seed development, BiP accumulates to high levels at developmental stages that coincide with the onset of active storage protein synthesis. We further characterized the association of BiP and β-conglycinin storage proteins though the two-hybrid system. In order to understand whether an increase in BiP levels would promote a concomitant increase in seed storage protein accumulation, we obtained tobacco transgenic seeds-overexpressing a soybean BiP gene. The effect of BiP overexpression on total protein accumulation was directly evaluated in Nicotiana tabacum homozygous transgenic seeds (T3 generation). The BiP protein levels detected in the transgenic seeds were significantly higher than those of wild type and antisense BiP-transformed seeds. Nevertheless, total protein from seeds-overexpressing BiP, as determined by Kjeldahl, did not differ significantly from that of wild type and antisense seeds. These results suggest, although do not prove, that the capacity of ER processing during seed development does not constitute a rate limiting process for storage protein synthesis and accumulation. / Dissertação importada do Alexandria

<p>The purpose of this study was to gain more knowledge about the deleterious effects of decreased muscle protein concentration on skeletal muscle function, by measuring the concentrations of myosin and actin in single pig muscle fibres. The pigs were earlier used in an experimental animal model to study the early stages of acute quadriplegic myopathy (AQM), a disease that is found in mechanically ventilated intensive care unit patients. Percutaneous biopsies were taken from these pigs and where now used in this study.</p><p>Even though the method used was accurately tested and theoretically working, certain problems arose. These problems were unexpected and caused problems to the study. The method used to measure the concentration of myosin and actin, an ELISA, gave no logical results. The reason could not be found and because of the time limit of this project no results from the AQM-pigs were gained. The efforts to make the method work is described and discussed.</p>

The purpose of this study was to gain more knowledge about the deleterious effects of decreased muscle protein concentration on skeletal muscle function, by measuring the concentrations of myosin and actin in single pig muscle fibres. The pigs were earlier used in an experimental animal model to study the early stages of acute quadriplegic myopathy (AQM), a disease that is found in mechanically ventilated intensive care unit patients. Percutaneous biopsies were taken from these pigs and where now used in this study. Even though the method used was accurately tested and theoretically working, certain problems arose. These problems were unexpected and caused problems to the study. The method used to measure the concentration of myosin and actin, an ELISA, gave no logical results. The reason could not be found and because of the time limit of this project no results from the AQM-pigs were gained. The efforts to make the method work is described and discussed.

In durum wheat (<i>Triticum turgidum</i> L. var <i>durum</i>), grain protein concentration (GPC) and gluten quality are among the important factors influencing pasta-making quality. Semolina with high protein content produces pasta with increased tolerance to overcooking and greater cooked firmness. However, genetic improvement of GPC is difficult largely because of its negative correlation with grain yield, and a strong genotype x environment interaction. Therefore, identification of quantitative trait loci (QTL) for high GPC and the associated markers is a priority to enhance selection efficiency in breeding durum wheat for elevated GPC. At a physiological level, GPC is influenced by several factors including nitrogen remobilization from vegetative organs and direct post-anthesis nitrogen uptake (NUP) from the soil. Understanding the relationship between elevated GPC and nitrogen remobilization, and post-anthesis NUP will enable durum wheat breeders to develop varieties that not only produce high yield and high GPC, but also exhibit better nitrogen use efficiency. The objectives of this study were: (1) to identify and validate QTL for elevated GPC in two durum wheat populations; and (2) to determine if elevated GPC is due to more efficient nitrogen remobilization and/or greater post-anthesis NUP. A genetic map was constructed with SSR and DArT® markers in a doubled haploid population from the cross Strongfield x DT695, and GPC data were collected in replicated trials in six Canadian environments from 2002 to 2005. Two stable QTL for high GPC, QGpc.usw-B3 on chromosome 2B and QGpc.usw-A3 on 7A, were identified. Strongfield, the high GPC parent, contributed the alleles for elevated GPC at both QTL. These two QTL were not associated with variation in grain weight (seed size) or grain yield. QGpc.usw-A3 was validated in a second Strongfield-derived population as that QTL was significant in all six testing environments. Averaged over five locations, selection for QGpc.usw-A3 resulted in a +0.4% to +1.0% increase in GPC, with only small effects on yield in most environments. A physiological study of grain protein accumulation revealed that regardless of the growing condition, nitrogen remobilization was the major contributor for grain nitrogen in durum genotypes evaluated, accounting for an average of 84.3% of total GPC. This study confirmed that introgression of Gpc-B1 into Langdon resulted in increased GPC, and this GPC increase was due to higher N remobilization. Strongfield expressed greater N remobilization than DT695 and the semi-dwarf cultivar Commander, but N remobilization was not the determining factor for Strongfields elevated GPC. Strongfield expressed greater post-anthesis NUP than DT695. Similarly, a selection of six high-GPC doubled haploid (DH) lines from the cross DT695 x Strongfield expressed significantly greater post-anthesis NUP than six low-GPC DH selections, supporting the hypothesis that elevated GPC in Strongfield is derived from greater post-anthesis NUP. All six high-GPC DH selections carried the Strongfield allele at QGpc.usw-A3, suggesting this QTL maybe associated with post-anthesis NUP.

In durum wheat (<i>Triticum turgidum</i> L. var <i>durum</i>), grain protein concentration (GPC) and gluten quality are among the important factors influencing pasta-making quality. Semolina with high protein content produces pasta with increased tolerance to overcooking and greater cooked firmness. However, genetic improvement of GPC is difficult largely because of its negative correlation with grain yield, and a strong genotype x environment interaction. Therefore, identification of quantitative trait loci (QTL) for high GPC and the associated markers is a priority to enhance selection efficiency in breeding durum wheat for elevated GPC. At a physiological level, GPC is influenced by several factors including nitrogen remobilization from vegetative organs and direct post-anthesis nitrogen uptake (NUP) from the soil. Understanding the relationship between elevated GPC and nitrogen remobilization, and post-anthesis NUP will enable durum wheat breeders to develop varieties that not only produce high yield and high GPC, but also exhibit better nitrogen use efficiency. The objectives of this study were: (1) to identify and validate QTL for elevated GPC in two durum wheat populations; and (2) to determine if elevated GPC is due to more efficient nitrogen remobilization and/or greater post-anthesis NUP. A genetic map was constructed with SSR and DArT® markers in a doubled haploid population from the cross Strongfield x DT695, and GPC data were collected in replicated trials in six Canadian environments from 2002 to 2005. Two stable QTL for high GPC, QGpc.usw-B3 on chromosome 2B and QGpc.usw-A3 on 7A, were identified. Strongfield, the high GPC parent, contributed the alleles for elevated GPC at both QTL. These two QTL were not associated with variation in grain weight (seed size) or grain yield. QGpc.usw-A3 was validated in a second Strongfield-derived population as that QTL was significant in all six testing environments. Averaged over five locations, selection for QGpc.usw-A3 resulted in a +0.4% to +1.0% increase in GPC, with only small effects on yield in most environments. A physiological study of grain protein accumulation revealed that regardless of the growing condition, nitrogen remobilization was the major contributor for grain nitrogen in durum genotypes evaluated, accounting for an average of 84.3% of total GPC. This study confirmed that introgression of Gpc-B1 into Langdon resulted in increased GPC, and this GPC increase was due to higher N remobilization. Strongfield expressed greater N remobilization than DT695 and the semi-dwarf cultivar Commander, but N remobilization was not the determining factor for Strongfields elevated GPC. Strongfield expressed greater post-anthesis NUP than DT695. Similarly, a selection of six high-GPC doubled haploid (DH) lines from the cross DT695 x Strongfield expressed significantly greater post-anthesis NUP than six low-GPC DH selections, supporting the hypothesis that elevated GPC in Strongfield is derived from greater post-anthesis NUP. All six high-GPC DH selections carried the Strongfield allele at QGpc.usw-A3, suggesting this QTL maybe associated with post-anthesis NUP.