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Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 85-97).

Yes / The high-performance liquid chromatography (HPLC) method employing stationary phases immobilized with plasma proteins was used for this study to investigate the structural properties governing drug-plasma protein binding. A set of 65 compounds with a broad range of structural diversity (in terms of volume, hydrogen-bonding, polarity and electrostatic force) were selected for this purpose. The Abraham linear free energy relationship (LFER) analyses of the retention factors on the immobilized HSA (human serum albumin) and AGP (α1-acid glycoprotein) stationary phases showed that McGowan’s characteristic molecular volume (V), dipolarity/polarizability (S) and hydrogen bond basicity (B) are the three significant molecular descriptors of solutes determining the interaction with immobilized plasma proteins, whereas excess molar refraction (E) is less important and hydrogen bond acidity (A) is not of statistical significance in both systems, for electrically neutral compounds. It was shown that ionised acids, as carboxylate anions, bind very strongly to the immobilized HSA stationary phase and that ionised bases, as cations bind strongly to the AGP stationary phase. This is the first time that the effect of ionised species on plasma protein binding has been determined quantitatively; the increased binding of acids to HSA is due almost entirely to acids in their ionised form.

Made available in DSpace on 2016-01-26T18:55:34Z (GMT). No. of bitstreams: 1 Ellyn.pdf: 424817 bytes, checksum: 14bf73c93a8a550f72aac6b4afc87223 (MD5) Previous issue date: 2012-09-20 / The objective of this study was to evaluate the framework sperm and protein seminal plasma and endocrine profile in deer bred in captivity. Four males aged between 12 and 36 months were evaluated. In four times at intervals of seven days, gave the weight (60.5 to 89.0 kg) and body mass index (93.07 kg/m2 to 126.56 kg/m2). There were four semen samples per animal, with an interval of seven days, by electroejaculation. Gave the fallowing values: ejaculate volume (0.50 ± 0.35 ml to 0.75 ± 0.28 ml), motility (87.75 ± 4.78% to 90.00 ± 7.07%) total defects (17.25 ± 5.81% and 47.72 ± 17.55%) and plasma testosterone (6.43 ± 4.33 ng/dL at 166.00 ± 64.48 ng/dL). Electrophoresis on SDS-PAGE revealed protein bands between 7.6 kDa and 142 kDa. Seminal plasma showed a wide range of proteins between 7.6 kDa and 142 kDa. Age influences the testosterone concentration, being higher in the range of 36 months. / O objetivo do trabalho foi avaliar o quadro espermático e protéico do plasma seminal e o perfil endócrino, em cervos criados em cativeiro. Quatro machos com idades entre 12 e 36 meses foram avaliados. Em quatro momentos com intervalos de sete dias, obteve-se o peso (60,5 a 89,0 kg) e o índice de massa corporal (93,07 kg/m2 a 126,56 kg/m2). Foram realizadas quatro colheitas de sêmen por animal, com intervalo de sete dias, por meio de eletroejaculador. Obteve-se os seguintes valores: volume do ejaculado (0,50±0,35 mL a 0,75±0,28 mL), motilidade espermática (87,75±4,78% a 90,00±7,07%), defeitos totais (17,25±5,81% a 47,72±17,55%) e testosterona plasmática (6,43±4,33 ng/dL a 166,00±64,48 ng/dL). A eletroforese em SDS-PAGE revelou bandas proteicas entre 7,6 kDa e 142 kDa. O plasma seminal apresentou uma grande gama de proteínas entre 7,6 kDa e 142 kDa. A idade influi na concentração de testosterona, sendo essa maior na faixa dos 36 meses.

Made available in DSpace on 2016-07-18T17:53:10Z (GMT). No. of bitstreams: 1 Ellyn.pdf: 424817 bytes, checksum: 14bf73c93a8a550f72aac6b4afc87223 (MD5) Previous issue date: 2012-09-20 / The objective of this study was to evaluate the framework sperm and protein seminal plasma and endocrine profile in deer bred in captivity. Four males aged between 12 and 36 months were evaluated. In four times at intervals of seven days, gave the weight (60.5 to 89.0 kg) and body mass index (93.07 kg/m2 to 126.56 kg/m2). There were four semen samples per animal, with an interval of seven days, by electroejaculation. Gave the fallowing values: ejaculate volume (0.50 ± 0.35 ml to 0.75 ± 0.28 ml), motility (87.75 ± 4.78% to 90.00 ± 7.07%) total defects (17.25 ± 5.81% and 47.72 ± 17.55%) and plasma testosterone (6.43 ± 4.33 ng/dL at 166.00 ± 64.48 ng/dL). Electrophoresis on SDS-PAGE revealed protein bands between 7.6 kDa and 142 kDa. Seminal plasma showed a wide range of proteins between 7.6 kDa and 142 kDa. Age influences the testosterone concentration, being higher in the range of 36 months. / O objetivo do trabalho foi avaliar o quadro espermático e protéico do plasma seminal e o perfil endócrino, em cervos criados em cativeiro. Quatro machos com idades entre 12 e 36 meses foram avaliados. Em quatro momentos com intervalos de sete dias, obteve-se o peso (60,5 a 89,0 kg) e o índice de massa corporal (93,07 kg/m2 a 126,56 kg/m2). Foram realizadas quatro colheitas de sêmen por animal, com intervalo de sete dias, por meio de eletroejaculador. Obteve-se os seguintes valores: volume do ejaculado (0,50±0,35 mL a 0,75±0,28 mL), motilidade espermática (87,75±4,78% a 90,00±7,07%), defeitos totais (17,25±5,81% a 47,72±17,55%) e testosterona plasmática (6,43±4,33 ng/dL a 166,00±64,48 ng/dL). A eletroforese em SDS-PAGE revelou bandas proteicas entre 7,6 kDa e 142 kDa. O plasma seminal apresentou uma grande gama de proteínas entre 7,6 kDa e 142 kDa. A idade influi na concentração de testosterona, sendo essa maior na faixa dos 36 meses.

Current screening tests for type 2 diabetes mellitus (T2DM) identify less than 50% of undiagnosed T2DM patients and provide no information about how the disease will develop in prediabetic patients. Here, twenty-nine protein glycation sites were quantified after tryptic digestion of plasma samples at the peptide level using tandem mass spectrometry and isotope-labelled peptides as internal standard. The glycation degrees were determined in three groups, i.e., 48 patients with a duration of T2DM exceeding ten years, 48 non-diabetic individuals matched for gender, BMI, and age, and 20 prediabetic men. In long-term controlled diabetic patients, 27 glycated peptides were detected at significantly higher levels, providing moderate diagnostic accuracies (ACCs) from 61 to 79%, allowing a subgrouping of patients in three distinct clusters. Moreover, a feature set of one glycated peptides and six established clinical parameters provided an ACC of 95%. The same number of clusters was identified in prediabetic males (ACC of 95%) using a set of eight glycation sites (mostly from serum albumin). All patients present in one cluster showed progression of prediabetic state or advanced towards diabetes in the following five years. Overall, the studied glycation sites appear to be promising biomarkers for subgrouping prediabetic patients to estimate their risk for the development of T2DM.

O fator VIII de coagulação (FVIII), recombinante ou purificado de plasma, é o biofármaco necessário para o tratamento da hemofília A, a doença hemorrágica mais freqüente em humanos. O método tradicional para a purificação de FVIII parte de crioprecipitado de plasma e precipitação alcoólica. No Instituto Butantan, foi proposto um método alternativo, utilizando somente cromatografia para esta purificação. Este projeto teve por objetivo comparar dois métodos cromatográficos de purificação do FVIII: 1 - gel filtração direta do plasma e 2 - pré-purificação de FVIII do plasma por cromatografia de troca aniônica, seguida de gel filtração. A purificação foi analisada por dosagens de atividade específica de FVIII e presença de outras proteínas da cascata de coagulação nas frações de cromatografia. Foram realizadas clonagem de fragmentos gênicos de FVIII e expressão de fragmentos protéicos para imunização de animais. Os soros com anticorpos policlonais anti-FVIII foram usados em ensaios de \"western blot\" para detectar as cadeias de FVIII ou degradação. / Coagulation factor VIII (FVIII), recombinant or purified from plasma, is the biopharmaceutical used for treatment of haemophilia A, the most frequent human hemorrhagic disorder. The traditional method used for purification of FVIII starts from plasma cryoprecipitate and alcoholic precipitation. The Instituto Butantan proposed an alternative methodology using only chromatography for FVIII purification. The main objective of this project was to compare two chromatographic methods for FVIII purification: 1 - direct plasma gel filtration and 2 - pre-purification of FVIII by anion exchange chromatography, followed by gel filtration. The purification process was analyzed by determination of FVIII specific activity and detection of other coagulation factors co eluting in chromatographic fractions. Fragments of FVIII gene were cloned and protein fragments were expressed for animal immunization. Sera with polyclonal antibodies anti-FVIII were used in western blots assays to detect FVIII chains or its degradation.