Thesis (Ph. D.)--Columbia University, 1942. / Vita. Bibliography: p. 14.

Thesis (Ph. D.)--Columbia University, 1941. / Vita. Bibliography: p. 20.

Thesis (Ph. D.)--Montana State University--Bozeman, 2006. / Typescript. Chairperson, Graduate Committee: David M. Dooley. Includes bibliographical references (leaves 204-216).

The prosthetic groups of cytochrome o, the terminal ubiquinol:dioxygen oxido-reductase of Escherichia coli, were investigated in the purified and in situ enzyme. The interactions between the redox-active centres, of which there are three, were characterised using optical, magnetic resonance and X-ray absorption spectroscopy techniques. The copper complement of this enzyme was investigated and the available data suggested a single copper centre associated with the ligand binding haem centre (haem o) forming a binuclear oxygen binding and reduction site. Two redox-active copper atoms are known to exist in the mammalian cytochrome c oxidase complex and the consequences of the different copper stoicheiometries, in these enzymes, are discussed. Spatial and organisational investigations are described and a model for the binuclear reaction site presented. The location of the haem centres was determined using low-temperature EPR spectroscopy by observing the effects on the relaxation behaviour of these centres in the presence of an extrinsic paramagnetic probe.

An in situ study was conducted of the cytochrome bd ubiquinol:oxygen oxidoreductase (cytochrome b558-b595-d) of Escherichia coli grown anaerobically on glycerol with fumarate as respiratory oxidant. Nitrite reacts with and is reduced by the oxidase, resulting in the formation of NO adducts to haems b595 and d. The kinetics of formation of these species indicate that the affinity of haem d for nitrite is higher than that of haem b595. CO also binds to the oxidase, resulting in the formation of CO adducts to haems d and b595. Binding titrations indicate that the affinity of haem d for CO is higher than that of haem b595. The steady state kinetics of the oxidase reaction in the presence of nitrite or CO are cooperative with respect to oxygen binding, suggesting that both haems d and b595 are involved in the reduction of oxygen. E.p.r. studies of the ferric oxidase indicate the presence of two high spin haem signals, one rhombic and one axial, which are assigned to haems b595 and d, respectively. These signals titrate potentiometrically with midpoint potentials similar to those published on the basis of optically followed titrations for haems b595 and d. The high spin ferric haem spectra are affected by oxygen, CO, cyanide, and pH. A low spin ferric haem signal is observed at g=3.3 and is assigned to haem b558. The sidedness with respect to the cytoplasmic membrane of ligand binding haems of the oxidase was determined by investigating the effect of the exogenous paramagnetic probe DyEDTA on the e.p.r. properties of the ferrous haems d-NO and b595-NO. These haems are located towards the inner aspect of the cytoplasmic membrane at around 8 and 12A° below the surface, respectively. Overall, the data supports a functional model for cytochrome bd with two oxygen binding sites, haems d and b595, forming the binuclear centre of the oxidase reaction. Possible mechanisms of this reaction are discussed.

A procedure for the purification of uricase from porcine liver is described, utilising the technique of bioaffinity chromatography as a major purification step. Bioaffinity support is prepared by coupling of urate to bisoxirane-activated Sepharose 4B. Purified uricase shows a single protein band corresponding to the activity band, when applied to polyacrylamide disc-gel electrophoresis. A single protein band but no activity band is obtained by SDS-acrylamide disc-gel electrophoresis. The enzyme has a pH optimum in the range of 8.9-9.1, with a Vmax of 12.6 U.mg^-1, a Km of 1 X 10^-5M and a molecular weight of 13 x 10⁴. Each enzyme molecule comprises 4 subunits of molecular weight 3 32-34 X 10³ each. Nylon tube is directly activated by alkaline glutaraldehyde solution to generate reactive centres for enzyme immobilisation. The optimal conditions for activation are studied. Purified uricase is immobilised to PEI-glutaraldehyde-nylon tube with about 20% activity retention. The derivatised enzyme has a pH optimum in the range of 9.0-9.2 and a Km of about 4 times that of the soluble enzyme. Immobilised uricase is incorporated into a continuous flow Auto-Analyser for use in the automated analysis of serum urate. For this purpose, the immobilised enzyme shows good storage and operational stability. Linear calibration plots can be obtained for a urate range of 2-20 mg.100ml^-1the method exhibiting a high degree of precision and accuracy. The results obtained also compare favourably with an established method of urate assay which employs soluble uricase.

CAVALCANTI, J. H. F. Caracterização da família multigênica da oxidase alternativa em plantas do gênero Medicago e avaliação da expressão em Medicago sativa sob condições de estresse. 2011. 75 f. Dissertação (Mestrado em Bioquímica) - Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2011. / Submitted by Daniel Eduardo Alencar da Silva (dealencar.silva@gmail.com) on 2015-01-05T20:09:02Z No. of bitstreams: 1 2011_dis_jhfcavalcanti.pdf: 988301 bytes, checksum: 031d26ae84ee8293165562c84ee42547 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa(jairo@ufc.br) on 2016-03-22T13:38:55Z (GMT) No. of bitstreams: 1 2011_dis_jhfcavalcanti.pdf: 988301 bytes, checksum: 031d26ae84ee8293165562c84ee42547 (MD5) / Made available in DSpace on 2016-03-22T13:38:55Z (GMT). No. of bitstreams: 1 2011_dis_jhfcavalcanti.pdf: 988301 bytes, checksum: 031d26ae84ee8293165562c84ee42547 (MD5) Previous issue date: 2011 / In plants, Alternative Oxidase (AOX) is enconded by small milti gene family located in genomc DNA. This multi gene family was studied in mono and dicots plants being clusterd in two subfamilies: Aox1 and Aox2. Aox1 gene are found in all angiosperms táxon presenting gene expression related to stress situations while Aox2 genes are found in dicots plants only and presenting a houkeeping expression. Many Aox1 genes e just one Aox2 are found in the most of dicots studied. However, in Fabales, as cowpea and soybean, is found a profile with two genes Aox2 (Aox2a , Aox2b) and one Aox1 gene. In this work was characterized a multi gene family of Aox genes in Medicago genus (Medicago truncatula e Medicago sativa) belonging to Fabales Order. Data mining in genBanks characterized four Aox genes (Aox1, Aox2a, Aox2b1 and Aox2b2) in Medicago truncatula genome revealing for first time a duplication of Aox2b genes. Specifics primers designed for each Aox gene and then were used to amplification by PCR, cloning and partial sequencing of these genes in Medicago sativa. Gene expression was carried out by semiquantitative RT-PCR in: germination seeds (0, 24, 48 hours of germination), roots and leaves from Medicago sativa grewth in Hoaglend’s medium and applied to disticts stress conditions (0, 6, 12 e 24 hs after treatment): control, salicylic acid (0,5mM), PEG (100g/L), H2O2 (10mM) e cisteíne (0,5mM). The results revealed that all four Aox genes were detected in seed of Medicago sativa. However, Aox1, Aox2b1 and Aox2b2 genes presented high expression during germination while Aox2a showed constitutive expression. In leaves, Aox1, Aox2a and Aox2b1 were detected in all conditions tested, but Aox2b2 was observed more strong in stress situations only. Simirily observed in germination, Aox1, Aox2b1 and Aox2b2 increased transcripts levels in response to all stress conditions. Aox2a, one more time, had constitutive, but very strong expression. In roots, all genes were detected and a similar induction profile of Aox1, Aox2b1 and Aox2b2 were confirmed less to PEG treatment where just Aox1 was responsive. Aox2a had constitutive expression too, but it was weakly expressed. In order to understand the co-expresion of Aox1, Aox2b1 and Aox2b2 genes, the promoter regions were cloned and sequenced revealing close motifs among them suggesting th involviment of cis-elements in their regulation. Theses resuls corroborate with co-expression stress-induced of Aox1/Aox2b found in cowpea. However, in Medicago sativa, it was observed tissue-specific exression. Aox1, Aox2a and Aox2b1 with characteristics constitutive and induced in seeds germination and roots tissue while in leaves Aox2b2 differed by present stress-induced expression only. / Oxidase Alternativa (AOX) em plantas é codificada por uma pequena família multigênica de origem nuclear (DNA genômico). Essa família multigênica foi bem estudada em mono e dicotiledôneas sendo subdividida em duas subfamílias: Aox1 e Aox2. Os genes Aox1 são encontrados em mono e eudicotiledôneas apresentando expressão mais relacionada a condições de estresses enquanto que os genes Aox2 são encontrados apenas em eudicotiledôneas com expressão constitutiva. Vários genes Aox1 e apenas hum gene Aox2 são encontrados na maioria das eudicotiledôneas estudadas. Nesse trabalho, caracterizou-se a família multigênica da Aox no gênero Medicago (Medicago truncatula e Medicago sativa) pertencente à ordem Fabales. Em Medicago truncatula, a família multigênica da Aox foi carcterizada através de busca por bioinformática em bancos de dados identificando-se 4 genes Aox (Aox1, Aox2a, Aox2b1 e Aox2b2) no genoma dessa espécie revelando pela primeira vez uma duplicação do gene Aox2b. Oligonucleotídeos específicos desenhados para cada um dos genes foram usados para amplificação por PCR, clonagem e seqüenciamento parcial dos referidos genes em Medicago sativa. A expressão gênica foi estudada, através de RT-PCR semiquantitativa, em sementes durante a germinação (0, 24 e 48 horas de após embebição) e em raízes e folhas de Medicago sativa crescidas em meio hidropônico de Hoagland e submetidas a diferentes condições de estresse (0, 6, 12 e 24 hs após tratamento): controle, ácido salicílico (0,5 mM), PEG (100 g/L), H2O2 (10 mM) e cisteína (0,5 mM). Os resultados revelaram que todos os 4 genes Aox foram detectados em sementes de Medicago sativa, entretanto os genes Aox1, 2b1 e 2b2 apresentaram aumento da expressão durante a germinação enquanto que o Aox2a mostrou-se constitutivo. Em folhas os genes Aox1, 2a e 2b1 foram detectados em todas as condições testadas já o Aox2b2 foi detectado mais intensamente apenas nas condições de estresse. De maneira semelhante ao observado durante a germinação os genes Aox1, 2b1 e 2b2 tiveram expressão aumentada em resposta a todas as condições de estresse. O Aox2a mostrou-se constitutivo, mas foi intensamente expresso. Em raízes, todos os genes foram detectados e um perfil semelhante de indução dos genes Aox1, 2b1 e 2b2 também foi observado com exceção do tratamento com PEG onde apenas Aox1 foi induzido. O Aox2a também se mostrou constitutivo, mas foi fracamente expresso. Com a finalidade de se compreender melhor a co-expressão dos genes Aox1, 2b1 e 2b2 em Medicago sativa os promotores foram clonados e seqüenciados revelando regiões idênticas entre eles indicando o envolvimento de elementos cis comuns na regulação. Esses resultados corroboram com a co-expressão induzida por estresse de Aox1/Aox2b observada anteriormente em feijão. Contudo, em Medicago sativa, observamos expressão diferencial entre tecidos: no tecido radicular os genes Aox1, Aox2a e 2b1 podem sem induzidos por estresses e/ou mostram uma característica de expressão. Por outro lado, em folhas o gene Aox2b2 diferenciou por apresentar apenas característica de gene induzido em condições de estresse.

DANTAS, Rodrigo Tavares. Avaliação dos efeitos renais da fração L-aminoácido oxidase isolada do veneno da serpente Bothrops marajoensis. 2010. 104 f. Dissertação (Mestrado em Farmacologia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2010. / Submitted by denise santos (denise.santos@ufc.br) on 2012-05-15T12:10:30Z No. of bitstreams: 1 2010_dis_rtdantas.pdf: 3573132 bytes, checksum: 27b964204fc5e26e329b4d9950fd7885 (MD5) / Approved for entry into archive by Eliene Nascimento(elienegvn@hotmail.com) on 2012-05-15T16:29:18Z (GMT) No. of bitstreams: 1 2010_dis_rtdantas.pdf: 3573132 bytes, checksum: 27b964204fc5e26e329b4d9950fd7885 (MD5) / Made available in DSpace on 2012-05-15T16:29:18Z (GMT). No. of bitstreams: 1 2010_dis_rtdantas.pdf: 3573132 bytes, checksum: 27b964204fc5e26e329b4d9950fd7885 (MD5) Previous issue date: 2010 / There are about 3.000 species of snakes worldwide, but only 10 to 14% are venomous. Among South American countries, Brazil is the one with the largest number of accidents, with approximately 20.000 snakebites each year. According to the Department of Health of Brazil, the genus Bothrops are the main involved in snakebites in the country. Acute renal failure (ARF) is a serious complication of snake poisoning. The fraction L-amino acid oxidase (LAAO) constitutes a main part of the total composition of snake venoms. In some cases this amount can reach 30% of total venom proteins. The renal effects of fraction L-amino acid oxidase isolated from the venom of Bothrops marajoensis (LAAOBM) was investigated in this study. Isolated perfused rat kidney and the cultured renal tubular cells line MDCK (Madin-Darby Canine Kidney) were used here. For the isolated perfused rat kidney method, we used Wistar rats weighing between 250 and 300. Their right kidneys were surgically excised and perfused with Krebs-Hanseleit containing 6% w/v bovine albumin previously dialyzed. The effects of LAAOBM (10 mg/mL, n=4) were analyzed on the Perfusion Pressure (PP), Renal Vascular Resistance (RVR), Urinary Flow (UF), Glomerular Filtration Rate (GFR) Percentage of Sodium Proximal Tubular Transport (%pTNa+), Percentage of Sodium (%TNa+), Potassium (%TK+) and Chloride (%TCl-) Tubular Transport. MDCK cells were cultured in RPMI 1640 medium supplemented with 10% v/v fetal bovine serum and incubated with LAAOBM at concentrations of 50, 25, 12.5, 6.25, 3.125 and 1.652 mg/mL. After 24 hours of incubation, assays were performed on cell proliferation and viability using the MTT method. The LAAOBM promoted a reduction of perfusion pressure at 90 minutes of the experiment and this reduction was even slightly higher at 120 minutes. It was also observed decrease in renal vascular resistance at 120 minutes. There was a sharp and sudden drop in urine flow at 90 minutes, despite the tendency of recovery observed at 120 minutes, still proved to be quite small when compared to the control group. The infusion of LAAOBM also promoted a reduction in glomerular filtration rate at 90 minutes compared to the control group and this parameter still remained at the same level of reduction at 120 minutes. The LAAOBM gradually reduced the percentage of sodium tubular transport at 90 and 120 minutes and the percentage of chloride tubular transport in the periods of 60, 90 and 120 minutes. Histological analysis of kidneys perfused with LAAOBM showed the presence of significant morphological changes such as accumulation of proteins in tubular and glomerular spaces. In the MDCK cell culture LAAOBM promoted a reduction in cell viability from concentrations of 3.25 mg / mL until 50μg/mL, with IC50 value of 2.43 mg/mL. Inverted light microscopy showed morphological changes of these cells, such as vacuolation, alteration of the state of confluence and detachment of the substrate culture. These results demonstrated that LAAOBM changed all the parameters evaluated in renal and vascular perfusion of isolated kidney and had cytotoxic activity on MDCK cells after 24 hours of incubation. / No mundo, existem cerca de 3.000 espécies de serpentes das quais 10 a 14% são peçonhentas. Dentre os países sul-americanos, o Brasil é o que apresenta maior número de aciden¬tes/ano com cerca de 20.000 acidentes ofídicos por ano. De acordo com o Ministério da Saúde, as serpentes do gênero Bothrops são as principais envolvidas nos acidentes ofídicos no país e a insuficiência renal aguda (IRA) é uma complicação grave dos envenenamentos produzidos por estas serpentes. Tendo em vista que a fração L-aminoácido oxidase (LAAO) constitui grande parte da composição total do veneno de serpentes, em algumas serpentes chegando a constituir mais de 30% do total de proteínas do veneno, neste trabalho, foram investigados os efeitos renais da fração L-aminoácido oxidase isolada do veneno da serpente Bothrops marajoensis (LAAOBM) em sistema de perfusão de rim isolado e em cultura de células tubulares renais da linhagem MDCK (Madin-Darby Canine Kidney). Para perfusão de rim isolado foram utilizados ratos Wistar pesando entre 250 e 300g, cujos rins foram excisados cirurgicamente e perfundidos com solução de Krebs-Hanseleit contendo 6%p/v de albumina bovina previamente dialisada. Foram investigados os efeitos da LAAOBM (10 µg/mL; n=4) sobre a Pressão de Perfusão (PP), Resistência Vascular Renal (RVR), Fluxo Urinário (FU), Ritmo de Filtração Glomerular (RFG), Percentual de Transporte Tubular Proximal de Sódio (%pTNa+),Percentual de Transporte Tubular de Sódio (%TNa+), de Potássio (%TK+) e de Cloreto (%TCl-). As células MDCK foram cultivadas em meio de cultura RPMI 1640 suplementado com 10% v/v de Soro Bovino Fetal e então incubadas com a LAAOBM nas concentrações de 50; 25; 12,5; 6,25; 3,125 e 1,652 µg/mL. Após 24 horas de incubação, foram realizados os ensaios de viabilidade e proliferação celular utilizando-se o método do MTT. A LAAOBM promoveu uma redução da pressão de perfusão aos 90 minutos de experimento e esta redução foi ainda discretamente maior aos 120 minutos. Observou-se também queda da resistência vascular renal aos 120 minutos. Houve uma queda abrupta e acentuada do fluxo urinário aos 90 minutos que, apesar da tendência à recuperação observada aos 120 minutos, ainda mostrou-se bastante reduzido quando comparado ao grupo controle. A infusão da LAAOBM também promoveu uma redução do ritmo de filtração glomerular aos 90 minutos quando comparada ao grupo controle e este parâmetro manteve-se ainda no mesmo patamar de redução aos 120 minutos. A LAAOBM reduziu gradativamente o percentual de transporte tubular de sódio aos 90 e 120 minutos e o percentual de transporte tubular de cloreto nos tempos de 60, 90 e 120 minutos. A análise histológica dos rins perfundidos com LAAOBM mostrou a presença de alterações morfológicas significativas, como acúmulo de proteínas nos espaços tubulares e glomerulares. Na cultura de células MDCK a LAAOBM promoveu uma redução da viabilidade celular a partir da concentração de 3,25µg/mL até a concentração de 50µg/mL, com valor da CI50 de 2,43µg/mL. Foram observadas também, através de microscópio óptico invertido, alterações morfológicas destas células, tais vacuolização citoplasmática, alteração do estado de confluência e desprendimento das mesmas do substrato de cultura. Estes resultados demonstram que a LAAOBM alterou todos os parâmetros vasculares e renais avaliados na perfusão de rim isolado e possui ação citotóxica sobre as células MDCK após 24 horas de incubação.

Increasing demand for chiral technology in industry has led to the rapid development of catalysts for enantioselective processes. In this respect biocatalysts are of particular interest due to their excellent regio- and stereo- selectivity and their ability to work under mild conditions. The development of catalysts for the selective oxidation of alcohols to aldehydes and ketones represents a major challenge in organic synthesis. Previous work showed that a variant of the enzyme galactose oxidase (GOase) was capable of oxidising a wide range of chiral secondary alcohols with high enantioselectivity. The aim of this work is to develop new applications for this variant. Two new stereoselective processes have been developed employing this variant: 1. Enzymatic desymmetrisation of proatropisomeric diaryl ethers and biaryls. Atropisomers are stereoisomers resulting from restricted rotation about an axis, where the rotational barrier is high enough for isolation of the conformers. Desymmetrisation by GOase has allowed the production of atropisomers with enantiomeric excess (ee) up to 99% in good yields. An alternative approach based upon asymmetric reduction of the corresponding dialdehyde using ketoreductases (KREDs) has also been explored. 2. Deracemisation of racemic secondary alcohols by a combination of enzyme and transition metal catalysts. Deracemisation is a method for the production of enantiopure compounds starting from racemic substrates. Combination of the enzyme GOase and a transition metal catalyst has allowed the production of a single enantiomer of a number of secondary alcohols from the corresponding racemic mixture (ee > 99%, yield > 98%). Primary amides are widely applied in the polymer industry. They are produced in large quantities each year. An efficient solvent-free process (yield > 90%) for the production of erucamide has been developed employing immobilised Candida antarctica Lipase B (CALB, Novozym® 435 ) under mild conditions (solid ammonia source, 90 °C).

The potential of using mushroom polyphenol oxidase (EC 1.14.18.1) as a biocatalyst for the biotransformation of phenols to produce catechols in an aqueous medium was investigated. Polyphenol oxidase is characterised by two distinct reactions i.e., the ortho-hydroxylation of phenols to catechols (cresolase activity) and the subsequent oxidation of catechols to orthoquinones (catecholase activity). In order to facilitate the development of a process to produce catechols, the accumulation of catechol as a true intermediate product released in the reaction system needed to be investigated, as its release had been disputed due to the oxidation of catechols to o-quinones. Using LC-MS, catechol products were successfully identified as true intermediate products formed during biocatalytic reactions in water.