No description available.

Thesis (M. Sc.)--Carleton University, 2008. / Includes bibliographical references (p. 116-125). Also available in electronic format on the Internet.

Thesis (doctoral)--Freie Universität Berlin, 1979.

Glutamate dehydrogenase a (GDHa) has been suggested as a potential drug target against the malaria parasite Plasmodium falciparum. GDHa knockout cell line was generated and needed a phenotypic description by means of molecular biology and biochemistry. The knockout cell line was tested for higher oxidative stress sensitivity, levels of relevant proteins and gene transcripts were quantified. Furthermore, concentrations of two key molecules enabling redox homeostasis, glutathione and NADPH, were attempted to quantify. Finally, we attempted to disrupt a gene of another glutamate dehydrogenase, gdhb, which did not result in formation of viable parasites. In conclusion, GDHa is not a suitable drug target and GDHb needs to be further elucidated.

No description available.

Clostridium difficile is the most common infectious cause of nosocomial diarrhea, affecting thousands of patients annually and exacting enormous costs on the U.S. health care system. Early diagnosis is critical to prevent transmission and reduce morbidity and mortality, yet sensitive and specific diagnostic tests with a quick turnaround time are lacking. The objective of this study was to determine if a new commercially available real time polymerase chain reaction (PCR) test would prove more rapid, sensitive and specific than standard methods for the diagnosis of C. difficile infection (CDI). BD GeneOhm Cdiff assay, a real-time PCR assay for detection of C. difficile toxin B (tcdB) gene, was compared with Tox A/B II ELISA and a two-step algorithm which includes C. Diff Chek-60 Glutamate Dehydrogenase (GDH)-antigen assay followed by cytotoxin neutralization. Four-hundred liquid or semisolid stools submitted for diagnostic C. difficile testing were selected: 200 GDH antigen-positive and 200 GDH antigen-negative. All samples were tested by the C. Diff Chek-60 GDH antigen, cytotoxin neutralization, Toxin A/B II ELISA, and BD GeneOhm Cdiff assay. Discrepant specimens were tested by toxigenic culture as an independent gold standard. Chart review was performed on patients with discrepant specimens. As BD GeneOhm Cdiff assay was not FDA-cleared at the time of study, PCR results were not clinically reported. Of 200 GDH-positive samples, 71 were positive by Tox A/B II, 88 were positive by the two-step method, 93 were positive by PCR, and 96 were positive by GDH-antigen only. Of 200 GDH-negative samples, 3 were positive by PCR only. Toxigenic culture was performed on 41 samples with discrepant results and 39 were culture-positive. After culture resolution of discrepants, Tox A/B II detected 70 (66.7%), the two-step method detected 87 (82.9%), and PCR detected 96 (91.4%) of 105 true positives. The BD Gene-Ohm Cdiff assay was more sensitive in detecting toxigenic C. difficile than Tox A/B II (p <0.0001); however, the difference between PCR and the two-step method was not significant (p=0.1237). The BD GeneOhm Cdiff assay took a similar amount of time to perform as the Tox A/B II and was more rapid than the two-step method. Chart review revealed that 18 patients with cytotoxin-negative, PCR-positive discrepant samples were given 1-2 days of therapy (n=8), or no treatment at all (n=10). Yet symptoms resolved and no further C. difficile testing was requested for 13 of 18 patients for 6-8 months after hospital discharge. Only one patient had a subsequent cytotoxin positive stool submitted 22 days after the study sample was tested. Enhanced sensitivity and rapid turnaround time make the BD GeneOhm Cdiff assay an important advance in the diagnosis of toxigenic C. difficile infection. The BD GeneOhm Cdiff assay is significantly more sensitive than a commonly-used ELISA toxin assay and has a sensitivity and specificity comparable to the two-step method. Its turnaround time is similar to ELISA toxin assays and more rapid than the two-step method. Disadvantages to implementation of BD GeneOhm Cdiff assay include increased cost and potential treatment of asymptomatic carriers and mild, self-resolving disease.

The gene gdhA from Escherichia coli, that encodes a NADPH-dependent glutamate dehydrogenase (GDH), directs a novel pathway in transgenic plants that potentially allows an increase in ammonium assimilation. Glufosinate leads to plant death by the irreversible inhibition of glutamate synthetase (GS) leading to a disruption of subsequent GS-related processes resulting in elevated ammonium and disruption of photorespiration. Therefore, it was speculated that the gdhA-transformed plants may exhibit a novel mechanism of resistance to glufosinate by altered activity of the GDH pathway and subsequently related processes. Studies were conducted in the greenhouse to evaluate 1) whole plant tolerance to glufosinate, 2) changes in absorption, translocation and metabolism of glufosinate, and 3) metabolic fingerprint changes in response to glufosinate treatment in tobacco plants containing the gdhA gene. Whole plant tolerance experiments showed that tobacco transformed with the gdhA gene expressed up to six fold increased resistance (GR50) to glufosinate compared with the non-gdhA control line. GDH enzyme activity among gdhA-transformed tobacco lines was highly correlated (r2 = 0.9903) with the amount of herbicide resistance. Thus, use of the E. coli gdhA gene in plant transformations can provide an additional mechanism for resistance to glufosinate. Foliar absorption and translocation of 14C from glufosinate was not altered to any large extent in gdhA-transformed plants which suggests these factors cannot fully explain the mechanism for whole-plant resistance to glufosinate. However, the metabolic fingerprint resulting from glufosinate treatment was significantly altered in gdhA tobacco. It was also shown that metabolic perturbation induced by glufosinate was lower in the high GDH activity tobacco line, +gdhA 9, than in the non-gdhA control tobacco line as evidenced by the reduced number of altered peaks recorded in leaves of these two tobacco lines. Thus, gdhA-transformed tobacco plants with low and high expression of GDH activity, exhibited greater overall stability of metabolism following the application of glufosinate, than recorded in non-gdhA control plants. This greater metabolic stability during GS inhibition was likely due to the amelioration of amino acid production through the increased activity of GDH. Therefore, the hypothesized mechanism of increased resistance to glufosinate in gdhA-transformed tobacco lines is by maintenance of amino acid production and maintenance of photorespiratory activity.

O protozoário flagelado Giardia duodenalis é um parasito intestinal que pode infectar uma ampla variedade de mamíferos, incluindo os humanos e os animais domésticos e selvagens. Devido à carência de informações a respeito da caracterização molecular de amostras de Giardia spp. no Brasil, o presente estudo teve como objetivo analisar 113 amostras de fezes positivas para Giardia spp. de origem humana e animal. Um segmento do gene que codifica a enzima glutamato desidrogenase (gdh) dos isolados provenientes de 37 humanos, 31 cães, 20 gatos, 12 bugios, cinco bezerros, três chinchilas, duas avestruzes, dois cachorros-do-mato e uma onça-pintada foi amplificado e seqüenciado. A análise da seqüência de nucleotídeos do gene gdh mostrou que estes isolados foram divididos em seis linhagens genéticas principais (Assemblages A, B, C, D, E, F). O genótipo AII foi identificado apenas nas amostras de humanos, enquanto o genótipo AI foi detectado nos isolados de origem animal (gato, bezerro, onça-pintada). Os isolados de Giardia spp. provenientes dos humanos, bugios, chinchilas e avestruzes foram caracterizados como pertencente ao genótipo BIV. Entretanto, os demais Assemblages foram identificados apenas em um grupo específico de hospedeiros. Assemblage C e D foram encontrados nas amostras de cães e cachorros-do-mato, Assemblage F nas amostras de gatos e Assemblage E nas amostras de bezerros. A caracterização molecular das amostras de Giardia spp. gera uma importante contribuição para o conhecimento da especificidade de hospedeiro dos diferentes genótipos. / The flagellated protozoan Giardia duodenalis is an intestinal parasite that can infect a broad variety of mammalian hosts, including humans and domestic and wild animals. Due the lack of information about the molecular characterization of Giardia spp. in Brazil, this study aimed to analyse 113 stool samples from human and animals, all positive to Giardia spp. A segment of the glutamate dehydrogenase gene (gdh) of isolates from 37 humans, 31 dogs, 20 cats, five calves, 12 wild monkeys, three chinchilas, two ostrichs, two crab-eating-foxes and one jaguar was amplified and sequenced. Nucleotide sequences analysis of gdh gene showed that these isolates could be divided into six main genetic lineage (Assemblages A, B, C, D, E, F). The genotype AII was identified only in human isolates, whereas genotype AI was detected in a variety of animals (cat, calf, jaguar). In adition, Giardia spp. isolates recovered from human, wild monkeys, chinchila and ostrichs were characterized as genotype BIV. However the other Assemblages identified were confined to restrict host species. Assemblages C and D were found in isolates from dogs and crab-eating-foxes, Assemblage F from cats and Assemblage E from calves. The molecular characterisation of Giardia spp. isolates has made a major contribution to our understanding of the host specificity of different genotypes.

Made available in DSpace on 2016-12-08T16:24:08Z (GMT). No. of bitstreams: 1 PGCA11MA068.pdf: 13593 bytes, checksum: bb2118cc19310d22381d61f71c63aee7 (MD5) Previous issue date: 2011-06-01 / The mitochondrial enzyme Glutamate Dehydrogenase (GDH: EC 1.4.1.2) catalyzes the reversible deamination of the L-glutamate for 2-oxoglutarate (&#945;-ketoglutarate) using NAD+ and NADP+ as coenzymes. It is a complex allosteric enzyme that consists of six identical subunits being its activity influenced by both, the ADP its positive modulator, and by the GTP, its negative modulator.It is one of the most important liver enzymes found in hepatocytes of cattle, sheep and goats. Infections by Fasciola spp or severe acute intoxication by toxins of plants such as Xanthium spp and Senecio spp as well as intoxication by copper result in the release of this enzyme in blood. The increase of the GDH indicates damage or hepatic necrosis in cattle and sheep. This is an enzyme of choice to evaluate the function of the ruminants. In the present study the cDNA, that codifies the GDH enzyme of the hepatocyte of sheep, was synthesized by means of the RT-PCR making use of mRNA extracted from the liver of sheep. Part of the region where the cDNA of the GDH of the ovine is codified was amplified by PCR from primers synthesized through the comparison of the aligned sequences of Ovis aries with those of the Bos taurus,. Homo sapiens, Rattus norvegicus and Mus musculus available in the database, where highly conserved regions, as well as variable regions were identified.The cDNA was cloned in the vector pGEM®-T Easy Vector Systems and inserted in competent cells of the Escherichia coli DH10B by means heat shock. The plasmid DNA was purified and after sequencing, the presence of an insert of 1292 pb was confirmed. The alignment of the sequence deduced of amino acid with other species revealed high homology among the GDH / A enzima mitocondrial Glutamato Desidrogenase (GDH : EC 1.4.1.2) catalisa a desaminação reversível do L- glutamato para 2-oxoglutarato (&#945;-cetoglutarato) usando o NAD+ e NADP+ como coenzimas . É uma enzima alostérica complexa que consiste em seis subunidades idênticas sendo sua atividade influenciada tanto pelo ADP, seu modelador positivo, como pelo GTP, seu modelador negativo. É uma das mais importantes enzimas hepáticas encontradas em hepatócitos de bovinos, ovinos e caprinos. Infecções por Fasciola spp ou intoxicação grave aguda por toxinas de plantas tais como Xanthium spp e Senecio spp e intoxicação por cobre resultam na liberação dessa enzima no sangue. O aumento da GDH indica danos ou necrose hepática em bovinos e ovinos. Esta é a enzima de escolha para avaliar a função hepática dos ruminantes. No presente trabalho o cDNA que codifica a enzima GDH do hepatócito de ovino foi sintetizado por meio de RT-PCR utilizando mRNA extraído do fígado de ovino. Parte da região de codificação do cDNA da GDH de ovino foi amplificada por PCR a partir de oligonucleotídeos iniciadores sintetizados através da comparação das sequências alinhadas de Ovis aries com de Bos taurus, Homo sapiens, Rattus norvegicus e Mus musculus disponíveis em banco de dados, onde foram identificadas regiões bastante conservadas e regiões variáveis. O cDNA foi clonado no vetor pGEM® -T Easy Vector Systems e inserido em células competentes de Escherichia coli DH10B através de choque térmico. O DNA plasmidial foi purificado e após o sequenciamento a presença de um inserto de 1292 pb foi confirmado. O alinhamento da sequência deduzida de aminoácidos com outras espécies revelou alta homologia entre as GDH

O protozoário flagelado Giardia duodenalis é um parasito intestinal que pode infectar uma ampla variedade de mamíferos, incluindo os humanos e os animais domésticos e selvagens. Devido à carência de informações a respeito da caracterização molecular de amostras de Giardia spp. no Brasil, o presente estudo teve como objetivo analisar 113 amostras de fezes positivas para Giardia spp. de origem humana e animal. Um segmento do gene que codifica a enzima glutamato desidrogenase (gdh) dos isolados provenientes de 37 humanos, 31 cães, 20 gatos, 12 bugios, cinco bezerros, três chinchilas, duas avestruzes, dois cachorros-do-mato e uma onça-pintada foi amplificado e seqüenciado. A análise da seqüência de nucleotídeos do gene gdh mostrou que estes isolados foram divididos em seis linhagens genéticas principais (Assemblages A, B, C, D, E, F). O genótipo AII foi identificado apenas nas amostras de humanos, enquanto o genótipo AI foi detectado nos isolados de origem animal (gato, bezerro, onça-pintada). Os isolados de Giardia spp. provenientes dos humanos, bugios, chinchilas e avestruzes foram caracterizados como pertencente ao genótipo BIV. Entretanto, os demais Assemblages foram identificados apenas em um grupo específico de hospedeiros. Assemblage C e D foram encontrados nas amostras de cães e cachorros-do-mato, Assemblage F nas amostras de gatos e Assemblage E nas amostras de bezerros. A caracterização molecular das amostras de Giardia spp. gera uma importante contribuição para o conhecimento da especificidade de hospedeiro dos diferentes genótipos. / The flagellated protozoan Giardia duodenalis is an intestinal parasite that can infect a broad variety of mammalian hosts, including humans and domestic and wild animals. Due the lack of information about the molecular characterization of Giardia spp. in Brazil, this study aimed to analyse 113 stool samples from human and animals, all positive to Giardia spp. A segment of the glutamate dehydrogenase gene (gdh) of isolates from 37 humans, 31 dogs, 20 cats, five calves, 12 wild monkeys, three chinchilas, two ostrichs, two crab-eating-foxes and one jaguar was amplified and sequenced. Nucleotide sequences analysis of gdh gene showed that these isolates could be divided into six main genetic lineage (Assemblages A, B, C, D, E, F). The genotype AII was identified only in human isolates, whereas genotype AI was detected in a variety of animals (cat, calf, jaguar). In adition, Giardia spp. isolates recovered from human, wild monkeys, chinchila and ostrichs were characterized as genotype BIV. However the other Assemblages identified were confined to restrict host species. Assemblages C and D were found in isolates from dogs and crab-eating-foxes, Assemblage F from cats and Assemblage E from calves. The molecular characterisation of Giardia spp. isolates has made a major contribution to our understanding of the host specificity of different genotypes.