Made available in DSpace on 2016-07-18T17:51:06Z (GMT). No. of bitstreams: 1 Dissertacao.pdf: 291613 bytes, checksum: 351209f8c0af1b2f460925dd83ce0a2d (MD5) Previous issue date: 2010-11-17 / Soybean is considered the main crop in Brazil due to both volume production and income. Many factors have contributed to a good performance of this crop in this country, such as adaptability of cultivars to many regions. The objective of this research was to evaluate soybean performance of early, semi-early and intermediate maturity cultivars in a sandy loam, clayey and very clayey Eutrudox. The experiment was carried out in Naviraí, Mato Grosso do Sul, Brazil, São Francisco Farm, in 2008/2009. The experimental design was the completely randomized block with four replications. The following soybean cultivars were evaluated: a) Early maturity: CD 202, CD 208 and CD 214RR; b) Semi early maturity: BRS 239, BRS CharruaRR and BRS 245RR; and c) Intermediate maturity: CD 219RR and Monsoy 8001. Sowing was conducted over maize residues on November, 15th, 2008 under no tillage system and 333 kg ha-1 of fertilizer was applied. The number of pods per plant, number of grains per pod, weight of 1,000 grains and yield (kg ha-1) were evaluated. Fertilization and maintenance of intermediate-high nutrient levels in Eutrudox soils of any textures provides conditions for soybean cultivars to express high yield potentials. / A soja é a principal cultura do País tanto em volume como em geração de renda, sendo que vários fatores tem contribuído para esse bom desempenho no Brasil, dentre eles podemos citar a adaptabilidade de cultivares ás diferentes regiões produtoras. O objetivo deste trabalho foi de avaliar o desempenho de cultivares de soja dos ciclos precoce, semiprecoce e médio em Latossolos Vermelhos com diferentes classes texturais. Latossolo Vermelho Eutrófico textura média, Latossolo Vermelho Eutrófico textura argilosa e Latossolo Vermelho Eutrófico textura muito argilosa. O experimento foi conduzido no município de Naviraí Estado de Mato Grosso do Sul, na Fazenda São Francisco, na safra agrícola 2008/2009. O delineamento experimental foi de blocos inteiramente casualisados com quatro repetições. Os cultivares de soja utilizados foram: a) Ciclo precoce: CD 202, CD 208 e CD 214RR; b) Ciclo semiprecoce: BRS 239, BRS CharruaRR, BRS 245RR e c) Ciclo médio: CD 219RR e Monsoy 8001. Em todos os solos o plantio ocorreu no dia 15 de novembro de 2008, em sistema de plantio direto sobre a resteva de milho com adubação de 333 kg ha-1. Foram avaliados os parâmetros de número de vagens por planta, número de grãos por vagens, peso de mil grãos e calculado produtividade em quilogramas por hectare. Independente da classe textural, a fertilização e manutenção de médios a altos teores de nutrientes nos LATOSSOLOS VERMELHOS Eutróficos de textura média a muito argilosa possibilita a expressão fenotípica de altas produtividades agrícolas de grãos pelas cultivares de soja.

Made available in DSpace on 2016-07-18T17:51:13Z (GMT). No. of bitstreams: 1 Kezia Aparecida Guidorizi.pdf: 1930989 bytes, checksum: 18ecb1b6355fc654b9a93762b1bcd9bb (MD5) Previous issue date: 2015-06-03 / Abiotic and biotic stresses have a negative effect on the physiological and biochemical processes that are associated with the growth and development of plants. Plants have several strategies to cope with environmental stresses, including expression level and modification of certain genes by introducing epigenetic modifications, such as DNA methylation. DNA methylation patterns in plants are dynamic and some earlier findings support the hypothesis that changes in methylation status of genes are due to the mechanisms of adaptation to environmental stress. In plants the 5-azacytidine is usually used as agent inhibiting methylation of DNA. The use of 5-azacytidine may increase hypomethylation and transcriptional reactivation of the genome of gene silencing and leads to alteration of the growth and development of the plant. The objective of this study was to evaluate in a systematic way the effects of 5-azacytidine in physiological processes of Glycine max (L.) Merrill under water stress. The experiment was conducted in a greenhouse, in a 2x4 factorial design, with two water regimes, 100% and 30% of daily replenishment of water compared to field capacity on the V4 stage, and four forms of 5-azacytidine application solution, seeds, plants in the V5 and R2 fase in seed plants in the V5 and R2 fase and control plants without application of the solution. The following parameters were evaluated: leaf water potential, chlorophyll content index, membrane leakage, photosynthetic potential in growth stages R2 and R5) and biomass yield at the end of the crop cycle. The data were submitted to ANOVA and mean values compared by Tukey test (p<0.05). In addition, a multivariate principal component analysis (PCA) was performed in an attempt to separate the different behavior. The level of disturbance from plants subjected to water stress was sufficient to reduce the values of the water status parameters, CRA, gas exchange and biomass. Plants under 100% daily replacement of the water with the treatment of 5-azacytidine in seed biomass statistically reduced parameters compared with other forms of 5-azac application under same water condition. However, in plants subjected to water stress, 5-azac application increased plant biomass over the plants without application of the product. Plants treated with 5-azacytidine also reduced phenotypic plasticity in relation to the plant without application of 5-azac. The physiological changes such as possible reduction in DNA methylation may have an impact on the average variability and plasticity of the characteristics of plants of Glycine max. These results can be useful for studying the molecular mechanism of DNA methylation induced by 5-azacytidine and establish a base for further studies of the relationship between methylation do DNA and phenotypic plasticity. / Estresses abióticos e bióticos têm efeitos negativos sobre os processos bioquímicos e fisiológicos, que estão associados com o crescimento e desenvolvimento das plantas. Plantas possuem várias estratégias para lidar com estresses ambientais, que incluem nível de expressão e alteração de alguns genes através da introdução de modificações epigenéticas, como a metilação do DNA. Os padrões de metilação do DNA em plantas são dinâmicos, e algumas descobertas anteriores suportam a hipótese de que variações no estado de metilação em genes são devido aos mecanismos de adaptação ao estresse ambiental Em plantas, a 5-azacitidina é normalmente utilizada como agente de inibição da metilação do DNA. O uso da 5-azacitidina pode aumentar a hipometilação e a reativação transcricional de genes e levar a alteração do crescimento e desenvolvimento da planta. O objetivo deste estudo foi avaliar de forma sistêmica os efeitos da 5-azacitidina nos processos fisiológicos de Glycine max (L.) Merril sob deficiência hídrica. O experimento foi conduzido em casa de vegetação, em esquema fatorial 2x4, ou seja, regimes hídricos, 100% e 30% da reposição diária de água em relação à capacidade de campo a partir do estádio V4 e quatro formas de aplicação de solução de 5-azacitidina: em sementes, em folhas no estádio V5 e R2, em sementes + folhas no estádio V5 e R2 e plantas controle sem aplicação da solução citada. Foram avaliados os seguintes parâmetros: potencial de água foliar, índice de conteúdo de clorofila, extravasamento de membrana, potencial fotossintético nos estádios fenológicos R2 e R5 e rendimento de biomassa no final do ciclo da cultura. Os dados foram submetidos à Análise de Variância e os valores médios comparados através do teste Tukey (p=0,05). Além disso, foi realizada uma análise multivariada por componentes principais (PCA) na tentativa de separar os diferentes comportamentos analisados. O nível de perturbação das plantas submetidas à deficiência hídrica foi suficiente para reduzir os valores dos parâmetros do estado hídrico, CRA, trocas gasosas e biomassa. As plantas com 100% da reposição diária de água e dos com 5-azacitidina na semente reduziram estatisticamente os parâmetros de biomassa em relação às outras formas de aplicação de 5-azac na mesma condição hídrica. Entretanto, em plantas submetidas à deficiência hídrica, as formas de aplicação de 5-azac influenciaram positivamente o aumento da biomassa em relação aquelas sem aplicação do produto. Plantas tratadas com 5-azacitidina também reduziram a plasticidade fenotípica em relação com a planta sem aplicação da solução de 5-azac. As mudanças fisiológicas como a possível redução da metilação do DNA, podem ter um impacto sobre a média, variabilidade e plasticidade das características das plantas de Glycine max (L.) Merril. Estes resultados podem ser úteis para estudar o mecanismo molecular da metilação do DNA afetada por 5-azacitidina e estabelecer uma base para um estudo mais aprofundado da relação entre metilação do DNA e plasticidade fenotípica.

O Brasil é o segundo maior produtor de soja (Glycine max, L.), sendo responsável por quase 30% da colheita mundial. Mais de 80% da produção total é destinada à extração de óleo e proteína para a alimentação humana, sendo que o consumo na forma de grão e de produtos industrializados vem crescendo, como alternativa a proteínas de origem animal e devido aos potenciais benefícios à saúde. Grande parte da literatura científica refere-se à soja amarela, enquanto estudos sobre soja preta ainda são escassos. Este projeto visou estudar e comparar as características físicas e de composição química, incluindo os compostos fenólicos e atividade antioxidante da soja preta e amarela, cultivadas no Brasil em condições climáticas e ambientais similares. Fez parte do escopo do projeto avaliar o efeito do cozimento sobre os compostos polifenólicos e a atividade antioxidante. Não foi observada diferença significativa no conteúdo de nutrientes e no perfil de ácidos graxos entre a soja preta e amarela, nem nas características físicas, como massa, capacidade de hidratação e tempo de cozimento dos grãos. Porém, a soja preta apresentou teores muito mais elevados de compostos fenólicos totais (CFT) e de flavonóides em relação à soja amarela. Os teores na soja preta foram, em média, de 4,78 mg eq. ácido gálico/g e de 1,75 mg eq. catequina/g, de CFT e de flavonoides,respectivamente. A soja amarela apresentou em média 40% a menos de CFT e aproximadamente 60% a menos de flavonóides. Ao contrário da soja amarela que é desprovida de antocianinas, na soja preta foi encontrado um teor médio de 0,92 mg eq. cianidina-3-O-glicosídeo/g. Empregando a técnica de HPLC-DAD-MS/MS foram identificadas duas antocianinas, a cianidina-3-O-glicosídeo e a peonidina-3-Oglicosídeo, além da cianidina. A soja preta apresentou atividade antioxidante 70% superior à soja amarela pelo método de DPPH e 50% superior, pelo método de ORAC. O cozimento dos grãos reduziu em pelo menos 40% o teor de compostos fenólicos e a atividade antioxidante, tanto na soja preta como na amarela. Foi observada uma forte correlação entre os teores de CFT, flavonóides e a atividade antioxidante, antes e após o cozimento. Assim, o consumo de soja preta ou mesmo a produção de novos alimentos a partir desses grãos poderá oferecer ao consumidor a possibilidade de diversificar a sua dieta, introduzindo um alimento com a mesma qualidade nutricional da soja amarela e, possivelmente, maiores benefícios à saúde. Vislumbra-se a possibilidade de expansão do cultivo da soja preta, inclusive por pequenos produtores, criando um novo nicho de mercado para um alimento diferenciado. / Brazil is the second largest producer of soybean (Glycine max, L.), accounting for almost 30% of the world\'s production. Over 80% of the total production is destined to oil extraction and protein for human consumption. Soybean and soy-product consumption has been rising as an alternative to animal protein and due to its potential health benefits. Most of the scientific literature refers to yellow soybeans, while studies on black soybeans are still scarce. This project aimed to study and compare black and yellow soybeans, cultivated in Brazil in similar climatic and environmental conditions, as to their physical characteristics and chemical composition, including phenolic compounds and antioxidant activity. The effect of cooking on phenolic compounds and antioxidant activity was also investigated. No significant difference was observed between black and yellow soybeans as to nutrient content, fatty acid composition and physical characteristics, such as seed-weight, hydration capacity and cooking time. However, black soybeans had a much higher content of total phenolic compounds (TPC) and flavonoids than yellow soybeans. TPC and flavonoid contents in black soybeans were 4.78 mg gallic acid equivalents/g and 1.75 mg (+)-catechin equivalents/g, respectively. Yellow soybeans had on average 40% less TPC and approximately 60% less flavonoids. Unlike yellow soybeans which lack anthocyanins, black soybeans were found to have an average content of 0.92 mg cyanidin-3-O-glucoside equivalents/g. High performance liquid chromatography coupled to photodiode array and mass spectrometry detectors (HPLCDADMS/MS) was used to identify two anthocyanins, namely cyanidin-3-Oglucoside and peonidin-3-O-glucoside, and cyanidin. Black soybeans also showed a 70% higher DPPH-free radical scavenging activity than yellow soybeans, and a 50% higher ORAC value. Cooking reduced at least 40% of phenolic compounds and antioxidant capacity in all soybean samples, both black and yellow. High correlations between phenolic compositions and antioxidant activities were observed, before and after cooking. These results suggest that black soybean consumption or new processed food products from these crops may offer consumers an opportunity to diversify their diet with food nutritionally equivalent to yellow soybeans, but possibly with greater health benefits. The possibility of expanding black soybean cultivation, including small-scale production, may create a new market niche for a value-added food product.

Submitted by Michele Mologni (mologni@unoeste.br) on 2018-05-11T19:30:03Z No. of bitstreams: 1 Eduardo Henrique Lima Mazzuchelli.pdf: 447873 bytes, checksum: 244bdede9e8c1f1e8a5f77effa4caa8a (MD5) / Made available in DSpace on 2018-05-11T19:30:03Z (GMT). No. of bitstreams: 1 Eduardo Henrique Lima Mazzuchelli.pdf: 447873 bytes, checksum: 244bdede9e8c1f1e8a5f77effa4caa8a (MD5) Previous issue date: 2016-05-10 / The microbial biomass of the soil is of great importance in the increase of crop productivity and its accompaniment reflects possible changes in the soil, being a good indicator of soil quality. The objective of this work was to evaluate the chemical and biological attributes of the soil and soybean yield succeeding coverage plants. The experiment was conducted in the municipality of Presidente Bernardes-SP, in a soil classified as Dystroferric Red Argisoil. The experimental design was completely randomized. The treatments were constituted by the adoption of cover species, being millet, fallow, seeded, sorghum, corn, pigeon pea and fertilized fallow. The cover plants were kept until the 85th day after sowing and were later withdrawn for silage and the entire area of the experiment left for the regeneration of Urochloa brizantha cv. Marandu and maintained for 160 days, afterwards the soybean was sown. The biomass production of cover and brachiaria plants, foliar and bromatological tissue analyzes were evaluated. Soil was also sampled to evaluate the chemical composition and microbiological characteristics, and evaluations of soy production components. Soybean yield increased after the pre-cultivation of pigeon pea. The biological attributes were influenced significantly by the cultivation of coverage crops, and the pre-cultivation with pigeon peas stood out in the improvement of such attributes. The species used as cover plants and green manure did not alter the chemical attributes of the soil, with the exception of sulfur in depth of 10 - 20 cm. / A biomassa microbiana do solo é de suma importância no aumento de produtividade das culturas e seu acompanhamento reflete possíveis mudanças no solo, sendo uma boa indicadora de qualidade dos solos. O objetivo deste trabalho foi avaliar os atributos químicos e biológicos do solo e produtividade da soja sucedendo plantas de cobertura. O experimento foi conduzido no município de Presidente Bernardes-SP, em um solo classificado como Argissolo Vermelho distroférrico. O delineamento experimental foi em faixas inteiramente casualizadas. Os tratamentos foram constituídos pela adoção de espécies de cobertura, sendo o milheto, pousio, braquiária semeada, sorgo, milho, feijão guandu e pousio adubado. As plantas de cobertura foram mantidas até o 85º dia após a semeadura e posteriormente foram retiradas para a realização de silagem e toda a área do experimento deixada para a regeneração do pasto de Urochloa brizantha cv. Marandu e mantida por 160 dias, posteriomente realizada a semeadura da soja. Foram avaliados a produção de biomassa das plantas de cobertura e da braquiária, análises do tecido foliar e bromatológica. Também foi amostrado o solo para avaliar a composição química e características microbiológicas, e avaliações dos componentes de produção da soja. A produtividade da soja apresentou incremento após o pré-cultivo de feijão guandu. Os atributos biológicos foram influenciados significativamente pelo cultivo de plantas de cobertura, sendo que o pré-cultivo com feijão guandu destacou-se na melhoria de tais atributos. As espécies utilizadas como plantas de cobertura e adubo verde não alteraram os atributos químicos do solo, com exceção do enxofre em profundidade de 10 – 20 cm.

Thorough investigation of the glycine conjugation pathway has been neglected over the last 30 years. Environmental factors, nutrition, and the chronic use of medications are increasing the exposure of humans to benzoate and drugs that are metabolized to acyl-CoA intermediates. Glycine conjugation of mitochondrial acyl-CoAs, catalysed by glycine N-acyltransferase (GLYAT, E.C. 2.3.1.13), is an important metabolic pathway responsible for maintaining adequate levels of free coenzyme A (CoASH). However, because of the small number of pharmaceutical drugs that are conjugated to glycine, the pathway has not yet been characterised in detail. Therefore, one of the objectives of this thesis was to develop a better understanding of glycine conjugation and its role in metabolism. In humans and animals a number of endogenous and xenobiotic organic acids are conjugated to glycine. Glycine conjugation has generally been assumed to be a detoxification mechanism, increasing the water solubility of organic acids in order to facilitate urinary excretion. However, recently it was proposed that the role of the amino acid conjugations, including glycine conjugation, is to regulate systemic levels of amino acids that are also utilised as neurotransmitters in the central nervous systems of animals. The glycine deportation hypothesis was based on the observation that, compared to glucuronidation, glycine conjugation does not significantly increase the water solubility of aromatic acids. A thorough review of the literature for this thesis showed that the major role of glycine conjugation, however, is to dispose of the end products of phenylpropionate metabolism. The review also introduced the new perspective that mitochondrial glycine conjugation prevents the accumulation of benzoate in the mitochondrial matrix by forming hippuric acid a less lipophilic conjugate that can be more readily transported out of the mitochondria. Although organic anion transporters can export benzoate from the matrix, this process would likely be futile because benzoic acid can simply diffuse back into the matrix. Hippurate, however, is significantly less lipophilic and therefore less capable of diffusing into the matrix. It is therefore not the transport out of the mitochondrial matrix that is facilitated by glycine conjugation, but rather the ability of the glycine conjugates to re-enter the matrix that is decreased. Lastly, glycine conjugation of benzoate also exacerbates the dietary deficiency of glycine in humans. Because the resulting shortage of glycine can negatively influence brain neurochemistry and the synthesis of collagen, nucleic acids, porphyrins, and other important metabolites, the risks of using benzoate as a preservative should not be underestimated. To date, no defect of the glycine conjugation pathway has been reported and this, together with the fact that GLYAT plays an important role in hepatic metabolism, suggests that this pathway is essential for survival. GLYAT activity affects mitochondrial ATP production, glycine availability, CoASH availability and the toxicity of various organic acids. Therefore, variation in the glycine conjugation pathway could influence liver cancer, musculoskeletal development and mitochondrial energy metabolism. Significant interindividual variation exists in glycine conjugation capacity. The molecular basis for this variability is not known. The main aim of this thesis was to investigate and characterise the genetic variation in the coding region of the GLYAT gene. This was accomplished by firstly, investigating the influence of non-synonymous single nucleotide polymorphisms (SNPs) on the enzyme activity of a recombinant human GLYAT and secondly, by analysing the level of genetic variation in the coding region of the GLYAT gene using existing worldwide population data. To investigate the influence of non-synonymous SNPs in the GLYAT gene on the enzyme activity, a recombinant human GLYAT was prepared, and characterised. Site-directed mutagenesis was used to generate six variants of the enzyme (K16N; S17T; R131H; N156S; F168L; R199C). The variants were expressed, purified, and enzymatically characterised. The enzyme activities of the K16N, S17T and R131H variants were similar to that of the wild-type, whereas the N156S variant was more active, the F168L variant less active, and the R199C variant was inactive. The results showed that SNP variations in the human GLYAT gene can influence the kinetic properties of the enzyme. The genetic variation data of the human GLYAT open reading frame (ORF) available on public databases was investigated by formulating the hypothesis that due to the essential nature of the glycine conjugation pathway, the genetic variation in the ORF of the GLYAT gene should be low and that deleterious alleles will be found at low frequencies. Data from the i) 1000 Genome Project, ii) the HapMap Project, and iii) the Khoi-San/Bantu Sequencing Project was downloaded from available databases. Sequence data of the coding region of a small cohort of South African Afrikaner Caucasian individuals was also generated and included in the analyses. In the GLYAT ORF of the 1537 individuals analysed, only two haplotypes (S156 and T17S156) out of 14 haplotypes were identified in all populations as having the highest haplotype frequencies (70% and 20% respectively). The S156C199 and S156H131 haplotypes, which have a deleterious effect on the enzyme activity of a recombinant human GLYAT, were detected at very low frequencies. The results of this study indicated that the GLYAT ORF is remarkably conserved, which supports the hypothesis that the glycine conjugation pathway is an essential detoxification pathway. The findings presented in this thesis highlight the importance that future investigations should determine the in vivo capacity of the glycine conjugation pathway for the detoxification of benzoate and other xenobiotics. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2015

Thorough investigation of the glycine conjugation pathway has been neglected over the last 30 years. Environmental factors, nutrition, and the chronic use of medications are increasing the exposure of humans to benzoate and drugs that are metabolized to acyl-CoA intermediates. Glycine conjugation of mitochondrial acyl-CoAs, catalysed by glycine N-acyltransferase (GLYAT, E.C. 2.3.1.13), is an important metabolic pathway responsible for maintaining adequate levels of free coenzyme A (CoASH). However, because of the small number of pharmaceutical drugs that are conjugated to glycine, the pathway has not yet been characterised in detail. Therefore, one of the objectives of this thesis was to develop a better understanding of glycine conjugation and its role in metabolism. In humans and animals a number of endogenous and xenobiotic organic acids are conjugated to glycine. Glycine conjugation has generally been assumed to be a detoxification mechanism, increasing the water solubility of organic acids in order to facilitate urinary excretion. However, recently it was proposed that the role of the amino acid conjugations, including glycine conjugation, is to regulate systemic levels of amino acids that are also utilised as neurotransmitters in the central nervous systems of animals. The glycine deportation hypothesis was based on the observation that, compared to glucuronidation, glycine conjugation does not significantly increase the water solubility of aromatic acids. A thorough review of the literature for this thesis showed that the major role of glycine conjugation, however, is to dispose of the end products of phenylpropionate metabolism. The review also introduced the new perspective that mitochondrial glycine conjugation prevents the accumulation of benzoate in the mitochondrial matrix by forming hippuric acid a less lipophilic conjugate that can be more readily transported out of the mitochondria. Although organic anion transporters can export benzoate from the matrix, this process would likely be futile because benzoic acid can simply diffuse back into the matrix. Hippurate, however, is significantly less lipophilic and therefore less capable of diffusing into the matrix. It is therefore not the transport out of the mitochondrial matrix that is facilitated by glycine conjugation, but rather the ability of the glycine conjugates to re-enter the matrix that is decreased. Lastly, glycine conjugation of benzoate also exacerbates the dietary deficiency of glycine in humans. Because the resulting shortage of glycine can negatively influence brain neurochemistry and the synthesis of collagen, nucleic acids, porphyrins, and other important metabolites, the risks of using benzoate as a preservative should not be underestimated. To date, no defect of the glycine conjugation pathway has been reported and this, together with the fact that GLYAT plays an important role in hepatic metabolism, suggests that this pathway is essential for survival. GLYAT activity affects mitochondrial ATP production, glycine availability, CoASH availability and the toxicity of various organic acids. Therefore, variation in the glycine conjugation pathway could influence liver cancer, musculoskeletal development and mitochondrial energy metabolism. Significant interindividual variation exists in glycine conjugation capacity. The molecular basis for this variability is not known. The main aim of this thesis was to investigate and characterise the genetic variation in the coding region of the GLYAT gene. This was accomplished by firstly, investigating the influence of non-synonymous single nucleotide polymorphisms (SNPs) on the enzyme activity of a recombinant human GLYAT and secondly, by analysing the level of genetic variation in the coding region of the GLYAT gene using existing worldwide population data. To investigate the influence of non-synonymous SNPs in the GLYAT gene on the enzyme activity, a recombinant human GLYAT was prepared, and characterised. Site-directed mutagenesis was used to generate six variants of the enzyme (K16N; S17T; R131H; N156S; F168L; R199C). The variants were expressed, purified, and enzymatically characterised. The enzyme activities of the K16N, S17T and R131H variants were similar to that of the wild-type, whereas the N156S variant was more active, the F168L variant less active, and the R199C variant was inactive. The results showed that SNP variations in the human GLYAT gene can influence the kinetic properties of the enzyme. The genetic variation data of the human GLYAT open reading frame (ORF) available on public databases was investigated by formulating the hypothesis that due to the essential nature of the glycine conjugation pathway, the genetic variation in the ORF of the GLYAT gene should be low and that deleterious alleles will be found at low frequencies. Data from the i) 1000 Genome Project, ii) the HapMap Project, and iii) the Khoi-San/Bantu Sequencing Project was downloaded from available databases. Sequence data of the coding region of a small cohort of South African Afrikaner Caucasian individuals was also generated and included in the analyses. In the GLYAT ORF of the 1537 individuals analysed, only two haplotypes (S156 and T17S156) out of 14 haplotypes were identified in all populations as having the highest haplotype frequencies (70% and 20% respectively). The S156C199 and S156H131 haplotypes, which have a deleterious effect on the enzyme activity of a recombinant human GLYAT, were detected at very low frequencies. The results of this study indicated that the GLYAT ORF is remarkably conserved, which supports the hypothesis that the glycine conjugation pathway is an essential detoxification pathway. The findings presented in this thesis highlight the importance that future investigations should determine the in vivo capacity of the glycine conjugation pathway for the detoxification of benzoate and other xenobiotics. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2015

An alcohol and anesthetic binding pocket is hypothesized to exist among transmembrane domains of the α1 glycine receptor (GlyR). Prior work has shown that amino acid residue serine-267 plays a significant role in the enhancing effects of alcohol and anesthetics and is theorized to form part of an alcohol and anesthetic binding cavity among subunit transmembrane domains. Propyl methanethiosulfonate (PMTS), an alcohol-like thiol, was previously shown to bind to a cysteine residue introduced at position 267 (S267C) and this resulted in permanent enhancement of GlyR function. If ethanol is binding to residue 267 in wildtype GlyR to potentiate receptor function then we hypothesized that covalent thiol labeling would produce receptor enhancement by the same mechanisms as ethanol. Using outside-out patch single channel electrophysiology we determined the open and closed dwell-times and burst properties of S267C GlyR in the absence and presence of PMTS. The primary consequence of PMTS binding to S267C GlyR was an increase in the lengths of burst durations, paralleling the main effect of ethanol on wildtype GlyR. Our findings thus provide a new line of evidence suggesting that ethanol is exerting its enhancing effects on the GlyR through its interactions with amino acid residue 267 in the second transmembrane domain. / text

No description available.

No description available.

Glycine-N-acyltransferase (GLYAT, EC 2.3.1.13) has been characterised in a number of species including: humans, chimpanzees, rhesus monkeys and bovines. The characterisation of GLYAT from various species contributes to a better understanding of the diversity of the enzyme which in turn might help improve the current understanding of detoxification in mammals. The GLYAT enzyme of both the chacma baboon and vervet monkey has not been characterised. In this project, tissue samples were obtained from a chacma baboon (Papio ursinus) and a vervet monkey (Chlorocebus pygerythrus) to determine the nucleic acid sequence that encodes GLYAT in these two species to broaden our current understanding on the diversity of GLYAT in primates. A liver of a chacma baboon was used to extract total RNA. Complementary DNA (cDNA) was synthesised using an oligo (dT) primer. An open reading frame (ORF) encoding GLYAT of the chacma baboon was amplified with a PCR (polymerase chain reaction) using primers designed from a human GLYAT transcript. The PCR product containing an ORF encoding GLYAT of the chacma baboon was cloned, sequenced and expressed. The recombinant GLYAT of the chacma baboon expressed well in bacteria, but was insoluble and did not have enzyme activity. A crude cytoplasmic extract was prepared from the liver of a chacma baboon. The objective was to compare enzyme activity between the native and recombinant GLYAT. The prepared liver extract from the chacma baboon was assayed for enzyme activity and compared to the activity in a liver extract from bovine, previously prepared by Ms M Snyders. Both the chacma baboon and bovine liver extracts had GLYAT enzyme activity. To obtain sequence information on vervet monkey GLYAT, leukocytes were isolated from blood obtained from a living vervet monkey. A human GLYAT gene sequence was used as a reference DNA sequence in the design of PCR primers that were used to amplify the exons of GLYAT of the vervet monkey. All six GLYAT exons were individually amplified and PCR products were sequenced. The sequences were combined to reconstruct an ORF encoding GLYAT of the vervet monkey. The ORFs coding the GLYAT of both chacma baboon and vervet monkey were found to be 888 bp long (excluding stop codon) and encoded a protein of 296 amino acids. A fragment of 1256 bp of the chacma baboon GLYAT transcript was sequenced. The two GLYAT ORF sequences were translated to amino acid sequences and aligned to that of GLYAT of primates obtained from the Ensembl sequence database. The GLYAT amino acid sequences of the chacma baboon, vervet monkey and rhesus monkey formed a related group, distinct from other primates. The chacma baboon and vervet monkey sequences were 99 % identical to the rhesus monkey sequence and 92.6 % identical to the human sequence. There were 4 new variations introduced by GLYAT amino acid sequences from the chacma baboon and the vervet monkey. The vervet monkey introduced an isoleucine in place of a valine at position 32 and an arginine in place of a histidine or glutamine at position 224. The chacma baboon introduced a tyrosine in place of isoleucine at position 201 and an arginine in place of histidine or glutamine at position 240. The knowledge generated in this project will broaden the understanding of GLYAT diversity relating to GLYAT in primates. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011