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Humans are continuously challenged by harmful endogenous and xenobiotic substances. Detoxification is the ability to neutralise and remove these substances from the body. Glycine N-acyltransferase, EC 2.3.1.13 (GLYAT) is a key enzyme in detoxification. GLYAT catalyses an amino acid (glycine) conjugation reaction in phase II of detoxification. It is expected that, similar to what has been observed in the Cytochrome P450 enzymes, variations within the GLYAT gene may lead to altered enzyme activity that may affect the efficacy of detoxification. The aim of this study was to identify genetic variations within the GLYAT gene of a cohort of individuals whose GLYAT activity has been biochemically characterized. Biochemical profiles of phase I and II detoxification of a number of individuals was screened to select those with possible aberrant GLYAT activity. Eighteen selected individuals agreed to participate in the study. The 23.21 kb GLYAT gene of the participants was amplified in four fragments and sent for pyrosequencing (Roche GS FLX titanium) at Inqaba Biotec. The results were analysed with the Lasergene software package from DNAStar (Madison, Wisconsin, USA). A total of 94 variations were identified from the Next Generation Sequencing data. Of these three found in the exons were known variations and four variations located in the exons were novel. A total of 62 known and 25 novel variations were identified in the introns of the GLYAT gene. Sanger sequencing verified 70.29% (68 in total) of the variation, which included 12 novel variations, of which one is located in exon six. Real-time quantitative PCR (qPCR) experiments were conducted and the data analysed using CopyCaller software to identify copy number variations within the cohort. It was found that participant 17 may have multiple copies of parts of the 3-terminal end of the gene (exons five and six), which might have an effect on GLYAT activity. Variations could possibly affect GLYAT activity, but the data was inconclusive and must be confirmed. Some of the variations could possibly affect GLYAT activity, but no correlation could be made between the variations identified during this study and the cohort’s detoxification ability. Further studies needs to be conducted to establish the effect of the variations in combination with one another on GLYAT activity. If some of these variations affect GLYAT activity such data might shed some light on variations observed between the glycine conjugation ability of individuals. Such information could eventually be of value in treatment of inborn errors of metabolism. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

Humans are continuously challenged by harmful endogenous and xenobiotic substances. Detoxification is the ability to neutralise and remove these substances from the body. Glycine N-acyltransferase, EC 2.3.1.13 (GLYAT) is a key enzyme in detoxification. GLYAT catalyses an amino acid (glycine) conjugation reaction in phase II of detoxification. It is expected that, similar to what has been observed in the Cytochrome P450 enzymes, variations within the GLYAT gene may lead to altered enzyme activity that may affect the efficacy of detoxification. The aim of this study was to identify genetic variations within the GLYAT gene of a cohort of individuals whose GLYAT activity has been biochemically characterized. Biochemical profiles of phase I and II detoxification of a number of individuals was screened to select those with possible aberrant GLYAT activity. Eighteen selected individuals agreed to participate in the study. The 23.21 kb GLYAT gene of the participants was amplified in four fragments and sent for pyrosequencing (Roche GS FLX titanium) at Inqaba Biotec. The results were analysed with the Lasergene software package from DNAStar (Madison, Wisconsin, USA). A total of 94 variations were identified from the Next Generation Sequencing data. Of these three found in the exons were known variations and four variations located in the exons were novel. A total of 62 known and 25 novel variations were identified in the introns of the GLYAT gene. Sanger sequencing verified 70.29% (68 in total) of the variation, which included 12 novel variations, of which one is located in exon six. Real-time quantitative PCR (qPCR) experiments were conducted and the data analysed using CopyCaller software to identify copy number variations within the cohort. It was found that participant 17 may have multiple copies of parts of the 3-terminal end of the gene (exons five and six), which might have an effect on GLYAT activity. Variations could possibly affect GLYAT activity, but the data was inconclusive and must be confirmed. Some of the variations could possibly affect GLYAT activity, but no correlation could be made between the variations identified during this study and the cohort’s detoxification ability. Further studies needs to be conducted to establish the effect of the variations in combination with one another on GLYAT activity. If some of these variations affect GLYAT activity such data might shed some light on variations observed between the glycine conjugation ability of individuals. Such information could eventually be of value in treatment of inborn errors of metabolism. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

A range-wide collection of Juglans regia seeds was undertaken in autumn 1997 from 12 countries, including 25 provenances and 375 half-sib progenies. 2200 seedlings were produced using innovative nursery techniques. The seedlings were planted in three provenance trials in southern England in 1999, the largest of which acted as a combined provenance/progeny trial. After one growing season, survival was 98.9 %, mean height growth 35 cm, and mean stem diameter increment 5 mm. Provenance differences for both height and stem diameter increment were highly significant (p<0.001). There were no significant genotype × environment interactions. Flushing assessments revealed few significant differences between provenances and flushing was complete by early April. Family heritability for tree height was 0.19 at one site and, with combined selection, genetic gain was estimated at 8 %. The effects of three types of treeshelter and a stumping treatment on walnut establishment were tested over three growing seasons. Treeshelters were found beneficial to height increment. However, 120 cm tall shelters promoted early flushing, and consequent risk of increased frost damage, and caused more stem die-back than 75 cm shelters. Stumping promoted rapid early height increment but gave no longer-term benefit. The crown (cd) and stem (dbh) diameter at breast height relationship of open growing trees in Britain was assessed and was highly significant (r2 = 0.96, p<0.001). The regression equation (cd = 2.71 + 17.6dbh) permitted the estimation of suitable planting densities for the provenance trials and the calculation of a thinning regime. Isozyme analysis of the 375 genotypes identified 20 loci in 15 enzyme systems with seed embryo extracts. Using young leaf extracts, the polymorphic locus Pgm-1 indicated low expected heterozygosity of 0.06 both within populations and at the species level. FST and GST estimates, both 0.05, indicated high uniformity among populations. Genetic distance estimates did not identify significant clustering consistent with geographic origin.

Twenty-two natural populations of the red shiner minnow, Notropis lutrensis were examined for morphological and genetic variation. This research was aimed at testing the hypothesis that morphological and genetic variation was primarily influenced by the degree of gene flow between populations. Ten linear measurements were taken from each of 1320 specimens. Morphological characters were adjusted for differential growth by least squares linear regression techniques. Genetic variability was estimated for each individual red shiner through the methods of starch gel electrophoresis. Twenty presumtive gene loci were resolved.

Neotoma micropus from Jack County, Texas, were studied over a 9-month period. Loci from blood and saliva were used to determine genetic variation within the population. Deviations from Hardy-Weinberg equilibrium were found at one locus. The average temporal F over all seven loci was 0.040. Genetic structuring was subtle, fluctuated on a seasonal basis, and was due to differential migration or predation on genotypes. Heterozygotes tended to move more than homozygotes, and a greater proportion of heterozygotes were lost from the population during each season. Genetic variation was maintained in the population by immigrant individuals. This differential in dispersal of genotypes fits current models of reorganization within the genome of populations.

Genetic variation was examined at 19 loci encoding enzymatic and general proteins Notropis lutrensis from the Brazos River in Texas. The thermal regime of the Brazos River below Possum Kingdom Reservoir is altered due to the release of water from the hypolimnion. Summer water temperatures fluctuate as much as 7^oC. Levels of heterozygosity at the malate dehydrogenase-2 locus were correlated with the degree of water temperature fluctuation at each locality. The isozymes from three homozygous patterns of supernatant malate dehydrogenase (Mdh-l, Mdh-2) exhibited different activities at different experimental temperatures.

M. Tech. (Biotechnology, Department of Health Sciences), Vaal University of Technology / Amaranthus tricolor (A. tricolor) is a nutritious vegetable crop that is used as a subsistence and cash crop in the rural areas in Africa. Its yield and production is severely limited by abiotic stresses such as drought. Mutation technology, using gamma irradiation, was previously employed as a tool to create genetic variation in order to select for lines with improved drought-tolerance. During irradiation, 160 Gy (Gray) was selected as the optimal dosimetry that allowed subsequent seed germination. The resulting mutant lines were screened over several generations under field and greenhouse conditions and seven promising drought-tolerant lines were selected. Here we report on physiological and morphological studies of two of these Amaranthus mutant lines (#2 and #5) to confirm the enganced drought-tolerance. Plants were grown in the greenhouse in plastic pots containing germination mix with fertiliser. They were exposed to 21 days of well-watered condition, 19 days of drought-stress conditions and 7 days of re-watering. shoot height, leaf area, protein content and relative water content (RWC) of the fresh and dry material were determined colorimetrically under well-watered and drought-stress conditions, while anthocyanin was only measured during well-watered conditions. Shoot height, leaf area, number of leaves per plant and the protein content were significantly reduced under water-stress conditions. Under well-watered condition mutant #5 grew faster with the shoot length significantly higher than mutant #2 and the wild type. Even though drought adversely affected shoot lenght, mutant#5 still performed better than mutant #2 and the wild type under drought-stress conditions. While under both well-watered and drought-stress conditions, the wild type plants had bigger leaf area compared to the two mutant lines. After 16 days of drought-stress conditions, all the leaves of the wild type plants were dried out, as a result no wild type plants recovered after 8 days re-watering. Meanwhile, both mutant #2 and #5 plants recovered significantly after 8 days of re-watering. The wild type was tolerant compared to the two mutant lines. Protein content for mutant #2 plants was higher under both well-watered and drought-stress conditions but was not significantly different from mutant #5 plants compared to the wild type plants after 19 days of drought-stress conditions. Furthermore, genetic diversity was examined in all the Amaranthus lines using random amplified polymorphic DNA (RAPD) analysis. Nineteen arbitrary RAPD markers were used of which two detected polymorphisms (OPA) 07 and OPA 16).

Made available in DSpace on 2017-07-21T19:59:57Z (GMT). No. of bitstreams: 1 Lucia Hoffmann.pdf: 1860358 bytes, checksum: d1a047a5d9cc402bd848a17fc42409b4 (MD5) Previous issue date: 2012-12-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Apolipoprotein B (apoB) is the principal protein from low density lipoprotein (LDL) involved in cholesterol metabolism and transport. Single nucleotide polymorphisms (SNPs) in the human apoB gene have been associated with cardiovascular disease risk, such as Coronary Artery Disease (CAD), dyslipidemia and atherosclerosis. These multifactorial diseases are generated by interaction between environmental and genetic factors. Previous works investigated the genetic causative components of these diseases and focused mainly on polymorphisms that occur in genes encoding structural proteins and enzymes related to lipid profile. The present study aimed to investigate the MspI polymorphism in the apoB gene association with cardiovascular risk factors in a case-control patients group from the Campos Gerais region (Paraná, Brazil) population, moreover to determine this polymorphism allele and genotype frequencies, and also to associate the obtained genetic data with cardiac risk related variables by using correlation analysis. We evaluated 66 patients, from which 55 patients formed the cardiovascular risk group, and 11 others composed the group without risk – control. The patients mean age was 60 ± 10.0 years in the group with risk and 53 ± 14.0 years in the control group. The MspI polymorphism (exon 26) on apoB gene was characterized by PCR-RFLP and, after restriction, DNA fragments were identified by electrophoresis using agarose gel. The normal allele was named M1 and the mutated allele was called M2. The obtained values for MspI polymorphism genotypic frequencies were 0.82, 0.09, 0.09 for the control group, and 0.76, 0.24, 0.00 for the CAD risk group, respectively for genotypes M1M1, M1M2, M2M2. It was showed that the M1M1 homozygous was dominant in the genotype distribution for both groups. There was no significant difference between the allele and genotype frequencies for MspI polymorphism in the apoB gene (2 = 5.90, P > 0.05; Contingency Test) when comparing the two groups. Therefore, the distribution of the studied polymorphism in the Campos Gerais region population presented to be in Hardy-Weinberg equilibrium both for the control group and for the CAD risk patients group, keeping stable the polymorphism, what was already previously reported for Chinese and Korean populations. The Pearson correlation analysis performed between the risk variables (age, diabetes mellitus, high LDL, high triglycerides, low HDL, hypothyroidism, weight, body mass index) showed correlation between the most of the variables for M1M1 and M1M2 genotypes. But for the total population (n = 66), the association of the apoB gene polymorphism was not correlated with cardiac risk variables. In the end, this research presented data for understanding the association of apoB gene polymorphism with lipid metabolism disorder in the development of risk factors for cardiovascular disease in the population of Campos Gerais (PR). / A apolipoproteína B (apoB) é a principal proteína da lipoproteína de baixa densidade (LDL) que está envolvida no transporte e metabolismo do colesterol. Polimorfismos de único nucleotídeo (SNPs) no gene da apoB humana têm sido associados ao risco para doenças cardiovasculares, como a Doença Arterial Coronariana (DAC), as dislipidemias e a aterosclerose. Estas doenças são caracterizadas como multifatoriais, causadas pela interação entre fatores ambientais e genéticos. Estudos de investigação dos componentes genéticos causadores destas doenças têm focado principalmente em polimorfismos nos genes que codificam proteínas estruturais e enzimas relacionadas ao perfil lipídico. Este trabalho teve como objetivo investigar a associação do polimorfismo MspI no gene da apoB com os fatores de risco cardiovascular em um grupo de pacientes caso-controle na população da Região dos Campos Gerais (Paraná, Brasil), além de determinar as frequências alélicas e genotípicas deste polimorfismo, e associar os dados genéticos obtidos com as variáveis relacionadas aos riscos cardíacos através da análise de correlação. Foram avaliados 66 pacientes, dos quais 55 compuseram o grupo com risco cardiovascular, e os 11 demais formaram o grupo sem risco – controle. A idade média dos pacientes foi de 60 ± 10,0 anos no grupo com risco e 53 ± 14,0 anos no grupo controle. O polimorfismo MspI (exon 26) no gene da apoB foi caracterizado por PCR-RFLP, sendo os fragmentos de DNA, após a restrição, identificados por eletroforese em gel de agarose. O alelo normal foi denominado M1 e o alelo mutado M2. As frequências genotípicas encontradas para o polimorfismo MspI foram 0,82, 0,09 e 0,09 para o grupo controle; e 0,76, 0,24 e 0,00 para o grupo de pacientes com risco, respectivamente para os genótipos M1M1, M1M2 e M2M2. O homozigoto M1M1 foi predominante na distribuição genotípica para ambos os grupos. Não houve diferença significativa entre as frequências genotípicas e alélicas do polimorfismo MspI no gene da apoB (2 = 5,90; P > 0,05; Teste de Contingência), quando comparados os 2 grupos em estudo. Assim, a distribuição do polimorfismo estudado na população da região dos Campos Gerais (PR) se mostrou no equilíbrio de Hardy-Weinberg, tanto no grupo controle quanto no grupo de pacientes com risco cardíaco, mantendo o polimorfismo estável, sendo estes resultados também relatados para as populações chinesa e coreana. Na análise de correlação de Pearson entre as variáveis de risco (idade, diabetes mellitus, LDL alto, triglicerídios alto, HDL baixo, hipotireoidismo, peso, índice de massa corporal), observou-se que a maioria das variáveis analisadas se correlacionaram para os genótipos M1M1 e M1M2. Já para a população total estudada (n = 66), a associação do polimorfismo gênico da apoB não apresentou correlação com as variáveis de risco cardíaco. Enfim, esta pesquisa apresentou dados para o entendimento da associação do polimorfismo do gene da apoB com o distúrbio de metabolismo lipídico no desenvolvimento de fatores de risco para doenças cardiovasculares na população dos Campos Gerais (PR).