Despite being the first biochemical reaction to be discovered, the glycine conjugation pathway remains poorly characterised. It has generally been assumed that glycine conjugation serves to increase the water solubility of organic acids, such as benzoic acid and isovaleric acid, in order to facilitate urinary excretion of these compounds. However, it was recently suggested that the conjugation of glycine to benzoate should be viewed as a neuroregulatory process that prevents the accumulation of glycine, a neurotransmitter, to toxic levels. The true importance of glycine conjugation in metabolism is therefore not well understood. However, no genetic defect of glycine conjugation has ever been reported. This seems to suggest that glycine conjugation is a fundamentally important metabolic process, whatever its function may be. Therefore, a major objective of this thesis was to develop a deeper understanding of glycine conjugation and its metabolic significance. A review of the literature on GLYAT and glycine conjugation suggested that the primary purpose of glycine conjugation is indeed to detoxify benzoate and other aromatic acids of dietary origin. However, the commonly held assumption, that glycine conjugation increases the water solubility of aromatic acids in order to facilitate urinary excretion, seems to be incorrect. A better explanation for the detoxification of benzoate by means of glycine conjugation is based on hydrophilicity, not water solubility. Because of its lipophilic nature, benzoic acid is capable of passively diffusing across the mitochondrial inner membrane into the matrix space, where it accumulates due to the pH gradient over the inner membrane. Although benzoate can be exported from the matrix by organic anion transporters, 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 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. The conversion of benzoate to hippurate is a two-step process. First, benzoate is activated by an ATP-dependent acid:CoA ligase (ACSM2A) to form the more reactive benzoyl-CoA. Second, glycine N-acyltransferase (GLYAT) catalyses the formation of hippurate and CoASH from benzoyl-CoA and glycine. Another major objective of this thesis was to gain a better understanding of the structure and function of the GLYAT enzyme. While the substrate selectivity and enzyme kinetics of GLYAT have been investigated to some extent, almost nothing has been published on the structure, active site, or catalytic mechanism of GLYAT. Furthermore, while interindividual variation in the rate of glycine conjugation has been reported by several researchers, it is not known if, or how, genetic variation in the human GLYAT gene contributes to this interindividual variation. To address these issues, systems for the bacterial expression of recombinant bovine GLYAT and recombinant human GLYAT were developed. Because no crystal structure of GLYAT has been reported, homology modelling was used to generate a molecular model of bovine GLYAT. By comparing the molecular model to other acyltransferases for which the catalytic residues were known, Glu227 of bovine GLYAT was identified as a potential catalytic residue. Site directed mutagenesis was used to generate an E227Q mutant recombinant bovine GLYAT lacking the proposed catalytic residue. Characterisation of this mutant suggested that Glu227 was indeed the catalytic residue, and the GLYAT catalytic mechanism was elucidated. The molecular model was also used to identify Asn131 of bovine GLYAT as a potential active site residue. Site-directed mutagenesis was used to generate an N131C mutant, which was sensitive to inhibition by the sulfhydryl reagent DTNB. This suggests that the Asn131 residue of bovine GLYAT may be situated in the active site of bovine GLYAT, but more work is needed to confirm this result. Finally, site-directed mutagenesis was used to generate variants of recombinant human GLYAT corresponding to six of the known SNPs in the human GLYAT gene. Expression and characterisation of the recombinant human GLYAT variants revealed that the enzyme activity and KM (benzoyl-CoA) parameter of the recombinant human GLYAT were influenced by SNPs in the human GLYAT gene. This suggests that genetic variation in the human GLYAT gene could partly explain the interindividual variation in the rate of glycine conjugation observed in humans. Interestingly, the SNPs that negatively influenced enzyme activity also had low allele frequencies, suggesting that there may be some selective advantage to having high GLYAT activity. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014

Despite being the first biochemical reaction to be discovered, the glycine conjugation pathway remains poorly characterised. It has generally been assumed that glycine conjugation serves to increase the water solubility of organic acids, such as benzoic acid and isovaleric acid, in order to facilitate urinary excretion of these compounds. However, it was recently suggested that the conjugation of glycine to benzoate should be viewed as a neuroregulatory process that prevents the accumulation of glycine, a neurotransmitter, to toxic levels. The true importance of glycine conjugation in metabolism is therefore not well understood. However, no genetic defect of glycine conjugation has ever been reported. This seems to suggest that glycine conjugation is a fundamentally important metabolic process, whatever its function may be. Therefore, a major objective of this thesis was to develop a deeper understanding of glycine conjugation and its metabolic significance. A review of the literature on GLYAT and glycine conjugation suggested that the primary purpose of glycine conjugation is indeed to detoxify benzoate and other aromatic acids of dietary origin. However, the commonly held assumption, that glycine conjugation increases the water solubility of aromatic acids in order to facilitate urinary excretion, seems to be incorrect. A better explanation for the detoxification of benzoate by means of glycine conjugation is based on hydrophilicity, not water solubility. Because of its lipophilic nature, benzoic acid is capable of passively diffusing across the mitochondrial inner membrane into the matrix space, where it accumulates due to the pH gradient over the inner membrane. Although benzoate can be exported from the matrix by organic anion transporters, 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 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. The conversion of benzoate to hippurate is a two-step process. First, benzoate is activated by an ATP-dependent acid:CoA ligase (ACSM2A) to form the more reactive benzoyl-CoA. Second, glycine N-acyltransferase (GLYAT) catalyses the formation of hippurate and CoASH from benzoyl-CoA and glycine. Another major objective of this thesis was to gain a better understanding of the structure and function of the GLYAT enzyme. While the substrate selectivity and enzyme kinetics of GLYAT have been investigated to some extent, almost nothing has been published on the structure, active site, or catalytic mechanism of GLYAT. Furthermore, while interindividual variation in the rate of glycine conjugation has been reported by several researchers, it is not known if, or how, genetic variation in the human GLYAT gene contributes to this interindividual variation. To address these issues, systems for the bacterial expression of recombinant bovine GLYAT and recombinant human GLYAT were developed. Because no crystal structure of GLYAT has been reported, homology modelling was used to generate a molecular model of bovine GLYAT. By comparing the molecular model to other acyltransferases for which the catalytic residues were known, Glu227 of bovine GLYAT was identified as a potential catalytic residue. Site directed mutagenesis was used to generate an E227Q mutant recombinant bovine GLYAT lacking the proposed catalytic residue. Characterisation of this mutant suggested that Glu227 was indeed the catalytic residue, and the GLYAT catalytic mechanism was elucidated. The molecular model was also used to identify Asn131 of bovine GLYAT as a potential active site residue. Site-directed mutagenesis was used to generate an N131C mutant, which was sensitive to inhibition by the sulfhydryl reagent DTNB. This suggests that the Asn131 residue of bovine GLYAT may be situated in the active site of bovine GLYAT, but more work is needed to confirm this result. Finally, site-directed mutagenesis was used to generate variants of recombinant human GLYAT corresponding to six of the known SNPs in the human GLYAT gene. Expression and characterisation of the recombinant human GLYAT variants revealed that the enzyme activity and KM (benzoyl-CoA) parameter of the recombinant human GLYAT were influenced by SNPs in the human GLYAT gene. This suggests that genetic variation in the human GLYAT gene could partly explain the interindividual variation in the rate of glycine conjugation observed in humans. Interestingly, the SNPs that negatively influenced enzyme activity also had low allele frequencies, suggesting that there may be some selective advantage to having high GLYAT activity. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014

The aim of this study is to confirm whether illegal immigration to South Africa is being regarded as a threat to national security and to analyse the implications thereof. The dissertation presents three assumptions to be explored and tested, namely: <ul> <li>Illegal immigration poses a growing threat to South Africa’s national security. The latter is due to an increase in the number of illegal immigrants entering the country as well as their seemingly increasing involvement in especially violent crime.</li> <li>The South African government does not adequately acknowledge the threat potential of illegal immigration with regard to the country’s national security status and as such adequate measures are not being taken to curb the influx of illegal immigrants.</li> <li>Violent attacks on foreigners in South Africa have been seen as xenophobia, however, specific factors exist that contributed to these attacks.</li> </ul> The study is conducted and contextualised in terms of the theory of national security, more specifically as it applies to developing countries. It aims to analyse the security implications of illegal immigration for South Africa, with specific reference to the involvement of foreigners in crime in South Africa and also focus on the political implications, more especially the role of foreigners in fomenting political violence. The study accepts the notion that the phenomenon is multi-dimensional and that it cannot be divorced from its socio-economic implications. Reference is therefore made to the political, social, economic and crime dimensions. The dissertation also focuses on the official and public views on illegal immigration and analyse measures adopted by the South African government to address the issue. The study predominantly focuses on the period 1994 to 2008 in an effort to demonstrate the extent of the increase in the number of illegal immigrants to South Africa following the advent of the new dispensation. It further attempts to ascertain whether this increase could be linked to the rise in incidences of violent crime in South Africa during the period in question. AFRIKAANS : Die doel van die studie is om te bepaal of onwettige immigrasie na Suid Afrika toenemend beskou word as ‘n bedreiging vir nasionale veiligheid en, tweedens, om die implikasies daarvan te analiseer. Die studie stel drie aannames voor vir toetsing en verdere bespreking, naamlik: <ul> <li>Onwettige immigrasie bedreig Suid Afrika se nasionale veiligheid toenemend. Laasgenoemde stelling is gebaseer op die toename in die getal onwettige immigrante wat Suid Afrika binnekom, sowel as aan die betrokkenheid van onwettige immigrante in misdaad (veral geweldadige misdade) in Suid Afrika.</li> <li>Die Suid Afrikaanse regering beskou die potensiële bedreiging van onwettige immigrasie vir nasionale veiligheid nie met die nodige erns nie en gevolglik word die nodige maatrëels nie ingestel om die toenemende instroming van omwettige immigrante na Suid Afrika te bekamp nie.</li> <li>Geweld teenoor vreemdelinge in Suid Afrika word alom gesien as xenofobies van aard, alhoewel spesifieke faktore teenwoordig is wat bydra tot sulke aanvalle.</li></ul> Die studie is gebaseer op die teoretiese beginsels van nasionale veiligheid en meer spesifiek soos dit van toepassing is op ontwikkelende lande. Die studie poog om die veiligheidsimplikasies van onwettige immigrasie na Suid Afrika te analiseer, met spesifieke verwysing na die betrokkenheid van onwettige immigrante by misdaad in Suid Afrika. Daar word ook gefokus op die politieke gevolge, insluitend die opvatting dat vreemdelinge betrokke is by, of bydra tot politieke spanning in sekere gemeenskappe. Die studie aanvaar die beginsel dat onwettige immigrasie multi-dimensioneel is en dat dit onlosmaaklik verbonde is aan die sosio-ekonomiese impak daarvan. Gevolglik word verwys na die politieke, sosiale, ekonomiese en misdaad impak van onwettige immigrasie. Die studie fokus verder ook op die amptelike en nie-amptelike standpunte oor onwettige immigrasie en analiseer die maatrëels deur die Suid Afrikaanse regering ingestel ten einde die vraagstuk aan te spreek. Die studie fokus oorwegend op die periode 1994 tot 2008 ten einde die toename in die getal omwettige immigrante wat die land sedert 1994 binnegekom het, aan te dui. Verder poog die studie om vas te stel of daar ‘n verband bestaan tussen die toename in onwettige immigrante en ‘n toename in geweldadige misdaad in Suid Afrika in die periode onder bespreking. Copyright / Dissertation (MSS)--University of Pretoria, 2009. / Political Sciences / unrestricted