Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 180-187).

Thesis (M.Sc.)--Memorial University of Newfoundland, 2003. / Bibliography: leaves 81-94. Also available online.

Thesis (Master's Degree (Medical Technology)-- Cape Technikon, Cape Town, 1997 / There is, at present, no recognised diagnostic biochemical marker of Alzheimer's Disease (AD). Recently, Gunnerson and Haley, (1992), reported that the presence of glutamine synthetase (GS) in cerebrospinal fluid (CSF) samples showed a 97% correlation with patients diagnosed as having AD. GS was detected by photolabelling with [y32P]2-azido-ATP or [y32P]8azido- ATP and visualisation following sodium dodecyl sulphate polyacrylamide gel electrophoresis (SOS-PAGE) and autoradiography. This study set out to reproduce Gunnerson and Haley's methodology for labelling sheep GS in CSF using [y32P]8-azidoATP, to develop this assay or possibly another, using a fluorescent probe of ATP binding sites, into a robust procedure suitable for a routine diagnostic laboratory, and finally to assess whether the presence of GS in CSF is indeed a marker of AD.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Please refer to full text for abstract / AFRIKAANSE OPSOMMING: Sien volteks vir opsomming

Estradiol (Compudose®, COM) implants are extensively used in beef cattle production systems to alter body composition and feed efficiency. Little information exists about the physiological mechanisms affected by COM treatment in growing, mature, and senescent female cattle. Moreover, no reports describe the level of blood estradiol resulting from COM treatment. The effect of COM on levels of plasma estradiol and blood metabolites and proteins, and relative content of glutamine synthetase (GS) and other amino acid nitrogen-metabolizing enzymes in liver tissue, was studied using three experimental models relevant to cow-calf production regimens: senescent cows (Trial 1), young mature (young) versus senescent (old) cows (Trial 2), and growing heifers (Trial 3). In Trial 1, plasma estradiol concentrations were 222 % more after 14 and 28 d in COM-implanted than sham implanted (Control) cows. COM treatment did not affect measured blood metabolites and enzymes, but increased hepatic GS protein expression by 350% after 14 d and 200% after 28 d of implantation. In contrast, protein expression of alanine transaminase, aspartate transaminase, glutamate dehydrogenase, and two glutamate transporters was not affected by COM. In Trial 2, plasma estradiol concentrations of COM implanted young and old cows were 48% higher than Control groups, whereas blood metabolites were not affected. COM implantation did not affect GS protein expression in young cows, but tended to increase GS expression in the old cows by 283% after 14 d and 41% after 28 d. GS mRNA content was increased about 38% in both young and old COM-treated cows. Hepatic content of beta-catenin and G protein-coupled receptor 30 (GPR30) content was not affected by COM treatment, indicating that estradiol-mediated GS expression was not regulated by beta-catenin- or GPR30-controlled pathways. In Trial 3, plasma estradiol levels in COM-treated heifers were 70% higher in COM heifers, concomitant with increased levels of total bilirubin and creatine kinase, and decreased creatinine. Correlation analysis of plasma estradiol levels and blood constituents only identified a positive correlation between plasma estradiol and potassium. Collectively, these data describe positive estradiol-mediated effects on hepatic metabolism and blood parameters in female cattle.

Abstract not available

Thesis (M.Tech.-Medical Technology)--Cape Technikon, 1997. / Bibliography: leaf 96-114. Also available online.

The bacterial cell needs ammonia for synthesis of glutamine from glutamate. Only one enzyme is able to catalyze this reaction, namely glutamine synthetase (GS). GS can be regulated both transcriptionally and post-translationally and it is present in all kingdoms of life. Our study has been focused on the post-translational regulation of GS in the diazotrophic bacterium Rhodospirillum rubrum. A number of proteins are involved in the covalent regulation of GS, among them are the regulatory PII proteins that depending on growth conditions also like GS are covalently modified. We have purified all proteins involved in GS regulation and developed several in vitro assays with the aim of understanding GS regulation in R. rubrum. Studies on the influence of the small metabolite effectors α-ketoglutarate and glutamine are also included together with the effect of divalent cations. In both R. rubrum and Escherichia coli, one of the enzymes participating in GS regulation is the bifunctional enzyme GlnE. GlnE is responsible for both the attachment and the removal of AMP groups from GS, which basically leads to a more inactive or active enzyme respectively. Apart from examining the above functions of GlnE, we have also found a novel third activity of R. rubrum GlnE, an antioxidant function, which is located in the C-terminal domain. We have examined this novel activity of GlnE in great detail, including site specific mutagenesis. We also generated and analyzed ΔglnE mutants in R. rubrum and the results from these studies show that suppressor mutations can occur within glnA, the gene encoding GS. We assume that the function of these suppressor mutations is to lower the specific activity of GS, which otherwise might be too high in a ΔglnE mutant since they lack the ability to adenylylate GS. In other words, it seems that ΔglnE mutants can not be generated without producing suppressor mutations.

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.

La détermination de la structure des protéines à une résolution atomique est cruciale pour la compréhension de leur fonction cellulaire. Actuellement, la cristallographie aux rayons X est la méthode la plus efficace pour la détermination à haute résolution de la structure de protéines monomériques allant 40 et 100 kDa. Par contre, elle est limitée par la croissance de cristaux de bonne qualité, qui est problématique pour nombreuses cibles. La cryo-microscopie électronique (cryoME) permet la détermination structurale à résolution quasi-atomique de larges structures protéiques, de préférence symétrique et en solution. Cependant, les images de cryoME sont très bruitées, car une faible dose d'électrons est appliquée de manière à limiter les dommages d'irradiation. En moyennant des dizaines d'images correspondant à la même orientation moléculaire, le rapport signal sur bruit est amélioré. La combinaison des images moyennées de plusieurs orientations permet l'obtention d'une carte de densité électronique 3D de la molécule d'intérêt. Si la taille et la symétrie de la molécule diminuent, l'analyse cryoME devient de moins en moins précise, il est alors impossible d'analyser des protéines monomériques de taille inférieure à 100 kDa. Le but de ce travail a été de développer une nouvelle approche pour réduire cette limite de poids moléculaire. Elle consiste à fusionner la protéine d'intérêt (cible) à une matrice homo-oligomérique, générant une particule symétrique et de taille importante adaptée à l'analyse par cryoME. Dans cette thèse, nous avons cherché à tester et démontrer la faisabilité de cette approche de symétrisation en utilisant des protéines cibles de structure connue.Pour mettre en place notre étude pilote, nous avons choisi différentes combinaisons de cibles et de matrices connectées par des peptides de liaison (linker) de longueur différentes. Nous avons caractérisé les fusions exprimées en bactéries par microscopie électronique après coloration négative et par plusieurs techniques biophysiques. Grace à ces techniques, nous avons trouvé que la meilleure combinaison est la fusion entre la protéine matrice glutamine synthétase (GS), un 12-mer de symétrie D6 et la cible maltose binding protein (Mbp), connectées par un linker contenant trois alanines, que nous avons appelée « Mag ». En jouant sur la longueur du linker nous avons ensuite sélectionné la fusion la plus compacte pour l'analyse cryoME: MagΔ5. Nous avons obtenu la carte cryoME à 10 Å de MagΔ5, qui présente une bonne corrélation avec les modèles atomiques de Mbp et GS. Plus particulièrement, le site catalytique et quelques hélices α sont identifiables. Ces résultats sont confirmés par l'étude cristallographique que nous avons conduite sur MagΔ5. L'ensemble de ce travail souligne que la présence d'une grande interface d'interactions cible-matrice stabilise la fusion et améliore la résolution en cryoME. Pour la symétrisation d'une cible inconnue, nous envisageons la même procédure expérimentale que celle développée pour MagΔ5. La matrice et le linker les plus adaptés devront être identifiés en utilisant les mêmes méthodes biophysiques.En conclusion, ce travail établit la preuve de concept que la méthode de symétrisation des protéines permet la détermination de la structure de protéines de poids moléculaire inférieur à 100 kDa par cryoME. Cette méthode a le potentiel d'être un nouvel outil prometteur, qui faciliterait l'analyse de cibles résistantes à l'analyse structurale conventionnelle. / Structural determination of proteins at atomic level resolution is crucial for unravelling their function. X-ray crystallography has successfully been used to determine macromolecular structures with sizes ranging from kDa to MDa, and currently remains the most efficient method for the high-resolution structure determination of monomeric proteins within the 40-100 kDa range. However, this method is limited by the ability to grow well diffracting crystals, which is problematic for several targets, such as membrane proteins. Single particle cryo electron microscopy (cryoEM) allows near atomic (3-4Å) resolution structural determination of large, preferably symmetric, assemblies in solution. Biological molecules scatter electrons weakly and, to avoid radiation damage, only low electron doses can be used during imaging. Consequently, raw cryoEM images are extremely noisy. However, averaging many molecular images aligned in the same orientation permits one to increase the signal-to-noise ratio, ultimately allowing the achievement of a 3D density map of the molecule of interest. Nevertheless, as the molecular size and degree of symmetry decrease, the individual images loose adequate features for accurate alignment. Currently, cryoEM analysis is practically impossible for monomeric proteins below ~100 kDa in mass. We propose to circumvent this obstacle by fusing such monomeric target proteins to a homo-oligomeric protein (template), thereby generating a self-assembling particle whose large size and symmetry should facilitate cryoEM analysis. In the present thesis we seek to test and demonstrate the feasibility of this ‘protein symmetrization' approach and to evaluate its usefulness for protein structure determination. To set up the pilot study we combined selected targets of known structure with two templates: Glutamine Synthetase (GS), a 12-mer with D6 symmetry and a helical N-terminus, and the E2 subunit of the pyruvate dehydrogenase complex, a 60-mer with icosahedral symmetry and an unstructured N-terminus. After recombinant production in E.coli we identified by negative stain EM a promising dodecameric chimera for structural analysis, comprising maltose binding protein (Mbp) connected to GS by a tri-alanine linker (denoted “Mag”). In order to optimize sample homogeneity we produced a panel of Mag deletion constructs by sequentially truncating the 17 residues between the Mbp and GS domains. A combination of biophysical techniques (thermal shift assay, dynamic light scattering, size exclusion chromatography) and negative stain EM allowed us to select the best candidate for cryoEM analysis, MagΔ5. By enforcing D6 symmetry we obtained a cryoEM map with a resolution of 10Å (FSC 0.5 criterion). The density of the symmetrized 40 kDa Mbp presents shape and features corresponding to the known atomic structure. In particular, the catalytic pocket and specific α-helical elements are distinguishable. The cryoEM map is additionally validated by a 7Å crystal structure of the MagΔ5 oligomer. The presence of a continuous helical connection between target (Mbp) and template (GS) likely contributed to the conformational homogeneity of MagΔ5. Moreover, comparing MagΔ5 with other chimeras studied in this work suggests that a large buried surface area and favorable interactions between the target and template limit the flexibility of the chimera and improve its resolution by cryoEM. For the symmetrization of a target of unknown structure, we envisage proceeding by a trial and error approach by fusing it to a panel of templates with helical termini and different surface properties, and subsequently selecting the best ones using biophysical assays. In conclusion, the present work establishes the proof-of-concept that protein symmetrization can be used for the structure determination of monomeric proteins below 100 kDa by cryoEM, thereby providing a promising new tool for analyzing targets resistant to conventional structural analysis.