Thesis (doctoral)--Ludwig Maximilians-Universität zu München, 1979.

Thesis (M.S.)--Montana State University--Bozeman, 2007. / Typescript. Chairperson, Graduate Committee: Michael Babcock. Includes bibliographical references (leaves 43-51).

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The synthesis of a new thiamine analogue, 2'-hydroxythiamine, is reported. Kinetic studies with thiamine pyrophosphate analogues and apopyruvate decarboxylase (EC 4.1.1.1) from brewer's yeast, gave the values of 2.3 X 10^-5 M as the K_m for thiamine pyrophosphate and 2.0 X 10^-5 M as the K_m for 2'-ethylthiamine pyrophosphate. The V_max for the latter was 14% that of thiamine pyrophosphate. Inhibitor constants, K_i, were determined for the following competitive inhibitors of thiamine pyrophosphate with the apoenzyme. All values are given for the pyrophosphate esters: tetrahydrothiamine, 0.65 X 10^-5 M; oxythiamine, 2.0 X 10^-5 M; 2'-n-butylthiamine, 4.5 X 10^-5 M; 2'-methoxythiamine, 7.0 X 10^-5 M; pyrithiamine, 7.8 X 10^-5; thiochrome, 15. X 10^-5 M; 2'-desmethylthiamine, 22. X 10^-5 M; 2'-hydroxythiamine, 38. X 10^-5 M. None of the inhibitors exhibited coenzyme activity. A hydrophobic interaction of the 2'-methyl group of thiamine pyrophosphate with the apoenzyme is suggested from these studies. The formation of a fluorescent complex at pH 6.7 between apopyruvate decarboxylase and thiochrome pyrophosphate was detected and found to be dependent upon Mg(II) ions. A similar complex between thiochrome and the apoenzyme could not be detected, demonstrating the importance of the pyrophosphate function in binding to the protein. The shift in the fluorescence emission spectrum of thiochrome pyrophosphate toward lower wavelengths upon complex formation with the apoenzyme, coincided with the behavior of thiochrome in solvents of decreasing dielectric constant. This latter observation suggests involvement of the thiochrome pyrophosphate with a hydrophobic region of the enzyme. A study of the pH dependency of the enzyme-coenzyme complex indicated considerable recombination of apoenzyme and coenzyme at alkaline pH, where dissociation of the coenzyme usually takes place. A rationale for the interpretation of the pH-behavior of the enzyme-coenzyme complex is offered. An amino acid analysis of a highly purified sample of pyruvate decarboxylase, considered to be essentially homogeneous, is reported. Assuming a molecular weight of 175,000 for the enzyme, a total of 1317 amino acid residues were calculated, of which 52.1% fall into the non-polar category. the half-cystine content was calculated as 10.3%, and the proline content, as 4.6%. The specific volume was calculated as 0.737 ml per g. A single low-angle X-ray diffraction study gave a value of 35.5 ± 1.5 A for the radius of gyration of pyruvate decarboxylase. Assuming a spherical shape, a diameter of 91.6 ± 4.0 A was calculated.

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Bioethanol, produced from organic waste as a second-generation biofuel, is an important renewable energy source. Here, recalcitrant carbohydrate sources, such as municipal and agricultural waste, and plants grown on land not suitable for food crops, are exploited. The thermophilic, Gram-positive bacterium Geobacillus thermoglucosidasius is naturally very flexible in its growth substrates and produces a variety of fermentation products, including lactate, formate, acetate and ethanol. TMO Renewables Ltd. used metabolic engineering to enhance ethanol production, creating the production strain TM242 (NCIMB 11955 ∆ldh, ∆pfl, pdhup). Ethanol yield has been increased to 82% of the theoretical maximum on glucose and up to 92% of the theoretical maximum on cellobiose. However, this strain still produces acetate, presumably derived from the overproduction of acetyl-CoA through the upregulated pdh gene encoding the pyruvate dehydrogenase complex. An alternative to the mixed fermentation pathway found in G. thermoglucosidasius is to introduce a homoethanologenic pathway. Yeast and a very limited range of mesophilic bacteria use the homoethanol fermentation pathway, which employs pyruvate decarboxylase (PDC) in conjunction with alcohol dehydrogenase (ADH), to convert pyruvate to ethanol. Despite extensive screening, no PDC has yet been identified in a thermophilic organism. Using the thermophile G. thermoglucosidasius as a host platform, we endeavoured to develop a thermophilic version of the homoethanol pathway for use in Geobacillus spp. This Thesis reports the in vitro characterization and crystal structure of one of the most thermostable bacterial PDCs from the mesophile Zymobacter palmae (ZpPDC) and describes strategies to improve expression of active PDC at high growth temperatures. This includes codon harmonization and the successful development of a PET (producer of ethanol) operon. Furthermore, ancestral sequence reconstruction was explored as an alternative engineering approach, but did not yield a PDC more thermostable than ZpPDC. In vitro ZpPDC is most active at 65°C with a denaturation temperature of 70°C, when sourced from a recombinant mesophilic host. Codon harmonization improved detectable PDC activity in G. thermoglucosidasius cultures grown up to 65°C by up to 42%. Pairing this PDC with G. thermoglucosidasius ADH6 produced a PET functional up to 65°C with ethanol yields of 87% of the theoretical maximum on glucose. This increase in yield at temperatures of up to 15°C higher than previously reported for any PDC expressed.

The number of newly detected autoantibodies (AB) targeting synaptic proteins in neurological disorders of the central nervous system (CNS) is steadily increasing. Direct interactions of AB with their target antigens have been shown in first studies but the exact pathomecha-nisms for most of the already discovered AB are still unclear. The present study investigates pathophysiological mechanisms of AB-fractions that are associated with the enigmatic CNS disease Stiff person syndrome (SPS) and target the synaptically located proteins amphiphysin or glutamate decarboxylase 65 (GAD65). In the first part of the project, effects of AB to the presynaptic endocytic protein amphiphysin were investigated. Ultrastructural investigations of spinal cord presynaptic boutons in an es-tablished in-vivo passive-transfer model after intrathecal application of human anti-amphiphysin AB showed a defect of endocytosis. This defect was apparent at high synaptic activity and was characterized by reduction of the synaptic vesicle pool, clathrin coated vesi-cles (CCVs), and endosome like structures (ELS) in comparison to controls. Molecular inves-tigation of presynaptic boutons in cultured murine hippocampal neurons with dSTORM microscopy after pretreatment with AB to amphiphysin revealed that marker proteins involved in vesicle exocytosis (synaptobrevin 2 and synaptobrevin 7) had an altered expression in GA-BAergic presynapses. Endophilin, a direct binding partner of amphiphysin also displayed a disturbed expression pattern. Together, these results point towards an anti-amphiphysin AB-induced defective organization in GABAergic synapses and a presumably compensatory rearrangement of proteins responsible for CME. In the second part, functional consequences of SPS patient derived IgG fractions containing AB to GAD65, the rate limiting enzyme for GABA synthesis, were investigated by patch clamp electrophysiology and immunohistology. GABAergic neurotransmission at low and high activity as well as short term plasticity appeared normal but miniature synaptic potentials showed an enhanced frequency with constant amplitudes. SPS patient IgG after preabsorption of GAD65-AB using recombinant GAD65 still showed specific synaptic binding to neu-rons and brain slices supporting the hypothesis that additional, not yet characterized AB are present in patient IgG responsible for the exclusive effect on frequency of miniature potentials. In conclusion, the present thesis uncovered basal pathophysiological mechanisms underlying paraneoplastic SPS induced by AB to amphiphysin leading to disturbed presynaptic architec-ture. In idiopathic SPS, the hypothesis of a direct pathophysiological role of AB to GAD65 was not supported and additional IgG AB are suspected to induce distinct synaptic malfunction. / Die Anzahl neu charakterisierter Autoantikörper (AAK) gegen synaptische Proteine bei Er-krankungen des zentralen Nervensystems (ZNS) ist stetig wachsend. Direkte Interaktionen der AAK mit ihren Zielantigenen konnten in ersten Studien belegt werden, jedoch besteht weiterhin Unklarheit über die exakten zugrunde liegenden Pathomechanismen. In der vorliegenden Arbeit wurden pathophysiologische Mechanismen von AAK gegen die synaptisch lokalisierten Proteine Amphiphysin und Glutamatdecarboxylase 65 (GAD65) untersucht, die mit der ZNS Erkrankung Stiff Person Syndrom (SPS) assoziiert sind. Im ersten Projektteil wurden die Effekte von AAK gegen das Endozytoseprotein Amphiphysin analysiert: in einem etablierten in-vivo Tiermodell konnten nach intrathekalem passiven Transfer von AAK gegen Amphiphysin ultrastrukturelle Untersuchungen von präsynaptischen Terminalen im Rückenmark eine Störung der Endozytose aufzeigen. Dieser Defekt, der bei hoher synaptischer Aktivität eintrat, war durch eine Verminderung synaptischen Vesikelpools, Clathrin-ummantelter Vesikel und endosomähnlicher Strukturen charakterisiert. Molekulare Untersuchungen präsynaptischer Terminale kultivierter hippokampaler Zellkulturen mit dSTORM Mikroskopie zeigten, dass an der Exozytose beteiligte synaptische Vesikelproteine (Synaptobrevin 2 und Synaptobrevin 7) ein verändertes Expressionsmuster innerhalb GA-BAerger Synapsen aufweisen. Die Expression von Endophilin, einem direkten Bindungs-partner von Amphiphysin, war ebenso verändert. Zusammengefasst weisen diese Ergebnis-se auf einen Organisationsdefekt GABAerger Synapsen hin, die durch anti-Amphiphysin AAK induziert sind und eine kompensatorische Umverteilung von Endozytoseproteinen vermuten lassen. Im zweiten Teil der Arbeit wurden die funktionellen Effekte von SPS AAK gegen GAD65, dem geschwindigkeitsbestimmenden Enzym der GABA-Synthese, mittels Patch-Clamp Mes-sungen und Immunhistologie untersucht. Die GABAerge synaptische Übertragung bei niedri-ger als auch hoher synaptischer Aktivität sowie die synaptische Kurzzeitplastizität wurden durch die IgG Fraktionen mit GAD65-AAK nicht beeinträchtigt. Die Frequenz von GABAergen Miniaturpotentialen war jedoch bei ansonsten gleichbleibender Amplitude erhöht. SPS-Patienten-IgG zeigte allerdings auch nach Präabsorbtion von GAD65-AAK mit Hilfe von rekombinanten GAD65 eine spezifische Anfärbung neuronaler Synapsen, was die Hypothese von weiteren, funktionell wirksamen, aber noch nicht identifizierten AAK im Patienten-IgG unterstützt. Zusammenfassend konnten in der vorliegenden Arbeit grundlegende pathophysiologische Mechanismen aufgezeigt werden, wie pathogene Antikörper gegen Amphiphysin die Struktur präsynaptischer Boutons beeinträchtigen können. Im Falle des idiopathischen SPS konnte keine unterstützenden Befunde für die Hypothese einer direkten pathophysiologischen Rolle von GAD65 AAK erhoben werden. Nach den vorliegenden Ergebnissen wird das Vorhandensein weiterer, derzeit noch nicht beschriebener IgG AAK postuliert, die die synaptische Fehlfunktion erklären können.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 110-120). Available also in a digital version from Dissertation Abstracts.