Nitric oxide (NO) is a key physiological messenger, but an excessive production of this molecule can be detrimental, leading to the onset or worsening of many pathological conditions. Dimethylarginine dimethylaminohydrolase (DDAH) is a key enzyme in the NO pathway, involved in the metabolism of asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA), which are both endogenous inhibitors of NO synthesis. Two isoforms of DDAH have been identified in humans, namely DDAH-1 and DDAH-2. DDAH inhibition represents a promising strategy in the treatment of NO overproduction under pathological conditions without affecting the homeostatic role of this messenger. In this work I described the design and synthesis of 12 novel potential DDAH inhibitors together with the development of a new UPLC-MS based assay to measure the activity of HEK293T cell lysates overexpressing recombinant human DDAH-1 in metabolizing ADMA into dimethylamine and L-citrulline. The same assay was used to assess the potential of the novel compounds, as well as of the well-known DDAH inhibitor L-257, to inhibit DDAH-1 catalyzed L-citrulline formation from ADMA. Three of the novel molecules (compounds 10a, 14a and 14b) showed very interesting inhibitory activity: in particular, the methylacylsulfonamide analogue of L-257 (10a) resulted in 13-fold higher inhibition potency than L-257 itself (98% of inhibition at 1mM, IC50 = 3±3 μM and Ki = 1±0 μM). This molecule was chosen for molecular dynamics simulations to study the putative mechanism for 10a inhibition of DDAH-1 activity. Furthermore, DDAH-1 and DDAH-2 were engineered introducing a FLAG-tag at the C-terminal of the proteins to allow their purification from the lysate components by immunoprecipitation. Although the purification protocol requires some further improvement, the fusion proteins did not show to be functionally affected by the modification.

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In der vorliegenden Arbeit wurde untersucht, auf welchem molekularem Weg die NOS1 Exon 1f VNTR – Länge eine Verhaltensmodifikation sowie die Auftrittswahrscheinlichkeit psychischer Krankheiten moderiert, zum einen direkt, zum anderen über Gen – Umwelt – Interaktionen. Zur weiteren Untersuchung der Gen – Umwelt – Interaktion wurde die Methylierung von CpG – Abfolgen einer genaueren Betrachtung unterzogen. Mit Hilfe des Bisulfit-PCR-Protokolls konnten keine gesicherten Aussagen zur Höhe der Methylierung in einzelnen CpG – sites getroffen werden. Mit Hilfe der ebenfalls angewandten Pyrosequenzierung konnten keine Unterschiede in der Methylierung in einem kleinen Sequenzbereich der beiden untersuchten Bereiche der NOS1 Exon 1f CGI gefunden werden. Als nächstes sollte also die NOS1 Exon 1f CGI mit einem verbesserten Bisulfit–PCR Sequenzierprotokoll analysiert werden, um dann Regionen mit in Abhängigkeit zum VNTR Die von Rife et al. aufgestellte These, dass die molekulare Ursache der unterschiedlichen NOS1 Exon 1f Expression in der unterschiedlichen Bildung von Z – DNA im NOS1 Exon 1f VNTR liegt, konnte weder bestätigt noch wiederlegt werden. Mit Hilfe der von Thomas et al. entwickelten Methode der Z–DNA–Detektion konnte nicht zwischen Z–DNA und anderen DNA-Sekundärstrukturen unterschieden werden. Mit der Überprüfung mit einer Positivkontrolle könnte die Validität der Methode und die Qualität des Z–DNA bindenden Antikörpers getestet werden. Vorherige Befunde die auf eine Erhöhung der NO3-- und NO2--Konzentrationen bei Patienten mit Bipolar-affektiver Störung hindeuten konnten in einer Stichprobe von 55 Personen repliziert werden. Die NOx--Konzentrationen waren bei Patienten in der manischen oder hypomanischen Phase am größten, gefolgt von euthymen Patienten bzw. solchen in einem Mischzustand und depressiven Patienten, ohne dass diese Unterschiede signifikant waren. In dieser Arbeit wurde die NOx--Bestimmungsmethode durch Entfernen von Serumproteinen und durch Verwenden eines Nitratstandards verändert, dies führt jedoch zu starken methodischen Ungenauigkeiten. NOS1 mRNA konnte im untersuchten Leukozytenlysat nicht detektiert werden. Die Unterschiede in der NOS3 Expression in Leukozyten waren nicht signifikant unterschiedlich zwischen den drei Gruppen. Durch die Replikation der erhöhten NOx--Konzentrationen im Serum von BPD – Patienten wird die Bedeutung des NO–Systems für psychische Erkrankungen nochmals unterstrichen. Auch wenn unsere Studie im Vergleich zu den schon mit methodischen Unabwägbarkeiten behafteten Vorstudien, weitere methodische Schwächen aufweist. Die Konzentrationserhöhung kann nicht durch eine gesteigerte Expression der verschiedenen NO-Synthasen in Leukozyten erklärt werden, was dafür sprechen könnte, dass die erhöhte NO–Produktion bei bipolar affektiven Patienten den NO-Metabolismus im Gehirn widerspiegelt. / In this Thesis we investigated molecular pathways of behavior modifiation through differential NOS1 Exon 1f VNTR Polymorphism length and differential CpG-methylation. Using Direct-sequencing after Bisulfite treatment, we were not able to gain further knowledge of differential methylation of CpG-Sites. Investigating smaller parts of the NOS1 Exon 1f CpG-Island with Pyroseuencing, we found no differential methylation. Furthermore, we investigated wetherZ-DNA-Formation is moderated by different NOS 1 Exon 1f VNTR-length. By different approaches (ELISA, DotBlot) we were not able to distinguish between Z-DNA and other secondary structures. In a sample of 55 subjects we could replicate findings that NO3- and NO2- -levels are increased in patients suffering of bipolar disorder. There was no difference in NOS3 expression in leukocytes of healthy subjects, bipolar patients and ADHD-patients. NOS1 was not expressed in leukocytes.

Bibliography: leaves 132-155. / viii, 155 leaves : ill., plates, charts (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / A putative callose synthase gene, designated HvGSL1 cDNA is siolated from barley and its involvement in callose biosynthesis is investigated. A near-full length HvGSL1 cDNA encoded a protein showing approximately 30% identitly with that of yeast FKS genes at the amino acid level. The function of this geen was investigated by heterologous expression, protein purification, immunochemistry and mass spectrometric analysis. Results provide strong evidence that the gene encodes a protein which is associated with callose synthase activity, and is likely to encode the catalytic subunit of the synthase complex. / Thesis (Ph.D.)--University of Adelaide, School of Agriculture and Wine, Discipline of Plant and Pest Science, 2003

During catalysis the flavoprotein domain of neuronal nitric-oxide synthase (nNOS) shuttles NADPH-derived reducing equivalents from FAD to FMN and then to the P450-heme enabling heme-based oxygen activation and subsequent NO-synthesis. The binding of Ca�����-activated calmodulin (Ca�����-CaM) to nNOS alleviates inhibition of flavin mediated electron transfer within the diflavin domain, which is demonstrated by the increase in the rate of 2,6-dichioroindoiphenol (DCIP) reduction by 2 to 3 fold and that of cytochrome c����� by 10 to 20 fold. To investigate the effect of the Ca�����-CaM on the nNOS reductase activity, the steady-state kinetics of basal and CaM-stimulated reduction of these two substrates was studied. Parallel initial velocity patterns indicated that both substrates are reduced in a ping-pong mechanism. Product and dead-end inhibition data with DCIP as the electron acceptor were consistent with a di iso ping-pong bi-bi mechanism. In contrast, product and dead-end inhibition studies with cytochrome c����� as the second substrate were consistent with an iso (two-site) ping-pong mechanism. Ca�����-CaM did not alter the proposed kinetic mechanisms; however, it did effect to varying degrees the (k[subscript cat]/K[subscript]m) for the various substrates. The pH-dependence of basal and CaM-stimulated reduction of DCIP revealed that ionizable groups involved in the binding of substrates and catalysis are not altered by Ca�����-CaM. However, the activated cofactor does influence catalytic rate constants and/or ionizable groups involved in cytochrome c����� reduction. nNOS was found to abstract the pro-R (A-side) hydrogen from NADPH. Primary deuterium isotope effects (NADP(D)) and solvent isotope effects (SKIE) suggests that of the two half reactions, the reductive half reaction involving NADPH oxidation limits the overall reaction rate, but that hydride transfer to FAD is not the slow step. A small value of [supercript D](V/K)[subscript NADPH] (1.2-1.6) suggests hydride transfer is not the rate-limiting step within the reductive half-reaction. Large solvent kinetic isotope effects (SKIE) were observed on (V/K)[subscript cytc] for basal and CaM stimulated reduction of cytochrome c����� suggesting that proton uptake from the solvent limits the rate of the oxidative half-reaction. A small SKIE on V and (V/K)[subscript NADPH] indicates that proton uptake does not limit the overall reaction rate. Proton inventory analysis revealed multiple transition-state protons contributed to the observed SKIE. / Graduation date: 2001