TY - THES A1 - Kersting, Sebastian T1 - Isothermal nucleic acid amplification for the detection of infectious pathogens Y1 - 2017 ER - TY - THES A1 - Castellanos, Reynel Urrea T1 - Functional characterization of FGT2, a positive regulator of heat stress memory Y1 - 2017 ER - TY - THES A1 - Moraes, Thiago Alexandre T1 - Exploring the role of the circadian clock in the regulation of starch turnover in changing light conditions in Arabidopsis Y1 - 2017 ER - TY - THES A1 - Grimm-Seyfarth, Annegret T1 - Effects of climate change on a reptile community in arid Australia T1 - Auswirkungen von Klimawandel auf eine Reptiliengemeinschaft im ariden Australien BT - exploring mechanisms and processes in a hot, dry, and mysterious ecosystem BT - eine Untersuchung von Mechanismen und Prozessen in einem heißen, trockenen, und rätselhaften Ökosystem N2 - Dies ist eine kumulative Dissertation, die drei Originalstudien umfasst (eine publiziert, eine in Revision, eine eingereicht; Stand Dezember 2017). Sie untersucht, wie Reptilienarten im ariden Australien auf verschiedene klimatische Parameter verschiedener räumlicher Skalen reagieren und analysiert dabei zwei mögliche zugrunde liegende Hauptmechanismen: Thermoregulatorisches Verhalten und zwischenartliche Wechselwirkungen. In dieser Dissertation wurden umfassende, individuenbasierte Felddaten verschiedener trophischer Ebenen kombiniert mit ausgewählten Feldexperimenten, statistischen Analysen, und Vorhersagemodellen. Die hier erkannten Mechanismen und Prozesse können nun genutzt werden, um mögliche Veränderungen der ariden Reptiliengesellschaft in der Zukunft vorherzusagen. Dieses Wissen wird dazu beitragen, dass unser Grundverständnis über die Konsequenzen des globalen Wandels verbessert und Biodiversitätsverlust in diesem anfälligen Ökosystem verhindert wird. N2 - This is a cumulative dissertation comprising three original studies (one published, one in revision, one submitted; Effective December 2017) investigating how reptile species in arid Australia respond to various climatic parameters at different spatial scales and analysing the two potential main underlying mechanisms: thermoregulatory behaviour and species interactions. This dissertation combines extensive individual-based field data across trophic levels, selected field experiments, statistical analyses, and predictive modelling techniques. Mechanisms and processes detected in this dissertation can now be used to predict potential future changes in the community of arid-zone lizards. This knowledge will help improving our fundamental understanding of the consequences of global change and thereby prevent biodiversity loss in a vulnerable ecosystem. KW - Australien KW - Reptilien KW - Australia KW - reptiles KW - Populationsökologie KW - population ecology KW - Thermoregulationsverhalten KW - thermoregulatory behaviour KW - interspezifische Wechselwirkungen KW - interspecific interactions KW - Vorhersagemodelle KW - predictive modelling KW - Klimawandel KW - climate change KW - Wüste KW - desert Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-412655 ER - TY - THES A1 - Foti, Alessandro T1 - Characterization of the human aldehyde oxidase T1 - Charakterisierung der menschlichen Aldehydoxidase BT - Studies on the FAD active site and ROS generation N2 - In this work the human AOX1 was characterized and detailed aspects regarding the expression, the enzyme kinetics and the production of reactive oxygen species (ROS) were investigated. The hAOX1 is a cytosolic enzyme belonging to the molybdenum hydroxylase family. Its catalytically active form is a homodimer with a molecular weight of 300 kDa. Each monomer (150 kDa) consists of three domains: a N-terminal domain (20 kDa) containing two [2Fe-2S] clusters, a 40 kDa intermediate domain containing a flavin adenine dinucleotide (FAD), and a C-terminal domain (85 kDa) containing the substrate binding pocket and the molybdenum cofactor (Moco). The hAOX1 has an emerging role in the metabolism and pharmacokinetics of many drugs, especially aldehydes and N- heterocyclic compounds. In this study, the hAOX1 was hetereogously expressed in E. coli TP1000 cells, using a new codon optimized gene sequence which improved the expressed protein yield of around 10-fold compared to the previous expression systems for this enzyme. To increase the catalytic activity of hAOX1, an in vitro chemical sulfuration was performed to favor the insertion of the equatorial sulfido ligand at the Moco with consequent increased enzymatic activity of around 10-fold. Steady-state kinetics and inhibition studies were performed using several substrates, electron acceptors and inhibitors. The recombinant hAOX1 showed higher catalytic activity when molecular oxygen was used as electron acceptor. The highest turn over values were obtained with phenanthridine as substrate. Inhibition studies using thioridazine (phenothiazine family), in combination with structural studies performed in the group of Prof. M.J. Romão, Nova Universidade de Lisboa, showed a new inhibition site located in proximity of the dimerization site of hAOX1. The inhibition mode of thioridazine resulted in a noncompetitive inhibition type. Further inhibition studies with loxapine, a thioridazine-related molecule, showed the same type of inhibition. Additional inhibition studies using DCPIP and raloxifene were carried out. Extensive studies on the FAD active site of the hAOX1 were performed. Twenty new hAOX1 variants were produced and characterized. The hAOX1 variants generated in this work were divided in three groups: I) hAOX1 single nucleotide polymorphisms (SNP) variants; II) XOR- FAD loop hAOX1 variants; III) additional single point hAOX1 variants. The hAOX1 SNP variants G46E, G50D, G346R, R433P, A439E, K1231N showed clear alterations in their catalytic activity, indicating a crucial role of these residues into the FAD active site and in relation to the overall reactivity of hAOX1. Furthermore, residues of the bovine XOR FAD flexible loop (Q423ASRREDDIAK433) were introduced in the hAOX1. FAD loop hAOX1 variants were produced and characterized for their stability and catalytic activity. Especially the variants hAOX1 N436D/A437D/L438I, N436D/A437D/L438I/I440K and Q434R/N436D/A437D/L438I/I440K showed decreased catalytic activity and stability. hAOX1 wild type and variants were tested for reactivity toward NADH but no reaction was observed. Additionally, the hAOX1 wild type and variants were tested for the generation of reactive oxygen species (ROS). Interestingly, one of the SNP variants, hAOX1 L438V, showed a high ratio of superoxide prodction. This result showed a critical role for the residue Leu438 in the mechanism of oxygen radicals formation by hAOX1. Subsequently, further hAOX1 variants having the mutated Leu438 residue were produced. The variants hAOX1 L438A, L438F and L438K showed superoxide overproduction of around 85%, 65% and 35% of the total reducing equivalent obtained from the substrate oxidation. The results of this work show for the first time a characterization of the FAD active site of the hAOX1, revealing the importance of specific residues involved in the generation of ROS and effecting the overall enzymatic activity of hAOX1. The hAOX1 SNP variants presented here indicate that those allelic variations in humans might cause alterations ROS balancing and clearance of drugs in humans. N2 - Aldehydoxidasen (AOX) sind Molybdo-enzyme, die durch breite Substratspezifität gekennzeichnet sind, aromatische/aliphatische Aldehyde in die entsprechenden Carbonsäuren oxidieren und verschiedene heteroaromatische Ringe hydroxylieren. Die Enzyme verwenden Sauerstoff als terminalen Elektronenakzeptor und produzieren reduzierte Sauerstoffspezies während des Umsatzes. Die physiologische Funktion von Säugetier-AOX-Isoenzymen ist noch unklar, aber menschliches AOX (hAOX1) ist ein Enzym von Phase-I-Wirkstoff-Metabolismus. Weiterhin, wurden zahlreiche Einzelnukleotidpolymorphismen (SNP) und weitere hAOX1-Mutanten im hAOX1-Gen identifiziert. SNPs sind eine Hauptquelle für die interindividuelle Variabilität in der menschlichen Population, und SNP-basierte Aminosäureaustausche in hAOX1 modulieren die katalytische Funktion des Enzyms entweder positiv oder negativ. In diesem Bericht haben wir zehn neue SNPs ausgewählt, die zu Aminosäureaustauschen in der Nähe der FAD-Cofaktor von hAOX1 führen und die gereinigten Enzyme nach heterologen Expression in Escherichia coli charakterisieren. Darüber hinaus haben wir zehn weitere FAD-Varianten produziert. Die hAOX1-Varianten wurden sorgfältig durch quantitative Unterschiede in ihrer Fähigkeit zur Herstellung von Superoxidradikal charakterisiert. ROS repräsentieren markante Schlüsselmoleküle in physiologischen und pathologischen Zuständen in der Zelle. Unsere Daten zeigen signifikante Veränderungen der Superoxid-Anionenproduktion unter den Varianten. Insbesondere führte der Rest L438 in der Nähe des Isoalloxanzinringes des FAD-Cofaktors zu einer erhöhten Superoxid-Radikalproduktion von 75-85%. In Anbetracht der hohen Toxizität des Superoxid-Anions in der Zelle ist die hAOX1-L438V SNP-Variante ein eventueller Kandidat für kritische oder pathologische Rollen dieser natürlichen Variante innerhalb der menschlichen Population. KW - aldehyde KW - oxidase KW - ROS KW - reactive oxygen species Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-410107 ER - TY - THES A1 - Radon, Christin T1 - Analyse der Funktion der dualen Lokalisation der 3-Mercaptopyruvat Sulfurtransferase im Menschen Y1 - 2017 ER - TY - THES A1 - Schedina, Ina-Maria T1 - Comparative genetic and transcriptomic analyses of the amazon molly, poecilia formosa and its parental species, poecilia mexicana and poecilia latipinna Y1 - 2017 ER - TY - THES A1 - Peng, Xingzhou T1 - Multiphase polymers based on polydepsipeptides as a multifunctional materials platform Y1 - 2017 ER - TY - THES A1 - Yang, Lei T1 - Verification of systemic mRNAs mobility and mobile functions Y1 - 2017 ER - TY - THES A1 - Martínez Jaime, Silvia T1 - Towards the understanding of protein function and regulation BT - organization of the mitochondrial protein complexome under different conditions and the role of SUM03 in Arabidopsis thaliana Y1 - 2017 ER -