Refine
Year of publication
- 2017 (51) (remove)
Document Type
- Doctoral Thesis (51) (remove)
Is part of the Bibliography
- yes (51)
Keywords
- Antibiotika (1)
- Australia (1)
- Australien (1)
- Autotrophie (1)
- Bakterien Sensor (1)
- Bioinformatik (1)
- Biosynthese (1)
- Constraint-basierte Modellierung (1)
- Cre Rekombinase (1)
- Cre recombinase (1)
- Cyanobakterien (1)
- DNA (1)
- DNA assembly (1)
- DNS Assemblierung (1)
- Datenintegration (1)
- Diversität (1)
- Durchflusszytometrie (1)
- El`gygytgyn Kratersee (1)
- El’gygytgyn Crater Lake (1)
- European bats (1)
- Europäische Fledermausarten (1)
- Eutrophierung (1)
- Evolutionsgenetik (1)
- Fotoschalter (1)
- GTPase (1)
- Grundwasser (1)
- Haptene (1)
- Huftiere (1)
- Hybridom (1)
- Immunfluoreszenz (1)
- Klimawandel (1)
- Kohlenhydrat-Protein Interaction (1)
- Kohlenstoffisotope (1)
- LAMP (1)
- Landschaftsanalyse (1)
- Licht (1)
- Litoral (1)
- Makrophyten (1)
- Microviridin (1)
- Mikrobiologie (1)
- NGS (1)
- Naturschutz (1)
- Naturstoffe (1)
- Nettoproduktion (1)
- Optimierung von Biosynthesewegen (1)
- Optogenetik (1)
- Pathogen (1)
- Periphyton (1)
- Pharmakodynamik (1)
- Phylogeographie (1)
- Populationsökologie (1)
- Protease-Inhibitoren (1)
- Pyrophosphat (1)
- ROS (1)
- Radiokarbon (1)
- Reptilien (1)
- RiPP (1)
- Rubisco (1)
- Southeast Asia (1)
- Stoffwechselnetze (1)
- Systemsbiologie (1)
- Südostasien (1)
- Talsperre (1)
- Thermoregulationsverhalten (1)
- Tiefe Biosphäre (1)
- Transkriptionsfaktor (1)
- Vorhersagemodelle (1)
- Wirkstoffinteraktionen (1)
- Wüste (1)
- aldehyde (1)
- antibiotic combinations (1)
- asymmetric competition (1)
- asymmetrische Konkurrenz (1)
- autotrophy (1)
- bacterial population growth (1)
- bacterial sensor (1)
- benthic primary producers (1)
- benthische Primärproduzenten (1)
- bifunctional enzyme (1)
- biogeography (1)
- bioinformatics (1)
- biosynthesis (1)
- carbohydrate-protein interaction (1)
- carbon isotopes (1)
- chloro-ribosome (1)
- climate change (1)
- conservation (1)
- constraint-based modeling (1)
- conventional agriculture (1)
- cyanobacteria (1)
- data integration (1)
- deep biosphere (1)
- degradation (1)
- desert (1)
- diversity (1)
- drug drug interactions (1)
- evolutionary history (1)
- flow cytometry (1)
- freshwater ecosystems (1)
- fusion (1)
- galactolipids (1)
- gelöster organischer Kohlenstoff (1)
- giant unilamellar vesicles (1)
- groundwater (1)
- hapten (1)
- high resolution mass spectrometry (1)
- hochauflösende Massenspektrometrie (1)
- hybridoma (1)
- immunofluorescence (1)
- interspecific interactions (1)
- interspezifische Wechselwirkungen (1)
- isothermal amplification (1)
- isothermale Amplifikation (1)
- konventionelle Landwirtschaft (1)
- landscape analysis (1)
- light (1)
- littoral eutrophication (1)
- macrophytes (1)
- mathematical modelling (1)
- mathematische Modellierung (1)
- metabolic networks (1)
- microbial decomposition (1)
- microbiology (1)
- microviridin (1)
- mikrobieller Abbau (1)
- mitochondrial transformation (1)
- monoclonal antibodies (1)
- monoklonale Antikörper (1)
- morphological changes (1)
- morphologischen Veränderungen (1)
- natural products (1)
- net production (1)
- optogenetics (1)
- organic carbon (1)
- organic matter quality (1)
- organischer Kohlenstoff (1)
- organisches Material (1)
- oxidase (1)
- pathogen (1)
- pathway engineering (1)
- peptide (1)
- periphyton (1)
- pharmacodynamics (1)
- photoswitches (1)
- photosynthesis (1)
- plastid transformation (1)
- population ecology (1)
- predictive modelling (1)
- protease inhibitor (1)
- pyrophosphate (1)
- radiocarbon (1)
- reactive oxygen species (1)
- reptiles (1)
- ribosomal dynamics (1)
- ribosome assembly (1)
- riesen unilamellare Vesikel (1)
- sulfadiazine (1)
- synthetic biology (1)
- synthetische Biologie (1)
- systems biology (1)
- thermoregulatory behaviour (1)
- time-kill curves (1)
- tobacco (1)
- tobramycin (1)
- transcription factor (1)
- translation (1)
- ungulates (1)
- water reservoir (1)
Institute
- Institut für Biochemie und Biologie (51) (remove)
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.
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.