Open Access

Eine Behandlung von Tumoren mit physikalischem Kaltplasma zeigt eine erhöhte Toxizität und ein reduziertes Tumorwachstum. Zeitgleich werden während einer Behandlung mit Plasma eine Vielzahl an reaktiven Sauerstoff- und Stickstoffspezies (RONS) generiert, welche Immunzellen stimulieren können. Viele neue Therapieansätze bestreben nicht nur eine Tumortoxizität, sondern auch eine Förderung der körpereigenen, da diese häufig durch Mechanismen der Tumorzellen unterdrückt wird. Zu solchen Therapien zählen checkpoint inhibitoren, Vakzinierungen oder ein adaptiver Zelltransfer mit transgenen oder vor-stimulierten Zellen. Die dadurch geförderte Antitumor-Immunantwort basiert grundlegend auf einem mehrphasigen Prozess. Dieser beginnt mit einer Antigen-unspezifischen frühen Phase, in der das innate Immunsystem aktiviert wird und zu einer Vermehrung und Differenzierung von Antigen-spezifischen CD4+ und CD8+ T-Zellen führt. Da während einer Entzündungsreaktion viele RONS gebildet werden, um Fremdkörper zu eliminieren und Immunzellen zu rekrutieren, ist eine Therapie mit RONS naheliegend. Durch die Anwendung von Kaltplasma können die gebildeten RONS zum Entzündungsgeschehen beitragen und Zellen des innaten und adaptiven Immunsystems stimulieren. Eine veränderte Immunogenität von Tumorzellen sowie eine daraus resultierende direkte Aktivierung von Immunzellen im Kontext einer Antitumor-Immunantwort wurden nach einer Behandlung mit Jet-Plasmen bislang nicht untersucht. In der vorliegenden Arbeit wurde die Kaltplasma-Behandlung von Hautkrebszellen und eines Modellantigens unter Berücksichtigung einer Antitumor-Immunantwort durch natürliche Killerzellen des innaten Immunsystems sowie adaptive Immunzellen in vitro und in vivo untersucht. Es konnte gezeigt werden, dass eine Behandlung mit Kaltplasma zu einer erhöhten Tumortoxizität führt und das Repertoire der Oberflächenmoleküle auf Tumorzellen verändert. In vivo wurde eine vermehrte Infiltration von Immunzellen in das Tumormikromilieu beobachtet, welche mit einer erhöhten Aktivierung von Lymphozyten und Konzentrationen immunstimulatorischer Zytokine einherging. Durch die zeitgleich reduzierten Tumorgrößen, ist eine durch Immunzellen vermittelte Tumortoxizität als Erklärung naheliegend. In zwei Vakzinierungsstudien konnte die Immunogenität von Plasma-behandelter Tumorzellen und einem Tumorassoziierten Modellantigen bestätigt werden.

The soluble blood protein beta2-glycoprotein I (beta2GPI; 326 aa, MW: 48 kDa, 5 domains) is one of the most abundant proteins in human serum and exhibits two main conformational states: the circular or closed conformation, where the first domain (DI) is bound to the last domain (DV) of the protein, and the linear or open conformation. The defined physiological function of beta2GPI is still unknown, though several roles in pro- and anticoagulation as well as oxidative stress protection were discovered. The open form is considered to play a crucial role in the systemic autoimmune disease antiphospholipid syndrome (APS), which is an acquired thrombophilia characterized by recurring thrombotic events and pregnancy morbidity. Beta2GPI constitutes the main antigen for APS autoantibodies which are supposed to bind a cryptic epitope within DI after a conformational change from closed to open form. However, the pathophysiological mechanism of APS is poorly understood. Therefore, investigating the structural dynamics of this protein in relation to its antigenicity is of high interest. Post-translational modifications (PTM) of a target protein often show an impact on the formation of neoantigens, for instance in the autoimmune-mediated diseases type 1 diabetes mellitus, rheumatoid arthritis, or multiple sclerosis. Such modified antigens may lead to immune tolerance breakdown as they are unknown to the immune system, which therefore could mistakes self for non-self proteins. In this thesis, two frequently occurring PTM were introduced to beta2GPI and their impact on the protein conformation was studied by biophysical tools (i.e. atomic force microscopy (AFM) imaging, transmission electron microscopy (TEM) imaging, dynamic light scattering (DLS), and circular dichroism (CD) spectroscopy). In order to examine immunopathophysiological relevance of these PTM, additional insights were gained from ELISA which was used to examine binding of anti-DI autoantibodies purified from the blood of APS patients to the modified beta2GPI species. A characteristic feature of beta2GPI is the high content of lysine residues. Previously, opening of beta2GPI was found to be triggered by a drastic shift in pH and salt concentration (pH 11.5 and 1.15 M NaCl), which results in reversible uncharging of the lysine residues. The aim of this study was to investigate the beta2GPI conformation after lysine acetylation as a model system, to elucidate the role of lysine residues on the conformational dynamics of this protein, and to examine anti-DI autoantibody binding to both the untreated as well as acetylated species. A strategy to permanently open up the closed form under physiological conditions by chemical acetylation of lysine residues utilizing the sensitive acetylation agent acetic acid N-hydroxysuccinimide ester (NHS-Ac) was established. Complete and specific lysine acetylation was verified by quantification of primary amines exerting a fluoraldehyde o-phthaldialdehyde (OPA) reagent assay, as well as by native PAGE and western blot analysis with an anti-acetylated lysine antibody. Beta2GPI acetylation revealed a partial opening of beta2GPI molecules. Compared to untreated, i.e. native beta2GPI which exhibited 93% of the molecules in closed and 7% in open form, complete lysine residue acetylation generated 39% of beta2GPI in closed and 61% in open conformation as shown by AFM high-resolution imaging. pH 11.5-treated beta2GPI was used as a reference in the applied methods and revealed 38% of the protein in closed and 62% in open conformation. Thus, a significant shift in beta2GPI conformation occurred upon lysine residue acetylation as well as basic pH-treatment. The data indicate that lysine residue acetylation destabilizes the closed form, leading to a facilitated opening of the structure. The closed conformation might be predominantly stabilized by electrostatic interactions of lysine residues, which potentially control the conformational dynamics of this glycoprotein. ELISA confirmed that anti-DI autoantibodies do not bind to untreated (closed) beta2GPI. Although acetylated beta2GPI was shown to have a substantial portion of open proteins, no binding of anti-DI autoantibodies to the acetylated species was found either. Hence, acetylated lysine residues may disrupt the immunorelevant epitope in DI which prevents antibody binding. This finding reveals a new hint for epitope organization. However, further detailed epitope mapping has to be performed. Beta2GPI carries two structural disulfide bonds per domain, whereas an additional disulfide bond Cys288/Cys326 is located at the C-terminus of DV near the putative contact interface of DI and DV in the closed conformation. It was previously shown that beta2GPI is a substrate of thiol oxidoreductases, including human thioredoxin-1 (Trx-1) generating different redox states of disulfide bond Cys288/Cys326, which might serve as a scavenger in oxidative stress protection in the blood stream. In APS patients, anti-DI antibody titers as well as an enhanced risk for thrombotic events are associated with an increase in the oxidized state of the protein. Hitherto, no structural study has been performed in order to prove a correlation of the redox state and the conformation of beta2GPI. Therefore, investigations of beta2GPI conformation in different redox states of disulfide bond Cys288/Cys326 were carried out. In addition, binding of anti-DI autoantibodies to the untreated (native) as well as reduced protein should be explored. At first, cysteine residues of untreated, i.e. native beta2GPI were confirmed to be completely in oxidized state using Ellman’s reagent assay and the absence of binding of a thiol-specific agent. Statistical analyses of AFM images revealed that untreated beta2GPI was mainly in closed conformation (80% in closed and 20% in open conformation) in the respective system. In this study, an optimized protocol for enzymatic reduction of disulfide bond Cys288/Cys326 was established. The agent TCEP was used to reduce human Trx-1, which in turn enzymatically reduced beta2GPI. To block reoxidation of free thiols and to facilitate product analysis, cysteine residues of reduced beta2GPI were subsequently labeled with the sensitive and thiol-specific reagent 3-(N-maleimidopropionyl) biocytin (MPB), which carries a biotin function. During protocol establishment, complete and specific reduction of disulfide bond Cys288/Cys326 was confirmed utilizing SDS-PAGE, streptavidin western blot, mass spectrometry (MS) analyses, and a biotin quantification assay. Protocol improvements constituted a homogenous system with remarkable decrease of unspecifically reduced beta2GPI. Upon beta2GPI reduction, AFM imaging revealed no significant shift in protein conformation (75% in closed and 25% in open conformation). These results were qualitatively confirmed by TEM imaging. Therefore, reduction of beta2GPI disulfide bond Cys288/Cys326 did not result in a major conformational change of the protein. Upon in vitro reduction, the closed form is still the main conformation and a direct correlation of beta2GPI redox state and conformation must be refused. Furthermore, beta2GPI reduction led to a strong and statistically highly significant increase in anti-DI autoantibody binding compared to untreated beta2GPI. Thus, the reduced form might be the antigenic form of the protein. In contrast to previous knowledge, these findings suggest that anti-DI autoantibodies may also bind to the closed conformation under certain conditions. Hypothetically, reduction of beta2GPI could induce a minor structural change in DV that might facilitate the binding of APS autoantibodies. Overall, this study reveals that PTM of beta2GPI may lead to a critical level of destabilization of the closed conformation (as in the case of acetylated beta2GPI) or significantly increase the binding of APS autoantibodies (as in the case of reduced beta2GPI), both of which could have a large impact on APS disease. However, further investigations are necessary to put these new findings in the context of APS immunopathophysiology.

This thesis focuses on the establishment of biocatalytic cascade reactions for the production and detection of industrially relevant flavor and fragrance compounds for food and cosmetic products. To meet the consumer’s demand for those products to be natural, environmentally friendly biocatalytic manufacturing processes that operate GMO-free must be established. Thus, this thesis presents such pathways for the production of an industrially relevant long-chain hydroxy fatty acid and the important flavor and aroma compound raspberry ketone. Furthermore, a biosensor for aldehyde detection was implemented to facilitate screening for suitable biocatalysts that produce industrially relevant aldehydes that are widely applied in the flavor and fragrance industry.

Ac(et)ylation is a post-translational modification present in all domains of life. First identified in mammals in histones to regulate RNA synthesis, today it is known that is regulates fundamental cellular processes also in bacteria: transcription, translation, metabolism, cell motility. Ac(et)ylation can occur at the ε-amino group of lysine side chains or at the α-amino group of a protein. Furthermore small molecules such as polyamines and antibiotics can be acetylated and deacetylated enzymatically at amino groups. While much research focused on N-(ε)-ac(et)ylation of lysine side chains, much less is known about the occurrence, the regulation and the physiological roles on N-(α)-ac(et)ylation of protein amino termini in bacteria. Lysine ac(et)ylation was shown to affect protein function by various mechanisms ranging from quenching of the positive charge, increasing the lysine side chains’ size affecting the protein surface complementarity, increasing the hydrophobicity and by interfering with other post-translational modifications. While N-(ε)-lysine ac(et)ylation was shown to be reversible, dynamically regulated by lysine acetyltransferases and lysine deacetylases, for N-(α)-ac(et)ylation only N-terminal acetyltransferases were identified and so far no deacetylases were discovered neither in bacteria nor in mammals. To this end, N-terminal ac(et)ylation is regarded as being irreversible. Besides enzymatic ac(et)ylation, recent data showed that ac(et)ylation of lysine side chains and of the proteins N-termini can also occur non-enzymatically by the high-energy molecules acetyl-coenzyme A and acetyl-phosphate. Acetyl-phosphate is supposed to be the key molecule that drives non-enzymatic ac(et)ylation in bacteria. Non-enzymatic ac(et)ylation can occur site-specifically with both, the protein primary sequence and the three dimensional structure affecting its efficiency. Ac(et)ylation is tightly controlled by the cellular metabolic state as acetyltransferases use ac(et)yl-CoA as donor molecule for the ac(et)ylation and sirtuin deacetylases use NAD+ as co-substrate for the deac(et)ylation. Moreover, the accumulation of ac(et)yl-CoA and acetyl-phosphate is dependent on the cellular metabolic state. This constitutes a feedback control mechanism as activities of many metabolic enzymes were shown to be regulated by lysine ac(et)ylation. Our knowledge on lysine ac(et)ylation significantly increased in the last decade predominantly due to the huge methodological advances that were made in fields such as mass-spectrometry, structural biology and synthetic biology. This also includes the identification of additional acylations occurring on lysine side chains with supposedly different regulatory potential. This review highlights recent advances in the research field. Our knowledge on enzymatic regulation of lysine ac(et)ylation will be summarized with a special focus on structural and mechanistic characterization of the enzymes, the mechanisms underlying non-enzymatic/chemical ac(et)ylation are explained, recent technological progress in the field are presented and selected examples highlighting the important physiological roles of lysine ac(et)ylation are summarized.

Abstract The known Schiff base compound, (E)1‐benzyl‐3‐((4‐methoxyphenyl)imino)‐5‐methylindolin‐2‐one, was prepared as before by reacting 1‐benzyl‐5‐methylindoline‐2,3‐dione with 4‐methoxyaniline. The product was unambiguously characterized using elemental analysis, 1H and 13C‐NMR spectroscopy, and its new single‐crystal X‐ray structural analysis. Molecular orbital calculations were conducted in order to investigate the structures and relative stabilities of the (E) and (Z) isomers of 1‐benzyl‐3‐([4 methoxyphenyl]‐imino)‐5‐methylindolin‐2‐one. Specific attention was paid to the (E) isomer. The available crystallographic experimental data for the latter ensured also validation of the model structures computationally derived at the theoretical B3LYP/6‐31G(d,p) level.

Scholz et al. developed an electrochemical assay to study the impact of reactive species on self-assembled monolayer (SAM). The aim of this thesis is to use this electrochemical assay with gold supported lipid bilayers instead of SAM to study the effect of reactive species on model membranes that mimic oxidative damage to the biological cell membrane. Here, three questions will be addressed: I) how specific substances such as lipophilic and hydrophilic antioxidants protect a membrane from oxidative damage, II) what are the lipid oxidation products after oxidative damage of the model membrane, and III) whether oxidative damage of the model membranes causes pore formation on lipid bilayer. Electrochemistry was first used to measure the oxidative damage over the entire lipid membrane. Then, mass spectroscopy was used to characterize how lipids as the molecular building blocks of the membrane, change when exposed to reactive species. Imaging the membrane with AFM showed how oxidative damage in the model membrane alters lipid self-assembly within the supported lipid bilayer in nanometer scale. In addition, cold physical plasma (CPP) was used to produce the biological relevant reactive species. This fundamental research demonstrates the great potential of supported lipid bilayers as model membranes and cold physical plasma as a source for the production of biologically relevant reactive species to study the effect of oxidative stress on cell membranes.

Abstract In the RNA world, the exchange of sequence patches between two RNAs is an intriguing evolutionary concept, allowing generation of new RNA molecules with novel functionality. Based on the hairpin ribozyme (HPR) with its unique cleavage‐ligation properties, we here demonstrate RNA supported RNA recombination as a possible scenario for the emergence of larger RNA molecules with more complex functionality. A HPR variant designed for the purpose of recombination is capable of cleaving two different RNA molecules, one being a hammerhead ribozyme (HHR) and the other an aptamer (A), and to subsequently recombine and ligate the resulting fragments to a hammerhead ribozyme that is allosterically controlled (HHA) by a cognate ligand. Two such recombination processes involving aptamers for either theophylline or flavine mononucleotide (FMN) are demonstrated with yields of functional recombination product of up to 34 %.

Bioactive lipids or lipid mediators influence numerous processes like the reproduction, the bone turnover, the pain perception, the cardiovascular function and the immune system. Eicosanoids and oxylipins are parts of the immunomodulatory lipid mediators, which can be synthesized from polyunsaturated fatty acids (PUFAs) by enzymatic and non-enzymatic reactions. Typical members of eicosanoids are prostaglandins and leukotrienes. The properties of bioactive lipids include the activation of inflammatory reactions as well as the support of resolution. Like hormones, they act locally restricted and in low concentrations. Further bioactive lipids exist i.e. intermediates of the sphingolipid class. The biosynthesis of some of these compounds like the prostaglandins can be influenced by different drugs whereas for other groups of lipid selective inhibitors are still missing. Their impact on inflammatory processes and against chronic diseases has already been analyzed, while studies in context with infection are largely limited. Infection of the upper respiratory tract caused by viral and bacterial pathogens constitute a huge burden for the human healthcare. The main pathogens are the Influenza A virus (IAV), Staphylococcus aureus (S. aureus), Streptococcus pneumoniae (S. pneumoniae) and Streptococcus pyogenes (S. pyogenes). Besides mono-infection with one of these pathogens, frequently occurring bacto-viral co-infections exist, which negatively influence the etiopathology. The main task of the immune system is the detection and the elimination of pathogens, which can essentially be affected by lipid mediators. Their instability due to oxidizability, the existence of regioisomers and the low abundance of eicosanoids and other oxylipins are the main problems for their analytical measurement. The mayor objective of this dissertation was the establishment of a suitable analytical method for selected lipid mediators and the detection of infection-related changes. The separation and detection was performed by using high-performance liquid chromatography (HPLC) coupled with triple quad mass spectrometry. This combination is called tandem mass spectrometry (MS/MS). The MS parameters were optimized for approximately 30 lipid mediators by use of chemical standards and the detection was achieved by dynamic multiple reaction monitoring (MRM). Furthermore, the spatial resolution of selected sphingolipids was analyzed in tissue samples using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MS-Imaging). Concerning the HPLC-MS/MS detection, an MS method was established and optimized with standard compounds. Another crucial part of the establishment was the extraction of bioactive lipids from the different sampling materials. Whereas well tested protocols exist for the extraction and detection of lipid mediators, such protocols for MALDI-MS-Imaging are still limited due to the novelty of this measurement. Ultimately, robust and reproducible protocols for both techniques that were used for the analysis of a broad array of samples from infection experiments were established for both techniques. The analyses of infected cell culture, mice and pigs revealed infection-related perturbations of host lipid mediator levels. Depending on the scientific issue, the sample types cell pellets, lungs, spleens, livers, blood plasmas, pawns including bones or bronchoalveolar lavages were analyzed. For MALDI-MS-Imaging, the spatial distribution of sphingolipids in lung and spleen was detected. The present dissertation includes four coherent research scopes, in which the pathogen impact on host-derived lipid mediators was detected with the above mentioned analytical methods. The infection models epithelial cells (article II), mouse (article III and IV) and pig (article I) – the latter as the most human like model - showed different aspects of the host-pathogen interaction. The analysis of samples from IAV infection for all three hosts revealed a couple of similarities for some oxylipins that were also described in human infections. Additionally, cell culture and mouse samples from mono-infections as well as co-infections with the pathogens S. aureus and S. pneumoniae were measured. In particular for the bacterial mono- and co-infections, these are the first published results with aspects of infection related changes of lipid mediators. The additional spatial resolution of the sphingolipid intermediates sphingosine 1-phosphate and ceramide 1-phosphate revealed important new insights into their tissue distribution and changes during co-infection. Article I describes the IAV-specific oxylipin changes in the pig (german landrace) as infection model. Therefore, the sample types lung, spleen, blood plasma, and bronchoalveolar lavage from infected animals at different time points after infection were analyzed and compared with samples from uninfected pigs. Mainly in the lung and the spleen, increased amounts of certain lipid mediators were observed. These changes coincide well with already described alterations in humans and mice. Furthermore, the analysis of different sample material provided an overview about appropriate sample types. Surprisingly, many perturbations were detected in the spleen, which itself was uninfected. Based on the local reaction of lipid mediators, most studies concentrate on sample material with close contact to side of infection. Therefore, this dissertation reveals new insights into a form of systemic immune response. Besides the use of animals with a complex immune system for infection experiments, human bronchial epithelial cells (16HBE) were mono- and co-infected with the pathogens S. aureus, S. pneumoniae and IAV as described in article II. Such cells are the initial barrier for and first contact site with pathogens and thus the comprehension of this host-pathogen interaction is of essential importance. Most changes were detected during pneumococcal infection. Furthermore, the analyzed infections with bacterial pathogens differed from IAV infection by an increased synthesis of 5-hydroxyeicosatetraenoic acid (HETE). For further infections with the above mentioned pathogens, the mouse was used as an infection model. Besides infections affecting the respiratory tract, also the impact of an S. pyogenes infection in different mice strains was analyzed and described in article III. Infection-related changes in prostaglandins, which are involved in bone turnover in swollen pawns as well as enhanced amounts of sepsis- and arthritis-associated lipid mediators were detected, in case arthritis had been induced prior to infection. Furthermore, increased amounts of 20-HETE could be observed for such severe infections. An enhanced biosynthesis of 20-HETE was further confirmed in a high-pathogenic S. aureus LUG2012 infection in article IV for all examined sample types. In this last article of this dissertation, bacterial and viral infections in mice were analyzed similar to those described in article II. Mainly IAV-specific lipid mediator alterations were detected, which are in accordance with the findings of the infected pigs. The additional MALDI-MS-Imaging measurements revealed so far unknown accumulation of ceramide 1-phosphate in lung and spleen as well as enrichment in the red pulp of the spleen. In summary, this dissertation provides substantial lipid mediator profiles for infections in three different model systems with selected bacterial and viral pathogens. The obtained data constitute a suitable basis for continuative research projects, in which the influence of single bioactive lipids on the course of infection could be examined in more detail.

Herein, we report the synthesis of a series of push–pull imines by considering cyclic diamino substituent at the C‐centre and fluoroaryl substituent at the N‐centre. This has been achieved by a selective aromatic nucleophilic substitution of different fluoroarenes by N‐H‐substituted N‐heterocyclic imines (NHIs) at ambient conditions without any additional reagent. Solid‐state molecular structure analysis reveals the elongation of the central C–N bond of the imine functionality, which is consistent with the push–pull nature of these imines. The push–pull nature of these imines is further validated by computational studies.

Abstract The 10–23 DNAzyme is an artificially developed Mg2+‐dependent catalytic oligonucleotide that can cleave an RNA substrate in a sequence‐specific fashion. In this study, new split 10–23 DNAzymes made of two nonfunctional fragments, one of which carries a boronic acid group at its 5′ end, while the other has a ribonucleotide at its 3′ end, were designed. Herein it is demonstrated that the addition of Mg2+ ions leads to assembly of the fragments, which in turn induces the formation of a new boronate internucleoside linkage that restores the DNAzyme activity. A systematic evaluation identified the best‐performing system. The results highlight key features for efficient control of DNAzyme activity through the formation of boronate linkages.

Blood platelets are primary major players in the coagulation cascade, that act upon damage in blood vessels at the subendothelial surface. During this process, platelets change their shape, release granules and aggregate by cross-linking of integrin αIIbβ3 via fibrinogen. The heterodimeric transmembrane receptor integrin αIIbβ3 is highly expressed on platelets and its regulation is bidirectional. Inside-out signaling leads to increased affinity for ligands due to dramatic rearrangements in the integrin conformation changing from an inactive bent conformation to an extended, high-affinity conformation. The swing-out motion of the integrin head domain enables binding of ligands, e.g. fibrinogen, resulting in outside-in signaling guiding kinase activation, shape change, platelet aggregation and spreading, subsequently. Agonists (e.g. thrombin) and other triggers (e.g. shear stress) promote the activity of platelets, making the study of specific proteins delicate. Therefore, this PhD thesis describes a biomimetic system used to study αIIbβ3 membrane receptors. Integrin αIIbβ3 was successfully reconstituted into liposomes and characterized by biophysical and molecular biological methods (e.g. dynamic light scattering, transmission electron microscopy, circular dichroism spectroscopy and flow cytometry). The fusion of liposomes to a solid substrate allows the analysis of potential activation triggers and interaction partners concerning their role in integrin αIIbβ3 activation in a lipid bilayer. Among others, quartz-crystal microbalance measurements show that divalent ions and clinically relevant drugs (e.g. unfractionated heparin and quinine), known to be involved in immune thrombocytopenia (ITP), are certainly candidates which induce integrin activation and minor changes in protein secondary structure. In addition, protein corona formation during contact of nanoparticles with blood components, such as fibrinogen, as well as their interaction with artificial platelet model membranes containing integrins were studied. Moreover, lipid environment can be strongly controlled as integrin activation is dependent on the ratio of liquid-ordered and disordered phases within the membrane. Eventually, by exclusion of disturbances of complex external and internal factors, the established system enables the interaction analysis of various substances with receptors under physiological conditions. In contrast, these disturbances are required to understand the complex machinery of cellular processes in vivo. Hence, an expression platform, on the basis of HEK293 cells, was established to study not only the interaction of integrin αIIbβ3 with cytoskeletal networks, but also the impact of mutations on integrin resulting in a disease-like phenotype. Mutations known to induce Glanzmann thrombasthenia (GT) symptoms, were introduced and led to different mechanical properties of integrin-expressing cells, especially during cell adhesion cells. Thereby, generation of biological and medically-relevant processes combined with the biophysical setup contribute to understand disease mechanisms as well as the action of therapeutic agents in diseases such as GT and ITP.

The aims of this thesis were the identification and development of whole-cell biocatalysts for the regio- and stereoselective hydroxylation of steroids, including hormones and bile acids by P450 monooxygenases. Steroids and their derivatives are applied as therapeutic agents. The chemical synthesis of such compounds depends on multi-step procedures, in a stereo- and regiospecific manner involving the protection and deprotection of functional groups and toxic reagents and intermediates. In this thesis, different P450 monooxygenases were investigated as ‘bio-based’ alternatives to chemical catalysts for the late-stage functionalization of steroids and bile acids and engineered by directed evolution procedures towards desired transformation activities. In Article I, the 16α-hydroxylation activity of the bovine CYP17A1 was enhanced by protein engineering to improve the transformation of progesterone into 16α-hydroxyprogesterone in Saccharomyces cerevisiae. Article II follows the same line of research and targets the selective synthesis of bile acid derivatives in Escherichia coli (E. coli) whole-cells. The P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus (S. antibioticus) was identified, which selectively hydroxylates bile acids like lithocholic acid (LCA) and deoxycholic acid (DCA) at the 6β-position, yielding murideoxycholic acid (MDCA), a gallstone solubilizing agent, and 3α-,6β-,12α-trihydroxy-5β-cholan-24-oic acid, respectively. The utilization of OleP as catalyst resulted in shorter synthesis routes for both compounds and additional in a higher yield for MDCA. Building on the results of Article II and the protein engineering approach from Article I, Article III deals with the switch of regioselectivity of the identified CYP107D1 from 6β- to 7β-hydroxylation to form the therapeutic agent ursodeoxycholic acid (UDCA) from LCA by direct hydroxylation. Following a rational protein engineering strategy, a variant with nearly perfect selectivity for UDCA formation was found. Until today, UDCA is either isolated from bile of catheterised farmed bears or produced semisynthetically through low-yielding multistep reactions starting from cholic acid (CA). Article III presents the first reported enzyme for the direct 7β-hydroxylation of LCA to UDCA.

This thesis deals with the process considerations and optimizations of a whole-cell enzyme cascade reaction for the synthesis of ɛ-caprolactone. The enzyme cascade synthesis of ɛ-caprolactone has been conceptualized and verified using a dehydrogenase and a monooxygenase. The advantage of this enzyme combination is the closed-loop co-factor regeneration. Dehydrogenase and monooxygenase expressed in discrete whole cells were applied in defined ratio to conceptualize the cascade reaction. This necessitates the use of separate co-factor regeneration system due to impermeability of the E. coli cell wall to the co-factor. Article I deal with the design and optimization of dehydrogenase and monooxygenase co-expression in a same E. coli cell. In Article II, the cascade reaction was upscaled and a fed-batch process was realized. Following which, the important reaction metrices were analyzed and optimized. Article III extends the two-enzyme cascade with a lipase. The use of lipase helps to overcome the product inhibition of monooxygenase by ɛ-caprolactone.

Die akute Pankreatitis ist eine der häufigsten nicht malignen gastrointestinalen Erkrankungen, die zu Krankenhausaufenthalten führt. Sie ist als Selbstverdau des Pankreas durch seine eigenen Proteasen wie z.B. Trypsin, Elastase und Chymotrypsin definiert. Als Ursprung der Erkrankung wird die frühzeitige intrazelluläre Aktivierung dieser Verdauungsenzyme angesehen. Dies führt zum Zelltod der Azinuszellen und zur Schädigung des Gewebes. Während der akuten Pankreatitis kommt es in 20% der Fälle zu einem schweren Verlauf der Erkrankung, der mit Organversagen in der Lunge und den Nieren assoziiert ist. Es ist bekannt, dass es zu einer Entzündungsreaktion kommt, bei der große Mengen an Zytokinen ausgeschüttet werden. Leukozyten infiltrieren das Pankreas und verstärken den Gewebeschaden. Es kommt zur Freisetzung von DAMPs, die das angeborene und adaptive Immunsystem aktivieren. Bislang ist nicht gut untersucht, wie das Immunsystem den schweren Verlauf der akuten Pankreatitis beeinflusst und es gibt wenig Theorien über den Organschaden in der Lunge und den Nieren. In dieser Arbeit lag der Fokus auf dem Organschaden in Lunge und Niere und die Wirkung von Interleukin 33 (IL33) auf die Zellen des angeborenen Immunsystems und deren Einwanderung in verschiedene Organe während der schweren akuten Pankreatitis im Mausmodell. Die schwere akute Pankreatitis wurde mittels Gangligatur und einmaliger Gabe von Caerulein an Tag 2 nach Gangligatur induziert. An Tag 3 nach Induktion wurden die Mäuse getötet und die Organe wurden für weitere Analysen entnommen. Am dritten Tag nach Induktion der Pankreatitis kam es zu einem Organschaden in der Lunge und den Nieren. In der Lunge fand sich eine Verdickung der Alveolarsepten und eine Verdichtung des Gewebes sowie eine Infiltration von Leukozyten und ein Ödem. In der Niere waren ebenfalls strukturelle Veränderungen zu finden und eine Infiltration von Leukozyten war zu beobachten. In durchflusszytometrischen Analysen der Lunge konnte beobachtet werden, dass CD11b+CD62L+ Monozyten während der akuten Pankreatitis signifikant anstiegen. Mittels RT-DC wurde gezeigt, dass diese Monozyten an Tag 3 signifikant an Größe zugenommen hatten. Mit einer CD11b Färbungen von Lungen und Nieren konnte die Infiltration durch Monozyten bestätigt werden. Unter einer Blockade von Monozyten durch systemische Gabe von anti-CCR2-Antikörpern verringerte sich die Schädigung in Lunge und Niere während der Pankreatitis signifikant. Diese Daten legen nahe, dass der Organschaden in der schweren akuten Pankreatitis durch infiltrierende Monozyten verursacht wird, die über CD62L (L-Selektin) an die Gefäßwände binden und über ihre Größe Gefäße verstopfen, was in den Kapillaren zur Ischämie führt. In vitro sezernierten Makrophagen, die mit CCK stimulierten Azinuszellen co-inkubiert wurden, IL33. Im Mausmodell wurde IL33 mittels sST2 blockiert, was die Schädigung des Pankreas in der Pankreatitis reduzierte. In IL33-depletierten Tieren fand sich im Vergleich zum Wildtyp ein geringerer Lungenschaden aber eine unveränderte Nierenschädigung. Somit scheint IL33 eine Rolle bei der Monozyten-vermittelten Organschädigung in der Pankreatitis zu spielen, die sich auf Grund von kompensatorischen Regulationsmechanismen im globalen IL33 Knock-out weniger gut belegen lässt als nach IL33 Inhibition. Die Hemmung von IL33 zur Behandlung der akuten Pankreatitis stellt somit ein vielversprechendes Therapieprinzip dar.

The leading idea of this thesis is to study the effects of (i) membrane composition and (ii) membrane environment (aqueous phases) on the redox properties of membrane-confined redox active compounds. For solutions, it is known since long, how strong solvents affect the redox properties of dissolved redox active species. However, for membranes this question has not yet been addressed, although it can be supposed that such effects may be important to understand the role of membrane-confined redox active compounds in biological systems. To interrogate this problem, a monolayer model was chosen. It consists of a lipid monolayer with embedded menaquinones on mercury electrodes. Since ion transfer across membranes is also a crucial question, in the first part of this project, 2,2-diphenyl-1-picrylhydrazyl (DPPH) was studied as a new redox probe for transferring anions and cation between an organic and an aqueous phase. The important findings of this thesis are: (i) accessing the ion pair equilibrium constant of anions and cations with DPPH redox probe as a model study using the three-phase electrochemistry, (ii) the redox potentials of menaquinone-4, -7, and -9 in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) monolayers and the acidity constants of menaquinones (MK’s) in membranes monolayer model, and (iii) the effects of membrane composition and the aqueous environment on the thermodynamics and kinetics of MK’s in membrane models.

G-quadruplexes (G4s) have been in the focus of research in the last decades for their regulatory roles in vivo and for their use in nano- and biotechnology. However, an understanding of the various factors that drive a particular quadruplex fold remains limited, challenging rational therapeutic targeting and design of these tetrahelical structures. In this regard, insights from modified G-quadruplexes may help to deepen our knowledge of G-quadruplex structure. In this dissertation, sugar-modified guanosine analogs are exploited for their altered conformational preferences regarding both glycosidic bond angle and sugar pucker by their incorporation into different syn positions of the G-core of a model G-quadruplex. Induced structural perturbations as characterized by NMR spectroscopy range from a local change in tetrad polarity to a complete refolding into an unusual structure with a V-shaped loop, a unique G4 structural element in the focus of this work. Detailed conformational analysis of the introduced G analogs and high-resolution structures of the modified quadruplexes reveal a complex interplay of glycosidic torsion angle, sugar pucker preferences and local interactions, which may all play a leading role in driving G4 folding.

Abstract Methylation of free hydroxyl groups is an important modification for flavonoids. It not only greatly increases absorption and oral bioavailability of flavonoids, but also brings new biological activities. Flavonoid methylation is usually achieved by a specific group of plant O‐methyltransferases (OMTs) which typically exhibit high substrate specificity. Here we investigated the effect of several residues in the binding pocket of the Clarkia breweri isoeugenol OMT on the substrate scope and regioselectivity against flavonoids. The mutation T133M, identified as reported in our previous publication, increased the activity of the enzyme against several flavonoids, namely eriodictyol, naringenin, luteolin, quercetin and even the isoflavonoid genistein, while a reduced set of amino acids at positions 322 and 326 affected both, the activity and the regioselectivity of the methyltranferase. On the basis of this work, methylated flavonoids that are rare in nature were produced in high purity.

Abstract Environmentally‐friendly processes for the manufacturing of valuable industrial compounds like ω‐hydroxy fatty acids (ω‐OHFAs) are highly desirable. Herein, we present such an approach by establishing a two‐step enzymatic cascade reaction for the production of 2,15,16‐trihydroxy hexadecanoic acid (THA). Starting with the easily accessible natural compound ustilagic acid (UA) that is secreted by the corn smut fungus Ustilago maydis, the recombinantly expressed esterase BS2 from Bacillus subtilis and the commercial β‐glucosidase from almonds were applied yielding 86 % product. Both hydrolases do not require expensive cofactors, making the process economically attractive. Additionally, no harmful solvents are required, so that the product THA can be labelled natural to be used in food and cosmetic products.

The aim of our research is a stereoselective synthesis development of 4-aminocyclohexanol by the application of a keto reductase (KRED) and an amine transaminase (ATA). 4-Aminocyclohexanol is a valuable precursor for active pharmaceutical ingredients, for example, lomibuvir (a HCV protease inhibitor), ambroxol (a secretolytic agent) and other bioactive molecules. Today, the trans-4-aminocyclohexanol is accessed via Ni-catalyzed synthetic procedure giving moderate yields. In our project we perform cis- and trans-4-aminocyclohexanol synthesis from 1,4-cyclohexanedione (a bio-based precursor) by an one-pot approach combining sequentially a KRED and an ATA as catalysts. For this, we envisaged two multistep enzymatic procedures. The route A would involve 4-hydroxycyclohexanone formation from 1,4-cyclohexanedione via a KRED-catalyzed monoreduction and a further transamination mediated by an ATA towards 4-aminocyclohexanol. The route B would consist of switching the steps of the previous sequential approach, that is, a monoamination of the diketone to yield 4-aminocyclohexanone, and the subsequent reduction of the remaining carbonyl group. Only route A turned out to be feasible, and we performed 4-aminocyclohexanol synthesis at the preparative scale in the sequential and tandem modes. Depending on the ATA, both isomers can be obtained.

Abstract A N‐heterocyclic olefin (NHO), a terminal alkene selectively activates aromatic C−F bonds without the need of any additional catalyst. As a result, a straightforward methodology was developed for the formation of different fluoroaryl‐substituted alkenes in which the central carbon–carbon double bond is in a twisted geometry.

Abstract Over the last years, there has been an enormous increase in the knowledge on koi herpesvirus (KHV), koi herpesvirus disease (KHVD), pathogenesis and virus variants. Different KHV lineages have clearly been identified, possible genomic changes during replication in different cell cultures at different temperatures but also in several hosts have been identified, a persistent stage of infection has been specified and it has been shown that infection with KHV is not host specific at all, but KHVD is. Additionally, it has been shown that it is possible to combat KHVD by immunization with inactivated and attenuated live vaccines using different delivery systems but also to benefit from alternative treatments with e.g. exopolysaccharids obtained from Arthrospira platensis.

The hirudin‐like factor 1 (HLF1) of Hirudo medicinalis belongs to a new class of leech‐derived factors. In previous investigations, HLF1 did not exhibit anticoagulatory activities. Here, we describe the analysis of natural and synthetic variants of HLF1 and HLF‐Hyb, a yet uncharacterized member of the HLF family. Modifications within the N terminus of HLF1 have a strong impact on its activity. Some variants of HLF1 exhibit thrombin‐inhibiting activity comparable to hirudins, whereas others have reduced or no activity. The analyses of HLF‐Hyb variants revealed a strong impact of the central globular domain on activity. Our results indicate a comparable mode of action of hirudins and thrombin‐inhibiting HLF variants. Finally, we propose and discuss criteria for classifying hirudins and HLFs.

Abstract A DNA G‐quadruplex adopting a (3+1) hybrid structure was modified in two adjacent syn positions of the antiparallel strand with anti‐favoring 2′‐deoxy‐2′‐fluoro‐riboguanosine (FrG) analogues. The two substitutions promoted a structural rearrangement to a topology with the 5′‐terminal G residue located in the central tetrad and the two modified residues linked by a V‐shaped zero‐nucleotide loop. Strikingly, whereas a sugar pucker in the preferred north domain is found for both modified nucleotides, the FrG analogue preceding the V‐loop is forced to adopt the unfavored syn conformation in the new quadruplex fold. Apparently, a preferred C3′‐endo sugar pucker within the V‐loop architecture outweighs the propensity of the FrG analogue to adopt an anti glycosidic conformation. Refolding into a V‐loop topology is likewise observed for a sequence modified at corresponding positions with two riboguanosine substitutions. In contrast, 2′‐F‐arabinoguanosine analogues with their favored south‐east sugar conformation do not support formation of the V‐loop topology. Examination of known G‐quadruplexes with a V‐shaped loop highlights the critical role of the sugar conformation for this distinct structural motif.

Abstract Erucic (22:1, cisΔ13) and gondoic acids (20:1, cisΔ11) are building blocks obtained from renewable sources for the oleochemical industry. Different biocatalytic strategies for the enrichment of these compounds with high recovery yields were developed in our group. Geotrichum candidum lipases (GCL) strongly discriminate against fatty acids longer than 18 carbon atoms. Thus, GCL‐I and ‐II were investigated using hydrolysis or ethanolysis reactions with Crambe and Camelina oils. Hydrolysis was also studied using fatty acid ethyl esters (FAEE) derived from the corresponding oil. Both isoforms were highly selective; however, interesting differences were observed. Although it has been reported that GCL‐I displays a higher preference toward 18 cisΔ9, which is present in the studied oils at high levels, GCL‐II showed higher enrichment values during hydrolysis independent of the substrate used. Hence, enrichments of 87% (Crambe oil) and 82% (Crambe FAEE) for erucic acid and 50% (Camelina oil) and 45% (Camelina FAEE) for gondoic acid, with recovery values between 89% and 99%, were achieved. On the contrary, the best enzyme for ethanolysis was GCL‐I (82% and 41% for erucic and gondoic acid, respectively). In this case, although GCL‐II also displayed good enrichment and recovery levels (77% and 28%, respectively), they were lower compared to the former reactions. In both ethanolysis reactions, the FAEE fraction contained between 92% and 97% of 18 unsaturated fatty acids.

Abstract The efficient multifunctionalization by one‐pot or cascade catalytic systems has developed as an important research field, but is often challenging due to incompatibilities or cross‐reactivities of the catalysts leading to side product formation. Herein we report the stereoselective preparation of cis‐ and trans‐4‐aminocyclohexanol from the potentially bio‐based precursor 1,4‐cyclohexanedione. We identified regio‐ and stereoselective enzymes catalyzing reduction and transamination of the diketone, which can be performed in a one‐pot sequential or cascade mode. For this, we identified regioselective keto reductases for the selective mono reduction of the diketone to give 4‐hydroxycyclohexanone. The system is modular and by choosing stereocomplementary amine transaminases, both cis‐ and trans‐4‐aminocyclohexanol were synthesized with good to excellent diastereomeric ratios. Furthermore, we identified an amine transaminase that produces cis‐1,4‐cyclohexanediamine with diastereomeric ratios >98 : 2. These examples highlight that the high selectivity of enzymes enable short and stereoselective cascade multifunctionalizations to generate high‐value building blocks from renewable starting materials. Introduction

Purpose: The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 may be involved in regulating re-entry of residual hepatocytes into the cell cycle upon loss of liver tissue by partial hepatectomy (PH). As yet, changes in Kip1 expression during the initial period following PH are not well-characterized. We investigated immediate changes in Kip1 mRNA and protein levels as well as changes in Kip1 phosphorylation in liver tissue within the relevant time window between surgery and the onset of DNA synthesis at 10–12 h. Methods: We used real-time PCR, quantitative Western blotting, and immune histochemistry on tissue samples of adult rats obtained during or between 2 and 10 h after surgical removal of two thirds of the liver to analyze Kip1 mRNA or protein levels, respectively, or to quantify nuclear expression of Kip1. Results: Kip1 mRNA was down-regulated within 4 h after PH by 60% and remained unchanged thereafter up to 10 h. With a lag phase of 2–3 h, Kip1-protein was down-regulated to a level of 40% of the control. The level of Thr187-phosphorylated Kip1 started to increase at 4 h and reached a maximum level at 8–10 h after PH. Kip1 immunoreactivity was observed in 30% of the hepatocytes before PH. Within 6–8 h after PH, more than half of the hepatocytes lost nuclear Kip1 signals. Kip1-specific micro-RNAs (miRNA221, miRNA222) were not changed upon PH. Conclusions: A portion of hepatocytes in adult rats constitutively express Kip1 and down-regulate Kip1 immediately upon PH. This response involves transcriptional processes (loss of Kip1 mRNA) as well as accelerated degradation of existing protein (increase in pThr187-phosphorylation mediating polyubiquitinylation and proteasomal degradation of Kip1). Kip1 down-regulation occurs precisely within the intervall between surgery and onset of DNA synthesis which supports the hypothesis that it mediates activation of G0/0S-phase Cdk/cyclin-complexes and re-entry of hepatocytes into the cell cycle.

Polykristallines Gold wurde bereits seit dem Ende des 19. Jahrhunderts elektrochemisch charakterisiert und seit Anfang des 20. Jahrhunderts regelmäßig als Arbeitselektrode in der elektrochemischen Analytik genutzt. Fälschlicherweise und trotz erster gegenteiliger Indizien, dominierte die Annahme, dass mechanisches Polieren die einzelnen Einkristallflächen des polykristallinen Materials freilegen würde, und dass deren statistisch gewichtetes elektrochemisches Verhalten reproduzierbar abgebildet werden könne. Mit dem Aufkommen neuer und verbesserter Verfahren zur Erzeugung hochwertiger Einkristallflächen parallel zur Entwicklung und Verbreitung leistungsstarker Techniken zur Oberflächenanalyse, konzentrierte sich die Goldforschung ab der Mitte des 20. Jahrhunderts auf die Charakterisierung der Einkristallflächen, ohne jedoch die neugewonnenen Erkenntnisse für die Interpretation des polykristallinen Materials zu nutzen. Gegenstand dieser Arbeit war daher die Kombination elektrochemischer Methoden (lineare und zyklische Voltammetrie) mit modernen Oberflächenanalysetechniken (Röntgendiffraktion, elektrochemische Unterpotentialabscheidung von Blei-Ionen) und bildgebenden Verfahren (AFM, STM, REM) zur Charakterisierung verschieden vorbehandelter polykristalliner Goldelektroden. Zudem sollte das elektrochemische Verhalten dieser Elektroden basierend auf dem bisherigen Wissen über das Verhalten der Einkristallflächen interpretiert werden. Der Großteil der erzielten Ergebnisse wurden in den drei Publikationen veröffentlicht, die den Hauptteil dieser Dissertation bilden. Zunächst konnte eine temporäre Aktivierung mittels mechanischer oder elektrochemischer Bearbeitung sowie eine Inaktivierung durch chemisches Ätzen in sauerstoffgesättigter Kaliumcyanidlösung, bezüglich der Sauerstoffreduktion als Referenzreaktion nachgewiesen werden, wobei Aktivierung und Inaktivierung relativ sind und im Zusammenhang mit der Anzahl sogenannter aktiver Zentren auf der Elektrodenoberfläche stehen (Publikation 1). Darüber hinaus erwiesen sich kontinuierliche Oxidations- und Reduktionszyklen an polierten polykristallinen Goldelektroden in schwefelsaurer Lösung als eine neue, Zusatzstoff freie Methode für die Goldnanopartikelsynthese, da diese wohldefinierte und immobilisierte Goldkristallite auf den Elektrodenoberflächen erzeugt (Publikation 2). Die sequenzielle Kombination aus Argon-Ionenstrahlätzen und thermischem Ausheizen hat sich hingegen als effiziente Methode zur Erzeugung sauberer und glatter Elektrodenoberflächen mit hoher atomarer Ordnung erwiesen (Publikation 3). Zugleich konnte gezeigt werden, dass polykristallines Gold ein eigenständiges Material ist, dessen Eigenschaften und Verhaltensweisen nicht ausschließlich auf das statistisch gewichtete elektrochemische Verhalten der einzelnen Einkristallflächen zurückzuführen sind, sondern auch von anderen energetischen Aspekten, wie beispielsweise der Koordination der Oberflächenatome im Kristallgitter, bedingt werden (Publikation 2 und 3).

In 2010, the identification of 17 novel (R)-ATAs represented a breakthrough for the biocatalytic asymmetric synthesis of chiral amines, because only one (R)-ATA was described before. These novel ATAs were identified in a bioinformatic approach by studying the substrate acceptance of BCATs and DATAs to deduce the unknown substrate coordination of (R)-ATAs. Article I describes an alternative approach for the identification of (R)-ATA activity by reengineering the substrate- recognition site of α-AATs. While the engineering of the eBCAT led to the formation of an initial (R)-amine acceptance only, the (R)-ATA activity was successfully introduced in the DATA scaffold. These results demonstrate the transformation of an α-AAT in a moderately active (R)-ATA for the first time and highlight the evolutionary relationship between α-AATs and ATAs. Despite the availability of different ATAs nowadays, their substrate spectrum is limited due to the natural composition of their active sites. Several protein-engineering studies showed the widening of the substrate spectrum and the acceptance of bulky substrates by screening large mutant libraries to identify beneficial variants. In Article II, we developed an in silico engineering approach for amine transaminases to improve the conversion of bulky substrates and to reduce the number of variants to be tested in the laboratory. The resulting double-mutants of the (S)-ATA from C. violaceum displayed a >200-fold improved activity towards the bulky benchmark substrate. These variants expand the available biocatalytic toolbox for the synthesis of bulky amines, and the developed framework paves the way for rational protein-engineering protocols. By studying unconventional transaminase substrates, we explored the potential of the available in- house transaminase toolbox in Articles III, IV, V, and VI. In Article III, we showed the transamination of a β-keto ester, leading to the synthesis of β-phenylalanine. The described cascade in Article IV enables the synthesis of amino carbohydrates. In addition, Article V describes an enzymatic cascade for the synthesis of amino fatty acids, which was extended in Article VI to obtain fatty amines. The findings of this thesis clearly contribute to the understanding of the substrate scope and specificity of amine transaminases and expand the application of this versatile biocatalyst beyond classical ketone substrates.

Given the central metabolic role of the liver, hepatic metabolites and transcripts reflect the organismal physiological state. Biochemical-clinical plasma biomarkers, hepatic metabolites, transcripts, and single nucleotide polymorphism (SNP) genotypes of some 300 pigs were integrated by weighted correlation networks and genome-wide association analyses. Network-based approaches of transcriptomic and metabolomics data revealed linked of transcripts and metabolites of the pentose phosphate pathway (PPP). This finding was evidenced by using a NADP/NADPH assay and HDAC4 and G6PD transcript quantification with the latter coding for first limiting enzyme of this pathway and by RNAi knockdown experiments of HDAC4. Other transcripts including ARG2 and SLC22A7 showed link to amino acids and biomarkers. The amino acid metabolites were linked with transcripts of immune or acute phase response signaling, whereas the carbohydrate metabolites were highly enrich in cholesterol biosynthesis transcripts. Genome-wide association analyses revealed 180 metabolic quantitative trait loci (mQTL) (p < 10-4). Trans-4-hydroxy-L-proline (p = 6 × 10-9), being strongly correlated with plasma creatinine (CREA), showed strongest association with SNPs on chromosome 6 that had pleiotropic effects on PRODH2 expression as revealed by multivariate analysis. Consideration of shared marker association with biomarkers, metabolites, and transcripts revealed 144 SNPs associated with 44 metabolites and 69 transcripts that are correlated with each other, representing 176 mQTL and expression quantitative trait loci (eQTL). This is the first work to report genetic variants associated with liver metabolite and transcript levels as well as blood biochemical-clinical parameters in a healthy porcine model. The identified associations provide links between variation at the genome, transcriptome, and metabolome level molecules with clinically relevant phenotypes. This approach has the potential to detect novel biomarkers displaying individual variation and promoting predictive biology in medicine and animal breeding.

Im Fokus dieser Arbeit lagen die Synthesen verschiedener Nukleosidderivate zur Aufklärung der Struktur und Funktion von RNA-Molekülen. Es wurden erfolgreich zwei Adenosinderivate synthetisiert und die für die post-synthetische Markierung benötigte Aminofunktion entweder mit Hilfe der Sonogashira-Kupplung an der Position C2 oder der Heck-Reaktion an der Position C8 eingebaut. Um auch Zugang zu modifizierten Cytidinen zu erhalten, wurde eine Synthesestrategie für ein aktiviertes Uridinderivat entworfen, um dieses nach der chemischen Synthese mittels Phosphoramiditverfahren, während der Reinigung, in das dazugehörige Cytidinderivat umzuwandeln. Hierzu wurden die funktionellen Gruppen erfolgreich für die chemische Oligonukleotidsynthese geschützt, die Modifikation an der Position C5 mit Hilfe der Sonogashira-Kupplung eingebaut und die Position C4 mit Hilfe von TIPS-Cl (2,4,6-Triisopropylbenzolsulfonylchlorid) aktiviert. In Vorversuchen konnte die erfolgreiche Umwandlung in das Cytidinderivat experimentell bestätigt werden. Im zweiten Teil der Arbeit wurde der Einfluss ausgewählter basenmodifizierter Nukleoside auf den Charakter einer doppelsträngigen RNA untersucht. Dazu wurden die Schmelzkurven und Schmelzpunkte modifizierter und unmodifizierter Oligonukleotide gemessen und ausgewertet. Die erhaltenen Daten lassen darauf schließen, dass der Einbau von basenmodifizierten Nukleosiden zur Senkung des Schmelzpunktes führt, jedoch nicht zur Veränderung des doppelsträngigen Charakters. Eine anschließende Markierung eines modifizierten Oligonukleotids mit dem Farbstoff ATTO 680 scheint nur einen marginalen Einfluss auf den Schmelzpunkt, im Vergleich zu den Schmelzpunkten der modifizierten Oligonukleotide, zu haben. Für die Untersuchung der Funktion und Struktur von größeren RNA-Molekülen, wie zum Beispiel ROSE-Elementen, wurde eine Strategie zu deren Herstellung mit Hilfe der T4 RNA Ligase I entwickelt und ex-perimentell bestätigt. Dazu wurde das ROSE-Element in drei Segmente geteilt, diese chemisch synthetisiert, gereinigt und mit Hilfe der T4 RNA Ligase I zum vollständigen Element ligiert. Dabei konnte das ROSE-Element erfolgreich vom 5´-Terminus aufgebaut werden. Es steht nun eine Methode zur Verfügung, um auch modifizierte Oligonukleotide zu einem ROSE-Element zu ligieren und dieses auf seine Funktion und Struktur hin zu untersuchen. Eine RNA 4-way-junction wurde durch Hybridisierung generiert und für strukturelle Untersuchungen verfügbar gemacht.

Bei der Arzneimittelentwicklung liegt der Fokus nicht nur auf der Wirksamkeit und Sicherheit einer pharmakologisch aktiven Substanz, sondern auch auf einer möglichst einfachen, idealerweise oralen Applikation. Um die benötigten Wirkstoffkonzentrationen im Zielorgan zu erreichen, wird die einzunehmende Dosis eines Medikaments in Abhängigkeit der präsystemischen Elimination ermittelt. Inzwischen ist bekannt, dass nicht ausschließlich der hepatische, sondern auch der intestinale Stoffwechsel die orale Bioverfügbarkeit eines Medikaments wesentlich beeinflussen kann. Arzneistoffe, die während der Darmpassage einer starken Metabolisierung unterliegen, sind zudem prädestiniert für unerwünschte Interaktionen mit anderen Substanzen, welche die entsprechenden Stoffwechselenzyme hemmen oder induzieren. Für die Abschätzung pharmakokinetischer Parameter eines neuen Wirkstoffs sind daher Kenntnisse zur Expression sowie Funktion klinisch relevanter intestinaler Stoffwechselenzyme von Bedeutung. Bisher publizierte Daten basieren größtenteils auf der Genexpression, obwohl aufgrund posttranskriptionaler Prozesse nicht zwingend Aussagen zur resultierenden Proteinmenge getroffen werden können. Die verfügbaren Daten zum intestinalen Proteingehalt wurden mittels immunologischer Methoden erhoben, die erhebliche Limitationen in Bezug auf Spezifität, Reproduzierbarkeit und Robustheit aufweisen. Diese Aspekte finden bei den inzwischen etablierten LC-MS/MS-basierten Targeted-Proteomics-Methoden Berücksichtigung. Dazu werden die Proteine einer Messprobe enzymatisch gespalten, um entstehende proteospezifische Peptide zur Quantifizierung der Proteine von Interesse zu nutzen. Ein Ziel der vorliegenden Arbeit bestand in der Entwicklung und Validierung einer entsprechenden Methode zur gleichzeitigen Bestimmung von CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, UGT1A1, UGT1A3, UGT2B7 sowie UGT2B15 in biologischen Matrices, welche die aktuell gültigen Leitlinien in Bezug auf Selektivität, Linearität, Richtigkeit, Präzision und Stabilität erfüllt. Bereits bei der ersten Anwendung der Methode zur Quantifizierung der Enzyme in kommerziell erhältlichen und selbst isolierten Mikrosomen zeigte sich, welchen erheblichen Einfluss die Probenvorbereitung auf die ermittelten Proteingehalte hat. Diese Erkenntnis wurde im Rahmen eines internationalen Projektes bestätigt, bei dem humane Leberproben desselben Ursprungs in diversen Laboren mit den dort etablierten Methoden prozessiert worden sind. Bezogen auf die eingesetzte Gewebemenge ergaben sich bei der Messung der Mikrosomen 6 - 30-fach geringere Enzymgehalte als bei der Analyse des nicht-fraktionierten Gewebes, da die subzelluläre Aufspaltung einer Probe mit erheblichen Proteinverlusten einhergeht. Folglich wurden alle weiteren Untersuchungen zur absoluten Enzymquantifizierung unter Verwendung von filterbasierten Zentrifugaleinheiten (filter aided sample preparation; FASP) mit Gesamtgewebelysatproben durchgeführt. Sowohl die optimierte Probenaufarbeitung als auch die validierte Targeted-Proteomics-Methode fanden bei der Untersuchung der Darmsegmente von 9 Spendern Anwendung, wobei jeweils Gewebe aus dem Duodenum, oberen und unteren Jejunum, Ileum sowie Colon zur Verfügung stand. Von den 13 untersuchten Enzymen wurden in allen Dünndarmabschnitten nur CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, UGT1A1, UGT1A3 und UGT2B7 nachgewiesen, deren Gehalt im Jejunum am höchsten war. Im Colon wurde auf Proteinebene keines der Metabolisierungsenzyme detektiert. Die entsprechenden Genexpressionsdaten dieser 8 Enzyme korrelieren signifikant mit den ermittelten Proteinwerten. Korrespondierend zur fehlenden Nachweisbarkeit der übrigen 5 Enzyme auf Proteinebene waren die Gene CYP2B6, CYP2C8, CYP2E1 sowie UGT2B15 nur sehr geringfügig und CYP1A2 gar nicht exprimiert. Zur Charakterisierung der metabolischen Aktivität der intestinalen Enzyme wurde eine weitere LC-MS/MS-basierte Methode entwickelt und validiert. Als Modellsubstrate fungierten Diclofenac (CYP2C9), Omeprazol (CYP2C19), Dextromethorphan (CYP2D6), Midazolam (CYP3A), Ezetimib (UGT1A) und Naloxon (UGT2B7). Die begrenzte Verfügbarkeit des intestinalen Gewebes sowie dessen sehr geringer mikrosomaler Proteingehalt stellten besondere Anforderungen an die Sensitivität der Methode. Ihre Eignung zur Charakterisierung der intestinalen Metabolisierungsaktivität wurde bei der Anwendung auf ein jejunales Mikrosomen-Gemisch gezeigt. Die im Rahmen dieser Arbeit generierten Daten zur Expression klinisch bedeutsamer Metabolisierungsenzyme entlang des humanen Darms tragen zu einem besseren Verständnis des intestinalen First-Pass-Metabolismus bei. Diese Kenntnisse können sowohl bei der Entwicklung neuer Arzneistoffe als auch für die Erstellung von Physiologie-basierten pharmakokinetischen Modellen (PBPK-Modellen) nützlich sein, um die orale Bioverfügbarkeit sowie das Interaktionspotential pharmakologisch aktiver Substanzen abzuschätzen.

Molybdenum dependent enzymes are involved in essential metabolic transformations in bacteria, plants, and human beings. The extreme instability of the molybdenum cofactor (Moco) prevents its use as an effective treatment for patients with a Moco deficiency. Therefore, the design, develop and execute the artificial molybdenum cofactor models are essential. In the present thesis, the asymmetric molybdopterin (mpt) model precursors with oxygen functionality and various electronic structures and their Moco model complexes mimicking the natural cofactor have been synthesized and comprehensively investigated through multi-nuclear NMR, MS, IR, resonance Raman, X-ray crystallography, UV-Vis, and electrochemical methods. Notably, the asymmetrically substituted dithiolenes in this thesis are confirmed through a significant push-pull effect, which is tuning its electronic structure. The redox behavior of Moco model complexes was investigated by temperature-dependent cyclic voltammetry. Electronic and vibrational spectral studies were investigated in detail to understand substituents effect on the electronic structure of model complexes and to elucidate roles of mpt in catalysis. Since the model complexes can be considered as structural models for the Moco dependent oxidoreductases, catalytic oxygen atom transfer (OAT) reactions in DMSO/PPh3 were investigated. The main focus of the present thesis was achieved through the development of various synthetic routes that address phosphonate bearing dithiolene ligands, inspiring the natural mpt. Simultaneously the Minisci protocol was applied for the synthesis of new pterin ketophosphonates, taking into consideration the essential aspects of the natural molybdopterin, including the phosphate anchor group. Even though some aspects of this protocol require further optimizations, but the mentioned synthetic route has exceptional potential and flexibility.

In acinar cells, cellular organelles like zymogene granule, mitochondria, endoplasmic reticulum and lysosome functions in coordinate way in order to synthesize and secrets large amounts of digestive enzyme. Dysfunction of this organelle, results into enzyme activation within acinar cell; ultimately, acute pancreatitis. While previous studies reported that mitochondrial function is disrupt but mechanism of clearance of these mitochondria remains unknown during pancreatitis. Here we reported that PINK1 and Parkin mediated pathway is activated during pancreatitis and clears dysfunctional mitochondria in-vivo. PINK1 or Parkin deficient acinar cell had energy crisis, decreased ATP production and altered acinar cell fate in-vitro. Inhibiting clearance of dysfunctional mitochondria aggravates experimental pancreatitis severity and delays regeneration/recovery of exocrine tissue after disease via PARIS-PGC-1α pathway. While an attempt to explore therapeutic target of PARIS-PGC-1α pathway by treatment of SRT1720 rescued experimental pancreatitis. Together, PINK1 and Parkin, restricts exocrine pancreatic damage in pancreatitis and accelerates tissue recovery after disease.

Oils and fats from natural origin are sustainable sources for a broad range of economically relevant products in food, feed, fuel, oleochemical, and cosmetic industries. Thereby, a huge variety of lipids or lipid-derived products exist which distinguish themselves by their unique physical properties making them suitable for their individual applications. To obtain such functional lipids in an environmentally friendly manner, enzymes can be employed. In that context, lipases have been proven to be valuable biocatalysts in lipid modification, which are broadly applied in industry. Even though they have been implemented successfully in the dairy, baking, and detergent industries, there is an increasing demand for the expansion of their utilization. New technologies like protein engineering and the implementation of process development are employed in solving this task. Within the enzymes in lipid modification, lipases are the most applied catalysts and in this thesis their utilization was expanded successfully to the implementation of novel separation processes and the production of improved drug delivery matrices.

The synthesis of pterin-dithiolene ligands was achieved by employing the radical nucleophilic substitution, i.e. the so-called “Minisci- Reaction”1. This protocol was used for the first time by Professor W. Pfleiderer on pterin substrates2 and proved a powerful method for the preparation of 6 acyl-pterins in course of this work. Subsequent construction of the dithiolene ring facilitates the synthesis of pterin-dithiolene ligands with completely unprotected pterin moieti. The molybdenum cofactor is probably one of the most relevant discoveries in the recent history of pterin chemistry and biochemistry. Many efforts have been made for the preparation of compounds able to mimic the features of the Moco ligand system called "Molybdopterin". In fact, the study of MPT models enables a deeper understanding of the “mechanism of function” of this cofactor and most importantly, lays the foundation for a potential treatment for the Moco related diseases MoCOD and iSOD.

Die akute Pankreatitis ist durch eine vorzeitige intraazinäre Proteasen-Aktivierung gekennzeichnet, wobei diese im Verlauf der Erkrankung durch eine zunehmende Immunantwort mit in das Pankreas infiltrierenden Immunzellen ergänzt wird. Eine besondere Bedeutung hat die intrazelluläre Aktivierung der Serinprotease Trypsinogen, die in Abhängigkeit der lysosomalen Hydrolase Cathepsin B (CTSB) verläuft. Wir konnten zeigen, dass verschiedene lysosomale Proteine (Cathepsin D (CTSD), Cathepsin C (CTSC)) nach pathologischem Stimulus in das sekretorische Kompartiment (Zymogengranula) umverteilt werden. Cathepsin D ist in der Lage, das Schlüsselenzym Cathepsin B zu aktivieren, indem es das Pro-Enzym zu aktivem Enzym spaltet. Der Ort dieser proteolytischen Aktivierung sind die sekretorischen Vesikel. Eine pharmakologisch induzierte Permeabilisierung der Lysosomen mit nachfolgendem Ausbleiben der Umverteilung der Enzyme in das sekretorische Kompartiment zeigte, dass die vorzeitige Zymogen-Aktivierung in der Frühphase der Pankreatitis erhalten geblieben ist und unabhängig vom Lysosom verläuft. Eine CTSB-Abhängigkeit bleibt jedoch bestehen. Ein Fehlen von CTSD in den Azinuszellen führt zu einem nur transient milderen Verlauf der akuten Pankreatitis, wie anhand von CTSDf/f/p48Cre/+ Mäusen demonstiert werden konnte, die einen Pankreas-spezifischen CTSD Knockout besitzen. Ein anhaltend milderer Verlauf der Pankreatitis fand sich in CTSD-/- Mäusen, der auf eine verminderte Sekretion pro-inflammatorischer Zytokine in Immunzellen zurückzuführen ist. Auch bei Defizienz von CTSC war der Schweregrad der akuten Pankreatitis milder, wie in CTSC-/- Mäusen experimentell demonstiert werden konnte. Ursächlich hierfür ist vor allem ein reduziertes Einwandern neutrophiler Granulozyten in das Pankreas und in die extrapankreatischen Organe (Lunge), die auf eine geringere Aktivität der Serinprotease Neutrophilen Elastase und verminderte Spaltung des Zell-Kontakt Moleküls E-Cadherin beruhen. Umgekehrt beeinflusste das Fehlen von CTSC in den Azinuszellen nicht die vorzeitige Proteasen-Aktivierung. Unsere Arbeit unterstreicht die Bedeutung lysosomaler Enzyme in der akuten Pankreatitis und zeigt, dass diese Enzyme maßgeblichen Einfluss auf die Funktion von Immunzellen haben, die den Verlauf der Erkrankung wesentlich mitbestimmen. Unsere Arbeit zeigt außerdem, dass der primäre Ort der intrazellulären und vorzeitigen Proteasen-Aktivierung alleinig im sekretorischen Kompartiment stattfindet und nicht von einer Fusion mit dem lysosomalen Kompartiment abhängig ist.

β-chirale Amine, wie zum Beispiel Pregabalin und Baclofen, sind Verbindungen von großem Interesse insbesondere für die pharmazeutische Industrie. Biokatalytische Herstellungsverfahren, vor allem Aminierungsreaktionen, sind bisher nur geringfügig untersucht worden und werden nach aktuellem Wissenstand bis auf die Synthese von Niraparib noch nicht in großtechnischem Maßstab eingesetzt. Wünschenswert ist die Etablierung einer Synthese, welche (S)-Pregabalin bzw. (R)-Baclofen in hohen Ausbeuten liefert, da diese beiden Enantiomere jeweils die höhere biologische Wirksamkeit aufweisen. Ziel dieser Arbeit war die Synthese von Pregabalin und Baclofen als Modellverbindungen für β-chirale Amine mit Hilfe einer selektiven Amintransaminase oder Amindehydrogenase. Zunächst wurde erfolgreich mit Hilfe der Gaschromatographie bzw. HPLC jeweils eine chirale Analytik für die beiden Reaktionsprodukte sowie die Baclofen-Derivate etabliert, die stabil reproduzierbar und auch zur Quantifizierung geeignet war. Auch für 3-(4-Chlorphenyl)-4-oxo-buttersäure-t-butylester konnte eine GC-Methode entwickelt werden, die Aufschluss über die Konzentration und den Enantiomerenüberschuss gab. Die vier zur Verfügung gestellten Amindehydrogenasen konnten erfolgreich exprimiert und mittels IMAC-Methode gereinigt werden. Trotz geringer Aktivitäten in einem photometrischen NADH-Assay konnte jedoch keine Produktbildung nachgewiesen werden. Eine Kollektion von ca. 150 Amintransaminasen wurde bezüglich der Desaminierung von Pregabalin und Baclofen mittels Dünnschichtchromatographie untersucht. In Richtung der Aminierung wurde ein photometrischer Acetophenon-Assay verwendet. Dabei wurden für Pregabalin sechs und für Baclofen 17 potenzielle Kandidaten ermittelt. Besonders vielversprechend war die Variante 3FCR 59W 87L 231A 382M 429A (3FCR_5M), welche 3-(4-Chlorphenyl)-4-oxo-buttersäure-t-butylester als Substrat akzeptierte. Nach der Ermittlung eines geeigneten Aminodonors und Optimierung der Reaktionsbedingungen konnten Umsätze bis zu 90% bei 99%ee (R) mit IMAC-gereinigter 3FCR_5M erzielt werden. Um Kosten für ein späteres großtechnisches Verfahren einzusparen, sollte die Reaktion ebenfalls für den Einsatz von Zellextrakt optimiert werden. Dabei wurde beobachtet, dass geringere Enantiomerenüberschüsse erzielt wurden als mit dem gereinigten Enzym und der Substratverbrauch höher als die Produktbildung war. Als mögliche Ursachen wurden der Umsatz des Substrats durch ein E. coli eigenes Enzym, beispielsweise eine Aldehydreduktase oder Aldehyddehydrogenase, sowie eine Beeinflussung der Enantioselektivität durch die veränderte chemische Umgebung oder den selektiven Entzug des gewünschten Substrat-Enantiomers durch eine selektive Nebenreaktion hypothetisiert. Dieses Phänomen konnte durch eine vorgeschaltete Reinigung mittels fraktionierender Ammoniumsulfat-Fällung jedoch erfolgreich umgangen werden. Mit dieser Methode konnten vergleichbar hohe Umsätze und Enantiomerenüberschüsse wie mit dem IMAC-gereinigten Enzym erreicht werden. Bei ersten Vorversuchen zum Up-Scaling der Reaktion wurde festgestellt, dass eine höhere Substratkonzentration nicht einen proportional höheren Umsatz zur Folge hatte, jedoch konnte der Umsatz durch eine versetzte Zugabe der Enzymlösung gesteigert werden, sodass ein Prozess mit diesem Biokatalysator in seiner aktuellen Form eine kontinuierliche Zugabe erfordern würde. Praktikabel wäre einer Verminderung der Substrat-Inhibierung und Erhöhung der Enzymstabilität durch weiteres Protein-Engineering. Auch zur Produktion von 3FCR_5M im größeren Maßstab wurden Experimente vorgenommen. Dabei konnte gezeigt werden, dass eine vielversprechende Expression im Bioreaktor bei einer kontinuierlichen Temperatur von 30°C und einer Expressionsdauer von sieben Stunden. Nach einigen Optimierungsschritten konnte im Bioreaktor die zwanzigfache volumetrische Aktivität im Vergleich zur Expression im Schüttelkolben erzeugt werden. Zusammenfassend ist zu sagen, dass in der vorliegenden Arbeit, trotz weiterem Optimierungsbedarf, eine sehr gute Grundlage für die Transaminase-vermittelte Synthese von (R)-Baclofen geschaffen wurde. In zukünftigen Arbeiten sollte die Optimierung der Reaktion in großem Maßstab im Fokus stehen.

The present work is a cumulative dissertation that covers the research work of the author at the Department of Analytical and Physical Chemistry of Chelyabinsk State University. It contains a short description of the study and a set of attached publications in peer-reviewed journals and conference proceedings. The phase and chemical equilibria in binary systems Me – Si (where Me is the 4th-period transition metal) as well as Mo – Si, Mn – Ge and Fe – Ge at low temperatures were considered. The solid solubility of silicon in vanadium, chromium, manganese, iron, nickel, cobalt and copper and that of germanium in manganese and iron was estimated. The phase equilibria in Me – Si – O, Mo – Si – O, Mn – Ge – O and Fe – Ge – O ternary systems at standard conditions were considered from a thermodynamic viewpoint. The atmospheric corrosion of transition metals silicides and manganese and iron germanides was discussed. The chemical and electrochemical equilibria in Me – Si – H2O, Mo – Si – H2O, Mn – Ge – H2O and Fe – Ge – H2O systems were considered from a thermodynamic viewpoint. Pourbaix diagrams for some 4th-period transition metals and molybdenum, as well as for silicon, were revised. The potential – pH diagrams for Me – Si – H2O, Mo – Si – H2O, Mn – Ge – H2O and Fe – Ge – H2O systems were plotted in the first time. The corrosion-electrochemical behaviour of transition metals silicides and manganese and iron germanides in aqueous media was discussed. The potential – pH diagrams for some siliceous brasses and bronzes (which are multicomponent alloys containing both transition metals and silicon) were plotted, and the corrosion of these alloys in aqueous media was discussed. Method of estimation of corrosion-electrochemical behaviour of multicomponent alloys, which takes into account both thermodynamic and kinetic data and is based on mutual construction of equilibrium and polarisation potential – pH diagrams, was described. Its usage was illustrated in the example of the structural steel 20KT.

In an aerobic environment the occurrence of reactive oxygen species (ROS) is a common phenomenon. The diverse roles of ROS in cellular function and in diseases make them a target of interest in many research areas. Substances capable of directly or indirectly reducing the (harmful) effects of ROS are referred to as “antioxidants”. However, the term is applied miscellaneously in the chemical and the biological context to describe different attributes of a substance. In this work the potential of an electrochemical assay to detect different ROS in-vitro was explored. The method was optimized to investigate the radical scavenging activities (antioxidant potential) of trolox and different plant compounds (ascorbic acid, caffeic acid, epigallocatechin gallate, ferulic acid, kaempferol, quercetin, rutin, and Gynostemma pentaphyllum extract) in-vitro. The obtained data was compared to established antioxidant in-vitro assays. Further, the impact of the plant substances on cellular parameters was evaluated with the electrochemical assay and established cell assays. The optimization of the electrochemical assay allowed the reproducible detection of ROS. The sensor electrode proved differently sensitive towards individual ROS species. The highest sensitivity was recorded for hydroxyl radicals while superoxide and hydrogen peroxide had little impact on the sensor. Extracellular ROS concentrations could be detected from cell lines releasing elevated ROS into the extracellular space. The antioxidant activity of the investigated plant substances could be demonstrated with all in-vitro assays applied. However, the absolute as well as the relative activity of the individual substances varied depending on the experimental parameters of the assays (pH, radical species, phase, detection method). The plant compounds modified redox related intracellular parameters in different cell lines. However, a direct correlation between intracellular and extracellular effects of the plant compounds could not be established. The work demonstrates the feasibility to use the electrochemical assay to sense ROS as well as to evaluate the radical scavenging activity of molecules. The in-vitro antioxidant activities demonstrated for the individual plant substances are not reliable to predict the cellular effects of the molecules.