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As an interface between an individual and its environment, the skin is a major site of direct exposure to exogenous substances. Once absorbed, these substances may interact with different biomolecules within the skin. The aryl hydrocarbon receptor (AhR) signaling pathway is one mechanism whereby the skin responds to exposures, predominantly through the induction or upregulation of metabolizing enzymes. One known physiological role of the AhR in many tissues is its involvement in the control of cell cycle progression. In skin, almost nothing is known about this physiological function. Moreover, the question whether frequently used naturally occurring phenolic derivatives like eugenol and isoeugenol impact on the AhR within the skin has rarely been studied so far. Eugenol and isoeugenol are due to their odour referred to as fragrances. The ubiquitous distribution of eugenol and isoeugenol results in an almost unavoidable contact with these substances in our daily lives. Despite this fact, their molecular mechanisms of action in skin are poorly understood. There is evidence supporting the hypothesis that these substances may impact on the AhR. On the one hand, eugenol is shown to induce cytochrome P450 1A1 (CYP1A1), a well-known target gene of the AhR. On the other hand, their known anti-proliferative properties might also be mediated by the AhR, based on its physiological function. In order to proof this hypothesis, it was investigated whether eugenol and isoeugenol impact on the AhR signaling pathway in skin cells. Results revealed that eugenol as well as isoeugenol impact on the AhR signaling pathway in skin cells. Both substances caused the translocation of the AhR into the nucleus, induced the expression of the well-known AhR target genes CYP1A1 and AhR repressor (AhRR) and exhibited impact on cell cycle progression. Both substances caused an AhR-dependent cell cycle arrest in skin cells, modulated protein levels of several cell cycle regulatory proteins, inhibited DNA synthesis and thereby reduced cell numbers. The comparison of wildtype cells to AhR knockdown cells revealed an influence of the AhR on cell cycle progression in skin cells in the absence of exogenous ligands. AhR knockdown cells exhibited a slower progression through the cell cycle caused by an accumulation of cells in the G0/G1 phase of the cell cycle and a decreased DNA synthesis rate. Modulation of cell cycle regulatory proteins involved in the transition from the G0/G1 to the S phase of the cell cycle was altered in AhR knockdown cells as well. To conclude, eugenol as well as isoeugenol were able to impact on the AhR signaling pathway in skin cells. Their molecular mechanisms of action are similar to those of classical AhR ligands, although their structural characteristics strongly differ from that of these ligands. In the absence of exogenous ligands the AhR promotes cell cycle progression in many tissues and this knowledge could be expanded on skin-derived cells within the scope of this thesis.
The skin is continuously challenged by environmental antigens that may penetrate and elicit a skin sensitization, which can develop into allergic contact dermatitis. Medical treatment for allergic contact dermatitis is limited - in fact only acute symptoms can be cured and for secondary prevention of the disease a lifelong avoidance of the allergen(s) is necessary. Therefore, the screening of the sensitization potential of substance used in commercially available products is indispensable to prevent such diseases. Hence, risk assessment is deduced from data obtained by murine local lymph node assay predominantly, but there exists a need to develop methods capable of providing the same information that do not require the use of animals in view of legislative initiatives such as REACH (registration, evaluation, authorization of chemicals) as well as the 7th Amendment to the Cosmetics Directive (2003/15/EC). Therefore, a number of promising in silico and in vitro approaches are being developed to address this need. In vitro test systems using the response of dendritic cells, which are the key player in the elicitation process of contact dermatitis, are established, but, although these novel methods for hazard identification might find application in the context of screening, it is not clear whether these approaches are useful for the purposes of risk assessment and risk management to predict allergic potency. Therefore, it was investigated whether on the one hand in vitro generated dendritic cells from primary blood monocytes (MoDC) and on the other hand a continuous monocytic cell line, the THP-1 cells, suggested as dendritic cell surrogate, react to a presumably weak allergen. Ascaridol, predicted as one of the possible causes for tea tree oil contact dermatitis, was studied and its effects in these two in vitro skin sensitization models were explored. Thus, the surface expression of CD86, HLADR, CD54, and CD40, which are known as activation markers in both in vitro models, were measured via flow cytometry. For MoDC, an augmented CD86 and HLADR surface expression in comparison to untreated cells were determined after 24 h exposure with ascaridol. An increased CD54 and CD40 surface expression were found only in some donors. After long term incubation of 96 h, ascaridol-treated MoDC still up-regulated CD86 and additionally an augmented CD40 expression was measured in all studied donors. An enhanced CD54 expression was determined for 50 percentage of all investigated donors. Furthermore, CD80, CD83 and CD209 protein expression were up-regulated in MoDC after 96 h of ascaridol incubation. In addition, it was determined that after 24 h ascaridol-treated MoDC showed an increased capacity to uptake antigens, whereas after 96 h this capacity got lost and antigen-capturing devices were reduced in comparison to non-treated MoDC. Moreover, the cytokine release of ascaridol-treated MoDC were measured after 24 h. Tumor necrosis factor (TNF)alpha, interleukin (IL)-1beta and IL 6 secretion were determined in some donors. Furthermore, IL-8 release was clearly increased after 24 h ascaridol treatment. By the same token, THP-1 cells were analyzed after ascaridol treatment for several activation markers. We found a similar response pattern as measured in MoDC. Ascaridol induced CD86 expression as well as CD54 after 24 h incubation. Additionally, the impact of ascaridol on phosphorylation of p38 mitogen-activated protein kinase, which had been shown to be involved in increased expression of activation markers like CD86 by others, were studied via Western blot analysis. A phosphorylation of p38 was determined after 15 min of ascaridol stimulation. Moreover, an augmented CD40 and HLADR surface expression were measured in a dose-response manner after 24 h ascaridol treatment. Also similar to MoDC an enhanced IL-8 secretion after ascaridol stimulation was observed in THP-1 cells. Hence, for the first time it was shown that ascaridol has immuno-modulating effects. The obtained data from both in vitro systems, MoDC and THP-1 cells, identified ascaridol as a sensitizer. Although for both systems there remain significant challenges to overcome for potency assessment, ascaridol is presumed to be a weak sensitizer probably. Interestingly, ascaridol treatment of THP-1 cells resulted also in an increased augmentation of CD184 and CCR2, two chemokine receptors expressed on monocyte. Therefore, these data encouraged the exploration of chemokine receptors as tools in skin sensitization prediction. Consequently, the combination of chemical assays with in vitro techniques may provide a useful surrogate to animal testing for skin sensitization. Due to the continuously changing environmental conditions, it is necessary to regularly monitor and update the spectrum of sensitizers that elicit contact dermatitis. Therefore, both debated in vitro test systems will become indispensable tools.
Exposure to fine and ultra-fine environmental particles is still a problem of concern in many industrialized parts of the world and the intensified use of nanotechnology may further increase exposure to small particles. Since many years air pollution is recognized as a critical problem in western countries, which led to rigorous regulation of air quality and the introduction of strict guidelines. However, the upper thresholds for particulates in ambient air recommended by the world health organization are often exceeded several times in newly industrialized countries. Such high levels of air pollution have the potential to induce adverse effects on human health. The response triggered by air pollutants is not limited to local effects of the respiratory system but is often systemic, resulting in endothelial dysfunction or atherosclerotic malady. The link between air pollution and cardiovascular disease is now accepted by the scientific community but the underlying mechanisms responsible for the pro-atherogenic potential still need to be unraveled in detail. Based on the results from in- vivo and in vitro studies the production of reactive oxygen species due to exposure to particles is the most important mechanism to explain the observed adverse effects. However, the doses that were applied in many in vivo and in vitro studies are far beyond the range of what humans are exposed to and there is the need for more realistic exposure studies. Complex in vitro coculture systems may be valuable tools to study particle-induced processes and to extrapolate effects of particles on the lung. One of the objectives of this PhD thesis was the establishment and further improvement of a complex coculture system initially described by Alfaro-Moreno et al. [1]. The system is composed of an alveolar type-II cell line (A549), differentiated macrophage-like cells (THP-1), mast cells (HMC-1) and endothelial cells (EA.hy 926), seeded in a 3D-orientation on a microporous membrane to mimic the cell response of the alveolar surface in vitro in conjunction with native aerosol exposure (VitrocellTM chamber). The tetraculture system was carefully characterized to ensure its performance and repeatability of results. The spatial distribution of the cells in the tetraculture was analyzed by confocal laser scanning microscopy (CLSM), showing a confluent layer of endothelial and epithelial cells on both sides of the Transwellâ„¢. Macrophage-like cells and mast cells can be found on top of the epithelial cells. The latter cells formed colonies under submerged conditions, which disappeared at the air-liquid-interface (ALI). The VitrocellTM aerosol exposure system was not significantly influencing the viability. Using this system, cells were exposed to an aerosol of 50 nm SiO2-Rhodamine nanoparticles (NPs) in PBS. The distribution of the NPs in the tetraculture after exposure was evaluated by CLSM. Fluorescence from internalized particles was detected in CD11b-positive THP-1 cells only. Furthermore, all cell lines were found to be able to respond to xenobiotic model compounds, such as benzo[a]pyrene (B[a]P) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with the upregulation of CYP1 mRNA. With this tetraculture system the response of the endothelial part of the alveolar barrier was studied in- vitro in a still realistic exposure scenario representing the conditions for a polluted situation without direct exposure of endothelial cells. After exposure to diesel exhaust particulate matter (DEPM) the expression of different anti-oxidant target genes and inflammatory genes such as NAD(P)H dehydrogenase quinone 1 (NQO1), superoxide dismutase 1 (SOD1) and heme oxygenase 1 (HMOX1), as well as the nuclear translocation nuclear factor erythroid-derived 2 (Nrf2) was evaluated. In addition, the potential of DEPM to induce the upregulation of CYP1A1 mRNA in the endothelium was analyzed. DEPM exposure led not to an upregulation of the anti-oxidant or inflammatory target genes, but to clear nuclear translocation of Nrf2. The endothelial cells responded to the DEPM treatment also with the upregulation of CYP1A1 mRNA and nuclear translocation of the aryl hydrocarbon receptor (AhR). Overall, DEPM triggered a response in the endothelial cells after indirect exposure of the tetraculture system to low doses of DEPM, underlining the sensitivity of ALI exposure systems. The use of the tetraculture together with the native aerosol exposure equipment may finally lead to a more realistic judgment regarding the hazard of new compounds and/or new nano-scaled materials in the future. For the first time, it was possible to study the response of the endothelial cells of the alveolar barrier in vitro in a realistic exposure scenario avoiding direct exposure of endothelial cells to high amounts of particulates.
Background: The growing production and use of engineered AgNP in industry and private households make increasing concentrations of AgNP in the environment unavoidable. Although we already know the harmful effects of AgNP on pivotal bacterial driven soil functions, information about the impact of silver nanoparticles (AgNP) on the soil bacterial community structure is rare. Hence, the aim of this study was to reveal the long-term effects of AgNP on major soil bacterial phyla in a loamy soil. The study was conducted as a laboratory incubation experiment over a period of 1 year using a loamy soil and AgNP concentrations ranging from 0.01 to 1 mg AgNP/kg soil. Effects were quantified using the taxon-specific 16S rRNA qPCR.
Results: The short-term exposure of AgNP at environmentally relevant concentration of 0.01 mg AgNP/kg caused significant positive effects on Acidobacteria (44.0%), Actinobacteria (21.1%) and Bacteroidetes (14.6%), whereas beta-Proteobacteria population was minimized by 14.2% relative to the control (p ≤ 0.05). After 1 year of exposure to 0.01 mg AgNP/kg diminished Acidobacteria (p = 0.007), Bacteroidetes (p = 0.005) and beta-Proteobacteria (p = 0.000) by 14.5, 10.1 and 13.9%, respectively. Actino- and alpha-Proteobacteria were statistically unaffected by AgNP treatments after 1-year exposure. Furthermore, a statistically significant regression and correlation analysis between silver toxicity and exposure time confirmed loamy soils as a sink for silver nanoparticles and their concomitant silver ions.
Conclusions: Even very low concentrations of AgNP may cause disadvantages for the autotrophic ammonia oxidation (nitrification), the organic carbon transformation and the chitin degradation in soils by exerting harmful effects on the liable bacterial phyla.
Global human population growth is associated with many problems, such asrnfood and water provision, political conflicts, spread of diseases, and environmental destruction. The mitigation of these problems is mirrored in several global conventions and programs, some of which, however, are conflicting. Here, we discuss the conflicts between biodiversity conservation and disease eradication. Numerous health programs aim at eradicating pathogens, and many focus on the eradication of vectors, such as mosquitos or other parasites. As a case study, we focus on the "Pan African Tsetse and Trypanosomiasis Eradication Campaign," which aims at eradicating a pathogen (Trypanosoma) as well as its vector, the entire group of tsetse flies (Glossinidae). As the distribution of tsetse flies largely overlaps with the African hotspots of freshwater biodiversity, we argue for a strong consideration of environmental issues when applying vector control measures, especially the aerial applications of insecticides.rnFurthermore, we want to stimulate discussions on the value of speciesrnand whether full eradication of a pathogen or vector is justified at all. Finally, we call for a stronger harmonization of international conventions. Proper environmental impact assessments need to be conducted before control or eradication programs are carried out to minimize negative effects on biodiversity.
Die Fauna-Flora-Habitat-Richtlinie (Richtlinie 92/43/EWG) stellt derzeit das umfangreichste Instrument des internationalen Naturschutzes zur Erhaltung der europäischen Biodiversität dar. Das Grundkonzept der FFH-RL beruht hierbei sowohl auf dem Schutz gefährdeter Arten als auch auf dem Erhalt natürlicher Lebensräume. Dieser ganzheitliche Ansatz verursacht jedoch infolge der großen Anzahl zu berücksichtigender Schutzgüter einen hohen personellen wie finanziellen Aufwand bei der Umsetzung der Richtlinienvorgaben. Daher wurde in der vorliegenden Arbeit am Beispiel der Schmetterlingsart Euphydryas aurinia (Anhang II FFH-RL) überprüft, inwieweit die Konzepte von ESUs (Evolutionarily Significant Units) und MUs (Management Units) geeignete Möglichkeiten bieten, um die Prioritätensetzung bei der Auswahl besonders schützenswerter Vorkommen gefährdeter Arten zu erleichtern. Zu diesem Zweck wurden mit drei verschiedenen Subspezies von E. aurinia (E. aurinia beckeri, E. aurinia debilis, E. aurinia aurinia) Fang-Markierung-Wiederfangstudien durchgeführt, sowie mit Hilfe von Allozym-Elektrophoresen populationsgenetische Parametern in europäischem Kontext und auf regionaler Ebene (Westtschechien) erfasst. Die drei untersuchten Subspezies zeigten hierbei spezifische ökologische Adaptationen an die jeweiligen Habitatbedingungen (z.B. bzgl. der Populationsdichte, Demographie und Mobilität). Ferner wiesen die genetischen Analysen starke Differenzierungen bei E. aurinia in Europa nach, die u.a. Antworten auf phylogeographische und taxonomische Fragestellungen ermöglichen. Auch auf regionaler Ebene (Westtschechien) konnten genetische Differenzierungen festgestellt werden. Auf Basis der erhobenen populationsökologischen und -genetischen Daten wird abschließend die generelle Anwendbarkeit und der Nutzen der Konzepte von ESUs und MUs bei der Etablierung von Schutzkonzepten für E. aurinia und andere Arten der FFH-RL diskutiert. rnDer zweite Teil der vorliegenden Arbeit beschäftigt sich exemplarisch mit dem aktuellen Schutzverfahren für deutsche E. aurinia-Vorkommen im Rahmen der FFH-RL und den damit verbundenen Problemen. Der Schwerpunkt lag hierbei auf der Schutzgebietsauswahl, dem Monitoring und dem Gebietsmanagement. In diesem Kontext werden u.a. Problematiken angesprochen, die sich aus der großen ökologischen Variabilität der Art ergeben bzw. die aufgrund von Koordinierungsschwierigkeiten zwischen einzelnen Bundesländern bestehen. Vor dem Hintergrund dieser Erkenntnisse werden Lösungsvorschläge unterbreitet, wie das aktuelle Schutzverfahren für E. aurinia in Deutschland weiter verbessert werden könnte.
Die vorliegende Arbeit untersucht zwei grundlegende Fragestellungen im Hinblick auf den Betrieb von Genbanken als Beitrag zur Sicherung der genetischen Diversität. Das erste Kapitel behandelt aus rechtlicher Sicht die juristischen Fragen nach dem Zugang zu genetischen Ressourcen, nach dem Ausgleich aus der Nutzung entstehender Vorteile (Access and Benefit Sharing, ABS) und nach den Möglichkeiten des Handels mit Proben gefährdeter Tierarten, dies sowohl im Hinblick auf die rein wissenschaftliche Forschung als auch in Bezug auf kommerziell orientierte Zwecke. Grundlegend für die Bearbeitung dieser Fragen war die detaillierte Betrachtung des Übereinkommens über die biologische Vielfalt (CBD) und des Übereinkommens über den internationalen Handel mit gefährdeten Arten freilebender Tiere und Pflanzen (CITES). Da das CBD im Hinblick auf den Zugang zu genetischen Ressourcen in der Bundesrepublik Deutschland bisher nicht umgesetzt ist, bleibt dem Betreiber einer Genbank zur rechtlichen Absicherung und Klarheit nur die Aushandlung von Materialübertragungsverträgen mit den Ursprungsländern der genetischen Ressourcen. Der nachfolgende Handel mit gelagerten Proben gefährdeter Tierarten ist möglich, wenn dies nicht-kommerziell zur wissenschaftlichen Forschung erfolgt und die beteiligten Institutionen bei ihren Regierungen als wissenschaftliche Institutionen registriert sind. Bei kommerziellen Absichten unterliegt der Handel streng den durch das CITES vorgegebenen Regulierungen. Ausnahmen des Handelsverbotes sind möglich, z.B. dann wenn es sich um Proben von Tieren handelt, die in Gefangenschaft gezüchtet worden sind. Das zweite Kapitel behandelt aus naturwissenschaftlicher Sicht die Möglichkeit der direkten Gewinnung genetischer Materialien von Tieren. Hierbei wurde die Isolierung adulter Stammzellen aus der Haut von Säugetieren als Zielzellen zur Lagerung in Genbanken fokussiert. Am Modellorganismus Hausschwein (Sus scrofa domestica) wurde eine Methode zur Isolation adulter stammzellähnlicher Zellen aus der Haut etabliert. Durch nachfolgende Laborversuche wurden die isolierten stammzellähnlichen Zellen (pSSCs, porcine skin derived stem cell-like cells) charakterisiert. Wie mesenchymale Stammzellen haben pSSCs eine fibroblasten-ähnliche Morphologie und exprimieren die Oberflächenproteine CD9, CD29, CD44 , CD105 und sehr gering CD90. Neben diesen konnte die Expression der stammzellasoziierten Gene Stat3, Oct3/4, Sox2, Nestin, Bcrp1/ABCG2 und Bmi1 nachgewiesen werden. Weitere Versuche zeigten die induzierte Differenzierung der pSSCs zu Zellen zweier embryonaler Keimblätter, dem Ektoderm (Neuronen, Astrozyten) und dem Mesoderm (glatte Muskelzellen und Adipozyten). Zur vollständigen Charakterisierung und gänzlichen Ermittlung des Differenzierungspotentials bedarf es allerdings weiterer Versuche, die wegen der Kostspieligkeit und einem erhöhten zeitlichen Aufwand hier nicht realisierbar waren. Die Expansion und die Kryokonservierung der pSSCs zeigten geringfügige Auswirkungen auf den Phänotyp und das Differenzierungspotential. In Bezug auf die Kryokonservierung konnte wegen der geringen Anzahl an Versuchen eine definitive Aussage über ihre Folgen nicht getroffen werden. Folglich bedarf es weiterer Untersuchungen zur Ermittlung der Auswirkungen der Kryokonservierung auf adulte Stammzellen, die aus der Haut gewonnen werden können. Durch ihre Lagerung in Genbanken eröffnen aus der Haut isolierbare Stammzellen neue Möglichkeiten für den Artenschutz, dies vor allem durch ihre Anwendbarkeit im Rahmen modernster Reproduktionsmethoden und als nahezu unendliche DNA-Quelle für phylogenetische Studien, die zum Populationsmanagement unerlässlich sind. Trotz der sich aus dieser Arbeit ergebenden neuen Fragen - im Hinblick auf den Zugang, die Gewinnung und auch die Lagerung genetischer Materialien gefährdeter Tierarten -, bleibt festzuhalten, dass Genbanken generell die Möglichkeit bieten, vitales, biologisches Material bereitzuhalten. Dies ist sowohl bedeutend für die Grundlagenforschung als auch für den Einsatz dieser Materialien zur Steigerung der Reproduktion gefährdeter Arten, letztlich mit dem Ziel, genetische Variationen zu erhalten, die anderenfalls verloren gehen würden.
Der Forschungsbereich der Systembiologie hat sich in den letzten Jahren mit unvergleichlicher Dynamik entwickelt und sich als interdisziplinäres Feld in den Biowissenschaften etabliert. Die Systembiologie verfolgt hierbei unter anderem das Ziel, biologische Systeme als Ganzes zu betrachten. Die analytische Erfassung der Stoffwechselzwischenprodukte, auch Metaboliten genannt, eröffnet hierbei neue Möglichkeiten. Metaboliten stellen Zwischenprodukte in vivo ablaufender biochemischer Reaktionen dar und stehen in enger Abhängigkeit zu Vorgängen, welche auf der Ebene von Transkriptom und Proteom gesteuert und ermöglicht werden. In dieser Arbeit wurden Zeitreihen von Metabolitkonzentrationen untersucht, welche im Rahmen von Fermentationsexperimenten mit dem nicht-pathogenen Bodenbakterium Corynebacterium glutamicum erfasst worden sind. Die Fermentationsexperimente wurden auf unterschiedlichen Ausgangssubstraten durchgeführt, wobei die Erfassung der Metabolitkonzentration in äquidistanten zeitlichen Abständen gehandhabt wurde. Zur Korrektur von Messfehlern und zur optimalen Vorverarbeitung der Daten wurde ein maßgeschneidertes System der Datenprozessierung entwickelt. Eine unüberwachte Datenstrukturanalyse ergab, dass sich die Metaboliten ihrer zeitlichen Ausprägung nicht uniform oder gar zufällig verhalten, sondern sich in Gruppen unterschiedlichen Prozessverhaltens einordnen lassen. Diese unüberwachte Eingruppierung anhand der in den Zeitreihen vorhandenen Strukturen erlaubte eine erste grundlegende funktionelle Zuordnung der Metaboliten. Weiterhin konnten in den Konzentrationsdaten Strukturen gefunden werden, welche deutliche Übereinstimmungen mit den physiologischen Phasen des bakteriellen Wachstums zeigten. Die Analyse der Metabolomdaten wurde in einem nächsten Schritt durch eine theoretische Betrachtungsweise erweitert. Hierzu wurde der Stoffwechsel von C. glutamicum rechnergestützt modelliert. Zu diesem Zweck wurde eine Genomannotation durchgeführt, mit dem Ziel einen möglichst umfangreichen und qualitativ hochwertigen Katalog über das enzymatische Repertoire von C. glutamicum aus Sequenzinformation abzuleiten. Generiertes Wissen über vorhandene Enzyme wurde in biochemische Reaktionen übersetzt, welche zu Reaktionsnetzwerken zusammengefügt wurden. Die erzeugten Reaktionsnetzwerke wurden unter Verwendung graphentheoretischer Ansätze analysiert. Die integrative Analyse experimenteller und theoretischer Daten ergab, dass sich Eigenschaften von Metabolitzeitreihen deutlich topologischen Merkmalen zuordnen lassen. So zeigt sich beispielsweise, dass ein auffälliger Zusammenhang zwischen der experimentell erfassten Sensitivität im Konzentrationsverlauf eines Metaboliten und seinem theoretischen Verknüpfungsgrad existiert. Weiterhin konnte gezeigt werden, dass eine hochsignifikante Prozessähnlichkeit zwischen Metaboliten sowohl in direkter Nachbarschaft als auch in größeren Reaktionsabständen auftreten kann, jedoch vorzugsweise dann existiert, wenn beide Metaboliten ihrerseits wenige Reaktionspartner haben. Die integrative Datenanalyse wurde in einem weiteren Schritt abermals erweitert, indem Transkriptominformation weiterer Studien integriert wurde. Im Detail wurde in dieser Analyse die Prozessähnlichkeit theoretisch benachbarter Metaboliten des Zentralstoffwechsels in Zusammenschau mit der Transkription enzymkodierender Gene untersucht. Die Ergebnisse zeigten deutlich, dass eine erhöhte Prozessähnlichkeit benachbarter Metaboliten dann existiert, wenn die entsprechenden enzymkodierenden Gene in Abhängigkeit des verwendeten Ausgangssubstrates signifikant exprimiert waren. Somit konnte ein Zusammenhang zwischen der Prozessähnlichkeit benachbarter Metaboliten in Abhängigkeit zur Genexpression als Resultat substratinduzierter Anpassungsvorgänge gezeigt werden. Es konnte folglich im systembiologischen Kontext belegt werden, dass auf der Ebene des Transkriptoms stattfindende Vorgänge sich deutlich bis in die Zeitreiheneigenschaften erfasster Metabolitkonzentrationen durchpausen können. Darüber hinaus zeigte sich, dass die Berechnung paarweiser Prozessähnlichkeiten das Potenzial zur Charakterisierung der zugrundeliegenden Systemeigenschaften besitzt. So ermöglichte die Betrachtung paarweiser Prozessähnlichkeiten aus allen untersuchten Fermentationsexperimenten, signifikante substrat-induzierte Veränderungen als auch invariante Merkmale im Stoffwechsel von C. glutamicum zu detektieren.
Global change, i.e. climate and land use changes, severely impact natural ecosystems at different scales. Poikilothermic animals as butterflies, amphibians and reptiles have proven to be useful indicators for global change impacts as their phenology, spatial distribution, individual fitness and survival strongly depend on external environmental factors. In this aspect, phenological changes in terms of advanced flight or breeding periods, immigrations of foreign species, range shifts concomitant with temperature increases and even local population declines have been observed in both species groups. However, to date much attention has been paid to global change impacts on the species or population level and analyses concerning entire ecosystems are scarce. Applying a novel statistical modelling algorithm we assessed future changes in the extent and composition of terrestrial ecoregions as classified by the World Wide Fund for Nature (WWF). They are defined as coarse-scale conservation units containing exceptional assemblages of species and ecological dynamics. Our results demonstrate dramatic geographical changes in the extent and location of these ecoregions across all continents and even imply a repriorisation of conservation efforts to cope with future climate change impacts on biodiversity. On the local scale, climate change impacts become unequivocal. Comparing historical to contemporary butterfly assemblages on vineyard fallows of the Trier Region, a significant decline in butterfly richness, but also a severe depletion in trait diversity was observed. Comparisons of community temperature indices reveal a striking shift in community composition leading to a replacement of sedentary and monophagous habitat specialists by ubiquitous species. Similar changes have been observed in nature reserves in the Saar-Mosel-area. Monitoring data reveal strong losses of species diversity and remarkable shifts of community compositions at the expense of habitat specialists. Besides climatic variability, these findings are largely attributed to changes in habitat structures, mostly due to eutrophication and monotonisation. Management activities are unlikely to counterbalance these effects, thus severely questioning current conservation strategies. Most dramatic global change impacts are suspected on closely associated species and disruptions of biotic interactions are often hold responsible for species declines. A strong host-parasite association has developed in Myrmica ants and Maculinea butterflies, the later crucially depending on specific host ants for their larval survival. Applying environmental niche models we determined considerable niche dynamics in the observed parasite-host relation with a pronounced niche plasticity in the butterfly species adapting to previous evasive niche shifts in their host ants. Moreover, the new emergence of species continuously expanding their northernmost range borders concomitant with global warming like the Short-tailed blue (Cupido argiades) is attributed to climate change. However, species distribution models predict a severe habitat loss and shifts of potentially suitable habitats of this species towards north-eastern Europe and higher altitudes under several IPCC scenarios making the presence of this species in the Trier region a contemporary phenomenon. Species distribution models have emerged as powerful tools to predict species distributions over spatial and temporal scales. However, not only the presence of a species, but also its abundance have significant implications for species conservation. The ability to deduce spatial abundance patterns from environmental suitability might more efficiently guide field surveys or monitoring programs over large geographical areas saving time and money. Although the application of species distribution models to deduce vertebrate abundances is well recognized, our results indicate that this method is not an adequate approach to predict invertebrate abundances. Structural and ecological factors as well as climatic patterns acting at the microscale are key drivers of invertebrate occurrence and abundances limiting conclusions drawn from modeling approaches. Population declines should be interpreted with care as in butterflies and amphibians various reasons are debated. Both species groups are acknowledged to be highly susceptible to land use changes and variations in landscape structure. Moreover, climate and land use are not independently operating factors. The combined impact of both is demonstrated in our study linking climate-driven changes in amphibian phenologies to temporal advanced applications of pesticides and fertilizers. Both environmental factors already represent severe threats to amphibians when standing alone, but linking their combined impacts may result in an potentiated risk for amphibian populations. As all amphibians and numerous butterfly species are legally protected under the Federal Nature Conservation Act, intensifications of agricultural land use in large parts of Germany as well as new agrarian practices (including genetically manipulated plants accompanied by new herbicide technologies) might severely challenge regional conservation activities in the future.
Geographic ranges of species and their determinants are of great interest in the field of biogeography and are often studied in terms of the species" ecological niches. In this context, the range of a species is defined by the accessibility of an area, abiotic factors and biotic interactions, which affect a species" distributions with different intensities across spatial scales. Parapatry describes a distributional pattern in which the ranges of two species meet along sharp range limits with narrow contact zones. Such parapatric range limits are determined by changing abiotic conditions along sharp environmental gradients or can result from interspecific resource competition. However, it has been shown that often the interplay of abiotic conditions and species interactions determine parapatry. The geographic ranges of the land salamanders, Salamandra salamandra and S. atra, narrowly overlap in the European Alps with only few syntopic localities and to date, the cause of parapatry is unknown. The goal of this thesis was thus to identify the importance of abiotic and biotic factors for their parapatric range limits at different spatial scales. On a broad spatial scale, the role of climate for the parapatric range limits of the species was investigated within three contact zones in Switzerland. Climatic conditions at species" records were analysed and species distribution modelling techniques were used to explore the species" climatic niches and to quantify the interspecific niche overlap. Furthermore, it was tested whether the parapatric range limit coincides with a strong climatic gradient. The results revealed distinct niches for the species as well as the presence of strong climatic gradients which could explain the parapatric range limits of the species. Yet, there was a moderate interspecific niche overlap in all contact zones indicating that the species may co-occur and interact with each other in areas where they both find adequate conditions. Comparison among contact zones revealed geographic variation in the species" niches as well as in the climatic conditions at their records suggesting that the species can occur in a much wider range of conditions than they actually do. These findings imply that climate represents a main factor for the species" parapatric range limits. Yet, interspecific niche overlap and the geographic variation provide indirect evidence that interspecific interaction may also affect their spatial distribution. To test whether competition restricts the species" ranges on the habitat scale and to understand local syntopic co-occurrence of the salamanders within their contact zones, site-occupancy modelling was used. This approach allowed to find the habitat predictors that best explain the species" local distribution. While the slope of the site positively affected the occupancy probability of S. salamandra, no tested predictor explained that of S. atra. Also, there was no effect of the occurrence of one species on the occupancy probability of the other providing no evidence for competition. Should competition occur, it does not lead to spatial segregation of the species on this scale. Because biotic interactions most significantly affect the ranges of species on small spatial scales, the microhabitat conditions at locations of the species within syntopic contact zones were compared and a null model analysis was applied to determine their niche overlap. Resource selection probability function models were used to assess those attributes that affect the species" habitat selections. The results revealed species-specific microhabitat preferences related to leaf litter cover, tree number and that the species were active at different temperatures as well as times of the day. The high degree of diurnal activity of S. atra may be due to its preference of forest floor microhabitats that long remain suitable during daytime. Besides, there was a great niche overlap for shelters indicating that the species may compete for this resource. Differential habitat selection and the use of the available shelters at different times of the day may minimize species interactions and allow their local co-occurrence within contact zones. To identify whether the potential infection with the pathogenic chytrid fungus could serve as an alternative biotic explanation for the range margins of S. atra, several populations throughout its range were screened for infection. Since the occurrence of this pathogen was detected mostly at lower altitudes of the Alps, it may confine the range of S. atra to higher elevations. Because chytrid was not detected in any of the samples, the pathogen unlikely plays a role in determining its range limits. Overall, these findings underline the complexity of mechanisms that determine the range margins of parapatric species and provide an important basis for subsequent studies regarding the determinants of the parapatric distribution of the two salamander species.
The availability of data on the feeding habits of species of conservation value may be of great importance to develop analyses for both scientific and management purposes. Stomach flushing is a harmless technique that allowed us to collect extensive data on the feeding habits of six Hydromantes species. Here, we present two datasets originating from a three-year study performed in multiple seasons (spring and autumn) on 19 different populations of cave salamanders. The first dataset contains data of the stomach content of 1,250 salamanders, where 6,010 items were recognized; the second one reports the size of the intact prey items found in the stomachs. These datasets integrate considerably data already available on the diet of the European plethodontid salamanders, being also of potential use for large scale meta-analyses on amphibian diet.
Allergische Kontaktdermatitis ist eine zellvermittelte verzögerte Überempfindlichkeitsreaktion auf niedermolekulare Substanzen, die häufig Hautreaktionen hervorrufen und dadurch sowohl persönliche als auch berufliche Probleme verursachen. Chemikalien wie para-Phenylendiamin (PPD) und andere verwandte para-Aminobenzole werden häufig in Farben eingesetzt und erlangen aufgrund ihrer starken Allergenität eine immer größere Aufmerksamkeit. Bis jetzt ist das Wissen über zelluläre Immunantworten auf PPD nur begrenzt verstanden. In dieser Studie haben wir das immunmodulatorische Potential vonrnPPD untersucht, vor allem hinsichtlich der Fragestellung, ob PPD selber naive T-Zellen sensibilisieren kann oder ob PPD letztendlich nur ein Allergen ist. In Anbetracht der Tatsache das dendritische Zellen (DZ) eine vorherrschende Rolle bei der allergischen Kontaktdermatitis spielen, haben wir untersucht, in wie weit PPD in der Lage ist, funktionelle Reifung zu modulieren. Um abschätzen zu können, welcher Anteil an PPD nach der Stimulation zur potentiellen Aktivierung tatsächlich zur Verfügung steht, haben wir die metabolische Kompetenz von "monocyte derived dendritic cells" (MoDC) untersucht, PPD zurnacetylieren. Dazu haben wir die N-Acetyltransferase 1 (NAT-1) und N-Acetyltransferase 2(NAT-2) mRNA Expression charakterisiert und weitergehend die metabolische Aktivität von NAT-1 Enzym bestimmt. Mit diesen Versuchen konnten wir zeigen, dass MoDC von 9 aus 10 Spendern das NAT-1 Enzym exprimieren und konnten in 4 von 6 Spendern acetyliertes PPD in Extrakten aus Zellkulturüberständen detektieren. In Anbetracht der Tatsache, dassrnacetyliertes PPD nicht in der Lage ist, MoDC zu aktivieren, können wir davon ausgehen,dass der Anteil an acetyliertem PPD zur DC Aktivierung nicht zur Verfügung steht. Um das sensibilisierende Potential von PPD herauszufinden, haben wir nach Stimulation mit verschiedenen PPD Konzentrationen die Expression von Oberflächenmolekülen, die für die Antigenpräsentation, Co-Stimulation, späte Migration und dendritische Zell-/T-Zell-Wechselwirkung entscheidend sind, zu verschiedenen Zeitpunkten mittels Durchflusszytometrie Messungen (FACS) gemessen. Zusammengefasst konnten wir zeigen,dass PPD signifikant die "chemokine receptor 7" (CCR7) Expression und nicht signifikant die "human leukocyte antigen " DR" HLA-DR, "DC-specific C-type lectin intercellular adhesion molecule-3-grabbing nonintegrin" (DC-SIGN) und "cluster domain" 11c (CD11c) Expression erhöht. Die erhöhte CCR7 Expression ermutigte uns, weiter nachzuforschen, ob PPD trotz der Tatsache, dass es keine traditionelle CD80 und CD86 Expression induzieren kann, fähig ist, DCs zu aktivieren. Ausgiebige Zytokinkinetikmessungen konnten diese Hypothese bestätigen. Mit Cytometric Beat Array (CBA) Messungen konnten wir gleichzeitig Mediatoren wie "Interleukin -1ß" (IL-1ß), "tumor necrosis factor-alpha" (TNF-α), IL-8, IL-6, IL-12P70 undrnIL-10 messen und damit demonstrieren, dass PPD in der Lage ist, bei allen Spendern "Danger Signals" zu induzieren. Zudem konnten wir zeigen, dass die PPD induzierte Zytokinausschüttung interindividuell schwankte und zu unterschiedlichen Zeitpunkten erfolgte. Überraschenderweise war die qualitative Zusammensetzung der Zytokine vergleichbar. Während in 3 von 4 Spendern die Zytokinexpression eher schwach erhöht war und von kurzer Dauer, haben wir in einem Spender vor allem mit den Konzentrationen von 10μM und 50μM PPD verblüffend hohe Zytokinausschüttungen gemessen. Dieser Spenderrnscheint eine Person zu repräsetieren, die empfänglicher für eine PPD Sensibilisierung ist als andere. Unabhängig davon, hat uns die Tatsache, dass PPD in allen Spendern "Danger Signals", aber keine gesteigerte Expressionsrate von traditionellen Reifungsmarkern (mit Ausnahme von CCR7) induzierte, veranlasst zu untersuchen, ob PPD anderweitig fähig ist,in MoDC Immunantworten zu modulieren. Daraufhin haben wir den Einfluss von PPD aufrnaktivierte DCs gemessen und die Ergebnisse bestätigten die Annahme. Weiterhin konnten wir zeigen, dass PPD immunogenes Potential aufweist. Aufgrund der Tatsache, dass die LPS induzierten Signaltransduktionswege in MoDC gut charakterisiert sind, konnten wir mit dieser Studie zusätzlich Hinweise über die PPD induzierte Signalgebung liefern.
Um die weiterhin ansteigende Weltbevölkerung auch zukünftig mit Nahrungsmitteln und anderen wichtigen Rohstoffen zu versorgen, bedarf es einer immer größeren Nutzungsausweitung der Landwirtschaft auf natürliche Flächen. Im Jahre 2009 wurde ein Drittel der Landoberfläche der Erde bereits landwirtschaftlich genutzt. In jüngster Zeit kommt der Landwirtschaft zusätzlich eine wichtige Bedeutung als Energielieferant zu. Ein weiter steigender Flächenbedarf ist die Konsequenz dieser Entwicklung. Dies führt zum Verlust von Habitaten und somit zu einer starken Fragmentierung der Landschaft. Die direkten und indirekten Auswirkungen dieser Entwicklung auf den weltweiten Biodiversitätsrückgang werden mittlerweile als eine der Hauptursachen für den Rückgang vieler Arten anerkannt. Auch der Gesetzgeber hat dieses Problem erkannt und versucht dem entgegenzuwirken. In der vorliegenden Arbeit wird durch einen multiplen Methodenansatz der Einfluss der Habitatfragmentierung auf die Konnektivität der Populationen von zwei unterschiedlich mobilen Insektenarten untersucht. Als Modelarten wurden eine wenig mobile Heuschreckenart (Chorthippus montanus) und ein hoch mobiler Tagfalter (Brenthis ino) ausgewählt, welche beide an feuchte Grünländer gebunden sind. Für C. montanus wurde gezeigt, dass die Art nur ein sehr eingeschränktes Mobilitätspotenzial besitzt und stark an ihr Habitat gebunden ist. Daher ist eine Durchquerung von ungeeigneten Lebensräumen als äußerst unwahrscheinlich zu erachten. Eine Studie zur Mobilität und dem Verhalten von Brenthis ino belegt eine starke Philopatrie für eine nicht territoriale Insektenart. Zusätzlich zu den Fang-Wiederfang-Untersuchungen im Freiland wurden populations- und landschaftsgenetische Analysen durchgeführt. Die populationsgenetischen Analysen zeigen eine starke Isolation der Populationen von C. montanus, während zwischen den untersuchten Populationen von B. ino eine gute Konnektivität besteht. Mittels der landschaftsgenetischen Analyse wird gezeigt, dass Wälder und bebaute Flächen für beide Arten substanzielle Genefluss-Barrieren darstellen. Basierend auf einer GIS gestützten Analyse wird dargelegt, dass eine lokal ausreichende Vernetzung von Grünländern in Rheinland-Pfalz durch den Biotopverbund existiert. Die Berücksichtigung der Konnektivität bei der Auswahl und Ausgestaltung der Instrumente zur rechtlichen Sicherung und Umsetzung des Biotopverbundes (-§ 21 BNatSchG) bergen großes Potenzial zum Schutz von Grünländern. Ferner wird gezeigt, dass die Landschaftsplanung zwar ein geeignetes Instrument zur Erarbeitung des Biotopverbundes ist, aber kein Instrument zur rechtlichen Sicherung desselbigen im Sinne des -§ 21 Abs. 4 BNatSchG sein kann.
Tropospheric ozone (O3) is known to have various detrimental effects on plants, such as visible leaf injury, reduced growth and premature senescence. Flux models offer the determination of the harmful ozone dose entering the plant through the stomata. This dose can then be related to phytotoxic effects mentioned above to obtain dose-response relationships, which are a helpful tool for the formulation of abatement strategies of ozone precursors. rnOzone flux models are dependant on the correct estimation of stomatal conductance (gs). Based on measurements of gs, an ozone flux model for two white clover clones (Trifolium repens L. cv Regal; NC-S (ozone-sensitive) and NC-R (ozone-resistant)) differing in their sensitivity to ozone was developed with the help of artificial neural networks (ANNs). White clover is an important species of various European grassland communities. The clover plants were exposed to ambient air at three sites in the Trier region (West Germany) during five consecutive growing seasons (1997 to 2001). The response parameters visible leaf injury and biomass ratio of NC-S/NC-R clone were regularly assessed. gs-measurements of both clones functioned as output of the ANN-based gs model, while corresponding climate parameters (i.e. temperature, vapour pressure deficit (VPD) and photosynthetic active radiation (PAR)) and various ozone concentration indices were inputs. The development of the model was documented in detail and various model evaluation techniques (e.g. sensitivity analysis) were applied. The resulting gs model was used as a basis for ozone flux calculations, which were related to above mentioned response parameters. rnThe results showed that the ANNs were capable of revealing and learning the complex relationship between gs and key meteorological parameters and ozone concentration indices. The dose-response relationships between ozone fluxes and visible leaf injury were reasonably strong, while those between ozone fluxes and NC-S/NC-R biomass ratio were fairly weak. The results were discussed in detail with respect to the suitability of the chosen experimental methods and model type.
Comparing the results of the phylogeographies of the four species included in this thesis, some accordances have been found, even though certain patterns are only represented in one or two species. In all cases, the findings of the studied species strongly support the existence of forests or forest-like ecosystems beyond the classic forest refugia in the Mediterranean areas (Iberian, Apennine and Balkan peninsulas) during glacial times. However, evidence of glacial refugial areas in Southeastern Europe, especially the Balkans, have been found in this study as well. The analysed populations of Aposeris foetida, Melampyrum sylvaticum and Erebia euryale showed high genetic diversity values and mostly higher private fragments in this area, which is a strong indicator for centres of glacial survival during Würm and, regarding the results of M. sylvaticum, even during the Riss ice age. Three of the analysed species (A. foetida, M. sylvaticum and Colias palaeno) supported a second main glacial refuge area located along the Northern Alps. Again, high genetic diversity values and the uniqueness of the populations living in this region today prove the importance of this area as a glacial centre of survival. Those results confirm several recently published studies on forest species and strongly indicate the persistence of forest-like structures or even forests during the ice ages along the foothills of the Northern Alps. Additionally, the persistence of C. palaeno in this area furthermore supports the existence of peatlands north of the Alps, at least during the last glacial. The results of M. sylvaticum and E. euryale further indicate the vicinity of the Tatra Mountains as core areas for glacial survival. However, the genetic patterns found for E. euryale are ambiguous. Due to an intermediate position of two genetic lineages (originating in the Eastern Alps and Southeastern Europe), the Tatras could also reflect a postglacial mixture zone of those lineages. Moreover, the glacial and postglacial importance of this area for woodland species was accentuated, supporting other phylogeographic studies published. Besides the congruities among the results of the study species, some unique patterns and therefore further potential glacial refugia have also been illuminated in this thesis. For instance, the calcicole species, A. foetida, most probably had further survival area at both sides of the Dinaric Alps, supported by high genetic diversity values and a high number of private fragments found in Croatian populations. Furthermore, the surroundings of the German Uplands and the margin of the Southern Alps provided suitable conditions for glacial survival for M. sylvaticum, while the Eastern and Southeastern Alpine region most probably sheltered the Large Ringlet E. euryale during ice ages. Additionally, this butterfly species survived at least the glaciation along the foothills of the Massif Central, whose present populations showed a unique genetic lineage and their genetic diversity values have been measurably higher than in other populations for this species. Finally, a large and continuous Würm distribution is highly likely south of the Fennoscandian glaciers in Central Europe for C. palaeno, which might indicate extended peatland areas during Würm glacial. With all the patterns found in this study, the understanding of glacial persistence of forest, respectively forest-like structures and peatlands during Würm or even Riss glacial in Europe could be advanced. The congruencies among the analysed woodland and bog species illustrate the importance and location of extra-Mediterranean refugia for European mountain forests and the glacial presence of Central European peatlands. Thus, already postulated theories could be supported and further pieces of the overall puzzle could be added. The varieties of the different survival centres once more clarified that further phylogeographic studies on mountain forest of different habitat requirements and especially peatland species have to be implemented to get a clearer picture of the glacial history of these habitats.
Climate change and habitat fragmentation modify the natural habitat of many wetland biota and lead to new compositions of biodiversity in these ecosystems. While the direct effects of climate are often well known, indirect effects due to biotic interactions remain poorly understood. The water meadow grasshopper, Chorthippus montanus, is a univoltine habitat specialist, which is adapted to permanently moist habitats. Land use change and drainage led to highly fragmented populations of this generally flightless species. In large parts of the Palaearctic Ch. montanus occurs sympatrically with its widespread congener, the meadow grasshopper Chorthippus parallelus. Due to their close relationship and their similar songs, hybridization is likely to occur in syntopic populations. Such a species pair of a habitat specialist and a habitat generalist represents an ideal model system to examine the role of ongoing climate change and an accumulation of extreme climatic events on the life history strategies, population dynamics and inter-specific interactions. In Chapter I a laboratory experiment was conducted to identify the impact of environmental factors on intra-specific life-history traits of Ch. montanus. Like other Orthoptera species, Ch. montanus follows a converse temperature size rule. In line with the dimorphic niche hypothesis, which states that sexual size dimorphism evolved in response to the different sexual reproductive roles, both sexes showed different responses to increasing density at lower temperatures. Males attained smaller body sizes at high densities, whereas females had a prolonged development time. This is the first evidence for a sex-specific phenotypic plasticity in Ch. montanus. Females benefit from the prolonged development as their reproductive success depends on the size and number of egg clutches they may produce. By contrast, the reproductive success of males depends on the chance to fertilize virgin females, which increases with faster development. This may become a disadvantage for Ch. montanus as an intraspecific phenology shift may increase hybridization risk with the sibling species. Despite the widespread assumption that hybridization between two sympatric species is rare due to complete reproductive barriers, the genetic analyses of 16 populations (Chapter II) provided evidence for wide prevalence of hybridization between both species in the wild. As no complete admixture was found in the examined population, it is assumed that hybridization only occurs in ecotones between wetlands and drier parts. Reproductive barriers (habitat isolation, behavior, phenology) seem to prevent the genetic swamping of Ch. montanus populations. Although a behavioral experiment showed that mate choice presents an important reproductive barrier between both species, the experiment also revealed that reproductive barriers could be altered by environmental change (e.g. increasing heterospecific frequency). Chapter III analyzes the impact of extreme climatic events on population dynamics and interspecific hybridization. A mark-recapture analysis combined with weather records over five years provides evidence that the embryonic development in Ch. montanus is vulnerable to extreme climatic events. Strong population declines in Ch. montanus lead to a disequilibrium between Ch. montanus and Ch. parallelus populations and increases the risk of hybridization. The highest hybridization risk was found in the first weeks of a season, when both species had an overlapping phenology. Furthermore, hybrids were generally localized at the edge of the Ch. montanus distribution with higher heterospecific encounter probabilities. The hybridization rate reached up to 19.6%. The genetic analyses in Chapter II and III show that hybridization differentially affects specialists and generalists. While generalists may benefit from hybridization by an increasing genetic diversity, such a positive correlation was not found for Ch. montanus. The results underline the importance of reproductive barriers for the co-existence of these sympatric species. However, climate change and other anthropogenic disturbances alter reproductive barriers and promote hybridization, which may threaten small populations by genetic displacement. As anthropogenic hybridization is recognized as a major threat to biodiversity, it should be considered in environmental law and policy. In Chapter IV the role of hybrids and hybridization in three levels of law and the historical backgrounds of hybrids becoming a part of legal instruments is analyzed. Due to legal uncertainties and the complexity of this topic a legal assessment of hybrids is challenging and argues for species-specific approaches. Nonetheless, existing legal norms provide a suitable basis, but need to be specified. Finally, this chapter discusses different opportunities for the management of hybrids and hybridization in a conservation perspective and their necessity.
N-acetylation by N-acetyltransferase 1 (NAT1) is an important biotransformation pathway of the human skin and it is involved in the deactivation of the arylamine and well-known contact allergen para-phenylenediamine (PPD). Here, NAT1 expression and activity were analyzed in antigen presenting cells (monocyte-derived dendritic cells, MoDCs, a model for epidermal Langerhans cells) and human keratinocytes. The latter were used to study exogenous and endogenous NAT1 activity modulations. Within this thesis, MoDCs were found to express metabolically active NAT1. Activities were between 23.4 and 26.6 nmol/mg/min and thus comparable to peripheral blood mononuclear cells. These data suggest that epidermal Langerhans cells contribute to the cutaneous N-acetylation capacity. Keratinocytes, which are known for their efficient N-acetylation, were analyzed in a comparative study using primary keratinocytes (NHEK) and different shipments of the immortalized keratinocyte cell line HaCaT, in order to investigate the ability of the cell line to model epidermal biotransformation. N-acetylation of the substrate para-aminobenzoic acid (PABA) was 3.4-fold higher in HaCaT compared to NHEK and varied between the HaCaT shipments (range 12.0"44.5 nmol/mg/min). Since B[a]P induced cytochrome p450 1 (CYP1) activities were also higher in HaCaT compared to NHEK, the cell line can be considered as an in vitro tool to qualitatively model epidermal metabolism, regarding NAT1 and CYP1. The HaCaT shipment with the highest NAT1 activity showed only minimal reduction of cell viability after treatment with PPD and was subsequently used to study interactions between NAT1 and PPD in keratinocytes. Treatment with PPD induced expression of cyclooxygenases (COX) in HaCaT, but in parallel, PPD N-acetylation was found to saturate with increasing PPD concentration. This saturation explains the presence of the PPD induced COX induction despite the high N-acetylation capacities. A detailed analysis of the effect of PPD on NAT1 revealed that the saturation of PPD N-acetylation was caused by a PPD-induced decrease of NAT1 activity. This inhibition was found in HaCaT as well as in primary keratinocytes after treatment with PPD and PABA. Regarding the mechanism, reduced NAT1 protein level and unaffected NAT1 mRNA expression after PPD treatment adduced clear evidences for substrate-dependent NAT1 downregulation. These results expand the existing knowledge about substrate-dependent NAT1 downregulation to human epithelial skin cells and demonstrate that NAT1 activity in keratinocytes can be modulated by exogenous factors. Further analysis of HaCaT cells from different shipments revealed an accelerated progression through the cell cycle in HaCaT cells with high NAT1 activities. These findings suggest an association between NAT1 and proliferation in keratinocytes as it has been proposed earlier for tumor cells. In conclusion, N-acetylation capacity of MoDCs as well as keratinocytes contribute to the overall N-acetylation capacity of human skin. NAT1 activity of keratinocytes and consequently the detoxification capacities of human skin can be modulated by the presence of exogenous NAT1 substrates and endogenous by the cell proliferation status of keratinocytes.
Caenorhabditis elegans Modelsysteme für Freiland und Labor Caenorhabditis elegans ist eines der am intensivsten untersuchten Tiermodelle, dabei weisen die nur unzureichenden Informationen über sein ursprüngliches Habitat eine Vielzahl offener Fragen auf. Deshalb beschäftigt sich der erste Teil vorliegender Arbeit mit einer Überprüfung der Freilandökologie und Biogeographie des Taxons. Nach den Untersuchungen spricht vieles dafür, dass die Ursprungsart der C. elegans Gruppe ein Begleiter von Gastropoden war, welche auch für ihre Ausbreitung häufig verantwortlich sind. Als Folge der Verschleppung durch Wirtstiere (u.a. die Weinbergschnecke Helix aspersa) erreichte sie ihr heutiges Areal, in allen Kontinenten. Ein nicht weniger wichtiges Ziel der vorliegenden Arbeit war die Etablierung von C. elegans als Biotest-System für die Indikation von Chemikalien-Wirkungen. In dieser Arbeit wurden ein ökotoxikologisches Testsystem entwickelt mit den C. elegans als Modellorganismus, das durch Messung von umweltbedingten intrazellulär gebildeten ROS im Gesamtorganismus eine Einschätzung der biologischen Aktivität von Chemikalien und Umweltmedien ermöglichen sollte. Als grundlegender biochemischer Parameter ist eine, die Kapazität der antioxidativen Schutzmechanismen übersteigende, ROS-Bildung ursächlich für oxidative Schädigungen der Zelle verantwortlich und damit Basis für zahlreiche pathologische Effekte. Aus den Resultaten als Bewertungsgrundlage lässt sich folgern, dass sich das erarbeitete Biotestsystem als adäquates Verfahren zur Messung von oxidativem Stress, als Folge exogener Belastung, einsetzen lässt. Damit leistet es einen Beitrag zur Beurteilung des Toxizitätspotentials von Chemikalien und Umweltproben. Zur Eignung des Einsatzes von C. elegans für Toxizitätstest wurden weitere Untersuchungen mit verschiedenen Chemikalien durchgeführt. Dabei ergibt sich bezogen auf molare Einheiten eine immer gleich bleibende Reihenfolge der Sensitivitäten gegenüber BaP > Cu2+ > AcL, ein Ergebnis welches mit Testergebnissen von anderen Versuchsorganismen zu vergleichen war. Die Wirkung von Atrazin auf die Entwicklung von C. elegans wurde auch untersucht. Es wurde festgestellt, dass Atrazin auf Wachstum und Reproduktion von C. elegans konzentrationsabhängig toxisch wirkt. Die bisherigen Ergebnisse zeigen jedoch, dass das Testsystem C. elegans hervorragend geeignet ist, um Chemikalienwirkungen frühzeitig sichtbar zu machen.
Besides well-known positive aspects of conservation tillage combined with mulching, a drawback may be the survival of phytopathogenic fungi like Fusarium species on plant residues. This may endanger the health of the following crop by increasing the infection risk for specific plant diseases. In infected plant organs, these pathogens are able to produce mycotoxins like deoxynivalenol (DON). Mycotoxins like DON persist during storage, are heat resistant and of major concern for human and animal health after consumption of contaminated food and feed, respectively. Among fungivorous soil organisms, there are representatives of the soil fauna which are obviously antagonistic to a Fusarium infection and the contamination with mycotoxins. Earthworms (Lumbricus terrestris), collembolans (Folsomia candida) and nematodes (Aphelenchoides saprophilus) provide a wide range of ecosystem services including the stimulation of decomposition processes which may result in the regulation of plant pathogens and the degradation of environmental contaminants. Several investigations under laboratory conditions and in the field were conducted to test the following hypotheses: (1) Fusarium-infected and DON-contaminated wheat straw provides a more attractive food substrate than non-infected control straw (2) the introduced soil fauna reduce the biomass of F. culmorum and the content of DON in infected wheat straw under laboratory and field conditions (3) the species interaction of the introduced soil fauna enhances the degradation of Fusarium biomass and DON concentration in wheat straw; (4) the degradation efficiency of soil fauna is affected by soil texture. The results of the present thesis pointed out that the degradation performance of the introduced soil fauna must be considered as an important contribution to the biological control of plant diseases and environmental pollutants. As in particular L. terrestris revealed to be the driver of the degradation process, earthworms contribute to a sustainable control of fungal pathogens like Fusarium and its mycotoxins in wheat straw, thus reducing the risk of plant diseases and environmental pollution as ecosystem services.
Leeches can parasitize many vertebrate taxa. In amphibians, leech parasitism often has potential detrimental effects including population decline. Most of studies on the host-parasite interactions involving leeches and amphibians focus on freshwater environments, while they are very scarce for terrestrial amphibians. In this work, we studied the relationship between the leech Batracobdella algira and the European terrestrial salamanders of the genus Hydromantes, identifying environmental features related to the presence of the leeches and their possible effects on the hosts. We performed observation throughout Sardinia (Italy), covering the distribution area of all Hydromantes species endemic to this island. From September 2015 to May 2017, we conducted >150 surveys in 26 underground environments, collecting data on 2629 salamanders and 131 leeches. Water hardness was the only environmental feature correlated with the presence of B. algira, linking this leech to active karstic systems. Leeches were more frequently parasitizing salamanders with large body size. Body Condition Index was not significantly different between parasitized and non-parasitized salamanders. Our study shows the importance of abiotic environmental features for host-parasite interactions, and poses new questions on complex interspecific interactions between this ectoparasite and amphibians.