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In the current era of anthropogenic climate change is the long-term survival of all organisms dependent on their ability to respond to changing environmental conditions either by (1) phenotypic plasticity, which allows species to tolerate novel conditions, (2) genetic adaptation, or (3) dispersal to more suitable habitats. The third option, dispersal, allows individuals to escape unfavorable conditions, the colonization of new areas (resulting in range shifts), and affects patterns of local adaptation. It is a complex process serving different functions and involving a variety of underlying mechanisms, but its multi-causality though has been fully appreciated in recent years only. Thus, the aim of this doctoral thesis was to disentangle the relative importance of the multiple factors relevant to dispersal in the copper butterfly Lycaena tityrus, including the individual condition (e.g. morphology, physiology, behavior) and the environmental context (e.g. habitat quality, weather). L. tityrus is a currently northward expanding species, which makes it particularly interesting to investigate traits underlying dispersal. In the first experiment, the influence of weather and sex on movement patterns under natural conditions was investigated. Using the Metatron, a unique experimental platform consisting of interconnected habitat patches, the second experiment aimed to examine the influence of environmental factors (resources, sun) on emigration propensity in experimental metapopulations. Human-induced global change (e.g. climate change, agricultural intensification) poses a substantial challenge to many herbivores due to a reduced availability or quality of feeding resources. Therefore, in the third experiment, the impact of larval and adult food stress on traits related to dispersal ability was investigated. Additionally, the effect of different ambient temperatures was tested. In the fourth experiment, core (Germany) and recently established edge (Estonia) populations were compared in order to explore variation in dispersal ability and life history traits indicative of local adaptation. Dispersal is often related to flight performance, and morphological and physiological traits, which was investigated in experiments 2-4. Butterflies were additionally subjected to behavioral experiments testing for the individual’s exploratory behavior (experiments 3 and 4).
Males and females differed substantially in morphology, with males showing traits typically associated with a better flight performance, which most likely result from selection on males for an increased flight ability to succeed in aerial combats with rivalling males and competition for females. This pattern could be verified by mobility measures under natural conditions and flight performance tests. Interestingly, although females showed traits associated with diminished flight performance, they had a higher emigration propensity than males (though in a context dependent manner). Reasons might be the capability of single mated females to found new populations, to spread their eggs over a wide range or to escape male harassment. Conditions indicative of poor habitat quality such as shade and a lack of resources promoted emigration propensity. The environmental context also affected condition and flight performance. The presence of resources increased the butterflies’ condition and flight performance. Larval and adult food stress in turn diminished flight performance, despite some reallocation of somatic resources in favor of dispersal-related traits. These detrimental effects seem to be mainly caused by reductions in body mass and storage reserves. A similar pattern was found for exploratory behavior. Furthermore, higher temperatures increased flight performance and mobility in the field, demonstrating the strong dependence of flight, and thus likely dispersal, on environmental conditions. Flight performance and exploratory behavior were positively correlated, probably indicating the existence of a dispersal syndrome. The population comparison revealed several differences between edge and core populations indicative of local adaptation and an enhanced dispersal ability in edge populations. For instance, edge populations were characterized by shorter development times, smaller size, and a higher sensitivity to high temperatures, which seem to reflect adaptations to the cooler Estonian climate and a shorter vegetation period. Moreover, Estonian individuals had an enhanced exploratory behavior, which can be advantageous in all steps of the dispersal process and may have facilitated the current range expansion.
In summary, these findings may have important implications for dispersal in natural environments, which should be considered when trying to forecast future species distributions. First, dispersal in this butterfly seems to be a highly plastic, context-dependent trait triggered largely by habitat quality rather than by individual condition. This suggests that dispersal in L. tityrus is not random, but an active process. Second, fast development and an enhanced exploratory behavior seem to facilitate the current range expansion. But third, while deteriorating habitat conditions are expected to promote dispersal, they may at the same time impair flight ability (as well as exploratory
behavior) and thereby likely dispersal rates. For a complete understanding of a complex process such as dispersal, further research is required.
In the 1940s cytochrome P450 monooxygenases have been discovered and have been the focus of many studies ever since. Although they catalyze very interesting reactions that might find applications in the production of fine chemicals or pharmaceuticals, their low activity and stability often reduces their economic value. Both properties, the activity and the stability, are influenced by the uncoupling of the catalytic cycle.
In this PhD thesis, an assay for the screening of activity and uncoupling of cytochrome P450 enzymes was successfully developed. After finding optimal conditions for the assay, concerning pH and enzyme concentration, the uncoupling of cytochrome P450 BM3 and five mutants (F87Y, R47L, Y51F, A82L and T268A) was investigated. With the results obtained, a comparison of data from literature was possible and revealed similarities. Additionally, through negative controls, the reliability of the assay could be further demonstrated. Although other methods have been described for the detection of hydrogen peroxide formation, the combination of NADPH consumption measurement and hydrogen peroxide formation in parallel was new and represents a very good basis for a pre-screening of large mutant libraries, followed by closer investigation of selected variants.
For the investigation of the activity of the CYP11A1 system, consisting of CYP11A1 and Adx and AdR as redox partner system, the expression and purification for all three proteins was investigated first. For the protein CYP11A1 and Adx, good expression levels were achieved, whereas for AdR the protein concentration obtained was very low. The purification of all three proteins was partially accomplished but left room for improvement. Therefore, in the Master thesis of Christopher Grimm, the pH and temperature stability of all three proteins was further investigated in order to improve conditions used for ion exchange chromatography and to investigate possible conditions for in vitro biocatalysis. As unfortunately even with further investigation of the expression of AdR, no improvement was achieved, a whole-cell system was further investigated. Here, the product formation could be increased 8-fold in comparison to the published data, from 0.27% conversion to 2.2% conversion over 24 h by using a different detergent for substrate solubilization, which might have led to a better substrate supply to the enzyme.
Due to the low activity and stability, a different P450 system, the CYP17A1 enzyme, was subsequently investigated, first by in vitro biocatalysis with the human CYP17A1 expressed in E. coli. Therefore, a suitable redox partner system needed to be found for efficient electron supply of the enzyme. In in vitro biocatalysis, in combination with the Pdx/PdR system of P. putida the CYP17A1 enzyme showed the highest conversion with 91% after 24 h. To investigate the activity of the enzyme further, all active site residues in 4 Å proximity to the bound substrate were exchanged with alanine. After expression of the variants, almost no correctly folded protein was obtained for the variants. Also, after investigating different buffers to possibly enhance the stability, no improvements were achieved. Therefore, a whole-cell approach with the bovine enzyme was chosen in order to investigate the activity of the alanine variants. Here the importance of positions N202, R239, G297, E305, and T306A, described in literature to be important for catalytic activity, was confirmed. Most importantly, three positions that alter the regioselectivity of the enzyme were identified. The reaction of the V483A mutant was therefore also further investigated by preparative biocatalysis. Afterwards the new product was separated by preparative HPLC and identified as 16α- hydroxyprogesterone as confirmed by NMR spectroscopy analysis.
In the last part of the thesis, another screening approach for possible high-throughput screening was investigated. In contrast to the other screening approach, here the investigation of the substrate conversion and the hydrogen peroxide formation were optimized for application in droplets. After finding that DCFH-DA was not sensitive enough towards hydrogen peroxide, the AmplifluTM Red probe was used. As both fluorescent products were found to stay in the aqueous phase above pH 7.4, the conditions investigated for the AmplifluTM Red assay were applied and only NADPH to substrate ratio was investigated by using an uncoupling variant, an active variant from literature and the cytochrome P450 BM3 wild-type enzyme. After finding a good ratio, the five variants used for the investigation of the AmplifluTM Red assay were investigated in the same concentration later on found in the droplets (1 cell per 4 pL), and one variant showed improved product formation compared to wild-type. This finding clearly shows the applicability of the assay for high-throughput screening in droplets.
Humanity is constantly confronted with the emergence and reemergence of infectious diseases. Many of them produce large or devastating epidemics, like AIDS (HIV) and Ebola. Others have been long neglected, yet pose immediate threats to global public health as evidences the abrupt emergence of Zika virus in South America and its association with microcephaly in babies. The examples illustrate, that many of these diseases are provoked by RNA viruses. One of the first steps in understanding and eliminating those threats is the development of sensitive and rapid diagnostic methods. A general and relatively rapid method is the direct detection and examination of the agent’s genome. However, the nature of (re)emerging RNA viruses poses a series of very specific problems for the design of such methods. Therefore, a systematic approach was proposed for the design of DNA-hybridization-base methods to detect and characterize RNA viruses that will have both a high sensitivity and a specificity sufficiently broad to detect, per reaction, down to a single copy of any of the possible variants of the viral genome.
Following this approach a series of assays were designed, developed or adapted and put into use for detection and characterization of important RNA viruses. One of those viruses is West Nile virus (WNV), which after its explosive introduction into USA become the most widespread flavivirus throughout the world and, consequently, many countries began an intensive monitoring. While existing assay detected predominantly the Lineage 1, in Europa Lineage 2 was expected. Two new RT-qPCR for the detection of both lineages were developed, and reportedly used by independent laboratories. Due to more than 50000 associated deaths per year, the Hepatitis E virus also received an increasing attention to elucidate novel routes of transmission. This virus (especially genotype 3) has the zoonotic potential of transmission from pigs and wild boar to humans. RT-qPCR and nested qPCR for detection and characterization of this virus as well as a methodology for subtyping were developed and the first detected case of subtype 3b in a German wild animal was documented. In addition a novel assay for flaviviruses conformed by a RT-qPCR coupled with a low density DNA microarray was developed, which enabled the identification of WNV in mosquitoes from Greece. A RT-qPCR suitable for surveillance and diagnostic of all known variants of Venezuelan equine encephalitis virus was developed too. A causative agent of hemorrhagic infections, the Ngari virus, was detected and characterized in animal samples from Mauritania. These achievements were supported by the development of software applications for selection and visualization of primers and probes from aligned DNA sequences and for modeling of DNA hybridizations using unaligned sequences.
In conclusion a general methodology for rapid development of sensitive diagnostic methods based in DNA-hybridization technics (PCR, sequencing and microarray) was stablished and successful applications are reported.
What shapes the prospect for democracy in the aftermath of civil conflicts? Some authors claim a successful transition from violence to elections mainly depends on the ability of political institutions, such as power-sharing arrangements, to mitigate the security dilemma among former battlefield adversaries. Drawing on a broader literature, others point to potential effects of foreign aid on democratic development.
This predominant focus on elections and the security dilemma, however, limits our understanding in a number of ways. We do not know how the choice of post-conflict elites to hold elections is strategically intertwined with their willingness to reform other state institutions. We also have only begun to understand how post-conflict power-sharing governments function as revenue source for elites. Knowing how this economic function drives or obstructs post-conflict democratic development is particularly helpful if we shift our attention to a major source of income for post-conflict elites: foreign aid, and the democratic conditions donors attach to it.
Addressing these gaps, I argue that both the economic utility from office as well as political conditionalities give rise to a rent-seeking/democracy dilemma for post-conflict elites: they can either hold elections and face uncertainty over their access to power, but secure economic rents from aid. Or they refuse to democratize, secure their hold on power, but risk losing revenues when donors withdraw aid. In this situation, their optimal strategy is to agree to democratic reforms in the area on which donors place most value, elections. But to maximize their chances of electoral victory and continued access to rents from office, elites simultaneously restrain an independent rule of law and narrowly distribute private goods to their supporters.
This rent-seeking/democracy dilemma is particularly prevalent in one of the most popular forms of post-conflict institutions: power-sharing governments. Including rebel groups in post-conflict cabinets increases the number of constituencies that need to be sustained from the government budget. In addition, the interim nature of transitional power-sharing cabinets leads elites to steeply discount the future and increase rent-seeking in the short term. My main hypothesis is therefore that large aid flows to extensive power-sharing governments should be associated with improved elections, but limits in the rule of law and more provision of private instead of public goods.
To test this prediction quantitatively, I combine data on aid flows and rebel participation in post-conflict cabinets between 1990 and 2010 with indicators for democratic development, election quality, rule of law, and public goods provision. Results from a wide range of regression models provide empirical support for my argument. Individually, extensive power-sharing governments and large aid flows do not seem to have strong effects. Models that introduce an interaction term between aid and power-sharing, however, yield strong evidence of a rent-seeking/democracy dilemma: Power-sharing and foreign aid jointly predict a positive, but small change in democracy scores as well as cleaner elections. At the same time, they are jointly associated with a limited rule of law and stronger distribution of private goods. For each indicator, I document evidence for mechanisms and changes in the effect over time.
The theory and empirical results presented in this dissertation have a number of implications for future research. They highlight the importance of moving away from a singular focus on post-conflict elections and looking also at other institutional dimensions of post-conflict politics. My political economy model of power-sharing also demonstrates the utility of explicitly including economic functions of post-conflict institutions into power-sharing and broader peacebuilding research. And I introduce novel evidence into research and practice of aid delivery; this helps not only to clarify academic debates under which conditions aid can be effective, but also informs practitioners who help conflict-affected countries in their transition from war to democracy.
Glacitectonic deformation in the Quaternary caused the tectonic framework of large-scale folds and displaced thrust sheets of Maastrichtian (Upper Cretaceous) chalk and Pleistocene glacial deposits in the southwestern Baltic Sea area.
A wide spectrum of methods has been compiled to unravel the structural evolution of the Jasmund Glacitectonic Complex. The analyses of digital elevation models (DEM) suggest a division into two structural sub-complexes – a northern part with morphological ridges striking NW–SE and a southern part with SW–NE trending ridges. Geological cross sections from the eastern coast (southern sub-complex) were constructed and restored using the software Move™ and the complementary module 2D Kinematic Modelling™.
The final geometric model of the southern sub-complex shows a small-scale fold-and-thrust belt. It includes three different orders of architectural surfaces (see PEDERSEN, 2014): erosional surfaces and the décollement (1st order), thrust faults (2nd order), and beds outlining hanging-wall anticlines as well as footwall synclines (3rd order). Thrust faults of the southern structural sub-complex are mainly inclined towards south, which indicates a local glacier push from the S/SE.
The glacitectonic structures have a surface expression in form of sub-parallel ridges and elongated valleys in between. Geomorphological mapping and detailed landform analyses together with the structural investigations provide an insight into the chronology of sub-complexes formation. The northern part of the glacitectonic complex is suggested to have been formed before the southern one, considering the partly truncated northerly ridges and their superimposition by the southern sub-complex.
Although there is a high number of scientific publications on the glacitectonic evolution of Jasmund, these presented models often lack a consistent theory for the development integrating all parts of the 100 km2 large complex. Therefore, the combination of all results leads to a more self-consistent genetic model for the entire Jasmund Glacitectonic Complex.
Self-affine tiles and fractals are known as examples in analysis and topology, as models of quasicrystals and biological growth, as unit intervals of generalized number systems, and as attractors of dynamical systems. The author has implemented a software which can find new examples and handle big databases of self-affine fractals. This thesis establishes the algebraic foundation of the algorithms of the IFStile package. Lifting and projection of algebraic and rational iterated function systems and many properties of the resulting attractors are discussed.
The Flavivirus genus (Flaviviridae family) comprises the most important arboviruses in the world such as dengue virus, West Nile virus (WNV), Zika virus (ZIKV), Japanese encephalitis virus and yellow fever virus (YFV). Every year, several outbreaks caused by flaviviruses are reported worldwide (i.e.: ZIKV and YFV outbreaks in South America) with a huge impact on economy and public health. In the last few decades, many aspects of the flavivirus biology and the interaction of flaviviruses with host cells have been elucidated. However, many underlying mechanisms concerning receptor usage, entry process and viral interaction with host cell factors are still not completely understood. Integrins, the major class of cell adhesion molecules have been implicated in the infectious cycle of different viruses including flaviviruses. A previous report proposed that a particular integrin, the αVβ3 integrin, might act as a cellular receptor for WNV. However, this hypothesis was not confirmed by other groups. In the present study, murine cell lines lacking the expression of one or more integrin subunits were used to evaluate the involvement of different integrins in the flavivirus infection cycle. Mouse fibroblasts lacking the expression of β1 integrin (MKF-β1-/-) or β3 integrin (MEF-β3-/-) subunits or αVβ3 integrin (MEF-αVβ3-/-) as well as their corresponding wild-type cells were utilized. A second model using Chinese hamster ovary cells (CHO-K1), a cell line that has been described to be refractory to some flaviviruses, were modified to express either αV (CHO-αV+/+) or β3 (CHO-β3+/+) integrin subunits. All cell lines were first characterized by confocal laser microscopy, flow cytometry and functional assays prior to infection to assess their integrin expression. The cell lines were then inoculated with different flaviviruses of public health relevance: WNV, YFV-17D, Usutu virus (USUV), Langat virus (LGTV) and ZIKV. Infection assays were designed in order to evaluate whether integrins influence i) cell susceptibility; ii) binding; iii) internalization and iv) replication of the investigated flaviviruses. Our findings clearly demonstrate that β1, β3 and αVβ3 integrins do not act as flavivirus cellular receptor or attachment factor since their ablation does not completely abrogate flavivirus infection in the investigated cell lines. Flavivirus binding to the cell surface of MEFs, MKFs and CHO cells was not disturbed by the genomic deletion of the above-mentioned integrins. The deletion of β1 and β3 integrin subunit did not affect internalization of any of the flaviviruses tested. In contrast to that, loss of αVβ3 integrin in the MEF-αVβ3-/- cells showed a statistically significant decrease in WNV and USUV internalization while ZIKV, YFV-17D and LGTV internalization remained unaffected suggesting that αVβ3 integrin might be involved in the internalization process of at least some flaviviruses. On the other hand, flavivirus replication was substantially impaired in the integrin-deficient cell lines in comparison to their corresponding wild-type cells. Both, MEF-β3-/- and MKF-β1-/- cells showed a statistically significant reduction on viral load for all flaviviruses tested in comparison to their respective wild-type cells. The MEF-αVβ3-/- cells in particular, showed a strong inhibition of flavivirus replication with a reduction of up to 99% on viral loads for all flaviviruses tested. Levels of flavivirus negative-strand RNA were substantially decreased in MEF-αVβ3-/- cells indicating that integrins might influence flavivirus RNA replication. The ectopic expression of either αV or β3 integrin subunits in CHO cells slightly increased the replication of all flaviviruses tested. Taken together, this is the first report highlighting the involvement of integrins in ZIKV, USUV, LGTV and YFV infection. The results strongly indicate that the investigated integrins play an important role in flavivirus infection and might represent a novel host cell factor that enhances flavivirus replication. Although the exact mechanism of interaction between integrins and flaviviruses is currently unknown, the results provided in this study deepen our insight into flavivirus - host cell interactions and open doors for further investigations.
Sexual selection favours traits that confer a competitive advantage in access to mates and to their gametes. This results in males evolving a wide array of adaptations that may be conflictual with female’s interests and even to collateral negative effects on female’s lifespan or reproductive success. Harmful male adaptations are diverse and can be extreme. For example, males of various species evolved adaptations that incur physical damage to the female during copulation, referred to as traumatic mating. Most of these adaptations provide males with a competitive fertilization advantage due to the injection of sperm or non-sperm compounds through the wound. In the spider taxonomical literature, alterations of external genital structures have been reported in females and may result from male inflicted damage during copulation. Contrarily to other cases of traumatic mating, the transfer of sperm or non-sperm compounds does not seem to be the target of selection for external female genital mutilation (EFGM) to evolve. Therefore, investigating EFGM may provide valuable information to extend our understanding of the evolution of harmful male adaptations. In this thesis, I explore this newly discovered phenomenon and combine empirical and theoretical approaches to investigate the causes and consequences of EFGM evolution from male and female perspectives. My findings suggest that EFGM is a natural phenomenon and is potentially widespread throughout spider taxa. I demonstrate the proximal mechanism by which the male copulatory organ mutilates the external female genitalia during genital coupling and show that the mutilation results in full monopolization of the female as mutilated females are unable to remate. Using a theoretical approach, I investigated the conditions for the evolution of EFGM. The model developed suggests that EFGM evolution is favoured for last male sperm precedence and for costs to females that can be relatively high as the male-male competition increases. I present the results of physiological measurements that suggest there is no physiological cost of genital mutilation resulting from healing and immune responses for the female. Finally, I report the results of a behavioural experiment that suggest that females have control over the mutilation and selectively allow or avoid mutilation. These findings suggest that EFGM benefits males by securing paternity, that males and females may have evolved to reduce the costs incurred by the female and that female choice may also play a role in EFGM evolution.
Amine transaminases are versatile biocatalysts for the production of pharmaceutically and agrochemically relevant chiral amines. They represent an environmentally benign alternative to waste intensive transition metal catalysed synthesis strategies, especially because of their high stereoselectivity and robustness. Therefore, they have been frequently used in the (chemo)enzymatic synthesis of amines and/or became attractive targets for enzyme engineering especially in the last decade, mainly in order to enlarge their substrate scope. Certainly, one of the most notable examples of amine transaminase engineering is the
manufacturing of the anti-diabetic drug Sitagliptin in large scale after several rounds of protein engineering. Thereby, the target amine was produced in asymmetric synthesis mode which is the most convenient and favored route to a target chiral amine, starting from the corresponding ketone. The choice of the amine donor is highly relevant for reaction design in terms of economical and thermodynamic considerations. For instance, the use of alanine as the natural amine donor is one of the most common strategies for the amination of target ketones but needs the involvement of auxiliary enzymes to shift the reaction equilibrium towards product formation. In fact, isopropylamine is probably one of the most favored donor molecules since it is cheap and achiral but it is supposed to be accepted only by a limited number of amine transaminases.
This thesis focusses on the optimization and application of amine transaminases for asymmetric synthesis reactions en route to novel target chiral amines using isopropylamine as the preferred amine donor.