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The correction of software failures tends to be very cost-intensive because their debugging is an often time-consuming development activity. During this activity, developers largely attempt to understand what causes failures: Starting with a test case that reproduces the observable failure they have to follow failure causes on the infection chain back to the root cause (defect). This idealized procedure requires deep knowledge of the system and its behavior because failures and defects can be far apart from each other. Unfortunately, common debugging tools are inadequate for systematically investigating such infection chains in detail. Thus, developers have to rely primarily on their intuition and the localization of failure causes is not time-efficient. To prevent debugging by disorganized trial and error, experienced developers apply the scientific method and its systematic hypothesis-testing. However, even when using the scientific method, the search for failure causes can still be a laborious task. First, lacking expertise about the system makes it hard to understand incorrect behavior and to create reasonable hypotheses. Second, contemporary debugging approaches provide no or only partial support for the scientific method. In this dissertation, we present test-driven fault navigation as a debugging guide for localizing reproducible failures with the scientific method. Based on the analysis of passing and failing test cases, we reveal anomalies and integrate them into a breadth-first search that leads developers to defects. This systematic search consists of four specific navigation techniques that together support the creation, evaluation, and refinement of failure cause hypotheses for the scientific method. First, structure navigation localizes suspicious system parts and restricts the initial search space. Second, team navigation recommends experienced developers for helping with failures. Third, behavior navigation allows developers to follow emphasized infection chains back to root causes. Fourth, state navigation identifies corrupted state and reveals parts of the infection chain automatically. We implement test-driven fault navigation in our Path Tools framework for the Squeak/Smalltalk development environment and limit its computation cost with the help of our incremental dynamic analysis. This lightweight dynamic analysis ensures an immediate debugging experience with our tools by splitting the run-time overhead over multiple test runs depending on developers’ needs. Hence, our test-driven fault navigation in combination with our incremental dynamic analysis answers important questions in a short time: where to start debugging, who understands failure causes best, what happened before failures, and which state properties are infected.
The nutrient exchange between plant and fungus is the key element of the arbuscular mycorrhizal (AM) symbiosis. The fungus improves the plant’s uptake of mineral nutrients, mainly phosphate, and water, while the plant provides the fungus with photosynthetically assimilated carbohydrates. Still, the knowledge about the mechanisms of the nutrient exchange between the symbiotic partners is very limited. Therefore, transport processes of both, the plant and the fungal partner, are investigated in this study. In order to enhance the understanding of the molecular basis underlying this tight interaction between the roots of Medicago truncatula and the AM fungus Rhizophagus irregularis, genes involved in transport processes of both symbiotic partners are analysed here. The AM-specific regulation and cell-specific expression of potential transporter genes of M. truncatula that were found to be specifically regulated in arbuscule-containing cells and in non-arbusculated cells of mycorrhizal roots was confirmed. A model for the carbon allocation in mycorrhizal roots is suggested, in which carbohydrates are mobilized in non-arbusculated cells and symplastically provided to the arbuscule-containing cells. New insights into the mechanisms of the carbohydrate allocation were gained by the analysis of hexose/H+ symporter MtHxt1 which is regulated in distinct cells of mycorrhizal roots. Metabolite profiling of leaves and roots of a knock-out mutant, hxt1, showed that it indeed does have an impact on the carbohydrate balance in the course of the symbiosis throughout the whole plant, and on the interaction with the fungal partner. The primary metabolite profile of M. truncatula was shown to be altered significantly in response to mycorrhizal colonization. Additionally, molecular mechanisms determining the progress of the interaction in the fungal partner of the AM symbiosis were investigated. The R. irregularis transcriptome in planta and in extraradical tissues gave new insight into genes that are differentially expressed in these two fungal tissues. Over 3200 fungal transcripts with a significantly altered expression level in laser capture microdissection-collected arbuscules compared to extraradical tissues were identified. Among them, six previously unknown specifically regulated potential transporter genes were found. These are likely to play a role in the nutrient exchange between plant and fungus. While the substrates of three potential MFS transporters are as yet unknown, two potential sugar transporters are might play a role in the carbohydrate flow towards the fungal partner. In summary, this study provides new insights into transport processes between plant and fungus in the course of the AM symbiosis, analysing M. truncatula on the transcript and metabolite level, and provides a dataset of the R. irregularis transcriptome in planta, providing a high amount of new information for future works.
A detailed description of the characteristics of antimicrobial peptides (AMPs) is highly demanded, since the resistance against traditional antibiotics is an emerging problem in medicine. They are part of the innate immune system in every organism, and they are very efficient in the protection against bacteria, viruses, fungi and even cancer cells. Their advantage is that their target is the cell membrane, in contrast to antibiotics which disturb the metabolism of the respective cell type. This allows AMPs to be more active and faster. The lack of an efficient therapy for some cancer types and the evolvement of resistance against existing antitumor agents make AMPs promising in cancer therapy besides being an alternative to traditional antibiotics. The aim of this work was the physical-chemical characterization of two fragments of LL-37, a human antimicrobial peptide from the cathelicidin family. The fragments LL-32 and LL-20 exhibited contrary behavior in biological experiments concerning their activity against bacterial cells, human cells and human cancer cells. LL-32 had even a higher activity than LL-37, while LL-20 had almost no effect. The interaction of the two fragments with model membranes was systematically studied in this work to understand their mode of action. Planar lipid films were mainly applied as model systems in combination with IR-spectroscopy and X-ray scattering methods. Circular Dichroism spectroscopy in bulk systems completed the results. In the first approach, the structure of the peptides was determined in aqueous solution and compared to the structure of the peptides at the air/water interface. In bulk, both peptides are in an unstructured conformation. Adsorbed and confined to at the air-water interface, the peptides differ drastically in their surface activity as well as in the secondary structure. While LL-32 transforms into an α-helix lying flat at the water surface, LL-20 stays partly unstructured. This is in good agreement with the high antimicrobial activity of LL-32. In the second approach, experiments with lipid monolayers as biomimetic models for the cell membrane were performed. It could be shown that the peptides fluidize condensed monolayers of negatively charged DPPG which can be related to the thinning of a bacterial cell membrane. An interaction of the peptides with zwitterionic PCs, as models for mammalian cells, was not clearly observed, even though LL-32 is haemolytic. In the third approach, the lipid monolayers were more adapted to the composition of human erythrocyte membranes by incorporating sphingomyelin (SM) into the PC monolayers. Physical-chemical properties of the lipid films were determined and the influence of the peptides on them was studied. It could be shown that the interaction of the more active LL-32 is strongly increased for heterogeneous lipid films containing both gel and fluid phases, while the interaction of LL-20 with the monolayers was unaffected. The results indicate an interaction of LL-32 with the membrane in a detergent-like way. Additionally, the modelling of the peptide interaction with cancer cells was performed by incorporating some negatively charged lipids into the PC/SM monolayers, but the increased charge had no effect on the interaction of LL-32. It was concluded, that the high anti-cancer activity of the peptide originates from the changed fluidity of cell membrane rather than from the increased surface charge. Furthermore, similarities to the physical-chemical properties of melittin, an AMP from the bee venom, were demonstrated.
Diet is a major force influencing the intestinal microbiota. This is obvious from drastic changes in microbiota composition after a dietary alteration. Due to the complexity of the commensal microbiota and the high inter-individual variability, little is known about the bacterial response at the cellular level. The objective of this work was to identify mechanisms that enable gut bacteria to adapt to dietary factors. For this purpose, germ-free mice monoassociated with the commensal Escherichia coli K-12 strain MG1655 were fed three different diets over three weeks: a diet rich in starch, a diet rich in non-digestible lactose and a diet rich in casein. Two dimensional gel electrophoresis and electrospray tandem mass spectrometry were applied to identify differentially expressed proteins of E. coli recovered from small intestine and caecum of mice fed the lactose or casein diets in comparison with those of mice fed the starch diet. Selected differentially expressed bacterial proteins were characterised in vitro for their possible roles in bacterial adaptation to the various diets. Proteins belonging to the oxidative stress regulon oxyR such as alkyl hydroperoxide reductase subunit F (AhpF), DNA protection during starvation protein (Dps) and ferric uptake regulatory protein (Fur), which are required for E. coli’s oxidative stress response, were upregulated in E. coli of mice fed the lactose-rich diet. Reporter gene analysis revealed that not only oxidative stress but also carbohydrate-induced osmotic stress led to the OxyR-dependent expression of ahpCF and dps. Moreover, the growth of E. coli mutants lacking the ahpCF or oxyR genes was impaired in the presence of non-digestible sucrose. This indicates that some OxyR-dependent proteins are crucial for the adaptation of E. coli to osmotic stress conditions. In addition, the function of two so far poorly characterised E. coli proteins was analysed: 2 deoxy-D gluconate 3 dehydrogenase (KduD) was upregulated in intestinal E. coli of mice fed the lactose-rich diet and this enzyme and 5 keto 4 deoxyuronate isomerase (KduI) were downregulated on the casein-rich diet. Reporter gene analysis identified galacturonate and glucuronate as inducers of the kduD and kduI gene expression. Moreover, KduI was shown to facilitate the breakdown of these hexuronates, which are normally degraded by uronate isomerase (UxaC), altronate oxidoreductase (UxaB), altronate dehydratase (UxaA), mannonate oxidoreductase (UxuB) and mannonate dehydratase (UxuA), whose expression was repressed by osmotic stress. The growth of kduID-deficient E. coli on galacturonate or glucuronate was impaired in the presence of osmotic stress, suggesting KduI and KduD to compensate for the function of the regular hexuronate degrading enzymes under such conditions. This indicates a novel function of KduI and KduD in E. coli’s hexuronate metabolism. Promotion of the intracellular formation of hexuronates by lactose connects these in vitro observations with the induction of KduD on the lactose-rich diet. Taken together, this study demonstrates the crucial influence of osmotic stress on the gene expression of E. coli enzymes involved in stress response and metabolic processes. Therefore, the adaptation to diet-induced osmotic stress is a possible key factor for bacterial colonisation of the intestinal environment.
Die Dissertation beschreibt die Herstellung von ringförmigen Verbindungen (Naphthalenophanen) mit Hilfe der Dehydro-Diels-Alder-Reaktion, wobei immer Enantiomerenpaare auftreten. Es wird der diastereoselektive Aufbau von Naphthalenophanen und der enantiomeren reine Aufbau von Biarylen untersucht. Desweiteren werden die physikalischen Eigenschaften der erhaltenen Verbindungen, wie die Phosphoreszenz, Trennbarkeit der entstehenden Enantiomere und die Ringspannung beschrieben.
Information flows in EU policy-making are heavily dependent on personal networks, both within the Brussels sphere but also reaching outside the narrow limits of the Belgian capital. These networks develop for example in the course of formal and informal meetings or at the sidelines of such meetings. A plethora of committees at European, transnational and regional level provides the basis for the establishment of pan-European networks. By studying affiliation to those committees, basic network structures can be uncovered. These affiliation network structures can then be used to predict EU information flows, assuming that certain positions within the network are advantageous for tapping into streams of information while others are too remote and peripheral to provide access to information early enough. This study has tested those assumptions for the case of the reform of the Common Fisheries Policy for the time after 2012. Through the analysis of an affiliation network based on participation in 10 different fisheries policy committees over two years (2009 and 2010), network data for an EU-wide network of about 1300 fisheries interest group representatives and more than 200 events was collected. The structure of this network showed a number of interesting patterns, such as – not surprisingly – a rather central role of Brussels-based committees but also close relations of very specific interests to the Brussels-cluster and stronger relations between geographically closer maritime regions. The analysis of information flows then focused on access to draft EU Commission documents containing the upcoming proposal for a new basic regulation of the Common Fisheries Policy. It was first documented that it would have been impossible to officially obtain this document and that personal networks were thus the most likely sources for fisheries policy actors to obtain access to these “leaks” in early 2011. A survey of a sample of 65 actors from the initial network supported these findings: Only a very small group had accessed the draft directly from the Commission. Most respondents who obtained access to the draft had received it from other actors, highlighting the networked flow of informal information in EU politics. Furthermore, the testing of the hypotheses connecting network positions and the level of informedness indicated that presence in or connections to the Brussels sphere had both advantages for overall access to the draft document and with regard to timing. Methodologically, challenges of both the network analysis and the analysis of information flows but also their relevance for the study of EU politics have been documented. In summary, this study has laid the foundation for a different way to study EU policy-making by connecting topical and methodological elements – such as affiliation network analysis and EU committee governance – which so far have not been considered together, thereby contributing in various ways to political science and EU studies.
Gegenstand dieser Arbeit sind sog. nicht-kanonische bzw. unintegrierte Nebensätze. Diese Nebensätze zeichnen sich dadurch aus, dass sie sich mittels gängiger Kriterien (Satzgliedstatus, Verbletztstellung) nicht klar als koordiniert oder subordiniert beschreiben lassen. Das Phänomen nicht-kanonischer Nebensätze ist ein Thema, welches in der Sprachwissenschaft generell seit den späten Siebzigern (Davison 1979) diskutiert wird und spätestens mit Fabricius-Hansen (1992) auch innerhalb der germanistischen Linguistik angekommen ist. Ein viel beachteter Komplex ist hierbei – neben der reinen Identifizierung nicht-kanonischer Satzgefüge – meist auch die Erstellung einer Klassifikation zur Erfassung zumindest einiger nicht-kanonischer Gefüge, wie dies etwa bei Fabricius-Hansen (1992) und Reis (1997) zu sehen ist. Das Ziel dieser Studie ist es, eine exhaustive Klassifikation der angesprochenen Nebensatztypen vorzunehmen. Dazu werden zunächst – unter Zuhilfenahme von Korpusdaten – alle potentiellen Subordinationsmerkmale genauer untersucht, da die meisten bisherigen Studien zu diesem Thema die stets gleichen Merkmale als gegeben voraussetzen. Dabei wird sich herausstellen, dass nur eine kleine Anzahl von Merkmalen sich wirklich zweifelsfrei dazu eignet, Aufschluss über die Satzverknüpfungsqualität zu geben. Die anschließend aufgestellte Taxonomie deutscher Nebensätze wird schließlich einzig mit der Postulierung einer nicht-kanonischen Nebensatzklasse auskommen. Sie ist darüber hinaus auch in der Lage, die zahlreich vorkommenden Ausnahmefälle zu erfassen. Dies heißt konkret, dass auch etwaige Nebensätze, die sich aufgrund bestimmter Eigenschaften teilweise idiosynkratisch verhalten, einfach in die vorgeschlagene Klassifikation übernommen werden können. In diesem Zuge werde ich weiterhin zeigen, wie eine Nebensatzklassifikation auch sog. sekundären Subordinationsmerkmalen gerecht werden kann, obwohl diese sich hinsichtlich der einzelnen Nebensatzklassen nicht einheitlich verhalten. Schließlich werde ich eine theoretische Modellierung der zuvor postulierten Taxonomie vornehmen, die auf Basis der HPSG mittels Merkmalsvererbung alle möglichen Nebensatztypen zu erfassen imstande ist.
Logging and large earthquakes are disturbances that may significantly affect hydrological and erosional processes and process rates, although in decisively different ways. Despite numerous studies that have documented the impacts of both deforestation and earthquakes on water and sediment fluxes, a number of details regarding the timing and type of de- and reforestation; seismic impacts on subsurface water fluxes; or the overall geomorphic work involved have remained unresolved. The main objective of this thesis is to address these shortcomings and to better understand and compare the hydrological and erosional process responses to such natural and man-made disturbances. To this end, south-central Chile provides an excellent natural laboratory owing to its high seismicity and the ongoing conversion of land into highly productive plantation forests. In this dissertation I combine paired catchment experiments, data analysis techniques, and physics-based modelling to investigate: 1) the effect of plantation forests on water resources, 2) the source and sink behavior of timber harvest areas in terms of overland flow generation and sediment fluxes, 3) geomorphic work and its efficiency as a function of seasonal logging, 4) possible hydrologic responses of the saturated zone to the 2010 Maule earthquake and 5) responses of the vadose zone to this earthquake. Re 1) In order to quantify the hydrologic impact of plantation forests, it is fundamental to first establish their water balances. I show that tree species is not significant in this regard, i.e. Pinus radiata and Eucalyptus globulus do not trigger any decisive different hydrologic response. Instead, water consumption is more sensitive to soil-water supply for the local hydro-climatic conditions. Re 2) Contradictory opinions exist about whether timber harvest areas (THA) generate or capture overland flow and sediment. Although THAs contribute significantly to hydrology and sediment transport because of their spatial extent, little is known about the hydrological and erosional processes occurring on them. I show that THAs may act as both sources and sinks for overland flow, which in turn intensifies surface erosion. Above a rainfall intensity of ~20 mm/h, which corresponds to <10% of all rainfall, THAs may generate runoff whereas below that threshold they remain sinks. The overall contribution of Hortonian runoff is thus secondary considering the local rainfall regime. The bulk of both runoff and sediment is generated by Dunne, saturation excess, overland flow. I also show that logging may increase infiltrability on THAs which may cause an initial decrease in streamflow followed by an increase after the groundwater storage has been refilled. Re 3) I present changes in frequency-magnitude distributions following seasonal logging by applying Quantile Regression Forests at hitherto unprecedented detail. It is clearly the season that controls the hydro-geomorphic work efficiency of clear cutting. Logging, particularly dry seasonal logging, caused a shift of work efficiency towards less flashy and mere but more frequent moderate rainfall-runoff events. The sediment transport is dominated by Dunne overland flow which is consistent with physics-based modelling using WASA-SED. Re 4) It is well accepted that earthquakes may affect hydrological processes in the saturated zone. Assuming such flow conditions, consolidation of saturated saprolitic material is one possible response. Consolidation raises the hydraulic gradients which may explain the observed increase in discharge following earthquakes. By doing so, squeezed water saturates the soil which in turn increases the water accessible for plant transpiration. Post-seismic enhanced transpiration is reflected in the intensification of diurnal cycling. Re 5) Assuming unsaturated conditions, I present the first evidence that the vadose zone may also respond to seismic waves by releasing pore water which in turn feeds groundwater reservoirs. By doing so, water tables along the valley bottoms are elevated thus providing additional water resources to the riparian vegetation. By inverse modelling, the transient increase in transpiration is found to be 30-60%. Based on the data available, both hypotheses, are not testable. Finally, when comparing the hydrological and erosional effects of the Maule earthquake with the impact of planting exotic plantation forests, the overall observed earthquake effects are comparably small, and limited to short time scales.
In the presence of a solid-liquid or liquid-air interface, bacteria can choose between a planktonic and a sessile lifestyle. Depending on environmental conditions, cells swimming in close proximity to the interface can irreversibly attach to the surface and grow into three-dimensional aggregates where the majority of cells is sessile and embedded in an extracellular polymer matrix (biofilm). We used microfluidic tools and time lapse microscopy to perform experiments with the polarly flagellated soil bacterium Pseudomonas putida (P. putida), a bacterial species that is able to form biofilms. We analyzed individual trajectories of swimming cells, both in the bulk fluid and in close proximity to a glass-liquid interface. Additionally, surface related growth during the early phase of biofilm formation was investigated. In the bulk fluid, P.putida shows a typical bacterial swimming pattern of alternating periods of persistent displacement along a line (runs) and fast reorientation events (turns) and cells swim with an average speed around 24 micrometer per second. We found that the distribution of turning angles is bimodal with a dominating peak around 180 degrees. In approximately six out of ten turning events, the cell reverses its swimming direction. In addition, our analysis revealed that upon a reversal, the cell systematically changes its swimming speed by a factor of two on average. Based on the experimentally observed values of mean runtime and rotational diffusion, we presented a model to describe the spreading of a population of cells by a run-reverse random walker with alternating speeds. We successfully recover the mean square displacement and, by an extended version of the model, also the negative dip in the directional autocorrelation function as observed in the experiments. The analytical solution of the model demonstrates that alternating speeds enhance a cells ability to explore its environment as compared to a bacterium moving at a constant intermediate speed. As compared to the bulk fluid, for cells swimming near a solid boundary we observed an increase in swimming speed at distances below d= 5 micrometer and an increase in average angular velocity at distances below d= 4 micrometer. While the average speed was maximal with an increase around 15% at a distance of d= 3 micrometer, the angular velocity was highest in closest proximity to the boundary at d=1 micrometer with an increase around 90% as compared to the bulk fluid. To investigate the swimming behavior in a confinement between two solid boundaries, we developed an experimental setup to acquire three-dimensional trajectories using a piezo driven objective mount coupled to a high speed camera. Results on speed and angular velocity were consistent with motility statistics in the presence of a single boundary. Additionally, an analysis of the probability density revealed that a majority of cells accumulated near the upper and lower boundaries of the microchannel. The increase in angular velocity is consistent with previous studies, where bacteria near a solid boundary were shown to swim on circular trajectories, an effect which can be attributed to a wall induced torque. The increase in speed at a distance of several times the size of the cell body, however, cannot be explained by existing theories which either consider the drag increase on cell body and flagellum near a boundary (resistive force theory) or model the swimming microorganism by a multipole expansion to account for the flow field interaction between cell and boundary. An accumulation of swimming bacteria near solid boundaries has been observed in similar experiments. Our results confirm that collisions with the surface play an important role and hydrodynamic interactions alone cannot explain the steady-state accumulation of cells near the channel walls. Furthermore, we monitored the number growth of cells in the microchannel under medium rich conditions. We observed that, after a lag time, initially isolated cells at the surface started to grow by division into colonies of increasing size, while coexisting with a comparable smaller number of swimming cells. After 5:50 hours, we observed a sudden jump in the number of swimming cells, which was accompanied by a breakup of bigger clusters on the surface. After approximately 30 minutes where planktonic cells dominated in the microchannel, individual swimming cells reattached to the surface. We interpret this process as an emigration and recolonization event. A number of complementary experiments were performed to investigate the influence of collective effects or a depletion of the growth medium on the transition. Similar to earlier observations on another bacterium from the same family we found that the release of cells to the swimming phase is most likely the result of an individual adaption process, where syntheses of proteins for flagellar motility are upregulated after a number of division cycles at the surface.