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Die Ca2+/Calmodulin-aktivierte Serin/Threonin-Phosphatase Calcineurin ist ein Schlüsselmolekül des T-Zell-Rezeptorabhängigen Signalnetzwerkes. Calcineurin aktiviert die Transkriptionsfaktoren der NFATc-Familie durch Dephosphorylierung und reguliert darüber die Expression wichtiger Zytokine und Oberflächenproteine. Die Aktivität von Calcineurin wird durch zahlreiche endogene Proteine moduliert und ist Angriffspunkt der immunsuppressiven Substanzen Cyclosporin A und FK506. In dieser Arbeit wurde der alternative niedermolekulare Calcineurin-NFATc-Inhibitor NCI3 hinsichtlich seiner Effekte auf T-Zell-Rezeptor-abhängige Signalwege charakterisiert. Die Ergebnisse zeigen, daß das Pyrazolopyrimidinderivat NCI3 nichttoxisch und zellmembranpermeabel ist. In T-Zell-Rezeptor-stimulierten primären humanen TH-Zellen unterdrückt NCI3 die Proliferation und IL-2-Produktion (IC50-Wert ~4 µM), da die Dephosphorylierung von NFATc und die anschließende nukleäre Translokation gehemmt wird. NCI3 inhibiert die calcineurinabhängige NFAT- und NF-κB-, aber nicht die AP-1-kontrollierte Reprtergenexpression, in mikromolaren Konzentrationen (IC50-Werte 2 bzw. 7 µM). Im Gegensatz zu Cyclosporin A stört NCI3 nicht die Phosphataseaktivität von Calcineurin, sondern interferiert mit der Calcineurin-NFATc-Bindung. Ein wichtiges endogenes Modulatorprotein für die Calcineurinaktivität ist RCAN1, das vermutlich den Calcineurin-NFATc-Signalweg über einen negativen Rückkopplungsmechanismus reguliert. Hier wurde gezeigt, daß RCAN1 in humanen TH-Zellen exprimiert wird. Die Spleißvariante RCAN1-1 ist in ruhenden T-Zellen basal exprimiert und wird nicht durch T-Zell-Rezeptor-Stimulierung in seiner Expression verändert. RCAN1-4 dagegen ist in ruhenden Zellen kaum zu detektieren und wird stimulierungsabhängig induziert. Durch die Verwendung Calcineurin-NFATc-spezifischer Inhibitoren wie NCI3 wurde gezeigt, daß die RCAN1-4-Induktion durch diesen Signalweg limitiert ist. Die in dieser Arbeit gewonnenen Daten und Erkenntnisse tragen dazu bei, das Verständnis der Funktion und Regulation von Calcineurin in T-Zellen zu vertiefen.
Analysis of structure and variability of major histocompatibility complex in different vertebrates
(2010)
Transport von Proteinen und mikro RNAs im Pholoem von Brassica napus und Arabidopsis thaliana
(2010)
Genexpression in Plastiden: Funktionen plastidärer Introns und Produktion Zellwand-abbauender Enzyme
(2010)
Studien zur Reinigung, Monomerisierung und Aggragation von Huntingtin : Exon 1 Proteinkonstrukten
(2010)
Investigating novel potential regulators of phosphate stress responses in Arabidopsis thaliana
(2010)
Proteostasis of the tonoplast : synthesis, sorting and turnover of the potassum channel AtTPK1
(2010)
Foraging in space and time
(2010)
All animals are adapted to the environmental conditions of the habitat they chose to live in. It was the aim of this PhD-project, to show which behavioral strategies are expressed as mechanisms to cope with the constraints, which contribute to the natural selection pressure acting on individuals. For this purpose, small mammals were exposed to different levels and types of predation risk while actively foraging. Individuals were either exposed to different predator types (airborne or ground) or combinations of both, or to indirect predators (nest predators). Risk was assumed to be distributed homogeneously, so changing the habitat or temporal adaptations where not regarded as potential options. Results show that wild-caught voles have strategic answers to this homogeneously distributed risk, which is perceived by tactile, olfactory or acoustic cues. Thus, they do not have to know an absolut quality (e.g., in terms of food provisioning and risk levels of all possible habitats), but they can adapt their behavior to the actual circumstances. Deriving risk uniform levels from cues and adjusting activity levels to the perceived risk is an option to deal with predators of the same size or with unforeseeable attack rates. Experiments showed that as long as there are no safe places or times, it is best to reduce activity and behave as inconspicuous as possible as long as the costs of missed opportunities do not exceed the benefits of a higher survival probability. Test showed that these costs apparently grow faster for males than for females, especially in times of inactivity. This is supported by strong predatory pressure on the most active groups of rodents (young males, sexually active or dispersers) leading to extremely female-biased operative sex ratios in natural populations. Other groups of animals, those with parental duties such as nest guarding, for example, have to deal with the actual risk in their habitat as well. Strategies to indirect predation pressure were tested by using bank vole mothers, confronted with a nest predator that posed no actual threat to themselves but to their young (Sorex araneus). They reduced travelling and concentrated their effort in the presence of shrews, independent of the different nutritional provisioning of food by varying resource levels due to the different seasons. Additionally, they exhibited nest-guarding strategies by not foraging in the vicinity of the nest site in order to reduce conspicuous scent marks. The repetition of the experiment in summer and autumn showed that changing environmental constraints can have a severe impact on results of outdoor studies. In our case, changing resource levels changed the type of interaction between the two species. The experiments show that it is important to analyze decision making and optimality models on an individual level, and, when that is not possible (maybe because of the constraints of field work), groups of animals should be classified by using the least common denominator that can be identified (such as sex, age, origin or kinship). This will control for the effects of the sex or stage of life history or the individual´s reproductive and nutritional status on decision making and will narrow the wide behavioral variability associated with the complex term of optimality.
Analysis of organellar genome dynamics and development of methods for organelle transformation
(2010)
In dieser Arbeit wurden die Möglichkeiten und Grenzen für Zirkulardichroismus-Messungen mit Synchrotronstrahlung untersucht. Dazu wurde ein Messaufbau für Zirkulardichroismus-Messungen an zwei Strahlrohren am Berliner Elektronenspeicherring für Synchrotronstrahlung eingesetzt, die für Messungen im Bereich des ultravioletten Lichts geeignet sind. Eigenschaften der Strahlrohre und des Messaufbau wurden in einigen wichtigen Punkten mit kommerziellen Zirkulardichroismus-Spektrometern verglichen. Der Schwerpunkt lag auf der Ausdehnung des zugänglichen Wellenlängenbereichs unterhalb von 180 nm zur Untersuchung des Zirkulardichroismus von Proteinen in diesem Bereich. In diesem Bereich ist es nicht nur die Lichtquelle sondern vor allem die Absorption des Lichts durch Wasser, die den Messbereich bei der Messung biologischer Proben in wässriger Lösung einschränkt. Es wurden Bedingungen gefunden, unter denen der Messbereich auf etwa 160 nm, in einigen Fällen bis auf 130 nm ausgedehnt werden konnte. Dazu musste die Pfadlänge deutlich reduziert werden und verschieden Probenküvetten wurden getestet. Der Einfluss der dabei auftretenden Spannungsdoppelbrechung in den Probenküvetten auf das Messsignal konnte mit einem alternativen Messaufbau deutlich reduziert werden. Systematische Fehler im Messsignal und auftretende Strahlenschäden begrenzen jedoch die Zuverlässigkeit der gemessenen Spektren. Bei Proteinfilmen schränkt die Absorption von Wasser den Messbereich kaum ein. Es wurden jedoch meist deutliche Unterschiede zwischen den Spektren von Proteinfilmen und den Spektren von Proteinen in wässriger Lösung festgestellt. Solange diese Unterschiede nicht minimiert werden können, stellen Proteinfilme keine praktikable Alternative zu Messungen in wässriger Lösung dar.
Lake ecosystems across the globe have responded to climate warming of recent decades. However, correctly attributing observed changes to altered climatic conditions is complicated by multiple anthropogenic influences on lakes. This thesis contributes to a better understanding of climate impacts on freshwater phytoplankton, which forms the basis of the food chain and decisively influences water quality. The analyses were, for the most part, based on a long-term data set of physical, chemical and biological variables of a shallow, polymictic lake in north-eastern Germany (Müggelsee), which was subject to a simultaneous change in climate and trophic state during the past three decades. Data analysis included constructing a dynamic simulation model, implementing a genetic algorithm to parameterize models, and applying statistical techniques of classification tree and time-series analysis. Model results indicated that climatic factors and trophic state interactively determine the timing of the phytoplankton spring bloom (phenology) in shallow lakes. Under equally mild spring conditions, the phytoplankton spring bloom collapsed earlier under high than under low nutrient availability, due to a switch from a bottom-up driven to a top-down driven collapse. A novel approach to model phenology proved useful to assess the timings of population peaks in an artificially forced zooplankton-phytoplankton system. Mimicking climate warming by lengthening the growing period advanced algal blooms and consequently also peaks in zooplankton abundance. Investigating the reasons for the contrasting development of cyanobacteria during two recent summer heat wave events revealed that anomalously hot weather did not always, as often hypothesized, promote cyanobacteria in the nutrient-rich lake studied. The seasonal timing and duration of heat waves determined whether critical thresholds of thermal stratification, decisive for cyanobacterial bloom formation, were crossed. In addition, the temporal patterns of heat wave events influenced the summer abundance of some zooplankton species, which as predators may serve as a buffer by suppressing phytoplankton bloom formation. This thesis adds to the growing body of evidence that lake ecosystems have strongly responded to climatic changes of recent decades. It reaches beyond many previous studies of climate impacts on lakes by focusing on underlying mechanisms and explicitly considering multiple environmental changes. Key findings show that climate impacts are more severe in nutrient-rich than in nutrient-poor lakes. Hence, to develop lake management plans for the future, limnologists need to seek a comprehensive, mechanistic understanding of overlapping effects of the multi-faceted human footprint on aquatic ecosystems.
This thesis contains quantum chemical models and force field calculations for the RuBisCO isotope effect, the spectral characteristics of the blue-light sensor BLUF and the light harvesting complex II. The work focuses on the influence of the environment on the corresponding systems. For RuBisCO, it was found that the isotopic effect is almost unaffected by the environment. In case of the BLUF domain, an amino acid was found to be important for the UV/vis spectrum, but unaccounted for in experiments so far (Ser41). The residue was shown to be highly mobile and with a systematic influence on the spectral shift of the BLUF domain chromophore (flavin). Finally, for LHCII it was found that small changes in the geometry of a Chlorophyll b/Violaxanthin chromophore pair can have strong influences regarding the light harvesting mechanism. Especially here it was seen that the proper description of the environment can be critical. In conclusion, the environment was observed to be of often unexpected importance for the molecular properties, and it seems not possible to give a reliable estimate on the changes created by the presence of the environment.
Pektatlyase (Pel-15) aus dem alkalophilen Bodenbakterium Bacillus spec. KSM-P15 ist mit 197 Aminosäuren eines der kleinsten, bekannten β-3-Solenoidproteine. Sie spaltet Polygalakturonsäurederivate in einem Ca2+-abhängigen β-Eliminierungsprozess. Wie bei allen Proteinen dieser Enzymfamilie ist auch die Polypeptidkette von Pel-15 zu einer einsträngigen, rechtsgängigen, parallelen β-Helix aufgewunden. In diesem Strukturmotiv enthält jede Windung drei β-Stränge, die jeweils durch flexible Schleifenbereiche miteinander verbunden sind. Insgesamt acht Windungen stapeln sich in Pel-15 übereinander und bilden entlang der Helixachse flächige, parallele β-Faltblätter aus. Im Bereich dieser β-Faltblätter existiert ein ausgedehntes Netzwerk von Wasserstoffbrückenbindungen, durch das der hydrophobe Kern, der sich im Inneren der β-Helix befindet, vom umgebenden Lösungsmittel abgeschirmt wird. Besondere Abschlussstrukturen an beiden Enden der β-Helix, wie sie typischerweise bei anderen Ver-tretern dieser Strukturklasse ausgeprägt werden, sind in Pel-15 nicht zu beobachten. Stattdessen sind die terminalen Bereiche der β-Helix über Salzbrücken und hydrophobe Seitenkettenkontakte stabilisiert. In der vorliegenden Dissertation wurde die Pektatlyase Pel-15 hinsichtlich ihres Faltungsgleichgewichtes, ihrer enzymatischen Aktivität und der Kinetik ihrer Strukturbildung charakterisiert. In eine evolutionär konservierte Helixwindung wurden destabilisierende Mutationen eingeführt, und deren Auswirkungen mittels spektroskopischer Methoden analysiert. Die Ergebnisse zeigen, dass Pel-15 in Gegenwart des Denaturierungsmittels Guanidiniumhydrochlorid einen hyperfluoreszenten Gleichgewichtsustand (HF) populiert, der nach Messungen von Faltungs- und Entfaltungskinetiken ein konformationelles Ensemble aus den Zuständen HFslow und HFfast darstellt. Diese HF-Zustände sind durch eine hohe Aktivierungsbarriere voneinander getrennt. In Rückfaltungsexperimenten populieren nur etwa 80 % der faltenden Moleküle den Zwischenzustand HFslow, der mit einer Zeitkonstante von ca. 100 s zu HFfast weiterreagiert. Die Denaturierungsmittelabhängigkeit dieser Reaktion ist sehr gering, was eine trans-/cis-Prolylisomerisierung als geschwindigkeitslimitierenden Schritt nahelegt. Die Existenz eines cis-Peptides in der nativen Struktur macht es erforderlich, den denaturierten Zustand als ein Ensemble kinetisch separierter Konformationen, kurz: DSE, zu betrachten, das durch die Spezies Ufast und Uslow populiert wird. Nach dem in dieser Arbeit aufgestellten „Minimalmodell der Pel-15 Faltung“ stehen die HF-Spezies (HFslow, HFfast) mit den Konformationen des DSE in einem thermodynamischen Kreisprozess. Das Modell positioniert HFfast und die native Konformation N auf die „native Seite“ der Aktivierungsbarriere und trägt damit der Tatsache Rechnung, dass die Gleichgewichtseinstellung zwischen diesen Spezies zu schnell ist, um mit manuellen Techniken erfasst zu werden. Die hochaffine Bindung von Ca2+ (Kd = 10 μM) verschiebt sich das Faltungsgleichgewicht bereits in Gegenwart von 1 mM CaCl2 soweit auf die Seite des nativen Zustandes, das HFfast nicht länger nachweisbar ist. Entgegen anfänglicher Vermutungen kommt einer lokalen, evolutionär konservierten Disulfidbrücke im Zentrum der β-Helix eine wichtige Stabilisierungsfunktion zu. Die Disulfidbrücke befindet sich in einem kurzen Schleifenbereich der β-Helix nahe dem aktiven Zentrum. Obwohl ihr Austausch gegen die Reste Val und Ala die freie Stabilisierungsenthalpie des Proteins um ca. 10 kJ/mol reduziert, lässt die Struktur im Bereich der Mutationsstelle keine gravierende Veränderung erkennen. Auch die katalytisch relevante Ca2+-Bindungsaffinität bleibt unbeeinflusst; dennoch zeigen Enzymaktivitätstests für VA-Mutanten eine Reduktion der enzymatischen Aktivität um fast 50 % an. Die evolutionär konservierte Helixwindung im Allgemeinen und die in ihr enthaltene Disulfidbrücke im Besonderen müssen nach den vorliegenden Ergebnissen also eine zentrale Funktion sowohl für die Struktur des katalytischen Zentrums als auch für die Strukturbildung der β-Helix während der Faltungsreaktion besitzen. Die Ergebnisse dieser Arbeit finden in mehreren Punkten Anklang an Faltungseigenschaften, die für andere β -Helixproteine beschrieben wurden. Vor allem aber prädestinieren sie Pel-15 als ein neues, β-helikales Modellprotein. Aufgrund seiner einfachen Topologie, seiner niedrigen Windungszahl und seiner hohen thermodynamischen Stabilität ist Pel-15 sehr gut geeignet, die Determinanten von Stabilität und Strukturbildung des parallelen β-Helix-Motivs in einer Auflösung zu studieren, die aufgrund der Komplexität bestehender β-helikaler Modellsysteme bislang nicht zur Verfügung stand.
Fire prone Mediterranean-type vegetation systems like those in the Mediterranean Basin and South-Western Australia are global hot spots for plant species diversity. To ensure management programs act to maintain these highly diverse plant communities, it is necessary to get a profound understanding of the crucial mechanisms of coexistence. In the current literature several mechanisms are discussed. The objective of my thesis is to systematically explore the importance of potential mechanisms for maintaining multi-species, fire prone vegetation by modelling. The model I developed is spatially-explicit, stochastic, rule- and individual-based. It is parameterised on data of population dynamics collected over 18 years in the Mediterranean-type shrublands of Eneabba, Western Australia. From 156 woody species of the area seven plant traits have been identified to be relevant for this study: regeneration mode, annual maximum seed production, seed size, maximum crown diameter, drought tolerance, dispersal mode and seed bank type. Trait sets are used for the definition of plant functional types (PFTs). The PFT dynamics are simulated annual by iterating life history processes. In the first part of my thesis I investigate the importance of trade-offs for the maintenance of high diversity in multi-species systems with 288 virtual PFTs. Simulation results show that the trade-off concept can be helpful to identify non-viable combinations of plant traits. However, the Shannon Diversity Index of modelled communities can be high despite of the presence of ‘supertypes’. I conclude, that trade-offs between two traits are less important to explain multi-species coexistence and high diversity than it is predicted by more conceptual models. Several studies show, that seed immigration from the regional seed pool is essential for maintaining local species diversity. However, systematical studies on the seed rain composition to multi-species communities are missing. The results of the simulation experiments, as presented in part two of this thesis, show clearly, that without seed immigration the local species community found in Eneabba drifts towards a state with few coexisting PFTs. With increasing immigration rates the number of simulated coexisting PFTs and Shannon diversity quickly approaches values as also observed in the field. Including the regional seed input in the model is suited to explain more aggregated measures of the local plant community structure such as species richness and diversity. Hence, the seed rain composition should be implemented in future studies. In the third part of my thesis I test the sensitivity of Eneabba PFTs to four different climate change scenarios, considering their impact on both local and regional processes. The results show that climate change clearly has the potential to alter the number of dispersed seeds for most of the Eneabba PFTs and therefore the source of the ‘immigrants’ at the community level. A classification tree analysis shows that, in general, the response to climate change was PFT-specific. In the Eneabba sand plains sensitivity of a PFT to climate change depends on its specific trait combination and on the scenario of environmental change i.e. development of the amount of rainfall and the fire frequency. This result emphasizes that PFT-specific responses and regional process seed immigration should not be ignored in studies dealing with the impact of climate change on future species distribution. The results of the three chapters are finally analysed in a general discussion. The model is discussed and improvements and suggestions are made for future research. My work leads to the following conclusions: i) It is necessary to support modelling with empirical work to explain coexistence in species-rich plant communities. ii) The chosen modelling approach allows considering the complexity of coexistence and improves the understanding of coexistence mechanisms. iii) Field research based assumptions in terms of environmental conditions and plant life histories can relativise the importance of more hypothetic coexistence theories in species-rich systems. In consequence, trade-offs can play a lower role than predicted by conceptual models. iv) Seed immigration is a key process for local coexistence. Its alteration because of climate change should be considered for prognosis of coexistence. Field studies should be carried out to get data on seed rain composition.
The genome can be considered the blueprint for an organism. Composed of DNA, it harbours all organism-specific instructions for the synthesis of all structural components and their associated functions. The role of carriers of actual molecular structure and functions was believed to be exclusively assumed by proteins encoded in particular segments of the genome, the genes. In the process of converting the information stored genes into functional proteins, RNA – a third major molecule class – was discovered early on to act a messenger by copying the genomic information and relaying it to the protein-synthesizing machinery. Furthermore, RNA molecules were identified to assist in the assembly of amino acids into native proteins. For a long time, these - rather passive - roles were thought to be the sole purpose of RNA. However, in recent years, new discoveries have led to a radical revision of this view. First, RNA molecules with catalytic functions - thought to be the exclusive domain of proteins - were discovered. Then, scientists realized that much more of the genomic sequence is transcribed into RNA molecules than there are proteins in cells begging the question what the function of all these molecules are. Furthermore, very short and altogether new types of RNA molecules seemingly playing a critical role in orchestrating cellular processes were discovered. Thus, RNA has become a central research topic in molecular biology, even to the extent that some researcher dub cells as “RNA machines”. This thesis aims to contribute towards our understanding of RNA-related phenomena by applying Bioinformatics means. First, we performed a genome-wide screen to identify sites at which the chemical composition of DNA (the genotype) critically influences phenotypic traits (the phenotype) of the model plant Arabidopsis thaliana. Whole genome hybridisation arrays were used and an informatics strategy developed, to identify polymorphic sites from hybridisation to genomic DNA. Following this approach, not only were genotype-phenotype associations discovered across the entire Arabidopsis genome, but also regions not currently known to encode proteins, thus representing candidate sites for novel RNA functional molecules. By statistically associating them with phenotypic traits, clues as to their particular functions were obtained. Furthermore, these candidate regions were subjected to a novel RNA-function classification prediction method developed as part of this thesis. While determining the chemical structure (the sequence) of candidate RNA molecules is relatively straightforward, the elucidation of its structure-function relationship is much more challenging. Towards this end, we devised and implemented a novel algorithmic approach to predict the structural and, thereby, functional class of RNA molecules. In this algorithm, the concept of treating RNA molecule structures as graphs was introduced. We demonstrate that this abstraction of the actual structure leads to meaningful results that may greatly assist in the characterization of novel RNA molecules. Furthermore, by using graph-theoretic properties as descriptors of structure, we indentified particular structural features of RNA molecules that may determine their function, thus providing new insights into the structure-function relationships of RNA. The method (termed Grapple) has been made available to the scientific community as a web-based service. RNA has taken centre stage in molecular biology research and novel discoveries can be expected to further solidify the central role of RNA in the origin and support of life on earth. As illustrated by this thesis, Bioinformatics methods will continue to play an essential role in these discoveries.
In a very simplified view, the plant leaf growth can be reduced to two processes, cell division and cell expansion, accompanied by expansion of their surrounding cell walls. The vacuole, as being the largest compartment of the plant cell, plays a major role in controlling the water balance of the plant. This is achieved by regulating the osmotic pressure, through import and export of solutes over the vacuolar membrane (the tonoplast) and by controlling the water channels, the aquaporins. Together with the control of cell wall relaxation, vacuolar osmotic pressure regulation is thought to play an important role in cell expansion, directly by providing cell volume and indirectly by providing ion and pH homestasis for the cytosoplasm. In this thesis the role of tonoplast protein coding genes in cell expansion in the model plant Arabidopsis thaliana is studied and genes which play a putative role in growth are identified. Since there is, to date, no clearly identified protein localization signal for the tonoplast, there is no possibility to perform genome-wide prediction of proteins localized to this compartment. Thus, a series of recent proteomic studies of the tonoplast were used to compile a list of cross-membrane tonoplast protein coding genes (117 genes), and other growth-related genes from notably the growth regulating factor (GRF) and expansin families were included (26 genes). For these genes a platform for high-throughput reverse transcription quantitative real time polymerase chain reaction (RT-qPCR) was developed by selecting specific primer pairs. To this end, a software tool (called QuantPrime, see http://www.quantprime.de) was developed that automatically designs such primers and tests their specificity in silico against whole transcriptomes and genomes, to avoid cross-hybridizations causing unspecific amplification. The RT-qPCR platform was used in an expression study in order to identify candidate growth related genes. Here, a growth-associative spatio-temporal leaf sampling strategy was used, targeting growing regions at high expansion developmental stages and comparing them to samples taken from non-expanding regions or stages of low expansion. Candidate growth related genes were identified after applying a template-based scoring analysis on the expression data, ranking the genes according to their association with leaf expansion. To analyze the functional involvement of these genes in leaf growth on a macroscopic scale, knockout mutants of the candidate growth related genes were screened for growth phenotypes. To this end, a system for non-invasive automated leaf growth phenotyping was established, based on a commercially available image capture and analysis system. A software package was developed for detailed developmental stage annotation of the images captured with the system, and an analysis pipeline was constructed for automated data pre-processing and statistical testing, including modeling and graph generation, for various growth-related phenotypes. Using this system, 24 knockout mutant lines were analyzed, and significant growth phenotypes were found for five different genes.