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For the elucidation of the dynamics of signal transduction processes that are induced by cellular interactions, defined events along the signal transduction cascade and subsequent activation steps have to be analyzed and then also correlated with each other. This cannot be achieved by ensemble measurements because averaging biological data ignores the variability in timing and response patterns of individual cells and leads to highly blurred results. Instead, only a multi-parameter analysis at a single-cell level is able to exploit the information that is crucially needed for deducing the signaling pathways involved. The aim of this work was to develop a process line that allows the initiation of cell-cell or cell-particle interactions while at the same time the induced cellular reactions can be analyzed at various stages along the signal transduction cascade and correlated with each other. As this approach requires the gentle management of individually addressable cells, a dielectrophoresis (DEP)-based microfluidic system was employed that provides the manipulation of microscale objects with very high spatiotemporal precision and without the need of contacting the cell membrane. The system offers a high potential for automation and parallelization. This is essential for achieving a high level of robustness and reproducibility, which are key requirements in order to qualify this approach for a biomedical application. As an example process for intercellular communication, T cell activation has been chosen. The activation of the single T cells was triggered by contacting them individually with microbeads that were coated with antibodies directed against specific cell surface proteins, like the T cell receptor-associated kinase CD3 and the costimulatory molecule CD28 (CD; cluster of differentiation). The stimulation of the cells with the functionalized beads led to a rapid rise of their cytosolic Ca2+ concentration which was analyzed by a dual-wavelength ratiometric fluorescence measurement of the Ca2+-sensitive dye Fura-2. After Ca2+ imaging, the cells were isolated individually from the microfluidic system and cultivated further. Cell division and expression of the marker molecule CD69 as a late activation event of great significance were analyzed the following day and correlated with the previously recorded Ca2+ traces for each individual cell. It turned out such that the temporal profile of the Ca2+ traces between both activated and non-activated cells as well as dividing and non-dividing cells differed significantly. This shows that the pattern of Ca2+ signals in T cells can provide early information about a later reaction of the cell. As isolated cells are highly delicate objects, a precondition for these experiments was the successful adaptation of the system to maintain the vitality of single cells during and after manipulation. In this context, the influences of the microfluidic environment as well as the applied electric fields on the vitality of the cells and the cytosolic Ca2+ concentration as crucially important physiological parameters were thoroughly investigated. While a short-term DEP manipulation did not affect the vitality of the cells, they showed irregular Ca2+ transients upon exposure to the DEP field only. The rate and the strength of these Ca2+ signals depended on exposure time, electric field strength and field frequency. By minimizing their occurrence rate, experimental conditions were identified that caused the least interference with the physiology of the cell. The possibility to precisely control the exact time point of stimulus application, to simultaneously analyze short-term reactions and to correlate them with later events of the signal transduction cascade on the level of individual cells makes this approach unique among previously described applications and offers new possibilities to unravel the mechanisms underlying intercellular communication.
An important goal in biotechnology and (bio-) medical research is the isolation of single cells from a heterogeneous cell population. These specialised cells are of great interest for bioproduction, diagnostics, drug development, (cancer) therapy and research. To tackle emerging questions, an ever finer differentiation between target cells and non-target cells is required. This precise differentiation is a challenge for a growing number of available methods.
Since the physiological properties of the cells are closely linked to their morphology, it is beneficial to include their appearance in the sorting decision. For established methods, this represents a non addressable parameter, requiring new methods for the identification and isolation of target cells. Consequently, a variety of new flow-based methods have been developed and presented in recent years utilising 2D imaging data to identify target cells within a sample. As these methods aim for high throughput, the devices developed typically require highly complex fluid handling techniques, making them expensive while offering limited image quality.
In this work, a new continuous flow system for image-based cell sorting was developed that uses dielectrophoresis to precisely handle cells in a microchannel. Dielectrophoretic forces are exerted by inhomogeneous alternating electric fields on polarisable particles (here: cells). In the present system, the electric fields can be switched on and off precisely and quickly by a signal generator. In addition to the resulting simple and effective cell handling, the system is characterised by the outstanding quality of the image data generated and its compatibility with standard microscopes. These aspects result in low complexity, making it both affordable and user-friendly.
With the developed cell sorting system, cells could be sorted reliably and efficiently according to their cytosolic staining as well as morphological properties at different optical magnifications. The achieved purity of the target cell population was up to 95% and about 85% of the sorted cells could be recovered from the system. Good agreement was achieved between the results obtained and theoretical considerations. The achieved throughput of the system was up to 12,000 cells per hour. Cell viability studies indicated a high biocompatibility of the system.
The results presented demonstrate the potential of image-based cell sorting using dielectrophoresis. The outstanding image quality and highly precise yet gentle handling of the cells set the system apart from other technologies. This results in enormous potential for processing valuable and sensitive cell samples.
A phagocyte-specific Irf8 gene enhancer establishes early conventional dendritic cell commitment
(2011)
Haematopoietic development is a complex process that is strictly hierarchically organized. Here, the phagocyte lineages are a very heterogeneous cell compartment with specialized functions in innate immunity and induction of adaptive immune responses. Their generation from a common precursor must be tightly controlled. Interference within lineage formation programs for example by mutation or change in expression levels of transcription factors (TF) is causative to leukaemia. However, the molecular mechanisms driving specification into distinct phagocytes remain poorly understood. In the present study I identify the transcription factor Interferon Regulatory Factor 8 (IRF8) as the specification factor of dendritic cell (DC) commitment in early phagocyte precursors. Employing an IRF8 reporter mouse, I showed the distinct Irf8 expression in haematopoietic lineage diversification and isolated a novel bone marrow resident progenitor which selectively differentiates into CD8α+ conventional dendritic cells (cDCs) in vivo. This progenitor strictly depends on Irf8 expression to properly establish its transcriptional DC program while suppressing a lineage-inappropriate neutrophile program. Moreover, I demonstrated that Irf8 expression during this cDC commitment-step depends on a newly discovered myeloid-specific cis-enhancer which is controlled by the haematopoietic transcription factors PU.1 and RUNX1. Interference with their binding leads to abrogation of Irf8 expression, subsequently to disturbed cell fate decisions, demonstrating the importance of these factors for proper phagocyte cell development. Collectively, these data delineate a transcriptional program establishing cDC fate choice with IRF8 in its center.
Actin is one of the most highly conserved proteins in eukaryotes and distinct actin-related proteins with filament-forming properties are even found in prokaryotes. Due to these commonalities, actin-modulating proteins of many species share similar structural properties and proposed functions. The polymerization and depolymerization of actin are critical processes for a cell as they can contribute to shape changes to adapt to its environment and to move and distribute nutrients and cellular components within the cell. However, to what extent functions of actin-binding proteins are conserved between distantly related species, has only been addressed in a few cases. In this work, functions of Coronin-A (CorA) and Actin-interacting protein 1 (Aip1), two proteins involved in actin dynamics, were characterized. In addition, the interchangeability and function of Aip1 were investigated in two phylogenetically distant model organisms. The flowering plant Arabidopsis thaliana (encoding two homologs, AIP1-1 and AIP1-2) and in the amoeba Dictyostelium discoideum (encoding one homolog, DdAip1) were chosen because the functions of their actin cytoskeletons may differ in many aspects. Functional analyses between species were conducted for AIP1 homologs as flowering plants do not harbor a CorA gene.
In the first part of the study, the effect of four different mutation methods on the function of Coronin-A protein and the resulting phenotype in D. discoideum was revealed in two genetic knockouts, one RNAi knockdown and a sudden loss-of-function mutant created by chemical-induced dislocation (CID). The advantages and disadvantages of the different mutation methods on the motility, appearance and development of the amoebae were investigated, and the results showed that not all observed properties were affected with the same intensity. Remarkably, a new combination of Selection-Linked Integration and CID could be established.
In the second and third parts of the thesis, the exchange of Aip1 between plant and amoeba was carried out. For A. thaliana, the two homologs (AIP1-1 and AIP1-2) were analyzed for functionality as well as in D. discoideum. In the Aip1-deficient amoeba, rescue with AIP1-1 was more effective than with AIP1-2. The main results in the plant showed that in the aip1-2 mutant background, reintroduced AIP1-2 displayed the most efficient rescue and A. thaliana AIP1-1 rescued better than DdAip1. The choice of the tagging site was important for the function of Aip1 as steric hindrance is a problem. The DdAip1 was less effective when tagged at the C-terminus, while the plant AIP1s showed mixed results depending on the tag position. In conclusion, the foreign proteins partially rescued phenotypes of mutant plants and mutant amoebae, despite the organisms only being very distantly related in evolutionary terms.
A systems biological approach towards the molecular basis of heterosis in Arabidopsis thaliana
(2011)
Heterosis is defined as the superiority in performance of heterozygous genotypes compared to their corresponding genetically different homozygous parents. This phenomenon is already known since the beginning of the last century and it has been widely used in plant breeding, but the underlying genetic and molecular mechanisms are not well understood. In this work, a systems biological approach based on molecular network structures is proposed to contribute to the understanding of heterosis. Hybrids are likely to contain additional regulatory possibilities compared to their homozygous parents and, therefore, they may be able to correctly respond to a higher number of environmental challenges, which leads to a higher adaptability and, thus, the heterosis phenomenon. In the network hypothesis for heterosis, presented in this work, more regulatory interactions are expected in the molecular networks of the hybrids compared to the homozygous parents. Partial correlations were used to assess this difference in the global interaction structure of regulatory networks between the hybrids and the homozygous genotypes. This network hypothesis for heterosis was tested on metabolite profiles as well as gene expression data of the two parental Arabidopsis thaliana accessions C24 and Col-0 and their reciprocal crosses. These plants are known to show a heterosis effect in their biomass phenotype. The hypothesis was confirmed for mid-parent and best-parent heterosis for either hybrid of our experimental metabolite as well as gene expression data. It was shown that this result is influenced by the used cutoffs during the analyses. Too strict filtering resulted in sets of metabolites and genes for which the network hypothesis for heterosis does not hold true for either hybrid regarding mid-parent as well as best-parent heterosis. In an over-representation analysis, the genes that show the largest heterosis effects according to our network hypothesis were compared to genes of heterotic quantitative trait loci (QTL) regions. Separately for either hybrid regarding mid-parent as well as best-parent heterosis, a significantly larger overlap between the resulting gene lists of the two different approaches towards biomass heterosis was detected than expected by chance. This suggests that each heterotic QTL region contains many genes influencing biomass heterosis in the early development of Arabidopsis thaliana. Furthermore, this integrative analysis led to a confinement and an increased confidence in the group of candidate genes for biomass heterosis in Arabidopsis thaliana identified by both approaches.
Iron-sulfur clusters are essential enzyme cofactors. The most common and stable clusters are [2Fe-2S] and [4Fe-4S] that are found in nature. They are involved in crucial biological processes like respiration, gene regulation, protein translation, replication and DNA repair in prokaryotes and eukaryotes. In Escherichia coli, Fe-S clusters are essential for molybdenum cofactor (Moco) biosynthesis, which is a ubiquitous and highly conserved pathway. The first step of Moco biosynthesis is catalyzed by the MoaA protein to produce cyclic pyranopterin monophosphate (cPMP) from 5’GTP. MoaA is a [4Fe-4S] cluster containing radical S-adenosyl-L-methionine (SAM) enzyme. The focus of this study was to investigate Fe-S cluster insertion into MoaA under nitrate and TMAO respiratory conditions using E. coli as a model organism. Nitrate and TMAO respiration usually occur under anaerobic conditions, where oxygen is depleted. Under these conditions, E. coli uses nitrate and TMAO as terminal electron. Previous studies revealed that Fe-S cluster insertion is performed by Fe-S cluster carrier proteins. In E. coli, these proteins are known as A-type carrier proteins (ATC) by phylogenomic and genetic studies. So far, three of them have been characterized in detail in E. coli, namely IscA, SufA, and ErpA. This study shows that ErpA and IscA are involved in Fe-S cluster insertion into MoaA under nitrate and TMAO respiratory conditions. ErpA and IscA can partially replace each other in their role to provide [4Fe-4S] clusters for MoaA. SufA is not able to replace the functions of IscA or ErpA under nitrate respiratory conditions.
Nitrate reductase is a molybdoenzyme that coordinates Moco and Fe-S clusters. Under nitrate respiratory conditions, the expression of nitrate reductase is significantly increased in E. coli. Nitrate reductase is encoded in narGHJI genes, the expression of which is regulated by the transcriptional regulator, fumarate and nitrate reduction (FNR). The activation of FNR under conditions of nitrate respiration requires one [4Fe-4S] cluster. In this part of the study, we analyzed the insertion of Fe-S cluster into FNR for the expression of narGHJI genes in E. coli. The results indicate that ErpA is essential for the FNR-dependent expression of the narGHJI genes, a role that can be replaced partially by IscA and SufA when they are produced sufficiently under the conditions tested. This observation suggests that ErpA is indirectly regulating nitrate reductase expression via inserting Fe-S clusters into FNR.
Most molybdoenzymes are complex multi-subunit and multi-cofactor-containing enzymes that coordinate Fe-S clusters, which are functioning as electron transfer chains for catalysis. In E. coli, periplasmic aldehyde oxidoreductase (PaoAC) is a heterotrimeric molybdoenzyme that
consists of flavin, two [2Fe-2S], one [4Fe-4S] cluster and Moco. In the last part of this study, we investigated the insertion of Fe-S clusters into E. coli periplasmic aldehyde oxidoreductase (PaoAC). The results show that SufA and ErpA are involved in inserting [4Fe-4S] and [2Fe-2S] clusters into PaoABC, respectively under aerobic respiratory conditions.
In the present thesis, AC electrokinetic forces, like dielectrophoresis and AC electroosmosis, were demonstrated as a simple and fast method to functionalize the surface of nanoelectrodes with submicrometer sized biological objects. These nanoelectrodes have a cylindrical shape with a diameter of 500 nm arranged in an array of 6256 electrodes. Due to its medical relevance influenza virus as well as anti-influenza antibodies were chosen as a model organism. Common methods to bring antibodies or proteins to biosensor surfaces are complex and time-consuming. In the present work, it was demonstrated that by applying AC electric fields influenza viruses and antibodies can be immobilized onto the nanoelectrodes within seconds without any prior chemical modification of neither the surface nor the immobilized biological object. The distribution of these immobilized objects is not uniform over the entire array, it exhibits a decreasing gradient from the outer row to the inner ones. Different causes for this gradient have been discussed, such as the vortex-shaped fluid motion above the nanoelectrodes generated by, among others, electrothermal fluid flow. It was demonstrated that parts of the accumulated material are permanently immobilized to the electrodes. This is a unique characteristic of the presented system since in the literature the AC electrokinetic immobilization is almost entirely presented as a method just for temporary immobilization. The spatial distribution of the immobilized viral material or the anti-influenza antibodies at the electrodes was observed by either the combination of fluorescence microscopy and deconvolution or by super-resolution microscopy (STED). On-chip immunoassays were performed to examine the suitability of the functionalized electrodes as a potential affinity-based biosensor. Two approaches were pursued: A) the influenza virus as the bio-receptor or B) the influenza virus as the analyte. Different sources of error were eliminated by ELISA and passivation experiments. Hence, the activity of the immobilized object was inspected by incubation with the analyte. This resulted in the successful detection of anti-influenza antibodies by the immobilized viral material. On the other hand, a detection of influenza virus particles by the immobilized anti-influenza antibodies was not possible. The latter might be due to lost activity or wrong orientation of the antibodies. Thus, further examinations on the activity of by AC electric fields immobilized antibodies should follow. When combined with microfluidics and an electrical read-out system, the functionalized chips possess the potential to serve as a rapid, portable, and cost-effective point-of-care (POC) device. This device can be utilized as a basis for diverse applications in diagnosing and treating influenza, as well as various other pathogens.
Assumed comparable environmental conditions of early Mars and early Earth in 3.7 Ga ago – at a time when first fossil records of life on Earth could be found – suggest the possibility of life emerging on both planets in parallel. As conditions changed, the hypothetical life on Mars either became extinct or was able to adapt and might still exist in biological niches. The controversial discussed detection of methane on Mars led to the assumption, that it must have a recent origin – either abiotic through active volcanism or chemical processes, or through biogenic production. Spatial and seasonal variations in the detected methane concentrations and correlations between the presence of water vapor and geological features such as subsurface hydrogen, which are occurring together with locally increased detected concentrations of methane, gave fuel to the hypothesis of a possible biological source of the methane on Mars.
Therefore the phylogenetically old methanogenic archaea, which have evolved under early Earth conditions, are often used as model-organisms in astrobiological studies to investigate the potential of life to exist in possible extraterrestrial habitats on our neighboring planet. In this thesis methanogenic archaea originating from two extreme environments on Earth were investigated to test their ability to be active under simulated Mars analog conditions. These extreme environments – the Siberian permafrost-affected soil and the chemoautotrophically based terrestrial ecosystem of Movile cave, Romania – are regarded as analogs for possible Martian (subsurface) habitats. Two novel species of methanogenic archaea isolated from these environments were described within the frame of this thesis.
It could be shown that concentrations up to 1 wt% of Mars regolith analogs added to the growth media had a positive influence on the methane production rates of the tested methanogenic archaea, whereas higher concentrations resulted in decreasing rates. Nevertheless it was possible for the organisms to metabolize when incubated on water-saturated soil matrixes made of Mars regolith analogs without any additional nutrients. Long-term desiccation resistance of more than 400 days was proven with reincubation and indirect counting of viable cells through a combined treatment with propidium monoazide (to inactivate DNA of destroyed cells) and quantitative PCR. Phyllosilicate rich regolith analogs seem to be the best soil mixtures for the tested methanogenic archaea to be active under Mars analog conditions. Furthermore, in a simulation chamber experiment the activity of the permafrost methanogen strain Methanosarcina soligelidi SMA-21 under Mars subsurface analog conditions could be proven. Through real-time wavelength modulation spectroscopy measurements the increase in the methane concentration at temperatures down to -5 °C could be detected.
The results presented in this thesis contribute to the understanding of the activity potential of methanogenic archaea under Mars analog conditions and therefore provide insights to the possible habitability of present-day Mars (near) subsurface environments. Thus, it contributes also to the data interpretation of future life detection missions on that planet. For example the ExoMars mission of the European Space Agency (ESA) and Roscosmos which is planned to be launched in 2018 and is aiming to drill in the Martian subsurface.
The rise of evolutionary novelties is one of the major drivers of evolutionary diversification. African weakly-electric fishes (Teleostei, Mormyridae) have undergone an outstanding adaptive radiation, putatively owing to their ability to communicate through species-specific Electric Organ Discharges (EODs) produced by a novel, muscle-derived electric organ. Indeed, such EODs might have acted as effective pre-zygotic isolation mechanisms, hence favoring ecological speciation in this group of fishes. Despite the evolutionary importance of this organ, genetic investigations regarding its origin and function have remained limited.
The ultimate aim of this study is to better understand the genetic basis of EOD production by exploring the transcriptomic profiles of the electric organ and of its ancestral counterpart, the skeletal muscle, in the genus Campylomormyrus. After having established a set of reference transcriptomes using “Next-Generation Sequencing” (NGS) technologies, I performed in silico analyses of differential expression, in order to identify sets of genes that might be responsible for the functional differences observed between these two kinds of tissues. The results of such analyses indicate that: i) the loss of contractile activity and the decoupling of the excitation-contraction processes are reflected by the down-regulation of the corresponding genes in the electric organ; ii) the metabolic activity of the electric organ might be specialized towards the production and turnover of membrane structures; iii) several ion channels are highly expressed in the electric organ in order to increase excitability, and iv) several myogenic factors might be down-regulated by transcription repressors in the EO.
A secondary task of this study is to improve the genus level phylogeny of Campylomormyrus by applying new methods of inference based on the multispecies coalescent model, in order to reduce the conflict among gene trees and to reconstruct a phylogenetic tree as closest as possible to the actual species-tree. By using 1 mitochondrial and 4 nuclear markers, I was able to resolve the phylogenetic relationships among most of the currently described Campylomormyrus species. Additionally, I applied several coalescent-based species delimitation methods, in order to test the hypothesis that putatively cryptic species, which are distinguishable only from their EOD, belong to independently evolving lineages. The results of this analysis were additionally validated by investigating patterns of diversification at 16 microsatellite loci. The results suggest the presence of a new, yet undescribed species of Campylomormyrus.
The ultimate aim of this study is to better understand the relevance of weak electricity in the adaptive radiation of the African mormyrid fish. The chosen model taxon, the genus Campylomormyrus, exhibits a wide diversity of electric organ discharge (EOD) waveform types. Their EOD is age, sex, and species specific and is an important character for discriminating among species that are otherwise cryptic. After having established a complementary set of molecular markers, I examined the radiation of Campylomormyrus by a combined approach of molecular data (sequence data from the mitochondrial cytochrome b and the nuclear S7 ribosomal protein gene, as well as 18 microsatellite loci, especially developed for the genus Campylomormyrus), observation of ontogeny and diversification of EOD waveform, and morphometric analysis of relevant morphological traits. I built up the first convincing phylogenetic hypothesis for the genus Campylomormyrus. Taking advantage of microsatellite data, the identified phylogenetic clades proved to be reproductively isolated biological species. This way I detected at least six species occurring in sympatry near Brazzaville/Kinshasa (Congo Basin). By combining molecular data and EOD analyses, I could show that there are three cryptic species, characterised by their own adult EOD types, hidden under a common juvenile EOD form. In addition, I confirmed that adult male EOD is species-specific and is more different among closely related species than among more distantly related ones. This result and the observation that the EOD changes with maturity suggest its function as a reproductive isolation mechanism. As a result of my morphometric shape analysis, I could assign species types to the identified reproductively isolated groups to produce a sound taxonomy of the group. Besides this, I could also identify morphological traits relevant for the divergences between the identified species. Among them, the variations I found in the shape of the trunk-like snout, suggest the presence of different trophic specializations; therefore, this trait might have been involved in the ecological radiation of the group. In conclusion, I provided a convincing scenario envisioning an adaptive radiation of weakly electric fish triggered by sexual selection via assortative mating due to differences in EOD characteristics, but caused by a divergent selection of morphological traits correlated with the feeding ecology.
Im Bereich der medizinischen Diagnostik spielen DNA-Chips eine immer wichtigere Rolle. Dabei werden Glas- oder Silikon-Oberflächen mit Tausenden von einzelsträngigen DNA-Fragmenten, sog. Sonden, bestückt, die mit den passenden DNA-Fragmenten in der zugefügten Patientenprobe verschmelzen. Die Auswertung solcher Messungen liefert die Diagnose für Krankheiten wie z.B. Krebs, Alzheimer oder für den Nachweis pathogener Erreger. Durch fortschreitende Miniaturisierung dieser Meßsysteme können bis zu 40.000 Genfragmente des Menschen in einer einzigen Messung analysiert werden. Neben den DNA-Fragmenten können Bio-Chips auch für andere biologische Komponenten wie Antikörper und Proteine eingesetzt werden, wobei bei letzteren neben der Bindung auch die Aktivität ein wichtiger Diagnoseparamter ist. Am Fraunhofer-Institut für medizinische Technik und am Lehrstuhl für Analytische Biochemie der Universität Potsdam wurden im Rahmen einer Doktorarbeit Methoden entwickelt, die es ermöglichen auf nukleinsäuremodifizierten Sensoroberflächen die Aktivität von Proteinen zu messen. Es wurden Nukleinsäuren auf Oberflächen optischer Sensoren verankert. Diese fungierten als Rezeptor für die Proteine sowie auch als Substrat für Restriktionsenzyme, die Nukleinsäuren schneiden und Polymerasen, die Nukleinsäuren synthetisieren und verlängern können. Seine Anwendung fand diese Messmethode in der Messung der Aktivität des Proteins Telomerase, das in 90% aller Tumore erhöhte Aktivität gegenüber gesunden Zellen aufweist. Die Vorteile dieses neuen Assays gegenüber älteren Methoden liegt im Verzicht auf radioaktiv-markierten Komponenten und einer deutlich verkürzten Analysezeit. Die Arbeit schliesst mit einem funktionsfähigen Nachweis der Telomeraseaktivität im Zellextrakt von gesunden und kranken Zellen. Der direkte Einfluß von Hemmstoffen auf die Aktivität konnte sichtbar gemacht werden, und steht daher bei der Entwicklung neuer Tumor-Diagnostika und Therapeutika zur Verfügung.
Over the last decades, the world’s population has been growing at a faster rate, resulting in increased urbanisation, especially in developing countries. More than half of the global population currently lives in urbanised areas with an increasing tendency. The growth of cities results in a significant loss of vegetation cover, soil compaction and sealing of the soil surface which in turn results in high surface runoff during high-intensity storms and causes the problem of accelerated soil water erosion on streets and building grounds. Accelerated soil water erosion is a serious environmental problem in cities as it gives rise to the contamination of aquatic bodies, reduction of ground water recharge and increase in land degradation, and also results in damages to urban infrastructures, including drainage systems, houses and roads. Understanding the problem of water erosion in urban settings is essential for the sustainable planning and management of cities prone to water erosion. However, in spite of the vast existence of scientific literature on water erosion in rural regions, a concrete understanding of the underlying dynamics of urban erosion still remains inadequate for the urban dryland environments.
This study aimed at assessing water erosion and the associated socio-environmental determinants in a typical dryland urban area and used the city of Windhoek, Namibia, as a case study. The study used a multidisciplinary approach to assess the problem of water erosion. This included an in depth literature review on current research approaches and challenges of urban erosion, a field survey method for the quantification of the spatial extent of urban erosion in the dryland city of Windhoek, and face to face interviews by using semi-structured questionnaires to analyse the perceptions of stakeholders on urban erosion.
The review revealed that around 64% of the literatures reviewed were conducted in the developed world, and very few researches were carried out in regions with extreme climate, including dryland regions. Furthermore, the applied methods for erosion quantification and monitoring are not inclusive of urban typical features and they are not specific for urban areas. The reviewed literature also lacked aspects aimed at addressing the issues of climate change and policies regarding erosion in cities. In a field study, the spatial extent and severity of an urban dryland city, Windhoek, was quantified and the results show that nearly 56% of the city is affected by water erosion showing signs of accelerated erosion in the form of rills and gullies, which occurred mainly in the underdeveloped, informal and semi-formal areas of the city. Factors influencing the extent of erosion in Windhoek included vegetation cover and type, socio-urban factors and to a lesser extent slope estimates. A comparison of an interpolated field survey erosion map with a conventional erosion assessment tool (the Universal Soil Loss Equation) depicted a large deviation in spatial patterns, which underlines the inappropriateness of traditional non-urban erosion tools to urban settings and emphasises the need to develop new erosion assessment and management methods for urban environments. It was concluded that measures for controlling water erosion in the city need to be site-specific as the extent of erosion varied largely across the city.
The study also analysed the perceptions and understanding of stakeholders of urban water erosion in Windhoek, by interviewing 41 stakeholders using semi-structured questionnaires. The analysis addressed their understanding of water erosion dynamics, their perceptions with regards to the causes and the seriousness of erosion damages, and their attitudes towards the responsibilities for urban erosion. The results indicated that there is less awareness of the process as a phenomenon, instead there is more awareness of erosion damages and the factors contributing to the damages. About 69% of the stakeholders considered erosion damages to be ranging from moderate to very serious. However, there were notable disparities between the private householders and public authority groups. The study further found that the stakeholders have no clear understanding of their responsibilities towards the management of the control measures and payment for the damages. The private householders and local authority sectors pointed fingers at each other for the responsibilities for erosion damage payments and for putting up prevention measures. The reluctance to take responsibility could create a predicament for areas affected, specifically in the informal settlements where land management is not carried out by the local authority and land is not owned by the occupants.
The study concluded that in order to combat urban erosion, it is crucial to understand diverse dynamics aggravating the process of urbanisation from different scales. Accordingly, the study suggests that there is an urgent need for the development of urban-specific approaches that aim at: (a) incorporating the diverse socio-economic-environmental aspects influencing erosion, (b) scientifically improving natural cycles that influence water storages and nutrients for plants in urbanised dryland areas in order to increase the amount of vegetation cover, (c) making use of high resolution satellite images to improve the adopted methods for assessing urban erosion, (d) developing water erosion policies, and (e) continuously monitoring the impact of erosion and the influencing processes from local, national and international levels.
The need to develop sustainable resource management strategies for semi-arid and arid rangelands is acute as non-adapted grazing strategies lead to irreversible environmental problems such as desertification and associated loss of economic support to society. In such vulnerable ecosystems, successful implementation of sustainable management strategies depends on well-founded under-standing of processes at different scales that underlay the complex system dynamic. There is ample evidence that, in contrast to traditional sectoral approaches, only interdisciplinary research does work for resolving problems in conservation and natural resource management. In this thesis I combined a range of modeling approaches that integrate different disciplines and spatial scales in order to contribute to basic guidelines for sustainable management of semi-arid and arid range-lands. Since water availability and livestock management are seen as most potent determinants for the dynamics of semi-arid and arid ecosystems I focused on (i) the interaction of ecological and hydro-logical processes and (ii) the effect of farming strategies. First, I developed a grid-based and small-scaled model simulating vegetation dynamics and inter-linked hydrological processes. The simulation results suggest that ecohydrological interactions gain importance in rangelands with ascending slope where vegetation cover serves to obstruct run-off and decreases evaporation from the soil. Disturbances like overgrazing influence these positive feedback mechanisms by affecting vegetation cover and composition. In the second part, I present a modeling approach that has the power to transfer and integrate ecological information from the small scale vegetation model to the landscape scale, most relevant for the conservation of biodiversity and sustainable management of natural resources. I combined techniques of stochastic modeling with remotely sensed data and GIS to investigate to which ex-tent spatial interactions, like the movement of surface water by run-off in water limited environments, affect ecosystem functioning at the landscape scale. My simulation experiments show that overgrazing decreases the number of vegetation patches that act as hydrological sinks and run-off increases. The results of both simulation models implicate that different vegetation types should not only be regarded as provider of forage production but also as regulator of ecosystem functioning. Vegetation patches with good cover of perennial vegetation are capable to catch and conserve surface run-off from degraded surrounding areas. Therefore, downstream out of the simulated system is prevented and efficient use of water resources is guaranteed at all times. This consequence also applies to commercial rotational grazing strategies for semi-arid and arid rangelands with ascending slope where non-degraded paddocks act as hydrological sinks. Finally, by the help of an integrated ecological-economic modeling approach, I analyzed the relevance of farmers’ ecological knowledge for longterm functioning of semi-arid and arid grazing systems under current and future climatic conditions. The modeling approach consists of an ecological and an economic module and combines relevant processes on either level. Again, vegetation dynamics and forage productivity is derived by the small-scaled vegetation model. I showed that sustainable management of semi-arid and arid rangelands relies strongly on the farmers’ knowledge on how the ecosystem works. Furthermore, my simulation results indicate that the projected lower annual rainfall due to climate change in combination with non-adapted grazing strategies adds an additional layer of risk to these ecosystems that are already prone to land degradation. All simulation models focus on the most essential factors and ignore specific details. Therefore, even though all simulation models are parameterized for a specific dwarf shrub savanna in arid southern Namibia, the conclusions drawn are applicable for semi-arid and arid rangelands in general.
Für ein tiefergehendes Verständnis von Entwicklung und Funktion der quergestreiften Muskulatur ist eine Betrachtung der am Aufbau der Myofibrillen, den kontraktilen Organellen, beteiligten Proteine essentiell. Die vorliegende Arbeit beschäftigt sich mit Myomesin, einem Protein der sarkomeren M-Bande. Zunächst wurde die cDNA des humanen Myomesins vollständig kloniert, sequenziert und nachfolgend die komplette Größe der aminoterminalen Kopfdomäne bestimmt. Es konnte gezeigt werden, daß Myomesin in vitro mit den Domänen 1 und 12 an Myosin bindet. Die muskelspezifische Isoform der Kreatinkinase bindet an die Domänen 7 und 8. Stimulations- und Inhibitionsexperimente belegen, daß Myomesin an Serin 618 in vivo durch die Proteinkinase A phosphoryliert wird und daß diese Phosphorylierung durch Aktivierung beta2-adrenerger Rezeptoren stimulierbar ist. In Muskelgewebeproben von Patienten, die an der Hypertrophen Kardiomyopathie, einer genetisch bedingten Herzmuskelkrankheit, erkrankt sind, konnte mit einem neu hergestellten phosphorylierungsabhängigen Antikörper eine Verminderung der Menge phosphorylierten Myomesins nachgewiesen werden. Mögliche Ursachen werden diskutiert. Myomesin bildet Dimere, wie durch hefegenetische und biochemische Experimente gezeigt werden konnte. Die Dimerisierung von Myomesin könnte eine zentrale Rolle für den Einbau der Myosinfilamente in die naszierende Myofibrille haben. Anhand der gewonnenen Daten wurde ein verbessertes Modell der zentralen M-Bande erstellt.
Ziel der vorliegenden Arbeit war die Entwicklung einer SNP-Genotypisierungsmethode mit auf Mikroarrays immobilisierten PCR-Produkten. Für die Analyse wurde ein faseroptischer Affinitätssensor bzw. ein Durchfluss-Biochip-Scanner mit integrierter Fluoreszenzdetektion verwendet. An den immobilisierten Analyten (PCR-Produkten) wurde eine Fluoreszenzoligonukleotidsonde hybridisiert und anschließend die Dissoziation der Sonde im Fluss verfolgt. Die Diskriminierung von Wildtyp- und Mutanten-DNA erfolgte durch die kinetische Auswertung der Dissoziationskurven sowie durch die Analyse der Fluoreszenzintensität. Die Versuche am faseroptischen Affinitätssensor zeigten, dass DNA-DNA-Hybride sowohl von Oligonukleotiden als auch von PCR-Produkten ein typisches Dissoziationsverhalten aufweisen, wobei fehlgepaarte Hybride eine signifikant schnellere Dissoziation zeigen als perfekt passende Hybride. Dieser Geschwindigkeitsunterschied lässt sich durch den Vergleich der jeweiligen kinetischen Geschwindigkeitskonstanten kD quantitativ erfassen. Da die Kopplung des Analyten an der Chipoberfläche sowie die Hybridisierungs- und Dissoziationsparameter essentiell für die Methodenentwicklung war, wurden die Parameter für ein optimales Spotting und die Immobilisierung von PCR-Produkten ermittelt. Getestet wurden die affine Kopplung von biotinylierten PCR-Produkten an Streptavidin-, Avidin- und NeutrAvidin-Oberflächen sowie die kovalente Bindung von phosphorylierten Amplifikaten mit der EDC/Methylimidazol-Methode. Die besten Ergebnisse sowohl in Spotform und -homogenität als auch im Signal/Rausch-Verhältnis wurden an NeutrAvidin-Oberflächen erreicht. Für die Etablierung der Mikroarray-Genotypisierungsmethode durch kinetische Analyse nach einem Hybridisierungsexperiment wurden Sondenlänge, Puffersystem, Spotting-Konzentration des Analyten sowie Temperatur optimiert. Das Analysensystem erlaubte es, PCR-Produkte mit einer Konzentration von 250 ng/µl in einem HEPES-EDTA-NaCl-Puffer auf mit NeutrAvidin beschichtete Glasträger zu spotten. In den anschließenden Hybridisierungs- und Dissoziationsexperimenten bei 30 °C konnte die Diskriminierung von homocygoter Wildtyp- und homocygoter Mutanten- sowie heterocygoter DNA am Beispiel von Oligonukleotid-Hybriden erreicht werden. In einer Gruppe von 24 homocygoten Patienten wurde ein Polymorphismus im SULT1A1-Gen analysiert. Sowohl durch kinetische Auswertung als auch mit der Analyse der Fluoreszenzintensität wurde der Genotyp der Proben identifiziert. Die Ergebnisse wurden mit dem Referenzverfahren, der Restriktionschnittstellenanalyse (PCR-RFLP) validiert. Lediglich ein Genotyp wurde falsch bestimmt, die Genauigkeit lag bei 96%. In einer Gruppe von 44 Patienten wurde der Genotyp eines SNP in der Adiponectin-Promotor-Region untersucht. Nach Vergleich der Analysenergebnisse mit denen eines Referenzverfahrens konnten lediglich 14 der untersuchten Genotypen bestätigt werden. Ursache für die unzureichende Genauigkeit der Methode war vor allem das schlechte Signal/Rausch-Verhältnis. Zusammenfassend kann gesagt werden, dass das in dieser Arbeit entwickelte Analysesystem für die Genotypisierung von Einzelpunktmutationen geeignet ist, homocygote Patientenproben zuverlässig zu analysieren. Prinzipiell ist das auch bei heterocygoter DNA möglich. Da nach aktuellem Kenntnisstand eine SNP-Analysemethode an immobilisierten PCR-Produkten noch nicht veröffentlicht wurde, stellt das hier entwickelte Verfahren eine Alternative zu bisher bekannten Mikroarray-Verfahren dar. Als besonders vorteilhaft erweist sich der reverse Ansatz der Methode. Der hier vorgestellte Ansatz ist eine kostengünstigere und weniger hoch dimensionierte Lösung für Fragestellungen beispielsweise in der Ernährungswissenschaft, bei denen meist eine mittlere Anzahl Patienten auf nur einige wenige SNPs zu untersuchen ist. Wenn es gelingt, durch die Weiterentwicklung der Hardware bzw. weiterer Optimierung, eine Verbesserung des Signal/Rausch-Verhältnisses und damit die Diskriminierung von heterocygoter DNA zu erreichen, kann diese Methode zukünftig bei der Analyse von mittelgroßen Patientengruppen alternativ zu anderen Genotypisierungsmethoden verwendet werden.
Metabolic systems tend to exhibit steady states that can be measured in terms of their concentrations and fluxes. These measurements can be regarded as a phenotypic representation of all the complex interactions and regulatory mechanisms taking place in the underlying metabolic network. Such interactions determine the system's response to external perturbations and are responsible, for example, for its asymptotic stability or for oscillatory trajectories around the steady state. However, determining these perturbation responses in the absence of fully specified kinetic models remains an important challenge of computational systems biology. Structural kinetic modeling (SKM) is a framework to analyse whether a metabolic steady state remains stable under perturbation, without requiring detailed knowledge about individual rate equations. It provides a parameterised representation of the system's Jacobian matrix in which the model parameters encode information about the enzyme-metabolite interactions. Stability criteria can be derived by generating a large number of structural kinetic models (SK-models) with randomly sampled parameter sets and evaluating the resulting Jacobian matrices. The parameter space can be analysed statistically in order to detect network positions that contribute significantly to the perturbation response. Because the sampled parameters are equivalent to the elasticities used in metabolic control analysis (MCA), the results are easy to interpret biologically. In this project, the SKM framework was extended by several novel methodological improvements. These improvements were evaluated in a simulation study using a set of small example pathways with simple Michaelis Menten rate laws. Afterwards, a detailed analysis of the dynamic properties of the neuronal TCA cycle was performed in order to demonstrate how the new insights obtained in this work could be used for the study of complex metabolic systems. The first improvement was achieved by examining the biological feasibility of the elasticity combinations created during Monte Carlo sampling. Using a set of small example systems, the findings showed that the majority of sampled SK-models would yield negative kinetic parameters if they were translated back into kinetic models. To overcome this problem, a simple criterion was formulated that mitigates such infeasible models and the application of this criterion changed the conclusions of the SKM experiment. The second improvement of this work was the application of supervised machine-learning approaches in order to analyse SKM experiments. So far, SKM experiments have focused on the detection of individual enzymes to identify single reactions important for maintaining the stability or oscillatory trajectories. In this work, this approach was extended by demonstrating how SKM enables the detection of ensembles of enzymes or metabolites that act together in an orchestrated manner to coordinate the pathways response to perturbations. In doing so, stable and unstable states served as class labels, and classifiers were trained to detect elasticity regions associated with stability and instability. Classification was performed using decision trees and relevance vector machines (RVMs). The decision trees produced good classification accuracy in terms of model bias and generalizability. RVMs outperformed decision trees when applied to small models, but encountered severe problems when applied to larger systems because of their high runtime requirements. The decision tree rulesets were analysed statistically and individually in order to explore the role of individual enzymes or metabolites in controlling the system's trajectories around steady states. The third improvement of this work was the establishment of a relationship between the SKM framework and the related field of MCA. In particular, it was shown how the sampled elasticities could be converted to flux control coefficients, which were then investigated for their predictive information content in classifier training. After evaluation on the small example pathways, the methodology was used to study two steady states of the neuronal TCA cycle with respect to their intrinsic mechanisms responsible for stability or instability. The findings showed that several elasticities were jointly coordinated to control stability and that the main source for potential instabilities were mutations in the enzyme alpha-ketoglutarate dehydrogenase.
Recent high-throughput technologies enable the acquisition of a variety of complementary data and imply regulatory networks on the systems biology level. A common approach to the reconstruction of such networks is the cluster analysis which is based on a similarity measure. We use the information theoretic concept of the mutual information, that has been originally defined for discrete data, as a measure of similarity and propose an extension to a commonly applied algorithm for its calculation from continuous biological data. We compare our approach to previously existing algorithms. We develop a performance optimised software package for the application of the mutual information to large-scale datasets. Furthermore, we design and implement a web-based service for the analysis of integrated data measured with different technologies. Application to biological data reveals biologically relevant groupings and reconstructed signalling networks show agreements with physiological findings.
For the first time the transcriptional reprogramming of distinct root cortex cells during the arbuscular mycorrhizal (AM) symbiosis was investigated by combining Laser Capture Mirodissection and Affymetrix GeneChip® Medicago genome array hybridization. The establishment of cryosections facilitated the isolation of high quality RNA in sufficient amounts from three different cortical cell types. The transcript profiles of arbuscule-containing cells (arb cells), non-arbuscule-containing cells (nac cells) of Rhizophagus irregularis inoculated Medicago truncatula roots and cortex cells of non-inoculated roots (cor) were successfully explored. The data gave new insights in the symbiosis-related cellular reorganization processes and indicated that already nac cells seem to be prepared for the upcoming fungal colonization. The mycorrhizal- and phosphate-dependent transcription of a GRAS TF family member (MtGras8) was detected in arb cells and mycorrhizal roots. MtGRAS shares a high sequence similarity to a GRAS TF suggested to be involved in the fungal colonization processes (MtRAM1). The function of MtGras8 was unraveled upon RNA interference- (RNAi-) mediated gene silencing. An AM symbiosis-dependent expression of a RNAi construct (MtPt4pro::gras8-RNAi) revealed a successful gene silencing of MtGras8 leading to a reduced arbuscule abundance and a higher proportion of deformed arbuscules in root with reduced transcript levels. Accordingly, MtGras8 might control the arbuscule development and life-time. The targeting of MtGras8 by the phosphate-dependent regulated miRNA5204* was discovered previously (Devers et al., 2011). Since miRNA5204* is known to be affected by phosphate, the posttranscriptional regulation might represent a link between phosphate signaling and arbuscule development. In this work, the posttranscriptional regulation was confirmed by mis-expression of miRNA5204* in M. truncatula roots. The miRNA-mediated gene silencing affects the MtGras8 transcript abundance only in the first two weeks of the AM symbiosis and the mis-expression lines seem to mimic the phenotype of MtGras8-RNAi lines. Additionally, MtGRAS8 seems to form heterodimers with NSP2 and RAM1, which are known to be key regulators of the fungal colonization process (Hirsch et al., 2009; Gobbato et al., 2012). These data indicate that MtGras8 and miRNA5204* are linked to the sym pathway and regulate the arbuscule development in phosphate-dependent manner.
The development of fast and reliable biochemical tools for on-site screening in environmental analysis was the main target of the present work. Due to various hazardous effects such as endocrine disruption and toxicity phenolic compounds are key analytes in environmental analysis and thus were chosen as model analytes. Three different methods were developed: For the enzymatic detection of phenols in environmental samples an enzyme-based biosensor was developed. In contrast to reported work using tyrosinase or peroxidases, we developed a biosensor based on glucose dehydrogenase as biorecognition element. This biosensor was devoted for an application in a laboratory flow system as well as in a portable device for on-site measurements. This enzymatic detection is applicable only for a limited number of phenols due to substrate specificity of the enzyme. For other relevant compounds based on a phenolic structure (i.e. nitrophenol, alkylphenols and alkylphenol ethoxylates) immunological methods had to be developed. The electrochemical GDH-biosensor was used as the label detector in these immunoassays. Two heterogeneous immunoassays were developed where ßGal was used as the label. An electrochemical method for the determination of the marker enzyme activity was processed. The separation step was realized with protein A/G columns (laboratory flow system) or by direct immobilization of the antibodies in small disposable capillaries (on-site analysis). All methods were targeted on the contemporary analysis of small numbers of samples.
Analysis of supramolecular assemblies of NE81, the first lamin protein in a non-metazoan organism
(2019)
Nuclear lamins are nucleus-specific intermediate filaments forming a network located at the inner nuclear membrane of the nuclear envelope. They form the nuclear lamina together with proteins of the inner nuclear membrane regulating nuclear shape and gene expression, among others. The amoebozoan Dictyostelium NE81 protein is a suitable candidate for an evolutionary conserved lamin protein in this non-metazoan organism. It shares the domain organization of metazoan lamins and is fulfilling major lamin functions in Dictyostelium. Moreover, field-emission scanning electron microscopy (feSEM) images of NE81 expressed on Xenopus oocytes nuclei revealed filamentous structures with an overall appearance highly reminiscent to that of metazoan Xenopus lamin B2. For the classification as a lamin-like or a bona fide lamin protein, a better understanding of the supramolecular NE81 structure was necessary. Yet, NE81 carrying a large N-terminal GFP-tag turned out as unsuitable source for protein isolation and characterization; GFP-NE81 expressed in Dictyostelium NE81 knock-out cells exhibited an abnormal distribution, which is an indicator for an inaccurate assembly of GFP-tagged NE81. Hence, a shorter 8×HisMyc construct was the tag of choice to investi-gate formation and structure of NE81 assemblies. One strategy was the structural analysis of NE81 in situ at the outer nuclear membrane in Dictyostelium cells; NE81 without a func-tional nuclear localization signal (NLS) forms assemblies at the outer face of the nucleus. Ultrastructural feSEM pictures of NE81ΔNLS nuclei showed a few filaments of the expected size but no repetitive filamentous structures. The former strategy should also be established for metazoan lamins in order to facilitate their structural analysis. However, heterologously expressed Xenopus and C. elegans lamins showed no uniform localization at the outer nucle-ar envelope of Dictyostelium and hence, no further ultrastructural analysis was undertaken. For in vitro assembly experiments a Dictyostelium mutant was generated, expressing NE81 without the NLS and the membrane-anchoring isoprenylation site (HisMyc-NE81ΔNLSΔCLIM). The cytosolic NE81 clusters were soluble at high ionic strength and were purified from Dictyostelium extracts using Ni-NTA Agarose. Widefield immunofluorescence microscopy, super-resolution light microscopy and electron microscopy images of purified NE81 showed its capability to form filamentous structures at low ionic strength, as described previously for metazoan lamins. Introduction of a phosphomimetic point mutation (S122E) into the CDK1-consensus sequence of NE81 led to disassembled NE81 protein in vivo, which could be reversibly stimulated to form supramolecular assemblies by blue light exposure.
The results of this work reveal that NE81 has to be considered a bona fide lamin, since it is able to form filamentous assemblies. Furthermore, they highlight Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope containing all rele-vant protein components known in animal cells.