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The cytoskeleton is an essential component of living cells. It is composed of different types of protein filaments that form complex, dynamically rearranging, and interconnected networks. The cytoskeleton serves a multitude of cellular functions which further depend on the cell context. In animal cells, the cytoskeleton prominently shapes the cell's mechanical properties and movement. In plant cells, in contrast, the presence of a rigid cell wall as well as their larger sizes highlight the role of the cytoskeleton in long-distance intracellular transport. As it provides the basis for cell growth and biomass production, cytoskeletal transport in plant cells is of direct environmental and economical relevance. However, while knowledge about the molecular details of the cytoskeletal transport is growing rapidly, the organizational principles that shape these processes on a whole-cell level remain elusive.
This thesis is devoted to the following question: How does the complex architecture of the plant cytoskeleton relate to its transport functionality? The answer requires a systems level perspective of plant cytoskeletal structure and transport. To this end, I combined state-of-the-art confocal microscopy, quantitative digital image analysis, and mathematically powerful, intuitively accessible graph-theoretical approaches.
This thesis summarizes five of my publications that shed light on the plant cytoskeleton as a transportation network: (1) I developed network-based frameworks for accurate, automated quantification of cytoskeletal structures, applicable in, e.g., genetic or chemical screens; (2) I showed that the actin cytoskeleton displays properties of efficient transport networks, hinting at its biological design principles; (3) Using multi-objective optimization, I demonstrated that different plant cell types sustain cytoskeletal networks with cell-type specific and near-optimal organization; (4) By investigating actual transport of organelles through the cell, I showed that properties of the actin cytoskeleton are predictive of organelle flow and provided quantitative evidence for a coordination of transport at a cellular level; (5) I devised a robust, optimization-based method to identify individual cytoskeletal filaments from a given network representation, allowing the investigation of single filament properties in the network context. The developed methods were made publicly available as open-source software tools.
Altogether, my findings and proposed frameworks provide quantitative, system-level insights into intracellular transport in living cells. Despite my focus on the plant cytoskeleton, the established combination of experimental and theoretical approaches is readily applicable to different organisms. Despite the necessity of detailed molecular studies, only a complementary, systemic perspective, as presented here, enables both understanding of cytoskeletal function in its evolutionary context as well as its future technological control and utilization.
Among the bloom-forming and potentially harmful cyanobacteria, the genus Microcystis represents a most diverse taxon, on the genomic as well as on morphological and secondary metabolite levels. Microcystis communities are composed of a variety of diversified strains. The focus of this study lies on potential interactions between Microcystis representatives and the roles of secondary metabolites in these interaction processes.
The role of secondary metabolites functioning as signaling molecules in the investigated interactions is demonstrated exemplary for the prevalent hepatotoxin microcystin. The extracellular and intracellular roles of microcystin are tested in microarray-based transcriptomic approaches. While an extracellular effect of microcystin on Microcystis transcription is confirmed and connected to a specific gene cluster of another secondary metabolite in this study, the intracellularly occurring microcystin is related with several pathways of the primary metabolism. A clear correlation of a microcystin knockout and the SigE-mediated regulation of carbon metabolism is found. According to the acquired transcriptional data, a model is proposed that postulates the regulating effect of microcystin on transcriptional regulators such as the alternative sigma factor SigE, which in return captures an essential role in sugar catabolism and redox-state regulation.
For the purpose of simulating community conditions as found in the field, Microcystis colonies are isolated from the eutrophic lakes near Potsdam, Germany and established as stably growing under laboratory conditions. In co-habitation simulations, the recently isolated field strain FS2 is shown to specifically induce nearly immediate aggregation reactions in the axenic lab strain Microcystis aeruginosa PCC 7806. In transcriptional studies via microarrays, the induced expression program in PCC 7806 after aggregation induction is shown to involve the reorganization of cell envelope structures, a highly altered nutrient uptake balance and the reorientation of the aggregating cells to a heterotrophic carbon utilization, e.g. via glycolysis. These transcriptional changes are discussed as mechanisms of niche adaptation and acclimation in order to prevent competition for resources.
Gene expression describes the process of making functional gene products (e.g. proteins or special RNAs) from instructions encoded in the genetic information (e.g. DNA). This process is heavily regulated, allowing cells to produce the appropriate gene products necessary for cell survival, adapting production as necessary for different cell environments. Gene expression is subject to regulation at several levels, including transcription, mRNA degradation, translation and protein degradation. When intact, this system maintains cell homeostasis, keeping the cell alive and adaptable to different environments. Malfunction in the system can result in disease states and cell death. In this dissertation, we explore several aspects of gene expression control by analyzing data from biological experiments. Most of the work following uses a common mathematical model framework based on Markov chain models to test hypotheses, predict system dynamics or elucidate network topology. Our work lies in the intersection between mathematics and biology and showcases the power of statistical data analysis and math modeling for validation and discovery of biological phenomena.
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.
Light-triggered release of bioactive compounds from HA/PLL multilayer films for stimulation of cells
(2016)
The concept of targeting cells and tissues by controlled delivery of molecules is essential in the field of biomedicine. The layer-by-layer (LbL) technology for the fabrication of polymer multilayer films is widely implemented as a powerful tool to assemble tailor-made materials for controlled drug delivery. The LbL films can as well be engineered to act as mimics of the natural cellular microenvironment. Thus, due to the myriad possibilities such as controlled cellular adhesion and drug delivery offered by LbL films, it becomes easily achievable to direct the fate of cells by growing them on the films.
The aim of this work was to develop an approach for non-invasive and precise control of the presentation of bioactive molecules to cells. The strategy is based on employment of the LbL films, which function as support for cells and at the same time as reservoirs for bioactive molecules to be released in a controlled manner. UV light is used to trigger the release of the stored ATP with high spatio-temporal resolution. Both physico-chemical (competitive intermolecular interactions in the film) and biological aspects (cellular response and viability) are addressed in this study.
Biopolymers hyaluronic acid (HA) and poly-L-lysine (PLL) were chosen as the building blocks for the LbL film assembly. Poor cellular adhesion to native HA/PLL films as well as significant degradation by cells within a few days were shown. However, coating the films with gold nanoparticles not only improved cellular adhesion and protected the films from degradation, but also formed a size-exclusion barrier with adjustable cut-off in the size range of a few tens of kDa.
The films were shown to have high reservoir capacity for small charged molecules (reaching mM levels in the film). Furthermore, they were able to release the stored molecules in a sustained manner. The loading and release are explained by a mechanism based on interactions between charges of the stored molecules and uncompensated charges of the biopolymers in the film. Charge balance and polymer dynamics in the film play the pivotal role.
Finally, the concept of light-triggered release from the films has been proven using caged ATP loaded into the films from which ATP was released on demand. ATP induces a fast cellular response, i.e. increase in intracellular [Ca2+], which was monitored in real-time. Limitations of the cellular stimulation by the proposed approach are highlighted by studying the stimulation as a function of irradiation parameters (time, distance, light power). Moreover, caging molecules bind to the film stronger than ATP does, which opens new perspectives for the use of the most diverse chemical compounds as caging molecules.
Employment of HA/PLL films as a nouvelle support for cellular growth and hosting of bioactive molecules, along with the possibility to stimulate individual cells using focused light renders this approach highly efficient and unique in terms of precision and spatio-temporal resolution among those previously described. With its high potential, the concept presented herein provides the foundation for the design of new intelligent materials for single cell studies, with the focus on tissue engineering, diagnostics, and other cell-based applications.
In this dissertation, an electric field-assisted method was developed and applied to achieve immobilization and alignment of biomolecules on metal electrodes in a simple one-step experiment. Neither modifications of the biomolecule nor of the electrodes were needed. The two major electrokinetic effects that lead to molecule motion in the chosen electrode configurations used were identified as dielectrophoresis and AC electroosmotic flow. To minimize AC electroosmotic flow, a new 3D electrode configuration was designed. Thus, the influence of experimental parameters on the dielectrophoretic force and the associated molecule movement could be studied. Permanent immobilization of proteins was examined and quantified absolutely using an atomic force microscope. By measuring the volumes of the immobilized protein deposits, a maximal number of proteins contained therein was calculated. This was possible since the proteins adhered to the tungsten electrodes even after switching off the electric field. The permanent immobilization of functional proteins on surfaces or electrodes is one crucial prerequisite for the fabrication of biosensors.
Furthermore, the biofunctionality of the proteins must be retained after immobilization. Due to the chemical or physical modifications on the proteins caused by immobilization, their biofunctionality is sometimes hampered. The activity of dielectrophoretically immobilized proteins, however, was proven here for an enzyme for the first time. The enzyme horseradish peroxidase was used exemplarily, and its activity was demonstrated with the oxidation of dihydrorhodamine 123, a non-fluorescent precursor of the fluorescence dye rhodamine 123.
Molecular alignment and immobilization - reversible and permanent - was achieved under the influence of inhomogeneous AC electric fields. For orientational investigations, a fluorescence microscope setup, a reliable experimental procedure and an evaluation protocol were developed and validated using self-made control samples of aligned acridine orange molecules in a liquid crystal.
Lambda-DNA strands were stretched and aligned temporarily between adjacent interdigitated electrodes, and the orientation of PicoGreen molecules, which intercalate into the DNA strands, was determined. Similarly, the aligned immobilization of enhanced Green Fluorescent Protein was demonstrated exploiting the protein's fluorescence and structural properties. For this protein, the angle of the chromophore with respect to the protein's geometrical axis was determined in good agreement with X-ray crystallographic data. Permanent immobilization with simultaneous alignment of the proteins was achieved along the edges, tips and on the surface of interdigitated electrodes. This was the first demonstration of aligned immobilization of proteins by electric fields.
Thus, the presented electric field-assisted immobilization method is promising with regard to enhanced antibody binding capacities and enzymatic activities, which is a requirement for industrial biosensor production, as well as for general interaction studies of proteins.
Savannas cover a broad geographical range across continents and are a biome best described by a mix of herbaceous and woody plants. The former create a more or less continuous layer while the latter should be sparse enough to leave an open canopy. What has long intrigued ecologists is how these two competing plant life forms of vegetation coexist.
Initially attributed to resource competition, coexistence was considered the stable outcome of a root niche differentiation between trees and grasses. The importance of environmental factors became evident later, when data from moister environments demonstrated that tree cover was often lower than what the rainfall conditions would allow for. Our current understanding relies on the interaction of competition and disturbances in space and time. Hence, the influence of grazing and fire and the corresponding feedbacks they generate have been keenly investigated. Grazing removes grass cover, initiating a self-reinforcing process propagating tree cover expansion. This is known as the encroachment phenomenon. Fire, on the other hand, imposes a bottleneck on the tree population by halting the recruitment of young trees into adulthood. Since grasses fuel fires, a feedback linking grazing, grass cover, fire, and tree cover is created. In African savannas, which are the focus of this dissertation, these feedbacks play a major role in the dynamics.
The importance of these feedbacks came into sharp focus when the notion of alternative states began to be applied to savannas. Alternative states in ecology arise when different states of an ecosystem can occur under the same conditions. According to this an open savanna and a tree-dominated savanna can be classified as alternative states, since they can both occur under the same climatic conditions. The aforementioned feedbacks are critical in the creation of alternative states. The grass-fire feedback can preserve an open canopy as long as fire intensity and frequency remain above a certain threshold. Conversely, crossing a grazing threshold can force an open savanna to shift to a tree-dominated state. Critically, transitions between such alternative states can produce hysteresis, where a return to pre-transition conditions will not suffice to restore the ecosystem to its original state.
In the chapters that follow, I will cover aspects relating to the coexistence mechanisms and the role of feedbacks in tree-grass interactions. Coming back to the coexistence question, due to the overwhelming focus on competition and disturbance another important ecological process was neglected: facilitation. Therefore, in the first study within this dissertation I examine how facilitation can expand the tree-grass coexistence range into drier conditions. For the second study I focus on another aspect of savanna dynamics which remains underrepresented in the literature: the impacts of inter-annual rainfall variability upon savanna trees and the resilience of the savanna state. In the third and final study within this dissertation I approach the well-researched encroachment phenomenon from a new perspective: I search for an early warning indicator of the process to be used as a prevention tool for savanna conservation. In order to perform all this work I developed a mathematical ecohydrological model of Ordinary Differential Equations (ODEs) with three variables: soil moisture content, grass cover and tree cover.
Facilitation: Results showed that the removal of grass cover through grazing was detrimental to trees under arid conditions, contrary to expectation based on resource competition. The reason was that grasses preserved moisture in the soil through infiltration and shading, thus ameliorating the harsh conditions for trees in accordance with the Stress Gradient Hypothesis. The exclusion of grasses from the model further demonstrated this: tree cover was lower in the absence of grasses, indicating that the benefits of grass facilitation outweighed the costs of grass competition for trees. Thus, facilitation expanded the climatic range where savannas persisted into drier conditions.
Rainfall variability: By adjusting the model to current rainfall patterns in East Africa, I simulated conditions of increasing inter-annual rainfall variability for two distinct mean rainfall scenarios: semi-arid and mesic. Alternative states of tree-less grassland and tree-dominated savanna emerged in both cases. Increasing variability reduced semi-arid savanna tree cover to the point that at high variability the savanna state was eliminated, because variability intensified resource competition and strengthened the fire disturbance during high rainfall years. Mesic savannas, on the other hand, became more resilient along the variability gradient: increasing rainfall variability created more opportunities for the rapid growth of trees to overcome the fire disturbance, boosting the chances of savannas persisting and thus increasing mesic savanna resilience.
Preventing encroachment: The breakdown in the grass-fire feedback caused by heavy grazing promoted the expansion of woody cover. This could be irreversible due to the presence of alternative states of encroached and open savanna, which I found along a simulated grazing gradient. When I simulated different short term heavy grazing treatments followed by a reduction to the original grazing conditions, certain cases converged to the encroached state. Utilising woody cover changes only during the heavy grazing treatment, I developed an early warning indicator which identified these cases with a high risk of such hysteresis and successfully distinguished them from those with a low risk. Furthermore, after validating the indicator on encroachment data, I demonstrated that it appeared early enough for encroachment to be prevented through realistic grazing-reduction treatments.
Though this dissertation is rooted in the theory of savanna dynamics, its results can have significant applications in savanna conservation. Facilitation has only recently become a topic of interest within savanna literature. Given the threat of increasing droughts and a general anticipation of drier conditions in parts of Africa, insights stemming from this research may provide clues for preserving arid savannas. The impacts of rainfall variability on savannas have not yet been thoroughly studied, either. Conflicting results appear as a result of the lack of a robust theoretical understanding of plant interactions under variable conditions. . My work and other recent studies argue that such conditions may increase the importance of fast resource acquisition creating a ‘temporal niche’. Woody encroachment has been extensively studied as phenomenon, though not from the perspective of its early identification and prevention. The development of an encroachment forecasting tool, as the one presented in this work, could protect both the savanna biome and societies dependent upon it for (economic) survival. All studies which follow are bound by the attempt to broaden the horizons of savanna-related research in order to deal with extreme conditions and phenomena; be it through the enhancement of the coexistence debate or the study of an imminent external threat or the development of a management-oriented tool for the conservation of savannas.
The human immunodeficiency virus (HIV) has resisted nearly three decades of efforts targeting a cure. Sustained suppression of the virus has remained a challenge, mainly due
to the remarkable evolutionary adaptation that the virus exhibits by the accumulation of drug-resistant mutations in its genome. Current therapeutic strategies aim at achieving and maintaining a low viral burden and typically involve multiple drugs. The choice of optimal combinations of these drugs is crucial, particularly in the background of treatment failure having occurred previously with certain other drugs. An understanding of the dynamics of viral mutant genotypes aids in the assessment of treatment failure with a certain drug
combination, and exploring potential salvage treatment regimens.
Mathematical models of viral dynamics have proved invaluable in understanding the viral life cycle and the impact of antiretroviral drugs. However, such models typically use simplified and coarse-grained mutation schemes, that curbs the extent of their application to drug-specific clinical mutation data, in order to assess potential next-line therapies. Statistical
models of mutation accumulation have served well in dissecting mechanisms of resistance evolution by reconstructing mutation pathways under different drug-environments. While these models perform well in predicting treatment outcomes by statistical learning, they do not incorporate drug effect mechanistically. Additionally, due to an inherent lack of
temporal features in such models, they are less informative on aspects such as predicting mutational abundance at treatment failure. This limits their application in analyzing the
pharmacology of antiretroviral drugs, in particular, time-dependent characteristics of HIV therapy such as pharmacokinetics and pharmacodynamics, and also in understanding the impact of drug efficacy on mutation dynamics.
In this thesis, we develop an integrated model of in vivo viral dynamics incorporating drug-specific mutation schemes learned from clinical data. Our combined modelling
approach enables us to study the dynamics of different mutant genotypes and assess mutational abundance at virological failure. As an application of our model, we estimate in vivo
fitness characteristics of viral mutants under different drug environments. Our approach also extends naturally to multiple-drug therapies. Further, we demonstrate the versatility of our model by showing how it can be modified to incorporate recently elucidated mechanisms of drug action including molecules that target host factors.
Additionally, we address another important aspect in the clinical management of HIV disease, namely drug pharmacokinetics. It is clear that time-dependent changes in in vivo
drug concentration could have an impact on the antiviral effect, and also influence decisions on dosing intervals. We present a framework that provides an integrated understanding
of key characteristics of multiple-dosing regimens including drug accumulation ratios and half-lifes, and then explore the impact of drug pharmacokinetics on viral suppression.
Finally, parameter identifiability in such nonlinear models of viral dynamics is always a concern, and we investigate techniques that alleviate this issue in our setting.