The cell is the central functional unit of life. Cell behaviors, such as cell division, movements, differentiation, cell death as well as cell shape and size changes, determine how tissues change shape and grow during regeneration and development. However, a generally applicable framework to measure and describe the behavior of the multitude of cells in a developing tissue is still lacking. Furthermore, the specific contribution of individual cell behaviors, and how exactly these cell behaviors collectively lead to the morphogenesis and growth of tissues are not clear for many developmental and regenerative processes. A promising strategy to fill these gaps is the continuing effort of making developmental biology a quantitative science. Recent advances in methods, especially in imaging, enable measurements of cell behaviors and tissue shapes in unprecedented detail and accuracy. Consequently, formalizing hypotheses in terms of mathematical models to obtain testable quantitative predictions is emerging as a powerful tool. Tests of the hypotheses involve the comparison of model predictions to experimentally observed data. The available data is often noisy and based on only few samples. Hence, this comparison of data and model predictions often requires very careful use of statistical inference methods. If one chooses this quantitative approach, the challenges are the choice of observables, i.e. what to measure, and the design of appropriate data-driven models to answer relevant questions. In this thesis, I applied this data-driven modeling approach to vertebrate morphogenesis, growth and regeneration. In particular, I study spinal cord and muscle regeneration in axolotl, muscle development in zebrafish, and neuron development and maintenance in the adult human brain. To do so, I analyzed images to quantify cell behaviors and tissue shapes. Especially for cell behaviors in post-embryonic tissues, measurements of some cell behavior parameters, such as the proliferation rate, could not be made directly. Hence, I developed mathematical models that are specifically designed to infer these parameters from indirect experimental data. To understand how cell behaviors shape tissues, I developed mechanistic models that causally connect the cell and tissue scales. Specifically, I first investigated the behaviors of neural stem cells that underlie the regenerative outgrowth of the spinal cord after tail amputation in the axolotl. To do so, I quantified all relevant cell behaviors. A detailed analysis of the proliferation pattern in space and time revealed that the cell cycle is accelerated between 3-4 days after amputation in a high-proliferation zone, initially spanning from 800 µm anterior to the amputation plane. The activation of quiescent stem cells and cell movements into the high-proliferation zone also contribute to spinal cord growth but I did not find contributions by cellular rearrangements or cell shape changes. I developed a mathematical model of spinal cord outgrowth involving all contributing cell behaviors which revealed that the acceleration of the cell cycle is the major driver of spinal cord outgrowth. To compare the behavior of neural stem cells with cell behaviors in the regenerating muscle tissue that surrounds the spinal cord, I also quantified proliferation of mesenchymal progenitor cells and found similar proliferation parameters. I showed that the zone of mesenchymal progenitors that gives rise to the regenerating muscle segments is at least 350 µm long, which is consistent with the length of the high-proliferation zone in the spinal cord. Second, I investigated shape changes in developing zebrafish muscle segments by quantifying time-lapse movies of developing zebrafish embryos. These data challenged or ruled out a number of previously proposed mechanisms. Motivated by reported cellular behaviors happening simultaneously in the anterior segments, I had previously proposed the existence of a simple tension-and-resistance mechanism that shapes the muscle segments. Here, I could verify the predictions of this mechanism for the final segment shape pattern. My results support the notion that a simple physical mechanism suffices to self-organize the observed spatiotemporal pattern in the muscle segments. Third, I corroborated and refined previous estimates of neuronal cell turnover rates in the adult human hippocampus. Previous work approached this question by combining quantitative data and mathematical modeling of the incorporation of the carbon isotope C-14. I reanalyzed published data using the published deterministic neuron turnover model but I extended the model by a better justified measurement error model. Most importantly, I found that human adult neurogenesis might occur at an even higher rate than currently believed. The tools I used throughout were (1) the careful quantification of the involved processes, mainly by image analysis, and (2) the derivation and application of mathematical models designed to integrate the data through (3) statistical inference. Mathematical models were used for different purposes such as estimating unknown parameters from indirect experiments, summarizing datasets with a few meaningful parameters, formalizing mechanistic hypotheses, as well as for model-guided experimental planning. I venture an outlook on how additional open questions regarding cell turnover measurements could be answered using my approach. Finally, I conclude that the mechanistic understanding of development and regeneration can be advanced by comparing quantitative data to the predictions of specifically designed mathematical models by means of statistical inference methods.

Fossil plants are regard to be excellent proxies to trace paleoclimatic and paleoatmospheric changes. The vegetational response to changing paleoclimate and paleoatmospheric conditions has already been known for a long time and is well documented for the Paleogene of central Europe. Methods such as the Coexistence Approach (CA) and the Climate Leaf Analyses Multivariate Program (CLAMP) analyze the composition of fossil plant assemblages. Changes in paleoclimate and CO2 through time can be tracked also via changes in morphometric parameters such as leaf area, leaf size and leaf shape or epidermal (cuticular) parameters as for instance stomata density (SD), stomata index (SI) and stomata size. The multivariate gas exchange model combines morphometric and cuticular parameters, together with assumed paleoclimate conditions and physiological data of nearest living equivalents to determine paleo-CO2. Plants show differences in morphological, morphometric and cuticular parameters, not only in response to overall changes in CO2 and climate, but also due to their immobility and dependency on light intensity, water availability and soil conditions at the respective site. In this study leaf traits of both Rhodomyrtophyllum reticulosum and Platanus neptuni from 23 sites in Germany, Austria and the Czech Republic covering a time span from the late early Eocene to the early Miocene of central Europe are investigated. Alongside the stratigraphic range of the data set, which allows for tracing long-term variations in the respective parameters, sites of different depositional facies types (maar deposits, marine deposits and fluvial-lacustrine deposits) were included. It has been proven that the investigation of single species and their correspondence to global and regional paleoclimatic and paleoatmospheric shifts has to be done considering differences in the respective depositional setting and thus habitat. Regional effects influence the peculiarity of leaf traits greatly which implies that regional and site related patterns partly overweigh global correspondences. The weak correlation of leaf trait changes to global changes in paleoclimate and CO2 implies that the long-lived species Rhodomyrtophyllum reticulosum and Platanus neptuni are not suitable to track these changes due their high plasticity and adaptability. The long stratigraphic range of the investigated species therefore point out the high adaption potential which by implication leads to a lower correspondence to global paleoclimatic changes. The determination of crucial leaf traits and their response to overall changes in paleoclimate and CO2 hampers the fact that the fossil record bears mainly elements present in azonal vegetation which is caused by predominantly burial of fossils in aquatic bodies. Hence, long-lived species could have been survived these remarkable changes in climate from the end of the Early Eocene Climatic Optimum to the Oligocene icehouse world due to their occurrence in azonal assemblages, buffering global effects in climate variability to a certain degree. The investigation of long-lived fossil species therefore has to be done by coincident consideration of the composition of the whole plant assemblage, which reflects both azonal and partly zonal vegetation of the respective time interval.

Kinesin-8 motors regulate the lengths of microtubules in cells. In previous studies, these motors have been shown to utilize their highly processive plus-end directed motility to reach microtubule plus-ends where they act as a microtubule depolymerase. The superprocessive motility importantly allows Kip3 motors to depolymerize microtubules in a length-dependent manner, the underlying mechanism of which has been described by an antenna model. During such long runs, motors in vivo are expected to frequently encounter roadblocks, such as microtubule associated proteins. The adaptions in the stepping mechanism that allow kinesin-8 motors to navigate around roadblocks to reach microtubule ends is not well understood. In this work, in vitro techniques were utilized to understand the navigation strategy of yeast kinesin-8, Kip3. Three-dimensional stepping motility of Kip3 on the surface of microtubules can be inferred (i) indirectly from rotational motion of microtubules gliding along a surface coated with Kip3 and (ii) directly by three-dimensional tracking of Kip3 on freely suspended microtubules. Firstly, an impact-free method to detect rotations of gliding microtubules was established based on fluorescent speckles within the microtubule structure in combination with fluorescent interference contrast microscopy. Secondly, a suspended microtubule assay was established to obtain three- dimensional trajectories of single Kip3 motors, using Parallax, a dual-focus imaging technique. The motility assays performed in this work revealed that Kip3 motors undergo left-handed helical motion around the microtubule lattice. This indicates that Kip3 employs a directed sidestepping strategy which is attributed to the motor having a flexible neck and/or a long neck linker. Interestingly, further analysis of the rotational motion revealed that the sidestepping of Kip3 is not directly coupled to the forward stepping. Based on these observations, it is hypothesized that the motor can transition from a two-head-bound conformation to a one-head-bound conformation while waiting for ATP. Whereas the motor can step forward from both states, sidestepping is strongly favored from the one-head-bound conformation. This hypothesis was confirmed through experiments as well as numerical simulations where the transition from the two-head-bound conformation to the one-head-bound conformation was enhanced by either prolonging the ATP waiting time or increasing the transition rate (by reducing the motor-microtubule interaction). Finally, Kip3 based motility assays were performed using microtubules decorated with rigor binding kinesin-1 motors acting as roadblocks. While gliding assays using roadblock-decorated microtubules indicated a left-biased sidestepping strategy for Kip3, stepping assays revealed an additional diffusive component in the stepping motility of Kip3, along with the leftward bias. Taken together, it is hypothesized that Kip3 has a dual-mode roadblock circumnavigation strategy. Upon encountering a roadblock, the motor circumnavigates it (i) by shifting to the adjacent left microtubule protofilament using the biased sidestepping mechanism or (ii) by shifting microtubule protofilaments in an unbiased diffusive manner upon switching out of the step cycle. Therefore, the biophysical properties of Kip3 are fine-tuned to ensure that the motor reaches the microtubule plus-end to perform its depolymerase activity.

Cells grow in environments that can change suddenly. To cope with unpredictable perturbations, they have evolved mechanisms to adjust their metabolism according to the various types of environmental stress. Cells experiencing starvation, for example, have low energy levels and are forced to lower their metabolism and enter a protective quiescent state to survive until nutrients become available again. Recently, it has been shown that starved yeast cells experience a marked acidification of the cytoplasm, due to a passive influx of protons. This pH drop causes multiple rearrangements in the cytoplasm: increased crowding, reduced mobility of intracellular components and formation of stress-induced non-membrane bound compartments of specific metabolic enzymes. Cytoplasm rearrangements are required for cell survival and can be reversed upon replenishment of energy. However, there is little understanding of how cytoplasmic components reorganize in stressed quiescent cells. Using high-pressure freezing, correlative light and electron microscopy (CLEM) and electron tomography, coupled to high-resolution 3D-reconstruction techniques, I investigate the structural modi cations that happen in situ in yeast cells undergoing quiescence. I observe that the cytoplasm becomes increasingly crowded, due to a massive rearrangement of membranous structures, including accumulation of intracellular vesicles and pronounced invaginations in the plasma membrane. This is proved by quantification of the difference in ribosome densities between stressed and not stressed cells. The increased crowding, coupled to cytoplasm acidification, leads to the formation of non-membrane bound enzyme compartments, that appear as foci and elongated structures of fluorescently tagged enzymes. I prove that the fluorescent structures correspond to bundles of filaments. Among many essential enzymes, known to form mesoscale structure in stressed yeast, I demonstrate that the eukaryotic translation initiation factor 2B (eIF2B) forms bundles of filaments in situ, and the evolutionary conserved glutamine synthetase (Gln1) self-assembles into filaments in vitro. The present study on the energy depleted cytoplasm and the structural analysis of filament-forming enzymes provides insights into an unexplored survival strategy that is used by yeast, as well as other organisms, to cope with extreme environmental conditions and stress.:1 Introduction 1 Stress, survival and quiescence 2 1.1 Cytoplasm and cellular compartments 2 1.2 Membraneless compartmentalization in the cell 3 1.3 Stress-induced non-membrane bound assemblies 4 The quiescent sleeping yeast 6 1.4 The yeast S.cerevisiae as model organism 7 1.5 Growth and metabolism of yeast 9 1.5.1 Yeast eukaryotic translation initiation factor 2B: eIF2B 11 1.5.2 Yeast glutamine synthetase: Gln1 12 3D electron microscopy 14 Aims of the Thesis 18 2 Materials and methods 21 Room temperature electron microscopy (EM) 21 2.1 Yeast strains, media and energy depletion 21 2.2 High-pressure freezing of yeast cells 22 2.2.1 EM sample preparation for untagged eIF2B yeast strains 22 2.2.2 EM sample preparation for GFP-tagged eIF2B yeast strains 23 2.3 Electron tomography 23 2.4 Subtomogram averaging 24 2.5 Fiji script for automated ribosome counting 25 2.6 Immunofluorescence of eIF2B in yeast 26 2.7 Western-blot on yeast ribosomes 27 Single particle procedures 30 2.8 Protein purification protocols 30 2.8.1 Baculovirus-insect cell expression and purification of eIF2B 30 2.8.2 Gradient of fixation for fragile complexes 31 2.8.3 Yeast expression and purification of Gln1 33 2.9 Negative staining 34 2.9.1 Image acquisition and analysis—eIF2B 35 2.9.2 Image acquisition and analysis—Gln1 36 Cryo-electronmicroscopy(cryo-EM) 37 2.10 Plunge freezing 37 2.11 Image acquisition and 3D reconstruction 37 3 Results Visualizing yeast’s cytoplasmic reorganization 39 3.1 Quiescence is accompanied by reorganization of the cytoplasm 40 3.2 Ribosome density proves cytoplasmic crowding in starved cells 42 3.3 eIF2B organizes in bundles of filaments in energy-depleted cells 45 3.4 eIF2B filaments are polymers of the eIF2B complex 47 3.5 Filaments are found in wild-type energy-depleted cells 49 Structural analysis of filament forming enzymes 51 3.6 Purification of eIF2B complexes 51 3.7 Single particle analysis of eIF2B 53 3.8 Purification of Gln1 complexes 55 3.9 Single particle analysis of Gln1 56 3.10 Gln1 forms filaments in vitro 58 4 Discussion and Outlook 59 4.1 Yeast cytoplasm reorganizes in response of stress 59 4.2 Ribosomes density is a measure of increased macromolecular crowding 60 4.3 eIF2B forms filaments as a survival strategy 62 4.4 Molecular analysis of filament forming enzymes 64 4.5 Outlook 65 Appendix 67 Bibliography 83

Embryonic development entails a series of morphogenetic events which require a precise coordination of molecular mechanisms coupled with cellular dynamics. Phyla such as arthropods show morphological and gene expression similarities during middle embryogenesis (at the phylotypic germband stage), yet early embryogenesis adopts diverse developmental strategies. In an effort towards understanding patterns of conservation and divergence during development, investigations are required beyond the traditional model systems. Therefore, in the past three decades, several insect species representing various insect orders have been established as experimental model systems for comparative developmental studies. Among these, the red flour beetle Tribolium castaneum has emerged as the best studied holometabolous insect model after the fruit fly Drosophila melanogaster. Unlike Drosophila, Tribolium is a short-germ insect that retains many ancestral characters common to most insects. The early embryogenesis of Tribolium shows dynamic epithelial rearrangements with an epibolic expansion of the extraembryonic tissue serosa over the embryo, the folding of the embryo in between the serosa and the second extra embryonic tissue amnion and the folding of the amnion underneath the embryo. These extensive tissues are evolutionarily conserved epithelia that undergo different tissue movements and are present in varying proportions in different insects, providing exceptional material to compare and contrast morphogenesis during early embryogenesis. However, most of the previous work on insects including Tribolium have largely focused on the conservation and divergence of gene expression patterns and on gene regulatory interactions. Consequently, very little studies on dynamic cell behaviour have been done and we lack detailed information about the cellular and tissue dynamics during these early morphogenetic events. During my PhD, I first established a live imaging and data analysis pipeline for studying Tribolium embryogenesis in 4-D. I combined live confocal and lightsheet imaging of transgenic or transiently labelled embryos with mechanical or genetic perturbations using laser ablations and gene knockdowns. Using this pipeline quantifications of cell dynamics and tissue behaviours can be done to compare different regions of the embryo as the development proceeds. In the second and third part of my thesis, I describe the actomyosin dynamics and associated cell behaviours during the stages of serosa epibolic expansion, amniotic fold formation and serosa window closure. I cloned and characterised the cellular dynamics of the Tribolium spaghetti squash gene (Tc-squash) - the non-muscle Myosin II regulatory light chain, which is the main molecular force generator in epithelial cells. Interestingly, the analysis of Tc-squash dynamics indicates a conserved role of Myosin II in controlling similar cell behaviours across short germ and long germ embryos. In the last part of the thesis, I report the dynamics of an actomyosin cable that emerges at the interface of the serosa and amnion. This cable increases in tension during development, concomitant with serosa tissue expansion and increased tensions in the serosa. It behaves as a modified purse string as it’s circumference shrinks due to a decrease in the number of cable forming cells over time. This shrinkage is an individual contractile property of the cells forming the cable. This indicates that a supracellular and contractile actomyosin cable might be functional during serosa window closure in insects with distinct serosa and amnion tissues. Further, the tension in the cable might depend on the relative proportion of the serosa, amnion and embryonic regions. Using these integrated approaches, I have correlated global cellular dynamics during early embryogenesis with actomyosin behaviours, and then performed a high-resolution analysis and perturbations of selected events. The established imaging, image processing and perturbation tools can serve as an important basis for future investigations into the tissue mechanics underlying Tribolium embryogenesis and can also be adapted for comparisons of morphogenesis in other insect embryos. More broadly, correlating the existing genetic, mechanical and biochemical understanding of developmental processes from Drosophila with species such as Tribolium, could help identify deeply conserved design principles that lead to different morphologies through differences in underlying regulation.:Page List of Tables v List of Figures vii 1 Introduction 1 1.1 Evo-Devo of insects 3 1.2 Tribolium castaneum 5 1.3 Fluorescence live imaging and lightsheet microscopy 10 1.4 Morphogenesis 15 1.5 Thesis objective 29 2 4D lightsheet imaging and analysis pipeline of Tribolium embryos 33 2.1 Standardisation of an injection protocol for sample mounting and imaging with the Zeiss LZ1 SPIM 35 2.2 Double labelling of Tribolium embryos 37 2.3 Image processing with Fiji 37 2.4 Long term timelapse imaging of Tribolium embryogenesis with SPIM 44 2.5 2D cartographic projections of 3D data as a method to visualise and analyse SPIM data 47 2.6 Summary 59 3 Cellular dynamics of the non muscle Myosin II regulatory light chain - Tc-Squash 61 3.1 Tc-Squash dynamics during Tribolium embryogenesis 64 3.2 Myosin drives basal cell closure during blastoderm cellularisation 66 3.3 Myosin shows planar polarity in the embryonic tissue 69 3.4 Myosin accumulation and apical constriction of putative germ cells at the posterior pole 71 3.5 Myosin pulses during apical constriction of mesoderm cells 74 3.6 Myosin accumulates at the extraembryonic-embryonic boundary to form a contractile supracellular cable 77 3.7 Summary 77 4 A supracellular actomyosin cable operates during serosa epiboly 79 4.1 Actin and Myosin accumulate at the extraembryonic-embryonic boundary 81 4.2 The actomyosin assembly migrates ventrally till it forms the rim of the serosa window 82 4.3 The actomyosin cable shows dynamic shape changes during serosa window closure 87 4.4 Serosa cells increase in area till circular serosa window stage 89 4.5 Tension in the serosa tissue increases during epibolic expansion 89 4.6 Serosa cells decrease their apical areas after laser ablation 92 4.7 Tension in the actomyosin cable increases during serosa epiboly 93 4.8 Myosin dynamics at the cable changes between early and serosa window stage 96 4.9 Individual cell membrane shrinkage and cell rearrangements decrease the cable circumference 98 4.10 Myosin dynamics at the cable during serosa window closure 101 4.11 Tension in the cable is not relieved after multiple laser cuts 103 4.12 Analysis of the actomyosin cable in Tc-zen 1 knockdown 105 4.13 Summary 109 5 Discussion 111 5.1 Reconstruction of insect embryogenesis using lightsheet microscopy and tissue cartography 111 5.2 Conserved Myosin II behaviours and its implications on morphogenesis across insects 114 5.3 A contractile supracellular actomyosin cable functions serosa window closure in Tribolium 119 6 Materials and Methods 123 6.1 Tribolium stock maintenance 123 6.2 RNA extraction and cDNA synthesis 124 6.3 Cloning of templates for mRNA synthesis and transgenesis 124 6.4 dsRNA synthesis for RNAi experiments 126 6.5 Capped, single stranded RNA synthesis 126 6.6 Fluorescence image acquisition 27 A Appendix 131 Bibliography 143

The disposal of large amounts of waste still containing energetic value is a central challenge in the waste management of the Brazilian sugarcane industry. As a sustainable solution, the biogas process appears to be a suitable technology for treating sugarcane waste products and for providing valuable commodities such as energy-rich biogas and digestate with fertilizer properties. Additionally, the proper treatment of the four major waste types (straw, bagasse, filter cake and vinasse) would avoid greenhouse gas emissions, air pollution and environmental contamination of soil and water. In order to investigate the feasibility and reliability of biogas production from sugarcane wastes, the microbial community dynamics of laboratory-scale reactors were assessed under different start-up strategies. Despite the promising results of the methane potential for all the waste products, chemical and physical pre-treatments were applied successfully to increase the methane yield of straw, bagasse and filter cake. The microbial community dynamics observed during co-digestion of filter cake and bagasse showed, together with the process parameters, that cattle manure can be effectively used as an inoculum for the start-up of a biogas process in the remote-located sugarcane industry. Monitoring methanogenic community dynamics at high organic loading rate of filter cake and bagasse demonstrated that the genera Methanosarcina and Methanobacterium are the major methanogens that produce biogas, even under process imbalances. Moreover, the results obtained from the process parameters and methanogenic community analyses revealed that the stable isotope fingerprinting technique may be a potential monitoring tool for quickly identifying changes in the methanogenic pathway, which indicates process disturbances. In conclusion, these studies established techniques for the efficient substrate processing and start-up procedure of a biogas process designed for the anaerobic digestion of sugarcane wastes, and by these means provided a highly detailed profile of the microbial community in relation to process parameters.

Eine Vielzahl von Krankheiten und Phänotypen im Menschen werden durch Mutationen in G-Protein-gekoppelten Rezeptoren (GPCR) verursacht. Die Klasse der Adhäsions-GPCR (aGPCR) ist bisher wenig erforscht, wird aber mit diversen Funktionen in der Immunität, Nervenentwicklung, Embryonalentwicklung und im Tumorwachstum in Zusammenhang gebracht. Ein Vertreter der Gruppe V der aGPCR ist der GPR133 (ADGRD1). Einige genomweite Assoziationsstudien konnten den gpr133-Lokus mit Veränderungen im Metabolismus, in der Körpergröße und der Herzfrequenz verknüpfen. Für diesen aGPCR wurde bereits die Signaltransduktion über Gαs und Gαi gezeigt, sodass eine funktionelle Charakterisierung des GPR133 und dessen Varianten über cAMP-, cAMP response element- (CRE) und CRE binding protein- (CREB) Assays in vitro möglich ist. Systematische Untersuchungen der Struktur des GPR133 konnten die gebundene agonistische „Stachelsequenz“ aufzeigen. Dies legt den Grundstein für die funktionelle Untersuchung von Mutationen im GPR133. Eine Analyse von mehr als 1000 sequenzierten humanen Genomen ergab über 9000 Einzelnukleotidpolymorphismen (SNP) im gpr133-Gen. Ungefähr 2,4 % der SNP liegen in kodierenden Genabschnitten und resultieren in 129 nicht-synonymen SNP (nsSNP) an 119 Aminosäurepositionen. Die funktionelle Relevanz dieser Missense-Varianten war unbekannt. Tiefergehende Analysen konnten nsSNP identifizieren, die zu einem vollständigen bzw. partiellen Funktionsverlust (A448D, Q600stop, C632fs [frame shift], A761E, N795K) oder zu einer erhöhten Basalaktivität (F383S, D453N) führen. Ein Vergleich der aGPCR Subklassen basierend auf diversen Orthologsequenzen konnte zudem stark konservierte Bereiche aufzeigen, deren Änderungen durch nsSNP im GPR133 in Funktionsänderungen münden. Das große im Menschen vorhandene funktionelle Spektrum von GPR133-Varianten könnte für klinisch relevante Phänotypen verantwortlich sein, auch wenn die bisher erfassten heterozygoten Individuen lebensfähig sind.

Biosensoren stellen technische Geräte dar, die im Zentrum ihres Wirkmechanismus ein Biomolekül, also ein Molekül mit einer biologischen Funktion, aufweisen. Eine Vielzahl von Biosensoren findet in der Wissenschaft bereits Verwendung. Viele dieser Sensoren werden an planen Grenzflächen mit hoher Steifigkeit angewandt, welche weit von der in vivo Situation entfernt sind. Hierdurch ist unklar, ob diese Vereinfachung von natürlichen Prozessen inkorrekte oder in der biologischen Funktionalität veränderte Ergebnisse liefert. Aus diesem Grund soll mit dieser Arbeit die Vielseitigkeit eines neuen Biosensorsystems aufgezeigt werden, mit dem physikalische Phänomene von biologischen Systemen mimetisch nachgestellt und untersucht werden können. Der auf Hydrogelmikropartikeln basierende Biosensor bewies hierbei seine Verwendbarkeit für die Quantifizierung von unspezifischen Adhäsionsvorgängen an Materialgrenzflächen, wie auch für spezifische Rezeptor-Ligand-Wechselwirkungen von lebenden Zellen. Weiterhin konnte mit zwei verschiedenen Nachweismethoden, einer auf Reflektionskontrastmikroskopie basierenden und einer kraftspektroskopischen Methode, die breitgefächerte Anwendung der Hydrogelmikropartikel für biophysikalische Fragestellungen aufgezeigt werden. Die multivalenten und biomimetischen Eigenschaften der Mikropartikel erlauben hierbei ein quantitatives Verständnis von Wechselwirkungsvorgängen von Biomolekülen mit Materialgrenzflächen und spezifischen Rezeptorstrukturen, wobei das Biosensorsystem selbst einfach und leicht zu kontrollieren bleibt.

Individuals endowed with a ‘Theory of Mind’ (‘ToM’) understand themselves and others as agents whose actions are driven by unobservabl e psychological states. How and when human infants come to such an understa nding has been extensively resear ched in the visual domain. In my dissertation, I addressed three gaps in the extant literature about what great apes’ know about others' visual perceptions and perceptual beliefs. In study 1, I investigated orangutans’ understanding of visual attenti on and others’ visual perspectives in a competitive situation. Overall, the results suggest that orangutans have a limited understanding of others’ perspectives, relying mainly on cues from facial and bodily orientation and egocentric ruleswhen making perspective judgements. In study 2, I explored whether apes and 20 month old human infants requesting a desired object from a human experimenter would use communicative means to direct visual attention towards the object. While infants used pointing to alter the experimenter’s focus of attention, we found no evidence that apes’ employ their point gestures in this way. In study 3, I examined chimpanzees’ and 5.5 year old human children’s understanding of perceptual beliefs. By designing two novel false belief tasks which required reduced executive functioning, I attempted to find out whether chimpanzees’ historical failure in explicit false belief tasks was due to their lack of inhibitory control Neither the chimpanzees nor the 5.5 year-olds succeeded in the novel tasks.

INTRODUCTION Increasing age of population is a great success of numerous breakthroughs in life science and improved health care. For a child born in 2015, for example, an average global life expectancy of meanwhile 71.4 years is assumed which increased by around 8% in the last decade [1]. In accordance with enhanced life expectancy, however, age-related health problems continuously rise. In this regard, the gap between patients awaiting transplantation and appropriate donors consequently will get larger in the future [2]. To this end, there is a need for new strategies in regenerative medicine [3]. Biomaterial matrices were developed to foster tissue regeneration by mimicking the key characteristics of the extracellular matrix (ECM) [4]. Modern biomaterial research focuses on 3D scaffolds, which can be adequately adapted toward specific requirements of the target tissue [5]. In this regard, flexible material platforms are wanted, whose properties can be adjusted over a wide range and independently of each other [6]. In this context, the macromer-based material concept is promising due to the high flexibility of macromers in chemical design and processability [7]. Macromers are reactive oligo- or polymeric molecules which act as monomers and can therefore be polymerized/cross-linked into a polymeric network [8]. The key principle of this approach is the synthesis of chemically well-defined structures which allows for a more precise control over the resulting properties of the cross-linked polymeric network when compared to conventional polymers. For example, macromer chemistry can be adjusted in terms of chemical macromer composition, valence, content of cross-linkable functionalities and molecular weight. The versatility of macromer-derived materials greatly increases when different macromer types are combined which potentially enables precise material tunability on multiple levels. The design flexibility of macromer-based networks motivated the investigation of two different macromer-based material concepts with regard to macromer processability and material adjustability. The following objectives were proposed: 1) To synthesize two sets of biodegradable, multi-valent macromers by using free-radical polymerization and ring-opening polymerization combined with established activation strategies. The synthesis setups will be tuned toward high macromer yields which will be required for processing into biomaterials with relevant sizes. 2) To physico-chemically characterize oligomeric macromers with regard to chemical composition, molecular weight and reactivity in order to yield well-defined macromer structures. NMR spectroscopy, gel permeation chromatography (GPC) and wet chemistry will be applied. 3) To characterize macromer processability into covalently cross-linked hybrid matrices. This work will focus on a soft macromer-cross-linked gelatin-derived hydrogel system for versatile biomedical applications as well as a rigid macromer/sol-gel glass hybrid material for hard tissue regeneration. Sets of different formulations will be investigated in order to characterize the range of macromer processability and to establish structure-property relationships. 4) To investigate strategies for the adjustment of material porosity. Besides the adaption via cross-linking density, porogen-leaching and 3D-printing approaches will be followed in order to introduce macroporosity and to enable a decoupling of porosity and chemical (nano)structure of the cross-linked network. 5) To determine key material properties relevant for regenerative applications, including mechanical properties by compression tests and oscillation rheology, in vitro matrix degradability, as well as material cytocompatibility in indirect and direct contact experiments. 6) To identify strategies for covalent functionalization of the hybrid materials. Post-fabrication functionalization via specifically introduced chemical functionalities is favored as it enables effective material decoration (almost) independent of the physico-chemical matrix properties. SUMMARY OF DISSERTATION The first material concept was based on anhydride-containing macromers which can be processed into hydrogel matrices by covalent cross-linking of amine-bearing macromolecules, such as gelatin [9–11]. The innovative aspect of this work was to decouple material functionalization from the physico-chemical properties of the cross-linked hydrogel network. To this end, a second chemical functionality was introduced which remained reactive in the hydrogel state and was therefore available for covalent post-fabrication functionalization strategies. Specifically, dual-functional macromers were synthesized by free-radical polymerization of maleic anhydride (MA) with diacetone acrylamide (DAAm) and pentaerythritol diacrylate monostearate (PEDAS) to yield oligo(PEDAS-co-DAAm-co-MA) (oPDMA) [12]. Amphiphilic oligomers (molecular weight (Mn) < 7.5 kDa) with anhydride contents of 7-20% were obtained. Fractions of chemically intact anhydrides of around 70% enables effective cross-linking with low molecular-weight gelatinous peptides (Collagel® type B, 11 kDa). Rigid two-component hydrogels (elastic modulus (E) = 4-13 kPa) with adjustable composition and physicochemical properties were formed. Reactivity of the incorporated methyl ketone functionality toward hydrazides and hydrazines was shown on the macromer level and in the cross-linked hydrogel by different strategies. Firstly, pre-fabricated hydrogels were successfully reinforced by secondary cross-linking with adipic acid dihydrazide (ADH). Secondly, pH-dependent immobilization of 2,4-dinitrophenylhydrazine (DNPH) to acid-soluble macromer derivatives as well as cross-linked oPDMA/COL matrices was demonstrated. Thirdly, reversible immobilization of a fluorescent hydrazide (AFH) was shown which was controlled by hydrogel ketone content, hydrazide ligand concentration and medium pH. This triple-tunability of hydrazide immobilization holds promise for adjustable and cost-effective hydrogel modification. Lastly, proof-of-concept experiments with hydrazido-functionalized hyaluronan (ATTO-hyHA) demonstrated the potential for covalent post-fabrication hydrogel decoration with ECM components. Hydrogel cytocompatibility was demonstrated and the introduction of DAAm into the hydrogel system resulted in superior cell material interactions when compared with previously established analogous ketone-free gels [13]. Limited ability of cells to migrate into deeper regions of these macromer-cross-linked gelatin-based gels further motivated the investigation of two different strategies to enhance hydrogel porosity [10,14]. On the one hand, the introduction of macropores was attempted by hydrogel fabrication in presence of poly(ethylene glycol) (Mn = 8000 Da, P8k). This polymer acted as porogen by phase separation during hydrogel formation. It was found that P8k was effectively extracted from the cross-linked matrix, while physico-chemical hydrogel properties remained unchanged. The second approach aimed at increasing mesh size of the cross-linked network by using hydrogel building blocks with increased molecular weights. In particular, high molecular-weight gelatin (160 Bloom, G160) was cross-linked by macromers with low MA content. Homogeneous and mechanically stable hydrogels were obtained and physico-chemical properties were determined. Successful optimization of hydrogel porosity was functionally shown by enhanced cell migration and improved release profile of incorporated nanoparticles [15]. In the second macromer-based material, hydrolytically degradable multi-armed macromers were covalently introduced into a tetraethoxysilane(TEOS)-derived silica sol in order to address the insufficient degradability of glass-based materials [16]. In detail, oligo(D,L-lactide) units were introduced into three- (TMPEO, Tx) and four-armed (PETEO, Px) ethoxylated alcohols by ring-opening polymerization, followed by activation with 3-isocyanatopropyltriethoxysilane (ICPTES) to yield TxLAy-Si and PxLAy-Si macromers [17,18]. A series of 18 oligomers (Mn: 1100-3200 Da) with different degrees of ethoxylation and varying length of oligoester units was synthesized. Applicability of a previously established indirect rapid prototyping method enabled fabrication of macromer/sol-gel-glass-derived class II hybrid scaffolds with controlled porosity [19]. Successful processability of a total of 85 different hybrid scaffold formulations allowed for identification of relevant structure-property relationships. In vitro degradation was analyzed over 12 months and a continuous linear weight loss (0.2-0.5 wt%/d) was detected which was controlled by oligo(lactide) content and matrix hydrophilicity. Compressive strength (2-30 MPa) and compressive modulus (44-716 MPa) were determined and total content, oligo(ethylene oxide) content, oligo(lactide) content and molecular weight of the oligomeric cross-linkers as well as material porosity were identified as the main factors determining hybrid mechanics by multiple linear regression. Cell migration into the entire scaffold pore network was indicated in cell culture experiments with human adipose tissue-derived stem cells (hASC) and continuous proliferation over 14 days was found. Overall, two macromer-based material platforms were established in which material versatility was realized by three main principles: I) synthesis of macromers with different chemical composition, II) combination of macromers with a second oligomeric building block, and III) flexible processability of these dual-component hybrid formulations into porous scaffold materials. 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