The recovery of metagenome-assembled genomes is biased towards the most abundant species in a given community. To improve the identification of species, even if only dominant species are recovered, we investigated the integration of flow cytometry cell sorting with bioinformatics tools to recover metagenome-assembled genomes. We used a cell culture of a wastewater microbial community as our model system. Cells were separated based on fluorescence signals via flow cytometry cell sorting into sub-communities: dominant gates, low abundant gates, and outer gates into subsets of the original community. Metagenome sequencing was performed for all groups. The unsorted community was used as control. We recovered a total of 24 metagenome-assembled genomes (MAGs) representing 11 species-level genome operational taxonomic units (gOTUs). In addition, 57 ribosomal operational taxonomic units (rOTUs) affiliated with 29 taxa at species level were reconstructed from metagenomic libraries. Our approach suggests a two-fold increase in the resolution when comparing sorted and unsorted communities. Our results also indicate that species abundance is one determinant of genome recovery from metagenomes as we can recover taxa in the sorted libraries that are not present in the unsorted community. In conclusion, a combination of cell sorting and metagenomics allows the recovery of MAGs undetected without cell sorting.

A doctoral thesis containing 3 studies addressing various aspects of human settlement strategy

Grazing by large herbivores has the potential to provide multiple ecosystem services, depending on multiple factors. Through a range of interdisciplinary methods, including literature reviews, case studies, and semi-structured interviews, I seek to assess how grazing can contribute to sustainable landscape management, as well as how sustainable practices can be incentivised. In chapter one, I focus on the role of grazing in wildfire prevention. Through a systematic literature review, I uncover new insights into the effectiveness of large herbivores in mitigating wildfires. I find that large herbivores can reduce wildfire frequency by promoting grass-dominated landscapes and reduce fire intensity by consuming vegetation and creating landscape features that reduce fuel loads. However, the effectiveness of large herbivores depends on the type of vegetation and diet preferences of the animals, and management practices associated with livestock grazing can also influence fire ignition. In chapter two, I investigate the relationship between grazing management and ecosystem services through a literature review, focusing on synergies and trade-offs between them. I identify management practices that are beneficial to multiple ecosystem services as well as policy mechanisms that can incentivise them. In the third chapter, I use case studies to investigate the role of domestic animals in rewilding projects. I describe how domestic and semi-wild herbivores can contribute to rewilding projects and make them more inclusive for rural and indigenous communities. In the fourth chapter, I aim to identify the challenges faced by land users in performing sustainable grazing management through semi-structured interviews with 88 land users from eight case studies in Europe. I gain insights into land users' determinants of behaviour towards sustainable grazing practices using the Behaviour Change Wheel framework. Overall I suggest that grazing can be a potential solution to the challenges of the Anthropocene, but only when done in the right way. I emphasize the importance of an interdisciplinary approach in grazing research and considering socio-ecological systems. I also highlight the potential of rewilding and semi-wild grazing systems especially in areas undergoing land abandonment. The results imply a shift in meat production and consumption and potential new pathways for human-livestock relations. Finally, agricultural policies such as the Common Agricultural Policy (CAP) can play a crucial role in incentivizing sustainable grazing management and should be improved to support extensive grazing and extensification.:Table of Contents i. General Introduction 6 i.i Context 7 i.i.i European grazed landscapes can provide high biodiversity and supply various ecosystem services 7 i.i.ii Challenges for European grazed systems 9 i.ii Thesis methods and outline 13 i.ii.i Literature reviews (chapters 1 & 2) 13 i.ii.ii Case studies (chapters 3 and 4) 13 i.ii.iii General approach and methodological considerations 18 i.ii.iv Research aims and questions 18 i.ii.v Thesis outline 19 i.iii Graphical abstract 21 1. Chapter One 22 1.1 Introduction 23 1.2 Literature search 25 1.3 Overall effects of herbivores on wildfire risks 26 1.3.1 Effects of herbivores on wildfire frequency 26 1.3.2 Effects of herbivores on fire intensity or severity 30 1.4 Effects of herbivores on fuel loads and fire hazard 30 1.5 Effects of vegetation characteristics on fire regimes 33 1.6 Effects of grazing-associated management on wildfires 33 1.7 Implications for management 34 1.8 Implications for policy 36 1.9 Conclusions and future challenges 37 2. Chapter Two 38 2.1 Introduction 39 2.2 Literature review: general methods 42 2.3 Results 44 2.3.1 Effects of grazing on biodiversity 44 2.3.2 Effects of grazing on climate change and its mitigation 46 2.3.3 Effects of grazing on soil quality 48 2.3.4 Effects of grazing on wildfire mitigation 49 2.3.5 Effects of grazing on cultural ecosystem services 51 2.3.6 Synergies and Trade-offs of different grazing practices and ecosystem services 52 2.3.7 Recommendations: optimizing ecosystem multifunctionality in grazed systems 55 2.4 Outlook 59 3. Chapter Three 60 3.1 Introduction 61 3.2 The Broader Theory of Rewilding and Potential Role for Livestock 64 3.2.1 Stochastic Disturbance Regimes 64 3.2.2 Trophic Complexity 65 3.2.3 Interacting Processes 66 3.3 General Case Studies 67 3.3.1 Reindeer Engineer in Swedish Lapland 67 3.3.2 Livestock Fire Brigade and Free Running Horses in the Côa Valley, Portugal 68 3.3.3 Ecotourism and Sustainable Meat at Knepp Estate, England 70 3.3.4 Oostvaardersplassen: The “Wild Experiment” 71 3.4 Conclusions 73 4. Chapter Four 77 4.1 Introduction 78 4.2 Methods 80 4.2.1 Interviews 80 4.3 Findings from interviews: Determinants of sustainable grazing decisions 81 4.3.1 Physical capability: the impact of the ‘rural exodus’ 81 4.3.2 Psychological capability: access to knowledge and understanding of administrative rules influences how land users conduct their management 82 4.3.3 Physical opportunity 83 4.3.4 Social opportunities 85 4.3.5 Motivation 85 4.4 Discussion 87 4.4.1 Intervention functions to support sustainable grazing, and associated policies 88 4.5 Conclusions and outlook 92 5. Discussion 93 5.1 Importance of the scientific, social and political context of the research 93 5.2 Societal relevance of the thesis 93 5.3 Policy relevance of grazing research 94 5.4 The value of transdisciplinary research 94 5.5 Contribution of the research 95 5.5.1 Contribution of chapter 1: large herbivores and wildfire mitigation 95 5.5.2 Contribution of chapter 2: grazing and multiple ecosystem services, trade-offs and synergies in a European context 96 5.5.3 Contribution of chapter 3: domestic livestock and rewilding 97 5.5.4 Contribution of chapter 4: drivers of decision making for sustainable grazing 98 5.5.5 Interdisciplinary approach to grazing research, grazing areas as socio-ecological systems 99 5.6 Limitations 100 5.6.1 The framework of ecosystem services 100 5.6.2 Limitations to rewilding research 101 5.6.3 The challenge of addressing different socioecological and administrative scales 101 5.6.4 Practical limitations 103 6. Outlook 104 6.1 Future research needs 104 6.2 Pathways for sustainable grazing 104 7. Conclusion 107 References 110 Summary 135 Zusammenfassung 139 Appendix 143 Appendix Chapter One 144 Appendix Chapter Two 145 Appendix Chapter Four 149 Acknowledgements 153 Author contribution statement 155

Amniotic membrane is applied to the diseased ocular surface to stimulate wound healing and tissue repair, because it releases supportive growth factors and cytokines. These effects fade within about a week after application, necessitating repeated application. Generally, amniotic membrane is fixed with sutures to the ocular surface, but surgical intervention at the inflamed or diseased site can be detrimental. Therefore, we have developed a system for the mounting of amniotic membrane between two rings for application to a diseased ocular surface without surgical intervention (sutureless amniotic membrane transplantation). With this system, AmnioClip, amniotic membrane can be applied like a large contact lens. First prototypes were tested in an experiment on oneself for wearing comfort. The final system was tested on 7 patients in a pilot study. A possible influence of the ring system on the biological effects of amniotic membrane was analyzed by histochemistry and by analyzing the expression of vascular endothelial growth factor-A (VEGF-A), hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF 2) and pigment epithelium-derived factor (PEDF) from amniotic membranes before and after therapeutic application. The final product, AmnioClip, showed good tolerance and did not impair the biological effects of amniotic membrane. VEGF-A and PEDF mRNA was expressed in amniotic membrane after storage and mounting before transplantation, but was undetectable after a 7-day application period. Consequently, transplantation of amniotic membranes with AmnioClip provides a sutureless and hence improved therapeutic strategy for corneal surface disorders.

The dual role of glycolytic enzymes in cytosol-located metabolic processes and in cell surface-mediated functions with an influence on virulence is described for various micro-organisms. Cell wall-less bacteria of the class Mollicutes including the common human pathogen Mycoplasma pneumoniae possess a reduced genome limiting the repertoire of virulence factors and metabolic pathways. After the initial contact of bacteria with cells of the respiratory epithelium via a specialized complex of adhesins and release of cell-damaging factors, surface-displayed glycolytic enzymes may facilitate the further interac-tion between host and microbe. In this study, we described detection of the four subunits of pyruvate dehydrogenase complex (PDHA-D) among the cytosolic and membrane-associated proteins of M.pneumoniae. Subunits of PDH were cloned, expressed and purified to produce specific polyclonal guinea pig antisera. Using colony blotting, fractionation of total proteins and immunofluorescence experiments, the surface localization of PDHA-C was demonstrated. All pecombinant PDH subunits are able to bind to HeLa cells and human plasminogen. These interactions can be specifically blocked by the corresponding polyclon-al antisera. In addition, an influence of ionic interactions on PDHC-binding to plasminogen as well as of lysine residues on the association of PDHA-D with plasminogen was confirmed. The PDHB subunit was shown to activate plasminogen and the PDHB-plasminogen complex induces degradation of human fibrinogen. Hence, our data indicate that the surface-associated PDH subunits might play a role in the pathogenesis of M.pneumoniae infections by interaction with human plasminogen.

The role of competition in tree communities is increasingly well understood, while little is known about the patterns and mechanisms of the interplay between above- and belowground competition in tree communities. This knowledge, however, is crucial for a better understanding of community dynamics and developing adaptive near-natural management strategies. We assessed neighbourhood interactions in an unmanaged old-growth European beech (Fagus sylvatica) forest by quantifying variation in the intensity of above- (shading) and belowground competition (crowding) among dominant and co-dominant canopy beech trees during tree maturation. Shading had on average a much larger impact on radial growth than crowding and the sensitivity to changes in competitive conditions was lowest for crowding effects. We found that each mode of competition reduced the effect of the other. Increasing crowding reduced the negative effect of shading, and at high levels of shading, crowding actually had a facilitative effect and increased growth. Our study demonstrates that complementarity in above- and belowground processes enable F. sylvatica to alter resource acquisition strategies, thus optimising tree radial growth. As a result, competition seemed to become less important in stands with a high growing stock and tree communities with a long continuity of anthropogenic undisturbed population dynamics. We suggest that growth rates do not exclusively depend on the density of potential competitors at the intraspecific level, but on the conspecific aggregation of large-diameter trees and their functional role for regulating biotic filtering processes. This finding highlights the potential importance of the rarely examined relationship between the spatial aggregation pattern of large-diameter trees and the outcome of neighbourhood interactions, which may be central to community dynamics and the related forest ecosystem services.

DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair.

Köcherfliegenlarven haben eine große Bedeutung im Nahrungsnetz der Bäche und Flüsse. Sie werden als Bioindikatoren für die Einschätzung der Gewässergüte verwendet. In Sachsen wurden bisher 221 Arten Köcherfliegen nachgewiesen. In der Artenliste und Roten Liste sind sie zusammengestellt und bewertet. Die Rote Liste informiert über die Gefährdungssituation der Arten und Lebensräume und stellt eine Grundlage für die Fachplanung im Naturschutz dar. Hiermit liegt eine Erstbearbeitung der Roten Liste der Köcherfliegen vor. Redaktionsschluss: 31.12.2019

Complexity in multi-cellular life arises from the ability of tissues to communicate with each other; tissue cross-talk during development coordinates growth and ultimately gives rise to organs with appropriate form and function. Drosophila larvae use systemic signaling mediated by endocrine factors and hormones to successfully grow and transition through developmental stages. Larvae sense nutrition primarily through the secretion of Drosophila insulinlike peptides (DILPs) 2 and 5 produced in the larval brain. Secreted DILPs bind to receptors in target tissues and activate the insulin/insulin-like signaling (IIS) pathway, which in imaginal tissues like the wing disc regulate adult wing size and cell number. The IIS pathway also directly regulates timing of developmental transitions through the production of the molting hormone ecdysone. Upon nutritional deprivation, growth is repressed but larvae manage to transition to pupal stages. This robust physiological adaptation allows Drosophila to successfully develop when grown on inadequate nutrient sources. However, the role of IIS in development under starvation is not clearly understood. In the first part of the thesis, I show that DILPs 2 and 5 are taken up in the corpora cardiaca (CC). No protein apart from the Drosophila insulin receptor (dInR) has been previously implicated in cellular uptake of DILPs in any tissue. I demonstrate that DILPs bind to non-canonical insulin-binding protein IMPL2 and are endocytosed in a clathrin-dependent manner. The CC produce adipo-kinetic hormone (AKH), a functional analog of glucagon. I show that up-taken DILPs are copackaged with AKH in secretory vesicles. CC is known to secrete AKH upon larval starvation. I find that upon starvation, CC cells also secrete stored DILPs to the prothoracic gland(PG) in a paracrine fashion, through neuronal projections running along the ‘lumen’ of the latter. DILP secretion from CC to PG reveals a novel type of hormonal signaling and provides a mechanism to ensure the insulin-dependent ecdysone production in the PG cells under nutritional deprivation. Based on this mechanism, I propose a model for how successful larval development may be achieved in the absence of sufficient nutrition; the model is further supported by the absence of ecdysone-mediated growth repression during starvation. Rearing temperature affects body size in all ectotherms including Drosophila. Previous work in the lab suggested a role for nutrient-dependent DILPs 2 and 5 in mediating body and tissue size changes with increased rearing temperature. In the second part of my thesis, I demonstrate that temperature does not have an effect on tissue IIS activity. I further show that it is not the absence of DILPs 2 and 5, but the abundance of DILP2 that abrogates temperature-dependent body size change. Upon closer inspection, I find that DILP2 up-regulation increases IIS activity specifically in the PG. I hypothesize that the observed growth inhibition in DILP2 over-expressing larvae might be due to an increase in ecdysone production by the PG cells. In a previously-published study from our group, dietary lipids derived from yeast (compared to those derived from plants) were shown to better facilitate hightemperature survival of Drosophila larvae, and acted by secreting higher levels of DILP2, followed by up-regulation of IIS activity in target tissues. In the third part of my thesis, I demonstrate that neither diet nor DILP2 level dictate larval survival at high temperatures. Using a wing disc explant culture system, I show that dietary lipids do however have an effect on tissue insulin sensitivity.

Die Broschüre „Phänologische Beobachtungen für den Lernort Wald“ richtet sich an alle, die mit Kindern und Jugendlichen im umweltpädagogischen Bereich arbeiten. Die Broschüre soll als Leitfaden dienen, der Anregungen für die Umsetzung jahreszeitbezogener Projekte im Wald bietet. Redaktionsschluss: 30.10.2019