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

Mit dem Leitfaden soll ein Überblick über gesetzliche Bestimmungen zur Gentechnik bei der Errichtung und dem Betrieb gentechnischer Anlagen gegeben werden. Im Fokus stehen dabei die dafür erforderliche Durchführung von Anzeige-, Anmelde- und Genehmigungsverfahren nach dem Gentechnikgesetz. Besonders Projektleitern und Projektleiterinnen sowie den Beauftragten für Biologische Sicherheit und allen, die es werden wollen, soll der Leitfaden als Hilfestellung dienen. Es werden darin häufig gestellte Fragen aufgegriffen. Redaktionsschluss: 30.10.2014

Early detection of pathogenic bacteria in food, drinking water and medicine products is one of the essential tasks of routine microbiological analysis. Through analytics, outbreaks can be discovered and consequently, countermeasures can be initiated to minimize health and economic damage. Cultivation of pathogens from contaminated specimens is routinely performed in microbiological laboratories worldwide. The procedure is easy to perform, requires little equipment and provides simple quantitative data in colony-forming units (CFUs) per sample volume. Only the time between preparation and confirmation of a positive (contaminated) sample usually extends over several days. The desired goal should be a technique that can retain the simplicity of cultivation while providing real-time information about the sample under investigation for early detection of potential contamination. Therefore, in the framework of this thesis, systematic heat flow measurements were performed on two model strains, Lactobacillus plantarum DSM 20205 and Pseudomonas putida mt-2 KT2440. The influence of cultivation techniques (in liquid, on solid and membrane filter placed onto solid medium) in static ampoule systems on calorimetric detection was investigated. In particular, the effect of contamination level (initial bacterial cell number), substrate amount (nutrients and oxygen), and detection limits were systematically evaluated. In addition, microcalorimetric measurements of Legionella pneumophila ATCC 33152, a waterborne pathogen, were conducted for the first time. Heat flow profiles demonstrated that high contamination levels (> 1000 CFU) were detected within 24 h. Compared to detection times of up to 10 days by ISO 11731:2017, calorimetric detection can serve as an early warning system. With this knowledge, a uniquely manufactured micro(bio-)calorimetric test system was designed to meet the requirements for detecting bacterial contaminations. In particular, the sample vessel geometry and the operating temperature perfectly matched the microbiological analysis. Within this development work, numerical models were established to investigate the temperature distribution of selected compounds as well as the complete calorimetric system. Based on these models, modifications to the test system were numerically simulated in advance to improve the instrument's performance stepwise. This thesis presents the methodological principles and a calorimetric test system designed as an early warning and detection tool for microbiological samples.

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A highly structured tissue is formed from an unstructured accumulation of cells during morphogenesis. The pioneering works by Thompson, Turing and Meinhardt introduced physical principles allowing the breaking of symmetry, i.e. the emergence of patterns. This started an ongoing effort to understand the physics behind morphogenesis. In this thesis I will analyze spatial and temporal aspects of morphogenesis for different biological systems in separate parts. The planarian is an ideal model animal to understand mechanisms of spatial body axis formation. This is due to the possibility to measure its body orientation field which utilizes the orientation of the cilia of the planarian’s ventral tissue. Moreover, their astonishing regeneration capabilities allow extensive perturbation experiments. I propose a minimal model which demonstrates the emergence of the wild type body orientation as well as the development of dual-head body orientation due to beta-Catenin RNAi treatment. The topological defects of the body orientation field are calculated on a lattice for simulations and lattice-free for experimental data. These topological defects are a robust way to analyze and compare experiments with simulations. My minimal model reveals sufficient components and mechanisms for robust body axis regeneration. The second important aspect of morphogenesis is the growth regulation of tissues which is often driven by cell proliferation. The regulation of growth is not only important during growth, but also to maintain homeostasis. As fast renewing tissues are very dynamic they may have more pathways of morphogenesis active than non-renewing tissues which points to mechanisms of morphogenesis. The in vivo measurement of this proliferation rate is a challenging task. In this thesis the analysis of DNA labelling assays and the carbon 14 dating method are extended. The carbon 14 dating method can be used to determine cell renewal rates on time scales as long as the lifetime of organism last. Moreover, this method can be applied for tissues in any terrestrial organism because it utilizes the change of carbon 14 in the atmosphere due to atmospheric nuclear bomb tests in the 1960s. The method is extended to gain a better understanding of the tissue dynamics of liver, muscles and amygdala. On the other hand, the DNA labelling assays are used to estimate cell cycle parameters for fast cycling cells. The measurements are fatal to the samples and involve plenty of labor resulting in few sampled data for a time series. The deterministic Nowakowski model is extended to a stochastic model accounting for cell-to-cell and sample-to-sample variability to fully exploit the information contained even in the fluctuations of the data points. A comprehensive parameter recovery study with synthetic ground truth data is performed to evaluate the models. The new stochastic model shows no bias, a good accuracy and scales well with the number of measurements in contrast to the deterministic text-book method. I conclude with proposed applications of my new models and methods that can advance our understanding of growth and pattern formation during morphogenesis. All python software developed in this thesis is shared as open source and a website makes the stochastic analysis of DNA labelling assays available to experimentalists in a user-friendly way.

Die vorliegende Arbeit befasst sich dem Glykosaminoglykan (GAG) Hyaluronan (HA) und dessen Einfluss auf die Entwicklung des malignen Melanoms (MM). Das in der extrazellulären Matrix (ECM) reichlich vorkommende HA wird schon seit dem späten 20. Jahrhundert genauerer im Zusammenhang mit der Tumorentwicklung untersucht. Gegensätzliche Eigenschaften von HA, die zum einen Tumore fördern und zum anderen ihnen entgegenwirken, wurden seither veröffentlicht. Allgemeiner Konsens ist, dass das HA-Molekulargewicht über die verschiedenen Effekte von HA entscheidet. Momentan ist jedoch nicht ausreichend belegt, wie HA auf das Tumorwachstum einwirkt und welche HA-Größen dies speziell begünstigen. Diese Untersuchung basiert auf einem in vivo Knockout von der Hyaluronan Synthase 2 (Has2) in der Maus, der die Produktion von hoch-molekulargewichtigen HA (HMW-HA) weitestgehend einschränkte. Das Wachstum vom experimentellem MM wurde in Abwesenheit der meisten HMW-HA untersucht. Diese Arbeit versuchte den Beweis zu erbringen, dass das lokale Wachstum und die Metastasenbildung der MM-Zellen abhängig von der vorhandenen HMW-HA in der Nähe des Tumors ist. Der Has2-knockout in der Mauslinie C57BL6 wurde verifiziert und nach einem veränderten Phänotyp der Mäuse untersucht. Obwohl nahezu dreiviertel der absoluten HA Menge durch den Knockout fehlte, zeigten die Mäuse keinen offensichtlichen Veränderungen. Erst eine Messung der Haut-Permeabilität deutete auf eine womöglich verstärkte Ausbildung der Stratum corneum hin. Eine direkte Auswirkung vom fehlendem HA auf das Tumorwachstum und der Metastasierungsrate konnte nicht ausreichend belegt werden. Das verwendete Mausmodell sowie die Wahl des experimentellen Tumors werden in dieser Arbeit kritisch diskutiert. Parallel durchgeführte in vitro Versuche mit teilweise artifiziellen 3D Matrizen, die unterschiedliche Mengen von HA enthielten, zeigten einen stärkeren Einfluss von niedermolekulare HA (LMW-HA) auf die Proliferation und Invasion von MM Zellen. Diese Beobachtungen stimmen mit dem derzeitigen Konsens überein, dass LMW-HA aktivierende Signaltransduktion auslöst und HMW-HA eher homöostatisch wirkt.:TABLE OF CONTENT Zusammenfassung Summary Table of content List of figures List of tables Abbreviations 1 Introduction 1.1 Structures of the skin 1.2 The malignant melanoma 1.3 The tumor microenvironment (TME) 1.4 Hyaluronan 1.4.1 HA Structure 1.4.2 HA anabolism 1.4.3 HA catabolism 1.4.4 HA binding proteins 1.4.5 HA cell surface receptors 1.5 Hyaluronan in malignant melanoma 1.6 Aim of the thesis 2 Methods 2.1 Murine malignant melanoma cell lines 2.2 Inducible Has2-knockout mice 2.2.1 The Cre/loxP System 2.2.2 Genetical modification for the Has2-knockout 2.2.3 4-Hydroxytamoxifen induction of knockout 2.2.4 Experimental tumor model in mouse 2.2.5 Intravenous tumor injection 2.3 Primary Has2-knockout fibroblasts for in vitro experiments 2.3.1 Isolation of primary fibroblast from mouse skin tissue 2.3.2 Induction of Has2-ko fibroblasts 2.4 Molecular analysis 2.4.1 PCR analysis of genome DNA 2.4.2 Quantification of gene expression 2.4.3 Microarray analysis 2.4.4 Quantification of Has2 protein levels 2.4.5 Quantification of HA 2.4.6 HA size analysis by agarose gel electrophoresis 2.5 Physical analysis of the skin 2.5.1 Skin elasticity measurement 2.5.2 Skin permeability measurement 2.5.3 Skin hydration and TEWL measurement 2.6 Histological analysis 2.6.1 Cryo-fixed samples 2.6.2 Immunofluorescence staining 2.6.3 FFPE samples 2.6.4 HA staining 2.6.5 Histochemical images 2.7 Physiological analysis of MM cell proliferation with BrdU-Assay 2.8 Statistical analysis 3 Materials 3.1 Mouse lines 3.2 Cell lines 3.3 Buffer and solution recipes 3.4 Chemicals 3.5 Molecular-biological reagents and enzymes 3.6 Primers 3.7 Antibodies 3.8 Kits 3.9 Devices and tools 3.10 Disposables 3.11 Software 4 Results 4.1 The Has2-knockout mouse model 4.1.1 Has2-knockout characterization 4.1.2 Incomplete Has2 deletion 4.1.3 Effects of the HA knockdown 4.1.4 Has2-knockout efficiency in other organs 4.1.5 Effects of Has2-knockout on gene expression 4.2 In vitro Has2-knockout and effect on MM cells 4.2.1 In vitro Has2- and HA-knockdown 4.2.2 Has2-ko fibroblast conditioned media decreased MM proliferation 4.2.3 MM conditioned media influenced fibroblast’s HA secretion 4.2.4 The transition towards in vivo tumor experiments 4.3 In vivo Tumor experiments 4.3.1 HA threshold, correlation, and exclusions 4.3.2 Effects of HA knockdown on primary tumor volume and weight 4.3.3 Tumor histology and HA localization 4.3.4 HA fragments in tumors, healthy-, and tumor-related-skin samples 4.3.5 Metastasis formation 5 Discussion 5.1 HA knockdown 5.2 HA knockdown phenotype 5.2.1 Skin stiffness 5.2.2 Skin water homeostasis 5.3 Paracrine interactions between MM and fibroblasts 5.4 HA thresholding 5.5 Tumor readouts 5.6 In vitro ECM models 5.7 Metastasis 5.8 Alternative tumor models with stronger stromal interaction 5.9 The presented results considering current HA-Tumor research 6 Conclusion 7 Literature Danksagung Lebenslauf Akademischer Werdegang Stipendium und Auszeichnung Publikation und Posterpräsentation Publikationen Vorträge und Posterpräsentationen Eigenständigkeitserklärung / The present work addresses the glycosaminoglycan (GAG) hyaluronan (HA) and its influence on the development of malignant melanoma (MM). HA, which is abundant in the extracellular matrix (ECM), has been studied closely in relation to tumor development since the late 20th century. Opposing properties of HA, on the one hand promoting tumors and the other hand counteracting them, have since been published. The general consensus is that HA molecular weight determines the various effects of HA. However, there is insufficient evidence on how HA affects tumor growth and which HA sizes specifically promote it. This study is based on an in vivo knockout of hyaluronan synthase 2 (Has2) in mice, which largely restricted the production of high molecular weight HA (HMW-HA). Growth from experimental MM was examined in the absence of most HMW-HA. This work sought to provide evidence that local growth and metastasis of MM cells is dependent on the presence of HMW-HA in the vicinity of the tumor. Has2 knockout in the mouse line C57BL6 was verified and examined for an altered phenotype of the mice. Although nearly three-quarters of the absolute HA amount was absent due to the knockout, the mice showed no obvious change. Only a measurement of skin permeability indicated a possible increased barrier function of the stratum corneum. A direct effect of the lack of HA on tumor growth and metastasis rate could not be sufficiently demonstrated. The applied mouse model and the choice of experimental tumor are critically discussed in this work. Parallel in vitro experiments with partially artificial 3D matrices containing different amounts of HA showed a stronger influence of low molecular weight HA (LMW-HA) on proliferation and invasion of MM cells. These observations are consistent with the emerging consensus that LMW-HA triggers activating signal transduction and HMW-HA is more homeostatic.:TABLE OF CONTENT Zusammenfassung Summary Table of content List of figures List of tables Abbreviations 1 Introduction 1.1 Structures of the skin 1.2 The malignant melanoma 1.3 The tumor microenvironment (TME) 1.4 Hyaluronan 1.4.1 HA Structure 1.4.2 HA anabolism 1.4.3 HA catabolism 1.4.4 HA binding proteins 1.4.5 HA cell surface receptors 1.5 Hyaluronan in malignant melanoma 1.6 Aim of the thesis 2 Methods 2.1 Murine malignant melanoma cell lines 2.2 Inducible Has2-knockout mice 2.2.1 The Cre/loxP System 2.2.2 Genetical modification for the Has2-knockout 2.2.3 4-Hydroxytamoxifen induction of knockout 2.2.4 Experimental tumor model in mouse 2.2.5 Intravenous tumor injection 2.3 Primary Has2-knockout fibroblasts for in vitro experiments 2.3.1 Isolation of primary fibroblast from mouse skin tissue 2.3.2 Induction of Has2-ko fibroblasts 2.4 Molecular analysis 2.4.1 PCR analysis of genome DNA 2.4.2 Quantification of gene expression 2.4.3 Microarray analysis 2.4.4 Quantification of Has2 protein levels 2.4.5 Quantification of HA 2.4.6 HA size analysis by agarose gel electrophoresis 2.5 Physical analysis of the skin 2.5.1 Skin elasticity measurement 2.5.2 Skin permeability measurement 2.5.3 Skin hydration and TEWL measurement 2.6 Histological analysis 2.6.1 Cryo-fixed samples 2.6.2 Immunofluorescence staining 2.6.3 FFPE samples 2.6.4 HA staining 2.6.5 Histochemical images 2.7 Physiological analysis of MM cell proliferation with BrdU-Assay 2.8 Statistical analysis 3 Materials 3.1 Mouse lines 3.2 Cell lines 3.3 Buffer and solution recipes 3.4 Chemicals 3.5 Molecular-biological reagents and enzymes 3.6 Primers 3.7 Antibodies 3.8 Kits 3.9 Devices and tools 3.10 Disposables 3.11 Software 4 Results 4.1 The Has2-knockout mouse model 4.1.1 Has2-knockout characterization 4.1.2 Incomplete Has2 deletion 4.1.3 Effects of the HA knockdown 4.1.4 Has2-knockout efficiency in other organs 4.1.5 Effects of Has2-knockout on gene expression 4.2 In vitro Has2-knockout and effect on MM cells 4.2.1 In vitro Has2- and HA-knockdown 4.2.2 Has2-ko fibroblast conditioned media decreased MM proliferation 4.2.3 MM conditioned media influenced fibroblast’s HA secretion 4.2.4 The transition towards in vivo tumor experiments 4.3 In vivo Tumor experiments 4.3.1 HA threshold, correlation, and exclusions 4.3.2 Effects of HA knockdown on primary tumor volume and weight 4.3.3 Tumor histology and HA localization 4.3.4 HA fragments in tumors, healthy-, and tumor-related-skin samples 4.3.5 Metastasis formation 5 Discussion 5.1 HA knockdown 5.2 HA knockdown phenotype 5.2.1 Skin stiffness 5.2.2 Skin water homeostasis 5.3 Paracrine interactions between MM and fibroblasts 5.4 HA thresholding 5.5 Tumor readouts 5.6 In vitro ECM models 5.7 Metastasis 5.8 Alternative tumor models with stronger stromal interaction 5.9 The presented results considering current HA-Tumor research 6 Conclusion 7 Literature Danksagung Lebenslauf Akademischer Werdegang Stipendium und Auszeichnung Publikation und Posterpräsentation Publikationen Vorträge und Posterpräsentationen Eigenständigkeitserklärung

Mainland Southeast Asia (MSEA) is an area with a long history of human occupation and great ethnolinguistic diversity. The earliest anatomically modern humans arrived ~65 thousand years ago, while presently it has a population size of ~263 million people speaking ~229 languages belonging to five major language families: Austroasiatic (AA), Austronesian (AN), Tai-Kadai (TK), Hmong-Mien (HM), and Sino-Tibetan (ST). Analyses of genome-wide data can provide rich insights into reconstructing human genetic population history, but there is a paucity of genome-wide data from MSEA (mostly limited to the majority groups such as the Kinh and Thai). The goal of this thesis was to analyze newly-genotyped genome-wide data (encompassing ~600 thousand SNPs) from an extensive, detailed sample of ethnolinguistic groups from Vietnam, Thailand, and Laos, encompassing all five major MSEA language families, in order to reconstruct their genetic history and relationships with cultural variation. In Chapter I, I analyzed data from 259 individuals from the Kinh and 21 Vietnamese ethnolinguistic groups. In contrast to previous studies suggesting that genetic diversity in Vietnam largely reflects internal diversification and isolation, I found evidence for different sources of genetic diversity in different linguistic groups, extensive contact between groups, and a likely case of language shift involving AN-speaking groups. In Chapter II, I analyzed data from 463 individuals from 33 ethnolinguistic groups together with 3 published groups (including the Thai), hence in total 36 groups from Thailand and Laos. I found fine-scale genetic structure for the major TK and AA groups according to their linguistic branches, and different levels of local interaction with other linguistic groups in geographical proximity. This diverse structure was also influenced by South Asian admixture, detected in several different linguistic groups from central and southern Thailand, dated to ~600-1000 years ago. This admixture date, together with the geographical distribution of these groups, suggests that the South Asian influence corresponds to the Ayutthaya kingdom period (1350-1767 AD), when there was extensive interactions and political and trading networks between people from MSEA and South Asia.:SUMMARY p.1 ZUSAMMENFASSUNG p.10 CHAPTER I p.20 Extensive ethnolinguistic diversity in Vietnam reflects multiple sources of genetic diversity CHAPTER II p.68 Reconstructing the human genetic history of mainland Southeast Asia: insights from genome- wide data from Thailand and Laos REFERENCES p.111 ACKNOWLEDGMENTS p.114 CURRICULUM VITAE p.115 DECLARATION OF INDEPENDENCE p.117 AUTHOR CONTRIBUTION STATEMENT p.118

Wolbachia (Alphaproteobacteria) is an inherited endosymbiont of arthropods and filarial nematodes and was reported to be widespread across insect taxa. While Wolbachia’s effects on host biology are not understood from most of these hosts, known Wolbachia-induced phenotypes cover a spectrum from obligate beneficial mutualism to reproductive manipulations and pathogenicity. Interestingly, data on Wolbachia within the most species-rich order of arthropods, the Coleoptera (beetles), are scarce. Therefore, we screened 128 species from seven beetle families (Buprestidae, Hydraenidae, Dytiscidae, Hydrophilidae, Gyrinidae, Haliplidae, and Noteridae) for the presence of Wolbachia. Our data show that, contrary to previous estimations, Wolbachia frequencies in beetles (31% overall) are comparable to the ones in other insects. In addition, we used Wolbachia MLST data and host phylogeny to explore the evolutionary history of Wolbachia strains from Hydraenidae, an aquatic lineage of beetles. Our data suggest that Wolbachia from Hydraenidae might be largely host genus specific and that Wolbachia strain phylogeny is not independent to that of its hosts. As this contrasts with most terrestrial Wolbachia–arthropod systems, one potential conclusion is that aquatic lifestyle of hosts may result in Wolbachia distribution patterns distinct from those of terrestrial hosts. Our data thus provide both insights into Wolbachia distribution among beetles in general and a first glimpse of Wolbachia distribution patterns among aquatic host lineages.

The traditional herbal medicinal product Myrrhinil-Intest® is a fixed herbal combination, which is marketed in Germany since 1959 and applied in medical practice for the treatment of gastrointestinal disorders such as functional diarrhea, irritable bowel syndrome and inflammatory bowel disease. It contains myrrh, which is described as the oleo-gum resin from mainly Commiphora molmol Engler (Burseraceae), coffee charcoal, which are the milled roasted to blackening outer seed parts of green dried Coffea Arabica Linné (Rubiaceae) fruits and chamomile flowers - the flower heads of Matricaria recutita Linné (Asteraceae). The clinical effectiveness of Myrrhinil-Intest® for the treatment of various gastrointestinal disorders was demonstrated in several clinical studies and is described in various experience reports, however its pharmacological profile is not fully elucidated. Within the present study the spasmolytic and anti-inflammatory potential of the components myrrh, chamomile and coffee charcoal was investigated. Therefore pharmacological, histological and molecular biological methods were utilised. Spasmolytic activity was characterised using isometric tension measurement with rat isolated small intestinal preparations. Anti-inflammatory potential was assessed with different methods using isolated rat small intestinal preparations and immune cell lines. Inflammation was induced with TNBS and LPS respectively. Additionally, the influence of the herbal components on the gene expression profile of native human macrophages after LPS/IFNγ stimulation was determined by microarray gene expression analysis. Chamomile flower and myrrh exerted spasmolytic effects, whereby the more pronounced spasmolytic effects of myrrh were mediated via calcium channel blockade. Myrrh and chamomile flower exerted anti-inflammatory effects.

For the last four decades, psycholinguistic research has dealt with the question to what extent elements of simple sentences like “The monk read the book” are planned ahead both on the abstract-lexical and phonological processing level. While a number of studies have shown that all up to the final element can be activated on these two levels, empirical evidence on the flexibility of the respective planning scopes is inconsistent, and a systematic delineation of the influence of different forms of cognitive load has not yet been provided. This thesis presents a series of 9 picture-word interference experiments in which participants produced subject-verb-object sentences while ignoring auditory distractor words. Advance planning was assessed at an abstract-lexical (lemma) level and at a phonological (word form) level under varying working memory load conditions (no load, or visuospatial load, or verbal load). In the absence of a concurrent working memory load and with a concurrent visuospatial working memory load, subject and object nouns were found to be activated at the abstract-lexical and the phonological level prior to speech onset. By contrast, with a concurrent verbal working load, the scope of advance planning at the phonological level was reduced, while the scope of advance planning at the abstract-lexical level remained unaffected. Moreover, sentence planning had a more disruptive effect on verbal working memory performance than on visuospatial working memory performance. Overall, these results suggest that advance planning at the phonological level is more adaptive to external factors than advance planning at the abstract-lexical level. Also, they indicate an overlap of resources allocated to phonological processing in speech production and verbal working memory.