Anthropogenic pollution of water resources and the environment by various hazardous compounds and classes of substances raises concerns about public health impacts and environmental damage. Commercially available, portable and easy-to-use devices to detect and quantify these compounds are rather sparse, but would contribute to comprehensive monitoring and reliable risk assessment. The Soft Colloidal Probe (SCP) assay is a promising platform for the development of portable analytical devices and thus has a great potential for a transfer to industry. This assay is based on the differential deformation of an elastic particle, i.e., the SCP, as a function of analyte concentration, which affects the extent of interfacial interactions between the SCP and a biochip surface. The objective of this work was to adapt this assay for the detection of anthropogenic pollutants. Biomimetic molecular recognition approaches were used based on naturally occurring target proteins that specifically bind the anthropogenic pollutants of interest. This adaptation included the elaboration of strategies for site-specific immobilization of the respective proteins and functionalization of SCPs. In this work, it is demonstrated that the SCP method can be employed for the highly specific and sensitive detection of the critically discussed pesticide glyphosate by using the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Furthermore, a specific detection scheme for estrogens and compounds with estrogenic and antiestrogenic activity was developed by harnessing estrogen sulfotransferase as the biomimetic recognition element. In the second part of the thesis, improvements of the SCP sensing methodology are described. These improvements were achieved by accelerating data analysis and developing a novel synthesis method for SCPs that ensures monodisperse particles with superior reproducibility. Rapid extraction of interaction energies is achieved by using a pattern matching algorithm that reduces the time required for data analysis to a fraction. The microfluidics-assisted synthesis of SCPs enables the production of highly monodisperse SCPs with adjustable size and mechanical properties. Various functionalization approaches have been developed that allow easy and modular introduction of functional groups and biomolecules for SCP-based sensing approaches.

The ability to recover ancient DNA from skeletal material has completely transformed the field of evolutionary anthropology, making it possible to sequence the genomes of individuals who lived thousands of years ago. In addition to solving the long-standing question of admixture between neanderthals and modern humans and uncovering evidence of dramatic migration events throughout human history, ancient DNA has become an important resource for understanding many facets of natural selection, which is often challenging using today's genetic variation alone. Chapter 1 examines the dynamics of negative selection acting against Neanderthal ancestry in modern humans and establishes its limits over long evolutionary timescales. It shows that the previously reported monotonic decline in Neanderthal ancestry over the last fifty-thousand years, thought to be a result of negative selection, is a statistical artifact caused by incorrect assumptions about modern human demographic history, in particular the gene flow between Africa and West Eurasia. Re-estimation of the Neanderthal ancestry proportions over time using a more robust statistic no longer infers a significant decline in Neanderthal ancestry, which is proven to be consistent with simulations of negative selection across a wide range of selection parameters. Chapter 2 describes the first comprehensive analysis of the Y chromosomes of neanderthals and Denisovans. Although Neanderthals and Denisovans form a sister group to modern humans at the autosomal level, Neanderthal Y chromosomes are more similar to modern humans than Denisovan Y chromosomes. In fact, the Y chromosomes of late neanderthals represent a lineage introgressed from an early modern human population. This introgression, which occurred hundreds of thousands of years, completely replaced the Y chromosomes of early neanderthals, reflecting the observations made from mitochondrial DNA. Population genetic simulations of selection and introgression show that although a complete replacement of both mitochondrial DNA and Y chromosomes is unlikely under neutrality, higher deleterious burden of neanderthals predicts a rapid replacement of both loci by their modern human counterparts. Finally, Chapter 3 presents an R package admixer, designed to facilitate the programming of automated, fully reproducible population genetic analyses using ADMIXTOOLS, a suite of programs widely used in ancient DNA research.

Background Within eukaryotes there is a complex cascade of RNA-based macromolecules that process other RNA molecules, especially mRNA, tRNA and rRNA. A simple example is the RNase MRP processing of ribosomal RNA (rRNA) in ribosome biogenesis. One hypothesis is that this complexity was present early in eukaryotic evolution; an alternative is that an initial simplified network later gained complexity by gene duplication in lineages that led to animals, fungi and plants. Recently there has been a rapid increase in support for the complexity-early theory because the vast majority of these RNA-processing reactions are found throughout eukaryotes, and thus were likely to be present in the last common ancestor of living eukaryotes, named here as the Eukaryotic Ancestor. Results We present an overview of the RNA processing cascade in the Eukaryotic Ancestor and investigate in particular, RNase MRP which was previously thought to have evolved later in eukaryotes due to its apparent limited distribution in fungi and animals and plants. Recent publications, as well as our own genomic searches have uncovered previously unknown RNase MRP RNAs, indicating that RNase MRP has a wide distribution in eukaryotes. Combining secondary structure and promoter region analysis of new and previously discovered RNase MRP RNAs along with analysis of the primary substrate (rRNA), allows us to discuss this distribution in the light of eukaryotic evolution. Conclusions We conclude that RNase MRP can now be placed in the RNA-processing cascade present in the Eukaryotic Ancestor. This highlights the complexity of RNAprocessing in early eukaryotes.

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Microtubule length control is indispensable for cytoskeletal functions such as mitotic spindle assembly and positioning. In vivo studies have shown that kinesin motor proteins can regulate microtubule length positively and negatively. The mechanisms by which kinesins act as depolymerases and catastrophe factors are well studied. By contrast, how kinesins promote microtubule growth is unknown. The aim of this work was to elucidate the mechanism by which budding yeast kinesin Kip2 regulates microtubule dynamics, using in vitro reconstitution assays combined with total internal reflection fluorescence (TIRF) and differential interference contrast (DIC) microscopy. Kip2 was shown to increase the mean length of microtubules through length-dependent polymerase and anti-catastrophe activities, both with porcine and yeast tubulin, in the absence of accessory proteins. Using single-molecule motility assays, Kip2 was shown to translocate in a highly processive, ATP-dependent manner and to processively target tubulin oligomers to microtubule plus-ends. Mutant studies to probe Kip2 structure-function relationships revealed that the N-terminus of Kip2 is dispensable for promotion of microtubule growth, while the C-terminus is not. An effort to functionally identify a tubulin/microtubule-binding domain in the Cterminus of Kip2 remained unfruitful. Finally, the combinatorial effect of Kip2 with interaction partners Bim1 and Bik1 on microtubule dynamics was reconstituted. This microtubule plus-end tracking complex promoted microtubule growth beyond the effect of Kip2 alone. Together, this work demonstrates that a kinesin motor can act directly as a length-dependent microtubule polymerase and anti-catastrophe factor in the absence of accessory proteins. Thereby, this work provides insight into how kinesins control microtubule length.

Climate change, pandemics, people searching for refuge from war and declining natural resources; the challenges within a globalized world cannot be solved without the cooperation between people from all over the world. This includes political cooperation, as well as grassroot movements working towards a sustainable and more equal society, and individuals changing the own consumer behavior or lifestyle. Unfortunately, both national and personal interests often conflict with behaviors and measures that would be necessary to mitigate crises. How to overcome those barriers to global responsible behavior? For almost seven decades, psychologists have shown that intergroup contact reduces prejudice against people from other groups. In the context of global environmental and social problems, we propose that contact has potential beyond that. We posit that international contact facilitates identification with the global ingroup of humanity and in turn induces globally responsible behavioral intentions and behaviors. In two manuscripts we present experimental as well as correlational evidence from nine studies (N=2147) supporting the “global contact” hypothesis. Both experimental induced contact (in six studies by the use of a simulated internet chat) and self-reported international contacts led to higher identification and solidarity with humanity compared to different control groups. Global identification and solidarity in turn, were related to higher global responsible attitudes and intentions. Those participants who had simulated contact with distant cultures reported a significantly higher level of identification with humanity compared to participants with close contacts. Climate change, people fleeing from war and poverty, pandemics – the challenges for the international community are enormous. However, the results of this dissertation suggest that this community also has the potential to face such crises. The promotion of positive contacts with people from other parts of the world can foster identities and engagement beyond national borders and interests. 'Know few, care for all.:Table of Contents 1 - Introduction 2 - Method Procedure The chat paradigm Contact condition Control conditions Imagined contact design Manipulations for additional research questions The questionnaire 3 - Get together, feel together, act together: International personal contact increases identification with humanity and global collective action Abstract Introduction International contact Processes underlying intergroup contact effects Contact and collective action The superordinate identity of humanity The model and preliminary results Study 1 Methods Results and discussion Study 2 Methods Results Discussion Empirical findings Study designs Advantages of contact interventions Superordinate categories Status groups Conclusion 4 – Know few, care for all. Does international contact increase global identification and responsible global action? Abstract Introduction Intergroup contact Contact and Pro-social action Generalization and recategorization Levels of identification The Present Research Overview of the Studies Method Procedure and measures Results Meta-Analysis Testing the salience of exclusive common ingroups and contact group similarity as moderators General Discussion The recategorization hypothesis Influence of lower level categories Conclusion 5 - Discussion Theoretical impulse Application in the field Conclusion and appeal Supplemental material Supplemental Material to Chapter 3 Supplemental Material to Chapter 4 References Appendix

Der Hirninfarkt ist einer der drei häufigsten Todesursachen in Deutschland. Er wird durch eine Unterversorgung des Gewebes mit Sauerstoff und Nährstoffen, häufig infolge von Gefäßverschlüssen, ausgelöst. Die bisher einzige Therapiemöglichkeit ist die Thrombolyse. Deshalb sind neue Therapieansätze nötig. Voraussetzung dafür sind geeignete Modelle. In dieser Arbeit wurden zellbasierte Hypoxiemodelle etabliert und charakterisiert. Es wurde der Einfluss von Sauerstoff- und/oder Glucoseentzug an humanen primären Makrophagen untersucht. Für Screeninguntersuchungen wurden neuronale und periphere (Makrophagen) Zelllinien von Ratte und Mensch verwendet. Im zweiten Teil der Arbeit wurden die Modelle genutzt, um die Wirkung von Adenin, eine rezeptorvermittelte Therapieoption, und des Phytopharmakons STW5 auf mögliche protektive Wirkungen zu untersuchen. Es wurden zellbiologische (MTT-Test, LDH-Test, DAPI-Färbung), immunologische (TNF α- ELISA, immunhistochemische Färbung), elektrophysiologische (Patch Clamp-Technik) und molekularbiologische (RT-PCR, Real-Time-PCR) Methoden angewendet. Es wurde erstmals der Adeninrezeptor an den untersuchten Zelllinien nachgewiesen und der pharmakologische Hinweis für eine bisher unbekannte humane Variante des Rezeptors erbracht. An neuronalen Zellen kam es zu einer Rezeptorinteraktion zwischen Adenin- und Adenosin-A1-Rezeptor. Antagonisten am Adeninrezeptor scheinen zur Behandlung hypoxiebedingter Zellschäden geeignet zu sein. STW5 hemmte unter hypoxischen Bedingungen die TNF α-Freisetzung humaner primärer Makrophagen. Der antiinflammatorischen Wirkung liegt die Blockierung erhöhter Kaliumströme zugrunde. An den untersuchten Zelllinien wirkte STW5 der hypoxieinduzierten Zellschädigung entgegen.

As one of the closest relatives of arthropods, Onychophora plays an important role in understanding the evolution of arthropod body plans. Currently there is controversy surrounding the evolution of the brain among the ecdysozoan clades, which shows a collar-shaped, circumoral organization in cycloneuralians but a ganglionic architecture in panarthropods. Based on the innervation pattern of lip papillae surrounding the mouth, the onychophoran brain has been interpreted as a circumoral ring, suggesting that this organization is an ancestral feature of Ecdysozoa. However, this interpretation is inconsistent with other published data. To explore the evolutionary origin of the onychophoran mouth and to shed light on the evolution of the ecdysozoan brains, we analyzed the innervation pattern and morphogenesis of the oral lip papillae in the onychophoran Euperipatoides rowelli using DNA labeling, immunocytochemistry, and neuronal tracing techniques. Our morphogenetic data revealed that the seven paired and one unpaired oral lip papillae arise from three anterior-most body segments. Retrograde fills show that only the first and the third nerves supplying the lip papillae are associated with cell bodies within the brain, whereas the second nerve exclusively receives fibers from somata of peripheral neurons located in the lip papillae. According to our anterograde fills and immunocytochemical data, the first nerve supplies the anterior-most pair of lip papillae, whereas the second and the third nerves are associated with the second to fifth and second to eighth lip papillae, respectively. These data suggest that the lip papillae of E. rowelli are mainly innervated by the proto- and deutocerebrum, whereas there are only a few additional cell bodies situated posterior to the brain. According to these findings, the overall innervation pattern of the oral lip papillae in E. rowelli is incompatible with the interpretation of the onychophoran brain as a modified circumoral ring.

Background Hedgehog signaling plays an important role in embryonic development, organogenesis and cancer. In the adult liver, Hedgehog signaling in non-parenchymal cells has been found to play a role in certain disease states such as fibrosis and cirrhosis. However, whether the Hedgehog pathway is active in mature healthy hepatocytes and is of significance to liver function are controversial. Findings Two types of mice with distinct conditional hepatic deletion of the Smoothened gene, an essential co-receptor protein of the Hedgehog pathway, were generated for investigating the role of Hedgehog signaling in mature hepatocytes. The knockout animals (KO) were inconspicuous and healthy with no changes in serum transaminases, but showed a slower weight gain. The liver was smaller, but presented a normal architecture and cellular composition. By quantitative RT-PCR the downregulation of the expression of Indian hedgehog (Ihh) and the Gli3 transcription factor could be demonstrated in healthy mature hepatocytes from these mice, whereas Patched1 was upregulated. Strong alterations in gene expression were also observed for the IGF axis. While expression of Igf1 was downregulated, that of Igfbp1 was upregulated in the livers of both genders. Corresponding changes in the serum levels of both proteins could be detected by ELISA. By activating and inhibiting the transcriptional output of Hedgehog signaling in cultured hepatocytes through siRNAs against Ptch1 and Gli3, respectively, in combination with a ChIP assay evidence was collected indicating that Igf1 expression is directly dependent on the activator function of Gli3. In contrast, the mRNA level of Igfbp1 appears to be controlled through the repressor function of Gli3, while that of Igfbp2 and Igfbp3 did not change. Interestingly, body weight of the transgenic mice correlated well with IGF-I levels in both genders and also with IGFBP-1 levels in females, whereas it did not correlate with serum growth hormone levels. Conclusions Our results demonstrate for the first time that Hedgehog signaling is active in healthy mature mouse hepatocytes and that it has considerable importance for IGF-I homeostasis in the circulation. These findings may have various implications for mouse physiology including the regulation of body weight and size, glucose homeostasis and reproductive capacity.

Alveolar fluid clearance is driven by vectorial Na+ transport and promotes postnatal lung adaptation. The effect of insulin on alveolar epithelial Na+ transport was studied in isolated alveolar cells from 18–19-day gestational age rat fetuses. Equivalent short-circuit currents (ISC) were measured in Ussing chambers and different kinase inhibitors were used to determine the pathway of insulin stimulation. In Western Blot measurements the activation of mediators stimulated by insulin was analyzed. The ISC showed a fast dose-dependent increase by insulin, which could be attributed to an increased ENaC (epithelial Na+ channel) activity in experiments with permeabilized apical or basolateral membrane. 5-(N-Ethyl-N-isopropyl)amiloride inhibition of ISC was not affected, however, benzamil-sensitive ISC was increased in insulin-stimulated monolayers. The application of LY-294002 and Akti1/2 both completely blocked the stimulating effect of insulin on ISC. PP242 partly blocked the effect of insulin, whereas Rapamycin evoked no inhibition. Western Blot measurements revealed an increased phosphorylation of AKT after insulin stimulation. SGK1 activity was also increased by insulin as shown by Western Blot of pNDRG1. However, in Ussing chamber measurements, GSK650394, an inhibitor of SGK1 did not prevent the increase in ISC induced by insulin. The application of IGF-1 mimicked the effect of insulin and increased the ENaC activity. In addition, an increased autophosphorylation of the IGF-1R/IR was observed after insulin stimulation. We conclude that insulin rapidly increases epithelial Na+ transport by enhancing the activity of endogenous ENaC through activation of PI3K/AKT in alveolar cells.