Exposure of hepatoma cell lines to the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) is serving as a model for a systems biological study concerning the response of cells to contaminant molecules. Several aspects of the cellular distribution of the aryl hydrocarbon receptor (AhR) and its ligand B[a]P have been addressed by different live cell imaging techniques: The intracellular distribution of the B[a]P/AhR complex is visualized by means of confocal laser scaning microscopy (cLSM) and the intracellular transport rates of the complex is investigated by fluorescence recovery after photobleaching (FRAP) technique. Furthermore, cLSM image stacks of living cells are generated for the modeling of three dimensional (3-D) cell geometries. In order to prevent photochemical damage of the living cells induced by UV excitation of B[a]P, visualization is done by B[a]P’s auto fluorescence using near infrared two-photon-excitation. Murine Hepatoma 1c1c7 cells are exposed to graded concentrations of B[a]P (50 nM to 20 μM) for different incubation time periods (15 minutes to 48 hours). The highest amounts of B[a]P were found in lipid droplets and lysosomes, where the B[a]P molecules are collected and form large aggregates. We were able to work with concentrations down to 50 nM corresponding to that used for genomic and proteomic investigations. Also, for the first time imaging of B[a]P metabolites inside lipid droplets is presented in this work. The data and the model developed in this study will provide new insights into the systematic regulation of the B[a]P, the AhR as well as the receptor-ligand-complex pathway and the study will also serve as a prototype for elucidating other stress response pathways in the future.

Development of methods for the characterization and retrieval of damaged DNA from ancient biological material Over course of the last decade, the field of ancient DNA has been transformed by the advent of highthroughput sequencing. In specimens with exceptional DNA preservation, this allowed whole nuclear genomes of extinct organisms to be sequenced and analyzed. To deal with material with suboptimal DNA preservation and high levels of external modern DNA contamination as is common in intensively handled museum specimens, significant challenges remain. In this thesis I developed laboratory and in silico methods to make highly contaminated biological material amenable to genetic research and explore the structure and decay mechanisms of ancient DNA. Chapter 1 illustrates the development of a library preparation method for high-throughput sequencing that selects for DNA molecules containing uracils, a damaged base typically found in ancent DNA, in order to enrich authentic ancient molecules against a background of modern contamination. The method was applied to Neanderthal samples from Gibraltar demonstrating its suitability for recovering DNA sequences from material that shows very poor DNA preservation and that is particularly strongly contaminated with modern human DNA. Chapter 2 describes a new method to reconstruct the native double-stranded DNA molecules and overhang structures in DNA isolated from a Neanderthal specimen by combining deep sequencing of low-input single-stranded DNA libraries with in silico sequence matching. I analyzed patterns of nucleotide substitutions and base composition separately in the different structures found and showed also that the method can be applied to modern sources of fragmented DNA, specifically to cell-free DNA isolated from present-day blood samples.

Zebrafish have been used as a model to study the vertebrate retina due to its functional and structural similarities to the human retina. Photoreceptor cells (PRCs) are highly specialised type of neurons present in the retina. In zebrafish, PRCs can be divided into 5 different subtypes, rods and green, red, blue and UV sensitive cones. Mature PRCs are composed of different morphological compartments (basal domain, inner segment and outer segment), which are essential for their phototransduction ability. During development, these cells are known to arise from columnar neuroepithelium precursor cells and undergo a maturation process to become compartmentalised10. However, a detailed characterisation of this process is lacking in zebrafish. In this project, I aimed to establish and characterise in detail the stages of PRC maturation in zebrafish. Next, I aimed to investigate the role of candidate genes in this PRC maturation process. To label the plasma membrane of all cells, a zebrafish transgenic line was utilised. Furthermore, a novel zebrafish transgenic line that labels the outer segments of red sensitive PRCs was generated. This transgenic line enabled visualisation and volume quantification of outer segments of red sensitive cones. The use of both transgenic lines in combination with antibody stainings indicated that, from 72 hours post fertilisation (hpf) onwards, subtypes of PRCs exhibit differences in growth rate and morphology of their cell compartments. Additionally, differences in mitochondrial clusters and nuclei positioning were observed during the maturation process. From 72 hpf to 120 hpf, rough endoplasmic reticulum accumulation emerged specifically in rod like PRCs. Changes in chromatin organisation were observed in UV sensitive cones like PRCs from 120 hpf onwards. This showed that a high degree of complexity was observed even within the cone PRC subtypes. Lastly, the role of a candidate gene, crb2b, was examined by comparing PRC maturation process in WT and crb2b mutants. My results indicate that loss of Crb2b does not show obvious defects in PRC maturation. Results obtained in this dissertation provided a comprehensive characterisation of six independent PRC maturation stages using the criteria of cell compartmentalisation and growth, organellar distribution and localisation of cell polarity related protein complexes. This defined developmental timeline provides a platform to further study PRC maturation and function.

Untersuchungen zu Pilzsporenemissionen aus Biofiltern großtechnischer Kompostieranlagen und aus Modellbiofilteranlagen mit Einschätzung einer möglichen gesundheitlichen Gefährdung.

Classically, the following three morphological states of microglia have been defined: ramified, amoeboid and phagocytic. While ramified cells were long regarded as “resting”, amoeboid and phagocytic microglia were viewed as “activated”. In aged human brains, a fourth, morphologically novel state has been described, i.e., dystrophic microglia, which are thought to be senescent cells. Since microglia are not replenished by blood-borne mononuclear cells under physiological circumstances, they seem to have an “expiration date” limiting their capacity to phagocytose and support neurons. Identifying factors that drive microglial aging may thus be helpful to delay the onset of neurodegenerative diseases, such as Alzheimer’s disease (AD). Recent progress in single-cell deep sequencing methods allowed for more refined differentiation and revealed regional-, age- and sex-dependent differences of the microglial population, and a growing number of studies demonstrate various expression profiles defining microglial subpopulations. Given the heterogeneity of pathologic states in the central nervous system, the need for accurately describing microglial morphology and expression patterns becomes increasingly important. Here, we review commonly used microglial markers and their fluctuations in expression in health and disease, with a focus on IBA1 low/negative microglia, which can be found in individuals with liver disease.

No description available.

No description available.

Tree root-associated microbiomes are shaped by geographic, soil physico-chemical, and host tree parameters. However, their respective impacts on microbiome variations in soils across larger spatial scales remain weakly studied. We out-planted saplings of oak clone DF159 (Quercus robur L.) as phytometer in four grassland field sites along a European North–South transect. After four years, we first compared the soil microbiomes of the tree root zone (RZ) and the tree root-free zone (RFZ). Then, we separately considered the total microbiomes of both zones, besides the microbiome with significant affinity to the RZ and compared their variability along the transect. Variations within the microbiome of the tree RFZ were shaped by geographic and soil physico-chemical changes, whereby bacteria responded more than fungi. Variations within both microbiomes of the tree RZ depended on the host tree and abiotic parameters. Based on perMANOVA and Mantel correlation tests, impacts of site specificities and geographic distance strongly decreased for the tree RZ affine microbiome. This pattern was more pronounced for fungi than bacteria. Shaping the microbiome of the soil zones in root proximity might be a mechanism mediating the acclimation of oaks to a wide range of environmental conditions across geographic regions.

The retina is the neural tissue situated at the back of the eyes that samples the visual scene and sends the processed information to the brain. Millions of people worldwide suffer from retinal diseases that affect mainly the light sensing photoreceptors or retinal ganglion cells, the output neurons projecting to the brain. Despite promising attempts in the fields of gene therapy and cell transplantation, a definitive cure for retinal diseases is still missing. Research on highly regenerative organisms like zebrafish (Danio rerio) offers an attractive perspective to inform gene as well as cell transplantation-based therapies to treat retinal pathologies. Indeed, the zebrafish retina has the same structure and function as the human retina, including the presence of all retinal neurons as well as Müller glia, glial cells that provide structural and metabolic support. Remarkably, and differently from mammalian species, zebrafish Müller glia behave additionally as stem cells upon tissue damage. In this context, Müller glia re-enter the cell cycle and generate retinal progenitors that eventually differentiate to all retinal neurons. In the last twenty years, there has been a considerable effort to understand the molecular mechanisms underlying zebrafish Müller glia reprogramming to pro-regenerative stem cells and retinal progenitor production. However, a comprehensive study of the molecular identity of Müller glia, Müller glia-derived retinal progenitors as well as regenerated progeny in uninjured and lesioned conditions is still missing. Furthermore, although retinal progenitors regenerate all retinal neurons, independently of the specific retinal cell type that has been mostly affected by the tissue damage, it is not known whether all regenerated progeny integrate and rewire into the existing circuitry. The present study had two aims: • First, it aimed to provide a comprehensive description of Müller glia, Müller glia-derived progenitors as well as regenerated progeny in uninjured and light-lesioned retina at 44 hours as well as at 4 and 6 days post-lesion. • Second, it aimed to establish a CreERT2 recombinase-based strategy to allow genetic access to follow and manipulate Müller glia-derived progenitors and their progeny during regeneration. To achieve the first aim, a short-term lineage tracing strategy was devised using the two fluorescent reporters Tg(gfap:mCherry) and Tg(pcna:EGFP) labelling Müller glia and proliferating cells, respectively. Double transgenic animals were employed to sort for Müller glia, Müller glia-derived progenitors as well as regenerated progeny from the uninjured and light-lesioned retina. Subsequently, 10x Genomics, single cell RNA sequencing was performed to characterize their transcriptome and to deduct their differentiation trajectories during retina regeneration. The sequencing experiment showed the presence of a glial and a neurogenic trajectory in the regenerating retina up to 6 days post-lesion. The glial trajectory starts with non-reactive Müller glia, characterized by canonical glial markers, and continues with injury-reactive Müller glia at 44 hours post-lesion, which upregulate genes associated with glia reprogramming and inflammation as well as proliferation. These early reactive Müller glia divide and generate cells belonging to a population with a hybrid identity that becomes eminent at 4 days post-lesion and is characterized by marker genes of both Müller glia and progenitors. A glial self-renewal and a neurogenic trajectory depart from the hybrid cell population. While the glial self-renewal trajectory feeds back to the non-reactive Müller glia cell population, the neurogenic trajectory continues with neurogenic progenitors, which progressively express markers of restricted fate competence and eventually regenerate several retinal neurons. The birthdate order of the regenerated progeny recapitulates the order observed during retinal development to a great extent. Indeed, retinal ganglion cells and red cone photoreceptors are born at 4 days post-lesion, followed by blue cones, amacrine and bipolar cells at 6 day post-lesion. Regenerated rod photoreceptors as well as horizontal cells were not detected among the sorted progeny, despite detection of their committed precursors. To achieve the second aim, genetic access to Müller glia-derived cells was established using the TgBAC(mmp9:CreETt2,cryaa:EGFP);Tg(Olactb:loxP-DsRed2-loxP-EGFP) double transgenic line. The injury-induced promoter mmp9 is expressed in reactive Müller glia and drives the expression of CreERT2. Upon administration of the metabolite 4-hydroxytamoxifen, CreERT2 catalyses recombination in the Cre-dependent reporter Tg(Olactb:loxP-DsRed2-loxP-EGFP), resulting in the expression of EGFP under the control of the broadly expressed Olactb promoter. Subsequently, recombined cells, which include progenitors and progeny, express EGFP permanently. Two time points of 4-hydroxytamoxifen intraperitoneal injection were tested to achieve efficient recombination: 6 hours post-lesion, corresponding to 2 hours prior to onset of mmp9 in reactive Müller glia, and 24 hours post-lesion. In both cases, a substantial number of EGFP-positive, Müller glia-derived cells was observed in the regenerating retina at 4 days post-lesion. The majority of the EGFP-positive cells co-localized with PCNA-positive nuclei and corresponded most likely to progenitors. Importantly, EGFP-positive cells were neither observed in the light-lesioned, ethanol injected controls nor in the uninjured, 4-hydroxytamoxifen-injected controls, indicating tight control of CreERT2. In conclusion, the current PhD thesis provides a comprehensive description of the transcriptome of Müller glia, Müller glia-derived retinal progenitors and regenerated progeny. Moreover, it establishes a CreERT2-based approach to study the composition as well as long-term integration of Müller glia-derived cells in the regenerated retina and allow their genetic manipulations in future studies.

We all know earthworms as important friends in our garden: they help plants to grow better by providing nutrients, water, and air in the soil. However, in some cases, earthworms have more negative effects. This is because other organisms need to be used to the activities of earthworms to benefit from their presence. Some regions of the world have developed without earthworms for over thousands of years. For example, in northern North America, earthworms have been absent for more than 10,000 years and have only been re-introduced over the past 400 years. In many cases, introduced earthworms find a perfect environment, because no other organisms have been able to use the resources that these earthworms now consume. As so-called ecosystem engineers, earthworms dramatically alter many ecosystem characteristics. In this article, we summarize the known consequences of earthworm invasion, report on how scientists study these, and highlight remaining knowledge gaps that you might help solving should you decide to become an ecologist.