Oomycetes (Stramenopiles, protists) are among the most severe plant pathogens, comprising species with a high economic and ecologic impact on forest ecosystems. Their diversity and community structures are well studied in terrestrial habitats, but tree canopies as huge and diverse habitats have been widely neglected. A recent study highlighted distinct oomycete communities in the canopy stratum compared to the ground region of three temperate deciduous trees (Quercus robur, Tilia cordata, Fraxinus excelsior). While the communities from the two strata were distinct when taking oomycete abundances into account, they were rather similar when only OTU presence/absence was considered. It remains, however, unknown if this homogeneity in the OTU presence also leads to a functional homogenisation among microhabitats within the two strata ground and canopy. In this study, we supplemented functional traits to oomycete communities in the tree microhabitats, which were determined over a time period of 2 years with a metabarcoding approach. Our results showed that even though most oomycetes occurred in all microhabitats, a strong discrepancy between the strata and correspondingly the distribution of oomycete lifestyles could be observed. This pattern was constant over several seasons. Obligate biotrophic species, exclusively feeding on living host tissue, dominated the canopy region, implying tree canopies to be a hitherto neglected reservoir for parasitic protists. OTUs assigned to the genus Hyaloperonospora—parasites highly specialised on hosts that were not sampled—could be determined in high abundances in the canopy and the surrounding air, challenging the strict host dependencies ruled for some oomycetes. Our findings further contribute to the understanding of oomycete ecosystem functioning in forest ecosystems

The demise of retinal ganglion cells (RGCs) is characteristic of diseases of the retina such as glaucoma and diabetic or ischemic retinopathies. Pigment epithelium-derived factor (PEDF) is a multifunctional secreted protein that mediates neuroprotection and inhibition of angiogenesis in the retina. We have studied expression and regulation of two of several receptors for PEDF, patatin-like phospholipase 2 gene product/PEDF-R and laminin receptor (LR), in serum-starved RGC under normoxia and hypoxia and investigated their involvement in the survival of retinal neuronal cells. We show that PEDF-R and LR are co-expressed in RGC and R28 retinal precursor cells. Expression of both receptors was enhanced in the presence of complex secretions from retinal glial (Müller) cells and upregulated by VEGF and under hypoxic conditions. PEDF-R- and LR-knocked-down cells demonstrated a markedly attenuated expression of anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-xL) and neuroprotective mediators (PEDF, VEGF, BDNF) suggesting that both PEDF-R and LR mediate pro-survival effects of PEDF on RGC. While this study does not provide evidence for a differential survival-promoting influence of either PEDF-R or LR, it nevertheless highlights the importance of both PEDF receptors for the viability of retinal neurons.

In systems biology, material balances, kinetic models, and thermodynamic boundary conditions are increasingly used for metabolic network analysis. It is remarkable that the reversibility of enzyme-catalyzed reactions and the influence of cytosolic conditions are often neglected in kinetic models. In fact, enzyme-catalyzed reactions in numerous metabolic pathways such as in glycolysis are often reversible, i.e., they only proceed until an equilibrium state is reached and not until the substrate is completely consumed. Here, we propose the use of irreversible thermodynamics to describe the kinetic approximation to the equilibrium state in a consistent way with very few adjustable parameters. Using a flux-force approach allowed describing the influence of cytosolic conditions on the kinetics by only one single parameter. The approach was applied to reaction steps 2 and 9 of glycolysis (i.e., the phosphoglucose isomerase reaction from glucose 6-phosphate to fructose 6-phosphate and the enolase-catalyzed reaction from 2-phosphoglycerate to phosphoenolpyruvate and water). The temperature dependence of the kinetic parameter fulfills the Arrhenius relation and the derived activation energies are plausible. All the data obtained in this work were measured efficiently and accurately by means of isothermal titration calorimetry (ITC). The combination of calorimetric monitoring with simple flux-force relations has the potential for adequate consideration of cytosolic conditions in a simple manner.

The human genome comprises 8% sequences of retroviral origin, so-called human endogenous retroviruses (HERVs). Most of these proviral sequences are defective, but some possess open reading frames. They can lead to the formation of viral transcripts, when activated by intrinsic and extrinsic factors. HERVs are thought to play a pathological role in inflammatory diseases and cancer. Since the consequences of activated proviral sequences in the human body are largely unexplored, selected envelope proteins of human endogenous retroviruses associated with inflammatory diseases, namely HERV-K18, HERV-K113, and HERV-Fc1, were investigated in the present study. A formation of glycosylated envelope proteins was demonstrated in different mammalian cell lines. Nevertheless, protein maturation seemed to be incomplete as no transport to the plasma membrane was observed. Instead, the proteins remained in the ER where they induced the expression of genes involved in unfolded protein response, such as HSPA5 and sXBP1. Furthermore, low expression levels of native envelope proteins were increased by codon optimization. Cell-free expression systems showed that both the transcriptional and translational level is affected. By generating different codon-optimized variants of HERV-K113 envelope, the influence of single rare t-RNA pools in certain cell lines was demonstrated. The mRNA secondary structure also appears to play an important role in the translation of the tested viral envelope proteins. In summary, the formation of certain HERV proteins is basically possible. However, their complete maturation and thus full biologic activity seems to depend on additional factors that might be disease-specific and await elucidation in the future.

For systems biology, it is important to describe the kinetic and thermodynamic properties of enzyme-catalyzed reactions and reaction cascades quantitatively under conditions prevailing in the cytoplasm. While in part I kinetic models based on irreversible thermodynamics were tested, here in part II, the influence of the presumably most important cytosolic factors was investigated using two glycolytic reactions (i.e., the phosphoglucose isomerase reaction (PGI) with a uni-uni-mechanism and the enolase reaction with an uni-bi-mechanism) as examples. Crowding by macromolecules was simulated using polyethylene glycol (PEG) and bovine serum albumin (BSA). The reactions were monitored calorimetrically and the equilibrium concentrations were evaluated using the equation of state ePC-SAFT. The pH and the crowding agents had the greatest influence on the reaction enthalpy change. Two kinetic models based on irreversible thermodynamics (i.e., single parameter flux-force and two-parameter Noor model) were applied to investigate the influence of cytosolic conditions. The flux-force model describes the influence of cytosolic conditions on reaction kinetics best. Concentrations of magnesium ions and crowding agents had the greatest influence, while temperature and pH-value had a medium influence on the kinetic parameters. With this contribution, we show that the interplay of thermodynamic modeling and calorimetric process monitoring allows a fast and reliable quantification of the influence of cytosolic conditions on kinetic and thermodynamic parameters.

The iliotibial band (ITB) is a suitable scaffold for anterior cruciate ligament (ACL) reconstruction, providing a sufficient mechanical resistance to loading. Hence, ITB-derived fibroblasts attract interest for ligament tissue engineering but have so far not been characterized. This present study aimed at characterizing ITB fibroblasts before, during, and after emigration from cadaveric ITB explants to decipher the emigration behavior and to utilize their migratory capacity for seeding biomaterials. ITB and, for comparison, ACL tissues were assessed for the content of alpha smooth muscle actin (αSMA) expressing fibroblasts and degeneration. The cell survival and αSMA expression were monitored in explants used for cell isolation, monolayer, self-assembled ITB spheroids, and spheroids seeded in polyglycolic acid (PGA) scaffolds. The protein expression profile of targets typically expressed by ligamentocytes (collagen types I–III, elastin, lubricin, decorin, aggrecan, fibronectin, tenascin C, CD44, β1-integrins, vimentin, F-actin, αSMA, and vascular endothelial growth factor A [VEGFA]) was compared between ITB and ACL fibroblasts. A donor- and age-dependent differing percentage of αSMA positive cells could be detected, which was similar in ITB and ACL tissues despite the grade of degeneration being significantly higher in the ACL due to harvesting them from OA knees. ITB fibroblasts survived for several months in an explant culture, continuously forming monolayers with VEGFA and an increased αSMA expression. They shared their expression profile with ACL fibroblasts. αSMA decreased during the monolayer to spheroid/scaffold transition. Using self-assembled spheroids, the migratory capacity of reversible myofibroblastic ITB cells can be utilized for colonizing biomaterials for ACL tissue engineering and to support ligament healing.

While cognitive impairment is common in peripheral diseases such as chronic kidney disease (CKD), mechanistic insights and effective therapies are lacking. Multiple toxins accumulating as a consequence of CKD have been identified, yet the consequences for cellular crosstalk in the brain and the mechanisms underlying the associated neuronal dysfunction remain largely elusive. In the case of CKD, more than 100 uremic toxins have been identified. Renal transplantation largely reverses the cognitive impairment associated with CKD, demonstrating that cognitive impairment in CKD can be reversed. This indicates that pharmaceutical approaches to target cognitive impairment in association with CKD may be feasible. However, it is unlikely that targeting a single toxin will be sufficient to combat neuronal dysfunction associated with peripheral diseases such as CKD, given the large number of toxins involved and since the pattern of accumulating toxins varies among affected patients. Rather than identifying single toxins, identifying a common mechanism inducing neuronal dysfunction and thus impairing cognition may identify new and feasible therapeutic approaches. One commonality of peripheral diseases such as liver or renal failure is sterile inflammation. Sterile inflammation has been linked with neurodegenerative diseases and associated cognitive impairment and inflammasome activation is one hallmark of chronic pathologies in the brain. Mutations in the inflammasome component NLRP3 show clinical manifestations of cryopyrin- associated periodic syndromes (CAPS), which are characterized by skin rash, fever and joint pain. Further, abnormal and constant NLRP3 signaling has been associated with some chronic and degenerative diseases such as Alzheimer’s disease (AD), atherosclerosis, arthritis or cancer. A causative function of the NLRP3 inflammasome for neurodegenerative processes is supported by preclinical studies. These pre-clinical studies used whole body knock out mice to demonstrate that deficiencies of NLRP3, caspase-1 or the primary receptor for IL-1β, IL-1R1, protect mice from neurodegenerative processes. While providing important insights into the role of the NLRP3-inflammasome in neurodegenerative processes, these studies did not identify the relevant cell types in which the inflammasome is activated, the mechanisms underlying inflammasome activation and the consequences thereof, e.g. for intracerebral cross-talk. In addition, whether sterile inflammation triggered by the NLRP3 inflammasome impairs cognition in the setting of primarily peripheral diseases such as CKD remains unknown. To address these open questions, I used a mouse model of CKD, in which I detected NLRP3 inflammasome in brains. Interestingly, despite inflammasome activation in the brain, microglial caspase-1 deficiency did not improve cerebral inflammation and cognition in CKD mice. I identified noncanonical IL-1β maturation in microglia in CKD conditions, which was cathepsin c – caspase-8 mediated. Restoring K+ homeostasis in microglia or genetic inhibition of neuronal IL-1R1 signaling abolished CKD-induced cognitive impairment. Mechanistically, noncanonical IL-1β maturation and secretion from microglia promotes via IL-1R signaling cognitive impairment in neurons. This identifies a molecular mechanism of sterile CNS inflammation and the associated intercellular signaling pathway, which may be therapeutically amendable. Microglial K+ dyshomeostasis and noncanonical microglial IL-1β maturation may be druggable targets in some forms of cognitive impairment.:Content 2 List of abbreviations 5 Graphical abstract 8 2 Introduction 9 2.1 Chronic kidney disease and cognition 11 2.2 Microglia cells 13 2.3 The inflammasome, potassium dyshomeostasis in brain cells and thallium autometallography 15 2.4 Sterile inflammation in neurodegenerative diseases 17 3 Aims of the study 19 4 Materials and Methods 20 4.1 Reagents 20 4.2 Mice 27 4.3 CKD mouse model (5/6 nephrectomy model) 30 4.4 Evans Blue extravasation assay 32 4.5 2-photon microscopy 32 4.6 Analysis of mice 33 4.7 In vivo interventions 33 4.8 Histology and immunohistochemical analysis 34 4.9 Cell culture 34 4.10 Dextran permeability assay 35 4.11 Thallium-AMG (TlAMG), ex vivo and in vitro 36 4.12 Protein extraction and Western blotting 38 4.13 IL-1β ELISA 38 4.14 Reverse Transcriptase Polymerase Chain Reaction (RT–PCR) 38 4.15 Proximity ligation assay (PLA) 39 4.16 Behavioral analysis 39 4.17 Cathepsin c substrate assay 40 4.18 snRNA-Seq 41 4.19 Statistical Analysis 42 5 Results 43 5.1 Chronically impaired renal function leads to cognitive decline 43 5.2 Blood brain barrier (BBB) disruption in chronic kidney disease 44 5.3 Potassium dyshomeostasis in brain cells in CKD 45 5.4 CKD leads to microglia activation 46 5.5 Priming of microglia in CKD depends on potassium dyshomeostasis and its restoration improves cognition in CKD 49 5.6 TRAM34 ameliorates potassium dyshomeostasis and behavior in CKD 51 5.7 Uremia-induced cognitive impairment depends on microglia- neuron crosstalk via IL-1R1 52 5.8 Deciphering the microglial molecular pathway in CKD 56 5.9 Microglia activation in CKD is independent of NLRP3 56 5.10 Microglial IL-1β maturation occurs independently of the NLRP3-Caspase-1 inflammasome in CKD 57 5.11 The role of caspase- 8 in microglia activation in CKD 60 5.12 Lysosomal cathepsin c promotes microglia activation pivotal for caspase-8 activation 62 5.13 Broader implication of the pathway in other chronic peripheric diseases 63 5.14 Microglia inflammasome activation and IL-1β release is sufficient to induce cognitive impairment 64 5.15 Tables 66 6 Discussion 69 7 Summary 75 8 Zusammenfassung 80 9 References 86 10 Declaration about the independent preparation of the work 97 11 Presentation of own contribution 98 12 Curriculum vitae 99 13 Publications 104 14 Acknowledgments 106

Serum levels of apolipoprotein ApoC1 have been described in a number of systemic tumor entities as potential biomarkers, but little is known about ApoC1 in neurosurgical patients. A total of 230 serum samples from 96 patients were analyzed using an ELISA technique. Patient diagnoses comprised 70 glioblastomas WHO IV◦ , 10 anaplastic astrocytomas III◦ , one anaplastic oligodendroglioma III◦ , one oligodendroglioma II◦ , one diffuse astrocytoma II◦ , one pilocytic astrocytoma I◦ , and a single case of a spindle cell tumor without WHO grading, as well as 11 spinal interventions. The mean ApoC1 level of the 230 samples was 132.03 µg/mL (median 86.83, SD 292.91). In the 176 glioblastoma samples, the mean ApoC1 level was 130.0 µg/mL (median 86.23, SD 314.9), which was neither different from the whole group nor from patients with spinal interventions (215.1 µg/mL, median 63.6, SD 404.9). In the postoperative samples, the mean ApoC1 level was significantly lower (85.81 µg/mL) than in the preoperative samples (129.64 µg/mL) and in samples obtained during adjuvant chemotherapy (168.44 µg/mL). While absolute ApoC1 serum levels in a patient do not allow for the distinction between neurosurgical histological entities, future analyses will examine whether the time course of ApoC1 in an individual patient can be related to certain treatment stages.

Eukaryotic cells are characterized by having a clearly defined nucleus that encloses genetic material, separating it from the rest of the cell. The regulation of protein partitioning between the nucleus and cytoplasm is crucial for controlling specific cellular functions; any imbalance in protein partitioning can lead to a range of diseases, highlighting its significance in cellular physiology. The major mechanism for establishing nuclear composition is the trafficking of proteins in and out of the nucleus by karyopherins after the formation of the nuclear envelope. Regulating protein partitioning is particularly challenging during early embryonic development due to the rapid and successive cell divisions that lead to a dramatic reduction in cell size. Further, the embryo is maternally deposited with vast quantities of proteins that must be appropriately incorporated into an exponentially increasing number of nuclei. How such large quantities of proteins are robustly partitioned into the nucleus during these dynamic developmental processes remains unclear. During my Ph.D., I studied the nuclear partitioning of the Xenopus laevis embryonic linker histone H1.8, a protein that alters the chromatin structure in a concentration-dependent manner. By using quantitative microscopy of Xenopus laevis egg extract, I provide evidence H1.8 is partitioned into the nucleus through a mechanism independent of nuclear import. H1.8 has already been shown to be one of the earliest proteins to partition within the nucleus during embryonic development. However, I demonstrate that the import kinetics of H1.8 is insufficient to account for its rapid nuclear partitioning. Further, I discovered that H1.8 is present in the nucleus and cytoplasm as liquid condensates. As the nucleus expands and grows in interphase, the nuclear condensates dissolve, suggesting that they act as reservoirs of proteins, potentially for DNA replication. The dissolution also suggests that most nuclear H1.8 is already present during the nuclear assembly, thus indicating that the partition of H1.8 inside the nucleus is not solely dependent on its import. Prior to the formation of the nucleus, I observe that the surface of the chromatid nucleates H1.8 as condensates similar to the formation of dew droplets on a cold surface. These condensates are then sequestered into the nucleus as the cell cycle progresses, leading to the protein reservoirs we observe in the nucleus. Such a mechanism allows for instantaneous enrichment of excess H1.8 inside the nucleus. Furthermore, I demonstrate that the cytoplasmic H1.8 condensates modulate the nucleation of H1.8 on the chromatid surface by buffering the soluble concentration of H1.8. This ensures that the amount of surface condensates is independent of the nucleus-to-cytoplasmic ratio, thus potentially allowing for a fixed enrichment of proteins despite the reduction in cell size during embryonic development. Such a mechanism would be crucial for key structural proteins, like H1.8, that maintain DNA packaging and structure in a concentration-dependent manner. Taken together, I propose that the nucleation of condensates on the surface of mitotic chromatids and subsequent wetting can provide an alternative mechanism to nuclear trafficking in regulating nuclear composition.:1) Introduction 2) This work – aim and scope 3) The dynamics of H1.8 nuclear localization 4) H1.8 undergoes liquid-liquid phase separation in Xenopus laevis egg extract. 5) H1.8 condenses on the surface of chromatids. 6) Cytoplasmic condensates buffer the amount of surface condensates on chromatids. 7) Summary. 8) Future perspectives. 9) Methods

Gradients of morphogens, secreted signaling molecules, are crucial for providing cells with positional information during animal development. While the processes of forma- tion and interpretation of these gradients have been extensively studied, the impact of morphogenetic events on patterning through morphogen gradients remains largely unex- plored. This thesis aims to understand the interplay and feedback mechanisms between tissue shape and morphogen gradients formation. To address this, we developed an analysis pipeline using MATLAB to accurately measure morphogen gradients in curved epithelia. By computationally deforming confocal images of curved tissues, we quantified the levels of a protein of interest at a specified distance from a reference point along the apico-basal axis. Applying our pipeline to the Hedgehog morphogen gradient in the Drosophila eye and wing imaginal discs, which serve as model systems for folded and flat epithelial tissues, respectively, we made an intriguing discovery. Despite the distinct morphologies of these tissues, the decay rate of the Hedgehog gradient remained com- parable. This led us to investigate the robustness of Hedgehog gradient formation by manipulating the morphology of the wing and eye discs. We induced ectopic fold forma- tion at the boundary between the source and receiver tissue of Hedgehog in the wing disc. We found that the decay rate of the Hedgehog gradient remained unchanged even in the wing disc with perturbed morphology, supporting the notion that the Hedgehog gradient is robust towards variability in tissue shapes. Additionally, we locally flattened the eye disc by introducing a mutation that inhibited depolymerization of F-actin. This resulted in the inability of cells to form the morphogenetic furrow and in an expansion of the Hedgehog range compared to the wild-type. However, according to our quantifica- tion, the expansion in the Hedgehog range is to be attributed to a shift in its source rather than a change in decay rate of the gradient. Overall, by developing quantitative methods to analyze the distribution of signaling proteins in curved tissues, we contribute to the understanding of the interplay between tissue morphology and pattern formation through morphogen gradients. Our findings highlight the robustness of the Hedgehog gradient formation towards diverse tissue morphologies. This observation leads us to hypothesize that this property of robustness could extend to other morphogens that employ transport mechanisms similar to Hedgehog.:Contents Summary . . . . . . . . . . i 1 Introduction . . . . . . . . . . 1 1.1 Basic principles of animal development: an intricated story . . . . . . . . . . 1 1.2 Epithelial folds: a fundamental building block for morphogenesis . . . . . . . . . . 3 1.3 Patterning via morphogen gradients . . . . . . . . . . 4 1.4 Hedgehog gradient in Drosophila imaginal discs as a model system. . . . . . . . . . 13 2 Aims of the Thesis . . . . . . . . . . 21 2.1 Developing an analysis pipeline to quantify morphogen gradients in curved epithelia. . . . . . . . . . 21 2.2 Assessing the robustness of the Hedgehog morphogen gradient in naturally folded and flat tissues: the eye and wing imaginal discs . . . . . . . . . . 22 2.3 Testing the robustness of the Hedgehog gradient by perturbing the morphology of the wing and eye discs . . . . . . . . . . 22 3 Materials and methods . . . . . . . . . . 25 3.1 Fly stocks. . . . . . . . . . 25 3.2 Immunohistochemistry. . . . . . . . . . 28 3.3 Imaging . . . . . . . . . . 30 3.4 Data analysis. . . . . . . . . . 30 4 Results . . . . . . . . . . 47 4.1 Analysis pipeline to computationally flatten curved epithelial tissues: limitations in applicability and comparison to other methodologies. . . . . . . . . . 47 4.2 The Hedgehog gradient is comparable between wing and eye disc in Drosophila . . . . . . . . . . 54 4.3 The extracellular basal gradient of Hedgehog has a decay rate comparable to the one of the internalized morphogen . . . . . . . . . . 62 4.4 Folds in the wing do not affect the Hedgehog gradient. . . . . . . . . . 66 4.5 Downregulation of ci leads to lower levels of the Hedgehog receptors Ptc, which in turn results in a longer Hedgehog gradient . . . . . . . . . . 71 4.6 Local flattening of the morphogenetic furrow expands the source of Hedge- hog but does not affect the decay rate of the gradient . . . . . . . . . . 74 5 Discussion . . . . . . . . . . 83 5.1 Developing quantitative methods to analyze morphogen gradients in curved epithelia opens new possibilities to study the interplay between morphogens gradients and morphogenesis . . . . . . . . . . 83 5.2 A methodological consideration: the decay rate as a relevant parameter for assessing the robustness of the Hedgehog morphogen gradient . . . . . . . . . . 85 5.3 The decay rate of the Hedgehog gradient is comparable between the wing and the eye disc . . . . . . . . . . 90 5.4 The transport mechanism underlying the formation of the Hedgehog gra- dient in the wing disc is robust towards deformations of the apical side of the tissue . . . . . . . . . . 91 5.5 The capt mutation in the eye disc affects the signaling for differentiation without affecting the decay rate of the Hedgehog gradient . . . . . . . . . . 94 5.6 Active transport and binding to heparan sulfate proteoglypicans allow the Hedgehog morphogen gradient formation to be robust towards variation in tissuemorphology . . . . . . . . . . 98 5.7 Tissue morphology: obstacle or aid to patterning via morphogens . . . . . . . . . . 99 6 Conclusion. . . . . . . . . . 103 7 Acknowledgments . . . . . . . . . . 105 8 References . . . . . . . . . . 107 9 Declaration according to §5.5 of the doctorate regulations . . . . . . . . . . 117