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

Living systems are subject to various types of spatial and temporal noise at all scales and stages. Nevertheless, evolving under the pressure of natural selection, biology has mastered the ability of dealing with stochasticity. This is particularly crucial because these systems encounter numerous situations which require taking robust and proper actions in the presence of noise. Due to the complexity and variability of these situations, it is impossible to have a prescribed plan for an organism that keeps it alive and fully functional. Therefore, they have to be active, rather than passive, by following three essential steps: I) gathering information about their fluctuating environment, II) processing the information and making decisions via circuits that are inevitably noisy, and finally, III) taking the appropriate action robustly with organizations crossing multiple scales. Although various aspects of this general scheme have been subject of many studies, there are still many questions that remain unanswered: How can a dynamic environmental signal be sensed collectively by cell populations? and how does the topology of interactions affect the quality of this sensing? When processing information via the regulatory network, what are the drawbacks of multifunctional circuits? and how does the reliability of the decisions decrease as the multifunctionality increases? Finally, when the right decision is made and a tissue is growing with feedbacks crossing different scales, what are the crucial features that remain preserved from one subject to another? How can one use these features to understand the mechanisms behind these processes? This thesis addresses the main challenges for answering these questions and many more using methods from dynamical systems, network science, and stochastic processes. Using stochastic models, we investigate the fundamental limits arising from temporal noise on collective signal sensing and context-dependent information processing. Furthermore, by combining stochastic models and cross-scale data analyses, we study pattern formation during complex tissue growth. / Lebende Systeme sind in allen Größenordnungen und Stadien verschiedenen Arten von räumlichem und zeitlichem Rauschen ausgesetzt. Dennoch hat die Biologie, die sich unter dem Druck der natürlichen Selektion entwickelt hat, die Fähigkeit gemeistert, mit stochastischen Fluktuationen umzugehen. Dies ist besonders wichtig, da Organismen auf zahlreiche Situationen stoßen, die es erfordern, in Gegenwart von Rauschen robuste und angemessene Maßnahmen zu ergreifen. Aufgrund der Komplexität und Variabilität dieser Situationen ist es unmöglich, einen vorgeschriebenen Plan für einen Organismus zu haben, der ihn überlebens- und funktionsfähig hält. Daher können Organismen sich nicht passiv verhalten, sondern befolgen aktiv drei wesentliche Schritte: I) Das Sammeln von Informationen über ihre dynamische Umgebung, II) Das Verarbeiten von Informationen und das Treffen von Entscheidungen über Regelnetzwerke, die unvermeidlich mit Rauschen behaftet sind, und schließlich, III) das robuste Funktionieren durch organisierte Maßnahmen, welche mehrere Größenordnungen überbrücken. Obwohl verschiedene Aspekte dieses allgemeinen Schemas Gegenstand vieler Studien waren, bleiben noch viele Fragen unbeantwortet: Wie kann ein dynamisches externes Signal kollektiv von Zellpopulationen wahrgenommen werden? Wie beeinflusst die Topologie der Interaktionen die Qualität dieser Wahrnehmung? Was sind die Nachteile multifunktionaler Schaltkreise bei der Verarbeitung von Informationen über das Regelnetzwerk? Wie nimmt die Zuverlässigkeit der Entscheidungen mit zunehmender Multifunktionalität ab? Und abschließend, wenn die richtige Entscheidung getroffen wurde und ein Gewebe wächst und dabei Rückkopplungen auf verschiedenen Größenordnungen erfährt, was sind die entscheidenden Merkmale, die von einem Versuchsobjekt zum anderen erhalten bleiben? Wie kann man diese Merkmale nutzen, um die Prozesse zu verstehen? Diese Arbeit befasst sich mit den wichtigsten Herausforderungen zur Beantwortung dieser und vieler weiterer Fragen mit Methoden aus dynamischen Systemen, Netzwerkforschung und stochastischen Prozessen.

Findings on executive functions (EFs) in binge-eating disorder (BED) are inconsistent and possibly biased by associated comorbidities. This study aimed to identify whether distinct levels of physical and mental comorbidity are related to EFs in BED. General and food-specific EFs in n = 77 adults with BED were compared to population-based norms and associations with weight status, depressive symptoms, and eating disorder psychopathology were analyzed. To detect within-sample patterns of EF performance, k-means clustering was applied. The results indicated that participants’ general EFs were within the average range with slight deficits in alertness. While depression and eating disorder psychopathology were unrelated to EFs, weight status was associated with food-specific attentional bias that was significantly higher in obesity class 2 than in overweight/obesity class 1 and obesity class 3. Four meaningful clusters with distinct strengths and impairments in general and food-specific EFs but without differences in clinical variables were identified. Altogether, adults with BED showed few specific deficits compared to normative data. Performance was unrelated to depression and eating disorder psychopathology, while weight status was associated with food-specific EFs only. The results highlight the need for longitudinal studies to evaluate the relevance of EFs in BED development and maintenance in neurologically healthy adults.

Malignant vascular tumors of the head and neck are rare neoplasms with variable clinical presentation, wide age distribution, and variable clinical courses. The heterogeneous presentation of angiosarcomas and epithelioid hemangioendothelioma often leads to misdiagnosis and unsuitable treatment. While risk factors for angiosarcomas are previous radiation, chronic lymphedema, and exposure to arsenic, thorium oxide, or vinyl chloride, there are only limited and retrospective data available on prognostic factors in EHE. In both angiosarcomas and EHE, surgery is the mainstay of treatment. There is limited evidence regarding the role of radiotherapy in EHE, although EHE is considered relatively radiosensitive. In angiosarcomas, adjuvant radiotherapy is recommended according to retrospective case series. A standard medical therapy for metastasized malignant vascular tumors is lacking. Chemotherapy, which is effective in angiosarcoma, is mostly ineffective in EHE. Targeted therapy, antiangiogenetic drugs and immunotherapy have been studied as new treatment options. The goal of this review is to summarize the current data regarding malignant vascular tumors along with their diagnosis and management.

Overexpression of members of the HER/erbB transmembrane tyrosine kinase family like HER2/erbB2/neu is associated with various cancers. Some heterodimers, especially HER2/HER3 heterodimers, are particularly potent inducers of oncogenic signaling. Still, from a clinical viewpoint their inhibition has yielded only moderate success so far, despite promising data from cell cultures. This suggests acquired resistance upon inhibitor therapy as one putative issue, requiring further studies in cell culture also aiming at rational combination therapies. In this paper, we demonstrate in ovarian carcinoma cells that the RNAi-mediated single knockdown of HER2 or HER3 leads to the rapid counter-upregulation of the respective other HER family member, thus providing a rational basis for combinatorial inhibition. Concomitantly, combined knockdown of HER2/HER3 exerts stronger anti-tumor effects as compared to single inhibition. In a tumor cell line xenograft mouse model, therapeutic intervention with nanoscale complexes based on polyethylenimine (PEI) for siRNA delivery, again reveals HER3 upregulation upon HER2 single knockdown and a therapeutic benefit from combination therapy. On the mechanistic side, we demonstrate that HER2 knockdown or inhibition reduces miR-143 levels with subsequent de-repression of HER3 expression, and validates HER3 as a direct target of miR-143. HER3 knockdown or inhibition, in turn, increases HER2 expression through the upregulation of the transcriptional regulator SATB1. These counter-upregulation processes of HER family members are thus based on distinct molecular mechanisms and may provide the basis for the rational combination of inhibitors.

The use of the plasticizer bis(2-ethylhexyl)phthalate (DEHP) and other plasticizers in the manufacture of plastic products has been restricted due to adverse health outcomes such as obesity, metabolic syndrome, and asthma, for which inflammation has been described to be a driving factor. The emerging alternative plasticizer 1,2-cyclohexanedioic acid diisononyl ester (DINCH) still lacks information regarding its potential effects on the immune system. Here, we investigated the effects of DINCH and its naturally occurring metabolite monoisononylcyclohexane-1,2-dicarboxylic acid ester (MINCH) on the innate immune response. Human THP-1 macrophages were exposed to 10 nM–10 μM DINCH or MINCH for 4 h, 16 h, and 24 h. To decipher the underlying mechanism of action, we applied an untargeted proteomic approach that revealed xenobiotic-induced activation of immune-related pathways such as the nuclear factor κB (NF-κB) signaling pathway. Key drivers were associated with oxidative stress, mitochondrial dysfunction, DNA damage repair, apoptosis, and autophagy. We verified increased reactive oxygen species (ROS) leading to cellular damage, NF-κB activation, and subsequent TNF and IL-1β release, even at low nM concentrations. Taken together, DINCH and MINCH induced cellular stress and pro-inflammatory effects in macrophages, which may lead to adverse health effects.

The heart works as a functional syncytium, which is realized via cell-cell coupling maintained by gap junction channels. These channels connect two adjacent cells, so that action potentials can be transferred. Each cell contributes a hexameric hemichannel (=connexon), formed by protein subuntis named connexins. These hemichannels dock to each other and form the gap junction channel. This channel works as a low ohmic resistor also allowing the passage of small molecules up to 1000 Dalton. Connexins are a protein family comprising of 21 isoforms in humans. In the heart, the main isoforms are Cx43 (the 43 kDa connexin; ubiquitous), Cx40 (mostly in atrium and specific conduction system), and Cx45 (in early developmental states, in the conduction system, and between fibroblasts and cardiomyocytes). These gap junction channels are mainly located at the polar region of the cardiomyocytes and thus contribute to the anisotropic pattern of cardiac electrical conductivity. While in the beginning the cell–cell coupling was considered to be static, similar to an anatomically defined structure, we have learned in the past decades that gap junctions are also subject to cardiac remodeling processes in cardiac disease such as atrial fibrillation, myocardial infarction, or cardiomyopathy. The underlying remodeling processes include the modulation of connexin expression by e.g., angiotensin, endothelin, or catecholamines, as well as the modulation of the localization of the gap junctions e.g., by the direction and strength of local mechanical forces. A reduction in connexin expression can result in a reduced conduction velocity. The alteration of gap junction localization has been shown to result in altered pathways of conduction and altered anisotropy. In particular, it can produce or contribute to non-uniformity of anisotropy, and thereby can pre-form an arrhythmogenic substrate. Interestingly, these remodeling processes seem to be susceptible to certain pharmacological treatment.

The pathophysiology of heart failure with preserved ejection fraction (HFpEF) is poorly understood and therapeutic strategies are lacking. This study aimed to identify plasma proteins with pathophysiological relevance in HFpEF and with respect to spironolactone-induced effects. We assessed 92 biomarkers in plasma samples from 386 HFpEF patients—belonging to the Aldo-DHF trial—before (baseline, BL) and after one-year treatment (follow up, FU) with spironolactone (verum) or a placebo. At BL, various biomarkers showed significant associations with the two Aldo-DHF primary end point parameters: 33 with E/e’ and 20 with peak VO2. Ten proteins including adrenomedullin, FGF23 and inflammatory peptides (e.g., TNFRSF11A, TRAILR2) were significantly associated with both parameters, suggesting a role in the clinical HFpEF presentation. For 13 proteins, expression changes from BL to FU were significantly different between verum and placebo. Among them were renin, growth hormone, adrenomedullin and inflammatory proteins (e.g., TNFRSF11A, IL18 and IL4RA), indicating distinct spironolactone-mediated effects. BL levels of five proteins, e.g., inflammatory markers such as CCL17, IL4RA and IL1ra, showed significantly different effects on the instantaneous risk for hospitalization between verum and placebo. This study identified plasma proteins with different implications in HFpEF and following spironolactone treatment. Future studies need to define their precise mechanistic involvement.

T cells are an essential part of the immune system. They determine the specificity of the immune response to foreign substances and, thus, help to protect the body from infections and cancer. Recently, T cells have gained much attention as promising tools in adoptive T cell transfer for cancer treatment. However, it is crucial not only for medical purposes but also for research to obtain T cells in large quantities, of high purity and functionality. To fulfill these criteria, efficient and robust isolation methods are needed. We used three different isolation methods to separate CD3-specific T cells from leukocyte concentrates (buffy coats) and Ficoll purified PBMCs. To catch the target cells, the Traceless Affinity Cell Selection (TACS®) method, based on immune affinity chromatography, uses CD-specific low affinity Fab-fragments; while the classical Magnetic Activated Cell Sorting (MACS®) method relies on magnetic beads coated with specific high affinity monoclonal antibodies. The REAlease® system also works with magnetic beads but, in contrast to MACS®, low-affinity antibody fragments are used. The target cells separated by TACS® and REAlease® are “label-free”, while cells isolated by MACS® still carry the cell specific label. The time required to isolate T cells from buffy coat by TACS® and MACS® amounted to 90 min and 50 min, respectively, while it took 150 min to isolate T cells from PBMCs by TACS® and 110 min by REAlease®. All methods used are well suited to obtain T cells in large quantities of high viability (>92%) and purity (>98%). Only the median CD4:CD8 ratio of approximately 6.8 after REAlease® separation differed greatly from the physiological conditions. MACS® separation was found to induce proliferation and cytokine secretion. However, independent of the isolation methods used, stimulation of T cells by anti CD3/CD28 resulted in similar rates of proliferation and cytokine production, verifying the functional activity of the isolated cells.