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Item type: Item , Charakterisierung eines Mausmodells der humanen idiopathischen pulmonalen Fibrose: Rolle des alveolären endoplasmatischen Retikulum Stresses (ER-Stress)(2025) Smeda, Mohamed SmaidaDie idiopathische pulmonale Fibrose (IPF) ist eine chronisch progrediente und letale interstitielle Lungenerkrankung, deren Pathogenese noch nicht vollständig verstanden ist. Insbesondere der endoplasmatische Retikulum (ER)-Stress in alveolären Epithelzellen wird als ein zentraler pathogenetischer Faktor diskutiert. Ziel dieser Arbeit war die Charakterisierung eines Mausmodells der humanen IPF mit besonderem Fokus auf die Rolle des ER-Stresses und des Transkriptionsfaktors Chop. Hierzu wurde ein experimentelles Mausmodell unter Verwendung des murinen Gammaherpesvirus 68 (MHV-68) etabliert und im Sinne eines „Two-hit“-Modells untersucht. Die Analysen umfassten molekularbiologische Methoden wie qPCR und Proteinanalysen zur Untersuchung von ER-Stress-assoziierten Signalwegen. Die Ergebnisse zeigen, dass die Aktivierung des ER-Stresses und die Überexpression von Chop allein nicht ausreichen, um eine ausgeprägte fibrotische Reaktion im Mausmodell zu induzieren. Erst in Kombination mit einer zusätzlichen Belastung, wie einer viralen Infektion, kommt es zu einer verstärkten fibrotischen Antwort im Sinne eines „Two-hit“-Mechanismus. Zusammenfassend deuten die Daten darauf hin, dass ER-Stress und Chop eine wichtige Rolle in der Pathogenese der pulmonalen Fibrose spielen, jedoch zusätzliche Faktoren erforderlich sind, um die Erkrankung vollständig auszulösen. Dies unterstreicht die Bedeutung komplexer pathogenetischer Mechanismen bei der IPF.Item type: Item , Size Constancy in Motion: Neural Signatures of Perception in Dynamic Visual Environments(2025) Schellen, ElefIntroduction Perceptual constancy refers to our ability to correctly perceive stable properties of the outside world, despite the fact that their corresponding sensory signals are in flux. Size constancy in particular refers to our visual system’s ability to correctly perceive an object’s size despite the fact that the retinal image may shrink or grow as our distance to it changes. This constancy affords us a stable impression of the physical world around us, and allows us to perform essential actions that we may take for granted like catching a ball, estimating the width of a doorway and being able to determine whether an approaching animal is a cat or a tiger. Much of the literature on size constancy and its neural underpinnings makes use of static displays, which leaves a gap in our understanding of what happens during ordinary, everyday episodes where the distance between an object and the observer changes. The overarching question of this thesis therefore relates to the neurophysiology of size perception when things are moving. Motivation and research Question The literature on moving stimuli is mixed. Under impoverished cues, motion can hinder size and distance perception and encourage reliance on retinal size. In richer scenes there is evidence that movement can help maintain size constancy, but studies often use ambiguous stimuli or illusory depth. The ambiguity in prior work motivates testing in depth cue rich environments that mimic real life viewing conditions, while measuring how neural representations in early visual areas behave when stimuli move in depth with either physical size held constant or retinal size held constant. The experiments described in this thesis address the neurophysiology of size perception right before and after movement, when the stimulus has recently been in motion in a depth cue rich environment. To probe the activity in the early visual cortex, and more specifically the size of object representations in these areas, we will make use of steady state visual evoked potentials (SSVEP) measured via electroencephalography (EEG), a method with proven effectiveness. These experiments will take place both in Virtual Reality, (VR), and in a physical setup that preserves the same geometry and depth cues. Experiments in this thesis will examine whether the primary visual cortex continues to encode physical size (as it does under static conditions), or whether it reverts to a purely retinotopic representation when motion is involved. Conceptual Advances Across matched experiments in VR and using a physical setup, we find the same neuroscientific result, namely that for objects that have just moved in depth, early visual cortex does not show size constant encoding. The SSVEP responses reflect retinal size after motion in both implementations of the task. By contrast, a control experiment using static but otherwise comparable stimuli shows partial size constancy of object representations in early visual cortex, replicating previously published findings. The conceptual advance is a clear dissociation between dynamic and static conditions. Following motion, early visual cortex represents the fixated object in a retinotopic way, whereas in a completely static condition, partial size constancy emerges in these same brain regions. This dissociation marks a key conceptual advance. It suggests that motion alters the balance between feedforward and feedback processing in early visual cortex, perhaps tipping it in favor of bottom-up input at the expense of perceptual (top-down) tuning. The result is that the same region of cortex (V1) that supports size constancy under static conditions fails to do so immediately after motion—even when behavioural responses indicate correct, size constant perception. This means that size-constant representations in V1 are not a fixed property, but dynamically modulated by viewing conditions. The static control experiment’s results also show that our SSVEP approach is sensitive to size constant signals when they are present, and demonstrates the replicability of earlier findings of this effect. This is to say that our findings are most likely related to motion rather than due to the specific visual environment being used or a failure to replicate earlier findings. A potential explanation for these findings is that during more dynamic viewing conditions, bottom up processing in early visual cortex dominates over feedback processing, at least in the cortical layers that contribute most to the SSVEP. Our findings highlight a need to understand the temporal dynamics and laminar specificity of feedforward and feedback influences in early vision—especially under ecologically valid, dynamic conditions. Methodological contributions A second major contribution is methodological. This work pairs a carefully controlled VR environment with a custom apparatus that moves a physical monitor along the sagittal axis, and equates stimulus geometry, timing and depth cues across the two. This design makes it possible to test the ecological validity of the VR environment and data quality in VR without substantially changing the paradigm. Two insights follow: 1. Behavioral validity: despite differences between VR and real life in size and distance judgements reported in the literature, we find VR to be an appropriate medium for experimentation, provided that the virtual scene is cue rich and closely mirrors the physical setup. In the behavioural tasks, errors are essentially identical between VR and the physical setup, with an average difference under one millimetre. 2. EEG signal quality: SSVEP data quality is much reduced in VR. Headset interference with electrodes results in remarkably worse signal-to-noise ratios. Although VR offers unmatched control over complex dynamic scenes, this comes at the cost of EEG data quality and necessitates careful attention to signal integrity. The thesis also catalogues VR specific pitfalls that matter for experimental design. Distance is often overestimated in sparsely cued scenes. To keep SSVEP contrast stable, some cues (like shading) cannot be used, so scenes must be constructed with careful anchoring to preserve depth information. Comparing VR to a physical counterpart helps validate that these design choices do not distort the effects of interest. All things considered, this work finds support for the use of VR in neuroscientific experiments in vision sciences. Broader implications The broader implication of these findings is that we should not assume neuroscientific findings related to perceptual constancy which are collected from experiments using static conditions generalize to more ecologically valid, dynamic contexts. While we demonstrate this distinction for size perception in particular, it should be noted that other forms of perceptual constancy (such as shape, colour or brightness constancy) tend to be studied in static conditions, while in real world viewing, these mechanisms emerge in dynamic visual environments. Although the precise temporal characteristics of this transition remain to be mapped, and our data do not by themselves prove that this transition is caused by a shift in feedforward versus feedback balance. They do, however, motivate future experiments that are sensitive to more specific cortical layers and temporal dynamics, which could give us a clearer picture of the neural underpinnings of size perception specifically and visual perception more broadly. Ultimately, the thesis invites a rethinking of perceptual neuroscience: one that more fully embraces the complexity and dynamism of real-world vision.Item type: Item , Molecular aspects of Besnoitia besnoiti-driven bovine neutrophil extracellular trap formation(2026) Espinosa, GabrielRecent studies have collectively advanced our understanding of bovine neutrophil responses against tachyzoite stages of Besnoitia besnoiti, an apicomplexan parasite responsible for the re-emerging cattle disease bovine besnoitiosis. A central focus of the current work was to fill some gaps on the molecular mechanisms underlying the parasite-driven formation of neutrophil extracellular traps (NETs), a key innate effector mechanism. Therefore, in a first investigation, the role of neutrophil purinergic signalling was studied, revealing that B. besnoiti tachyzoites significantly induce NET formation without altering intracellular or extracellular ATP concentrations in bovine PMN. Despite this finding, extracellular supplementation with ATPγS, a non-hydrolyzable ATP analog, significantly enhanced NET release - specifically anchored NETs - whilst native ATP failed to do so. This response depended on the P2X1 purinergic receptor since treatments with the specific antagonist NF449 (IC50 = 1.27 μM) inhibited both NET formation and PMN clustering triggered by tachyzoite exposure. In contrast, pharmacological blockade of other purinergic receptors (P2Y2, P2Y6, P2X4, P2X7) did not affect NET formation, thereby highlighting a selective role for the P2X1 puringergic receptor in B. besnoiti tachyzoite-driven NET formation. Metabolic assays using Seahorse technology further revealed increased oxygen consumption rates (OCR) in tachyzoite-exposed PMN, while ATPγS treatment led to enhanced extracellular acidification rates (ECAR), suggesting mitochondrial involvement and metabolic adaptation during the early steps of the NETotic process. A second study complemented above-mentioned findings by focusing on the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK)/AMPK signalling axis and its link to autophagy. B. besnoiti and Toxoplasma gondii tachyzoite exposure induced rapid phosphorylation of AMPK in bovine PMN within 30 minutes, a response mirrored by the AMPK activator AICAR. Referring to signalling pathways, AMPK phosphorylation correlated with an activation of the upstream regulator CAMKK in both B. besnoiti and T. gondii-exposed PMN and upregulation of the downstream autophagy-related protein ULK-1 (but not Beclin- 1) in the case of B. besnoiti-exposed PMN, indicating a selective autophagic signature. Notably, AICAR treatments alone led to enhanced NET formation without compromising PMN viability. However, in B. besnoiti tachyzoite-exposed PMN, AICAR co-treatments failed to affect oxidative response. Moreover, AICAR co-treatments induced additive effects on tachyzoite-induced NET formation. In the case of T. gondii, this NET formation response further depended on MAPK and store-operated calcium entry (SOCE) pathways, as shown by reduced DNA release upon MAPK- and SOCE inhibition. These findings suggest that autophagy and AMPK signaling signify parallel, ROS-independent pathways involved in the support of B. besnoiti-driven NET formation. A third line of investigation explored the role of extracellular vesicles (EVs) as potential modulators of B. besnoiti-driven bovine neutrophil responses. To obtain EVs from differential sources, EVs were isolated from B. besnoiti tachyzoites, infected and non-infected bovine umbilical vein endothelial cells (BUVEC), and tachyzoite-exposed PMN. Their identity was confirmed via nano-flow cytometry, by EV markers like CD9 and CD81, and morphologically by transmission electron microscopy. When bovine PMN were exposed to differential EVs, particularly those derived from tachyzoites and infected BUVECs induced a significant NET release. This was validated microscopically by the presence of extracellular DNA structures adorned with histones and neutrophil elastase - hallmarks of classical NETs. Interestingly, PMN-derived EVs failed to trigger NET formation. Moreover, none of the different EV types drove changes in the neutrophil metabolic profile (oxidative and glycolytic responses) or ROS production in exposed bovine neutrophils, as assessed by Seahorse analysis and chemiluminescence assays, respectively. These findings indicated a NADPH oxidase (NOX)- independent mechanism of EV-induced NET formation. Furthermore, cytokine analyses revealed that EVs from infected BUVECs selectively induced IL-1β and IL-6 secretion in PMN, without influencing CXCL8 production, thereby pointing towards a tailored inflammatory response modulated by EVs of distinct cellular origin.Item type: Item , The role of remote sensing and geospatial modelling in mapping ecosystem condition und functioning(2024) Große-Stoltenberg, AndréThe main scope of this habilitation thesis is to present a broad range of approaches on how remote sensing and geospatial modelling can be applied to map condition and degradation of different types of ecosystems. Data from both active and passive remote sensing instruments acquired by aircraft and satellite platforms at single dates or in multitemporal mode were used in the presented studies. Main drivers of ecosystem degradation such as Land Use/Land Cover changes, climate change and invasive alien species are addressed, and different types of ecosystems such as urban areas, temperate forests, Central European agricultural landscapes, tropical high-mountain grassland and dryland rangelands are examined. Beyond technical remote sensing aspects, the studies give insights on the importance of spatial modeling approaches in ecological applications, e.g. to account for the spatial heterogeneity of ecosystem properties. However, the increasing availability of remote sensing data poses both new opportunities and challenges in ecological or ecosystem studies, which are addressed in the conceptual and review paper of this thesis, e.g., dealing with large data volumes during processing and analysis. Future progress can be expected in fast evolving fields such as the use of drones, sensor-networks and crowd-sourced data together with interpretable artificial intelligence to study ecosystems. Further, very high resolution multispectral, thermal and radar satellite data is becoming more and more available, adding to the already filled toolbox from a data point of view. Nevertheless, a profound understanding of the ecological processes or mechanisms to be studied remains a fundamental requirement, as technological progress does not per se imply progress in using sensor-based approaches to study ecological phenomena. The increasing amount of studies and initiatives joining knowledge from different disciplines demonstrate that if we succeed in integrating expertise from different backgrounds, then we can create an innovative atmosphere that can lead to progress in using novel technologies to tackle urgent challenges regarding the degradation of our ecosystems.Item type: Item , Unveiling Hematopoietic Stem Cell Dynamics: Identification and Isolation of Hematopoietic Stem Cells at Single-Cell Level(2025-06) Schmachtel, TessaThe human bone marrow constitutes the principal site of hematopoiesis, sustaining the continuous generation of platelets, erythrocytes and immune cells through the activity of hematopoietic stem and progenitor cells (HSPCs) residing within specialized microenvironmental niches. Within this compartment, a rare subset of hematopoietic stem cells (HSCs) maintains lifelong hematopoiesis by precisely regulating the balance between self-renewal and differentiation, predominantly persisting in a quiescent state under homeostatic conditions but capable of rapid activation in response to acute physiological stressors such as infection or hemorrhage. The equilibrium between steady-state and emergency hematopoiesis is fundamental to hematopoietic homeostasis; however, chronic inflammation and aging can disrupt this balance, leading to impaired blood cell production and increased risk of developing hematologic malignancies. Although substantial progress has been made in the characterization and prospective isolation of distinct human HSPC subsets using surface marker-based strategies, current strategies often yield in heterogeneous populations and several differentiation models have been proposed. To address this challenge and map differentiation trajectories, the presented research employed a single cell Transcriptomic/AbSeq approach, simultaneously quantifying the expression of 596 genes at mRNA level and 46 surface markers, on over 62,000 FACS-enriched HSPCs from 15 healthy donors across different age groups. Comprehensive computational analysis revealed four main lineage pathways, supporting a stepwise model of differentiation with an early branching point for megakaryocyte-erythroid progenitors. Notably, HSPCs from older donors exhibited a higher proportion of undifferentiated cells and diminished differentiation across all lineages. A key finding of this study is the identification of Programmed death ligand 2 (PD-L2/CD273) as a surface marker highly expressed on the most primitive HSPCs. CD273/PD-L2-positive HSPCs, isolated from mobilized PB samples, demonstrated a distinct molecular signature characterized by the enrichment of stemness genes such as Thy1, DLK1 and MPL, as well as delayed entry into the cell cycle, reduced mitochondrial activity and delayed in vitro differentiation indicating a deeper quiescent state. Functional assays, including in vitro colony forming assays and in vivo xenograft experiments, confirmed that CD273/PD-L2high HSPCs possess the capacity for multi-lineage reconstitution. Beyond their stem cell properties, CD273/PD-L2-expressing HSPCs exhibited notable immunomodulatory functions. In allogeneic lymphocyte reaction assays, these cells suppressed CD8+ T-cell proliferation and activation. Blocking PD-L2 promoted the secretion of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-6), showed a reduced abundance of regulatory T-cells and a shift toward myeloid lineage bias in HSPCs, implying an increased inflammatory response reaction. These findings highlight a dual role for PD-L2: First as a marker of more quiescent, primitive HSPCs and second as a mediator of immune regulation within the hematopoietic compartment. In summary, this integrated study provides valuable insights into the organization of human hematopoiesis and the identification of PD-L2/CD273 as a defining marker of a particularly quiescent, immunomodulatory HSPC subset. These discoveries may have important implications for stem cell transplantation and the treatment of blood malignancies, as understanding and manipulating these pathways may improve therapeutic outcomes and help maintaining healthy hematopoiesis throughout life.