@phdthesis{Schlegel2021,
  author    = {Schlegel, Jan},
  title     = {Super-Resolution Microscopy of Sphingolipids and Protein Nanodomains},
  doi       = {10.25972/OPUS-22959},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-229596},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The development of cellular life on earth is coupled to the formation of lipid-based biological membranes. Although many tools to analyze their biophysical properties already exist, their variety and number is still relatively small compared to the field of protein studies. One reason for this, is their small size and complex assembly into an asymmetric tightly packed lipid bilayer showing characteristics of a two-dimensional heterogenous fluid. Since membranes are capable to form dynamic, nanoscopic domains, enriched in sphingolipids and cholesterol, their detailed investigation is limited to techniques which access information below the diffraction limit of light. In this work, I aimed to extend, optimize and compare three different labeling approaches for sphingolipids and their subsequent analysis by the single-molecule localization microscopy (SMLM) technique direct stochastic optical reconstruction microscopy (dSTORM). First, I applied classical immunofluorescence by immunoglobulin G (IgG) antibody labeling to detect and quantify sphingolipid nanodomains in the plasma membrane of eukaryotic cells. I was able to identify and characterize ceramide-rich platforms (CRPs) with a size of ~ 75nm on the basal and apical membrane of different cell lines. Next, I used click-chemistry to characterize sphingolipid analogs in living and fixed cells. By using a combination of fluorescence microscopy and anisotropy experiments, I analyzed their accessibility and configuration in the plasma membrane, respectively. Azide-modified, short fatty acid side chains, were accessible to membrane impermeable dyes and localized outside the hydrophobic membrane core. In contrast, azide moieties at the end of longer fatty acid side chains were less accessible and conjugated dyes localized deeper within the plasma membrane. By introducing photo-crosslinkable diazirine groups or chemically addressable amine groups, I developed methods to improve their immobilization required for dSTORM. Finally, I harnessed the specific binding characteristics of non-toxic shiga toxin B subunits (STxBs) and cholera toxin B subunits (CTxBs) to label and quantify glycosphingolipid nanodomains in the context of Neisseria meningitidis infection. Under pyhsiological conditions, these glycosphingolipids were distributed homogenously in the plasma membrane but upon bacterial infection CTxB detectable gangliosides accumulated around invasive Neisseria meningitidis. I was able to highlight the importance of cell cycle dependent glycosphingolipid expression for the invasion process. Blocking membrane accessible sugar headgroups by pretreatment with CTxB significantly reduced the number of invasive bacteria which confirmed the importance of gangliosides for bacterial uptake into cells. Based on my results, it can be concluded that labeling of sphingolipids should be carefully optimized depending on the research question and applied microscopy technique. In particular, I was able to develop new tools and protocols which enable the characterization of sphingolipid nanodomains by dSTORM for all three labeling approaches.},
  subject      = {Sphingolipide},
  language  = {en}
}
@phdthesis{Erbacher2023,
  author    = {Erbacher, Christoph},
  title     = {Systemic and local mechanisms of small fiber pathology in female patients with fibromyalgia syndrome},
  doi       = {10.25972/OPUS-29020},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-290203},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Fibromyalgia syndrome (FMS) is a largely heterogeneous chronic pain syndrome of unclear pathophysiology, which lacks objective diagnostics and specific treatment. An immune-related shift towards a pro-inflammatory profile is discussed at a systemic level. Small fiber pathology (SFP) and local participation of non-neuronal skin cells like keratinocytes in cutaneous nociception are potential peripheral contributors. Small RNAs, particularly microRNAs (miRs) and newly described tRNA fragments (tRFs) act as posttranscriptional key regulators of gene expression and may modulate systemic and peripheral cell pathways. On cellular level, the exact mechanisms of keratinocyte-intraepidermal nerve fiber (IENF) interaction in the skin are insufficiently understood. Via small RNA sequencing and quantitative real-time PCR, we investigated miR and tRF signatures in whole blood cells and skin biopsy-derived keratinocytes of female FMS patients versus healthy controls. We applied gene target prediction analysis to uncover underlying cellular pathways affected by dysregulated small RNAs. Altered FMS small RNAs from blood were compared with their expression in disease controls, i.e. Parkinson`s patients and patients with major depression and chronic pain. Association of SFP with small RNAs was investigated via correlation with clinical parameter. To explore keratinocyte-nerve fiber interactions with high relevance for SFP and cutaneous nociception, we adapted a super-resolution array tomography (srAT) approach and expansion microscopy (ExM) for human skin samples. Further, we created a fully human 2D co-culture model of primary keratinocytes and induced pluripotent stem cell derived sensory neurons. Blood miR deregulation indicated systemic modulation of immune processes exerted by CholinomiRs and by miRs targeting the FoxO signaling pathway. Short sized tRFs were associated with mRNA metabolism and splicing. This supports the hypothesis of an inflammatory/autoimmunity component in FMS. Expression of blood small RNAs in FMS were discriminative against disease controls, highlighting their potential as objective biomarker. Blood small RNAs were predominantly upregulated and correlations between miR and clinical parameter reflected rather pain in general than SFP. In FMS keratinocytes, a downregulation of miRs and tRFs was evident. Pathways for adenosine monophosphate-activated protein kinase (AMPK), adherens junction, and focal adhesion were predicted to be affected by miRs, while tRFs may influence proliferation, migration, and cell growth. Similar to blood miRs, altered miRs in keratinocytes correlated mostly with widespread pain and pain severity parameter. TRFs were partially associated with more severe IENF loss. Small RNAs in FMS keratinocytes may modulate pathways that define how keratinocytes interact with each other and with IENF. These interactions include nerve fiber ensheathment, a conserved epithelial mechanism, which we visualize in human epidermis and a fully human co-culture model. Additionally, we revealed plaques of connexin 43, a pore forming protein involved in intercellular communication, at keratinocyte- nerve fiber contact sites. Objective quantification of these morphological findings in FMS and other diseases with SFP may inherit diagnostic value similar to IENF density. We provide evidence for distinct miR and tRF signatures in FMS with implications for systemic immune regulation and local cell-cell interaction pathways. In the periphery we explored novel keratinocyte-nerve fiber interactions relevant for SFP and cutaneous nociception.},
  subject      = {Fibromyalgiesyndrom},
  language  = {en}
}
@phdthesis{Ehmann2015,
  author    = {Ehmann, Nadine},
  title     = {Linking the active zone ultrastructure to function in Drosophila},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-118186},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Accurate information transfer between neurons governs proper brain function. At chemical synapses, communication is mediated via neurotransmitter release from specialized presynaptic intercellular contact sites, so called active zones. Their molecular composition constitutes a precisely arranged framework that sets the stage for synaptic communication. Active zones contain a variety of proteins that deliver the speed, accuracy and plasticity inherent to neurotransmission. Though, how the molecular arrangement of these proteins influences active zone output is still ambiguous. Elucidating the nanoscopic organization of AZs has been hindered by the diffraction-limited resolution of conventional light microscopy, which is insufficient to resolve the active zone architecture on the nanometer scale. Recently, super-resolution techniques entered the field of neuroscience, which yield the capacity to bridge the gap in resolution between light and electron microscopy without losing molecular specificity. Here, localization microscopy methods are of special interest, as they can potentially deliver quantitative information about molecular distributions, even giving absolute numbers of proteins present within cellular nanodomains. This thesis puts forward an approach based on conventional immunohistochemistry to quantify endogenous protein organizations in situ by employing direct stochastic optical reconstruction microscopy (dSTORM). Focussing on Bruchpilot (Brp) as a major component of Drosophila active zones, the results show that the cytomatrix at the active zone is composed of units, which comprise on average ~137 Brp molecules, most of which are arranged in approximately 15 heptameric clusters. To test for a quantitative relationship between active zone ultrastructure and synaptic output, Drosophila mutants and electrophysiology were employed. The findings indicate that the precise spatial arrangement of Brp reflects properties of short-term plasticity and distinguishes distinct mechanistic causes of synaptic depression. Moreover, functional diversification could be connected to a heretofore unrecognized ultrastructural gradient along a Drosophila motor neuron.},
  subject      = {Taufliege},
  language  = {en}
}