Naturwissenschaften

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    Biophysical characterization and analysis of different mycobacterial WXG100 proteins to study possible inter-loci complex formation
    (2025) Kallenberg, Christina Jana Luise
    Tuberculosis has remained a global public health concern for the past 30 years. The causative agent Mycobacterium tuberculosis has developed several strategies to evade the host immune system and for intracellular survival. Extensive genomic and proteomic studies have revealed hundreds of genes involved in the bacterium’s resistance and survival. The first known secreted virulence factors, EsxA and EsxB, are the paragons of a group of heterologous proteins, termed the “WXG100 family”. M. tuberculosis has 23 WXG100 proteins (EsxA-EsxW), five of such protein pairs are found within a genomic cluster making up a type-VII-secretion system (ESX-1 to ESX-5). Mycobacterial WXG100 proteins appear to share a similar secondary structure and form complexes with neighboring co-expressed proteins. The question arises as to whether Esx proteins from different genomic loci, i.e. not within the same operon, can form complexes. If this were the case, even only for some, the recombination of individual complex partners could greatly increase the versatility of the bacterium in reacting to the numerous host defense mechanisms. In this work, ten different mycobacterial Esx proteins were recombinantly expressed and purified in order to evaluate the possibility of inter-loci complex formation and their structural properties. The results suggest that inter-loci complex formation between several Esx proteins is possible. Special focus was put on the complex formation between EsxB and EsxT, which was evaluated with chemical crosslinking studies and single-molecule FRET experiments. Kinetic evaluations suggest a rather low binding constant between EsxB and EsxT (mM range), compared to the high binding constant between the native partners EsxB and EsxA (nM range). The proteins EsxF and EsxE were found to be highly heat resistant, both in complex and monomeric form, with a high structural recovery of over 90 % after heating to 96 °C. In addition, evidence of homo-dimer or homo-oligomer formation was found for EsxF and EsxU, which has not been reported previously.
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    Robotische, endoskopische und dynamische Megahertz-Optische Kohärenztomographie und deren Anwendungen in Klinik und Forschung
    (2025) Göb, Madita
    Die Optische Kohärenztomographie (OCT, engl. optical coherence tomography) hat sich als leistungsstarke Technik zur dreidimensionalen Visualisierung von Gewebestrukturen etabliert. Forschungsergebnisse zeigen das enorme Potenzial dieser Technologie zwischen gesundem und pathologischem Gewebe zu unterscheiden. Im Gegensatz zur Biopsie ist OCT nicht-invasiv und liefert sofortige diagnostische Ergebnisse. Dennoch stoßen handelsübliche OCT-Systeme aufgrund langsamer Scangeschwindigkeiten und kleiner Bildfelder im klinischen Alltag an ihre Grenzen. Die MHz-OCT, eine Weiterentwicklung mit Linienraten im Megahertz-Bereich, überwindet diese Einschränkungen und ermöglicht ultraschnelle 3D-Bildgebung in Echtzeit. Um die hohe Geschwindigkeit der MHz-OCT gezielt nutzbar zu machen, wurden im Rahmen dieser Arbeit drei neuartige Systeme entwickelt und ihre Anwendungen in spezifischen medizinischen und wissenschaftlichen Bereichen erforscht: 1. Robotische MHz-OCT: Eine robotergestützte Plattform wurde für die automatisierte Bildgebung großer Bereiche entwickelt. Mit zwei verschiedenen Robotersystemen wird eine präzise Abtastung ganzer Hautareale in vivo demonstriert sowie die Eignung als automatisiertes Hochdurchsatz-Messgerät für innovative ex vivo Forschungsansätze in der Dermatologie. 2. Endoskopische MHz-OCT: Ein 4D-Echtzeit-OCT-Endoskop wurde konzipiert, das die Lumen-Topografie abbildet und mikroskopische Einblicke in die Gewebemorphologie liefert. Kolorektale Bildgebungsexperimente an Körperspendern zeigen die mögliche Anwendung zur Früherkennung gastrointestinaler Läsionen und der Beurteilung tiefer Gewebeschichten. 3. Dynamische MHz-OCT: Zur Analyse physiologischer und metabolischer Prozesse wurde die MHz-OCT um ein Verfahren zur dynamischen Bildgebung erweitert. Diese Technologie bietet durch zusätzlichen funktionellen Kontrast detaillierte Einblicke in zelluläre Strukturen, ähnlich der Histologie, und deren Funktion. Die Methodik wird an ex vivo Gewebe demonstriert. Diese Ansätze zeigen das Potenzial der MHz-OCT, diagnostische Verfahren nicht nur zu ergänzen, sondern auch neue Anwendungsfelder in der medizinischen Bildgebung zu erschließen. Die vorliegende Arbeit beschreibt die Entwicklung, Validierung und Anwendung dieser Methoden sowie deren Beitrag zur Integration der MHz-OCT in die klinische Praxis.
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    One miR to rule them all
    (2025) Naujack, Alison-Michelle
    Metabolic dysfunction-associated steatotic liver disease (MASLD) affects 30% of the global population. With rising numbers it presents a risk for global health and health care systems. The development of MASLD and the more severe stage, metabolic dysfunction-associated steatohepatitis (MASH), is driven not only by comorbidities like obesity and type 2 diabetes, but also by hypothyroidism. Independent of the systemic thyroid hormone levels, dysregulation of hepatic thyroid hormone signaling plays an important role in MASLD pathogenesis, with the only approved medication for MASH treatment, resmetirom, being a thyroid hormone receptor beta (THRB) agonist. While the effect of thyroid hormone signaling dysregulation on the hepatic metabolism has been investigated thoroughly there is little information on whether epigenetic regulations contribute to this dysregulation in MASLD. Therefore, the aim of this thesis was the investigation of epigenetic regulations on hepatic thyroid hormone signaling in MASLD. Since the expression of THRB negatively correlates with the disease progression marker MASLD activity score (MAS) and since miRNAs have inhibiting effects on their target genes, miRNAs that potentially bind to THRB were identified. The expression of miRNAs and mRNAs potentially involved in the regulation of thyroid hormone action in the liver was measured in human and mouse liver tissue using qPCR. Verification of increased expression of potential THRB targeting miRNAs was performed in a cohort of obese individuals (BMI > 30 kg/m 2) which was divided into a non-MASH group (n = 41, MAS ≤ 3) and MASH group (n = 27, MAS ≥ 4), as well as in multiple mouse models mimicking different stages of MASLD. Binding of miRNA to target mRNAs was verified using luciferase reporter assays, as well as RNA-interacting protein immunoprecipitation-sequencing. Furthermore, cell culture models were metabolically stimulated to induce a MASLD-like state in the cells and investigate the effect on the expression of thyroid hormone metabolism genes. Additionally, DNA methylation was measured at THRB and miRNA genes, to further investigate involvement of additional epigenetic regulators. Unfortunately, no suitable THRB antibody was available to verify the results of mRNA measurements on the protein level. To address this, plasmids were designed to identify tags, that when added to THRB do not interfere with thyroid hormone signaling. miR-34a-5p was identified to be increased in MASLD and was predicted to bind not only THRB but also the thyroid hormone receptor alpha, the thyroid hormone activating enzyme DIO1 as well as the thyroid hormone transporters SLC10A1 and SCL16A2. The binding to THRB and DIO1 was verified and induced overexpression of miR-34a-5p reduced the levels of THRB regulated genes after stimulation with thyroid hormones. Cell culture experiments further showed induction of miR-34a-5p by stimulation with fatty acids. Interestingly, the results of the overexpression of miR-34a-5p on potential target genes could also be induced by stimulation with fructose and insulin without miR-34a- 5p involvement. Repression of thyroid hormone signaling through miR-34a-5p, reducing the expression of THRB as well as DIO1 which reduces the amount of available active thyroid hormone further, is exacerbated by increased DNA methylation in the THRB gene, which negatively correlates with THRB expression. Finally, treatment of high-fat diet fed mice with metformin reduced the expression level of miR-34a-5p. This reduced dysregulation of the miRNA could potentially recover THRB expression and signaling and might also be of use to patients unresponsive to resmetirom due to low THRB levels.
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    The role of sleep on enhancing the efficacy of psychotherapy
    (2025-10-13) Ehsanifard, Mojgan
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    The reactive pyruvate metabolite dimethylglyoxal in experimental disease models and the role of Ilvbl in its generation in the brain
    (2025) Costalunga, Riccardo
    Reactive glucose metabolites are involved in neurological complications of diabetes by the generation of advanced glycated end products (AGEs), which are formed by the interaction between α-dicarbonyls and proteins. AGEs induce oxidative stress, inflammation and tissue damage and have been associated with neuropathy, nephropathy and retinopathy. So far, only the α-dicarbonyls 3-deoxyglucosone, glyoxal and methylglyoxal have been recognized as reactive glucose metabolites and associated with complications in diabetes. Only recently, the α-dicarbonyl dimethylglyoxal (DMG) has been categorized as mammal cellular pyruvate metabolite, and its levels were higher in the plasma of type-1 diabetic mice and in the serum of patients with diabetes. In this thesis, I investigated the α-dicarbonyl DMG in mouse models under pathological conditions and its metabolic pathway in mammals using liquid chromatography coupled to tandem mass spectrometry (LC-MS2). Here, plasma DMG concentrations were upregulated in mouse experimental models of type-1 and type-2 diabetes. In contrast, high-fat diet did not influence the serum levels of DMG, suggesting that diabetic conditions – but not obesity itself – are associated with plasma DMG upregulation and accumulation. Moreover, DMG levels were enormously augmented in the ipsilateral side of a mouse model of hyperglycemic stroke compared to sham control. Interestingly, DMG levels were also higher in the contralateral hemisphere of the brain of mice with stroke compared to sham controls, showing that the contralateral hemisphere is also affected by ischemic stroke. Furthermore, the current study confirmed that DMG is a post-glycolytic product in mammals and its levels are increased under hypoxic conditions in vitro. Then, I investigated the role of the gene ilvB acetolactate synthase-like (Ilvbl), the mammal orthologue gene of acetolactate synthase that generates DMG from pyruvate in bacteria, yeast and plants. Importantly, Ilvbl knockout reduced DMG levels in the brain of mice with type-1 diabetes or hyperglycemic stroke, showing that Ilvbl has conserved its role in DMG generation in mammals. Moreover, DMG interacted with lysine, generating the glycated amino acid Nε-3-hydroxy-2-butanonelysine (HBL), confirming that DMG is a reactive compound and can glycate proteins generating AGEs. Thus, DMG induced oxidative stress and neuroinflammation in mouse hippocampal neuronal (HT-22) cells, probably based on protein glycation. In conclusion, this study characterizes the α-dicarbonyl DMG in experimental models under pathological conditions, showing that DMG could play a significant role in diabetic complications and ischemic stroke complications. DMG is the only α-dicarbonyl generated after glycolysis, and the current discovery could explain why cells consuming lactate – such as neurons – are not protected by dicarbonyl stress. Importantly, this study shows that ILVBL plays a significant role in the DMG generation from pyruvate in the brain of acute or chronic hyperglycemic mice. Further investigation on DMG and the attenuation of its concentrations may lead to improvement in diabetic and ischemic stroke complications.
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    High-content screen for cardiovascular modulators in zebrafish (Danio rerio)
    (2024) Vedder, Viviana Luisa
    Over the past two decades, cardiovascular diseases (CVDs) have remained the leading cause of death worldwide. Among these diseases, congenital heart disease is the most common type of human birth defect, affecting 1 in 100 live-born infants. It is crucial to identify new therapeutic targets and treatments for CVDs. Zebrafish are a suitable model for this task, as they can provide new insights into CVDs. Further, their small size and translucency make them an ideal model for high-content drug screenings. In this study, a phenotype-based high-content screening approach in zebrafish was established to identify cardiovascular modulators. Utilizing the ‘Acquifer Imaging Machine‘, 1,280 compounds of the Prestwick Chemical Library were screened for angiogenesis and heartbeat phenotypes. The previously established type III receptor tyrosine kinase inhibitor sunitinib malate, which inhibits angiogenesis in zebrafish, was used as a positive control. Additionally, the competitive 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) inhibitor atorvastatin, which was previously shown to cause severe heart phenotypes in zebrafish, served as a positive control for heart rate (HR) and heart morphology. HR was selected as a screening phenotype under the assumption that morphological and functional changes in the heart during development would result in altered HR. In search of angiogenesis modulators, blood vessel diameters of treated fish were measured for 320 of the 1,280 tested compounds. Application of different filters for known toxicities, previously published results and unknown targets, led to the identification of 11 promising hit compounds. Three of these compounds, namely desonide, oxibendazole, and azlocillin sodium salt, directly targeted angiogenesis by significantly reducing the diameter of intersegmental vessels. The remaining nine compounds affected the diameter of the dorsal aorta and/or dorsal vein. Of the 134 compounds significantly altering HR, 21 compounds repeatedly induced significant HR alterations in two different transgenic zebrafish lines. In this study, two main target clusters were identified for HR modulators: the histaminergic receptor H1 (HRH1) and the glucocorticoid receptor (NR3C1). While HRH1 is commonly associated with allergic reactions, it has also been linked to CVDs. All antagonistic hit compounds targeting HRH1 caused significant bradycardia with moderate to severe dose-dependent teratogenicity. In situ hybridization for various hrh probes revealed that hrh1 was the only detectable hrh in the heart at 3 days post fertilization (dpf), indicating its involvement in heart development. Additionally, the heart analysis tool pyHeart4Fish showed chamber-specific effects of HRH1 antagonists. Collectively, the data suggests that hrh1 plays a role in heart contractility and heart size. However, further investigation on the mechanism of action is necessary to comprehend how Hrh1 is involved in cardiogenesis and what long-term effects HRH1 antagonists can have on the developing embryo to determine drug safety for pregnant women. NR3C1 is a ligand-activated transcription factor known to be involved in inflammation, metabolism, and stress response. In this study involving zebrafish larvae, a group of six NR3C1 agonists was found to significantly increase HR at 48 hours post fertilization. While previous research had identified nr3c1's involvement in heart development in zebrafish, its role in arrhythmias remained unclear. Here, expression of nr3c1 was detected in the larval heart at 5 dpf using in situ hybridization. Further analysis using pyHeart4Fish revealed that NR3C1 agonists induced chamber-specific phenotypes, including atrioventricular block. A homology model of zebrafish Nr3c1 was also developed, which suggested that the agonists known to bind in humans may also bind in zebrafish. These findings support the use of zebrafish as a screening model for cardiovascular phenotypes and highlight the potential role of nr3c1 in CVDs.
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    Single cell sequencing in development and disease
    (2025-05-20) Balachandran, Saranya
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    850 nm Fourier domain mode-locked laser for ophthalmic optical coherence tomography imaging
    (2025) Klufts, Marie
    Non-invasive imaging techniques have become essential in medical diagnostics over the past few decades. Among these, Optical Coherence Tomography (OCT) offers micrometer resolution with millimeter-scale depth penetration, making it particularly valuable in ophthalmology. OCT captures backscattered light to generate 3D volumes. For eye imaging, wavelengths around 850 nm are ideal due to minimal absorption by the vitreous and high scattering in the upper retinal layers. Imaging speed is also critical, as faster speeds reduce motion artifacts. Swept-source OCT, using wavelength-tunable lasers, enables high-speed imaging. Fourier Domain Mode-Locked (FDML) lasers providing megahertz-level scan rates are ideal for this purpose. This thesis explores the development and application of FDML lasers for ophthalmic imaging. Unlike other tunable lasers, FDML lasers have a unique design that stores a full sweep in their fiber cavity for hundreds of round trips, avoiding rebuilding of lasing from spontaneous emission after tuning to new wavelengths offering high phase stability and long coherence length necessary for high quality OCT images. A new megahertz FDML laser at 850 nm would merge the unique advantages of this wavelength with the proven benefits of FDML lasers allowing for a low latency, dynamic view of the retina, opening new doors for real-time diagnostics. The first part delves into the challenges of developing an FDML laser around 850 nm, addressing issues like polarization mode dispersion, chromatic dispersion, and low gain/loss ratios. These factors contribute to the complexity of managing short wavelength OCT lasers, which explain their scarcity to date. The second part presents in-vivo ophthalmic OCT imaging results, with comparisons to other imaging techniques. The newly designed FDML laser demonstrates strong performance for OCT imaging, achieving an axial resolution below 10 µm, sensitivity above 84 dB, and a ranging depth of 1.4 cm. Also, its high phase stability, with a time jitter of 25 ps over 1,000 sweeps, makes it suitable for phaseresolved techniques. Retinal images were captured at 414,000 axial scans per second using a master-slave based calibration technique, at 828 kHz with bidirectional sweeping, and at 1.7 MHz using optical buffering with a single-k-calibration technique. While increased scattering at 850 nm limits choroidal imaging, most retinal layers of interest are clearly visible. This FDML laser highlights the advantages of short-wavelength, high-speed imaging and paves the way for new applications.
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    Multi-omics analysis uncovers split formation and keratinocyte detachment as key drivers of long-lasting cellular effects in pemphigus vulgaris
    (2025-08-11) Guo, Sen
    Pemphigus Vulgaris (PV) is an autoimmune disease in which antibodies mistakenly target the adhesion proteins Desmoglein 1 (DSG1) and/or Desmoglein 3 (DSG3) on skin cells, leading to the loss of cell-cell adhesion and causing blistering. While the molecular changes following antibody binding in PV remain poorly understood, we investigated these downstream effects by analyzing gene and protein responses in two experimental models: human primary epidermal keratinocytes (HPEKs) and human skin organ culture (HSOC). Samples were treated with either PX43, a human-derived antibody fragment targeting DSG1 and DSG3, or AK23, a mouse-derived DSG3 antibody, alongside control treatments. In the HPEK model, PV antibody treatments did not trigger notable changes compared to controls at 5, 10, or 24 hours. However, in the HSOC model, only PX43 induced tissue splitting and significant changes in gene and protein expression, particularly in pathways linked to inflammation and immune signaling (e.g., TNFα, Interferon α/γ, IL2-STAT5, IL5-STAT3). Importantly, these molecular changes resembled those seen in wounded or inflamed skin, suggesting that physical damage from blister formation—not the antibody binding itself—is the primary driver of downstream cellular responses. This study reveals that tissue injury may be the main trigger for disease progression in PV, pointing toward new therapeutic targets that focus on modulating the wound response and inflammation, rather than the antibodies alone.
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    Exploring structure-based discovery of antivirals targeting Human cytomegalovirus (HCMV)
    (2025-07-28) Thiyagaraj, Dinesh
    Human cytomegalovirus (HCMV) causes widespread infections globally, typically remaining asymptomatic and latent. However, under conditions of immunosuppression, HCMV can reactivate and cause severe disease. The United States Food and Drug Administration (USFDA) has approved six antivirals for prevention and/or treatment of HCMV infection: Ganciclovir, Valganciclovir, Cidofovir, Foscarnet, Letermovir (only prophylaxis), and Maribavir. Despite their efficacy, these antivirals face one or more of the following challenges: toxicity, efficacy, and the emergence of drug resistance. This highlights the urgent need for novel antivirals that are both effective and safer, targeting essential viral proteins to minimize resistance. This study aimed to extend the available HCMV drugs by establishing structure-based drug design pipeline for three distinct HCMV protein targets, namely the pUL94/pUL99 complex, pUL98 and pUL77. I worked towards the structural characterization of two HCMV targets, employing crystallization methods in combination with crystallization enhancing scaffolds as “crystallization chaperones” as well as alternative methodologies like cryo-EM single particle analysis. The resulting atomic structures will pave the way for virtual screening of small molecules to identify potential antiviral candidates. Additionally, we aimed to establish a semi-automated virtual screening workflow and screen for protein-protein interaction inhibitors against HCMV pUL77. I initially expressed pUL98 in E.coli and purified it to homogeneity. Since initial robotic crystallization experiments remained unsuccessful, I employed sybodies and megabodies as “crystallization chaperones” for pUL98. Unfortunately, crystallizing pUL98 with sybodies or megabodies did not yield diffraction-quality crystals. For the pUL94/pUL99 complex sybodies were already available at the beginning of this thesis, and as crystallization also remained unsuccessful, we started a collaboration with the group of Maya Topf at CSSB; Hamburg, to characterize the complex structure using cryo-EM single particle analysis. To increase particle contrast in cryo-EM, I used the existing sybodies to engineer legobodies binding pUL94/pUL99, but the complex showed significant compositional and conformational heterogeneity, limiting the obtained resolution to ~6Å. Finally, I used the reported cryo-EM structure of the pUL77 pentamer to establish a consensus virtual screening workflow for pUL77 using multiple scoring functions, docking tools and additional screening parameters. This allowed us to identify consensus poses from two docking tools and attempt to improve probability of true-positive hits. Clustering algorithms were used to scrutinize the molecules further, enhancing the hit identification rate and lead selection. The shortlisted molecules from this screening workflow have been ordered and will be tested by our collaboration partners in cell culture models for HCMV infection. In conclusion, this work established a workflow for virtually screening molecules with improved hit rates, moving beyond reliance on docking scores alone. This workflow aims to enhance the efficiency of structure-based antiviral discovery targeting HCMV infection.
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    Variability in biological correlates of nightmares and implications for the development of new treatment options
    (2024) Sayk, Clara Anna Luise
    are very common in both the general population and individuals with psychiatric disorders. They can cause distress, sleep disturbance and impairment in daily functioning. Several biological correlates of nightmares have been investigated in the last decades, such as brain activity during sleep, changes in heart rate, electrodermal activity or cortisol levels. All of these measures indicate some form of hyperarousal which, together with impaired fear extinction, is indeed one of the central components of the integrative etiology model for nightmares. However, it is still unclear, how exactly hyperarousal and nightmares interact, i.e., whether hyperarousal actually is a causal mechanism or rather a byproduct of other processes. Likewise, it is still unknown whether it can be influenced through interventions for nightmares, such as imagery rehearsal therapy or - directly during sleep - with some form of stimulation or targeted memory reactivation (TMR). The aim of the three studies that constitute my thesis was to examine biological correlates of nightmares, especially cortical hyperarousal in individuals with frequent nightmares from different samples and the effects of imagery rehearsal therapy (IRT) and TMR on physiological correlates and nightmare symptoms using polysomnography and high-density EEG-measurements. The first two studies focused on physiological correlates of nightmares and the effects of IRT. Study 1 addressed how physiological correlates differ between individuals with frequent nightmares and healthy controls and how an 8-week IRT group intervention influences nightmare symptoms and physiological correlates. Participants with frequent nightmares showed increased beta and gamma activity compared to healthy controls and their gamma activity during REM was reduced after the intervention. In study 2, a similar paradigm was used to investigate the effects of imagery rehearsal therapy as an add-on to inpatient treatment for individuals with borderline personality disorder. While this study did not show any changes in physiological correlates, the intervention group experienced a significantly more pronounced reduction in anxiety, intrusions and hyperarousal. Study 3 investigated the effects of experimentally manipulating these biological correlates. More specifically, whether reactivating a relaxation exercise with TMR in participants with frequent nightmares reduced hyperarousal and nightmare symptoms. While the reactivation did lead to a reduction in spindle count and density, another form of cortical hyperarousal, there was no influence on nightmare symptoms. Taken together, I could demonstrate that i) increased (cortical) arousal plays an important role as biological correlate and shows sensitivity to treatment in various samples with frequent nightmares (participants with frequent nightmares but no severe co-morbidities as well as individuals with borderline personality disorder and frequent nightmares), ii) IRT is successful in reducing gamma activity and psychological arousal alongside nightmare symptoms and iii) the reactivation of relaxation associated contents influences spindle activity which is even associated with changes in subjective sleep disturbance, indicating that this is a promising novel technique to target hyperarousal. Future research should therefore focus on further attempts to experimentally manipulate cortical hyperarousal by stimulation methods or by reactivating IRT and should also include other factors of the integrative model of nightmare etiology, especially fear extinction.
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    Untersuchung des Einflusses dreidimensionaler Kultivierung auf Neuronen und Schweißdrüsenzellen mit Fokus auf elektrophysiologischer Funktionalität
    (2025) Panzer, Jörg
    Tissue-Engineering und innovative Kulturmethoden haben das Potential, in vielen Bereichen der klinischen Forschung zu neuen Therapiemöglichkeiten zu führen. Dazu gehören unter anderem die Schmerzforschung mit der Suche nach molekularen Zielen für Medikamente, sowie der Forschungsbereich der Wundheilung und Hautrekonstruktion. Die Umgebung der in vitro kultivierten Zellen ist dabei ein essentieller Faktor für deren Differenzierung und Funktion. In der vorliegenden Arbeit wurde für die kommerzielle neuronale Zelllinie F11, sowie für humane Schweißdrüsen-abgeleitete Stammzellen (sweat gland derived stem cells, hSGSCs), untersucht, ob die 3D-Kultivierung in Form von multizellulären Sphäroiden einen Einfluss auf die gewebsspezifische Differenzierung hat. Um multizelluläre Sphäroide zu erzeugen, wurden die Zellen mit der liquid overlay Methode in Mikromulden aus Agarose kultiviert, was bei beiden Zelltypen zu einer spontanen Aggregation führte. Beide Zelltypen wurden in verschiedenen Medien in 2D und 3D kultiviert. Mittels RT-qPCR wurden auf mRNA-Ebene zelltypspezifische Genpanels analysiert, die Differenzierungsstatus und elektrophysiologische Aktivität ausgerichtet waren. Für die F11-Zellen zeigte sich bei zwei untersuchten Differenzierungsmarkern eine deutlich veränderte Expression. Das neuronale Strukturprotein Neurofilament M (Nefm) wurde in der 3D-Kultur vermehrt exprimiert, der Ca2+-Sensor Synaptotagmin 1 (Syt1) hingegen vermindert. Die Expression der weiteren Differenzierungsmarker sowie der untersuchten Ionenkanäle zeigte keine Unterschiede zwischen 2D- und 3D-Kultur. Diese Ergebnisse lassen nicht auf eine veränderte Funktionalität der Zellen durch 3DKultivierung in Sphäroiden schließen. Um die elektrophysiologische Funktionalität der Zellen zu untersuchen, wurde automatisiertes Patch-Clamping angewendet. An dissoziierten F11-Zellen konnten erfolgreich Messungen durchgeführt werden, die Sphäroide jedoch ließen sich mit dieser Methode nicht analysieren. Die Frage nach einer veränderten elektrophysiologischen Funktionalität durch 3D-Kultivierung konnte somit nicht abschließend geklärt werden. In den untersuchten hSGSCs konnte die Expression von wichtigen funktionellen Markern sekretorischer Schweißdrüsenzellen gezeigt werden. Die 3D-Kultivierung hatte einen deutlichen Effekt auf einige dieser Marker. Der muskarinerge Acetylcholinrezeptor M3 (CHRM3) und der calciumgesteuerte Calciumkanal ORAI1 wurden in 3D-Kultur verringert exprimiert, der calciumgesteuerte Chloridkanal Anoctamin 1 (ANO1, auch TMEM16A) wurde verstärkt exprimiert. Die Ergebnisse der Expressionsanalyse ließen auf eine veränderte Funktionalität in 3D- gegenüber 2D-Kultur schließen. Im PatchClamping konnten, wie bei F11-Zellen, nur Einzelzellen analysiert werden. Zur weiteren Untersuchung der Funktionalität wurde ein Calcium-Imaging-Assay durchgeführt. Dabei konnten keine signifikanten Unterschiede in der maximalen Calciumkonzentrationszunahme beobachtet werden. Der Anteil an stimulierbaren Zellen unterschied sich ebenfalls nicht signifikant voneinander. Die Kultivierung in einem Differenzierungsmedium führte bei hSGSCs zu einer erhöhten CHRM3-Expression in der Zellpopulation sowie einem erhöhten Anteil an MCH-reaktiven Zellen.
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    Speech comprehension through the lens of auditory and motor brain rhythms
    (2025) Lubinus, Christina Anna-Lena

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