Naturwissenschaften

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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.
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.
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.
Speech comprehension through the lens of auditory and motor brain rhythms
(2025) Lubinus, Christina Anna-Lena
Metabolic and cellular factors determining the therapeutic effects of dimethyl fumarate
(2025-06-12) Kosińska, Joanna
Investigating the phenotypic plasticity and functional potential of gut bacteria to various IBD relevant stressors
(2025) Schäfer, Kathrin
Lipidomic and structural characterization of the cell wall of Streptococcus pneumoniae
(2025-05-13) Alshaar, Belal
Streptococcus pneumoniae (Spn) is a Gram-positive bacterium that naturally colonizes various niches within the human body, but can also lead to infections with varying degrees of symptoms. Despite the availability of vaccines, Spn infections remain a significant health concern, exacerbated by the rising threat of antibiotic resistance. This growing challenge underscores the urgent need to explore new drug targets that are common across different strains. A particularly promising target is the pneumococcal cell wall, which has become a central point of structure-focused research. The cellular membrane, a key component of this wall, plays a crucial role in host-pathogen interactions. Nevertheless, the precise quantities of its components are still not well understood. This thesis presents a first-of-its-kind quantitative analysis of the pneumococcal lipidome, conducted in close collaboration with the group of Prof. Dr. Sven Hammerschmidt from the University of Greifswald. The implementation of a standardized pipeline applicable for in-depth microbial lipidomics on pneumococcal samples is described. Further, I examined how the knockout of selected genes influenced the membrane lipid composition. As a proof of concept, the wild-type serotype 2 strain S. pneumoniae D39 was compared to three mutant strains with single gene knockouts. The targeted mutant strains investigated here are: 1) tacL: TacL is responsible for the attachment of the polymeric teichoic acid chain on the glycolipid anchor, thus forming the lipoteichoic acid (LTA); 2) lgt: Lgt (diacylglyceryl transferase) catalyzes the diacylation of preprolipoproteins via a thioester bond by transferring a diacylglycerol moiety from a phosphatidylglycerol (PG) precursor; 3) cps: enzymes of the cps cluster are responsible for capsule polysaccharide formation. To date, such quantitative lipidome studies have not been performed for Spn. Methodically, shotgun lipidomics was employed and the development of a customized mix of internal standards ensured accurate quantitative analysis. The analysis allowed quantification of more than 100 lipids across the following six classes: diacylglycerol, glucosyldiacylglycerol (GlcDAG), galactosylglucosyldiacylglycerol (GalGlcDAG), cardiolipin, phosphatidylcholine and PG, with GlcDAG and GalGlcDAG being the predominant classes. Gas Chromatography-Mass Spectrometry (GC-MS) and Thin Layer Chromatography in conjunction with high-resolution tandem mass spectrometry were further utilized for the validation of identified lipid species. The lipid composition of tacL and lgt strains showed no significant changes compared to the wild type, while the non-encapsulated strain exhibited significant adaptations. Another aim of this thesis was to optimize a GC-MS method for the accurate quantification of pneumococcal wall glycopolymers, particularly wall teichoic acid and lipoteichoic acid. This is a challenging task for researchers in the field since decades. This quantification technique is anticipated to provide a basis for studying enzymes that affect teichoic acid levels without using radioactive materials in cultures and may also be used for capsule polysaccharide quantification, advancing our understanding of pneumococcal cell wall regulation. Preliminary results from this optimized method have already provided insights into how LytR, which plays an essential role in attaching the wall teichoic acid to the peptidoglycan, influences the teichoic acids ratio. In summary, a robust quantitative method for lipidome analysis has been established, along with a developed method which delivered the first successful proof of concept for teichoic acids quantification in Spn. These approaches enable future investigations into membrane homeostasis and biosynthesis regulation in other mutants or under various growth conditions.
Antibody and B cell development after vaccination against COVID-19
(2024) Buhre, Jana Sophia
During the COVID-19 pandemic, new mRNA- and adenovirus-based vaccine formats encoding the viral spike protein (S) were developed. These newly developed vaccine formats have helped to successfully combat the pandemic. However, there is a high rate of breakthrough infections, which is why the vaccine induced immune responses should be investigated in detail. Both vaccine formats induce antibodies that have neutralizing functions via their Fab-part. The effect of IgG antibodies also depends on Fc-mediated effector functions, which are influenced by the IgG subclass and its Fc N-glycosylation pattern. At the beginning of this work, it was known that both vaccines induce IgG1 and IgG3 antibodies initially and especially after two vaccinations, but information on long-term antibody responses was lacking. The aim of this work was therefore to longitudinally describe and characterize the vaccine-induced IgG subclasses, their Fc N-glycosylation and their Fc-mediated effector functions. It was shown that the mRNA vaccines produced significantly higher IgG1 and IgG3 antibody levels in the short term compared to adenovirus-based vaccination, but that these levels equalized in the long term. In contrast to this equalization, late emerging IgG4 antibodies were only observed in people who received two mRNA vaccinations. It is generally assumed that IgG4, in contrast to IgG1 and IgG3, has a more inhibitory function. In functional assays, anti-S IgG1 was confirmed as a strongly activating subclass. In contrast, IgG4 antibodies, which increased with every vaccination, showed activating but also inhibitory immunomodulatory effects. These need to be further investigated in the future in order to better assess their role in the long-term vaccination response. In addition, a dynamic development of glycosylation was observed: Highly galactosylated and sialylated IgG antibodies appeared shortly after each vaccination, while significantly lower galactosylated and sialylated IgG antibodies were found in the long term. This wave pattern was described for all IgG subclasses and indicates that differently glycosylated antibodies exert different effector functions, which could mean a new general understanding of early and late antibody responses after vaccination. In contrast to this dynamic, the new vaccines were only able to induce afucosylated antibodies temporarily and only after the first vaccination. By investigating a cohort of patients with inflammatory diseases who received tumor necrosis factor (TNF) inhibitors during vaccination, the influence of TNF on the longitudinal antibody response and thus also on B-cell differentiation could be described. The patients showed lower IgG levels, altered antibody glycosylation patterns and reduced Fc-mediated effector functions both in the short and long term after repeated vaccination. Anti-TNF therapy not only reduced the long-lived IgG response, but also the short-lived IgG response resulting from the re-activation of memory cells. Accordingly, an increased risk of breakthrough infection was observed in patients treated with anti-TNF. These results suggest that patients receiving anti-TNF therapy may benefit little from a booster vaccination.
Aparecium: Revealing the role of the zona incerta in central thyroid hormone action
(2025) Maier, Julia
The importance of thyroid hormones (THs) for regulation of body temperature and energy metabolism as well as brain functions such as mood is well established. This is most evident in hypo- or hyperthyroidism, which have severe consequences on these parameters. THs exert their effects via thyroid hormone receptors α (TRα) and β (TRβ). Similar to altered ligand availability, mutations in these receptors lead to several deficits, such as defaults in neuronal development and regulation of body temperature, as well as psychomotor alterations. However, it often remains unknown whether defects are caused by TH actions directly on tissue level or centrally in the brain, and in the latter case, which brain regions mediate the central effects of THs. To address this question, PET/CT scans of mice undergoing treatment with T3 were conducted and showed activation of the Zona Incerta (ZI), a region of the subthalamus, indicating its role in mediating effects of THs. To further investigate the effect of THs in the ZI, TH signalling was inhibited by introducing a dominant-negative TRα1 via adeno-associated virus (AAV) injection into the ZI. In a separate set up, inhibition was specific to dopaminergic neurons by injecting the AAVs into tyrosine hydroxylase-Cre mice. Analysing these mice revealed that inhibited TH signalling in the ZI caused an increase in basal metabolic rate (BMR) with increased fasting weight loss without affecting body temperature. In addition, it resulted in a chronic stress-like state, in which serum corticosterone was elevated, and a partial anxiety phenotype was present, as habituation to stressful situations was impaired. However, none of these effects were observed in the conditional model, demonstrating that they were not mediated by dopaminergic neurons. Taken together, the study identifies a new brain region for central actions of THs, the ZI, and implicate it as a key brain region in TH-meditated control of behaviour and energy metabolism.
Gene-environment modifiers in Parkinson's disease
(2025) Gabbert, Carolin
Comparative analysis of host response in tuberculosis across species
(2024-10-24) Tijani, Hawanot Olaitan
In tuberculosis (TB) studies, the extent to which host genome as well as transcriptome contribute to susceptibility and pathology has not been clearly defined. Globally still one of the most abundant infectious disease, numbers of TB cases caused by drug resistant agents of the Mycobacterium tuberculosis (M. tuberculosis) complex are increasing especially with onset of emerging and re-emerging viral diseases and other comorbidities. Our study aims to identify host specific differences in TB disease susceptibility and pathogenesis. Previously, we and others have demonstrated the detrimental role of neutrophils in necrosis, tissue damage and bacterial spread. We hypothesized that cell death modalities during the interaction of neutrophils and other myeloid cells in the lung determines pathogen restriction or spread and ultimately, disease exacerbation. We therefore designed an integrative systems biology approach from genome to cellular, tissue and animal model to better understand TB in vitro and in vivo. Inbred female congenic mice differing in their susceptibility to M. tuberculosis infection were studied before, during and after infection and compared to human leukocytes and RNA samples from excised lung tissue. Murine bone marrow derived macrophages from wild-type B6 in comparison to susceptible C3HeBFeJ and B6.C3Hsst1 mice showed differential responses to M. tuberculosis. Upon aerosol infection with M. tuberculosis, immune-competent but susceptible B6.I-9.3.19.8 and B6.C3Hsst1 had bigger lung lesions and higher bacterial loads than resistant B6 mice. Immune cell phenotyping of murine lung samples characterized by flow cytometry and immunohistochemistry revealed distinct composition of immune cells between susceptible and resistant mouse strains. Importantly, human PMN and PBMCs showed infection specific transcriptional profiles in vitro skewed towards active phagocytosis. Comparative analyses of both murine and human transcriptome identified unique genes for each data set and shared genes across the samples. Taken together, our findings will contribute to better understand TB disease processes in order to break disease transmission, reducing bacterial burden in active TB patients, shortening treatment duration, and eventually preventing disease development in latent TB cases.
Multidrug resistance, population structure, and epidemiology of Mycobacterium tuberculosis complex strains from West Africa
(2025-02-25) Blankson, Harriet Naa Afia
Tuberculosis (TB) remains a global public health challenge, impacting millions of individuals and placing a substantial burden on healthcare systems worldwide. The causative agents of TB, Mycobacterium tuberculosis complex (MTBC) strains, engage in a complex interplay of factors that influence its transmission, evolution, and the emergence of drug-resistant strains. Despite commendable progress in TB control efforts, the persistent threat of multidrug-resistant TB(MDR-TB), characterized by resistance to both isoniazid (INH) and rifampicin (RIF), has significantly complicated treatment protocols. Recent recommendations by the World Health Organization (WHO) advocate for a new all-oral 6-month regimen to address MDR-TB. However, concerns arise with the development of resistance to fluoroquinolones (FQs) and bedaquiline (BDQ), potentially jeopardizing the regimen's efficacy. Documented outbreaks of MTBC strains, particularly of MDR strains, underscore the urgency of monitoring the transmission of MTBC strains, as it can impact diagnostics and treatment strategies in affected regions. Specific strains within MTBC lineages may exhibit unique traits, such as varying minimum inhibitory concentrations (MIC) or inherent resistance to specific drugs. Understanding the circulating MTBC strains in a country, their drug resistance profiles, and their evolutionary dynamics is imperative for effective TB management. To close these knowledge gaps, this thesis seeks to explore the multifaceted landscape of TB through epidemiological, molecular, and clinical perspectives. The research aims to unravel the genomic intricacies of MTBC strains, by investigating the transmission dynamics of rifampicin-resistant (RR)/MDR-TB in Sierra Leone and exploring the transmission dynamics and drug resistance patterns of the sublineage 4.6.2.2 Cameroon. By shedding light on these aspects, this thesis seeks to contribute to enhanced diagnostics and more effective treatment strategies and ultimately support the global mission to eradicate TB. In the study on RR/MDR-TB in Sierra Leone, whole genome sequencing (WGS) revealed high levels of drug resistance, with one in four strains being resistant to all first-line anti-TB drugs. While no FQ resistance was detected, five strains exhibited resistance to BDQ/clofazimine (CFZ) due to mutations in the Rv0678 gene. The study also revealed a greater diversity of drug resistance mutations, including borderline INH and RIF resistance mutations, which can potentially influence treatment options. The high cluster rate of over 40% indicated ongoing transmission of RR/MDR-TB strains, contributing to the burden of RR/MDR-TB in the country. Analysis of the 238 MTBC strains revealed a high diversity of strains in Sierra Leone. The presence of six major lineages (L) of MTBC strains (L1= 4%, L2 = 9%, L3= 0.8%, L4= 62%, L5= 2.9% and L6= 21%) were identified in Sierra Leone. Mycobacterium tuberculosis (Mtb) strains constitute 56%, while Mycobacterium africanum (Maf) L6 strains, account for 21% of MDR MTBC strains, which suggest a longitudinal outbreak with specific branches exhibiting resistance to multiple drugs, including BDQ/CFZ. Despite the high diversity, strains of certain sublineages 4.1.2.1. Haarlem, 4.8 mainly T and 2.2.1 Beijing Ancestral 3 and 6.3.3 West Africa 2 were implicated in the ongoing MDR transmissions. The global population structure and phylogeography of strains of the Cameroon sublineage were also investigated using WGS. The strains were classified into eight distinct clades, with strains originating from 24 countries across Africa, Asia, Australia, and Europe. A fourth of the strains were identified as transmission strains. The strains of two clades, C.5 and C.8, exhibited high clustering rates, indicating higher transmission potential. Drug resistance was also observed, with over 10% of the strains classified as MDR. The strains remained susceptible to BDQ/CFZ, except for two strains displaying resistance. Overall, these findings contribute to a comprehensive understanding of the transmission dynamics and drug resistance patterns of MTBC strains in Sierra Leone and the sublineage 4.6.2.2 Cameroon. The clades of strains that were defined, clusters, and global distribution emphasize the role of migration in the spread of these strains locally and beyond Africa. The insights gained from this research can inform and improve TB surveillance and control measures, both within Sierra Leone, West Africa, and in other parts of the world where the Cameroon sublineage strains have been identified.
Dynamische mikroskopische Optische Kohärenztomografie
(2024-11-07) Münter, Michael
Die vorliegende Arbeit beschreibt ein Verfahren zur kontrastreichen Bildgebung von zellulären und subzellulären Strukturen mittels mikroskopischer Optischer Kohärenztomografie (OCT). Die auch als dynamischer OCT Kontrast bezeichnete Methode basiert auf der Detektion von Bewegungen zellulärer und subzellulärer Strukturen und einer anschließenden Auswertung. Im Theorieteil werden die biologischen und physikalischen Grundlagen als auch die OCT beschrieben. Die physikalischen und mathematischen Grundlagen der für den dynamischen OCT Kontrast wichtigen Schritte werden in den darauffolgenden Kapitel genauer untersucht. Zuerst werden Ergebnisse mit einem mikroskopischen OCT Aufbau bei einer A Scan Rate von 248 kHz vorgestellt. Hierbei konnte anhand von Volumen und B Scan Aufnahmen von frischen ex vivo Gewebeproben eine Bildgebung mit dynamischen OCT Kontrast mittels scannender Spectral-Domain OCT demonstriert werden. Zur Evaluation der Ergebnisse wurden diese mit einem Full Field Time Domain OCT Aufbau verglichen. Die Abhängigkeit der axialen Auflösung durch Anpassung der Hann Fensterung bei der Rekonstruktion auf den dynamischen OCT Kontrast wurde untersucht. Zudem erfolgt eine Betrachtung des Kontrast Rausch Verhältnisses in Abhängigkeit zur Aufnahmezeit. Es konnte gezeigt werden, dass bereits eine Aufnahmezeit von 100 ms ausreichend ist, um zelluläre und subzelluläre Strukturen mittels dynamischen OCT Kontrast zu visualisieren. In Zeitserien mit dynamischen OCT Kontrast konnten Immunzellen in der Bewegung beobachtet werden, die mit herkömmlicher OCT-Bildgebung nicht hätten visualisiert werden können. Um die Bildgebungsgeschwindigkeit zu erhöhen, wurde eine Zeilenkamera mit einer Auslesegeschwindigkeit von 600 kHz für den mOCT Aufbau eingesetzt. Hierfür wurde eine angepasste OCT Software zur Online-Darstellung von B Scans und eine Auswertungssoftware geschrieben. Anhand einer technischen Charakterisierung wurde die Zeilenkamera mit 248 kHz Ausleserate mit der 600 kHz Zeilenkamera verglichen. Die Vorteile einer schnelleren Bildgebung wurden durch die Darstellung dynamischer 4 dimensionaler Transportvorgänge in ex vivo Trachea gezeigt als auch an dynamischen OCT Aufnahmen von unfixierten ex vivo Proben. Dynamische OCT erweitert die OCT durch die bisher nicht erreichte kontrastreiche Darstellung von Zellen und subzellulären Strukturen und stellt damit eine wichtige Applikation für hochauflösende OCT dar.
Thyroid hormone receptor beta in MASH
(2024) López Alcántara, Nuria
Metabolic dysfunction-associated steatotic liver disease (MASLD) and steatohepatitis (MASH) are widespread diseases influenced by thyroid hormone (TH). Hypothyroidism pro-motes hyperlipidemia, obesity, and insulin resistance, while TH replacement therapy and thyromimetics show promising results in improving disease progression. However, recent studies indicate that is not the systemic levels but the local hepatic TH metabolism that plays a crucial role. This is primarily controlled in the liver by the TH beta receptor (TRβ) and TH-activating deiodinase type I (DIO1). In humans, the severity of MASH correlates with reduced hepatic TRβ expression, indicating developing TH resistance, which could therapeutically diminish the efficacy of thyromimetics. This dissertation investigates the role of hepatic TRβ and DIO1 during MASH development. Using TRβ knockout mice and viral overexpression of TRβ, the influence of receptor levels on MASH progression is examined. The experimental approach also included the development of a novel MASH mouse model by combining a choline-deficient high-fat diet with optimized methionine concentration and thermoneutral housing, reflecting important human charac-teristics such as a reduction in TRβ during disease progression. Additionally, in vitro studies with primary mouse hepatocytes were conducted to identify specific factors that can selec-tively modulate deiodinase type 1 or TRβ to find new therapeutic approaches for improving local TH availability. The main results showed that thermoneutrality not only mitigates the usual hyperthyroidism of TRβ knockout mice, allowing the study of TRβ without confounding effects, but also that such a mouse model better reflects the human situation compared to studies at room tem-perature. The lack of TRβ in the animals led to increased fat mass but improved fibrosis and less inflammation, while TRβ overexpression had no effect. Increased Dio1 mRNA after MASH induction indicated a protective compensatory mechanism. The findings clearly demonstrate that a reduction in the TRβ signaling pathway can be beneficial for certain dis-ease pathologies, thus questioning the use of thyromimetics in later stages of fibrosis.
BAT, can you feel the stream of dopamine?
(2025) Raffaelli, Francesca-Maria
Combatting overweight and obesity is one of the major concerns in healthcare in the world at the moment. Over two thirds of the world’s population present with an above average BMI, which is the root cause for metabolic diseases, sky rocketing costs in healthcare, and eventually premature death. Recent advances in the field include injections of antidiabetics that promote weightloss, but are essentially black boxes regarding long term side effects, sustainability, and overall consumer safety. An even deeper understanding of adipose tissue biology and its effects on whole body metabolism must be achieved to uncover safe ways for individuals to manage their body weight and therefore lead a healthier life – however long it may be. One mechanism that has been investigated as a potential route to aid weight loss is brown adipose tissue (BAT) thermogenesis activation. Brown adipocytes differ from white adipocytes in cell size, fat vacuole distribution, mitochondrial density and a protein unique to their cell type, called uncoupling protein 1 (UCP1). This protein enables brown adipocytes to combust excess energy into heat, instead of intracellular storage, as is the case in white adipocytes. The current state of the knowledge is, that BAT thermogenesis is activated through cold exposure and food intake which lead to hypothalamic signalling via the sympathetic nervous system and increase the release of norepinephrine in BAT, which consequently initiates G protein-coupled receptor-dependent molecular pathways leading to increased UCP1 expression. Interestingly, observations in the 1980s have described dopamine to have similar effects on BAT temperature increases in rodents, as norepinephrine does. A more recent study by Kohlie et al. followed this lead and investigated this observation on a molecular level in rodent cells in vitro; concluding dopamine and dopamine receptor D1-agonists did increase thermogenesis in brown adipocytes. This study therefore aims to further investigate the role of dopamine and dopamine receptors in BAT thermogenesis ex vivo and in vivo in mice using direct tissue treatment, single injection short term approaches, as well as repeated injection and constant release methods over the course of one week in wild type C57BL/6NCrl mice, respectively. Furthermore, the presence of dopamine receptors in BAT of mice was investigated meticoulously in a proteomics approach. Neither BAT thermogenesis activation, nor dopamine receptor presence were detected in these experiments, leading to the conclusion, that dopamine or dopamine receptors D1 and D2 do not directly contribute to BAT thermogenesis. However, an indirect role of dopamine, as the direct metabolic precursor of norepinephrine in the catecholamine synthesis pathway, via the central axis, can not be excluded and should be investigated further.
Mycobacterium tuberculosis resistance evolution
(2025) Walz, Teresa
With nearly half a million new multidrug resistant (MDR) tuberculosis (TB) cases estimated annually, TB is a major contributor to the current antimicrobial resistance crisis. MDR-TB is defined as an infection with a Mycobacterium tuberculosis complex (MTBC) strain resistant to the two most effective first-line drugs rifampicin (RIF) and isoniazid. To effectively address this public health crisis, it is essential to un-derstand resistance mechanisms and evolution. This will better guide the development of new antibiot-ics and treatment strategies, and avoid rapid resistance development, as seen with bedaquiline (BDQ). This work explores resistance mechanisms to one key MDR-TB treatment drug, namely BDQ, and new evolutionary medicine informed treatment strategies based on negative hysteresis. BDQ, one of the most important drugs for treating MDR-TB, is facing increasing resistance in clinical MTBC strains in several high incidence settings. Clinical MTBC strains acquire BDQ resistance through mutations in the atpE gene, which encodes the ATP synthase (primary target of BDQ), and in the Rv0678 gene, which regulates the mmpS5-mmpL5 efflux pump system. Most clinical resistance is associated with mutations in Rv0678, leading to increased expression of the efflux pump genes. In this work, RNA sequencing (RNAseq) analysis of BDQ-resistant mutants, harbouring different mutations in Rv0678 (to-tal loss-of-function mutations and reduced-function mutations), was conducted. The results revealed that efflux pump expression varied among clones, with baseline mmpS5-mmpL5 efflux pump gene ex-pression upregulated without BDQ exposure. Surprisingly, under BDQ challenge, the wild-type H37Rv strain upregulated the mmpS5-mmpL5 efflux pump to similar levels as the Rv0678 mutant clones, point-ing to other mechanisms involved in resistance development. Analysis of the transcriptional response of Rv0678 mutant clones and H37Rv at different BDQ exposure timepoints revealed a complex response indicating that resistance is mediated not by a single mechanism, but rather by an enhanced metabolic plasticity of the Rv0678 mutants, which allows them to shift to a dormant-like state under stress while maintaining some cellular functions. The second focus of this work was to explore evolution-informed treatment strategies aimed at en-hancing drug efficacy and circumvent resistance evolution by using negative hysteresis as potential mechanism in sequential treatments to limit the evolutionary potential of MTBC strains. An in vitro model was designed based on short pre-exposure with one drug, followed by main-exposure with the second drug utilizing sub-lethal concentrations which potentially induces cellular hysteresis, a phenom-enon where antibiotic effectiveness is enhanced based on the order of drug administration. Sequential exposure of MTBC reference strain H37Rv with ethambutol (EMB) followed by RIF led to a sequence-dependent reduction in bacterial load, attributed to the negative hysteresis effect, where EMB pre-exposure enhanced RIF's efficacy. RNAseq was employed to investigate the underlying mechanisms, indicating that the cellular hysteresis effect, induced by the respective drug sequence, was likely driven by a cell surface stress response and metabolic remodelling by a danK-mediated stress response. Taken together, the results obtained in this thesis contradicts common knowledge that BDQ resistance induced by Rv0678 mutations is solely conferred by upregulation of the mmpS5-mmpL5 efflux pump system only, but rather points to the role of an enhanced metabolic plasticity in antibiotic responses as important survival strategy. It also shows, that comprehensive studies based on mutant generation combined with OMICS tools are needed to understand pathogen drug response, and escape mecha-nisms. Further, to the best of our knowledge, for the first time the potential of negative hysteresis in-duced by fast drug switching for enhancing drug efficacy even at sub-minimum inhibitory concentration (MIC) conditions was observed in M. tuberculosis. This has great potential for optimizing TB treatment concepts especially for drug resistant MTBC strains, where drug options are limited and cycling thera-pies with a lower number of antibiotics significantly enhance treatment possibilities. Accordingly, evolu-tionary informed treatment strategies should be urgently further exploited.
The impact of the pro-atherogenic MRAS gene in smooth muscle cells
(2025-03-07) Shah, Pashmina Wiqar
Cardiovascular disease (CVDs) including coronary artery disease (CAD), cardiomyopathy, myocardial infarction and stroke, are the leading cause of death globally. The preeminent cause of CVDs is atherosclerosis, an inflammatory thickening of the vessel wall. Numerous risk factors including smoking, alcohol consumption, obesity, hypertension, stress, age, gender and genetic predisposition, contribute to its development. Genome wide association studies (GWAS) have pinpointed more than 393 loci marked by single nucleotide variants significantly associated with CAD. However, the causal mechanisms behind these associations remain unclear for which functional follow-up studies are very essential to identify the causal genetic variation or gene, to better understand the link between these loci and the disease. A study by Erdmann et al. in 2009, revealed a region on 3q22.3, which encompasses the MRAS gene as a risk factor for CAD. In this doctoral dissertation, the CAD-risk gene MRAS was investigated. MRAS encodes muscle Ras, a small GTPase that acts as a signal transducer in tumour necrosis factor (TNF) signalling and other related acute phase response signalling pathways. According to eQTL data, MRAS risk variants for CAD increase MRAS mRNA levels primarily in the arterial tissue. Recently, it has been indicated that functional MRAS variants are specific to vascular smooth muscle cells (SMCs). The exact role of MRAS in atherogenesis, the underlying mechanism of CAD, and the therapeutic potential of targeting MRAS is still elusive. Therefore, we investigated the function of MRAS in vascular SMCs, one of the key cell types in the etiology of atherosclerosis and plaque stabilization. Human primary aortic SMCs transfected with MRAS-specific siRNA and murine aortic SMCs derived from our Mras-/-ApoE-/- double knockout (dKO) mouse model was subjected to functional assays including proliferation, migration and apoptosis. The siRNA mediated knockdown of MRAS in human SMCs increased migration and proliferation with and without stimulation of TNF, IL-6 and IL-1ß. In line with that, the absence of Mras in murine SMCs led to significant increase in proliferation, enhanced migration and reduced apoptotic activity compared to control B6.ApoE-/- SMCs. Stimulation with TNF, IL-6 and IL-1ß enhanced the proliferative effect of Mras deficiency, indicating an interplay of these cytokines and MRAS pathways. Moreover, bulk RNA sequencing revealed that Mras knockout in murine SMCs led to differential gene expression of numerous genes involved in cGMP and cAMP signalling, suggesting that SMCs upregulate cGMP/cAMP signalling to compensate for the loss of Mras. The in vivo atherosclerosis study on plaque characteristic revealed that the impact of Mras deficiency was sex-specific as significant differences in plaque size, collagen content and macrophage staining were only observed in male mice. Male Mras-/-ApoE-/- dKO mice showed significantly higher levels of collagen content, MoMa content and SMC content as compared to B6.ApoE-/- male mice. In conclusion, our data indicates that lower MRAS levels in the vessel wall, particularly in SMCs, provides protection against CAD by stabilizing atherosclerotic plaques, and thereby decreasing the risk of plaque rupture and subsequent hospitalization.
Modulation der elektrischen Erregbarkeit sensorischer Neurone durch Funktionsveränderungen von NaV1.9-Kanälen
(2025-02-27) Kühs, Samuel
Spannungsgesteuerte Natriumkanäle (NaVs) sind von entscheidender Bedeutung für die Signalübertragung in elektrisch erregbaren Zellen, wie Muskel- und Nervenzellen. Im Menschen kodieren neun Gene (SCN1A-SCN5A, SCN8A-SCN11A) für die NaV-Subtypen NaV1.1-NaV1.9. NaV1.9 ist neben NaV1.7 und NaV1.8 einer der wesentlichen NaV-Kanäle in sensorischen Afferenzen und nimmt hinsichtlich seiner Funktionseigenschaften eine Sonderstellung innerhalb der NaV-Kanalfamilie ein. Anders als die übrigen NaV-Subtypen sind NaV1.9-Kanäle bereits beim Ruhemembranpotential aktiv und generieren aufgrund ihrer ausgeprägt langsamen Inaktivierungskinetik persistente Na+-Ströme. Die physiologische Relevanz der NaV1.9-Kanäle wird durch zahlreiche Mutationen im dafür kodierenden SCN11A-Gen unterstrichen, die beim Menschen kongenitale Analgesie oder periphere Schmerzattacken auslösen können und oftmals mit ausgeprägten gastrointestinalen Beschwerden einhergehen. Interessanterweise steigern die bekannten und funktionell charakterisierten pathogenen NaV1.9-Varianten die Kanalaktivität und zeigen damit gain-of-function-Eigenschaften. Die Zunahme der Kanalaktivität beruht dabei entweder auf einer verstärkten Kanalaktivierung, einer eingeschränkten Kanalinaktivierung oder der Kombination beider Funktionsveränderungen. Die Datenlage suggeriert, dass insbesondere das Inaktivierungsverhalten der NaV1.9-Varianten entscheidend an der Ausprägung des klinischen Phänotyps beteiligt ist. Die vorliegende Dissertationsschrift ist in drei Themenkomplexe gegliedert, in denen verschiedene Aspekte der Modulation von NaV1.9 untersucht werden. Der erste Teil dieser Arbeit beschäftigt sich mit der Bedeutung des NaV1.9-C-Terminus für die Kanalinaktivierung. Mittels systematisch erzeugter Kanalchimären und deren detaillierter Analyse in Patch-Clamp-Experimenten wurde ein 100-Aminosäuren umfassender Abschnitt im C-Terminus identifiziert, der die Inaktivierungseigenschaften der Kanäle maßgeblich mitbestimmt. Im zweiten Teil der Arbeit wurden die Funktionseigenschaften mehrerer potenziell pathogener NaV1.9-Varianten untersucht, die bei Patienten mit veränderter Schmerzwahrnehmung identifiziert wurden, und die Varianten hinsichtlich ihrer Krankheitsrelevanz bewertet. Zusätzlich zu einer veränderten Schmerzwahrnehmung gehen pathogene NaV1.9-Varianten beim Menschen regelhaft mit gastrointestinalen Beschwerden einher. Dieser Zusammenhang ist zwar bekannt, doch aufgrund der schwierigen experimentellen Zugänglichkeit enterischer Neurone, kaum untersucht. Um die Auswirkungen krankheitsassoziierter Varianten auf enterische Neurone systematisch untersuchen zu können, wurde im Rahmen dieser Arbeit ein Verfahren entwickelt, dass die Isolation, Transfektion und systematische elektrophysiologische Analyse enterischer Neurone ermöglicht. Mit Hilfe dieser Methodik wurde die mit Analgesie- und Obstipation assoziierte Variante NaV1.9-L396P erstmals in enterische Neurone eingebracht und funktionell charakterisiert. Es konnte gezeigt werden, dass die NaV1.9-L396P-Kanäle nahezu vollständig inaktivierungsdefizient und somit vermutlich entscheidend an der Ausprägung des klinischen Phänotyps beteiligt sind. Im dritten Teil der Arbeit wurden die Auswirkungen reaktiver Sauerstoffspezies (ROS), die mit Schmerzen unterschiedlichster Ätiologie in Verbindung stehen, auf die Funktion Nozizeptor-spezifischer NaV-Kanäle untersucht. Einerseits bewirken ROS eine Reduktion des Na+-Peakstroms. Andererseits verzögern sie aber auch die physiologisch wichtige Kanalinaktivierung. Diese potenziell proexzitatorische Funktionsveränderung ist bei NaV1.8 besonders stark ausgeprägt, weshalb dieser Kanal lange Zeit als ROS-sensitivster NaV Subtyp galt. Daten zur ROS-Sensitivität der NaV1.9-Kanäle existieren bislang nicht. In der vorliegenden Arbeit wurde erstmals NaV1.9 hinsichtlich seiner ROS-Sensitivität analysiert und gezeigt, dass er eine ähnlich hohe ROS-Sensitivität wie NaV1.8 aufweist und somit ebenfalls zu den stark ROS-regulierten NaV-Kanälen gezählt werden muss.
Effect of different vaccine adjuvants and checkpoint inhibitors on antibody responses and tumor growth
(2025-02-18) Lehrian, Selina
Vaginal tract microbiota in health and disease
(2025-02-07) Lupatsii, Mariia

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