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Item Multidrug resistance, population structure, and epidemiology of Mycobacterium tuberculosis complex strains from West Africa(2025-02-25) Blankson, Harriet Naa AfiaTuberculosis (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.Item Dynamische mikroskopische Optische Kohärenztomografie(2024-11-07) Münter, MichaelDie 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.Item Thyroid hormone receptor beta in MASH(2024) López Alcántara, NuriaMetabolic 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.Item BAT, can you feel the stream of dopamine?(2025) Raffaelli, Francesca-MariaCombatting 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.Item Mycobacterium tuberculosis resistance evolution(2025) Walz, TeresaWith 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.Item The impact of the pro-atherogenic MRAS gene in smooth muscle cells(2025-03-07) Shah, Pashmina WiqarCardiovascular 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.Item Modulation der elektrischen Erregbarkeit sensorischer Neurone durch Funktionsveränderungen von NaV1.9-Kanälen(2025-02-27) Kühs, SamuelSpannungsgesteuerte 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.Item Effect of different vaccine adjuvants and checkpoint inhibitors on antibody responses and tumor growth(2025-02-18) Lehrian, SelinaItem Vaginal tract microbiota in health and disease(2025-02-07) Lupatsii, MariiaItem Molecular characterization of disease-causing TUBB4A mutations reflecting the phenotypic spectrum(2025-01-13) Krajka, Victor PeterWhile the broad phenotypic spectra or even pleiotropy caused by pathogenic variants in the same gene remains a poorly understood enigma, they are of great translational importance for a better understanding of potential differential mechanisms on the one hand, and for more individualized patient counseling on the other. In this thesis, TUBB4A mutations with a clinical spectrum ranging from dystonia (DYT-TUBB4A) to hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) were functionally characterized. Whether this broad spectrum represents a phenotypic continuum or a pleiotropic pattern is controversial. While the former would imply that a common pathway is impaired to varying degrees, the latter could imply that different cellular functions are impaired to varying degrees. Another unresolved question is how different TUBB4A mutations affect the dynamic properties of microtubules in affected cells such as oligodendrocytes. To this end, mutations representative of the phenotype were examined for their specific effects at the molecular and cellular levels. For this thesis, an advanced cell lysate method was established that allows detailed characterization of microtubule dynamics, combining the advantages of live-cell imaging and TIRF microscopy - without the need to isolate tubulin beforehand. In addition, in silico simulations were performed to determine the influence of TUBB4A mutations on heterodimer conformations, which has not been done before for this tubulin isoform. Furthermore, a CRISPR/Cas9-generated heterozygous TUBB4A knockout iPSC line was established. The derived neurons showed severe impairment of mitochondrial motility, providing an ideal positive control to compare TUBB4A mutations regarding intracellular trafficking. The major finding of this thesis is the demonstration of a differential effect of TUBB4A mutations (H-ABC vs. DYT-TUBB4A) on microtubule growth, possibly indicating a pleiotropic manifestation. The comprehensive combination of in vitro and in silico approaches enabled to obtain a holistic picture of the potential effects of TUBB4A mutations and provided mechanistic insights into their differential phenotypic expression. In particular, the thesis reveals that: 1) The analyzed TUBB4A variants exert distinct effects on microtubule dynamics. Importantly, the H-ABC-associated TUBB4A variants (p.R2W and p.D249N) severely disrupt microtubule polymerization compared to variants causing the milder dystonic phenotype. 2) Mutants are less likely to adopt a straight conformation in silico. Instead, the bent conformation appears to be sterically favored in H-ABC mutants, consistent with the impaired microtubule polymerization of these mutants. 3) Anti-morphic variants per se do not prevent the morphological maturation of oligodendrocytes. Instead, these mutations may disrupt mitosis during the proliferation of oligodendrocyte progenitors, most likely leading to hypomyelination in H-ABC patients in infancy. Therefore, altered microtubule growth appears to be differentially affected by TUBB4A mutations, which may interfere with various cellular functions, suggesting a pleiotropic spectrum rather than a phenotypic continuum in TUBB4A-related diseases. The work was mainly performed at the University of Lübeck at the Institute of Neurogenetics (laboratory of Prof. Christine Klein, supervised by Dr. Aleksander Rakovic). For specific methods, collaborations were established at the University of Lübeck with the Institute of Cardiogenetics (laboratory of Prof. Jeanette Erdmann, supervised by Dr. Stephanie Tennstedt), the Institute of Experimental and Clinical Pharmacology and Toxicology (laboratory of Prof. Markus Schwaninger, supervised by Dr. Helge Müller-Fielitz) and the Institut Curie, Université PSL, CNRS UMR3348, Orsay, France (laboratory of Dr. Carsten Janke "Control of microtubule dynamics and function with the tubulin code").Item Sex differences in vaccine induced immunity and protection against Mycobacterium tuberculosis(2025-01-24) Harikumar Parvathy, GishnuTuberculosis (TB), a disease killing over a million people per annum, shows a strong male preponderance in disease development. Although increased male affliction for TB has long been known from an epidemiological perspective, the mechanistic understanding of those differences is relatively recent. The only approved vaccine for TB, Bacillus Calmette Guérin (BCG), shows high variability in its protective efficacy – necessitating the development of effective vaccine candidates. However, whether the male-biased susceptibility to TB also applies to the efficacy of the BCG vaccine, has been scarcely explored. In the current study, a male specific failure of BCG is demonstrated in the C57BL/6 mouse model. However, two recombinant derivatives of BCG (rBCGs) - VPM1002 and BCGΔBCG1419c - were found to ameliorate this male specific vulnerability of BCG by significantly improving survival rates in males upon Mycobacterium tuberculosis (Mtb) challenge. The disparities in survival between rBCGs and BCG vaccinated males were not attributable to their ability to reduce lung colony forming units (CFUs). Further analysis revealed that BCGΔBCG1419c, used as a representative of VPM1002 and BCGΔBCG1419c, significantly enhances CD8 T cell responses 90 days post vaccination compared to BCG, specifically in males. This enhancement shows a strong positive correlation with improved survival following Mtb challenge. In addition, significant positive correlations were identified between the CD4 T cell response on day 28 post-vaccination and the CD8 T cell response on day 90 post-vaccination, as well as between the CD4 T and B cell responses on day 28 post-vaccination. The CD4 T cell response at day 28 post-vaccination also showed a significant direct correlation with survival following Mtb challenge. Lastly, 28 days post-vaccination, CD8 T cell populations in the spleen showed distinct global differences between sexes, with specific clusters varying between males and females, independent of vaccine type. In summary, the current study identified a male specific failure of BCG in the C57BL/6 mouse model of TB and the ability of rBCGs to significantly improve the protective efficacy specifically in males. The underlying differences in post-vaccine immune responses that correlate with vaccine efficacy, as well as, those differences between sexes were identified. Elucidating how sex-specific differences in CD8 T cell responses influence vaccine efficacy, as well as the potential role of CD4 T cells and B cells in the sex-specific development of different CD8 T cell populations open new avenues for future studies.Item Item Interplay between diet and hippocampal circadian clocks in the regulation of memory performance(2024) Schmidt, Cosima XeniaItem The effects of social feedback and underlying neuropsychological mechanisms(2024) Sobczak, AlexandraItem B cell metabolism in autoimmunity(2024) Lindemann, TimoMetabolic reprogramming has been recognised as a major hallmark of immune cell activation. While researchers have gained detailed insight about this process in many different immune cells like T cells and macrophages, the B cell lineage lacks this level of understanding. In this thesis, I aimed at elucidating the metabolic profile of B cells and plasma cells to modulate their function by pharmaceutical inhibition of their respective metabolic profile. Using flow cytometry, plasma cells and B cells were analysed with regard to the basic metabolic parameters glucose uptake and mitochondrial mass, as well as antibody synthesis and the activity of the unfolded protein response (UPR) on the single cell level. These parameters were analysed after treatment with glycolysis inhibitor 2-deoxyglucose (2-DG) in vitro in either isolated B cells activated with lipopolysaccharides (LPS) or whole spleen cells without stimulant. In addition, B6.NZM-Sle1NZM2410/Aeg Sle2NZM2410/Aeg Sle3NZM2410/Aeg/Lmoj (BcN) mice, a mouse strain developing lupus-like symptoms due to the deletion of three lupus susceptibility loci, were injected with 2-DG to analyse its effect on B lineage cells in spleen and bone marrow in vivo. Plasma cells from spleen showed high glucose uptake, but low mitochondrial mass. Furthermore, 2-DG treatment lead to a distinct decrease in splenic plasma cell numbers both in vitro and in vivo, indicating a preference for aerobic glycolysis. However, the number of bone marrow plasma cells was not affected by 2-DG treatment. In addition, 2-DG treatment in vitro resulted in a decrease in the concentration of IgM, IgG and IgA antibodies in cell culture supernatant, while only the number of IgM+ plasma cells was decreased. Instead of a decrease in numbers, IgG+ and IgA+ plasma cells showed an increase in the amount of intracellular antibodies and UPR activity, indicating defective protein synthesis or secretion due to 2-DG. Some (but not all) of the effects of 2-DG could be prevented by adding mannose to the cell culture, an effect that could not be found when replacing 2-DG with the structurally similar glycolysis inhibitor 2-fluorodeoxyglucose (2-FDG). In conclusion, these data show that plasma cells adopt aerobic glycolysis after differentiation, a metabolic profile similar to other mTOR-dependent immune cells like Type 1 or Type 17 T helper cells (Th1, Th17) or M1 macrophages. The dependence on aerobic glycolysis makes plasma cells susceptible to treatment with glycolysis inhibitors. While the impact of 2-DG on plasma cells is in part due to its function as a glycolysis inhibitor, it also inhibits mannose metabolism, leading to defective protein folding, flawed lysosome assembly thereby eventually inducing UPR activity. Due to its specificity towards plasma cells, 2-DG has potential to become a novel therapy approach in autoimmune diseases, reducing autoantibody production by plasma cells while simultaneously increasing the expression of the anti-inflammatory cytokine interleukin 10 (IL-10) in B cells.Item Functional and structural changes in the progression of Alzheimer's disease(2024-12-18) Mieling, MartheItem microRNAs – size doesn’t matter!(2024) Britsemmer, Jan HendricItem B cell receptor physical properties affect relative IgG1 and IgE responses in mouse egg allergy(2023) Udoye, Chinweike Christopher