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Outside-in or inside-out: Analyzing the assembly pathway of the type III secretion injectisome of Salmonella
No full textWith the aim to reveal the assembly pathway of Salmonella Typhimurium T3SS, we used in vivo photocrosslinking to test interactions between different proteins in various genetic backgrounds with a focus on components of the base and export apparatus. Based on the results, we propose a model in which assembly is carried out from two nucleation points. Assembly starts simultaneously at the outer membrane with the secretin and at the inner membrane with the export apparatus, onto which PrgK (SctJ) assembles. Initially, di- and trimers of PrgH (SctD) are attached to the secretin, but they can only form their complete ring in the presence of the export apparatus - PrgK intermediate; PrgK seems to be necessary as a template for the oligomerization of PrgH. A decision in favor of inside-out or outside-in assembly is therefore not possible.Die Dissertation ist gesperrt bis zum 31. März 2027
Impact of drug-microbiome interactions on the intestinal colonization with pathogenic Gammaproteobacteria
No full textThe human gut microbiome has garnered scientific interest due to its key role in health. Maintaining a balanced microbiome is crucial for preventing harmful pathogens from colonizing the intestine and causing disease. It has been shown that drugs beyond classical antibiotics, inhibit growth of gut bacteria in monoculture. To translate these findings to a community context, we developed the synthetic model community Com20, consisting of 20 prevalent and abundant species of the human gut microbiome for which the individual direct growth effects of over 1000 non-antibiotic drugs have already been described. We identified S. perfringens as a key member required for Com20 community stability in vitro and explored the impact of individual community members when challenged with diverse pathogenic Gammaproteobacteria. Among them, Salmonella typhimurium showed the greatest variability in growth when a single strain was absent from the community. Com20 was able to colonize mice long-term, making the model suitable for investigating a chronic treatment regimen followed by a subsequent pathogen challenge. This allows us to explore the impact of non-antibiotic drugs on disrupting colonization resistance. To identify drugs which affect the community in vitro, we used Com20 and human stool-derived communities and treated them for 24h with >50 non-antibiotic drugs in different concentrations, before challenging the drug-treated communities with Salmonella typhimurium. Approximately 35% of the tested drugs increased the growth of the pathogen, which can be explained by shifts in community composition or a decrease in community biomass. We then challenged drug-treated communities with various other pathogenic Gammaproteobacteria and observed an increase in pathogen growth. We tested 5 drugs in our chronic treatment animal model in both defined colonized and conventional mice to demonstrate the risk of non-antibiotic drugs in disrupting colonization resistance. Drugs that promoted the growth of Salmonella typhimurium and other Gammaproteobacteria in the in vitro screen resulted in an increase of fecal Salmonella counts in both animal models, suggesting a possible influence of non-antibiotics on infection risk. In summary, our study provides a model which allows for a detailed examination of the effects of non-antibiotic drugs within a controlled context and highlights a critical and overlooked issue regarding the implications of non-antibiotic drug usage on microbial dynamics and pathogen proliferation.Dissertation ist gesperrt bis 9. April 2027
Neue Perspektiven zur Direktverstromung von Methanol
No full textDie Dissertation ist gesperrt bis zum 27. Februar 2027 !Der Umsatz von Methanol zur Energiegewinnung in Direktmethanol-Brennstoffzellen verläuft nach dem jetzigen Stand der Technik unter signifikanten Einbußen in der Nutzspannung. Im Rahmen der vorliegenden Dissertation wurde die Möglichkeit einer Effizienzsteigerung der katalytischen oxidativen Methanolspaltung durch die Neustrukturierung der Katalysereaktion untersucht. Hierzu wurde das Reaktionsprinzip des Enzyms Galaktoseoxidase (GO) genutzt.
Nach dem Vorbild literaturbekannter katalytisch aktiver Modellstrukturen für GO wurden funktionalisierte Salankomplexe des Kupfers entwickelt. Ziel war es, die Geschwindigkeit des α-Wasserstoffatomtransfers (HAT) im geschwindigkeitsbestimmenden Schritt der Methanoloxidation zu erhöhen. Dabei sollte die Übertragung der Reaktion in Wasser mit pH>7 zusätzlich unterstützend auf den HAT einwirken. Die Redox-Aktivität und Reaktivität der Komplexe wurde mittels Cyclovotammetrie, UV-vis-NIR-Absorptionsspektroskopie und magnetischer Messungen untersucht. Die Cu(II)-Komplexe zeigen für Salan-Komplexe typische Absorptionsspektren, ein für Cu(II)-Ionen typisches magnetisches Moment sowie ESR-Spektrum und eine quasireversible Elektrochemie. Infolge der Reaktion der Cu(II)-Salankomplexe mit einem Oxidationsmittel entsteht jedoch nicht, wie in der Literatur beschrieben, ein Cu(II)-Monophenoxylradikal, stattdessen bilden die Komplexe Cu(II)-Bisphenoxylradikale. Dabei stabilisieren sie sich durch Dissoziation der Alkalimetallkationen vermutlich als Zwitterionen.
Untersuchungen der katalytischen Reaktivität der zweifach oxidierten Komplexe zeigen zudem keine HAT-Aktivität. Stattdessen reagieren sie mit Hydroxidionen, wobei die reduzierten Komplexe und vermutlich Wasserstoffperoxid entsteht. Dieses Verhalten wird auf die Vermeidung des einfach oxidierten Zustandes der Komplexe zurückgeführt. Die vorliegende Arbeit liefert einen Beitrag zum Verständnis der Reaktionssteuerung katalytisch aktiver Metallkomplexe und zeigt auf, dass die flexible Dissoziation und Assoziation geladener Gruppen in Abhängigkeit vom Oxidationsstatus eines Komplexes wesentlichen Einfluss auf ihre Reaktivität haben kann
Structural Investigation of Bacterial Cell Wall Modifying and Recycling Enzymes
No full textDie Dissertation ist gesperrt bis zum 28. Juli 2027 !The bacterial cell wall with its main structural component, peptidoglycan, is essential for the
survival of bacteria as it maintains cell integrity by countering the internal turgor pressure and
provides protection against environmental stresses as an outer defense barrier. Continuous
synthesis, remodeling, and degradation of the peptidoglycan macromolecule is required during
the bacteria's life cycle in order to allow growth, division, and separation. In the context of
increasing antibiotic resistance in many bacterial strains, some of which are human pathogens
with major implications for clinical healthcare, these processes and the peptidoglycan molecule
itself have been the subject of numerous studies.
In the course of peptidoglycan biosynthesis, many bacteria introduce modifications to the
archetypical peptidoglycan structure to evade host defense mechanisms and to improve rigidity
and stability of the peptidoglycan scaffold. One of the most common modifications is the
amidation of the second amino acid residue in the peptidoglycan stem peptide, which directly
influences the peptidoglycan cross-linking efficiency and contributes to antibiotic resistance. In
the first project of this thesis, the MurT/GatD enzyme complex that performs this amidation in
important human pathogenic bacteria such as Staphylococcus aureus, Mycobacterium
tuberculosis, and Streptococcus pneumoniae was investigated. Following up on two previously
determined structures, a new crystal packing for S. aureus MurT/GatD was identified that
allows crystallization experiments with peptidoglycan precursor ligands. Moreover, a third
homologous structure from Streptococcus pyogenes was obtained enabling a more detailed
comparison of two distinct arrangements of the protein complex and confirming the conserved
binding site for ATP MurT in a ligand-bound structure. The structures determined in this work
in combination with enzyme activity and ligand binding assays expanded and improved our
knowledge on bacterial peptidoglycan amidation by the MurT/GatD complex. Together with
the structures from S. aureus and S. pneumoniae, they provide a basis for future structural
studies on MurT/GatD with the long-term goal of developing specific, small molecule inhibitors
for this enzyme complex in order to combat antimicrobial resistant pathogens.
To preserve resources when degrading the cell wall, most bacteria recycle the individual
building blocks of the peptidoglycan molecule, either for reuse in cell wall synthesis or in case
of starvation as a nutrient source. Recycling pathways influence the viability of bacterial species
and are often linked to antibiotic resistance mechanisms. While peptidoglycan recycling in
Gram-negative bacteria has been studied in more detail, less information is available for Gram-
Ipositive species. The second project of this thesis focused on two peptidases, YkfA and YkfC,
from the Gram-positive model organism Bacillus subtilis that cleave amide bonds in
peptidoglycan-derived peptides. Protein expression, purification, and crystallization
experiments were conducted for the L,D-carboxypeptidase YkfA, that cleaves the bond
between the third and fourth residue in the peptidoglycan stem peptide, with the aim of
determining the first atomic structure of this enzyme. The experiments provide a starting point
for further studies to elucidate the structure and mechanism of YkfA in order to generally
improve the understanding of serine peptidases from the S66 family that are involved in cell
wall recycling. The D,L-endopeptidase YkfC cleaves between the second and third residue in
the stem peptide and belongs to the major class of NlpC/P60 cysteine peptidases. In this work,
a high yield expression and purification protocol of YkfC was established followed by X-ray
crystallographic experiments. To analyze substrate specificity and engagement on an atomic
level and for comparison to a homologous structure from Bacillus cereus, structures of YkfC
in the apo state, covalently bound to a dipeptide product, and in complex with a pentapeptide
substrate were determined. Investigation of the structures revealed a conserved specificity to
peptides with a free N-terminal L-alanine residue and the formation of an oxyanion hole by a
tyrosine residue, which is unusual for cysteine peptidases, in the YkfC subfamily. Moreover,
the structures gave insights into the orientation of important residues during catalysis and the
engagement of the C-terminal residues in the peptide substrate by YkfC. Residues potentially
forming contacts to the peptide substrate were additionally investigated in a mutation activity
experiment. With the here laid structural foundation, further experiments including the natural
mDAP-containing peptide substrate are now straightforward. Afterwards, the focus could be
shifted towards related NlpC/P60 peptidases that, in contrast to B. subtilis YkfC, are essential
for the bacterium.
In summary, the structural studies on peptidoglycan modification and recycling enzymes
presented in this work improved our understanding of important processes associated with the
bacterial cell wall. The information gathered could serve as a basis for comparison in future
studies on functionally related enzymes and help in the development of strategies to inhibit
these enzymes and thus combat bacterial pathogens
Erste klinische Anwendung von [18F]FPyGal als Seneszenz-Tracer im Rahmen einer Phase 1/2-Studie
No full textDie Dissertation ist gesperrt bis zum 17. Juni 2027 !Mit Auswertungen der Patientensicherheit und Dosimetrie-Analysen
an gesunden Probanden
sowie ersten Erkenntnissen an onkologischen Patienten/-inne
Cognitive and brain mechanisms of semantic control in creative problem-solving
No full textDie Dissertation ist gesperrt bis zum 14. April 2027 !Creativity has been defined as the capacity to generate novel and appropriate ideas, a core skill for innovation and adaptive functioning. Yet, the cognitive and neural underpinnings of creativity remain elusive. This dissertation examines the neurocognitive mechanisms underlying creative problem-solving, with a particular focus on how semantic processing shapes idea generation. Combining computational modelling and neuroimaging, it investigates how both internal manipulations (e.g., semantic control) and external contexts (e.g., immersive virtual reality illusions) shape the emergence of creative solutions. First, the findings reveal that VR-induced visual disruptions foster cognitive flexibility, thereby enhancing creative problem-solving performance. Second, this thesis demonstrates that even young children engage in goal-directed semantic exploration optimally balancing novelty and appropriateness. Third, neural findings with fMRI shed light on how the brain supports story generation, particularly creative storytelling, integrating whole-brain and region-specific analyses. Finally, EEG results link semantic distances to dynamic reconfigurations of brain activity. Collectively, these findings elucidate the neural and cognitive foundations of creative thought, showing how both internal processes and external influences shape problem-solving. They underscore the inherent complexity of creative process and the need for advanced computational methods to capture their dynamic evolution over time and across diverse contexts. Ultimately, this thesis sets the stage for future research on human creativity, an area essential for scientific progress and for addressing our rapidly evolving world's challenges
The Cellular Roles of the PII-like Protein SbtB and its Effector Molecule 3′,5′-c-di-Adenosine-5′-Monophosphate (c-di-AMP)
No full textThe signaling transduction proteins of the PII superfamily, such as the PII-like protein SbtB,
represent an ancient and exceptionally well-preserved protein family ubiquitously across all
domains of life. Within cyanobacteria, SbtB serves as the main regulator of the carbon
concentrating mechanism (CCM) and inorganic carbon (Ci) acclimation and is co-expressed
from a bicistronic operon with the sodium-dependent bicarbonate transporter (SbtA). SbtB
regulates bicarbonate uptake by direct interaction with all three cyanobacterial bicarbonate
transporters: SbtA, BicA, and BCT1, in response to fluctuations in Ci levels and the adenylate
energy charge (AEC). This study proposes a novel model for SbtB-mediated regulation of SbtA,
emphasizing the significance of the flexible SbtB T-loop structure for this interaction, akin to
the canonical PII T-loop in target protein interactions. In the AMP-bound state, corresponding
to low Ci (LC) supply, SbtB inserts its T-loop into the inter-domain cleft of SbtA, inducing an
inward-open conformation that facilitates substrate secretion into the cytoplasm while
inhibiting backward bicarbonate transport. However, the cellular function of SbtB extends
beyond bicarbonate uptake, significantly impacting central carbon metabolism and Ci
acclimation. In the absence of SbtB in the slr1513 knockout mutant (ΔsbtB), CCM-associated
gene expression, including the master Ci acclimation regulator NdhR, is broadly affected,
resulting in a constitutively LC pre-acclimated state.
In Synechocystis sp. PCC 6803, SbtB has been identified as the primary receptor of the
second messenger, c-di-AMP. The SbtB:c-di-AMP complex regulates glycogen synthesis
through its interaction with the glycogen branching enzyme (GlgB), which is essential for
nighttime cyanobacterial survival.
While SbtB primarily influences cellular functions related to the central carbon metabolism,
c-di-AMP plays a multifaceted role in governing various cellular processes. These encompass
the maintenance of ion homeostasis, notably K+
, Na+
, and Mg2+, thus regulating
osmoprotection. C-di-AMP also exerts control over the central nitrogen metabolism, either
directly or indirectly, by modulating the expression of NtcA, which in turn impacts on the
process of chlorosis. Additionally, c-di-AMP plays a pivotal role in regulating the uptake of
glutamine by regulating the activity of the basic amino acid and glutamine transporter complex
BgtAB. Moreover, it is indispensable for facilitating chromatic acclimation by influencing the
expression of CpcL through Rbp2.Die Dissertation ist gesperrt bis zum 24. Juli 202
Characterization of a new human in vitro model of the optic nerve based on assembloid technique
No full textDie Dissertation ist gesperrt bis zum 16. Januar 2027 !The eyes give us the ability to see, to interact with our environment and with other people being fundamental for an independent and social live. Vision loss leads to a great impairment of our live, including the loss of autonomy and self- determination. Generally, various neurodegenerative diseases have an impact on the optic nerve leading to damage and cell death of the RGC and their axons forming the optic nerve. This results in the deterioration of vision up to the complete vision loss. One of the most common causes of blindness worldwide is the glaucoma. Predominantly in elderly people an elevated intraocular pressure mechanically damages the axons of the RGC ending in RGC death. Unfortunately, due to the lack of apparent symptoms until advanced disease stages, treatment is often late and insufficient. Currently, the only possible treatment available is decreasing the intraocular pressure which can lead to a slowdown in diseases progress. Consequently, the treatment possibilities are very limited for the glaucoma and other neurodegenerative diseases resulting in vision impairment or blindness of the patient.
Currently, the pathophysiology of the optic nerve as well as treatment possibilities are studied in animal models predominantly using for instance the optic nerve crush model. Animal models have certain limitations compared to human models. A limitation of the animal model is that due to numerous differences between animals and humans predominantly in the biology and in genetics, the results obtained in animal models are hardly transferable to patients. As an example, treatments tested in mice are uncertain to work in humans the same way. This leads to the conclusion that human models of the optic nerve are necessary to overcome these limits and to obtain an increased comparability and significance of the results.
In this thesis, the main goal was to develop a new human in vitro model of the optic nerve using the assembloid technique. The created and characterized optic nerve assembloids (ONA) are based on hiPSC derived organoids: a retinal organoid represents the retina and a thalamic organoid mimicking the target areas of the retinothalamic fibers in the thalamus, both connected by a 3D matrigel based sphere containing astrocytes. This “nerve-like compartment” is formed by the RGC axonal projections which grow out from the RGC layer of the RO and pass through the 3D matrigel based sphere into the TO. Additionally, the RGC axonal projections are ensheathed by astrocytes and form synapses to the thalamic neurons. This leads to the conclusion that the development of the RGC and the outgrowth of the RGC axonal projections can be studied in the ONA model. On top of that, the ONA model can be produced patient derived in order to perform drug testing in the setting of individualized medicine.
Furthermore, the nerve crush model was established in the ONA model providing the opportunity to study the pathophysiology of neurodegenerative diseases of the optic nerve and the glaucoma. The pathological changes take place particularly in the unmyelinated part of the optic nerve which is represented by the ONA model. RGC death as well as astrocyte activation and the inflammatory response were analyzed after the optic nerve crush treatment. The immune system was represented through the integration of microglia cells into the 3D matrigel based spheres which allows to study the immune reaction after crush treatment.
In summary, results obtained in the ONA crush/cut model showed a significant increase in the expression of Caspase 3 and BRN3a double positive cells indicating the RGC loss particularly at D1 after cut treatment in immunostainings. Besides, in the RNA expression level analysis a trend towards downregulation of RGC markers like NEFM was found. In addition to that, astrocyte activation was shown via a significant increase of the RNA expression levels of various astrocyte markers like GFAP, S100b and SLC1A3 and a trend towards a higher expression of the astrocyte marker GFAP was visible in the immunostaining analysis. Furthermore, different inflammation markers like NFkB1, CD44 and APOE showed a significant increase in their expression levels after the crush/cut treatment.
As the summarized results from the ONA model are overall comparable to those described in optic nerve crush and glaucoma studies in animal models, the ONA can be used to reduce or replace the animals needed in this research field. Additionally, benefits from the ONA model compared to the animal model are the human biology and that the ONA model can be produced in a high number needing less space and care and is easier to handle. Despite the variability within the ONA batches this provides further advantages particularly for drug testing in the glaucoma and neurodegenerative optic neuropathy research field
Entwicklung eines Rasterionenleitfähigkeitsmikroskops zur Untersuchung morphologischer und mechanischer Besonderheiten von menschlichen Thrombozyten
No full textDissertation gesperrt bis zum 06.07.2027!Um zellbiologische Prozesse unter physiologischen Bedingungen hochauflösend und nicht-invasiv untersuchen zu können, wurde ein neuer Aufbau für die Rasterionenleitfähigkeitsmikroskopie (SICM) entwickelt. SICM ermöglicht die quantitative, kontaktfreie Erfassung topographischer und mechanischer Eigenschaften lebender Zellen mittels einer Nanopipette, deren Ionenstrom in Abhängigkeit vom Abstand zur Zelloberfläche gemessen wird.
Der entwickelte Aufbau zeichnet sich durch verbesserte Benutzerfreundlichkeit und hohe Bildraten aus (<7 s/Bild bei 40×40 Pixeln), unter anderem durch die Integration eines Annäherungsmotors und eines Systems zum Flüssigkeitsaustausch während laufender Messungen. Damit konnten erstmals dynamische Volumenänderungen einzelner menschlicher Thrombozyten unter hypotonischem Schock mit einer Auflösung von 0.1 fL analysiert werden. Die Zellen zeigten hierbei eine aktive Volumenregulation, deren Geschwindigkeit und Effizienz quantitativ beschrieben wurde. Zudem wurde der Einfluss verschiedener Aktivatoren (Thrombin, Kollagen, ADP) sowie eines Volumenregulationsinhibitors auf dieses Verhalten untersucht. Aktivierte Thrombozyten zeigten dabei ein ausbleibendes Regulationsverhalten. Besonders interessant war die Beobachtung, dass Thrombin- und ADP-aktivierte Zellen trotz Reduktion des nicht-osmotischen Zellanteils ihr Gesamtvolumen konstant hielten – ein Hinweis auf wasservermittelte Volumenkompensation.
Zusätzlich wurde gezeigt, dass das Zellvolumen mit der mechanischen Steifigkeit der Thrombozyten korreliert: Zunehmendes Volumen ging mit abnehmender Steifigkeit einher. Diese Korrelation blieb auch unter verschiedenen Bedingungen (z. B. bei Zellspreading oder unter aktinhemmender Behandlung mit Cytochalasin D) erhalten. Damit konnte eine robuste, behandlungsunabhängige Beziehung zwischen Volumen und Steifigkeit etabliert werden.
Insgesamt belegt diese Arbeit die Leistungsfähigkeit des neuen SICM-Systems zur hochauflösenden Echtzeit-Charakterisierung lebender Zellen und liefert neue Erkenntnisse zur Volumenregulation und mechanischen Eigenschaften von Thrombozyten – mit potenzieller Relevanz für die Untersuchung krankheitsrelevanter Prozesse
Investigating Antibiotic Efficacy and Resistance in Gram-Negative Bacteria
No full textDie Dissertation ist gesperrt bis zum 18. November 2027 !The rising threat of antibiotic resistance, mainly driven by the indiscriminate use of antibiotics, has compromised the effectiveness of many treatments and placed an increasing burden on global health systems. This study investigates how different Gram-negative bacterial species, namely Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae, respond to individual antibiotics and antibiotic combinations. My thesis aims to better understand the species-specific mechanisms underlying drug action and resistance, with the goal of informing more targeted therapeutic strategies in the future. The first project focused on combinations of β-lactams and aminoglycosides across various species. The results demonstrated that the effectiveness of this combination is highly species-specific and driven by the binding specificity of β-lactams. These findings suggest that while antibiotic combinations hold clinical promise, their application must be guided by a deeper understanding of species-specific interactions and cannot yet be generalised across bacterial taxa. The second project explored the emergence of antibiotic-resistant populations following short-term treatments, especially in E. coli. Short-term treatments were chosen to avoid biases inherent in long-term evolution studies, which often miss low-fitness resistant mutants. The study found that aminoglycosides uniquely enabled the rapid development of resistance, but only in E. coli. Survivor isolates showed reduced growth rates, indicating the presence of low-fitness mutants. The results highlight the importance of using alternative experimental designs to capture the full spectrum of resistance mechanisms. The third project examined how non-antibiotic-related elements, such as the phage defence system CBASS, modulate antibiotic susceptibility. The CBASS system was found to sensitise bacteria to antifolate antibiotics, transforming these typically bacteriostatic agents into bactericidal ones. However, this effect depended on the presence of CBASS components, in this case, the CD-NTase, underlining the complexity and species-specific nature of this interaction. Interestingly, our data suggest a co-occurrence of CBASS systems with antifolate resistance genes, pointing to a trade-off between phage defence and antibiotic resistance markers. Together, these projects reinforce that antibiotic efficacy and resistance are highly dependent on bacterial species and genetic context, way beyond the antibiotic target. A more personalised, species-level approach to antibiotic therapy, rather than generalising by Gram stain or taxonomy, may be necessary to improve clinical outcomes and combat resistance more effectively