40639 research outputs found
Sort by
The Space in Language – Do Children’s Early Words Link to Their Sensorimotor Experiences of Vertical Space?
No full textDie Dissertation ist gesperrt bis zum 26. März 2027 !Mehrere Studien haben gezeigt, dass die Verarbeitung von Substantiven (z. B. „Mond“), die eine bestimmte vertikale Position implizieren (z. B. oben), nachfolgende Reaktionen verzerrten. Erwachsene reagierten in der Regel schneller nach oben, wenn sie ein implizites „oben Wort“ lasen, als nach einem impliziten „unten Wort“ (z. B. „Stein“). Solche Belege für Sprach-Raum-Assoziationen wurden als Argumentation für die Aktivierung sensorischer und/oder motorischer Systeme während des Sprachverstehens angeführt. Einige solcher verkörperten Kognitionstheorien argumentieren, dass sensomotorische Erfahrungen während des anfänglichen Wortlernens relevant sind. Allerdings wurde diese Annahme in wesentlich weniger Arbeiten mit Kindern experimentell überprüft. Die vorliegende Dissertation versuchte diese Forschungslücke zu schließen, indem Sprach-Raum-Assoziationen über einen breiten Entwicklungsbereich hinweg untersucht wurden. Die zentrale Forschungsfrage war: Sind frühe Wörter von Kindern bereits mit sensomotorischen Erfahrung des vertikalen Raums verknüpft?
Studie I untersuchte, wann Kinder ihre ersten Substantive verstehen, also entsprechende lexikalische Einträge für Wörter wie „Hut“ in ihrem Proto-Lexikon etablieren. In Anlehnung an Arbeiten aus anderen Sprachen konnten die getesteten Deutsch lernenden Kinder im Alter von 6 bis 14 Monaten konkrete Substantive den dargestellten Referenten zuordnen. Interessanterweise beruhten diese lexikalischen Einträge auf festen eins-zu-eins-Zuordnungen zwischen Substantiven und Referenten und waren damit robuster als bisher angenommen.
Studie II untersuchte Sprach-Raum-Assoziationen und stellte die Frage, ob die Verarbeitung von Substantiven die Augenbewegungen von Säuglingen (11 bis 14 Monate), Kleinkindern (2 bis 4 Jahre) und Erwachsenen in die Richtung der impliziten vertikalen Position verzerrte. Die Augenbewegungen von Kleinkindern und Erwachsenen waren während der Substantivverarbeitung entsprechend den Sprach-Raum-Assoziationen verzerrt. Dies war bei den getesteten Säuglingen nicht der Fall. Studie III zeigte, dass etwas ältere Kinder (18 bis 24 Monate) gerade erst damit begannen, Substantive (von denen ihre Eltern berichten, dass sie diese verstehen) mit dem typischen vertikalen Raum zu verbinden.
Studie IV untersuchte, ob zusätzliche Informationen in der sprachlichen Umgebung eines Kindes die Erfahrung des vertikalen Raums weiter verstärken können. Diese Erfahrungen gehen über das bloße Erleben von Referenten in der jeweiligen vertikalen Position hinaus. Hier wurde getestet, ob Eltern Objekte im oberen vertikalen Raum mit einer höheren Tonhöhe benennen als Objekte im unteren vertikalen Raum. Es ist bekannt, dass bereits Neugeborene einen Zusammenhang zwischen Tonhöhe und vertikalem Raum wahrnehmen.
Insgesamt ist diese Dissertation die erste wissenschaftliche Arbeit, die das Verständnis von Substantiven bei Deutsch lernenden Kindern im Alter von unter einem Jahr nachweist. Darüber hinaus lieferte diese Dissertation, den bis jetzt jüngsten Hinweis für Sprach-Raum-Assoziationen: Kinder im Alter von zwei bis vier Jahren verknüpften Substantive mit den sensomotorischen Erfahrungen des vertikalen Raums. Entgegen einigen theoretischen Behauptungen wurden solche Verbindungen jedoch nicht während des anfänglichen Wortlernens hergestellt. Kinder unter zwei Jahren verknüpften Substantive nicht systematisch mit dem typischen Raum – obwohl die Kinder alle Substantive verstanden. Besonders hervorzuheben ist die Entwicklung eines neuen Sprach-Raum-Experiment innerhalb dieser Arbeit. Dieses zeichnet sich durch eine hohe praktische Durchführbarkeit aus und könnte in Zukunft auch auf andere kognitive Bereiche (neben der Sprache) ausgedehnt werden. Schließlich bot diese Arbeit eine alternative Perspektive auf die Entwicklung von Sprach-Raum-Assoziationen, indem sie andere Aspekte – wie Tonhöhenmodulationen – innerhalb der Sprachumgebung von Kindern beleuchtete.Several studies demonstrated that processing of nouns (e.g., “moon”) that imply a specific vertical position (e.g., up-response) biased subsequent responses. Adults were typically faster to respond with an up-response after reading an implicit “up word” rather than an implicit “down word” (e.g., “stone”). Such evidence for language-space associations has been used as a line of reasoning for the activation of sensory and/or motor systems during language comprehension. While many of embodied theories argue that such sensorimotor experiences are relevant during initial word learning, substantially less work has experimentally tested young children. By investigating the associations between language and space across a broad developmental range, this dissertation addresses this research gap. The central research question was: Do children’s early words link to their sensorimotor experiences of vertical space?
Study I investigated when children understand their first nouns, hence, established lexical entries for words such as “hat” within their proto-lexicon. Aligned to work from other languages, the tested German-learning infants between 6 to 14 months could map concrete nouns to depict referents. Intriguingly, this mapping relied on firm one-on-one mappings between nouns and referents, thus, were more robust than previously assumed.
Study II probed language-space associations, asking the question: whether noun processing biased the eye movements of infants (11 to 14 months), toddlers (2 to 4 years) and adults towards the implicit vertical position. Whereas toddlers and adults eye movements were biased during noun processing in line with language-space associations. This was not the case for the tested infants. Study III revealed that slightly older children (18 to 24 months) just started to link nouns (that their parents report them to understand) to typical vertical space.
Study IV investigated whether additional information in a child’s language environment could further reinforce the experience of vertical space, above and beyond the experience of referents in the respective vertical position. Here, I tested whether parents introduced objects in upper vertical space with a higher pitch than objects in lower vertical space. Mappings between pitch and vertical space are already known to be perceived by newborns.
In summary, this dissertation is the first scientific work to demonstrate the understanding of nouns in German-learning children under the age of one. This dissertation also provided evidence of language-space associations in the youngest age group so far: Children between two to four years linked nouns to their sensorimotor experience of vertical space. Contrary to theoretical claims, however, such connections were not made during initial word learning. Children under the age of two did not systematically link nouns to typical space – although they understood all nouns. Furthermore, this dissertation developed a new language-space experiment characterized by a high particle feasibility which could be extended to other cognitive domains besides language in the future. Finally, this work offered an alternative perspective on the development of language-space associations by exploring other aspects – such as pitch modulations – within children’s language environments
A Role for WIPI2 in Mitophagy
No full textDie Dissertation ist gesperrt bis zum 20. Januar 2027 !Autophagie ist ein konservierter intrazellulärer Recyclingweg in Eukaryonten, der essenziell für die Aufrechterhaltung der Homöostase ist. Eine dysregulierte Autophagie wird beim Menschen mit zahlreichen Krankheiten in Verbindung gebracht.
Die menschlichen WIPI-Proteine WIPI1 bis WIPI4 erfüllen entscheidende und individuelle Funktionen in der Autophagie. WIPI ist die Abkürzung für WD-repeat protein interacting with phosphoinositides und beschreibt ihre Rolle als PI3P-Effektoren: die Fähigkeit, an PI3P in autophagosomalen Membranen zu binden, aber auch Protein-Protein-Interaktionen zur Regulierung und Förderung der Autophagosomenbildung zu ermöglichen.
Im ersten Kapitel zeige ich, wie WIPI2 am Prozess der Mitophagie beteiligt ist, die durch den Multi-Kinase-Inhibitor Sorafenib ausgelöst wird, der die Standardtherapie für fortgeschrittene hepatozelluläre Karzinome ist. Sorafenib induzierte eine mitochondriale Depolarisierung und Spaltung. WIPI2 kolokalisierte mit beschädigten Mitochondrien, wo Mitophagie aktiviert wurde. In diesem Zusammenhang wurde WIPI2 an Ser413 dephosphoryliert, was auch durch mTOR-Inhibition und Mitophagie-Aktivierung über CCCP beeinflusst wurde. Diese noch nicht erforschte Phosphorylierungssteelle könnte Mitophagie-spezifisch sein und einen negativen Regulationsmechanismus von WIPI2 und Mitophagie über mTOR darstellen.
Kapitel 2 behandelt ein menschliches Kinom-Screening durchgeführt, um regulatorische Netzwerke der WIPI-Proteine zu identifizieren, wobei eine neue Signalachse für die Kontrolle von WIPI1 gefunden. Dieser Signalweg funktioniert über DDR1, die Abl-Kinasen, dann ERK2 und MYC-MAX, was die Genexpression von WIPI1 und damit seine Funktion hemmt. Darüber hinaus konnten wir WIPI1 als Verstärker der Autophagie charakterisieren, was Auswirkungen auf die Verlängerung der Lebensspanne hat.
Im nächsten Kapitel beschreibe ich, wie wir eine Autophagie-Analysemethode mit Hilfe von Immunfluoreszenzbildern von GFP-WIPI1 als Autophagie-Marker, konfokaler Laser-Scanning-Mikroskopie und automatischer Bildanalyse mit der Open-Source-Software CellProfiler entwickelt haben.
Im letzten Kapitel wurde unsere CellProfiler-Methode unter Verwendung von Giant Unilamellar Vesicles in Kombination mit nativen Proteinextrakten von GFP-WIPI2 Zellen, für die Analyse der Protein-Lipid-Bindung erweitert.Autophagy is a conserved intracellular recycling pathway in eucaryotes, which is essential for the maintenance of homeostasis. Dysregulated autophagy is associated with multiple pathologies in humans.
The human WIPI proteins WIPI1 through WIPI4 fulfill crucial and individual functions in autophagy. WIPI is short for WD-repeat protein interacting with phosphoinositides and describes their role as PI3P effectors: the ability to bind to PI3P in autophagosomal membranes, but also protein-protein interactions to regulate and foster autophagosome formation.
In the first chapter, I show how WIPI2 is involved in mitophagy induced by the multi-kinase inhibitor Sorafenib, which is the therapy standard for advanced hepatocellular carcinomas. Sorafenib induced mitochondrial depolarization and fission. WIPI2 translocated to damaged mitochondria, where mitophagy was activated. In this context, WIPI2 was dephosphorylated at the Ser413 site, which was also affected by mTOR inhibition and mitophagy induction via CCCP. This underexplored phosphosite could be mitophagy-specific and represent a negative regulatory mechanism of WIPI2 and mitophagy via mTOR.
In chapter 2, we performed a human kinome screen to identify regulatory networks of the WIPI proteins and found a novel signaling axis for the control of WIPI1. This signaling pathway functions via DDR1, the Abl kinases, then ERK2 and MYC-MAX to inhibit the gene expression of WIPI1 and therefore its function. In addition, we could characterize WIPI1 as an autophagy enhancer, which has implications for lifespan extension.
In the next chapter, I describe how we established an autophagy analysis method using immunofluorescence images of GFP-WIPI1 as an autophagy marker, confocal laser-scanning microscopy and automated image analysis with the open-source software CellProfiler.
In the final chapter, using giant unilamellar vesicles combined with native protein extracts from cells stably expressing GFP-WIPI2, our CellProfiler method was extended for the analysis of protein-lipid binding
The molecular pathophysiology of WIPI4 mutation in BPAN disease
No full textAutophagy is a highly conserved cell renewal process that consists on the formation of a double membraned autophagosome that grows and engulfs a portion of the cytosol for the degradation of its components to yield anabolic precursors. The WIPI proteins, WIPI1 to WIPI4 are key autophagy effectors with distinct functions supporting the cellular process at various levels. This study will focus on WIPI4 and its involvement in human disease. BPAN is a neurodegeneration with brain iron accumulation (NBIA) subtype characterized by the presence of iron deposits in the Substantia nigra and Globus pallidus of patients’ brains. The symptoms associated with the disease include early onset parkinsonism, dystonia, intellectual disability and seizures. The condition is caused by the de novo mutation of WDR45, the gene encoding human WIPI4. Although loss of WIPI4 function is known to be the underlying cause of BPAN pathophysiology, the specific cellular mechanisms disrupted by WIPI4 mutation remain largely unknown. Several studies propose that loss of WIPI4 results in autophagy and hence, ferritinophagy disruption. Nevertheless, none has described the specific role of WIPI4 in this process. The aim of this study was to unravel the involvement of WIPI4 in ferritinophagy and iron homeostasis in the context of BPAN disease.Die Dissertation ist gesperrt bis zum 10. Juli 2027
Studying the Intracellular Engagement of Nucleic Acids
No full textA large portion of the human proteome (~2000 proteins) binds to RNA. RNA-protein interactions influence the functions of all RNA species, e.g. messenger RNA (mRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), and long non-coding RNA (lncRNA) and in all cellular compartments, e.g. the nucleus, the cytoplasm, and in organelles like mitochondria and endosomes. An RNA’s fate is dictated by these interactions throughout its entire life span from biogenesis and processing over transport and subcellular localization to the modulation of the biological activity and ultimately its degradation. During these processes, accumulation effects of RNAs and proteins can occur that lead to the formation of aggregates. These fulfill important biological functions like gene expression and splicing regulation, but can also be a source of cellular toxicity by aberrant protein recruitment e.g. in triplet repeat expansion disorders like myotonic dystrophy. Moreover, pathogenic RNA identification by pattern recognition receptors is a key element of the innate immunity. Together, this highlights the importance of RNA-protein interactions on a molecular level as well as for the whole organism. Hence it is crucial to understand RNA-protein interactions in a scenario as lifelike as possible.
Currently, interactions between RNAs and proteins and the protein composition of their aggregates are typically identified by enrichment assays or proximity biotinylation and subsequent mass spectrometry (MS). These assays are often limited to highly abundant RNAs, require large input amounts and/or genetic manipulation of the target cells. Applying proximity biotinylation in situ – as described here by the FISH ID protocol – removes these limitations. On top of that, it enables to identify an RNA’s interactome under different cellular states such as the absence or presence of oxidative stress and with sub-compartment specificity. Here, a recombinant biotin ligase was recruited to the RNA-of-interest (ROI) by target-specific chemically modified antisense probes that are bound covalently by the enzyme’s tag.
Formation of the previously mentioned intracellular aggregates has also been observed after administration of chemically modified (RNA-like) therapeutic antisense oligonucleotides (ASOs). Interactions between proteins and ASOs are on the one hand responsible for their function, e.g. for cellular uptake and recruitment of effector proteins. On the other hand, the toxicity of an ASO drug is often connected to its protein interactions. Most of these interactions were identified by pulldown assays coupled to MS as well. The obtained interactome is independent from the subcellular localization of proteins and oligonucleotides. Hence it cannot reflect the intracellular interactions and does not allow any connection to other drug properties like efficacy and toxicity. This was overcome by the development of in situ ASO identification (isASO-ID): A purified biotin ligase construct was redirected to the ASO-of-interest by a coupling moiety. Proximal proteins were identified by MS after biotinylation and streptavidin enrichment. This allowed deciphering the interactomes with respect to chemical modifications, ASO concentration, the uptake pathway and under additional stress induction. Ultimately, the assay was applied to uncover the molecular basis of toxicity for an emerging class of ASO drugs: ADAR-recruiting ASOs. They impaired regular RNA processing upon their interaction with RNA processing enzymes. A better understanding of this will pave the way towards the engineering of safer ADAR-recruiting ASOs.
Taken together, FISH-ID and isASO-ID allow de novo identification of protein-RNA interactions in the cellular and thus – compared to existing methods – more relevant environment. They promise to be readily transferable to other cell types and will help to understand the roles of endogenous RNAs in disease and impact the development of safe and efficient ASO therapeutics, respectively.Die Dissertation ist gesperrt bis zum 06. Mai 2027
Exploring the Effects of Aurantimycin A, DCAP and ADEP on bacterial cell envelope functions
No full textDissertation ist gesperrt bis 09.05.2027
Metacognitive reinforcement learning: a theory of how people learn how to plan
No full textPlanning is a fundamental cognitive process that enables individuals to navigate complex decision-making scenarios, from daily activities to long-term strategic objectives. It can be conceptualised as a search tree that expands exponentially with the number of available options and the depth of foresight. While computers can leverage increasing processing power to explore vast decision spaces and find optimal solutions, the human mind operates within strict cognitive limitations. Despite these constraints, people routinely plan and make effective decisions - whether it be arranging the next meal or managing their long-term careers. This raises a crucial question: How do individuals plan so efficiently given their cognitive limitations? Prior research suggests that humans rely on heuristics and adaptive planning strategies, yet the mechanisms by which they learn these strategies remain unclear. This dissertation posits that these strategies are acquired through experience via a process known as metacognitive reinforcement learning. To test this proposition, this dissertation investigated two primary sets of hypotheses.
The first set of hypotheses asserted that experience-driven learning governs the adaptation of the amount of planning and planning strategies, as well as the discovery of novel planning strategies. To test these hypotheses, three experiments were conducted. Experiment 1 examined how people regulate the amount of planning, while Experiment 2 explored the adaptation of planning strategies across three different environmental structures. Experiment 3 focused on the discovery of novel planning strategies through learning, as one potential source of where planning strategies originate. Findings from these experiments consistently demonstrated that participants adjusted their planning in accordance with environmental structure, supporting the prediction of experience-driven adaptation.
The second set of hypotheses evaluated whether metacognitive reinforcement learning provides a superior explanation for the observed adaptation compared to alternative theories such as mental habit formation and rational strategy selection learning. To this end, a set of computational models was employed, encompassing purely model-based, purely model-free, and hybrid metacognitive reinforcement learning mechanisms, which contains both model-based and model-free features. Across all three experiments, metacognitive reinforcement learning consistently accounted for a larger proportion of participants' adaptation better compared to alternative theories. Moreover, individual differences in learning were observed, with learners best described by metacognitive reinforcement learning outperforming those best fitted by the mental habit formation model.
To explore whether people employ additional cognitive mechanisms to facilitate planning, additional cognitive mechanisms - intrinsically generated pseudo-reward, subjective effort of planning, and termination deliberation - were integrated into one of the hybrid metacognitive reinforcement learning model resulting in 8 different model variants. Results indicated that participants who were better described by a model variant using pseudo-reward, subjective effort and learning the value of termination exhibited better performance than their counterparts.
Furthermore, subsequent Experiments 4 and 5 validated the methodological approach and examined the effectiveness of model-based metacognitive reinforcement learning. Experiment 4 demonstrated that individuals engaged in model-based learning when direct interaction with the environment was restricted, though hybrid approaches proved to be more effective. Experiment 5 confirmed the robustness of the findings by replicating results in a more naturalistic planning environment.
Overall, this dissertation provides compelling evidence that metacognitive reinforcement learning offers a reasonable explanation of human planning adaptability. The findings contribute to a deeper understanding of the computational principles underlying metacognitive learning and have implications for both cognitive science and artificial intelligence, informing the development of more efficient and human-like planning algorithms.Die Dissertation ist gesperrt bis zum 23. September 2027
Nutrient- and Secondary Metabolite-Mediated Interactions in the Human Nasal Microbiome
No full textDie Dissertation ist gesperrt bis zum 22. Dezember 2027
Unraveling Human Cytomegalovirus Immune Evasion Mechanisms and Advancing Antiviral Therapeutic Strategies
No full textHuman cytomegalovirus (HCMV) is a large, double-stranded DNA virus belonging to the Herpesviridae family. Its seroprevalence is estimated to reach up to 100% in certain geographical areas. The virus has been co-evolving with humans for thousands of years, during which time it has established a complex mechanism to evade antiviral immunity and to successfully establish an infection. Although an HCMV infection is usually asymptomatic for immunocompetent individuals, it poses high risk for immunosuppressed patients, including AIDS patients and transplant recipients. Furthermore, it is the primary cause of congenital infections, which can result in severe complications for the newborn. Currently, there is a treatment available for HCMV, but it is accompanied by certain limitations including toxicity and the emergence of resistant strains. This highlights the urgent need for the development of new treatments to combat HCMV and minimize its complications.
The initial section of this thesis examines the potential of novel antiviral compounds. The antiviral host cell protein SAMHD1, is subject to downregulation by HCMV. One of the mechanisms by which HCMV achieves this is through the induction of SAMHD1 phosphorylation by a viral kinase, pUL97. The current thesis demonstrates that phosphorylation occurs during the early stages of infection and is a conserved mechanism among clinical isolates of HCMV. These findings highlight the importance of this process for viral fitness. Therefore, it was investigated as a potential therapeutic target. Subsequently, Abemaciclib (AC), a CDK4/6 inhibitor, was identified as an antiviral compound that targets HCMV replication in macrophages. It is noteworthy that AC was observed to specifically inhibit the pUL97-mediated phosphorylation of SAMHD1, which provides insight into the mechanism of its antiviral activity. Moreover, LDC4297, a CDK7 inhibitor, has been demonstrated to exhibit antiviral activity against HCMV, specifically by blocking SAMHD1-phosphorylation in the context of HCMV infection. Furthermore, another antiviral compound, the human defensin HD5, was investigated. It was observed that an HD5-derived peptide, HD5(1-9), exhibits antiviral effect against HCMV by enhancing the innate immune response upon infection.
The second part of the study focused on CD84, CD164 and CD180. These are three surface receptors that undergo downregulation upon HCMV infection. The objective was to analyze the functional importance of this modulation. Although the knockout of CD84, CD164, and CD180 did not significantly affect viral replication, overexpression of CD164 indicated a potential restriction in viral replication, thereby suggesting a vital role for CD164 in HCMV restriction.
Taken together, the findings of this study elucidate new mechanisms through which HCMV circumvents the host immune response and offers insights into the mode of action of emerging antiviral compounds.Dissertation ist gesperrt bis 7. Februar 2027
Design, synthesis and development of novel SARS-CoV-2 and its variants main protease inhibitors with potent antiviral activity
No full textDie Dissertation ist gesperrt bis zum 09. Januar 2027 !The SARS-CoV-2 virus, a member of the β-coronavirus family, first emerged in Wuhan, China, in December 2019. This resulted in a pandemic with unprecedented socio-economic damage, with over 760 million people infected worldwide and 6.9 million deaths. Infection via droplet transmission is the primary means of transmission, resulting in the development of the disease known as COVID-19. This disease can lead to life-threatening respiratory complications and multi-organ failure. The main protease (Mpro) represents a promising pharmacological target in the treatment of coronavirus diseases, such as COVID-19. This is due to the fact that the Mpro cleaves the substrate in an unique manner. Moreover, Mpro plays a pivotal role in viral transcription (cleavage of non-structural proteins 5-16), which is essential for replication.
In this work, two different structural classes of Mpro inhibitors were synthesized, characterized and optimized. For this purpose, the nucleophilic Cys145 of the catalytically active Cys-His dyad of Mpro should be addressed by using a covalent design approach. The successful covalent addressing of Cys145 was verified by X-ray crystallography on three structures, which provided valuable insights into the unique binding mode. Furthermore, in a broad structure-activity relationship (SAR) study (138 compounds), important parameters for the identification of several promising Mpro inhibitors with IC50 values in the single-digit nanomolar range were derived and the knowledge gained was applied by sequential optimization of the P1-P3 residues or the P1' residue. Moreover, a number of selected potent inhibitors, demonstrated remarkable inhibitory activity at the cellular level against a range of naturally occurring nirmatrelvir-resistant mutants, such as His172Gln. Furthermore, potent inhibitors against the related coronaviruses SARS-CoV-1 , MERS-CoV, and HCoV-NL63 were successfully developed. In addition, the identification of compounds with dual (CatL + Mpro, Scaffold A) or selective (Mpro, Scaffold B) inhibitory properties led to the their antiviral properties. These observations were corroborated by robust in vitro antiviral activity against the wild-type strain of SARS-CoV-2 (in Calu-3 cells) and its variants, such as omicron (in Vero-E6 TMRSS2 cells). Due to their advantageous metabolic stability in human liver microsomes (HLM) and favorable pharmacokinetic properties, some representatives are suitable for further preclinical development
Investigation of molecular interactions between Clostridium perfringens and human gastrointestinal bacteria
No full textDie Dissertation ist gesperrt bis zum 05. Juni 2027 !One of modern medicine’s most innovative and emerging fields in response to complex, multifactorial diseases is to unravel molecular dependencies from a systems point of view. One key factor driving this research is the growing recognition of the critical role in human health and disease played by the microbiome, an aspect that was previously little appreciated and understood. In addition, the overuse of antibiotics in our society is leading to a growing number of antibiotic-resistance-associated infections and, consequently, deaths. Further, antibiotic treatment is linked with dysbiosis, resulting in diseases. Often, those drugs are not pathogen-specific and also collaterally damage commensal bacteria. One approach to face these challenges, which is gaining more attention, is modulation of the microbiome to specifically eradicate pathogens and restore health in the host. Several strategies, such as probiotics, prebiotics, and fecal microbiota transplantation (FMT) are well-recognized already in supporting the host’s health. However, while it is fully acknowledged that microbiome-modulation holds much more potential to counteract many diseases, applications are still in an early stage. Currently, the investigation of the complex interplay of the microbiome and the host attracts scientific attention and expands the understanding of the microbiome’s far-reaching influence on the body. Its impact extends beyond colonized body regions, creating connections between different organs as well as linking the host’s environment to internal physiological processes. A deeper understanding of these intricate networks, including the key metabolites involved and the microbes that influence them, could enable us to precisely modulate relevant microbial populations. This knowledge holds the potential to prevent or treat diseases in a targeted manner while minimizing secondary unintended effects on the host. To harness this potential, we must understand how to precisely regulate microbes – either by modulating their abundance or by selectively activating or inhibiting metabolic pathways – without disrupting the homeostasis of the microbiome and the host. This requires insight into both microbe-host interactions and the complex network of microbial interactions within a community. Screenings of patient samples from healthy and diseased donors are valuable for finding correlations and identifying key species for disorders and diseases but cannot elucidate the interactions between microbes. For that, microbial communities need to be investigated in more detail.
This dissertation investigates the interactions between human gut commensals and the pathobiont Clostridium perfringens. One part focuses on the development of tools to study C. perfringens and members of a synthetic community in co-cultures or communities. In this section, I focused on developing strain-specific primers for quantifying strain abundance using quantitative polymerase chain reaction (qPCR), worked on the development of a real-time fluorescence reporter system, and established a multiple bioreactor system (MBS) that was used to study microbes under controlled pH conditions and continuous nutrient supply. I show the suitability of this MBS for microbial community studies by using it to investigate the effects of a proton-pump inhibitor (PPI) and pH changes on communities and how this affects colonization resistance against Clostridioides difficile.
In another part of this dissertation, I combined the developed tools with proteome and metabolome analysis to elucidate the molecular response of C. perfringens to the presence of each member of our synthetic community and vice versa. To further investigate these molecular responses and, consequently, the nature of these inter-species interactions, a novel approach was pursued to genetically modify C. perfringens. A newly developed CRISPR-Cas9 application for C. perfringens demonstrated its functionality through the successful deletion of two genes. This culminated in the insight into interspecies level interaction on physiological, metabolic, and proteomic levels, relevant for C. perfringens growth and virulence, providing leads for future therapeutic applications.
This study demonstrates the relevance and functionality of the developed tools for investigating microbial interactions. While C. perfringens is used here as a model microbe, the approaches presented can be applied to other communities and strains