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CD96: a new co-stimulatory receptor on primary human CD4+ T cells - Elucidating its downstream pathways and contribution to HIV-1 infection
No full textDie Dissertation ist gesperrt bis zum 04. Februar 2027 !One strategy of HIV-1 to evade the host immune system is the manipulation of several receptors of the host cell plasma membrane. In this regard, CD96 (TACTILE = T cell activation, increased late expression) a member of the immunoglobulin superfamily, was identified in previous work by our group to be downregulated by HIV-1 accessory proteins Nef and Vpu. Although the mechanism of CD96 downregulation is well characterized, the biological relevance for HIV-1 pathogenesis is unclear.
Together with the two receptors CD226 (DNAM-1 = DNAX accessory molecule-1) and TIGIT (T cell immunoglobulin and ITIM domain), as well as the commonly shared ligand CD155 (PVR = Poliovirus receptor), CD96 belongs to a complex receptor-ligand network. Whereas the function of CD226 and TIGIT is well characterized, the function of CD96, especially on primary human CD4+ T cells, is unclear. Thus, the biological function of CD96 on primary CD4+ T cells was investigated, as well as the contribution of its downregulation to HIV-1 replication.
By performing co-immunoprecipitation experiments, it was observed that Grb2 and Lck differentially regulate CD96 expression. Co-expression of Grb2 and CD96 in HEK293T cells resulted in increased CD96 protein levels, whereas co-transfection of Lck resulted in decreased CD96 protein levels that could be restored if transfected cells were additionally treated with the lysosomal degradation inhibitor Bafilomycin A1. Yet, a direct interaction between CD96 and Grb2, or Lck, as well as previously suggested direct interaction partners MKK7 and Gab1, were not observed. Using primary CD4+ T cells it was possible to show that CD96 is phosphorylated and that this is independent of TCR stimulation. Finally, stimulation experiments with primary CD4+ T cells revealed a co-stimulatory function for CD96, resulting in increased numbers of CD69 and CD25 expressing cells, as well as enhanced secretion of pro-inflammatory cytokines, chemokines and growth factors. This effect was dependent on the activation of CD3 and CD28 signaling pathways in a time-dependent manner.
Regarding HIV-1 infection, it was shown that primary HIV-1 isolates similar to lab-adapted
HIV-1 strain NL4-3 downregulated CD96 from the cell surface in a Nef and Vpu dependent manner. Although it was previously shown by our group that CD96 is incorporated into viral particles if not removed from the cell surface, targeting virion incorporated CD96 with soluble CD155 did not impair particle infectivity. Moreover, co-stimulation experiments with HIV-1 infected resting and pre-stimulated CD4+ T cells suggest a positive effect of CD96 co-stimulation on HIV-1 replication.
Altogether, further research on the contribution of CD96 downregulation to HIV-1 pathogenesis is required. Nevertheless, the obtained results show that CD96 functions as a co-stimulatory receptor on primary human CD4+ T cells, leading to increased numbers of activated T cells, enhanced secretion of pro-inflammatory cytokines and thus, enhanced T cell activation
Microaeration for Valorization of Hydrothermal Liquefaction Process Water
No full textDie Dissertation ist gesperrt bis zum 16. Mai 2027 !Hydrothermal liquefaction (HTL) converts wet organic feedstocks (e.g., lignocellulosic biomass, food waste, sewage sludge, or algae) into bio-crude oil under subcritical water conditions (250 – 374°C, 5 – 20 MPa, 5 – 60 min reaction time), simultaneously addressing waste valorization and renewable energy production. However, the resulting HTL process water retains over 30% of feedstock carbon and contains complex organic compounds (e.g., phenols and nitrogen-containing species), posing environmental risks if untreated. Anaerobic digestion (AD) offers a subsequent solution by recovering energy through methane production while mitigating pollutant release. This dissertation systematically investigates the toxicity, biodegradability, and microaeration potential for enhancing the AD of HTL process water.
In the first part, a critical review reveals that a higher HTL severity (≥ 300°C) generates more recalcitrant and inhibitory organic compounds in HTL process water, with feedstock composition dictating relative concentrations of organic compounds. Comparative batch tests were conducted using HTL process water that was derived from two typical feedstocks at 350°C for 15 minutes – a food-waste proxy (i.e., dog food, rich in proteins) and wheat straw (rich in lignocellulose). Results demonstrated feedstock-dependent inhibition patterns: wheat-straw process water (WSPW) exhibited higher toxicity due to condensed aromatic compounds but achieved greater methane yields (54% of theoretical potential) compared to food-waste process water (FWPW) (37%). Notably, microaeration acclimation significantly enhanced methane yield from FWPW by 27% compared to strict anaerobic conditions, yet showed negligible effects on WSPW.
The second part evaluates continuous bioreactor performance under anaerobic versus microaerobic conditions. Three representative organics exhibited distinct degradation behaviors: 2-pyrrolidinone was fully degraded regardless of oxygen, while methyl-pyrazine persisted in both systems. Microaeration accelerated 4-hydroxyacetophenone removal initially, but fully anaerobic systems caught up after microbial acclimation. Under incremental FWPW loading from 0.92 to 2.76 g COD·L⁻¹, the microaerobic bioreactor exhibited progressive methane production rate enhancements of 3.9% (0.92 g COD·L⁻¹), 7.8% (1.84 g COD·L⁻¹), and 13% (2.76 g COD·L⁻¹), contrasting with the limited 8.3% increase exclusively at the maximum load (2.76 g COD·L⁻¹) in the anaerobic digester.
These findings demonstrate that microaeration selectively enhances anaerobic treatment efficiency for protein-rich HTL process water while preferentially degrading structurally susceptible organics. This feedstock- and compound-specificity highlights the importance of tailored strategies to optimize waste-to-energy conversion in HTL-AD systems
Molecular Imaging of Pulmonary Fibrosis
No full textProgressive pulmonary fibrosis (PF) is a rare yet fatal disease that causes a rapid reduction of lung function due to the formation and accumulation of extracellular matrix (ECM) fibres. Since progressive PF is an age-related and diffuse disease, improvement in life span and better clinical standards in diagnostics in industrial countries have led to an increased number of accurately diagnosed cases for this disease, while numbers from developing countries remain unreliable. Despite sustained research efforts, many unanswered questions regarding the cause, progression as well as diagnosis and therapy of PF remain unanswered. In addition to the disruption of the ECM, recent findings seem to point at senescence as a promising biomarker and a potential therapeutic target in progressive PF. The role of senescence in disease development and the success of senolytic treatment remains however debated.
To improve the understanding of PF, preclinical molecular imaging was utilized to provide novel insights into the in vivo progression of PF and its treatment monitoring by using novel PET tracers in a bleomycin mouse model. First, ECM disruption was accurately and selectively detected in vivo over a time course of several weeks using the ECM fibre targeting [64Cu]Cu-NOTA-GPVI-Fc tracer. In addition, longitudinal imaging of PF progression using a novel senescence-targeted radiotracer, which targets the senescence-associated (SA) enzyme β-Galactosidase, could show a crucial role of senescence in disease onset and development. While the detection of ECM disruption and senescence yielded encouraging results, therapy monitoring proved to be more challenging. It was not possible to detect a change in the progression of ECM disruption following the administration of either the approved anti-fibrotic and anti-inflammatory drug pirfenidone or of the novel senolytic combination dasatinib/quercetin using our ECM targeting [64Cu]Cu-NOTA-GPVI-Fc tracer.
Through the in-depth evaluation of two new radiotracers for progressive PF, this study provides new perspectives for in vivo PF diagnosis and therapy monitoring in a pre-clinical mouse model of the disease. Furthermore, clinical translation of the research performed during this PhD could be achieved in the short term as both tracers are currently used in clinical studies, albeit in different application fields. This enables the investigation of their diagnostic potential in patients and depicts the implementable “from bench to bedside” feasibility of this study.Die Dissertation ist gesperrt bis zum 01. Oktober 2027
Greater than the Sum of its Parts: Social Cognition Research beyond its Individual Learning Roots
No full textHumans excel at learning from the ever-present social stimuli in their environment. To understand this cognitive capacity, computational models of social learning often borrow from the longstanding tradition of computationally modelling individual learning; reinforcement learning models can be augmented with social information to model imitation behaviour, and planning models can be inverted to model inference behaviour. While a fruitful approach, this influence comes at a cost: social cognition research commonly adapts experimental designs and analysis methods from individual learning, treating social information as little more than a reward signal. This strips social cognition of the richness of stimuli we experience in even the most banal everyday situations, and risks a systematic underestimation of our social learning capabilities. To address this, I applied findings from three key domains of individual learning to social learning contexts: generalization, learning rate biases, and cost-benefit strategy arbitration. I modified existing paradigms to bring them closer to naturalistic social learning settings, and critically examined to what extent findings from the individual learning literature can be applied to social learning, and how they differ in their application.
In the first project, we examined how humans integrate social information into individual decision-making when learning from others with similar, though not identical, reward functions. Prior studies in this domain have focused on cases where the demonstrator and the observer share the same reward function, limiting our understanding about the flexibility of our social learning abilities. We extended the spatially correlated multi-armed bandit paradigm, which is commonly used to investigate individual generalization, to social settings. This allowed us to better capture how humans may share some general preferences, while maintaining specific individual tastes. Participants used social information more flexibly than previous paradigms indicated, with our novel Social Generalization model, which treats social information as noisier individual information, providing the best fit. Social information was used as an exploration tool, outsourcing costly individual exploration to others – a resource-rational approach consistent with findings from individual learning literature showing that humans often use heuristics to simplify cognitively complex tasks. Additionally, participants performed better in group versus solo settings, in line with existing results on collective intelligence.
In the second project, we investigated how learning rate biases differ between individual and social learning settings. We tested whether the positivity bias frequently reported in individual reinforcement learning also manifests in observational learning settings, and whether this bias benefits agent performance. In individual learning, positivity bias refers to the tendency to weight positive outcomes more strongly than negative outcomes; in social learning, it translates to weighting a demonstrator’s positive feedback more strongly than their negative feedback. While we replicated the persistent positivity bias found in individual learning, we only found a social positivity bias in environments where it was adaptive. This shows higher flexibility of learning rate biases in social settings compared to individual contexts.
In the third project, we tested whether the resource-rational cost-benefit arbitration between strategies observed in individual learning extends to social learning. Inspired by distinctions between model-free learning and model-based learning, we designed a task to investigate how humans switch between imitation, value inference, and model-based inference when relative costs and benefits of each strategy vary. This extends previous literature on arbitration between levels of social learning by incorporating a distinction between value inference and model-based inference, and accounting for cognitive cost in addition to strategy reliability. Participants adjusted their learning strategy based on task demands, favouring imitation when higher level strategies were more costly. This mirrors findings from individual settings, where humans favour simpler strategies when planning is complex and unreliable. However, some participants persisted in their higher level strategy use even when it was not beneficial for them, hinting at a general bias towards social inference. Neural analysis provides insight into how humans make this trade-off: participants imitated when they were uncertain about the structure of the environment and still attempting to infer it. When they were more certain about the environment, they no longer relied on social inference, instead basing their choices on the information they had inferred previously.
Across three domains, this thesis demonstrates the way social learning is characterized by distinct behavioural patterns when testing individual learning principles in settings that preserve some of the richness of our social environments. Participants often performed better in social contexts, underscoring the importance of investigating human intelligence in the settings we evolved to excel in. More broadly, this thesis contributes to descriptions of human behaviour by providing computational models, contrasted with normative strategies and individual learning in the same settings, which help us better understand the unique aspects of human social cognition. [untranslated]Die Dissertation ist gesperrt bis zum 20. November 2027
Effects of normobaric oxygen treatment and the combination of normobaric oxygen treatment and intracarotid cold infusion in an animal model of transient focal cerebral ischemia
No full textBackground Although NBO therapy has been demonstrated to exert significant neuroprotective effects in both animal and clinical trials, research into the combination of NBO with thrombectomy remains in its early stages. The efficacy of combining oxygen therapy with hypothermia treatment has thus far been studied in only a handful of animal experiments 149. The combination of NBO and selective brain hypothermia may represent an ideal early stroke treatment method for preserving the ischaemic penumbra. Our hypothesis is that the neuroprotective effects of NBO are verifiable and can be enhanced through combined application of NBO and ICCI hypothermia.
Methods Upon establishing a 60-min MCAO rat model, rats were randomly allocated into three groups (24h group: n=4/group; 2 weeks group: n=12/group): CTR (no treatment), NBO (mask inhalation of 100% oxygen) and CB (combination treatment of NBO and ICCI ， 0-1 ℃ 0.9% saline infusion via ICA). The primary outcome parameter chosen for this study was the volume of cerebral infarction detected using T2-MRI at 14 days. We selected the infarct volume and cerebral oedema on the ADC map 24 h after MCAO as secondary outcome parameters. Using conventional H&E staining to examine the extent of damage. Subsequently, some staining methods were employed based on the duration post-stroke: 1) The 24-hour group received H&E staining, along with GFAP, Hypoxyprobe and NeuN; 2) The 14-day group was subjected to NeuN, GFAP, CD31, and Iba1. Additionally, neuroscore tests and blood gas analysis were conducted.
Results Analysis of the experimental results revealed that, compared with that in control group, the striatal infarct volume in NBO group was significantly reduced according to the striatal infarct volume obtained from T2-MRI at 14 days. Additionally, for the cerebral cortical infarct volume measured by the apparent diffusion coefficient (ADC) at 24 h, the cortical infarct volume in the combined treatment group was significantly reduced compared to that in NBO group. Moverover, in the hypoxyprobe analysis, the use of NBO, ICCl, or a combination of both treatments significantly reduced hypoxia in the cortex following stroke. Additionally, significant differences in post treatment pH, PCO2 and PO2 were observed among the three groups of rats.
Discussion We confirmed the potent neuroprotective effect of NBO, and showed augmentation of effectiveness when combined with ICCI. Thus, early combination treatment may potentially be useful in ischaemic stroke to further reduce resulting infarct volumes. Nevertheless, the observed respiratory acidosis observed during combined use of ICCI and NBO, as well as the trend towards aggravated cerebral oedema, remains concerning and warrants caution. Future studies should incorporate mechanically ventilation for enabling the control of blood gases.Die Dissertation ist gesperrt bis zum 15. August 2027
On the Tuning of Singlet Exciton Fission in Pentacene Using Solid Solutions
No full textDie Dissertation ist gesperrt bis zum 01. Juli 2027 !Diese Dissertation befasst sich mit der Kontrolle und Optimierung der Singulett-Aufspaltung (singlet exciton fission, SF) in organischen Halbleitern, insbesondere in Pentacen, zur Steigerung der Effizienz organischer Solarzellen. Durch die Verwendung statistisch mischender binärer Systeme, hergestellt mittels organischer Molekularstrahlabscheidung, wird gezeigt, dass sich der SF-Prozess gezielt beeinflussen lässt. Experimentelle Untersuchungen mit transienter Absorptionsspektroskopie werden mit quantenmechanischen Simulationen kombiniert, um den Zusammenhang zwischen molekularer Struktur, lokaler Ordnung und photophysikalischen Eigenschaften zu analysieren. Die Ergebnisse verdeutlichen die zentrale Rolle der intramolekularen vibronischen Kopplung bei der ultraschnellen Singulett-Aufspaltung und liefern neue Ansätze zur Feinabstimmung struktureller und optischer Eigenschaften organischer Mischsysteme. Damit trägt die Arbeit zum besseren Verständnis und zur Weiterentwicklung effizienter, nachhaltiger organischer Photovoltaiktechnologien bei
Transport von Insulin in den humanen Liquor
No full textDie Dissertation ist gesperrt bis zum 13. Oktober 2027 !Die Insulinwirkung im Gehirn spielt bei der Regulierung der Nahrungsaufnahme eine
sehr wichtige Rolle. Insulin zeigt peripher eine anabole Wirkung während es im
zentralen Nervensystem eine katabole Wirkung hat. Das Hormon kann
Nahrungsmittelreize über eine negative Rückkopplungsschleife beeinflussen. Das
bedeutet, dass Insulin ein Signal aus dem Körper ist, das durch seine Wirkung im
Gehirn die Nahrungsaufnahme beendet. Passend hierzu zeigt eine Studie eine
Fettleibigkeit von Mäusen, bei ausgeschalteter Insulinrezeptorsignalgebung.
In Deutschland zeigt sich eine hohe Anzahl von übergewichtigen bis adipösen
Menschen, die bereits eine periphere Insulinresistenz aufweisen. Das menschliche
Gehirn reagiert dann häufig ebenfalls weniger sensibel auf Insulin.
Ziel der Arbeit ist es die Reaktion des menschlichen Körpers bezüglich Insulin, C-
Peptid und Glucose nicht nur im Blut, sondern auch im Liquor besser zu verstehen und
die Studienlage soweit auszubauen, dass im Verlauf die Adipositasprävention und so
auch im weiteren Sinne die Prävention des Diabetes mellitus als mögliche entstehende
Pathologie weiter vorangetrieben werden kann. Die Studie teilte sich hierfür in drei
Studienarme auf. Es wurden Messwerte während eines oralen Glucosetoleranztests,
nach intranasaler Insulingabe und während circadianer Rhythmik mittels
Blutentnahmen über eine periphere Venenverweilkanüle und Liquorentnahmen über
eine einliegende Liquordauerdrainage erstellt. Im Anschluss erfolgte dann die
Auswertung der Ergebnisse der Liquor- und Plasma-Proben bezüglich Insulin, C-
Peptid und Glucose. So konnte bereits bestehendes Wissen über das Verhalten dieser
Werte im Plasma nochmals bestätigt werden. Die Liquorproben ergaben jedoch neue
Ergebnisse.
Laut Born et al. aus dem Jahr 2002 hätte das Peptidhormon Insulin nach nasaler
Insulingabe einen schnellen Anstieg zeigen und mindestens 80 min im menschlichen
Liquor in erhöhter Konzentration nachweisbar sein müssen. Dies konnten wir in
unserer Studie an einer anderen Patientenpopulation mit einer anderen Methodik so
nicht reproduzieren. Im Gegenteil, es kam initial sogar zu einem Abfall des Insulins in
den Liquorproben und anschließend zu keiner merklichen Erhöhung innerhalb von 240
min.
Ein zusätzlicher Befund ist, dass der Quotient von Glucose aus Liquor und Plasma
sich als deutlich schwankend erwiesen hat. Häufig wird dieser Quotient neben anderen
Laborparametern jedoch bei der Diagnose einer Meningitis hinzugezogen und zeigt
sich beispielweise bei einer bakteriellen Meningitis laut Literatur konstant bei <0,3. Die
Miteinbeziehung des Quotienten von Glucose aus Liquor und Plasma sollte also
möglicherweise in Abhängigkeit von der vorangegangenen Nahrungsaufnahme
gesehen werden.
Aufgrund der Coronapandemie konnten leider nur weniger Patienten als ursprünglich
geplant untersucht werden. Die Ergebnisse liegen daher aus einer deutlich kleineren
Probandenanzahl als zunächst geplant vor. Es bedarf daher eines Ausbaus der
Studienarme bzw einer nachfolgenden Studie, um die Ergebnisse zu untermauern.
Zudem zeigt sich die Probeentnahme aus der Liquordauerdrainage als erschwert, da
die Peptide möglicherweise an der Oberflächeninnenseite des Plastikschlauchs haften
könnten, was die Messungen vor allem bei wiederholten Abnahmen verfälschen
könnte. Auch die vorangegangene neurochirurgische Operation könnte eine
Veränderung der Messwerte durch veränderte Schrankenverhältnisse und
Transportprozesse hervorrufen. Trotzdem kann man als zentrale Erkenntnis dieser
Arbeit festhalten, dass es keinen Anhalt zur intracerebralen Insulinproduktion gibt und
die Glucosewerte im Liquor weitaus mehr Schwankungen über den Tag unterliegen
als bisher angenommen. Möglicherweise könnte dies zukünftig beispielsweise in der
Meningitisdiagnostik berücksichtigt werden.
Weiter zu erforschen bleiben unbedingt die Messparameter von Glucose, C-Peptid und
Insulin aus Liquorproben in einer größeren Probandenzahl. Dabei sollten Alter und
Geschlecht nicht außen vor bleiben und die mögliche Beeinflussung der
Messparameter durch eine Abnahme per Liquordrainage aus Plastik ausgeschlossen
werden.
Zusammenfassend zeigt diese Arbeit, dass Insulintransport in das zentrale
Nervensystem und Insulinwirkung im Gehirn komplexer ist als bisher angenommen.
Diese Erkenntnisse tragen zum besseren Verständnis der zentralen Insulinwirkung bei
und legen den Grundstein für zukünftige Studien, die letztendlich darauf abzielen, die
Prävention von Adipositas und Diabetes durch gezielte Beeinflussung des
Insulintransports ins Gehirn zu verbessern
The Function of Septal Junctions Mediated Cell-Cell Communication in Cell Differentiation and Stress Response in Filamentous Cyanobacteria of the Genus Nostoc
No full textSingle cells within the filaments of certain multicellular cyanobacteria exchange molecules through cell-cell connections known as junctions (SJs). These SJs consist of cytoplasmic cap and plug modules and a tube module positioned in nanopores, allowing connection through the septal peptidoglycan. SJs display reversible gating and are analogous to metazoan gap junctions, despite predating them by a billion years. Mutants in the fraD and sepN genes lack structural components of SJs and are therefore impaired in SJ closure. SJ-mediated cell-cell communication is important for differentiation of nitrogen-fixing heterocysts and metabolite exchange during diazotrophic growth. However, its role in the differentiation of other cell types – such as motile hormogonia and dormant akinetes, remains unexplored. Additionally, the coordination mechanism for hormogonia phototaxis, where filaments rapidly change direction in response to light conditions, is not well understood. The importance of gating for stress survival and heterocyst formation has been speculated but lacked strong evidence.
In this work, by studying the communication-impaired fraI mutant of Nostoc punctiforme, we show that, despite highly impaired cell-to-cell communication the differentiation of hormogonia and akinetes is still possible, unlike heterocyst differentiation. The heavily fragmenting fraI mutant filaments showed altered hormogonia formation with significantly reduced motility. However, the loss of SJ-mediated cell-to-cell communication did not affect hormogonia phototaxy, suggesting the need for an alternative hypothesis for the high degree of synchronization among the hormogonia cells.
By studying the gating-impaired fraD and sepN— we showed that the closure of SJs is important for whole-filament survival under stress conditions such as UV-C light treatment, where many individual cells undergo cell death. However, SJ gating was not necessary for heterocyst differentiation. Instead, we suggest the idea that peptidoglycan remodelling is crucial in the heterocyst differentiation process.
This work contributed to the understanding of the mode of action, the function under stress conditions and cell differentiation of SJs, and added new insights in the SJ architecture.Die Dissertation ist gesperrt bis zum 28. Januar 2027
Reading Illustrated Texts: How Pictures Influence Activation, Integration, and Validation Processes
No full textReading and comprehending texts illustrated with pictures is an essential part of our daily lives. Newspaper articles, internet blogs, and textbooks are just some examples of written works that use illustrated texts to communicate information. In order to facilitate and improve comprehension of illustrated texts, it is important to understand the underlying cognitive processes (Mayer, 2021b).
This dissertation project aimed to specify the ongoing cognitive processes when processing illustrated texts. To this end, the research presented in this project focused on the Resonance-Integration-Validation model of text comprehension (RI-Val, O’Brien & Cook, 2016a, 2016b) in combination with the Cognitive Theory of Multimedia Learning (CTLM; Mayer 2021a, 2021b). The RI-Val model assumes that readers passively activate information stored in long-term memory that overlaps with the current content that is being read, integrate the activated and currently read content, and validate the newly formed linkages, that is, check them for consistency. The CTML does not yet consider these passive processes.
The overarching research questions investigated in this dissertation were whether activation, integration, and validation processes apply to illustrated texts, and, if so, whether pictures might influence these processes. To answer these questions, three research packages (A-C), each consisting of three experiments, were conducted (total N = 2,496). All nine experiments used adapted versions of what is known as the contradiction paradigm (Albrecht & O’Brien, 1993). In this paradigm, reading times for a target and the following sentence (i.e., spillover sentence) are measured. It was varied whether the information presented beforehand was consistent or inconsistent with the target sentence and whether the previously mentioned consistent or inconsistent information was illustrated with a picture or not.
Overall, the results show that activation, integration, and validation processes apply to illustrated text and that these processes occur across modalities (i.e., text and picture). Furthermore, a summary with meta-analytic measures showed that pictures when presented beforehand slightly accelerated the reading times of the target sentences. These findings broaden the scope of the RI-Val model and further specify the cognitive processes identified within the CTML, by showing that passive activation, integration, and validation processes apply to illustrated texts and that they occur across modalities (i.e., for information from both texts and pictures).Dissertation ist gesperrt bis 25. März 2027
Target validation and mode-of-action of C10, a broadly acting flavivirus capsid inhibitor
No full textC10 was identified in a FACS-FRET-based screen designed to identify small compounds that interfere
with HCV capsid protein (CP) interaction. Previous experiments demonstrated that C10 exhibited
antiviral activity against all tested members of the Flaviviridae family, exhibiting a favorable
therapeutic index (TI) of 400 to 600 and a specific crosslinking effect on the CP. Subsequent testing of
different C10 derivatives, revealed C45, a compound that exhibited enhanced antiviral efficacy in
reinfection, while maintaining comparable toxicity (TI=2800). C45 was found to also induce higher
molecular weight species of CP.
The objective of this project was to elucidate the mode of action of C10 and C45, regarding their ability
in the viral life cycle and to analyze the compound-CP-interaction.
To study the impact of C10 and C45 on different steps of the flavivirus replication cycle, cell-based
infection assays were employed and quantified by Western Blot (WB), qPCR, and immunofluorescence
microscopy. To analyzing the compound binding site, a method to purify compound-bound viral
particles from unbound particles was established, and compound-resistant DENV was generated and
sequenced to identify acquired mutations.
In this thesis it was observed that C10 and C45 can bind to assembled DENV and HCV particles reducing
their infectivity. Next, it was demonstrated that treatment with C10 or C45 had no impact on the viral
particle release and that the high antiviral activity of both compounds cannot be explained by
diminished genome packaging. Infection with C10-treated DENV particles resulted in capsid
accumulation without co-localized E-protein in cells, suggesting that C10 interferes with the uncoating
of the viral life cycle, thereby inhibiting viral replication and translation. The impact of C45 on the
uncoating and viral fusion has not yet been studied, however as both compounds are closely related it
is hypothesized that they act similarly. The acquired data supports a model in which C10 and C45 target
the flavivirus CP during viral assembly or in assembled viral particles, inducing crosslinking of CPs,
which leads to an inhibition of the CP disassociation during viral uncoating.
Upon selection of C10-resistant viruses, different mutations were mapped to all three structural
proteins and the non-structural proteins NS4B and NS5. Within the target protein CP, mutations of the
amino acids K9 and N93 were identified. Utilizing a published an in-silico prediction model of different
N-terminal orientations picked up during distinct phases of the viral life cycle, the hypothesis that C10-
binding to the residues K9 and N93 stabilizes the N-terminal part in a conformation acquired for
assembly was made. This hypothesis is supported by experimental evidence from multiple studies,
which demonstrate that the binding of C10 to the nucleocapsid hinders the disassembly process,
thereby preventing genome release. However, further validation is required to substantiate this
hypothesis, particularly through the identification of compound interaction sites.Dissertation ist gesperrt bis zum 4. Juni 2027