Biologische Hochschulschriften (Goethe-Universität)
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Die Zahl der gramnegativen Bakterien auf der WHO-Liste der Antibiotikaresistenzen hat in den letzten Jahrzehnten erheblich zugenommen. Schätzungen zufolge wird die Antibiotikaresistenz bis 2050 tödlicher sein als Krebs. Die äußere Membran gramnegativer Bakterien ist aufgrund ihres wichtigsten Strukturbestandteils, des Lipopolysaccharids (LPS), sehr anpassungsfähig an Umweltveränderungen. Das LPS macht gramnegative Bakterien von Natur aus resistent gegen viele Antibiotika und führt somit zu Antibiotikaresistenz. Der bakterielle ATP-bindende Kassettentransporter (ABC-Transporter) MsbA spielt eine entscheidende Rolle bei der Regulierung der bakteriellen Außenmembran, indem er das Kern-LPS durch ATP-Hydrolyse über die Innenmembran von gramnegativen Bakterien flockt. Darüber hinaus fungiert diese Floppase als Efflux-Pumpe, indem sie Medikamente durch die innere Membran transportiert, was sie zu einem interessanten Ziel für Medikamente macht. Vor kurzem wurden zwei verschiedene Klassen von MsbA-Inhibitoren entdeckt: (1) Tetrahydrobenzothiophene (TBT), die den LPS-Transport aufheben, und (2) Chinolinderivate, die sowohl die ATP-Hydrolyse als auch die LPS-Translokation blockieren. Darüber hinaus hat die Bestimmung der 3D-Struktur von MsbA durch Rontgen- und Kryo-EM mehrere interessante Zustände der Floppase ergeben. Die Kernspinresonanzspektroskopie ist eine hervorragende biophysikalische Methode zur Ergänzung der vorhandenen 3D-Strukturdaten. Insbesondere ermöglicht die Festkörper-NMR die Untersuchung von Membranproteinen in einer nativen Umgebung (z. B. in einer Lipiddoppelschicht). In der Vergangenheit hat unser Labor mithilfe der Festkörper-NMR einige detaillierte Mechanismen von MsbA aufgedeckt. Trotz der zahlreichen Fortschritte bei der Untersuchung der ABC-Transporterprotein-Superfamilie ist der spezifische Prozess der Substrattranslokation von MsbA noch immer unbekannt. Es wird angenommen, dass dieser Translokationsprozess über die Kopplungshelices (CHs) erfolgt, die sich zwischen der Transmembranregion (TMD) und der Nukleotidbindungsdomäne (NBD) befinden. Nukleotid-Bindungsdomäne (NBD). Zu diesem Zweck wird dem Zusammenspiel zwischen der TMD und der NBD über die CHs besondere Aufmerksamkeit gewidmet, mit dem Ziel, den Prozess der Substrattranslokation mithilfe von funktionellen Assays und Festkörper-NMR zu verstehen. Bei letzterem wurden spezifische Reporter in die CHs eingeführt, um Konformationsänderungen in 2D-spektroskopischen Daten zu verfolgen. Darüber hinaus wurde zeitaufgelöste NMR eingesetzt, um die Auswirkungen verschiedener Substrate in der TMD während der ATP-Hydrolyse in der NBD sichtbar zu machen. Die einzigartigen Reporter in den CHs haben Konformationsänderungen in bestimmten katalytischen Zuständen gezeigt. Darüber hinaus scheinen verschiedene Substrate die Kinetik der ATP-Hydrolyse zu beeinflussen. Die Ergebnisse zeigten, dass einige Substrate einen bevorzugten katalytischen Zustand innerhalb des ATP-Hydrolyse Zyklus aufweisen, der möglicherweise einen gekoppelten oder ungekoppelten Kinasemechanismus hat. Diese Ergebnisse könnten verschiedene Einblicke in die molekulare Struktur potenzieller neuer Antibiotika liefern.
The simultaneous inhibition of HDACs and BET proteins has shown promising anti-proliferative effects against different cancer types, including the difficult to treat pancreatic cancer. In this work, the strategy of concurrently targeting HDACs and BET proteins was pursued by developing different types of dual inhibitors.
By developing a novel scaffold that selectively inhibits HDAC1/2 together with BET proteins in cells, an effective tool for the investigation of pancreatic cancer, and other diseases which are sensitive to epigenetic processes, was created. The compound’s small size further gives the opportunity to further develop the inhibitor towards optimized pharmacokinetic properties, potentially resulting in a drug for cancer treatment.
A second novel approach that was pursued, was the development of a small-molecule degrader, targeting HDACs and BET proteins. Through synthesizing a variety of different molecules, a compound that was capable of lowering BRD4 levels and, at the same time, increasing histone acetylation was developed. While additional mechanistic investigations are needed to verify the degradation, the potent antiproliferative effects in pancreatic cancer cells encourage further studies following this alternative new strategy.
The role of USP22 in nucleic acid sensing pathways and interferon-induced necroptotic cell death
(2023)
Every day, living organisms are challenged by internal and external factors that threaten to bring imbalance to their tightly regulated systems and disrupt homeostasis, leading to degeneration, and ultimately death. More than ever, we face the challenge of combating diseases such as COVID-19 caused by infection with the SARS-CoV-2 coronavirus. It is therefore crucial to identify host factors that control antiviral defense mechanisms. In addition, in the fight against cancer, it is becoming increasingly important to identify markers that could be used for targeted therapy to influence cellular processes and determine cell fate.
As a deubiquitylating enzyme, ubiquitin specific peptidase 22 (USP22) mediates the removal of the small molecule ubiquitin, which is post-translationally added to target proteins, thereby regulating several important processes such as protein degradation, activation or localization. Through its deubiquitylating function, USP22 controls several biological processes such as cell cycle regulation, proliferation and cancer immunoresistance by modulating key proteins involved in these pathways. Lately, USP22 was reported to positively regulate TNFα-mediated necroptosis, an inflammatory type of programmed cell death, in various human tumor cell lines by affecting RIPK3 phosphorylation. In addition, USP22 as a part of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcription complex is known to regulate gene expression by removing ubiquitin from histones H2A and H2B. However, little is known about the role of USP22 in global gene expression.
In this study, we performed a genome-wide screen in the human colon carcinoma cell line HT-29 and identified USP22 as a key negative regulator of basal interferon (IFN) expression. We further demonstrated that the absence of USP22 results in increased STING activity and ubiquitylation, both basally and in response to stimulation with the STING agonist 2'3'-cGAMP, thereby affecting IFNλ1 expression and basal expression of antiviral ISGs. In addition, we were able to establish USP22 as a critical host factor in controlling SARS-CoV-2 infection by regulating infection, replication, and the generation of infectious virus particles, which we attribute in part to its role in regulating STING signaling.
In the second part of the study, we connected the findings of USP22-dependent regulation of IFN signaling and TNFα-induced necroptosis and investigated the role of USP22 during necroptosis induced by the synergistic action of IFN and the Smac mimetic BV6 in caspase-deficient settings. We identified USP22 as a negative regulator of IFN-induced necroptosis, which does not depend on STING expression, but relies on a yet unknown mechanism.
In summary, we identify USP22 as an important regulator of IFN signaling with important implications for the defense against viral infections and regulation of the necroptotic pathway that could be exploited for devising targeted therapeutic strategies against viral infections and related diseases like COVID-19, and advancing precision medicine in cancer treatment.
Necroptosis is an immunogenic form of programmed cell death characterized by plasma membrane accumulation of activated mixed lineage kinase domain-like (MLKL) that eventually leads to membrane disruption and release of danger-associated molecular patterns (DAMPs). Necroptotic cell death is tightly controlled by checkpoints, including compartmentalization as well as post-translational modifications (PTMs), like phosphorylation and ubiquitination of receptor-interacting protein kinase (RIPK) 1, RIPK3 and MLKL. Removal of plasma membrane-located activated MLKL via endocytosis or exocytosis can counteract necroptosis, but up till now, the exact mechanisms by which necroptosis is regulated downstream of MLKL activation and oligomerization are not fully understood.
Ubiquitination is a key post-translational modification that regulates various cellular processes including cell survival and cell death signaling via ubiquitination of RIPK1, RIPK3 and MLKL. M1-linked (linear) poly-ubiquitination is mediated exclusively by the linear ubiquitin chain assembly complex (LUBAC) which critically regulates cell fate and immune signaling via death receptors such as TNF receptor 1 (TNFR1).
In this study, we demonstrate that M1 poly-Ubiquitin (poly-Ub) increases during necroptosis which can be blocked by inhibition of LUBAC activity with the small-molecule HOIL-1-interacting protein (HOIP) inhibitor HOIPIN-8 or by loss of LUBAC catalytic subunit HOIP. Intriguingly, HOIPIN-8, as well as the HOIP inhibitor gliotoxin, and HOIP knockdown effectively prevent TNFα/smac mimetic/zVAD.fmk-induced necroptotic cell death in cells of human origin, without affecting necroptotic RIPK1 and RIPK3 phosphorylation, necrosome formation and oligomerization of phosphorylated MLKL. We demonstrate that HOIPIN-8 treatment inhibits MLKL translocation to intracellular membranes and accumulation in plasma membrane hotspots as well as MLKL exocytosis. We further confirm that HOIPIN-8 treatment suppresses necroptotic cell death in primary human pancreatic organoids (hPOs). Using time-lapse imaging and live/dead staining, we demonstrate loss of organoid structure and hPO cell death induced by smac mimetics and caspase inhibitors, thus providing a novel platform to investigate necroptosis in near physiological settings. Inhibition of LUBAC activity with HOIPIN-8 prevents hPO collapse and extends cell viability. Of note, loss of the M1 Ub-targeting deubiquitinating enzymes (DUBs) OTU DUB with linear linkage specificity (OTULIN) and cylindromatosis (CYLD) in human cell lines does not affect necroptosis induction and HOIPIN-8-mediated rescue of necroptosis. Intriguingly, inhibition of LUBAC activity with HOIPIN-8 does not block necroptotic cell death in murine cell lines.
Using massive analyses of cDNA ends (MACE)-seq-based global transcriptome analysis we confirm that necroptosis induces a pro-inflammatory cytokine profile which is dependent on LUBAC function and necroptotic signaling. Loss of LUBAC activity prevents the MLKL-dependent production and release of pro-inflammatory cytokines and chemokines.
Finally, we identify Flotillin-1 and -2 (FLOT1/2) as putative targets of necroptosis-induced M1 poly-Ub. Ubiquitin-binding in ABIN and NEMO (UBAN)-based pulldowns of M1 poly-ubiquitinated proteins revealed enrichment of FLOTs after necroptosis induction which is dependent on LUBAC activity and can be blocked with necroptosis inhibitors Nec-1s, GSK’872 and NSA, targeting RIPK1, RIPK3 and MLKL, respectively. Of note, loss of FLOT1/2 potentiates necroptosis suppression induced by LUBAC inhibition with HOIPIN-8.
Together, these findings identify LUBAC-mediated M1 poly-Ub as an important mediator of necroptosis and identify FLOTs as novel putative targets of LUBAC-mediated M1 poly-Ub during necroptosis. In addition, by modeling necroptosis in primary human organoids, we further expand the spectrum of experimental models to study necroptosis in human cellular settings.
Chapter I of this work addressed the piggyBac (PB) transposon system, a non-viral genome engineering tool that is capable of efficiently performing stable integration of DNA sequences into a target cells genome and has already been used in clinical trials. However, the PB transposase has the problematic property of preferentially integrating transposons near transcriptional start sites (TSSs). This increases the likelihood of causing genotoxic effects, limiting its potential use as a tool in clinical applications. It has been shown in the past that the PB transposase shows physical interactions with BET proteins (e.g. BRD4) through Co-IP experiments. Representatives of these proteins are part of the transcriptional activation complex and are abundant at TSSs. Accordingly, it was previously proposed that this interaction is the underlying cause for the biased integration preference. For the first chapter of this thesis, the goal was to disrupt this interaction potentially modifying said integration preference. A secondary structure hypothesized to be mainly responsible for said interaction was extensively mutated resulting in several PB variants that were analyzed for their interaction capacity through a series of Co-IP experiments with BRD4. In total, seven substitutions were identified (E380F, V390K, T392Y, M394R, K407C, K407Q, and K407V) which exhibited reduced interaction capacity with BRD4. Each of the aforementioned mutants were used to generate integration libraries and, through NGS, it was determined if the integration preferences of the respective mutants had changed. In the immediate range 200 base pairs up- and downstream from known TSSs all mutants used exhibited a reduced integration bias. At a wider observation window 3 kbp up- and downstream from TSSs, further mutants with the substitutions M394R, T392Y and V390K showed a reduction in integration frequency of 17.3%, 1.5% and 5.4%, respectively, compared to the wildtype. Of particular note was the M394R mutant, which showed a reduction in all window sizes analyzed with a maximum of 65% less integration preference in the immediate vicinity of TSSs, theoretically generating a safety advantage over the wildtype transposase.
Chapter II was dedicated to the overall safety improvement for transposon-based gene modification and addresses the time point after the transgene has already been integrated and serious side effects may not be preventable. With this in mind, the aim was to develop a novel suicide-switch that can be stably introduced into cells via transposition, and reliably leads to cell death of the modified cells once activated. A system based on CRISPR/Cas9 was developed, where single guide RNAs were used to guide the Cas9 nuclease to Alu elements. These are short, repetitive sequences, which are distributed over the human genome in more than one million copies. Inducing double strand breaks within these elements would lead to genomic fragmentation and cell death. To be inducible, a transcriptional as well as post- translational control mechanism was added. Transcription of the Cas9 nuclease was regulated using a tet-on system, making expression dependent on doxycycline (DOX) supplementation. Furthermore, a version of the Cas9 nuclease called arC9 was used that allows double strand break generation only in the presence of 4-Hydroxytamoxifen (4-HT). Together with an expression cassette for the Alu-specific guide RNA and an expression cassette for the reverse tetracycline controlled transactivator all components were arranged between transposase-specific recognition sequences on a plasmid to allow transposon-system based gene transfer. The system was tested in HeLa cells. First, conditional expression of the arC9 nuclease was confirmed by addition of 1 μg/ml DOX. Second, the suicide-switch was further induced by adding 200 nM 4-HT and protein extracts were assayed for the KAP1 phosphorylation. Only upon induction with DOX and 4-HT phosphorylated KAP1 was detected, indicating DNA damage. Further, extensive growth and survival experiments were conducted to determine the effect of suicide-switch induction on cell proliferation and survival. Between 24 and 48 hours after induction, a halt in cell division was detected, after which extensive cell death was observed. Within 5 days post induction, >99% of all cells were eliminated. In the absence of both inducers, no significant differences in survival were observed compared to control cells line lacking Alu-specific guide RNAs. Microscopic examinations of the <1% surviving cell fraction revealed a senescence-associated phenotype and showed no signs of resumption of the cell division process. Accordingly, the second chapter of this thesis also achieved its goal in developing a functional suicide-switch that can be inserted into human cells via transposition, is highly dependent on the necessary induction signals, and exhibits excellent elimination capabilities in the context tested.
Synaptic transmission is a fundamental process that involves the transfer of information from a presynaptic neuron to a target cell through the release of neurotransmitters. The SV cycle is a complex series of events that enables the recycling of SVs, allowing for the sustained release of neurotransmitters. This process is mediated by a variety of proteins and enzymes, and its regulation is critical for maintaining proper synaptic function. Despite extensive research efforts, many aspects of the SV cycle and the underlying synaptic proteins remain poorly understood, highlighting the need for continued investigation into this important process. During this work, multiple aspects of synaptic transmission were studied by performing
behavioural, pharmacological, optogenetic, electrophysiological and ultrastructural assays on Caenorhabditis elegans. First, the role of two proteins (ERP-1 and RIMB-1) were analysed in the synaptic vesicle cycle. Second, a new optogenetic tool, the pOpsicle assay was described, which enables the direct visualization of synaptic vesicle (SV) release.
Activity-dependent bulk endocytosis (ADBE) enables the endocytosis of SV membrane and proteins in a fast manner during intense stimulation, resulting in bulk endosomes (also so-called large vesicles, LVs). Recycling proteins can be characterized by its site of action, whether they act at the plasma membrane (participating at the LV formation), or at the LV membrane (participating at the SV formation). ERP-1 (the C. elegans ortholog of Endophilin B) was recently identified as a possible SV recycling factor, its contribution to synaptic transmission has not been analysed before. During this project the function and possible cooperation of three proteins, ERP-1, UNC-57 (the C. elegans ortholog of Endophilin A) and CHC-1 (the C. elegans ortholog clathrin heavy chain) were studied, with a special emphasis of the site of action. It has been confirmed that these proteins participate together in synaptic vesicle recycling. Endophilins (ERP-1 and UNC-57) act both at the PM and the LV level, but while UNC-57 has been identified as the main player, ERP-1 rather has a minor role and acts as a back-up protein. CHC-1 functions the LV level in the first place, but it can compensate for the loss of UNC-57 and acts as a back-up protein at the PM.
RIM-binding protein is an evolutionarily conserved active zone protein, which interacts directly with RIM and N, P/Q, as well as L-type Ca2+ channels. RIM-BP and RIM have redundant functions in different model organisms including C. elegans, however, while the loss of UNC-10 (the C. elegans ortholog of RIM) led to drastic behavioural defects, the loss of RIMB-1 (the C. elegans ortholog of RIM-BP) led only to mild phenotypes. During this work the synaptic function of RIMB-1 and its interaction with UNC-10 and UNC-2 (C. elegans ortholog of the CaV2 1 subunit) were extensively investigated. It has been shown that RIMB-1 contributes to the precise localization of VGCCs in cooperation with UNC-10. Furthermore, it has been demonstrated, that RIMB-1 plays different roles in cholinergic and GABAergic neurons, thus it contributes to maintain a proper excitation/inhibition balance.
There are numerous available assays, which enable the indirect analysis of synaptic transmission, however, a tool, that enables the direct visualization of SV release, is highly desired. pOpsicle is a method which combines the optogenetic stimulation of cholinergic neurons with real-time visualization of SV release. A pH-sensitive fluorescence protein, pHuji, was inserted into the second intravesicular loop of the synaptic vesicle membrane protein, synaptogyrin (SNG-1). The fluorescence of pHuji is quenched inside the vesicles, but once they are released, the pH increases and pHuji can be detected. pOpsicle enables not only the direct visualization of SV exo-, and endocytosis events, but also the identification of putative SV recycling proteins.
Die Kernspinresonanz(NMR)-Spektroskopie ist ein leistungsstarkes analytisches Werkzeug. Allerdings ist ihre Empfindlichkeit aufgrund geringer Wechselwirkungs-energie zwischen den Kernspins und dem externen Magnetfeld begrenzt. Die dynamische Kernpolarisation (DNP) erhöht DNP die Empfindlichkeit der NMR, indem sie die Polarisation von ungepaarten Elektronenspins auf die benachbarten Kernspins überträgt. In den letzten Jahrzehnten hat die DNP bei hohen Magnetfeldern erneut an Aufmerksamkeit gewonnen, bedingt durch die Verfügbarkeit leistungsstarker Gyrotron-Mikrowellen(mw)-Quellen. Jedoch wurde die Anwendung von DNP für Flüssigkeiten im Vergleich zu Festkörperproben bei niedrigen Temperaturen (≈100 K) weit weniger erforscht. Zwei Gründe können dafür hauptsächlich benennt werden. Bei hohen Magnetfeldern (entsprechend hohen mw-Frequenzen) wird die mw-Strahlung sehr stark von Flüssigkeiten absorbiert, was zu einer starken Erwärmung führt. Darüber hinaus sind die Translations- und Rotationsdynamik der Radikale und Target-Molekülen nicht schnell genug, um Spectraldichten bei den hohen mw-Frequenzen zu erzeugen, die für eine Overhauser-Effekt (OE) DNP Verstärkung benötigt werden. In dieser Arbeit wird gezeigt, Flüssigzustands-DNP bei hohen Magnetfeldern, insbesondere bei 9,4 T, mit hocheffizienten DNP-Probenköpfen möglich ist.
Der von skalaren Hyperfein-Wechselwirkung (hfWW) angetriebene OE ist für Flüssigzustands-DNP-Forschungen von besonderem Interesse, da der von der Theorie vorhergesagte Mechanismus auch bei hohen Magnetfeldern noch effizient ist. In der vorliegenden Arbeit wurde eine Methode zur Vorabprüfung potenzieller DNP-Kandidaten durch Messungen ihrer paramagnetischen NMR-Verschiebungen vorgeschlagen und untersucht. Wir beobachtete signifikante 13C-skalare OE DNP-Verstärkungen bis zu 50 bei den ausgewählten kleinen Biomolekülen, einschließlich Imidazol, Indol, verschiedene Aminosäuren und Kohlenhydraten. Das Lösungssystem wurde auch von organischen Lösungsmitteln auf Wasser erweitert.
Im Kontext von dipolarer OE DNP haben wir den Beitrag der Rotation des Radikals neben der Translationsbewegung zwischen Radikal und Target-Molekül zur OE DNP-Effizienz systematisch untersucht, indem wir verschiedene Nitroxidderivate mit unterschiedlichen Ringgeometrien und Substituenten verwendet haben. Mithilfe eines Models, das eine 'out-sphere' Translationsbewegung und eine 'inner-sphere' Rotationsbewegung des Radikal-Lösungsmittel-Komplexes enthält, konnte unsere Beobachtungen quantitativ simuliert werden. Außerdem wurde ein anderes Model untersucht, das eine Translationsbewegung mit der Rotation von Radikalen, bei denen das ungepaarte Elektron nicht im Zentrum sitzt, kombiniert.
Eine weitere neue Entdeckung in der DNP bei hohen Magnetfeldern waren der beobachtete SE (Solid-Effekt) an Lipidmolekülen mit BDPA-Radikal oberhalb der Lipidphasen-übergangstemperatur. Die neue Anwendung von SE DNP bietet einen alternativen Mechanismus zur OE DNP in Flüssigkeiten bei hohen Magnetfeldern und könnte möglicherweise auf Makromoleküle mit relativ langsamer Rotationsbewegung angewendet werden.
Wir haben zusätzliche Untersuchungen an den Lipiddoppelschichten mit Nitroxid-radikale durchgeführt, basierend auf dem beobachteten 1H DNP-Verstärkungen in einer viskosen Lipidumgebung bei 9,4 T . Durch Messung des Feldprofils wurden DNP-Verstärkungen durch OE und SE in Abhängigkeit ihrer relativen Verschiebungen von der Elektronen-Larmor-Frequenz bestimmt. Die individuelle OE DNP-Effizienzen für Protonen des Wassers, der Lipid-Cholin-Kopfgruppen oder der Lipid-Acylketten wurde bestimmt. Dadurch wird ein quantitativer Vergleich mit MD-Simulationen ermöglicht. Obwohl die von der MD-Simulationen vorhergesagten DNP Kopplungsfaktoren noch deutliche Abweichungen von den experimentellen Beobachtungen aufweisen, wird die schnelle Dynamik nahe der Elektronen-Larmor-Frequenz, die für einen erfolgreichen OE DNP Transfer erforderlich ist, von den MD-Simulationen gut erfasst.
In der Arbeit wurden auch zwei unterschiedliche Dreifachresonanz-DNP-Experimente durchgeführt. Zum einen wurde 13C OE DNP unter 1H-Entkopplung in wässriger Natriumpyruvatlösung, und zum anderen 13C-NMR von Glycin, verstärkt durch SE DNP an 1H zusammen mit einem 1H-13C INEPT-Polarisationstransfer, im Rahmen dieser Doktorarbeit durchgeführt.
The role of lncRNAs in the CVS and the endothelium is highly diverse and has been subject to a substantial amount of research over the last decade. The identification of lncRNAs as clinically relevant biomarkers and as co-regulatory molecules let to the appreciation of the functional relevance of lncRNAs.
In the present study, LINC00607 was identified as an endothelial-enriched, human-specific lncRNA. With its distinct functions, LINC00607 maintains and supports the endothelial homeostasis especially in response to VEGF-A signalling.
In the first part of this study, LINC00607 was functionally characterized in human endothelial cells. LINC00607 is highly and specifically expressed in endothelial cells and is differentially regulated in CVDs. Depletion of LINC00607 resulted in decreased angiogenic sprouting, reduced integration of ECs in a newly formed vascular network in vivo, enhanced endothelial migration and differential expression of many important genes for endothelial cell homeostasis. Functionally, LINC00607 maintains ERG-driven endothelial gene expression programs through BRG1. BRG1 secures stably accessible enhancer regions as well as TSS of ERG target genes, thus enabling transcription of endothelial gene programs.
The second part of this study proposes an additional mode of action for LINC00607. The strongly impaired response to VEGF-A after LINC00607 KO can only be partially explained by its’ expression control of ERG target genes. It rather appears that LINC00607 is involved in the control of alternative splicing of VEGF receptor FLT1. The differential splicing of FLT1 produces the anti-angiogenic soluble isoform of FLT1. Even though further validation is needed to uncover the underlying mechanism, there is the potential of a more general role of LINC00607 in splicing control through BRG1. As AS of FLT1 is a clinical marker in preeclampsia, LINC00607 might qualify to be an additional marker for the onset and manifestation of the pregnancy disorder.
Taken together, LINC00607 is a target in future for molecular therapy in CVD to restore a healthy endothelial phenotype and has the potential to serve as a biomarker in preeclampsia.
This cumulative dissertation examines learning in chemistry laboratories, focusing on the challenges and benefits of problem-based learning (PBL) for novices in the lab. It addresses the lack of consistent understanding about what should be learned in labs and why it's important. The research aims to understand what students learn, how they learn, and how lab learning can be improved.
A central concept in PBL labs is Information Literacy, defined as a sociocultural practice enabling learners to identify and use information sources within a specific context as legitimized by the practice community.
The first publication, Wellhöfer and Lühken (2022a), investigates the relationship between PBL and learner motivation. It identifies factors that can foster students' intrinsic motivation in a PBL lab. Autonomy is found to be a key factor, increasing student motivation and presenting a model of the autonomous scientific process. This model involves four steps: information acquisition, designing and applying experimental procedures, experimental feedback, and autonomous process optimization. The results suggest that intrinsic motivation in PBL labs can be enhanced by enabling students to independently execute these steps.
The second publication, Wellhöfer and Lühken (2022b), examines the information process students undergo during their first PBL lab. Using a sociocultural framework, it explores Information Literacy to understand students' handling of information and their perceptions of the information process. The findings reveal that in PBL labs, developing a practical, applicable experimental procedure is crucial for problem-solving and significantly shapes the information-acquisition process. This process is iterative, influenced by new information, leading to more precise information needs. Students assess information quality based on its usefulness for their problem, implementability (considering cognitive understanding, available equipment, and psychomotor skills), and safety.
Furthermore, the role of privileged knowledge forms in evaluating the quality of text sources is explored. Students viewed non-scientific sources as "poor" and scientific sources as "good," yet used both for information gathering. There were discrepancies between their assessment of source quality and actual use, indicating that perception of source quality doesn't always affect their practical decisions.
The third publication, Wellhöfer, Machleid, and Lühken (2023), investigates students' information practices in the lab, focusing on discourse between novice learners and experienced assistants. It shows that theoretical knowledge isn't sufficient for independent practical action, and students need actionable social information from experienced community members. The results highlight that information literacy in the lab for newcomers to a community of practice has distinctive features, and physical experience and tacit knowledge are crucial for learning the methods and group-specific knowledge of the practice community. The article demonstrates how learning information literacy in a practice community requires a social and physical experience and provides insights on how educators can support this process.
Mechanistic characterization of photoisomerization reactions in organic molecules and photoreceptors
(2023)
In dieser Arbeit wurden verschiedene Einflüsse auf die Dynamik von Photoisomerisierungen in Phytochromen und indigoiden Photoschaltern untersucht. Beide Forschungsgebiete teilen wesentliche Aspekte wie die Kontrolle durch sterische Wechselwirkungen und den starken Einfluss der Polarität oder der ionischen Umgebung.
Auf dem Gebiet der Phytochrome wurde die relative Positionierung der knotenlosen Phytochrome innerhalb der Superfamilie der Phytochrome in Bezug auf ihre Photodynamik und den Effekt von Grundzustandsheterogenität herausgearbeitet. Es wurde anhand von ultraschnellen, zeitaufgelösten Anrege-Abtast-Experimenten der einzelnen GAF-Domäne All2699g1 im Vergleich mit dem vollständigen knotenlosen Phytochrom All2699g1g2 und dem strukturell ähnlichen knotenlosen Phytochrom SynCph2 gezeigt, dass knotenlose Phytochrome in ihrer Vorwärtsdynamik eine komplexe mehrphasige Kinetik mit einem langlebigen angeregten Zustand (~100 ps) aufweisen. Die beobachtete mehrphasige Kinetik konnte einer initialen Chromophordynamik sowie einer nicht exponentiellen Reorganisation der chromophor-umgebenden Proteinmatrix zugeordnet werden. Dies steht im starken Kontrast zur im Gebiet der Phytochrome etablierten Beschreibung derartiger mehrphasiger Kinetiken mittels heterogener Grundzustände. Stattdessen wurde ein konserviertes kinetisches Muster identifiziert, welches die mehrphasige Dynamik beschreibt und in allen in dieser Arbeit untersuchten Phytochrome beobachtet wurde. Zudem konnte dieses Muster in einem Phytochrom der Gruppe I und einem Phytochrom der Gruppe III, die einen ähnlichen Pr Dunkelzustand aufweisen, gezeigt werden, was eine breite Anwendbarkeit des damit verbundenen Mechanismus vermuten lässt. Weiterhin konnte die zentrale Rolle eines konservierten Tyrosins in der Photoisomerisierung anhand von Mutationsstudien in All2699g1 herausgearbeitet werden. Diese konservierte Aminosäure muss im Rahmen der Reorganisation der Proteinmatrix vom Chromophor weggezogen werden, damit die sterische Blockade abgebaut werden kann, die die Isomerisierung des Chromophors zunächst verhindert. Da diese Bewegung von diversen Faktoren in der den Chromophor umgebenden Proteinmatrix abhängt, weist sie eine nicht exponentielle Kinetik auf, die je nach Phytochrom, der spezifischen Flexibilität und dem vorhandenen Raum in der Bindetasche unterschiedliche Lebenszeiten aufweist.
Die Rückreaktion knotenloser Phytochrome konnte ebenfalls im Rahmen dieser Arbeit charakterisiert werden, welche im Pikosekundenbereich abläuft, und damit signifikant schneller ist als die Vorwärtsreaktion. Im Gegensatz zur Vorwärtsreaktion nimmt Grundzustandsheterogenität in der Rückreaktion eine weitaus bedeutendere Rolle ein. Hier weisen die in All2699g1 vorhandenen heterogenen Grundzustandspopulationen jeweils eine eigene Kinetik ihres angeregten Zustands auf, während die homogenen Grundzustände von All2699g1g2 und SynCph2 jeweils nur einen Zerfall des angeregten Zustands zeigen. Der Ursprung dieser Heterogenität konnte im Wasserstoffbrückennetzwerk des Chromophors lokalisiert und mit dem konservierten Tyrosin und einem konservierten Serin in der PHY-Domäne verknüpft werden. Die Anwesenheit der PHY-Domäne sorgt demnach für eine Verringerung der Grundzustandsheterogenität und des vorhandenen Raums in der Bindetasche, wodurch die Effizienz der Photoreaktion optimiert wird.
Zuletzt konnte die Millisekundendynamik knotenloser Phytochrome und der Einfluss der PHY-Domäne auf diese aufgeklärt werden. Die PHY-Domäne sorgt hierbei durch den verringerten Raum in der Bindetasche dafür, dass die zunächst stattfindende thermische Relaxation des Chromophors signifikant verlangsamt wird, während spätere Änderungen im Photozyklus nur wenig beeinflusst werden.
Auf dem Gebiet der indigoiden Photoschalter konnte, anhand eines sterisch überladenen Hemithioindigo Photoschalters, der Photoisomerisierungsmechanismus des Hula-Twists beobachtet und eine starke Lösungsmittelabhängigkeit der entsprechenden Kinetik aufgezeigt werden. Aus den durchgeführten zeitaufgelösten Anrege-Abtast-Experimenten in verschiedenen Lösungsmitteln konnte ein Modell für die Photodynamik des verwendeten Hemithioindigo Photoschalters entwickelt werden. In unpolaren Lösungsmitteln muss eine hohe Barriere zur produktiven konischen Durchschneidung überwunden werden, was zu Lebenszeiten des angeregten Zustands im Nanosekundenbereich führt. Der Weg zur produktiven konischen Durchschneidung folgt dabei dem Hula-Twist Mechanismus. Dieser Pfad ist in polaren Lösungsmitteln unerreichbar, weshalb eine schnelle Relaxation über eine unproduktive konische Durchschneidung stattfindet.
Im zweiten Projekt auf dem Gebiet der indigoiden Photoschalter wurde anhand der neuartigen Klasse der Iminothioindoxyl Photoschalter ein Schwingungsenergiedonor für Schwingungsenergietransferstudien entwickelt. Das daraus entwickelte Modellsystem, bestehend aus einer künstlichen Aminosäure auf Basis des Iminothioindoxyl Photoschalters und einem daran gekoppelten Schwingungsenergiesensor, wurde charakterisiert und die primäre Photoreaktion untersucht. Es konnte gezeigt werden, dass der angeregte Zustand des Modellsystems kurzlebig ist und unter Abgabe von großen Mengen an Schwingungsenergie zerfällt, unabhängig von der Anregungswellenlänge und dem verwendeten Lösungsmittel. Somit zeigt das entwickelte System vorteilhafte Eigenschaften für Schwingungsenergietransferstudien.
Insgesamt konnten somit die Mechanismen der Photoisomerisierungsreaktionen in knotenlosen Phytochromen und indigoiden Photoschaltern aufgeklärt und daraus die Relevanz der Umgebung für derartige Reaktionen herausgearbeitet werden.