Refine
Has Fulltext
- yes (55)
Is part of the Bibliography
- yes (55)
Year of publication
Document Type
- Doctoral Thesis (52)
- Journal article (2)
- Master Thesis (1)
Keywords
- Epigenetik (3)
- Genexpression (3)
- Angiogenese (2)
- Biofilm (2)
- Cancer (2)
- Exomsequenzierung (2)
- Guanylatcyclase (2)
- Immunotherapy (2)
- Konsanguinität (2)
- Krebs <Medizin> (2)
- Maus (2)
- Melanom (2)
- Therapie (2)
- Tissue Engineering (2)
- Vaskularisierung (2)
- microbiome (2)
- 3D (1)
- 3D Tumormodell (1)
- 3D cell culture (1)
- 3D tumour model (1)
- ADHD (1)
- APOBEC3G (1)
- Alternative methods (1)
- Alternativmethoden (1)
- Alzheimer's disease (1)
- Alzheimerkrankheit (1)
- Aminobuttersäure <gamma-> (1)
- Angeborene Immunität (1)
- Animal model (1)
- Anthropocene (1)
- Aorta (1)
- Arbeitsteilung (1)
- Aspergillus fumigatus (1)
- Aussterbedynamik (1)
- Automation (1)
- BRAF-mutant (1)
- BRAF-mutiert (1)
- Bacterial infection (1)
- Bakteriophagen (1)
- Biene (1)
- Bioengineering (1)
- Bioinformatik (1)
- Bleomycin (1)
- C. difficile (1)
- CDI (1)
- CNBP (1)
- Calciumkanal (1)
- Calpain (1)
- Cataglyphis (1)
- Catenin (1)
- Catenine (1)
- Cavβ subunit (1)
- Ceramide (1)
- Channelrhodopsin 2 (1)
- Chlamydia trachomatis (1)
- Control centrality (1)
- Craniosynostosis (1)
- Cyclo-AMP (1)
- Cyclo-GMP (1)
- DG diacyglycerol (1)
- DNA Reparatur (1)
- DNAse (1)
- Danio rerio (1)
- Darmkrebs (1)
- Deep learning (1)
- Dickdarmtumor (1)
- Dispersal (1)
- Dopamin (1)
- Drosophila melanogaster (1)
- E. coli Nissle 1917 (1)
- EHEC (1)
- Elektrophysiologie (1)
- Enterohemorrhagic Escherichia coli (1)
- Entwicklung (1)
- Evolution (1)
- Exozytose (1)
- Expansion microscopy (1)
- Expansionsmikroskopie (1)
- FOSL1 (1)
- FOXP2 (1)
- Fanconi Anämie (1)
- Farbensehen (1)
- Fetale Programmierung (1)
- Fluoreszenzmikroskopie (1)
- Folliculin (1)
- G protein-coupled receptors (1)
- GABA (1)
- GAD1 (1)
- GRM8 (1)
- Genetic etiology (1)
- Genetic regulatory networks (1)
- Genetik (1)
- Genetisches Imprinting (1)
- Genmutation (1)
- Genregulation (1)
- Glioblastom (1)
- Glioblastoma (1)
- Glucosetransportproteine (1)
- Gonococcal invasion (1)
- Guanylyl-Cyclase (1)
- HIV-1 (1)
- Hair follicle (1)
- Haut (1)
- Hautmodell (1)
- Herzhypertrophie (1)
- Herzmuskelzelle (1)
- Himmelskompass (1)
- Hirntumor (1)
- Histogenese (1)
- Histogenesis (1)
- Hochauflösungsmikroskopie (1)
- Homing (1)
- Host defense (1)
- Host-pathogen interaction (1)
- Hyperactivity (1)
- Hörstörung (1)
- Hörstörungen (1)
- Immunologie (1)
- Immunsuppression (1)
- Implantat (1)
- In vitro (1)
- Individual based model (IBM) (1)
- Induzierte Mutation (1)
- Innate immunity (1)
- Integrin (1)
- Intracellular bacteria (1)
- Intrazelluläre Bakterien (1)
- Japankärpfling (1)
- Knochenimplantat (1)
- Knochenregeneration (1)
- Knock-Out <Molekulargenetik> (1)
- Komplexauge (1)
- Kraniosynostose (1)
- Kryoelektronenmikroskopie (1)
- L-type calcium channels (1)
- LTCC-independent function of Cavβ (1)
- Lambdoide Prophagen (1)
- Locomotor activity (1)
- Lungenfibrose (1)
- MAX (1)
- MYCN (1)
- Marine sponge-derived actinomycetes (1)
- Marine sponges (1)
- Medizinprodukt (1)
- Merkel cell carcinoma (1)
- Merkel-Zellkarzinom (1)
- Messenger-RNS (1)
- Microbubbles (1)
- Mikrowellen (1)
- Mitochondria (1)
- Mitochondrien (1)
- Mitochondrium (1)
- Model Organism (1)
- Modellorganismus (1)
- Modellsystem (1)
- ModulationTregs (1)
- Murine embryonale Stammzellen (1)
- Mutation (1)
- Mycoplasma pneumoniae (1)
- Myeloid-derived suppressor cells (1)
- Myofibroblast (1)
- NO-sensitive Guanylyl-Cyclase (1)
- NO-sensitive guanylyl cyclase (1)
- Natrium/Glukose Cotransporter (1)
- Neisseria gonorrhoeae (1)
- Nephroblastom (1)
- Netzhaut (1)
- Neue Fanconi Anämie Gene (1)
- Neuroepigenomics (1)
- Neuroethologie (1)
- Neuromodulation (1)
- Neuronale Plastizität (1)
- Neuropeptide (1)
- Neuropeptidom (1)
- Neutrophils (1)
- Next Generation Sequencing (NGS) (1)
- Nichtlineare Differentialgleichung (1)
- PE Phosphoethanolamine (1)
- PRC1.6 (1)
- PTI signalling (1)
- Paternal age and BMI effects (1)
- Pcgf6 (1)
- Perizyt (1)
- Pflanzenaussterben (1)
- Pharmaceutische Biologie (1)
- Phosphodiesterase 3 (1)
- Polyomavirus (1)
- Probiotikum (1)
- Proliferation (1)
- Protein interactor (1)
- Proteinbiosynthese (1)
- Pulmonary fibrosis (1)
- Pulswellengeschwindigkeit (1)
- Quorum-Sensing (1)
- RNA binding potein CNBP (1)
- RNAlater (1)
- Rechtsmedizin (1)
- Regenerative Medizin (1)
- Rhodopsin (1)
- Riesensynapsen (1)
- SGLT (1)
- STD patients (1)
- Schlaf (1)
- Schwangerschaftsdiabetes (1)
- Schädel-Hirn-Trauma (1)
- Serotonin (1)
- Shigella flexneri (1)
- Simulation (1)
- Slice Culture (1)
- Sodium-Glucose Transporter 2 (1)
- Soziale Insekten (1)
- Spatial heterogeneity (1)
- Spatiotemporal analysis (1)
- Staphylococci (1)
- Staphylococcus aureus (1)
- Stickstoffmonoxid (1)
- Strategies to Obtain Tumor-Reactive Cells (1)
- Streptomyces (1)
- Stress (1)
- Superresolution microscopy (1)
- TIAR (1)
- TOP mRNA (1)
- Targeted therapies (1)
- Taufliege (1)
- Temporal heterogeneity (1)
- Terminal Oligopyrimidine Tract (1)
- Theoretical Ecology (1)
- Thigmotaxis (1)
- Tisuue Engineering (1)
- Tomografie (1)
- Transkriptionsfaktor (1)
- Translationsinitiation (1)
- Translationskontrolle (1)
- Trypanosoma brucei (1)
- Tumor models (1)
- USP10 (1)
- Ubiquitin (1)
- Ubiquitinligase (1)
- Ultraschall (1)
- VLA-1 (1)
- VSG (1)
- Vaskularisation (1)
- Verhaltensplastizität (1)
- Verwandtenehe (1)
- Vif (1)
- Wilms Tumor (1)
- Wnt-Proteine (1)
- Xiphophorus Melanom (1)
- Zebrafish (1)
- Zellassay (1)
- agr (1)
- antigene Variation (1)
- assay (1)
- autophagocytose (1)
- autophagy (1)
- autosomal rezessiver Erbgang (1)
- bees (1)
- bleomycin (1)
- bone (1)
- chlamydia trachomatis (1)
- circadian clock (1)
- color vision (1)
- colorectal cancer (1)
- compound eyes (1)
- cryo-EM (1)
- cryo-ET (1)
- deep learning (1)
- dopamine (1)
- extinction dynamics (1)
- fish model (1)
- flash freezing (1)
- fluoreszenz (1)
- implant (1)
- infant (1)
- insects (1)
- kolorektales Karzinom (1)
- lambdoid prophage (1)
- mRNA (1)
- mechanistische Modellierung (1)
- membrane protein (1)
- metabolic modelling (1)
- metabolic theory of ecology (1)
- metagenomics (1)
- metaproteomics (1)
- metatranscriptome (1)
- microbiota (1)
- monoallele Expression (1)
- mycoplasma (1)
- myofibroblast (1)
- neurobiology (1)
- particle picking (1)
- pericyte (1)
- plant immunity (1)
- pneumoniae (1)
- polarized epithelium (1)
- probiotica (1)
- protease (1)
- proteomics (1)
- rapid evolution (1)
- retinal development (1)
- sae (1)
- sample storage (1)
- sar (1)
- schnelle Evolution (1)
- screening (1)
- serotonin (1)
- skin (1)
- sleep (1)
- sodium-dependend glucose transporter (1)
- stx-Phagen (1)
- stx-phages (1)
- targeted therapy (1)
- tomography (1)
- transient dynamics (1)
- translation (1)
- traumatic brain injury (1)
- tumour (1)
- uORF (1)
- vascularization (1)
- vesicle trafficking (1)
- visual proteomics (1)
- zielgerichtete Behandlung (1)
Institute
- Fakultät für Biologie (55) (remove)
Sonstige beteiligte Institutionen
- EMBL Heidelberg (2)
- Fachhochschule Kaiserslautern, Campus Zweibrücken (1)
- Institut für Medizinische Mikrobiologie und Hygiene der Eberhard-Karls-Universität Tübingen (1)
- Lehrstuhl für Biochemie und molekulare Biologie (1)
- Lehrstuhl für Bioinformatik (1)
- Maastricht University, Maastricht, the Netherlands (1)
- Medizinische Universität Innsbruck (1)
- Queensland University of Technology (1)
EU-Project number / Contract (GA) number
Lung cancer is the main cause of cancer-related deaths worldwide. Despite the availability of several targeted therapies and immunotherapies in the clinics, the prognosis for lung cancer remains poor. A major problem for the low benefit of these therapies is intrinsic and acquired resistance, asking for pre-clinical models for closer investigation of predictive biomarkers for refined personalized medicine and testing of possible combination therapies as well as novel therapeutic approaches to break resistances.
One third of all lung adenocarcinoma harbor mutations in the KRAS gene, of which 39 % are transitions from glycine to cysteine in codon 12 (KRASG12C). Being considered “undruggable” in previous decades, KRASG12C-inhibitors now paved the way into the standard-of-care for lung adenocarcinoma treatment in the clinics. Still, the overall response rates as well as overall survival of patients treated with KRASG12C-inhibitors are sobering. Therefore, 3D KRASG12C-biomarker in vitro models were developed based on a decellularized porcine jejunum (SISmuc) using commercial and PDX-derived cell lines and characterized in regards of epithelial-mesenchymal-transition (EMT), stemness, proliferation, invasion and c-MYC expression as well as the sensitivity towards KRASG12C-inhibiton. The phenotype of lung tumors harboring KRAS mutations together with a c-MYC overexpression described in the literature regarding invasion and proliferation for in vivo models was well represented in the SISmuc models. A higher resistance towards targeted therapies was validated in the 3D models compared to 2D cultures, while reduced viability after treatment with combination therapies were exclusively observed in the 3D models. In the test system neither EMT, stemness nor the c-MYC expression were directly predictive for drug sensitivity. Testing of a panel of combination therapies, a sensitizing effect of the aurora kinase A (AURKA) inhibitor alisertib for the KRASG12C-inhibitor ARS-1620 directly correlating with the level of c-MYC expression in the corresponding 3D models was observed. Thereby, the capability of SISmuc tumor models as an in vitro test system for patient stratification was demonstrated, holding the possibility to reduce animal experiments.
Besides targeted therapies the treatment of NSCLC with oncolytic viruses (OVs) is a promising approach. However, a lack of in vitro models to test novel OVs limits the transfer from bench to bedside. In this study, 3D NSCLC models based on the SISmuc were evaluated for their capability to perform efficacy and risk assessment of oncolytic viruses (OVs) in a pre-clinical setting. Hereby, the infection of cocultures of tumor cells and fibroblasts on the SISmuc with provided viruses demonstrated that in contrast to a wildtype herpes simplex virus 1 (HSV-1) based OV, the attenuated version of the OV exhibited specificity for NSCLC cells with a more advanced and highly proliferative phenotype, while fibroblasts were no longer permissive for infection. This approach introduced SISmuc tumor models as novel test system for in vitro validation of OVs.
Finally, a workflow for validating the efficacy of anti-cancer therapies in 3D tumor spheroids was established for the transfer to an automated platform based on a two-arm-robot system. In a proof-of-concept process, H358 spheroids were characterized and treated with the KRASG12C-inhibitor ARS-1620. A time- and dose-dependent reduction of the spheroid area after treatment was defined together with a live/dead-staining as easy-to-perform and cost-effective assays for automated drug testing that can be readily performed in situ in an automated system.
Laut des aktuellen Reports der Weltgesundheitsorganisation sind ca. 466 Millionen Menschen weltweit von einer Hörstörung (HS) betroffen. Durch die enorme Heterogenität und die klinische Variabilität, die diese Erkrankung ausmacht, und viele bisher nicht mit HS assoziierte Gene, bleibt ein großer Teil der erblich bedingten HS in vielen Familien unaufgeklärt. Die Entwicklung moderner Techniken, wie die Next-Generation Sequenzierung (NGS) und der Fortschritt bei der Untersuchung von Modellorganismen trugen jedoch in den letzten Jahren immens dazu bei, neue Gene zu identifizieren, die innerhalb des auditorischen Signalwegs oder damit assoziierten Strukturen beteiligt sind. Die vorliegende Arbeit umfasst Ergebnisse dreier Veröffentlichungen, in denen iranische und pakistanische Familien und eine deutsche Familie mit erblich bedingter HS untersucht und neue, krankheitsverursachende Varianten identifiziert und funktionell charakterisiert wurden. Im ersten Abschnitt konnten zwei neue rezessive Varianten im CDC14A-Gen als krankheitsverursachend identifiziert werden, die zu einem potentiellen Funktionsverlust des kodierten Proteins in einer iranischen und einer pakistanischen Familie führen. Mit Hilfe einer funktionellen Charakterisierung auf RNA-Ebene (Spleiß-Assay und RT-qPCR) konnte der Funktionsverlust beider Varianten bestätigt werden. Der zweite Abschnitt umfasst eine deutsche Familie mit sieben von einer HS betroffenen Familienmitgliedern, in der eine heterozygote missense Variante in MYO3A identifiziert wurde. In der vorliegenden Arbeit konnte somit die erste autosomal dominante Variante in einer europäischen Familie mit einer bilingualen, sensorineuralen Hochtonschwerhörigkeit beschrieben werden und der dominante Charakter von MYO3A bestätigt werden. Im dritten Abschnitt konnten die krankheitsverursachenden Varianten in 13 Familien aus einer Kohorte mit 21 pakistanischen Familien mit einer syndromalen und nicht-syndromalen HS ausfindig gemacht werden. Hierbei wurden sowohl bekannte, als auch bisher nicht beschriebene Varianten detektiert. Die Aufklärungsrate innerhalb dieser Kohorte betrug 61,9% und es konnte somit das Spektrum syndromaler und nicht-syndromaler HS erweitert werden. Der letzte Abschnitt dieser Arbeit beschreibt eine iranische Familie mit einer milden HS und milden Intelligenzminderung, in der eine homozygote missense Variante im Kandidatengen DBN1 ausfindig gemacht wurde. Um die Funktion und die Auswirkungen eines potentiellen Verlusts des codierten Proteins Drebrin zu untersuchen, wurden immunhistochemische Färbungen und auditorische Messungen an Dbn1 Knockout (KO)-Mäusen durchgeführt. Hierbei konnte eine Expression innerhalb der Nervenfasern, die innere Haarzellen innervieren, nachgewiesen werden. Eine leicht verlängerte Latenz für die ABR-Welle IV in KO-Mäusen im Vergleich zum Wildtyp ergab den Hinweis auf einen Defekt innerhalb des zentralen auditorischen Signalwegs, der möglicherweise mit einer Sprachverarbeitungsstörung im Menschen korreliert.
Regulatory T cells (Tregs) are the masters of immune regulation controlling inflammation and tolerance, tissue repair and homeostasis. Multiple immunological diseases result from altered Treg frequencies and Treg dysfunction. We hypothesized that augmenting Treg function and numbers would prevent inflammatory disease whereas inhibiting or depleting Tregs would improve cancer immunotherapy.
In the first part of this thesis, we explored whether in vivo activation and expansion of Tregs would impair acute graft-versus-host disease (aGvHD). In this inflammatory disease, Tregs are highly pathophysiological relevant and their adoptive transfer proved beneficial on disease outcome in preclinical models and clinical studies. IL-2 has been recognized as a key cytokine for Treg function. Yet, attempts in translating Treg expansion via IL-2 have remained challenging, due to IL-2s extremely broad action on other cell types including effector T cells, NK cells, eosinophils and vascular leakage syndrome, and importantly, due to poor pharmacokinetics in vivo. We addressed the latter issue using an IL-2-IgG-fusion protein (irrIgG-IL-2) with improved serum retention and demonstrated profound Treg expansion in vivo in FoxP3-luciferase reporter mice. Further, we augmented Treg numbers and function via the selective-TNF based agonists of TNFR2 (STAR2). Subsequently, we tested a next-generation TNFR2 agonist, termed NewSTAR, which proved even more effective. TNFR2 stimulation augmented Treg numbers and function and was as good as or even superior to the IL-2 strategy. Finally, in a mouse model of aGvHD we proved the clinical relevance of Treg expansion and activation with irrIgG-IL-2, STAR2 and NewSTAR. Notably, the TNFR2 stimulating constructs were outstanding as we observed not the IL-2 prototypic effects on other cell populations and no severe side effects.
In the second part of this thesis, we explored Tregs in pancreatic ductal adenocarcinoma (PDAC) and developed targeting strategies. Among several tumor entities in which Tregs impact survival, preclinical and clinical data demonstrated their negative role on PDAC. In our studies we employed the orthotopic syngeneic Panc02 model in immunocompetent mice. Based on flow cytometric analysis of the tumor microenvironment we propose TIGIT and TNFRSF members as novel therapeutic targets. Surprisingly, we found that blocking TNFR2 did not interfere with intratumoral Treg accumulation. However, we decreased the highly abundant intratumoral Tregs when we disrupted the tumor extracellular matrix. In PDAC, Treg manipulation alone did not lead to tumor regression and we propose that an additional immune boost may be necessary for efficient tumor immune surveillance and cancer clearance. This contrasts with aGvHD, in which Treg manipulation alone was sufficient to improve disease outcome.
Conclusively, we demonstrated the enormous medical benefit of Treg manipulation. Our promising data obtained with our newly developed powerful tools highlight the potential to translate our findings into clinical practice to therapeutically target human Tregs in patients. With novel TNFR2 agonists (STAR2, NewSTAR) we augmented Treg numbers and function as (or even more) effectively than with IL-2, without causing adverse side effects. Importantly, exogenous in vivo Treg expansion protected mice from aGvHD. For the therapy of PDAC, we identified novel targets on Tregs, notably TIGIT and members of the TNFRSF. We demonstrated that altering the extracellular tumor matrix can efficiently disrupt the Treg abundance in tumors. These novel targeting strategies appear as attractive new treatment options and they may benefit patients suffering from inflammatory disease and cancer in the future.
The research that is compiled in this thesis can be divided in two parts. The first part, consisting of four chapters, is centered around the role of epigenetic dysregulation in the etiopathophysiology of sporadic alzheimer's disease (sAD). In addition to providing insights into the most recent developments in neuroepigenomic studies of this disease, the first part of the thesis also touches upon remaining challenges, and provides a future outlook on possible developments in the field. The second part, which includes three more chapters, is focused on the application of induced pluripotent stem cell (iPSC)-based disease models for the study of AD, including but not limited to mechanistic studies on epigenetic dysregulation using this platform. Aside from outlining the research that has been conducted using iPSC-based models for sAD to date, the second part of the thesis also provides insights into the acquisition of disease-relevant neural cultures based on directed differentiation of iPSCs, and furthermore includes an experimental approach for the establishment of such a model system.
Glioblastoma (GBM) sind bösartige hirneigene Tumore, deren schlechte Prognose einer innovativen Therapie bedarf. Aus diesem Grund wurde ein neuer Therapieansatz entwickelt, der auf einer lokalen Ultraschall-vermittelten Zytostatika Applikation beruht. Hierfür wurden stabile Microbubbles (MB) bestehend aus Phospholipiden synthetisiert. Es konnte gezeigt werden, dass MB als auch fokussierter Ultraschall niedriger Intensität (LIFU) keinen negativen Einfluss auf GBM-Zellen hat. MB hingegen konnten mittels LIFU destruiert werden, wodurch das in den MB eingeschlossene Chemotherapeutikum freigesetzt werden kann. Es wurden verschiedene Platin(II)- und Palladium(II)-Komplexe auf GBM Zellen getestet. Zur Beladung der MB wurde Doxorubicin (Dox) verwendet. Es konnte eine Beladungseffizienz der MB mit Dox von 52 % erreicht werden, auch eine Aufreinigung dieser mittel Ionenaustausch-Chromatographie und Dialyse war erfolgreich. Die Austestung der mit Dox beladenen MB (MBDox) erfolgte auf GBM-Zellen in 2D- und 3D-Zelkulturmodellen. Dabei zeigte sich, dass die Behandlung mit MBDox und LIFU für 48 h eine zytotoxische Wirkung hatte, die sich signifikant von der Behandlung mit MBDox ohne LIFU unterschied. Zur Austestung der MBDox in 3D-Zellkulturmodellen wurden zwei Scaffold-Systeme eingesetzt. Es zeigte sich in den Versuchen, dass MBDox mit LIFU im Vergleich zu MBDox ohne LIFU Applikation einen zytotoxischen Effekt auf GBM-Zellen haben. Somit konnte die Wirksamkeit der Zytostatika Applikation mittels MB und LIFU in 2D- und 3D-Zellkulturmodellen erfolgreich etabliert werden. Als weiterer Schritt wurden zwei 3D in vitro Modelle erarbeitet. Dabei wurden zunächst organotypische hippocampale Slice Kulturen (organotypic hippocampal brain slice cultures, OHSC) aus der Maus hergestellt und anschließend mit fluoreszent-markierten Mikrotumoren aus GBM-Zelllinien, Primärzellen (PZ) und aus Patienten generierten GBM-Organoiden hergestellt. Diese GBM-Modelle wurden mit Tumor Treating Fields (TTFields) behandelt. Dabei war eine Abnahme der Tumorgröße von Mikrotumoren aus GBM-Zellen und PZ unter TTFields-Behandlung für 72 h messbar. Als weiteres in vitro Modell wurden humane Tumorschnitte aus intraoperativ entferntem GBM-Patientenmaterial hergestellt. Die Schnitte wiesen ein heterogenes Ansprechen nach 72 h TTFields-Applikation auf. Dies spiegelt die Heterogenität des GBM sehr gut wider und bestärkt die Eignung des Modelles zur Untersuchung von neuen Therapieansätzen zur Behandlung von GBM.
MDSCs are suppressive immune cells with a high relevance in various pathologies including cancer, autoimmunity, and chronic infections. Surface marker expression of MDSCs resembles monocytes and neutrophils which have immunostimulatory functions instead of suppressing T cells. Therefore, finding specific surface markers for MDSCs is important for MDSC research and therapeutic MDSC manipulation. In this study, we analyzed if the integrin VLA-1 has the potential as a novel MDSC marker. VLA-1 was expressed by M-MDSCs but not by G-MDSCs as well as by Teff cells. VLA-1 deficiency did not impact iNOS expression, the distribution of M-MDSC and G-MDSC subsets, and the suppressive capacity of MDSCs towards naïve and Teff cells in vitro. In mice, VLA-1 had no effect on the homing capability of MDSCs to the spleen, which is a major reservoir for MDSCs. Since the splenic red pulp contains collagen IV and VLA-1 binds collagen IV with a high affinity, we found MDSCs and Teff cells in this area as expected. We showed that T cell suppression in the spleen, indicated by reduced T cell recovery and proliferation as well as increased apoptosis and cell death, partially depended on VLA-1 expression by the MDSCs. In a mouse model of multiple sclerosis, MDSC injection prior to disease onset led to a decrease of the disease score, and this effect was significantly reduced when MDSCs were VLA-1 deficient. The expression of Sema7A by Teff cells, a ligand for VLA-1 which is implicated in negative T cell regulation, resulted in a slightly stronger Teff cell suppression by MDSCs compared to Sema7A deficient T cells. Live cell imaging and intravital 2-photon microscopy showed that the interaction time of MDSCs and Teff cells was shorter when MDSCs lacked VLA 1 expression, however VLA-1 expression had no impact on MDSC mobility. Therefore, the VLA-1-dependent interaction of MDSC and Teff cells on collagen IV in the splenic red pulp is implicated MDSC-mediated Teff cell suppression.
The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular restriction factors APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutation during reverse transcription. Vif counteracts A3G at several levels (transcription, translation, and protein degradation) that altogether reduce the levels of A3G in cells and prevent its incorporation into viral particles. How Vif affects A3G translation remains unclear. Here, we uncovered the importance of a short conserved uORF (upstream ORF) located within two critical stem-loop structures of the 5′ untranslated region (5′-UTR) of A3G mRNA for this process. A3G translation occurs through a combination of leaky scanning and translation re-initiation and the presence of an intact uORF decreases the extent of global A3G translation under normal conditions. Interestingly, the uORF is also absolutely required for Vif-mediated translation inhibition and redirection of A3G mRNA into stress granules. Overall, we discovered that A3G translation is regulated by a small uORF conserved in the human population and that Vif uses this specific feature to repress its translation.
The holy grail of structural biology is to study a protein in situ, and this goal has been fast approaching since the resolution revolution and the achievement of atomic resolution. A cell's interior is not a dilute environment, and proteins have evolved to fold and function as needed in that environment; as such, an investigation of a cellular component should ideally include the full complexity of the cellular environment. Imaging whole cells in three dimensions using electron cryotomography is the best method to accomplish this goal, but it comes with a limitation on sample thickness and produces noisy data unamenable to direct analysis. This thesis establishes a novel workflow to systematically analyse whole-cell electron cryotomography data in three dimensions and to find and identify instances of protein complexes in the data to set up a determination of their structure and identity for success. Mycoplasma pneumoniae is a very small parasitic bacterium with fewer than 700 protein-coding genes, is thin enough and small enough to be imaged in large quantities by electron cryotomography, and can grow directly on the grids used for imaging, making it ideal for exploratory studies in structural proteomics. As part of the workflow, a methodology for training deep-learning-based particle-picking models is established.
As a proof of principle, a dataset of whole-cell Mycoplasma pneumoniae tomograms is used with this workflow to characterize a novel membrane-associated complex observed in the data. Ultimately, 25431 such particles are picked from 353 tomograms and refined to a density map with a resolution of 11 Å. Making good use of orthogonal datasets to filter search space and verify results, structures were predicted for candidate proteins and checked for suitable fit in the density map. In the end, with this approach, nine proteins were found to be part of the complex, which appears to be associated with chaperone activity and interact with translocon machinery.
Visual proteomics refers to the ultimate potential of in situ electron cryotomography: the comprehensive interpretation of tomograms. The workflow presented here is demonstrated to help in reaching that potential.
Attention-deficit/hyperactivity disorder (ADHD) is the most prevalent neurodevelopmental disorder described in psychiatry today. ADHD arises during early childhood and is characterized by an age-inappropriate level of inattention, hyperactivity, impulsivity, and partially emotional dysregulation. Besides, substantial psychiatric comorbidity further broadens the symptomatic spectrum. Despite advances in ADHD research by genetic- and imaging studies, the etiopathogenesis of ADHD remains largely unclear. Twin studies suggest a heritability of 70-80 % that, based on genome-wide investigations, is assumed to be polygenic and a mixed composite of small and large, common and rare genetic variants. In recent years the number of genetic risk candidates is continuously increased. However, for most, a biological link to neuropathology and symptomatology of the patient is still missing. Uncovering this link is vital for a better understanding of the disorder, the identification of new treatment targets, and therefore the development of a more targeted and possibly personalized therapy.
The present thesis addresses the issue for the ADHD risk candidates GRM8, FOXP2, and GAD1. By establishing loss of function zebrafish models, using CRISPR/Cas9 derived mutagenesis and antisense oligonucleotides, and studying them for morphological, functional, and behavioral alterations, it provides novel insights into the candidate's contribution to neuropathology and ADHD associated phenotypes. Using locomotor activity as behavioral read-out, the present work identified a genetic and functional implication of Grm8a, Grm8b, Foxp2, and Gad1b in ADHD associated hyperactivity. Further, it provides substantial evidence that the function of Grm8a, Grm8b, Foxp2, and Gad1b in activity regulation involves GABAergic signaling. Preliminary indications suggest that the three candidates interfere with GABAergic signaling in the ventral forebrain/striatum. However, according to present and previous data, via different biological mechanisms such as GABA synthesis, transmitter release regulation, synapse formation and/or transcriptional regulation of synaptic components. Intriguingly, this work further demonstrates that the activity regulating circuit, affected upon Foxp2 and Gad1b loss of function, is involved in the therapeutic effect mechanism of methylphenidate. Altogether, the present thesis identified altered GABAergic signaling in activity regulating circuits in, presumably, the ventral forebrain as neuropathological underpinning of ADHD associated hyperactivity. Further, it demonstrates altered GABAergic signaling as mechanistic link between the genetic disruption of Grm8a, Grm8b, Foxp2, and Gad1b and ADHD symptomatology like hyperactivity. Thus, this thesis highlights GABAergic signaling in activity regulating circuits and, in this context, Grm8a, Grm8b, Foxp2, and Gad1b as exciting targets for future investigations on ADHD etiopathogenesis and the development of novel therapeutic interventions for ADHD related hyperactivity. Additionally, thigmotaxis measurements suggest Grm8a, Grm8b, and Gad1b as interesting candidates for prospective studies on comorbid anxiety in ADHD. Furthermore, expression analysis in foxp2 mutants demonstrates Foxp2 as regulator of ADHD associated gene sets and neurodevelopmental disorder (NDD) overarching genetic and functional networks with possible implications for ADHD polygenicity and comorbidity. Finally, with the characterization of gene expression patterns and the generation and validation of genetic zebrafish models for Grm8a, Grm8b, Foxp2, and Gad1b, the present thesis laid the groundwork for future research efforts, for instance, the identification of the functional circuit(s) and biological mechanism(s) by which Grm8a, Grm8b, Foxp2, and Gad1b loss of function interfere with GABAergic signaling and ultimately induce hyperactivity.
Clostridioides difficile is a bacterial species well known for its ability to cause C. difficile
infection (also known as CDI). The investigation of the role of this species in the human
gut has been so far dominated by a disease-centred perspective, focused on studying
C. difficile in relation to its associated disease.
In this context, the first aim of this thesis was to combine publicly available
metagenomic data to analyse the microbial composition of stool samples from patients
diagnosed with CDI, with a particular focus on identifying a CDI-specific microbial
signature.
However, similarly to many other bacterial species inhabiting the human gut, C.
difficile association with disease is not valid in absolute terms, as C. difficile can be
found also among healthy subjects. Further aims of this thesis were to 1) identify
potential C. difficile reservoirs by screening a wide range of habitats, hosts, body sites
and age groups, and characterize the biotic context associated with C. difficile
presence, and 2) investigate C. difficile within-species diversity and its toxigenic
potential across different age groups.
The first part of the thesis starts with the description of the concepts and
definitions used to identify bacterial species and within-species diversity, and then
proceeds to provide an overview of the bacterial species at the centre of my
investigation, C. difficile. The first Chapter includes a detailed description of the
discovery, biology and physiology of this clinically relevant species, followed by an
overview of the diagnostic protocols used in the clinical setting to diagnose CDI.
The second part of the thesis describes the methodology used to investigate
the questions mentioned above, while the third part presents the results of such
investigative effort. I first show that C. difficile could be found in only a fraction of the
CDI samples and that simultaneous colonization of multiple enteropathogenic species
able to cause CDI-like clinical manifestations is more common than previously
thought, raising concerns about CDI overdiagnosis. I then show that the CDIassociated
gut microbiome is characterized by a specific microbial signature,
distinguishable from the community composition associated with non-CDI diarrhea.
Beyond the nosocomial and CDI context, I show that while rarely found in adults, C.
difficile is a common member of the infant gut microbiome, where its presence is
associated with multiple indicators typical of a desirable healthy microbiome
development.
In addition, I describe C. difficile extensive carriage among asymptomatic
subjects, of all age groups and a potentially novel clade of C. difficile identified
exclusively among infants.
Finally, I discuss the limitations, challenges and future perspectives of my
investigation.
Im Zellkern eukaryotischer Zellen werden Gene in mRNAs transkribiert, welche umfangreich prozessiert und aus dem Zellkern exportiert werden. Im Zytoplasma erfolgt die Translation der mRNAs in Proteine, ein Prozess, welcher viel Energie benötigt und daher mittels vielfältiger Mechanismen streng reguliert wird. Ein Beispiel hierfür stellt die Klasse der TOP-mRNAs dar, eine RNA-Spezies, welche hauptsächlich Transkripte von Genen umfasst, die selbst in die Translation involviert sind. Die prominentesten Vertreter dieser Klasse sind die Proteine der kleinen und großen ribosomalen Untereinheiten. TOP-mRNAs zeichnen sich durch ein gemeinsames Sequenz-Motiv am Anfang Ihrer 5’-UTR aus, welches aus einem Pyrimidinstrang besteht und unmittelbar nach dem Cap mit einem Cytosin beginnt. Dieses allen TOP-RNAs gemeinsame Motiv ermöglicht die zeitgleiche Translationskontrolle dieser RNA-Klasse. So kann die Translation der TOP-mRNAs unter Stressbedingungen wie z.B. Nährstoffmangel koordiniert inhibiert werden, wodurch Energie eingespart wird.
Bereits lange wird nach einem Regulator gesucht, der an dieses TOP-Motiv bindet und die koordinierte Regulation ermöglicht. Man kann sich hier einen Inhibitor oder auch einen Aktivator vorstellen. Verschiedene Proteine wurden bereits in Erwägung gezogen. In dieser Arbeit wurde das Protein TIAR mittels Massenspektrometrie als TOP-interagierender Faktor identifiziert und dessen Bindungseigenschaften mit dem TOP-Motiv durch Shift Assays untersucht. Hierbei konnten Minimalkonstrukte verschiedener Organismen sowie RNA-TOP – Sequenzen identifiziert werden, welche sich für Strukturanalysen eignen würden. Als weiterer TOP-interagierender Faktor wurde über verschiedene sequenzielle Reinigungsschritte das Protein 14-3-3ε identifiziert.
Weiterhin wurden die TOP-Motiv-bindenden Proteine LARP1 und LARP7 auf Ihre Bindungseigenschaften mit Ihren Zielsequenzen untersucht. Während gezeigt werden konnte, dass LARP1 einen inhibierenden Einfluss auf TOP-RNAs hat, wurde in weiteren Shift-Assays die Bindungseigenschaften von LARP7 mit 7SK untersucht, wobei ebenfalls ein minimales LARP7–Konstrukt sowie 7SK-Konstrukte für Strukturanalysen identifiziert werden konnten. Weiterhin konnte gezeigt werden, dass verschiedene Substanzen wie tRNA und Arginin einen starken Einfluss auf die LARP7-7SK – Interaktion ausüben, welcher in weiteren Studien berücksichtigt werden sollte.
Wilms tumor (WT) is the most common renal tumor in childhood. Among others, MYCN copy number gain and MYCN P44L and MAX R60Q mutations have been identified in WT. The proto-oncogene MYCN encodes a transcription factor that requires dimerization with MAX to activate transcription of numerous target genes. MYCN gain has been associated with adverse prognosis. The MYCN P44L and MAX R60Q mutations, located in either the transactivating or basic helix-loop-helix domain, respectively, are predicted to be damaging by different pathogenicity prediction tools. These mutations have been reported in several other cancers and remain to be functionally characterized.
In order to further describe these events in WT, we screened both mutations in a large cohort of unselected WT patients, to check for an association of the mutation status with certain histological or clinical features. MYCN P44L and MAX R60Q revealed frequencies of 3 % and 0.9 % and also were significantly associated to higher risk of relapse and metastasis, respectively. Furthermore, to get a better understanding of the MAX mutational landscape in WT, over 100 WT cases were analyzed by Sanger sequencing to identify other eventual MAX alterations in its coding sequence. R60Q remained the only MAX CDS alteration described in WT to date.
To analyze the potential functional consequences of these mutations, we used a doxycycline-inducible system to overexpress each mutant in HEK293 cells. This biochemical characterization identified a reduced transcriptional activation potential for MAX R60Q, while the MYCN P44L mutation did not change activation potential or protein stability. The protein interactome of N-MYC-P44L was likewise not altered as shown by mass spectrometric analyses of purified N-MYC complexes. However, we could identify a number of novel N-MYC partner proteins, several of these known for their oncogenic potential. Their correlated expression in WT samples suggested a role in WT oncogenesis and they expand the range of potential biomarkers for WT stratification and targeting, especially for high-risk WT.
Der WNT-Signalweg ist ein hochkonservierter Signalweg, dessen zentraler
intrazellulärer Regulationsschritt die Proteinstabilität des Proteins β-Catenin ist.
Deregulierende Mutationen in diesem sind frühe Ereignisse bei der Entstehung von
Darmtumoren. Ist der Abbau von β-Catenin gestört, so ist unabhängig von äußerer
Kontrolle der Signalweg konstitutiv aktiviert und liefert ein Wachstumssignal.
Untersuchungen haben aber gezeigt, dass beim Vorliegen solcher Mutationen immer
noch eine – unzureichende – Ubiquitinylierung und ein Abbau von β-Catenin stattfindet.
Ziel dieser Studie war Deubiquitinasen (DUBs) zu finden, die durch ihre
Aktivität den Abbau von β-Catenin verhindern. Mithilfe eines siRNA Screens in der
Vorarbeit konnten DUBs als Kandidaten für einen CRISPR Ansatz ausgewählt werden.
APC Wildtyp HEK293T Zellen und Darmkrebszellen wurden mit lentiviralen
CRISPR/Cas9 Vektoren infiziert, in welche sgRNAs gegen exonische Sequenzen von
DUBs geklont waren. Einzelne Zellklone von USP10 CRISPR Zellen wurden weiter
untersucht. In Western Blots und Immunofluoreszenz zeigte sich bei den USP10 CRISPR
Zellen eine verminderte Expression von USP10 und damit einhergehend β-Catenin.
Proteinstabilitätsversuche mit MG132 und Cycloheximid zeigten einen erhöhten Abbau
von β-Catenin in HEK293T USP10 CRISPR Zellen, vor allem nach Stimulierung des
WNT-Signalwegs durch LiCl. In Aktivierungsassays (Luciferase und TOP-GFP FACS)
des WNT-Signalwegs zeigte sich in HEK293T Zellen nach Behandlung mit LiCl eine
geringere Aktivierung in den USP10 CRISPR Zellen. In einem Wachstumsassay zeigten
HT29 USP10 CRISPR ein geringeres Wachstum als Kontrollzellen. Während in einer
histologischen Färbung von Mausgewebe eine erhöhte Expression von USP10
nachweisbar war, zeigten sich in einer TMA Färbung kein eindeutiger Unterschied
zwischen gesundem Gewebe und Tumorgewebe.
Die Studie identifiziert USP10 als eine mögliche DUB für β-Catenin und potenzielles
Ziel für eine Beeinflussung des mutierten WNT-Signalwegs in Darmkrebszellen.
The interaction between circadian clocks and metabolism is of increasing interest, since clock dysfunction often correlates with metabolic pathologies. Many research articles have been published analysing the impact of factors such as circadian clock, light, feeding time and diet-type on energy homeostasis in various tissues/organs of organisms with most of the findings done in mammals. Little is known about the impact of circadian clock and the above-mentioned factors on circulating lipids, especially the transport form of lipids - diacylglycerol (DG) and membrane lipids such as phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in the Drosophila hemolymph. The fruit fly Drosophila is a prime model organism in circadian, behaviour and metabolism research.
To study the role of circadian clock and behaviour in metabolism, we performed an extensive comparative hemolymph lipid (diacylglycerol: DG, phosphatidylethanolamine: PE, phosphatidylcholine: PC) analysis using ultra performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC-MS) between wild-type flies (WTCS) and clock disrupted mutants (per01). In addition, clock controlled food intake– feeding behaviour was investigated. Time-dependent variation of transport (DG) and membrane lipids (PE and PC) were not rhythmic in WTCS under constant darkness and in per01 under LD, suggesting an impact of light and clock genes on daily lipid oscillations. Day-time and night-time restriction of food led to comparable lipid profiles, suggesting that lipid oscillations are not exclusively entrained by feeding but rather are endogenously regulated. Ultradian oscillations in lipid levels in WTCS under LD were masked by digested fatty acids since lipid levels peaked more robustly at the beginning and end of light phase when flies were fed a lipid- and protein-free diet. These results suggest that metabolite (DG, PE and PC) oscillation is influenced by complex interactions between nutrient-type, photic conditions, circadian clock and feeding time.
In conclusion, the results of this thesis suggest that circadian clocks determine transport and membrane lipid oscillation in Drosophila hemolymph in complex interactions between nutrient-type, photic conditions and feeding behaviour.
With the technological advances of the last decade, it is now feasible to analyze microbiome samples, such as human stool specimens, using multi-omic techniques. Given the inherent sample complexity, there exists a need for sample methods which preserve as much information as possible about the biological system at the time of sampling. Here, we analyzed human stool samples preserved and stored using different methods, applying metagenomics as well as metaproteomics. Our results demonstrate that sample preservation and storage have a significant effect on the taxonomic composition of identified proteins. The overall identification rates, as well as the proportion of proteins from Actinobacteria were much higher when samples were flash frozen. Preservation in RNAlater overall led to fewer protein identifications and a considerable increase in the share of Bacteroidetes, as well as Proteobacteria. Additionally, a decrease in the share of metabolism-related proteins and an increase of the relative amount of proteins involved in the processing of genetic information was observed for RNAlater-stored samples. This suggests that great care should be taken in choosing methods for the preservation and storage of microbiome samples, as well as in comparing the results of analyses using different sampling and storage methods. Flash freezing and subsequent storage at −80 °C should be chosen wherever possible.
Im Rahmen dieser Arbeit wurde zur Untersuchung der Rolle von PCGF6 und E2F6 in murinen embryonalen Stammzellen (mESCs) und zu Beginn der Differenzierung Knockout-Zelllinien beider Proteine und in Kombination durch das CRISPR/Cas9n Systems erstellt. Die Charakterisierung dieser Knockout-Zelllinien erfolgte durch Wachstumsanalysen in mESCs und differenzierenden murinen Stammzellen (EBs). Es konnte festgestellt werden, dass Zellen des Pcgf6 Knockout (KO) kleinere Ebs bildeten, die zudem nicht über einen längeren Zeitraum in Kultur gehalten werden konnten. Zur Klärung dieses spezifischen Phänotyps wurden weitere molekulare Analysen mittels Durchflusszytometrie durchgeführt. Zellen des Pcgf6 KO wiesen während der Differenzierung einen erhöhten Anteil an Zellen in der G1-Phase sowie eine erhöhte apoptotische Frequenz auf. Unterstützend zur Annahme eines Zellzyklusdefekts wurden RNASeq-Daten analysiert. Die Auswertung ergab, dass Zellen des Pcgf6 KO zeitlich unkontrolliert differenzierten. Die Auswertung differenziell exprimierter Gene ergab zudem, dass die Expression von E2f6, ein Regulator des Zellzyklus und weitere Untereinheit des nicht-kanonischen PRC1.6, in mESC und EB-Kulturen herunter reguliert war, während Zellzyklus-spezifische Targets der E2F6-abhängigen Genregulation an Tag 2 der Differenzierung hochreguliert waren. Diese Ergebnisse deuteten darauf hin, dass eine Deletion von Pcgf6 zu Beginn der Differenzierung Auswirkungen auf eine E2F6-abhängige Zellzyklusregulation haben muss. Auf Grund einer zu diesem Zeitpunkt aufgetretenen Mykoplasmenkontamination in der Zellkultur musste die Pcgf6 KO-Zelllinie neu erstellt werden. Zusätzlich wurden KO-Zelllinien von E2f6 in Wt und in Pcgf6 KO mESCs erstellt. Die anschließende Wiederholung der zellulären Charakterisierung des Phänotyps ergab, dass EB-Kulturen des Pcgf6 KO und des Doppelknockout von Pcgf6 und E2f6 (dKOPcgf6/E2f6) während der Differenzierung eine verringerte Zellzahl aufwiesen. Die molekularen Charakterisierungen des Phänotyps ergaben, dass der erhöhte Anteil an Zellen in der G1-Phase des Pcgf6 KO, welche vor der Mykoplasmenkontamination detektiert wurde, nicht reproduziert werden konnte. Es wurde jedoch eine erhöhte Frequenz an Zellen in der G2-Phase des dKOPcgf6/E2f6 in der mESC-und EB-Kultur ermittelt. Die Analyse der apoptotischen Frequenz in allen KO-Zelllinien zeigte einen Anstieg während der Differenzierung. Zur Unterstützung der bis dahin durchgeführte Analysen wurden RNASeq-Daten zweier Publikationen zu PCGF6 und E2F6 herangezogen (Qui et al., 2021; Dahlet et al, 2021). Gene Ontology Enrichtment Analysen dieser Daten ergaben, dass in beiden KO-Zelllinien in mESCs unabhängig voneinander Keimbahngene hochreguliert waren. Beide KO-Zelllinien zeigten aber auch eine Schnittmenge gemeinsam hochregulierter Keimbahngene. In Anlehnung an diese Veröffentlichungen, ergaben Genexpressionsanalysen einzelner Keimbahngene, dass ein Verlust von E2f6 zu einer De-Repression von Genen führt, die eine Bindestelle für E2F6 besitzen. Der Verlust von Pcgf6 hingegen hatte keine Auswirkung auf Expression dieser Targets. Diese Ergebnisse unterstützen die Vermutung, dass es unterschiedliche Subkomplexe gibt, die die Expression von Keimbahngenen in mESC- und EB-Kulturen regulieren.
According to the “canonical” paradigm of GPCR signaling, agonist-bound GPCRs only signal to the downstream adenylyl cyclase enzyme when they are seated at the plasma membrane. Upon prolonged binding of an agonist, receptor internalization usually takes place, leading to the termination of this downstream signaling pathway and activation of alternative ones. However, a set of recent studies have shown that at least some GPCRs (e.g. thyroid stimulating hormone receptor) continue signaling to adenylyl cyclase after internalization. In this study, I aimed to investigate canonical signaling by internalized μ opioid receptors (MORs), which are Gi-coupled receptors, using a fluorescence resonance energy transfer (FRET) sensor for cyclic AMP (cAMP) known as Epac1-camps. My results show that the cyclic AMP inhibition signal induced by the binding of DAMGO, a MOR agonist, persists after agonist washout. We hypothesized that this persistent signal might come from internalized DAMGO-bound receptors located in the endosomal compartment. To test this hypothesis, I used dynasore and Dyngo 4a, two dynamin inhibitors that are known to prevent clathrin-mediated endocytosis. Interestingly, dynasore but not Dyngo 4a pretreatment largely blunted the response to MOR activation as well as to adenylyl cyclase activation with Forskolin (FSK). In addition, DAMGO-induced cAMP signal remained persistent even in the presence of 30 M Dyngo 4a. These results might point to a complex interplay between clathrin-mediated internalization and MOR signaling. Further experiments are required to elucidate the mechanisms underlying the persistent MOR signaling and to fully clarify whether MORs are capable of Gi signaling in the endosomal compartment.
Die Entwicklung des Schädeldachs beginnt beim Menschen bereits in der frühen Embryogenese und ist erst im Erwachsenenalter abgeschlossen. Das Wachstum der Schädelknochen muss sich während der Entwicklung fortwährend dem Gehirnwachstum anpassen. An den Stellen, wo zwei Schädelknochen aufeinandertreffen, formen sich Schädelnähte, die aus mesenchymalem Bindegewebe bestehen und als Wachstumsfugen des Schädels dienen. Tritt eine frühzeitige Verknöcherung innerhalb einer oder mehrerer Schädelnähte auf, spricht man von einer Kraniosynostose. Als Konsequenz wird ein weiteres Knochenwachstum verhindert, sodass sich das Neurokranium in dieser Region nicht dem expansiven Wachstum des Gehirns anpassen kann. Dies geht in der Regel mit einem kompensatorischen Wachstum des Schädels und infolgedessen mit kraniofazialen Dysmorphien und einem erhöhten intrakraniellen Druck einher. Klinische Studien und Forschungen an Modellorganismen konnten bereits eine Vielzahl an Genen mit der Entstehung von Kraniosynostosen assoziieren, darunter die Transkriptionsfaktoren TCF12 und TWIST1. Beim Menschen sind heterozygote Mutationen in TCF12 und TWIST1 mit Kraniosynostosen der Koronarnaht assoziiert. Bei Mäusen hingegen führt eine heterozygote Tcf12 Mutation nur in Kombination mit einer heterozygoten Twist1 Mutation zu Fusionen der Koronarnaht.
Der Zebrabärbling (Danio rerio, überwiegend auch Zebrafisch genannt) weist eine bemerkenswerte Ähnlichkeit bezüglich der Anatomie und Morphologie des Schädeldachs zum Menschen auf. Um die genaue Funktion von TCF12 bei der Ausbildung der Schädelnähte zu untersuchen, wurde im Rahmen dieser Arbeit der Zebrafisch als in vivo Modell für die Entstehung tcf12-induzierter Kraniosynostosen etabliert. Zu Beginn der Arbeit wurde das Expressionsmuster von tcf12 über die Entwicklung hinweg analysiert. Ein besonderer Fokus lag dabei auf einem Expressionsnachweis während der Entwicklung der Schädelplatten und der Schädelnähte. Ein erster Expressionsnachweis von tcf12 mittels PCR-Analysen und Whole-mount RNA in-situ Hybridisierungen zeigte eine breite Expression von tcf12 ab dem 1-3 Somiten Stadium an. Für tiefergehende in vivo Analysen wurden im Zuge dieser Arbeit tcf12:EGFP Reportergenlinien generiert. Mit diesen gelang ein Nachweis der tcf12 Expression entlang der Wachstumsfronten der Schädelplatten, innerhalb der Schädelnähte sowie im Periost und der Dura mater.
Mit den tcf12:EGFP Fischen als Referenz wurde in weiterführenden Experimenten die Aktivität drei hochkonservierter CNEs (engl. conserved non-coding elements) in vivo im Zebrafisch untersucht. Zwei der CNEs konnten als tcf12 Enhancer verifiziert werden, die eine Genexpression während der Neurogenese des zentralen Nervensystems (ZNS) steuern. Die beiden Enhancer-Elemente zeichnen sich durch eine hohe Konservierung vom Menschen bis hin zum Zebrafisch aus.
Aufgrund der unterschiedlichen Sensitivität gegenüber einem Funktionsverlust von TCF12 und TWIST1 in Mensch und Maus sollte die Auswirkung eines Knockouts der orthologen Gene auf die Entwicklung der Schädelnähte des Zebrafisches untersucht werden. Mittels CRISPR/Cas9 wurden verschiedene Knockout-Linien für die Gene tcf12, twist1a und twist1b generiert. Analysen der Knockoutmutanten zeigten, dass ein heterozygoter Verlust von tcf12 und twist1b in seltenen Fällen zu partiellen Fusionen der Koronarnähte im Zebrafisch führt. Des Weiteren konnte bei tcf12 und twist1b Einzel- und Doppelmutanten ein abnormes Wachstum der Schädelplatten im Bereich der Suturen beobachtet werden. Die Expressionsstudien und die Analysen der Knockoutmutanten deuten auf eine Regulation von TCF12 bei der Differenzierung der Stammzellen sowie der Proliferation der Osteoblasten innerhalb der Schädelnähte hin.
Um die Auswirkung von TCF12 Mutationen auf funktioneller Ebene zu untersuchen wurden im Verlauf dieser Arbeit Luciferase-Reporter Assays durchgeführt. Anhand dieser konnte nachgewiesen werden, dass Mutationen, die die basic helix-loop-helix (bHLH)-Domäne beeinträchtigen, die Transaktivierungsfähigkeit von TCF12 aufheben. Co-Transfektions-Experimente mit TWIST1 offenbarten eine Regulation der Transaktivierung von TCF12 durch TWIST1, sowohl im Menschen, als auch im Zebrafisch. Im Rahmen dieser Arbeit konnten die genauen Expressionsorte von TCF12 während der Morphogenese des Schädeldachs nachgwiesen und die Funktion von TCF12 und seinem Interaktionspartner TWIST1 bei der Entstehung von Kraniosynostosen weiter aufgeklärt werden.
Neisseria gonorrhoeae (GC) is a human specific pathogenic bacterium. Currently, N. gonorrhoeae developed resistance to virtually all the available antibiotics used for treatment. N. gonorrhoeae starts infection by colonizing the cell surface, followed by invasion of the host cell, intracellular persistence, transcytosis and exit into the subepithelial space. Subepithelial bacteria can reach the bloodstream and disseminate to other tissues causing systemic infections, which leads to serious conditions such as arthritis and pneumonia. A number of studies have well established the host-pathogen interactions during the initial adherence and invasion steps. However, the mechanism of intracellular survival and traversal is poorly understood so far. Hence, identification of novel bacterial virulence factors and host factors involved in the host-pathogen interaction is a crucial step in understanding disease development and uncovering novel therapeutic approaches. Besides, most of the previous studies about N. gonorrhoeae were performed in the conventional cell culture. Although they have provided insights into host-pathogen interactions, much information about the native infection microenvironment, such as cell polarization and barrier function, is still missing.
This work focused on determining the function of novel bacterial virulence factor NGFG_01605 and host factor (FLCN) in gonococcal infection. NGFG_01605 was identified by Tn5 transposon library screening. It is a putative U32 protease. Unlike other proteins in this family, it is not secreted and has no ex vivo protease activity. NGFG_01605 knockout decreases gonococcal survival in the epithelial cell. 3D models based on T84 cell was developed for the bacterial transmigration assay. NGFG_01605 knockout does not affect gonococcal transmigration.
The novel host factor FLCN was identified by shRNA library screening in search for factors that affected gonococcal adherence and/or internalization. We discovered that FLCN did not affect N. gonorrhoeae adherence and invasion but was essential for bacterial survival. Since programmed cell death is a host defence mechanism against intracellular pathogens, we further explored apoptosis and autophagy upon gonococcal infection and determined that FLCN did not affect apoptosis but inhibited autophagy. Moreover, we found that FLCN inhibited the expression of E-cadherin. Knockdown of E- cadherin decreased the autophagy flux and supported N. gonorrhoeae survival. Both non-polarized and polarized cells are present in the cervix, and additionally, E-cadherin represents different polarization properties on these different cells. Therefore, we established 3-D models to better understand the functions of FLCN. We discovered that FLCN was critical for N. gonorrhoeae survival in the 3-D environment as well, but not through inhibiting autophagy. Furthermore, FLCN inhibits the E-cadherin expression and disturbs its polarization in the 3-D models. Since N. gonorrhoeae can cross the epithelial cell barriers through both cell-cell junctions and transcellular migration, we further explored the roles FLCN and E-cadherin played in transmigration. FLCN delayed N. gonorrhoeae transmigration, whereas the knockdown of E-cadherin increased N. gonorrhoeae transmigration.
In summary, we revealed roles of the NGFG_01605 and FLCN-E-cadherin axis play in N. gonorrhoeae infection, particularly in relation to intracellular survival and transmigration. This is also the first study that connects FLCN and human-specific pathogen infection.
The importance of understanding species extinctions and its consequences for ecosystems and human life has been getting increasing public attention.
Nonetheless, regardless of how pressing the current biodiversity loss is, with rare exceptions, extinctions are actually not immediate.
Rather, they happen many generations after the disturbance that caused them.
This means that, at any point in time after a given disturbance, there is a number of extinctions that are expected to happen.
This number is the extinction debt.
As long as all the extinctions triggered by the disturbance have not happened, there is a debt to be paid.
This delay in extinctions can be interpreted as a window of opportunity, when conservation measures can be implemented.
In this thesis, I investigated the relative importance of ecological and evolutionary processes unfolding after different disturbances scenarios, to understand how this knowledge can be used to improve conservation practices aiming at controlling extinctions.
In the Introduction (chapter 1), I present the concept of extinction debts and the complicating factors behind its understanding.
Namely, I start by presenting i) the theoretical basis behind the definition of extinction debts, and how each theory informed different methodologies of study, ii) the complexity of understanding and predicting eco-evolutionary dynamics, and iii) the challenges to studying extinctions under a regime of widespread and varied disturbance of natural habitats.
I start the main body of the thesis (chapter 2) by summarizing the current state of empirical, theoretical, and methodological research on extinction debts.
In the last 10 years, extinction debts were detected all over the globe, for a variety of ecosystems and taxonomic groups.
When estimated - a rare occurrence, since quantifying debts requires often unavailable data - the sizes of these debts range from 9 to 90\% of current species richness and they have been sustained for periods ranging from 5 to 570 yr.
I identified two processes whose contributions to extinction debts have been studied more often, namely 1) life-history traits that prolong individual survival, and 2) population and metapopulation dynamics that maintain populations under deteriorated conditions.
Less studied are the microevolutionary dynamics happening during the payment of a debt, the delayed conjoint extinctions of interaction partners, and the extinction dynamics under different regimes of disturbances (e.g. habitat loss vs. climate change).
Based on these observations, I proposed a roadmap for future research to focus on these less studies aspects.
In chapters 3 and 4, I started to follow this roadmap.
In chapter 3, I used a genomically-explicit, individual-based model of a plant community to study the microevolutionary processes happening after habitat loss and climate change, and potentially contributing to the settlement of a debt.
I showed that population demographic recovery through trait adaptation, i.e. evolutionary rescue, is possible.
In these cases, rather than directional selection, trait change involved increase in trait variation, which I interpreted as a sign of disruptive selection.
Moreover, I disentangled evolutionary rescue from demographic rescue and show that the two types of rescue were equally important for community resistance, indicating that community re-assembly plays an important role in maintaining diversity following disturbance.
The results demonstrated the importance of accounting for eco-evolutionary processes at the community level to understand and predict biodiversity change.
Furthermore, they indicate that evolutionary rescue has a limited potential to avoid extinctions under scenarios of habitat loss and climate change.
In chapter 4, I analysed the effects of habitat loss and disruption of pollination function on the extinction dynamics of plant communities.
To do it, I used an individual, trait-based eco-evolutionary model (Extinction Dynamics Model, EDM) parameterized according to real-world species of calcareous grasslands.
Specifically, I compared the effects of these disturbances on the magnitude of extinction debts and species extinction times, as well as how species functional traits affect species survival.
I showed that the loss of habitat area generates higher number of immediate extinctions, but the loss of pollination generates higher extinction debt, as species take longer to go extinct.
Moreover, reproductive traits (clonal ability, absence of selfing and insect pollination) were the traits that most influenced the occurrence of species extinction as payment of the debt.
Thus, the disruption of pollination functions arose as a major factor in the creation of extinction debts.
Thus, restoration policies should aim at monitoring the status of this and other ecological processes and functions in undisturbed systems, to inform its re-establishment in disturbed areas.
Finally, I discuss the implications of these findings to i) the theoretical understanding of extinction debts, notably via the niche, coexistence, and metabolic theories, ii) the planning conservation measures, including communicating the very notion of extinction debts to improve understanding of the dimension of the current biodiversity crisis, and iii) future research, which must improve the understanding of the interplay between extinction cascades and extinction debts.