Refine
Has Fulltext
- yes (102)
Is part of the Bibliography
- yes (102)
Year of publication
- 2023 (102) (remove)
Document Type
- Doctoral Thesis (102)
Language
- English (102) (remove)
Keywords
- Thrombozyt (5)
- Tissue Engineering (5)
- Angststörung (4)
- Biene (4)
- Funktionelle Kernspintomografie (4)
- Zellzyklus (4)
- Maus (3)
- Mikroskopie (3)
- Platelet (3)
- Small RNA (3)
- Zellskelett (3)
- fMRI (3)
- platelets (3)
- ADHD (2)
- Alzheimerkrankheit (2)
- Aufmerksamkeit (2)
- Biofabrication (2)
- Biomarker (2)
- Brain-derived neurotrophic factor (2)
- CRISPR/Cas-Methode (2)
- CRISPR/Cas9 (2)
- Campylobacter jejuni (2)
- Cement (2)
- Cytoskeleton (2)
- Expansion Microscopy (2)
- Expositionstherapie (2)
- Fibromyalgie (2)
- Gephyrin (2)
- Immunologie (2)
- Immunsuppression (2)
- Induzierte pluripotente Stammzelle (2)
- Inhibitorische Synapse (2)
- Inhibitory synapse (2)
- Kognition (2)
- Krebs <Medizin> (2)
- MMB (2)
- Monarchfalter (2)
- Orientierung (2)
- Peptidsynthese (2)
- Pflanzen (2)
- Regulatorischer T-Lymphozyt (2)
- Ribosom (2)
- SPECT (2)
- Spinale Muskelatrophie (2)
- Zement (2)
- bee (2)
- division of labor (2)
- gephyrin (2)
- honeybee (2)
- 18S rRNA (1)
- 3 D bioprinting (1)
- 3D Gewebemodelle (1)
- 3D Printing (1)
- 3D cell culture (1)
- 3D tissue model (1)
- 3D-Druck (1)
- 3D-Zellkultur (1)
- ATR-FTIR (1)
- Actin cytoskeleton-related protein (1)
- Adjuvans (1)
- Adrenocortical Carcinoma (1)
- Affinity probe (1)
- Agoraphobie (1)
- Aktivierungsenergie (1)
- Akzeptanz (1)
- Akzeptanz- und Commitment Therapie (1)
- Allogenic hematopoietic stem cell transplantation (1)
- Altern (1)
- Alzheimer's Dementia (1)
- Alzheimer's disease (1)
- AmGr1, AmGr2, AmGr3 (1)
- Amygdala (1)
- Angst (1)
- Angst als Zustand (1)
- Angsterkrankung (1)
- Animal model (1)
- Anxiety disorder (1)
- Anxiety disorders (1)
- Apis mellifera (1)
- Apoptosis (1)
- Atemwege (1)
- Atemwegsschleimhaut (1)
- Aufmerksamkeitsdefizit-Syndrom (1)
- Autoantikörper (1)
- Autoinhibition (1)
- B-MYB (1)
- B-Zell-Lymphom (1)
- BDNF stimulation (1)
- BOLD signal (1)
- BRET (1)
- Bacterial infection (1)
- Baghdadite (1)
- Bakterielle Infektion (1)
- Bandwürmer (1)
- Bauchspeicheldrüse (1)
- Bauchspeicheldrüsenkrebs (1)
- Bed nucleus of stria terminalis (1)
- Benzimidazolderivate (1)
- Bestärkendes Lernen <Künstliche Intelligenz> (1)
- Bildgebendes Verfahren (1)
- Bimolekulare Lipidschicht (1)
- Bindegewebe (1)
- Bioinformatics (1)
- Biologie (1)
- Biomechanics (1)
- Biomechanik (1)
- Bioreactor (1)
- Blick (1)
- Blood nerve barrier (1)
- Blut-Hirn-Schranke (1)
- Bone-replacement (1)
- Bortezomib (1)
- Brownsche Bewegung (1)
- CD28 Superagonist (1)
- CIDP (1)
- CMV (1)
- CVT (1)
- Calcium Phosphate (1)
- Calciumphosphat (1)
- Cell adhesion (1)
- Cell cycle (1)
- Characterization (1)
- Chromatin (1)
- Circadian (1)
- Clostridium difficile (1)
- Comorbidity (1)
- Contactin-1 (1)
- Corpus amygdaloideum (1)
- Correlative microscopy (1)
- Coxiella burnetii (1)
- Crosstalk (1)
- Cytomegalie-Virus (1)
- Cytotoxic T cell (1)
- DHX30 (1)
- DNS-Reparatur (1)
- DNS-Schädigung (1)
- Danaus plexippus (1)
- Deep-sequencing (1)
- Dephosphorylierung (1)
- Depression (1)
- Diabetic painful neuropathy (1)
- Diagnostik (1)
- Diffusion coefficient (1)
- Dorsal root ganglion (1)
- Dual-setting (1)
- Dynamics of ribosome assembly (1)
- Dünndarm (1)
- ER dynamics in axon terminals (1)
- Echtzeit (1)
- Ecological Momentary Assessment (1)
- Einfühlung (1)
- Einfühlung <Motiv> (1)
- Einsamkeit (1)
- Einzelzellanalyse (1)
- Elektrophysiologie (1)
- Emotionsregulation (1)
- Endocytose (1)
- Endocytosis (1)
- Endophänotypen (1)
- Endoplasmatisches Retikulum (1)
- Endosomes (1)
- Endothel (1)
- Endothelium (1)
- Enterobacteriaceae (1)
- Entzündung (1)
- Epigenetik (1)
- Erregbarkeit (1)
- Excitatory/inhibitory imbalance (1)
- Exposure treatment (1)
- Extinktion (1)
- Extrazellulärmatrix (1)
- FAT10ylation (1)
- FOXP2 (1)
- FRET (1)
- Fabry disease (1)
- Fabry-Krankheit (1)
- Fibromyalgia syndrome (1)
- Fibromyalgiesyndrom (1)
- Flavonoids (1)
- FluidFM (1)
- Fluorescence Microscopy (1)
- Fluorescence microscopy (1)
- Fluorescent probes (1)
- Fluoreszenz-Resonanz-Energie-Transfer (1)
- Fluoreszenzmikroskopie (1)
- Fluoreszenzsonde (1)
- Forkhead Transcription Factors (1)
- Forkhead-Box-Proteine (1)
- FoxO transcription factors (1)
- Freies Molekül (1)
- Fructosebisphosphat-Aldolase (1)
- Frühe Gene (1)
- Fusobacterium nucleatum (1)
- Förster Resonanz Energie Transfer (1)
- G-Protein gekoppelter Rezeptor (1)
- G2/M genes (1)
- GAD1 (1)
- GC1 cells (1)
- GLA KO mouse model (1)
- GPCR (1)
- GRM8 (1)
- Galectin 1 (1)
- Galectine (1)
- Ganzkörperbestrahlung (1)
- Gas chromatography (1)
- Gaschromatographie (1)
- Gehirn (1)
- Genetic etiology (1)
- Genexpression (1)
- Genomics (1)
- Genotyping (1)
- Genregulation (1)
- Gewebemodell (1)
- Gewebemodelle (1)
- Glioblastoma (1)
- Glucocorticoids (1)
- Glycocalyx (1)
- Glykobiologie (1)
- Glykokalyx (1)
- Graft-versus-host-disease (1)
- Graft-versus-leukemia (1)
- Gram-positive (1)
- Granulozyten (1)
- Guanine nucleotide exchange factor (GEF) (1)
- HASH (1)
- HCMV (1)
- HPLC-MS (1)
- Habituationstraining (1)
- Hautleitfähigkeit (1)
- Helicobacter pylori (1)
- Herpes (1)
- Herzfrequenz (1)
- Herzfunktion (1)
- Herzinfarkt (1)
- Herzmuskel (1)
- Heterogenität von Mikroorganismen (1)
- Hfq (1)
- Homing (1)
- Humanparasitologie (1)
- Hybrid-Molecules (1)
- Hyperactivity (1)
- Hypothalamus (1)
- IE3 (1)
- IL-2 (1)
- IP6 (1)
- Ibrutinib (1)
- Image Quality (1)
- Immune Checkpoint Therapy (1)
- Immune System (1)
- Immunkardiologie (1)
- Immunocardiology (1)
- Immunotherapy (1)
- Immunsystem (1)
- Immuntherapie (1)
- Impulsivität (1)
- In-vitro-Kultur (1)
- Induced pluripotent stem cells (1)
- Infection models (1)
- Infektionsmodell (1)
- Infektionsstudien (1)
- Inhibitor (1)
- Inhibitory-postsynapse (1)
- Injectability (1)
- Insekten (1)
- Integrin (1)
- Interaktion (1)
- Interleukin 2 (1)
- Isolation and Characterization (1)
- K-Ras (1)
- Kardiologie (1)
- Keratinozyt (1)
- Kernspintomografie (1)
- Knochenersatz (1)
- Knochenmark (1)
- Knochenmarktransplantation (1)
- Kolloid (1)
- Komorbidität (1)
- Korrelative Mikroskopie (1)
- Krebsforschung (1)
- Kutikula (1)
- Lebensmittelchemie (1)
- Library of Phytochemicals (1)
- Library of plant species (1)
- Lidschlag (1)
- Locomotor activity (1)
- Lungenkrebs (1)
- Lysosome (1)
- MAP-Kinase (1)
- MCMV (1)
- MEK5/ERK5 cascade (1)
- MYC (1)
- Macrophages (1)
- Makrophage (1)
- Malignant melanoma (1)
- Mantle cell lymphoma (1)
- Maschinelles Lernen (1)
- Massenspektrometrie (1)
- Masspectrometry (1)
- Megakaryozyt (1)
- Melanom (1)
- Melt electrowriting (1)
- Metabolic Glycoengineering (1)
- Microscopy (1)
- Microwave Assisted Extraction (1)
- Mikrotubuli (1)
- Mikrotubuli-assoziiertes Protein (MAP) (1)
- Mitose (1)
- Modellierung (1)
- Moderator (1)
- Modification (1)
- ModulationTregs (1)
- Molekulare Bildgebung (1)
- Morbus Fabry (1)
- Motoneuron (1)
- Motoneuron-Krankheit (1)
- Multi-Unit Aufnahmen (1)
- Multidrugresistant (1)
- Multilayered skin tissue model (1)
- Myc (1)
- Myeloid-derived suppressor cells (1)
- Myocardial infarction (1)
- Myosin IIA (1)
- NA (1)
- NEDMIAL (1)
- NFATc1 (1)
- NaV1.9 (1)
- Narrow escape problem (1)
- Natriumkanal (1)
- Nature-Insipired Synthesis (1)
- Naturstoff (1)
- Nebenniere (1)
- Nebennierenrindenkarzinom (1)
- Nebennierentumor (1)
- Nervenstimulation (1)
- Neurodegeneration (1)
- Neurodevelopmental diseases (1)
- Neuroepigenomics (1)
- Neurofascin (1)
- Neuromodulation (1)
- Neuropathic pain (1)
- Neuroprotection (1)
- Neuroscience (1)
- Neurowissenschaften (1)
- Neutrophils (1)
- Nicht-kleinzelliges Bronchialkarzinom (NSCLC) (1)
- Nociceptor (1)
- Non-coding RNA (1)
- Olfaktorik (1)
- Onchocerca volvulus (1)
- Onchozerkose (1)
- Oncogenes (1)
- Optogenetics (1)
- Optogenetik (1)
- Organische Synthese (1)
- Oxytocin (1)
- PAR-CLIP (1)
- PEG (1)
- PER (1)
- PTH1R (1)
- Panikstörung (1)
- Pankreas (1)
- Parasitology (1)
- Parkinson's disease (1)
- Parkinson-Krankheit (1)
- Pathogenesis (1)
- Pathogenität (1)
- Pathophysiologie (1)
- Peptide (1)
- Peripheral eosinophils (1)
- Peripheres Nervensystem (1)
- Pesticide (1)
- Phosphoglykolatphosphatase (1)
- Phospholipide (1)
- Phylogenie (1)
- Phylogeny (1)
- Plant extracts (1)
- Plants (1)
- Plastin 3 (1)
- Platelets (1)
- Polymere (1)
- Polymers (1)
- Polyneuropathie (1)
- Prognose (1)
- Programmed Cell Death 1 (1)
- Programmed Cell Death Ligand 1 (1)
- Proliferation (1)
- Prosoziales Verhalten (1)
- Proteasom (1)
- Proteasome (1)
- Pseudouridin (1)
- RAMP (1)
- RIL-seq (1)
- RNA helicase (1)
- RNA metabolism (1)
- RNA modifications (1)
- RNA-Protein Interaktom (1)
- RNA-RNA interactions (1)
- RNA-Sequenzierung (1)
- RNA-bindendes Protein (1)
- RNA-protein interactome (1)
- RNS-Bindungsproteine (1)
- RNS-Viren (1)
- Random Forest (1)
- Random-walk simulations (1)
- Ranvier-Schnürring (1)
- Rbm8a (1)
- Reaktive Sauerstoffspezies (1)
- Regenerative Medizin (1)
- Regulation (1)
- Regulatory T Cells (1)
- Rekonsolidierung (1)
- Reproducibility challenges (1)
- Reprogrammming (1)
- Rezeptor (1)
- Rezeptor-Tyrosinkinasen (1)
- Rheology (1)
- Rho GTPasw (1)
- Rodents (1)
- SARS-CoV-2 (1)
- SPECT/CT (1)
- SUMOylation (1)
- Salmonella (1)
- Satellite glial cell (1)
- Scherstress (1)
- Schlaf (1)
- Schlaganfall (1)
- Schmerz (1)
- Schmerzforschung (1)
- Schmetterlinge (1)
- Sehen (1)
- Sekundärkrankheit (1)
- Sequence-Structure (1)
- Sequenzdaten (1)
- Serotonin (1)
- Setting Control (1)
- Shear Stress (1)
- Silica precursor (1)
- Single-cell RNA-sequencing (1)
- Sinusthrombose (1)
- Skin Tissue Engineering (1)
- Sleep (1)
- Social Buffering (1)
- Social Distancing (1)
- Sol-gel (1)
- Solid-phase peptide synthesis (1)
- Sozialer Kontakt (1)
- Specialized pro resolving mediators (1)
- Spinal Muscular Atrophy (1)
- Spinalganglion (1)
- Spo0A (1)
- Sporenbildung (1)
- Sporulation (1)
- Steroide (1)
- Steroidhormon (1)
- Strumpellin (1)
- Super-Resolution Microscopy (1)
- Synthese (1)
- Synthesis (1)
- T Helper Cell (1)
- T Lymphocyte (1)
- TLR3 (1)
- TRRAP (1)
- Tagesrhythmus (1)
- Targeted therapy (1)
- Temporal predictability (1)
- Theory of Mind (1)
- Therapie (1)
- Therapieresistenz (1)
- Thigmotaxis (1)
- Thrombose (1)
- Thrombosis (1)
- Tissue staining (1)
- Transkription <Genetik> (1)
- Transkriptomanalyse (1)
- Traumatic neuropathy (1)
- Treg (1)
- Trem2 (1)
- TruD (1)
- Trypanosoma (1)
- Trypanosoma brucei (1)
- Trypanosomiasis (1)
- Tumor (1)
- UBA6 (1)
- UBE2Z (1)
- Ubiquitylation (1)
- Ultraschall (1)
- VLA-1 (1)
- Vasopressin (1)
- Verwundbarkeit (1)
- Virus infection (1)
- Visuelle Orientierung (1)
- Visuelle Wahrnehmung (1)
- Vorhersage (1)
- WASH complex (1)
- Wahrnehmung (1)
- Waiting Impulsivity (1)
- Web services (1)
- YAP (1)
- Zebrafish (1)
- Zelldifferenzierung (1)
- Zellkern (1)
- Zellmigration (1)
- Zellteilung (Zytokinese) (1)
- acceptance-based strategies (1)
- adrenal incidentaloma (1)
- adrenocortical adenoma (1)
- adrenocortical carcinoma (1)
- aging (1)
- allografts (1)
- alpha-IIb beta-3 (1)
- amygdala (1)
- anaerobe (1)
- anterior insula (1)
- anxiety disorders (1)
- artificial human skin (1)
- attention (1)
- bee-lining (1)
- benzimidazole (1)
- beta cell (1)
- biofabrication (1)
- bioink (1)
- biology (1)
- bitter taste (1)
- blood brain barrier (1)
- bone marrow (1)
- brain (1)
- calcium activity (1)
- cell migration (1)
- central complex (1)
- chromatin accessibility (1)
- cine loop (1)
- closing of chromatin (1)
- cognitive decline (1)
- collybistin (1)
- conformational activation (1)
- developmental differentiation (1)
- dexamethasone suppression test (1)
- diet (1)
- differential RNA-seq (1)
- drift-diffusion model (1)
- early detection (1)
- electrophysiology (1)
- endosomal trafficking (1)
- enhancers (1)
- exposure therapy (1)
- extinction (1)
- extracellular matrix (1)
- eye contact (1)
- fMRI time series (1)
- feral bees (1)
- fluorescence resonance energy transfer (FRET) (1)
- fmri activity (1)
- forager (1)
- forest landscape (1)
- functional neuroimaging (1)
- functional resting-state connectivity (1)
- funktionelle Kernspintomographie (1)
- funktionelle Magnetresonanztomographie (1)
- funktionelle Resting-State Konnektivität (1)
- genetic modification (1)
- genetic screen (1)
- haloacid dehalogenase phosphatase (1)
- honey bee density (1)
- hyaluronic acid (1)
- hydrogel (1)
- iPSCs (1)
- immediate early genes (1)
- immune evasion (1)
- inferior frontal gyrus (1)
- inflammatory pain (1)
- inhibitory postsynapse (1)
- innate immunity (1)
- ischemic stroke (1)
- juvenile hormone (1)
- kinase (1)
- late enhancement (1)
- late gadolinium-enhancement (1)
- luciferase assay (1)
- lytic infection (1)
- macrophages (1)
- magnetic resonance imaging (1)
- malnourishment (1)
- mean first passage time (1)
- mesenchymal stem cells (1)
- metabolite repair (1)
- miRNA (1)
- miRNA target (1)
- miRNS (1)
- mild cognitive impairment (1)
- molecular mechanism (1)
- monarch butterfly (1)
- multi-pinhole collimation (1)
- multi-unit recording (1)
- myeloablation (1)
- neuroinflammation (1)
- neurologin-2 (1)
- neuronal excitability (1)
- neuropaticher Schmerz (1)
- nurse bee (1)
- olfaction (1)
- opening of chromatin (1)
- orientation (1)
- p53 (1)
- pain regulation (1)
- pancreas (1)
- pancreatic cancer (1)
- pancreatic differentiation (1)
- perception (1)
- performance evaluation (1)
- peripheral nervous system (1)
- pharmacology (1)
- phosphoglycolate phosphatase (1)
- platelet (1)
- post-transcriptional regulation (1)
- potenzielles therapeutisches Target (1)
- quiescence (1)
- real-time imaging (1)
- reciprocity (1)
- regenerative medicine (1)
- regulatory RNA (1)
- reporter screen (1)
- responsiveness (1)
- reversible oxidation (1)
- ribosome biogenesis (1)
- self-targeting CRISPR-Cas (1)
- sigma factor (1)
- single particle tracking (1)
- single-cell RNA sequencing (1)
- single-molecule localization microscopy (1)
- skin equivalent (1)
- small RNA expression (1)
- small-animal SPECT (1)
- social understanding (1)
- specific phobia (1)
- spezifische Phobie (1)
- spider phobia (1)
- spiral trajectory (1)
- spontaneous blinks (1)
- sugar perception (fructose, sucrose) (1)
- sugar receptor (1)
- super-resolution microscopy (1)
- systemic inflammation (1)
- tapeworm (1)
- temporo-parietal junction (1)
- thrombo-inflammation (1)
- thrombopoiesis (1)
- time-correlated single photon counting (TCSPC) (1)
- tissue engineering (1)
- transcription (1)
- transcriptional termination site (1)
- vasp (1)
- viral genome packaging (1)
- viral miRNAs (1)
- vision (1)
- visual cue (1)
- visual perception (1)
- waggle dance decoding (1)
- wax (1)
- wild-living honey bees (1)
- zentrale Spindel und Mittelkörper (1)
Institute
- Graduate School of Life Sciences (102) (remove)
Sonstige beteiligte Institutionen
- Helmholtz Institute for RNA-based Infection Research (HIRI) (2)
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg (2)
- Carl-Ludwig-Institut für Physiologie, Universität Leipzig (1)
- Chair of Experimental Biomedicine I (1)
- Evangelisches Studienwerk e.V. (1)
- Helmholtz Center for RNA-based Infection Research (1)
- Helmholtz Institute for RNA-based Infection Research (1)
- Helmholtz-Institut für RNA-basierte Infektionsforschung (1)
- Maastricht University, Maastricht, the Netherlands (1)
- Max Delbrück Center for Molecular Medicine (1)
This thesis aimed at searching for new effective agents against Multidrug-Resistant Enterobacteriaceae. This is necessitated by the urgent need for new and innovative antibacterial agents addressing the critical priority pathogens prescribed by the World Health Organization (WHO). Among the available means for antibiotics discovery and development, nature has long remained a proven, innovative, and highly reliable gateway to successful antibacterial agents. Nevertheless, numerous challenges surrounding this valuable source of antibiotics among other drugs are limiting the complete realization of its potential. These include the availability of good quality data on the highly potential natural sources, limitations in methods to prepare and screen crude extracts, bottlenecks in reproducing biological potentials observed in natural sources, as well as hurdles in isolation, purification, and characterization of natural compounds with diverse structural complexities.
Through an extensive review of the literature, it was possible to prepare libraries of plant species and phytochemicals with reported high potentials against Escherichia coli and Klebsiella pneumnoniae. The libraries were profiled to highlight the existing patterns and relationships between the reported antibacterial activities and studied plants’ families and parts, the type of the extracting solvent, as well as phytochemicals’ classes, drug-likeness and selected parameters for enhanced accumulation within the Gram-negative bacteria. In addition, motivations, objectives, the role of traditional practices and other crucial experimental aspects in the screening of plant extracts for antibacterial activities were identified and discussed.
Based on the implemented strict inclusion criteria, the created libraries grant speedy access to well-evaluated plant species and phytochemicals with potential antibacterial activities. This way, further studies in yet unexplored directions can be pursued from the indicated or related species and compounds. Moreover, the availability of compound libraries focusing on related bacterial species serves a great role in the ongoing efforts to develop the rules of antibiotics penetrability and accumulation, particularly among Gram-negative bacteria. Here, in addition to hunting for potential scaffolds from such libraries, detailed evaluations of large pool compounds with related antibacterial potential can grant a better understanding of structural features crucial for their penetration and accumulation. Based on the scarcity of compounds with broad structural diversity and activity against Gram-negative bacteria, the creation and updating of such libraries remain a laborious but important undertaking.
A Pressurized Microwave Assisted Extraction (PMAE) method over a short duration and low-temperature conditions was developed and compared to the conventional cold maceration over a prolonged duration. This method aimed at addressing the key challenges associated with conventional extraction methods which require long extraction durations, and use more energy and solvents, in addition to larger quantities of plant materials. Furthermore, the method was intended to replace the common use of high temperatures in most of the current MAE applications. Interestingly, the yields of 16 of 18 plant samples under PMAE over 30 minutes were found to be within 91–139% of those obtained from the 24h extraction by maceration. Additionally, different levels of selectivity were observed upon an analytical comparison of the extracts obtained from the two methods. Although each method indicated selective extraction of higher quantities or additional types of certain phytochemicals, a slightly larger number of additional compounds were observed under maceration. The use of this method allows efficient extraction of a large number of samples while sparing heat-sensitive compounds and minimizing chances for cross-reactions between phytochemicals.
Moreover, findings from another investigation highlighted the low likelihood of reproducing antibacterial activities previously reported among various plant species, identified the key drivers of poor reproducibility, and proposed possible measures to mitigate the challenge. The majority of extracts showed no activities up to the highest tested concentration of 1024 µg/mL. In the case of identical plant species, some activities were observed only in 15% of the extracts, in which the Minimum Inhibitory Concentrations (MICs) were 4 – 16-fold higher than those in previous reports. Evaluation of related plant species indicated better outcomes, whereby about 18% of the extracts showed activities in a range of 128–512 μg/mL, some of the activities being superior to those previously reported in related species.
Furthermore, solubilizing plant crude extracts during the preparation of test solutions for Antibacterial Susceptibility Testing (AST) assays was outlined as a key challenge. In trying to address this challenge, some studies have used bacteria-toxic solvents or generally unacceptable concentrations of common solubilizing agents. Both approaches are liable to give false positive results. In line with this challenge, this study has underscored the suitability of acetone in the solubilization of crude plant extracts. Using acetone, better solubility profiles of crude plant extracts were observed compared to dimethyl sulfoxide (DMSO) at up to 10 %v/v. Based on lacking toxicity against many bacteria species at up to 25 %v/v, its use in the solubilization of poorly water-soluble extracts, particularly those from less polar solvents is advocated.
In a subsequent study, four galloylglucoses were isolated from the leaves of Paeonia officinalis L., whereby the isolation of three of them from this source was reported for the first time. The isolation and characterization of these compounds were driven by the crucial need to continually fill the pre-clinical antibiotics pipeline using all available means. Application of the bioautography-guided isolation and a matrix of extractive, chromatographic, spectroscopic, and spectrometric techniques enabled the isolation of the compounds at high purity levels and the ascertainment of their chemical structures.
Further, the compounds exhibited the Minimum Inhibitory Concentrations (MIC) in a range of 2–256 µg/mL against Multidrug-Resistant (MDR) strains of E. coli and K. pneumonia exhibiting diverse MDR phenotypes. In that, the antibacterial activities of three of the isolated compounds were reported for the first time. The observed in vitro activities of the compounds resonated with their in vivo potentials as determined using the Galleria mellonella larvae model. Additionally, the susceptibility of the MDR bacteria to the galloylglucoses was noted to vary depending on the nature of the resistance enzymes expressed by the MDR bacteria. In that, the bacteria expressing enzymes with higher content of aromatic amino acids and zero or positive net charges were generally more susceptible. Following these findings, a plausible hypothesis for the observed patterns was put forward.
The generally challenging pharmacokinetic properties of galloylglucoses limit their further development into therapeutic agents. However, the compounds can replace or reduce the use of antibiotics in livestock keeping as well as in the treatment of septic wounds and topical or oral cavity infections, among other potential uses.
Using nature-inspired approaches, a series of glucovanillin derivatives were prepared following feasible synthetic pathways which in most cases ensured good yields and high purity levels. Some of the prepared compounds showed MIC values in a range of 128 – 512 μg/mL against susceptible and MDR strains of Klebsiella pneumoniae, Methicillin-Resistant Staphylococcus aureus (MRSA) and Vancomycin-Resistant Enterococcus faecium (VRE). These findings emphasize the previously reported essence of small molecular size, the presence of protonatable amino groups and halogen atoms, as well as an amphiphilic character, as crucial features for potential antibacterial agents.
Due to the experienced limited success in the search for new antibacterial agents using purely synthetic means, pursuing semi-synthetic approaches as employed in this study are highly encouraged. This way, it is possible to explore broader chemical spaces around natural scaffolds while addressing their inherent limitations such as solubility, toxicity, and poor pharmacokinetic profiles.
The recent pandemic has reminded the public that basic research in virology is pivotal for human health. Understanding the mechanisms of successful viral replication and the role of host factors can help to combat viral infections and prevent future pandemics.
Our lab has published the first SARS-CoV-2 RNA-protein interaction atlas, laying the foundation to investigate the interplay between viral RNA and host RNA binding proteins (RBP). Based on this, my project created the largest collection of binding profiles of host and viral RBPs on SARS-CoV-2 RNA to date. This revealed the host protein SND1 as the first human RBP that specifically binds negative sense viral RNA at the 5´ end, a region associated with viral transcription initiation. The binding profile shares similarities with the viral RBP nsp9, which binds the 5´ ends of positive and negative sense SARS-CoV-2 RNA. Depletion of SND1 shows reduced levels of viral RNA revealing it as a proviral host factor. To decode the underlying molecular mechanism, I characterized the protein-protein interactions of SND1 in SARS-CoV-2 infected and uninfected cells. Infection remodels the protein interactors of SND1 from general RNA biology to membrane association and viral RNA synthesis. Upon infection, SND1 specifically interacts with nsp9, the RBP that shares the same binding region on the negative strand of SARS-CoV-2 RNA. Recent work demonstrates that nsp9 is NMPylated in vitro suggesting a functional role of nsp9 in priming of viral RNA synthesis. I was able to show that nsp9 is covalently linked to the 5´ ends of SARS-CoV-2 RNA during infection of human cells. Analysing the covalent bond of nsp9 with the viral RNA on nucleotide level shows close proximity to the initiation sites of viral RNA synthesis, suggesting that nsp9 acts as a protein-primer of SARS-CoV-2 RNA synthesis. SND1 modulates the distribution of nsp9 on the viral RNA, since depletion of SND1 results in imbalanced occupancy of nsp9 at the 5´ends of viral RNA.
This study is the first to provide evidence for the priming mechanism of SARS-CoV-2 in authentic viral replication and further reveals how this mechanism is modulated by the host RBP SND1. Detailed knowledge about priming of viral RNA synthesis can help to find targeted antivirals that could be used to fight coronaviral infections.
Single-molecule dynamics at a bottleneck: a systematic study of the narrow escape problem in a disc
(2023)
Diffusion facilitates numerous reactions within the biological context of a cell. It is remarkable how the cost-efficient random process of Brownian motion promotes fast reactions. From the narrow escape theory, it is possible to determine the mean first passage time of such processes based on their reaction space and diffusion coefficient. The narrow escape theory of Brownian particles is characterized by a confining domain with reflective boundaries and a small reaction site. In this thesis, the mean first passage time was systematically tested in a disc as a function of the escape opening size in vitro and in silico. For the in vitro experiments, a model system of patterned supported-lipid bilayers (SLB) was established. Such a model is prepared by a combined colloid metalization approach, where a gold scaffold on glass facilitates assembly of SLB patches of distinct sizes through vesicle fusion. The model setup was evaluated and found to match all necessary requirements to test the nar- row escape problem in vitro. In particular, the reflectivity of the boundaries, the unhindered, free diffusion of the tracer lipids, and the distinct area were assessed. Observed results of the mean first passage time agreed with the theory of the narrow escape problem. There was excellent agreement in both absolute values and across a range of small escape opening sizes. Additionally, I developed a straightforward method, a correction factor, to calculate the mean first passage time from incomplete experimental traces. By re-scaling the mean first passage time to the fraction of particles that escaped, I was able to overcome the lifetime limitations of fluorescent probes. Previously inaccessible measurements of the mean first passage time relying on fluorescent probes will be made possible through this approach. The in vitro experiments were complemented with various in silico experiments. The latter were based on random walk simulations in discs, mimicking the in vitro situation with its uncertainties. The lifetime of single particles was either set sufficiently long to allow all particles to escape, or was adjusted to meet the lifetime limitations observed in the in vitro experiments. A comparison of the mean first passage time from lifetime-unlimited particles to the corrected, lifetime-limited particles did support the use of the correction factor. In agreement with the narrow escape theory, it was experimentally found that the mean first passage time is independent of the start point of the particle within the domain. This is when the particle adheres to a minimum distance to the escape site. In general, the presented random walk simulations do accurately represent the in vitro experiments in this study. The required hardware for the establishment of an astigmatism-based 3D system was installed in the existing microscope. The first attempts to analyze the obtained 3D imaging data gave insight into the potential of the method to investigate molecule dynamics in living trypanosome cells. The full functionality will be realized with the ongoing improvement of image analysis outside of this thesis.
Allogenic hematopoietic stem cell transplantation (allo-HCT) is a curative therapy for the treatment of malignant and non-malignant bone marrow diseases. The major complication of this treatment is a highly inflammatory reaction known as Graft-versus-Host Disease (GvHD). Cyclosporin A (CsA) and tacrolimus are used to treat GvHD which limits inflammation but also interferes with the anticipated Graft-versus-Leukemia (GvL) effect. These drugs repress conventional T cells (Tcon) along with regulatory T cells (Treg), which are important for both limiting GvHD and supporting GvL. Both of these drugs inhibit calcineurin (CN), which dephosphorylates and activates the nuclear factor of activated T-cells (NFAT) family of transcription factors. Here, we make use of our Cd4cre.Cas9+ mice and developed a highly efficient non-viral CRISPR/Cas9 gene editing method by gRNA-only nucleofection. Utilizing this technique, we demonstrated that unstimulated mouse T cells upon NFATc1 or NFATc2 ablation ameliorated GvHD in a major mismatch mouse model. However, in vitro pre-stimulated mouse T cells could not achieve long-term protection from GvHD upon NFAT single-deficiency. This highlights the necessity of gene editing and transferring unstimulated human T cells during allo-HCT. Indeed, we established a highly efficient ribonucleoprotein (RNP)-mediated CRISPR/Cas9 gene editing for NFATC1 and/or NFATC2 in pre-stimulated as well as unstimulated primary human T cells. In contrast to mouse T cells, not NFATC1 but NFATC2 deficiency in human T cells predominantly affected proinflammatory cytokine production. However, either NFAT single-knockout kept cytotoxicity of human CD3+ T cells untouched against tumor cells in vitro. Furthermore, mouse and human Treg were unaffected upon the loss of a single NFAT member. Lastly, NFATC1 or NFATC2-deficient anti-CD19 CAR T cells, generated with our non-viral ‘one-step nucleofection’ method validated our observations in mouse and human T cells. Proinflammatory cytokine production was majorly dependent on NFATC2 expression, whereas, in vitro cytotoxicity against CD19+ tumor cells was undisturbed in the absence of either of the NFAT members. Our findings emphasize that NFAT single-deficiency in donor T cells is superior to CN-inhibitors as therapy during allo-HCT to prevent GvHD while preserving GvL in patients.
In this work, dRNA-seq (differential RNA sequencing) and RNAtag-seq were applied to first define the global transcriptome architecture of C. difficile, followed by Hfq RIP-seq (RNA immunoprecipitation followed by RNA-seq) and RIL-seq (RNA interaction by ligation and sequencing) to characterize the Hfq-mediated sRNA interactome on a transcriptome-wide scale. These approaches resulted in the annotation of > 60 novel sRNAs. Notably, it not only revealed 50 Hfq-bound sRNAs, but also > 1000 mRNA-sRNA interactions, confirming Hfq as a global RNA matchmaker in C. difficile. Similar to its function in Gram-negative species, deletion of Hfq resulted in decreased sRNA half-lives, providing evidence that Hfq affects sRNA stability in C. difficile. Finally, several sRNAs and their function in various infection relevant conditions were characterized. The sRNA nc085 directly interacts with the two-component response regulator eutV, resulting in regulation of ethanolamine utilization, an abundant intestinal carbon and nitrogen source known to impact C. difficile pathogenicity. Meanwhile, SpoY and SpoX regulate translation of the master regulator of sporulation spo0A in vivo, thereby affecting sporulation initiation. Furthermore, SpoY and SpoX deletion significantly impacts C. difficile gut colonization and spore burden in a mouse model of C. difficile infection.
Forkhead box O transcription factors are a family of proteins involved in cellular processes downstream of the Insulin-PI3K-PKB pathway. In response to extra- or intracellular stresses, for example starvation or oxidative stress, FoxOs are required to direct cell cycle progression and apoptosis. In endothelial cells, they induce apoptosis, and their deregulation is linked to diseases involving the insulin pathway, such as diabetes. FoxOs also exhibit a complex role in tumour transformation: here their main function is to suppress tumorigenesis. In both physiological and cancer contexts, FoxO activation leads to the transcription of some general targets, such as p27kip1 or IGFBP1. The FoxOs can also induce tissue-specific genes, as ANGPT2 and BIM in the endothelium.
In endothelial cells, another pathway with a pivotal function is the MEK5/ERK5 MAPK signalling way. Its activation promotes cell survival and proliferation in stressful conditions, e.g., when blood vessels are exposed to the shear forces exerted by the blood stream. Furthermore, recent data described ERK5 as a kinase directing tumour resistance upon therapy-induced stress.
Comparing their reported roles in various tumours and in the endothelium, FoxO proteins and the MEK5/ERK5 MAPK cascade appear to exert opposite functions. First non-published data confirmed the hypothesis that FoxO factors are subject to a negative modulation by the MEK5/ERK5 pathway. Hence, one goal of this PhD project was to further characterise this crosstalk at molecular level. The major mechanism of FoxO regulation is the balance among several post translational modifications, such as phosphorylation, acetylation, and ubiquitination. Most importantly, the PKB dependent phosphorylation of FoxOs negatively controls their activity, and it is critical for their subcellular localization. Therefore, the regulation of FoxO localization as mechanism of ERK5 dependent suppression was studied, but the results presented in this thesis argue against this hypothesis. However, additional experiments are required to explore the impact of ERK5 activity on FoxO post-translational modifications.
FoxO activity can also be modulated by the interaction with other proteins, which in turn could explain general- and tissue-specific gene expression. Thus, another objective of this work was to investigate FoxO3-interactome in endothelial cells and the impact of MEK5/ERK5 activation on it. As published in (Fusi et al. 2022) and presented here, this analysis unveiled TRRAP as new FoxO bound protein in several cell types. Moreover, the interaction did not rely on the capacity of the FoxOs to bind their consensus DNA sequences at the promoter of target genes. Functional data demonstrated that TRRAP is required for FoxO-dependent gene transcription in endothelial and osteosarcoma cells. In addition, TRRAP expression in the endothelium is important for FoxO induced apoptosis. In summary, the interaction between FoxO factors and TRRAP revealed a new regulatory mechanism of FoxO-dependent gene transcription. It remains to be analysed whether the MEK5/ERK5 cascade may exert its suppressive effect on FoxO activity by interfering with their binding to TRRAP and whether such a mechanism may be relevant for tumorigenesis.
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.
Many arthropods such as mosquitoes, ticks, bugs, and flies are vectors for the transmission of pathogenic parasites, bacteria, and viruses. Among these, the unicellular parasite Trypanosoma brucei (T. brucei) causes human and animal African trypanosomiases and is transmitted to the vertebrate host by the tsetse fly. In the fly, the parasite goes through a complex developmental cycle in the alimentary tract and salivary glands ending with the cellular differentiation into the metacyclic life cycle stage. An infection in the mammalian host begins when the fly takes a bloodmeal, thereby depositing the metacyclic form into the dermal skin layer. Within the dermis, the cell cycle-arrested metacyclic forms are activated, re-enter the cell cycle, and differentiate into proliferative trypanosomes, prior to dissemination throughout the host.
Although T. brucei has been studied for decades, very little is known about the early events in the skin prior to systemic dissemination. The precise timing and the mechanisms controlling differentiation of the parasite in the skin continue to be elusive, as does the characterization of the proliferative skin-residing trypanosomes. Understanding the first steps of an infection is crucial for developing novel strategies to prevent disease establishment and its progression.
A major shortcoming in the study of human African trypanosomiasis is the lack of suitable infection models that authentically mimic disease progression. In addition, the production of infectious metacyclic parasites requires tsetse flies, which are challenging to keep. Thus, although animal models - typically murine - have produced many insights into the pathogenicity of trypanosomes in the mammalian host, they were usually infected by needle injection into the peritoneal cavity or tail vein, bypassing the skin as the first entry point. Furthermore, animal models are not always predictive for the infection outcome in human patients. In addition, the relatively small number of metacyclic parasites deposited by the tsetse flies makes them difficult to trace, isolate, and study in animal hosts.
The focus of this thesis was to develop and validate a reconstructed human skin equivalent as an infection model to study the development of naturally-transmitted metacyclic parasites of T. brucei in mammalian skin. The first part of this work describes the development and characterization of a primary human skin equivalent with improved mechanical properties. To achieve this, a computer-assisted compression system was designed and established. This system allowed the improvement of the mechanical stability of twelve collagen-based dermal equivalents in parallel through plastic compression, as evaluated by rheology. The improved dermal equivalents provided the basis for the generation of the skin equivalents and reduced their contraction and weight loss during tissue formation, achieving a high degree of standardization and reproducibility. The skin equivalents were characterized using immunohistochemical and histological techniques and recapitulated key anatomical, cellular, and functional aspects of native human skin. Furthermore, their cellular heterogeneity was examined using single-cell RNA sequencing - an approach which led to the identification of a remarkable repertoire of extracellular matrix-associated genes expressed by different cell subpopulations in the artificial skin. In addition, experimental conditions were established to allow tsetse flies to naturally infect the skin equivalents with trypanosomes.
In the second part of the project, the development of the trypanosomes in the artificial skin was investigated in detail. This included the establishment of methods to successfully isolate skin-dwelling trypanosomes to determine their protein synthesis rate, cell cycle and metabolic status, morphology, and transcriptome. Microscopy techniques to study trypanosome motility and migration in the skin were also optimized. Upon deposition in the artificial skin by feeding tsetse, the metacyclic parasites were rapidly activated and established a proliferative population within one day. This process was accompanied by: (I) reactivation of protein synthesis; (II) re-entry into the cell cycle; (III) change in morphology; (IV) increased motility. Furthermore, these observations were linked to potentially underlying developmental mechanisms by applying single-cell parasite RNA sequencing at five different timepoints post-infection.
After the initial proliferative phase, the tsetse-transmitted trypanosomes appeared to enter a reversible quiescence program in the skin. These quiescent skin-residing trypanosomes were characterized by very slow replication, a strongly reduced metabolism, and a transcriptome markedly different from that of the deposited metacyclic forms and the early proliferative trypanosomes. By mimicking the migration from the skin to the bloodstream, the quiescent phenotype could be reversed and the parasites returned to an active proliferating state. Given that previous work has identified the skin as an anatomical reservoir for T. brucei during disease, it is reasonable to assume that the quiescence program is an authentic facet of the parasite's behavior in an infected host.
In summary, this work demonstrates that primary human skin equivalents offer a new and promising way to study vector-borne parasites under close-to-natural conditions as an alternative to animal experimentation. By choosing the natural transmission route - the bite of an infected tsetse fly - the early events of trypanosome infection have been detailed with unprecedented resolution. In addition, the evidence here for a quiescent, skin-residing trypanosome population may explain the persistence of T. brucei in the skin of aparasitemic and asymptomatic individuals. This could play an important role in maintaining an infection over long time periods.
Adrenocortical carcinoma (ACC) is a rare, but highly aggressive endocrine malignancy. Tumor-related hypercortisolism is present in 60 % of patients and associated with worse outcome. While cancer immunotherapies have revolutionized the treatment of many cancer entities, the results of initial studies of different immune checkpoint inhibitors in ACC were heterogeneous. Up to now, five small clinical trials with a total of 121 patients have been published and demonstrated an objective response in only 17 patients. However, one of the studies, by Raj et al., reported a clinically meaningful disease control rate of 52 % and a median overall survival of almost 25 months suggesting that a subgroup of ACC patients may benefit from immunotherapeutic approaches. Following the hypothesis that some ACCs are characterized by a glucocorticoid-induced T lymphocytes depletion, several studies were performed as part of the presented thesis. First, the immune cell infiltration in a large cohort of 146 ACC specimens was investigated. It was demonstrated for the first time, and against the common assumption, that ACCs were infiltrated not only by FoxP3+ regulatory T cells (49.3 %), but also that a vast majority of tumor samples was infiltrated by CD4+ TH cells (74 %) and CD8+ cytotoxic T cells (84.3 %), albeit the immune cell number varied heterogeneously and was rather low (median: 7.7 CD3+ T cells / high power field, range: 0.1-376). Moreover, the presence of CD3+-, CD4+- and CD8+ ACC-infiltrating lymphocytes was associated with an improved recurrence-free (HR: 0.31 95 % CI 0.11-0.82) and overall survival (HR: 0.47 96 % CI 0.25-0.87). Particularly, patients with tumor-infiltrating CD4+ TH cells without glucocorticoid excess had a significantly longer overall survival compared to patients with T cell-depleted ACC and hypercortisolism (121 vs. 27 months, p = 0.004). Hence, the impact of glucocorticoids might to some extent be responsible for the modest immunogenicity in ACC as hypercortisolism was reversely correlated with the number of CD4+ TH cells. Accordingly, CD3+ T cells co-cultured with steroidogenic NCI-H295R ACC cells demonstrated in vitro an enhanced anti-tumoral cytotoxicity by secreting 747.96 ±225.53 pg/ml IFN-γ in a therapeutically hormone-depleted microenvironment (by incubation with metyrapone), versus only 276.02 ±117.46 pg/ml IFN-γ in a standard environment with glucocorticoid excess.
Other potential biomarkers to predict response to immunotherapies are the immunomodulatory checkpoint molecules, programmed cell death 1 (PD-1) and its ligand PD-L1, since both are targets of antibodies used therapeutically in different cancer entities. In a subcohort of 129 ACCs, expressions of both molecules were heterogeneous (PD-1 17.4 %, range 1-15; PD-L1 24.4 %, range 1 - 90) and rather low. Interestingly, PD-1 expression significantly influenced ACC patients´ overall (HR: 0.21 95 % CI 0.53-0.84) and progression- free survival (HR: 0.30 95 % CI 0.13-0.72) independently of established factors, like ENSAT tumor stage, resection status, Ki67 proliferation index and glucocorticoid excess, while PD-L1 had no impact.
In conclusion, this study provides several potential explanations for the heterogeneous results of the immune checkpoint therapy in advanced ACC. In addition, the establishment of PD-1 as prognostic marker can be easily applied in routine clinical care, because it is nowadays anyway part of a detailed histo-pathological work-up. Furthermore, these results provide the rationale and will pave the way towards a combination therapy using immune checkpoint inhibitors as well as glucocorticoid blockers. This will increase the likelihood of re-activating the immunological anti-tumor potential in ACC. However, this will have to be demonstrated by additional preclinical in vivo experiments and finally in clinical trials with patients.
Infectious diseases caused by pathogenic microorganisms are one of the largest socioeconomic burdens today. Although infectious diseases have been studied for decades, in numerous cases, the precise mechanisms involved in the multifaceted interaction between pathogen and host continue to be elusive. Thus, it still remains a challenge for researchers worldwide to develop novel strategies to investigate the molecular context of infectious diseases in order to devise preventive or at least anti-infective measures. One of the major drawbacks in trying to obtain in-depth knowledge of how bacterial pathogens elicit disease is the lack of suitable infection models to authentically mimic the disease progression in humans. Numerous studies rely on animal models to emulate the complex temporal interactions between host and pathogen occurring in humans. While they have greatly contributed to shed light on these interactions, they require high maintenance costs, are afflicted with ethical drawbacks, and are not always predictive for the infection outcome in human patients. Alternatively, in-vitro two-dimensional (2D) cell culture systems have served for decades as representatives of human host environments to study infectious diseases. These cell line-based models have been essential in uncovering virulence-determining factors of diverse pathogens as well as host defense mechanisms upon infection. However, they lack the morphological and cellular complexity of intact human tissues, limiting the insights than can be gained from studying host-pathogen interactions in these systems.
The focus of this thesis was to establish and innovate intestinal human cell culture models to obtain in-vitro reconstructed three-dimensional (3D) tissue that can faithfully mimic pathogenesis-determining processes of the zoonotic bacterium Campylobacter jejuni (C. jejuni). Generally employed for reconstructive medicine, the field of tissue engineering provides excellent tools to generate organ-specific cell culture models in vitro, realistically recapitulating the distinctive architecture of human tissues. The models employed in this thesis are based on decellularized extracellular matrix (ECM) scaffolds of porcine intestinal origin. Reseeded with intestinal human cells, application of dynamic culture conditions promoted the formation of a highly polarized mucosal epithelium maintained by functional tight and adherens junctions. While most other in-vitro infection systems are limited to a flat monolayer, the tissue models developed in this thesis can display the characteristic 3D villi and crypt structure of human small intestine.
First, experimental conditions were established for infection of a previously developed, statically cultivated intestinal tissue model with C. jejuni. This included successful isolation of bacterial colony forming units (CFUs), measurement of epithelial barrier function, as well as immunohistochemical and histological staining techniques. In this way, it became possible to follow the number of viable bacteria during the infection process as well as their translocation over the polarized epithelium of the tissue model. Upon infection with C. jejuni, disruption of tight and adherens junctions could be observed via confocal microscopy and permeability measurements of the epithelial barrier. Moreover, C. jejuni wildtype-specific colonization and barrier disruption became apparent in addition to niche-dependent bacterial localization within the 3D microarchitecture of the tissue model. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D host environment deviated from those obtained with conventional in-vitro 2D monolayers but mimicked observations made in vivo. Furthermore, a genome-wide screen of a C. jejuni mutant library revealed significant differences for bacterial factors required or dispensable for interactions with unpolarized host cells or the highly prismatic epithelium provided by the intestinal tissue model. Elucidating the role of several previously uncharacterized factors specifically important for efficient colonization of a 3D human environment, promises to be an intriguing task for future research.
At the frontline of the defense against invading pathogens is the protective, viscoelastic mucus layer overlying mucosal surfaces along the human gastrointestinal tract (GIT). The development of a mucus-producing 3D tissue model in this thesis was a vital step towards gaining a deeper understanding of the interdependency between bacterial pathogens and host-site specific mucins. The presence of a mucus layer conferred C. jejuni wildtype-specific protection against epithelial barrier disruption by the pathogen and prevented a high bacterial burden during the course of infection. Moreover, results obtained in this thesis provide evidence in vitro that the characteristic corkscrew morphology of C. jejuni indeed grants a distinct advantage in colonizing mucous surfaces.
Overall, the results obtained within this thesis highlight the strength of the tissue models to combine crucial features of native human intestine into accessible in-vitro infection models. Translation of these systems into infection research demonstrated their ability to expose in-vivo like infection outcomes. While displaying complex organotypic architecture and highly prismatic cellular morphology, these tissue models still represent an imperfect reflection of human tissue. Future advancements towards inclusion of human primary and immune cells will strive for even more comprehensive model systems exhibiting intricate multicellular networks of in-vivo tissue. Nevertheless, the work presented in this thesis emphasizes the necessity to investigate host-pathogen interactions in infection models authentically mimicking the natural host environment, as they remain among the most vital parts in understanding and counteracting infectious diseases.