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Im Rahmen dieser Arbeit wurden verschiedene metabolische Anpassungsmechanismen des humanpathogenen Bakteriums Acinetobacter baumannii an seinen Wirt untersucht. Im ersten Teil wurde die Rolle von verschiedenen Trimethylammoniumverbindungen (Cholin, Glycinbetain und Carnitin) und den zugehörigen Aufnahmesystemen, sowie ihren Stoffwechselwegen während dieses Prozesses analysiert. Für die Analyse der Transportsysteme wurde eine markerlose Vierfachmutante (Δbcct) von A. baumannii generiert, sodass alle bekannten Transportsysteme für die genannten Verbindungen deletiert vorlagen. Wachstumsversuche mit dieser Mutante zeigten, dass es in A. baumannii keine weiteren Transporter für die Aufnahme von Cholin gibt, jedoch weitere primär aktive oder sekundär aktive Transporter für die Aufnahme von Glycinbetain. Weiterhin konnten innerhalb dieser Arbeit die KM-Werte der Transporter bestimmt werden. Verschiedene Virulenz- und Infektionsanalysen führten zu dem Schluss, dass die Transporter keine Rolle bei der Virulenz von A. baumannii spielen. In Genomanalysen konnten die Gene, die für die Enzyme des Oxidationsweges von Cholin zu Glycinbetain kodieren identifiziert werden (Cholin-Dehydrogenase (betA), GlycinbetainAldehyd-Dehydrogenase (betB) und ein potenzieller Regulator (betI)). Es wurden Deletionsmutanten innerhalb dieses Genclusters generiert, mit dessen Hilfe gezeigt werden konnte, dass Cholin unter Salzstress ausschließlich als Vorläufer für das kompatible Solut Glycinbetain fungiert und nicht als kompatibles Solut von A. baumannii genutzt werden kann. Virulenz- und Infektionsstudien mit den Deletionsmutanten zeigten, dass der Cholin-Oxidationsweg keine Rolle bei der Virulenz von A. baumannii spielt.
Die Cholin-Dehydrogenase BetA wurde zusätzlich in E. coli produziert und anschließend mittels NiNTA-Affinitätschromatographie aufgereinigt. Die biochemische Charakterisierung des Enzyms zeigte, dass BetA membranständig ist und die höchste Aktivität bei einem pH-Wert von 9,0 hat. Salze wie NaCl oder KCl hatten keinen Effekt auf die Aktivität des Enzyms, während Glutamat die Aktivität stimulierte.
Weiterhin konnte FAD als Cofaktor identifiziert werden und der KM-Wert ermittelt werden. Zudem konnte gezeigt werden, dass die Oxidation von Cholin zu Glycinbetain unter isoosmotischen Bedingungen zu einem Anstieg der ATP-Konzentration in A. baumannii-Zellsuspensionen führt und damit, dass Cholin als alternative Energiequelle genutzt wird. Das Phospholipid Phosphatidylcholin konnte als natürliche Cholinquelle identifiziert werden. Eine Rolle der Phospholipasen D bei der Abspaltung der Cholin-Kopfgruppe des Phosphatidylcholins konnte ausgeschlossen werden. Die Gene für die Oxidation von Cholin zu Glycinbetain werden ausschließlich in Anwesenheit von Cholin exprimiert, jedoch unabhängig von der extrazellulären Salzkonzentration. Diese Studien zeigten, dass der Cholin-Oxidationsweg eine Rolle in der metabolischen Adaptation von A. baumannii an den Wirt spielt. Phosphatidylcholin kann hier als natürliche Cholinquelle im Wirt genutzt werden, da die Wirtsmembranen aus bis zu 70 % Phosphatidylcholin bestehen. Transportstudien mit Carnitin führten zu dem Schluss, dass der Transporter Aci01347 aus A. baumannii neben Cholin ebenfalls Carnitin transportiert. Wachstumsversuche mit einer aci01347-Mutante bestätigen, dass Aci01347 essenziell für die Aufnahme und anschließende Verwertung von Carnitin als Kohlenstoffquelle ist. Es konnte weiterhin gezeigt werden, dass das Transportergen mit essenziellen Genen für den Carnitin-Abbau in einem Operon liegt. Für die Analyse des Abbauweges von Carnitin wurden markerlose Deletionsmutanten innerhalb des Operons generiert. In Wachstumsstudien mit diesen Mutanten konnte der Abbauweg aufgeklärt werden und der Regulator des Operons identifiziert werden. Carnitin wird hier über Trimethylamin und Malat-Semialdehyd zu D-Malat umgewandelt und anschließend über Pyruvat in den TCA-Zyklus eingespeist. Der Regulator wurde zusätzlich in E. coli produziert und mittels Ni-NTA-Affinitätschromatographie aufgereinigt. Mithilfe von EMSA-Studien konnte die Bindestelle des Regulators auf eine 634 Bp lange DNA-Sequenz stromaufwärts des CarnitinOperons eingegrenzt werden. Durch Transkriptomanalysen konnte gezeigt werden, dass bei Wachstum mit Acetylcarnitin, Carnitin und D-Malat die Expression des Carnitin-Operons induziert wurde. Darüber hinaus wurden die Gene konservierter Aromatenabbauwege wie z. B. des Homogentisatweges, des Phenylacetatweges und des Protocatechuat-Abbaus, verstärkt exprimiert. In G. mellonellaVirulenzstudien konnte eine Rolle des Abbaus von Carnitin bei der Virulenz von A. baumannii nachgewiesen werden. Zusätzlich konnte dieser Effekt dem entstehenden Trimethylamin zugesprochen werden...
Macroautophagy, herein referred to as autophagy, is an evolutionarily conserved homeostatic process that normally occurs inside eukaryotic cells which involves degradation of cytoplasmic substances via lysosomes. It can be induced by various conditions such as starvation and drug exposure, as well as be inhibited by numerous compounds. Under normal conditions, the doublemembrane autophagosomes engulf the cytosolic substrates and deliver them to lysosomes for digestion. These substrates include unnecessary or dysfunctional cell components, such as faulty macromolecules, organelles and even invading pathogens. Autophagosomes are formed through the co-operative work of various autophagy-related (ATG) proteins organized into complexes. Upon closure of the autophagosomes, they fuse with the acidic lysosomes, resulting in formation of autolysosomes and the delivery of lysosomal hydrolases to degrade the engulfed contents. The fusion of the autophagosome with lysosome is carried out by specific SNARE proteins, small GTPases and their effectors including tethers, adaptors and motor proteins. Autophagy is impaired in many human diseases including cancer, neurodegenerative diseases, aging and inflammation. Therefore, manipulation of autophagy pathway holds a great promise for new therapeutic applications ...
In Europe, the sugar refinery is largely based on sugar beets. This route for obtaining household sugar results in a large amount of biomass waste, consisting mainly of the insoluble beet resi-dues, e.g., cell wall fragments. To a vast moiety this debris consists of the polymer pectin (up to 20% in the dry total solids). The structure of pectin is based on a backbone of D-galacturonic acid units (GalA), but also contains various other sugar monomers, predominantly L-arabinose, D-galactose, L-rhamnose and D-xylose. The amount of GalA adds up to a moiety of up to 70% with-in this sugar cocktail. So far, this debris is only fed to cattle or simply burnt. In nature, pectin is a common substrate for various organisms. The degradation of pectin-rich biomass is often per-formed by filamentous fungi like Hypocrea jecorina (also known as Trichoderma reesei) and As-pergillus niger, which evolved pectinases to degrade the pectin backbone and pathways to con-sume the monomer GalA as a sole carbon source. The fungal catabolism of pectin residues starts with the reduction of GalA to L-galactonate (GalOA) by a GalA-reductase. Even though filamen-tous fungi are native hosts of the GalA-catabolism and certain engineering approaches have al-ready been demonstrated, this class of organisms remains challenging with regard to bioreactor cultivation and tedious genetic accessibility. In contrast, the yeast S. cerevisiae is well known in fermentation processes and easily modified by a versatile set of genetic tools. So far, first ap-proaches have already been conducted to transfer the GalA utilization pathways into S. cerevisiae, but these approaches indicated limitations regarding GalA-uptake and redox cofac-tor replenishment due to the relatively high oxidative state of GalA compared to other sugars like glucose and galactose. Furthermore, the generally strongly increased demand for redox co-factors must be met by GalA reduction by finding new cofactor sources or redirecting reactions of the core metabolism.
This work aimed at the production of GalOA, which is the first intermediate of the fungal GalA catabolism. This compound shows an interesting range of potential applications, for instance as a food and cosmetic additive. To overcome the oxidized character of GalA, the presence of a more reduced co-substrate as a redox donor and as a carbon and energy source was required. To further enhance the reduction of GalA, modulation of the redox-cofactor supply and enzyme engineering were performed.
The Southern Ocean (SO) is one of the most pristine regions of our Planet, characterised by high levels of biodiversity (5% of the global diversity) (David and Saucède 2015) and hosting a unique fauna (up to 90% of SO species are endemic) (De Broyer and Danis 2011; Chown et al. 2015). Yet, the knowledge on SO biodiversity is still far from being completed. In addition, the knowledge on the impact that changing environments have on SO species-richness is very little and for some groups, it is still totally unknown. For instance, most of studies generally focus on one single species such as Antarctic krill (Kawaguchi et al. 2011), Clio pyramidata Linnaeus, 1767 (Orr et al. 2005), Globigerina bulloides d'Orbigny, 1826 (Moy et al. 2009), or only on a high taxonomic level (e.g. phylum, class): Echinodermata, Crustacea, Mollusca, Porifera, Bryozoa, Brachiopoda, Hydrozoa, Ascidiacea, Holoturoidea
(Barnes 1999; Rowden et al. 2015; Post et al. 2017; Gutt et al. 2019; Vause et al. 2019; Pineda-Metz et al. 2020). Ultimately, the influence of sea-ice coverage on benthic species diversity was totally unknown prior to this study. In light of this, the objectives of the thesis are:
1. To expand the knowledge on shelf and deep-sea peracarid assemblage structure and abundance on a small regional (Weddell Sea) and on a large regional (Atlantic sector of the SO and South Atlantic Ocean) geographic scale.
2. To assess the environmental variables driving peracarid assemblage structure and abundance from the above mentioned areas.
3. To investigate SO benthic isopod species diversity from the Atlantic sector of the SO and assess the influence of environmental variables on their species-richness and composition.
4. To describe new possible peracarid species by means of integrative taxonomy, using morphological descriptions and whole genome sequencing analyses to support the species identification.
Objective outcomes: The present thesis provides new information on the abundance and assemblage structure based on 64766 peracarid crustaceans from different 28 locations within the Atlantic sector of the SO continental shelf and deep sea (Chapters I-II). These locations are characterised by different environmental conditions, for instance different sea-ice concentrations. Results from Chapters I-II confirmed the dominance of peracarid assemblages in the benthos, with amphipods being the most abundant group, followed by isopods. Sea ice was identified as the main driver shaping benthic peracarid assemblage structure (Chapter I). On a larger geographic scale and wider bathymetric range (e.g. including sampling locations from previous studies performed in the South Atlantic Ocean
and at a depth range from 160 to ~6000 m), depth was the main physical variable driving peracarid assemblage structure (Chapter III). In addition, 16157 isopod specimens from the Atlantic sector of the SO were identified to species level at a smaller scale (Chapter IV). In this case, sea ice was identified as the main physical driver affecting isopod diversity and composition among sampling locations (Chapter IV). Reduced concentration of sea ice
causes a decrease in isopod biodiversity, thus climate change was identified as a huge threat for this taxon and for SO benthos in general. During the identification process, two new isopod species were discovered (Chapter V). The two new species (Notopais sp.1 n. sp. and Notopais sp.2 n. sp.) were accurately described and identified by means of integrative taxonomy. This provided the first whole genome sequencing of benthic isopods from the SO and the first complete mitochondrial genome of the genus Notopais (Chapter V). Thanks to the collaboration with the University of Genoa (Dipartimento di Scienze della Terra dell'Ambiente e della Vita, DISTAV, Italy) and the National Antarctic Museum (MNA) in Genoa, two new SO species of the suborder Valvifera G. O. Sars, 1883 were described by means of classical taxonomy. In this case, a molecular approach could not be used because both new species were represented by a single specimen, therefore it was important to preserve the integrity of the holotypes (Chapters VI-VII).
Generally speaking, protein import into mitochondria and chloroplasts is a post-translational process during which the precursor proteins destined for mitochondria or chloroplasts are translated with cytosolic ribosomes and targeted. The previous results showed that the isolated chloroplasts can import in vitro synthesized proteins and the absence of ribosomes in the immediate area around chloroplasts in electron microscopy (EM) images. However, none of the EM images were recorded in the presence of a translation elongation inhibitor. Also, the observation showed that ribosomes stably bind to purified liver mitochondria in vitro, and the first indication of chloroplast localization of mRNAs encoding plastid proteins in Chlamydomonas rheinhardtii, which challenge the post-translational import and support the co-translational process. Therefore, in this study, the association of the ribosomes to the isolated chloroplasts were analyzed, a binding assay was established and showed that naked ribosomes are not considerably bound to chloroplasts. Additionally, mRNA localize in close vicinity to mitochondria also challenged post-translation protein import. Global analysis of transcripts bound to mitochondria in yeast or human revealed that around half of the transcripts of mitochondrial proteins displayed a high mitochondrial localization. The observed association of mRNAs with chloroplast fractions and the in vivo analysis of the distribution of mRNAs was used as base to formulate the hypothesis that mRNA can bind to chloroplast surface. Therefore, in this study, the mRNA binding assay was established and revealed that mRNAs coding for the mitochondrial cytochrome c oxidase copper chaperone COX17 showed unspecific binding to the chloroplasts. The mRNA coding for chloroplast outer envelope transport protein OEP24 and mRNA coding for the essential nuclear protein 1 (ENP1) showed specific binding, and OEP24 has a 3-fold higher affinity than ENP1 mRNA. Moreover, the BY2-L (Nicotiana tabacum non-green cell culture) could confer the highest enhancement of OEP24 mRNA binding efficiency than the COX17 and ENP1 mRNA and the preparation of the BY2-L was optimized. Afterwards, the feasibility to fix the interaction between mRNA and the proteins on the surface of chloroplasts was confirmed. OEP24 mRNA showed more efficiency in the UV-crosslinking. Following, the pull-down with antisense locked nucleic acid (LNA)/DNA oligonucleotides was established which could be used for the further investigation of the proteins involved in the mRNA binding to the chloroplasts.
Plastic pollution is a pervasive problem. In the environment, both the physical and chemical aspects of the material contribute to pollution. For instance, discarded plastic is useless waste that is fragmented upon degradation and so-called microplastics <5 mm are formed. Besides, the chemicals added into plastics are usually customized for specific functions, but these can easily transfer from the polymer into an ambient medium. This work examined both of these aspects. Moreover, the question of whether ecotoxicological effects are more likely to appear because of the microparticle properties or the chemicals transferring from the microplastics was addressed. A special focus was laid on the UV-weathering-induced chemical release.
First, conventional and biodegradable plastics made from fossil and bio-based resources were chosen. The different materials (pre-production and recycled pellets as well as final products)were weathered and their leachates evaluated in vitro. The leachates were analyzed with nontarget screening in order to measure the number of transferred chemicals. Plastics identified as toxic were subjected to further investigations in vivo. A biodegradable shampoo bottle was processed to microplastics and the particles’ physical and chemical properties were assessed with the freshwater worm Lumbriculus variegatus. Here, commonly used endpoints such as mortality, reproduction and weight were tested via different exposure routes. Moreover, the freshwater shrimp Neocaridina palmata was exposed to microplastic beads and fragments to clarify if the shape of the particles affects the ingestion and egestion, respectively. Thereafter, two materials that displayed the strongest toxic responses in vitro within the first study were weathered and leached. Finally, the shrimps were exposed to the leachates and the locomotor behavior was used as an ecologically relevant but less frequently studied endpoint.
The results of the studies highlight that plastics are chemically complex mixtures, containing a wide range of chemicals in terms of the number and functionality. These chemicals induced oxidative stress, baseline toxicity and endocrine activities. This shows that pellets represent a processing state that comprises chemically heterogenous materials. Moreover, it was shown that a degradation initiator is not necessarily relevant to trigger inherent substances to leach out from plastics. Despite this, the UV-weathering resulted in increasingly released chemicals and exacerbated the in vitro toxicities. Even plastics assessed as toxicologically harmless prior to weathering released toxic chemical mixtures once they were weathered. One recycled and all of the biodegradable plastics were toxicologically most concerning. This means that such materials are currently not better than conventional, virgin plastics in terms of their toxicity.
To clarify the source of the microplastic toxicity, L. variegatus was exposed to biodegradable microplastics. The particles were ingested by the worms and adversely affected the examined endpoints. In comparison, microplastics that were depleted from their chemicals via a solvent treatment were less toxic. Kaolin as a natural particle control was evaluated alongside and positively affected the weight of the worms. This emphasizes the ecological relevance of fine-sized matter for the test species. The chemicals extracted from the microplastics induced a 100% mortality. A chemical analysis of the material revealed two ecotoxicologically relevant biocides. The physically-mediated effects of the microplastics seemed to be less of a concern for the worms, which is probably linked to their adaptation to high concentrations of naturally occurring particles in the environment. However, the effects related to the chemicals of plastic cannot be ignored, especially for materials that are claimed to be environmentally friendly.
In the third study, the role of the particle shape in the gut passaging of N. palmata was studied. While the particle size was a determinant factor for the ingestion, the ingestion and egestion of the beads and fragments did not differ, respectively. The shrimps ingested less fragments when food was provided than in the absence of food. As for the worms, the shrimps are known to ingest many naturally occurring particles. Their unselective feeding behavior towards the particle shape could indicate that microplastics as a physical pollutant are negligible for the shrimps. That is why the chemicals of the two most toxic in vitro materials were tested with N. palmata. However, no trend towards elevated or reduced movements of the shrimps was observed, even though the leachates contained baseline toxicants. This shows that the in vitro toxicities of plastics are not necessarily indicative for effects to occur at the in vivo level...
Until quite recently, stem cell technology mainly focused on pure populations of embryonic stem cells (ES) derived from the inner cell mass of the blastocyst and induced pluripotent stem cells (iPS). Using organoids, a newly established culture technique, it is now possible to culture also organ and patient-specific adult stem (AS) and induced pluripotent stem (IPS) cells in vitro. Furthermore, it has been shown that adult stem cells, grown as organoids, are genetically stable, proliferate and maintain their multi-potency (often a bi-potency) for months. This is possible by providing conditions that recapitulate the stem cell niche of the corresponding organ. Particularly, defined growth factors and a physiological scaffold, which is provided by an extracellular matrix (ECM). Because of increasing research activities, organoids became influential in the recent years. Wide-ranging interest also led to a clearer definition: organoids must contain multiple organ-specific cell types, must be able to recapitulate some organ specific functions, and the cells must be spatially organized in a way similar to the organ they are derived from. The excitement about organoids is based on their high potential as a model to understand wound healing, cellular behaviour and differentiation processes in organogenesis. Furthermore, high potential in the drug development and in personalized stem cell therapeutic approaches has been shown. Specifically, for personalized stem cell therapy, one potential application is for chronic autoimmune diseases such as Diabetes type 1 (T1D). T1D is characterized by the immune-mediated destruction of ß-cells in the Pancreas that leads to absolute insulin deficiency. In T1D the first-line therapeutic approach is exogenous insulin replacement therapy, which always implicates the risk of high fluctuations in blood-sugar levels and therefore the risk of hypoglycaemia. Another therapeutic approach is the xenotransplantation of islets from human donors. A successful islet transplantation allows patients a years-long insulin independence. However, the therapeutic value of islet transplantation is highly limited by the availability of organ donors and by the need for chronic administration of immune suppressive medication. The use of pancreas organoids offers a promising alternative as a personalized cell therapeutic approach to treat T1D without the hypoglycaemia risks of the established therapies. In 2013 Meritxell Huch and colleagues established for the first-time organoids from the exocrine, ductal part of the pancreas. These pancreas organoids are characterized by a monolayered, spherical cell epithelium which comprises a liquid filled lumen. In addition, they showed that after transplantation of these cells into immunodeficient mice, they differentiate into ß-cells and cure T1D. However, basic knowledge of the culture growth behaviour is still lacking: to date, no growth parameters are defined and reliable and robust investigation approaches are still missing. Furthermore, basic knowledge about the organoid development and biochemical/biophysical mechanisms that generate the phenotypic structure are not identified. For a clinical approach these parameters are fundamental and therefore must be defined pre-clinically.
The aim of this study is the preclinical characterization of the hPOs...
Interleukin-11 signaling is a global molecular switch between regeneration and scarring in zebrafish
(2022)
The two diametrically opposing outcomes after tissue damage are regeneration and fibrotic scarring. After injury, adult mammals predominantly induce fibrotic scarring, which most often leads to patient lethality. Fibrotic scarring is the deposition of excessive extracellular matrix that matures and hinders tissue function. The scarring response is mainly orchestrated by myofibroblasts, which arise only upon tissue damage, from various cellular origins, including tissue resident fibroblasts, endothelial cells and circulating blood cells. On the contrary, species like zebrafish, possess the remarkable capacity to regenerate their damaged tissues. After injury, instead of inducing a myofibroblast-mediated fibrogenic gene program, cells in these species undergo regenerative reprogramming at the transcriptional level to activate vital cellular processes needed for regeneration, including proliferation, dedifferentiation, and migration. Several pro-regenerative mechanisms have been identified to date. Most of them, if not all, are also important for tissue homeostasis and hence, are not injury specific. Therefore, the central aim of this study is to identify injury-specific mechanisms that not only induce regeneration, but also limit fibrotic scarring.
To test the notion that fibrotic scarring limits regeneration, I first compared the scarring response in the regenerative zebrafish heart after cryoinjury with what is known in the non-regenerative adult mouse heart. I found that zebrafish display ~10-fold less myofibroblast differentiation compared to adult mouse after cardiac injury. With these findings, I hypothesized that zebrafish employ mechanisms to actively suppress scarring response. Using a novel comparative transcriptomic approach coupled with genetic loss-of-function analyses, I identified that Interleukin-6 (Il-6) cytokine family-mediated Stat3 is one such pro-regenerative pathway in zebrafish.
Il-6 cytokine family consists of Il-6, Interleukin-11 (Il-11), Ciliary neurotrophic factor, Leukemia inhibitory factor, Oncostatin M, and Cardiotrophin-like cytokine factor 1. Il-6 family ligands signal through their specific receptors and a common receptor subunit (Il6st or Gp130). Using gene expression analyses after adult heart and adult caudal fin injuries in zebrafish, I identified that both the Il-11 cytokine encoding paralogous genes (il11a and il11b) are the highest expressed and induced among the Il-6 family cytokines. Hence, I chose Il-11 signaling as a candidate pathway for further analysis. To investigate the role of Il-11 signaling, I generated genetic loss-of-function mutants for both the ligand (il11a and il11b) and the receptor (il11ra) encoding genes. Using various tissue regeneration models across developmental stages in these mutants, I identified that Il-11/Stat3 signaling is indispensable for global tissue regeneration in zebrafish.
To investigate the cellular and molecular mechanisms by which Il-11 signaling promotes regeneration, I performed transcriptomics comparing the non-regenerative il11ra mutant hearts and fins with that of the wild types, respectively. I identified that Il-11 signaling orchestrates both global and tissue-specific aspects of regenerative reprogramming at the transcriptional level. In addition, I also found that impaired regenerative reprogramming in the il11ra mutant hearts and fins resulted in defective cardiomyocyte and osteoblast repopulation of the injured area, respectively.
On the other hand, by deep phenotyping the scarring response in il11ra mutant hearts and fins, I identified that Il-11 signaling limits myofibroblast differentiation. Furthermore, I found that cardiac endothelial cells and fibroblasts are one of the major responders to injury-induced Il-11 signaling. Using lineage tracing, I found that both the endothelial and fibroblast lineages in the non-regenerative il11ra mutants commit to a myofibroblast fate, spearheading the scarring response. In addition, using cell type specific manipulations, I showed that Il-11 signaling in cardiac endothelial cells allows cardiomyocyte repopulation of the injured area. Finally, using human endothelial cells in culture, I uncovered a novel feedback mechanism by which Il-11 signaling limits fibrogenic gene expression by inhibiting its parent activator and a master regulator of tissue fibrosis, TGF-β signaling.
Overall, I identified Interleukin-11/Stat3 signaling as the first global regulator of regeneration in zebrafish. Briefly, I showed that Interleukin-11 signaling promotes regeneration by regulating two crucial cellular aspects in response to injury – (1) it promotes regenerative reprogramming, thereby allowing cell repopulation of the injured area and (2) it limits mammalian-like fibrotic scarring by inhibiting myofibroblast differentiation and TGF-β signaling. Altogether, these zebrafish data, together with the contradicting mammalian data strongly indicate that the secrets of tissue regeneration lie downstream of IL-11 signaling, in the differences between regenerative and non-regenerative species. Furthermore, I establish the non-regenerative il11ra mutant as an invaluable zebrafish model to study mammalian tissue fibrosis.
The heart is the first functional organ that develops in the embryo. To become a functional organ, it undergoes several morphogenetic processes. These morphogenetic events involve different cell types, that interact with each other and respond to the surrounding extracellular matrix, as well as intrinsic and extrinsic mechanical forces, assuming different behaviors. Additionally, transcription factor networks, conserved among vertebrates, control the development.
To have a better understanding of cell behavior during development, it is necessary to find a model system that allows the investigation in vivo and at single-cell resolution. Thanks to the common evolutionary origin of the different cardiac structures, together with the conserved molecular pathways, the two-chambered zebrafish heart offers many advantages to study cell behavior during cardiac morphogenesis. Here, using the zebrafish heart as a model system, I uncovered the cell behavior behind two of the main cardiac morphogenetic events: cardiac wall maturation and cardiac valve formation.
In the first part of this study, I investigated how the cardiac wall is maintained at the molecular level. Using genetic, transcriptomic, and chimeric analyses in zebrafish, we find that Snai1b is required for myocardial wall integrity. Global loss of snai1b leads to the extrusion of CMs away from the cardiac lumen, a process we show is dependent on cardiac contractility. Examining CM junctions in snai1b mutants, we observed that N-cadherin localization was compromised, thereby likely weakening cell-cell adhesion. In addition, extruding CMs exhibit increased actomyosin contractility basally, as revealed by the specific enrichment of canonical markers of actomyosin tension - phosphorylated myosin light chain (active myosin) and the α-catenin epitope α-18. By comparing the transcriptome of wild-type and snai1b mutant hearts at the early stages of CM extrusion, we found the dysregulation of intermediate filament genes in mutants including the upregulation of desmin b. We tested the role of desmin b in myocardial wall integrity and found that CM-specific desmin b overexpression led to CM extrusion, recapitulating the snai1b mutant phenotype. Altogether, these results indicate that Snai1 is a critical regulator of intermediate filament gene expression in CMs and that it maintains the integrity of the myocardial epithelium during embryogenesis, at least in part by repressing desmin b expression.
In the second part of this study, I focused on the behavior of valve cells during cardiac development. Using the zebrafish atrioventricular valve, I focus on the valve interstitial cells which confer biomechanical strength to the cardiac valve leaflets. We find that initially AV endocardial cells migrate collectively into the cardiac jelly to form a bilayered structure; subsequently, the cells that led this migration invade the extracellular matrix (ECM) between the two EC monolayers, undergo an endothelial-to-mesenchymal transition as marked by loss of intercellular adhesion, and differentiate into VICs. These cells proliferate and are joined by a few neural crest-derived cells. VIC expansion and a switch from a pro-migratory to an elastic ECM drive valve leaflet elongation. Functional analysis of Nfatc1 reveals its requirement during VIC development. Zebrafish nfatc1 mutants form significantly fewer VICs due to reduced proliferation and impaired recruitment of endocardial and neural crest cells during the early stages of VIC development. Analysis of downstream effectors reveals that Nfatc1 promotes the expression of twist1b, a well-known regulator of epithelial-to-mesenchymal transition. This study shows for the first time that Nfatc1 regulates zebrafish VICs formation regulating valve EMT in part by regulating twist1b expression. Moreover, it proposes the zebrafish valve as an excellent model to study the cellular and molecular process that regulate VIC development and dysfunction.
In conclusion, my work: 1) identified an unsuspected role of Snai1 in maintaining the integrity of the myocardial epithelium, opening new avenues in its role in regulating cellular contractility; 2) uncovered the function of Nfatc1 in the establishment of the VIC, establishing a new model to study valve development and function.
Despite all advancements in cancer research and clinical practice, cancer remains a life- threatening disease with an increasing incidence. According to a 2018 WHO forecast, cancer incidence will double to approximately 37 million new cancer cases by 2040. Today, clinical management of cancer is based on a "one-fits-all" strategy. Most cancers are still treated by surgical therapy followed by adjuvant or neoadjuvant chemotherapy based on rather strict guidelines (S3 guidelines in Europe) which are based on studies of large cohorts of patients with the same tumor entity. While this approach has led to substantial increases in progression-free survival and overall patient survival, most patients do not benefit from the administered treatment regimen. One reason for this is intra-tumor heterogeneity, which results from clonal evolution between cancer cells and their environment. This means that cancer patients may respond differently to a particular drug due to the different mutation patterns of their tumor cells. Therefore, patients should be screened in advance for reliable cancer biomarkers that definitively predict whether they will respond to a particular therapy. This would increase the probability of a successful treatment.
Colorectal cancer (CRC) is the third most diagnosed cancer and the second leading cause of cancer deaths worldwide. The main cause of death in CRC is a metastatic disease, which is presented in 20 % of patients and eventually develops in more than 30 % of early-stage patients. Despite the significant increase (to more than 30 months) in median survival with the development of cytotoxic agents and the introduction of targeted therapy, the progression-free survival in the first-line setting has remained largely unchanged over the past decade.
The heterogeneity in CRC is characterized by alterations in multiple signaling pathways that affect cellular functions such as cell proliferation or apoptosis. Commonly affected signaling pathways include the mitogen-activated protein kinase (MAPK)- and the transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP)-pathway. Alterations in the TGF-β/BMP pathway, due to mutations in the SMAD4 gene (mothers against decapentaplegic homolog 4), are associated with different drug response and promote resistance to chemotherapy. In addition, they are associated with a higher recurrence rate.
SMAD4 is one of the most common cancer driver genes, and mutations occur in up to 15 % of CRC cases. Therefore, there is an urgent need for therapeutic agents that can specifically target SMAD4-mutated tumors.
The aim of the present study was the identification of the clinical relevance of the SMAD4 gene and the investigation of its suitability as a potential biomarker in CRC.
For this purpose, I investigated sibling patient-derived organoids (PDOs) derived from different regions of a chemo-naïve CRC tumor. PDOs are 3D cell cultures that reliably recapitulate the architecture of the tissue of origin, as well as preserve the genomic background and intra-tumor heterogeneity. The sibling PDOs (R1R361H and R4wt) shared the most common CRC mutations, such as KRASG12D (kirsten rat sarcoma), PIK3CAH1047R (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha), and TP53C242F (tumor protein 53), but differed in a SMAD4R361H mutation and showed a different drug response. The single nucleotide variant R361H of the SMAD4 gene is among the most common pathogenic alterations in various cancers, including CRC.
The sibling PDOs showed significant differences in response to the MEK-inhibitors cobimetinib, trametinib, and selumetinib. MEK-inhibitors are antineoplastic agents that inhibit the function of MEK1 and MEK2, preventing phosphorylation of transcription factors, which leads to inhibition of tumor cell proliferation. MEK-inhibitors are approved for the treatment of malignant melanoma. Currently, they are in phase-III clinical trials for the treatment of patients with metastatic CRC.
To investigate whether SMAD4R361H is responsible for sensitivity to MEK-inhibitors, Iestablished three syngeneic PDOs harboring a SMAD4R361H mutation using the CRISPR/Cas9 genome editing system. All CRISPR-PDOs were significantly more sensitive to the MEK-inhibitors, compared to R4wt. I have shown that the SMAD4R361H mutation is responsible for sensitivity to MEK inhibition in CRC models and may be a predictive biomarker.
To test this hypothesis, I examined 62 CRC PDO models and treated them with the MEK-inhibitors cobimetinib, trametinib, and selumetinib. All models that had a pathogenic mutation or deletion in the SMAD4 gene (15 %) were sensitive to cobimetinib, 10 % of models were sensitive to trametinib, and 8 % were sensitive to selumetinib.
I performed transcriptome (RNA sequencing) and proteome analyses using the DigiWest® method to investigate the mechanism underlying MEK-inhibitor sensitivity.
DigiWest® is a Luminex® bead-based analysis that allows the simultaneous analysis of over 100 (phospho-)proteins. The transcriptome and proteome data support the observation that MEK inhibition primarily affects SMAD4R361H PDOs. Furthermore, I have shown that activation of the BMP signaling pathway in organoids with wild-type SMAD4 appears to be responsible for resistance to MEK-inhibitors. Thus, a genetic alteration in the BMP signaling pathway, beyond SMAD4, could lead to sensitivity to MEK-inhibitors.
I identified four genes involved in the TGF-β/BMP signaling pathway that are frequently mutated in CRC and grouped them into the so-called SFAB-signature (SMAD4, FBXW7 (F-box/WD repeat-containing protein 7), ARID1A (AT-rich interactive domain-containing protein 1A), or BMPR2 (Bone morphogenetic protein receptor type II). Clinical data show that approximately 36 % of CRC patients have at least one pathogenic mutation in these genes.
I tested all 62 CRC PDO models and found a significant positive prediction for sensitivity to cobimetinib (95 %) and selumetinib (70 %) for the SFAB-signature. Trametinib and the newly approved MEK-inhibitor binimetinib showed a similar trend. Therefore, the SFAB-signature has high predictive power for response to MEK-inhibitors and could be used as a predictive biomarker panel.
The current clinically used biomarkers for CRC are based on the mutation status of driver genes KRAS and BRAF, which are present in up to 50 % and 10 % of CRC, respectively. Investigation of molecular alterations in CRC revealed that mutations in the KRAS gene, which is downstream of EGFR (epidermal growth factor receptor) in the MAPK-pathway, interfere with an anti-EGFR-antibody therapy (e.g., cetuximab). Therefore, cetuximab is only relevant for RAS wild-type tumors. However, approximately 40 % of patients with RAS wild-type status do not respond to this treatment.
About 53 % of CRC PDO models carry a pathogenic RAS mutation, about 10 % harbor a pathogenic BRAF mutation. Both, the RAS and RAF status alone as well as the combination of RAS and RAF status with SFAB-signature did not provide a better prediction of sensitivity to MEK inhibition.