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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.
Eine große Gruppe von Aptameren sind die Guanosintriphosphat (GTP) Aptamere. Diese zeigt sehr eindrücklich, wie RNA unterschiedliche Strategien nutzt, um denselben Liganden zu erkennen. Die komplette Struktur des GTP Klasse II Aptamers wird in der ersten Publikation gezeigt. Interessanterweise zeichnet die Struktur ein stabil protoniertes Adenine unterhalb der GTP-Bindestelle aus. Dieses wurde durch eine Kombination aus weiterführenden NMR- und ITC-Experimente untersucht und charakterisiert. Es zeigte sich, dass die protonierte Base einen pKs-Wert hat, der weit von der Neutralität verschoben ist. Die Protonierung ist auch noch bei sehr basischen Puffern stabil.
Eine Art der funktionellen Protonierung wird von den zyklischen di-Nukleotiden (CDN) bindenden Riboswitches genutzt, um zwei CDN mit ähnlicher Affinität zu binden. c-di-GMP Riboswitches wurden als regulatorische Einheit beschrieben und deren Kristallstruktur aufgeklärt. Mutationsexperimente führten dazu, dass bei einer G-zu-A Mutation an der Gα-Bindestelle die Selektivität des Riboswitches verändert wurde. Die Mutante bindet sowohl c-di-GMP als auch cGAMP mit ähnlichen Bindungsaffinitäten. Riboswitche, die cGAMP binden wurden auch in der bakteriellen Genomen gefunden. Hierbei ist die Promiskuität unterschiedlich stark ausgeprägt. Die Untersuchung des Bindungsmodus und der damit verbundenen Promiskuität ist in der zweiten Publikation beschrieben. Hier wurde gezeigt, dass die Riboswitche beide Liganden nur binden können, wenn zur Bindung von c-di-GMP das Ligand bindende A protoniert vorliegt. Auch diese Protonierung konnte mit weiterführenden NMR- und ITC-Experimenten charakterisiert werden. Die Untersuchungen einer solch großen RNA sind mit NMR Spektroskopie herausfordernd. Hierbei wurde ausgenutzt, dass die Kristallstruktur bereits bekannt war, welche allerdings die Protonierung nicht zeigte. Auch diese Protonierung zeigt einen pKs-Wert, der weit von der Neutralität verschoben ist und außerdem bei unterschiedlichen pH stabil ist.
In den beiden untersuchten Beispielen wurden zwei verschiedene Arten von Protonierung gezeigt: eine strukturelle und eine funktionelle. Das GTP Klasse II Aptamer benutzt die Protonierung als strukturelle Basis für die Basis der Ligandenbindungsstelle. Hierbei werden durch die Protonierung des Adenines mehr nutzbare Wasserstoffbrücken ausgebildet und damit die Tertiärstruktur stabilisiert. Im Unterschied dazu nutzen die promiskuitiven CDN Ribsowicthes die Protonierung, um verschiedene Liganden binden zu können und es kommt damit zu einer Verschiebung der Funktionalität. Der regulatorische Nutzen dafür ist allerdings noch unbekannt.
Auch bei den SAM Riboswitches wurde ein promiskuitiver Vertreter beschrieben. SAM Riboswitches gehören zu den am längsten bekannten Klassen der Riboswitches. Bis heute sind hier die meisten unterschiedlichen Klassen bekannt. SAM wird häufig als Donor für funktionelle Gruppen benutzt, besonders häufig als Methlygruppendonor für die Methylierung einer Reihe unterschiedlicher Substrate (z.B. DNA, Proteine, Metabolite etc.). Bei dieser Reaktion entsteht SAH als Nebenprodukt. Zusätzlich ist SAH zelltoxisch, da es affin an Methyltransferasen bindet und damit diese essenzielle Reaktion inhibiert. Eine enge Kontrolle der SAH-Konzentration ist daher kritisch. SAM bindende Riboswitches haben zu SAM eine bis zu 1000-fach höhere Bindungsaffinität im Vergleich zu SAH. Die Beschreibung eines translationalen OFF-Riboswitches, der SAM und SAH mit ähnlicher Affinität bindet, ist daher überraschend. Zumal seine Genassoziation fast ausschließlich zu SAM Synthetasen ist, deren Regulation durch SAH wenig sinnvoll erscheint. Um ein besseres Verständnis für die Funktion des SAM/SAH Riboswitches zu erhalten, wurde seine 3D-Struktur mittels NMR-Spektroskopie aufgeklärt, wie in der vierten Publikation beschrieben. Dafür mussten zunächst alle Resonanzen der Sequenz und dem Liganden zugeordnet werden, wie in der dritten Publikation beschrieben. Dabei wurde als Ligand SAH gewählt, da dieser chemisch stabiler und damit für die teils tagelangen NMR-Messungen besser geeignet ist. Zusätzlich wurden Mutanten bzw. verwandte Liganden mittels ITC Experimente auf ihre Bindungseigenschaften untersucht, um die Bedeutung der Linkerlänge, einzelner Basenpaare und funktionelle Gruppen des Liganden zu untersuchen. Bei anderen bekannten SAM Riboswitches umschließt die RNA den Liganden fast komplett. Dabei wird zum einem das Sulfoniumion spezifisch durch die Carboxylgruppen verschiedener Uracil-Nukleotide erkennt und koordiniert. Außerdem bildet sich eine Bindetasche aus, die genug Platz für die stabile Bindung der Methylgruppe hat. Beim SAH Riboswitch wird die Selektivität für SAH dadurch erreicht, dass die Bindetasche sterisch keinen Platz für die Methylgruppe von SAM bereitstellt.
Zusammenfassend wurden in dieser Arbeit drei verschiedene Ligand bindende RNA-Strukturen untersucht, die alle sehr unterschiedliche Strategien zur Bindung der Liganden nutzen. Obwohl Portionierungen bei Aptameren und Riboswitches selten beschrieben wurden, haben sie eine maßgebliche Funktion in den beiden zuerst untersuchten Strukturen. Obwohl bisher im Hinblick auf alle bekannten RNA Strukturen eher selten beschrieben, gibt es doch neben den genannten zwei, einige Beispiele für strukturelle oder funktionelle Protonierungen. Auch in Hinblick auf zukünftige bzw. Verbesserung bestehender RNA-Strukturvorhersage-Programme ähnlich wie sie für Proteine schon lange nutzt werden, müssen protonierte Nukleobasen ernsthaft in Betracht gezogen werden. Außerdem konnte gezeigt werden, dass zwei der untersuchten Riboswitches zwei Liganden mit ähnlicher Affinität binden. Die genutzte Strategie ist hierbei unterschiedlich. Während bei den promiskuitiven CDN Riboswitches der regulatorische Nutzen noch unbekannt ist, konnte für den SAM/SAH Ribsowitch gezeigt werden, dass SAH nur zufällig aufgrund der wahrscheinlich sehr niedrigen intrazellulären Konzentration gebunden wird und dieser daher wahrscheinlich später in der evolutionären Entwicklung entstanden ist. Riboswitches halten es weiterhin spannend.
The intensive use of the North Sea area through offshore activities, sand mining, and the spreading of dredged material is leading to increasing pollution of the ecosystem by chemicals such as hydrophobic organic contaminants (HOCs). Due to their toxicological properties and their ability to accumulate in the environment, HOCs are of particular concern. The contaminants partition between aqueous (pore water, overlying water) and solid phases (sediment, suspended particulate matter, and biota) within these systems. The accumulated contaminants in the sediment are of major concern for benthic organisms, who are in close contact with sediment and interstitial water. It is thus particularly important to better understand how contaminants interact with biota, as these animals may contribute to trophic transfer through the food web. Furthermore, sediments are a crucial factor for the water quality of aquatic systems. They not only represent a sink for contaminants but also determine environmental fate, bioavailability, and toxicity. The Marine Strategy Framework Directive (MSFD) was introduced to protect our marine environment across Europe and includes the assessment of pollutant concentrations in the total sediment, which, however, rarely reflects the actual exposure situation. The consideration of the pollutant concentrations in the pore water is not implemented, although this is needed for the evaluation of bioavailability and risk assessment. For this reason, special attention is given to further development, implementation, and validation of pollutant monitoring methods that can determine the bioavailable fraction in sediment pore water. For risk assessment purposes, it is furthermore important to use biological indicators in addition to classical analytics to determine the effect of pollutants on organisms. The main objective of this thesis was to gain insight into the pollution load and the potential risk of hydrophobic organic chemicals (HOCs) in the sediment of the North Sea and to evaluate these results with regard to possible risks for benthic organisms and the ecosystem. The following five aims are covered within these studies to gain a holistic assessment of sediment contamination:
1. Assessment of the pore water concentrations of PAHs and PCBs
2. Determination of the bioturbation potential by macrofauna analysis
3. Application of the SPME method on biological tissue
4. Assessment of recreated environmental mixtures in passive dosing bioassays
5. Development of SPME method for DDT in sediments
The thesis is comprised of three main studies supported by three additional studies ...
Coupling between epidermis and amphid morphogenesis during embryonic development of C. elegans
(2021)
Sensory organs are fundamental for survival of animal populations, since the detection of environmental stimuli is crucial for localization of nourishment, predators or mating partners. In nematodes, the amphid (AM) sensilla are the largest sensory organs for detection of chemical compounds.
This study investigates how the AM sensilla acquire their special elongated shape during lima-bean to 1.5-fold embryonic stages of C. elegans head development. The dissertation also examines events facilitating the morphogenesis of other head sensilla (IL/OL/CEP) and addresses aspects of general embryonic head morphogenesis. Using high resolution live-cell imaging techniques with different combinations of markers highlighting specific tissues, this study shows that epidermal head enclosure, migration of AM socket cells (pores) and translocation of AM dendrite tips are coupled processes, facilitating the elongation of AM dendrites. Importantly, during AM dendrite elongation the AM neural cell bodies are staying stationary. Manipulation through conducting UV-Laser ablation (epidermis close to pore/pore) and RPN-6.1 dsRNA interference resulted in compromised AM pore migration and impaired dendrite elongation. This leads to the conclusion that AM pores need to be physically attached (through C. elegans apical junctions, CeAJ) to the migrating epidermal sheet and to AM dendrite tips for successful AM morphogenesis. This study infers that RPN-6.1 plays an important role for correct AM pore morphogenesis and AM pore to AM dendrite tip attachment. Our results lead to the conclusion that head enclosure drives AM pore migration and AM dendrite elongation with AM neural cell bodies staying stationary. Thereby, CeAJ are interconnecting AM dendrite tips to AM pores and CeAJ link the sensillar ending to the migrating epidermis. Thus, migration of attached target tissue (pore), with neural cell bodies staying stationary (constituting an abutment), creates a pulling force facilitating AM dendrite elongation. This passive neurite elongation procedure is coined dendrite towing in this study.
Additionally, this study discovers that translocation of IL, OL and CEP head sensilla pores is influenced by apical constriction. This conclusion was made based on the findings that IL/OL/CEP pores migrate towards the prospective mouth anterior to the epidermal leading edge, separated from AM pores and irrespective of highly impaired AM sensilla morphogenesis after strong RPN-6.1 depletion. Also, concurrent with translocation of IL/OL/CEP pores, bottle-shaped cells occur and non-muscle-myosin and apical polarity factors are getting enriched at the anterior most part of the head, indicating de-novo manifestation of apical constriction. It is furthermore assumed that apical constriction in arcade cells might contribute to early pharynx development. All in all, this study reveals two force-generating events: Head enclosure-driven AM sensilla morphogenesis via dendrite towing and, otherwise, apical constriction-facilitated translocation of IL/OL/CEP sensilla pores. These events can get separated by graded depletion of the proteasome activator RPN-6.1.
Autism spectrum disorder (ASD) is a common neurodevelopmental disorder with a multifarious clinical presentation. Even though many genetic risk factors have been identified and studied in mouse models, the neurophysiological mechanisms underlying the autistic phenotype are still unclear. Based on the high rates of comorbidity with epilepsy, it was hypothesized that the balance between excitation and inhibition in neural circuits may be disrupted in autistic individuals.
In this dissertation, synaptic and network activity was measured in three different genetically modified mouse models that exhibit the characteristic behavioral abnormalities of the disorder: the Neurobeachin (Nbea) haploinsufficient mouse, the Neuroligin-3 (Nlgn3) knockout (KO) mouse, and the Neuroligin-4 (Nlgn4) KO mouse. Each of the affected proteins is involved in the formation and/or function of synapses in the central nervous system. Therefore, it was posited that the reduction or deletion of these proteins might alter the balance of excitatory to inhibitory synaptic transmission in individual neurons and in neural circuits. Extracellular recordings in the hippocampal dentate gyrus of anesthetized mice revealed that the excitation-inhibition (E-I) balance was reduced in Nbea haploinsufficient and Nlgn4 KO mice, but unchanged in Nlgn3 KO mice despite a reduction in excitatory synaptic transmission to dentate granule cells. Unexpectedly, the intrinsic excitability of dentate granule cells was altered in all three mouse models. These results imply that a homeostatic increase in the intrinsic excitability is able to compensate for the decreased excitatory transmission in Nlgn3 KO mice, whereas the decreased intrinsic excitability in the Nbea haploinsufficient and Nlgn4 KO mice leads to a reduction in the E-I balance. Taken together, these findings suggest that the influence of genetic factors on the E-I balance might be a potential common mechanism underlying the development of ASD.