Biologische Hochschulschriften (Goethe-Universität)
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Trait-dependent effects of biotic and abiotic filters on plant regeneration in Southern Ecuador
(2024)
Tropical forests have always fascinated scientists due to their unique biodiversity. However, our understanding of ecological processes shaping the complexity of tropical rainforests is still relatively poor. Plant regeneration is one of the processes that remain understudied in the tropics although this is a key process defining the structure, diversity and assembly of tropical plant communities. In my dissertation, I combine experimental, observational and trait-based approaches to identify processes shaping the assembly of seedling communities and compare associations between environmental conditions and plant traits across plant life stages. By working along a steep environmental gradient in the tropical mountains of Southern Ecuador, I was able to investigate how processes of plant regeneration vary in response to biotic and abiotic factors in tropical montane forests.
My dissertation comprises three complementary chapters, each addressing an individual research question. First, I studied how trait composition in plant communities varies in relation to the broad- and local-scale environmental conditions and across the plant life cycle. I measured key traits reflecting different ecological strategies of plants that correspond to three stages of the plant life cycle (i.e., adult trees, seed rain and recruiting seedlings). I worked on 81 subplots along an elevational gradient covering a large climatic gradient at three different elevations (1000, 2000 and 3000 m a.s.l.). In addition, I measured soil and light conditions at the local spatial scale within each subplot. My findings show that the trait composition of leaves, seeds and seedlings changed similarly across the elevational gradient, but that the different life stages responded differently to the local gradients in soil nutrients and light availability. Consequently, my findings highlight that trait-environment associations in plant communities differ between large and small spatial scales and across plant life stages.
Second, I investigated how seed size affects seedling recruitment in natural forests and in pastures in relation to abiotic and biotic factors. I set up a seed sowing experiment in both habitat types and sowed over 8,000 seeds belonging to seven tree species differing in seed size. I found that large-seeded species had higher proportions of recruitment in the forests compared to small-seeded species. However, small-seeded species tended to recruit better in pastures compared to large-seeded species. I showed that high surface temperature was the main driver of differences in seedling recruitment between habitats, because it limited seedling recruitment of large-seeded species. The results from this experiment show that pasture restoration requires seed addition of large-seeded species and active protection of recruiting seedlings in order to mitigate harmful conditions associated with high temperatures in deforested areas.
Third, I examined the associations between seedling beta-diversity and different abiotic and biotic factors between and within elevations. I applied beta-diversity partitioning to obtain two components of beta-diversity: species turnover and species richness differences. I associated these components of beta-diversity with biotic pressures by herbivores and fungal pathogens and environmental heterogeneity in light and soil conditions. I found that species turnover in seedling communities was positively associated with the dissimilarity in biotic pressures within elevations and with environmental heterogeneity between elevations. Further, I found that species richness differences increased primarily with increasing environmental heterogeneity within elevations. My findings show that the associations between beta-diversity of seedling communities and abiotic and biotic factors are scale-dependent, most likely due to differences in species sorting in response to biotic pressures and species coexistence in response to environmental heterogeneity.
My dissertation reveals that studying processes of community assembly at different plant life stages and spatial scales can yield new insights into patterns and processes of plant regeneration in tropical forests. I investigated how community assembly processes are governed by abiotic and biotic filtering across and within elevations. I also experimentally explored how the process of seedling recruitment depends on seed size-dependent interactions, and verified how these effects are associated with abiotic and biotic filtering. Identifying such processes is crucial to inform predictive models of environmental change on plant regeneration and successful forest restoration. Further exploration of plant functional traits and their associations with local-scale environmental conditions could effectively support local conservation efforts needed to enhance forest cover in the future and halt the accelerating loss of biodiversity.
Neurodevelopmental psychiatric disorders (NPDs) like attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), and schizophrenia, affect millions of people worldwide. Despite recent progress in NPD research, much remains to be discovered about their underpinnings, therapeutic targets, effects of biological sex and age. Risk factors influencing brain development and signalling include prenatal inflammation and genetic variation. This dissertation aimed to build upon these findings by combining behavioural, molecular, and neuromorphological investigations in mouse models of such risk factors, i.e. maternal immune activation (MIA), neuron-specific overexpression (OE) of the cytoplasmatic isoforms of the RNA-binding protein RBFOX1, and neuronal deletion of the small Ras GTPase DIRAS2.
Maternal infections during pregnancy pose an increased risk for NPDs in the offspring. While viral-like MIA has been previously established elsewhere, this study was the first in our institution to implement the model. I validated NPD-relevant deficits in anxiety- and depression-like behaviours, as well as dose- and sex-specific social deficits in mouse offspring following MIA in early gestation. Proteomic analyses in embryonic and adult hippocampal (HPC) synaptoneurosomes highlighted novel and known targets affected by MIA. Analysis of the embryonic dataset implicated neurodevelopmental disruptions of the lipid, polysaccharide, and glycoprotein metabolism, important for proper membrane function, signalling, and myelination, for NPD-pertinent sequelae. In adulthood, the observed changes encompassed transmembrane trafficking and intracellular signalling, apoptosis, and cytoskeletal organisation pathways. Importantly, 50 proteins altered by MIA in embryonic and adult HPC were enriched in the NPD-relevant synaptic vesicle cycle. A persistently upregulated protein cluster formed a functional network involved in presynaptic signalling and proteins downregulated in embryos but upregulated in adults by MIA were correlated with observed social deficits. 49/50 genes encoding these proteins were significantly associated with NPD- and comorbidity-relevant traits in human phenome-wise association study data for psychiatric phenotypes. These findings highlight NPD-relevant targets for future study and early intervention in at-risk individuals. MIA-evoked changes in the neuroarchitecture of the NPD-relevant HPC and prefrontal cortex (PFC) of male and female mice highlighted sex- and region-specific alterations in dendritic and spine morphology, possibly underlining behavioural phenotypes.
To further investigate genetic risk factors of NPDs, I performed a study based on the implications of RBFOX1’s pleiotropic role in neuropsychiatric disorders and previous preclinical findings. Cytoplasmatic OE of RBFOX1, which affects the stability and translation of thousands of targets, was used to disseminate its role in morphology and behaviour. RBFOX1 OE affected dendritic length and branching in the male PFC and led to spine alterations in both PFC and HPC. Due to previously observed ASD-like endophenotypes in our Rbfox1 KO mice and the importance of gene × environment effects on NPD susceptibility, I probed the interaction of cytoplasmatic OE and a low-dose MIA on offspring. Both RBFOX1 OE alone and with MIA led to increased offspring loss during the perinatal period. Preliminary data suggested that RBFOX1 OE × MIA might increase anxiety- and anhedonia-like behaviours. Morphological changes in the adult male OE HPC and PFC suggested increased spine density and reduced dendritic complexity. A small post-mortem study in human dorsolateral PFC of older adults did not reveal significant effects of a common risk variant on RBFOX1 abundance.
To expand upon NPD genetic risks, I evaluated the effects of a homo- (KO) or heterozygous (HET) Diras2 deletion in a novel, neuron-specific mouse model. DIRAS2’s function is largely unknown, but it has been associated with ADHD in humans and neurodevelopment in vitro. In adult mice, there were subtle sex-specific effects on behaviour, i.e. more pronounced NPD-relevant deficits in males, in keeping with human data. KO mice had subtly improved cognitive performance, while HET mice exhibited behaviours in line with core ADHD symptoms, e.g. earning difficulties (females), response inhibition deficits and hyperactivity (males), suggesting Diras2 dose-sensitivity and sex-specificity. The morphological findings revealed multiple aberrations in dendritic and spine morphology in the adult PFC, HPC, and amygdala of HET males. KOs changes in spine and dendritic morphology were exclusively in the PFC and largely opposite to those in HETs and NPD-like phenotypes. Region- and genotype-specific expression changes in Diras2 and Diras1 were observed in six relevant brain regions of adult HET and KO females, also revealing differences in the survival and morphology regulator mTOR, which might underlie observed differences.
In conclusion, the effects of MIA and partial Diras2 knockdown resembled each other in core, NPD-associated behavioural and morphological phenotypes, while cytoplasmatic RBFOX1 OE and full Diras2 KO differed from those. My findings suggest complex dose- and sex-dependent relationships between these prenatal and genetic interventions, whose NPD-relevant influences might converge onto neurodevelopmental molecular pathways. An assessment of such putative overlap, based on available data from the MIA proteomic analyses of embryonic and adult HPC, suggested the three models might be linked via downstream targets, interactions, and upstream regulators. Future studies should disseminate both distinct and shared aspects of MIA, RBFOX1, and DIRAS2 relevant to NPDs and build upon these findings.
Research on the human and animal microbiome has become increasingly important in recent years. It is now widely accepted the gut microbiome is of crucial importance to health, as it is involved in a large number of physiological processes. The term ‘microbiome’ refers to the all living microorganisms including their genes and metabolites in a defined environment, while the specific composition of microorganisms consisting of bacteria, archaea and protozoa is referred to as the ‘microbiota’ (Lane-Petter, 1962; Lederberg and McCray, 2001).
In recent years, research has focused on various of these communities in the soil (Fierer, 2017), water (Sunagawa et al., 2015), air (Leung et al., 2014) and especially in the human gut. However, this topic is also becoming increasingly relevant for the conservation of endangered species. In the face of global mass extinctions and the listing of over 42,000 animal species as ‘critically endangered’, conservation breeding programmes are more important than ever (Díaz et al., 2019; IUCN, 2022). The responsibility for these tasks lies with zoological institutions, which are dedicated to animal conservation and the continuous monitoring of animal welfare. Microbiome research offers a non-invasive method to support species conservation. By analysing faecal samples, microbial markers can be identified that provide important information about the health status and reproductive cycle of animals (Weingrill et al., 2004; Antwis et al., 2019). Zoological facilities also provide an ideal research environment for comparing individuals from different habitats. In addition, all necessary metadata such as age, sex, kinship or medical treatment are documented and can be used for the analysis.
This is the starting point for this thesis. In order to identify such microbial markers, it is necessary to understand the microbiome of a variety of animal species. The first aim is therefore to characterise the faecal microbiota of 31 mammalian species, focusing on herbivores and carnivores. It could be shown that they differ significantly in terms of both microbial diversity and microbiota composition. Herbivorous species express a very diverse microbial composition, consisting mainly of cellulose-degrading taxa of the families Fibrobacteraceae or Spirochaetaceae. In contrast, the microbiota of carnivorous species is less diverse and is dominated by protein-degrading Fusobacteriaceae and Clostridiaceae. In addition, this thesis proves that the microbiota of herbivorous species is highly consistent, whereas the microbiota of carnivorous species is highly variable. The results of this study provide important insights for the sampling scheme of future projects. Especially when analysing carnivorous species, single samples are not sufficient to capture the full variability of the microbiome.
These results lead to the question of whether this variability can be explained by daily fluctuations in the individual microbiome and whether this can be used to distinguish between species or individuals. Using individual longitudinal data and a combined approach of clustering algorithms and dynamic time warping, it is shown that such a distinction is possible at the species and individual level. This was confirmed for both a carnivorous (Panthera tigris) and a herbivorous (Connochaetes taurinus) species. These results confirm the influence of the host individual on the faecal microbiota, in addition to the often described influence of diet (Ley et al., 2008a; Kartzinel et al., 2019).
Based on the knowledge gained from these studies, a methodology has been developed that will enable the conservation of species in the field to be supported by microbiome research in the future. The focus here lays on the identification of host-specific metadata based on the faecal microbiota. The developed regression model is able to distinguish between carnivorous, herbivorous and omnivorous hosts with up to 99% accuracy. In addition, a more accurate phylogenetic classification of the family (Canidae, Felidae, Ursidae, Herpestidae) can be made for carnivorous hosts. For herbivorous hosts, the model can predict the respective digestive system with up to 100% accuracy, distinguishing between ruminants, hindgut fermenters and a simple digestive system. The acquisition of host-specific metadata from an unknown faecal sample is an important step towards establishing microbiome research in species conservation. Field studies in particular will benefit from such new methods. Usually, costly microsatellite analysis and high-quality host DNA are required to obtain host-specific information from faecal samples. The newly developed method offers a less costly and labour-intensive alternative to conventional techniques and opens up a more accessible field for microbiome research in the field.
Exploring strategies to improve the reverse beta-oxidation pathway in Saccharomyces cerevisiae
(2024)
Microbes are the most diverse living organisms on Earth, with various metabolic adaptations that allow them to live in different conditions and produce compounds with different chemical complexity. Microbial biotechnology exploits the metabolic diversity of microorganisms to manufacture products for different industries. Today, the chemical industry is a significant energy consumer and carbon dioxide emitter, with processes that harm natural ecosystems, like the extraction of medium-chain fatty acids (MCFAs). MCFAs are used as precursors for biofuels, volatile esters, surfactants, or polymers in materials with enhanced properties.
However, their current extraction process uses large, non-sustainable monocultures of coconut and palm trees. Therefore, the microbial production of MCFAs can help reduce the current environmental impact of obtaining these products and their derivatives.
In nature, fatty acids are mostly produced via fatty acid biosynthesis (FAB). However, the reverse β-oxidation (rBOX) is a more energy-efficient pathway compared to FAB. The rBOX pathway consists of four reactions, which result in the elongation of an acyl-CoA molecule by two carbon units from acetyl-CoA in each cycle. In this work we used Saccharomyces cerevisiae, an organism with a high tolerance towards toxic compounds, as the expression host of the rBOX pathway to produce MCFAs and medium-chain fatty alcohols (MCFOHs).
In the first part of this work, we expanded the length of the products from expressing the rBOX in the cytosol and increased the MCFAs titres. First, we deleted the major glycerol-3-phosphate dehydrogenase (GPD2). This resulted in a platform strain with significantly reduced glycerol fermentation and increased rBOX pathway activity, probably due to an increased availability of NADH. Then, we tested different combinations of rBOX enzymes to increase the length and titres of MCFA. Expressing the thiolase CnbktB and β-hydroxyacyl-CoA dehydrogenase CnpaaH1 from Cupriavidus necator, Cacrt from Clostridium acetobutylicum and the trans-enoyl-CoA reductase Tdter (Treponema denticola) resulted in hexanoic acid as the main product.
Expressing Cncrt2 (C. necator) or YlECH (Y. lipolytica) as enoyl-CoA hydratases resulted in octanoic acid as the main product. Then, we integrated the octanoic (Cncrt2 or YlECH) and the hexanoic acid (Cacrt)-producing variants in the genome of the platform strain and we achieved titers of ≈75 mg/L (hexanoic acid) and ≈ 60 mg/L (octanoic acid) when growing these strains in a complex, highly buffered medium. These are the highest titers of octanoic and hexanoic acid obtained in S. cerevisiae with the rBOX. Additionally, we deleted TES1 and FAA2 to prevent competition for butyryl-CoA and degradation of the produced fatty acids, respectively.
However, these deletions did not improve MCFA titers. In addition, we tested two dual acyl-CoA reductase/alcohol dehydrogenases (ACR/ADH), CaadhE2 from C. acetobutylicum and the putative ACR/ADH EceutE from Escherichia coli, in an octanoyl-CoA-producing strain to produce MCFOH. As a result, we produced 1-hexanol and 1-octanol for the first time in S. cerevisiae with these two enzymes. Nonetheless, the titres were low (<10 mg/L and <2 mg/L, respectively), and four-carbon 1-butanol was the main product in both cases (>80 mg/L). This showed the preference of these two enzymes for butyryl-CoA.
In the second part of this work, we expressed the rBOX in the mitochondria of S. cerevisiae to benefit from the high levels of acetyl-CoA and the reducing environment in that organelle. First, in an adh-deficient strain, we mutated MTH1, a transcription factor regulating the expression of hexose transporters, and deleted GPD2. This resulted in a strain with a reduced Crabtree effect and, therefore, an increased carbon flux to the mitochondria. We partially validated the increased flux to the mitochondria by expressing the ethanol-acetyltransferase EAT1 from Kluyveromyces marxianus in this organelle. This resulted in a higher isoamyl acetate production in the MTH1-mutant strain. Isoamyl acetate is synthesised by Eat1 from acetyl-CoA and isoamyl alcohol, a product of the metabolism of amino acids in the mitochondria. Then, we targeted different butyryl-CoA-producing rBOX variants to the mitochondria, and we used the production of 1-butanol and butyric acid as a proof-of-concept. The strong expression of all the enzymes was toxic for the cell, and the highest butyric acid titres (≈ 50 mg/L) in the mitochondria from the rBOX were obtained from the weak expression of the pathway. The highest 1-butanol titers (≈ 5 mg/L) were obtained with the downregulation of the mitochondrial NADH-oxidase NDI1. However, this downregulation led to a non-desirable petite phenotype.
In summary, we produced hexanoic and octanoic acid for the first time in S. cerevisiae using the rBOX and achieved the highest reported titers of hexanoic and octanoic acid so far using this pathway in S. cerevisiae. In addition, we successfully compartmentalised the rBOX in the mitochondria. However, competing reactions, some of them essential for the viability of the cell, limit the use of this organelle for the rBOX.
Subject of this thesis was the investigation of the actin-interacting and glucocorticoid-sensitive Protein DRR1 (or Fam107a) and its role in promoting stress resilience in the murine hippocampus.
We proposed the hypothesis that DRR1 through its actin-binding properties specifically modulates neuronal actin dynamics and promotes resilience through synaptic plasticity leading to subsequently improvement of cognitive performance and social behavior. The accompanied AMPA-receptor transport could create an efficient way regulating neural function and complex behavior during stress episodes.
By utilizing fluorescent immunohistochemistry, we showed basal expression of DRR1 primarily in the murine cerebellum and hippocampal CA3 and CA1 area. Co-staining with different cell marker proteins showed DRR1 expression in neurons, microglia and especially in astrocytic end-feet, which create contact to the brain vasculature.
To test whether DRR1 and AMPA receptor function correlate to modulate stress-associated consequences, primary hippocampal neuron cultures were transduced with adeno-associated virus (AAV) for overexpression or suppression of the protein. Western Blot analysis showed a positive correlation between the AMPA-receptor subunit GluR2 and DRR1 amounts. Further the application of the proximity ligation assay (PLA) in untreated neural cultures indicated interaction between DRR1 and the AMPA receptor subunit GluR2. To address whether DRR1 even affects AMPAR trafficking we performed the “newly inserted assay” after AAV-treatment of primary hippocampal neuron cultures. Suppression of DRR1 revealed less newly inserted GluR2 subunits as compared to controls. Inconclusive were the results upon DRR1 overexpression, however they point to no changes.
In the second part we correlated behavioral phenotypes originating from in vivo overexpression and suppression of DRR1 in the murine hippocampus with potential alterations in neuronal morphology. Therefore, in vitro analysis was performed utilizing AAV transduced primary hippocampal cultures overexpressing or suppressing DRR1. Synchronously the viral vector included a green fluorescent protein (GFP) being expressed throughout the complete neural cell. GFP staining was used to verify successful transfection and for reconstruction of dendritic arbors and dendritic stretches for spine classification. DRR1 suppression showed reduced total spine numbers especially evoked by reduced numbers of immature spine classes – namely long thin spines and filopodia. Whereas mature mushroom spines and stubby spines were unaffected. By overexpressing DRR1, tendencies inclined against higher total dendritic lengths, branch points and increased dendritic arbors in comparison to controls. In regard of spines, total numbers were unaffected. However, mature mushroom spines were significantly declined in numbers, but compensated by increased numbers of immature long thin spines and filopodia.
Chronic social defeat stress (CSDS) is widely used in mouse models to study the effects of stress and resilience. We exposed C57Bl/6J mice expressing GFP under the Thy1 promoter CSDS and categorized them into resilient (R+/-), susceptible (R-/-) and non-learning (R+/+) mice following a modified social interaction test (MSIT). We found alterations in CA1 spine compositions with resilient animals resembling the untreated phenotype. Stress susceptible and non-learning animals displayed reduced numbers in stubby spines with simultaneous increases in mature mushroom spines. In addition, we could detect a tendency towards more immature spines in susceptible animals and non-learners, mirroring our in vitro results.
Finally, we present a different investigative approach in this thesis. Sequenced acute stress was previously found to compromise cognition including spine loss.
We aimed to investigate the implication of acute stress on DRR1 levels and its occurrence in diverse cell types of the brain. We subjected one group of C57Bl/6J mice to acute stress and injected another group with the artificial glucocorticoid DEX. Six hours post stress, animals were perfused and brains were subsequently immunobiologically analyzed. We found DRR1 protein levels elevated in the hippocampus of stressed and DEX-treated animals compared to controls. Interestingly, DRR1 seemed was especially elevated in endothelial cells. This coincides with our investigations finding DRR1 present in astrocytic end-feet under basal conditions and might claim a participation of DRR1 in the blood-brain-barrier integrity.
Our results show DRR1 as actin-interacting and glucocorticoid-sensitive gene affecting structural plasticity of hippocampal spines. Moreover, DRR1 directly interacts with AMPA glutamate receptors and presumably is involved in AMPA trafficking to the postsynaptic membrane. In addition, this study could demonstrate that DRR1 is expressed by other cell types of the brain. Of special interest is DRR1’s occurrence in astrocytic end-feet and endothelial cells suggesting a role as integrator of cell-cell communication and to this end also acting as modifier of stress-induced consequences at the neurovascular unit.
In vivo data of chronically stressed mice displayed no phenotypic differences in hippocampal pyramidal neurons of resilient animals as compared to unstressed mice. Morphological alterations of spine structures were particularly visible in stress susceptible and non-learning animals. Integrating our findings with existing behavioral data, we can conclude that DRR1 plays a role in stress resilience whereby it needs to be expressed in a tightly managed homeostatic equilibrium.
The functional and molecular role of transglutaminase 2 in hematopoietic stem and progenitor cells
(2023)
Long-term repopulating hematopoietic stem cells (LT-HSCs) that reside in the bone marrow (BM) give rise to all blood cell types including erythrocytes, leukocytes and platelets. LT-HSCs are mainly quiescent during steady state hematopoiesis. LT-HSCs can process self-renewal to expand and maintain stemness, or commit to differentiation into short-term (ST) repopulating HSC and multipotent progenitors (MPPs). MPPs differentiate into oligopotent lineagerestricted progenitors which eventually produce all mature blood cell lineages, and thereby regenerate hematopoietic system.
Previous studies have shown in transcription profiles and quantitative PCR (qPCR) analysis that transglutaminase 2 (Tgm2) is one of the most upregulated genes in quiescent LT-HSCs in comparison to active HSCs, mobilized HSCs, ST-HSCs, MPPs, as well as leukemic stem cells (LSC). However, the reason why Tgm2 is strongly upregulated in dormant mouse LTHSCs and what the role of Tgm2 is in LT-HSCs has not been investigated yet.
Tgm2, encoded by the Tgm2 gene, is a multi-functional protein within the transglutaminase family. It has been found to be widely expressed inside and outside the cells. It consists of four domains and two functionally exclusive forms that are regulated by the Ca2+ and GTP concentration. Besides the most well-known transglutaminase enzymatic activity for transamidation, deamidation and crosslinking, Tgm2 acts also as a GTPase/ATPase, kinase, adhesion/scaffold protein, as well as disulfide isomerase. The role of Tgm2 in hematopoiesis remains elusive. Accordingly, the aim of this dissertation is to investigate the role of Tgm2 in murine hematopoiesis, especially in murine LT-HSCs.
Firstly, the expression of Tgm2 was analyzed in highly purified murine hematopoietic stem and progenitor cell (HSPC) populations. Low input label-free mass spectrometric proteomics and WES protein analysis confirmed the highly specific expression of Tgm2 in LT-HSCs at protein level. Already at the state of MPPs, Tgm2 protein was almost absent with further decline towards oligopotent progenitors. These results indicated Tgm2 as a specific protein marker for LT-HSCs, justifying the future generation of a fluorescent reporter mouse line based on endogenous Tgm2 tagging.
To delineate the functional and molecular role of Tgm2 in LT-HSCs, a conditional Tgm2 knockout mouse model was generated using the Mx1-Cre/loxP system, with the loxP sites flanking the coding exons of the catalytic domain of Tgm2. After PolyIC-mediated induction, a more than 95% knockout efficiency was observed in purified LT-HSCs and the protein expression of Tgm2 was confirmed to be vanished in the purified LT-HSCs from conditional Tgm2-KO mice. Conditional knockout mice are viable and show no aberrant organ functions.
In steady state condition, the distribution of mature blood cell lineages and immunophenotypically-defined HSPC populations within the BM, the mitochondrial potential of HSPCs reflected by the non-invasive cationic dye JC-1, as well as the cell cycle status of HSPCs mirrored by the intracellular Ki67 staining did not show any significant variations upon loss of Tgm2. However, the in vitro continuous observation of prospectivly isolated LT-HSCs by time-lapse microscopy-based cell tracking revealed a delayed entry into cell cycle with a two fold increased apoptosis rate after knocking out Tgm2, indicating Tgm2 expression might be essential for survival of LT-HSCs. Moreover, while the absence of Tgm2 in LT-HSCs did not influence differentiation and lineage choice in vitro, overexpression of Tgm2 in LT-HSCs resulted in an increase of the most immature subpopulation upon cultivation. All these features were not observed in Tgm2-deleted MPPs, suggesting Tgm2 playing a specific function at the level of LT-HSCs. Upon stress hematopoiesis, induced by the administration of 5-fluorouracil (5-FU), there was a trend towards delayed recovery of LT-HSCs lacking Tgm2. Although Tgm2 express specificly in LT-HSCs, two rounds of competitive BM serial transplantation displayed an equal overall engraftment and multi-lineage reconstitution of LT-HSCs from Tgm2-WT and Tgm2-KO mice in peripheral blood (PB), BM and spleens. Interestingly, LT-HSCs from Tgm2-KO mice reconstituted to more myeloid cells and fewer B cells in the first four weeks after primary transplantation, which disappeared at later time points.
Gene expression profiling and simultaneous single cell proteo-genomic profiling indicated that HSPCs and LT-HSCs from Tgm2-KO mice were transcriptionally more active. A heterogeneity of Tgm2 expression within Tgm2-WT LT-HSCs was revealed by single cell data. Commonly up-regulated genes in Tgm2-KO LT-HSCs and MPPs were significantly involved in regulation of transcription from RNA polymerase II promoter in response to stress, positive regulation of cell death as well as negative regulation of mitogen-activated protein kinase (MAPK) signaling pathways. In Tgm2-KO LT-HSCs, 136 up-regulated genes demonstrated an enrichment of genes involved in apoptosis, as well as negative regulation of MAPK signaling pathway.
Taken together, this dissertation shows that Tgm2 protein is highly specifically expressed in LT-HSCs, but not in subsequent progenitor populations. However, Tgm2 is not essential for differentiation and maturation of myeloid lineages, the proliferation and the long-term multilineage reconstitution potential of LT-HSCs after transplantation. Tgm2 might be involved in accurate stress response of LT-HSCs and the transition from LT-HSCs into MPPs, meaning that the absence of Tgm2 results in poor survival, myeloid bias upon transplantation, as well as slower recovery upon chemotherapeutic treatment.
Sphingolipids are not only structural components of cell membranes but can also act as signalling molecules in different pathways. Sphingolipid precursors, Ceramides (Cer), are synthesized de novo by six different synthases (CerS1-6) which generate Cer of different chain lengths. Cer can be further synthesized to glycosphingolipids and sphingomyelin. Cell membrane parts that are enriched in glycosphingolipids are so-called lipid rafts and can function as signalling platforms for different receptors, such like the T cell receptor (TCR). CD4+ T cells play a crucial role in the development of ulcerative colitis, a chronic inflammatory disease of the colon. As CerS3 expression was increased in the white blood cells of human colitis patients, the role of CerS3 in the TCR signalling and colitis was investigated in this dissertation. By lenti-viral transduction of a CerS3-shRNA into a CD4+ Jurkat cell line, it was shown that CerS3 has an impact on activated T cells. A decrease of different sphingolipids after T cell activation via CD2/3/28 activation beads and IL2 treatment was observed that was accompanied by an inhibition of Zap70 phosphorylation, an important protein of the TCR signalling. The impaired TCR signalling led to a diminished NFAT1 translocation into the nucleus which subsequently led to a reduced NFAT1- dependent TNFα release. Downregulation of CerS3 in primary CD4+ T cells, obtained from the blood of healthy volunteers, also showed a reduced release of pro-inflammatory cytokines after activation. This dissertation demonstrates a pivotal role for CerS3 in T cell function and highlights CerS3 as potential new target for T cell driven colitis.
Gravitropism is a fundamental process in plants that allows shoots to grow upward and roots to grow downward. Protein phosphorylation has been postulated to participate in the intricate signaling cascade of gravitropism. In order to elucidate the underlying mechanisms governing the gravitropic signaling and unearth novel protein constituents, an exhaustive investigation employing microgravity-induced phosphoproteomics was undertaken. The significantly phosphorylated proteins unraveled in this study can be effectively divided into two groups through clustering analysis. Furthermore, the elucidation of Gene Ontology (GO) enrichment analysis disclosed the conspicuous overrepresentation of these clustered phosphoproteins in cytoskeletal organization and in hormone-mediated responses intimately intertwined with the intricate phenomenon of gravitropism. Motif enrichment analysis unveiled the overrepresentation of [-pS-P-] and [-R-x-x-pS-] motifs. Notably, the [-pS-P-] motif has been suggested as the substrate for the Casein kinase II (CK II) and Cyclin-dependent kinase (CDK). Kinase-inhibitor assays confirmed the pivotal role played by CK II and CDK in root gravitropism. Mutant gravitropism assays validated the functional significance of identified phosphoproteins, with some mutants exhibiting altered bending kinetics using a custom-developed platform. The study also compared phosphoproteomics data from different platforms, revealing variations in the detected phosphopeptides and highlighting the impact of treatment differences. Furthermore, the involvement of TOR signaling in microgravity-induced phosphorylation changes was uncovered, expanding the understanding of plant gravitropism responses.
To fulfill the large-scale verification of interesting candidates from the phosphoproteomics study, a novel root and hypocotyl gravitropism phenotyping platform was developed. This platform integrated cost-effective hardware, including Raspberry Pi, a high-quality camera, an Arduino board, a rotation stage (obtained from Prof. Dr. Maik Böhmer), and programmable green light (modified by Sven Plath). In addition, through collaboration with a software developer, machine-learning-based software was developed for data analysis. This platform tested the gravitropic response of candidate mutants identified in the phosphoproteomics study. Furthermore, the capabilities of this platform were expanded to investigate tropisms in other species and organs. To find novel proteins that might act as partners of a key protein that is involved in gravitropism signaling, ALTERED RESPONSE TO GRAVITY 1 (ARG1), immunoprecipitation coupled with Mass Spectrometry (IP-MS) was performed and identified ARG1-LIKE1 (ARL1) as a potential interacting protein with ARG1. This interaction was further confirmed through in vivo pull-down assays and bimolecular fluorescence complementation assays. In addition, the interaction between ARG1 and HSP70-1 was also validated.
Overall, this thesis sheds light on the molecular components and signaling events involved in plant gravitropism. It contributes to existing knowledge and opens up new ways to investigate this fascinating area of plant biology.
The role of Apelin signaling and endocardial protrusions during cardiac development in zebrafish
(2023)
During cardiac development, cardiomyocytes (CMs) are delaminated from the compact muscle wall to increase the muscle mass of the heart. This process is also known as cardiac trabeculation. It has been shown that growth factors produced by endocardial cells (EdCs) are required for myocardial morphogenesis and growth. In particular, Neuregulin produced by EdCs promotes myocardial trabeculation. The deficiency of Neuregulin signaling leads to hypotrabeculation. Endocardial protrusions project from the endocardium to the myocardium are also essential for the trabeculae onset. Yet current studies only introduce the function of endocardial sprouts descriptively. This article first reports the mechanisms of endocardial sprouting during myocardial trabeculation. By living imaging, we first demonstrate that EdCs interact with CMs through membrane protrusions in zebrafish embryos. More interestingly, these protrusions stay in close contact with their target CMs in spite of the cardiac contraction. We utilize loss-of-function strategies to report the importance of myocardial apelin, which induces endocardial protrusion formation. Zebrafish lacking Apelin signaling exhibit defects in endocardial protrusion formation as well as excessive deposition of cardiac jelly and hypotrabeculation. Notably, we also present data that blocking protrusion formation in endocardial cells phenocopies the trabeculation defects in apelin mutants. Mechanistically, endocardial-derived Neuregulin requires Apelin signaling mediated endocardial protrusions, and Neuregulin dependent pERK expression is attenuated in the condition of reduced endocardial protrusion formation. Together, our data suggest that endocardial-myocardial communication through endocardial protrusions acts as an underlying principle allowing myocardial growth.
Discrepancies between knockdown and knockout animal model phenotypes have long stood as a perplexing phenomenon. Several mechanisms explaining such observations have been proposed, namely the toxicity or the off-target effects of the knockdown reagents, as well as, in certain cases, genetic robustness – an organism's ability to maintain its phenotype despite genetic perturbations. In addition to these explanations, transcriptional adaptation (TA), a phenomenon defined as an event whereby a mutation in one gene leads to transcriptional upregulation or downregulation of another, adapting, gene or genes expression, has been recently proposed as an alternative explanation for the conflicting knockdown and knockout phenotype paradox.
Since its discovery in 2015, TA's precise mechanism remains a subject of ongoing research. Majority of evidence suggests that mutant mRNA degradation plays a central in TA. Epigenetic remodeling is also thought to play a role, as evidenced by an increase in active histone marks at the transcription start sites of the adapting genes. Whether mRNA degradation is indeed the key player in TA remains debated. Furthermore, it is still unknown how exactly TA develops, what adapting genes it targets, and whether genomic mutations that render mutant mRNA sensitive to degradation are required for TA to occur.
Throughout the experiments described in this Dissertation, I have designed an inducible TA system where TA can be triggered on demand and its effects on the cell’s transcriptome followed through time. I have demonstrated that degradation-prone transgenes, once induced and expressed, can be efficiently degraded, resulting in the protein loss-independent upregulation of adapting genes via TA. Adapting genes with higher degree of sequence similarity become upregulated faster than genes with lower degree of sequence similarity. Further functionality of this approach to study TA is limited by the leakiness of the inducible gene expression system; however, constitutively expressed degradation-prone transgenes were used to demonstrate TA in human cells.
In addition, I have developed an approach to target wild-type cytoplasmic mRNAs without altering the cell’s genome and reported a TA-like phenomenon, which manifested as adapting gene upregulation not relying on mutations in other genes. Cytoplasmic mRNA cleavage with CRISPR-Cas13d triggered a TA-like response in three different gene models: Actg1 knockdown, Ctnna1 knockdown, and Nckap1 knockdown. After comparing two different modes of triggering TA, CRISPR-Cas9 knockout versus CRISPR-Cas13d knockdown, I reported little overlap between the dysregulated genes and suggested that diverse mRNA degradation modes led to distinct TA responses. In addition, the transcriptional increase of Actg2 caused by CRISPR-Cas13d-mediated Actg1 mRNA cleavage did not require chromatin accessibility changes.
Experiments and genetic tools described in this dissertation investigated how TA develops from its earliest onset, how it affects the global transcriptome of the cell, as well as provided compelling evidence for an mRNA degradation-central TA mechanism. I have created tools to study both direct and indirect TA gene targets and unveiled important insights into the temporal dynamics of TA. Genes with higher sequence similarity were found to be upregulated more rapidly than those with lower similarity. Furthermore, it was revealed that the epigenetic properties of TA responses vary depending on the triggering mechanism. Cas13d-mediated degradation of wild-type mRNAs led to immediate transcriptional enhancement independent of epigenetic changes, which stood in contrast to previously measured alterations in chromatin accessibility in CRISPR-Cas9 mutants. This research has thus significantly advanced our knowledge of TA and provided valuable tools and findings that contribute to the broader understanding of gene expression regulation in response to mRNA degradation.
Bei den meisten erwachsenen Säugetieren führt ein Herzinfarkt zu Fibrose und Verlust von funktionellem Herzgewebe. Einige Wirbeltiere, wie der Zebrabärbling, besitzen jedoch die bemerkenswerte Fähigkeit, nach einer Schädigung ihres Herzgewebes verlorenes Gewebe zu regenerieren und so schädliche Folgen zu verhindern. Die lokale Immunantwort auf eine Verletzung wird zunehmend als eine wichtige Determinante für das regenerative Potential eines Gewebes gesehen. Das Komplementsystem ist Teil des humoralen Immunsystems. Historisch ist es als eine Sammlung von Protein bekannt, den Komplementkomponenten, die in der Leber synthetisiert werden und im Blutkreislauf zirkulieren. Bei Exposition gegenüber einem Auslöser, wie z. B. einem Pathogen, wird eine Komplementkomponentproteinspaltungskaskade initiiert, die dazu führen kann, dass Immunzellen rekrutiert werden, und, dass die Phagozytose erleichtert, ggf. die Zielzelle lysiert wird. Studien legen nahe, dass das Komplementsystem an zellulären Prozessen beteiligt sei, die für Entwicklungs- und Krankheitsprozesse entscheidend sind, wie etwa Proliferation und Dedifferenzierung. Es gibt Hinweise, dass das Komplementsystem eine Rolle bei Krebserkrankungen und bei regenerativen Prozessen spielen könnte. In verschiedenen Arten wurde eine lokale verletzungsinduzierte Expression von komplementkomponentkodierenden Genen in regenerierendem Gewebe beobachtet.
Einzelne Studien legen nahe, dass Funktionsverlust einzelner Komplementkomponenten regenerative Prozesse beeinträchtigt.
Offene Fragen bleiben jedoch: Ist die lokale Expression von mehreren komplementkomponentkodierenden Genen ein Merkmal von regenerierendem Gewebe, das sie von Geweben unterscheidet, welchem die Fähigkeit zur Regeneration fehlt? Und welche Rolle könnte das Komplementsystem und seine Komponenten während des regenerativen Prozesses spielen? Um diesen Fragen nachzugehen, wurde eine Expressionsanalyse von Zebrabärblingsgewebe nach Verletzung mittels RT-qPCR und in situ Hybridisierung durchgeführt: kardiale Kryoverletzung, Larvenrumpfamputation und Schwanzflossenamputation. Ich beobachtete, dass mehrere komplementkomponentkodierende Gene in diesen Geweben nach Verletzung induziert wurden. Die Interpretation veröffentlichter single cell RNAseq Datensätze legt nahe, dass diese komplementkomponentenkodierenden Gene von verschiedenen Zelltypen exprimiert werden, darunter Immunzellen, Epikardzellen und Fibroblasten. Um transkriptionelle Unterschiede zwischen regenerierendem und nicht regenerierendem Gewebe zu identifizieren, verwendete ich ein nicht regeneratives Zebrabärblingmodell, die il11ra- Mutante. Dieser Mutante fehlt die Fähigkeit, verschiedene Organe zu regenerieren, das ist der Fall beim Herzen, dem larvalen Rumpf, und der Schwanzflosse. Ich stellte fest, dass die Mehrheit der verletzungsinduzierten komplementkomponentkodierenden Gene il11ra nachgeschaltet war. Darüber hinaus zeigten Experimente unter Verwendung chemischer Inhibitoren, dass speziell die Expression der komplementkomponentkodierenden Gene c3a.1,
c4b und c7a im Larvenrumpfamputationsmodell durch den Il11-Stat3-Signalweg moduliert wird.
Zur Klärung der Frage, ob das Komplementsystem und/ oder seine Komponenten eine Rolle während der Regeneration spielen, wurden verschiede Funktionsverlustmodelle generiert und im larvalen Rumpfamputationsmodell auf mögliche Aberrationen getestet. Zum einen generierte ich Überexpressionslinien von endogenen Inhibitoren der Komplementproteinspaltungskaskade. Überexpression eines etablierten Komplementsysteminhibitors rca2.1/ tecrem führte zu einer im Vergleich zu Wildtyp- Geschwistern verringerten Regeneration des larvalen Rumpfs. Zum anderen generierte ich Funktionsverlustmutanten von individuellen Komplementkomponenten durch CRISPR/Cas9 vermittelter Mutagenese, und zwar für masp1, masp2, cfd, c1s, c4b, c5 und c9. Die larvale Rumpfregeneration war in diesen Mutanten unauffällig. Allerdings zeigten c4b Mutanten eine verringerte Kardiomyozytenproliferation und eine differenzielle Expression von einigen Markergenen, einschließlich einer erhöhten Expression von inflammatorischen Zytokinen.
Meine Studien führten zu neuen Einblicken in das Komplementsystem im Kontext der Regeneration. Ich fand heraus, dass mehrere komplementkomponentenkodierenden Gene in regenerierendem Zebrabärblinggewebe exprimiert werden, und zwar im Herzgewebe, im larvalen Rumpf und in der adulten Flosse. Darüber hinaus zeige ich, dass die verletzungsinduzierte Expression von komplementkodierenden Genen in regenerierendem Gewebe dem Regenerationsmasterregulator il11ra nachgeschaltet ist. Speziell c3a.1, c4b und c7a wurden durch il11/ stat3 reguliert...
Compaction and spheroid formation modulates stemness and differentiation of human pancreas organoids
(2023)
The incidence of diabetes type 1 (T1D) in children and young adults is increasing worldwide. T1D is well treated by insulin administration. However, there is currently no long-lasting cure for this ailment. The success rate of pancreatic islet transplantation to treat T1D is limited by the availability of patient-matched islets and the necessity of using life-long immunosuppressive medication. The difficulties caused by transplantation can be overcome by generating bio-engineered pancreatic islets from patient-derived progenitor cells. Aim of this thesis is to establish new strategies for the generation and analysis of pancreatic lineages derived from human progenitor cells. It reports on the optimization of a technique to form human pancreatic spheroids from hollow monolayered human pancreas organoids (hPOs) to investigate how cell-cell and cell-matrix interaction can be leveraged to induce endocrine differentiation of the pancreas progenitor cell organoids. We introduce cell aggregation protocols to generate endocrine pancreas cell lineages from ductal pancreatic cells. Next, we study the effect of co-culture with stromal and endothelial cells to promote cell differentiation toward a pancreatic fate enhancing β cells productivity.
This thesis has focused on identifying the differences in gene expression along with phenotypical transformation during differentiation of human pancreatic organoids (hPOs) towards human β cells to be used in the future of cellular therapeutics in treating T1D patients.
Baleen whales (Mysticeti) are a clade of highly adapted carnivorous marine mammals that can reach extremely large body sizes and feature characteristic keratinaceous baleen plates used for obligate filter feeding. From a conservation perspective, nearly all baleen whale species were hunted extensively over a roughly 100 years lasting time period that depleted many of the respective whale stocks with so far unknown consequences for e.g. their molecular viability. From an evolutionary perspective, the lack of fossil records together with conflicting molecular patterns resulted in a still unclear and debated phylogeny of modern baleen whales, particularly in rorquals (Balaenopteridae). In this dissertation, I will demonstrate the application of baleen whale genomes to tackle these open questions by using modern approaches of conservation and evolutionary genomics.
Conservation genomic aspects of baleen whales were addressed in two projects, both using whole genome data of either an Icelandic fin whale (Balaenoptera physalus) population or multiple blue whale (Balaenoptera musculus) populations to evaluate the impact of the industrial whaling era on their molecular viability. The results suggest a substantial drop in effective population size of both species but also a lack of manifestation in genotypes of the fin whale population when compared to the blue whale populations. Especially the rare and short runs of homozygosity (ROH), usually indicative for inbreeding, suggest frequent outcrossing in fin whales while all analyzed blue whale populations featured long and frequent ROH. In addition to these analyses, genome data of blue whale populations was further used to evaluate if northern hemisphere blue whales diverged into different subspecies. Population genetic and gene flow analyses showed clearly separated and well isolated populations in accordance with their assumed geographical distance. In contrast, the genome-wide divergence between all blue whale populations was low compared to other cetacean populations and to the next closely related sei whale species. Because this includes the morphologically different and well recognized pygmy blue whale subspecies, a proposal was made to equally categorize the two northern-hemisphere blue whale populations as subspecies.
Evolutionary aspects were addressed in a third project, by constructing the genome of the pygmy right whale (Caperea marginata) and testing its potential in phylogenetics and cancer research. Phylogenomic analyses using fragments of a whole-genome alignment featuring nearly all extant baleen whales, allowed the revision of the complex evolutionary relationships of rorquals by quantifying and characterizing the amounts of conflicts in early diverging branches. These relationships were further used to identify phylogenetically independent pairs of baleen whales with a maximum of diverging body size differences to compare rates of positive selection between their genomes. The results suggest nearly evenly distributed frequencies of alternative topologies which supports the representation of the early divergence of rorquals as a hard polytomy with high amounts of introgression and incomplete lineage sorting. Within the set of available genomic data, three independent pairs of baleen whales with diverging body sizes were found and comparisons of positive selection rates resulted in many potentially body size and cancer related genes. The lack of conserved selection patterns, however, suggest a more convergent evolution of size and cancer resistance like previously discussed in paleontology.
In conclusion, the application of whole genome data using methods of conservation genetics allowed for a comprehensive estimation about the molecular viability of blue and fin whales as well as an assessment of the taxonomic status of northern-hemisphere blue whale populations. The rather different results between blue and fin whales underlines the importance of genomic monitoring of baleen whales because different species show rather different molecular consequences of their potentially varying depletions. Furthermore, as showcased for the northern-hemisphere blue whale, many important isolated populations of baleen whales may still be unknown to conservation management and genome-wide comparisons will most likely contribute to overcome this under-classification problem. The application of whole genome data in evolutionary research allowed the characterization of the complex patterns of molecular conflicts within baleen whales and especially rorquals that will contribute to the still rather unclear understanding of their evolution. The here found molecular support for the idea of convergent evolution of gigantism in whales will further guide the search for molecular patterns responsible for Peto’s paradox.
Xylose, an abundant sugar fraction of lignocellulosic biomass, is a five-carbon skeleton molecule. Since decades, utilization of this sugar has gained much attention and has been in particular focus as a substrate for production of biofuels like ethanol by microbial hosts, including Saccharomyces cerevisiae. In this yeast, xylose is naturally not used as a carbon source, but its utilization could be achieved by metabolic engineering either via the oxidoreductive route or through the isomerase pathway. Both pathways share xylulose as a common intermediate that must be phosphorylated before entering the endogenous metabolism via the non-oxidative pentose phosphate pathway (noxPPP). Besides this, in some bacteria a non-phosphorylating oxidative pathway for xylose degradation exists, known as Weimberg pathway, where a molecule of xylose is converted by a series of enzymes - xylose dehydrogenase (XylB), xylonate dehydratase (XylD), 3-keto-2-deoxy-xylonate dehydratase (XylX) and α-ketoglutarate semialdehyde dehydrogenase (KsaD) - to form α-ketoglutarate (AKG). Besides having several useful properties as a product, AKG could also be used for cell growth as an intermediate of the tricarboxylic acid (TCA) cycle. One target of the present study is to establish a functional Weimberg pathway in S. cerevisiae. Previous studies have shown that this task is not trivial, for instance due to the toxicity of xylonate (the first metabolite of the pathway) and the involvement of an iron-sulfur cluster dependent enzyme, the D-xylonate dehydratase. The assembly of iron-sulfur clusters on a heterologous protein in yeast is known to be challenging.
To establish the Weimberg pathway in yeast, the genes xylB, xylD, and xylX were obtained from Caulobacter cresentus and ksaD was from Corynebacterium glutamicum. In a variant, the dehydratase xylD was replaced with orf41 from Arthrobacter nicotinovorans, which is believed to be independent of iron-sulfur clusters. Growth of yeast cells on xylose as a sole carbon source was expected as an indicator of a functional Weimberg pathway. However, the heterologous expression of the codon optimized genes was not sufficient to reach this goal. Due to the complexity of the interactions of the heterologous pathway with the endogenous cellular processes, it was assumed that potential limitations could be overcome by adaptive laboratory evolution, using xylose as a sole source of carbon. Increasing selection pressure was applied on a strain with Weimberg pathway genes integrated into the genome over several generations. As a variant of the evolutionary engineering approach, mutator strains were generated. For this, RAD27 and MSH2 genes were deleted, which are involved in nucleotide excision and mismatch repair mechanisms, respectively. Some of the resulting strains PRY24, PRY25, PRY27 and PRY28 were able grow in xylose as a sole carbon source after evolutionary engineering. As a control, a non-mutator strain PRY19 was also included. Strikingly, only the mutator strains were able to consume xylose as a sole carbon source, which shows the feasibility of the approach.
In addition to the mutator strain strategy, a further approach employed in the present study was the simultaneous expression of the Weimberg pathway in the cytosol and mitochondria. This was based on the reasoning that the iron-sulfur cluster biogenesis on XylD may be improved in the organelle and that the AKG is an intermediate of the TCA cycle. In the strain AHY02, all enzymes of the pathway were tagged with mitochondrial targeting signals in addition to a full cytosolically localized pathway. The localization of the mitochondrial variants was confirmed by fluorescence microscopy. Together with AHY02, CEN.PK2-1C wild type strain was also included as a control for evolution. When a selection pressure on xylose was applied, both strains - AHY02 and CEN.PK2-1C - were able to grow in the course of evolution. Deletion of the xylulokinase (XKS1) gene was found to be detrimental for both evolved strains in xylose-containing media. This suggests that the evolution of the endogenous oxidoreductive and noxPPP genes is responsible for growth of the evolved cells. For the evolved strain AHY02, it could also be possible that the Weimberg pathway genes supported to growth in addition to the oxidoreductive route. To elucidate the underlying molecular mechanisms, genome sequencing and reverse engineering approaches would be necessary in future.
In addition to screening for growth on xylose as a sole carbon source, a less stringent screening system was created to examine even a minor flux of xylose towards AKG. For this, all genes necessary for conversion of isocitrate to AKG where deleted, yielding a glutamate auxotrophic strain. In this system, the cells can grow on other carbon sources, whereas xylose is only provided as a source of AKG for the synthesis of glutamate...
Identification of new natural products from nematode-associated bacteria using mass spectrometry
(2023)
This work aims to find unknown natural products produced by bacteria, that live in close association with nematodes and to elucidate their structure by using mass spectrometry.
The first chapter of this work is dedicated to the detection of hitherto unknown natural products by using a metabolomics approach and subsequent structure elucidation of said compounds. This chapter includes metabolomics analysis of Xenorhabdus szentirmaii wild type and knockout mutants, overproduction of the target compound, identification of derivatives from other strains and MS based structure elucidation.
The second and third chapters are about natural products that protect C. elegans from B. thuringiensis infections.
The second chapter deals with natural products that protect the nematode host without killing the pathogen. I deployed molecular biology methods to generate deletion and overproduction strains of a target compound, identified it via LC-MS/MS analysis and used LC-MS/MS and lipidomics to analyse the chemical properties of the active compound.
The third chapter aims at finding natural products, which are produced by Pseudomonas strains MYb11 and MYb12, respectively. These natural products display the ability to protect C. elegans by killing B. thuringiensis. I identified said compounds via fractionation and subsequent bioactivity testing. After identification, I generated production strains of the target compounds and elucidated the structure of the bioactive derivative.
The last chapter deals with the structure elucidation of peptides produced by an unusual GameXPeptide synthetase in Xenorhabdus miraniensis. I analysed producer strains of GameXPeptides using LC-MS and elucidated the structural differences between the known GameXPeptides, produced by P. luminescens TT01, and the unusual ones produced by X. miraniensis.
This work addresses the investigation of the biosynthesis mechanisms of type II polyketide synthase (PKS) and fatty acid synthase (FAS) derived specialized metabolites (SMs) from Photorhabdus laumondii.
The elucidation of the biosynthetic pathway of the bacterial 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS) was one of the major topics of this thesis. IPS exhibits several bioactive characteristics as it inhibits the phenoloxidase of insects, acts antibacterial, but also influences the soluble epoxide hydrolase which is involved in inflammatory reactions. It was recently approved as a treatment against psoriasis by the FDA and is the first Photorhabdus derived drug.
The stilbene generation in Photorhabdus requires the formation of the two acyl-carrier-protein (ACP) bound 5-phenyl-2,4-pentadienoyl- and isovaleryl-β-ketoacyl-moieties. The ketosynthase (KS)/cyclase StlD catalyzes a ring formation via a Michael-addition between the two intermediates which is then further processed by an aromatase. The formation of 5-phenyl-2,4-pentadienoyl-ACP was shown via in vitro assays with purified proteins by proving the influence of the KS FabH, ketoreductase FabG and dehydratase FabA or FabZ of the fatty acid metabolism. While E. coli was able to complement most of these enzymes in attempts to produce IPS in the heterologous host, the Photorhabdus derived FabH was not replaceable despite 73 % sequence identity with the E. coli based isoenzyme, acting as a gatekeeper enzyme for cinnamic acid (CA) moieties. Furthermore, the ability to incorporate meta-substituted halogenated CA-derivatives was shown in order to produce 3-chloro- and 3-bromo-IPS. While studying the stilbene biosynthesis, the ability of Photorhabdus and Xenorhabdus to produce hydrazines was also discovered.
The second investigated biosynthesis was the formation of benzylideneacetone (BZA). BZA is produced by Photorhabdus and Xenorhabdus strains acting as a suppressor for the immune cascade of insects and has also antibiotic activities towards Gram-negative bacteria. Due to its structural similarity towards CA and the intermediates during the stilbene formation, a shared mechanism for Photorhabdus and Xenorhabdus budapestensis was proposed due to their ability to produce CA. The production of BZA was also dependent on the stilbene related CoA-ligase, the ACP and FabH. It was verified in vitro and in vivo in E. coli yielding a 150-fold increase of the BZA production compared to the Photorhabdus and Xenorhabdus wildtype (WT) strains.
The second part of this work deals with the optimization of P. laumondii strains regarding the production titers of IPS. Therefore, several deletions of other SM related genes as well as promoter exchanges in front of stilbene related genes were carried out. These approaches were combined with the upregulation of the phenylalanine by heterologous plasmid expression, since it is the precursor of CA. Another approach applied in parallel was the optimization of the cultivation conditions with different media and supplementation with XAD-resins. It was proved that media rich on fatty acids or peptides led to higher optical densities of the cultures and thus to higher titers of stilbenes. Since IPS is inhibiting the phenoloxidase, an enzyme important for the insect immunity, it was hypothesized that cultivation in media containing insects might enhance the output of this SM. Starting from 23 mg/l of IPS in the P. laumondii WT strain, it was possible to increase the production levels to more than 860 mg/l by utilizing the mentioned approaches.
The last topic of this thesis focuses on the production of epoxidated IPS (EPS) and its derivatives. Under laboratory conditions, only a low titer of EPS was observed for the wildtype strain. However, the optimized IPS strains and IPS-production conditions could also be applied for EPS which led to higher productions and also to the detection of many new derivatives. Most of the EPS derivatives were amino acid or peptide derived acting as nucleophiles to open the epoxide ring and yielding β-amino-alcohols. However, purification and chemical synthesis attempts to obtain EPS failed due to its poor stability. Epoxides were utilized in in vitro assays with amino acids, peptides and proteins to get insights whether epoxidations might act as posttranslational modification in Photorhabdus. The reactions were performed with styrene oxide and stilbene oxide replacing EPS based on their structural similarity. The modifications were executed successfully although proteomics approaches with in vivo data are required to confirm these findings. During the purification attempts of EPS, further derivatives were detected. The structures of dimerized stilbenes, a cis-isomer of IPS and another derivative that might incorporate an amino-group in the resveratrol ring were proposed on the basis of the HPLC-MS data.
Anthropogenic interventions have altered all ecosystems around the world. One of those ecosystems are forests, the main resource for timber. They have been strongly transformed in their structure with large consequences on forest biodiversity. Especially the decrease in dead-wood volume due to the timber extraction and alternation of natural forest structures with even-aged stands of less diverse tree species composition has put especially saproxylic, i.e., dead-wood dependent species, under threat, which comprise about 20% of all forest species. Beetles, fungi and bacteria are three functional important groups for decomposition processes but we still lack much information about their sampling and the drivers of their diversity, thus it is difficult to comprehensively protect their diversity. Saproxylic fungi are a highly diverse species group and the main drivers of dead-wood decomposition; hence they play a major role in the global carbon cycle. Due to their cryptic lifestyle, many species are still unknown, but the recent advances in environmental DNA barcoding methods (metabarcoding) shed light on the formerly underestimated diversity. Yet, this method's accuracy and suitability in detecting specific species have not been assessed so far, limiting its current usefulness for species conservation. On the other hand, these methods are a convenient tool to study highly diverse areas with high numbers of unknown species, enabling the study of global diversity and its drivers, which are unknown for saproxylic fungi, but important to assess to predict the future impacts of global change. Since nature conservation concepts are usually not applied on a global scale, the drivers of diversity must also be assessed on smaller scales. Besides understanding the drivers of diversity, to identify focus scales to create comprehensive, evidence-based conservation concepts must utilize multi-taxonomic studies since saproxylic species are differently sensitive towards environmental variables and closely interact with each other. Filling these knowledge gaps is utterly needed to protect the high saproxylic diversity and ensure the functional continuity of decomposition processes, especially regarding the global change.
To address the usefulness of metabarcoding for fungal species conservation, I compared the traditional method of fruit body sampling with metabarcoding and their efficiency in detecting threatened fungal species in the first chapter of this thesis. Both methods have advantages and disadvantages. Their ability to detect threatened saproxylic fungal species and their dependencies on detecting specific fungal groups have not been compared, albeit they are important to inform species conservation like Red Lists properly. I found metabarcoding to generally detect more threatened fungal species than fruit body sampling with a higher frequency than fruit body sampling. Moreover, fruit body sampling detected a unique set of species, while fruit body sampling missed large parts of fungal diversity due to species-specific fruiting characteristics. Metabarcoding with high sampling intensity is thus a viable method to assess threatened saproxylic fungal diversity and inform nature conservation like Red Lists about distribution and abundances. Nevertheless, a complementary approach with fruit body sampling is indispensable for assessing all threatened fungal species.
In order to analyse the global diversity of saproxylic fungi and its drivers, I examined whether fungal species richness increases from the poles towards the equator and thus follows the latitudinal diversity gradient already found in many other species groups. I further investigated whether such an increase is caused by increasing ecological specialisation, i.e., niche partitioning, or local tree diversity, i.e., niche space. Gamma diversity per biome increased from the boreal, over the temperate to the tropics and thus confirmed the latitudinal diversity for saproxylic fungi. Contrastingly, alpha diversity at the log level did not significantly increase towards the tropics, suggesting a grain size dependency of the observed pattern and an equal niche space within dead-wood across latitudes. Ecological specialisation on the plot level was globally on a high level but did not increase significantly towards the equator. Additionally, I found local tree species richness to drive plot-based fungal diversity. Further analysis of gamma diversity against the total number of sampled tree species strengthened the assumption that tree species diversity and not increased ecological specialisation was the main driver of the latitudinal diversity gradient, as there was no significant difference between the gamma diversity of the temperate and tropical biome. Nonetheless, as the gamma diversity of the boreal biome was still significantly smaller, my results do not allow a complete neglection of the ecological specialisation hypothesis. The overall results indicate a strong dependency of saproxylic fungi diversity with host tree species diversity and that the global loss of tree species threatens saproxylic fungi with an unpredictable impact on carbon and nutrient cycling.
To support saproxylic conservation, I conducted two analyses. First, I compared the beta diversity of the three main decomposer groups (beetles, fungal fruit bodies, mycelial fungi (metabarcoding), and bacteria (metabarcoding)) across different scales to assess the impact of different environmental variables on their overall diversity. I used an experimental design to disentangle two different spatial scales, influenced by differences in macroclimate, forest microclimate and spatial distance, and two host scales, driven by differences between tree lineages and tree species. I set these beta diversities in relation to the gamma diversity of the three main decomposer groups to identify whether a unified conservation concept could be applied to one scale to optimally protect the diversity of all three species groups. Second, I identified whether diversity and community composition of fungi and bacteria differed among climate and land use gradients. Further I explored whether specialisation and niche packing could explain the expected pattern. To do so I used an experimental design disentangling climate and land use across a large gradient in Germany. The results differed among the species groups, denying a unified conservation concept focusing on one scale. Saproxylic beetle and fruit body beta diversity was equally high on each scale, as they are more sensitive towards environmental factors like macro- and microclimate. On the other hand, mycelial fungi and bacteria beta diversity was highest on the host scale, especially the host tree scale, indicating a high host specificity of the two groups. The second study also identified tree species as the main driver of diversity and community composition of these two study groups. Specialisation of fungi was not influenced by land use or climate. Bacterial specialisation and diversity were under a strong influence of mean precipitation. Comprehensive conservation of multi-taxonomic diversity across regions thus requires the integration of several scales. Within different macroclimatic regions, forests of varying microclimates, i.e., forest management, must be implemented. In these forests, dead-wood of different tree lineages, i.e., angio- and gymnosperms and tree species, must be provided.
Taken together, I could demonstrate that metabarcoding is an efficient method to sample threatened fungal species and identify differing drivers of fungal diversity present as fruit bodies or mycelium. Its usefulness will further increase due to the ongoing improvement of sequencing databases and thus better inform conservation concepts. Using metabarcoding, I could demonstrate that high host specialisation of saproxylic fungi is not a European but a global phenomenon and identify tree species loss under global change as one major concern for saproxylic diversity. My dissertation further highlighted the importance of multi-taxonomic studies for evidence-based nature conservation, as different species groups require varying concepts. These results were especially important for saproxylic bacteria as the drivers of their diversity are still largely unknown. Howbeit, large research gaps still exist regarding the impacts of global change on species and processes. Moreover, the spatial coverage of studies is needed to confirm or neglect the generality of current research especially concerning the highly diverse tropical areas. An increased focus on the drivers of diversity in these areas is crucial to ensure a globally comprehensive saproxylic conservation and the various ecosystem functions they control.
The capacity of pathogenic bacteria to adhere to host cells and to avoid subsequent clearance by the host´s immune response is the initial and most decisive step leading to infections. Human pathogenic bacteria circulating in the bloodstream need to find ways to interact with endothelial cells (ECs) lining the blood vessels to infect and colonise the host. The extracellular matrix (ECM) of ECs might represent an attractive initial target for bacterial interaction, as many bacterial adhesins have reported affinities to ECM proteins, particularly fibronectin (Fn). Trimeric autotransporter adhesins (TAA) have been described as important pathogenicity factors of Gram-negative bacteria. The TAA from human pathogenic Bartonella henselae, Bartonella adhesin A (BadA), is one of the longest and best characterised adhesin and represents a prototypic TAA due to its domain architecture. B. henselae, the causative agent of cat scratch disease, endocarditis, and bacillary angiomatosis, adheres to ECs and ECM proteins via BadA interaction.
In this research, it was determined that the interaction between BadA and Fn is essential for B. henselae host cell adhesion. BadA interactions were identified within the heparin-binding domains of Fn, and the exact binding sites were revealed by mass spectrometry analysis of chemically crosslinked whole-cell bacteria and Fn. It turned out that specific BadA interactions with defined Fn regions represent the molecular basis for bacterial adhesion to ECs. These data were confirmed by using BadA-deficient bacteria and CRISPR-Cas FN1 knockout ECs. It was also identified that BadA binds to Fn from both cellular and plasma origin, suggesting that B. henselae binding to Fn might possibly take part in other infection processes apart from bacterial adherence, e.g. evasion from the host cell immune system.
Interactions between TAAs and Fn represent a key step for adherence of B. henselae to ECs. Still, Fn-mediated binding is of more significant importance for pathogenic bacteria than broadly recognised. Fn removal from the ECM environment of ECs, also reduced adherence of Staphylococcus aureus, Borrelia burgdorferi, and Acinetobacter baumannii to host cells Interactions between adhesins and Fn might therefore represent a crucial step for the adhesion of human-pathogenic Gram-negative and Gram-positive bacteria targeting the ECs as a niche of infection or as means for persistence.
This research demonstrated that combining large-scale analysis approaches to describe protein-protein interactions with supportive functional readouts (binding assays) allows for the discrimination of crucial interactions involved in bacterial adhesion to the host. The herein-described experimental approaches and tools might guide future research for other pathogenic bacteria and represent an initial point for the future generation of anti-virulence strategies to inhibit bacterial binding to host cells.
In our rapidly changing world, land use has been recognized as having one of the strongest impacts on species and genetic diversity. The present state of temperate forests in Europe is a product of decisions made by former and current management and policy actions, rather than natural factors. Alterations of crown projection areas, structural complexity of the forest stand caused by thinning and cuttings, and changes in tree species composition caused by regeneration or plantings not only affect forest interior buffering against warming, but also the understorey light environment and nutrient availability. Ultimately, current silvicultural management practices have deep impact on the forest ecosystems, microenvironmental changes and forest floor understorey herbs. In response to environmental changes, plants rely on genetically heritable phenotypic variation, an important level of variation in the population, as it is prerequisite for adaptation. However, until now most studies on plant adaptation to land use focus on grassland management. Yet, studies on the adaptation of forest understorey herbs to forest management have been absent so far. This is important because understanding adaptation of understorey herbs is crucial for biodiversity conservation, forest restoration, and climate change mitigation. Studying current adaptation of understorey herbs to forest management yields insights into the evolutionary consequences of management practices, which could be employed to improve sustainable use of forest habitat.
In sum, my conducted experiments complement each other well and managed to fill in research gaps on the topic of genetically heritable phenotypic variation in understorey herbs and how it is affected by forest management and related microenvironmental variables. I showed that forest management has direct evolutionary consequences on the genetic basis of understorey herbs, but also indirectly through the microenvironment. Furthermore, I revealed that local adaptation and phenotypic plasticity of understorey herbs to forest structural attributes act along continuous gradients. And lastly, I highlighted the important role of intra-individual variation by revealing plastic responses to drought and shading, urging researchers to not ignore this important level of trait variation. Ultimately, understorey herbs in temperate forests employ phenotypic plasticity as a flexible strategy to adapt to varying environmental conditions. By adjusting their leaf characteristics, reproductive investment, and phenology, they can optimize their fitness and survival in response to changes in light availability, resource availability, and seasonal cues. The anthropogenic impact on temperate forests and understorey herbs will continue and likely increase in the future. This should urge foresters to adapt their silvicultural management decisions towards the long-term preservation of genetic diversity and, through this, the evolvability and adaptability of forest understorey herbs and associated organisms. Based on the results shown in my dissertation, variation in forest management regimes and types could be beneficial for promoting genetic diversity within several species of forest understorey herbs. Lastly, in the face of future climatic changes, the mechanisms by which plants can cope with increasing stressful environmental conditions might very well rely heavily on intra-individual variation, providing the necessary rapid plastic adjustment to changing microclimatic conditions within populations and thus increase climate change resilience.
Brain development is a complex and highly organized process that relies on the coordinated interaction between neurons and vessels. These cell systems form a neurovascular link that involves the exchange of oxygen, ions, and other physiological components necessary for proper neuronal and vascular function. This physiologically coupled process is executed through analogous structural and molecular signaling mechanisms shared by both cell types. At the neurovascular interface, the cellular crosstalk via these shared signaling mechanisms allows for the synchronized expansion and integration of neurons and vessels into complex cellular networks. This study investigated the role of VEGFR2, a receptor for vascular endothelial growth factor (VEGF), during postnatal neuronal development in the mouse hippocampus. Prior studies have revealed physiological roles of VEGF, a pro-angiogenic morphogen, in nervous system development. However, it was unclear if VEGF signaling had a direct effect on neuronal physiology and function through neuronal-expressing receptors. In this investigative work, we identified a previously unknown function of VEGFR2, whereby VEGF-induced signaling coordinates the development and circuitry integration of CA3 pyramidal neurons in the early postnatal mouse hippocampus. Mechanistically, we found that VEGFR2 signaling requires receptor endocytosis, a process mediated by ephrinB2. We also found that VEGF-induced cooperative signaling between VEGFR2 and ephrinB2 is functionally required for the dendritic arborization and spine maturation of developing CA3 neurons during the first few postnatal weeks. Moreover, in a collaborative effort with the research group of Carmen Ruiz de Almodovar, formerly at the University of Heidelberg, we simultaneously studied VEGF-induced VEGFR2 signaling in CA3 axonal development. Together, we aimed to gain a comprehensive understanding of the complex interplay between VEGF and VEGFR2 signaling during the early postnatal development of CA3 neurons. Ruiz de Almodovar’s research group found that, unlike the branch and spine development of CA3 dendrites, VEGF-VEGFR2 signaling promotes axonal development through mechanisms that are independent of ephrinB2 function. Our findings on CA3 dendritic development are reported in the published manuscript, Harde et al. (2019), and the complementary work on CA3 axonal development from Ruiz de Almodovar's group is presented in the co-published manuscript, Luck et al. (2019). Although the totality of Ruiz de Almodovar's group's work on CA3 axons is not fully discussed here, it is referenced where noted to provide biological context for our findings on CA3 dendritic development.
VEGFR2 signaling within neurovascular niches is known to play a role in the neurogenesis of neural progenitor cells during embryonic development and within the adult brain. However, the precise localization of neuronal VEGFR2 expression and functional role within the nervous system during postnatal brain development was unknown. To investigate this, we used immunohistochemistry to identify the spatial expression of VEGFR2 within the mouse hippocampus during the first few weeks after birth. Our results showed that VEGFR2 was predominantly expressed within the hippocampal vasculature, consistent with prior studies. However, we also observed localized VEGFR2 expression in pyramidal cell neurons of the hippocampal CA3 region by postnatal day 10 (P10). This spatially restricted postnatal expression of VEGFR2 in CA3 neurons suggested a potential role in the development of these neurons during this developmental stage.
The first two weeks after birth in the mouse hippocampus is a critical period for the development of neuronal circuits, as neurons undergo extensive dendritic arborization and spine formation. To explore the role of VEGFR2 in the postnatal nervous system, we used a Nes-cre VEGFR2lox/- mouse line to target the deletion of VEGFR2 expression within the nervous system while preserving normal receptor expression in all other cell types. We also generated corresponding control mice that were negative for Nes-cre. By breeding these mice with Thy1-GFP reporter mice, we could analyze the functional consequences of VEGFR2 by assessing the morphologies of CA3 dendritic trees and spine density and maturation at P10 and P15, respectively. Our analysis showed that CA3 neurons in Nes-cre VEGFR2lox/- mice had less complex dendritic arbors compared to control mice. There were significant reductions in total length and branch points, particularly in areas located 100-250 μm from the cell soma within the stratum radiatum layer. Additionally, Nes-cre VEGFR2lox/- mice exhibited a significant decrease in spine density accompanied by an increased proportion of immature spines. These findings suggest that VEGFR2 plays a crucial role in the proper development of CA3 dendrites and spines during the early postnatal weeks.
For thousands of years, S. cerevisiae has been employed by humans in brewing and baking. Nowadays, this budding yeast is more than that: it is a well investigated model organism and an established workhorse in biotechnology. S. cerevisiae serves as a production host for various applications such as i) bioethanol production ii) the biosynthesis of hormones including insulin or iii) cannabinoid biosynthesis. Hereby, the robustness of S. cerevisiae and its high tolerances regarding pH and salt concentrations qualifies it for a wide range of industrial applications. Moreover, products of S. cerevisiae are generally recognised as safe (GRAS), enabling diverse biotechnological applications. Various mechanisms for genetic engineering of S. cerevisiae are applicable and the engineering process itself is straightforward since methods are established and widely known. Due to the wide range of industrial applications of S. cerevisiae, this organism is an ideal candidate for applied research and implementation of the recombinant biosynthesis of tocochromanols in this study.
Tocochromanols encompass tocotrienols and tocopherols, which are lipid-soluble compounds that are commonly associated with vitamin E activity. Hereby, α-tocopherol is the most prevalent form, as it is an essential nutrient in the diet of humans and animals. Naturally, tocochromanols are almost exclusively synthesised by photoautotrophic organisms such as plants or cyanobacteria. They consist of an aromatic head group and a polyprenyl side chain which is saturated in tocopherols and 3-fold unsaturated in tocotrienols. The methylation status of the chromanol ring distinguishes α-, β-, γ- and δ-tocochromanol. All forms of tocochromanols represent a group of powerful antioxidants, scavenging reactive oxygen species (ROS) and preventing the propagation of lipid oxidation in lipophilic environments. Recently, attention has been drawn to tocotrienols, due to their benefits in neuroprotection as well as cholesterol-lowering and anti-cancer properties. Consequently, tocochromanols are valuable additives in the food, feed, cosmetic and pharmaceutical industries.
The metabolic engineering strategy of S. cerevisiae to enable tocochromanol biosynthesis was started in a preceding master thesis with the provision of the aromatic moiety, homogentisic acid (HGA), from the aromatic amino acid biosynthesis. Hereby, the upregulation and redirection of the native pathway was essential. Therefore, a strain with an engineered aromatic amino acid pathway for improved 4 hydroxyphenylpyruvate (HPP) production (MRY33) was utilised from Reifenrath and Boles (2018). Furthermore, a heterologous hydroxyphenylpyruvate dioxygenase (HPPD) was required to convert HPP into HGA. Thus, several heterologous HPPDs were expressed and characterised regarding their HGA production within the previous study. The best variant originated from Yarrowia lipolytica, YlHPPD, and was integrated into the genome of MRY33. The resulting strain JBY2, produced 435 mg/L HGA in a shake flask fermentation.
This work was started with the genetically highly modified strain JBY2, whose genome already contained a large number of genes artificially expressed behind strong promoters. For further strain development, it was advantageous to maintain a high degree of sequence variability in order to prevent genomic instabilities due to sequence homologies. Thus, 17 artificial promoters (AP1-AP17) were characterised regarding their strength of expression by the yellow fluorescent protein (YFP). These sequences were also part of a patent that was filed during this work (WO2023094429A1).
The key point of this study was the development of a metabolic engineering strategy for the strain JBY2. First, the sufficient supply of the second precursor, the polyprenyl side chain, was investigated. Natively, S. cerevisiae produces the precursor, geranylgeranyl diphosphate (GGPP), from the isopentenyl diphosphate pathway. However, without further engineering, GGPP was barely detectable in JBY2 (< 0.1 mg/L). Thus, engineering of the isopentenyl diphosphate biosynthesis was necessary. The limiting enzyme of the mevalonate pathway was the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), which is encoded by HMG1. Therefore, a truncation for feedback-resistance and its overexpression by a promoter exchange was performed. Furthermore, the promoter of the gene for the squalene synthase (pERG9) was exchanged by the ergosterol sensitive promoter pERG1 to limit the metabolic flux of the mevalonate pathway into the ergosterol pathway. The native GGPP synthase (BTS1) was another limitation that was observed throughout this study. To overcome this bottleneck, plasmid-based and integrative overexpression of the native BTS1 and a codon optimised BTS1 were investigated. Other strategies to improve GGPP production were the deletion of the gene for the diacylglycerol pyrophosphate phosphatase (DPP1) to prevent excessive dephosphorylation of GGPP to geranylgeraniol (GGOH), and the overexpression of the farnesyl pyrophosphate synthetase, encoded by ERG20. However, the best improvements of the GGPP biosynthesis, inferred through GGOH measurements, were achieved from the screening of several heterologous GGPP synthases in S. cerevisiae. The best performing strain was JBY61 (JBY2, hmg1Δ::pTDH3-HMG1tr[1573–3165], pERG9Δ::pERG1, ChrIV-49293-49345Δ::pTDH3-XdcrtE-tSSA1_LEU2), bearing the heterologous GGPP synthase crtE of Xanthophyllomyces dendrorhous and produced 64.23 mg/L GGOH. Consequently, this engineering strategy improved the GGOH production by a factor of 642 compared to the parent strain JBY2.
Seed dispersal is a key ecosystem function for plant regeneration, as it involves the movement of seeds away from the parental plants to particular habitats where they can germinate and transition to seedlings and ultimately adult plants. Seed dispersal is shaped by a diversity of abiotic and biotic factors, particularly by associations between plants and climate and between plants and other species. Due to the ongoing loss of biodiversity and changing global conditions, such interactions are prone to change and pose a severe threat to plant regeneration. One way to address this challenge is to study associations between plant traits and abiotic and biotic factors to understand the potential impacts of global change on plant regeneration. Plant communities have long been analyzed through the lens of vegetative traits, mainly ignoring how other traits interact and respond to the environment. For instance, while associations between vegetative traits (e.g., specific leaf area, leaf nitrogen content) and climate are well studied, there are few case studies of reproductive traits in relation to trait-environment associations in the context of global change.
Thus, the overarching aim of this dissertation is to explore how trait-environment associations, with a special focus on reproductive traits, can improve our understanding of the effect that global change may have on seed dispersal, and ultimately on plant regeneration. To this end, my research focuses on studying associations between plant traits and abiotic and biotic factors along an elevational gradient in both forests and deforested areas of tropical mountains. This dissertation addresses three principal research objectives.
First, I investigate the extent to which reproductive (seed and fruit traits) and vegetative traits (leaf traits) are related to abiotic and biotic factors for communities of fleshy-fruited plants in the Ecuadorian Andes. I used multivariate analyses to test associations between four (a)biotic factors and seven reproductive traits and five vegetative traits measured on 18 and 33 fleshy fruited plant species respectively. My analyses demonstrate that climate and soil conditions are strongly associated with the distribution of both reproductive and vegetative traits in tropical tree communities. The production of “costly” vs. “cheap” seeds, fruits and leaves, i.e., the production of few rewarding fruits and acquisitive leaves versus the production of many less-rewarding fruits and conservative leaves, is primarily limited by temperature, whereas the size of plant organs is more related to variation in precipitation and soil conditions. My findings suggest that associations between reproductive and vegetative traits and the abiotic environment follow similar principles in tropical tree communities.
Second, I assess how climate and microhabitat conditions affect the prevalence of endozoochorous plant species in the seed rain of tropical montane forests in southern Ecuador. I analyzed seed rain data for an entire year from 162 traps located across an elevational gradient spanning of 2000 m. I documented the microhabitat conditions (leaf area index and soil moisture next to each seed trap) at small spatial scale as well as the climatic conditions (mean annual temperature and rainfall in each plot) at large spatial scale. After a one-year of sampling, I counted 331,838 seeds of 323 species/morphospecies. My analyses demonstrate that the prevalence of endozoochorous plant species in the seed rain increases with temperature across elevations and with leaf area index within elevations. These results show that the prevalence of endozoochory is shaped by the interplay of both abiotic and biotic factors at large and small spatial scales.
Third, I examine the potential of seed rain to restore deforested tropical areas along an elevational gradient in southern Ecuador. For this chapter, I collected seed rain using 324 seed traps installed in 18 1-ha plots in forests (nine forest plots) and in pastures (nine deforested plots) along an elevational gradient of 2000 m. After a sampling period of three months, I collected a total of 123,039 seeds of 255 species/morphospecies from both forests and pastures along the elevational gradient. I did not find a consistent decrease in the amount and richness of seed rain between forests and pastures, but I detected a systematic change in the type of dispersed seeds, as heavier seeds and a higher proportion of endozoochorous species were found in forests compared to pastures at all elevations. This finding suggests that deforestation acts as a strong filter selecting seed traits that are vital for plant regeneration.
Understanding the role that trait-environment associations play in how plant communities regenerate today could serve as a basis for predicting changes in regeneration processes of plant communities under changing global conditions in the near future. Here, I show how informative the measurement of reproductive traits and trait environment associations are in facilitating the conservation of forest habitats and the restoration of deforested areas in the context of global change.
In view of a growing world population and the finite nature of fossil resources, the development of eco-friendly production processes is essential for the transition towards a sustainable industry. Methanol, which can be produced both petrochemically and from renewable resources, offers itself as bridging technology and attractive alternative raw material for biotechnological processes. This work describes developments for the progress of the well-studied methylotrophic α proteobacterium Methylorubrum extorquens AM1 towards an efficient methylotrophic cell factory. Although many homologous and heterologous production routes have already been described and realized for M. extorquens in a laboratory scale, no industrial process has yet been realized. Three major reasons can be identified for this: (1) A limited choice of tools for genetic modifications, (2) a lack of understanding of carbon fluxes and side reactions occurring in modified strains, such as product reimports, and (3) the lack of tailored production strains for profitable target products and optimized bioprocessing protocols. The aim of the present work was to achieve developments for the mentioned areas. As a model application, the high-level production of chiral dicarboxylic acids from the substrate methanol was chosen. Enantiomerically pure chiral compounds are of great interest, e.g., as building blocks for chiral drugs. The ethylmalonyl CoA metabolic pathway (EMCP) which is part of the primary metabolism of M. extorquens, harbors unique chiral CoA-ester intermediates. Their acid derivatives can be released by cleavage of the CoA-moiety using heterologous enzymes. The dicarboxylic acids 2 methylsuccinic acid and mesaconic acid were produced in a previous study by introducing the heterologous thioesterase YciA into M. extorquens. In the said study, a combined product titer of 0.65 g/L was obtained in shake flask experiments. These results serve as the basis for the developments in the present work.
First, the previously described reuptake of products was thoroughly investigated and dctA2, a gene encoding for an acid transporter, was identified as target for reducing the product reuptake. In addition, reuptake of mesaconic acid was prevented by converting it to (S)-citramalic acid, a product not metabolizable by M. extorquens, by the introduction of a heterologous mesaconase. Together with 2-methylsuccinic acid, for which a high enantiomeric excess of (S)-2-methylsuccinic acid was determined, a second chiral molecule was thus added to the product spectrum. For the release of dicarboxylic acid products, YciA, a broad-range thioesterase that accepts a variety of CoA-esters with different chain lengths as substrates, was chosen. The enzyme should theoretically be able to hydrolyze all CoA-esters of interest present in the EMCP. However, in culture supernatants of M. extorquens strains that were overexpressing the corresponding yciA gene, only mesaconic acid and 2 methylsuccinic acid could be detected. To expand the substrate spectrum of YciA thioesterase with respect to other EMCP intermediates, semi-rational enzyme engineering was attempted. Screening of the corresponding strains carrying the respective YciA variants did not result in strains capable of producing new dicarboxylic acid products. However, the experiments revealed an amino acid position that strongly affected the production of mesaconic acid and 2-methylsuccinic acid in vivo. By substituting the according amino acid in YciA, the maximum titers of mesaconic acid and 2-methylsuccinic acid could be increased substantially. Application of an improved thioesterase variant in a second E. coli-based process confirmed the enhanced activity of the enzyme. The desired extension of the product spectrum by another chiral molecule (2-hydroxy-3-methylsuccinic acid, presumably the (2S,3R)-form) was finally achieved by using an alternative thioesterase. Tailored fermentation strategies were developed for the high-level production of the above-mentioned products.
As second part of the work, two novel genetic tools for M. extorquens were developed and characterized. The pBBR1-derived plasmid pMis1_1B was shown to be stably maintained in M. extorquens cells. In addition, its suitability for co-transformations with other plasmids was demonstrated. The second tool, the cumate-inducible promoter Ps6, is tailored for expression of pathways with toxic products, as the transcription of genes controlled by Ps6 is strongly repressed in the absence of an inducer.
Overall, the present work demonstrates the enormous potential of using M. extorquens as a methylotrophic cell factory. In the applications shown, the biotechnological production of high-priced chiral molecules is combined with the use of an attractive alternative substrate. In addition, new achievements and approaches are presented to facilitate the development of future M. extorquens production strains.
How the brain evolved remains a mystery. The goal of this thesis is to understand the fundamental processes that are behind the evolutionary history of the brain. Amniotes appeared 320 million years ago with the transition from water to land. This early group bifurcated into sauropsids (reptiles and birds) and synapsids (mammals). Amniote brains evolved separately and display obvious structural and functional differences. Although those differences reflect brain diversification, all amniote brains share a common ancestor and their brains show multiple derived similarities: equivalent structures, networks, circuits and cell types have been preserved during millions of years. Finding these differences and similarities will help us understand brain historical evolution and function. Studying brain evolution can be approached from various levels, including brain structure, circuits, cell types, and genes. We propose a focus on cell types for a more comprehensive understanding of brain evolution. Neurons are the basic building blocks and the most diverse cell types in the brain. Their evolution reflects changes in the developmental processes that produce them, which in turn may shape the neural circuits they belong to. However, there is currently a lack of a unified criteria for studying the homology of connectivity and development between neurons. A neuron’s transcriptome is a molecular representation of its identity, connectivity, and developmental/evolutionary history. Hence the comparison of neuronal transcriptomes within and across species is a new and transformative development in the study of brain evolution. As an alternative, comparing neuronal transcriptomes across different species can provide insights into the evolution of the brain. We propose that comparing transcriptomes can be a way to fill this gap and unify these criteria. In previous studies, published in Science (Tosches et al., 2018) and Nature (Norimoto et al., 2020), we leveraged scRNAseq in reptiles to re-evaluate the origins and evolution of the mammalian cerebral cortex and claustrum. Motivated by the success of this approach, in this thesis we have now expanded single-cell profiling to the entire brain of a lizard species, the Australian dragon Pogona vitticeps, with a special focus in thalamus and prethalamus of. This approach allowed us to study the evolution of neuron types in amniotes. Therefore, we aimed to build a multilevel atlas of the lizard brain based on histology and transcriptomic and compare it to an equal mouse dataset (Zeisel et al., 2018).
Our atlas reveals a general structure that is consistent with that for other amniote brains, allowing us to make a direct comparison between lizard and mouse, despite their evolutionary divergence 320 million years ago. Through our analysis of the transcriptomes present in various neuron types, we have uncovered a core of conserved classes and discovered a fascinating dichotomy of new and conserved neuron types throughout the brain. This research challenges the traditional notion that certain brain regions are more conserved than others.
Our research also has uncovered the evolutionary history of the lizard thalamus and prethalamus by comparing them to homologous brain regions of the mouse. This pioneering research sheds new light on our understanding of the evolutionary history of the lizard brain. We propose a new classification of the lizard thalamic nuclei based on
transcriptomics. Our research revealed that the thalamic neuron types in lizards can be grouped into two large, conserved categories from the medial to lateral thalamus. These categories are encoded by a common set of effector genes, linking theories based on connectivity and molecular studies of these areas. In our data we have seen that there is a conservation of the medial-lateral transcriptomic axis in mouse and lizard, this conservation was most likely already present in the common ancestor. Although there is a shared medial-lateral axis, a deeper study of the thalamic cell types has allowed us to see the existence of a partial diversification of the thalamic population, specifically in the sensory-related lateral thalamus; in opposition, the medial thalamic nuclei neuron-types have been preserved.
On the other hand, the comparison with the mammalian prethalamus allowed us to confirm that the lizard ventromedial thalamic neuron types are homologous to mouse reticular thalamic neuron types (Díaz et al., 1994), even if they do not express the classical Reticular thalamic nucleus (RTn) marker PV/pvalb. We also discovered that there has been a simplification in the mammalian prethalamic neuron types in favor of an increase in the number of Interneurons (IN) types within their thalamus. We suggest that the loss of GABAergic neuronal types in the mammalian prethalamus is linked to the need for a more efficient control of the thalamo-pallial communication in mammals, while in lizards, where thalamo-pallial communication is probably simpler, the diversity prethalamus presents a higher diversity.
Epithelial cells enable essential physiological functions, including absorption, morphogenesis, secretion, and transport. To execute these functions, epithelial cells often form three-dimensional shapes that include curved sheets of cells surrounding a pressurized fluid-filled lumen. These three-dimensional tissues (called domes) are essential for organ function, but when they are not working properly, developmental defects, inflammation, and cancer can ensue. Recently, it has been shown that the cells that form domes show active superelasticity on micropatterned plates.
We show here that the immortalized renal proximal tubule epithelial cell line, LLC-PK1, stereotypically forms tubules in 10 days. Tubule formation takes place in 4 stages. When cells are plated on a culture dish, they form a monolayer on the 1st day; on the 3rd day, three-dimensional structures are formed, called domes; and after the 4.5th day, these domes start fusing to begin the transition stage and transit to the tubule stage. At the end of the 10th day, differentiated, elongated, and matured tubes form (Figure 3.1). Therefore, tubule formation is a self-organized, stereotypic morphogenetic program under long-term, unperturbed tissue culture conditions.
We propose that tubulogenesis is a two-step process in proximal tubules by doming and wrapping. The process begins with dome formation, and as the cell layers come together in the transition stage at the edge of the dome, this leads to the formation of the lumen of the eventual tubule. We also found that F-actin provides the mechanical strength during the formation of these three-dimensional structures during tubule formation. To better understand this 4-step process on a molecular level, we performed proteomics of tubule formation to identify the different proteins that play a significant role in proximal tubule development. Importantly, we identified proximal tubule markers like synaptopondin, angiotensin 1-10, collectrin, polycystin 1, and polycystin 2. These proteins play an important role in renal tube formation and differentiation.
Cell division is carried out by highly conserved cyclin-CDK complexes, which phosphorylate various cellular components. Cyclin-CDKs act differently depending on the cell cycle phase and work cooperatively to create DNA replication and cytokinesis. Therefore, we identified that cyclin-B1, marker of proliferation Ki-67, the RAD51 recombinase, and proliferating cell nuclear antigen (PNCA) are upregulated in the monolayer stage, and the expression decreases as tubule formation takes place. The proximal tubule reabsorbs 60-65% of the glomerulus filtrate. Therefore, it requires a lot of energy generated by using the fatty acid oxidation (FAO) pathway. In our model, we found FAO expression is higher than that of the other metabolic pathways.
We found expression of an intricate protein network in mitochondria, which we interpret as a sign of mitochondrial homeostasis being vital for the FAO pathway to work. Furthermore, we also identified different types of transporters at each stage of proximal tubule formation, and we could recognize different cytoskeletal components playing a significant role in each stage of proximal tubule formation, for instance, at the monolayer stage, vimentin expression is high, and its expression is reduced as tubules form. Hence, this 2D system, at this step of characterization, seems suitable to use to study differential transport protein expression and how this might relate to physiological functions and syndromes.
Next, we inhibited different transporters using specific inhibitors and analyzed the effect on dome and tubule formation. We identified that Na+/K+ ATPase and vacuolar H+ ATPase play a significant role in the process of epithelial dynamics. Digoxin (a Na+/K+ ATPase inhibitor) treatment inhibits dome and tubule formation. Bafilomycin (a v-ATPase inhibitor) treatment demonstrated a delay in dome and tube formation. Therefore, this study shows that this 2D proximal tubule novel system can be used for screening of pharmacological leads in the context of specific aspects of kidney physiology.
Despite the recent success in growing kidney organoids, they are not well suited to investigate various pathophysiological conditions in vitro for several reasons: They grow in 3D and form a tissue that later needs to be dissected/cleared and stained to investigate pathophysiological changes. Moreover, organoids require complex and expensive protocols for generation and are challenging to use in screening approaches. Therefore, we set out to demonstrate feasibility for our 2D system using normal renal epithelial cells, which are the origin of various pathological conditions, to study pathophysiological conditions.
Biotechnological processes offer better production conditions for a wide variety of goods of industrial interest. The production of aromatic compounds, for example, involves molecules of great value for cosmetic, plastic, agrochemical and pharmaceutic industries. However, the yield of such processes frequently prevents a proper implementtation that would allow the replacement of traditional production processes.
Numerous rational engineering approaches have been attempted to enhance metabolic pathways associated with desired products. Unfortunately, genetic modifications and heterologous pathway expression often lead to a higher metabolic burden on the producing organisms, ultimately leading to reduced production levels and fitness.
This project utilised adaptive laboratory evolution to better understand the development of synthetic cooperative consortia, using S. cerevisiae as a model organism. Specifically, a synthetic cooperative consortium was developed around the exchange of lysine and tyrosine, which was subjected to adaptive laboratory evolution aiming to induce mutations that would improve the system’s fitness either by enhanced production or upgraded stress resistance. Consequently, the mutant strains isolated after the evolution rounds were sequenced to identify relevant variations that could be related to the growth and production phenotypes observed.
The insights derived from this project are expected to contribute to further developing synthetic cooperative consortia with utilitarian purposes.
Hyperparasitic fungi on black mildews (Meliolales, Ascomycota) : hidden diversity in the tropics
(2023)
Meliolales (Sordariomycetes, Ascomycota) is a group of obligate plant parasitic microfungi mainly distributed in the tropics and subtropics. Meliolalean fungi are commonly known as “black mildews”, as they form black, superficial hyphae on the surface of vegetative and reproductive organs of vascular plants. They are considered biotrophic parasites, and the infections caused by black mildews can lead to a decrease in the photosynthetic activity of plants, as well as to an increase in the temperature and respiration rate of their leaves.
Meliolales are frequently parasitized by hyperparasitic fungi, i.e., parasitic fungi that have parasitic hosts. These hyperparasites are all Ascomycota and belong mainly to the Dothideomycetes and Sordariomycetes. Although hyperparasites represent a megadiverse group, species were only described by morphology until 1980, and the systematic position of more than 60 % of known species is still unclear. In addition, there are no DNA reference sequences available in public databases for any of the species of hyperparasites of Meliolales, and no ecological studies have been done up to now.
Before this study, no exact number of hyperparasitic fungi growing on colonies of black mildews existed. Here, we present a checklist including 189 species of fungi known to be hyperparasitic on Meliolales, but the number of existing species is likely to be even higher. The elaboration of this species checklist laid the foundations for this investigation, as it helped to understand the present state of knowledge of hyperparasitic fungi on Meliolales worldwide.
For the present study, fresh specimens of leaves infected with colonies of Meliolales and hyperparasites were opportunistically collected at 32 collection sites in Western Panama and Benin, West Africa, in 2020 and 2022, respectively. In total, 100 samples of plant specimens infected with black mildews were collected, of which 58 samples were parasitized by hyperparasitic fungi. 31 species and morphospecies of hyperparasitic fungi were identified. In addition, 35 historical specimens, including 12 type specimens, were examined for the present work.
DNA of hyperparasitic fungi was isolated directly from conidia, synnemata, apothecia, perithecia or pseudothecia of fresh and dried specimens. The main challenges faced by scientists in doing molecular studies of hyperparasitic fungi are related to the fact that the hyperparasitic fungi are intermingled with tissues of the meliolalean hosts and other organisms present in a given sample. This makes the isolation of DNA exclusively from the hyperparasite difficult. Moreover, hyperparasitic fungi on Meliolales are biotrophs and cannot be grown axenically. The hosts themselves are also biotrophic, further complicating DNA isolation from either partner. These factors have contributed to a lack of reference sequences in public databases. After more than 100 attempts, DNA of 20 specimens of hyperparasitic fungi, representing seven species, has been isolated in the context of the present investigation. Three partial nuclear gene regions were amplified and sequenced: nrLSU, nrSSU and nrITS. The datasets were assembled for phylogenetic analyses applying Maximum Likelihood (ML) and Bayesian inference (BI) methods. DNA sequences of hyperparasitic fungi on Meliolales were generated for the first time in the context of the present investigation.
Hyperparasitic fungi on Meliolales do not represent a single systematic group, but a polyphyletic ecological guild of fungi. Because of this huge diversity, only the systematics of species of perithecioid hyperparasites, as well as of the species of the genera Atractilina and Spiropes known to be hyperparasitic on black mildews was discussed in this thesis, as they represented the most common groups of fungi found in Benin and Panama. The results indicated, for example, the systematic position of Dimerosporiella cephalosporii and Paranectriella minuta in the Sordariomycetes and Dothideomycetes, respectively. In addition, the first record of a hyperparasitic fungus of black mildews in the Lecanoromycetes, namely Calloriopsis herpotricha, is reported here. The systematics of Atractilina parasitica and of some species of Spiropes is also discussed here.
In the context of the present investigation, four species new to science were described. They are presented with detailed descriptions, photos and scientific illustrations. Taxonomic studies of this thesis also generated seven new synonyms, nine new records for Benin, seven for Panama, one for Africa and two for mainland America, as well as the confirmation of one anamorph-teleomorph connection by molecular sequence data.
The ecology of hyperparasitic fungi on Meliolales is complex and far from being completely understood. The hypothesis of host specificity between hyperparasitic fungi, their meliolalean hosts and their plant hosts was tested for the first time, through a tritrophic network analysis. Results indicate that hyperparasites of Meliolales are generalists concerning genera of Meliolales, but apparently specialists at the level of order. In addition, hyperparasitic fungi tend to be found alongside their meliolalean hosts, suggesting a pantropical distribution.
Anthropogenic activities have a major impact on our planet and rapidly drive biodiversity loss in ecosystems at a global scale. Particularly over the last century, rising CO2 emissions significantly raised global temperatures and increased the intensity and frequency of droughts and heatwaves. Additionally, agricultural land use and fossil fuel combustion contribute to the continuous release of nitrogen (N) and phosphorus (P) into ecosystems worldwide through extensive fertilization and deposition from the atmosphere. It is important to understand how these rapid changes affect the evolution of plant populations and their adaptive potential. Adaptation by natural selection (i.e., adaptive evolution) within a few generations is an essential process as a response to rapid environmental changes. Rapid evolution of plant populations can be detected by using the so-called resurrection approach. Here, diaspores (i.e., seeds) from a population are collected before (ancestors) and after (descendants) a potential selection pressure (e.g., consecutive years of drought or changes in nutrient supply). Comparing phenotypes of ancestors and descendants in a common environment such as an outside garden, greenhouse, or climate chamber, may then reveal evolutionary changes. Ideally, plants are first grown in a common environment for an intermediate refresher generation to reduce parental and storage effects.
The aim of this thesis was to investigate the occurrence of adaptive evolution in natural plant populations in response to rapidly changing environments over the past three decades. I conducted three experiments using the resurrection approach to generate comprehensive data on the adaptive processes that acted on three plant populations from three different species over the last three decades. Furthermore, I filled knowledge gaps in plant evolutionary ecology and conceptually developed the resurrection approach further.
In Chapter I, I performed a novel approach by testing for adaptive evolution in natural plant populations using the resurrection approach in combination with in-situ transplantations. I cultivated seedlings from ancestors (23 – 26 years old) and contemporary descendants of three perennial species (Melica ciliata, Leontodon hispidus and Clinopodium vulgare) from calcareous grasslands in the greenhouse and In Chapter III, I assessed the reproducibility of phenotypic differences between genotypes among three different growth facilities (climate chamber, greenhouse, and outdoor garden). I also evaluated differences in phenotypic expression between plants grown after one vs. two intermediate generations (i.e., refresher generations). I performed this experiment within the framework of the resurrection approach and compared ancestors and descendants of the same population of Leontodon hispidus.
I observed very strong differences among plants growing in the different growth facilities. I found a significant interaction between the growth facility and the temporal origin (ancestors vs. descendants): descendants had significantly larger rosettes than ancestors only in the greenhouse and they flowered significantly later than ancestors exclusively in the climate chamber. I did not find significant differences between intermediate generations within the growth facilities. Overall, Chapter III shows that the use of a particular experimental system can dictate the presence and magnitude of phenotypic differences. This implies that absence of evidence is not evidence of absence when it comes to investigating genetically based trait differentiation among plant origins (in space or time). Experimental systems should be carefully designed to provide meaningful conditions, ideally mimicking the environmental conditions of the population’s origins. Finally, growing a second intermediate generation did not impact the genetic differences of ancestors and descendants within the environments, supporting the idea that only one intermediate generation may be sufficient to reduce detectable parental and storage effects.
The resurrection approach allows a better understanding of rapid plant adaptation, but some limitations deserve to be highlighted. I only studied one population per species, and Chapters II and III only focus on one population of L. hispidus, which is also hampering generalizations, as adaptive potential can vary greatly among populations of the same species. I only compared the ancestral genotypes to one descendant sample with a long time span in between (26 – 28 years), which makes it hard to pinpoint the selection agents that caused the genetic differentiation among the sampling years. Hence, closely monitoring biotic and abiotic factors of the studied populations between the ancestral and descendant sampling in future studies, would make identifying the responsible selection pressures more precise. I also recommend sampling multiple populations over consecutive years to improve the robustness of results and make generalizations more approachable.Furthermore, combining the resurrection approach with other methods such as in-situ transplantations will be valuable to offset the limitation that adaptations cannot be proven under artificial conditions (e.g., in the greenhouse).
The attention on the protein PURA has increased recently following the discovery of the rare PURA Syndrome. This neurodevelopmental disorder is caused by de novo mutations in the PURA gene. Notably, our collaborators could show that the protein PURA can bind DNA and RNA in vitro. As a result, I was motivated to explore PURA's cellular RNAbinding activity. Furthermore, I inquired on the connection of PURA-RNA binding to the cellular effect of a reduction of functional PURA as present in PURA Syndrome patients.
To investigate the binding of PURA and the impact of PURA de ciency on cellular RNA and protein expression, I performed an integrative computational analysis of multimodal data from complementary high-throughput experiments. An essential component was the examination of UV Crosslinking and immunoprecipitation (CLIP) experiments, which can query the global RNA-binding behaviour of a given protein in a cellular context. As the processing and analysis of CLIP data are rather complex, I introduce an automated command line tool for the processing of CLIP data named racoon_clip as part of this dissertation. Therefore, this dissertation comprises two major segments. Firstly, I describe the implementation and usage of racoon clip for CLIP data analysis. Secondly, I discuss my research on the protein PURA, demonstrating its global RNA-binding properties, the effects of PURA depletion and its association with neuronal functions and P-bodies, among others.
racoon_clip is a command line application that I have developed for processing of individualnucleotide resolution CLIP (iCLIP) and enhanced CLIP (eCLIP) experiments - two of the most commonly used types of CLIP experiments - in a comparable and user-friendly way.
For this, I built racoon_clip as an automated work how that encompasses all CLIP processing steps from raw data to single-nucleotide resolution crosslink events. racoon_clip is available as a command line tool that users can run with a single command. The work how is implemented with Snakemake work how management providing computational advantage tages including parallelisation, scalability and portability of the work how. The main task of racoon_clip is to extract single-nucleotide crosslink events from iCLIP, iCLIP2, eCLIP and similar data types. To strike a balance between being highly customisable and easy to use, racoon_clip supplies pre-set options for the most common types of experiments.
Additionally, it is possible for users to create a custom setup of barcode and adapter architectures, which allows them to use the software for other types of CLIP data. While accounting for the different architectures in the reads, the performed central processing steps remain the same. This leads to a high degree of comparability between the different experiment types, which I demonstrate in the exemplary processing of U2AF2 iCLIP and eCLIP data. Taken together, I am confident that racoon_clip will be beneficial to numerous researchers interested in RNA-Protein interactions as it offers easily accessible processing for CLIP data and enhances the comparability of multiple CLIP datasets across di erent experiment types.
In the second part of this dissertation, I focus on the cellular function of the RNAbinding protein PURA. Through in-depth computational analysis of one iCLIP data set of endogenous PURA and two iCLIP data sets of overexpressed PURA in HeLa cells, I establish that PURA is a global RNA-binding protein. It preferentially binds RNAs in either the coding sequence (CDS) or the 3' untranslated region (3'UTR) of mature protein-coding transcripts by recognising a Purine-rich degenerated sequence motif. Even though overexpression of PURA results in less specific binding behaviour, the same overall binding patterns as from endogenous PURA persist. Overall characteristics of PURA binding remain similar in three distinct PURA iCLIP data sets with and without PURA overexpression.
To learn about the molecular consequences of a depletion of functional PURA in a cellular context, I used a 50% reduction of PURA in HeLa cells as a model for the heterozygous loss of PURA in PURA Syndrome and evaluated its impact on global RNA and protein expression. The results demonstrate that PURA depletion globally a ects RNA and protein expression. Additionally, I integrate PURA RNA binding with the changes in expression of RNAs and proteins in the context of PURA depletion. This reveals 234 targets of PURA that are bound by PURA and are impacted at both RNA and protein levels by the PURA protein. RNAs that are bound by PURA or change in abundance upon PURA depletion are enriched in neuronal development factors, RNA lifecycle regulators, and mitochondrial factors, among others. Consistent with a possible role of PURA in neuronal transport, there is considerable overlap between PURA bound transcripts and transcripts, that are transported to the dendritic end of neurons.
Notably, there is a link between PURA and P-bodies, as documented by the enrichment of PURA-bound RNAs in both the P-body and stress granule transcriptome. Further, PURA was found by our collaborators to be localised within P-bodies and P-body numbers were strongly reduced in cells that are depleted of PURA. This absence might be attributed to the downregulation of the proteins encoded by the PURA targets LSM14A and DDX6 as both of them were previously identified as essential for P-body formation.
Overall, the reduction of P-body numbers in PURA depletion, the neuronal function of PURA, and its association with mitochondria and RNA lifecycle regulation may indicate the cellular foundation of both PURA Syndrome and related neuronal diseases.
In summary, I present a versatile and user-friendly computational tool for the analysis of CLIP data. Subsequently, I conduct a thorough computational analysis of CLIP and other high-throughput data in the context of the RNA-binding protein PURA, which offers valuable insights into the cellular functions of PURA. These insights advance our understanding of the impact of PURA loss in PURA Syndrome and other disease contexts.
The nucleus reuniens drives hippocampal goal‑directed trajectory sequences for route planning
(2023)
Goal-directed spatial navigation requires accurate estimates of one’s position and destination, as well as careful planning of a route between them to avoid known obstacles in the environment. Despite its general importance across species, the neural circuitry supporting the ability for route planning remains largely unclear. Previous studies described that place cells in the hippocampal CA1 encode the animal's next movement direction (Wood et al., 2000; Ito et al., 2015) and upcoming navigational routes (Pfeiffer & Foster, 2013). However, it has been shown that part of the CA1 activity representing the animal’s future behaviors is not necessarily generated in the hippocampus, but is derived from the medial prefrontal cortex (PFC) via the nucleus reuniens of the thalamus (RE) (Ito et al., 2015). Notably, the importance of the PFC in navigation has been demonstrated in several studies, including the recent finding of a goal map in the orbitofrontal cortex (Basu et al., 2021). Therefore, I hypothesized that information flow from the PFC to CA1 via the RE plays a key role in route planning.
To assess the animals' route planning ability, I designed a new navigation task in which a rat has to navigate to a fixed target location from various starting positions in an arena. Furthermore, by adding an L-shaped wall in the maze and removing all light sources in the experimental room, this task forced the animals to plan a wall-avoiding route without relying on direct sensory perceptions. I confirmed that rats could learn this task successfully, memorizing the wall location and taking a smooth wall-avoidance route. To test the role of the RE, I inactivated RE neurons by expressing the inhibitory opsin SwiChR++, which resulted in a significant deficit in the animal’s route planning ability, taking a longer non-smooth path to the destination. By contrast, this manipulation did not affect navigation performance when a straight goal-directed route was available, suggesting a specific role of the RE in route planning. I further found that DREADDs-mediated inactivation of neurons in the bilateral hippocampi resulted in a similar deficit in route planning ability, implying cooperation between the RE and the hippocampus.
I finally examined the activity of hippocampal CA1 neurons with and without RE inactivation. While neurons in the hippocampus exhibited brief trajectory sequences corresponding to the animal’s subsequent goal-directed journey, I found that this goal-directed bias of trajectory events was significantly reduced by RE inactivation, likely associated with route-planning deficits in these animals.
Altogether, this dissertation demonstrates the role of the RE from both behavioral and neural coding perspectives, identifying a pivotal circuit element supporting the animal’s route-planning ability.
The prefrontal cortex (PFC) is considered the cognitive center of the mammalian brain. It is involved in a variety of cognitive functions such as decision making, working memory, goal-directed behavior, processing of emotions, flexible action selection, attention, and others (Fuster, 2015). In rodents, these functions are associated with the medial prefrontal cortex (mPFC). Experiments in mice and rats have shown that neurons in the mPFC are necessary for successful performance of many cognitive tasks. Moreover, measurements of neural activity in the mPFC show excitation or inhibition in different cells in relation to specific aspects of the tasks to be solved. To date, however, it is largely unknown whether prefrontal neurons are stably activated during the same behaviors within a task and whether similar aspects are represented by the same neurons in different tasks. In addition, it is unclear how specifically neurons are activated, for example, whether cells that are activated in response to reward are activated in a different task without reward in a different situation or remain inactive. To address these questions, we recorded the same neurons in the mPFC of mice over the course of several weeks while the animals performed various behaviors.
To do this, we expressed GCaMP6 in pyramidal neurons in the mPFC of mice. A small lens was implanted in the same location and a miniature microscope ("miniscope") was used to record neural activity. Later the extracted neurons got aligned based on their shape and position across multiple days and sessions. The mice performed five different behavioral tests while neural activity was measured: A spatial working memory test in a T-maze, exploration of the elevated plus maze (EPM), a novel object recognition (NO) test including free open field (OF) exploration, a social interaction (SI) test and discriminatory auditory fear conditioning (FC). Each task was repeated at least twice to check for stable task encoding across sessions. Behavioral performance and neural correlates to specific task events were similar to earlier studies across all tasks. We utilized generalized linear models (GLM) to determine which behavioral variables most strongly influence neural activity in the mPFC. The position of the mouse in the environment was found to explain most of the variance in neural activity, together with movement speed they were the strongest predictors of neural activity across all tasks. Reward time points in the working memory test, the conditioned stimulus after fear conditioning, or head direction in general were also strongly encoded in the mPFC.
Many of the recorded neurons showed a stable spatial activity profile across multiple sessions of the same task. Similarly, cells that coded for position in one task tended to code for position in other tasks. Not only did the same cells code for position across multiple tasks, but cells also coded for movement speed and head direction. This indicates that at least these general behavioral variables are each represented by the same neurons in the mPFC. Interestingly, the stability of position or speed coding did not depend on the time between two sessions, but only on whether it was within the same or across different tasks. Within the same task, stability was slightly higher than across different tasks.
To find out whether task-specific behavioral aspects were also stably encoded in the mPFC, difference scores as the difference in neural activity between two task aspects like left- and right-choice trials or exposed and enclosed locations were calculated. Many cells encoded these aspects stably across different sessions of each task. Both the left-right differences in the different phases of the working memory test, the open-closed-arm differences in the elevated plus maze, the different activity between center and corners in the open field, the social target-object differences in the social interaction test, and the differences between the two tones during fear conditioning were all stably encoded across the population of mPFC cells. Only the distinction between the novel and the familiar object during object recognition was not stably encoded, but also the preference for the novel object was not present in the second session of novel object exploration.
There was also an overlap in coding for different aspects within a task across multiple sessions. For example, cells stably encoded left-right differences in the T-maze between different sessions as a function of walking direction across different phases of working memory, an aspect that we could already show within one session (Vogel, Hahn et al., 2022). During fear conditioning, the same cells showed a discrimination between CS+ and CS- that also responded to the start of CS+.
Consistency in the neurons activity across different tasks was also found, but only between tasks with similar demands, the elevated plus-maze and free exploration of the open field. Cells that were more active in the open arms also showed more activity in the center of the open field and vice versa. This could be an indicator that the cells were coding for anxiety or exposure across those tasks, indicating that neurons in the mPFC also stably encode general task aspects independent of the specific environment. However, it remains unclear what exactly these neurons encode; in the case of a general fear signal, one would also expect activation during fear conditioning which could not be found.
Overall, we found that neurons in the mPFC of mice encoded multiple general behavioral variables across multiple tasks and task-specific variables were encoded stably within each of the tested tasks. However, we found little task-specific variables that were systematically encoded by the same neurons with the exception being the elevated plus-maze and open field exploration, two tasks with similar features.
Precise regulation of gene expression networks is required to develop and maintain a healthy organism before and after birth and throughout adulthood. Such networks are mostly comprised of regulatory proteins, but meanwhile many long non-coding transcripts (lncRNAs) are shown to participate in these regulatory processes. The functions and mechanisms of these lncRNAs vary greatly, however they are often associated with transcriptional regulation. Three lncRNAs, namely Sweetheart RNA (Swhtr), Fetal-lethal noncoding developmental regulatory RNA / Foxf1 adjacent non-Coding developmental regulatory RNA (Fendrr) and lncFsd2, were studied in this work to demonstrate the variety of cellular and biological processes that require lncRNA-mediated fine-tuning, in regard to the cardiopulmonary system.
Swhtr was found to be expressed exclusively in cardiomyocytes and became critical for regeneration after myocardial injury. Mice lacking Swhtr did not show issues under normal conditions, but failed to undergo compensatory hypertrophic remodeling after injury, leading to increased mortality. This effect was rescued by re-expressing Swhtr, demonstrating importance of the RNA. Genes dependent on Swhtr during cardiac stress were found to likely be regulated by NKX2-5 through physical interaction with Swhtr. Fendrr was found to be expressed in lung and interacted with target promoters through its RNA:dsDNA binding domain, the FendrrBox, which was partially required for Fendrr function. Fendrr, together with activated WNT signaling, regulated fibrosis related target genes via the FendrrBox in fibroblasts. LncFsd2, an ubiquitously expressed lncRNA, showed possible interaction with the striated muscle specific Fsd2, but its exact function and regulatory role remain unclear in muscle physiology. Immunoprecipitation and subcellular fractionation experiments suggest that lncFsd2 might be involved in nuclear retention of Fsd2 mRNA, thus fine-tuning FSD2 protein expression. These investigations have shed light on the roles of these lncRNAs in stress responses, fibrosis-related gene regulation, and localization processes, advancing our understanding of cardiovascular and pulmonary maintenance, reaction to injury, and diseases. The diverse and intricate roles of these three lncRNAs highlight how they influence various cellular processes and disease states, offering avenues for exploring lncRNA functions in different biological contexts.
Regulatory required, classical toxicity studies for environmental hazard assessment are costly, time consuming, and often lack mechanistic insights about the toxic mode of action induced through a compound. In addition, classical toxicological non-human animal tests raise serious ethical concerns and are not well suited for high throughput screening approaches. Molecular biomarker-based screenings could be a suitable alternative for identifying particular hazardous effects (e.g. endocrine disruption, developmental neurotoxicity) in non-target organisms at the molecular level. This, however, requires a better mechanistic understanding of different toxic modes of action (MoA) to describe characteristic molecular key events and respective markers.
Ecotoxicgenomics, which uses modern day omic technologies and systems biology approaches to study toxicological responses at the molecular level, are a promising new way for elucidating
the processes through which chemicals cause adverse effects in environmental organisms. In this context, this PhD study was designated to investigate and describe MoA-characteristic
ecotoxicogenomic signatures in three ecotoxicologically important aquatic model organisms of different trophic levels (Danio rerio, Daphnia magna and Lemna minor).
Applying non-target transcriptomic and proteomic methodologies post chemical exposure, the aim was to identify robust functional profiles and reliable biomarker candidates with potential
predictive properties to allow for a differentiation among different MoA in these organisms. For the sublethal exposure studies in the zebrafish embryo model (96 hpf), the acute fish embryo toxicity test guideline (OECD 236) was used as conceptual framework. As different test compounds with known MoA, the thyroid hormone 3,3′,5-triiodothyronine (T3) and the thyrostatic 6-propyl-2-thiouracil (6-PTU), as well as six nerve- and muscle-targeting insecticides (abamectin, carbaryl, chlorpyrifos, fipronil, imidacloprid and methoxychlor) were evaluated. Furthermore, a novel sublethal immune challenge assay in early zebrafish embryos (48 hpf) was evaluated for its potential to assess immuno-suppressive effects at the gene expression level. Therefore, toxicogenomic profiles after an immune response inducing stimulus with and without prior clobetasol propionate (CP) treatment were compared. For the aquatic invertebrate D. magna, the study was performed with previously determined low effect concentrations (EC5 & EC20) of fipronil and imidacloprid according to the acute immobilization test in water flea (OECD 202). The aim was to compare toxicogenomic signatures of the GABA-gated chloride channel blocker (fipronil) and the nAChR agonist (imidacloprid). With similar low effect concentrations, a shortened 3 day version of the growth inhibition test with L. minor (OECD 221) was conducted to find molecular profiles differentiating between photosynthesis and HMG-CoA reductase inhibitory effects. Here, the biological interpretation of the molecular stress response profiles in L. minor due to the lack of functional annotation of the reference genome was particularly challenging. Therefore, an annotation workflow was developed based on protein sequence homology predicted from the genomic reference sequences.
With this PhD work, it was shown how transcriptomic, proteomic and computational systems biology approaches can be coupled with aquatic toxicological tests, to gain important mechanistic insights into adverse effects at the molecular level. In general, for the different investigated adverse effects for the different organisms, biomarker candidates were identified, which describe a potential functional link between impaired gene expressions and previously reported apical effects. For the assessed chemicals in the zebrafish embryo model, biomarker candidates for thyroid disruption as well as developmental toxicity targeting the heart and central nervous system were described. The biomarkers derived from nerve- and muscletargeting insecticides were associated with three major affected processes: (1) cardiac muscle cell development and functioning, (2) oxygen transport and hypoxic stress and (3) neuronal development and plasticity. To our knowledge, this is the first study linking neurotoxic insecticide exposure and affected expression of important regulatory genes for heart muscle (tcap, actc2) and forebrain (npas4a) development in a vertebrate model. The proposed immunosuppression assay found CP to affect innate immune induction by attenuating the response of genes involved in antigen processing, TLR signalling, NF-КB signalling, and complement activation ...
Ischemic heart disease caused by occlusion of coronary vessels leads to the death of downstream tissues, resulting in a fibrotic scar that cannot be resolved. In contrast to the adult mammalian heart, the adult zebrafish heart can regenerate following injury, enabling the study of the underlying cellular and molecular mechanisms. One of the earliest responses that take place after cardiac injury in adult zebrafish is coronary revascularization. Previous transcriptomic data from our lab show that vegfc, a well-known regulator of lymphatic development, is upregulated early after injury and peaks at 96 hours post cryoinjury, coinciding with the peak of coronary endothelial cell proliferation. To test the hypothesis that vegfc is involved in coronary revascularization, I examined its expression pattern and found that it is expressed by coronary endothelial cells after cardiac damage. Using a loss-of-function approach to block Vegfc signaling, I found that it is required for coronary revascularization during cardiac regeneration. Notably, blocking Vegfc signaling resulted in a significant reduction in cardiomyocyte regeneration. Using transcriptomic analysis, I identified the extracellular matrix component gene emilin2a and the chemokine gene cxcl8a as effectors of Vegfc signaling. During cardiac regeneration, cxcl8a is expressed in epicardium-derived cells, while the gene encoding its receptor cxcr1 is expressed on coronary endothelial cells. I found that overexpressing emilin2a increases coronary revascularization, and induces cxcl8a expression. Using loss-of-function approaches, I observed that both cxcl8a and cxcr1 are required for coronary revascularization after cardiac injury.
Altogether, my findings indicate that Vegfc acts as an angiocrine factor that plays an important role in regulating cardiac regeneration in zebrafish. Mechanistically, Vegfc promotes the expression of emilin2a, which promotes coronary proliferation, at least in part by enhancing Cxcl8a-Cxcr1 signaling. This study helps in understanding the mechanisms underlying coronary revascularization during cardiac regeneration, with promising therapeutic applications for human heart regeneration.
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.
With 5-10 newly diagnosed patients per 100,000 people every year, glioblastoma is the most common malignant primary brain tumor. Despite extensive research activity in the last decades, clinical effectiveness of the currently available therapy standard of surgery, radiochemotherapy and tumor-treating fields is still limited and mean survival rates in unselected collectives are only about one year. Accordingly, there is an urgent need to explore new therapeutic options. The current standard of care includes surgery followed by radiation therapy in combination with the alkylating chemotherapeutic agent Temozolomide. Even with successful initial therapy, tumor recurrence is still inevitable. Currently, there are no defined recommendations for clinical management of the disease in the event of tumor recurrence. Only 20-30% of patients qualify for a second surgical resection, while other options include retreatment with Temozolomide, CCNU (Lomustine) or Regorafenib and enrollment in a clinical trial.
The development of immunotherapies for glioblastoma, in particular, has been the focus of intense preclinical and clinical efforts. However, low numbers of mutations and a highly immunosuppressive tumor microenvironment result in glioblastoma being considered an immunologically “cold” tumor. Strategies successfully established in mutagen-induced tumors with antibodies directed against the PD-1, PD-L1 or CTLA-A4 immune checkpoints have therefore failed in glioblastoma.
Cellular immunotherapies based on chimeric antigen receptor (CAR)-technology have emerged as an alternative powerful option to tackle immunologically “cold” tumors. Several CAR-T cell products targeting glioma antigens have been developed and some evidence of clinical activity has been demonstrated. Natural killer (NK) cells as carriers of CAR constructs have several advantages over T cells, including a much lower risk of neurotoxicity and better interaction with immune cells in the microenvironment. Based on the human NK cell line NK-92, a clinical-grade product, suitable as an off-the-shelf therapeutic, has been developed. The NK-92/5.28.z clone (CAR-NK) expresses a CAR based on the HER2-specific antibody FRP5 in addition to signal-enhancing CD28 and CD3ζ domains. Similar to several other tumor entities, overexpression of the growth factor receptor HER2 is often found in glioblastoma patients. Because of its substantial role in the regulation of cell proliferation, survival, differentiation, angiogenesis and invasion, this receptor is classified as an oncogene. HER2 overexpression plays a major role in the malignant transformation of cells and its oncogenic potential has been studied in detail in breast cancer. However, HER2 expression was also found in up to 80% of glioblastomas, which correlates with an impaired probability of survival. Under physiological conditions, HER2 is not expressed in the adult central nervous system, making it a promising target antigen for glioblastoma immunotherapy.
In previous projects, it has already been shown that these CAR-NK cells exhibit a high and specific lytic activity towards HER2+ glioblastoma cells. While repetitive intratumoral injections of CAR-NK cells already significantly extended symptom-free survival in murine orthotopic xenograft models, CAR-NK cell therapy in immunocompetent mice promotes an endogenous anti-tumor immune response which improves tumor control and provides persisting anti-tumor immunity after therapy of early-stage tumors. However, in more advanced tumor models, efficacy is limited and induction of the checkpoint-molecule PD-L1 in response to CAR-NK-cell therapy was identified as a key mechanism of therapy resistance.
Immunotherapy employing the intravenous administration of checkpoint inhibitors has already revolutionized the treatment of various malignant diseases such as melanoma or lung cancer. In particular, the approach of cancer immunotherapy has focused on the systemic administration of antibodies directed against immune checkpoints such as PD-1, PD-L1 and CTLA-4. In glioblastoma, both tumor cells and microglia, the brain-resident macrophages, express PD-L1, which hinders the activation of CD8+ and CD4+ T cells. Therefore, immunotherapy directed against the PD-1/PD-L1 axis represents a promising approach for the treatment of glioblastoma. One problem, however, is the severe toxicity caused by the systemic effects of checkpoint inhibitors, since the immune response is stimulated not only in tumor tissue but also in healthy organs. Serious side effects such as colitis, hepatitis, pancreatitis or hypophysitis, including numerous deaths, have been reported.
This study aimed to improve the efficacy of CAR-NK cell therapy by combining it with adeno-associated virus (AAV)-mediated transfer of anti-PD-1 antibodies as a strategy to enable local combination therapy to control intracranial tumors.
AAVs carrying a payload coding for an anti-PD-1 immunoadhesin (aPD-1) retargeted to HER2-expressing cells by fusion of so-called Designed Ankyrin Repeat Proteins (DARPins) with a viral capsid protein were employed for this to focus checkpoint inhibitor therapy to the tumor area, resulting in high intratumoral and low systemic drug concentrations. ...
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.
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...
Heart development is a dynamic process modulated by various extracellular and intracellular cues. Cardiac progenitors in vertebrates such as the zebrafish, migrate over to the midline after differentiation from the epiblast (Bakkers, 2011; Rosenthal & Harvey, 2010; Stainier et al., 1996; Trinh & Stainier, 2004). These progenitors form a cardiac disc at the midline which elongates into the linear heart tube. The differentiation and migration of cardiac precursors is modulated by signaling interactions between cardiac precursor cells and their extracellular environment known as the Extracellular Matrix (ECM). Studies have shown that Cell-ECM interactions play a crucial role in sculpting the heart during early morphogenic events (Davis CL, 1924; Männer & Yelbuz, 2019; Rosenthal & Harvey, 2010). One key factor to these processes is the presence of a specialized ECM known as the Basement Membrane (BM). Extracellular basement membrane proteins such as Fibronectin have been shown to modulate these very early migration processes of the cardiomyocyte progenitors (Trinh & Stainier, 2004). As the heart develops further, the linear heart tube is composed of myocardial cells with an inner endothelial cell lining separated by a layer of thick jelly like substance called the cardiac jelly (Barry A, 1948; Davis CL, 1924; Little et al., 1989). The cardiac jelly also called the cardiac basement membrane, has been shown to regulate distinct developmental events during cardiogenesis. This early CJ contains components of the basal lamina such as laminins, fibronectin, hyaluronan as well as non-fibrillar collagens such as Collagen IV (Little et al., 1989). In this study, I aimed to identify ECM molecules of the Basement Membrane in the heart and identify their role in the modulation of cardiac development and regeneration using the zebrafish as my model organism.
I identified genes belonging to the Zebrafish Matrisome expressed during cardiac developmental and regeneration and performed CRISPR/Cas9 sgRNA mediated mutagenesis. I also developed overexpression tools for these genes.
Agrinp168 mutants exhibited no obvious gross morphology defects during cardiac development and were adult viable. Adult mutants exhibited reduced cardiomyocyte proliferation, but no significant difference in cardiomyocyte dedifferentiation post cardiac cryoinjury.
Decorin overexpression through mRNA injections led to increased myocardial wall thickness and DN dcn overexpression through mRNA injections led to loss of cardiac looping during early development.
Mutants for Small Leucine Rich Proteoglycan (SLRP) prelp generated using CRISPR/Cas9 mutagenesis exhibited cardiovascular defects. Close observation of prelp mutant hearts revealed a reduced heart rate and impaired fractional shortening of the ventricle. prelp mutants exhibited an enlarged atrium at 48 hpf and 72 hpf as well as a reduced ventricle size at 72 hpf. Chamber size in the mutant hearts were enlarged irrespective of contractility of the heart. Mutants showed an increased number of Atrial cardiomyocytes, but no change in cell size. On the molecular level, extracellular Laminin localization was disrupted in prelp mutants along with an increase in thickness and volume of the cardiac HA in the CJ suggesting a potential compensatory role, or retention of immaturity of the cardiac jelly in the prelp mutants. Transcriptomics analysis on the prelp mutant hearts revealed downregulation of ECM organization and ECM-Receptor interaction processes in the mutants. Gene Ontology analysis on prelp mutants hearts transcriptome revealed increased MAPK signaling. Interestingly, genes related to degradation of cardiac HA and maturation of cardiac jelly were downregulated, and genes related to epithelial identity of cardiomyocytes were upregulated. Analysis of the mutant hearts at single cell resolution revealed increased number of mutants exhibiting rounded up cardiomyocytes and loss of apical Podocalyxin. Truncated forms of prelp were generated to identify domain specific roles for Prelp, and reintroduction of N-terminal truncated Prelp into the mutants rescued the basal lamina localization and cardiac jelly volume phenotypes. Myocardium specific re-establishment of prelp expression revealed a marked rescue of the mutant cardiovascular phenotype suggesting that tissue specific expression of prelp is not required so long as Prelp is secreted into the CJ. With these data, I’ve elucidated the role of ECM SLRPs in modulation of cardiac chamber morphogenesis process and regeneration of the heart.
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.
Chemical pollution is one of the main contributors to the degradation of lotic ecosystems and their biodiversity. Among chemicals driving lotic biodiversity decline are anthropogenic organic micropollutants (AOM), which affect the survival and functioning of freshwater organisms. Continuous exposure of freshwater organisms to AOM leads to adverse effects that sometimes cannot be traced with standard toxicity methods such as standard toxicity testing or biodiversity indices. Among these effects of AOM are selective or mutagenic effects that cause impaired species genetic diversity. Thus, the correlation between different levels of AOM and genetic diversity of species is still poorly understood. However, it can be explored by applying population genetics screening.
In Chapter 1 of this thesis, background information on environmental pollution, genetic screening, and the detection of evolutionary-relevant AOM effects in freshwater organisms are described and the thesis goals are identified. The main goal of the thesis is to study whether AOM exposure occurring in European rivers causes a significant evolutionary footprint in freshwater species and leads to a selection of more tolerant geno-and phenotypes. Therefore, population genetics indices together with high-resolution chemical exposure screening of a widespread indicator invertebrate species, Gammarus pulex (Linnaeus, 1758), living in polluted and pristine European rivers were investigated.
In Chapter 2, the development of a genetic screening method for G. pulex (microsatellites) is described. Due to genetic differentiation and the presence of morphologically cryptic lineages, the available sets of target loci do not enable a reliable population genetic characterization of G. pulex from central Germany. Thus, a novel set of microsatellite loci for a high-precision assessment of population genetic diversity was here applied. Eleven loci were first identified and thereafter amplified in G. pulex from three rivers. The new loci reliably amplified and indicated polymorphisms in the studied amphipods. The amplification resulted in the successful identification of genetically distinct populations of G. pulex from the analyzed rivers. Moreover, the microsatellite loci were amplified in other genetic lineages of G. pulex and another Gammarus species, G. fossarum, promising a broader applicability of the loci in related amphipod species.
In Chapter 3, the effects of AOM on species genetic differentiation and sensitivity to toxic chemicals in a typical central European river with pristine and AOM-polluted sections was investigated. The river’s site-specific concentrations of AOM were assessed by chemical analysis of G. pulex tissue and water samples. To test, whether different levels of AOM in the river select for pollution-dependent genotypes, the genetic structure of G. pulex from the river was analyzed. Finally, the toxicokinetics of and sensitivity to the commonly used insecticide imidacloprid were determined for amphipods sampled at pristine and polluted sections to assess whether various levels of AOM in the river influence sensitivity of G. pulex to imidacloprid. The results indicated that different levels of AOM did not drive genetic divergence of G. pulex within the river but led to an increased sensitivity of exposed amphipods to imidacloprid. The amphipods living in polluted river sections were more sensitive to the insecticide due to chronic exposure to toxic levels of AOM.
In Chapter 4, the relationship between site-specific pollution levels of AOM and genetic diversity parameters of G. pulex was analyzed at the regional scale within six rivers in central Germany. The genetic structure of G. pulex in the studied area was tested for relatedness to the waterway distance between sites. Gammarus pulex genetic diversity parameters, including allelic richness and inbreeding rate, were tested against environmental pollution parameters using linear mixed-effect- and structural-equation models. According to the results, G. pulex genetic diversity parameters were significantly associated with the detected AOM levels. At sites with high concentrations of AOM and toxicity potential G. pulex showed reduced genetic diversity and increased rates of inbreeding. These results suggest that AOM play a major role in shaping the genetic diversity of G. pulex in rivers.
According to the findings presented here, the applied microsatellites can be used to successfully detect changes in genetic patterns in freshwater amphipods facing increased levels of AOM. The findings indicate that levels of AOM representative for European rivers do not lead to the separation of genotypes among G. pulex as the connectivity between sites majorly contributes to species’ genetic structure. However, the chronic exposure to increased levels of toxic AOM leads to a reduction of species genetic diversity and increases the sensitivity of G. pulex to the toxic chemical effects.
A promising strategy to reduce the dependency from fossil fuels is to use the yeast Saccharomyces cerevisiae to bioconvert renewable non-food feedstocks or waste streams, like lignocellulosic biomass, into bioethanol and other valuable molecule blocks. Lignocellulosic feedstocks contain glucose and significant fractions of the pentoses xylose and arabinose in varying proportions depending on the biomass type. S. cerevisiae is an efficient glucose consumer, but it cannot metabolize xylose and arabinose naturally. Therefore, extensive research using recombinant DNA techniques has been conducted to introduce and improve the biochemical pathways necessary to utilize these non-physiological substrates. However, any functional pathway capable of metabolizing D xylose and L arabinose in S. cerevisiae requires the transport of these sugars across the plasma membrane. The endogenous sugar transport system of S. cerevisiae can conduct a limited uptake of D-xylose and L-arabinose; this uptake enables only basal growth when the enzymatic pathways are provided. For this reason, the uptake of D xylose and L-arabinose has been recognized as a limiting step for the efficient utilization of these non-physiological substrates.
Gal2, a member of the major facilitator superfamily, is one of the most studied hexose transporters in S. cerevisiae. Although its expression is repressed in the presence of glucose, it also transports this sugar with high affinity when constitutively expressed. Recent efforts to engineer yeast strains for the utilization of plant biomass have unraveled the ability of Gal2 to transport non-physiological substrates like xylose and arabinose, among others. Improving Gal2 kinetic and substrate specificity, particularly for pentoses, has become a crucial target in strain engineering. The main goal of this study is to improve the utilization of xylose and arabinose by increasing the cell permeability of these non physiological substrates through the engineering of the galactose permease Gal2.
GAL2 gene expression depends on galactose, which acts as an inducer; nevertheless, even in the presence of galactose, glucose act as a strict repressor; consequently, GAL2 gene is usually placed under the control of a constitutive promoter. However, the presence of glucose additionally triggers the Gal2 degradation, which is mediated by the covalent attachment of the small 76 amino acid protein ubiquitin (Ub) to the targeted transporter; in a multi-step process called ubiquitination.
Ubiquitination of hexose permeases involves the activation of the Ub molecule by the E1 Ub-activating enzyme using ATP; then, the activated Ub is transferred to a specific Ub-conjugating enzyme E2, which donates the Ub indirectly through a specific HECT E3 enzyme (Rsp5) to a lysine residue of the substrate, with the aid of an adaptor protein which recognizes the target (Rsp5-adaptor). Ubiquitinated permeases are sent by membrane invagination to early endosomes, where they encounter ESCRTs (endosomal sorting complex required for transport). The targeted permeases are sorted in intralumenal vesicles (ILV) inside of the endosome, which after several cycles, turns into a multivesicular body (MVB) that subsequently fuses with the vacuole to expose the protein content of the ILVs to lumenal hydrolases for degradation.
Gal2 contains 30 lysine residues that may accept the ubiquitin molecule, which targets its degradation. It is known that mono-ubiquitination by Rsp5 on multiple lysine residues is necessary to internalize Gal2 (Horak & Wolf, 2001). However, the authors did not identify the specific lysine residues involved in the ubiquitination processes. This study screened several Gal2 variants where lysine residues were mutated or removed from the protein sequence to discover which lysine residues are likely involved in ubiquitination and consequent turnover of the transporter. The results of the screening showed that mutation of the N terminal lysine residues 27, 37, and 44 to arginine (Gal23KR) produced a functional transporter that, when fused with GFP (Gal23KR_GFP), showed an exclusive localization at the plasma membrane in cells growing in galactose or glucose as a sole carbon source (Tamayo Rojas et al., 2021b).
This study furthermore evaluated upstream signals caused by phosphorylation which triggers ubiquitination and consequent turnover of the targeted protein; using similar screening approaches to assess the stabilization of Gal2 by lysine residue modifications, it was possible to identify that N terminal serine residues 32, 35, 39, 48, 53, and 55 are likely involved in the internalization of Gal2, since a Gal2 construct where all these serines were mutated to alanine residues and tagged with GFP (Gal26SA_GFP) exhibited practically complete localization at the plasma membrane in cells growing in galactose or glucose as a sole carbon source (Tamayo Rojas et al., 2021b)...
Mutational analysis of ribosomal DNA and maturation-scheme analysis of ribosomal RNA in A. thaliana
(2022)
Ribosome biogenesis is a fundamental cellular process beginning with long precursor rRNA transcription from multi-copies of repetitive 45S ribosomal DNAs. At the subunit level, the primary pre-rRNA transcript encapsuled in 90S protein-RNA complex undergoes decisive splitting in two chief ways for further maturation into large (LSU) and small (SSU) ribosomal subunit. The usage of specific rDNA copies from defined chromosomes and their selective role during growth and development have been a topic of interest owing to its contribution to specialized ribosome theory which proposes non-monolithic functions for ribosomes and thereby their mRNA translation potential. Dual-guide CRISPR/Cas9 mediated disruption of rDNA regions resulted in stable disruption of up to 2.5% and 5% of all rDNA copies in hetero- and homozygous (ploop KD) conditions, respectively. At the RNA level, the mutation excised a critical structural element, P-loop on the LSU 25S rRNA. Mutation caused a dosage dependent defect with homozygosity leading to severe developmental defects through vegetative and reproductive growth phases which is manifested in their proteome by means of disregulation through both increase and decrease of several gene ontological categories of proteins in mutants. Interestingly, the mutation on chromosome 4 triggered dosage compensation through rRNA expression from chromosome 2 further compounded by ectopic rRNA biogenesis defects. The mutated copies however are not incorporated in the translating ribosomes and as a direct or indirect consequence led to elevated basal autophagic levels in the mutants.
The primary 35S transcript is known to undergo two modes of initial cleavages at the pre-rRNA level that aid in their subsequent maturation. Root cell culture (RCC) studies shows that these cells contain a novel ITS2-first cleaved precursor even under control growth conditions, P-C2 adding a third maturation means for the 35S pre-rRNA. This maturation path is further known to be triggered under elevated growth temperature forming a novel adaptive response in Arabidopsis and two other crop plants, tomato, and rice. Taken together, the pulse-chase labeling analysis of control and stressed tissues uncovers the fine-tuned pre-rRNA schematics with crossovers between multiple maturation paths.
Lipopolysaccharide (LPS) is a major glycolipid component in the outer leaflet of the outer membrane of Gram-negative bacteria and known as endotoxin exhibited by the lipid A moiety, which serves as a membrane anchor. The effective permeability barrier properties of the outer membrane contributed by the presence of LPS in the extracellular layer of the outer membrane confer Gram-negative bacteria a high resistance against hydrophobic compounds such as antibiotics, bile salts and detergents to survive in harsh environments. The biogenesis of LPS is well studied in Escherichia coli (herewith E. coli) and the LPS transport (Lpt) is carried out by a transenvelope complex composed of seven essential proteins (LptABCDEFG), which are located in the three compartments of the cell such as the outer membrane, the inner membrane and the periplasm. The Lpt system also exists in Anabaena sp. PCC 7120 (herewith Anabaena sp.), however, homologues of LptC and LptE are still missing. BLAST search failed to identify a homologue of LptC, in contrast, the secondary structure analysis using the Pfam database based on the existing ecLptC secondary structure identified one open reading frame All0231 as the putative Anabaena sp. homologue of LptC, which is designated anaLptC. Despite the low sequence similarity, the secondary structure alignment between anaLptC and ecLptC using the HHpred server showed that both proteins share high secondary structural similarities. The genotypic analysis of the insertion mutant anaLptC did not identify a fully segregated genome and its phenotypic analysis revealed that it was sensitive against chemicals, suggesting that the analptC gene is essential for the growth of Anabaena sp. and involved in the outer membrane biogenesis. This is further supported by the observation of the small cell phenotype in the anaLptC mutant via transmission electron microscopy. Moreover, physical interactions between the anaLptC periplasmic domain with anaLptA as well as with anaLptF were established, indicating that the anaLptC periplasmic domain is correctly folded and alone functional and that the transmembrane helix is not required for the interaction with anaLptA and anaLptF. Furthermore, the reduction of the O-antigen containing LPS was observed in the insertion mutant anaLptC and the dissociation constant Kd of the anaLptC periplasmic domain for ecLPS was determined.The three-dimensional structure of the periplasmic domain of anaLptC was solved by X-ray crystallography with a resolution of 2.8 Å. The structural superposition between the ecLptC crystal structure (PDB number 3my2) and the crystal structure of anaLptC periplasmic domain obtained by this study showed the similarity in the folding of the two proteins with a Cα r.m.s.d value of about 1 Å and confirmed that the length of anaLptC is more than two times longer than that of ecLptC. The structural comparison also revealed that both structures share the typical β-jellyroll fold and conserved amino acids, which were shown in ecLptC to bind to LPS in vivo and found in anaLptC. Overall, these data strongly suggest that anaLptC is involved in the transport of LPS and support the model whereby the bridge spanning the inner membrane and the outer membrane would be assembled via interactions of the structurally conserved β-jellyroll domains shared by five (LptACDFG) out of seven Lpt proteins.
Non-ribosomal peptide synthetases (NRPSs) are modular biosynthetic megaenzymes producing many important natural products and refer to a specific set of peptides in bacteria’s and fungi’s secondary metabolism. With the actual purpose of providing advantages within their respective ecological niche, the bioactivity of the structurally highly diverse products ranges from, e.g., antibiotic (e.g., vancomycin) to immunosuppressive (e.g., cyclosporin A) to cytostatic (e.g., echinomycin or thiocoralin) activity.
An NRPS module consists of at least three core domains that are essential for the incorporation of specific substrates with the 'multiple carrier thiotemplate mechanism' into a growing peptide chain: an adenylation (A) domain selects and activates a cognate amino acid; a thiolation (T) domain shuffles the activated amino acid and the growing peptide chain, which are attached at its post-translationally 4ʹ-phosphopantetheine (4'-PPant) group, between the active sites; a condensation (C) domain links the upstream and downstream substrates. NRPS synthesis is finished with the transfer of the assembled peptide to the C-terminal chain-terminating domain. Accordingly, the intermediate is either released by hydrolysis as a linear peptide chain or by an intramolecular nucleophilic attack as a cyclic peptide.
The NRPS’s modular character seems to imply straightforward engineering to take advantage of their features but appears to be more challenging. Since the pioneering NRPS engineering approaches focused on the reprogramming and replacement of A domains, several working groups developed advanced methods to perform a complete replacement of subdomains or single or multiple catalytic domains.
The first part of this work focusses parts of the publication with the title 'De novo design and engineering of non-ribosomal peptide synthetases', which follows up assembly line engineering with the development of a new guideline. Thereby, the pseudodimeric V-shaped structure of the C domain is exploited to separate the N-terminal (CDSub) and C-terminal (CASub) subdomains alongside a four-AA-long linker. This results in the creation of self-contained, catalytically active CASub-A-T-CDSub (XUC) building blocks. As an advantage over the previous XU concept, the characteristics (substrate- and stereoselectivity) assigned to the C domain subunits are likewise exchanged, and thus, no longer represent a barrier. Furthermore, with the XUC concept, no important interdomain interfaces are disrupted during the catalytic cycle of NRPS, allow to expect much higher production titers. Moreover, the XUC concept shows a more flexible application within its genus origin of building blocks to create peptide libraries. Additionally, with this concept only 80 different XUC building blocks are needed to cover the entire proteinogenic amino acid spectrum.
The second part of this work addresses the influence of the C domain on activity and specificity of A domains. In a comprehensive analysis, a clear influence of different C domains on the in vitro activation rate and the in vivo substrate spectrum could be observed. Further in situ and in silico characterizations indicate that these influences are neither the result of the respective A domains promiscuity nor the C domain’s proofreading, but due to an 'extended gatekeeping' function of the C domain. This novel term of an 'extended gatekeeping' function describes the very nature of interfaces that C domains can form with an A domain of interest. Therefore, the C-A interface is assumed to have a more significant contribution to a selectivity filter function.
The third part of this work combines the NRPS engineering with phylogenetic/evolutionary perspectives. At first, the C-A interface could be precisely defined and further identified to encode equivalent information corresponding to the complete C-A didomain. Moreover, the comparison of NRPSs topology reveals hints for a co-evolutionary relatedness of the C-A didomain and could be shown to reassemble even after separation. In this regard, based on a designed CAopt.py algorithm, the reassembling-compatibility of hybrid interfaces could be determined by scoring of the co-expressed NRPS hybrids. This algorithm also enables the randomization of the interface sequences, thus, leading to the identification of more functional interface variant, which cause significantly higher peptide production and could even be applied to other native and hybrid interfaces.
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 ...
The increasing demand of the high value ω-3 fatty acids due to its beneficial role for human health, explains the huge need for alternative production ways of ω-3 fatty acids. The oleaginous alga Phaeodactylum tricornutum is a prominent candidate and has been investigated as biofactory for ω-3 fatty acids, e.g. the synthesis of eicosapentaenoic acid (EPA). In general, the growth and the lipid content of diatoms can be enhanced by genetic engineering or are influenced by environmental factors, e.g. nutrients, light or temperature.
In this study, the potential of P. tricornutum as biofactory was improved by heterologously expressing the hexose uptake protein 1 (HUP1) from the Chlorophyte Chlorella kessleri.
An in situ localization study revealed that only the full length HUP1 protein fused to eGFP was correctly targeted to the plasma membrane, whereas the N-terminal sequence of the protein is only sufficient to enter the ER. Protein and gene expression data displayed that the gene-promoter combination was relevant for the expression level of HUP1, while only cells expressing the protein under the light-inducible fcpA promoter showed a significant expression. In these mutants an efficient glucose uptake was detectable under mixotrophic growth condition, low light intensities and low glucose concentrations leading to an increased cell dry weight.
In a second approach, the growth and lipid content of wildtype cells were analyzed in a small 1l photobioreactor. Here, a commercial F/2 medium and a common culture medium, ASP and modified versions were compared. There was neither a significant impact on the growth and lipid content in P. tricornutum cells due to the supplemention of trace elements nor due to elevated salt concentrations in the media. In a modified version of ASP medium, with adapted nitrate and phosphate concentration a constantly high biomass productivity was achieved, yielding the highest value of 82 mg l-1 d-1 during the first three days. This was achieved even though light intensity was reduced by 40%. The differences in biomass productivity as well as the lipid content and the lipid composition underlined the importance of the choice of culture medium and the harvest time for enhanced growth and EPA yields in P. tricornutum.
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.
Die Vorläuferform der eukaryotischen mRNA (prä-mRNA) durchläuft, eine Reihe von Prozessierungs-Schritte, die schließlich zu der Synthese einer „reifen“ und Exportkompetenten mRNA führt. prä-mRNA Spleißen ist ein essentieller Teilschritt dieser Reifung bei der intragene Sequenzen, sogenannte Introns, von der prä-mRNA entfernt werden, während Exons legiert werden. Das prä-mRNA Spleißen wird durch das Spleißosom katalysiert. Dieser Mega-Dalton Komplex, besteht aus fünf Sub-Komplexen, die sich wiederum aus katalytisch aktiven „kleinen nukleären Ribonukleinsäuren“ (snRNAs) und einer Vielzahl von proteinogenen Faktoren zusammensetzen. Diese Subkomplexe, bezeichnet als snRNPs (small nuclear Ribonucleoprotein Particles), binden die prä-mRNA an charakteristischen Sequenzen und richten die prä-mRNA durch eine Reihe von Konformations-Änderungen so aus, dass benachbarte Exons in Kontakt treten und über eine biochemische Ligations-Reaktion verbunden werden können.
Die Exon- bzw Intronerkennung der snRNPs wird durch zahlreiche Spleißfaktoren reguliert. Eine Proteinfamilie, die essentiell für die Regulierung des Spleißens ist, sind Serin/Arginin-reiche Proteine (SR-Proteine). Diese binden vorzugsweise an das 3‘ oder 5’ Ende von Exons, rekrutieren snRNPs und stimulieren dadurch die Exon-Inklusion. Durch diese Stimulierung können Spleiß-Events reguliert und gezielt spezifische Exons ausgeschlossen oder eingeschlossen werden. Dieser Prozess, der als alternatives Spleißen (AS) bezeichnet wird, tritt in 95% des menschlichen Transkriptoms auf und erweitert die Diversität eines Organismus, da verschiedene Transkripte von demselben Gen erzeugt werden können und folglich die Translation unterschiedlicher Proteine mit distinkten Funktionen ermöglicht wird.
Darüber hinaus verfügt die Zelle durch das AS über eine weitere posttranskriptionale Genregulationsebene, die insbesondere unter zellulären Stressbedingungen zur Expression von alternativen Protein-Isoformen von der Zelle genutzt wird. Eine in medizinischer Hinsicht besonders relevante Stressbedingung ist die sogenannte Hypoxie, die eine Sauerstoff-Unterversorgung von Zellen oder Gewebebereichen beschreibt. Hypoxie bzw. hypoxische Bereiche finden sich in Krebszellen und treten in 90% aller soliden Tumoren auf. Als Teil der Hypoxie Stress-Antwort, verfügt die Zelle über einen Adaptations-Mechanismus, der durch Hypoxieinduzierbare Faktoren (HIF) vermittelt wird. Diese Faktoren induzieren die Transkription zahlreicher Gene und stimulieren die Expression von Stressfaktoren, die an der zellulären Adaption der Hypoxie beteiligt sind. Einer dieser Faktoren ist der vaskuläre endotheliale Wachstumsfaktor A (VEGFA), welcher unter hypoxischen Bedingungen sekretiert wird und dadurch die Proliferation von Endothelzellen, die Neubildung von Blutgefäßen und damit die Vaskularisation des hypoxischen Bereichs stimuliert.
Die zelluläre Anpassung ist jedoch nicht nur auf die transkriptionelle Regulation des HIF-vermittelten Hypoxie Signalwegs beschränkt, sondern wird auf multiplen Genexpressions-Ebenen reguliert. Obwohl bekannt ist, dass tausende Transkripte unter hypoxischen Bedingungen alternativ gespleißt werden, sind die Faktoren, die die zelluläre Stress-Antwort durch AS regulieren, sowie deren molekularer Mechanismus jedoch weitestgehend unbekannt.
Diese Arbeit umfasst die Identifizierung und Charakterisierung von AS Events, sowie den Einfluss und die Regulation von Spleißfaktoren auf AS unter hypoxischen Bedingungen. Hierzu führten wir globale Genexpressions- und AS-Analysen in HeLaKarzinomzelllinien unter Normoxie (21% O2) und Hypoxie (0.2% O2) durch und zeigen, dass 7962 Gene nach 24h Hypoxie unterschiedlich exprimiert werden. Über AS-Analysen konnten 4434 Transkripte identifiziert werden, die bei Hypoxie über AS reguliert sind. Dabei trat „Exon-Skipping“ als das am häufigsten auftretende AS-Events auf. Über PCR basierte Validierungs-Experimente konnten 5 regulierte Transkripte nachgewiesen werden. Dabei weisen Exon 3 und 4 in BORA, Exon 6 in MDM4 und Exon 4-5 in CSSP1 Exon-Skipping Events auf, während Exon-Inklusionen in CEP192 Exon 28 und in der 3’UTR von EIF4A2 validiert werden konnten.
Darüber hinaus wurde im Rahmen der AS-Analyse die Regulation des sogenannten „backsplicings“ bei Hypoxie untersucht. Im Gegensatz zum linearen Spleißens, wird beim backsplicing das 5’Ende und das 3’Ende von Exons verbunden, was die Bildung von sogenannten zirkulären RNAs (circRNAs) zufolge hat. Obwohl nur wenige Funktionen dieser RNA-Klasse bekannt sind, wurde die Regulation von circRNAs während der Zell-Differenzierung sowie in diversen Krebszellen beschrieben. Dabei können circRNAs als microRNA- oder Protein-Schwämme fungieren oder dienen als Protein-Interaktion Plattform und regulieren dabei die Genexpression.