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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.
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.
Influenza is a contagious respiratory disease caused by influenza A and influenza B viruses. The World Health Organisation (WHO) reports that annual influenza epidemics result in approximately 1 billion infections, 3 to 5 million severe cases, and 300 to 650 thousand deaths. Understanding hidden mechanisms that lead to optimal vaccine efficacy and improvement antiviral treatment strategies remain continuous and central tasks. First, regarding the immune response to vaccines and natural infections, the antibody response echoes the dynamics of diverse immune elements such as B-cells, and plasma cells. Also, responses reflect the processes for B-cells to gain and adapt affinity for the virus. Antibodies (Abs) that respond to the virus surface proteins, particularly to the hemagglutinin (HA), have been identified to protect against infection. The Abs responses binding to HA can be broadly protective as this protein is considerably accessible on the virion. When following sequential infections with similar influenza strains, i.e. two infections with different strains of a subtype, an enhanced breadth and magnitude of Abs response is developed, mainly after the second infection. The effect of being effective to new strains is called Abs cross-reaction.
On the other hand, as for antiviral treatment, the WHO currently approves the use of neuraminidase inhibitors (NIs) such as zanamivir and oseltamivir. Diverse research areas such as system biology, learning-based methods, control theory, and systems pharmacology have guided the development of modern treatment schemes. To do so, mathematical models are used to describe a wide range of phenomena such as viral pathogenesis, immune responses, and the drug's dynamics in the body. Drug dynamics are usually expressed in two phases, pharmacokinetics (PK) and pharmacodynamics (PD) - the PK/PD approach. These schemes leverage pre-clinical and clinical data through modeling and simulation of infection and drug effects at diverse levels. Under such a framework, control-based scheduling systems seek to tailor optimal antiviral treatment for infectious diseases. Thus, influenza treatment can be theoretically studied as a control-based optimization duty (about systems stability, bounded inputs, and optimality). Finally, towards real-world implementation, learning-based methods such as neural networks (NNs) can guide solving issues on the control-based performance. Using NNs as identifiers provide a setting to deal with infrequent measures and uncertain parameters for the control systems.
This thesis theoretically explores central mechanisms in influenza infection via modeling and control approaches. In the first project, we explore how and to what extent antibody-antigen affinity flexibility could guide the Abs cross-reaction in two sequential infections using a hypothetical family of antigens. The set of antigens generally represent strains of influenza, such as those of a subtype. Each antigen is composed of a variable and a conserved area, generically representing the structures of the HA, head, and stalk, respectively. We test diverse scenarios of affinity thresholds in the conserved and variable areas of the antigens. The Abs response reaches a high magnitude when using equivalent affinity thresholds in the conserved and variable areas during the first infection. However, improved cross-reaction is developed when slightly increasing the affinity threshold of the variable area for the second infection. Key mutations via affinity maturation is a feature that, together with affinity flexibility between infections, guides Abs cross-reaction in the model outcome. These results could correlate with studies pointing out that broad responses might be dependent on reaching specific mutations for getting affinity to a newly presented antigen while broadly reaching related antigens. The general platform may serve as a proof-of-concept for exploring fundamental mechanisms that favor the Abs cross-reaction.
In a second project, theoretical schemes are developed to combine impulsive and inverse optimal control strategies to address antiviral treatment scheduling. We present results regarding stability, passivity, bounded inputs, and optimality using impulsive action. The study is founded on mathematical models of the influenza virus (target-cell limited model) adjusted to data from clinical trials. In these studies, participants were experimentally infected with influenza H1N1 and treated with NIs. Results show that control-based strategies could tailor dosage and reduce the amount of medication by up to 44%. Also, control-based treatment reaches the efficacy (98%) of the current treatment recommendations by the WHO. Monte Carlo simulations (MCS) disclose the robustness of the proposed control-based techniques. Using MCS, we also explore the applicability to the individualized treatment of infectious diseases through virtual clinical trials. Furthermore, bounded control strategies are applied directly in drug dose estimation accounting for overdose prevention. Finally, due to the limitations of the available technology intended for clinical practice, we emphasize the necessity of developing system identifiers and observers for real-world applications.
In the third project, the problem of data scarcity and infrequent measures in the real world is handled by means of learning-based methods. System identification is derived using a Recurrent High Order Neural Network (RHONN) trained with the Extended Kalman filter (EKF). Lessons learned from impulsive control frameworks are taken to develop a neural inverse optimal impulsive control --neurocontrol. The treatment efficacy is tested for early (one day post-infection) and late (2 to 3 days post-infection) treatment initiation. The neurocontrol reaches an efficacy of up to 95% while saving almost 40% of the total drug in the early treatment. Robustness is tested via virtual clinical trials using MCS.
Lastly, taking all together, the schemes developed in this thesis for modeling the Abs cross-reaction and control-based treatment tailoring can be extended and adapted to explore similar phenomena in different respiratory pathogens, such as SARS-CoV-2.
Unter den weltweit in ständigem Gebrauch befindlichen Chemikalien befinden sich nicht nur Verbindungen mit akuter toxischer Wirkung, sondern auch solche mit Wirkung auf das endokrine System. Eine große Rolle spielt hier vor allem die Störung der Geschlechtsdifferenzierung und der Reproduktion, ausgelöst durch natürliche oder synthetische Chemikalien mit endokrinem Potential, sogenannte endokrine Disruptoren (ED). Diese Chemikalien können über unterschiedliche Eintragspfade in die Umwelt gelangen. Seit Mitte des 20. Jahrhunderts werden mehr und mehr Fälle bekannt, in denen anthropogene Chemikalien die Pflanzen- und Tierwelt belasten, darunter zahlreiche Befunde zu Störungen des Hormonsystems von Mensch und Tier.
Im Rahmen der Gefahren- und Risikobewertung steht bereits eine Vielzahl harmonisierter Prüfrichtlinien für die Identifizierung und Evaluierung der Effekte von (potentiellen) ED zur Verfügung. Um die Gesamtheit aller potentiellen Interaktionen von ED mit dem Hormonsystem detektieren zu können, ist die In-vivo-Untersuchung an Vertebraten in der Chemikalienregistrierung bisher unabdingbar. Bei der Untersuchung endokriner Potentiale in höheren Vertebraten spielen vor allem nager- und vogelbasierte Testsysteme eine wichtige Rolle. Diese bergen jedoch einen hohen zeitlichen, personellen und finanziellen Aufwand und erfordern eine massive Zahl an Versuchstieren, die für diese Tests benötigt werden. Darüber hinaus beinhalten Tierversuche eine Vielzahl von Problemen einschließlich ethischer Bedenken, die sich als Konsequenz der Tierhaltung unter Versuchsbedingungen ergeben. Ein sehr interessanter und vielversprechender Ansatz zur Reduktion von Tierversuchen ist die Entwicklung eines standardisierten Verfahrens für die Untersuchung potentieller ED in Vogelembryonen. Auf Vogelembryonen basierende In-ovo-Modelle stellen einen Mittelweg zwischen In-vitro- und In-vivo-Testsystemen dar. Mit dem Vogeleitest wird der sich entwickelnde Embryo, das für ED sensitivste Entwicklungsstadium im Leben eines Organismus, berücksichtigt.
Das Ziel der vorliegenden Arbeit war die Entwicklung und Eignungsuntersuchung eines auf dem Embryo des Haushuhns (Gallus gallus domesticus) basierenden Testsystems für den Nachweis von ED. Das resultierende Testsystem soll als Alternativmethode zu bisher etablierten nager- und vogelbasierten Testsystemen für die Untersuchung der Effekte hormonell aktiver Substanzen auf die Geschlechtsdifferenzierung in höheren Wirbeltieren eingesetzt werden.
Die im Rahmen der vorliegenden Dissertation durchgeführten Arbeiten umfassten sowohl die Charakterisierung der Normalentwicklung des Hühnerembryos, unbeeinflusst durch ED, als auch die morphologisch-histologischen Veränderungen der Gonaden von substanzexponierten Embryonen. Für die Untersuchung substanzbedingter Effekte, welche den Schwerpunkt der vorliegenden Arbeit darstellen, wurden die Embryonen gegenüber verschiedenen (anti)estrogenen und (anti)androgenen Substanzen exponiert. Unter Einfluss der Estrogene Bisphenol A (BPA) und 17α-Ethinylestradiol (EE2) entwickelten sich die Keimdrüsen der Männchen zu Ovotestes, während Weibchen ein Ovar mit deutlich schmalerem Cortex ausbildeten. Unter Einfluss der Antiestrogene Fulvestrant und Tamoxifen blieben Effekte auf die Gonaden männlicher Embryonen aus, eine durch das potente Estrogen EE2 hervorgerufene Feminisierung männlicher Gonaden konnte durch beide Substanzen jedoch effektiv antagonisiert werden. Weibchen bilden unter Einfluss von Tamoxifen deutlich schmalere linke Gonaden mit einem missgebildeten Cortex aus. Unter Einfluss der Androgene Tributylzinn (TBT) und 17α-Methyltestosteron (MT) blieben die Effekte auf männliche Embryonen aus, während die Weibchen anatomisch virilisierte Gonaden und eine Reduktion des linken gonadalen Cortex aufwiesen. Allein die untersuchten antiandrogenen Versuchssubstanzen Cyproteronacetat (CPA), Flutamid und p,p´-Dichlorodiphenyldichloroethen (p,p´-DDE) hatten keinen Effekt auf die gonadale Geschlechtsdifferenzierung männlicher und weiblicher Hühnerembryonen.
Es konnte gezeigt werden, dass der Embryo von G. gallus domesticus einen sensitiven Organismus innerhalb des Tierreichs darstellt und hinreichend sensitiv auf eine Reihe von endokrin wirksamen und reproduktionstoxischen Chemikalien reagiert. Anatomische und histologische Änderungen der Gonaden können daher als Biomarker für die Wirkung von ED bei Vögeln nützlich sein. Die untersuchten Endpunkte beziehen sich jedoch auf apikale Effekte und liefern keine mechanistischen Informationen zu den untersuchten Substanzen. Der
Hühnereitest ist eine sinnvolle Ergänzung zur bestehenden OECD-Testbatterie und zeichnet sich besonders durch seine kostengünstige und einfache Handhabung im Labor sowie einfach durchzuführende Tests aus. Durch die vergleichsweise kurze Versuchsdauer von nur 19 Tagen ist ein schnelles Substanzscreening möglich, welches zeitlich deutliche Vorteile gegenüber den etablierten nager- und vogelbasierten Testsystemen hat. Als Alternative zu bisherigen Assays könnte der vorgeschlagene Hühnereitest dazu beitragen, im Rahmen der (öko)toxikologischen Gefährdungs- und Risikobewertung von Chemikalien künftig weniger Versuchstiere zu verwenden.
Seit Jahrzehnten finden Kunststoffe aufgrund ihrer vorteilhaften Materialeigenschaften wie z. B. Formbarkeit und im Vergleich zu Glas oder Metall geringe Kosten und leichtes Gewicht, vermehrt Anwendung in allen Bereichen des täglichen Lebens. Einhergehend gelangen Kunststoffe zunehmend in die Umwelt, und reichern sich dort an. Besondere Aufmerksamkeit erfahren Partikel im Größenbereich von 1-1000 µm, sogenanntes Mikroplastik (MP), welches entweder direkt eingetragen wird oder in der Umwelt durch Fragmentierung größerer Plastikteile entsteht. Lange Zeit fokussierte sich die MP Forschung vorrangig auf aquatische Ökosysteme, obwohl Schätzungen davon ausgehen, dass die Kunststoffeinträge in terrestrischen Ökosystemen um ein Vielfaches höher sind. Besonders relevante Eintragspfade sind neben der unsachgemäßen Entsorgung von Abfällen, die landwirtschaftliche Klärschlamm- und Kompostdüngung und der zunehmende Einsatz von Mulchfolien, sowie der im Straßenverkehr generierte Reifenabrieb.
Für eine Abschätzung und Bewertung der MP-Belastung in Böden sind analytische Messungen von MP in Umweltproben essenziell, derzeit jedoch kaum existent, da MP im Boden partikulär und heterogen verteilt vorliegt und deshalb nur schwierig zu detektieren ist. Die für viele Analyseverfahren notwendige Isolation der Kunststoffpartikel, sowie die für repräsentative Messungen erforderliche Aufbereitung großer Probenvolumina stellen besondere analytische Herausforderungen mit großem Kosten- und Zeitaufwand dar. Chromatografische Verfahren finden wenig Anwendung, bieten aber vorteilhafte Voraussetzungen als Screeningverfahren für die Untersuchung von Böden, da sie nicht zwangsweise eine Partikelisolation verlangen, und zudem als Ergebnis einen Massegehalt liefern.
Diese Dissertation zeigt drei Anwendungen Chromatografie basierter Analyseverfahren zur Charakterisierung von MP im Boden. Erstmalig wurde die Thermo-Extraktion-Desorption-Gaschromatografie-Massenspektrometrie (TED-GC/MS) für die Analytik von Reifenabrieb in realen Umweltproben angewandt bei minimaler Probenaufbereitung. Dafür wurde ein Straßenrandboden umfangreich beprobt und analysiert, und es konnte neben der Eignung der analytischen Methode auch eine repräsentative Probenahmestrategie und räumliche Verteilungsmuster von Reifenabrieb im Boden demonstriert werden.
Der zweite Forschungsschwerpunkt lag auf der Methodenentwicklung und validierung eines neuartigen chemischen Extraktionsverfahrens für die Bestimmung von Polyestern in Bodenproben. Das Verfahren basiert auf der hydrolytischen Spaltung von Polyestern in ihre Monomere, deren flüssigchromatografische Abtrennung von Matrixbestandteilen und der Detektion mittels UV-Absorption. Das Verfahren verlangt neben der Extraktion keine weiteren Probenaufbereitungsschritte, ist für unterschiedliche Umweltmatrizes geeignet und ist damit z. B. prädestiniert für den Nachweis von Polyesterfasern auf gedüngten landwirtschaftlichen Flächen.
MP ist nicht nur aufgrund seiner Persistenz problematisch, sondern auch, weil es hydrophobe organische Schadstoffe aus dem Umweltmedium anreichern und transportieren kann. Maßgeblich für das Sorptionsverhalten sind die Materialeigenschaften des zugrunde liegenden Kunststoffes, welche Änderungen durch Alterungsprozessen unterliegen. Der Zusammenhang zwischen Materialalterung und Sorptionsverhalten wurde in früheren Studien kontrovers diskutiert und ist der dritte Teil dieser Arbeit. In einem Sorptionsexperiment konnte mittels Headspace-Gaschromatografie mit Flammenionisations-Detektion die Aufnahme von Aromaten an den Kunststoffen Polypropylen und Polystyrol quantifiziert werden. Die Kunststoffe wurden materialwissenschaftlich charakterisiert, teilweise künstlich gealtert und die daraus resultierende Änderungen der Materialeigenschaften sowie einhergehenden Änderungen des Sorptionsverhaltens erfasst. Dadurch war es möglich den Einfluss einzelner Materialeigenschaften auf das Sorptionsverhalten zu bewerten, Rückschlüsse auf zugrunde liegende Sorptionsmechanismen zu treffen und zu zeigen, dass in vorliegendem Experiment die Polymeralterung bei MP nicht zu einer erhöhten Schadstoffsorption führte.
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.