92-XX BIOLOGY AND OTHER NATURAL SCIENCES
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Pervasive human impacts rapidly change freshwater biodiversity. Frequently recorded exceedances of regulatory acceptable thresholds by pesticide concentrations suggest that pesticide pollution is a relevant contributor to broad-scale trends in freshwater biodiversity. A more precise pre-release Ecological Risk Assessment (ERA) might increase its protectiveness, consequently reducing the likelihood of unacceptable effects on the environment. European ERA currently neglects possible differences in sensitivity between exposed ecosystems. If the taxonomic composition of assemblages would differ systematically among certain types of ecosystems, so might their sensitivity toward pesticides. In that case, a single regulatory threshold would be over- or underprotective.
In this thesis, we evaluate (1) whether the assemblage composition of macroinvertebrates, diatoms, fishes, and aquatic macrophytes differs systematically between the types of a European river typology system, and (2) whether these taxonomical differences engender differences in sensitivity toward pesticides. While a selection of ecoregions is available for Europe, only a single typology system that classifies individual river segments is available at this spatial scale - the Broad River Types (BRT).
In the first two papers of this thesis, we compiled and prepared large databases of macroinvertebrate (paper one), diatom, fish, and aquatic macrophyte (paper two) occurrences throughout Europe to evaluate whether assemblages are more similar within than among BRT types. Additionally, we compared its performance to that of different ecoregion systems. We employed multiple tests to evaluate the performances, two of which were also designed in the studies. All typology systems failed to reach common quality thresholds for the evaluated metrics for most taxa. Nonetheless, performance differed markedly between typology systems and taxa, with the BRT often performing worst. We showed that currently available, European freshwater typology systems are not well suited to capture differences in biotic communities and suggest several possible amelioration.
In the third study, we evaluated whether ecologically meaningful differences in sensitivity exist between BRT types. To this end, we predicted the sensitivity of macroinvertebrate assemblages across Europe toward Atrazine, copper, and Imidacloprid using a hierarchical species sensitivity distribution model. The predicted assemblage sensitives differed only marginally between BRT types. The largest difference between
median river type sensitivities was a factor of 2.6, which is far below the assessment factor suggested for such models (6), as well as the factor of variation commonly observed between toxicity tests of the same species-compound pair (7.5 for copper). Our results don’t support the notion that a type-specific ERA might improve the accuracy of thresholds. However, in addition to the taxonomic composition the bioavailability of chemicals, the interaction with other stressors, and the sensitivity of a given species might differ between river types.
Northwest Africa is predicted to undergo a climatic shift from a temperate to an arid climate resulting in increased aridity, water salinity, and river intermittency. These changes have the potential to impact freshwater communities, ecosystem functioning, and related ecosystem services. However, there is still limited data on the impact of climate change and salinity on river ecosystems and the people depending on it, particularly in understudied regions such as Northwest Africa. In this dissertation, I focus on the Draa River basin in southern Morocco to assess the primary factors shaping and altering macroinvertebrate communities. A particular focus is placed on the impacts of salt on the ecosystem and the consequences for human well-being. We conducted a meta-analysis covering 195 sites in Northwest Africa to examine the responses of insect communities and their trait profiles to climate change and anthropogenically induced stressors. To exclude large-scale geographic patterns such as variations in climate conditions we conducted a confluence-based study focusing on tributaries and their joint downstream sections near three confluences in the Draa River basin. Additionally, we investigated the water and biological quality of 17 further sites, aiming to explore the relationship between human well-being and the ecosystem. Our approach involved conducting water measurements, biological monitoring, and household surveys to create water, biological, and human satisfaction indices. Our findings revealed that insect family richness in arid sites of Northwest Africa was, on average, 37 % lower than in temperate sites. Among the strongest factors contributing to reduced richness and low biological quality were low flow and high water salinity. Based on the results of the confluence study only around five taxa comprised over 90 % of specimens per site, with a higher proportion of salt-tolerant generalist species in saline sites. Resistance and resilience traits such as small body size, aerial dispersal, and air breathing were found to promote survival in arid and saline sites. However, low γ-diversity in the basin caused minimal differences in macroinvertebrate community composition suggesting that the community was generally adapted to the arid climate. We observed positive associations between river water quality and biological quality indices. However, no significant associations were found between these indices and human satisfaction. Human satisfaction was particularly low in the Middle Draa, where 89 % of respondents reported emotional distress due to water salinity and scarcity. Inhabitants in areas characterized by higher levels of water salinity and scarcity generally rated drinking and irrigation water quality lower. Considering that large parts of Northwest Africa will become arid by the end of the century, we can expect a loss of macroinvertebrate diversity affecting the entire ecosystem, which might potentially affect human well-being negatively. To protect the integrity of the ecosystem in the face of ongoing climate change, it is crucial to limit anthropogenic stressors such as secondary salinization and the pressures on water resources. Protecting both more and less saline rivers, preserving natural water flow, and maintaining connectivity between habitats will allow to maintain the Draa River biodiversity, ensure ecosystem functioning, and benefit inhabitants through ecosystem services. Future policies and action plans should consider the interdependence between ecosystems and human inhabitants to enhance overall well-being.
Toxicology, the study of the adverse effects of chemicals and physical agents on living organisms, is a critical process in chemical and drug development. The low throughput, high costs, limited predictivity and ethical concerns related to traditional animal-based toxicity studies render them impractical to assess the growing number and complexity of both existing and new compounds and their formulations. These factors together with the increasing implementation of more demanding regulations, evidence the current need to develop innovative, reliable, cost effective and high throughput toxicological methods.
The use of metabolomics in vitro presents the powerful combination of a human relevant system with a multiparametric approach that allows assessing multiple endpoints in a single biological sample. Applying metabolomics in a cell-based system offers an alternative to both, the ethical concerns and relevance of animal testing and the restraining nature of single endpoint evaluations characteristic of conventional toxicological in vitro assays. However, there are still challenges that hamper the expansion of metabolomics beyond a research tool to a feasible and implementable technology for toxicology assessment.
The aim of this dissertation is to advance the applications of in vitro metabolomics in toxicology by addressing three major challenges that have limited its widespread implementation in the field. In chapter 2 the restrictive high cost and low throughput of in vitro metabolomics was addressed through the development, standardization and proof of concept of a high throughput targeted LC-MS/MS in vitro metabolomics platform for the characterization of hepatotoxicity. In chapter 3, the use of the developed in vitro metabolomics system was expanded beyond hazard identification, to its implementation for deriving dose- and time response metrics that were shown useful for Point of departure (PoD) estimations for human risk assessment. Finally, in chapter 4 in order to increase the reliance and confidence of using in vitro metabolomics data for risk assessment, the human relevance of the metabolomics in vitro assays was attempted to be improved by the implementation and evaluation of in vitro metabolomics in a hiPSCs-derived 3D liver organoid system.
The work developed here demonstrates the suitable of in vitro metabolomics for mechanistic-based hazard identification and risk assessment. By advancing the applications of metabolomics in toxicology, this work has significantly contributed to the aim of toxicology of the 21st century for a human-relevant non-animal toxicological testing, supporting the toxicology task of protecting human health and the environment.
Phycobilisomes (PBS) are the major light-harvesting complexes for the majority of cyanobacteria
and allow these organisms to absorb in the so-called green gap. They consist of smaller units called
phycobiliproteins (PBPs), which are composed of an α- and a β-subunit with covalently bound
linear tetrapyrroles (phycobilins). The latter are attached to the apo-PBPs by phycobiliprotein
lyases. Interestingly, cyanobacteria of the genus Prochlorococcus lack complete PBS and instead
use prochlorophyte chlorophyll-binding proteins (Pcbs), which effectively utilize the energy of the
blue light region. The low-light-adapted (LL) strain Prochlorococcus marinus SS120 has a single
PBP, phycoerythrin-III (PE-III). It has been postulated that PE-III is chromophorylated with the
phycobilins phycourobilin (PUB) and phycoerythrobilin (PEB) in a 3:1 ratio. Thereby, the function
of PE-III remains unclear so far, so that light-gathering function and also photoreceptor function
are discussed.
The main goal of this work was to characterize the assembly of PE-III and thus the function of the
six putative phycobiliprotein lyases of P. marinus SS120. Previous work found that the individual
lyases could not be produced in soluble form, so we switched to a dual pDuet™ plasmid system in
E. coli, which was successfully established. Investigation of the binding of PEB to Apo-PE
revealed that the CpeS lyase specifically chromophorylated Cys82 with 3Z-PEB. Unfortunately,
additional chromophorylation could not be observed using the pDuet system. Therefore, in a
second part of the work, the entire PE gene cluster from P. marinus SS120 was to be introduced
into E. coli and expressed. Although the gene cluster was successfully transcribed within E. coli,
no translation was observed, possibly due to incompatible translation initiation between
Prochlorococcus and E. coli. The introduction of a mini PE cluster (CpeAB) into the
cyanobacterium Synechococcus sp. PCC 7002 was also successfully performed, in which case
production of CpeB but not CpeA from Prochlorococcus was detected. Recombinant CpeB was
also detected together with intrinsic PBP in Synechococcussp. 7002, indicating structural similarity
and incorporation into PBS in Synechococcus sp. 7002. Overall, the obtained results suggest that a
cyanobacterial host is a good option for the studies on the assembly of PE-III from P. marinus and,
based on this, future work could aim at generating an artificial operon using synthetic biology to
achieve efficient translation of all genes.
Synaptic transmission is controlled by re-uptake systems that reduce transmitter concentrations in the synaptic cleft and recycle the transmitter into presynaptic terminals. The re-uptake systems are thought to ensure cytosolic concentrations in the terminals that are sufficient for reloading empty synaptic vesicles (SVs). Genetic deletion of glycine transporter 2 (GlyT2) results in severely disrupted inhibitory neurotransmission and ultimately to death. Here we investigated the role of GlyT2 at inhibitory glycinergic synapses in the mammalian auditory brainstem. These synapses are tuned for resilience, reliability, and precision, even during sustained high-frequency stimulation when endocytosis and refilling of SVs probably contribute substantially to efficient replenishment of the readily releasable pool (RRP). Such robust synapses are formed between MNTB and LSO neurons (medial nucleus of the trapezoid body, lateral superior olive). By means of patch-clamp recordings, we assessed the synaptic performance in controls, in GlyT2 knockout mice (KOs), and upon acute pharmacological GlyT2 blockade. Via computational modeling, we calculated the reoccupation rate of empty release sites and RRP replenishment kinetics during 60-s challenge and 60-s recovery periods. Control MNTB-LSO inputs maintained high fidelity neurotransmission at 50 Hz for 60 s and recovered very efficiently from synaptic depression. During 'marathon-experiments' (30,600 stimuli in 20 min), RRP replenishment accumulated to 1,260-fold. In contrast, KO inputs featured severe impairments. For example, the input number was reduced to ~1 (vs. ~4 in controls), implying massive functional degeneration of the MNTB-LSO microcircuit and a role of GlyT2 during synapse maturation. Surprisingly, neurotransmission did not collapse completely in KOs as inputs still replenished their small RRP 80-fold upon 50 Hz | 60 s challenge. However, they totally failed to do so for extended periods. Upon acute pharmacological GlyT2 inactivation, synaptic performance remained robust, in stark contrast to KOs. RRP replenishment was 865-fold in marathon-experiments, only ~1/3 lower than in controls. Collectively, our empirical and modeling results demonstrate that GlyT2 re-uptake activity is not the dominant factor in the SV recycling pathway that imparts indefatigability to MNTB-LSO synapses. We postulate that additional glycine sources, possibly the antiporter Asc-1, contribute to RRP replenishment at these high-fidelity brainstem synapses.
Synapses are connections between different nerve cells that form an essential link in neural signal transmission. It is generally distinguished between electrical and chemical synapses, where chemical synapses are more common in the human brain and are also the type we deal with in this work.
In chemical synapses, small container-like objects called vesicles fill with neurotransmitter and expel them from the cell during synaptic transmission. This process is vital for communication between neurons. However, to the best of our knowledge no mathematical models that take different filling states of the vesicles into account have been developed before this thesis was written.
In this thesis we propose a novel mathematical model for modeling synaptic transmission at chemical synapses which includes the description of vesicles of different filling states. The model consists of a transport equation (for the vesicle growth process) plus three ordinary differential equations (ODEs) and focuses on the presynapse and synaptic cleft.
The well-posedness is proved in detail for this partial differential equation (PDE) system. We also propose a few different variations and related models. In particular, an ODE system is derived and a delay differential equation (DDE) system is formulated. We then use nonlinear optimization methods for data fitting to test some of the models on data made available to us by the Animal Physiology group at TU Kaiserslautern.
Linking protistan community shifts along salinity gradients with cellular haloadaptation strategies
(2019)
Salinity is one of the most structuring environmental factors for microeukaryotic communities. Using eDNA barcoding, I detected significant shifts in microeukaryotic community compositions occurring at distinct salinities between brackish and marine conditions in the Baltic Sea. I, furthermore, conducted a metadata analysis including my and other marine and hypersaline community sequence data to confirm the existence of salinity-related transition boundaries and significant changes in alpha diversity patterns along a brackish to hypersaline gradient. One hypothesis for the formation of salinity-dependent transition boundaries between brackish to hypersaline conditions is the use of different cellular haloadaptation strategies. To test this hypothesis, I conducted metatranscriptome analyses of microeukaryotic communities along a pronounced salinity gradient (40 – 380 ‰). Clustering of functional transcripts revealed differences in metabolic properties and metabolic capacities between microeukaryotic communities at specific salinities, corresponding to the transition boundaries already observed in the taxonomic eDNA barcoding approach. In specific, microeukaryotic communities thriving at mid-hypersaline conditions (≤ 150 ‰) seem to predominantly apply the ‘low-salt – organic-solutes-in’ strategy by accumulating compatible solutes to counteract osmotic stress. Indications were found for both the intracellular synthesis of compatible solutes as well as for cellular transport systems. In contrast, communities of extreme-hypersaline habitats (≥ 200 ‰) may preferentially use the ‘high-salt-in’ strategy, i. e. the intracellular accumulation of inorganic ions in high concentrations, which is implied by the increased expression of Mg2+, K+, Cl- transporters and channels.
In order to characterize the ‘low-salt – organic-solutes-in’ strategy applied by protists in more detail, I conducted a time-resolved transcriptome analysis of the heterotrophic ciliate Schmidingerothrix salinarum serving as model organism. S. salinarum was thus subjected to a salt-up shock to investigate the intracellular response to osmotic stress by shifts of gene expression. After increasing the external salinity, an increased expression of two-component signal transduction systems and MAPK cascades was observed. In an early reaction, the expression of transport mechanisms for K+, Cl- and Ca2+ increased, which may enhance the capacity of K+, Cl- and Ca2+ in the cytoplasm to compensate possibly harmful Na+ influx. Expression of enzymes for the synthesis of possible compatible solutes, starting with glycine betaine, followed by ectoine and later proline, could imply that the inorganic ions K+, Cl- and Ca2+ are gradually replaced by the synthesized compatible solutes. Additionally, expressed transporters for choline (precursor of glycine betaine) and proline could indicate an intracellular accumulation of compatible solutes to balance the external salinity. During this accumulation, the up-regulated ion export mechanisms may increase the capacity for Na+ expulsion from the cytoplasm and ion compartmentalization between cell organelles seem to happen.
The results of my PhD project revealed first evidence at molecular level for the salinity-dependent use of different haloadaptation strategies in microeukaryotes and significantly extend existing knowledge about haloadaptation processes in ciliates. The results provide ground for future research, such as (comparative) transcriptome analysis of ciliates thriving in extreme-hypersaline habitats or experiments like qRT-PCR to validate transcriptome results.
Poor posture in childhood and adolescence is held responsible for the occurrence
of associated disorders in adult age. This study aimed to verify whether body
posture in adolescence can be enhanced through the improvement of neuromuscular
performance, attained by means of targeted strength, stretch, and body perception
training, and whether any such improvement might also transition into adulthood. From
a total of 84 volunteers, the posture development of 67 adolescents was checked
annually between the age of 14 and 20 based on index values in three posture
situations. 28 adolescents exercised twice a week for about 2 h up to the age of 18, 24
adolescents exercised continually up to the age of 20. Both groups practiced other
additional sports for about 1.8 h/week. Fifteen persons served as a non-exercising
control group, practicing optional sports of about 1.8 h/week until the age of 18,
after that for 0.9 h/week. Group allocation was not random, but depended on the
participants’ choice. A linear mixed model was used to analyze the development
of posture indexes among the groups and over time and the possible influence of
anthropometric parameters (weight, size), of optional athletic activity and of sedentary
behavior. The post hoc pairwise comparison was performed applying the Scheffé test.
The significance level was set at 0.05. The group that exercised continually (TR20)
exhibited a significant posture parameter improvement in all posture situations from
the 2nd year of exercising on. The group that terminated their training when reaching
adulthood (TR18) retained some improvements, such as conscious straightening of the
body posture. In other posture situations (habitual, closed eyes), their posture results
declined again from age 18. The effect sizes determined were between Eta² = 0.12 and
Eta² = 0.19 and represent moderate to strong effects. The control group did not exhibit
any differences. Anthropometric parameters, additional athletic activities and sedentary
behavior did not influence the posture parameters significantly. An additional athletic
training of 2 h per week including elements for improved body perception seems to
have the potential to improve body posture in symptom free male adolescents and
young adults.
SDE-driven modeling of phenotypically heterogeneous tumors: The influence of cancer cell stemness
(2018)
We deduce cell population models describing the evolution of a tumor (possibly interacting with its
environment of healthy cells) with the aid of differential equations. Thereby, different subpopulations
of cancer cells allow accounting for the tumor heterogeneity. In our settings these include cancer
stem cells known to be less sensitive to treatment and differentiated cancer cells having a higher
sensitivity towards chemo- and radiotherapy. Our approach relies on stochastic differential equations
in order to account for randomness in the system, arising e.g., by the therapy-induced decreasing
number of clonogens, which renders a pure deterministic model arguable. The equations are deduced
relying on transition probabilities characterizing innovations of the two cancer cell subpopulations,
and similarly extended to also account for the evolution of normal tissue. Several therapy approaches
are introduced and compared by way of tumor control probability (TCP) and uncomplicated tumor
control probability (UTCP). A PDE approach allows to assess the evolution of tumor and normal
tissue with respect to time and to cell population densities which can vary continuously in a given set
of states. Analytical approximations of solutions to the obtained PDE system are provided as well.
Increasing costs due to the rising attrition of drug candidates in late developmental phases alongside post-marketing withdrawal of drugs challenge the pharmaceutical industry to further improve their current preclinical safety assessment strategies. One of the most common reasons for the termination of drug candidates is drug induced hepatotoxicity, which more often than not remains undetected in early developmental stages, thus emphasizing the necessity for improved and more predictive preclinical test systems. One reason for the very limited value of currently applied in vitro test systems for the detection of potential hepatotoxic liabilities is the lack of organotypic and tissue-specific physiology of hepatocytes cultured in ordinary monolayer culture formats.
The thesis at hand primarily deals with the evaluation of both two- and three-dimensional cell culture approaches with respect to their relative ability to predict the hepatotoxic potential of drug candidates in early developmental phases. First, different hepatic cell models, which are routinely used in pharmaceutical industry (primary human hepatocytes as well as the three cell lines HepG2, HepaRG and Upcyte hepatocytes), were investigated in conventional 2D monolayer culture with respect to their ability to detect hepatotoxic effects in simple cytotoxicity studies. Moreover, it could be shown that the global protein expression levels of all cell lines substantially differ from that of primary human hepatocytes, with the least pronounced difference in HepaRG cells.
The introduction of a third dimension through the cultivation of spheroids enables hepatocytes to recapitulate their typical native polarity and furthermore dramatically increases the contact surface of adjacent cells. These differences in cellular architecture have a positive influence on hepatocyte longevity and the expression of drug metabolizing enzymes and transporters, which could be proven via immunofluorescent (IF) staining for at least 14 days in PHH and at least 28 days in HepaRG spheroids, respectively. Additionally, the IF staining of three different phase III transporters (MDR1, MRP2 and BSEP) indicated a bile canalicular network in spheroids of both cell models. A dose-dependent inducibility of important cytochrome P450 isoenzymes in HepaRG spheroids could be shown on the protein level via IF for at least 14 days. CYP inducibility of HepaRG cells cultured in 2D and 3D was compared on the mRNA level for up to 14 days and inducibility was generally lower in 3D compared to 2D under the conditions of this study. In a comparative cytotoxicity study, both PHH and HepaRG spheroids as well as HepaRG monolayers have been treated with five hepatotoxic drugs for up to 14 days and viability was measured at three time points (days 3, 7 and 14). A clear time- and dose-dependent onset of the drug-induced hepatotoxic effects was observable in all conditions tested, indicated by a shift of the respective EC50 value towards lower doses by increasing exposure. The observed effects were most pronounced in PHH spheroids, thus indicating those as the most sensitive cell model in this study. Moreover, HepaRG cells were more sensitive in spheroid culture compared to monolayers, which suggests a potential application of spheroids as long-term test system for the detection of hepatotoxicities with slow onset. Finally, the basal protein expression levels of three antigens (CYP1A2, CYP3A4 and NAT 1/2) were analyzed via Western Blotting in HepaRG cells cultured in three different cell culture formats (2D, 3D and QV) in order to estimate the impact of the cell culture conditions on protein expression levels. In the QV system enables a pump-driven flow of cell culture media, which introduces both mechanical stimuli through shear and molecular stimuli through dynamic circulation to the monolayer. Those stimuli resulted in a clearly positive effect on the expression levels of the selected antigens by an increased expression level in comparison to both 2D and 3D. In contrast, HepaRG spheroids showed time-dependent differences with the overall highest levels at day 7.
The studies presented in this thesis delivered valuable information on the increased physiological relevance in dependence on the cell culture format: three-dimensionality as well as the circulation of media lead to a more differentiated phenotype in hepatic cell models. Those cell culture formats are applicable in preclinical drug development in order to obtain more relevant information at early developmental stages and thus help to create a more efficient drug development process. Nonetheless, further studies are necessary to thoroughly characterize, validate and standardize such novel cell culture approaches prior to their routine application in industry.