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Quantitative studies of mesenchymal cell motion are important to elucidate cytoskeleton function and mechanisms of cell migration. To this end, confinement of cell motion to one dimension (1D) significantly simplifies the problem of cell shape in experimental and theoretical investigations. Here we review 1D migration assays employing micro-fabricated lanes and reflect on the advantages of such platforms. Data are analyzed using biophysical models of cell migration that reproduce the rich scenario of morphodynamic behavior found in 1D. We describe basic model assumptions and model behavior. It appears that mechanical models explain the occurrence of universal relations conserved across different cell lines such as the adhesion-velocity relation and the universal correlation between speed and persistence (UCSP). We highlight the unique opportunity of reproducible and standardized 1D assays to validate theory based on statistical measures from large data of trajectories and discuss the potential of experimental settings embedding controlled perturbations to probe response in migratory behavior., Peer Reviewed

Genetic tools form the basis for the study of molecular mechanisms. Despite many recent advances in the field of genetic engineering in bacteria, genetic toolsets remain scarce for non-model organisms, such as the obligatory human pathogen Streptococcus pyogenes. To overcome this limitation and enable the straightforward investigation of gene functions in S. pyogenes, we have developed a comprehensive genetic toolset. By adapting and combining different tools previously applied in other Gram-positive bacteria, we have created new replicative and integrative plasmids for gene expression and genetic manipulation, constitutive and inducible promoters as well as fluorescence reporters for S. pyogenes. The new replicative plasmids feature low- and high-copy replicons combined with different resistance cassettes and a standardized multiple cloning site for rapid cloning procedures. We designed site-specific integrative plasmids and verified their integration by nanopore sequencing. To minimize the effect of plasmid integration on bacterial physiology, we screened publicly available RNA-sequencing datasets for transcriptionally silent sites. We validated this approach by designing the integrative plasmid pSpy0K6 targeting the transcriptionally silent gene SPy_1078. Analysis of the activity of different constitutive promoters indicated a wide variety of strengths, with the lactococcal promoter P23 showing the strongest activity and the synthetic promoter PxylS2 showing the weakest activity. Further, we assessed the functionality of three inducible regulatory elements including a zinc- and an IPTG-inducible promoter as well as an erythromycin-inducible riboswitch that showed low-to-no background expression and high inducibility. Additionally, we demonstrated the applicability of two codon-optimized fluorescent proteins, mNeongreen and mKate2, as reporters in S. pyogenes. We therefore adapted the chemically defined medium called RPMI4Spy that showed reduced autofluorescence a, Peer Reviewed

Komplexe Lebenszyklen sind eine häufige, aber anspruchsvolle Lebensweise von Parasiten. Parasiten mit komplexen Lebenszyklen infizieren nacheinander mehrere Wirte und passen sich an verschiedene ökologische Nischen an. Werden in diesem Szenario dieselben Gene in allen Wirten einheitlich exprimiert? Die Hypothese der adaptiven Entkopplung besagt, dass verschiedene Stadien in einem komplexen Lebenszyklus genetisch und evolutionär unabhängig sind, was bedeutet, dass die Selektion auf Parasitenmerkmale in einem Wirt keine Auswirkungen auf Merkmale in anderen Wirten hat. Wir haben diese Hypothese anhand von zwei Parasitenarten getestet: dem Bandwurm Schistocephalus solidus und dem Fadenwurm Camallanus lacustris. Die Transkriptomsequenzierung wurde an Proben während ihrer komplexen Lebenszyklen durchgeführt. Die Genexpressionsanalyse wurde durchgeführt, um Gene zu identifizieren, die zwischen den Wirten, zwischen den Funktionsstadien und zwischen den Bedingungen unterschiedlich exprimiert werden. Darüber hinaus wurde mit einer Analyse der Anreicherung von Gensätzen untersucht, ob ähnliche biologische Funktionen von ähnlichen Genen in verschiedenen Stadien kodiert werden. Unsere Ergebnisse zeigen signifikante Unterschiede in der Genexpression zwischen den verschiedenen Stadien, wobei keine positive Korrelation zwischen Stadien mit derselben Aufgabe oder demselben Wirt beobachtet wurde. Gene, die in einem Stadium hochreguliert (oder herunterreguliert) werden, werden in anderen Stadien nicht in ähnlicher Weise exprimiert. Dies spricht für die Unabhängigkeit der einzelnen Stadien bei der Genexpression, die es den Parasiten ermöglicht, ihren Phänotyp als Reaktion auf unterschiedlichen Selektionsdruck zu modulieren. Diese Ergebnisse bestätigen die Hypothese der adaptiven Entkopplung bei parasitären Würmern und bieten Einblicke in den evolutionären Erfolg dieser Lebensweise., Complex life cycles are a common but demanding lifestyle among parasites. Parasites with complex life cycles sequentially infect multiple hosts, adapting to various ecological niches using information encoded within a single genome. In this scenario, are the same genes expressed consistently across all hosts, as might occur when parasite stages perform similar functions? The adaptive decoupling hypothesis posits that different stages in a complex life cycle are genetically and evolutionarily independent, meaning selection on parasite traits in one host does not affect traits in other hosts. We tested this hypothesis using two species of parasites: the tapeworm Schistocephalus solidus and the nematode Camallanus lacustris, both of which have three-host life cycles. Transcriptome sequencing was performed on samples throughout their complex life cycles, generating transcriptomes from 10 stages of S. solidus and 7 stages of C. lacustris. Gene expression analysis was conducted to identify genes differentially expressed between hosts, between functional stages, and between conditions. Additionally, gene set enrichment analysis assessed whether similar biological functions are encoded by similar genes across stages. Our findings demonstrate significant differences in gene expression across stages, with no positive correlation observed between stages sharing the same task or host. The highest correlation occurred between stages sampled close in time. In conclusion, the lack of positive correlation between different life cycle stages indicates that genes up-regulated (or down-regulated) in one stage are not similarly expressed in other stages. This evidence supports the independence of each stage in gene expression, enabling parasites to modulate their phenotype in response to different selective pressures. These findings corroborate the adaptive decoupling hypothesis in parasitic worms with complex life cycles, offering insights into the evolutionary success of this lifesty

The article processing charge was funded by the Open Access Publication Fund of Humboldt-Universität zu Berlin., We explored the behavioral flexibility of Commissaris’s long-tongued bats through a spatial serial reversal foraging task. Bats kept in captivity for short periods were trained to obtain nectar rewards from two artificial flowers. At any given time, only one of the flowers provided rewards and these reward contingencies reversed in successive blocks of 50 flower visits. All bats detected and responded to reversals by making most of their visits to the currently active flower. As the bats experienced repeated reversals, their preference re-adjusted faster. Although the flower state reversals were theoretically predictable, we did not detect anticipatory behavior, that is, frequency of visits to the alternative flower did not increase within each block as the programmed reversal approached. The net balance of these changes was a progressive improvement in performance in terms of the total proportion of visits allocated to the active flower. The results are compatible with, but do not depend on, the bats displaying an ability to ‘learn to learn’ and show that the dynamics of allocation of effort between food sources can change flexibly according to circumstances., Peer Reviewed

Identifying factors that influence age-related cognitive decline is crucial, given its severe personal and societal impacts. However, studying aging in human or animal models is challenging due to the significant variability in aging processes among individuals. Additionally, longitudinal and cross-sectional studies often produce differing results. In this context, home-cage-based behavioral analysis over lifespans has emerged as a significant method in recent years. This study aimed to explore how prior experience affects cognitive performance in mice of various age groups (4, 12, and 22 months) using a home-cage-based touchscreen test battery. In this automated system, group-housed, ID-chipped mice primarily obtain their food during task performance throughout the day, motivated by their own initiative, without being subjected to food deprivation. Spatial working memory and attention were evaluated using the trial unique non-matching to location (TUNL) and the five-choice serial reaction time task (5-CSRTT), respectively. The same set of mice learned both of these demanding tasks. While signs of cognitive decline were already apparent in middle-aged mice, older mice exhibited poorer performance in both tasks. Mice at both 12 and 22 months displayed an increase in perseverance and a decrease in the percentage of correct responses in the TUNL test compared to the 4-month-old mice. Furthermore, during the 5-CSRTT, they exhibited higher rates of omissions and premature responses compared to their younger counterparts. Additionally, the correct response rate in 22-month-old mice was lower than that of the 4-month-old ones. However, mice that had undergone cognitive training at 4 months maintained high-performance levels when re-tested at 12 months, showing an increase in correct responses during TUNL testing compared to their untrained controls. In the 5-CSRTT, previously trained mice demonstrated higher correct response rates, fewer omissions, and reduced premature re, Peer Reviewed

Many folding enzymes use separate domains for the binding of substrate proteins and for the catalysis of slow folding reactions such as prolyl isomerization. FKBP12 is a small prolyl isomerase without a chaperone domain. Its folding activity is low, but it could be increased by inserting the chaperone domain from the homolog SlyD of E. coli near the prolyl isomerase active site. We inserted two other chaperone domains into human FKBP12: the chaperone domain of SlpA from E. coli, and the chaperone domain of SlyD from Thermococcus sp. Both stabilized FKBP12 and greatly increased its folding activity. The insertion of these chaperone domains had no influence on the FKBP12 and the chaperone domain structure, as revealed by two crystal structures of the chimeric proteins. The relative domain orientations differ in the two crystal structures, presumably representing snapshots of a more open and a more closed conformation. Together with crystal structures from SlyD-like proteins, they suggest a path for how substrate proteins might be transferred from the chaperone domain to the prolyl isomerase domain., Fonds der Chemischen Industrie and the Deutsche Forschungsgemeinschaft, Peer Reviewed

We created a database of lost and rediscovered tetrapod species, identified patterns in their distribution and factors influencing rediscovery. Tetrapod species are being lost at a faster rate than they are being rediscovered, due to slowing rates of rediscovery for amphibians, birds and mammals, and rapid rates of loss for reptiles. Finding lost species and preventing future losses should therefore be a conservation priority. By comparing the taxonomic and spatial distribution of lost and rediscovered tetrapod species, we have identified regions and taxa with many lost species in comparison to those that have been rediscovered—our results may help to prioritise search effort to find them. By identifying factors that influence rediscovery, we have improved our ability to broadly distinguish the types of species that are likely to be found from those that are not (because they are likely to be extinct). Some lost species, particularly those that are small and perceived to be uncharismatic, may have been neglected in terms of conservation effort, and other lost species may be hard to find due to their intrinsic characteristics and the characteristics of the environments they occupy (e.g. nocturnal species, fossorial species and species occupying habitats that are more difficult to survey such as wetlands). These lost species may genuinely await rediscovery. However, other lost species that possess characteristics associated with rediscovery (e.g. large species) and that are also associated with factors that negatively influence rediscovery (e.g. those occupying small islands) are more likely to be extinct. Our results may foster pragmatic search protocols that prioritise lost species likely to still exist., Israel Science Foundation http://dx.doi.org/10.13039/501100003977, Leibniz‐Institut für Gewässerökologie und Binnenfischerei, Alexander von Humboldt‐Stiftung, Pontificia Universidad Católica del Ecuador http://dx.doi.org/10.13039/501100011749, Peer Reviewed

In ant-aphid interactions, various aphid species offer honeydew to the ant partner and increase their density by ant attendance, whilst others never attend ant species, in which case ants tend to treat them as prey. In this regard, ants should have the ability to distinguish myrmecophile aphid species from non-mutualistic species, so that mutualistic aphids will be accepted as partners rather than prey. Although ant-aphid interactions are now the focus of chemical ecology studies, the role of the different chemical stimuli in determining mutualistic interactions has not been completely clarified. Therefore, we have investigated the cuticular hydrocarbon profile of four myrmecophiles aphid species using GC-MS. We also investigated the behavior of the worker-ants (Lasius niger L., Hymenoptera: Formicidae) to different chemical stimuli derived from aphids. We applied four treatments and found that the behavior of the ant workers varied depending on the source of the different treatments. In particular, the real aphid Aphis pomi and the sugar solution proved to be the most attractive to the ants, while the presence of pure extract of the individuals is enough to disturb the behavior of the ants. We provide evidence that the key stimuli of the tending behavior could be the CHC patterns of the aphids and the CHC profile of the aphids tends to be genus specific. This research will promote further investigations to test the behavior of ant workers towards other species of aphids and treatment combinations., Peer Reviewed

The article processing charge was funded by the Open Access Publication Fund of Humboldt-Universität zu Berlin., Background: Biological adaptation manifests itself at the interface of different biologically relevant ‘levels’, such as ecology, performance, and morphology. Integrated studies at this interface are scarce due to practical difficulties in study design. We present a multilevel analysis, in which we combine evidence from habitat utilization, leaping performance and limb bone morphology of four species of tamarins to elucidate correlations between these ‘levels’. Results: We conducted studies of leaping behavior in the field and in a naturalistic park and found significant differences in support use and leaping performance. Leontocebus nigrifrons leaps primarily on vertical, inflexible supports, with vertical body postures, and covers greater leaping distances on average. In contrast, Saguinus midas and S. imperator use vertical and horizontal supports for leaping with a relatively similar frequency. S. mystax is similar to S. midas and S. imperator in the use of supports, but covers greater leaping distances on average, which are nevertheless shorter than those of L. nigrifrons. We assumed these differences to be reflected in the locomotor morphology, too, and compared various morphological features of the long bones of the limbs. According to our performance and habitat utilization data, we expected the long bone morphology of L. nigrifrons to reflect the largest potential for joint torque generation and stress resistance, because we assume longer leaps on vertical supports to exert larger forces on the bones. For S. mystax, based on our performance data, we expected the potential for torque generation to be intermediate between L. nigrifrons and the other two Saguinus species. Surprisingly, we found S. midas and S. imperator having relatively more robust morphological structures as well as relatively larger muscle in-levers, and thus appearing better adapted to the stresses involved in leaping than the other two. Conclusion: This study demonstrates the complex ways, Peer Reviewed

In sight of a growing urban population and intensified extreme weather events, cities must integrate in their urban planning elements to both reduce their impact (i.e., air and water pollution, degradation of habitats, loss of biodiversity) and increase their resilience to climate change. In contrast to engineering solutions, which normally not only fail to adequately address these issues but often also exacerbate them, Nature-based Solutions are an efficient strategy which can help cities become more sustainable. Aqua-Nature-based Solutions (aNBS) tackle water-related hazards by enhancing water regulation and mitigating flood and drought impacts. However, under a warming climate, aNBS are expected to often dry-out, changing biodiversity and the ecosystem services they support. The aim of this study is to compare the biodiversity of temporarily and permanently wet urban waterbodies which function as aNBS. We selected two pond complexes with different hydroperiod (i.e., different duration, amplitude and frequency of inundation) and studied the riparian vegetation and aquatic macroinvertebrate biodiversity. The Multimetric Macroinvertebrate Index of Flanders was used to determine the macroinvertebrate biodiversity and to assess water quality of the ponds. Using water stable isotopes and piezometers, the hydrological dynamics were studied in order to identify the water regulating ecosystem services these ponds deliver. The results showed that the selected pond complexes have a high plant biodiversity, particularly in temporary ponds. Water quality ranged from moderate to poor and macroinvertebrate biodiversity tended to be greater in permanent ponds. Plant and macroinvertebrate alien species were also found in the aNBS. Regarding water regulating ecosystem services, the pond complexes enhanced infiltration and groundwater recharge, providing resilience to both flooding and drought. Our findings corroborate previous studies on the need of diversifying urban ponds’ hydro, Peer Reviewed

The origin of cynodonts, the group ancestral to and including mammals, is one of the major outstanding problems in therapsid evolution. One of the most troubling aspects of the cynodont fossil record is the lengthy Permian ghost lineage between the latest possible divergence from its sister group Therocephalia and the first appearance of definitive cynodonts in the late Permian. The absence of cynodonts and dominance of therocephalians in middle Permian strata has led some workers to argue that cynodonts evolved from within therocephalians, rendering the latter paraphyletic, but more recent analyses support the reciprocal monophyly of Cynodontia and Therocephalia. Furthermore, although a fundamental dichotomy in the derived subclade Eucynodontia is well‐supported in cynodont phylogeny, the relationships of more stemward cynodonts from the late Permian and Early Triassic are unresolved. Here, we provide a re‐evaluation of the phylogeny of Eutheriodontia (Cynodontia + Therocephalia) and an assessment of character evolution within the group. Using computed tomographic data derived from extensive sampling of the earliest known (late Permian and Early Triassic) cynodonts and selected exemplars of therocephalians and later (Middle Triassic onwards) cynodonts, we describe novel aspects of the endocranial anatomy of these animals. These data were incorporated into a new phylogenetic data set including a comprehensive sample of early cynodonts. Our phylogenetic analyses support some results previously recovered by other authors, but recover therocephalians as paraphyletic with regards to cynodonts, with cynodonts and eutherocephalians forming a clade to the exclusion of the “basal therocephalian” families Lycosuchidae and Scylacosauridae. Though both conservatism and homoplasy mark the endocranial anatomy of early non‐mammalian cynodonts, we were able to identify several new endocranial synapomorphies for eutheriodont subclades and recovered generally better‐supported topolo, Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659, Peer Reviewed

Die embryonale Gonade ist bei Säugetieren das einzige bipotente Organ, das sich während der Geschlechtsbestimmung in Hoden oder Eierstock differenziert. Nach der Spezifikation produziert sie geschlechtsspezifische Hormone, die wichtige morphologische, physiologische und Verhaltensänderungen auslösen und schließlich zu reifen Fortpflanzungsorganen führen, die für die Fortpflanzung der Art unerlässlich sind. Trotz der evolutionären Bedeutung der Gonadenfunktion gibt es erhebliche Entwicklungsunterschiede zwischen den Arten, deren molekulare Mechanismen weitgehend unerforscht sind. Zur Untersuchung dieser Mechanismen wurden Einzelzell-Omics-Techniken (scRNA- und scATAC-seq) an sich entwickelnden Gonaden eingesetzt, um molekulare Faktoren für interspezifische Unterschiede während der Geschlechtsdifferenzierung zu analysieren. Bekannte Zelltypen wurden in Hoden und Eierstöcken von Schweinen (mit medullären Strängen), Mäusen (ohne medulläre Stränge), Kaninchen (mit verzögerter Meiose der Keimzellen) und Maulwürfen (mit Ovotestes) zu geschlechtsdimorphen Zeitpunkten charakterisiert. Interartspezifische Vergleiche zeigten sowohl konservierte als auch artspezifische Ereignisse auf den Ebenen der dynamischen Genexpression, Co-Expressionsnetzwerke und zellulären Kommunikation. Expressionsdaten wurden mit epigenomischen Daten integriert, um genregulatorische Netzwerke (GRNs) abzuleiten und die regulatorischen Mechanismen zu klären. Analysen zeigten die Einbeziehung artenspezifischer Transkriptionsfaktoren in konservierte GRNs und identifizierten mutmaßliche cis-regulatorische Elemente, die mit artspezifisch exprimierten Genen verknüpft sind. Die Studie legt nahe, dass unterschiedliche Kontrollmechanismen die Meiose in ovariellen Keimzellen über Arten hinweg initiieren und dass der Metabolismus steroidogener Enzyme zu einzigartigen Entwicklungsmerkmalen bei Kaninchen beitragen könnte., In mammals, the embryonic gonad is the only bipotential organ, differentiating into either testis or ovary during sex determination. Once specified, it produces sex-specific hormones that induce key morphological, physiological, and behavioral changes, leading to mature reproductive organs essential for species perpetuation. Despite the evolutionary importance of gonadal function, significant developmental plasticity exists across species, with underlying molecular mechanisms largely unexplored. To address this, single-cell omics techniques (scRNA- and scATAC-seq) were employed on developing gonads to investigate molecular contributors to interspecies differences during sex differentiation. Known cell types were characterized in testes and ovaries of pigs (exhibiting medullary cords), mice (lacking medullary cords), rabbits (displaying delayed meiosis of germ cells), and moles (forming ovotestes) at sexually dimorphic time points. Interspecies comparative analyses revealed both conserved and species-specific events at the levels of dynamic gene expression, co-expression networks, and cellular communication. Expression data were integrated with epigenomic data to infer gene regulatory networks (GRNs), clarifying regulatory mechanisms governing these events. Analyses revealed species-specific transcription factors in conserved GRNs and identified putative cis-regulatory elements linked with species-specific expressed genes. The study suggests diverse controls initiate meiosis in ovarian germ cells across species and that steroidogenic enzyme metabolism may contribute to unique developmental features in rabbits. Overall, this study advances the understanding of mammalian gonad differentiation and highlights how gene expression program evolution has contributed to mammalian phenotype diversity.

Die mitotische Spindel ist ideal für die Erforschung der Selbstorganisation und Plastizität molekularer Kollektive im Zytoplasma. Die Geometrie der Spindel ist entscheidend für die korrekte Chromosomentrennung, muss sich aber an die Zellgröße anpassen. Es ist unbekannt, ob und wie Zellen während ihrer Differenzierung die Spindelarchitektur anpassen, was insbesondere während der Gehirnentwicklung relevant ist. Wir untersuchten dies mit Maus-Embryonalstammzellen, die in frühe neuronale Vorläuferzellen differenziert wurden. Wir entwickelten ein automatisiertes Mikroskopieprotokoll um einen umfassenden Datensatz von mitotischen Zellen zu generieren. Außerdem entwickelten wir Spindle3D, ein Werkzeug zur dreidimensionalen Analyse von Spindeln. Überraschenderweise waren die Spindelvolumina in differenzierenden Zellen bis zu 24% kleiner als in pluripotenten Zellen. Während die Wachstumsgeschwindigkeit der Mikrotubuli unverändert blieb, verschob sich in sich differenzierenden Zellen die Nukleation von Mikrotubuli zugunsten der astralen Population. Diese Veränderung der Spindelarchitektur basierte auf der differenzierungsbedingten Verdünnung des Zytoplasmas. Dies aktivierte CPAP, ein Protein, das die Zentrosomenreifung reguliert, was zur Superskalierung des perizentriolären Materials und verstärkte Rekrutierung von gamma-Tubulin an den Zentrosomen und somit zur Umlagerung von Mikrotubuli innerhalb der Spindel führte. Diese Veränderungen der mitotischen Architektur konnten durch externe osmotische Einwirkung in undifferenzierten Zellen nachgestellt werden. Insgesamt verbinden unsere Ergebnisse zelltypspezifische zytoplasmatische Materialeigenschaften mit der Spindelarchitektur., The mitotic spindle provides an excellent system in which to study the plasticity of molecular collectives. To segregate chromosomes accurately, the spindle’s geometry must be adaptive to changes in cell size. It is unknown whether and how differentiating cells adjust spindle architecture, specifically during brain development when spindle defects have severe pathological consequences. Using murine embryonic stem cells, we recapitulated the transition from pluripotency to early neural cell identities in vitro. Aiming at a systematic exploration of spindle and cell morphology throughout this process, we developed an automated microscopy protocol and Spindle3D, a morphometric tool for the analysis of spindles in confocal images. Intriguingly, in cells with comparable cell volume, spindle volumes were up to 24% smaller in cells undergoing differentiation. While microtubule growth speed remained equal, we measured increased nucleation of astral microtubules at the expense of the spindle bulk in differentiating cells. The shift in spindle architecture was explained by the differentiation-driven cytoplasmic dilution. This activated the centrosomal regulator CPAP, causing the superscaling of the pericentriolar material and the concomitant increased recruitment of gamma-tubulin to the centrosomes, redistributing microtubule numbers within the spindle. Mimicking the dilution effect by osmotic challenge reproduced the same mitotic architecture in undifferentiated cells. Collectively, our results link cell state-specific cytoplasmic material properties to spindle architecture.

Freshwater megafauna, such as sturgeons, giant catfishes, river dolphins, hippopotami, crocodylians, large turtles, and giant salamanders, have experienced severe population declines and range contractions worldwide. Although there is an increasing number of studies investigating the causes of megafauna losses in fresh waters, little attention has been paid to synthesising the impacts of megafauna on the abiotic environment and other organisms in freshwater ecosystems, and hence the consequences of losing these species. This limited understanding may impede the development of policies and actions for their conservation and restoration. In this review, we synthesise how megafauna shape ecological processes in freshwater ecosystems and discuss their potential for enhancing ecosystem restoration. Through activities such as movement, burrowing, and dam and nest building, megafauna have a profound influence on the extent of water bodies, flow dynamics, and the physical structure of shorelines and substrata, increasing habitat heterogeneity. They enhance nutrient cycling within fresh waters, and cross-ecosystem flows of material, through foraging and reproduction activities. Freshwater megafauna are highly connected to other freshwater organisms via direct consumption of species at different trophic levels, indirect trophic cascades, and through their influence on habitat structure. The literature documenting the ecological impacts of freshwater megafauna is not evenly distributed among species, regions, and types of ecological impacts, with a lack of quantitative evidence for large fish, crocodylians, and turtles in the Global South and their impacts on nutrient flows and food-web structure. In addition, population decline, range contraction, and the loss of large individuals have reduced the extent and magnitude of megafaunal impacts in freshwater ecosystems, rendering a posteriori evaluation more difficult. We propose that reinstating freshwater megafauna populations h, Villum Fonden http://dx.doi.org/10.13039/100008398, Leibniz‐Gemeinschaft http://dx.doi.org/10.13039/501100001664, Danmarks Frie Forskningsfond http://dx.doi.org/10.13039/501100004836, Danmarks Grundforskningsfond http://dx.doi.org/10.13039/501100001732, Deutscher Akademischer Austauschdienst http://dx.doi.org/10.13039/501100001655, Chinese Academy of Sciences http://dx.doi.org/10.13039/501100002367, Peer Reviewed

The article processing charge was funded by the Open Access Publication Fund of Humboldt-Universität zu Berlin., The Berlin Fat Mouse Inbred line (BFMI) is a model for obesity and metabolic syndrome. The sublines BFMI861-S1 and BFMI861-S2 differ in weight despite high genetic similarity and a shared obesity-related locus. This study focused on identifying additional body weight quantitative trait loci (QTLs) by analyzing weekly weight measurements in a male population of the advanced intercross line BFMI861-S1 x BFMI861-S2. QTL analysis, utilizing 200 selectively genotyped mice (GigaMUGA) and 197 males genotyped for top SNPs, revealed a genome-wide significant QTL on Chr 15 (68.46 to 81.40 Mb) for body weight between weeks 9 to 20. Notably, this QTL disappeared (weeks 21 to 23) and reappeared (weeks 24 and 25) coinciding with a diet change. Additionally, a significant body weight QTL on Chr 16 (3.89 to 22.79 Mb) was identified from weeks 6 to 25. Candidate genes, including Gpt, Cbx6, Apol6, Apol8, Sun2 (Chr 15) and Trap1, Rrn3, Mapk1 (Chr 16), were prioritized. This study unveiled two additional body weight QTLs, one of which is novel and responsive to diet changes. These findings illuminate genomic regions influencing weight in BFMI and emphasize the utility of time series data in uncovering novel genetic factors., Peer Reviewed

The article processing charge was funded by the Open Access Publication Fund of Humboldt-Universität zu Berlin., Background: GOLDEN-like (GLK) transcription factors are central regulators of chloroplast biogenesis in Arabidopsis and other species. Findings from Arabidopsis show that these factors also contribute to photosynthetic acclimation, e.g. to variation in light intensity, and are controlled by retrograde signals emanating from the chloroplast. However, the natural variation of GLK1-centered gene-regulatory networks in Arabidopsis is largely unexplored. Results: By evaluating the activities of GLK1 target genes and GLK1 itself in vegetative leaves of natural Arabidopsis accessions grown under standard conditions, we uncovered variation in the activity of GLK1 centered regulatory networks. This is linked with the ecogeographic origin of the accessions, and can be associated with a complex genetic variation across loci acting in different functional pathways, including photosynthesis, ROS and brassinosteroid pathways. Our results identify candidate upstream regulators that contribute to a basal level of GLK1 activity in rosette leaves, which can then impact the capacity to acclimate to different environmental conditions. Indeed, accessions with higher GLK1 activity, arising from habitats with a high monthly variation in solar radiation levels, may show lower levels of photoinhibition at higher light intensities. Conclusions: Our results provide evidence for natural variation in GLK1 regulatory activities in vegetative leaves. This variation is associated with ecogeographic origin and can contribute to acclimation to high light conditions., Peer Reviewed

Computertomographie (CT) ist eine nicht-destruktive bildgebende Methode, die in den letzten Jahren häufig bei fossilen Wirbeltierschädeln angewendet wird und Paläontologen ermöglicht bisher unbekannte interne Schädelmerkmale zu untersuchen. Letztere sind bedeutend um die Gehör- und Gehirnevolution, Gesichtsinnervation, den Zahnwechsel und den Ursprung von Endothermie entlang der Stammlinie der Säugetiere (Synapsida) zu verdeutlichen. Obwohl interne Schädelmerkmale in den letzten Jahren bei zahlreichen Therapsiden beschrieben wurden, sind sie bisher von der Wissenschaft weitestgehend vernachlässigt worden, verglichen mit den gut erforschten äußeren craniodentalen Merkmalen bei Synapsiden und wurden daher in phylogenetischen Analysen überwiegend vernachlässigt. In dieser Doktorarbeit stelle ich eine Neubewertung der der Phylogenie der Eutheriodontier (die Synapsidengruppe bestehend aus Cynodontia+Therocephalia) und eine Beurteilung der Merkmalsevolution innerhalb dieser Gruppe bereit. Der Ursprung der Cynodontier, die Ahnen der Säugetiere, stellt einen der bedeutendsten, bisher ungelösten Probleme in der Evolution der Therapsiden dar. Es ist bekannt, dass die Cynodontier am engsten mit den Therocephaliern verwandt sind, jedoch sind die genauen Verwandtschaftsverhältnisse bisher nicht eindeutig geklärt. Obwohl eine wesentliche Dichotomie in der abgeleiteten triassischen Subklade Eucynodontia in der Phylogenie der Cynodontia gut unterstützt wird, bleiben die Verwandtschaftsverhältnisse basalerer Cynodontier derzeit ungelöst. Anhand von CT Daten, die anhand eines ausgiebigen Datensatzes von Exemplaren der am frühesten bekannten (oberpermischen und untertriassischen) Cynodontier, sowie einiger ausgewählter Therocephalier und höherer (mitteltriassischer) Cynodontier (Eucynodontia) gewonnen wurden, konnte ich neue interne Schädelmerkmale dieser Tiere beschreiben. Diese Daten wurden anschließend in einem neuen, umfassenden phylogenetischen Datensatz dieser Gruppe integriert., Computed tomography (CT) is a non-destructive imaging technique that has been widely used on fossil vertebrate crania in recent years and allowed paleontologists to investigate previously-obscure endocranial data. The latter have been instrumental in elucidating patterns of ear and brain evolution, facial innervation, tooth replacement and the origin of endothermy along the mammalian stem lineage (Synapsida). Although endocranial characters have been described for various synapsids in recent years, they remain understudied compared to the well-known external craniodental features in synapsids and have been largely ignored in phylogenetic analyses. In this thesis, I provide a re-evaluation of the phylogeny of Eutheriodontia (the synapsid subclade consisting of Cynodontia+Therocephalia) and an assessment of character evolution within the group. The origin of cynodonts, the group ancestral to (and including) mammals, is one of the major outstanding problems in therapsid evolution. It is well-established that cynodonts are most closely related to Therocephalia among the major synapsid groups, but the exact nature of this relationship is uncertain. Furthermore, although a fundamental dichotomy in the derived Triassic subclade Eucynodontia is well-supported in cynodont phylogeny, the relationships of more stemward cynodonts are currently unresolved. Using CT data derived from extensive sampling of the earliest known (late Permian and Early Triassic) cynodonts and selected exemplars of therocephalians and later (Middle Triassic onwards) cynodonts (eucynodonts), I described novel aspects of the endocranial anatomy of these animals. These data were incorporated into a new, taxonomically comprehensive phylogenetic dataset of the group.

There is a lack of systematic research exploring cross-species variation in liver lobular geometry and zonation patterns of critical drug-metabolizing enzymes, a knowledge gap essential for translational studies. This study investigated the critical interplay between lobular geometry and key cytochrome P450 (CYP) zonation in four species: mouse, rat, pig, and human. We developed an automated pipeline based on whole slide images (WSI) of hematoxylin-eosin-stained liver sections and immunohistochemistry. This pipeline allows accurate quantification of both lobular geometry and zonation patterns of essential CYP proteins. Our analysis of CYP zonal expression shows that all CYP enzymes (besides CYP2D6 with panlobular expression) were observed in the pericentral region in all species, but with distinct differences. Comparison of normalized gradient intensity shows a high similarity between mice and humans, followed by rats. Specifically, CYP1A2 was expressed throughout the pericentral region in mice and humans, whereas it was restricted to a narrow pericentral rim in rats and showed a panlobular pattern in pigs. Similarly, CYP3A4 is present in the pericentral region, but its extent varies considerably in rats and appears panlobular in pigs. CYP2D6 zonal expression consistently shows a panlobular pattern in all species, although the intensity varies. CYP2E1 zonal expression covered the entire pericentral region with extension into the midzone in all four species, suggesting its potential for further cross-species analysis. Analysis of lobular geometry revealed an increase in lobular size with increasing species size, whereas lobular compactness was similar. Based on our results, zonated CYP expression in mice is most similar to humans. Therefore, mice appear to be the most appropriate species for drug metabolism studies unless larger species are required for other purposes, e.g., surgical reasons. CYP selection should be based on species, with CYP2E1 and CYP2D6 being the, Peer Reviewed

African savanna ecosystems are home to the world's richest large herbivore (LH) assemblages. However, its landscapes are changing faster than any other region on Earth due to human activities and natural events. Therefore, understanding the factors influencing the distribution of LH in these human-dominated environments is crucial for decision-making on wildlife and habitat management. We combined ecological aerial surveys, camera trap, and dung count data to investigate how ecological (habitat types, perennial rivers, and rainfall) and anthropogenic (human settlements and cattle grazing areas) factors influence the distribution of LH species in Limpopo National Park (LNP). We used generalized linear models fitting binary logistic regression models to distinguish 25-km2 cells occupied (where the species were detected) by elephants (Loxodonta africana), buffalos (Syncerus caffer), zebras (Equus quagga), kudus (Tragelaphus strepsiceros), nyalas (Tragelaphus angasii), and impalas (Aepyceros melampus) from unoccupied regions (where the species were not detected) in the LNP landscape. We found that habitat type and rainfall were the most influential factors shaping the pattern of LH distribution in the LNP, except for elephants, whose prevalence was not associated with rainfall. The prevalence of zebras was positively associated with the proximity to perennial rivers, while kudus avoided these areas. While some species (zebras, kudus, and impalas) tended to avoid settlements, others (elephants, buffalos, and nyala) seemed attracted to settlements. Cattle grazing areas were the worst predictors of the distribution of all study species. Our results disclosed the role of ecological factors for the distribution of LH and showed that anthropogenic disturbances seemed to either (partially) prevent the occurrence of LH or show the potential for human–wildlife conflict risk in the study area. Therefore, the results highlight the need to investigate/quantify the potential human–w, German Development Bank, Zwillenberg‐Tietz Foundation, Limpopo National Park Administration, Peer Reviewed

Aquaponic systems have potential to improve water and nutrient use efficiency compared to separately operated recirculating aquaculture systems (RAS) and hydroponic facilities. However, fish feeds are optimized for fish performance and low effluent nutrient discharge instead of integrated production of fish and plants, often leading to deficiencies of certain dissolved plant nutrients in RAS water. This study tested the effect of gradually replacing a blend of animal by-products (poultry by-product meal, catfish by-product meal, poultry blood meal) in a marine ingredient-free and entirely animal protein-based diet with a blend of common plant protein sources in four quasi-realistic isonitrogenous (42% crude protein) and isocaloric (20 MJ/kg) diets for African catfish on growth performance, body composition and nutrient release in dissolved and solid form into RAS water. Reducing the contribution of animal proteins to dietary protein from 100% over 75% and 50% to 25% resulted in the best biomass gain (%), feed conversion ratio (dry matter basis) and protein efficiency ratio for the 50% level, with differences, however, non-significant compared to the 100% and 75% level. At the 25% level, i.e. the highest level of plant protein inclusion, significant impairments regarding these growth indices as well as body ash, P and Ca were recorded compared to all other diets. No differences were observed for total dissolved 4 release per unit of feed between diets. Higher animal protein contribution lead to significantly higher release of dissolved P and Ca into RAS water, whereas increasing plant protein contribution caused significantly higher release of dissolved K, Mg and B. A mass balance of dietary nutrient fate revealed that N, P, K, S and Mg primarily ended up in fish biomass and in dissolved form in RAS water rather than in collected solids, while Ca, Fe, Mn, Zn and Cu predominantly ended up in solids and fish biomass rather than in dissolved form in RAS water. Modeling, Peer Reviewed

The article processing charge was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – 491192747 and the Open Access Publication Fund of Humboldt-Universität zu Berlin., The mitochondrial genomes of apicomplexans comprise merely three protein-coding genes, alongside a set of thirty to forty genes encoding small RNAs (sRNAs), many of which exhibit homologies to rRNA from E. coli. The expression status and integration of these short RNAs into ribosomes remains unclear and direct evidence for active ribosomes within apicomplexan mitochondria is still lacking. In this study, we conducted small RNA sequencing on the apicomplexan Toxoplasma gondii to investigate the occurrence and function of mitochondrial sRNAs. To enhance the analysis of sRNA sequencing outcomes, we also re-sequenced the T. gondii mitochondrial genome using an improved organelle enrichment protocol and Nanopore sequencing. It has been established previously that the T. gondii genome comprises 21 sequence blocks that undergo recombination among themselves but that their order is not entirely random. The enhanced coverage of the mitochondrial genome allowed us to characterize block combinations at increased resolution. Employing this refined genome for sRNA mapping, we find that many small RNAs originated from the junction sites between protein-coding blocks and rRNA sequence blocks. Surprisingly, such block border sRNAs were incorporated into polysomes together with canonical rRNA fragments and mRNAs. In conclusion, apicomplexan ribosomes are active within polysomes and are indeed assembled through the integration of sRNAs, including previously undetected sRNAs with merged mRNA-rRNA sequences. Our findings lead to the hypothesis that T. gondii’s block-based genome organization enables the dual utilization of mitochondrial sequences as both messenger RNAs and ribosomal RNAs, potentially establishing a link between the regulation of rRNA and mRNA expression., Peer Reviewed

Viruses frequently contain overlapping genes, which encode functionally unrelated proteins from the same DNA or RNA region but in different reading frames. Yet, overlapping genes are often overlooked during genome annotation, in particular in DNA viruses. Here we looked for the presence of overlapping genes likely to encode a functional protein in human parvovirus B19 (genus Erythroparvovirus), using an experimentally validated software, Synplot2. Synplot2 detected an open reading frame, X, conserved in all erythroparvoviruses, which overlaps the VP1 capsid gene and is under highly significant selection pressure. In a related virus, human parvovirus 4 (genus Tetraparvovirus), Synplot2 also detected an open reading frame under highly significant selection pressure, ARF1, which overlaps the VP1 gene and is conserved in all tetraparvoviruses. These findings provide compelling evidence that the X and ARF1 proteins must be expressed and functional. X and ARF1 have the exact same location (they overlap the region of the VP1 gene encoding the phospholipase A2 domain), are both in the same frame (+1) with respect to the VP1 frame, and encode proteins with similar predicted properties, including a central transmembrane region. Further studies will be needed to determine whether they have a common origin and similar function. X and ARF1 are probably translated either from a polycistronic mRNA by a non-canonical mechanism, or from an unmapped monocistronic mRNA. Finally, we also discovered proteins predicted to be expressed from a frame overlapping VP1 in other species related to parvovirus B19: porcine parvovirus 2 (Z protein) and bovine parvovirus 3 (X-like protein)., Peer Reviewed

The use of DNA barcoding is well established for specimen identification and large‐scale biodiversity discovery, but remains underutilized for time‐sensitive applications such as rapid species discovery in field stations, identifying pests, citizen science projects, and authenticating food. The main reason is that existing express barcoding workflows are either too expensive or can only be used in very well‐equipped laboratories by highly‐trained staff. We here show an alternative workflow combining rapid DNA extraction with HotSHOT, amplicon production with NextGenPCR thermocyclers, and sequencing with low‐cost MinION sequencers. We demonstrate the power of the approach by generating 250 barcodes for 285 specimens within 6 h including specimen identification through BLAST. The workflow required only the following major equipment that easily fits onto a lab bench: Thermocycler, NextGenPCR, microplate sealer, Qubit, and MinION. Based on our results, we argue that simplified barcoding workflows for species‐level sorting are now faster, more accurate, and sufficiently cost‐effective to replace traditional morpho‐species sorting in many projects., Bundesministerium für Bildung und Forschung http://dx.doi.org/10.13039/501100002347, Carlsbergfondet http://dx.doi.org/10.13039/501100002808, Peer Reviewed

Bivalvia is a class of freshwater and marine mollusks such as scallops, clams, oysters, or mussels which are bilaterally compressed and enclosed in a shell consisting of two hinged parts. The current qualitative study was designed to assess the biodiversity of Bivalvia in selected habitats at Helgoland, an island in the south-eastern part of the North Sea. Samples were collected by hand in the north-eastern intertidal rocky shore as well as by dredging in the “Tiefe Rinne”, an area south of Helgoland 50 to 60 m deep. The number of species identified was 14. Nine species were collected in the intertidal rocky shore Cochlodesma praetenue, Dosinia exoleta, Dosinia lupinus, Mya arenaria, Mytilus edulis, Parvicardium scabrum, Polititapes aureus, Timoclea ovata and Venerupis corrugata. Whilst the dredged species were Modiolus modiolus, Aequipecten opercularis, Mimachlamys varia and Ostrea edulis. Magallana gigas was only observed by chance in the south harbor and Mytilus edulis was found underneath a pontoon in the north-east harbor. The study identified fewer species as compared to previous studies. This may be due to differences in sampling period and sampling sites and might also reflect the dynamics in parameters that influence the fluctuations in species diversity like tides and anthropogenic impacts., Peer Reviewed

The balance between excitation and inhibition is essential to the proper function of cortical circuits. To maintain this balance during dynamic network activity, modulation of the strength of inhibitory synapses is a central requirement. In this study, we aimed to characterize perisomatic inhibition and its plasticity onto pyramidal cells (PCs) in the subiculum, the main output region of the hippocampus. We performed whole-cell patch-clamp recordings from the two main functional PC types, burst (BS) and regular spiking (RS) neurons in acute rat hippocampal slices and applied two different extracellular high-frequency stimulation paradigms: non-associative (presynaptic stimulation only) and associative stimulation (concurrent pre-and postsynaptic stimulation) to induce plasticity. Our results revealed cell type-specific differences in the expression of inhibitory plasticity depending on the induction paradigm: While associative stimulation caused robust inhibitory plasticity in both cell types, non-associative stimulation produced long-term potentiation in RS, but not in BS PCs. Analysis of paired-pulse ratio, variance of IPSPs, and postsynaptic Ca2+ buffering indicated a dominant postsynaptic calcium-dependent signaling and expression of inhibitory plasticity in both PC types. This divergence in inhibitory plasticity complements a stronger inhibition and a higher intrinsic excitability in RS as compared to BS neurons, suggesting differential involvement of the two PC types during network activation and information processing in the subiculum., Peer Reviewed

When exposed to low temperatures, plants undergo a drastic reprogramming of their transcriptome in order to adapt to their new environmental conditions, which primes them for potential freezing temperatures. While the involvement of transcription factors in this process, termed cold acclimation, has been deeply investigated, the potential contribution of chromatin regulation remains largely unclear. A large proportion of cold-inducible genes carries the repressive mark histone 3 lysine 27 trimethylation (H3K27me3), which has been hypothesized as maintaining them in a silenced state in the absence of stress, but which would need to be removed or counteracted upon stress perception. However, the fate of H3K27me3 during cold exposure has not been studied genome-wide. In this study, we offer an epigenome profiling of H3K27me3 and its antagonistic active mark H3K4me3 during short-term cold exposure. Both chromatin marks undergo rapid redistribution upon cold exposure, however, the gene sets undergoing H3K4me3 or H3K27me3 differential methylation are distinct, refuting the simplistic idea that gene activation relies on a switch from an H3K27me3 repressed chromatin to an active form enriched in H3K4me3. Coupling the ChIP-seq experiments with transcriptome profiling reveals that differential histone methylation only weakly correlates with changes in expression. Interestingly, only a subset of cold-regulated genes lose H3K27me3 during their induction, indicating that H3K27me3 is not an obstacle to transcriptional activation. In the H3K27me3 methyltransferase curly leaf (clf) mutant, many cold regulated genes display reduced H3K27me3 levels but their transcriptional activity is not altered prior or during a cold exposure, suggesting that H3K27me3 may serve a more intricate role in the cold response than simply repressing the cold-inducible genes in naïve conditions., Peer Reviewed

The autocatalyzed ethanolic organosolv process is gaining increasing attention for the sulfur-free isolation of lignin, which is subsequently used as a renewable substitute for various fossil-based applications. For the first time, the mechanochemical influence of seven different particle sizes of two different biomasses on the respective organosolv lignin structure is examined. Wine pruning (Pinot Noir) and wine pomace (Accent) are used for organosolv process with particle sizes ranging from 2.0-1.6 mm to less than 0.25 mm. As particle size decreases, the weight-average molecular weight increases, while the total phenol content decreases significantly. Additionally, the distribution of the lignin-typical monolignols and relevant substructures, as determined by two-dimensional heteronuclear nuclear magnetic resonance spectra single quantum coherence (HSQC), is observed. The degree of grinding of the biomass has a clear chemical-structural influence on the isolated HG and HGS organosolv lignins. Therefore, it is crucial to understand this influence to apply organosolv lignins in a targeted manner. In the future, particle size specifications in the context of the organosolv process should be expressed in terms of distribution densities rather than in terms of a smaller than specification.

The goal of this study was to explore a route for introducing functionalities into agarose-based hydrogels to tune the physical, chemical, and biological properties. Several agarose derivatives were prepared by homogeneous synthesis, including anionic agarose sulfates (ASs), reactive azido agaroses (AZAs), and cationic agarose carbamates (ACs), as well as agarose tosylates (ATOSs) and agarose phenyl carbonates (APhCs). The products were characterized in terms of their molecular structure and solubility behavior. The results suggest that the native gel-forming ability of agarose is retained if the introduced functionalities are hydrophilic, and the overall degree of substitution is low (DS < 0.5). Thus, functional hydrogels from several agarose derivatives could be obtained. The mechanical stability of the functional hydrogels was decreased compared to native agarose gels but was still in a range that enables safe handling. An increase in mechanical strength could be achieved by blending functional agarose derivatives and agarose into composite hydrogels. Finally, it was demonstrated that the novel functional agarose hydrogels are biocompatible and can potentially stimulate interactions with cells and tissue.

Die Schriftenreihe informiert über Forschungsergebnisse zur Entstehung und Transport von gelösten organischen Kohlenstoffverbindungen (DOC, dissolved organic carbon) im Einzugsgebiet der Talsperre Sosa. DOC entsteht in den Humusauflagen der Wälder, in degradierten Mooren und in mineralischen Böden. Es wird insbesondere bei Starkregen und während der Schneeschmelze ausgetragen. Der Eintrag in die Gewässer hängt ab von der Freisetzung des DOC, von der Fließverbindung des Bodenwassers und von Rückhalteprozessen im Boden. Hieraus ergeben sich mögliche Optionen zur Verminderung des DOC-Eintrags in Trinkwassertalsperren, da DOC hier erhebliche Probleme verursacht. Die Veröffentlichung richtet sich an Forschungseinrichtungen, Fach- und Vollzugsbehörden und interessierte Bürgerinnen und Bürger. Redaktionsschluss: 30.09.2023

Phosphorus (P) is one of the fundamental chemical elements and one of the building blocks of life. In nature, there are two major P flows. One P flow is natural, where P over thousands and millions of years gets mobilised from P rock and circulates, “changing hands” many times before it gets deposited and biologically unavailable. The second is the anthropogenic flow, three times larger than the natural P cycle. To feed the anthropogenic flow, P is mined from the P rock and processed towards the final product - phosphoric acid. From here, P is used in chemical industry, food and beverage industry, pharmacy, but mostly in agriculture as a fertiliser. Because fertilisers are applied on farmlands on a yearly basis, the turnover of P in agriculture, the largest consumer of the man-made P circle, occurs only once, therefore, it can be considered a primarily linear flow. From the man-made P flow, P leaches into the natural cycle. The consequence of this can be seen in the environment through eutrophication, and the formation of dead zones in lakes and the ocean, causing environmental damage. The importance of P and the danger it poses for the environment were recognised in EU regulations as well as in member states laws. Today, P is classified as a critical raw material at the EU level, and legal incentives are in place on both levels to reduce P emissions into the environment and to begin recovering P from human-made waste streams. There are several possible sources of P in human waste streams, including agricultural runoff, animal manures, food and food processing waste, wastewater and wastewater sludge, sewage sludge ash, but also already existing, immobilised, and unavailable P in the soil. This research focuses on wastewater and wastewater sludge for P recovery. It is estimated that if all the P in wastewater were recovered, this would cover 20% of the world's annual fertiliser needs. In wastewater treatment plants (WWTP), P recovery is usually done using chemically, Phosphor (P) ist eines der grundlegenden chemischen Elemente und einer der Bausteine des Lebens. In der Natur gibt es zwei große P-Ströme. Ein P-Strom ist natürlich, wenn P über Tausende und Millionen von Jahren aus P-Gestein mobilisiert und zirkuliert, dabei „durch mehrere Hände wandert“, bevor es abgelagert und biologisch unverfügbar wird. Der zweite ist der anthropogene Strom, der dreimal größer ist als der natürliche P-Kreislauf. Um den anthropogenen Strom zu speisen, wird P aus dem P-Gestein abgebaut und zum Endprodukt - Phosphorsäure - verarbeitet. Dieser P wird in der chemischen, in der Lebensmittel- und Getränkeindustrie, in der Medizin, aber hauptsächlich in der Landwirtschaft als Düngemittel verwendet. Da Düngemittel jährlich auf landwirtschaftlichen Flächen ausgebracht wird, findet der Umsatz von P in der Landwirtschaft, nur einmal statt und kann daher als überwiegend linearer Fluss betrachtet werden. Aus dem anthropogenen P-Fluss gelangt P in den natürlichen Kreislauf. Die Folgen davon sind in der Umwelt durch Eutrophierung und die Bildung von Todeszonen in Seen und im Ozean zu sehen, was Umweltschäden zur Folge hat. Die Bedeutung von P und die Gefahr, die es für die Umwelt darstellt, wurden in EU-Verordnungen sowie in den Gesetzen der Mitgliedstaaten anerkannt. Heute ist P auf EU-Ebene als kritischer Rohstoff eingestuft und es bestehen rechtliche Anreize auf beiden Ebenen, um die P-Emissionen in die Umwelt zu reduzieren und die Rückgewinnung von P aus anthropogenen Abfallströmen zu beginnen. Es gibt mehrere mögliche Quellen von P in anthropogenen Abfallströmen, einschließlich landwirtschaftlicher Abflüsse, tierischer Exkremente, Lebensmittel- und Lebensmittelverarbeitungsabfälle, Abwasser und Klärschlamm, Klärschlammasche, aber auch bereits vorhandenes, immobilisiertes und nicht verfügbares P im Boden. Diese Forschung konzentriert sich auf die P-Rückgewinnung aus Abwasser und Abwasserschlamm. Schätzungen ergaben, dass eine erfolgreiche P-Rückgewinnung a

Background and Purpose. Tibial head fracture (THF) rehabilitation is still a challenge in clinical practice. Short-term use of continuous passive motion (CPM) postoperatively for THFs can increase knee range of motion (ROM) immediately, and its effect on enhanced rehabilitation also ended when the CPM application was discontinued. The aim of this study was to investigate the effect on the recovery of prolonged use of CPM in the postoperative treatment of THFs. Methods. 60 patients with THFs were randomly and equally divided into the CPM group and non-CPM group. Both groups immediately received CPM and conventional physical therapies during hospitalization. After discharge, the non-CPM group was treated with conventional physical therapy alone, while the CPM group received conventional physical training in combination with CPM treatment. At 6 weeks and 6 months postoperatively, the primary outcome which was knee ROM and the secondary outcome which was knee functionality and quality of life were evaluated. Results. The CPM group had a significantly increased ROM at both follow-up time points. The Knee Society Score, UCLA activity score, and the EuroQoL as well as the pain analysis showed significantly better results of the CPM group than the non-CPM group. Conclusions. The prolonged application of CPM therapy is an effective method to improve the postoperative rehabilitation of THFs.

Metabolic coupling between glial cells and neurons is essential for neuronal function. It is a well-conserved and vital feature of the bilaterian nervous system as well. Under normal and adverse conditions, glial cells act as a major metabolic hub fueling neuronal oxidative metabolism by producing lactate or ketone bodies. I now ask the question of whether such metabolic coupling is only necessary for preserving brain homeostasis or if it could also have implications in decision making such as food choice behavior. Choosing an appropriate food source is key for the survival of an organism. Carbohydrates are the preferred source of energy and thus evaluation of their nutritive content is essential. Several studies have demonstrated that Drosophila melanogaster larvae and adults, as mammals, can distinguish between nutritious and non-nutritious carbohydrates independent of their taste. Two groups of neurons, Diuretic Hormone 44 (Dh44)-expressing neurons and gustatory receptor 43a (Gr43a)-expressing neurons, have been implicated in postprandial sugar sensing in adult flies. Gr43a- expressing neurons are narrowly fine-tuned for sensing fructose in both adults and larvae. Nonetheless, in the larva, central nervous system (CNS) Gr43a neurons have been shown to act as the main sugar sensor. This raises the question of how CNS fructose-sensing neurons are involved in sensing non-fructose dietary sugars. To decipher this post-ingestive mechanism, I used frustrated total internal reflection (FTIR) - based larval tracking to investigate larval food choice behavior. The results present in this thesis suggest that besides Gr43a-expressing neurons, glial cells are indispensable for sensing nutritive non-fructose dietary sugars such as glucose and sorbitol. I show that post-ingestive carbohydrate sensing involves carbohydrate conversion into fructose locally in the glial cells. Glia-derived fructose then enables Gr43a-dependent postprandial carbohydrate sensing in the CNS and driv

Novizen fällt es häufig schwer, die fachbiologische Perspektive und die Alltagsvorstellungen der Lernenden gleichermaßen in ihrer Unterrichtsplanung mit einzubeziehen (z.B. DANNEMANN et al., 2014; RICHTER & KOMOREK, 2017). Dieser Befund steht im Kontrast zu den fachdidaktischen Forschungsbemühungen im Bereich der Vorstellungsforschung (LABUDDE & MÖLLER, 2012; REINISCH, HELBIG & KRÜGER, 2020). In dieser Studie wird eine Ursache des skizzierten Problemfeldes näher untersucht: das Planungsverhalten von Novizen im Vergleich zu einer Planung mit Alltagsvorstellungen. Eine Planung mit Vorstellungen berücksichtigt themenspezifische Lernchancen und -hürden, orientiert sich an zentralen Konzepten der Fachleute zum Phänomen und bezieht sich auf Didaktische Leitlinien für den Lehr- Lernprozess (GROPENGIEßER & MAROHN, 2018). Diese Planungsphilosophie findet sich im Modell der Didaktischen Rekonstruktion (MDR; DUIT et al., 2012) wieder und wird als Standard zur Planung in dieser Studie angenommen. Bestehende Studien führen die eingangs benannte Schwierigkeit von Novizen vor allem auf fehlendes Wissen über Lernendenvorstellungen und deren Berücksichtigung zurück (z.B. DANNEMANN et al., 2014; KIND, 2016). Dies beklagen auch die Novizen selbst (z.B. SCHNEBEL & KREIS, 2014). Deshalb wurde in dieser Studie für Biologie Lehramtsstudierende (7. Fachsemester) und eng angekoppelt an ein fachwissenschaftliches Pflichtmodul (Neurobiologie) ein „MDR-Training“ entwickelt und durchgeführt. Im MDR-Training wurde insbesondere geübt, Unterricht unter Berücksichtigung der Alltagsvorstellungen zu planen. Die begleitende qualitative Studie geht grundlegend der Frage nach, inwiefern das MDR den Studierenden bei ihrer Unterrichtsplanung hilft und wo Schwierigkeiten liegen. Ferner lassen sich aus den Forschungsergebnissen und den Erfahrungen im MDR-Training 1.0 Rückschlüsse für ein MDR-Training 2.0 ziehen. Die während dieses Trainings erhobenen Daten umfassen je zwei Einzelinterviews mit jedem Proband, Science teaching that promotes understanding is oriented toward the learner's perspective. Although preservice biology teachers (PST) generally attach great importance to this perspective for learning processes. But it is difficult for them to consistently take it into the lesson planning. A deeper understanding of the planning processes of PST as well as of the difficulties associated with learner-oriented lesson planning enables the modification of subject-specific didactic courses to the prerequisites of PST and further helps to develop their planning competencies. This empirical study reconstructs planning difficulties of biology PST (N=8). The study context is a biology didactics seminar on lesson planning which complements a subject-specific neurobiology course. The qualitative content analysis of the planning difficulties is based on the planning products of the PST as well as on semi-structured individual interviews. The results, validated by experts, show that the PST i) focus too much on subject-specific details without clarifying the content structure for instruction, ii) anticipate the learners’ generic experiences instead of referring to their conceptions, and iii) already assume a kind of blanket learning effectiveness as soon as they refer to student conceptions. Possible causes for the reconstructed planning difficulties are discussed against the background of MER, a constructivist paradigm, and with reference to the PST’s beliefs. Finally, for the subject-specific didactic courses, we recommend a stronger reflection on the paradigms of learning, a gradual introduction of the subject matter clarification, as well as the further development of the MER.:1 EINFÜHRUNG I THEORETISCHER TEIL 2 LERNENDENVORSTELLUNGEN BEI DER UNTERRICHTSPLANUNG BERÜCKSICHTIGEN 2.1 Erkenntnistheoretische Grundannahmen, Definitionen und Vorgehen 2.2 Vorstellungen als Konstrukt verstehen 2.3 Lerngegenstand rekonstruieren 2.4 Lernendenvorstellungen verändern: conceptual change

Die jüngsten Fortschritte in der Behandlung haben das Gesamtüberleben von Kindern mit akuter lymphoblastischer Leukämie (ALL) verbessert. Dennoch erleiden 20 % der Kinder mit ALL Rückfälle, was die Notwendigkeit neuer zielgerichteter Behandlungsstrategien erhöht. Ein Empfindlichkeitsprofil wurde in ALL-Zelllinien (n=18) und patientenabgeleiteten Zellen (n=14) mit heterogenem genetischem Hintergrund unter Verwendung einer Wirkstoffbibliothek (n=46 Verbindungen) erstellt. Dies identifizierte neuartige Arzneimittelempfindlichkeitsmuster, wie z.B. für den H3K27me3-Demethylase-Inhibitor GSKJ4, dessen Empfindlichkeit mit der CREBBP-Expression im Kontext der Dexamethason-Resistenz korrelierte. In einem Screening von Wirkstoffkombinationen (n=12 Wirkstoffe) wurde Venetoclax als vielversprechendster Kombinationspartner identifiziert. Die höchsten synergistischen Effekte wurden bei der Kombination von Venetoclax mit dem CK2-Inhibitor Silmitasertib, dem PARP-Inhibitor Olaparib und dem MEK-Inhibitor Selumetinib beobachtet. Die Induktion von Apoptose und ein synergistischer Effekt wurden in zahlreichen BCP-ALL-Zelllinien, PDX und Primärproben validiert, wobei diese Kombination besonders wirksam in den venetoclax-resistentesten Proben war. Silmitasertib sensibilisierte die Zellen für Venetoclax durch die Induktion des MCL1-Proteasom-Abbaus über die Herabregulierung der AKT-Signalübertragung und die Hochregulierung der GSK3B-Aktivität. Ein neuartiges In-vivo-Zebrafisch-Embryomodell validierte die Wirksamkeit von Venetoclax und Silmitasertib sowie die Herabregulierung von MCL1. Insgesamt wurde eine Plattform für das Screening von Einzelwirkstoffen und Wirkstoffkombinationen vorgestellt, die neuartige Arzneimittelempfindlichkeitsmuster sowie vielversprechende synergistische Wirkstoffpartner identifizierte, die im Fall von Silmitasertib und Venetoclax funktionell charakterisiert und deren Wirkung in klinisch nahen Proben sowie in einem In-vivo-Zebrafischmodell validiert wurden., Recent advances in treatment have improved overall survival for children with acute lymphoblastic leukemia (ALL). However, 20% of children with ALL still relapse, raising the need for the implementation of novel targeted treatment strategies. Drug sensitivity profiling was performed in ALL cell lines (n=18) and patient derived cells (n= 14) with heterogeneous genetic backgrounds using a candidate drug library (n=46 compounds). This identified novel drug sensitivity patterns such as for the H3K27me3 demethylase inhibitor GSKJ4, which sensitivity correlated to CREBBP expression in the context of dexamethasone resistance. In a drug combination screening (n=12 drugs), venetoclax was identified as the most promising combination partner. Highest synergistic effects where seen when combining venetoclax with CK2 inhibitor silmitasertib, PARP inhibitor Olaparib and MEK inhibitor selumetinib. Induction of apoptosis and a synergistic effect was validated across numerous BCP-ALL cell lines, patient derived xenografts (PDX) and primary samples, being particularly effective in the most venetoclax resistant samples. Silmitasertib sensitized cells to venetoclax by the induction of MCL1 proteasome degradation via downregulating AKT signaling and thus upregulating GSK3B activity. A novel in vivo zebrafish embryo model validated the effectivity of venetoclax and silmitasertib and MCL1 downregulation. Overall, a single drug and drug combination screening platform is presented, which identified novel drug sensitivity patterns as well as promising synergistic drug partners that, in the case of silmitasertib and venetoclax, were functionally characterized and its effect validated in clinically near samples as well as in an in vivo zebrafish model.

Next-generation sequencing (NGS) ermöglicht das molekulare Profiling von Zellen mit beispiellos hohem Durchsatz. Allerdings ist der Fokus oftmals auf proteinkodierende Proteine beschränkt, wodurch die vollständige Diversität des Transkriptoms übersehen wird. Nicht-kodierende RNA-Moleküle variieren stark in ihrer Biogenese, Struktur und Funktion, wodurch ihre unverzerrte Inklusion in die Analyse erschwert wird. Diese Promotion fokussiert sich auf das Verständnis nicht-kodierender RNA und navigiert durch drei aufeinander aufbauende Säulen in der Analyse, um Beobachtungen in Wissen zu verwandeln: Generierung von Daten, Quantifizierung und Interpretation. Diese drei Säulen werden in den drei Kapiteln der Dissertation aus der bioinformatischen Perspektive adressiert, indem Schlüsselherausforderungen beschrieben und neue Lösungen vorgestellt werden, um die Analyse des gesamten Transkriptoms mit NGS-Techniken zu verbessern. Zunächst wird ein vollautomatischer Algorithmus vorgestellt, welcher die verschiedenen Quellen von aus der Vorberei- tung von Bibliotheken resultierenden Artefakten mittels unüberwachtes Lernen erkennt, was anschließend zur Optimierung der Protokolle zur Vorbereitung von total-RNA-seq-Bibliotheken genutzt werden kann. Zudem werden die primären Herausforderungen der Quantifizierung von total-RNA-seq behandelt: die Prozessierung von Reads, die mehreren, möglicherweise überlappenden Loci zugeordnet werden können, wie auch die Tatsache, dass manche Loci mehrfach im Genom vorkommen und ein Read zu all diesen Loci passen kann. Diese beiden Fälle können auch gleichzeitig vorkommen, was die Analyse von nicht-kodierender RNA mit üblichen Methoden erschwert. Um diese Problematik anzugehen, wird eine neue Software namens Multi-Graph count (MGcount) vorgestellt. Diese ordnet hierarchisch Reads Transkripten zu, um unter anderem eine Diskrepanz zwischen der Loci-Länge von small und long RNA zu berücksichtigen. Wenn Reads konsistent mehrfach alignieren, fasst MGcount, Next-generation sequencing (NGS) techniques enable the molecular profiling of cells with unprecedented high throughput. Yet, in transcriptome analysis, the focus is often restricted to protein-coding RNA, overlooking the transcriptome in its entire diversity. Non-coding RNA molecules largely vary in biogenesis, structure and function and this challenges their unbiased inclusion into the analyses. This doctoral research places non-coding RNA understanding at the focus spot and navigates through the three workflow pillars that must align effectively to turn observations into knowledge: data generation, quantification, and interpretation. Throughout three chapters, this Thesis addresses these pillars from a Bioinformatics perspective, by outlining key challenges and introducing novel solutions to improve whole-transcriptome analysis through NGS techniques. First, we introduce a fully automatic algorithm that identifies sources of library preparation artifacts in an unsupervised manner and we demonstrate its utility within the development and optimization of total-RNA-seq library preparation protocols. Secondly, we address a major challenge in total-RNA-seq quantification; processing reads that align to multiple loci that overlap within the same genomic region or/and multiple loci that are present in high copy numbers. Such ambiguous alignments commonly arise due to the inherent characteristics of non-coding RNA. To tackle this, we introduce a novel software, named Multi-Graph count (MGcount), that hierarchically assigns reads to transcripts to account for loci length disparity between small-RNA and long-RNA and subsequently collapses loci where reads consistently multi-map into communities defined in a data-driven fashion. We show that these cohesive communities allow the quantification of biologically meaningful RNA entities (single-transcripts or locus-families) and estimate their abundance more accurately. Finally, we apply the developed method to investigate non-co

Multizelluläre Mikrogewebe in Form von unregelmäßig geformten oder kugelförmigen Clustern können Zell-Zell-Interaktionen abbilden und gewinnen in verschiedenen Bereichen, darunter auch in der Herz-Kreislauf-Forschung, zunehmend an Bedeutung. Kardiale Mikrogewebe entwickeln sich zu hervorragenden Modellsystemen für die Arzneimittelprüfung in vitro („organ-on-a-chip“), werden als Gewebebausteine in 3D Druckverfahren verwendet und ebnen den Weg für verbesserte Zellersatztherapien in vivo. Mikrogewebe werden zum Beispiel im hängenden Tropfen oder speziellen Mikrotiterplatten kultiviert. Wirklich skalierbare Methoden zur Herstellung von Mikrogeweben sind bisher nicht verfügbar. In dieser Studie wird eine neue Methode zur Verkapselung von Zellen in Sphäroiden aus Poly N Isopropylacrylamid (PNIPAAm) vorgestellt. PNIPAAm fällt oberhalb seiner unteren kritischen Lösetemperatur (LCST) zu einem wasserunlöslichen, physikalisch verknüpften Gel aus und schrumpft durch den Ausstoß von Wasser. Aus murinen induziert pluripotenten Stammzellen gewonnene Kardiomyozyten und aus murinem Knochenmark gewonnene mesenchymale Stammzellen wurden in einem Mikrofluidiksystem in PNIPAAm verkapselt. Indem die Temperatur über die (LCST) von 32 °C erhöht wurde, wurden die Zellen in einem kollabierenden Polymernetzwerk eingeschlossen und die Zelldichte in den Polymerkapseln um den Faktor 2,7 erhöht. Innerhalb von 24 Stunden wurden zunächst stabile Herzmikrogewebe gebildet und später durch Auswaschen des PNIPAAm bei Temperaturen unterhalb der LCST aus ihrer Polymerhülle gelöst. Rhythmisch kontrahierende Mikrogewebe zeigten eine homogene Zellverteilung, eine altersabhängige Sarkomerorganisation und die Erzeugung von Aktionspotenzialen. Der neuartige Ansatz ist für die Bildung vitaler Mikrogewebe homogener Eigenschaften geeignet. Außerdem können verschiedene Zelltypen durch das Polymer in ein Gewebe zusammengeführt werden. Das ermöglicht eine Steigerung in der Vielfalt der modellierbaren Gewebe. In Zuku

Cell death is a fundamental biological process for tissue homeostasis and tissue remodelling during development and for the defence against a vast range of stressors. However, uncontrolled cell death can impair these programs causing severe pathological consequences. Thus, the regulation of cell death is a critical step to trigger proper immune responses and healing processes following stress conditions. Tumour necrosis factor (TNF), a proinflammatory cytokine secreted during different stress conditions, can determine cell fate, either cell survival and proliferation or cell death, the latter via the formation of a cytosolic, cell death-inducing complex, called complex II. Checkpoints are specific mechanisms in TNF signalling that control the decisison between cell survival and death and, within the latter, control the amplitude of cell death. Therefore, these checkpoints have a high pathophysiological relevance. Hence, our goal is to gain a deeper molecular understanding of how these mechanisms regulate the cytotoxic potential of TNF. Cellular FLICE inhibitory protein (cFLIP) is a crucial regulator of TNF-induced cell death. cFLIP is cleaved at D377 by Caspase-8. Yet, the physiological role of cFLIP cleavage in the regulation of TNF-mediated cell death outcomes remains elusive. Here, we discovered that cFLIP cleavage counterbalances complex II assembly and cytotoxicity. Consequently, abrogation of cFLIP cleavage by the D377A mutation sensitizes cells to TNF-induced cell death by favouring complex II formation. The cell death sensitizing effect of D377A mutation depends on the C-terminal residues of the caspase-like domain on cFLIP, in particular the Q469 resiude. To address the physiological funtions of cFLIP cleavage, we generated a novel mouse line bearing a non-cleavable version of cFLIP (CflipD377A/D377A). These mice exhibited increased tissue damage caused by Sharpin deficiency, impaired skin wound healing and increased lethality upon SARS CoV-1 infection. The

To date, a plethora of artificial nucleic acid analogs with enhanced functional properties have been described in the literature. However, most of the currently described artificial nucleic acids comprise a modification in only one structural element of the tripartite structure of nucleic acids, which is comprised of a phosphate backbone, a sugar moiety, and a nucleobase. This work explores the enzymatic synthesis of a novel artificial nucleic acid variant with modifications in the sugar and the nucleobase moieties. Therefore, the nuclease-resistant alpha-L-threofuranosyl nucleic acid (TNA) was combined with the unnatural hydrophobic base pair TPT3:NaM. In this context, the alpha-L-threofuranosyl NaM triphosphate and the TNA nucleotides bearing the canonical nucleobases were synthesized. The enzymatic incorporation of the highly modified nucleotide building blocks bearing the unnatural TPT3 and NaM nucleobases into TNA was investigated using different TNA-compatible polymerases. The enzymatic synthesis of TPT3-modified TNA was successfully achieved, whereas the preparation of NaM-modified TNA presented greater challenges. This study marks the first enzymatic synthesis of TNA with an expanded genetic alphabet (exTNA), opening promising opportunities in nucleic acid therapeutics, particularly for the selection of nuclease-resistant, high-affinity aptamers.

For non-sexually reproducing organisms such as bacteria, natural transformation, a common mode of horizontal gene transfer (HGT), plays an important role in increasing genetic diversity, spreading beneficial mutations and curing deleterious ones. However, little is known about the genome-wide barriers, the effects of transformation on fitness and the role of the genetic background of the strains. In the first project, we elucidate the genome-wide barriers of transformation and their dependence on the genetic distance between donor and recipient. We conduct replacement accumulation experiments with Bacillus subtilis as recipient and different Bacillus species as well as a Geobacillus species as donors. For 20 cycles, we allow the recipient to transform for two hours in presence of donor DNA, ensure minimal selection by plating the hybrids directly after transformation, and apply a single cell bottleneck by selecting a monoclonal hybrid for the start of the next cycle. Following cycle 10 and 20, the DNA of the hybrids is sent for whole genome sequencing and the fitness is measured by competition experiments. By pooling all hybrids, we find 96% of core genes affected by replacements when supplying B. subtilis with Bacillus spizizenii DNA, indicating an almost unrestricted exchange between closely related species. In the less-related species Bacillus atrophaeus, we find replacement primarily in regions with high sequence identity, with an overrepresentation of ribosomal genes. A comparison of all donor-recipient pairs reveals an exponential relationship between core genome identity and transfer rate. Core genome identity also affects the length of replaced segments. At a low rate, the replacements entail insertions and deletions of accessory regions. We conclude that transformation between closely related species is strongly dependent on sequence identity and is dominated by replacements. In the second project, we characterize the fitness effects of transformation. To t

Crop evolution, especially that of amaranth, has long been thought to be a linear progression from wild to domesticated. However, amaranth's incomplete domestication and extensive cultivation history make it an excellent model for studying the complex genome dynamics of crops. This thesis aims to unravel the intricate genomic mosaic of the domestication process in five amaranth sub-species, wild and domesticated. Through population genomics and computational biology, this study investigates the genetic impact on amaranth populations, including gene flow and the potential for speciation. The primary objective of this thesis is to explore the genomic effects of post-domestication contact on five amaranth populations, both wild and domesticated, found in the Americas. The study shows that not only do amaranth crop species exchange genetic material after domestication, but contact with their wild ancestor also contributes to maintaining their genetic diversity and, consequently, evolutionary rescue. Moreover, this research creates computational tools and resources to handle and analyze the vast amount of genomic data generated. The thesis introduces PopAmaranth, a novel genome browser for population genetics. PopAmaranth provides an intuitive interface integrating various features such as selection signals, gene annotation, and variant calls, enabling interdisciplinary researchers to explore population-scale genomic data and facilitate investigations in population genomics and crop breeding. In summary, this doctoral thesis provides novel insights into the evolutionary history of amaranth, unraveling the intricate interplay between domesticated and wild populations. The research highlights the importance of computational tools like Genome Browsers in facilitating population-scale genomic analyses. These findings contribute to the field of population genomics, advancing our understanding of the genetic dynamics underlying species diversification.

The conjugating green algae (Zygnematophyceae) inhabit a wide range of freshwater fed systems worldwide – including lakes, rivers, ephemeral ponds and moorlands. Some species even thrive in extreme habitats, for example, on terrestrial surfaces or glacial ice. Zygnematophytes have a rather simple cellular organization and have been traditionally divided into three morphologically defined groups: the placoderm desmids (semi-symmetrical unicells with ornamented cell walls), the saccoderm desmids (rod-shaped unicells with smooth cell walls), and the filamentous forms (also with smooth cell walls). Surprisingly, the Zygnematophyceae were found to be the closest relatives of land plants (Embryophyta) – despite their rather simple organization. Since this discovery, the number of studies on zygnematophytes has increased rapidly, and they are now very popular study objects for understanding the evolution of land plants. And yet, the evolutionary relationships between major zygnematophyte groups are unclear and the zygnematophyte taxonomy is outdated. In particular, the saccoderm desmids are under-studied and consist of only a few polyphyletic genera, for example Mesotaenium and Cylindrocystis. Interestingly, these algae colonize various extreme habitats and have been reported to accumulate colorful specialized compounds, whose inducing factors, biological function and chemical identity remain largely unknown. During this doctoral study, saccoderm desmids were isolated from freshwater and terrestrial habitats, resulting in axenic laboratory cultures. Based on these cultures, the cell morphology as well as vegetative and sexual processes were studied with different microscopy techniques. In addition, established marker genes (rbcL and 18S rRNA) were used to localize the new strains in the tree of zygnematophytes by molecular phylogenetics. Furthermore, available datasets from 46 taxonomically diverse zygnematophytes were used to infer a multigene phylogeny (326 nuclear loci)

The pluripotency of embryonic stem cells (ESCs) is defined by their capacity for self-renewal and differentiation into any cell type of an organism. These two characteristics, combined with the ability to induce pluripotency, harbor great potential to apply pluripotent cells in different fields of research and medicine. In contrast to the well-studied state of pluripotency, the cellular networks that disrupt the self-renewal program and promote the exit from pluripotency are poorly characterized. This thesis aims to investigate the networks that promote differentiation and break the self-renewal circuit, thus leading to cells transitioning from naïve to formative pluripotency. We conducted a comprehensive analysis of multiple datasets reflecting the naïve to formative transition to elucidate the mechanisms that drive differentiation. A dense 32-hour wildtype (WT) differentiation time course was utilized to quantify the differentiation delay of 73 mouse knockout (KO) cell lines exhibiting altered differentiation. The comparison of the delays of the naïve and formative networks suggested a decoupling between both networks in their exit from naïve pluripotency. Furthermore, we investigated gene regulatory dependencies between genes in the naïve to formative transition. For this purpose, we developed two methods to infer gene regulatory dependencies from the KO data and an independent single-cell (sc) WT differentiation time course. The resulting dependencies of both approaches showed high concordance and revealed that the transcriptional adaptation of the formative network is independent of the naïve network. Additionally, we observed that parts of the formative network had already adapted to differentiation despite inhibiting fibroblast growth factor/extracellular signal-regulated kinase (FGF/ERK) signaling, indicating FGF/ERK independent regulation of the formative network. Moreover, we provided potential upstream regulators of the formative network based on integrat

CRISPR-Cas9 gene editing is a promising therapy for pathogenic mutations, but current editing approaches are mainly aimed at relatively small groups of patients with specific mutations. Here, we introduce a new application of gene editing to develop an antifibrotic strategy that could potentially be used to address a wide range of organs with fibrosis. In our research on adenine base editing, we successfully targeted the CCAAT box inside the promoter of Col1a1gene promoter in fibroblasts to substitute a twin adenine (AA) with guanine (GG). This innovative modification led to reduction in collagen indicating the precision and efficiency of our editing approach.To assess the broader implications of this intervention; we employed high-throughput multi-omics technologies. Through transcriptomics (RNA-seq) and proteomics (tandem mass spectrometry), we aimed to evaluate our gene editing approach on the molecular signature. Notably, our investigation revealed that the reduction in Col1a1 expression influencing in PI3K-Akt signalling pathway which is linked with various organ fibrosis such as liver, kidney, lung and heart. This suggests the specificity and efficacy of our editing approach for possible antifibrosis goals. By targeting CCAAT promoter region, our method presents a versatile alternative to the CRISPR interference (CRISPRi) system. This addresses its key drawbacks, such as reversible and transient outcomes, which are suboptimal especially in the context of therapeutic applications where permanent cure is desired. Col1a1 promoter editing may thus represent a permanent and optimal therapeutic strategy across diverse fibrotic disorders.