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Brassica napus, a crop that greatly contributes to global agricultural economies, originated approximately 7,500 years ago through interspecific hybridization between Brassica rapa and Brassica oleracea, resulting in a complex genome with A and C sub-genomes. Flowering time is a crucial phenological trait that directly affects the yield potential and economic sustainability of B. napus. In Arabidopsis thaliana, the FLOWERING LOCUS T (FT) gene plays a pivotal role in flowering regulation. It encodes florigen, a mobile signal that is synthesized in leaves and moves to the apical meristem to trigger the transition from vegetative to reproductive growth. Despite the well-characterized functions and regulatory mechanisms of FT in A. thaliana, knowledge about the FT homologues in B. napus, a species belonging to the same family as A. thaliana, is relatively limited. To address this, we identified and characterized eight FT homologues in B. napus. Among these genes, four BnFT genes show synteny among A. thaliana, Schrenkiella parvula and B. napus, two out of the three NEW SISTER OF FT AND TSF (NFT) genes that possess synteny only among the B. napus and S. parvula were predicted to be pseudogenes, and a C-GENOME SISTER OF FT AND TSF (CFT) gene is unique to B. napus and the C-genome parent, B. oleracea, but absent from S. parvula and A. thaliana. In inter-species complementation experiments, six functional FT homologues all exhibited weaker complementation ability compared to FT, but when expressed directly in the shoot apical meristem, four BnFTs exhibited a similar and strong florigen function as FT, whereas BnNFT.A7 and BnCFT.C4 still showed reduced florigen activity. Furthermore, four BnFT genes contain conserved sequence Block A, Block C and Block E in their flanking sequences, which are known to be involved in the transcriptional regulation of FT. The four BnFT genes are expressed predominantly in long-day conditions, showing an upward parabola of diurnal expression wi

Background: Degenerative cervical myelopathy (DCM) is the most common cause of spinal cord dysfunction in adults and shows growing prevalence due to demographic trends. Nevertheless, the surgical decision-making is still challenging due to the lack of sufficiently reliable predictors of disease progression and surgical outcome. Recently, the compensatory potential of reorganization processes within the cerebrospinal motor network has been discussed. Somatosensory impairment as an early but often overlooked symptom of DCM, however, was not in the main focus of research so far. In this thesis, the functional adaptation of the somatosensory system in DCM patients was investigated using blood oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI). Materials and Methods: 18 right-handed participants, including 9 DCM patients (age 56 ± 12 years, 7 male) and 9 age- and gender-matched healthy control subjects (age 57 ± 12 years, 7 male) underwent a block design fMRI (3 Tesla) session. The fMRI procedure consisted of alternating, passive somatosensory stimulation of the subjects' dorsal hands and feet using a wooden stick with a felt tip. By means of MATLAB® 2019a and the SPM12 software package, a region of interest (ROI)-wise analysis of BOLD-response was performed at the group level using a three-way analysis of variance (ANOVA), including the factors “group” (patients/controls), “limb” (hand/foot) and “side” (left/right). Functional hemispheric lateralization was assessed by calculating lateralization indices for individual ROIs. Furthermore, a comprehensive clinical assessment including clinical tests and scores, neurological examination, and somatosensory evoked potentials (SSEPs) of the tibial nerve was performed. An SPM multiple regression analysis was performed to determine correlations between BOLD signal change and clinical impairment. Results: Both, patients and controls featured a strongly left-lateralized cortical response in S1 and S2 (

The human gut microbiota harbors untapped potential for biotechnological applications. Within the phylum of Bacteroidota, Phocaeicola vulgatus stands out as a promising candidate for sustainable production of key platform chemicals like succinate. However, genetic engineering of Phocaeicola sp. remains challenging due to its intricate molecular landscape. This study lays the groundwork for manipulating the central carbon pathways in Phocaeicola vulgatus, offering insights into overcoming genetic hurdles for increased succinate yields.

The mechanistic target of Rapamycin (mTOR) serves as the central regulator of cell growth and metabolism, operating through two distinct complexes, mTORC1 and mTORC2. Abnormal mTORC1 activity is a common feature in various diseases including cancer, metabolic disorders, neurodegenerative conditions, as well as ageing. Therefore, it is crucial to understand the regulatory mechanisms of mTORC1 activity. In this work, I focused on elucidating the regulation of mTORC1 by amino acids as well as the small compound Rapamycin in complex with FKBPs. Amino acids play a dominant role in the regulation of mTORC1 activity and recent discoveries revealed an amino acid sensing machinery centered around the Rag GTPases. Although mammalian cells express four different Rags, RRAGA-D, the Rag paralogs were considered functionally redundant until recently. Furthermore, Rapamycin, a naturally-occuring mTOR inhibitor, selectively targets mTORC1 by forming a tertiary complex with the small immunophilin FKBP12. However, potential mTOR-independent effects of Rapamycin prompted further investigation. In the context of my PhD work, I demonstrated that there are indeed functional differences between RagA and RagB as well as RagC and RagD. Whereas RagA/B determines the response to amino acid depletion, RagC/D determines substrate specificity downstream of mTORC1. Moreover, I showed that Rapamycin selectively inhibits mTORC1 and that all observed effects can be attributed to mTOR inhibition. My data indicate that Rapamycin-treatment minimally alters the interactome of FKBPs, confirming its specificity towards mTOR. Additionally, I discovered a novel FKBP-interacting protein called PRR14L which may function as a transcriptional regulator with implications in cancer and vesicle exocytosis. Collectively, my findings offer valuable insights into the regulation of mTORC1 by amino acids and the specificity of Rapamycin towards mTOR inhibition. A comprehensive understanding of mTOR regulation holds imm

One of the unique characteristics of meiosis is the formation of meiotic crossovers (CO) between homologous chromosomes that leads to the reciprocal exchange of DNA and eventually diverse haploid gametes. Meiotic CO leads to generation of genetic variation and therefore creates diversity at the level of the individual organism and population. Research and knowledge on the fundamental mechanisms of meiotic CO could ultimately expedite the improvement of crop plant varieties and animal breeds. As has been reported previously in many types of organisms, the class I CO pathway is usually responsible for the majority of CO events, while the class II CO pathway is the minor CO pathway. The genetic knock-out of some factors that facilitate non-crossover (NCO) repair have been found to lead to increased activity of the class II CO pathway. Information on the regulation of meiotic CO for both class I CO and NCO are previously extensively described in the model plant, A. thaliana. However, there is a lack of information available in other dicot plants especially those used as a food crops. Therefore, this thesis described the generation of mutants in the class I CO (SlMLH1 and SlZIP4) and NCO repair (SlRECQ4) genes, the role of those genes and their products in promoting and regulating meiotic CO, and also the importance of those genes for the fertility in tomato. CRISPR/Cas9 mutagenesis was applied to produce three different mutants in the dwarf tomato variety, Micro-Tom. Slmlh1 and Slzip4 mutants are associated with reduced activity of the class I CO pathway, while the Slrecq4 mutant is a NCO repair gene. All three mutants exhibited a significant reduction in plant fertility. The average percentage of viable pollen is 1.49%, 13.4% and 37.3% for Slzip4, Slmlh1 and Slrecq4 mutants respectively, when compared to wildtype Micro-Tom (97.6%). This showed that both class I CO genes and NCO genes are essential to ensure tomato fertility probably due to their important functions in

Seit der Erfindung des Mikroskops haben mikroskopische Bilder zu neuen Erkenntnissen in der biomedizinischen Forschung geführt. Moderne Mikroskope sind in der Lage große Bilddatensätze von zunehmender Komplexität zu erzeugen, was eine manuelle Analyse ineffizient, wenn nicht gar undurchführbar macht. In dieser Arbeit stelle ich zwei neue Methoden für die automatische Bildanalyse von Mikroskopiedaten vor. 1. Die Fourier-Ringkorrelations-basierte Qualitätsschätzung (FRC-QE), ist eine neue Metrik für die automatisierte Bildqualitätsschätzung von 3D-Fluoreszenzmikroskopieaufnahmen, hier getestet am Beispiel von menschlichen Hirnorganoiden. FRC-QE automatisiert die Qualitätskontrolle, eine Aufgabe, die häufig manuell durchgeführt wird und somit einen Engpass bei der Skalierung bildbasierter Experimente auf tausend oder mehr Bilder darstellt. Die Methode kann die Clearing-Effizienz über experimentelle Replikate und Protokolle quantifizieren. Sie ist auf verschiedene Mikroskopiemodelle übertragbar und lässt sich effizient auf Tausende von Bildern skalieren. 2. Der von mir entwickelte "WormObserver" ermöglicht Langzeitaufnahmen, verarbeitet automatisch die aufgenommenen Videos und erleichtert die Datenintegration über Tausende von Individuen hinweg, um Verhaltensmuster zu entschlüsseln. Darauf aufbauend, habe ich mich auf ein Beispiel für die Plastizität des Nervensystems konzentriert: Die Verhaltenstrajektorie des "C. elegans Dauer Exits". Um den Entscheidungsmechanismus beim Verlassen des Dauer Larvenstadiums zu charakterisieren, habe ich Zeitrafferdaten von Larvenpopulationen in verschiedenen Umgebungen erfasst, analysiert und wichtige Entscheidungspunkte identifiziert. Indem ich die Verhaltensanpassung mit der Genexpression kontextualisiert habe, konnte ich neue Erkenntnisse gewinnen, wie ein sich entwickelndes Nervensystem externe Stimuli robust integrieren und das Verhalten des Organismus an neue Umgebungen anpassen kann., Since the invention of the microscope, microscopy images have generated new insights in biomedical research. While in the past these images were used for illustrative purposes, state-of-the-art microscopy images provide quantitative measurements. Moreover, modern microscopes are capable of autonomously producing large image datasets of increasing complexity, rendering manual analysis inefficient if not infeasible. Thus, extracting biologically relevant information from these datasets requires computational analysis using appropriate algorithms and software. While some analysis methods generalize to different microscope set-ups and types of images, others need to be well tailored to a particular problem. In this work, I present two new methods for automated image analysis of microscopy data. First, Fourier ring correlation-based quality estimation (FRC-QE) is a new metric for automated image quality estimation of 3D fluorescence microscopy acquisitions. I benchmarked the method in the context of evaluating clearing efficiency in human brain organoids. FRC-QE automates image quality control, a task that is often performed manually and thereby represents a bottleneck when scaling image-based experiments to thousand or more images. The method can estimate clearing efficiency across experimental replicates and clearing protocols. It generalizes to different microscopy modalities and efficiently scales to thousands of images. Second, I have developed a new method for behavioral imaging of C. elegans larvae. The “WormObserver” enables long-term imaging (>12h, >80k images/experiment), automatically processes the acquired videos a

In den letzten Jahren gab es Fortschritte im Verständnis der Gefäßbildung bei Ereignissen wie Keimen, Lumenbildung und Gefäßstabilisierung. Nach der Bildung eines primitiven Plexus ist die Gefäßoptimierung und hierarchische Umwandlung der morphologischen Gefäße in einen reifen Plexus durch vaskuläres "Pruning" wenig verstanden. Unterschiedliche Blutflussprofile in nebeneinander angeordneten Gefäßen können Asymmetrien in der Scherspannung verursachen, was die Zellmigration in Bereichen mit höherem Fluss fördert und die Destabilisierung von Segmenten mit geringem Fluss induziert. Diese Studie basiert auf der Hypothese, dass funktionelle Gefäßnetzwerke und Umbildung durch flussgesteuerte Endothelzellmigration ausgelöst werden. Wie die zelluläre Erfassung physikalischer Kräfte integriert ist, um Informationen zu übertragen und das Verhalten von Zellen zu modifizieren, ist noch unbekannt. Die Studie untersucht die Regulation und Koordination durch RhoGTPase-Signale während der kollektiven endothelialen Migration aufgrund von Flüssigkeitskräften. RhoGTPasen ermöglichen die räumlich-zeitliche Koordination, langfristige Anpassung an den Fluss und morphologische Umgestaltung. Beeinträchtigungen von RhoGTPasen zeigen Defekte bei Zellmigration und kollektiver Koordination in Umgebungen mit freiem Rand und strömungsgetriebener Migration. Die Studie erläutert den Einfluss der RhoGTPase-Regulation der Verbindungsdynamik in Verbindung mit der Aktinorganisation, die für die mechanische Kopplung und endotheliale Reaktionsfähigkeit erforderlich ist. Insgesamt betont die Studie die Relevanz der räumlich-zeitlichen RhoGTPase-Kontrolle und der Aufrechterhaltung der mechanischen Kopplung zwischen Strömung und Migration für die kollektive Koordination als Reaktion auf hämodynamische Kräfte., In recent years, there have been significant advances in understanding how new vessels form during events like sprouting, lumen formation, and vessel stabilization. Yet, after the formation of a basic network, the crucial step of rearranging vessels into a mature structure, known as vascular pruning, needs further investigation. It's suggested that different blood flow profiles in nearby vessels create imbalances in shear stress, leading to cell migration toward higher flow regions, destabilizing low-flow segments, and causing the collapse of redundant segments. This study, in line with existing literature, proposes that functional vascular networks and remodeling result from the flow-driven migration of endothelial cells. However, how cells precisely sense physical forces to regulate their behavior and coordinate migration in response to flow remains unknown. I explore the regulation and coordination of Rho GTPases during collective endothelial migration under fluid forces. Rho GTPases' coordination allows long-term adaptation to flow and morphological remodeling. Impairments in Rho GTPases reveal defects in cell migration and collective coordination during free-edge and flow-driven migration. Finally, I explain how Rho GTPases' regulation influences junctional dynamics and actin organization, crucial for mechanical coupling and endothelial responsiveness to flow. Overall, this study emphasizes the importance of controlling Rho GTPases over time and maintaining mechanical coupling between flow and migration for collective coordination in response to fluid forces.

Über die letzten Jahre haben sich Einzelzelldaten als Trend in der Bioinformatik etabliert, was zu umfangreichen Datensätzen führte. Die Entwicklung von Analysemethoden für solche Daten hat jedoch nicht mit deren Produktion Schritt gehalten. Diese Arbeit befasst sich mit einigen Problemen, die beim Analysieren von Einzelzelldaten auftreten. Das zweite Kapitel enthält eine Analyse von scRNA-seq-Daten von Darmkrebspatienten und Organoiden. Es werden Entwicklungstrajektorien des Darms beschrieben. Anschließend werden Signalgradienten dieser Achsen charakterisiert, insbesondere MAPK- und WNT-Signale. Weiter wird gezeigt, wie RNA velocity basierend auf metabolischen labeling ähnliche Trajektorien in Organoiden aufzeigen kann. Schließlich werden Auswirkungen von Signalwegenhemmung auf die Entwicklungstrajektorien im Detail beschrieben. Das dritte Kapitel bietet einen noch nie dagewesenen Einblick in frühe Stadien von COVID-19 in der Lunge. Verwendete scRNA-seq Daten stammen aus Lungengewebeproben etablierter Hamstermodelle, die mehrere Spezies, SARS-CoV-2-Dosen und Zeitpunkte umfassen, und die ich mit entsprechenden Daten von menschlichen Patienten vergleiche. Für die Analyse zentraler Zelltypen, die den unterschiedlichen Krankheitsverläufen zugrunde liegen, wende ich post-hoc Interpretation auf sonst unzugängliche latent spaces von diffusionmaps an, die neue Einblicke in die zelluläre Pathogenese von COVID-19 bieten. Im letzten Kapitel stelle ich scperturb vor, die größte Sammlung von perturbierten Einzelzelldaten. Ich zeige, wie E-Statistik verwendet werden kann, um solche Daten auf statistisch fundierte Weise zu analysieren. Verzerrungen werden mittels eines neuen Term zur Korrektur der E-Distanz beseitigt. Anschließlich untersuche ich Robustheit der E-Statistiken für einige Analyseszenarien, wie die COVID-19 Daten aus vorherigen Kapitel. Schließlich leite ich Richtlinien für die experimentelle Planung von Einzelzell-Perturbationsstudien mit robuster Statistik ab., In recent years, single-cell data has emerged as leading trend in bioinformatics, resulting in the generation of substantial datasets. However, development of analysis methods for single-cell data has not kept pace with its production, presenting challenges for analysts. This thesis addresses some of the most pressing issues encountered during the analysis of single-cell data. After a short introductory chapter, the second chapter deals with the problem of arranging single-cell transcriptomes based on biological trajectories, and how these correlate with signaling pathways, specifically those relevant as targets for potential treatments. This thesis demonstrates how RNA velocity based on metabolic labeling can recover similar trajectories in organoids, identifying WNT and MAPK as underlying signaling pathways for development in normal and colon cancer organoids. The third chapter provides an unprecedented view into early stages of COVID-19 in the lungs. For the analysis of key cell types underlying divergent COVID-19 outcomes I apply post-hoc interpretation methods to otherwise inaccessible latent spaces of diffusion maps, revealing new insights into the cellular pathogenesis of COVID-19. Used scRNA-seq data is derived from lung tissue samples of established hamster models, encompassing multiple species, varying SARS-CoV-2 doses, and time points, which I then compare to data from human patients. In the fourth chapter, I present scperturb, the largest collection of single-cell perturbation data. I show how E-statistics can be used to analyze such data in a statistically sound way. After introducing a new bias-correction term to the calculation of E-distances, I investigate the robustness of resulting E-statistics for various analysis scenarios, such as the COVID-19 data from the previous chapter. Finally, I derive guidelines for the experimental design of single-cell perturbation studies such that robust statistics can be achieved.

Chapter I of this work addressed the piggyBac (PB) transposon system, a non-viral genome engineering tool that is capable of efficiently performing stable integration of DNA sequences into a target cells genome and has already been used in clinical trials. However, the PB transposase has the problematic property of preferentially integrating transposons near transcriptional start sites (TSSs). This increases the likelihood of causing genotoxic effects, limiting its potential use as a tool in clinical applications. It has been shown in the past that the PB transposase shows physical interactions with BET proteins (e.g. BRD4) through Co-IP experiments. Representatives of these proteins are part of the transcriptional activation complex and are abundant at TSSs. Accordingly, it was previously proposed that this interaction is the underlying cause for the biased integration preference. For the first chapter of this thesis, the goal was to disrupt this interaction potentially modifying said integration preference. A secondary structure hypothesized to be mainly responsible for said interaction was extensively mutated resulting in several PB variants that were analyzed for their interaction capacity through a series of Co-IP experiments with BRD4. In total, seven substitutions were identified (E380F, V390K, T392Y, M394R, K407C, K407Q, and K407V) which exhibited reduced interaction capacity with BRD4. Each of the aforementioned mutants were used to generate integration libraries and, through NGS, it was determined if the integration preferences of the respective mutants had changed. In the immediate range 200 base pairs up- and downstream from known TSSs all mutants used exhibited a reduced integration bias. At a wider observation window 3 kbp up- and downstream from TSSs, further mutants with the substitutions M394R, T392Y and V390K showed a reduction in integration frequency of 17.3%, 1.5% and 5.4%, respectively, compared to the wildtype. Of particular note was the M394R

The mTOR (mechanistic target of Rapamycin) signaling pathways are at the center of cellular physiology. As such, their dysregulation is a common characteristic of many conditions, such as cancer, neurodegeneration and aging. mTORC1 (mTOR complex 1) regulates cellular growth by controlling protein synthesis, autophagy and lysosomal biogenesis. On the other hand, mTORC2 (mTOR complex 2) is involved in the regulation of cell survival and proliferation. mTOR responds to upstream cues that control its function via the activity of key GTPases that regulate its localization, and via alterations in PTMs (post-translational modifications) on several signaling components. In particular, AAs (amino acids) regulate mTORC1 via changes in its subcellular localization, promoting mTORC1 recruitment to lysosomes. However, the reasoning for the lysosomal localization of mTORC1 and whether mTORC1 is active in different subcellular locations is an open question. Here, using multiple approaches to disrupt lysosomal function, I show that mTORC1 localization to lysosomes is linked to lysosomal activity. This observation indicates that mTORC1 is found at lysosomes likely due to basal protein degradation in the lysosomal lumen and subsequent nutrient release. Unexpectedly, under AA sufficiency, delocalization of mTORC1 from lysosomes led to a complete loss of TFEB phosphorylation, with very mild effects on the canonical substrates S6K1 and 4E-BP1. PTMs primarily include phosphorylation and ubiquitination events. Although the former is well-described in the control of mTOR complexes, the latter is less well-understood. Nonetheless, work over the recent years established that ubiquitination of several components of the mTORC1 and mTORC2 pathways is also an important part of their regulation. mTOR, the catalytic component of both mTORC1 and mTORC2, is itself heavily ubiquitinated. Hence, the identification of proteins able to modulate mTOR ubiquitination is of great importance to understand mT

Myeloperoxidase (MPO), formerly termed Verdoperoxidase, is a lysosomal protein produced mostly by activated neutrophils and monocytes. It functions in response to pathogens during bacterial and fungicidal infections and contributes to pathogen clearance both within the phagosome and upon secretion. Clinical studies identified both MPO deficiency and over presentation in a wide range of diseases, including cardiovascular diseases. However, patients lacking MPO were found healthy besides minor bacterial infection [111]. Hence, the demand to MPO functional studies apart from its role in the host defense has risen. Previous study had described the accumulation of MPO in the plasma upon secretion, as well as the afterwards uptake by endothelial cells (ECs) [123]. This finding opens up a promising aspect to dissect the vascular remodelling potential of MPO. In this dissertation, EC remodelling upon MPO transcytosis was studied systematically by hiring RNA-seq and ATAC-seq techniques. Both transcriptional regulations and chromatin structural changes were identified in the MPO treated cells. For the first time, MPO association with EC chromatin was identified and verified via using multiple molecular biology methods, and its occupying regions realised via ChIP-seq. Moreover, by treating ECs with inactivated MPO, the EC remodelling was recapitalised, indicating that the effect is independent of MPO enzymatic activity. With the help of mass-spectrometry analysis, potential MPO interacting proteins were identified, including ILF3, which previously was described in the context of angiogenesis. A significant loss of cytoplasmic and gain of nuclear ILF3 was detected. In the end, genes regulated by MPO via ILF3, and MPO-ILF3 synergistical effect on RNA stabilisation were studied via ILF3 knock down in ECs.

Dissertation über die dynamische Darstellung und Analyse der regulären Oberlidanatomie mittels Hochfrequenz-Ultraschall (50 MHz, bezeichnet als "Ultraschall Biomikroskopie") und anschließender Vergleich der erhobenen Parameter zwischen unterschiedlichen Bevölkerungskohorten und Ethnien.

Tuberculosis (TB) is a communicable disease that is responsible for serious health problems around the globe. TB is caused by Mycobacterium tuberculosis (Mtb) and can be effectively treated with an extensive treatment regimen. However, incompetent application of this regimen is the cause for the prevalent emergence of multidrug-resistant Mtb strains, which are no longer sensitive to first-line antibiotics. Furthermore, the current drug development pipeline for novel antibiotics targeting Mtb, while promising, still lacks the diversity of targets needed to introduce a variety of novel mechanisms of action. This circumstance is partly caused by the robustness of Mtb against bioactive compounds. The major reason for this is the unique, impermeable cell wall containing additional layers of mycolic acids. An alternative approach to conventional antibiotics is the adjunct application of anti-virulence drugs. These compounds target specific mycobacterial components or substrates (virulence factors) that are essential for the host cell infection by Mtb. As a result, the pathogen’s efficacy in infection is compromised and it can potentially be more easily cleared by the host. The most notable virulence factor of Mtb is the type VII secretion system ESX-1 and its substrates EsxA and EsxB. In this thesis, 60,000 compounds were tested in a medium-throughput screening for anti-Mtb activity. This screening utilized different combinatorial applications of the screening platform by combining testing for growth inhibitory activity and host cell survival (cytoprotection) during Mtb infection. This includes both conventional antibiotically active compounds as well as anti virulence compounds, which show no antibiotic activity but still exhibit cytoprotective activity against Mtb. Investigation of the genome of Mtb mutants resistant to the antibiotics revealed putative modes of action associated with cell wall biogenesis, amino acid production and the respiratory chain. One notable com

The ER (Endoplasmic reticulum) of mammalian cells is an important intracellular calcium storage and the place where lipids are generated. Furthermore, the maturation and folding of transmembrane proteins as well as secretory proteins, which constitute around one third of all synthesized proteins within the cell, takes place within the ER. The Sec61 translocon constitutes the main channel for protein translocation across the ER membrane. Upon translocation into the ER lumen, proteins associate with chaperones that assist their substrate proteins in reaching their final conformation. Under conditions of nutrient deficiency, hypoxia or disturbed calcium homeostasis, protein folding and maturation can be impaired. ER-stress develops as a consequence of the increased burden of ER luminal unfolded or misfolded proteins. The cellular response to ER-stress is called the Unfolded Protein Response (UPR). During the UPR, general protein translation is downregulated and transcription and translation of chaperones is upregulated in order to maintain ER luminal proteostasis. Terminally misfolded proteins are retrotranslocated to the cytosol for proteasomal degradation. If these mechanisms are insufficient to restore ER homeostasis, apoptosis is initiated by the cell. An important ER-resident chaperone is the Hsp70 family member BiP/GRP78. HSP70 chaperones bind to hydrophobic domains of unfolded or misfolded proteins, promote folding and prevent aggregation of their substrates. In order to promote folding of substrate proteins, an interaction of Hsp70 chaperones with Hsp40 co-chaperones is necessary. Hsp40 co-chaperones possess a conserved J-domain. The J-domain binds to Hsp70 chaperones and stimulates the ATPase activity of the Hsp70 chaperones. Currently, eight members of the Hsp40 family (ERdj1 to ERdj8) are known to be localized within the ER of mammalian cells. ERdj1 to ERdj7 have been shown to bind to BiP and stimulate the ATPase activity of BiP. As ERdj8 has only recently b

Nitrogen is a key component to life. The bioavailability of nitrogen (N), in the form of ammonium (NH4+) and nitrate (NO3-), in soil ecosystem varies strongly and it often is a growth limiting factor for plants. Therefore, plants have evolved an arsenal of nitrogen signalling and uptake mechanisms to forage nitrogen from their environment. Once taken up by the root, nitrogen amongst other nutrients, is transported to other organs of the plant through the central vascular cylinder. To prevent the leakage of nutrients out of the vasculature, it is surrounded by a barrier tissue: the endodermis. We have only recently started to understand how the reinforcement of endodermal cell walls by lignin and suberin is coordinated with the plant nutrient status and especially the role of nitrogen remained unresolved. Here we found that, in Arabidopsis thaliana, nitrogen starvation leads to an increase of endodermal suberization and that endodermal nitrogen signalling and suberization are required to facilitate microbe-dependent growth promotion after nitrogen fertilizer application to agricultural soil. Tissue-specific post-transcriptional analysis of nitrogen starved plants revealed, that in the mature primary root, the high affinity nitrate transporter NRT2.1 and the high affinity ammonium transporter AMT1.5 are upregulated in the outer cortex layer above the suberized endodermis. Similarly, coumarin biosynthesis appears to be distinctively regulated in the mature root under nitrogen starvation. The scopoletin synthesizing gene F6’H1 and the fraxetin synthesizing gene S8H are upregulated in the mature cortex, while the sideretin synthesizing gene CYP82C4 is downregulated. Moreover, the nitrate transceptor NPF6.3 and the transcription factor NIGT1.3 are upregulated specifically in the endodermis of nitrogen starved plants. Manipulation of nitrogen signalling by overexpressing the nitrate responsive transcription factor NLP7, in the endodermis specifically, leads to higher sensi

Aging organisms show a pervasive decline in cellular function, with important implications for healthspan and lifespan. Aging associated impairment of gene transcription from Pol II are believed to underlie a large part of this phenotype. Elongation is a particularly important step of transcription since it regulates a lot of cotranscriptional processes, however the exact molecular mechanisms involved in its changes with aging remain unclear. In this thesis, we report the effects of aging in various transcriptional processes across different eukaryotes. We use a combination of previously published and newly-generated next generation sequencing data to understand the mechanisms of aging associated changes in Pol II speed and fidelity We profiled and analyzed genome-wide, aging-related changes in transcriptional processes across different organisms: nematode worms, fruit flies, mice, rats and humans. The average transcriptional elongation speed (Pol II speed) increased with age in all five species. Along with these changes in elongation speed, we observed changes in co-transcriptional processes that are partially regulated by elongation, including splicing alterations, the formation of more circular RNAs and loss of transcriptional fidelity. Two lifespan-extending interventions, dietary restriction and lowered insulin/Igf signaling, both partially reversed some of these aging-related changes. Remarkably, genetic variants of Pol II that reduced its speed in worms and flies increased their lifespan, which proves the importance of elongation rate for organismal longevity. Similarly, reducing Pol II speed by overexpressing histone components, to counter age-associated changes in nucleosome positioning, also extended lifespan in flies and the division potential of human cells. Our findings uncover fundamental molecular mechanisms underlying animal aging and lifespan-extending interventions, and point to possible preventative measures. Furthermore, we developed a new comput

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

Einzelzellomics bieten unvoreingenommene Einblicke in Transkriptionsprogramme und Genom-Zugänglichkeiten auf zellulärer Ebene, auch wenn der zelluläre Kontext verloren geht. Wir haben einen virtuellen Multi-omic Embryo der Drosophila melanogaster erstellt, basierend auf den Datentypen RNA (Transkriptom) und ATAC (Zugänglichkeit der DNA), welche gleichzeitig auf Einzelzell Ebene erhoben wurden. Mithilfe des Tools novoSpaRc, welches den räumlichen Ursprung der Zellen rekonstruiert, konnte ein regulatorischen Bauplan erstellt werden, der die Genexpression und die Zugänglichkeit von Enhancern widerspiegelt. Diese Ressource hilft beim Verständnis der regulatorischen Dynamik in der Entwicklung. Bei der Untersuchung von ATAC-Peaks konnten wir Überschneidungen zwischen den Mustern der Chromatin Zugänglichkeit und der Aktivität unabhängiger getesteter Enhancer feststellen, was die Bedeutung der Zugänglichkeit unterstreicht. Die nicht-negative Matrixfaktorisierung identifizierte Archetypen der Genexpression und der Chromatin-Zugänglichkeit. Archetypen, die möglicherweise durch Transkriptionsfaktoren (TFs) reguliert werden, wurden einer Motiv-Anreicherungsanalyse für Archetyp-assoziierte CRMs unterzogen. Ein Ansatz zur Vorhersage von Enhancern, ordnete die Enhancer den Genen auf der Grundlage partieller Ähnlichkeit der Muster zu. Zusammenfassend dient unser multimodaler virtueller Embryo als Ressource und präsentiert zum ersten Mal räumliche Chromatin-Zugänglichkeiten für genomische Regionen für einen ganzen Organismus. Die Ergebnisse geben Aufschluss über die Prinzipien der Genregulation und zeigen den regulatorischen Einfluss von Transkriptionsfaktoren auf den Chromatinzustand von Enhancern., Single-cell-omics techniques provide unbiased insights into transcriptional programs and genomic accessibility patterns at the cellular level despite sacrificing spatial information. We created a multi-omic virtual Drosophila melanogaster stage 6 embryo by simultaneously assessing genome accessibility and transcriptional states in individual cells. Using novoSpaRc, a spatial mapping tool, we accurately reconstructed the spatial origin of cells, yielding a regulatory blueprint reflecting gene expression and enhancer accessibilities. This resource aids in understanding developmental regulatory dynamics. Examining ATAC-peaks, we observed overlapping chromatin accessibility patterns with the activity of independently testes enhancers, emphasizing accessibility's importance. Non-negative matrix factorization identified archetypes in gene expression and chromatin accessibility. Accessibility archetypes, potentially regulated by transcription factors (TFs), were subjected to motif enrichment analysis for archetype-associated CRMs. An enhancer prediction approach, utilizing a generalized linear model, assigned enhancers to genes based on partial pattern similarity. In summary our multi-modal virtual embryo serves as a resource and presents for the first time single-cell chromatin accessibilities for genomic regions reconstructed in space for a whole organism in a single developmental stage. The results shed light on gene regulatory principles, highlighting the regulatory impact of TFs on chromatin states of enhancers.

mTOR signaling is a crucial pathway involved in numerous biological and disease processes. Hyperactive mTOR signaling has been linked to several diseases including cancer, diabetes, metabolic and neurological syndromes as well as the aging process. Inhibition of mTOR signaling is an important experimental and therapeutic tool to study mTOR biology and treat medical conditions involving hyperactive mTOR signaling. Several modes of mTOR inhibition exist including pharmacological inhibitors such as rapamycin and Torin1 as well as other interventions like amino acid (AA) starvation and genetic deletion of mTOR regulators. Despite the wide use of these interventions, a comprehensive understanding of the differential effects elicited by different modes of mTOR inhibition on cellular physiology is still lacking. Moreover, researchers outside the mTOR field tend to use these interventions interchangeably to probe a variety of biological questions, which can lead to incomplete or erroneous findings. Here, to comprehensively study the differences between different modes of mTOR inhibition, we measured the effects that four interventions (rapamycin, Torin1, AA starvation and loss of RagA/B GTPases) elicited on the cellular proteome via a combination of proteomics and systems biology approaches. Our findings reveal that different modes of mTOR inhibition, while sharing a common set of protein targets, lead to unique proteomic signatures. Specifically, we find that Torin1 regulates mRNA splicing and lipogenesis-related proteins to a much greater extent than rapamycin. Additionally, we show how modeling AA starvation with Torin1 treatment may lead to inaccurate results, especially when studying AA-induced transcriptional responses. Moreover, we show that, while rapamycin, Torin1 and AA starvation are all associated with an increase in oxidative-related stress proteins, differential regulation of mitochondrial proteins suggests that different modes of mTOR inhibition induce ROS pr

Plants and animals have similarities in their immune system. A new protein family was reported to be conserved across seed plants that structurally resembles animal mixed lineage kinase domain-like (MLKL), a mediator of necroptotic cell death. The Arabidopsis MLKLs (AtMLKLs) play a role in disease resistance mediated by TIR-type nucleotide binding–leucine-rich repeats (NLRs). Cryo-EM structures of Arabidopsis MLKLs (AtMLKLs) reveal a tetrameric configuration in contrast with the monomeric structure of animal MLKL. To understand the biological meaning of the AtMLKL tetramer, I performed a structure-guided study. Together with a newly established disease resistance assay in N. benthamiana, the data imply that the AtMLKL tetramers represent an auto-repressed conformation of plant MLKLs and the exposure of the N-terminal HeLo-domain from the tetramer is an important activation step, which could be mediated by a phosphorylation in the activation loop of the pseudokinase domain. To elucidate the genetic relationship between helper NLRs and plant MLKLs, the disease resistance activities of AtMLKL1 variants were examined in helper NLR null background and eds1 pad4 sag101 null background. The plant MLKL-mediated immunity was partially retained in the helperless mutant background but no longer retained in the eds1 pad4 sag101 mutant in N. benthamiana. It was found that the AtMLKL1 HeLo domain elicits Ca2+ influx in human cell line and in N. benthamiana. These data demonstrate that plant and animal MLKLs commonly utilize the N-terminal HeLo-domain as a signaling domain for immune responses. Furthermore, to study the activation step, I purified N-terminal HeLo domain of AtMLKL1 and a gain-of-function variant of AtMLKL1 carrying a phosphomimetic mutation in the activation loop of the pseudokinase domain. The purified recombinant N-terminal HeLo domain of AtMLKL1 autonomously formed a higher-order oligomer with a 10 nm ring-like structure. Additionally, the p

The repair of DNA damage is a key process to maintain the integrity of the genome and prevent dysregulation resulting in diseased states, such as cancer. To ensure efficient repair, this process is carried out by dedicated DNA repair pathways. The signaling cascade orchestrated to promote DNA repair relies not only on recruitment dynamics of specific repair factors, but also on the enzymatic synthesis of several post-translational modifications (PTMs). ADP-ribosylation (ADPr) is one of the most common PTMs in DNA repair, synthesized by the poly(ADP-ribosyl)polymerases PARP1 and PARP2. The biological and clinical relevance of this PTM is underscored by the therapeutic use of four FDA-approved PARP1/2 inhibitors against cancer. However, the chemical and structural complexity of ADPr resulted in a dearth of research tools, which severely limits our ability to study it. In this thesis, we address this key issue by the development of new research tools and methodologies for the study of ADPr. We then leverage these tools to investigate the regulation and functional outcome of this PTM in the DNA damage response. In the first study, we describe the development of a phospho-guided, chemoenzymatic strategy for the preparation of ADP-ribosylated peptides. We apply these peptides to develop a repertoire of new recombinant antibodies against several forms of ADPr, including site-specific and broad-specificity antibodies against mono-ADPr. We use these antibodies to discover that mono-ADPr is prevalent upon DNA damage, and describe its dependence on the hydrolases ARH3 and PARG. In the second study, we further improve the versatility and sensitivity of our antibodies by applying affinity maturation and the SpyTag/SpyCatcher technology. We leverage these reagents to discover that, in DNA damage, ADPr forms a two-phase signaling pathway characterized by a rapid but transient poly-ADPr signal followed by a delayed, long-lived mono-ADPr signal. We find that persistent mono-ADPr on

Die dreidimensionale Organisation des Chromatins verändert sich während der Zelldifferenzierung als Reaktion auf die Umgebung und ist bei Krankheiten oftmals verändert. Das Zusammenspiel zwischen Chromatinzustand, Chromatinorganisation und Genexpression ist insbesondere bei Neuronen nach wie vor nur geringfügig erforscht. In dieser Arbeit untersuchte ich die Organisation und den Zustand des Chromatins im Zusammenhang mit der Transkription in embryonalen Stammzellen (ESCs) und dopaminergen Neuronen (DNs) der Maus. Dazu habe ich die Organisation des Chromatins mittels Genome Architecture Mapping (GAM) bestimmt und zelltypspezifische Genexpressionsprofile zur Klassifizierungen von Promotoren, Enhancern und Super-Enhancern (SEs) erzeugt. Anschließend habe ich diese linearen Chromatinprofile mit den verschiedenen Stufen der Chromatinorganisation kombiniert und konnte so Unterschiede zwischen den 3D-Genomstrukturen von ESCs und DNs aufzeigen. Zudem konnte ich verstärkt Dreifach-Wechselwirkungen zwischen zelltypspezifischen SEs und/oder exprimierten Genen nachweisen, die bei DNs besonders oft neuronale Signalgene darstellen und oftmals bei neurologischen Störungen betroffen sind. Ich fand auch heraus, dass die Grenzen topologisch assoziierter Domänen (TADs) oft mit Genen zur zellulären Differenzierung zusammen fallen und zudem zelltyp-spezifische Eigenschaften aufweisen, was von Bedeutung für zukünftige funktionelle Untersuchungen solcher Grenzen sein dürfte. Schließlich konnte ich zeigen, dass Chromatinkompartimente zwischen ESCs und DNs in Abhängigkeit vom Chromatinzustands und der Chromatinexpression variieren und dass eine Gruppe transkriptionell aktiver DN Gene, die für die neuronale Aktivität wichtig sind, in B-Kompartimenten liegt. Mit diesen neuen Erkenntnissen erweitert meine Arbeit das Verständnis der Chromatinorganisation bei der Regulierung der Genexpression in Maus ESCs und DNs., The three-dimensional organization of chromatin changes during cell differentiation, in response to the environment, and is often altered in disease. The interplay between chromatin state, chromatin organization and gene expression remains poorly understood, particularly in neurons. In this work, I examined the organization and state of chromatin associated with transcription in mouse embryonic stem cells (ESCs) and dopaminergic neurons (DNs). To do this, I determined the organization of chromatin using genome architecture mapping (GAM) and generated cell type-specific gene expression profiles to classify promoters, enhancers and super-enhancers (SEs). I then combined these linear chromatin profiles with the different levels of chromatin organization and was able to show differences between the 3D genome structures of ESCs and DNs. In addition, I was able to demonstrate increased triple interactions between cell type-specific SEs and/or expressed genes, which are often neuronal signalling genes in DNs and affected in neurological disorders. I also found that the boundaries of topologically associated domains (TADs) often coincide with cellular differentiation genes and also exhibit cell type-specific properties, which may be important for future functional studies of such boundaries. Finally, I was able to show that chromatin compartments between ESCs and DNs vary depending on chromatin state and chromatin expression, and that a group of transcriptionally active DN genes important for neuronal activity are located in B compartments. With these new findings, my work expands the understanding of chromatin organization in regulating gene expression in mouse ESCs and DNs.

Echinodermata are ecologically important in marine ecosystems and play a central role in the marine food web. The aim of this study is to provide an overview on the current biodiversity of Echinodermata on the coasts of Heligoland and compare our findings to historical data of the last 150 years from. We collected samples from the rocky shore next to “Lange Anna”, the “Tiefe Rinne” and the north-east harbor of Heligoland. We identified eight different species from three classes: Asteroidea, Ophiuroidea, and Echinoidea. In Asteroidea, we identified Asterias rubens and Asterina phylactica. Within Ophiuroidea, three species were identified: Amphipholis squamata, Amphiura chiajei, and Ophiuocten affinis. In Echinoidea, we found three species: Echinocyamus pusillus, Psammechinus miliaris, and Strongylocentrotus droebachiensis. Generally, low numbers of Asteroidea and Echinoidea were found, while most individuals were Ophiuroidea. With A. phylactica we have been able to identify a new species that had not been found in Heligoland before. We suggest that rising water temperatures around Heligoland may have led to this new species that is normally found more southerly occurring in this habitat., Peer Reviewed

“Proceedings in Marine Biology” is an international journal publishing original research by graduate students on all aspects of marine biology. Subjects covered include: ecological surveys and population studies of oceanic, coastal and shore communities; physiology and experimental biology; taxonomy, morphology and life history of marine animals and plants. Papers are also published on techniques employed at sea for sampling, recording, capture and observation of marine organisms. This volume deals exlusively with the results of the research field trip of the Humboldt-Universität zu Berlin to the AWI - Biologische Anstalt Helgoland (Germany) from 26.09. - 05.10.2023, Peer Reviewed

To investigate the species diversity of polychaetes on Helgoland, we collected samples from a total of three different locations including rocky shore intertidal, the subtidal „Tiefe Rinne“ and plankton samples in the open sea. In this context, a total of 14 different species were identified, which were then compared with historical records. One species was found that had not previously been documented on Helgoland. Anthropogenic factors were examined, and future research possibilities were discussed., Peer Reviewed

Global warming, pollution and other anthropogenic influences severely affect species richness and alter community structure and composition of marine environments globally. To better understand how marine invertebrate communities changed over the last centuries in the southern North Sea, we conducted a field study on and around the shores of the German island of Helgoland to observe selected marine environments. This study focuses on Anthozoa, a sessile group of cnidarians, because of their sensitivity to environmental changes such as global warming and pollution. We observed which anthozoan species are currently present in the rocky shore and the offshore benthic environment “Tiefe Rinne” of Helgoland and how this compares to previous estimates (37-87 years ago). The two localities observed are inhabited by different species, except one species (Sagartiogeton laceratus) that was found in both environments. Our results show that the community composition of anthozoans changed in both environments over time. While in the „Tiefe Rinne“ the species richness remained the same, the species richness in the rocky shore decreased. Our results could be explained by the displacement of certain anthozoan species due to anthropogenic impacts and the introduction of neobiotic taxa through increased global shipping. However, sampling was only conducted for a short period of time, and the possibility of misidentifications cannot be excluded. Further sampling and the addition of molecular data to improve species identification, would greatly enhance this study., Peer Reviewed

The subphylum Crustacea is characterized by a huge range of morphological, developmental, and adaptive diversity across virtually all marine, freshwater, and various terrestrial habitats. They are very abundant on North Sea shores, including on the intertidal rocky mud flats of the German archipelago of Heligoland. We collected a total of 32 species on two distinct localities in proximation to the main island: the rocky mud flats on the North tip and the “Tiefe Rinne” southwest of the main island. We compared our survey with different datasets for crustacean macrofaunae on Heligoland from different time periods (1850-1950, 1984, 1950-2004, 2022) and found 12 species that were not documented in these datasets but are known for the North Sea region., Peer Reviewed

During a comprehensive study of the biodiversity of marine invertebrates on the island of Heligoland in September 2023, I identified 14 species of tunicates. I observed that some of these species occur only in specific locations on the island during this period and This agrees well with the results of previous studies on the distribution of species in different locations on the island. I suggest, that Molgula manhattensis is a relatively recently introduced species in this region., Peer Reviewed

Marine Gastropoda and their distribution around the island of Helgoland (North Sea) were studied at two different locations; the intertidal zone of the northeastern rocky shore and the so-called Tiefe Rinne, a depression situated on the edge of the island with an average depth of 50 m. 31 species of gastropods were identified and the findings were compared to previous data from the same locations. The most common species in the intertidal are Littorina littorea, Littorina obtusata, Littorina saxatilis, Steromphala cineraria, and Lacuna pallidula. In the intertidal, species composition and dynamics compared to previous studies remain largely unchanged. In the area of the Tiefe Rinne, the most common species are Crepidula fornicata, Rissoa parva, Candiella plebeia, and Atalodoris inconspicua, none of which were found in this habitat in a previous study., Peer Reviewed

Ziel dieser Doktorarbeit ist eine umfassende Untersuchung der Heterogenität von Mikroglia aus morphologischer, elektrophysiologischer und transkriptioneller Perspektive mit dem Schwerpunkt auf Unterschiede zwischen weißer und grauer Substanz. Im ersten Kapitel diskutiere ich die morphologische Heterogenität von Mikroglia mit dem Fokus auf Satelliten- und Parenchymale-Mikroglia. Wir führten eine eingehende Analyse mehrerer Hirnareale durch und quantifizierten die Anzahl der Satellitenmikroglia, die mit verschiedenen neuronalen Subtypen in Kontakt stehen. Wir fanden heraus, dass die Anzahl der Satellitenmikroglia stark mit der neuronalen Dichte eines bestimmten Bereichs korreliert. Im zweiten Kapitel dieser Arbeit untersuche ich die transkriptionelle Heterogenität von Mikroglia aus weißer und grauer Substanz, wobei ich die in Gliazellen neu etablierte Patch-seq-Methode anwende. Diese Methode ermöglicht es eine Kombination aus morphologischen, lektrophysiologischen und transkriptionellen Profilen einzelner Zellen zu erhalten, die es erlauben, zelluläre Unterschiede zu charakterisieren. Wir identifizieren einen zellulären Subtyp, wenn wir den Patch-seq-Datensatz mit FACS-basierter Einzelzell-RNA-seq-Datensätzen vergleichen. Dieser Subtyp gehört eindeutig zu dissoziierten Gewebeproben und ist durch die Expression von Stress-assoziierten Genen charakterisiert. Im dritten Kapitel wende ich mich der Frage zu, wie Transkripte mittels SLAM-seq nachverfolgt werden können, die während der Dissoziation des Gewebes entstehen. Das Verfahren ermöglicht es mRNA, die während der Dissoziation der Probe entsteht, metabolisch zu markieren, rechnerisch zu identifizieren und zu entfernen. Indem wir die markierten Transkripte aus dem Mikroglia “entfernen”, beobachten wir, dass ein „aktivierter Mikroglia“-Subtyp zur allgemeinen Mikroglia-Population gehört., The aim of this doctoral work is to provide a comprehensive study and overview on the topic of the heterogeneity of microglia in the normal adult mouse brain from the morphological, electrophysiological and transcriptional perspective with the focus on differences between white and grey matters. In the first Chapter, I discuss the morphological heterogeneity of microglia in the brain with the focus on two morphologically distinct classes: satellite and parenchymal microglia. We performed an in-depth analysis of multiple brain areas and quantified the number of satellite microglia which is in contact with different neuronal subtypes. We found that satellite microglia numbers are highly correlated with neuronal densities of a certain area, while showing no preferences for any of the neuronal types. In Chapter two of this work, I study transcriptional heterogeneity of microglia from white and grey matters. For this I am employing Patch-seq, which we newly established in glial cells. This method allows a combination of morphological, electrophysiological and transcriptional profiles of single cells to assess their differences. When comparing Patch-seq dataset to the previously published FACS isolated single cell RNA-seq microglia datasets, we find a subtype of cells which uniquely belongs to FACS sample and is characterized by expression of stress-associated genes. This finding points out to the fact of dissociation-related artifacts in the single cell RNA-seq data which are not present in situ. In the third chapter, I identified transcripts which are induced during the dissociation of the tissue by employing the SLAM-seq method. This procedure allows to metabolically label newly transcribed mRNA and computationally remove transcripts from the sample. By removing the labeled transcripts from the dataset of cells isolated from the hippocampus via enzymatic dissociation, we observe that an “activated microglia” subtype merges with the general microglia population.

Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain, and the conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins with a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a literal central role in ISGylation, underscoring it as an important drug target for boosting innate antiviral immunity. Depending on the type of conjugated protein and the ultimate target protein, E2 enzymes have been shown to function as monomers, dimers, or both. UbcH8 has been crystalized in both monomeric and dimeric forms, but the functional state is unclear. Here, we used a combined approach of small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy to characterize UbcH8′s oligomeric state in solution. SAXS revealed a dimeric UbcH8 structure that could be dissociated when fused with an N-terminal glutathione S-transferase molecule. NMR spectroscopy validated the presence of a concentration-dependent monomer-dimer equilibrium and suggested a backside dimerization interface. Chemical shift perturbation and peak intensity analysis further suggest dimer-induced conformational dynamics at ISG15 and E3 interfaces - providing hypotheses for the protein′s functional mechanisms. Our study highlights the power of combining NMR and SAXS techniques in providing structural information about proteins in solution.

The ubiquitin (Ub) code denotes the complex Ub architectures, including Ub chains of different length, linkage-type and linkage combinations, which enable ubiquitination to control a wide range of protein fates. Although many linkage-specific interactors have been described, how interactors are able to decode more complex architectures is not fully understood. We conducted a Ub interactor screen, in humans and yeast, using Ub chains of varying length, as well as, homotypic and heterotypic branched chains of the two most abundant linkage types – K48- and K63-linked Ub. We identified some of the first K48/K63 branch-specific Ub interactors, including histone ADP-ribosyltransferase PARP10/ARTD10, E3 ligase UBR4 and huntingtin-interacting protein HIP1. Furthermore, we revealed the importance of chain length by identifying interactors with a preference for Ub3 over Ub2 chains, including Ub-directed endoprotease DDI2, autophagy receptor CCDC50 and p97-adaptor FAF1. Crucially, we compared datasets collected using two common DUB inhibitors – Chloroacetamide and N-ethylmaleimide. This revealed inhibitor-dependent interactors, highlighting the importance of inhibitor consideration during pulldown studies. This dataset is a key resource for understanding how the Ub code is read.

Discrepancies between knockdown and knockout animal model phenotypes have long stood as a perplexing phenomenon. Several mechanisms explaining such observations have been proposed, namely the toxicity or the off-target effects of the knockdown reagents, as well as, in certain cases, genetic robustness – an organism's ability to maintain its phenotype despite genetic perturbations. In addition to these explanations, transcriptional adaptation (TA), a phenomenon defined as an event whereby a mutation in one gene leads to transcriptional upregulation or downregulation of another, adapting, gene or genes expression, has been recently proposed as an alternative explanation for the conflicting knockdown and knockout phenotype paradox. Since its discovery in 2015, TA's precise mechanism remains a subject of ongoing research. Majority of evidence suggests that mutant mRNA degradation plays a central in TA. Epigenetic remodeling is also thought to play a role, as evidenced by an increase in active histone marks at the transcription start sites of the adapting genes. Whether mRNA degradation is indeed the key player in TA remains debated. Furthermore, it is still unknown how exactly TA develops, what adapting genes it targets, and whether genomic mutations that render mutant mRNA sensitive to degradation are required for TA to occur. Throughout the experiments described in this Dissertation, I have designed an inducible TA system where TA can be triggered on demand and its effects on the cell’s transcriptome followed through time. I have demonstrated that degradation-prone transgenes, once induced and expressed, can be efficiently degraded, resulting in the protein loss-independent upregulation of adapting genes via TA. Adapting genes with higher degree of sequence similarity become upregulated faster than genes with lower degree of sequence similarity. Further functionality of this approach to study TA is limited by the leakiness of the inducible gene expression system; howe

Die vorliegende Doktorarbeit beschäftigt sich mit der Untersuchung von molekularen Systemen, die aus mehreren Chromophoren bestehen und über einen Zweiphotonen-Prozess aktiviert werden können. Die Zweiphotonen-Absorption (2PA) beschreibt die nahezu simultane Absorption zweier Photonen, deren Summe die Energie ergibt, die für den entsprechenden elektronischen Übergang nötig ist. Da für die Anregung somit zwei niederenergetische Photonen benötigt werden, kann für die 2PA Nahinfrarot-Licht (NIR-Licht) verwendet werden, welches eine geringe Phototoxizität aufweist und eine tiefe Gewebedurchdringung ermöglicht. Weiterhin wird durch die intrinsische dreidimensionale Auflösung der 2PA eine hohe Ortsauflösung der Photoaktivierung erzielt. Photolabile Schutzgruppen (PPGs) bzw. Photocages sind chemische Verbindungen, die der vorübergehenden Maskierung der biologischen Funktion eines (Makro-)Moleküls dienen. Sie können durch Licht geeigneter Wellenlängen abgespalten werden (uncaging), wodurch die Aktivität des geschützten Substrats wiederhergestellt wird. Leider weisen viele der etablierten PPGs schlechte Zweiphotonen-Eigenschaften auf. Um die 2P-Aktivität einer PPG zu erhöhen, kann sie kovalent mit einem guten Zweiphotonen-Absorber verknüpft werden, der bei Bestrahlung das Licht über einen Zweiphotonen-Prozess absorbiert und anschließend mittels Energietransfer auf die photolabile Schutzgruppe überträgt. Dies führt schließlich zur Uncaging-Reaktion. Im Zuge von Projekt I dieser Dissertation wurde eine solche molekulare Dyade für verbessertes Zweiphotonen-Uncaging bestehend aus einem Rhodamin-Fluorophor als Zweiphotonen-Absorber und einem Rotlicht-absorbierenden BODIPY als photolabile Schutzgruppe hergestellt und charakterisiert. Die Zweiphotonen-Aktivität des Fluorophors wurde mittels TPEF-Messungen (two-photon excited fluorescence) untersucht. Anschließend wurde das Rhodamin an einen 3,5-Distyryl-substituierten BODIPY-Photocage gekuppelt. Der Energietransfer innerhalb dieser

Tropical forests have always fascinated scientists due to their unique biodiversity. However, our understanding of ecological processes shaping the complexity of tropical rainforests is still relatively poor. Plant regeneration is one of the processes that remain understudied in the tropics although this is a key process defining the structure, diversity and assembly of tropical plant communities. In my dissertation, I combine experimental, observational and trait-based approaches to identify processes shaping the assembly of seedling communities and compare associations between environmental conditions and plant traits across plant life stages. By working along a steep environmental gradient in the tropical mountains of Southern Ecuador, I was able to investigate how processes of plant regeneration vary in response to biotic and abiotic factors in tropical montane forests. My dissertation comprises three complementary chapters, each addressing an individual research question. First, I studied how trait composition in plant communities varies in relation to the broad- and local-scale environmental conditions and across the plant life cycle. I measured key traits reflecting different ecological strategies of plants that correspond to three stages of the plant life cycle (i.e., adult trees, seed rain and recruiting seedlings). I worked on 81 subplots along an elevational gradient covering a large climatic gradient at three different elevations (1000, 2000 and 3000 m a.s.l.). In addition, I measured soil and light conditions at the local spatial scale within each subplot. My findings show that the trait composition of leaves, seeds and seedlings changed similarly across the elevational gradient, but that the different life stages responded differently to the local gradients in soil nutrients and light availability. Consequently, my findings highlight that trait-environment associations in plant communities differ between large and small spatial scales and across plant, Tropische Regenwälder haben Wissenschaftler aufgrund ihrer einzigartigen Artenvielfalt schon immer fasziniert. Allerdings ist unser Verständnis der oftmals komplexen ökologischen Prozesse in diesen Wäldern, wie zum Beispiel der Verjüngung von Pflanzenpopulationen und Gemeinschaften, immer noch relativ gering. Die Pflanzenverjüngung ist ein der Schlüsselprozess, der die Struktur, Vielfalt und Zusammensetzung von Pflanzengemeinschaften bestimmt. Die Pflanzenverjüngung umfasst die Abfolge von einem keimenden Samen zu einem etablierten Keimling, bis hin zu einer reproduzierenden Pflanze. Die Etablierung und das Überleben von Pflanzen wird hierbei von sogenannten Filtern, also den gegebenen Umweltbedingungen, beeinflusst. Abiotische Filter sind z.B. Temperatur oder Niederschlag, während biotische Filter sich auf die Interaktionen zwischen der Pflanzen mit anderen Organismen bezieht (z.B. Herbivorie, Befall von Pathogenen Pilzen) beziehen. Diese Filter sind entscheidend für die Zusammensetzung von Pflanzengemeinschaften und wirken auf unterschiedlichen räumlichen und zeitlichen Skalen. Bisherige Studien haben die frühen Stadien im Lebenszyklus von Pflanzen, wie die Etablierung von Keimlingen, oft nur wenig untersucht. Dabei sind es insbesondere diese frühen Stadien, die die Zusammensetzung einer Pflanzengemeinschaft über Raum und Zeit hinweg verändern können. Ein umfassendes Verständnis der Pflanzenverjüngung ist daher erforderlich, um die derzeitigen Diversitätsmuster zu verstehen und künftige Veränderungen in Ökosystemen besser vorhersagen zu können. Merkmalsbasierte Forschung ist besonders nützlich, um die Auswirkungen abiotischer und biotischer Faktoren auf die Pflanzenverjüngung und die Zusammensetzung von Artengemeinschaften zu entflechten. Funktionelle Merkmale sind die Merkmale eines Individuums, die sich auf Wachstum, Fortpflanzung und Überleben auswirken und somit indirekt die Fitness eines Individuums beeinflussen. Zum Beispiel kann die Samengröße einer Pflanze

Targeted protein degradation (TPD) has recently emerged as an exciting new drug modality. However, the strategy of developing small molecule-based protein degraders has evolved over the past two decades and has now established molecular tags that are already in clinical use, as well as chimeric molecules, PROteolysis TArgeting Chimeras (PROTACs), based mainly on ligand systems developed for the two E3 ligases CRBN and VHL. The large size of the human E3 ligase family suggests that PROTACs can be developed by targeting a large diversity of E3 ligases, some of which have restricted expression patterns with the potential to design disease- or tissue-specific degraders. Indeed, many new E3 ligands have been published recently, confirming the druggability of E3 ligases. This review summarises recent data on E3 ligases and highlights the challenges in developing these molecules into efficient PROTACs rivalling the established degrader systems.

This work investigated cyclic-di-nucleotide (CDN) riboswitches, gaining a detailed understanding of their folding during transcription and the feasibility of 19F-NMR fragment-based screening for RNA binding lead structures for riboswitch aptamers.

The lipid content of skin plays a determinant role in its barrier function with a particularly important role attributed to linoleic acid and its derivatives. Here we explored the consequences of interfering with the soluble epoxide hydrolase (sEH) on skin homeostasis. sEH; which converts fatty acid epoxides generated by cytochrome P450 enzymes to their corresponding diols, was largely restricted to the epidermis which was enriched in sEH-generated diols. Global deletion of the sEH increased levels of epoxides, including the linoleic acid-derived epoxide; 12,13-epoxyoctadecenoic acid (12,13-EpOME), and increased basal keratinocyte proliferation. sEH deletion (sEH-/- mice) resulted in thicker differentiated spinous and corneocyte layers compared to wild-type mice, a hyperkeratosis phenotype that was reproduced in wild-type mice treated with a sEH inhibitor. sEH deletion made the skin sensitive to inflammation and sEH-/- mice developed thicker imiquimod-induced psoriasis plaques than the control group and were more prone to inflammation triggered by mechanical stress with pronounced infiltration and activation of neutrophils as well as vascular leak and increased 12,13-EpOME and leukotriene (LT) B4 levels. Topical treatment of LTB4 antagonist after stripping successfully inhibited inflammation and neutrophil infiltration both in wild type and sEH-/- skin. While 12,13-EpoME had no effect on the trans-endothelial migration of neutrophils, like LTB4, it effectively induced neutrophil adhesion and activation. These observations indicate that while the increased accumulation of neutrophils in sEH-deficient skin could be attributed to the increase in LTB4 levels, both 12,13-EpOME and LTB4 contribute to neutrophil activation. Our observations identify a protective role of the sEH in the skin and should be taken into account when designing future clinical trials with sEH inhibitors.

Isothermal titration calorimetry (ITC) is a widely used technique for the characterization of protein-protein and protein-ligand interactions. It provides information on the stoichiometry, affinity, and the thermodynamic driving forces of interactions. This chapter exemplifies the use of ITC to investigate interactions between human autophagy modifiers (LC3/GABARAP proteins) and their interaction partners, the LIR motif containing sequences. The purpose of this report is to present a detailed protocol for the production of LC3/GABARAP-interacting LIR peptides using E. coli expression systems. In addition, we outline the design of ITC experiments using the LC3/GABARAP:peptide interactions as an example. Comprehensive troubleshooting notes are provided to facilitate the adaptation of these protocols to different ligand-receptor systems. The methodology outlined for studying protein-ligand interactions will help to avoid common errors and misinterpretations of experimental results.

Here, we introduce racoon_clip, a sustainable and fully automated pipeline for the complete processing of iCLIP and eCLIP data to extract RNA binding signal at single-nucleotide resolution. racoon_clip is easy to install and execute, with multiple pre-settings and fully customizable parameters, and outputs a conclusive summary report with visualizations and statistics for all analysis steps.

Determining the structure and mechanisms of all individual functional modules of cells at high molecular detail has often been seen as equal to understanding how cells work. Recent technical advances have led to a flush of high-resolution structures of various macromolecular machines, but despite this wealth of detailed information, our understanding of cellular function remains incomplete. Here, we discuss present-day limitations of structural biology and highlight novel technologies that may enable us to analyze molecular functions directly inside cells. We predict that the progression toward structural cell biology will involve a shift toward conceptualizing a 4D virtual reality of cells using digital twins. These will capture cellular segments in a highly enriched molecular detail, include dynamic changes, and facilitate simulations of molecular processes, leading to novel and experimentally testable predictions. Transferring biological questions into algorithms that learn from the existing wealth of data and explore novel solutions may ultimately unveil how cells work.

Zinc finger (ZnF) domains appear in a pool of structural contexts and despite their small size achieve varying target specificities, covering single-stranded and double-stranded DNA and RNA as well as proteins. Combined with other RNA-binding domains, ZnFs enhance affinity and specificity of RNA-binding proteins (RBPs). The ZnF-containing immunoregulatory RBP Roquin initiates mRNA decay, thereby controlling the adaptive immune system. Its unique ROQ domain shape-specifically recognizes stem-looped cis-elements in mRNA 3’-untranslated regions (UTR). The N-terminus of Roquin contains a RING domain for protein-protein interactions and a ZnF, which was suggested to play an essential role in RNA decay by Roquin. The ZnF domain boundaries, its RNA motif preference and its interplay with the ROQ domain have remained elusive, also driven by the lack of high-resolution data of the challenging protein. We provide the solution structure of the Roquin-1 ZnF and use an RBNS-NMR pipeline to show that the ZnF recognizes AU-rich elements (ARE). We systematically refine the contributions of adenines in a poly(U)-background to specific complex formation. With the simultaneous binding of ROQ and ZnF to a natural target transcript of Roquin, our study for the first time suggests how Roquin integrates RNA shape and sequence specificity through the ROQ-ZnF tandem.

Nicotinamide adenine dinucleotide (NAD) serves as a cap-like structure on cellular RNAs (NAD-RNAs) in all domains of life including the bacterium Escherichia coli. NAD also acts as a key molecule in phage-host interactions, where bacterial immune systems deplete NAD to abort phage infection. Nevertheless, NAD-RNAs have not yet been identified during phage infections of bacteria and the mechanisms of their synthesis and degradation are unknown in this context. The T4 phage that specifically infects E. coli presents an important model to study phage infections, but a systematic analysis of the presence and dynamics of NAD-RNAs during T4 phage infection is lacking. Here, we investigate the presence of NAD-RNAs during T4 phage infection in a dual manner. By applying time-resolved NAD captureSeq, we identify NAD-capped host and phage transcripts and their dynamic regulation during phage infection. We provide evidence that NAD-RNAs are – as reported earlier – generated by the host RNA polymerase by initiating transcription with NAD at canonical transcription start sites. In addition, we characterize NudE.1 – a T4 phage-encoded Nudix hydrolase – as the first phage-encoded NAD-RNA decapping enzyme. T4 phages carrying inactive NudE.1 display a delayed lysis phenotype. This study investigates for the first time the dual epitranscriptome of a phage and its host, thereby introducing epitranscriptomics as an important field of phage research.

Meliolales (Sordariomycetes, Ascomycota) is a group of obligate plant parasitic microfungi mainly distributed in the tropics and subtropics. Meliolalean fungi are commonly known as “black mildews”, as they form black, superficial hyphae on the surface of vegetative and reproductive organs of vascular plants. They are considered biotrophic parasites, and the infections caused by black mildews can lead to a decrease in the photosynthetic activity of plants, as well as to an increase in the temperature and respiration rate of their leaves. Meliolales are frequently parasitized by hyperparasitic fungi, i.e., parasitic fungi that have parasitic hosts. These hyperparasites are all Ascomycota and belong mainly to the Dothideomycetes and Sordariomycetes. Although hyperparasites represent a megadiverse group, species were only described by morphology until 1980, and the systematic position of more than 60 % of known species is still unclear. In addition, there are no DNA reference sequences available in public databases for any of the species of hyperparasites of Meliolales, and no ecological studies have been done up to now. Before this study, no exact number of hyperparasitic fungi growing on colonies of black mildews existed. Here, we present a checklist including 189 species of fungi known to be hyperparasitic on Meliolales, but the number of existing species is likely to be even higher. The elaboration of this species checklist laid the foundations for this investigation, as it helped to understand the present state of knowledge of hyperparasitic fungi on Meliolales worldwide. For the present study, fresh specimens of leaves infected with colonies of Meliolales and hyperparasites were opportunistically collected at 32 collection sites in Western Panama and Benin, West Africa, in 2020 and 2022, respectively. In total, 100 samples of plant specimens infected with black mildews were collected, of which 58 samples were parasitized by hyperparasitic fungi. 31 species and mor

The spike protein of SARS-CoV-2 is a highly flexible membrane receptor that triggers the translocation of the virus into cells by attaching to the human receptors. Like other type I membrane receptors, this protein has several extracellular domains connected by flexible hinges. The presence of these hinges results in high flexibility, which consequently results in challenges in defining the conformation of the protein. Here, We developed a new method to define the conformational space based on a few variables inspired by the robotic field’s methods to determine a robotic arm’s forward kinematics. Using newly performed atomistic molecular dynamics (MD) simulations and publicly available data, we found that the Denavit-Hartenberg (DH) parameters can reliably show the changes in the local conformation. Furthermore, the rotational and translational components of the homogenous transformation matrix constructed based on the DH parameters can identify the changes in the global conformation of the spike and also differentiate between the conformation with a similar position of the spike head, which other types of parameters, such as spherical coordinates, fail to distinguish between such conformations. Finally, the new method will be beneficial for looking at the conformational heterogeneity in all other type I membrane receptors.

MicroRNAs (miRNAs) are critical post-transcriptional regulators in many biological processes. They act by guiding RNA-induced silencing complexes to miRNA response elements (MREs) in target mRNAs, inducing translational inhibition and/or mRNA degradation. Functional MREs are expected to predominantly occur in the 3’ untranslated region and involve perfect base-pairing of the miRNA seed. Here, we generate a high-resolution map of miR-181a/b-1 (miR-181) MREs to define the targeting rules of miR-181 in developing murine T-cells. By combining a multi-omics approach with computational high-resolution analyses, we uncover novel miR-181 targets and demonstrate that miR-181 acts predominantly through RNA destabilization. Importantly, we discover an alternative seed match and identify a distinct set of targets with repeat elements in the coding sequence which are targeted by miR-181 and mediate translational inhibition. In conclusion, deep profiling of MREs in primary cells is critical to expand physiologically relevant targetomes and establish context-dependent miRNA targeting rules. Key Points: * Deep profiling identifies novel targets of miR-181 associated with global gene regulation. * miR-181 MREs in repeat elements in the coding sequence act through translational inhibition. * High-resolution analysis reveals an alternative seed match in functional MREs.

Highlights • TAM polarization induces CP RNA. • CP RNA expression is regulated by HIF-2 and STAT1. • CP RNA is transferred from TAMs to HT1080 cells. • CP RNA is translated by HT1080 cells and protects from ferroptosis. • Co-cultured HT1080 cells decrease iron and lipid peroxidation. Abstract Solid tumors are characterized by hypoxic areas, which are prone for macrophage infiltration. Once infiltrated, macrophages polarize to tumor associated macrophages (TAM) to support tumor progression. Therefore, the crosstalk between TAMs and tumor cells is of current interest for the development of novel therapeutic strategies. These may comprise induction of an iron- and lipid peroxidation-dependent form of cell death, known as ferroptosis. To study the macrophage - tumor cell crosstalk we polarized primary human macrophages towards a TAM-like phenotype, co-cultured them with HT1080 fibrosarcoma cells, and analyzed the tumor cell response to ferroptosis induction. In TAMs the expression of ceruloplasmin mRNA increased, which was driven by hypoxia inducible factor 2 and signal transducer and activator of transcription 1. Subsequently, ceruloplasmin mRNA was transferred from TAMs to HT1080 cells via extracellular vesicles. In tumor cells, mRNA was translated into protein to protect HT1080 cells from RSL3-induced ferroptosis. Mechanistically this was based on reduced iron abundance and lipid peroxidation. Interestingly, in naïve macrophages also hypoxia induced ceruloplasmin under hypoxia and a co-culture of HT1080 cells with hypoxic macrophages recapitulated the protective effect observed in TAM co-cultures. In conclusion, TAMs provoke tumor cells to release iron and thereby protect them from lipid peroxidation/ferroptosis.

Nukleinsäuren und Proteine bilden zusammen mit den Kohlenhydraten und Lipiden die vier großen Gruppen der Biomoleküle. Dabei setzen sich Nukleinsäuren aus einer variierenden Abfolge von Nukleotiden zusammen. Gleiches trifft auf die Proteine zu, wobei deren Bausteine als Aminosäuren bezeichnet werden. Die Reihenfolge der Bausteine bestimmt zusammen mit der Interaktion, die die einzelnen Bestandteile untereinander eingehen, deren Funktion. Um deren Wirkungsweise verstehen und nachverfolgen zu können, wurden unterschiedliche Methoden entwickelt, zu welchen auch die EPR-Spektroskopie gehört. Durch den Einbau modifizierter Nukleotide oder Aminosäuren lassen sich Spinlabel in die sonst EPR-inaktiven Nukleinsäuren und Proteine einführen. Diese Marker lassen sich grundsätzlich in drei Klassen unterteilen (Metallionen, Nitroxidradikale und TAMs), weisen aber immer mindestens ein ungepaartes Elektronenpaar auf. Die Festphasensynthese ist eine Standardprozedur zur Herstellung von markierten Nukleinsäuren und Proteinen. Allerdings führen die Bedingungen dieser Methode zumindest teilweise zur Zersetzung der Nitroxidradikale, die dieser Arbeit zugrunde liegen, wenn sie direkt während der Synthese eingebaut werden. Der direkte Einbau ist aber in vielen Fällen essenziell, um bestimmte Eigenschaften zu erzielen. Um den Abbau des Nitroxidradikals während der Festphasensynthese zu verhindern, kann dieses vorübergehend mit einer Schutzgruppe versehen werden, welche sich anschließend wieder abspalten lässt. Der Schwerpunkt dieser Arbeit liegt hierbei auf der Darstellung neuer photolabil geschützter Spinlabel zur Synthese markierter Proteine und Nukleinsäuren. Basierend auf den Nukleotiden Uridin und Cytidin konnten zwei für die RNA-Synthese vorgesehene Phosphoramidite synthetisiert werden, welche jeweils an der 5-Position des Pyrimidinrings mit einem photolabil geschützten Spinlabel auf Basis von TPA versehen waren. Durch Einbau des Uridinderivats in das Neomycin-Aptamer konnte zudem de

Highlights • Seed size mediates seedling recruitment in tropical forests and pastures. • Large-seeded species recruited better than small-seeded species in the forest. • Recruitment of large-seeded species in pastures was limited by surface temperature. • Large-seeded species should be protected against drought in regenerating pastures. Abstract Seedling recruitment is a key process of plant regeneration that often depends on plant functional traits, such as seed size. To optimize forest restoration efforts, we need to better understand how seedling recruitment of different seed sizes varies along environmental gradients with strong variation in abiotic and biotic factors. To understand these interacting effects, we conducted a sowing experiment with different-sized seeds in forests and pastures in the tropical mountains of southern Ecuador. We quantified seedling recruitment in relation to temperature, soil moisture and biotic pressures. We sowed seeds of five tree species of varying seed size at three elevations (1000, 2000 and 3000 m a.s.l.) in primary forest and pastures. We tested (1) how habitat type influences the recruitment of seedlings belonging to three small- and two large-seeded species, and (2) how abiotic and biotic factors limit seedling recruitment of species with different seed sizes. We found that seedlings of the two large-seeded species recruited better than seedlings of the three small-seeded species, but only in the forest habitat. Seedling recruitment of large seeds was primarily limited by high surface temperature, which explains lower recruitment of large seeds in pastures compared to forests. Our study shows that seed size can be a key trait mediating variability in seedling recruitment in tropical ecosystems. We conclude that restoration measures should aim to mitigate extreme temperatures in tropical pastures to aid the natural regeneration of large-seeded tree species.