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Item type: Item , Development of a pipeline for spatial transcriptome analysis of samples from Hodgkin's lymphoma patients(2025-12-17) Melanie KeckOVERVIEW Automated pipeline for spatial transcriptomics analysis of classical Hodgkin lymphoma samples using 10x Genomics Visium low-density platform. Includes preprocessing (Space Ranger), quality control, batch correction (Harmony, scVI), and downstream analysis. Master's thesis, Justus Liebig University Giessen (2026). CONTENTS - Bash script for automated Space Ranger preprocessing - JupyterLab notebooks for quality control, batch correction, and downstream analysis - PDF reports documenting all analysis steps and results - HTML tables with QC metrics, correlation matrices, and differential expression results - Figures and visualizations from all analyses - Test dataset for pipeline validation (mouse spleen, GEO: GSE254652) TECHNICAL REQUIREMENTS - Ubuntu 24.04.2 LTS, Space Ranger v3.1.3, Python 3.12.10 - Python packages: Scanpy (v1.10.4), Squidpy (v1.6.5), harmonypy (v0.0.10), scVI (v1.3.1.post1) - Hardware: Minimum 40 GB RAM, 4 CPU cores recommended - Reference files: refdata-gex-GRCh38-2020-A, Visium_Human_Transcriptome_Probe_Set_v2.0_GRCh38-2020-A USAGE 1. Prepare input files (SampleSheet.csv, Aggregation.csv) 2. Configure paths in script.sh and collect_output.py 3. Run preprocessing: bash script.sh 4. Generate metadata: python collect_output.py 5. Update base paths in JupyterLab notebooks 6. Execute JupyterLab notebooks sequentially For detailed documentation, see README.pdfItem type: Item , Deciphering Self-Assembly Mechanisms and Chemical Reactions of Organic Building Blocks on Metal Surfaces by Chemical Bond Imaging(2025-09-24) Wiche, MiguelIn recent years, the novel field of on-surface synthesis has been established as one of the main tools for constructing customized, low-dimensional organic nanostructures via bottom-up approaches on atomically flat metal substrates. The self-assembly of the molecular precursors, a process that often serves as pre-step of the on-surface reaction, determines the precise local arrangement of atoms and bonds in neighboring molecules, thus playing a decisive role in product formation. Thereby, intermolecular interactions between hydrogen and fluorine atoms have proven to be a valuable tool to steer molecular alignments. In this work, the mainly unexplored intermolecular hydrogen-fluorine interaction is systematically investigated on inert Au(111) and reactive Cu(111) substrates, using a linear, unilaterally fluorinated 1,2,10,11,12,14-hexafluoropentacene molecule as a model system. In the combined scanning tunneling microscopy and chemical bond imaging study, the local arrangement of hydrogen and fluorine atoms in neighboring molecules is determined in the picometer range and angular variations of a few degrees. While on Au(111) the intermolecular interactions between the molecules are the main contributor to the self-assembly, the higher reactivity of Cu(111) results in different adsorption geometries and molecular arrangements. The highly precise self-assembly study provides new insights into the on-surface interaction of hydrogen and fluorine atoms, thereby highlighting its significance for the field of on-surface synthesis. An on-surface reaction that solely takes place using halogenated precursors is the on-surface Ullmann coupling reaction, which enables the formation of covalent carbon-carbon bonds with the underlying metal surface as a catalyst. However, when applying Ullmann coupling reaction steps, complex self-assembly mechanisms can occur when halogenated precursors interact with the metal surface. Further, cleaved halogens adsorbed on the surface may potentially inhibit the reaction steps. Hence, there is a growing demand for halogen-free precursors for on-surface reactions. Recently, it has been demonstrated that halogen-free (6)Cycloparaphenylene ((6)CPP) molecules are suited to thermally induce a ring-opening polymerization reaction for the synthesis of graphene and biphenylene nanoribbons of confined widths. However, the mechanism of the ring-opening polymerization and the use of cycloparaphenylenes as precursors have not been investigated sufficiently. To contribute to the fundamental understanding of this remarkable reaction, in this thesis we systematically investigate the influence of the ring strain, which decreases with increasing ring size, using a set of cycloparaphenylenes of different sizes ((6)CPP vs. (8)CPP vs. (10)CPP). Our results demonstrate that the ring-opening polymerization is facilitated when using smaller, highly strained cycloparaphenylenes. For larger molecules with lower strain energies, the initial ring-opening is hampered, leading to only partial polymerization for (8)CPP and no polymerization in case of (10)CPP. Additionally, dehydrogenation of individual phenyl rings in intact molecules is observed for (8)CPP and (10)CPP, which further impedes the polymerization reaction.Item type: Item , The Effects of DNA Repair Pathway Engineering on CRISPR-Mediated Genome Editing in Neuronal Cells(2025) Alabudeeb, FatimahInherited retinal dystrophies are a group of genetically and clinically heterogeneous disorders that vary in their clinical presentation and progression, possibly leading to blindness. So far, about 332 genes, most expressed in photoreceptors, have been identified to be associated with these diseases. The relatively new genome editing field, particularly CRISPR-Cas9 mediated genome editing technology, which introduces DNA double-strand breaks (DSBs) that are subsequently repaired by the cells' repair systems, has recently drawn much attention. The major repair pathways are non-homologous end-joining (NHEJ) and homology-directed repair (HDR). One of the main obstacles to its application in the retina is the limited knowledge of DNA repair in photoreceptors. This work aims to analyze the DNA repair mechanisms, improve CRISPR Cas9 genome editing efficacy by modulating and engineering the DNA repair pathways in mature neurons using the human inducible Neurogenin iPS (iNGN) cell line, and validate it as an in vitro model system. These cells are human-induced pluripotent stem cells that differentiate into mature neurons within 4 days. In addition, the iNGN TET3KO cell line was also investigated to determine the impact of the TET3 protein at various differentiation stages. To achieve this, the iNGN cell lines and a control cell line, HEK293T cells, were transfected with BRET reporter assay plasmid using Cas9 and the inducible Cas systems (iCas). Promoter optimizations were done by replacing the CMV promoter, which can be silenced and suppressed in certain cell types, with a sustained EF1α promoter. Applying the Cas9 system, iNGN WT and iNGN TET3KO cells were tested throughout the differentiation process. Moreover, the modifications of PARP1 protein levels using PARP1 overexpression or knockout plasmids were necessary to study its influence on the DNA repair pathways of different cell lines. The BRET reporter assay was the primary quantitative technique used. The results showed decreased frameshift rates for the undifferentiated iNGN using the iCas system compared to the Cas9 system, and along with failure to use its timing control advantage, the Cas9 system was mainly used for the remaining experiments. The undifferentiated iNGN TET3KO cells' frameshift rates, as well as at the beginning of differentiation, were significantly higher than iNGN WT ones. Furthermore, the frameshift rate results of the TET3KO overexpressed cells resembled those of iNGN WT cells. After PARP1 modulation, the frameshift rates of PARP1 downregulation were greater throughout the differentiation process of the iNGNs, regardless of the HDR template addition. Interestingly, the results of the iNGN TET3KO cells were higher than those of the WT cells. For future assessment, the generation and application of iNGNs TLR3 cell lines is essential to verify the results obtained using the BRET reporter assay at the genomic level.Item type: Item , Charakterisierung des Down-Syndrom-Zelladhäsionsmoleküls Dscam in der angeborenen Immunantwort von Manduca sexta (L.)(2025) Speckmann, MartinDie Spezifität angeborener Immunmechanismen wird entscheidend von genetischer Diversifizierung und selektiver Anpassung in der Evolution von Wirt-Mikroben-Interaktionen beeinflusst. Angeborene Immunantworten von Insekten werden demnach allgemein als effizient, jedoch bedingt durch Keimbahn-kodierte Mustererkennungsrezeptoren (PRR) als eingeschränkt spezifisch beschrieben. In Zusammenhang mit exklusiver molekularer Diversität des Down-Syndrom-Zelladhäsionsmoleküls (Dscam) bei Vertretern der Pancrustacea verdichten sich Belege, die für eine Funktion als hypervariabler, Pathogen-spezifischer PRR der Immunglobulin-Superfamilie sprechen. Obwohl experimentelle Daten erregerspezifische Dscam-Spleißformen in Hämozyten von Insekten und Krebstieren sowie deren Beteiligung an In-vitro-Phagozytose von Mikroorganismen zeigen, sind Genexpressionsmuster in Hämozyten und deren Relevanz im Rahmen angeborener Immunantworten in vivo noch größtenteils unbekannt. Anhand eines In-vivo-Modells bakterieller Stimulation und monoklonaler Antikörper für spezifische Hämozytentypen wurden die Genaktivität und das Proteinmuster von Manduca sexta Dscam (MsDscam) in zirkulierenden, larvalen Hämozyten charakterisiert. Trotz geringer quantitativer Veränderungen der relativen mRNA-Level konnte eine qualitative Veränderung des differenziellen Transkriptionsmusters von MsDscam in Zusammenhang mit Hämocoel-Injektion von fixierten E. coli K12 festgestellt werden. In Abwesenheit von Immunstimulation ist MsDscam-Transkript ausschließlich in einer seltenen Population sphärischer Hämozyten mit exzentrischen Zellkernen lokalisiert. Prophenoloxidase (proPO) enthaltende Oenozytoide mit gleichen morphologischen Eigenschaften fallen unabhängig als einzige Population in Zirkulation mit membranassoziiertem MsDscam-Muster auf. Nach E. coli-Injektion ist der prozentuale Anteil von Hämozyten mit aktiver MsDscam-Transkription hingegen signifikant um das Zehnfache erhöht (p < 0,001), darunter auch Subpopulationen von Plasmatozyten und granulären Zellen. Damit vermittelt diese Dissertation zum ersten Mal in zwei Jahrzehnten immunologischer Dscam-Forschung ein fundiertes Bild immunstatusabhängiger Genaktivität in drei distinkten Hämozytentypen. Zusätzlich ergeben sich kumulative Hinweise, die eher für funktionelle Relevanz von MsDscam im Rahmen einer zweiten Immunantwort sprechen als in der Akutphase-Immunantwort nach erstmaliger mikrobieller Stimulation. Dementsprechend liefert diese Arbeit erstmals eine Grundlage für die Analyse Dscam-basierter molekularer Diversität in proPO-produzierenden Hämozyten, was zu einem erweiterten Verständnis von Oenozytoide als immunologische Masterregulatoren in Manduca beitragen kann. Typspezifische Transkriptomanalyse von Manduca-Hämozyten in Homöostase und Immunstimulation kann somit nicht nur zur Validierung der Hypothese von funktioneller Relevanz von Dscam beim Immunpriming in Insekten, sondern auch zur Aufklärung der immer noch unbekannten Regulation zellulärer Dscam-Spleißsignaturen beitragen.Item type: Item , Microbes in bee-plant networks: Composition characterization and their ecological implications(2025) Shi, HaoranHoney bees are vital pollinators in ecosystems around the world, and microbes play key roles in connecting bees and plants. Collectively, microbes, bees and plants form intricate tripartite interactions networks. Through a co-evolution, many bee- and plant-associated microbes have developed functions that benefit their hosts, including promoting growth, enhancing pathogen resistance, and aiding digestion. Microbiome associated with different hosts tend to be specific, and within bee-microbe-plant networks, both beneficial and pathogenic microbes are dynamically transmitted among hosts. In the current study, honey bee corbicular samples were collected over a two-year period from beehives at Justus-Liebig-University Giessen. Corbicular pollen is able to reflect both microbes and plants encountered by honey bees during foraging activity. Plant and microbial communities in honey bee corbicular pollen were profiled using 16S rRNA gene and plant ITS2 metabarcoding. The results indicated that the corbicular pollen microbiome exhibited clear seasonal variations, and was affected by multiple environmental factors as well as choices of forage plants. Co-occurrence network analysis further revealed specific plant-microbe associations and identified several hub plant taxa that may serve as hotspots for microbial transmissions. Following this study, we characterized bacterial and fungal microbiome of flowers from a highly insect-visited hub plant, bramble (genus Rubus), using 16S rRNA gene and fungal ITS2 metabarcoding. The data showed that insect visitation increased microbial loads on flowers and enriched specific microbial groups including fermentative and pathogenic microbes, highlighting the role of bramble flowers as hotspots for microbial transmission. In addition, insect visitation altered floral microbiome structure, potentially through the introduction of several hub microbial taxa and by increasing the centralization of the microbial interaction networks. Honey bees were collected from beehives, and common bacterial, fungal and viral honey bee pathogens were screened. The expression levels of several immunity-related genes (defensin-1, lysozyme-like, vitellogenin, glucose oxidase) and the composition of the bee microbiome were examined to assess honey bee health status. Black queen cell virus (BQCV) was detected in almost all individuals, while Vairimorpha pathogens were only partially detected. Paenibacillus larvae, Melissococcus plutonius, Kashmir bee virus (KBV), Acute Bee Paralysis Virus (ABPV), Chronic bee paralysis virus (CBPV), Deformed Wing Virus (DWV), and Sacbrood bee virus (SBV) were not detected in the samples. The data indicated that BQCV and Vairimorpha infections had no significant impact on the expression of immunity-related gene. Since the microbiome composition was assessed at the hive level and BQCV was present in every hive, its potential influence on the microbiome remains to be further clarified. In addition, a bacterial isolate from birch pollen was phenotypically, genotypically, and chemotaxonomically characterized using a polyphasic approach. Based on 16S rRNA gene phylogeny and comparative genomic analysis, the isolate was identified as a novel species of the genus Robbsia. The bacterium was rod-shaped, non-motile, facultative anaerobic and grew optimally at 28 °C and pH 6–7. Unlike its closest relative Robbsia andropogonis, which is a known phytopathogen, the isolate exhibited no phytopathogenic traits such as flagellum formation, rhizobitoxine production, or induction of plant hypersensitive response. The proposed and accepted name of the isolate is Robbsia betulipollinis Bb-Pol-6T.