12033 research outputs found
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Perturbative and non-perturbative theories of structure formation
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Mechanism of Action Analyses of Cell Wall Synthesis Inhibitors and Lipophilic Derivatives with Enhanced Antibiotic Activity
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Role of GluA2-containing AMPARs in Oligodendrocyte Lineage Cells
No full textOligodendrocytes precursor cells (OPC), also called NG2 cells, proliferate or differentiate into myelin-producing oligodendrocytes throughout life. Myelination depends on neuronal activity. Although NG2 cells receive neuronal glutamatergic synaptic input throughout the CNS, factors mediating activity-dependent control of the oligodendrocyte lineage cell fate have not been resolved yet. Glutamatergic signalling in NG2 cells is mainly mediated by AMPARs. During the postnatal period, AMPARs are calcium impermeable due to the expression of the GluA2 subunit. Later in the adult period, NG2 cells downregulate the GluA2 subunit, therefore they express GluA2-lacking AMPARs, permeable to calcium. Thus, the decline of the proliferation rate of NG2 cells observed during the transition from the postnatal to the adult period is mirrored by an increase in the calcium-permeability of synaptic AMPA receptors. We hypothesized that the postnatally expressed GluA2 subunit enables the proliferation of NG2 cells by suppressing calcium entry during synaptic activity. Here, we assessed the impact of postnatal GluA2 subunit deletion on the oligodendrocyte lineage cell fate. In order to induce a conditional deletion of the GluA2 subunit in NG2 cells, we crossed the three following mouse lines. NG2CreER mouse line conditionally express Cre under the NG2 promotor. R26REYFP mouse line express the yellow fluorescent protein reporter after Cre expression. GluA2lox mouse line undergoes a deletion of Gria2 allele responsible for GluA2 expression. First, we followed the oligodendrocyte lineage cell fate with the BrdU and PCNA markers to examine NG2 cell proliferation, the cleaved caspase-3 marker to probe for cell death, and the CC1 marker to analyse differentiation of NG2 cells into oligodendrocytes. We found that GluA2 deletion in early postnatal NG2 cells increased BrdU uptake in NG2 cells without increasing cell density or cell death in the oligodendrocyte lineage cell population. Secondly, we investigated the role of GluA2 subunit on myelination establishment by measuring the internodes length during myelination onset and at a later timepoint during the postnatal phase. GluA2 deletion in NG2 cells shortened immature internodes during myelination onset. Thirdly, the role of the GluA2 subunit in NG2 cells was investigated in a motor learning task requiring newly formed oligodendrocytes. GluA2 deletion altered the motor learning performance in mice. Although it did not affect the myelination properties investigated or NG2 cells proliferation. Overall, the GluA2 subunit in postnatal NG2 cells regulates DNA synthesis in NG2 cells and myelination onset through the regulation of internode elongation. Our study highlights the prominence of glutamatergic synaptic input integration in postnatal NG2 cells to regulate oligodendrocyte lineage cell fate
Cellular and molecular defence mechanisms against Legionella infection
No full textThe two most prevalent causes of Legionnaires’ disease are L. pneumophila and L. longbeachae. The rising incidence of the Legionnaires’ disease over the past two decades together with the increasing prevalence of L. longbeachae in the Northern hemisphere highlights the necessity to gain a more detailed insight into the L. longbeachae-induced immune response. The aim of this thesis was to investigate the cellular and molecular mechanisms required for protection, focussing on immune cell responses and intracellular host-bacterial interactions.
By combining a murine mouse model, a novel fluorescent reporter to track L. longbeachae, and cell depletion experiments, I uncovered the differential contribution of tissue-resident alveolar macrophages (AM) and infiltrating neutrophils to the defence against L. longbeachae. Early during infection, AM contained most of the bacteria. AM numbers sharply decreased during infection, which was accompanied by a large influx of neutrophils that also internalized bacteria. Comparative analysis of bacterial viability revealed that neutrophils were more efficient at killing and clearing of L. longbeachae than AM. In contrast, the results presented here indicated that lung-resident AM promoted infection, most likely by serving as a replicative niche.
Defence against L. pneumophila is known to require IFN-gamma but not IL-18, a strong IFN-gamma inducer. However, Il18r1–/– mice were less able to clear L. longbeachae and had significantly impaired IFN-gamma levels in the lung. Furthermore, IL-18R signalling was critical for the efficient bacterial killing by neutrophils via production of reactive oxygen species. Previous bone marrow chimera experiments in our laboratory suggested that IL-18R expression in epithelial cells was necessary and sufficient for protection against L. longbeachae. However, genetic in vivo experiments using the Cre-lox-system revealed that IL-18R+ NK cells and T cells were central players in the IL-18-dependent anti-L. longbeachae defence via IFN-gamma secretion, whereas IL-18R expression by ciliated bronchiolar epithelial cells did not confer protection.
Finally, our results highlighted key mechanisms by which Legionella subverts host macrophages to form an intracellular endoplasmic reticulum (ER)-like vacuole as its intracellular replicative niche. Establishment of the Legionella-containing vacuole (LCV) by recruitment of ER-derived vesicles induces ER stress. Although the relevance of ER stress in this process is unclear, effector proteins secreted by L. pneumophila are known to inhibit onset of the ER stress response. Using the pharmacological ER stress inducer thapsigargin, we showed that ER stress induces a protective host response promoting the secretion of pro-inflammatory cytokines, limiting intracellular L. pneumophila replication, and improving host survival. Mechanistically, ER stress induced a novel non-canonical activation of the transcription factor STAT1 via the IRE1 kinase driving transcription of the IFN-gamma-induced chemokine CXCL10. These results highlighted a potential role of the host ER stress response in the initiation of a protective cellular immune response towards L. pneumophila
Tissue cell-type composition changes during aging in mice
No full textAging is linked to progressive alterations in organ functionality and a heightened susceptibility to various diseases. These age-related functional deteriorations are likely, in part, due to microstructural changes within organs, even in the absence of pathological conditions. Examining cellular composition at the organ-level across tissues traditionally posed significant challenges due to the high workload associated with the application of unbiased cell counting methods across a range of histological samples. Here, we employed bulk RNA-sequencing, coupled with deconvolution based on celltype specific markers, to generate datasets and derive estimates regarding agingassociated changes in cell-type composition across multiple tissues (brain, heart, lung, skeletal muscle, kidney, testis) in male C57BL/6J mice, covering much of their lifespan (3, 5, 8, 14, 20 and 26 month). We show that with advanced age, immune cell types showed predominant changes among all tested tissues. Across organs, most immune cells increased with advancing age while parenchymal cells showed a decreasing trend during aging. In addition, we mined publicly available RNA-seq datasets in mice to identify experimental conditions that either counteract or phenocopy aging-associated changes in cellular tissue composition. These analyses revealed, for instance, that diabetes phenocopies aging associated changes in the mouse kidney, and calorie restriction counteract aging in mice muscle. Additional well-established cell counting methodologies were used to confirm findings obtained using our deconvolution approach
Return of the first inflammasome : Elucidation of NLRP1 inflammasome activation by p38-mediated phosphorylation and ubiquitination
No full textThe assembly of inflammasomes is linked to the detection of pathogens and other danger signals by intracellular pattern-recognition receptors of the mammalian innate immune system. The human inflammasome sensor NLRP1 is activated by N-terminal proteolytic cleavage and subsequent degradation, causing the release of the C-terminal NLRP1UPA-CARD fragment and the recruitment of the adaptor protein ASC and caspase-1, resulting in the processing of IL-1β/IL-18 and pyroptotic cell death.To study NLRP1 inflammasomes, I characterized HEK 293T and N/TERT-1 keratinocyte inflammasome reporter cell lines, and I identified two NLRP1PYD-specific nanobodies which, combined with the E3 ligase receptor VHL, allowed the precise stimulation of endogenous NLRP1 by targeted NLRP1PYD ubiquitination and subsequent N-terminal degradation.Using the reporter cell lines, I found that various stimuli of the ribotoxic stress response activate human NLRP1 in a p38-dependent manner. In addition, infection with alphaviruses, including Semliki Forrest virus and Chikungunya virus, caused p38-dependent NLRP1 activation. p38 kinases directly phosphorylate the N-terminal linker region of the inflammasome sensor, in which serine 107 represents a critical phosphorylation site. I propose that phosphorylation of the N-terminal linker generates a phospho-degron which is recognized by cullin RING E3 ligases, causing the ubiquitination of NLRP1PYD, N-terminal degradation, and inflammasome assembly.In addition to delineating p38-mediated NLRP1 activation, I identified novel viral NLRP1 stimuli and established lymphocytes as NLRP1-competent cell types
Importance of root-soil contact conditions for the microbiota in the soil-rhizosphere-plant continuum
No full textThe contact zone between a root and the surrounding soil mediates processes such as nutrient and water uptake and the interaction with microorganisms. Despite the importance of the contact zone, there is a lack of understanding of the specific characteristics and their relevance to plant performance and microbial colonization. The objective of this thesis was, therefore, to investigate the effect of the extent and characteristics of this root-soil contact zone on plant performance and microbial abundance, and community structure. In particular, I addressed the following research questions: I.) How do specific root traits impact the establishment of a contact zone and how do variations in the root-soil contact zone influence corresponding plant and microbial parameters?, II.) How do root-soil contact zone conditions furthermore modulate these responses? and III.) How do root-soil contact zone conditions modulate these responses in the field? In order to answer these questions, I conducted a literature review, two microcosms, and one field experiment. To modify the root-soil contact zone, I grew maize plants in treatments with artificial pores (abiogenic pores), with pores incubated with earthworms (biogenic pores), and, as a control, without additional pores. To analyze the microbial abundance and community diversity and composition, qPCR, fluorescence microscopy, and amplicon sequencing were used. Plant performance was assessed by endoscopy and measurements of plant height and weight and shoot nutrient contents.
My results confirmed that the root-soil contact significantly influenced the occurrence of microorganisms on the root surface, as well as the diversity and composition of the bacterial community in the rhizosphere. The root-soil contact was especially important for nitrogen cycling microorganisms in the rhizosphere, as nitrifying bacteria were less abundant when the root established only partial soil contact. However, the level of root-soil contact had no effects on early plant performance in the microcosm experiments (research question I). When the contact zone between root and soil was enriched in nutrients, due to soil faunal activity in pores, the abundance of microorganisms at the pore wall as well as in the corresponding rhizosphere was increased, and microbial community composition was influenced (Research question II). The same trends can be seen in the field. Moreover, in the field experiment, which also covered later growth stages, reduced root-soil contact due to the presence of abiogenic pores resulted in growth deficits compared with the presence of biogenic pores and the absence of soil pores (Research question III).
In summary, these findings underline the importance of root-soil contact and contact zone characteristics for rhizosphere processes and plant performance. Agricultural management should, therefore, aim to minimize cracks in the soil to guarantee root-soil contact and to improve plant performance by enhancing biological activity in pores
3D culture conditions instruct an in vivo-like phenotype in primary microglia
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Design and Multistep Synthesis of Ligands for the GPR18 and GPR183: Related Orphan G Protein-Coupled Receptors with Immunoregulatory Functions
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Synthesis, Properties, and Reactivity of Ni-Group 13 and Group 14 Bimetallic Complexes
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