Identification of presynaptic proteins as neddylation targets

Reversible post-translational modifications are essential for the regulation of cell functions. One such modification is the attachment of ubiquitin and ubiquitin-like proteins, including Nedd8 (neural-precursor-cell-expressed developmentally down- regulated 8) (Enchev et al., 2015). It is well established, that neddylation is involved in the post-translational modification of synaptic proteins. As inhibition of neddylation has been demonstrated to decrease the probability of transmitter release (Brockmann, 2019), it can be hypothesised that proteins involved in synaptic transmission are neddylated. However, the target proteins of Nedd8 remain largely unexplored. The main focus of my work was the identification of presynaptic proteins modified by neddylation. The investigation focused on three key criteria: the covalent binding of Nedd8 to the target protein, the contribution of the neddylation-specific machinery, and the identification of neddylated lysines. For this, an Avi-tagged Nedd8 was used to detect all covalently attached target proteins of Nedd8 with magnetic streptavidin beads. The use of MLN-4924 as a control ensured that the attachment of Nedd8 occurred through the neddylation-specific machinery. In my thesis, I identified Stx1a and the homologous proteins Stx3 and Stx4 as targets for neddylation. By mutating lysine residues to arginine, I revealed that lysine 117 or 126 play an essential role in the neddylation of Stx1a. In the case of Stx4, lysines 123, 140 and 161 were identified as neddylated residues, while lysines 102, 108, 124 and 151 are suggested to be irrelevant for neddylation. Additionally, I also identified RIM1α as a target protein for Nedd8. Experiments using truncated proteins indicate that the neddylated lysine residue is located within the N-terminal region up to PRM 1. I unexpectedly discovered that the carboxylases are prospective Nedd8 candidates as well. As my work does not provide any additional information, a precise conclusion cannot be drawn at present. In conclusion, I identified four new neddylation targets: Stx1a, Stx3, Stx4 and RIM1α. The identification of Stx and RIM1α as Nedd8 targets will require further investigation of the function of Nedd8 in the synapse. In particular, the discovery of the neddylated lysines of Stx1a and Stx4, and the resulting availability of non-neddylatable variants, increases the potential for investigation of the function of Nedd8

Programmed cell death regulation during <em>Salmonella</em> Typhimurium infections

Programmed cell death (PCD) is a highly regulated process that is vital for the development and homeostasis of multicellular organisms. It functions as an essential mechanism to remove dispensable or unfavourable cells in a coordinated manner under physiological as well as pathophysiological conditions. Importantly, this type of cellular suicide also serves as an effective defence strategy to control intracellular pathogens that aim to repurpose host cells as replicative niche and to evade extracellular immune responses. Recent findings indicate that host cells utilise multiple PCD pathways to fight invading pathogens. However, the organisation and regulation of this complex cell death network consisting of pyroptosis, apoptosis and necroptosis and their relative importance for the control and clearance of intracellular infection is not completely understood. In this thesis, we systematically investigated the cellular and molecular requirements for PCD induction and the contribution of different PCD pathways to immunity against intracellular bacteria. We infected novel genetically modified mouse strains deficient for various combinations of PCD mediators, such as different cysteine-dependent aspartate-directed proteases (caspases), with the bacterial model organism Salmonella enterica serovar Typhimurium (S. Typhimurium) to analyse their ability to control intracellular infections. Additionally, we established in vitro assays to disentangle the complex interactions of the PCD network by analysing the cell death kinetics of bone-marrow derived macrophages obtained from various mouse strains in real-time and determining the intracellular bacterial burden following infection with S. Typhimurium. Our findings unveiled that the PCD pathways pyroptosis and apoptosis are highly interconnected and regulated with a remarkable level of redundancy. We identified that pyroptosis and apoptosis are essential for the control of S. Typhimurium and that molecular components of these pathways, such as caspase-1 and -8, can be used interchangeably to counteract bacterial evasion strategies. Furthermore, we investigated the underlying mechanisms that coordinate the flexible induction of pyroptosis and apoptosis during S. Typhimurium infection. We demonstrated that caspase-2 neither plays a significant primary nor compensatory role in the regulation of cell death and control of intracellular infections. Upon excluding caspase-2 as link between PCD pathways, we evaluated means of extracellular cell death induction. The results presented in this thesis imply critical functions for cytotoxic CD4+ T cells and interferon gamma (IFN-γ) in extrinsic apoptosis induction of S. Typhimurium infected cells. In conclusion, our results highlight the relative importance, interconnectivity and redundancy of different PCD pathways. This study provides detailed insights into the highly complex network of PCD by unravelling new functions for several of its components and thereby defining novel mechanisms of cell death induction during infections with S. Typhimurium. These findings aid to discover new drug targets and develop novel treatment strategies to fight intracellular infections by enhancing essential host immune responses

The German Hydrogen Sector in Global Production Networks : Structures, Agency, and Uneven Development

(noch nicht zugänglich / not yet accessible

<em>Hordeum erectifolium</em>, a physiological and genomic study of drought-adaptive traits

(noch nicht zugänglich / not yet accessible

Importance of root-soil contact conditions for the microbiota in the soil-rhizosphere-plant continuum

The 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

(noch nicht zugänglich / not yet accessible

Studies towards the total synthesis and structure revision of neaumycin B

(noch nicht zugänglich / not yet accessible

Return of the first inflammasome : Elucidation of NLRP1 inflammasome activation by p38-mediated phosphorylation and ubiquitination

The 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-1&#946;/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

Die Rolle von Fraktalkin in der Aristolochiasäure-induzierten Nephropathie

Die Fraktalkin (CX3CL1/R1) Achse spielt eine bedeutende Rolle in der Rekrutierung von Immunzellen in verschiedenste Organe. In dieser Arbeit untersuchten wir die Rolle von CX3CR1 exprimierenden Immunzellen in der Aristolochiasäure-induzierten Nephropathie im Mausmodell. Diese Erkrankung zeichnet sich durch eine fulminante interstitielle Inflammation, einen proximalen Tubulusepithelzellschaden sowie eine subsequente Fibrose aus. Wir konnten zeigen, dass eine bestimmte Makrophagen-Subpopulation (sog. Typ M2a Makrophagen) sowohl in vitro als auch in vivo eine CX3CL1/R1 abhängige Protektion der Nieren vermittelt

The role of production method and country-of-origin labelling in consumers' food choice : Empirical study based on meta-analyses and stated preference choice experiments

Organic production and domestic origin of food serve for consumers as extrinsic cues for food safety and food quality dimensions. With consumers' growing interest in food quality and their increasing concern regarding food safety organic and domestic origin labeling has gained in relevance for businesses and policy makers as well as in research. Accordingly, numerous studies have investigated the effects of organic labeling (OL) and domestic origin labeling (DOL), arriving at heterogeneous results. This leads to the question whether there are any systematic patterns that can explain the difference in the magnitude and in some cases even the sign of willingness to pay (WTP) estimates for OL and DOL. The objective of this research is twofold. First, by conducting two meta-regression analyses (MRA) the thesis identifies the main determinants that drive consumers' WTP a premium for OL and DOL as derived from previous literature. Second, for Taiwan—a newly industrialized country—three empirical field studies are conducted investigating the role of trust and attitude as well as the impact of information on consumers' choice of products differentiated by OL and DOL. The studies use red sweet pepper as study object and make use of Discrete Choice Experiments (DCE) (chapters 3, 4 and 5), Best Worst Scaling (BWS) (chapter 5) as well as Propensity Score Matching (chapter 4). After a brief introduction (chapter 1), two MRA analyses are conducted (chapter 2), one in which the results of 74 WTP estimates for OL and another in which the findings of 31 WTP estimates related to DOL are considered. The research finds evidence for the presence of publication bias in both MRAs. In addition, the results reveal systematic variation of WTP estimates across underlying study characteristics such as the location the survey was conducted as well as the elicitation methods used to derive WTP estimates. Chapter 3 examines the relative importance of product and process information—country of origin, production methods, chemical residue testing (CRT) and price—for Taiwanese consumers' product choice based on a convenience sample of 290 valid responses. Results show that respondents focus mostly on country of origin labeling, followed by price. Furthermore, interaction between attributes matter and consumers' preference for attribute levels depend on socioeconomic characteristics. Given the increasing relevance of organic imported products on the Taiwanese market the impact of an equality of organic standards information treatment on Taiwanese consumers' food preference and purchasing behavior is examined in chapter 4 based on a valid sample of 800 respondents. Results indicate that information on the equality of organic standards significantly increased the purchase likelihood for Taiwan-origin organic products, while the opposite effect is detected for Chinese organic products. In addition, an increase in the utility of the opt-out option in the information treatment group was revealed. Provision of information thus failed to assure consumers about the equality of organic standards. Making use of data obtained from the same survey as in chapter 4 chapter 5 combines two elicitation techniques–DCE and BWS–to provide information about the role of consumers' attitude and trust in influencing food choice decisions. Results of linking the DCE with the BWS attitudinal dimensions reveals that consumers' attitude and trust significantly explain consumers' membership to a specific consumer segment and therefore, is of importance for understanding food purchase behavior. The main results, limitations and conclusions are summarized in chapter 6. The research presented in this thesis adds to the literature twofold. From a content point of view the studies conducted in the framework of this thesis provide a better understanding of consumer food choice behavior, specifically with regard to OL and DOL. Some of the insights are obtained by the combination of methods that so far have not been applied jointly in the literature. Therefore, the thesis also adds to the literature from a methodological point of view