Open Access

Gram-negative bacteria secrete lipopolysaccharides (LPS), leading to a host immune response of proinflammatory cytokine secretion. Those proinflammatory cytokines are TNF-α and IFN-γ, which induce the production of indoleamine 2,3-dioxygenase (IDO). IDO production is increased during severe sepsis, and septic shock. High IDO levels are associated with increased mortality. This enzyme catalyzes the degradation of tryptophan (TRP) to kynurenine (KYN) along the kynurenine pathway (KP). KYN is further degraded to kynurenic acid (KYNA). Increased IDO levels accompany with increased levels of KYNA, which is associated with immunoparalysis. Due to its central role, the KP is a potential target of therapeutic intervention. The degradation of TRP to KYN by IDO was intervened by 1-Methyltryptophan (1- MT), which is assumed to inhibit IDO. By administering 1-MT, the survival of 1-MT-treated mice suffering from sepsis increased compared to mice not treated with 1-MT. The levels of downstream metabolites such as KYN and KYNA were expected to be decreased. Surprisingly, in healthy mice and pigs, an increase in KYNA after 1-MT administration was reported. Those unexpected metabolite alterations after 1-MT administration, and the mode of action, were not the focus of recent research. Hence, there is no explanation for KYNA increase, while KYN did not change. This thesis aims to postulate a possible degradation pathway of 1-MT along the KP with the help of ordinary differential equation (ODE) systems. Moreover, the developed ODE models were used to determine the ability of 1-MT to inhibit IDO in vivo. Therefore, a multiplicity of ODE models were developed, including a model of the KP, an extension by lipopolysaccharide (LPS) administration, and 1-MT administration. Moreover, seven ODE models were developed, all considering possible degradation pathways of 1-MT. The most likely degradation pathway was combined with the ODE model of LPS administration, including the inhibitory effects of 1-MT. Those models consist of several dependent equations describing the dynamics of the KP. For each component of the KP, one equation describes the alterations over time. Equations for TRP, KYN, KYNA, and quinolinic acid (QUIN) were developed. Moreover, the alterations of serotonin (SER) were also included. All together belong to the TRP metabolism. They include the degradation of TRP to SER and to KYN, which is further degraded to KYNA and QUIN. Every degradation is catalyzed by an enzyme. Therefore, Michaelis-Menten (MM) equations were used employing the substrate constant Km and the maximal degradation velocity Vmax. To reduce the complexity of parameter calculation, Km values of the different enzymes were fixed to literature values. The remaining parameters of the equations were determined so that the trajectories of the calculated metabolite levels correspond to data. The parameters of different models were determined. To propose a degradation pathway of 1-MT leading to increased KYNA levels, seven models were developed and compared. The most likely model was extended to test whether the inhibitory effects of 1-MT on IDO can be determined. Three different approaches determined the ODE model parameters of the different hypothesis of 1-MT degradation. In the first approach, ODE model parameters were fixed to values fitted to an independent data set. In the second approach, parameters were fitted to a subset of the data set, which was used for simulations of the different hypotheses. The third approach calculated ODE model parameters 100 times without fixed parameters. The parameter set ending up in trajectories of the TRP metabolites, which have the smallest distance to the data, was assumed to be the most likely. The ODE model parameters were fitted to data measured in pigs. Two different experimental models delivered data used in this thesis. The first experimental model activates IDO by LPS administration in pigs. The second one combines the IDO activation by LPS with the administration of 1-MT in pigs. The most likely hypothesis, according to approach 1 was the degradation of 1-MT to KYNA and TRP. For the second data set the most likely one was the direct degradation of 1-MT to KYNA. With approach 2 the most likely degradation pathways were the combination of all degradation pathways and the degradation of 1-MT to TRP and TRP to KYNA. With approach 3 the most likely way of KYNA increase was given by the direct degradation of 1-MT to KYNA. In summary, the three approaches revealed hypothesis 2, the direct degradation of 1-MT to KYNA most frequently. A cell-free assay validated this result. This experiment combined 1-MT or TRP with or without the enzyme kynurenine aminotransferase (KAT). KAT was already shown to degrade TRP directly to KYNA. The levels of TRP, KYN and KYNA were measured. The highest KYNA levels were yielded with an assay adding KAT to 1-MT, corresponding to hypothesis 2. The models describing the inhibitory effects of 1-MT revealed that the model without inhibitory effects of 1-MT on IDO was more likely for all three approaches. The correctness of hypothesis 2 has to be confirmed by further in vitro experiments. It also has to be investigated which reactions promote the degradation of 1-MT to KYNA. The missing inhibitory properties of 1-MT on IDO, determined by the in silico ODE models, align with previous research. It was shown that the saturation of 1-MT was too low, e.g. in pigs, to inhibit IDO efficiently. In this study, the first possible degradation pathway of 1-MT along the KP is proposed. The reliability of the results depends on the quality of the experimental data, and the season, when data were measured. Moreover, the results vary between the different approaches of parameter fitting. Different approaches of parameter fitting have to be included in the analysis to get more evidence for the correctness of the results.

Alternative splicing (AS) is a major mechanism for gene expression in eukaryotes, increasing proteome diversity but also regulating transcriptome abundance. High temperatures have a strong impact on the splicing profile of many genes and therefore AS is considered as an integral part of heat stress response. While many studies have established a detailed description of the diversity of the RNAome under heat stress in different plant species and stress regimes, little is known on the underlying mechanisms that control this temperature-sensitive process. AS is mainly regulated by the activity of splicing regulators. Changes in the abundance of these proteins through transcription and AS, post-translational modifications and interactions with exonic and intronic cis-elements and core elements of the spliceosomes modulate the outcome of pre-mRNA splicing. As a major part of pre-mRNAs are spliced co-transcriptionally, the chromatin environment along with the RNA polymerase II elongation play a major role in the regulation of pre-mRNA splicing under heat stress conditions. Despite its importance, our understanding on the regulation of heat stress sensitive AS in plants is scarce. In this review, we summarize the current status of knowledge on the regulation of AS in plants under heat stress conditions. We discuss possible implications of different pathways based on results from non-plant systems to provide a perspective for researchers who aim to elucidate the molecular basis of AS under high temperatures.

The innate immune system relies on families of pattern recognition receptors (PRRs) that detect distinct conserved molecular motifs from microbes to initiate antimicrobial responses. Activation of PRRs triggers a series of signaling cascades, leading to the release of pro-inflammatory cytokines, chemokines and antimicrobials, thereby contributing to the early host defense against microbes and regulating adaptive immunity. Additionally, PRRs can detect perturbation of cellular homeostasis caused by pathogens and fine-tune the immune responses. Among PRRs, nucleotide binding oligomerization domain (NOD)-like receptors (NLRs) have attracted particular interest in the context of cellular stress-induced inflammation during infection. Recently, mechanistic insights into the monitoring of cellular homeostasis perturbation by NLRs have been provided. We summarize the current knowledge about the disruption of cellular homeostasis by pathogens and focus on NLRs as innate immune sensors for its detection. We highlight the mechanisms employed by various pathogens to elicit cytoskeleton disruption, organelle stress as well as protein translation block, point out exemplary NLRs that guard cellular homeostasis during infection and introduce the concept of stress-associated molecular patterns (SAMPs). We postulate that integration of information about microbial patterns, danger signals, and SAMPs enables the innate immune system with adequate plasticity and precision in elaborating responses to microbes of variable virulence.

Simple Summary Paratuberculosis is a disease which affects ruminants worldwide. Many countries have implemented certification and monitoring systems to control the disease, particularly in dairy herds. Monitoring herds certified as paratuberculosis non-suspect is an important component of paratuberculosis herd certification programs. The challenge is to detect the introduction or reintroduction of the infectious agent as early as possible with reasonable efforts but high certainty. In our study, we evaluated different low-cost testing schemes in herds where the share of infected animals was low, resulting in a low within-herd prevalence of animals shedding the bacteria that causes paratuberculosis in their feces. The test methods used were repeated pooled milk samples and fecal samples from the barn environment. Our study showed that numerous repetitions of different samples are necessary to monitor such herds with sufficiently high certainty. In the case of herds with a very low prevalence, our study showed that a combination of different sampling approaches is required. Abstract An easy-to-use and affordable surveillance system is crucial for paratuberculosis control. The use of environmental samples and milk pools has been proven to be effective for the detection of Mycobacterium avium subsp. paratuberculosis (MAP)-infected herds, but not for monitoring dairy herds certified as MAP non-suspect. We aimed to evaluate methods for the repeated testing of large dairy herds with a very low prevalence of MAP shedders, using different sets of environmental samples or pooled milk samples, collected monthly over a period of one year in 36 herds with known MAP shedder prevalence. Environmental samples were analyzed by bacterial culture and fecal PCR, and pools of 25 and 50 individual milk samples were analyzed by ELISA for MAP-specific antibodies. We estimated the cumulative sensitivity and specificity for up to twelve sampling events by adapting a Bayesian latent class model and taking into account the between- and within-test correlation. Our study revealed that at least seven repeated samplings of feces from the barn environment are necessary to achieve a sensitivity of 95% in herds with a within-herd shedder prevalence of at least 2%. The detection of herds with a prevalence of less than 2% is more challenging and, in addition to numerous repetitions, requires a combination of different samples.

Self-similar sets with the open set condition, the linear objects of fractal geometry, have been considered mainly for crystallographic data. Here we introduce new symmetry classes in the plane, based on rotation by irrational angles. Examples without characteristic directions, with strong connectedness and small complexity, were found in a computer-assisted search. They are surprising since the rotations are given by rational matrices, and the proof of the open set condition usually requires integer data. We develop a classification of self-similar sets by symmetry class and algebraic numbers. Examples are given for various quadratic number fields.