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Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory infectious disease responsible for global economic losses in the pig industry. From a monitoring perspective as well as due to the different courses of disease associated with the various serovars, it is essential to distinguish them in different herds or countries. In this study, we developed a novel high resolution melting (HRM) assay based on reference strains for each of the 19 known serovars and additional 15 clinical A. pleuropneumoniae isolates. The novel HRM comprises the species-specific APP-HRM1 and two serovar-specific HRM assays (APP-HRM2 and APP-HRM3). APP-HRM1 allowed polymerase chain reaction (PCR) amplification of apxIV resulting in an A. pleuropneumoniae specific melting curve, while nadV specific primers differentiated biovar 2 from biovar 1 isolates. Using APP-HRM2 and APP-HRM3, 13 A. pleuropneumoniae serovars can be determined by inspecting the assigned melting temperature. In contrast, serovar 3 and 14, serovar 9 and 11, and serovar 5 and 15 have partly overlapping melting temperatures and thus represent a challenge to accurately distinguish them. Consequently, to unambiguously ensure the correct assignment of the serovar, it is recommended to perform the serotyping HRM assay using a positive control for each serovar. This rapid and user-friendly assay showed high sensitivity with 1.25 fg-125 pg of input DNA and a specificity of 100% to identify A. pleuropneumoniae. Characteristic melting patterns of amplicons might allow detecting new serovars. The novel HRM assay has the potential to be implemented in diagnostic laboratories for better surveillance of this pathogen.

Blood and plasma proteins are heavily investigated as biomarkers for different diseases. However, the post-translational modification states of these proteins are rarely analyzed since blood contains many enzymes that rapidly remove these modifications after sampling. In contrast to the well-described role of protein ADP-ribosylation in cells and organs, its role in blood remains mostly uncharacterized. Here, we discovered that plasma phosphodiesterases and/or ADP-ribosylhydrolases rapidly demodify in vitro ADP-ribosylated proteins. Thus, to identify the in vivo whole blood and plasma ADP-ribosylomes, we established a mass-spectrometry-based workflow that was applied to blood samples collected from LPS-treated pigs (Sus scrofa domesticus), which serves as a model for human systemic inflammatory response syndrome. These analyses identified 60 ADP-ribosylated proteins, 17 of which were ADP-ribosylated plasma proteins. This new protocol provides an important step forward for the rapidly developing field of ADP-ribosylation and defines the blood and plasma ADP-ribosylomes under both healthy and disease conditions.

Summary Protein ADP-ribosylation is a post-translational modification (PTM) that consists of the addition of ADP-ribose moieties to target proteins. In the nucleus, the modification is catalyzed by members of the diphtheria toxin-like ADP-ribosyl- transferases (ARTDs), of which ARTD1 is the nuclear most abundant and best studied. ADP-ribosylation has been implicated in a variety of cellular processes, ranging from maintenance of genome integrity and DNA repair and gene transcription. However, very little is known about the localization and molecular function of chromatin-associated ADP-ribosylation. In this thesis, we have investigated the mechanistic role of ARTD1-mediated chromatin poly-ADP-ribosylation (PARylation) for different cellular conditions. In the first paradigm, we investigated the role of ADP-ribosylation for the transcription of SRY (sex determining region Y)-box 2 (SOX2) target genes during the early phase of fibroblasts reprogramming to induced pluripotent stem cells (iPSC). We could show that ARTD1 and PARylation are necessary for iPS colony formation and that PAR formation keeps SOX2 associated to the DNA to efficiently transcribe SOX2 target genes, including fibroblast growth factor 4 (Fgf4). We found that exogenous FGF4 administration functionally rescues ARTD1 ablation or inhibition of PARylation. In addition, we have developed a novel chromatin affinity precipitation (ChAP) protocol to enrich ADP-ribosylated chromatin for elucidating where ADP- ribosylation is localized. We have applied this protocol to an oxidative stress paradigm and found that ADP-ribosylation induced by hydrogen peroxide (H2O2) preferentially localizes to ARTD1 and nucleosome-dense heterochromatic regions, as well as to repetitive elements within the genome. Chromatin ADP-ribosylation induced at these sites was correlated with a higher accessibility. Moreover during in vitro induced adipogenesis, ADP-ribosylation was tightly associated with peroxisome proliferator-activate