570 Biowissenschaften; Biologie
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With the advent of highthroughput sequencing (HTS), profiling immunoglobulin (IG) repertoires has become an essential part of immunological research. The dissection of IG repertoires promises to transform our understanding of the adaptive immune system dynamics. Advances in sequencing technology now also allow the use of the Ion Torrent Personal Genome Machine (PGM) to cover the full length of IG mRNA transcripts. The applications of this benchtop scale HTS platform range from identification of new therapeutic antibodies to the deconvolution of malignant B cell tumors. In the context of this thesis, the usability of the PGM is assessed to investigate the IG heavy chain (IGH) repertoires of animal models. First, an innovate bioinformatics approach is presented to identify antigendriven IGH sequences from bulk sequenced bone marrow samples of transgenic humanized rats, expressing a human IG repertoire (OmniRatTM). We show, that these rats mount a convergent IGH CDR3 response towards measles virus hemagglutinin protein and tetanus toxoid, with high similarity to human counterparts. In the future, databases could contain all IGH CDR3 sequences with known specificity to mine IG repertoire datasets for past antigen exposures, ultimately reconstructing the immunological history of an individual. Second, a unique molecular identifier (UID) based HTS approach and network property analysis is used to characterize the CLLlike CD5+ B cell expansion of A20BKO mice overexpressing a natural short splice variant of the CYLD gene (A20BKOsCYLDBOE). We could determine, that in these mice, overexpression of sCYLD leads to unmutated subvariant of CLL (UCLL). Furthermore, we found that this short splice variant is also seen in human CLL patients highlighting it as important target for future investigations. Third, the UID based HTS approach is improved by adapting it to the PGM sequencing technology and applying a custommade data processing pipeline including the ImMunoGeneTics (IMGT) database error detection. Like this, we were able to obtain correct IGH sequences with over 99.5% confidence and correct CDR3 sequences with over 99.9% confidence. Taken together, the results, protocols and sample processing strategies described in this thesis will improve the usability of animal models and the Ion Torrent PGM HTS platform in the field if IG repertoire research.
Caenorhabditis elegans Modelsysteme für Freiland und Labor Caenorhabditis elegans ist eines der am intensivsten untersuchten Tiermodelle, dabei weisen die nur unzureichenden Informationen über sein ursprüngliches Habitat eine Vielzahl offener Fragen auf. Deshalb beschäftigt sich der erste Teil vorliegender Arbeit mit einer Überprüfung der Freilandökologie und Biogeographie des Taxons. Nach den Untersuchungen spricht vieles dafür, dass die Ursprungsart der C. elegans Gruppe ein Begleiter von Gastropoden war, welche auch für ihre Ausbreitung häufig verantwortlich sind. Als Folge der Verschleppung durch Wirtstiere (u.a. die Weinbergschnecke Helix aspersa) erreichte sie ihr heutiges Areal, in allen Kontinenten. Ein nicht weniger wichtiges Ziel der vorliegenden Arbeit war die Etablierung von C. elegans als Biotest-System für die Indikation von Chemikalien-Wirkungen. In dieser Arbeit wurden ein ökotoxikologisches Testsystem entwickelt mit den C. elegans als Modellorganismus, das durch Messung von umweltbedingten intrazellulär gebildeten ROS im Gesamtorganismus eine Einschätzung der biologischen Aktivität von Chemikalien und Umweltmedien ermöglichen sollte. Als grundlegender biochemischer Parameter ist eine, die Kapazität der antioxidativen Schutzmechanismen übersteigende, ROS-Bildung ursächlich für oxidative Schädigungen der Zelle verantwortlich und damit Basis für zahlreiche pathologische Effekte. Aus den Resultaten als Bewertungsgrundlage lässt sich folgern, dass sich das erarbeitete Biotestsystem als adäquates Verfahren zur Messung von oxidativem Stress, als Folge exogener Belastung, einsetzen lässt. Damit leistet es einen Beitrag zur Beurteilung des Toxizitätspotentials von Chemikalien und Umweltproben. Zur Eignung des Einsatzes von C. elegans für Toxizitätstest wurden weitere Untersuchungen mit verschiedenen Chemikalien durchgeführt. Dabei ergibt sich bezogen auf molare Einheiten eine immer gleich bleibende Reihenfolge der Sensitivitäten gegenüber BaP > Cu2+ > AcL, ein Ergebnis welches mit Testergebnissen von anderen Versuchsorganismen zu vergleichen war. Die Wirkung von Atrazin auf die Entwicklung von C. elegans wurde auch untersucht. Es wurde festgestellt, dass Atrazin auf Wachstum und Reproduktion von C. elegans konzentrationsabhängig toxisch wirkt. Die bisherigen Ergebnisse zeigen jedoch, dass das Testsystem C. elegans hervorragend geeignet ist, um Chemikalienwirkungen frühzeitig sichtbar zu machen.
Besides well-known positive aspects of conservation tillage combined with mulching, a drawback may be the survival of phytopathogenic fungi like Fusarium species on plant residues. This may endanger the health of the following crop by increasing the infection risk for specific plant diseases. In infected plant organs, these pathogens are able to produce mycotoxins like deoxynivalenol (DON). Mycotoxins like DON persist during storage, are heat resistant and of major concern for human and animal health after consumption of contaminated food and feed, respectively. Among fungivorous soil organisms, there are representatives of the soil fauna which are obviously antagonistic to a Fusarium infection and the contamination with mycotoxins. Earthworms (Lumbricus terrestris), collembolans (Folsomia candida) and nematodes (Aphelenchoides saprophilus) provide a wide range of ecosystem services including the stimulation of decomposition processes which may result in the regulation of plant pathogens and the degradation of environmental contaminants. Several investigations under laboratory conditions and in the field were conducted to test the following hypotheses: (1) Fusarium-infected and DON-contaminated wheat straw provides a more attractive food substrate than non-infected control straw (2) the introduced soil fauna reduce the biomass of F. culmorum and the content of DON in infected wheat straw under laboratory and field conditions (3) the species interaction of the introduced soil fauna enhances the degradation of Fusarium biomass and DON concentration in wheat straw; (4) the degradation efficiency of soil fauna is affected by soil texture. The results of the present thesis pointed out that the degradation performance of the introduced soil fauna must be considered as an important contribution to the biological control of plant diseases and environmental pollutants. As in particular L. terrestris revealed to be the driver of the degradation process, earthworms contribute to a sustainable control of fungal pathogens like Fusarium and its mycotoxins in wheat straw, thus reducing the risk of plant diseases and environmental pollution as ecosystem services.
Leeches can parasitize many vertebrate taxa. In amphibians, leech parasitism often has potential detrimental effects including population decline. Most of studies on the host-parasite interactions involving leeches and amphibians focus on freshwater environments, while they are very scarce for terrestrial amphibians. In this work, we studied the relationship between the leech Batracobdella algira and the European terrestrial salamanders of the genus Hydromantes, identifying environmental features related to the presence of the leeches and their possible effects on the hosts. We performed observation throughout Sardinia (Italy), covering the distribution area of all Hydromantes species endemic to this island. From September 2015 to May 2017, we conducted >150 surveys in 26 underground environments, collecting data on 2629 salamanders and 131 leeches. Water hardness was the only environmental feature correlated with the presence of B. algira, linking this leech to active karstic systems. Leeches were more frequently parasitizing salamanders with large body size. Body Condition Index was not significantly different between parasitized and non-parasitized salamanders. Our study shows the importance of abiotic environmental features for host-parasite interactions, and poses new questions on complex interspecific interactions between this ectoparasite and amphibians.