@phdthesis{Westermann2014,
  author    = {Westermann, Alexander J.},
  title     = {Dual RNA-seq of pathogen and host},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-112462},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {The infection of a eukaryotic host cell by a bacterial pathogen is one of the most intimate examples of cross-kingdom interactions in biology. Infection processes are highly relevant from both a basic research as well as a clinical point of view. Sophisticated mechanisms have evolved in the pathogen to manipulate the host response and vice versa host cells have developed a wide range of anti-microbial defense strategies to combat bacterial invasion and clear infections. However, it is this diversity and complexity that makes infection research so challenging to technically address as common approaches have either been optimized for bacterial or eukaryotic organisms. Instead, methods are required that are able to deal with the often dramatic discrepancy between host and pathogen with respect to various cellular properties and processes. One class of cellular macromolecules that exemplify this host-pathogen heterogeneity is given by their transcriptomes: Bacterial transcripts differ from their eukaryotic counterparts in many aspects that involve both quantitative and qualitative traits. The entity of RNA transcripts present in a cell is of paramount interest as it reflects the cell's physiological state under the given condition. Genome-wide transcriptomic techniques such as RNA-seq have therefore been used for single-organism analyses for several years, but their applicability has been limited for infection studies. The present work describes the establishment of a novel transcriptomic approach for infection biology which we have termed "Dual RNA-seq". Using this technology, it was intended to shed light particularly on the contribution of non-protein-encoding transcripts to virulence, as these classes have mostly evaded previous infection studies due to the lack of suitable methods. The performance of Dual RNA-seq was evaluated in an in vitro infection model based on the important facultative intracellular pathogen Salmonella enterica serovar Typhimurium and different human cell lines. Dual RNA-seq was found to be capable of capturing all major bacterial and human transcript classes and proved reproducible. During the course of these experiments, a previously largely uncharacterized bacterial small non-coding RNA (sRNA), referred to as STnc440, was identified as one of the most strongly induced genes in intracellular Salmonella. Interestingly, while inhibition of STnc440 expression has been previously shown to cause a virulence defect in different animal models of Salmonellosis, the underlying molecular mechanisms have remained obscure. Here, classical genetics, transcriptomics and biochemical assays proposed a complex model of Salmonella gene expression control that is orchestrated by this sRNA. In particular, STnc440 was found to be involved in the regulation of multiple bacterial target mRNAs by direct base pair interaction with consequences for Salmonella virulence and implications for the host's immune response. These findings exemplify the scope of Dual RNA-seq for the identification and characterization of novel bacterial virulence factors during host infection.},
  subject      = {Transkriptomanalyse},
  language  = {en}
}
@phdthesis{Terhoeven2020,
  author    = {Terhoeven, Niklas},
  title     = {Genomics of carnivorous Droseraceae and Transcriptomics of Tobacco pollination as case studies for neofunctionalisation of plant defence mechanisms},
  doi       = {10.25972/OPUS-18971},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-189712},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Plants have evolved many mechanisms to defend against herbivores and pathogens. In many cases, these mechanisms took other duties. One example of such a neofunction- alisation would be carnivory. Carnivory evolved from the defence against herbivores. Instead of repelling the predator with a bitter taste, the plant kills it and absorbs its nutrients. A second example can be found in the pollination process. Many of the genes involved here were originally part of defence mechanisms against pathogens. In this thesis, I study these two examples on a genomic and transcriptomic level. The first project, Genomics of carnivorous Droseraceae, aims at obtaining annotated genome sequences of three carnivorous plants. I assembled the genome of Aldrovanda vesiculosa, annotated those of A. vesiculosa, Drosera spatulata and Dionaea muscipula and com- pared their genomic contents. Because of the high repetitiveness of the D. muscipula genome, I also developed reper, an assembly free method for detection, classification and quantification of repeats. With that method, we were able to study the repeats without the need of incorporating them into a genome assembly. The second large project investigates the role of DEFL (defensin-like) genes in pollen tube guidance in tobacco flowers. We sequenced the transcriptome of the SR1 strain in different stages of the pollination process. I assembled and annotated the transcriptome and searched for differentially expressed genes. We also used a method based on Hidden- Markov-Models (HMM) to find DEFLs, which I then analysed regarding their expression during the different stages of fertilisation. In total, this thesis results in annotated genome assemblies of three carnivorous Droser- aceae, which are used as a foundation for various analyses investigating the roots of car- nivory, insights into the role of DEFLs on a transcriptomic level in tobacco pollination and a new method for repeat identification in complex genomes.},
  subject      = {Droseraceae},
  language  = {en}
}
@phdthesis{Reuter2023,
  author    = {Reuter, Christian Steffen},
  title     = {Development of a tissue-engineered primary human skin infection model to study the pathogenesis of tsetse fly-transmitted African trypanosomes in mammalian skin},
  doi       = {10.25972/OPUS-25114},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-251147},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Many arthropods such as mosquitoes, ticks, bugs, and flies are vectors for the transmission of pathogenic parasites, bacteria, and viruses. Among these, the unicellular parasite Trypanosoma brucei (T. brucei) causes human and animal African trypanosomiases and is transmitted to the vertebrate host by the tsetse fly. In the fly, the parasite goes through a complex developmental cycle in the alimentary tract and salivary glands ending with the cellular differentiation into the metacyclic life cycle stage. An infection in the mammalian host begins when the fly takes a bloodmeal, thereby depositing the metacyclic form into the dermal skin layer. Within the dermis, the cell cycle-arrested metacyclic forms are activated, re-enter the cell cycle, and differentiate into proliferative trypanosomes, prior to dissemination throughout the host. Although T. brucei has been studied for decades, very little is known about the early events in the skin prior to systemic dissemination. The precise timing and the mechanisms controlling differentiation of the parasite in the skin continue to be elusive, as does the characterization of the proliferative skin-residing trypanosomes. Understanding the first steps of an infection is crucial for developing novel strategies to prevent disease establishment and its progression. A major shortcoming in the study of human African trypanosomiasis is the lack of suitable infection models that authentically mimic disease progression. In addition, the production of infectious metacyclic parasites requires tsetse flies, which are challenging to keep. Thus, although animal models - typically murine - have produced many insights into the pathogenicity of trypanosomes in the mammalian host, they were usually infected by needle injection into the peritoneal cavity or tail vein, bypassing the skin as the first entry point. Furthermore, animal models are not always predictive for the infection outcome in human patients. In addition, the relatively small number of metacyclic parasites deposited by the tsetse flies makes them difficult to trace, isolate, and study in animal hosts. The focus of this thesis was to develop and validate a reconstructed human skin equivalent as an infection model to study the development of naturally-transmitted metacyclic parasites of T. brucei in mammalian skin. The first part of this work describes the development and characterization of a primary human skin equivalent with improved mechanical properties. To achieve this, a computer-assisted compression system was designed and established. This system allowed the improvement of the mechanical stability of twelve collagen-based dermal equivalents in parallel through plastic compression, as evaluated by rheology. The improved dermal equivalents provided the basis for the generation of the skin equivalents and reduced their contraction and weight loss during tissue formation, achieving a high degree of standardization and reproducibility. The skin equivalents were characterized using immunohistochemical and histological techniques and recapitulated key anatomical, cellular, and functional aspects of native human skin. Furthermore, their cellular heterogeneity was examined using single-cell RNA sequencing - an approach which led to the identification of a remarkable repertoire of extracellular matrix-associated genes expressed by different cell subpopulations in the artificial skin. In addition, experimental conditions were established to allow tsetse flies to naturally infect the skin equivalents with trypanosomes. In the second part of the project, the development of the trypanosomes in the artificial skin was investigated in detail. This included the establishment of methods to successfully isolate skin-dwelling trypanosomes to determine their protein synthesis rate, cell cycle and metabolic status, morphology, and transcriptome. Microscopy techniques to study trypanosome motility and migration in the skin were also optimized. Upon deposition in the artificial skin by feeding tsetse, the metacyclic parasites were rapidly activated and established a proliferative population within one day. This process was accompanied by: (I) reactivation of protein synthesis; (II) re-entry into the cell cycle; (III) change in morphology; (IV) increased motility. Furthermore, these observations were linked to potentially underlying developmental mechanisms by applying single-cell parasite RNA sequencing at five different timepoints post-infection. After the initial proliferative phase, the tsetse-transmitted trypanosomes appeared to enter a reversible quiescence program in the skin. These quiescent skin-residing trypanosomes were characterized by very slow replication, a strongly reduced metabolism, and a transcriptome markedly different from that of the deposited metacyclic forms and the early proliferative trypanosomes. By mimicking the migration from the skin to the bloodstream, the quiescent phenotype could be reversed and the parasites returned to an active proliferating state. Given that previous work has identified the skin as an anatomical reservoir for T. brucei during disease, it is reasonable to assume that the quiescence program is an authentic facet of the parasite's behavior in an infected host. In summary, this work demonstrates that primary human skin equivalents offer a new and promising way to study vector-borne parasites under close-to-natural conditions as an alternative to animal experimentation. By choosing the natural transmission route - the bite of an infected tsetse fly - the early events of trypanosome infection have been detailed with unprecedented resolution. In addition, the evidence here for a quiescent, skin-residing trypanosome population may explain the persistence of T. brucei in the skin of aparasitemic and asymptomatic individuals. This could play an important role in maintaining an infection over long time periods.},
  subject      = {Trypanosoma brucei},
  language  = {en}
}
@phdthesis{MikaGospodorz2022,
  author    = {Mika-Gospodorz, Bozena},
  title     = {Development and application of bioinformatics tools for analysis of dual RNA-seq experiments},
  doi       = {10.25972/OPUS-28126},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-281264},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Dual RNA-seq captures both host and pathogen transcriptomes at the site of infection, facilitating an exploration of processes that play an essential role in pathogenesis and the host defense. This work presents an application of this technique to explore processes occurring during the infection of the human endothelial cells with two clinical isolates of Orientia tsutsugamushi (Ot) — the causative agent of scrub typhus. Combining comparative genomics, transcriptomics, and proteomics, we investigated the transcriptional architecture of Ot and identified non-coding RNAs, operon structures, and widespread antisense transcription, that may have a role in regulation of repetitive genes that are abundant in the Ot genome. In addition, the comparative analysis of bacterial and eukaryotic transcriptomes allowed us to investigate factors that drive the difference in virulence between Karp and UT176 and the host response to these two Ot strains. The host and pathogen transcriptional profiles in each dual RNA-seq study are obtained in‑silico by adopting tools developed for RNA-seq data analysis. The Dualrnaseq pipeline presented in the second part of this work is the first publicly available, highly reproducible, scalable, and user‑friendly workflow developed for processing dual RNA‑seq data of any eukaryotic and bacterial organisms with a reference genome and annotation. It provides three mapping and quantification strategies: (i) alignment-based mapping of reads onto the chimeric genome with STAR followed by counting of uniquely mapped reads with HTSeq; (ii) a fast transcriptome quantification method handling multi‑mapped reads (Salmon with Selective Alignment); (iii) and Salmon alignment-based mode which uses a STAR‑derived alignment combined with Salmon quantification. Performing an initial benchmark analysis of the employed methods we provided recommendations ensuring accurate estimation of host and pathogen transcript expression.},
  subject      = {Transkriptomanalyse},
  language  = {en}
}
@phdthesis{Herz2021,
  author    = {Herz, Michaela},
  title     = {Genome wide expression profiling of Echinococcus multilocularis},
  doi       = {10.25972/OPUS-20380},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-203802},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Alveolar echinococcosis, which is caused by the metacestode stage of the small fox tapeworm Echinococcus multilocularis, is a severe zoonotic disease with limited treatment options. For a better understanding of cestode biology the genome of E. multilocularis, together with other cestode genomes, was sequenced previously. While a few studies were undertaken to explore the E. multilocularis transcriptome, a comprehensive exploration of global transcription profiles throughout life cycle stages is lacking. This work represents the so far most comprehensive analysis of the E. multilocularis transcriptome. Using RNA-Seq information from different life cycle stages and experimental conditions in three biological replicates, transcriptional differences were qualitatively and quantitatively explored. The analyzed datasets are based on samples of metacestodes cultivated under aerobic and anaerobic conditions as well as metacestodes obtained directly from infected jirds. Other samples are stem cell cultures at three different time points of development as well as non-activated and activated protoscoleces, the larval stage that can develop into adult worms. In addition, two datasets of metacestodes under experimental conditions suitable for the detection of genes that are expressed in stem cells, the so-called germinative cells, and one dataset from a siRNA experiment were analyzed. Analysis of these datasets led to expression profiles for all annotated genes, including genes that are expressed in the tegument of metacestodes and play a role in host-parasite interactions and modulation of the host's immune response. Gene expression profiles provide also further information about genes that might be responsible for the infiltrative growth of the parasite in the liver. Furthermore, germinative cell-specific genes were identified. Germinative cells are the only proliferating cells in E. multilocularis and therefore of utmost importance for the development and growth of the parasite. Using a combination of germinative cell depletion and enrichment methods, genes with specific expression in germinative cells were identified. As expected, many of these genes are involved in translation, cell cycle regulation or DNA replication and repair. Also identified were transcription factors, many of which are involved in cell fate commitment. As an example, the gene encoding the telomerase reverse transcriptase (TERT) was studied further. Expression of E. multilocularis tert in germinative cells was confirmed experimentally. Cell culture experiments indicate that TERT is required for proliferation and development of the parasite, which makes TERT a potentially interesting drug target for chemotherapy of alveolar echinococcosis. Germinative cell specific genes in E. multilocularis also include genes of densoviral origin. More than 20 individual densovirus loci with information for non-structural and structural densovirus proteins were identified in the E. multilocularis genome. Densoviral elements were also detected in many other cestode genomes. Genomic integration of these elements suggests that densovirus-based vectors might be suitable tools for genetic manipulation of tapeworms. Interestingly, only three of more than 20 densovirus loci in the E. multilocularis genome are expressed. Since the canonical piRNA pathway is lacking in cestodes, this raises the question about potential silencing mechanisms. Exploration of RNA-Seq information indicated natural antisense transcripts as a potential gene regulation mechanism in E. multilocularis. Preliminary experiments further suggest DNA-methylation, which was previously shown to occur in platyhelminthes, as an interesting avenue to explore in future. The transcriptome datasets also contain information about genes that are expressed in differentiated cells, for example the serotonin transporter gene that is expressed in nerve cells. Cell culture experiments indicate that serotonin and serotonin transport play an important role in E. multilocularis proliferation, development and survival. Overall, this work provides a comprehensive transcription data atlas throughout the E. multilocularis life cycle. Identification of germinative cell-specific genes and genes important for host-parasite interactions will greatly facilitate future research. A global overview of gene expression profiles will also aide in the detection of suitable drug targets and the development of new chemotherapeutics against alveolar echinococcosis.},
  subject      = {Fuchsbandwurm},
  language  = {en}
}