@phdthesis{Ye2023,
  author    = {Ye, Liqing},
  title     = {RNA-RNA interactions in viral genome packaging},
  doi       = {10.25972/OPUS-29636},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-296361},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {RNA is one of the most abundant macromolecules and plays essential roles in numerous biological processes. This doctoral thesis consists of two projects focusing on RNA structure and RNA-RNA interactions in viral genome packaging. In the first project I developed a method called Functional Analysis of RNA Structure (FARS-seq) to investigate structural features regulating genome dimerization within the HIV-1 5'UTR. Genome dimerization is a conserved feature of retroviral replication and is thought to be a prerequisite for binding to the viral structural protein Pr55Gag during genome packaging. It also plays a role in genome integrity and evolution through recombination, and is linked to a structural switch that may regulate genome packaging and translation within cells. Despite its importance for HIV-1 replication, the RNA signals regulating genome dimerization, and the molecular mechanism leading to the selection of the genome dimer over the monomer for packaging are incompletely understood. The FARS-seq method combines RNA structural information obtained by chemical probing with single nucleotide resolution profiles of RNA function obtained by mutational interference. In this way, we found nucleotides that were critical for dimerization, especially within the well-characterized dimerization motif within stem-loop 1 (SL1). We also found stretches of nucleotides that enhanced genome dimerization upon mutation, suggesting their role in negatively regulating dimerization. A structural analysis identified distinct structural signatures within monomeric and dimeric RNA. The dimeric conformation displayed the canonical transactivation response (TAR), PolyA, primer binding site (PBS), and SL1-SL3 stem-loops, and contained a long range U5-AUG interaction. Unexpectedly, in monomeric RNA, SL1 was reconfigured into long- and short-range base-pairings with PolyA and PBS, respectively. Intriguingly, these base pairings concealed the palindromic sequence needed for dimerization and disrupted the internal loop in SL1 previously shown to contain the major packaging motif for Pr55Gag. We therefore rationally introduced mutations into PolyA and PBS, and showed how these regions regulate genome dimerization, and the binding of Pr55Gag in vitro, as well as genome packaging into virions. These findings give insights into late stages of the HIV-1 life cycle and a mechanistic explanation for the link between RNA dimerization and packaging. In the second project, I developed a proximity ligation and high-throughput sequencing-based method, RNA-RNA seq, which can measure direct (RNA-RNA) and indirect (protein-mediated) interactions. In contrast to existing methods, RNA-RNA seq is not limited by specific protein or RNA baits, nor to a particular crosslinking reagent. The genome of influenza A virus contains eight segments, which assemble into a "7+1" supramolecular complex. However, the molecular details of genome assembly are poorly understood. Our goal is to use RNA-RNA seq to identify the sites of interaction between the eight genomic RNAs of influenza, and to use this information to define the quaternary RNA architecture of the genome. We showed that RNA-RNA seq worked on model substrates, like the HIV-1 Dimerization Initiation Site (DIS) RNA and purified ribosome, as well as influenza A virus infected cells.},
  subject      = {RNS-Viren},
  language  = {en}
}
@phdthesis{Vafadarnejad2022,
  author    = {Vafadarnejad, Ehsan},
  title     = {Implementation and application of bioinformatics methods to analyze and visualize single-cell RNA-sequencing data},
  doi       = {10.25972/OPUS-26925},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-269258},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {RNA sequencing (RNA-seq) has become a transformative method to profile genome-wide gene expression and whole transcriptome analysis over the last decade. In recent years, with the development of new technologies, it has become possible to study gene expression at single-cell level. This new advances in single-cell RNA-sequencing has revolutionized the way scientists study biological processes. Single-cell RNA-sequencing has been used in different areas to better understand the underlying mechanisms of biological processes. In particular, single-RNA-sequencing is a suitable method to study infectious diseases. Infection is composed of heterogeneous mechanisms on either the host or pathogen side and the best way to understand the heterogeneity of these mechanisms and how they interact with each other is to study infectious diseases at the single-cell level. Studying infection processes at the single-cell level can reveal not only the heterogeneity but also the dynamics of infection and the interplay between the host and pathogen at the molecular level. In this thesis, we implemented and applied different single-cell RNA-seq technologies to better understand infectious diseases. In the present work, we conducted four independent but related research works to shed light on different aspects of infection biology: ● We took advantage of this novel technology to study the consequences of RSV infection on primary human epithelial cells. The primary human epithelial cells were collected from six donors and cultured in air liquid interface (ALI) cell culture inoculated with respiratory syncytial virus (RSV). In this project, we discovered ciliated cells as the susceptible cell types in RSV infection. We applied viral load as an indicator of infection progression and used it to reconstruct the dynamics of host response to RSV infection. Reconstruction of the dynamics of infection revealed many host genes and pathways that were suppressed or induced as a result of RSV infection. Pathways related to innate immune response and interferon response were suppressed during the progression of infection and on the other hand pathways like protein targeting to endoplasmic reticulum and apoptosis were induced. ● We developed a new method which is capable of sequencing the transcriptome of a bacterium at the single-cell level and potentially can help us to characterize the bacterial heterogeneity during the course of infection. In this research project, bacteria were cultured in three different culture conditions namely Late stationary phase, Anaerobic shock and NaCl shock and we used a poly(A)-independent single-cell RNA-sequencing protocol to sequence bacteria at the single-cell level. In this work, we report the faithful capture of growth-dependent gene expression patterns in individual Salmonella and Pseudomonas bacteria. The results of our analysis showed that not only we could capture transcripts across different RNA classes but also our method is capable of discerning the transcriptome of bacteria across different culture conditions. ● We used single-cell RNA-sequencing technology to characterize the immune cells landscape over the course of atherosclerosis. Atherosclerosis is considered a cardiac disease which is highly related to infections and previous infections with bacteria or viruses is considered as a risk factor for atherosclerosis. We performed single-cell RNA sequencing of aortic CD45+ cells extracted from healthy and atherosclerotic aorta of mice. We managed to find certain cell populations which were specifically present in atherosclerotic mice. One of the atheroschelorotic populations was previously undescribed TREM2high macrophages showing enrichment in Trem2 gene expression. This population of macrophages seemed to be involved in functions like lipid metabolism and catabolism and lesion calcification. This work revealed the phenotypic heterogeneity and immune cells landscape of different immune cell populations at different stages of atherosclerosis. Our work paves the way to better describe the relation between different infectious diseases and cardiovascular diseases. ● We developed a web-based platform called Infection Atlas to browse and visualize single-cell RNA-sequencing data. Infection Atlas platform provides a user-friendly interface to study different aspects of infectious diseases at the single-cell level and can potentially promote targeted approaches to intervene in infectious diseases. This platform which is available at infection-atlas.org in the short term provides a user-friendly interface to browse and visualize different aspects of infectious diseases and in the long-term is expected to be a comprehensive atlas of infection in human and mouse across different tissues and different pathogens. Overall, in this thesis we provide a framework to study infectious diseases at the single cell level with providing novel data analysis methods and this thesis paves the way for future studies to study host-pathogen encounters at the single-cell level.},
  subject      = {Einzelzellanalyse},
  language  = {en}
}
@phdthesis{Pekarek2024,
  author    = {Pek{\´a}rek, Luk{\´a}š},
  title     = {Single-Molecule Approaches To Study Frameshifting Mechanisms},
  doi       = {10.25972/OPUS-34611},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-346112},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {The RNAs of many viruses contain a frameshift stimulatory element (FSE) that grants access to an alternate reading frame via -1 programmed ribosomal frameshifting (PRF). This -1PRF is essential for effective viral replication. The -1PRF efficiency relies on the presence of conserved RNA elements within the FSE, such as a slippery sequence, spacer, and a downstream secondary structure - often a hairpin or a pseudoknot. The PRF efficiency is also affected by trans-acting factors such as proteins, miRNAs and metabolites. The interactions of these factors with the RNA and the translation machinery have not yet been completely understood. Traditional ensemble methods used previously to study these events focus on the whole population of molecular species. This results in innate averaging of the molecular behavior and a loss of heterogeneity information. Here, we first established the experimental workflow to study the RNA structures and the effect of potential trans-acting factors using single-molecule force spectroscopy technique, optical tweezers. Additionally, to streamline the data analysis, we developed an algorithm for automatized data processing. Next, we harnessed this knowledge to study viral RNA elements responsible for stimulation of PRF and how the presence of trans-acting factors affects the RNA behavior. We further complemented these single-molecule structural data with ensemble functional assays to gain a complex view on the dynamics behind the programmed ribosomal frameshifting. Specifically, two different viral RNA elements have been studied in the presented work. First, the dynamics of SARS-CoV-2 FSE and the role of extended sequences have been explored. Then, the mode of action of the host-encoded trans-acting factor ZAP-S inhibition of SARS-CoV-2 PRF has been examined. Finally, the mechanism of the trans-acting viral factor induced PRF in Encephalomyocarditis virus (EMCV) has been uncovered.},
  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{Imdahl2023,
  author    = {Imdahl, Fabian Dominik},
  title     = {Development of novel experimental approaches to decipher host-pathogen interaction at the single-cell level},
  doi       = {10.25972/OPUS-28943},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-289435},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Abstract: COVID-19 has impressively shown how quickly an emerging pathogen can have a massive impact on our entire lives and show how infectious diseases spread regardless of national borders and economic stability. We find ourselves in a post-antibiotic era and have rested too long on the laurels of past research, so today more and more people are dying from infections with multi-resistant germs. Infections are highly plastic and heterogeneous processes that are strongly dependent on the individual, whether on the host or pathogen side. Improving our understanding of the pathogenicity of microorganisms and finding potential targets for a completely new class of drugs is a declared goal of current basic research. To tackle this challenge, single-cell RNA sequencing (scRNA-seq) is our most accurate tool. In this thesis we implemented different state of the art scRNA-seq technologies to better understand infectious diseases. Furthermore, we developed a new method which is capable to resolve the transcriptome of a single bacterium. Applying a poly(A)-independent scRNA-seq protocol to three different, infection relevant growth conditions we can report the faithful detection of growth-dependent gene expression patterns in individual Salmonella Typhimurium and Pseudomonas aeruginosa bacteria. The data analysis shows that this method not only allows the differentiation of various culture conditions but can also capture transcripts across different RNA species. Furthermore, using state of the art imaging and single-cell RNA sequencing technologies, we comprehensively characterized a human intestinal tissue model which in further course of the project was used as a Salmonella enterica serovar Typhimurium infection model. While most infection studies are conducted in mice, lacking a human intestinal physiology, the in vitro human tissue model allows us to directly infer in vivo pathogenesis. Combining immunofluorescent imaging, deep single-cell RNA sequencing and HCR-FISH, applied in time course experiments, allows an unseen resolution for studying heterogeneity and the dynamics of Salmonella infection which reveals details of pathogenicity contrary to the general scientific opinion.},
  subject      = {Salmonella},
  language  = {en}
}