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Agrobacterium tumefaciens is the causal agent of crown gall disease in a wide range of plants via a unique interkingdom DNA transfer from bacterial cells into the plant genome. Agrobacterium tumefaciens is capable of transferring its T-DNA into different plant parts at different developmental stages for transient and stable transformation. However, the plant genes and mechanisms involved in these transformation processes are not well understood. We used Arabidopsis thaliana Col-0 seedlings to reveal the gene expression profiles at early time points during Agrobacterium infection. Common and differentially expressed genes were found in shoots and roots. A gene ontology analysis showed that the glucosinolate (GS) biosynthesis pathway was an enriched common response. Strikingly, several genes involved in indole glucosinolate (iGS) modification and the camalexin biosynthesis pathway were up-regulated, whereas genes in aliphatic glucosinolate (aGS) biosynthesis were generally down-regulated, on Agrobacterium infection. Thus, we evaluated the impacts of GSs and camalexin during different stages of Agrobacterium-mediated transformation combining Arabidopsis mutant studies, metabolite profiling and exogenous applications of various GS hydrolysis products or camalexin. The results suggest that the iGS hydrolysis pathway plays an inhibitory role on transformation efficiency in Arabidopsis seedlings at the early infection stage. Later in the Agrobacterium infection process, the accumulation of camalexin is a key factor inhibiting tumour development on Arabidopsis inflorescence stalks. In conclusion, this study reveals the differential roles of GSs and camalexin at different stages of Agrobacterium-mediated transformation and provides new insights into crown gall disease control and improvement of plant transformation.
Im gleichen Maße wie informatisches Wissen mehr und mehr in den wissenschaftlichen Alltag aller Lebenswissenschaften Einzug gehalten hat, hat sich der Schwerpunkt bioinformatischer Forschung in stärker mathematisch und informatisch-orientierte Themengebiete verschoben. Bioinformatik heute ist mehr als die computergestützte Verarbeitung großer Mengen an biologischen Daten, sondern hat einen entscheidenden Fokus auf der Modellierung komplexer biologischer Systeme. Zur Anwendung kommen hierbei insbesondere Theorien aus dem Bereich der Stochastik und Statistik, des maschinellen Lernens und der theoretischen Informatik. In der vorliegenden Dissertation beschreibe ich in Fallstudien die systematische Modellierung biologischer Systeme aus einem informatisch - mathematischen Standpunkt unter Anwendung von Verfahren aus den genannten Teilbereichen und auf unterschiedlichen Ebenen biologischer Abstraktion. Ausgehend von der Sequenzinformation über Transkriptom, Metabolom und deren regulatorischer Interaktion hin zur Modellierung von Populationseffekten werden hierbei aktuelle biologische Fragestellungen mit mathematisch - informatischen Modellen und einer Vielzahl experimenteller Daten kombiniert. Ein besonderer Augenmerk liegt dabei auf dem Vorgang der Modellierung und des Modellbegriffs als solchem im Rahmen moderner bioinformatischer Forschung. Im Detail umfassen die Projekte (mehrere Publikationen) die Entwicklung eines neuen Ansatzes zur Einbettung und Visualisierung von Multiplen Sequenz- und Sequenz-Strukturalignments, illustriert am Beispiel eines Hemagglutininalignments unterschiedlicher H5N1 Varianten, sowie die Modellierung des Transkriptoms von A. thaliana, bei welchem mit Hilfe einer kernelisierten nicht-parametrischen Metaanalyse neue, an der Infektionsabwehr beteiligten, Gene ausfindig gemacht werden konnten. Desweiteren ist uns mit Hilfe unserer Software YANAsquare eine detaillierte Untersuchung des Metabolismus von L. monocytogenes unter Aktivierung des Transkriptionsfaktors prfA gelungen, dessen Vorhersagen durch experimentelle 13C Isotopologstudien belegt werden konnten. In einem Anschlußprojekt war der Zusammenhang zwischen Regulation des Metabolismus durch Regulation der Genexpression und der Fluxverteilung des metabolischen Steady- State-Netzwerks das Ziel. Die Modellierung eines komplexen organismischen Phänotyps, der Zellgrößenentwicklung der Diatomee Pseudo-nitzschia delicatissima, schließt die Untersuchungen ab.
Altered metabolic processes contribute to carcinogenesis by modulating proliferation, survival and differentiation. Tumours are composed of different cell populations, with cancer stem-like cells being one of the most prominent examples. This specific pool of cells is thought to be responsible for cancer growth and recurrence and plays a particularly relevant role in glioblastoma (GBM), the most lethal form of primary brain tumours. Here, we have analysed the transcriptome and metabolome of an established GBM cell line (U87) and a patient-derived GBM stem-like cell line (NCH644) exposed to neurosphere or monolayer culture conditions. By integrating transcriptome and metabolome data, we identified key metabolic pathways and gene signatures that are associated with stem-like and differentiated states in GBM cells, and demonstrated that neurospheres and monolayer cells differ substantially in their metabolism and gene regulation. Furthermore, arginine biosynthesis was identified as the most significantly regulated pathway in neurospheres, although individual nodes of this pathway were distinctly regulated in the two cellular systems. Neurosphere conditions, as opposed to monolayer conditions, cause a transcriptomic and metabolic rewiring that may be crucial for the regulation of stem-like features, where arginine biosynthesis may be a key metabolic pathway. Additionally, TCGA data from GBM patients showed significant regulation of specific components of the arginine biosynthesis pathway, providing further evidence for the importance of this metabolic pathway in GBM.
Viral infections induce a significant impact on various functional categories of biological processes in the host. The understanding of this complex modification of the infected host immune system requires a global and detailed overview on the infection process. Therefore it is essential to apply a powerful approach which identifies the involved components conferring the capacity to recognize and respond to specific pathogens, which in general are defeated in so-called compatible virus-plant infections. Comparative and integrated systems biology of plant-virus interaction progression may open a novel framework for a systemic picture on the modulation of plant immunity during different infections and understanding pathogenesis mechanisms. In this thesis these approaches were applied to study plant-virus infections during two main viral pathogens of cassava: Cassava brown streak virus and African cassava mosaic virus.
Here, the infection process was reconstructed by a combination of omics data-based analyses and metabolic network modelling, to understand the major metabolic pathways and elements underlying viral infection responses in different time series, as well as the flux activity distribution to gain more insights into the metabolic flow and mechanism of regulation; this resulted in simultaneous investigations on a broad spectrum of changes in several levels including the gene expression, primary metabolites, and enzymatic flux associated with the characteristic disease development process induced in Nicotiana benthamiana plants due to infection with CBSV or ACMV.
Firstly, the transcriptome dynamics of the infected plant was analysed by using mRNA-sequencing, in order to investigate the differential expression profile according the symptom developmental stage. The spreading pattern and different levels of biological functions of these genes were analysed associated with the infection stage and virus entity. A next step was the Real-Time expression modification of selected key pathway genes followed by their linear regression model. Subsequently, the functional loss of regulatory genes which trigger R-mediated resistance was observed. Substantial differences were observed between infected mutants/transgenic lines and wild-types and characterized in detail. In addition, we detected a massive localized accumulation of ROS and quantified the scavenging genes expression in the infected wild-type plants relative to mock infected controls.
Moreover, we found coordinated regulated metabolites in response to viral infection measured by using LC-MS/MS and HPLC-UV-MS. This includes the profile of the phytohormones, carbohydrates, amino acids, and phenolics at different time points of infection with the RNA and DNA viruses. This was influenced by differentially regulated enzymatic activities along the salicylate, jasmonate, and chorismate biosynthesis, glycolysis, tricarboxylic acid cycle, and pentose phosphate pathways, as well as photosynthesis, photorespiration, transporting, amino acid and fatty acid biosynthesis. We calculated the flux redistribution considering a gradient of modulation for enzymes along different infection stages, ranging from pre-symptoms towards infection stability.
Collectively, our reverse-engineering study consisting of the generation of experimental data and modelling supports the general insight with comparative and integrated systems biology into a model plant-virus interaction system. We refine the cross talk between transcriptome modification, metabolites modulation and enzymatic flux redistribution during compatible infection progression. The results highlight the global alteration in a susceptible host, correlation between symptoms severity and the alteration level. In addition we identify the detailed corresponding general and specific responses to RNA and DNA viruses at different stages of infection. To sum up, all the findings in this study strengthen the necessity of considering the timing of treatment, which greatly affects plant defence against viral infection, and might result in more efficient or combined targeting of a wider range of plant pathogens.
Background: The transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is mediated by dormant sexual precursor cells, the gametocytes, which become activated in the mosquito midgut. Because gametocytes are the only parasite stages able to establish an infection in the mosquito, they play a crucial role in spreading the tropical disease. The human-to-mosquito transmission triggers important molecular changes in the gametocytes, which initiate gametogenesis and prepare the parasite for life-cycle progression in the insect vector.
Results: To better understand gene regulations during the initial phase of malaria parasite transmission, we focused on the transcriptome changes that occur within the first half hour of parasite development in the mosquito. Comparison of mRNA levels of P. falciparum gametocytes before and 30 min following activation using suppression subtractive hybridization (SSH) identified 126 genes, which changed in expression during gametogenesis. Among these, 17.5% had putative functions in signaling, 14.3% were assigned to cell cycle and gene expression, 8.7% were linked to the cytoskeleton or inner membrane complex, 7.9% were involved in proteostasis and 6.4% in metabolism, 12.7% were cell surface-associated proteins, 11.9% were assigned to other functions, and 20.6% represented genes of unknown function. For 40% of the identified genes there has as yet not been any protein evidence. For a subset of 27 genes, transcript changes during gametogenesis were studied in detail by real-time RT-PCR. Of these, 22 genes were expressed in gametocytes, and for 15 genes transcript expression in gametocytes was increased compared to asexual blood stage parasites. Transcript levels of seven genes were particularly high in activated gametocytes, pointing at functions downstream of gametocyte transmission to the mosquito. For selected genes, a regulated expression during gametogenesis was confirmed on the protein level, using quantitative confocal microscopy.
Conclusions: The obtained transcriptome data demonstrate the regulations of gene expression immediately following malaria parasite transmission to the mosquito. Our findings support the identification of proteins important for sexual reproduction and further development of the mosquito midgut stages and provide insights into the genetic basis of the rapid adaption of Plasmodium to the insect vector.
Staphylococcus aureus ist ein bedeutender opportunistischer Krankheitserreger, der eine Vielzahl von Infektionen in Menschen und Tieren hervorrufen kann. Das Krankheitsbild reicht von leichten Hautinfektionen bis hin zu lebensbedrohlichen Infektionen wie Endokarditis, Sepsis oder Pneumonien. S. aureus ist ein Haupterreger nosokomialer Infektionen. Besonders die Antibiotikaresistenzentwicklung von S. aureus–Stämmen ist problematisch. Als wirksame Antibiotika können zur Zeit oft nur noch Vancomycin, Synercid oder Linezolid zur Therapie eingesetzt werden. Die alarmierende Resistenzentwicklung in S. aureus verdeutlicht, dass die Entwicklung neuer Antibiotika und die Identifizierung neuer bakterieller Angriffsstrukturen dringend erforderlich ist. Gängige antiinfektive Therapeutika sind gegen die bakterielle Zellwandsynthese, den DNA- und RNA-Stoffwechsel oder die Proteinbiosynthese gerichtet. In dieser Arbeit sollten Virulenz-relevante Zielstrukturen für die Entwicklung neuer Antibiotika untersucht werden. Insgesamt wurden sieben Gene analysiert, von denen vier zu Anfang dieser Arbeit in S. aureus noch nicht charakterisiert waren. Die Zielgene (clpP, purH, ssrA und smpB) in S. aureus sollten deletiert werden, um ihre Überlebensnotwendigkeit in vitro- und in vivo zu überprüfen. Eine Deletion gelang bei den Genen clpP und purH, die somit als nicht essenziell in S. aureus zu betrachten sind. Die bereits zuvor als nicht-essenziell charakterisierten Gene arlR, arlS und putP wurden deletiert und die Mutanten dclpP, darlR, darlS, dpurH und dputP wurden phänotypisch in Hinsicht auf ihren Einfluss auf die Pathogenität in S. aureus analysiert. Die differenzielle Genexpression der Mutanten dclpP und darlR wurde mit Hilfe von Microarray-Hybridisierungsexperimenten untersucht. Die ∆clpP-Mutante zeigte einen starken Wachstumsdefekt bei verschiedenen Temperaturen (30, 37, 42°C) und war nicht mehr in der Lage bei 20°C zu wachsen. Ebenso war das Wachstum unter anaeroben Bedingungen stark beeinträchtigt. Der Stamm dclpP wies eine verringerte hämolytische Aktivität sowie eine verminderte Adhärenz an Polystyren auf. Außerdem konnte eine stark erhöhte autolytische Aktivität in einem Triton X-100-Assay beobachtet werden. In einem Invasions-Zellkulturassay mit 293T-Epithelzellen konnte eine ~10-fach erhöhte Invasivität im Vergleich zu dem isogenen Wildtyp festgestellt werden. Die Komplementierung der ∆clpP-Mutante durch Einführung eines clpP-Expressionsvektors führte nahezu bei allen getesteten Bedingungen zur Wiederherstellung des wildtypischen Phänotyps. Die Transkriptomanalyse der dclpP-Mutante ergab eine deutliche Veränderung in der Genexpression (15 % aller Gene). Eine computerunterstützte Analyse der Upstreambereiche der deregulierten Gene führte zu der Identifizierung verschiedener Regulons, die bei der bakteriellen Antwort auf verschiedene Stressbedingungen eine Rolle spielen. Die clpP-Deletion betrifft besonders Regulatoren, deren Aktivität in Abhängigkeit zu veränderten Redox-Bedingungen reguliert wird, wie z. B. verschiedenen Stressbedingungen und Anaerobiose. Die Konstruktion der darlR- und darlS-Mutanten führte zu einer gesteigerten hämolytischen Aktivität, einer erhöhten Adhärenz an Polystyren sowie einer erhöhten autolytischen Aktivität in Triton X-100-Assays. Die Internalisierungsrate durch 293T-Epithelzellen war vermindert. Die darlR-Mutante wurde in einem Katheter-assoziierten Infektionsmodell in Ratten eingesetzt. Die kompetitive Infektion mit Mutante und Wildtyp ergab einen deutlichen Nachteil bei der Etablierung einer Infektion durch die Mutante. Die Transkriptomanalyse der 8325darlR-Mutante in der exponenziellen und in der stationären Phase unterstreicht den großen Einfluss des ArlRS-Zwei-Komponenten-Systems auf die Regulation der Genexpression in S. aureus. In der exponenziellen Phase wurden insgesamt 5 % und in der stationären Phase 15 % der Gene differenziell exprimiert. dpurH- und dputP-Mutanten wiesen in vitro keine Veränderungen im Wachstums-verhalten, der Biofilmbildung oder hämolytischen Aktivität auf. In einem Infektionsmodell in Ratten führte die Deletion von purH in dem S. aureus-Stamm MA12 zu einer signifikanten Verminderung der Virulenz. Die Herstellung von smpB- und ssrA-Deletionsmutanten verlief ohne Erfolg. Es wurde versucht, einen direkten Nachweis für den essenziellen Charakter dieser Gene durch den Einsatz konditional letaler Expressionssysteme zu erbringen. Weder der Austausch des wildtypischen durch einen regulierbaren Promotor noch eine Antisense-RNA-Strategie war für eine eindeutige Klärung dieser Frage ausreichend. Es konnte durch diese Arbeit jedoch gezeigt werden, dass die Antisense-RNA-Strategie eine Beeinträchtigung des Wachstums von S. aureus bewirkt.
(1) Background: C-X-C Motif Chemokine Receptor 4 (CXCR4) and Fibroblast Activation Protein Alpha (FAP) are promising theranostic targets. However, it is unclear whether CXCR4 and FAP positivity mark distinct microenvironments, especially in solid tumors. (2) Methods: Using Random Forest (RF) analysis, we searched for entity-independent mRNA and microRNA signatures related to CXCR4 and FAP overexpression in our pan-cancer cohort from The Cancer Genome Atlas (TCGA) database — representing n = 9242 specimens from 29 tumor entities. CXCR4- and FAP-positive samples were assessed via StringDB cluster analysis, EnrichR, Metascape, and Gene Set Enrichment Analysis (GSEA). Findings were validated via correlation analyses in n = 1541 tumor samples. TIMER2.0 analyzed the association of CXCR4 / FAP expression and infiltration levels of immune-related cells. (3) Results: We identified entity-independent CXCR4 and FAP gene signatures representative for the majority of solid cancers. While CXCR4 positivity marked an immune-related microenvironment, FAP overexpression highlighted an angiogenesis-associated niche. TIMER2.0 analysis confirmed characteristic infiltration levels of CD8+ cells for CXCR4-positive tumors and endothelial cells for FAP-positive tumors. (4) Conclusions: CXCR4- and FAP-directed PET imaging could provide a non-invasive decision aid for entity-agnostic treatment of microenvironment in solid malignancies. Moreover, this machine learning workflow can easily be transferred towards other theranostic targets.
Fungal infections are a major global health burden where Candida albicans is among the most common fungal pathogen in humans and is a common cause of invasive candidiasis. Fungal phenotypes, such as those related to morphology, proliferation and virulence are mainly driven by gene expression, which is primarily regulated by kinase signaling cascades. Serine-arginine (SR) protein kinases are highly conserved among eukaryotes and are involved in major transcriptional processes in human and S. cerevisiae. Candida albicans harbors two SR protein kinases, while Sky2 is important for metabolic adaptation, Sky1 has similar functions as in S. cerevisiae. To investigate the role of these SR kinases for the regulation of transcriptional responses in C. albicans, we performed RNA sequencing of sky1Δ and sky2Δ and integrated a comprehensive phosphoproteome dataset of these mutants. Using a Systems Biology approach, we study transcriptional regulation in the context of kinase signaling networks. Transcriptomic enrichment analysis indicates that pathways involved in the regulation of gene expression are downregulated and mitochondrial processes are upregulated in sky1Δ. In sky2Δ, primarily metabolic processes are affected, especially for arginine, and we observed that arginine-induced hyphae formation is impaired in sky2Δ. In addition, our analysis identifies several transcription factors as potential drivers of the transcriptional response. Among these, a core set is shared between both kinase knockouts, but it appears to regulate different subsets of target genes. To elucidate these diverse regulatory patterns, we created network modules by integrating the data of site-specific protein phosphorylation and gene expression with kinase-substrate predictions and protein-protein interactions. These integrated signaling modules reveal shared parts but also highlight specific patterns characteristic for each kinase. Interestingly, the modules contain many proteins involved in fungal morphogenesis and stress response. Accordingly, experimental phenotyping shows a higher resistance to Hygromycin B for sky1Δ. Thus, our study demonstrates that a combination of computational approaches with integration of experimental data can offer a new systems biological perspective on the complex network of signaling and transcription. With that, the investigation of the interface between signaling and transcriptional regulation in C. albicans provides a deeper insight into how cellular mechanisms can shape the phenotype.
The blunt snout bream Megalobrama amblycephala is the economically most important cyprinid fish species. As an herbivore, it can be grown by eco-friendly and resource-conserving aquaculture. However, the large number of intermuscular bones in the trunk musculature is adverse to fish meat processing and consumption. As a first towards optimizing this aquatic livestock, we present a 1.116-Gb draft genome of M. amblycephala, with 779.54 Mb anchored on 24 linkage groups. Integrating spatiotemporal transcriptome analyses, we show that intermuscular bone is formed in the more basal teleosts by intramembranous ossification and may be involved in muscle contractibility and coordinating cellular events. Comparative analysis revealed that olfactory receptor genes, especially of the beta type, underwent an extensive expansion in herbivorous cyprinids, whereas the gene for the umami receptor T1R1 was specifically lost in M. amblycephala. The composition of gut microflora, which contributes to the herbivorous adaptation of M. amblycephala, was found to be similar to that of other herbivores. As a valuable resource for the improvement of M. amblycephala livestock, the draft genome sequence offers new insights into the development of intermuscular bone and herbivorous adaptation.
Background
Prokaryotes have relatively small genomes, densely-packed with protein-encoding sequences. RNA sequencing has, however, revealed surprisingly complex transcriptomes and here we report the transcripts present in the model hyperthermophilic Archaeon, Thermococcus kodakarensis, under different physiological conditions.
Results
Sequencing cDNA libraries, generated from RNA isolated from cells under different growth and metabolic conditions has identified >2,700 sites of transcription initiation, established a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements. The primary transcription start sites (TSS) upstream of 1,254 annotated genes, plus 644 primary TSS and their promoters within genes, are identified. Most mRNAs have a 5'-untranslated region (5'-UTR) 10 to 50 nt long (median = 16 nt), but ~20% have 5'-UTRs from 50 to 300 nt long and ~14% are leaderless. Approximately 50% of mRNAs contain a consensus ribosome binding sequence. The results identify TSS for 1,018 antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mRNA, and confirm the presence of transcripts from all three CRISPR loci, the RNase P and 7S RNAs, all tRNAs and rRNAs and 69 predicted snoRNAs. Two putative riboswitch RNAs were present in growing but not in stationary phase cells. The procedure used is designed to identify TSS but, assuming that the number of cDNA reads correlates with transcript abundance, the results also provide a semi-quantitative documentation of the differences in T. kodakarensis genome expression under different growth conditions and confirm previous observations of substrate-dependent specific gene expression. Many previously unanticipated small RNAs have been identified, some with relative low GC contents (≤50%) and sequences that do not fold readily into base-paired secondary structures, contrary to the classical expectations for non-coding RNAs in a hyperthermophile.
Conclusion
The results identify >2,700 TSS, including almost all of the primary sites of transcription initiation upstream of annotated genes, plus many secondary sites, sites within genes and sites resulting in antisense transcripts. The T. kodakarensis genome is small (~2.1 Mbp) and tightly packed with protein-encoding genes, but the transcriptomes established also contain many non-coding RNAs and predict extensive RNA-based regulation in this model Archaeon.