4 research outputs found

    Die Rolle von SPON2 im Rahmen der epithelial-mesenchymalen Transition.

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    Epithelial-mesenchymal transition (EMT) describes a process of differentiation of epithelial cells into cells with mesenchymal properties. EMT is indispensable for normal development, including formation of mesoderm and neural crest, and the same mechanism is also abused by tumour cells to promote metastasis and generation of tumour derived supportive stroma. Analysis of a human small cell lung carcinoma cell line that exhibited simultaneous adherent and suspensive growth demonstrated a mesenchymal gene expression signature in the adherent fraction, which was absent from the suspensive subpopulation. In xenograft transplantation experiments it turned out that only the mesenchymal subpopulation was able to invade the host tissue. SPON2 was among the few differentially expressed genes in the two subpopulations, suggesting this gene as a cause or early consequence of EMT in this cell line. The few reports published till now describe SPON2 as an extra cellular matrix protein involved in axon guidance, immune response and adhesion. More recently it has also been reported as a prognostic marker for ovarian and prostate cancer. A fetal lung fibroblast cell line characterized by high SPON2 expression was chosen to broaden the understanding of the biological function of this protein. In interphase cells SPON2 was observed as fibrous meshwork in the cytoplasm and as distinct speckles within the nucleus. During mitosis and cytokinesis SPON2 expression was limited to the spindle apparatus and the midbody, respectively. The molecular consequences of transient siRNA knockdown were monitored at the RNA level. Genes deregulated by reduced levels of SPON2 were significantly enriched for Gene Ontology terms such as EMT, adhesion and motility. Pathway analysis indicated that these changes were likely to be mediated by triggering the TGF-beta signalling pathway. In silico prediction of potential disease association highlighted fibrosis, rheumatoid arthritis and tumour progression, all of which are known to be related to EMT. Co-immunoprecipitation followed by mass spectrometry significantly linked SPON2 to migration and the cytoskeleton, which nicely corroborated the results obtained by immunofluorescence. Both gene expression and co-immunoprecipitation data were subjected to network analysis. The vast majority of potential SPON2 interaction partners, as highlighted by these two independent experiments, were integrated in one comprehensive network indicating the plausibility of the results. In conclusion, data generated in this thesis suggest that SPON2 has an intracellular function, and that deregulation of this protein in tumour cells may enhance their metastatic capacity via EMT. Furthermore congenital or somatic mutations of SPON2 may be responsible for EMT associated late onset disorders including rheumatoid arthritis and fibrosis.Der Prozess in dem Epithelzellen in Zellen mit mesenchymalen Eigenschaften übergehen wird allgemein als Epitheliale-Mesenchymale Transition (EMT) bezeichnet. EMT ist unerlässlich für normales Wachstum, einschließlich der Bildung des Mesoderms und der Neuralleiste, allerdings wird derselbe Mechanismus von Tumorzellen bei der Metastasenbildung und zur Entstehung von tumorösem Stroma missbraucht. Die Untersuchung einer humanen kleinzelligen Lungenkarzinomzelllinie, die gleichzeitig adhärentes und suspensives Wachstum zeigt, hat für die adhärierende Fraktion ein mesenchymales Genexpressionsmuster ergeben, dass sich bei der suspensiven Subpopulation nicht beobachten lässt. In Xenograftexperimenten zeigte sich, dassnur die mesenchymale Subpopulation zur Invasion des Wirtsgewebes fähig war.Genexpressionsanalysen beider Subpopulationen identifizierten SPON2 als eines der wenigen Gene mit unterschiedlicher Expressionshöhe, was darauf hinweist das SPON2 die Ursache oder eine frühe Folge von EMT für diese Zelllinie darstellt. Die wenigen bisher erschienenen Arbeiten beschreiben SPON2 als ein Protein der extrazellulären Matrix, das in die Axon Leitung/Zielfindung, die Immunantwort und die Adhäsion involviert ist. Jüngsten Berichten zufolge ist es auch prognostischer Marker für Ovarial- und Prostatakrebs. Eine fötale Lungenfibroblastenzelllinie, die sich durch hohe SPON2-Expression auszeichnet, wurde für das vertiefte Studium der biologischen Funktionen dieses Proteins ausgewählt. In der Interphase ließ sich SPON2 als fibröses Netzwerk im Zytoplasma und als wohlabgegrenzte Flecken im Zellkern beobachten. Während der Mitose und Zytokinese war die Lokalisation von SPON2 auf den Spindelapparat bzw. die Zellteilungsfurche beschränkt. Die molekularen Konsequenzen eines transienten siRNA-Knockdown wurden auf RNA-Ebene beobachtet. Gene die durch den Knockdown von SPON2 dereguliert wurden wiesen eine signifikante Anreicherung der „Gene Ontology“ Begriffen wie EMT, Adhäsion und Motilität auf. Signalwegsanalysen ließen vermuten, dass diese Veränderungen durch das Auslösen des TGF-beta Signalwegs vermittelt werden. In silico Vorhersagen möglicher Krankheitsassoziationen verwiesen auf Fibrose, rheumatoide Arthritis und Tumorprogression, von denen alle bekanntermaßen mit EMT zusammenhängen. Koimmunopräzipitation, gefolgt von Massenspektrometrie, stellte die Verbindung zwischen SPON2 zu Migration und Zytoskelett her, was die Ergebnisse der Immunofluoreszenzuntersuchungen noch zusätzlich bekräftigten. Die Datenkonvolute aus den Expressionsstudien und der Koimmunopräzipitation, wurden unabhängigen Netzwerkanalysen unterzogen durch welche potentielle Interaktionspartner von SPON2 identifiziert wurden. Die überwiegende Mehrheit der hierdurch identifizierten Interaktionspartner konnte im Anschluss in ein umfassendes Netzwerk integriert werden, welches die Plausibilität der Ergebnisse unterstreicht. Zusammenfassend deuten die Ergebnisse dieser Arbeit auf eine intrazelluläre Funktion von SPON2 hin, dessen Deregulation in Tumorzellen, deren metastatische Fähigkeit über EMT steigern kann. Darüberhinaus könnten erbliche oder somatische Mutationen von SPON2 für EMT-assoziierte, spät manifestierende Krankheiten wie rheumatoide Arthritis und Fibrosen verantwortlich sein

    Development of modern design solutions to increase the stone buildings’ seismic resistance in the reconstruction process

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    The article discusses the possible design solutions to improve the seismic resistance of stone buildings. In particular - the device of anti-seismic belts at the floor level, the device of dowels and nodes for connecting the floor slabs with the walls and among themselves, the reinforcement of the walls with steel clips, as well as the installation of transverse frames, if the maximum distances between the transverse walls’ axes are not observed

    Design of mobile medical testing laboratories with the focus on COVID-19 testing procedures

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    The COVID-19 pandemic caused by corona virus in 2019 has forced all the health and public organi-zations, across the globe, to take different measures for identification, prevention, and control of the disease. Early and accurate detection of the corona virus in human body being one of the im-portant measures to prevent the disease spread in population and successful eradication. However, available and known methods and technologies and project management systems in this context have not been used to the extent necessary to adequately control infection events (Lehrach, 2020; Glitscher, 2021). Today different testing methods are being used to check the COVID-19 infection for isolation of in-fected from uninfected population. The accuracy, time between sample collection to results declara-tion, testing cost, etc. depend on the type of testing method. In addition to the use of accurate testing method, it becomes necessary to ensure that the test centers are mobile and accessible to a maximum extent, so that the testing intensity can be regulated depending on the rate of spread, potential sudden outbreaks, areas of concern, etc. In this context, a new high throughput genome -based testing pipeline for SARS-CoV-2 is proposed (Lehrach, 2022) which uses the combination of state-of-the art biotechnology and in-formatics. This proposed new method is claimed to deliver faster and cheaper test results at the population scale, without compromising the testing quality. This study is aimed to develop a project plan to design such a container-based COVID- 19 testing facility. Literature study shows a lack of frameworks to develop a design for mobile medical facility (Carl Heneghan et al. 2020; Bridges et al. 2014). On the other hand, at least in Germany, companies like centogene have deployed container-based mobile test stations. However, based on available experience, these are limited in terms of use and throughput of tests (Centogene 2020; Kyoug Ho Roh 2022). Thus, this study proposes a framework to design a mobile medical test facility, in addition to propos-ing a project plan to design a mobile container-based facility to implement the new COVID-19 test-ing method. The framework proposed is based on the combined adaptation of facility design meth-ods, namely - Systematic Layout Planning and project-based approach as per VDI 5200. The resulting outputs in the form of a project plan include - capacity plan, material flow plan and financial plan. It is clear from these plans that the industrialization of the new testing facility is feasi-ble in terms of testing throughput - approximately 450,000 tests per day (when the line is operated in all three shifts) - and cost per test being 3 Euros, spatial requirements as proposed in the draft layout, based on the inputs as published (Lehrach, 2022). The identified risks do not pose alarming threats to continue with the implementation, however contingency plans should be considered for every worst-case scenario

    Efficient parameter estimation enables the prediction of drug response using a mechanistic pan-cancer pathway model

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    Mechanistic models are essential to deepen the understanding of complex diseases at the molecular level. Nowadays, high-throughput molecular and phenotypic characterizations are possible, but the integration of such data with prior knowledge on signaling pathways is limited by the availability of scalable computational methods. Here, we present a computational framework for the parameterization of large-scale mechanistic models and its application to the prediction of drug response of cancer cell lines from exome and transcriptome sequencing data. This framework is over 104 times faster than state-of-the-art methods, which enables modeling at previously infeasible scales. By applying the framework to a model describing major cancer-associated pathways (>1,200 species and >2,600 reactions), we could predict the effect of drug combinations from single drug data. This is the first integration of high-throughput datasets using large-scale mechanistic models. We anticipate this to be the starting point for development of more comprehensive models allowing a deeper mechanistic insight
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