Analysis of structural changes and RNA release in human rhinoviruses

Rhinoviren sind die Hauptursache der gewöhnlichen Erkältung. Die Viren werden durch eine Rezeptor-vermittelter Endozytose internalisiert. Rezeptorbindung und/oder Ansäuerung der frühen Endosomen führen zu einer Konformationsänderung des nativen Virus in ein verändertes, subvirales A-Partikel. Dieses Partikel heftet sich über die Innenseite der Endosomenmembran an und erzeugt eine kleine Pore, durch welche die genomische RNA in das zelluläre Zytoplasma freigesetzt wird. Es ist daher möglich verschiedene Antikörper, die spezifisch gegen diese viralen Konformationen gerichtet sind, zu nutzen. Um den aktuellen Internalisierungsprozess in vitro visualisieren zu können, wurden Antikörper für die Immunofluoreszenz verwendet. Leider wurde bisher kein spezifischer Antikörper gegen das A-Partikel gefunden, der in der Immunofluoreszenz verwendbar wäre. Im ersten Teil dieser Thesis versuchte ich einen solchen Antikörper zu identifizieren. Niclosamid verhindert die Azidifizierung der frühen Endosomen, wodurch kein A-Partikel entsteht. Dieser chemische Eingriff wurde genutzt, um die Spezifität von einer Reihe unterschiedlicher Antikörper zu testen. Um die Geschwindigkeit und Richtung des viralen RNA Austritts durch eine der Poren im A-Partikel untersuchen zu können, wurden intermediäre A-Partikel mit partiell freigesetzter RNA, durch Infektion von HeLa Zellen, in vivo gebildet (Teil 2). Alternativ, wurde der Vorgang in vitro durch Inkubation in Puffern mit niedrigem pH ausgelöst (Teil 3). In beiden Fällen wurde die Freisetzung der RNA zu unterschiedlichen Zeitpunkten gestoppt, jener Teil der RNA, der bereits freigesetzt wurde, verdaut und die A-Partikel mittels Immunopräzipitation isoliert. Das noch im Kapsid vorhandene RNA Fragment wurde mittels RT-PCR und RT-qPCR unter Zuhilfenahme von Primern, die an bestimmte Regionen des viralen Genoms binden, untersucht und mit dem vollständigen Genom des nativen Virus verglichen.Rhinoviruses are the major causative agents of the upper respiratory tract infections which cause the common cold. Upon infection, virus is internalized by receptor-mediated endocytosis. Receptor binding and/or acidification of the early endosomes trigger conformational modifications of the native virus into the subviral A-particle. This particle attaches to the inner surface of endosomes generating a small pore through which the genomic RNA is released into the cellular cytoplasm. These modifications allow certain antibodies to bind specifically to antigens of the different viral conformations. To visualize the current internalization process in vitro, different antibodies are available, which allow detection of the virus via immunofluorescence. Unfortunately, there is no antibody available yet which binds specifically to the A-particle and allows its detection by fluorescence microscopy. The first part of the thesis focused on finding such an antibody. ^The agent niclosamide hinders acidification of the early endosome; therefore, no A-particle can be generated. This manipulation of the pathway is used to generate information about the antibodies specificity. To investigate the speed and the direction of the movement of the viral RNA through the pore in the shell of the subviral particle, intermediate forms of the virion with partially released RNA were generated in vivo by infecting HeLa cells (part 2). A-particles can also be generated in vitro by using low pH buffer (part 3). In both cases the uncoating process was stopped at different times, the part of the viral RNA that had been released was digested and these intermediate A-particles were isolated by immunoprecipitation. The unreleased viral RNA strand was analyzed via RT-PCR and RT-qPCR by using a set of different primers binding to different positions on the viral genome. ^The length of the viral RNA remaining intact can be distinguished and compared with the full-length genome of the native virus.verfasst von Jakob Weinzierl, BScZusammenfassungen in Deutsch und EnglischKarl-Franzens-Universität Graz, Masterarbeit, 2017(VLID)224599

Hematoxylin binds to mutant calreticulin and disrupts its abnormal interaction with thrombopoietin receptor.

Somatic mutations of calreticulin (CALR)have been identified as one of the main disease drivers of myeloproliferative neoplasms (MPNs), suggesting that developing drugs targeting mutant CALR is of great significance. Site-directed mutagenesis in the N-glycan binding domain (GBD)abolishes the ability of mutant CALRto oncogenically activate the thrombopoietin receptor (MPL).We thus hypothesized that a small molecule targeting the GBD might inhibit the oncogenicity of the mutant CALR. Using an in-silico molecular docking study, we identified candidate binders to the GBD of CALR. Further experimental validation of the hits identified a group of catechols inducing selective growth inhibitory effect on cells that depend on oncogenic CALRs for survival and proliferation. Apoptosis-inducing effects by the compound were significantly higher in the CALR mutated cells than in CALR wild type cells. Additionally, knockout or C-terminal truncation of CALR abolished the drug hypersensitivity in CALR mutated cells. We experimentally confirmed the direct binding of the selected compound to CALR, the disruption of the mutant CALR-MPL interaction, the inhibition of the JAK2-STAT5 pathway, and reduction of intracellular level of mutant CALR upon the drug treatment. Our data conclude that small molecules targeting the GBD of CALR can selectively kill CALR mutated cells by disrupting the CALR-MPL interaction and inhibiting the oncogenic signaling

Analytic and algorithmic aspects of generalized harmonic sums and polylogarithms

In recent three-loop calculations of massive Feynman integrals within Quantum Chromodynamics (QCD) and, e.g., in recent combinatorial problems the so-called generalized harmonic sums (in short S-sums) arise. They are characterized by rational (or real) numerator weights also different from ±1. In this article we explore the algorithmic and analytic properties of these sums systematically. We work out the Mellin and inverse Mellin transform which connects the sums under consideration with the associated Poincaré iterated integrals, also called generalized harmonic polylogarithms. In this regard, we obtain explicit analytic continuations by means of asymptotic expansions of the S-sums which started to occur frequently in current QCD calculations. In addition, we derive algebraic and structural relations, like differentiation with respect to the external summation index and different multi-argument relations, for the compactification of S-sum expressions. Finally, we calculate algebraic relations for infinite S-sums, or equivalently for generalized harmonic polylogarithms evaluated at special values. The corresponding algorithms and relations are encoded in the computer algebra package HarmonicSums