Stability of possible Earth-like planets in multiplanetary Systems

Ziel dieser Magisterarbeit ist die Untersuchung der Stabilität von möglichen Planeten mit Erdmassen in zwei extrasolaren Systemen, in denen bereits zwei bzw. drei Planeten entdeckt wurden. Die verwendeten Methoden sind n-Körper Integrationen sowie säkulare Störungstheorie. Die betrachteten Systeme sind HD 60532 und HD 40307, beide wurden im Jahr 2008 entdeckt und haben massereiche Körper nahe dem Zentralstern, im Falle des ersten in Jupitergrößenordnung, im letzteren Mini-Neptun oder Super-Erden. Die Integration wurde mit einem Lie-Integratior durchgeführt.This thesis investigates the stability of possible Earth-mass planets in two extrasolar systems where two and three planets respectively had already been found. Methods used were both n-body integration, as well as secular perturbation theory. The systems involved were HD 60532 and HD 40307. Both systems were discovered in 2008, and have massive bodies close to the central star, in case of the former Jupiter-sized, while in the latter mini Neptunes or super Earths. Integration was performed with a Lie integrator

Dissecting the role of putative CD81 binding regions of E2 in mediating HCV entry: Putative CD81 binding region 1 is not involved in CD81 binding

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) encodes two transmembrane glycoproteins E1 and E2 which form a heterodimer. E1 is believed to mediate fusion while E2 has been shown to bind cellular receptors including CD81. In this study, alanine substitutions in E2 were generated within putative CD81 binding regions to define residues critical for viral entry. The effect of each mutation was tested by challenging susceptible cell lines with mutant HCV E1E2 pseudotyped viruses generated using a lentiviral system (HCVpp). In addition to assaying infectivity, producer cell expression and HCVpp incorporation of HCV E1 and E2 proteins, CD81 binding profiles, and E1E2 association of mutants were examined.</p> <p>Results</p> <p>Based on these characteristics, mutants either displayed wt characteristics (high infectivity [≥ 50% of wt HCVpp], CD81 binding, E1E2 expression, association, and incorporation into viral particles and proper conformation) or segregated into 4 distinct low infectivity (≤ 50% of wt HCVpp) mutant phenotypes: (I) CD81 binding deficient (despite wt E1E2 expression, incorporation and association and proper conformation); (II) CD81 binding competent, but lack of E1 detection on the viral particle, (despite adequate E1E2 expression in producer cell lysates and proper conformation); (III) CD81 binding competent, with adequate E1E2 expression, incorporation, association, and proper E2 conformation (i.e. no defect identified to explain the reduced infectivity observed); (IV) CD81 binding deficient due to disruption of E2 mutant protein conformation.</p> <p>Conclusion</p> <p>Although most alanine substitutions within the putative CD81 binding region 1 (amino acids 474–492) displayed greatly reduced HCVpp infectivity, they retained soluble CD81 binding, proper E2 conformation, E1E2 association and incorporation into HCVpp suggesting that region 1 of E2 does not mediate binding to CD81. In contrast, conformationally correct E2 mutants (Y527 and W529) within the second putative CD81 binding region (amino acids 522–551) disrupted binding of E2 to CD81-GST, suggesting that region 2 is critical to CD81 binding. Likewise, all conformationally intact mutants within the third putative CD81 binding region (amino acids 612–619), except L615A, were important for E2 binding to CD81-GST. This region is highly conserved across genotypes, underlining its importance in mediating viral entry.</p

CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cells

Hepatitis C virus (HCV) infects cells by the direct uptake of cell-free virus following virus engagement with specific cell receptors such as CD81. Recent data have shown that HCV is also capable of direct cell-to-cell transmission, although the role of CD81 in this process is disputed. Here, we generated cell culture infectious strain JFH1 HCV (HCVcc) genomes carrying an alanine substitution of E2 residues W529 or D535 that are critical for binding to CD81 and infectivity. Co-cultivation of these cells with naïve cells expressing enhanced green fluorescent protein (EGFP) resulted in a small number of cells co-expressing both EGFP and HCV NS5A, showing that the HCVcc mutants are capable of cell-to-cell spread. In contrast, no cell-to-cell transmission from JFH1ΔE1E2-transfected cells occurred, indicating that the HCV glycoproteins are essential for this process. The frequency of cell-to-cell transmission of JFH1W529A was unaffected by the presence of neutralizing antibodies that inhibit E2–CD81 interactions. By using cell lines that expressed little or no CD81 and that were refractive to infection with cell-free virus, we showed that the occurrence of viral cell-to-cell transmission is not influenced by the levels of CD81 on either donor or recipient cells. Thus, our results show that CD81 plays no role in the cell-to-cell spread of HCVcc and that this mode of transmission is shielded from neutralizing antibodies. These data suggest that therapeutic interventions targeting the entry of cell-free HCV may not be sufficient in controlling an ongoing chronic infection, but need to be complemented by additional strategies aimed at disrupting direct cell-to-cell viral transmission

Hepatitis C Virus Infection in Phenotypically Distinct Huh7 Cell Lines

In 2005, the first robust hepatitis C virus (HCV) infectious cell culture system was developed based on the HCV genotype 2a JFH-1 molecular clone and the human-derived hepatoma cell line Huh7. Although much effort has been made to dissect and expand the repertoire of JFH-1-derived clones, less attention has been given to the host cell despite the intriguing facts that thus far only Huh7 cells have been found to be highly permissive for HCV infection and furthermore only a limited number of Huh7 cell lines/stocks appear to be fully permissive. As such, we compiled a panel of Huh7 lines from disparate sources and evaluated their permissiveness for HCV infection. We found that although Huh7 lines from different laboratories do vary in morphology and cell growth, the majority (8 out of 9) were highly permissive for infection, as demonstrated by robust HCV RNA and de novo infectious virion production following infection. While HCV RNA levels achieved in the 8 permissive cell lines were relatively equivalent, three Huh7 lines demonstrated higher infectious virion production suggesting these cell lines more efficiently support post-replication event(s) in the viral life cycle. Consistent with previous studies, the single Huh7 line found to be relatively resistant to infection demonstrated a block in HCV entry. These studies not only suggest that the majority of Huh7 cell lines in different laboratories are in fact highly permissive for HCV infection, but also identify phenotypically distinct Huh7 lines, which may facilitate studies investigating the cellular determinants of HCV infection

Human Monoclonal Antibodies to a Novel Cluster of Conformational Epitopes on HCV E2 with Resistance to Neutralization Escape in a Genotype 2a Isolate

The majority of broadly neutralizing antibodies to hepatitis C virus (HCV) are against conformational epitopes on the E2 glycoprotein. Many of them recognize overlapping epitopes in a cluster, designated as antigenic domain B, that contains residues G530 and D535. To gain information on other regions that will be relevant for vaccine design, we employed yeast surface display of antibodies that bound to genotype 1a H77C E2 mutant proteins containing a substitution either at Y632A (to avoid selecting non-neutralizing antibodies) or D535A. A panel of nine human monoclonal antibodies (HMAbs) was isolated and designated as HC-84-related antibodies. Each HMAb neutralized cell culture infectious HCV (HCVcc) with genotypes 1–6 envelope proteins with varying profiles, and each inhibited E2 binding to the viral receptor CD81. Five of these antibodies neutralized representative genotypes 1–6 HCVcc. Epitope mapping identified a cluster of overlapping epitopes that included nine contact residues in two E2 regions encompassing aa418–446 and aa611–616. Effect on virus entry was measured using H77C HCV retroviral pseudoparticles, HCVpp, bearing an alanine substitution at each of the contact residues. Seven of ten mutant HCVpp showed over 90% reduction compared to wild-type HCVpp and two others showed approximately 80% reduction. Interestingly, four of these antibodies bound to a linear E2 synthetic peptide encompassing aa434–446. This region on E2 has been proposed to elicit non-neutralizing antibodies in humans that interfere with neutralizing antibodies directed at an adjacent E2 region from aa410–425. The isolation of four HC-84 HMAbs binding to the peptide, aa434–446, proves that some antibodies to this region are to highly conserved epitopes mediating broad virus neutralization. Indeed, when HCVcc were passaged in the presence of each of these antibodies, virus escape was not observed. Thus, the cluster of HC-84 epitopes, designated as antigenic domain D, is relevant for vaccine design for this highly diverse virus

In vitro Kulturen von Sinapis alba : morphologische und physiologische Charakterisierung

Diese Arbeit befasste sich mit der Herstellung und der anschließenden Untersuchung von in vitro Kulturen von Sinapis alba, dem weißen Senf. Die durchgeführten Versuche inkludierten die Etablierung einer dauerhaften Calluskultur von Sinapis alba auf MS-Medium mit den Hormonen 2,4-D und Kinetin und die Untersuchung einer Calluskultur über den Zeitraum von 6 Wochen, wobei hier in der dritten Woche das größte Wachstum nachweisbar war und die Kulturen spätestens in Woche 6 abzusterben begannen. Des Weiteren wurde ein Regenerationsversuch durchgeführt, wobei es nach 6 Wochen nur zur Ausdifferenzierung von Wurzelgewebe kam. Mithilfe der Calli wurden Suspensionskulturen angesetzt und in weiterer Folge wurde versucht eine photoautotrophe Suspensionskultur zu erhalten. Trotz spezieller Kulturgefäße und dem Variieren der Kulturbedingungen konnte weder eine photoautotrophe noch eine photomixotrophe Suspensionskultur etabliert werden. Die Charakterisierung der in vitro Kulturen wurde anschließend mit Hellfeld- und fluoreszenzmikroskopischen Methoden durchgeführt. Die Färbung mit Jod-Jodkalium zeigte Stärkedepots in Callus- und Suspensionskulturen, die Färbung mit FCA zeigte verholzte Zellwände in der Callus-, jedoch nicht in der Suspensionskultur. Die Färbung mit Anilinsulfat wiederholte diese Ergebnisse. Die Untersuchungen des Zellkerns sollten Aufschluss über das Wachstum der Zellen geben, weder die Fluoreszenzfärbung mit DAPI, noch die Färbung mit Schiffschem Reagenz ermöglichten die Erstellung eines Mitoseindex.The subject of this thesis was to generate an in vitro culture of Sinapis alba, yellow mustard, and the characterisation of the callus and suspension culture. The first subject included establishing a permanent callus culture on MS-media with the addition of the phytohormones 2,4-D and Kinetin. The next step was the monitoring of the development of the callus culture over a period of 6 weeks. The results showed, that the cultures had the highest growth in week 3, but started to die from week 6 onward. To conclude the callus in vitro culture testing, a regeneration of the calli was induced. No plants were regenerated, but some rooting was observed. The calli were used to generate a suspension culture. The main goal was to establish a photoautotrophic culture, which was not accomplished during this experiment. Neither a photomixotrophic nor a durable photoheterotrophic culture could be obtained. The characterisation of the cultures was performed with bright field and fluorescent microscopy. The stain with Lugol's iodine presented starch granula in callus and suspension culture, the FCA stain showed lignification in callus but not in suspension culture. The anilinsulfate stain supported these findings. The analysis of the nucleus should have yielded an insight into the growth of the cells but showed only the miniscule size of the nucleus. Neither the fluorescent stain with DAPI, nor the stain with Feulgen enabled the analysis of the mitotic state of the nucleus.eingereicht von Ursula Rothwangl, BScZusammenfassungen in Deutsch und EnglischAbweichender Titel laut Übersetzung des Verfassers/der VerfasserinKarl-Franzens-Universität Graz, Masterarbeit, 2017(VLID)230409

Characterization of hepatitis C virus entry and potential small molecule inhibitors.

Characterization of hepatitis C virus entry and potential small molecule inhibitors

Ausgewählte Fragen zu Internet-Suchmaschinen aus kartell- und wettbewerbsrechtlicher Sicht : Suchmaschinenindex, Ranking, Deep-Linking, Hot-Linking

Diese Arbeit untersucht bestimmte Verhaltensweisen von Internet-Suchmaschinenbetreibern aus der Sicht des europäischen und österreichischen Kartell- und Wettbewerbsrechts. Zum einen werden Fragen behandelt, die im Zusammenhang mit Internet-Suchmaschinen und der europäischen und österreichischen Missbrauchskontrolle stehen. Im Speziellen geht es dabei um die Beurteilung von Verhaltensweisen, wie der Nichtaufnahme in den Suchmaschinenindex, dem Ausschluss aus diesem oder der Manipulation der Suchergebnisse durch Internet-Suchmaschinenbetreiber. Gegenstand der Untersuchungen sind dabei Fragen, die sich mit der marktbeherrschenden Stellung von Internet-Suchmaschinenbetreibern auseinandersetzen. Es wird beurteilt, ob Betreiber von Internet-Suchmaschinen überhaupt in den Anwendungsbereich der einschlägigen Bestimmungen fallen. Darauf aufbauend wird untersucht, ob es einen eigenen Markt für Internet-Suchmaschinen gibt, ob derzeit marktbeherrschende Internet-Suchmaschinenbetreiber existieren und welche Verpflichtungen aus einer marktbeherrschenden Stellung eines Internet-Suchmaschinenbetreibers erwachsen. Dabei werden auch die Unterschiede zwischen dem europäischen und dem österreichischen Kartell- und Wettbewerbsrecht herausgearbeitet. Die Relevanz der einschlägigen Bestimmungen des europäischen und österreichischen Kartell- und Wettbewerbsrechts für den Internet-Suchmaschinensektor wird am Beispiel der Internet-Suchmaschine Google dargestellt. Zum anderen wird die Verwendung ausgewählter Verlinkungsmethoden durch Internet-Suchmaschinenbetreiber aus dem Blickwinkel des österreichischen UWG beleuchtet. Der Schwerpunkt liegt dabei auf den Verlinkungsmethoden des Deep- und Hot-Linking bzw Inline-Linking.This work investigates certain behaviour patterns of Internet search engine operators from the perspective of the European and Austrian antitrust and competition law. On the one hand there are questions which stand in connection with search engines and the European and Austrian control of abusive practices. Specifically, it is about the assessment of behaviors, such as the non-inclusion in the search engines index, the exclusion from the index or the manipulation of search results through search engine operators. Subject of the investigations are issues that deal with the dominance of Internet search engine operators. It is judged whether operators of Internet search engines fall generally in the scope of the appropriate regulations. On this basis it is examined whether there is a separate market for Internet search engines, whether dominant Internet search engines currently exist, and which obligations arise from a market-dominating position of a Internet search engine operator. In addition, the investigations outline the differences between the European and Austrian antitrust and competition law. The relevance of the appropriate regulations of the European and Austrian antitrust and competition law for the search engine sector is shown at the example of the search engine Google. On the other hand the use of different types of hyperlinks by Internet search engine operators is examined from the point of the Austrian UWG. In this regard the main focus lies on deep-linking and hot-linking, respectively inline-linking.von Andreas RothwanglAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersGraz, Univ., Dipl.-Arb., 2011(VLID)21718

Analysis of a conserved RGE/RGD motif in HCV E2 in mediating entry

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) encodes two transmembrane glycoproteins E1 and E2 which form a heterodimer. E1 is believed to mediate fusion while E2 has been shown to bind cellular receptors. It is clear that HCV uses a multi-receptor complex to gain entry into susceptible cells, however key elements of this complex remain elusive. In this study, the role of a highly conserved RGE/RGD motif of HCV E2 glycoprotein in viral entry was examined. The effect of each substitution mutation in this motif was tested by challenging susceptible cell lines with mutant HCV E1E2 pseudotyped viruses generated using a lentiviral system (HCVpp). In addition to assaying infectivity, producer cell expression and HCVpp incorporation of HCV E2 proteins, CD81 binding profiles, and conformation of mutants were examined.</p> <p>Results</p> <p>Based on these characteristics, mutants either displayed wt characteristics (high infectivity [≥ 90% of wt HCVpp], CD81 binding, E1E2 expression, and incorporation into viral particles and proper conformation) or very low infectivity (≤ 20% of wt HCVpp). Only amino acid substitutions of the 3^rdposition (D or E) resulted in wt characteristics as long as the negative charge was maintained or a neutral alanine was introduced. A change in charge to a positive lysine, disrupted HCVpp infectivity at this position.</p> <p>Conclusion</p> <p>Although most amino acid substitutions within this conserved motif displayed greatly reduced HCVpp infectivity, they retained soluble CD81 binding, proper E2 conformation, and incorporation into HCVpp. Our results suggest that although RGE/D is a well-defined integrin binding motif, in this case the role of these three hyperconserved amino acids does not appear to be integrin binding. As the extent of conservation of this region extends well beyond these three amino acids, we speculate that this region may play an important role in the structure of HCV E2 or in mediating the interaction with other factor(s) during viral entry.</p