Cell and Receptor Tropism of γ2-Herpesviruses

Das Kaposi-Sarkom-Herpesvirus (KSHV), das einzige Rhadino- oder γ2-Herpesvirus im Menschen, ist mit dem Kaposi Sarkom (KS) und zwei B-Zell-Lymphomen, dem primären Effusionslymphom und der multizentrischen Castleman-Erkrankung, assoziiert. Da der Mechanismus der Primärinfektion, die Ausbreitung im Wirt und die Entstehung Virus-assoziierter Erkrankungen zumindest teilweise durch den viralen Zell- und Gewebetropismus bestimmt werden, ist es entscheidend den Beitrag spezifischer Interaktionen zwischen viralen Glykoproteinen und zellulären Rezeptoren zur Infektion unterschiedlicher Zelltypen zu verstehen. Der Fokus dieser Arbeit liegt hierbei auf der Familie der Eph Rezeptortyrosinkinasen, die an der Infektion diverser adhärenter Zelllinien beteiligt sind. Indes der KSHV gH/gL Glykoproteinkomplex die höchste Affinität für EphA2 aufweist, wurden weitere Eph Rezeptoren des A-Typs als Bindepartner von KSHV beschrieben. Obwohl die gH/gL-Eph Interaktion Gegenstand verschiedener Studien war, gibt es weiterhin unbeantwortete Fragen bezüglich der Rolle von Eph Rezeptoren für den KSHV Tropismus und KSHV-assoziierte Pathologien. Unsere Zielsetzung lag demnach in der Identifikation von Aminosäuren in KSHV gH/gL, welche essentiell für die Eph-Interaktion sind, in der Konstruktion von rekombinanten Viren mit Mutationen in den identifizierten Aminosäuren, sowie in der Charakterisierung der Eph Rezeptor Nutzung auf BJAB Zellen, einem Modell für die Zell-assoziierte KSHV Infektion von B-Zellen. Analog zu KSHV interagiert das verwandte Rhesusaffen Rhadinovirus (RRV) mit Eph Rezeptoren, zeigt hierbei jedoch abweichende Affinitäten für einzelne Mitglieder der Eph Familie. Durch Vergleiche zwischen beiden Viren konnten wir konservierte Aminosäuren in der N-terminalen Region von gH identifizieren, welche essentiell für die gH/gL-Eph Interaktion sind. Mutation dieser Aminosäuren in KSHV und RRV verhinderte die Interaktion mit Eph Rezeptoren und ermöglichte uns die Analyse des Zelltypspezifischen Beitrags von Eph Rezeptoren zur KSHV und RRV Infektion. Dieses System kam außerdem in unserer Studie zum Einsatz, die zwei weitere Eph Rezeptoren des A-Typs als funktionelle KSHV und RRV Rezeptoren auf BJAB Zellen charakterisierte. Die Funktion von EphA5 und EphA7 in der KSHV Zell-Zell Übertragung, sowie in der zellfreien RRV Infektion wurde durch Knockout mit Hilfe der CRISPR/Cas9 Methode nachgewiesen. Ferner beschäftigten wir uns mit der Frage, ob weitere, Eph-unabhängige Interaktionspartner des gH/gL Komplexes die Infektion verschiedener Zelltypen durch Rhadinoviren beeinflussen. Wir identifizierten die Plexin domain containing Proteine 1 und 2 (Plxdc1/2) als spezifische Interaktionspartner von RRV im Gegensatz zu KSHV und beschrieben ein essentielles Plxdc-Interaktionsmotiv nahe dem Eph-Interaktionsmotiv in RRV gH. Die Plxdc-Rezeptorfunktion wurde mittels lentiviraler Überexpression sowie mit Hilfe von zur Plxdc-Bindung unfähigen RRV Deletionsmutanten nachgewiesen. Zusammengenommen beschreiben die vorliegenden Studien weitere Eph Rezeptoren vom A-Typ als funktionelle Rezeptoren für KSHV und RRV, charakterisieren die Funktion einer neuen Rezeptorfamilie für die RRV Infektion und verdeutlichen die Bedeutung der N-terminalen Region des rhadinoviralen gHs als konservierte Rezeptorbindedomäne, welche die Interaktion von KSHV und RRV mit Eph Rezeptoren und die Interaktion von RRV mit Plxdc Rezeptoren vermittelt.Kaposi’s sarcoma-associated herpesvirus (KSHV), the only rhadino- or γ2-herpesvirus of humans, is associated with Kaposi’s sarcoma (KS) and two B cell proliferative malignancies, primary effusion lymphoma (PEL) and a variant of multicentric Castleman’s disease (MCD). As routes of primary infection, dissemination through the host, and development of virus-associated pathologies are at least partially shaped by viral cell and tissue tropism, it is crucial to understand the contribution of distinct viral glycoproteins and cellular receptor interactions to cell type-specific infection. In this context, the present thesis focuses on members of the Eph family of receptor tyrosine kinases, which were shown to play a role in KSHV infection of various adherent cell lines. While the KSHV gH/gL glycoprotein complex exhibits the highest affinity for EphA2, additional A-type Ephs have been described as interaction partners of KSHV. Even though the gH/gL-Eph interaction was subject of various studies, key questions regarding the role of Ephs in KSHV tropism and pathology remained unanswered. We therefore aimed to identify amino acid residues on the KSHV gH/gL complex that critically mediate the Eph interaction, create Eph detargeted virus recombinants mutated in the identified amino acid residues, and characterize the Eph usage on BJAB cells, as model for cell-to-cell transmission of KSHV into B cells. Similar to KSHV, the related rhesus monkey rhadinovirus (RRV) interacts with Eph receptors while exhibiting differing affinities for individual Eph family members. Comparison of the two viruses allowed us to identify conserved amino acid residues in the N-terminal domain of gH which are critical for the gH/gL-Eph interaction. Mutation of these amino acids in KSHV and RRV recombinants abrogated the viral interaction with Eph receptors and allowed us to analyze the cell type-specific contribution of the Eph family to KSHV and RRV infection. This system was also employed in our second study which identified two additional A-type Ephs as functional KSHV and RRV receptors on BJAB cells. The role of EphA5 and EphA7 in KSHV cell-to-cell transmission and RRV cell-free infection was demonstrated using CRISPR/Cas9-mediated knockout. We furthermore addressed the question whether additional cellular, Eph-independent interaction partners of the gH/gL complex shape the rhadinoviral infection of different cell types. We identified the Plexin domain containing proteins 1 and 2 (Plxdc1/2) as specific interactors for RRV, but not KSHV, and characterized a crucial Plxdc-interaction motif in close proximity to the identified Eph-interacting residues on RRV gH. Receptor function of Plxdcs was demonstrated by lentiviral overexpression of Plxdc1 and 2 in target cells and a Plxdc-detargeted RRV deletion mutant. Collectively, the present studies identify additional A-type Eph members as functional receptors for KSHV and RRV, characterize the role of a novel family of gH/gL-interacting proteins for RRV infection, and underline the importance of the N-terminal domain of the rhadinoviral gH as conserved receptor-binding domain, which mediates the interaction of KSHV and RRV with Eph receptors and the independent interaction of RRV with Plxdc family members

Direct and indirect costs of dinitrogen fixation in Crocosphaera watsonii WH8501 and possible implications for the nitrogen cycle

The recent detection of heterotrophic nitrogen (N2) fixation in deep waters of the southern Californian and Peruvian OMZ questions our current understanding of marine N2 fixation as a process confined to oligotrophic surface waters of the oceans. In experiments with Crocosphaera watsonii WH8501, a marine unicellular diazotrophic (N2 fixing) cyanobacterium, we demonstrated that the presence of high nitrate concentrations (up to 800 μM) had no inhibitory effect on growth and N2 fixation over a period of 2 weeks. In contrast, the environmental oxygen concentration significantly influenced rates of N2 fixation and respiration, as well as carbon and nitrogen cellular content of C. watsonii over a 24-h period. Cells grown under lowered oxygen atmosphere (5%) had a higher nitrogenase activity and respired less carbon during the dark cycle than under normal oxygen atmosphere (20%). Respiratory oxygen drawdown during the dark period could be fully explained (104%) by energetic needs due to basal metabolism and N2 fixation at low oxygen, while at normal oxygen these two processes could only account for 40% of the measured respiration rate. Our results revealed that under normal oxygen concentration most of the energetic costs during N2 fixation (∼60%) are not derived from the process of N2 fixation per se but rather from the indirect costs incurred for the removal of intracellular oxygen or by the reversal of oxidative damage (e.g., nitrogenase de novo synthesis). Theoretical calculations suggest a slight energetic advantage of N2 fixation relative to assimilatory nitrate uptake, when oxygen supply is in balance with the oxygen requirement for cellular respiration (i.e., energy generation for basal metabolism and N2 fixation). Taken together our results imply the existence of a niche for diazotrophic organisms inside oxygen minimum zones, which are predicted to further expand in the future ocean

Winfried Marotzki / Lothar Wigger (Hrsg.): Erziehungsdiskurse. Bad Heilbrun: Klinkhardt 2008 (24 S.) [Rezension]

Rezension von: Winfried Marotzki / Lothar Wigger (Hrsg.): Erziehungsdiskurse. Bad Heilbrun: Klinkhardt 2008 (24 S.; ISBN 978-3-7815-1583-3; 19,80 EUR)

In-depth analysis of elements and properties of hydrated subsurface layers on optical surfaces of a SiO2-BaO-B2O3 glass with SIMS, IBSCA, RBS and NRA Part 1. Experimental procedures and results

The formation of thin subsurface layers was studied which occurred during the chemical interaction of polished and cleaned optical surfaces with different slurries before and after covering them with λ/4-MgF2 coatings. By a suitable selection of the parameters for these chemical interactions a thickness of the subsurface layers was produced which allowed to meet the requirements of the various surface analysis methods. The thicknesses and the refractive indices of the subsurface layers could be calculated from the measured spectral reflectances. Slurries with a pH value < 9 were applied so that a leaching of glass components from the subsurface layers occurred. This was indicated by the refractive indices and was studied in detail by analyzing the in-depth distributions of the glass components. Distinct matrix effects could be disclosed from the in-depth profiles for SIMS and IBSCA by a comparison with the results of the quantitative analysis with RBS and NRA. These matrix effects were different within the subsurface layers from those observed for the bulk glass. The quantitative analysis of the hydrogen in-depth distributions by NRA allows for the first time to link differences in the matrix effects with a different hydrogen content within the subsurface layers

Investigations on three species of Diptera associated with hawkweeds in Europe and their potential for biological control of alien invasive Hieracium spp. in New Zealand and North America

Hieracium spp. (Asteraceae) of Eurasian origin have become weeds in New Zealand, Australia and North and South America. They are particularly a problem in low production areas, nature reserves, and forest margins. Since traditional management efforts are either uneconomic or impractical, a project for biological control was initiated. In this thesis, the biology and host-specificity of the phytophagous hoverflies Cheilosia urbana (Meigen) and Cheilosia psilophthalma (Becker), and of the gall midge Macrolabis pilosellae (Binnie) were investigated. The aim of this study was to evaluate the suitability and potential effectiveness of these selected herbivores as biological control agents of alien invasive hawkweeds