Die Rolle des AF4 Proteins in der Transkription und in der Infektion durch Herpesviren

Die Transkription ist ein entscheidender Schritt in der Transition der genetischen Information, welche durch die DNA codiert und im Genom hinterlegt ist, zu dreidimensionalen Funktionseinheiten in der Zelle, den Proteinen. Während der Transkription wird die Information von der Ebene der DNA in RNA umgewandelt, welche in der Zelle zusätzlich zu dessen Rolle als Informationsmediator in Form der mRNA eine Vielzahl von Funktionen ausübt. Die Transkription benötigt in Hinblick auf ihre essentielle Rolle in der Errichtung des Proteoms und der notwendigen Adaption von Genexpressionsprogrammen an externe zelluläre Stimuli, den Zellzyklus etc. eine präzise und gleichzeitig flexible Regulation. Besonders für die Transkription von mRNA dient die eukaryotische RNA-Polymerase II (RNAP II) in diesem Prozess als eine zentrale Einheit, die einer Vielzahl regulativer Mechanismen wie post-translationaler Modifikationen und der Assemblierung dynamischer Proteinkomplexe unterliegt. Während Komponenten dieser Regulation wie die Zusammensetzung und Dynamik des Prä-Initiationskomplex bereits seit Jahrzehnten beschrieben sind, ist eine besondere Form der RNAP II-abhängigen Regulation erst in den letzten Jahren Gegenstand genauerer Untersuchungen geworden. So erfährt die RNAP II bei einer Vielzahl von Genen unmittelbar nach der Initiation einen Arrest, der das Enzym nicht weiter über die DNA prozessieren lässt und somit die produktive Elongation des Gens blockiert. Die Aufhebung dieser Blockade wird durch den positiven Transkriptions-elongationsfaktor b (P-TEFb) dominiert, der durch distinkte post-translationale Modifikationen der C-terminalen Domäne der RNAP II und assoziierter Faktoren die produktive Elongation ermöglicht. P-TEFb selbst unterliegt dabei einer strengen Regulation durch eine inaktivierende Assoziation mit Speicherkomplexen. P-TEFb wurde abseits dieser Komplexe in einer Vielzahl von Elongations-assoziierten Proteinkomplexen identifiziert, der Mechanismus der Transition aus dem inaktiven Speicherkomplex zur aktiven Form an der RNAP II war jedoch unbekannt. Ein zentrales Element aller aktiven Komplexe ist die Anwesenheit von Proteinen der AF4/FMR2-Familie, darunter das AF4 Protein. Bemerkenswerterweise war die genaue Rolle dieses Proteins in den Komplexen bisher unbekannt oder wurde lediglich auf die strukturelle Integrität der Komplexe beschränkt. AF4 und speziell dessen N-Terminus ist über diese Rolle hinaus als Bestandteil des Fusionsproteins AF4-MLL eng mit der onkogenen Zelltransformation im Falle einer durch die t(4;11)(q21;q23) chromosomalen Translokation bedingter, akuter lymphoblastischer Leukämie assoziiert. In dieser Arbeit konnte gezeigt werden, dass das AF4 Protein und im Speziellen sein N-Terminus in der Lage ist, die zelluläre Transkription durch die Aktivierung und Rekrutierung von P-TEFb zu aktivieren. In Anwesenheit von AF4 wird die Kinase-Untereinheit CDK9 des P-TEFb post-translational an Lysinresten modifiziert und damit aktiviert sowie die C-terminale Domäne der RNAP II im Kontext stärker phosphoryliert. Gleichzeitig wurde das P-TEFb inaktivierende Protein HEXIM1 stärker exprimiert. AF4 und AF4-MLL waren weiterhin in der Lage ein Elongations-kontrolliertes Reportergen zu aktivieren. Gleichzeitig führte die Überexpression des AF4 zu einer Erhöhung der zellulären RNA Menge. Zur genaueren Untersuchung der AF4-abhängigen Mechanismen wurden zwei Zelllinien erstellt, die zum Einen eine induzierbare und reproduzierbare Überexpression und Reinigung des AF4 erlaubten (TCZP-AF4ST) und zum Anderen durch lentiviralen knock-down eine an AF4-Mangelsituation nachstellten (AF4kd V100). Es konnte so gezeigt werden, dass AF4 über P-TEFb hinaus eine regulative Funktion gegenüber Transkription-assoziierten Faktoren wie CDK7, MENIN und NF?B besitzt und dass diese Faktoren vorrangig, analog zu P-TEFb, mit dem N-Terminus des AF4 interagieren. Die Überexpression von AF4 führte über die Bindung an die 7SK snRNA und deren Degradation zur Rekrutierung des P-TEFb aus den Speicherkomplexen in distinkte AF4-assoziierte Komplexe und zu einer Umverteilung des Faktors auf distinkte Loci im Zellkern, wobei der AF4 N-Terminus für sich alleine jedoch nicht in der Lage war, diese Funktion auszuüben. Im Falle eines Mangels an AF4 kam es zur Wachstumsretardierung der Zellen sowie zu einem völligen Aktivitätsverlust in Reportergenversuchen. Die Tatsache, dass AF4 ein zentrales Element in der Elongationskontrolle darstellt führte zu der weitergehenden Vermutung, dass virale immediate early (IE) Proteine zur Kontrolle viraler Genexpression auf der Ebene der Elongation ebenfalls auf dieses Wirtsprotein zugreifen können. Es konnte vor diesem Hintergrund gezeigt werden, dass AF4 tatsächlich mit den IE-Proteinen IE1 (HCMV) und Zta (EBV) aus der Familie der Herpesviren interagiert und durch die Stabilisierung des AF4 Proteins eine kooperative, transaktivierende Funktion auf ein ALOX5 Reportergen ausgeübt wurde. Es wurde gezeigt, dass die viralen IE-Proteine dabei Komponenten der AF4 Komplexe sind und in der Zelle zur epigenetischen Regulation des ALOX5 Gens führen. Weiterhin konnte in diesen Experimenten dargestellt werden, dass AF4 über seine Rolle in der Elongationskontrolle hinaus auch distinkte Effekte in der Aktivierung von Promotoren und damit in der Initiation der Transkription zeigt. Damit konnte in dieser Arbeit zum ersten Mal die essentielle Rolle des AF4 Proteins in der Elongationskontrolle und der Initiation der Transkription als auch in der Infektion durch Herpesviren gezeigt werden

On the lifetime of discs around late type stars

We address the question of whether protoplanetary discs around low mass stars (e.g. M-dwarfs) may be longer lived than their solar-type counterparts. This question is particularly relevant to assess the planet-making potential of these lower mass discs. Given the uncertainties inherent to age-dating young stars, we propose an alternative approach that is to analyse the spatial distribution of disc-bearing low-mass stars and compare it to that of disc-bearing solar-type stars in the same cluster. A significant age difference between the two populations should be reflected in their average nearest neighbour distance (normalised to the number of sources), where the older population should appear more spread out. To this aim, we perform a Minimum Spanning Tree (MST) analysis on the spatial distribution of disc-bearing young stellar objects (YSOs) in six nearby low mass star forming regions. We find no evidence for significant age differences between the disc-bearing low-mass (later than M2) and 'solar-type' (earlier than M2) stars in these regions. We model our results by constructing and analysing synthetic fractal distributions that we evolve for typical values of the velocity dispersions. A comparison of simple models to our MST analysis suggests that the lifetime of discs around M-stars is similar to that of discs around solar-type stars. Furthermore, a model-independent spatial analysis of the observations robustly shows that any age differences between the two samples must be smaller than the average age difference between disc-bearing classical T-Tauri stars and disc-less Weak-Lined T-Tauri stars.Comment: 8 pages, 3 Figures, 3Tables; Accepted MNRA

What drives gamer toxicity? Essays from players

Negative online behaviors, such as toxicity, continue being issues in several popular multiplayer online games. Related research suggests that there are individual differences in how players understand the concept, and that various interconnected variables are relevant in understanding the emergence of toxicity. To explore this topic further, in this study, we gathered 16 essays from gamers regarding their experiences of toxicity in online games. Using the Gioia method for qualitative analysis, we divided the concepts described in the essays broadly into characteristics related to (1) the socio-technological setting in which the playing takes place; (2) the stakeholders' individual disposition including personality and player relationships; and (3) situational drivers, meaning events and actions that transpire during gameplay. As an important meta-level implication, our findings raise concerns regarding the lack of a universally shared view on toxicity, which were visible even with the rather homogenous sample of participants in this study.Peer reviewe

Spectroscopy of new brown dwarf members of rho Ophiuchi and an updated initial mass function

To investigate the universality hypothesis of the initial mass function in the substellar regime, the population of the rho Ophiuchi molecular cloud is analysed by including a new sample of low-mass spectroscopically confirmed members. To that end, we have conducted a large spectroscopic follow-up of young substellar candidates uncovered in our previous photometric survey. The spectral types and extinction were derived for a newly found population of substellar objects, and its masses estimated by comparison to evolutionary models. A thoroughly literature search was conducted to provide an up-to-date census of the cluster, which was then used to derive the luminosity and mass functions, as well as the ratio of brown dwarfs to stars in the cluster. These results were then compared to other young clusters. It is shown that the study of the substellar population of the rho Ophiuchi molecular cloud is hampered only by the high extinction in the cluster ruling out an apparent paucity of brown dwarfs. The discovery of 16 new members of rho Ophiuchi, 13 of them in the substellar regime, reveals the low-mass end of its population and shows the success of our photometric candidate selection with the WIRCam survey. The study of the brown dwarf population of the cluster reveals a high disk fraction of 76 (+5-8)%. Taking the characteristic peak mass of the derived mass function and the ratio of brown dwarfs to stars into account, we conclude that the mass function of rho Ophiuchi is similar to other nearby young clusters.Comment: Accepted to A&A (30 December 2011); v2 includes language editin

Toxicity of Tungsten Carbide and Cobalt-Doped Tungsten Carbide Nanoparticles in Mammalian Cells in Vitro

BACKGROUND: Tungsten carbide nanoparticles are being explored for their use in the manufacture of hard metals. To develop nanoparticles for broad applications, potential risks to human health and the environment should be evaluated and taken into consideration. OBJECTIVE: We aimed to assess the toxicity of well-characterized tungsten carbide (WC) and cobalt-doped tungsten carbide (WC-Co) nanoparticle suspensions in an array of mammalian cells. METHODS: We examined acute toxicity of WC and of WC-Co (10% weight content Co) nanoparticles in different human cell lines (lung, skin, and colon) as well as in rat neuronal and glial cells (i.e., primary neuronal and astroglial cultures and the oligodendrocyte precursor cell line OLN-93). Furthermore, using electron microscopy, we assessed whether natioparticles can be taken up by living cells. We chose these in vitro systems in order to evaluate for potential toxicity of the nanoparticles in different mammalian organs (i.e., lung, skin, intestine, and brain). RESULTS: Chemical-physical characterization confirmed that WC as well as WC-Co natioparticles with a mean particle size of 145 nm form stable suspensions in serum-containing cell culture media. WC nanoparticles were not acutely toxic to the studied cell lines. However, cytotoxicity became apparent when particles were doped with Co. The most sensitive were astrocytes and colon epithelial cells. Cytotoxicity of WC-Co nanoparticles was higher than expected based on the ionic Co content of the particles. Analysis by electron microscopy demonstrated presence of WC nanoparticles within mammalian cells. CONCLUSIONS: Our findings demonstrate that doping of WC nanoparticles with Co markedly increases their cytotoxic effect and that the presence of WC-Co in particulate form is essential to elicit this combinatorial effect

A large spectroscopic sample of L and T dwarfs from UKIDSS LAS : peculiar objects, binaries, and space density

This is a pre-copyedited, author-produced PDF of an article accepted for publication in Monthly Notices of the Royal Astronomical Society following peer review. The version of record [F. Marocco, et al, 'A large spectroscopic sample of L and T dwarfs from UKIDSS LAS: peculiar objects, binaries, and space density', MNRAS, Vol. 449)4): 3651-3692, April 2015] is available online at: https://doi.org/10.1093/mnras/stv530.We present the spectroscopic analysis of a large sample of late-M, L, and T dwarfs from UKIDSS. Using the YJHK photometry from ULAS and the red-optical photometry from SDSS we selected a sample of 262 brown dwarf candidates and we followed-up 196 of them using X-shooter on the VLT. The large wavelength coverage (0.30-2.48 $\mu$m) and moderate resolution (R~5000-9000) of X-shooter allowed us to identify peculiar objects including 22 blue L dwarfs, 2 blue T dwarfs, and 2 low gravity M dwarfs. Using a spectral indices-based technique we identified 27 unresolved binary candidates, for which we determined the spectral type of the potential components via spectral deconvolution. The spectra allowed us to measure the equivalent width of the prominent absorption features and to compare them to atmospheric models. Cross-correlating the spectra with a radial velocity standard, we measured the radial velocity for our targets, and we determined the distribution of the sample, which is centred at -1.7$\pm$1.2 km s$^{-1}$ with a dispersion of 31.5 km s$^{-1}$. Using our results we estimated the space density of field brown dwarfs and compared it with the results of numerical simulations. Depending on the binary fraction, we found that there are (0.85$\pm$0.55) x 10$^{-3}$ to (1.00$\pm$0.64) x 10$^{-3}$ objects per cubic parsec in the L4-L6.5 range, (0.73$\pm$0.47) x 10$^{-3}$ to (0.85$\pm$0.55) x 10$^{-3}$ objects per cubic parsec in the L7-T0.5 range, and (0.74$\pm$0.48) x 10$^{-3}$ to (0.88$\pm$0.56) x 10$^{-3}$ objects per cubic parsec in the T1-T4.5 range. There seem to be an excess of objects in the L/T transition with respect to the late T dwarfs, a discrepancy that could be explained assuming a higher binary fraction than expected for the L/T transition, or that objects in the high-mass end and low-mass end of this regime form in different environments, i.e. following different IMFs.Peer reviewe

The formation of very wide binaries during the star cluster dissolution phase

Over the past few decades, numerous wide (>1000 au) binaries in the Galactic field and halo have been discovered. Their existence cannot be explained by the process of star formation or by dynamical interactions in the field, and their origin has long been a mystery. We explain the origin of these wide binaries by formation during the dissolution phase of young star clusters: an initially unbound pair of stars may form a binary when their distance in phase-space is small. Using N-body simulations, we find that the resulting wide binary fraction in the semi-major axis range 1000 au - 0.1 pc for individual clusters is 1-30%, depending on the initial conditions. The existence of numerous wide binaries in the field is consistent with observational evidence that most clusters start out with a large degree of substructure. The wide binary fraction decreases strongly with increasing cluster mass, and the semi-major axis of the newly formed binaries is determined by the initial cluster size. The resulting eccentricity distribution is thermal, and the mass ratio distribution is consistent with gravitationally-focused random pairing. As a large fraction of the stars form in primordial binaries, we predict that a large number of the observed 'wide binaries' are in fact triple or quadruple systems. By integrating over the initial cluster mass distribution, we predict a binary fraction of a few per cent in the semi-major axis range 1000 au - 0.1 pc in the Galactic field, which is smaller than the observed wide binary fraction. However, this discrepancy may be solved when we consider a broad range of cluster morphologies.Comment: 14 pages, 12 figures, accepted by MNRA