Die Rolle von APOBEC3G in der angeborenen Immunität gegenüber HIV-Infektionen primärer Zellen

In the past few years the regulation of HIV-1 replication by cellular cofactors has been a major topic of ongoing research. These factors potentially represent new targets for antiviral therapy as resistance will be minimized. However this requires a better understanding of the interaction of HIV-1 with these cellular factors and the immune system. The virus infects the cells of the immune system, beginning with macrophages and dendritic cells as primary target cells during transmission. The cellular cofactor, APOBEC3G was found to be an antiviral factor in macrophages, dendritic cells and primary T cells. APOBEC3G is a cytidindeaminase which causes G->A hypermutations in the HIV-Genome. Another protein which has a strong inhibitory effect on the HIV infection is Interferon alpha (IFN-alpha), however the exact reason for this has not yet been elucidated. The bacterial protein, Lipopolysaccharide (LPS) also induces a strong antiviral state in macrophages. In micro-array analysis it was shown that APOBEC3G was upregulated after the stimulation with both IFN-alpha and LPS in macrophages. The goal of this work was to investigate the role of APOBEC3G in the innate immune response to APOBEC3G. For this, the expression of APOBEC3G was examined in HIV-1 target cells after stimulation with IFN-alpha or LPS and the effect of the protein on the viral infection was examined. In the first experiments it could be shown through real time quantitative PCR that APOBEC3G was overexpressed after the stimulation with IFN-alpha or LPS. This result could be shown in monocytes derived macrophages from different blood donors. It was also shown that the overexpression of APOBEC3G correlated directly with the concentration of IFN-alpha. Through mutational analysis it could be then shown that the overexpressed APOBEC3G protein was also functional in the cells. In order to show that this was the result of APOBEC3G, the protein was the regulated through lentiviral vectors. After transduction of cell lines with lentiviral vectors containing APOBEC3G, the infection was inhibited by up to 70%. The infection was restored after the addition of shRNAs against APOBEC3G. For the further experiments, CD34+ stem cells were used. The cells were transduced the day after thawing with lentiviral vectors containing an eGFP marker gene and either APOBEC3G or shRNAs against APOBEC3G. The CD34+ cells were then cultivated and differentiated to macrophages. The cells transduced with Lentiviral vectors containing APOBEC3G had a very high expression of APOBEC3G in the cells, however the cells transduced with shRNA against APOBEC3G did not show a reduction in the protein expression. The infectivity of the transduced CD34+ and CD34 derived macrophages was then examined. It was expected that the cells transduced with APOBEC3G would show a reduced HIV-1 infection, and the cells transduced with shRNA against APOBEC3G would show an increase in infection. After the transduction and differentiation the CD34+ cells from the 3 donors were stimulated and infected with wild type HIV-1 and Vif defective HIV-1 virus. Vif is a viral protein that can bind to APOBEC3G leading it to the proteasome for degradation. The cells from the first donor transduced with APOBEC3G, were very difficult to infect. In general the shRNA against APOBEC3G had little effect on the course of infection; presumably, the shRNA against APOBEC3G was not active in most of these cells. Only the cells from the first donor showed an increase in HIV infection after the transduction with the shRNAs against APOBEC3G, this was most notably the case in the cells stimulated with IFN-alpha, which usually show very little infection. This work showed that APOBEC3G plays an important role in the innate immune response to HIV-1. The effect of APOBEC3G is both cell type as well as donor dependent. Recently, an interesting study also showed that there is a correlation between the expression of APOBEC3G in HIV infected individuals and their progression to AIDS. A better understanding of the role that APOBEC3G plays in the innate immune response would help in the search of new therapeutic possibilities. This could be done by inhibiting the Vif-APOBEC3G interaction in order to increase the amount of active APOBEC3G in the cells or increasing the APOBEC3G concentration in the cells in some manner.Das HI-Virus stellt mit zahlreichen Neuinfektionen pro Jahr immer noch ein großes gesundheitliches und gesellschaftliches Problem dar. Daher ist die ständige Entwicklung neuer, fortschrittlicher Medikamente wichtig. Um dies zu ermöglichen, muss man das Zusammenspiel zwischen dem Virus und dem Immunsystem sowie mit den zellulären Cofaktoren besser verstehen. Das Virus greift zuerst die Zellen des Immunsystems an, allen voran die Makrophagen und die dendritischen Zellen. Der zelluläre Cofaktor APOBEC3G wurde in genau diesen Zelltypen als antiviraler Faktor gefunden. Die Cytidindeaminase APOBEC3G verursacht unter anderem G->A hypermutierte HIV-Genome, die nicht funktionell sind. Ein weiteres zelluläres Protein, das speziell in Makrophagen eine antivirale Wirkung gegenüber HIV hat, ist Interferon alpha (IFN-alpha). Auch bakterielles Lipopolysaccharid (LPS) übt einen starken Hemmeffekt gegenüber der HIV-Infektion aus; der genaue Grund hierfür ist jedoch nicht bekannt. Es wurde in Mikroarrayanalysen festgestellt, dass das APOBEC3G-Protein durch die Stimulierung mit IFN-alpha oder LPS in Makrophagen hochreguliert wurde. Daher sollte in dieser Arbeit untersucht werden, ob ein Zusammenhang zwischen diesem zellulären Faktor und der HIV-Restriktion besteht. Dazu wurde die Expression von APOBEC3G in HIV-Zielzellen (Makrophagen, :dendritischen Zellen und CD34+-Vorläuferzellen) nach Stimulierung mit IFN-alpha oder LPS sowie die Steuerung der Expression von APOBEC3G in diesen Zellen untersucht. Des Weiteren wurde die Korrelation mit der Infektiösität der Zellen mit HIV-1 untersucht. Zuerst konnte durch quantitative PCR bestätigt werden, dass APOBEC3G nach der Zugabe von IFN-alpha oder LPS überexprimiert war. Dieses Ergebnis wurde in den von Monozyten abgeleiteten Makrophagen mehrerer Blutspender nachgewiesen. Es wurde auch gezeigt, dass die Überexpression von APOBEC3G direkt mit der Konzentration von IFN-alpha korreliert. Danach wurde durch Mutationsanalyse der in diesen Zellen produzierten HIV-Genome die Funktionalität des APOBEC3G gezeigt. Um zu zeigen, dass dieser Effekt auf APOBEC3G zurückzuführen ist, wurde das Protein direkt durch ein lentivirales Vektorsystem reguliert. Bei der Transduktion von APOBEC3G in Zelllinien wurde die HIV-Infektion zu über 70% gehemmt, was durch die zusätzliche Expression von shRNAs gegen APOBEC3G aufgehoben wurde. In den weiteren Versuchen wurden menschliche blutbildende CD34+-Stammzellen von drei Spendern eingesetzt. Die CD34+-Zellen wurden am ersten Tag nach dem Auftauen mit lentiviralen Vektoren mit einem eGFP-Markergen transduziert und dann kultiviert. Nach der Transduktion nahm die Menge der Zellen, die eGFP exprimierten, unvermuteterweise mit der Zeit ab. Trotz dieses starken Rückgangs war die Expression von APOBEC3G in den Zellen, die mit dem APOBEC3G-Gen transduziert waren, immer sehr hoch. Allerdings hatte die shRNA gegen APOBEC3G, die in Zelllinien aktiv war, sowohl in den CD34+-Zellen als auch in den von CD34+-Zellen abgeleiteten Makrophagen keine reduzierende Wirkung auf die APOBEC3G-Expression. Als nächstes wurde die HIV-Infizierbarkeit der CD34+-Zellen und der von CD34+-Zellen abgeleiteten Makrophagen analysiert. Erwartet wurde, dass die Infizierbarkeit in den Zellen mit dem zusätzlich exprimierten APOBEC3G geringer ist als in den Zellen mit shRNAs gegen APOBEC3G. Nach der Transduktion und Ausdifferenzierung der CD34+-Zellen von drei Spendern wurden die Zellen stimuliert und mit Wild-Typ-HIV-1 und Vif-defektem HIV-1 infiziert. Vif ist ein virales Protein, welches APOBEC3G bindet und dieses zum Proteinabbau zum Proteasom führt. Die Zellen des ersten Spenders, die mit APOBEC3G transduziert worden waren, waren sehr schwer infizierbar, während die Zellen der anderen zwei Spender unabhängig von der Zugabe von APOBEC3G infizierbar waren. Der Effekt der shRNA gegen APOBEC3G auf die HIV-Infektion war, wie aufgrund der Expressionsanalyse zu erwarten, sehr gering. Offenbar war die shRNA in diesen Zellen nicht aktiv. Lediglich die Zellen des ersten Spenders, die mit einem Vif-defektiven Virus infiziert wurden, waren nach der Transduktion mit shRNA gegen APOBEC3G wieder infizierbar. Dies gilt insbesondere auch für diejenigen Zellen, die mit IFN-alpha stimuliert worden waren. Sonst waren die CD34+-Zellen nach Stimulierung mit IFN-alpha nicht mehr infizierbar. Diese Arbeit zeigt, dass APOBEC3G eine wichtige Rolle in der Immunität gegen HIV-1 spielt. Der Zusammenhang ist sowohl vom Zelltyp als auch Spender abhängig. Interessanterweise wurde kürzlich ein Artikel über den Zusammenhang zwischen der Expression von APOBEC3G in HIV-Patienten und der Progression ihres Krankheitsverlaufs publiziert. Das bessere Verständnis dieser Zusammenhänge wäre eventuell eine Möglichkeit, APOBEC3G therapeutisch einzusetzen, indem zum Beispiel durch Hemmung der Vif-APOBEC3G-Interaktion die Menge von aktivem APOBEC3G in der Zelle erhöht wird

Computational studies of glutamate transporters and receptors

In this thesis we use molecular dynamics simulations to study glutamate transporters and receptors, as well as toxin binding to human potassium channels. We apply a variety of techniques in computational biology such as ligand docking, parameterization of molecules, homology modeling, free energy perturbation and potential of mean force. We first study the archaeal aspartate transporter GltPh, which is a homolog of the human glutamate transporters (EAATs). We locate the third sodium ion binding site and show that this site is also conserved in the EAATs. We then perform molecular dynamics simulations in the outward and inward facing states of GltPh, calculating the ligand affinities in both conformations, the order of ligand binding/unbinding, and the differences in the gating mechanism between the inward and outward states. Using the GltPh crystal structures, we build models for the human glutamate transporters. We use our models to locate the potassium and proton binding sites in EAATs, investigate the gating mechanism, and elucidate the mechanism of proton transport. We also calculate the standard binding free energy of five small molecules to the ligand binding domain of the GluA2 receptor, using a free energy perturbation approach that produces results with very good agreement to experimental values. We show that this method is effective for polar and charged ligands, and can be applied to many biological systems, providing an useful tool in computer aided drug design. Finally, we develop a method based on free energy perturbation that can be used to calculate the binding free energy differences of peptide toxin mutants to Kv potassium channels, producing very good agreement to potential of mean force and experimental results. We use it to obtain a ShK mutant that is selective for the Kv1.1 channel over Kv1.3, and therefore may be used in the treatment of auto-immune diseases

Olten Meeting – A Contact Forum for Biotech - November 19, 2008: Conference Report

The annual Olten Meeting organized by biotechnet switzerland and the Swiss Biotech Association is a place for biotech specialists from the universities and Empa to meet up with people from the private sector to network and initiate joint projects. A look at the presentations given at the event on 19 November 2008

The Entirely Improbable, Daring, Quiet Food Revolt: Imaginary Feasts at Nazi Concentration Camps as shown in Anne Georget\u27s Documentary Festins Imaginaires

Festins Imaginaires by the French documentary filmmaker Anne Georget premiered at the Culinary Cinemas series of the Berlin film festival in 2015. This was proof that eating and cooking could create a bond even in its virtual form as people talked about food but could also invigorate and nourish even in its absence. The 70 minutes documentary is about a quiet revolt. The starving women dream up a communal kitchen and are bold enough to write it down

Fake Pastorals: Where is the Nature in Cheese?: a Critical Look at Modern Vertical Pastoralism—Alpine Cheeses, Mountain Pastures, and Cultural Diversity—Presenting Two Examples of Best Practice in Austria and Anatolia

We cherish the pictures of happy cows on luscious green mountain pastures. As informed ‘foodies’ we know about the better quality of pastured milk, and the artisanal cheesemaking traditions at all those small summer mountain dairies, conveying its special character to each single wheel. But is that really what is happening? In most cases it isn’t. Why would it even be important? Presenting two examples of best practice, I hope to show why real alpine cheese matters and why it is important to ask the right questions

Visualising the Prophet – Rhetorical and Graphic Aspects of Three Ottoman-Turkish Poems

Narrative and panegyric poems about the Prophet Muḥammad were among the most widely read texts in the Ottoman Empire. They made the Prophet accessible to a broad readership; they were recited during rites; and the acts of copying them with one’s own hand, reading them, and presenting them as endowments promised reward on the day of judgement. There also was a strong visual aspect to the production and usage of these manuscripts: the beauty of the Prophet, the beauty of the (spoken) word, and the beauty of the handwriting were interrelated. Much can therefore be learned about the image of the Prophet Muḥammad in Ottoman society, and specifically in Ottoman book culture, by analysing this interrelation. In my study I focus on three poems, which are preserved in large numbers in manuscript collections all over the world, and which were published in several printed editions – Süleymān Çelebi’s Vesīlet en-Necāt (812/1409), Yazıcıoğlı Muḥammed’s Muḥammedīye (853/1449), and Ḫāḳānī’s Ḥilye (1007/1598–9). These have several features in common: their topic (the biography and/or the physiognomy of Muḥammad); their form (poetry); the fact that the texts are transmitted with the signature of their author; and a precise date. However, the authors employed very different strategies to visualise the Prophet, and so did the copyists, calligraphers, and illustrators. The three texts are analysed successively. Each section begins with a short survey of the intention of the authors as described in prefaces or colophons, and, if relevant, later biographical and hagiographical texts. Then the structure of the texts and narrative, along with their poetical and rhetorical characteristics, will be described, and, in a concluding step, the interrelation with graphic aspects of selected manuscripts and printed copies is explored

The German-Jewish paediatrician Albert Eckstein (1891-1950) exiled to Turkey: Pioneering modern paediatric care and social hygiene (health sciences) during World War II

During the thirties of the twentieth century, German medical doctors immigrated to Turkey. Among them, was the German-Jewish paediatrician Albert Eckstein. In this short biography, the richness of the literature, written by or about Eckstein, will be presented, and altogether combined. Starting from 1937 and further on, Albert Eckstein undertook scientific surveys on children’s state of health and health care in the most remote areas of Anatolia. The value of the social-hygienic approach could be recognized, even in this early stage, starting with epidemiological analysis and followed by basic comprehensive health care. Social hygiene, as a young branch of health sciences at the time, was in the position even then to model the health care system for large population groups, at least in countries actively developing health care, as was Turkey of that time. Albert Eckstein and his co-workers, such as Ihsan Dogramaci, stand out as founders of the modern Turkish health care system today and health sciences in this country