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The PANDA experiment will be one of the flagship experiments at the future Facility for Antiproton and Ion Research (FAIR) in Darmstadt, Germany. It is a versatile detector dedicated to topics in hadron physics such as charmonium spectroscopy and nucleon structure. A DIRC counter will deliver hadronic particle identification in the barrel part of the PANDA target spectrometer and will cleanly separate kaons with momenta up to 3.5 GeV/c from a large pion background. An alternative DIRC design option, using wide Cherenkov radiator plates instead of narrow bars, would significantly reduce the cost of the system. Compact fused silica photon prisms have many advantages over the traditional stand-off boxes filled with liquid. This work describes the study of these design options, which are important advancements of the DIRC technology in terms of cost and performance. Several new reconstruction methods were developed and will be presented. Prototypes of the DIRC components have been built and tested in particle beam, and the new concepts and approaches were applied. An evaluation of the performance of the designs, feasibility studies with simulations, and a comparison of simulation and prototype tests will be presented.
Im Rahmen des FAIR Projektes wurde ein neuartiger Prototyp eines nicht strahlzerstörenden Bunch Struktur Monitors (BSM) am GSI UNILAC entwickelt. Ziel ist es, ein zuverlässiges Diagnosegerät zu entwickeln, welches die longitudinale Struktur der Ionenbunche innerhalb des LINACs untersuchen kann. Notwendig ist hierbei eine effektive Zeitauflösung deutlich unter 100 ps, bei möglichst wenigen Makropuls Mittelungen. Nach der erfolgreichen Inbetriebnahme soll der BSM Prototyp dazu dienen, die Umsetzbarkeit eines weiteren nichtinvasiven Geräts für den geplanten Proton-LINAC bei FAIR mit einer notwendigen Zeitauflösung von 10 ps zu beurteilen.
Die numerische Simulation von Materialien, welche dem Hochstrom-Ionenstrahl ausgesetzt sind, zeigten einen sehr hohen thermischen Stress. Daher wurde der Ansatz eines nicht strahlzerstörenden Diagnosegerätes verfolgt. Das Design beruht auf der Erzeugung von Sekundärelektronen durch Strahl-Restgas Kollisionen im Strahlrohr. Durch das Anlegen eines homogenen Hochspannungspotentials von bis zu -31 kV, wird ein Elektronenstrahl erzeugt, welcher die zeitliche Struktur des Ionenbunches trägt. Die zeitliche Information des Elektronenstrahles wird beim Durchfliegen eines HF-Ablenkers, welcher resonant an die 36 MHz des Beschleunigers gekoppelt ist, in eine räumliche Intensitätsverteilung umgewandelt. Anschließend wird die Elektronenverteilung auf einem bildgebenden MCP-Phosphor-Detektor durch eine CCD-Kamera detektiert und in die Bunch Struktur überführt.
Intensive Untersuchungen der BSM Eigenschaften ergaben eine höchste Auflösung von 37 ±6.3 ps bei gleichzeitig akzeptabler Intensität auf dem MCP-Detektor. Unter anderem wurden auch stabile Einzelschussmessungen durchgeführt, welche für die Profilmessung nur einen einzelnen Makropuls benötigten, statt über typischerweise 8-32 Pulse zu mitteln.
Durch die systematische Manipulation der Bunchlänge durch einen Rebuncher sind nicht gaußförmige Profile von 280 ps bis 650 ps detektiert worden, welche als Studie für eine Emittanzbestimmung genutzt worden sind. In Abhängigkeit des Analyseverfahrens sind Werte von εGauss = 1.42 ±0.14 keV/u ns bis εSD = 3.03 ±0.33 keV/u ns für die Emittanz bestimmt worden.
Des Weiteren ist ein Finite-Elemente Modell erstellt worden, um die Zeitstruktur der Sekundärelektronen innerhalb des elektronenoptischen Systems zu bestimmen. Für das Setup mit der höchsten Auflösung von 37 ps ergab sich eine zusätzliche Zeitverbreiterung von 5.6 ps, welche nur geringfügig die experimentell bestimmte Auflösung verschlechtert.
Der nicht strahlzerstörende BSM liefert eine ausreichend hohe zeitliche Auflösung für detailreiche Untersuchung der longitudinalen Bunchstruktur, ohne negative Einflüsse auf den Ionenstrahl auszuüben. Fortgeschrittene Messungen, wie longitudinale Emittanzbestimmung und Makropulsanalysen, sind möglich und werden dazu beitragen, die LINAC Strukturen besser zu verstehen und weiter zu optimieren.
Obwohl bei der Umsetzung des Arbeitsprinzips für den geplanten Proton-LINAC die veränderten Strahlparameter berücksichtigt werden müssen, zeigen die Ergebnisse, wie die Zeitstrukturuntersuchung und die erreichte Phasenauflösung von 0.5° bei 36 MHz, dass zeitliche Auflösungen bei Aufrechterhaltung der Phasenauflösung von bis zu 10 ps für einen neuen BSM Prototypen möglich sind.
Xenorhabdus and Photorhabdus bacteria are gaining more and more attention as a subject of research because of their unique yet similar life cycle with nematodes and insects. This work focused on the secondary metabolites that are produced by Xenorhabdus and Photorhabdus. With the help of modern HPLC-MS methodologies and increasingly available bacterial genome sequences, the structures of unknown secondary metabolites could be elucidated and thus their biosynthesis pathways could be proposed, too.
The first paper reported 17 depsipeptides termed xentrivalpeptides produced by the bacterium Xenorhabdus sp. 85816. Xentrivalpeptide A could be isolated from the bacterial culture as the main component. The structure of xentrivalpeptide A was elucidated by NMR and the Marfey´s method. The remaining xentrivalpeptides were exclusively identified by feeding experiments and MS fragmentation patterns.
The second paper described the discovery and isolation of xenoamicin A from Xenorhabdus mauleonii DSM17908. Additionally, other xenoamicin derivatives from Xenorhabdus doucetiae DSM17909 were analyzed by means of feeding experiments and MS fragmentation patterns. The xenoamicin biosynthesis gene cluster was identified in Xenorhabdus doucetiae DSM17909.
The manuscript for publication focused on the biosynthesis of anthraquinones in Photorhabdus luminescens. The Type II polyketide synthase for the biosynthesis of anthraquinone derivatives was discovered in P. luminescens in a previous publication by the Bode group,1 in which a partial reaction mechanism for the biosynthesis has been proposed. The manuscript reported in this thesis however elucidated the biosynthetic mechanisms in a greater detail as compared to the previous publication. Particularly, the biosynthetic mechanism was deciphered through heterologous expression of anthraquinone biosynthesis (ant) genes in E. coli. Additionally, deactivation of the genes antG encoding a putative CoA ligase and antI encoding a putative hydrolase, was performed in P. luminescens. Selected ant genes were over-expressed in E. coli as well as the corresponding proteins purified for in vitro assays. Model compounds were chemically synthesized as possible substrates of AntI and were used for in vitro assays. Here, it was revealed that the CoA ligase AntG played an essential role in the activation of the ACP AntF. Furthermore, a chain shortening mechanism by the hydrolase AntI was identified and was further confirmed by in vitro assays using model compounds. Additionally, this chain shortening mechanism was supported by homology based structural modeling of AntI.
Metal-organic frameworks (MOFs) have emerged as a promising class of crystalline porous inorganic-organic hybrid materials showing a wide range of applications. In order to realize the integration of MOFs into specific devices, this thesis mainly focuses on the controlled growth and the properties of highly oriented surface-mounted metal-organic frameworks (SURMOFs).
The stepwise layer-by-layer (LbL) growth method exhibits vast advantages for the controllable growth of SURMOFs regarding the crystallite orientation, film thickness and homogeneity. However, up to date, only a few MOFs have been demonstrated to be suited for this protocol. So the first project of this thesis was designed to extend the applicability of the LbL growth. To this end, a semi-rigid linker based [Cu2(sdb)2(bipy)] (sdb = 4,4’-sulfonylbiphenyl dicarboxylate; bipy = 4,4’-bipyridine) MOF was chosen. Employing the LbL growth, [Cu2(sdb)2(bipy)] SURMOFs were successfully grown onto both pyridyl- and carboxyl-terminated surfaces at the temperature range of 15-65 °C. Interestingly, the orientation of the SURMOFs largely depends on temperature on both surfaces. At low temperatures (below 40 °C), SURMOFs with exclusive [010] orientation are obtained. In contrast, at high temperatures (40-65 °C), [001] oriented SURMOF growth is favored. A novel growth mode was demonstrated, which is, instead of surface chemistry, the temperature-induced ripening processes and the tendency to minimize surface energies can dominate the SURMOF growth.
Inspired by the advantages of LbL deposition of isoreticular SURMOFs, the second project was conceived to grow multivariate SURMOFs (MTV-SURMOFs) using mixed dicarboxylate linkers. We advance a hypothesis that linker acidity (expressed by the pKa values) may have an influence on the oriented growth of MTV-SURMOFs. To test the hypothesis, seven isoreticular [Cu2L2(dabco)] (L = single kind of dicarboxylate linker; dabco = 1,4-diazabicyclo[2.2.2]octane) SURMOFs were grown onto pyridyl-terminated surfaces at 60 °C. The quality of [001] orientation is greatly affected by the acidity of the linkers. With this observation, we deposited a series of [Cu2Lm2(dabco)] (Lm = mixed dicarboxylate linkers) SURMOFs under the same conditions. [Cu2Lm2(dabco)] SURMOFs with exclusive [001] orientation are obtained when the growth solution contains two linkers of relatively high pKa value or more than two kinds of linkers (independent of the pKa values), while the mixtures of ligands with relatively low pKa values or a high content of low pKa valued linkers can result in mis-oriented growth of SURMOFs with unexpected [100] orientation.
Moreover, the LbL growth shows enormous potential in the rational construction of functional SURMOFs. Therefore, the third project of this thesis was devised to deposit SURMOFs containing redox-active species. For this, the 4,4’-biphenyldicarboxylic acid (H2(bpdc)) linker was functionalized with ferrocene (Fc) and dimethyl ferrocene (Me2Fc) moieties. [Cu2(bpdc-amide-Fc)2(dabco)] SURMOF (Fc-SURMOF) is perfectly grown along the [100] direction, while mis-oriented growth of [Cu2(bpdc-amide-Me2Fc)2(dabco)] SURMOF (Me2Fc-SURMOF) was observed. Surprisingly, Fc-SURMOF shows excellent electrochemical properties due to the reversible oxidation and reduction of the ferrocene moieties in the oriented pores, while the Me2Fc-SURMOF was found to be a closely packed insulating layer since no extensive charge transfer is observed. A diffusion controlled mechanism of redox reaction is proposed, where the diffusion of the counter anions in the pores limits the current.
Besides the LbL growth protocol, the spin-coating technique is also promising for the oriented growth of SURMOFs. Driven by the specific applications, the fourth project of this thesis was planned to grow functional SURMOFs containing catalytically active units. The Keggin-type polyoxometalates (POMs) with high catalytic activities were chosen to functionalize the HKUST-1 SURMOFs. Combining the technique with methanol vapor induced growth, a series of POM functionalized HKUST-1 SURMOFs (denoted as POM@HKUST-1 SURMOFs) were controllably deposited onto pyridyl-terminated surfaces. The SURMOFs exhibit great potential as electrocatalysts in electrochemical devices due to the excellent redox properties of POMs. In addition, the PTA@HKUST-1 (PTA = phosphotungstic acid) SURMOF can be employed as an ideal platform for the selective loading of methylene blue (MB) dye with high efficiency. Owing to the strong binding between the dye molecules and the framework, the MB dye cannot be desorbed by ion exchange and MB loaded PTA@HKUST-1 SURMOF shows reliable redox properties under inert conditions, further confirming the application potential in electrochemical devices.
RNA modifications are widespread in the RNA world. Nevertheless, their functions remain enigmatic. Recent analysis in tRNAs, mRNAs and rRNAs have revealed that apart from enriching their topological potential, these chemical modifications provide an added significant regulatory level to gene expression...
Das Hauptziel dieser Dissertation lag in der Verbesserung einzelner Schritte im Prozess der automatischen Proteinstrukturbestimmung mittels Kernmagnetischer Resonanz (NMR). Dieser Prozess besteht aus einer Reihe von sequenziellen Schritten, welche zum Teil bereits erfolgreich automatisiert wurden. CYANA ist ein Programmpaket, welches routinemäßig zur automatischen Zuordnung der chemischen Verschiebungen, der Nuclear Overhauser Enhancement (NOE) Signalen und der Strukturrechnung von Proteinen verwendet wird. Einer der Schritte, der noch nicht erfolgreich automatisiert wurde, stellt die Signalidentifizierung von NMR Spektren dar. Dieser Schritt ist besonders wichtig, da Listen von NMR-Signalen Grundlage aller Folgeschritte sind. Fehler in den Signallisten pflanzen sich in allen Folgeschritten der Datenauswertung fort und können am Ende in falschen Strukturen resultieren. Daher war ein Ziel dieser Arbeit, einen robusten und verlässlichen Algorithmus zur Signalidentifizierung von NMR Spektren in CYANA zu implementieren. Dieser Algorithmus sollte mit dem in FLYA implementierten Ansatz zur automatischen Resonanzzuordnung, der automatischen NOE-Zuordnung und der Strukturrechnung mit CYANA kombiniert werden. Der in CYANA implementierte CYPICK Algorithmus ahmt den von Hand durchgeführten Ansatz nach. Bei der manuellen Methode schaut sich der Wissenschaftler zweidimensionale Konturliniendarstellungen der NMR Spektren an und entscheidet anhand verschiedener Geomtrie- und Ähnlichkeitskriterien, ob es sich um ein Signal des Proteins oder um einen Artefakt handelt. Proteinsignale sind ähnlich zu konzentrischen Ellipsen und erfüllen bestimmte geometrische Kriterien, wie zum Beispiel ungefähr kreisförmiges Aussehen nach entsprechender Skalierung der spektralen Achsen und gänzlich konvexe Formen, die Artefakte nicht aufzeigen. CYPICK bewertet die Konturlinien lokaler Extrema nach diesen Bedingungen und entscheidet anhand dieser, ob es sich um ein echtes Signal handelt oder nicht. Das zweite Ziel dieser Arbeit war es ein Maß zur Quantifizierung der Information von strukturellen NMR Distanzeinschränkungen zu entwickeln. Der sogenannte Informationsgehalt (I) ist vergleichbar mit der Auflösung in der Röntgenkristallographie. Ein weiteres Projekt dieser Dissertation beschäftigte sich mit der strukturbasierten Medikamentenentwicklung (SBDD). SBDD wird meist von der Röntgenkristallographie durchgeführt. NMR hat jedoch einige Vorteile gegenüber der Röntgenkristallographie, welche interessant für SBDD sind. Daher wurden Strategien entwickelt, die NMR für SBDD zugänglicher machen sollen.
Water is scarce in semi-arid and arid regions. Using alternative water sources (i.e. non-conventional water sources), such as municipal reuse water and harvested rain, contributes to using existing water resources more efficiently and productively. The aim of this study is to evaluate the two alternative water sources reuse water and harvested rain for the irrigation of small-holder agriculture from a system perspective. This helps decision and policy makers to have proper information about which system and technology to adopt under local conditions. For this, the evaluation included ecologic, societal, economic, institutional and political as well as technical aspects. For the evaluation, the study area in central-northern Namibia was chosen in the frame of the research and development project CuveWaters. The main methods used include a mathematical material flow analysis, the computation and modelling of crop requirements, a multi-criteria decision analysis using the Analytical Hierarchy Process (AHP) method and a financial cost-benefit analysis. From a systemic perspective, the proposed novel systems were compared to the exciting conventional infrastructure. The results showed that both water reuse and rainwater harvesting systems for the irrigation of small-holder horticulture offer numerous technological, ecologic, economic, societal, institutional and political benefits. Rainwater harvesting based gardens have a positive benefit-cost ratio under favorable conditions. Government programs could fund the infrastructure investment costs, while the micro-entrepreneur can assume a micro-credit to finance operation and maintenance costs. Installing sanitation in informal settlements and reusing municipal water for irrigation reduces the overall water demand of households and agriculture by 39%, compared to improving sanitation facilities in informal settlements without reusing the water for agriculture. Given that water is the limiting factor for crop fertigation, the generated nutrient-rich reuse water is sufficient to annually irrigate about 10 m2 to 13 m2 per sanitation user. Compared to crop nutrient requirements, there are too many nutrients in the reuse water. Thus when using nutrient-rich reuse water, no use of fertilizers and a careful salt management is necessary. When comparing this novel system with improved sanitation, advanced wastewater treatment and nutrient-rich water reuse to the conventional and to two adapted systems, results showed that the novel CuveWaters system is the best option for the given context in a semi-arid developing country. Therefore, the results of this study suggest a further roll-out of the novel CuveWaters system. The methodology developed and the results of this study demonstrated that taking sanitation users into consideration plays a major role for the planning of an integrated water reuse infrastructure because they are the determinant factor for the amount of available nutrient-rich reuse water. In addition, it could be shown that water reuse and rainwater harvesting systems for the irrigation of small-scale gardens provide a wide range of benefits and can be key to using scarce water resources more efficiently and to contributing to the Sustainable Development Goals.
Light scalar mesons can be understood as dynamically generated resonances. They arise as 'companion poles' in the propagators of quark-antiquark seed states when accounting for hadronic loop contributions to the self-energies of the latter. Such a mechanism may explain the overpopulation in the scalar sector - there exist more resonances with total spin J=0 than can be described within a quark model.
Along this line, we study an effective Lagrangian approach where the isovector state a_{0}(1450) couples via both non-derivative and derivative interactions to pseudoscalar mesons. It is demonstrated that the propagator has two poles: a companion pole corresponding to a_{0}(980) and a pole of the seed state a_{0}(1450). The positions of these poles are in quantitative agreement with experimental data. Besides that, we investigate similar models for the isodoublet state K_{0}^{*}(1430) by performing a fit to pion-kaon phase shift data in the I=1/2, J=0 channel. We show that, in order to fit the data accurately, a companion pole for the K_{0}^{*}(800), that is, the light kappa resonance, is required. A large-N_{c} study confirms that both resonances below 1 GeV are predominantly four-quark states, while the heavy states are quarkonia.
In dieser Arbeit wurden die Strukturen von drei Membranproteinen mittels Einzelpartikel-Kryo‑Elektronenmikroskopie (Kryo‑EM) gelöst. Bei den Membranproteinen handelt es sich um den humanen TRP-Kanal Polycystin‑2, den sekundär-aktiven Transporter BetP aus Corynebacterium glutamicum und den Rotor-Ring der N‑Typ ATPase aus Burkholderia pseudomallei.
Kanäle sind Membranproteine, die Ionen durch eine Pore über die Membran diffundieren lassen. Durch einen präzisen, kanalabhängigen Regulationsmechanismus wird die Pore nur bei Bedarf geöffnet. TRP (transient receptor potential) Kanäle sind anhand von DNA-Sequenzvergleichen identifiziert worden und kommen ausschließlich in Eukaryonten vor. In dieser Arbeit lag der Fokus auf der Strukturbestimmung des humanen TRP Kanals Polycystin‑2 (PC‑2). PC‑2 wurde in einer Studie entdeckt, in der Patienten mit der autosomal dominanten Erbkrankheit „polyzystische Nierenerkrankung“ untersucht wurden. Patienten mit dieser Krankheit tragen eine Mutation in einem der beiden Gene PKD1 oder PKD2, welche für die Proteine Polycystin‑1 und ‑2 kodieren. In dieser Arbeit wurden verschiedene Deletionsmutanten von PC‑2 hergestellt und in das Genom menschlicher HEK293 GnTI‑ Zellen inseriert. Die Zellen, die PC‑2 bzw. die Deletionskonstrukte am stärksten synthetisierten, wurden isoliert und für die rekombinante Proteinherstellung verwendet. Die Expression von PC‑2 führte zu der Entstehung von kristalloidem endoplasmatischem Retikulum. Mutationsstudien in dieser Arbeit zeigen, dass diese morphologische Veränderung durch die Akkumulation von Membranproteinen, die mit sich selbst interagieren, begünstigt wird. Weiter ist es in dieser Arbeit gelungen, PC‑2 zu reinigen und die Struktur des Proteins mit Hilfe von Einzelpartikel Kryo-EM mit einer Auflösung von 4.6 Å zu bestimmen. Die Membrandomäne von PC‑2 ist sehr ähnlich zu den bekannten TRP Kanal Strukturen. Ein Vergleich der PC‑2 Struktur mit dem offenen und geschlossenen TRPV1 Kanal legt nahe, dass PC‑2 in seiner offenen Konformation gelöst wurde.
Der sekundär aktive Transporter BetP von C. glutamicum gehört zu der Familie der BCC- (betaine-carnitine-choline) Transporter und wird durch osmotischen Schock aktiviert. Nach seiner Aktivierung importiert BetP zwei Natriumionen und ein Glycinbetain Molekül. Durch die Akkumulierung von Glycinbetain in der Zelle steigt das osmotische Potential des Zytoplasmas, was den Wasserausstrom aus der Zelle stoppt. Viele Strukturen, die BetP in unterschiedlichen Stadien des Transportprozesses zeigen, konnten bereits mittels Röntgenkristallographie gelöst werden. Allerdings ist die N‑terminale Domäne für die Kristallisation entfernt worden und die C‑terminale Domäne, die komplett aufgelöst ist, ist an einem wichtigen Kristallkontakt beteiligt. Um strukturelle Informationen über die N‑ und C‑terminale Domäne ohne Kristallisationsartefakte zu erhalten, wurde in dieser Arbeit die Struktur von BetP mittels Einzelpartikel Kryo‑EM bestimmt. Die Struktur mit einer Auflösung von 6.8 Å zeigt BetP in einem zum Zytoplasma geöffneten Zustand. Der größte Unterschied zu allen Kristallstrukturen ist die Position der C‑terminalen α‑Helix, die um ~30° rotiert ist und dadurch deutlich enger am Protein zu liegen kommt. Da BetP in Abwesenheit von aktivierenden Stoffen analysiert wurde, wird vermutet, dass es sich bei der gelösten Struktur um den inaktiven Zustand von BetP handelt.
Rotierende ATPasen sind membrangebunden Enzymkomplexe, die bei der zellulären Energieumwandlung eine entscheidende Rolle einnehmen. Sie bestehen aus einem löslichen und einem membrangebundenen Teil. Während in dem löslichen Teil der zelluläre Energieträger Adenosintriphosphat (ATP) entweder synthetisiert oder hydrolysiert wird, baut der membrangebundene Teil entweder einen Ionengradienten auf oder nutzt die Energie eines existierenden Gradienten für die ATP Synthese. Ein wesentlicher Bestandteil des membrangebundenen Teils einer rotierenden ATPase ist der Rotor-Ring. Dieser transportiert Ionen über die Membran und rotiert dabei um seine eigene Achse. In dieser Arbeit wurde eine Studie fortgesetzt, die den Rotor-Ring der N‑Typ ATPase von B. pseudomallei mittels Kryo‑EM untersuchte und zeigte, dass der Rotor-Ring aus 17 identischen Untereinheiten aufgebaut ist. Damit hat die N‑Typ ATPase das größte Ionen-zu-ATP-Verhältnis aller bisher charakterisierten ATPasen. In dieser Arbeit wurde die c17 Stöchiometrie des N‑Typ ATPase Rotor-Rings bestätigt und die Struktur mittels Kryo‑EM bestimmt. Im besonderen Fokus lag dabei der Einfluss von Detergenzien auf die Strukturbestimmung. Es konnte gezeigt werden, dass die beiden Parameter Dichte und Mizellengröße der verwendeten Detergenzien ausschlaggebend für den Erfolg der Strukturbestimmung dieses sehr kleinen Membranproteins sind.
This PhD thesis has been carried out within an interdisciplinary cooperational project between the Deutsches Bergbau-Museum Bochum and the Goethe-Universität Frankfurt, which is dedicated to ancient Pb-Ag mining and metal production in the hinterland of the municipium Ulpiana in central Kosovo. Geochemical analysis (OM, XRD, EMP, MC-ICP-MS) of ores, metallurgical (by-) products and metal artefacts allowed to reconstruct the local chaîne opératoire and to decipher significant chronological differences between presumably Roman/late antique and medieval/early modern metallurgical processing. Pb isotope provenance studies documented the relevance of local metal production within the Roman Empire and confirmed the actual existence of a Metalla Dardanica district, which until now solely has been suspected on basis of epigraphy.
The predominant abundance of the by-products matte (Cu, Pb, Fe and Zn sulphides) and speiss (ferrous speiss: Fe-As compounds; base metal speiss: ~(Cu,Ni,Fe,Ag )x(Sb,Sn,As )y ) at smelting sites with a preliminary Roman/late antique dating points to treatment of complex polymetallic ore. Pb isotope analysis demonstrated that the mining district of Shashkoc-Janjevo (partially) supplied six of the ten investigated metallurgical sites. In this mineralisation, parageneses with elevated Cu, As and Sb abundances comprise significant proportions of particularly tennantite-tetrahedrite minerals, chalcopyrite, arsenopyrite and were generated during the early and main stages of ore formation. Later precipitated ore in contrast is marked by a significantly less versatile mineralogy and consists almost exclusively of galena, sphalerite and pyrite/marcasite. Besides increased Cu, As and Sb contents, ore from the main formation stage also exhibits generally higher Ag abundances, which are mainly hosted by fahlore and locally abundant secondary Cu sulphides (chalcocite, digenite and covellite) and oxidised phases (e.g. malachite, azurite). The higher precious metal grades of this ore type, whose geochemical signature (i.e. higher proportions of Cu, As and Sb) is mirrored by the abundance of the metallurgical by-products matte and speiss (almost exclusively found at potentially Roman/late antique smelting sites; see above), presumably were a pivotal factor leading to its preferential exploitation in earlier times. Matte and base metal-rich speiss contain notable amounts of Ag, which are mainly present in Cu-(Fe) sulphides and particularly antimonides ((Cu,Ni)2Sb, Ag3Sb), respectively. While the speiss compounds due to their close association with Pb bullion presumably were cupelled automatically, the metallurgical treatment of matte could not have been proven unambiguously, but overall certainly is highly likely.
The beneficiated ore (i.e. crushed and sorted, potentially also treated by more lavish techniques such as grinding, sieving or wet-mechanical methods) possibly was partially roasted and subsequently together with fluxes and charcoal submitted to the furnaces. The working temperatures approximately ranged between 1100 and 1400 °C. Slags from all presumably Roman/late antique dated and few of their potentially medieval/early modern analogues were produced from smelting of (partially roasted) ore with charcoal and added siliceous material, thus resulting in fayalite-dominant phase assemblages or rarely observed glassy parageneses. Even though several subtypes of fayalite slags have been established on basis of the abundance of Fe-rich oxide phases (i.e. spinel ss and wüstite), late clinopyroxene and the general solidification sequence of the slags, the process conditions (i.e. temperature, fO2, added fluxing agents) must have been widely similar; chemical variations could be explained by varying degrees of interaction of the slag melt with charcoal ash and furnace material. The other investigated metallurgical remains indicate employment of a calcareous flux, which led to formation of Ca-rich olivine-, olivine+clinopyroxene-, clinopyroxene- or melilite-type slags. These types as well as glassy slags were generated at more oxidising conditions outside the fayalite stability field (FMQ buffer equilibrium, cf. Lindsley, 1976) than their olivine-dominant analogues. Conclusions on the furnace construction could be drawn on basis of the typology of the slags, which mostly were tapped into a basin located outside the furnace, but partially (at two presumably medieval/early modern sites) also accumulated in a reservoir within the smelter.
Lead artefacts excavated in Ulpiana could be isotopically related to ores from mineralisations in its vicinity and demonstrate that the resources were at least utilised for local metal production. However, also ship wreck cargo from Israel - including several lead ingots with the inscription 'MET DARD' (Raban, 1999) - and late antique lead-glazed pottery from Serbia and Romania (Walton & Tite, 2010) could be related to a possible Kosovarian/Serbian provenance of the raw material and thus indicate flourishing trade of metal from the Metalla Dardanica district within the Roman Empire.
References:
Lindsley, D. H. (1976). Experimental studies of oxide minerals. In D. Rumble, III (Hrsg.), Oxide minerals (61-88). Reviews in Mineralogy, Volume 3. Washington, DC: Mineralogical Society of America.
Raban, A. (1999). The lead ingots from the wreck site (area K8). Journal of Roman Archaeology, Supplementary Series, 35, 179-188.
Walton, M. S., & Tite, M. S. (2010). Production technology of Roman lead-glazed pottery and its continuance into late antiquity. Archaeometry, 52(5), 733-759.
Embryonale Stammzellen (ESCs) sind ein wichtiges Werkzeug zur Untersuchung der frühen embryonalen Entwicklung. ESCs können mit Hilfe neuer Technologien zur Modifikation von Genen (z.B. mit dem CRISPR/Cas9 System) genetisch manipuliert werden. Daraus resultierende „knockout“ ES Zelllinien können helfen, die physiologische Rolle von Proteinen während der Differenzierung zu verstehen.
Transkriptionsfaktoren, die schnell und spezifisch Signalwege regulieren, spielen während der Embryonalentwicklung und während der Differenzierung von ESCs in vielen verschiedenen Zelltypen eine essentielle Rolle. Der Transkriptionsregulator „Far Upstream Binding Protein 1“ (FUBP1) ist ein Protein, welches eine ganz bestimmte einzelsträngige DNA Sequenz, das „Far Upstream Sequenz Element“, erkennt, bindet, und dadurch Gene wie z.B c-myc oder p21 reguliert. Mit der Entwicklung zweier Fubp1 Genfallen Mausstämme (Fubp1 GT) sollte die Frage nach der physiologischen Funktion von FUBP1 beantwortet werden. Die homozygoten FUBP1-defizienten GT Embryonen sterben im Mutterleib ungefähr am Tag E15.5 der Embryonalentwicklung. Sie sind kleiner als Wildtypembryonen und zeigen ein anämisches Aussehen. Daher wurden diese Mausmodelle hinsichtlich der Hämatopoese untersucht, die zu diesem Zeitpunkt vor allem in der Leber stattfindet. Es konnte eine signifikante Reduktion der hämatopoetischen Stammzellen (HSCs) festgestellt werden und zusätzlich war die langfristige Repopulation der FUBP1-/--Stammzellen im Knochenmark in Transplantationsexperimenten reduziert.
In der vorliegenden Arbeit wurde die Rolle von FUBP1 in einem weiteren Stammzellsystem analysiert und gleichzeitig seine Bedeutung in anderen Zelltypen der frühen Embryonalentwicklung untersucht.
Die Quantifizierung der FUBP1 Expression in den ESCs und während der Differenzierung zu sogenannten `embryoid bodies` (EBs) zeigten eine starke Expression auf mRNA- und auf Proteinebene. Nach der erfolgreichen Optimierung der Differenzierung von murinen ESCs wurden Fubp1 „knockout“ (KO) ESC Klone mit Hilfe der CRISPR/Cas9 Technologie etabliert. Die molekularbiologische Analyse der ESCs zeigte eine signifikante Erhöhung der Oct4 mRNA-Expression, während Nanog und die Differenzierungsmarker Brachyury, Nestin und Sox17 unverändert und in vergleichbarer Menge zu den Kontrollen vorhanden waren. Während der Differenzierung der Fubp1 KO Klone zu EBs zeigte sich eine signifikante Reduktion mesodermaler Marker wie Flk-1, SnaiI, Snai2, Bmp4 und FgfR2. Mit Hilfe durchflusszytometrischer Analysen bestätigte sich die verzögerte Bildung mesodermaler Zellen (Brachyury- und Flk-1-exprimierender Zellen) in den Fubp1 KO Klonen der EBs an den Tagen 3, 4 und 5 nach Beginn der Differenzierung.
Die Anwendung einer Ko-Kultivierung auf OP9 Zellen zur Differenzierung der ESCs in hämatopoetische Linien sollte zeigen, ob der Fubp1 KO ESCs ein Defekt in der frühen Entwicklung hämatopoetischer Stammzellen zu beobachten ist. Erneut konnte am Tag 5 der ESC-Differenzierung in der OP9 Ko-Kultur eine signifikante Reduktion der mesodermalen (Flk-1+) Zellen festgestellt werden. Die weitere Differenzierung zu hämatopoetischen CD45+ Zellen zeigte jedoch keinen Unterschied im prozentualen Anteil CD45+ Zellen am Tag 12 der Differenzierung. Auch die gezielte Differenzierung zu erythroiden Zellen durch Zugabe des Zytokins EPO zum Medium zeigte keinen signifikanten Unterschied im Differenzierungsgrad der erythroiden Zellen zwischen Kontroll- und Fubp1 KO Klonen.
In weiteren Experimenten habe ich in dieser Arbeit die Expression von FUBP1 in WT Embryos an den Tagen E9.5 und E13.5 der Embryonalentwicklung untersucht. Hierbei zeigte sich in beiden Entwicklungsstadien eine immunhistochemische Anfärbung von FUBP1 in den meisten Zellen des Embryos. Die Annahme, dass die Abwesenheit von FUBP1 in der Embryonalentwicklung zu verstärkten apoptotischen Vorgängen führen könnte und gleichzeitig die massive Expansion von Zellen gestört sein könnte wurde mit Hilfe immunhistochemischer Färbung von „cleaved Caspase 3“ (Apoptosemarker) und „Ki-67“ (Proliferationsmarker) in den homozygoten Fubp1 GT Embryos an den Tagen E9.5 und E13.5 nicht bestätigt.
Die Ergebnisse dieser Arbeit lassen darauf schließen, dass die Regulation von Apoptose und Proliferation durch FUBP1 während der Embryonalentwicklung nicht die Hauptrolle von FUBP1 darstellt. Es zeigte sich jedoch, dass FUBP1 als Transkriptionsregulator wichtig für die mesodermale Differenzierung von ESCs ist. Zu beobachten war, dass es in den FUBP1-defizienten ESCs zu einer Verzögerung der mesodermalen Differenzierung kommt. Es konnte bereits gezeigt werden, dass FUBP1 essenziell für die Selbsterneuerung von HSCs ist. Dies macht deutlich, dass FUBP1 neben der Proliferation und Apoptose ein breiteres Spektrum an Signalwegen reguliert, die für Stammzellen und deren Differenzierung von Bedeutung sind.
Photoinduzierte Energietransferprozesse und -reaktionen spielen in vielen Gebieten von Chemie, Physik und Biologie eine wichtige Rolle. Zu den prominentesten Beispielen zählen der Lichtsammelprozess in der Photosynthese und der Anregungsenergietransfer in funktionellen Materialien. Der Fokus dieser Arbeit liegt auf letzterem Bereich, genauer auf organischer Elektronik und flexiblen Donor-Akzeptor-Bausteinen und Schaltern. Im Besonderen werden hier zwei verschiedene Typen von funktionellen organischen Systemen betrachtet: zum einen oligomere Fragmente organischer halbleitender Polymere wie Oligo-p-Phenylen-Vinylen (OPV) und Oligo-Thiophen (OT), welche als Bausteine für neuartige organische Solarzellen dienen, und zum anderen kleine funktionelle Donor-Akzeptor-Einheiten wie Dithienylethen-Bordipyrromethen (DTE-BODIPY). Letzteres wurde in Kooperation mit den experimentellen Gruppen von K. Rück-Braun (TU Berlin) und J. Wachtveitl (Goethe Universität) untersucht. Um die relevanten Energietransfermechanismen genauer zu verstehen, wurden an diesen Systemen elektronische Strukturrechnungen und quantendynamische Untersuchungen durchgeführt. Hierzu wurden mittels ab initio-Methoden Modell-Hamiltonians parametrisiert und mit hochdimensionalen quantendynamischen oder semiklassischen Methoden kombiniert. Während die Parametrisierung für kleinere Fragmente durchgeführt wurde, lässt sich der so parametrisierte Hamiltonian ohne Weiteres auf größere Systeme erweitern. Die dynamischen Studien der betreffenden Systeme wurden mittels der Multikonfigurationellen Zeitabhängigen Hartree (MCTDH) Methode durchgeführt, welche eine vollständige quantendynamische Beschreibung des Systems zulässt. Für größere Systeme wurde die semiklassische Ehrenfest Methode in Verbindung mit dem Langevin-Ansatz zur Beschreibung von Umgebungseffekten genutzt. Hierzu wurde ein eigens für diese Methode und Systeme geschriebenes Programm eingesetzt. Im Falle der OT- und OPV-Oligomere wurde die Dynamik bei Vorliegen eines strukturellen Defekts untersucht. Ziel war es hierbei, die dynamischen Phänomene, welche durch die Photoanregung induziert werden, zu untersuchen. Des Weiteren wurde untersucht, ob das Konzept von „spektroskopischen Einheiten“, welche die Lokalisierung der Anregung durch strukturelle Defekte beschreibt, in diesen Systemen zutrifft. Hierzu wurden die Systeme in einer Frenkel-Basis definiert, welche ein auf einem Monomer lokalisiertes Elektron-Loch-Paar beschreibt. Delokalisierte elektronische Anregungen können somit als Superposition solcher Frenkel-Zustände beschrieben werden. Neben der Frenkel-Basis wurde aber auch eine verallgemeinerte Elektron-Loch-Basis verwendet, welche über zusätzliche Ladungstransferzustände eine räumliche Separation von Elektronen und Löchern erlaubt.Die Parametrisierung des OPV- und OT-Hamiltonians erfolgte mittels der Algebraischen Diagrammatischen Konstruktions (ADC(2))-Methode, welche in Kombination mit einer Übergangs-Dichte-Matrix-Analyse eine sehr akkurate Beschreibung der Frenkel- und Ladungstransferzustände basierend auf den supermolekularen Zuständen erlaubt. Um vibronische Effekte auf die Dynamik miteinzubeziehen,wurden nieder- und hochfrequente Torsions- und alternierende Bindungslängenmoden des Systems im Hamiltonian berücksichtigt. Hierzu wurden eindimensionale Schnitte der Potentialflächen entlang dieser Koordinaten berechnet und mittels einer Transformation in diabatische Potentialflächen überführt. Mit diesem Setup wurden die quantendynamischen und semiklassischen Simulationen für ein OPV/OT-Hexamer und ein 20-mer durchgeführt. Die Ergebnisse dieser Simulationen zeigen, dass der Energietransfer auf einer Subpikosekunden-Zeitskala stattfindet und eine starke Abhängigkeit vom Vorliegen eines strukturellen Defekts aufweist. Des Weiteren konnte auf einer Zeitskala von 100 Femtosekunden eine Lokalisierung des Exzitons beobachtet werden. Fluktuationseffekte werden zudem über Quantenfluktuationen im Falle von MCTDH bzw. über thermische Fluktuationen im Falle des Ehrenfest-/Langevin-Ansatzes berücksichtigt. Letzterer ist jedoch nicht in der Lage, die kohärente Charakteristik der mit den Schwingungsmoden gekoppelten Exziton- und Lokalisierungsdynamik wiederzugeben. Dagegen kann dieser Ansatz erfolgreich genutzt werden, um eine fluktuationsgetriebene „Hopping“-Dynamik des quasi- stationären Zustandes auf einer längeren Zeitskala in Abhängigkeit von der Temperatur zu beschreiben. Die Beschreibung der Photodynamik der DTE-BODIPY-Dyade zielt darauf ab, experimentell beobachtete vibrationelle Schwingungen des BODIPY-Fragments zu erklären, die ohne eine direkte Anregung dieses Fragments zustande kommen. Diese wurden nach einer selektiven Anregung des DTE-Fragments in zeitaufgelösten UV/Vis Anreg-Abtast-Experimenten beobachtet. Der Fokus der Untersuchung liegt daher auf der Beschreibung der photoinduzierten intramolekulare Energieumverteilung (IVR) auf einer Subpikosekunden-Zeitskala. Die DTE-BODIPY Dyade wurde mittels eines Hamiltonians, welcher durch TDDFT Rechnungen parametrisiert wurde, dargestellt. Basierend auf den Normalmoden des Systems, wurden lokale DTE- und BODIPY-Moden konstruiert, wobei einige dieser Moden miteinander gekoppelt sind und die Photoanregung des DTE auf das BODIPY-Fragment übertragen. Hierbei zeigte sich, dass die Zeitskala und die charakteristischen Frequenzen des Experiments mittels der hochdimensionalen MCTDH-Methode gut reproduziert wurden. Aus den Simulationen ergab sich zudem, dass der beobachtete Energietransfer stark von einem Reservoir von vibrationell angeregten lokalen DTE-Moden beeinflusst wird. Der untersuchte IVR- Prozess zeigt zudem eine ausgeprägte Abhängigkeit von lokalen Kopplungen und der Kopplung an eine Umgebung.
Nanomaterials, i.e., materials that are manufactured at a very small spatial scale, can possess unique physical and chemical properties and exhibit novel characteristics as compared to the same material without nanoscale features. The reduction of size down to the nanometer scale leads to the abundance of potential applications in different fields of technology. For instance, tailoring the physicochemical properties of nanomaterials for modification of their interaction with a biological environment has been reflected in a number of biomedical applications.
Strategies to choose the size and the composition of nanoscale systems are often hindered by a limited understanding of interactions that are difficult to study experimentally. However, this goal can be achieved by means of advanced computer simulations. This thesis explores, from a theoretical and a computational viewpoints, stability, electronic and thermo-mechanical properties of nanoscale systems and materials which are related to biomedical applications.
We examine the ability of existing classical interatomic potentials to reproduce stability and thermo-mechanical properties of metal systems, assuming that these potentials have been fitted to describe ground-state properties of the perfect bulk materials.
It is found that existing classical interatomic potentials poorly describe highly-excited vibrational states when the system is far from the potential energy minimum. On the other hand, construction of a reliable computational model is essential for further development of nanomaterials for applications. A new interatomic potential that is able to correctly reproduce both the melting temperature and the ground-state properties of different metals, such as gold, platinum, titanium, and magnesium, by means of classical molecular dynamics simulations is proposed in this work. The suggested modification of a many-body potential has a general nature and can be utilized for similar numerical exploration of thermo-mechanical properties of a broad range of molecular and solid state systems experiencing phase transitions.
The applicability of the classical interatomic potentials to the description of nanoscale systems, consisting of several tens-hundreds of atoms, is also explored in this study. This issue is important, for instance, in the case of nanostructured materials, where grains or nanocrystals have a typical size of about a few nanometers. We validate classical potentials through the comparison with density-functional theory calculations of small
atomic clusters made of titanium and nickel. By this analysis, we demonstrate that the classical potentials fitted to describe ground-state properties of a bulk material can describe the energetics of nanoscale systems with a reasonable accuracy.
In this work, we also analyze electronic properties of nanometer-size nanoparticles made of gold, platinum, silver, and gadolinium; nanoparticles composed of these materials are of current interest for radiation therapy applications. We focus on the production of low-energy electrons, having the kinetic energy from a few electronvolts to several tens of electronvolts. It is currently established that the low-energy secondary electrons of such energies play an important role in the nanoscale mechanisms of biological damage resulting from ionizing radiation. We provide a methodology for analyzing the dynamic response of nanoparticles of the experimentally relevant sizes, namely of about several nanometers, exposed to ionizing radiation. Because of a large number of constituent atoms (about 1000 −10000 atoms) and consequently high computational costs, the electronic properties of such systems can hardly be described by means of ab initio methods based on a quantum-mechanical treatment of electrons, and this analysis should rely on model approaches. By comparing the response of smaller systems (of about 1 nm size) calculated within the ab initio- and the model framework, we validate this methodology and make predictions for the electron production in larger systems.
We have revealed that a significant increase in the number of the low-energy electrons emitted from nanometer-size noble metal nanoparticles arises from collective electron excitations formed in the systems. It is demonstrated that the dominating mechanisms of electron yield enhancement are related to the formation of plasmons excited in a whole system and of atomic giant resonances formed due to excitation of valence d electrons in individual atoms of a nanoparticle. Being embedded in a biological medium, the noble metal nanoparticles thus represent an important source of low-energy electrons, able to produce a significant irrepairable damage in biological systems.
A general methodology for studying electronic properties of nanosystems is used to make quantitative predictions for electron production by non-metal nanoparticles. The analysis illustrates that due to a prominent collective response to an external electric field, carbon nanoparticles embedded in a biological medium also enhance the production of low-energy electrons. The number of low-energy electrons emitted from carbon nanoparticles is demonstrated to be several times higher as compared to the case of liquid water.
The phenomenon of magnetism has been known to humankind for at least over 2500 years and many useful applications of magnetism have been developed since then, starting from the compass to modern information storage and processing devices. While technological applications are an important part of the continuing interest in magnetic materials, their fundamental properties are still being studied, leading to new physical insights at the forefront of physics. The magnetism of magnetic materials is a pure quantum effect due to the electrons that carry an intrinsic spin of 1/2. The physics of interacting quantum spins in magnetic insulators is the main subject of this thesis.We focus here on a theoretical description of the antiferromagnetic insulator Cs2CuCl4. This material is highly interesting because it is a nearly ideal realization of the two-dimensional antiferromagnetic spin-1/2 Heisenberg model on an anisotropic triangular lattice, where the Cu(2+) ions carry a spin of 1/2 and the spins interact via exchange couplings. Due to the geometric frustration of the triangular lattice, there exists a spin-liquid phase with fractional excitations (spinons) at finite temperatures in Cs2CuCl4. This spin-liquid phase is characterized by strong short-range spin correlations without long-range order. From an experimental point of view, Cs2CuCl4 is also very interesting because the exchange couplings are relatively weak leading to a saturation field of only B_c=8.5 T. All relevant parts of the phase diagram are therefore experimentally accessible. A recurring theme in this thesis will be the use of bosonic or fermionic representations of the spin operators which each offer in different situations suitable starting points for an approximate treatment of the spin interactions. The methods which we develop in this thesis are not restricted to Cs2CuCl4 but can also be applied to other materials that can be described by the spin-1/2 Heisenberg model on a triangular lattice; one important example is the material class Cs2Cu(Cl{4-x}Br{x}) where chlorine is partially substituted by bromine which changes the strength of the exchange couplings and the degree of frustration.
Our first topic is the finite-temperature spin-liquid phase in Cs2CuCl4. We study this regime by using a Majorana fermion representation of the spin-1/2 operators motivated by theoretical and experimental evidence for fermionic excitations in this spin-liquid phase. Within a mean-field theory for the Majorana fermions, we determine the magnetic field dependence of the critical temperature for the crossover from spin-liquid to paramagnetic behavior and we calculate the specific heat and magnetic susceptibility in zero magnetic field. We find that the Majorana fermions can only propagate in one dimension along the direction of the strongest exchange coupling; this reduction of the effective dimensionality of excitations is known as dimensional reduction.
The second topic is the behavior of ultrasound propagation and attenuation in the spin-liquid phase of Cs2CuCl4, where we consider longitudinal sound waves along the direction of the strongest exchange coupling. Due to the dimensional reduction of the excitations in the spin-liquid phase, we expect that we can describe the ultrasound physics by a one-dimensional Heisenberg model coupled to the lattice degrees of freedom via the exchange-striction mechanism. For this one-dimensional problem we use the Jordan-Wigner transformation to map the spin-1/2 operators to spinless fermions. We treat the fermions within the self-consistent Hartree-Fock approximation and we calculate the change of the sound velocity and attenuation as a function of magnetic field using a perturbative expansion in the spin-phonon couplings. We compare our theoretical results with experimental data from ultrasound experiments, where we find good agreement between theory and experiment.
Our final topic is the behavior of Cs2CuCl4 in high magnetic fields larger than the saturation field B_c=8.5 T. At zero temperature, Cs2CuCl4 is then fully magnetized and the ground state is therefore a ferromagnet where the excitations have an energy gap. The elementary excitations of this ferromagnetic state are spin-flips (magnons) which behave as hard-core bosons. At finite temperatures there will be thermally excited magnons that interact via the hard-core interaction and via additional exchange interactions. We describe the thermodynamic properties of Cs2CuCl4 at finite temperatures and calculate experimentally observable quantities, e.g., magnetic susceptibility and specific heat. Our approach is based on a mapping of the spin-1/2 operators to hard-core bosons, where we treat the hard-core interaction by the self-consistent ladder approximation and the exchange interactions by the self-consistent Hartree-Fock approximation. We find that our theoretical results for the specific heat are in good agreement with the available experimental data.
Modern mobile devices offer a great variety of data that can be recorded. This broad range of information offers the possibility to tailor applications more to the needs of a user. Several context information can be collected, like e.g. information about position or movement. Besides integrated sensors, a broad range of additional sensors are available which can be connected to a mobile device. These additional sensors offer for example the possibility to measure physiological signals of a user.The human body offers a broad range of different signals. These signals have been used in several examples to conclude on the state of a user. The different signals allow to get a deeper insight into emotional or mental state of a user. Electrodermal activity gives feedback about the current arousal level of a user. Heart rate and heart rate variability can give an estimation about valence and mental load of a user. Several models exist to conclude from information like valence and arousal on different emotional states. Russell defined a two dimensional model, using valence and arousal to define affective states. Yerkes and Dodson developed a curve that expresses the relationship between arousal and performance of a user. Different examples exist, that use physiological signals to determine the user state for tailoring and adapting of applications. At the time of this work most of these examples did not address the usage of physiological signals for user state estimation in mobile applications and in mobile scenarios. Mobile scenarios lead to several challenges that need to be addressed. Influencing factors on physiological signals, like e.g. movement have to be controlled. Furthermore a user might be interrupted and influenced by environmental aspects. The combination of physiological data and context information might improve the interpretation of user state in mobile scenarios. In this work, we present a model that addresses the challenges of usage in mobile scenarios to offer an estimation of user state to mobile applications. To address a broad range of mobile applications, affective and cognitive state are provided as output. As input heart rate and electrodermal activity are used, as well as context information about movement and performance. Electrodermal activity is measured by a simple sensor that can be worn as a wristband. Heart rate is measured by a chest strap as used in sports. The input channels are transformed to affective and cognitive state based on a fuzzy rule based approach. With help of fuzzy logic, uncertainty can be expressed and the data continuously being processed. At the start, input channels are fuzzified by defined functions. After a that, a first fuzzy rule set transforms the input signals into values for valence, arousal and mental load. In a second step, these values and context information are transformed with another fuzzy rule set to values for affective and cognitive state. Affective state is based on the model of Russell, where valence and arousal are used to determine different emotional states. The output of the model are eight different affective states (alarmed, excited, happy, relaxed, tired, bored, sad and frustrated), which can have a high, medium, low or very low value as output. Cognitive state is determined based on mental load and context information about performance and movement. The output value can be very high, high, medium or low. The model was implemented as background service for Android devices. Different applications have been used for evaluation of the model. The model has been integrated in a multiplayer space shooter game, called ”Zone of Impulse”, which mainly benefits from the affective state. Cognitive state is more addressed in applications like a simple vocable trainer, which adapts difficulty based on user state. A study to evaluate different aspects of the model has been conducted. The study was designed to investigate the suitability of the model for mobile scenarios. The game ”zone of impulse” and the vocable trainer have been investigated in different configurations. Versions with integrated model have been compared to version of the applications without model, as well as versions of the model without context information. In total 41 participants took part in the study. A part of the participants had to do the tasks of the study in a mobile scenario, walking around several streets. The remaining participants had to do the tasks in a controlled environment in a sitting position. Different aspects were collected with ratings and questionnaires. Overall, participants rated that they did not feel impaired by the sensors they had to wear. The results showed, that the combination of physiological data and context information had an advantage against versions without context information in part of the ratings. A comparison between versions with and without model showed, that the subjective mental load ratings were significantly better for the version with model. Subjective ratings for aspects like fun, overstrain and support were mixed. When comparing the application versions in indoor and outdoor scenarios, no significant difference could be found, which leads to the assumption that there is no loss of interpretation quality in outdoor scenarios. The results also showed that the model seems to be robust enough to compensate the loss of an input channel, as there was no significant difference between application versions with full integrated model and versions with one channel lost. With the model developed in this work, context information and physiological data were combined to improve user state estimation. Furthermore pitfalls of user state estimation in mobile scenarios are overcome with this combination. However, the model has only been evaluated with a limited amount of applications and situations that mobile scenarios offer.
Für die Optimierung sowie Entwicklung lichtsteuerbarer Systeme für biologische Anwendungen oder neue Materialien ist ein detailliertes Verständnis der zugrunde liegenden komplexen, lichtinduzierten Prozesse eine Voraussetzung. Die Verwendung von Photoschaltern in Makromolekülen ermöglicht eine zeitliche und örtliche Kontrolle über strukturelle Änderungen sowie die entsprechend folgenden (biologischen) Funktionen durch die Verwendung von Licht als externem Auslöser.
Ein wichtiger Bestandteil dieser Arbeit befasst sich mit der Entwicklung eines auf Licht reagierenden Riboschalters, welcher die gezielte Kontrolle über Genexpression ermöglicht. Hierzu wurde eine spektroskopische Charakterisierung von verschiedenen Photoschaltern bezüglich einer Verwendung als biologischer Ligand sowie der Wechselwirkungen zwischen Azobenzolen und RNA, auch hinsichtlich ihrer Bindungsdynamiken durchgeführt. Zunächst wurde die hohe Abhängigkeit der (photo-)chemischen Eigenschaften der Azobenzole von der Wahl der Substituenten untersucht, wobei besonders die Anwendung in wässrigem Milieu betrachtet wurde. In einer detaillierten (zeitaufgelösten) Studie wurde der positionsabhängige Einfluss der Hydroxy-Substitution von Azobenzolen auf die Photoisomerisierung in wässriger Lösung untersucht. Für eine ortho-Substitution ergab sich hierbei ein alternativer Deaktivierungskanal nach Photoanregung, welcher stärker ausgeprägt ist als die Isomerisierung. Hierbei wird ein intramolekularer Protontransfer im angeregten Zustand (ESIPT) beobachtet, welcher mit einer Zeitkonstante von 0.3 ps beschrieben werden kann und in einer Keto-Spezies resultiert. Eine Keto-Enol-Tautomerie konnte für die para-Hydroxy-Substitution schon im Grundzustand beobachtet werden. Somit können beide Spezies gezielt adressiert werden. Durch Acetylierung der Hydroxygruppe verlangsamt sich die thermische Relaxation des cis-Isomer zu dem entsprechenden trans-Isomer signifikant ohne die Isomerisierung zu beeinträchtigen. Dementsprechend ermöglicht eine solche Acetylierung die Verwendung von bekannten Azobenzolderivaten als Photoschalter.
Zudem werden in dieser Arbeit zwei verschiedene Herangehensweisen in der Entwicklung eines Riboschalters beschrieben, welcher sich durch Licht regulieren lässt.
Diese sind durch kovalentes bzw. nicht-kovalentes Einbringen eines Azobenzolderivats in die RNA Struktur charakterisiert. Ein neuer Linker, welcher auf einer Desoxyribose-Struktur beruht, wird für die kovalente Anbindung des Azobenzols an den RNA Strang präsentiert, welcher eine licht-induzierte Dehybridisierung ermöglichen soll. Eine außergewöhnlich hohe Schaltamplitude mit einem cis-Gehalt von etwa 90% konnte für das Azobenzol im RNA Einzelstrang schon bei Raumtemperatur ermittelt werden. Zudem wurde der Einfluss des Photoschalters sowie der benachbarten Nukleotide in der RNA auf die Stabilität der RNA Doppelhelix untersucht. Die zweite Vorgehensweise beruht auf einer nicht-kovalenten Bindung zwischen einem Azobenzolderivat und einem RNA-Aptamer, welche lediglich für eines der Photoisomere ermöglicht wird, wodurch eine örtliche und zeitliche Kontrolle der Ligandenbindung der RNA erfolgt. Im Rahmen dieser Arbeit war es möglich zwei verschiedene photoschaltbare RNA Aptamere zu identifizieren und zu untersuchen, welche eine hohe Spezifität und Affinität aufweisen. Zudem wurde die Photoisomerisierung des Azobenzols innerhalb der RNA-Struktur sowie daraus resultierende lichtinduzierte Konformationsänderungen der RNA mittels zeitaufgelöster Anreg-/Abtastspektroskopie untersucht. Die daraus resultierende Dynamik der photoinduzierten Ligandenbindung sollte eine weitere gezielte Optimierung lichtschaltbarer biologischer Systeme erlauben.
Der zweite Teil dieser Arbeit beschäftigt sich mit der zeitaufgelösten Untersuchung eines photoschaltbaren Foldamers. Speziell wurde der strukturelle Übergang des OmPE-Foldamers 10-5 zwischen einer definierten helikalen und einer ungefalteten Konformation auf Grund der Photoisomerisierung der, in das Rückgrat integrierten, Azobenzole untersucht.
Dabei konnten die frühen (Ent-)Faltungsmechanismen des Foldamers im sub-Nanosekunden-Zeitbereich beobachtet werden, welche durch quantenmechanische Rechnungen unterstützt werden konnten. Darüberhinaus, war es möglich einen Anregungsenergietransfer vom PE-Rückgrat des Foldamers auf die Azobenzole nachzuweisen, welcher die Lebensdauer der angeregten Zustände des Systems signifikant verkürzt.
Diese Arbeit liefert wichtige Informationen zu den Reaktionspfaden, den gezielten Wechselwirkungen zwischen Photoschaltern und größeren organischen Molekülen, sowie den daraus resultierenden lichtinduzierten strukturellen Änderungen durch die Anwendung einer Vielzahl an (zeitaufgelösten) spektroskopischen Methoden. Diese Ergebnisse tragen zum weiteren Verständnis komplexer Prozesse in biologischem sowie nicht-biologischem Zusammenhang und somit zu einer weiterführenden Entwicklung neuer Systeme bei.
In the adult mammalian brain stem cells within defined neurogenic niches retain the capacity for lifelong de novo generation of neurons. The subventricular zone (SVZ) of the lateral ventricles and the subgranular layer (SGL) of the hippocampal dentate gyrus (DG) have been identified as the two major sites of adult neurogenesis. Moreover, the third ventricle in the hypothalamus is emerging as a new neurogenic niche in the adult brain. Extracellular purine and pyrimidine nucleotides are involved in the control of both embryonic and adult neuro-genesis. These nucleotides act via ionotropic P2X or metabotropic P2Y receptors and studies of the adult SVZ and the DG provide strong evidence that ATP promotes progenitor cell proliferation in this stem cell rich regions. Previous studies have shown that the extracellular nucleotide-hydrolyzing enzyme NTPDase2 is highly expressed by adult neural stem and progenitor cells of the SVZ and the rostral migratory stream (RMS), the hippocampal SGL, and the third ventricle. NTPDase2 preferentially hydrolyzes extracellular nucleoside triphosphates (NTPs) and, to a lower extent, diphosphates, thus modulating their effect on nearby nucleotide receptors. Deletion of the enzyme increases extracellular NTP concentrations, and might indicate roles of purinergic signaling in adult neurogenesis. As shown by enzyme histochemistry, genetic deletion of NTPDase2 essentially eliminates ATPase activity in neurogenic niches but does not affect protein expression levels and activity of other ectonucleotidases. Lack of NTPDase2 leads to expansion of the hippocampal stem cell pool as well as of the inter-mediate progenitor type-2 cells. Cell expansion is lost at around type-3 stage, paralleled by increased labeling for caspase-3, indicating increased apoptosis, and decreased levels in CREB phosphorylation in doublecortin-expressing cells, diminishing survival in this cell population. In line with increased cell death, P2Y12 receptor-expressing microglia is enriched at the hilus orientated side of the granule cell layer. These data strongly suggest that NTPDase2 functions as central homeostatic regulator of nucleotide-mediated neural progenitor cell proliferation and expansion in the adult brain by balancing extracellular nucleotide concentrations and activation of purinergic receptors.
In order to further characterize the role of purinergic signaling in adult neurogenesis, the ADP-sensitive P2Y13 receptor was identified as a potential candidate whose activation might inhibit neurogenesis in the hippocampal dentate gyrus and the newly identified neurogenic niche at the third ventricle. Deletion of P2ry13 increased progenitor cell proliferation and long-term progenitor survival as well as new neuron formation in the hippocampal neurogenic niche. This was further paralleled by increased thickening of the granule cell layer, CREB phosphorylation, and expression of the neuronal activity marker c-Fos. Increased progenitor cell proliferation and progenitor survival persist in aged P2ry13 knockout animals. However, in the ventral dentate gyrus proliferation and expansion levels of progenitor cells did not differ significantly from the wild type. This study strongly supports the notion that extracellular nucleotides significantly contribute to the control of adult neurogenesis in the dentate gyrus in situ. Data in this work suggest that activation of the P2Y13 receptor dampens progenitor cell proliferation, new neuron formation, and neuronal activity. In contrast to several in vitro studies and studies in the SVZ in situ, a contribution of the ATP/ADP-sensitive P2Y1 receptor could not be confirmed in the dentate gyrus in vivo.
To unravel implications of purinergic signaling and P2Y13 receptor action in the control of adult hypothalamic neurogenesis a pilot study was performed. Mice null for P2ry13 revealed increased progenitor cell proliferation at the third ventricle as well as long-term progeny survival and new neuron formation in the hypothalamus. In contrast to results obtained in the dentate gyrus expression of the neuronal activity marker c-Fos was significantly decreased in hypothalamic nuclei, indicating increased inhibition of appetite-regulating neuronal circuits by surplus neurons in knockout animals. These data provide first evidence that extracellular nucleotide signaling contributes to the control of adult hypothalamic neurogenesis in situ. Activation of the P2Y13 receptor inhibits progenitor cell proliferation, long-term survival and neuron formation and therefore controls inhibition of appetite-regulating circuits in the adult rodent hypothalamus.
Juvenile neuronal ceroid-lipofuscinosis (JNCL) is a rare lysosomal storage disease in children with lethal outcome and no therapy. The origin of JNCL has been traced to autosomal recessive mutations in the CLN3 gene, and ~85% of the JNCL patients harbor a 1.02 kb deletion that removes the exons 7 and 8 and the surrounding intronic DNA (CLN3Δex7/8). So far, structure, function and localization of the CLN3 protein remain elusive. However, there is strong evidence that CLN3 modulates a process or condition that is essential in many cellular pathways. Lipid metabolism and antero-/retrograde transport, two mechanisms CLN3 was previously implicated in, fulfill these requirements. Notably, also a bioactive group of glycosphingolipids referred to as gangliosides is tightly interrelated with these functions. Furthermore, a-series gangliosides have been shown to be involved in the development and sustenance of the brain, where they are essential for neurite outgrowth and cell survival. Defects in ganglioside metabolism were shown to play a crucial role in many lysosomal storage disorders. However, the contribution of gangliosides to NCL pathology is largely unknown.
The present study analyzed central enzymes and metabolites of the a-series ganglioside pathway in a JNCL cell model. The core finding was, thereby, the reduced amount of the neuroprotective ganglioside GM1 in homozygous CbCln3Δex7/8 cells. This was caused by the enhanced action of the GM1-degrading multimeric enzyme complex and in particular, by the upregulation of protein levels and increased enzyme activity of β-galactosidase (Glb1).
Improved binding of Glb1 to substrate-carrying membranes was provided by an increase in LBPA levels. In combination with other smaller alterations in the ganglioside pattern, a shift towards less complex gangliosides became present. The resulting loss of neuroprotection may be the reason for the multifocal pathology in homozygous CbCln3Δex7/8 cells.
The second part of the present study investigated the cellular mechanisms behind the altered ganglioside profile with regard to the potential role of CLN3. Here, the anterograde transport of GM1 to the plasma membrane presented a positive correlation with the amount of full-length CLN3. In case of the truncated protein this correlation was missing, resulting in reduced PM staining with CTxB-FITC. However, transfection of full-length CLN3 in these cells restored the CTxB-FITC intensity. Based on the neuroprotective role of GM1, the corresponding increase in GM1 levels may be the cause for the restoration effects observed in previous studies using full-length CLN3. Hence, administration of GM1 was expected to improve cell viability of homozygous CbCln3Δex7/8 cells and beyond that to rescue potentially some disease phenotypes. However, no effect could be observed. The reason for this may be reduced caveolar uptake and the mislocalization of ganglioside GM1 to the trans-Golgi network (TGN) and redirection towards degradative compartments.
Both are in line with the idea of an impaired endocytic flux in CLN3 deficiency. The observed localization of CLN3 in the TGN suggests a potential role for CLN3 in the lipid sorting machinery, subsequently altering membrane composition and its regulatory functions. The resulting imbalance may affect many of the cellular processes impaired in JNCL.
In this thesis, we study some features of the quantum chromodynamics (QCD) phase diagram at purely imaginary chemical potential using lattice techniques. This is one of the possible methodologies to get insights about the situation at finite density, where the sign problem prevents direct investigations from first principles.
We focus, in particular, on the Roberge-Weiss plane, where the phase structure with two degenerate flavours is studied both in the light and in the heavy quark mass limit. On the lattice, any result is affected by cut-off effects and so are the positions of the two tricritical points m_{tric}^{1,2} separating the second-order intermediate mass region from the first-order triple light and heavy mass regions. Therefore, changing the lattice spacing 'a', the values of m_{tric}^1 and m_{tric}^2 will change. In order to find their position in the continuum limit – i.e. for 'a' going to 0 – they have to be located on finer and finer lattices. Typically, in lattice QCD (LQCD) simulations, the temperature T is tuned through the bare coupling β, on which 'a' depends, while keeping Nt fixed. Hence, it is common to implicitly refer to how fine the lattice is just mentioning its temporal extent.
Using both Wilson and staggered fermions, we simulate Nf=2 QCD on Nt=6 lattices, varying the quark bare mass from the chiral (m_{u,d} going to 0) to the quenched (m_{u,d} going to infinity) limit. For each quark mass, a thorough finite scaling analysis is carried out, taking advantage of two different but consistent methods. In this way we identify the order of the phase transition locating, then, the position of the tricritical points. In order to convert our measurements to physical units we fix the scale measuring the lattice spacing as well as the pion mass corresponding to the quark bare mass used. This allows a comparison between different discretisation, getting a first idea of how serious are cut-off effects.
To be able to make a comparison between two different discretisations, we added an RHMC algorithm with staggered fermions to the CL2QCD software, a GPU code based on OpenCL, which we released in 2014. A considerable part of our work has been invested in ameliorating and optimising CL2QCD, as well as in developing new analysis tools regularly used next to it. Just to mention one, the multiple histogram method has been implemented in a completely general way and we took advantage of it in order to obtain more precise results. Finally, in order to efficiently handle and monitor the hundreds of simulations that are typically concurrently run in finite temperature LQCD, a completely new Bash library of tools has been developed. We plan to release it as a byproduct of CL2QCD in the near future.
In this thesis, the production of charged kaons and Φ mesons in Au+Au collisions at sqrt sAuAu = 2.4 GeV is studied. At this energy, all particles carrying open and hidden strangeness are produced below their respective free nucleon-nucleon threshold with the corresponding so-called excess energies: sqrt sK+ exc = -0.15 GeV, sqrt sK- exc = -0.46 GeV, sqrt sΦ exc = -0.49 GeVGeV. As a consequence, the production cross sections are very sensitive to medium effects like momentum distributions, two- or multistep collisions, and modification of the in-medium spectral distribution of the produced states [1]. K+ and K- mesons exhibit different properties in baryon dominated matter, since only K- can be resonantly absorbed by nucleons. Although strangeness exchange reactions have been proposed to be the dominant channel for K- production in the analyzed energy regime, the production yield and kinematic distributions could also be explained in smaller systems based on statistical hadronization model fits to the measured particle yields, including a canonical strangeness suppression radius RC, and taking the Φ feed-down to kaons into account [2, 3]. For the first time in central Au+Au collisions at such low energies, it is possible to reconstruct and do a multi differential analysis of K- and Φ mesons. In principle, this should be the ideal environment for strangeness exchange reactions to occur, as the particles are produced deeply sub-threshold in a large and long-living system. Therefore, it is the ultimate test to differentiate between the different sources for K- production in HIC.
In total 7.3x10exp9 of the 40% most central Au(1.23 GeV per nucleon)+Au collisions are analyzed. The data has been recorded with the High Acceptance DiElectron Spectrometer HADES located at Helmholtzzentrum für Schwerionenforschung GSI in April/May 2012. A substantially improved reconstruction method has been employed to reconstruct the hadrons with high purity in a wide phase space region.
The estimated particle multiplicities follow a clear hierarchy of the excess energy: 41.5 ± 2.1|sys protons at mid-rapidity per unit in rapidity, 11.1 ± 0.6|sys ± 0.4|extrapol π-, (3.01 ± 0.03|stat ± 0.15|sys ± 0.30|extrapól) x10 exp -2 K+, (1.94 ± 0.09|stat ± 0.10|sys ± 0.10|extrapol)x10 exp -4 K- and (0.99 ± 0.24|stat ± 0.10|sys ± 0.05|extrapol)x10 exp -4 Φ per event. The multiplicities of the strange hadrons increase more than linear with the mean number of participating nucleons hAparti, supporting the assumption that the necessary energy to overcome the elementary production threshold is accumulated in multi-particle interactions. Transport models predict such an increase, but are overestimating the measured particle yield and are not able to describe the kinematic distributions of K+ mesons perfectly. However, the best description is given by the IQMD model with a density dependent kaonnucleon potential of 40 MeV at nuclear ground state density.
The K-=K+ multiplicity ratio is constant as a function of centrality and follows with (6.45 ± 0.77)x10 exp -3 the trend of increasing with beam energy indicated from previous experiments [4]. The effective temperature of K- TK+eff = (84 ± 6) MeV is found to be systematically lower than the one of K+ TK+eff = (104 ± 1) MeV, which has also been observed by the other experiments.
The Φ=K- ratio is with a value of 0.52 ± 0.16 higher than the one obtained at higher center-of-mass energies and smaller systems. This behavior is predicted from a tuned version of the UrQMD transport model [5], when including higher mass baryonic resonances which can decay into Φ mesons and from statistical hadronization models when suppressing open strangeness canonically. The found ratio is constant as a function of centrality and results with a branching ratio of 48.9%, that ~ 25% of all measured K- originate from Φ feed-down decays. A two component PLUTO simulation, consisting of a pure thermal and a K- contribution originating from Φ decays, can fully explain the observed lower effective temperature in comparison to K+ and the shape of the measured rapidity distribution of K-. As a result, we find no indication for strangeness exchange reactions being the dominant mechanism for K- production in the SIS18 energy regime, if taking the contribution from Φ feed-down decays into account.
The hadron yields for the 20% most central collisions can be described by a statistical hadronization model fit with the chemical freeze-out temperature of Tchem = (68 ± 2) MeV and baryochemical potential of μB = (883 ± 25) MeV, which is higher than expected from previous parameterizations. The analysis of the transverse mass spectra of protons indicate a kinetic freeze-out temperature of Tkin = (70 ± 4) MeV and radial flow velocity of βr = 0.43 ± 0.01, which is in agreement with the parameters obtained from the linear dependence of the effective temperatures on the particle mass Tkin = (71.5 ± 4.2) MeV and βr = 0.28 ± 0.09.
Rhythms, i.e. periodic sequences of events or states, are a ubiquitous feature of physiological systems such as the heart, the lungs or the brain. For the brain in particular, the diversity of rhythms is remarkable, ranging from low frequency rhythms in the slow/delta band (0.5-4 Hz) during sleep to gamma band oscillations (30-120 Hz) rhythms during alert behavior, all expressed in various brain areas and at various spatial scales. To understand whether these rhythms subserve a function for the organism it is important to also understand the underlying mechanisms that generate them. While the generation of some rhythms appear to be well-understood, e.g. sleep spindles, others such as the cortical beta rhythm (13-30 Hz) have remained elusive.
Understanding the generation of a brain rhythm involves multiple spatial scales, from identifying intracellular mechanisms such as the contribution of individual transmembrane currents to studying how specific neuronal populations or areas affect the full physiological rhythm present in the intact, highly interconnected brain. The aim of this work has been to delineate the mechanistic contributions of individual brain areas to the in vivo generation of two particular rhythms present in efferent areas: (1) The first part of this work studies the influence of thalamocortical neurons on cortical slow/delta waves (0.5-4 Hz) of sleep that are sometimes also present in awake animals. (2) The second part is about the contribution of primary visual cortex to the beta rhythm (13-30 Hz) in extrastriate cortex of awake behaving animals.
Soil fungal communities are an essential element in the terrestrial ecosystem, however their response to ongoing anthropogenic climate change is currently poorly understood. Fungi are one of the most abundant groups of microbes in soil, they are mainly responsible for the decomposition of organic matter (Baldrian et al., 2012; Buée et al., 2009). By binding carbon in soil, fungi thus maintain an important role in the global carbon cycle (Bardgett et al., 2008). Future climates are likely to influence the communities of belowground microbial organisms (Castro et al., 2010; Deacon et al., 2006). However, how these communities are affected in their diversity, composition, and function after environmental perturbation is insufficiently known.
Molecular techniques using high-throughput sequencing are presently revolutionizing the analysis of complex communities, such as soil fungi. High-throughput metabarcoding enables the recovery of DNA sequence data directly from environmental samples, and DNA sequences from entire communities present in these samples can be simultaneously recovered through massively parallel sequencing reactions (Bik et al., 2012; Taberlet et al., 2012b). This results in more accurate estimation of diversity and community composition and thus provides unprecedented insight into cryptic communities (Lindahl and Kuske, 2014). Yet, challenges associated with these novel techniques include the bioinformatic processing, and the ecological analyses of the large amount of sequence data generated. Most biologists without explicit training in bioinformatics spend a fair amount of time learning how to filter raw sequence data, and customize bioinformatics pipelines specific to their project. To improve the quality of data treatment, and decrease the time needed for the analyses, it is desirable to have bioinformatics pipelines that are easy to use, well explained to researchers not trained in bioinformatics, and adaptable to individual research needs...
Despite mounting evidence of the anthropogenic influence on the Earth's climate, underlying mechanisms of climate change often remain elusive. The investigation of periods of rapid climate change from geological archives may provide crucial information about magnitude, duration, teleconnections of and regional responses to global and hemispheric scale climate perturbations. Thus, paleoclimate reconstructions may help in mitigating and adapting to the challenges of the coming decades. The '8.2 kyr B.P. climatic event' has previously been proposed as a possible analogue for the future climatic scenario of a reduced Atlantic Meridional Overturning Circulation (AMOC). The catastrophic drainage of the Laurentide meltwater lakes through the Hudson Bay and into the Labrador Sea, that occurred ca. 8.47 kyr B.P., caused the slowdown of the AMOC around 8.2 kyr B.P.. Subsequently, reduced heat transfer towards Europe triggered a substantial decline in (winter) temperature and pronounced changes in atmospheric circulation patterns in many regions of the northern hemisphere, especially the North Atlantic realm and Europe. Among the regions affected by the 8.2 kyr B.P. climatic event, the Eastern Mediterranean region is of particular interest for both past and future climate developments. Traditionally characterized as a region highly sensitive to variations in the climate systems of the high and low latitudes, abrupt climate changes have the potential to strongly alter atmospheric circulation patterns and thus precipitation distribution in the region that may have severe socioeconomical consequences. The analysis of stable hydrogen (δD) and oxygen isotopes (δ18O) in precipitation is an excellent tool to trace changes in atmospheric circulation. Here, we present a comparative study of δD and δ18O in precipitation from the Eastern Mediterranean region both in a present day scenario and during the 8.2 kyr B.P. climatic event. We analyze the influences of topography, air mass trajectory, climate and seasonality among others the stable isotopic compositions of meteoric waters from the Central Anatolian Plateau (CAP), Turkey, in order to create a first-order template which may serve as a reference against which paleoenvironmental proxy data may be more accurately interpreted and tested. Further, we employ a multiproxy approach on the early Holocene peat deposits of the classical site of Tenaghi Philippon (TP), NE Greece, to investigate paleoenvironmental responses to northern hemisphere cooling during the 8.2 kyr B.P. climatic event and aim to determine changes atmospheric circulation from δD of leaf wax n-alkanes (δDwax).
Based on δD and δ18O data from more than 480 surface water samples from the CAP, we characterize moisture sources affecting the net isotopic budget of precipitation, manifesting in a systematic north-south difference in near-sea level moisture compositions. Rainout, induced by the major orographic barriers of the plateau, the Pontic Mountains to the north and the Taurus Mountains to the south, strongly shape the modern patterns of δD and δ18O. Stable isotope data from the semi-arid plateau interior provide clear evidence for an evaporitic regime that drastically affects surface water compositions. Strong evaporative enrichment contrasts rainfall patterns along the plateau margins, in part obfuscating the effects of topography and air mass trajectory.
Consequently, in order to address possible influences of evaporation on δD and δ18O in paleoprecipitation from TP, we analyze n-alkane abundances and distributions along with stable carbon isotope compositions of total organic carbon (δ13CTOC) and palynological data to estimate surface moisture conditions during the early Holocene (ca. 8.7 - 7.5 kyr B.P.) and especially during the 8.2 kyr B.P. climatic event. A period of relatively dry surface conditions from ca. 8.7 to 8.2 kyr B.P., indicated by low values of the 'aquatic index' (Paq ) and by elevated Average Chain Length (ACL) values, in concert with elevated δ13CTOC values, precedes the 8.2 kyr B.P. climatic event. The event itself is characterized by slightly wetter, more humid conditions, as suggested by an increase in Paq values as well as reduced ACL and δ13CTOC values between ca. 8.2 and 7.9 kyr B.P.. In the upper section of the core, a distinct change in paleohydrology becomes. A steep increase in Paq and a decrease in ACL values as well as variations in δ13CTOC from 7.9 kyr B.P. onwards imply considerably elevated surface moisture levels, likely caused by the increased activity of the karstic system of the surrounding mountains. Collectively, the biomarker proxies presented here, reveal a concise picture of changing moisture conditions at TP that is consistent with palynological data and provide detailed paleoenvironmental information for the analysis of δDwax as a paleoprecipitation proxy. The long-term decline in δDwax values characterizes the lower section of the core until ca. 8.2 kyr B.P.. The 8.2 kyr B.P. climatic event itself is connected to two distinct positive hydrogen isotope excursions: a minor shift in δDwax around 8.2 kyr B.P. and a major shift in δDwax between ca. 8.1 and 8.0 kyr B.P.. The upper part of the section shows a progressive trend towards higher δDwax values. With no indication of increased evaporitic conditions at TP during the 8.2 kyr B.P. climatic event, as evident in biomarker proxies and pollen data, we link shifts in δDwax to changes in Mediterranean air mass trajectories supplying precipitation to northeastern Greece, with variations in the relative contributions of northerly derived, D-depleted moisture and southerly-derived, D-enriched moisture. Possible control mechanisms include changes in the influence of the Siberian High and differences in the influence of the African and Asian monsoon circulations on anticyclonic conditions in the Mediterranean region as well as regional inflow of moist air from the Aegean Sea.
This thesis describes the adaptation of Acinetobacter species to dry environments with the soil bacterium A. baylyi and the opportunistic hospital pathogen A. baumanii in its focus. The adaptation of A. baylyi and A. baumannii to osmotic stress was investigated. Compatible solutes that were uptaken from the environment or synthesized de novo to cope with the loss of water at high salinity were identified. The corresponding transporters and enzymes involved were characzerized. In addition, the desiccation resistance of A. baumannii was analyzed to elucidate its survival in hospital environments. The usage of compatible solutes during desiccation stress was analyzed and proteins that were produced were identified.
The availability of water is essential for bacterial life and if environmental conditions are awkward, bacteria have to cope with high salinitiy to prevent loss of water. In this thesis it was shown that A. baylyi synthesizes glutamate and mannitol de novo as compatible solutes in response to osmotic stress to balance the osmotic potential. The pathway for mannitol biosynthesis from Fructose-6-Phosphate (F-6-P) via Mannitol-1-Phosphate (Mtl-1-P) was elucidated and the isolation and characterization of a novel type of biofunctional enzyme was described. Interestingly, the unique bifunctional enzyme MtlD, acting as dehydrogenase and phosphatase, mediates both steps of the mannitol biosynthesis pathway. This enzyme catalyzes the reduction of F-6-P to Mtl-1-P with NADPH as reducing equivalent. The dehydrogenase activity of MtlD was salt dependent and the phosphatase activity was dependent on Mg2+ as cofactor. Phylogenetic analyses revealed that MtlD is broadly distributed among other Acinetobacter strains but not in other phylogenetic tribes.
In this thesis it is also described that, besides de novo synthesis of compatible solutes, A. baylyi takes up glycine betaine (GB) or its precursor choline by different transport systems and uses this solutes as osmoprotectants. The uptake of GB occurs via a secondary transporter (ACIAD3460) of the BCCT family. Choline is taken up as precursor and oxidized to GB by two dehydrogenases. The uptake and use of choline as GB precursor involves two transporters, whose genes are encoded in the bet cluster (BetT1, BetT2), two dehydrogenases (BetA, BetB) and a regulatory protein (BetI). Both transporters differ from each other in structure and function: BetT1 is osmo-independent and active independently of osmotic stress. BetT2 contains - in contrast to BetT1 - a long C-terminal domain for osmo-sensing and its activity highly increases in the presence of high osmolarity. The oxidation of choline occurs independently of the osmolarity of the medium but in the absence of salt stress, GB is exported. In contrast, in the presence of high salinity, GB is accumulated in the cytoplasm to balance the osmotic potential in order to prevent loss of water. The regulation of both transporters, the uptake of choline independently of the osmolarity and the export of GB under isoosmotic conditions are regulated by the transcriptional regulator BetI.
A. baumannii ATCC 19606 was also shown to cope with high salinity. Analogously to A. baylyi, A. baumannii ATCC19606 synthesizes glutamate and mannitol de novo in response to osmotic stress. The genes for the synthesis of these compatible solutes are identical to those found in A. baylyi. This suggests that the solute biosynthesis pathways of A. baumannii and A. baylyi are identical. A. baumannii was also able to take up GB and choline in response to osmotic stress and growth at high salinity was restored upon addition of GB and its precursor choline. The bet cluster was also present in the genome A. baumannii and also contains the two different choline transporters BetT1 and BetT2.
Our suggestion that choline or GB or the utilization of phosphatidylcholine as carbon source led to an increase in the survival under desiccation stress was not confirmed. However, 2D analysis of proteins produced during desiccation stress in A. baumannii led to elevated amounts of proteins implicated in biofilm formation, regulation, cell morphology and general stress response, such as Hsp60 or superoxide dismutase, both might play a role in general stress protection.
The behaviour of electronic circuits is influenced by ageing effects. Modelling the behaviour of circuits is a standard approach for the design of faster, smaller, more reliable and more robust systems. In this thesis, we propose a formalization of robustness that is derived from a failure model, which is based purely on the behavioural specification of a system. For a given specification, simulation can reveal if a system does not comply with a specification, and thus provide a failure model. Ageing usually works against the specified properties, and ageing models can be incorporated to quantify the impact on specification violations, failures and robustness. We study ageing effects in the context of analogue circuits. Here, models must factor in infinitely many circuit states. Ageing effects have a cause and an impact that require models. On both these ends, the circuit state is highly relevant, an must be factored in. For example, static empirical models for ageing effects are not valid in many cases, because the assumed operating states do not agree with the circuit simulation results. This thesis identifies essential properties of ageing effects and we argue that they need to be taken into account for modelling the interrelation of cause and impact. These properties include frequency dependence, monotonicity, memory and relaxation mechanisms as well as control by arbitrary shaped stress levels. Starting from decay processes, we define a class of ageing models that fits these requirements well while remaining arithmetically accessible by means of a simple structure.
Modeling ageing effects in semiconductor circuits becomes more relevant with higher integration and smaller structure sizes. With respect to miniaturization, digital systems are ahead of analogue systems, and similarly ageing models predominantly focus on digital applications. In the digital domain, the signal levels are either on or off or switching in between. Given an ageing model as a physical effect bound to signal levels, ageing models for components and whole systems can be inferred by means of average operation modes and cycle counts. Functional and faithful ageing effect models for analogue components often require a more fine-grained characterization for physical processes. Here, signal levels can take arbitrary values, to begin with. Such fine-grained, physically inspired ageing models do not scale for larger applications and are hard to simulate in reasonable time. To close the gap between physical processes and system level ageing simulation, we propose a data based modelling strategy, according to which measurement data is turned into ageing models for analogue applications. Ageing data is a set of pairs of stress patterns and the corresponding parameter deviations. Assuming additional properties, such as monotonicity or frequency independence, learning algorithm can find a complete model that is consistent with the data set. These ageing effect models decompose into a controlling stress level, an ageing process, and a parameter that depends on the state of this process. Using this representation, we are able to embed a wide range of ageing effects into behavioural models for circuit components. Based on the developed modelling techniques, we introduce a novel model for the BTI effect, an ageing effect that permits relaxation. In the following, a transistor level ageing model for BTI that targets analogue circuits is proposed. Similarly, we demonstrate how ageing data from analogue transistor level circuit models lift to purely behavioural block models. With this, we are the first to present a data based hierarchical ageing modeling scheme. An ageing simulator for circuits or system level models computes long term transients, solutions of a differential equation. Long term transients are often close to quasi-periodic, in some sense repetitive. If the evaluation of ageing models under quasi-periodic conditions can be done efficiently, long term simulation becomes practical. We describe an adaptive two-time simulation algorithm that basically skips periods during simulation, advancing faster on a second time axis. The bottleneck of two-time simulation is the extrapolation through skipped frames. This involves both the evaluation of the ageing models and the consistency of the boundary conditions. We propose a simulator that computes long term transients exploiting the structure of the proposed ageing models. These models permit extrapolation of the ageing state by means of a locally equivalent stress, a sort of average stress level. This level can be computed efficiently and also gives rise to a dynamic step control mechanism. Ageing simulation has a wide range of applications. This thesis vastly improves the applicability of ageing simulation for analogue circuits in terms of modelling and efficiency. An ageing effect model that is a part of a circuit component model accounts for parametric drift that is directly related to the operation mode. For example asymmetric load on a comparator or power-stage may lead to offset drift, which is not an empiric effect. Monitor circuits can report such effects during operation, when they become significant. Simulating the behaviour of these monitors is important during their development. Ageing effects can be compensated using redundant parts, and annealing can revert broken components to functional. We show that such mechanisms can be simulated in place using our models and algorithms. The aim of automatized circuit synthesis is to create a circuit that implements a specification for a certain use case. Ageing simulation can identify candidates that are more reliable. Efficient ageing simulation allows to factor in various operation modes and helps refining the selection. Using long term ageing simulation, we have analysed the fitness of a set of synthesized operational amplifiers with similar properties concerning various use cases. This procedure enables the selection of the most ageing resilient implementation automatically.
Given an Abelian semi-group (A, +), an A-valued curvature measure is a valuation with values in A-valued measures. If A = R, complete classifications of Hausdorff-continuous translation-invariant SO(n)-invariant valuations and curvature measures were obtained by Hadwiger and Schneider, respectively. More recently, characterisation results have been achieved for curvature measures with values in A = Sym^p R^n and A = Sym^2 Λ^q R^n for p, q ≥ 1 with varying assumptions as for their invariance properties.
In the present work, we classify all smooth translation-invariant SO(n)-covariant curvature measures with values in any SO(n)-representation in terms of certain differential forms on the sphere bundle S R^n and describe their behaviour under the globalisation map. The latter result also yields a similar classification of all continuous SO(n)-module-valued SO(n)-covariant valuations. Furthermore, a decomposition of the space of smooth translation-
invariant scalar-valued curvature measures as an SO(n)-module is obtained. As a corollary, we construct explicit bases of continuous translation-invariant scalar-valued valuations and smooth translation-invariant scalar-valued curvature measures.
Magnetism is a beautiful example of a macroscopic quantum phenomenon. While known at least since the ancient Greeks, a microscopic theoretical explanation of magnetism could only be achieved with the advent of quantum mechanics at the beginning of the 20th century. Then it was understood that in a certain class of solids the famous Pauli exclusion principle leads to an effective interaction between the microscopic magnetic moments, i.e., the spins, which favors an ordered, and hence macroscopically magnetic, state. Nowadays, magnetic phenomena are used in a host of applications, and are especially relevant for information storage and processing technologies.
Despite the long history of the field, magnetic phenomena are still an active research topic. In particular, in the last decade the fields of spintronics and spin-caloritronics emerged, which manipulate the microscopic spins via charge and heat currents respectively. This opens new avenues to potential applications; including the possibility to use the magnetic spin degrees of freedom instead of charges as carriers of information, which could provide a number of advantages such as reduced losses and further miniaturization.
In this thesis we do not delve any further into the realm of possible applications. Instead we use sophisticated theories to explore the microscopic spin dynamics which is the basis of all such applications. We also focus on a particular compound: Yttrium-iron garnet (YIG), which is a ferrimagnetic insulator. This material has been widely used in experiments on magnetism over the last decades, and is a popular candidate for spintronic devices. Microscopically, the low-energy magnetic properties of YIG can be described by a ferromagnetic Heisenberg model. For spintronics and spin-caloritronics applications, it is however insufficient to only consider the magnetic degrees of freedom; one should also include the coupling of the spins to the elastic lattice vibrations, i.e., the phonons. Besides giving an overview on techniques used throughout the thesis, the introductory Ch. 1 provides a discussion of the microscopic Hamiltonian used to model the coupled spin-phonon system in the subsequent chapters.
The topic of Ch. 2 are the consequences of the magnetoelastic coupling on the low-energy magnon excitations in YIG. Starting from the microscopic spin-phonon Hamiltonian, we rigorously derive the magnon-phonon hybridization and scattering vertices in a controlled spin wave expansion. For the experimentally relevant case of thin YIG films at room temperature, these vertices are then used to compute the magnetoelastic modes as well as the magnon damping. In the course of this work, the damping of magnons in this system was also investigated experimentally using Brillouin light scattering spectroscopy. While comparison to the experimental data shows that the magnetoelastic interactions do not dominate the total magnon relaxation in the experimentally accessible regime, we are able to show that the spin-lattice relaxation time is strongly momentum dependent, thereby providing a microscopic explanation of a recent experiment.
In the final Ch. 3, we investigate a different phenomenon occurring in thin YIG films: Room temperature condensation of magnons. Prior work attributed this condensation process to quantum mechanics, i.e., it was interpreted as Bose-Einstein condensation. However, this is not satisfactory because at room temperature, the magnons in YIG behave as purely classical waves. In particular, the quantum Bose-Einstein distribution reduces to the classical Rayleigh-Jeans distribution in this case. In addition, the effective spin in YIG is very large. Therefore we start from the hypothesis that the room temperature magnon condensation is actually a new example of the kinetic condensation of classical waves, which has so far only been observed by imaging classical light in a photorefractive crystal. To distinguish this classical condensation from the quantum mechanical Bose-Einstein one, we refer to it as Rayleigh-Jeans condensation. To prove our claim, we consider the classical equations of motion of the coupled spin-phonon system. By eliminating the phonon degrees of freedom, we microscopically derive a non-Markovian stochastic Landau-Lifshitz-Gilbert equation (LLG) for the classical spin vectors. We then use this LLG to perform numerical simulations of the magnon dynamics, with all parameters fixed by experiments. These simulations accurately reproduce all stages of the magnon time evolution observed in experiments, including the appearance of the magnon condensate at the bottom of the magnon spectrum. In this way we confirm our initial hypothesis that the magnon condensation is a classical Rayleigh-Jeans condensation, which is unrelated to quantum mechanics.
From an early understanding of organisational theorist (Bartlett & Ghosal, 1989; 1990), the function of global teams in transnational organisations has been conceptualised as the transformation of different embedded cultural practices for the development of a global strategy, products and services. Simultaneously, in the field, from the beginning of the 1990ies to the edge of the new millennium neo-liberal political developments enforced a free flow of capital on a global level (cf. Turner, 2006). In line with the development of respective connectivity via the internet the form of globally distributed team work was spread (Maznevski & Chudoba, 2000). In a study by Biggs (2000), published just after the millennial change, it was shown that 60% of tasks in multinational companies are accomplished by virtual teams. More recent data (Society for Human Resource Management, 2012) showed that the prevalence of such teams stayed more or less constant in the last 10 years. According to the survey 66% of companies are working with distributed global teams.
Globally distributed teams were already described by Bartlett and Ghosal (1989) in their functions of articulation and translation of differing market practices for the integration of requirements and needs on a global level. From a European perspective the importance to further develop innovation capabilities in order to compete in the global market is stressed today (Imp3rove, 2012). In a globalised economy not only the big multinational companies are involved in globally distributed research and development activities (R&D). On the level SMEs, for example, in Switzerland the involvement in global development processes is increasing (Gassmann, 2009). From my own experiences in working with Swiss SMEs, the macro-economic processes in regard to the strong Swiss Franc may accelerate such processes. Thus, the form of globally distributed teams, and their functional task in global development processes, can be viewed as highly relevant, in a globalised economy.
The crucial question for companies at the moment is, if teams can be enabled for innovative project work, which enables the integration of diverging perspectives in a globally distributed setting? Or, if such teams have to be collocated for more innovative, interdependent task work? Requirements for integrating embedded knowledge from different regionally defined clusters into global innovations at least, seems to indicate for the relevance of interdependent globally distributed team work (cf. Li, Eden, Hitt, Ireland, & Garrett, 2012). Bilateral practices of partnering, for example in the Swiss pharmaceutical sector, lead to the integration of selected subsidiaries in the R&D process of the company (Festel et al., 2010). Thus, the form of dispersion for project teams becomes more critical for effective global R&D practices (Boyer O’Leary & Cummings, 2007). So called partially distributed teams integrating balanced subgroups between two sites, hence, become an important subject of inquiry with practical relevance.
The context of partially distributed team work represents by virtue a context involving multiple perspectives influenced by the involvement of actors stemming from different cultural contexts (Dekker, Rutte, & Van den Berg, 2008). It thereby provides the synergetic potential for integrating different perspectives in the resolution of complex problems on a global level (Janssens & Brett, 2006). Simultaneously, cultural diversity engenders challenges for collaboration. Challenges, like different understandings and interpretations regarding tasks, the structuring of communication (Maznevski & Chudoba, 2000) and unexpected events occurring in the collaboration between the actors (Dekker et al., 2008; Oertel & Antoni, 2014) were identified in respective empirical studies.
Opportunities and challenges of partially distributed global teams can be compared with the problematic of face-to-face (f2f) teams with a moderate amount of diversity. Studies have shown (see Thatcher & Patel, 2011 for a meta-analysis) that when the distribution of diversity characteristics is aligned to potentially form culture specific subgroups, so-called diversity faultlines (Lau & Murnighan, 1998), negative subgroup dynamics are reinforced.
To achieve the above mentioned synergetic potentials it seems important to effectively cope with such negative dynamics and allow for a balanced participation in partially distributed teams (Janssens & Brett, 2006). In the research on faultline teams, especially the structuring of task-related interdependences across respective subgroups has been identified as an important impediment for the mentioned subgroup dynamics. Task interdependences, which cross functional roles across respective group faultlines (Bettencourt, Molix, Talley, & Eubanks, 2007; Marcus-Newhall, Miller, Holtz, & Brewer, 1993), are able to unlock the inherent potentials of globally distributed teams on more complex tasks that require the integration of different perspectives. From a work group diversity perspective (van Knippenberg & Schippers, 2007), partially distributed global teams represent a research object for studying the interaction between social categorisation processes involved in the above mentioned subgroup dynamics, and processes of task-related information processing required for innovative team outputs. The exploration of effects of task structures on the interaction between categorisation processes and task-related information processing (van Knippenberg et al., 2004), will be in the main explorative research focus of this thesis. The research thesis represents a heuristic explorative inquiry (Kleining & Witt, 2001) of respective dynamics and structural as well as process-related enablers.
The thesis starts with the theoretical part, in which the historical development of the understanding of teams as open, complex and temporally dynamic systems (Arrow et al., 2005, 2000), will be outlined. A sound definition of partially distributed global teams, including the respective contextual characteristics will be delineated. In a sensitizing framework (Blumer, 1954) which guided the explorative research process, the central boundary condition of task interdependence (Wageman, 2001) and respective episodic theories for explaining global task-related dynamics in teams (Marks et al., 2001), the dynamics of social categorisation (Gaertner, Dovidio, Anastasio, Bachman, & Rust, 1993; Gaertner & Dovidio, 2000), as well as the interaction between social categorisation processes and task-related information processing will be integrated (van Knippenberg et al., 2004). According to the framework, empirical studies on effects of task interdependence on interactions between task-related information processing and social categorisation processes will be addressed (van Knippenberg et al., 2004).
The empirical part of the contribution will be split in two parts. In the first heuristic exploratory study eleven partially distributed global teams are followed up during the time of relevant innovation projects. The approach allowed the study of task interdependence, productive interactions with social categorisation processes and there effects on team innovation. In the second empirical step, the developed hypotheses, were tested in an experimental simulation (Arrow et al., 2005, 2000) in undergraduate courses.
As a conclusion of the two exploratory studies, an episodic team process model will be outlined. The model specifies interdependence dynamics, which allows for team innovation. Furthermore, on a process level, the episodic categorisation-elaboration model (van Knippenberg et al., 2004) proposes three critical team performance episodes. Dynamics in the interplay between task-related information processing and social categorisation processes allow for the development of hypothesis for further research projects. Finally the implications for theory and the practical relevance of the heuristic model will be discussed.
Fungi are an important component of every ecosystem but hardly considered in biodiversity monitoring projects. This thesis aims at characterizing fungal diversity, with an emphasis on epigeous fungi, encompassing different biogeographic zones and points in time. A main sampling area was established in the Taunus mountain range in Germany, which was sampled monthly over three years.
For testing species richness on spatial scale, the Taunus transect was compared with four other areas, which were assessed with lower sampling effort. One of these areas was Bulau in Germany, in which four excursions were made. Furthermore, two sampling events were performed in Somiedo in Spain and one sampling event in Kleinwalsertal in Austria. Already existing data of a two-year monitoring project in Panama next to the river Majagua were additionally used for comparison.
All these areas were investigated with a standardized sampling protocol focusing on macroscopically evident fungi and vascular plants using a time-restricted transect design. The transects consisted of strips, which were 500 m long and about 20 m broad, and were sampled for 2 hours at each single sampling event....
The transition from the marine to the terrestrial realm is one of the most fascinating issues in evolutionary biology for it required the appearance, in different organisms, of several novel adaptations to deal with the demands of the new realm. Adaptations include, for instance, modifications in different metabolic pathways, development of body structures to facilitate movement and respiration, or tolerance to new conditions of stress. The transition to the land also gives an extraordinary opportunity to study whether evolution used similar changes at the genomic level to produce parallel adaptations in different taxa. Mollusks are among taxa that were successful in the conquest of the land. For instance, several lineages of the molluscan clade Panpulmonata (Gastropoda, Heterobranchia) invaded the intertidal, freshwater and land zones from the marine realm. In my dissertation, using tools from bioinformatics, phylogenetics, and molecular evolution, I used panpulmonates as a suitable model group to study the independent invasions into the terrestrial realm and the adaptive signatures in genes that may have favored the realm transitions. My work includes two peer-reviewed published papers and one manuscript under review. In Publication 1 (Romero et al., 2016a), I used mitochondrial and nuclear molecular markers to resolve the phylogeny of the Ellobiidae, a family that possesses intertidal and terrestrial species. The phylogeny provided an improved resolution of the relationships within inner clades and a framework to study the tempo and mode of the land transitions. I showed that the terrestrialization events occurred independently, in different lineages (Carychiinae, Pythiinae) and in different geological periods (Mesozoic, Cenozoic). In addition, the diversification in this group may not have been affected by past geological or climate changes as the Cretaceous-Paleogene (K-Pg) event or the sea-level decrease during the Oligocene. In Publication 2 (Romero et al., 2016b), I generated new mitochondrial genomes from terrestrial species and compared them with other panpulmonates. I used the branch-site test of positive selection and detected significant nonsynonymous changes in the terrestrial lineages from Ellobioidea and Stylommatophora. Two genes appeared under positive selection: cob (Cytochrome b) and nad5 (NADH dehydrogenase 5). Surprisingly, I found that the same amino acid positions in the proteins encoded by these genes were also under positive selection in several vertebrate lineages that transitioned between different habitats (whales, bats and subterranean rodents). This result suggested an adaptation pattern that required parallel genetic modifications to cope with novel metabolic demands in the new realms. In Manuscript 1 (Romero et al., under review), I de novo assembled transcriptomes from several panpulmonate specimens resulting in thousands of genes that were clustered in 702 orthologous groups. Again, I applied the branch-site test of positive selection in the terrestrial lineages from Ellobioidea and Stylommatophora and in the freshwater lineages from Hygrophila and Acochlidia. Different sets of genes appeared under positive selection in land and freshwater snails, supporting independent adaptation events. I identified adaptive signatures in genes involved in gas-exchange surface development and energy metabolism in land snails, and genes involved in the response to abiotic stress factors (radiation, desiccation, xenobiotics) in freshwater snails. My work provided evidence that supported multiple land invasions within Panpulmonata and provided new insights towards understanding the genomic basis of the adaptation during sea-to-land transitions. The results of my work are the first reports on the adaptive signatures at the codon level in genes that may have facilitated metabolic and developmental changes during the terrestrialization in the phylum Mollusca. Moreover, they contribute to the current debate on the conquest of land from the marine habitat, a discussion that has been only based in vertebrate taxa. Future comparative genome-wide analyses would increase the number of genes that may have played a key role during the realm transitions.
Ischemic injuries of the cardiovascular system are still the leading cause of death worldwide. They are often accompanied by loss of cardiomyocytes (CM) and their replacement by non-functional heart tissue. Cardiac fibroblasts (CF) play a major role in the recovery after ischemic injury and in the scar formation. In the last few years researchers were able to reprogram fibroblasts into CM in vitro and in murine models of myocardial infarction using various protocols including a cocktail of microRNAs (miRs). These miRs can target hundreds of messenger RNAs and inhibit their translation into proteins, potentially regulating multiple cellular signaling pathways. Because of this, there has been a rising interest in the use of miRs for therapeutic purposes. However, as different miRs have different effects in different cells, there is the danger of causing serious side effects. These could be alleviated by enacting a cell-specific transport of miRs, for example by using aptamers. Aptamers are usually short strands of DNA or RNA, which can fold into a specific three-dimensional confirmation which allows them to bind specifically to target molecules. Aptamers are commonly selected from a large library for their ability to bind to target molecules using a procedure called SELEX. Aptamers have already been used to transport miRs into cancer cells.
In this thesis, we first established the transport of miRs into cells of the cardiovascular system using aptamers. MiR-126 is an important part of the signaling in endothelial cells (EC), protects from atherosclerosis and supports angiogenesis, which is why we chose it as a candidate to transport into the vasculature. We first tested two aptamers for their ability to internalize into EC and fibroblasts. Both the aptamer for the ubiquitously expressed transferrin receptor (TRA) and a general internalizing RNA motif, but not a control construct, could internalize efficiently into all cell types tested. We then designed three chimeras (Ch) using different strategies to connect TRA to miR-126. While all chimeras could internalize efficiently, only Ch3, which connects TRA to Pre-miR-126 using a sticky bridge structure, had functional effects in EC. Ch3 reduced the protein expression of VCAM-1 in EC and increased the VEGF induced sprouting of EC in a spheroid-sprouting assay. Treatment of breast cancer cells with Ch3 emulated the effects of treatment with classical miR-126-3p and miR-126-5p mimics. In the SK-BR3 cell line Ch3 and miR-126-3p reduce the viability of the cells while they reduce recruitment of EC by the MCF7 cell line. miR-126-5p had no apparent effect in the SK-BR3 line, but increased viability of MCF7 cells, as did Ch3. This implies that Ch3 can be processed to both functional miR-126-3p and miR-126-5p in treated cells.
We were unable to achieve a reprogramming of adult murine cardiac fibroblasts into cells resembling CM using the cocktail of 4 miRs. This indicates that the miR-mediated transdifferentiation is only possible in neonatal fibroblasts. The effects in mice after an AMI might possibly be caused by an enhanced plasticity of fibroblasts in and close to the infarcted area.
We also screened to find aptamers specifically binding to cells of the cardiovascular system. We used two oligonucleotide libraries in a cell-SELEX to select candidates which bind to CF, but not EC. We observed that only the library which contains two randomized regions of 26 bases showed an enrichment of species binding to fibroblasts. We then sequenced rounds 5-7 of the SELEX and analyzed the data bioinfomatically to select 10 candidate aptamers. All candidates showed a strong binding not only to CF, but also EC. This indicates that the selection pressure against species binding to EC was not high enough and would have to be increased to find true CF-aptamers. Four promising candidates were also analyzed for their potential to be internalized and we surprisingly found that all of them were internalized by EC and CF more efficiently than TRA. The similar behavior of the candidates implies that they possibly share a ligand, which is expressed both by EC and CF, but more prominently by the latter.
This work demonstrates the possibility of using aptamers to transport miRs into cells of the cardiovascular system. It also shows that it is possible to select aptamers for non-cancerous mammalian cells, which has not been done before. It is reasonable to assume that a refinement of the cell-SELEX will allow selection of cell-specific aptamers. Due to the failure of reprogramming of adult fibroblasts into induced cardiomyocytes we were unable to test whether a miR-mediated reprogramming might be inducible using aptamer transported-miRs. Ultimately, aptamer mediated transport of miRs is a feasible and promising therapeutic option for the treatment of cardiovascular diseases and other disorders like cancer.
Die Etablierung der Festphasensynthese innerhalb der letzten Jahrzehnte macht hoch modifizierte Oligonukleotide verfügbar. Damit werden Methoden wie Einzelmolekül-aufgelöstes Tracking möglich, um beispielsweise den Weg einer einzelnen RNA von der Transkriptionsstelle im Nukleus bis zur Proteinbiosynthese im Cytoplasma verfolgen und kritische Stelle verstehen zu können. In den letzten Jahren entwickelten sich auch vermehrt Fragen zur lokalen Proteinsynthese. Dabei nimmt man besonders im Fall von polaren Zellen wie Neuronen an, dass die Proteinbiosynthese nicht global im Cytosol stattfindet, sondern es einen Transport der „ruhenden“ RNA bis zu dem Ort geben muss, an dem das entsprechende Protein lokal benötigt wird. In dieser vorliegenden Arbeit sollen nun in zwei Hauptprojekten molekulare Werkzeuge entwickelt werden, mit deren Hilfe oben genannte Fragestellungen in Zukunft beantwortet werden könnten. Im ersten Hauptprojekt wurde dazu eine neue Generation lichtaktivierbarer Molecular Beacons (von engl.: molekulare Leuchtfeuer) entwickelt. Dabei handelt es sich um Oligonukleotide, die komplementär zu einer intrazellulären RNA-Sequenz (Target-RNA) sind und mit Fluorophor und Fluoreszenz-Quencher modifiziert werden. Bei den lichtaktivierbaren Designs kann Fluoreszenz detektiert werden, wenn der Molecular Beacon an seine Targetsequenz gebunden und zusätzlich zuvor eine Lichtaktivierung stattgefunden hat. Im Gegensatz zu früheren Designs wurde bei diesem hier vorgestellten Molecular Beacon der Fluorophor mit Hilfe eines zweiten photoabspaltbaren Quenchers verbunden. Dadurch kann der Beacon an seine Targetsequenz binden, obwohl noch keine Lichtaktivierung stattgefunden hat. Fluoreszenz kann allerdings erst nach photoinduzierter Abspaltung des zusätzlichen Quenchers detektiert werden. In der vorliegenden Studie konnten dadurch extrem gute Signal-zu-Rausch-Verhältnisse von bis zu 170:1 erreicht werden. Zusätzlicher Vorteil dieses Designs ist die Tatsache, dass eine Vielzahl kommerziell erhältlicher Fluorophor-Quencher-Paare verwendet werden kann. Dabei ist es nicht relevant, ob der entsprechende Farbstoff co-synthetisch während der Festphasensynthese oder post-synthetisch durch die Modifikation funktioneller Gruppen angebracht wird. Nach anfänglichen in vitro Tests wurden die besten Molecular Beacons in vivo in der Zuckmücken-Art Chironomus tentans getestet. Dieser Organismus ist aufgrund seiner Polytänchromosomen, der sog. Balbiani Ringe, interessant. Dabei handelt es sich um ein Chromosom, das viele Chromatiden mit jeweils identischen Gensequenzen enthält. Diese Balbiani Ringe haben eine sehr charakteristische Struktur. Die Molecular Beacons wurden in den Zellkern injiziert und anschließend photoinduziert. Auch in den in vivo Messungen zeigte sich die Überlegenheit des neuen Design mit Signal-zu-Rausch-Verhältnissen von bis zu 80:1. Im zweiten Hauptprojekt war es das Ziel, lokale mikroRNA-Reifung in Neuronen nachzuweisen bzw. sichtbar zu machen. MikroRNA (kurz miRNA) ist einer der wichtigsten zellulären Werkzeuge, um Genregulation auf post-transkriptioneller Ebene zu ermöglichen. Für dieses Projekt wurde eine Sonde entwickelt, die den nativen miRNA-Vorläufer – die sog. prä-miRNA – nachbildet. Der enzymatische Reifungsprozess durch die RNase Dicer sollte durch Fluoreszenz nachweisbar sein. Dies gelang durch Modifikationen der Sequenz um die enzymatische Schnittstelle herum. Durch den Dicer-vermittelten, enzymatischen Verdau wurde ein Fluorophor von einem Quencher getrennt, wobei der fluoreszente Farbstoff an der reifen mikroRNA verblieb. Nach der Etablierung der in vitro Tests und Auswahl des optimalen Fluorophor-Quencher-Paars zeigte sich in einem Kontrollexperiment, dass bei Verwendung von neuronalen Ganglien aus Dicer-Knock-Out Mäusen kein Fluoreszenzanstieg zu beobachten war. Dieses Experiment bewies, dass bisher beobachtete Fluoreszenzanstiege Dicer-spezifisch waren. Im nächsten Schritt wurden in vivo Messungen durchgeführt. Es zeigte sich dabei, dass die sog. Patch Clamp Technik herkömmlichen Transfektionsmethoden überlegen war. Unter normalen Bedingungen zeigte sich sowohl im Soma als auch in den Dendriten ein Fluoreszenzanstieg. Durch Depolarisation des Neurons konnte dieser Effekt noch verstärkt werden, wobei das somatische Signal grundsätzlich als höher einzustufen war. Interessanterweise führte eine Blockade der NMDA-Rezeptoren auch bei gleichzeitiger Depolarisation zu einer verringerten Fluoreszenz. Dies lässt darauf schließen, dass die Reifung der untersuchten prä-miRNA in Dendriten von der Aktivität des NMDA-Rezeptors bzw. einem als Konsequenz ansteigenden Ca2+-Spiegels in der Zelle abhängig ist. In einem weiteren Experiment wurde nach „Beladung“ eines Neurons mit der prä-miRNA-Sonde Dendriten punktuell aktiviert. Dies konnte durch Licht-aktivierbares Glutamat erreicht werden. Im zentralen Nervensystem gilt Glutamat als der wichtigste aktivierende Neurotransmitter. Es konnte beobachtet werden, wie einerseits Fluoreszenz lokal an der aktivierten Stelle anstieg und gleichzeitig sog. dendritische Spines wuchsen. Zum Teil war auch ein Wachstum benachbarter Spines zu beobachten. Dabei handelt es sich um pilzförmige Aussackungen der Dendriten an Stellen, an denen Vernetzungen zu Synapsen anderer Neuronen existieren. Als Ergebnis kann geschlussfolgert werden, dass es eine lokale Reifung der untersuchten prä-miRNA durch Dicer in Dendriten gibt. Dieser Prozess kann sehr spezifisch und lokal durch die Aktivierung einzelner synaptischer Verbindungen initiiert werden.
Functional dynamics of ribonucleic acids : development and application of spectroscopic tools
(2016)
Im Rahmen der vorliegenden Dissertation wird der Aufbau eines zeitaufgelösten Fluorimeters, die photophysikalische Grundcharakterisierung der drei 2-(Pyrenylethinyl)-Adenosine (PyAs) und das Wechselwirkungsgeflecht des tetracyclinbindenden Aptamers (TC-Aptamer) mit seinem Liganden Tetracycyclin (TC) und Mg2+ dargestellt.
Das zeitaufgelöste Fluorimeter basiert auf der experimentellen Technik des zeitkorrelierten Einzelphotonenzählens. Es verfügt über zwei Anregungsquellen: gepulste UV-LEDs und einen frequenzverdoppelten titandotierten Saphirlaser. Diese Quellen Decken einen Wellenlängenbereich von (310 - 550) nm ab. Das Spektrometer kann unter günstigen Umständen eine Zeitauflösung von 50 ps erreichen bei einer zeitlichen Messungenauigkeit von weniger als 0,02 %.
Die Leistungsfähigkeit des Aufbaus wird anhand einer umfangreichen Studie an den drei PyAs demonstriert.
Die drei PyAs 2-(1-Pyrenylethinyl)-Adenosine (1PyA), 2-(2-Pyrenylethinyl)-Adenosine (2PyA) und 2-(4-Pyrenylethinyl)-Adenosine (4PyA) sind eine Gruppe fluoreszierender RNA-Nukleosidanaloga, welche die Gesamtheit aller möglichen Konfigurations-isomere der Grundverbindung PyA umfassen. Ihre zeitabhängigen Fluoreszenzzerfallseigenschaften werden ergänzt von stationären Absorptions- und Fluoreszenzspektren, ultraschneller transienter Absorptionsspektroskopie und quantenchemischen Rechnungen. Die Fluoreszenz von 1PyA und 4PyA gehorchen der Regel von Kasha, wohingegen 2PyA einen triexponentiellen Zerfall mit ausgeprägter Abhängigkeit von der Anregungswellenlänge zeigt. Die transienten Absorptionsspektren aller drei Isomere weisen im gesamten Spektrum dominante, wenig strukturierte Absorptionsbanden des ersten angeregten Zustands auf, welche im nahen UV in unterschiedlichem Maße vom Grundzustandsbleichen und stimulierter Emission überlagert werden. 2PyA zeigt eine deutlich ausgeprägte Signatur für eine interne Umwandlung hin zum S1, wenn es in höhere angeregte Zustände angeregt wird.
Das Fluoreszenzverhalten von 2PyA wird mithilfe eines lokal angeregten (LE) und zweier intramolekularer Ladungstransferzustände, von denen einer der koplanaren Orientierung von Pyren und Adenin (MICT) und der andere einer um 90 ° verdrehten Orientierung (TICT) entspricht. Der LE-Zustand ist hierbei verknüpft mit dem S2 von 2PyA, welcher einer rein pyrenlokalisierten Anregung entspricht. Dieser Zustand existiert so in 1PyA und 4PyA nicht. Der verdrehte TICT-Zustand ist nur in 2PyA bevölkerbar, weil für 2PyA die Barriere zur Bildung von Rotameren am niedrigsten ist und das Molekül nach Anregung daher in diese Geometrie kommen kann und dann durch die stärkere elektrostatische Anziehung stabilisiert wird. 1PyA und 4PyA emittieren hingegen nur aus dem MICT-Zustand.
Die Komplexbildung des TC-Aptamers mit seinem Liganden TC in Lösung wird empfindlich beeinflusst durch die-Konzentration von Magnesiumkationen. Dies wird untersucht durch Bindungs- und Faltungs- und Denaturierungsstudien mit verschiedenen Mg- und Harnstoffkonzentrationen. Als experimentelle Observable dienen hierbei die konformationsabhängige Nukleobasenabsorption und ihr Zirkulardichroismus im fernen UV, die Fluoreszenz des Liganden TC und die freiwerdende Wärme der exothermen Bindungsreaktion des Aptamers mit Mg in An- und Abwesenheit von TC.
Ohne Mg ist eine Interaktion des TC-Aptamers mit TC nicht nachweisbar. Dies liegt daran, dass Mg die notwendige elektrostatische Abschirmung der negativen elektrischen Ladung am RNA-Rückgrat zur Verfügung stellt. Die Abschirmung erlaubt es dem Aptamer kompakte Strukturen mit tertiären Kontakten auszubilden. Wenn die Mg-Konzentration die Faltung des Aptamers vollständig unterstützt (> 1 mM), so befindet sich das Aptamer weitgehend in einer vorgefalteten Konformation, welche der bindungskompetenten stark ähnelt. In diesem Zustand kann das Aptamer seinen Liganden extrem schnell, nämlich annähernd diffusionslimitiert binden. Unter diesen Bedingungen hat TC kaum Einfluss auf die Konformation seines Aptamers.
Bei physiologischen Mg-Konzentrationen (0,2 - 0,8 mM) kann das Aptamer kompakte Konformationen mit tertiären Strukturen einnehmen. Diejenige Konformation, welche der bindenden sehr stark ähnelt, dominiert das konformationelle Gleichgewicht jedoch noch nicht vollständig, es ist lediglich eine Konformation von vielen möglichen. Daher eröffnen physiologische Mg-Konzentrationen dem TC-Aptamer Teile des Konformationsraumes, welche andernfalls nicht zugänglich wären und TC stabilisiert selektiv die native Konformation. Diese konformationelle Verschiebung liefert kann hierbei zur robusten Signalgebung für die Funktion als Riboschalter dienen.
The Hepatitis C virus (HCV) infects more than 170 million individuals worldwide and causes challenging HCV-related diseases. Unfortunately, there is no vaccine available. Therefore, a better understanding of the HCV life cycle is urgently needed to develop more effective and better tolerated therapies.
It has been reported that the secretory pathway plays an essential role for the release of HCV, and the SNARE complexes are a central factor controlling intracellular vesicular trafficking. Recently, our group observed that α-taxilin that binds to free syntaxin 4 prevents the SNARE complex formation and exerts an inhibitory effect on the release of HCV particles. Therefore, it was analyzed whether the t-SNARE protein syntaxin 4 is involved in the HCV life cycle.
An increased intracellular amount of syntaxin 4 was found in HCV-positive cells, while the level of syntaxin 4-specific transcripts was decreased as observed in HCV-positive Huh7.5 cells and in HCV-infected primary human hepatocytes (PHH). Since in HCV-positive cells a significant longer half-life of syntaxin 4 was found, the decreased expression is overcompensated, leading to the elevated amount of syntaxin 4. Overexpression of syntaxin 4 increases the amount of secreted infectious viral particles, while silencing of syntaxin 4 expression decreases the number of released viral particles, which indicates that HCV could use the SNARE-dependent secretory pathway for viral release. Confocal immunofluorescence microscopy and co-immunoprecipitation experiments revealed that syntaxin 4 interacts with HCV core and NS5A. To identify the binding domain, various mutants of syntaxin 4 were generated. Based on these mutants, it was found that the H3 domain of syntaxin 4 interacts with core. These data show that the t-SNARE protein syntaxin 4 is an essential cellular factor for HCV morphogenesis and secretion.
HCV induces autophagy, and in HCV-infected cells a major fraction of the de novo synthesized viral particles is not released but intracellularly degraded. Syntaxin 17 is an autophagosomal SNARE required for the fusion of autophagosomes with lysosomes to form autolysosomes and thereby to deliver the enclosed contents for degradation. Therefore, we aim to investigate whether syntaxin 17 is a relevant factor for the HCV life cycle by regulating the fusion between autophagosomes and lysosomes. It was found that HCV-positive cells possess a decreased amount of syntaxin 17, and HCV reduces the intracellular level of syntaxin 17 by NS5A-mediated interruption of c-Raf signaling, which triggers the syntaxin 17 transcription, and by HCV-dependently induced autophagy. Overexpression of syntaxin 17 decreases the intracellular amount of viral particles and reduces the number of released infectious viral particles by favoring the formation of autolysosomes, in which HCV particles can be degraded. Vice versa, inhibition of syntaxin 17 expression by specific siRNAs results in an elevated amount of intracellular viral particles and increases the number of released viral particles by impaired autophagosome-lysosome fusion. Confocal immunofluorescence microscopy analyses show a fraction of core protein in autophagosomes as stained by lysotracker and the autophagy maker p62. These data identify syntaxin 17 as a novel factor controlling the release of HCV and reveal the autophagosome-autolysosome fusion as an essential step affecting the equilibrium between the release of infectious viral particles and lysosomal degradation of intracellular viral particles.
Taken together, these data identify the t-SNARE proteins syntaxin 4 and syntaxin 17 as essential cellular factors for HCV morphogenesis and secretion.
The condensation phase transition and the number of solutions in random graph and hypergraph models
(2016)
This PhD thesis deals with two different types of questions on random graph and random hypergraph structures.
One part is about the proof of the existence and the determination of the location of the condensation phase transition. This transition will be investigated for large values of $k$ in the problem of $k$-colouring random graphs and in the problem of 2-colouring random $k$-uniform hypergraphs, where in the latter case we investigate a more general model with finite inverse temperature.
The other part deals with establishing the limiting distribution of the number of solutions in these structures in density regimes below the condensation threshold.
In view of the diverse functionalities of RNA, the search for tools suitable for regulating and understanding RNA grows continuously. Dysfunction of RNA controlled processes can lead to diseases, calling for external regulation mechanisms – a difficult task in view of the complexity of biological systems. One of the recently developed methods that aim to systematically control RNA relates to photoregulation. Here, the RNA functions are triggered by photochromic molecules – for example, azobenzene or spiropyran – which are bound either covalently or non-covalently to the target RNA. This is a flexible approach, which can be improved by using suitably substituted chromophores. However, many issues regarding the details of photocontrol are still open. A detailed understanding of the mechanism of photocontrol is therefore of crucial importance.
The present thesis explores theoretical approaches to the photocontrol of RNA, focussing upon azobenzene chromophores covalently bound to RNA. The aim of the thesis is to characterize, at a molecular level, the effect of trans-to-cis isomerization of the azobenzene chromophore on RNA, and thus understand the mechanism of RNA unfolding triggered by azobenzene isomerization. In particular, we attempt to answer the following questions:
How does azobenzene isomerization happen in an RNA environment, i.e., how is
the isomerization influenced by the local RNA environment?
Conversely, how is RNA dynamics, on a longer time scale, affected by azobenzene attachment and photoisomerization?
Further, can regulation be enhanced by substituted azobenzenes? And, does simulation yield a picture that is consistent with experiment?
Due to the very different times scales of azobenzene isomerization (femtoseconds to picoseconds) and the much slower RNA response (nanoseconds to milliseconds), complementary techniques have been chosen: (i) hybrid quantum-classical approaches, i.e., on-the-fly Quantum Mechanics/Molecular Mechanics (QM/MM), to characterize the isomerization and RNA response on an ultrafast time scale, and (ii) molecular dynamics with enhanced sampling techniques, in particular, Replica Exchange MD (REMD), to explore longer time scales where the effect of RNA unfolding becomes manifest. Furthermore, substituent effects on azobenzene were separately investigated, in collaboration with two experimental groups.
The first part of this thesis is focused on the conformational influence of azobenzene on a small RNA hairpin on longer time scales using REMD simulations. In accordance with experiment, it is found that both the trans and cis form of azobenzene destabilize the RNA system. Trans azobenzene stays stacked in the double strand, whereas the cis form flips out of the RNA. These stacking interactions are the main reason why a trans azobenzene-RNA-complex is more stable than a cis-azobenzene-RNA-complex. Furthermore, the loop region of the RNA hairpin is highly destabilized by the intercalation of azobenzene.
In the second part, on-the-fly QM/MM simulations of the same azobenzene substituted hairpin are undertaken. These simulations use a surface hopping (SH) algorithm in conjunction with hybrid QM/MM electronic structure calculations to give a complete picture of the isomerization process on a picosecond time scale. It is shown that, due to the constraints of the RNA environment, the isomerization time of the azobenzene chromophore is significantly increased (from 300 femtoseconds in the gas phase to around 20 picoseconds in the RNA environment), and the isomerization yield is low. To the best of our knowledge, these are the first QM/MM simulations reported for azobenzene in a nucleic acid environment.
In the third and final part of this thesis, the properties of substituted azobenzenes have been explored, in collaboration with two experimental groups at the department. In particular, para- and meta-hydroxy substituted azobenzenes were suggested as improved photoswitches for the photoregulation of RNA, but spectroscopic investigations showed that isomerization was inefficient in some of the investigated species. Therefore, we investigated the photoisomerisation pathway of the keto/enol-form of para- and meta-hydroxy-azobenzenes by Time-Dependent Density Functional Theory (TDDFT) calculations. These calculations show that the competing keto/enol-tautomerism can result in an unstable cis form, making these substituted chromophores unsuitable as photoswitches.
Overall, the present thesis has contributed to obtaining a molecular-level understanding of photocontrol in azobenzene substituted RNAs, showing that theory and simulations can provide useful guidance for new experiments.
The elliptic flow of heavy-flavour decay electrons is measured at midrapidity |eta| < 0.8 in three centrality classes (0-10%, 10-20% and 20-40%) of Pb-Pb collisions at sqrt(sNN) = 2.76TeV with ALICE at LHC. The collective motion of the particles inside the medium which is created in the heavy-ion collisions can be analyzed by a Fourier decomposition of the azimuthal anisotropic particle distribution with respect to the event plane. Elliptic flow is the component of the collective motion characterized by the second harmonic moment of this decomposition. It is a direct consequence of the initial geometry of the collision which is translated to a particle number anisotropy due to the strong interactions inside the medium. The amount of elliptic flow of low-momentum heavy quarks is related to their thermalization with the medium, while high-momentum heavy quarks provide a way to assess the path-length dependence of the energy loss induced by the interaction with the medium.
The heavy-quark elliptic flow is measured using a three-step procedure.
First the v2 coefficient of the inclusive electrons is measured using the event-plane and scalar-product methods. The electron background from light flavours and direct photons is then simulated, calculating the decay kinematics of the electron sources which are initialised by their respective measured spectra. The final result of this work emerges by subtracting the background from the inclusive measurement. A significant elliptic flow is observed after this subtraction. Its value is decreasing from low to intermediate pT and from semi-central to central collisions.
The results are described by model calculations with significant elastic interactions of the heavy quarks with the expanding strongly-interacting medium.
The Earth’s surface condition we find today is a result of long exposure to metabolism of life forms. Particularly, molecular oxygen in the atmosphere is a feature which developed over time. The first substantial and lasting rise of atmospheric oxygen level happened ≈ 2.5 Ga ago, but localities are reported where transiently elevated oxygen levels appeared before this time-point. To trace the timing and circumstances of the earliest availability of free oxygen in the atmosphere is important to understand the habitats of early microbial life forms on Earth.
This thesis focuses to obtain information of oxygen levels and the related atmospheric cycling of metals in sediments of the 3.5 to 3.2 Ga Barberton Greenstone Belt. First, as iron was a ubiquitous constituent of Archean seawater, I investigated its isotopic composition in minerals of chemical sediments. Hereby, I tried to resolve the changes within the water basin on small scale sedimentary sequence cycles. Second, I focused on the minor constituents of Archean seawater. The Re-Os geochronologic system and the abundance patterns of the platinum-group elements were chosen to integrate information of oxygen promoted weathering of a large source area. To integrate information of a large time interval, the isotopes of uranium were investigated over a large stratigraphic section.
The two key findings of this thesis are:
• Quantitative oxidation of ferrous iron in surface layers of Paleoarchean seawater occurred during the onset and termination of hydrothermal FeIIaq delivery into shallow waters.
• Paleoarchean sedimentary successions of the Barberton Greenstone Belt lack any evidence of transient basin-scale oxygenation.
The Manzimnyama Iron Formation (IF, Fig Tree Group, Barberton Greenstone Belt, South Africa) has been deciphered to exist of cyclic stacks of lithostratigraphic units with varying amounts of iron oxide and carbonate minerals. In-situ femtosecond-Laser-Ablation ICP-MS iron isotope measurements showed that the majority of siderite (γ56Fe ≈ −0.5 ‰) precipitated directly from seawater of γ56Fe ≈ 0 ‰. Ferric iron from the surface layers is preserved in ≤ 1μ m hematite and in magnetite that has been grown within the consolidated sediment. During FeIIaq events, fine-grained hematite (γ56Fe ≈ 2.2 ‰) and magnetite (γ56Fe 0.5 to 0.8 ‰) indicate oxygen levels in surface waters of lower than 0.0002 μM. Upon onset and termination of iron oxide abundance, magnetite with γ56Fe ≈ 0 ‰ indicates that low concentrations of FeIIaq in surface waters were oxidized quantitatively. These observations demonstrate the existence of iron oxidation in Paleoarchean surface waters independent of FeIIaq concentration. This is the first investigation of Paleoarchean IF showing that lithostratigraphic cyclicity can be traced in iron isotopic composition of oxide minerals.
ID-ICP-MS measurement of Re, Ir, Ru, Pt and Pd, trace element (SF-ICP-MS) and ID-MCICP- MS uranium isotope determination have been applied to carbonaceous shale of the Mapepe Fm. (Fig Tree Group) after inverse Aqua Regia leaching and bulk digestion. The sediments reveal a silicified fraction which exhibits a seawater REE signature and a mixture of detrital and meteoritic PGE. Neither enrichment of the redox-sensitive elements Re or Mo nor fractionated uranium isotopes have been found on a stratigraphic interval of several hundred meters. The non-silica fraction shows no depletion of Re which indicates that the detrital material had no contact to oxidizing fluids. ID-TIMS measurements of Re and Os after the CrO3-SO4 Carius Tube method of two sample intervals showed that the Re-Os isotopic systems of the non-silica fractions are identical to two komatiite occurrences. Weltevreden Fm. and Komati Fm. rocks were uplifted, eroded and transported to the deep part of the sedimentary basin without any change to the Re-Os system. Negative fractionated uranium isotopes (γ238U = −0.41 ± 0.01 ‰) associated with detrital Ba-Cr-U occurrences suggest the existence of distal redox-processes that involve uranium species. This study demonstrates that over the time of exposure and deposition of the Mapepe Fm. sedimentation, free oxygen was not available for weathering in the catchment area.
Clouds and precipitation are essential climate variables. Because of their high spatial and temporal variability, their observation and modeling is difficult. In this thesis multiple observational data sources, including satellite data and station data are globally analyzed to understand the distribution and variability of clouds and precipitation, while a special focus is on the diurnal cycle of both variables. Substantial diurnal cycles of clouds and precipitation are observed in the tropics, with different properties over land and ocean. But also in Europe cloud diurnal cycles are observed in the summer season. Overall the maximum cloud cover and also the maximum precipitation is observed in the afternoon over land, and in the morning over ocean. The analyzed climate model simulations and the model-based reanalysis fail to simulate the observed diurnal cycles. Owing to their limited resolution, models can not fully resolve the processes responsible for the existence of diurnal cycles of clouds and precipitation.
Identification of disease modulating compounds in juvenile neuronal ceroid lipofuscinosis (JNCL)
(2016)
Mutationen im CLN3 Gen verursachen die neurodegenerative Erkrankung juvenile neuronale Zeroidlipofuszinose (JNCL). Bei dieser Erkrankung sind die Autophagie, der lysosomale pH Wert und der mitochondriale Metabolismus beeinträchtigt. Störungen dieser Prozesse führen zu einer erhöhten Verletzlichkeit neuronaler Zellen gegenüber alters- und umweltbedingten Schäden, einer Anhäufung von Autophagosomen und lysosomalem Speichermaterial, Zelltod und Neurodegeneration. Um die JNCL zu erforschen bedienen wir uns eines Zellmodels aus der Maus, welches die häufigste krankheitsauslösende CLN3 Mutation im Menschen, die Deletion der Exons 7 und 8, nachbildet. Die aus dem Kleinhirn dieser Mäuse stammenden cerebellaren Körnerstammzellen werden als CbCln3Δex7/8/Δex7/8 Zellen, solche aus wild-typ Mäusen als CbCln3+/+ Zellen bezeichnet. Die JNCL ist nicht heilbar und die Entwicklung von Wirkstoffen steht noch am Anfang.
Die vorliegende Arbeit befasst sich mit der Durchführung eines Hochdursatzscreenings um Wirkstoffe zu identifizieren, welche eine Anhäufung von Autophagosomen in CbCln3Δex7/8/Δex7/8 Zellen verhindern können. Unter 1750 verschiedenen untersuchten Wirkstoffen konnten wir 28 aktive „Hits“ identifizieren und stellten fest, dass Kalziumkanalblocker, Östrogene und HMG-CoA-Reduktase Inhibitoren gehäuft vertreten waren. Eine sorgfältige Untersuchung die möglichen Interaktionen der aktiven Wirkstoffe mit zellulären Signalwegen und die Analyse ihrer Dosis-Wirkungskurven unterstützte uns bei der Auswahl von Verapamil, Nicardipin und Fluspirilen zur näheren Untersuchung. Diese Wirkstoffe sind Kalziumkanalblocker und Fluspirilen blockt auch D2 Dopaminrezeptoren.
Außerdem untersuchten und quantifizierten wir mitochondriale Phänotypen in CbCln3Δex7/8/Δex7/8 Zellen. Unsere Untersuchungen ergaben, dass Mitochondrien in CbCln3Δex7/8/Δex7/8 Zellen einer signifikanten Hyperfusion unterliegen und ein schwächeres Membranpotenzial aufweisen. Weiterhin fanden wir eine Verringerung der maximalen der mitochondrialen Elektronentransportkapazität und eine verringerte Aktivität des Enzyms Zitratsynthase, welches die Effizienz des Zitratzyklus bestimmt.
Fluspirilen, Verapamil und, in geringerem Ausmaß, Nicardipin, verbesserten einige krankheitsbedingte lysosomale und mitochondriale Phänotypen. Des Weiteren konnten Verapamil und Nicardipin, nicht aber Fluspirilen, den erhöhten zellulären Kalziumspiegel in CbCln3Δex7/8/Δex7/8 Zellen absenken. Erniedrigungen im Kalziumgehalt können durch die Inhibition der kalziumabhängigen Protease Calpain 1 zu einer Induktion der Autophagie führen. Wir untersuchten, ob eine chemische Inhibition der Calpain 1-Protease die Anzahl der Autophagosomen in CbCln3Δex7/8/Δex7/8 Zellen senkt, und stellten fest, dass dies nicht der Fall ist. Eine Inhibition von Calpain 1 führte lediglich zu einem Anstieg der Zahl zellulärer Autophagosomen. Als Nächstes untersuchten wir die Auswirkung der Wirkstoffbehandlung auf den Autophagiefluss. Verapamil und Nicardipin hatten keinen Einfluss auf den Autophagiefluss in der getesteten Konzentration in CbCln3Δex7/8/Δex7/8 Zellen während Fluspirilen die Autophagie induzierte. Gleichzeitig stellten wir fest, dass hohe Dosen von Nicardipin und Verapamil teilweise vor einem Verlust des lysosomalen pH-Werts durch eine Behandlung mit Bafilomycin A1 schützen konnten. Da Fluspirilen auch ein Dopaminrezeptorblocker ist, untersuchten wir die Auswirkung einer erhöhten Dosis von Dopamin auf die Zahl der Autophagosomen. Wir fanden, dass eine mittlere Dosierung von Dopamin einen Trend zu einer leichten Verringerung von Autophagosomen in CbCln3Δex7/8/Δex7/8 Zellen zur Folge hat.
Wir vermuten, dass die Kalziumkanalblocker Verapamil und Nicardipin und der Dopaminrezeptorblocker Fluspirilen unterschiedliche zelluläre Signalwege benutzen, aber letztendlich um ähnliche Botenstoffe verwenden, um die Funktion der Lysosomen in CbCln3Δex7/8/Δex7/8 Zellen zu verbessern. Die Verringerung des intrazellulären Kalziumgehalts durch Verapamil und Nicardipin führt zu einer Aktivierung von Adenylatzyklasen, welche eine Erhöhung des intrazellulären cAMP Spiegels herbeiführen. Fluspirilen inhibiert Dopaminrezeptoren vom Typ D2 (D2DR), was zu einer selektiven Aktivierung von Dopaminrezeptoren des Typs D5 (D5DR) führen könnte. Im Gegensatz zu D2 führen D5D Rezeptoren zu einer Aktivierung von Adenylatzyklasen und einer Erhöhung des cAMP Spiegels. cAMP aktiviert die Protein Kinase A (PKA), welche durch eine Proteinphosphorylierung von lysosomalen Chloridkanälen und Protonenpumpen die lysosomale Aktivität erhöht. Dies führt zu einer Verbesserung des Abbaus von Autophagosomen und lysosomalem Speichermaterial und zu einer verbesserten Zellgesundheit in CbCln3Δex7/8/Δex7/8 Zellen.
Eine Verbesserung der lysosomalen Funktion in der JNCL kann einen wirksamen Therapieansatz ergeben. Wir hoffen, dass die hier vorgestellten Methoden und Ergebnisse einen ersten Schritt in diese Richtung darstellen.
The baker’s yeast Saccharomyces cerevisiae is a valuable and increasingly important microorganism for industrial applications (Hong and Nielsen, 2012). Its robustness concerning process conditions like low pH, osmotic and mechanical stress as well as toxic compounds is an advantage. Moreover, S. cerevisiae is ‘generally regarded as safe’ (GRAS). The model organism has been studied intensively. The collected data, including genomic, proteomic and metabolic information, can be used to genetically modify and improve its metabolism. Fatty acids and fatty acid derivatives have wide applications as biofuels, biomaterials, and other biochemicals. Several studies have been dealing with the overproduction of fatty acids and derivatives thereof in S. cerevisiae. The fatty acid biosynthesis starting with acetyl-CoA requires two enzymes, the acetyl-CoA carboxylase (Acc1p) and the fatty acid synthase complex (FAS), to produce acyl-CoA esters with predominantly 16 to 18 carbon atoms chain length (Lynen et al., 1980). For the synthesis of monounsaturated fatty acids in S. cerevisiae the ER bound acyl-CoA desaturase, Ole1p is essential (Tamura et al., 1976; Certik and Shimizu, 1999).
Using S. cerevisiae, the first section of this work dealt with the heterologous characterization of potential ω1-desaturases. Due to the fact that unsaturated fatty compounds can be modified further by hydrosilylations, hydrovinylations, oxidations to epoxides, acids, aldehydes, ketones or metathesis reactions, the interest in ω1-fatty acids is tremendous (Behr and Gomes, 2010). With the intention to find enzymes in fungi, that have a terminal desaturase activity a search in different genome databases was performed. The sequences of Pex-Desat3 and Obr-TerDes were used as reference sequences. The analysed proteins from Schizophyllum commune (EFI94599.1), Schizosaccharomyces octosporus (EPX72095.1), Wallemia mellicola (EIM20316.1), Wallemia ichthyophaga (EOR00207.1) and Agaricus bisporus var. bisporus (EKV44635.1), however, finally turned out to be Δ9 desaturases. A fungal desaturase with ω1-activity could not be found. The Δ9 desaturase SCD1 from Mus musculus was crystallized by Bai et al. (2015) and the information for specific amino acids responsible for the substrate specificity or enzyme activity were allocated. In combination with sequence and enzyme activity data form ChDes1 from Calanus hyperboreus, Desat2 from Drosophila melanogaster, Pex-Desat3 from Planotortrix excessana and Obr-TerDes from Operophtera brumata single amino acid exchanges were performed in the Δ9 desaturase Ole1p from S. cerevisiae. For all mutants, only fatty acids (C16 - C18) with a double bond between carbon C9 and C10 could be found. This indicates, that all inserted amino acid exchanges do not affect the substrate specificity or the position of the introduced double bond.
In the second section the focus was in the development of a production system for fatty acids in S. cerevisiae with regard to the previously established procedures by metabolic engineering. The combination of cytosolic malate dehydrogenase (MDH3), cytosolic malate enzyme (MAE1) and a citrate- α-ketoglutarate- carrier (YHM2) should improve the availability of acetyl-CoA in the cytosol, which is an important precursor for the fatty acid biosynthesis. If the major pathway (acetyl-CoA carboxylase and fatty acid synthase) was already optimized by high expression levels than no positive effect on increased fatty acid synthesis was detectable. Only non-optimized strains, with the additional overexpression of ATP-citrate lyase and cytosolic malate dehydrogenase, lead to a 41 % (20 mg/g dcw) improvement of fatty acid synthesis. In order to increase the fatty acid content further, the additional overexpression of DGA1 and TGL3 was performed. Hence, the highest amount of fatty acids could be observed with the strain S. cerevisiae WRY1ΔFAA1ΔFAA4 (2.5 g/L ± 0.8 g/L). The additional elimination of acyl-CoA synthetase Fat1p did not improve the yield.
It was recently reported, that chain length control of the fatty acid synthesis of bacterial FAS can be changed by rational engineering (Gajewski et al., 2017a). The knowledge about bacterial FAS was transferred in this work to S. cerevisiae FAS. Mutating up to five amino acids in the FAS complex enabled S. cerevisiae to produce medium chain fatty acids (C6 - C12). Further improvement was done by metabolic pathway engineering (promoter of alcohol dehydrogenase II from S. cerevisiae (pADH2), deletion of acyl-CoA synthetase FAA2) and optimization of fermentation conditions (YEPD-bacto medium buffered with potassium phosphate). The production of medium chain fatty acids resulted in the highest yield of 464 mg/L (C6 to C12 fatty acids). Furthermore, strains were created specifically overproducing hexanoic acid (158 mg/L) and octanoic acid (301 mg/L). The characterization of transferases, which could be responsible for the de-esterification of CoA-bound fatty acids, was analysed in an additional approach. It could be shown, that the genes EHT1, EEB1 and MGL2 have an influence on the MCFA yield in the supernatant. Generally speaking, the data from the single and double deletion strains suggest that Eeb1p has a selective hydrolytic activity for hexanoic acid-CoA ester, while Eht1p shows selective hydrolytic activity for octanoic acid-CoA ester, which is in line with Saerens et al. (2006).
Ziel der Arbeit war es, die Flugzeitmassenspektrometrie als neue Analysemethode für die instrumentelle Analytik halogenierter Spurengase in der Luft zu etablieren. Die grundle-gende Motivation dafür ist, dass anthropogene Emissionen vieler Vertreter dieser Sub-stanzklasse einen negativen Einfluss auf die Umwelt zeigen: in der Atmosphäre agieren die Substanzen bzw. ihre Abbauprodukte als Katalysatoren für den stratosphärischen Ozonab-bau und verstärken den Strahlungsantrieb der Erde durch Absorption elektromagnetischer Strahlung im sogenannten atmosphärischen Fenster. Um diese Effekte und deren Auswir-kung quantifizieren zu können, ist es notwendig, Konzentrationen und Trends der Substan-zen in der Atmosphäre zu überwachen. Nur so können Gegenmaßnahmen wie Produktions-reglementierungen geplant und bewertet werden. In Kombination mit inverser Modellie-rung können zudem Rückschlüsse über tatsächlich emittierten Mengen gezogen werden. Dies stellt den Anspruch an die Analytik, sehr geringe Mengen dieser Gase sehr präzise quantifizieren zu können, um auch schwache Trends zu erkennen. Zudem muss die Analy-semethode die Möglichkeit zu bieten, mit der wachsenden Anzahl bekannter und zu über-wachender Substanzen Schritt zu halten. Besonders für letzteren Aspekt bietet die Flug-zeitmassenspektrometrie einen entscheidenden Vorteil gegenüber der „konventionellen“ Methode, der Quadrupolmassenspektrometrie: sie zeichnet das gesamten Massenspektrum auf ohne dadurch an Empfindlichkeit einzubüßen. Um das atmosphärische Mischungsver-hältnis von Substanzen im Bereich von pmol mol−1 bis fmol mol−1 bestimmen zu können, muss das Quadrupolmassenspektrometer im Single Ion Monitoring Modus betrieben wer-den – so wird zwar eine hohe Sensitivität erreicht, es wird aber auch nur die Intensität eines bestimmten Masse zu Ladungsverhältnisses (kurz: Masse) zu einem Zeitpunkt aufgezeich-net. Ein Flugzeitmassenspektrometer hingegen extrahiert Ionen mit einer Frequenz im Ki-loherzbereich und zeichnet für jede Extraktion das vollständige Flugzeitspektrum und da-mit Massenspektrum auf.
Aufgabe dieser Arbeit war es, ein Flugzeitmassenspektrometer mit vorgeschalteter Pro-benanreicherungseinheit sowie Gaschromatograph zur Trennung des Subtanzgemisches vor der Detektion aufzubauen und Werkzeuge zur Datenauswertung zu entwickeln. Um einen zukünftigen Feldeinsatz vorzubereiten, sollte der Aufbau möglichst kompakt, mobil und vollständig automatisiert sein. Anschließend sollte Empfindlichkeit, Präzision und dynami-scher Messbereich geprüft, optimiert und die Anwendbarkeit zur Analyse halogenierter Spurengase gezeigt werden. Die Ergebnisse aus der in der vorliegenden Arbeit präsentier-ten Geräteentwicklung finden sich in drei Publikationen wieder, welche in thematischer Reihenfolge die Probenanreicherung (Obersteiner et al., 2016b), den Vergleich von Quadrupol- und Flugzeitmassenspektrometrie (Hoker et al., 2015) sowie Eigenschaften und Anwendung des neuen Aufbaus (Obersteiner et al., 2016a) behandeln. Mit den genannten Aufsätzen ist die Arbeitsgruppe Engel weltweit die erste, welche hochpräzise Analytik ha-logenierter Spurengase routinemäßig mittels Flugzeitmassenspektrometrie durchführt. Der nächste Schritt ist der Übergang von der Laboranwendung zur Feldmessung, z.B. in Form von bodenbasierter in situ Analyse troposphärischer Luftmassen am Taunus Observatorium auf dem Kleinen Feldberg. Da es bisher keine Messstation für die hier beschriebene analy-tische Fragestellung in Deutschland gibt, könnte eine deutliche Verbesserung der Überwa-chung halogenierter Treibhausgase und ozonzerstörender Substanzen in Europa erzielt wer-den. Weiterhin wäre eine Flugzeugapplikation in Zukunft denkbar, welche neben der durch das Flugzeitmassenspektrometer abgedeckten Substanzbandbreite auch von dessen hoher möglicher Spektrenrate profitieren könnte. In Kombination mit Hochgeschwindigkeitsgas-chromatographie könnte eine bisher unerreichte Zeitauflösung der Beprobung der Atmo-sphäre mittels Gaschromatographie-Massenspektrometrie erzielt werden.
For the transport of high-intensity hadron beams in low-energy beam lines of linear accelerators, the compensation of space charge forces by the accumulation of particles of opposite charge is an important effect, reducing the required focusing strength and potentially the emittance growth due to space charge forces. In this thesis, space charge compensation was studied by including the secondary particles in particle-in-cell simulations.
For this purpose, a new electrostatic particle-in-cell code named bender was developed. The software was tested using known self-consistent solutions for an electron plasma confined in an external potential as well as for a KV distributed beam in a periodic focusing lattice. For the simulation of compensation, models for residual gas ionisation by proton and electron impact were implemented.
The compensation process was studied for a 120 keV, 100 mA proton beam transported through a short drift section. Various features in the particle distributions were identified, which can not explained by a uniform reduction in the electric field of the beam. These were tied to the presence of thermal electrons confined within the beam potential. Using the Poisson-Boltzmann equation, their distribution could be reproduced and their influence on the beam for a wider range of parameters studied. However, the observed temperatures show a significant numerical influence. The hypothesis was formed, that stochastical heating present in particle-in-cell simulations is the mechanism leading to the formation of the observed (partial) thermal equilibrium.
For the low-energy beam transport line of the Frankfurt neutron source FRANZ, bender was used to predict the pulse shaping in the novel ExB chopper system. The code was also used for the design and the study of an electron lens for the Integrable Optics Test Accelerator at Fermi National Accelerator Laboratory. Aberrations due to guiding center drifts and the strong electric field of the electron beam as well as the current limits in such a system were investigated.
This thesis primarily covers a systematic assessment of quantum chemical methods to predict accurate 19F NMR shifts for fluoroarenes and magnetic exchange coupling constant (J) in organic spin dimers which are basic building blocks for rational designing of organic magnetic materials.
One of the most important goals in chemistry is to design and synthesize molecules with optimum properties. This thesis is divided into two parts: the first part comprises of a systematic effort to find an inexpensive quantum chemical method to predict accurate 19F NMR chemical shifts (within an accuracy of 2 ppm) for perfluoraromatics. Essentially, these strenuous efforts have been devoted to find best DFT functional and basis set combination to predict accurate 19F shifts. In addition,the influence of geometrical parameters, solvents, chemical environment was also analyzed. Various correction approaches were tested to correct the calculated shifts. The influence of various functionals and basis sets was also analyzed on the correction efficiency of an individual scheme. All the NMR calculation methods already being used and correction approaches were verified to predict shifts of three different fluorine-substituted molecular sets. These structure sets include fluorobenzenes, substituted benzenes and fluorine substituted aromatic fused rings (e.g. fluorine substituted anthracene).
In the second part of this thesis, we investigated the accurate prediction of magnetic exchange couplings (J) for organic spin dimers using quantum chemical methods. We analyzed the performance of various DFT methods and various post-HF methods, such as the CASSCF, CASPT2, MSTDISD, DDCI1, DDCI2, DDCI3, and FCI to predict magnetic exchange couplings (J).
Overview of the Chapters:
Chapter 1, presents a brief theoretical introduction to the Schrödinger equation and its application in quantum mechanical calculations, the Hartree-Fock approximation, basis sets, electron correlation energy, and density functional theory (using pure and hybrid functionals).
In chapters 2 and 3, an introduction is given for quantum chemical approaches used to calculate NMR parameters and magnetic exchange coupling constants. We discuss an effective spin Hamiltonian, the Breit-Pauli Hamiltonian (BPH), chemical shielding tensor and total energy relationship, measuring of the NMR spectra, and different techniques to deal with gauge origin problem. In addition, the theoretical background of magnetic exchange coupling constant calculation for spin dimers, the Heisenberg-Dirac-van-Vleck Hamiltonian (HDVV) and the Noodelman's broken-symmetry approach for calculating J values are briefly discussed.
Chapter 4, presents a benchmark study of various DFT functionals and basis sets to calculate accurate C-F bond lengths and 19F chemical shifts. High-resolution NMR spectral data of complex molecules are often difficult to interpret. Great scientific efforts have been devoted to search for a computational approach to interpret experimental NMR data. Quantum chemical methods such as the CCSD(T) method offer high accuracy in calculation of NMR parameters but being computationally too demanding they cannot be applied to large chemical systems. On the other hand, density functional theory (DFT) is achieving a steady progress among diversity of computational techniques. An accuracy within 2 ppm deviation from the experimental values in 19F chemical shifts can be achieved if the NMR calculation is performed using accurate equilibrium geometries, GIAO is used to tackle gauge origin problem and electron correlation is properly treated by employing a high level of theory (e.g. CCSD (T)/cc-pVQZ). We found that the calculation of 19F shielding tensors with the density-functional theory does not provide any noticeable improvement over the HF method. Post-HF theory demands too much computational resources that makes them impossible to use for large systems [35] .
We found that a quantitative prediction of NMR shifts can be made as the errors introduced by theoretical methods are cancelled out while calculating shifts. Various benchmark studies in this thesis show that 19F chemical shifts calculated for perfluoraromatics with the M06-L, BHandH, BHandHLYP in combination with the 6-311+G (2d,p) basis set are within 4 ppm deviation from the experiments. Furthermore, we noted that NMR calculations on accurate
C-F (e.g. PBE/6-311G (d, p)) bond lengths does not show any improvement if the NMR calculation and optimization are performed at the same level of theory. A significant improvement can be achieved on calculated 19F NMR shifts, if some correction schemes are used.
In chapter 4 we discuss various correction schemes applied to correct the calculated 19F chemical shifts. A multi-standard approach (MSTD) was used to minimize the error that may occur due to the difference in the nature of the reference compound and test molecules [122]. We propose another approach to correct shielding constants which is the reference corrected approach. This approach makes a correction similar to the MSTD. We also tested a Linear Regression Correction Approach and we noted that this is the best approach amongst all. This is found to be less dependent on the theoretical method. We use conformation averaging corrections to correct the calculated shifts[126].
...
Development and implementation of novel optogenetic tools in the nematode Caenorhabditis elegans
(2016)
Optogenetics, though still only a decade old field, has revolutionized research in neurobiology. It comprises of methods that allow control of neural activity by light in a minimally-invasive, spatio-temporally precise and genetically targeted manner. The optogenetic actuators or the genetically encoded light sensitive elements mediate light driven manipulation of membrane potential, intracellular signalling, neuronal network activity and behaviour (Fenno et al. 2011; Dugué et al. 2012). These techniques have been particularly useful for dissecting neural circuits and behaviour in the transparent and genetically amenable nematode model system Caenorhabditis elegans (Husson et al. 2013; Fang-yen et al. 2015).
In fact, C. elegans was the first living organism in which microbial rhodopsin based optogenetic tools (Channelrhodopsin-2 or ChR2, and Halorhodopsin or NpHR) were successfully implemented and bimodal 'remote' control of behaviour was achieved (Nagel et al. 2005; Zhang et al. 2007). Since then it has been a prominent model for the development and application of novel optogenetic tools and techniques, especially in the nervous system which comprises of 302 neurons and is organised in a hierarchical organization. The environmental stimuli are sensed by the sensory neurons, leading to the processing of information by the downstream interneurons, that relay to motor neurons which in-turn synapse onto muscles that drive the movement-based responses.
The microbial rhodopsins like ChR2 and NpHR mediate light driven depolarization and hyperpolarization, respectively and thereby activate or inhibit neural activity. However, they do not allow local control of membrane potential as they are expressed all over the plasma membrane of the cell rather than being restricted to specific domains, for example synaptic sites. Moreover, they completely over-ride the intrinsic activity of the cell, completely bypassing the signal transduction processes inside the cell. Thus, in order to study intracellular signalling and to answer questions pertaining to the endogenous role of receptors and channels in an in-vivo context, the optogenetic tool-kit needs to be expanded.
This thesis aimed at developing and implementing novel optogenetic tools in C. elegans that allow for sub-cellular signalling control as well as endogenous receptor control. These are: two light activated guanylyl cyclases (bPGC and BeCyclOp) to modify cyclic guanosine monophosphate (cGMP) mediated signalling in the sensory neurons, as well as attempts towards rendering endogenous C. elegans receptors - glutamate receptor (GLR-3/-6), acetylcholine receptor (ACR-16), glutamate gated chloride channel (GLC-1) light switchable and to understand their biological function in-vivo.
Organisms respond to sensory cues by activation of a primary receptor followed by relay of information downstream to effector targets by secondary signalling molecules. cGMP is a widely used 2nd messenger in cellular signaling, acting via protein kinase G or cyclic nucleotide gated (CNG) channels. In sensory neurons, cGMP allows for signal modulation and amplification, before depolarization. Chemo-, thermo-, and oxygen-sensation in C. elegans involve sensory neurons that use cGMP as the main 2nd messenger. For example, ASJ is the pheromone sensing neuron regulating larval development, AWC is the chemosensory neuron responding to volatile odours and BAG senses oxygen and carbon dioxide in the environment. In these neurons, cGMP acts downstream of the GPCRs and functions by activating cationic TAX-2/-4 CNG channels, thereby depolarising the sensory neuron. Manipulating cGMP levels is required to access signalling between sensation and sensory neuron depolarization, thereby provide insights into signal encoding. We achieve this by implementing two photo-activatable guanylyl cyclases - 1) a mutated version of Beggiatoa sp. bacterial light-activated adenylyl cyclase, with specificity for GTP (Ryu et al. 2010), termed BlgC or bPGC (Beggiatoa photoactivated guanylyl cyclase) and 2) guanylyl cyclase rhodopsin (Avelar et al. 2014) from Blastocladiella emersonii (BeCyclOp).
bPGC is a BLUF (blue light sensing using flavin) domain containing cyclase which uses FAD as the co-factor and catalyses the synthesis of cGMP from GTP upon activation by blue light. Prior to implementation in sensory neurons, a simpler heterologous system with co-expression of the TAX-2/-4 CNG channel in C. elegans body wall muscle (BWM) was used. The cGMP generated by the light activated cyclases activates the CNG channel leading to the muscle depolarization, thereby causing changes in body length which can be easily scored.
NOSTRIN belongs to the family of F-BAR proteins, which are multi-domain adaptor proteins that have emerged as important regulators of membrane remodeling and actin dynamics in a variety of vital cellular processes. They have been analyzed structurally and biochemically and overexpression studies have revealed their potential in inducing membrane curvature and tubulation. Several studies have begun to decipher the function of individual proteins, but the understanding of F-BAR protein functions in vivo is still quite limited. The F-BAR protein NOSTRIN is mainly expressed in endothelial cells and has originally been described as interaction partner of the endothelial nitric oxide synthase (eNOS), modulating eNOS subcellular localization. The phenotypic characterization of NOSTRIN knockout mice revealed decreased nitric oxide (NO) and cGMP levels, an increase in systolic blood pressure and an impairment of the acetylcholine-induced, NO-dependent relaxation of aortic rings from mice with global as well as endothelial cell-specific knockout of the NOSTRIN gene (ECKO) . These findings implied that NOSTRIN plays a role in regulating NO production in vivo, but the underlying molecular mechanisms were unclear. Therefore, this study was aimed at addressing the mechanism causing the inhibited vasodilation specifically upon stimulation with acetylcholine in NOSTRIN KO and ECKO mice, and at exploring additional roles of NOSTRIN in the signal transduction of endothelial cells.
The major acetylcholine receptor that mediates vessel relaxation upon stimulation with acetylcholine is the muscarinic acetylcholine receptor subtype M3 (M3R). In the present study NOSTRIN was identified as novel interaction partner of the M3R and important factor for the correct spatial distribution and functionality of the M3R. Moreover, it provides the first example of an F-BAR protein regulating a GPCR. Confocal immunofluorescence microscopy analysis of isolated aortae from NOSTRIN KO and WT mice indicated that NOSTRIN was necessary for the proper subcellular localization of the M3R and targeted it to the plasma membrane. A series of pulldown experiments revealed a direct interaction of NOSTRIN with the M3R. The binding required the SH3 domain of NOSTRIN and the third intracellular loop of the M3R, which has a recognized role in receptor regulation. The interaction of NOSTRIN with the M3R was confirmed by co-localization of NOSTRIN and the M3R upon overexpression in mammalian cells. Expression levels of the M3R as well as eNOS were not affected by the loss of NOSTRIN in accordance with the finding, that NOSTRIN impacts on the acetylcholine/eNOS signaling axis through regulation of the subcellular trafficking of its binding partners.
Furthermore, there were first indications for a role of NOSTRIN in facilitating the carbachol-induced calcium response in M3R-expressing cells, suggesting that NOSTRIN might influence M3R activation. in the absence of NOSTRIN, the function of the M3R in mammalian cells overexpressing the M3R was markedly impaired, resulting in abolition of the calcium response to the M3R agonist carbachol. In accordance, the activated eNOS fraction associated with the Golgi complex was markedly reduced in aorta explants from NOSTRIN knockout and ECKO mice. Moreover, NOSTRIN knockout inhibited the carbachol-induced, activating phosphorylation of eNOS in murine aortae as well as primary mouse lung endothelial cells confirming its role as important regulator of eNOS activity in vivo.
This thesis addresses the reconstruction of the topographic evolution and the climate dynamics of the Early Cenozoic North American Cordillera through integrated geochronology, sedimentology, stable isotope, and clumped isotope thermometry studies. It encompasses the scientific disciplines of geochemistry, tectonics, and Earth surface processes.
Die Dissertation befasst sich mit den kognitiven Prozessen die Intelligenz ausmachen und ist in drei Teile aufgeteilt. Der erste Teil rezensiert den Beitrag kognitiver und nicht-kognitiver Variablen zur Vorhersage von Hochbegabung in einer Altersspanne die von der Geburt bis zur Einschulung reicht. Aus dem nicht-kognitiven Bereich stammen Konstrukte wie Schlafverhalten, motivationale Faktoren wie Neugier und Interesse, und deren Interaktion mit dem sozialen Umfeld. Kognitive Variablen stellen frühe, außergewöhnliche Sprach-, Lese-, Schreib- und Rechenfähigkeiten dar, sowie Intelligenzquotienten, die mit den gängigsten testpsychologischen Verfahren ermittelt werden, und Komponenten der Informationsverarbeitung wie Habituation und Arbeitsgedächtnis. Trotz der Berichte über mittlere Korrelation ist die aktuelle Datenlage kritisch zu betrachten und weist eine niedrige Vorhersagevalidität der Frühprognose von Hochbegabung auf. Es wird mit dem dynamischen Modell der Intelligenz argumentiert, nach dem die mangelnde prognostische Validität und die Unzuverlässigkeit der kognitiven Vorhersageindikatoren auf die Annahme zurückzuführen sind, dass kognitive Prozesse unabhängig voneinander sind. Erst im Laufe des Lebens werden diese immer häufiger miteinander verknüpft und korreliert (Mutualismus). Das bedeutet, es findet eine zunehmende Integration kognitiver Prozesse statt und daraus resultiert der g-Faktor.
Doch das Arbeitsgedächtnis spielt weiterhin eine zentrale Rolle in Bezug auf Intelligenz, wenn nicht im Säuglingsalter, dann zumindest ab dem Vorschulalter und besonders im Erwachsenenalter. Arbeitsgedächtniskapazität stellt das Ausmaß an Fähigkeit dar, Informationen simultan zu speichern und zu verarbeiten, ohne die Notwendigkeit auf Vorwissen zurückzugreifen. Es kann damit als eng umschriebener kognitiver Prozess aufgefasst werden. Vorherige Forschungsarbeiten haben bereits deutlich gezeigt, dass Arbeitsgedächtnis und Intelligenz korrelativ stark zusammenhängen. Das ist durchaus überraschend, denn die Aufgaben und Tests mit denen die Konstrukte jeweils erfasst werden können sich oberflächlich stark unterscheiden. Um fluide Intelligenz valide erfassen zu können, sind Aufgaben zum induktiven Denken, wie zum Beispiel Matrix-Aufgaben, sehr beliebt. Im Rahmen der zweiten Arbeit hat man sich dem Einfluss der Prozesse Zielmanagement und Regel-Induktion auf den Zusammenhang zwischen Arbeitsgedächtnis und Problemlösung der Matrix-Aufgaben gewidmet. Zielmanagement wurde bereits zuvor mit dem Arbeitsgedächtnis in Verbindung gebracht jedoch war die Befundlage hinsichtlich der Regel-Induktion unklar. Daher sollte die Hypothese ob Regel-Induktion unabhängig vom Arbeitsgedächtnis ist, in einem kritischen Experiment überprüft werden. Bei Neutralisierung der Notwendigkeit für Regelinduktion, indem den Probanden die Regeln im Voraus erklärt wurden, konnte man einen erhöhten Zusammenhang zwischen dem Arbeitsgedächtnis und der Leistung in der Matrix-Aufgabe erkennen. Das deutete in der Tat darauf hin, dass Regel-Induktion nicht vom Arbeitsgedächtnis abhängig ist, zumindest nicht im gleichen Maße wie Zielmanagement. Darüber hinaus zeigte sich: Die Kenntnis der Regeln beeinflusst den Problemlöseprozess. Eye-Tracking-Messungen weisen auf eine konstruktive Anpassungsstrategie in der Experimentalbedingung (mit Regelwissen) hin. Basierend auf den Erkenntnissen aus vier Experimenten, wird mit zwei möglichen Mechanismen argumentiert, die die Steigerung des Zusammenhangs zwischen Arbeitsgedächtnis und Matrix-Problemlösen in der Experimentalgruppe erklären könnten.
Alternativ zur zuvor angenommenen Unabhängigkeit von Regel-Induktion und Arbeitsgedächtnis, könnte die erhöhte Korrelation bei bekannten Regeln auch mit der beobachteten Strategieänderung erklärbar sein. Die dritte und letzte Arbeit widmete sich deshalb erneut dieser Fragestellung. Erneut kamen Matrix-Aufgaben zum Einsatz und es wurden Testleistung, Augenbewegung und Reaktionszeiten erhoben, um den Einfluss von Regelwissen zu erfassen. Es zeigte sich die Anwendung einer effektiveren Lösungsstrategie in der Experimentalbedingung. Anhand der Eye-Tracking Messung wurde gezeigt, dass Probanden mit Regelwissen über einen längeren Zeitraum das problemrelevante Areal der Matrix-Aufgabe fixieren, und eine niedrigere Frequenz an Sakkaden zwischen diesem Areal und den Antwortalternativen aufwiesen.
Weitere Einflussvariablen auf die Lösestrategie stellen Schwierigkeit der Aufgabe und Fähigkeiten des Probanden dar. Diese weisen einen differenziellen Einfluss auf zwei Subgruppen von Indikatoren der Augenbewegungsmessung auf, die in Relation zu den Reaktionszeiten gesetzt wurden um ein besseres Verständnis dieser Variablen zu erzielen. Es wird vermutet, dass Variablen wie Augenbewegungen und Reaktionszeiten das Ausmaß des Entstehens von mentalen Modellen während des logischen Denkens widerspiegeln. Unter der Annahme dass die Komplexität von Mentalen Modellen mit einer gewissen Belastung für das Arbeitsgedächtnis einhergeht, lassen sich auch vorherige Ergebnisse mit dieser Hypothese in Einklang bringen. Abschließend werden die grundlegenden kognitiven Prozesse des induktiven Denkens diskutiert und ein Ausblick auf zukünftige Intelligenzmessung angeboten.
Great interest has emerged recently in the search for Kitaev spin liquid states in real materials. Such states rely on strongly anisotropic magnetic interactions, which have been suggested to exist in a number of candidate materials based on Ir and Ru. This thesis concentrates on two priority purposes. The first is the investigation of electronic and magnetic properties of candidate materials Na2IrO3, α-Li2IrO3, α-RuCl3, γ-Li2IrO3, and Ba3YIr2O9 for Kitaev physics where both spin-orbit coupling and correlation effects are important. The second is the method development for the microscopic description of correlated materials combining many-body methods and density functional theory (DFT). ...
Die Populationsgenetik beschäftigt sich mit dem Einfluss von zufälliger Reproduktion, Rekombination, Migration, Mutation und Selektion auf die genetische Struktur einer Population.
In dieser Arbeit mit dem englischen Titel "Ancestral lines under mutation and selection" wird das Zusammenspiel von zufälliger Reproduktion, gerichteter Selektion und Zweiwegmutation untersucht.
Dazu betrachten wir eine haploide Population in der jedes Individuum zu jedem Zeitpunkt genau einen von zwei Typen aus S:={0,1} trägt. Dabei sei 1 der neutrale und 0 der selektiv bevorzugte Typ. Im Diffusionslimes sehr großer Populationen modellieren wir den Prozess der Frequenz der Typ-0-Individuen durch eine Wright-Fisher-Diffusion X:=(X_t) mit Mutation und gerichteter Selektion.
Zu jedem Zeitpunkt s gibt es genau ein Individuum, dessen Nachkommen ab einem bestimmten zukünftigen Zeitpunkt t>s die gesamte Population ausmachen werden. Wir nennen dieses Individuum den gemeinsamen Vorfahren zum Zeitpunkt s, da alle Individuen zu allen Zeitpunkten r>t von ihm abstammen. Sei R_{s} dessen Typ zum Zeitpunkt s. Wir nehmen an, dass der Prozess X zum Zeitpunkt 0 im Gleichgewicht ist und definieren die Wahrscheinlichkeit, dass der gemeinsame Vorfahre zum Zeitpunkt 0 Typ 0 hat, durch h(x):= P(R_{0}=0|X_{0}=x). Eine Darstellung von h(x) wurde bereits von Fearnhead (2002) und Taylor (2007) gefunden und dort mit vorwiegend analytischen Methoden bewiesen. In dieser Arbeit entwickeln wir in Kapitel 3 ein neues Teilchenbild, den pruned lookdown ancestral selection graph (pruned LD-ASG), der für sich selbst genommen interessant ist und eine neue probabilistische Interpretation der Darstellung von h(x) liefert.
Durch Erweiterung des Teilchenbildes auf Nachkommenverteilungen mit schweren Tails und mit Hilfe einer Siegmund Dualität gelingt es uns in Kapitel 4 das Resultat für h(x) von klassischen Wright-Fisher-Diffusionen auf Lambda-Wright-Fisher-Diffuison zu erweitern.
Eine Verbindung zwischen Ideen von Taylor (2007), der den gemeinsamen Prozess (X,R) untersucht hat, und einem von Fearnhead (2002) betrachteten Prozess (R,V), der die Entwicklung des Typs R des gemeinsamen Vorfahren in einer Umgebung von V sogenannten virtuellen Linien beschreibt, stellen wir in Kapitel 6 her. Wir bestimmen die gemeinsame Dynamik des Tripels (X,R,V). In Kapitel 7 betrachten wir ein diskretes Bild mit endlicher Populationsgröße N und schlagen dort eine Brücke zu Resultaten von Kluth, Hustedt und Baake (2013).
Des Weiteren entwickeln wir in Kapitel 5 dieser Arbeit einen Algorithmus zur Simulation der Typen einer Stichprobe von m Individuen, die aus einer Wright-Fisher-Population mit Mutation und Selektion im Gleichgewicht gezogen wird. Mittels dieses Algorithmus illustrieren wir die Typenverteilung für verschiedene Parameterwerte und Stichprobengrößen.