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The TTL is the transition layer between the tropical troposphere and stratosphere, and is the main region where tropospheric air enters the stratosphere. In this thesis different transport processes are studied by using in situ measurements of tracers. Long-lived tracers were measured with the High Altitude Gas Analyzer (HAGAR) on board the M55 Geophysica aircraft. The instrument was developed by the University of Frankfurt and measures the long-lived tracers CO2, N2O, CFC-12, CFC-11, H-1211, SF6, CH4 and H2 with two gas chromatographic channels and a CO2 sensor (LICOR). The measurements are supported by CO and O3 measurements of other instruments. Two campaigns were conducted to obtain measurements in the TTL: SCOUT-O3 (November/December 2005 in Darwin, Australia) and AMMA-SCOUT-O3 (August 2006 in Ouagadougou, Burkina Faso). After a general introduction of the thesis in chapters one and two, the third chapter describes the findings during this last campaign. Five local flights are analyzed to study the different transport processes that occur in the tropical tropopause layer above West-Africa: deep convection up to the level of main convective outflow, vertical mixing after overshooting of air in deep convection, horizontal inmixing from the extratropical lower stratosphere, and horizontal transport across the subtropical barrier. Main findings are that the TTL over West-Africa is mostly influenced by remote convection. The subtropical barrier is not a strong barrier but more a region of transition between the extratropical and the tropical stratosphere. Chapter 4 presents the results obtained during the SCOUT-O3 campaign. From the eight local flights the last four flights (051129, 051130a, 051130b, 051205) show enhanced values of ozone, CO and CO2 between 355 and 380 K potential temperature in comparison with the first four flights (051116, 051119, 051123, 051125). Horizontal inmixing from the extra-tropical stratosphere and influence of the local convective system Hector cannot explain the enhanced values of the two flights on 30 November Therefore, other possible explanations for these enhanced CO, CO2 and ozone levels are proposed. The first explanation is vertical mixing in the vicinity of the jet stream. However, the jet cannot explain the differences between the flights on 30 November and the flights on 29 November and 5 December. Another possible explanation is influence of polluted boundary layer air masses from the Indonesian region. Especially air sampled during the flights on November 30 crossed large parts of northern Indonesia between 8 and 10 days before the measurements. Convective uplift of biomass burning and other pollution plumes can transport CO and ozone precursors into the upper troposphere, where they can significantly enhance the ozone production. The last chapter deals with the vertical ascent rate in the TTL and uses measurements of both the SCOUT-O3 and AMMA-SCOUT-O3 campaign as well as data from previous aircraft campaigns (TROCCINOX and APE-THESEO). Time scales and residence times for mean vertical transport in the background TTL are estimated for different seasons and over different geographic regions using in situ observations of CO2 and long-lived tracers. The vertical transport time scales are constrained using the seasonal variation of CO2 in the tropical troposphere as a “tracer clock” for vertical ascent. Two methods are applied to calculate the residence time in the layer between 360 and 390 K potential temperature. The first method uses the slope of the CO2 index, the second method fits the CO2 index directly to the measurements assuming a constant ascent rate. The first method yields residence times for Australia,West Africa, and Brazil of the same order, 35-45 days to 380 K and 50 days to 390 K (where no value can be derived for Australia as the slope is changing approximately one month before the campaign). For APE-THESEO, the method does not yield reasonable results. The best estimates using the second method show moderate residence times between 360 and 390 K of 60±25 days SCOUT-O3 (NH autumn) and 43±8 days for AMMA/SCOUT-O3 (NH summer). These results agree well with the results calculated using the first method. For APE-THESEO and TROCCINOX the best fits yield shorter residence times of 23±7 and 40±10 days, respectively, both during winter. These results correspond well to the expectations based on the seasonal variation of the Brewer-Dobson circulation.
Although often depicted as rigid structures, proteins are highly dynamic systems, whose motions are essential to their functions. Despite this, it is difficult to investigate protein dynamics due to the rapid timescale at which they sample their conformational space, leading most NMR-determined structures to represent only an averaged snapshot of the dynamic picture. While NMR relaxation measurements can help to determine local dynamics, it is difficult to detect translational or concerted motion, and only recently have significant advances been made to make it possible to acquire a more holistic representation of the dynamics and structural landscapes of proteins. Here, we briefly revisit our most recent progress in the theory and use of exact nuclear Overhauser enhancements (eNOEs) for the calculation of structural ensembles that describe their conformational space. New developments are primarily targeted at increasing the number and improving the quality of extracted eNOE distance restraints, such that the multi-state structure calculation can be applied to proteins of higher molecular weights. We then review the implications of the exact NOE to the protein dynamics and function of cyclophilin A and the WW domain of Pin1, and finally discuss our current research and future directions.
Sommergrüne Laubwälder Südwest-Europas umfassen eine größere Breite von Vegetationstypen, abhängig von verschiedenen edaphischen und klimatischen Faktoren. Von diesen Wäldern werden hier nur mesophytische und submediterrane (subhumide) Eichen- und Eschenwälder besprochen; sowohl Buchenwälder als auch alle Gesellschaften azidotoleranter Wälder sind ausgeschlossen. Es können zwei Haupttypen unterschieden werden:
Die erste Gruppe wird gebildet von Quercus robur, Fraxinus excelsior, Corylus avellana, Acer pseudoplatanus und A. campestre; sie wächst auf basenreichen Böden (pH gewöhnlich zwischen 6 und 7) aus Kalken, Mergeln oder entsprechenden Sedimenten. Die Böden sind oft reich an Ton oder Schlick und besitzen eine reiche Wirbellosenfauna, insbesondere aus Regenwürmern. Die Wälder sind verbunden mit Waldmänteln der Prunetalia. Ihr Unterwuchs enthält zahlreiche Arten, z.B. Polystichum setiferum, Dryopteris borreri, Brachypodium sylvaticum, Mercurialis perennis, Carex sylvatica u.a. Sie sind in Kalkgebieten der Britischen Inseln, Westfrankreichs, der Pyrenäen und des Kantabrischen Gebirges weit verbreitet. Ihr Areal reicht von Schottland bis ins westliche Asturien (sie fehlen in Galizien und Nord-Portugal).
Die zweite Gruppe besteht vorwiegend aus Quercus pubescens (Q. humilis) und Q. faginea. Sie wächst ebenfalls auf kalkreichen Böden unter submediterranem Klimaeinfluss. Weitere Baumarten sind Abies pinsapo, Acer granatense, A. monspessulanus, A. opalus, Colutea arborescens, Fraxinus ornus, Sorbus torminalis und S. domestica. Die Strauchschicht ist infolge hohen Lichtgenusses und guter Böden artenreich und dicht; in ihr wachsen viele Arten der Prunetalia, vor allem Prunus spinosa, Rosa- und Rubus-Arten. Spezifisch sind weit verbreitete submediterrane Arten wie Amelanchier ovalis, Coronilla emerus, Lonicera etrusca, Prunus mahaleb, Viburnum lantana u.a. Die Krautschicht ist ebenfalls artenreich. Sie teilt viele Arten mit anderen Laubmischwäldern, hat aber auch einige charakteristische Eigenheiten wie Helleborus foetidus, Campanula persicifolia, Digitalis lutea, Melittis melissophyllum, Viola alba u.a. Solche Wälder sind in Südeuropa weit verbreitet, vom Balkan über Alpen und Apennin bis zur Iberischen Halbinsel. Im Südwestteil des Kontinents kommen sie von Süd-Frankreich bis Süd-Spanien (nicht in Portugal) vor. In Süd-Spanien sind sie auf Kalkgebirge beschränkt, wo die extremen Bedingungen des mediterranen Klimas etwas abgeschwächt sind.
This thesis describes the structural characterization of interactions between biological relevant ribonucleic acid biomacromolecules (RNAs) and selected ligands to optimize the methodologies for the design of pharmacological lead compounds. To achieve this aim, not only the structures of the RNA, the ligand and their complexes need to be known, but also information about the inherent dynamics, especially of the target RNA, are necessary. To determine the structure and dynamics of these molecules and their complexes, liquid state nuclear magnetic resonance spectroscopy (NMR) is a suitable and powerful method. The necessity for these investigations arises from the lack of knowledge in RNA-ligand interactions, e.g. for the development of new medicinal drugs targeting crucial RNA sequences. In the first chapters of this thesis (Chapters II to IV), an introduction into RNA research is given with a focus on RNA structural features (Chapter II), into the interacting molecules, the biology of the specific RNA targets and the further development of their ligands (Chapter III) and into the NMR theory and methodologies used within this thesis (Chapter IV). Chapter II begins with a description of RNA characteristics and functions, placing the focus on the increasing attention that these biomacromolecules have attracted in recent years due to their diverse biological functionalities. This is followed by a detailed description of general structural features of RNA molecules. The biological functions of the RNAs investigated in this thesis (Human immunodeficiency virus PSI- and TAR-RNA and Coxsackievirus B3 Stemloop D in the 5’-cloverleaf element), together with their known structural characteristics are introduced in Chapter III. Furthermore, a description of the investigated ligands is given, focusing on the methods how their affinity and specificity were determined. The introduction is completed in Chapter IV, where the relevant NMR theory and methodologies are explained. First, kinetics and thermodynamics of ligand binding are summarized from an NMR point of view. Subsequently, a detailed description of the resonance assignment procedures for RNAs and peptidic ligands is given. This procedure mainly concentrates on the assignment of the proton resonances, which are essential for the later structure calculation from NMR restraints. The procedure for NMR structure calculation of RNA and its complexes follows with a short introduction into the programs ARIA and HADDOCK. The final part of this chapter explains the relaxation theory and the methodology to extract dynamic information from autocorrelated relaxation rates via the model-free formalism. In the Chapters V to VII of this thesis, the original publications are included and grouped into three topics. Chapter V comprehends the publications on the investigations of HIV PSI-RNA and its hexapeptidic ligand. These three publications[1-3] focus on the characterization of the ligand and its binding properties, its structure and the optimization of its composition aiming to improve its usage for further spectroscopic investigations.
Motions on planetary spatial scales in the atmosphere are governed by the planetary geostrophic equations. However, little attention has been paid to the interaction between the baroclinic and barotropic flows within the planetary geostrophic scaling. This is the focus of the present study, which utilizes planetary geostrophic equations for a Boussinesq fluid supplemented by a novel evolution equation for the barotropic flow. The latter is affected by meridional momentum flux due to baroclinic flow and drag by the surface wind. The barotropic wind, on the other hand, affects the baroclinic flow through buoyancy advection. Via a relaxation towards a prescribed buoyancy profile the model produces realistic major features of the zonally symmetric wind and temperature fields. We show that there is considerable cancellation between the barotropic and the baroclinic surface zonal mean zonal winds. Linear and nonlinear model responses to steady diabatic zonally asymmetric forcing are investigated, and the arising stationary waves are interpreted in terms of analytical solutions. We also study the problem of baroclinic instability on the sphere within the present model.
The following thesis is concerned with the elucidation of structural changes of RNA molecules during the time course of dynamic processes that are commonly denoted as folding reactions. In contrast to the field of protein folding, the concept of RNA folding comprises not only folding reactions itself but also refolding- or conformational switching- and assembly processes (see chapter III). The method in this thesis to monitor these diverse processes is high resolution liquid-state NMR spectroscopy. To understand the reactions is of considerable interest, because most biological active RNA molecules function by changing their conformation. This can be either an intrinsic property of their respective sequence or may happen in response to a cellular signal such as small molecular ligand binding (like in the aptamer and riboswitch case), protein or metal binding. The first part of the thesis (chapters II & III) provides a general overview over the field of RNA structure and RNA folding. The two chapters aim at introducing the reader into the current status of research in the field. Chapters II is structured such that primary structure is first described then secondary and tertiary structure elements of RNA structure. A special emphasis is given to bistable RNA systems that are functionally important and represent models to understand fundamental questions of RNA conformational switching. RNA folding in vitro as well as in vivo situations is discussed in Chapter III. The following chapters IV and V also belong to the introduction part and review critically the NMR methods that were used to understand the nature and the dynamics of the conformational/structural transitions in RNA. A general overview of NMR methods quantifying dynamics of biomolecules is provided in chapter IV. A detailed discussion of solvent exchange rates and time-resolved NMR, as the two major techniques used, follows. In the final chapter V of the first part the NMR parameters used in structure calculation and structure calculation itself are conferred. The second part of the thesis, which is the cumulative part, encompasses the conducted original work. Chapter VI reviews the general NMR techniques applied and explains their applicability in the field of RNA structural and biochemical studies in several model cases. Chapter VII describes the achievement of a complete resonance assignment of an RNA model molecule (14mer cUUCGg tetral-loop RNA) and introduces a new technique to assign quaternary carbon resonances of the nucleobases. Furthermore, it reports on a conformational analysis of the sugar backbone in this RNA hairpin molecule in conjunction with a parameterization of 1J scalar couplings. Achievements: • Establishment of two new NMR pulse-sequences facilitating the assignment of quaternary carbons in RNA nucleobases • First complete (99.5%) NMR resonance assignment of an RNA molecule (14mer) including 1H, 13C, 15N, 31P resonances • Description of RNA backbone conformation by a complete set of NMR parameters • Description of the backbone conformational dependence in RNA of new NMR parameters (1J scalar couplings) Chapters VII & VIII summarize the real-NMR studies that were conducted to elucidate the conformational switching events of several RNA systems. Chapter VIII gives an overview on the experiments that were accomplished on three different bistable RNAs. These molecules where chosen to be good model systems for RNA refolding reactions and so consequently served as reporters of conformational switching events of RNA secondary structure elements. Achievements: • First kinetic studies of RNA refolding reactions with atomic resolution by NMR • Application of [new] RT-NMR techniques either regarding the photolytic initiation of the reaction or regarding the readout of the reaction • Discovery of different RNA refolding mechanisms for different RNA molecules Deciphering of a general rule for RNA refolding methodology to conformational switching processes of RNA tertiary structure elements. The models for these processes were a) the guanine-dependent riboswitch RNA and b) the minimal hammerhead ribozyme. Achievements: • NMR spectroscopic assignment of imino-resonances of the hypoxanthine bound guanine-dependent riboswitch RNA • Application of RT-NMR techniques to monitor the ligand induced conformational switch of the aptamer domain of the guanine-dependent riboswitch RNA at atomic resolution • Translation of kinetic information into structural information • Deciphering a folding mechanism for the guanine riboswitch aptamer domain • Application of RT-NMR techniques to monitor the reaction of the catalytically active mHHR RNA at atomic resolution In the appendices the new NMR pulse-sequences and the experimental parameters are described, which are not explicitly treated in the respective manuscripts.
„Naturnähe“ ist kein naturschutzfachliches Dogma, sondern ein anwendungsorientierter Maßstab für eine klare und differenzierte Ableitung von naturschutzfachlichen Zielen. Dazu werden verschiedene Maßstabsebenen (z. B. Waldgebiete, Waldbestände, Einzelbaumstrukturen) und verschiedene Bezugsgrößen der Naturnähe benötigt wie 1. Flora und Vegetation, 2. Strukturen und Dynamik, 3. Biotoptradition und Habitatkontinuität. Die Geobotanik liefert für die 1. Bezugsgröße die entscheidenden Grundlagen (Nullpunkt: heutige potenzielle natürliche Vegetation). Für die 2. und 3. Bezugsgröße liefern neben floristischen besonders faunistische Artengruppen maßgebliche Indikatoren („Urwaldreliktarten“, die an spezifische Urwaldstrukturen und -ressourcen gebunden sind und häufig hohe Totholzmengen benötigen). Somit bringt eine Naturnähebewertung des Waldes den Standort, die Pflanzen- und Tierwelt in einen Gesamtzusammenhang. In Bezug auf eine Naturnäheentwicklung gibt es unterschiedliche Ausgangsbedingungen, verschiedene Qualitätsziele und Prioritäten. In der Kulturlandschaft kann nicht immer, aber möglichst oft eine große Naturnähe im Wirtschaftswald das vorrangige naturschutzfachliche Ziel sein.
Cell-free expression represents an attractive method to produce large quantities of selectively labeled protein for NMR applications. Here, cell-free expression was used to label specific regions of the growth hormone secretagogue receptor (GHSR) with NMR-active isotopes. The GHSR is a member of the class A family of G protein-coupled receptors. A cell-free expression system was established to produce the GHSR in the precipitated form. The solubilized receptor was refolded in vitro and reconstituted into DMPC lipid membranes. Methionines, arginines, and histidines were chosen for 13C-labeling as they are representative for the transmembrane domains, the loops and flanking regions of the transmembrane α-helices, and the C-terminus of the receptor, respectively. The dynamics of the isotopically labeled residues was characterized by solid-state NMR measuring motionally averaged 1H-13C dipolar couplings, which were converted into molecular order parameters. Separated local field DIPSHIFT experiments under magic-angle spinning conditions using either varying cross polarization contact times or direct excitation provided order parameters for these residues showing that the C-terminus was the segment with the highest motional amplitude. The loop regions and helix ends as well as the transmembrane regions of the GHSR represent relatively rigid segments in the overall very flexible receptor molecule. Although no site resolution could be achieved in the experiments, the previously reported highly dynamic character of the receptor concluded from uniformly 13C labeled receptor samples could be further specified by this segmental labeling approach, leading to a more diversified understanding. of the receptor dynamics under equilibrium conditions
Cell-free-synthesized voltage-gated proton channels: Approaches to the study of protein dynamics
(2018)
We often only realize how important health is when diseases manifest themselves through their symptoms and, ultimately, in a diagnosis. Over time, we suffer from many diseases starting with the first childhood disease to colds to gastrointestinal infections. Most diseases pass harmlessly and symptoms fade away. However, not all diseases are so harmless. Alzheimer’s disease, breast cancer, Parkinson’s disease, and colorectal cancer usually cause severe illness with high mortality rates. In pharmaceutical research, efforts are therefore being made to determine the molecular basis of them in order to provide patients with potential relief and, at best, healing. A special group of regulators, involved in the previously mentioned diseases, are voltage-gated proton channels. Thus, the understanding of their structure, function, and potential drug interaction is of great importance for humanity.
Voltage-gated proton channels are localized in the cell membrane. As their name indicates, they are controlled by voltage changes. Depolarization of the cell membrane induces conformational changes that open these channels allowing protons to pass through. Here, the transfer is based on a passive process driven by a concentration gradient between two individual compartments separated by the cell membrane. Voltage-gated proton channels are highly selective for protons and show a temperature- and pH-dependent gating behavior. However, little is known about their channeling mechanism. Previous experimental results are insufficient for understanding the key features of proton channeling.
In this thesis, for the first time, the cell-free production of voltage-sensing domains (VSD) of human voltage-gated proton channels (hHV1) and zebrafish voltage-sensing phosphatases (DrVSP) is described. Utilizing the cell free approach, parameters concerning protein stability, folding and labeling can be easily addressed. Furthermore, the provision of a membrane mimetic in form of detergent micelles, nanodiscs, or liposomes for co-translational incorporations of these membrane proteins is simple and efficient. Both VSDs were successfully produced up to 3 mg/ml. Furthermore, the cell-free synthesis enabled for the first time studies of lipid-dependent co-translational VSD insertions into nanodiscs and liposomes. Cell-free produced VSDs were shown to be active, and to exist mainly as dimers. In addition, also their activation was stated to be lipid-dependent, which has not been described so far. Solution-state NMR experiments were performed with fully and selectively labeled cell-free produced VSDs. With respect to the development of potential drug candidates, I could demonstrate the inhibition of the VSDs by 2-guanidinobenzimidazole (2GBI). Determined KD values were comparable to literature data for the human construct. For the first time, a low affinity for 2GBI of the zebrafish VSD could be described.
In future, the combination of a fast, easy and cheap cell-free production of fully or selectively labeled VSDs and their analysis by solution state NMR will enable structure determinations as well as inhibitor binding studies and protein dynamic investigations of those proteins. The results of these investigations will serve as a basis for example for the development of new drugs. In addition, a detailed description of the lipid-dependent activity might be helpful in controlling the function of voltage-gated proton channels in cancer cells and thereby reducing their growth or disturbing their cell homeostasis in general.
Der vorliegende Aufsatz gibt eine Übersicht über die Vegetation und die Vegetationsbedingungen bodensaurer Eichen- und Eichenmischwälder (Verband Quercion roboris Malcuit 1929) in Europa. Kennzeichnend für die Physiognomie und Struktur dieser Wälder ist das Vorherrschen der Lichtbaumarten Quercus robur oder Qu. petraea in der Baumschicht. Beide Arten haben, als einzige Vertreter der Gattung Quercus, eine europaweite Verbreitung. Andere Baumarten (bspw. Quercus pyrenaica, Fagus sylvatica, Pinus sylvestris, Betula pendula, B. pubescens, Castanea sativa) treten in Abhängigkeit von großklimatischen, edaphischen oder syndynamischen Verhältnissen zur Baumschicht hinzu. Da die Lichtverhältnisse im Bestandesinnern günstig sind, ist eine - mitunter artenreiche - Strauchschicht häufig entwickelt. Die Bodenvegetation wird, neben Zwergsträuchern, von lichtliebenden, azidophytischen Kräutern, Gräser und Kryptogamen gebildet. - Übereinstimmendes Merkmal der von bodensauren Eichen(misch)wäldern besiedelten Böden ist deren geringes Basen- und Stickstoffangebot. Während in den Tieflagen Nordmitteleuropas pleistozäne Lockersedimente und deren Umlagerungsprodukte vorherrschen, dominieren in den Mittelgebirgsregionen silikatische Festgesteine. Die Böden sind durch biologisch ungünstige Humusformen charakterisiert, zumeist Rohhumus, Moder oder feinhumusreicher Moder. - Die Vorkommensschwerpunkte und auch das Mannigfaltigkeitszentrum bodensaurer Eichen(misch)wälder liegen in Westeuropa, wo sie besonders viele, gebietspezifische Kenn- und Trennarten aufweisen: Auf der iberischen Halbinsel treten zahlreiche Phanerogamen und auf den Britischen Inseln verschiedene Kryptogamen auf, die bodensauren Eichen(misch)wäldern im übrigen Europa fehlen. Im östlichen Mitteleuropa und im nordöstlichen Europa werden bodensaure Eichen(misch)wälder von Gesellschaften des Dicrano-Pinion abgelöst. Mit zunehmender Sommerwärme im subkontinentalen, submediterranen südöstlichen Mitteleuropa treten thermophile Eichenmischwälder (Quercetalia pubescenti-petraeae-Gesellschaften) an die Stelle der Quercion roboris-Gesellschaften. - Abschließend werden Aspekte der vegetationsgeographischen Gliederung sowie Fragen der Gefährdung und des Schutzes bodensaurer Eichen(misch)wälder erörtert.