Microtubule associated molecular motors are involved in a multitude of fundamental cellular processes such as intracellular transport and spindle positioning. During these movements multiple motor proteins often work together and are, therefore, able to exert high forces. Thus force generation and sensing are common mechanisms for controlling motor driven movement. These mechanisms play a pivotal role when motor proteins antagonize each other, e.g. to facilitate oscillations of the spindle or the nucleus. Single motor proteins have been characterized in depth over the last two decades, our understanding of the collective behavior of molecular motors remains, however, poor. Since motor proteins often cooperate while they walk along microtubules, it is necessary to describe their collective reaction to a load quantitatively in order to understand the mechanism of many motor-driven processes. I studied the antagonistic action of many molecular motors (of one kind) in a gliding geometry. For this purpose I crosslinked two microtubules in an antiparallel fashion, so that they formed \"doublets\". Then I observed the gliding motility of these antiparallel doublets and analyzed the gliding velocity with respect to the relative number of motors pulling or pushing against each other. I observed that the antiparallel doublets gliding on conventional kinesin-1 (from Drosophila melanogaster) as well as cytoplasmic dynein (from Saccharomyces cerevisae) exhibited two distinct modes of movement, slow and fast, which were well separated. Furthermore I found a bistability, meaning, that both kinds of movement, slow and fast, occurred at the same ratio of antagonizing motors. Antiparallel doublets gliding on the non-processive motor protein Ncd (the kinesin-14 from D. melanogaster) showed, however, no bistability. The collective dynamics of all three motor proteins were described with a quantitative theory based on single-motor properties. Furthermore the response of multiple dynein motors towards an external, well-defined load was measured in a gliding geometry by magnetic tweezing. Examples of multi-motor force-velocity relationships are presented and discussed. I established, furthermore, a method for counting single surface immobilized motors to guide the evaluation of the tweezing experiments.

A fundamental problem in biology is the reconstruction of the relatedness of all (extant) species. Traditionally, systematists employ visually recognizable characters of organisms for classification and evolutionary analysis. Recent developments in molecular and computational biology, however, lead to a whole different perspective on how to address the problem of inferring relatedness. The discovery of molecules, carrying genetic information, and the comparison of their primary structure has, in a rather short period of time, revolutionized our understanding of the phylogenetic relationship of many organisms. These novel approaches, however, turned out to bear similar problems as previous techniques. Moreover, they created new ones. Hence, taxonomists came to realize that even with this new type of data not all problematic relationships could be unambiguously resolved. The search for complementary approaches has led to the utilization of rare genomic changes and other characters which are largely independent from the primary structure of the underlying sequence(s). These “higher order” characters are thought to be evolutionary conserved in certain lineages and largely unaffected by primary sequence data-based problems, allowing for a better resolution of the Tree of Life. The central aim of this thesis is the utilization of molecular characters of higher order in connection with their consistent and comparable extraction from a given data set. Two novel methods are presented that allow such an inference. This is complemented with the search for and analysis of known and novel molecular characteristics to study the relationships among Metazoa, both intra- as well as interspecific. The first method tackles a common problem in phylogenetic analyses: the inference of reliable data set. As part of this thesis a pipeline was created for the automated annotation of metazoan mitochondrial genomes. Data thus obtained constitutes a reliable and standardized starting point for all downstream analyses, e.g. genome rearrangement studies. The second method utilizes a subclass of gaps, namely those which define an approximate split of a given data set. The definition and inference of such split-inducing indels (splids) is based on two basic principles. First, indels at the same position, i.e. sharing the same end points in two sequences, are likely homologous. Second, independent single-residue insertions and deletions tend to occur more frequently than multi-residue indels. It is shown that trees based on splids recover most of the undisputed monophyletic groups while influence of the underlying alignment algorithm is relatively small. Mitochondrial markers are a valuable tool for the understanding of small and large scale population structure. The non-coding control region of mitochondrial DNA (mtDNA) often contains a higher amount of variability compared to genes encoding proteins and non-coding RNAs. A case study on a small scale population structure investigates the control region of the European Fire-bellied Toad in order to find highly variable parts which are of potential importance to develop informative genetic markers. A particular focus is placed on the investigation of the evolutionary dynamics of the repetitive region at an inter- and intraspecific level. This includes understanding mechanisms underlying its evolution, i.e. by exploring the impact of secondary structure on slipped strand mispairing during mtDNA replication. The 7SK RNA is a key player in the regulation of polymerase II (Pol-II) transcription, interacting with at least three known proteins: It mediates the inhibition of the Positive Transcription Elongation Factor b (P-TEFb) by the HEXIM1/2 proteins, thereby repressing transcript elongation by Pol-II. A highly specific interaction with LARP7 (La-Related Protein 7), on the other hand, regulates its stability. 7SK RNA is capped at its 5’ end by a highly specific methyltransferase MePCE (Methylphosphate Capping Enzyme). Employing sequence and structure similarity it is shown that the 7SK RNA as well as its protein binding partners have a much earlier evolutionary origin than previously expected. Furthermore, this study presents a good illustration of the pitfalls of using markers of higher order for phylogenetic inference.

Referat: Das lyotrope Phasenverhalten und die molekulare Architektur von Membranen ausgewählter Dienlipi-de wird mittels Infrarot-(IR)-Spektroskopie untersucht. Doppelschichten aus synthetischen Lipiden haben sich als Strukturmodell biologischer Membranen bewährt. Durch den Einbau reaktiver Diengruppen können die Lipidmoleküle polymerisiert werden. Mögliche Anwendungen als biokompatible Materialien oder als Mikro-Hüllen für die Wirkstoffüber-tragung machen polymerisierte Lipidstrukturen und insbesondere Membranen interessant. Darüber hinaus stellen die Diengruppen eine definierte Störung der Membran dar, die die Untersuchung grundlegender Fragen der Membranarchitektur erlaubt. In der Arbeit wird ein besonderer Schwerpunkt auf die Hydratation von Lipidsystemen gelegt. Das lyotrope Phasenverhalten und die molekulare Struktur im polaren und hydrophoben Bereich von Lipi-den mit Diengruppen am Ende und am Anfang der Fettsäureketten werden detailliert analysiert. Der Einfluß der molekulargeordneten Umgebung auf die chemische Stabilität der Diengruppen in den Li-pidaggregaten, ihrer Konfiguration und die Struktur des polymeren Produkts werden untersucht. Abstoßende, kurzweitreichende Wechselwirkungen zwischen polaren, hydratisierten Oberflächen sind für die Stabilität kolloidaler Systeme und für biologisch relevante Prozesse, wie die Fusion von Zell-membranen, von großer Bedeutung. Die Untersuchung dieser Hydratationskraft vor und nach der Po-lymerisation der Lipidmembranen, sowie in Systemen unterschiedlicher Struktur im Kopfgruppenbe-reich der Membranen, lieferte Informationen über spezifische Beiträge, die durch die Bewegung ein-zelner Moleküle oder die intermolekularen Wechselwirkungen im polaren Bereich der Membranen hervorgerufen werden. Zur Aufklärung der molekularen Struktur der Lipidsysteme wird in erster Linie ihr IR-Lineardichroismus analysiert. Obwohl die Theorie der Absorption polarisierter elektromagnetischer Strahlung seit langem ausgearbeitet ist, fehlte bislang ein allgemeiner Formalismus zur Behandlung lamellarer, biaxialer Strukturen. Diese Lücke wird in dieser Arbeit geschlossen. Im theoretischen Teil wird der Zusammenhang zwischen der Absorption polarisierten Lichtes und der molekularen Ordnung betrachtet. Die Meßgröße, der IR-Ordnungsparameter, wird in kompakter Form als Funktion moleku-larer Ordnungsparameter ausgedrückt. Besondere Aufmerksamkeit wird der Technik der abge-schwächten Totalreflexion (ATR) gewidmet. Der potentielle Informationsgehalt ausgewählter Schwingungsmoden langkettiger Moleküle wird anhand der bekannten Struktur von Stearinsäurekri-stallen und DPPC-Membranen überprüft. Eine Reihe von für die praktische Anwendung der ATR-Spektroskopie wichtigen Details, wie der Einfluß der Doppelbrechung und die effektive Eindringtiefe des IR-Lichtes in die Probe, wird anhand von Beispielen untersucht. Eine zentrale Stellung nimmt der Lineardichroismus der intensiven Banden der C–H-Streckschwingungen der Fettsäureketten und vor allem der IR-aktiven Moden der Diengruppen ein.

Regensburg, Univ., Diss., 2008

Regensburg, Univ., Diss., 2009.

Techn. Universiẗat, Diss., 2003--Dresden.

Molecule-based magnets (molecular magnets) have attracted much interest in recent decades both from an experimental and from a theoretical point of view, not only because of their interesting physical effects, but also because of their potential applications: e.g., molecular spintronics, quantum computing, high density information storage, and nanomedicine. Molecular magnets are at the very bottom of the possible size of nanomagnets. On reducing the size of objects down to the nanoscale, the coexistence of classical properties and quantum properties in these systems may be observed. In additional, molecular magnets exist with structural variability and permit selective substitution of the ligands in order to alter their magnetic properties. Therefore, these characteristics make such molecules suitable candidates for studying molecular magnetism. They can be used as model systems for a detailed understanding of interplay between structural and magnetic properties of them in order to optimize desired magnetic properties. This thesis considers the investigation of magnetic properties of several new transition metal molecular compounds via different experimental techniques (continuous wave electron spin resonance (CW ESR), pulse ESR, high-field/high-frequency ESR (HF-ESR) and static magnetization techniques). The first studied compounds were mono- and trinuclear Cu(II)-(oxamato, oxamidato)/bis(oxamidato) type compounds. First, all components of the g-tensor and the tensors of onsite ACu and transferred AN HF interactions of mononuclear Cu(II)- bis(oxamidato) compounds have been determined from CW ESR measurements at 10 GHz and at room temperature and pulse ELDOR detected NMR measurements at 35 GHz and at 20 K. The spin density distributions of the mononuclear compounds have been calculated from the experimentally obtained HF tensors. The magnetic exchange constants J of their corresponding trinuclear compounds were determined from susceptibility measurements versus temperature. Our discussion of the spin density distribution of the mononuclear compounds together with the results of the magnetic characterization of their corresponding trinuclear compounds show that the spin population of the mononuclear compounds is in interplay with the J values of their corresponding trinuclear compounds. The second studied compounds were polynuclear Cu(II)-(bis)oxamato compounds with ferrocene and ferrocenium ligands. The magnetic properties of these compounds were studied by susceptibility measurements versus temperature to determine J values. In addition, the ESR technique is used to investigate the magnetic properties of the studied compounds because they contain two different magnetic ions and because only the ESR technique can selectively excite different electron spin species. These studies together with geometries of the ferrocenium ligands determined by crystallographic studies indicate that the magnetic interaction between a central Cu(II) and a Fe(III) ions changed from the antiferromagnetic coupling to the ferromagnetic coupling when a stronger distortion of the axial symmetry in the feroccenium cation exists. Therefore, the degree of the distortion of the feroccenium cation is a control parameter for the sign of the interaction between the central Cu(II) ion and the Fe(III) spins of the studied compounds. The last two studied molecular magnets were a binuclear Ni(II) compound (Ni(II)-dimer) and a cube-like tetranuclear compound with a [Fe4O4]-cube core (Fe4-cube). HF-ESR measurements enabled us to determine the g-factor, the sign, and the absolute value of the magnetic anisotropy parameters. Using this information together with static magnetization measurements, the J value and the magnetic ground state of the studied compounds have been determined. In Ni(II)-dimer, two Ni(II) ions, each having a spin S = 1, are coupled antiferromagnetically that leads to a ground state with total spin Stot = 0. An easy plane magnetic anisotropy with a preferable direction for each Ni(II) ion is found. For Fe4-cube, a ground state with total spin Stot = 8 has been determined. The analysis of the frequency dependence and temperature dependence of HF-ESR lines reveals an easy axis magnetic anisotropy (Dcube = -22 GHz (-1 K)) corresponding to an energy barrier of U = 64 K for the thermal relaxation of the magnetization. These results indicate that Fe4-cube is favorable to show single molecular magnet (SMM) behavior.

No description available.

Microtubule associated molecular motors are involved in a multitude of fundamental cellular processes such as intracellular transport and spindle positioning. During these movements multiple motor proteins often work together and are, therefore, able to exert high forces. Thus force generation and sensing are common mechanisms for controlling motor driven movement. These mechanisms play a pivotal role when motor proteins antagonize each other, e.g. to facilitate oscillations of the spindle or the nucleus. Single motor proteins have been characterized in depth over the last two decades, our understanding of the collective behavior of molecular motors remains, however, poor. Since motor proteins often cooperate while they walk along microtubules, it is necessary to describe their collective reaction to a load quantitatively in order to understand the mechanism of many motor-driven processes. I studied the antagonistic action of many molecular motors (of one kind) in a gliding geometry. For this purpose I crosslinked two microtubules in an antiparallel fashion, so that they formed \"doublets\". Then I observed the gliding motility of these antiparallel doublets and analyzed the gliding velocity with respect to the relative number of motors pulling or pushing against each other. I observed that the antiparallel doublets gliding on conventional kinesin-1 (from Drosophila melanogaster) as well as cytoplasmic dynein (from Saccharomyces cerevisae) exhibited two distinct modes of movement, slow and fast, which were well separated. Furthermore I found a bistability, meaning, that both kinds of movement, slow and fast, occurred at the same ratio of antagonizing motors. Antiparallel doublets gliding on the non-processive motor protein Ncd (the kinesin-14 from D. melanogaster) showed, however, no bistability. The collective dynamics of all three motor proteins were described with a quantitative theory based on single-motor properties. Furthermore the response of multiple dynein motors towards an external, well-defined load was measured in a gliding geometry by magnetic tweezing. Examples of multi-motor force-velocity relationships are presented and discussed. I established, furthermore, a method for counting single surface immobilized motors to guide the evaluation of the tweezing experiments.:1 Introduction to the functions of molecular motors 1 1.1 How molecular motors move 1 1.1.1 Of muscles and molecules 1 1.1.2 Kinesin-1, the working horse of single-molecule research 3 1.1.3 Kinesin-14, an unusual kinesin with a new twist 6 1.1.4 Cytoplasmic dynein, the molecule with many qualities 7 1.2 Structure and function of microtubules 8 1.3 The directionality of molecular motors 9 1.4 Force regulation in cell biology via molecular motors 10 1.4.1 Bidirectional cargo transport 10 1.4.2 Dynein drives intracellular oscillations 13 1.4.3 Control of spindle length 15 2 Introduction to the collective behavior of molecular motors in vitro 19 2.1 Cooperativity of molecular motors 19 2.2 How multiple motors work against a load 21 2.2.1 Theoretical concepts 21 2.2.2 Optical tweezing of multiple motors 22 2.2.3 Alternative experimental approaches 23 2.2.4 Membrane tube dynamics 24 2.3 Antagonizing molecular motors 25 2.3.1 Competition between dissimilar motors 25 2.3.2 Competition between identical motors 26 2.4 Aim of the project 28 3 Characterization of molecular motors 31 3.1 Results: The run length of processive motors 31 3.1.1 Run length of kinesin-1 at different ATP concentrations 31 3.1.2 The run length of cytoplasmic dynein 34 3.2 Results for multi-motor gliding assays 37 3.2.1 The effect of ATP on the gliding motility 37 3.2.2 The effect of temperature on the gliding motility 39 3.2.3 Bead transport does not influence gliding motility 42 3.3 Discussion 43 4 Magnetic tweezing of multiple molecular motors 45 4.1 Concepts of the magnetic tweezing setup 45 4.1.1 Theoretical concepts 45 4.1.2 Implementation 48 4.1.3 Calibration 51 4.2 Results of multi-motor force measurements 53 4.2.1 External force leads to microtubule re-orientation 53 4.2.2 Cytoplasmic dynein is able to withstand high opposing loads 55 4.2.3 Force-velocity curves at very low motor densities 56 4.2.4 Averaging of multi-motor force-velocity relationships 58 4.3 Discussion 60 5 Reconstitution of antagonizing motor activity 63 5.1 The doublet assay 63 5.2 Experimental results of the doublet assay 65 5.2.1 Kinesin-1 driven doublets move in discrete velocity regimes 65 5.2.2 Velocity affects the shape of the bistability curve 68 5.2.3 Dynein\'s processivity allows bistability at low velocity 69 5.2.4 Ncd does not exhibit a bistability curve 70 5.3 Theoretical results of the doublets assay 71 5.3.1 General concepts 71 5.3.2 Theory for processive motors 73 5.3.3 Theory for non-processive motors 75 5.3.4 The emergence of bistability 78 5.3.5 Model for single-motor force-velocity relationships 81 5.4 Comparison between theoretical and experiment results 83 5.5 Discussion 87 6 Materials and Methods 91 6.1 List of chemicals and equipment 91 6.2 Buffer recipes 92 6.3 Protein purification 93 6.4 Preparation of microtubules 95 6.5 Preparation of flow cells 96 6.6 Fluorescence microscopy 98 6.7 Errors computation 100 6.8 Software 100 7 References 103 8 Acknowledgement 113

No description available.