Práca sa zaoberá vývojom ďalekej infračervenej spektroskopie v silnom magnetickom poli. Kombinácia ďalekej infračervenej spektroskopie a silného magnetického poľa je veľmi dôležitým nástrojom pri charakterizácii materiálov, ako sú jedno-molekulové magnety. Predstavuje tiež ideálnu experimentálnu techniku, ktorá dokáže skúmať a objasniť vlastnosti nových 2D materiálov. Ďaleká infračervená spektroskopia v magnetickom poli taktiež umožňuje študovať elektrónovú paramagnetickú rezonanciu (EPR) jedno-molekulových magnetov s veľmi veľkým delením pri nulovom poli, hlavne na báze komplexov prechodných kovov alebo lantanoidov, v ktorých bežne používané EPR systémy neposkytujú experimentálny prístup k magnetickým rezonančným prechodom. V práci sú podrobne popísané dve zostavy ďaleko infračervených spektrometrov pripojené k supravodivým magnetom. Prvá opísaná zostava, ktoré sa nachádza na univerzite v Stuttgarte, je už zmontovaná a jej výkon je diskutovaný. Magneto-optické merania jedno-molekulových magnetov vykonané na tejto zostave sú predstavené. Druhá magneto-optická zostava čerpá zo skúseností získaných pri prvej zostave a je určená pre CEITEC.

This work describes theoretical studies on molecular and electronic structures of lanthanide-based single-molecule magnets focusing on their magnetic properties. In this work, two main problems are covered: the structural ordering of endohedral fullerenes in different supramolecular arrangements, and the magnetic anisotropy of lanthanides in different charge coordinations. The basic methodes used in this work are density functional theory and multiconfigurational self-consistent field.:CHAPTER 1. THEORETICAL FOUNDATIONS OF RARE-EARTH MAGNETISM 12 1.1. Single-molecule magnetism and 4f-elements 14 1.1.1. Electronic structure of 4f-elements 16 1.1.2. LS-coupling scheme 19 1.1.3. Parameterization of the Crystal-Field splitting effect. 20 1.1.4. Zeeman splitting for a free ion 24 1.1.5. Spin Hamiltonian and pseudospin approximation 24 1.1.6. Kramers theorem 25 1.1.7. Weak and strong molecular interactions. 26 1.2. Computational methods in application to Ln-based SMMs 27 1.2.1. Density functional theory (DFT). 28 1.2.2. Multiconfigurational methods in quantum chemistry 33 1.3. Role of molecular structure in single-molecular magnetism 41 1.3.1. Complexes of SMMs with organic molecules 45 1.3.2. SMMs deposited on surfaces 46 CHAPTER 2. STRUCTURAL ORDERING IN COCRYSTALS OF EMFs AND Ni(OEP) 49 2.1. Ordering in endohedral metallofullerenes 49 2.2. Modeling details 51 2.3. Conformer analysis 54 2.4. Electrostatic potential 58 CHAPTER 3. ISOMERISM OF Dy2ScN@C80 DEPOSITED ON SURFACES 61 3.1. Modeling details 62 3.2. Cluster conformation analysis 69 3.3. Charge density analysis 75 CHAPTER 4. Ho|MgO AS A SINGLE-ATOMIC MAGNET. FV-MAGNETISM. 80 4.1. DFT description of Ln|MgO 85 4.2. Ho|MgO system: ab initio calculations 92 4.3. Magnetic properties of lanthanides with FV magnetism 99 4.4. Generalized ligand field and spin Hamiltonians for FV systems. 101 CHAPTER 5. FV-MAGNETISM IN [Ln2+] METALLOCENE COMPLEXES 107 5.1. TbII(CpiPr5)2 DFT-model 108 5.2. FV-interaction in terms of ab initio multiconfigurational approach 113 5.3. Point-charge model 115

Telomeres are nucleoprotein structures that cap the physical ends of eukaryotic chromosomes. They consist of repetitive DNA sequences 5’-TTAGGG-3’ assembled with proteins which form the shelterin complex. This complex protects the ends of chromosomes by inhibiting DNA repair pathways at telomeres and avoid their recognition as double-strand breaks. Telomeres have been identified as a transcriptionally silent zone until 2007 when a noncoding RNA called TERRA (TElomeric Repeat containing RNA) transcribed from telomeres was discovered. This RNA gave rise to many questions: How is TERRA regulated? How is TERRA expressed? Does TERRA interact with proteins, DNA or RNA? After several studies, we know that TERRA is frequently expressed in cancer cells and it interacts with a large proteome. Nevertheless, its specific function remains unknown. In this thesis, we studied this RNA in human cancer cells using live-cell microscopy which allowed us to get information on TERRA’s dynamics, localization and its interactome. Moreover, we used single-molecule imaging on TERRA 15q labeled by the MS2-GFP system, which allowed the visualization of TERRA transcripts. This study resulted in the discovery of two types of TERRA population from telomere 15q: one of the population is characterized by the formation of clusters and a second population is constituted of unique molecules more dynamic in the nucleus. Finally, in order to better understand TERRA’s functions, we developed a new approach which consists on immunoprecipitating TERRA using the MS2 stem-loops as a tag to identify TERRA-interacting proteins such as the telomeric factor TRF2 or RNA-binding proteins like hnRNP -A1 or FUS. / Les télomères forment les extrémités des chromosomes chez les eucaryotes. Ces séquences répétées en tandem 5’-TTAGGG-3’ font partie d’un complexe nucléoprotéique appelé shelterin. En effet, cet assemblage de protéines télomériques permet la protection des extrémités des chromosomes, permettant à celles-ci de ne pas être reconnues comme des cassures dans l’ADN et d’activer les voies de réparation de l’ADN. Les télomères ont longtemps été reconnus comme étant des zones de transcription inactives, ce jusqu’en 2007 lorsqu’une équipe de recherche découvrit un ARN non codant appelé TERRA (Telomeric Repeat containing RNA). Ce dernier a suscité de nombreuses questions : quel est le rôle de cet ARN? Comment est-il exprimé et régulé? Interagit-il avec d’autres facteurs cellulaires? Les différentes recherches menées sur cet ARN ont permis de conclure que celui-ci était fréquemment induit dans les cellules de cancer, que ses partenaires d’interactions sont nombreux, mais que ses fonctions restent encore mal définies. Par ailleurs, ces différentes études ont toujours été ou presque réalisées sur des cellules fixées, sur une population totale d’ARN télomérique TERRA. Afin d’apporter de nouvelles réponses et de mieux caractériser cet ARN, nous avons étudié ce transcrit dans des cellules de cancer humain en utilisant la technique de microscopie en temps réel, qui permet de récolter des données sur la dynamique, la localisation de cet ARN et ses éventuels partenaires. De plus, nous nous sommes intéressés à des molécules uniques de TERRA issues du télomère 15q en exploitant la technique de marquage avec des tiges-boucles MS2 (MS2-GFP). Cette étude de microscopie a permis de découvrir deux types de population de l’ARN TERRA 15q : une population caractérisée par des assemblages d’ARN dit clusters (agrégats d’ARN) et une population plus singulière qui semble avoir une diffusion plus importante dans le noyau de la cellule. Par ailleurs, l’expression de l’ARN TERRA semble être différente d’un type cellulaire à un autre et nous avons donc cherché à connaître le niveau d’expression de cet ARN au sein de la lignée étudiée au cours de ce projet de recherche. Enfin, afin de découvrir de nouveaux rôles pour cet ARN, nous avons développé une approche de co-immunoprécipitation de l’ARN TERRA pour identifier des interactions avec des protéines du complexe shelterin comme TRF2, ou des protéines liant l’ARN comme hnRNP-A1 ou encore FUS.

Die molekulare Elektronik setzt sich zum Ziel, passive und aktive Bausteine in integrierten Schaltkreisen auf molekularer Ebene zu realisieren. Dabei ist entscheidend, dass sich der elektrische Leitwert der molekularen Bauelemente hinreichend regulieren lässt. Um zu belegen, dass dies möglich ist, wird in dieser Dissertation die gezielte Leitwertkontrolle einzelner über Nanoelektroden kontaktierter Moleküle nachgewiesen. Die erzielten Ergebnisse ergänzen dabei nahtlos aktuellste Studien. Zum einen werden kontaktierte molekulare Schalter durch Bestrahlung mit Licht einer bestimmten Wellenlänge in-situ von einem nicht-leitenden in einen leitenden Zustand geschaltet, wobei der Einfluss unterschiedlicher Seitengruppen für eine zusätzliche Modifikation des Leitwerts sorgt. Ausschlaggebend ist hierbei die elektronische Anbindung des Moleküls an die Elektroden. Zum anderen werden Molekül-Metall-Komplexe durch die Einbindung eines Übergangsmetallions von einem isolierenden in einen leitenden Zustand versetzt. In diesem Fall lässt sich der leitende Zustand durch die Wahl des Ions innerhalb einer Größenordnung variieren, was eine völlig neue Möglichkeit der Leitwertkontrolle in molekularen Bausteinen darstellt. Das Ion bestimmt dabei sowohl die mechanische Stabilität als auch die elektronische Struktur des Moleküls. Für die Kontaktierung einzelner Moleküle kommt die Technik des mechanisch kontrollierten Bruchkontakts zum Einsatz. So lassen sich feine Goldnanoelektroden herstellen, an die Moleküle anbinden. Um eine präzise Analyse durchzuführen, werden über zwei unabhängige Messstrategien Informationen über das elektrische Transportverhalten sowie über die elektronische Struktur der Moleküle erworben. In dieser Arbeit sind echte Neuentwicklungen auf dem Gebiet der molekularen Elektronik gelungen, die einen wesentlichen Beitrag für die Umsetzung integrierter molekularer Schaltkreise leisten.

Die vorliegende Promotionsarbeit “Density functional study of the electronic and magnetic properties of selected transition metal complexes” beschäftigt sich mit dem Zusammenhang zwischen strukturellen Merkmalen sowie elektronischen und magnetischen Eigenschaften von Einzelmolekül-Magneten. Im Wesentlichen konnte dabei gezeigt werden, dass die magnetischen Eigenschaften sowohl von strukturellen Merkmalen als auch von den elektronischen Eigenschaften bestimmt werden. Des Weiteren ergab sich, dass verschiedene Kenngrößen der magnetischen Eigenschaften (im speziellen der magnetische Grundzustand S sowie die magnetische Anisotropie D) miteinander korreliert sind. Dies ist im Besonderen für eine mögliche Anwendung von Einzelmolekül-Magneten im Bereich der Datenspeicherung von Bedeutung.

Single-molecule force spectroscopy (SMFS) with atomic force microscope (AFM) has advanced our knowledge of the mechanical aspects of biological processes, and helped us take big strides in the hitherto unexplored areas of protein (un)folding. One such virgin land is that of membrane proteins, where the advent of AFM has not only helped to visualize the difficult to crystallize membrane proteins at the single-molecule level, but also given a new perspective in the understanding of the interplay of molecular interactions involved in the construction of these molecules. My PhD work was tightly focused on exploiting this sensitive technique to decipher the intra- and intermolecular interactions in membrane proteins, using bacteriorhodopsin and bovine rhodopsin as model systems. Using single-molecule unfolding measurements on different bacteriorhodopsin oligomeric assemblies - trimeric, dimeric and monomeric - it was possible to elucidate the contribution of intra- and interhelical interactions in single bacteriorhodopsin molecules. Besides, intriguing insights were obtained into the organization of bacteriorhodopsin as trimers, as deduced from the unfolding pathways of the proteins from different assemblies. Though the unfolding pathways of bacteriorhodopsin from all the assemblies remained the same, the different occurrence probability of these pathways suggested a kinetic stabilization of bacteriorhodopsin from a trimer compared to that existing as a monomer. Unraveling the knot of a complex G-protein coupled receptor, rhodopsin, showed the existence of two structural states, a native, functional state, and a non-native, non-functional state, corresponding to the presence or absence of a highly conserved disulfide bridge, respectively. The molecular interactions in absence of the native disulfide bridge mapped onto the three-dimensional structure of native rhodopsin gave insights into the molecular origin of the neurodegenerative disease retinitis pigmentosa. This presents a novel technique to decipher molecular interactions of a different conformational state of the same molecule in the absence of a high-resolution X-ray crystal structure. Interestingly, the presence of ZnCl2 maintained the integrity of the disulfide bridge and the nature of unfolding intermediates. Moreover, the increased mechanical and thermodynamic stability of rhodopsin with bound zinc ions suggested a plausible role for the bivalent ion in rhodopsin dimerization and consequently signal transduction. Last but not the least, I decided to dig into the mysteries of the real mechanisms of mechanical unfolding with the help of well-chosen single point mutations in bacteriorhodopsin. The monumental work has helped me to solve some key questions regarding the nature of mechanical barriers that constitute the intermediates in the unfolding process. Of particular interest is the determination of altered occurrence probabilities of unfolding pathways in an energy landscape and their correlation to the intramolecular interactions with the help of bioinformatics tools. The kind of work presented here, in my opinion, will not only help us to understand the basic principles of membrane protein (un)folding, but also to manipulate and tune energy landscapes with the help of small molecules, proteins, or mutations, thus opening up new vistas in medicine and pharmacology. It is just a matter of a lot of hard work, some time, and a little bit of luck till we understand the key elements of membrane protein (un)folding and use it to our advantage.

The atomic force microscope (AFM) provides a powerful instrument for investigating and manipulating biological samples down to the subnanometer scale. In contrast to other microscopy methods, AFM does not require labeling, staining, nor fixation of samples and allows the specimen to be fully hydrated in buffer solution during the experiments. Moreover, AFM clearly compares in resolution to other techniques. In general, the AFM can be operated in an imaging or a force spectroscopy mode. In the present work, advantage was taken of this versatility to investigate single biomolecules and biomolecular assemblies. A novel approach to investigate the visco-elastic behavior of biomolecules under force was established, using dextran as an example. While a molecule tethered between a solid support and the cantilever tip was stretched at a constant velocity, the thermally driven oscillation of the cantilever was recorded. Analysis of the cantilever Brownian noise provided information about the visco-elastic properties of dextran that corresponded well to parameters obtained by alternative methods. However, the approach presented here was easier to implement and less time-consuming than previously used methods. A computer controlled force-clamp system was set up, circumventing the need for custom built analogue electronics. A commercial PicoForce AFM was extended by two computers which hosted data acquisition hardware. While the first computer recorded data, the second computer drove the AFM bypassing the manufacturer's microscope control software. To do so, a software-based proportional-integral-differential (PID) controller was implemented on the second computer. It allowed the force applied to a molecule to be held constant over time. After tuning of the PID controller, response times obtained using that force-clamp setup were comparable to those of the recently reported analogue systems. The performance of the setup was demonstrated by force-clamp unfolding of a pentameric Ig25 construct and the membrane protein NhaA. In the latter case, short-lived unfolding intermediates that were populated for less than 10 ms, could be revealed. Conventional single-molecule dynamic force spectroscopy was used to unfold the serine:threonine antiporter SteT from Bacillus subtilis, an integral membrane protein. Unfolding force patterns revealed the unfolding barriers stabilizing structural segments of SteT. Ligand binding did not induce new unfolding barriers suggesting that weak interactions with multiple structural segments were involved. In contrast, ligand binding caused changes in the energy landscape of all structural segments, thus turning the protein from a brittle, rigid into a more stable, structurally flexible conformation. Functionally, rigidity in the ligand-free state was thought to facilitate specific ligand binding, while flexibility and increased stability were required for conformational changes associated with substrate translocation. These results support the working model for transmembrane transport proteins that provide alternate access of the binding site to either face of the membrane. Finally, high-resolution imaging was exploited to visualize the extracellular surface of Cx26 gap junction hemichannels (connexons). AFM topographs reveal pH-dependent structural changes of the extracellular connexon surface in presence of HEPES, an aminosulfonate compound. At low pH (< 6.5), connexons showed a narrow and shallow channel entrance, which represented the closed pore. Increasing pH values resulted in a gradual opening of the pore, which was reflected by increasing channel entrance widths and depths. At pH > 7.6 the pore was fully opened and the pore diameter and depth did not increase further. Importantly, coinciding with pore gating a slight rotation of the subunits was observed. In the absence of aminosulfonate compounds, such as HEPES, acidification did not affect pore diameters and depths, retaining the open state. Thus, the intracellular concentration of taurine, a naturally abundant aminosulfonate compound, might be used to tune gap junction sensitivity at low pH.

Each organism has to maintain the integrity of its genetic code, which is stored in its DNA. This is achieved by strongly controlled and regulated cellular processes such as DNA replication, -repair and -recombination. An essential element of these processes is the unwinding of the duplex strands of the DNA helix. This biochemical reaction is catalyzed by helicases that use the energy of nucleoside triphophate (NTP) hydrolysis. Although all helicases comprise highly conserved domains in their amino acid sequence, they exhibit large variations regarding for example their structure, their function and their target nucleic acid structures. The main objective of this thesis is to obtain insight into the DNA unwinding mechanisms of three helicases from two different organisms. These helicase vary in their structures and are involved in different pathways of DNA metabolism. In particular the replicative, hexameric helicase Large Tumor-Antigen (T-Antigen) from Simian virus 40 and the DNA repair helicases RecQ2 and RecQ3 from Arabidopsis thaliana are studied. To observe DNA unwinding by these helicases in real-time on the single molecule level, a biophysical technique, called magnetic tweezers, was applied. This technique allows to stretch single DNA molecules attached to magnetic particles. Simultaneously one can measure the DNA end-to-end distance. Special DNA hairpin templates allowed to characterize different parameters of the DNA unwinding reaction such as the unwinding velocity, the length of unwound DNA (processivity) or the influence of forces. From this mechanistic models about the functions of the helicases could be obtained. T-Antigen is found to be one of the slowest and most processive helicases known so far. In contrast to prokaryotic helicases, the unwinding velocity of T-Antigen shows a weak dependence on the applied force. Since current physical models for the unwinding velocity fail to describe the data an alternative model is developed. The investigated RecQ helicases are found to unwind and close short stretches of DNA in a repetitive fashion. This activity is shown for the first time under external forces. The experiments revealed that the repetitive DNA unwinding is based on the ability of both enzymes to switch from one single DNA strand to the other. Although RecQ2 and RecQ3 perform repetitive DNA unwinding, both enzymes differ largely in the measured DNA unwinding properties. Most importantly, while RecQ2 is a classical helicase that unwinds DNA, RecQ3 mostly rewinds DNA duplexes. These different properties may reflect different specific tasks of the helicases during DNA repair processes. To obtain high spatial resolution in DNA unwinding experiments, the experimental methods were optimized. An improved and more stable magnetic tweezers setup with sub-nanometer resolution was built. Additionally, different methods to prepare various DNA templates for helicase experiments were developed. Furthermore, the torsional stability of magnetic particles within an external field was investigated. The results led to selection rules for DNA-microsphere constructs that allow high resolution measurements. / Jeder Organismus ist bestrebt, die genetischen Informationen intakt zu halten, die in seiner DNA gespeichert sind. Dies wird durch präzise gesteuerte zelluläre Prozesse wie DNA-Replikation, -Reparatur und -Rekombination verwirklicht. Ein wesentlicher Schritt ist dabei das Entwinden von DNA-Doppelsträngen zu Einzelsträngen. Diese chemische Reaktion wird von Helikasen durch die Hydrolyse von Nukleosidtriphosphaten katalysiert. Obwohl bei allen Helikasen bestimmte Aminosäuresequenzen hoch konserviert sind, können sie sich in Eigenschaften wie Struktur, Funktion oder DNA Substratspezifität stark unterscheiden. Gegenstand der vorliegenden Arbeit ist es, die Entwindungsmechanismen von drei verschieden Helikasen aus zwei unterschiedlichen Organismen zu untersuchen, die sich in ihrer Struktur sowie ihrer Funktion unterscheiden. Es handelt sich dabei um die replikative, hexamerische Helikase Large Tumor-Antigen (T-Antigen) vom Simian-Virus 40 und die DNA-Reparatur-Helikasen RecQ2 und RecQ3 der Pflanze Arabidopsis thaliana. Um DNA-Entwindung in Echtzeit zu untersuchen, wird eine biophysikalische Einzelmolekültechnik, die \"Magnetische Pinzette\", verwendet. Mit dieser Technik kann man ein DNA-Molekül, das an ein magnetisches Partikel gebunden ist, strecken und gleichzeitig dessen Gesamtlänge messen. Mit speziellen DNA-Konstrukten kann man so bestimmte Eigenschaften der Helikasen bei der DNA-Entwindung, wie z.B. Geschwindigkeit, Länge der entwundenen DNA (Prozessivität) oder den Einfluß von Kraft, ermitteln. Es wird gezeigt, dass T-Antigen eine der langsamsten und prozessivsten Helikasen ist. Im Gegensatz zu prokaryotischen Helikasen ist die Entwindungsgeschwindigkeit von T-Antigen kaum kraftabhängig. Aktuelle Modelle sagen dieses Verhalten nicht vorraus, weshalb ein alternatives Modell entwickelt wird. Die untersuchten RecQ-Helikasen zeigen ein Entwindungsverhalten bei dem permanent kurze Abschnitte von DNA entwunden und wieder zusammengeführt werden. Dieses Verhalten wird hier zum ersten Mal unter dem Einfluß externer Kräfte gemessen. Es wird gezeigt, dass die permanente Entwindung auf die Fähigkeit beider Helikasen, von einem einzelen DNA-Strang auf den anderen zu wechseln, zurückzuführen ist. Obwohl RecQ2 und RecQ3 beide das Verhalten des permanenten Entwindens aufzeigen, unterscheiden sie sich stark in anderen Eigenschaften. Der gravierendste Unterschied ist, dass RecQ2 wie eine klassische Helikase die DNA entwindet, während RecQ3 eher bestrebt ist, die DNA-Einzelstränge wieder zusammenzuführen. Die unterschiedlichen Eigenschaften könnten die verschieden Aufgaben beider Helikasen während DNA-Reparaturprozessen widerspiegeln. Weiterhin werden die experimentellen Methoden optimiert, um möglichst hohe Auflösungen der Daten zu erreichen. Dazu zählen der Aufbau einer verbesserten und stabileren \"Magnetischen Pinzette\" mit sub-nanometer Auflösung und die Entwicklung neuer Methoden, um DNA Konstrukte herzustellen. Außerdem wird die Torsions\\-steifigkeit von magnetischen Partikeln in externen magnetischen Feldern untersucht. Dabei finden sich Auswahlkriterien für DNA-gebundene magnetische Partikel, durch die eine hohe Auflösung erreicht wird.

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Knowledge of the properties of single molecule diffusion is important for controlling dynamic self-assembly of molecular structures. A powerful experimental technique for determining diffusion coefficients is the recording of diffusion-induced signal fluctuations by a locally fixed point-like probe. Here, the signal becomes modified, whenever a molecule enters a certain detection area on the surface under the probe. The technique is minimal invasive and has a very good time resolution, enabling the investigation of highly mobile molecules. Theories are necessary for the analysis of the fluctuations and the extraction of diffusion properties. In this thesis, three methods are presented, which are based on the autocorrelation function, the distribution of peak widths and the distribution of interpeak intervals. Analytical expressions are derived for the distributions and the autocorrelation function in case of molecules, which can be described by circular or rectangular shapes. For rectangular shaped molecules, rotational diffusion can influence the recorded fluctuations. To allow for a simultaneous determination of rotational and translational diffusion coefficients the analytical treatment is extended. Furthermore, new methods are developed to determine the diffusion tensor for anisotropic stochastic molecular motion, using either one linearly extended probe or two individual probes. Coarse-graining the signal recorded by a point-like probe, which repeatedly moves on a line or a circle, is suggested for experimental implementation. All facets of the evaluation methods are verified against kinetic Monte Carlo simulations. Applications to experimental data, recorded by a locally fixed scanning tunneling microscope tip, are demonstrated for copperphthalocyanine and PTCDA molecules diffusing on Ag(100).