Le ribosome est un organite essentiel de la cellule qui assure la synthèse des protéines. C'est une structure très conservée, composée d'ARN et de protéines ribosomiques organisés en deux sous-unités. Les expériences de reconstitution in vitro du ribosome d'E. coli ont montré que l'assemblage est un processus coordonné impliquant de nombreuses interactions entre les différents constituants. En particulier, les premières étapes de l'assemblage de la grande sous-unité dépendent fortement de la fixation coopérative de cinq protéines ribosomiques à l'ARN 23S, mais les mécanismes moléculaires sous-jacents sont mal connus.Cette étude à l'échelle de la molécule unique vise à préciser ces mécanismes et porte sur un fragment constitué des hélices H18, H19 et H20 du domaine I de l'ARN ribosomique 23S contenant les sites de fixation des protéines uL24 et uL4. Ce fragment d'ARN a été préparé dans une configuration qui permet la mesure de force via un double piège optique. Les courbes de force obtenues ont permis de dresser une cartographie de la stabilité des structures du fragment d'ARN.Ces cartes ont été comparées en absence et en présence des protéines ribosomiques uL24 et/ou uL4, démontrant ainsi que le fragment d'ARN est stabilisé par la fixation des protéines uL24 et/ou uL4. Leur fixation est coopérative et la présence conjointe des deux protéines sur-stabilise les structures du fragment d'ARN.Ces résultats sont discutés dans la perspective de préciser le rôle du fragment d'ARN et des protéines ribosomiques uL24 et uL4 dans l'assemblage de la grande sous-unité du ribosome. / Ribosomes are essential organelles of the cell responsible for the synthesis of proteins. Their well conserved structure made of RNA and proteins is organized into two subunits. In vitro reconstitution of E. coli ribosomes showed that their assembly is a coordinated process which involves many interactions between the components. More specifically, the early stages of the large subunit assembly depend strongly on the cooperative binding of five ribosomal proteins to the 23S RNA. The underlying molecular mechanisms however remain poorly understood.The aim of this study is to shine new light on these mechanisms at the single molecule level. It focuses on a 23S ribosomal RNA fragment composed of the helices H18, H19 and H20 in domain I which encompasses the binding sites of the ribosomal proteins uL24 and uL4. This RNA fragment has been prepared in a dumbbell configuration and force versus displacement measurements have been performed using a dual optical trap. From these measurements, a map summarizing the mechanical stability of the RNA fragment has been determined.The maps obtained in absence and in presence of the ribosomal proteins uL24 and/or uL4 have been compared consequently demonstrating mechanical stabilization of the RNA fragment induced by the binding of uL24 and/or uL4. Moreover, their binding is cooperative and when both are present, the mechanical stabilization of the RNA fragment is enhanced.These results are discussed to specify the role of the RNA fragment and proteins uL24 and uL4 in the large ribosomal subunit assembly.

Desde o desenvolvimento dos primeiros métodos de controle do movimento e posição de partículas usando lasers, ainda no início da década de 1970, até o reconhecimento com o prêmio Nobel de Física de 2018, uma das principais e mais versáteis ferramentas de manipulação óptica, as chamadas pinças ópticas, têm sido usadas majoritariamente para explorar objetos em dois regimes de tamanho: o limite das partículas sub-nanométricas (átomos e moléculas simples) e o limite das partículas micrométricas (com aplicações especialmente em sistemas biológicos). Nesse trabalho, foi desenvolvido e construído um aparato experimental para aprisionar micro e nanopartículas numa pinça óptica, que pode ser controlada de forma dinâmica usando modulação acusto-óptica do feixe de aprisionamento. A calibração da pinça óptica foi feita por diversos métodos, incluindo o método de equipartição de energia e análise do potencial óptico, resultando em forças de aprisionamento da ordem de piconewtons por micrometros. Ademais, simulações computacionais de modelos estocásticos foram realizadas com o intuito de comparar os resultados experimentais com àqueles previstos teoricamente e guiar estudos futuros. / Since the development of early methods for controlling the motion and position of particles using lasers, in the 1970s, to the recognition with the 2018 Nobel Prize for Physics, one of the most versatile optical manipulation tools, the so-called optical tweezers, have been used mostly to explore objects in two limits of sizes: the sub nanometric particles (atoms and simple molecules) and the micrometric particles (with applications especially in biological systems). In this work, an experimental apparatus was developed and built to trap micro and nanoparticles in an optical tweezer that can be dynamically controlled, using acoustic-optical modulation of the trapping beam. The calibration of the optical tweezer was done using several methods, including the energy equipartition method and optical potential analysis, resulting in trapping forces on the order of piconewtons per micrometers. In addition, computational simulations of stochastic models were performed with the purpose of comparing the experimental results with those predicted theoretically and guiding future studies.

In an effort to develop an optical-acoustical understanding of ultrasound contrast agent microbubble dynamics in a micro-environment that resembles blood vessels, this thesis presents experimental work on optical trapping and acoustical probing of ultrasound contrast agent microbubbles confined in regenerated cellulose capillaries. First, we showed by acoustical means that the pressure threshold of an individual microbubble shell rupture increases significantly when confined in regenerated cellulose capillaries. We report that the shell rupture threshold in regenerated cellulose capillaries increased by at least 0.3 MPa from 0.8 MPa for unconfined microbubbles. Second, we achieved optical trapping and manipulation of ultrasound contrast agent microbubbles confined in capillaries using Hermite-Gaussian laser beams. / Graduation date: 2012

Troger's base was the first amine to be resolved where the chirality was due to solely very high inversion barrier around nitrogen atom. Though the molecule was known for over a century, work done during the past decade has shown that Troger's base and its analogues could be used as chiral solvating agent, DNA binding ligand and for the construction of biomimetic molecular receptors and clathrare hosts. Asymmetric synthesis of the Troger’s base analogues has also been achieved recently. Because of the rigid, 'V'-shaped chiral nature of this molecule, there is growing interest for the use of this unit in the design of potential host systems. This section briefly describes the chemistry of Troger's base developed over a century.

In dieser Arbeit wurden Nanopartikel mit einer Laserpinzette gespeichert und untersucht. Ein Schwerpunkt war speziell die Untersuchung der bei der Speicherung beteiligten Kräfte. Diese Abhängigkeiten wurden bei variablen Drücken im Bereich von einigen mbar betrachtet. Als Testpartikel dienten µm große Agglomerate aus Nanodiamanten. Aus den gemachten Beobachtungen wurden Erkenntnisse über das Verhalten und die Wechselwirkung von Streukraft, Gradientenkraft und photophoretischer Kraft gewonnen. Ein zweiter Schwerpunkt war die Charakterisierung der Partikel. Mit einem einfachen optischen Aufbau konnten Fluoreszenzspektren von Nanodiamanten mit N-V-Zentren aufgenommen werden. Zusätzlich wurden einige der Partikel mit dem Laserfarbstoff Rhodamin 6G benetzt und spektroskopiert.

Optical tweezers have been widely used by biophysicists to measure forces in single molecular processes, such as the force of a motor molecule walking and the force of a DNA molecule winding and unwinding. In these and similar force measurements, the usual assumption is that the force applied to a particle inside the tweezers is proportional to the displacement of the particle away from the trap center like Hookean springs, which would imply that the force field is conservative. However, the Gaussian beam model has indicated that the force field generated by optical tweezers is actually nonconservative, yet no experiments have measured or accounted for this effect. We introduce an experimental method -- the local drift method -- that can measure the force field in optical tweezers with high precision without any assumptions about the functional form of the force field. The force field is determined by analyzing the Brownian motion of a trapped particle. We successfully applied this method to different sizes of particles and measured the three dimensional force field with 10 nm spatial resolution and femtonewton precision in force. We find that the force field is indeed nonconservative. The nonconservative contribution increases radially away from the optical axis for both small and large particles. The curl vector field -- a measurement of the nonconservative force field -- reverses direction from counter-clockwise for small particles in the Rayleigh regime to clockwise for large particles in the ray optics regime, consistent with the different scattering force profiles in the two distinct scattering regimes. Together with the thermal fluctuations of the trapped particle, the nonconservative force can cause a complex flux of energy into the system. Optically-confined Brownian motion is further used to probe nanostructures such as a biopolymer network. This technique -- thermal noise imaging -- uses a Brownian particle as a "natural scanner" to explore a biopolymer network by moving the Brownian particle through the network with optical tweezers. The position fluctuations of the probe particle reflect the location of individual filaments as excluded volumes. The resolution of thermal noise imaging is directly coupled to the size of the probe particle. A smaller probe is capable of exploring smaller pore sizes formed by dense network. Previously, a 200 nm polystyrene particle had been used to probe an agar network. In this work, 100 nm gold probe particles are used to enhance the resolution. A 100 nm particle explore a network with mesh 2³ times smaller and therefore enhance the network resolution by 2³ times. A 100 nm particle also improves the imaging speed by a factor of 2 because of its faster diffusion. Three-dimensional thermal noise images of agarose filaments are obtained and a resolution of 10 nm for the position of the filaments is achieved. In addition, a gold particle is trapped with significantly less power than a polystyrene particle of the same size, indicating the possibility for using even smaller gold particles to further improve the resolution. / text

This dissertation combines various holographic techniques with application on the two- and three-dimensional imaging of ophthalmic tissue, fingerprints, and microsphere samples with micrometer resolution. Digital interference holography (DIH) uses scanned wavelengths to synthesize short-coherence interference tomographic images. We used DIH for in vitro imaging of human optic nerve head and retina. Tomographic images were produced by superposition of holograms. Holograms were obtained with a signal-to-noise ratio of approximately 50 dB. Optic nerve head characteristics (shape, diameter, cup depth, and cup width) were quantified with a few micron resolution (4.06 -4.8 microns). Multiple layers were distinguishable in cross-sectional images of the macula. To our knowledge, this is the first report of DIH use to image human macular and optic nerve tissue. Holographic phase microscopy is used to produce images of thin film patterns left by latent fingerprints. Two or more holographic phase images with different wavelengths are combined for optical phase unwrapping of images of patent prints. We demonstrated digital interference holography images of a plastic print, and latent prints. These demonstrations point to significant contributions to biometry by using digital interference holography to identify and quantify Level 1 (pattern), Level 2 (minutia points), and Level 3 (pores and ridge contours). Quantitative studies of physical and biological processes and precise non-contact manipulation of nanometer/micrometer trapped objects can be effectuated with nanometer accuracy due to the development of optical tweezers. A three-dimensional gradient trap is produced at the focus position of a high NA microscope objective. Particles are trapped axially and laterally due to the gradient force. The particle is confined in a potential well and the trap acts as a harmonic spring. The elastic constant or the stiffness along any axis is determined from the particle displacements in time along each specific axis. Thus, we report the sensing of small particles using optical trapping in combination with the digital Gabor holography to calibrate the optical force and the position and of the copolymer microsphere in the x, y, z direction with nm precision.

Eukaryotic genomes are densely compacted into chromatin, so that they can be contained in the nucleus. Despite the tight packaging genes need to be accessible for normal metabolic activities to occur, such as transcription, repair and replication. These processes are regulated by a vast number of proteins but also by the level of compaction of chromatin. The translocation of motor proteins along DNA produces torsional stress which in turn alters chromatin compaction both upstream and downstream. Few single-molecule studies have investigated the behaviour of nucleosomes when subjected to torsion. The inability to measure the applied torque though represented a major limitation to those reports. The implementation of the rotor bead assay, which allows to directly measure the torque applied in magnetic tweezers experiments, has been hindered by a difficult sample preparation procedure. In order to overcome this limitation an efficient protocol for the insertion of chemical or structural modifications in long DNA substrates was developed. This was then further expanded to allow the introduction of labels in multiple loci and/or both strands and has been used successfully in a number of studies. Furthermore this is the first report of tensile experiments performed on nucleosomes with a histone variant. H2AvD nucleosomes were studied due to the interest in the biological role of H2A.Z-family proteins. Interestingly, the variant nucleosomes appear to bind less DNA and to be evicted from the DNA at lower forces than those observed for canonical nucleosomes. These findings show an important role for the H2A-H2B dimers in the mechanical stability of nucleosomes. Furthermore these results are in agreement with recently proposed models of a dynamic nucleosome, in contrast to the long-standing view of nucleosomes as static structures.

Von Willebrand factor (VWF) is a multimeric glycoprotein that critically supports platelet aggregation in hemostasis. Disordered VWF function causes both thrombotic and bleeding disorders, and genetic defects in VWF are responsible for von Willebrand’s disease (VWD), the most common inherited bleeding disorder in humans. Very large VWF multimers exhibit the greatest thrombogenic activity, which is attenuated by ADAMTS13 cleavage in the A2 domain. A2 cleavage is regulated by mechanical force, and pathologically high shear forces are known to enhance proteolysis and cause bleeding in patients. Enhanced cleavage is also described in patients with VWD 2A mutations. In contrast, VWF A2 is stabilized against cleavage by a calcium binding site within A2. Single molecule studies have demonstrated that mechanical unfolding is required for A2 cleavage to expose the scissile bond. In this dissertation, we aim to better understand the mechanosensitivity of A2 cleavage by characterizing the force sensitivity of A2 unfolding and refolding. We first characterized the interaction between VWF A2 and calcium using bulk isothermal calorimetry and thermal denaturation assays. In parallel, we used single molecule optical tweezers to characterize A2 unfolding and refolding. Calcium was found to bind A2 with high affinity, stabilize A2 against thermal denaturation, and enhance domain refolding. In contrast, we found that VWD 2A mutations destabilize the A2 domain against thermal denaturation. R1597W, the most common VWD 2A mutation, lies within the calcium binding loop and exhibited diminished calcium stabilization against thermal denaturation. Using optical tweezers, we found that R1597W also diminished A2 refolding. R1597W refolding in the presence of calcium was similar to that of wild-type A2 in the absence of calcium, suggesting that loss of calcium stabilization contributes to the disease mechanism of R1597W. Other VWD 2A mutations lying outside the calcium binding loop also destabilized A2, but retained calcium mediated stabilization. These studies provide a better understanding of VWD 2A pathophysiology and offer structural insights into A2 unfolding and refolding pathways. By exploring the role of mechanical force in regulating VWF cleavage, this work moves towards a better understanding of how hydrodynamic forces within the vasculature regulate VWF function in hemostasis and thrombosis.

In dieser Dissertation werden die Bindungseigenschaften von Rezeptor-Ligand-Komplexen mit Hilfe von Optischen Pinzetten untersucht. Aufgrund ihrer außerordentlichen Orts- (2nm) und Kraftauflösung (0,2pN) ist es möglich, diese spezifischen Interaktionen anhand einzelner Bindungsereignisse zu charakterisieren. Als Modellsysteme dienen die Wechselwirkungen zwischen den phosphorylierungsspezifischen, monoklonalen Antikörpern HPT-101 und HPT-104 und dem Morbus Alzheimer relevanten Tau-Peptid. Dieses pathogen veränderte Peptid wird krankheitsspezifisch an den Aminosäuren Threonin231 und Serin235 phosphoryliert, sodass die Detektion dieses Phosphorylierungsmusters mit Hilfe von monoklonalen Antikörpern eine mögliche Früherkennung der Alzheimer-Krankheit darstellt. Eine notwendige Voraussetzung dafür ist jedoch die exakte Kenntnis der Bindungsstellen des Liganden am Rezeptor. Ziel des ersten Teils dieser Arbeit ist es, das Epitop des monoklonalen Antikörpers HPT-101 zu bestimmen. Dazu werden mögliche bindungsrelevante Aminosäuren durch ein Alanin ausgetauscht (Alanin-Scan) und so insgesamt sieben neue Tau-Isoformen aus dem ursprünglichen doppelt-phosphorylierten Peptid Tau[pThr231/pSer235] hergestellt. Die jeweiligen Interaktionen zwischen den modifizierten Peptiden und dem Antikörper werden mit der dynamischen Kraftspektroskopie untersucht und mit Hilfe eines literaturbekannten Modells analysiert. Die sich daraus ergebenden Bindungsparameter (Lebensdauer der Bindung, charakteristische Bindungslänge, freie Aktivierungsenergie und Affinitätskonstante) werden zusammen mit den relativen Bindungshäufigkeiten erstmals genutzt, um Kriterien für essentielle, sekundäre und nicht-essentielle Aminosäuren im Tau-Peptid zu definieren. Bemerkenswerterweise existieren für insgesamt drei dieser Parameter (Bindungslebensdauer, Bindungslänge und Affinitätskonstante) scharfe Klassengrenzen, mit denen eine objektive Einteilung des Epitops von Antikörper HPT-101 möglich ist. Die erhaltenen Ergebnisse sind in überzeugender Weise im Einklang mit ELISA-Messungen zu diesem Antikörper-Peptid-Komplexen, sie liefern jedoch einen tieferen Einblick in die Natur einer spezifischen Bindung, da den kraftspektroskopischen Messungen auch die Bindungskinetik zugänglich ist. Das zweite Projekt der vorliegenden Dissertation etabliert eine Methodik, um die Datenvarianz in der Bestimmung der relativen Bindungshäufigkeit zu reduzieren. Anhand einer Kombination aus Fluoreszenz- und kraftspektroskopischen Messungen werden die Wechselwirkungen zwischen dem monoklonalen Antikörper HPT-104 und dem fluoreszenzmarkierten Peptid Tau[Fl-pThr231] untersucht. Es wird gezeigt, dass durch Vorsortieren der Peptid-beschichteten Kolloide, entsprechend ihrer Oberflächenbeladung, die Datenvarianz in den Bindungshäufigkeitsmessungen signifikant reduziert wird.