Biological membranes are heterogeneous two-dimensional fluids composed of lipids, sterols and proteins that act as complex gateways and define the cell boundary. The functions of these interfaces are diverse and specific to individual organisms, cell types, and tissues. Membranes must take up nutrients and small molecules, release waste products, bind ligands, transmit signals, convert energy, sense the environment, maintain cell adhesion, control cell migration, and much more while forming a tight barrier around the cell. The molecular mechanisms and structural details responsible for this diverse set of functions of biological membranes are still poorly understood, however. Developing new tools capable of probing and determining the local molecular organization, structure, and dynamics of membranes and their components is critical for furthering our knowledge about these important cellular processes that are often linked to health and diseases. Combinatorial microscopy takes advantage of the rich properties of light (intensity, wavelength, polarization, etc.) to create new forms of imaging that quantify the motions, orientations, and binding kinetics of the sample’s biomolecular constituents. These new optical imaging modalities can also be further combined with other types of microscopy to produce spatially correlated micrographs that provide complementary pieces of information about the sample under investigation that would otherwise remain hidden from the observer if the two imaging techniques were applied independently. The first part of this thesis provides a detailed account of the construction of a specialized hybrid microscopy platform that combines polarized total internal reflection fluorescence microscopy (pTIRFM) with atomic force microscopy (AFM) for the purpose of studying fundamental sterol-lipid and antimicrobial peptide-lipid interactions in model membranes. The second half describes a combined pTIRFM and Förster resonance energy transfer (FRET) imaging method to elucidate the oligomeric state and spatial distribution of carcinoembryonic-antigen-related cell-adhesion molecules (CEACAMs) in the membranes of living cells.

Biological membranes are heterogeneous two-dimensional fluids composed of lipids, sterols and proteins that act as complex gateways and define the cell boundary. The functions of these interfaces are diverse and specific to individual organisms, cell types, and tissues. Membranes must take up nutrients and small molecules, release waste products, bind ligands, transmit signals, convert energy, sense the environment, maintain cell adhesion, control cell migration, and much more while forming a tight barrier around the cell. The molecular mechanisms and structural details responsible for this diverse set of functions of biological membranes are still poorly understood, however. Developing new tools capable of probing and determining the local molecular organization, structure, and dynamics of membranes and their components is critical for furthering our knowledge about these important cellular processes that are often linked to health and diseases. Combinatorial microscopy takes advantage of the rich properties of light (intensity, wavelength, polarization, etc.) to create new forms of imaging that quantify the motions, orientations, and binding kinetics of the sample’s biomolecular constituents. These new optical imaging modalities can also be further combined with other types of microscopy to produce spatially correlated micrographs that provide complementary pieces of information about the sample under investigation that would otherwise remain hidden from the observer if the two imaging techniques were applied independently. The first part of this thesis provides a detailed account of the construction of a specialized hybrid microscopy platform that combines polarized total internal reflection fluorescence microscopy (pTIRFM) with atomic force microscopy (AFM) for the purpose of studying fundamental sterol-lipid and antimicrobial peptide-lipid interactions in model membranes. The second half describes a combined pTIRFM and Förster resonance energy transfer (FRET) imaging method to elucidate the oligomeric state and spatial distribution of carcinoembryonic-antigen-related cell-adhesion molecules (CEACAMs) in the membranes of living cells.

L'étude des fluctuations dans les milieux confinés constitue un domaine de recherche très récent, que ce soit du point de vue théorique ou expérimental. Afin d'analyser le rôle du confinement sur les propriétés des fluctuations de densité dans un mélange binaire, nous avons développé un système de mesure d'une grande sensibilité, où l'intensité des fluctuations et leur longueur de corrélation peuvent être amplifiées. L'idée consiste à travailler proche du point critique d'une transition de phase de démixion d'un mélange binaire. En effet, la longueur de corrélation augmente exponentiellement lorsqu'on s'approche de la température Tc du point critique de démixion.Nous avons développé un montage permettant de confiner le mélange entre un échantillon plan et une sphère colloïdale attachée à l'extrémité d'un levier de Microscope à Force Atomique (AFM). D'après les prédictions de Fisher et De Gennes, un effet intéressant émerge lorsque la longueur de corrélation est comparable avec la taille du confinement: les deux surfaces vont soit s'attirer, soit se repousser suivant les préférences d'adsorption des composants du mélange pour chacune des surfaces. On nomme cet effet l'effet Casimir critique, en référence à la force de Casimir électrodynamique qui résulte du confinement des fluctuations quantiques du champ électromagnétique.Durant cette thèse, nous avons mesuré la force de Casimir électrodynamique avec le système de mesure que nous avons développé, d'abord dans une atmosphère d'azote puis dans l'éthanol. Ces mesures prouvent que notre appareil de mesure est assez sensible pour mesurer des forces très faibles de l'ordre de la dizaine de pN. Les forces mesurées sont comparées à la théorie de Lifshitz, où les effets de conductivité finie des surfaces sont considérées. / The study of density fluctuations inside confined liquid systems has received the attention of recent theoretical and experimental papers. In order to analyze the role of confinement on the statistical properties of fluctuations, we developed a highly sensitive system where the intensity of fluctuations, as well as their spatial correlation length can be simply tuned. The idea will be to enhance the role of fluctuations working close to the critical temperature Tc of a second order phase transition in a binary mixture. Indeed, the correlation length dramatically increases when one approaches the critical demixion point.The confinement is obtained by using a sphere-plane geometry with a colloidal particle attached to the cantilever of an Atomic Force Microscope (AFM). When the correlation length is comparable with the distance of confinement, Fisher and De Gennes predicted the existence of an interesting effect: the two surfaces will be submitted to either an attracting or a repelling force, depending on boundary conditions. This effect is called the critical Casimir force in reference to the quantum Casimir force resulting from the confinement of quantum fluctuations of the electromagnetic field.During this thesis, we measured the quantum Casimir force between the sphere and the plate, first in a nitrogen atmosphere and then in ethanol, showing that the developed instrument is sufficiently sensible to measure very weak force, of the same order of magnitude or even weaker than the critical Casimir force. Measurements are compared to Lifshitz theory, taking into account the finite conductivity of surfaces.

No description available.

<p>Peptides, specifically cell-penetrating peptides (CPP), have become wonderful research tools due to their enhanced stability, solubility, and ease of synthesis. They have been used for a wide range of biomedical applications, from insecticides to biosensors and drug-delivery scaffolds. The work presented in this dissertation characterizes the biophysical properties of two different CPPs. The first is the cationic amphiphilic polyproline helix (CAPH) peptide, P14LRR. In addition to cell penetration, this CPP has demonstrated broad spectrum antibacterial properties. Fluorescence polarization (FP) and SEC-MALS were conducted to understand the dissociation constant (KD) and oligomerization effects of P14LRR with respect to its putative molecular target in Staphylococcus aureus (S. aureus). A biotinylated derivative of this peptide was also used as a drug-delivery scaffold to transport fluorescently conjugated streptavidin into mammalian cells. A second CPP, DAN13, was also developed as a biosensor for phosphoinositide lipids, specifically PI(4,5)P2. This was effected through careful calibration using stacked supported lipid bilayers (SSLB) in combination with total internal reflection fluorescence (TIRF) microscopy. This was then used to determine the absolute densities and spatial distribution of PIP2 in live KRas mutant cells.</p>

No description available.

Das IceCube Neutrino-Observatorium ist ein am geografischen Südpol im Eis installierter Neutrinodetektor. In IceCube werden Neutrinos mit Tscherenkow-Strahlung von Sekundärteilchen aus Neutrino Interaktionen detektiert. Für den Nachfolgedetektor IceCube-Gen2, werden neue und verbesserte Lichtdetektoren gesucht. Die vorliegende Arbeit beschreibt die Entwicklung eines dieser Lichtdetektoren. Dieser basiert auf Wellenlängen schiebenden und Licht leitenden Technologien. Der Detektor mit dem Namen "Wavelength-shifting Optical Module" (WOM) verwendet eine transparente Röhre, mit wellenlängenschiebender Farbe, als passiver Photonendetektor. Das in der Wellenlänge verschobene Licht wird durch Totalreflexion, zu kleinen PMTs an beiden Enden geleitet. Die Auswahl dieses Designs reduziert die Kosten und verbessert das Signal-Rausch-Verhältnis wesentlich, möglicherweise können mit dieser Lösung extragalaktische Supernova in zukünftigen Detektoren beobachtet werden. Als eine Kernkomponente wird die wellenlängenschiebende Röhre ausführlich untersucht. Verschiedene Messaufbauten und Auswertungsmethoden werden entwickelt, um diese im Anschluss zu untersuchen und zu bewerten. Iterative Verbesserungen der Materialien und des Farbauftrageverfahren als auch Messmethoden, resultieren in einer kombinierten Einfang-, Wellenlängenschiebe- und Transporteffizienz von 28,1 +/- 5,4 % der Röhre. Ein Model zur Beschreibung des Lichtverhaltens in der Röhre wird entwickelt um eine Diskrepanz zwischen Theorie und Messung zu untersuchen. Die Kombination zwischen Messung und Model, bestätigt die Aussagekraft des Models und zeigt, dass ein Großteil der Verluste beim Lichttransport zustande kommen. Darüber hinaus werden die physikalischen Eigenschaften des WOM in die IceCube Simulationsumgebung eingebaut. Der Vergleich zu einem Konkurrenzmodul zeigt eine Überlegenheit des WOM um den Faktor 1,05 +/- 0,07. Es werden Vorschläge und Ausblicke für Verbesserungen der Leistungsfähigkeit des WOMs gegeben. / The IceCube Neutrino Observatory is an in ice neutrino detector located at the geographic South Pole. In IceCube neutrinos are detected via Cherenkov light produced by secondary particles in neutrino interactions. For the upgraded detector IceCube-Gen2, new and improved light detectors are sought-after. This work describes the development of one of those light detectors based on a novel combination of wavelength-shifting and light-guiding technology. The detector named the Wavelength-shifting Optical Module (WOM) utilizes a large transparent tube, coated with wavelength-shifting paint as a passive photon detector. The wavelength-shifted light is guided via total internal reflection towards small active light detectors, at each end of the tube. This design reduces costs and improves the signal to noise ratio significantly, thereby potentially enabling extragalactic supernova detections in future detectors. As a core component, the wavelength-shifting tube is extensively investigated. Different measurement setups and evaluation techniques are developed and investigated. Iterative improvement of materials and coating techniques as well as measurement methods currently result in a combined photon capture, shift and transport efficiency of 28.1 +/- 5.4 % for the tube. Those results contrast the theoretical maximum of 74.5 %. A model is developed to describe the light propagation and loss processes in the tube and to understand the discrepancies between theory and measurement. The combination of the measurements with the model, validate the descriptive qualities of the model and show that most of the light is lost during the light propagation in the tube. Additionally, the physical properties of the WOM are included in the IceCube simulation framework. A comparison to a competing module showed that the WOM outperforms by a factor of 1.05 +/- 0.07 in photon detection numbers. Where applicable, suggestions and outlooks are given to enhance the performance of the WOM.

No description available.