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This thesis focuses on the study of UV light-generated zinc complexes with cadmium and organic molecules SAM, SAH, CYS, HCYS and GSSG, specifically at 375 nm. Furthemore, the aim of the work is to characterize the precursors spectrally and temporally before and after irradiation in the transilluminator at 250 nm. Study of genesis these complexes was performed by FLIM. Thanks to this method, it was found that the formation of complexes occurs only with Zn:SAH, Zn:GSSG and Zn:Cd. The formation of complexes is influenced by the method of preparation. The spectral characteristic was performed on a fluorimeter where the increase in fluorescence intensity of the irradiated solution with the precursors was expected. These were turbid solutions where sedimentation of the particles was observed and the intensity of fluorescence was changed. In the Zn:SAM and Zn:CYS sample, the sedimentation increased in intensity, while in Zn:SAH and Zn:HCYS decreased. The Zn:Cd precursor solution was clear and there was no change in intensity. Zn:Cd showed the best spectral properties, while the Zn:SAM sample, whose excitation and emission maxima are very close to each other, appeared to be the worst. A sample with Zn:CYS and Zn:HCYS showed almost the same spectra and respective peak results. Based on lifetime characteristics by TCSPC, the sample with Zn:CYS, Zn:HCYS and Zn:GSSG, which showed 3 lifetimes, was best treated. Lifetime could not be unambiguously determined for SAM and SAH samples. Zn:Cd had 4 lifetimes

The theoretical part of this master's thesis is focused on research of confocal microscopy and FLIM method. There are a principles and types of confocal microscopy and the use of broad-spectrum laser as a basic light source of these microscopes. It gives what the FLIM method and its use not only in cell biology. The practical part thesis includes the acquisition of three sets of fluorescence intensity images with use of applications tunable pulsed laser, function TimeGate and detection of hybrid detectors. For practical elaboration of this thesis is in the software Fiji created a plugin, which is the source code in the Java programming language. The types of plugins and their uses are described in the third chapter of the thesis. This plugin including the graphical user interface in the form of the dialog box, proceses the fluorescence intensity images and creates a graphical representation of data showing the fluorescence lifetime, so called pseudocolor map.

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Fluorescence lifetime imaging microscopy (FLIM) is an imaging modality that is able to provide key insights into subcellular processes. When used to measure Förster resonance energy transfer (FRET), for instance, it can discern protein-protein interactions and conformational changes. This kind of information is highly useful in the drug screening process in order to determine the effectiveness of drug leads and their mechanisms of action. FLIM has yet to be successfully translated to high-content screening (HCS) platforms due to the high throughput and fine temporal and spatial resolution requirements of HCS. Our prototype HCS FLIM system uses a time-resolving instrument called a streak camera to multiplex the FLIM scanning process, allowing for 100 confocal spots to be simultaneously scanned across a sample. There have been a few major advancements to the prototype. First the fiber array used to connect the fluorescence channels to the streak camera was characterized. Its alternating fiber delay scheme was successful in greatly reducing optical crosstalk between adjacent channels. Next, an optical beam scanner for parallel excitation beams was designed and implemented, greatly improving the possible scan speeds of the system. The streak camera was upgraded to a higher repetition rate sweep, and modifications to system components and reconstruction procedures were made to accommodate the new sweep unit. A single-photon avalanche diode array was also tested as a possible replacement for the streak camera, and was found to offer photon detection efficiency advantages. Finally, improvements were made to the excitation power and optical throughput of the system in order to reduce the required exposure time. These advances to the prototype system bring it closer to realizing the requirements of HCS FLIM, and provide a clear picture for future improvements and research directions. / Thesis / Doctor of Philosophy (PhD) / Fluorescent proteins are commonly used to tag subcellular targets so that they can easily be distinguished with a fluorescence microscope. While this can help visualize where different organelles and proteins are located in the cell, a great deal more information can be gained by measuring the fluorescence lifetime at each point in the sample, which is highly sensitive to the microenvironment. Fluorescence lifetime imaging microscopy (FLIM) has the potential to be a powerful technique for testing drug leads in the drug discovery process, although current FLIM systems are not able to provide the high throughput speeds and high temporal resolution required for drug screening. This thesis project has succeeded in improving a highly parallel FLIM microscope by reducing inter-channel crosstalk, implementing an optical scanner, improving power and optical throughput, and investigating future time-resolving instruments. This progress has brought the prototype setup closer to being used in a drug screening environment.

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This diploma thesis deals with the analysis of structural and dynamic organization of thyrotropin releasing hormone receptor (TRH-R) and δ-opioid receptor (DOR) within plasma membrane (PM) in relation to the specific sub-compartments of PM denominated as domains or membrane rafts. Modern fluorescence microscopy techniques FLIM, FRAP and RICS were used for this purpose. The experiments were performed on the live cells derived from HEK293 cell line. To reach the main goal of this work, the integrity of PM structure was altered by depletion of cholesterol which was performed by incubation of cells with β cyclodextrin. Results clearly support our previously suggested idea that the vast majority of TRH-R is localized in non-raft regions of plasma membrane. This work also compared different modes of performance of FRAP and results obtained by FRAP and RICS because these methods are to some extent analogous. This is one of the first works that used the RICS approach to characterize the G protein-coupled receptors. In the second part of this work, the setup of transient transfection of the HEK293 cells with DOR-ECFP and DOR EYFP constructs was established. Simultaneously, the functionality of these constructs, i.e. the ability of DOR to activate the cognate G protein was determined. Powered by TCPDF (www.tcpdf.org)

In der vorliegenden Arbeit wird ein neuer Ansatz vorgestellt, um Lipiddomänen, die Bindungsorte peripherer und integraler Membranproteine darstellen können, zu charakterisieren. Insbesondere wurde die Analyse der Fluoreszenzlebenszeiten von NBD-markierten Lipidanaloga benutzt, um Lipiddomänen in Giant unilamellar vesicles (GUV) und darauf aufbauend, in der Plasmamembran von Säugerzellen zu untersuchen. Das typische Zeitfenster von Fluoreszenzlebenszeiten im Bereich von Nanosekunden ermöglicht es, auch sehr kurzlebige Lipiddomänen nachzuweisen. Mit Hilfe des Fluorescence lifetime imaging (FLIM) wurden für die liquid disordered (ld) und liquid ordered (lo) Domänen in GUV jeweils spezifische Werte für das Abklingen der Fluoreszenz gemessen. Sogar die Existenz von submikroskopischen Domänen in GUV konnte nachgewiesen werden. Die Fluoreszenzlebenszeit des Lipidanalogs C6-NBD-PC zeigte in der Plasmamembran von Säugerzellen eine breite Verteilung. Dies legt in Übereinstimmung mit FLIM-Experimenten an aus der Plasmamembran von HeLa-Zellen gewonnenen Giant vesicles nahe, dass in der Plasmamembran von Zellen eine Vielzahl verschiedener submikroskopischer Lipiddomänen existiert. Darauf aufbauend wurde die Fluoreszenzmikroskopie an GUV angewendet, um die Bindung von fluoreszenzmarkiertem alpha-Synuclein an mittels FLIM charakterisierte Lipiddomänen zu untersuchen. Die Experimente zeigten, dass das Protein mit hoher Affinität an negativ geladene Phospholipide unter der Vorraussetzung bindet, dass diese sich in ld Domänen befinden. Im Gegensatz dazu erfolgt keine Bindung wenn diese Lipide in lo Domänen lokalisiert sind. Im Vergleich zum wildtypischen alpha-Synuclein zeigte die Variante A30P eine geringere Affinität zur Membran, während die E46K-Variante eine stärkere Bindung zeigte. Dies deutet darauf hin, dass bei den erblichen Formen des Morbus Parkinson eine veränderte Assoziation des alpha-Synucleins mit der Membran eine Rolle spielen kann. / In the present study a novel approach to characterize lipid domains, which may provide binding sites for peripheral or integral membrane proteins, is demonstrated. In particular, analysis of fluorescence lifetimes of NBD-labeled lipid analogues was used to study lipid domains in Giant unilamellar vesicles (GUV) and – based on the GUV results – in the plasma membrane of mammalian cells. As fluorescence decays in a few nanoseconds it is possible to to detect also very short-lived lipid domains. Fluorescence Lifetime Imaging (FLIM) revealed that the fluorescence decay of NBD-lipid analogues showed domain dependent decay times in the liquid disordered (ld) and the liquid ordered (lo) phase of GUV. Even the existence of submicroscopic domains in lipid membranes could be detected by FLIM. A broad distribution of the fluorescence lifetime was found for C6-NBD-PC inserted in the plasma membrane of mammalian cells. In agreement with FLIM studies on lipid domain forming Giant vesicles derived from the plasma membrane of HeLa-cells this may suggest that a variety of submicroscopic lipid domains exists in the plasma membrane of intact mammalian cells. Based on that, fluorescence microscopy was used on GUV to study the binding of fluorescently labeled alpha-synuclein at lipid domains previously characterized by FLIM. The experiments suggested that alpha-synuclein binds with high affinity to negatively charged phospholipids, when they are embedded in a ld as opposed to a lo environment. When compared with wildtype alpha-synuclein, the disease-causing alpha-synuclein variant A30P bound less efficiently to anionic phospholipids, while the variant E46K showed enhanced binding. This suggests that an altered association of alpha-synuclein with membranes may play a role in the inherited forms of Parkinson’s disease.

Der ABC-Transporter ABCA1 ist unmittelbar in die zelluläre Lipidhomeostasie einbezogen, in dem er die Freisetzung von Cholesterol an plasmatische Rezeptoren, wie ApoA-I, vermittelt. Trotz intensiver Untersuchungen ist dieser molekulare Mechanismus nicht verstanden. Verschiedene Studien deuten daraufhin, dass durch die Aktivität von ABCA1 bedingte Veränderungen in der Lipidphase der äußeren Hälfte der Plasmamembran (PM) wichtig für die Freisetzung des Cholesterols sind. In der vorliegenden Arbeit wird die Lipidumgebung von ABCA1 in der PM lebender Säugetierzellen unter Anwendung der Fluoreszenzlebenszeitmikroskopie von fluoreszierenden Lipidsonden untersucht. Es wurde eine breite Verteilung der Fluoreszenzlebenszeiten der Sonden gefunden, die sensitiv gegenüber Veränderungen der lateralen und transversalen Organisation der Lipide ist. Im Einklang mit Studien an riesengroßen unilamellaren Vesikeln und Plasmamembranvesikeln weisen unsere Ergebnisse die Existenz einer größeren Vielfalt submikroskopischer Lipiddomänen auf. Die FLIM-Untersuchungen an ABCA1 exprimierenden HeLa-Zellen weisen eine die Lipidphase destabilisierende Funktion des Transportes aus. Dieses wurde unterstützt durch die Lipidanalyse von Fraktionen der PM. Auf der Basis unserer Untersuchungen und früheren Daten stellen wir die Hypothese auf, dass die Exponierung von Phosphatidylserin (PS) auf der Zelloberfläche ein zentrales Ereignis der ABCA1 bedingten Veränderungen ist. Allerdings zeigen vergleichende Studien an ABCA7 exprimierenden Zellen, dass dies nicht ausreicht, um die ABCA1 verursachten Veränderungen in der Lipidpackung der PM zu erklären. Unsere Ergebnisse beweisen, dass die Fähigkeit von ABCA1, den Cholesterolefflux zu vermitteln, auf durch den Transporter bedingte Veränderungen in der LP der PM zurückzuführen sind, die unabhängig von der Bindung von ApoA-1 sind und dieser vorausgehen. Diese Veränderungen sind notwendig für die Lipidierung von ApoA-1 und der Generierung von HDL-Partikeln. / The ABCA1 transporter organizes cellular lipid homeostasis by promoting the release of cholesterol to plasmatic acceptors such as ApoA-I. Despite intensive investigation, the molecular mechanism of such a process has not yet been clarified. In the present study we report on the analysis of the ABCA1 lipid microenvironment at the plasma membrane of living cells, by a novel approach based on fluorescence lifetime imaging microscopy (FLIM). In the plasma membrane of mammalian cells, a broad fluorescence lifetime distribution sensitive to treatments interfering with the membrane lateral and transbilayer organization was found. In agreement with investigations in giant unilamellar vesicles and giant plasma membrane vesicles, our results are consistent with the existence of a large variety of submicroscopic lipid domains. Based on that, FLIM in HeLa cells expressing ABCA1 revealed the destabilizing function of the transporter on the lipid arrangement at the membrane, indicating that lipid packing was a primary target of ABCA1 activity. This was corroborated by the analysis of plasma membrane fractions isolated by density fractionation. On the basis of our analysis and previous data, we speculate that the exposure of phosphatidylserine on the cell surface is a central event for ABCA1-dependent modifications. However, a comparative study of cells expressing ABCA7, the member of the ABCA subfamily with the highest homology to ABCA1, revealed that exposure of PS alone cannot account for the detected effects. Collectively, our data suggest that the ability of ABCA1 to promote cholesterol efflux is independent and precedes its actual binding to ApoA-I. Rather, ABCA1-induced plasma membrane modifications are necessary for the lipidation of ApoA-I and the generation of high density lipoprotein particles.