Fluoreszenzmikroskopische Untersuchungen zur Temperatur- und Cholesterol-abhängigen Membrandynamik lebender Zellen
Auch gedruckt in der BibliothekZ: J-H 13.891; W: W-H 12.359
Ressourcen- / MedientypDissertation, Text
Datum der Freischaltung2011-01-12
Wide-field fluorescence microscopy is an important tool in biological and medical research. In this thesis different microscopic methods are combined for describing membrane dynamics - including mechanical stiffness and fluidity - of living cells. Membrane behaviour has a large impact on cell function and may be an important factor for pathogenesis of, e.g., cancer or Alzheimer’s disease. In addition, the influence of cholesterol, temperature and cell aging on membrane dynamics was examined. The membrane marker 6-dodecanoyl-2-dimethylamino naphthalene (laurdan) whose electronic excitation energy is different in polar and non-polar environments, proved to be useful for membrane studies. Its fluorescence spectrum shows a shift toward longer wavelengths when the laurdan molecules come into contact with adjacent water molecules of the cytoplasm, e.g., when membrane stiffness decreases or when a phase transition from the tightly packed gel phase to the liquid crystalline phase of membrane lipids occurs. Plasma membranes were selected by illumination with an evanescent electromagnetic field and distinguished from intracellular membranes assessed by whole-cell illumination. Membrane stiffness was found to increase with decreasing temperature and increasing amounts of cholesterol and was always higher for the plasma membrane than for intracellular membranes. These effects may have some clinical relevance in the research of drug resistance or cell aging. The methods established in this thesis were also used to study possible interactions of fluorescent derivatives of amyloid precursor protein (APP) and ß-secretase (BACE) which are relevant for Alzheimer’s disease.
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