Design and synthesis of anionic and photoactivatable fluorophores for super-resolution microscopy

Small-molecule fluorophores have been recognized as efficient fluorescent reporters in the visualisation of living cells by super-resolution fluorescence microscopy. Much of the ongoing research focuses on positively charged fluorophores derived from rhodamine cores, which show good photostability, live cell permeability and brightness, but suffer from undesired off-targeting and accumulation in certain cell compartments, most typically in mitochondria. Furthermore, their derived photoactivatable labels often rely photocleavable caging groups as bulky nitrobenzene-type groups or α-diazoketones, which may generate toxic or non-fluorescent by-products. Negatively charged fluorophores derived from fluorescein present a valuable alternative with no off-targeting and accumulation. Fluorescein, one of the oldest synthetic fluorophores, is a brightly emitting fluorophore, but suffer from low photostability and cell membrane-impermeability compared to rhodamines. Therefore, the rational design of negatively charged small-molecule fluorophores with improved photostability and cell membrane-permeability is desired. Furthermore, photoactivatable fluorescein analogs may rely on photocleavable groups less bulky than the nitrobenzene-types, which favor the generation of non-toxic by-products, and hence useful for super-resolution microscopy. Within this thesis, the chemical design and synthetic methods to access these fluorophores are discussed and the photophysical properties and application in live-cell super- resolution fluorescence microscopy examined. As a result of this work, N-cyanorhodamines have been identified as bathochromically shifted fluorescein analogs with improved photostability and cell-permeability. Three different synthetic methods have been developed to access these fluorophores from known aryl triflates or iodides. Unlike tetramethylrhodamine, N-cyanorhodamines demonstrate no off-targeting affinity. Based on these fluorophores, fluorescent ligands for self-labeling protein tags HaloTag and SNAP-tag have been prepared and applied in combination with commercially available labels in multi-color confocal and stimulated emission depletion microscopy. The second part of this thesis explores several different strategies to access photoactivatable fluorescein analogs bearing no or smallest possible photocleavable group. Protection of negatively charged fluorophores with photocleavable groups resulted in Ntetrazinylrhodamines and mercaptofluorans as unstable or non-photoactivatable compounds. On the other hand, a synthetic route towards 3,6-dihydroxyxanthones on previously described caging-group-free photoactivatable PaX dyes has been established. Efficient photoactivation of these compounds indicated their potential for future application in single-molecule localisation imaging and as enzymatic sensor activity probes. In conclusion, it was demonstrated that negatively charged fluorescein analogs represents a valuable addition to the toolkit of live cell-compatible fluorescent dyes. Within this work, N-cyanorhodamines as highly photostable and cell membrane-permeable fluorescein derivatives and hydroxy analogs of PaX dyes as photoactivatable analogs of fluoresceins have been prepared. The application of such dyes as fluorophores in live cell imaging has been demonstrated

Recherchieren im Internet : Konzeption und Realisation von Schulungsmaterialien für Benutzer

Diese Diplomarbeit beschäftigt sich mit dem Verfassen von Anleitungen für das Surfen im Internet für Benutzer Öffentlicher Bibliotheken. Daher handelt ein Teil von dem Erstellen von Anleitungen im allgemeinen, ein weiterer Teil beschäftigt sich mit der Suche im Internet. In diesem Teil ist beschrieben, wie man was wo am effektivsten sucht, und wie das Gefundene ausgewertet wird. Es befinden sich in dieser Arbeit mehrere Beispiele, wie eine Anleitung aussehen kann

The Influence of Dietary Factors on the Risk of Urinary Stone Formation

The action of various beverages and foods on the composition of the urine in the circadian rhythm and in the 24-hour urine has been investigated under standardized conditions. Orange juice leads to a significant increase of urinary pH and citric acid excretion. Black tea leads to a raised excretion of oxalic acid by only 7.9%. In the short term, beer increases diuresis, but afterwards leads to a compensatory antidiuresis with increased risk of stone formation. Depending on their composition, mineral waters have very different effects on the urinary constituents. Milk as well as cocoa beverage significantly increase calcium excretion; moreover, cocoa causes an increase in the oxalic acid excretion. The leafy vegetable foods containing oxalate, e.g., spinach and rhubarb, lead to peaks of oxalate excretion of 300-400% in the circadian excretion curve. Cheese leads to a significant rise of calcium excretion with acidification of the urine and lowering of citrate excretion. Calcium excretion is increased by 30% by sodium chloride. Foods containing purine result in an increased uric acid excretion over several days. Depending on their phytic acid content, brans bind calcium, but lead to an increased oxalic acid excretion. Analysis of the urine indicates that average diet in Germany entails a high risk of urinary stone formation. As a result of the change to a balanced mixed or vegetarian diet, according to the requirements, significant alterations in urinary pH, calcium, magnesium, uric acid, citric acid, cystine, and glycosaminoglycan excretion are measured, resulting in a drastic reduction in the risk of urinary stone formation

Analysis of Compound Synergy in High-Throughput Cellular Screens by Population-Based Lifetime Modeling

Despite the successful introduction of potent anti-cancer therapeutics, most of these drugs lead to only modest tumor-shrinkage or transient responses, followed by re-growth of tumors. Combining different compounds has resulted in enhanced tumor control and prolonged survival. However, methods querying the efficacy of such combinations have been hampered by limited scalability, analytical resolution, statistical feasibility, or a combination thereof. We have developed a theoretical framework modeling cellular viability as a stochastic lifetime process to determine synergistic compound combinations from high-throughput cellular screens. We apply our method to data derived from chemical perturbations of 65 cancer cell lines with two inhibitors. Our analysis revealed synergy for the combination of both compounds in subsets of cell lines. By contrast, in cell lines in which inhibition of one of both targets was sufficient to induce cell death, no synergy was detected, compatible with the topology of the oncogenically activated signaling network. In summary, we provide a tool for the measurement of synergy strength for combination perturbation experiments that might help define pathway topologies and direct clinical trials

Mutations and Deregulation of Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Cascades Which Alter Therapy Response

The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Certain components of these pathways, RAS, NF1, BRAF, MEK1, DUSP5, PP2A, PIK3CA, PIK3R1, PIK3R4, PIK3R5, IRS4, AKT, NFKB1, MTOR, PTEN, TSC1, and TSC2 may also be activated/inactivated by mutations or epigenetic silencing. Upstream mutations in one signaling pathway or even in downstream components of the same pathway can alter the sensitivity of the cells to certain small molecule inhibitors. These pathways have profound effects on proliferative, apoptotic and differentiation pathways. Dysregulation of components of these cascades can contribute to: resistance to other pathway inhibitors, chemotherapeutic drug resistance, premature aging as well as other diseases. This review will first describe these pathways and discuss how genetic mutations and epigenetic alterations can result in resistance to various inhibitors

N-Cyanorhodamines: cell-permeant, photostable and bathochromically shifted analogues of fluoresceins

Fluorescein and its analogues have found only limited use in biological imaging because of the poor photostability and cell membrane impermeability of their O-unprotected forms. Herein, we report rationally designed N-cyanorhodamines as orange- to red-emitting, photostable and cell-permeant fluorescent labels negatively charged at physiological pH values and thus devoid of off-targeting artifacts often observed for cationic fluorophores. In combination with well-established fluorescent labels, self-labelling protein (HaloTag, SNAP-tag) ligands derived from N-cyanorhodamines permit up to four-colour confocal and super-resolution STED imaging in living cells