Development and application of molecular and computational tools to image copper in cells

Copper is a trace element which is essential for many biological processes. A deficiency or excess of copper(I) ions, which is its main oxidation state of copper in cellular environment, is increasingly linked to the development of neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease (PD and AD). The regulatory mechanisms for copper(I) are under active investigation and lysosomes which are best known as cellular “incinerators” have been found to play an important role in the trafficking of copper inside the cell. Therefore, it is important to develop reliable experimental methods to detect, monitor and visualise this metal in cells and to develop tools that allow to improve the data quality of microscopy recordings. This would enable the detailed exploration of cellular processes related to copper trafficking through lysosomes. The research presented in this thesis aimed to develop chemical and computational tools that can help to investigate concentration changes of copper(I) in cells (particularly in lysosomes), and it presents a preliminary case study that uses the here developed microscopy image quality enhancement tools to investigate lysosomal mobility changes upon treatment of cells with different PD or AD drugs. Chapter I first reports the synthesis of a previously reported copper(I) probe (CS3). The photophysical properties of this probe and functionality on different cell lines was tested and it was found that this copper(I) sensor predominantly localized in lipid droplets and that its photostability and quantum yield were insufficient to be applied for long term investigations of cellular copper trafficking. Therefore, based on the insights of this probe a new copper(I) selective fluorescent probe (FLCS1) was designed, synthesized, and characterized which showed superior photophysical properties (photostability, quantum yield) over CS3. The probe showed selectivity for copper(I) over other physiological relevant metals and showed strong colocalization in lysosomes in SH-SY5Y cells. This probe was then used to study and monitor lysosomal copper(I) levels via fluorescence lifetime imaging microscopy (FLIM); to the best of my knowledge this is the first copper(I) probe based on emission lifetime. Chapter II explores different computational deep learning approaches for improving the quality of recorded microscopy images. In total two existing networks were tested (fNET, CARE) and four new networks were implemented, tested, and benchmarked for their capabilities of improving the signal-to-noise ratio, upscaling the image size (GMFN, SRFBN-S, Zooming SlowMo) and interpolating image sequences (DAIN, Zooming SlowMo) in z- and t-dimension of multidimensional simulated and real-world datasets. The best performing networks of each category were then tested in combination by sequentially applying them on a low signal-to-noise ratio, low resolution, and low frame-rate image sequence. This image enhancement workstream for investigating lysosomal mobility was established. Additionally, the new frame interpolation networks were implemented in user-friendly Google Colab notebooks and were made publicly available to the scientific community on the ZeroCostDL4Mic platform. Chapter III provides a preliminary case study where the newly developed fluorescent copper(I) probe in combination with the computational enhancement algorithms was used to investigate the effects of five potential Parkinson’s disease drugs (rapamycin, digoxin, curcumin, trehalose, bafilomycin A1) on the mobility of lysosomes in live cells.Open Acces

Phthaolocyanin - Koordinationsverbindungen

Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheAscorbinsäure (Vitamin C) ist gut bekannt als wichtiger Nährstoff in Bezug auf biologische Funktionen wie beispielsweise die Carnitin Biosynthese, als Antioxidationsmittel oder als Anti-aging Mittel. Speziell vor kurzem hat Ascorbinsäure große Aufmerksamkeit auf sich gezogen durch seine Verwendung in der modernen Krebstherapie. Daher ist es äußerst wichtig, Fluoreszenz Proben zu entwickeln, die Ascorbinsäure in deren biologischen Rollen aufklären können. Fluorophore verbunden mit Nitroxidradikal (FN-Systeme) scheinen vielversprechende Kandidaten für die Detektion von Ascorbinsäure zu sein. Das Nitroxidradikal verursacht eine effiziente Fluoreszenz-Quenchung und reagiert vorzugsweise mit Ascorbinsäure. Daher ist zu erwarten, dass die FN-basierten fluoreszierenden Proben gut für die quantitative Bestimmung von Ascorbinsäure in wässrigen Lösungen geeignet sind. Für den Einsatz dieses Detektionsverfahrens von Ascorbinsäure im biologischen Umfeld müssen noch Verbesserungen an den FN Systemen vorgenommen werden. Der Grund ist der, dass neben Ascorbinsäure noch einige andere Stoffe wie die mitochondriale NADH oder Superoxide mit den Radikalen reagieren können. In diesem Forschungsprojekt wurde ein bereits erforschtes System aus Fluorophor und Nitroxidradikalen, welches in der Lage ist Vitamin C durch Fluoreszenz zu detektieren, verbessert. Es wurde ein einfach sulfonierter SiPc Komplex neu synthetisiert. Dieser wurde als Fluoreszenzprobe für die Ascorbinsäuredetektion in wässriger Lösung angewendet. Dieser Komplex wurde neu synthetisiert, und mittels ESI-MS, Absorptionsmessung, ESR und MCD gemessen. Als letzter Schritt wurde das Fluoreszenzverhalten mit dem der ersten Generation dieses Komplexes verglichen. Da die hydrophoben SiPc-TEMPO Derivate in Wasser unlöslich sind, wurden die Komplexe in Liposome für die Messung in wässriger Lösung eingekapselt. Die Fluoreszenz von liposomalen R2cS1 und R2c stieg für beide Komplexe nach der Zugabe von Ascorbinsäure. Die Sensitivität des neu synthetisierten R2cS1 Komplexes war jedoch 10 Mal höher als die des alten R2c Komplexes. Die erhöhte Sensibilität von R2cS1 gegenüber R2c wurde durch die erhöhte Polarität durch die installierte Sulfatgruppe der neuen Verbindung verursacht. Diese führt zu einem näheren Aufenthaltsortes zur polaren Außenwand im Liposom des Komplexes, welcher dadurch leichter mit dem ebenfalls polaren Vitamin C reagieren kann. - Außerdem wurde noch eine DFT-Berechnung durchgeführt, welche zusammen mit der MCD Messung eine mysteriöse zweite Q-Bande im Absorptionsspektrum des R2cS1 Komplexes erklären konnte. Diese wurde durch die neuentstandene Asymmetrie des Komplexes durch die neue Sulfatgruppe verursacht, da diese die Energie der Orbitale aufspaltete.Ascorbic acid (Vitamin C) is a well-known and essential nutrient in relation to biological functions such as carnitine biosynthesis, as anti-oxidizing agents or as anti-aging agents. In particular, recently, ascorbic acid has attracted considerable attention for its uses in modern cancer therapy. Thus, it is extremely important to develop fluorescence probes to detect ascorbic acid for clarifying the biological roles. Fluorophores linked to a nitroxide radical (FN systems) are promising candidates for detecting ascorbic acid. The nitroxide radical provides efficient fluorescence quenching and preferably reacts with ascorbic acid. Thus, the FN-based probes have been expected to be applied to the quantitative determination of ascorbic acid in aqueous solutions. However, in contrast to aqueous solutions, improvements are required to apply the FN probes to the detection of ascorbic acid in biological environments, since, in addition to ascorbic acid, the nitroxide radical easily reacts with some biological reductants such as mitochondrial NADH and superoxide. [1] In this study, an already existing system of fluorophore with nitroxid radicals which was capable in detecting vitamin C by fluorescence was improved. One times sulfonated SiPc covalently linked to two TEMPO radicals was applied as fluorescence probe for detecting ascorbic acid in aqueous solution. This complex was synthesized and measured by ESI-MS, absorption-spectroscopy, ESR, MCD and the fluorescence capabilities were compared with the first generation of the complex which already showed these fluorescence properties. Since the hydrophobic SiPc-TEMPO derivatives are insoluble in aqueous solutions, they were encapsulated in liposomes for measurement in aqueous solutions. The fluorescence of liposomal R2cS1 and R2c increased for both after ascorbic acid addition. The sensitivity, however, of the new synthesized R2cS1 complex was 10-times better than for the R2c complex. The increased sensibility of R2cS1 over R2c is caused by the stronger polarity of the new complex due to the sulfate group. This results in a closer position to the polar regions of the liposomes which makes it easier to react with the also quite polar ascorbic acid. Furthermore a DFT calculation was carried out to explain a mysterious Q-band splitting in the absorption spectra of the R2cS1 complex. Together with the results of the MCD measurement it was discovered that the additional peak in the Q-band region is caused by a split of the orbital energies due to the asymmetry of the complex because of the sulfate group.11

HSQC Spectra Simulation and Matching for Molecular Identification

In the pursuit of improved compound identification and database search tasks, this study explores Heteronuclear Single Quantum Coherence (HSQC) spectra simulation and matching methodologies. HSQC spectra serve as unique molecular fingerprints, enabling a valuable balance of data collection time and information richness. We conducted a comprehensive evaluation of four HSQC simulation techniques: ACD-Labs (ACD), MestReNova (MNova), Gaussian NMR calculations (DFT), and a graph-based neural network (ML). For with the latter two techniques, we developed a reconstruction logic to combine proton and carbon 1D spectra into HSQC spectra. The methodology involved the implementation of three peak-matching strategies (Minimum-Sum, Euclidean-Distance, and Hungarian-Distance) combined with three padding strategies (zero-padding, peak-truncated, and nearest-neighbor double assignment). We found that coupling these strategies with a robust simulation technique facilitates the accurate identification of correct molecules from similar analogues (regio- and stereoisomers) and allows for fast and accurate large database searches. Furthermore, we demonstrated the efficacy of the best-performing methodology by rectifying the structures of a set of previously misidentified molecules. This research indicates that effective HSQC spectra simulation and matching methodologies significantly facilitate molecular structure elucidation. Furthermore, we offer a Google Colab notebook for researchers to use our methods on their own data