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In order to understand the structural changes in myosin S1, fluorescence polarization and computational dynamics simulations were used. Dynamics simulations on the S1 motor domain indicated that significant flexibility was present throughout the molecular model. The constrained opening versus closing of the 50 kDa cleft appeared to induce opposite directions of movement in the lever arm. A sequence called the "strut" which traverses the 50 kDa cleft and may play an important role in positioning the actomyosin binding interface during actin binding is thought to be intimately linked to distant structural changes in the myosin's nucleotide cleft and neck regions. To study the dynamics of the strut region, a method of fluorescent labeling of the strut was discovered using the dye CY3. CY3 served as a hydrophobic tag for purification by hydrophobic interaction chromatography which enabled the separation of labeled and unlabeled species of S1 including a fraction labeled specifically at the strut sequence. The high specificity of labeling was verified by proteolytic digestions, gel electrophoresis, and mass spectroscopy. Analysis of the labeled S1 by collisional quenching, fluorescence polarization, and actin-activated ATPase activity were consistent with predictions from structural models of the probe's location. Although the fluorescent intensity of the CY3 was insensitive to actin binding, its fluorescence polarization was notably affected. Intriguingly, the mobility of the probe increases upon S1 binding to actin suggesting that the CY3 becomes displaced from interactions with the surface of S1 and is consistent with a structural change in the strut due to cleft motions. Labeling the strut reduced the affinity of S1 for actin but did not prevent actin-activated ATPase activity which makes it a potentially useful probe of the actomyosin interface. The different conformations of myosin S1 indicated that the strut is not as flexible as several other key regions of myosin as determined by the application of force constraints to elastic portions of the myosin structure.

Using the technology afforded by Winschel et al., cyclen-1, a high affinity, strong complexation agent for 8-hydroxypyrene-1,3,6 trisulfonate and derivatives, a new assay has been developed for fluorescently labeling proteins of interest (POIs). Ligation of the endogenous ligand for nicotinic acetylcholine receptors (nAChRs), acetylcholine, using click chemistry afforded the triazole derivative of an alkynyl-acylcholine (compound 1) with 8-azidopyrene-1,3,6 trisulfonate (compound 2). Liposomes encapsulated with Rhodamine B were used to strengthen the initial fluorophore response of compound 2, using an anchored form of cyclen-1 complex. Using a palmitoyl tail as the lipophilic moiety for liposomal amplification, the subsequent response has a fluorophore ratio of up to 1:1 million, compound 2:Rhodamine B molecules. in vitro assay using compound 2 and cyclen-1 anchored liposomes with HEK-293 cells produced a positive binding response, allowing brightly colored fluorescent images of nAChRs upon the cellular membrane. A control for nAChR binding was performed using a co-culture of HEK-293 and endothelial cell lines. Control experiments show compound 2 and liposomes weak binding endothelial cells, however, this could be do to accumulation from another mechanism, more work is necessary to prove whether or not this is correct.

Rapid isolation and identification of spores from various environmental samples is necessitated because anthrax spores can be used as biological weapons. The hydrophobic nature of spores may allow for their rapid concentration and partial purification from contaminating materials. In this study, spores from four taxonomic groups of Bacillaceae were isolated, purified and characterized for hydrophobicity by hexadecane partitioning, surface morphology by scanning electron microscopy, and steady-state fluorescence by spectroscopy. The morphology of spores was similar within taxonomic groups and dissimilar between groups. Spore hydrophobicity ranged from 0.3% to 65% and all spores had fluorescence emission peaks at 335 nm and 450 nm. The excitation maxima for the peak at 450 nm were shifted to higher wavelengths for the least hydrophobic spores. Regression analysis demonstrated a correlation between the taxonomic identity, as established by fatty acid analyses, and hydrophobicity. Hydrophobicity can be used to help isolate spores from complex environmental samples and intrinsic fluorescence is helpful in discriminating the taxonomic groups.

For applications from biosensor generation to synthetic biology, the ability to accurately and quantitatively generate input-output data from biological systems over a wide dynamic range is becoming increasingly important. This study has demonstrated that a simple approach utilising multiple promoters, recognising the same transcriptional activator but with differing affinities, linked to compatible reporter genes can achieve this goal. This simple system highlights that even for complex promoters with multiple binding sites we can use the ratio of two reporters to obtain accurate and reproducible input-output data for reporters contained on a high copy number plasmid without the need for any background subtraction or accurate determination of cell number. It has also demonstrated that the precise promoter strengths are not too crucial to the design, provided they are significantly different, as taking the ratio of either the high affinity/low affinity or medium affinity/low affinity channels give similar improvements over any single channel alone. The modularity of this system means that it should be possible to exchange the IPTG inducible promoter and hence place the ntrC* gene under the regulation of any environmentally important promoter. It should also be possible to simply construct different strength promoters for the same transcriptional activator by standard DNA synthesis techniques, therefore allowing direct ratiometric detection of specific active transcription factors. Finally, there is considerable interest in the biosensor field in the use of immobilised cells for field based applications. In these cases, determining the exact number of viable cells present at the time of use may be problematic and obtaining high signal levels may also be essential. In this regard, the use of ratiometric reporters from constructs on high copy number plasmids could alleviate many of these potential problems and significantly simplify the required biosensor detection equipment.

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department: Department of Biophysics and Physical Chemistry Candidate: Lenka Hrubá Supervisor: Assoc. Prof. Veronika Nováková, Ph.D. Title of Thesis: Optimization of methods for determination of singlet oxygen production and fluorescence emission of azaphthalocyanine derivatives Photodynamic therapy (PDT) with a singlet oxygen as an essential agent is believed to be an alternative way of cancer treatment or treatment of some cutaneous diseases. The principle of PDT is based on excitation of a photosensitizer by light absorption, followed by transfer of energy to tissue oxygen (3 O2) forming cytotoxic singlet oxygen (1 O2). The efficiency by which photosensitizer transforms absorbed energy to singlet oxygen is characterized by singlet oxygen quantum yields (ΦΔ). The aim of this thesis was to develop and optimize absolute method for determination of ΦΔ. In comparison to a relative method, no reference is needed in this case, which enables accurate results with lower error. Verification of the new method was performed in N,N- dimethylformamide with a zinc phthalocyanine as a model photosensitizer because of its well-known ΦΔ and with 1,3-diphenylisobenzofuran as a chemical quencher of 1 O2. Different sources of light for excitation...

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department Department of Biophysics and Physical Chemistry Candidate Jiří Demuth Supervisor Assoc. Prof. Veronika Nováková, PhD. Title of Thesis Synthesis and photophysical study of hydrophilic tetrapyridoporphyrazines bearing charged substituents on the periphery Phthalocyanines are planar organic molecules, which have a metal cation coordinated in their centre. This work deals with their aza-analogues - tetra-3,4-pyridoporphyrazines (TPyPz). TPyPz can absorb light in red part spectrum and then produce singlet oxygen. Due to this ability, they may be used in photodynamic therapy (PDT) of cancer. PDT's mechanism is based on three components: photosensitizer, light and singlet oxygen. Photosensitizer transfers energy of absorbed light to oxygen making, thus, cytotoxic singlet oxygen. The goal of this thesis was to synthesize water soluble TPyPzs absorbing in red part of absorption spectrum. TPyPzs bearing different charged substituents will be compared within the series. The synthesis consisted of preparation of 2-chloro-5,6-dimethylpyridine-3,4-dicarbonitrile (1), which was the starting precursor for other reactions. Nucleophilic substitution of 1 was used for the introducing of various hydrophilic substituents. Prepared precursors...

Suwannee River fulvic acid (SRFA) and humic acid (SRHA) were used as dissolved organic matter (DOM) and were applied to probe the effect of DOM. Addition of DOM resulted in decreased first order rate constants for all species selected. The inhibition became more significant as the hydrophobicity of the species increased. The decrease could not be simply attributed to the binding of hydrophobic species to DOM. This can be explained by the physical isolation of iron (II), which binds to hydrophilic sites of DOM and is the hydroxyl radical generation site, from hydrophobic pollutants which bind to hydrophobic sites of DOM. Accordingly, species which could compete agains t this physical isolation by DOM and bring iron (II) closer to target species could increase the degradation rates. This was observed with application of carboxymethyl-ß-cyclodextrin (CMßCD). Effects from concentration, structure of the target species and acidity etc., were studied. The increased degradation rates were observed even in the presence of DOM. Studies on ternary complexes of hydrophobic pollutants, iron (II) and CMßCD were carried with ESMS, UV and Fluorescence experiments and further calix[6]arene derivatives. Along with the fact that CMßCD can increase the solubility of hydrophobic species and remove them from contaminated sites, this indicates a potential application to in-situ degradation systems. Initial two -phase studies were carried out with quartz sand deposited with polycholobiphenyl (PCBs) and polycyclic aromatic hydrocarbons (PAHs). Successful degradations were observed with PCBs but not PAHs. The difference is attributed to the slow equilibrium of sorbed PAHs with dissolved CMßCD and the higher PAH loading used in these experiments. A halide sensor-molecule (1, 8-diphenylureaylnaphthalene), which performs with increasing fluorescence in the presence of fluoride and decreasing fluorescence with all other halides, was synthesized and reported. Studies using NMR and computer modeling with SPARTAN were carried out to compare the sensor-molecule with an analog, 2, 3-diphenylureaylnaphthalene. Both studies indicated that only fluoride can be accommodated in the space between the urea group protons to form a strong interaction. The sensor-molecule could to lead to improved sensors that overcome limitations with current fluorescence-quenching based anion sensors.

Quantum dots (QDs), especially colloidal semiconductor QDs, possess properties including high quantum yields, narrow fluorescence spectra, broad absorption and excellent photostability, making them extremely powerful in bioimaging. In this thesis, we studied the fluorescence properties of QDs and attempted multiple ways to boost applications of QDs in bioimaging field. By time-correlated single photon counting (TCSPC) measurement, we quantitatively interpreted the fluorescence mechanism of colloidal semiconductor QDs. To enhance QD fluorescence, we used a porous alumina membrane as a photonic crystal structure to modulate QD fluorescence. We studied the acid dissociation of 3-mercaptopropionic acid (MPA) coated QDs mainly through electrophoretic mobility of 3-MPA coated CdSe QDs and successfully demonstrated the impact of pH change and Ca2+ ions. Blinking phenomena of both CdSe-CdS/ZnS core-shell QDs and 3C-SiC nanocrystals (NCs) were studied. A general model on blinking characteristics relates the on-state distribution to CdSe QD surface conditions. The energy relaxation pathway of fluorescence of 3C-SiC NCs was found independent of surface states. To examine QD effect on ciliated cells, we conducted a 70-day long experiment on the bioelectric and morphological response of human airway epithelial Calu-3 cells with periodic deposition of 3-MPA coated QDs and found the cytotoxicity of QDs was found very low. In a brief summary, our study of QD could benefit in bioimaging and biosensing. Especially, super-resolution fluorescent bioimaging, such as, stochastic optical reconstruction microscopy (STORM) and photo-activated localization microscopy (PALM), may benefit from the modulation of the QD blinking in this study. And fluorescence lifetime imaging (FLIM) microscopy could take advantage of lifetime modulation based on our QD lifetime study. / <p>QC 20160905</p>

In this work a system-level approach was taken to the single-molecule fluorescence microscopy of living cells. This primarily involved the unification of relevant information within appropriately structured artefacts that were used to inform and enhance experimentation. Initially the diversity of emerging single-molecule techniques was reviewed and presented with a novel article structure to suit the purpose of designing an experiment (Harriman and Leake 2011). Techniques were grouped by the type of information they could access, rather than the standard organisation centred on the techniques themselves. A bespoke microscope was conceived and built with reference to knowledge and tools from the fields of Architecture and Systems-Engineering. The microscope layout would enable multiple experiment types through independent control of multiple illumination beams. A technique was developed enabling the prescription of evanescent field penetration depth for each incident beam. The various empirical and theoretical results that are used to understand and modify a microscopy experiment were integrated into an internally consistent simulation model (Harriman and Leake. 2013). This was used to inform the selection of experimental components and parameters and ultimately acquire higher data quality as measured by functions such as signal-to-noise ratio (SNR). The combined experimental system of microscope and simulation model was applied in two live-cell investigations. In Escherichia coli, the spatial distribution of membrane bound proteins was investigated and a novel technique was applied to the analysis of colocalisation. Results indicate that NADH dehydrogenase and ATP synthase follow uncorrelated trajectories. This supports the hypothesis of spatial decoupling of molecules that energise the membrane and molecules that use membrane energy. In human carcinoma cells, the mechanism of ligand-receptor binding was investigated. Data was collected prior to and periodically after the addition of ligands, and fluorescence images were acquired of both ligands and receptors. Analyses based on single particle tracking are currently being carried out by a collaborator to extract information on stoichiometry and dynamics at the single-molecule level.