No description available.

Phosphorus is an element that is essential for life, and is used in the synthesis of many proteins, carbohydrates and deoxyribonucleic acids. Phosphorus often exists in the form of phosphate when found in the biological systems. Clinical development of possible pharmaceutical agents have used phosphorus in the form of phosphonates to increase the metabolic stability of the potential drug. Some of these phosphonates target the isoprenoid biosynthetic pathway (IBP). The IBP plays an important role in the synthesis of cholesterol and in other aspects of cellular metabolism. The enzymes of the IBP have been the target of possible therapeutic agents for treatment of multiple diseases, including cancer. Often these phosphonic acids are masked by an enzymatically cleavable group in order to increase their bioavailability and activity. Phosphoantigens are small organo-phosphorus molecules that stimulate the expansion of Vγ9Vδ2 T-cells which detect and eliminate infected cells. Both natural and non-natural phosphoantigens have exhibited a wide range of effective concentrations (EC50) for γδ T-cells. The most potent phosphoantigen is E-4-hydroxy-3-methylbut-2-enyl pyrophosphate (HMBPP), which is an intermediate in the bacterial IBP. Nanomolar concentration of this compound stimulate T-cell proliferation. While HMBPP is highly potent, it undergoes rapid decomposition when injected into the blood stream. Synthesis of more stable phosphonate analogues can show better activity for expansion of the γδ T-cell population. Increased activity was observed in T-cell assays after masking the phosphonic acids to increase the bioavailability of the active phosphoantigen. Because some phosphoantigen showed strong activity with masked phosphonic acids, families of phosphonate analogues now have been prepared. Most use selenium dioxide mediated oxidation to incorporate the terminal alcohol and ester exchange to provide prodrugs to study the structure-activity-relationships. The biological activity of these compounds has been investigated and new phosphoantigens were shown to be strong activators of γδ T-cells. Furthermore, the phosphoantigens have been shown to bind to the protein butyrophilin 3A1 (BTN3A1) at an intracellular domain. A second family of phosphoantigen derivatives, masked by a new pH fluorescent cell-cleavable ester, were prepared and tested by our collaborators to explore the compounds’ activity and to investigate the mechanism of action. Finally, a new class of phosphorus compounds alkyl 1, 1, 1-trisphosphonates has been studied to obtain salts that might be biologically active. Trisphosphonates contain a unique arrangement of phosphonate groups on a single carbon and could provide charge states unseen in the more traditional bisphosphonates. A general route to asymmetric trisphosphonates through a step-wise phosphorylation of each phosphonate has been developed. Selective phosphonate ester cleavage would allow for the ability to obtain a multitude of charge states and possible biological activity.

abstract: Proteins function as molecular machines which perform a diverse set of essential jobs. To use these proteins as tools and manipulate them with directed engineering, a deeper understanding of their function and regulation is needed. In the studies presented here, the chemical mechanism of a fluorescent protein and the assembly behavior of a chemo-mechanical protein were explored to better understand their operation. In the first study a photoconvertible fluorescent protein (pcFP) was examined which undergoes a photochemical transformation upon irradiation with blue light resulting in an emission wavelength change from green to red. Photo-transformable proteins have been used in high resolution, subcellular biological imaging techniques, and desires to engineer them have prompted investigations into the mechanism of catalysis in pcFPs. Here, a Kinetic Isotope Effect was measured to determine the rate-limiting step of green-to-red photoconversion in the reconstructed Least Evolved Ancestor (LEA) protein. The results provide insight on the process of photoconversion and evidence for the formation of a long-lived intermediate. The second study presented here focuses on the AAA+ protein Rubisco activase (Rca), which plays a critical role in the removal of inhibitors from the carbon-dioxide fixing enzyme Rubisco. Efforts to engineer Rubisco and Rca can be guided by a deeper understanding of their structure and interactions. The structure of higher plant Rca from spinach, and its interactions with its cognate Rubisco, were investigated through negative-stain electron microscopy (EM) and cryo-EM experiments. Multiple types of higher-order oligomers of plant Rca were imaged which have never been structurally characterized, and the AAA+ core of plant Rca was shown to bind Rubisco side-on, similar to bacterial Rca’s. Higher resolution structures of these aggregates and complexes are needed to make definitive observations on protein-protein interactions. However, the results presented here provide evidence for the formation of regulatory structures and an experimental foundation for future exploration of plant Rca through cryo-EM. / Dissertation/Thesis / Masters Thesis Biochemistry 2020

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1 Abstract The goals of the thesis were to synthesise novel fluorescently labelled nucleotides and the corresponding nucleic acids for bio-analytical applications as well as their delivery into cells. The thesis also aimed at the development of an effective method to inhibit non-templated incorporation of nucleotides. The problematic non-templated enzymatic incorporation of nucleotides is addressed by using several commercially available 5'-modified-oligonucleotides. The oligonucleotides (ONs) that we tested bore ortho twisted intercalating nucleic acid (oTINA), a trityl group, or biotin at the 5'-end. The modified ONs were used as templates in the enzymatic primer extension (PEX) experiments in the presence of either modified nucleotides or only natural deoxynucleoside triphosphates (dNTPs). The oTINA templates underwent PEX reaction using natural dNTPs and different DNA polymerases of the A or B family. In parallel, two types of fluorescent nucleoside derivatives were independently designed and synthesised. Firstly, we envisaged new fluorescent nucleotide tags containing the hexamethylated BODIPY moiety as a bright fluorescent label. Conversely, we focused on the improvement of fluorescent nucleotide probes sensitive to the viscosity or polarity. The fluorescently labelled methylated BODIPY nucleotides...

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21418号 / 医博第4408号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 木村 剛, 教授 髙橋 良輔, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Research in fluorescent solar concentrators (FSCs) commenced in mid-1970’s to lower the cost of solar cells through the reduction of the required solar cell active area, and by incorporating them in-to buildings, thereby offsetting installation costs. In FSCs, light penetrates the top surface of a waveguide, is absorbed by the fluorescent material (FM) and is emitted at a longer wavelength that is then internally reflected towards edge solar cells. In this project, the use of cerium doped yttrium aluminum garnet (Ce:YAG) was explored as an FM, from which the optical transport of fluorescence must be enhanced towards solar cell edges. Optical spectroscopic techniques were conducted to characterize a sample of Ce:YAG with a doping level of 0.180 mol%. An excitation and emission profile indicated a strong absorption at 476 nm and a strong radiation at 530 nm, where the fluorescence process lasted for only 62.3 ns, with a conversion efficiency of 80.0%. Meanwhile, x-ray analysis concluded that this material had a density of 4.56 g/cm3. An acrylic concentrator with local islands of Ce:YAG was successfully fabricated. A lens sheet was used to provide strong sunlight coupling to Ce:YAG. The concentrator was analysed using Optics Lab, Monte Carlo simulations (MCS) and through experimental flux measurements, the percentage of light that waveguided to the edges was determined. Optics Lab yielded 56.10%, MCS yielded 59.20% and flux measurements resulted in 58.22% (without lenses) and 57.14% with lenses. Also, an overall experimental optical efficiency was determined to be 32.45% without lenses and 53.53% with lenses. These results can be improved by modifying the fabrication techniques and using substrates with higher refractive indices. / Thesis / Master of Applied Science (MASc)

Over the past several decades the phenomenon of luminescence (divided into fluorescence and phosphorescence) has received great attention in the field of biological science. This quest has motivated scientists for a variety of applications, including fluorescence imaging. Fluorescence microscopy techniques that provide unique advantages, such as high spatial resolution and superior sensitivity, have been regarded as attractive tools in biophotonics. With the progress of ultrafast laser sources, two-photon absorption (2PA), in which a molecule absorbs two photons simultaneously, has opened possibilities of using it for various applications. Two-photon fluorescence microscopy (2PFM), which affords deeper tissue penetration and excellent three-dimensional (3D) images, is now being widely employed for bioimaging. This dissertation focuses on the design, synthesis, and photophysical characterization of new fluorophores, as well as desirable applications. Chapter 1 gives an account of a brief introduction of luminescence and 2PA, as well as their utilities in biological applications. In chapter 2, a series of new BODIPY derivatives are presented along with their comprehensive linear and nonlinear characteristics. They exhibited excellent photophysical properties including large extinction coefficients, high fluorescence quantum yields, good photostability, and reasonable two-photon absorption cross sections. Two promising compounds were further evaluated as NIR fluorescent probes in one-photon and two-photon fluorescence imaging. Chapter 3 provides the design, synthesis, and photophysical characterization of two BODIPY dyes. In order to assess the potential of using the dye as a fluorescent probe, Lysotracker Red, a commercial lysosomal marker, was investigated for comparison purposes. The results indicate that figure of merit of both compounds were three orders of magnitude higher than that of Lysotracker Red. With an eye towards applications, one of the compounds was encapsulated in silica-based nanoparticles for in vitro and ex vivo one-photon and two-photon fluorescence imaging, in which the surface of the nanoparticle was modified with RGD peptides for specific targeting. The nanoprobe exhibited good biocompatibility and highly selective RGD-mediated uptake in ?V?3 integrin-overexpressing cancers, while maintaining efficient fluorescence quantum yield and high photostability. In chapter 4, the synthesis and photophysical properties of a novel photosensitizer with heavy atoms (halogen) were presented. The dye exhibited low fluorescence quantum yield, resulting in high singlet oxygen generation quantum yield. In vitro photodynamic studies demonstrated that photosensitization of the agent can induce cellular damage, subsequently leading to cell death by a necrotic cell death mechanism, supporting the therapeutic potential of using the agent for photodynamic therapy.

Transgenic techniques have rapidly evolved in recent years. However, the efficiency of these techniques to produce viable offspring is still disappointingly low. The purpose of this study was to assess in vitro development, transgene expression, and integration following pronuclear or cytoplasmic microinjection of condensed or linear green fluorescent protein DNA into murine embryos using electroporation. In experiment 1, the effect of embryo orientation (group or linear) within the electroporation chamber on development was evaluated using zygotes which received one pulse duration (10 msec), and one of two voltages (250 or 400 V). Zygotes that received 400 V had the lowest development score (Group, 2.06 ? 0.12; Linear, 1.97 ? 0.13), irrespective of orientation. Embryos that received 250 V had the highest development of the voltage treated groups (Group 3.42 ? 0.12; Linear 3.32 ? 0.12), irrespective of orientation, and development was lower than the control embryos (Control 4.28 ? 0.12; Mannitol control 4.36 ? 0.18). In experiment 2, the efficiency of utilization of the prepared enhanced green fluorescent protein (EGFP) construct as a visual marker of protein expression was evaluated using pronuclear microinjection. Embryo development and fluorescence were evaluated following pronuclear injection of EGFP at a concentration of 3 μg/ml and compared to an uninjected control. Embryos injected with the EGFP had lower development scores (3.85 ± 0.15) than uninjected control embryos (5.72 ± 0.2). Of the embryos injected, 32.4% fluoresced due to expression of EGFP. Experiment 3 evaluated the effect of combining cytoplasmic injection of EGFP (425 μg/ml) with electroporation at 250 V on EGFP expression. The non-manipulated control embryos had significantly higher (P < 0.01) 4 d development scores (5.57 ± 0.11) than manipulated control embryos (4.6 ± 0.18), where the injection needle was inserted into the cytoplasm and no DNA was injected. Combining cytoplasmic DNA injection and electroporation caused a significant (P < 0.01) decrease in development scores, irrespective of DNA construct, when compared to embryos injected with a DNA construct alone. The mechanical effects of needle insertion combined with electroporation were not significantly different (P > 0.05) from embryos injected with DNA alone, irrespective of construct injected. Cytoplasmic injection of condensed DNA (0.38%), linear DNA (0.38%), and condensed DNA combined with electroporation (0.36%) resulted in one fluorescent embryo respectively. Cytoplasmic injection of linear DNA when combined with electroporation (3.57%) resulted in 13 fluorescent embryos. Pronuclear injection of the prepared EGFP construct results in lower development than control embryos. Electrical stimulation of zygotes reduces early embryo development. However, low amounts of electrical stimulation may allow for enhancement of gene integration in transgenic embryos. / Master of Science

<p> The mineral eudialyte from Seal Lake, Labrador, was analyzed by means of x-ray fluorescence analysis for the rare earths La, Ce, Pr, Nd, Sm, Gd, Y and also for the element Zr. Samples were prepared by two methods: a) whole mineral powder being compressed into pellets, and b) the dissolved mineral being put through an ionic exchange process to eliminate interfering ions before compressing into pellets for analysis. The results are compared with published data.on other eudialyte occurrences. </p> / Thesis / Bachelor of Science (BSc)