The main theme of this thesis is to develop a fluorescent probe for imaging the subcellular distribution of kinetically labile copper pools that might play a critical role in copper homeostasis. Various copper-selective sensors were designed by combining 1,3,5-triaryl-2-pyrazoline fluorophores with polythioethers as receptor moieties. A series of donor-substituted 1,3,5-triaryl-2-pyrazoline fluorophores were synthesized and characterized in terms of their photophysical and electrochemical properties. Interestingly, the aryl substituents attached to the 1- and 3-position of the pyrazoline ring influence the photophysical properties of the fluorophore in distinctly different ways. The excited-state equilibrium energy is primarily influenced by changes of the substituent in the 1-position, whereas the reduction potential of the fluorophore is determined by the 3-aryl group. Results from computational analyses agree well with the experimental data. A pyrazoline fluorophore library was synthesized, and their photophysical and electrochemical properties were studied. The compounds cover a broad range of excited state energies and reduction potentials, and allow for selective and differential tuning of these two parameters. A series of thiazacrownethers and tripodal aniline copper(I) receptors were synthesized and their copper binding stoichiometries, stability constants, and copper-self-exchange kinetics were investigated. The measured self-exchange activation parameters revealed for all studied ligands a negative activation entropy, suggesting a predominant associative exchange mechanism. With detailed knowledge of the fluorophore platform and copper receptors, sensor CTAP-1 was designed, synthesized and characterized. The probe shows a 4.6-fold emission enhancement and reaches a quantum yield of 14% upon saturation with Cu(I). The sensor exhibits excellent selectivity towards Cu(I) and is insensitive towards millimolar concentrations of Mg(II) or Ca(II). Mouse fibroblast cells (3T3) incubated with the sensor produced a copper-dependent perinuclear staining pattern, which colocalizes with the subcellular location of the mitochondria and the Golgi apparatus. The subcellular topography of copper was further determined by synchrotron-based x-ray fluorescence (SXRF) microscopy. Furthermore, microprobe x-ray absorption measurements at various subcellular locations showed a near-edge feature that is characteristic for low-coordinate monovalent copper. The data provide a coherent picture with evidence for a kinetically labile copper pool, which is predominantly localized in the mitochondria and the Golgi apparatus.

This dissertation describes the development of a new method for increasing the resolution of the current protein microarray technology, down to the single molecule detection level. By using a technique called size-dependent self-assembly, different proteins can be bound to different sized fluorescent nanostructures, and then located on a patterned silicon substrate based on the sized pattern which is closest to the size of the bead diameter.The protein nanoarray was used to detect antibody-antigen binding, specifically anti-mouse IgG binding to mouse IgG. The protein nanoarray is designed with the goal of analyzing rare proteins. However, common proteins, such as IgG, are used in the initial testing of the array functionality. Mouse IgG, representing rare proteins, is conjugated to fluorescent beads and the beads are immobilized on a patterned silicon surface. Then anti-mouse IgG binds to the mouse IgG on the immobilized beads. The binding of the antibody, anti-mouse IgG, to the antigen, mouse IgG is determined by fluorescent signal attenuation.The first objective was to bind charged nanoparticles, conjugated with proteins, to an oppositely charged silicon substrate. Binding of negatively charged gold nanoparticles (AuNP), conjugated with mouse IgG, to a positively charged silicon surface was successful.The second objective was to demonstrate the method of size-dependent self-assembly at the nanometer scale (<100 >nm). Different-sized, carboxylated, fluorescent beads and AuNP, which were conjugated with proteins, were serially added to a patterned polymethyl methacrylate (PMMA) coated silicon surface. Size-dependent self-assembly was successfully demonstrated, down to the nanometer scale.The final objective was to obtain a signal from antibody-antigen binding within the protein array. Conjugated fluorescent beads were bound to e-beam patterns and signal attenuation was measured when the antibodies bound to the conjugated beads. The size-dependent self-assembly is a valuable new method that can be used for the detection and quantification of proteins.

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 143-151). Also available for download via the World Wide Web; free to University of Oregon users.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

The synthesis of proteins bearing unnatural amino acids has the potential to enhance and elucidate many processes in biochemistry and molecular biology. There are two primary methods for site specific unnatural amino acid incorporation, both of which use the cell’s native protein translating machinery: in vitro chemical acylation of suppressor tRNAs and the use of orthogonal amino acyl tRNA synthetases. Total chemical synthesis is theoretically possible, but current methods severely limit the maximum size of the product protein. In vivo orthogonal synthetase methods suffer from the high cost of the unnatural amino acid. In this thesis I sought to address this limitation by increasing cell density, first in shake flasks and then in a bioreactor in order to increase the yield of protein per amount of unnatural amino acid used. In a parallel project, I used the in vitro chemical acylation system to incorporate several unnatural amino acids, key among them the fluorophore BODIPYFL, with the aim of producing site specifically fluorescently labeled protein for single molecule FRET studies. I demonstrated successful incorporation of these amino acids into the trial protein GFP, although incorporation was not demonstrated in the final target, FEN1. This also served to confirm the effectiveness of a new procedure developed for chemical acylation.

L'epicocconone est un produit naturel tricyclique, de la famille des azaphilones, isolé en 2003 d'un champignon Epicoccum nigrum. Ce composé se lie de façon covalente aux amines, conduisant à la formation d'une énamine fluorescente. Cette réaction, réversible en fonction du pH, fait de ce composé un excellent marqueur de protéines pour la détection sur gels d'électrophorèse compatible avec une analyse de spectrométrie de masse. La synthèse de ce produit naturel a été débutée au sein de notre laboratoire en s'appuyant sur les travaux réalisés précédemment et mettant en jeu une étape clé de désaromatisation oxydante à l'aide d'iode hypervalent. Une étude méthodologique de réaction clé a permis de comparer l'efficacité et la diastéréosélectivité de l'oxydation effectuée par l'iode (III) ou l'iode (V). / Epicocconone is a tricyclic natural product of the azaphilone family, isolated from the fungus Epicoccum nigrum. This compound covalently binds to primary amines, leading to a protein conjugate which is highly fluorescent. This reaction, reversible according to the pH, make this compound an excellent protein stain compatible with mass spectrometry analysis. The synthesis of this natural product has been started in our laboratory based on the previous work in involves a key oxidative dearomatization using hypervalent iodine. Methodological studies of this key reaction allowed us to compare the efficiency and diastereoselectivity of iodine (III) and iodine (V) mediated oxidations.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18299号 / 工博第3891号 / 新制||工||1597(附属図書館) / 31157 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 濵地 格, 教授 梅田 眞郷, 教授 森 泰生 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(理学) / 甲第24442号 / 理博第4941号 / 新制||理||1706(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)准教授 板東 俊和, 教授 深井 周也, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第23998号 / エネ博第434号 / 新制||エネ||82(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 森井 孝, 教授 片平 正人, 教授 佐川 尚 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DFAM

The interior of the cell is a densely crowded and complex arena, full of a vast and diverse array of molecules and macromolecules. A fundamental understanding of cellular physiology will depend not only upon a reductionist analysis of the chemistry, structure, and function of individual components and subsystems, but also on a sagacious exegesis of the dynamic and emergent properties that characterize the higher-level system of living cells. Here, we present work on two aspects of the supramolecular organization of the cell: the controlled assembly of the mitotic spindle during cell division and the regulation of cellular metabolism through the formation of multienzyme complexes. During division, the cell undergoes a profound morphological and molecular reorganization that includes the creation of the mitotic spindle, a process that must be highly controlled in order to ensure that accurate segregation of hereditary material. Chapter 2 describes results that implicate the kinase, Zeste-white3/Shaggy (Zw3/Sgg), as having a role in regulating spindle morphology. The congregation of metabolic enzymes into macromolecular complexes is a key feature of cellular physiology. Given the apparent pervasiveness of these assemblies, it seems likely that some of the mechanisms involved in their organization and regulation might be conserved across a range of biosynthetic pathways in diverse organisms. The Winkel laboratory makes use of the flavonoid biosynthetic pathway in Arabidopsis as an experimental model for studying the architecture, dynamics, and functional roles of metabolic complexes. Over the past several years, we have accumulated substantive and compelling evidence indicating that a number of these enzymes directly interact, perhaps as part of a dynamic globular complex involving multiple points of contact between proteins. Chapter 3 describes the functional analysis of a predicted flavonol synthase gene family in Arabidopsis. The first evidence for the interaction of flavonoid enzymes in living cells, using fluorescent lifetime imaging microscopy fluorescent resonance energy transfer analysis (FLIM-FRET), is presented in Chapter 4. / Ph. D.