Amyloidogenesis refers to a process of protein misfolding and aggregation that leads to the formation of highly stable amyloid fibers. Amyloidogenesis may lead to loss of physiological protein function and/or formation of toxic intermediates, which are linked to mutliple human diseases. Amyloidogenesis is inhibited by plasma proteins, which function as extracellular chaperones by binding to stressed and misfolded proteins, including amyloidogenic peptides, and preventing their aggregation. This thesis focuses on the ability of human serum albumin (HSA), the main protein in human plasma, to inhibit amyloidogenesis, with emphasis on the molecular nature of the interactions between HSA and the amyloid β peptide (Aβ) associated with Alzhemier’s disease. HSA is as a key amyloidogenic regulator, a novel function for this protein that goes beyond the traditional HSA roles as plasma osmotic pressure regulator and as binder and carrier of endogenous and exogenous low molecular weight ligands. As a first step towards understanding the detailed molecular nature of these interactions, this thesis will focus on defining the key binding determinants in the interaction between HSA and Aβ peptides. Primarily, we will try to answer two main questions. First, which HSA residues are critical for the recognition of Aβ peptides and the prevention of Aβ aggregation? Second, which Aβ residues are mostly affected by HSA binding? Starting form our knowledge about the stoichiometry and affinity of the Aβ interactions at the level of HSA domains, Chapter 2 addresses the first question through successful applications of a reductionist approach, based on a combination of mutational comparative analyses and fatty acid (FA) competition. This strategy allowed us to identify a short HSA derived peptide that specifically recognizes Aβ and prevents its aggregation. In Chapter 3, we examine the effect of HSA on the pseudo-equilibrium state between Aβ monomers and protofibrils. Using Dark state Exchange Saturation Transfer (DEST), Saturation Transfer Difference (STD) and 15N T2 relaxation experiments, we show that Aβ peptides interact with HSA via a dual mechanism. First, selected residues in Aβ (1-40) monomers bind specifically but weakly to HSA (Kd = 0.1 - 1 mM). Second, HSA competes with Aβ monomers for the binding to the protofibrils, as indicated by an HSA-dependent decrease in the direct vs. tethered probabilities for contacts between Aβ monomer residues and the protofibril surface. The effect of HSA mimics that of dilution for the majority of the Aβ (1-40) residues involved in the cross-beta strands of amyloid fibrils. Finally, Chapter 4 will outline future investigations to address currently open questions about HSA dynamics, HSA-Aβ and HSA-FA interactions, for which we acquired preliminary data. / Thesis / Master of Science (MSc)

Non-invasive techniques to measure bone fluorine levels in vivo are few and not well studied. These techniques would prove useful for longitudinal studies of fluorine accumulation and treatment optimization for patients with poor bone health. Two measurement techniques were analyzed and improvements to each technique attempted with bone samples and bone-mimicking phantoms. The first method analyzed was neutron activation analysis (NAA), a technique previously studied in our laboratory. A previous detector setup consisting of nine sodium iodide detectors was re-tested and a new detector setup consisting of two high-purity germanium detectors was also tested. The detection limit of the sodium iodide setup was found to be higher than previously reported by a factor of 4, and the new high-purity germanium detector setup was found to result in a higher detection limit by a factor of 5 compared to the sodium iodide setup. The second method analyzed was nuclear magnetic resonance (NMR). Magic angle spinning was performed on a human bone sample, and a novel probe was constructed for future in vivo measurements. MAS NMR measurement of the human bone sample showed it to have an appropriate chemical shift and shape consistent with previous research on substances similar to bone. The constructed probe successfully resonated at the appropriate frequency, however there were potential contamination problems which prevented a measurable fluorine signal from being obtained. Both the NAA and NMR techniques may be optimized further, though with the results obtained, NAA remains the more sensitive technique for measuring bone fluorine in vivo. / Thesis / Master of Science (MSc) / Fluorine is an element which accumulates in bones and teeth. High levels of fluorine have been shown to be unhealthy, causing both dental and skeletal fluorosis. Low levels of fluorine have been shown to reduce dental cavities, however, their effect on bone health is not well understood. Currently, fluorine can be measured in bone samples from either biopsies or cadavers. Having a non-invasive way to measure fluorine concentrations in living humans without the need for surgery would be invaluable. These measurements could be used to optimize treatment for osteoporosis patients or to determine if emergency measures are necessary in cases of high accidental doses to members of the public. Additionally, long-term studies examining fluorine metabolism and bone health could be performed on population groups of interest. For these reasons, two different non-invasive methods for determining fluorine content in bone were analyzed and enhancements to each measurement technique attempted.

This dissertation summarizes my efforts and research in the Roy group to study the tunability of superatoms through ligand effects, create microporous structures from molecular cluster precursors to act as battery materials, and understand the electronic structure governing the interesting magnetic properties of Fe₆S₈(CN)₆, as well as efforts to design novel extended structures utilizing Fe₆S₈(CN)₆. Chapter 1 serves as an introduction to superatoms. It briefly discusses the quantum nature of small materials and how this gives rise to properties exhibited by superatoms. Properties which will prove important to this dissertation and methods of altering those properties through core composition and ligand choice are explored. Next, an overview of many methods to create extended structures is provided. Select examples of how superatomic clusters have already been used to increase our knowledge of fundamental concepts in science are then discussed. Finally, a brief summary and explanation of how these concepts will be explored in later chapters is given. This chapter is meant to serve as a targeted review with plenty of further reading cited for any incoming students with interest in continuing my projects. Chapter 2 discusses studies to understand the effects of either replacing PEt₃ ligands with CO ligands or the removal of PEt₃ ligands in the Co₆S₈(PEt₃)ₓ(CO)₆₋ₓ and Co₆S₈(PEt₃)ₓ systems, respectively. It presents a collaborative approach to synthesize a series of clusters for analysis by anion photoelectron spectroscopy and evaluation of results using computational chemistry. A drastic change in the donor/acceptor behavior of the cluster is observed, but surprisingly little change in the HOMO-LUMO gap is observed as the HOMO and LUMO experience similar energetic changes upon ligand removal or substitution. Chapter 3 presents a practical application for ligand removal of superatomic clusters. I present a synthesis of microspherical, highly porous materials derived from superatomic clusters. These microsphere materials display very different morphology from typical materials made using the same elemental ratio. This altered morphology results in a material which is favorable for use as a battery electrode. Its increased porosity improves its capacity retention upon cycling and at high power. The Co₆S₈(PEt₃)₆ derived material also shows promise as a Na+ ion battery material. In this chapter I also discuss unfinished studies on mixed chalcogenide materials. Chapter 4 explores the electronic basis for the high magnetic moment of the Fe₆S₈(CN)₆ cluster. Through collaboration with computational chemists, I present evidence of a phenomenon known as dual-subshell filling allowing for two spin channels holding different number of electrons resulting in many unpaired electrons. This cluster is also uniquely prepared for use as an extended material due to its cyanide ligands which may readily be used to form Prussian blue analogs. Chapter 5 describes efforts to design extended structures using the Fe₆S₈(CN)₆ cluster. Attempts towards Prussian blue analogs, covalently bound clusters using DCNQI, and EDT-TTF-CONH2 utilizing structures are discussed. Detailed notes on the synthesis of [NEt₄]₅[Fe₆S₈(CN)₆] are also provided. 2 structures which have successfully been synthesized, a 4 bridging ligand and a 2 bridging ligand “wire” are described in detail. In Chapter 6, collaborative efforts to increase our understanding of the cluster building blocks which can function as nanoscale atoms that assemble to form superatomic solids are described. We characterize a representative superatomic cluster, Co₆S₈(PEt₃)₆, in terms of structural, electronic, and magnetic properties using Solid State Nuclear Magnetic Resonance (SSNMR), Density Function Theory (DFT) calculations, and Superconducting Quantum Interference Device (SQUID) measurements. Evidence of delocalized HOMO orbitals and a delocalized spin in the oxidize cluster is shown. The findings presented in this chapter will assist the design of superatomic clusters and state-of-the-art applications, such as single-electron devices. Finally, Chapter 7 is much shorter than the other chapters as it is used to describe smaller projects which do not fit in the scope of the overall thesis. Magnetic measurements on a compound designed in the Norton lab are described.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第24640号 / 工博第5146号 / 新制||工||1983(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 杉野目 道紀, 教授 生越 友樹, 教授 松田 建児 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

In the present study, interactions at the liquid/liquid and liquid/solid interfaces have been investigated by the combination of both nuclear magnetic resonance (NMR) and dynamic nuclear polarization (DNP) techniques. The ¹³C and ¹⁵N paramagnetic contact shifts, and ¹H, ¹³C, and ¹⁵N relaxation times in CH₃CN/2,2,6,6,-tetramethyl-1-piperidinyloxy (TEMPO) and CH₃CONH₂/TEMPO systems have been measured at high magnetic field (B₀ = 1.9-9.4 T). The 13 DNP enhancements at low magnetic field (0.33 T) in the CH₃CONH₂/TEMPO system have been determined by the flow liquid-liquid intermolecular transfer (LLIT) DNP technique. The data can be understood in terms of transient hydrogen bond formation between closed shell diamagnetic molecules and the open shell free radical TEMPO. A set of static and dynamic parameters, such as hyperfine coupling constants, correlation times, and free radicalnuclear internuclear distances in the hydrogen bonding complex, have also been determined. The scalar and dipolar contributions derived from the NMR study have been subsequently employed to predict the corresponding ¹H, ¹³C, and ¹⁵N low magnetic field (0. 33 T) DNP enhancements. Good agreement has been obtained between the NMR predicted and experimentally measured low magnetic field DNP results. The dynamic electron-nuclear intermolecular interactions between the newly discovered fullerene, C₆₀, and the free radical TEMPO have been characterized by flow LLIT and solid liquid intermolecular transfer (SLIT) DNP techniques. A dipolar dominated ultimate DNP enhancement (-250) at 0.33 T magnetic field has been observed. The results are consistent with a model for C₆₀/TEMPO interactions involving nonspecific complex formations. In addition to DNP studies in the liquid state, the solid/liquid surface intermolecular interactions in solid samples of various activated carbon specimens have been monitored by using flow SLIT 'H and ¹³C DNP experiments. The activated carbon samples were prepared by pyrolysis of cellulose, and commercial samples were also employed. The surface-liquid interaction in these studies were monitored with the solvent benzene ( or d₆-benzene). Both time dependent (Overhauser) and time-independent (solid-state) DNP enhancements were observed in these studies. Both chemisorption and physisorption processes of oxygen to the activated carbon were also monitored using the DNP approach. / Ph. D.

The preparation of palladium methoxide complexes was attempted by reacting LiOCH3 with precursors such as Me2- bpyPdCl2 , [(Me2-bpy)Pd(CH3CN)2 J (BF4 )2 and (Me2-bipy)Pd(OAC)2 which have been synthesized for this purpose. Typically the reactions were done on small scale in an NMR tube and monitored spectroscopically. The precursors do not react with methanol, however a broadening peaks is observed at the aromatic region. In deuterated methylene chloride, the precursors and lithium methoxide show similar results. The reaction with LiOCH3 proceeds smoothly in methanol, however it appears to be in equilibrium. In a mixed solvent system, such as methanol/chloroform or methylene chloride,the product appears to decompose. / Master of Science

Ultrasound can be used to create free radicals by growth and collapse of cavitation bubbles. These free radicals have potential use in various fields. The formation of free radicals can be monitored by decrease in T1 during NMR experiments due to paramagnetic effects. Our goal is to develop a method in which ultrasound is used to enhance NMR. By irradiating the sample during analytical measurements, we can decrease T1 which can be used as a non-toxic contrast agent1 producing hydroxyl radicals from the water in the body, invoke NMR enhancement using dynamic nuclear polarization2, control and understand polymer reactions3,4, and study the formation of radicals in chemical systems with EPR5. The experiments conducted indicated a decrease in T1 when ultrasound was applied. A maximum decrease was observed when 104 W of ultrasound power was applied and with higher concentrations of radical producing species. Through the experiments it was evident that the sample temperature increased during sonication. To counter this, gated sonication was used to minimize temperature increase. During sonication, the sample was vigorously mixed. Experiments where the sample was mixed through alternate means and theoretical simulations indicate that sample mixing gives an apparent decrease in T1. In situ sonication to decrease T1 shows promise. The question remains if the decrease is due to a combination of radical production and mixing or just an artifact of sample mixing. This is a difficult parameter to determine but future experiments will attempt to supply further conclusions. / Master of Science

The use of ¹⁹F observation using MLEV ¹H decoupling in LC/¹⁹F {¹H} NMR was investigated as an alternative to LC/¹H NMR for fluorine containing mixtures and in order to avoid the solvent background problems associated with LC/¹H NMR. P-fluorobenzoate derivatives of various alcohols were analyzed by both LC/¹⁹F {¹H} and LC/¹H NMR. Another alternative exists in supercritical fluid chromatography. A delivery system was assembled and an NMR flow probe was developed and demonstrated practical for directly coupled SFC/¹H NM. The alkane substituents of a model fuel mixture were identified using SFC/¹H NMR in contrast to using the normal phase LC/¹H NMR approach. The relaxation behavior and molecular motion of dilute solutions of benzene and acetonitrile in sub- and supercritical CO₂ were determined using stopped flow ¹H and ¹⁴N NM. The nuclear spin-lattice relaxation times (T₁) for ¹H and ¹⁴N were measured through inversion recovery and linewidth, respectively. Relaxation was found to be dominated by spin-rotation interactions with molecular correlation times (r_sr and _rc) being determined directly from the ¹H and ¹⁴N T₁ over a wide range of viscosities and temperatures. Line-narrowing improvements of ¹⁴N averaged 3-fold as a result of the increased molecular motion. The increased molecular motion as a result of supercritical CO₂ resulted in improved signal enhancement using flow dynamic nuclear polarization. The observed enhancements were two times greater than that typically achieved for the same system and configuration using normal liquid solutions. Through observed NMR enhancements, relative microwave magnetic field values in the vicinity of the NMR coils were measured for typical flow and static DNP-NMR configurations. The advantages of the former were noted. / Ph. D.

A method has been developed whereby various physical properties of middle distillate fuels may be rapidly and accurately calculated by a group property approach from data obtained from a directly coupled Liquid Chromatograph -⁻¹H Nuclear Magnetic Resonance Spectrometer (LC-⁻¹H NMR). The physical properties include cetane number, cetane index, density, specific gravity, pour point, flash point, viscosity, filterability, heat of combustion, cloud point, volume percent aromatics, residual carbon content, and initial, 10%, 50%, 90%, and end boiling points. These property predictions have accuracies approaching the error for measurement of the experimental physical property and require less than two hours analysis time per fuel. An interface was developed between the NMR spectrometer and a personal computer to aid in automation of the LC-⁻¹H NMR data collection and to perform off-line analysis of the LC·⁻¹H NMR data. This interface and all associated software is described. Also presented is a series of model compound studies in which the physical properties of pure hydrocarbons (i.e., alkanes, monocyclic and dicyclic aromatics) were predicted by a similar group property approach. / Ph. D.

The intramolecular carbonyl exchange which occurs in solution for the dinuclear and tetranuclear cobalt complexes containing the bisphosphines DPM, bis(diphenylphosphino)methane; DMPM, (dimethyl-diphenylphosphino)methane; and DMM, bis(dimethylphosphino)methane is very fast at temperatures down to -80°C. For the tetranuclear clusters Co₄(CO)₈(DPM)₂, Co₄(CO)₈(DMPM)₂, Co₄(CO)₈(DMM)₂, and Rh₄(CO)₈(DPM)₂, this exchange is slow at -80°C on the NMR time scale. The postulated mechanism for carbonyl exchange is based on a previously proposed mechanism, which is the expansion of the ligand icosahedron into a cubooctohedron. Because of the constraints imposed by the bisphosphine ligands, only one ligand icosahedron can be formed that is consistent with the known structure. Racemization of enantiomers by rotation of the ligands on the apical metal atom can occur, and thus enables complete carbonyl exchange to take place. The series of binary metal carbonyls: Co₄(CO)₈, Fe₃(CO)₁₂, Co₄(CO)₁₂, Co₃Rh(CO)₁₂, Co₂Rh₂(CO)₁₂ and Rh₄(CO)₁₂, has been studied by variable temperature MAS (magic angle spinning) ¹³C NMR spectroscopy. All of these molecules except for Rh₄(CO)₁₂ show dynamic behavior as evidenced by their solid state ¹³C NMR spectra. Since carbonyl ligands cannot move within the crystalline lattice to an extent sufficient to render bridging and terminal carbonyls equivalent, then the dynamic behavior observed for the binary metal carbonyls must be described as metal atom movement within the carbonyl cage. The tetranuclear clusters which contain rhodium show a higher coalescence temperature in their NMR spectra than Co₄(CO)₁₂. As the rhodium content increases the activation energy for carbonyl exchange for exchange increases. cluster Rh₄(CO)₁₂ does not exhibit dynamic behavior in the solid state. lt is concluded that the rhodium tetrahedron is too large to move within the carbonyl cage. The cobalt “A-Frame" complexes Co₂(CO)₃,(DPM)₂I₂, Co₂(CO)₃(DMM)(DPM)I₂, and Co₂(CO)₃(DPM)₂S were synthesized but show no dynamic behavior in solution. The crystal structure of Co₂(CO)₃(DMM)(DPM)l₂, shows that this "A-Frame" complex is coordinatively saturated around the cobalt atoms. Thus, these molecules are relatively inert, and show no evidence of carbonyl scrambling. / Ph. D. / incomplete_metadata