京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18303号 / 工博第3895号 / 新制||工||1598(附属図書館) / 31161 / 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 森 泰生, 教授 跡見 晴幸 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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Optimization of Semiempirical Quantum Mechanical Methods for in Silico Drug Design Doctoral thesis Kristian Kříž The subject of this thesis is the optimization of semiempirical quantum mechanical methods (SQM) for their use in in silico drug design. The thesis covers two topics - COSMO2 solvation model optimization part and PLF547, PLA15 dataset development part. The first part is devoted to the optimization of COSMO solvation model by addition of a nonpolar term and reparametrization of the model for SQM methods PM6 and PM7. We have shown that the accuracy of the resulting "COSMO2" optimized model improved on all the tested datasets and we have compared it to other selected SQM solvation models. The method has also been tested on the protein ligand complexes as a part of a scoring function, where it provides better preditction of binding affinity of drug candidates for their target protein. The second part of the thesis describes the construction of datasets for noncovalent interactions aimed speicificly to represent an environment of an enyzme active site complexed with a ligand with reliable benchmark values of interaction energies in vacuum and solvent (water). The developed PLF547 and PLA15 datasets are suitable for testing and development of methods for the use in drug design. We have...

A study was conducted on sol-gel synthesis of an adsorbent (phosphotungstic acid embedded in silica gel, H-PTA/SiO­2) of radioactive cesium. A novelty of this work is covalent bonding of PTA to the surface of solid support that prevents leaching from the surface of the material. The sample was granulated with a binder, aluminium oxide (γ-Al2O3). Solid-state NMR and FT-IR spectroscopy were used to confirm the presence of Keggin units of PTA in the bound materials. Thermal analysis of H-PTA/SiO­2 - γ-Al2O3 (50 %) showed that the water content in the bound material was appreciably lower than in the pure adsorbent. Quantitative determination of surface acidity of porous materials is an important analytical problem in characterization of the adsorbents. This problem was solved by reversed titration after saturation of the materials by anhydrous solution of pyridine. Batch and column adsorption tests showed that the adsorbent demonstrated high adsorption capacities towards cesium.

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<p>Prothrombinase is composed of a proteinase, factor Xa (Xa), its cofactor Va (Va), Ca²⁺ and a zymogen, prothrombin (II), assembled on a phospholipid surface. During coagulation, prothrombinase accelerates II to thrombin conversion; but during anticoagulation, it protects the proteinase from inhibition by antithrombin (AT) ± unfractionated heparin (UFH). Although the degree of Xa protection by prothrombinase varies according to the reports in literature, moderate to significant protective effects have been consistently reported by most investigators. To overcome the limitations of UFH, our laboratory has developed a covalent complex of AT and UFH (ATH) with superior anticoagulant responses. To further understand the mechanisms of enhanced anticoagulant activity of ATH, we proceeded to study inhibition of the prothrombinase complex<em> </em>on synthetic vesicles, activated platelets and red blood cells (RBCs). Using discontinuous inhibition assays, we determined the rate of inhibition of prothrombinase-complexed Xa compared to control Xa. With synthetic vesicles, Xa was protected from inhibition by AT+UFH when in prothrombinase, while only a mild protective effect was observed with ATH. Omission of various components of the prothrombinase led to a reduction in Xa protection for AT+UFH. However, an increased Xa protection against ATH was observed when II was omitted from the prothrombinase. In comparison to the synthetic vesicle system, activated platelets showed a similar trend for protection of Xa in reactions involving prothrombinase ± components, while no protection of Xa was observed for ATH reactions. Alternatively, RBCs showed differences relative to vesicles in that increased protection of Xa occurred with omission of II and Va for AT+UFH, whereas omission of Va increased protection against ATH inhibition. In addition, ATH had improved inhibition of thrombin generation, fibrin formation and plasma coagulation compared to AT+UFH. Studies of fluorescently labelled Xa and inhibitors detailed binding interactions with prothrombinase subunits. Overall, the results suggest that a covalent linkage between AT and heparin improves inactivation of prothrombinase complexed-Xa leading to down-regulation of prothrombinase function.</p> / Doctor of Philosophy (Medical Science)

Phosphonium ions readily compare to ammonium ions in regards to their aggregate characteristics, thermal stability, and antibacterial activity. Ionic aggregation in phosphonium-based polymers provides thermoreversible crosslinks, ideal for reversible self-assembly, self-healing, and smart response. In polymers, these ionic functionalities aggregate, providing improved moduli, and altering the size and structure of ionic aggregates regulates polymer melt processability. This dissertation highlights phosphonium-based chemistry for the synthesis of novel step-growth ionomers and structure-property relationships in ionic polymers. The synthesis of phosphonium endcapping reagents for melt polyester reactions afforded a thermally stable ionic functionality that controlled molecular weight. Weak association was present with phosphonium ions at low ion concentrations below 7.7 mole %. The use of novel ionic bisacetoacetate monomers in the formation of networks from Michael addition reactions led to the synthesis of ionic networks with increased and broadened glass transitions and improved tensile stresses at break and strains at break compared to those in the non-ionic networks. The first electrospun fibers from Michael addition crosslinking reactions are reported, and equilibrium ionic liquid uptake experimental results indicated that ionic functional networks absorb close to three times the amount of ionic liquid as non-ionic, poly(ethylene glycol)-based films. Chain-extending polyurethanes with a phosphonium diol and subsequently varying the hard segment content led to changes in ionic aggregation, crystallinity, and thermal transitions in the polymers. Additionally, novel phosphonium-based methacrylate monomers incorporated into diblock copolymers with styrene exhibited microphase separation. Overall, the inclusion of phosphonium ions pendant to or in the main chain of various types of polymers led to changes in morphology, improved tensile properties, enhanced moduli, broadened transitions, changes in crystalline melting points, changes in solubility, and appearance of ionic aggregation. / Ph. D.

<p dir="ltr">Humans encode approximately 100 deubiquitinating enzymes (DUBs) which are categorized into seven distinct subfamilies. Each family and representative has a unique expression, function and binding topology to ubiquitin. In addition to human DUBs, parasites, bacteria, and viruses contain DUBs with unique structures and functions. One subfamily of DUBs, the ubiquitin C-terminal hydrolases (UCH), has four structurally similar human members and two known members within the <i>Plasmodium falciparum</i> genome. Human UCHL1 and UCHL3 are genetically validated targets in oncology and <i>Plasmodium falciparum</i><i> </i>UCHL3 (PfUCHL3) is a prospective target for antimalarial drug development. Though these three UCH enzymes have potential as therapeutic targets, there is a significant lack of quality small molecule chemical probes to understand the underlying biology and function of the enzymes, pharmacologically validate the targets, and serve as leads for drug development in oncology and malaria.</p><p dir="ltr">The UCH enzymes are cysteine proteases, which our lab has leveraged to identify novel covalent small molecule inhibitors of each enzyme. The workflow for each hit identification and optimization campaign is similar. Covalent fragment screening of electrophilic small molecule libraries against the respective recombinant enzyme was performed to identify chemical space around each enzyme. Subsequent medicinal chemistry hit-to-lead optimization was undertaken to improve upon the moderately potent hit molecules to provide improved small molecule inhibitors for each enzyme. Inhibitor identification and optimization for UCHL1 is described in Chapter 2, revealing a novel scaffold and a cocrystal structure reveals a unique binding pose for UCHL1 inhibitors. These molecules were also characterized in breast cancer cells to validate UCHL1 as a therapeutic target in breast cancer. First-in-class covalent inhibitors of UCHL3 are described in Chapter 3. Medicinal chemistry optimization along with a cocrystal structure of the initial hit has revealed the molecular interactions of this novel inhibitory scaffold. PfUCHL3 inhibitor identification is described in Chapter 4. Characterization of these molecules against Plasmodium falciparum is described along with a comparison to a recently identified reversible PfUCHL3 inhibitor. Finally, conclusions and future directions toward the development of potent, drug-like inhibitors of each UCH enzyme is presented in Chapter 5.</p>

Coiled coils are a well-known protein structure prevalent in eukaryotic function, synthetic applications, and de novo protein design. Coiled-coil folding is often described using heptad repeat positions labeled abcdefg where a and d positions occupy the interface between the coils, e and g positions flank the interface, and the b, c, and f positions face the solvent-exposed surface. The a, d, e, and g positions have been extensively studied in the coiled-coil literature. There is a lack of investigation on the impact of the b, c, and f positions on coiled-coil folding. Chapter 1 is an introduction to the heptad repeat of coiled coils and the impact on folding of each heptad repeat position. In Chapter 2 we introduce a non-covalent interaction among the b, c, and f positions of a coiled-coil trimer that significantly enhances thermodynamic stability. We identify characteristics of the f-position residue (hydrogen bond donating ability and hydrophobicity) that lead to the greatest amount of stability. Chapter 3 introduces crystal structures and molecular dynamic simulations of the interaction to identify the mechanism of stabilization. Further thermodynamic studies find a key salt-bridge interaction between the b and c positions that are influenced by the f-position residue. Chapter 4 explores the impact of salt on the non-covalent interaction and determines that the interaction is sensitive to salt screening and is ionic in nature. It also explores more characteristics of the f-position amino acid, in particular the hydrogen bond donating component. In Chapter 5 we insert the solvent-exposed interaction into helix bundles of differing length and oligomeric state. We find that stability is not only dependent upon amino acid identity but also the length and stoichiometry of a coiled coil.

Tesis por compendio / [ES] En la presente tesis doctoral se ha evaluado el uso de nuevos soportes celulósicos y silíceos como sistemas de filtración para la estabilización y conservación de alimentos líquidos con el fin de afrontar dos grandes retos de la industria de bebidas. Por un lado, evitar o minimizar los cambios en las propiedades nutricionales, estructurales y organolépticas de los alimentos, ocasionados por la pasteurización térmica tradicional, y ofrecer una alternativa al problema de la baja viabilidad debida a los altos costos de inversión/producción al aplicar nuevas tecnologías no térmicas. Por ello, esta tesis doctoral se centra en el desarrollo y evaluación de una nueva tecnología no térmica de conservación de alimentos líquidos basada en la filtración. Se han desarrollado sistemas de filtración a partir de soportes celulósicos y silíceos, sin funcionalizar o funcionalizados con compuestos antimicrobianos. En el primer capítulo se evaluó el uso de materiales de celulosa como soportes filtrantes para el tratamiento de alimentos líquidos. Como primera aproximación se desarrolló un material poroso nano-micro tubular a partir de la extracción y deslignificación del material celulósico presente en el corazón o raquis de la mazorca de maíz. El uso de este soporte resultó ser efectivo como material filtrante para el tratamiento de agua y zumo de naranja, en un sistema de flujo continuo, eliminando la carga microbiana. La aplicación de este soporte como sistema de filtración presenta diversas ventajas como su capacidad de retención microbiana, la reutilización de sub-productos del maíz y, por tanto, su respeto al medioambiente. Sin embargo, sería necesario optimizar el proceso de filtrado para evitar la frecuente obturación de sus poros que requirió varios ciclos de lavado durante el proceso, así como establecer un método de regeneración del material para incrementar su vida útil. Además, este sistema afectó al color del zumo filtrado, que no se mantuvo constante durante el proceso, lo que supone una importante desventaja que es necesaria abordar. Como segunda aproximación, se evaluó el potencial de la inmovilización de una molécula bioactiva sobre membranas de celulosa, para mejorar la capacidad de retención microbiana del material celulósico, así como permitir su reutilización. Los filtros de celulosa funcionalizados con poliaminas demostraron ser eficaces en la eliminación de patógenos en agua, debido a las cargas positivas generadas por los grupos amina inmovilizados en la superficie de las membranas, que atraen y retienen las bacterias cargadas negativamente. Dada la fácil preparación y procedimiento de uso de las membranas de celulosa funcionalizadas con poliaminas, éstas podrían ser consideradas una buena opción para el desarrollo de sistemas de tratamiento de aguas in situ, rápidos, de fácil manejo y de bajo coste. El segundo capítulo describe el desarrollo y aplicación de partículas de sílice funcionalizadas con compuestos de aceites esenciales, con el fin de diseñar coadyuvantes de filtración con actividad antimicrobiana. La filtración de diversas matrices alimentarias (agua, cerveza y zumo de manzana) a través de los soportes funcionalizados con los antimicrobianos naturales demostró ser eficaz en la reducción del recuento de la cepa patógena Escherichia coli, así como frente a la microflora endógena de la cerveza y el zumo (bacterias acidolácticas, aerobios mesófilos, psicrófilos, mohos y levaduras). La eficacia en el control microbiano se debe a la combinación de la adsorción física y la inactivación por contacto con los compuestos de aceites esenciales inmovilizados. Además, la evaluación de las propiedades físico-químicas y sensoriales de los alimentos líquidos demostró un efecto poco significativo, éste depende del tamaño de las partículas de sílice usadas y de la molécula bioactiva inmovilizada. Por lo tanto, el sistema de conservaci� / [CA] En la present tesi doctoral s'ha avaluat l'ús de nous suports cel·lulòsics i silicis com a sistemes de filtració per a l'estabilització i conservació d'aliments líquids, amb la finalitat d'afrontar dos grans reptes de la indústria de begudes. D'una banda, evitar o minimitzar els canvis en les propietats nutricionals, estructurals i organolèptiques dels aliments, ocasionats per la pasteurització tèrmica tradicional, i oferir una alternativa al problema de la baixa viabilitat deguda als alts costos d'inversió/producció en aplicar noves tecnologies no tèrmiques. Per això, aquesta tesi doctoral es centra en el desenvolupament i avaluació d'una nova tecnologia no tèrmica de conservació d'aliments líquids basada en la filtració. S'han desenvolupat sistemes de filtració a partir de suports cel·lulòsics i silicis, sense funcionalitzar o funcionalitzats amb compostos antimicrobians. En el primer capítol es va avaluar l'ús de materials de cel·lulosa com a suports filtrants per al tractament d'aliments líquids. Com a primera aproximació es va desenvolupar un material porós nano-micro tubular a partir de l'extracció i deslignificació del material cel·lulòsic present en el cor o raquis de la panolla de dacsa. L'ús d'aquest suport va resultar ser efectiu com a material filtrant per al tractament d'aigua i suc de taronja, en un sistema de flux continu, eliminant la càrrega microbiana. L'aplicació d'aquest suport com a sistema de filtració presenta diversos avantatges com la seua capacitat de retenció microbiana, la reutilització de subproductes de la dacsa i, per tant, el seu respecte al medi ambient. No obstant això, seria necessari optimitzar el procés de filtrat per a evitar la freqüent obturació dels seus porus que va requerir diversos cicles de rentada durant el procés, així com establir un mètode de regeneració del material per a incrementar la seua vida útil. A més, aquest sistema va afectar el color del suc filtrat, que no es va mantenir constant durant el procés, la qual cosa suposa un important desavantatge que és necessari abordar. Com a segona aproximació, es va avaluar el potencial de la immobilització d'una molècula bioactiva sobre membranes de cel·lulosa, per a millorar la capacitat de retenció microbiana del material cel·lulòsic, així com permetre la seua reutilització. Els filtres de cel·lulosa funcionalitzats amb poliamines van demostrar ser eficaces en l'eliminació de patògens en aigua, a causa de les càrregues positives generades pels grups amina immobilitzats en la superfície de les membranes, que atrauen i retenen els bacteris carregats negativament. Donada la fàcil preparació i procediment d'ús de les membranes de cel·lulosa funcionalitzades amb poliamines, aquestes podrien ser considerades una bona opció per al desenvolupament de sistemes de tractament d'aigües in situ, ràpids, de fàcil maneig i de baix cost. El segon capítol descriu el desenvolupament i aplicació de partícules de sílice funcionalitzades amb compostos d'olis essencials, amb la finalitat de dissenyar coadjuvants de filtració amb activitat antimicrobiana. La filtració de diverses matrius alimentàries (aigua, cervesa i suc de poma) a través dels suports funcionalitzats amb els antimicrobians naturals va demostrar ser eficaç en la reducció del recompte del cep patogen Escherichia coli, així com enfront de la microflora endògena de la cervesa i el suc (bacteris àcid làctics, aerobis mesòfils, psicròfils, floridures i llevats). L'eficàcia en el control microbià es deu a la combinació de l'adsorció física i la inactivació per contacte amb els compostos d'olis essencials immobilitzats. A més, l'avaluació de les propietats fisicoquímiques i sensorials dels aliments líquids estudiats va demostrar un efecte poc significatiu, aquest depèn de la grandària de les partícules de sílice usades i de la molècula bioactiva immobilitzada. Per tant, el sistema de conserv / [EN] In the present doctoral thesis the use of new cellulosic and silica supports as filtering systems for the stabilization and preservation of liquid foods has been evaluated to overcome two major challenges of the beverage industry. On the one hand, avoid or minimize the changes in the nutritional, structural and organoleptic properties of food caused by traditional thermal pasteurization, and offer an alternative to the problem of low viability due to high investment/production costs when applying new non-thermal technologies. Therefore, this doctoral thesis focuses on the development and evaluation of a new non-thermal technology for the preservation of liquid foods based on filtration. The filtering systems have been developed from cellulosic and silica supports, non-modified or functionalized with antimicrobial compounds. In the first chapter, the use of cellulose materials as filtering supports for the treatment of liquid foods was evaluated. As first approximation, a porous nano-micro tubular material was developed from the extraction and delignification of the cellulosic material present in the corn stalk. The use of this support was effective as filtering material for the treatment of water and orange juice, in a continuous flow system, eliminating the microbial load. The application of this support as filtering system has several advantages, such as its microbial retention capacity, the reuse of corn by-products and, therefore, its respect for the environment. However, it would be necessary to optimize the filtering process to avoid the frequent clogging of its pores that required several washing cycles during the process, as well as to establish a method of material regeneration to increase its life. In addition, this system affected the color of the filtered juice, which did not remain constant during the process, representing an important disadvantage that must be addressed. As a second approach, the potential of the immobilization of a bioactive molecule on cellulose membranes was evaluated to improve the microbial retention capacity of the cellulosic material, as well as to allow its reuse. The cellulose filters functionalized with polyamines proved to be effective in eliminating pathogens in water, due to the positive charges generated by the amine groups immobilized on the surface of the membranes, which attract and retain the negatively charged bacteria. Given the easy preparation and usage of the polyamines-functionalized cellulose membranes, these could be considered a good option for the development of fast, easy to use and low cost in situ water treatment systems. The second chapter describes the development and application of silica particles functionalized with essential oil components to design filtering aids with antimicrobial activity. The filtration of various food matrices (water, beer and apple juice) through the supports functionalized with natural antimicrobials proved to be effective in reducing the load of the pathogenic strain Escherichia coli, as well as reducing the endogenous microflora of beer and the juice (lactic acid bacteria, mesophilic, psychrophilic, mold and yeast). The removal capability is due to the combination of physical adsorption and contact inactivation with the essential oil compounds immobilized. In addition, the evaluation of the physicochemical and sensory properties of the liquid foods studied showed a not significant effect, it depends on the size of the silica particles used and the immobilized bioactive molecule. Therefore, the proposed preservation system has a high potential for cold beverage pasteurization processes. / N. Peña-Gomez would like to thank for financial support in the frame of her PhD project to Operational Programme of the European Social Fund (ESF) 2014-2020, the Agencia Estatal de Investigación, Generalitat Valenciana and FEDER-EU (Projects RTI2018-101599-B-C21 and AGL2015-70235-C2-1-R). The authors also thank the Electronic Microscopy & Microanalysis Laboratory at Patras University for support. / Peña Gomez, N. (2020). Development of polymeric and silica filtering materials functionalized with antimicrobial compounds for the elimination of microorganisms in liquid food [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/137041 / Compendio