Cryopreservation of adherent cells may be advantageous for cell types that are difficult to preserve in suspension or when it is necessary to preserve characteristics of the adherent cultured cells. Vitrification is a promising procedure for the preservation of adherent cells that prevents ice crystal formation and the resulting dissociation and morphological damage. To successfully vitrify adherent cells, high concentrations of CPA are required which increases the likelihood of osmotic and toxic damage. In this dissertation, we describe a rational design strategy that predicts mathematically optimized CPA addition and removal procedures based on the minimization of a toxicity cost function. These rationally designed procedures rely on the accurate knowledge of cell biophysical parameters. We validate an in situ calcein fluorescence quenching method for the determination of membrane permeability parameters for adherent cells. We also describe the determination of osmotic tolerance limits for adherent cells. We use rational design strategies to determine CPA addition and removal procedures for adherent endothelial cells, neuronal cells, and induced pluripotent stem cells as well as oocytes. Also, we provide experimental support for the feasibility of these methods using adherent endothelial cells. The mathematical methods and experimental procedures outlined in this dissertation are important tools for the design of addition and removal procedures for concentrated CPA solutions. This dissertation is an important step toward successful design and implementation of vitrification strategies for adherent cells and tissues. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Feb. 14, 2013 - Feb. 14, 2015

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No. of bitstreams: 2 Tese - Marcelo do Nascimento Gomes - 2017.pdf: 17453741 bytes, checksum: 2207111e32956fbf831d9eedf261a49e (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-04-06 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / In view of the current panorama of tuberculosis (TB) pandemic in the world, aggravated by co-infection with the HIV virus and the emergence of resistant strains of Mycobacterium tuberculosis (M. tb.), The research and discovery of new Anti-TB drugs. The present work aimed at the planning, synthesis and biological evaluation of new compounds with anti-TB activity, candidates for TB drugs. Structure and activity relationship (SAR) studies were developed using Matched Pair Molecular Analysis (MMPA) and binary models of quantitative relations between structure and activity (QSAR) using a combination of molecular fingerprints and machine learning methods. Bioisosteric replacement were proposed to plan new aryl and heteroaryl chalcones using the information obtained from SAR and QSAR analyses. Thirty-three compounds were selected by the consensus QSAR model for the synthesis. These compounds were synthesized and their structures confirmed by infrared (IR), hydrogen nuclear magnetic resonance (1H NMR) and carbon (13C NMR) spectroscopic methods and mass spectrometry (MS). Compounds which showed high purity (≥95% in HPLC) were tested against strains of M. tb. H37Rv (sensitive) and resistant to rifampicin (RMP) or isoniazid (INH). In addition, they were also tested in mammalian cell cytotoxicity assays and activity spectrum. We identified 22 hits with anti-TB activity, with minimum inhibitory concentration (MIC) in M. tb. H37Rv in aerobic conditions (MABA) <10 μM. Of these, 12 compounds exhibited potent M. tb. replication activity on nanomolar scale, with MIC values in MABA <1 μM and in micromolar under anaerobic conditions (LORA) with MIC <10 μM. In addition, these compounds also showed potent inhibitory activity against monoresistant strains at RMP or INH (MIC <1 μM and MIC <10 μM, respectively). Hits also demonstrated low cytotoxicity in mammalian cells (Vero cells) and selectivity index between 11 and 545 for M. tb. The same selectivity was verified in the activity spectrum assay against four commensal strains and six strains of non-tuberculosis mycobacteria (NTMs), in which the compounds presented broad spectrum against the NTMs strains. These results demonstrated that the combination of in silico strategies for the design of aryl and heteroaryl chalcones was efficient in identifying new compounds that proved to be potent, selective and promising candidates for prototypes of anti-TB drugs. / Face ao panorama atual da pandemia de tuberculose (TB) no mundo, agravado pela co-infecção com o vírus HIV e o surgimento de cepas de Mycobacterium tuberculosis (M. tb.) resistentes aos fármacos utilizados, é urgente a pesquisa e descoberta de novos fármacos anti-TB. O presente trabalho objetivou o planejamento, a síntese e avaliação biológica de novos compostos com atividade anti-TB, candidatos a fármacos para TB. Foram desenvolvidos estudos de relação entre estrutura e atividade (SAR) utilizando o método Matched Pair Molecular Analysis (MMPA) e modelos binários de relações quantitativas entre estrutura e atividade (QSAR) utilizando combinação de descritores (fingerprints) moleculares e métodos de aprendizado de máquina. Substituições bioisostéricas foram propostas para planejar novas aril e heteroaril chalconas utilizando as informações obtidas nas análises de SAR e QSAR. Trinta e três compostos foram selecionados pelo modelo de QSAR por consenso para a síntese. Estes compostos foram sintetizados e suas estruturas foram confirmadas por métodos espectroscópicos no infravermelho (IV), de ressonância magnética nuclear de hidrogênio (RMN de 1H) e de carbono (RMN de 13C) e espectrometria de massas (EM). Os compostos que apresentaram elevado grau de pureza (≥95% em CLAE) foram testados contra cepas de M. tb. H37Rv (sensíveis) e resistentes a rifampicina (RMP) ou isoniazida (INH). Além disso, também foram testados em ensaios de citotoxicidade em células de mamíferos e espectro de atividade. Foram identificados 22 hits com atividade anti-TB, com concentração inibitória mínima (MIC) em M. tb. H37Rv em ensaios em condições aeróbias (MABA) <10 μM. Destes, 12 compostos exibiram potente atividade na replicação de M. tb em escala nanomolar, com valores de MIC em MABA <1 μM e em micromolar em condições anaeróbias (LORA) com MIC <10 μM. Ademais, esses compostos também apresentaram potente atividade inibitória contra cepas monoresitentes a RMP ou INH (MIC<1 μM e MIC<10 μM, respectivamente). Os hits também demonstraram baixa citotoxicidade em células de mamífero (células Vero) e índice de seletividade entre 11 e 545 para M. tb. A mesma seletividade foi verificada no ensaio de espectro de atividade frente a quatro cepas comensais e seis cepas de micobactérias não-tuberculose (NTMs), em que os compostos apresentaram amplo espectro contra as cepas NTMs. Estes resultados demonstraram que a combinação das estratégias in silico para o planejamento de aril e heteroaril chalconas foi eficiente na identificação de novos compostos que se mostraram potentes, seletivos e promissores candidatos a protótipos de fármacos anti-TB.

Les peptides antimicrobiens sont considérés comme une nouvelle classe prometteuse d'agents anti-infectieux. Afin de développer de nouveaux agents efficaces et non toxiques, des stratégies de conception rationnelle peuvent être utilisées. Dans cette perspective, nous avons utilisé une approche computationnelle pour concevoir trois peptides synthétiques ([I5, R8] MP, EcDBS1R6 et PaDBS1R1). En déterminant la concentration minimale inhibitrice, nous avons montré que tous les peptides sont actifs contre les bactéries Gram-négatif et -positif. Seul [I5, R8] MP a montré une activité antifongique. La mesure de la concentration de peptide provoquant 50 % de mortalité cellulaire a permis de montrer que les peptides étaient faiblement ou non hémolytiques, sans toxicité vis-à-vis des cellules embryonnaires rénales humaines HEK-293. La cinétique bactéricide a révélé que PaDBS1R1 et [I5, R8] MP tuent rapidement E. coli en comparaison à S. aureus et que EcDBS1R6 élimine rapidement les deux souches. Des études de perméabilisation et de dépolarisation combinées à de la microscopie électronique à haute résolution (FEG-SEM) ont montré un mécanisme membranolytique des peptides. L'analyse de la structure des peptides par spectroscopie de dichroïsme circulaire et résonance magnétique nucléaire, ainsi que par modélisation moléculaire lors de leur interaction avec une membrane modèle, révèle une conformation en hélice alpha amphipatique. En conclusion, notre étude indique que l'évaluation structurale et fonctionnelle de peptides antimicrobiens synthétiques conçus de manière rationnelle représente une stratégie prometteuse pour le développement de nouveaux agents antimicrobiens. / Antimicrobial peptides (AMPs) have been considered as a potential novel class of antimicrobial compounds. In order to generate new potent and non-toxic antimicrobial agents, rational design strategies may be employed. In this view, we used a computational method to design three synthetic AMPs ([I5, R8] MP, EcDBS1R6 and PaDBS1R1). By determining the minimum inhibitory concentration, we found that all the peptides were active against Gram-negative and -positive bacteria. Only [I5, R8] MP was found to display antifungal activity. The determination of the peptide concentration producing 50% of cell lysis revealed low or no hemolytic activity, with no cytotoxicity towards human embryonic kidney cells HEK-293. During time-kill assays more rapid bactericidal effects were observed for PaDBS1R1 and [I5, R8] MP against E. coli compared to S. aureus. For the peptide EcDBS1R6, identical killing curves were obtained for both bacterial strains. Membrane permeabilization and depolarization studies combined with field emission gun scanning electron microscopy (SEM-FEG) revealed that a membranolytic mechanism occurs for these peptides. When analyzed by circular dichroism and nuclear magnetic resonance microscopy or by molecular dynamics simulations during interaction with a membrane model, peptides were shown to adopt an amphipathic alpha-helical conformation. In conclusion, our results indicate that the structural and functional evaluation of rationally designed synthetic AMPs represents a promising strategy for the development of potent novel antimicrobial agents.

This thesis describes the rational design of a novel enzyme, a thiolester hydrolase, derived from human glutathione transferase (GST) A1-1 by the introduction of a single histidine residue. The first section of the thesis describes the design and the determination of the reaction mechanism. The design was based on the crystal structure of human GST A1-1 complexed with S-benzylglutathione. The resulting enzyme, A216H, catalyzed the hydrolysis of the non-natural substrate GSB, a thiolester of glutathione and benzoic acid. The reaction followed saturation kinetics with a kcat of 0.00078 min-1 and KM of 5 μM. The rate constant ratio, (kcat/KM)/kuncat, was found to be more than 107 M-1. The introduction of a single His residue in position 216 opened up a novel reaction pathway in human GST A1-1 and is a nice example of catalytic promiscuity. The substrate requirements were investigated and A216H was found to be selective since only two out of 18 GS-thiolesters tested were substrates for A216H. The reaction mechanism of the A216H-catalyzed hydrolysis of GSB was determined and found to proceed via an acyl intermediate at Y9. The hydrolysis was catalyzed by H216 that acts as a general base and the deacylation was found to be the rate-determining step. The Y9-intermediate could be selectively trapped by oxygen nucleophiles and primary alcohols, in particular 1-propanol and trifluoroethanol, were the most efficient. In addition, saturation kinetics was obtained in the acyl transfer reaction with 1-propanol indicating the presence of a second binding site in A216H. The second section of this thesis describes the site-specific covalent modification of human GST A1-1. The addition of GSB to the wild-type protein results in a site-specific benzoylation of only one tyrosine residue, Y9, out of ten present in the protein (one out of totally 51 nucleophiles). The reaction was tested with five GST classes (Alpha, Mu, Pi, Theta and Omega) and found to be specific for the Alpha class isoenzymes. The covalent modification reaction was further refined to target a single lysine residue, K216, providing a more stable linkage in the form of an amide bond. The reaction was found to be versatile and approximately 50% of the GS-thiolesters tested acylated K216, including a fluorophore. / <p>On the day of the public defence the status of article II was: Submitted and article IV was: In press.</p>

RNAs have become increasingly recognized as possible drug targets due to their involvement in important biochemical functions, as well as their unique but well-defined structures. Recently published crystal structures depict the binding of a series of aminoglycosides- or more specifically- 2-deoxystretamine (2-DOS), the most preserved central scaffold of aminoglycosides, to a conserved 5'-GU-3'region on their target RNAs. A novel unnatural γ-amino acid, 1, has been synthesized using 2-deoxystreptamine as a template through structure-based rational design. The unnatural amino acid has been designed to replace a glycosidic linkage with an amide bond, which may limit the promiscuous binding characteristics of aminoglycosides through increased rigidity of the ligands and additional hydrogen bonding. The binding selectivity and affinity will be studied in the future through a fluorescence assay.

Cette thèse propose une approche rationnelle pour le design de polymères à empreintes moléculaires (MIPs) pour la détection de nitro-explosifs. Les polymères à empreintes moléculaires qui miment la reconnaissance moléculaire biologique, ont l’avantage d’être stables dans des environnements sévères et peuvent adopter différentes formes physiques pour le couplage avec des transducteurs. Leur synthèse est basée sur la co-polymérisation de monomères fonctionnels et réticulants en présence de la molécule cible, ou comme dans cette thèse, d’un analogue ayant une structure proche de celle de la molécule cible. Cela conduit à la formation d’un réseau polymérique tridimensionnel rigide avec des sites de liaison complémentaires en taille, forme et position des groupes fonctionnels de la molécule cible ou de l’analogue. Pour identifier le meilleur monomère fonctionnel pour notre molécule cible, une approche rationnelle basée sur la modélisation moléculaire, la résonance magnétique nucléaire (RMN) et le titrage par calorimétrie isotherme (ITC) a été utilisée. Elle permet d’optimiser le mélange de pré-polymérisation pour identifier le monomère fonctionnel interagissant le plus fortement avec la molécule cible. Les résultats obtenus ont été confrontés à des études de liaison à partir de polymères synthétisés. La formulation polymérique ainsi conçue est intégrée aux surfaces du transducteur sous forme de nanoparticules, de films et de nanoparticules incorporés dans des films de polydopamine électropolymérisés. En plus des polymères traditionnels obtenus par polymérisation radicalaire classique sous forme de particules, des films de MIP à base de polydopamine électropolymérisés ont été étudiés en tant qu'approche alternative pour la détection électrochimique de nitro-explosifs. / This thesis proposes a rational design approach towards molecularly imprinted polymers (MIPs) for sensing nitro-explosives. Molecularly imprinted polymers are mimicking biological molecular recognition. They have the advantage to be stable in harsh environments and can be tailored into different physical forms for interfacing with transducers. Their synthesis is based on the co-polymerization of functional and cross-linking monomers in the presence of the target analyte or, as in this thesis, with a structural analogue leading to a rigid three-dimensional polymer network with binding sites complementary to the template in size, shape and position of the functional groups. The choice of the functional monomer was carried out with a rational design approach combining molecular modelling, nuclear magnetic resonance (NMR) and isothermal calorimetry (ITC) studies. This allows to optimize the pre-polymerization mixture in order to get strong complexation between the functional monomer and the template. The obtained results were confronted with binding studies performed on synthesized polymers. The thus designed polymer formulation was interfaced with transducer surfaces in form of nanoparticles, films and nanoparticles embedded into electro-polymerized polydopamine films. In addition to the traditional MIPs by free radical polymerization, molecularly imprinted in-situ electro-polymerized polydopamine films were investigated as an alternative approach for sensing nitro-explosives electrochemically.

La présence croissante de diverses substances dans notre environnement, conséquencedes activités anthropiques de ces dernières décennies, a entraîné un besoingrandissant et urgent de nouveaux matériaux et dispositifs dans la quête de senseurschimiques efficaces et fiables. D'énormes progrès technologiques ont permis de mettreà disposition toute une gamme d'outils techniques pour leur développement, enprenant en compte les exigences à respecter en terme de sélectivité ou de rapidité deréponse, entre autres. Dans ce contexte, les méthodes de chimie quantique permettentune compréhension fondamentale des processus en jeu dans la détection des espèceschimiques, et par extension, l'élaboration de manière rationnelle de nouveauxmatériaux sensibles. Certaines molécules organiques pouvant être largementfonctionnalisées, elles constituent un point de départ idéal en raison des importantesmodulations possibles de leurs propriétés par des modifications structuralesappropriées.Cette étude vise à développer de manière rationnelle des chromoionophores pour lacomplexation de cations par une approche combinant méthodes de chimiecomputationnelles et caractérisation par spectroscopie optique. Deux pointsprincipaux ont été traités à l'aide de la Théorie de la Fonctionnelle de la Densité(DFT) et son extension dépendante du temps (TD-DFT): d'une part les relationsstructure moléculaire-propriétés optiques de chromophores, d'autre part le phénomènede complexation. En particulier, la détection de l'ion Zn2+, démontrée de manièrethéorique et expérimentale, est finalement réalisée après intégration du senseurmoléculaire dans un dispositif à fibre optique. / The increasing presence of various substances in our environment has brought abouta growing need for rapid emergence of new materials and devices in the quest forefficient and reliable chemical sensors. Massive technological progress have madeavailable an extensive range of technical tools to serve their development, accountingfor the requirements to be fulfilled (selectivity, quick response..). In this context,quantum chemistry methods provide a fundamental understanding of the processes atstake in the detection of chemical species and allow for rational design of sensingmaterials. Certain organic molecules can be extensively functionalised and thusconstitute an evident starting point owing to the tunability of their propertiesprovided by appropriate choice of structural modifications. The versatility of somechromophores associated to the selectivity offered by receptor units constitute theresearch playground for the development of ever better chemosensors.The present research aims at the rational development of chromoionophores for thecomplexation of cations, combining computational chemistry methods with basicspectroscopic characterisation. Using Density Functional Theory (DFT) and its timedependentextension (TD-DFT), two main aspects were treated, namely therelationship between molecular structure and optical properties of organicchromophores featuring valuable characteristics, and the complexation phenomenon.Photophysics of Zn2+ ion detection were more specifically studied, and recognitionwas demonstrated with both quantum-chemical calculations and experiments,accounting for the future integration of the chemical sensor in an optical fibre device.

This thesis presents the application of different protein engineering methods on enzymes and non-catalytic proteins that act upon xyloglucans. Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glucans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glucans such as cellulose. One important group of xyloglucan-active enzymes is encoded by the GH16 XTH gene family in plants, including xyloglucan endo-transglycosylases (XET) and xyloglucan endo-hydrolases (XEH). The molecular determinants behind the different catalytic routes of these homologous enzymes are still not fully understood. By combining structural data and molecular dynamics (MD) simulations, interesting facts were revealed about enzyme-substrate interaction. Furthermore, a pilot study was performed using structure-guided recombination to generate a restricted library of XET/XEH chimeras. Glycosynthases are hydrolytically inactive mutant glycoside hydrolases (GH) that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Different enzymes with xyloglucan hydrolase activity were engineered into glycosynthases, and characterised as tools for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns. Carbohydrate-binding modules (CBM) are non-catalytic protein domains that bind to polysaccharidic substrates. An important technical application involves their use as molecular probes to detect and localise specific carbohydrates in vivo. The three-dimensional structure of an evolved xyloglucan binding module (XGBM) was solved by X-ray diffraction. Affinity-guided directed evolution of this first generation XGBM resulted in highly specific probes that were used to localise non-fucosylated xyloglucans in plant tissue sections. / QC 20100902

This thesis details research performed in modeling, simulation, and rational design of porous SOFC cathodes via development, extension, and use of the key tools to aid in the fundamental understanding and engineering design of cathode materials. Phenomenological modeling of triple phase boundary (TPB) reactions and surface transport on La₁₋ₓSrₓMnO₃ (LSM) was conducted, providing insight into the role of the bulk versus surface oxygen reduction pathway and the role of sheet resistance in thin-film patterned electrode measurements. In response to observation of sheet resistance deactivation, a modeling study was conducted to design thin-film patterned electrodes with respect to sheet resistance. Additionally, this thesis outlines the application of phenomenological chemical kinetics to describe and explain the performance and stability enhancements resulting from surface modification of La₁₋ₓSrₓCo₁₋yFeyO₃₋delta (LSCF) with a conformal LSM coating. The analysis was performed in close coordination with electrochemical experiments and transmission electron microscopy. Finally, the thesis describes conformal modeling of porous cathode microstructures using chemical kinetics and transport models. A novel application of conservative point defect ensembles was developed to allow simulations with complicated chemical surface kinetics to be efficiently coupled with bulk transport within the porous structure. The finite element method was employed to simulate electrochemical response conformal to sintered porous ceramic structures using actual 3D microstructural reconstructions obtained using x-ray microtomography. Mesh refinement, linear, and nonlinear reaction rate kinetics were employed to study the bulk versus surface oxygen reduction pathways and the effect of near-TPB nanostructure.

Theory has been used to complement experiment in the development of both drugs and delivery systems. Theoretical methods are capable of identifying the molecular basis of drug formulation inadequacies and systematic theoretical studies may suggest fruitful avenues for material modification. This thesis highlights the utility of computer-based theoretical calculations for guiding the design of drug formulations and enhancing material-drug compatibility and stability. Specifically, the present work explores the applications of semi-empirical methods and atomistic molecular dynamics (MD) simulations to enhance the performance of nano-emulsions and polymer micelle formulations for the delivery of hydrophobic drugs. This work includes three separate studies preceded by an introductory summary of available theoretical techniques. The first study evaluates the accuracy and reliability of semi-empirical methods and MD simulations as means to select suitable excipients to formulate the anti-cancer drug docetaxel in an emulsion. Here, simulations accurately predict the rank order of drug solubility in various excipients, suggesting that simulation is useful for library enrichment. In the second study, a drug conjugation approach is used to further improve the stability and solubility of docetaxel in a triglyceride-based nano-emulsion. Here, optimal conjugates are identified with computer-based theoretical calculations and conjugates with formulation-compatible moieties are synthesized. As predicted, the conjugates exhibit enhanced solubility and loading efficiency in a nano-emulsion. The goal of the third study is to rationally design a stable unimolecular star copolymer that, as a unimer, does not disassemble upon the dilution that accompanies intravenous injection. Here, MD simulation is used to systematically investigate the solution properties of differently composed star copolymers. Overall, star copolymers with a hydrophobic PCL core ≤ 2 kDa and hydrophilic PEG blocks approaching 14.6 kDa per arm are predicted to form unimolecular micelles that remain unimeric at high concentrations. The studies presented in this thesis demonstrate that theoretical approaches are useful for fast pre-screening of drug formulation materials and for the development of delivery systems and drug derivatives.