Thesis (Ph.D.)--Texas Christian University, 2008. / Title from dissertation title page (viewed Feb. 26, 2009). Includes abstract. Includes bibliographical references.

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The main goal of this thesis was to study cancer immunotherapy based on combination of ligands of TLR and phagocytic receptor agonists. This study is focused on looking for proper and save TLR stimulation and specific anchoring of phagocytic ligands.

Cytotoxic T lymphocytes (CTL) are major effector cells in the adaptive immune response against intracellular pathogens and cancers, killing targets with high precision. Precision is achieved through the specificity of the clonally expressed T cell receptor (TCR). TCRs recognise a specific peptide chain loaded into a major-histocompatability complex, triggering signalling, inducing the CTL to attach and kill target cells. Key stages in this attack are the initial conjugation followed by polarisation and docking of the centrosome to the junction of the two cells, the immune synapse (IS). This focuses secretion of the cytolytic components, perforin and granzyme, from modified lysosomes to kill the target cell. My PhD has utilised amino acid substitutions in the target peptide to alter its signal strength and shown this alters the subsequent killing efficiency of a target population. I developed new imaging and analysis techniques to investigate the effect of TCR signal strength at each step of the killing process. I show the first step, conjugation, is reduced for a percentage of cells with dwell times decreasing as TCR signal strength decreased. The next key step of centrosome polarisation and docking at the IS was also impaired for an increasing proportion of cells as TCR signalling reduced. Impaired centrosome docking reduced efficient granule recruitment to the IS, necessary for target killing. Centrosome docking was linked with the TCR-induced intracellular calcium flux, the duration of which increases with the strength of TCR signalling. This demonstrates how the process of CTL killing can be fine-tuned by the quality of antigen.

Complexos de coordenação de Cu(II), Ni(II), Zn(II) e Nb(V) foram obtidos com os íons acetato, citrato e oxalato como ligantes (complexos ex-situ) para serem aplicados em banhos de eletrodeposição. O objetivo é usar estes complexos como fontes de metais sem adição de aditivos os quais são os ligantes que estão coordenados. Como estudos de referência, foram investigados banhos de eletrodeposição contendo sais de sulfatos dos íons metálicos na presença de ligantes nas razões metal:ligante de 10:1 e 1:5; assim, complexos de coordenações similares podem ser gerados em solução (complexos in-situ). Os complexos ex-situ foram analisados por análise elementar, espectroscopias nas regiões do infravermelho e ultravioleta-visível e voltametria cíclica (em Pt). As eletrólises foram realizadas em substrato de aço 1020 com um ou dois complexos metálicos presentes no banho. Banhos de eletrodeposição de nióbio usando como substrato aço 1020 coberto com Cu e/ou Zn foram bem sucedidos. Os depósitos foram analisados por microscopia eletrônica de varredura (MEV), espectroscopia de dispersão de raios X (EDX), reflectância difusa, espectroscopia de difração de raios X (DRX) e por curvas de polarização anódica (estudos de corrosão). Os banhos com complexos in-situ 10:1 fomeceram depósitos com aspectos morfológicos ruins em relação aos depósitos obtidos de banhos com complexos in-situ 1:5 e ex-situ. As morfologias dos depósitos obtidos dos banhos com acetato foram dendríticos e com baixa aderência. Os depósitos dos banhos com complexos citrato e oxalato foram lisos, compactos, com boa aderência e brilhantes. Diferentes fases estruturais foram observadas nos depósitos e a maioria deles protege contra a corrosão. Os estudos concluíram que os ligantes analisados influenciam diferentemente nos processos de eletrodeposição. / Cu(II), Ni(II), Zn(II) and Nb(V) coordination complexes were obtained with acetate, citrate and oxalate ions as ligands (ex-situ complexes) to be applied in electrodeposition baths. The aim is to use these complexes as metal sources without further addition of additives which are the coordinated ligands. As reference study, electroplating baths with sulfate salts of the metal ions in presence of the ligands were investigated in the metal:ligand ratio of 1:5 and 10:1; then, similar coordination complexes can be generate in solution (in-situ complexes). The ex-situ complexes were characterized by elemental analysis, infrared and UV-vis spectroscopies and cyclic voltammetry (in Pt). The electrolyses were performed in 1020 steel as substrate with one or two metal complexes present in the bath. Niobium electroplating baths with 1020 steel previously covered with Cu and/or Zn as substrates were also successfully performed. The deposits were analyzed by scanning electron microscopy (SEM), energy dispersive spectroscopy X-ray (EDS), diffuse reflectance, X-ray diffraction (XRD) and polarization curves (corrosion studies). The bath with 10:1 in-situ complexes provided deposits with poor morphologic aspects in relation to the deposits obtained from bath with 1:5 in-situ and ex-situ complexes. The morphologies of the deposits obtained from bath with the acetate complexes were dendritics and with low adherence. The bath with both citrate- and oxalate-complexes were smoothes, compacts, with good adherence and brightness. Different structural phases were observed in the deposits and the most of them provided protection against corrosion. It was concluded that coordinated ligands influence differently in the electrodeposition processes.

Descritores moleculares são essenciais em muitas aplicações de física e química computacional, como na análise de similaridade entre ligantes baseada em sua estrutura. Harmônicos esféricos têm sido utilizados como descritores da superfície molecular por serem uma forma compacta de descrição geométrica e por possuírem um descritor invariante por rotação. Assim, este trabalho propõe um método de análise de similaridade estrutural entre ligantes no qual se modela a superfície de uma molécula através de uma expansão em harmônicos esféricos realizada pelo programa LIRA. Os coeficientes encontrados são utilizados para percorrer o banco de dados DUD-E, com descritores previamente calculados, utilizando Distância Euclidiana e diversos valores de corte para selecionar compostos mais semelhantes. O potencial do método é avaliado usando o Ultrafast Shape Recognition (USR) como método padrão, pelo fato de ser uma excelente e rápida métrica para análise da similaridade de ligantes. Foram selecionadas 50 moléculas de diferentes tamanhos e composição de forma a representar todos os grupos moleculares presentes na DUD-E. Em seguida, cada molécula foi submetida à busca de similares variando-se valores de corte para o LIRA em que o conjunto de moléculas selecionadas foi comparado com as selecionadas pelo USR através de um processo de classificação binária e criação e interpretação de curvas ROC. Além do benchmarking, foi realizada a análise das componentes principais para determinar quais descritores são os mais importantes e carregam as melhores informações utilizadas na descrição da superfície da molécula. A partir das componentes principais, foi realizado um estudo do uso de funções peso, associando mais importância aos descritores adequados, e a redução da dimensionalidade do banco de dados, seleção de um novo conjunto de autovetores que formam as bases do espaço vetorial e uma nova descrição das moléculas para o novo espaço, no qual cada variação foi avaliada através de um novo benchmarking. O LIRA se mostrou tão rápido quanto o USR e apresentou grande potencial de seleção de moléculas similares, para a maioria das moléculas testadas, pois as curvas ROC apresentaram pontos acima da linha do aleatório. Tanto a redução da dimensionalidade quanto o uso de funções de ponderação agregaram valor à métrica deixando-a mais veloz, no caso da redução da quantidade de descritores, e seletiva, em ambos os casos. Dessa forma, o método proposto se mostrou eficiente em mensurar a similaridade entre ligantes de forma seletiva e rápida utilizando somente informações a respeito da superfície molecular. / Molecular descriptors are essential for many applications in computational chemistry and physics, such as ligand-based similarity searching. Spherical harmonics have previously been suggested as comprehensive descriptors of molecular structure due to their properties, orthonormality and rotationally invariant. Here we proposed a ligand similarity analysis method where molecule\'s surface is modeled by an expansion in Spherical Harmonics, called LIRA, whose coefficient are used to perform a search in DUD-E database, with all descriptors previously calculated, measured by Euclidian Distance and different cutoff\'s values to select similar compounds. Method\'s potential is evaluated against Ultrafast Shape Recognition (USR), due to it is an excellent a fast metric to ligand similarity analysis, in a benchmarking. Fifty molecules are selected varying chemical composition and size to represent all molecular groups of DUD-E. After that, which one was submitted in a search with different values of cutoff for LIRA and the subset selected was compared with the ones selected by USR through binary classification and ROC curves analysis. Beyond benchmarking, it was performed a principal component analysis to identify which are the most valuable coefficient for shape description. Using principal components two other studies are made, weight functions are applied to descriptors, providing more value for those carry more information, and dimensionality reduction, where a subset of eigenvectors are select to form the new basis of the vector space and the new molecule\'s description was made in the new space, which variation was tested in a new benchmarking. Lira showed to be as fast as USR and a big potential to select similar molecules, for the majority of the molecules tested, because ROC curves had points over the random line. Dimensionality reduction and weight functions improved LIRA results raising velocity, due to the use of less descriptors to model molecule\'s surface, and the selection power, for both cases. In summary, the proposed method showed to be an efficient and fast tool for measure similarity between ligands based in molecular shape.

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No. of bitstreams: 2 Dissertação - Dayane Kelly Sabec Pereira - 2015.pdf: 7161660 bytes, checksum: 9e90c47a6af39120173e8142ccb64d21 (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) Previous issue date: 2015-09-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The increased bacterial resistance is an important factor in the number of deaths in hospitals, hampering the cure and elevating spending on assistance. Thus it is urgent to develop new drugs, synthetic or natural origin. A group of compounds that have been prominent are the thiosemicarbazones (TSCs) and semicarbazones (SCs) due to low cost of synthesis and broad spectrum of action, antifungal, antibacterial, anti-inflammatory and antiviral. In this study tests were performed with four synthesized chemical binders, denominated: H2L0; H2L1; H2L2 and H2L3 and H2L3 the four compounds complexed ligand named: Bipy-fenil-Sn, Bipy-nit-Cu, (H2L3)butil-Sn and (H2L3)bipy-Sn submitted to quantitative tests to determine the Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) and also evaluated for cytotoxicity cardiomyocytes and peritoneal macrophages. The standard strains ATCC (American Type Culture Collection) used were bacteria Staphylococcus aureus and Klebsiella pneumoniae, yeast and fungi: Candida albicans, Candida tropicalis and Candida parapsilosis. The tests were performed in triplicate in 96 well plates, incubated for 24 hours at 35 ° C, with specific medium. The observed antibacterial activity of the compounds H2L1, H2L2 and H2L3 on S. aureus, presented MIC in range from 3.7 to 19.7 μg/mL; to K. pneumoniae the MICs of the compounds were over 500 μg/mL. The compound H2L3, inhibited the growth of three species of Candida tested MIC between 5.6 - 6.2 μg/mL. Because of this, the binder was chosen for complexation with metals. The complexed compounds obtained MICs 1.6 to 245.8 μg/mL. MFC tests with the compounds H2L0, H2L1 and H2L2 showed no change in the growth curve of the colonies to the three species of Candida. As for the compounds complexed MFC tests indicated a fungicidal effect of bipy-nit-Cu, bipy-fenil-Sn, only on C. albicans at concentrations of 250 μl/mL and less than 62.5 μl/mL. Cytotoxicity tests revealed that the compounds exhibit cytotoxicity under moderate conditions. In the compounds complexed with metals, antifungal activity showed up similar to H2L3 isolated compound, obtaining a satisfactory result for further research with compounds under study, presenting a great antimicrobial potential which is no prospect of a future metalodrug. / O aumento da resistência bacteriana é um fator importante no número de ocorrência de mortes em ambientes hospitalares, dificultando a cura e elevando os gastos com a assistência. Desta forma faz-se urgente o desenvolvimento de novos fármacos, sintéticos ou de origem natural. Um grupo de compostos que tem se destacado são as tiossemicarbazonas (TSCs) e as semicarbazonas (SCs), por apresentarem baixo custo de síntese e amplo espectro de ação, antifúngica, antibacteriana, antiinflamatória e antiviral. Neste trabalho foram utilizados quatro ligantes químicos sintetizados, denominados: H2L0; H2L1; H2L2 e H2L3 e quatro compostos complexados ao ligante H2L3 denominados: Bipy-fenil-Sn, Bipy-nit-Cu, (H2L3)butil-Sn e (H2L3)bipy-Sn submetidos a testes quantitativos para determinação da Concentração Inibitória Mínima (MIC) e da Concentração Fungicida Mínima (MFC) e também avaliada a citotoxidade em cardiomiócitos e macrófagos peritoneais. As cepas de padrão ATCC (American Type Culture Collection) utilizadas foram de bactérias Staphylococcus aureus e Klebsiella pneumoniae, e fungos leveduriformes: Candida albicans, Candida tropicalis e Candida parapsilosis. Os testes foram realizados em triplicata, em placas 96 poços, incubadas por 24 horas a 35ºC, com meio específico. A atividade antibactericida observada dos compostos H2L1, H2L2 e H2L3, sobre S. aureus, apresentou MIC na faixa de 3,7 a 19,7 μg/mL; para K. pneumoniae os MICs dos compostos foram superiores a 500 μg/mL. O composto H2L3, inibiu o crescimento das três espécies de Candida testadas com MIC entre 5,6 – 6,2 μg/ml. Devido a isso, este foi o ligante escolhido para a complexação com metais. Os compostos complexados obtiveram valores de MIC 1,6 a 245,8 μg/mL Os testes de MFC com os compostos H2L0, H2L1 e H2L2 não apresentaram alteração na curva de crescimento para as três espécies de Candida. Quanto aos compostos complexados, os testes de MFC apontaram um efeito fungicida do bipy-nit-Cu, bipy-fenil-Sn, somente sobre C. albicans, nas concentrações de 250 μl/mL e menores que 62,5 μl/mL. Os testes de citotoxidade revelaram que os compostos apresentam citotoxidade em condições moderadas. Nos compostos complexados com metais, a atividade antifúngica apresentou-se semelhante ao composto H2L3 isolado, obtendo um resultado satisfatório para novas pesquisas com os compostos em estudo, apresentando um ótimo potencial antimicrobiano ao qual há perspectiva de uma futura metalodroga.

The use of bidentate ligands in organometallic chemistry is best illustrated by the successful application in homogeneous catalysis1 and bio-medical2 systems. For catalytic systems the use of bidentate phosphines have revolutionised the way in which homogeneous catalysis is viewed, with the use of functionalised organic ligands to induce favourable catalytic product formation such as the production of L-Dopa3. To this end, the investigation of tropolone type ligands and complexes will be discussed further in this chapter. The possible application of tropolone systems will be investigated in the context of possible catalytic and bio-medical applications. In 1945 Dewar4 predicted that 2-hydroxy-2,4,6-cycloheptatrien-one (Tropolone, Eq. 1.1.I), would show certain aromatic properties; he specifically referred to the natural products known at that time, stipitatic acid and colchicine5 as representative of this class of compounds. Thus Dewar was the first person to point to a new field in organic chemistry, which to date has not been fully explored due to the diversity of uses of tropolone and its derivatives. / Prof. A. Roodt

M.Sc. / This study focused on the synthesis and evaluation of phosphine ligands with multiple functional ities. Polar ligands suitable for use in homogeneous catalysis in aqueous/organic systems were synthesised, as were ligands incorporating a boron atom in an intramolecular Lewis acid-Lewis base interaction with the phosphorus atom. A malonate moiety was readily incorporated into a phosphorus starting material, and derivatives were obtained by reduction of the ester groups. The polar diol products were reacted with 1,4-butanesultone which made them water soluble and thus ideal for biphasic catalysis. Wittig chemistry was employed to introduce alkenes of varying electronic nature into a phosphorus-containing aldehyde starting material. The catalysed hydroboration reaction making use of diboron reagents was used to introduce a boron functionality into the resulting a, n-unsaturated ester phosphine I igands. All of the ligands produced were tested in transition metal-catalysed reactions, namely the Heck reaction, the Suzuki reaction, the Stille reaction, the carboxymethylation reaction and the hydroformylation reaction. The polar and water soluble ligands all showed comparable or improved yields to the standard benchmark triphenylphosphine ligand in organic, biphasic and ionic liquid media. The electronic nature of the alkene ligands largely dictated the activities observed in the Heck, Suzuki and Stille reactions. The electron rich ligands showed improved activities in the Heck reaction, while the electron poor ligands showed improved activities in the Suzuki reaction. In contrast, the Stille reaction seemed to be more affected by the steric demands of the ligands rather than by electronic considerations. It was also found that the boron containing ligands showed an enhanced activity in comparison to the boron free unsaturated and saturated ester ligands. This enhancement was directly ascribable to the Lewis acidic boron atom. This study allowed the synthesis of a range of functionally varying phosphine ligands which where shown to influence transition metal-catalysed reactions based specifically on the functionality present.

The duel objective of the research described in this dissertation, was the optimisation of reaction conditions for various palladium(0)-catalysed Heck-type reactions, and the development and application of bidentate iminophosphine ligands for some of these reactions. The efficiency of Heck and Heck-type reactions was evaluated by comparison of various parameters, e.g. solvent, palladium salt, reaction temperature and ligands for these palladium salts. In most cases, DMF was used as solvent, and palladium acetate served as a source of catalyst. Reactions were mostly carried out at 110 ºC, and triphenylposphine was used as the bench mark monodentate ligand, while 1,3-bis(diphenylphos-phino)propane (dppp) was applied as a bench mark for the performance of bidentate ligands. Apart from the Heck reaction between aryl halides and olefins, palladium-catalysed esterification reactions, transmetallation reactions and intramolecular reactions were investigated. A series of iminophosphine ligands was synthesised via the condensation of 2-(diphenylphosphino)benzaldehyde with primary amines. Although these ligands are thermally stable, they are extremely susceptible towards oxidation. This problem of oxidation, together with their unstability on silica, led us to the development of specialised techniques to isolate and purify the ligands. Distillation of these ligands under vacuum, together with the simultaneous subjection of the distillation apparatus to an inert argon atmosphere, resulted in the isolation of pure, unoxidised iminophosphine ligands. Application of the synthesised iminophosphine ligands to the optimised Heck reaction, indicated that these ligands were catalytically active. A series of Heck reactions employing different aryl halides, was chosen to test the efficiency of the newly synthesised ligands. In the cases of iodobenzene and o-nitroiodobenzene, similar results were obtained for triphenylphosphine, the iminophosphine ligands, and dppp. However, in the case of p-bromobenzonitrile, where the oxidative addition of palladium to the C-Br bond is slower than for corresponding C-I bonds, the efficiency of the iminophosphine ligands could clearly be seen in the higher yields that were obtained in comparison to the bench mark ligands. Further development of bidentate P-N ligands with different electronic characteristics, and application of these ligands with palladium and other transition metals for catalysis, is a field of research that should not be left unexpored. / Prof. D.B.G. Williams