Thesis (Ph. D.)--Florida State University, 2004. / Advisors: Dr. Naresh Dalal, Dr. Johan van Tol, Florida State University, Arts and Sciences, Dept. of Chemistry and Biochemistry. Title and description from dissertation home page (viewed Jan. 18, 2005). Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 160-166). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 160-166). Also available on the Internet.

Undergraduate honors paper--Mount Holyoke College, 2005. Dept. of Chemistry. / Includes bibliographical references (leaves 107-111).

Honors Project--Smith College, Northampton, Mass., 2010. / Includes bibliographical references (p. 102-105).

Orientador: Eduardo Maffud Cillli / Banca: Ana Marisa Fusco Almeida / Banca: Vani Xavier de Oliveira Júnior / Resumo: Neste trabalho foi avaliado o efeito da dimerização e do comprimento/polaridade do espaçador utilizado na formação dos dímeros, na estrutura e atividade biológica do peptídeo antimicrobiano: Gly-Trp-Leu-Asp-Val-Ala-Lys-Lys-Ile-Gly-Lys-Ala-Ala-Phe-Asn-Val-Ala-Lys-Asn-Phe-Leu-CONH2. Nesse sentido, o monômero e três dímeros com diferentes espaçadores foram sintetizados pelo método de síntese de peptídeo em fase sólida (SPFS) utilizando uma combinação das químicas Fmoc e Boc. Análises por cromatografia de fase reversa e espectrometria de massas confirmaram o sucesso das sínteses e das purificações. Os ensaios de atividade antimicrobiana, em termos de CIM mostraram que a dimerização não aumentou a capacidade do peptídeo de inibir o crescimento de bactérias e fungos. No entanto, quando analisada a capacidade dos peptídeos de matar E. coli, um menor tempo para produzir o efeito inibitório foi observado para os dímeros. Adicionalmente, a atividade hemolítica dos peptídeos também foi avaliada, encontrando-se um aumento significativo, aproximadamente 40 vezes, para os dímeros em relação ao monômero. Com o objetivo de tentar explicar esses efeitos, o teste de proteção osmótica foi realizado. No entanto, o tamanho dos poros foi semelhante, o que não permite explicar as diferenças em termos desta variável. Conhecendo seus diâmetros, foi possível determinar que o poro é formado por 6 moléculas monoméricas ou 3 diméricas. Estudos de permeabilização mostraram que a porcentagem e a velocidade de liberação de carboxifluoresceína foram maiores para os dímeros quando comparados com o monômero, especialmente em vesículas contendo esfingomielina. Este ensaio também mostrou que a duplicação da concentração de monômeros não é suficiente para atingir a capacidade de permeabilização dos dímeros, confirmando que a proximidade das cadeias é um fator... (Resumo completo clicar acesso eletrônico abaixo) / Abstract: This work analyzed the effect of dimerization and the length/polarity of the spacer used in the formation of dimers, in the structure and biological activity of the antimicrobial peptide: Gly-Trp-Leu-Asp-Val-Ala-Lys-Lys-Ile-Gly-Lys-Ala-Ala-Phe-Asn-Val-Ala-Lys-Asn-Phe-Leu-CONH2. In this way, the monomer and three dimers (Lys-branched) with different spacer groups were synthesized by solid phase peptide synthesis methodology using a combination of Fmoc and Boc chemical approaches. Analysis by reverse phase chromatography and mass spectrometry confirmed the success of the synthesis and purifications. The antimicrobial activity assay, in terms of MICs showed that dimerization did not increase the ability of the peptides to inhibit the growth of bacteria and fungi. Nevertheless, when analyzing the peptides activity against E. coli in terms of kinetics, an increased velocity was observed for the dimers. The hemolytic activity of peptides was also evaluated, finding a very significant difference, approximately 40 times greater for dimers. Aiming to explain this difference, the osmotic protection test was performed, but the pore size was similar, which can not explain the differences in terms of this variable. Knowing the diameter of the pores, it was possible to determine that the pore is formed by six monomeric or three dimeric molecules. Additionally, permeabilization studies showed that the percentage and rate of carboxyfluorescein release were larger for the dimers compared with monomer, especially in vesicles containing sphingomyelin. This test also showed that the use of two times more monomer concentration's is not sufficient to reach dimers permeabilization capacity, confirming that the proximity of the chains is an important factor in the activity of this peptide. Analysis by circular dichroism revealed that peptides in aqueous solution are in random coil, whereas... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-06-11T19:23:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-24Bitstream added on 2014-06-13T19:49:48Z : No. of bitstreams: 1 nicolaslorenzon_e_me_araiq.pdf: 1141028 bytes, checksum: 6886093f73fd039fe1ed73b26a3d43dd (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Neste trabalho foi avaliado o efeito da dimerização e do comprimento/polaridade do espaçador utilizado na formação dos dímeros, na estrutura e atividade biológica do peptídeo antimicrobiano: Gly-Trp-Leu-Asp-Val-Ala-Lys-Lys-Ile-Gly-Lys-Ala-Ala-Phe-Asn-Val-Ala-Lys-Asn-Phe-Leu-CONH2. Nesse sentido, o monômero e três dímeros com diferentes espaçadores foram sintetizados pelo método de síntese de peptídeo em fase sólida (SPFS) utilizando uma combinação das químicas Fmoc e Boc. Análises por cromatografia de fase reversa e espectrometria de massas confirmaram o sucesso das sínteses e das purificações. Os ensaios de atividade antimicrobiana, em termos de CIM mostraram que a dimerização não aumentou a capacidade do peptídeo de inibir o crescimento de bactérias e fungos. No entanto, quando analisada a capacidade dos peptídeos de matar E. coli, um menor tempo para produzir o efeito inibitório foi observado para os dímeros. Adicionalmente, a atividade hemolítica dos peptídeos também foi avaliada, encontrando-se um aumento significativo, aproximadamente 40 vezes, para os dímeros em relação ao monômero. Com o objetivo de tentar explicar esses efeitos, o teste de proteção osmótica foi realizado. No entanto, o tamanho dos poros foi semelhante, o que não permite explicar as diferenças em termos desta variável. Conhecendo seus diâmetros, foi possível determinar que o poro é formado por 6 moléculas monoméricas ou 3 diméricas. Estudos de permeabilização mostraram que a porcentagem e a velocidade de liberação de carboxifluoresceína foram maiores para os dímeros quando comparados com o monômero, especialmente em vesículas contendo esfingomielina. Este ensaio também mostrou que a duplicação da concentração de monômeros não é suficiente para atingir a capacidade de permeabilização dos dímeros, confirmando que a proximidade das cadeias é um fator... / This work analyzed the effect of dimerization and the length/polarity of the spacer used in the formation of dimers, in the structure and biological activity of the antimicrobial peptide: Gly-Trp-Leu-Asp-Val-Ala-Lys-Lys-Ile-Gly-Lys-Ala-Ala-Phe-Asn-Val-Ala-Lys-Asn-Phe-Leu-CONH2. In this way, the monomer and three dimers (Lys-branched) with different spacer groups were synthesized by solid phase peptide synthesis methodology using a combination of Fmoc and Boc chemical approaches. Analysis by reverse phase chromatography and mass spectrometry confirmed the success of the synthesis and purifications. The antimicrobial activity assay, in terms of MICs showed that dimerization did not increase the ability of the peptides to inhibit the growth of bacteria and fungi. Nevertheless, when analyzing the peptides activity against E. coli in terms of kinetics, an increased velocity was observed for the dimers. The hemolytic activity of peptides was also evaluated, finding a very significant difference, approximately 40 times greater for dimers. Aiming to explain this difference, the osmotic protection test was performed, but the pore size was similar, which can not explain the differences in terms of this variable. Knowing the diameter of the pores, it was possible to determine that the pore is formed by six monomeric or three dimeric molecules. Additionally, permeabilization studies showed that the percentage and rate of carboxyfluorescein release were larger for the dimers compared with monomer, especially in vesicles containing sphingomyelin. This test also showed that the use of two times more monomer concentration´s is not sufficient to reach dimers permeabilization capacity, confirming that the proximity of the chains is an important factor in the activity of this peptide. Analysis by circular dichroism revealed that peptides in aqueous solution are in random coil, whereas... (Complete abstract click electronic access below)

This dissertation describes my efforts in the Nuckolls lab to expand synthetic methods of wet-chemistry superatom synthesis, superatom surface ligand and core modification, and assembly of superatoms into materials with useful, cumulative properties. This work builds off of previous work from the Nuckolls lab describing photolabile ligand substitution and use of this technique to covalently bind superatoms to form various materials such as polymers and weaved sheets. This work will focus on the Chevrel-type M₆E₈L₆ metal-chalcogenide cluster Co₆Se₈, modification of its outer stabilizing ligands, and fusion of its core with other Co₆Se₈ superatoms to form fused dimers. Chapter 1 consists of a review of background material that forms a foundational basis for this work. The field of superatoms and superatomic materials will first be covered to contextualize this work in the field at large. Then, the prior work on wet-chemistry synthesis of Co₆Se₈ superatoms with replaceable, photolabile carbonyl (CO) ligands will be discussed. Finally, previous dimensionally-controlled assembly of materials using these carbonylated superatoms will be covered. Chapter 2 consists of the discovery of a masking carbene ligand generated from trimethylsilyl diazomethane (TMSD) and its use to create a new, electronically-coupled superatom dimer species (Co₁₂Se1₆(PEt₃)₁₀) that shows evidence of quantum confinement akin to nanoparticles and nanoparticle assemblies. Chapter 3 consists of new ligand substitution and methods to synthetically functionalize the fused dimer introduced in Chapter 2. The reactive carbene-ligated cluster is used to add new functional groups that were previously inaccessible to these cobalt-selenide clusters. New multi-carbene clusters are demonstrated as well as the use of site-differentiated clusters to form functionalized fused dimers from bis-carbonyl clusters. Chapter 4 consists of an investigation of the carbene cluster and insights that may be used in the future to finally expand cluster fusion into a chain. A reversible bridging of the carbene ligand based on temperature and oxidation state is analyzed experimentally and computationally. This information is used to synthesize a series of new carbene clusters which are used to try and assemble electronically-coupled, fused Co₆Se₈ superatomic materials.

The receptor dimers that mediate bacterial chemotaxis form high-order signaling complexes with CheW and the kinase CheA. From the packing arrangement in two crystal structures of different receptor cytoplasmic fragments, two different models have been proposed for receptor signaling arrays: the trimers-of-dimers and hedgerow models. We identified an interdimer distance that differs substantially in the two models, labeled the atoms defining this distance through isotopic enrichment, and measured it with 13C-19F REDOR. This was done in two types of receptor samples: first, isolated bacterial membranes containing overexpressed, intact receptor, and second, soluble receptor fragments reconstituted into kinase-active signaling complexes. In both cases, the distance found was not compatible with the receptor dimer−dimer contacts observed in the trimers-of-dimers or in the hedgerow models. Comparisons of simulated and observed REDOR dephasing were used to deduce a closest-approach distance at this interface, which provides a constraint for the possible arrangements of kinase-competent receptor assemblies. An alternate model of receptor signaling is proposed, which reconciles this result with existing structural and biochemical data. Additionally, two advances to solid-state NMR methodology are described. The first is a set of strategies to protect protein samples against degradation by solid-state NMR analysis. Biochemical and spectroscopic techniques are prescribed to identify and isolate specific challenges to protein stability, allowing them to be addressed individually. For this purpose a new pulse sequence (Thermal Calibration Under Pulseload, or TCUP) is employed, which allows sample temperature to be measured with exceptional time resolution. The second NMR advance describes the creation and characterization of a 13C-19F REDOR distance-calibration standard. The inclusion compound of 4-fluorotoluene and tert-butylcalix[4]arene was used for this purpose. The compound is easily synthesized from commercially available materials, and provides a long, isolated 13C-19F distance of 4.1 Å. Dynamics within the compound allow direct observation of 19F resonances without 1H-decoupling, and provide exceptionally sharp 13C resonances; these characteristics speed the routine setup of REDOR experiments. Both methodological developments were important to performing accurate distance measurements on functionally relevant chemotaxis signaling complexes; they also pave the way for making similar measurements in other proteins of interest.

No description available.