Thesis (Ph. D. in Chemistry)--Vanderbilt University, Dec. 2005. / Title from title screen. Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:27:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-13Bitstream added on 2014-06-13T20:56:17Z : No. of bitstreams: 1 goncalesgarcia_l_me_sjrp.pdf: 353281 bytes, checksum: 6786716aa7970dd6c603e25cad2d5709 (MD5) / As atividades realizadas compreenderam a produção de cDNA utilizando primer anti-senso e RNA purificado, amplificação do gene codificador da proteína 25K do Cole latent virus (CoLV25K), purificação do fragmento amplificado, ligação em vetor de multiplicação pGEM-T, transformação em células competentes de Escherichia coli linhagem TOP 10, purificação do vetor, digestão enzimática com as enzimas Bam HI e Hind III, subclonagem no vetor de expressão pET28a, transformação em células competentes de E. coli linhagem BL21-RIL, sequenciamento do gene no vetor de expressão e expressão da proteína a 37 o C . Quando expressa a 37 ºC, a proteína, de 25 kDa, foi encontrada em sua totalidade nos corpos de inclusão. Dessa forma, a proteína foi purificada sob condição desnaturante (utilizando 8 M de uréia) e submetida à diálise para seu reenovelamento. Após o reenovelamento, a proteína foi concentrada para aproximadamente 3 mg/mL e foram realizadas medidas de dicroísmo circular, para verificar o seu conteúdo de estrutura secundária, e o espalhamento dinâmico de luz, para estimar a distribuição de tamanho das populações de partículas que estão presentes na solução. Os dados da deconvolução do experimento de dicroísmo circular indicam um percentual de 40-46% α-hélice, 12-14% folha-β, 15-22% voltas e 24-28% de outras estruturas, indicando que a proteína está estruturada; e os dados do espalhamento dinâmico de luz mostraram que a proteína encontra-se estável, monodispersa, mas apresenta um complexo de partículas que deve ser removido para que fique nas condições ideais de cristalização. Foram utilizadas também outras técnicas para tentar alcançar a solubilidade da proteína expressa: abaixando a temperatura... / The experiments performed were, the production of cDNA using anti-sense primers and purified RNA, amplification of the gene encoding the 25K protein of Cole latent virus (CoLV25K), purification of the amplified fragment, cloning in multiplication vector pGEM-T for cell transformation into competent Escherichia coli strain TOP 10, vector purification, enzymatic digestion using the enzymes Bam HI and Hind III, subcloning in expression vector pET28a, transformation into competent cells of E. coli strain BL21-RIL, sequencing of the gene cloned into the expression vector and protein expression at 37 o C. When expressed at 37 °C, the protein with a molecular mass of 25 kDa was detected in inclusion bodies. Thus, the protein was purified under denaturing conditions (using 8 M urea) and subjected to dialysis to stimulate refolding. After refolding, the protein was concentrated to approximately 3 mg / mL and the circular dichroism assay was performed to verify the content of secondary structure, and dynamic light scattering, to estimate the size distribution of particle populations which are present in solution. The data from the deconvolution of circular dichroism experiments indicate a percentage of 40-46% α-helix, 12-14% β-sheet, 15-22% turns and 24-28% of other structures, indicating a structured protein; and the data of the dynamic light scattering showed that the protein is stable, monodispersive, but forms a large complex of particles which must be separated to before crystallization experiments. Other techniques were used to solubilize the expressed protein: lowering the temperature of expression to 18 °C, using the method... (Complete abstract click electronic access below)

Orientador: Eduardo Maffud Cilli / Banca: José Roberto Ernandes / Banca: Patricia Targon Campana / Banca: Antonio José da Costa Filho / Banca: Vani Xavier de Oliveira Junior / Resumo: A diidroorotato desidrogenase é uma enzima que apresenta um papel central na biossíntese de pirimidinas e catalisa a oxidação do diidroorotato a orotato. A enzima atua durante a via "de novo" de síntese de pirimidinas e está presente em quase todos os organismos vivos. A diidroorotato desidrogenase humana (HsDHODH) pode representar um importante alvo para o tratamento de doenças hiperproliferativas e inflamatórias, já que sua inibição bloqueia a síntese de ácidos nucléicos, impedindo a sua proliferação. Esta enzima tem uma estrutura monomérica e está associada com a membrana interna das mitocôndrias pela sua extensão N-terminal. Assim, entender em detalhes como esta enzima interage com a membrana poderia elucidar um alvo seletivo para drogas antiproliferativas, antiparasíticas e imunossupressivas. Esta região está também envolvida com a catálise central da enzima, sequestrando moléculas de ubiquinona presentes na membrana, fundamentais para as reações de oxirredução feitas pela enzima. Deste modo, para um melhor entendimento destes aspectos, neste trabalho foram sintetizados, por meio da Síntese de Peptídeos em Fase Sólida (SPFS) o peptídeo Ac-GDERFYAEHLMPTLQGLLDPESAHRLAVRFTSLG-NH2, que corresponde ao microdomínio existente na porção N-terminal da HsDHODH, entre os resíduos 33 e 66. Três análogos marcados com o aminoácido paramagnético TOAC nas posições 0, 12 ou 20, além de dois análogos duplamente marcados também foram obtidos. Ambos os peptídeos com dupla marcação possuem uma cisteína ligada ao spin MTSSL na posição 35 (C-terminal) se diferenciando pela posição do segundo marcador: um contendo outra cisteína ligada ao MTSSL na posição 12 e o segundo possuindo o TOAC na posição 0 (ou N-terminal). Estes peptídeos foram estudados por técnicas... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The dihydroorotate dehydrogenase is an enzyme that has a central role on the pyrimidine biosynthesis and catalyses the oxidation of dihydrorotate to orotate. The enzyme acts on "de novo" pyrimidines nucleotides pathway and it is present in almost all the live organisms. The human dihydroorotate dehydrogenase (HsDHODH) can represent an important target for the treatment of hiperproliferative and inflammatory diseases, since its inhibition blocks the nucleic acid synthesis, which restrains the cell proliferation. This enzyme has a monomeric structure and it is associated into the inner mitochondrial membrane by the N-terminal extension. Thus, understanding in details how this enzyme interacts with the membrane could help to elucidate a selective target for antiproliferative, antineoplasic and immunosuppressive drugs. This region is also involved with the central enzyme catalysis, harboring quinones molecules that are in the membranes, which is essential for the oxidation-reduction reactions made by the HsDHODH. In this way, for a better evaluation of these aspects, in this work we synthesized through the Solid Phase Peptide Synthesis (SPPS) the peptide Ac-GDERFYAEHLMPTLQGLLDPESAHRLAVRFTSLG-NH2, which corresponds to the HsDHODH N-terminal microdomain, between the residues 33 to 66. Three analogues labeled with the paramagnetic amino acid TOAC in the positions 0, 12 or 20 and two doubly labeled analogues were also synthesized. Both doubly labeled peptides contain a MTSSL-attached cysteine residue bounded to the position 35 (C-terminus), differing by the position of the second spin label: one possessing a cysteine with MTSSL at position 12 and the other contains TOAC at position 0 (N-terminus). These peptides were studied by spectroscopy techniques in order to obtain information about... (Complete abstract click electronic access below) / Doutor

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All biological processes are fundamentally inter-molecular interactions. In order to understand, and hence control, biomolecular structure and function, methods are required that probe biological systems at the molecular level, ideally with those molecules being in their native environment. The research summarized herein has at its core the development and application of ultra violet (UV)-visible spectrophotometric techniquies for this prupose, in particular circular dichrosim (CD) and linear dichrosim (LD) but also absorbance, fluorescence and resonance light scattering. The spectroscopy is complemented by fundamental theoretical work on molecular structure and reactivity that forms the basis for designing molecules to bind to biomolecules for a particular structural or functional effect. A brief summary of the contributions of the listed publications to our understanding of 'Molecular aspects of biololecule structure and function' is given below under five headings: Circular dichroism theory Molecular geometry and reactivity Small molecule-macromolecule interactions: spectroscopic probes of inter-molecular geometries Molecular design for nucleic acid structure and control Spectroscopic probes of biomolecule structure: instrumentation and application In general terms these correspond to successive phases of the research programme, however, all areas have been present since the first publications in 1983 and can be traced weaving through all subsequent activity.

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains xv, 190 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 153-180).

All biological processes are fundamentally inter-molecular interactions. In order to understand, and hence control, biomolecular structure and function, methods are required that probe biological systems at the molecular level, ideally with those molecules being in their native environment. The research summarized herein has at its core the development and application of ultra violet (UV)-visible spectrophotometric techniquies for this prupose, in particular circular dichrosim (CD) and linear dichrosim (LD) but also absorbance, fluorescence and resonance light scattering. The spectroscopy is complemented by fundamental theoretical work on molecular structure and reactivity that forms the basis for designing molecules to bind to biomolecules for a particular structural or functional effect. A brief summary of the contributions of the listed publications to our understanding of 'Molecular aspects of biololecule structure and function' is given below under five headings: Circular dichroism theory Molecular geometry and reactivity Small molecule-macromolecule interactions: spectroscopic probes of inter-molecular geometries Molecular design for nucleic acid structure and control Spectroscopic probes of biomolecule structure: instrumentation and application In general terms these correspond to successive phases of the research programme, however, all areas have been present since the first publications in 1983 and can be traced weaving through all subsequent activity.

Back in 1894 Lord Kelvin coined the term "chiral" in order to refer to molecules whose mirror images were not superimposable with themselves. Over the years, research has demonstrated the important role that chiral molecules play in life, chemistry, and biology as well as their importance in the development of new drugs and technologies. The efforts to understand chiral systems have been mainly driven by spectroscopic methods that leverage on the opposite responses that enantiomers have to linear or circularly polarized light of both handedness. More specifically, Electronic Circular Dichroism (ECD) which measures the differences in linear absorption of left and right circularly polarized light has been the method par excellence for the spectroscopic characterization of chiral compounds. Unfortunately, the fact that ECD is based on linear absorption severely limits the use of this method in the near to far UV region. This is mainly due to the interferences generated by the strong linear absorption of common organic solvents and buffers in this portion of the light spectrum. Nevertheless, the fact remains that many chiral biomolecules of interest related to deceases like Alzheimer and Parkinson, exhibit most of their linear absorption in the near to far UV region where ECD cannot be employed for their study. Therefore, it has become an urgent necessity to develop spectroscopic methods to study chiral molecules that can circumvent the limitations of ECD at shorter wavelengths. In order to overcome the existent limitations in linear chiral spectroscopy, the nonlinear equivalent of ECD arises as a promising alternative, i.e. Two-Photon Circular Dichroism (TPCD). Although, this phenomenon was theoretically predicted in 1975, it was not until 2008, with the introduction of the double-L scan, that a reliable and versatile method for the measurement of TPCD was introduced. The high sensitivity of this method is based on the use of "twin" pulses that allow accounting for fluctuations in the excitation source that prevented the experimental realization of the measurement. The first measurement of a full TPCD spectrum was performed on BINOL enantiomers and the results were supported and discussed with the help of theoretical calculations. After that seminal work, we embarked in expanding the understanding of the structure-property relationship of TPCD by performing, systematically, a series of theoretical-experimental studies in chiral biaryl derivatives and compounds with helical chirality. In Chapter 2 we present the theoretical-experimental study of the effect of the π-electron delocalization curvature on the TPCD of molecules with axial chirality. The targeted molecules for this part of our investigation were S-BINOL, S-VANOL, and S-VAPOL. Our findings revealed that an increase in the TPCD signal, within this series of compounds, was related to the curvature of the π–electron delocalization. The contributions of the different transition moments to the two-photon rotatory strength support our outcomes. Then, in Chapter 3 we introduce the development of the Fragment-Recombination Approach (FRA) for the calculation of the TPCD spectra of large molecules. This simple but powerful method is based on the additivity of the TPCD signal, and is subject to a strict conditional fragmentation approach. FRA-TPCD is demonstrated, theoretically, in two hypothetical molecular systems from the biaryl derivatives family. Afterward, in Chapter 4 we show the first experimental demonstration of FRA-TPCD through the conformational analysis of an axially-chiral Salen ligand in solution (AXF-155). The FRA-TPCD spectra calculated for the different isomers of AXF-155 allowed narrowing the number of possible isomers of this complex molecule in THF solution to only two. This represents a significant improvement from previously reported results using ECD. Subsequently, in Chapter 5 we present the study of the effect of intramolecular charge transfer (ICT) in S-BINAP, an axially dissymmetric diphosphine ligand with strong ICT. The evaluation of the performance of two different exchange-correlation functional (XCF) confirmed that in order to properly predict the theoretical TPCD spectrum of a molecule exhibiting strong ICT, it is required to use an XCF such as CAM-B3LYP. In addition, our findings revealed the importance of considering an adequate number of excited states in order to be able to fully reproduce the experimental TPCD spectrum, thus avoiding wrong assignments of theoretical transitions to experimental spectral features. Finally, and expanding on our previous study, in Chapter 6 we investigated the effect of the nature of ICT on two hexahelicene derivatives. Our investigation demonstrated that the TPCD signal of chiral molecules with strong ICT does not only depend on the strength of this effect but on its nature, i.e. extension of the π–electronic delocalization increasing beyond (EXO-ICT) or within (ENDO-ICT) the helicene core. In summary, with the results presented in this thesis we closed a first loop in the understanding of the structure-property relationship of TPCD. In the future, we expect to deepen in our knowledge of the structure-property relationship of this phenomenon by studying further helicene derivatives with donor-acceptor motif, and through the application of FRA-TPCD to the conformational analysis of amino acids in peptides. We foresee numerous applications of TPCD for the study of optically active molecules with implications in biology, medicine, and the drug and food industry, and applications in nanotechnology, asymmetric catalysis and photonics.

The induced circular dichroism (ICD) of several chiral nitrosamines and various chiral reagents has been investigated. The interaction is attributed to a 1:1 hydrogen bonded complex between the NO group of the nitrosamine and the hydroxyl groups of alcohols and polyols, or the amino group of amines. Only those chiral reagents possessing large differences in size of the groups about the hydrogen bonding site contributed to CD anomalies. The acyclic 2-octanols did not give observable Cotton effects, presumably due to the similarity in size of the methyl and methylene groups and rotational freedom of the acyclic system. The signs of the Cotton effects could be correlated with the absolute configuration of the sterically hindered alcohols and amines. Only the alpha, axial hydrogens of conformationally biased, heterocyclic nitrosamines were found to undergo selective hydrogen-deuterium exchange, suggesting that this feature is critical for nitrosamine carcinogenicity.