Bovine pancreatic ribonuclease (RNase A) and its homologues are pyrimidine-specific ribonucleases widely present in mammals, birds, amphibians, reptiles, and fish. RNases recognise a specific sequence – an adenosine 3' to a pyrimidine – on RNA, and cleave the molecule on the 3' side of the 3'-phosphate of the pyrimidine base. Extensive studies have been carried out on the RNase A homologues, including eosinophil-derived neurotoxin (EDN; RNase 2), eosinophil cationic protein (ECP; RNase 3), and angiogenin (ANG; RNase 5), and revealed distinct biological functions: EDN and ECP are involved in neurotoxicity, and ANG possesses angiogenic activity. RNase 4, although being discovered for a long time, is not as well characterised as much as other RNases. RNase 4 has been found in several mammalian species including a few primates, porcine, bovine, and rodents. The mature protein of RNase 4 consists of 119 amino acids, making it the shortest amongst all RNase A homologues. It has a higher inter-species similarity than its homologues, and such high evolutionary conservation suggests that RNase 4 has a more specialised function than RNA degradation. While RNase A, EDN, ECP, and ANG show cytidine preference, RNase 4 has a strong preference for uridine, which can be reversed back to cytidine by a single amino acid substitution of Asp-80, as shown by studies performed with porcine RNase 4 (also known as PL3). In this study, we used PL3 as a model to study the substrate specificity of RNase 4, and have solved four structures, including PL3, PL3 D80A mutant, and these two proteins in complex with dUMP and dCMP respectively. PL3 adopts the classic kidney-shaped RNase A fold, and residues forming the substrate binding subsites occupy similar positions as those in human RNase 4 and the prototypic RNase A. The structure of PL3 D80A mutant resembles that of the wild type iii PL3, and only hydrogen bond interactions between the side chains of Asp-80 and Arg-101 are lost. The structure of PL3·dUMP complex revealed interactions between the dUMP and residues Arg-7, His-12, Thr-44 and Phe-117 of PL3, which were also observed in the structure of human RNase 4 in complex with dUp. The additional hydrogen bonds identified between dUMP and residues Gln-11, Lys-40, Asn-43, and Lys-119 of PL3, as well as the absence of the interactions between Arg-101 of PL3 and the ligand that were present in the hRNase 4·dUp structure, could be due to the flexibility of the mononucleotide ligand. The crystal structure of PL3 D80A·dCMP complex presents a small number of hydrogen bond interactions between the protein and the dCMP ligand, which might be sufficient to stabilise the ligand in the B1 subsite, as the repulsion force on the dCMP ligand from the side chain of Arg-101 is absent in the PL3 D80A mutant. This is because in the D80A mutant, Ala-80 cannot provide hydrogen bonding that would hold the side chain of Arg-101 towards the B1 subsite. The activities of RNases can be inhibited by a 50 kDa cytosolic protein, the natural ribonuclease inhibitor (RI). RI binds to all the members of the RNase A superfamily, thus regulating the cytoplasmic RNA levels and protecting cells from inappropriately secreted RNases. The interactions between RNase and RI are tight, reversible, and in a 1:1 ratio. Several complex structures of RNase·RI from various species have been determined, and the residues in the interfaces between RNase and RI are conserved in all of the complexes. Studies revealed a 17-fold tighter interaction between PL3 and human RI than RNase A, making it very interesting to study the structure of the PL3·RI complex and characterise the interactions between PL3 and RI proteins. iv To date, we have established purification protocols for both proteins, and the next step towards the structure of PL3·RI would be to prepare and purify the protein complex, subject the protein complex to crystallisation experiments, and eventually lead us to the structural determination of PL3·RI complex.

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Digitized by Kansas Correctional Industries

No description available.

Orientadores: Stephenson Caticha Ellis, Iris Concepcion Linares de Torriani / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-09-27T18:22:52Z (GMT). No. of bitstreams: 1 Retz_ClaudiaMaria_M.pdf: 1955996 bytes, checksum: 41377763b5920390a6eabd188461baf1 (MD5) Previous issue date: 1978 / Resumo: Não informado / Abstract: Not informed / Mestrado / Física / Mestre em Física

Orientador: Stephenson Caticha-Ellis / Tese (doutorado) - Universidade Estadualde Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-09-27T18:40:21Z (GMT). No. of bitstreams: 1 Bicalho_HamiltonAraujo_D.pdf: 7138235 bytes, checksum: a04e7af2999c5478c975659de8db6905 (MD5) Previous issue date: 1991 / Resumo: A formação de imagens com raios X fluorescentes não tinha sido desenvolvida até o presente. Neste trabalho são apresentados dois novos métodos de formação de imagens com raios X fluorescentes. Eles produziram imagens que consideramos como o primeiro passo de um novo campo de pesquisa. Eles apresentam algumas vantagens com relação aos tradicionais devido à natureza desta radiação, especialmente a de ser emitida sob forma de raias características. a) Radiografia de um corpo com um feixe de radiação característica que denominamos de Radiografia Monocromática. (Tempos de exposição: da ordem do mseg.). b) Imagem obtida com a fluorescência emitida pelo próprio corpo, excitada com radiação escolhida convenientemente, que denominamos de Imagem Fluorescente. (Tempos de exposição: um par de minutos). Consegue-se um feixe adequado mediante o uso de um elemento fluorescedor, que converte de forma simples, prática e eficiente um certo espectro de radiação (até as bordas de absorção do fluorescedor) em outro, bem determinado, formado pelas linhas características do elemento. Deste modo, radiação branca de um gerador convencional de raios X pode ser transformada em radiação característica de comprimento de onda numa região predeterminada. O método (b) permite obter várias imagens simultaneamente. Duas destas constituem um par estereoscópico e permitem visualizar a imagem tridimensional do interior da amostra. Os métodos desenvolvidos decrescem consideravelmente o risco de irradiação ja que a radiação característica incidente, escolhida convenientemente para excitar o elemento opacificador, é pouco absorvida pelos tecidos animais. A quantidade estimada de opacificador é duas ordens de grandeza menor que nos métodos tradicionais devido a que ele é excitado preferencialmente / Abstract: Imaging by using X-Ray fluorescent radiation had not been developed till now. In this work two new methods of X-ray fluorescent imaging were developed. They have produced images which are to be considered as the first step in a new field. a) "Monochromatic Radiography" (Typical exposure times are of about 1 msec.) .Radiography obtained by using characteristic radiation. b) "Fluorescent Image Method" (Exposure time of about two minutes). The image obtained of a body by using the characteristic radiation emitted by an atom selectively excited by another properly chosen characteristic radiation. An adequate characteristic beam is obtained from a plate made out of a pure element irradiated with a full white spectrum of a conventional generator. This fluorescent element converts it up until the absorption edge, in a practical, efficient and simple way in another spectrum made out of discrete characteristic lines of the element. Thus, white radiation from a conventional generator is transformed to characteristic radiation of wavelengths in a given region. Method (b) may produce several simultaneous images. A couple of them is an stereoscopic pair and the three dimensional internal structure of the body can then be visualized. The new methods have some implicit advantages when compared with traditional methods, with regard to decreased body irradiation since the hard monochromatic radiation is nearly not absorved by the light elements of the animal tissues. In addition, the estimated amount of opacifier is about two orders of magnitude smaller than in classical methods since it is selectively excited here / Doutorado / Física / Doutor em Ciências

Orientador: Iris Concepcion Linares de Torriani / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-09-27T18:30:59Z (GMT). No. of bitstreams: 1 Santos_DeniseRibeirodos_M.pdf: 1895377 bytes, checksum: 6a4dbc0fd4b0ba660c304372792426ad (MD5) Previous issue date: 1992 / Resumo: É bem sabido que a forma de linha de difração de raios-x está relacionada com a cristalinidade do material, e que podemos estimar o tamanho médio dos grãos cristalinos (L) em várias direções na rede analisando o pico de raios-x correspondente. Um efeito equivalente ocorre no espectro de espalhamento Raman de um semicondutor microcristalino, conforme pode ser observado em publicações de vários grupos experimentais e teóricos nos últimos anos. Com base na teoria proposta por Richter e outros [Ri1] para o espectro Raman devido a fônons semi-confinados no volume de pequenos grãos esféricos, obtemos uma expressão analítica para a forma de linha Raman de germânio microcristalino. Utilizando uma subrotina de ajuste de curvas baseada no método de mínimos quadrados, ajustamos a expressão obtida aos dados experimentais de vários filmes de mc-Ge, obtendo como resultado do ajuste o diâmetro médio dos grãos. Considerando que estes obedecem uma distribuição gaussiana de tamanhos, determinamos também a largura aL dessa distribuição para cada amostra, conseguindo uma concordância muito boa entre a expressão teórica e os dados experimentais. Comparando os valores encontrados com os resultados obtidos pela técnica usual de raios-x, concluímos que o novo método de cálculo de L é viável, sendo que os resultados têm uma boa concordância com L medido por raios-x nos casos em que se pode dizer que a aproximação de grãos esféricos é válida / Abstract: It is well known that the X-ray diffraction line shape is related to the crystallinity of the sample material. The average size of the crystallites (L) in certain lattice directions can be evaluated from the broadening of the corresponding X-ray line. A similar effect is observed in the Raman spectrum of micro-crystalline semiconductors. This subject has been discussed and applied in sever al publications of experimental and theoretical groups during the last few years. Based on the theory proposed by Richter et at [Ri1] for the Raman spectrum due to partially confined fonons in the volume of small spherical grains we can obtain an analytical expression for the Raman line of micro-crystalline Ge. Using a curve fitting sub-routine based on the least squares method we proceeded to fit the experimental data obtained from several mc-Ge samples with the proposed analytical expression. The average crystallite size was obtained directly from the fitting parameters. Assuming a Gaussian size distribution we determined the corresponding aL parameters for each sample, getting to a very good agreement between the experimental data and the theoretical calculation. Making a comparison of the values obtained using this analysis of the Raman line and the usual X-ray diffraction technique, we were able to conclude that both techniques will give similar results for the crystallite measurement only in those cases in which the approximation of "spherical" grains is valid / Mestrado / Física / Mestre em Física

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Tese de mestrado. Faculdade de Engenharia. Universidade do Porto. 1998

The analysis of broadened x-ray diffraction profiles provides a useful insight into the structural properties of materials, including crystallite size and inhomogenous strain. In this work a general method for analysing broadened x-ray diffraction profiles is developed. The proposed method consists of a two-fold maximum entropy (MaxEnt) approach. Conventional deconvolution/inversion methods presently in common use are analysed and shown not to preserve the positivity of the specimen profile; these methods usually result in ill-conditioning of the solution profile. It is shown that the MaxEnt method preserves the positivity of the specimen profile and the underlying size and strain distributions, while determining the maximally noncommital solution. Moreover, the MaxEnt method incorporates any available a priori information and quantifies the uncertainties of the specimen profile and the size and strain distributions. Numerical simulations are used to demonstrate that the MaxEnt method can be applied at two levels: firstly, to determine the specimen profile, and secondly to calculate the size or strain distribution, as well as their average values. The simulations include both sizeand strain-broadened specimen profiles. The experimental conditions under which the data is recorded are also simulated by introducing instrumental broadening, a background level and statistical noise to produce the observed profile. The integrity of the MaxEnt results is checked by comparing them with the traditional results and examining problems such as deconvolving in the presence of noisy data, using non-ideal instrument profiles, and the effects of truncation and background estimation in the observed profile. The MaxEnt analysis is also applied to alumina x-ray diffraction data. It is found that the problems of determining the specimen profile, column-length and strain distributions can be solved using the MaxEnt method, with superior results compared with traditional methods. Finally, the issues of defining the a priori information in each problem and correctly characterising the instrument profile are shown to be critically important in profile analysis.