Made available in DSpace on 2014-06-11T19:23:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-29Bitstream added on 2014-06-13T19:28:55Z : No. of bitstreams: 1 barros_ac_me_botib_parcial.pdf: 177767 bytes, checksum: 6e03eeb7f2b4d288bf363856776ce73f (MD5) Bitstreams deleted on 2015-06-03T11:42:50Z: barros_ac_me_botib_parcial.pdf,. Added 1 bitstream(s) on 2015-06-03T11:44:13Z : No. of bitstreams: 1 000691671_20150729.pdf: 177273 bytes, checksum: 1165399c11c2160349be3e18d5484858 (MD5) Bitstreams deleted on 2015-08-03T12:21:12Z: 000691671_20150729.pdf,. Added 1 bitstream(s) on 2015-08-03T12:22:25Z : No. of bitstreams: 1 000691671.pdf: 1177243 bytes, checksum: 866f3caf560a4a70badf707c949ccf65 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A estabilidade e integridade do material genético são fundamentais para manutenção e continuação da vida. O genoma humano é composto por três bilhões de pares de bases, os quais codificam entre 30.000 – 40.000 genes, e é constantemente atacado por metabólitos reativos endógenos, drogas terapêuticas e uma infinidade de agentes mutagênicos ambientais que afetam sua integridade. Assim, é evidente que a estabilidade do genoma deve estar sob vigilância contínua. Isso é conseguido através de mecanismos de reparo de DNA, que se desenvolveram para remover ou tolerar lesões e erros no DNA. Dentre os mecanismos de reparo de DNA presentes nos organismos, eles podem ser divididos em: i) reparo por excisão de bases (BER), ii) excisão de nucleotídeos (NER), iii) mal-pareamento de bases (MMR) e iv) reparo de DNA por junção de terminais não homólogos (NHEJ). Para o que esses mecanismos funcionem de maneira adequada, é evidente a importância da interação entre proteínas responsáveis pela função de reparo, bem como é essencial a regulação da importação nuclear para localização correta das proteínas responsáveis pelos mecanismos mencionados. Dentre os mecanismos responsáveis pela regulação da importação nuclear, a via clássica constituída pelo heterodímero Importina-α/β é um dos principais mecanismos de deslocamento. Algumas proteínas de reparo parecem interagir somente com algumas isoformas da Importina-α (Imp), indicando uma regulação adicional do processo de reparo, porém, pouco se sabe a respeito do reconhecimento das sequências de localização nuclear (NLS) dessas proteínas. Esse trabalho trata especificamente do estudo do complexo estrutural de complexos de Imp com peptídeos NLSs de proteínas relacionadas ao reparo de DNA utilizando... / The stability and integrity of the genetic material are essential for the maintenance and continuation of life. The human genome, comprising three billion base pairs coding for 30.000 – 40.000 genes, is constantly attacked by endogenous reactive metabolites, therapeutic drugs and a plethora of environmental mutagens that impact its integrity. Thus it is obvious that the stability of the genome must be under continuous surveillance. This is accomplished by DNA repair mechanisms, which were developed to remove or tolerate injuries and mistakes in DNA. In the DNA repair mechanisms available in the organisms, they can be divided in: i) base excision repair (BER), ii) nucleotide excision repair (NER), iii) mismatch repair (MMR) and iv) DNA repair by non-homologous end joining (NHEJ). To an adequate operation of these mechanisms, it is evident the importance of the interaction between proteins responsible for the function of DNA repair, as well as the regulation for nuclear import is essential for the correct localization of the proteins responsible for the mentioned mechanisms. In the group of the mechanisms responsible for nuclear import regulation, the classical pathway composed by Importin-α/β heterodimer is one of the major mechanisms of transport. Some DNA repair proteins seem to interact only with certain isoforms of Importin-α (Imp), indicating an additional regulation of the repair process. However, little information is known about the recognition of nuclear localization sequences (NLS) of these proteins. This work concerns specifically the structural studies of complexes with Imp and NLSs peptides from proteins related to DNA repair using crystallographic techniques. The expression and purification of Impα from Mus musculus N-terminally truncated were performed as well as the co-crystalization of... (Complete abstract click electronic access below)

Orientador: Marcos Roberto de Mattos Fontes / Coorientador: Agnes Alessandra Sekijima Takeda / Banca: Maria Célia Bertolini / Banca: André Luis Berteli Ambrósio / Resumo: A estabilidade e integridade do material genético são fundamentais para manutenção e continuação da vida. O genoma humano é composto por três bilhões de pares de bases, os quais codificam entre 30.000 - 40.000 genes, e é constantemente atacado por metabólitos reativos endógenos, drogas terapêuticas e uma infinidade de agentes mutagênicos ambientais que afetam sua integridade. Assim, é evidente que a estabilidade do genoma deve estar sob vigilância contínua. Isso é conseguido através de mecanismos de reparo de DNA, que se desenvolveram para remover ou tolerar lesões e erros no DNA. Dentre os mecanismos de reparo de DNA presentes nos organismos, eles podem ser divididos em: i) reparo por excisão de bases (BER), ii) excisão de nucleotídeos (NER), iii) mal-pareamento de bases (MMR) e iv) reparo de DNA por junção de terminais não homólogos (NHEJ). Para o que esses mecanismos funcionem de maneira adequada, é evidente a importância da interação entre proteínas responsáveis pela função de reparo, bem como é essencial a regulação da importação nuclear para localização correta das proteínas responsáveis pelos mecanismos mencionados. Dentre os mecanismos responsáveis pela regulação da importação nuclear, a via clássica constituída pelo heterodímero Importina-α/β é um dos principais mecanismos de deslocamento. Algumas proteínas de reparo parecem interagir somente com algumas isoformas da Importina-α (Imp), indicando uma regulação adicional do processo de reparo, porém, pouco se sabe a respeito do reconhecimento das sequências de localização nuclear (NLS) dessas proteínas. Esse trabalho trata especificamente do estudo do complexo estrutural de complexos de Imp com peptídeos NLSs de proteínas relacionadas ao reparo de DNA utilizando... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The stability and integrity of the genetic material are essential for the maintenance and continuation of life. The human genome, comprising three billion base pairs coding for 30.000 - 40.000 genes, is constantly attacked by endogenous reactive metabolites, therapeutic drugs and a plethora of environmental mutagens that impact its integrity. Thus it is obvious that the stability of the genome must be under continuous surveillance. This is accomplished by DNA repair mechanisms, which were developed to remove or tolerate injuries and mistakes in DNA. In the DNA repair mechanisms available in the organisms, they can be divided in: i) base excision repair (BER), ii) nucleotide excision repair (NER), iii) mismatch repair (MMR) and iv) DNA repair by non-homologous end joining (NHEJ). To an adequate operation of these mechanisms, it is evident the importance of the interaction between proteins responsible for the function of DNA repair, as well as the regulation for nuclear import is essential for the correct localization of the proteins responsible for the mentioned mechanisms. In the group of the mechanisms responsible for nuclear import regulation, the classical pathway composed by Importin-α/β heterodimer is one of the major mechanisms of transport. Some DNA repair proteins seem to interact only with certain isoforms of Importin-α (Imp), indicating an additional regulation of the repair process. However, little information is known about the recognition of nuclear localization sequences (NLS) of these proteins. This work concerns specifically the structural studies of complexes with Imp and NLSs peptides from proteins related to DNA repair using crystallographic techniques. The expression and purification of Impα from Mus musculus N-terminally truncated were performed as well as the co-crystalization of... (Complete abstract click electronic access below) / Mestre

Common crystallization procedures, X-ray diffraction method and crystallographic software to determine and refine the structure of haloalkane dehalogenase enzyme were used in this thesis.

Some of the challenges faced when developing novel functional materials, cannot be resolved without the correct understanding of their structure‐property relationships. Metal‐organic frameworks (MOFs) constitute a representative example where in-depth structural knowledge can greatly help improve and optimise their application into industrially relevant settings. Fortunately, the inherent crystalline nature of MOFs allows for analysis using the wide range of crystallographic experimental techniques that are currently available. This work covers the study of the structural properties of a particular family of MOFs, which have shown significant potential as molecular sieves and for gas storage. Sc-based MOFs first attracted attention for their particularly robust and inert nature, bypassing some of the physical challenges many MOFs have when undergoing industrial implementation. After an initial review of the state of the art in the field of MOFs and the techniques utilised to analyse their properties, this work then focuses on the mechanical properties of a series of functionalised and unfunctionalised Sc‐dicarboxylate MOFs. Using nano‐indentation techniques and high‐pressure crystallography, the hardness and elasticity of these materials are correlated to their different structural features, confirming their relative robustness when compared to other MOFs in the literature. An interesting property of Sc2BDC3 is its selective uptake of CO2 over other fuel-related gases such as CH4 and CO. In this context, the in situ adsorption crystallographic analysis of Sc2BDC3 and its amino‐functionalised derivative Sc2(BDC‐NH2)3 (BDC‐NH2 = 1,4‐amino‐2‐benzenedicarboxylate) is described, as performed using the gas cell set up of beamline I19 at the Diamond Light Source synchrotron. This study is the first example of a mixed gas atmosphere experiment using single‐crystal diffraction, which in conjunction with in silico, adsorption and breakthrough experiments, provides direct insight into the interactions that drive the selective behaviour of both frameworks. Following this, the MOF Sc2BDC3 (BDC = 1,4‐benzenedicarboxylate), is selected as a case study for branched and unbranched alkane separation. Here, high‐pressure crystallography shows how these relatively oversized guest molecules, can be forced at thousands of atmospheres of pressure inside the narrow triangular channels (< 4 Å diameter) of the framework. It is also possible to resolve the structural changes the framework undergoes upon uptake of the different guests, as well as locate the adsorption sites of the hydrocarbons in the pores of Sc2BDC3, which can be then correlated to the gas adsorption behaviour of the different guests. To conclude, the high‐pressure inclusion study of both CO2 and CH4 inside Sc2BDC3 shows how combining cryoloading techniques and molecular crystallography for the first time, can provide improved models of the adsorbed gaseous guests inside Sc2BDC3. This example not only provides a novel alternative in which to study more easily the adsorption sites in MOFs via diffraction techniques, but also reveals some of the interesting structural behaviour MOFs can have in these extreme conditions.

Abstract Multifunctional enzyme type 2 (MFE-2) is a peroxisomal enzyme participating in the breakdown of fatty acids in eukaryotes. Depending on the organism, MFE-2 is composed of two to four functional units, out of which the two enzymatic ones, 2-enoyl-coenzyme A (CoA) hydratase 2 and (3R)-hydroxyacyl-CoA dehydrogenase, are found in the all MFE-2s. These units are responsible for the catalysis of the second and third steps of the peroxisomal β-oxidation of various CoA thioesters of fatty acids and fatty acyl derivatives. Their (R)-stereospecificity and ability to accept a broad range of fatty acid CoA esters as substrates, in addition to the fact that they do not share any sequence similarity with the classical mitochondrial counterparts, make the enzymatic units of MFE-2 structurally very interesting. In this study, the three-dimensional structures of the (3R)-hydroxyacyl-CoA dehydrogenase and 2-enoyl-CoA hydratase 2 units were solved by crystallographic methods. The crystal structure of the (3R)-hydroxyacyl-CoA dehydrogenase unit of rat MFE-2 reveals a dimeric enzyme with an α/β short-chain alcohol dehydrogenase/reductase (SDR) fold. A unique feature of (3R)-hydroxyacyl-CoA dehydrogenase, however, is the separate C-terminal domain, which completes the active site cavity of the adjacent monomer and extends the dimeric interactions. The 2-enoyl-CoA hydratase 2 unit is a dimer with a unique two-domain structure proposed to evolve via gene duplication. The fold consists of two side-by-side arranged repeats of the hot-dog fold motifs, thus being highly reminiscent of the tertiary structures of the (R)-specific 2-enoyl-CoA hydratase of the polyhydroxyalkanoate synthesis pathway and the β-hydroxydecanoyl thiol ester dehydrase of fatty acid synthesis type II, both from prokaryotic sources. The importance of the N-domain in the binding of bulky substrates was shown by the enzyme-product complex structure, which also indicates the active site. For the first time, it was shown that the eukaryotic hydratase 2 uses an Asp/His catalytic dyad in catalysis. Moreover, a novel catalytic mechanism was proposed for (R)-specific hydration/dehydration. The solved structures also provide a molecular basis for understanding the effects of the patient mutations of MFE-2. They also allow disussion of the possible organisation of the three units in full-length MFE-2 of mammals.

M.Sc. / The main objective of the work presented in this dissertation was to investigate the application of the phosphorus atom as a probe to evaluate stereo-electronic effects in arylphosphines. Traditionally, electronic effects are described as having inductive or resonance origins. In addition to the aforementioned mechanisms, the possibility of an additional field effect pathway was also investigated. For this purpose, a series of ortho, meta and para mono-substituted triaryl phosphines, i.e. Ph2(C6H4-X), were synthesised using a lithium-halogen exchange pathway. This series included a selection of electron-withdrawing and electron-donating substituents (X = F, CN, COOtBu, Me, OMe, NMe2) as well as combinations of these. Most of these ligands are crystalline which allowed analysis of their electronic nature by means of X-ray crystallography. From these ligands a representative range of electron-donating and electron-withdrawing aryl substituted phosphines was chosen to collect high-resolution (d=0.5 Å) data. An aspherical multipole refinement was carried out on each of the high-resolution data sets by employing the Hansen Coppens multipole formalism. This was followed by an experimental charge density analysis of each phosphine by employing the principles of QTAIM employed in WinXD. From topological analysis of the Laplacian of the electron density, properties at the (3,-3) lone pair critical points were evaluated. Similarly, the density properties at the (3,-1) bond critical points of the P-Cipso bond were evaluated by analysis of the topology of the electron density. In addition, several integrated properties including the volume, charge and electron population of the phosphorus atom were evaluated. All of the above properties showed very good linear correlations with the infrared CO stretching frequencies of the Rh-Vaska-type complexes corresponding to these phosphines. Furthermore, computational chemistry was employed as a complementary investigation tool to the X-ray crystallographic study. A theoretical charge density study was conducted for the complete range of phosphines described above in paragraph 1 of this Synopsis by employing the principles of QTAIM employed in AIMAll. All of the properties mentioned in the above paragraph were also calculated. In addition, the calculated molecular electrostatic potential properties of the phosphorus lone pair (Vmin and dcp), the integrated substituent bond dipole and NBO (Natural bond orbital) analysis was used to evaluate substituent electronic effects. All of the calculated properties (with the exception of the charge and electron population of the phosphorus atom calculated from NBO analysis) showed good linear correlations with the infrared CO stretching frequencies of the Vaska-type complexes corresponding to these phosphines within a particular electron-withdrawing/electron-donating or ortho/meta and para series. In addition, very ii good linear correlations were obtained between the experimental and theoretical properties within a particular electron-withdrawing/electron-donating or ortho/meta and para series. As additional investigation tools, the ligands were characterised by several techniques including infrared CO stretching frequency measurements performed on Rh Vaska-type compounds derived from the synthesised ligands, 31P NMR chemical shift measurements as well as 103Rh-31P coupling constant measurements to evaluate the effect of various substituents on the electron density at the phosphorus lone pair. In conclusion, it was found that the phosphorus atom is a sensitive probe of substituent electronic effects. Furthermore, it was found that high-resolution X-ray crystallography, computational chemistry, 31P NMR and infrared spectroscopy are all excellent techniques that can be employed to obtain a better understanding of the nature and transmission of substituent effects. From this study, it appeared that the electronic effects in phosphine ligands could not be rationalised by an inductive mechanism alone, but seemingly more correctly by an additional field effect mechanism.

M.Phil. (Energy Studies) / Non-destructive investigations of unusual natural polycrystalline diamond samples are reported. The samples originate from various international locations discovered in soils and sediments and may have been formed by di erent mechanisms which are the subject of debate. Analysis techniques include scanning electron microscopy, with energy dispersive spectroscopy and X-ray di raction. Sample composition, structure and surface stresses were investigated. The samples (two Egyptian nodules, two Brazillian carbonados, two Venetian ballas diamonds and a polycrystalline diamond compact sample consisting of a polycrystalline diamond layer sintered onto a Co-cemented tungsten carbide support) are cubic diamonds, with varying amounts of minority phases. In most cases the minority phases could not be identi ed unambiguously due to their low intensities and the overlap of Bragg peaks. The Egyptian nodule samples in particular showed a large presence of oxygen that could not be linked to speci c metal oxides. The Ballas samples had the lowest impurity content. Using a Rietveld based quanti cation analysis, the crystalline cubic diamond contents were determined respectively as 99 wt.%, 98 wt.% and 76 wt.% in the Ballas, Brazilian carbonado and Egyptian nodule samples. A signi cant result from this study is the substantial inhomogeneous peak broadening observed in the X-ray diffraction patterns of the Egyptian samples. This is indicative of the presence of nano-crystalline diamond grains in addition to substantial mechanical deformation with extensive accumulation of dislocations and crystalline defects. These observations are supportive of a recently proposed impact mechanism for their formation.

The crystal structure of dibenzothiophene has been determined by X-ray diffraction. Mo-Kα [subscript omitted] scintillation counter data were used for this analysis; the sulfur atom position was determined by means of a Patterson function; the carbon atoms were located from a Fourier synthesis, and the hydrogen atoms, from a difference synthesis. Refinement of positional and thermal parameters was by least-squares methods. The molecule is slightly folded, the dihedral angles between the five-membered ring and the six-membered rings being 0.4° and 1.2°. The bond distances and valency angles are similar to those in related molecules. The C-S bond length is 1.740 A, and the C-S-C angle is 91.5°. The crystal structure of DL-ornithine hydrobromide has been determined by means of visual Cu-Kα [subscript omitted] data. The bromine ion position was found by Patterson methods; carbon, nitrogen, and oxygen atoms were located on Fourier summations and the hydrogen atoms, on a difference synthesis. The positional and thermal parameters were refined by least-squares. The ornithine molecule is a zwitterion, with both nitrogens accepting protons. The mean bond distances are C-0, 1.249 A; C-N, 1.469 A; C-C, 1.532 A. The structure is held together by a system of N—H …… 0 (2.84, 2.84, 2.89 A) and N—H…..Br (3.29, 3.36,.3.46 A) hydrogen bonds. The crystal and molecular structure of histamine diphosphate monohydrate has been determined with scintillation counter Mo-Kα [subscript omitted] data. The positions of the phosphorus atoms were determined by Patterson methods; the carbon, nitrogen and oxygen atoms were located by means Fourier syntheses; the hydrogen atoms were found on a difference synthesis. The thermal and positional parameters were refined by least-squares. The atoms of this histamine cation lie in two almost perpendicular planes, the plane of the imidazole ring and that of the side chain. The bond lengths and angles are similar to the corresponding values in histidine hydrochloride monohydrate. The dimensions of the two P0₂(OH) ₂⁻ ions are P-0 1.51 A, P-OH 1.57 A, O-P-0 115.5°, and HO-P-OH 107.0°. The most important feature of the packing is a complex system of 0-H.....0 and N-H...0 hydrogen bonds. / Science, Faculty of / Chemistry, Department of / Graduate

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy / In this dissertation the physical and chemical alterations induced by mastication and manipulation of wax by the worker bee in honeycomb construction, and the subsequent contribution afforded the structural integrity of the nest, are elucidated. In comb building, the freshly secreted wax scales are mandibulated together with a frothy salivary emulsion, and added piece-meal to form honeycomb. Textural modifications were revealed using X-ray crystallography. While virgin scale wax is highly structured, with the crystallites aligned approximately perpendicular to the planar surface, comb wax has a random crystallographic arrangement. This reflects a disruption of the crystallite structure following the mechanical insult of mastication. Chemical analyses included investigation of both lipid and proteinaceous elements. Lipid composition was evaluated by enzyme-catalyzed as well as thin-layer and gas-liquid chromatographic methods. The results indicate a reduction in scale diacylglycerols with a corresponding increase in comb saturated monoaeylglycerols. Such modifications are highly suggestive of lipase activity within the salivary addition. The proteins of comb and scale wax were analyzed electrophoretically, under reduced conditions. Each wax possesses unique polypeptide fractions, in addition to sharing common protein species, It is speculated that those in common represent integral proteins, such as transport molecules, while the disparities noted may be due to salivary enzymatic degradation, or even glycosylation. The effects of these textural and chemical alterations on the mechanical behaviour of the waxes was assessed. Tensile tests were performed on a variety of scale and comb wax preparations over the range of temperatures likely to impinge on the honeybee nest. These investigations reveal the specific structural contributions made by each of the physico-chemical alterations described. Further, they demonstrate that while scales are ideal moulding materials due to their high distensibility and low stiffness, the greater resistance to deformation and lower potential for extension makes comb wax a superior structural material. The mechanical advantage for including propolis and cocoon silk within the comb structure was also investigated. Tensile testing indicates that the resultant composite material is structurally superior, largely due to the presence of silk reinforcement. / Andrew Chakane 2018

Thesis: M.S., Massachusetts Institute of Technology, Department of Materials Science and Engineering, 1977 / Includes bibliographical references. / M.S. / M.S. Massachusetts Institute of Technology, Department of Materials Science and Engineering