The atomic resolution structure of a low populated and transiently formed on-pathway folding intermediate of the FF domain from human HYPA/FBP11 has recently been reported[1]. The structure was determined on the basis of backbone chemical shift and bond vector orientation restraints measured on the ‘invisible’ intermediate state using relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy that were subsequently input into the data-base structure determination program CS-Rosetta. This thesis focuses on the cross-validation of the structure so produced. We present here the solution NMR structure of a mimic of the folding intermediate that is highly populated in solution, obtained from the wild-type domain by protein mutagenesis. The ensemble of structures generated of the mimic are within 2Å of the relaxation dispersion/CS-Rosetta structures of the intermediate, with the non-native interactions in the intermediate also observed in the mimic. The results presented in this thesis strongly confirm the structure of the FF domain folding intermediate, in particular, and validate the use of relaxation dispersion derived restraints in structural studies of invisible excited states, in general.

The atomic resolution structure of a low populated and transiently formed on-pathway folding intermediate of the FF domain from human HYPA/FBP11 has recently been reported[1]. The structure was determined on the basis of backbone chemical shift and bond vector orientation restraints measured on the ‘invisible’ intermediate state using relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy that were subsequently input into the data-base structure determination program CS-Rosetta. This thesis focuses on the cross-validation of the structure so produced. We present here the solution NMR structure of a mimic of the folding intermediate that is highly populated in solution, obtained from the wild-type domain by protein mutagenesis. The ensemble of structures generated of the mimic are within 2Å of the relaxation dispersion/CS-Rosetta structures of the intermediate, with the non-native interactions in the intermediate also observed in the mimic. The results presented in this thesis strongly confirm the structure of the FF domain folding intermediate, in particular, and validate the use of relaxation dispersion derived restraints in structural studies of invisible excited states, in general.

Soluble guanylyl cyclase (sGC) is the receptor for nitric oxide and a highly sought-after therapeutic target for the management of cardiovascular diseases. New compounds that stimulate sGC show clinical promise, but where these stimulator compounds bind and how they function remains unknown. Here, using a photolyzable diazirine derivative of a novel stimulator compound, IWP-051, and MS analysis, we localized drug binding to the 1 heme domain of sGC proteins from the hawkmoth Manduca sexta and from human. Covalent attachments to the stimulator were also identified in bacterial homologs of the sGC heme domain, referred to as H-NOX domains, including those from Nostoc sp. PCC 7120, Shewanella oneidensis, Shewanella woodyi, and Clostridium botulinum, indicating that the binding site is highly conserved. The identification of photoaffinity-labeled peptides was aided by a signature MS fragmentation pattern of general applicability for unequivocal identification of covalently attached compounds. Using NMR, we also examined stimulator binding to sGC from M. sexta and bacterial H-NOX homologs. These data indicated that stimulators bind to a conserved cleft between two subdomains in the sGC heme domain. L12W/T48W substitutions within the binding pocket resulted in a 9-fold decrease in drug response, suggesting that the bulkier tryptophan residues directly block stimulator binding. The localization of stimulator binding to the sGC heme domain reported here resolves the longstanding question of where stimulators bind and provides a path forward for drug discovery.

Ces dernières décennies, les xénolites de péridotites provenant des kimberlites et des basaltes ont contribué de manière déterminante à la compréhension de l’état physico-chimique du manteau supérieur de la Terre dont dépend la modélisation directe de déformation telle que le rebond post-glaciaire. Une telle approche requiert a priori la détermination des conditions physiques P, T et σ dont témoignent la chimie minérale et la texture de ces roches. Un protocole original d’inversion thermodynamique de données expérimentales pour des pyroxènes en équilibre mutuel, a d’abord été développé dans le but d’obtenir un géothermobaromètre fiable. D’autre part, l’étude menée par spectroscopie IRTF de pyroxènes montre que ces minéraux nominalement anhydres (NAMs) contiennent en fait de l’eau sous forme dissoute, de 38 à 450 ppm pour les cpx, et de 19 à 184 ppm pour les opx.Par son rôle perturbateur du réseau cristallin, cette eau affecte directement les propriétés physico-chimiques du manteau terrestre (viscosité, diagrammes de phase, conductivité électrique). La dissolution de cette eau est correlée à fO2, P et T, et seulement indirectement à la chimie des minéraux. Des analyses 1H, 27Al et 29Si MAS RMN sur ces phases contenant des teneurs non négligeables en fer, ainsi que sur des mélanges kaolinite+magnétite, permettent, entre autres, d’authentifier les signatures spectrales de ces pyroxènes naturels mais aussi de quantifier les espèces Al en termes de sites d’occupation. La complémentarité de ces outils, appliqués ici avec succès, ouvre de nouvelles perspectives de compréhension et modélisation des processus profonds. / Over the last decades, peridotite xenoliths brought up by kimberlites and basalts have largely and critically contributed to the understanding of the physical and chemical state of the Earth’s upper-mantle, a prerequisite for direct modelling of deep deformations such as those at work in post-glacial rebound. Such an approach first requires quantification of the physical conditions (P, T, σ) in control of mineral chemistry and rock textures. An original protocol for thermodynamic inversion of experimental data on mutually-equilibrated pyroxenes, was first developed to obtain a reliable geothermobarometer. Moreover, FTIR spectroscopic studies of pyroxenes show that these nominally anhydrous minerals (NAMs) actually contain dissolved water, from 38 to 450 ppm for cpx’s and from 19 to 184 ppm for opx’s. According to its disruptive action onto the crystalline network, such water directly affects the physical and chemical properties of the Earth’s mantle (viscosity, phase diagrams, electrical conductivity). Dissolution of this water component is correlated to fO2, P and T, but only indirectly to mineral composition. Last, 1H, 27Al and 29Si MAS NMR analyses on the same pyroxenes (as iron-bearing minerals), as well as on kaolinite+magnetite mixes, have authenticated NMR spectra for natural pyroxenes, but have also yielded constraining data on site occupancies, specially for Al. These complementary tools applied here with some success, open new prospects for the understanding and modelling of deep processes.

Thesis (MScEng (Electrical and Electronic Engineering))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Assessing the quality of fruit has become vitally important for farmers and growers. With retailers placing ever stricter requirements on fresh produce, growers have to spend a greater amount of time and effort sorting and grading their harvest. Increasingly, vendors are placing requirements not only on external factors like colour, and firmness, but on internal quality factors such as sugar content and acidity, because, although consumers buy fruit based on their external appearances, the taste of the fruit is what determines whether the consumer will buy again. Different techniques exist that probe the internal quality of fruit non-destructively. The technique most widely used today is Near Infrared Spectroscopy. This technique is powerful, but has certain limitations such as poor reliability and the need for constant recalibration. This thesis suggests an alternative method for evaluating internal fruit quality based on low-field nuclear magnetic resonance detected by superconducting quantum interference devices. It introduces the theory of SQUIDs and NMR, and evaluates the use of SQUID detected NMR spectroscopy as a method for determining the internal quality of fruit. The fabrication techniques and processes are explained in detail and a design for a SQUID detected NMR spectrometer is given. Relevant simulations and simulation results are also given. No working SQUID could be fabricated and, as such, no working NMR spectrometer was demonstrated. This thesis serves as a reference work for future research. / AFRIKAANSE OPSOMMING: Bepaling van die gehalte van vrugte het vir boere uiters belangrik geword. Met kleinhandelaars wat al strenger vereistes plaas op vars produkte moet boere meer tyd en inspanning bestee met die sortering en gradering van hul oes. Handelaars plaas nie net vereistes op eksterne kwaliteitsfaktore soos kleur en fermheid nie, maar begin al hoe strenger vereistes plaas op interne kwaliteitsfaktore soos suikerinhoud en suurgehalte, want, hoewel verbruikers vrugte koop op grond van hul eksterne kwaliteitsfaktore, is dit die smaak van die vrug wat bepaal of die verbruiker weer die vrug sal koop. Verskillende tegnieke bestaan wat die interne kwaliteit van vrugte op ’n nie-destruktiewe manier kan bepaal. Die mees algemene tegniek is Naby Infrarooi Spektroskopie. Hierdie tegniek is kragtig maar het sekere beperkings soos swak betroubaarheid en die noodsaaklikheid van konstante herkalibrasie. Hierdie tesis stel ’n alternatiewe metode vir die evaluering van interne vrugkwaliteit gebaseer op lae-veld kernmagnetiese resonans waargeneem deur supergeleidende kwantum inmenging toestelle voor. Dit stel die teorie van SKWITs en KMR bekend, en evalueer die gebruik van SKWIT-bespeurde KMR spektroskopie as ’n metode vir die bepaling van die interne kwaliteit van vrugte. Die fabrikasie tegnieke en prosesse word in detail verduidelik en ’n ontwerp vir ’n SWKIT opgevangde KMR spektrometer word gegee. Toepaslike simulasies en simulasie resultate word ook gegee. Geen werkende SKWIT kon vervaardig word nie en as gevolg daarvan kon geen werkende KMR spektrometer gedemonstreer word nie. Hierdie tesis dien as ’n naslaan werk vir toekomstige navorsing.

No description available.

Study of thermoresponsive porphyrins and their supramolecular complexes Abstract: We studied the water-soluble artificial compound meso-tetrakis{3,4,5-tris[2-(2-(2- methoxyethoxy)ethoxy)ethoxy]phenyl}porphyrin prepared at NIMS, Japan, using the high-resolution NMR spectroscopy experimental method. We observed its LCST-type phase separation and applied the Flory-Huggins theory of polymer solutions in order to find its phase diagram (binodal and spinodal curves of the phase separation) and we also obtained molar enthalpies, entropies and critical temperatures of its phase separation; from the Flory-Huggins theory we discovered that its molecules form dimers in aqueous solutions. We also studied its host-guest interactions with the S-camphorsulfonic acid; we learned that the porphyrin binds cations and the porphyrin dimers break down when dissolved cations are available for complexation. We observed no phase separation in chloroform. We obtained no proof of the existence of molecular stacks larger than dimers. 1

O silício (Si) é benéfico ao desenvolvimento das plantas por proporcionar aumento na capacidade de defesa contra o ataque de pragas e de doenças, além de garantir porte ereto das plantas, o que aumenta a capacidade fotossintética dos vegetais. Fontes silicatadas, como silicatos de cálcio e de magnésio, fornecem Si ao solo e podem funcionar como corretivos de acidez. O acúmulo de resíduos vegetais e fertilizantes, mesmo no sistema semeadura direta (SSD), acelera o processo de acidificação do solo em curto prazo, principalmente em sua camada mais superficial. Esse processo é contínuo em solos tropicais úmidos e tem por consequência a baixa disponibilidade de nutrientes e elevados teores tóxicos de Al+3. Assim como no sistema convencional de manejo do solo, o SSD também demanda a aplicação de insumos, especialmente materiais corretivos de acidez. A aplicação de silicato pode interferir na disponibilidade de Si às plantas e nas formas desse elemento na solução do solo. O ácido silícico (H4SiO4) é a espécie de Si predominante em solos ácidos; e o aumento do pH pode causar sua desprotonação, gerando o ânion silicato (H3SiO4-). Estudos ligados ao Si em solos sob SSD são escassos, principalmente em experimentos de longa duração conduzidos sob condições de clima tropical úmido. Desde 2002 está sendo conduzido experimento de campo em um Latossolo Vermelho distroférrico sob SSD que recebeu aplicação superficial de calcário e de silicato de cálcio e de magnésio. Foram determinados os teores disponíveis de macronutrientes e silício, pH, soma de bases, saturação por bases, capacidade de troca catiônica, matéria orgânica, H+Al e Al3+, minerais silicatados e óxidos de Fe e Al, 24 meses após a reaplicação superficial dos corretivos. Foi feita especiação iônica de Al, Ca, Mg e Si em extrato aquoso e realizada a quantificação de Al por espectroscopia de ressonância magnética nuclear (27Al-NMR). Os principais óxidos encontrados foram a gibbsita, goethita e hematita. Diferentemente do Ca e do Mg, o Al não estava presente, em sua maioria, na forma livre Al3+, mas formando complexos com o carbono orgânico dissolvido. O silício estava quase que totalmente na forma de H4SiO4, sendo que a atividade dessa espécie foi semelhante à sua concentração em solução. A análise quantitativa de 27Al-NMR forneceu somente picos do complexo de alumínio hexa-hidratado ([Al(H2O)6]3+), não havendo espécies de alumínio complexadas com ácidos orgânicos. Amostras com teores moderados a alto de Al (27Al-NMR) se correlacionaram com as concentrações totais de Al provenientes da análise de espectroscopia de emissão atômica (ICP-AES). / Silicon (Si) is an element which is beneficial to plant development in that it provides a line of defense against attack by pests and disease as well, as ensure a plant posture that is more erect that increases their photosynthetic capacity. Silicon sources such as calciummagnesium silicates provide Si to the soil and act as acidity correctives. The accumulation of plant residues and fertilizers, even under a no-till system (NT), accelerates the process of soil acidification in the short term, especially in the uppermost surface layer. This process is continuous in moist tropical soils and results in a low level of nutrients and a high level of Al3+. As is the case under the conventional system of soil management, NT also requires the application of additives, especially acidity corrective materials. The application of silicate can interfere with the availability of Si to plants and the forms in which this element is found in soil solution. Silicic acid (H4SiO4) is the predominant form of Si found in acidic soils; and increases in the pH can cause deprotonation, which generates silicate anions (H3SiO4-). Studies related to Si in soils under NT are scarce, especially long-term experiments carried out under humid tropical conditions. Since 2002, a field experiment has been conducted on a dystroferric Oxisol under NT, where lime and calcium-magnesium silicate were surface applied. The contents of macronutrients and silicon available, as well as pH, basis total and basis saturation, cationic exchange capacity, organic matter, H+Al and Al3+, silicate minerals and Fe and Al oxides, were all determined 24 months after the correctives were applied. The ionic speciation of Al, Ca, Mg and Si took place in aqueous extract as well as the quantification of Al by nuclear magnetic resonance spectroscopy (27Al-NMR). The main oxides found were gibbsite, goethite and hematite. Unlike Ca and Mg, Al was not, for the most part, present in its free form Al3+ but complexed with dissolved organic carbon. Silicon was almost entirely in the form of H4SiO4, and its activity was similar to the concentration of this specie in solution. Quantitative analysis of 27Al-NMR has provided only peaks of Hexaaquaaluminium complex ([Al(H2O) 6]3+), with no species of organic-aluminum complexes. Samples with moderate to high levels of Al (27Al-NMR) were positively correlated with the total concentration of Al derived from the atomic emission spectroscopy analysis (ICP-AES).

A Morte Súbita dos Cítros (MSC) é uma doença que afeta plantas de laranjeira-doce enxertadas em limão Cravo ou Volkameriano e ainda não tem o agente causal determinado. Para entender quais são as principais mudanças fisiológicas causadas pela MSC, analisou-se as alterações nos metabolitos primários, tanto da casca da copa quanto do cavalo. Fez-se análises dos extratos hexânico e alcoólico com RMN em alta resolução e do extrato hexânico com cromatografia gasosa. Também analisou-se os metabolitos diretamente nas cascas com técnicas de RMN em alta e baixa resolução. Com essas análises foi possível observar que a MSC altera a deposição de triacilglicerídeos e sacarose nas cascas. A MSC também modifica o perfil de ácidos graxos, com o decréscimo dos teores de ácido oléico e linolênico e aumento dos ácidos cáprico, láurico, mirístico, plamítico, esteárico e linoléico, com a evolução da doença. Com esses resultados foi possível entender as principais alterações bioquímicas causadas pela MSC, como também demonstrar que elas podem ser usadas de forma complementar no diagnóstico da doença. Dentre todos os métodos avaliados, as análises das cascas com RMN em baixa resolução, com as técnicas CPMG e de precessão livre em onda contínua (CWFP), foram as mais rápidas (em alguns segundos) e eficientes para discriminar as plantas assintomáticas das sintomáticas. A discriminação das plantas pelos dados de RMN foi realizada com métodos quimiométricos como análise de componentes principais (PCA), análises de agrupamentos hierárquicos (HCA) e regressão por mínimos quadrados parciais (PLS). / Citrus Sudden Death (CSD) is a new graft-transmissible disease of sweet orange grafted on Rangpur lime and Citrus volkameriana rootstocks. The causal agent is unknown. To understand the main physiological changes caused by the CSD, we analyzed the variations in the primary metabolites content in the bark of scion and rootstock. The hexanic and hydroalcoholic extracts were analyzed by high resolution NMR. The hexanic extracts were also analyzed by gas chromatography. The metabolites were also analyzed directly in the barks with high and low resolution NMR techniques. With these analyses it was possible to observe that CSD modifies the content of triglycerides and sucrose in the barks. The disease also changes the fatty acids profile, with a decrease in the oleic and linolenic and an increase in the capric, lauric, miristic, palmitic, stearic and linoleic content. With these results it was possible to understand the main biochemical disorders caused for the CSD, as well as to demonstrate that they can be used as complementary information in the disease diagnosis. The analysis of the barks with low resolution NMR techniques CPMG and continuous wave free precession (CWFP), had been the fastest (few seconds) and the most efficient one to discriminate between the symptomless and symptomatic plants. The discrimination of the plants by NMR data had been carried with chemometric methods such as principal component analysis (PCA), hierarchic cluster analysis (HCA) and partial least square regression (PLS).

O silício (Si) é benéfico ao desenvolvimento das plantas por proporcionar aumento na capacidade de defesa contra o ataque de pragas e de doenças, além de garantir porte ereto das plantas, o que aumenta a capacidade fotossintética dos vegetais. Fontes silicatadas, como silicatos de cálcio e de magnésio, fornecem Si ao solo e podem funcionar como corretivos de acidez. O acúmulo de resíduos vegetais e fertilizantes, mesmo no sistema semeadura direta (SSD), acelera o processo de acidificação do solo em curto prazo, principalmente em sua camada mais superficial. Esse processo é contínuo em solos tropicais úmidos e tem por consequência a baixa disponibilidade de nutrientes e elevados teores tóxicos de Al+3. Assim como no sistema convencional de manejo do solo, o SSD também demanda a aplicação de insumos, especialmente materiais corretivos de acidez. A aplicação de silicato pode interferir na disponibilidade de Si às plantas e nas formas desse elemento na solução do solo. O ácido silícico (H4SiO4) é a espécie de Si predominante em solos ácidos; e o aumento do pH pode causar sua desprotonação, gerando o ânion silicato (H3SiO4-). Estudos ligados ao Si em solos sob SSD são escassos, principalmente em experimentos de longa duração conduzidos sob condições de clima tropical úmido. Desde 2002 está sendo conduzido experimento de campo em um Latossolo Vermelho distroférrico sob SSD que recebeu aplicação superficial de calcário e de silicato de cálcio e de magnésio. Foram determinados os teores disponíveis de macronutrientes e silício, pH, soma de bases, saturação por bases, capacidade de troca catiônica, matéria orgânica, H+Al e Al3+, minerais silicatados e óxidos de Fe e Al, 24 meses após a reaplicação superficial dos corretivos. Foi feita especiação iônica de Al, Ca, Mg e Si em extrato aquoso e realizada a quantificação de Al por espectroscopia de ressonância magnética nuclear (27Al-NMR). Os principais óxidos encontrados foram a gibbsita, goethita e hematita. Diferentemente do Ca e do Mg, o Al não estava presente, em sua maioria, na forma livre Al3+, mas formando complexos com o carbono orgânico dissolvido. O silício estava quase que totalmente na forma de H4SiO4, sendo que a atividade dessa espécie foi semelhante à sua concentração em solução. A análise quantitativa de 27Al-NMR forneceu somente picos do complexo de alumínio hexa-hidratado ([Al(H2O)6]3+), não havendo espécies de alumínio complexadas com ácidos orgânicos. Amostras com teores moderados a alto de Al (27Al-NMR) se correlacionaram com as concentrações totais de Al provenientes da análise de espectroscopia de emissão atômica (ICP-AES). / Silicon (Si) is an element which is beneficial to plant development in that it provides a line of defense against attack by pests and disease as well, as ensure a plant posture that is more erect that increases their photosynthetic capacity. Silicon sources such as calciummagnesium silicates provide Si to the soil and act as acidity correctives. The accumulation of plant residues and fertilizers, even under a no-till system (NT), accelerates the process of soil acidification in the short term, especially in the uppermost surface layer. This process is continuous in moist tropical soils and results in a low level of nutrients and a high level of Al3+. As is the case under the conventional system of soil management, NT also requires the application of additives, especially acidity corrective materials. The application of silicate can interfere with the availability of Si to plants and the forms in which this element is found in soil solution. Silicic acid (H4SiO4) is the predominant form of Si found in acidic soils; and increases in the pH can cause deprotonation, which generates silicate anions (H3SiO4-). Studies related to Si in soils under NT are scarce, especially long-term experiments carried out under humid tropical conditions. Since 2002, a field experiment has been conducted on a dystroferric Oxisol under NT, where lime and calcium-magnesium silicate were surface applied. The contents of macronutrients and silicon available, as well as pH, basis total and basis saturation, cationic exchange capacity, organic matter, H+Al and Al3+, silicate minerals and Fe and Al oxides, were all determined 24 months after the correctives were applied. The ionic speciation of Al, Ca, Mg and Si took place in aqueous extract as well as the quantification of Al by nuclear magnetic resonance spectroscopy (27Al-NMR). The main oxides found were gibbsite, goethite and hematite. Unlike Ca and Mg, Al was not, for the most part, present in its free form Al3+ but complexed with dissolved organic carbon. Silicon was almost entirely in the form of H4SiO4, and its activity was similar to the concentration of this specie in solution. Quantitative analysis of 27Al-NMR has provided only peaks of Hexaaquaaluminium complex ([Al(H2O) 6]3+), with no species of organic-aluminum complexes. Samples with moderate to high levels of Al (27Al-NMR) were positively correlated with the total concentration of Al derived from the atomic emission spectroscopy analysis (ICP-AES).