No description available.

Ethanolamine kinase (EC 2.7.1.82) catalyses the reaction of ethanolamine and Mg^2+-ATP to produce phosphoethanolamine and Mg^2+ -ADP. For spinach (Spinacia oleracea) the activity of ethanolamine kinase is increased in leaf extracts of salinized plants. A comparison of ethanolamine kinase activity between extracts from control and salinized plants after native polyacrylamide gel electrophoresis shows that ethanolamine kinase activity migrates to the same position on a gel. This observation suggests that salinization does not induce the activity of a novel ethanolamine kinase isozyme. Ethanolamine kinase has been purified 6,537 fold to apparent homogeneity from spinach leaves by ammonium sulphate fractionation and sequential fractionation by both open-bed and HPLC chromatography, using ion-exchange and hydrophobic interaction matrices. The enzyme has an estimated molecular weight of 80,000 D by gel filtration chromatography and a subunit size of 38,000 D by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Ethanolamine kinase has a broad pH optimum between pH 7 and 9 and the optimal ratio of Mg^2+:ATP for the reaction is 1: 1 at 5 mM. The apparent K^m value for the substrate ethanolamine is 16 μM and the V^max is 438 nmol • min^-1 •mg^-l protein. Monomethylethanolamine and dimethylethanolamine serve as substrates for. ethanolamine kinase but not trimethylethanolamine (ie choline). Enzyme activity is slightly stimulated by NaCl and KCl and inhibited to varying degrees by phosphate, ammonium, phosphoethanolamine and phosphodimethylethanolamine. Not surprisingly enzyme activity is also inhibited by ADP and to varying degrees by the divalent cations Mn^2+, Ca^2+, Co^2+, Ba^2+ and Ni^2+. This work is the first purification and biochemical characterization of ethanolamine kinase in spinach and is the first step towards understanding the contribution ethanolamine kinase makes towards the synthesis of choline. / Thesis / Master of Science (MSc)

No description available.

The mitofusin 1 and 2 (MFN and MFN2) proteins reside in the outer mitochondrial membrane and have been shown to regulate mitochondrial network architecture by mediating tethering and fusion of mitochondria. Mitochondria normally form a tubular and branched reticular network dynamically regulated by a balance of fusion and fission events. Absence of either Mfn1 or Mfn2 results in a fragmented mitochondrial network. Züchner et al. previously described mutations in the gene mitofusin 2 (MFN2) as the cause of the major autosomal-dominant, axonal form of Charcot-Marie-Tooth neuropathy (CMT2A). CMT type 2 (CMT2) is characterized by chronic axonal degeneration of peripheral nerves leading to the loss of functional nerve fibers. Mutations in MFN2 are the most common cause of CMT2, and in Chapter 2 we report the results from a genetic screen of MFN2 in a CMT2 patient cohort. The original finding that mutations in MFN2 cause CMT2A led to investigations focused on deficiencies of mitochondrial fusion and transport, specifically in the context of long axonal processes affected in CMT. While some experimental work supports disrupted mitochondrial transport in the etiology of CMT2A, other studies on CMT2A patient fibroblasts and cell models suggest abnormal mitochondrial fusion and dynamics do not underlie the etiology of this. In the first half of Chapter 3, we present some of our initial investigations prior to de Brito and Scorrano’s report published in 2008 regarding a novel role for Mfn2 in tethering the endoplasmic reticulum (ER) to mitochondria. In Mfn2 null mouse embryonic fibroblasts (MEFs) regions of contact between mitochondria and the endoplasmic reticulum (ER) are significantly reduced. These regions of contact are thought to form specialized subdomains of the ER, called mitochondrial associated membranes (MAM). Besides observing a fragmented ER network in Mfn2 knockout (KO) mouse embryonic (MEF) cells, de Brito and Scorrano presented several lines of evidence which suggest that the underlying pathogenic mechanism in CMT2A stems from disrupted ER-mitochondria. As this observation had not been replicated in the literature, we describe our attempts to replicate these finding in the last half of Chapter 3. The MAM represents a sub-domain of the ER in close association with the mitochondrial outer membrane. The movement of phosphatidylserine (PS) from the MAM domains of the ER to mitochondria and its subsequent decarboxylation to phosphatidylethanolamine (PE) by the enzyme PS decarboxylase (Pisd) has been well characterized and is known to depend on the existence of an outer mitochondrial membrane protein. As PE has curvature inducing and fusogenic biophysical characteristics, a deficiency in PE would be an attractive mechanism contributing to the morphological and fusion defects observed in Mfn2 null cell models. We hypothesized that loss of Mfn2 would lead to specific decreases in mitochondrial and cellular levels of PE. Chapter 4 describes experiments designed to test this hypothesis. We observed significantly lower levels of PE in Mfn2 null cells, yet observe similar changes in Mfn1 null cells. Likewise, other lipid species such as ether linked PE (ePE) are decreased. To investigate how CMT2A mutations in MFN2 influence cellular phospholipid profiles, we then profiled cellular phospholipids of CMT2A patients and control lymphoblasts. We hypothesized that mutations in MFN2 would result in decreased levels of PE. In Chapter 5, we report the results of a phospholipid screen which reveal changes in ePE in CMT2A patient lymphoblasts, without the drastic decreases in PE previously observed in Mfn2 null lines. In conclusion, our data indicates an important role for both mitofusins in the mitochondrial synthesis of PE. In the context of CMT2A mutations, ePE levels are specifically reduced. Future studies may reveal how deficiencies in ePE might have important functional consequences in the pathogenesis of CMT2A.

No description available.

Separation processes are fundamental to all chemical engineering industries. Solvent separation, either liquid-liquid extraction or extractive distillation, is a specialised segment of separation processes. Solvents can be used either to optimise conventional distillation processes or for azeotropic systems, which can not be separated by conventional means. This work focuses on the performance of monoethanolamine (MEA) as a solvent in extractive distillation. Furthermore, the methodology of solvent evaluation is also studied. The preliminary assessment of solvent selection requires the determination of selectivity factors. The selectivity factor is defined as follows: P• = y,." . y, where y" is the activity coefficient at infinite dilution of the solute in the solvent. Subscript 1 and 2 refer to solute 1 and 2. A large selectivity factor implies enhanced separation of component 1 from 2 due to the solvent. Activity coefficients at infinite dilution were determined experimentally (gas-liquid chromatography) and predicted theoretically (UNIFAC group contribution method) for twenty-four solutes at three temperatures. Solutes used were alkanes, alkenes, alkynes, cyclo-alkanes, aromatics, ketones and alcohols. Most of this experimental work comprises data for systems which have not been measured before. Predicted and experimental values for y' were compared. For systems such as these (with polar solvents and non-polar solutes), UNIFAC results are not accurate and experimentation is vital. The experimental selectivity factors indicated tihat MEA could be an excellent solvent for hydrocarbon separation. Three binary azeotropic systems were chosen for further experimentation with MEA n-hexane (1) - benzene (2): fJ,~ = 31. Compared to other industrial solvents this is one of the largest values and MEA could serve as an excellent solvent. cyclohexane (1) - ethanol (2): fJ,~ = 148. This high value indicates an excellent solvent for this system. Acetone (1) - methanol (2): fJ,~ = 7.7. Further work involved vapour-liquid equilibrium experimentation at sub-atmospheric pressures in a dynamic recirculating stil l. The binary components with a certain amount of MEA were added to the still. The vapour and liquid mole fractions for the binary azeotropic components were measured and plotted on a solvent-free basis. The results are summarised below: n-hexane - benzene: Amount MEA added to still feed: 2%. MEA improved separability slightly. Further addition of MEA resulted in two liquid phases forming. cyclohexane - ethanol: Amount MEA added to still feed: 5% and 10%. Two liquid phases were formed for cyclohexane rich mixtures. Addition of MEA improved separability but did not remove the azeotrope. acetone - methanol: Amount MEA added to still feed : 5%, 10% and 20%. The ternary mixture remained homogenous and separability improved with addition of MEA. The binary azeotrope was eliminated. Due to the hetrogenous nature of the cyclohexane - ethanol system liquid-liquid equilibrium experimentation was performed to complete the analysis. Viable separation processes are possible for (a) cyclohexane - ethanol mixtures and for (b) acetone - methanol mixtures using MEA as the solvent. Comparison of various solvents used for the separation of acetone from methanol was possible by constructing equivolatility curves for the ternary systems. Results showed that MEA may possibly be the best solvent for this extractive distillation process. This study provides the following results and conclusions: • New thermodynamic data, important for the understanding of MEA in the field of solvent separations, was obtained. • Results show that the UNIFAC contribution method cannot be used to accurately predict polar solvent - non-polar solute y«> values. Experimentation is essential. • Selectivity factors indicate that MEA could be an excellent solvent for hydrocarbon separation. • The separation of the azeotropic cyclohexane - ethanol mixture is possible with a combination of extractive distillation and liquid-liquid extraction or simply liquid-liquid extraction using MEA as the solvent. • The separation of the azeotropic acetone methanol mixture is possible with extractive distillation using MEA as the solvent. The solvent MEA is possibly the best solvent for this separation. / Thesis (M.Sc.Eng.)-Univeristy of Natal, Durban, 2000.

The present research involves two projects: a surface science study of the room-temperature adsorption and thermal evolution of allylamine and ethanolamine on Si(100)2×1, studied by using temperature-dependent X-ray photoelectron spectroscopy (XPS) and thermal desorption spectrometry (TDS), as well as Density Functional Theory (DFT) calculations; and a materials science study on the shape control of copper nanoparticles (Cu NPs) deposited on H-terminated Si(100) substrate with an extended size regime of 5-400 nm, by using a simple, one-step electrochemical method. The Cu NPs of three primary shapes were characterized with scanning-electron microscopy (SEM), glancing-incidence X-ray diffraction (GIXRD) and XPS. In the first surface science study, the presence of broad N 1s XPS features at 398.9-399.1 eV, corresponding to N–Si bonds, indicates N–H dissociative adsorption for both allylamine and ethanolamine on Si(100)2×1. For allylamine, the presence of C 1s features at 284.6 eV and 286.2 eV, corresponding to C=C and C−N, respectively, and the absence of the Si−C feature expected at 283.5 eV show that the reactions involving the ethenyl group such as the [2+2] C=C cycloaddition or those producing the [N, C, C] tridentate adstructures do not occur at room temperature. For ethanolamine, the O 1s feature at 533.1 eV indicates the formation of Si−O bond and O−H dissociation, which confirms an [O, N] bidentate adstructure and excludes the N−H and O−H dissociation unidentate structures. These XPS data are consistent with the N−H unidentate, and N−H and O−H double dissociation [O, N] bidentate adstructures for allylamine and ethanolamine, respectively, as predicted by the DFT calculations. TDS and temperature-dependent XPS data further show the desorption of propene and ethylene at 580 K and of acetylene at 700 K for allylamine and the desorption of ethylene at 615 K for ethanolamine, while the lack of N- or O-containing desorbates suggests that the dissociated N and O species are likely bonded to multiple surface Si atoms or diffused into the bulk at elevated temperatures (as confirmed by the corresponding temperature-dependent XPS spectra). Unlike the multidentate allyl alcohol and allylamine adstructures that have been found to be not favored kinetically, the present [O, N] bidentate ethanolamine adstructure appears to be kinetically favored on Si(100)2×1. In the second materials science study, Cu NPs of three primary shapes have been deposited on H-terminated Si(100) by a simple, one-step electrochemical method. By precisely manipulating the electrolyte concentration [CuSO4.5H2O] below their respective critical values, cubic, cuboctahedral, and octahedral Cu NPs of ranges of average sizes and number densities can be easily obtained by varying the deposition time. Combined GIXRD and depth-profiling XPS studies show that these Cu NPs have a crystalline core-shell structure, with a face-centered cubic metallic Cu core and a simple cubic Cu2O shell with a CuO outerlayer. The shape control of Cu NPs can be understood in terms of a progressive growth model under different kinetic conditions as dictated by different [CuSO4.5H2O] concentration regimes. The two studies in the present work lay the foundation for future investigation of surface biofunctionalization of these fascinating Cu NPs with different shapes and therefore different surface chemistries as controlled by the relative amounts of the (100) and (111) facets, and their boundaries.

The present research involves two projects: a surface science study of the room-temperature adsorption and thermal evolution of allylamine and ethanolamine on Si(100)2×1, studied by using temperature-dependent X-ray photoelectron spectroscopy (XPS) and thermal desorption spectrometry (TDS), as well as Density Functional Theory (DFT) calculations; and a materials science study on the shape control of copper nanoparticles (Cu NPs) deposited on H-terminated Si(100) substrate with an extended size regime of 5-400 nm, by using a simple, one-step electrochemical method. The Cu NPs of three primary shapes were characterized with scanning-electron microscopy (SEM), glancing-incidence X-ray diffraction (GIXRD) and XPS. In the first surface science study, the presence of broad N 1s XPS features at 398.9-399.1 eV, corresponding to N–Si bonds, indicates N–H dissociative adsorption for both allylamine and ethanolamine on Si(100)2×1. For allylamine, the presence of C 1s features at 284.6 eV and 286.2 eV, corresponding to C=C and C−N, respectively, and the absence of the Si−C feature expected at 283.5 eV show that the reactions involving the ethenyl group such as the [2+2] C=C cycloaddition or those producing the [N, C, C] tridentate adstructures do not occur at room temperature. For ethanolamine, the O 1s feature at 533.1 eV indicates the formation of Si−O bond and O−H dissociation, which confirms an [O, N] bidentate adstructure and excludes the N−H and O−H dissociation unidentate structures. These XPS data are consistent with the N−H unidentate, and N−H and O−H double dissociation [O, N] bidentate adstructures for allylamine and ethanolamine, respectively, as predicted by the DFT calculations. TDS and temperature-dependent XPS data further show the desorption of propene and ethylene at 580 K and of acetylene at 700 K for allylamine and the desorption of ethylene at 615 K for ethanolamine, while the lack of N- or O-containing desorbates suggests that the dissociated N and O species are likely bonded to multiple surface Si atoms or diffused into the bulk at elevated temperatures (as confirmed by the corresponding temperature-dependent XPS spectra). Unlike the multidentate allyl alcohol and allylamine adstructures that have been found to be not favored kinetically, the present [O, N] bidentate ethanolamine adstructure appears to be kinetically favored on Si(100)2×1. In the second materials science study, Cu NPs of three primary shapes have been deposited on H-terminated Si(100) by a simple, one-step electrochemical method. By precisely manipulating the electrolyte concentration [CuSO4.5H2O] below their respective critical values, cubic, cuboctahedral, and octahedral Cu NPs of ranges of average sizes and number densities can be easily obtained by varying the deposition time. Combined GIXRD and depth-profiling XPS studies show that these Cu NPs have a crystalline core-shell structure, with a face-centered cubic metallic Cu core and a simple cubic Cu2O shell with a CuO outerlayer. The shape control of Cu NPs can be understood in terms of a progressive growth model under different kinetic conditions as dictated by different [CuSO4.5H2O] concentration regimes. The two studies in the present work lay the foundation for future investigation of surface biofunctionalization of these fascinating Cu NPs with different shapes and therefore different surface chemistries as controlled by the relative amounts of the (100) and (111) facets, and their boundaries.

Nous avons caractérisé cinq protéines bovines qui sont potentiellement impliquées dans la reproduction.Un travail de clonage a été initié qui permettra à terme de purifier les GPA2 et GPB5 recombinantes puis naturelles pour étudier leurs structures. GPA2 et GPB5 sont considérés comme les ancêtres moléculaires des sous-unités α et β des hormones glycoprotéiques. Nous avons montré la relative fragilité thermique de la structure quaternaire de la FSH bovine par rapport aux FSH ovine et humaine et nous avons étudié les propriétés enzymatiques de la PDI (Protein Disulfide Isomerase) en préalable à l’étude de l’activité PDI de GPA2/GPB5. Nous avons aussi purifié la phosphatidyl-ethanolamine-binding protein (PEBP) et l’ubiquitine testiculaires par chromatographie hydrophobe à très haute concentration de sulfate d’ammonium. A partir de la PEBP purifiée, on a produit des anticorps spécifiques chez le lapin qui nous ont permis d’être les premiers à développer un dosage ELISA fiable pour cette protéine. / We characterized five bovine proteins that are potentially involved in reproduction. We started with the cloning of gpa2 and gpb5 cDNAs in order to eventually purify recombinant and natural GPA2 and GPB5 to study their possible quaternary structure. GPA2 and GPB5 are the evolutionary ancestors of Glycoprotein hormones α and β subunits respectively. Meanwhile, we have shown the relative quaternary structure fragility of bovine FSH compared to human and sheep FSH. We also studied the effect of endocrine disruptors on PDI (Protein Disulfide Isomerase) before addressing GPA2/GPB5 PDI activity of GPA2/GPB5 once purified.We succeeded to purify the phosphatidyl-ethanolamine-binding protein (PEBP) and ubiquitin from bovine testis by hydrophobic interaction chromatography at very high ammonium sulfate concentration and we produced specific antibodies (anti-PEBP) in rabbits that allowed us to be the first to develop a reliable Elisa assay for this protein.

Foram preparados produtos da reação do ácido algínico com a monoetanolamina (MEA), dietanolamina (DEA) e trietanolamina (TEA) sob refluxo em meio de clorofórmio, os quais foram denominados MEA, DEA e TEA-produtos. Os compostos foram caracterizados por Análise Elementar e Espectroscopia Vibracional na Região do Infravermelho. As técnicas termoanalíticas Termogravimetria (TG), Termogravimetria Derivada (DTG), Análise Térmica Diferencial (DTA) e Calorimetria Exploratória Diferencial (DSC) foram utilizadas para avaliar o comportamento e estabilidade térmica, as etapas envolvidas e determinar os parâmetros cinéticos da decomposição térmica do Halg e dos produtos de reação. A 13C RMN foi utilizada para propor uma possível estrutura para o MEA-produto e também para estimar o grau de conversão de ácido a produto. A organização estrutural do Halg e de um de seus produtos de reação foi avaliada usando difração de raios X. A Microscopia Eletrônica de Varredura foi utilizada para estudar a morfologia do Halg e dos produtos de reação. O MEA-produto foi misturado aos fármacos Paracetamol, Tioconazol e Ramipril e essas misturas foram caracterizadas por Espectroscopia Vibracional na Região do Infravermelho e por Análise Térmica, com o objetivo de verificar a interação de fármacos com o material biopolimérico. / The products of the reaction between alginic acid and monoethanolamine (MEA), diethanolamine (DEA) and triethanolamine (TEA) were prepared in chlroform under reflux, named MEA, DEA e TEA-products. These compounds were characterized by Elemental Analysis and Spectroscopy in the Infrared region. The Thermal analytical techiniques Thermogravimetry (TG), Derivative Thermogravimetry (DTG), Differential Thermal Analysis (DTA) and Differential Scanning Calorimetry (DSC) were used to evaluate the thermal behavior and stability of the compounds as well as the steps and the kinetic parameters involved in the thermal decomposition of Halg and the MEA, DEA e TEA-products.<br /> The 13C NMR was used to propose a possible structure for MEA-product and to estimate the degree of the conversion. The structure order of Halg and MEA-product was evaluated by X-ray diffraction. The scanning electron microscopy was used to investigate the morphology of Halg and the reaction products. The MEA-product was mixed with the drugs Paracetamol, Tioconazole and Ramipril. The mixtures were characterized by Infrared spectroscopy and Thermal Analysis, in order to verify the interaction of drugs with the biopolymeric material