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No description available.

1. The molecular weights of the G6PD subunits from the three yeast sources, bovine adrenals and rat liver were estimated by SDS-PAGE and found to be in the region of 55-59 K. An estimate of 46 K was similarly made for the G6PD subunit from the prokaryote, L. mesenteroides. 2. Two close bands or sometimes one diffuse band appeared on the polyacrylamide slab gel following electrophoresis and Coomassie blue staining for rat liver G6PD. These corresponded to the molecular weight region of about 57-59 K. So far, the evidence from peptide sequences does not reveal more than one subunit sequence. 3. G6PD from bakers' yeast was inactivated with sodium [1-. C] acetylsalicylic acid, following whichit was shown that 1.1 mole of 14 C moiety had been incorporated per enzyme subunit and that a lysine residue, essential to enzyme activity, had been modified. 4. Inactivation of bakers' yeast G6PD in the presence of high concentrations of substrate or coenzyme indicated that the acetylsalicylic acid was binding to the enzyme at a site which was directly or indirectly involved in substrate binding. The observation that high concentrations of coenzyme in the incubation mixture did not offer protection against acetylsalicylic acid inhibition was supplemented by TRNOE studies which showed no significant change in the conformation of coenzyme bound to bakers' yeast G6PD inactivated with acetylsalicylic acid compared with the active enzyme. 5. Acetylsalicylic acid did not inactivate rat liver G6PD to the same extent as the bakers' yeast enzyme. Inactivation of rat liver 14 G6PD with C-HIAB was relatively slow and resulted in the partial labelling of many cysteine residues. 6. Sequence studies on rat liver G6PD resulted in the isolation of a peptide which was homologous in sequence to the bakers' yeast G6PD tryptic peptide containing the reactive lysine residue, although no evidence for special reactivity of the corresponding lysine residue in the rat liver enzyme was found. 7. A high degree of sequence homology was noted between rat liver G6PD and human erythrocyte G6PD. It was also established that the human erythrocyte G6PD sequence published by Beutler (1983) was incorrect due to the misalignment of tryptic peptides.

This thesis outlines work carried out in the last three years concerning the development of novel, atom-economic, catalytic syntheses of amide bonds, determination of the ranges of these reactions through substrate screenings and investigations into the mechanisms by which the reactions are operating. In Chapter 1, an introduction to amide bonds is given, after which a discussion on the synthesis of amide bonds is presented, covering direct coupling methods, enzymatic methods and both non-metal catalysts and metal catalysts used for amide bond synthesis. In the Results and Discussion section, an iron catalysed coupling of nitriles and amines is presented in Chapter 2. In Chapter 3, firstly the development of an indium and zinc catalysed rearrangement of aldoximes into primary amides is discussed, followed by development of a novel, nickel catalysed coupling of aldehydes and amines. Detailed mechanistic studies using 18O labelled substrates are then presented for both of these reactions. Chapter 4 details work on a novel, hydroxylamine hydrochloride catalysed transamidation of primary amides with amines, including 1H NMR studies to attempt to elucidate the mechanism of the reaction. Finally, in Chapter 5, work investigating the direct coupling of unactivated carboxylic acids and amines is discussed, followed by an investigation into suitable catalysts to improve the efficiency of the reaction and a comparison of the rates in the catalysed and uncatalysed reactions for a wide range of substrates. In the Results and Discussion section, an iron catalysed coupling of nitriles and amines is presented in Chapter 2. In Chapter 3, firstly the development of an indium and zinc catalysed rearrangement of aldoximes into primary amides is discussed, followed by development of a novel, nickel catalysed coupling of aldehydes and amines. Detailed mechanistic studies using 18O labelled substrates are then presented for both of these reactions. Chapter 4 details work on a novel, hydroxylamine hydrochloride catalysed transamidation of primary amides with amines, including 1H NMR studies to attempt to elucidate the mechanism of the reaction. Finally, in Chapter 5, work investigating the direct coupling of unactivated carboxylic acids and amines is discussed, followed by an investigation into suitable catalysts to improve the efficiency of the reaction and a comparison of the rates in the catalysed and uncatalysed reactions for a wide range of substrates.

The Integrator complex (INT) is a metazoan-specific group of proteins associated with RNA polymerase II (Pol II) that has important functions in the 3'-end processing of noncodng RNAs, including uridine-rich small nuclear RNA (UsnRNA) and enhancer RNA (eRNA). Recently, INT has also been reported to be involved in Pol II transcriptional regulation of protein-encoding genes. INT contains at least 14 subunits, but the function of each subunit is difficult to predicted, because most subunits lack identifiable domains and display little similarity with other proteins. The endonuclease activity of INT is carried out by its subunit 11 (IntS11), which belongs to the metallo--lactamase superfamily and is a paralog of CPSF-73, the endonuclease for pre-mRNA 3'-end processing. IntS11 forms a stable complex with INT subunit 9 (IntS9) through their C-terminal domains (CTDs). This dissertation describes the crystal structure of the IntS9-IntS11 CTD complex at 2.1-Å resolution and summaries the structure-based biochemical and functional studies. The complex is composed of a continuous nine-stranded -sheet with four strands from IntS9 CTD and five from IntS11 CTD. Highly conserved residues are located in the interface between the two CTDs. The structural observations on the complex are confirmed by yeast two-hybrid assays and coimmunoprecipitation experiments. Functional studies demonstrate that the Int9-IntS11 interaction is crucial for proper INT function in snRNA 3'-end processing. The dissertation also presents the structural studies of a newly found mammalian mRNA deNADding enzyme, Nudt12. We determined the crystal structure of mouse Nudt12 in complex with the deNADding product AMP and three Mg2+ ions at 1.6-Å resolution. The structure provides exquisite insights into the molecular basis of the deNADding activity within the NAD pyrophosphate. Previous studies have reported that NAD-capped mRNAs in mammalian cells are hydrolyzed by the DXO deNADding enzyme. Together with biochemical and functional studies, we demonstrate that Nudt12 is a second mammalian deNADding enzyme structurally and mechanistically distinct from DXO and targets different RNAs.

Chronic Myeloid Leukaemia (CML) is a clonal haemopoietic stem cell (HSC) disorder characterised by the presence of a disease-specific gene, BCR-ABL, which leads to the production of a bcr-abl mRNA transcript. CML is an ideal candidate for gene therapy using ribozymes (Rz), catalytic RNA molecules that cleave and inactivate target RNA in a sequence specific manner. Limited data is available on the activity of ribozymes in human CML cells. In this study, hammerhead ribozyme sequences directed against the b3a2 bcr-abl mRNA sequence (Rz6-Rz10) were cloned into several retroviral vectors. Initial experiments using MSCVHSA based retroviral constructs failed to express the sequences in cell lines. Rz cDNA fragments were then cloned into an LNL6 based retroviral vector (LGL1) encoding a GFP reporter gene and stable LGLRz constructs produced. Using cell sorting, high-titre PA317 producer cell line clones were isolated. Transcriptional silencing of the LGLRz6 producer cell line occurred with prolonged culture, with partial reversal on treatment with the demethylating agent 5' azacytidine. To assess the activity of these constructs in human cells, CD34+ HSC were isolated from newly diagnosed b3a2 Ph+ CML patients. Cells were transduced with either control LGL vector or the LGLRz6 construct. Transduced human cells were sorted based on GFP expression and placed into long-term HSC culture (LTC-IC assays). Using a common cDNA, RT-PCR was performed to detect the expression of both the transgene and bcr-abl in individual colonies derived from the LTCIC assay at various time points, allowing assessment of the effect of transgene expression on bcr-abl expression. LGLRz transgene expression was detectable for up to 6 weeks in culture. Colony RT-PCR results from 3 patients showed that expression of the LGLRz6 construct was associated with decreased bcr-abl expression. It also appeared that the reduced bcr-abl expression decreased the proliferation of Ph+ cells leading to their loss from culture. In summary, these results appear to show an effect of a retroviral vector containing a bcr-abl Rz sequence on human CML HSC. Targeting of bcr-abl remains a valid therapeutic goal in the Imatinib era, particularly if problems related to effective ribozyme delivery and targeting can be overcome.

Stringent legislation on control of vehicle exhaust emissions has led to consideration of alternative means of reducing emissions, with hydrogen fuel cell powered vehicles being accepted as one favoured possibility. However, the difficulties of storing and distributing hydrogen as a fuel are such that the conversion of more readily available fuels to hydrogen on board the vehicle may be required. The production of hydrogen by the partial oxidation of isooctane over Rh/Al2O3, Rh/CeO2-?l2O3 and Rh/CeO2-ZrO2 catalysts has been investigated. Oxidation was initiated at temperatures between 200 ?220 oC. The yield of hydrogen was 100%. CeO2-ZrO2 was found to be the best support. The production of hydrogen by the autothermal reforming of artificial gasoline has been studied. Part of gasoline is oxidised to produce heat and steam to promote the steam reforming of unburnt gasoline to produce hydrogen. The use of platinum impregnated on ceria supports (active for oxidation) and a commercial nickel based catalyst (Ni-com), for steam reforming of gasoline have been explored. Initiation of oxidation of artificial gasoline over unreduced platinum based catalysts occurred at temperature as low as 150 oC, depending on the oxygen:carbon ratio and the liquid hydrocarbon used. Detailed kinetic studies of the steam reforming of isooctane and artificial gasoline (a mix of cyclohexane, isooctane and octane) over pre-reduced Ni-com catalysts showed that the reaction was 0.17 order in isooctane and 0.54 order in steam, whilst the reaction was 0.08 order in artificial gasoline and 0.23 order in steam. Mechanisms have been proposed to account for the dual site surface reaction with dissociative adsorption of isooctane or artificial gasoline and steam. Combined oxidation and steam reforming systems (autothermal reforming) using Pt/CeO2 as a front catalyst bed and Ni-com as the rear bed at the feed conditions of oxygen:carbon (O:C) ratio of ca.1.2 and steam:carbon (S:C) ratio of ca.2, produces ca. 3.5 moles of hydrogen per mole of gasoline fed. The system reaction temperature could be controlled by adjusting the O:C and S:C ratios in feed.

This research was aimed at investigating a counter-diffusive catalytic reactor for mitigation of methane and BTEX emissions from the natural gas dehydration process. A commercial radiant heater unit was used in the experiments and the effect of methane flow rate on its conversion was studied. Methane conversion decreased with increasing methane feed rate. It was found that the external diffusion of oxygen through the boundary layer was the limiting factor in the system. Complete methane conversion was achieved when the oxygen diffusion limitation was overcome by inducing convective air flux in the boundary layer in front of the catalyst pad. To simulate natural gas dehydration emissions, which contain excess amount of water, the effect of addition of liquid water and water vapor on methane combustion was also studied. Small volumes of liquid water did not affect the methane combustion, however, at 2 g/min liquid water, which is comparable to the amount of water produced during the reaction, combustion was inhibited. Added water vapor did not show any influence on combustion efficiency. The presence of pentane and toluene, representing the non-aromatic hydrocarbons and BTEX substances in the emissions, inhibited methane conversion due to the competition for oxygen since pentane and toluene are easier to oxidize compared to methane. Two-dimensional modeling of the radiant heater system was conducted using the COMSOL Multiphysics software package. Comparing the model data for methane conversion with experimental results revealed similar decreasing trend in conversion with increasing the methane flow rate; however, the model under-predicted the conversion. Increasing the mass transfer coefficient, resulted in improved methane conversion, confirming the dominance of mass diffusion limitation in the system. In fact, the real mass transfer coefficient was 1.5-2 times higher than the values originally used in the model. Changing the kinetic parameters did not significantly improve the conversion leading to the conclusion that the catalytic radiant heater system is not kinetically controlled. Developing the three-dimensional model of the system in Fluent revealed that the fuel distribution in the system is not a significant factor, in agreement with experimental observation. / Chemical Engineering

In this study, a two-bed electrically-heated regenerative catalytic oxidizer (RCO) was used to test the destruction characteristics in burning toluene-borne air streams. The RCO contained two 0.152 m¡Ñ0.14 m¡Ñ1.0 m (L ¡Ñ W ¡Ñ H) beds, both packed with gravel particles with an average diameter of around 0.0111 m and a height of up to 0.875 m with a void fraction of 0.42 in the packed section. In addition, in each column catalytic particles with an average diameter of around 0.008 m were packed over the gravel particles to a height of 0.125 m. Gas temperature rise and the gas pressure drop over the beds were also studied. Experimental results reveal that, with a valve shifting time (ts) of 1.5 min, superficial gas velocities (Ug) of 0.39 and 0.86 m/s (evaluated at an influent air temperature of around 30 oC) and preset maximum destruction temperatures (TS) of 300-400 oC, only around 25% of the influent toluene (Co = 200-400 ppm) was thermally destructed with no catalyst in both beds. With the cartalyst packings and operation conditions of Ug = 0.39 m/s and Co = 200-800 ppm, destruction efficacies of around 80.9¡Ó0.8, 94.6¡Ó1.8, and 98.1¡Ó0.2 % were observed, respectively, at TS of 250, 300, and 400 oC. At Ug = 0.86 m/s and Co = 200-800 ppm, destruction efficacies of around 69.7¡Ó3.1, 93.9¡Ó1.7, and 97.8¡Ó0.4 % were observed, respectively, at TS of 250, 300, and 400 oC. It is suggested that operation conditions with Ug = 0.39-0.86 m/s (equivalent to empty-bed-residence times of 0.29-0.64 s for the gas at 30oC through the catalyst beds) and TS = 300 oC are suitable for the destruction of around 98% of the influent air with 200-800 ppm toluene. Gas temperature rises of 21 and 26 oC, respectively, were found for Ug = 0.39 and 0.86 m/s with TS = 300 oC. The Ergun equation was found to suffice in the estimation of the pressure drop when the gas flowed over the packing beds.