This project was carried out in order to complete the requirements of the Master of Engineering Management degree at the University of Canterbury. The project objective was to examine the reasons for Attainment to Plan (ATP) misses at the Meadow Fresh Christchurch plant, specifically the Fresh Beverages division. ATP is a measure of how closely the production team follows the daily packing plan, and is a site Key Performance Indicator (KPI). This report describes the action plans that were developed to decrease the number of misses to the target value, an analysis of the success of these plans, and recommendations which were made around the purchase of plant equipment in order to further improve the ATP results.

Approved for public release; distribution is unlimited / The aim of this study was to fabricate tin/carbon (Sn/C) battery anodes using a novel approach, reduction expansion synthesis (RES), and test their performance as electrodes in lithium or sodium batteries. A second preparation route, the Aerosol-Through-Plasma (ATP) method, was also employed for comparison. The specimens generated were characterized, before and after cycling, using techniques such as X-ray diffraction, scanning, and transmission electron microscopy. The RES technique was successful in creating remarkably small (ca. <5 nm) nano-scale particles of tin dispersed on the carbon support. The use of the electrodes as part of coin cell batteries resulted in capacitance values of 320 mAh/g and 110 mAh/g for lithium-ion and sodium-ion batteries, respectively. Nano-sized Sn particles were found before and after cycling. It is believed that bonds between metal atoms and dangling carbon produced via the reduction of the carbon surface during RES were responsible for the materials' ability to withstand stresses during lithiation, avoid volumetric expansion, and prevent disintegration after hundreds of cycles. When tin loading in Sn/C was increased from 10% to 20%, an increase of capacitance from 280 mAh/g to 320mAh/g was observed; thus, increased tin loading is recommended for future studies. Tin/carbon produced using ATP presented morphology consistent with stable electrodes, although battery testing was not completed because of the difficulty of producing the material in sufficient quantity. / Military Expert 5, Republic of Singapore Navy

The glyoxalase system mediates the conversion of methylglyoxal, a toxic ketoaldehyde, to D-lactic acid. The system is composed of two enzymes, glyoxalase I (Glo-I) and glyoxalase II (Glo-II), and exhibits an absolute requirement for a catalytic quantity of glutathione (GSH). Glo-I catalyzes the isomerization of a hemithioacetal, formed non-enzymatically from methylglyoxal and GSH, to the corresponding a -D-hydroxyacid thioester, s-D-lactoylglutathione (SLG). Glo-II catalyzes the irreversible breakdown of SLG to D-lactate and GSH. We have observed that ATP or GTP significantly inhibits the Glo-II activity of tissue homogenates from various sources. We have developed a rapid, one step chromatography procedure to purify Glo-II such that the purified enzyme remains "sensitive" to inhibition by ATP or GTP (Glo-II-s). Studies indicate that inhibition of Glo-II-s by nucleotides is restricted to ATP, GTP, ADP, and GDP, with ATP appearing most effective. Kinetics studies have shown that ATP acts as a partial non-competitive inhibitor of Glo-II-s activity, and further suggest that two kinetically distinguishable forms of the enzyme exist. The sensitivity of pure Glo-II-s to nucleotide inhibition is slowly lost on storage even at -80° C. This loss is accelerated at higher temperatures or in the presence of ATP. Kinetics studies on the resultant "insensitive" enzyme (Glo-II-i) show that a significant reduction of the affinity of the enzyme for the substrate, SLG, occurs and further suggest that only one form of the enzyme is kinetically distinguishable after "de-sensitization". Tryptophan fluorescence studies of the two enzyme preparations suggest that a subtle conformational change in the enzyme has occurred during de-sensitization. We have also observed that Glo-II-i is "resensitized" to nucleotide inhibition after incubation in the presence of a reagent that reduces disulfide bonds. The resensitized enzyme exhibits an increased KM value similar to that of the original Glo-II-s. Kinetics studies show that ATP or GTP again act as partial non-competitive inhibitors of the resensitized enzyme and suggest that only one form of the enzyme is present. The physiological significance of the two enzyme forms is discussed.

Thesis advisor: Steven D. Bruner / Peptidic natural products are produced by diverse organisms ranging from bacteria to humans. These secondary metabolites can be assembled by the ribosome or by nonribosomal peptide synthetase (NRPS) enzymatic assembly lines. The architectural complexity and biological activity of such compounds make them interesting targets for study. Frequently, nonribosomal peptides contain nonproteinogenic amino acid building blocks, and the biosynthetic routes to both ribosomal and nonribosomal peptides often utilize tailoring enzymes. These specialized enzymes catalyze mechanistically challenging reactions and provide peptidic natural products with structural motifs not normally found in proteins. Structural studies of these tailoring enzymes will further our understanding of biosynthetic pathways, and engineered tailoring enzymes could find use as promiscuous catalysts for the chemoenzymatic synthesis of natural product analogs. The L-tyrosine 2,3-aminomutase <italic>Sg</italic>TAM catalyzes the formation of &beta;-tyrosine from L-tyrosine, and is used in the biosynthetic pathway to the enediyne antitumor antibiotic C-1027. This enzyme contains the rare electrophilic prosthetic group 4-methylideneimidazole-5-one (MIO) and is homologous to the histidine ammonia lyase family of enzymes. While lyases form &alpha;,&beta;-unsaturated carboxylates as products, <italic>Sg</italic>TAM catalyzes additional chemical steps that result in an overall 2,3-amino shift. The precise mechanistic role of MIO in the ammonia lyase and aminomutase families of enzymes was actively debated for over 50 years. Here, we report the first x-ray crystal structure of an MIO-dependent aminomutase and detail the synthesis and characterization of mechanistic probes for this enzyme. Furthermore, we report several structures of <italic>Sg</italic>TAM bound to substrate analogs. These co-crystal structures reveal how <italic>Sg</italic>TAM achieves substrate recognition and suggest a specific role for MIO in catalysis. The results of our studies allow for the rational engineering of MIO-based aminomutases and ammonia lyases with altered physical properties and substrate specificities. Additionally, we are currently studying several enzymes involved in the biosynthesis of the tricyclic depsipeptide microviridin J. This ribosomal peptide natural product contains two lactones and one lactam, which are introduced by two enzymes belonging to the ATP-grasp ligase superfamily of proteins. Here, we detail the overexpression of these enzymes, MdnJ-B and MdnJ-C, in <italic>E. coli</italic> and report the optimization of conditions which lead to the crystallization of both enzymes. The structural characterization of MdnJ-B and MdnJ-C will lead to a greater understanding of macrocycle formation in ribosomal peptide biosynthesis, and engineered variants of these enzymes may find use as macrocylcization catalysts. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

by Wen-Liang Zhou. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (p. 125-144). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

The Escherichia coli vitamin B12 transporter BtuCD-F is a type II importer belonging to the ABC transporter superfamily. Available data suggest both exporters and type I importers in the ABC superfamily employ similar transport mechanism in which the transmembrane (TMDs) are open to cytoplasm in the resting state, and ATP binding induces a major conformational change resulting in opening of the TMDs instead to the periplasm. However, the crystal structures of BtuCD from E. coli and recent EPR spectroscopy studies indicate that this type II importer employs a substantially different mechanism in which the TMDs are open to the periplasm in the resting state and to the cytoplasm after ATP binding. We have developed robust methods to study the conformation and transport mechanism of BtuCD-F reconstituted into lipid bilayers using single molecule fluorescence resonance energy transfer (smFRET) measurements. Fluorescent probes have been introduced at a variety of diagnostic sites, enabling smFRET to be used to measure distance changes in different conformational states as well as to observe the transitions between these states in real time. These data suggest that thermal fluctuations enable the transporter to explore different functional conformational states in the absence of ATP or other ligands. They also suggest that the ATP-bound state is indeed open to the cytoplasm and ATP binding/hydrolysis increases the rate of transition between open and closed states. Efforts are currently underway to observe the transport of vitamin B12 through a single BtuCD-F oligomer in real-time.

Philosophiae Doctor - PhD / Background: Extended-spectrum beta-lactamase-producing (ESBL) and carbapenemaseproducing Gram-negative bacilli showing resistance to cephalosporins and carbapenems respectively, have been reported from several countries globally and recently among Libyan combatants who have been transferred to European countries for advanced medical care. However, there is a lack of data about their presence in Misrata and in Libya in general. This is the first documented study aimed at investigating the prevalence and resistance mechanisms of ESBL and carbapenemase-producing K. pneumoniae isolates from Misrata. Materials and Methods: Two hundred Gram-negative bacillus isolates were collected and identified from hospitals and pathology laboratories in Misrata. Following antimicrobial susceptibility screening, those showing resistance to cephalosporin and carbapenem were tested for ESBL activity using the Modified double disc synergy test, Sensititer ESBL confirmatory MIC plates and MAST AmpC detection sets D52C and D68C. Carbapenemase activity was detected using RAPIDEC CARBA NP test, Modified Hodge test (MHT), carbapenem inactivation methods (CIM), carbapenem combined test (CCT), and by MAST carba puls set. ESBL and carbapenemases genes were detected using multiplex PCR. Results: K. pneumoniae was the predominant species (85/200) of the 14 species identified, with 56 (65.8%) showing carbapenem resistance, 16 (18.8%) were cephalosporin-resistant carbapenem-susceptible and 13 (15.2%) were susceptible to all antibiotics except ampicillin. OXA-48 was the only carbapenemase detected, with SHV, TEM and CTX-M group 1 found in almost all carbapenem and cephalosporin resistant K. pneumoniae. Rep-PCR analysis revealed multiple clones and some K. pneumoniae strains were genetically related or indistinguishable despite differences in ESBL genes or carbapenemase activity. Conclusion: The findings of this study show that carbapenemase- and ESBL-producing K. pneumoniae are prevalent in Misrata and emphasize the urgent need for optimized infection control and antibiotic stewardship programmes in the Libyan hospitals to prevent further spread of these organisms.

Nitrogenase is the enzyme that catalyzes the reduction of nitrogen to ammonia in a reaction requiring MgATP hydrolysis. Two component proteins of nitrogenase are the iron protein (Fe protein) and the molybdenum-iron protein (MoFe protein). Nitrogenase contains two nucleotide binding sites. During catalysis, the Fe protein binds two MgATP first. The confonnational changes induced upon MgA TP binding allow the Fe protein to associate with the MoFe protein. After the formation of the Fe protein-MoFe protein complex, a single electron is transferred from the Fe protein to the MoFe protein, an event that is coupled to MgATP hydrolysis in the Fe protein. The wild-type Fe protein and all the altered Fe proteins studied so far are homodimeric. In order to assess the contribution of each nucleotide binding site in the Fe protein to the events occurring during nitrogenase catalysis, a heterodimeric Fe protein was constructed that has Asp 39 substituted by Asn in one subunit and the other subunit the same as the wild-type Fe protein. Characterization of this heterodimeric Fe protein showed that alterations in the properties of the [4Fe-4S] cluster that occur upon nucleotide binding to the Fe protein are due to the additive effect of each nucleotide binding to the Fe protein. The rates of MgATP hydrolysis and MgATP-dependent primary electron transfer of this heterodimeric Fe protein are intermediate between those of the homodimeric wild-type Fe protein and D39N Fe protein. These observations suggested that each ATP binding site contributes to the rate acceleration of primary electron transfer. After electron transfer, this heterodimeric Fe protein forms a tight complex with the MoFe protein, demonstrating that alteration in one subunit is enough for the formation of a tight nitrogenase complex. When this heterodimeric Fe protein was combined with the MoFe protein, no substrate reduction was detected. Therefore, two functional subunits of the Fe protein are necessary for reduction of substrates. The mechanism of ATP hydrolysis in the Fe protein was also investigated. Using site-directed mutagenesis, the role of lysine 10 of the Azotobacter vinelandii nitrogenase Fe protein in MgATP hydrolysis was examined. Changing Lys 10 of the protein to Arg resulted in an Fe protein that hydrolyzed MgATP at a rate 3% that of the wild-type Fe protein. The affinities of the K10R Fe protein for nucleotides and the changes in the properties of the [4Fe-4S] cluster of the K10R Fe protein upon nucleotide binding were compared with those of the wild-type Fe protein. These results indicated that in the absence of the MoFe protein, the interactions of the Kl OR Fe protein with nucleotides are similar to the wild-type Fe protein. After the Fe protein-MoFe protein complex formation, the dramatic decrease in the rate of MgATP hydrolysis of the K10R Fe protein indicated a role of Lys 10 in ATP hydrolysis. This conclusion is consistent with the X-ray crystal structure of the nitrogenase complex stabilized by the AlF4-•ADP, where Lys 10 is proposed to facilitate product formation in ATP hydrolysis.

The sphincter of Oddi (SO) is a neuromuscular structure located at the junction of the bile and pancreatic ducts with the duodenum. The primary functions of the SO are to regulate the delivery of bile and pancreatic juice into the duodenum, and to prevent reflux of duodenal contents into the biliary and pancreatic systems. Neural, hormonal or functional disturbances of biliary motility can lead to painful and sometimes life threatening clinical conditions, such as SO dysfunction and acute pancreatitis. Clearly understanding the regulation of biliary and duodenal motility patterns is necessary and may provide useful pharmacological sites for drug development to aid in the treatment of these diseases. Spontaneous activity of the SO is regulated by complex interactions between the enteric nervous system, hormones, possibly interstitial cells of Cajal and other bioactive agents, together with modulation via neural reflexes between the duodenum, common bile duct/gallbladder, and stomach. Purines are one group of neurotransmitters/regulatory agents that have been shown to effect gastrointestinal motility, however their functions in the regulation of SO motility have not been elucidated. The studies described in this thesis used in vitro organ bath techniques and in vivo preparations to determine the effects of exogenous purines on possum SO and duodenal motility. The possum SO has been extensively characterized and is an excellent model for motility studies. In vitro, exogenous adenosine was found to decrease spontaneous activity in both the SO and duodenum. In contrast exogenous ATP induced both excitatory and inhibitory responses in the SO and duodenum. Interestingly, the adenosine and ATP-induced effects were predominantly exhibited by the proximal portion of the SO (proximal-SO), with no or little effect observed in the distal portion of the SO (distal-SO). These data support the hypothesis that the SO is comprised of different functional components that can act differently in response to certain stimuli, and highlights the importance of studying each of the SO components. Agonists and antagonists, together with immunohistochemical studies, were used in an attempt to identify the P1 and P2 receptor sub-types responsible for mediating the adenosine- and ATP-induced responses. In the duodenum the adenosine-induced decrease in spontaneous activity was likely to be mediated by A2A and A3 receptors, but the receptors mediating the proximal-SO response could not be identified. In the duodenum ATP induced a complex non-neural response consisting of a P2X1, and P2Y2 and/or P2Y4 mediated immediate inhibition. This was followed by a return to baseline activity or small excitation. The response concluded with a late inhibitory response, likely to be mediated by P2Y1 receptors, but the effects of other P2Y receptors could not be excluded. In contrast, ATP application to the proximal-SO evoked a partially neurally mediated early excitation, likely via P2X receptors, followed by an inhibition of activity, likely via activation of non-neural P2Y2 and/or P2Y4 receptors. In vivo studies with exogenous application of adenosine and ATP to the SO activated neural pathways to produce increased motor activity. Characterisation of these neural pathways found ATP and/or adenosine to activate excitatory cholinergic motor neurons. ATP also activated an inhibitory nicotinic/nitrergic pathway. This is the first comprehensive investigation of the possible involvement of purines in the regulation of SO motility. These studies demonstrate that exogenous purines influence SO and duodenal motility, inducing complex neural and non-neural responses, acting via multiple P1 and P2 receptors. It now remains to be determined if endogenously released purines induce similar responses, together with elucidation and location of the receptor sub-types involved.

ABCA1 is a key plasma membrane protein required for the efflux of cellular cholesterol to extracellular acceptors, particularly to apoA-I. This process is essential to maintain cholesterol homeostasis in the body. The detailed molecular mechanisms, however, are still insufficiently understood. Also, the molecular identity of ABCA1, i.e. channel, pump or flippase, remains unknown. In this study we analyzed the extracellular ATP levels in the medium of ABCA1-expressing BHK cells and RAW macrophages and compared them to the medium of relevant non-expressing cells. We found that the extracellular ATP concentrations are significantly elevated when cells express ABCA1. Importantly, a dysfunctional ABCA1 mutant (A937V), when expressed similarly as WT-ABCA1, is unable to raise extracellular ATP concentration. This suggests a causal relationship between functional ABCA1 and elevated extracellular ATP. To explore the physiological role of elevated extracellular ATP, we analyzed ABCA1-mediated cholesterol efflux under the conditions where extracellular ATP levels were modulated. We found that increasing extracellular ATP within the physiological range, i.e. < μM, promotes cholesterol efflux to apoA-I. On the other hand, removing extracellular ATP, either by adding apyrase to the medium or by expressing a plasma membrane bound ecto-nucleotidase CD39, abolishes cholesterol efflux to apoA-I. Based on these results we conclude that, through direct or indirect mechanisms, ABCA1 functions to raise ATP levels in the medium. This elevated extracellular ATP is required for ABCA1-mediated cholesterol efflux to apoA-I.