The shikimate pathway is a biosynthetic pathway that is responsible for producing a variety of organic compounds that are necessary for life in plants and microorganisms. The pathway consists of seven enzyme catalysed reactions beginning with the condensation reaction between D-erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP) to give the seven-carbon sugar DAH7P. This thesis describes the design, synthesis and evaluation of a range of alternative non-natural four-carbon analogues of E4P (2- and 3-deoxyE4P, 3-methylE4P, phosphonate analogues of E4P) to probe the substrate specificity of different types of DAH7P synthases [such as Mycobacterium tuberculosis (a type II DAH7PS), Escherichia coli (a type Ialpha DAH7PS) and Pyrococcus furiosus (a type Ibeta DAH7PS)].

The enzyme 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyses the stereospecific aldol-like condensation between phosphoenolpyruvate (PEP) and the five-carbon sugar D-arabinose 5-phosphate (A5P). This is the first biosynthetic step in the formation of 3-deoxy-D-manno-octulosonate (KDO), an essential lipopolysaccharide component of all Gram-negative bacteria. KDO8PS is evolutionarily related to the shikimate pathway enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), which catalyses a similar condensation reaction between PEP and the four-carbon sugar D-erythrose 4-phosphate (E4P), in the first step of the shikimate pathway to aromatic compounds in plants and microorganisms. As well as being a one-carbon shorter substrate, E4P has the opposite C2-OH configuration to A5P. While there are both metal-dependent and metal-independent forms of KDO8PS, in contrast, all DAH7PS are metal-dependent enzymes. Little is understood about the key sequence features that distinguish KDO8PS and DAH7PS. These features, particularly those that contribute to A5P or E4P binding, are thought to be responsible for the differences in substrate specificity between the two enzymes. This thesis describes the functional and structural studies of KDO8PS mutants to examine the roles of these residues, using the metal-dependent KDO8PS from Acidithiobacillus ferrooxidans and the metal-independent KDO8PS from Neisseria meningitidis. In Chapter 2 an extensive KDO8PS and DAH7PS sequence analysis is presented. The results, which identify sequence conservation in both enzymes, are discussed in the context of the (β/α)8 TIM-barrel structure. Some of the differences in conservation between the two enzymes were highlighted as being obvious in having a role or contributing to the different substrate selection preferences of the two enzymes, such as an extended β7α7 loop in KDO8PS, and motif differences on the β2α2 and β4α4 loops. A similar analysis was also used to compare metal-dependent and metal-independent KDO8PSs, and it was found the two forms differ in the conservation of only three residues. Chapter 3 describes the characterisation of A. ferrooxidans KDO8PS (AfeKDO8PS) and investigates aspects of metal dependency in KDO8PS. The enzyme was found to be metal dependent, and like all other KDO8PS enzymes, to possess a tetrameric quaternary structure, and display tight substrate specificity. The β8α8 loop was found to have a critical role in binding and positioning the substrates, and AfeKDO8PS could not be engineered to be a metal-independent enzyme. The role of the KDO8PS-conserved KANRS motif, present on the β2α2 loop and one of the main contributors to the A5P binding site, is probed in Chapter 4. Individual residues of the motif were mutated to investigate function, and the motif was converted to the equivalent motif found in DAH7PS (KPRS). It was found that the Lys plays a critical role in enzymatic catalysis, and is likely intimately involved in the enzyme mechanism. The Asn residue of the motif in KDO8PS was found to be an important contributor to KDO8PS stereospecificity. The work described in Chapter 5 investigates the role of the β7α7 loop in KDO8PS. This long active-site loop, which exists in a shorter version in DAH7PS, was found not to be essential for catalysis in KDO8PS, but was necessary for efficient catalysis. The two conserved residues on the loop provide interactions to A5P, but the presence of the extended loop as a whole was found to be most important for catalytic efficiency. In Chapter 6 a conserved residue on the re face of PEP is investigated. In KDO8PS the residue is conserved as Asp, and in DAH7PS the same residue is conserved as a Glu. Mutational analysis found that in KDO8PS the Asp residue appears to be important for enzyme activity but unimportant for PEP binding. Mutating this Asp in KDO8PS to Glu was accommodated by KDO8PS, but it was found its introduction could potentially be optimised by coupling the change with mutation to other conserved differences. In KDO8PS, one of the interfaces between adjacent subunits in the tetrameric structure is partially composed of a conserved sequence motif, PAFLxR. In Chapter 7, the roles of the residues in this motif are explored. The Arg of the motif was found to be important for A5P binding. The equivalent (and also conserved) motif in DAH7PS is GARNxQ, and mutation of residues in the KDO8PS motif to the equivalent residues in DAH7PS was tolerated by KDO8PS, but negatively impacted upon the enzyme kinetic parameters. The sequence features investigated in the other chapters were combined with those to the subunit interface to create a DAH7PS-like protein. This extensively engineered protein lost all KDO8PS activity, but nor did it gain DAH7PS activity. Lastly, in Chapter 8 the results from all chapters are reviewed and ideas are discussed for advancing the research presented in this thesis.

Plants use several strategies to defend themselves against microbial pathogens. These include basal resistance, which is induced in response to pathogen encoded effector proteins, and resistance (R) protein-mediated resistance that is activated upon direct or indirect recognition of pathogen encoded avirulence protein(s). The activation of Rmediated signaling is often associated with generation of a signal, which, upon its translocation to the distal uninfected parts, confers broad-spectrum immunity against related or unrelated pathogens. This phenomenon known as systemic acquired resistance (SAR) is one of the well-established forms of induced defense response. However, the molecular mechanism underlying SAR remains largely unknown. Induction of plant defense is often associated with a fitness cost, likely because it involves reprogramming of the energy-providing metabolic pathways. Glycerol metabolism is one such pathway that feeds into primary metabolism, including lipid biosynthesis. In this study, I evaluated the role of glycerol-3-phosphate (G3P) in host-pathogen interaction. Inoculation with the hemibiotrophic fungal pathogen Colletotrichum higginsianum led to increased accumulation of G3P in wild-type plants. Mutants impaired in biosynthesis of G3P showed enhanced susceptibility, suggesting a correlation between G3P levels and basal defense. Conversely, increased biosynthesis of G3P correlated with enhanced resistance. The Arabidopsis genome encodes one copy of glycerol kinase (GK), which catalyzes phosphorylation of glycerol to G3P, and five copies of G3P dehydrogenase (G3Pdh), which catalyze reduction of dihydroxyacetone phosphate to G3P. Analysis of plants mutated in various G3Pdh's showed that plastidal lipid biosynthesis was only dependent on the GLY1 isoform but the pathogen induced G3P pool required the function of GLY1 and two other G3Pdh isoforms. Interestingly, compromised G3P biosynthesis in GK and G3Pdh mutants also compromised SAR, which was restored when G3P was provided exogenously. Detailed biochemical analysis showed that G3P was transported to distal tissues and that this process was dependent on a lipid transfer protein, DIR1. Together, these results show that G3P plays an important role in both basal- and induced-defense responses.

No description available.

Many of the physiological responses concomitant with exercise are understood. Similarly, many of the changes characterizing the aging process have been established. However, the combination of the two (ie. effects of aging on exercise or vice versa) presents a myriad of questions, of which many remain unanswered.The objective of this study was to establish the differences between previously untrained young and adult male Fischer 344 rats following an exhaustive swim for the following parameters: 1) muscle glycogen, an essential fuel substrate; 2) Glucose-6-phosphate dehydrogenase (G6PDH), a marker of inflammation and tissue damage; 3) citrate synthase (CS), an integral enzyme of the Kreb's cycle and a respiratory chain marker; 4) muscle protein; and 5) percent muscle dry weight.The rats were divided into two groups by age. Young (3 mo., n=16) and adult (12 mo., n=17) rats were randomly divided into sedentary (young sed (YSD) n=7 and adult sed (ASD) n=9) or exercised groups (young swimmers (YSW) n=8 and adult swimmers (ASW) n=8). Rats in the swimming groups were given a brief exposure to the water one week prior to their exhaustive swim to minimize the stress and confusion during the actual exercise bout. On the study days one randomly selected swimmer from each age group was swum to exhaustion and sacrificed via pneumothorax. One animal from each of the respective sedentary age groups was also randomly selected and sacrificed as above. The plantaris, rectus femoris, red vastus, soleus, triceps, and liver were surgically excised from each animal and frozen in liquid nitrogen for later analysis.While the younger animals had lower glycogen stores initially, following the exhaustive swim their reduction in muscle glycogen was approximately 150% that of the adult animals for any given muscle. Muscle glycogen levels in ASD and YSD rats were significantly higher than those of the YSW animals for all muscles with the exception of the YSD's soleus. However, the percent decrease in liver glycogen following the swim for the two age groups was almost identical (a reduction of 55.05% and 58.59% for the adult and young age groups, respectively).Although the adult animals were significantly heavier than the younger rats, this did not appear to cause a significant difference in their swim time to exhaustion. No significant differences were observed between the groups for muscle protein or G6PDH. Levels of CS were significantly higher in the YSD plantaris when compared to the ASW. Similarly, the ASD rectus femoris CS levels were significantly greater than those of the ASW. Although significant differences between groups in percent muscle dry weight existed for the plantaris, rectus femoris, and triceps such differences seemed to have little bearing on the two age group's swim to exhaustion times.On the basis of this study it was concluded that although starting with greater glycogen stores prior to exercise, adult animals use less of this substrate prior to exhaustion than do younger animals. While the mechanism for such a phenomenon was not discovered it is believed to be enzymatic in nature. Furthermore, the adult animals do not appear to exhibit significantly more tissue damage following an exhaustive swim than that seen in younger animals. / School of Physical Education

The aqueous solubility of an active pharmaceutical ingredient (API) is an important property that requires evaluation during early development and prior to formulation of the final product. With general, experimental, solubility testing of different APIs, the question always arises as to whether particle size had been determined beforehand or not. All available literature suggests that particle size, for pharmaceutical powders, does not significantly affect equilibrium solubility. The dissolution rate will differ according to different particle sizes, but the overall results should be identical after equilibrium is established. This study was therefore planned to investigate as to whether different particle size fractions of the same API, dissolving at different rates, would all reach solubility equilibrium within 24 hours. Also, APIs from different solubility classes were investigated, because poorly soluble substances would most likely require a longer period of time to equilibrate. The time period of 24 hours was selected, because many published solubility studies report using that interval and is it the standard for our research group also. Available APIs were selected to determine the influence (if any) of particle size on their equilibrium solubilities and the time required for attaining that status. For the purpose of this investigation, five APIs were selected from compounds at our disposal in-house, ranging from freely soluble to poorly soluble in the order: chloroquine phosphate > pyrazinamide > mefloquine hydrochloride > closantel sodium > roxithromycin. Solubility studies were successfully completed on four of the five APIs selected. For closantel sodium, pyrazinamide and roxithromycin it was demonstrated that the 24 hour test period was sufficient for the attainment of equilibrium solubility, regardless of the particle size fractions tested. Surprisingly, the only API in this study for which 24 hours was an insufficient test period was mefloquine HCl, which was not the least soluble compound tested. Further testing would be required to clarify this anomaly. What was evident from the outcomes of this investigation was that although the ubiquitous 24 hour solubility test may work well in many cases, its suitability should be reviewed on a case-by-case basis and not just for the most poorly soluble compounds. Researchers testing solubility at temperatures lower than 37°C should be especially cautious of using a standardised test period, because equilibrium solubility would take longer to achieve with less energy available to the system. / Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013

The aqueous solubility of an active pharmaceutical ingredient (API) is an important property that requires evaluation during early development and prior to formulation of the final product. With general, experimental, solubility testing of different APIs, the question always arises as to whether particle size had been determined beforehand or not. All available literature suggests that particle size, for pharmaceutical powders, does not significantly affect equilibrium solubility. The dissolution rate will differ according to different particle sizes, but the overall results should be identical after equilibrium is established. This study was therefore planned to investigate as to whether different particle size fractions of the same API, dissolving at different rates, would all reach solubility equilibrium within 24 hours. Also, APIs from different solubility classes were investigated, because poorly soluble substances would most likely require a longer period of time to equilibrate. The time period of 24 hours was selected, because many published solubility studies report using that interval and is it the standard for our research group also. Available APIs were selected to determine the influence (if any) of particle size on their equilibrium solubilities and the time required for attaining that status. For the purpose of this investigation, five APIs were selected from compounds at our disposal in-house, ranging from freely soluble to poorly soluble in the order: chloroquine phosphate > pyrazinamide > mefloquine hydrochloride > closantel sodium > roxithromycin. Solubility studies were successfully completed on four of the five APIs selected. For closantel sodium, pyrazinamide and roxithromycin it was demonstrated that the 24 hour test period was sufficient for the attainment of equilibrium solubility, regardless of the particle size fractions tested. Surprisingly, the only API in this study for which 24 hours was an insufficient test period was mefloquine HCl, which was not the least soluble compound tested. Further testing would be required to clarify this anomaly. What was evident from the outcomes of this investigation was that although the ubiquitous 24 hour solubility test may work well in many cases, its suitability should be reviewed on a case-by-case basis and not just for the most poorly soluble compounds. Researchers testing solubility at temperatures lower than 37°C should be especially cautious of using a standardised test period, because equilibrium solubility would take longer to achieve with less energy available to the system. / Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013

Le phosphogypse est un sous-produit de la synthèse de l'acide phosphorique (utilisé pour la fabrication d'engrais). Ce travail de thèse a consisté à étudier l'influence des impuretés majoritaires, à savoir les ions monohydrogénophosphates HPO₄^2- et fluorophosphates FPO₃^2- sur les propriétés des phosphogypses et sur la réactivité des phosphoplâtres, de manière à les valoriser dans l'industrie plâtrière. <br />Pour cela, nous avons cherché : <br /> * à déterminer le rôle de ces ions sur le diagramme de phases CaO-SO₃-H₂0 à température ambiante ; <br /> * à déterminer l'effet de la température (simulation de la cuisson de gypse en plâtre) sur ce diagramme de phases ; <br /> * à comprendre la différence de réactivité entre les différents échantillons après cuisson.<br />Les ions HPO₄^2- et FPO₃^2- peuvent se substituer aux ions sulfates et entraînent une cristallisation du gypse avec une morphologie en "roses des sables". Lorsque la totalité des ions sulfates est substituée par les ions HPO₄^2- (ou FPO₃^2-), de nouvelles phases apparaissent : l'ardéalite (Ca(SO₄)_1-x(HPO₄)_x.2H₂O avec 0.42< x < 0.54 puis la brushite : CaHPO₄.2H₂O (ou le fluorophosphate de calcium dihydraté : CaFPO₃.2H₂O). La déshydratation des phases pures (brushite et fluorophosphate de calcium dihydraté) et solutions solides a été étudiée. Nous avons retenu que lors de la calcination de la brushite à 150°C, il n'y a ni déshydratation totale, ni passage par une phase sous-hydratée mais obtention d'un mélange de deux phases : la brushite (24%) et la monétite (76%) ayant des propriétés différentes de la brushite et de la monétite seules. A cette même température, la déshydratation du fluorophosphate de calcium dihydraté provoque un dégagement d'acide fluorhydrique (gaz responsable de la corrosion prématurée des fours) et de l'apparition d'une phase de phosphate de calcium amorphe. La réactivité de nos échantillons après calcination (sous forme de plâtre) a pu être interprétée à partir des résultats obtenus par couplage de la conductimétrie avec la pH-métrie et ponctuellement des mesures d'extraction de solution et analyse du solide résiduel au cours de l'hydratation. Nous avons pu conclure que : <br />- les ions HPO₄²⁻ syncristallisées ou appartenant aux produits de déshydratation de la brushite (soit un mélange brushite et monétite) entraînent un retard à l'hydratation (ce retard est maximal pour une température de calcination de la brushite à 160°C). Ces retards sont accentués si l'on opère en milieu alcalin. (Ainsi, les traitements de neutralisation à la chaux des phosphogypses contenant des impuretés phosphatées sont néfastes pour lhydratation). <br />- les ions FPO₃²⁻ appartenant à la phase fluorophosphate de calcium augmentent la durée d'hydratation, mais syncristallisés, ils accélèrent les temps de prise s'ils sont issus d'une cuisson "flash", ou bien se transforment en ions HPO₄²⁻ syncristallisés (avec dégagement d'acide fluorhydrique) pour un mode de calcination plus lent et lon est alors ramené au cas précédent. Ces travaux de thèse ont donc permis de mettre en place des méthodes de caractérisation des phosphogypses, de comprendre certains phénomènes observés en usines telles que des fins de prise très longues, la corrosion rapide des fours, et d'établir un classement pour connaître leur aptitude à une valorisation dans l'industrie plâtrière.

Palyginus klampos modifikatorių savybes nustatyta, kad didėjant hidroksipropilkrakmolo fosfato koncentracijai brinkimo laikas reikšmingai ilgėja, todėl siekiant sumažinti laiko sąnaudas tikslingiau rinktis akrilato/stearato-20 itakonato kopolimerą, kuris yra skystame būvyje arba polietilenglikolio hidroksipropil hidroksietil etilceliuliozės polimerą, kuris lyginant su modifikuotu krakmolu išbrinksta reikšmingai greičiau. Sudarant emulsijų receptūras pastebėta, kad glicerolis klampos modifikatorių brinkimo laikui įtakos neturi, tačiau produktui įtakos turi brinkinimo temperatūra ir pagaminimo metodas: norint pagaminti didelės klampos produktą reiktų tirštiklį brinkinti 25±2oC temperatūroje ir homogenizuoti aparatu „Unguator“. Sudarant emulsinių pagrindų sudėtis, pastebėta, kad klampos modifikatorių pakanka 0,2-2%, išskyrus hidroksipropilkrakmolo fosfato, kurio, norit pasiekti 8,45 ±0,02 Pa*s klampą, reikėjo 6%. Centrifugavimo testo metu nustatyta, kad stresinėmis sąlygomis didžiausiu stabilumu pasižymi emulsijos, pagamintos naudojant klampą reguliuojančias medžiagas, kadangi tyrime nagrinėjami emulsiniai pagrindai testo metu neišsisluoksniavo ir išliko stabilūs 14 dienų. Mikroskopinio tyrimo metu nustatytas monodispersiškiausias pagrindas su 6% hidroksipropilkrakmolo fosfatu, skirtumas tarp maksimaliųjų ir minimaliųjų dalelių yra 1,1 μm. Analizuojant emulsinių pagrindų tekstūrą nustatyta, kad kiečiausias ir lipniausias pagrindas gautas naudojant, akrilato/stearato-20... [toliau žr. visą tekstą] / While comparing the properties of viscosity modifiers it was established that with an increase in concentration of hydroxypropyl starch phosphate the time of swelling extends on condition p<0,05, because of that to reduce the time its beter to choose acrylates/steareth-20 itaconate copolymer, which is in liquid state or polyethylene glycol hydroxy propyl hydroxyethyl ethyl cellulose polymer, which swells noticeably faster. It was observed that glycerol does not make an impact on the time thickeners swel, but the product is influenced by the temperature of swelling and the production method. To produce high-viscosity product viscosity modifiers should be soak in water at room temperature and homogenized device called "Unguator". It was observed that in order to reach 8.45 ± 0.02 Pa*s viscosity you would need 6 % of hydroxypropyl starch phosphate, while others viscosity regulators would make it with percentage 0,2-2%. Emulsion bases together with viscosity modifiers, which were used in the research, prevent droplet aggregation after formation and remained stable for 14 days and didn‘t split into layers during the centrifuge test (thereby increasing long-term stability). In microskopic test it was revealed that the smallest difference in the particle size of emulsions is the base 6% hydroxypropyl starch phosphate (1,1 μm). For the purposes of analyzing the texture is established that the hardest and the most viscous base was produced using an acrylates/steareth-20 itaconate... [to full text]

Increasing pressure from environmental, political and economic sources are driving the development of an electric vehicle powertrain. The advent of hybrid electric vehicles (HEVs), plug-in hybrid electric vehicles (PHEVs), and battery electric vehicles (BEVs) bring significant technological and design challenges. The success of electric vehicle powertrains depends heavily on the robustness and longevity of the on-board energy storage system or battery. Currently, lithium-ion batteries are the most suitable technology for use in electrified vehicles. The majority of literature and commercially available battery performance data assumes a working environment that is at room temperature. However, an electrified vehicle battery will need to perform under a wide range of temperatures, including the extreme cold and hot environments. Battery performance changes significantly with temperature, so the effects of extreme temperature operation must be understood and accounted for in electrified vehicle design. In order to meet the aggressive development schedules of the automotive industry, electrified powertrain models are often employed. The development of a temperature-dependent battery model with an accompanying vehicle model would greatly enable model based design and rapid prototyping efforts. This paper empirically determines the performance characteristics of an A123 lithium iron-phosphate battery, re-parameterizes the battery model of a vehicle powertrain model, and estimates the electric range of the modeled vehicle at various temperatures. The battery and vehicle models will allow future development of cold-weather operational strategies. As expected the vehicle range is found to be far lower with a cold battery back. This effect is seen to be much more pronounced in the aggressive US06 drive cycle where the all-electric range was found to be 44% lower at -20°C than at 25°C. Also it was found that there was minimal impact of temperature on range above 25°C