Thesis (M.B.A.)--North-West University, Potchefstroom Campus, 2009.

Carnocyclin A (CclA) is a remarkably stable, potent bacteriocin produced by Carnobacterium maltaromaticum UAL307. Elucidation of the amino acid and genetic sequences revealed that CclA is a circular bacteriocin. Preliminary structural studies (dynamic light scattering, NMR, circular dichroism, stereochemical analysis) indicated that CclA is monomeric and alpha-helical in aqueous conditions and composed of L-residues. The 3D structure of [13C,15N]CclA was solved by NMR, revealing a compact arrangement of four helices. To examine the structure of the precursor peptide (pCclA) several fusion proteins were constructed and overexpressed; however, pCclA could not be isolated. To investigate the requirements for cyclization, several internally hexahistidine-tagged (His6) pCclA mutants were constructed. Expression conditions are underway. PisI was heterologously expressed and confirmed to impart protection against piscicolin 126 (PisA). Labeled and unlabeled PisA and PisI were purified following overexpression as maltose-binding protein fusions (MalE-fusions) and Factor Xa cleavage. NMR studies indicated that PisI and PisA do not physically interact. The 3D structure of PisI was solved by NMR, confirming that the four-helix bundle is a conserved motif for the immunity proteins of type IIa bacteriocins. The putative receptor proteins for these bacteriocins were cloned and overexpressed as His6-fusion proteins. Experiments are underway to optimize the expression and purification of these membrane proteins. The peptidase domain of the ABC-transporter protein (CbnTP) for carnobacteriocin B2 (CbnB2) was overexpressed as a His6-fusion protein. Active protease could not be purified from inclusion bodies, but was obtained as soluble protein following low-temperature overexpression. The CbnB2 precursor pCbnB2 (and a truncated derivative pCbnB2-RP) was purified following overexpression as a MalE-fusion and Factor Xa cleavage. pCbnB2 was incubated with CbnTP and MALDI-TOF and activity testing confirmed that CbnTP cleaved the leader peptide from pCbnB2. Five CysSer CbnTP mutants were constructed. Crystallographic studies of CbnTP are underway. Six bacteriocins (nisin, gallidermin, lacticin 3147, CclA, PisA, enterocin 710C) were tested against Gram-negative bacteria (E. coli DH5, Pseudomonas aeruginosa ATCC 14207, Salmonella typhimurium ATCC 23564) in the absence and presence of EDTA. PisA and lacticin 3147 exhibited minimal activity, whereas the other bacteriocins killed at least one strain, in the presence of EDTA.

The present study aimed to examine factors that predict glycemic control in Hispanic youths. Secondary aims included developing and evaluating Spanish translations of three measures commonly used in research with youths with type 1 diabetes and examining factors associated with diabetes self-management in this population. Data was collected at three sites through interviews, questionnaires, and medical chart review. Participants included 117 Hispanic diabetic youths (10 to 17 years old) and their caregiver. 15% of the youths who participated were born outside of the continental U.S. and 57% were first generation born in the U.S. Translations of the Diabetes Self-Management Profile, Diabetes Social Support Questionnaire-Family version, and the Family Environment Scale?s Organization subscale had adequate reliability and showed evidence of concurrent validity. Primary analyses indicated that parent education, occupation, and household income are all significant predictors of glycemic control in Hispanic youth. In addition, global ratings of diabetes self-management on the DSMP predicted control in this population. Family factors did not contribute directly to glycemic control after controlling for sociodemographic and disease variables. However, older age, more acculturation, and language barriers were associated with poorer self-management. Additionally, family support and organization significantly contributed to self-management.

Modern problems in astrophysics tend to require large, complex computational frameworks to solve many aspects of the system simultaneusly. Calculation of the energy production through nuclear reactions is typically one of those aspects. The use of standard nuclear burning algorithms will take up the majority of the computational time with all but the smallest of networks. The explicit asymptotic method has shown promise in computing large networks faster than existing methods in various environments while retaining accuracy. The purpose of this thesis is to show that this method can be successfully used to solve complex systems using a network of realistic size in a reasonable amount of time, and to investigate some problems in the flame propagation for a Type Ia, which have never been investigated with a realistic network.

We hypothesized that the interaction between tumor necrosis factor alpha(TNF)/nuclear factor-kappaB (NFkB) via activation of IKK may amplify one anotherresulting in the evolution of vascular disease and insulin resistance associated withdiabetes. The interaction between TNFa and monocyte chemoattractant protein-1 (MCP-1) may contribute to the evolution of vascular inflammation and endothelial dysfunctionin coronary arterioles in type 2 diabetes. To test this hypothesis, endothelium-dependent(ACh) and –independent (SNP) vasodilation of isolated, pressurized coronary arterioles(40-100 μm) from mLeprdb (heterozygote, normal), Leprdb (homozygote, diabetic) andLeprdb mice null for TNF (dbTNF-/dbTNF-) were examined. Although dilation of vesselsto SNP was not different between Leprdb and mLeprdb mice, dilation to ACh was reducedin Leprdb mice. The NFkB antagonist, MG-132, IKK inhibitor, sodium salicylate(NaSal), or Anti-MCP-1 partially restored endothelium-dependent coronary arteriolardilation in Leprdb mice. Protein expression of IKK and IKK were higher in Leprdb thanin mLeprdb mice. The expression of IKK, but not the expression of IKK was increasedin dbTNF-/dbTNF- mice. Leprdb mice showed increased insulin resistance, but NaSal improved insulin sensitivity. Protein expression of TNFa, NFkB, phosphorylation ofIKK and JNK were greater in Leprdb mice, but NaSal attenuated protein expression ofthem in Leprdb mice. The ratio of phosphorylated IRS-1 at Ser307 (pIRS-1)/IRS-1protein expression was elevated in Leprdb mice; both NaSal and JNK inhibitor SP600125reduced pIRS-1/IRS-1 in Leprdb mice. MG-132 or neutralization of TNF reducedsuperoxide production in Leprdb mice. Anti-MCP-1 attenuated superoxide productionand protein expression of nitrotyrosine (N-Tyr), which is an indicator of peroxynitriteproduction, in isolated coronary arterioles of Leprdb mice. Immunostaining resultsshowed that expression of MCP-1 and vascular cellular adhesion molecule-1 (VCAM) isco-localized with endothelial cells and macrophages. Anti-TNFa or anti-MCP-1markedly reduced macrophage infiltration and the number of MCP-1 positive cells.Neutralization of TNFa or anti-MCP-1 reduced the expression of adhesion molecules. Inconclusion, our results indicate that the interaction between NFkB and TNFa signalinginduces activation of IKKb. In addition, TNFa and TNFa-related signaling, includingthe expression of MCP-1 and adhesion molecules, further exacerbates oxidative stressleading to endothelial dysfunction in type 2 diabetes.

Les NAD(P)H déshydrogénases de type II (Ndh-II) sont des enzymes de faible poids moléculaire capables doxyder le NAD(P)H et de transférer les électrons à un groupement quinone (plastoquinone ou ubiquinone). On les appelle « de type II » par opposition aux déshydrogénases de type I qui correspondent au complexe I mitochondrial. Chez Arabidopsis thaliana, des protéines Ndh-II ont été identifiées sur les faces interne et externe de la membrane interne mitochondriale, sur la membrane des peroxysomes, et au niveau de la membrane thylacoïdale du chloroplaste. Au niveau de la chaîne de transport délectrons mitochondriale, les protéines Ndh-II constituent une voie alternative aux complexes I et II pour lapport des électrons au pool dubiquinones. Cette voie alternative permettrait une adaptation de la chaîne de transport délectrons en fonction du métabolisme de lalgue. Au niveau de la chaîne de transport délectrons chloroplastique, les protéines Ndh-II participeraient à plusieurs mécanismes dadaptation de la chaîne à la quantité et à la qualité de la lumière disponible : transitions détats, transport cyclique délectrons autour du photosystème II. Leur fonction serait de catalyser la réduction non-photochimique du pool de plastoquinones. En 2005, sept open reading frame correspondant à des NAD(P)H déshydrogénases de type II hypothétiques (NDA1 à NDA7) ont été identifiées dans le génome nucléaire de Chlamydomonas. Ces séquences étaient cependant largement incomplètes du fait de régions non séquencées dans le génome de Chlamydomonas. Les données récoltées au cours de ce travail ont permis lobtention dune version complète de la séquence codante des gènes NDA de Chlamydomonas. Ces analyses ont démontré que le gène putatif NDA4 correspondait, en fait, à des régions internes non attribuées au gène NDA2. Chez Arabidopsis thaliana et Solanum tuberosum, une corrélation entre le positionnement phylogénétique des gènes NDH-II et la localisation subcellulaire de la protéine correspondante a été mise en évidence. Lanalyse phylogénétique des séquences des protéines Nda de Chlamydomonas montre que les gènes NDA1, 2 et 3 seraient proches phylogénétiquement et seraient à positionner dans le clade des protéines Ndh-II mitochondriales des plantes supérieures. A linverse, la protéine Nda5 serait dorigine cyanobactérienne et se positionne dans le même clade que les protéines identifiées dans le chloroplaste des plantes supérieures. Les protéines Nda6 et 7 sont très proches du point de vue de la séquence, suggérant une duplication récente des gènes NDA6 et 7. Ces deux protéines se positionnent dans un nouveau clade, apparemment intermédiaire entre le domaine eucaryote et le domaine procaryote. Une étude dexpression des gènes NDA de Chlamydomonas a permis de mettre en évidence lexpression apparemment majoritaire du gène NDA2. Pour étudier la fonction spécifique de NDA2, nous avons inactivé lexpression de ce gène par RNA interférence afin détudier le phénotype des mutants obtenus. Contrairement aux prédictions in silico, il est apparu que la protéine Nda2 se localise au niveau du chloroplaste. Létude de la fluorescence chlorophyllienne de deux mutants montre que la capacité de ces mutants à réduire de manière non-photochimique le pool de plastoquinones est largement diminuée. Dautre part, les mutants sont largement affectés dans leur capacité à modifier la distribution de lénergie dexcitation entre les deux photosystèmes (transition détat) lorsque la respiration mitochondriale est inhibée. Il est connu que les transitions détat sont initiées par des changements de létat rédox du pool de plastoquinones, qui est lui-même dépendant de létat rédox de la cellule. Dans ce cadre, nous proposons que la protéine Nda2 pourrait servir de « senseur » du métabolisme cellulaire de lalgue et permettrait dadapter les flux délectrons chloroplastiques en réponse aux changements du contexte énergétique cellulaire.

This thesis focuses on host and pathogen specific interactions during invasive disease. We have investigated the role and impact of different virulence factors of Neisseria gonorrhoeae, N. meningitidis and Streptococcus pyogenes on host epithelial cells and in vivo. N. gonorrhoeae cause the sexually transmitted disease gonorrhoea and N. meningitidis is the most common cause of bacterial meningitis and may be leathal to the host within hours of infection. The neisserial type IV pili were shown to have an important impact on host cells for the induction of pro-inflammatory and other cellular defence transcriptional responses. Furthermore, N. meningitidis generally induced an earlier response compared to N. gonorrhoeae, probably as a result of the meningococcal capsule. The role of N. meningitidis serogroup B lipooliogsaccharide was investigated during invasive disease. Bacterial invasion of host cells and blood survival as well as virulence in vivo was dependent on the integrity of the LOS structure. S. pyogenes may cause a variety of diseases ranging from uncomplicated diseases such as 'strep-throat' to more severe invasive diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome. S. pyogenes ScpC protease degrade interleukin 8 during necrotizing fasciitis. We investigated the role of ScpC in systemic disease and observed enhanced virulence by bacteria unable to degrade IL-8. Following an intravenous infection of mice pro-inflammatory cytokines and complement activation was induced by the ScpC negative mutant compared to the wild-type and correlated with higher bacteremia. These data indicate that the precense of the ScpC protease has an important impact on the host for the outcome of streptococcal sepsis. Another phagocytic escape mechanism of S. pyogenes is their ability to coat themselves with host proteins. We observed that released complement control protein, CD46, bound to the streptococcal cell surface. CD46 has been shown to interact with the streptococcal M protein and have now been found to bind to the surface of the bacteria in a growth phase dependent manner. We observed a more aggressive disease development in CD46 transgenic mice after an intravenous infection with an M6 serotype, resulting in higher mortality of CD46 transgenic mice compared with control mice. These data indicate that CD46 may confer a protection to the streptococci during early stage of systemic infection and contributes to the understanding of immune evsion of S. pyogenes.

Using web-based knowledge management systems has become the mainstream when enterprises adopt the strategy of knowledge management. With the accumulated amount of materials, the issue of information reuse is emphasized, and how to properly design a knowledge management system to improve the efficiency of information reuse is becoming extremely important. In the past there were two approaches for improving the efficiency of information reuse. One is knowledge map that focuses on information retrieval to promote the efficiency, and the other is usability of Human-Computer Interaction that focuses on the effect of different information representation styles and the user's liking. According to Task-Technology Fit, the greater the degree of adherence to an ideal fit profile, the better the performance. In this study we discussed the impact of information presentation styles on information reuse with different reuse tasks on knowledge management systems. The study is intended as an experimental design to investigate the impact of information presentation styles on information reuse within a knowledge management system. We use an experiment involving 83 subjects. The results of this study indicate that information presentation styles have a significant impact on information reuse. We found that for retrieval tasks, the impact on information reuse would be enhanced with the hierarchical presentation style. Additionally, the result also shows that when increasing the complexity of the task, information presentation styles have no differences to the impact on information reuse. And according to the results of two kinds of questionnaires, the users all had higher liking and partialities for the hierarchical presentation style. Finally, suggestions for the vision information design based on the results of this study are provided.

Carbon nanotubes were dispersed and functionalized with polyethylene imine (PEI) before incorporation in a polyvinyl acetate matrix. The resulting samples exhibit air-stable N-type characteristics with electrical conductivities as great as 1600 S/m and thermopowers as high as 100 microV/K. Thermopowers and electrical conductivities correlate, in a reversal of the trend found in typical materials. This phenomenon is believed to be due to the increase in the number of tubes that are evenly coated in a better dispersed sample. Increasing the amount of PEI relative to the other constituents positively affects thermopower but not conductivity. Air exposure reduces both thermopower and conductivity, but a stable value is reached within seven days following film fabrication. The atmospheric effects on the electrical conductivity prove to be reversible. Oxygen is believed to be the primary contributor to the decay.

There are two main human pathogens in the family of Chlamydiaceae. Different serovars of Chlamydia trachomatis cause sexually-transmitted disease and eye infections whereas C. pneumoniae (TWAR) is a common cause of community-acquired respiratory infection. Chlamydia species are obligate, intracellular bacteria sharing a unique developmental cycle that occurs within a protected vacuole termed an inclusion. These microorganisms can be distinguished by two different forms: the infectious, metabolically inert elementary body (EB) and the reproducing non-infectious form, termed the reticulate body (RB). The cycle is terminated when re-differentiation of RBs back to infectious EBs occurs. Chlamydia possesses a type III secretion system (T3SS) essential for delivery of effector proteins into the host for host-cell interactions. This virulence system has been systematically characterized in several mammalian pathogens. Due to lack of a tractable genetic system for Chlamydia species, we have employed chemical genetics as a strategy to investigate molecular aspects of the T3SS. We have identified that the T3S-inhibitors INP0010 and INP0400 block the developmental cycle and interfere with secretion of T3S effector proteins in C. pneumoniae and C. trachomatis, without any cytotoxic effect. We have further shown that INP0010 decreases initiation of transcription in C. pneumoniae during the early mid-developmental cycle as demonstrated by a novel calculation, useful for measurement of transcription initiation in any intracellular pathogen. The mechanism regulating the signal(s) for primary as well as terminal differentiation of RBs has not been defined in Chlamydia. We show using T3S-inhibitors that INP0010 targets the T3SS and thereby arrests RB proliferation as well as RB to EB re-differentiation of C. pneumoniae as where INP0400 targets the T3SS and provokes a bacterial dissociation from the inclusion membrane presumed to mimic the natural occurrence of terminal differentiation. The effect of INP0010 on iron-responsive genes indicates a role for T3S in iron acquisition. Accordingly, our results suggest the possibility that C. pneumoniae acquires iron via the intracellular trafficking pathway of endocytosed transferrin. Moreover, we have for the first time presented data showing generalized bone loss from C. pneumoniae infection in mice. The infection was associated with increased levels of the bone resorptive cytokines IL-6 and IL-1beta. In addition, an increased sub-population of T-cells expressed RANKL during infection. Additionally, C. pneumoniae established an infection in a human osteoblast cell line in vitro with a similar cytokine profile as seen in vivo, supporting a causal linkage. Collectively, these data may indicate a previously unknown pathological role of C. pneumoniae in generalized bone loss.