Bibliography: leaves 120-145. Reports Bt crystal protein toxicity to a phthirapteran species. Although Bt strain WB3516 may produce other unidentified toxins effective against B. ovis, the results provide strong evidence that the [delta]-endotoxin crystal proteins of strain WB3516 significantly contribute to the lousicidal toxicity of this strain.

ABSTRACT Insufficient pain management continues to be problematic for hospitalized patients throughout the country. It significantly interferes with a person's quality of life making it an issue of great concern to nurses in any setting. However, nurses do not do a good job of managing pain. The purpose of this evaluation was to assess graduate nursing students' knowledge and attitudes toward pain management. Forty (n=40) graduate nursing students were asked to participate in the evaluation. All students agreed and completed the Demographic Data Form, Nurses Attitude Survey and the Pain Management and Principles Assessment. Thirty-eight females and two males participated in this study. The mean age was 35 years old (SD=9.77) with a range between 24 and 62. The majority of the participants were Non-Hispanic white (70%), followed by African American (10%), Hispanic (10%), Asian and others. The mean years of nursing experience was 10 years with a standard deviation 7.31. The data showed that nursing students demonstrated inadequate knowledge regarding pain management. The mean score on the PMPAT was 66% (SD= 3.61). The mean score on the Nurse Attitude Survey was 77 (SD=5.8) on a survey with scores that could range from 25 to 100. The higher the score the more favorable that nurse is towards pain management. The scores ranged from 69 to 91. Knowledge and attitude scores had room for improvement, suggesting that the curriculum of the college could be improved. In addition, continuing education courses could be developed to support nurses' knowledge of pain management.

Vibrio cholerae, a natural inhabitant of aquatic environments and the causative agent of the diarrheal disease cholerae, requires iron for survival. Since one of the key factors in the survival of V. cholerae in the environment is the formation of biofilms, we determined the effect of iron on this aspect of the pathogens lifestyle. Since wild type V. cholerae forms a much more robust biofilm in the presence of exogenous iron we tested mutants in iron transport and regulation and found that a mutation in the gene encoding an iron-regulated small RNA, RyhB, was clearly attenuated in the biofilm assay. We determined through microarray analysis that the ryhB mutant has altered regulation of genes involved in many systems that may be involved in biofilm formation including amino acid biosynthesis, the TCA cycle, motility and chemotaxis, and the expression of outer membrane proteins. Due to the pleiotropic regulatory effects of RyhB, it is unlikely that any one individual gene or system regulated by RyhB is the cause of the biofilm defect, but rather the sum effect of the regulatory changes is decreased biofilm formation. Additionally, we discovered that the outer membrane protein, OmpT, is positively regulated by iron and Fur. Generally, when Fur has acted as a positive regulator in previous studies, it has been ultimately shown to do so by negatively regulating the negative regulator, RyhB. However, the positive regulation of ompT by Fur is independent of RyhB. While CRP, a positive regulator of ompT expression, did not affect iron-dependent regulation of ompT, over-expression of the negative regulator ToxR abolishes the iron and Fur dependent regulation. Sequence analysis has revealed a possible Fur box approximately 70 base pairs upstream of the transcriptional start site in a region that overlaps both a ToxR binding site and a CRP binding site in the ompT promoter. We propose the model that in iron-replete environments under ToxR repressing conditions, such as when amino acids are limiting, Fur can further increase the expression of ompT. / text

The Gram-negative bacterium Pseudomonas aeruginosa is a notorious opportunistic pathogen of individuals with the genetic disease cystic fibrosis (CF). Pseudomonas aeruginosa establishes a chronic infection within the CF lung, where the sputum accumulation characteristic of CF provides a complex and copious growth substrate. P. aeruginosa can grow to high densities in vivo (>10⁹ cells/ml lung sputum), and exacerbations associated with P. aeruginosa high density in vivo growth are primary contributors to CF morbidity and mortality. Surprisingly little is known about the catabolic processes that underlie P. aeruginosa in vivo growth. Unfortunately, nutritional modeling of the CF lung environment in animal models is difficult, as current animal models fail to mimic the sputum accumulation characteristic of CF. In this dissertation, I describe the use of expectorated CF sputum as a P. aeruginosa in vitro growth medium. Using global expression analysis, I show that P. aeruginosa up-regulates genes important for amino acid and lactate metabolism during growth in CF sputum as compared to a laboratory medium. P. aeruginosa also demonstrates enhanced production of the cell-cell communication signal 2-heptyl-3-hydroxy-4-quinolone (the Pseudomonas quinolone signal, PQS), a critical regulator of virulence factor production, during growth in CF sputum. Further, I use chemical analyses of CF sputum samples to develop a defined, synthetic medium that can be used to nutritionally model in vivo conditions. Using this medium, I show that PQS biosynthesis and aromatic amino acid metabolism are intimately linked and that cell-cell communication mediated by PQS is strikingly dependent upon the growth environment of P. aeruginosa. In addition, I demonstrate that P. aeruginosa preferentially consumes specific carbon sources present in the CF sputum milieu during rapid growth. I also describe the use of in vivo-relevant nutrient concentrations to evaluate the potential for P. aeruginosa anaerobic growth in CF sputum. Finally, I describe the purification and characterization of the aromatic amino acid-responsive transcriptional regulator PhhR and discuss its potential role in regulation of P. aeruginosa in vivo carbon substrate preference. / text

Background and objectives: Nonalcoholic steatohepatitis (NASH), which is characterized by concurrent existence of hepatic steatosis and predominantly lobular necroinflammation, represents the more advanced stage in the spectrum of nonalcoholic fatty liver disease (NAFLD). NASH exhibits dramatically increased risk of progression to end-stage liver diseases than simple steatosis. Therefore, the progression of hepatic steatosis to steatohepatitis is the crucial step in the development of obesity-related NASH. Toll like receptor 4 (TLR4), a master regulator of innate immunity, is the principal receptor for endotoxin, which is a central mediator of liver inflammation associated with both alcoholic and nonalcoholic liver disease. However, due to a lack of suitable animal models which fully recapitulate the natural history of obesity-induced NASH, the precise pathophysiological function of TLR4 signaling in the development of this disease remains poorly understood. The objective of this study is to investigate the role of TLR4 in mediating inflammatory responses in obesity-induced NASH using both in vivo and ex vivo approaches, and to unveil cellular and molecular mechanisms responsible for TLR4 actions. Key findings: 1. To address the role of TLR4 in the pathogenesis of NASH, we crossed ApoEdeficient mice (ApoE-/-) with TLR4 mutant mice (TLR4-/-) to generate ApoE-/- /TLR4 wild type mice (ApoE-/-/TLR4-WT) and ApoE-/-/TLR4-/- mice. Noticeably, when fed with high fat high cholesterol (HFHC) diet, ApoE-/-/TLR4-WT mice developed the typical pathology of NASH (hepatic steatosis, lobular inflammation, and hepatocyte ballooning) in the context of obesity and metabolic syndrome, suggesting HFHC-fed ApoE-/- mice as a suitable animal model for NASH. 2. TLR4 inactivation protected ApoE-/- mice against HFHC diet-induced liver injury, as indicated by a significant improvement in liver histology, a a marked reduction in serum ALT activity, a dramatic repression of inflammatory infiltrates, as well as an obvious decrease in hepatic production of pro-inflammatory cytokines. 3. In ApoE-/-/TLR4-WT mice, TLR4 expression was selectively elevated in Kupffer cells in response to HFHC diet feeding. 4. The activation of XBP1, a transcription factor involved in endoplasmic reticulum stress, was markedly elevated in liver of ApoE-/-/TLR4-WT mice fed with HFHC diet, whereas this change was abrogated in HFHC diet-fed ApoE-/-/TLR4-/- mice. 5. In rat primary Kupffer cells, treatment with anti-oxidants blocked endotoxininduced activation of XBP1 and NF-κB, leading to decreased cytokine production. In addition, siRNA-mediated knockdown of XBP1 inhibited NF-κB activation and cytokine production resulted from the treatment with the TLR4 agonist LPS. 6. In ApoE-/-/TLR4-WT mice, adenovirus-mediated expression of dominant negative XBP1 had no obvious effect on HFHC diet-induced hepatic steatosis and ROS production, but markedly decreased lobular inflammation, NF-κB activation, cytokine production in the liver and significantly reduced serum levels of ALT. Conclusions: These findings support the role of TLR4 in Kupffer cells as a key player in mediating the progression of simple steatosis to NASH, by inducing ROS-dependent activation of XBP1. In light of the obligatory role of XBP1 in TLR4-induced liver inflammation and injury, therapeutic interventions that inhibit TLR4/XBP1 activation may represent a promising strategy for treatment of NASH. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

The cystic fibrosis transmembrane conductance regulator (CFTR) was identified to be involved in acidosis-induced ATP release from skeletal myocytes in vitro and from contracting muscle in vivo. My PhD studies aimed to investigate the underlying mechanism and identify the pathway for ATP release in acidosis-induced CFTR-regulated ATP release. Lactic acid (10 mM) decreased the intracellular pH of L6 skeletal myocytes to 6.87 ± 0.12 after 3 hours, and the lowered pH resulted in the elevation of ATP release from skeletal myocytes. The acidosis-induced ATP release was totally abolished by GlyH-101 (40 μM), an open-channel CFTR blocker, suggesting that CFTR was involved. The cAMP/PKA signaling pathway was involved in the CFTR-regulated ATP release from skeletal myocytes: 1). Forskolin increased the extracellular ATP and the phosphorylation of CFTR; IBMX, a phosphodiesterase inhibitor, further enhanced the forskolin-induced extracellular ATP and phosphorylation of CFTR; 2). Inhibition of PKA by its selective inhibitor KT-5720 abolished the acidosis-induced ATP release and the forskolin-induced phosphorylation of CFTR. In addition, the inhibition of Na+/H+ exchanger (NHE) by amiloride, or inhibition of Na+/Ca2+ exchanger (NCX) by its specific inhibitors SN-6 and KB-R7943 abolished the lactic-acid-induced ATP release from skeletal myocytes, indicating that NHE and NCX might be involved. Previous studies demonstrated that Connexin hemichannels and Pannexin channels were able to conduct ATP in response to stimuli. This study found that connexin 43 (Cx43) was strongly expressed on skeletal myocytes, while Pannexin 1 (Panx1) showed a strong expression in gastrocnemius muscle. Investigation of the role that Cx43 may play in acidosis-induced cAMP/PKA-activated CFTR-regulated ATP release from myocytes showed that: 1). Cx43 was immunoprecipitated with CFTR suggesting a physical interaction; 2). The opening of Cx hemichannels was increased by lactic acid and this lactic-acid-induced opening was inhibited by CFTRinh-172, suggesting the mediation of CFTR; 3). Inhibition of Cxs and Panxs with carbenoxolone abolished the acidosis-induced ATP release; moreover, specific silencing of the Cx43 gene using siRNA decreased both basal and acidosis-induced ATP release, suggesting that Cx43 was involved; 4). Overexpression of CFTR alone did not elevate the acidosis-induced ATP release, while overexpression of Cx43 alone doubled the acidosis-induced ATP, and co-overexpression of CFTR and Cx43 further elevated the acidosis-induced ATP release, supporting the concept that Cx43 functionally interacted with CFTR to induce the acidosis-induced ATP release. Panx1 was studied in native skeletal muscle, and found to be coimmunoprecipitated with CFTR. Inhibition of Panxs with gadolinium or probenecid abolished the muscle-contraction-induced ATP release, while inhibition with carbenoxolone or quinine reduced it to less than 10% of control, suggesting that Panx1 may be involved in the acidosis-induced ATP release during muscle contraction. All the in vitro and in vivo studies suggested that Cxs and Panx were involved in the acidosis-induced CFTR-regulated ATP release from skeletal myocytes and skeletal muscle. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Medicine / Doctoral / Doctor of Philosophy

The information processing approach was used as a basis for studying some brief visual memory processes in reading disabled children. Three aspects of processing were examined, viz. (i) Duration of icon persistence; (ii) Performance under different backward masking conditions; and (iii) Processing of information into a more durable short-term visual memory store. It was found that there were no differences in the duration of icon persistence in reading disabled children, but that these children exhibited marked impairment in performance in the tasks used in the latter two experiments. The reasons for the reading disabled children's poorer performance in these tasks were not apparent. Speculations about the strategies used by these children in approaching the tasks are made. Possible implications and directions for future research are discussed. / Thesis (M.Soc.Sc.)-University of Natal, Durban, 1980.

Thesis (M.Med.)--University of Natal, Durban, 1997.

The global burden of heart disease increases every year. It has been estimated that by the year 2020, coronary artery disease (CAD) will be the number one cause of death worldwide. Indian populations throughout the world have the highest prevalence of CAD and early onset of the disease compared to other ethnic groups. Glutathione S-transferases (GSTs) detoxify environmental agents which influence the onset and progression of disease. Dysfunctional detoxification enzymes are responsible for prolonged exposure to reactive molecules and can contribute to endothelial damage, an underlying factor in CAD. Uncoupling proteins (UCPs) 2 and 3 play an important role in the regulation of oxidative stress which contributes to chronic inflammation. Coronary artery disease is a chronic inflammatory disorder characterized by elevated levels of C-reactive protein (CRP) and pro-inflammatory cytokines such as interleukin 6 (IL-6). Polymorphisms of these genes have been linked to CAD and other chronic diseases. Statins, metabolised in the liver, are the most commonly used drug to control atherosclerosis progression in CAD patients. The pleiotropic effects of statins have been attributed to both favourable and adverse outcomes in CAD patients particularly related to myopathy and hepatotoxicity. All patients (n=102) recruited into this study were South African Indian males. A corresponding age-, gender- and ethnicity-matched control group (n=100) was also recruited. The frequency of the GSTM1 +/0, GSTP1 A105/G105, IL6 -174G/C and CRP -390C/A/T genotypes was assessed by polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (PCR-RFLP). For the in vitro study, the biological effect of atorvastatin on HepG2 cells was assessed. The metabolic activity, cytotoxicity, oxidative stress and nitric oxide production was assessed by the ATP, lactate dehydrogenase (LDH), thiobarbituric acid reactive substance (TBARS) and Griess assays, respectively. The profile of 84 microRNA (miRNA) species was evaluated using the miRNA Pathway Finder PCR SuperArray. The predicted targets of up-regulated miRNAs were determined using the online software, Targetscan. The mRNA levels of guanidinoacetoacetate (GAMT), arginine glycine aminotransferase (AGAT) and spermine oxidase (SMO) were determined using quantitative PCR. Western blotting was used to determine GAMT and phosphorylated p53 levels in treated cells. The GSTM1 0/0 and GSTP1 A105/A105 genotypes occurred at higher frequencies in CAD patients compared with the control group (36% vs. 18% and 65% vs. 48%, respectively). A significant association with CAD was observed in GSTM1 0/0 (odds ratio (OR)=2.593; 95% confidence interval (CI) 1.353 - 4.971; p=0.0043) and GSTP1 A105/A105 OR=0.6011; 95% CI 0.3803 - 0.9503; p=0.0377). We found a significant association between smoking and CAD; the presence of either of the respective genotypes together with smoking increased the CAD risk (GSTP1 A105 relative risk (RR)=1.382; 95% CI 0.958 - 1.994; p=0.0987 and GSTM1 null RR=1.725; 95% CI 1.044 - 2.851; p=0.0221). The UCP2 -866G/A and UCP3 -55C/C genotypes occurred at highest frequency in CAD patients (59% vs. 52% and 66% vs. controls: 63% respectively) and did not influence the risk of CAD. Homozygous UCP3 -55T/T genotype was associated with highest fasting glucose (11.87±3.7mmol/L vs. C/C:6.11±0.27mmol/L and C/T:6.48±0.57mmol/L, p=0.0025), HbA1c (10.05±2.57% vs. C/C:6.44±0.21% and C/T:6.76±0.35%, p=0.0006) and triglycerides (6.47±1.7mmol/Lvs. C/C:2.33±0.17mmol/L and C/T:2.06±0.25mmol/L, p<0.0001) in CAD patients. A significant association between the G allele of the IL6 -174 polymorphism and non-diabetic CAD patients was found (p=0.0431 odds ratio: 1.307, 95% CI: 1.047-1.632). A significant association with the C allele of the -390 CRP triallelic variants and CAD (p=0.021 odds ratio: 1.75, 95% CI: 1.109-2.778) was also found using a contingency of the C allele vs. the minor A and T allele frequencies. The strength of the association of the C allele with non- diabetic CAD subjects was much higher (p=0.0048 odds ratio: 2.634, 95% CI: 1.350-5.138). Circulating median levels of IL-6 (0.9 (0.90, 0.91) pg/ml and 0.9 (0.87, 0.92) pg/ml) and CRP (5.65 (1.9, 8.2) mg/l and 2.90 (1.93, 8.35) mg/l) were similar between CAD patients and controls, respectively. A similar finding was observed between controls and non-diabetic CAD subjects. Levels of IL-6 and CRP in CAD subjects were not significantly influenced by polymorphic variants of IL-6 and CRP. In the control group, the level of IL-6 was significantly influenced by the IL6 -174 G allele (p=0.0002) and the CRP -390 C allele (p=0.0416), where subjects with the homozygous GG (0.9 (0.9, 1,78) pg/ml) and CC (0.9 (0.9, 0.95) pg/ml) genotype had higher levels than the C allele carriers (0.9 (0.64, 0.91) pg/ml) or A and T carriers (0.9 (0.69, 0.91) pg/ml) combined. The lowest measure of proliferation/metabolism in HepG2 cells was observed at 20μM atorvastatin, with 82±9.8% viability. The level of cytotoxicity was increased in statin treated cells from 0.95±0.02 units to 1.11±0.03 units (p=0.001) and malondialdehyde levels was reduced from 0.133±0.003 units to 0.126±0.005 units (p=0.009) whilst nitrite levels were elevated (0.0312±0.003 units vs. control: 0.027±0.001 units, p=0.044). MicroRNAs most significantly upregulated by atorvastatin included miR-302a-3p (3.05-fold), miR-302c-3p (3.61-fold), miR-124-3p (3.90-fold) and miR-222-3p (4.4-fold); miR-19a-3p, miR-101-3p and let-7g were downregulated (3.63-fold, 2.92-fold, 2.81-fold, respectively). A list of miRNA targets identified included those with a role in metabolism and inflammation. The miR-124a specifically targets the mRNA of GAMT and SMO. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2012.