Glioblastoma multiforme (GBM) is the most malignant form of brain cancer. Therapies targeting glioblastoma have not consistently been able to give those diagnosed the best prognosis. Treatments that directly infiltrate into the tumor are highly sought after. Indirubins have been used to treat various types of cancers and are a promising avenue for future glioma research. In the current study, we further researched several key GSK-3 inhibitors, BIO (an indirubin) and CHIR99021, in addition to LiCl, to see their effects on the translocation of β-catenin to the nucleus, and the invasion and migration of cells in both a sphere assay and an aortic ring assay. Here we studied anti-invasive therapies that may have a future role in GBM treatment. It is thought that combining conventional treatments with anti-invasive therapies will create cytotoxicity in and reduce migration of the tumor. Three types of cells were used throughout the experiments: HBMEC, HUVEC, and U251 glioma cells. We reported that GSK-3 inhibitors might have a valuable role in the treatment of GBM. The selected inhibitors (BIO, CHIR99021, and LiCl) all were shown to lessen cell migration and invasion in vitro in a range of assays and in all cell lines tested. All inhibitors tested cause a dose-dependent, reversible inhibition of glioma cell invasion in spheroid assays. BIO was shown to cause a rapid upregulation of total and nuclear β-catenin. BIO, at higher concentrations, also created a toxic environment for cells, sometimes killing them. This shows that a more in-depth experiment involving different BIO concentrations is needed to test the optimal concentration for treatment. Each of the experimented GSK-3 inhibitors also showed a change in the junctions between cells. NaCl as a control showed normal, spikey, junctions, while CHIR99021 and BIO caused the junctions to become more smooth. This suggests that GSK-3 inhibition has a role in either maintaining the ECM and/or in communication between cells. Also in this assay, there was a heterogeneity between cells treated with the same inhibitor and in the same dish, indicating that not all cells respond to each drug the same way. The reasons for this are not known and further investigation is required. A new construct was also made to report β-catenin transcriptional coactivation using luciferase expression as the reporter in response to these selected GSK-3 inhibitors.With the combined results of these experiments, we concluded that GSK-3 inhibitors may be a promising approach to the treatment of GBM. Further investigation is required before any treatments can be administered to those diagnosed.

Magister Scientiae - MSc / Selective serotonin re-uptake inhibitors (SSRIs) are a new class of antidepressants used mainly for the treatment of depression and other forms of related disorders. There are a number of side effects associated with these drugs which include loss of weight, sexual dysfunction, nervousness and nausea. A fast and reliable detection method such as biosensing for the determination of the SSRIs metabolic profile is therefore essential for the appropriate dosing of these drugs. Biosensors for the determination of the SSRIs biotransformation were prepared with cytochrome P450 (2D6) isoenzyme and poly (anilinonapthalene sulfonic acid) film electrochemically deposited on gold. / South Africa

M.Sc. (Botany) / Please refer to full text to view abstract

Class of 2005 Abstract / Objective: The purpose of this study was to retrospectively determine if individuals within the SAVAHCS home-based patient population with a diagnosis of diabetes mellitus are receiving an angiotensin-converting-enzyme inhibitor (ACE-I) based on recent evidence supporting its use in these patients. Research Design: A retrospective, chart review of 41 patients with a diagnosis of diabetes mellitus from November 1, 2004 to December 31, 2005. Methodology: This was a retrospective, chart review of all patients within the SAVAHCS home-based population with an active diagnosis of diabetes mellitus. Once the patients were identified, their clinical profiles were extracted from the VISTA computer system. The patients had data regarding age, gender, diabetes type, diagnoses of heart disease and hypertension, type of ACE- Inhibitor prescribed, blood pressure, HgbA1c, and height and weight in order to calculate body mass index (BMI). The patients were classified as either having or not having ACE-Inhibitor therapy. Results: The total number of diabetic patients currently receiving an ACE-Inhibitor was 24 (58.5%). This was significantly lower than the value of 80% predetermined (p=0.0352). Thirty-one patients were also found to have a diagnosis of hypertension (75.6%), with 18 of these patients having a prescription for an ACE-Inhibitor (43.9%). Four patients (9.8%) who were not currently on an ACE- Inhibitor had a documented history of cough induced by the use of these drugs. Clinical Relationships: ACE-Inhibitors are drug agents used to treat hypertension. They have also been shown to be of significant clinical value in diabetic patients, in both renal protective effects as well as to reduce cardiovascular risk, the most common cause of morbidity and mortality in diabetic patients.

It is suggested that there is a world of research on the design and implementation of performance management and the effect it has on overall organisational results, but there appears to be a gap in knowledge on the enablers and inhibitors of performance management, or causes of its positive and negative effects. This study aimed to investigate the perceptions relating to performance management of chosen respondents at Multichoice (Pty) Ltd. A literature review was performed to identify research that was previously conducted pertaining to performance management. The literature review analysed the factors perceived to enable and inhibit performance management with the question remaining as to whether the perceptions of the sample population differ or are congruent with the literature summary. The research involved using a formal quantitative causal and explanatory design with a questionnaire being used to gather information from a sample of employees. The sample comprised of 60 employees, 20 from each category of performers, namely A players, B players and C players. Statistical techniques, namely descriptive and inferential statistics were used to extract key constructs from the raw data obtained from the questionnaires. The outcome of the research resulted in the enablers and the inhibitors of performance management at Multichoice (Pty) Ltd. These findings have been used to develop a framework which should be considered by organisations to achieve high performance. Further to this, the analysis revealed the strength of the different forces which were ranked in an attempt to determine whether the enabling forces outweigh the inhibiting forces or vice versa. The reason for this was to strengthen the enabling forces supporting the performance management systems at Multichoice (Pty) Ltd and reduce the impact of opposition to it. / Dissertation (MBA)--University of Pretoria, 2012. / Gordon Institute of Business Science (GIBS) / unrestricted

The major serine proteinase inhibitors Kunitz and Bowman-Birk-type trypsin are key anti-nutrients responsible for the low nutritional value of raw soy cake, the by product of oil expression from soybean. Traditionally, proteinase inhibitors are eliminated from soy cake through intensive heating, which is highly costly. The long term goal is to generate soybean seeds devoid of trypsin inhibitors through tissue culture and genetic modification of soybean. The RNAi technology has been selected in this study as a technique for down-regulation or silencing these two major serine trypsin inhibitors. Conserved regions, which have been identified by searching NCBI and EMBL database, were targeted for down regulation. Seed specific promoters were also isolated to drive the expression of hairpin constructs designed to down-regulate selected conserved regions of the inhibitors in soybean seeds. RNAi silencing constructs were designed for use in soybean transformation. Ultimately, a tissue culture and transformation protocol for a local soybean variety PAN 512 was established for transformation with two designed RNAi constructs. Suitability of selected promoters was tested by attaching promoters to the gus gene and evaluating specificity of seed expression after soybean transformation using the Agrobacterium tumefaciens strain EHA101. Future work will focus on further optimisation of the transformation protocol and generation of transformed plants carrying the designed silencing vectors. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Plant Science / unrestricted

The metastatic process requires that tumour cells are capable of traversing various micro-environmental barriers, such as the basement membrane. There are various proteolytic mechanisms which could contribute to the process, plasminogen activation by tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) is one such mechanism. Extensive reports in the literature (reviewed in the introduction) indicate that most tumour cells synthesize uPA and that it is this enzyme, particularly when receptor-bound, which plays a role in invasion. UCT-Mel 3 is a human malignant melanoma cell line which was established in our laboratory, and has been shown to be highly metastatic in the nude mouse. This cell line is typical of many melanomas in that it synthesizes only tPA and not uPA. In part 1 of this thesis I further investigated the plasminogen activator production by these cells (at the level of mRNA as well as activity) as well as expression of plasminogen activator inhibitor PAl-1 and receptors for tPA and uPA (uPAR). UCT-Mel 3 cells expressed uPAR although uPA was not detected. I also examined cells cultured from two metastatic deposits. Interestingly, the metastatic cells produced PAl-1 which was undetected in the parent cells. After confirming that UCT-Mel 3 do not express detectable levels of uPA, I attempted (in part 2) to determine whether tPA could play a comparable role to that of uPA in the invasive process. My strategy was to inhibit the expression of tPA via two different methods, namely the use of antisense RNA and ribozyme. I then hoped to isolate clones producing no tPA, which would have been injected into nude mice in order to assay for metastasis. Unfortunately, neither of these methods proved to be successful in abrogating tPA expression. I was thus unable to achieve the ultimate aim of the project. However, during the course of the study a number of unforeseen problems arose. Firstly, the clonal variation within the cell population, and secondly, my inability to obtain antisense transfectants. I have speculated that a possible reason for the latter may be that the cells are in fact unable to grow in the absence of tPA.

No description available.

The naturally occuring polyamines play an essential role in cell growth and proliferation. N8-Acetylspermidine is excreted in rat urine in significant levels (75 nmole/24 hours) but tissue levels of this compound are very low (1.0 nmole/gram), close to the limits of detection. This may be due to rapid degradation of N8-acetylspermidine by an enzyme in the cytoplasm of virtually all rat tissues. This enzyme N8-acetylspermidine deacetylase catalyzes the deacetylation of N8-acetylpermidine to spermidine and acetic acid. In order to study the physiologicalf unction of N8-acetylspermidine, we need a means of altering tissue levels of this compound, and inhibition of this deacetylation reaction could produce such an alteration. In our study, we have used an in vitro deacetylase assay to determine the types of compounds which can inhibit this reaction. The Km for this reaction was 10.5 microM. Studies on the structural requirements for the inhibitory activity indicated that: (1) increasing the size of the acetamido group leads to a decrease in inhibitory activity, (2) separation of the two primary amine groups with (CH2)8 produces the best inhibitory activity, and (3) removal of the nitrogen in the 4-position decreases the inhibitory activity slightly but increases the stability with respect to a competing enzyme, polyamine oxidase. Studies on the nature of the active site indicated that: (1) a sulfhydryl group is apparently essential for optimal activity as shown by inhibition with N-ethylmaleimide and also by dithiothreitol and 5,5'-dithio-bis-(nitro-benzoic acid), (2) a seryl hydroxyl group does not appear to be an essential part of the active site since diisopropyl-fluorophosphate has a relatively high Ki. (However, the organophosphate echothiophate is a very effective inhibitor. It has a quartarnary nitrogen which may orient this molecule to its site of action.), and (3) a divalent metal ion may be involved in the enzyme activity as is shown by inhibition with the chelating agents, EDTA and diethylmalonate.

Organophosphates (OPs), such as nerve agents and insecticides, potently inhibit acetylcholinesterase (AChE). Oximes, such as the currently FDA approved oxime 2-PAM, remove the OP from the inhibited enzyme. 2-PAM is effective against select OPs and cannot effectively pass the blood-brain barrier to attenuate OP induced CNS damage. Our laboratory has synthesized a series of substituted phenoxyalkyl pyridinium oximes (Patent number: 9,227,937) that have demonstrated increased survival rates compared to 2-PAM. This research investigated 1) in vitro oxime reactivation of rat, human, and guinea pig serum BChE after inhibition by nerve agent and insecticidal OPs; 2) in vitro determination of reactivation kinetic rate constants for OP inhibited human and rat serum BChE and electric eel AChE after inhibition by a sarin surrogate and paraoxon; 3) intranasal delivery of oximes to reactivate brain AChE in vivo after inhibition by a sarin surrogate. Novel oxime 15 demonstrated significant broad spectrum reactivation of OP-inhibited rat serum BChE while novel oxime 20 demonstrated significant broad spectrum reactivation of OP-inhibited human serum BChE. All tested oximes were poor reactivators of OP-inhibited guinea pig serum BChE. Kinetic analysis of reactivation for NIMP and paraoxon human and rat serum BChE and electric eel AChE demonstrated differences in the second order rate constants. Oxime 20 demonstrated reactivation efficiency for both NIMP and paraoxon inhibited rat and human serum BChE and electric eel AChE more effectively than 2-PAM. Intranasal delivery of either oxime 20 or 2-PAM showed attenuation of NIMP-inhibited brain AChE inhibition in select brain regions and select time points. Oxime 20 demonstrated a larger window of effectiveness but neither oxime attenuated brain AChE inhibition in the hindbrain for any time point or for any brain region at the ten minute time point. These data suggest that reactivation of OP-inhibited BChE may be contributing to the observed increases in survival seen with our oximes. Novel oxime 20 demonstrated reactivation efficacy towards both BChE and AChE inhibited enzyme and a rapid entry into the brain after intranasal delivery. Having an oxime that can be effective in a multitude of ways would be of great value to medical and military personnel.