Doctoral Degrees (Haematology and Cell Biology)
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Browsing Doctoral Degrees (Haematology and Cell Biology) by Author "Pretorius, G. H. J."
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Item Open Access Recombinant production and evaluation of a multifunctional haemostatic fusion protein(University of the Free State, 1999-10) Van Zyl, Walda Brenda; Pretorius, G. H. J.; Kotzé, H. F.Platelets and coagulation both play a pivotal role in thrombosis, one of the major life-threatening diseases in our society. We have thus experienced a drastic increase in the development of potent and secure antithrombotic, antiplatelet and fibrinolytic agents during the past decade. Recently, much research has been devoted to the development of chimeric proteins, where haemostasis is simultaneously targeted at different levels. For the purpose of this study, such a chimera, named PLATSAK, was designed. A 29 amino acid antithrombotic and antiplatelet peptide, comprising three inhibitory regions, was linked to staphylokinase via a cleavable factor Xa recognition sequence. The overall activities of PLATSAK should include inhibition of thrombin, prevention of platelet aggregation and activation of fibrinolyis. The gene encoding PLATSAK was expressed in E. coli cells under controlled conditions. PLATSAK was produced as a strongly expressed protein of 18 kDa and was purified form native E. coli proteins using metal affinity chromatography. In vitro analysis of PLATSAK activity revealed strong inhibition of thrombin and potent fibrin degradation. However, no effect on platelet aggregation could be observed. Several attempts at producing more potent antiplatelet variants were unsuccessful. According to its in vitro activity, PLATSAK appeared to be a potent novel haemostatic agent and was prone to be evaluated in an in vivo system. The in vivo activity of PLATSAK was evaluated by assessing its effect on platelet deposition in a baboon model of arterial and venous thrombosis. Dacron vascular graft segments and expansion chambers, inserted as extensions into permanent femoral arteriovenous shunts, were used to simulate arterial and venous thrombosis, respectively. PLATSAK (3.68 mg/kg) was administered as a bolus. Platelet deposition onto the graft surface and in the expansion chamber was imaged in real time with a scintillation camera as the deposition of 111ln-labelled platelets. After two hours, platelet deposition in the graft segments and expansion chambers was inhibited by 50% and 85% respectively when compared to control studies. The aPTT was lengthened to >120 seconds. Interestingly, the level of FOP in plasma did not increase after administration of PLATSAK. These results demonstrate that PLATSAK effectively inhibited platelet deposition in both arterial- and venous-type thrombosis an animal model. This is in contrast to the lack of the antiplatelet activity of PLATSAK in vitro. This illustrates that in vitro platelet aggregation results can not be directly applied to an in vivo situation. In summary, the recombinant production of a multifunctional haemostatic fusion protein, PLATSAK, was successful. In vitro PLATSAK showed significant antithrombin and fibrinolytic activity, but trivial antiplatelet activity. In vivo studies revealed that PLATSAK is a potent antithrombin and also prevented platelet deposition on thrombogenic material. The strong immuun response of PLATSAK however needs to be investigated and a variant with a weak immunogenic nature needs to be produced.Item Open Access Telomeres and telomerase in cancer of esophagus(University of the Free State, 2001-11) Van den Heever, Wilhelmina Maria Jacoba; Pretorius, G. H. J.English: Squamous cell carcinoma of the esophagus is a cancer with a high incidence in South Africa. We have investigated the prognostic value of telomerase activity in tumors as well as in nearby normal tissue. Biopsies from 98 patients were analyzed using an adaptation of the TRAP assay. We found all tumor biopsies to have moderate to high telomerase activity, while one third of biopsies from normal mucosa were negative. The telomerase activity level of the tumors had no prognostic value (P=0.95) as determined by the log rank test. A P-value of 0.02 was found when the telomerase-negative and moderately positive normal biopsies were grouped together and compared to those with high activity. Our results show that telomerase activity of normal mucosa in the vicinity of the tumor can identify a population of patients with significantly worse prognosis, even in late stage patients. Telomerase has attracted intense interest as a possible target for cancer therapeutics. Previous attempts at inhibiting telomerase activity utilized antisense oligonucleotides targeted at the template region of the RNA subunit of the enzyme. Although it worked well in cell extracts, getting the oligonucleotides into intact cells are difficult. We attempted a peptide-based approach to overcome the transport problem. A phage-display selection strategy was designed to isolate peptides (7 and 12 amino acids) capable of binding to the RNA template with high affinity and in so doing preventing the enzyme from elongating existing telomeres. Both libraries showed no high affinity binding to the RNA. The most probable explanation is that such short peptides are incapable of binding sequence-specifically to nucleic acids. Many studies have indicated the importance of telomerase activity as an independent prognostic indicator in a variety of cancers, but recent results have shown that telomere length is actually a better indicator, as it shows the final result oftelomerase activity. We thus decided to develop a flow cytrometric method that would allow us to analyze telomere length in small tissue samples. After the initial technique had been established on lymphocytes, it was tested on the SNO cell line before we switched to solid tissue. The background of cellular debris and cell clusters was much worse than for lymphocytes, but differences in signal strength could be seen. From the results obtained it is clear that this method is not optimized yet. The results also indicate that at this stage of development, flow-FISH is inferior to telomerase activity as prognostic indicator. The best way to validate the current method is to analyze the same samples by Southern hybridization and flow-FISH, preferably well defined tumor and normal tissue.