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Masters Degrees (Medical Biochemistry)

Permanent URI for this collectionhttps://hdl.handle.net/10413/7039

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Browsing Masters Degrees (Medical Biochemistry) by Subject "Antibody-dependent cell cytotoxicity."

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    Antiproliferative effect of a novel synthesized carbazole compound on A549 lung cancer cell line.
    (2014) Molatlhegi, Refilwe P.; Chuturgoon, Anil Amichund.
    Increased death rates due to lung cancer have necessitated the search for potential novel anticancer compounds such as carbazole derivatives. Carbazoles are aromatic heterocyclic compounds with anticancer, antibacterial and anti-inflammatory activity. The study investigated the ability of the novel carbazole compound (Z)-4-[9-ethyl-9aH-carbazol-3-yl) amino] pent-3-en-2-one (ECAP) to inhibit the proliferation of lung cancer cells and its mechanism of action. ECAP was synthesized as a yellow powder with melting point of 240-247 °C. The 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), lipid peroxidation and comet assays were used to assess the anti-proliferative effects of the compound on A549 lung cancer cell line. Protein expression was determined using western blots, apoptosis was measured by luminometry for caspase-3/7, -8 and -9 and flow cytometry was used to measure phosphatidylserine externalisation. ECAP induced a p53 mediated apoptosis of lung cancer cells by significantly down-regulating the expression of antioxidant defense proteins, Hsp70 (p < 0.02) and Bcl-2 (p < 0.0006), thereby up-regulating reactive oxygen species (ROS) production. This resulted in DNA damage (p < 0.0001) and subsequent up-regulation of Bax and caspase activity consequently inducing apoptosis of lung cancer cells. These results demonstrate the potential anticancer effects of ECAP on cultured lung cancer cells. However, further investigation and characterization is required to fully understand the possible use of carbazole compound (Z)-4-[9-ethyl-9aH-carbazol-3-yl) amino] pent-3- en-2-one as potential lung cancer treatment.
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    An investigation into marine bacterial species found in shark mouths in the Indian Ocean and their implications for human health.
    (2015) Ramlakhan, Yathisha.; Chuturgoon, Anil Amichund.; Phulukdaree, Alisa.; Bester, Linda Antionette.; Singh, Sanil Duleep.
    There is an ever increasing amount of pollution and waste being released into the environment. This is due to the increase in population, urbanisation and people migrating into cities. Approximately 2.4 billion people living in urban and rural areas have no access to basic sanitation. In the next 20 years, there will be a further increase of 2 billion people who will lack basic sanitation. In developing countries, 90% of untreated sewage is released into rivers, lakes and coastal waters. Apart from sewage, waste such as petroleum products, heavy metals and organochlorine also contribute to marine pollution. Companies that manufacture sugar/artificial sweeteners etc. and farming activities that utilize fertilizers for crops can cause eutrophication, as un-used fertilizers get washed into rivers. The marine water is a different environment to other aquatic and terrestrial environments. This then forces microbes to adapt, so they can be able to survive in the marine environment. The difference in the marine environment allows for the production of distinct bioactive metabolites such as secondary metabolites. These secondary metabolites come from algae and marine bacteria and these secondary metabolites are then exclusive to the marine waters. These secondary metabolites can be used for medical purposes, cosmetics, personal-care products etc. There is a huge problem with antibiotic resistance and research needs to be done to solve this resistance issue. Two common bacterial strains were isolated and identified from the mouth of sharks. The bacteria were identified as Bacillus cereus and Vibrio alginolyticus. They were isolated and cultured in broth for 3 days, till they reached the log phase of growth. The broth was then extracted for metabolites which the bacteria produced, using ethyl acetate. These metabolites were tested for cytotoxicity in the human liver hepatocellular carcinoma (Hep G2) cells. The concentrations that were determined to cause 50% cell death (IC50) in the cell viability assay on Hep G2 cells were 0.764 mg/ml and 0.918 mg/ml for B. cereus and V. alginolyticus, respectively. These values were then used for subsequent assays. Antibacterial testing was done for the bacterial extracts of Bacillus cereus and Vibrio alginolyticus. There was no antibacterial activity against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853. Assays that used flow cytometry was used to show if apoptosis/necrosis occurred. These were assays such as Annexin V and propidium staining. While assays that used luminometry showed the levels of ATP and determined whether apoptosis of the cells occurred. These were assays such as the ATP assay, mitochondrial depolarisation assay and determination of the caspase activities of caspase 3/7, 8 and 9. Additional assays, like the comet and TBARS assays, were done to show DNA fragmentation and oxidative stress of the cells, respectively. The results for the Annexin V/ propidium staining showed the control had a mean of 11.20 ± 1.0. Extract 1 (20.83 ± 0.8737) and extract 2 (25.37 ± 1.050) showed a higher percentage when compared to the control. Extract 2 was significant against the control (p<0.0273). For propidium staining, the control had a mean of 6.033 ± 0.4524. Extracts 1(11.57 ± 1.387) and 2 (11.43 ± 0.3215) showed a higher percentage when compared to the control. The Annexin V and propidium staining suggested that extract 1 and 2 had undergone both apoptosis and necrosis. For luminometry assays, the ATP assay showed that the control had a mean of 1.83x106 ± 5.82x104. Extracts 1 (1.5x106 ± 9.4x104) and extract 2 (1.4x106 ± 8.3x104) showed a decrease in ATP with reference to the control. In the mitochondrial depolarisation assay, the control had a mean of 14.83 ± 1.350. Extracts 1 (30.57 ± 0.75) and extract 2 (20.53 ± 8.56) showed a decrease in polarisation with reference to the control. For caspase 8 analysis, the control, extract 1 and extract 2 had means that were 4.23x104 ± 3.37x103, 52x103 ± 10.1x103 and 40x103±5.2x103, respectively. For caspase 9 analysis, the control, extract 1 and extract 2 had means that were 8.6x104 ± 4.6x103, 5.6x104 ± 4x103and 9.6x104 ± 5.6x104, respectively. The caspase 3/7 analysis showed that the control, extract 1 and extract 2 had means of 4.4x103 ± 0.57x103, 5.5x103 ± 0.19x103 and 5.8x103 ± 2 x103, respectively. Caspase 3/7 showed that apoptosis had occurred with the cells for all extracts used. Extract 1 showed a high caspase activity for caspase 8. This suggested that it followed the extrinsic pathway of apoptosis. Extracts 2 showed a high activity for caspase 9 which suggested that it followed the intrinsic pathway of apoptosis. The comet assay showed that the means of the control, extract 1 and extract 2 were 35.91 ± 21.93, 75.85 ± 11.43 and 60.48 ± 11.86, respectively. The extracts were significantly higher than the control (extract 1 and 2 p<0.0001). Extract 1 and 2 were compared to each other and had shown a significance between them (p<0.0001). The TBARS assay obtained the following MDA concentrations for the control, extract 1, extracts 2, negative and positive samples: 0,137, 0,132, 0,150, 0,088 and 20,502, respectively. The MDA concentration gives an indication of oxidative stress of the cells. From the cell viability assay, the secondary metabolites produced by B. cereus needed a lower concentration of extract to determine an IC50 value. This suggested that the secondary metabolites produced by B. cereus were more toxic than the secondary metabolites produced by V. alginolyticus. This was then further supported by assays such as mitochondrial depolarisation and the comet assay. The secondary metabolites that could be the reason why there were apoptosis and necrosis, are the toxins the bacteria produce. This is the enterotoxin or cereulide produced by B. cereus and TLH by V. alginolyticus. However, further studies need to be done to confirm if these toxins are the cause of cell death.