Browsing by Author "Bernert, Martin"

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    Investigating telomere dynamics in oesophageal squamous carcinoma cells using standard and gold nanoparticle-based assays
    (2017) Bernert, Martin
    Cancer is characterised by abnormal cell proliferation and is one of the leading causes of death in first world countries and the second leading cause in developing countries. In 2012 alone, over 14 million cases were reported and over 8 million deaths were attributed to cancer worldwide, with sub-Saharan Africa, especially South Africa having one of the highest oesophageal cancer rates in the world. An important aspect of cancer is the telomeres, which are 10-15kbp of TTAGGG DNA repeats in humans at the ends of chromosomes. These repeats are maintained by the enzyme telomerase. Up to 90% of all cancers show increased telomerase activity to overcome the "end-replication" problem in which the telomeres shorten after each cell division. This eventually leads to cellular senescence. Due to the high number of cancers relying on increased telomerase activity to bypass senescence, telomerase could be a viable target for anti-cancer therapies. The limiting factor of the multi-subunit telomerase enzyme is its telomerase reverse transcriptase component (hTERT). hTERT has also been shown to migrate to the mitochondria during times of high oxidative stress caused by reactive oxygen species (ROS). Here it confers protection to the mitochondria against ROS, potentially preventing the cell form undergoing apoptosis and reaching senescence. This can potentially be detrimental, as cells become damaged by the ROS and continue dividing. This could lead to further genetic damage. Metformin, a drug used for the treatment of type-2 diabetes, has been linked to lower incidences of cancer. The mode of action of metformin is not yet fully understood, however it is known that it affects the mitochondria. Since hTERT and metformin could co-localise, the drug may influence hTERT and potentially telomerase activity. This makes metformin an anticancer candidate to be used in conjunction with traditional anticancer therapies. To determine telomerase activity in metformin treated oesophageal carcinoma cells, qPCR based telomerase activity assays must be used. These assays can be very expensive and time consuming, so a faster and cheaper alternative would be beneficial. Therefore, the aim of this project was to alter and improve a nanoparticle based detection method for telomerase activity, by decreasing the time required to prepare the DNA functionalised nanoparticles as well as determining a more rapid method of data measurement, and compare it to conventional qPCR based techniques (TRAPeze RT Telomerase Activity Kit – Merck). Thereafter the effects of the metformin treatment on telomere dynamics, such as telomere length, telomerase activity and hTERT mRNA expression, in oesophageal squamous carcinoma cells were determined. Gold nanoparticles were synthesised and functionalised with thiolated-DNA (telomerase substrate). These functionalised particles were characterised using transmission electron microscopy. To assess telomerase activity the extracted protein was added to the functionalised nanoparticle solution and allowed to elongate the coupled DNA. A characteristic of gold nanoparticles is that the size of the particles as well as their proximity to one another determines the colour of the nanoparticle solution. Due to the steric hindrance caused by the now elongated DNA, a distinct colour change was observable. The change in absorption spectra of the nanoparticle solution was recorded after the enzyme elongated the substrate. This nanoparticle based assay was then compared to TRAPeze RT Telomerase detection kit (Merck-Millipore) as a positive control. Using the conventional qPCR based telomerase activity assay, it was found that metformin significantly decreased telomerase activity in oesophageal cancer cell lines, however this was not seen using the nanoparticle assay. A colour change was observed with the nanoparticle assay compared to the negative control reflecting detection of telomerase activity. However, no significant decrease in telomerase activity could be detected due to metformin treatment. More optimisation is required, however this technique has great potential, as nanoparticle based assays are also known for their high sensitivity. This technique is also far more rapid and significantly cheaper that the qPCR based method. The gold nanoparticle based telomerase activity assay could become an alternative to conventional qPCR based techniques.
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    Investigating telomere dynamics using standard and AuNP-based assays and developing an LRP-based nanoparticle drug
    (University of the Witwatersrand, Johannesburg, 2023) Bernert, Martin; van der Merwe, Eloise; Weiss, Stefan FT
    Telomere dynamics, specifically telomerase activity have been implicated in age-related diseases, such as CVD, Alzheimer’s disease, and cancer. This makes the accurate detection of telomerase activity within cell cultures and tissue samples a necessity. Conventional techniques have many drawbacks, including their very high cost. Therefore, this research aimed to develop a gold nanoparticle (AuNP)-based assay to determine telomerase activity. In the assay the extracted telomerase leads to a colour change in the solution through the addition of telomeric repeats and subsequent elongation of the synthetic telomeres attached to the AuNPs. This colour change is detectable using spectrophotometric readings and represents telomerase activity. This assay would be useful as an alternative to expensive existing telomerase activity kits as large batches of AuNPs can be synthesised inexpensively. Telomerase activity was successfully detected in both HEK-293 and WHCO-5 cells using this novel technique, although the sensitivity of the AuNP-based telomerase activity assay is currently lower than a commercially available qPCR-based telomerases activity kit. In addition, telomerase activity is directly affected by the LRP protein, a highly conserved non-integrin transmembrane receptor, which has been shown to have therapeutic effects in ageing, Alzheimer’s disease, Parkinson’s disease, diabetes, and cardiovascular disease models. Recently it has been found that overexpression of LRP::FLAG, by plasmid transfection, leads to a significant increase in telomerase activity in cell culture models. This may indicate that upregulation of LRP can be used to treat various age-related diseases, however, transfection is not a viable treatment strategy and therefore, a protein-based drug was created. For a protein-based drug, a suitable delivery system needed to be developed and nano-capsules, such as those synthesised using Poly(lactic-co-glycolic acid) (PLGA), are able to contain the therapeutic protein. The molecules contained within the nanoparticles also gain the benefit of having increased stability compared to unprotected molecules and the capsules have the capacity for surface modifications for targeted therapy. These polymer- based nanoparticles are also biodegradable and biocompatible, making them a safe delivery agent. Thus, this research further aimed to develop a PLGA-based LRP drug delivery system for the 37 kDa Laminin receptor protein. Both synthesis of the nanoparticles and encapsulation of the LRP protein were successfully optimised and the completed drug was tested in a cell culture model, where treatment increased cell viability and telomerase activity in HEK-293 cells. Therefore, this LRP drug delivery system has great potential to assist in the translation of our in vitro studies into an in vivo context. Due to the wide range of applications elevating LRP levels has in the treatment of different disorders, this could represent a safer alternative to plasmid transfection treatment and could potentially be used for the treatment of age-related diseases, through its ability to increase telomerase activity