Dissertation submitted in fulfilment of the requirements for the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2012. / Apoptosis, or programmed cell death, is a process which is pivotal in eliminating damaged, infected, or unwanted cells from the body. It has been studied in numerous types of cell lines ranging from normal to infected cell lines, and there have been a wide range of studies on apoptosis in laryngeal cancer because this type of cancer has become one of the most common types of head or neck cancer due to the high incidence of alcohol consumption, tobacco smoking and chewing of betel quid amongst populations. Laryngeal cancer is usually treated with radiotherapy or is surgically removed, but due to the loss of the function of the larynx after surgery, it has been suggested that alternative strategies or ways of treating laryngeal cancer are required. This has prompted the use of, and research in the field of, plant medicine to combat laryngeal cancer. Plant medicine has been used for centuries by the Chinese, Indian and Arabian population in Uhani, Ayurveda and Siddha as a form of replacing conventional medicine with complementary and alternative medicine, these include many plants from the family Asphodelaceae, which have become marketable commodities owing to their medicinal values and traditional uses. Amongst these plants, the genus Bulbine has been used as a form of natural medicine in rural Africa and they are also exploited for their aloe vera properties as well as their possession of phytochemical compounds such as isoflavanoids, nor-lignans, naphthalene derivatives, anthracene and poly prenylated flavonoids. There has been a compelling amount of literature on the traditional uses of the Bulbine spp. because these are linked to the Bulbine spp. having secondary metabolites such as pyroles, chromones, coumarins, bianthraceane, benzene as well as alkaloids. However, for Bulbine natalensis and B. frutescens, the plants of interest in this study, the location of anticancer compounds in them are the only amounts of information available. It has been reported, traditionally, that B. natalensis possesses the anticancer potential in the roots, while the anticancer potential for B. frutescens is in the leaves. However, this requires scientific clarification. Therefore, this study was conducted to assess programmed cell death or apoptosis by analysing the responses of the human laryngeal carcinoma cell line (HEp-2) to crude aqueous and organic (50% and 100% ethanol) fractions of B. natalensis and B. frutescens. In order to have achieved this, the HEp-2 cell line was exposed to the above mentioned fractions at three different final concentrations (20, 2 and 1μg/ml) and assessed for cytotoxicity using the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay as an indicator of cell death after fraction utilisation (3 days) for 5 and 8 days. The differences in the potency of the Bubline spp. fractions were confirmed using the non-parametric ANOVA test. Thereafter, selected fractions were screened for apoptotic potential using reverse transcriptase-polymerase chain reaction (RT-PCR) to determine the expression of bax and caspase-3 biomarkers, which are the biomarkers that participate in mitochondrial, endoplasmic reticulum and receptor-ligand mechanism of apoptosis. The fractions of B. frutescens were selected relative to those of B. natalensis for the RT-PCR procedure (read section 3.4.1. for the selection procedure) and links between the cytotoxicity and gene expression results were analysed. It was found that the B. natalensis fractions had a much greater cytotoxic effect on the HEp-2 cell line compared to fractions of B. frutescens by the fifth day of the MTT assay. On the eight day of incubation, there was an increase in HEp-2 cell line proliferation by the fractions of both plant species administered. The fractions selected for bax and caspase-3 gene expression analysis for B. natalensis were the: 20 μg/ml root and corm aqueous fractions, 20 μg/ml leaf and corm 100% ethanol fractions, 20 μ g/ml corm 50% ethanol fraction, 2 μg/ml root aqueous fraction, 2 μg/ml leaf 100% ethanol fraction and the corm 1 μg/ml aqueous and 50% ethanol fractions. The fractions that were compared to B. natalensis were the 20 μg/ml root and leaf aqueous and 100% ethanol fractions respectively, the 2 μg/ml root aqueous fraction and the 2 μg/ml leaf 100% ethanol fraction. It was found from RT-PCR analysis that all of the B. natalensis fractions tested induced expression of caspase-3, which indicated that those fractions were capable of inducing apoptosis in laryngeal carcinoma in vitro, since caspase-3 is the molecular indicator of apoptosis. The aqueous B. frutescens root fraction, did not induce expression of caspase-3 gene, although it caused expression of bax. This implied that the root aqueous B. frutescens fraction, may be involved in some other form of cell death, other than apoptosis. It was also found that there was variability in the response of the HEp-2 cell line to the Bulbine spp. fractions because of the variation in bax expression among fractions of different concentration. It was difficult, from this study, to classify fractions into categories for their mechanism of action, because not all of the fractions that caused the expression of capase-3, induced bax gene expression. Hence, proper conclusions were unable to be made, more so, because all the mechanisms of apoptosis mentioned, involve bax gene activation in order to proceed to completion. Therefore for those Bulbine spp. fractions to which the HEp-2 cell line exhibited a variable response to, it was postulated that cell death or apoptosis occurred through some other unknown mechanism. Overall, the cytotoxicity result didn’t correlate to the gene expression results because fractions that promoted HEp-2 cell line growth by day five, expressed apoptotic markers, which highlighted the sensitivity and accuracy of the cells-to-cDNATM II kit for detecting a few possibly apoptosed cells. This was confirmed by the fact that the HEp-2 cell line used in the MTT cytotoxicity assay and gene expression study had the same passage number and were viable, the latter being achieved because the MTT assay only measures the cytotoxicity of compounds once they have been taken up by viable cells – measuring mitochondrial activities expressed as absorbances. Therefore, the deduction that HEp-2 cell death may be due to bax/caspase-3 expression was valid because the mRNA was isolated from viable HEp-2 cells that had been killed by Bulbine spp. fractions of different polarity. Furthermore, the lack of correlation between the cytotoxicity and gene expression results indicated the amount of HEp-2 cell line proliferation by the fraction out-competes those that died, thereby producing a negative cytotoxicity result. There was a relationship between the traditional information about the anticancer potential for B. natalensis and B. frutescens. For example, the aqueous root fractions of B. natalensis were found to be non-toxic to the HEp-2 cell line, but did express caspase-3, which indicated the possibility of apoptosis. Similarly, the 100% ethanol leaf B. frutescens fractions were non-toxic to the HEp-2 cell line, but were able to induce apoptosis as well. This emphasised that the MTT cytotoxicity assay should be compared with other methods of measuring cytotoxicity when performing studies like this, because although literature has emphasised many advantages of using the MTT cytotoxicity assay in apoptotic studies, this study proved otherwise. When identical HEp-2 cells were treated with the same extract, only some cells were killed (apoptosis) whereas others proliferated. This was because although the cells were identical phenotypically, they were all probably at different phases of the cell cycle resulting in the HEp-2 cells responding variably to the same fraction at different concentrations. It was also found that the responses were concentration independent. For example, the 1 μg/ml B. natalensis corm fraction exhibited the highest toxicity of the three concentrations administered. The lowest cytotoxicity was achieved for the 20 μg/ml fraction – showing a proliferative effect on the HEp-2 cell line. Similarly, the 2 μg/ml aqueous B. natalensis leaf fraction induced the highest cytotoxicity level in the HEp-2 cell line followed by the 1 μg/ml and then the 20 μg/ml fractions. Apart from the genetic variation in identical HEp-2 cells; this indicated that the HEp-2 cell line was selective to particular fractions of the Bulbine spp. for utilisation. Concentration independence and HEp-2 cell preferential selection has been reported in many other studies involving plant fractions/extracts and natural products. This study demonstrated that although all the tested B. natalensis fractions were capable of inducing HEp-2 cell death possibly via. apoptosis (caspase-3 induction), a lack of any link between apoptosis and the cytotoxicity results (hence the 20 μg/ml corm fraction had a negative cytotoxicity but expressed both apoptotic markers), indicated the need for phytochemical screening of both Bulbine spp. in future, to determine the compounds that are responsible for the cytotoxicity and gene expression result outcomes of both Bulbine spp. fractions. Furthermore, procaspase genes also have to be analysed since genes are expressed to form procaspases, which then form active caspases. Although normal cells also express caspase-3 genes during apoptosis, this study focused exclusively on the effect of Bulbine natalensis and B. frutescens fractions (selected relative to the cytotoxicity results of B. natalensis) on the HEp-2 cell line (read cell culture and cytotoxicity discussion for selection of HEp-2 cell line). The validity of this study is confirmed by similar experimental designs that assayed the cytotoxicity of plant-derived or natural compounds on cancer cell lines only, and the detection of apoptosis through caspase- 3 induction and other unrelated methods. This is the first study to report the induction of apoptosis in cancer cell lines by Bulbine spp. fractions using cytotoxicity and the expression of bax and caspase-3 apoptotic markers. It provides insight into the interaction between the HEp-2 cell line and the aqueous and organic fractions of B. natalensis and B. frutescens by analyzing links between cytotoxicity and bax and caspase-3 gene expression; which could probably contribute to drug design with selected Bulbine spp. fractions. Further investigations are required in future, to confirm the possible drug targets of the studied Bulbine spp. fractions in an attempt of assaying their therapeutic importance. / National Research Foundation

The Bunyaviridae family is one of the largest among RNA viruses, comprising more than 350 serologically distinct viruses. The family is classified into five genera, Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus. Orthobunyaviruses, nairoviruses and phleboviruses are maintained in nature by a propagative cycle involving blood-feeding arthropods and susceptible vertebrate hosts. Like most arthropod-borne viruses, bunyavirus replication causes little damage to the vector, whereas infection of the mammalian host may lead to death. This situation is mimicked in the laboratory: in cultured mosquito cells no cytopathology is observed and a persistent infection is established, whereas in cultured mammalian cells orthobunyavirus infection is lytic and leads to cell death. Bunyaviruses encode four common structural proteins: an RNA-dependent RNA polymerase, two glycoproteins (Gc and Gn), and a nucleoprotein N. Some viruses also code for nonstructural proteins called NSm and NSs. The NSs protein of the prototype bunyavirus, Bunyamwera virus, seems to be one of the factors responsible for the different outcomes of infection in mammalian and mosquito cell lines. However, only limited information is available on the growth of bunyaviruses in cultured mosquito cell lines other than Aedes albopictus C6/36 cells. Here, I compared the replication of Bunyamwera virus in two additional Aedes albopictus cell clones, C7-10 and U4.4, and two Aedes aegypti cell clones, Ae and A20, and investigated the impact of virus replication on cell function. In addition, whereas the vertebrate innate immune response to arbovirus infection is well studied, relatively little is known about mosquitoes’ reaction to these infections. I investigated the immune responses of the different mosquito cells to Bunyamwera virus infection, in particular antimicrobial signaling pathways (Toll and IMD) and RNA interference (RNAi). The data obtained in U4.4 cells suggest that NSs plays an important role in the infection of mosquitoes. Moreover infection of U4.4 cells more closely resembles infection in Ae and A20 cells and live Aedes aegypti mosquitoes. My data showed that the investigated cell lines have various properties, and therefore they can be used to study different aspects of mosquito-virus interactions.

This thesis investigates the application of a variety of optical techniques for the manipulation of single cells and their local micro-environment. The methodologies developed provide enhanced control over a single cell under study affording exquisite spatial and temporal control over biological processes of interest. The work presented within the thesis can be split into three distinct categories. The first of these provides an investigation in light activated “caged” molecular probes. This work generated several new compounds which were then applied to providing control over processes involved in pain, mitochondrial intracellular signalling and memory processes in the central nervous system. Application of caged neurotransmitters then demonstrates the first in vitro wavelength orthogonal photolysis of biologically relevant substances. Such a technique has great potential in the study of fundamental interactions within the processes underpinning memory and cognitive function. Secondly the application of optical injection techniques for the introduction of membrane impermeable species of interest is presented. An exploration of laser sources and optical systems has yielded two new strategies for optical injection. The targeted introduction of fluorescent stains, nucleic acids and gold nanoparticles to the interior of live mammalian cells demonstrates the power of these techniques. Thirdly, an investigation in optical trapping and optical injection provides simplified micromanipulation techniqes for application to biological studies. The use of capillaries as reservoirs for reagents of interest has realised a procedure for the reduction of large-scale chemical assays to a single cell level in static flow. When this technique is combined with intelligent control over the trapping laser source’s temporal behaviour, the interaction with the sample under study can be tailored for biological amiability or sample ablation. In this way a single laser source can be employed for the optical trapping and nanosurgery of a biological sample. A final study is presented demonstrating initial results for the targeted optical injection of caged compounds into mammalian cells. This methodology draws on the strengths of optical injection and caging technologies and presents a significant step forward in the level of control afforded over a biological system under study by optical techniques. The studies presented highlight the level of control and flexibility afforded by the application of optical manipulation and excitation strategies. Such optical methodologies extend the photonic tools available for enhanced studies in the life sciences.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

Forming tissues in the laboratory to replace diseased or dysfunctional tissue or act as models for drug treatment is the goal of tissue engineering. The large intestine epithelium (colon surface) is a tissue which could benefit from both diseased and non-diseased models for the purpose of tackling colon cancer causes and treatments. Scaffolds (cell supports) are a pivotal part in many tissue engineering strategies. This thesis describes the design and production of two separate scaffolds based on the degradable polymer poly(lactic-co-glycolic acid) (PLGA). The first was a two dimensional scaffold to mimic the intestinal basement membrane which was modified with an oxygen plasma. The changes to the surface due to plasma and the degradation properties of the scaffold were extensively studied with SEM, XPS, AFM and GPC. The data showed that the oxygen plasma induced surface porosity and associated changes to surface roughness. The surface chemistry as detected by XPS was unchanged by both plasma treatment and degradation in buffered solution. The plasma treatment did lead to a dramatic loss in molecular weight but the degradation profile of both the untreated and etched films was similar. Extensive cell studies with SEM, live/dead, alamarBlue and Hoechst DNA assays showed that intestinal cells on the plasma treated scaffold was enhanced in terms of morphology, metabolic activity and proliferation. Finally, a two dimensional co-culture model using epithelial and myofibroblasts cell lines on the modified PLGA scaffold was achieved. The second scaffold was a three dimensional scaffold bearing the crypt like architecture of the colon. An accurate mould produced through electron beam lithography using dimensions measured from mouse histological sections. PLGA particles were used to fill the mould and sintered to produce the scaffold. A unique cell seeding approach using cell sheets was used. The cell sheets were produced on plasma polymers of acrylic acid and the discharge power was shown to affect surface wettability, chemistry and cell viability. The cell sheet approach proved to enhance cell attachment to the scaffold compared to individual cell seeding. Finally, a bilayer scaffold with model protein to mimic Wnt protein presence in the lower half of the crypt was studied with ToF-SIMS.

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