M. Tech. / Stem cells are defined as undifferentiated cells that can proliferate indefinitely and have the capacity of both self-renewal and differentiation to one or more types of specialised cells. Traumatic tissue injury and age-related degenerative diseases are a major problem in South Africa and worldwide. Stem cells could be used for tissue engineering and reconstructive surgery. In treating these conditions, the main principle of stem cell therapy is the replacement of damaged and dead cells in injured tissues and organs with new healthy ones expanded in vitro from stem cells (Orlic et al., 2002). These cells can be isolated from adipose tissue in significant numbers and exhibit stable growth and proliferation kinetics in culture and could be differentiated into bone, fat, cartilage and muscle when treated with established lineage-specific factors (Zuk et al., 2002). Low Level Laser Therapy (LLLT) is currently applied in the treatment of numerous diseases and pathological conditions (Gasparyan et al., 2004). LLLT produces positive effects on irradiated cells and tissues such as proliferation of cells, capillary growth and adenosine triphosphate (ATP) activation (Schindl et al., 1998). Low level laser radiation at different intensities has been shown to stimulate as well as to inhibit cellular processes (Moore et al., 2005). Epidermal growth factor (EGF) is a growth factor that plays important roles in the regulation of cell growth, proliferation and differentiation. This study investigated the effect of low level laser radiation alone as well as in combination with EGF on adult adipose derived stem cells (ADSCs) isolated from human adipose tissue. ADSCs were isolated from human adipose tissue through collagenase digestion and cultured in DMEM-F12 containing 10% FBS and antibiotics and incubated at 37°C in a humidified atmosphere of 5% CO2 (Zuk et al., 2001). iii Semi-confluent monolayers of ADSCs were exposed to low level laser at 5 J/cm2 using 636 nm diode laser with a power density of 12.1 mW/cm2 at room temperature in the dark. Cell morphology was monitored at 0, 24 and 48 h using an inverted light inverted microscope. Cell viability was evaluated at 0, 24 and 48 h using the Trypan Blue exclusion test and an adenosine triphosphate (ATP) luminescence assay. bFGF (basic fibroblast growth factor) indirect ELISA and optical density assays were used to monitor cell proliferation at 0, 24 and 48 h post irradiation. In addition the expressions of stem cell markers, β1-integrin and Thy-1, were monitored by immunocytochemical live cell surface labelling and Western blot analysis. Cells were incubated with EGF to enhance proliferation and differentiation and the cell morphology, viability and proliferation were monitored as well as the expressions of stem cell markers, β1-integrin and Thy-1. Morphology of the cells was not altered by irradiating them with 5 J/cm2 using diode laser at 0, 24 and 48 h. Cell viability and proliferation showed an increase at 24 and 48 h post irradiation. At 0 h, there was no significant difference between irradiated and non-irradiated cells in cell viability and proliferation. There was an increase in the expression of β1-integrin and Thy-1 after irradiation as shown by Western blot analysis and immunocytochemical live cell surface labelling. Cell viability and proliferation showed a significant increase at all time points post irradiation with the addition of EGF. There was no noticeable change in cellular morphology at any time point. Low level laser irradiation of human ADSC’s at 636 nm with 5 J/cm2 and 12.1 mW/cm2 increased the viability and proliferation of these cells in vitro. Furthermore, low level laser irradiation appeared to increase the expression of stem cell markers, β1-integrin and Thy-1. In addition, laser irradiation did not alter the morphology of the cultured cells. The addition of EGF to the cells also increased their viability and proliferation as well the expression of the markers, β1-integrin and Thy-1. The study showed that laser irradiation stimulates two important cellular responses namely cell viability and proliferation which indicates that ADSCs may be suitable for tissue engineering and future cell differentiation studies.

M.Tech. / Stem cells possess self-renewal capacity, long-term viability, and multilineage potential. Stem cells play important roles in normal physiological and disease processes, they also have great therapeutic potential. However, there have been controversies surrounding stem cells in political, religious and ethical arenas. Although the use of certain stem cells (i.e. embryonic stem cells) and the means by which they are obtained contravene certain basic ethical laws, researchers have developed methods with which to ethically obtain and create stem cell lines. Stem cells can be classified as either: totipotent, pluripotent, multipotent, oligopotent and unipotent (Moore, 2007). Totipotent cells have the ability to differentiate into all cell types of an embryo, including the extra-embryonic and post embryonic tissues and organs. Pluripotent cells have the potential to differentiate into almost all tissues found in an embryo (including germ cells), but are not capable of giving rise to supporting cells and tissues. Multipotent stem cells have progeny of several differentiated cell types - but all within a particular tissue, organ, or physiological system. A good example of multipotent cells, are the haematopoietic stem cells that produce blood cell-restricted progenitors, as well as all cell types and elements, such as platelets, that are normal components of blood. Oligopotent stem cells produce two or more lineages within a specific tissue, such as neural stem cells that are able to produce subsets of neurons in the brain. Unipotent cells self-renew, as well as give rise to a single mature cell type, a prime example being the spermatogonial stem cells, that give rise to spermatozoa (Moore, 2007). Adult human subcutaneous adipose tissue contains cells with multilineage developmental plasticity like marrow-derived mesenchymal stem cells (Strem et al., 2005, Tong et al., 2000). Adipose derived stem cells can be obtained in abundance and can differentiate into osteogenic, adipogenic, myogenic and chondrogenic lineages when treated with appropriate growth factors.

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The Cancer Stem Cell (CSC) theory suggests that cancers arise from and are maintained by a subpopulation of cancer cells with stem cell properties. Molecular chaperones are key components of cellular regulation. The overexpression of chaperones has become synonymous with cancer cells with chaperones being recognized as bona fide anti-cancer drug targets. Although chaperone activity has been characterized in cancer cells, very little is known about the cellular functions of chaperones in cancer stem cells. We set out to compare the expression of selected molecular chaperones in non-stem cancer cell and cancer stem cell enriched populations isolated from breast cancer lines, in order to identify chaperones differentially expressed between the two populations for further biological characterization. In order to isolate breast cancer stem cells from the MCF-7 and MDA-MB-231 breast cancer cell lines, three cancer stem cell isolation and identification techniques were utilized based on (1) cell surface marker expression (CD44+/CD24- and CD44+/CD24-/EpCAM+ phenotypes), (2) aldehyde dehydrogenase enzyme activity (ALDHHi) and (3) ability to grow in anchorage-independent conditions. The MDA-MB-231 and MCF-7 breast cancer cell lines displayed CD44+/CD24- cell populations with the MCF-7 cell line additionally displaying a large CD44+/CD24-/EpCAM+ population. Although both cell lines showed similar ALDHHi populations, they differed substantially with respect to anchorage-independent growth. MCF-7 cells were able to form anchorage-independent colonies while the MDA-MB-231 cell line was not. Anchorage-independent MCF-7 cells showed enrichment in CD44+/CD24- and CD44+/CD24-/EpCAM+ cells compared to adherent MCF-7 cells, and were selected for gene expression studies. Gene expression studies identified 22 genes as being down-regulated at the mRNA level in the anchorage-independent MCF-7 cells, while only 2 genes (BAG1 and DNAJC12) were up-regulated. The down-regulation of selected chaperones in anchorage independent MCF-7 cells was confirmed at the protein level for selected chaperones, including DNAJB6, a type II DNAJ protein shown to be involved in the regulation of Wnt signaling. In order to characterize the effect of DNAJB6 expression on BCSCs we developed a pCMV mammalian expression plasmid for both DNAJB6 isoforms (DNAJB6L and DNAJB6S). We successfully constructed mutants of the conserved histidine-proline-aspartic acid (HPD) motif of the J domain of DNAJB6S and DNAJB6L. These constructs will allow the analysis of the role of DNAJB6 in cancer stem cell function. To the best of our knowledge, this is the first report to focus on the comparative expression of molecular chaperones in normal and cancer stem cell enriched breast cancer populations.

Biological pre- and postharvest disease control strategies depend on successful colonisation and survival of the introduced antagonists on the infection court. Effective and economical applications involve targeting the antagonists where they are critically needed i.e. the infection court. Honeybee dispersal is one method of antagonist application to such specific sites. In view of this, an in vitro experiment was conducted to investigate attachment, colonisation and survival of Bacillus subtilis on avocado flowers. Scanning electron microscopy studies showed that the bacterium could attach and colonise avocado flower surfaces. It can also survive on the flowers for longer periods of time. In vivo mode of action of the antagonist against stem-end rot (SER) pathogens was also studied where results showed lysis and degradation of hyphae and conidia. However, no viable colonies were retrieved from bee antagonist dispersal under field conditions. Bee antagonist dissemination was compared with antagonist and fungicide spray applications in terms of SER control and the added effect on other diseases such as Cercospora spot and anthracnose. Spray applications of the antagonist were more effective in reducing the incidence of SER than bee dissemination. Integrated sprays of the antagonist and fungicides significantly reduced the incidence of both pre- and postharvest diseases. The identity of Dothiorella aromatica, one of the most important SER pathogens, was investigated at a molecular level. RAPD techniques using the discriminatory OPC02 primer successfully separated isolates into three groups based on banding profiles. A further study using RFLP identified the pathogen as a Botryosphaeria spp. The most dominant specie was B. parva followed by B. rhodina. Further studies should focus on assessing the distribution of these pathogens within avocado-growing regions of South Africa. / Dissertation (MSc (Plant Pathology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Pluripotent embryonic stem cells hold a great promise as an unlimited source of tissue for treatment of chronic diseases such as Type 1 diabetes and chronic liver disease. Various attempts have been made to produce cells that can serve as precursors for pancreas and liver. By using all-trans-retinoic acid, basic fibroblast growth factor, dibutyryl cAMP, and cyclopamine, an attempt has been made to produce definitive endoderm and subsequently cells that can serve as pancreatic and hepatocyte precursors from mouse embryonic stem cells. By using retinoic acid and basic-FGF, in the absence of embryoid body formation, mouse embryonic stem cells were differentiated at different culture periods. Four protocols of varying lengths of culture and reagents and their cells were analyzed by quantitative PCR, immunohistochemistry and static insulin release assay for markers of trilaminar embryo, pancreas and hepatocytes. Inclusion of DBcAMP and extension of culture time resulted in cells that display features of definitive endoderm by expression of Sox 17 and FOXA2 and minimal expression of primitive endoderm and other germ cell layers such as ectoderm and mesoderm. These cells produced insulin and C-peptide and secreted insulin in a glucose responsive manner. However, they seem to lack mature insulin secretion mechanism. There was a production of hepatocyte markers (AFP-2 and transthyretin) but there was insufficient data to assess for convincing production of hepatocytes. In summary, one of the protocols produced cells that displayed characteristics of definitive endoderm and they may serve as pancreatic endocrine precursors. / Surgery, Department of / Medicine, Faculty of / Graduate

Telomeres are the repetitive DNA sequence and associated proteins found at the ends of linear chromosomes. They have a role in biological processes including meiosis and aging as well as implications in a number of genomic instability disorders and cancers. Telomeres maintain genomic stability by protecting chromosome ends from terminal fusions and misidentification as DNA damage sites. Their wide range of functions has resulted in an increased interest in developing tools to study the dynamics of telomeres in live cells. To do this, current studies use the ubiquitously expressed protein Telomere Repeat Factor 1 (TRF1) tagged with a fluorescent protein. TRF1 is a negative regulator of telomere length that binds exclusively to telomere repeats. Over-expression of the fluorescent protein fused to TRF1 has been a useful tool to track telomere movement. The foci formed by the tagged TRF1 protein accurately represent the number of telomeres expected in the cells and the localization is maintained throughout the cell cycle. A caveat with this system is that over-expression of TRF1 leads to accelerated telomere shortening, as well as replication defects that can stall telomere replication. These caveats make it difficult to draw conclusions about telomere dynamics based solely on observations of cells over-expressing fluorescently tagged TRF1. To eliminate problems associated with protein over-expression, I have tried to develop knock-in embryonic stem (ES) cells expressing fluorescently tagged TRF1 from the endogenous Trf1 promoter. To do this, I have used a recombineering technique using Bacterial Artificial Chromosomes (BACs). BAC recombineering allows for the direct knock-in of a fluorescent tag into the mouse Trf1gene locus. Genetic constructs with the correct sequence inserts have been obtained and have been used for transfection of ES cells. While no correctly targeted ES cells have been identified so far, the expectation is that ES cell lines with correctly targeted fluorescently tagged TRF1 will be obtained in the near future. Such lines will be used to study telomere dynamics in ES cells, differentiated cells generated from ES cells, as well as to generate mice. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

A central tenet of stem cell biology has been that proliferating tissues are maintained through a cellular hierarchy comprising of self-renewing stem cells at the apex, multiple lineage-restricted short-lived progenitor cells, and post-mitotic differentiated cells. The wide range of colony sizes in cultured human keratinocytes has been taken to support this hypothesis. Contrary to this model, researchers using genetic lineage tracing in mouse epidermis have inferred a single progenitor population for homeostasis, and a quiescent stem cell population activated upon wounding or genetic mutation. To study the proliferative behaviour of human keratinocytes, I used live imaging in vitro at single cell resolution. This shows two modes of proliferation: Type 1 cell division is stochastic with equal odds of generating dividing or non-dividing progeny, while Type 2 cell division predominantly produces two dividing daughters. These two modes are sufficient to explain the entire range of colony sizes seen after 7-12 days of culture and does not require a spectrum of proliferative ability. This insight provides a simple way to study the effects of external factors on cell fate. To exemplify this, I observed the effects of epidermal growth factor (EGF) and the Wnt agonist R-spondin on proliferation. Here I find proliferation in type 2 colonies changes by changing the proportion of cells dividing. This has implications for the limited success of EGF therapies in clinical trials following burns. To examine clonal contributions to wound repair, I used the mouse oesophageal epithelium which is exclusively composed of, and maintained by, a single progenitor population. I developed a micro-endoscopic wounding technique that produced localised superficial wounds. Here, I found that these wounds healed by uniform contribution from surrounding keratinocytes, demonstrating that reserve stem cells are not obligatory for wound repair. In summary, my work shows that human keratinocytes in vitro have two, and only two, modes of proliferation: a stochastic mode that is insensitive to external EGF signalling, and a EGF-sensitive exponential mode. Additionally, proliferation during wound repair can occur with stochastically dividing progenitors, and does not obligate stem cell recruitment in vivo.

The eukaryotic cell nucleus is a highly organised organelle, with distinct specialised sub- compartments responsible for specific nuclear functions. Within the context of this functional framework, the genome is organised, allowing contact between specific genomic regions and sub-compartments. Previous work has shown that genes in both cis and trans can make specific contacts with each other. I hypothesise that such a preferred juxtaposition may impact the propensity for specific cancerinitiating chromosomal translocations to occur. In this thesis, I describe how I have extended and developed a ligation based proximity assay known as enriched 4C. I have coupled this technique with high throughput sequencing to determine genomic regions that spatially co-associate with the proto-oncogenes MLL, ABL1 and BCR. In addition to further developing the laboratory protocol, I have created bioinformatics tools used in the analysis of the sequencing data. I find that the association profiles of the three genes show strong correlation to the binding profile of RNA polymerase II and other active marks, suggesting that transcribed genes have a propensity to associate with other transcribed regions of the genome. Each gene also exhibits a unique repertoire of preferred associations with specific regions of the genome. Significantly, I find that the most frequent trans association of BCR is telomeric chromosome 9, encompassing its recurrent translocation partner gene ABL1. Interestingly, ABL1 is not at the maximum point of interaction. I use DNA-fluorescence in-situ hybridisation to validate the e4C association. My data supports a hypothesis that gene transcription has a direct role on genome organisation. I suggest that preferred co-associations of genes at transcription factories may promote the occurrence of specific chromosomal translocations.

Upon activation in epidermal stem cells, the proto-oncogene c-Myc triggers their exit from the stem cell compartment resulting in an increase in progenitor cell proliferation and an induction in terminal differentiation. Whether c-Myc plays a direct transcriptional role in epidermal stem cell differentiation was unknown. The exploration of c-Myc's transcriptional roles at the epidermal differentiation complex (EDC), a locus essential for skin maturation demonstrated that binding of c-Myc to the EDC can simultaneously recruit and displace specific sets of differentiation-specific transcriptional regulators to EDC genes. Among these factors, Sin3A acts as a transcriptional co-repressor and was initially discovered via its direct interaction with Mxi1 and Mxd1, which are antagonists of the Myc family network. As such, I concentrated on the role of Sin3A as a potential opposing factor to c-Myc activity in the epidermis. To analyse the role of Sin3A in regulating epidermal stem cell fate in vivo, I generated a number of transgenic mouse models. To determine whether Sin3A functions in hair follicle stem cells, I inducibly deleted Sin3A in the hair follicle bulge, where quiescent stem cells reside. However, lack of Sin3A in the hair bulge did not cause any aberrant phenotype and I concluded that Sin3A is dispensable for hair follicle homeostasis. I next analysed a mouse model in which Sin3A is inducibly deleted in the basal layer of the epidermis. Deletion of Sin3A resulted in a severe disruption of epidermal homeostasis-namely due to increases in proliferation and differentiation. Further investigation demonstrated that this phenotype is driven by enhanced genomic recruitment of c-Myc to the epidermal differentiation complex and reactivation of c-Myc target genes involved in cellular proliferation. I found that Sin3A causes de-acetylation of the c-Myc protein to directly repress c-Myc’s transcriptional activity and is antagonistic to c-Myc in the interfollicular epidermis. I hypothesised that simultaneous deletion of Sin3A and c-Myc might return the skin to normality. Indeed, when Sin3A and Myc are concurrently deleted, proliferation and differentiation levels returned to normal. These results demonstrate how levels of Sin3A and c-Myc must be carefully balanced for epidermal homeostasis to be maintained. Decreased expression of Sin3A has been linked to tumour susceptibility in other tissues for example in non-small cell lung carcinoma making Sin3A a candidate tumour suppressor gene. I therefore considered that loss of Sin3A may lead to increased susceptibility to skin cancer. To investigate this I performed pilot experiments using UVB irradiation of skin that has one copy of Sin3A deleted in the basal layer of the epidermis. Under normal conditions, these mice have no identifiable phenotype, but pilot experiments demonstrated that after short term and long term UVB irradiation, they exhibit increased epidermal thickness and proliferation relative to controls. This recapitulated the phenotype observed when Sin3A is inducibly deleted in the interfollicular epidermis and further demonstrates the role of SinA as an inhibitor of proliferation in this tissue. Overall, these results demonstrate that an interplay between the opposing functions of Sin3A and c-Myc are necessary to ensure that there is balanced homeostasis in the interfollicular epidermis.