Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by expression of defined embryonic factors. My thesis is focused on exploring the mechanisms underlying reprogramming using a secondary mouse embryonic fibroblast model that forms iPS cells with high efficiency upon inducible expression of Oct4, Klf4, c-Myc and Sox2. My analyses of the temporal changes in gene expression reveal that reprogramming is a multi-step process characterized by initiation, maturation and stabilization phases. Using functional RNAi screening, I discovered a key role for BMP signaling and the induction of mesenchymal-to-epithelial transition (MET) during the initiation phase. I showed that MET induction was linked to BMP-dependent induction of miR-205 and the miR-200 family of microRNAs. These studies thus defined a multi-step mechanism that incorporates a BMP-miRNA-MET axis during somatic cell reprogramming. Next I focused on the two later phases of reprogramming, maturation and stabilization. I showed the stabilization phase and acquisition of pluripotency is dependent on removal of transgene expression late in the maturation phase. Clonal analysis of reprogramming cells revealed subsets of stabilization competent (SC) versus stabilization incompetent (SI) cells. SC clones robustly entered the pluripotent state upon transgene withdrawal in the late, but not early maturation phase, whereas SI clones failed to reprogram at either stage. Transcriptome profiling by RNA-Seq revealed that SC clones acquire a competency gene expression signature late in the maturation phase. Functional RNAi screening of SC signature genes further identified regulators of transition to the stabilization phase, while screening of the same signature in iPS cells revealed a distinct subset of genes required for maintenance of pluripotency. These studies reveal that the acquisition and subsequent maintenance of pluripotency are controlled by distinct molecular networks and uncover a novel regulatory program that is required for transition to transgene independence.

Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by expression of defined embryonic factors. My thesis is focused on exploring the mechanisms underlying reprogramming using a secondary mouse embryonic fibroblast model that forms iPS cells with high efficiency upon inducible expression of Oct4, Klf4, c-Myc and Sox2. My analyses of the temporal changes in gene expression reveal that reprogramming is a multi-step process characterized by initiation, maturation and stabilization phases. Using functional RNAi screening, I discovered a key role for BMP signaling and the induction of mesenchymal-to-epithelial transition (MET) during the initiation phase. I showed that MET induction was linked to BMP-dependent induction of miR-205 and the miR-200 family of microRNAs. These studies thus defined a multi-step mechanism that incorporates a BMP-miRNA-MET axis during somatic cell reprogramming. Next I focused on the two later phases of reprogramming, maturation and stabilization. I showed the stabilization phase and acquisition of pluripotency is dependent on removal of transgene expression late in the maturation phase. Clonal analysis of reprogramming cells revealed subsets of stabilization competent (SC) versus stabilization incompetent (SI) cells. SC clones robustly entered the pluripotent state upon transgene withdrawal in the late, but not early maturation phase, whereas SI clones failed to reprogram at either stage. Transcriptome profiling by RNA-Seq revealed that SC clones acquire a competency gene expression signature late in the maturation phase. Functional RNAi screening of SC signature genes further identified regulators of transition to the stabilization phase, while screening of the same signature in iPS cells revealed a distinct subset of genes required for maintenance of pluripotency. These studies reveal that the acquisition and subsequent maintenance of pluripotency are controlled by distinct molecular networks and uncover a novel regulatory program that is required for transition to transgene independence.

The South African Sugarcane production is one of the world’s leading sugarcane (Saccharum spp. Hybrid) producers. However, in recent years Eldana saccharina Walker has been the most destructive pest in South African sugarcane production, causing great crop loses per annum and is the most important factor limiting sugarcane productivity. The pest has been monitored using a traditional visual approach whereby a representative sample of stalks is taken from a field and split longitudinally to assess damage and count the number of E. saccharina larvae and pupae. However, this approach is time-consuming, labour intensive and sometimes biased as only easily accessible areas are often surveyed. In order to investigate a more economical but equally effective survey methodology, this study aimed to determine the potential of using hyperspectral remote sensing (spectroradiometry) for identifying sugarcane attacked by E. saccharina. A hand-held spectroradiometer ASD Field Spec® 3 was used to collect leaf spectral measurements of sugarcane plants from a potted-plant trial taking place under shade house conditions at the South African Sugarcane Research Institute (SASRI). In this trial, nitrogen (N) and silicon (Si) fertilizers were applied at known levels to sugarcane varieties. Varieties were either resistant or intermediate resistant or susceptible to E. saccharina attack. In addition, watering regimes and artificial infestation of E. saccharina were carefully controlled. Results illustrated that severe E. saccharina infestation increased spectral reflectance throughout the whole spectrum range (400 – 2500 nm) and caused a red-edge shift to the shorter wavelength. Eldana saccharina stalk damage was also linearly related to modified normalized difference vegetation index (mNDVI) using R2025 and R2200 (R2 = 0.69). It was concluded that hyperspectral data has a potential for use in monitoring E. saccharina in sugarcane rapidly and non-destructively under controlled conditions. A followup study is recommended in field conditions and using airborne and/or spaceborne hyperspectral sensors. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2009.

Quantitative trait loci (QTL) analysis for Sclerotinia stem rot resistance was carried out in five doubled haploid (DH) populations of Brassica napus. Sclerotinia stem rot is caused by the necrotrophic fungus Sclerotinia sclerotiorum (Lib.) de Bary. Sclerotinia stem rot has worldwide occurrence and causes significant yield losses in many crop species. Several screening methods have been recommended in the literature to evaluate plant resistance to Sclerotinia stem rot. Four controlled environment based screening methods: 1) excised leaf assay, 2) cotyledon assay, 3) mycelial stem inoculation technique and 4) petiole inoculation technique compared for their ability to differentiate between plant susceptibility/resistance, their reliability and suitability for large scale screening using eight B. napus cultivars/lines of varying reaction to S. sclerotiorum. The petiole inoculation technique and the mycelium stem inoculation technique were identified as reliable methods in this study. Previously developed, five B. napus DH populations (H1, H2, H3, DH179 and DH180) segregating for resistance to Sclerotinia stem rot were used in this study. The petiole inoculation technique was used to evaluate resistance to Sclerotinia stem rot. Data on days to wilting was recorded for a two week period. Twelve plants per line were screened in each evaluation and each population was evaluated three times. Two to three day-old mycelial cultures of S. sclerotiorum isolate Canada 77 was used. QTL analyses were carried out using a LOD threshold value of 2.5 on each individual replicate and on the average of all the replicates. In the H1 population, the number of QTL detected ranged from four to six in each analysis. In the H2 population, there were three to six QTL in each analysis. There were two to six QTL in each analysis of the H3 population. In the DH179 population, the number of QTL detected ranged from three to five in each analysis. In DH180 population, the number of QTL identified varied from three to six in each analysis. A number of common QTL were found between the replicates of each population. Five common QTL were identified between these populations. The markers linked to these QTL are now available for marker assisted selection.

This thesis includes two parts related to hydrogels as therapeutically useful constructs: a biomimetic hydrogel carrying stem cells for bone regeneration and an acid-sensitive hydrogel carrying drugs for cancer therapy. In tissue engineering, one of the biggest difficulties is the control of stem cell fate on scaffolds. A biodegradable and cell attachable cross-linker was synthesized by one-step Michael additional reaction, and was used to fabricate a novel hydrogel to control the stem cell fate. For anti-cancer therapy, releasing drug on tumor cells or organs while having low effects on health cells under physiological conditions is a critical requirement. Two nature polymers are modified to achieve loading anti-cancer drug while forming hydrogels which can selectively release the drug in tumor environment by acid-sensitive linkages.

Human embryonic stem cells (hESC) present a potentially unlimited supply of hematopoietic progenitors for cell-based therapies. However, current protocols for generating these progenitors typically also generate undesired cell types due to imprecise control of the hESC microenvironment and poor understanding of the signalling networks regulating mesoderm differentiation (the germ layer from which hematopoietic cells emerge). This report demonstrates that activation of the downstream effectors of Activin/Nodal and bone morphogenetic protein (BMP) signalling (Smad2 (composite of Sma (smaller) and Mad (mothers against decapentaplegic) and Smad1, respectively) are both required for mesoderm differentiation. It is further shown that microcontact printing-mediated control of hESC colony size creates local microenvironments that guide differentiation, via a Smad1-dependent mechanism, preferentially towards the mesoderm lineage. These findings demonstrate the need for precise control of the microenvironment in order to effectively guide hESC differentiation to produce specific cell types for potential therapeutic applications.

This thesis shows that p53-deficient mouse glioma brain tumour stem cells (BTSCs), which fail to express pluripotency factors, can be reprogrammed with specific transcription factors to generate iPS cell lines (GNS-iPS) expressing endogenous pluripotency factors (Nanog, Oct4, and Rex1). GNS-iPS cell lines formed embryoid bodies (EBs) in vitro and undifferentiated growths in vivo that phenotypically did not resemble tumours derived from non-reprogrammed BTSCs. EBs formed from one GNS-iPS cell line expressed markers of mesoderm, endoderm, and ectoderm. Tumours produced from GNS-iPS cells had reduced astrocytic marker (GFAP) expression compared to those generated from control iPS cell lines or non-reprogrammed BTSCs. Preliminary results suggest that the reprogrammed cells can be re-differentiated into cells that show neural precursor phenotype. These findings suggest that BTSCs can acquire aspects of the pluripotent state with a defined set of transcription factors, opening the door for further exploration of reprogramming strategies to attenuate the cancer phenotype.

Adult mammopoiesis occurs in close synchronization with reproductive development when the hypothalamic-pituitary-ovarian axis delivers integral systemic hormone cues to propel mammary morphogenesis during puberty, remodeling during reproductive cycles and functional differentiation following pregnancy. While hormones remain the driving force behind normal glandular development, increased life-time hormone exposure is a strong risk factor for breast cancer. Breast cancer heterogeneity has been attributed to different cells of origin and/or different mutation repertoires. Stem/progenitor cells are intensely investigated as cells of origin given their regenerative and self-renewal properties that provide conceivable advantage in cancer. Although hormones have a fundamental influence in breast cancer, their capacity to regulate stem/progenitor cells was unknown, and presents the central directive in this thesis. Employing mouse models, we show that mammary epithelial subpopulations and in particular, stem cells, are highly responsive to ovarian hormones and depend on key molecular events. A progesterone peak during the luteal phase of reproductive cycles results in a significant increase in stem cell-enriched basal cells and an expansion of stem cells measured by in vivo transplantation assays, with rapid development of lobuloalveoli. Progesterone was found to stimulate expression of mitogenic ligands, RANKL and Wnt4, in ER+PR+ luminal epithelial niche cells concomitant with increased expression of their receptors and target genes in the ER-PR- basal stem cell population, suggesting a cross-talk between luminal and basal cells that elicits stem cell expansion within the niche. The requirement of RANKL signaling for hormone-induced mammary stem cell dynamics was further explored utilizing mice deficient for its receptor, RANK, and by pharmacological inhibition of RANKL. Disruption of RANKL/RANK signaling resulted in abrogated activation of the basal stem cell-enriched population and alveolar progenitor cells in response to progesterone. This was accompanied by a marked reduction in cell proliferation, cell cycle regulators, alveolar lineage determinants and notably, in epithelial Wnt responsiveness. Thus, progesterone orchestrates a series of molecular events in the mammary stem cell niche where RANK is effectively positioned to deliver instructive signals to stem cells, culminating in stem cell recruitment and alveolar regeneration, processes which when deregulated have considerable potential to promote breast cancer pathogenesis.

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