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Indiana University-Purdue University Indianapolis (IUPUI) / In order to develop effective cures for diseases and decipher disease pathology, the need exists to cultivate a better understanding of human development. Existing studies employ the use of animal models to study and model human development and disease phenotypes but the evolutionary differences between humans and other species slightly limit the applicability of such animal models to effectively recapitulate human development. With the development of human pluripotent stem cells (hPSCs), including Human induced Pluripotent stem cells (hiPSCs) and Human Embryonic Stem cells (hESCs), human development can now be mirrored and recapitulated in vitro. These stem cells are pluripotent, that is, they possess the potential to generate any cell type of the body including muscle cells, nerve cells or blood cells. One of the major focuses of this study is to use hiPSCs to better understand and model human retinogenesis. The retina develops within the first three months of human development, hence rendering it inaccessible to investigation via traditional methods. However, with the advent of hiPSCs, retinal cells can be generated in a culture dish and the mechanisms underlying the specification of a retinal fate can be determined. Additionally, in order to use hiPSCs for successful cell replacement therapy, non-xenogeneic conditions need to be employed to allow for fruitful transplantation tests. Hence, another emphasis of this study has been to direct hiPSCs to generate retinal cells under non-xenogeneic conditions to facilitate their use for future translation purposes.

This thesis considers the understudied issue of genetic engineering as it has been deployed in the literature of the late 20th century. With reference to the concept of the enlightened gender hybridity of Cyborg theory and an eye to ecocritical implications, I read four texts: Joan Slonczewski's 1986 science fiction novel A Door Into Ocean, Octavia Butler's science fiction trilogy Lilith's Brood – originally released between 1987 and 1989 as Xenogenesis – Simon Mawer's 1997 literary novel Mendel's Dwarf, and the first two books in Margaret Atwood's speculative fiction MaddAddam series: 2003's Oryx and Crake and 2009's The Year Of the Flood. I argue that the inclusion of genetic engineering has changed as the technology moves from science fiction to science fact, moving from the fantastic to the mundane. Throughout its recent literary history, genetic engineering has played a role in complicating questions of sexuality, paternity, and the division between nature and culture. It has also come to represent a nexus of potential cultural change, one which stands to fulfill the dramatic hybridity Haraway rhapsodized in her "Cyborg Manifesto" while also containing the potential to disrupt the ecocritical conversation by destroying what we used to understand as nature. Despite their four different takes on the issue, each of the texts I read offers a complex vision of utopian hopes and apocalyptic fears. They agree that, for better or for worse, genetic engineering is forever changing both our world and ourselves.

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Il s’agit de montrer que les technosciences, rencontrant l’humanitude comme leur condition de possibilité et leur moteur propulsif, risquent d’actualiser une potentialité aporétique associée à une enclave organique perméable et à une maintenance identitaire inscrite dans le devenir - associée à une intériorité se dépliant en extériorité et à un monisme substantiel s’exprimant dans la dualité (en soi, comme soi et hors de soi). Pour ce faire, nous observons et confrontons :les techniques (des PMA aux manipulations d’embryons, des diagnostics génétiques aux thérapies géniques, du clonage à la transgenèse) à l’individu, l’individu à ses latitudes (en ce compris par la voie d’une «consultation» des personnes souffrantes, des scientifiques et de la population dite «générale») et celles-ci à l’humanitude. <p>Par ailleurs, nous définissons l’individu d’une indéfinition :où l’homme est point de convergence (entre divers états, plans et déterminants) et force d’émergence (hors ses donnés) - équilibre en construction incessante et incessible. Où sa spécificité tient à l’occupation corporelle et pensée d’un espace et d’un temps :équilibre en soutenance entre stase et métabolisme, non-soi et soi, subordinations et libérations, centralisations identitaires et extériorisations identifiantes. Par suite, toute intervention pesant en déséquilibre sur ces articulations devrait être évitée – en principe. En principe car, du fait de la multiplicité et de la diversité des intervenants, du fait même de leur liberté et des incertitudes plurielles, il y aura toujours pression ou déséquilibre. Néanmoins, il importe de mesurer l’acte aux conditions de la liberté et de l’humanité :conscience, autonomie, libre disposition de soi, sensibilité, émotivité, souci, malléabilité principielle (contre une assignation à demeure spécielle ou existentielle) et réappropriation essentielle (contre un déterminisme global – une appropriation par tiers). Et il convient de préserver le lien du corps et de l’esprit :où l’esprit transcende le corps qui le forme et l’informe – selon une mise à distance au sein d’une unité (et d’une unicité). <p>Or, nous constatons que l’humanitude, prise en charge par les techniques qu’elle produit, édifie un domaine existentiel caractérisé par une biographie de l’arrachement ou du désinvestissement. Et que l’homme, être de l’entre-deux défini par la négation de tout Etat advenu, produit un processus technique propre à l’extraire de cet «entre-deux». Déjà, l’individu libère ses tendances dispersives et ses tendances confusionnelles :mise à distance de l’entité corporelle (en sa force référentielle ou définitoire), identité décisionnelle et puissance volitive plus dispersive que centralisatrice. Où donc néoténie, imparfaite assignation, distanciation et in-essentialité ouvrent à la liberté tout en autorisant l’incorporation du non-soi, l’opérativité de la volonté et l’évanescence des états de l’organisme et de la psyché (de la personnalité comme tout identitaire). Cependant, si les techniques font exploser ces équilibres, reste la dissipation :où le «soi serait amené à se reconnaître comme pure et simple concept construit. Dans ce contexte, tout s’abandonne aux modifications. Et l’homme de jouer avec le donné et le donné en lui - donné qui est lui mais se décompose dorénavant en fonds «sacral» de puissances, en substrats géniques, mnésiques et morphologiques modelables et en constituants interchangeables. Semblable attitude recouvre une fuite hors de la condition humaine :tantôt vers «autre chose», tantôt vers un alignement démissionnaire sur un Décideur Transcendant. Et pour la première fois aussi globalement et intensément, instinct et volonté de survivre se soumettent à leur propre négation :pour qu’il subsiste «quelque chose plutôt que rien» dans le futur lointain, l’espèce conspire à sa propre fin. Pour la première fois (première fois aussi proche d’une réalisation), l’individu aspire à une dispersion de conscience, à une évanescence personnale et à une fin de l’Histoire sous couvert d’une histoire sans fin et d’un devenir incessant – gardant du devenir la seule processualité mécanique. Le danger est alors considérable car l’homme sort de l’animalité dans le champ où s’entrechoquent libre arbitre et déterminisme, références identitaires et décentrages, appartenances et abstractions. Car cet animal-là exprime sa spécificité à l’extérieur de l’enceinte biologique mais à partir d’une densité individuale :dans l’élaboration sociale et culturelle. Dès lors, quand l’existence précède l’essence et face aux possibles technoscientifiques, il importe de préserver l’homme d’une existenciation illusoire et d’une personnalisation évanescente. Et de pondérer la totalité individuale de ses dimensions temporelles :où l’individu est un être perpétuellement devenant ;où l’existence crée, investit et signifie une durée ;où l’identité est continuité d’unicité en devenir ;où l’humanité est construction d’Histoire et invention de sens. Cela oblige l’individu à se soutenir eu égard à des doubles nœuds référentiels :anthropique et autobiographique, culturel et familial, spirituel ou décisionnel et charnel ou factuel, symbolique et opératoire, autoréférentiel et relationnel. Mais aussi, en matière éthique, entre principe et casuistique, idéal et exception, collectif et individuel.<p>Doubles nœuds contestés par les techniques. Aussi, face à l’opérativité croissante de celles-ci, nous nous interrogeons sur ce qu’il pourrait advenir de la condition humaine et des conditions de possibilité de l’homme - considéré ici comme individu (spécimen défini en ses spécificités) au milieu du monde, conscience (sensible et émotionnelle) en situation d’interrelation, et subjectivité (volitive) face à ses semblables. <p> / Doctorat en philosophie et lettres, Orientation bioéthique / info:eu-repo/semantics/nonPublished

Text in English / South Africa is one of the biggest producers of genetically modified crops in the world. However, recent studies in South Africa show a low public willingness to consume genetically modified crops and accept modern agricultural biotechnology. The study analysed public perception towards consuming genetically modified crops and the acceptance of modern agricultural biotechnology in South Africa. 220 participants (N = 220) were sampled from the city of Kempton Park and the Chi-square formula was used to determine how well the sample represented the population under study. Data was collected using a 7-point Likert scale questionnaire designed following the guidelines for developing a theory of planned behaviour questionnaire in Ajzen (1991, 2001). Data analyses were carried out using the Statistical Package for Social Sciences (SPSS). The Cronbach’s alpha and Exploratory Factor Analysis were both used to determine the internal consistency and validity of the questionnaire. Correlations, independent sample t-tests, ANOVA, linear regression, and path analysis were also conducted. Findings of the study confirmed that there is low public willingness to consume genetically modified crops and to accept modern agricultural biotechnology in South Africa. / Development Studies / M.A. (Development Studies)

Genetically modified (GM) white and yellow maize, Zea mays, has been commercially released and cultivated in South Africa since 1997/1998. The traits expressed are insect resistance and herbicide tolerance conferred by the bacteria Bacillus thuringiensis (Bt) Cry genes and Agrobacterium 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase gene, respectively. The Cry genes have been used widely to control lepidopteran insect pests but insect resistance to GM Bt crops has been a concern since the introduction of this technology. A management strategy includes refugia planting of 5% non-Bt plants, with no insecticide application, and 20%, where insecticide application is allowed. These refugia are designed to allow the survival of insect pests within restricted planted zones. However, in South Africa there are reports of Bt-resistant stem borer (Busseola fusca) (Fuller) (Lepidoptera: Noctuidae) and non-compliance with refuge planting. The aims of this study were two-fold: 1. To conduct a survey among KwaZulu-Natal (KZN) GM maize growers to ascertain information such as level of compliance with refuge planting and to determine which refugia were predominantly planted and reasons thereof; 2. To conduct a replicated field trial to determine yield, insect borer damage and economic benefit of the 5% unsprayed and 20% sprayed refuge options (including three configurations namely strip, perimeter and block and a 5 and 20% ‘refuge-in-a-bag’ option). The survey indicated that 28 out of 29 (96.6%) KZN Bt maize growers plant the 5% non-sprayed refuge with 27 (96.4%) of those respondents planting the strip configuration for the purpose of insect management (75%) and ease of planting (32.2%). The survey also showed that 7 (seven) i.e. 21.9% of KZN Bt maize growers observed borer damage and although growers are now fully compliant with refugia planting requirements, initially 7 respondents (24.1%) did not comply with or plant refugia correctly. Furthermore, 7 respondents reported insect borer damage in their maize with 4 of the 7 instances (57.1%) likely stemming from incorrectly planted refugia. vii No significant differences in yield or insect damage were observed between the 5 and 20% refugia for any of the planting configurations in the field trial. However due to costs involved with insecticide application and labour required for the operation in the 20% option, these treatments were less economically advantageous than the non-Bt control. The 20% block and strip configurations had a cost benefit ratio of ZAR 7.21 and ZAR 6.67 respectively, earned per R1 spent by the grower compared with ZAR 7.76 in the sprayed control. The cost-benefit comparison for the 5% block and strip configurations was ZAR 8.48 and ZAR 7.71, respectively compared with ZAR 9.44 in the unsprayed control. In addition, the 20% seed mixture limited borer damage to 4.95% when compared with 15.77% damage in the sprayed control (ANOVA, F pr = 0.124). The seed mixtures are not available commercially and the results from the survey indicated that some education and marketing by the seed companies would be advisable prior to their release to the farming community. In order to determine which of the refuge options between 5 and 20% would be more advantageous for growers overall, regardless of the planting configuration; data were grouped and analysed. There were no significant differences in either the yield or insect damage for the 5 and 20% refugia, but the cost-benefit calculations indicated that the 5% option was more cost effective – for the 5 and 20% refugia, ZAR 7.97 and ZAR 7.15 respectively, earned per ZAR 1 spent by the grower (ANOVA, F pr. = 0.03). This is because no insecticide was used in the 5% treatments. Mean ear damage comparisons between the 5 and 20% refugia showed that the 20% refuge in the perimeter configuration incurred the least damage (2.65% ear damage) compared with 5% perimeter (10.86% ear damage), although the reasons for this are not clear. While the results of the field trials showed no significant differences in insect damage and yield with regard to choice of refuge configuration, monitoring insect resistance management remains an integral part of Bt maize crops in South Africa, in order to delay further resistance development and to prolong the viability of Bt technology. / Agriculture and  Animal Health / M. Sc. (Agriculture)

Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus. Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%). Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×10⁶ g⁻¹FW was obtained when cell suspensions were used as the tissue source, with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots. The highest protoplast yield (7×10⁶ g⁻¹FW) of L. corniculatus was achieved from cotyledons also with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L). Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm² for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants. The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.