Polyamines play an important role in DNA, RNA and protein synthesis as well as a variety of other biological processes (cell division, differentiation and death) as outlined in Chapter 1. Assaraf and co-workers (1984) demonstrated that treatment with DFMO resulted in the inhibition of polyamine biosynthesis as well as schizogony arrest in P. falciparum. However, they did not elaborate on any other consequences that polyamine depletion could exert on the parasite. This dissertation aims to elucidate the significance of the inhibition of polyamine biosynthesis within P. falciparum by using differential transcriptome profiling. Suppression subtractive hybridisation generated transcripts which were potentially up-and down-regulated due to endogenous polyamine depletion within the human malaria parasite P. falciparum. The resulting transcripts were subjected to a restriction enzyme analysis and those with unique digestion profiles were selected and sequenced. The sequences were analysed using PlasmoDB to identify the genomic sequences to which they were best matched. To confirm that the selected transcripts were indeed differentially expressed a reverse virtual Northern dot blot was performed. Transcripts for proteins involved in protein processing, methionine and polyamine metabolism, various transporters, proteins involved in cellular differentiation and signal transduction were found to be upregulated in the absences of polyamines. This could be suggestive of a metabolic response induced by the parasite in order to overcome this deficiency. Polyamines seem to influence protein synthesis and haemoglobin degradation as well since depletion of endogenous polyamines within the parasite seems to result in increased food vacuole acidification, haemoglobin degradation, transport of proteins to the cytoplasm and protein synthesis and stabilisation. The majority of downregulated transcripts were found to be involved in cell-cell adhesion and erythrocyte invasion, protein processing and transport indicating that these processes are dependent on polyamines. Further validation of these findings by microarray as well as proteomic analysis will need to be undertaken. These results validate that polyamines do play an essential role in the cellular biology of the parasite. They also confirm that the inhibition of polyamine biosynthesis is a viable route to undertake in the search for new and improved antimalarial targets. This would be especially useful if it was combined with other antimalarials and their synergistic effects were investigated by transcriptomic, proteomic and bioinformatic analysis / Dissertation (MSc (Biochemistry))--University of Pretoria, 2007. / Biochemistry / MSc / unrestricted

No / In the past two decades comparative genomic hybridization (CGH) and array CGH have become crucial and indispensable tools in clinical diagnostics. Initially developed for the genome-wide screening of chromosomal imbalances in tumor cells, CGH as well as array CGH have also been employed in genotoxicology and most recently in toxicogenomics. The latter methodology allows a multi-endpoint analysis of how genes and proteins react to toxic agents revealing molecular mechanisms of toxicology. This chapter provides a background on the use of CGH and array CGH in the context of genotoxicology as well as a protocol for conventional CGH to understand the basic principles of CGH. Array CGH is still cost intensive and requires suitable analytical algorithms but might become the dominating assay in the future when more companies provide a large variety of different commercial DNA arrays/chips leading to lower costs for array CGH equipment as well as consumables such as DNA chips. As the amount of data generated with microarrays exponentially grows, the demand for powerful adaptive algorithms for analysis, competent databases, as well as a sound regulatory framework will also increase. Nevertheless, chromosomal and array CGH are being demonstrated to be effective tools for investigating copy number changes/variations in the whole genome, DNA expression patterns, as well as loss of heterozygosity after a genotoxic impact. This will lead to new insights into affected genes and the underlying structures of regulatory and signaling pathways in genotoxicology and could conclusively identify yet unknown harmful toxicants.

Denaturation and renaturation is indispensable for the biological function of nucleic acids in many cellular processes, such as for example transcription for the synthesis of RNA and DNA replication during cell division. However, the reversible hybridisation of complementary nucleic acids is equally crucial in nearly all molecular biology technologies, ranging from nucleic acid amplification technologies, such as the polymerase chain reaction, and DNA biosensors to next generation sequencing. For nucleic acid amplification technologies, controlled DNA denaturation and renaturation is particularly essential and achieved by cycling elevated temperatures. Although this is by far the most commonly used method, the management of rapid temperature changes requires bulky instrumentation and intense power supply. These factors so far precluded the development of true point-of-care tests for molecular diagnostics. This Thesis explored the possibility of using electrochemical means to control reversible DNA hybridisation by using electroactive intercalators. First, fluorescence-based melting curve analysis was employed to gain an in depth understanding of the reversible process of DNA hybridisation. Fundamental properties, such as stability of the double helix, were investigated by studying the effect of common denaturing agents, such as formamide and urea, pH and monovalent salt concentration. Thereafter, four different electroactive intercalators and their effect on the thermodynamic stability of duplex DNA were screened. The intercalators investigated were methylene blue, thionine, daunomycin and adriamycin. Absorbance-based melting curve analysis revealed a significant increase of the melting temperature of duplex DNA in the presence of oxidised daunomycin. This was not observed in the presence of chemically reduced daunomycin, which confirmed the hypothesis that switching of the redox-state of daunomycin altered its properties from DNA binding to non-binding. Accordingly this altered the thermodynamic stability of duplex DNA. The difference in the stability of duplex DNA, as a direct result of the redox-state of daunomycin, was exploited to drive cyclic electrochemically controlled DNA denaturation and renaturation under isothermal conditions. This proof-of-principle was demonstrated using complementary synthetic 20mer and 40mer DNA oligonucleotides. Analysis with in situ UV–vis and circular dichroism spectroelectrochemistry, as two independent techniques, indicated that up to 80 % of the duplex DNA was reversibly hybridised. Five cycles of DNA denaturation and renaturation were achieved and gel electrophoresis as well as NMR showed no degradation of DNA or daunomycin. As no extreme conditions were implicated, no covalent modification of DNA was required and isothermal conditions were kept, this finding has great potential to simplify future developments of miniaturised and portable bioanalytical systems for nucleic acid-based molecular diagnostics.

Cette thèse aborde l’écologie et l’évolution des relations qu’entretiennent les orchidées du genre Ophrys avec leurs pollinisateurs. L’approche comparative et la combinaison (i) d’analyses chimiques de phéromones sexuelles et de parfums floraux, (ii) d’analyses génétiques avec des outils moléculaires, et (ii) de tests de comportement réalisés sur les insectes in situ nous ont permis d’éclairer certains aspects méconnus de ces interactions inter-spécifiques. La pollinisation des orchidées du genre Ophrys est assurée par des mâles d'abeilles ou de guêpes solitaires qui opèrent une tentative d’accouplement (pseudocopulation) sur le labelle des fleurs. L'attraction des pollinisateurs est généralement hautement spécifique, régie par un mimétisme des signaux (chimiques, visuels, tactiles) des femelles des espèces d'insectes concernés. Malgré cette spécificité, des hybrides se forment occasionnellement en conditions naturelles, témoignant de la perméabilité partielle des barrières d'isolement reproductif entre espèces. Au cours de ce programme de recherche, nous avons entrepris l’étude des interactions Ophryspollinisateurs en mettant l’accent sur trois aspects spécifiques, à savoir (i) la sélection des caractères floraux par les pollinisateurs, (ii) l'isolement reproductif entre espèces d'Ophrys sympatriques, et enfin (iii) l'évolution des caractères floraux au sein d'un complexe d'espèces-soeurs d'Ophrys associées à différents pollinisateurs. Les principaux résultats de ce travail sont repris ci-dessous, ponctués de références aux articles qui rassemblent l’intégralité des études réalisées.

Hybridisation between introduced and endemic species causes conservation concerns, but also provides us with an opportunity to study the dynamics of gene flow between two species as they first meet. Japanese sika deer (Cervus nippon) were introduced to the British Isles at a number of locations at the beginning of the 20th century. In the intervening time, sika have spread and their range now extends across approximately 40% of Scotland, where they overlap with that of native red deer (C. elaphus), with which they hybridise. In this study we focus on the consequences of one particular introduction that took place at Carradale, on the Kintyre Peninsula in 1893. First, I assessed the current state of hybridisation using a sample of 735 red and sika deer samples collected in 2006/7 from forestry blocks throughout the Kintyre Peninsula. Genetic analysis was conducted with a panel of 22 highly differentiated microsatellite loci and one mtDNA marker. Population admixture analysis of the microsatellite data was conducted with the Bayesian clustering programme STRUCTURE. Over most of the study area, levels of introgression into red and sika deer were low and were consistent with a scenario of very occasional F1 hybridisation followed by backcrossing. There was, however, one forestry block where 43% of individuals could be defined as hybrids. Second, I developed a branching process model of introgression via backcrossing, to assess whether variation in introgression across microsatellite loci could be interpreted as a signature of selection, or could in fact be attributed to stochastic processes. If only a few hybridisation events have contributed to the hybridising population, the pattern of introgression, even with a large number of genetic markers, will be highly stochastic. This pattern of neutral variation in introgression can have high enough variance that it could be mistaken for selection. Therefore, even if strong selection is acting, it may not be possible to distinguish its effects from neutral variation. Third, I analysed trends in hybridisation and introgression over 15 years on the peninsula, through analysis of a dataset of 1513 red and sika deer samples at 20 microsatellite and a mtDNA marker. There was little evidence of change in the extent of hybridisation and introgression over time. MtDNA introgression was predominantly from red deer into sika. Recent introgression into sika on the peninsula can be explained by a very small number of F1 hybridisation events (~10) via analysis of the number of alleles that have introgressed from polymorphic red deer into the genetically homogenous sika population (a similar analysis cannot be conducted for introgression into red deer). Finally, I conducted a regression analysis of genetic hybrid scores against phenotypic traits to assess the effect of hybridisation on phenotype. Hybridisation has caused changes in the weight of sika-like deer and red-like females. Hybridisation has caused changes in incisor arcade breadth of both populations and jaw length (a proxy for skeletal size) in sika-like females. However, there is no evidence that hybridisation has caused changes in kidney fat (a measure of condition) or pregnancy rates in either population. In conclusion, even a small number of F1 hybridisation events can lead to extensive introgression and the timing and spatial distribution of these events is likely to have a large impact on the structure of a recently hybridising population - stochastic factors dominate both the distribution of hybrid individuals and the distribution of the genes that introgress following a hybridisation event. In red deer and sika deer, increasing phenotypic similarities of the two populations caused by hybridisation are likely to facilitate further breakdown between the two species. It is possible that breakdown in assortative mating between the two species could occur across their range.

An education that does not recognise schools as places for the mere transmission and assimilation of knowledge, but as places for critical and creative inquiry, is quality education. Philosophising with children in schools assumes that children are actively and deliberately encouraged in seeking responses to the questions about reality they raise at a very early age. The practice of philosophy is undoubtedly one of the underpinnings of a quality education for all. By contributing to opening children‘s minds, building their critical reflection and autonomous thinking, philosophy contributes to the protection against manipulation and exclusion at the hands of adults. If education in general must open up to children the maps of an intricate world in a continuous state of tension, then philosophy is a compass for navigating that world. Hence children, irrespective of their geographical location and regardless of their social milieu or state of development of their country, deserve to be equipped with the tools so motivated for. Using conceptual analysis as a tool, I explore the Lipman method of Philosophy for Children by presenting a case for an African perspective of the same. I situate doing philosophy with children in the context of the African philosophy debate. While Lipman‘s model provides the case for the role of rational, logical and systematic thinking in children, the African background promises the raw materials on which the said instruments work. I therefore settle for a hybridised Philosophy for Children programme that marries the universalist and the particularist views of doing philosophy. I argue that the traditional African notion of community plays a significant role in our understanding of the community of inquiry as pedagogy of doing philosophy with children. Embedded in African ―community‖ is the concept of ukama qua relationality, which constitutes a keystone in the envisaged African perspective of Philosophy for Children. I conclude that doing philosophy with children in schools in Africa contributes to the interpretation of the cultural, economic and circumstances of the African situation.

Student Number : 0419128X - MSc Dissertation - School of Animal, Plant and Environmental Sciences - Faculty of Science / Reproductive isolation was investigated in two chromosomally distinct populations of Rhabdomys on the Gauteng highveld. The two populations, Midrand (2n = 48) and Irene (2n = 46), occur 15 km apart, with no known contact or hybrid zone between them. Behavioural experiments, comprising male-female dyadic encounters and female preference tests for same-and different-population male odour, were used to test for premating barriers. Aggression levels were highest in different-population than same-population dyads, and females spent more time with odours of males from their own population than of those of the other population. Breeding and postnatal development studies were conducted to establish postmating barriers. Compared to different-population pairs, reproductive success was markedly reduced in different-population pairings, and the few hybrids that were produced did not breed. My studies indicate that behavioural isolation is well-developed between the Midrand and Irene striped mice, and suggest that the mate recognition system has diverged in allopatry, which would reduce gene flow between the two populations. Such divergence supports the findings of mtDNA studies by other workers who proposed that the two chromosomal forms used in my study represent two subspecies of R. dilectus. Previous studies showed that distant striped mice populations (>900km) displayed behavioural divergence and intermediately located populations (~80km) were behavioural compatible but had hybrid failure; the Irene population was used in both studies. In comparison, the behavioural incompatibility between the closely-located Midrand and Irene populations provides support for the reinforcement of previous postmating isolation seen in the intermediately located populations, particularly since no contact or hybrid zone exists between the two forms. However, I cannot rule out other explanations, such as dissimilar ecological conditions, influencing interfertility.

Esse estudo se dedica a fazer uma reflexão sobre a arte transmidiática através da apropriação das tecnologias digitais criadas a partir do advento do computador e da internet. Apesar de não ser novo o uso de múltiplas plataformas, a conexão entre as pessoas e o grande fluxo de informação trocada pela internet possibilita um novo ambiente criativo e uma nova relação entre público e arte. O trabalho tem seu início analisando a perspectiva histórica dessas novas tecnologias, que se tornaram ubíquas, e disserta sobre sua hibridação com o universo artístico, levando em conta a interatividade com o público e a cocriação. Para compreensão deste cenário tecnossocial, a pesquisa discorre sobre a penetração das novas tecnologias no cotidiano e seu impacto na sociedade. Finalmente, o estudo apresenta e descreve uma seleção de obras que podem ser definidas ou foram criadas como transmidiáticas, e examina sua produção e circulação na sociedade. / This study reflects on transmedia art through the use of the digital technologies that emerged with the advent of the computer and the internet. Even though the use of multiple platforms isnt new, the interpersonal links and the significant flux of data and information exchanged through the internet enables a new creative environment and a new relationship between art and its public. Observing these new and ubiquitous technologies from a historic viewpoint, this work examines its hybridization with the art world, its interaction with the public and collaborative co-creation. To comprehend this techno-social scenario, the research explains the advance of new technologies in everyday life and its impact on society. Finally, the study presents and describes a selection of artworks that could be defined or were created as transmedia art, its production process and the way society reaches and absorbs it.

Les systèmes cyber-physiques sont une classe de systèmes complexe, de grande échelle, souvent critiques de sûreté, qui apparaissent dans des applications industrielles variées. Des approches de vérification formelle sont capable de fournir des garanties pour la performance et la sûreté de ces systèmes. Elles nécessitent trois éléments : un modèle formel, une méthode de vérification, ainsi qu’un ensemble de spécifications formelles. En revanche, les modèles industriels sont typiquement informels, ils sont analysés dans des environnements de simulation informels et leurs spécifications sont décrits dans un langage naturel informel. Dans cette thèse, nous visons à faciliter l’intégration de la vérification formelle dans le processus industriel de la conception basé sur modèle.Notre première contribution clé est une méthodologie de transformation de modèle. A partir d’un modèle de simulation standard, nous le transformons en un modèle de vérification équivalent, plus précisément en un réseau d’automates hybrides. Le processus de transformation prend en compte des différences de syntaxes, sémantique et d’autres aspects de la modélisation. Pour cette classe de modèle formel, des algorithmes d’atteignabilité peuvent être appliqués pour vérifier des propriétés de sûreté. Un obstacle est que des algorithmes d’atteignabilité se mettent à l’échelle pour des modèles affines par morceaux, mais pas pour des modèles non linéaires. Pour obtenir des surapproximations affines par morceaux des dynamiques non linéaires, nous proposons une technique compositionnelle d’hybridisation syntaxique. Le résultat est un modèle très compact qui retient la structure modulaire du modèle d’origine de simulation, tout en évitant une explosion du nombre de partitions.La seconde contribution clé est une approche pour encoder des spécifications formelles riches de façon à ce qu’elles peuvent être interprétées par des outils d’atteignabilité. Nous prenons en compte des spécifications exprimées sous forme d’un gabarit de motif (pattern template), puisqu’elles sont proche au langage naturel et peuvent être compris facilement par des utilisateurs non experts. Nous fournissons (i) des définitions formelles pour des motifs choisis, qui respectent la sémantique des automates hybrides, et (ii) des observateurs qui encodes les propriétés en tant qu’atteignabilité d’un état d’erreur. En composant ces observateurs avec le modèle formel, les propriétés peuvent être vérifiées par des outils standards de vérification qui sont automatisés.Finalement, nous présentons une chaîne d’outils semi-automatisée ainsi que des études de cas menées en collaboration avec des partenaires industriels. / Cyber-Physical Systems form a class of complex, large-scale systems of frequently safety-critical nature in various industrial applications. Formal verification approaches can provide performance and safety guarantees for these systems. They require three elements: a formal model, a formal verification method, and a set of formal specifications. However, industrial models are typically non-formal, they are analyzed in non-formal simulation environments, and their specifications are described in non-formal natural language. In this thesis, we aim to facilitate the integration of formal verification into the industrial model-based design process.Our first key contribution is a model transformation methodology. Starting with a standard simulation model, we transform it into an equivalent verification model, particularly a network of hybrid automata. The transformation process addresses differences in syntax, semantics, and other aspects of modeling. For this class of formal models, so-called reachability algorithms can be applied to verify safety properties. An obstacle is that scalable algorithms exist for piecewise affine (PWA) models, but not for nonlinear ones. To obtain PWA over-approximations of nonlinear dynamics, we propose a compositional syntactic hybridization technique. The result is a highly compact model that retains the modular structure of the original simulation model and largely avoids an explosion in the number of partitions.The second key contribution is an approach to encode rich formal specifications so that they can be interpreted by tools for reachability. Herein, we consider specifications expressed by pattern templates since they are close to natural language and can be easily understood by non-expert users. We provide (i) formal definitions for select patterns that respect the semantics of hybrid automata, and (ii) monitors which encode the properties as the reachability of an error state. By composing these monitors with the formal model under study, the properties can be checked by off-the-shelf fully automated verification tools.Furthermore, we provide a semi-automated toolchain and present results from case studies conducted in collaboration with industrial partners.

Cardiac hypertrophy defines an adaptive process brought about in response to sustained increases in haemodynamic work. Cardiomyocytes undergo an initial compensatory phase in which enlargement and contractility alterations normalise wall stress and maintain adequate perfusion of organs. In pathological hypertrophy, this deteriorates to a decompensated state characterised by ventricular dysfunction and predisposition to heart failure. In contrast, physiological hypertrophy and associated enhanced cardiac functioning arising from chronic exercise training does not progress to heart failure. Determination of the molecular pathways underlying myocardial hypertrophy remains a challenge for cardiovascular research. The objective of the work presented in this thesis was to identify genes differentially expressed during pathological and physiological hypertrophy in order to enhance our knowledge of the mechanistic processes involved. A reverse Northern hybridisation method was applied to profile the expression of specifically selected genes in the hypertrophic models examined. Functional categories represented in the gene panel assembled included cardiac contractile and cytoskeletal markers, matrix metalloproteinases, vasoactive pathway factors, calcium handling genes, ion channels, cardiac regulatory factors, signalling pathway intermediates, apoptotic factors and histone deacetylases. In order to investigate pathological hypertrophy, a deoxycorticosterone acetate-salt (DOCA-salt) rat model was utilised. DOCA-salt treated rats used in this study demonstrated a 1.4-fold increase in heart weight to body weight ratio compared to controls. Impaired cardiac function indicative of a decompensated pathological phenotype in the DOCA-salt treated group was demonstrated by way of decreased chamber size, impaired myocardial compliance and significantly reduced cardiac output. Reverse Northern hybridisation analysis of 95 selected genes identified a number of candidates with differential expression in hearts of DOCA-salt treated rats. Increased gene expression was demonstrated for the collagenase MMP1 and stress-activated signal transduction factor Sin1. In contrast, the sarcoplasmic reticulum calcium ATPase SERCA-2 and anti-apoptotic factor BCL2l-10 genes exhibited decreased expression. To investigate changes in gene expression associated with physiological hypertrophy, use was made of an endurance run-trained rat model. The run-trained rats used in this study demonstrated a 24.1% increase in heart weight to body weight ratio and improvements in performance consistent with physiological cardiac adaptation. These performance indicators included improvements in systolic volume, cardiac output, myocardial compliance and bio-energetic function. Reverse Northern hybridisation expression analysis of 56 genes identified a number of differentially expressed mRNA transcripts in run-trained hypertrophied hearts. Four genes shown to demonstrate reduced expression in the run-trained rat model were interleukin-1 receptor associated kinase (IRAK1) and the developmentally expressed transcription factors Nkx-2.3, dHAND, and IRX-2. Based upon the reverse Northern hybridisation results, four genes were selected for Western blotting analysis of rat cardiac tissue. Of these, MMP1 and a putative isoform of Sin1 exhibited increased levels in DOCA-salt treated hypertrophic left ventricular tissue, results that correlate with the findings of increased mRNA expression for these two genes. Therefore, this study identified MMP1 and Sin1 as candidates involved in pathological but not physiological hypertrophy. This finding is in accord with other recent investigations demonstrating that pathological hypertrophy and physiological hypertrophy are associated with distinct molecular phenotypes. An aside to the major objective of identifying genes differentially regulated in left ventricular hypertrophy involved the application of the P19CL6 cell in vitro model of cardiomyogenesis to compare protein expression during hypertrophy and development. The Sin1 isoform, found to be up-regulated during DOCA-salt induced hypertrophy, was also shown to be more abundant in differentiating, than non-differentiating, P19CL6 cells. This result is consistent with the developing paradigm that implicates 'fetal' genes in the hypertrophic remodelling process.