The first chapter gives an overall introduction to Arachis hypogaea L., the crop that was studied in this thesis. The literature reviewed touched on various aspects of the crop in general such as the importance of peanut as a crop, its botanical description, origin and distribution world-wide. Different marker studies done on A. hypogaea L. and their extents have also been reviewed. The chapter also looked at the oryzacystatin-1 (OC-1) gene as a possible candidate gene for peanut improvement in South Africa (Chapter I). Agronomic data was collected in an attempt to observe and evaluate variability amongst the 18 commercially cultivated peanut lines from South Africa. The growth habits, growth seasons, testa colours, total oil percentages and oleic and linoleic acid ratios were the agronomic characters used to study variation (Chapter II). One South African commercially cultivated peanut line, JL 24, was transformed with the oryzacystatin-1 (OC-1) gene, which was isolated from rice. Three to four week old plantlets were vacuum infiltrated with Agrobacterium during the transformation process and various methods were used to analyze the putative transformants (Chapter III). Two types of DNA markers along with phynotypic data were used to examine polymorphism among 18 South African peanut lines and a transformed line. The amplified fragment length polymorphisms (AFLPs) and simple sequences repeats (SSRs) were the two DNA markers used in this study to determine the level of variation amongst these lines (Chapter IV). / Dissertation (MSc (Genetics))--University of Pretoria, 2008. / Genetics / unrestricted

Reproduction de : Thèse doctorat : Odontologie. Sciences biologiques. Biologie cellulaire : Reims : 2005. / Titre provenant de l'écran-titre. Bibliogr. p.139-160.

5' Nucleotidase (5NU) in <i>Dictyostelium discoideum</i> is an enzyme that shows high substrate specificity to 5'AMP. The enzyme has received considerable attention in the past because of the critical role played by cyclic AMP in cell differentiation in this organism. Degradation of cAMP by cAMP phosphodiesterase (PDE) produces 5'AMP, the substrate of 5NU. Dictyostelium switches its genetic program from growth to cellular differentiation when nutrients become limited. During the time course of development, the activity of 5NU is high and becomes restricted to a narrow band of cells that form the interface between the prestalk/prespore zones. Understanding how this gene is regulated will provide knowledge underlying the process of cell differentiation. In order to understand the functional significance of the 5NU, I first purified of the 5NU protein using an artificial substrate p-nitrophenol phosphate (pNPP). An activity stain on non-denaturing gels with Nitro Blue Tetrazolium (NBT) and 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP) as the substrate was also used. A polypeptide of approximately 90 kDa was associated with 5NU enzyme activity after gel filtration chromatography and denaturing gel electrophoreses. Protein sequence of this peptide was obtained from Mass Spectrometry and Edmund Degradation. Various databanks were searched for similar sequences, but no matches with high identity were obtained. However, a search of the sequences of an ongoing cDNA project at the University of Tsukuba in Japan revealed a clone that corresponded to the peptide sequence of 5NU. In addition, a clone was found that corresponded to the classical "alkaline phosphatase" found in several organisms. Analysis of the expression of the 5NU and AP during Dictyostelium development by Northern blotting determined that the 5NU is developmentally regulated while the AP is expressed at all stages of the life cycle. Southern blot analysis showed a single form of the gene for both 5NU and AP. Targeted gene disruption and knockout mutagenesis using the 5NU sequences flanking a blasticidin-resistant cassette was attempted. Analysis of the transformants showed the 5NU gene was not disrupted, and that the blasticidin-resistant cassette was randomly inserted into the genome. / Ph. D.

In contrast to plant and animal cells, the fungal cells are multinucleate. A consequence of their multinucleate condition is heterokaryosis — the occurrence of genetically different nuclei in a common cytoplasm. In nature this condition occurs because of spontaneous mutations in the haploid nuclei in the coenocytic mycelium. Inspite of heterokaryosis being a fundamental aspect of fungal biology, the behaviour and dynamics of nuclei in fungal mycelium are little understood. This study was prompted by the following questions: (1) Why does a fungus need so many nuclei? (2) Are they all active simultaneously? (3) Does the proportion of the different nuclear types in fungal mycelium alter in response to change in conditions of growth? (4) Is the activity of an enzyme related to the dose of nuclei containing the encoding gene? Experimental approach. The approach taken was to generate heterokaryons in which one of the nuclear types carries a mutant allele for a specific enzyme while the other nuclear type carries the functional allele, introduced by transformation. Because in filamentous fungi, the transforming DNA commonly integrates randomly into the chromosomal DNA, the transformants would be genetic 'variants' in which the ratios of transformed to non-transformed nuclei might be controlled differently. The transformants could thus be useful in investigating the relationship between the frequency of transformed nuclei and the activity of encoded enzyme. In addition the transformants might be useful for studying nuclear behaviour. The availability of developmental information, genetic and molecular methodology, and biochemical mutant in Neurospora crassa made this fungus a material of choice for this investigation. Strain construction. A histidinol dehydrogenase (his-3) mutant strain was used into which an albino colour marker and a biochemical marker, inositoL were introduced by crossing. The latter two markers served as check against possible laboratory contamination. In addition, a gene mem, was introduced into the strain. In the mem genetic background, the strain has a wild-type morphology on agar medium but when grown in liquid shake culture it produces uninucleate microconidia that are useful in estimating nuclear ratio. Protoplasts of a constructed strain (his-3 al-1; mem; inl) were transformed with a plasmid containing the wild-type his-3 allele, thereby converting the original strain into a heterokaryotic strain having a mixture of transformed (his-3+t) and untransformed (his-3) nuclei. [The superscript +/ is used here to denote an his~3+ allele ectopically introduced by transformation]. Integration of plasmid DNA sequence in three selected transformants, 2T5, 3T3 and 4T12, was confirmed by genomic Southern analysis using the vector DNA as probe. The exponential growth rate of all three transformants was similar (~0.08mgh"1). Nuclear ratio. Assuming a uniform distribution of nuclei in mycelium, and a correspondence between nuclear ratio in mycelium and conidia, the ratio his-3* {: his-3 was estimated by plating microconidia. In transformant 3T3, the nuclear ratio was 7:1. In 2T5, all nuclei were his-3n. Transformant 4T12 did not produce microconidia. The nuclear ratio in this transformant was therefore estimated by macroconidial plating and found to be 1:5, in favour of his-3 nuclei. Behaviour of transformants in vegetative and sexual phase. Although the transformants had originally been selected for the expression of his-3+T gene, a majority of macroconidia produced in cultures of 3T3 and 2T5 required histidine to trigger their germination. This condition, referred to as cphenotypic lag', led to a gross underestimation of the proportion of prototrophic macroconidia by the direct plating method and biased the estimation of nuclear ratios. Therefore nuclear ratio was estimated by first germinating macroconidia on histidine supplemented medium before testing colonies in histidine dropout slants and comparing the numbers of auxotrophic and prototrophic mycelia. Phenotypic lag was not observed in 4T12. The variation in the degree of expression of phenotypic lag among the transformants was ascribed to transgene position effect. The transformants differed also in meiotic instability of the transforming DNA — the transforming DNA in 3T3 was passed through unchanged but it was deleted or modified in4T12and2T5. Experimental alteration of nuclear ratio. The transformants differed with respect to the self-adjusted ratio of transformed to non-transformed nuclei and also to the degree to which their nuclear ratio could be altered by nutritional manipulation of the growth medium, i.e., by growing the transformants in the presence or absence of histidine in the medium. In 3T3, the proportion of his-3+t nuclei progressively decreased by 3.5-fold in the sixth subculture on histidine medium. The change in 4T12 was even more striking: in the sixth serial subculture, the proportion of his-3+t nuclei decreased from 17-20% to -0.05%.However, when it was propagated again in medium that lacked histidine, the frequency of his-3+t nuclei was immediately restored to original level (-17%). That drastic alterations in nuclear ratio occurred upon nutritional manipulation was verified by Southern analysis. The intensity of signal specific for transformed DNA (nuclei) in cultures grown without histidine supplement was strong, but barely detectable in cultures grown with histidine. The signal reappeared when 4T12 was propagated in medium lacking histidine. Histidine induced change in nuclear ratio in 4T12 was further confirmed by three tests: (i) inoculum test using conidia, (ii) hyphal tip analysis, and (iii) genetic test using colour markers. Nuclear ratio and enzyme activity. Because in 4T12 changes in nuclear ratio could be manipulated, this transformant was used to investigate whether the proportion of his-3+t nuclei is correlated with the levels of encoded enzyme, histidinol dehydrogenase. Surprisingly, the specific activity of histidinol dehydrogenase was the same regardless of the percentage of his-3+t nuclei. This observation suggested that the physiological demand of a metabolite may be satisfied with only a few nuclei carrying the relevant gene. Or in other words the majority of nuclei in the coenocytic mycelium may, perhaps, not be active simultaneously. Silencing of transforming DNA in nuclei. Two experiments were done to test the possibility that in a majority of nuclei, the transforming DNA is selectively silenced by methylation of cytosine: (1) Southern analysis of chromosomal DNA digested with isoschizomers, and (2) Reactivation by growth of transformants in presence of 5-azacytidine, an inhibitor of methylation. The results suggested that a majority of transformed nuclei may, perhaps, be inactive. The results of Northern analysis suggested that the amount of his-3+t transcript was correlated (but 5-azacytidine experiment indicated that only few his-3+t nuclei may be active) with the proportion of his-3+t nuclei, but not histidinol dehydrogenase activity. The above results suggested that expression of his-3+t gene was controlled both at the levels of transcription and posttranscription. Nuclear selection. To study competition between nuclei containing mutant (his-3) nuclei and prototrophic nuclei containing his-3+ gene at its normal chromosomal location or at the ectopic location, heterokaryons were synthesized using strains in which the nuclear types had been marked by non-allelic genetic colour markers, al-1 and al-2. The results suggested that in heteronuclear mixture, the replication rate of the transformed nuclei is affected as compared to the nuclei having the gene in normal chromosomal location. Major contributions. This study generated (his-3 + his-3+) heterokaryons by transformation. The behaviour of transformants differed in some respects both in the vegetative and sexual phases. It was demonstrated that nuclear ratio could be experimentally altered. However, there was no correlation between nuclear ratio and enzyme activity. The observations imply asynchronous division rate among nuclei and raise the possibility that not all nuclei in the coenocytic mycelium are active simultaneously.

The Hox gene family encodes transcription factors that play an essential role in embryo patterning and organogenesis. Hoxa5 mutant mice have been produced and majority of Hoxa5" mice die at birth from respiratory failure. Surviving Hoxa5'1 ' dams are unable to feed their pups properly, revealing abnormal mammary gland development. The Hoxa5 null mutation results in increased proliferation and precocious differentiation in the mammary epithelium, thus causing the formation of collapsed alveoli unable to secrete milk. In order to find Hoxa5 targets involved in mammary gland development, I performed microarray analyses combined with a candidate gene expression analysis approach. This work confirmed the secretory defects observed in Hoxa5'1 ' and demonstrated a new role for Hoxa5 in extracellular matrix organisation. It also identified the TGFp signaling pathway as a potential effector of Hoxa5 function. Finally, this work suggests interdependence between Hoxa5 and the ovarian hormones in controlling mammary gland proliferation. / Les gènes Hox sont des facteurs de transcription essentiels à la spécification des axes embryonnaires et à l'organogenèse. L'étude d'une lignée de souris mutante pour le gène Hoxa5 a mis en évidence une augmentation de la prolifération et un développement lobullo-alvéolaire précoce dans les glandes mammaires Hoxa5~f ~, accompagnés de l'expression précoce des protéines du lait. Afin de caractériser les mécanismes moléculaires contrôlés par Hoxa5 dans la glande mammaire, une étude utilisant les micropuces d'ADN combinée à l'analyse de l'expression de gènes candidats ont été réalisées. Ces travaux ont confirmé les problèmes de sécrétion et mis en évidence la désorganisation de la matrice extracellulaire dans les glandes mammaires Hoxa5~'~. Ils ont également permis d'identifier la voie de signalisation du TGFp comme un effecteur potentiel du gène Hoxa5. Enfin, ces travaux suggèrent une interdépendance entre les hormones ovariennes et le gène Hoxa5 dans la régulation de la prolifération de la glande mammaire

Contexte: Les cellules endothéliales cornéennes humaines (CECH) peuvent adopter un phénotype fibroblastique en culture, les rendant inutilisables en génie tissulaire de la cornée. Le facteur de croissance transformant β (TGF-β) serait impliqué dans ce phénomène, mais il est aussi connu pour maintenir les CECH en état de quiescence in vivo. Objectifs: Comparer l’effet du TGF-β1 sur le phénotype des CECH lors des phases de prolifération ou de maturation des cultures et optimiser les conditions de culture des CECH. Méthodes: Des CECH ont été cultivées en présence ou non de TGF-β1 et de facteur de croissance épidermal (EGF) dans une culture à deux phases. La morphologie, l’expression de marqueurs de fonctionnalité, la résistance trans-endothéliale et la perméabilité des cultures ont été analysées à l’approche de la confluence (phase de prolifération) et suivant la phase de maturation post-confluence. Résultats: L’ajout de TGF-β1 lors de la prolifération a généré des CECH fibroblastiques et la perte des marqueurs de jonctions cellulaires, indépendamment de la présence d’EGF. En phase de maturation, les CECH présentaient un phénotype endothélial et des jonctions cellulaires fonctionnelles. Elles avaient une forte résistance trans-endothéliale et une faible perméabilité. Les résultats démontrent que le TGF-β1 favorise la formation d’une barrière endothéliale semi-perméable lors de la maturation des cultures de CECH, se rapprochant ainsi de l’état physiologique des CECH in vivo. / Background: Human corneal endothelial cells (HCEC) easily become fibroblastic when cultured, rendering them unsuitable for tissue engineering of the cornea. Transforming growth factor β (TGF-β) could be a key factor in this phenomenon; however, it is also known to maintain the endothelium in a quiescent state in vivo. Purpose: To compare the effects of TGF-β1 on HCEC’s phenotype during either the proliferation or the maturation phase of the cultures and optimize culture conditions for HCECs. Morphology, functionality markers, trans-endothelial resistance and permeability of the cultures were analyzed at confluency (proliferation phase) and after the post-confluence maturation phase. Results: Adding TGF-β1 during proliferation produced fibroblastic HCECs and loss of the cell junction’s markers, independently from the presence of EGF. On contrast, during the maturation phase, HCECs had an endothelial phenotype and functional cell junctions. They produced a high trans-endothelial resistance and a low permeability. Overall, results show that TGF-β1 promotes formation of a typical leaky endothelial barrier during the maturation phase of cultured HCECs, thus approaching the physiological properties of in vivo HCECs.

La dystrophie musculaire de Ouchenne (OMO) est une maladie héréditaire récessive caractérisée par une dégénérescence progressive des muscles squelettiques. Aucun traitement curatif n'est actuellement disponible. La thérapie cellulaire pourrait être une solution potentielle pour les patients OMO. Elle est basée sur la transplantation de cellules myogéniques normales afin de restaurer l'expression de la dystrophine, protéine absente dans cette maladie. Une période de régénération limitée suivant un dommage fait au muscle dystrophique réduirait le nombre de fibres hybrides formées diminuant ainsi le succès de la greffe. Notre hypothèse est d'augmenter le temps de régénération musculaire en inhibant l'action de la myostatine à l'aide d'un récepteur tronqué. Les travaux de ce mémoire démontrent l'incorporation d'un récepteur tronqué de l'activine de type IIA, IIB ou les deux dans des cellules myogéniques humaines. La transplantation de ces cellules semble améliorer le succès de la greffe de myoblastes donc plus de fibres exprimant la dystrophine humaine.

La sclérodermie est une maladie auto-immune du tissu conjonctif caractérisée par une fibrose de la peau et des organes internes. L'objectif principal du projet était d'utiliser un modèle de derme reconstruit par auto-assemblage pour vérifier la capacité intrinsèque des fibroblastes sclérodermiques à sécréter et organiser une matrice extracellulaire in vitro. L'utilisation de fibroblastes de peau lésée ou non, de patients atteints de sclérodermie diffuse depuis moins d'un an ou depuis plus de dix ans nous a permis d'établir une nouvelle hypothèse. Au début de la maladie, les fibroblastes nécessiteraient la présence d'un ou de facteurs extrinsèques pour induire une fibrose. Avec le temps et lorsque la maladie s'aggrave, les fibroblastes deviendraient indépendants de ces stimuli externes. De plus, le TGF-βl pourrait être un des facteurs importants pour induire des lésions fibrotiques. Finalement, ce même TGF-β1 pourrait être responsable des changements phénotypiques stables des fibroblastes au stade précoce de la maladie.

La dystrophie musculaire de Duchenne est une maladie héréditaire causée par la mutation du gène DYS qui code pour la dystrophine, une protéine exprimée au niveau des fibres musculaires et qui y joue un rôle important dans le maintient de la stabilité et de l'intégrité des membranes. Elle est caractérisée par la dégénérescence progressive des fibres musculaires ce qui aboutit à l'atrophie de la plupart des muscles. Il n'existe pas pour le moment un remède pour la maladie, mais plusieurs types de thérapies sont étudiés pour mettre au point un traitement. Dans notre laboratoire, on s'intéresse à développer une thérapie cellulaire basée sur la transplantation de myoblastes normaux dans le muscle dytrophique, afin de permettre la restauration de l'expression de la dystrophine au niveau des fibres réparées par la fusion des myoblastes transplantés. Cependant, le succès de cette approche évalué par le nombre de fibres formées après la transplantation qui expriment la dystrophine, est limité. Ce succès limité est dû entre autre à la durée limitée de la période de régénération qui suit le dommage fait au muscle dystrophique. Afin de remédier à ce problème, notre hypothèse de travail est d'augmenter la régénération après le dommage induit par la transplantation de myoblastes, en augmentant la capacité proliférative et la capacité de fusion de ces derniers. Pour cela, l'inhibition du signal induit par une protéine présente normalement dans le muscle, la myostatine, pourrait être bénéfique. Ainsi, les travaux dont fait l'objet la présente thèse démontrent que le blocage du signal de la myostatine dans le muscle de la souris dystrophique, en utilisant des approches différentes, permet en effet d'améliorer le succès de la greffe de myoblastes normaux, mais aussi d'entraîner une hypertrophie musculaire et une augmentation de la force musculaire. / Duchenne muscular dystrophy is a hereditary disease caused by the mutation of DYS gene, which codes for dystrophin, a protein expressed in the myofibers where it plays an important role in the stability and the integrity of membranes. The disease is characterized by the progressive degeneration of the muscle fibers, which induces the atrophy of most of the muscles. To date, there is no cure for this pathology, but several types of therapies are studied to develop a treatment. In our laboratory, we are interested to develop a cell therapy based on the transplantation of normal myoblasts in the dystrophic muscle, to allow the restoration of the expression of dystrophin in fibers repaired by the fusion of the transplanted myoblasts. However, the success of this approach evaluated by the number of fibers formed after the transplantation, which express dystrophin, is limited due in part to the short period of regeneration following the muscle damage. To bypass this problem, our research work presented in the present thesis aimed to increase the regeneration after the damage caused by the myoblast transplantation procedure, by improving the proliferative and fusion capacities of the transplanted myoblasts. This was achieved by inhibiting a protein normally present in the muscle, i.e., myostatin. Thus, the results of this thesis show that the blockade of the myostatin signal in dystrophic mice, using different approaches, not only improved the success of normal myoblast graft, but also induced fiber hypertrophy and increased muscle strength.

Les facteurs de croissances tels que le Transforming Growth Factor-ß₂ (TGF-ß₂) sont des polypeptides qui agissent sur la régulation de la prolifération cellulaire. Les analyses immunohistochimiques révèlent leurs multiples fonctions physiologiques régulatrices dans la croissance de plusieurs types de cellules incluant les kératinocytes. Plusieurs études ont démontré le potentiel thérapeutique et nutraceutique des extraits de lait bovin riches en TGF-ß₂, cependant peu de travaux ont porté sur la concentration de cette molécule à grande échelle. Le but de ce travail est de développer une approche applicable industriellement pour la production d'un ingrédient protéique enrichi en TGF-ß₂ en vue d'une application en dermatologie. Cette étude démontre que le traitement thermique du lait a un impact crucial sur la distribution, la concentration, et le potentiel d'interaction du TGF-(32 dans le lait particulièrement entre 66 °C et 76 °C. Une pasteurisation industrielle entraîne plus de 85% des pertes en TGF-ß₂ dans le lactosérum après décaséination du lait, et induit un changement du ratio IGF-I/TGF-ß₂ de 3500 à 17 durant la préparation des isolats de protéines de lactosérum (IPL). Un IPL natif (non chauffé) induit un maximum de 25% d'inhibition de la prolifération lymphocytaire comparativement à 8.5% obtenue par des IPLs issus de laits pasteurisés. Un ingrédient contenant plus de 19 ng de TGF-ß₂ par milligramme de poudre a été obtenu par agrégation acide de l'IPL natif, ce produit induit une activité inhibitrice splénocytaires supérieure à 60%. Ainsi, l'utilisation de la microfiltration (MF) et Pultrafïltration (UF) pour générer et concentrer un lactosérum natif (non chauffé) représente une approche très prometteuse pour générer des ingrédients à haute teneur en TGF-ß₂ La migration isoélectrique libre ainsi que les techniques d'immunoréaction dans un système modèle révèlent que le chauffage favorise les interactions entre TGF-ß₂ et la ß₂-Lactoglobuline ainsi qu'avec la caséine-K par la formation de ponts disulfures intermoléculaires formés par la réactivité des groupements SH. L'ensemble des résultats générés permettent de maitriser la teneur et le rendement en TGF-ß₂ lors des différentes étapes du procédé développé. En perspective, Il serait complémentaire d'étudier la possibilité de concentrer le TGF-ß₂ à partir du babeurre et comprendre le mécanisme d'interaction concerné.