The research presented in this thesis is concerned with optimising the structure of Artificial Neural Networks. These techniques are based on computer modelling of biological evolution or foetal development. They are known as Evolutionary, Genetic or Embryological methods. Specifically, Embryological techniques are used to grow Artificial Neural Network topologies. The Embryological Algorithm is an alternative to the popular Genetic Algorithm, which is widely used to achieve similar results. The algorithm grows in the sense that the network structure is added to incrementally and thus changes from a simple form to a more complex form. This is unlike the Genetic Algorithm, which causes the structure of the network to evolve in an unstructured or random way. The thesis outlines the following original work: The operation of the Embryological Algorithm is described and compared with the Genetic Algorithm. The results of an exhaustive literature search in the subject area are reported. The growth strategies which may be used to evolve Artificial Neural Network structure are listed. These growth strategies are integrated into an algorithm for network growth. Experimental results obtained from using such a system are described and there is a discussion of the applications of the approach. Consideration is given of the advantages and disadvantages of this technique and suggestions are made for future work in the area. A new learning algorithm based on Taguchi methods is also described. The report concludes that the method of incremental growth is a useful and powerful technique for defining neural network structures and is more efficient than its alternatives. Recommendations are also made with regard to the types of network to which this approach is best suited. Finally, the report contains a discussion of two important aspects of Genetic or Evolutionary techniques related to the above. These are Modular networks (and their synthesis) and the functionality of the network itself.

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Magister Chirurgiae Dentium (MChD) / The author is interested in the morphogenesis of the oral region including the nasopalatine complex. With the intention of undertaking a study of the· embryological development in this area, perusal of available literature failed to reveal a single compreh.ensive description of the reception and handling of embryonic and foetal material, mensuration and preparation for miscroscopy. Human material for embryological study is relatively scarce in· the Republic of South Africa. According to the literature there is, however, a distinct similarity between human and domestic pig development in certain regions, notably the palate. Furthermore, pig embryos and foetuses are available in comparative abundance from sows slaughtered at abattoirs. As a consequence of the above-mentioned factors it was,decided to undertake a -preparatory study in order to firstly evaluate existing methods of handling of embryonic and foetal material and secondly, to statistically evaluate data relating to mass and measurements.'· The aim was to draw a comparison with existing information and to select a sample for investigation. Embryos and foetuses were removed from slaughtered sows in a fresh state and removed to the laboratory immersed in 10 per cent neutral buffered formol saline. In the laboratory foetal membranes were removed, umbilical cords cut and the specimens weighed. They were then placed in Bouin's solution for final fixation and decalcification. Instruments were designed to measure crown-tailroot length, crown-rump length and dorsal profile length. After one day in Bouin's solution all specimens were measured. In order to determine the accuracy of the weighing and measuring procedures ten fixed specimens were weighed and measured on seven consecutive days. Statistical analysis of this data indicated that crown-rump length was the most accurately determinable linear measurement, judged by both the coefficient of variation and the standard deviation. On this basis crown-rump length was chosen as the criterion for selecting the sample to be studied. Correlation between linear measurements and between linear measurements and mass for the entire series showed a very strong positive relationship between all the parameters indicating that a dimensional relationship was maintained during growth. After measuring, the small specimens were embedded whole while larger embryos and foetuses were decapitated. A method was described for trimming and embedding these heads in such a way that subsequent sectioning would take place in a standardised transverse plane. In larger specimens this procedure had to be delayed until demineralization had taken place. Conclusions based on a consideration of data for the entire population included the following: 1. The mean number of specimens per litter was 6,475. 2. The number of pigs per litter stayed relatively constant throughout the period of gestation. 3. Mass showed a greater intra-litter variation than any of the three linear measurements recorded. 4. Relatively, lengths appeared to vary less in older than in younger Ldtt.ers-, irrespective of litter size

La transition épithélio-mésenchymateuse (EMT) est un processus incontournable dans de nombreux contextes normaux et pathologiques, tels que gastrulation, organogenèse, fibroses et cancers. Cette transformation de cellule épithéliale en cellule mésenchymateuse est indissociable de l'acquisition de propriétés migratoires et est généralement associée à un changement de destin cellulaire. Différentes voies moléculaires sont impliquées selon le contexte de l'EMT concernée. Récemment, notre laboratoire a mis en évidence que la voie Notch cytoplasmique contrôle l'EMT des cellules de la lèvre dorso-médiale du somite (DML). Les crêtes neurales exprimant DLL1 activent « en passant » le récepteur NOTCH, liberant ainsi le domaine intra-cytoplasmique de NOTCH (NICD). Dans le cytoplasme, NICD inhibe la kinase GSK3ß, conduisant à la stabilisation de SNAIL, un gène maître de la transition épithélio-mésenchymateuse. Il en résulte une libération de la βcaténine des jonctions adhérentes qui, après translocation dans le noyau, active la transcription des gènes de la myogénèse (Myf5). Ainsi, l'activation de la voie Notch cytoplasmique permet une induction concomitante de l'EMT et de la myogénèse. La fonction cytoplasmique de Notch reste controversée et le mécanisme par lequel NICD inhibe GSK3ß reste obscur. Au cours de ma thèse j'ai cherché à élucider le mécanisme par lequel NICD inhibe l'activité kinase de GSK3ß. J'ai confirmé l'interaction de GSK3ß et de NICD en démontrant leur interaction via CoIP. Après avoir démontré l'implication de la sérine-thréonie kinase AKT dans la myogenèse des cellules de la DML, j'ai mis en évidence, via CoIP et électroporation, que l'inhibition GSK3ß par NICD est très certainement médiée par AKT, connue pour être impliquée dans l'EMT et inhiber GSK3ß par phosphorylation. En comparant le NICD1 de poulet et les 4 NICD de souris, j'ai montré que l'expression exogène de ces 5 molécules induit l'EMT et la différenciation myogénique de manière similaire. J'ai aussi montré que parmi des différents domaines de NICD, le domaine RAM, connu pour se lier à l'ADN (via RBPJ), est nécessaire et suffisant à l'inhibition de GSK3ß. Un second axe de ma thèse a été de tester l'implication de la voie Notch cytoplasmique dans d'autres contextes d'EMT. Pour ce faire, j'ai mis en évidence que cette voie est impliquée dans les autres lèvres du dermomyotome mais aussi dans les crêtes neurales qui délaminent du toit du tube neural. J'ai en particulier mis en évidence une co-activation des voies Wnt et Notch, une inhibition de la kinase GSK3ß par NICD cytoplasmique ainsi qu'une inhibition de la différenciation en présence d'une ß-caténine mutée, retenue à la membrane, ou en présence d'une molécule SNAIL2 dominant-négative. Le dernier axe de ma thèse a consisté à élucider le mécanisme de régulation de l'induction de l'EMT et de la myogenèse via l'activation de NICD. Il a été mis en évidence que toutes les cellules de la DML peuvent être activées via DLL1 et que la surexpression massive de NICD dans la DML provoque une différenciation massive et une déplétion du groupe de cellules progénitrices. Afin de déterminer si la régulation de cette initiation se fait avant ou après induction de NICD, j'ai créé un plasmide permettant de répondre à cette question et afin de visualiser son expression in vivo, j'ai initié une collaboration avec une équipe de l'ILM afin de créer un microscope vertical SPIM biphoton permettant l'observation d'embryon de poulets vivants [etc...] / The epithelio-mesenchymal transition (EMT) is a well-known mechanism by which epithelial cells lose their adherent connections and gain migratory properties, associated with a gain of a mesenchymal phenotype. This EMT is required in numerous processes as gastrulation, organogenesis, fibrosis and cancers. Various molecular pathways orchestrate the EMT depending on the EMT biological context. Recently, our laboratory highlighted the implication of the cytoplasmic Notch pathway in the dorso-medial lip (DML) EMT. In the DML tissue, theEMT is synchronized with differentiation pathways, to generate cells forming the primary myotome. Our laboratory showed that neural crests cells expressing DLL1 activate NOTCH receptor of the DML cells, via a “kiss and run” model. This leads to NOTCH cleavage, releasing an activated intra-cytoplasmic NOTCH domain (NICD). In the cytoplasm, NICD inhibits the GSK3ß kinase, leading to the stabilization of SNAIL and the free cytoplasmic ßcatenin. These molecules translocate into the nucleus and lead to the activation of MRF as Myf5 (ß-catenin) and to the repression of adherent genes (SNAIL). Therefore, Notch cytoplasmic pathway allows a synergized induction of both, the EMT and myogenic programs. This pathway remains controversial and the precise mechanism how NICD inhibits GSK3ß needs to be elucidated. Therefore, the aim of my thesis project was to clarify how NICD inhibits GSK3ß activity. First, I confirmed that NICD and GSK3ß physically interact by CoIP. Moreover, I demonstrated that the serin-threonin kinase AKT, known to inhibit GSK3ß by phosphorylation and also to mediate EMT in cancer, can physically interact with NICD in the cytoplasm. I have also shown that AKT mediates the induction of the myogenic program through the inhibitory phosphorylation of GSK3ß and that SNAIL is downstream of AKT. Together, these experiments indicate that AKT mediates, through phosphorylation, the cytoplasmic NICD inhibition of GSK3ß leading to myogenesis. A comparison of the chicken NICD1 and the 4 isoforms of mouse NICD highlighted that these 5 proteins induce EMT and myogenesis similarly. The dissection of the different conserved domains in the 5 different NICD proteins demonstrated that the RAM domain, known to activate transcription by binding to RBPJ, is necessary and sufficient for GSK3ß inhibition. A second axis of the thesis has been to test the involvment of the cytoplasmic Notch pathway in other EMT contexts. First, I highlighted that this pathway induces myogenesis, showing that NICD inhibits GSK3ß activity in the ventro-lateral lip. I further demonstrated that the cytoplasmic Notch pathway is implicated in the EMT and differentiation of the neural crests cells delaminating from the dorsal neural tube. Particularly, I have shown a co-activation of the Wnt and Notch pathway in premigratory and migratory neural crests. Moreover, I demonstrated a cytoplasmic inhibition of the kinase activity of GSK3ß by NICD, as well as the induction of the differentiation by cytoplasmic ß-catenin or SNAIL2. In a third axis of my thesis, I tried to clarify the regulatory mechanism involved in Notch activation. Previously it has been demonstrated that in all the DML cells Notch can be activated by an overexpression of DLL1 and that an ectopic expression of NICD in the DML cells induce a massive differentiation and depletion of the progenitor pool. To determine if the regulation of this initiation of the myogenic program occurs before or after Notch activation, I designed a plasmid to visualize Notch activation in vivo. In order to be able to follow the DLM cells and Notch activation in vivo, I initiated a collaboration with an ILM team to create a vertical SPIM biphoton microscope. In the future, this microscope will allow us to follow cells in living chicken embryos [etc...]

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