La morphogenèse crâniofaciale chez les vertébrés est un phénomène important, strictement régulé dans l’espace et dans le temps. Elle est basée sur une série complexe d'événements moléculaires et morphogénétiques qui implique un réseau interactionnel de gènes et de facteurs de transcriptions, tels les homéoboîtes. La crête neurale (CN) est au cœur de ce processus. Cette dernière fournit la principale source du mésenchyme crâniofacial. Cette population de cellules embryonnaires transitoires va subir une transition épithélio-mésenchymateuse et migrer en plusieurs vagues vers des sites prédéfinis puis se différencier en divers types cellulaires. La CN est à l’origine de plusieurs structures : une grande partie du squelette facial dont le maxillaire, la mandibule, l’os alvéolaire qui entoure les dents ainsi qu’une partie des tissus conjonctifs crâniofaciaux. Les cellules issues des CN sont pluripotentes et offrent un espoir en régénération osseuse et cartilagineuse. Ces caractéristiques ont généré un intérêt particulier des chercheurs pour les utiliser en thérapie cellulaire afin de réparer les défauts osseux des mâchoires. Parmi les tissus crâniofaciaux, nous avons choisi d’étudier plus avant la gencive et les cellules gingivales car leur accès est le plus facile et leurs capacités de différenciation autorisent l’observation d’autres phénotypes cellulaires.La gencive est un tissu kératinisé qui entoure les dents et recouvre l’os alvéolaire. Ce tissu est composé principalement de fibroblastes gingivaux (GFs). Parmi ces cellules, se trouvent des cellules souches gingivales (GSCs) caractérisées par leur auto-renouvellement et leur multipotence. Les GSCs sont facilement recueillies chez les patients adultes, elles montrent une plasticité importante et une activité immunomodulatrice qui en font un outil de choix pour la thérapie cellulaire. De plus, la biopsie se fait sans douleur et n’entraîne ni cicatrice ni problème fonctionnel.La première partie de mon travail de doctorat avait pour objectif d’évaluer le rôle de Msx1 dans la morphogenèse crâniofaciale et par la suite d’analyser l’os alvéolaire après une extraction dentaire afin d’analyser les mécanismes associés à ce processus et l’impact de Msx1 sur la cicatrisation osseuse.La deuxième partie de mon travail est axé sur la gencive et avait pour objectif de mettre en évidence l’origine embryologique des cellules souches orales, dont les GSCs, et de déterminer si elles proviennent des crêtes neurales, du mésoderme ou d’une mosaïque des deux. Enfin, pour appliquer nos connaissances sur l’origine embryologique des cellules souches gingivales, nous avons étudié le profil immunitaire des cellules dérivées des CN. Pour cela, nous avons déterminé la capacité phagocytaire des cellules souches gingivales murines dérivées des CN et comparé à des cellules de CN d’autres espèces vertébrées. / Craniofacial morphogenesis in vertebrates is an important phenomenon, strictly regulated in space and in time. It is based on a complex series of molecular and morphogenetic events involving an interactional network of genes and transcription factors, such as the homeobox. Neural crest (NC) is at the heart of this process. The latter provides the main source of craniofacial mesenchyme. This transient population of embryonic cells will undergo epithelial-mesenchymal transition and migrate in waves to predefined sites and to differentiate into various cell types. NC is the source of several structures: a large part of the facial skeleton including the maxillary, mandibular alveolar bone around the teeth as well as connective tissue in craniofacial portion. Cells from NC are pluripotent and offer hope for bone and cartilage regeneration. These characteristics have generated particular interest to researchers for use in cell therapy to repair bone defects of the jaw. Among the craniofacial tissues, we decided to further investigate the gums and gingival cells because their access is easier and differentiation capabilities allow observation of other cellular phenotypes.The gum is a keratinized tissue around the teeth and covers the alveolar bone. This tissue is composed mainly of populations of gingival fibroblasts (GFs). Among these populations, there are gingival stem cells (GSCs) characterized by their self-renewal and pluripotency. The GSCs are easily collected in adult patients, they show significant plasticity and immunomodulatory activity that make it a tool of choice for cell therapy. In addition, the biopsy is painless and involves neither scar nor functional problem.The first part of my PhD work was to evaluate the Msx1 role in craniofacial morphogenesis and subsequently analyse the alveolar bone after tooth extraction to analyse the mechanisms involved in this process and the impact of Msx1 on bone healing.The second part of my work focuses on the gingiva and was intended to highlight the embryological origin of oral stem cells, including GSCs and determine if they come from the neural crest, mesodermal or mosaic two. Finally, to apply our knowledge of the embryological origin of gum stem cells, we studied the immune profile derived NC cells. For this, we determined the phagocytic capacity gingival murine stem cells derived from CN and compared to cells of CN other vertebrate species.

Conventional rotating machine vibration monitoring techniques are based on the assumption that changes in the measured structural response are caused by deterioration in the condition of the rotating machine. However, due to changing rotational speed, the measured signal may be non-stationary and difficult to interpret. For this reason, the order tracking technique was introduced. One of main advantages of order tracking over traditional vibration monitoring techniques, lies in its ability to clearly identify non-stationary vibration data, and to a large extent exclude the influences from varying rotational speed. Several order tracking techniques have been developed and researched during the past 20 years. Among these techniques, Fourier Transform Based Order Tracking (FT-OT), Angle Domain Sampling Based Order Tracking (AD-OT) and Vold-Kalman Filter Order Tracking (VKF-OT) are the three most popular techniques and have been commercialised in software. While the VKF-OT is comparatively new, and both its theory and application are different from the other two techniques, the unique advantages of this technique has led to increased research attention in this field. This growing interest in research on the application of the VKF-OT technique on real machines, and its comparative advantages with respect to other order tracking techniques, inspired the present research. With this work, a comprehensive literature of electrical machine condition monitoring was surveyed, which gives a broad perspective of electrical machine monitoring methods ranging through electrical techniques, vibration techniques, temperature techniques and chemical techniques. To simply the process of applying VKF-OT in initial investigations, simulated single-degree-of freedom and two-degree-of freedom rotor models were established, and the application of the VKF-OT technique on these simulated models was explored. Because most of the current research draws significantly on an understanding of the VKF-OT theory, it was also necessary to review and summarize the current status of VKF-OT theory from previous work, as well as explore the procedures for selection of its filter bandwidth when dealing with real data. An experimental set-up for monitoring an electrical alternator was constructed. Real experimental data were subsequently used to compare the advantages and disadvantages of the three popular order tracking techniques. The unique time domain advantage of VKF-OT was implemented, using crest factor and kurtosis values as indictors of the fault condition of the machine. This gave encouraging results. / Dissertation (MSc)--University of Pretoria, 2008. / Mechanical and Aeronautical Engineering / unrestricted

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Differentiation of the head ectoderm is crucial for the evolutionary diversification of vertebrates. Expression of the genes responsible for this process is orchestrated troughout complex gene regulatory networks that are induced and modulated by Wnt, FGF and BMP signaling pathways. In addition, Wnt/β-catenin signaling, in combination with expression of the Wnt antagonists from the rostral-most part of the head ectoderm, represent a key source of information for the regionalization of the tissue along the antero-posterior axis. This allows the differentiation of the anterior ectoderm that gives rise to the anterior neural fold (ANF) and anterior part of the presumptive placodal region (PPR), and more posterior ectoderm where higher levels of active Wnt/β-catenin signaling promote differentiation into the neural crest (NC) and posterior PPR. Although the requirement of Wnt/β-catenin signalling for ANF, PPR and NC development has been intensively studied in non-mammalian vertebrate model organisms, we lack a clear picture about the situation in mammals. Furthermore, current knowledge in mammals has been gathered via experiments on the level of β-catenin and very little is known about the individual roles of the Tcf/Lef transcription factors. Thereby, we decided to manipulate the Tcf7l1, member of the...

Meis transcription factors belong to the group of TALE (three amino acids loop extension) homeodomain proteins. Meis2 proteins have a potential role in regulation of neural crest cells development and in differentiation of their derivates. Zebrafish genome has two paralogues of meis2 gene, meis2a and meis2b. CRISPR/Cas9 technology was used to prepare mutant lines of both paralogues, meis2a and meis2b, for the purpose of study of function of Meis2 transcription factors. Specific morpholinos that reduce the expression of meis2a and meis2b were used as controls. Craniofacial and cardiac development in mutant fish was analyzed in the meis2a line by RNA in situ hybridization, histological cartilage staining, and computed tomography. While we observed impaired craniofacial and cardiac development after injection of specific Morpholinos, we did not detect similar changes in the meis2a KO line. Our genetic approach has not clearly shown that the meis2a paralogue itself plays an important role in craniofacial development and cardiac development. For more detailed analysis, further experiments on fish lines with combined meis2a and meis2b knock-outs are needed. Key words Mutagenesis CRISPR, Danio rerio, neural crest cells, Meis2, transcription factor

Despite the fact that some aspects of single studies differ, there's a generally accepted view that the whole cartilaginous viscerocranium of vertebrates is neural crest derived. By the series of isotopic transplantation experiments of presumptive neural crest on the model organism Ambystoma mexicanum I partly specify this oppinion and prove that the most ventro-caudal cartilage, the second basibranchial, is of a different origin. Furher I mention the level of the presumptive neural crest where the single parts of cartilaginous viscerocranium arise from. Moreover there is one element, the first basibranchial, which has double origin. I discuss also some other neural crest derivatives such as head and outer gills mesenchyme, the trabeculae cranii, part of the cartilaginous otic capsule or the connective tissue in the head. I have performed 179 transplantations between transgenic and normal axolotl embryos. My final analysis is composed of 65 embryos of stage 40 - 42 and 7 larvae of lenght of 15 - 17 mm.

La crête neurale est une population de cellules migratoires multipotentes qui se délaminent du tube neural et se différencient en plusieurs types cellulaires. Des altérations du réseau génique de régulation (GRN) de la CNengendrent des maladies congénitales, peu comprises. Cette thèse a pour but d’approfondir la compréhension du rôle de PAX3, un gène crucial dans le GRN de la CN, pendant le développement normal ou pathologique de la CN. Tout d’abord, nous avons caractérisé deux lignées transgéniques de X. laevis, Pax3:GFP etSox10:GFP qui permettent d’étudier l’induction et la spécification précoce de la CN ou sa migration, respectivement. Ensuite, en utilisant des analyses à large échelle, RNAseq et ChIPseq,nous avons défini le premier CN-GRN centré surPax3 chez X. laevis et avons notamment identifié quatre nouveaux gènes régulés par Pax3 :pcdh8l, ercc1 (directement) et fhl3, mmp14(indirectement). Des analyses par perte et gain de fonction de Pax3 in vivo ont permis de vérifier lapertinence de ces cibles.Puis, nous avons analysé le rôle des cibles, Fhl3,pendant le développement de la CN. Fhl3 s’est avéré être un stimulateur intracellulaire de la voie BMP qui, de manière contrôlée spatio-temporellement,est indispensable pour que les cellules cibles de BMP activent la production de WNT à un niveau suffisant pour le développement de la CN.Finalement, nous avons généré les premières lignées iPSC dérivées de patients atteints du syndrome de Waardenburg de type 1 qui ont un allèle de Pax3 muté et nous avons pu les différencier en CN. L’ensemble de ce travail apporte de nouveaux outils et de nouveaux gènes d’intérêt à étudier la CN tant chez X. laevis que chez l’humain. / The neural crest (NC) is a population of multipotent migratory cells that delaminate from the neural tube and differentiate into several cell types. Alterations in NC regulatory gene network (GRN) result in congenital diseases that are poorly understood. This thesis aims to better understand the role of Pax3, a crucial gene in NC GRN, during the normal orpathological NC development. First, we characterized two transgenic lines of X. laevis,Pax3:GFP and Sox10:GFP that allowed us to study the induction and early specification of NC or its migration, respectively. Then, using large scale analyzes, RNAseq and ChIPseq, we defined the first NC-GRN centered on Pax3 inX. laevis and identified in particular four new genes regulated by Pax3 : pcdh8l, ercc1(directly) and fhl3, mmp14 (indirectly). The relevance of these targets was verified by Pax3loss- and gain-of-function in vivo.Then, we analyzed the role of one target, Fhl3,during NC development. We have shown thatFhl3 is an intracellular stimulator of the BMP pathway, which, in a spatiotemporally controlled manner, is essential for BMP target cells to activate the production of WNT at a sufficient level for the development of NC.Finally, we generated the first iPSC lines derived from Waardenburg syndrome type 1patients with a heterozygous Pax3 loss-of function mutation and we were able to differentiate them into NC. All of this work brings new tools and new genes of interest to study NC in both X. laevis and humans.

Glaucoma is a heterogeneous group of diseases that is currently considered to be the leading cause of irreversible blindness worldwide. Of the identified risk factors, elevated intraocular pressure remains the only modifiable risk factor that can be targeted clinically. Ocular hypertension is often a result of dysregulation of aqueous humour fluid dynamics in the anterior eye segment. Aqueous humour drainage is regulated by structures located in the anterior chamber of the eye. In some circumstances dysregulation occurs due to developmental abnormalities of these structures. The malformation of structures in the anterior segment is thought to be due to a defect in the differentiation and/or migration of the periocular mesenchyme during development. Unique to vertebrates, the neural crest cell (NCC) population contributes to the periocular mesenchyme and is instrumental to the proper development of structures in the anterior segment. For many years our laboratory has examined the role of the Activating Protein-2 (AP-2) transcription factors that are expressed in the neural crest and vital during the development of the eye. The purpose of this research project is to investigate the role of AP-2β in the NCC population during the development of the anterior segment of the eye. Conditional deletion of AP-2β expression in the NCC population demonstrated that mutants have dysgenesis of structures in the anterior segment including defects of the corneal endothelium, corneal stroma, ciliary body and a closed iridocorneal angle. Loss of retinal ganglion cells and their axons was also observed, likely due to the disruption of aqueous outflow, suggesting the development of glaucoma. The data generated from this research project will be critical in elucidating the role of AP-2β in the genetic cascade dictating the development of the anterior eye segment in addition to providing scientific research with a novel model of glaucomatous optic neuropathy. / Thesis / Master of Science (MSc)

Extensively migrating population of neural crest cells, which contributes to many tissues and builds up most of craniofacial vertebrate structures, has a crucial role in embryonic development of vertebrate body. The migratory pathways of neural crest cells are thought to be very conserved throughout the vertebrates and cranial neural crest migration is defined by progression of three migratory streams: trigeminal, hyoid and a common branchial stream. In this diploma thesis, migration of cranial neural crest was analysed using embryos of the Senegal bichir (Polypterus senegalus) and of sterlet (Acipenser ruthenus), which represent two basal-most lineages of extant ray-finned fishes. A combination of several techniques was used in both species in order to study cranial neural crest cells from their sites of origin to post- migratory stages and the pattern of migration was compared and discussed in revealed embryonic context. In the Senegal bichir the hyoid neural crest stream was shown to migrate first and it is also the most abundant; this heterochrony shift is apparently related to formation of external gills, which in bichir are situated on the hyoid arch only. In sterlet, neural crest cells migrate in a classic pattern of three progressive streams but their dynamics and patterning is influenced by...

La crête neurale (CN) est une population transitoire de cellules multipotentes qui émerge à la frontière entre l’ectoderme neural et non-neural, dans une région appelée la bordure neurale (BN). Lorsque la BN se soulève pour former le tube neural, les cellules de la CN subissent une transition épithélium-mésenchyme (TEM), et migrent de façon intensive dans l’ensemble de l’embryon pour atteindre leur destination finale et se différencier. Elles sont à l’origine de nombreux types de dérivés : neurones, cellules gliales, cartilage de la tête, os et tissus connectifs, cellules pigmentaires, cellules sympatho-adrenales. Tous ces processus sont régulés par l’action coordonnée de nombreux gènes qui forment un réseau de régulations génétiques complexe, au sein duquel de nombreuses interactions ont été décrites, même si de nombreuses relations restent à élucider à ce jour. Une mauvaise régulation de gènes normalement impliqués dans la formation de la CN provoque des malformations congénitales appelées neurocristopathies. Par ailleurs, la TEM subie par les cellules de CN avant leur migration est également observée dans les cellules cancéreuses acquérant des propriétés métastatiques. Les événements moléculaires et de nombreux gènes impliqués dans la TEM sont communs au développement de la CN et au cancer.Les liens existant entre le développement de la CN et les neurocristopathies, ainsi que les métastases, soulignent l’importance de l’étude du réseau de régulations génétiques permettant la formation de la CN et l’EMT.Au laboratoire, nous nous intéressons aux événements précoces d’induction et de spécification de la CN. Dans le but d’identifier les gènes préférentiellement impliqués dans le développement précoce de la CN et non dans la formation de l’ectoderme neural et non-neural, un crible a été effectué sur le transcriptome de différents tissus embryonnaires micro-disséqués. La validation des résultats de ce crible a permis d’identifier plusieurs gènes intéressants possédant une fonction potentielle dans la formation de la CN. Nous nous sommes particulièrement intéressés à deux d’entre eux, en raison de leur fonction originale comparée à la majorité des gènes impliqués dans le développement de la CN : serca1 et pfkfb4, un régulateur de l’homéostasie calcique et un régulateur de la glycolyse respectivement.Nous avons analysé les patrons d’expression des gènes des familles serca et pfkfb au cours du développement de Xenopus laevis. En raison de son expression spécifique dans la CN, nous avons étudié plus en détails le rôle de pfkfb4 dans la formation de la CN. Cette analyse a montré que pfkfb4 est nécessaire pour la spécification neurale et de la crête neurale.Toutefois, malgré son rôle documenté dans la glycolyse, le phénotype des morphants pfkfb4 dans l’embryon de Xenopus laevis n’est pas dû à une altération de la glycolyse.En conclusion, nos résultats démontrent l’existence d’un nouveau rôle non glycolytique pour Pfkfb4 au cours du développement embryonnaire de Xenopus Laevis. / Neural Crest (NC) is a transient population of multipotent cells that arises at the border between neural and non-neural ectoderm, in a region named the neural border (NB). As the neural border elevates to form the neural tube, NC cells undergo an Epithelial-To-Mesenchymal Transition (EMT), migrate extensively into the whole body to reach their final destinations and differentiate. They give rise to multiple derivatives: neurons and glia, head cartilage, bones and connective tissue, pigment cells, sympatho-adrenal cells. All these processes are regulated by the concerted actions of several genes that form a complex Gene Regulatory Network (GRN), in which many interactions have been elucidated, but even more relationships still need to be understood. Misregulation of genes normally involved in NC formation causes birth defects called neurocristopathies. Moreover, the EMT that NC cells undergo before migration also takes place when cancer cells become metastatic: the molecular events and many of the genes involved in EMT and migration are shared between NC development and cancer. The links with metastasis, neurocristopathies and the fact that still little is known about the earliest steps of NC formation, highlight the importance and the interest in understanding the Gene Regulatory Network (GRN) leading to NC formation and EMT.In the laboratory, we are interested in the early steps of NC induction and specification. In order to identify genes preferentially involved in early NC development compared to genes involved in neural and non-neural ectoderm formation, a transcriptome screen on different microdissected embryonic tissues has been performed. The validation of the results of the screen revealed several interesting genes with a potential function in NC formation. We focused particularly on two of them, due to their original function compared to the majority of the genes involved in NC development: serca1 and pfkfb4, a calcium homeostasis regulator and a glycolysis regulator respectively. We analysed the expression patterns of serca and pfkfb family genes during Xenopus laevis development. Then, due to its specific expression in NC, we studied more in details the role of pfkfb4 in NC formation. This analysis revealed that pfkfb4 is necessary for neural and neural crest specification. However, despite its known role in glycolysis, pfkfb4 morphant phenotype in Xenopus laevis embryos is not due to an alteration of the glycolytic pathway.In conclusion, our results reveal a novel extra-glycolytic role for Pfkfb4 during Xenopus laevis embryonic development.