In this work, the evolution of sex determination gene networks is inves tigated using a modelling approach. Recent evidence indicates that an in crease in the complexity of interactions has played an important role in gene network evolution. Sex determination mechanisms offer a good model for studying gene network evolution because, among other reasons, they evolve rapidly. In chapter 2, the potential for evolutionary change of the existing Drosophila sex determination gene network is considered. With the aid of a synchronous logical model, theoretical concepts such as a network-specific form of mutation are defined, as well as a notion of functional equivalence between networks. Applying this theoretical framework to the sex deter mination mechanism, it is found that sex determination networks generally exist within large sets of functionally equivalent networks all of which satisfy the sex determination task. These large sets are in turn composed of sub sets which are mutationally related, suggesting a high degree of flexibility is available without compromising the core functionality. The technique for finding functional equivalence between networks suggests a general method for gene network reconstruction, which is explored in chapter 3. Lastly, in chapters 4 and 5, a hierarchical model is presented which integrates popu lation genetics techniques with network dynamics. This model consists of a core population genetics simulation within which parameters such as the sex and fitness of the genotype are calculated from the corresponding network dynamics. The model is used to investigate the early evolution of sex deter mination networks. Following from a hypothesis proposed by Wilkins (1995), the assumption is made that sex determination networks have evolved in a retrograde manner from bottom to top. Starting from the simplest possible ancestral system, based on a single locus, we explore the way in which more complex systems, involving two or three loci, could have evolved.

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Addendum pasted inside front fly leaf. / Bibliography: leaves 168-247 / xxv, 269, [15] leaves, [4] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1996

Somitogenesis, the sequential formation of a periodic pattern along the antero-posterior axis of vertebrate embryos, is one of the most obvious examples of the segmental patterning processes that take place during embryogenesis and also one of the major unresolved events in developmental biology. The principal aim of this thesis is to develop a series of mathematical models for somite formation. We begin by reviewing the current models for somitogenesis in the light of new experimental evidence regarding the presence of a segmentation clock and graded expression of FGF8. We conduct a preliminary investigation into the wavefront of FGF8 along the antero-posterior axis and integrate this model into the framework of an existing model for a signalling process. We demonstrate that this new “Clock and Wavefront” model can produce coherent series’ of somites in a manner that is tightly regulated in both space and time, and that it can also mimic the effects seen when FGF8 expression is perturbed locally. We then use the model to make some experimentally testable predictions. The latter part of the thesis concentrates on building more biologically accurate model for the FGF8 gradient. We move to consider a model for the FGF8 gradient which involves a complex network of biochemical interactions with negative feedback between FGF8 and retinoic acid. The resulting system of seven coupled non-linear equations, including both ordinary and partial differential equations, is difficult to analyse. To facilitate our understanding of the non-linear interactions between FGF8 and retinoic acid, we finally consider a reduced model which can display travelling wavefronts of opposing FGF8 and retinoic acid concentrations moving down the antero-posterior axis. The model allowed us to calculate a minimum wave speed for the wavefronts as a function of key model parameters such as the rate of FGF8 and retinoic acid decay; strong dependence on the values of these parameters is a result that is hypothesised to occur in vivo.

Deux états d'autorenouvellement des cellules souches pluripotentes (PSCs) ont été définis, à savoir les états naïf et amorcé. De nombreuses différences existent entre ces deux états dont la plus marquante est la capacité unique des PSCs à l'état naïf, de coloniser l'embryon préimplantatoire et former des chimères. L'objectif de mon projet doctoral a été d'étudier la pluripotence chez le lapin. Dans ce cadre, j'ai d'abord entrepris de fabriquer et de caractériser des cellules souches pluripotentes induites (RbiPSCs), puis d'évaluer leur capacité à coloniser l'embryon et à former des chimères. Trois lignées de RbiPSCs dépendantes du FGF2 ont été obtenues par reprogrammation de fibroblastes de lapin. Leur caractérisation moléculaire et fonctionnelle a révélé des caractéristiques mixtes, naïves et amorcées. En revanche, sur le plan fonctionnel, elles sont incapables de coloniser l'embryon de lapin, une caractéristique de la pluripotence amorcée. La seconde partie de mon projet doctoral a consisté à reprogrammer des RbiPSCs vers l'état naïf. Dans ce but, j'ai surexprimé KLF2 et KLF4, deux gènes appartenant au réseau de pluripotence naïf, et utilisé les conditions de culture des PSCs de souris. Les cellules ainsi obtenues présentent un profil d'expression génique plus proche de celui de l'ICM de lapin, dû notamment à la réactivation de marqueurs spécifiques de la pluripotence naïve. Enfin, les cellules ainsi reprogrammées présentent une capacité accrue pour la colonisation de l'embryon préimplantatoire de lapin. Mes travaux constituent le premier exemple de reprogrammation de cellules souches pluripotentes vers l'état naïf chez le lapin. Les cellules ainsi produites ouvrent la voie à la fabrication de chimères somatiques et germinales / Pluripotent stem cells (PSCs) can self-renew at two distinct states, the naive and primed states. Many differences exist between these two states, the most striking is the unique ability of PSCs naïve to colonize the preimplantation embryo and form chimeras. The purpose of my doctoral project was to study pluripotency in rabbits. In this context, I initially manufactured and characterized induced pluripotent stem cells (RbiPSCs) and then evaluated their ability to colonize the embryo and form chimeras. Three RbiPSCs lines were obtained by rabbit fibroblasts reprogramming. Their molecular characterization revealed mixed characteristics, naïve and primed. However, functionally, they are unable to colonize the rabbit embryo, a feature of primed pluripotency. The second part of my doctoral project was to reprogram RbiPSCs to the naïve state. To this end, I have overexpressed Klf2 and Klf4, two genes belonging to the naïve pluripotency network and the mouse PSCs culture conditions. These new cell lines have a gene expression profile closer to that of the rabbit ICM, particularly due to the reactivation of specific markers of naïve pluripotency. Finally, the reverted cells have an increased capacity of colonization of the preimplantation embryo rabbit. My work represents the first example of pluripotent stem cells reprogramming toward the naive state in rabbits. The cells thus produced pave the way for the production of somatic and germline chimeras

In this thesis, I explore four different ways in which relatedness affects sexual interactions in the red junglefowl Gallus gallus ssp., and the fruit fly Drosophila melanogaster. First, I show that in both species, inbreeding depression is sex-specific and modulated by parental age and gametic age. However, the sex that suffers higher inbreeding depression was trait- and species-dependent. Second, I examined patterns of inbreeding avoidance. I found no evidence of inbreeding avoidance in the fruit fly, but in the red junglefowl both males and females avoided mating with relatives, independently from sex-ratio of the social group. Third, I investigated whether relatedness amongst members of one sex affects mate choice in members of the opposite sex. Male fruit flies preferentially courted females unrelated to females with whom they had previously mated, while female flies displayed a weak preference for males related to their previous mates. In the red junglefowl, females exposed to male trios of two males related to each other and one unrelated male, displayed a marked preference for mating with the male unrelated to the other two males, and might also bias postcopulatory sperm utilization in favour of the unrelated male. Fourth, I explored the implications of male relatedness on the intensity of male-male competition. Male red junglefowl were less aggressive towards related competitors, but invested more sperm in females that had previously mated with a related male rather than with an unrelated male. In fruit flies, male relatedness had a strong impact on female life-history and offspring viability, although I found no evidence that these effects were modulated by changes in male-male competition. Collectively, the findings of these studies demonstrate the complex relationship between relatedness and other important biological phenomena as such senescence and sexual conflict.

De nos jours, la cryoconservation est une technique très largement utilisée dans les protocoles d’assistance à la reproduction ou comme outil pour la sauvegarde des ressources génétiques. Toutefois, la chienne est un modèle animal complexe pour l’application des biotechnologies de la reproduction du fait de ses nombreuses singularités anatomiques et physiologiques. L’objectif de notre travail était d’étudier et de développer une méthode de cryoconservation des ressources génétiques chez la chienne par le biais de deux types de ressources : les embryons et le tissu ovarien. Après avoir mis au point une méthode de collecte d’embryons, nous nous sommes appliqués à la constitution d’un stock d’embryons cryoconservés en prévision d’un transfert embryonnaire. L’étude et le développement d’un protocole de cryoconservation du tissu ovarien ont été abordés après avoir adapté et validé nos méthodes d’analyses in vitro. L’utilisation de plans d’expériences factoriels fractionnaires a permis de mettre en évidence les facteurs les plus influents sur la qualité de la réserve folliculaire (nature du cryoprotecteur pénétrant, cinétique de congélation, étapes d’équilibration) et de proposer un protocole de cryoconservation. La combinaison du DMSO incorporé en un seul bain d’équilibration avec une vitesse de congélation de 0,3°C/min est apparue comme la combinaison la plus appropriée à la cryoconservation de tissu ovarien chez la chienne et a permis d’observer, après xénogreffe de tissu ovarien cryoconservé, une reprise de la croissance folliculaire et de l’activité hormonale du tissu greffé / Nowadays, cryopreservation is widely used in animal assisted reproduction or safeguarding of genetic resources. Nevertheless, the bitch is a complex animal model concerning the use of this biotechnology, due to numerous anatomical and physiological peculiarities. The aim of our research work was to investigate and develop a method of cryopreservation of genetic resources in the bitch by exploring two kinds of resources: embryos and ovarian tissue. After the setting up of a method for embryo collection, we have built up a stock of cryopreserved embryo for subsequent embryo transfer. After a preliminary validation of our in vitro assessment methods, the investigation and development of a cryopreservation protocol has been conducted. The use of fractional experimental design allowed us to highlight the main factors affecting the follicular pool quality (CPA nature, freezing rate and equilibration steps). The combination of DMSO incorporated in a unique equilibration bath with a freezing rate of 0.3°C/min appeared to be suitable for the cryopreservation of bitch ovarian tissue. Finally, Follicular growth and hormonal activity resumption have been observed after xenotransplantation of cryopreserved bitch ovarian tissue