Bibliography: p. 96-108. / 108, [31] p., [9] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The major aim of this thesis is the identification and characterisation of Polycomblike (PCL) protein interactors. The study analyses the ability of PCL to bind directly to DNA anchoring the Pc-G complex to the genes which they repress. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1997

This thesis was done in collaboration with Sorbonne University as part of a double degree Cotutelle. / During development, cell differentiation frequently occurs upon signaling from concentration or activity gradients of molecules called morphogens. These molecules control in a dose-dependent manner the expression of sets of target genes that determine cell identity. A simple paradigm to study morphogens is the Bicoid gradient, which determines antero-posterior patterning in fruit fly embryos. The Bicoid transcription factor allows the rapid step-like expression of its major target gene hunchback, expressed only in the anterior half of the embryo. The general goal of my thesis was to understand how the information contained in the Bicoid morphogen gradient is rapidly interpreted to provide the precise expression pattern of its target. Using the MS2 system to fluorescently tag specific RNA in living embryos, we were able to show that the ongoing transcription process at the hunchback promoter is bursty and likely functions according to a two-state model. At each nuclear interphase, transcription is first observed in the anterior and it rapidly spreads towards the posterior, as expected for a Bicoid dose-dependent activation process. Surprisingly, it takes only 3 minutes from the first hints of transcription at the anterior to reach steady state with the setting of a sharp expression border in the middle of the embryo. Using modeling taking into account this very fast dynamics, we show that the presence of only 6 Bicoid binding sites (known number of sites in the hunchback promoter) in the promoter, is not sufficient to explain the establishment of a sharp expression border in such a short time. Thus, either more Bicoid binding sites or inputs from other transcription factors could help reconcile the model to the data. To better understand the role of transcription factors other than Bicoid in this process, I used a two-pronged strategy involving synthetic MS2 reporters combined with the analysis of the hunchback MS2 reporter in various mutant backgrounds. I show that the pioneer factor Zelda and the Hunchback protein itself are also critical for hunchback expression, maternal Hunchback acting at nuclear cycle 11-12, while zygotic Hunchback is acting later at nuclear cycle 13-14. The synthetic reporter approach indicate that in contrast to Hunchback and Caudal, Bicoid is able to activate transcription on its own when bound to the promoter. However, the presence of 6 Bicoid binding sites only leads to stochastic activation of the target loci. Interestingly, the binding of Hunchback to the Bicoid-dependent promoter reduces this stochasticity while Caudal might act as a posterior repressor gradient. Confronting these experimental data to theoretical models is ongoing and should allow to better understand the role of transcription factors, other than Bicoid, in hunchback expression at the mechanistic level. / Thesis / Doctor of Philosophy (PhD) / Have you ever wondered how a single cell can become a full grown organism? Well it starts when an egg and sperm fuse together. As time passes this single cell divides over and over again until an organism is formed. During this developmental process, somehow the cells know exactly where they are and what they need to become so that they form the organism. However, we don’t fully understand this process and this is what we hope to answer with our research: How do the cells know where they are and what they need to become during development? We study this process in the fruit fly. Although fruit flies might not look a lot like us, during early embryonic development we are quite similar, so we can try to answer these questions in fruit flies and what we find might be relevant to other organisms like us. During development, the first element that an embryo needs to know is the orientation of its body, where the head and tail, the left and right and the back and front of the body will be. We concentrate on studying how the head to tail axis, which we call the anterior-posterior axis, is formed. To know where the head is going to be, the embryo releases proteins called morphogens that broadcast instructions to other genes so that cells know where they are and what they should become. We study a morphogen called Bicoid. Its concentration is high in the anterior, the region that will become the head of the embryo, and lower as you move towards the posterior where the tail will form. Bicoid activates a gene called hunchback, which ends up dividing the embryo in two large parts, the top and the bottom. However, Bicoid’s message fades away during each cell division and needs to be read again at the beginning of each new nuclear cycle. So how is the message read and how long does this process take? This last question is particularly critical during the period of very fast cell division. My thesis tries to answer this question. We found out that it takes 3 minutes for a nuclei to read the Bicoid concentration, activate hunchback and express it correctly. However, in contrast to what was believed before, or namely, that only Bicoid was involved in this process, we found out that other players are involved in helping relay this message. This way hunchback can accurately divide the body in two parts exactly in the middle and without mistake in such a short period of time.

Au cours de ma thèse j’ai étudié les néprilysines (Nep), des protéinases connues pour dégrader de petits neuropeptides, en particulier les peptides amyloïdes (Aβ). Lors de la maladie d’Alzheimer, les peptides Aβ s’agrègent pour former des plaques toxiques. Il a été montré que l’expression des Nep module l’effet toxique d’Aβ sur la mémoire chez les modèles murins. Néanmoins, le rôle des Nep dans la mémoire dans des conditions physiologiques reste à ce jour inconnu.La drosophile exprime 4 Nep dans le système nerveux central adulte. Nous avons analysé leur rôle dans la mémoire olfactive. Les 4 Nep sont requises pour 2 phases spécifiques de mémoire: à moyen terme (MTM) et à long terme (LTM). De plus, nous avons identifié les neurones dans lesquels elles sont requises : les Mushroom Bodies (MB) ainsi qu’une paire de neurones afférents, les Dorsal Paired Medial neurons (DPM). Nous avons ensuite cherché à savoir si Aβ était l’une des cibles des Nep. Nous avons montré que l’expression d’Aβ dans les DPM n’altère la MTM que lorsque l’expression de Nep1 est inhibée. De plus, le défaut de LTM de drosophiles exprimant Aβ dans les DPM est sauvé par la surexpression de Nep1. En conclusion, nos résultats suggèrent qu’Aβ est dégradé par Nep1 au cours des processus de mémorisation, et qu’Aβ est une cible de Nep1 en conditions non pathologiques.Enfin, nous nous sommes intéressés au neuropeptide amnesiac, décrit comme étant requis pour la mémoire dans les DPM. Nos travaux démontrent qu’amnesiac est en fait requis dans les DPM pour leur développement, et chez l’adulte dans les MB pour activer l’adénylate cyclase responsable de la détection de coïncidence permettant la formation de la MTM. / During my PhD, I studied neprilysins, proteinases known to degrade small neuropeptides, in particular mammalian amyloid-β peptides (Aβ). During Alzheimer’s disease, Aβ peptides aggregate to form toxic plaques. It has been shown that neprilysins expression modulates toxic effects of Aβ on memory in murine models of the disease. However, the role of neprilysins in memory under physiological conditions is still unknown. Drosophila expresses 4 neprilysins in the adult central nervous system. First we have analyzed their role in olfactive memory. We have shown that all of them are required for 2 specific memory phases: Middle-Term Memory (MTM) and Long-Term Memory (LTM). We also have identified the neurons in which they are required: the Mushroom Bodies (MB) and a pair of afferent neurons, the Dorsal Paired Medial (DPM) neurons. Then we investigated whether Aβ peptides could be one of the neprilysins’ targets. We have shown that Aβ expression in DPM neurons alters MTM only when Nep1 expression is inhibited. Furthermore, the LTM deficit of flies expressing Aβ in DPM neurons is rescued by Nep1 overexpression. To conclude, our results suggest that Nep1 degrades endogenous Aβ peptides during memory processes, and that Aβ is a physiological target for Nep1 under non-pathological condition.Finally, we became interested in the amnesiac neuropeptide, described as being required for memory in DPM neurons. Our work shows that amnesiac is actually required in DPM neurons for their development, and in the MB of adult flies in order to activate the adenylate cyclase responsible for coincidence detection leading to MTM formation.

La profondeur possible d’imagerie en laser-scanning microscopie est limitée non seulement par la distance de travail des lentilles de objectifs mais également par la dégradation de l’image causée par une atténuation et une diffraction de la lumière passant à travers l’échantillon. Afin d’étendre cette limite, il est possible, soit de retourner le spécimen pour enregistrer les images depuis chaque côté, or couper progressivement la partie supérieure de l’échantillon au fur et à mesure de l‘acquisition. Les différentes images prises de l’une de ces manières doivent ensuite être combinées pour générer un volume unique. Cependant, des mouvements de l’échantillon durant les procédures d’acquisition engendrent un décalage non seulement sur en translation selon les axes x, y et z mais également en rotation autour de ces même axes, rendant la fusion entres ces multiples images difficile. Nous avons développé une nouvelle approche appelée 2D-SIFT-in-3D-Space utilisant les SIFT (scale Invariant Feature Transform) pour atteindre un recalage robuste en trois dimensions de deux images. Notre méthode recale les images en corrigeant séparément les translations et rotations sur les trois axes grâce à l’extraction et l’association de caractéristiques stables de leurs coupes transversales bidimensionnelles. Pour évaluer la qualité du recalage, nous avons également développé un simulateur d’images de laser-scanning microscopie qui génère une paire d’images 3D virtuelle dans laquelle le niveau de bruit et les angles de rotations entre les angles de rotation sont contrôlés avec des paramètres connus. Pour une concaténation précise et naturelle de deux images, nous avons également développé un module permettant une compensation progressive de la luminosité et du contraste en fonction de la distance à la surface de l’échantillon. Ces outils ont été utilisés avec succès pour l’obtention d’images tridimensionnelles de haute résolution du cerveau de la mouche Drosophila melanogaster, particulièrement des neurones dopaminergiques, octopaminergiques et de leurs synapses. Ces neurones monoamines sont particulièrement important pour le fonctionnement du cerveau et une étude de leur réseau et connectivité est nécessaire pour comprendre leurs interactions. Si une évolution de leur connectivité au cours du temps n’a pas pu être démontrée via l’analyse de la répartition des sites synaptiques, l’étude suggère cependant que l’inactivation de l’un de ces types de neurones entraine des changements drastiques dans le réseau neuronal. / Although laser scanning microscopy is a powerful tool for obtaining thin optical sections, the possible depth of imaging is limited by the working distance of the microscope objective but also by the image degradation caused by the attenuation of both excitation laser beam and the light emitted from the fluorescence-labeled objects. Several workaround techniques have been employed to overcome this problem, such as recording the images from both sides of the sample, or by progressively cutting off the sample surface. The different views must then be combined in a unique volume. However, a straightforward concatenation is often not possible, because the small rotations that occur during the acquisition procedure, not only in translation along x, y and z axes but also in rotation around those axis, making the fusion uneasy. To address this problem we implemented a new algorithm called 2D-SIFT-in-3D-Space using SIFT (scale Invariant Feature Transform) to achieve a robust registration of big image stacks. Our method register the images fixing separately rotations and translations around the three axes using the extraction and matching of stable features in 2D cross-sections. In order to evaluate the registration quality, we created a simulator that generates artificial images that mimic laser scanning image stacks to make a mock pair of image stacks one of which is made from the same stack with the other but is rotated arbitrarily with known angles and filtered with a known noise. For a precise and natural-looking concatenation of the two images, we also developed a module progressively correcting the sample brightness and contrast depending on the sample surface. Those tools we successfully used to generate tridimensional high resolution images of the fly Drosophila melanogaster brain, in particular, its octopaminergic and dopaminergic neurons and their synapses. Those monoamine neurons appear to be determinant in the correct operating of the central nervous system and a precise and systematic analysis of their evolution and interaction is necessary to understand its mechanisms. If an evolution over time could not be highlighted through the pre-synaptic sites analysis, our study suggests however that the inactivation of one of these neuron types triggers drastic changes in the neural network.