Genetic screens provide information on phenotype interactions as a step to uncovering functional interactions in vivo. In this study, I conducted genetic modifier screens in Drosophila that might reveal genes expressed in the embryonic CNS that interact genetically with slit. These screens include a blind genetic screen, stable line double mutant analysis, transheterozygote interactions and a dosage-sensitive dominant modifier screen. Differences in CNS phenotypes between the blind screen and the double mutant analysis were uncovered for some of the crosses, indicating background effects of genotype can influence phenotypes observed. A group of candidate genes were chosen based on motif composition for potential interaction with slit, and spatial and temporal expression patterns coinciding with Slit. The majority of these genes show a mutant CNS phenotype on their own. slit-interacting genes were identified by their ability to alter midline axon guidance, as assayed with antibodies specific to CNS-expressing proteins. The interacting genes include those encoding receptor and second messengers thought to function in growth cones, integrins and extracellular matrix proteins. From these interactions, I could then propose models of function in axon guidance. The first model I propose adds to a currently known repressor-derepressor model of axon guidance at the midline of the CNS, involving the Netrin, Commissureless and Robo signaling molecules, whereby Slit is locally suppressed by Netrin function to allow axons to cross the midline via Comrnissureless-mediated intemalisation of Robo. In my second model, Slit may also be involved in integrin signaling, especially through the αPS3 and βPS integrins, in concert with the Laminin molecule. Included in this model is the intersection of cytoplasmic Dock signaling within the growth cone. Thirdly, I demonstrate a genetic interaction with other molecules expressed by the midline glia, Masquerade, Toll and Neurexin. Masquerade and Toll may function in a common pathway, but in parallel to Slit function. Likewise, Neurexin may function in a parallel pathway to Slit. Slit has shown to interact genetically with a number of molecules that appear to be involved in different aspects of axon guidance. This study was meant to provide a survey of molecules that may functionally interact with Slit and provides a good basis upon which to explore the interactions in detail in future work. / Thesis / Master of Science (MSc)

No description available.

Healthy pre-natal development of the mammalian visual system requires that retinal ganglion cell (RGC) axons navigate a precise path to their targets in the thalamus and superior colliculus by making a precise series of turns determined by the complex interactions between growth cone and extracellular environment. One important choice point for RGC axons is the crossing of the midline at the optic chiasm, where ipsilateral/ contralateral sorting takes place. In this thesis a novel image analysis method using steerable filters for quantifying the gross orientation and turning of axons from a static image (such as from DiI filled axons) is presented. This method was applied to understanding Slit dependent axon guidance at the mouse optic chiasm. It was possible to quantify the differences at the chiasm between the wildtype and various classes of mutants involving heterzygous or homozygous knockout of the Slit1 and the Slit2 genes. Assessment was in terms of the spatial distributions in axon density and axon orientation as derived from DiI labeled RGCs originating from one eye. The animals were assessed at embryonic day 13.5. To my knowledge this is the first quantification of its kind in the field of axon guidance. It was found that there were strong statistical differences from wildtype in both the Slit1-/-;Slit2-/- and Slit1+/+;Slit2-/- knockouts in terms of both axon density and axon orientation across large extents of the chiasm. In both these knockouts it was found that the changes in axon density were localised to the anterior region of the chiasm, but the changes in axon orientation were spread across almost the entire extent of the chiasm. No other combination of the Slit1 and Slit2 knockouts for which embryos could be generated showed significant differences from wildtype in terms of spatial changes in axon density or axon orientation. No embryos were generated for the Slit1+/-;Slit2-/- combination. No changes in the spatial distribution of axon density or axon orientation were found between the Slit1-/-;Slit2-/- and Slit1+/+;Slit2-/- knockouts, suggesting that in terms of these two quantities, the two phenotypes are indistinguishable. This evidence suggests that the role of Slit2 is more important than the role of Slit1 at the optic chiasm in terms iii of axon guidance. In addition, the gradients of mRNA expression of Slit1 and Slit2 were quantified using in situ hybridisation, and these data were used to compare the mRNA gradients with the orientation and turning of axons in both the wildtype and Slit1/Slit2 knockout chiasms. Although this provided a powerful visualisation tool, no simple mathematical relationship was found between the mRNA gradient of Slit1 or Slit2 and the orientation or turning of axons at the optic chiasm. These approaches now provide an important suite of methods for spatial analysis of axon tracts and molecular gradients in axon guidance.

Slit valves play an important role in semiconductor manufacturing industry. They enable creation of a vacuum environment required for wafer processing. Due to the high volume of production in the modern semiconductor industry, slit valves experience severe degradation over their useful lifetime. If maintenance is not applied in due time, degraded valves may lead to defects in end products because of pressure loss and particle generation. In this thesis, we proposed methods for signal processing and feature extraction for analysis of slit valve vibration signatures. These methods would be used to demonstrate the ability of reliably, accurately and efficiently distinguish between each individual valve via a multi-class classification procedure. Such ability is a clear illustration of the feasibility of vibration based monitoring of the slit valve conditions. / text

Le sang est une suspension concentrée (45 % en volume) de cellules déformables, les globules rouges, dans un liquide newtonien, le plasma. Dans la microcirculation, i.e. le sous-ensemble du système de circulation sanguine où s'effectuent les échanges de matière entre le sang et les tissus, les tailles de vaisseaux sont comparables à la taille d'un globule rouge (environ 10 µm). En conséquence, les effets dynamiques liés à la présence de ces cellules induisent des comportements rhéologiques complexes, qui jouent un rôle important dans le transport de l'oxygène vers les tissus. En particulier, aux bifurcations microvasculaires divergentes, les débits de globules rouges et de plasma peuvent se répartir de façon non proportionnelle entre les deux branches filles. La fraction volumique de globules rouges (hématocrite) dans l'une des branches filles est alors plus élevée que celle de la branche d'entrée, et la fraction volumique dans l'autre branche y est plus faible. Cet effet, connu sous le nom d'effet de séparation de phase, induit une très grande hétérogénéité de l'hématocrite d'un vaisseau à l'autre dans la microcirculation. Il induit également un couplage entre l'architecture du réseau microvasculaire et la dynamique de l'écoulement sanguin dans ce réseau. L'objectif de ce travail de thèse est d'étudier finement l'effet de séparation de phase in vitro, dans un régime représentatif des conditions physiologiques, au moyen de dispositifs microfluidiques modélisant les bifurcations microvasculaires et de suspensions de globules rouges. Dans ce but, un dispositif expérimental microfluidique a d'abord été élaboré. Puis, les aspects métrologiques spécifiques aux suspensions concentrées ont été abordés afin de quantifier les paramètres de l'écoulement. En particulier, la technique de dual-slit a été comprise et optimisée, assurant une mesure précise de profils de vitesse de globules rouges en microcanaux. Des métrologies spécifiques à nos conditions expérimentales ont également été mises en place pour déterminer l'hématocrite. Ces techniques ont été validées par vérification du principe de conservation de la masse entre les trois branches d'une bifurcation, et elles nous ont permis de caractériser les écoulements de globules rouges dans des micro-canaux de différentes tailles (10 à 100 µm), et ce pour de larges gammes de débits et de concentrations. Enfin, l'écoulement de suspensions de globules rouges a été étudié au niveau de micro-bifurcations, dans l'objectif de caractériser l'effet de séparation de phase pour des tailles de canaux et des gammes d'hématocrites qui n'ont pas été étudiés auparavant en conditions d'écoulement maîtrisées. / Blood is a concentrated suspension (45% by volume) of deformable red blood cells, flowing in a Newtonian fluid called plasma. The microcirculation is the part of the blood circulation system where the exchanges of material (e.g. nutrients, oxygen) between the blood and tissues take place. The microvessels are characterized by diameters less than 100 microns, which is similar in size to the size of a red blood cell ( 10 microns). As a result, the presence of these cells considerably influences the dynamics of microvascular flows and induces complex rheological behaviors. In particular, at diverging microvascular bifurcations, red blood cells and plasma may be nonproportionally distributed between two daughter vessels : one gets a higher red blood cell volume fraction (hematocrit) than the feeding vessel, while the other gets a lower one. This effect, known as the phase separation effect, causes a tremendous heterogeneity of the hematocrit among vessels in microvascular networks and induces a coupling between the microvascular architecture and the blood flow dynamics. The aim of this thesis is to investigate the phase separation effect in vitro, in physiological conditions, using red blood cell suspensions and microfluidic devices modeling microvascular bifurcations. For this purpose, a microfluidic experimental device was first developed. Then the metrological aspects specific to concentrated suspensions were addressed in order to quantify all the flow parameters. In particular, the dual-slit technique has been understood and optimized, ensuring accurate measurement of velocity profiles of red blood cells in microchannels. Measurement methods for our experimental conditions were also implemented to determine the hematocrit. All these techniques have been validated by verification of the principle of mass conservation between the three branches of a bifurcation. They allowed us to characterize the flow of red blood cells in microchannels of different sizes (10 to 100 microns) and for wide ranges of flow rates and concentrations. Finally, the flow of red blood cell suspensions was investigated at micro-bifurcations, with the aim of characterizing the phase separation effect for channel sizes and hematocrit ranges never studied in controlled flow conditions.

Digital imaging systems for medical applications must bebased upon highly efficient detectors to ensure low patientdose. This is particularly important in screening mammographybecause of the large number healthy women that is examined. Amammography system must also provide high spatial and contrastresolution. Different approaches are compared in this thesis,and it is argued that a system based on photon countingdetectors in a scanned multi-slit geometry provides aperformance superior to established technologies. The system is realized using silicon strip detectorsirradiated at a small angle relative to the wafer surface,thereby offering large absorption depth. A linear pixelarray isscanned across the breast to obtain the complete image.Pulse-processing electronics rejecting all detector andelectronics noise count the number of photons that aredetected, forming the pixel values of the image. Optimization of the detector design is discussed in detail.The detector has been carefully simulated to investigate chargemotion and signal formation after photoninteraction. Based onthese simulations, the impact of the detector characteristicson the image quality has been evaluated. Detectors have been manufactured and evaluated both assingle components and as part of experimental imaging devicesincluding custom readout electronics. Presented in this thesisare the measured detector characteristics including a verifi-cation of charge collection efficiency and confirmation thatthe quantum efficiency is 90% for a typical mammographyspectrum. Modulation transfer functions and noise power spectrawere recorded and the detective quantum efficiency calculated.A prototype mammography system was also assembled and themodulation transfer function recorded. The interpretation ofthe modulation transfer function and detective quantumefficiency is discussed for digital systems in general and fora scanned multi-slit system in particular. <b>Keywords:</b>x-ray, imaging, silicon, detector, digital,mammography, scanning, photon counting.

<p>Digital imaging systems for medical applications must bebased upon highly efficient detectors to ensure low patientdose. This is particularly important in screening mammographybecause of the large number healthy women that is examined. Amammography system must also provide high spatial and contrastresolution. Different approaches are compared in this thesis,and it is argued that a system based on photon countingdetectors in a scanned multi-slit geometry provides aperformance superior to established technologies.</p><p>The system is realized using silicon strip detectorsirradiated at a small angle relative to the wafer surface,thereby offering large absorption depth. A linear pixelarray isscanned across the breast to obtain the complete image.Pulse-processing electronics rejecting all detector andelectronics noise count the number of photons that aredetected, forming the pixel values of the image.</p><p>Optimization of the detector design is discussed in detail.The detector has been carefully simulated to investigate chargemotion and signal formation after photoninteraction. Based onthese simulations, the impact of the detector characteristicson the image quality has been evaluated.</p><p>Detectors have been manufactured and evaluated both assingle components and as part of experimental imaging devicesincluding custom readout electronics. Presented in this thesisare the measured detector characteristics including a verifi-cation of charge collection efficiency and confirmation thatthe quantum efficiency is 90% for a typical mammographyspectrum. Modulation transfer functions and noise power spectrawere recorded and the detective quantum efficiency calculated.A prototype mammography system was also assembled and themodulation transfer function recorded. The interpretation ofthe modulation transfer function and detective quantumefficiency is discussed for digital systems in general and fora scanned multi-slit system in particular.</p><p><b>Keywords:</b>x-ray, imaging, silicon, detector, digital,mammography, scanning, photon counting.</p>

Chez les bilatériens, les connexions permettant de relier la partie droite et gauche du système nerveux central (SNC) sont appelées commissures cérébrales. Comprendre les mécanismes moléculaires permettant la mise en place de ces circuits constituent un enjeu majeur en neurobiologie du développement. Le guidage des commissures cérébrales repose sur des paires de ligands-récepteurs telles que Netrine-1/DCC et Slits/Robos. Nétrine et Slits sont exprimés au niveau de la plaque du plancher tandis que leurs récepteurs respectifs, DCC et Robo1/2, sont exprimés dans les neurones et axones commissuraux de la moelle épinière et du tronc cérébral au cours du développement. L'étude des souris déficientes pour ces gènes a permis d'établir un modèle : Nétrine-1, agirait à distance afin d'attirer les axones commissuraux par gradient vers la ligne médiane ventrale, puis, les Slits permettraient de repousser les axones en dehors de la plaque du plancher. Au cours de ma thèse, j'ai utilisé des modèles génétiques murins afin d'étudier in vivo la mise en place des commissures et la migration des neurones précérébelleux. Nous avons inactivé de façon spécifique l'expression des molécules Nétrine-1, Slits et Robo1/2 dans différentes régions du SNC. Nous avons montré que la présence de Nétrine-1 dans la plaque du plancher n'était pas nécessaire à l'attraction des axones commissuraux mais que la source principale de Nétrine-1 était la glie radiaire. Nous avons aussi montré que le couple Slit/Robo n'était pas essentiel à la migration des neurones précérébelleux. Ces données remettent en cause le modèle classiquement proposé et suggèrent l'existence de d'autres mécanismes moléculaires de guidage. / In all bilaterians, the connections linking the left and the right part of the central nervous system (CNS) are called the commissures. Understanding molecular mechanisms involved in the formation of these circuits is a major issue in developmental neurobiology. The guidance of commissural projections lay on two major couples of ligand- receptors: Netrin-1/DCC and Slits/Robos. Netrin-1 and Slits are expressed in the floor plate whereas their respective receptors, DCC and Robo1/2, are present in the neurons and commissural axons of the spinal cord and the hindbrain during the development. Analysis of mice deficient for these genes lead to establish a model: a Netrin-1 gradient would act through long distances in order to attract commissural axons to the ventral midline, then Slits would repulse axons outside the floor plate. During my thesis, I used genetic mouse models to study the setting of commissural axons and the migration of precerebellar neurons in vivo. We have specifically inactivated the expression of Netrin-1 Slits and Robo1/2 genes in different region of the CNS. We showed that floor plate-derived Netrin-1 was not necessary for the attraction of commissural axons and that the principal source of Netrin-1 was the radial glia. We also showed that the Slit/Robo couple was not essential for the migration of precerebellar neurons. These results reassess the model firstly provided and suggest the presence of other molecular mechanisms involved in commissural axon guidance.

CubeSat development has seen a rise since the first launch in 2003 due to faster design process and low launch costs. It has played a vital role in providing access to space to small start-ups and academic organizations with low budgets. It has also enabled the testing of different upcoming technologies in space and has helped in providing hands-on experience to students taking part in design of such platforms. University of Pisa, in collaboration with SITAEL, has also taken an initiative to design and develop a CubeSat to test the FEEP thruster, design of which is presented in the thesis. A FEEP system was designed to fit within 1U dimensions and with a dry mass of approximately 820 grams. The system is based on slit emitter which provides an advantage over already available technologies in the market which uses needle emitters. Slit emitter scan achieve multiple Taylor cones without the need of clustering as used in needle emitters and also have a higher Thrust to Power Ratio. A propellant comparison was done considering all the properties required for an ideal propellant for a FEEP system. This comparison led to the selection of indium as working propellant which has an atomic mass of 114.8 u and a melting point of 156.6 °C. The FEEP system was designed keeping in mind easy assembling and modularity of thruster for ease in changing parts. The design consists of three different modules that are assembled separately and then joined together to complete the assembling of the system. The propellant tank, which also houses the emitter, has an internal volume of 32.75 cm3 and can hold approximately 240 grams of indium, which has a density of 7.31 g/cm3. During mission analysis, a 600km altitude orbit was proposed by analyzing the amount of propellant required for drag compensation and de-orbit maneuver at different altitudes with worst case values for ballistic coefficient and Thrust to Weight Ratio. At this altitude, the propellant requirement is 254.4 grams, 14.4 grams more than that of what can fit in the propellant tank of the designed thruster. However, both design of the system and mission analysis are ongoing processes and changes would be made in the future to either one or both to meet the requirements.

The Slit family (Slit1-3) of secreted glycoproteins and their cognate Roundabout family (Robo1-4) of transmembrane receptors provide important repulsive signals to guide cell migration during development and postnatal life. The dynamic regulation of Slit/Robo signaling is poorly understood in vertebrates. In this study, we identified a novel role for endocytosis in regulating Slit2 /Robo1 expression. Using heterologous expression systems, Slit2 was found be endocytosed in a Robo1-dependent manner and subsequently degraded in the lysosome, while Robo1 was found to be primarily recycled. An AP-2 consensus binding site, which mediates clathrin-dependent endocytosis, was identified in the Robo1 cytoplasmic tail and found to be required for Slit2 down-regulation and Slit2-induced endocytosis of Robo1. Preliminary data suggests that Slit2-induced endocytosis of Robo1 may be required for downstream signaling. These findings have important implications for how Slit/Robo signaling may be dynamically regulated during cell migration.