Aluminium sheet production is a multi-stage process in which altering processing conditions can drastically alter the size and type of second phase particles found in the final product. The properties of these second phase particles also affects deformation and annealing processes, meaning that any attempt to create a through process model would require the ability to predict both how the particles would develop in the material, and how these particles then affect the alloy moving forward. This project first focuses on gaining insight into how the particles in a model aluminium alloy change during homogenisation heat treatment and hot rolling. This has been accomplished by utilising serial block face scanning electron microscopy (SBF-SEM), a technique which allows the capture of 3D data sets at sub micron resolutions. This has allowed the populations of primary (constituent) and secondary (dispersoid) particles to be analysed at different stages of sheet production, and thus allowing the effects of homogenisation and hot rolling on particle populations to be quantified. To discover how the particles would go on to affect further processing, digital image correlation has been used to examine the localised strain in the alloy near to a selection of particle configurations. This highlighted the heterogeneity in slip behaviour within the alloy and illustrated that plumes of rotation develop near to non deformable regions. Rotation plumes have previously been modelled using a crystal plasticity model, and so further work is also presented expanding upon this model to simulate a variety of particle configurations. This has shown that in the case of single particles, local deformation is dependent on both the aspect ratio of the particle and how it is aligned to the active slip system. With the incorporation of a second particle, the interparticle spacing must also be considered.

Philosophiae Doctor - PhD / The survival of free-ranging (in situ) African elephant and Southern white rhinoceros populations are currently being challenged on a daily basis in Africa. Reproductive health is considered a vital component of species conservation. Conservation of the last mega land mammals may ultimately require intervention by breeding management or combined with assisted reproductive technologies (ART). There is a strong case for gathering baseline information, both physiological and biological, of any species, as opportunities arise. During this study a total number of 21 ejaculates collected over two seasons from 12 free-ranging African elephant bulls were characterised, as well as 10 ejaculates collected from 10 free-ranging Southern white rhinoceros bulls from two populations. Ejaculates were collected from adult bulls by means of electroejaculation under anaesthesia. Routine semen analysis was combined with Computer-aided sperm analysis (CASA), Computer-aided sperm morphology analysis (CASMA), Transmission electron microscopy (TEM) and Genomics as diagnostic tools. Additionally, sperm functionality within different media was investigated and sperm subpopulation classification according to the motion pattern displayed. The results presented is based on the evaluation and classification of ≈ 45 000 individual African elephant spermatozoa and ≈ 18 000 individual Southern white rhinoceros spermatozoa. The average elephant ejaculate contained a total number of 47 x 10⁹ spermatozoa (volume of 56 ± 38mL x concentration of 818 ± 750 x 10⁶/mL) that recorded a total motility of 81 ± 29% of which 62 ± 26% were progressively motile. CASA recorded velocities for curvilinear velocity (VCL 241 ± 58μm/s), straight-line velocity (VSL 173 ± 181μm/s) and average path velocity (VAP 201 ± 54μm/s), and kinematics at straightness of track (STR 86 ± 85%), linearity of track (LIN 67 ± 16%), amplitude of lateral head displacement (ALH 4 ± 0.75μm) and beat cross frequency (BCF 21 ± 3Hz). Structural analysis revealed 68 ± 11% of the spermatozoa were viable (intact plasma membrane) and 77 ± 11% maintained acrosome integrity. Ejaculates contained 55 ± 14% morphologically normal spermatozoa, CASMA measured sperm head lengths at 6.83 ± 0.26μm and width 3.32 ± 0.18μm (total head area of 20.17 ± 1.96μm²) of which 38.95 ± 0.92% is covered by an acrosomal cap. The average rhinoceros ejaculate contained a total number of 1.1 x 10⁹ spermatozoa (volume of 24 ± 24mL x concentration of 83 ± 96 x 10⁶/mL) that recorded a total motility at 82 ± 8% of which 28 ± 23% were progressively motile. CASA recorded velocities for VCL (85 ± 29μm/s), VSL (44 ± 25μm/s) and VAP (69 ± 30μm/s, and kinematics at STR (63 ± 14%), LIN (51 ± 16%), ALH (2 ± 0.16μm) and BCF (16 ± 6Hz). Structural analysis revealed 73 ± 10% of the spermatozoa were viable (intact plasma membrane) and 76 ± 4% maintained acrosome integrity. Ejaculates contained 62 ± 14% morphologically normal spermatozoa, CASMA measured sperm head lengths at 5.5 ± 0.17μm and width 2.9 ± 0.19μm (total head area of 14.8 ± 1.43μm²) of which 36.3 ± 0.59% is covered by an acrosomal cap. Based on a Boolean argument and CASA data exploration it was possible to derive elephant and rhinoceros CASA cut-off criteria to sort between activated and hyperactivated motile spermatozoa. For the genomic component of this study, the CatSper1 (Loxodonta africana) gene was identified,sequenced and verified in a free-ranging (natural) African elephant population. Multivariate analysis(MVA) was applied to examine the associations between the semen and sperm parameters and the traits they accounted for in this study. Our understanding of wildlife reproductive sciences can substantially progress as the analytical techniques applied and the combination thereof is expanded. This investigation presents a new set of comprehensive semen and sperm threshold values for future investigations.

La cryo-microscopie électronique a connu une révolution majeure ces dernières années, liée à des évolutions technologiques importantes, tant au niveau des microscopes, que des caméras ou des logiciels de traitement d’images. Ainsi, avec l’arrivée de microscopes électroniques plus stables mécaniquement et électroniquement, il est possible d’enregistrer plusieurs milliers d’images de façon automatique en quelques jours, et par conséquent d’obtenir un jeu initial d’images conséquent des particules des complexes protéiques étudiés. Sauf que maintenant, il ne s’agit plus d’image au sens strict, mais de film. En effet, les nouvelles caméras, dites à détection directe d’électrons, permettent de décomposer l’image en plusieurs fractions d’images au cours de l’exposition, et ce, avec une sensibilité dix fois supérieure par rapport à celle des anciennes caméras. L’analyse de ces fractions d’images a permis de montrer que les particules protéiques, bien que piégées dans une mince pellicule de glace, bougeaient sous l’effet du faisceau d’électrons. L’alignement des fractions et leur sommation permettent ainsi de corriger ces mouvements, améliorant la qualité du signal contenu dans chaque image. Ainsi, alors que pendant des dizaines d’années, les informations structurales extraites des images des microscopes électroniques étaient limitées à la moyenne résolution, c’est à dire entre 5 et 15 Å de résolution, nous voyons apparaître depuis ces deux-trois dernières années, de très nombreuses cartes de densités électroniques de complexes protéiques issues de la microscopie électronique, à des résolutions inférieures à 4 Å, permettant de construire des modèles atomiques à l’aide d’outils jusqu’alors réservés à la cristallographie aux rayons X. C’est dans ce contexte, que j’ai étudié par cryo-microscopie électronique et traitement d’images, l’organisation structurale de trois virus de plante :- Le virus de la mosaïque de l’arabette (ArMV), un Nepovirus uniquement transmis par le nématode Xiphinema diversicaudatum, qui est responsable de la maladie du court-noué de la vigne. -Le virus de la tâche de la fève (BBSV), un Comovirus transmis par les coléoptères, responsable de la dégénération chez les légumineuses.- Le virus de la mosaïque du chou-fleur (CaMV), un Caulimovirus servant de virus modèle pour l’étude de la transmission des virus non circulants.Les virus sont des parasites endocellulaires obligatoires, dont l’efficacité dépend de leur capacité de réplication au sein de la cellule infectée et de leur transmission vers de nouveaux hôtes. En raison de l’immobilité des plantes, les phytovirus font souvent appel à des vecteurs pour la transmission plante à plante, qui sont principalement des insectes, des nématodes, des champignons ou des acariens. Les phytovirus sont généralement responsables d’une baisse importante de croissance de la plante et des fruits, voire de la mort de l’hôte infecté. Les dégâts ainsi causés engendrent des pertes de rendement dans les cultures partout dans le monde, se traduisant par d’énormes pertes économiques pour les cultivateurs. Ce travail de thèse présente les structures atomiques de l’ArMV et du BBSV obtenues par cryo-microscopie électronique, ainsi que les premiers résultats obtenus sur la structure de la capside du CaMV, et sa protéine de transmission P2. / A revolution has taken over the world of cryo-electron microscopy for the last years, by dint of a major breakthrough both in technology, with the rise of new microscopes and cameras, and in image processing. With the advent of high-end microscopes, mechanically and electronically more stable, one can expect to record an initial data set of thousand images in few days, thanks to automated acquisition. Besides, the new direct electron detectors can not only record images, but also movies with a better sensitivity than the one we used to have. The movie processing revealed the existence of a beam-induced motion occurring during acquisition. The correction of the motion through frame alignment improves significantly the quality of data. Thus, cryo-electron microscopy was only limited to a middle resolution range (5 to 15 Å) until two or three years ago, when several density maps above 4 Å started to appear, allowing the building of atomic model using tools that were only restricted to X-ray crystallography.In this context, I have studied the structural organization of three plant viruses, using cryo-electron microscopy and image processing:- Arabis Mosaic Virus (ArMV), it’s a Nepovirus only transmitted by the nematode Xiphinema diversicaudatum, responsible for disease of vineyards.- Broad Bean Stain Virus (BBSV), it’s a Comovirus transmitted by beetles, responsible for the degeneration of leguminous plants.- Cauliflower Mosaic Virus (CaMV), it’s a Caulimovirus used as model to characterize the transmission of non circulative viruses.Viruses are obligate intracellular parasites, which efficiency is directly related to its replicative capacity inside the infected cell, and its transmission to new hosts. Due to the immobility of plants, plant viruses often use vectors for the transmission plant to plant, which are mainly insects, nematodes, fungi or mites. Plant viruses are generally responsible for a significant decrease in plant and fruit growth, and even the death of the plant. The plant viruses are devasting fields worldwide, causing huge loss in crop yield each year. This study highlights the atomic structures of ArMV and BBSV, as well as the first data about the CaMV capsid and its transmission protein.

Depuis les années 80, Les matériaux composites stratifié verre résine (SVR) ont été utilisés pour la construction des tuyaux et des réservoirs dans l'industrie chimique, y compris pour le stockage d’acides. Ce matériau composite présente une résistance supérieure à la corrosion. Cependant, des auteurs ont observé des ruptures accidentelles de réservoirs (horizontaux et verticaux) contenant des acides (chlorhydrique et sulfurique). Ces ruptures sont attribuées au mécanisme de corrosion sous contrainte (CSC). La corrosion des fibres de verre dans une solution acide est moins connue mais reste très importante. Ce mécanisme de corrosion, appelée désalcalinisation de la fibre peut provoquer la fissuration de la fibre de verre.Des essais de corrosion avec de l’acide chlorhydrique (37%) ont été effectués sur éprouvette SVR. Ces essais de corrosion ont été suivis par émission acoustique. Les observations au microscope électroniques à balayage (MEB) et les analyses physico-chimiques confirment la corrosion de fibres de verre dans une solution de HCl. L’utilisation de la micro-tomographie nous montre que cette technique permet d’avoir une information sur la profondeur d’attaque du matériau.Une approche statistique est utilisée pour caractériser les paramètres de la salve d’émission acoustique afin de les séparer. Le Clustering est fait en utilisant la méthode des k-moyennes. Trois classes d’émission acoustique distinctes ont ainsi été identifiées. L’analyse croisée de l’émission acoustique et des observations ont permis de relier les classes observées aux conséquences de la corrosion du SVR. / Since the 1980, Glass Reinforced Plastic (GRP) has been used for construction of pipes and tanks in the chemical industry, including the storage of mineral acids. This composite material offers superior and cost effective corrosion resistance. However, authors found accidental breakage of tanks (horizontal and vertical) containing mineral acids (hydrochloric and sulphuric). These failures are attributed to environmental stress-corrosion cracking (ESCC) mechanism. The corrosion of glass fibers in mineral acid solution is less known but very important. The mechanism of the corrosion, called leaching, is thought to induce tensile stresses in the surface of the glass. These stresses could be large enough to cause cracking of the fiber glass.Corrosion tests have been performed on GRP specimen. Aggressive environments used are hydrochloric acid (37%) This environment is known to react with E-glass. Corrosion tests have been monitored by acoustic emission.SEM observations and physicochemical analysis confirm the corrosion of glass fibers in HCl solution. The use of micro - tomography allows to have information on the depth of degradation of the material.Statistical approaches are used to characterize hit’s parameters. Clustering is made by using k-mean’s method. Three distinct acoustic emission classes are identified. Thanks to SEM observations and acoustic emission results, clusters can be assigned to the appearance of minor defects in the material.

No description available.

The objective of this research was to develop a novel technique for mechanical treatment to manipulate the microstructure of Nickel-Manganese-Gallium Hesuler alloys to increase anisotropy, which can lead to higher magnetocaloric properties. Ni2+xMn1-xGa intermetallics have the potential to be employed in magnetic refrigeration devices including residential refrigerators, heat pumps, and air conditioning. Solid-state magnetic refrigeration systems are smaller, quieter, and reduce energy consumption by 20% compared to existing conventional vapor-cycle refrigeration devices which rely on harmful hydro-fluorocarbon gases and pump millions of tons of greenhouse gases into the atmosphere. The magnetic refrigeration market is predicted to reach US$ 315.7 Million by 2022. Magnetic refrigeration systems can also be used in electronic systems and the space industry. The current state-of-the-art magnetic refrigeration systems use expensive rare earth elements including Gadolinuim (Gd). The need to replace Gd and other rare earth elements with cheaper and more available elements led to other alloys including Ni-Mn-Ga. By understanding the processing-microstructure-property relationship of Ni-Mn-Ga alloy, it is possible to manipulate the microstructure in order to obtain higher refrigeration capacity. It is a promising alternative to rare earth elements and improves national security by minimizing foreign dependence on the import of rare earth metals. This novel in situ study establishes that twin boundaries can be manipulated in a polycrystalline Ni-Mn-Ga alloy. This results in a change in magnetocrsytalline anisotropy, which leads to a higher magnetic cooling power. Mechanical loading in a preferred direction, traditionally referred to as a training process, was able to move the twin boundaries, and the combination of focused ion beam imaging linked specific movement with mechanical loading. This technique, in situ monitoring process, can be utilized to devise training procedures for future iterations of magnetocaloric and shape memory alloys.

No description available.

No description available.

Les synapses chimiques sont des structures spécialisées permettant une transmission d'information unidirectionnelle, d’un élément présynaptique vers un élément postsynaptique. L’organisation des terminaisons présynaptiques permet la conversion d’un potentiel d’action en signal chimique. Elles se présentent sous la forme de varicosités axonales contenant des vésicules synaptiques (VSs) qui concentrent le neurotransmetteur (NT). Une partie des VSs sont apposées (ancrées) à une région de la membrane plasmique, la zone active (ZA ; Bennett et al. 1992, Siksou et al. 2009). La ZA est située face à l’accumulation postsynaptique des récepteurs au NT. La dépolarisation d’une terminaison par un potentiel d’action active des canaux calciques dépendants du voltage. L’influx de calcium qui en résulte entraîne la fusion d’une fraction des VSs ancrées avec la membrane plasmique en moins d’une milliseconde, permettant la libération de NT (Sabatini et Regehr 1996, Lisman et al. 2007). Une endocytose compensatoire permet ensuite la reformation de VSs (Rizzoli et Betz, 2005). Des questions se posent encore quant à ce trafic régulé. Nous avons étudié la régulation de l’ancrage des VSs et développé un outil pour analyser le cycle des VSs en microscopie électronique (ME).La première partie de mon travail de thèse a porté sur la régulation du nombre de VSs ancrées à la ZA. Notre objectif était de savoir si l’ancrage était régulé spécifiquement ou en coordination avec les autres paramètres morphologiques du bouton, et de déterminer le rôle de Rab3-Interacting Molecules (RIMs), protéines centrales de la ZA, dans cette régulation. La régulation de l’ancrage a été étudiée sur un modèle de cultures organotypiques de tranches d’hippocampe dont l’activité est bloquée 3 jours par l’application de tétrodotoxine. Ces tranches ont été immobilisées par congélation sous haute pression (CHP) pour être observées en ME sans les artefacts induits par les fixations aldéhydiques. Le blocage d’activité entraîne une augmentation du nombre de VSs ancrées, de la taille de la ZA, et du nombre de récepteurs postsynaptiques au glutamate de type GluA2. Le nombre de VSs total dans le bouton et la taille du bouton ne changent pas. En immunocytochimie Nous n’avons pas observé de modification de la quantité moyenne de protéines RIM1/2 dans les terminaisons présynaptiques sous l’effet du blocage d’activité. Enfin, les enregistrements électrophysiologiques ne révèlent pas de modification de fréquence des courants excitateurs miniatures malgré l’augmentation du nombre de VSs ancrées. Ces résultats montrent une régulation spécifique de la taille de la jonction synaptique par l’activité neuronale et indiquent que le nombre de VSs ancrées n’est par régulé par la quantité de RIM.La deuxième partie de mon travail a consisté à développer un outil d’étude du cycle des VSs. En effet, la ME ne permet pas d’observer des évènements dynamiques, tandis que la résolution spatiale des microscopes optiques est insuffisante pour observer directement les VSs. Nous avons voulu associer une stimulation optogénétique de neurones avec leur immobilisation rapide par CHP, afin de pouvoir observer en ME les VSs à des temps précis après la stimulation. Nous avons travaillé sur des cultures dissociées de neurones d’hippocampe de rat. Ces neurones ont été infectés avec un Adeno-Associated Virus exprimant une protéine, la ChannelRhodopsine2, pour les rendre activables par des stimulations lumineuses. Une collaboration avec Leica Microsystems a permis de modifier l’appareil de congélation (HPM) pour (i) stimuler les neurones dans l’HPM, et (ii) synchroniser cette stimulation avec la congélation. Ce nouvel outil devrait permettre dans le futur une analyse du trafic des VSs à très haute résolution temporelle et spatiale. / Chemical synapses are highly specialized structures that convey information unidirectionnally, from a presynaptic to a postsynaptic element. Presynaptic terminals convert action potentials into chemical signals. These axonal varicosities contain synaptic vesicles (SVs) filled with neurotransmitter (NT) molecules. A fraction of the SVs are apposed (docked) to a part of the plasma membrane called the active zone (AZ; Bennett et al. 1992, Siksou et al. 2009b). The AZ is located in front of the postsynaptic accumulation of NT receptors. Depolarization of a terminal by an AP activates voltage dependent calcium channels. The resulting calcium influx induces the fusion of a fraction of the docked SVs in less than a millisecond, which release their NT content (Sabatini & Regehr 1996, Lisman et al. 2007). New SVs are then produced through a process of compensatory endocytosis (Rizzoli & Betz, 2005). Unanswered questions remain about the mechanism of this regulated traffic of SVs. We studied the regulation of SVs docking and developed a tool to analyze the cycle of SVs with electron microscopy (EM). The first part of my PhD work was focused on the regulation of the number of docked SVs at the AZ. Our objective was to determine whether SVs docking was regulated specifically or together with other morphological parameters of the bouton. We also investigated the role of Rab3-Interacting Molecules (RIMs), central proteins of the AZ, in this regulation. We worked on organotypic culture of hippocampal slices in which we blocked neuronal activity with tetrodotoxin for 3 days. Slices were immobilized using high pressure freezing (HPF) to avoid artifacts due to chemical fixation, and studied with EM. Activity blockade induced an increase in the number of docked SVs, in the size of the AZ and in the number of GluA2 postsynaptic glutamate receptors. However, the total number of SVs in the bouton and the size of the bouton did not change. With immunocytochemistry we did not detect any change in the mean amount of RIM in presynaptic terminals after chronic activity blockade. Furthermore, electrophysiology recordings showed no increase of the mean frequency of mEPSCs despite the increase in the number of docked SVs. Together these results show a specific regulation of the size of the presynaptic junction by neuronal activity, and indicate that the amount of RIMs does not regulate the number of docked SVs. The second part of my work consisted in the development of a new tool to study the cycle of SVs. Indeed, EM does not allow the visualization of dynamic phenomenon, whereas optical microscopes do not have a sufficient spatial resolution to observe SVs with the required precision. We wanted to associate optogenetic stimulations of neurons with their rapid immobilization by HPF, in order to visualize SVs at precise moments after stimulation. We worked on rats hippocampal dissociated neurons cultures. These neurons were infected with an Adeno-Associated Virus encoding a light sensitive protein channel, the ChannelRhodopsin2, in order to be able to activate them with light stimulations. We collaborated with Leica Microsystems to modify our high pressure machine (HPM), so that we can (i) stimulate the neurons within the HPM, and (ii) synchronize this stimulation with the freezing. In the future, this new tool this system should allow us to analyze the traffic SVs with high temporal and spatial resolution.

Electrical trees are micrometre-size tubular channels of degradation in high voltage polymeric insulation, a precursor to failure of electrical power plant. Hence, electrical trees critically affect the reliability of power systems and the performance of new insulation designs. Imaging laboratory-grown electrical trees has been an important tool for studying how trees develop. Commonly, electrical trees prepared in transparent or translucent polymers are imaged using traditional optical methods. Consequently, most of the analysis has been based on two-dimensional (2D) images of trees, thus, valuable information may be lost. However, electrical trees are complex interconnected structures that require a tree-dimensional (3D) approach for more complete analysis. This thesis investigates a method for imaging and analysis of electrical trees to characterise their 3D structure and provide a platform for further modelling. Laboratory created electrical trees were imaged using X-ray Computed Tomography (XCT) and Serial Block-Face Scanning Electron Microscopy (SBFSEM), 3D imaging techniques that provide sub-micrometre spatial resolution. Virtual replicas of the trees, which are the 3D geometrical models representing the real electrical trees, were generated and new indices to characterise the 3D structure of electrical trees were developed. These parameters were indicative of differences in tree growth and thus, they can be used to investigate patterns and classify the structure of electrical trees. The progression of the tree was analysed using cross-sections of the tree that are orthogonal to the growth: the number of tree channels and area covered by them were measured. The fractal dimension of the tree was calculated from the 3D model and from the 2D projections, the latter being lower for all the tree-type structures studied. Parameters from the skeleton of the tree such as number of nodes, segment length, tortuosity and branch angle were measured. Most of the mean segment lengths ranged 6-13 µm, which is in accordance to the 10µm proposed by various tree-growth models. The capabilities of XCT and SBFSEM imaging techniques were evaluated in their application to electrical trees. Bush and branch trees, including early-growth electrical trees (of length 20-40 µm), were analysed and compared using the comprehensive tool of visualisation and characterisation developed. A two-stage tree-growth experiment was conducted to analyse the progression and development of tree branches using XCT: tree channels after the second stage of growth were wider than after the first, while the fractal dimension remained the same. The capabilities of XCT and SBFSEM were tested for imaging electrical trees in optically-opaque materials such as micro and nano-filled epoxy compounds. The general structure of trees in epoxy filled up to 20 wt% micro-silica was observed using both techniques. The use of a virtual replica as the 3D geometrical model for the simulation of the electric field distribution using Finite Element Analysis (FEA) was preliminary explored. A combination of the imaging techniques is proposed for a more complete structural analysis of trees. It is believed that a great impact towards understanding electrical treeing will be achieved using the 3D technical platform developed in this thesis.