In mature chloroplasts, the DNA (cpDNA) is surrounded by a potentially genotoxic environment that would make the mitochondrial DNA milieu look like a “nadree” (picnic). And yet, the slower evolution of cpDNA compared to other cellular genomes suggests that this organelle must have efficient mechanisms for repairing DNA. Unfortunately, those mechanisms have been barely noted, much less studied. This dissertation describes a novel approach that was developed to study how chloroplasts of Arabidopsis repair the most severe form of DNA damage, a double-strand break (described in Chapter 2). The success with this approach also prompted the development of a new method for site-specific modification of tobacco cpDNA that is described in Chapter 3. To study the consequences and repair of a break in the circular plastid genome, we developed an inducible system based on a psbA-intron endonuclease from Chlamydomonas (I-CreII) that specifically cleaves the psbA gene of Arabidopsis. The protein was targeted to the chloroplast using the rbcS1 transit peptide, and activation of the nuclear gene was made dependent on an exogenous inducer (β-estradiol). In Chlamydomonas, I-CreII cleavage at psbA was repaired, in the absence of the intron, by homologous recombination between repeated sequences (20-60 bp) that are abundant in that genome. By comparison, Arabidopsis cpDNA is very repeat-poor. Nonetheless, phenotypically strong and weak transgenic lines were obtained, and shown to correlate with I-CreII expression levels. Southern blot hybridizations indicated a substantial loss of psbA, but not cpDNA as a whole, in the strongly-expressing line. PCR analysis identified deletions nested around the I-CreII cleavage site that were indicative of repair using microhomology (6-12 bp perfect repeats, or 10-16 bp with mismatches) or no homology. The results provide evidence of alternative repair pathways in the Arabidopsis chloroplast that resemble the nuclear microhomology-mediated and nonhomologous end-joining pathways, in terms of the homology requirement. Moreover, when taken together with the results from Chlamydomonas, plus other considerations, the data suggests that an evolutionary relationship may exist between the repeat structure of cpDNA and the organelle’s ability to repair broken chromosomes. Taking advantage of the inducible I-CreII system, I developed a method to delete defined regions of cpDNA in tobacco, which was named DREEM (for direct repeat and endonuclease mediated). Chloroplast transformation was used to introduce an I-CreII cleavage site adjacent to an aadA:gfp marker and flanked by a direct repeat of 84 bp. When chloroplast-targeted I-CreII was induced with β-estradiol during germination, complete loss of the aadA:gfp marker occurred by SSA-type repair involving the 84-bp direct repeat. I obtained additional evidence for DREEM effectiveness by deleting 3.5 kb of native cpDNA that included part of the large ycf1 gene. DREEM can be used for other modifications besides gene deletions, partly because it is seamless and leaves no trace of introduced DNA. Since expression of the endonuclease is controlled by steroid application (and concentration), and the deleted cpDNA is probably destroyed during the SSA process, this inducible gene-ablation technique could enable the study of essential chloroplast genes in vivo. / text

Estimating human motion is the topic of this thesis. We are interested in accurately estimating the motion of a human body using only video images capturing the subject in motion. Video images from up to two cameras are considered. The first main topic of the thesis is to investigate a new type of input data. This data consists of some sort of texture. This texture can be added to the human body segment under study or it can be the actual texture of the skin. In paper I we investigate if added texture together with the use of a two camera system can provide enough information to make it possible to estimate the knee joint center location. Evaluation is made using a marker based system that is run in parallel to the two camera video system. The results from this investigation show promise for the use of texture. The marker and texture based estimates differ in absolute values but the variations are similar indicating that texture is in fact usable for this purpose. In paper II and III we investigate further the usability in images of skin texture as input for motion estimation. Paper II approaches the problem of estimating human limb motion in the image plane. An image histogram based mutual information criterion is used to decide if an extracted image patch from frame k is a good match to some location in frame k+1. Eval- uation is again performed using a marker based system synchronized to the video stream. The results are very promising for the application of skin texture based motion estimation in 2D. In paper III, basically the same approach is taken as in paper II with the substantial difference that here estimation of three dimensional motion is addressed. Two video cameras are used and the image patch matching is performed both between cameras (inter-camera) in frame k and also in each cameras images (intra-camera) for frame k to k+1. The inter-camera matches yield triangulated three dimensional estimates on the approximate surface of the skin. The intra-camera matches provide a way to connect the three dimensional points between frame k and k+1 The resulting one step three dimensional trajectories are then used to estimate rigid body motion using least squares methods. The results show that there is still some work to be done before this texture based method can be an alternative to the marker based methods. In paper IV the second main topic of the thesis is discussed. Here we present an investigation in using model based techniques for the purpose of estimating human motion. A kinematic model of the thigh and shank segments are built with an anatomic model of the knee. Using this model, the popular particle filter and typical simulated data from the triangulation in paper III, an estimate of the motion variables in the thigh and shank segment can be achieved. This also includes one static model parameter used to describe the knee model. The results from this investigation show good promise for the use of triangulated skin texture as input to such a model based approach.

Dans le domaine du raffinage, les mesures morphologiques de particules sont devenues indispensables pour caractériser les supports de catalyseurs. À travers ces paramètres, on peut remonter aux spécificités physico-chimiques des matériaux étudiés. Une des techniques d'acquisition utilisées est la tomographie électronique (ou nanotomographie). Des volumes 3D sont reconstruits à partir de séries de projections sous différents angles obtenues par microscopie électronique en transmission (MET). Cette technique permet d'acquérir une réelle information tridimensionnelle à l'échelle du nanomètre. Les projections sont obtenues en utilisant les possibilités d'inclinaison du porte objet d'un MET. À cause des limitations mécaniques de ce porte objet (proximité avec les lentilles magnétiques et déplacement nanométrique), on ne peut acquérir qu'un nombre assez restreint de projections, celles-ci étant limitées à un intervalle angulaire fixe. D'autre part, l'inclinaison du porte objet est accompagnée d'un déplacement mécanique nanométrique non parfaitement contrôlé. Ces déplacements doivent être corrigés après l'acquisition par un alignement des projections suivant le même axe 3D de rotation. Cette étape est un pré-requis à la reconstruction tomographique. Nous suggérons d'utiliser une méthode de reconstruction tomographique par une approche de type "problème inverse". Cette méthode permet d'aligner des projections et de corriger les lacunes de l'acquisition de l'objet observé en introduisant de façon pertinente des informations a priori. Ces informations sont donc basées à la fois sur la physique de l'acquisition (nature physique des images MET, géométrie et limitation spécifique de l'acquisition des projections, etc...) et sur la nature des objets à reconstruire (nombre et répartition des phases, critères morphologiques de type de connexité, etc...). L'algorithme proposé permet de réaliser la reconstruction nanotomographique avec une grande précision et un temps de calculs réduit considérablement par rapport à la technique classique. Nous avons testé avec succès notre méthode pour les projections réelles de différents supports de catalyseur / In oil refining industry, morphological measurements of particles have become an essential part in the characterization of catalyst supports. Through these parameters, one can infer the specific physicochemical properties of the studied materials. One of the main acquisition techniques is electron tomography (or nanotomography). 3D volumes are reconstructed from sets of projections from different angles made by a transmission electron microscope (TEM). This technique provides a real three-dimensional information at the nanometric scale. Projections are obtained by tilting the specimen port in the microscope. The tilt mechanism has two drawbacks: a rather limited angular range and mechanical shifts, which are difficult to deal with, knowing that these shifts must be corrected after the acquisition by an alignment of projections. This alignment step is a prerequisite for the tomographic reconstruction. Our work deals with a wholly "inverse problem" approach for aligning projections and reducing artifacts due to missing projections by introducing in a relevant way certain a priori informations. These informations are jointly based on the physics of acquisition (physical nature of the TEM images, geometry and specific limitation on the acquisition of projections...) and on the nature of objects to be reconstructed (number and distribution of phases, morphological criteria such as connectivity ...). This approach is described in an algorithmic way. The implementation of this algorithm shows higher precision reconstruction and smaller computation time compared to earlier techniques. We successfully tested our method for real projections of different catalyst supports

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