Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 4 uppsatser.

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La gradation de lames de biopsie fournit des informations pronostiques essentielles pour le diagnostic et le traitement. La détection et le comptage manuel des mitoses est un travail fastidieux, sujet à des variations inter-et intra- observateur considérables. L'objectif principal de cette thèse de doctorat est le développement d'un système capable de fournir une détection des mitoses sur des images provenant de différents types de scanners rapides automatiques, ainsi que d'un microscope multispectral. L'évaluation des différents systèmes proposés est effectuée dans le cadre du projet MICO (MIcroscopie COgnitive, projet ANR TecSan piloté par notre équipe). Dans ce contexte, les systèmes proposés ont été testés sur les données du benchmark MITOS. En ce qui concerne les images couleur, notre système s'est ainsi classé en deuxième position de ce concours international, selon la valeur du critère F-mesure. Par ailleurs, notre système de détection de mitoses sur images multispectrales surpasse largement les meilleurs résultats obtenus durant le concours. / Digital pathology represents one of the major and challenging evolutions in modernmedicine. Pathological exams constitute not only the gold standard in most of medicalprotocols, but also play a critical and legal role in the diagnosis process. Diagnosing adisease after manually analyzing numerous biopsy slides represents a labor-intensive workfor pathologists. Thanks to the recent advances in digital histopathology, the recognitionof histological tissue patterns in a high-content Whole Slide Image (WSI) has the potentialto provide valuable assistance to the pathologist in his daily practice. Histopathologicalclassification and grading of biopsy samples provide valuable prognostic information thatcould be used for diagnosis and treatment support. Nottingham grading system is thestandard for breast cancer grading. It combines three criteria, namely tubule formation(also referenced as glandular architecture), nuclear atypia and mitosis count. Manualdetection and counting of mitosis is tedious and subject to considerable inter- and intrareadervariations. The main goal of this dissertation is the development of a framework ableto provide detection of mitosis on different types of scanners and multispectral microscope.The main contributions of this work are eight fold. First, we present a comprehensivereview on state-of-the-art methodologies in nuclei detection, segmentation and classificationrestricted to two widely available types of image modalities: H&E (HematoxylinEosin) and IHC (Immunohistochemical). Second, we analyse the statistical and morphologicalinformation concerning mitotic cells on different color channels of various colormodels that improve the mitosis detection in color datasets (Aperio and Hamamatsu scanners).Third, we study oversampling methods to increase the number of instances of theminority class (mitosis) by interpolating between several minority class examples that lietogether, which make classification more robust. Fourth, we propose three different methodsfor spectral bands selection including relative spectral absorption of different tissuecomponents, spectral absorption of H&E stains and mRMR (minimum Redundancy MaximumRelevance) technique. Fifth, we compute multispectral spatial features containingpixel, texture and morphological information on selected spectral bands, which leveragediscriminant information for mitosis classification on multispectral dataset. Sixth, we performa comprehensive study on region and patch based features for mitosis classification.Seven, we perform an extensive investigation of classifiers and inference of the best one formitosis classification. Eight, we propose an efficient and generic strategy to explore largeimages like WSI by combining computational geometry tools with a local signal measureof relevance in a dynamic sampling framework.The evaluation of these frameworks is done in MICO (COgnitive MIcroscopy, ANRTecSan project) platform prototyping initiative. We thus tested our proposed frameworks on MITOS international contest dataset initiated by this project. For the color framework,we manage to rank second during the contest. Furthermore, our multispectral frameworkoutperforms significantly the top methods presented during the contest. Finally, ourframeworks allow us reaching the same level of accuracy in mitosis detection on brightlightas multispectral datasets, a promising result on the way to clinical evaluation and routine.

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Most existing chemotherapeutics lack adequate specificity for transformed cells and therefore have high rates of collateral damage to normal tissue. Moreover, such therapies often depend on p53 to induce cell death and are ineffective on the large number of human cancers that have lost p53 function. The discovery of novel p53-independent cancer therapies is therefore of significant interest. The Chicken Anemia Virus protein Apoptin selectively induces apoptosis in transformed cells in a p53-independent manner while leaving normal primary cells unaffected. This selectivity is thought to be largely due to cell type-specific localization: in primary cells Apoptin is cytoplasmic, whereas in transformed cells the protein localizes to the nucleus. The basis for this cell type-specific localization remains to be determined. In this study, Apoptin is revealed to be a nucleo-cytoplasmic shuttling protein whose localization is mediated by an N-terminal nuclear export signal (NES) and a C-terminal nuclear localization signal (NLS). Both signals are required for cell type-specific localization, as Apoptin fragments containing either the NES or NLS fail to localize differently between transformed and primary cells. Significantly, cell type-specific localization can be rescued in trans by co-expression of the two separate fragments, which are able to interact through an Apoptin multimerization domain. Interestingly, this multimerization domain overlaps with the NES suggesting that these two activities may be functionally coupled in cytoplasmic retention in primary cell types. Factors present in transformed cells induce localization of Apoptin to the nucleus where a biochemically distinct, more soluble form of the protein exists. Using affinity-purification and mass spectroscopy it was found that, specifically in transformed cells, Apoptin is associated with APC1, a subunit of the anaphase-promoting complex/cyclosome (APC/C). The APC/C is required to establish a mitotic cell-cycle checkpoint, and its inhibition results in G2/M arrest and apoptosis. Expression of wild type Apoptin in transformed cells inhibits APC/C function and induces G2/M arrest and apoptosis, whereas Apoptin mutants that are unable to associate with APC1 have no effect. In p53 null cells, ablation of APC1 by RNA interference induces a G2/M arrest and apoptosis analogous to that observed following Apoptin expression. Furthermore, Apoptin was found to induce the formation of PML bodies and to recruit APC/C subunits to these nuclear structures suggesting a mechanism involving sequestration and subsequent inhibition of the APC/C. Thus, the results of this study clarify Apoptin cell type-specific localization behavior and explain the ability of Apoptin to induce apoptosis in transformed cells in the absence of p53. This study advances a newly emerging field of viral mechanisms of apoptosis involving G2/M arrest and APC/C modulation. The resultant p53-independent apoptosis suggests that the APC/C may be an attractive target for the development of anti-cancer drugs.

Avancement dans l'acquisition d'image et le progrès dans les méthodes de traitement d'image ont apporté les mathématiciens et les informaticiens dans les domaines qui sont d'une importance énorme pour les médecins et les biologistes. Le diagnostic précoce de maladies (comme la cécité, le cancer et les problèmes digestifs) ont été des domaines d'intérêt en médecine. Développement des équipements comme microscope bi-photonique à balayage laser et microscope de fluorescence par réflexion totale interne fournit déjà une bonne idée des caractéristiques très intéressantes sur l'objet observé. Cependant, certaines images ne sont pas appropriés pour extraire suffisamment d'informations sur de cette image. Les méthodes de traitement d'image ont été fournit un bon soutien à extraire des informations utiles sur les objets d'intérêt dans ces images biologiques. Rapide méthodes de calcul permettent l'analyse complète, dans un temps très court, d'une série d'images, offrant une assez bonne idée sur les caractéristiques souhaitées. La thèse porte sur l'application de ces méthodes dans trois séries d'images destinées à trois différents types de diagnostic ou d'inférence. Tout d'abord, Images de RP-muté rétine ont été traités pour la détection des cônes, où il n'y avait pas de bâtonnets présents. Le logiciel a été capable de détecter et de compter le nombre de cônes dans chaque image. Deuxièmement, un processus de gastrulation chez la drosophile a été étudié pour observer toute la mitose et les résultats étaient cohérents avec les recherches récentes. Enfin, une autre série d'images ont été traités où la source était une vidéo à partir d'un microscopie photonique à balayage laser. Dans cette vidéo, des objets d'intérêt sont des cellules biologiques. L'idée était de suivre les cellules si elles subissent une mitose. La position de la cellule, la dispersion spatiale et parfois le contour de la membrane cellulaire sont globalement les facteurs limitant la précision dans cette vidéo. Des méthodes appropriées d'amélioration de l'image et de segmentation ont été choisies pour développer une méthode de calcul pour observer cette mitose. L'intervention humaine peut être requise pour éliminer toute inférence fausse. / Advancement in Image Acquisition Equipment and progress in Image Processing Methods have brought the mathematicians and computer scientists into areas which are of huge importance for physicians and biologists. Early diagnosis of diseases like blindness, cancer and digestive problems have been areas of interest in medicine. Development of Laser Photon Microscopy and other advanced equipment already provides a good idea of very interesting characteristics of the object being viewed. Still certain images are not suitable to extract sufficient information out of that image. Image Processing methods have been providing good support to provide useful information about the objects of interest in these biological images. Fast computational methods allow complete analysis, in a very short time, of a series of images, providing a reasonably good idea about the desired characteristics. The thesis covers application of these methods in 3 series of images intended for 3 different types of diagnosis or inference. Firstly, Images of RP-mutated retina were treated for detection of rods, where there were no cones present. The software was able to detect and count the number of cones in each frame. Secondly, a gastrulation process in drosophila was studied to observe any mitosis and results were consistent with recent research. Finally, another series of images were treated where biological cells were observed to undergo mitosis. The source was a video from a photon laser microscope. In this video, objects of interest were biological cells. The idea was to track the cells if they undergo mitosis. Cell position, spacing and sometimes contour of the cell membrane are broadly the factors limiting the accuracy in this video. Appropriate method of image enhancement and segmentation were chosen to develop a computational method to observe this mitosis. Cases where human intervention may be required have been proposed to eliminate any false inference.

Transcription has turned out to be a discontinuous process when imaged at a single cell level. This observation is referred to as transcriptional bursting or pulsing and has been detected in a variety of organisms ranging from bacteria to mammalian cells. The dynamics of transcriptional pulsing are influenced by the properties intrinsic to the transcriptional process, as well as by upstream factors: chromatin environment, signalling molecules, cell cycle stage etc. In the first part of this thesis, we focused on the regulation of transcriptional pulsing in the nucleolus. Using imaging of living cells, we detected pulsatile transcription of a transgene with nucleolar localization whose expression was mediated by RNA polymerase II. In the second part of the thesis, we investigated the relationship between chromatin decondensation and transcriptional dynamics. We used hyperosmotic medium to induce global condensation of chromatin and revealed that upon chromatin decondensation, a transient spike in transcriptional intensity occurs in induvial living cells. Next, we analysed expression of TFRC and POLR2A genes in several cell cycle stages using single molecule RNA FISH. We detected increase in both frequency and size of transcriptional pulses during a limited time window which coincided with chromatin...

L'exigence principale du cycle cellulaire est une coordination étroite entre l'achèvement du processus de réplication et l'entrée des cellules en mitose, afin de maintenir l'intégrité génétique, l'identité et à la survie cellulaire. Toutes les cellules somatiques exécutent de manière reproductible une phase G2 intermédiaire, d'une durée constante pendant les divisions cellulaires successives au sein d'un type cellulaire donné. Cependant, les mécanismes moléculaires qui contrôlent précisément sa durée au cours d'un cycle cellulaire non perturbé, restent mal caractérisés. La kinase Cycline B1-Cdk1, facteur universel permettant l'entrée en mitose, est sous le contrôle, chez les mammifères, d'un ensemble de régulateurs directs, les kinases inhibitrices Wee1 et Myt1 et les phosphatases activatrices Cdc25A, B et C. Son activation soudaine, en fin de phase G2, révèle qu'une modification rapide de l'équilibre entre ses régulateurs opposés prend place par des mécanismes moléculaires qui restent à élucider. Dans ce cadre, j'ai étudié le rôle potentiel de la kinase Polo-like kinase 1 (Plk1) pour l'initiation de l'activation de Cycline B1-Cdk1. Bien que les rôles de Plk1 au cours de la mitose soient bien caractérisés, sa contribution dans la régulation de l'entrée des cellules en mitose reste controversée. Au niveau moléculaire, Plk1 phosphoryle au moins in vitro, plusieurs régulateurs de Cycline B1-Cdk1, tels que Cdc25B & C, et Wee1 et Myt1. Cependant, il reste largement inconnu si ces événements de phosphorylation se produisent in vivo et s'ils contribuent de manière significative au processus de l'activation de Cycline B1-Cdk1 permettant l'entrée en mitose. / A main requirement of the cell cycle is a tight coordination between the completion of the replication process and entry into mitosis in order to maintain genetic integrity and the identity and survival of cell progeny. All somatic cells reproducibly execute an intermediate G2 phase of constant duration during successive cell divisions in a given cell type. However the molecular mechanisms controlling precisely its duration during unperturbed cell cycle remains poorly characterized. In this context, the main objective of my PhD project was to decipher signaling pathways controlling entry into mitosis during normal cell cycles as well as their spatiotemporal regulation. CyclinB1-Cdk1, the universal master mitotic driver, is under the control of direct inhibitors (Wee1 and Myt1) and activators (Cdc25A, B and C). Previously, it was determined that CyclinB1-Cdk1 is suddenly activated in very late G2 phase, suggesting that a rapid modification in the equilibrium between its opposite regulators is reproducibly taking place in late G2 by poorly elucidated mechanisms. During my PhD, I investigated the potential role of Polo-like kinase 1 (Plk1) in the initial activation of CyclinB1-Cdk1. Even though its roles during mitosis are well characterized, its contribution for the regulation of entry into mitosis remains controversial. At the molecular level, Plk1 was shown to phosphorylate at least in vitro, several regulators of CyclinB1-Cdk1 including Cdc25B&C, and Wee1 and Myt1. However, it remains largely unknown if these phosphorylation events are taking place in vivo and whether they significantly contribute to the activation process of CyclinB1-Cdk1 leading to mitotic entry.

L’ubiquitination est une modification post-traductionnelle impliquée dans de nombreux mécanismes cellulaires. L’E3-ubiquitine ligase CULLIN 3 (CUL3) est un régulateur essentiel de la progression mitotique, ubiquitinant d’importants régulateurs mitotiques et contrôlant leur localisation subcellulaire. Plus particulièrement, notre travail décrit le rôle de la nouvelle E31 ligase CUL3/KLHL22 dans la régulation de l’activité localisée de Polo-like kinase 1 (PLK1) et de ce fait dans l’établissement d’une progression mitotique précise. Néanmoins, les mécanismes moléculaires qui régissent la reconnaissance de son substrat par CUL3 demeurent inconnus. L’activité catalytique de PLK1 ne semble pas être nécessaire à son interaction avec KLHL22, mais aussi bien son domaine kinase que Polo-box (PBD) suffisent à co-purifier KLHL22. Des mutations au niveau du motif DFG, situé en amont du domaine kinase,et du tryptophane 414 au sein du PBD semblent influer sur la reconnaissance de KLHL22. Les résultats obtenus montrent les premières indications biochimiques du mode d’interaction du complexe CUL3/KLHL22/PLK1. / Ubiquitination is a post-translational modification involved in many cellular processes. The E3 ubiquitin-ligase based on CULLIN 3 protein (CUL3) is an essential regulator of mitotic division in human cells by ubiquitinating several important mitotic regulators and controlling their subcellular localization. In particular, our work described the role of novel CUL3/KLHL22 E3-ligase in regulation of localized activity of Polo-like kinase 1 (PLK1) and there by faithful mitotic progression. However, the molecular mechanisms of substrate recognition by CUL3 remain unknown. The catalytic activity of PLK1 may not be required for binding KLHL22 but both the kinase and the Polo-box domains are sufficient to co-purify KLHL22. Mutating the DFG motif within the kinase domain and the tryptophan 414 within the PBD influence the binding to KLHL22. These results provide first insights into molecular mechanisms of CUL3/KLHL22/PLK1complex.

L’objectif de ces travaux de thèse est l’étude des modifications post-traductionnelles de la protéine ATF7. Cette protéine est phosphorylée sur les résidus thréonine (T) 51, T53 et T112 lorsque les cellules subissent un stress (UVs, choc osmotique). Cela permet à la protéine d’être active transcriptionnellement. En absence de stress une fraction de la protéine ATF7 est sumoylée et cela induit une inhibition de son activité transcriptionnelle. Le premier projet consiste à développer des outils qui permettent l’étude de la forme sumoylée de la protéine ATF7. Ces travaux ont permis l’élaboration de scFv-bispécifiques tétramériques qui permettent la reconnaissance simultanée de la protéine ATF7 et de la protéine SUMO1. L’autre projet principal était l’étude du rôle de la phosphorylation de la T112 en absence de stress. Les travaux menés au laboratoire ont permis de montrer que cette thréonine est phosphorylée pendant la phase M du cycle cellulaire et que cet événement est conduit par une autre voie de phosphorylation que la voie du stress et met en jeu la CDK1. / The objective of this thesis work was is the study of post-translationnal modifications (PTM) of the protein ATF7. This protein is phosphorylated on several threonine (T) residues upon stress (UVs, osmotic chock). This allows the protein to be transcriptionally active. In the absence of stress, a fraction of the protein is sumoylated resulting in an inhibition of its transcriptional activity. The first project raise to the development of tools that will enable the study of the sumoylated form of ATF7 protein. This work raise to the development of tetrameric bispecific scFv possessing a simultaneously recognizing ability of the proteins ATF7 and SUMO1. The other main project was the study of ATF7 T112 phosphorylation in the absence of stress. The experiments drove in the lab have shown that thus threonine is phosphorylated during mitosis by a specific pathway, which includes the CDK1.