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Investigations of DNA damage response (DDR) mechanisms in normal tissues have implications for both cancer prevention and treatments. The accumulating knowledge about protein function and molecular markers makes it possible to directly trace and interpret cellular DDR in a tissue context. Using immunohistochemical techniques and digital image analysis, we have examined several principal DDR events in epidermis from patients undergoing fractionated radiotherapy. Acquiring biopsies from different regions of the skin provides the possibility to determine in vivo dose response at clinically relevant dose levels throughout the treatment. A crucial event in cellular DDR is the repair of DNA double strand breaks (DSBs). These serious lesions can be directly visualised in cells by detecting foci forming markers such as γH2AX and 53BP1. Our results reveal that DSB-signalling foci can be detected and quantified in paraffin-embedded tissues. More importantly, epidermal DSB foci dose response reveals hypersensitivity, detected as elevated foci levels per dose unit, for doses below ~0.3Gy. The low-dose hypersensitive dose response is observed throughout the treatment course and also in between fractions: at 30 minutes, 3 hours and 24 hours following delivered fractions. The dose response at 24 hours further reveals that foci levels do not return to background levels between fractions. Furthermore, a low-dose hypersensitive dose response is also observed for these persistent foci. Investigations of end points further downstream in the DDR pathways confirmed that the low-dose hypersensitivity was preserved for: the checkpoint regulating p21 kinase inhibitor; mitosis suppression; apoptosis induction and basal keratinocyte reduction. Our results reveal preserved low-dose hypersensitivity both early and late in the DDR pathways. A possible link between the dose-response relationships is therefore suggested. The preserved low-dose hypersensitivity is a cause for re-evaluation of the risks associated with low-dose exposure and has implications for cancer treatments, diagnostics and radiation protection.

Symmetric cell division in gram-negative bacteria is essential for generating two equal-sized daughter cells, each containing cellular material crucial for growth and future replication. The Min system, comprised of proteins MinC, MinD and MinE, is particularly important for this process since its deletion leads to minicells incapable of further replication. This thesis focuses on the interactions involving MinE that are important for allowing cell division at the mid-cell and for directing the dynamic localization of MinD that is observed in vivo. Previous experiments have shown that the MinE protein contains an N-terminal region that is required to stimulate MinD-catalyzed ATP hydrolysis in the Min protein interaction cycle. However, MinD-binding residues in MinE identified by in vitro MinD ATPase assays were subsequently found to be buried in the hydrophobic dimeric interface in the MinE structure, raising the possibility that these residues are not directly involved in the interaction. To address this issue, the ability of N-terminal MinE peptides to stimulate MinD activity was studied to determine the role of these residues in MinD activation. Our results implied that MinE likely undergoes a change in conformation or oligomerization state before binding MinD. In addition we performed circular dichroism spectroscopy of MinE. The data suggest that direct interactions between MinE and the lipid membrane can lead to conformational changes in MinE. Using NMR spectroscopy in an attempt to observe this structure change, different membrane-mimetic environments were tested. However the results strongly suggest that structural studies on the membrane-bound state of MinE will pose significant challenges. Taken together, the results in this thesis open the door for further exploration of the interactions involving MinE in order to gain a better understanding of the dynamic localization patterns formed by these proteins in vivo.

The mitotic spindle is a bipolar microtubule-based structure that mediates proper cell division by segregating the genetic material and by positioning the cytokinesis cleavage plane. Spindle assembly is a complex process, involving the modulation of microtubule dynamics, microtubule focusing at spindle poles and the formation of stable microtubule attachments to chromosomes. The cellular events leading to spindle formation are highly regulated, and mitotic kinases have been implicated in many aspects of this process. However, little is known about their counteracting phosphatases. A screen for genes required for early embryonic cell divisions in C. elegans identified rsa-1 (for regulator of spindle assembly 1), a putative Protein Phosphatase 2A (PP2A) regulatory subunit whose silencing causes defects in spindle formation. Upon rsa-1(RNAi), spindle poles collapse onto each other and microtubule amounts are strongly reduced. My thesis work demonstrates that RSA-1 indeed functions as a PP2A regulatory subunit. RSA-1 associates with the PP2A enzyme and recruits it to centrosomes. The centrosome binding of PP2A furthermore requires the new protein RSA-2 as well as the core centrosomal protein SPD-5 and is based on a hierarchical protein-protein interaction pathway. When PP2A is lacking at centrosomes after rsa-1(RNAi), the centrosomal amounts of two critical mitotic effectors, the microtubule destabilizer KLP-7 and the kinetochore microtubule stabilizer TPXL-1, are altered. KLP-7 is increased, which may account for the reduction of microtubule outgrowth from centrosomes in rsa-1(RNAi) embryos. TPXL-1 is lost from centrosomes, which may explain why spindle poles collapse in the absence of RSA-1. TPXL-1 physically associates with RSA-1 and RSA-2, suggesting that it is a direct target of PP2A. In summary, this work defines the role of a novel PP2A complex in mitotic spindle assembly and suggests a model for how different microtubule re-organization steps might be coordinated during spindle formation.

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.

La division cellulaire est un événement fondamental, indispensable au développement embryonnaire animal et à l’homéostasie des organismes adultes. Il s’agit d’un processus complexe qui doit être précisément contrôlé dans le temps et l’espace pour permettre la formation de deux cellules filles, au contenu génétique identique à celui de la cellule mère. Ceci requiert une coordination entre la ségrégation des chromosomes, opérée par les microtubules, et le clivage de la cellule, engageant une réorganisation dynamique du cytosquelette d’Actine. La modification de la forme des cellules en cours de division est en effet due au remodelage du cortex cellulaire, incluant la membrane plasmique et le réseau de filaments d’Actine sous-jacent. Bien que cette série de modifications du cortex soit indispensable au déroulement correct de la division cellulaire, les mécanismes moléculaires du contrôle l’organisation corticale en mitose restent mal caractérisés. Le PI(4,5)P2 est un phosphoinositide constituant de la membrane plasmique, notamment nécessaire à la division cellulaire. Nos travaux chez la drosophile mettent en évidence que ce phospholipide présente une distribution dynamique, homogène sur l’ensemble du cortex à l’entrée en mitose, puis se concentrant à l’équateur des cellules après la séparation des deux lots de chromosomes. Nous montrons que le PI(4,5)P2 est nécessaire au contrôle de la stabilité corticale et du fuseau mitotique, au moins en partie par son rôle favorisant l’activation de la dMoésine. La dMoésine régule l’interaction entre les filaments d’Actine et la membrane plasmique, jouant un rôle clé dans l’organisation locale du cortex des cellules en mitose et ses propriétés mécaniques. Nous montrons que l’interaction PI(4,5)P2/dMoésine participe à la contraction cellulaire à l’entrée en mitose, puis à l’élongation cellulaire caractéristique des étapes plus tardives de la division. A la fin de la mitose, nous montrons que la phosphatase pP1-87B inhibe l’activation de la dMoésine, indispensable à la relaxation du cortex des cellules en interphase. Par un crible fonctionnel systématique, nous avons recherché l’ensemble des facteurs indispensables à la production et à l’enrichissement localisé du PI(4,5)P2 au cortex mitotique. Nous montrons le rôle majeur de deux voies de biosynthèse, qui collaborent pour produire localement le PI(4,5)P2 à la membrane plasmique au cours de la mitose. Leur absence prévient l’activation et le recrutement membranaire de la dMoésine, et conduit à une instabilité corticale associée à des défauts du fuseau mitotique. Une troisième voie, nécessitant l’activité de la protéine dOcrl, contribue à l’homéostasie de ce phosphoinositide, en dégradant le PI(4,5)P2 présent sur les membranes internes de la cellule. L’inactivation de dOcrl empêche la formation normale et l’ingression du sillon de clivage. Ensemble, ces résultats identifient donc des régulateurs importants de la membrane plasmique et de son interaction avec le cytosquelette, permettant de mieux comprendre les mécanismes de la réorganisation de la forme cellulaire au cours de la mitose. / Cell division must be accurately controlled in time and space to permit the formation of two daughter cells whose genetic content is identical to that of the mother cell. This process requires successive modifications of cell shape, induced by cortical remodelling. Molecular mechanisms controlling cortical reorganization during mitosis remain partially uncharacterized. Our work in Drosophila cells demonstrates that PI(4,5)P2, a phosophoinositide of the plasma membrane, is enriched at the equatorial region at the onset of anaphase. This PI(4,5)P2 is necessary for the cortical stability of mitotic cells, and requires dMoesin activation. The dMoesin, linking actin to the plasma membrane, plays a critical role in the cortical organization of mitotic cells and in the regulation of its mechanical properties. We show that the interaction PI(4,5)P2/dMoesin participates in cellular contraction at the beginning of mitosis, then in cell elongation characteristic of subsequent steps. At the end of mitosis, the Pp1-87B phosphatase inactivates the dMoesin. By a systematic functional screen, we characterize the key role of two pathways acting in synergy to locally produce PI(4,5)P2, Skittles- and Pten-dependent, and the role of a third pathway requiring dOcrl activity to control PI(4,5)P2 homeostasis. Altogether, these results allow us to better understand the mechanisms controlling cortical remodelling and modifications of cell shape that occur during mitosis. / Doctorat réalisé en cotutelle avec le laboratoire de François Payre au Centre de Biologie du Développement à Toulouse, France (Université de Toulouse III - Paul Sabatier)

Les dinoflagellés sont des eucaryotes unicellulaires qui composent une grande partie du phytoplancton et qui jouent un rôle important au niveau de la photosynthèse, de la production primaire et de la conservation des écosystèmes marins. Les dinoflagellés se distinguent des autres eucaryotes par leur biologie et leur organisation nucléaire unique. Lors de la mitose, leur membrane nucléaire demeure intacte et la ségrégation des chromosomes se fait à partir de fuseaux mitotiques formés dans le cytoplasme et qui traversent le noyau au travers de canaux spécialisés Aussi, leurs chromosomes sont condensés en permanence et le processus utilisé pour y arriver est encore très mal compris puisque les dinoflagellés ne possèdent aucunes histones détectables. Lingulodinium polyedrum est un dinoflagellé photosynthétique marin utilisé comme organisme modèle en ce qui concerne l’étude des rythmes circadiens (bioluminescence, migration verticale, mitose et photosynthèse). La découverte et l’étude des éléments régulateurs du cycle cellulaire peuvent nous amener à comprendre le mécanisme, l’influence et la portée du contrôle circadien sur le cycle cellulaire. De plus, l’étude du cycle cellulaire pourrait permettre de révéler des indices quant aux caractéristiques singulières des dinoflagellés qui sont pour le moment énigmatiques. Par le passé, une étude chez Lingulodinium polyedrum a permis d’identifier la cycline impliquée dans la mitose, LpCyc1, le premier régulateur du cycle cellulaire a être découvert chez les dinoflagellés. La présente étude s’attarde sur la caractérisation de la LpCyc1, soit son expression, sa localisation, sa phosphorylation. Ces trois éléments concordent de façon à synchroniser l’activité de la LpCyc1 (et ainsi la mitose) de façon circadienne. Cette étude présente aussi la création et le développement d’un outil majeur pour l’étude future de Lingulodinium polyedrum, le transcriptome des ARNm à partir d’un iv séquençage Illumina. C’est d’ailleurs avec cet outil que nous avons découvert la CDK responsable du contrôle de la phase M, LpCdk1. Cette CDK possède tous les domaines d’une CDK classique, un site de liaison des substrats, un site de liaison à l’ATP, une boucle activatrice, et une interface de liaison avec la cycline. Le transcriptome de Lingulodinium polyedrum a aussi permis de recenser toutes les protéines conservées normalement retrouvées dans le contrôle du cycle cellulaire, qui nous a permis de faire une ébauche préliminaire du cycle cellulaire de L. polyedrum. Cette analyse est une première chez Lingulodinium polyedrum et peut s’étendre pour l’étude d’une multitude d’autres processus métaboliques. / Dinoflagellates are unicellular eukaryotes that constitute a large part of the phytoplankton. They are major contributors to the global photosynthesis and primary production and they possess an important role in conservation of marine ecosystems. Dinoflagellates are distincted from other eukaryotes by their unique biology and nuclear organization. During mitosis, their nuclear envelope stays intact and chromosome segregation is done by a mitotic spindle that passed through the nucleus inside several specialized cytoplasmic channels. In addition, the chromosomes are permanently condensed and are not thought to have histones. Lingulodinium polyedrum is a marine photosynthetic dinoflagellate widely used to study the control mechanisms of circadian rhythms, because many aspects of its physiology (bioluminescence, mitosis, photosynthesis and vertical migration) are circadian. The discovery of cell cycle regulators is essential for understanding the mechanism and the circadian control over the cell cycle. A previously study identified the M-phase cyclin, LpCyc1, the first dinoflagellate cell cycle regulator to be discovered. The present study presents the characterization of the LpCyc1, with respect to expression levels and phosphorylation patterns. These elements act together to ensure the synchronization of the LpCyc1 activity (and the mitosis) within the day. This study also presents the creation and the development of the transcriptome, a major tool for the upcoming studies of Lingulodinium polyedrum. With this tool, we identified the Lingulodinium polyedrum M-CDK, LpCdk1. The LpCdk1 has all the domains of a classic M-CDK, a substrate binding site, an ATP binding site, an activation loop and a cyclin binding interface. vi With the Lingulodinium polyedrum transcriptome, we also made a census of all the conserved proteins normally found in the cell cycle control of yeast. The identification of these proteins had provided a rough shape of L. polyedrum cell cycle. This kind of analysis is the first to be made with Lingulodinium polyedrum and could be expanded to other metabolic processes.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Dissertation (Ph.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references (leaves 166-187). Also available online.

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