Thesis (Ph. D.)--University of Wisconsin--Madison, 1984. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 182-193).

No description available.

No description available.

No description available.

The centrosome is the main microtubule-organising centre in animal cells; important to assemble a bipolar mitotic spindle ensuring proper chromosome segregation and genomic stability. Whereas correct centrosome number (1-2) is tightly maintained in normal cells, cancer cells usually have an increased number of centrosomes (>2), termed centrosome ampli cation. Centrosome ampli cation has been correlated with aneuploidy, increased tumour grade, chemoresistance and overall poor prognosis. Cancer cells primarily adapt to supernumerary centrosomes by clustering them into two poles resulting in a `normal' pseudo-bipolar mitosis. Undermining centrosome clustering is a potential target for cancer-speci c treatment. Indeed, depleting the kinesin HSET has already been shown to speci cally kill cancer cells by impairing the centrosome clustering mechanism. However, it is unclear whether this process requires adaptation or it is inherent to all cell types. Using a panel of non-transformed cell lines, we observed that cells expressing Ecadherin have ine cient clustering mechanisms compared to cell lines without E-cadherin. Loss of E-cadherin (siRNA/CRISPR) promotes centrosome clustering and survival of epithelial cells with multiple centrosomes. In addition, loss of DDR1, involved in regulating cortical contractility downstream of E-cadherin, increases centrosome clustering in epithelial cells. Using Atomic Force Microscopy we con rmed that indeed loss of E-cadherin leads to increased cortical contractility in mitotic cells. Inhibition of actomyosin contractility prevents e cient clustering in cells that do not express E-cadherin, further suggesting that it is important for this process. Loss of E-cadherin and DDR1 is strongly correlated with high levels of centrosome ampli cation in breast cancer cell lines suggesting that these changes are an important adaptation mechanism to centrosome amplification.

No description available.

No description available.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 107-123). Also available in electronic version.

Fbxl13 (F-box and leucine-rich repeat protein 13) is an orphan F-box protein. Fbox proteins are a family of substrate-targeting specificity factors for the SCF superfamily of E3 ubiquitin ligases. Since their discovery, many F-box proteins have been shown to have oncogenic and tumour suppressive roles. The importance of Fbxl13 itself in tumourigenesis is reflected in several genome-wide shRNA screens. Fbxl13 depletion in human cancer cells correlates with increased ionising radiation sensitivity and increased genomic instability. Furthermore, Fbxl13 depletion reduces proliferation in mouse embryonic epidermis. Conversely, Fbxl13 amplification is frequently observed in several cancer patient cohorts. However, the main function of Fbxl13 is unknown and its biochemical mechanism of action remains uncharacterised. The aim of this study was to identify the interactors, substrates, and functions of Fbxl13, in order to elucidate its role in tumourigenesis. In this study, I identify and validate Fbxl13 interactors Centrin-2, Centrin-3, Cep152, and Cep192. I show that Fbxl13 is enriched at the centrosome, and present evidence that Fbxl13 targets Cep192-3 for ubiquitin mediated proteolysis. In line with this, Fbxl13 overexpression downregulated centrosomal Cep192 and γ-tubulin, and disrupted the microtubule nucleation activity at the centrosome. Finally, Fbxl13 amplification in U2OS cells is associated with increased cell motility. Thus, we propose that Fbxl13 is a novel regulator of centrosome microtubule nucleation activity.

Les récepteurs d'antigènes des lymphocytes T (TCR) sont assemblés au cours du développement précoce de ces cellules dans le thymus suite à des recombinaisons complexes de gènes. Le réarrangement d'une chaine beta des TCR fonctionnelle (pré-TCR) déclenche des voies de signalisation intracellulaires qui entrainent la survie, l'expansion et la maturation des thymocytes. Par ailleurs, l'engagement des TCR à la surface des lymphocytes T (LT) matures par des antigènes conduit également à des cycles de prolifération qui permettent le développement de réponses immunitaires efficaces. Ces évènements cellulaires s'accompagnent de remaniements importants du réseau de microtubules et une redistribution des moteurs moléculaires, tels que la dynéine, qui véhiculent les structures cellulaires sur ces réseaux. Les mécanismes moléculaires et les conséquences physiologiques de ces remaniements sont peu connus dans les LT. Lis1 est un régulateur de la dynéine qui est mis à contribution dans la migration neuronale et la prolifération des cellules souches au cours du développement neural. Son rôle au sein du tissu lymphoïde est peu connu. Dans ce travail, nous avons utilisé des modèles de souris spécifiquement déficients en Lis1 dans les LT afin d'étudier les fonctions moléculaires, cellulaires et physiologiques de cette protéine dans ces cellules. Nous montrons que Lis1 joue un rôle essentiel dans le développement précoce des LT et dans l'homéostasie des LT matures. La déficience en Lis1 n'affecte pas le réarrangement de la chaine beta ou les évènements de signalisation déclenchés par le pré-TCR ou le TCR. Cependant, la prolifération des thymocytes ayant passé la beta-sélection ou des LT matures dont le TCR a été engagé, est fortement impactée. L'analyse fine de la mitose indique que la déficience en Lis1 ralentit fortement le processus mitotique, contrarie les remaniements intracellulaires conduisant à la métaphase et entraîne la répartition asymétrique du matériel génétique dans les cellules filles. L'analyse des réseaux de microtubules montre que l'absence de Lis1 entraîne l'amplification du nombre de centrosomes et l'augmentation des cellules multipolaires au cours de la mitose. Enfin, nous montrons que Lis1 favorise l'interaction de la dynéine avec la dynactine, indiquant que Lis1 joue un rôle important dans les LT pour relier la dynéine aux structures cellulaires qu'elle véhicule. En conclusion, nous avons montré que Lis1 est importante dans la distribution du matériel génétique au cours de la prolifération des thymocytes doubles négatifs et des lymphocytes T périphériques. / The T cell receptor (TCR) is assembled during the early development of T lymphocytes in the thymus after complexe genetic recombinations. The rearrangement of a functional TCR beta-chain (pre-TCR) triggers intracellular signaling pathways that cause the survival, expansion and maturation of thymocytes. The commitment of the TCR to the surface of mature T cells after antigen recognition also leads to proliferation allowing the development of effective immune responses. These cellular events go along with significant reorganization of the microtubule networks and a redistribution of molecular motors, such as dynein, which transport the cellular structures via this network. The molecular mechanisms and physiological consequences of the reorganization are poorly understood in T cells. Lis1 is a dynein regulator involved in neuronal migration and stem cells proliferation during neural development. Its role in lymphoid tissue is still unknown. In this study, we used mouse models specifically Lis1-deficient in T cells to study the molecular, cellular and physiological functions of this protein in T cells. We identifiy that Lis1 plays an essential role in the early development of T cells and in the homeostasis of mature cells. Lis1 deficiency does not affect beta-chain rearrangement or signaling events triggered by pre-TCR or TCR, but leads to the blockage of thymocyte cell division that have undergone beta-selection or mature T cells stimulated. Fine analysis of mitosis indicates that the deficiency of Lis1 strongly slows down the mitotic process, counteracts the cell changes leading to the metaphase and leads to asymmetric distribution of the genetic material in the daughter cells. Microtubule networks analysis shows that the absence of Lis1 induces centrosomes amplification and increase of multipolar cells during mitosis. Finally, we show that Lis1 promotes the dynein-dynactin interaction, indicating that Lis1 plays an important role in T cells to bind dynein to the cell structures it carries. In conclusion, we here described that Lis1 is important for the distribution of genetic material during double negative thymocyte and peripheral lymphocyte proliferation.