In two separate experiments, weight adjusted doses of nocodazole and acrylamide were injected intraperitoneally at various time intervals into twelve week old female mice. Within the nocodazole experiment, the doses were injected at varying time intervals before and after mating. On day seventeen of gestation, the female mice were sacrificed and their uterine contents examined. Nocodazole induced a significant increase in reproductive pathology per total implants when administered one hour after mating to the (SECxC57BL)F, stock: 5.00% total deads, 70.23% moles, and 3.41% abnormal fetuses. Acrylamide treatment produced a significant reduction in live births when administered six hours after mating: 50.86% moles and 46.46% living fetuses per total implants.

O estudo de agentes que interferem no funcionamento das proteínas relacionadas com o ciclo celular é importante para a compreensão dos processos de transformação e de morte celular. Alterações de ploidia, embora presentes na maioria dos tumores humanos, não têm ainda seu papel conhecido no processo de oncogênese. A alteração do número cromossômico é conseqüência primária de erros que envolvem o fuso mitótico e o cinetócoro. Dessa maneira, drogas que agem sobre os microtúbulos são consideradas aneugênicas potenciais. O presente trabalho enfocou o estudo do mecanismo pelo qual drogas que atuam sobre microtúbulos levam ao aparecimento de células multinucleadas e o significado biológico destas. Os resultados mostraram que a vincristina induziu bloqueio em mitose das células BBnt e MDCK, com conseqüente entrada em interfase no estado multinucleado. As células multinucleadas não apresentaram sinais de morte celular por apoptose, entretanto, quando em prófase apresentaram vários centrossomos, que poderiam originar divisões celulares com fusos multipolares. Estes resultados indicam que essas linhagens celulares possuem pontos de checagem mitótico funcionais e células multinucleadas são viáveis e capazes de prosseguir no ciclo celular. A presença de mitoses com fusos multipolares é indício de que as células multinucleadas passam por divisões anormais, que progrediriam para apoptose resultando na eliminação desta população. / The study of agents that interfere in the functionality of proteins related to cell cycle is important for the understanding of the transformation and cell death processes. Although ploidy alterations are presented in the majority of human tumors, their role in oncogenic process is not understood yet. The alteration on chromosomal number is the primary consequence of errors involving the mitotic spindle and kinetocore. Thus, drugs acting on the microtubules are considered as potentially aneugenic agents. The present work aimed to study the mechanism of multinucleated cells induction by action of antimicrotubule drug and biological meaning of these cells. The results showed that vincristine induced mitotic arrest of both BBnt and MDCK cells, with consequent entrance into interphasic-multinucleated status. Multinucleated cells did not present features of cell death by apoptosis; they were still viable and able to go further in cell-cycle progression. The presence of many structures suggested microtubule enucleation, centrosomes-like were detected on treated cells and could be responsible for the multi-spindle assembling that leads the multinucleated cells to abnormal divisions. Later on, when the multinucleated cells accumulated more abnormalities they were eliminated from the cell population by apoptosis.

La transmission correcte du matériel génétique au cours de la mitose requiert l’attachement correct des chromosomes aux microtubules du fuseau mitotique. Les centromères au niveau des chromosomes servent de site d’assemblage aux kinétochores, interfaces multiprotéiques permettant la liaison des microtubules. Cependant, nous avons récemment mis en évidence chez la drosophile un mécanisme par lequel les fragments acentriques ségrégent normalement. Celui-­‐ci fonctionne grâce à un « tether », un filament d’ADN, qui relie les fragments acentriques à leurs partenaires centriques. L’intégrité du tether dépend de la fonction de BubR1, qui s’accumule au tether pendant de la mitose. BubR1 est une protéine clé dans le point de contrôle d’assemblage du fuseau mitotique, ou SAC (Spindle Assembly Checkpoint), qui contrôle l’attachement correct des kinétochores aux microtubules et inhibe l’entrée en anaphase. Nous avons voulu déterminer comment BubR1 est recrutée au tether, et nous avons montré que ce recrutement est dépendant du Bub3 Binding Domain de BubR1 et plus précisément de l’acide aminé E481 dans ce domaine. L’interaction Bub3-­‐BubR1 par l’intermédiaire de ce domaine est nécessaire à la localisation du complexe au tether. Nous avons également montré que BubR1 recrute à son tour Fzy par l’intermédiaire de son domaine KEN.Nous proposons un modèle dans lequel le recrutement successif de Bub3-­‐BubR1 et Fzy au niveau des chromosomes endommagés est nécessaire à leur bonne ségrégation en mitose. / Accurate transmission of genome during mitosis requires proper chromosomes attachment to microtubules of the mitotic spindle. Centromeres of chromosomes are assembly sites for kinetochores, multiproteic interfaces for microtubule binding. However, we recently discovered in Drosophila a mechanism that permits proper acentric chromosomes segregation. This mechanism works through a DNA « tether » that binds together acentric chromosomes to their centric counterparts. Tether integrity depends on BubR1 function, which accumulates on the tether during mitosis. BubR1 is a key protein in the Spindle Assembly Checkpoint (SAC), which monitors proper kinetochore-­‐microtubule attachment, and inhibits anaphase onset until all kinetochores are properly bound to microtubules. We wanted to determine how BubR1 is recruted to the tether, and we showed that this recruitment is dependant on the Bub3-­‐Binding Domain of BubR1, and more precisely the E481 amino acid. Bub3-­‐BubR1 interaction mediated by this domain is necessary for complex localisation on the tether. We also discovered that BubR1 then recruits Fzy via its KEN domain. We propose a model where successive recruiting of Bub3-­‐BubR1 and Fzy at the broken chromosome level is mandatory to their proper segregation in mitosis.

No description available.

Menopause is characterized by prolonged lifespan beyond the point of reproductive cessation. Defined so that at least 25% of adulthood is nonreproductive, humans and some toothed whale species are the only groups that have been found to exhibit menopause. Menopause is a puzzling trait that seems to contradict classical evolutionary theory that equates selection operating on reproduction to selection operating on survival. I created two computational models to gain better understanding of the evolution of menopause. The first model explored why menopause is not observed in elephants despite their being characterized by key features in common with menopausal species, specifically offspring care from older females and longevity. Simulations allowed testing the effects of varying age at reproductive cessation and levels of offspring care, modeled by decreases in interbirth intervals. I found that hypothetical populations with greatest post-reproductive lifespans, characterized by longer interbirth intervals and earlier reproductive cessation, were most likely to be out-competed by contemporary elephants. Conversely, hypothetical populations that were most reproductively competitive, those with shorter interbirth intervals and older ages of reproductive cessation, returned post-reproductive lifespans that failed to meet the 25% post-reproductive lifespan criterion for menopause. I identified a small region in the parameter space where populations that were both menopausal and reproductively competitive evolved, but the majority of that region corresponds to biologically unrealistic scenarios. The scenario that is most feasible involves an interbirth interval of 4 years and an age at reproductive cessation of 40 years. The second model studied how menopause might have evolved in humans through a behavioural strategy of ending reproduction early to avoid risk of aneuploidy later in life and diverting resources toward extant kin. I found that populations that ceased reproduction earlier and exhibited greater post-reproductive lifespan returned lower reproductive success. The model also demonstrated that the aneuploidy avoidance behaviour is most successful when reproduction ends at approximately age 50. These concepts have never been explored computationally before, so these experiments contribute a novel simulation-based perspective to the growing body of knowledge surrounding the origin and evolution of menopause. / Thesis / Master of Science (MSc) / Menopause can be defined generally for a group as a life history characterized by prolonged post-reproductive lifespan. Defined specifically so that at least 25% of adulthood is nonreproductive, menopause has been recorded in only humans and some species of toothed whales. This trait presents an evolutionary puzzle, as it appears to contradict classical evolutionary theory, which suggests that reproduction should continue until the end of life. In this thesis, I use computational modeling to explore why elephants have not evolved menopause despite sharing key features with menopausal species and how aneuploidy might have contributed to the evolution of menopause in humans.

At any given time there are on the order of one hundred million cells undergoing mitosis in the human body. To accurately segregate chromosomes, the cell forms the bipolar mitotic spindle, a molecular machine that distributes chromosomes equally to the daughter cells. To this end, microtubules of the mitotic spindle must appropriately attach the kinetochores: protein structures that form on each chromatid of each mitotic chromosome. The majority of the time correct kinetochore microtubule attachments are formed. However, mis-attachments can and do form. Mis-attachments that are not corrected before chromosome segregation can give rise to aneuploidy, an incorrect number of chromosomes. Aneuploidy occurring in the germ line can cause both miscarriage and genetic diseases. Furthermore, aneuploidy is a major characteristic of cancer cells, and aneuploid cancer cells frequently mis-segregate chromosomes at high rates, a phenotype termed chromosomal instability (CIN). CIN has been correlated with both advanced tumorigenesis and poor patient prognosis and over the years there have been many hypotheses for what causes CIN. In this study, we identified two distinct mechanisms that are responsible for CIN. Both of these mechanisms cause a transient, abnormal geometric arrangement of the mitotic spindle. Specifically, cancer cells possess supernumerary centrosomes, which lead to the assembly of multipolar spindles during early mitosis when attachments between kinetochores and microtubules are forming. Supernumerary centrosomes facilitate the formation of merotelic attachments, in which a single kinetochore binds microtubules from more than one centrosome. As mitosis progresses the supernumerary centrosomes cluster, giving rise to a bipolar spindle by the time of chromosome segregation. However, the high rates of merotelic attachments formed during the transient multipolar stage result in high rates of chromosome mis-segregation. The second geometric defect characterized is caused by failure of centrosomes to separate before kinetochore-microtubule attachments begin to form. This mechanism, too, leads to high rates of kinetochore mis-attachment formation and high rates of chromosome mis-segregation. Finally, this study shows that the mechanisms characterized here are prevalent in human cancer cells from multiple organ sites, thus revealing that both mechanisms are a common cause of CIN. / Ph. D.

Muitas evidências indicam que a tumorigênese em humanos é um processo com várias etapas. A progressão de um tumor em direção a malignidade ocorre de maneiras muito distintas entre os diferentes tipos de câncer. Entender os processos celulares que levam a tumorigênese é importante para se delinear tratamentos mais adequados contra os diversos tipos de câncer. Drogas antimitóticas (ou venenos de fuso) são utilizadas no tratamento de alguns cânceres, e entre eles está o câncer de mama. A ação dessas drogas reside sobre a mitose, e têm como alvo os fusos mitóticos, estruturas essenciais que dirigem o ciclo celular na divisão das células. Recentemente, pesquisadores têm delineado possíveis respostas da célula aos venenos de fuso, e essas respostas são dependentes da concentração da droga. Baixas concentrações de venenos de fuso provocam o aparecimento de populações celulares aneuplóides, que ocorrem quando a célula sai do bloqueio mitótico. O presente trabalho utilizou as drogas vincristina e paclitaxel sobre linhagens celulares de mama humana (células normais e tumorais) para pesquisar se as drogas são capazes de induzir células aneuplóides. Os resultados obtidos no presente trabalho indicam que baixa concentração de vincristina e paclitaxel pode induzir o aparecimento de população aneuplóide através de mitoses aberrantes. Os venenos de fuso induzem a formação de mitoses contendo múltiplos fusos mitóticos, e essas mitoses não ficam bloqueadas, dando origem a células aneuplóides. O papel da aneuploidia na tumorigênese não foi ainda estabelecido, mas indiferente da sua importância no desenvolvimento do tumor, encontrar maneiras de inibir sua formação ou de super induzir seu aparecimento podem ter implicações significativas para as terapias contra o câncer. / Several lines of evidence indicate that tumorigenesis in humans is a multistep process and that these steps reflect genetic alterations that drive the progressive transformation of normal cells into highly malignant derivatives. Understanding the cellular processes that lead to tumorigenesis is important to devise more appropriate treatments against various types of cancer. Antimitotic drugs are used in the treatment of some cancers, and among them is breast cancer. The action of these drugs lies on the mitotic spindles, essential structures that drive the cell cycle in the division of cells. Recently, researchers have outlined possible responses to the antimitotic drugs on cells, and these responses are dependent on the concentration of the drug. Low concentrations of drugs can cause the appearance of aneuploid population, which occur when a cell leaves the mitotic block. This study used the drug vincristine and paclitaxel on human breast cancer cell lines (normal and tumoral cells) to find if the drugs are capable of inducing cell aneuploidy. The results of this study indicate that low concentration of vincristine and paclitaxel can induce the emergence of aneuploidy population through aberrant mitosis. The drugs induce the formation of mitosis containing multiple mitotic spindles, resulting in aneuploidy population of cells. The role of aneuploidy in tumorigenesis has not yet been established, but indifferent to this is important find ways to inhibit its formation or the super-induce their appearance. These questions may have significant implications for therapies against cancer.

L’avortement spontané (AS) désigne la perte du produit de conception avant sa viabilité, c'est-à-dire avant la 22e semaine d’aménorrhée, ou un poids fœtal inférieur à 500 g. La cause génétique est à l’origine de plus des deux tiers des AS, les aneuploïdies autosomiques, représentant à elles seules jusqu’à 70% des pertes fœtales du 1er trimestre. Le caryotype présente une très bonne sensibilité en ce qui concerne le dépistage des trisomies autosomiques (13, 18 et 21) et des aneuploïdies affectant les chromosomes sexuels, mais il montre d’importantes limites, d’une part en raison des échecs de culture cellulaire et d’autre part en raison de l’existence de remaniements non détectables au caryotype standard. Actuellement plusieurs techniques moléculaires de dépistage rapides des aneuploïdies liées aux échecs de grossesses ont été vérifiées : 1°) la fluorescence in situ par hybridation (FISH) 2°) l’amplification multiplex de sondes nucléiques dépendant des ligatures (MLPA). Ces deux méthodes présentent l’avantage d’être réalisables, sans culture préalable, sur noyaux en interphase ou sur ADN extrait et de permettre la détection d’anomalies cryptiques. Notre étude repose sur l’étude cytogénétique des produits d’AS pour mettre en évidence les anomalies chromosomiques les plus fréquentes à l’origine de ces pertes fœtales et d’en mieux appréhender les mécanismes de survenue. Elle a été réalisée sur 220 patientes âgées de 19 à 45 ans, et était fondée sur l’analyse directe par FISH sur noyaux interphasiques (AneuVysionTM) de prélèvements de villosités choriales et sur l’analyse de l’ADN extrait de tissus fœtaux par MLPA afin de révéler d’éventuelles aneuploïdies et micro-remaniements. L’âge gestationnel au moment des prélèvements était compris entre 7 et 38 semaines d’aménorrhée. Sur un total de 151 échantillons analysés par AneuVysionTM, 10 anomalies chromosomiques ont été observées: 3 trisomies 21, 1 trisomie 18, 1 trisomie 13, 1 mosaïque 46,XX/47,XX+21, 3 triploïdies et 1 monosomie X (Turner). Par ailleurs, sur les 69 autres échantillons analysés par MLPA, 6 étaient ininterprétables. Les anomalies trouvées par cette technique étaient: 2 monosomies X. Pour les échantillons restants, la MLPA a été négative. Nous avons en parallèle réalisé une étude rétrospective fondée sur l’analyse comparative d’un échantillon recruté à Sidi Bel Abbès, de femmes ayant subi un AS et admises à la maternité del’hôpital Hassani Abdelkader de Sidi Bel Abbès et d’un échantillon recruté à Clermont-Ferrand de femmes ayant subi un AS et pour lesquelles un prélèvement pour établir le caryotype du produit de fausse-couche avait été adressé dans le service de cytogénétique du CHU Estaing de Clermont-Ferrand. Cette étude a couvert une période de six années, allant de janvier 2005 à décembre 2010. Les techniques de FISH et de MLPA représentent des outils simples, rapides et sensibles pour la détection des remaniements chromosomiques. Elles représentent une alternative très intéressante à la culture cellulaire, et permettent le diagnostic de désordres génomiques indécelables par les techniques conventionnelles. / Spontaneous abortion (SA) is the loss of the product of fertilization before its viability, that is, before22 weeks of gestation or fetal weight less than 500 g. Genetic causes account for more than two thirds of SA, autosomal aneuploidies alone accounting for up to 70% fetal loss. Chromosomal cytogenetic techniques show significant limitations on the one hand because of the failures of cell culture, and secondly because of the existence of undetectable alterations to the standard karyotype. It was therefore planned to use molecular techniques :- Fluorescent in situ hybridization (FISH)- Multiplex ligation-dependent probe amplification (MLPA). Both techniques have the advantage of being achievable without prior culture of cores interphase or DNA extracted and to enable detection of cryptic abnormalities. The project is based on cytogenetic study of AS products to highlight the most frequent chromosomal abnormalities causing fetal losses, and to better understand their occurrence. Our study was performed on 220 patients from 19 to 45 years, and was based on the direct analysis by FISH on interphase nuclei (AneuVysionTM) of chorionic villus sampling and analysis of DNA extracted fetal tissue by MLPA to reveal any aneuploidy and rearrangements. The gestational age of the samples ranged from the 7th to the 38th week of gestation. In a total of 151 samples analyzed by AneuVysionTM, 10 chromosomal abnormalities were observed: three trisomies 21, one trisomy 18, one trisomy 13, one mosaic 46,XX/47,XX+21, 3 triploidies and one monosomy X (Turner). In addition, among the other 69 samples analyzed by MLPA, 6 were uninterpretable. The abnormalities found by this technique were 2 monosomies X. For the remaining samples, the MLPA was negative. We conducted a retrospective parallel study based on the analysis of a sample recruited in Sidi Bel Abbes, women who have had an AS and were admitted to the maternity hospital Abdelkader Hassani, Sidi Bel Abbes ; and a sample recruited in Clermont-Ferrand : women who underwent AS for which a levy to establish the karyotype product miscarriage had been addressed in the Department of Cytogenetics of CHU Estaing, Clermont-Ferrand. This study covered a period of six years, from January 2005 to December 2010. The techniques of FISH and MLPA are simple, rapid and sensitive tools for the detection of chromosomal rearrangements. They represent a very interesting alternative to cell culture and allow diagnosis for genomic disorders undetectable by conventional techniques.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 44-53).

The cohesin complex is responsible for the fidelity of chromosomal segregation during mitosis. It consists of four core subunits namely Rad21/Mcd1/Sccl, Smc1, Smc3 and one of the yeast Scc3 orthologs SA1 or SA2. Sister chromatid cohesion is formed by the cohesin complex during DNA replication and maintained until the onset of anaphase. Among the many proposed models of how cohesin holds sister chromatids together, the 'core' cohesin subunits Smc1, Smc3 and Rad21/Mcd1/Scc1 are almost universally displayed as forming a contiguous ring. However, other than its supportive role in the cohesin ring, little is known about the fourth core protein SA1/SA2 - despite its physical association to the cohesin ring. To gain deeper insight into how physically and physiologically SA2 interacts with the cohesin complex, we performed structural characterization of SA2 and Rad21 and mapped the interaction region of the two proteins in vitro and ex vivo . We found SA2 interacts with Rad21 at multiple domains while Rad21 only interacts with SA2 through a 10 amino acid α-helical motif from 383-392aa. Deletion of these 10 amino acids or mutation of three conserved amino acids (L385, F389, and T390) in this α-helical motif prevents Rad21 from physically interacting with SA2/SA1 and causes premature sister chromatid separation in mitotic cells that often leads to aneuploidy. Our studies provide a model for how SA2 structurally strengthens the cohesin ring through its interaction with Rad21. Results from our structural characterization of these two proteins also provided directions for further investigation of the structural basis of protein-protein interaction in the cohesin complex.