No description available.

No / Ran is a small ras-related GTPase that controls the nucleocytoplasmic exchange of macromolecules across the nuclear envelope. It binds to chromatin early during nuclear formation and has important roles during the eukaryotic cell cycle, where it regulates mitotic spindle assembly, nuclear envelope formation and cell cycle checkpoint control. Like other GTPases, Ran relies on the cycling between GTP-bound and GDP-bound conformations to interact with effector proteins and regulate these processes. In nucleocytoplasmic transport, Ran shuttles across the nuclear envelope through nuclear pores. It is concentrated in the nucleus by an active import mechanism where it generates a high concentration of RanGTP by nucleotide exchange. It controls the assembly and disassembly of a range of complexes that are formed between Ran-binding proteins and cellular cargo to maintain rapid nuclear transport. Ran also has been identified as an essential protein in nuclear envelope formation in eukaryotes. This mechanism is dependent on importin-β, which regulates the assembly of further complexes important in this process, such as Nup107–Nup160. A strong body of evidence is emerging implicating Ran as a key protein in the metastatic progression of cancer. Ran is overexpressed in a range of tumors, such as breast and renal, and these perturbed levels are associated with local invasion, metastasis and reduced patient survival. Furthermore, tumors with oncogenic KRAS or PIK3CA mutations are addicted to Ran expression, which yields exciting future therapeutic opportunities.

Die mitotische Spindel ist ideal für die Erforschung der Selbstorganisation und Plastizität molekularer Kollektive im Zytoplasma. Die Geometrie der Spindel ist entscheidend für die korrekte Chromosomentrennung, muss sich aber an die Zellgröße anpassen. Es ist unbekannt, ob und wie Zellen während ihrer Differenzierung die Spindelarchitektur anpassen, was insbesondere während der Gehirnentwicklung relevant ist. Wir untersuchten dies mit Maus-Embryonalstammzellen, die in frühe neuronale Vorläuferzellen differenziert wurden. Wir entwickelten ein automatisiertes Mikroskopieprotokoll um einen umfassenden Datensatz von mitotischen Zellen zu generieren. Außerdem entwickelten wir Spindle3D, ein Werkzeug zur dreidimensionalen Analyse von Spindeln. Überraschenderweise waren die Spindelvolumina in differenzierenden Zellen bis zu 24% kleiner als in pluripotenten Zellen. Während die Wachstumsgeschwindigkeit der Mikrotubuli unverändert blieb, verschob sich in sich differenzierenden Zellen die Nukleation von Mikrotubuli zugunsten der astralen Population. Diese Veränderung der Spindelarchitektur basierte auf der differenzierungsbedingten Verdünnung des Zytoplasmas. Dies aktivierte CPAP, ein Protein, das die Zentrosomenreifung reguliert, was zur Superskalierung des perizentriolären Materials und verstärkte Rekrutierung von gamma-Tubulin an den Zentrosomen und somit zur Umlagerung von Mikrotubuli innerhalb der Spindel führte. Diese Veränderungen der mitotischen Architektur konnten durch externe osmotische Einwirkung in undifferenzierten Zellen nachgestellt werden. Insgesamt verbinden unsere Ergebnisse zelltypspezifische zytoplasmatische Materialeigenschaften mit der Spindelarchitektur. / The mitotic spindle provides an excellent system in which to study the plasticity of molecular collectives. To segregate chromosomes accurately, the spindle’s geometry must be adaptive to changes in cell size. It is unknown whether and how differentiating cells adjust spindle architecture, specifically during brain development when spindle defects have severe pathological consequences. Using murine embryonic stem cells, we recapitulated the transition from pluripotency to early neural cell identities in vitro. Aiming at a systematic exploration of spindle and cell morphology throughout this process, we developed an automated microscopy protocol and Spindle3D, a morphometric tool for the analysis of spindles in confocal images. Intriguingly, in cells with comparable cell volume, spindle volumes were up to 24% smaller in cells undergoing differentiation. While microtubule growth speed remained equal, we measured increased nucleation of astral microtubules at the expense of the spindle bulk in differentiating cells. The shift in spindle architecture was explained by the differentiation-driven cytoplasmic dilution. This activated the centrosomal regulator CPAP, causing the superscaling of the pericentriolar material and the concomitant increased recruitment of gamma-tubulin to the centrosomes, redistributing microtubule numbers within the spindle. Mimicking the dilution effect by osmotic challenge reproduced the same mitotic architecture in undifferentiated cells. Collectively, our results link cell state-specific cytoplasmic material properties to spindle architecture.

Indiana University-Purdue University Indianapolis (IUPUI) / Fanconi anemia (FA) is a heterogenous genetic syndrome characterized by progressive bone marrow failure, aneuploidy, and cancer predisposition. It is incompletely understood why FA-deficient cells develop gross aneuploidy leading to cancer. Since the mitotic spindle assembly checkpoint (SAC) prevents aneuploidy by ensuring proper chromosome segregation during mitosis, we hypothesized that the FA signaling network regulates the mitotic SAC. A genome-wide RNAi screen and studies in primary cells were performed to systematically evaluate SAC activity in FA-deficient cells. In these experiments, taxol was used to activate the mitotic SAC. Following taxol challenge, negative control siRNA-transfected cells appropriately arrested at the SAC. However, knockdown of fourteen FA gene products resulted in a weakened SAC, evidenced by increased formation of multinucleated, aneuploid cells. The screen was independently validated utilizing primary fibroblasts from patients with characterized mutations in twelve different FA genes. When treated with taxol, fibroblasts from healthy controls arrested at the mitotic SAC, while all FA patient fibroblasts tested exhibited weakened SAC activity, evidenced by increased multinucleated cells. Rescue of the SAC was achieved in FANCA patient fibroblasts by genetic correction. Importantly, SAC activity of FANCA was confirmed in primary CD34+ hematopoietic cells. Furthermore, analysis of untreated primary fibroblasts from FA patients revealed micronuclei and multinuclei, reflecting abnormal chromosome segregation. Next, microscopy-based studies revealed that many FA proteins localize to the mitotic spindle and centrosomes, and that disruption of the FA pathway results in supernumerary centrosomes, establishing a role for the FA signaling network in centrosome maintenance. A mass spectrometry-based screen quantifying the proteome and phospho-proteome was performed to identify candidates which may functionally interact with FANCA in the regulation of mitosis. Finally, video microscopy-based experiments were performed to further characterize the mitotic defects in FANCA-deficient cells, confirming weakened SAC activity in FANCA-deficient cells and revealing accelerated mitosis and abnormal spindle orientation in the absence of FANCA. These findings conclusively demonstrate that the FA signaling network regulates the mitotic SAC, providing a mechanistic explanation for the development of aneuploidy and cancer in FA patients. Thus, our study establishes a novel role for the FA signaling network as a guardian of genomic integrity.

Master of Science / Department of Biochemistry / Qize Wei / Proper regulation of mitosis and cytokinesis is fundamentally important for all living organisms. During anaphase, antiparallel microtubules are bundled between the separating chromosomes, forming the central spindle (also called the spindle midzone), and the myosin contractile ring is assembled at the equatorial cortex. Regulators of central spindle formation and myosin contractile ring assembly are mostly restricted to the interdigitated microtubules of central spindles and they can be collectively called midzone components. It is thought that characteristic microtubule configurations during mitosis and cytokinesis are dictated by the coordinated action of microtubule-stabilizing and -destabilizing factors. Although extensive investigations have focused on understanding the roles of microtubule-bundling/stabilizing factors in controlling central spindle formation, efforts have been lacking in aiming to understand how microtubule-destabilizing factors regulate the assembly of central spindles. This dissertation describes the role of a novel microtubule-destabilizing factor termed CCDC69 (coiled-coil domain-containing protein 69) in controlling the assembly of central spindles and the recruitment of midzone components. Endogenous CCDC69 was localized to the nucleus during interphase and to the central spindle during anaphase. Exogenous expression of CCDC69 in HeLa cells destabilized microtubules and disrupted the formation of bipolar mitotic spindles. RNA interference (RNAi)-mediated knockdown of CCDC69 led to the formation of aberrant central spindles and interfered with the localization of midzone components such as aurora B kinase, protein regulator of cytokinesis 1 (PRC1), MgcRacGAP/HsCYK-4, and pololike kinase 1 (Plk1) at the central spindle. CCDC69 knockdown also decreased equatorial RhoA staining, indicating that CCDC69 deficiency can impair equatorial RhoA activation and ultimately lead to cytokinesis defects. Four coiled-coil domains were found in CCDC69 and the C terminal coiled-coil domain was required for interaction with aurora B. Disruption of aurora B function in HeLa cells by treatment with a small chemical inhibitor led to the mislocalization of CCDC69 at the central spindle. Further, vitro kinase assay showed that Plk1 could phosphorylate CCDC69. Taken together, we propose that CCDC69 acts as a scaffold and a microtubule-destabilizing factor to control the recruitment of midzone components and the assembly of central spindles.

La mitose est une phase importante du cycle cellulaire. Les mécanismes de surveillance s'assurent de l'ordre et de l'exécution correcte des événements du cycle cellulaire dont les erreurs peuvent conduire à l'aneuploïdie. Pendant la mitose, la séparation des chromatides sœurs est régulée par le point de contrôle du fuseau mitotique qui s'assure que tous les chromosomes sont correctement alignés sur la plaque métaphasique. L'entrée et la sortie de mitose sont régulées par l'activation et l'inactivation du complexe cycline B/Cdk1. Cette fine régulation fait intervenir de nombreuses kinases et phosphatases. Dans ce projet nous nous sommes intéressés plus particulièrement à deux AGC kinases : RSK2 et Greatwall (Gwl).Au cours de cette étude nous nous sommes proposés d'analyser l'implication de RSK2, substrat majeur de la MAPK, dans le point de contrôle du fuseau mitotique. Nos résultats montrent que RSK2 est essentielle pour l'activité du point de contrôle du fuseau mitotique dans les extraits d'œufs de xénope ainsi que pour la localisation des autres protéines de ce mécanisme de surveillance localisées aux kinétochores. Nous montrons également que RSK2 participe au point de contrôle dans les cellules humaines. En effet, RSK2 est nécessaire à la localisation aux kinétochores de Mad1, Mad2 et Cenp-E, protéines essentielles à l'activité de ce checkpoint. L'entrée et la sortie de mitose sont régulées par le complexe cycline B/Cdk1 et des phosphatases. Gwl est une nouvelle kinase essentielle à l'entrée en mitose et au maintien de l'état mitotique dans les extraits d'œufs de xénope. En effet, nos résultats montrent que Gwl maintient l'état mitotique indépendamment du complexe cycline B/Cdk1, en régulant négativement PP2A, une phosphatase responsable de la déphoshorylation des substrats mitotiques. / Mitosis is an important phase of cell cycle. The Spindle Assembly Checkpoint (SAC) verifies the orders and the events correct execution of the cell cycle, as errors may lead to aneuploidy. During the mitosis, the checkpoint delays the anaphase onset until all chromosomes are correctly attached to the spindle‘s microtubules. Entry and Exit of mitosis are regulated by the activation and inactivation of cyclin B/Cdk1. A lot of kinases and phosphatases are involved in this fine regulation. In this project, we are particularly focusing on two AGC kinases: RSK2 and Greatwall (Gwl).In this study, we analyzed RSK2, a major substrates of MAPK, involvement in SAC. Our results show that RSK2 is essential to the activation of SAC in xenopus egg extracts and for the localization at the kinétochores of the others SAC components. We also show that RSK2 participate in the maintenance of the SAC in human cells. Indeed, RSK2 is necessary for Mad1, Mad2 and Cenp-E localization, essential proteins for SAC activation.Entry and exit of mitosis are regulated by cyclin B/Cdk1 complex and phosphatases. Gwl is a new kinase essential to the entry into mitosis and maintenance of the mitotic state in xenopus egg extracts. Indeed, our results showed that Gwl maintains the mitotic state independently of cyclin B/Cdk1 but with the negative regulation of PP2A, which dephosphorylate the mitotic substrates

Precise regulation of cell cycle events by the Cdk-control network is essential for cell proliferation and the perpetuation of life. The unidirectionality of cell cycle progression is governed by several critical irreversible transitions: the G1-to-S transition, the G2-to-M transition, and the M-to-G1 transition. Recent experimental and theoretical evidence has pulled into question the consensus view that irreversible protein degradation causes the irreversibility of those transitions. A new view has started to emerge, which explains the irreversibility of cell cycle transitions as a consequence of systems-level feedback rather than of proteolysis. This thesis applies mathematical modelling approaches to test this proposal for the Mto- G1 transition, which consists of two consecutive irreversible substeps: the metaphase-to-anaphase transition, and mitotic exit. The main objectives of the present work were: (i) to develop deterministic models to identify the essential molecular feedback loops and to examine their roles in the irreversibility of the M-to-G1 transition; (ii) to present a straightforward and reliable workflow to translate deterministic models of reaction networks into stochastic models; (iii) to explore the effects of noise on the cell cycle transitions using stochastic models, and to compare the deterministic and the stochastic approaches. In the first part of this thesis, I constructed a simplified deterministic model of the metaphase-to-anaphase transition, which is mainly regulated by the spindle assembly checkpoint (the SAC). Based on the essential feedback loops causing the bistability of the transition, this deterministic model provides explanations for three open questions regarding the SAC: Why is the SAC not reactivated when the kinetochore tension decreases to zero at anaphase onset? How can a single unattached kinetochore keep the SAC active? How is the synchronized and abrupt destruction of cohesin triggered? This deterministic model was then translated into a stochastic model of the SAC by treating the kinetochore microtubule attachment at prometaphase as a noisy process. The stochastic model was analyzed and simulation results were compared to the experimental data, with the aim of explaining the mitotic timing regulation by the SAC. Our model works remarkably well in qualitatively explaining experimental key findings and also makes testable predictions for different cell lines with very different number of chromosomes. The noise generated from the chemical interactions was found to only perturb the transit timing of the mitotic events, but not their ultimate outcomes: all cells eventually undergo anaphase, however, the time required to satisfy the SAC differs between cells due to stochastic effects. In the second part of the thesis, stochastic models of mitotic exit were created for two model organisms, budding yeast and mammalian cells. I analyzed the role of noise in mitotic exit at both the single-cell and the population level. Stochastic time series simulations of the models are able to explain the phenomenon of reversible mitotic exit, which is observed under specific experimental conditions in both model organisms. In spite of the fact that the detailed molecular networks of mitotic exit are very different in budding yeast and mammalian cells, their dynamic properties are similar. Importantly, bistability of the transitions is successfully captured also in the stochastic models. This work strongly supports the hypothesis that uni-directional cell cycle progression is a consequence of systems-level feedback in the cell cycle control system. Systems-level feedback creates alternative steady states, which allows cells to accomplish irreversible transitions, such as the M-to-G1 transition studied here. We demonstrate that stochastic models can serve as powerful tools to capture and study the heterogeneity of dynamical features among individual cells. In this way, stochastic simulations not only complement the deterministic approach, but also help to obtain a better understanding of mechanistic aspects. We argue that the effects of noise and the potential needs for stochastic simulations should not be overlooked in studying dynamic features of biological systems.

Quelques évidences suggèrent que Bcl-xL, un membre anti-apoptotique de la famille Bcl-2, possède également des fonctions au niveau du cycle cellulaire et de ses points-contrôle. Pour étudier la régulation et fonction de Bcl-xL au cours du cycle cellulaire, nous avons généré et exprimé dans des cellules humaines une série de mutants de phosphorylation incluant Thr41Ala, Ser43Ala, Thr47Ala, Ser49Ala, Ser56Ala, Ser62Ala et Thr115Ala. L'analyse de cette série de mutants révèle que les cellules exprimant Bcl-xL(Ser62Ala) sont moins stables au point-contrôle G2 du cycle cellulaire comparées aux cellules exprimant le type sauvage ou les autres mutants de phosphorylation incluant Thr41Ala, Ser43Ala, Thr47Ala, Ser56Ala et Thr115Ala. Les études de cinétiques de phosphorylation et de localisation de phospho-Bcl-xL(Ser62) dans des cellules synchronisées et suite à l'activation du point-contrôle en G2 médié par l'étoposide (VP16), nous indiquent que phospho-Bcl-xL(Ser62) migre dans les corps nucléolaires durant l'arrêt en G2 dans les cellules exposées au VP16. Une série d'expériences incluant des essais kinase in vitro, l'utilisation d'inhibiteurs pharmacologiques et d'ARN interférant, nous révèlent que Polo kinase 1 (PLK1) et MAPK9/JNK2 sont les protéines kinase impliquées dans la phosphorylation de Bcl-xL(Ser62), et pour son accumulation dans les corps nucléolaires pendant le point-contrôle en G2. Nos résultats indiquent que durant le point-contrôle en G2, phospho-Bcl-xL(Ser62) se lie et se co-localise avec CDK1(CDC2), le complexe cycline-kinase qui contrôle l'entrée en mitose. Nos résultats suggèrent que dans les corps nucléolaires, phospho-Bcl-xL(Ser62) stabilise l'arrêt en G2 en séquestrant CDK1(CDC2) pour retarder l'entrée en mitose. Ces résultats soulignent également que les dommages à l'ADN influencent la composition des corps nucléolaires, structure nucléaire qui émerge maintenant comme une composante importante de la réponse aux dommages à l'ADN. Dans une deuxième étude, nous décrivons que les cellules exprimant le mutant de phosphorylation Bcl-xL(Ser62Ala) sont également plus stables au point-contrôle de l'assemblage du fuseau de la chromatine (SAC) suite à une exposition au taxol, comparées aux cellules exprimant le type sauvage ou d'autres mutants de phosphorylation de Bcl-xL, incluant Thr41Ala, Ser43Ala, Thr47Ala, Ser56Ala. Cet effet est indépendent de la fonction anti-apoptotique de Bcl-xL. Bcl-xL(Ser62) est fortement phosphorylé par PLK1 et MAPK14/SAPKp38α à la prométaphase, la métaphase et à la frontière de l'anaphase, et déphosphorylé à la télophase et la cytokinèse. Phospho-Bcl-xL(Ser62) se trouve dans les centrosomes avec γ-tubuline, le long du fuseau mitotique avec la protéine moteure dynéine et dans le cytosol mitotique avec des composantes du SAC. Dans des cellules exposées au taxol, phospho-Bcl-xL(Ser62) se lie au complexe inhibiteur CDC20/MAD2/BUBR1/BUB3, alors que le mutant Bcl-xL(Ser62Ala) ne se lie pas à ce complexe. Ces résultats indiquent que durant le SAC, la phosphorylation de Bcl-xL(Ser62) accélère la résolution du SAC et l'entrée des cellules en anaphase. Des expériences bloquant l'expression de Bcl-xL révèlent ègalement un taux très élevé de cellules tétraploïdes et binuclées après un traitement au nocodazole, consistant avec une fonction de Bcl-xL durant la mitose et dans la stabilité génomique. Dans la troisième étude, l'analyse fonctionnelle de cette série de mutants de phosphorylation indique également que les cellules exprimant Bcl-xL(Ser49Ala) sont moins stables durant le point-contrôle G2 et entre en cytokinèse plus lentement dans des cellules exposées aux inhibiteurs de la polymérisation/dépolymérisation des tubulines, composantes des microtubules. Ces effets de Bcl-xL(Ser49Ala) sont indépendents de sa fonction anti-apoptotique. La phosphorylation de Bcl-xL(Ser49) est dynamique au cours du cycle cellulaire. Dans des cellules synchronisées, Bcl-xL(Ser49) est phosphorylé en phase S et G2, déphosphorylé à la prométaphase, la métaphase et à la frontière de l'anaphase, et re-phosphorylé durant la télophase et la cytokinèse. Au cours du point-contrôle G2 induit par les dommages à l'ADN, un pool important de phospho-Bcl-xL(Ser49) se trouve aux centrosomes, un site important pour la régulation de l'entrée en mitose. Durant la télophase et la cytokinèse, phospho-Bcl-xL(Ser49) se trouve le long des microtubules avec la protéine moteure dynéine et dans le cytosol mitotique. Finalement, nos résultats suggèrent que PLK3 est responsable de la phosphorylation de Bcl-xL(Ser49), une protéine kinase impliquée pour l'entrée des cellules en mitose et pour la progression de la mitose jusqu'à la division cellulaire. / Accumulating evidence suggest that Bcl-xL, an anti-apoptotic member of the Bcl-2 family, also functions in cell cycle progression and cell cycle checkpoints. To further understand Bcl-xL regulation and function in cell cycle progression, we first expressed a series of single-point Bcl-xL cDNA phospho-mutants, including Thr41Ala, Ser43Ala, Thr47Ala, Ser49Ala, Ser56Ala, Ser62Ala and Thr115Ala in human cancer cell lines and investigated their impact on cell cycle progression. Analysis of this series of phosphorylation mutants reveals that cells expressing Bcl-xL(Ser62Ala) mutant are less stable at the G2 checkpoint and enter mitosis more rapidly than cells expressing wild type Bcl-xL or Bcl-xL phosphorylation mutants, including Thr41Ala, Ser43Ala, Thr47Ala, Ser56Ala and Thr115Ala. Dynamic phosphorylation and location studies on phospho-Bcl-xL(Ser62) in unperturbed, synchronized cells and during DNA damage-induced G2 arrest revealed that phospho-Bcl-xL(Ser62) translocates into nucleolar structures in VP16-exposed cells during G2 arrest. Using in vitro kinase assays, pharmacological inhibitors and specific siRNAs experiments, we found that Polo kinase 1 and MAPK9/JNK2 are major protein kinases involved in Bcl-xL(Ser62) phosphorylation and accumulation into nucleolar structures during the G2 checkpoint. In nucleoli, phospho-Bcl-xL(Ser62) binds to and co-localizes with CDK1(CDC2), the key cyclin-dependent kinase required for entry into mitosis. These data indicate that, during G2 checkpoint, phospho-Bcl-xL(Ser62) stabilizes G2 arrest by timely trapping CDK1(CDC2) in nucleolar structures to slow mitotic entry. It also highlights that DNA damage affects the dynamic composition of the nucleolus, which now emerges as a key event in the DNA damage response. In a second study, we describe that cells expressing Bcl-xL(Ser62Ala) are also more stable at a sustained spindle-assembly checkpoint (SAC) after exposure to taxol than cells expressing wild-type Bcl-xL or other mutants, an effect that appears to be independent of its anti-apoptotic activity. Bcl-xL(Ser62) is strongly phosphorylated by PLK1 and MAPK14/SAPKp38α at prometaphase, metaphase and the anaphase boundary, while it is dephosphorylated at telophase and cytokinesis. Phospho-Bcl-xL(Ser62) localizes in centrosomes with γ-tubulin, along the mitotic spindle with dynein motor protein and in cytosol with SAC signaling components. In taxol-exposed cells, phospho-Bcl-xL(Ser62) binds to the CDC20/MAD2/BUBR1/BUB3 complex, while Bcl-xL(Ser62Ala) does not. The data indicate that during SAC, Bcl-xL(Ser62) phosphorylation accelerates SAC resolution and cell entry into anaphase, even in the presence of unattached or misaligned chromosomes. Silencing Bcl-xL expression also leads nocodazole-exposed cells to tetraploidy and binucleation, consistent with a Bcl-xL function in SAC and genomic stability. In the third study, the functional analysis of a Bcl-xL phosphorylation mutant series has revealed that cells expressing Bcl-xL(Ser49Ala) mutant are less stable at G2 checkpoint after DNA damage and enter cytokinesis much more slowly after microtubule poisoning than cells expressing wild-type Bcl-xL. These effects of Bcl-xL(Ser49Ala) mutant seem to be distinct from Bcl-xL function in apoptosis. Bcl-xL(Ser49) phosphorylation is cell cycle-dependent. In synchronized cells, phospho-Bcl-xL(Ser49) appears during the S phase and G2, whereas it disappears rapidly in early mitosis during prometaphase, metaphase and early anaphase, and re-appears during telophase and cytokinesis. During DNA damage-induced G2 arrest, an important pool of phospho-Bcl-xL(Ser49) accumulates in centrosomes which act as essential decision centers for progression from G2 to mitosis. During telophase/cytokinesis, phospho-Bcl-xL(Ser49) is found along microtubules and at midbody with dynein motor protein. In a series of in vitro kinase assays, specific small interfering RNA and pharmacological inhibition experiments, polo kinase 3 (PLK3) was implicated in Bcl-xL(Ser49) phosphorylation. These data indicate that during G2 checkpoint phospho-Bcl-xL(Ser49) is another downstream target of PLK3, acting to stabilize G2 arrest. Bcl-xL phosphorylation at Ser49 also correlates with essential PLK3 activity and function, enabling cytokinesis and mitotic exit.

Les avancées techniques et méthodologiques de la neuroscience ont permis de caractériser le sommeil comme un état actif et dynamique où des événements neuronaux cohésifs organisent les fonctions cérébrales. Les fuseaux de sommeil et les ondes lentes sont les marqueurs électroencéphalographiques de ces événements, et la mesure de leurs paramètres reflète et nuance les interactions neuronales à l’oeuvre pendant le sommeil lent. Considérant leur implication dans les fonctions hypniques et cognitives, les événements du sommeil lent sont particulièrement pertinents à l’étude du vieillissement, où l’intégrité de ces fonctions est mise au défi. Le vieillissement normal s’accompagne non seulement de réductions importantes des paramètres composant les événements du sommeil lent, mais aussi de modifications précises de l’intégrité anatomique et fonctionnelle du cerveau. Récemment, les études ont souligné la régulation locale des événements du sommeil lent, dont l’évolution avec l’âge demeure toutefois peu explorée. Le présent ouvrage se propose de documenter les liens unissant la neurophysiologie du sommeil, le vieillissement normal et l’activité régionale du cerveau par l’évaluation topographique et hémodynamique des événements du sommeil lent au cours du vieillissement. Dans une première étude, la densité, la durée, l’amplitude et la fréquence des fuseaux de sommeil ont été évaluées chez trois groupes d’âge au moyen de l’analyse topographique et paramétrique de l’électroencéphalogramme. Dans une seconde étude, les variations hémodynamiques associées à l’occurrence et modulées par l’amplitude des ondes lentes ont été évaluées chez deux groupes d’âge au moyen de l’électroencéphalographie combinée à l’imagerie par résonance magnétique fonctionnelle. Globalement, les résultats obtenus ont indiqué : 1) une dichotomie des aires corticales antérieures et postérieures quant aux effets d’âge sur les paramètres des fuseaux de sommeil; 2) des variations de la réponse hémodynamique associées aux ondes lentes dans une diversité de régions corticales et sous-corticales chez les personnes âgées. Ces résultats suggèrent la réorganisation fonctionnelle de l’activité neuronale en sommeil lent à travers l’âge adulte, soulignent l’utilité et la sensibilité des événements du sommeil lent comme marqueurs de vieillissement cérébral, et encouragent la recherche sur l’évolution des mécanismes de plasticité synaptique, de récupération cellulaire et de consolidation du sommeil avec l’âge. / As demonstrated by recent advancements in the field of neuroscience, sleep is an active and dynamic state in which cohesive neural oscillations organize brain functions. Sleep spindles and slow waves are hallmarks of non-rapid eye movement (NREM) sleep and are used as markers on the electroencephalogram to characterize the underlying neural activity. Because of their implication in sleep and cognitive processes, these oscillations are particularly relevant in aging research, as functional challenges to sleep and memory are well known among this population. Normal aging not only reduces the characteristics of NREM sleep oscillations, but it also modifies anatomical and functional measures of brain integrity. Local regulation of NREM sleep oscillations have recently been described, yet few evidence is currently available on this process in aging. The present work aims to characterize the relationship between sleep neurophysiology, normal aging and regional brain activity with the assessment of the topography and hemodynamics of NREM sleep oscillations throughout adulthood. In a first study, sleep spindle density, duration, amplitude and frequency will be assessed in three age groups in relation to brain topography using electroencephalography. In a second study, hemodynamic responses to slow wave events and their modulation by amplitude will be assessed in two age groups using electroencephalography combined with functional magnetic resonance imaging. Our results can be summarized as follows: 1) age effects on sleep spindle characteristics showed an intriguing dichotomy between anterior and posterior cortical areas; 2) hemodynamic variations related to slow waves were observed in a wide array of cortical and subcortical regions in older individuals. These results suggest the functional reorganization of neural activity during NREM sleep throughout adulthood, support NREM sleep oscillations as useful and sensible biomarkers of brain aging, and promote further research on age-related changes in synaptic plasticity, cell restoration and sleep maintenance.

Introdução: Apesar de controverso, questiona-se um papel deletério da endometriose nos resultados de procedimentos de reprodução assistida, o que pode estar relacionado ao comprometimento da qualidade oocitária. Para que o oócito maduro esteja preparado para a fertilização, é necessário que o fuso meiótico mantenha a sua integridade e funcionabilidade. Objetivos: Comparar a presença e localização do fuso meiótico e o estágio de maturação nuclear de oócitos com o primeiro corpúsculo polar (CP) visível de pacientes inférteis sem e com endometriose. Comparar os resultados de Injeção Intracitoplasmática de espermatozóides (ICSI) entre os oócitos em telófase I e metáfase II, e entre aqueles com e sem fuso celular visível, nos grupos analisados. Metodologia: Estudo prospectivo e controlado com pacientes inférteis, submetidas à estimulação ovariana para realização de ICSI, selecionadas consecutivamente e divididas em dois grupos: Controle (fator tubário e/ou masculino) e Endometriose (subdividido em endometriose mínima e leve I/II versus moderada e severa III/IV). Os oócitos com extrusão do primeiro CP foram avaliados pela microscopia de polarização imediatamente antes da realização da ICSI e caracterizados quanto à presença/localização do fuso celular em relação ao primeiro CP e ao estágio de maturação nuclear (telófase I ou metáfase II). Foram analisados as taxas de fertilização, clivagem, número de embriões de boa qualidade no segundo (D2) e terceiro (D3) dia de desenvolvimento oriundos dos oócitos em telófase I versus metáfase II, e metáfase II com fuso visível versus sem fuso visível, nos grupos controle, endometriose, endometriose I/II e endometriose III/IV. Resultados: Foram analisados 441 oócitos, sendo 254 do grupo controle e 187 do grupo endometriose (115 do grupo endometriose I/II e 72 do grupo endometriose III/IV). Não observamos diferença significativa entre a percentagem de oócitos em metáfase II com fuso celular visível e não visível (88,6%, 91,3%, 88,2%, respectivamente, nos grupos controle, endometriose I/II e endometriose III/IV) e entre a percentagem de oócitos com fuso celular nas diferentes localizações nos grupos avaliados. Entre os oócitos aparentemente maduros, observamos um aumento significativo de oócitos em telófase I no grupo endometriose III/IV (5,6%) quando comparado ao grupo endometriose I/II (0%). Observamos uma tendência a menores taxas de fertilização dos oócitos injetados em telófase I quando comparados aos em metáfase II, nos grupos controle (p=0,08), endometriose (p=0,05) e endometriose III/IV (p=0,09). Comparando-se os oócitos com e sem fuso celular visível, não observamos diferença significativa nos resultados de ICSI entre os grupos analisados. Conclusão: Não observamos diferença significativa entre os grupos analisados quanto à visualização e localização do fuso celular em oócitos maturados in vivo com o primeiro CP visível. Todavia, observamos um aumento significativo de oócitos em telófase I nas portadoras de endometriose moderada e severa, sugerindo um retardo ou comprometimento na conclusão da meiose I. Considerando que os oócitos injetados em telófase I apresentam piores taxas de fertilização do que os injetados em metáfase II, este achado poderia justificar o comprometimento dos resultados de reprodução assistida em mulheres inférteis com endometriose moderada e severa, além de ser utilizado com ferramenta prognóstica pós-ICSI. / Introduction: Although it has been a controversial issue for decades, a deleterious role of endometriosis on assisted reproductive techniques (ART) outcomes is questioned, which may be related to oocyte quality. For a mature oocyte be prepared for fertilization is necessary that the meiotic spindle keeps its integrity and its function. Objectives: To compare the presence and localization of the meiotic spindle and the oocyte nuclear maturation with the visible first polar body of infertile patients with and without endometriosis. To compare ICSI outcomes between oocytes on telophase I and metaphase II, and the ones with and without visible meiotic spindle, on those two groups. Methodology: A prospective and controlled study with infertile patients who underwent ovarian stimulation for purposes of ICSI, selected consecutively and divided into two groups: control (tubal and/or male factor) and endometriosis (subdivided in minimum and mild stage I/II versus moderate and severe stage III/IV). The oocytes with the first polar body extruded (in vivo matured oocytes) were imaged using a polarization microscopy immediately before ICSI and characterized according to the presence and localization of meiotic spindle and its relation to the first polar body and the nuclear maturation stage (telophase I and metaphase II). We have analyzed the fertilization rates, clivage, number of good quality embryos on the second (D2) and third (D3) day of development from oocytes on telophase I versus the ones on metaphase II, and metaphase II visible spindle versus the non-visible ones, on the control groups, endometriosis, endometriosis stage I/II and endometriosis stage III/IV. Results: A total of 441 oocytes were analyzed, 254 oocytes form the control group and 187 from the endometriosis one (115 from endometriosis stage I/II and 72 from endometriosis stage III/IV). No significant differences between the percentage of metaphase II with visible and non-visible meiotic spindle were found (88,6%, 91,3%, and 88,2%, in the control, endometriosis I/II and endometriosis III/IV groups, respectively). Among the apparently matured oocytes, we have observed a significant increase of oocytes on telophase I on the endometriosis III/IV group (5,6%) when compared with the endometriosis I/II group (0%). We have observed a tendency to fewer fertilization rates from the injected oocytes on telophase I when compared with the ones on metaphase II, on the control group (p=0,08), endometriosis (p=0,05) and endometriosis III/IV group (p=0,09). When we compared oocytes with and without visible meiotic spindle, we found no significant difference on ICSI outcomes among the studied groups. Conclusions: We have found no significant difference among the studied groups regarding the visualization and localization of the meiotic spindle from in vivo matured oocytes with a visible first polar body. However, we have observed a significant increase on the number of oocytes on telophase I from patients with moderate and severe endometriosis, suggesting a delay or an impairment in the completion of meiosis I. Since the injected oocytes on telophase I present a worse fertilization rates than the ones injected on metaphase II, this finding could explain the impairment on the outcomes of ART in infertile women with moderate and severe endometriosis, besides it could be used as a prognosis tool after ICSI procedures.