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.

The purpose of this thesis was to study mitosis in Drosophila, from a network biology perspective. The primary aim was to develop and test a network-based prediction model that could integrate available data in public databases (like Flybase) and, based on that, predict potential mitotic proteins. The approach taken to design the protein interaction network included the use of a priori knowledge about the microtubule composition of the mitotic spindle and the higher likelihood of microtubule-associated proteins (MAPs) to have a putative mitotic function. The design also included the integration of different complementary datasets, from gene expression and functional RNAi screens to cross species conservation of MAPs for fitting a network-based model for predicting mitotic proteins. I begin with the creation of the MAP interactome based on a MAP dataset in Drosophila. This initial network was extended by transferring homologs and interologues of MAP datasets from four other species, i.e. human, mouse, rat and Arabidopsis. These proteins were then used as seed proteins to conduct a virtual pull-down experiment, by adding indirect interactors into the network, i.e. proteins that directly bind to two or more MAPs within the network, which completed the MAP interactome. Data from genome-wide studies in Drosophila were gathered for each node in the MAP interactome. These ‘layers’ of data were then used as features to fit a prediction model that could score each node in the network, based on the likelihood of its role in mitosis. The final model performed with 96% accuracy after 10-fold cross validation and was used to rank all the proteins in the MAP interactome. By analysing the top 100 high scoring predicted mitotic proteins, a highly connected cluster of 33 proteins was identified that was subject to experimental validation in the lab. The first approach was to conduct an in vitro analysis using an RNAi screen to test for any spindle, chromosome or centrosome phenotypes upon gene knockdown. After two independent RNAi screens, around 80% of the proteins produced mutant mitotic phenotypes strongly supporting the results of the MAP prediction model. The second approach was to conduct an in vivo analysis by expressing GFP- fusion constructs of selected genes from the subcluster. These were expressed in Drosophila early embryos to study their subcellular localization during interphase and mitosis. A variety of localizations were observed ranging from chromatin and microtubules to more generic cytoplasmic localizations. These results suggested not all predicted proteins were co-localizing with microtubules, and therefore might not necessarily be microtubule associated proteins but can possibly be functioning as microtubule associated regulator proteins. Proteomics analysis of a subset of these genes showed a large proportion of false positive interactions but also picked new interactions between member proteins that highlighted a module within the subcluster. The RNAi hits from the in vitro analysis and the members of the module within subcluster-16 from the in vivo analysis provide interesting subjects for further characterization.

Les centrosomes dont le rôle principal est d’organiser le cytosquelette de microtubules et le fuseau mitotique servent aussi de sites d’interaction pour plusieurs protéines régulatrices du cycle cellulaire et de la réponse aux dommages à l’ADN. Une de ces protéines est la kinase CHK2 et plusieurs publications montrent une sous-population de CHK2 localisée aux centrosomes dans les cellules en interphase et en mitose. Toutefois, la localisation de CHK2 aux centrosomes demeure controversée, car des doutes subsistent en ce qui concerne la spécificité des anticorps utilisés en immunocytochimie. En utilisant des lignées cellulaires du cancer du côlon, les cellules HCT116 sauvages et HCT116 CHK2-/- ainsi que différentes lignées d’ostéosarcome humain dans lesquelles l’expression de CHK2 a été inhibée par ARN interférence, nous montrons que les anticorps anti-CHK2 qui donnent un signal centrosomal sont non spécifiques et reconnaissent un antigène inconnu sur les centrosomes. Cependant, par des expériences d’immunofluorescence réalisées avec des cellules U2OS qui expriment les protéines de fusion GFP-CHK2 ou FLAG-CHK2, nous révélons une localisation centrosomale de CHK2 dans les cellules en mitose, mais pas en interphase. Ce résultat a été confirmé par vidéomicroscopie dans les cellules vivantes exprimant GFP-CHK2. Pour déterminer le ou les rôles potentiels de CHK2 en mitose nous avons réalisé des expériences pour explorer le rôle de CHK2 dans la progression de la mitose, la nucléation des microtubules aux centrosomes et la progression de la mitose en présence de problèmes d’attachement des chromosomes où de lésions génotoxiques. Nos données suggèrent que CHK2 n’est pas impliquée dans la régulation de la mitose dans les cellules U2OS. / Centrosomes function primarily as microtubule-organizing centres and play a crucial role during mitosis by organizing the bipolar spindle. In addition to this function, centrosomes act as reaction centers where numerous key regulators meet to control cell cycle progression. One of these factors involved in genome stability, the checkpoint kinase CHK2, was shown to localize at centrosomes throughout the cell cycle. Here, we clarify that CHK2 only localized at centrosomes during mitosis. Using wild-type and CHK2-/- HCT116 human colon cancer cells, or human osteosarcoma U2OS cells depleted for CHK2 with small hairpin RNAs, we show that several CHK2 antibodies are non-specific for immunofluorescence and cross-react with an unknown centrosomal protein(s). To analyse further CHK2 localization, we established cells expressing inducible GFP-CHK2 and Flag-CHK2 fusion proteins. We show that CHK2 localizes to the nucleus in interphase cells but that a fraction of CHK2 associates with centrosomes in mitotic cells, from early mitotic stages until cytokinesis. In contrast to previous data obtained by A. Stolz and colleagues with the human colon carcinoma HCT116 cell line, our experiments exploring the possible functions for CHK2 during mitosis did not support a role for CHK2 in the bipolar spindle formation and the timely progression of mitosis in human osteosarcoma U2OS cells.

La mitose est une étape essentielle du cycle cellulaire à l’issue de laquelle le génome répliqué de la cellule mère est ségrégé de façon équitable entre les deux cellules filles. Pour cela, la cellule assemble une structure hautement dynamique et composée de microtubules, appelée le fuseau mitotique. En plus d’assurer la bonne ségrégation des chromosomes, le fuseau mitotique détermine l’axe de division, un phénomène particulièrement important pour la division asymétrique où des déterminants d’identité cellulaire doivent être distribués de façon inéquitable entre les deux cellules filles. L’assemblage et la dynamique de ce fuseau sont finement régulés par de nombreuses protéines qui sont associées aux microtubules. Au cour de ma thèse, nous avons identifié 855 protéines constituant l’interactome des microtubules de l’embryon de Drosophile par spectrométrie de masse puis criblé par ARNi 96 gènes peu caractérisés pour un rôle en mitose dans le système nerveux central larvaire. Par cette approche, nous avons identifié 18 candidats sur la base de leur interaction aux microtubules et de leur phénotype mitotique, dont Ensconsine/MAP7. Nous avons montré qu’Ensconsine est capable de s’associer aux microtubules du fuseau et favorise leur polymérisation. De plus, les neuroblastes des larves mutantes présentent des fuseaux raccourcis et une durée de mitose prolongée. Ce délai en mitose est dû à une activation prolongée du point de contrôle du fuseau mitotique qui est essentiel pour une ségrégation correcte des chromosomes en l’absence d’Ensconsine. D’autres part, en association avec la Kinésine-1, son partenaire fonctionnel en interphase, nous avons montré qu’Ensconsine est également impliquée dans la séparation des centrosomes au cours de l’interphase. Ceci entraine une distribution aléatoire des centrosomes pères et fils dans cellules filles. Grâce à cette étude, nous avons révélé deux nouvelles fonctions pour Ensconsine : elle favorise la polymérisation des microtubules et participe donc à l’assemblage du fuseau mitotique et est impliquée, avec la Kinésine-1 dans la dynamique des centrosomes. / Mitosis is a key step of the cell cycle that allows the mother cell to segregate its replicated genome equally into the two daughter cells. To do so, the cell assembles a highly dynamic structure composed of microtubules called the mitotic spindle. Additionally to its role in the faithful segregation of chromosomes, the mitotic spindle defines the axis of cell division. This phenomenon is particularly important for the asymmetric cell division in which cell fate determinants have to be unequally distributed between the two daughter cells. Spindle assembly and dynamics are subtly regulated by numerous microtubules-associated proteins. During my PhD, we identified using mass spectrometry, 855 proteins establishing the Drosophila embryo microtubule interactome. An RNAi screen was performed in the larval central nervous system for 96 poorly described genes, in order to identify new mitotic regulators. Based on microtubule interaction and mitotic phenotype, among 18 candidates we focused on Ensconsin/MAP7. We have shown that Ensconsin is associated with spindle microtubules and promotes their polymerization. Neuroblasts from mutant larvae display shorter spindles and a longer mitosis duration. This mitotic delay is a consequence of an extended activation of the spindle assembly checkpoint, which is essential for the proper chromosome segregation in the absence of Ensconsin. This study also showed that, in association with its interphase partner Kinesin-1, Ensconsin is involved in centrosome separation during interphase. As a result, mother and daughter centrosomes are randomly distributed between the daughter cells. In conclusion, we highlighted two news functions of Ensconsin : first, this protein promotes microtubule polymerization and is involved in spindle assembly ; second, Ensconsin and its partner Kinesin-1 regulate centrosome dynamics.

A maioria dos tumores sólidos apresentam características aneuplóides. Porém a relação entre aneuploidia e transformação maligna, ainda não está definida. Nos últimos anos diversas proteínas têm sido descritas como reguladoras de eventos durante a divisão celular, principalmente as relacionadas com a formação do fuso bipolar e segregação equacional dos cromossomos. Neste estudo propomo-nos a analisar os efeitos da interferência em dois pontos críticos da mitose, a segregação cromossômica e a citocinese, em relação à aneuploidia e à instabilidade genética tumoral. Nossos dados mostraram que o tratamento sequencial de Monastrol e Blebistatina determinou o surgimento de fusos mitóticos anormais, amplificação centrossômica, localização ectópica de Aurora A e aumento de micronúcleos. Esta interferência pode levar a um quadro de instabilidade genética e, consequentemente a progressão tumoral, abrindo novas possibilidades para o estudo dos mecanismos moleculares envolvidos na regulação do ponto de checagem mitótico e resistência a quimioterápicos. / Most solid tumors have aneuploid feature. Therefore the relationship between aneuploidy and malignant transformation is not yet understood. In the last years it has been described many proteins involved in regulation of mitosis, mainly those related to bipolar spindle and chromosome segregation. In this work we propose to study the effects of the interference on two mitotic critical points, the chromosome segregation and cytokinesis, in relation to aneuploidy and genetic tumor instability. Our data showed that sequential treatment with Monastrol and Blebbistatin led to abnormal mitotic spindle, centrosome amplification, Aurora A ectopic and micronucleus increased. This interference can lead to genetic instability and may be involved in a tumor progression, opening news possibilities to study the molecular mechanisms involved in regulation the checkpoint mitotic and resistance to chemotherapy found in genetically unstable cells.

Une réponse rapide et adaptée est nécessaire à la survie des cellules soumises à un stress. La réponse cellulaire au stress (HSR pour Heat-Shock response) médié par le facteur de transcription HSF1 est induite par les contextes environnementaux (chaleur, hypoxie, …) et les processus biologiques normaux et pathologiques (vieillissement, inflammation, …) associés à une accumulation de protéines endommagées (Morimoto, 1998). Ces protéines endommagées forment des agrégats toxiques aux conséquences létales pour les cellules.Conservé chez tous les eucaryotes, HSF1 orchestre les actions nécessaires à la survie et à la croissance des cellules malgré le stress. Ses cibles les mieux connues sont les gènes codants pour les Heat Shock Protein (HSP) qui font office de chaperon moléculaire. Une caractéristique de la HSR chez l’Homme est l’accumulation massive du facteur HSF1 en foyers nucléaires nommés Nuclear Stress Bodies (nSBs). Curieusement, ces foyers ciblent l’hétérochromatine péricentrique composée de séquences répétées en tandem de type Satellite III (SATIII), particulièrement au niveau du locus 9q12. HSF1 induit une forte transcription en ARN SATIII Sens (Jolly et al., 2004). Le rôle des nSBs est une des problématiques majeures de notre équipe cependant jusqu’à présent aucune fonction n’a été confirmée pour ces structures.Les nSBs, spécifiques aux cellules humaines, n’ont été décrits que dans des cellules en culture. Mon projet de thèse a consisté dans un premier temps à montrer la présence des nSBs in vivo chez l’Homme. Cette étude, réalisée sur du tissu testiculaire nous a également permis d’identifier une nouvelle cible SATIII majeure pour HSF1, la région Yq12. Dans les testicules, les nSBs sont associés à des processus méiotiques et post-méiotiques, suggérant un rôle dans le remodelage de l’hétérochromatine. Dans un deuxième temps, nous avons cherché à mieux comprendre le rôle des nSBs lors de la HSR. Nous avons pu montrer que l’étape de transcription des SATIII induit une déstabilisation de l’hétérochromatine péricentrique caractérisée par une dissociation des facteurs HP1 (Heterochromatin Protein 1) alpha et beta et une perte de la marque répressive H3K9me3. Au cours de la période de récupération qui accompagne la reformation de l’hétérochromatine, une transcription séquentielle d’ARN SATIII Sens puis Anti-sens précède la restructuration des loci 9q12. Nous avons également pu montrer que la transcription des SATIII est associée à un blocage de la mitose. Nous montrons que dans les cellules stressées, une altération de ce point de contrôle par un Knock down des ARN sat III par des approches LNA conduisent à une l’instabilité génomique des cellules tumorales avec apparition de cellules polynucléées. / A rapid and well-adapted response is required for cell survival upon stress. The cellular stress response (HSR) is mediated by the transcription factor Heat Shock Factor 1 (HSF1) (Morimoto, 1998). It is activated by environmental stress (heat, hypoxia, ...) and by a series of patho-physiological contexts (aging, inflammation, ...) involving protein damages.The best-characterized targets of HSF1 are genes encoding for Heat Shock Protein (HSP) acting as molecular chaperone. A specific feature of the HSR in human cells is the presence of HSF1 nuclear foci named Nuclear Stress Bodies (NSBs). Surprisingly, nSBs target pericentric heterochromatin consisting in tandem repeats of type III Satellite (SATIII) sequences, primarily at the 9q12 locus. HSF1 triggers a strong transcriptional activation of this locus (Jolly et al., 2004). The role of nSBS is a major issue since no function related to these structures has been reported so far.So far, nSBs have been only identified in cells in culture. My thesis project has been to further explore whether these structures also existed in normal tissues. Indeed, we have been able to identify the presence of nSBs in testis where they were found to be associated to meiotic and post-meiotic stages, suggesting a role related to heterochromatin remodeling. Moreover, we have identified the Yq12 locus as a new target of nSBs in these tissues. Secondly, we have brought new evidence that sat III sequences triggers a transient dissociation of HP1 (heterochromatin Protein 1) α and β as well as a loss of the repressive epigenetic H3K9me3 histone mark at pericentric heterochromatin. Interestingly we have also found that, following stress, a sequential accumulation of SATIII RNA in a Sense and Antisense orientation occurs, suggesting that this specific pattern of expression plays an important role in heterochromatin reformation. Finally, we have found that the accumulation of SATIII RNA is associated with a slowdown of mitosis. Indeed we have found that in stressed cells, accumulation of sat III impcats the progression of mitosis and that a knock down of sat III RNA using LNA approaches releases this blockade, leading to genomic instability of tumor cells and to the appearance of poly nucleated cells.

La Liver Kinase B1 (LKB1) est une protéine pléiotrope, impliquée dans divers processus biologiques. Dans le foie, LKB1 est notamment connue pour être un régulateur clé du métabolisme et de la polarité cellulaire. Au cours de notre étude, nous avons investigué l’implication de LKB1 dans le contrôle de la prolifération des hépatocytes au cours du processus de régénération hépatique physiologique (hépatectomie partielle des 2/3). Nous avons démontré que la perte de Lkb1, spécifiquement dans les hépatocytes, favorise la récupération de la masse hépatique après hépatectomie partielle, en induisant une augmentation drastique de la réponse proliférative hépatocytaire, indépendamment de la balance métabolique/énergétique. Ainsi, LKB1 agit comme un senseur négatif de la prolifération et régule la transition G0/G1, en particulier en contrôlant la signalisation de l’EGFR (Epidermal Growth Factor Receptor). Par ailleurs, plus tard pendant la régénération, LKB1 garantit également l’intégrité mitotique. En effet, la suppression de Lkb1 entraîne des altérations majeures de la formation du fuseau mitotique. Nos résultats établissent également que LKB1 contrôle la polarité de la division cellulaire, indépendamment de l'activité de l’AMPK (AMP-activated protein kinase), une cible clé de LKB1. Par conséquent, la perte de LKB1 conduit à une altération majeure du profil de ploïdie, au stade tardif du processus de régénération. L’ensemble de notre étude souligne le double rôle de LKB1, au cours de la régénération hépatique, en tant que gardien de la prolifération hépatocytaire et de l'intégrité génomique. / Liver Kinase B1 (LKB1) is involved in pleiotropic biological processes and known to be a key regulator of hepatic metabolism and polarity. Here, we investigated the contribution of LKB1 in hepatocyte proliferation and liver regeneration process. We demonstrated that loss of hepatic Lkb1 promotes liver mass recovery, through an increase of hepatocytes proliferation, independently on metabolic/energetic balance. LKB1 regulates G0/G1 progression, specifically by controlling Epidermal Growth Factor Receptor (EGFR) signaling. In addition, later during regeneration, LKB1 controls mitotic fidelity. Deletion of Lkb1 results in major alterations of mitotic spindle formation, along the polarity axis, independently of AMP- activated protein kinase (AMPK) activity, a key target of LKB1. Consequently, LKB1 deficiency leads to an alteration of ploidy profile, at late stage of regenerative process. Overall our study highlights the dual role of LKB1, during liver regeneration, as a guardian of hepatocyte proliferation and genomic integrity.

The trimorphic fungus Candida albicans is the leading cause of systemic candidiasis, a disease with poor prognosis affecting immunocompromised patients. The capacity to switch between growth morphologies is tightly coupled to its ability to cause life-threatening infection. Recently, the molecular chaperone Heat Shock Protein 90 (Hsp90) has been implicated as a major regulator of C. albicans morphogenesis via the Ras1-PKA pathway. In model organisms from plant, animal and fungal kingdoms, Hsp90 stabilizes regulators of cell signaling and participates in many important cellular processes. Hsp90’s roles in C. albicans are beginning to be dissected. This thesis represents a comprehensive overview of the morphological response of C. albicans to compromised Hsp90 function, illuminating previously unidentified roles for this chaperone in cell cycle progression, cytokinesis and vacuole maintenance. This work sheds light on the importance of Hsp90 in fungal development and the therapeutic potential of Hsp90 inhibitors in the treatment of fungal infections.

The trimorphic fungus Candida albicans is the leading cause of systemic candidiasis, a disease with poor prognosis affecting immunocompromised patients. The capacity to switch between growth morphologies is tightly coupled to its ability to cause life-threatening infection. Recently, the molecular chaperone Heat Shock Protein 90 (Hsp90) has been implicated as a major regulator of C. albicans morphogenesis via the Ras1-PKA pathway. In model organisms from plant, animal and fungal kingdoms, Hsp90 stabilizes regulators of cell signaling and participates in many important cellular processes. Hsp90’s roles in C. albicans are beginning to be dissected. This thesis represents a comprehensive overview of the morphological response of C. albicans to compromised Hsp90 function, illuminating previously unidentified roles for this chaperone in cell cycle progression, cytokinesis and vacuole maintenance. This work sheds light on the importance of Hsp90 in fungal development and the therapeutic potential of Hsp90 inhibitors in the treatment of fungal infections.

This thesis project is focused on the study of the signaling module formed by the NIMA-related protein Nek6, Nek7, and Nek9 and their function during early mitosis, with particular interest in centrosome separation and maturation. Nek9/Nercc1 was identified by Dr. Joan Roig. Nek9 is expressed in all cell lines and tissues studied is inactive during interphase while during mitosis is activated through phosphorylation by Plk1 which is in fact able to bind Nek9 and subsequently phosphorylates Nek9 on its activation loop. During mitosis Nek6 and Nek7 bind the C-terminal of Nek9. Once active, Nek9 can phosphorylate Nek6 and Nek7, thus activating them. Active Nek9 localizes at centrosome, suggesting that Nek9/Nek6-7 has important functions in the organization of microtubules during cell division. Confirming this idea, it has been shown that the microinjection of anti-Nek9 module induces arrest in prometaphase with disorganized spindle structures and misaligned chromosomes, or leads to abnormal mitosis resulting in aneuploidy. In the same direction, interference with the function of Nek7 or Nek6 leads to abnormal mitotic progression and spindle formation. We described how the Nek9/Nek6-7 module could provide a link connecting Plk1 and Eg5 in the context of centrosome separation. we analyzed the effects of Plk1, Eg5, Nek9, Nek6 or Nek7 down-regulation by RNAi on the extent of separation of duplicated centrosomes in prophase cells and we observed how this downregulation was affecting centrosome separation. We determine whether the activation of Nek9 or Nek6 could induce centrosome separation trasfecting cells with the active form of these two kinases; a considerable amount of cells that were in interphase shown separate centrosome demonstrating that Nek9/Nek6 are sufficient to induce centrosome separation. To test whether active Nek9 and Nek6 exerted their effect through the regulation of Eg5 we simultaneously transfected the cells with Eg5 siRNAs and we completely lost the centrosome separation described above. We demonstrated by immunofluorescence that the key event during centrosome separation was the recruitment of Eg5 at centrosomes and that the down-regulation of Plk1, Nek6, Nek7 or Nek9 resulted in prophase cells with unseparated centrosomes because Eg5 was not properly recruited. To prove whether the phosphorylation on Ser-1033 controls the accumulation of Eg5 to centrosomes and centrosome separation during early mitosis we transfected cells with wild type Eg5 or Eg5 S1033A; the wild type form of the kinesin was able to localize at centrosome and rescue the normal phenotype while Eg5 S1033A was not able to localize and resulted in cells delayed in mitosis. Plk1, the Nek9 activator, is involved in the regulation of centrosome maturation during early mitosis. Centrosome maturation refers to the process through which centrosomes increase size and microtubule nucleation activity and requires the accumulation of γ-TuRC complexes at centrosome. This recruitment depends on Nedd1 that acts as γ-Tubulin targeting factor. Plk1 depletion prevents accumulation of Nedd1 at centrosome. Our experiments show the importance of Nek9 in the regulation of centrosome maturation downstream of Plk1. Depletion of Nek9 by siRNA determined a decrease of γ-Tubulin and Nedd1 at centrosome. Further we investigated the upstream role of Plk1 depleting Plk1 and trasfecting active Nek9 and it was able to rescue the normal phenotype. Nek9 can interact with Nedd1 during mitosis and phosphorylates it provoking its accumulation at centrosome. The no-phosphorylable form of Nedd1 was not able to accumulate at centrosome and support the accumulation of γ-Tubulin there, determining a delay of the cells in prometaphase. Our results show that Nek9 is the link between Plk1 activity and the recruitment of Nedd1 to the centrosome and that the pathway formed by Plk1/Nek9/Nedd1 can be a key element in the control of mitotic centrosome maturation.