Aperçu du contrôle de la dégradation des ARNm par les protéines YTHpendant la transition de la méiose à la mitose chez les levures.Le cycle cellulaire est contrôlé par des processus complexes et interconnectés. Un gène est transcrit en ARNm qui est traduit en protéines mais de nombreux processus de régulation travaillent pour contrôler chaque étape de ce processus apparemment simple. Parmi ces points de contrôle, la régulation post-transcriptionnelle est importante, et la formation d'un complexe protéine-ARN peut diriger le destin cellulaire. Parmi ces protéines de liaison à l'ARN, les protéines contenant des domaines YTH n’ont été découvertes qu’à la fin des années 90. Les protéines contenant des domaines YTH sont abondantes chez les eucaryotes et absentes chez les procaryotes. Elles constituent la majorité des protéines « readers » capables de reconnaître spécifiquement la modification m6A. L’Homme possède cinq protéines YTH, YTHDF1-3, YTHDC1,2 (Hazra, D., C. Chapat, et Graille, M. (2019). Destin de l'ARNm de m6A : enchaînés au rythme par les protéines contenant de la YTH. , 10 (1), 49.). Bien qu'il soit évident que ces protéines contrôlent le destin cellulaire, la fonction de chaque protéine et son réseau d’interaction restent à élucider. Chez les levures, une seule protéine YTH est présente: Pho92 chez Saccharomyces cerevisiae et Mmi1 chez Schizosaccharomyces pombe. Hormis le domaine YTH, il n'y a pas d'homologie de séquence entre ces deux protéines mais leur fonction cellulaire est similaire.Il est bien établi que Mmi1 est responsable de la dégradation des transcrits spécifiques de la méiose au cours de la croissance végétative des cellules chez la levure S. pombe. Mmi1 forme un complexe stable avec une petite protéine, Erh1 (complexe Erh1-Mmi1 ou EMC). Le complexe EMC peut physiquement interagir avec la sous-unité Not1 du complexe CCR4-Not et la recruter pour la dégradation des ARNm contenant des motifs DSR (déterminant de l'élimination sélective). L'action de Mmi1 est à son tour régulée par une protéine possédant un domaine RRM, Mei2. Au cours de la méiose, Mei2, avec l’aide d’un lncRNA meiRNA, séquestre Mmi1 dans un point nucléaire, le rendant inactif et assurant la continuité de la méiose. Ces trois protéines, Mmi1-Erh1-Mei2, jouent un rôle clé dans la transition de la mitose vers la méiose.Chez S. cerevisiae, Pho92 est impliquée dans la dégradation des transcrits de PHO4, contribuant à la voie du métabolisme du phosphate, pendant la privation en phosphate et participe également à la dégradation des ARNm contenant les marques épitranscriptomiques de N6-méthyladénosine (m6A). Comme pour S. pombe Mmi1, Pho92 recrute le complexe CCR4-Not via une interaction physique avec Not1.Au cours de ma thèse, j'ai tenté d'élucider le rôle de ces deux protéines du domaine YTH de deux organismes modèles, S. cerevisiae et S. pombe, dans la dégradation de l'ARNm et la régulation du cycle cellulaire par des approches biochimiques et structurales.Pho92 de S. cerevisiae interagit physiquement avec Not1 du complexe CCR4-Not, nous avons pu déterminer les limites des domaines impliqués dans cette interaction. L’interaction entre ces deux protéines a été étudiée par anisotropie de fluorescence. Le complexe protéique a été purifié avec succès et des essais de cristallisation sont en cours.Chez S. pombe, la structure de Mei2-RRM3 a été résolue avec et sans ARN. Les propriétés de liaison à l'ARN de Mei2-RRM3 ont été étudiées par ITC. La structure de Erh1 a également été résolue révélant une organisation en homodimere. Nous avons montré que la formation de cet homodimere est important pour la fonction biologique de Mmi1. Des essais de co-cristallisation ont été réalisés avec de l'ARN et les protéines Mmi1 et Mei2, mais sans succès et nous avons obtenu des cristaux de Mmi1. / Insights into the control of mRNA decay by YTH proteinsduring the transition from meiosis to mitosis in yeasts.Keywords: Epitranscriptomics, mRNA decay, meiosis, multi-protein complexes, YTH domainCell cycle is controlled by multi-layered processes. A gene is transcribed in mRNA which is translated in proteins but innumerable regulation processes are working to control every step of this apparently simple process. Among these regulatory check points, post-transcriptional regulation is an important one, where formation of a protein-RNA complex may direct the cellular fate. Among these RNA binding proteins, YTH domain proteins are most novel, discovered in late 90s. YTH domain proteins are abundant in eukaryotes and absent in prokaryotes. YTH domain proteins constitute the majority of reader proteins that can specifically identify m6A modification. Human beings have five YTH domain proteins YTHDF1-3, YTHDC1-2 (Hazra, D., Chapat, C., & Graille, M. (2019). m6A mRNA Destiny: Chained to the rhYTHm by the YTH-Containing Proteins. Genes, 10(1), 49.). Although it is evident that these proteins are controlling cellular fate, the function of each protein and their network is yet to be elucidated. In yeast, there is only one YTH domain protein present: Pho92 in Saccharomyces cerevisiae and Mmi1 in Schizosaccharomyces pombe. Apart from the YTH domain there is no sequence homology between these two proteins but their cellular function is similar.It is well established that Mmi1 is responsible for degradation of meiosis specific transcripts during vegetative growth of the cell. Mmi1 forms a tight complex with a small protein, Erh1 (Erh1-Mmi1 complex or EMC). EMC can physically interact with Not1 of CCR4-Not complex and recruit it for degradation of DSR (determinant of selective removal) containing RNAs. The action of Mmi1 is in turn regulated by an RRM domain protein, Mei2. During meiosis, Mei2, along with a lncRNA meiRNA sequesters Mmi1 in a nuclear dot, rendering it inactive and ensuring smooth continuance of meiosis. These three proteins, Mmi1-Erh1-Mei2 play a key role in mitosis to meiosis switch.In S. cerevisiae, Pho92 is involved in the degradation of PHO4 transcripts contributing to phosphate metabolism pathway, during phosphate starvation and also participates in the degradation of mRNAs containing the N6-methyladenosine (m6A) epitranscriptomics marks. Similarly, to S. pombe Mmi1, Pho92 recruits CCR4-Not complex by physical interaction with Not1.During my PhD, I have tried to elucidate the role of these two YTH domain proteins from two model organisms, S. cerevisiae and S. pombe, in mRNA degradation and cell cycle regulation using biochemical and structural approaches.Pho92 of S. cerevisiae physically interacts with Not1 of CCR4-Not complex, we were able to determine the boundaries of this interaction. The interaction between these two proteins was studied by Fluorescence anisotropy. The protein complex was successfully purified and crystallization trials are ongoing.From S. pombe, structure of Mei2-RRM3 was solved with and without an RNA. RNA binding properties of Mei2-RRM3 was studied by ITC. The structure of Erh1 was also solved and we tried to elucidate its importance for biological function of Mmi1. A co-crystallization trial was performed with Mmi1-Mei2-RNA but it was unsuccessful and we ended up with Mmi1 crystals.

Hybrid sterility is one of the reproductive isolation mechanisms restricting gene flow between the related species and leading to speciation. PR domain containing 9 (Prdm9), the only known vertebrate hybrid sterility gene, determines the sites of programmed DNA double-strand breaks (DSBs) and thus specifies hotspots of meiotic recombination but in hybrids between two mouse subspecies causes failure of meiotic chromosome synapsis and hybrid male sterility. In the present study on sterile hybrids, the five smallest autosomes were more prone to asynapsis. To manipulate with the synapsis rate, random stretches of consubspecific homology were inserted into several autosomal pairs. Twenty seven or more megabases of consubspecific sequence fully restore synapsis in a given autosome. Further, at least two symetric DN double-strand breaks per chromosome were necessary for successful synapsis. Moreover, F1 hybrids had sperm when synapsis was rescued in at least three of four segregating chromosomes. To verify the assumption of a lack of symmetric DSBs in meiotic chromosomes of sterile males the chemotherapeutic drug cisplatin was used to induce exogenous DNA DSBs. Cells treated with 5 mg/kg and 10 mg/kg of cisplatin showed increased number of DSBs monitored by immunostaining of RPA and DMC1 sites and...

The production of germ cells is an essential process in all sexually reproducing eukaryotes. During male meiosis, four haploid sperm cells are formed from one primary spermatocyte, thereby undergoing two consecutive cell divisions after only one round of chromosome duplication. This process was studied in the nematode Caenorhabditis elegans, as this model organism offers a number of experimental advantages to simultaneously analyze spindle dynamics and ultrastructure. The worm is easy to cultivate, completely sequenced and numerous mutants are available, the worm is small and thus ideal for light and electron microscopic investigations, and the transparent body allows live-cell imaging within living animals. Importantly, meiotic spindles in C. elegans males are organized by centrosomes and show a lagging X-chromosome, which is always segregated after the autosomes have been partitioned to the newly forming secondary spermatocytes. The aim of this thesis was to systematically investigate this characteristic feature of chromosome segregation in male meiotic spindles. For that, spindle dynamics in the first and second meiotic division was analyzed with fluorescence light microscopy. Furthermore, the spindle ultrastructure was investigated in spindles of various stages of meiosis I using electron tomography. Light microscopy revealed a shortening of the distance between centrosomes and chromosomes (anaphase A) and an increase in the pole-to-pole distance (anaphase B). Moreover, spindles in male meiosis I and II showed differences in certain aspects of spindle dynamics. In addition it was demonstrated that spindles in metaphase II in the presence of a single X-chromosome were shorter compared to spindles without the X-chromosome. In addition, it was found that the process of aging had an impact on spindle length in both metaphase I and II. By manipulating the number of unpaired chromosomes, it could be demonstrated that the lagging behavior of univalent chromosomes is caused by the incapability of pairing in meiotic prophase. After performing a quantitative analysis of the light microscopic data it was further shown that a dynamic microtubule bundle is connecting the X-chromosome to the spindle poles. Using laser microsurgery it could be demonstrated that this bundle exerts a pulling force to the univalent chromosome throughout anaphase. Unexpectedly, electron tomography showed that anaphase-type movements of the autosomes were not accompanied by a shortening of the kinetochore microtubules. Instead, three findings indicated a shortening of the centrosome-chromosome distance itself: (1) upon anaphase onset, tension is released on the beforehand stretched autosomes; (2) centrosomes shrink in preparation for meiosis II and (3) the attachment angle of end-on microtubules changes. Interestingly, microtubules connecting the X-chromosome to the spindle poles showed a high curvature around the kinetochore region of the X-chromosome, suggesting an involvement of motor proteins in the process of segregation. Taken together, this thesis gives the first detailed quantitative analysis of spindle dynamics and architecture during male meiosis in the nematode C. elegans. This wild-type data will serve as a basis for future mutant analyses and should help to further understand the complex dynamic and ultrastructural aspects of spindle organization in the meiotic divisions in C. elegans males.

Over the course of more than two decades, numerous studies have meticulously explored the fundamental roles of the cohesin complex, unraveling its intricate functions in sister chromatid cohesion, DNA recombination and repair, gene expression regulation, telomere protection, and regulatory mechanisms in cell division processes. However, the detailed and multifaceted roles of individual subunits within the cohesin complex during meiosis remain poorly understood. During my PhD, I focused my attention on two principal subunits that complete the tripartite ring: the meiotic isoform of SMC1, namely SMC1β, and a kleisin subfamily protein, RAD21. While RAD21 is the sole kleisin during mitosis, it is accompanied by two other kleisin subfamily proteins, REC8 and RAD21L, during meiosis. From past research, it is known that SMC1β is crucial for telomere protection in both spermatocytes and oocytes. Interestingly, while several major phenotypes of SMC1β-deficient spermatocytes were rescued by SMC1α, telomere abnormalities were not. The expression of telomerase and shelterin components appeared usual in SMC1β-deficient spermatocytes. This study highlights SMC1β's role in safeguarding telomeres at chromosome ends from damage and abnormalities by regulating the expression of a long non-coding RNA transcribed from the subtelomeres of different chromosomes, known as TERRA (Telomeric repeat–containing RNA). TERRA comprises repetitive sequence motifs transcribed from the telomeric DNA strand that is complementary to the DNA sequence of the telomere itself. SMC1β suppresses the expression of TERRA strongly in spermatocytes and mildly in oocytes, increasing the number of foci and intensity at the ends of spermatocyte chromosomes corresponding to visually elevated telomeric damage in the absence of SMC1β. This suggests the strong role of SMC1β in regulating TERRA at chromosome ends. TERRA, with a similar sequence to that of telomeres, has the potential to form RNA-DNA hybrids, often referred to as open R-loops, which may make telomeres more susceptible to damage. This study demonstrates that SMC1β-deficient mice exhibited increased staining for R-loops at both autosomal chromosome ends and sex chromosomes, which was mitigated upon treatment with RNase H endonuclease. In our recent publication, Biswas et al., 2023, it is confirmed that SMC1β helps maintain close chromatin at telomeric ends with support from ATAC sequencing and RNA sequencing data, thereby protecting the chromosome ends. One pertinent question that remains unanswered is what distinguishes SMC1β from SMC1α in maintaining these telomere functions. To address this, we generated a CRISPR-Cas9 mice strain with a deletion of the DNA binding domain at the carboxy terminal of SMC1β, which marks the major difference in sequence between SMC1β and SMC1α. While a reduced stability of these truncated proteins was observed at both RNA and protein levels in spermatocytes, yet it hints that the majority of SMC1β’s functions were abolished upon deletion of this C-tail. Interestingly, these mutant spermatocytes exhibit elevated telomere abnormalities, albeit not to the extent seen in full knockouts. This suggests that the C-terminal tail, along with additional components, participates in protecting telomeres, considering that a minimal level of SMC1β protein is sufficient to maintain telomeres in germ cells. Another objective of my thesis is to emphasize the significance of RAD21 as a kleisin protein in female meiosis. While there is limited research on RAD21 in spermatocytes, it has been described to transiently appear during late prophase I of male meiosis. However, studies on RAD21 in oocytes are lacking. RAD21 exhibits an appearance on the chromosome axis during mid to late pachytene in embryonic oocytes before being depleted in the diplotene stage. As RAD21 is the only kleisin protein in somatic cells, generating a constitutive Rad21 knockout mouse would be lethal. Therefore, using the Cre-LOX system, conditional Rad21 knockout mouse models were created, where RAD21 was specifically eliminated in oocytes at various stages of maturation. Early excision of Rad21 during the embryonic diplotene stage or in pups shortly after birth had a significant impact on ovary development and oocyte count with age, while oocyte sizes were reduced, indicating potential stress or onset of apoptosis. Conversely, late excision of Rad21 in activated germinal vesicle oocytes showed no notable differences in ovary size or oocyte number, and only mild differences in oocyte diameter, underscoring the significant role of RAD21 in the pre metaphase prophase stage. RAD21 does not actively participate in long-term arrest centromeric cohesin protection, chiasma maintenance, or DNA damage repair in heterozygous mice. However, young adult Rad21 conditional knockout mice with early excision exhibit a trend of delayed and less efficient oocyte maturation when exposed to DNA damage. These findings suggest a potential role of RAD21 in nonprogrammed DNA repair before metaphase I, which may ensure chromosome integrity after programmed recombination. Further investigation is necessary to study the mechanism of DNA repair by RAD21 through Homologous Recombination or End Joining pathways. In summary, these studies provide insights into the role of cohesin subunit SMC1β in telomere maintenance through TERRA regulation in spermatocytes, as well as the role of RAD21 in preserving ovarian reserve and oocyte health by potentially contributing to non-programmed DNA damage repair.

Dans l’histoire évolutive des végétaux, la polyploïdisation a été un phénomène récurrent qui a façonné les génomes, aurait contribué à l’avènement de grandes étapes évolutives et aurait favorisé la survie de nombreuses lignées lors de crises écologiques majeures. Le principal mécanisme d’apparition d’espèces polyploïdes est la polyploïdisation sexuelle, qui implique la formation de gamètes 2n résultant de modifications de la division méiotique. Récemment plusieurs mutants produisant des taux élevés de gamètes 2n ont été identifiés chez A. thaliana. Chez cette espèce, la perte de fonction du gène AtPS1 conduit à la mise en place de fuseaux parallèles en méiose II et celle du gène AtCYCA1;2/TAM à l’omission de la seconde division méiotique. L’objectif de cette thèse a été de déterminer des facteurs et mécanismes responsables de la formation de gamètes 2n, en utilisant le rosier comme modèle végétal. Ces travaux ont permis : (i) d’identifier un facteur abiotique, la température élevée, comme inducteur de production de forts taux de gamètes 2n, (ii) de montrer que la fenêtre de sensibilité à ce facteur est restreinte à la méiose et (iii) de révéler que ces gamètes 2n produits sont principalement issus de la mise en place de fuseaux parallèles en méiose II. Afin de déterminer les mécanismes moléculaires à l’origine de leur formation, deux gènes candidats, RhPS1 et RhCYCA1 ont été identifiés chez Rosa. L’analyse de leur expression a révélé : (i) en condition non inductible, leur forte expression dans les étamines au stade méiose et (ii) la répression rapide de leurs niveaux de transcrits en condition d’induction de gamètes 2n. La fonction méiotique du gène RhPS1 a été validée par complémentation du mutant atps1-1 d’A. thaliana et par l’obtention d’une lignée rosier transgénique p35S::ARNi-RhPS1. Compte tenu de ces résultats, l’étude de la polyploïdisation et de ses mécanismes peut désormais être replacée dans le contexte actuel de changement climatique. / In the evolutionary history of plants, polyploidization has been a recurring phenomenon that has shaped the genomes, might have contributed to the occurrence of major evolutionary step and might have facilitated the survival of many plant families during major ecological crises. The main mechanism of polyploidization is sexual polyploidization, which involves the formation of 2n gametes resulting from meiotic division changes. Recently, mutants highly producing 2n gametes have been isolated in A. thaliana. Loss of AtPS1 gene function leads to parallel spindles orientation in meiosis II and loss of AtCYCA1;2/TAM gene function leads to the omission of the second meiotic division. The aim of this PhD project was to identify factors and mechanisms responsible for the 2n gametes formation, using Rosa as a model. This work permitted to: (i) discover an abiotic factor, high temperature, that can induce a high production of 2n gametes, (ii) show that the sensitivity window to this factor is narrow and restricted to meiosis and (iii) reveal that 2n gamete production in inductive condition, results from parallel spindle orientation in meiosis II. To determine molecular mechanisms responsible for their formation, two candidate genes, RhPS1 and RhCYCA1 were identified in Rosa. Analysis of their expression revealed: (i) their high expression level in stamens at meiosis stage in non-inductive condition and (ii) the rapid repression of their transcript levels under inductive condition. Meiotic gene function of RhPS1 was validated by complementation of atps1-1 mutant and by generating a rose transgenic line p35S:: RNAi-RhPS1. According to these results, polyploidization and its mechanisms can now be replaced in the context of the current climate.

Au cours des dernières décennies, un contexte alarmant de déclin des paramètres du sperme etd’accroissement constant des pathologies génitales masculines a été décrit dans de nombreuxpays industrialisés.Notre travail évalue dans un premier temps l’évolution des paramètres spermatiques chez11 330 hommes ayant consulté pour infertilité conjugale au Centre de ProcréationMédicalement Assistée du CHU de Marseille entre 1988 et 2007. Les données ont étérecueillies de manière rétrospective et analysées par régression linéaire multi-variée afin detenir compte de l’effet lié à l’âge. Nous montrons une diminution significative des principauxparamètres du sperme: concentration en spermatozoïdes (-1.4% par an), numération totale enspermatozoïdes (-1.5% par an), mobilité progressive rapide (-5.6% par an) et morphologienormale (-1.9% par an). Les possibles biais de selection ont été discutés. L’effet del’environnement sur notre fonction de reproduction est l’une des hypothèses principalesévoquées pour expliquer ce phénomène.Nous montrons ensuite comment un modèle de culture de tubes séminifères de rat en chambrebicamérale, couplé à des techniques d’étude de la méiose en immunocytochimie peut êtreutilisé comme nouvel outil de reprotoxicologie. Nous décrivons tout d’abord avec l’anticorpsanti-SCP3 la prophase de première division méiotique chez le rat in-vivo et nous validons lemodèle de culture par comparaison entre les résultats des cellules obtenues in vivo et ceux descellules obtenues ex-vivo. Nous montrons ensuite grâce à ce système l’impact du ChromeHexavalent et du Cadmium sur la méiose du rat. Ces 2 métaux lourds, largement présent dansnotre environnement, peuvent être responsables d’atteintes importantes des cellulesméiotiques, y compris à de faibles doses pouvant correspondre à celles retrouvées chez desindividus exposés dans leur vie quotidienne et/ou professionnelle. / In recent decades, an alarming decline of sperm parameters and a constant increase in malegenital diseases has been described in many industrialized countries.In a first time, our study evaluates the evolution of sperm parameters in 11 330 menconsulting for infertility of their couple in the Reproduction Biology Laboratory of anUniversity Hospital in Marseilles between 1988 and 2007. Data were collected retrospectivelyand analyzed by multivariate linear regression to take into account the effect related to age.We show a significant decrease of the main sperm parameters: sperm concentration (-1.4%per year), total sperm count (-1.5% per year), rapid progressive motility (-5.6% per year) andnormal morphology (-1.9 % per year). Possible selection bias were discussed. The effect ofenvironment on our reproductive function is one of the main hypothesis to explain thisphenomenon.Secondly, we show how a rat seminiferous tubule culture in a bicameral chamber system,coupled with study of meiosis by immunocytochemistry can be used as a new tool ofreprotoxicology. We first describe with an anti-SCP3 antibody the first prophase of ratmeiosis and we validate the model of culture by comparing the results obtained on cellsobtained after in vivo spermatogenesis and those of cells obtained ex-vivo. Then, we showusing this system, the impact of hexavalent chromium and cadmium on rat meiosis. These twoheavy metals, widely present in our environment, may cause substantial impairment ofmeiotic cells, including low doses which can fit with those found in individuals exposed intheir personnal life and / or occupational training.

La mise en place de la lignée germinale au cours du développement constitue une des étapes fondamentales conditionnant la fertilité de l’individu adulte. Au cours des dernières décennies, le nombre croissant de couples consultant pour une aide médicale à la procréation a fait émerger l’hypothèse d’une altération des fonctions de reproduction chez l’Homme qui pourrait trouver son origine dans la perturbation du développement précoce. Dans l’ovaire fœtal, les cellules germinales s’orienteront vers la voie de l’ovogénèse, caractérisée entre autres par l’entrée en méiose de ces cellules. La majorité des données actuelles relatives à ces évènements sont issues du modèle murin alors que le développement de l’ovaire humain est significativement diffèrent de celui de la souris. Il est donc nécessaire d’approfondir nos connaissances du développement ovarien humain et d’identifier ses éventuelles perturbations. L’objectif de mon travail a été de mettre au point un outil d’étude du développement ovarien et d’identifier de nouvelles voies impliquées dans la régulation de l’entrée en méiose des cellules germinales fœtales humaines et leurs perturbations éventuelles.Nous avons mis au point un nouveau modèle de xénogreffe d’ovaires fœtaux humains du premier trimestre de gestation (au moment de l’apparition des premières cellules méiotiques). Ce modèle nous a permis d’observer un développement de l’organe et une différenciation des cellules germinales similaires à ceux observés in vivo. Ce modèle permettra des travaux à des âges auxquels le matériel d’étude est peu accessible. En couplant ce modèle de xénogreffe à une stratégie d’ARN-interférence, il nous a été possible d’inhiber l’expression d’un gène spécifiquement exprimé dans les cellules germinales ovariennes, DMRTA2, et de mettre en évidence un potentiel rôle de ce gène dans leur différenciation pré-méiotique. Nous avons observé une diminution du nombre de cellules ayant initié la méiose après inhibition de l’expression de ce gène. Par ailleurs, nous avons également identifié la présence dans l’ovaire fœtal de nombreux marqueurs décrits comme testiculaires chez la souris (PLZF, DNMT3L, FGF9, NANOS2 ou CYP26B1). L’expression de ces marqueurs pourrait expliquer la présence de cellules mitotiques tardives dans l’ovaire fœtal humain que nous avons pu observer jusqu’à 30 semaines de gestation. En parallèle de ces travaux, nous avons testé la sensibilité des cellules germinales à la dexaméthasone, glucocorticoïde pouvant être administré au cours de la grossesse. Il a été observé une augmentation de l’expression de PLZF, gène cible de l’activation des récepteurs aux glucocorticoïdes, pouvant expliquer la diminution du nombre de cellules germinales.En conclusion, ce travail de thèse a permis d’identifier un nouveau gène potentiellement régulateur de la transition mitose/méiose dans l’ovaire humain, et d’affiner nos connaissances sur le développement de l’ovaire humain et l’entrée en méiose des cellules germinales. Toutefois, de nombreuses questions restent posées ainsi de futures études devront clarifier si les cellules germinales mitotiques observées à des stades tardifs sont capables de se différencier en ovocytes compétents. / Woman fertility is partially dictated by the set up of the human female germ line. During the last ten years, which saw an increased number of couples consulting for assisted reproductive cares, the hypothesis of an early alteration in reproduction functions has emerged.In the fetal ovary, germ cells enter the path of oogenesis differentiation characterized by meiotic initiation. On this subject, vast majority of the scientific data are obtained from the mouse model, even if differences with human ovarian physiology are widely acknowledged. Therefore it is necessary to extend our knowledge on human ovarian development and identify its perturbations. The objective of my work was to assess a new model to study ovarian growth, studying regulation of meiotic entry and perturbation of germ line differentiation.We sat up a new xenograft model of early human fetal ovaries, when very early meiotic germ cells appear. Organ growth and germ cells differentiation were comparable with in vivo observations. Using this model with an RNA-interference strategy, we inhibited the expression of an oogonia germ cell gene, DMRTA2. This inhibition conducted to a significantly reduced number of germ cells gene that initiated meiosis and DMRTA2 seemed to be required for mitotic-meiotic transition. In another hand, we identified, in the ovary, the expression of germ cells markers described as specifically male in rodent (PLZF, DNMT3L, FGF9, NANOS2 ou CYP26B1). The expression of these markers in the human ovary could explain the observation of mitotic germ cells in late fetal ovaries (30 wpf).In parallel, we tested germ cells sensibility to a synthetic glucocorticoid, dexamethasone, administrated during pregnancy in some justified pathologies. We observed an increased expression of PLZF that could explain the decreased number of germ cells observed in treated ovaries.In conclusion, we identified a new gene expressed in human fetal ovaries, potentially involved in the meiotic entry, and we extended our knowledge to characterized human germ line development. However, many points have to be clarified, as the possible competence of late mitotic germ cells to form oocytes.

Made available in DSpace on 2014-06-11T19:30:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-04-27Bitstream added on 2014-06-13T21:01:36Z : No. of bitstreams: 1 araujo_d_dr_rcla.pdf: 1858376 bytes, checksum: dbeb43d42be45d0e0d9524437faa5d74 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Dentre todas as ordens de aracnideos conhecidas taxonomicamente, Araneae e a segunda mais diversa, com numero de especies menor somente em relacao a Acari. Atualmente, 39.725 especies ja foram descritas, sendo que centenas de novas descricoes sao feitas a cada ano em diversas familias de aranhas. O conhecimento citogenetico sobre a ordem restringe-se a analise de 638 especies (ca 2%) do total descrito do ponto de vista taxonomico. Este trabalho tem como objetivos fornecer uma compilacao dos dados citogeneticos existentes para a ordem na literatura ate a presente data, bem como caracterizar e estabelecer as estrategias de diferenciacao cromossomica em 13 especies de aranhas pertencentes ao grupo das haploginas, clado que corresponde a somente 3.257 especies (ca 8%) do total da ordem e a apenas 41 especies (ca 6%) do total cariotipado ate os dias atuais. Aliado a baixa representatividade dos dados cariologicos, outros pontos que fazem das haploginas um grupo interessante para estudos sao a predominancia de cromossomos meta/submetacentricos e de sistemas cromossomicos de determinacao sexual simples e multiplos, muitas vezes incluindo um cromossomo Y, ambas caracteristicas raras entre os outros clados de Araneae. As especies analisadas pertencem a tres familias de haploginas, Pholcidae (Mesabolivar luteus e Micropholcus fauroti), Sicariidae (Loxosceles amazonica, Loxosceles gaucho, Loxosceles hirsuta, Loxosceles intermedia, Loxosceles laeta, Loxosceles puortoi, Loxosceles similis e Sicarius tropicus) e Scytodidae (Scytodes fusca, Scytodes globula e Scytodes itapevi). Em Pholcidae, os resultados ineditos para os dois generos mostraram... / Mesabolivar luteus (Keyserling 1891) and Micropholcus fauroti (Simon 1887) specimens were collected in Ubatuba and Rio Claro, both in the state of São Paulo, Brazil. Mesabolivar luteus showed 2n(.) = 15 = 14 + X and 2n(.) = 16 = 14 + XX in mitotic metaphases and 7II + X in diplotenic cells. During late prophase I, all bivalents presented a ring shape, evidencing two chiasmata per bivalent. In this species, some diplotenic cells appear in pairs, maybe due to specific characteristics of the intercellular bridges. The metaphases II showed n = 7 or n = 8 = 7 + X chromosomes. Micropholcus fauroti evidenced 2n(.) = 17 = 16 + X in spermatogonial metaphases and 8II+X in diplotenic cells, with only one chiasma per bivalent, contrasting with M. luteus. In both species, all chromosomes were metacentrics. The X sexual chromosome was the largest element and appeared as a univalent during meiosis I. These are the first cytogenetical data for the genera Mesabolivar and Micropholcus. Additionally, M. luteus is the first chromosomally analyzed species of the New World clade and the observed diploid number for M. fauroti had not yet been recorded in Pholcidae.

Saccharomyces cerevisiae HOP1, which encodes a component of the synaptonemal complex, plays an important role in both gene conversion and crossing over between homologs, as well as enforces the meiotic recombination checkpoint control over the progression of recombination intermediates. The zinc-finger motif (Znf) 348CX2CX19CX2C374) of Hop1 is crucial for its function in meiosis, since mutation of conserved Cys371 to Ser in this motif results in a temperature-sensitive phenotype, which is defective in sporulation and meiosis. The direct role for Hop1 or its ZnF in the formation of joint molecules and checkpoint control over the progression of meiotic recombination intermediates is unknown. To understand the underlying biochemical mechanism, we constructed a series of recombination intermediates. Hop1 or its ZnF were able to bind different recombination intermediates. Interestingly, the binding affinity of Hop1 and its ZnF was much higher for the Holliday junction as compared to other recombination intermediates. The complexes of Hop1 or its ZnF with the Holliday junction were stable and specific as shown by NaCl titration and competition experiment. Hop1 and its ZnF blocked BLM helicase-induced unwinding of the Holliday junction, indicating that the interaction between Hop1 and its ZnF with the Holliday junction is specific. DNase I footprinting experiment showed that Hop1 or its ZnF bind to the center of the Holliday junction. 2-aminopurine fluorescence and KMnO4 experiments showed that Hop1 or its ZnF can distort the Holliday junction in a 2-fold symmetrical manner. The molecular modeling study showed that Hop1 ZnF folded into unique helix-loop-helix motif and bound to center of the Holliday junction. In summary, this study shows that Hop1 protein or its ZnF interact specifically with the Holliday junction and distort its structure. Taken together, these results implicate that Hop1 protein might coordinate the physical monitoring of meiotic recombination intermediates during the process of branch migration and that Hop1 ZnF acts as a structural determinant of Hop1 protein functions.

Since antiquity figures have been widely studied within the framework of rhetoric, although contemporary rhetoric has tended to disregard their importance and relegate their study to the domain of literary criticism. However, since the 1980s, there has been a renewed interest in figurative language not only in literary studies, but also in other fields of research. Indeed, research on figuration has emerged as a new and distinct discipline, namely figurative language studies. However, within this framework, metaphor and irony have received the greatest attention, while other non-literal forms have been largely ignored. This is certainly the case of hyperbole, a long neglected trope despite its ubiquity in discourse. The present study aims to make a contribution to the literature on exaggeration and so by extension to figurative language theories in general.Not all aspects of figuration have attracted equal interest among researchers. With a few exceptions, most attention has been directed at explaining how figures are comprehended, given their non-literal nature. In contrast to understanding, the question of production has been largely ignored. Similarly, although the reception process, in terms of understanding, has been widely studied, almost no attention has been devoted to listeners' responses to figures and their collaboration in a joint construction of non-literal frames.Rather than addressing comprehension, this study concentrates on the production process and usage of exaggeration, since these fields of research have been largely ignored in the literature on the subject. It aims to provide a general framework for the description and understanding of hyperbole in interaction, mainly from a production viewpoint but without totally disregarding the reception process, since special attention is devoted to the interactive dimension of exaggeration. This aim is formulated in terms of the following objectives:Objective 1: to provide an adequate definition of the notion of hyperbole; to list the criteria for identifying the trope so that non-exaggerated uses of expressions can be excluded.Objective 2: to set up a classification of overstated items according to semantic field, grammatical category, auxesis or meiosis, and interactivity with other figures.Objective 3: to explore the long neglected production process of hyperbole, both in terms of usage and functions.Objective 4: to examine the interactive nature of the trope, as an activity collaboratively constructed between speaker and hearer, by studying listeners' reactions and their own further contributions to overstatement.As for the theoretical framework, this study combines pragmatic and conversational-analytical methods with a corpus-based approach to the study of hyperbole. Exaggeration is a purely pragmatic phenomenon since it is entirely dependent on context. On the other hand, since overstatements are not one-off but complex lexico-grammatical items, they need to be examined within the constraints of placement, sequencing and turn-taking of conversational analysis. Finally, a major benefit of corpus-based research, only recently applied to the study of figures, is that it grounds its theorising on empirical observation rather than linguistic intuition. Besides, the use of corpora grants certain benefits, such as the use of naturalistic data, automatic access to context, evidence of interactivity and hyperbolic cues, wide coverage of genres, etc.The data examined consists of naturalistic spoken texts, totalling 52,000 words, extracted from the British National Corpus. The focus is on oral discourse, since not a great amount of research exists into everyday spoken hyperbole. The bulk of research has been conducted in written language or relies on artificial and elicited data. The aim was to demonstrate that although traditionally relegated to the literary text, hyperboles are rather ubiquitous strategies in everyday language. This idea adheres to a prevailing view among figurative language researchers, namely that cognition is partly figurative. / El lenguaje figurado ha sido, desde antiguo, ampliamente estudiado en el marco de la retórica. Más recientemente, las figuras retóricas parecen haber despertado un nuevo interés en otras disciplinas. De hecho, dichos estudios están empezando ya a ser considerados como una disciplina en sí: las teorías del lenguaje figurado. Dentro de este marco, metáfora e ironía han sido ampliamente estudiadas y como consecuencia de su estudio intensivo, otras figuras han sido marginadas. Éste es el caso de la hipérbole, figura retórica largo tiempo olvidada a pesar de su ubicuidad y tradicionalmente relegada al dominio de la crítica literaria.El presente estudio, en lugar de examinar el proceso de comprensión, se centra en la producción y uso de la exageración, temas escasamente tratados en la literatura existente. Intenta proporcionar un marco general para la descripción y comprensión de la hipérbole en la interacción, principalmente desde el punto de vista de la producción, pero sin descartar del todo el proceso de recepción, dado que su dimensión interactiva como figura creada conjuntamente entre hablante y oyente cobra especial relevancia. Dicho propósito se materializa en los siguientes objetivos:Objetivo 1: proporcionar una definición adecuada de la noción de exageración que nos permita diferenciarla de figuras afines y excluir los usos no exagerados de las expresiones.Objetivo 2: clasificar los elementos hiperbólicos de acuerdo con los siguientes parámetros: campo semántico, categoría gramatical, auxesis o meiosis, e interacción con otras figuras.Objetivo 3: explorar el descuidado proceso de producción de la figura en términos de uso y función.Objetivo 4: examinar la naturaleza interactiva del tropo, como actividad conjunta entre hablante y oyente, mediante el estudio de las respuestas y demás contribuciones del receptor al acto hiperbólico del hablante.Este estudio fusiona tres tipos de enfoque: pragmática, análisis conversacional y lingüística de corpus, a la hora de estudiar la exageración en el discurso oral, dado que son escasos los estudios de esta figura en el habla cotidiana. El corpus examinado abarca unas 52.000 palabras y es una selección de textos orales extraídos del British National Corpus.