Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-10-21T01:01:09Z No. of bitstreams: 1 shaianysabrinalopesgomes.pdf: 8345225 bytes, checksum: 345de5fe2a909ceb0a04bbb96016c1ff (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-10-21T13:13:03Z (GMT) No. of bitstreams: 1 shaianysabrinalopesgomes.pdf: 8345225 bytes, checksum: 345de5fe2a909ceb0a04bbb96016c1ff (MD5) / Made available in DSpace on 2017-10-21T13:13:03Z (GMT). No. of bitstreams: 1 shaianysabrinalopesgomes.pdf: 8345225 bytes, checksum: 345de5fe2a909ceb0a04bbb96016c1ff (MD5) Previous issue date: 2017-03-22 / As orquídeas do complexo "Zygopetalum maculatum" (Kunth) Garay são típicas dos campos rupestres e de altitude do leste do Brasil. Atualmente são reconhecidas seis espécies para este grupo que, por compartilharem características morfológicas diagnósticas, são reconhecidas aqui como um complexo de espécies. De forma a contribuir para entender o padrão de variação fenotípica observado entre as populações e com isso tentar esclarecer a formação deste complexo, este trabalho caracterizou do ponto de vista genético ar 22 populações de "Z. maculatum", sendo 21 provenientes do Brasil e uma da Bolívia. As análises compreenderam estimativa da quantidade de DNA nuclear, determinação do número cromossômico, comportamento meiótico e diversidade genética identificada por marcadores do tipo ISSR. O conteúdo médio de DNA revelou três níveis de ploidia no complexo, 7,36 pg, 10,52 pg e 14,09 pg de DNA, sendo estes relacionados a números cromossômicos de 2n=48; 2n=72; 2n=96, respectivamente. Tal variação ocorreu entre e dentro das populações. Dados cariotípicos (morfometria, DNAr 5S e 45S) evidenciaram similaridade entre os três níveis de ploidia. A meiose mostrou-se irregular, com ocorrência de atraso e perda cromossômica, pontes, “cromossomos aderentes”, micronúcleos e assincronia em todos os níveis de ploidia. O dendrograma baseado em marcadores ISSR revelou tendência de agrupamento de indivíduos com o mesmo nível de ploidia e originados das mesmas regiões geográficas. Os resultados sugerem que as populações possuem baixos índices de diversidade genética entre indivíduos e que a maior diversidade encontra-se entre as populações. De forma geral, os resultados indicam que eventos de poliploidização estejam relacionados à diversificação e especiação de “Z. maculatum”, sendo um processo importante para explicar as variações morfológicas observadas dentro do complexo. / The orchids of the "Zygopetalum maculatum" (Kunth) Garay complex are typical of the “campos rupestres” of the east side of Brazil. Currently, six species are recognized for this group which, due to their morphological similarity, is recognized here as a species complex. In order to understand the phenotypic pattern observed among the populations and contribute to clarify the formation of this complex, this study characterized 22 populations of “Z. maculatum”, being 21 populations from Brazil and one population from Bolivia. The study included DNA amount estimation, determination of the chromosome number, meiotic behavior and genetic diversity estimation. The average of DNA content revealed three 2C values, 7.36 pg, 10.52 pg and 14.09 pg of DNA, which were associated to 2n = 48; 2n = 72 and 2n = 96 chromosomes, respectively. The variation was observed within and among populations. Karyotypic data (morphometry, 5S and 45S rDNA) showed similarity among all ploidy levels. Meiosis behavior was not regular being observed chromosome delay and loss, bridges, micronuclei, chromosomal stickiness and asynchrony. The dendrogram based on ISSR revealed similarity regarding the ploidy level and the geographical distribution of the individuals. The data also suggested low genetic diversity within populations being most of the diversity observed among populations. In general, the results indicate that polyploidization events are probably related to the Zygopetalum diversification and speciation, being an important process to explain the morphological variations observed within the complex.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The economic potential of the medicinal species native to Brazil is huge, rendering it important to maintain the available plant genetic diversity via studies characterizing germplasm. Between these studies, highlights the characterization meiotic and pollen viability, genotoxic and antiproliferative activitie, beyond determination of phenolic compounds. Sambucus australis Cham. & Schltdl. ( sabugueiro ) numbers among the native species with medicinal potential, being widely used in the treatment of symptoms of skin eruptions, influenzas and common colds, for its diaphoretic, anti-inflammatory and analgesic actions. The objective of the present study was to investigate the antiproliferative, genotoxic and antigenotoxic activity of aqueous extracts of two accesses of S. australis, using the Allium cepa test, and to determine the phenolic compounds present in these extracts, as well as to analyze the meiotic behavior and estimate the pollen viability of different accesses of S. australis collected in Rio Grande do Sul state. Antiproliferative, genotoxic and antigenotoxic activities were determined by assessing the effect of aqueous extracts from the inflorescences and leaves of two accesses of S. australis, at concentrations of 3 g.L-1 and 12 g.L-1, on the cellular cycle of A. cepa. High performance liquid chromatography (HPLC) was employed to determine the phenolic compounds present in the extracts. For the meiosis analysis, slides were prepared by squashing technique of the anthers removed from the flower buds. The phases of association and distribution of the chromosomes were observed and meiotic indexes determined. To estimate pollen viability, the slides were prepared by squashing the anthers, comparing three stains: 2% acetic-orcein, 2% acetic-carmine, and Alexander´s reaction. All statistical analyses of the data were performed using the Scott-Knott test (p<0.05). Results showed that the aqueous extracts of S. australis exerted antiproliferative activity on the cellular cycle of A. cepa. The extracts prepared from S. australis (12g.L-1) leaves, for both accesses, also exhibited antigenotoxic activity. Chromatographic analysis disclosed the presence of the following compounds: gallic acid, chlorogenic acid, caffeic acid, ellagic acid, rutin, quercitrin, isoquercitrin, quercetin and kaempferol. Ultimately, the accesses of S. australis studied exhibited regular meiotic behavior, gametic number n=19, meiotic index > 90% and high pollen viability, being stain Alexander´s reaction the most efficient to estimate pollen viability in the specie. / O potencial econômico de espécies medicinais nativas no Brasil é imenso, sendo necessário conservar a diversidade genética vegetal disponível através de estudos de caracterização de germoplasma. Entre esses estudos, destacam-se a caracterização meiótica, a viabilidade polínica, a análise da atividade genotóxica e antiproliferativa, além da determinação dos compostos fenólicos. Dentre as espécies nativas com potencial medicinal, Sambucus australis Cham. & Schltdl. (sabugueiro) tem grande popularidade no tratamento sintomático de moléstias eruptivas, gripes e resfriados, por suas ações diaforética, anti-inflamatória e analgésica. O presente trabalho teve por objetivo verificar a atividade antiproliferativa, genotóxica e antigenotóxica de extratos aquosos de dois acessos de S. australis, através do teste de Allium cepa, bem como determinar os compostos fenólicos presentes nesses extratos, além de analisar o comportamento meiótico e estimar a viabilidade polínica de diferentes acessos de S. australis coletados no Rio Grande do Sul. Para determinação das atividades antiproliferativa, genotóxica e antigenotóxica, foi avaliado o efeito dos extratos aquosos das inflorescências e folhas de dois acessos de S. australis, nas concentrações de 3 g.L-1 e 12 g.L-1, sobre o ciclo celular de A. cepa. Os compostos fenólicos presentes nesses extratos foram determinados por meio da técnica de cromatografia líquida de alta eficiência (CLAE). Para a análise da meiose, foram preparadas lâminas pela técnica de esmagamento das anteras retiradas dos botões florais, observando-se as fases de associação e distribuição dos cromossomos, sendo também determinados os índices meióticos. Para estimativa da viabilidade polínica, as lâminas foram preparadas por esmagamento das anteras, comparando-se três corantes: orceína acética 2%, carmim acético 2% e reativo de Alexander. Todos os dados foram analisados estatisticamente pelo teste de Scott-Knott (p<0,05). A partir dos resultados, pode-se observar que os extratos aquosos de S. australis apresentaram atividade antiproliferativa sobre o ciclo celular de A. cepa. Os extratos preparados a partir das folhas de S. australis (12g.L-1), em ambos acessos, também apresentaram atividade antigenotóxica. A partir da análise cromatográfica foi possível determinar a presença dos seguintes compostos: ácido gálico, ácido clorogênico, ácido cafeico, ácido elágico, rutina, quercitrina, isoquercitrina, quercetina e canferol. Por fim, os acessos de S. australis estudados possuem comportamento meiótico regular, número gamético n=19, índice meiótico superior a 90% e alta viabilidade polínica, sendo o corante reativo de Alexander o mais eficiente para estimar a viabilidade polínica na espécie.

Meiosis is a specialized type of cell division where two rounds of chromosome segregation follow a single round of DNA duplication resulting in the formation of four haploid daughter cells. Once the DNA replication is complete, the homologous chromosomes pair and recombine during the meiotic prophase I, giving rise to genetic diversity in the gametes. The process of homology search during meiosis is broadly divided into recombination-dependent (involves the formation of double-strand breaks) and recombination-independent mechanisms. In most eukaryotic organisms, pairing of homologs, recombination and chromosome segregation occurs in the context of a meiosis-specific proteinaceous structure, known as the synaptonemal complex (SC). The electron microscopic visualization of SC has revealed that the structure is tripartite with an electron-dense central element and two lateral elements that run longitudinally along the entire length of paired chromosomes. Transverse filaments are protein structures that connect the central region to the lateral elements. Genetic analyses in budding yeast indicate that mutations in SC components or defects in SC formation are associated with chromosome missegregation, aneuploidy and spore inviability. In humans, defects in SC assembly are linked to miscarriages, birth defects such as Down syndrome and development of certain types of cancer. In Saccharomyces cerevisiae, genetic screens have identified several mutants that exhibit defects in SC formation culminate in a decrease in the frequency of meiotic recombination, spore viability and improper chromosome segregation. Ten meiosis-specific proteins, viz. Hop1, Red1, Mek1, Hop2, Pch2, Zip1, Zip2, Zip3, Zip4 and Rec8, have been shown to be the bona fide components of SC and/or associated with SC function. S. cerevisiae HOP1 (HOmolog Pairing) gene was isolated in a genetic screen for mutants that showed defects in homolog pairing and, consequently, reduced levels of interhomolog recombination (10% of wild-type). Amino acid sequence alignment together with genetic and biochemical analyses revealed that Hop1 is a 70 kDa protein with a centrally embedded essential zinc-finger motif (Cys2/Cys2) and functions in polymeric form. Previous biochemical studies have also shown that Hop1 is a structure-specific DNA binding protein, which exhibits high affinity for the Holliday junction (HJ) suggesting a role of this protein in branch migration of the HJ. Furthermore, Hop1 displays high affinity for G-quadruplex structures (herein after referred to as GQ) and also promotes the formation of GQ from unfolded G-rich oligonucleotides. Strikingly, Hop1 promotes pairing between two double-stranded DNA molecules via G/C-rich sequence as well as intra- and inter-molecular pairing of duplex DNA molecules. Structure-function analysis suggested that Hop1 has a modular organization consisting of a protease-sensitive N-terminal, HORMA domain (characterized in Hop1, Rev7, Mad2 proteins) and protease-resistant C-terminal domain, called Hop1CTD. Advances in the field of DNA quadruplex structures suggest a significant role for these structures in a variety of biological functions such as signal transduction, DNA replication, recombination, gene expression, sister chromatid alignment etc. GQs and i-motif structures that arise within the G/C-rich regions of the genome of different organisms have been extensively characterized using biophysical, biochemical and cell biological approaches. Emerging studies with guanine- and cytosine-rich sequences of several promoters, telomeres and centromeres have revealed the formation of GQs and i-motif, respectively. Although the presence of GQs within cells has been demonstrated using G4-specific antibodies, in general, the in vivo existence of DNA quadruplex structures is the subject of an ongoing debate. However, the identification and isolation of proteins that bind and process these structures support the idea of their in vivo existence. In S. cerevisiae, genome-wide survey to identify conserved GQs has revealed the presence of ~1400 GQ forming sequences. Additionally, these potential GQ forming motifs were found in close proximity to promoters, rDNA and mitosis- and meiosis-specific double-strand break sites (DSBs). Meiotic recombination in S. cerevisiae as well as humans occurs at meiosis-specific double-strand break (DSBs) sites that are embedded within the G/C-rich sequences. However, much less is known about the structural features and functional significance of DNA quadruplex motifs in sister chromatid alignment N during meiosis. Therefore, one of the aims of the studies described in this thesis was to investigate the relationship between the G/C-rich motif at a meiosis-specific DSB site in S. cerevisiae and its ability to form GQ and i-motif structures. To test this hypothesis, we chose a G/C-rich motif at a meiosis-specific DSB site located between co-ordinates 1242526 to 1242550 on chromosome IV of S. cerevisiae. Using multiple techniques such as native gel electrophoresis, circular dichroism spectroscopy, 2D NMR and chemical foot printing, we show that G-rich motif derived from the meiosis-specific DSB folds into an intramolecular GQ and the complementary C-rich sequence folds into an intramolecular i-motif, the latter under acidic conditions. Interestingly, we found that the C-rich strand folds into i-motif at near neutral pH in the presence of cell-mimicking molecular crowding agents. The NMR data, consistent with our biochemical and biophysical analyses, confirmed the formation of a stable i-motif structure. To further elucidate the impact of these quadruplex structures on DNA replication in vitro, we carried out DNA polymerase stop assay with a template DNA containing either the G-rich or the C-rich sequence. Primer extension assays carried out with Taq polymerase and G-rich template blocked the polymerase at a site that corresponded to the formation of an intramolecular GQ. Likewise, primer extension reactions carried out with KOD-Plus DNA polymerase and C-rich template led to the generation of a stop-product at the site of the formation of intramolecular I -motif under acidic conditions (pH 4.5 and pH 5.5). However, polymerase stop assay performed in the presence of single-walled carbon nanotubes (SWNTs) that stabilize I -motif at physiological pH blocked the polymerase at the site of intramolecular I -motif formation, indicating the possible existence of i-motif in the cellular context. Taken together, these results revealed that the G/C-rich motif at the meiosis-specific DSB site folds into GQ and i-motif structures in vitro. Our in vitro analyses were in line with our in vivo analysis that examined the ability of the G/C-rich motif to fold into quadruplex structures in S. cerevisiae cells. Qualitative microscopic analysis and quantitative analysis with plasmid constructs that harbour the GQ or i-motif forming sequence revealed a significant decrease in the GFP expression levels in comparison to the control. More importantly, all the assays performed with the corresponding mutant sequences under identical experimental conditions did not yield any quadruplex structures, suggesting the involvement of contagious guanine and cytosine residues in the structure formation. Prompted by our earlier results that revealed high binding affinity of Hop1 for GQ, we wished to understand the role of the GQ and i-motif structures during meiosis by analysing their interaction with Hop1 and its truncated variants (HORMA and Hop1CTD). In agreement with our previous observations, Hop1 and Hop1CTD associated preferentially with GQ DNA. Interestingly, whereas the full-length Hop1 showed much weaker binding affinity for i-motif DNA, Hop1 C-terminal fragment but not its N-terminal fragment exhibited robust i-motif DNA binding activity. We have previously demonstrated that Hop1 promotes intermolecular synapsis between synthetic duplex DNA molecules containing a G/C-rich sequence. Hence, to understand the functional role of the quadruplex structures formed at the meiosis-specific G/C-rich motif, we examined the ability of Hop1 to promote pairing between linear duplex DNA helices containing the G/C-rich motif. DNA pairing assay indicated that binding of Hop1 to the G/C-rich duplex DNA resulted in the formation of a side-by-side synapsis product. Under similar conditions, Hop1 was unable to pair mutant duplex DNA molecules suggesting the involvement of the G/C-rich motif in the formation of the synapsis product. Our results were substantiated by the observation that yeast Rad17 failed to promote pairing between duplex DNA molecules with a centrally embedded G/C-rich motif. Altogether, these results provide important structural and functional insights into the role of quadruplex structures in meiotic pairing of homologous chromosomes. The second part of the thesis focuses on the biochemical and functional properties of Red1 protein, a component of S. cerevisiae lateral element. RED1 was identified in a screen for meiotic lethal, sporulation proficient mutants. Genetic, biochemical and microscopic analyses have demonstrated the physical interaction between Hop1 and Red1. Given this, hop1 and red1 mutants display similar phenotypes such as chromosome missegregation and spore inviability and thus are placed under the same epistasis group. However, unlike hop1 mutants, red1 mutants show complete absence of SC. RED1 overexpression suppressed certain non-null hop1 phenotypes, indicating that these proteins may have partially overlapping functions. Further, although the functional significance is unknown, chromatin immunoprecipitation studies have revealed the localization of Red1 to the GC-rich regions (R-bands) in the genome, considered to be meiotic recombination hotspots. Although the aforementioned genetic studies suggest an important role for Red1 in meiosis, the exact molecular function of Red1 in meiotic recombination remains to be elucidated. To explore the biochemical properties of Red1, we isolated the S. cerevisiae RED1 gene, cloned, overexpressed, and purified the protein to near homogeneity. Immunoprecipitation assays using meiotic cells extracts suggested that Red1 exists as a Homodimer linked by disulphide-bonds under physiological conditions. We characterized the DNA binding properties of Red1 by analysing its interaction with recombination intermediates that are likely to form during meiotic recombination. Protein-DNA interaction assays revealed that Red1 exhibits binding preference for the Holliday junction over replication fork and other recombination intermediates. Notably, Red1 displayed ~40-fold higher binding affinity for GQ in comparison with HJ. The observation that Red1 binds robustly to GQs prompted us to examine if Red1 could promote pairing between duplex DNA helices with the G/C-rich sequences similar to Hop1. Interestingly, we found that Red1 failed to promote pairing between dsDNA molecules but potentiated Hop1 mediated pairing between duplex DNA molecules. Our AFM studies with linear and circular DNA molecules along with Red1 suggested a possible role of Red1 in DNA condensation, bridging and pairing of double-stranded DNA helices. Bioinformatics analysis of Red1 indicated the lack of sequence or structural similarity to any of the known proteins. To elucidate structure-function relationship of Red1, we generated several N- and C-terminal Red1 truncations and studied their DNA binding properties. Our results indicated that the N-terminal region comprising of 678 amino acid residues constitutes the DNA-binding region of Red1. The N-terminal region, called RNTF-II, displayed similar substrate specificity comparable to that of full-length Red1. Interestingly, site-directed mutagenesis studies with the Red1 C-terminal region revealed the involvement of two cysteine residues at position 704 and 707 in the disulfide bond mediated intermolecular dimer formation. Finally, to understand the functional significance of Red1 truncations we analyzed the subcellular localization of Red1 and its truncations. We made translation fusions of RED1 and its truncations by placing their corresponding nucleotide sequences downstream of GFP coding sequence in yeast expression vector. Confocal microscopy studies with S. cerevisiae cells transformed with the individual plasmid constructs indicated that the N-terminal variants localized to the nucleus, whereas the C-terminal variants did not localize to the nucleus. These results suggest that NLS-like motifs are embedded in the N-terminal region of the protein. Furthermore, other results indicated that the N-terminal region contains functions such as DNA-binding and intermolecular bridging of non-contiguous DNA segments. Altogether, these findings, on the one hand, provide insights into the molecular mechanism underlying the functions of Hop1 and Red1 proteins and, on the other, support a role for DNA quadruplex structures in meiotic chromosome synapsis and recombination.

(English) Meiotic homologous recombination, homologous chromosomes synapsis, and F1 hybrid sterility (enabling formation of species) are mutually interconnected phenomenons, one being the prerequisite to the latter. In the present thesis, these phenomenons were investigated on a genetic and mechanistic level using a mouse subspecies as a model. Noncrossovers (NCOs, gene conversions), 90% prevalent resolution of Prdm9- determined meiotic double-strand breaks (DSBs), were uniquely identified and characterized on a chromosome-wide level. The mean gene conversion tract length, based on 94 NCOs events, was calculated to be 32 bp. On a local level, the NCOs overlapped the known hotspots of PRDM9-controlled histone trimethylation and DSB formation, indicating their origin in the standard meiotic DSB repair pathway. On chromosome-wide level, NCO and CO distributions differed, in particular COs being relatively preferred over NCOs in subtelomeric regions. A specific subset of nonparental/asymmetric NCOs and COs was underrepresented in our datasets, proposing their problematic repair, hypothetically enabled by sister chromatids, and thus not contributing to indispensable homologous synapsis. Genome-wide crossover (CO) rates, genetically and mechanistically crucial ~10% of DSB repair, were proven to be...

La recombinaison méiotique joue un rôle essentiel dans la ségrégation des chromosomes pendant la méiose et dans la création de nouvelles combinaisons du matériel génétique des espèces. Ses effets cause une déviation du principe de l'assortiment indépendant de Mendel; cependant, les mécanismes moléculaires impliqués restent partiellement incompris jusqu'à aujourd'hui. Il s'agit d'un processus hautement régulé et de nombreuses protéines sont impliquées dans son contrôle, dirigeant la recombinaison méiotique dans des régions génomiques de 1 à 2 kilobases appelées « hotspots ». Au cours des dernières années, l'apprentissage profond a été appliqué avec succès à la classification des séquences génomiques. Dans ce travail, nous appliquons l'apprentissage profond aux séquences d'ADN humain afin de prédire si une région spécifique d'ADN est un hotspot de recombinaison méiotique ou non. Nous avons appliqué des réseaux de neurones convolutifs sur un ensemble de données décrivant les hotspots de quatre individus non-apparentés, atteignant une exactitude de plus de 88 % avec une précision et un rappel supérieur à 90 % pour les meilleurs modèles. Nous explorons l'impact de différentes tailles de séquences d'entrée, les stratégies de séparation des jeux d'entraînement/validation et l’utilité de montrer au modèle les coordonnées génomiques de la séquence d'entrée. Nous avons exploré différentes manières de construire les motifs appris par le réseau et comment ils peuvent être liés aux méthodes classiques de construction de matrices position-poids, et nous avons pu déduire des connaissances biologiques pertinentes découvertes par le réseau. Nous avons également développé un outil pour visualiser les différents modèles afin d'aider à interpréter les différents aspects du modèle. Dans l'ensemble, nos travaux montrent la capacité des méthodes d'apprentissage profond à étudier la recombinaison méiotique à partir de données génomiques. / Meiotic recombination plays a critical role in the proper segregation of chromosomes during meiosis and in forming new combinations of genetic material within sexually-reproducing species. For a long time, its side effects were observed as a deviation from the Mendel’s principle of independent assortment; however, its molecular mechanisms remain only partially understood until today. We know that it is a highly regulated process and that many molecules are involved in this tight control, resulting in directing meiotic recombination into 1-2 kilobase genomic pairs regions called hotspots. During the past few years, deep learning was successfully applied to the classification of genomic sequences. In this work, we apply deep learning to DNA sequences in order to predict if a specific stretch of DNA is a meiotic recombination hotspot or not. We applied convolution neural networks on a dataset describing the hotspots of four unrelated male individuals, achieving an accuracy of over 88% with precision and recall above 90% for the best models. We explored the impact of different input sequence lengths, train/validation split strategies and showing the model the genomic coordinates of the input sequence. We explored different ways to construct the learnt motifs by the network and how they can relate to the classical methods of constructing position-weight-matrices, and we were able to infer relevant biological knowledge uncovered by the network. We also developed a tool for visualizing the different models output in order to help digest the different aspects of the model. Overall, our work shows the ability for deep learning methods to study meiotic recombination from genomic data.

No description available.

Charles University in Prague Faculty of Science Ph.D. study program: Molecular and Cellular Biology, Genetics and Virology Abstract Genetics and genomics of hybrid sterility Mgr. Tanmoy Bhattacharyya Supervisor: Prof. MUDr. Jiří Forejt, DrSc. Praha 2013 Abstract Male-limited hybrid sterility restricts gene flow between the related species, an important pre- requisite of speciation. The F1 hybrid males of PWD/Ph female (Mus m. musculus subspecies) and C57BL/6J or B6 male (Mus m. domesticus) are azoospermic and sterile (PB6F1), while the hybrids from the reciprocal (B6PF1) cross are semi fertile. A disproportionately large effect of the X chromosome (Chr) on hybrid male sterility is a widespread phenomenon accompanying the origin of new species. In the present study, we mapped two phenotypically distinct hybrid sterility loci Hstx1 and Hstx2 to a common 4.7 Mb region on Chr. X. Analysis of meiotic prophase I of PB6F1 sterile males revealed meiotic block at mid-late pachynema and the TUNEL assay showed apoptosis of arrested spermatocytes. In sterile males over 95% of pachytene spermatocytes showed one or more unsynapsed autosomes visualized by anti SYCP1, HORMAD2 and SYCP3 antibodies. The phosphorylated form of H2AFX histone, normally restricted only to XY chromosome containing sex body decorated unsynapsed...

La division cellulaire est essentielle pour la survie de chaque être vivant. Au cours de ce processus, les chromosomes de la cellule en division sont transmis aux deux cellules filles. La répartition des chromosomes est orchestrée par une structure cellulaire transitoire appelée fuseau mitotique (ou fuseau méiotique dans les cellules reproductrices). Le fuseau est composé de microtubules, de nombreuses protéines et de moteurs moléculaires, qui interagissent de manière complexe et précise aboutissant à l’organisation d’une structure bipolaire dynamique. Comme certains mécanismes moléculaires restent mal compris, nous avons choisi d'aborder la question de l'assemblage du fuseau méiotique dans des extraits d'oeufs de grenouille. Xenopus laevis est un organisme modèle car il est proche, d’un aspect phylogénétique, de l'homme, et il est particulièrement adapté à l’étude de la division cellulaire. Nous avons également utilisé une méthode in vitro (appelée spindle array ou puce à fuseaux) qui a été développée au sein du groupe de recherche auparavant, et qui offre certains avantages par rapport aux approches existantes. Une puce à fuseaux est composée de billes recouvertes de chromatine immobilisées selon des micro-motifs géométriques obtenus selon une technique d’impression par microcontact. L'assemblage des fuseaux méiotiques a été visualisé par microscopie confocale à fluorescence. Grâce à ces outils, nous avons, lors d’un premier projet, abordé le rôle de l’Augmin dans l'assemblage des fuseaux. L’Augmin est un complexe protéique récemment identifié grâce à son hypothétique rôle dans la nucléation de microtubules à partir de microtubules existants. Après déplétion de l’Augmin, nous avons constaté que la nucléation des microtubules était réduite et que les fuseaux avaient une morphologie anormale. De plus, ces derniers qui étaient essentiellement multipolaires sont progressivement devenus bipolaires grâce à une voie de nucléation des microtubules, découverte lors de notre étude, émanant des pôles acentrosomaux et qui est indépendante de l’Augmin. Nos résultats révèlent que l’Augmin est essentiel pour l’assemblage et la bipolarité du fuseau acentrosomal. Au cours d’un second projet, nous avons étudié les fonctions des chromokinésines kinésine-4 (Xklp1) et kinésine-10 (Xkid) dans l'assemblage des fuseaux et leurs mouvements. Xkid participe à la force d’éjection polaire nécessaire à la congression des chromosomes alors que Xklp1 contribue principalement à la régulation de la dynamique des microtubules. En étudiant l'assemblage de fuseaux dans des extraits après déplétion de Xkid, Xklp1 ou les deux, nous avons démontré que Xkid limite la dynamique des mouvements longitudinaux des fuseaux, contribue à la mise en place de la bipolarité et régule la longueur des fuseaux. Nous avons également quantifié la cinétique de nucléation des microtubules et confirmé le rôle de Xklp1 dans la régulation de la dynamique des microtubules. L’ensemble de nos travaux contribuent à une meilleure compréhension des mécanismes d’assemblage du fuseau méiotique et confirme la pertinence de notre méthode pour l'étude de sa morphogenèse. / Cell division is essential for the survival of every living organism. During this process, the chromosomes of the dividing cell are transmitted to the two daughter cells. The partition of the chromosomes is orchestrated by a transient sub-cellular structure called the mitotic spindle (or meiotic spindle in gamete cells). The spindle is composed of microtubules, numerous proteins and molecular motors, which interact in an intricate and yet precise manner leading to a highly dynamic and complexstructure. As some molecular mechanisms remain elusive, we have chosen to address the question of meiotic spindle assembly in Xenopus egg extracts. Xenopus laevis is a model system that is evolutionary close to human, and suitable for cell division studies. We have combined this with an in vitro assay - spindle array - which we developed prior to this work, and which provides advantages over existing approaches. A spindle array is composed of chromatin-coated beads that are immobilized according to geometrical patterns obtained by microcontact printing. The assembly of meiotic spindles wasvisualized by time-lapse fluorescence confocal microscopy. Using these tools, we first addressed the role of augmin in the assembly of meiotic spindles. Augmin is a recently identified protein complex that has been hypothesized to induce microtubule nucleation from the side of preexisting microtubules. By depleting augmin, we found that microtubule nucleationwas reduced and that spindles were morphologically impaired. Spindles were predominantly multipolar but finally reached bipolarity as a result of a newly uncovered augmin-independent microtubule nucleation pathway from acentrosomal poles. Our results thus reveal that augmin is essential for the proper establishment of the microtubule scaffolding and the bipolarity ofacentrosomal spindles. Secondly, we investigated the functions of the chromokinesins kinesin-4 (Xklp1) and kinesin-10 (Xkid)in acentrosomal spindle architecture and motions. Xkid plays a major role in the polar ejection forces leading chromosome movements during congression while the main function of XKlp1 is to regulate microtubule dynamics. We studied spindle assembly in depleted extracts and we report that Xkid limits the dynamics of spindle longitudinal movements, contributes to spindle bipolarity and affects spindle length while XKlp1 controls the spindle microtubule mass. Altogether these findings contribute to a better understanding of meiotic spindle assembly and confirm the pertinence of our method to study spindle morphogenesis.

Theory predicts that sexually antagonistic mutations will be over- or under-represented on the X and Z chromosomes, depending on the average dominance coefficient of the mutations. However, as little is known about the dominance coefficients for new mutations, the effect of sexually antagonistic selection is difficult to predict. To elucidate the role of sexually antagonistic selection in the evolution of Z chromosome gene content in chicken, we analyzed publicly available microarray data from several somatic tissues as well as somatic and germ cells of the ovary. We found that the Z chromosome is enriched for genes showing preferential expression in ovarian somatic cells, but not for genes with preferential expression in primary oocytes or non-sex-specific somatic tissues. Our results suggest that sexual antagonism leads to higher abundance of female-benefit alleles on the Z chromosome. No bias towards Z-linkage of oocyte-enriched genes can be explained by lower intensity of sexually antagonistic selection in ovarian germ cells compared to ovarian somatic cells. An alternative explanation would be that meiotic Z chromosome inactivation hinders accumulation of oocyte-expressed genes on the Z chromosome. Our results are consistent with findings in mammals and indicate that recessive rather than dominant...

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