Made available in DSpace on 2014-06-11T19:29:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-04-20Bitstream added on 2014-06-13T20:59:29Z : No. of bitstreams: 1 crocomo_lf_me_botfmvz.pdf: 25649116 bytes, checksum: 05169c2ff45ce468070cb0f57b50ab2f (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Este estudo visou avaliar e comparar a eficiência e reversibilidade da inibição meiótica promovida pela roscovitina e ciclohexemida nos COCs de ovinos cultivados in vitro, assim como analisar o efeitos desses inibidores da meiose sobre a ultraestrutura dos COCs e expansão das células do cumulus. Para isto, COCs grau 1 e 2 recuperados de ovários de ovelhas, obtidos em frigoríficos, foram cultivados, por 24 horas a 38,5ºC e atmosfera com 5% de CO2, em meio de maturação constituído por TCM 199 suplementado com 0,1UI/mL de FSH; 0,1UI/mL de LH; 0,3mM de piruvato; 75µg/mL de penicilina/estreptomicina; 10% SFB e 100µM/mL de cisteamina (Grupo Controle) acrescido de 100μM de roscovitina ou 1μg/mL de ciclohexemida (Grupos Tratamentos). Para retomada e progressão da maturação nuclear, os COCs foram cultivados por mais 22 horas em meio de maturação livre de inibidores da meiose, nas mesmas condições acima descritas. Após 24 e 46 horas de cultivo in vitro, os COCs foram avaliados quanto à expansão das células cumulus em estereomicroscópio, quanto à maturação nuclear em microscópio de fluorescência e quanto à maturação citoplasmática, em microscópio eletrônico de transmissão. Foi constatado adequado bloqueio meiótico em 53,07% e 89,67% dos COCs tratados com roscovitina e ciclohexemida, respectivamente. Em ambos os tratamentos, o bloqueio meiótico foi reversível, com aproximadamente 60% dos oócitos em MII ao final de 46 horas de cultivo in vitro. No entanto, o tratamento com roscovitina inibiu de maneira irreversível a expansão do cumulus em aproximadamente 90% dos COCs e também afetou a ultraestrutura oocitária. Em contrapartida, no tratamento com ciclohexemida, 84,23% dos COCs apresentaram expansão total do cumulus ao final de 46 horas de cultivo in vitro, e houve adequada progressão da maturação citoplasmática, o que denota que a ciclohexemida confirmou a hipótese proposta no presente estudo / This study aimed to evaluate and compare the efficiency and reversibility of meiotic inhibition promoted bv roscovitine and cycloheximide in ovine COCs in vitro cultured, as well as, to assess the effects of these inhibitors on the ultrastructure of COCs and on the cumulus cells expansion. To this, grades I and II COCs recovered from ovines ovaries, obtained from slaughterhouse, were cultured, for 24 hours at 38.5ºC in a 5% CO2 humidified air atmosphere, in a specific maturation medium consisting of TCM 199 supplemented with 0,1UI/mL FSH, 0,1UI/mL LH, 1µg/mL estradiol, 0,3mM sodium pyruvate, 75µg/mL penicillin/ streptomycin, 10% FBS and 100µM/mL cysteamine (Control Group) plus 100 μM roscovitine or 1μg/ml cycloheximide (Treatment Groups). For the resumption and progression of nuclear maturation, COCs were cultured for another 22 hours in maturation medium free of meiosis inhibitors in the same conditions (as described before). After 24 and 46 hours of in vitro culture, COCs were evaluated according to cumulus cells expansion under stereomicroscope, to nuclear maturation under fluorescence microscope and to cytoplasmic maturation under electronic transmission microscope. There was adequate meiotic arrest in 53,07% and 89,67% of COCs treated with roscovitine and cycloheximide, respectively. In both treatments, the meiotic arrest was reversible, with approximately 60% MII oocytes at the end of 46 hours of in vitro culture. However, treatment with roscovitine irreversibly inhibited the cumulus expansion by approximately 90% of COCs and also affected the ultrastructure of COC. In contrast, in the treatment with cycloheximide, 84,23% of COCs showed total cumulus expansion at the end of 46 hours of in vitro culture, and there was adequate progression of cytoplasmic maturation, which indicates that the cycloheximide confirmed the hypothesis proposed in this study

Les crossing-overs (CO) sont issus d’échange réciproque de matériel génétique entre les chromosomes homologues. Les COs produisent de la diversité génétique et sont essentiels chez la plupart des eucaryotes, pour la distribution équilibrée des chromosomes lors de la méiose. Malgré leur importance, et un large excès de précurseurs moléculaires, le nombre de CO est très limité dans la grande majorité des espèces (Typiquement 1 à 4 par paire de chromosomes). Cela suggère que les COs sont étroitement régulés, mais les mécanismes sous-jacents sont mal connus. Pour identifier les gènes qui limitent la formation des CO, l’équipe a mené un crible génétique chez Arabidopsis thaliana. Ces travaux ont mené à l’identification de plusieurs facteurs anti-CO, définissant trois voies : (i) L’hélicase FANCM et ses co-facteurs ; (ii) L’AAA-ATPase FIDGETIN-LIKE-1 (FIGL1) ; (iii) Le complexe RECQ4-Topoisomerase 3α-RMI1.Le premier objectif de ma thèse est d’explorer les relations entre ces trois voies en s’attachant aux questions suivantes ; (1) Jusqu’où peut-on augmenter la recombinaison en combinant les mutations dans FANCM, FIGL1 et RECQ4 ? Nous avons montré que la plus forte augmentation de recombinaison était obtenue dans recq4 figl1, atteignant 7,5 fois la fréquence du sauvage en moyenne sur le génome. (2) Quel est la distribution de ces extra-COs ? L’augmentation de recombinaison n’est pas homogène le long du génome : Les fréquences de CO augmente fortement des centromères vers les télomères, avec les plus hautes fréquences observées dans les régions distales. (3) La modification des fréquences de recombinaison est-elle identique lors des méioses mâles et femelle ? Chez le sauvage, la fréquence de recombinaison est plus élevée lors de la méiose mâle que femelle. Au contraire, la recombinaison femelle devient plus élevée que la recombinaison mâle chez les mutants recq4 et recq4 figl1. Ceci suggère que des contraintes qui s’appliquent sur la formation des CO lors de la méiose femelle sont relâchées chez ces mutants. En poursuivant le crible génétique, un nouveau mutant hyper-recombinant a été identifié. Le second objectif de ma thèse fut d’identifier et de caractériser fonctionnellement le gène correspondant. Une cartographie génétique et des études d’interactions protéine-protéine, ont mené à l’identification d’un facteur qui interagit directement avec FIGL1 et semble former un complexe conservé depuis les plantes jusqu’au mammifères. Nous avons baptisé cette protéine FLIP (Fidgetin-like-1 interacting protein). Les fréquences de recombinaisons sont augmentées dans flip-1, confirmant que FLIP1 limite la formation des COs. Des études d’épistasie ont montré que FLIP et FIGL1 agissent dans la même voie. De plus les protéines FIGL1/FLIP d’Arabidopsis ou humaine, interagissent avec RAD51 et DMC1, les deux protéines qui catalyse une étape clef de la recombinaison, l’invasion d’un ADN homologue. Finalement, dans flip comme dans figl1, la dynamique de DMC1 est modifiée. Nous proposons donc un modèle dans lequel le complexe FLIP-FIGL1 régule négativement l’activité de RAD51/DMC1 pour limiter la formation des COs. L’étude du complexe conservé FLIP-FIGL1 a mis en évidence un nouveau mode de régulation de la recombinaison, qui agit vraisemblablement à l’étape clé de l’échange de brin homologue. De plus, l’augmentation des CO sans précédent obtenues chez recq4 figl1 peut être d’un grand intérêt pour l’amélioration des plantes en permettant de diversité de nouvelles combinaisons alléliques. / Meiotic crossovers (CO) are formed by reciprocal exchange of genetic material between the homologous chromosomes. CO generate genetic diversity and are essential for the proper segregation of chromosomes during meiosis in most eukaryotes. Despite their significance and a large excess of CO precursors, CO number is very low in vast majority of species (typically one to three per chromosome pair). This indicates that COs are tightly regulated but the underlying mechanisms of this limit remain elusive. In order to identify genes that limit COs, a genetic screen was performed in Arabidopsis thaliana. This led to the identification and characterization of several anti-CO factors belonging to three different pathways: (i) The FANCM helicase and its cofactors (ii) The AAA-ATPase FIDGETIN-LIKE-1 (FIGL1) (iii) The RECQ4 -Topoisomerase 3α-RMI1 complex. The first objective was to understand the functional relationship between these three pathways and to address following questions: (1) how far can we increase recombination when combining mutations in FANCM, FIGL1 and RECQ4? We show that the highest increase in recombination was obtained in figl1 recq4, reaching to 7.5 fold the wild type level, on average genome wide. (2) How is the distribution of recombination events genome wide in mutants? The increased CO frequency in the mutants was not uniform throughout the genome. CO frequency rises from the centromere to telomeres, with distal intervals having highest COs (3) is the recombination frequency increase same in both male and female? In Arabidopsis wild type, male has higher recombination than female meiosis. In contrast, in recq4 and recq4 figl1, female recombination was higher than male. This suggests that certain constraints that apply to CO formation in wild type females are relieved in the mutant. By continuing the same genetic screen, a novel anti-CO mutant was identified. The second objective was to identify and functionally characterize the corresponding gene. Genetic mapping and protein interaction studies led to the identification of a factor that directly interacts with FIGL1 and appears to form a conserved complex both in Arabidopsis and humans. Hence, the factor was named FLIP (Fidgetin-like-1 interacting protein). Recombination frequency is increased in flip, confirming that FLIP limit COs. Epistasis studies showed that FLIP and FIGL1 act in same pathway. Further, FIGL1/FLIP proteins of Arabidopsis and humans directly interact with the recombinases RAD51 and DMC1 which catalyze a crucial step of homologous recombination, the inter homolog strand invasion. In addition flip like figl1 modifies dynamics of DMC1. We thus propose a model wherein the FLIP-FIGL1 complex negatively regulates RAD51/DMC1 to limit CO formation. Studying the conserved FIGL1-FLIP complex led to the identification of a novel mode of regulation of recombination, that likely acts at the key step of homologous strand invasion. Further the unprecedented level of CO increase in recq4figl1 in hybrids could be of great interest for crop improvement, allowing the production of novel allele combinations.

T. vaginalis is a protozoan parasite without an observed sexual stage in its life cycle. However, T. vaginalis has genes, such as Dmc1, known to be involved in meiosis in other organisms. In order to look at the expression of these genes in T. vaginalis, RT-PCR was done using purified mRNA. It shows that Dmc1 is expressed in both normal and drug treated cells. However, relative levels are unclear. Localization studies were done in T. vaginalis using immunofluorescence against Dmc1 protein with an HA tag. These studies showed that recombinant Dmc1 remained in the cytoplasm upon treatment with DNA damaging drugs. Additionally, T.vaginalis Dmc1 protein was expressed and purified from E.coli to have polyclonal antibodies made to use in further immunofluorescence studies.

Meiosis is a specialized form of cell division in sexually reproducing eukaryotes. Crossovers are physical connections formed between homologous chromosomes during meiosis; these connections help ensure normal segregation of homologous chromosomes at meiosis I. However, the yeast Saccharomyces cerevisiae and other eukaryotes can still segregate homologs properly even in the absence of some crossovers. This is due to a backup mechanism known as distributive segregation, which correctly segregates non-crossover chromosomes at a higher rate than if segregation were completely random. To study distributive segregation, we have generated diploid yeast with one homeologous chromosome pair consisting of a Saccharomyces cerevisiae chromosome V and a Saccharomyces carlsbergensis chromosome V. This pair of chromosomes rarely recombine resulting in crossing over occurring in less than 3% of meiosis. Appropriate segregation of this chromosome pair during meiosis will depend on distributive segregation; we can then assess the possible roles of candidate proteins in distributive segregation through determination of the effect of mutation on segregation of this chromosome pair. Our work has focused on the roles of three proteins, Ndj1, Tid1, and Spo16. These three proteins affect meiosis in many ways, including the efficiency of crossover regulation and the overall timing of meiosis, but their roles during distributive segregation are not fully known. A comparison of spore viability among WT, ndj1, and tid1 strains reveals an elevated incidence of 2-spore-viable tetrads (suggestive of chromosome nondisjunction) in ndj1, but not tid1; these results suggest that the Ndj1 protein, but not the Tid1 protein, plays some role in distributive segregation. spo16 strains seem to also show elevated levels of 2-spore-viable tetrads, but due to a lack of data no deductions can be made about the role of Spo16 in distributive segregation.

Yes / We investigated the meiotic role of Srs2, a multi-functional DNA helicase/translocase that destabilises Rad51-DNA filaments and is thought to regulate strand invasion and prevent hyper-recombination during the mitotic cell cycle. We find that Srs2 activity is required for normal meiotic progression and spore viability. A significant fraction of srs2 mutant cells progress through both meiotic divisions without separating the bulk of their chromatin, although in such cells sister centromeres often separate. Undivided nuclei contain aggregates of Rad51 colocalised with the ssDNA-binding protein RPA, suggesting the presence of persistent single-strand DNA. Rad51 aggregate formation requires Spo11-induced DSBs, Rad51 strand-invasion activity and progression past the pachytene stage of meiosis, but not the DSB end-resection or the bias towards interhomologue strand invasion characteristic of normal meiosis. srs2 mutants also display altered meiotic recombination intermediate metabolism, revealed by defects in the formation of stable joint molecules. We suggest that Srs2, by limiting Rad51 accumulation on DNA, prevents the formation of aberrant recombination intermediates that otherwise would persist and interfere with normal chromosome segregation and nuclear division. / Biotechnology and Biological Sciences Research Council (BB/K009346/1)

<p> The mode of chromosome duplication during meiosis and mitosis in Haplopappus gracilis was investigated. Tritiated thymidine was incorporated into the pollen mother cells during premeiotic interphase, and the cells were allowed to reach the tetrad stage. The autoradiographs prepared from the tetrads showed an unequal distribution of grains over their nuclei, suggesting a conservative mode of chromosome duplication during meiosis. Seedlings were fed with tritiated thymidine for the duration of one cell cycle and also for the duration of several cell cycles. The autoradiographs prepared from the root tip cells, thus treated, showed both labelled and unlabeled chromatids in the anaphases of all the experiments, thus again suggesting a conservative mode of chromosome duplication. A chromosome model to explain the results is discussed. </p> / Thesis / Doctor of Philosophy (PhD)

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Meiotic recombination is both a fundamental biological process required for proper chromosomal segregation during meiosis and a fundamental genomic parameter that shapes major features of the genomic landscape. While there is strong evidence of fitness costs of low rates of recombination, the possible fitness costs of high rates of recombination are less defined. To determine whether a single lower fitness bound can explain the variation in recombination rate observed in human populations, we simulated the evolution of recombination rate as a quantitative trait using empirically-derived parameters. For our fitness function, we implemented a hyperbolic tangent curve with flexible parameters to capture a wide range of existing hypotheses. We found that both a lower and upper bound are necessary to explain the observed variation in recombination rate, and we describe a parameter space for an upper bound on recombination rate that produces results consistent with empirical observations.

No description available.

Testicular tissue removed from 10 month old fire-brats, Thermobia domestica Pack., was prepared for cytogenetic examination by the squash technique. Chromatin staining with acetic orcein and the use of phase contrast microscopy simplified preparatory procedures and preserved the greatest amount of cell integrity. Photomicrographs taken of the complete meiotic cycle in the male indicated 34 chromosomes in reduction division -- 32 autosomes + X₁X₂. While all autosomes followed normal division patterns, the X chromosomes remained united by a chromatin strand and moved as a unit to one pole. Resultant products of this uneven division were daughter cells containing either 16 or 18 chromosomes. All diads divided normally during the second phase. Thus, cells with 16 chromosomes produced daughter cells with 16, and cells with 18 chromosomes produced daughter cells with 18, respectively. Spermatozoa produced by this maturation division were of two types -- those with heterochromatin and those without. Both are apparently viable, and no evidence of degeneration of either type was found. All studies, thus far, indicate that in Thermobia individual spermatozoa are non-motile. Two must intertwine for approximately 1/3 their total length in order to produce translocatory movements. / Master of Science