Submitted by Angela Maria de Oliveira (amolivei@uepg.br) on 2017-10-18T13:46:42Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Barbara de Melo.pdf: 1643171 bytes, checksum: abd718922152e41d663027fa3f15740b (MD5) / Made available in DSpace on 2017-10-18T13:46:42Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Barbara de Melo.pdf: 1643171 bytes, checksum: abd718922152e41d663027fa3f15740b (MD5) Previous issue date: 2013-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A subfamília Alticinae é considerada moderna com grande variação de número diplóide e sistema de determinação sexual. Considerando a tribo Systenini apenas seis espécies foram analisadas citogeneticamente e essas espécies possuem grande variação no número cromossômico e no sistema de determinação do sexo. Oedionychina, por outro lado, apresenta espécies com número diploide e sistema de determinação sexual conservados de 2n=22=20+X+y com cromossomos sexuais gigantes e assinápticos. A análise das Regiões Organizadoras de Nucléolo (RONs) tem sido realizada principalmente através de Impregnação pelo Íon Prata, poucas espécies de Alticinae têm sido analisadas através de Hibridação in situ Fluorescente até o presente momento. O mapeamento do gene ribossomal 5S foi analisado apenas em espécies de Scarabaeidae. Com relação ao cístron 45S somente 3 espécies de Alticinae foram descritas. Assim, o objetivo desse trabalho foi caracterizar citogeneticamente seis espécies de Alticinae (Omophoita octoguttata, Omophoita personata, Omophoita magniguttis, Alagoasa coccinelloide, Alagoasa florigera e Systena tenuis), visando estabelecer as diferenças cariotípicas e as estratégias de diferenciação cromossômicas, para compreender as relações evolutivas entre as espécies. A análise citogenética das espécies A. coccinelloide, A. florigera e S. tenuis estão concordantes com os dados da literatura. O número diploide das espécies do gênero Alagoasa é de 2n=22=20+X+y com cromossomos sexuais gigantes e assinápticos que segregam corretamente na anáfase I. S. tenuis apresentou o número diplóide de 2n=32=15II+neoXY. O uso de fluorocromo em S. tenuis mostrou à presença de regiões pericentrométicas ricas em repetições AT. A Hibridação in situ do Genoma confirmou o mecanismo cromossômico neoXY. A realização da FISH com sonda de 18S e 5S evidenciou que em O. octoguttata, O. personata e A. coccinelloide ocorre a presença de um par autossômico portador dos genes ribossomais. O. magniguttis e A. florigera mostraram uma derivação no cariótipo apresentando dois e três pares de cromossomos portadores dos genes ribossomais respectivamente. A dupla FISH mostra que, nas 5 espécies os genes estão colocalizados. S. tenuis evidenciou múltiplos sítios desses genes, e a dupla FISH mostrou os genes estão em cromossomos separados ou colocalizados. A FISH com fibras estendidas mostrou que em todos os casos os genes estão dispostos de forma interespaçada. / The Alticinae subfamily is considered modern with wide variation of the diploid number and sex determination system. Considering the Systenini tribe only 6 species were cytogenetically analyzed and these species exhibit wide variation in the chromosome number and the system of sex determination. Oedionychina, on the other hand, present species with diploid number and sex determination system conserved of 2n=22=20+X+y, with extremely large and asynaptic sex chromosomes. The analysis of Nucleolus Organizer Regions (NORs) has been attempted mainly by impregnation of the Ag-NOR, and a few species of Alticinae have been examined by using Fluorescent in situ Hybridization to date. The mapping of 5S ribosomal gene was analyzed only in Scarabaeidae species. In relation to the 45S cistron only three species of Alticinae have been described. The aim of the present study was characterize cytogenetically 6 species of Alticinae (Omophoita octoguttata, Omophoita personata, Omophoita magniguttis, Alagoasa coccinelloide, Alagoasa florigera e Systena tenuis), in order to determine the karyotype differentiation and the strategies of chromosome differentiation to understand the evolutionary relation among the species. The cytogenetic analysis of A. coccinelloide, A. florigera and S. tenuis are in accordance with the literature data. The diploid number of the Alagoasa species is 2n=22=20+X+y with large sex chromosomes, which are asynaptic although have regular segregation in anaphase I. Systena tenuis presented the diploid number of 2n=32=15II+neoXY. The fluorocrome analysis in S. tenuis showed the presence of pericentromeric region rich in AT repetitions. The Genome In situ Hybridization confirmed the system of sex determination of the type neoXY. Performing FISH with 18S and 5S probe revealed the presence of one autosomal pair carrier the ribosomal genes in O. octoguttata, O. personata and A. coccinelloide. Derivations on karyotype showing two or three pairs of chromosomes carrying of the ribosomal genes have been observed in O. magniguttis and A. florigera, respectively. The double FISH showed that genes are colocalized in the five species. S. tenuis showed multiple sites of these genes, and the double FISH showed the genes are in separate chromosomes or colocalized. The extended fiber FISH showed that in all cases the genes are interspersed arranged.

Made available in DSpace on 2014-06-11T19:26:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-21Bitstream added on 2014-06-13T20:33:40Z : No. of bitstreams: 1 pires_wl_me_sjrp.pdf: 611018 bytes, checksum: 961d88c40b2d3d5b170049ad3384f050 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Os triatomíneos são vetores do protozoário Typanosoma cruzi, agente etiológico da moléstia de Chagas. Esses insetos são hematófagos e pertencem à ordem Heteroptera e à família Reduviidae. Disseminada por grandes extensões do Brasil e de outros países latinoamericanos, a doença de Chagas representa um grave e importante problema de saúde pública, caracterizando limitações e dificuldades aos tratamentos. Isso ocorre devido à precariedade apresentada pela vida dos contingentes humanos mais expostos à infecção. Atualmente, a parasitose tem grande participação entre as doenças cardíacas na América do Sul. Segundo a Organização Mundial de Saúde, estima-se que, até a década de 90, por volta de 20 milhões de indivíduos estavam infectados pelo Trypanosoma cruzi nas áreas endêmicas. Dados atuais revelam que o número de pessoas infectadas foi reduzido para 9,8 milhões graças à intensa erradicação desses insetos. No entanto, a vigilância deve continuar, pois é fundamental para se evitar novos casos. Citogeneticamente, o interesse sobre os triatomíneos está em seus cromossomos holocêntricos e no processo incomum da meiose, cuja segregação dos sexuais é pós-reducional. O número básico de cromossomos nos triatomíneos é de 2n=22. No presente trabalho foi analisado a espermatogênese de duas espécies do gênero Triatoma (Triatoma maculata e Triatoma pseudomaculata), com ênfase aos seguintes aspectos: fases da espermatogênese; estrutura cromatínica e dos cromossomos meióticos e acompanhamento do ciclo nucleolar. Essas espécies estão distribuídas principalmente nos estados do Nordeste brasileiro e são consideradas potencialmente vetores do T. cruzi. As espécies analisadas foram cedidas pelo insetário do Serviço Especial de Saúde de Araraquara (SESA), pertencente ao Departamento de Epidemiologia da Faculdade de Saúde Pública da USP. Os... / The triatomines are vectors of the protozoan Typanosoma cruzi, etiological agent of Chagas' disease. These insects are hematophages that belong to the order Heteroptera and to the family Reduviidae. Disseminated through large portions of Brazil and of other Latin American countries, Chagas' disease represents a grave and important public health problem, characterizing limitations and difficulties in treatments. This occurs due to the precariousness presented by life contingent to humans most exposed to the infection. Currently, parasitosis presents high participation among cardiac diseases in South America. According to the World Health Organization, it is estimated that, until the 1990s, approximately 20 million individuals were infected by Trypanosoma cruzi in endemic areas. Current data reveal that the number of persons infected was reduced to 9.8 million by virtue of intense eradiaction of these insects. Nevertheless, vigilance must continue, since it is fundamental to avoiding new cases. Cytogenetically, the interest in triatomines is in its holocentric chromosomes and in its uncommon meiosis process, whose sexual segregation is post-reductional. The basic number of chromosomes in triatomines is 2n=22. The present work analyzed the spermatogenesis of two species of the genus Triatoma (Triatoma maculata and Triatoma pseudomaculata), with emphasis on the following aspects: spermatogenesis phases; structure of chromatin and of meiotic chromosomes and accompaniment of the nucleolar cycle. These species are distributed principally in the states of northeastern Brazil and are considered potential vectors for T. cruzi. The species analyzed were supplied by the insectary of the Special Health Service of Araraquara (SESA), belonging to the Department of Epidemiology in the School of Public Health at USP. The organs studied were...(Complete abstract click electronic access below)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The process of meiotic resumption in oocytes, arrested since fetal life, and the expansion of compact layers of cumulus cells is triggered by intrafollicular mediators stimulated by LH. These events are extremely complex. In mice, among system components of natriuretic peptides (NP), only the C-type NP (CNP) has a role to inhibit the resumption of meiosis. However, little is know about the function of NPs on resumption of meiosis, nuclear maturation and cumulus expansion in monovular species. The aim of this study was to characterize the natriuretic peptide system, studing its role in the resumption of meiosis and cumulus expansion. We also proposed a new model to study cumulus expansion. Initially, we detected the presence of mRNA for the ANP, CNP, natriuretic peptide receptor 1 (NPR-1), NPR-2 and NPR-3 in the cumulus cells and NPR-2 mRNA in the oocyte. Using an in vitro model, in which the oocytes are arrested in germinal vesicle (VG) by the action of forskolin (100 μM), we demonstrated that ANP, BNP and CNP, alone or in combination, induce resumption of meiosis after 12 h of maturation. In another experiments, we observed that the concentration of 100 μM forskolin inhibited cumulus expansion stimulated by FSH for12 h, which was reversed by adding ANP, BNP and CNP in the COC culture system. Thus, we demonstrated for the first time the localization of mRNA for the NP system in COCs. Furthermore, we found that the ANP, BNP and CNP are likely mediators of LH to induce meiotic resumption and cumulus expansion in monovuluar species, using the bovine as the animal model. / O processo de retomada da meiose no oócito, bloqueada desde a vida fetal, e a expansão de compactas camadas de células do cumulus que o envolvem é desencadeado por mediadores intrafoliculares estimulados pelo LH, sendo eventos extremamente complexos. Em camundongos, dentre os componentes do sistema peptídeos natriuréticos (NP) somente o NP tipo C (CNP) apresenta função, bloqueando a retomada da meiose. Entretanto, em espécie monovular, o conhecimento sobre a ação dos NP, na maturação nuclear de oócitos e expansão do cumulus, é extremamente escasso. O objetivo do presente estudo foi caracterizar o sistema peptídeo natriurético, demonstrar sua função na retomada da meiose e expansão do cumulus, além de propor um novo modelo para estudo da expansão do cumulus. Inicialmente, demonstramos a presença de RNAm para ANP, CNP, receptor peptídeo natriurético 1 (NPR-1), NPR-2 e NPR-3 nas células do cumulus, sendo que no oócito somente foi detectado RNAm do NPR-2. Utilizando um modelo in vitro, no qual os oócitos permanecem bloqueados em vesícula germitava (VG) por ação do forskolin (100 μM), demonstramos que os ANP, BNP e CNP, isoladamente ou em associação, induzem o reinício da meiose após 12 h de maturação. Em outros experimentos, observamos que a concentração de 100 μM de forskolin inibiu, por 12 h, a expansão das células do cumulus estimulada por FSH e que o ANP, BNP e CNP revertem o efeito inibitório do forskolin sobre a expansão do cumulus. Dessa forma, demonstramos pela primeira vez a localização de RNAm para o sistema NP em CCOs. Além disso, foi demonstrado que em espécie monovuluar, utilizando o bovino como modelo animal, os peptídeos natriuréticos (ANP, BNP e CNP) apresentam função de mediadores intrafoliculares do LH, na qual estimulam a retomada da meiose e expansão do cumulus.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The objectives of this study were: to determine the presence of prorenin and the receptor of (pro)renin [(P)RR] in cumulus cells (CC) and oocytes; evaluate the role of prorenin in the resumption of oocyte meiosis, ovulation and on levels of plasma progesterone (P4). The mRNA expression of prorenin and (P) RR was evaluated in oocytes and cumulus cells of the bovine species. The mRNA of prorenin and (P)RR was detected in CC, in oocytes only mRNA expression of (P)RR was found. The role of prorenin in the resumption of meiosis was determined from 3 experiments using a co-culture of cumulus-oocyte complex (COC) and follicular halves. In the first experiment, three different concentrations of prorenin (10-10, 10-9, and 10-8M) were tested, which stimulated the resumption of meiosis similar to the positive control and angiotensin II (Ang II). In experiment 2, the co-culture was supplemented with prorenin (10-10M) and aliskiren (direct inhibitor of renin, 10-3M, 10-5M and 10-7M). The aliskiren blocked the resumption of meiosis-induced by prorenin in all concentrations tested. The aliskiren (10-5M and 10-7M) was also evaluated in isolation in a culture system of COCs without follicular halves and the rate of oocytes that resumed meiosis was not different of the positive control. The third experiment was conducted to evaluate the stimulation of meiosis resumption by prorenin independent of Ang II using the combination of prorenin (10-10M) and saralasin [nonspecific Ang II receptor antagonist - Sar (10-5M)], which obtained a rate of oocytes at metaphase I (MI) similar to the positive control and treatments of AngII and prorenin. The prorenin also resumed the oocyte meiosis in a culture system of COCs suplemmented with forskolin, wicth blocks the meiosis by intracellular cAMP accumulation. To evaluate the regulation of (P)RR mRNA in the theca and granulosa cells by LH, cows who achieved follicular diameter ≥12mm after synchronization were submitted to the ovariectomy 0, 3, 6, 12 and 24 hours after GnRH analogue treatment. There was a higher mRNA expression of (P)RR in the theca cells at hour 6; and at hour 3 in the granulosa cells. The effect of (P)RR in ovulation and plasma P4 during luteinization was observed cows that were synchronized and induced by GnRH analogue (IM) associated for intrafollicular aliskiren (10-5M) or PBS (0hour/0day) and the ovulation was evaluated at 24, 48 and 72 hours by ultrasound, wicth was not different of the control (PBS). The leves of plasma P4 was analysed at day 6 and 8 after GnRH and intrafollicular treatment only in the cows that ovulated. The intrafollicular (P)RR blocks decreased plasma P4 at day 6. It was concluded that the prorenin / (P)RR participates in the peri-ovulatory period: in the resumption of oocyte meiosis independently of AngII, in the ovulation by LH stimulation to (P)RR mRNA and in the P4 synthesis during luteinization. / Os objetivos deste estudo foram: determinar a presença de prorenina e receptor de (pro)renina [(P)RR] nas células do cumulus (CC) e em oócitos; avaliar o papel da prorenina na retomada da meiose oocitária; na ovulação e sobre os níveis de progesterona plasmática (P4). A expressão do RNAm de prorenina e (P)RR foi avaliada em oócitos e CC da espécie bovina. O RNAm de prorenina e (P)RR foi detectado nas células do cumulus; nos oócitos ocorreu apenas expressão de RNAm de (P)RR. O papel da prorenina na retomada da meiose foi determinado a partir de 3 experimentos utilizando um sistema de co-cultivo de complexo-cumulus oócito (COC) e metades foliculares. No primeiro experimento, foram testadas três concentrações de prorenina (10-10; 10-9; e 10-8M), as quais estimularam a retomada da meiose semelhante ao controle positivo e angiotensina II (Ang II). No experimento 2, o co-cultivo foi suplementado com prorenina (10-10M) e alisquireno (inibidor direto de renina; 10-3M, 10-5M e 10-7M). O alisquireno bloqueou a retomada da meiose induzida pela prorenina em todas concentrações testadas. O alisquireno (10-5M e 10-7M) também foi avaliado isoladamente em sistema de cultivo de COC sem metades foliculares e a taxa de oócitos que retomaram a meiose não diferiu do controle positivo. O experimento 3 foi realizado para avaliar o estímulo da retomada da meiose pela prorenina independente da Ang II utilizando a associação de prorenina (10-10M) e saralasina [antagonista do receptor da Ang II - Sar (10-5M)], a qual obteve taxa de oócitos em metáfase I (MI) semelhante ao controle positivo e aos tratamentos AngII e prorenina. A prorenina também retomou a meiose oocitária em cultivo de COCs suplementado com forskolin, que bloqueia a meiose pelo acumulo de AMPc intracelular.Para avaliar a possível influência do pico de LH na expressão do RNAm de (P)RR nas células da teca e granulosa, vacas que apresentaram folículo ≥12mm de diâmetro após a sincronização farmacológica do ciclo estral foram ovariectomizadas 0, 3, 6, 12 e 24 horas após aplicação do análogo do GnRH. Houve maior expressão de RNAm de (P)RR nas células da teca na hora 6; e na hora 3 nas células da granulosa. O papel do (P)RR na ovulação e sobre a P4 plasmática durante a luteinização foi verificado em vacas sincronizadas farmacológicamente, induzidas pelo análogo de GnRH (IM) associado ao alisquireno (10-5M) ou PBS intrafolicular (hora 0/ dia 0) e foram avaliadas por ultrason 24, 48 e 72 horas quanto a ovulação, a qual não diferiu do controle (PBS). Os níveis de P4 plasmática foram analisados nos dias 6 e 8 após GnRH e tratamento intrafolicular apenas nas vacas que ovularam, nas quais o bloqueio de (P)RR reduziu P4 no dia 6. Concluiu-se que a prorenina/(P)RR participa do periodo peri-ovulatório: na retomada da meiose oocitária de forma independente da AngII, na ovulação pelo estímulo de LH sobre RNAm do (P)RR e na síntese de P4 durante a luteinização.

Orientador: Maria Tercília Vilela de Azeredo Oliveira / Banca: Alba Regina de Abreu Lima Catelani / Banca: Lilian Castiglioni / Resumo: Os triatomíneos são vetores do protozoário Typanosoma cruzi, agente etiológico da moléstia de Chagas. Esses insetos são hematófagos e pertencem à ordem Heteroptera e à família Reduviidae. Disseminada por grandes extensões do Brasil e de outros países latinoamericanos, a doença de Chagas representa um grave e importante problema de saúde pública, caracterizando limitações e dificuldades aos tratamentos. Isso ocorre devido à precariedade apresentada pela vida dos contingentes humanos mais expostos à infecção. Atualmente, a parasitose tem grande participação entre as doenças cardíacas na América do Sul. Segundo a Organização Mundial de Saúde, estima-se que, até a década de 90, por volta de 20 milhões de indivíduos estavam infectados pelo Trypanosoma cruzi nas áreas endêmicas. Dados atuais revelam que o número de pessoas infectadas foi reduzido para 9,8 milhões graças à intensa erradicação desses insetos. No entanto, a vigilância deve continuar, pois é fundamental para se evitar novos casos. Citogeneticamente, o interesse sobre os triatomíneos está em seus cromossomos holocêntricos e no processo incomum da meiose, cuja segregação dos sexuais é pós-reducional. O número básico de cromossomos nos triatomíneos é de 2n=22. No presente trabalho foi analisado a espermatogênese de duas espécies do gênero Triatoma (Triatoma maculata e Triatoma pseudomaculata), com ênfase aos seguintes aspectos: fases da espermatogênese; estrutura cromatínica e dos cromossomos meióticos e acompanhamento do ciclo nucleolar. Essas espécies estão distribuídas principalmente nos estados do Nordeste brasileiro e são consideradas potencialmente vetores do T. cruzi. As espécies analisadas foram cedidas pelo insetário do Serviço Especial de Saúde de Araraquara (SESA), pertencente ao Departamento de Epidemiologia da Faculdade de Saúde Pública da USP. Os...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The triatomines are vectors of the protozoan Typanosoma cruzi, etiological agent of Chagas' disease. These insects are hematophages that belong to the order Heteroptera and to the family Reduviidae. Disseminated through large portions of Brazil and of other Latin American countries, Chagas' disease represents a grave and important public health problem, characterizing limitations and difficulties in treatments. This occurs due to the precariousness presented by life contingent to humans most exposed to the infection. Currently, parasitosis presents high participation among cardiac diseases in South America. According to the World Health Organization, it is estimated that, until the 1990s, approximately 20 million individuals were infected by Trypanosoma cruzi in endemic areas. Current data reveal that the number of persons infected was reduced to 9.8 million by virtue of intense eradiaction of these insects. Nevertheless, vigilance must continue, since it is fundamental to avoiding new cases. Cytogenetically, the interest in triatomines is in its holocentric chromosomes and in its uncommon meiosis process, whose sexual segregation is post-reductional. The basic number of chromosomes in triatomines is 2n=22. The present work analyzed the spermatogenesis of two species of the genus Triatoma (Triatoma maculata and Triatoma pseudomaculata), with emphasis on the following aspects: spermatogenesis phases; structure of chromatin and of meiotic chromosomes and accompaniment of the nucleolar cycle. These species are distributed principally in the states of northeastern Brazil and are considered potential vectors for T. cruzi. The species analyzed were supplied by the insectary of the Special Health Service of Araraquara (SESA), belonging to the Department of Epidemiology in the School of Public Health at USP. The organs studied were...(Complete abstract click electronic access below) / Mestre

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-08-15T17:53:44Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) A_Marques_PhD_thesis_2016_FINAL.pdf: 9324057 bytes, checksum: 92c1de27f6fa947d7e6b3e74ae09a849 (MD5) / Made available in DSpace on 2016-08-15T17:53:46Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) A_Marques_PhD_thesis_2016_FINAL.pdf: 9324057 bytes, checksum: 92c1de27f6fa947d7e6b3e74ae09a849 (MD5) Previous issue date: 2015-02-15 / capes / Cromossomos holocêntricos são caracterizados pela ausência de constrição primária e apresentam normalmente a proteína centromérica CENH3 distribuída ao longo de um eixo em cada cromátide. Embora muitos organismos com cromossomos monocêntricos apresentem sequências de DNA centroméricas específicas e associadas com a CENH3, nenhuma sequência centromérica havia sido identificada em organismos com cromossomos holocêntricos até o momento. Além disso, vários estudos reportam adaptações meióticas em espécies com cromossomos holocêntricos. Sendo observada em alguns casos uma inversão da ordem dos eventos meióticos (meiose invertida ou pós-reducional). Assim, o presente trabalho objetivou estudar a organização centromérica e a meiose de espécies com cromossomos holocêntricos do gênero Rhynchospora (Cyperaceae). Foi realizada uma análise citogenômica da organização e composição dos holocentrômeros de Rhynchospora pubera (2n = 10), sendo reportada a primeira descoberta de sequências centroméricas em espécies com cromossomos holocêntricos. Foi observado que os holocentrômeros de R. pubera são compostos principalmente por arranjos de DNA satélite (Tyba) e retroelementos centroméricos (CRRh) distribuídos pelo genoma. A análise detalhada da sucessão dos eventos meióticos de R. pubera e R. tenuis (2n = 4) reportou uma prófase inicial semelhante a de monocêntricos. No entanto, foi verificado que as cromátides-irmãs separam para polos opostos durante a anáfase I e os homólogos segregam somente durante a meiose II, comprovando uma meiose invertida para ambas as espécies. Curiosamente, durante a meiose de R. pubera foi observado uma organização diferencial dos centrômeros. Ao contrário do observado em mitose, durante meiose não foi observado a formação de holocentrômeros em forma de linha, sendo, na verdade, observado estruturas centroméricas aglomeradas. O restabelecimento de holocentrômeros em forma de linha se deu durante a primeira mitose do pólen. / Holocentric chromosomes are characterized by the absence primary constriction and normally show the centromeric protein CENH3 distributed along the axis of each chromatid. Although many monocentric organisms show centromere-specific DNA sequences associated to CENH3, no centromeric sequences had been identified in any holocentric organism so far. Furthermore, many studies report meiotic adaptations in holocentric species. In some cases is observed an inversion of the order of meiotic events. This type of meiosis has been named of inverted or post-reductional meiosis and would be exclusive of holocentric organisms. Thus, the present work aimed to study the centromere organization and meiosis of holocentric species of the genus Rhynchospora (Cyperaceae). A cytogenomic analysis of the composition and organization of the holocentromeres of Rhynchospora pubera (2n = 10) has been performed, being reported the first centromeric sequences from a holocentric species. It was observed that the holocentromeres of R. pubera are composed mainly by arrays of satellite DNA (Tyba) and centromeric retrotransposons (CRRh) distributed genomewide. The detailed analysis of the succession of meiotic events of R. pubera and R. tenuis (2n = 4) demonstrated an early meiotic prophase similar to that of monocentric. However, it was verified that sister chromatids separate to opposite poles during anaphase I, while homologs only segregate at meiosis II. These results prove the inverted meiosis for both species. Curiously, it was observed during meiosis of R. pubera a differential organization of centromere units. In contrast to the observed in mitosis, during meiosis we did not observed the formation of line-like holocentromeres, being in fact observed the formation of cluster-like holocentromeres. The reestablishment of a line-like holocentromere occurred during the first pollen mitosis.

O óxido nítrico (NO) é um mensageiro químico detectado em vários tipos celulares como células endoteliais, neurônios e macrófagos, desempenhando também funções variadas como vasodilatação, neurotransmissão e indução de morte celular. O NO é gerado pela atividade da enzima óxido nítrico sintase (NOS), a qual foi detectada em vários órgãos incluindo o sistema reprodutor. O sistema NOS/NO parece desempenhar papel importante na maturação oocitária entre outras funções. No entanto, apesar das evidências, há poucos estudos sobre o possível papel desse sistema em oócitos da espécie bovina. O presente estudo teve por objetivo investigar a importância do sistema NOS/NO na maturação in vitro (MIV) de oócitos bovinos. Para isso, foram avaliados os efeitos da inibição da NOS durante a MIV na presença de concentrações crescentes do inibidor de NOS (L-NAME, 0-1mM) e do aumento do NO durante a MIV na presença de concentrações crescentes do doador de NO (SNAP, 0-1mM) sobre a taxa de maturação (metáfase II) e formação de blastocistos após a fecundação in vitro. Também foi avaliado o efeito da pré-maturação com butirolactona (10µM BLI), seguida de MIV, na presença ou não de doador ou inibidor de NO em cada fase de cultivo, sobre o desenvolvimento de blastocistos. Após 22h de MIV os grupos tratados com L-NAME apresentaram uma taxa de metáfase II (MII) inferior (~80%, P<0,05) ao controle (95,5%). A taxa de blastocisto foi similar entre os grupos (34 a 41,5%, P>0,05). Apenas 7,2% (P<0,05) dos oócitos maturados com a maior concentração de SNAP (10-3M), atingiram o estádio de MII. Os demais tratamentos (71,6; 72,4 e 54,8% para controle, 10-7 e 10-5M, respectivamente) não diferiram entre si (P>0,05). Altos níveis de SNAP (10-3M) bloquearam o desenvolvimento embrionário, enquanto os demais tratamentos apresentaram cerca de 38% de blastocistos (P>0,05). As taxas de blastocistos (26,3 a 34,1%) e de eclosão (14,8 a 22,0%) adicionando ou não L-NAME em baixa concentração (10-7M) durante a pré-maturação, a maturação ou ambas as fases foram similares (P>0,05). Também não houve diferença (P>0,05) na taxa de blastocistos (25,5 a 39,7%) com a adição ou não de baixa concentração de SNAP (10-7M) durante a pré-maturação, a maturação ou ambas as fases. O grupo com SNAP (10-7M) na pré-maturação e MIV superou a taxa de eclosão (27,5%, P<0,05) dos demais grupos que tiveram SNAP adicionado somente no bloqueio, na MIV ou sem adição de SNAP (14,2 a 18,6 %, P>0,05). Esses resultados exibiram o duplo efeito do NO sobre oócitos bovinos, que tiveram a maturação reduzida com a inibição da síntese de NO e a maturação nuclear e citoplasmática bloqueadas pelo excesso de NO. / Nitric oxide (NO) is a chemical messenger detected in several cell types such as endothelial cells, neurons and macrophages, performing also varied functions as vasodilatation, neurotransmission and induction of cell death. NO is generated by the activity of the enzyme nitric oxide synthase (NOS), which has been detected in several organs including the reproductive system. The NOS/NO system seems to play an important role in oocyte maturation besides other functions. However, despite the evidence, there are few studies on the possible role of this system in bovine oocytes. The present study aimed to investigate the importance of the NOS/NO system on in vitro maturation (IVM) of bovine oocytes. The effects of NOS inhibition during IVM in the presence of increasing concentrations of NOS inhibitor (L-NAME, 0-1mM) and of the increase in NO during IVM with the NO donor SNAP (0-1mM) on maturation rates (metaphase II) and on blastocyst development after in vitro fertilization were assessed. The effect of prematuration with butyrolactone I (10µM BLI) followed by IVM, in the presence or not of NO inhibitor or donor in each culture period on blastocyst development was also investigated. After 22h IVM, L-NAME treated groups showed a lower (~80%, P<0.05) metaphase II (MII) rate when compared with controls (95.5%). Blastocyst rates were similar among all groups (34 to 41.5%, P>0.05). Only 7.2% (P<0.05) of oocytes matured with the highest SNAP concentration (10-3M) reached MII. The other treatments (71.6; 72.4 and 54.8% for control, 10-7 and 10-5M, respectively) were similar among them (P>0.05). High SNAP concentration (10-3M) blocked blastocyst development, while the other treatments presented about 38% blastocyst rates (P<0.05). Blastocyst (26.3 to 34.1%) and hatching rates (14.8 and 22.0%) were similar (P>0.05) when low L-NAME concentration (10-7M) was added or not during prematuration, maturation or both. Blastocyst rates (25.5 to 39.7%) were also similar (P>0.05) whether SNAP (10-7M) was added or not during prematuration, IVM or both. When low concentration of SNAP (10-7M) was added during both prematuration and IVM, hatching rates were increased (27.5%, P<0.05) when compared with oocytes cultured in the presence of SNAP only during prematuration or IVM or without it (14.2 to 18.6 %, P>0.05). These results shoe the dual effect of NO on bovine oocytes, which had maturation rates decreased when NO synthesis was inhibited and nuclear maturation and blastocyst development were blocked by excess NO.

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.

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.