Made available in DSpace on 2014-06-11T19:26:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-19Bitstream added on 2014-06-13T20:54:01Z : No. of bitstreams: 1 castanhole_mmu_me_sjrp.pdf: 2031630 bytes, checksum: 3da893a45e11184e2cff1d69ff7649e4 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os aspectos da espermatogênese e do comportamento nucleolar foram analisados em Brachymetra albinerva, Cylindrostethus palmaris, Halobatopsis platensis, Limnogonus aduncus (Gerridae), Martarega sp. (Notonectidae), Rhagovelia whitei e Rhagovelia sp. (Veliidae). Os testículos são arredondados (Veliidae), alongados (Gerridae) ou espiralados (Notonectidae) e apresentam membrana transparente recobrindo-os. O complemento cromossômico encontrado foi de 2n = 23 (22A + X0, L. aduncus e R. sp.); 25 (24A + X0, B. albinerva e H. platensis); 26 (22A + 2m + XY, Martarega sp.); 29 (28A + X0, C. palmaris) ou 39 (38A + X0, R. whitei) cromossomos, sendo que a única espécie com sistema cromossômico do sexo diferente foi M. sp., que apresentou sistema XY, além de ser, também, a única espécie com m-cromossomos. O comportamento meiótico de todas as espécies foi semelhante, isto é, apresentaram: cromossomos holocêntricos, material heteropicnótico na prófase; quiasmas intersticiais e/ou terminais; primeira divisão reducional para os autossomos e o inverso para os cromossomos sexuais. A única diferença observada foi com relação ao tamanho extremamente maior das células de M. sp., em todas as fases da espermatogênese. Com relação ao comportamento nucleolar as espécies não apresentaram diferenças, somente M. sp. que possui nucléolos maiores que as demais espécies. A única espécie na qual foi possível identificar com clareza a região da RON foi L. aduncus, na região terminal de um autossomo. Confirmou-se, também, através da espécie L. aduncus, que as associações teloméricas não ocorrem ao acaso. Nas demais espécies a marcação da RON foi bastante discreta, não sendo possível afirmar com clareza onde ela está localizada. / The aspects of spermatogenesis and nucleolar behaviour were analyzed in Brachymetra albinerva, Cylindrostethus palmaris, Halobatopsis platensis, Limnogonus aduncus (Gerridae), Martarega sp. (Notonectidae), Rhagovelia whitei and Rhagovelia sp. (Veliidae). The testicles are rounded (Veliidae), elongated (Gerridae) or spiral (Notonectidae) and have a transparent membrane covering them. The complement chromosome was 2n = 23 (22A + X0, L. aduncus and R. sp.), 25 (24A + X0, B. albinerva and H. platensis), 26 (22A + 2m + XY, Martarega sp.), 29 (28A + X0, C. palmaris) or 39 (38A + X0, R. whitei) chromosomes, and the only specie with different sex chromosome system was M. sp., which presented XY system and m-chromosomes. The meiotic behavior of all species was similar: holocentric chromosomes, heteropicnotic material at prophase; chiasmas interstitial and/or terminal; first reductional division for the autosomes and the reverse for the sex chromosomes. The only difference observed was related to the very largest size of the cells M. sp. in all stages of spermatogenesis. With regard to the nucleolar behavior, the species did not show differences, only M. sp. which has nucleoli larger than the other species. The only species in which it was clearly possible to identify the region of NOR was L. aduncus, in the region of a terminal autosome. It was also confirmed that the telomeric associations do not occur at random. In other species the marking was very discreet, no one can clearly say where NOR is located.

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The nucleolus is the site for rRNA synthesis, a process requiring the recruitment of many proteins involved in ribosomal biogenesis. Nrap is a novel nucleolar protein found to be present in all eukaryotes. Preliminary characterisation of Nrap suggested it was likely to participate in ribosome biogenesis but as with many other nucleolar proteins, the functional role of Nrap is largely unknown. In this study, the role of mammalian Nrap in the nucleolus and in ribosome biogenesis was explored. Initially, a number of tools were generated to investigate Nrap function. This involved raising and purifying a polyclonal antibody against the N-terminal region of Nrap. The anti-Nrap antibody was found to detect two Nrap bands in mouse fibroblast cells, possibly corresponding to the two mouse Nrap isoforms, and . In addition, mammalian expression vectors containing the full Nrap sequence as well as deletion constructs were created. The subcellular localisation of each construct was observed by fluorescent microscopy. It was revealed that recombinant Nrap did not localise to the nucleolus, possibly because it was exported to undergo degradation by the 26S proteasome. Two putative NLSs were found to be responsible for directing Nrap to the nucleus but a region accountable for nucleolar localisation was not identified. The data indicated that multiple domains working together are likely to direct Nrap to the nucleolus. Nrap was also observed to co-localise with nucleolar proteins B23 and p19ARF. Moreover, it was shown by reciprocal immunoprecipitation that these three nucleolar proteins existed in a complex in unsynchronised mouse fibroblast cells. Recent reports demonstrated a complex relationship between B23 and p19ARF although the functional significance remained unclear. Nrap's in vivo association with B23 and p19ARF indicated a specific functional role in the nucleolus. Nrap knockdown using siRNA significantly increased B23 protein levels in a dose-dependent manner and down-regulated p19ARF protein levels at higher siRNA concentration. Preliminary studies also implicated Nrap in cell proliferation through these novel interactions. Both endogenous and recombinant Nrap were found to be highly unstable suggesting that Nrap might regulate B23 and p19ARF through its own tightly regulated stability. Finally, the role of Nrap in rRNA processing was investigated by northern blot analysis. Nrap knockdown was found to affect the levels of 45S, 32S and 28S rRNAs. The changes found may be a consequence of the concurrent perturbation in the levels of B23 and p19ARF caused by Nrap knockdown. As the results were not consistent with previous reports, it was likely that changes to rRNA processing could be contributed to Nrap loss of function. This study demonstrated for the first time a functional role of Nrap in rRNA processing possibly through its association with B23 and p19ARF.

Ribosomal proteins (RPs), synthesized in the cytoplasm, need to be transported from the cytoplasm to the nucleolus (a nuclear compartment), where a single molecule of each RP assembles with rRNAs to form the large and small ribosomal subunits. The objectives of this research were to identify nuclear/nucleolar localization signals (NLSs/NoLSs; generally basic motifs) that mediate the transport of Arabidopsis RPL23aA, RPL15A and RPS8A into the nucleus and nucleolus, and to study transcriptional regulation and subcellular localization of RPs. While all previous research has shown that nucleolar localization of proteins is mediated by specific basic motifs, in this study, I showed that a specific number of basic motifs mediated nucleolar localization of RPL23aA, rather than any specific motifs. In this protein, single mutations of any of its eight putative NLSs (pNLSs) had no effect on nucleolar localization, however, the simultaneous mutation of all eight completely disrupted nucleolar localization, but had no effect on nuclear localization. Furthermore, mutation of any four of these pNLSs had no effect on localization, while mutation of more than four increasingly disrupted nucleolar localization, suggesting that any combination of four of the eight pNLSs is able to mediate nucleolar localization. These results support a charge-based system for the nucleolar localization of RPL23aA. While none of the eight pNLSs of RPL23aA were required for nuclear localization, in RPS8A and RPL15A, of the 10 pNLSs in each, the N-terminal two and three NLSs, respectively, were absolutely required for nuclear/nucleolar localization. Considering the presence of only a single molecule of each RP in any given ribosome, which obligates the presence of each RP in the nucleolus in equal quantities, I studied transcriptional regulation of Arabidopsis RP genes and the subcellular localization of five RP families to determine the extent of coordinated regulation of these processes. Variation of up to 300-fold was observed in the expression levels of RP genes. However, this variation was drastically reduced when the expression level was considered at the RP gene family level, indicating that coordinate regulation of expression of RP genes, coding for individual RP isoforms, is more stringent at the family level. Subcellular localization also showed differential targeting of RPs to the cytoplasm, nucleus and nucleolus, together with a significant difference in the nucleolar import rates of RPS8A and RPL15A. Although one could expect coordinated regulation of the processes preceding ribosomal subunit assembly in the nucleolus, my results suggest differential regulation of these processes.

Ribosomal proteins (RPs), synthesized in the cytoplasm, need to be transported from the cytoplasm to the nucleolus (a nuclear compartment), where a single molecule of each RP assembles with rRNAs to form the large and small ribosomal subunits. The objectives of this research were to identify nuclear/nucleolar localization signals (NLSs/NoLSs; generally basic motifs) that mediate the transport of Arabidopsis RPL23aA, RPL15A and RPS8A into the nucleus and nucleolus, and to study transcriptional regulation and subcellular localization of RPs. While all previous research has shown that nucleolar localization of proteins is mediated by specific basic motifs, in this study, I showed that a specific number of basic motifs mediated nucleolar localization of RPL23aA, rather than any specific motifs. In this protein, single mutations of any of its eight putative NLSs (pNLSs) had no effect on nucleolar localization, however, the simultaneous mutation of all eight completely disrupted nucleolar localization, but had no effect on nuclear localization. Furthermore, mutation of any four of these pNLSs had no effect on localization, while mutation of more than four increasingly disrupted nucleolar localization, suggesting that any combination of four of the eight pNLSs is able to mediate nucleolar localization. These results support a charge-based system for the nucleolar localization of RPL23aA. While none of the eight pNLSs of RPL23aA were required for nuclear localization, in RPS8A and RPL15A, of the 10 pNLSs in each, the N-terminal two and three NLSs, respectively, were absolutely required for nuclear/nucleolar localization. Considering the presence of only a single molecule of each RP in any given ribosome, which obligates the presence of each RP in the nucleolus in equal quantities, I studied transcriptional regulation of Arabidopsis RP genes and the subcellular localization of five RP families to determine the extent of coordinated regulation of these processes. Variation of up to 300-fold was observed in the expression levels of RP genes. However, this variation was drastically reduced when the expression level was considered at the RP gene family level, indicating that coordinate regulation of expression of RP genes, coding for individual RP isoforms, is more stringent at the family level. Subcellular localization also showed differential targeting of RPs to the cytoplasm, nucleus and nucleolus, together with a significant difference in the nucleolar import rates of RPS8A and RPL15A. Although one could expect coordinated regulation of the processes preceding ribosomal subunit assembly in the nucleolus, my results suggest differential regulation of these processes.

The nucleolus of eukaryotic cells contain a number of the intron-coding small nucleolar RNAs (snoRNAs), which functions are related to covalent modification of pre-rRNAs. The snoRNA that from long, phylogenetically conserved sequence complementarity to 28S, 18S, 5.8S and 5S rRNAs are designated as 28S, 18S, 5.8S and 5S snoRNAs, respectively. In the present study, studying on human 5S snoRNAs had been carried out. The human genome encoding candidate 5S snoRNAs were searched using database mining. The transcripts of 5S snoRNA genes were identified by RT-PCR analyses and DNA sequencing. No appreciable diversities of 5S snoRNA genes were observed as evidenced by single strand conformation polymorphism (SSCP) and high resolution agarose gel. Moreover, sequence conservation of 5S snoRNAs reflects a requirement for maintaining their secondary structure on exerting their function. The results of RT-PCR analyses revealed a tissue-specific transcription of 5S snoRNAs. A 5S snoRNA designated as N117 was identified to be highly expressed in normal brain. On the contrary, its expression markly decreased in brain tumor (meningioma). This seems to be associated with the expression of host gene, which encodes a protein similar to synapsin III protein. Consequently, this may implicate that the use of snoRNA as a potential index for the transcription of its host gene.

Orientador: Shirlei Maria Recco Pimentel / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T19:35:01Z (GMT). No. of bitstreams: 1 Carvalho_KleliaAparecida_M.pdf: 5095277 bytes, checksum: d39b0879e4a22b528220f0642206dc20 (MD5) Previous issue date: 2005 / Resumo: A família Hylidae possui quatro subfamílias, sendo Hylinae constituída por 27 gêneros, dentre eles o gênero Hyla que possui muitas espécies alocadas em grupos relacionados entre si. O grupo de Hyla albomarginata, também conhecido por "hylas verdes" é subdividido em três complexos: "albomarginata", "albosignata" e "albofrenata".O gênero Aplastodiscus está envolvido em discussão taxonômica desde sua descrição, tendo sido sinonimizado e retirado de Hyla mais de uma vez. Como há grande semelhança morfológica e comportamental entre as espécies do grupo de H albomarginata e as espécies de Aplastodiscus (A Lutz in B. Lutz, 1950), o relacionamento filogenético e taxonômico entre esses gêneros ainda é bastante discutido. No presente trabalho foi realizado um estudo citogenético das duas espécies de Aplastodiscus (A. perviridis e A. cochranae), de três populações de H albomarginata (complexo "albomarginata"), H albosignata e H leucopygia (complexo "albosignata") e H albofrenata, H arildae, H ehrhardti e Hyla eugenioi (complexo "albofrenata"), além de H faber, uma espécie proximamente relacionada não pertencente ao grupo, com o objetivo de contribuir para a sistemática desse grupo e para o entendimento da problemática em questão envolvendo os relacionamentos intra e intergenérico de Aplastodiscus. As metáfases mitóticas e meióticas foram obtidas por suspensão de células de epitélio intestinal e testículos e submetidas aos métodos de coloração com Giemsa, bandamento C, impregnação pela prata e hibridação in situ com sondas de DNA ribossomal e de seqüência telomérica. O número diplóide de 2n=24 foi encontrado nas duas espécies de Aplastodiscus, Hyla albomarginata e Hyla faber, enquanto Hyla albosignata apresentou 2n=20 e Hyla leucopygia 2n=18. As quatro espécies do complexo "albofrenata" apresentaram 2n=22 cromossomos. As duas espécies de Aplastodiscus apresentaram o mesmo padrão de heterocromatina entre si, mas diferiram das espécies de Hyla. A região organizadora do nuc1éolo (NOR) foi localizada no telômero do par 12 em ambas as espécies de Aplastodiscus, do par 09 em H albosignata e H leucopygia e do par 11 em H. faber, e intersticialmente no braço curto do par 02, com heteromorfismo de tamanho, em H albomarginata. Em dois dos oito indivíduos de Hyla albofrenata, a NOR está presente na região te1omérica do braço longo no par 07 e em seis desses indivíduos foi encontrado uma interessante variante em que a NOR está presente nos pares 01 e 07, mas em apenas um dos homólogos de cada par. Em H. ehrhardti, a técnica de Ag-NOR marcou os pares 06 e 10, mas apenas a do par 06 foi confirmada por hibridação in situo Em H arildae, a NOR foi localizada no par 10 e em Hyla eugenioi no par 07, também nos telômeros. Em H albofrenata e H arildae foram observados anéis multivalentes na prófase I da meiose, constituídos de quatro a seis bivalentes, com a participação dos cromossomos portadores da NOR. Eventos de translocação durante a evolução das espécies H albofrenata e H arildae podem ser responsáveis pela formação desses anéis na meiose. O envolvimento dos cromossomos portadores da NOR pode ter facilitado a ocorrência do polimorfismo observado em H albofrenata. A sonda de seqüências teloméricas marcou os telômeros em todas as espécies e também as regiões centroméricas dos cromossomos de H albofrenata e H arildae, coincidente com a heterocromatina centromérica. Em anfíbios, seqüências localizadas fora dos te1ômeros tinham sido detectadas apenas em regiões intersticiais. A presença dessas seqüências no centrômero pode ter origem em amplificação de seqüências (TTAGGG)n relacionada com a amplificação de regiões de heterocromatina. Os dados do presente trabalho permitiram concluir que A. perviridis e A. cochranae não podem ser diferenciadas cito geneticamente. O par 09, portador da NOR, de H albosignata e H leucopygia é muito semelhante na morfologia (metacêntricos) e na localização (região te1omérica) da NOR, ao par 12 das espécies de Aplastodiscus e ao par 11 de H faber, sugerindo serem esses cromossomos homeólogos. Embora Hyla albomarginata diferencie-se das demais espécies analisadas pela localização da NOR no par 2, apresentou o mesmo número diplóide e a morfologia dos cromossomos semelhante a Aplastodiscus. A morfologia cromossômica conservada, principalmente dos sete primeiros pares, entre as espécies dos complexos "albofrenata" (presente trabalho) "albosignata" e "albomarginata" e de Aplastodiscus, sugere que um cariótipo ancestral comum deve ter originado todas essas espécies atuais e que rearranjos cromossômicos levaram à diferenciação desses cariótipos, com mudança de número diplóide, envolvendo principalmente o grupo de cromossomos menores, e dispersão de NOR e de heterocromatina / Abstract: The Hylidae family includes four subfamilies, with Hylinae comprising 27 genera. The Hyla genus has many species divided into related groups. The Hyla albomarginata group, as known as "green Hylas" is subdivided into 3 complexes: "albomarginata","albosignata" and "albofrenata". The Aplastodiscus genus has been the object oftaxonomic discussion since its description, having been synonymyzed to and removed from Hyla more than once. Since there are great morphological and behavioral similarities between the species of the H albomarginata group and the species of Aplastodiscus, the phylogenetic and taxonomic relationships of these genera are still under discussion. In the present research, a cytogenetic study was carried out evaluating two species of Aplastodiscus (A.perviridis and A. cochranae), three populations of H albomarginata ("albomarginata" complex), H albosignata and H leucopygia ("albosignata" complex) and H albofrenata, H arildae, H ehrhardti and Hyla eugenioi ("albofrenata" complex), as well as H faber, a very closely related species not pertaining to the group. The aim of this research was to contribute to the systematics of this group and to the understanding of the intra- and intergeneric relationships of Aplastodiscus. Mitotic and meiotic metaphases were obtained from suspension of intestinal epithelial cells, stained with Giemsa and submitted to the techniques of C-banding, silver impregnation and in situ hybridization with probes of ribosomal DNA and telomeric sequence. The diploid number of 2n=24 was found in the two species of Aplastodiscus, Hyla albomarginata and Hyla faber, while Hyla albosignata had 2n=20 and Hyla leucopygia had 2n=18. The four species of the complex "albofrenata" had 2n=22 chromosomes. The two species of Aplastodiscus had the same heterochromatin pattern, but they differ from the Hyla species. The nucleolus organizing region (NOR) was located on the telomeric regions or pair 12 in both species of Aplastodiscus, in pair 09 of H albosignata and H leucopygia, in pair 11 in H faber, and intersticially on the short arms of pair 02, with size heteromorphism in H albomarginata. In two of the eight individuals of Hyla albofrenata, the NOR was present in the telomeric region on the long arm in pair 07 and, in six of these individuals, a interesting pattern for Anura was found. The NOR was found in the pairs 01 and 07, but in only one of the homologues of each pair. In H ehrhardti, the Ag-NOR technique marked the pairs 06 and 10, but only that on pair 06 was confirmed by in situ hybridization. In H arildae, the NOR was located in the 10 pair and in Hyla eugenioi in the pair 07, also in the telomeric regions. In H albofrenata and H arildae multivalent rings were observed in the prophase I of meiosis, constituted of 4 to 6 bivalents, being one of them the NOR-bearing chromosomes. Translocation events during the evolution of H albofrenata and H arildae could be responsible for the ring formation in meiosis. The participation of the NOR bearing chromosomes in the multivalente rings could have facilitated the occurrence of polymorphism of NOR, as observed in H albofrenata. The telomeric sequence probe marked the telomeric region in all species studied as well as the centromeric regions of the chromosomes of H albofrenata and H arildae, at the centromeric heterochromatin. In amphibians, telomeric sequences located outside the telomeric regions had only been detected in interstitial regions. The presence of these sequences in centromeric regions may have been originated by the amplification of the (TTAGGG)n sequences related to heterochromatic amplification. The data of the present work a11ow the conc1usion that A. perviridis and A. cochranae could not be differentiated. The NOR bearing chromosomes 9 of H leucopygia and H albosignata are very similar in size, morphology (metacentric) and NOR localization (telomeric region) to the pair 12 of the species Aplastodiscus and also to the pair 11 of H faber, suggesting that these are homologous chromosomes. Although Hyla albomarginata differs from all species analyzed for NOR localization on pair 2, it presented the same diploid number and chromosome morphology of Aplastodiscus. The conserved chromosomal morphology, mainly in the first seven pairs, between the species of "alboftenata" (present work), "albosignata" and "albomarginata" complexes and Aplastodiscus, suggests that one ancestral karyotype in common must have originated all these current species. The chromosomal rearrangements could lead to the differentiation of these karyotypes, with changes in diploid number, involving mainly the short chromosome group and the dispersion of heterochromatin and NOR / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

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O presente trabalho teve por objetivo compreender e analisar os mecanismos genéticos e epigenéticos da expressão diferencial de regiões organizadoras do nucléolo - RONs através do estudo de dois acessos (CRT-1 e CRT-2) de Crotalaria retusa. O acesso CRT-1 é uma cultivar, enquanto que o acesso CRT-2 é proveniente de uma população periférica da orla marítima de Ilhéus BA. Por serem temporalmente e espacialmente separados, acredita-se que os acessos foram submetidos a pressões seletivas diferentes, resultando em alterações dos padrões epigenéticos, principalmente nas RONs. Para o desenvolvimento deste trabalho foram realizadas medidas cromossômicas e nucleolares a partir de células coradas pelo método de Feulgen e por nitrato de prata, coloração com fluorocromos específicos às regiões cromossômicas ricas em nucleotídeos GC e AT, mapeamento físico dos locos de DNA ribossômico 45S por hibridação in situ fluorescente, análise qualitativa e quantitativa de modificações pós-traducionais de histonas por Western blot e eletroforese bidimensional de extrato protéico radicular com enfoque em proteínas envolvidas nos mecanismos epigenéticos. As análises citológicas demonstraram uma grande semelhança nos cariótipos dos dois acessos, diferindo apenas no tamanho do segmento proximal do braço curto do cromossomo 1. Em ambos os acessos foi observada uma expressão nucleolar diferencial em, aproximadamente, 50% das células; contudo, a expressão diferencial em CRT-2 apresentou-se consideravelmente maior. Além disso, os dois acessos demonstraram diferenças quantitativas nas modificações pós-traducionais de histonas e em proteínas possivelmente envolvidas em mecanismos epigenéticos. Uma vez que as variações epigenéticas podem ser modificadas por fatores ambientais, sugere-se que as diferenças nos padrões de modificações de histonas e nos perfis protéicos encontradas entre os acessos, como também a expressão diferencial mais expressiva em CRT-2, sejam devidas às diferentes pressões seletivas as quais as populações originais foram submetidas. O estudo dos mecanismos genéticos e epigenéticos na dominância nucleolar possibilita uma maior compreensão da ação do remodelamento da cromatina no controle da expressão gênica do rDNA, como também da expressão gênica em geral. / The aim of this present work was to understand and analyze the genetic and epigenetic mechanisms of differential expression of the nucleolus organizer regions - NORs through the study of two accesses (CRT-1 and CRT-2) of Crotalaria retusa. Access CRT-1 is a cultivar, while access CRT-2 is from a peripheral population of the shoreline of Ilhéus BA. Because they are temporally and spatially separated, it is believed that the accesses were submitted to different selective pressures, resulting in changes in epigenetic patterns, primarily in NORs. To develop this work, it was carried out chromosomal and nucleolar measurements from cell stained by Feulgen method and silver nitrate, staining with specific fluorochromes to chromosomal regions rich in GC and AT nucleotides, physical mapping of 45S ribosomal DNA loci by fluorescent in situ hybridization, qualitative and quantitative analysis of post-translational histone modifications by western blot, and two-dimensional electrophoresis of root extract protein focusing on proteins involved in epigenetic mechanisms. The cytological analysis showed a great similarity in karyotypes of two accessions, differing only in size of the proximal segment of the sort arm of chromosome 1. In both accesses, it was observed a differential nucleolar expression in approximately 50% of the cells; however, the differential expression in CRT-2 showed considerably larger. Furthermore, the two accesses showed quantitative differences in the posttranslational histone modifications, and in a protein possibly involved in epigenetic mechanisms. Since epigenetic variations can be modified by environmental factors, it is suggested that differences in patterns of histone modifications and protein profiles found between the accesses, but also the most significant differential expression in CRT-2, are due to different selective pressures to which the original populations were submitted. Studies of the epigenetic mechanisms in nucleolar dominance allows a better understanding of the action of the remodeling of chromatin in controlling the dosage of rRNA genes, but also in the control of gene expression in general.

Made available in DSpace on 2014-06-11T19:32:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-26Bitstream added on 2014-06-13T20:43:02Z : No. of bitstreams: 1 peruquetti_rl_dr_sjrp.pdf: 4798980 bytes, checksum: 183f2f2646027bae2081cc27764e1c82 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O corpo cromatóide (CB) é uma organela citoplasmática que, aparentemente, possui um papel no estoque de RNA e proteínas para a diferenciação final dos espermatozóides. Existem algumas teorias que tentam explicar a origem do material que compõe essa organela. Uma dessas teorias, proposta por alguns autores, sugere que o CB se origine a partir de material nucleolar, que se fragmenta nas etapas iniciais da espermatogênese e, em seguida, migra para o citoplasma. O objetivo do presente estudo foi acompanhar o ciclo nucleolar por meio de análises citoquímicas – hematoxilina-eosina (HE); azul de toluidina (AT); variante da concentração crítica de eletrólitos (CEC); reação de Feulgen; impregnação por íons prata (AgNOR); citogenéticas – impregnação por íons prata (AgNOR), e análises ultra-estruturais – microscopia eletrônica de transmissão (MET), para verificar a relação da fragmentação do material nucleolar com a formação do corpo cromatóide (CB), em algumas espécies de vertebrados: Tilapia rendalli (Teleostei, Cichlidae); Dendropsophus minutus (Amphibia, Anura); Phrynops geoffroanus (Reptilia, Testudines) e coelho albino da raça Nova Zelândia – Oryctolagus cuniculus (Mammalia, Lagomorpha). Por meio das análises citoquímicas foi possível observar que ocorre uma fragmentação do material nucleolar no início da prófase I, em todas as espécies analisadas, e uma posterior reorganização do nucléolo no núcleo de espermátides iniciais, com uma área significantemente menor do que a área do nucléolo das espermatogônias. Três fenômenos podem contribuir para essa diferença significante entre as áreas nucleolar de espermatogônias e espermátides: a) Modificação no estado funcional da célula; b) Diminuição no número de RONs nas espermátides; c) Migração de material nucleolar fragmentado... / The chromatoid body (CB) is a cytoplasmic organelle that has a function related to RNA and protein accumulation and ⁄ or storage for later germ-cell differentiation. Many theories have been postulated in order to explain the origins of the CB material. One of the most accepted theory describes that it originates from a nucleolar material, where it was fragmented in the early spermatogenesis, and finally, this fragmented nucleolar material migrates to cytoplasm. The aims of the present study were: 1) monitoring the nucleolar material distribution by means of cytochemical techniques (hematoxylin–eosin (HE), toluidine blue (TB), modified Critical Electrolyte Concentration for detecting RNA (CEC), silver-ion impregnation (AgNOR) and Feulgen reaction), and by ultrastructural analysis (Transmission Electron Microscopy – TEM); and 2) comparing the nucleolar material distribution with the formation of CB in some vertebrate species: Tilapia rendalli (Teleostei, Cichlidae); Dendropsophus minutus (Amphibia, Anura); Phrynops geoffroanus (Reptilia, Testudines); and Oryctolagus cuniculus (Mammalia, Lagomorpha). For all analyzed species, the cytochemical techniques showed that the nucleolar fragmentation occurred during the beginning of prophase I, and the nucleolus reorganization occurred in the early spermatids nucleus. Statistical tests evidenced that area of the early spermatids nucleolus were smaller than the spermatogonia nucleolus area. Three phenomena can contribute for the statistical difference between the spermatogonia nucleolar area and the early spermatids nucleolar area: a) Modification of cell activity; b) Decrease of the number of NORs in the spermatids; c) Migration of the fragmented nucleolar material from the nucleus to the cytoplasm. This nucleolar material will participate in the CB formation process. The ultrastructural analysis showed an ...(Complete abstract click electronic access below)