<p>The relatively simple innate immune system of <italic>Caenorhabditis elegans</italic> and the number of traits that facilitate genetic and genomic analysis using this organism have nurtured rapid advances into the understanding of <italic>C. elegans</italic> innate immunity during the last few years. However, traditional methods of isolating and mapping <italic>C. elegans</italic> mutants exhibiting aberrant immune responses to pathogen infection are often labor intensive and time consuming. Therefore, a simple and rapid means of isolating and mapping <italic>C. elegans</italic> immune mutants will increase the number of mutants that can be studied. <italic>Salmonella enterica</italic>, as well as other bacterial pathogens, has been described to cause a significant distension of the <italic>C. elegans</italic> intestinal lumen, which correlates with death of the nematode. <italic>C. elegans</italic> mutants which exhibit a weakened immune response would therefore be expected to develop intestinal distension at an earlier time point than wild type. Likewise, mutants which exhibit an enhanced immune response would be expected to develop intestinal distension at a later time point than wild type. Taking advantage of this correlation, we designed a novel approach to isolating <italic>C. elegans</italic> mutants which exhibit aberrant immune responses to the bacterial pathogen, <italic>S. enterica</italic>. Furthermore, we validated and optimized the use of Amplifluor®, a high-throughput genotyping system, for use in <italic>C. elegans</italic> single nucleotide polymorphism (SNP) mapping. </p><p>To date, the only known negative regulators of innate immunity in <italic>C. elegans</italic> are dependent on the FOXO transcription factor, DAF-16 and regulate lifespan in addition to immunity. Therefore, we focused our efforts on identifying additional negative regulators of innate immunity by screening for mutants which display a reduced accumulation of <italic>S. enterica</italic> at a time point when wild-type nematodes are packed with bacteria. In a genetic screen for <italic>C. elegans</italic> mutants which display reduced accumulation of <italic>S. enterica</italic>/GFP, we identified a mutation in <italic>nol-6</italic>, a nucleolar protein containing a nucleolar RNA-associated protein (Nrap) domain which is conserved across eukaryotic organisms. <italic>nol-6</italic> is implicated in ribosomal RNA (rRNA) processing during the early stages of ribosome biogenesis. We show that knockdown of <italic>nol-6</italic> as well as other nucleolar genes leads to a reduction of pathogen accumulation and enhanced resistance to killing by pathogen. In addition, we demonstrate that enhanced resistance is dependent on p53/<italic>cep-1</italic>. Furthermore, microarray analysis shows a significant enrichment of upregulated genes that have previously been shown to be dependent on p53/<italic>cep-1</italic> for induction following ultraviolet radiation. These results represent the first evidence that <italic>C. elegans</italic> innate immunity is regulated by the nucleolus through a p53/<italic>cep-1</italic>-dependent mechanism.</p> / Dissertation

Chromatin structure is a fundamental determinant of eukaryotic gene expression and it is composed of two chromatin environments, euchromatin and heterochromatin. Euchromatin provides an accessible platform for transcription factors; hence it is permissive for gene expression. Heterochromatin on the other hand is highly compacted and inaccessible, which in most cases leads to transcriptional repression. A locus that is composed of both of these environments is the ribosomal DNA (rDNA). In eukaryotes the rDNA is composed of hundreds to thousands of tandemly repeated genes where maintaining both silent and active copies is fundamental for the stability of the genome. The aim of this research was to investigate the role of the rDNA in gene expression in Drosophila melanogaster. In D. melanogaster the rDNA loci are present on the X and Y chromosomes. This research used the Y-linked rDNA array to investigate the role of this locus on gene expression. A genetic and molecular strategy was designed to create and quantify specific, graded and isogenic Y- linked rDNA deletions. Then the deletions were used to address the effect of rDNA deletions on gene expression using reporter genes sensitive to Position Effect Variegation (PEV). In addition, the effect of the deletions in nucleolus size and structure as well as the effect of spontaneous rDNA deletions on gene expression were tested in this study. This research found that changes in rDNA size change the chromatin balance, which resulted in increased expression of the reporter genes, decreased nucleolus volume, and altered nucleolus structure. These findings prompted a further research question on whether this effect on gene expression occured globally in the genome. This was addressed by performing microarray analysis where the results showed that rDNA deletions affect about half of the genes on the genome. Presented in this dissertation is evidence that suggest a novel role for the rDNA is a global modulator of gene expression and also is a contributor to the gene expression variance observed in natural populations.

Dictyostelium is a model eukaryote for the study of a multitude of fundamental cellular processes as well as several human diseases. Despite its extensive study relatively little is known about its nucleolus. Only three nucleolar proteins have been identified. The nucleolus in Dictyostelium is different than that of other eukaryotes since it is neither bipartite nor tripartite, possessing no visible subcompartments at the ultrastructural level. Moreover, it exists as two to four patches adjacent to the inner nuclear envelope instead of within the nucleoplasm. The aim of this study was thus to identify and characterize novel nucleolar proteins in Dictyostelium in order to better understand the structure and function of its nucleolus. Previous work had shown that NumA1, a protein linked to cell cycle in Dictyostelium, localizes to similar intranuclear patches suggesting it may be nucleolar. NumA1-binding partners Ca2+-binding protein (CBP) 4a and puromycin-sensitive aminopeptidase A may therefore also reside in the nucleolus. Based on the function of a potential NumA1 homologue in other organisms, BRG1-associated factor 60a homologue Snf12 and checkpoint kinase 2 (Rad53 in yeast) homologue forkhead-associated kinase (Fhk) A were chosen as potential nucleolar proteins in Dictyostelium that may also be involved in cell cycle events. Using a diversity of approaches, this study found that NumA1, CBP4a, Snf12, and FhkA are nucleolar proteins in Dictyostelium while puromycin-sensitive aminopeptidase A is nucleoplasmic. Several nuclear localization signals (NLSs) were identified in these proteins some of which also act as nucleolar localization signals (NoLSs). These NLS/NoLSs (within NumA1 and Snf12) represent the first NoLSs and first NLS/NoLSs identified in Dictyostelium. Treatment with the rDNA transcription inhibitor AM-D led to the budding of nucleolar CBP4a, Snf12, and FhkA from the nucleus to the cytoplasm, a phenomenon not previously observed in any organism. This study also examined for the first time the redistribution of nucleolar proteins during mitosis, a time when the nucleolus disassembles into its component parts. The nuclear envelope was also shown to become permeable at this time. Finally, multiple nucleolar subcompartments were identified suggesting compartmentalization of different functions in the Dictyostelium nucleolus.

Dictyostelium is a model eukaryote for the study of a multitude of fundamental cellular processes as well as several human diseases. Despite its extensive study relatively little is known about its nucleolus. Only three nucleolar proteins have been identified. The nucleolus in Dictyostelium is different than that of other eukaryotes since it is neither bipartite nor tripartite, possessing no visible subcompartments at the ultrastructural level. Moreover, it exists as two to four patches adjacent to the inner nuclear envelope instead of within the nucleoplasm. The aim of this study was thus to identify and characterize novel nucleolar proteins in Dictyostelium in order to better understand the structure and function of its nucleolus. Previous work had shown that NumA1, a protein linked to cell cycle in Dictyostelium, localizes to similar intranuclear patches suggesting it may be nucleolar. NumA1-binding partners Ca2+-binding protein (CBP) 4a and puromycin-sensitive aminopeptidase A may therefore also reside in the nucleolus. Based on the function of a potential NumA1 homologue in other organisms, BRG1-associated factor 60a homologue Snf12 and checkpoint kinase 2 (Rad53 in yeast) homologue forkhead-associated kinase (Fhk) A were chosen as potential nucleolar proteins in Dictyostelium that may also be involved in cell cycle events. Using a diversity of approaches, this study found that NumA1, CBP4a, Snf12, and FhkA are nucleolar proteins in Dictyostelium while puromycin-sensitive aminopeptidase A is nucleoplasmic. Several nuclear localization signals (NLSs) were identified in these proteins some of which also act as nucleolar localization signals (NoLSs). These NLS/NoLSs (within NumA1 and Snf12) represent the first NoLSs and first NLS/NoLSs identified in Dictyostelium. Treatment with the rDNA transcription inhibitor AM-D led to the budding of nucleolar CBP4a, Snf12, and FhkA from the nucleus to the cytoplasm, a phenomenon not previously observed in any organism. This study also examined for the first time the redistribution of nucleolar proteins during mitosis, a time when the nucleolus disassembles into its component parts. The nuclear envelope was also shown to become permeable at this time. Finally, multiple nucleolar subcompartments were identified suggesting compartmentalization of different functions in the Dictyostelium nucleolus.

NEDD8, molécule de la famille de l’ubiquitine est essentielle au développement, à la croissance et à la viabilité d’un organisme, de plus c’est une cible prometteuse en thérapeutique. Nous avons découvert que l’inhibiteur spécifique de la NEDDylation, MLN4924 altère la morphologie sans fragmentation et augmente la surface du nucléole de cellules humaines et de noyaux de la lignée germinale de Caenorhabditis elegans. Une approche de protéomique quantitative (SILAC) combiné à l’analyse de la production des ARNr et des ribosomes montrent que MLN4924 change la composition protéique du nucléole sans affecter l’activité transcriptionnelle de l’ARN pol I. Notre analyse montre que MLN4924 active p53 par la voie RPL11/RPL5-Mdm2 caractéristique d’un stress du nucléole. Cette étude identifie le nucléole comme une cible intéressante dans l’utilisation d’inhibiteurs de la NEDDylation et apporte un nouveau mécanisme d’activation de p53 par inhibition de la voie NEDD8.Dans une seconde étude nous avons adapté la méthode de FLIM-FRET (« Fluorescence Lifetime Imaging Microscopy – Förster Resonance Energy Transfer ») à l’étude de la compaction de la chromatine à l’échelle du nanomètre dans un organisme vivant. Le nématode Caenorhabditis elegans s’est révélé être un modèle de choix. Au sein des chromosomes méiotiques, nous avons identifié différentes régions de compaction, de niveau variable par mesure du FRET entre histones fusionnées à des protéines fluorescentes. Par une approche originale d’ARN interférence et injection d’un « extra-chromosome » nous avons défini l’architecture à une nano-échelle de différents états de l’hétérochromatine et montré que cette organisation est contrôlée par les protéines HP1 « Heterochromatin Protein 1 » et SETDB1, une protéine « H3-Lysine 9 methyl transferase ». Nous avons également montré que la compaction de l’hétérochromatine est dépendante des condensines I et II et plus particulièrement la condensine I contrôle l’état faiblement compacté de la chromatine.Nos travaux ont confirmé que C. elegans est un modèle d’intérêt majeur pour l’étude des compartiments nucléaires et parfaitement adapté pour des études pré-clinique. / The ubiquitin-like molecule NEDD8 is conserved and essential for viability, growth and development; its activation pathway is a promising target for therapeutic intervention. We found that the small molecule inhibitor of NEDDylation, MLN4924, alters the morphology and increases the surface size of the nucleolus in human cells and Caenorhabditis elegans germ cells in the absence of nucleolar fragmentation. Through SILAC proteomic analysis and rRNA production, processing and ribosome profiling, we show that MLN4924 changes the composition of the nucleolar proteome but does not inhibit RNA Pol I transcription. Further analysis demonstrates that MLN4924 activates the p53 tumour suppressor through the RPL11/RPL5-Mdm2 pathway, with characteristics of nucleolar stress. The study identifies the nucleolus as a target of the NEDDylation pathway and provides a mechanism for p53 activation upon NEDD8 inhibition.Then we adapted a quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) approach to assay the nano-scale chromatin compaction in a living organism, the nematode Caenorhabditis elegans. By measuring FRET between histone-tagged fluorescent proteins, we visualized distinct chromosomal regions and quantified the different levels of nanoscale compaction in meiotic cells. Using RNAi and repetitive extrachromosomal array approaches, we defined the heterochromatin state and showed that its architecture presents a nanoscale-compacted organization controlled by Heterochromatin Protein-1 (HP1) and SETDB1 H3-lysine-9 methyl-transferase homologs in vivo. Next, we functionally explored condensin complexes. We found that condensin I and condensin II are essential for heterochromatin compaction and that condensin I additionally controls lowly compacted regions. Our data show that, in living animals, nanoscale chromatin compaction is controlled not only by histone modifiers and readers but also by condensin complexes.We confirm that C. elegans is an interesting model to study nuclear signalling and perfectly adapt to be a platform for pre-clinical studies.

The nucleolus is a multifunctional organelle that, in addition to its primary role in ribosome biogenesis, has emerged as a critical stress sensor and coordinator of stress response. However, the molecular nature of how nucleoli sense stress and coordinate downstream cellular consequence remains poorly understood. NF-κB signalling is a critical regulator of stress response. Many cellular stresses that disrupt nucleolar function also stimulate the NF-κB pathway. However, the role of NF-κB as a downstream effector of nucleolar stress has not yet been examined. Aspirin, a known chemopreventative agent, stimulates the NF-κB pathway to mediate apoptosis but the upstream mechanisms are unclear. In this thesis, I identified a novel nucleolar stress response pathway that culminates in activation of NF-κB signalling, and demonstrated the significance of this nucleolar pathway in the anti-tumour effects of aspirin. Using multiple approaches, I made the novel observations that disruption of the Pol I complex activates the cytoplasmic NF-κB signalling pathway. I show that multiple stress stimuli of NF-κB pathway induce degradation of the crucial Pol I complex component, rDNA transcription initiation factor IA (TIF-IA). I identified the tumour suppressor, p14ARF and the Pol I complex component, upstream binding factor (UBF) as mediators of this degradation. I revealed that inhibition of CDK4 activity lies upstream of UBF/p14ARF-facilitated TIF-IA degradation. Furthermore, using different approaches I show that blocking aspirin/CDK4i-mediated degradation of TIF-IA blocks the effects of these agents on nucleolar morphology and NF-κB signalling. Finally, I show this nucleolar stress response pathway, containing a UBF/p14ARF/TIF-IA axis, is utilized by aspirin to kill colon cancer cells. Taken together, this data presented in this thesis advances understanding of nucleolar stress response, and has therapeutic implications with regard to the anti-tumour effects of aspirin.

Made available in DSpace on 2014-06-11T19:26:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-26Bitstream added on 2014-06-13T19:12:50Z : No. of bitstreams: 1 bardella_vb_me_sjrp.pdf: 973541 bytes, checksum: 2642d082f6fe5ee2cb77ab3b60832684 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Os heterópteros apresentam a meiose cística nos túbulos seminíferos. Esses possuem o cisto espermatogonial envolto pelas células císticas, as quais desenvolvem a função de nutrição das células em divisão celular. Quanto às características citogenéticas, esses insetos apresentam cromossomos holocinéticos, baixa variabilidade cariotípica e meiose invertida dos cromossomos sexuais. No presente trabalho foram caracterizadas as células císticas quanto a sua localização, ultraestrutura e citogenética e, também, foram analisados os aspectos citogenéticos de quatro espécies do gênero Triatoma. Foram utilizadas as técnicas de microscopia eletrônica de transmissão, citogenética convencional (orceína e AgNOR), bandamento C CMA3/DAPI e a técnica de hibridização in situ fluorescente (FISH), com sonda de DNAr 45S de Drosophila melanogaster. Os resultados indicaram que a célula cística envolve um cisto espermatogonial e apresenta um grande núcleo com invaginações citoplasmáticas. Em todas as espécies foram observados vários graus de ploidia da célula cística. Triatoma infestans e T. infestans melanosoma apresentaram vários blocos heterocromáticos com a periferia CMA3 + e o interior DAPI+. Associada às bordas dos blocos heterocromáticos foram observados os segmentos de DNAr 45S, além da presença de vários nucléolos em cada núcleo. Triatoma matogrossensis, T. rubrovaria e T. brasiliensis apresentaram apenas um bloco heterocromático com as mesmas características, com exceção de T. brasiliensis, que apresentou em algumas células vários blocos CMA3 + dispersos. Nessas espécies foi observado apenas um nucléolo com similaridade na localização dos sítios de DNAr. Quanto aos aspectos citogenéticos, todas as espécies apresentaram 2n = 20A + XY, com decréscimo do tamanho relativo dos cromossomos. Em T. infestans melanosoma os cromossomos foram... / Heteroptera, or true bugs, exhibit meiosis in their seminiferous tubules. They posses the spermatogonial cysts that are enclosed by cyst cells, which develop the nutritional function of the cells during cell division. In terms of cytogenetic characteristics, these insects possess holokinetic chromosomes, low karyotype variability, and inverted meiosis in the sex chromosomes. In this study, cyst cells from four species of the genus Triatoma were characterized by their location, superstructure, and cytogenetic makeup. Electronic transmission microscopy techniques were used, as well as conventional cytogenetic techniques (Orcein and AgNOR), C-banding with CMA3 and DAPI banding, and Fluorescence in situ Hybridization (FISH) with a 45S DNA probe of Drosophila melanogaster. The results indicated that the the spermatogonial cyst is enclosed by the cyst cell, and that the cyst cell possesses a large nucleus with cytopasmic invaginations. In all species studied, varying degrees of ploidy were observed in the cyst cells. Triatoma infestans and T. infestans melanosoma presented with various heterochromatic blocks, with CMA3 + at the periphery and DAPI+ at the interior. Segments of rDNA 45S were found along the edges of the heterochromatic blocks, along with the presence of various nucleoli in each nucleus. Triatoma matogrossensis, T. rubrovaria and T. brasiliensis presented with only one heterochromatic block with the same characteristics (with the exception of T. brasiliensis, which presented with various dispersed CMA3 + blocks). In these species, only one nucleolus that was similar to the localization of the rDNA sites was found. All species presented with 2n = 20A + XY, with a decrease in size relative to the chromosomes. In the case of T. infestans melanosoma, the chromosomes were split into groups based on their relative sizes. The heterochromatin of this species presented... (Complete abstract click electronic access below)

O núcleo é uma estrutura organizada e possui verdadeiras organelas nucleares. Entre elas, estão os nucléolos e os corpúsculos de Cajal (CBs). Estes compartimentos nucleares são estruturas dinâmicas, mantidos pela associação de macromoléculas envolvidas na expressão gênica que interagem entre si delimitando-as. A principal proteína encontrada nos CBs é a p-80-coilin e, portanto, o principal epítopo capaz de marcar essas estruturas. Suas funções específicas ainda tem sido alvo de estudo. Existe uma proteína em comum a ambas as estruturas, a fibrilarina que participa no processamento de rRNA. Estes corpúsculos já foram descritos na periferia dos nucléolos ou mesmo fisicamente ligados a ele. Acredita-se que os corpúsculos de Cajal participem da síntese de rRNA, maturação, transporte e associação das subunidades ribossômicas.Diante esta relação, este trabalho visa estudar a inter-relação entre estas estruturas em células normais e as respectivas linhagens de células tumorais em cultura antes e após tratamentos com actinomicina D. Esta droga se usada em baixas concentrações, bloqueia a transcrição dos genes que foram decodificados pela RNA polimerase I e II, e -amanitin, por sua vez bloqueia a transcrição de genes decodificado pela RNA polimerase II . Além disso, também visa investigar uma relação entre a proliferação das linhagens estudadas e freqüência dos corpúsculos de Cajal nas células controle e tratadas. O microscópio confocal de varredura a laser permitiu o estudo dessas estruturas em preparações imunofluorescência fornecendo uma análise tridimensional destas estruturas quando utilizados anticorpos específicos. Linhagens de células que apresentaram um crescimento mais lento foram aquelas que tinham uma maior freqüência de corpúsculos por núcleo. Por outro lado, aquelas que apresentaram um crescimento mais intenso, foram aquelas que apresentaram maior variação no número de corpúsculos por núcleo. Após o tratamento com inibidores de síntese de RNA, tanto os corpúsculos de Cajal quanto os nucléolos, apresentaram alterações morfológicas, às vezes apresentando um grande acúmulo na região dos corpúsculos ou desorganizando os nucléolos. Mudanças no tamanho e forma também puderam ser destacadas. / The nucleus is a structure that has sub-compartments which can be called nuclear organelles. Among them, may be cited the nucleoli and the Cajal bodies (CBs). These nuclear compartments are dynamic structures, maintained by association and stock of macromolecules involved in gene expression. The main protein found in the CB is a p-80-coilin and therefore the main epitope able to label these structures. Their functions are still to be clarified. There is a protein in common to the nucleolus and Cajal bodies, the fibrillarin that takes part in the processing of rRNA. The CBs can be found at the periphery of the nucleoli or even physically connected to them. It is believed that the CBs may have role in the synthesis of rRNA and maturation, transport and association of ribosome subunits. In view of this relationship between Cajal bodies and nucleoli, this work aims to study the interrelationship between these structures in normal cells and their respective tumor cell line in culture before and after treatments with actinomycin D, which in low concentrations, blocks the transcription of genes that were decoded by the RNA polymerase I and II and -amanitin, which is responsible for blocking the transcription of genes decoded by the RNA polymerase II and find out a relationship between cell proliferation and Cajal bodies frequencies in control and treated cells. The confocal microscope of laser scanning enabled the study of these structures in preparations immunofluorescent providing a three-dimensional analysis of these structures when used specific antibodies before and after treatment. Cell lines that shown low cell grow, appears to have lass CB/ nucleus in the other hand, cell lines that have fastest grow shown nuclei with more Cajal bodies frequencies and more variation in the number of Cajal/nucleus After treatment with inhibitors, both Cajal bodies as nucleoli, made quite clear morphological changes, sometimes giving large accumulation of proteins in organelles and sometimes appeared disorganized in the nucleoplasm. Changes in the size and shape were also highlighted. The tumor cell lines also showed changes compared to their normal cell type.

Orientador: Shirlei Maria Recco-Pimentel / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-05T12:50:04Z (GMT). No. of bitstreams: 1 Busin_CarmenSilvia_D.pdf: 6669737 bytes, checksum: 7a1f9be2cc98550bffd4b6b919b57406 (MD5) Previous issue date: 2005 / Resumo: A posição taxonômica, as relações filogenéticas, a sinonimização de espécies consideradas distintas, a existência de subespécies e as propostas de agrupamentos intragenéricos dos gêneros Pseudis e Lysapsus sempre foram bastante discutidas entre os herpetólogos. Os gêneros Pseudis e Lysapsus já foram considerados membros da família Pseudidae e também da subfamília de Hylidae. Recentemente, foram alocados na subfamília Hylinae como gêneros distintos. Pseudis paradoxa, P. minuta e Lysapsus limellus, já analisados citogeneticamente por outros autores, apresentam 2n=24 cromossomos e Pseudis sp. (aff. minuta), hoje confirmada citogeneticamente como a recentemente descrita P. cardosoi, apresenta 2n=28 cromossomos, com quatro pares adicionais de cromossomos telocêntricos. No presente trabalho foram analisadas, através de coloração convencional dos cromossomos, padrão de distribuição de heterocromatina, número e localização das regiões organizadoras de nucléolo (NOR), espécies do gênero Pseudis e do gênero Lysapsus, exceto L. laevis, com o objetivo de contribuir com caracteres citogenéticos para a sistemática e para os estudos de filogenia dos dois gêneros, além de buscar evidências para a compreensão dos processos envolvidos na evolução cromossômica nesses grupos. As análises citogenéticas revelaram que o complemento 2n=24 cromossomos é a condição plesiomórfica tanto no gênero Pseudis quanto no gênero Lysapsus e corroboraram a hipótese de que o cariótipo 2n=28 cromossomos tenha uma origem comum ao cariótipo 2n=24 de P. minuta, pois as bandas heterocromáticas marcadoras dos dois cariótipos não foram detectadas em nenhuma das espécies analisadas no presente trabalho. As análises da morfologia cromossômica e do padrão de distribuição de heterocromatina permitiram a separação inter- e intragenérica nos dois gêneros, exceção feita entre as subespécies Pseudis paradoxa paradoxa e P. p. platensis que apresentaram os dados citogenéticos comuns. As diferenças detectadas no padrão de distribuição de heterocromatina além de permitir a separação das espécies de Lysapsus e de Pseudis permitiu, também, sugerir uma reavaliação do status taxonômico das subespécies L. limellus limellus e L. ,. bolivianus, especialmente da população de L. I. bolivianus de Guajará-Mirim, que apresentaram diferenças na morfologia e padrão de bandamento dos cromossomos 7 e 8, também em relação às outras populações da mesma subespécie. A presença da região organizadora de nucléolo (NOR) nos braços longos dos cromossomos do par 7 é o caráter plesiomórfico tanto no gênero Pseudis como no gênero Lysapsus e a posição que a mesma ocupa ao longo do braço é um dado citogenético importante na separação das espécies de Pseudis. A morfometria cromossômica, padrão de bandamento e posição da NOR nos cromossomos 7 permitiram também verificar a presença de cromossomos sexuais heteromórficos no sistema ZZIZW em P. tocantins, com evidências de que mecanismos de inversão e de ganho de heterocromatina tenham ocasionado a diferenciação dos cromossomos Z e W / Abstract: The taxonomic position, phylogenetic relationships, synonymization of species considered to be distinct, existence of subspecies, and the proposals of intrageneric groups of the genera Pseudis and Lysapsus have always been a matter of discussion among herpetologists. The genera Pseudis and Lysapsus have already been included in the family Pseudidae and also in the subfamily Hylidae. Recently, these two genera have been allocated to the subfamily Hylinae as distinct genera. Pseudis paradoxa, P. minuta and Lysapsus limellus, cytogenetically analyzed by other investigators, show a chromosome number of 2n=24, and Pseudis sp. (aff. minuta), now cytogenetically confirmed as the recently described P. cardosoi, has 2n=28 chromosomes, including four additional pairs of telocentric chromosomes. In the present study, we analyzed the pattem of heterochromatin distribution and the number and location of the nucleolar organizer region (NOR) by conventional chromosome staining in species of the genera Pseudis and Lysapsus, except for L. laevis, in order to add cytogenetic traits to the systematics and to the study of the phylogeny of the two genera, in addition to providing evidence for the understanding of the processes involved in the chromosome evolution of these groups. Cytogenetic analysis revealed that the complement of 2n=24 chromosomes is a plesiomorphic condition both in the genus Pseudis and in the genus Lysapsus, and confirmed the hypothesis that the origin of the 2n=28 karyotype is the same as that of the 2n=24 karyotype of P. minuta since the heterochromatic marker bands in the two karyotypes were not detected in any of the species analyzed in the present study. Analysis of the chromosome morphology and the pattem of heterochromatin distribution permitted the inter- and intrageneric separation of the two genera, except for the subspecies Pseudis paradoxa paradoxa and P. p. platensis which presented common cytogenetic data. In addition to permitting the separation of Lysapsus and Pseudis species, the differences detected in the pattem of heterochromatin distribution also suggested the reassessment of the taxonomic status of the subspecies L. limellus limellus and L. I. bolivianus, especially of the L. I. bolivianus population from Guajará-Mirim, which differed in the morphology and banding pattem of chromosomes 7 and 8 in relation to the other populations of the same subspecies. The presence of the NOR on the long arms of the chromosomes of pair 7 was a plesiomorphic trait both in the genus Pseudis and in the genus Lysapsus, and the position the NOR occupies on the long arm is an important cytogenetic characteristic for the separation of Pseudis species. Chromosome morphometry, banding pattern and NOR position on chromosomes 7 also permitted the detection of heteromorphic sex chromosomes in the ZZlZW system of P. tocantins, with evidence that mechanisms of inversion and heterochromatinization caused the differentiation of the Z and W chromosomes / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Orientador: Luciana Bolsoni Lourenço / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T06:46:34Z (GMT). No. of bitstreams: 1 Quindere_YedaRumiSerraDouglas_M.pdf: 8609021 bytes, checksum: 5a91bb6ce1e8fff91e49eb0b18f73d2d (MD5) Previous issue date: 2007 / Resumo: O gênero Physalaemus é composto por 40 espécies divididas em sete grupos: ¿albifrons¿, ¿cuvieri¿, ¿deimaticus¿, ¿gracilis¿, ¿henselii¿, ¿olfersii¿ e ¿signifer¿. Nove espécies compõem o grupo "cuvieri" e dessas apenas P. cuvieri já teve seu cariótipo descrito com detalhes, tendo apresentado expressiva variação intra e interpopulacional em relação à localização de regiões organizadoras de nucléolo (NOR). Dado que P. Cuvieri apresenta ampla distribuição geográfica e que a variação mencionada foi encontrada em populações do sul e do sudeste do Brasil, no presente trabalho ampliamos seu estudo, com a análise cromossômica de quatro populações da região nordeste, uma da região norte e uma da região sudeste do Brasil. Adicionalmente uma população da Argentina também foi estudada. Com o intuito de comparar cariotipicamente P. cuvieri com outras espécies de Physalaemus, também foram estudados os cariótipos de P. albonotatus, P. centralis, P. cuqui e P. ephippifer, pertencentes ao grupo "cuvieri", P. albifrons, espécie recentemente removida desse grupo, e P. santafecinus atualmente do grupo de P. albifrons. Todas as populações de P. cuvieri aqui estudadas apresentaram cariótipo com 2n=22 cromossomos e grande variação em relação às NORs pôde ser observada. Duas populações de P. cuvieri (Urbano Santos-MA e Crateús-CE) apresentaram as NORs nos pares 8 e 9. No par 8, a NOR, de localização intersticial, mostrou-se adjacente a uma região de heterocromatina, enquanto a NOR presente no par 9 foi coincidente com um bloco heterocromático. Na população de São Pedro da Água Branca (MA), além dos pares 8 e 9, o par 7 foi também portador de NOR. Na população de Palmeiras (BA) e Uberlândia (MG), apenas um par cromossômico (8) foi portador de NOR. Na população mineira, diferenças intra-individuais foram encontradas principalmente em relação ao tamanho da NOR. Já nas populações da Argentina, a NOR intersticialmente localizada no o par cromossômico 8 não foi encontrada. Em todos os exemplares dessas populações argentinas, o par 11 foi portador de NOR e NORs adicionais foram encontradas nos cromossomos 1, 7 ou 8 (em posição pericentromérica) em alguns indivíduos. É interessante notar que o morfo 11 dessa população argentina é muito semelhante ao cromossomo 11 encontrado na população de Santa Maria (RS) analisada anteriormente. Já na população do estado de Tocantins, NORs múltiplas foram visualizadas em pelo menos cinco cromossomos (pertencentes aos pares 1, 3, 4 e 10), padrão que difere bastante daqueles encontrados nas outras populações de P. cuvieri. Também em relação ao padrão de distribuição de bandas heterocromáticas no cariótipo, a população de Tocantins analisada difere das demais, fato que intriga e corrobora a necessidade de uma revisão taxonômica da espécie em questão. A morfologia cariotípica de P. albifrons, P. albonotatus, P. centralis, P. cuqui, P. ephippifer e P. santafecinus foi muito semelhante, principalmente em relação aos primeiros pares cromossômicos, embora em P. albonotatus e P. cuqui a ordenação de alguns pares tenha sido diferente. Todas essas espécies puderam ser diferenciadas pela localização da NOR e em P.ephippifer um interessante heteromorfismo foi observado no par 8 portador de NOR em todas as fêmeas analisadas. Não foi descartada a hipótese de tal heteromorfismo estar relacionado à determinação sexual nessa espécie, no entanto, futuros estudos são necessários para sua comprovação. Exceto no cariótipo dos indivíduos de P. cuvieri de Tocantins, nos cariótipos aqui descritos a principal (ou única) NOR foi encontrada nos últimos pares cromossômicos, alterando a morfologia desses, o que dificultou a inferência de homeologias interespecíficas e dos possíveis rearranjos que envolveram a NOR durante a diferenciação das espécies em análise. Já com o bandamento C, algumas homeologias interespecíficas foram claramente notadas. Uma banda intersticial no braço curto do par 5, outras na região pericentromérica do braço curto dos pares 3 e 7, por exemplo, foram encontradas em P. cuvieri, P. centralis e P. ephippifer, e parecem homeólogas às encontradas nos pares classificados como 3, 5 e 7 no cariótipo de P. albonotatus / Abstract: The genus Physalaemus is composed by 41 species distributed in seven groups: ¿albifrons¿, ¿cuvieri¿, ¿deimaticus¿, ¿gracilis¿, ¿henselii¿, ¿olfersii¿ and ¿signifer¿. Nine species compose the group ¿cuvieri¿ but just P. cuvieri had already been karyotyped in details. Expressive intra and interpopulational variation related to the localization of the nucleolus organizer regions (NOR) was described for this species. Physalaemus cuvieri is widely geographically distributed and NOR variation was described based on populations from Southern and Southeastern Brazil. In the present work we analyzed the cytogenetic of four populations from Northeastern, one from Northern and one from Southeastern Brazil. Additionally, three Argentinian populations were also included. To cytogenetically compare P. cuvieri with other species of Physalaemus, species belonging to P. cuvieri group (i.e. P. albonotatus, P. centralis, P. cuqui and P. ephippifer), P. albifrons, recently removed from this group, and P. santafecinus, currently alocated in P. albifrons group, were also analyzed. All populations of P. cuvieri studied here showed diploid number of 22 chromosomes and high intraspecific variation was observed related to the NORs. Two populations of P. cuvieri (Urbano Santos, state of Maranhão (MA) and Crateús, state of Ceará (CE)) had pairs 8 and 9 as NOR-bearing chromosomes. In pair 8 the interstitial NOR was adjacent to C-bands whereas the NOR at the nineth pair was coincident with a heterochromatic block. In the population from São Pedro da Água Branca, state of Maranhão (MA), besides pairs 8 and 9, the seventh pair was also a NOR-bearing one. The specimens from Palmeiras, state of Bahia (BA) and Uberlândia, state of Minas Gerais (MG) showed only one NOR, which was located at pair 8. In the population from Uberlândia (MG) intraindividual differences was found related to the NOR size. In the populations from Argentina, the interstitial NOR in chromosome pair 8 was not found. In all the specimens of these Argentinean populations, pair 11 was the NOR-bearing chromosome pair and additional NORs were also found in chromosomes 1, 7 or 8 (in a pericentromeric position) in some of the individuals. The morph 11 of specimens from Argentina was very similar to the NOR-bearing eleventh pair described for specimens from Santa Maria (RS) previously analyzed. In contrast, in the population from Porto Nacional, state of Tocantins (TO), multiple NORs were visualized at least at five chromosomes (belonging to pair 1, 3, 4 and 10), a pattern that greatly differed from those found in the others populations of P. cuvieri. Also concerning the C-band distribution, the karyotype found in the population of Tocantins differed from the others. These findings are very interesting and can be useful for future taxonomic studies of this taxon. Regarding to chromosome morphology, P. albifrons, P. albonotatus, P. centralis, P. cuqui, P. ephippifer and P. santafecinus were very similar, specially for the seven first chromosome pairs, although in P. albonotatus and P. cuqui the position of some chromosome pairs was different. All the species could be differentiated by NOR localization and in P. ephippifer an interesting heteromorphism was detected in NOR bearing pair 8 of all the females. We do not discard the hypothesis that such heteromorphism could be related to sex determination in this species, but future studies are necessary to test it. Except for the karyotype of P. cuvieri from Tocantins, the karyotypes described here the principal (or only) NOR was found among the last chromosome pairs, resulting in different chromosome morphologies, what impaired interespecific inferences of homeologies and the recognition of possible rearrengements involving the NOR during the differentiation of the species analyzed. On the other hand, C-banding tecnique permitted to notice some interspecific homeologies. A band at an interstitial region in the short arm of pair 5, and pericentromeric C-bands in the short arm of pairs 3 and 7 were detected in P. cuvieri, P. centralis and P. ephippifer which seemed to be homeologous to C-bands found in pair 3, 5 and 7 of P. albonotatus / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural