Orientadores: Shirlei Maria Recco-Pimentel, Ana Cristina Prado Veiga-Menoncello / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T04:20:05Z (GMT). No. of bitstreams: 1 Favero_EduardoRondelli_M.pdf: 1364067 bytes, checksum: 03d34437fbe08a46a8cdd3631aaedcc9 (MD5) Previous issue date: 2008 / Resumo: O gênero Pseudopaludicola, pertencente à família Leiuperidae, compreende atualmente 12 espécies de rãs de pequeno tamanho, distribuídas pela América do Sul, sendo a ocorrência de oito delas foi relatada para o Brasil. Devido à grande semelhança morfológica entre espécies, algumas ocorrendo em simpatria, confusões taxonômicas são freqüentes. Alguns estudos morfológicos acerca deste gênero foram realizados, mas as relações de parentesco inter- e intragenéricas de Pseudopaludicola permanecem pouco esclarecidas. As poucas informações citogenéticas para o gênero Pseudopaludicola restringiam-se apenas à determinação do número de cromossomos e análise do cariótipo por métodos de coloração convencional. Para Pseudopaludicola falcipes, em especial, foi descrita uma variação intra-específica do número de cromossomos, de 2n=16 à 2n=20. O presente estudo visa contribuir com dados citogenéticos para a caracterização de espécies de Pseudopaludicola e para o entendimento dos processos envolvidos na evolução cariotípica do gênero. Foram analisados os cariótipos de exemplares de Pseudopaludicola falcipes, P. ameghini (sensu Cope, 1887) e P. mystacalis de suas respectivas localidades-tipo (região de Porto Alegre, RS e Chapada dos Guimarães, MT), de P. mystacalis e de P. ternetzi, de Uberlândia (MG), de Pseudopaludicola aff. falcipes I, II, III e IV, da região noroeste do estado de São Paulo (municípios de Santa Fé do Sul, Vitória Brasil, Palestina e Icém), de Pseudopaludicola aff. mystacalis I, II, III e IV, dos municípios de Icém (SP), Barreirinhas (MA) e Urbano Santos (MA) e Pseudopaludicola sp. 1, 2 e 3, sendo as duas primeiras provenientes de Poconé (MT) e a terceira de Santa Terezinha (MT). As metáfases foram obtidas de suspensões de células de epitélio intestinal e testículo, e coradas com Giemsa ou submetidas às técnicas impregnação por prata (Ag-NOR) para detecção de NOR e de bandamento C, para a localização de heterocromatina. Os dados obtidos revelaram uma variação interespecífica quanto ao número de cromossomos. Dentre os espécimes provenientes de Poconé, MT, havia dois cariótipos distintos, com 2n=22 e com 2n=16 cromossomos (Pseudopaludicola sp. 1 e 2) e os de Icém, SP, com indivíduos 2n=20 e 2n=16 cromossomos (Pseudopaludicola aff. mystacalis, respectivamente I e II). Pseudopaludicola falcipes e Pseudopaludicola sp.1, de Poconé, apresentaram 2n=22 e a NOR localizada na região pericentromérica do braço longo do par 8. Estas espécies diferiram, na morfologia da NOR, sendo heteromórfica em P. falcipes e homomórfica em Pseudopaludicola sp. 1, e na localização de algumas bandas heterocromáticas. Pseudopaludicola ameghini (sensu Cope, 1887), P. ternetzi e Pseudopaludicola aff. mystacalis I de Icém apresentaram 2n=20 cromossomos e a NOR localizada na região telomérica do braço longo do par 9. O cariótipo de P. ternetzi diferiu do de P. ameghini tanto pela classificação morfológica distinta do par 7 quanto pelo padrão de distribuição de heterocromatina. Pseudopaludicola mystacalis, bem como todos os espécimes de Pseudopaludicola aff. falcipes I, II, III e IV, Pseudopaludicola aff. mystacalis II, III e IV e Pseudopaludicola sp. 2 e 3 apresentaram 2n=16 cromossomos metacêntricos e submetacêntricos, com a NOR localizada na região pericentromérica do braço curto do par 4. Vários espécimes apresentaram um heteromorfismo de tamanho em relação aos homólogos do par 4 (morfo 4 e morfo 4'), alterando a classificação desse cromossomo para metacêntrico em algumas populações. Em P. mystacalis, P. aff. falcipes I, II, III e IV, P. aff. mystacalis II, III e IV e Pseudopaludicola sp 2 e 3 foram detectados blocos de heterocromatina fortemente marcados nas regiões pericentroméricas no braço curto do par 1 e longo do par 2. Os resultados obtidos mostram que P. ameghini (sensu Cope, 1887) com 2n=20 e P. mystacalis com 2n=16, são unidades taxonômicas distintas e que os espécimes tidos como Pseudopaludicola aff. falcipes) e como Pseudopaludicola sp. mostraram-se citogeneticamente relacionados à P. mystacalis e não à P. falcipes que possui 2n=22 cromossomos. Desta forma, os nossos dados sugerem a retirada de P. ameghini da sinonímia de P. mystacalis e reforçam a necessidade de uma revisão taxonômica no gênero. / Abstract: The genus Pseudopaludicola (family Leiuperidae) comprises 12 species of small sized frogs, which are widely distributed in South America. In Brazil, eight species are described within this genus, and several of them are sympatric. The relevant morphological similarities among the Pseudopaludicola species have contributed to the still poor understanding of many aspects of their taxonomy, including inter- and intrageneric relationships. Cytogenetic data on Pseudopaludicola have been restricted to karyotype analyses using conventional Giemsa staining. Variation in intraspecific chromosomal number was described in P. falcipes, ranging from 2n=16 to 2n=20. In the present work, Brazilian Pseudopaludicola species were submitted to cytogenetic analysis aiming at their further characterization and attempting to better understanding the karyotypical evolution of this genus. The analyzed species were Pseudopaludicola falcipes (Porto Alegre, RS), P. ameghini (sensu Cope, 1887) and P. mystacalis (Chapada dos Guimarães, MT), P. mystacalis and P. ternetzi (Uberlândia, MG), P. aff. falcipes I, II, III and IV (respectively from Santa Fé do Sul, Vitória Brasil, Palestina and Icém, SP), P. aff. mystacalis I, II, III and IV (Icém, SP, Barreirinhas, MA, and Urbano Santos, MA), and Pseudopaludicola sp. 1 and sp. 2 (Poconé, MT) and sp. 3 (Santa Terezinha, MT). Metaphases were obtained from suspensions of intestinal epithelium and testicular cells, and stained with Giemsa or submitted to silver staining technique in order to detect the nucleolus organizing regions (Ag-NOR), and C-banding, for heterochromatin localization. The results revealed interspecific chromosomal number variation. In the Pseudopaludicola sp. 1 and 2 specimens (Poconé MT), two distinct karyotypes were identified, respectively with 2n=22 and 2n=16 chromosomes. Within the Pseudopaludicola aff. mystacalis from Icém, SP, the analyzed specimens had 2n=20 and 2n=16 chromosomes, being nominated I and II, respectively. These data clearly indicated two criptic species of Pseudopaludicola within each of those two localities. The P. falcipes and Pseudopaludicola sp.1 (Poconé, MT) had 2n=22 and the NOR was located at the pericentromeric region in the long arm of the pair 8. The species differed in the NOR morphology, which was heteromorphic in P. falcipes and homomorphic in Pseudopaludicola sp.1, as well as in the localization of some C-bands. Pseudopaludicola ameghini (sensu Cope, 1887), P. ternetzi and Pseudopaludicola aff. mystacalis I (Icém, SP) had 2n=20 chromosomes and the NOR was located on the telomeric region in the long arm of the pair 9. The karyotypes of P. ternetzi and P. ameghini differed in the pair 7 morphology and in the heterochromatin distribution pattern. All analyzed specimens of P. mystacalis, P. aff. falcipes I, II, III and IV, P. aff. mystacalis II, III and IV, and Pseudopaludicola sp. 2 and 3, showed 2n=16 chromosomes, which were all metacentric and submetacentric, with the NOR located in the pericentromeric region of the short arm of the pair 4. In several of those specimens, the size heteromorphism of the pair 4 altered, from submetacentric to metacentric, the classification of one of the homologous of that pair. Strong pericentromeric C-bands were detected on the short arm of the pair 1 and on the long arm of the pair 2 in P. mystacalis, P. aff. falcipes I, II, III and IV, P. aff. mystacalis II, III and IV, and Pseudopaludicola sp. 2 and 3. As based on the cytogenetic data, P. ameghini (sensu Cope, 1887), with 2n = 20, and P. mystacalis, with 2n = 16, are distinct taxonomic units, and the specimens formerly identified as P. aff. falcipes and as Pseudopaludicola sp. were indeed cytogenetically closely related to P. mystacalis and not to P. falcipes, which has 2n = 22 chromosomes. Hence, our data suggest that P. ameghini is not a P. mystacalis synonymy and emphasize the importance of a taxonomic review of the genus Pseudopaludicola. / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

Comme le nucléole joue un rôle fondamental dans l’expression des protéines, via la synthèse des ARN ribosomiques, il n’est donc pas surprenant que des études aient révélé un lien étroit, entre des dysfonctionnements nucléolaires et l’origine de certaines maladies humaines. La découverte, il y a plusieurs années, d’un taux anormalement élevé de protéines nucléolaires dites argyrophiles ou AgNORs, dans les cellules tumorales, a permis d’envisager leur utilisation comme outil diagnostique ou pronostique du cancer. Détectées, de manière in vitro grâce à leur affinité pour l’argent, l’identification de quelques protéines AgNORs n’a pourtant pas permis d’établir une caractéristique commune à toutes les protéines argyrophiles détectées dans les extraits nucléolaires. Ainsi, bien que le test colorimétrique AgNOR soit utilisé dans de nombreux laboratoires académiques, l’absence d’identification de protéines AgNORs spécifiques du processus de cancérisation, a limité son utilisation en laboratoire clinique. Comme certaines limites technologiques et expérimentales ont limité leur caractérisation chez l’humain, nous avons donc décidé de reprendre les recherches sur ce sujet et de le réactualiser grâce aux avancées technologiques et scientifiques. Les protéines AgNORs étant étroitement liées à la biogenèse des ribosomes, nous avons donc décidé d’amorcer nos recherches chez la levure Saccharomyces cerevisiae, dans laquelle, la voie de biosynthèse des ribosomes a été particulièrement bien décrite. Devant l’intérêt biologique et médical de ces protéines, l’objectif de ce projet a donc été triple : 1-identifier des protéines AgNORs chez la levure 2-caractériser les propriétés physico-fonctionnelles et physico-chimiques de ces protéines AgNORs. 3-utiliser ces caractéristiques physico-chimiques pour rechercher de nouvelles AgNORs humaines, spécifiques de processus de cancérisation et potentiellement utilisables comme marqueurs tumoraux./The nucleolus is a subnuclear compartment that organized around ribosomal gene (rDNA) repeats NORs, which encode for ribosomal RNA. A peculiar group of acidic proteins which are highly argyrophilic are also localized at the same sites as NORs, thus allowing NORs to be very clearly and rapidly visualized by silver nitrate staining procedures. However, if three human argyrophilic proteins, UBF, C23 (nucleolin) and B23 (nucleophosmin), have been associated for staining of NOR, the exact number of AgNOR proteins and their intrinsic biochemical feature are unclear. Here, we have performed an heterologous screen in a genetically tractable eukaryotic organism (budding yeast) for the identification of novel AgNOR proteins and in vitro characterized an intrinsic feature that underlies silver binding and offers a strong predictive value for the identification of novel human AgNOR proteins.

Comme le nucléole joue un rôle fondamental dans l’expression des protéines, via la synthèse des ARN ribosomiques, il n’est donc pas surprenant que des études aient révélé un lien étroit, entre des dysfonctionnements nucléolaires et l’origine de certaines maladies humaines. La découverte, il y a plusieurs années, d’un taux anormalement élevé de protéines nucléolaires dites argyrophiles ou AgNORs, dans les cellules tumorales, a permis d’envisager leur utilisation comme outil diagnostique ou pronostique du cancer. Détectées, de manière in vitro grâce à leur affinité pour l’argent, l’identification de quelques protéines AgNORs n’a pourtant pas permis d’établir une caractéristique commune à toutes les protéines argyrophiles détectées dans les extraits nucléolaires. Ainsi, bien que le test colorimétrique AgNOR soit utilisé dans de nombreux laboratoires académiques, l’absence d’identification de protéines AgNORs spécifiques du processus de cancérisation, a limité son utilisation en laboratoire clinique. Comme certaines limites technologiques et expérimentales ont limité leur caractérisation chez l’humain, nous avons donc décidé de reprendre les recherches sur ce sujet et de le réactualiser grâce aux avancées technologiques et scientifiques. Les protéines AgNORs étant étroitement liées à la biogenèse des ribosomes, nous avons donc décidé d’amorcer nos recherches chez la levure Saccharomyces cerevisiae, dans laquelle, la voie de biosynthèse des ribosomes a été particulièrement bien décrite. Devant l’intérêt biologique et médical de ces protéines, l’objectif de ce projet a donc été triple :<p>1-identifier des protéines AgNORs chez la levure<p>2-caractériser les propriétés physico-fonctionnelles et physico-chimiques de ces protéines AgNORs.<p>3-utiliser ces caractéristiques physico-chimiques pour rechercher de nouvelles AgNORs humaines, spécifiques de processus de cancérisation et potentiellement utilisables comme marqueurs tumoraux./The nucleolus is a subnuclear compartment that organized around ribosomal gene (rDNA) repeats NORs, which encode for ribosomal RNA. A peculiar group of acidic proteins which are highly argyrophilic are also localized at the same sites as NORs, thus allowing NORs to be very clearly and rapidly visualized by silver nitrate staining procedures. However, if three human argyrophilic proteins, UBF, C23 (nucleolin) and B23 (nucleophosmin), have been associated for staining of NOR, the exact number of AgNOR proteins and their intrinsic biochemical feature are unclear. Here, we have performed an heterologous screen in a genetically tractable eukaryotic organism (budding yeast) for the identification of novel AgNOR proteins and in vitro characterized an intrinsic feature that underlies silver binding and offers a strong predictive value for the identification of novel human AgNOR proteins. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished