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Promyelocytic leukemia protein (PML) is a tumour suppressor which is frequently downregulated in human tumours. PML plays a role in many cellular processes including DNA damage response, senescence and apoptosis and is mainly localized in special structures called PML nuclear bodies (PML NBs). The nucleolus is a key nuclear compartment, where transcription of ribosomal DNA and biogenesis of ribosomes take place. The nucleolus is also called a stress sensor because of its role, for instance, in stabilization of tumour suppressor p53. Localization of PML to the nucleolar periphery appears to be prominent after disturbance of nucleolar functions - for example inhibition of rRNA transcription or processing. Thus the relationship between the nucleolus and PML nuclear bodies may be important for cellular response to stress. However, the role of PML nucleolar associations in nucleolar function including mechanism of formation of these structures remain unclear. Here we characterised PML nucleolar structures and mechanism of their formation. We showed that formation of PML nucleolar structures is not caused by replication stress, is not dependent on any specific phase of cell cycle and is not caused by DNA damage response but is induced by topological stress due to inhibition of toposiomerase function....

Despite the existence of various molecular marker systems there are still limitations in distinguishing between closely related species based on molecular divergence, especially when hybridization events have occurred in the past. The characterisation of plant small nucleolar RNA (snoRNA) genes and their organisation into multigene clusters provides a potential nuclear marker system which could help in resolving the phylogenetic history of plants and might be applicable in DNA barcoding. Using closely and distantly related Senecio species, I investigated a combination of fragment length and sequence variation of snoRNA genes/snoRNA gene clusters to assess the utility of this marker system for barcoding and resolving species relationships. SnoRNA gene and gene cluster sequences identified in Arabidopsis thaliana were used to find homologues in other species and subsequently used for the design of universal primers. Most of the universal primer pairs designed were successful in amplifying snoRNA fragments in most Senecio species and fragment length variation between and within species could be detected. Furthermore, the combination of some fragment length datasets produced by different primer pairs enabled the separation of species and the detection of reticulate evolution indicating a high potential of snoRNA gene/gene cluster fragment length polymorphisms (SRFLPs) for phylogenetic reconstructions in Senecio and other plant genera. Most of the examined gene clusters showed a similar gene order in Senecio and Arabidopsis. However, the majority of these clusters appeared to exhibit more copies in Senecio, some of which were distinguishable by a combined sequencing/fragment profiling approach, and shown to be putative single copy regions with the potential to be used as co-dominant markers. However, a high number of paralogues and possible differences in copy number between species excludes these regions from being used in DNA barcoding. This is because specific primers would have to be developed for specific copies which would preclude development of a universal application for barcoding. None of the regions showed enough sequence variation to delimit distinctly closely related Senecio species and were therefore also considered to be unsuitable for DNA barcoding. Although most snoRNA genes and gene clusters might be inapplicable for DNA barcoding, they are likely to be valuable for phylogenetic studies of species groups, genera and families. On this scale, specific primers might act universally and the number of paralogous copies is likely to be equal across the species group of interest.

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)

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

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

Orb-weavers (Araneidae) are a diversified spider family comprising more than 3,100 species in more than 170 genera. Together with 13 other families, they con- stitute to superfamily Araneoidea. The presented thesis focuses on karyotype evo- lution of Araneidae, including its comparison with a related family Tetragnathidae. The results obtained from 19 araneid and four tetragnathid species confirm previ- ously postulated hypothesis that the ancestral karyotype of Araneoidea (including Araneidae) consists of 24 acrocentric chromosomes in males, including two acro- centric X chromosomes of system X1X20. However, there is a tendency of 2n decrease in some araneids due to centric fusions. In these cases, centric fusions affected most autosomes (and sometimes gonosomes as well); number of chromosome pairs de- creased from 11 to six. Three independent reduction events were detected in this thesis. Furthermore, pattern of nucleolar organizer regions (NORs) was studied in this thesis using fluorescent in situ hybridization, since data on evolution of this marker are scarce in spiders. Striking variability in NORs number was discovered, ranging from one to 13 loci. Remarkably, multiple centric fusions were always ac- companied by considerable increase of NORs number. In araneids and tetragnathids possessing...

Orb-weavers (Araneidae) are a diversified spider family comprising more than 3,100 species in more than 170 genera. Together with 13 other families, they con- stitute to superfamily Araneoidea. The presented thesis focuses on karyotype evo- lution of Araneidae, including its comparison with a related family Tetragnathidae. The results obtained from 19 araneid and four tetragnathid species confirm previ- ously postulated hypothesis that the ancestral karyotype of Araneoidea (including Araneidae) consists of 24 acrocentric chromosomes in males, including two acro- centric X chromosomes of system X1X20. However, there is a tendency of 2n decrease in some araneids due to centric fusions. In these cases, centric fusions affected most autosomes (and sometimes gonosomes as well); number of chromosome pairs de- creased from 11 to six. Three independent reduction events were detected in this thesis. Furthermore, pattern of nucleolar organizer regions (NORs) was studied in this thesis using fluorescent in situ hybridization, since data on evolution of this marker are scarce in spiders. Striking variability in NORs number was discovered, ranging from one to 13 loci. Remarkably, multiple centric fusions were always ac- companied by considerable increase of NORs number. In araneids and tetragnathids possessing...

Platinum-based chemotherapeutics are used for the treatment of a wide range of cancers and a number of attempts have been made toward developing compounds with better cellular stability and similar or enhanced cytotoxicity as compared to their predecessors. The first part of the work reported here focuses on the cellular effects of the metabolically stable dinuclear platinum compound, BBR3610-DACH. Comet assay showed this compound to form interstrand crosslinks, a highly toxic DNA lesion in HCT116 cells, at equimolar concentrations to its parental compound, BBR3610. Cell cycle studies showed that BBR3610-DACH causes G1/S and G2/M cell cycle arrest with S phase depletion, which was p21 dependent and partially p53 dependent in contrast to BBR3610 which showed initial S phase accumulation followed by a classical G2/M arrest. BBR3610-DACH-induced G1/S and G2/M cell cycle arrest interestingly was found to be independent of the DNA damage response mediated via the activation of ATM and ATR kinases. Also, the cell cycle arrest culminated in apoptosis, although apparently through a non-canonical pathway. The second project explores the cellular effects of trans-4-NBD which is a fluorescent derivative of transplatin. Like cisplatin, trans-4-NBD induced interstrand crosslinks in HCT116 cells as detected by the comet assay. Treatment with trans-4-NBD showed a G2/M arrest in HCT116 cells and a transient S phase accumulation in A2780 cells, with a marked increase in p53 and p21 protein levels. A robust apoptotic response is also seen via caspase activation and PARP cleavage in both the cell lines. Finally, the focus is shifted toward the nucleolar targeting platinum complex, TriplatinNC. Confocal studies in TriplatinNC-treated HCT116 and A2780 cells showed disruption of rRNA transcription as an early event followed by a robust G1 cell cycle arrest. Apoptotic induction was observed with the onset of cellular morphological changes and apparent caspase activation which was independent of the p53 status of the cells. Overall, these studies explore novel platinum based compounds that show promising anti-cancer activities by affecting various facets of cellular signaling.

Resumo: Os triatomíneos são insetos hematófagos de grande importância para a parasitologia humana, pois são transmissores do Trypanosoma cruzi, protozoário causador da doença de Chagas. Além de sua importância médico-sanitária, os triatomíneos destacam-se pela sua citogenética, pois possuem cromossomos holocêntricos e um modelo de meiose incomum, com meiose invertida para os cromossomos sexuais. Recentes pesquisas com marcadores moleculares em triatomíneos tentam compreender a ancestralidade do grupo. Uma das formas de se compreender a evolução entre espécies é a partir da análise de seqüências do DNA ribossômico (DNAr). Por pertencer a famílias multigênicas, cópias individuais do DNAr não acumulam mutações independentemente, resultando em pequena variação intra-específica e relevante diferenciação interespecífica. No presente trabalho foi realizado um estudo comparativo entre as espécies Triatoma maculata e Triatoma pseudomaculata, com base no uso das técnicas citogenéticas convencionais de orceína lacto-acética, impregnação por íons prata, bandamento-C, reação de Feulgen; da técnica de citogenética molecular de bandamento- C CMA/DAPI; e também por meio da análise da região ITS-1 do DNAr, com base no sequenciamento, com o objetivo de avaliar o grau de homologia entre as espécies estudadas. Os cariogramas das duas espécies indicaram dez pares de autossomos (um deles de tamanho maior) e um par de cromossomos sexuais (2n = 22). No ciclo meiótico foi possível observar a fragmentação da região nucleolar no final do estágio difuso. Corpúsculos nucleolares foram observados em alguns dos núcleos em metáfases meióticas de T. pseudomaculata, evidenciando a persistência nucleolar. A técnica de bandamento-C revelou que o cromossomo Y é heterocromático em ambas as espécies. O sequencimento da região ITS-1, indicou que as espécies apresenta... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The triatomines are hematophagous insects of great concern in public health because they are vectors of Trypanosoma cruzi, a protozoan that causes Chagas disease. Triatomines are also of great genetic interest, because that they present holocentric chromosomes and an unusual form of meiosis with post-reductional segregation of sex chromosomes. Recent studies based on molecular markers try to understand the evolutionary history of triatomines. To understand the evolution of a given species, ribosomal DNA (rDNA) analyses are frequently used, which can help to infer evolutionary relationships among species. Individual copies of rDNA do not accumulate mutations independently because they belong to multigene families, resulting in slight intraspecific and important interspecific variation. In this study, a comparative analysis was performed between the species Triatoma maculata and Triatoma pseudomaculata, based on the cytogenetic techniques of lacto-acetic orcein, silver ion impregnation, Cbanding, Feulgen reaction; and CMA/DAPI C-banding. We also compared the species by sequencing the ITS-1 rDNA internal transcribed region in order to evaluate the degree of homology among the studied species. The cariograms of the two species revealed ten autosomes and one pair of sexual chromosomes (2n= 22). In the meiotic cycle, nucleolar fragmentation during the final stages of meiotic prophase I was found. Nucleolar corpuscles were found in some meiotic metaphases of T. pseudomaculata, which is evidence of nucleolar persistence. The C-banding technique revealed that the Y chromosome is heterochromatic in both species. The ITS-1 rDNA sequences showed that the species presented a discharge proximity to each other, and had a high degree of homology (98.5%). The knowledge obtained in this study contributes to the understanding of the interrelation and distribution of those species, and offers... (Complete abstract click electronic access below) / Orientador: Maria Tercília Vilela de Azeredo Oliveira / Coorientador: Lilian Castiglioni / Banca: João Aristeu da Rosa / Banca: Carlos Roberto Ceron / Mestre