Orientador: Luciana Bolsoni Lourenço Morandini / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T20:53:49Z (GMT). No. of bitstreams: 1 Rodrigues_DeboraSilva_M.pdf: 3296545 bytes, checksum: 2a2ccc454f9a1df9f09173c2127af330 (MD5) Previous issue date: 2012 / Resumo: O gênero Engystomops apresenta ampla distribuição geográfica e constitui um interessante grupo de anuros para estudos cariotípicos. As populações de Engystomops encontradas na Amazônia têm sua identificação taxonômica ainda controversa. Análises genéticas e citogenéticas apoiam hipóteses que sugerem a existência de um complexo de espécies crípticas e especiação incipiente. Muitas vezes a variação citogenética observada entre diferentes populações estudadas dificultou o reconhecimento de homeologias cromossômicas entre os cariótipos. Uma caracterização cromossômica mais detalhada poderia auxiliar no possível reconhecimento de homeologias cromossômicas e, dessa forma, contribuir para o estudo dos processos envolvidos na divergência desses anuros. Já que o gene do DNAr 5S tem sido importante marcador genético e citogenético para estudos evolutivos e para a identificação e comparação de espécies em diversos grupos, no presente trabalho o DNAr 5S de Engystomops freibergi e de exemplares de Engystomops petersi de duas localidades Equatorianas (Puyo e Yasuní) foi estudado. Em todos os casos, dois tipos de DNAr 5S, facilmente diferenciados pelo tamanho e composição da sequência do seu espaçador não transcrito, foram isolados. A provável região promotora do gene do RNAr 5S (ICR) foi localizada nos dois tipos de sequências de DNAr 5S e a presença de possíveis sequências regulatórias adicionais foi discutida. No cariótipo de E. freibergi, sonda contendo a unidade repetitiva do DNAr 5S tipo I hibridou na região pericentromérica do braço curto dos cromossomos do par 3, e o DNAr 5S tipo II foi mapeado na região distal do braço longo dos cromossomos do par 6. A sonda formada somente pela região de NTS do DNAr 5S tipo I claramente detectou a região pericentromérica de 3p nos cariótipos de E. freibergi e E. petersi (Puyo) e de 5p no cariótipo de E. petersi (Yasuní), porém nenhum sinal distal ou intersticial foi observado. A sonda formada pela região de NTS do DNAr 5S tipo II detectou apenas a região distal de 6q nos três cariótipos estudados, corroborando a distribuição diferencial dos dois tipos de DNAr 5S nesses cariótipos. Tais sítios de DNAr 5S constituem novos marcadores cromossômicos, os quais permitem sugerir a homeologia entre o cromossomo 6 dos cariótipos de E. freibergi e de E. petersi, e entre o cromossomo 5 do cariótipo de E. petersi de Yasuní e o cromossomo 3 dos cariótipos de E freibergi e de E. petersi de Puyo. Já que os dois tipos de DNAr 5S encontrados em Engystomops são relacionados àqueles de Physalaemus tanto quanto à composição nucleotídica quanto à localização cromossômica, é ainda possível inferir que a origem desses dois tipos de sequências tenha antecedido a divergência evolutiva desses gêneros / Abstract: The Engystomops genus is widely distributed geographically and constitutes an interesting group for karyotypic studies. The taxonomic identifications of Amazonian populations of Engystomops is still controversial. Genetic and cytogenetic analyses suggest the existence of a complex of cryptic species and incipient speciation. The cytogenetic variations found among some populations prevent the recognition of chromosomal homeologies between the described karyotypes. A more detailed chromosomal characterization could help in the recognition of chromosome homeologies and, therefore, could contribute to the study of the processes involved in the divergence of these anurans. Since the 5S rDNA gene has been an important genetic and cytogenetic marker for evolutionary studies and even for the identification and comparison of species in diverse groups, in the present work the 5S rDNA of Engystomops freibergi and exemplars of Engystomops petersi from two Ecuadorian locations (Puyo and Yasuní) was studied. In all cases, two types of 5S rDNA, easily differed by size and compositon of their non-transcribed spacer, were isolated. A putative promoting region of the 5S rRNA gene (ICR) was recognized in the two types of 5S rDNA sequences and the presence of possible additional regulatory sequences was discussed. In the E. freibergi karyotypes, the entire type I 5S rDNA repeating unit hybridized to the pericentromeric region of 3p, whereas the entire type II 5S rDNA repeating unit mapped to the distal region of 6q, suggesting a differential localization of these sequences. The type I NTS probe clearly detected the 3p pericentromeric region in the karyotypes of E. freibergi and E. petersi from Puyo and the 5p pericentromeric region in the karyotype of E. petersi from Yasuní, but no distal or interstitial signals were observed. Interestingly, this probe also detected many centromeric regions in the three karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA. The type II NTS probe detected only distal 6q regions in the three karyotypes, corroborating the differential distribution of the two types of 5S rDNA. Because the 5S rDNA types found in Engystomops are related to those of Physalaemus with respect to their nucleotide sequences and chromosomal locations, their origin likely preceded the evolutionary divergence of these genera. In addition, our data indicated homeology between chromosome 5 in E. petersi from Yasuní and chromosomes 3 in E. freibergi and E. petersi from Puyo. In addition, the chromosomal location of the type II 5S rDNA corroborates the hypothesis that the chromosomes 6 of E. petersi and E. freibergi are homeologous despite the great differences observed between the karyotypes of the Yasuní specimens and the others / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

Le complexe Scc2-Scc4 est essentiel pour l’association du complexe cohésine sur l’ADN. Les proteines Cohésine génèrent la cohésion entre les chromatides sœurs, ce qui est essentiel pour la ségrégation des chromosomes. Scc2 (également connu sous le nom NIPBL) est muté chez les patients atteints du syndrome de Cornelia de Lange, une maladie multi-organique caractérisée par des anomalies du développement du visage, de la developpement mental cardiaque et du tractus gastro-intestinal. Comment les mutations localisées au niveau du gène codant pour la proteine Scc2 conduisent à des anomalies du développement chez les patients n’a pas encore été élucidé. Une des hypothèses est que la liaison de Scc2 / cohésine à différentes régions du génome a une incidence sur la transcription. Chez la levure de bière, il a été montre que Scc2 se lie aux genes transcrits par l'ARN Pol III (les ARNt et spliceosomals) , ainsi qu‘aux gènes transcrits par l'ARN Pol II codant pour des petits ARN nucléolaires et nucléaires (snARN et snoARNs ) et des gènes de protéines ribosomiques. Nous rapportons ici que Scc2 est important pour l'expression de ces gènes. Scc2 et le régulateur transcriptionnel Paf1 collaborent pour promouvoir la production de Box H / ACA snoARNs qui guident la pseudouridylation des ARN y compris l'ARN ribosomal. Une mutation de Scc2 a été associée à des défauts dans la production d'ARN ribosomal, la biogenèse des ribosomes, et del’épissage. Alors que le mutant Scc2 n'a pas de défaut général de la synthèse protéique, il montre un déphasage accrue et une réduction de l’utilisation du site interne d'entrée ribosomale (IRES)/ coiffe-indépendante. Ces résultats suggèrent que Scc2 favorise normalement un programme d'expression génétique qui prend en charge la fidélité de la traduction. Nous émettons l'hypothèse que le dysfonctionnement de traduction peut contribuer au syndrome de Cornelia de Lange, qui est causé par des mutations dans Scc2. / The Scc2-Scc4 complex is essential for loading the cohesin complex onto DNA. Cohesin generates cohesion between sister chromatids, which is critical for chromosome segregation. Scc2 (also known as NIPBL) is mutated in patients with Cornelia de Lange syndrome, a multi-organ disease characterized by developmental defects in head, limb, cognition, heart, and the gastrointestinal tract. How mutations in Scc2 lead to developmental defects in patients is yet to be elucidated. One hypothesis is that the binding of Scc2/cohesin to different regions of the genome will affect transcription. In budding yeast, Scc2 has been shown to bind to RNA Pol III transcribed genes (tRNAs, and spliceosomal), as well as RNA Pol II-transcribed genes encoding small nuclear and nucleolar RNAs (snRNAs and snoRNAs) and ribosomal protein genes. Here, we report that Scc2 is important for gene expression. Scc2 and the transcriptional regulator Paf1 collaborate to promote the production of Box H/ACA snoRNAs which guide pseudouridylation of RNAs including ribosomal RNA. Mutation of Scc2 was associated with defects in the production of ribosomal RNA, ribosome biogenesis, and splicing. While the scc2 mutant does not have a general defect in protein synthesis, it shows increased frameshifting and reduced internal ribosomal entry site (IRES) usage/cap-independent translation. These findings suggest Scc2 normally promotes a gene expression program that supports translational fidelity. We hypothesize that translational dysfunction may contribute to the human disorder Cornelia de Lange syndrome, which is caused by mutations in Scc2.

Neste trabalho foram caracterizadas molecularmente e inferidas as relações filogenéticas de 14 isolados brasileiros de Acanthamoeba, provenientes de casos de ceratite, e 8 isolados da ATCC (4 de ceratite e 4 ambientais). Foram utilizados inicialmente os métodos de RAPD, RFLP de DNA genômico total e RFLP do SSU rDNA. Apesar de revelar a alta variabilidade genética em Acanthamoeba, estes métodos permitiram estabelecer grupos bem definidos de isolados mais similares geneticamente. O seqüenciamento do SSU rDNA permitiu a inferência da filogenia entre os isolados utilizados nesse estudo em relação àqueles presentes na literatura, que estão distribuídos em doze tipos de seqüência deste gene. Dentre os 17 isolados de ceratite presentes em nosso estudo, 16 apresentaram SSU rDNA tipo T4 (anteriormente já fortemente correlacionado à ceratite) e um deles constitui um novo tipo de seqüência. Dois dos 4 isolados ATCC (ambientais) cujas seqüências ainda não haviam sido determinadas também apresentaram novos tipos de SSU rDNA, enquanto outros 2 apresentaram o tipo T4. / In this work we performed the molecular phylogeny and characterization of 22 Acanthamoeba isolates, 14 Brazilian keratitis isolates and 8 from ATCC, 4 keratitis and 4 environmental isolates. In spite of the extensive genetic variability disclosed by RAPD, total genomic DNA RFLP and SSU rDNA RFLP techniques, these methods enabled us to group some isolates in well defined clusters of genetically more related organisms. Sequencing of SSU rDNA allowed inference of the phylogeny of our isolates with those present in the literature, which are distributed through 12 sequence types of this gene. Among the 17 keratitis isolates of our study, 16 presented SSU rDNA of type T4 (previously found to be strongly correlated to keratitis), and one was assigned to a new sequence type. Of the 4 isolates from ATCC whose sequences were previously undetermined, the two environmental isolates also constituted new sequence types, while the two keratitis isolates were assigned to type T4.

Le nucléole est le site de biogenèse des ribosomes, qui commence par la transcription des gènes d’ARN ribosomique (ARNr). Cependant, le nucléole est également impliqué dans d'autres processus cellulaires, comme l’organisation 3D du génome. Ainsi, des régions génomiques appelées NADs pour Nucleolus-Associated chromatin Domains, ont été identifiées dans des cellules animales et végétales. Ces régions sont surtout hétérochromatiques et les gènes associés ont tendance a être peu ou pas transcrits. Un des objectifs de ma thèse a été d’étudier l’implication du nucléole dans l’organisation de la chromatine au sein du noyau et la régulation transcriptionnelle de gènes transcrits par l’ARN Polymérase II chez Arabidopsis thaliana. Par ailleurs, parmi les centaines de copies de gènes d’ARNr, uniquement une fraction participe au processus de biogenèse des ribosomes. Dans un second temps, j’ai donc étudié le rôle de ces copies inactives. On a pu démontrer que l’absence des gènes d’ARNr inactifs n’engendre pas de changements majeurs dans la fonction nucléolaire. Par contre, ces copies participent à la stabilité du génome. En effet, en leur absence, des duplications génomiques allant jusqu’à plusieurs centaines de kilobases s’accumulent, entraînant des duplications de gènes et des différences du niveau d’expression de ces derniers. Finalement, les effets de ces changements structuraux sur la biologie de la plante sont discutés. / The nucleolus is the site of ribosome biogenesis, which begins with the transcription of ribosomal RNA (rRNA) genes. However, the nucleolus is also involved in other cellular processes, such as the 3D genome organization. Thus, genomic regions called NADs for Nucleolus-Associated chromatin Domains, have been identified in animal and plant cells. These regions are mostly heterochromatic and the associated genes tend to be poorly transcribed. One of the objectives of my thesis was to study the involvement of the nucleolus in the 3D genome organization and the transcriptional regulation of genes transcribed by RNA Polymerase II in Arabidopsis thaliana. In addition, only a fraction of rRNA gene copies participates in the process of ribosome biogenesis. In a second time, I studied the role of the inactive rRNA gene copies. We show that in their absence, there is no major changes in the nucleolus function. However, these copies contribute to genome stability. Indeed, in their absence, up to several hundred of kilobases long duplication events accumulate, resulting in the duplication and the differential expression of hundreds of genes. Finally, the impact of these structural changes on the plant biology are discussed.

RNAs associate with chromatin through various ways and carry out diverse functions. One mechanism by which RNAs interact with chromatin is by the complementarity of RNA with DNA, forming a three-stranded nucleic acid structure named R-loop. R-loops have been shown to regulate transcription initiation, RNA modification, and immunoglobulin class switching. However, R-loops accumulated in the genome can be a major source of genome instability, meaning that they must be tightly regulated. This thesis aims to identify R-loop-binding proteins systemically and study their regulation of R-loops. Using immunoprecipitation of R-loops followed by mass spectrometry, with or without crosslinking, a total of 364 proteins were identified. Among them RNA-interacting proteins were prevalent, including some already known R-loop regulators. I found that a large fraction of the R-loop interactome consists of proteins localized to the nucleolus. By examining several DEAD-box helicases, I showed that they regulate rRNA processing and a shared set of mRNAs. Investigation of an R-loop-interacting protein named CEBPZ revealed its nucleolar localization, its depletion caused down-regulation of R-loops associated with rRNA and mRNA. Characterization of the genomic distribution of CEBPZ revealed its colocalization with insulator-regulator CTCF. When studying if CEBPZ recruits CTCF, I found that instead of regulating CTCF binding, CEBPZ depletion has a major effect on the performance of CUT&RUN, a technique for identifying DNA binding sites of proteins. How CEBPZ affects CUT&RUN is still under investigation, the study of which may help us understand the roles of CEBPZ in regulation of global chromatin structure and genome integrity.

Presented here is a comprehensive computational survey of evolutionarily conserved secondary structure motifs in the genomic RNAs of the family Flaviviridae. This virus family consists of the three genera Flavivirus, Pestivirus and Hepacivirus and the group of GB virus C/hepatitis G virus with a currently uncertain taxonomic classification. Based on the control of replication and translation, two subgroups were considered separately: the genus Flavivirus, with its type I cap structure at the 5′ untranslated region (UTR) and a highly structured 3′ UTR, and the remaining three groups, which exhibit translation control by means of an internal ribosomal entry site (IRES) in the 5′ UTR and a much shorter less-structured 3′ UTR. The main findings of this survey are strong hints for the possibility of genome cyclization in hepatitis C virus and GB virus C/hepatitis G virus in addition to the flaviviruses; a surprisingly large number of conserved RNA motifs in the coding regions; and a lower level of detailed structural conservation in the IRES and 3′ UTR motifs than reported in the literature. An electronic atlas organizes the information on the more than 150 conserved, and therefore putatively functional, RNA secondary structure elements.

Dbp2p, a member of the large family of DEAD-box proteins and a yeast homolog of human p68, was shown to interact with Upf1p, an essential component of the nonsense-mediated mRNA decay pathway. Dbp2p:Upf1p interaction occurs within a large conserved region in the middle of Upf1p that is largely distinct from its Nmd2p and Sup35/45p interaction domains. Deletion of DBP2, or point mutations within its highly conserved DEAD-box motifs, increased the abundance of nonsense-containing transcripts, leading us to conclude that Dbp2p also functions in the nonsense-mediated mRNA decay pathway. Dbp2p, like Upf1p, acts before or at decapping, is predominantly cytoplasmic, and associates with polyribosomes. Interestingly, Dbp2p also plays an important role in rRNA processing. In dbp2Δ cells, polyribosome profiles are deficient in free 60S subunits and the mature 25S rRNA is greatly reduced. The ribosome biogenesis phenotype, but not the mRNA decay function, of dbp2Δ cells can be complemented by the human p68 gene. We propose a unifying model in which Dbp2p affects both nonsense-mediated mRNA decay and rRNA processing by altering rRNA structure, allowing specific processing events in one instance and facilitating dissociation of the translation termination complex in the other.

No description available.

Flera olika arter av mjölksyrabakterier som ingår i släktena Lactobacillus och Bifidobacterium har hittats hos bin och i deras honung. Idag finns ingen effektiv metod för identifiering av bakterierna. Syftet med detta projekt är att utveckla en metod för snabb identifiering genom att hitta lämpliga primers till olika mjölksyrabakterier och därmed få fram en Polymeraskedjereaktion (PCR) metod. Ribosomal ribonukleinsyra (rRNA) generna eller 16S-23S rRNA intergenic spacer region (ISR) används ofta vid design av primers, som därefter används i PCR för att identifiera olika bakterier. Deoxiribonukleinsyra (DNA) visualiseras i agarosgelen med hjälp av SYBRgreen I som fluorescens på ultraviolett (UV)-ljusbord. I detta projekt har 16S rRNA och 16S-23S rRNA ISR amplifierats i enkel PCR och multiplex PCR och visualiserats i agarosgel i försök att identifiera mjölksyrabakterierna. 16S rRNA har visat sig ha mycket liten variation mellan bakterierna och ansågs därför inte lämplig att använda för identifiering av närbesläktade arter. 16S-23S rRNA ISR visade större variation, fram för allt mellan lactobacillerna och bifidobakterierna. Gruppering av bakterierna med hjälp av multiplex PCR gjordes med viss framgång, med undantag av några bakterier som inte hamnade i den förväntade gruppen. Dock behövs fler försök för att stödja dessa resultat. / Several different lactic acid bacterium (LAB) species from the genera Lactobacillus and Bifidobacterium was discovered in bees and in their honey. Today there is no rapid and reliable method to identify these LAB. Therefore a rapid polymerase chain reaction (PCR) method to identify the LAB is needed. The aim of this project is to find primers suitable for the different LAB. Ribosomal ribonucleic acid (rRNA) genes or 16S-23S rRNA intergenic spacer region (ISR) are often used to designing of primers followed by PCR assays, for identification of different bacteria. To visualize deoxyribonucleic acid (DNA) in agarose gels, SYBRgreen I was used as fluorescence and then viewed under ultraviolet (UV) light. In this project the 16S rRNA and 16S-23S rRNA ISR was used as a target in a PCR and a multiplex PCR amplification. The PCR product was analyzed in agarose gel in an attempt to identify the LAB. 16S rRNA sequence have to little variation and is not suitable to identify closely related species. 16S-23S rRNA ISR sequence exhibits greater variations, especially between Lactobacillus and Bifidobacterium. Differentiation of the bacteria into groups by multiplex PCR was done with good result, except for some of the bacteria that did not end up in the expected group. More studys is needed to support these results.

Antibiotics have been commonly used in medical practice for over 40 years. However, the misuse and overuse of current antibiotics is thought to be the primary cause for the increase in antibiotic resistance. Many current antibiotics target the bacterial ribosome. Antibiotics such as aminoglycosides and macrolides specifically target the 30S or 50S subunits to inhibit bacterial growth. During the assembly of the bacterial ribosome, ribosomal RNA of the 30S and 50S ribosomal subunits is processed by bacterial ribonucleases (RNases). RNases are also involved in the degradation and turnover of this RNA during times of stress, such as the presence of an antibiotic. This makes ribonucleases a potential target for novel antibiotics. It was shown that Escherichia coli mutants that were deficient for RNase III, RNase E, RNase R, RNase G, or RNase PH had an increase in ribosomal subunit assembly defects. These mutant bacterial cells also displayed an increased sensitivity to neomycin and paromomycin antibiotics. My research has also shown that an inhibitor of RNases, vanadyl ribonucleoside complex, potentiated the effects of an aminoglycoside and a macrolide antibiotic in wild type Escherichia coli, methicillin sensitive Staphylococcus aureus, and methicillin resistant Staphylococcus aureus. RNases are essential enzymes in both rRNA maturation and degradation. Based on this and previous work, the inhibition of specific RNases leads to an increased sensitivity to antibiotics. This work demonstrates that the inhibition of RNases might be a new target to combat antibiotic resistance.