The thesis introduces two developed methods to quantify extracellular vesicles (EVs) isolated from cancer cell lines and healthy human saliva by using capillary zone electrophoresis. In the first chapter, the importance of EVs, as well as the existing EV isolation, characterization, and quantification methods, are described. The general principle of capillary electrophoresis (CE) is explained for a better understanding of these two methods. Chapter II describes the idea and concepts of Extracellular Vesicles quantitative Capillary Electrophoresis (EVqCE). The method evolved from the previous study carried out in our research group for the quantification of viruses. After the isolation of EVs from different cell lines, the characterization and quantification of EVs were performed using nanoparticle tracking analysis (NTA) and flow cytometry. EVqCE consists of four steps for EV quantification. In this study, EVqCE was employed to know the concentrations of EVs in unknown samples, followed by calculation of the average mass of the RNA present in EVs. In the next chapter, one of the human body fluids, i.e., saliva, was chosen for the quantification of EVs. Salivary EVqCE was developed in a similar way as EVqCE for cell lines since the general theories and procedures are practically the same. However, human saliva contains an abundant amount of viscous proteins and ribonuclease (RNase), that were the major obstacles for salivary EVs detection and quantification. The method for the isolation of EVs from the saliva was optimized, and the quantification was performed successfully. The average mass of RNA in saliva EVs was also calculated and analyzed. The concentration of saliva EVs in unknown samples were compared with the results from NTA and flow cytometry to validate the salivary EVqCE. In the last chapter, I described the application of EVqCE to study the quality control of EVs. The method calculates the degradation level of EVs samples under different conditions, providing a potential way for real-time monitoring of the EVs status in the body fluid sample.

Les origines de réplication sont les sites à travers le génome où est initiée la synthèse de l’ADN. Les multiples cartographies des origines de réplication dans des cellules de vertébrés ont identifié une association entre origines de réplication et motifs G4. Les motifs G4 sont des séquences ayant le potentiel de se replier en G-quadruplexe. Des travaux menés précédemment au laboratoire ont montré que la capacité d’un motif G4 à se replier en G-quadruplexe est essentielle pour l’activité de deux origines de réplication modèles dans la lignée cellulaire de poulet DT40. Cependant, le motif G4 n’est pas suffisant pour spécifier une origine de réplication. Dans l’origine modèle βA, un élément cis de 227 pben 3’ du motif G4 est également nécessaire pour l’initiation de la réplication. L’analyse de la séquence de cet élément indique qu’il comporte plusieurs motifs connus pour être des sites de fixation de facteurs de transcription. Nous avons testé le rôle potentiel de ces motifs en évaluant l’effet de leurs délétions individuelles sur l’activité de l’origine βA. Ces travaux ont identifié les boites TATA et CCAAT, pouvant être liées par le facteur TBP (TATA Binding Protein) et NFY (Nuclear Factor Y) respectivement, comme étant les éléments cruciaux avec le motif G4 pour l’initiation de la réplication. Nous avons cherché à éclaircir de quelle manière ces éléments permettent la spécification d’une origine de réplication. A cet effet, nous avons émis l’hypothèse selon laquelle les motifs G4 qui se trouvent au niveau des origines de réplication sont ceux qui in vivo sont capables de former un G-quadruplexe. La formation du G-quadruplex dans l’origine βA dépendrait alors de la présence des boites TATA et CCAAT qui peuvent recruter des facteurs de transcription favorisant l’ouverture de la double hélice de l’ADN et le repliement du G-quadruplex. Cette hypothèse prévoit que la stabilisation d’un G-quadruplexe in vivo est nécessaire et suffisante pour former une nouvelle origine de réplication. Nous avons donc testé cette hypothèse de deux manières. D’abord, nous avons entrepris de stabiliser un G-quadruplex à une position donnée du génome en induisant la transcription d’un motif G4. Ensuite, nous avons déterminé les effets d’une stabilisation globale des G-quadruplexes à travers le génome sur la position des origines de réplication. Pour cela, nous avons cartographié les origines de réplication dans des lignées de cellules DT40 dans lesquelles des facteurs impliqués dans la linéarisation des G-quadruplexes ont été inactivés. Selon notre hypothèse, en l’absence de tels facteurs, comme l’hélicase FancJ ou l’ADN polymérase translésionnelle Rev1, davantage de motifs G4 pourraient se replier et former une origine de réplication. Les résultats obtenus avec chacune des deux approches indiquent que la stabilisation de G-quadruplexes ne permet pas de produire de nouvelles origines de réplication. L’ensemble de nos données indique que l’activité de l’origine βA dépend d’un motif G4 et des boites TATA et CCAAT. La manière par laquelle l’ensemble de ces éléments permettent l’initiation de la réplication reste à éclaircir / Replication origins are the position where DNA synthesis is initiated. Mapping of replication origins across the genome showed a link between origins and G4 motifs. G4 motifs are sequences the potential for forming G-quadruplexes. Works carried out previously in the laboratory showed that the ability to fold into G-quadruplex is critical for the activity of two model origin in the DT40 cell line. However, the G4 motif is not enough to specify a replication origin. In the βA model origin, a 227 bp cis element is required for the initiation of replication. The analysis of this sequence indicates the presence of several motifs known to be binding sites for transcription factors. We tested the potential roles of these motifs by evaluating the effect of their individual deletion on the activity of the βA origin. This work identified the TATA and CCAAT boxes who bind TBP (TATA Binding Protein) and NFY (Nuclear Factor Y) respectively as the crucial elements, with the G4 motifs, pour the initiation of replication.We endeavored to shed light on the manner by which these elements enable the specification of a replication origin. We hypothesized that the G4 motifs associated with replication origins are those able to form a G-quadruplex in vivo. The formation of the G-quadruplex of the βA origin would require the presence of the TATA and CCAAT boxes who could recruit transcription factors facilitating the opening of the double helix and G-quadruplex folding. We tested this hypothesis by two different manners. First, we undertook to stabilize a G-quadruplex at a given position in the genome by inducing the transcription of a G4 motif. Then, we observed the effects of a genome wide stabilization of G-quadruplexes on the position of replication origins. For that, we mapped replication origins in DT40 cell lines in which factors implicated in G-quadruplex linearization are inactivated. According to our hypothesis, without such factors, like the FancJ helicase of the translesional DNA polymerase Rev1, more G4 motifs could fold into G-quaduplexes and specify replication origins

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BRCA2 is a highly penetrant breast cancer predisposing gene. The protein product of the BRCA2 gene mediates repair of breaks in DNA, through Homologous Recombination (HR). Understanding the mechanism(s) behind BRCA2 involvement in HR will help clarify its clinical importance and may pave the way for possible therapy. In this work we show that BRCA2 affects telomere maintenance in mammalian cells. Telomeres are physical ends of chromosomes implicated in cell senescence and carcinogenesis. In particular, the enzyme telomerase that synthesizes telomeric DNA is highly active in ~90% cancers and it is considered one of the cancer markers. The remaining 10% of cancers do not show telomerase activity and they maintain their telomeres by an alternative pathway known as Alternative Lengthening of Telomeres (ALT). We observed telomere shortening, loss of telomere function in the form of end chromosome fusions and increased incidence of Telomere Sister Chromatid Exchanges (T-SCE), one of the recognized markers of ALT, in 3 sets of Chinese hamster and human BRCA2 defective cell lines, all of which maintained telomeres by conventional mechanisms. We have also inhibited BRCA2 expression in ALT positive cells by transfecting them with si (short interfering) RNA oligonucleotides specific for BRCA2 and monitored its expression by Real Time-PCR and Western blot. Results indicate that BRCA2 knock-down in ALT positive human cells that causes reduction in T-SCE frequencies, thus suggesting that ALT cells and those that maintain telomeres by conventional mechanisms differ in this respect. One interesting scenario that emerges from these results is that BRCA2 deficiency could potentially suppress the ALT pathway. We wanted to explore this possibility further by creating a permanent BRCA2 knock-down. Our preliminary results suggest that our method for the permanent BRCA2 knock-down based on the SMARTvector 2.0 system and sh (short hairpin) iv RNA approach is still not working effectively. We identified hyper-methylation of the promoter within the vector as a possible cause. Finally, we examined repair kinetics of interstitial telomeric sites (ITSs) in BRCA2 deficient Chinese hamster cells in order to test the hypothesis that defective DNA double strand break repair may be responsible for their increased sensitivity to DNA damaging agents. Our results indicate that DNA damage within ITSs is repaired effectively thus disproving the above hypothesis. In conclusion, this work demonstrates the involvement of BRCA2 in telomere maintenance.

O estudo de prevalência do vírus rábico foi realizado em uma amostra de morcegos capturados na Mata Atlântica da região sudeste do Brasil. Os morcegos são um dos principais reservatórios silvestres do vírus rábico. No Brasil existem aproximadamente 144 espécies de morcegos, e pouco se sabe sobre a circulação do vírus rábicos nessas espécies. Foram realizadas estimativas – com duas metodologias de isolamento - para detectar a presença do vírus rábico na população estudada. Os resultados foram obtidos pelo cruzamento entre a variável infectividade (presença de vírus rábico) e as variáveis epidemiológicas (espécies de morcegos, sexo, idade, local de captura ). Observou-se que o método de isolamento que utiliza as células McCoy isolou com maior facilidade vírus de morcegos insetívoros, além de apresentar maior capacidade de detectar a infecção na fase latente (subclínica). Já as células N2A foram mais eficientes na detecção do vírus rábico em morcegos hematófagos D. rotundus. As duas metodologias utilizadas apresentaram maior proporção de isolamento do vírus rábico em morcegos insetívoros, nectarívoros e fitófagos. Tais resultados sugerem que os morcegos insetívoros desempenham importante papel na manutenção do vírus no reservatório cuja população foi estudada. Também foi possível constatar que a circulação do vírus ocorre inter e intra-espécies, mas estudos especificamente desenhados para avaliar esse aspecto devem ser implementados. / The prevalence study of the rabies virus was carried out in a sample of bats captured in the Brazilian southeastern São Paulo. Bats are one of the main wild reservoirs of the rabies virus. Brazil holds 144 species of bats and little is know about the circulation of such virus in these species. Two metodologies were used for the estimates of the presence of the rabies virus in the captured in the Parque Estadual Intervales. The results were obtained crossing the variable (presence of rabies virus) with epidemiological variables (bat species, sex, age, site of capture). The McCoy cell line method proved isolating more easily the virus of insectivorous bats besides presenting more capability of detection of infection in the latent phase (sub-clinic phase). On the other hand the N2A cell line were more efficient in detecting the rabies virus in D. rotundus hematophagous bats. It was also observed that for both cells the insectivorous, nectarivorous and phytophagous bats presented higher rabies virus isolation proportion. These results suggest that insectivorous bats play in important role in the maintence of the virus in this reservoir. Although could also be observed that the circulation of the virus occurs intra and inter species, but studies specially designed to asses this issue must be re-evaluated.

A presença de Ad com competência para replicar (RCA, replication-competent adenovirus) nas preparações é um dos maiores problemas para a produção de Ad em larga escala. RCAs são gerados pela recombinação entre seqüência do vetor e seqüência homóloga do gene E1 presente nas células helper. Objetivo: desenvolver uma nova linhagem auxiliar para produção de Ad livre de RCA - LEY79 - derivada da linhagem de retinoblastoma humano Y79, tratando-se da primeira linhagem empacotadora de adenovírus com inativação mutacional da proteína supressora de tumor pRb, que crescem em suspensão. Células Y79 foram infectadas com o retrovírus pCLDE1A/E1BSN, selecionadas com G418. A eficiência de produção de AdeGFP na linhagem LEY79 foi testada e comparada com a HEK293A. Células Y79 foram adaptadas em meio livre de soro. Esperamos com a linhagem LEY79SF inovar no campo de processos para a produção de Ad recombinante. / The presence of Ad with the ability to replicate (RCA, replication-competent adenovirus) in preparations is a major problem in the large-scale production of Ad. RCAs are generated by recombination between the vector sequence and sequence of the homologous gene in E1 helper cells. Objective: To develop a new helper cell line for the production of RCA-free Ad., called LEY79, derived from the Y79 of human retinoblastoma line, the first line Packer adenovirus with mutational inactivation of the tumor suppressor protein pRb, which are adapted to grow in suspension. Y79 cells were infected with the retrovirus pCLDE1A/E1BSN, selected with G418. The efficiency of production of AdeGFP in the LEY79 was tested and compared with the HEK293A. Y79 cells were adapted to grow in serum-free medium. We hope that use of the the LEY79SF cell line will promote innovation in the processing and production of recombinant Ad.