Dans le contexte prébiotique du " monde ARN ", les ribonucléotides sont considérés comme étant les premières espèces à avoir émergé sur Terre. En milieu aqueux, leur formation est défavorable thermodynamiquement. Les voies de synthèse de nucléotides décrites en phase homogène impliquent l'utilisation des molécules activées. En 1951, Bernal a introduit une autre voie de synthèse impliquant des surfaces minérales. Cependant, dans cette voie, les effets thermodynamique et/ou catalytique des surfaces minérales restent inexplorés. Dans le cadre de l'hypothèse de Bernal, notre travail présente pour la première fois une étude in-situ de la réactivité thermique des " briques élémentaires " des nucléotides adsorbés sur des surfaces minérales avec comme objectif de réaliser la synthèse des nucléotides sans activation chimique. Ce travail a montré dans un premier temps que les surfaces minérales sont capables de déclencher la formation de polyphosphates inorganiques à partir de monophosphates à des températures modérées. D'autre part, l'adsorption du ribose sur la surface de la silice a permis d'améliorer sa stabilisation thermique : alors qu'il est instable dès 90°C en milieux aqueux, il devient stable jusqu'à 200°C après adsorption sur la silice. Dans un deuxième temps, nous avons mis en évidence la formation de PRPP, un intermédiaire réactionnel très important, par co-adsorption du ribose avec du phosphate inorganique sur la surface de la silice. Enfin, on a pu montrer la glycosylation de l'adénine et la formation après co-adsorption de ses composants sur les deux surfaces minérales utilisées. Une étude préliminaire suggère même la possibilité de dimérisation des nucléotides. / In the « RNA world» prebiotic scenario, ribonucleotide polymers are considered as the first biochemical species to have emerged. However, in aqueous solution, their formation through conventional mechanisms of condensation is thermodynamically forbidden. Several synthesis pathways of nucleotides have been described in aqueous solution; most often, they involve chemically activated molecules. Another pathway to nucleotides implies mineral surfaces, which have been considered in prebiotic processes at least since the work of Bernal in 1951. However, these studies have hardly tried to understand surface-molecule interactions and consequently, thermodynamic and/or catalytic effects of mineral surfaces are not well rationalized. In the context of Bernal's hypothesis, we present for the first time an in-situ study of the thermal reactivity of nucleotides “building blocks” adsorbed on mineral surfaces (amorphous silica, saponite) emphasizing the synthesis of nucleotides without chemical activation. In our work, we first show that mineral surfaces are able to trigger the formation of inorganic polyphosphates from monophosphates at moderate temperatures. On the other hand, adsorption of ribose on silica surface improves its thermal stabilization. While ribose decomposes at 90°C in aqueous solutions, it is stable up to 200°C on silica (in the presence of ZnCl2). Secondly, we have demonstrated the formation of PRPP, as important reaction intermediate, by co-adsorption of ribose and inorganic phosphate on the silica surface. Finally, we showed the glycosylation of adenine to adenosine and the formation of AMP (i.e. simultaneous glycosylation and phosphorylation) after co-adsorption of their components on both mineral surfaces employed. A preliminary study even suggests that nucleotide dimerisation can occur in the same conditions.

Ce projet porte sur la synthèse d'oligonucléotides modifiés décorés par des groupements chimiques rappelant les chaines latérales des acides aminés impliqués dans la catalyse enzymatique des protéases à sérine, à savoir l'acide aspartique (Asp), la sérine (Ser) et l'histidine (His) afin d'en mimer l'activité protéolytique. L'approche synthétique de type phosphoramidite a été mise en oeuvre. De ce fait nous avons synthétisé des phosphoramidites fonctionnalisés avec une fonction acide carboxylique rappelant l'Asp, une fonction hydroxyle pour la Ser ou un imidazole pour l'His, correctement protégés pour être ensuite incorporés au sein de séquences oligonucléotidiques, et des phosphoramidites convertibles, qui portent une fonction chimique réactive permettant une conjugaison post synthèse supportée et automatisée de l'oligonucléotide. Ces deux types de phosphoramidites offrent la possibilité de les combiner et donc d'obtenir des séquences hautement fonctionnalisées. Les séquences oligonucléotidiques ont été choisies afin d'apporter un contrôle topologique structurant. Les structures secondaires envisagées sont de type bulge, épingle à cheveux et jonction trois voies car stables grâce aux appariements Watson et Crick entre les bases tout en ayant une flexibilité importante due à la présence de bases non appariées. Les travaux présentés dans ce manuscrit décrivent la synthèse des phosphoramidites modifiés ainsi que l'incorporation du nucléotide convertible de type alcyne au sein de séquences qui ont été conjuguées par CuAAC, et qui ont permi d'obtenir une jonction trois voies qui a été caractérisée par dichroïsme circulaire, gel de polyacrylamide et par dénaturation thermique. / We are interested in the synthesis of modified oligonucleotides decorated by chemical fonctions mimicking the amino acids side chains involved in enzymatic catalysis of serine proteases, acid aspartic (Asp), serine (Ser) and histidine (His), in order to mimic proteolytic activity. The phosphoramidite synthetic approach was choosen. We synthesised functionnalized phosphoramidites bearing either a carboxylic acid function reminding Asp, a hydroxyle function for Ser or an imidazole for His properly protected for their incorporation in oligonucleotides sequences, and convertible phosphoramidites, bearing a reactive function that permits a conjugation post automated oligonucleotides solid phase synthesis. Oligonucleotides sequences have been choosen in order to have a perfect structural control. The envisaged secondary structures are bulge, hairpin and three way junction because of their stability, thanks to Watson et Crick base pairing, and their flexibility thanks to the unpaired bases. The work presented here regards the synthesis of modified phosphoramidites and their incorporation of the convertible alkyne in sequences then conjugated by CuAAC reaction to form a three way junction whose stability and physico-chemical behavior have been evaluated.

No description available.

The aspartate transcarbamoylase (ATCase) was purified from Burkholderia cepacia 25416. In the course of purification, three different ATCase activities appeared namely dodecameric 550 kDa holoenzyme, and two trimeric ATCases of 140 kDa (consists of 47 kDa PyrB subunits) and 120 kDa (consists of 40 kDa PyrB subunits) each. The 120 kDa PyrB polypeptide arose by specific cleavage of the PyrB polypeptide between Ser74 and Val75 creating an active polypeptide short by 74 amino acids. Both the 40 and 47 kDa polypeptides produced active trimers. To compare the enzyme activity of these trimers, an effector assay using nucleotides was performed. The 140 kDa trimer showed inhibition while the 120 kDa polypeptide showed less inhibition. To verify the composition of the pyrBC holoenzyme complex, B. cepacia dihydroorotase (DHOase, subunit size of 45 kDa) was purified by the pMAL protein fusion and purification system and holoenzyme reconstruction was performed using purified ATCase and DHOase. Both the 140 kDa and the 120 kDa trimers could produce holoenzymes of 550 kDa and 510 kDa, respectively. The reconstructed ATCase holoenzyme from cleaved ATCase showed better reconstruction compared to that from uncleaved ATCase in the conventional ATCase activity gel assay. To characterize the relationship between pyrimidine pathway and virulence factor production, motility tests and biofilm assays were conducted using pyrC- mutant. Even though no significant difference in growth rates was observed, there were significant differences between the wild type and mutant in the production of biofilm and virulence factors. This study will help us to understand the structure and regulation of ATCase holoenzyme with DHOase, and facilitate the use of B. cepacia as an applicable bio-tool. Additionally, we can potentially pursue more efficient drug targets for B. cepacia.

Neste trabalho caracterizou-se a família das nucleotídeo pirofosfatases/ fosfodiesterases do parasita S. mansoni visando avaliá-los como antígenos vacinais. O parasita possui quatro proteínas desta família (SmNPP-5a, SmNPP-5b, SmNPP-5c e SmNPP-6), sendo que duas delas possuem maior expressão gênica nos estágios que infectam o homem. Dos anticorpos produzidos, apenas o anti-SmNPP-5a apresentou especificidade para a proteína nativa e utilizando-os demonstrou-se que a SmNPP-5a é uma glicoproteína associada às membranas do tegumento dos vermes adultos. Também se verificou que esses anticorpos inibiram parcialmente a atividade da enzima em parasitas vivos. Assim avaliamos a SmNPP-5a recombinante como antígeno vacinal juntamente com uma apirase (SmATPDase) e com a fosfatase alcalina (SmAP), outras duas nucleotidases presentes no tegumento de parasitas adultos. A SmNPP-5 e a SmNTDPase apresentaram menor imunogenicidade que a SmAP. Porém só verificamos a redução da carga parasitária em animais imunizados com a SmAP e tratados com doses subcurativas de praziquantel. / In this work the family of nucleotide pyrophosphatases/phosphodiesterases (NPP) from S. mansoni was characterized in order to evaluate them as vaccine antigens. The parasite has four proteins of this family (SmNPP-5a, SmNPP-5b, SmNPP-5c and SmNPP-6), whereas two of them present higher gene expression in stages that infect humans. Only anti-SmNPP-5a anitbodies showed specificity for the native protein. We use them to characterize SmNPP-5a as a glycoprotein associated with tegument membranes from adult worms. It was also found that these antibodies were able to partially inhibit the activity of the enzyme in live parasites. Thus we evaluated SmNPP-5a as recombinant vaccine antigen together with an apyrase (SmATPDase) and alkaline phosphatase (SmAP), two other nucleotidases involved in nucleotide metabolism and present in the tegument of adult parasites. SmNTDPase and SmNPP-5 were less immunogenic than SmAP. However, we only verified the reduction of parasite burden in mice immunized with SmAP, when the animals also received a subcurative treatment with praziquantel.

Baculovírus são vírus específicos de inseto que infectam principalmente membros da ordem Lepidoptera. Diatraea saccharalis granulovírus (DsGV) foi isolado de larvas de Diatraea saccharalis (Lepidoptera: Crambidae), a broca-da-cana-de-açúcar, um dos insetos-praga de maior importância na cultura de cana-de-açúcar no Brasil. O genoma completo de DsGV foi obtido através do sequenciamento 454 (Roche). O genoma de DsGV apresentou 98.463 pb e potencialmente codifica 116 genes. Foram identificados os 37 genes conservados em todos os baculovírus, 19 genes específicos de betabaculovírus e 17 genes únicos. DsGV é o primeiro betabaculovírus que possui o gene gp64, que codifica uma proteína de fusão, originalmente encontrado apenas em alfabaculovírus do grupo I. A análise filogenética utilizando a concatenação das sequências deduzidas de aminoácidos de 30 genes conservados em 61 baculovírus totalmente sequenciados sugere que DsGV está inserido no clado b do grupo dos betabaculovírus e parece estar mais estritamente relacionado a 5 GVs (ChocGV, PiraGV, ClanGV, CpGV e CrleGV). / Baculoviruses are insect specific viruses that infect mainly members of the Order Lepidoptera. Diatraea saccharalis granulovirus (DsGV) was isolated from Diatraea saccharalis (Lepidoptera: Crambidae), one of the most important insect pest of the sugar cane culture in Brazil. The genome of DsGV was obtained by the 454 sequencing system (Roche). Our results showed that the nucleotide sequence of the DsGV genome is 98.463 bp in length and potentially encodes 116 putative genes. It contains the 37 baculovirus core genes, a set of 19 betabaculovirus-specific genes and 17 putative DsGV genes were not found in any genome of the baculoviruses sequenced up to the present. DsGV is the first betabaculovirus sequenced so far that has the gp64 envelope fusion protein gene, originally found only in alphabaculovirus group I. Phylogenetic analysis performed with concatamers of 30 conserved proteins from 61 fully sequenced baculovirus genomes suggests that DsGV is a member of clade b of the betabaculovirus and seems to be closer to 5 GVs (ChocGV, PiraGV, ClanGV, CpGV e CrleGV).

The overall objective of this study has been to address some of the longstanding questions concerning functional organization of the interphase nucleus. This was achieved by using recently developed high-resolution fluorescence in situ hybridization techniques for a precise localization of specific DNA and RNA sequences in conjunction with immunocytochemistry and biochemical fractionation. This study is based on the philosophy that new insights may be gained by an approach that attempts to interrelate genomic organization, spatial arrangement of RNA metabolism, and nuclear substructure within the mammalian cell nucleus. The nuclear distribution of an exogenous, viral RNA (Epstein-Barr Virus, EBV) within nuclear matrix preparations was studied by developing an approach which couples in situhybridization with biochemical fractionation procedures. EBV RNA molecules accumulate in highly localized foci or elongated tracks within the nucleus of lymphoma cells. These RNA tracks were retained with spatial and quantitative fidelity in nuclear matrix preparations even after biochemical fractionation which removes 95% of cellular protein, DNA, and phospholipid. This provided direct evidence that the primary transcripts are localized via their binding to, or comprising part of, a non-chromatin nuclear substructure. Then the nuclear distribution of RNA from an endogenous gene, fibronectin, was investigated using fluorescence techniques modified for more sensitive detection of endogenous RNAs within nuclear morphology. A series of in situhybridization experiments were performed using different combinations of intron, cDNA, and genomic probes for RNA/RNA or RNA/DNA analysis in intact cells. Fibronectin RNAs were highly localized in the nucleus, forming foci or tracks. Both intron and exon sequences were highly concentrated at the same site within the nucleus, indicating the presence of primary unspliced transcripts. Double-color hybridization using a nontranscribed 5' flanking sequence probe and a genomic DNA probe showed that the gene and RNA track for fibronectin were spatially overlapped, with the gene consistently towards one end of the track. These results provided evidence that the accumulation of RNA molecules occurs directly at or near the site of transcription, and further indicated a structural polarity to the RNA track formation with the gene towards one end. It was further discovered that within a single cell, cDNA probes produced longer tracks than those formed with intron probes, i.e. intron signals were generally confined to a smaller part of the track than the exon signals, indicating that splicing occurs within the RNA track. Additional experiments using poly(A) RNA hybridization or anti-SC-35 antibody staining combined with fibronectin RNA hybridization have shown that the fibronectin tracks were associated with recently discovered transcript domains enriched in poly(A) RNA and splicing factors. To further determine whether other specific genes and RNAs are functionally organized within the nucleus, the nuclear distribution of several active or inactive genes was analyzed in terms of their spatial relationship to transcript domains. The results indicated that in addition to fibronectin, the genes or their primary transcripts from two other active genes, collagen and actin, were also closely associated with the domains. For both of these, over 90% of the gene/RNA sites were either overlapping or directly contacting the domains. In contrast. for two inactive genes, cardiac myosin heavy chain and neurotensin, it was found that both genes were separated from the domains in the majority of nuclei. Histone genes, which have several unique features, showed a relatively complex result with about half of the gene signals extremely close to the domains. Therefore, three actively expressed genes were demonstrated to be tightly associated with the domains and, moreover, their RNAs showed distinct and characteristic spatial relationships with the domains. In contrast, two inactive genes were not associated with the domains. One potential implication of these finding is that active genes may be preferentially localized in and around these transcript domains. The nuclear localization of another RNA, XIST, standing for X-inactivation specific transcript, was studied because of its potentially unique biological role. XIST is the only gene which is known to be expressed from the inactive human X chromosome but not from the active X chromosome, and was believed to be important in X inactivation. Using fluorescence in situhybridization, it was found that XIST RNA was highly localized within the nucleus and always completely overlapped the Barr body which is the condensed, inactive X chromosome. The different fine distribution pattern of XIST RNA within the nucleus as compared to other protein coding RNAs suggested a unique function for this RNA, possibly involving a structural role in inactivating the X chromosome. The final area of my thesis research was to study and acquire expertise in the applications of fluorescence in situ hybridization in gene mapping and cancer genetics. A retinoblastoma (RB)-related putative tumor suppressor gene, p107, was mapped to human chromosome 20 in band q11.2. Localization of p107 to 20q11.2 was of particular interest because of the correlation of breakpoints in this area with specific myeloid disorders such as acute nonlymphocytic leukemia and myelodysplastic syndrome. Other applications of in situ hybridization including the search for unknown genes at a known chromosomal breakpoint, detection of deletions, translocations or other chromosomal rearrangements associated with specific tumors were also explored and reviewed.

In previous work I described the purification and properties of the microtubule-based mechanochemical ATPase cytoplasmic dynein. Cytoplasmic dynein was found to produce force along microtubules in the direction corresponding to retrograde axonal transport. Cytoplasmic dynein has been identified in a variety of eukaryotes including yeast and human, and there is a growing body of evidence suggesting that this "molecular motor" is responsible for the transport of membranous organelles and mitotic chromosomes. The first part of this thesis investigates the molecular basis of microtubule-activation of the cytoplasmic dynein ATPase. By analogy with other mechanoenzymes, this appears to accelerate the rate-limiting step of the cross-bridge cycle, ADP release. Using limited proteolysis, site-directed antibodies, and N-terminal microsequencing, I identified the acidic C-termini of α and β-tubulin as the domains responsible for activation of the dynein ATPase. The second part of this thesis investigates the structure of the 74 kDa subunit of cytoplasmic dynein. The amino acid sequence deduced from cDNA clones predicts a 72,753 dalton polypeptide which includes the amino acid sequences of nine peptides determined by microsequencing. Northern analysis of rat brain poly(A) revealed an abundant 2.9 kb mRNA. However, PCR performed on first strand cDNA, together with the sequence of a partially matching tryptic peptide, indicate the existence of three isoforms. The C-terminal half is 26.4% identical and 47.7% similar to the product of the Chlamydomonas ODA6 gene, a 70 kDa subunit of flagellar outer arm dynein. Based on what is known about the Chlamydomonas70 kDa subunit, I suggest that the 74 kDa subunit is responsible for targeting cytoplasmic dynein to membranous organelles and kinetochores of mitotic chromosomes. The third part of this thesis investigates a 50 kDa polypeptide which co-purifies with cytoplasmic dynein on sucrose density gradients. Monoclonal antibodies were produced against the 50 kDa subunit and used to show that it is a component of a distinct 20S complex which contains additional subunits of 45 and 150 kDa. Moreover, like cytoplasmic dynein, the 50 kDa polypeptide localizes to kinetochores of metaphase chromosomes by light and electron microscopy. The 50 kDa-associated complex is reported to stimulate cytoplasmic dynein-mediated organelle motility in vitro. The complex is, therefore, a candidate for modulating cytoplasmic dynein activity during mitosis.

Members of the Argonaute family of proteins, which interact with small RNAs, are the key players of RNAi and other related pathways. The C. elegans genome encodes 27 members of the Argonaute family. During this thesis research, we sought to understand the functions of the members of this gene family in C. elegans. Among the Argonaute family members, rde-1 and alg-1/2have previously been shown to be essential for RNAi and development, respectively. In this work, we wanted to assign functions to the remaining members of this large family of proteins. Here, we describe the phenotype of 31 deletion alleles representing all of the previously uncharacterized Argonaute members. In addition to rde-1, our analysis revealed that two other Argonaute members csr-1 and prg-1 are also essential for development. csr-1 is partially required for RNAi, and essential for proper chromosome segregation. prg-1, a member of PIWI subfamily of Argonaute genes, exhibits reduced brood size and temperature-sensitive sterile phenotype, implicating that it is required for germline maintenance. Additionally, we showed that RDE-1 interacts with trigger-derived sense and antisense siRNAs (primary siRNAs) to initiate RNAi, while several other Argonaute proteins, SAGO-1, SAGO-2, and perhaps others, functioning redundantly, interact with amplified siRNAs (secondary siRNAs) to mediate downstream silencing. Moreover, our analysis uncovered that another member of Argonaute gene family, ergo-1, is essential for the endogenous RNAi pathway. Furthermore, we built an eight-fold Argonaute mutant, MAGO8, and analyzed its developmental phenotype and sensitivity to RNAi. Our analysis revealed that the genes deleted in the MAGO8 mutant function redundantly with each other, and are required for RNAi and the maintenance of the stem cell totipotency.

The long-term goal of this project is to develop novel methods for the detection of nucleotides, oligonucleotides, and modified nucleotides such as DNA adducts by mass spectrometry. DNA adducts are important because they are formed during chemical carcinogenesis as well as during anti-cancer chemotherapy. However, DNA adducts are not routinely monitored due to difficulties associated with their detection. Mass spectrometry is a promising method for the detection of DNA adducts because it can detect almost any type of adduct, and in addition mass spectrometers can provide structural information. The work presented here shows successful detection of nucleotides and oligonucleotides of various sizes. Specific sizes detected include mononucleotides, 6-mer, 8-mer, 1 O-rner, and 16-mer oligonucleotides, and enzyme digests of genomic DNA and oligonucleotides. Through researchinvolving several separation methods (HPLC, TLC, and PAGE) and alternative detection methods (32P postlabeling and mass spectrometry), a novel method for the separation and detection of DNA adducts has been developed. The present research has shown promising results for tracking nucleotides in TLC using biomimetic dyes in order to eliminate the need for radioactive isotopes. In addition, progress has been made involving elution of nucleotides from a TLC plate and subsequent detection of these nucleotides by mass spectrometry. Together, these results will facilitate future studies that involve testing samples that contain altered DNA by different mass spectrometers, which are expected to be particularly useful for the detection and identification of mixed or novel DNA ' modifications.