Magister Scientiae - MSc / Nevirapine (NVP) is a cheap anti-retroviral drug used in poor countries worldwide, administered to pregnant women at the onset of labour to inhibit HIV enzyme reverse transcriptase. Viruses which may get transmitted to newborns are deficient in this enzyme, and HIV-1 infection cannot be established, thereby preventing mother to child transmission (MTCT). In some cases, babies get infected and positive selection for viruses resistant to nevirapine may be inferred. Positive selection can be inferred from sequence data, when the rate of nonsynonymous substitutions is significantly greater than the rate of synonymous substitutions. Unfortunately, it is found that available positive selection methods should not be used to analyse before- and after- NVP treatment sequence pairs associated with MTCT. Methods which use phylogenetic trees to infer positive selection trace synonymous and nonsynonymous substitutions further back in time than the short time duration during which selection for NVP occurred. The other group of methods for inferring positive selection, the pairwise methods, do not have appreciable power, because they average susbtituion rates over all codons in a sequence pair and not just at single codons. We introduce a simple counting method which we call the Pairwise Homologous Codons (PHoCs) method with which we have inferred positive selection resulting from NVP treatment in longitudinal HIV-1 reverse transcriptase sequences. The PHoCs method estimates rates of substitutions between before- and after- NVP treatment codons, using a simple pairwise method. / South Africa

Retrons are genetic elements encoding reverse transcriptase (RT) usually located on the chromosome of a wide variety of mostly Gram-negative bacteria. Here we describe a new retron, designated Ne144, found in the chromosome of the myxobacterium Nannocystis exedens. This element codes for a 515-amino-acid RT that is most closely related to those found in other myxobacterial retrons. The RT is responsible for the production of a small satellite DNA called msDNA. This msDNA is composed of a 144 base, single-stranded DNA that is linked to a 72 base single-stranded RNA. The RNA strand is joined to the 5′ end of the DNA chain via a 2′-5′ linkage that occurs from the 2′ position of an internal guanosine residue in the RNA. In addition to the retron element, the chromosome of N. exedens also contains several partial copies of the msDNA sequence as revealed by DNA hybridization experiments using msDNA as a probe. One of these partial copies was characterized from a chromosome restriction fragment and found to contain a sequence that matches the last 82 bases of the DNA strand and five bases of the RNA strand in msDNA-Ne144. This partial copy of msDNA is very likely a retrotransposed sequence that was generated by reverse transcription using an RNA (the primer-template RNA for msDNA) as a template and the 3′ end of a nick in the chromosome as a primer, followed by incorporation into an open reading frame. The presence of this truncated copy of msDNA is strong evidence of retrotransposition in N. exedens causing an alteration in the bacterial genome.

Human Immunodeficiency Virus 1 (HIV-1) is a lentivirus that forms persistent latently infected reservoirs that are the remaining major hurdle for current HIV-1 treatments. APOBEC3 (A3) proteins are intrinsic retroviral restriction factors that introduce GA mutations during reverse transcription, while Reverse Transcriptase (RT) introduces on average 2-3 mutations every reverse transcription cycle due to a lack of proofreading ability. The goal of this research is to characterize the infectivity and activation of mutated HIV-1 viruses that display reduced transcription upon infection, viruses that we term latency prone viruses (LPVs). We hypothesize that GA transition mutations in the HIV-1 Long Terminal Repeat (LTR) region of the LPVs introduced through Reverse Transcriptase and low levels of A3 protein activity can create HIV-1 sequences that display a reversible, latency-like phenotype. Variable levels of transcription and promoter activation were seen among the LPVs when tested against four classes of Latency Reversing Agents (LRAs). Subsequently, three tested LPVs demonstrated an initial latency-like phenotype before rebounding in infectivity. This project demonstrates for the first time that HIV-1 latency is not simply a byproduct of the infection timing and cellular conditions, but that replication-competent HIV-1 latent viruses can also be created through sublethal mutagenesis of their viral promoter sequence introduced through A3 and RT exposure. The characterization of the complete mechanism of HIV-1 latency induction, maintenance, and reversal is critical in the development of sterilizing and functional cures for HIV-1 infection.

No description available.

This research focuses on non-Long Terminal Repeat (non-LTR) retrotransposons in the African malaria mosquito, Anopheles gambiae and other mosquito species. An unprecedented diversity of non-LTRs was discovered by genome analysis of the An. gambiae genome assembly. One hundred and four families were found by a reiterative and comprehensive search using the conserved reverse transcriptase domains of known non-LTRs from a number of organisms as the starting queries. These families range in copy number from a few to approximately 2000 and occupy at least 3% of the genome. An. gambiae non-LTRs represent 8 of the 15 previously defined clades, plus two novel clades, Loner and Outcast, raising the total number of known clades to 17. The first invertebrate L1 clade representatives were also found. All clades except one have families with sequence characteristics suggesting recent activity. Juan, a non-LTR of the Jockey clade originally discovered in the mosquito Culex pipiens quinquefasciatus (Mouches et al. 1991), has been implicated in horizontal transfer in three non-sibling species of the Aedes genus (Mouches, Bensaadi, and Salvado 1992). PCR was used to obtain sequences from 18 mosquito species of six genera. Phylogenetic analysis demonstrates predominant vertical inheritance of Juan elements among these species. There is strong evidence from sequence analysis supporting the recent activity of Juan in several divergent species. We hypothesize that the sustained activity (versus quick inactivation) of non-LTRs in mosquitoes may contribute to the diversity we observe in the An. gambiae genome today. Promoter and transcriptional analyses were performed for several families previously identified as potentially active elements based on sequence analysis. RT-PCR results indicate that transcripts are present in An. gambiae cell lines that contain sequences corresponding to 13 of 15 tested non-LTR families. The 5' UTRs of An. gambiae non-LTRs from the I, Jockey, and L1 clades support basal transcription in divergent mosquito cell lines from 3 species. The Jen-1 5'UTR did not support transcription in Ae. aegypti and had low activity in Ae. albopictus. In summary, this research shows that Non-LTRs have been highly successful genomic elements that have flourished in many divergent mosquito species. / Ph. D.

No description available.

No description available.

Moquiniastrum e Richterago possuem 21 e 16 espécies, respectivamente. Nesse trabalho foi descrita a composição química de três espécies de Moquiniastrum (M. floribundum, M. blanchetianum e M. oligocephalum) e duas de Richterago (R. discoidea e R. campestres), avaliado as atividades citotóxica, antirradicalar, antileishmania, antitripanossoma e a inibição enzimática da transcriptase reversa (HIV-1) pelos metabólitos isolados, e as informações químicas dos metabólitos isolados e de literatura foram analisados como caracteres quimiotaxonômicos na segregação dos gêneros estudados. Dessas espécies foram identificadas 109 substâncias, sendo vinte e dois componentes graxos, um derivado de tocoferol, dezessete triterpenos, uma flavanona, quatro flavonas derivadas de apigenina, seis flavonas derivadas de luteolina, oito flavonóis derivados de caempferol, três flavonóis derivados de quercetina, três flavonóis acilados, quatro flavonóis glicosilados, dois ácidos fenólicos, quize derivados de ácido cinâmico, cinco lactonas sesquiterpênicas, seis diterpenos e doze sesquiterpenos de esqueleto bisabolano, totalizando 19 componentes inéditos em literatura. Moquiniastrum e Richterago apresentam como caracteres compartilhados a presença de triterpenos, flavonas derivadas de apigenina e luteolina, flavonóis acilados, ácidos cafeoil-quínicos e ácidos C6-C3. Adicionalmente, as espécies de Moquiniastrum caracterizam-se pela produção de flavonóis 3-O-metoxilados derivados de caempferol além de germacranolídeos, eudesmanolídeos e guaianolídeos lactonizados na posição 6,12. Por outro lado, as espécies de Richterago acumulam flavonóis 3-O-glicosilados derivados de quercetina, além de germacranolídeos lactonizados na posição 8,12. Dessa forma, os dados permitem a distinção química entre os gêneros e corroboram a segregação proposta para os mesmos. Na atividade antirradicalar os ácidos monocafeoilquinicos apresentaram mais de 100% Tx (comparativamente ao Trolox) e tanto os ácidos como os ésteres di- e tricafeoilquinicos mostraram mais de 213%Tx, evidenciando um grande potencial antirradicalar. No ensaio antileishmania nenhuma das substâncias isoladas apresentou atividade considerável. No ensaio antitripanossoma a genkwanina e o éster metílico do ácido 3,4,5-tricafeoilquínico apresentaram atividade frente a forma tripromastigota de Trypanossoma cruzi. No ensaio citotóxico a fase DCM de M. floribundum apresentou um grande potencial bioativo (> 90% na concentração de 50,0 µg.mL-1) porém as flavonas isoladas dessa fase foram testadas não apresentando atividade e as substâncias inéditas estão em avaliação. No ensaio anti HIV-1 os ácidos clorogenicos e as flavonas mostraram potencial como inibidores da transcriptase reversa do HIV-1. / Moquiniastrum and Richterago have 21 and 16 species, respectively. This work describes the chemical composition of three species of Moquiniastrum (M. floribundum, M. blanchetianum and M. oligocephalum) and two Richterago (R. discoidea and R. campestris). The isolated metabolites were evaluated as cytotoxic, antiradicalar, antileishmania, antitrypanosome and enzymatic reverse transcriptase inhibition (HIV-1) activities and the chemical data from the isolated compounds and literature data were analyzed as chemotaxonomic characters in the segregation of both genera. From these species 109 compounds were identified, including twenty-two fatty components, a tocopherol derivative, seventeen triterpenes, a flavanone, four flavones derived from apigenin, six flavones derived from luteolin, eight flavonols derived from caempferol, three flavonols derived from quercetin, three acylated flavonols, four glycosylated flavonols, two phenolic acids, fifteen cinnamic acid derivatives, five sesquiterpene lactones, six diterpenes and twelve sesquiterpenes pertaining to the bisabolane skeleton. Among these compounds 19 metabolites were unpublished in literature. Moquiniastrum and Richterago show as shared characters the presence of triterpenes, flavones derived from apigenin and luteolin, acylated flavonols, caffeoylquinic acids and C6-C3 acids. In addition, Moquiniastrum species are characterized by the production of 3-O-methoxylated flavonols derived from kaempferol, and germacranolides, eudesmanolides and guaianolides lactonized at 6,12 position. On the other hand, Richterago species accumulate 3-O-glycosylated flavonols derived from quercetin and germacranolides lactonized at 8,12 position. Therefore, the data allow the chemical distinction and corroborate the proposed segregation between both genera. For antiradical activity, the monocaffeoylquinic acids showed more than 100% Tx (compared to Trolox) and both di- and tricaffeoylquinic acids and esters exhibited more than 213% Tx, showing a great antiradical potential. None of isolated compounds showed considerable activity in the antileishmania assay, however for antitrypanosome assay, genkwanin and 3,4,5-tricaffeoylquinic acid methyl ester showed activity against the promastigote form of Trypanosoma cruzi. For cytotoxic assay the DCM phase from M. floribundum showed a high bioactive potential (> 90% at concentration of 50.0 µg.mL-1), however the isolated flavones were tested and showed no activity. The new compounds are under evaluation. Finally, for anti-HIV-1 assay the chlorogenic acids and flavones showed potential as inhibitors of HIV-1 reverse transcriptase.

A genotipagem do vírus da hepatite C (VHC) é a principal ferramenta para prognóstico e tempo de tratamento. Dependendo do genótipo infectado existem diferentes esquemas e tempo de tratamento. O objetivo deste trabalho foi desenvolver padronizar e validar um método de genotipagem por PCR em tempo real com base na análise da região NS5B. Esta região apresenta um grau de polimorfismo que permite identificar de modo mais acurado tanto os tipos como os subtipos do VHC. Para isto foram desenhados dois conjuntos de primers e sondas. Neste trabalho desenvolvemos um método one-step modificado em uma reação triplex em que ocorre a identificação dos genótipos (1a, 1b, 3a) e em outro set a identificação dos genótipos (2a, 2b, 2c). Os resultados obtidos pelo método de genotipagem em tempo real concordaram em 100% com os resultados de seqüênciamento da região NS5B quando excluímos amostras que foram identificados como mistura de genótipos no método desenvolvido e classificados somente como um único genótipo no seqüênciamento. Houve uma boa concordância entre o método desenvolvido e o seqüênciamento da região NS5B pelo coeficiente de Kappa (k= 0,6222; p=0,0020). O método de desenvolvido conseguiu detectar 97,93% (190/194) do genótipo 1, 86,11% (31/36) do genótipo 2 e 100% (80/80) do genótipo 3. A média da sensibilidade foi de 97%. Quando comparamos a genotipagem por PCR em tempo real e LiPA nas 310 amostras analisadas não houve resultados discordantes em relação ao genótipo. Entretanto, 26,24% (79/301) das amostras analisadas apresentaram resultados discordantes em relação ao subtipo quando comparados os dois métodos. Foi determinada a sensibilidade analítica do método através de um ponto do painel da OptiQuant que foi diluído de modo seriado e a sensibilidade relativa foi realizada através de amostras de plasma de pacientes com carga viral determinada pelo Cobas Amplicor. O limite mínimo de detecção determinado foi de 125UI/ml para o genótipo 3a, 250 UI/ml para o genótipo 1b e 2b e 500 UI/ml para o genótipo 1a. Somados a isso, o método desenvolvido tem um custo de R$ 58,00, um valor nove vezes menor que o método comercial utilizado em nosso laboratório. Além disso, no método desenvolvido, o tempo trabalhado cai para menos de 2 horas sem a necessidade de manipulação constante em comparação com LiPA que necessita em torno de 16 horas, devido ao vários passos de hibridizações e lavagens. No presente trabalho o método de genotipagem por PCR em tempo real para o VHC mostrou-se eficiente e capaz de identificar de um modo acurado os diferentes genótipos e seus subtipos e contribuir para um entendimento melhor do verdadeiro papel dos genótipos e seus subtipos e da variabilidade genética na história natural da infecção do VHC. / Hepatitis C virus (HCV) genotyping is the most significant predictor of response to antiviral therapy. Depending on the infecting HCV genotyping different antiviral regimens have been proposed as well as the length of different treatment. The aim of this study was to develop and evaluate a new real time PCR of HCV genotyping based in NS5B region. This region has sequencing heterogeneity and can accurately identify both type and subtype of HCV. Furthermore, we compared the real time PCR with LiPA and sequencing of NS5B region. We developed a new one-step modified method in triplex reaction where we identified in two sets genotypes (1a, 1b, 3a) and (2a, 2b, 2c). Results obtained by real time PCR agreed 100% with those obtained by NS5B sequencing when excluded samples with mixed of HCV genotypes identified by real time PCR genotyping and in NS5B sequencing all samples were classified only as only one genotype. We found a good concordance for the analysis of genotype concordance between genotyping by real time and sequencing of NS5B region through the coefficient kappa (k= 0,6222; p=0,0020). The method developed detected 97,93% (190/194) of genotype 1, 86,11% (31/36) of genotype 2 and 100% (80/80) of genotype 3, with the overall sensitivity of this new method being 97%. Among 310 samples only two samples had discordant results at type level when comparing real time PCR and LiPA. However, 26,24% (79/301) had discordant results at subtype level when comparing LiPA and real time PCR genotyping of HCV. In order to measure the analytical sensitivity of the real time assay, one member of the panel OptiQuant HCV RNA was diluted. The relative sensitivity was determined by analysis the clinical specimens based upon the initial HCV RNA concentration determined by Cobas Amplicor. The lower limit of detection was estimated to be 125 IU/ml for genotype 3a, 250 IU/ml for genotype 1b and 2b, and 500 IU/ml for genotype 1a. Finally, the cost of each reaction are about R$ 58,00 nine fold lower than the commercial method available in Brazil. Manipulation time of real time PCR genotyping is about 2 hours, in comparison to LiPA that requires about 16 hours due to various hybridization steps and washing. This study was demonstrated an efficient method of identification in a accurate way. HCV genotyping which is important to understand the role of genotypes and subtypes, as well as of genomic variability in the natural history of HCV infection.

Les éléments LINE-1 (L1) sont les seuls éléments transposables autonomes et actifs, constituant 20% de notre ADN. Ils prolifèrent via un intermédiaire ARN dans un processus appelé rétrotransposition. Les L1s encodent deux protéines, ORF1p et ORF2p, qui s’associent avec l’ARN du L1 pour former une particule ribonucléoprotéique (RNP), constituant l’intermédiaire fonctionnel de la rétrotransposition. Les L1s « sautent » activement dans les cellules germinales, les cellules souches embryonnaires et dans l’embryon précoce, conduisant occasionnellement à des maladies génétiques. Ils sont également exprimés et mobiles dans un certain nombre de cancers. Les sites d’intégration des L1s sont généralement considérés comme aléatoires, les déterminants moléculaires de leur insertion demeurant mal connus. Afin d’éclaircir ce processus, nous avons d’abord exploré les propriétés biochimiques des RNPs du L1, en mesurant leur activité transcriptase inverse in vitro sur une collection variée de substrats d’ADN. Nous avons observé que des substrats, qui diffèrent par leur séquence ou leur structure, ne sont pas tous utilisés efficacement par les RNPs du L1 pour amorcer la transcription inverse. Notre travail suggère que la spécificité et la flexibilité de l’initiation de la transcription inverse du L1 participe aux choix du site d’insertion. Dans un second temps, nous avons recherché des partenaires cellulaires de la RNP du L1 qui pourraient contribuer à la rétrotransposition et/ou la réguler, par des cribles double-hybride chez la levure. Nous avons découvert que la protéine ORF2p interagit avec un groupe de récepteurs nucléaires. Ces derniers possèdent un domaine de liaison à l’ADN qui reconnaît des séquences d’ADN spécifiques réparties dans le génome, et un domaine de liaison du ligand, qui permet d’activer la transcription des gènes cibles. Nos données suggèrent que ces facteurs participent à la rétrotransposition des L1s, possiblement en ciblant leurs RNPs dans certaines régions du génome. Dans leur ensemble, nos travaux ont contribué à améliorer notre compréhension de la relation entre éléments transposables et génome hôte, et de leur impact sur la plasticité du génome humain. / LINE-1 (L1) elements are mobile genetic elements, comprising up to 20% of the contemporary human genome, in which they are the only autonomously active element. They replicate through an RNA intermediate in a process named retrotransposition. Replication-competent L1 copies code for two proteins, ORF1p and ORF2p, that associate in cis with their own RNA to form a ribonucleoprotein complex (RNP), the functional intermediate of retrotransposition. L1s « jump » actively in germ cells, embryonic stem cells and in the early embryo, leading occasionally to genetic diseases. These elements are also expressed and mobile in a number of cancers. L1 insertion sites are generally considered as random. The molecular determinants of L1 insertion, as well as many steps of the retrotransposition cycle, remain uncertain. To get further insight in the molecular mechanisms of L1 retrotransposition, we first explored the biochemical properties of the L1 RNP, by measuring their reverse transcriptase activity in vitro on various DNA substrates. Using this approach, we observed that L1 RNPs do not equally extend DNA substrates, which differ in sequence or structure, to initiate cDNA synthesis. Our work suggests that the specificity and flexibility of L1 reverse transcription priming contribute to the choice of target sites. In a second approach, we performed yeast two-hybrid screens in order to discover cellular partners of the L1 RNP, which could contribute and/or regulate retrotransposition, We found that ORF2p interacts with a group of nuclear receptors. These proteins contain a DNA binding domain, which recognizes specific DNA sequences spread in the genome, and a ligand binding domain, driving transcriptional regulation of target genes. Our data suggest that these factors participate to L1 retrotransposition, potentially by tethering L1 RNPs to specific genomic regions. Altogether, this work has contributed to a better understanding of the relationship between mobile genetic elements and their host genome, and their impact on human genome plasticity.