A leptospirose é uma doença endêmica causada por Leptospiras. O genoma da Leptospira interrogans sorovar Copenhageni foi analisado para seleção de potenciais antígenos vacinais. Oito genes foram selecionados e clonados para expressão e purificação dos antígenos. A salmonela SL3261 foi usada como carregadora dos genes de leptospira em vetor pAEsox para expressão das proteínas in vivo. As salmonelas recombinantes induziram resposta imune quando administradas em camundongos por via intraperitoneal. Hamsters foram imunizados com as salmonelas, observando-se que a SLLIC10191 induziu proteção parcial no desafio com L. interrogans sorovar Pomona. Vetores híbridos foram construídos para expressão simultânea de dois antígenos em salmonelas in vivo. Observamos indução de anticorpos específicos, porém, os ensaios de desafio não foram conclusivos. Vários parâmetros do desafio com sorovar Copenhageni foram estudados, como contagem das bactérias e ajuste de dose, variação de virulência por passagens em cultivo e interferência da idade dos animais. / Leptospirosis is an endemic disease caused by Leptospira. The genome of Leptospira interrogans serovar Copenhageni was analyzed for screening potential vaccine antigens. Eight genes were selected and cloned for expression and purification. Salmonella SL3261 was used as carrier of the genes of leptospira in pAEsox vector for in vivo proteins expression of proteins in vivo. Recombinant Salmonella induced immune response when administered intraperitoneally in mice intraperitoneally. Hamsters were immunized with salmonella, resulting we observed that the SLLIC10191 induced partial protection against on challenge with L. interrogans serovar Pomona. Hybrid vectors were constructed for expression of two antigens simultaneously by salmonella in vivo. We observed induction of specific antibodies, however, the challenge tests were not conclusive. Several parameters of the challenge assay with serovar Copenhageni were studied, such as the counting of bacteria count and dose adjustment, changes in virulence by passages in culture and interference backgroundof from the age of animals.

O P3HB é um tipo de poliéster sintetizado por bactérias como reserva de carbono e energia, e mobilizado na escassez destes. Um estudo com mutantes de Burkholderia sacchari, indicou o envolvimento de PhaZa1 e LonA na mobilização de P3HB. Este estudo avaliou o papel de PhaZa1 e LonA neste processo. A complementação heteróloga dos mutantes de B. sacchari, a partir dos genes phaZa1 e lonA de Ralstonia eutropha, restabeleceu a capacidade de mobilização dessas cepas, confirmando o envolvimento de seus produtos no processo. A partir de cepas recombinantes de Escherichia coli, abrigando tanto os genes de acúmulo de P3HB como de mobilização de R. eutropha, obteve-se cepas abrigando os genes phaZa1 e lonA isoladamente e outra abrigando os dois genes simultaneamente. Quando separados, tanto phaZa1 quanto lonA, não apresentaram papel significante na mobilização porém, quando os dois genes são expressos simultaneamente, as taxas de mobilização atingem mais de 50%, indicando que deva ter uma interação entre PhaZa1 e LonA para que o processo de mobilização seja efetivo. / The P3HB is a type of polyester synthesized by bacteria like carbon and energy source, and is mobilizated when there is a shortage of these. A study with mutants of Burkholderia sacchari, showed the involvement of PhaZa1 and LonA in mobilization of P3HB. The present study evaluated the role of PhaZa1 and LonA in this process. The heterologous complementation of mutants of B. sacchari, with phaZa1 and lonA genes from Ralstonia eutropha, reestablished the mobilization capacity in these strains, confirming the involvement of their products in this process. From the recombinant strain of Escherichia coli, harboring accumulation and mobilization genes from R. eutropha, we obtained strains harboring phaZa1 and lonA genes singly and another harboring both genes simultaneously. When expressed singly, both phaZa1 as lonA, had no significant role in mobilization but, when both genes were expressed simultaneously, the rates of mobilization reached more than 50%, appointing that an interaction must occur between PhaZa1 and LonA for the mobilization process to be effective.

No description available.

DNA methylation regulates gene expression through a complex network of protein/protein and protein/DNA interactions in chromatin. The maintenance methylase, DNA methyltransferase 1 (DNMT1), is a prominent enzyme in the process that is linked to DNA replication and drives the heritable nature of epigenetic modifications in somatic cells. The mechanistic details that explain how DNMT1 catalytic action is directed in a chromatin setting are not well understood. We hypothesize that post translational modifications and a variety of protein-protein interactions processes are key regulatory elements that set the methylation of CpG elements essential for normal growth behavior in somatic cells. These fundamental processes can be disrupted by DNA damage leading to inappropriate gene silencing and loss of growth control in somatic cells. First, we show that DNMT1 is post-translationally modified by sumoylation and we have mapped these sumoylation sites by defined mutations. Sumoylated DNMT1 is catalytically active on genomic DNA in vivo and substantially increases the enzymatic activity of DNMT1 both in vitro and in chromatin. These data establish that sumoylation modulates the endogenous activity of a prominent epigenetic maintenance pathway in cells. Second, we investigated novel mechanisms whereby somatic cells can erase then reset DNA methylation events in somatic cells. In this study, the relationship between DNA damage and gene silencing was explored. To this end, we generated a HeLa cell line containing a specialized GFP reporter cassette (DRGFP) containing two mutated GFP genes and a unique ISceI restriction endonuclease site. These cells do not express GFP. A unique double strand break is then delivered by transfecting in the gene for I-SceI. About 4% of the cells produced a functional GFP by gene conversion and homologous recombination (HR); however roughly half iv of the GFP recombinants expressed the gene poorly and this was attributed to gene silencing. Silencing of the GFP expressing cell clones was due to DNA methylation and could be reversed using a drug that inhibits global methylation (5-aza-2'-deoxycytidine). Approximately half of the repaired genes were heavily methylated, and half were hypomethylated. That is, a key intermediate methylation state after HR repair is hemimethylated DNA, defined as methylation limited to one strand. Evidence is given that DNMT1 is acting as a de novo methylase at the HR repair patches in cells. Moreover, the DNA damage inducible protein, GADD45, interacts specifically with the catalytic domain of DNMT1 and GADD45 binds with extremely high affinity to hemimethylated DNA sites. Thus, GADD45 is a key regulatory element in silencing of HR repaired DNA segments and appears to inhibit the activity of DNMT1. Consistent with these results, we found that GADD45 increased the expression of recombinant GFP following HR repair, further suggesting its role in orchestrating strand specific DNA methylation by DNMT1. Since these experiments were performed in live cells, there is strong physiological relevance. We propose that DS DNA damage and the resulting HR process involves precise, strand selected DNA methylation mediated by the prominent methylase enzyme, DNMT1. Moreover, DS DNA break repair through HR and gene conversion, may potentially erase and reset DNA methylation patterns and therefore alter the expression of repaired genes. The overall process is tightly regulated by the DNA damage inducible protein GADD45, which may coordinate strand specific methylation by recruiting DNMT1 to HR repair templates. The ability of GADD45 to modulate DNMT1 catalytic activity may explain its role as a passive mediator of demethylation that has been reported by other groups. The overall process of silencing post DNA repair is a strong evolutionary force that may predispose cells to malignant transformation

The understanding of basic cellular processes has been greatly facilitated through investigation of the behaviour of mutant forms. In a similar way the mechanisms of genetic recombination may be clarified by a study of strains which are known to show inherited differences in recombination behaviour at meiosis. The haploid fungus Neurospora crassa is particularly well suited to such an investigation since recombination frequency heterogeneity has been extensively reported in that organism, and the differences are believed to be, to a large extent, under genetic control. Strains showing recombination frequency heterogeneity over a marked genetic region have been extensively analysed in the present work and the mode of action of the factors controlling recombination frequency has been investigated by combining differing strains in heterokaryons. / Thesis / Doctor of Philosophy (PhD)

DNA double-strand breaks (DSBs) are one of the most dangerous lesions cells encounter, given that DSBs can lead to genomic instability and cell death if not repaired properly. Cells have two primary pathways to repair DSBs: Homologous recombination (HR) and nonhomologous end-joining (NHEJ). HR is the high-fidelity branch of the DSB repair pathway since it employs a process of homology search and synthesis from a homologous template. The homology search is carried out by ssDNA that is generated on either side of the DSB by end resection. End resection occurs via a two-step mechanism involving resection initiation, followed by long-range resection. Previous work has revealed that long-range resection is dispensable for some cases of HR; however, it is currently unclear why the requirement for long-range resection is context- dependent. Furthermore, it is not completely clear how the mechanisms of HR, including requirements for long-range resection, apply to single-ended DSBs (seDSBs) arising during replication. Therefore, we defined the role of long-range resection in two-ended DSB repair in different chromosomal contexts. We also established a Cas9 nickase (Cas9n) system to study seDSB repair and defined genetic requirements for repair. To study the requirement for long-range resection in HR, we employed inter- and intrachromosomal genetic recombination assays in haploid yeast. We found that long-range resection is required for interchromosomal HR, but not for intrachromosomal HR. This difference is linked to the observation that the DNA damage checkpoint, which is deficient in the absence of long-range resection, is activated in interchromosomal HR, but not intrachromosomal HR. The DNA damage checkpoint has also previously been implicated in promoting chromosome mobility. Therefore, we reason that the requirement for long-range resection in interchromosomal repair is due to a need to activate the DNA damage checkpoint and chromosome mobility, specifically during slower repair events. To study seDSB repair, we implemented Cas9n, which creates nicks that can cause replication fork collapse. We demonstrated that expression of Cas9n with an efficient gRNA can induce replication fork collapse and that repair of these seDSBs breaks is dependent on the HR machinery. A genome-wide screen using Cas9n revealed a requirement for replication-coupled nucleosome assembly (RCNA) in repair of seDSBs, specifically in replication origin-deplete regions of the genome. Consistent with the model of seDSB repair, we found that Cas9n-induced seDSBs preferentially undergo sister chromatid recombination. This preference was altered in the absence of Mre11, which we hypothesize is due to a role of MRX in sister chromatid tethering. Altogether, the results presented in this thesis offer a different perspective on the role of long-range resection in two-ended DSB repair and establish a Cas9n-based system to better study single-ended DSB repair.

In eukaryotes, recombination plays a critical role in both the production of viable gametes and as a population genetic process. Here, we are interested in studying recombination as it provides insight into a process that has shaped variation. To this end, we study the evolution of cross-over rates in chimpanzees and humans through two experiments. Components of the recombination machinery are well described in yeast and C. elegans, but less so in other species. In humans, cross-over rates vary across physical scales and occur predominantly in narrow ∼2 kb regions called hotspots, where hotspot usage differs considerably between individuals. Differential hotspot usage is associated with specific DNA motifs, and DNA-contacting zinc finger array variants in the transacting PRDM9 H3K4 trimethyltransferase. The precise relationship between DNA motifs, PRDM9 and hotspot activity is not completely understood. Experiment 1. To investigate the importance of PRDM9 motif recognition, which is predicted be different between humans and chimpanzees, and the effect of PRDM9 on the evolution of fine-scale cross-over rates, we sequenced 10 unrelated Pan troglodytes verus (Western chimpanzee) genomes to moderate coverage (∼10×). I validate the approach by demonstrating that fine-scale maps estimated from 10 human genomes of each African and European ancestry recapitulate independently estimated maps. Then I characterise the error modes in sequencing data arising from errors in chemistry, alignment, variant calling, and genotyping. I identify several cryptic error modes missed by state-of-the-art filters and develop methods to counteract them. To guard against genotype error arising from stochastic variation in low to moderate coverage sequencing, I develop methods to incorporate the underlying statistical uncertainty into recombination analyses, evaluate the approaches through simulation (estimated 11% improvement) and empirical assessment (estimated 4% improvement), and discover that the reported genotype uncertainty is poorly calibrated, which limits the approaches. Consequently, a filtering approach was applied to the hard-called chimpanzee genotypes. I estimate recombination rates in chimpanzees through an existing LD-based method. In contrast to humans, there is no increased cross-over localisation around chimpanzee PRDM9 binding predictions, nor motifs consistently associated with activity. Hotspots do not overlap between the two species, indicating that rates evolved rapidly and consistent with PRDM9 localising all hotspots. In contrast, gene pro- moters and CpG islands are common attractors of recombination (2.7-fold increase in rate in chimpanzee, 1.5-fold increase in human), suggesting chromatin state influences hotspot placement but to varying degree in the species. I discuss the potential implications for PRDM9 mechanism. Experiment 2. To enable a more representative characterisation of the spectrum of genome changes occurring in chimpanzee genomes, I analyse data from an extended three generation Western chimpanzee pedigree sequenced at high coverage (∼30×). I use Mendel transmission to filter variants, infer haplotypes, and identify recombination events through a Hidden Markov Model approach. We detect 375 recombination events, of which 3 are double cross-over events. Sex-specific recombination rate estimates in chimpanzees mirror sex differences in humans (N♂/N♀ = 0.58) and have similar levels of total recombination. We resolve recombination events typically at ∼ 856 base-pair resolution. Additionally, analyses of Mendel inconsistencies suggest that extended pedigree sequencing opens the door on studying complex genome changes. These experiments demonstrate the power of comparative analyses, the utility of high throughput sequencing in enabling the study of recombination in almost any species of interest, the challenges in sifting signal from noise in these data, and the need for experimental and algorithmic methods to guard against error.

Durant la méiose, il se produit des échanges réciproques entre fragments de chromosomes homologues par recombinaison génétique. Les chromosomes parentaux ainsi modifiés donnent naissance à des gamètes uniques. En redistribuant les mutations génétiques pour générer de nouvelles combinaisons, ce processus est à l’origine de la diversité haplotypique dans la population. Dans cette thèse, je présente des résultats décrivant l’implication de la recombinaison méiotique dans les maladies chez l’humain. Premièrement, l'analyse statistique de données de génotypage de familles québécoises démontre une importante hétérogénéité individuelle et sexe-spécifique des taux de recombinaisons. Pour la première fois chez l’humain, nous avons observé que le taux de recombinaison maternel diminue avec l'âge de la mère, un phénomène potentiellement impliqué dans la régulation du taux d’aneuploïdie associé à l’âge maternel. Ensuite, grâce à l’analyse de données de séquençage d’exomes de patients atteints de leucémie et de ceux de leurs parents, nous avons découvert une localisation anormale des évènements de recombinaison chez les enfants leucémiques. Le gène PRDM9, principal déterminant de la localisation des recombinaisons chez l’humain, présente des formes alléliques rares dans ces familles. Finalement, en utilisant un large spectre de variants génétiques identifiés dans les transcriptomes d’individus Canadiens Français, nous avons étudié et comparé le fardeau génétique présent dans les régions génomiques à haut et à faible taux de recombinaison. Le fardeau génétique est substantiellement plus élevé dans les régions à faible taux de recombinaison et nous démontrons qu’au niveau individuel, ce fardeau varie selon la population humaine. Grâce à l’utilisation de données génomiques de pointe pour étudier la recombinaison dans des cohortes populationnelles et médicales, ce travail démontre de quelle façon la recombinaison peut affecter la santé des individus. / The intergenerational mixing of DNA through meiotic recombination of homologous chromosomes is, along with mutation, a major mechanism generating diversity and driving the evolution of genomes. In this thesis, I use bioinformatics and statistical approaches to analyse modern genomic data in order to study the implication of meiotic recombination in human disease. First, using high-density genotyping data from French-Canadian families, we studied sex- and age-specific effects on recombination patterns. These analyses lead to the first observation of a significant decrease in recombination rates with advancing maternal age in humans, with potential implications for understanding trisomic conceptions. Second, using next-generation sequencing of exomes from families of children with leukemia, we discovered unusual distributions of recombination breakpoints in some leukemia patients, which implicates PRDM9, a protein involved in defining the location of recombination breakpoints, in leukemogenesis. Third, using single nucleotide polymorphisms (SNPs) called from RNA sequencing data, we present a detailed comparison of the mutational burden between high and low recombining regions in the human genome. We further show that the mutational load in regions of low recombination at the individual level varies among human populations. In analysing genomic data to study recombination in population and disease cohorts, this work improves our understanding of how recombination impacts human health. Furthermore, these results provide insights on how variation in recombination modulates the expression of phenotypes in humans.

L’étude de la structure génétique des populations d’agents pathogènes à grandes échelles reste très important dans la contexte de nouvelles invasions. Puccinia striiformis f.sp. tritici (PST), responsable de la rouille jaune du blé, constitue un modèle fongique d’intérêt pour les études d’invasion étant donné sa capacité de migration et l’apparition récurrente de nouvelles souches localement. Nous avons analysé la structure des populations de PST à l’échelle mondiale, à l’aide de marqueurs microsatellites sur un échantillon de 409 isolats issus des six continents. Les génotypes ont été répartis en six groupes génétiques correspondant à leur origine géographique. Les analyses indiquent une forte hétérogénéité géographique de diversité génotypique, avec des signatures de recombinaison dans les régions de l'Himalaya (Népal et Pakistan) et à proximité en Chine. La structure reste clonale pour les populations des autres régions. L’assignation des isolats aux différents groupes génétiques a permis de déterminer l’origine des invasions (récentes ou anciennes). Ainsi, les souches agressives adaptées à de hautes températures, répandues de par le monde depuis 2000, sont originaires de Mer rouge-Moyen Orient ; les isolats d'Amérique du Nord et du Sud et d’Australie proviennent d’Europe du Nord-Ouest. Par ailleurs, les isolats d'Afrique du Sud appartiennent au groupe génétique de la zone méditerranéenne. La subdivision marquée entre les différentes zones géographiques indique qu’elles ne sont pas fortement marquées par les migrations récentes. De plus, les voies de migration identifiées attestent de l'importance des activités humaines dans la dispersion de PST à longue distance. La biologie des populations des zones les plus diverses (Chine et Pakistan) a été finement étudiée à l’aide d‘échantillonnages réalisés deux années consécutives. Une population échantillonnée en 2004 et 2005 dans la vallée de Tianshui, (province de Gansu, Chine), s’est révélée très diverse, fortement recombinante et non structurée spatialement et temporellement. L’observation de clones identiques entre les deux échantillons temporels a permis de développer un estimateur du taux de sexualité, i.e. du rôle relatif de la reproduction sexuée par rapport à celui de la reproduction asexuée dans le maintien de la population. Ce taux de reproduction sexuée est estimé à 74 %, alors que la taille efficace de la population est de 1735, ce qui donne les premières indications du rôle du cycle sexué. L’échantillonnage réalisé au Nord du Pakistan a permis de décrire quatre groupes génétiques ayant tous une grande diversité génotypique et une structure recombinante. Le très faible taux de ré-échantillonnage de génotypes identiques au cours de deux années suggère le rôle prédominant de la reproduction sexuée dans le maintien temporel des populations locales. La forte diversité génétique et génotypique, la signature de recombinaison et la capacité à la reproduction sexuée de PST dans la région himalayenne suggèrent que cette zone est le centre d'origine potentielle de PST. Les analyses d’approximations bayésiennes confirment la thèse d’une dispersion à partir de l’Himalaya vers les autres régions du monde. La variabilité pour la capacité à produire des téleutosores, spores indispensables à l’initiation de la phase sexuée, a été analysée (56 isolats mondiaux), et s’avère liée à la variabilité génotypique et au taux de recombinaison. Ce résultat conforte la thèse de l'apparition de la sexualité dans la zone himalayenne et à proximité de cette zone et de la perte de sexualité lors de migrations dans les zones où l’hôte alternant est absent et où le cycle épidémique est essentiellement asexué. La description de l'origine, des voies mondiale de migration de PST ainsi que de son centre de diversité contribue à la compréhension du potentiel évolutif de PST et à la construction de stratégies de gestion de lutte contre l’agent pathogène. / Analyses of the large-scale population structure of pathogens enable the identification of migration patterns, diversity reservoirs or longevity of populations, the understanding of current evolutionary trajectories and the anticipation of future ones. A detailed analysis of populations in centre of diversity should enable to infer the adaptive capacity of the pathogen and identify potential sources for new invasions. Puccinia striiformis f.sp. tritici (PST) is the causal agent of wheat yellow/stripe rust, and despite a worldwide distribution, this fungus remains a model species for invasion studies, due to its long-distance migration capacity and recurrent local emergence of new strains. Little is known about the ancestral relationship of the worldwide PST population with unknown center of origin. We used multilocus microsatellite genotyping to infer the worldwide population structure of PST and the origin of new invasions, analysing a set of isolates representative of sampling performed over six continents. Bayesian and multivariate clustering methods partitioned the isolates into six distinct genetic groups, corresponding to distinct geographic areas. The assignment analysis confirmed the Middle East-Red Sea Area as the most likely source of newly spreading, high-temperature-adapted strains; Europe as the source of South American, North American and Australian populations; and Mediterranean-Central Asian populations as the origin of South African populations. The existence of strong population subdivision at worldwide level shows that major genetic groups are not markedly affected by recent dispersal events. However, the sources for recent invasions and the migration routes identified emphasize the importance of human activities on the recent long-distance spread of the disease. The analyses of linkage disequilibrium and genotypic diversity indicated a strong regional heterogeneity in levels of recombination, with clear signatures of recombination in the Himalayan (Nepal and Pakistan) and near-Himalayan (China) regions and a predominant clonal population structure in other regions. To explain the variability in diversity and recombination of worldwide PST populations, we assessed their sex ability in terms of telial production, the sex-specific structures that are obligatory for PST sexual cycle, in a set of 56 isolates representative of these worldwide geographical origins. We confirmed that the variability in genotypic diversity/ recombination was linked with the sex ability, pinpointing the Himalayan region as the possible center of origin of PST, from where it then spread worldwide. The reduced sex ability in clonal populations certainly reflects a loss of sexual function, associated to migration in areas where sexual alternate host is lacking, or not necessary for the completion of epidemic cycle. Approximate Bayesian computation analyses confirmed an out of Himalaya spread of PST, with Pakistan and China being the most ancestral population. A detailed analysis of Pakistani population at regional level revealed the existence of a strong population subdivision, a high genotypic diversity and the existence of recombination signature at each location reflecting the role of sexual recombination in the temporal maintenance at local level. A time spaced sampling of PST in the valley of Tianshui (China) inspired the development of a new estimator, allowing to quantify the relative contribution of sexual reproduction and effective population size on the basis of clonal resampling within and between years. A sexual reproduction rate of 74% (95% confidence interval [CI]: 38-95%) and effective population size of 1735 (95% CI: 675-2800) was quantified in Chinese PST population. The description of the origin and migration routes of PST populations worldwide and at its centre of diversity contributes to our understanding of PST evolutionary potential, and is helpful to build disease management strategies.

A recombinação durante a transcrição reversa é um fator importante no aumento da diversidade genética e adaptação do HIV-1, permitindo que mutações vantajosas presentes em diferentes linhagens sejam combinadas em um mesmo genoma. No Brasil, vários recombinantes foram descritos e seis formas recombinantes circulantes (CRFs) já foram identificados, demonstrando a relevância destes recombinantes na epidemia brasileira. Portanto, um dos objetivos desta tese foi analisar os dados gerados pela Rede de Diversidade Genética Viral (VGDN) (sequências parciais de gag, pol e env), a fim de identificar recombinantes inter-subtipos de HIV-1 e avaliar a frequência e distribuição geográfica destes vírus. Utilizando diferentes técnicas foram identificados 152/1083 pacientes portadores de recombinantes BF. A frequência destes recombinantes foi maior em cidades como São Vicente (30%) e Sorocaba (22,6%), sendo que os recombinantes circulantes em São Vicente foram geralmente relacionados às CRF28 e CRF29, enquanto que os vírus presentes na região de Sorocaba comumente apresentam um envelope subtipo F1, independente do subtipo nos demais genes. Além disso, o gene da integrase de 159 pacientes foi amplificado e sequenciado. A análise deste gene revelou mais 10 pacientes infectados com recombinantes BF e nenhuma mutação de resistência primária aos inibidores da integrase foi encontrada. O segundo objetivo foi isolar e caracterizar recombinantes BF in vitro. O isolamento viral foi realizado por co-cultivo e ao final foram obtidos 10 isolados primários. O sequenciamento do genoma quase completo desses dez isolados primários revelou que três isolados primários pertencem ao grupo da CRF28_BF, três ao grupo da CRF29_BF e quatro foram classificados como formas recombinantes únicas (URFs). Ainda, o uso de correceptores desses isolados foi avaliado in vitro em ensaios com as células GHOST(3), e revelou três duplo-trópicos (X4/R5) vírus, quatro CXCR4 (X4) e três isolados utilizaram apenas CCR5 (R5). Em suma, uma alta frequência de URFs foi encontrada em algumas cidades do Estado de São Paulo, e também foi desenvolvido e caracterizado um painel de isolados primários representando as CRF28_BF, CRF29_BF e algumas URFs. / Recombination during reverse transcription is an important factor promoting HIV-1 diversity and adaptive change, allowing advantageous mutations arising on different genomes to undergo linkage in the same progeny recombinant genome more frequently than what would be expected under random mutation alone. In Brazil, several recombinant viruses were reported, and six circulating recombinant forms (CRFs) have already been identified. Therefore, the first objective of this Thesis was to analyze the data generated by the Viral Genetic Diversity Network (VGDN) (gag, pol and env partial sequences), in order to identify HIV-1 intersubtype recombinants and evaluate the frequency and geographical distribution of these viruses. Using different techniques we identified 152/1083 patients harboring BF recombinants. The frequency of these recombinants was higher in cities like São Vicente (30%) and Sorocaba (22.6 %). The recombinant viruses circulating in São Vicente were generally related to CRF28 and CRF29, while those viruses circulating in Sorocaba commonly presented an envelope region of subtype F1, irrespective the subtype composition on the remaining genes. Additionally, the integrase gene of HIV-1 from 159 patients was further amplified and sequenced. The analysis of this viral gene revealed ten more patients infected with BF recombinants and no primary mutations related to integrase inhibitor resistance were found. The second objective was to isolate and characterize BF recombinants in vitro, which resulted in ten primary HIV-1 isolates. The near full-length genomes of these ten primary isolates revealed that three were related to CRF28_BF, three to CRF29_BF and four were unique recombinant forms (URFs), according to their breakpoints profile determined with the jpHMM program. Additionally, the coreceptor usage of these isolate was investigated in vitro using GHOST assays, which revealed three dual-tropic (X4/R5) viruses, four CXCR4 (X4) viruses and three CCR5 (R5) viruses. In sum, we report a high frequency of URFs in some cities of São Paulo State, and also developed a well-characterized panel of viruses representing CRF28_BF, CRF29_BF and URFs.