O desenvolvimento de uma vacina contra malária causada por P. falciparum é prejudicado pelo alto nível de polimorfismo apresentado pelos antígenos desse parasito. O dimorfismo alélico é um padrão no qual os alelos observados de um gene se encontram divididos em duas famílias. A proteína dimórfica MSP-6 se associa à proteína MSP-1 (também dimórfica) na superfície do merozoíto. Genes de msp-6 de 21 isolados obtidos de pacientes do Brasil, mais 2 isolados da Tanzânia, África, foram seqüenciados para estudo da diversidade nucleotídica e distribuição geográfica dos alelos. As duas famílias possuem distribuição global. Não foi verificada associação entre o dimorfismo de MSP-1 e MSP-6. O gene ortólogo de msp-6 em P. reichenowi, grupo irmão de P. falciparum, foi seqüenciado para estudos evolutivos. Os alelos dimórficos de MSP-6 aparentam ter surgido de uma população ancestral polimórfica, tendo sido mantidos no presente por seleção balanceada. O alto grau de conservação encontrado dentro de cada família alélica torna MSP-6 um potencial alvo de uma vacina contra a malária. / The development of a vaccine against malaria caused by Plasmodium falciparum has been hampered by the high level of antigen polymorphism exhibited by this parasite. Allelic dimorphism is a pattern in which every observed allele of a gene is clearly grouped into one of two families. The dimorphic protein MSP-6 forms a complex with MSP-1 (also dimorphic) on merozoite surface. The msp-6 genes were sequenced in isolates obtained from 21 patients from Brazil, plus 2 isolates from Tanzania, Africa, to study nucleotide diversity and geographic distribution of alleles. Both families are globally distributed. Moreover, no association was observed between the MSP-1 and MSP-6 allelic types. Orthologous gene of msp-6 in P. reichenowi, chimpanzee parasite and sister group of P. falciparum, was sequenced for evolutionary studies. Dimorphic alleles of MSP-6 seem to have originated from an ancestral polymorphic population and are maintained by balancing selection. The high degree of conservation observed within each allelic family makes MSP-6 an promising target for vaccine development.

Jatropha curcas L., também conhecida no Brasil como pinhão-manso, é uma oleaginosa que atraiu a atenção do mundo para bioenergia dado a qualidade e o alto conteúdo de óleo na semente. Contudo, a espécie necessita de programas de melhoramento genético para fixar características de interesse em bancos de germoplasma. Este trabalho objetivou identificar e analisar a diversidade alélica, metabólica e físico-química relacionada à síntese de óleo e ésteres de forbol em uma amostra de 22 genótipos do BAG do Instituto Agronômico de Campinas para seleção de genótipos elite. Os genes MFP2, KASIII e 3-N-D, envolvidos na biossíntese de óleo e de taxol, foram selecionados a partir de Gomes et al. (2010). Os resultados mostraram que há baixa diversidade alélica para os genes KAS III e MFP2, por outro lado, a expressão desses mesmos genes e a composição de ácidos graxos foi bastante variável entre os genótipos e ao longo do desenvolvimento da semente de J. curcas indicando uma provável regulação diferenciada da via de óleo. O estudo realizado permite combinar a diversidade alélica, expressão dos genes, conteúdo metabólico e poder calorífico para a seleção de genótipos e identificação de parentais direcionando cruzamentos no quadro do programa de melhoramento do IAC. / Jatropha curcas L., also know in Brazil as physic nut, is an oilseed that call world´s attention to bioenergy due to quality and high oilseed content. However, this species needs of breeding programs so that interest traits may to be fixed in germplasm banks. This study aimed to identify and analyze allelic, metabolic and physicochemical diversity related to oil and phorbol esters synthesis in a 22 genotypes sample from Instituto Agronômico de Campinas-BAG for elite genotypes selection. The MFP2, KASIII and 3-ND genes involved in oil and taxol biosynthesis were selected from Gomes et al. (2010). The results showed that there is low allelic diversity for KAS III and MFP2 genes, on the other hand, the expression of these same genes and fatty acid composition was quite variable between genotypes through J. curcas seed development indicating a putative differential regulation in oil pathway. This study allows combining allelic diversity, gene expression, metabolic content and calorific values for parental genotypes identification and selection driving crosses within IAC breeding program.

The bacteria of genus Streptococci are among the most significant producers of hyaluronic acid in industrial scale. One of the typical representatives of that group is Streptococcus equi subsp. zooepidemicus. The production of hyaluronic acid in Streptococcus equi subsp. zooepidemicus is heavily influenced by cultivation conditions and by genetic alterations. The present work describes the deletion of genes covR and covS responsible for transcriptional regulation of stress response. According to Galeas a kol. [35] the deletion of these genes in S. pyogenes led to the hyaluronic acid capsule increase. As the S. pyogenes and S. equi subsp. zooepidemicus share approx. 80 % of genome, it was assumed, that the deletion of genes covR and covS in Streptococcus equi subsp. zooepidemicus genome would lead to the higher hyaluronic acid production. The new strain SEZ covRS was obtained by allelic replacement mutagenesis. The cultivations performed in laboratory-scale fermenters in rich Wheat E1 medium showed approx. 9% higher production over parental strain. Therefore, the covRS regulation system plays the same role in Streptococcus equi subsp. zooepidemicus and indirectly regulates the biosynthesis of hyaluronic acid.

Les thérapies ciblées ont amélioré la survie des patients atteints de mélanome cutané métastatique, toutefois certains patients échappent au traitement. La réponse au traitement met en jeu des paramètres dépendants du patient, de la biologie de la tumeur, et de la survenue de mécanismes de résistance largement étudiés.Le but de ce travail est d’étudier deux marqueurs mesurables en pratique clinique lors du suivi des patients, pour leur potentiel prédictif de réponse aux inhibiteurs de BRAF en monothérapie, l’un biologique et l’autre pharmacologique : la charge allélique mutée (CAM), définie par le rapport muté/total, des oncogènes BRAF et NRAS, et la concentration plasmatique de vemurafenib (CPV).Nous rapportons pour la première fois la prévalence et les mécanismes du déséquilibre allélique des oncogènes BRAF et NRAS, défini par une CAM élevée (>60%) ou faible (≤30%), par opposition à une CAM « hétérozygote » équilibrée (entre 30 et 60%). Des arguments in vitro et in vivo soutiennent l’hypothèse qu’une CAM BRAFV600E >60% puisse être associée à une meilleure réponse aux inhibiteurs de BRAF. La CAM de NRAS ne semble pas être un marqueur pronostique au moment du diagnostic de mélanome, toutefois sa valeur prédictive sur la réponse aux inhibiteurs de MEK sera intéressante à étudier.Nous montrons pour la première fois l’impact du dosage de la CPV lors du suivi des patients traités en monothérapie. Une CPV basse serait prédictive d’une progression ; ainsi, la CPV semble être un marqueur prédictif original de réponse, pour lequel la valeur seuil ainsi que les mécanismes pharmacogénétiques impliqués restent à déterminer. / Targeted therapies have improved survival in patients with metastatic cutaneous melanoma, but some patients escape treatment. Response to treatment involves parameters dependent of the patient, the tumor biology, and the occurrence of resistance mechanisms extensively investigated. The aim of this work is to study two measurable markers in clinical practice in patient monitoring, for their predictive potential of response to BRAF inhibitors monotherapy; the first one is biological and the other one is pharmacological: mutant allele burden (MAB), defined by the ratio mutant/total, of BRAF and NRAS oncogenes, and plasma vemurafenib concentration (PVC).We report for the first time the prevalence and the mechanisms of BRAF and NRAS oncogenic allelic imbalance, defined by a high (>60%) or a low (≤30%) MAB, as opposed to a balanced “heterozygous” MAB (30 to 60%). In vitro experiments and in vivo data support the hypothesis that a BRAFV600E MAB >60% may be associated with a better response to BRAF inhibitors. NRAS MAB does not seem to be a baseline prognostic marker, but its predictive value of response to MEK inhibitors will be interesting to investigate.We demonstrated for the first time the impact of CPV assay during the monitoring of patients treated with monotherapy. A low CPV would be predictive of progression; thus, CPV appears to be an original predictive marker of response, for which the threshold value and the involved pharmacogenetic mechanisms remain to be determined.

No description available.

Equine herpesvirus-1 (EHV-1) can cause acute upper respiratory tract disease, abortion, neonatal death and neurological disease in horses. Rapid, accurate and timely diagnosis of EHV-1 infection in horses is important to curtail the spread of this pathogen. It has been reported that the neuropathogenic phenotype of EHV-1 can result from a single non-synonymous nucleotide substitution at position 2254 (A→G2254) in open reading frame 30 (ORF30). This was the basis for the development of an allelic discrimination, real-time PCR assay to distinguish between potential neuropathogenic and non-neuropathogenic EHV-1 strains. However, PCR analysis of a panel of EHV-1 abortion isolates revealed that other point mutations within ORF30 could produce false negative results with this previously described assay. Therefore, one of the objectives of this dissertation project was to develop a more sensitive and specific allelic discrimination real-time PCR assay for the detection of EHV-1. This was achieved by redesigning the primers and probes targeting ORF30. The new assay was ten times more sensitive than the original assay, with a lower detection limit of 10 infectious virus particles. While mutations within EHV-1’s genome can hinder diagnosis, they can also impact the virulence of the virus. Objective two, therefore, was to determine if sequential cell passage of T953 would induce sufficient attenuation of the EHV-1 genome to produce a low virulence phenotype. Two separate groups of 28 BALB/c mice were inoculated with either the parental strain or passage 135 (T953 P135) of EHV-1 strain T953. The animals were observed for fourteen days, euthanized and their tissues analyzed for the presence of EHV-1. At the conclusion of the fourteen day observation period, all of the mice infected with T953 P135 survived and regained their pre-inoculation body condition. Furthermore, there were significant differences in virus titer and viral DNA concentrations between T953 P135 and the parental strain, further confirming the attenuated phenotype of the virus. Taken together, data from this study clearly demonstrates that sequential cell culture passage of the neuropathogenic T953 strain of EHV-1 results in attenuation for young adult BALB/C mice.

En 1975, Wilson et King ont proposé que l'évolution opère non seulement via des changements affectant la structure des protéines, mais aussi via des mutations qui modifient la régulation génétique. L'étude des éléments régulateurs de l'expression génétique a un rôle important dans la compréhension de l'expression de différentes maladies et de la réponse thérapeutique. Nous avons développé un algorithme bio- informatique qui nous permet rapidement de trouver des sites de régulation génétique à travers tout le génome et pour une grande quantité de gènes. Notre approche consiste à trouver des sites polymorphes (SNPs) qui sont en déséquilibre de liaison avec le débalancement allélique (AI) afin de cartographier la région régulatrice et le site responsable. Notre méthode est avantageuse par rapport à d'autres méthodes, car elle n'a pas besoin des données « phasées». De plus, les données de débalancement allélique ne sont pas affectées par des facteurs externes étant donné qu'ils sont mesurés dans la même cellule. Nous avons démontré que notre approche est fiable et qu'elle peut détecter des sites loin du gène. De plus, il peut être appliqué à des données de génotypage sans avoir besoin de les « phaser » . / Wilson and King (1975) proposed that evolution frequently operates through mutations affecting genetic regulation. Likewise, it is expected that genetic variation responsible for inter-individual differences will be due to variation in regulatory sites. Identifying such sites is thus important in the genetic and medical research. We have developed a new bioinformatics algorithm to find genome-wide regulatory sites for a big number of genes. Individuals carrying different alleles at a regulatory site will exhibit allelic imbalance(AI) due to differential expression of the two copies the same locus. Our approach consists of searching polymorphic sites (SNPs) in linkage disequilibrium with AI in order to map regulatory regions. We have detected many SNPs associated to the regulation of different genes pointed in previous studies. We have also found regulatory regions far from the transcription start site (TSS). The major advantage of this method is that phased data is not needed. In addition, AI data has the benefit of not being affected by external factors since it is measured in the same cell. The results show that our approach is reliable and it can detect sites far from the gene.

A presença de DNA fetal livre de células no plasma materno possibilitou o surgimento de novas tecnologias para o diagnóstico pré natal não invasivo. Existem técnicas de genotipagem fetal já estabelecidas em investigações de sequências exclusivas fetais, mas a sua aplicação para a identificação humana em testes de paternidade ainda é pouco conhecida. A metodologia de genotipagem fetal não invasiva foi padronizada com o uso de três lócus InDels e iniciadores inserção-específicos. Entretanto, para que se alcance o poder satisfatório, é necessário investigar elevado número de lócus informativos. Para suprir a ausência de informações suficientes sobre lócus deste tipo, são descritas, no presente trabalho, as condições laboratoriais para PCR e as frequências alélicas de um conjunto de lócus InDels, ainda inéditos, em uma amostra representativa da população urbana brasileira, visando aumentar a robustez e a confiabilidade da genotipagem fetal não invasiva na investigação de paternidade. Foram selecionados 20 lócus, um em cada cromossomo. Em um dos lócus polimórficos foi encontrado um alelo novo, não registrado no banco de dados do NCBI. O poder de discriminação e o poder de exclusão dos 16 lócus polimórficos combinados (0,999 e 0,937, respectivamente) tiveram valores dentro do esperado para um conjunto com essa quantidade de lócus bialélicos. Alguns lócus apresentaram elevado número de indivíduos que não responderam à amplificação, indicando a necessidade de reajustes na padronização dos procedimentos laboratoriais e a possibilidade de variabilidade das sequências complementares aos iniciadores empregados, o que exigirá o sequenciamento completo das regiões. Para a maioria dos lócus, entretanto, os parâmetros forenses atingiram valores esperados e sugerem que podem ser úteis na composição de painel robusto e eficiente para ser usado na genotipagem fetal não invasiva. / The presence of cell-free fetal DNA in maternal plasma allows for new non-invasive prenatal diagnosis technologies to develop. Even though there are already well-established fetal genotyping techniques in fetal-exclusive sequence investigations, their application to human identification in paternity tests is not yet well-known. Non-invasive fetal genotyping methodology was previously standardized using three InDel loci and insertion-specific primers. However, in order to attain satisfactory power, it is necessary to investigate large number of informative loci. To compensate for the absence of sufficient information about this type of locus, this work describes the PCR conditions and determinates allelic frequencies for a set of unpublished InDel loci, in a representative group of Brazilian population, raising the robustness and reliability of non-invasive fetal genotyping in paternity investigation. We selected 20 loci, one in each chromosome. Not previously registered on NCBI database allele was found in one of the polymorphic loci. Discrimination and exclusion power of the 16 polymorphic loci combined (0.999 and 0.937, respectively) had expected values for an ensemble with such an amount of biallelic loci. Some loci showed many non-amplified individuals, vincing the need of corrections in the standardizing process and the possible variability of the complementary sequences to the primers used. However, for most loci, the forensic parameters reached the expected values and suggest that they may be useful in the more robust panel for utilization in non-invasive fetal genotyping.

Submitted by admin tede (tede@pucgoias.edu.br) on 2016-08-18T13:03:27Z No. of bitstreams: 1 Jonas Garcia de Almeida.pdf: 3226956 bytes, checksum: 9d13dc0a531d222433b4267892b08ac2 (MD5) / Made available in DSpace on 2016-08-18T13:03:27Z (GMT). No. of bitstreams: 1 Jonas Garcia de Almeida.pdf: 3226956 bytes, checksum: 9d13dc0a531d222433b4267892b08ac2 (MD5) Previous issue date: 2015-03-26 / For human identification purposes in Forence area, the United States created the database (DB) of DNA Index System Combined (CODIS), containing 13 loci: CSF1PO, FGA, TH01, TPOX, VWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51 and D21S11. Then the DBs of the markers of the Y-STR X-STRs haplotypes were created. Comparison of the obtained data was conducted from allele frequencies (AF), paternity index (PI) by marker and paternity probabilities (PPs) obtained from nine BD population, one of those national (BR), using 241 cases containing the PP 99.99 using 13 loci STRs (CODIS) and 2 more STRs markers of Penta D- and E, granted by the BDs of Núcleo de Pesquisas Replicon, of the Pontifícia Universidade Católica de Goiás (PUC - GO) and LaGene, Laboratório Central de Saúde Pública (SES - GO). 241 cases were analyzed in Trio (alleged father, son and progenitor) and Duo (alleged father and son) situations, using 13 STRs markers (CODIS) by Bayes theorem and the Likelihood Ratio (LR). The results were submitted to the Kruskal-Wallis, chi-square, Spearman correlation and Fishers exact tests and SPSS software (2010). Static analysis of RV cases containing the Trio resulted in 1,148 results PPs 99.99% and 21 results PPs <99.99%, and in a position to Duo 1686 results with PPs 99.99% and 483 results PPs <99.99%. The analysis of the IP average of each marker showed the D21S11 of STRs markers and FGA with the highest power of inclusion and TH01 and D3S1358 with the lowest power of inclusion. The PPs did not show significantly different, containing mostly positive correlation, of moderate to strong, between 8 BDs compared to the BR population databases. This study demonstrated the statistics interference that each allele frequencies DB can have in PP calculations using only 13 loci of genetic, thus making it more significant in cases Duo situation. According to the information available in databases of gene frequencies of the different geographical regions of Brazil, it became possible to conclude that the allele frequencies obtained and the IPs per marker and PPs obtained, suggest strong similarities to those found in the national database. / Para fins de identificação humana na área forense os Estados Unidos criou o Banco de Dados (BD) do Sistema de Índice de DNA Combinado (CODIS), contendo 13 loci: CSF1PO, FGA, TH01, TPOX, VWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51 e D21S11. Foi realizado no presente estudo, a comparação dos dados obtidos das frequências alélicas (FA), índices de paternidade (IP) por marcador e probabilidades de paternidade (PPs) obtidas a partir de 9 BD populacionais, sendo 1 nacional (BR), utilizando 241 casos contendo a PP 99,99 com o uso de 13 loci STRs (CODIS) e mais 2 marcadores STRs do Penta-D e E, cedidos pelos BDs do Núcleo de Pesquisa Replicon, da Pontifícia Universidade Católica de Goiás (PUC-GO) e do LaGene, Laboratório Central de Saúde Pública (SES-GO). Os 241 casos foram analisados nas situações de Trio (suposto pai, filho e genitora) e Duo (suposto pai e filho) usando 13 marcadores STRs (CODIS) através do teorema de Bayes e pela Razão de Verossimilhança (RV). Os resultados foram submetidos aos testes de Kruskal-Wallis, Qui-quadrado, Correlação de Spearman e Exato de Fisher usando o software SPSS (2010). As análises estáticas da RV dos casos contendo o Trio resultaram em 1.148 resultados com PPs 99,99% e 21 resultados com PPs < 99,99%, e em situação de Duo 1.686 resultados com PPs 99,99% e 483 resultados com PPs < 99,99%. As análises das médias dos IPs de cada marcador demonstraram os marcadores STRs do D21S11 e FGA com os maiores poder de inclusão e TH01 juntamente com o D3S1358 os menores poderes de inclusão. As PPs não apresentaram diferenças estatísticas significativas contendo em sua maioria correlações positivas de moderadas a fortes entre os 8 BDs comparados ao BDs populacional do BR. Este estudo demonstrou as interferências estatísticas que cada BDs de frequências alélicas pode exercer nos cálculos de PP quando se utiliza apenas 13 loci para confirmação de vínculo genético, tornando-se assim mais significativas nos casos em situação de Duo. De acordo as informações disponíveis nos BDs de frequências alélicas das diferentes regiões geográficas do Brasil, tornou-se possível concluir, que as frequências alélicas obtidas, bem como os IPs por marcador e as PPs obtidas, sugerem fortes similaridades às encontradas no banco de dados nacional.

O NER é uma via de reparo de DNA capaz de lidar com uma ampla variedade de lesões. Participam do NER diversas proteínas, entre elas a endonuclease XPG. Pacientes que possuem mutações no gene XPG apresentam a síndrome XP, e em alguns casos XP/CS. Investigamos a natureza genética de dois pacientes XP-G, que são irmãos e apresentam fenótipo moderado. As células destes pacientes demonstraram alta sensibilidade à luz UVC. Quando expostas a um agente oxidativo, apenas células XP-G/CS exibiram sensibilidade. Identificamos duas mutações missense no gene XPG desses pacientes, e comparamos com outras mutações existentes. Observamos que as mutações possuem um impacto negativo na funcionalidade de XPG. A proteína com a mutação p.Ala28Asp exibiu uma atividade residual em testes de complementação. Os resultados indicam que o fenótipo XP-G desses pacientes é causado por duas mutações missense em heterozigose composta, e que células portadoras dessas alterações exibem respostas diferenciadas frente aos estresses genotóxicos causados pela luz UV e pelo agente oxidativo utilizado. / NER is the most flexible of all known DNA repair mechanisms. XPG is an endonuclease that participates in the final steps of NER. Mutations in this gene may result in the human syndrome XP and, in some cases, in the XP/CS. We investigated the genetic nature in two XP-G patients, siblings and mildly affected. The cells from these patients demonstrated the high UV sensitivity typical of this syndrome. When exposed to an oxidative agent, only XP-G/CS cells exhibited sensitivity. We identified two missense mutations in the XPG gene of these patients, and a comparison with other known mutations is presented. These mutations have a negative impact in the function of XPG. The protein harboring the mutation p.Ala28Asp exhibited residual activity in complementation tests. These results indicate that the phenotype of XP-G patients is caused by two missense mutations in a compound heterozygous manner, and that the cells carrying these alterations exhibit different responses against genotoxic stress caused by the UV light and by the oxidative agent used.