Orientadores: Reginaldo Bruno Gonçalves, Cristiane Duque / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-16T07:13:26Z (GMT). No. of bitstreams: 1 Sardi_JanainadeCassiaOrlandi_D.pdf: 1215494 bytes, checksum: 51470817af2a54ba8f45aa4374a2e52f (MD5) Previous issue date: 2010 / Resumo: Candida spp. são leveduras comensais que habitam diferentes sítios da cavidade bucal. Em indivíduos saudáveis, sem alterações imunológicas, esses microrganismos não causam doença. Entretanto, diante de condições imunossupressoras, essas leveduras podem se tornar mais virulentas e expressar patogenicidade. Espécies de Candida apresentam diversos fatores de virulência, incluindo mecanismos de adesão e invasão celular associado à produção de enzimas que auxiliam na degradação tecidual e facilitam sua proliferação na mucosa bucal. Estudos têm demonstrado a presença de Candida sp. em sítios periodontais de pacientes com periodontite crônica, principalmente quando estes são imunologicamente comprometidos. Entretanto, ainda é desconhecido o papel desses microrganismos na patogênese da doença periodontal. Os objetivos do presente trabalho foram: 1) identificar a presença de espécies de Candida e periodontopatógenos por PCR em sítios bucais de pacientes diabéticos ou não com periodontite crônica; 2) isolar cepas de Candida albicans desses pacientes e avaliá-las quanto à atividade das enzimas proteinase, fosfolipase e hemolisina e os graus de hidrofobicidade da superfície celular, sob diferentes condições atmosféricas, além de realizar a análise genotípica desses isolados; 3) avaliar a capacidade de adesão e invasão de cepas de Candida albicans com diferentes graus de hidrofobicidade, em fibroblastos gengivais humanos. As reações de PCR mostraram que os diabéticos tiveram maior prevalência de Candida spp. principalmente C. albicans e C. dubliniensis, e menor freqüência de Tannerella forsythia, quando comparado aos pacientes não diabéticos, para bolsa periodontal e furcas. C. glabrata e C. tropicalis não foram encontradas em sítios periodontais de pacientes não diabéticos. Dos pacientes diabéticos, foram isoladas 128 cepas de C. albicans, das quais 51.6% foram determinadas como genótipo B e 48.4% como genótipo A. As condições ambientais consideradas neste estudo, níveis reduzidos de oxigênio ou anaerobiose, não modificaram o tipo de hemólise realizado pelo microrganismo, sendo que a maioria das cepas foi alfa-hemolítica. Nesses ensaios, 100% das cepas em anaerobiose apresentaram as colônias rugosas, enquanto que em ambiente de oxigênio reduzido, houve variação em relação à morfologia e a maioria delas apresentou colônia lisa. Com relação à atividade de proteinase e fosfolipase, cepas de C. albicans não produziram as enzimas na ausência total de oxigênio. Em ambiente com nível reduzido de oxigênio, a maioria das cepas de C. albicans foram fortemente produtoras de proteinase e a maioria das cepas foi positiva para fosfolipase. A hidrofobicidade foi mais alta na condição de anaerobiose. A partir desses resultados, foram selecionadas 16 cepas com alta ou baixa hidrofobicidade e avaliadas quanto à capacidade de adesão e invasão em fibroblastos gengivais humanos. Foi verificado que ambos os processos foram maiores nas cepas com alta hidrofobicidade. A produção de óxido nítrico foi maior para as cepas mais hidrofóbicas. Os resultados demonstraram que as espécies de Candida podem ser encontradas, em grande proporção, em bolsas periodontais e furcas de pacientes portadores de periodontite crônica, principalmente naqueles acometidos por diabetes mellitus. A maioria das cepas de C. albicans apresentou atividade enzimática, que atuaria diretamente na degradação tecidual. Além disso, a hidrofobicidade das cepas de C. albicans mostrou estar relacionada à maior capacidade de adesão e invasão em fibroblastos. Todos esses fatores de virulência aumentam a patogenicidade da Candida, que poderia colaborar na progressão da doença periodontal, principalmente em pacientes imunodeficientes / Abstract: Candida spp. are commensal yeasts that inhabit different sites of the oral cavity. In healthy subjects, without immunological alterations, these microorganisms do not cause disease. However, in immunosuppressive conditions, these yeasts can become more virulent and express pathogenicity. Candida species have different virulence factors, including mechanisms of cell adhesion and invasion associated with the production of enzymes that facilitate tissue degradation and their proliferation in oral mucosa. Studies have shown the presence of Candida spp. in periodontal sites of patients with chronic periodontitis, especially when they are immunologically compromised. However is still unknown their role in the pathogenesis of periodontal disease. The objectives of this study were: 1) to identify the presence of Candida species and putative periodontopathogens by PCR in periodontal sites of diabetic or non-diabetic patients with chronic periodontitis; 2) to isolate strains of Candida albicans in these patients and evaluate the proteinase, phospholipase and haemolysin activities and degrees of cell surface hydrophobicity under different atmospheric conditions, besides to performe the genotypic analysis of these isolates; 3) to evaluate the ability of adhesion and invasion of Candida albicans strains with different degrees of hydrophobicity, in human gingival fibroblasts. The PCR reactions revealed that diabetics had higher prevalence of Candida spp., mainly C. albicans and C. dubliniensis, and lower T. forsythia frequency, when compared to non-diabetic patients, for both periodontal sites. C. glabrata and C. tropicalis were not found in periodontal pockets and furcation sites of non-diabetic patients. From diabetic patients, it was isolated 128 strains of C. albicans and 51.6% were determined as genotype B and 48.4% as genotype A. The atmospheric conditions, reduced oxygen and anaerobiosis, did not change the type of hemolysis, and the most of strains were alpha-hemolytic. From these assays, 100% of the strains under anaerobiosis showed rough colonies, whereas in an environment with reduced oxyen was no change in relation to morphology and most of them had smooth colony. Considering proteinase and phospholipase activities, C. albicans strains did not produce the enzymes in the total absence of oxygen. In reduced oxygen, the majority of C. albicans strains were strong proteinase producers and most strains were positive for phospholipase. Hydrophobicity was higher in anaerobic condition. From these results, 16 hydrophobic or hydrophilic strains were selected and evaluated their ability of adhesion and invasion in human gingival fibroblasts. Both processes were greater in strains with high hydrophobicity. The production of nitric oxide was higher for hydrophobic strains. The results showed that Candida species can be found in large proportion, in periodontal pockets and furcation of patients with chronic periodontitis, especially diabetics. The most of C. albicans strains showed enzymatic activity, which could act directly on tissue degradation. Moreover, the hydrophobicity of C. albicans seems to be related to higher capacity of adhesion and invasion in fibroblasts. All these virulence factors enhance the pathogenicity of Candida that could collaborate for the progression of periodontal disease / Doutorado / Microbiologia e Imunologia / Doutor em Biologia Buco-Dental

The effect of genotype and slaughter age on gonadal development, body development and the correlations between these measurements were studied in five pig genotypes (Genotype 1, Genotype 2, Genotype 3, Genotype 4 and Genotype 5) consisting of 112 gilts and 112 boars with initial live weights varying between 25 – 30 kg. The pigs were group-housed in commercial type grower houses and fed a diet consisting of 14 MJ/kg energy, 18 % CP and 1.1 % lysine during the growth period up to a live weight of 65 kg, followed by a diet consisting 13.5 MJ/kg, 16 % CP and 0.9% lysine from 65 kg to 90 kg and then a diet consisting of 13.2 MJ/kg, 15 % CP and 0.7% lysine from 90 kg onwards. Pigs were slaughtered at 116, 130, 144, 158, 172, 186, 200 and 214 days of age. Gonadal growth and development were measured in gilts (ovary length, ovary width, ovary thickness, ovary weight, ovary volume, follicle number, and size of the largest follicle), boars (testis length, testis width, testis weight and testis volume) and body development parameters (slaughter weight, warm carcass weight, carcass length, chest depth, dressing percentage and P2 backfat thickness) were compared. Differences between means were tested using breed, sex and slaughter age as fixed effects, while the relationships between gonadal and body development parameters were evaluated by means of correlation analysis. Genotype 5 had a significantly shorter ovary length than Genotype 4 and Genotype 2. Genotype 2 gilts also had heavier ovaries and larger ovary volumes than Genotype 5 gilts. In boars, Genotype 2 had significantly heavier testes weights than Genotype 5 boars. Genotype 5 boars also tended to have smaller testis volumes than Genotype 2 boars. In body development, Genotype 2 gilts and boars were superior to the Genotype 5 in terms of slaughter and warm carcass weights, while Genotype 3 seconded Genotype 2. The average P2 backfat thicknesses were 11.88 mm and 13.68 mm for boars and gilts respectively. Correlations between gonadal and body development parameters were low to moderate in the gilts (r = -0.305 to 0.555) and moderate to high in boars (r = 0.560 to 0.871). However, dressing percentage, follicle number and size of the largest follicle correlated poorly with all other measurements. It is concluded from the study that although Genotype 5 do not grow to the same size and at the same rate compared to the other genotypes, they appear to be the most suitable for the production of top quality pork in terms of its low backfat thickness. Genotype 5 pigs were also characterized with a slower gonadal growth and body development compared to Genotype 2 pigs. Results from this study suggest that selecting against backfat may delay gonadal development and sexual maturation in pigs. / Dissertation (MSc (Agric) Production Physiology)--University of Pretoria, 2007. / Animal and Wildlife Sciences / unrestricted

Background: Gastroenteritis by rotavirus is responsible for approximately 810 annual deaths/year in children under 5 years in Peru and emerging rotavirus genotypes have led to concerns regarding cross-protection by the vaccines available. Moreover, there are no reports on the molecular-epidemiology of rotavirus diarrhea in Peru Methodology: A total of 131 stool samples were obtained from children under 5 years old hospitalized from January 2010 to December 2012 in the Hospital Regional de Cajamarca, Peru. ELISA and RT-PCR techniques were performed for rotavirus detection. G and P typing of rotavirus-positive samples were obtained by semi-nested multiplex RT-PCR and sequencing was performed to confirm the PCR results. Results: Of the 117 samples available, 18.80% (22/117) tested positive for rotavirus by ELISA and 35.90% (42/117) by RT-PCR. Among the G-genotype identified, G9 in 35.71% (15/42) and G12 in 33.33% (14/42) were the most prevalent. With the most common combination being G12/P6 in 23.81% (10/42). Conclusions: A high prevalence of the G12/P6 genotype was detected. It is know that this genotype is not covered by the current vaccines available. More in depth studies are needed to know the current rotavirus genotypes presents in Peru. / Tesis

Taste impacts the palatability and intake of food, which is influenced by several factors such as cultural and genetic factors. Individual variations in taste perception may be important risk factors for poor eating habits and development of obesity. The differences in taste perception which impact dietary intake may lead to better understanding of obesity development and prevention of diet-related diseases. Obesity is one of the main causes for various health conditions in the United States as well as in the world. Genetic inheritance plays an important role in individual variations to taste and food choices. This study explored associations between two single nucleotide polymorphisms (SNPs, rs713598 and rs10246939) in the TAS2R38 bitter taste receptor gene, dietary intake, and body fat percentage. Five hundred presumably healthy students aged 18-25 years, including 86 (17%) males and 414 (83%) females from Mississippi State University participated in the study. Saliva was collected for genetic analysis, participants completed dietary history questionnaires and body composition was measured using bioelectrical impedance analysis. All statistical analysis of data was conducted using SPSS software to examine associations between SNPs, food intake, and percentage of body fat. Our results did not show a significant association between the SNPs; rs713598 and rs10246939 in the TAS2R38 bitter taste receptor gene and dietary intake of vegetables and fruits as well as percentage of body fat in this group of participants. However, alcohol and caffeine intakes were significantly different between genotypes in rs713598; p< 0.01, p< 0.05, respectively.

Hybridization refers to mating between species or between genetically differentiated populations of the same species. Although hybrid offspring may exhibit sterility and/or inviability, hybridization can generate novel genotypic and phenotypic diversities, leading to the origin of new traits and new species, the expansion into ecological niches outside of the parental range (e.g., host range), and altered virulence properties in pathogens. However, the relationship between parental genetic divergence and hybrid performance remains largely unknown. The human pathogenic Cryptococcus (HPC) is an ideal model to study the impacts of parental divergence on the genetic and phenotypic consequences of hybridization. HPC consists of a group of divergent lineages with various degrees of interfertility. These yeasts are the etiologic agents of cryptococcosis, a potentially lethal disease in humans and animals. In this thesis, I examined the effects of parental divergence on cryptococcal hybrids from multiple aspects. I conducted genetic crosses between different lineages to evaluate the mating success and the germination of sexual spores (i.e., basidiospores) under various environmental conditions. Then, I investigated the genotypic and phenotypic diversities among the hybrids under different environmental conditions. Furthermore, I examined the genome stability of diploid inter-lineage hybrids through laboratory experimental evolution and the effect of antifungal drug stress on the loss of heterozygosity (LOH) in these hybrids. We found that parental genetic divergence plays an important role in genotypic and phenotypic diversities among hybrid progeny in HPC. However, our results indicate that parental genetic di-vergence alone can’t explain most of the observed variations. Instead, genetic divergence along with specific parental strains, environmental factors, and their interactions all contributed to hybridization success and to hybrid genotypic and phenotypic variations. My findings will broaden the current understanding of the phenotypic and genotypic consequences of hybridization and explore the connection between genetic architecture and hybrid speciation in the human pathogenic Cryptococcus. / Thesis / Doctor of Science (PhD) / The role of hybridization in evolution can vary widely, giving rise to hybrid vigor and hybrid weakness. Hybridization plays an important role in plants and animals, especially crops, with advantages of increased yield and quality of products. However, the emergence of hybrid vigor in pathogens with increased virulence is an increasing threat to plant, animal, and human healths. My PhD thesis aimed at understanding the effects of parental divergence on hybridization and hybrids in the human pathogenic Cryptococcus. Here, I investigated basidiospore germination rate and hybrid progeny genotypes and phenotypes from diverse genetic crosses in this group of pathogens. My findings contribute towards understanding cryptococcal hybrids and establishing treatment plans against infections by these hybrids.

Temperature is a major abiotic stress limiting plant growth. Thermotolerance evaluation during germination and early growth may help identify adaptable genotypes of new crops. Two studies were conducted to evaluate temperature effects on 12 Brassica carinata genotypes during germination and early growth. During germination, genotype AX17004 was both the most cold- and heat-tolerant. During early-season growth (35 d after seeding), there were temperature and genotype effects on shoot, root, and physiological components. Cumulative low- and high-temperature response indices, and cumulative root and shoot response indices were related, indicating the importance of these traits. Genotype AX17006 was identified as heat tolerant, and AX17009 as cold tolerant during early-season growth. When genotypes were grouped according to breed types, hybrids generally had better responses than the inbred lines, and double haploids and the check responses were intermediate. These studies provided rapid results that will reduce the number of genotypes assessed in field studies.

La gastroenteritis ocasionada por rotavirus se encuentra entre las principales causas de morbilidad y mortalidad infantil. En el Perú rotavirus representa el 30% de las muertes por diarrea y 4% del total de defunciones, afectando principalmente a niños menores de 5 años. Desde el año 2009 el Perú implementó la vacuna contra rotavirus en su programa de vacunación universal, sin embargo, debido a la aparición de genotipos emergentes, es importante monitorear la diversidad del virus para evaluar los posibles efectos sobre la eficacia de la vacuna. Pese a esto, existen escasos reportes de la epidemiología de rotavirus en nuestro país. El objetivo del presente estudio es determinar la presencia y los genotipos G y P de Rotavirus en muestras provenientes de niños con gastroenteritis aguda atendidos en un hospital de Lima entre Octubre del 2013 y Octubre del 2014. Como metodología se utilizó el RT-PCR en tiempo real (qRT- PCR) para la detección del rotavirus y el RT PCR convencional para la genotipificación de las muestras positivas, asimismo se secuenciaron algunas muestras para determinar los genotipos finales. De las 448 muestras analizadas en el estudio, 45 (10%) tuvieron resultado positivo para rotavirus. Entre los genotipos identificados, G12 (40.9%), G2 (25%) y P[8] (77.3%) fueron los más frecuentes y la combinación G/P más dominante fue G12P[8] (54.5%). Los resultados evidenciaron una alta prevalencia de G12P[8]entre los casos positivos, genotipo no reportado previamente en el Perú. Se recomienda realizar nuevos estudios para evaluar las variaciones en la diversidad de rotavirus circulantes en el Perú y los posibles efectos ante la presión selectiva de la vacuna.Rotavirus gastroenteritis is one of the leading causes of morbidity and mortality in children. In Peru rotavirus represents 30% of deaths due to diarrhea and is responsible of 4% of the total deaths, affecting mostly children under 5 years. Peru implemented the rotavirus vaccine in its universal vaccination program since 2009. Due to the appearance of emerging genotypes, it is important to evaluate the diversity of the virus in order to determine possible effects on vaccine efficacy. However there are few reports on the molecular epidemiology of rotavirus in our country. The aim of this study was to determine the presence and Rotavirus G and P genotypes in samples from children with acute gastroenteritis attended in a hospital in Lima between October 2013 and October 2014. The method usedfor the detection of rotavirus was real time RT-PCR (qPCR) and conventional RT-PCR to determine the genotype of positive samples. Additionally, sequencing was used to confirm the final genotype of some samples. Of the 448 samples analyzed, 45 (10%) were positive for rotavirus.G12 (40.9%), G2 (25%) and P[8] (77.3%) were the most frequent genotypes and the most prevalent G/P combination was G12P[8] (54.5%). The results showed a high prevalence of G12P[8] among the positive cases. G12P[8] has not been previously reported in Peru. We recommend more in deep studies to identify the diversity of circulating genotypes in Peru.

Os rotavírus, membros da família Reoviridae, são uma importante causa de diarreia em mamíferos e aves. São partículas icosaédricas não envelopadas e seu genoma é formado por 11 segmentos de RNA fita dupla que codificam seis proteínas estruturais e seis proteínas não estruturais. O objetivo deste estudo foi caracterizar os genes codificadores das proteínas não estruturais (NSP1-5) e estruturais (VP1-4, VP6-7) dos rotavírus do grupo A em aves de criações comerciais (corte, postura, matrizeiros e avozeiros) localizadas em 12 estados brasileiros, seguido de análises de recombinação e pressão de seleção das amostras definidas. Um total de 226 amostras fecais foram triadas através de reações de RT-PCR tendo como alvo a amplificação da VP6 e NSP5. A frequência de ocorrência, baseada em cada uma destas provas, variou de 9,7% a 18,14%, respectivamente. Em seguida, 10 das amostras positivas foram processadas com primers específicos visando a amplificação dos demais genes, seguido do sequenciamento nucleotídico e filogenia baseada no método de maximum likelihood, tendo como modelos de substituição GTR (NSP1-3, VP1-3, VP4, VP6, VP7) e HKY (NSP4, NSP5) e 1.000 repetições de bootstrap. Foram definidas sequências parciais para os genes codificadores da VP1-4, VP6-7 e NSP1-4 e sequências completas para NSP5. As respectivas árvores demonstraram que as dez amostras definidas se agruparam em clados aviários previamente descritos. Duas constelações genotípicas foram caracterizadas: G19-P[31]-I11-R6-C6-M7-A16-N6-T8-E10-H8 e G19-P[31]-I4-R4-C4-M4-A16-N4-T4-E4-H4. Estes genotipos são tipicamente encontrados em aves, mas quando analisados em conjunto, esta é a primeira descrição destas constelações. Eventos de recombinação foram observados nos genes NSP2, VP1, VP3 e VP7. Pelo menos um códon com pressão de seleção positiva foi encontrado nos genes codificadores das proteínas NSP1, VP2 e VP3. Este estudo propicia um melhor entendimento acerca da epidemiologia e diversidade viral circulante nas criações aviárias brasileiras, servindo de base para o estabelecimento de medidas profiláticas mais eficazes. / Rotaviruses are members of the Reoviridae family and they are a common cause of acute diarrhea in several mammalian and avian species. They are non-enveloped icosahedral particles and its genome comprises 11 segments of double-stranded RNA, which encodes six structural proteins (VP1-4, VP6-7) and six nonstructural proteins (NSP1-6). The objective of this study was to characterize the RVA nonstructural and structural proteins coding genes (NSP1-NSP5, VP1-VP4, VP6 and VP7) from fecal samples from avian farms (broiler breeders, poultry, laying hens, and grandparents) raised in Brazilian commercial farms from 12 states, followed by recombination and selection pressure analysis from samples defined here. A total of 226 fecal samples were screened using a RT-PCR technique targeting the amplification of the VP6 and NSP5. The frequency of occurrence, using these techniques, ranging from 9.7% to 18,14%, respectively; and from these, ten samples were further processed with specific primers to amplify the remaining genes, followed by respective nucleotide sequencing of the amplicons and phylogeny based on method maximum likelihood, as substitutions models GTR (NSP1-3, VP1-3, VP4, VP6, VP7) and HKY (NSP4, NSP5) and 1.000 bootstrap repetitions. Partial nucleotide sequences of VP1-4, VP6-7, and NSP1-4, and complete from NSP5, were obtained in this study. The phylogenetic trees depicted that the ten Brazilian rotavirus strains segregated with previous avian RVA described elsewhere. Two avian genotype constellations have been characterized here: G19-P[31]-I11-R6-C6-M7-A16-N6-T8-E10-H8, and G19-P[31]-I4-R4-C4-M4-A16-N4-T4-E4-H4. These genotypes are typically found in avian species, although when analyzed together, this is the first report of such constellations. Recombination events were observed in NSP2, VP1, VP3, and VP7 coding genes. At least on positive selected site was observed in NSP1, VP2, and VP3 genes. This study provides a better understanding of rotavirus epidemiology, by the definition of genetic variability of circulating strains.

As enzimas CYP450 são as principais enzimas metabolizadoras de fármacos. Elas são codificadas por genes que apresentam polimorfismos gênicos que lhes confere características fenotípicas diversas. Estes fenótipos são: Metabolizadores Lentos ou PM, Metabolizadores Normais ou EM, Metabolizadores Intermediários ou IM e Metabolizadores Ultra-rápidos ou UM. A Farmacogenética é a ciência que estuda estas variações gênicas e sua relação com a resposta terapêutica no organismo. Entre as enzimas CYP450 se encontram as enzimas CYP2D6, que são responsáveis pelo metabolismo de 25% dos fármacos clinicamente prescritos. O objetivo principal deste estudo foi a identificação dos polimorfismos mais importantes deste gene: CYP2D6*1, CYP2D6*2, CYP2D6*3, CYP2D6*4, CYP2D6*5, CYP2D6*6, CYP2D6*10, CYP2D6*17 e CYP2D6*41 pelos métodos de Genotipagem (PCR Tetra Primer e Seqüenciamento) e Fenotipagem (analisado pelo Índice Metabólico) em 75 voluntários sadios da região de Campinas. Para a caracterização da Fenotipagem foi usada a substância teste Dextrometorfano (DM). Esta foi monitorada por espectrometria de massa mediante a determinação da concentração do seu principal metabólito o Dextrorfano (DX), que foi extraída das amostras de urina. Os resultados foram comparados entre estas duas metodologias e apresentaram alta correlação. Os resultados obtidos são a identificação das freqüências dos alelos *1, *3 e *4 pelo método PCR Tetra Primer (30.66%, 1.3% e 14%, respectivamente). O método de seqüenciamento detectou também outros alelos que não foram detectados pela PCR Tetra Primer. A avaliação do número de cópias do gene CYP2D6 também foi avaliada, detectando em um voluntário 3 cópias do gene CYP2D6, característica de metabolizadores Ultra-rápidos. Podemos afirmar que os métodos usados forneceram perfis dos polimorfismos de maneira rápida e prática. / The CYP450 enzymes are the major drug metabolizing enzymes. They are encoded by genes that show genetic polymorphisms which gives them several phenotypic characteristics. These phenotypes are Poor Metabolizers or PM, or Extensive Metabolizers or EM, Intermediate Metabolizers or IM, and finally, Ultra-rapid Metabolizers or UM. Pharmacogenetics is the science that studies these genetic variations and its relationship to therapeutic response in the body. One of CYP450 enzymes is CYP2D6 enzyme, which are responsible for the metabolism of 25% of clinically prescribed drugs. The main objective of this study was to identify the most important polymorphisms of this gene: CYP2D6 * 1, CYP2D6 * 2, CYP2D6 * 3, CYP2D6 * 4, CYP2D6 * 5, CYP2D6 * 6, CYP2D6 * 10, CYP2D6 * 17 and CYP2D6 * 41 by genotyping methods (PCR Tetra Primer and Sequencing) and phenotyping (by metabolic rate monitoring) in 75 healthy volunteers in Campinas region.To characterize the phenotyping was used to test substance Dextromethorphan (DM). This was monitored by mass spectrometry by determining the concentration of its major metabolite the Dextrorphan (DX), which was extracted from urine samples. The results were compared between these two methods and showed high correlation. We can obtain the identification of allelic frequencies of alleles * 1, * 3 and * 4 by Tetra Primer PCR (30.66%, 1.3% and 14% respectively). The sequencing method has also detected other alleles that were not detected by PCR Tetra Primer. The assessment of the number of copies of the CYP2D6 gene was also assessed. This method detected a volunteer which carrying three copies of CYP2D6 gene, characteristic of Ultra-rapid metabolizers. We can say that the methods used in this study provide polymorphism profiles quickly and conveniently.

Os sistemas de grupos sanguíneos eritrocitários e plaquetários têm grande importância na medicina transfusional. Os serviços de hemoterapia têm investido cada vez mais em protocolos profiláticos à aloimunização contra antígenos eritrocitários, sendo o surgimento das plataformas de genotipagem em larga escala um avanço muito importante nesse âmbito. Este estudo tem por objetivo a padronização e a validação da plataforma OpenArray, por meio da técnica de microarranjos líquidos para genotipagem de antígenos eritrocitários e plaquetários em larga escala para futura implementação na rotina de doadores de sangue. Tal genotipagem permitirá a análise da frequência genotípica encontrada nos doadores de sangue da Fundação Pró-sangue/Hemocentro de São Paulo. Métodos: Foram analisadas 400 amostras de sangue total coletadas de doadores de sangue entre outubro e novembro de 2011. A genotipagem dos polimorfismos de troca de um nucleotídeo (Single nucleotide polymorphism - SNPs) que codifica os principais antígenos eritrocitários e plaquetários de relevância transfusional foi realizada utilizando a tecnologia OpenArray para as 400 amostras. Dessas, 242 também foram analisadas pela plataforma de genotipagem BLOODchip, em larga escala. Procedeu-se à comparação entre as técnicas em termos de acurácia, reprodutibilidade, número de resultados incorretos, número de resultados não amplificados ou indeterminados, possibilidade de customização dos ensaios, tempo total de duração da reação e número de amostras processadas por bateria. Foi também calculada a frequência genotípica dos SNPs e feita a comparação com dados prévios de literatura. Resultados e discussão: as amostras que foram testadas em ambas as plataformas apresentaram acurácia de 99,9% pela técnica OpenArray e 100% pela técnica BLOODchip, sendo os resultados discrepantes analisados por sequenciamento direto (técnica Sanger), confirmando os resultados obtidos pela genotipagem realizada no BLOODchip. Além disso, a técnica OpenArray apresentou maior número de resultados não amplificados ou indeterminados (no call), o que representa uma grande desvantagem do método, em decorrência da perda de insumos. Entretanto, a técnica OpenArray apresentou outras vantagens em relação ao BLOODchip, como a possibilidade de customização do ensaio, menor tempo para processamento das amostras, considerando a possibilidade de automatização total do processo e número de amostras testadas por bateria superior ao método comparativo. Os resultados da frequência alélica e genotípica dos polimorfismos analisados pelo método OpenArray foram comparados com as frequências encontradas na literatura, observando-se prevalência de doadores com perfil genotípico mais próximo da população caucasoide, porém, com a presença de alelos encontrados na população negra. Entretanto, esse perfil genotípico dos doadores predispõe à aloimunização de doentes falciformes, com perfil fenotípico negroide, reiterando a necessidade de transfusões com fenótipo compatível como profilaxia à aloimunização. / The erythrocyte and platelets blood groups are extremely important for transfusional medicine. Hemotherapy services have increasingly invested in prophylactic protocols for alloimmunization against erythrocyte antigens and the emergence of high-throuput genotyping platforms are a very prominent advance in this area. The aim of this study was to validate and to standardize the OpenArray platform, which is based on the microarray technolgy for the erythrocyte antigens genotyping on large scale, to further implementation in blood bank routine. This tecnique also allowed to asses the genotype frequencies in blood donors from Fundação Pró-sangue/Hemocentro de São Paulo. Method: We examined 400 blood donor samples collected from October to November 2011. The SNPs were detected using OpenArray technology. From the 400 blood samples, 272 were also tested using BLOODchip platform, to compare the results between both tecniques and evaluate the accuracy, reproducibility, number of incorrect results, failed samples, possibility of assays customization, duration of procedures, and number of samples that can be processed per batch. The genotype frequency of SNPs was also assesed and the findings were compared to previous studies. Results and Discussion: the OpenArray method showed accuracy of 99.9% and the BLOODchip of 100%. The inconsistent results were confirmed by Sanger Sequencing which showed that BLOODchip analysis was accurate. Besides, the OpenArray method showed a higher number of non-amplification or failed results (no call), which may be a major disavantage, due to reagent loss. However, the OpenArray platform showed other advantages when compared to BLOODchip such as the possibility of assay customization and full automation of the process, it was less time-consuming and allowed a higher number of samples per batch. The genotypic and allelic frequencies of each SNP tested in the blood donor population were calculated by the OpenArray method and the results were compared to reports from previous studies. We observed a prevalence of genotypic profiles closest to the Caucasian population, however, there was the presence of alleles found in the black population as well. This genotypic profile donors predispose to alloimmunization of sickle cell patients, with negroid phenotypic profile, reiterating the need for compatible phenotype transfusions and prophylaxis to alloimmunization.