Footrot is a contagious hoof disease of ruminants. It is endemic in New Zealand and throughout sheep and goat farming regions of the world. The disease results from a mixed bacterial infection, but the essential agent is Dichelobacter nodosus, a Gram-negative, anaerobic bacterium that possesses type-IV fimbriae on its surface. Genetic variation in the fimbriae of D. nodosus was investigated in this study. Using the polymerase chain reaction (PCR), the variable region of the gene encoding the fimbrial subunit (fimA) was amplified from bacterial DNA extracted from footrot lesions. Different fimA amplimers were differentiated by single-strand conformation polymorphism (SSCP) analysis. In conjunction with DNA sequencing, 15 unique sequences of D. nodosus fimA were obtained from 14 footrot samples taken from 6 farming regions throughout New Zealand. When these sequences were compared to fimA of known serogroups, it revealed that there were at least 15 D. nodosus strains, representing 8 serogroups, present on New Zealand farms. The predominant serogroup was B which contained 6 strains, followed by serogroups F, H and G. No strains from serogroups D and I were detected in this investigation. Twelve out of the 15 New Zealand D. nodosus strains had fimbriae different to those previously reported and the presence of multiple strains on a single hoof was common (86% of samples). The fimA sequences from the 12 D. nodosus strains incorporated into the footrot vaccine currently available in New Zealand were determined. A primer set targeting the relatively conserved fimA regions and based on the published sequence of serogroup M Nepalese isolates (designated M-Nep), failed to amplify fimA from the vaccine serotype M strain (designated as M-SPAHL). When the downstream primer was substituted with a primer that was specific for other serogroups of D. nodosus, the fimA gene was successfully amplified. Cloning followed by DNA sequencing, revealed that M-SPAHL fimA was different to M-Nep fimA. The predicted amino acid sequence of M-SPAHL fimA did not show homology to any known serogroups or serotypes. The most similar sequence was from serotype F1, and not M-Nep. The sequence difference between M-SPAHL and M-Nep was larger than that expected within a serogroup. The consequences of serological relatedness and sequence dissimilarity are discussed. Only eight of the 15 New Zealand field strains had fimbriae identical to those of the vaccine strains, while the remaining seven strains possessed different fimbriae. In addition, the vaccine contained two more D. nodosus strains, representing two sera groups, that were not found on the New Zealand farms investigated in this study. This may, to some extent, explain why the current footrot vaccine is at times less efficient in New Zealand. Another 17 footrot samples were screened for new or additional D. nodosus strains. Two PCR amplimers (designated X and Y) derived from footrot samples generated SSCP patterns different to those of previously identified strains. DNA sequencing revealed that these two fragments possessed novel sequences. The upstream of X (nt 1-183) was identical to serotype M1 while its downstream (nt 223-414) was identical to serotype F1; the upstream of Y (nt 1-116) was identical to serotype E1 whereas its downstream (nt 148-423) was identical to serotype F1. A 14-mer sequence consisting of two partially overlapping Chi-like sequences, 5'-GCTGGTGCTGGTGA-3', was also found in these fragments. Two primer sets with the downstream primer specific for serotype Fl and the upstream primer specific for serotype M1 or E1, produced PCR products of the expected sizes from the footrot samples from which fragments X and Y were isolated, respectively. These primer sets did not appear to amplify artificially mixed genomic DNA from serotypes M1 and F1 or E1 and F1. However, when the reactions were re-amplified, PCR recombination artefacts were observed, suggesting that PCR recombination does occur, but at a low frequency. It therefore seems more likely that fragments X and Y reflect genuine fimA sequences of D. nodosus which have resulted from in vivo DNA recombination, than from a PCR recombination artifact. The genetic capability for recombination at the fimbrial subunit locus may therefore endow D. nodosus with the ability to alter its antigenic appearance. D. nodosus strains present in footrot lesions can be genotyped using a PCR-SSCP/sequencing technique. However, this typing technique requires cloning and screening of D. nodosus fimA sequences, which is both laborious and costly. A rapid molecular typing system for D. nodosus was therefore developed in this study. A close examination of available D. nodosus fimA sequences revealed regions that appear to be specific for serogroups and serotypes. These regions were used to design a panel of sequence-specific oligonucleotide probes (SSOPs), and a rapid and accurate D. nodosus typing system using PCR and reverse dot-blot hybridisation (PCR/oligotyping) was subsequently developed. The variable region of D. nodosus fimA, amplified and labelled with digoxigenin (DIG) in a single multiplex PCR amplification, was hybridised to a panel of group- and type-specific, poly-dT tailed oligonucleotides that were immobilised on a nylon membrane strip. A mixture of positive control poly-dT tailed oligonucleotides was also included on the membrane. After hybridisation the membrane was washed to a defined specificity, and DIG-labelled fragments that had hybridised were detected. The specificity of the oligonucleotides was verified by the lack of cross-reactivity with D. nodosus fimA sequences that had a single base difference. DNA from 14 footrot samples previously genotyped by PCR-SSCP/sequencing, was assayed using the PCR/oligotyping technique. All types of D. nodosus which had been detected previously with a PCR-SSCP/sequencing method were detected by this procedure. However, for three of the 14 footrot samples, PCR/oligotyping detected additional types of D. nodosus. Further PCR amplification using type-specific primers, confirmed that these types were present in the original footrot samples. These results indicate that PCR/oligotyping is a specific, accurate, and useful tool for typing footrot samples. In combination with a rapid DNA extraction protocol, D. nodosus present in a footrot sample can be accurately genotyped in less than two days. Individual animals from the same farm, or the same paddock, were often infected by different strains of D. nodosus. This suggests a host role in mediating footrot infection, or that the interaction between the pathogen and the host is important. In order to better understand the interaction between the bacterium and the host, two polymorphic ovine class II MHC genes DQA1 and DQA2, which have been previously shown to be important in footrot infection, were also investigated in this study. PCR-SSCP/sequencing analysis of the DQA1 locus revealed ten unique ovine DQA1 sequences, with five of them being newly identified. This increases the number of known ovine DQA1 alleles from 8 to 13 (including a null allele), implying a high level of polymorphism at the ovine DQA1 locus. D. nodosus present on 20 footrot infected sheep from the same flock were genotyped, together with the ovine DQA1 and DQA2 genotypes of their hosts. Preliminary results showed that sheep with the same DQA1 and DQA2 genotypes tended to be infected by similar types of D. nodosus. Different types of D. nodosus were generally found on sheep with different genotypes at either the DQA1 or the DQA2 locus. This suggests the diversity in D. nodosus infection may be associated with the heterogeneity in the host MHC. However, as only a small number of animals from the same sire were analysed, further investigation is needed to gain a better understanding of the interaction between D. nodosus and the host MHC.

Made available in DSpace on 2015-12-06T22:54:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2009 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Neste estudo, 151 isolados brasileiros de Sporothrix schenckii foram caracterizados quanto as suas características fenotípicas e genotípicas. Quanto às características fenotípicas, a maioria dos isolados apresentou conídios ovais, sendo que os de origem animal são maiores que os de origem humana (animal: 2,96 ± 1,07 versus forma linfocutânea: 2,37 ± 0,43; forma fixa: 2,33 ± 0,53). Os isolados provenientes da região Norte do Brasil são os mais termotolerantes e os isolados do Nordeste são os mais termossensíveis. Os isolados de origem animal são mais tolerantes à temperatura quando comparados com isolados obtidos de pacientes. Os isolados brasileiros apresentaram grande variabilidade genética quando avaliados por RAPD. Por Southern Blot, utilizando a enzima APA I para o estudo do polimorfismo das regiões ITS, três genótipos foram obtidos. Não houve correlação entre o perfil obtido por RAPD ou Southern Blot com a origem clínica ou geográfica dos isolados. A secreção de proteínas/glicoproteínas é variável entre os isolados, sofrendo influência direta do meio de cultura. Os isolados brasileiros avaliados são produtores das enzimas DNAse e urease, 15,78% produzem gelatinase, 26,31% produzem proteinase e 21,05% produzem caseinase. Todos os isolados avaliados toleram pressão osmótica de 16,6% de glicerol, 89,47% toleram 20% e todos são inibidos a 23% e 28,5 % de glicerol. Todos os isolados avaliados toleram pressão salina de 6% e 8%; 42,10% toleram pressão de 9% e 10% de sal e todos são inibidos a 12% de sal. Todos os isolados avaliados crescem na faixa de pH de 2,2 a 12,5. A patogenia e virulência são variáveis entre os isolados, podendo estar relacionada ao genótipo do isolado. Diferentes proteínas são reconhecidas pelos soros dos camundongos infectados, sendo a molécula de 60 kDa, a mais reconhecida neste sistema. Em relação à esporotricose humana, as moléculas de 70 kDa e 38 kDa são comumente reconhecidas por soros de pacientes com esporotricose fixa e linfocutânea. O antígeno bruto do isolado Ss 118 e a fração SsCBF são eficientes para diagnóstico da esporotricose felina através da técnica de ELISA. A fração SsCBF é capaz de elicitar resposta imune do tipo HTT em camundongos experimentalmente infectados. Em geral, os resultados indicam que os isolados brasileiros são heterogêneos, apresentando características genéticas e fenotípicas individuais, independentes de sua origem. Somente a área conidial e a tolerância a temperatura foram características fenotípicas relacionadas à origem. / In this study, we analyzed 151 Brazilian S. schenckii isolates by phenotyping and genotyping aspects. About phenotyping aspects, most of isolates showed oval conidia and the mean conidial area of S. schenckii animal isolates was greater than clinical isolates (animal: 2.96 ± 1.07 versus lymphocutaneous form: 2.37 ± 0.43; fixed form: 2.33 ± 0.53). Isolates from the Northeast region exhibited the lowest thermotolerance and the Northern isolates exhibited the highest thermotolerance. Animal isolates are better thermotolerants than clinical isolates. The Brazilian isolates showed great genetic diversity when analyzed by RAPD. By Southern blot using the endonuclease APA I to study polymorphism of ITS regions, three genotypes were obtained. There was no association between RAPD and Southern profiles with clinical forms origin or geographic origin. The protein/glycoprotein secretion was variable among isolates and was directly influenced by the medium composition. All Brazilian isolates studied produced DNAse and urease exoenzymes, 15.78% produced gelatinase, 26.31% produced proteinase and 21.05% produced caseinase. All analyzed isolates grew at 16,6% of glycerol, 89,47% grew at 20% of glycerol and all of them were inhibited at 23% and 28,5 % of glycerol. All analyzed isolates grew at 6% and 8% of NaCl, 42.10% grew at 9% and 10% of NaCl and all of them were inhibited at 12% of NaCl. All of them were able to grow at pH 2.2 to 12.5. The pathogenicity and virulence were variable among isolates and it can be related to the genotype. Different proteins were recognized by sera of infected mice and the 60 kDa molecule was the most one recognized in murine system. In relation to human sporotrichosis, 70 kDa and 38 kDa molecules were common recognized by sera from patients with fixed and lymphocutaneous sporotrichosis. The crude exoantigen of Ss 118 isolate and the SsCBF fraction are able to be used in the diagnosis of feline sporotrichosis by ELISA. The SsCBF fraction is able to elicit DTH immune response in infected mice. In general, our results indicate that Brazilian isolates are heterogeneous, which present individual phenotyping and genotyping characteristics, independent of origin. Only the conidial area and thermotolerance were related to origin. / BV UNIFESP: Teses e dissertações

Made available in DSpace on 2016-12-23T13:49:01Z (GMT). No. of bitstreams: 1 Mariana Penha de Nadai Sartori.pdf: 1901445 bytes, checksum: b8d41cb1f38aa4dfcc9f35c781bbff06 (MD5) Previous issue date: 2012-03-09 / Human papillomavirus (HPV) is the most common sexually transmitted virus in the world, being a cause of genital warts (Low-Risk HPV) and cervical cancer (High-Risk HPV). The virus is found in sexually active adults and adolescents, however, it has been detected in girls victims of sexual violence or not. The worldwide incidence of HPV is increasing in girls and viral transmission modes are still not completely defined. Currently, HPV prevention is based on two available vaccines only for the age group 9-26 years old and there are no available vaccines or other prevention methods for patients under nine years old, since it is not considered a risk group by competent health authorities. There are no studies of the Espírito Santo state about the HPV prevalence in girls under 9 years old and therefore their importance is not established. Because of these reasons, the aim of this study was to detect HPV in girls under nine years old, as well as finding possible routes of transmission in order to provide information for the development of preventive practices for this risk group. A total of 43 samples extracted from girls under 9 years old were analyzed using PCR, RFLP and DNA sequencing methods for viral detection and typing. Human papillomavirus was detected in 13.9% of patients, mostly low risk genotypes. Clinical and personal evaluation suggested that girls were infected by horizontal transmission and via fomites. Due to our findings, we propose that HPV infection prevention through vaccination should be extended to girls under 9 years old, especially in specific high risk populations / O papilomavírus humano (HPV) é considerado a doença sexualmente transmissível prevalente mundialmente, sendo responsável por causar verrugas genitais (HPV de baixo risco) e o câncer de colo de útero (HPV de alto risco). O vírus é encontrado em adultos e adolescentes sexualmente ativos, entretanto, tem sido também detectado em meninas vítimas de violência sexual ou não. Em todo o mundo, a incidência do HPV vem aumentando em meninas e as formas de transmissão viral ainda não são totalmente conhecidas. Atualmente, a prevenção do HPV é baseada em dois tipos de vacinas, disponíveis somente para um grupo de indivíduos na idade entre 9 26 anos, não estando disponível nenhuma vacina ou outra forma de prevenção para pacientes abaixo de nove anos, uma vez que este grupo não é considerado um grupo de risco pelos órgãos de saúde pública. Não há estudos no estado do Espírito Santo relacionando a prevalência do HPV em meninas abaixo de nove anos de idade, portanto, a sua importância não é conhecida. Devido a estas razões, o objetivo da pesquisa foi detectar o HPV em meninas na faixa etária até nove anos, bem como determinar as possíveis vias de transmissão viral com o intuito de fornecer informações para o desenvolvimento de atitudes preventivas para esse grupo de risco. O DNA viral foi extraído de um total de 43 amostras de meninas na faixa etária estabelecida, e a análise molecular foi realizada através dos métodos de PCR, RFLP e sequenciamento para detecção e genotipagem viral. O papilomavírus humano foi detectado em 13,9% das pacientes, com prevalência dos genótipos virais de baixo risco para o desenvolvimento de neoplasias. A avaliação clínica e epidemiológica propõe que as meninas são infectadas por transmissão horizontal e via fômites. De acordo com nossos resultados, propomos que a prevenção da infecção do HPV através da vacinação deve compreender meninas com idade abaixo de 9 anos, especialmente nas populações de alto risco

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O Cryptosporidium é um parasito coccídeo reconhecido por causar diarréia em humanos e animais em todo o mundo. O gênero compreende pelo menos 20 espécies confirmadas, sendo o C. hominis e C. parvum as principais espécies causadoras de criptosporidiose em humanos. Ferramentas moleculares têm sido desenvolvidas para detectar e diferenciar espécies/genótipos e subgenótipos de Cryptosporidium. O objetivo do trabalho foi avaliar a heterogeidade molecular de Cryptosporidium sp. obtidos de amostras clínicas provenientes dos municípios do Rio de Janeiro e de Salvador, através da PCR em tempo real e seqüenciamento automático. Foram analisadas 85 amostras, distribuídas em 3 grupos distintos, sendo 45 delas do município do Rio de Janeiro e 40 amostras provenientes de Salvador, Bahia. Todas as amostras foram positivas para Cryptosporidium sp. pelo método de coloração de Kinyoun a frio. O ensaio da PCR em tempo real combinou uma reação multiplex para a detecção do gênero Cryptosporidium e da espécie C. parvum e uma reação simples para a detecção de C. hominis. Na detecção do gênero Cryptosporidium foram utilizados par de primers e uma sonda TaqMan desenhados a partir do alinhamento de seqüências conservadas do gene 18S rRNA de várias espécies de Cryptosporidium disponíveis no GenBank. Para a detecção das espécies C. parvum e C. hominis foram utilizados primers e sondas específicos obtidos a partir de seqüências de cada espécie disponíveis no GenBank. A detecção do gênero Cryptosporidium através da sonda 18S rRNA, na reação duplex, foi visualizada em 63 de 85 amostras totais. Destas, a sonda TaqMan específica para C. parvum detectou 6 amostras e a sonda TaqMan específica para C. hominis detectou 42 amostras. Quinze amostras não puderam ser detectadas pelas sondas C. hominis ou C. parvum. Nos ensaios da PCR para o gene 18S, 31 amostras foram positivas e 27 delas sequenciadas. As análises filogenéticas confirmaram a presença de C. hominis, C. parvum e C. felis nas amostras estudadas. Na análise da topologia da árvore filogenética obtida por Neighbor Joining, observou-se-se que as sequências obtidas neste estudo se agruparam com espécies do gênero Cryptosporidium já descritas, com 99% ou 100% de similaridade. Conclui-se que os dois métodos utilizados são importantes ferramentas para o diagnóstico da criptosporidiose e diferenciação de C. hominis e C. parvum em investigações epidemiológicas, assim como avaliação da heterogeneidade molecular de Cryptosporidium sp / Cryptosporidium is a coccidia parasite known to cause diarrhea in humans and animals worldwide. The genus comprises at least 20 confirmed species, with C. hominis and C.parvum major species that cause cryptosporidiosis in humans. Molecular tools have been developed to detect and differentiate Cryptosporidium at the species/genotypes and subtype levels. The objective of this study was to evaluate the molecular heterogeneity of Cryptosporidium sp. clinical samples obtained from Rio de Janeiro and Salvador (Bahia), through real-time PCR and automatic sequencing. We analyzed 85 samples, distributed in three distinct groups, 45 of them in the city of Rio de Janeiro and 40 samples from Salvador. All samples were positive for Cryptosporidium sp. by modified Kinyoun acid-fast staining technique. The real-time PCR procedure combined a duplex reaction for the detection of Cryptosporidium species and C. parvum and a simple reaction for the detection of C. hominis. The detection of the genus Cryptosporidium have been used a pair of primers and TaqMan probe designed from the alignment of conserved sequences of the 18S rRNA gene of several species of Cryptosporidium available in GenBank. For the detection of species C. parvum and C. hominis were used specific primers and probes derived from sequences of each species available in the GenBank. Detection of Cryptosporidium sp. by 18S rRNA probe, in the duplex reaction, was visualized in 63 of 85 total samples. Of these, the C. parvum TaqMan probe detected 6 samples and the C. hominis TaqMan probe detected 42 samples. Fifteen samples could not be detected by C. hominis or C. parvum probes. In the 18S PCR assays, 31 samples were positive and 27 of them sequenced. Phylogenetic analysis confirmed the presence of C. hominis, C. parvum and C. felis. The analysis of the phylogenetic tree obtained by Neighbor Joining showed that the sequences obtained in this study were grouped with Cryptosporidium species described in the GenBank, with 99% or 100% similarity. Both methods are important tools for the diagnosis of cryptosporidiosis and differentiation of C. hominis and C. parvum in epidemiological investigations, as well as evaluation of Cryptosporidium sp. molecular heterogeneity

Doctor of Philosophy / Genetics - Plant Pathology / Jesse A. Poland / The global population is estimated to reach 9.1 billion by 2050. Together with climate change, insuring food security for this population presents a significant challenge to agriculture. In this context, a large number of breeding objectives must be targeted. The focus of the work presented here is to explore genomic approaches for tapping exotic germplasm for valuable alleles to increased yield, disease resistance and abiotic stress tolerance. The loss of genetic diversity in bread wheat (Triticum aestivum L.) due to bottlenecks during polyploidization, domestication and modern plant breeding can be compensated by introgressing novel exotic germplasm. Here, the potential of genomic selection (GS) for rapid introgression of synthetic derived wheat is evaluated in field trials. Overall, the GS models had moderate predictive ability. However, prediction accuracies were lower than expected likely due to complex and confounding physiological effects. As such, implementation of rapid cycle GS for introgression of exotic alleles is possible but might not perform very well with synthetic derived wheat. Disease resistance is another important trait affecting grain yield. Stem rust (Puccinia graminis f. sp. tritici) has historically caused severe yield loss of wheat worldwide. In a quantitative trait loci (QTL) mapping study with a synthetic-derived mapping population, QTLs for resistance to stem rust races TRTTF and QTHJC were identified on chromosomes 1AS, 2BS, 6AS and 6AL. Some of these genes could be new resistance genes and useful for marker-assisted selection (MAS). In addition to food insecurity through lack of sufficient source of calories, nutrient deficiency is considered the ‘hidden hunger’ and can lead to serious disorders in humans. Through biofortification, essential nutrients are increased in staple crops for improved quality of food and human health. A high-throughput elemental profiling experiment was performed with the same synthetic derived mapping population to study the wheat ionome. Twenty-seven QTL for different elements in wheat shoots and two QTL in roots were identified. Four “hotspots” for nutrient accumulation in the shoots were located on chromosomes 5AL, 5BL, 6DL and 7AL. Overall, exotic germplasm is a valuable source of favorable alleles, but improved breeding methodologies are needed to rapidly utilize this diversity.

Submitted by Vasti Diniz (vastijpa@hotmail.com) on 2017-09-06T12:44:39Z No. of bitstreams: 1 arquivototal.pdf: 1491913 bytes, checksum: 48f18b769a4ddee1245aef6c202388b1 (MD5) / Made available in DSpace on 2017-09-06T12:44:39Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1491913 bytes, checksum: 48f18b769a4ddee1245aef6c202388b1 (MD5) Previous issue date: 2017-02-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Basal Cell Carcinoma (BCC) is a frequent neoplasm in humans and its main etiological factor is exposure to solar radiation. Although genetic and epigenetic changes can activate proto-oncogenes, inactivate tumor suppressor genes and repair mechanism genes, the cell has several mechanisms that contribute to the maintenance of genomic stability. Mutations in repair genes can lead to tumor progression and loss of genome integrity leading to the onset of cancer. Nucleotide excision repair (NER) is an important mechanism primarily used to repair injuries caused by UV. The objective of this study was to evaluate single nucleotide polymorphisms (SNP) of XPA and XPC genes and the risk of developing BCC. One hundred samples of paraffined tissue from patients from the State of Paraíba with histopathological diagnosis of BCC were analyzed for each polymorphism. The results were obtained by a newly developed genotyping method, the Dideoxy Unique Allele Specific - PCR, a method that presents high sensitivity and low cost. Graphpad Prism 6.01 software was used for the statistical analysis and application of Chi-square and Fisher's exact test. The SNP rs535425175 of the XPC gene showed a significant association with the BCC in the analyzed samples (X2 = 14.51 and P <0.005). Whereas the SNPs rs745769173 of the XPA gene and rs761106780 of the XPC gene are in the Hardy-Weinberg equilibrium, not showing any association with the neoplasia. The results suggest that the SNP rs535425175 of the XPC gene may be considered a risk factor associated with the development of BCC. / O Carcinoma Basocelular (CBC) é uma neoplasia frequente em seres humanos e seu principal fator etiológico é a exposição à radiação solar. Embora alterações genéticas e epigenéticas possam ativar proto-oncogenes, inativar genes supressores de tumor e genes do mecanismo de reparo, a célula apresenta vários mecanismos que contribuem para a manutenção da estabilidade genômica. Mutações em genes de reparo podem levar a progressão tumoral e à perda da integridade do genoma levando ao surgimento do câncer. O reparo por excisão de nucleotídeo (NER) é um importante mecanismo utilizado principalmente para reparar lesões causadas por UV. O objetivo deste trabalho foi avaliar polimorfismos de nucleotídeo único (SNP) dos genes XPA e XPC e o risco de desenvolver CBC. Foram analisadas 100 amostras de tecido parafinado de pacientes do Estado da Paraíba com diagnóstico histopatológico de CBC para cada polimorfismo. Os resultados foram obtidos por um método de genotipagem recentemente desenvolvido, o Didesoxi Único Alelo Específico – PCR, método que apresenta alta sensibilidade e de baixo custo. O software Graphpad Prism 6.01 foi utilizado para as análises estatísticas e aplicação de teste Qui-quadrado e Exato de Fisher. O SNP rs535425175 do gene XPC apresentou associação significativa com o CBC nas amostras analisadas (X2=14,51 e P<0,005). Enquanto que os SNP rs745769173 do gene XPA e rs761106780 do gene XPC estão no equilíbrio de Hardy-Weinberg, não apresentando associação com a neoplasia. Os resultados sugerem que o SNP rs535425175 do gene XPC pode ser considerado um fator de risco associado ao desenvolvimento de CBC. Palavras-chaves: Carcinoma Basocelular, Família XP, Reparo por excisão de nucleotídeo, Polimorfismo de nucleotídeo único, Genotipagem. VII

Made available in DSpace on 2015-03-26T13:42:17Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1309318 bytes, checksum: 2bf5ec79170b4c80eee708ade23f9897 (MD5) Previous issue date: 2010-06-11 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The grapevine is a perennial fruit of greater economic importance worldwide. Although not among the world's largest producer, Brazil has been showing a growing development in the industry, and production area has increased markedly with the renovation of the vineyards and new plantings. For the deployment and renewal of vineyards wine growers in Brazil have faced serious problems related to lack of plantlets with the genetic and phytossanitari quality tested by national especialisaded companies. Thus, the lack of a private or official certification program of plants, in order to prevent the spread of propagation material of dubious genetic and sanitary quality, has hindered the development of the national viticulture. The aim of this work was (I) the genetic caracterization (DNA fingerprinting) and the analysis of the genetic diversity of 27 grape varieties cultivated in Minas Gerais, Brazil, using SSR molecular markers; and (II) to identify and map the occurrence of viruses in the grapevine Core collection of EPAMIG Grape and Wine, which are used for production of grafted plants, and analyzing the evolution of viral titer of infected plants along the grape phenological cycle by means of DASELISA serological method. Among the 27 cultivars analyzed, 26 different SSR profiles were detected. In this work were identified 101 alleles in 7 loci analyzed with average of 14.43 alleles per locus, confirming the high power of these markers for genetic polymorphism detection. The expected heterozigosity ranged from 0.832 to 0.9205 with average 0.8873 while the observed heterozigosity ranged from 0.7692 to 1.000 with average of 0.8943. The high number of heterozygous individuals might be due the breeding selection followed by genotype maintenance through vegetative propagation. Grapes are known to be very sensitive to inbreeding depression and the higher performance is mainly observed in heterozygous individuals. The loci with highest polymorphisms content (PIC) were VVS2 (0.92), VVMD27 (0.918) and VrZAG62 (0.918) while the loci with lowest values of PIC were VVMD25 (0.832) and VrZAG79 (0.845). Considering the 7 loci analyzed the PI accumulation was very low (1.27 x 10-12) indicating the high efficiency of these loci for grapevine genotypes differentiation. The dendrogram showed four main groups and they were in agreement with the genetic similarity (pedigree) of these varieties. In the principal coordinate analysis, the group formed for 4 Americans varieties showed the highest level of genetic structuration. Despite no clear division was observed between vinifers and rootstocks groups during the structuration, the results indicates a tendency of clustering among the varieties belonging to the same group. Regarding the study of viruses plants of six canopy varieties ('Syrah', 'Chardonnay', 'Cabernet Sauvignon', 'Merlot', 'Bordo' and 'Niagara Rosada'), four rootstock varieties ('Traviú', '1103 Paulsen, 'I'AC 572' and 'IAC 766') and nine combinations of seedlings produced from grafted plants. Twelve 'Bordo' clones belonging to a clonal selection breeding program based of EPAMIG were also analyzed. The plants were tested for the following viruses: GLRaV-1, GLRaV-2, GLRaV-3, GLRaV-6, GVA, GFkV and GFLV. Bordo clones had a high rate of infection with GLRaV-2, which was detected in 91.66% of samples. The GLRaV-1 were detected in two clones (3 and 17), whereas the GLRaV-3 were also detected in two clones (3 and 07). Analyzing mother plants and seedlings produced from them, from a set of 7 seven virus studied, only GLRaV-3 was detected, being present in Niagara Rosada mother plants and in two rootstocks ( IAC 572 and IAC 766 ). The variation in viral titer along the phenological cycle showed an increase in the concentration of viral particles of the three viruses analyzed (GLRaV-1, GLRaV-2, and GLRaV-3). / A videira é a frutífera perene de maior importância econômica a nível mundial. Apesar de não figurar entre os maiores produtores mundiais, o Brasil vem apresentando um crescente desenvolvimento no setor, e a área de produção tem aumentado acentuadamente com a renovação dos vinhedos e novos plantios. Para a implantação e renovação dos vinhedos, os viticultores brasileiros têm enfrentado sérios problemas relacionados à falta de mudas com qualidade genética e fitossanitária testadas por empresas nacionais especializadas. Assim, a inexistência de um sistema público ou privado de certificação de mudas, com a finalidade de impedir a difusão de material propagativo de qualidade genética e fitossanitária duvidosas, dificulta o desenvolvimento da viticultura nacional. Os objetivos desse trabalho foram (I) a identificação genética ( DNA fingerprinting ) e estudo da diversidade genética de variedades copa e de porta-enxerto de videira cultivados no estado de Minas Gerais, utilizando marcadores moleculares do tipo SSR, para dar suporte ao programa de certificação genética de mudas de videira da EPAMIG; e (II) identificar e mapear a ocorrência de viroses na coleção de plantas matrizes de videira do Núcleo Tecnológico EPAMIG Uva e Vinho, que são utilizadas para produção de mudas enxertadas e analisar a evolução da carga viral de plantas infectadas ao longo do ciclo produtivo das plantas. Os sete marcadores microssatélites utilizados permitiram a diferenciação de 26 genótipos entre as 27 variedades estudadas. Foram identificados 101 alelos com uma média de 14,43 alelos por locus, confirmando a eficiência dos marcadores para a detecção de polimorfismos genéticos. A heterozigosidade esperada variou de 0,832 a 0,9205, com média 0,8873, enquanto a heterozigosidade observada variou de 0,7692 a 1,000, com média 0,8943. O excesso de heterozigotos se explica pelo fato da seleção de indivíduos superiores para qualidade e produtividade em videira ter sido realizada precocemente durante o processo de melhoramento da cultura e perpetuado pela multiplicação vegetativa. Além disso, a videira é sensível à depressão endogâmica sendo as melhores performances obtidas com indivíduos heterozigotos. Os loci que apresentaram os maiores valores de conteúdo de informação polimórfica (PIC) foram VVS2 (0,92), VVMD27 (0,918) e VrZAG62 (0,918). Já os que apresentaram os menores valores foram o VVMD25 (0,832) e o VrZAG79 (0,845). Considerando os 7 loci analisados, a probabilidade de identidade atingiu um valor relativamente baixo (1,27 x 10-12), demonstrando uma grande eficiência dos loci utilizados para a diferenciação das variedades. A análise agrupou corretamente a maioria das variedades de acordo com o pedigree. Na análise das coordenadas principais, o grupo formado pelas 4 variedades copa americanas apresentou o maior nível de estruturação. Apesar de não ter ocorrido uma clara estruturação em relação aos grupos das viníferas e dos porta-enxertos, foi possível notar uma tendência de agrupamento das variedades pertencentes ao mesmo grupo. Em relação ao estudo das viroses, foram analisadas plantas matrizes de seis variedades copa, ( Syrah , Chardonnay , Cabernet Sauvignon , Merlot , Bordô e Niágara Rosada ), quatro variedades de porta-enxertos ( Traviú , 1103 Paulsen , I AC 572 e IAC 766 ) e 9 combinações de mudas enxertadas produzidas a partir das plantas matrizes. Foram também analisados 12 clones da variedade Bordô pertencentes a um programa de seleção clonal desenvolvido na EPAMIG. As plantas foram testadas para os seguintes vírus: GLRaV-1, GLRaV-2, GLRaV-3, GLRaV-6, GVA, GFkV e GFLV. Os clones Bordô apresentaram uma alta taxa de infecção pelo GLRaV-2, que foi detectado em 91,66 % das amostras avaliadas. O GLRaV-1 foi detectado nos clones 3 e 17 e o GLRaV-3 nos clones 3 e 7. Analisando plantas matrizes e as mudas produzidas a partir delas, apenas o GLRaV-3 foi detectado, estando presente nas matrizes de Niágara Rosada, nos portaenxertos IAC 572 e IAC 766, e nas mudas Niágara Rosada/IAC 766 e Niágara Rosada/IAC 572. A análise da variação da carga viral ao longo do ciclo vegetativo evidenciou um aumento da concentração de partículas virais ao longo do ciclo vegetativo da videira para os 3 vírus analisados (GLRaV-1, GLRaV-2 e GLRaV-3).

Made available in DSpace on 2015-03-26T13:42:19Z (GMT). No. of bitstreams: 1 texto completo.pdf: 525415 bytes, checksum: 988d62e0043ef473ad492ea9ce941239 (MD5) Previous issue date: 2006-09-29 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / In the genetic mapping in outbred populations not always it is possible to determine the linkage phase of the alleles. Thus, heterozygous individuals are discarded from these analyses due to the lack of information, once it is not possible, through their genotype, to distinguish the origin of their parental alleles. In this way, the main objective of this work was to propose the endosperm genotyping as a strategy to identify the allelic origin of those heterozygotes individuals. Initially, fragments from the endosperm representing 10, 25 and 50% of the corn seeds weight were extracted and the seeds were submitted to the germination test. The results suggest that the elimination of up to 50% of the endosperm did not affected the seed germination. The methodology of semiquantitative PCR was optimized to differentiate doses of the alleles in the mixtures of DNA derived from leaves of two maize inbred lines (L3 and L1113- 01). It was represented different allelic proportions observed in the endosperm of their reciprocal crosses, based on the maximum amount of endosperm that could be used for DNA extraction. SSR markers were generated by semiquantitative PCR technique and the amplified fragments were evaluated in both agarose gels treated with ethidium bromide and poliacrylamide gels using fluorescently labeled primers. Gel resolution using agarose did not allow the differentiation of the mixtures of parental DNAs. However, through the regression analysis and comparison of the band intensity corresponding to the same allele in the different mixtures, the initial concentration of each one of the alleles could be inferred. The requirement of an allelic pattern limited the use of this technique to QTL analysis in populations where at least one of the genitors is known. Although the resolution of poliacrylamide gels using fluorescent markers was more efficient in the endosperm genotyping, once it was allowed to differentiate the maternal origin of reciprocal hybrids seed´s. So, the strategy of endosperm genotyping using fluorescent SSR primer amplified by semiquantitative PCR allowed the determination of allelic origin in the heterozygous offspring derived from outbred populations, including these individuals in the QTL detection, and consequently, increasing the precision of this analysis. / No mapeamento genético e detecção de QTLs em populações exogâmicas nem sempre é possível a determinação da fase de ligação dos alelos. Assim, indivíduos heterozigotos são descartados dessas análises por serem não informativos, uma vez que não é possível, por meio do seu genótipo, distinguir a origem de seus alelos em relação aos dois genitores. Dessa forma, o objetivo principal deste trabalho foi propor a genotipagem do endosperma para identificar a origem alélica dos indivíduos heterozigotos. Inicialmente, fragmentos do endosperma representando 10, 25 e 50% do peso das sementes de milho foram retirados, sendo as sementes submetidas ao teste de germinação. Observou-se que a remoção de até 50% do endosperma não afetou a taxa de germinação das sementes. A metodologia de PCR semiquantitativo foi otimizada para diferenciar doses dos alelos nas misturas de DNA foliar de duas linhagens de milho (L3 e L1113-01), representando as diferentes proporções alélicas observadas no tecido endospermático dos seus cruzamentos recíprocos, tendo como base a quantidade máxima de endosperma que podia ser utilizada na extração do DNA. Marcadores SSR foram gerados pela técnica de PCR semiquantitativo, e os fragmentos amplificados foram avaliados tanto em gel de agarose tratado com brometo de etídio quanto em gel de poliacrilamida, usando-se primers fluorescentes. A resolução do gel de agarose não possibilitou a diferenciação das misturas dos DNAs parentais. No entanto, por meio da análise de regressão e da comparação da intensidade da banda correspondente a um mesmo alelo nas diferentes misturas, pôde-se inferir a concentração inicial de cada um dos alelos. A necessidade de um padrão de alelos limitou o uso dessa técnica nas análises de QTLs em populações nas quais pelo menos um dos genitores é conhecido. Já a resolução do gel de poliacrilamida utilizando marcadores fluorescentes foi mais eficiente na genotipagem de endospermas, uma vez que possibilitou a diferenciação da origem materna das sementes dos híbridos recíprocos. Assim, a estratégia de genotipagem do endosperma utilizando primers SSR fluorescentes amplificados pela técnica de PCR semiquantitativo possibilitou a determinação da origem dos alelos dos descendentes heterozigotos derivados de populações exogâmicas, permitindo a inclusão destes na detecção de QTLs e, conseqüentemente, aumentando a precisão das análises.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O Cryptosporidium é um parasito coccídeo reconhecido por causar diarréia em humanos e animais em todo o mundo. O gênero compreende pelo menos 20 espécies confirmadas, sendo o C. hominis e C. parvum as principais espécies causadoras de criptosporidiose em humanos. Ferramentas moleculares têm sido desenvolvidas para detectar e diferenciar espécies/genótipos e subgenótipos de Cryptosporidium. O objetivo do trabalho foi avaliar a heterogeidade molecular de Cryptosporidium sp. obtidos de amostras clínicas provenientes dos municípios do Rio de Janeiro e de Salvador, através da PCR em tempo real e seqüenciamento automático. Foram analisadas 85 amostras, distribuídas em 3 grupos distintos, sendo 45 delas do município do Rio de Janeiro e 40 amostras provenientes de Salvador, Bahia. Todas as amostras foram positivas para Cryptosporidium sp. pelo método de coloração de Kinyoun a frio. O ensaio da PCR em tempo real combinou uma reação multiplex para a detecção do gênero Cryptosporidium e da espécie C. parvum e uma reação simples para a detecção de C. hominis. Na detecção do gênero Cryptosporidium foram utilizados par de primers e uma sonda TaqMan desenhados a partir do alinhamento de seqüências conservadas do gene 18S rRNA de várias espécies de Cryptosporidium disponíveis no GenBank. Para a detecção das espécies C. parvum e C. hominis foram utilizados primers e sondas específicos obtidos a partir de seqüências de cada espécie disponíveis no GenBank. A detecção do gênero Cryptosporidium através da sonda 18S rRNA, na reação duplex, foi visualizada em 63 de 85 amostras totais. Destas, a sonda TaqMan específica para C. parvum detectou 6 amostras e a sonda TaqMan específica para C. hominis detectou 42 amostras. Quinze amostras não puderam ser detectadas pelas sondas C. hominis ou C. parvum. Nos ensaios da PCR para o gene 18S, 31 amostras foram positivas e 27 delas sequenciadas. As análises filogenéticas confirmaram a presença de C. hominis, C. parvum e C. felis nas amostras estudadas. Na análise da topologia da árvore filogenética obtida por Neighbor Joining, observou-se-se que as sequências obtidas neste estudo se agruparam com espécies do gênero Cryptosporidium já descritas, com 99% ou 100% de similaridade. Conclui-se que os dois métodos utilizados são importantes ferramentas para o diagnóstico da criptosporidiose e diferenciação de C. hominis e C. parvum em investigações epidemiológicas, assim como avaliação da heterogeneidade molecular de Cryptosporidium sp / Cryptosporidium is a coccidia parasite known to cause diarrhea in humans and animals worldwide. The genus comprises at least 20 confirmed species, with C. hominis and C.parvum major species that cause cryptosporidiosis in humans. Molecular tools have been developed to detect and differentiate Cryptosporidium at the species/genotypes and subtype levels. The objective of this study was to evaluate the molecular heterogeneity of Cryptosporidium sp. clinical samples obtained from Rio de Janeiro and Salvador (Bahia), through real-time PCR and automatic sequencing. We analyzed 85 samples, distributed in three distinct groups, 45 of them in the city of Rio de Janeiro and 40 samples from Salvador. All samples were positive for Cryptosporidium sp. by modified Kinyoun acid-fast staining technique. The real-time PCR procedure combined a duplex reaction for the detection of Cryptosporidium species and C. parvum and a simple reaction for the detection of C. hominis. The detection of the genus Cryptosporidium have been used a pair of primers and TaqMan probe designed from the alignment of conserved sequences of the 18S rRNA gene of several species of Cryptosporidium available in GenBank. For the detection of species C. parvum and C. hominis were used specific primers and probes derived from sequences of each species available in the GenBank. Detection of Cryptosporidium sp. by 18S rRNA probe, in the duplex reaction, was visualized in 63 of 85 total samples. Of these, the C. parvum TaqMan probe detected 6 samples and the C. hominis TaqMan probe detected 42 samples. Fifteen samples could not be detected by C. hominis or C. parvum probes. In the 18S PCR assays, 31 samples were positive and 27 of them sequenced. Phylogenetic analysis confirmed the presence of C. hominis, C. parvum and C. felis. The analysis of the phylogenetic tree obtained by Neighbor Joining showed that the sequences obtained in this study were grouped with Cryptosporidium species described in the GenBank, with 99% or 100% similarity. Both methods are important tools for the diagnosis of cryptosporidiosis and differentiation of C. hominis and C. parvum in epidemiological investigations, as well as evaluation of Cryptosporidium sp. molecular heterogeneity

A Enfuvirtida(ENF), único inibidor de fusão disponível, representa uma opção interessante aos pacientes com infecção pelo HIV quando utilizada em combinação com outros antirretrovirais, principalmente no tratamento de multiexperimentados com falha virológica e poucas opções terapêuticas. Sua eficácia já comprovada em ensaios clínicos esbarra nas barreiras impostas por sua administração parenteral. Impulsionado por estes dados, avaliamos durante 48 semanas a resposta virológica, a evolução de células T CD4 a possível resistência primária a ENF e o impacto para a adesão do uso subcutâneo da droga em dez pacientes que fazem acompanhamento ambulatorial no Hospital Universitário Pedro Ernesto e que tinham história de mais de dez anos de infecção pelo HIV e uso de ENF no seu esquema terapêutico sugerido por teste de resistência. Todos os pacientes alcançaram ao final do seguimento sucesso terapêutico, mantendo carga viral não detectada, e um incremento médio significativo de linfócitos T CD4. Em relação a uma possível resistência primária, em nenhum dos testes, genotipagem da glicoproteína 41, foi visualizado mutações naturais que pudessem diminuir a ação da ENF. Sobre o manejo do medicamento, preparo e aplicação, observamos que é imprescindível um apoio multidisciplinar para que não haja descontinuação na sua utilização / Enfuvirtide (ENF) is the only fusion inhibitor available. It is an interesting option for patients with HIV infection when used in combination with other antiretroviral drugs, especially in the treatment of multi-experienced patients with virological failure and few therapeutic options. Its effectiveness confirmed in clinical trials finds the barriers in its parenteral administration. Using these data, we evaluated, for 48 weeks, the virological response, evolution of CD4 T cells, the possible primary resistance to ENF and the impact to the subcutaneous use of the drug in ten patients undergoing outpatient monitoring at Hospital Universitário Pedro Ernesto with a history of more than ten years of HIV infection and use of ENF in their therapy, as suggested by resistance testing. All patients have successfully completed the therapy by the end of follow-up with an undetected viral load and a significant average increase of CD4 T lymphocytes. As for a possible primary resistance, neither the genotyping nor the glycoprotein 41 revealed natural mutations that could diminish the effect of ENF. Concerning the management, preparation and application of the drug, we found that a multidisciplinary support is essential to avoid that the drug be discontinued