Campylobacter jejuni is the commonest cause of bacterial foodborne gastroenteritis in industrialised countries. Despite its significance, it remains unclear how C. jejuni is disseminated in the environment, whether particular strains are more pathogenic than others, and by what routes this bacterium is transmitted to humans. One major factor hampering this knowledge is the lack of a standardised method for fingerprinting C. jejuni. Therefore, the overall aim of this project was to develop systematic and novel genotyping methods for C. jejuni. Chapter Three describes the use of single nucleotide polymorphisms (SNPs) derived from the multilocus sequence typing (MLST) database of C. jejuni and the closely related Campylobacter coli for genotyping these pathogens. The MLST database contains DNA sequence data for over 4000 strains, making it the largest comparative database available for these organisms. Using the in-house software package "Minimum SNPs", seven SNPs were identified from the C. jejuni/C. coli MLST database that gave a Simpson's Index of Diversity (D), or resolving power, of 0.98. An allele-specific real-time PCR method was developed and tested on 154 Australian C. jejuni and C. coli isolates. The major advantage of the seven SNPs over MLST is that they are cheaper, faster and simpler to interrogate than the sequence-based MLST method. When the SNP profiles were combined with sequencing of the rapidly evolving flaA short variable region (flaA SVR) locus, the genotype distributions were comparable to those obtained by MLST-flaA SVR. Recent technological advances have facilitated the characterisation of entire bacterial genomes using comparative genome hybridisation (CGH) microarrays. Chapter Four of this thesis explores the large volume of CGH data generated for C. jejuni and eight binary genes (genes present in some strains but absent in others) were identified that provided complete discrimination of 20 epidemiologically unrelated strains of C. jejuni. Real-time PCR assays were developed for the eight binary genes and tested on the Australian isolates. The results from this study showed that the SNP-binary assay provided a sufficient replacement for the more laborious MLST-flaA SVR sequencing method. The clustered regularly interspaced short palindromic repeat (CRISPR) region is comprised of tandem repeats, with one half of the repeat region highly conserved and the other half highly diverse in sequence. Recent advances in real-time PCR enabled the interrogation of these repeat regions in C. jejuni using high-resolution melt differentiation of PCR products. It was found that the CRISPR loci discriminated epidemiologically distinct isolates that were indistinguishable by the other typing methods (Chapter Five). Importantly, the combinatorial SNP-binary-CRISPR assay provided resolution comparable to the current 'gold standard' genotyping methodology, pulsed-field gel electrophoresis. Chapter Six describes a novel third module of "Minimum SNPs", 'Not-N', to identify genetic targets diagnostic for strain populations of interest from the remaining population. The applicability of Not-N was tested using bacterial and viral sequence databases. Due to the weakly clonal population structure of C. jejuni and C. coli, Not-N was inefficient at identifying small numbers of SNPs for the major MLST clonal complexes. In contrast, Not-N completely discriminated the 13 major subtypes of hepatitis C virus using 15 SNPs, and identified binary gene targets superior to those previously found for phylogenetic clades of C. jejuni, Yersinia enterocolitica and Clostridium difficile, demonstrating the utility of this additional module of "Minimum SNPs". Taken together, the presented work demonstrates the potentially far-reaching applications of novel and systematic genotyping assays to characterise bacterial pathogens with high accuracy and discriminatory power. This project has exploited known genetic diversity of C. jejuni to develop highly targeted assays that are akin to the resolution of the current 'gold standard' typing methods. By targeting differentially evolving genetic markers, an epidemiologically relevant, high-resolution fingerprint of the isolate in question can be determined at a fraction of the time, effort and cost of current genotyping procedures. The outcomes from this study will pave the way for improved diagnostics for many clinically significant pathogens as the concept of hierarchal combinatorial genotyping gains momentum amongst infectious disease specialists and public health-related agencies.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen that is endemic in hospitals all over the world. It has more recently emerged as a serious threat to the general public in the form of community-acquired MRSA. MRSA has been implicated in a wide variety of diseases, ranging from skin infections and food poisoning to more severe and potentially fatal conditions, including; endocarditis, septicaemia and necrotising pneumonia. Treatment of MRSA disease is complicated and can be unsuccessful due to the bacterium's remarkable ability to develop antibiotic resistance. The considerable economic and public health burden imposed by MRSA has fuelled attempts by researchers to understand the evolution of virulent and antibiotic resistant strains and thereby improve epidemiological management strategies. Central to MRSA transmission management strategies is the implementation of active surveillance programs, via which unique genetic fingerprints, or genotypes, of each strain can be identified. Despite numerous advances in MRSA genotyping methodology, there remains a need for a rapid, reproducible, cost-effective method that is capable of producing a high level of genotype discrimination, whilst being suitable for high throughput use. Consequently, the fundamental aim of this thesis was to develop a novel MRSA genotyping strategy incorporating these benefits. This thesis explored the possibility that the development of more efficient genotyping strategies could be achieved through careful identification, and then simple interrogation, of multiple, unlinked DNA loci that exhibit progressively increasing mutation rates. The baseline component of the MRSA genotyping strategy described in this thesis is the allele-specific real-time PCR interrogation of slowly evolving core single nucleotide polymorphisms (SNPs). The genotyping SNP set was identified previously from the Multi-locus sequence typing (MLST) sequence database using an in-house software package named Minimum SNPs. As discussed in Chapter Three, the genotyping utility of the SNP set was validated on 107 diverse Australian MRSA isolates, which were largely clustered into groups of related strains as defined by MLST. To increase the resolution of the SNP genotyping method, a selection of binary virulence genes and antimicrobial resistance plasmids were tested that were successful at sub typing the SNP groups. A comprehensive MRSA genotyping strategy requires characterisation of the clonal background as well as interrogation of the hypervariable Staphylococcal Cassette Chromosome mec (SCCmec) that carries the β-lactam resistance gene, mecA. SCCmec genotyping defines the MRSA lineages; however, current SCCmec genotyping methods have struggled to handle the increasing number of SCCmec elements resulting from a recent explosion of comparative genomic analyses. Chapter Four of this thesis collates the known SCCmec binary marker diversity and demonstrates the ability of Minimum SNPs to identify systematically a minimal set of binary markers capable of generating maximum genotyping resolution. A number of binary targets were identified that indeed permit high resolution genotyping of the SCCmec element. Furthermore, the SCCmec genotyping targets are amenable for combinatorial use with the MLST genotyping SNPs and therefore are suitable as the second component of the MRSA genotyping strategy. To increase genotyping resolution of the slowly evolving MLST SNPs and the SCCmec binary markers, the analysis of a hypervariable repeat region was required. Sequence analysis of the Staphylococcal protein A (spa) repeat region has been conducted frequently with great success. Chapter Five describes the characterisation of the tandem repeats in the spa gene using real-time PCR and high resolution melting (HRM) analysis. Since the melting rate and precise point of dissociation of double stranded DNA is dependent on the size and sequence of the PCR amplicon, the HRM method was used successfully to identify 20 of 22 spa sequence types, without the need for DNA sequencing. The accumulation of comparative genomic information has allowed the systematic identification of key MRSA genomic polymorphisms to genotype MRSA efficiently. If implemented in its entirety, the strategy described in this thesis would produce efficient and deep-rooted genotypes. For example, an unknown MRSA isolate would be positioned within the MLST defined population structure, categorised based on its SCCmec lineage, then subtyped based on the polymorphic spa repeat region. Overall, by combining the genotyping methods described here, an integrated and novel MRSA genotyping strategy results that is efficacious for both long and short term investigations. Furthermore, an additional benefit is that each component can be performed easily and cost-effectively on a standard real-time PCR platform.

No description available.

Made available in DSpace on 2016-12-23T13:55:58Z (GMT). No. of bitstreams: 1 Solange Alves Vinhas.pdf: 4494197 bytes, checksum: c05b3a06ae983246a835977f36eb32e3 (MD5) Previous issue date: 2013-08-30 / Background: Based on the hypothesis that genetic variability of Mycobacterium tuberculosis (MTB) could influence virulence and immunopathology we analyzed genetic profiles of different MTB strains in order to detect relatedness between genetic diversity and presence of cavity (disease severity). Methods: We conducted a retrospective molecular study in Vitória ES, based on TB strains (2003 to 2006, n = 214) from patients with pulmonary cavitary and non-cavitary TB using IS6110-RFLP, Spoligotyping and MIRU-VNTR methodologies. RESULTS: Initially, we compared the association of the demographic and clinical characteristics of patients with the presence of cavities. After logistic regression the variables that most contributed to explain the model of the disease were smear positive (ORajust = 5.96; IC= 2.58-13.73) and sputum production (ORajust = 4.55; IC= 1.28-16.12), there was no statistically significant association with the remaining variables. The LAM family was the most frequent within the samples of the two groups analyzed, representing 65 (62%) of the isolates in the cavitary group and 40 isolates (38%) of the non-cavitary. After comparing the proportions of LAM and other spoligotyping families there was no statistically significant difference between the groups (p=0.17). In relation to deletions RDRio (p=0.65) and RD174 (p=0.65) there were no statistically significant difference between the groups. Amongst the 205 isolates analyzed, 25 (12%) belonging to the non-cavitary group and 43 (21%) belonging to the cavitary group, were grouped in clusters. The statistical analysis of the association of the occurence of clusters with the presence of cavity showed no statistically significant difference between the quantity of clusters and the groups that were analyzed, (p= 0.4). Conclusion: The genotipic profile for the isolates from patients with cavitary and non-cavitary disease was determined. Our data showed that LAM9 was the most frequent among the strains between cavitary and noncavitary groups, corroborating findings that this family is the most frequent in Brasil. There were no statistical differences that could show association among the variables analyzed related to presence of cavity or disease severity / Introdução: Baseado na hipótese de que a variabilidade genética de Mycobacterium tuberculosis (MTB) pode influenciar a virulência e a gravidade da doença os perfis genéticos de isolados clínicos de MTB foram avaliados para detectar associação entre diversidade genética e gravidade da doença. Objetivos: Analisar características genéticas de isolados de MTB e verificar sua possível associação com a gravidade da TB pulmonar. Métodos: Estudo retrospectivo, caso controle, conduzido em Vitória-ES, utilizando isolados de MTB (2003 a 2006, n=214) de pacientes com TB pulmonar, cavitária (127) e não cavitária (87). Realizou-se genotipagem por meio de RFLP-IS6110, Spoligotyping, MIRU-VNTR 24 loci, e a análise de deleções e inserções, como RDRio, RD174 utilizando PCR multiplex, bem como a detecção do Ag85C103. Realizou-se análise estatística, para verificação dos padrões de distribuição das variáveis, seguida de análises bivariadas para verificação de associações entre elas, empregando-se os teste exato de Fisher ou Chi-quadrado, ambos com 95% de intervalo de confiança e nível de significância (&#61554;) < 0,05. Resultados: Após a regressão logística, as variáveis que contribuíram no modelo explicativo da doença foram baciloscopia (ORajust = 5,96; IC= 2,58-13,73) e produção de escarro (ORajust = 4,55; IC= 1,28- 16,12). Não houve associação estatisticamente significativa com o restante das variáveis.A família LAM foi a mais frequente entre os dois grupos analisados, representando 65 (62%) dos isolados no grupo cavitário e 40 isolados (38%) do grupo não cavitário. Não houve diferença estatisticamente significativa entre os grupos em relação à deleção RDRio (p=0,65) e com relação à deleção RD174 (p=0,65). Dentre os 205 isolados analisados, 25 (12%) isolados do grupo não cavitário e 43 (21%) do grupo cavitário, estavam em cluster. não houve diferença estatisticamente significativa entre a quantidade de clusters e os grupos analisados (p= 0,4). Conclusões: Foi determinado o perfil genotípico dos isolados de pacientes com doença pulmonar, cavitária e não cavitária. Não houve associação entre a presença de cavidade e os genótipos encontrados. Não houve associação do genótipo com nenhum dos marcadores moleculares avaliados

Made available in DSpace on 2014-12-17T14:16:35Z (GMT). No. of bitstreams: 1 WalicyranisonPS_DISSERT.pdf: 2780035 bytes, checksum: 20a70f53e533bfb0816b6d9b02b8af32 (MD5) Previous issue date: 2013-06-11 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Candida albicans is a diploid yeast that in some circumstances may cause oral or oropharyngeal infections. The investigation of natural products is mandatory for the discovery of new targets for antifungal drugs development. This study aimed to determine the genotypes of 48 clinical isolates of C. albicans obtained from the oral cavity of kidney transplant patients from two distinct geographic regions of Brazil. In addition, we investigated three virulence factors in vitro: phospholipase activity, morphogenesis and the ability to evade from polymorphonuclear neutrophils. The expression of these virulence factors in vitro was also investigated in the presence of the crude extract of Eugenia uniflora. The genotype A was the most prevalent (30 isolates; 62.5%), followed by genotype C (15 isolates; 31.5%) and genotype B (3 isolates; 6.25%). When microsatellite technique with primer M13 was applied, 80% of the isolates from the South were placed within the same cluster. All Genotype C strains were grouped together within two different clusters. Genotype C was considered more resistant to PMNs attack than genotypes A and B. Strains isolated from the South of Brazil showed higher ability to combat PMNs phagocytosis. We found a high rate of genotype C strains isolated from the oral cavity of this group of patients. The crude extract of E. uniflora inhibited proper hypha formation and phagocytosis by PMNs, but had no significant effect on phospholipase activity. This study characterized oral C. albicans strains isolated from kidney transplant recipients and will contribute for the better understanding of the pathogenesis and alternative therapeutics for oral candidiasis / Candida albicans ? uma levedura dipl?ide que em certas circunst?ncias pode causar infec??es da cavidade oral e da orofaringe. A investiga??o de produtos naturais ? fundamental para a descoberta de novos alvos para o desenvolvimento de drogas antif?ngicas. Este estudo objetivou determinar os gen?tipos de 48 isolados cl?nicos de C. albicans obtidos da cavidade oral de pacientes transplantados renais de duas distintas regi?es geogr?ficas do Brasil. Al?m disso, foram investigados tr?s fatores de virul?ncia in vitro: atividade de fosfolipase, morfog?nese e a capacidade de escapar do ataque de neutr?filos polimorfonucleares. A express?o destes fatores de virul?ncia tamb?m foi investigada na presen?a do extrato bruto de Eugenia uniflora. O gen?tipo A foi o mais prevalente (30 isolados; 62,5%), seguido do gen?tipo C (15 isolados; 31,5%) e do gen?tipo B (3 isolados; 6,25%). Quando a t?cnica do microssat?lite com o primer M13 foi empregada, 80% dos isolados da regi?o Sul foram agrupados no mesmo cluster. Todos os isolados do gen?tipo C foram agrupados juntos em dois diferentes clusters bem definidos. Isolados do gen?tipo C foram considerados mais resistentes ? a??o de PMNs do que os dos gen?tipos A e B. As cepas isoladas do Sul do Brasil demonstraram maior habilidade em combater a fagocitose por PMNs. Encontrou-se uma alta taxa de isolados do gen?tipo C da cavidade oral deste grupo de pacientes. O extrato bruto de E. uniflora inibiu a forma??o de hifa e fagocitose por PMNs, mas n?o apresentou efeito significativo na atividade de fosfolipase. Este estudo caracterizou isolados cl?nicos de C. albicans da cavidade oral de pacientes transplantados renais, contribuindo para um melhor entendimento da patog?nese e terap?utica alternativa para a candid?ase oral

INTRODUÇÃO: Aproximadamente 170 milhões de pessoas são portadores de hepatite C crônica, sendo esta atualmente a principal causa de transplantes hepáticos no mundo. Os pacientes com hepatite crônica C são atualmente tratados com interferon e ribavirina (IFN/RBV). Estudos de associação do genoma associaram à resposta ao tratamento com IFN/RBV a um polimorfismo de nucleotídeo único (SNP) nas proximidades do gene da Interleucina 28B, que codifica a interferona-?. OBJETIVOS: Padronizar nova metodologia de baixo custo e menor tempo de execução para a genotipagem do polimorfismo rs12979860. Investigar a frequência do polimorfismo rs12979860, em uma coorte de pacientes com hepatite crônica C e sua associação com a resposta ao tratamento no Hospital das Clínicas da Universidade de São Paulo. MÉTODOS: A genotipagem foi realizada, por novo método diagnóstico, PCR multiplex CTPP (confronto de dois pares de iniciadores) e validado através de sequenciamento direto e PCR em tempo real. Um estudo retrospectivo foi realizado em 248 portadores de hepatite C crônica, tratados com interferon e ribavirina; 138 portadores de hepatite C crônica, virgens de tratamento e 240 doadores de sangue. Foi analisado o DNA, dados clínicos e demográficos, juntamente com dados sobre a resposta ao tratamento. RESULTADOS: O método de PCR CTPP foi padronizado e mostrou-se mais rápido e de menor custo comparado ao sequenciamento e PCR em tempo real. Pacientes com resposta virológica sustentada (RVS) apresentaram uma frequência de 33/61 (54,1%) para o genótipo C/C, de 21/61 (34,4%) para o genótipo C/T e 7/61 (11,5%) para o genótipo T/T. Pacientes que não tiveram RVS (Não RVS) apresentam uma frequência de 44/185 (23,8%) para o genótipo C/C, de 102/185 (55,1%) para o genótipo C/T e de 39/185 (21,1%) para o genótipo T/T. Os Não RVS estão associados ao genótipo C/T (p=0,002) e ao genótipo T/T (p=0,001) quanto comparados com o grupo de RVS. CONCLUSÕES: Esta dissertação descreve um método inovador, rápido e de baixo custo, o PCR CTPP, que detecta o polimorfismo rs12979860. O ensaio é internamente controlado e não requer a utilização de endonucleases de restrição ou equipamento especial. O polimorfismo rs12979860 é um preditor significativo da resposta ao tratamento com IFN/RBV em pacientes com infecção crônica pelo vírus da hepatite C. A genotipagem deste, em conjunto com os indicadores já existentes, pode identificar prováveis não respondedores ao tratamento / INTRODUCTION: Approximately 170 million people are chronic carriers of hepatitis C virus (HCV). Chronic HCV patients are currently treated with pegylated interferon and ribavirin (PEG- IFN/RBV). A genome-wide association with PEG-IFN/RBV treatment response and a single nucleotide polymorphism (rs12979860) has been identified near the interleukin 28B gene that encodes interferon-?-3. AIM: Describe an innovative, fast, and low-cost multiplex polymerase chain reaction with confronting two-pair primers that detects the rs12979860 polymorphism. Investigate the frequency of polymorphism rs12979860, among patients with chronic hepatitis C and association with to response treatment at the Hospital das Clínicas da Universidade de São Paulo. METHODS: Genotyping was performed by new diagnostic method, multiplex PCR CTPP (confronting two-pairprimers) and validated by direct sequencing and real time PCR. Retrospective study was conductedin 248 patients with hepatitis C chronic treated with interferon and ribavirin, 138 patients with chronic hepatitis C treatment-naïve and 240 blood donors. We analyzed DNA, clinical and demographic data, along with data onthe response to treatment. RESULTS AND CONCLUSIONS: The CTPP method was standardized and proved faster and lower cost compared to sequencing andreal time PCR. Patients with sustained virologic response (SVR) showed a frequency of 33/61 (54.1%) for the genotype C/C of 21/61 (34.4%) for the genotype C/T and 7/61 (11.5%) for genotype T/T. Patients with out sustained virologic response (Non SVR) have a frequency of 44/185 (23.8%) for the genotype C/C,102/185 (55.1%) for the genotype C/T and 39/185 (21.1%) for genotype T/T.The Non SVR are associated with genotype C/T (p = 0.002) and T/T genotype (p= 0.001) as compared with the group of SVR. Today, the IL28B genotyping is recomended by in the American Association for the Study of Liver Diseases and the European Association for the Study of the Liver guidelines. As a result, physicians should consider testing IL28B in patients with hepatitis C; however, the implementation of routine genotyping has been halted in tertiary care hospitals because of the need for molecular biology tools that are expensive and highly complex. The CTPP multiplex assay described here detects in a single reaction the genotypes C/C, C/T, and T/T. This method allows rapid genotyping of the polymorphism rs12979860, which is reproducible in minimally equipped laboratories; it does not require any special equipment and is a relatively low-cost procedure. The PCR-CTPP method can be used for large testing arrays and is also suitable for genotyping a small number of samples

O genótipo e a carga parasitária constituem dois dos principais fatores associados à patogênese na toxoplasmose congênita. Na Europa e nos EUA, o genótipo II é o mais prevalente em infecções congênitas, enquanto que na América do Sul existem evidências apontando uma maior frequência de genótipos atípicos ou recombinantes, associados a casos mais graves. A carga parasitária também parece atuar como fator de risco independente associado ao prognóstico fetal. Os objetivos do estudo foram padronizar uma amplificação quantitativa (qPCR) com iniciadores do gene B1 para avaliar a carga parasitária; determinar o genótipo parasitário por multiplex-nested-PCR-RFLP dos marcadores 5\'-SAG2, 3\'-SAG2, SAG3 e GRA6, seguido de sequenciamento para confirmação da RFLP e análise de mutações; e verificar se existiria associação entre a carga parasitária e os genótipos parasitários nas mesmas gestações. Foram analisadas 76 amostras de líquido amniótico de gestações com toxoplasmose e 31 amostras controle. A qPCR apresentou LOD de 10 parasitos/mL, detectou as 76 amostras de estudo e nenhum controle. As cargas parasitárias variaram de 222 a 808.328 parasitos/mL. Houve duas amostras com valores acima de 104 parasitos/mL, apesar de todas as gestantes serem tratadas. Na genotipagem, SAG3 amplificou 55 amostras (54 tipo III e uma tipo II); 5\' e 3\'-SAG2 amplificaram 54 amostras (todas tipo I), e GRA6, amplificou 20 amostras (todas tipo III). A única amostra com genótipo parasitário SAG3-tipo II foi a que apresentou mais mutações (n=4), carga parasitária de 958 parasitos/mL, porém o recém-nascido foi assintomático. Houve diferença do número de amostras amplificadas por SAG3, e 5\' e 3\'-SAG2 em relação a GRA6 (McNemar, p<0,001). Os sequenciamentos confirmaram 100% dos resultados de RFLP, e foram encontradas 24 amostras com e 52 sem mutações, não existindo diferenças entre as cargas parasitárias dos dois grupos (Mann-Whitney, p= 0,085). Mais de uma mutação foi observada em cinco amostras. Foram detectadas 37 mutações no estudo: 26 heterozigotas/sinônimas e 11 homozigotas/sinônimas, não havendo regiões hot spot. Quanto à correlação clínico-laboratorial, dos 76 recém-nascidos, todos apresentaram IgM positiva ao nascimento, e 75 eram assintomáticos. O único recém-nascido sintomático apresentava tríade de Sabin e uma das duas cargas parasitárias mais elevadas do estudo (309.574 parasitos/mL), porém o genótipo não foi discriminante e não havia mutações. A outra amostra com carga parasitária acima de 104 parasitos/mL pertencia a recém-nascido assintomático, com genótipo não discriminante, e sem mutações. O estudo concluiu que a técnica de Real Time PCR (qPCR) foi padronizada com sucesso, usando os iniciadores B22 e B23 do gene B1 do parasito, podendo ser empregada na rotina diagnóstica. Além disso, foi possível realizar a genotipagem das amostras incluídas no estudo, com melhor desempenho de SAG3 e 5\' e 3\'-SAG2. O sequenciamento confirmou a confiabilidade da técnica de RFLP, e encontrou frequência elevada de mutações, todas sinônimas, sem regiões hot spot, e aparentemente sem associação com a carga parasitária. Houve elevada variabilidade das cargas parasitárias, porém grande homogeneidade dos genótipos parasitários, não tendo sido observada associação entre a carga parasitária e os genótipos de T. gondii no estudo. / The genotype and the parasite load are two of the main factors associated with pathogenesis in congenital toxoplasmosis. In Europe and the USA, genotype II is the most prevalent in congenital infections, while in South America there is evidence pointing to a higher frequency of atypical or recombinant genotypes associated with more severe cases. The parasite load also appears to act as an independent risk factor associated with fetal prognosis. The study objectives were to standardize a quantitative amplification (qPCR) with B1 gene primers to assess the parasite load; determine the genotype by multiplex-nested-PCR-RFLP of 5\' and 3\'-SAG2, SAG3 and GRA6 markers, followed by sequencing to confirm RFLP and analyze mutations, verifying whether there is association between parasite load and parasite genotypes in the same pregnancies. We analyzed 76 amniotic fluid samples from pregnancies with toxoplasmosis and 31 controls. The qPCR presented LOD of 10 parasites/mL, detected the 76 study samples and no control. Parasite loads ranged from 222 to 808,328 parasites/mL. There were two samples with values above 104 parasites/mL, despite all pregnant women be treated. In genotyping, SAG3 amplified 55 samples (54 type III and 1 type II); 5 \'and 3\'-SAG2 amplified 54 samples (all type I); and GRA6, amplified 20 samples (all type III). The only sample with genotype SAG3-type II showed the highest number of mutations (n=4), parasite load of 958 parasites/mL, but the newborn was asymptomatic. There were differences in the number of samples amplified by SAG3, and 5 \'and 3\'-SAG2 over GRA6 (McNemar test, p <0.001). Sequencing confirmed 100% of the RFLP results; and found 24 samples with and 52 without mutations, with no difference between the parasite load of these two groups (Mann-Whitney, p= 0.085). More than one mutation was observed in five samples. A total of 37 mutations were detected in this study: 26 heterozygotes/synonymous and 11 homozygous/synonyms, with no hot spot regions. Regarding the clinical-laboratory correlation, among the 76 newborns, all showed positive IgM at birth, and 75 were asymptomatic. The only symptomatic newborn presented the Sabin\'s triad and one of the two higher parasite loads in the study (309,574 parasites/mL). However, the genotype was not discriminant and no mutations were detected. The other sample with parasite load above 104 parasites/mL belonged to an asymptomatic newborn with a non-discriminating genotype, and no mutations. The study concluded that the Real Time PCR (qPCR) was successfully developed with primers B22 and B23 of the parasite B1 gene, and can be used in routine practice. Moreover, it was possible to perform the samples genotyping, with better performance of SAG3 and 5 \'and 3\'-SAG2. Sequencing results confirmed the RFLP reliability, and found a high frequency of mutations, all synonymous, with no hot spot regions, and apparently not associated with the parasite load. There was a high variability in parasite load, however great homogeneity of parasite genotypes, with no association between the parasite load and T. gondii genotypes in the study.

Neste estudo, geramos ensaios de espectrometria de massa para detecção de 111 mutações nos genes RET, BRAF, NRAS, HRAS, KRAS, PIK3CA e AKT1 e avaliamos inúmeras linhagens celulares e tumores tiroidianos. Mostramos que as mutações dos genes BRAF e RAS refletem prognósticos distintos e que as mutações BRAF são altamente prevalentes em câncer metastático. Mutações dos genes PIK3CA e AKT1, esta última sendo reportada pela primeira vez no câncer de tiróide, são relativamente frequentes neste câncer. Avaliamos ainda a função do microRNA let-7 neste câncer. Mostramos que a ativação do rearranjo RET/PTC3 em células de tiróide PCCl3 reduz a expressão de let-7. Além disso, a transfecção deste microRNA em células TPC-1, que apresentam o rearranjo RET/PTC1, inibe a fosforilação de ERK, o crescimento celular e modula a expressão de genes do ciclo celular e diferenciação. Estes dados contribuem na aplicação de terapias dirigidas a efetores das vias PI3K e MAPK no câncer de tiróide, além de salientar o envolvimento do miRNA let-7 como um gene supressor tumoral nesta doença. / In this study, we designed an assay panel for genotyping 111 mutations by mass spectrometry in RET, BRAF, NRAS, HRAS, KRAS, PIK3CA, AKT1 and other related genes in many thyroid cancer cell lines and tumors. We show that patients with BRAF and RAS mutations have distinct prognosis and that BRAF mutations are highly prevalent in metastatic thyroid cancers. Mutations of PIK3CA and AKT1, the latter not previously described in this disease, are comparatively frequent in thyroid cancers. In addition, we evaluated the role of let-7 microRNA in this cancer. We show that RET/PTC3 activation in PCCl3 thyroid cells reduces let-7 expression. Moreover, transfection of let-7 in TPC-1 cells, which harbor RET/PTC1 rearrangement, inhibits MAPK activation, reduces cell growth and modulates the expression of cell cycle and differentiation genes. These findings may contribute to the design of therapies directed at MAPK and PI3K effectors and to highlight the function of let-7 as tumor suppressor gene in thyroid cancer.

Made available in DSpace on 2014-07-29T15:16:37Z (GMT). No. of bitstreams: 1 AnaClaraferraz.pdf: 1870286 bytes, checksum: d17e1fe96ee32ca1852c51743beff160 (MD5) Previous issue date: 2010-01-12 / The progress of Molecular Biology and Genetics provided the appearance of several molecular markers that detect the genetic polymorphism directly at DNA. Among these markers are the microsatellites (SSR), which are distinguished by their high degree of polymorphism. The use of these markers for individual genotyping has evolved into multiplex systems, which allow many SSR fragments to be detected and analyzed simultaneously. Currently there are several articles in literature discussing the criteria to be used in the primer design for use in PCR, as well as various softwares are available for this end. However, there are few studies and tools for the analysis of compatibility between pairs of primers for use in multiplex systems, where multiple fragments are simultaneously amplified using PCR. This paper evaluated different criteria for compatibility between pairs of primers. A set of 74 combinations of pairs of primers, involving the amplification of 94 SSR loci were evaluated in duplex systems. The same combinations were evaluated according to different criteria, including the degree of complementarity between primers, the magnitude of differences of denaturation temperatures (Tm) and the tendency to annealing between pairs of primers based on the Gibbs free energy resulting from the association between them. The comparison between the different criteria allowed the identification of a set of criteria with positive predictive value equal to 94%. These criteria were implemented for use in a software called Multiplexer, which from the analysis in sequence of pairs of primers, suggests compatible combinations for use in multiplex genotyping systems. Using this tool can significantly reduce the costs related to laboratory activities for genotyping using PCR. / Os avanços da Biologia Molecular e da Genética proporcionaram o surgimento de diversos marcadores moleculares que detectam o polimorfismo genético diretamente no DNA. Entre estes marcadores se encontram os microssatélites (SSR), que se destacam pelo seu elevado grau de polimorfismo. O uso desses marcadores para fins de genotipagem individual tem evoluído para sistemas multiplex, os quais permitem que vários fragmentos SSR sejam detectados e analisados simultaneamente. Atualmente são abundantes na literatura artigos que discutem os critérios a serem utilizados no desenho de pares de primers para aplicação em PCR, bem como estão disponíveis diversos softwares para este fim. No entanto, ainda são escassos os estudos e ferramentas destinados à análise de compatibilidade entre pares de primers para aplicação em sistemas multiplex, onde vários fragmentos são amplificados simultaneamente por PCR. Neste trabalho são avaliados diferentes critérios de compatibilidade entre pares de primers. Um conjunto de 74 combinações de pares de primers, envolvendo a amplificação de 94 locos SSR foram avaliados em sistemas duplex. As mesmas combinações foram avaliadas segundo diferentes critérios, incluindo o grau de complementariedade entre primers, magnitude das diferenças de temperaturas de desnaturação (Tm) e a tendência ao anelamento entre pares de primers com base na energia livre de Gibbs resultante da associação entre eles. A comparação entre os diferentes critérios permitiu a identificação de um conjunto de critérios com valor preditivo positivo igual a 94%. Estes critérios foram implementados para utilização em um software denominado Multiplexer, que a partir da análise de sequências de pares de primers, sugere combinações compatíveis para a utilização em sistemas de genotipagem multiplex. O uso dessa ferramenta pode reduzir consideravelmente os custos laboratoriais relativos às atividades de genotipagem utilizando PCR.

Introdução: estudos com Enfuvirtida (ENF) mostraram que mutações na HR1 da gp41 levam à resistência primária de cepas em pacientes sem tratamento prévio com inibidores de fusão (Derdeyn et al., 2000; Rimsky et. al., 1998; Sista et. al., 2002). Outros, que o uso contínuo de HAART leva à falha virológica (Shafer et. al., 1998; Shafer & Vuitton, 1999). Para a melhor escolha terapêutica recomenda-se usar a Genotipagem do HIV1 (Ministério da Saúde, 2008b; Perez-Elias et. al., 2003; Shafer et. al., 2001). Objetivos: avaliar o perfil de resistência do HIV1 ao ENF pelo sequenciamento da HR1 da gp41 em pacientes sob HAART, sem uso de inibidor de fusão. Realizar fenotipagem virtual da pol do HIV1 em trinta e duas cepas com resistência aos NRTI, NtRTI , NNRTI e PI para verificar indicação do ENF. Métodos: investigamos 877 prontuários de pacientes do CRT-DST/AIDS entre 5/2002 a 7/2005 e verificamos que 92 que haviam realizado o Teste de Genotipagem do HIV1 em nosso laboratório, sem tratamento prévio com inibidores de entrada poderiam participar do nosso estudo. O primeiro grupo, com 60 pacientes apresentou cepas sensíveis à pelo menos um antirretroviral (ARV) e o segundo com 32 cujas cepas apresentaram resistência parcial e completa às drogas. A região HR1/HR2 da gp41 foi seqüenciada com o kit Big Dye Terminator vs. 3.1. Alinhamos as seqüências e as comparamos com HXB2 através do programa BioEdit vs.7.0.9.0. As mutações de resistência ao ENF foram determinadas pelo algoritmo Francês (ANRS) e pelo da Universidade de Stanford. Submetemos as sequências ao Virtual Phenotype Assay (Virco, Bélgica) para obtermos a Fenotipagem Virtual. Os subtipos da HR1/HR2 da gp41 e do pol foram determinados por análises filogenéticas e os recombinantes, pelo Simplot v 3.5.1. Resultados: 89(97%) amostras foram seqüenciadas, 2 (2%) não amplificaram e 1 (1%) não tinha material suficiente. Destas, 82 (92%) eram do subtipo B, 6(7%) do F1 e 1(1%), do BF na gp41. No pol, 80(87%) eram B, 4(4%) eram F e 7(8%), eram FB. Três (3%) apresentaram resistência primária ao ENF, determinada pela N42D (1%), N43H (1%) e L44M (1%). Dez amostras tinham os polimorfismos Q39R(10%), N42H(10%), N42R(10%), N42N/S(10%) e N42S(60%). Na HR2, as mutações N126N/K, N126K/Q/E, E137E/K, E137K e S138S/A estavam presentes em 26% das cepas. Apesar das cepas apresentarem resistência aos ARV pelo teste de genotipagem, a fenotipagem virtual mostrou que 10% dos ARV ainda atuavam sobre elas, enquanto que 75% delas apresentaram resistência parcial e 15%, completa às drogas. Conclusão: nosso estudo sugere a introdução do teste de genotipagem da HR1/HR2 da gp41 antes de indicar do ENF em pacientes com falha terapêutica e que a Fenotipagem Virtual pode ser usada para a escolha do melhor esquema antirretroviral que permita uma supressão viral sustentada. / Background: studies using Enfuvirtide (ENF) have showed that mutations in the HR1 (gp41) have been associated with primary resistance in strains of the patients without previous treatment with fusion inhibitors. (Rimsky et. al.,1998; Derdeyn et al.,2000, Sista et. al.,2002). The widespread use of HAART may achieve maximal suppression of the viral load, but the viral rebound may occur, leading to virologic failure (Shafer et. al.,1998; Shafer & Vuitton,1999). To optimize therapeutic choices it is recommended to use the HIV1 Genotypic Assay. Objective: to evaluate the resistance profile in the HR1 in patients submitted to HAART, but naïve entry inhibitors. To make the Virtual Phenotypic Assay in the pol of the HIV1 strains isolated of the 32 patients with resistance to the antiretroviral (ARV) and to verify the possibility of using the ENF. Methods: we investigated 877 handbook patients of the CRT-DST/AIDS from May/2002 to July/2005. We identified 92 naïve patients to entry inhibitors included in our cohort to do genotypic assay; they were included in our research. The first group was composed by 60 persons with sensible strains to only one ARV. The second, with 32 presented strains with resistance to all ARV. HR1/HR2 was sequenced (ABIPrism BigDye Terminator vs 3.1). Bioedit Software vs.7.0.9.0 was used to compare sequences with the HIVHXB2. The resistance mutations to ENF were analyzed using French ANRS and Stanford HIV Drug Resistance Algorithm. The results by Virtual Phenotype Assay of 32 patients that presented regimen failure by Genotypic Assay predicted HIV phenotypic resistance. Subtype analysis were did by Phylogeny and the recombinants strains results, confirmed by Simplot v.3.5.1. Results: 89(97%) out of 92 samples were sequenced. Two of them (2%) could not be amplified and one (1%) we did not have enough material to use. In the HR1/HR2 82(92%) of the samples were typed as B, 6(7%), as F and 1(1%) as FB. In the pol 80(87%) were classified as B, 4(4%), as F and 7(8%) as FB. According to algorithms used, 3(3%) out of 89 ENF naïve patients presented complete resistance to this drug. One virus harbored the N42D mutation (1%), another, the N43H(1%) and the other one the L44M(1%). In 10 samples we observed the polymorphisms Q39R(1%), N42H(1%), N42R(1%), N42N/S(1%) and N42S(7%). In the HR2 domain the substitutions N126N/K, N126K/Q/E, E137E/K, E137K and S138S/A were present in 26% of the samples. Although strains presented resistance to the ARV by Genotypic Assay, in the Virtual Phenotypic Assay we verified that 10% of the drugs had some effect upon them. 75% of the strains presented partial and 15% completed resistance to ARV. Conclusion: it is important to introduce the HR1/HR2 genotyping test to verify resistance mutations to ENF before indicating this drug to patients with virologic failure. The virtual phenotypic assay can be used to select the better combination therapy to obtain viral suppression sustained response.