Position weight matrices (PWMs) have become a tool of choice for the identification of transcription factor binding sites in DNA sequences. PWMs are compiled from experimentally verified and aligned binding sequences. PWMs are then used to computationally discover novel putative binding sites for a given protein. DNA-binding proteins often show degeneracy in their binding requirement, the overall binding specificity of many proteins is unknown and remains an active area of research. Although PWMs are more reliable predictors than consensus string matching, they generally result in a high number of false positive hits. A previous study introduced a novel method to PWM training based on the known motifs to sample additional putative binding sites from a proximal promoter area. The core idea was further developed, implemented and tested in this thesis with a large scale application. Improved mono- and dinucleotide PWMs were computed for Drosophila melanogaster. The Matthews correlation coefficient was used as an optimization criterion in the PWM refinement algorithm. New PWMs keep an account of non-uniform background nucleotide distributions on the promoters and consider a larger number of new binding sites during the refinement steps. The optimization included the PWM motif length, the position on the promoter, the threshold value and the binding site location. The obtained predictions were compared for mono- and dinucleotide PWM versions with initial matrices and with conventional tools. The optimized PWMs predicted new binding sites with better accuracy than conventional PWMs.

INTRODUÇÃO: O papilomavírus humano (HPV) encontra-se amplamente distribuído na população mundial. Apesar da grande maioria das infecções serem transientes, assintomáticas e passíveis de regressão espontânea, a infecção persistente por tipos de alto risco de HPV é necessária para o desenvolvimento de neoplasias intraepiteliais cervicais. Uma vez que apenas uma pequena parcela das infecções progride à lesões malignas após um longo período desde o diagnóstico inicial de lesões precursoras, tem-se iniciado a busca por fatores que possam influenciar na progressão ou na eliminação destas manifestações iniciais. A variabilidade genética viral tem sido apontada como um dos fatores que interagem neste processo. Embora virtualmente todos os tumores da cérvice uterina apresentem o DNA viral, neoplasias em outros sítios anatômicos têm sido apenas em parte correlacionadas com a presença viral, sendo o HPV proposto como um dos agentes causadores de tumores em sítios de cabeça e pecoço. MÉTODOS: Espécimens clínicos de tumores de cabeça e pescoço, fixados em formalina e contidos em parafina (FFPE), provenientes do Instituto do Câncer do Estado de São Paulo (n=79) e da Santa Casa de Misericórida de São Paulo (n=94), tiveram seu DNA extraído, seguido de diagnóstico e genotipagem de HPV pela metodologia de Inno-LiPA. Análises de linhagens moleculares foram realizadas nas amostras HPV-16 positivas. Análise imunohistoquímica de P16INK4a foi realizada em todas as amostras. RESULTADOS: A presença do DNA viral foi encontrada em 24,1% (19/79) dentre a série de tumores provenientes do ICESP, sendo a cavidade oral o sítio em que foi observada a maior proporção de DNA viral (27,1%), enquanto que 13,8% (13/94) dentre os espécimens provenientes da Santa Casa apresentaram-se positivos para HPV, sendo a cavidade oral o sítio em que foi observada a maior proporção do DNA viral (18,1%). O HPV-16 foi o tipo mais prevalente, detectado em 73,4% das amostras HPV positivas provenientes do ICESP e 61,5% das amostras provenientes da Santa Casa. Independente da Instituição, as amostras foram alocadas no clado das linhagens Asiático-Americana e Europeia em 50%, cada uma, entre os 18 tumores HPV-16 positivos em que as análises de linhagem foram possíveis. Não foi observada, nestas séries, correlação entre a superexpressão de P16INK4a e a presença do DNA viral. CONCLUSÃO: Nas amostras analisadas, o DNA de HPV foi detectado em 18,5% dos 173 espécimens. O HPV-16 foi o tipo mais prevalente. Isolados da linhagem Europeia e da linhagem Asiatico-Americano foram detectados em 50% dos casos, cada uma, dentre as amostras HPV-16 analisadas por este estudo / INTRODUCTION: Human papillomaviruses (HPV) are widely distributed worldwide. Although the majority of infections are usually transient, asymptomatic and frequently regress spontaneously, persistent infections by high-risk HPVs are necessary for the development of cervical intraepithelial neoplasia. Once only a small proportion of infections progress to malignant lesions after a long period of time since the initial diagnosis of precursor lesions, the search for factors that might influence the progression or clearence of these early manifestations are currently under way. Viral genetic variability has been proposed as one of the factors interacting in this process. Although virtually all cervix tumors present the viral DNA, neoplasias from other anatomical sites have been only in part correlated with viral presence, and HPV has been proposed as one causative agent in tumors from head and neck sites. METHODS: Clinical specimens of formalin-fixed paraffin embedded head and neck tumors, provided by the Cancer Institute of São Paulo (n=79) and also by the Santa Casa de Misericórdia de São Paulo (n=94), were submitted to DNA extraction and further HPV diagnostic and genotyping by the Inno-LiPA methodology. Molecular lineages analyses were performed in all HPV-16 positive samples. P16INK4a immunohistochemical analyses were conducted in all samples. RESULTS: HPV DNA was detected among 24.1% (19/79) of samples provided by ICESP, tumors from oral cavity presented the highest viral positivity (27.1%), whereas 13,8% (13/94) of the samples from Santa Casa presented HPV DNA, tumors from the oral cavity also presented the highest HPV positivity with 18.1% of viral DNA presence. HPV-16 was the most prevalent type detected in 73.4% and 61.5% of HPV positive ICESP and Santa Casa samples, respectively. Irrespective of the Institution, samples submitted to lineage analyzes were allocated in the Asiatic-American and European phylogenetic branches in 50%, each one, among the 18 tumors HPV-16 positive for which lineage analysis was possible. No correlation between P16INK4a overexpression and HPV DNA presence was observed. CONCLUSION: In this study, HPV DNA was detected in 18.5% among 173 head and neck tumor specimens. HPV-16 was the most prevalent type. The European and the Asiatic-American lineage were detected in 50% of the cases, each one, among the cases HPV-16 positive analyzed

Position weight matrices (PWMs) have become a tool of choice for the identification of transcription factor binding sites in DNA sequences. PWMs are compiled from experimentally verified and aligned binding sequences. PWMs are then used to computationally discover novel putative binding sites for a given protein. DNA-binding proteins often show degeneracy in their binding requirement, the overall binding specificity of many proteins is unknown and remains an active area of research. Although PWMs are more reliable predictors than consensus string matching, they generally result in a high number of false positive hits. A previous study introduced a novel method to PWM training based on the known motifs to sample additional putative binding sites from a proximal promoter area. The core idea was further developed, implemented and tested in this thesis with a large scale application. Improved mono- and dinucleotide PWMs were computed for Drosophila melanogaster. The Matthews correlation coefficient was used as an optimization criterion in the PWM refinement algorithm. New PWMs keep an account of non-uniform background nucleotide distributions on the promoters and consider a larger number of new binding sites during the refinement steps. The optimization included the PWM motif length, the position on the promoter, the threshold value and the binding site location. The obtained predictions were compared for mono- and dinucleotide PWM versions with initial matrices and with conventional tools. The optimized PWMs predicted new binding sites with better accuracy than conventional PWMs.

INTRODUÇÃO: O papilomavírus humano (HPV) encontra-se amplamente distribuído na população mundial. Apesar da grande maioria das infecções serem transientes, assintomáticas e passíveis de regressão espontânea, a infecção persistente por tipos de alto risco de HPV é necessária para o desenvolvimento de neoplasias intraepiteliais cervicais. Uma vez que apenas uma pequena parcela das infecções progride à lesões malignas após um longo período desde o diagnóstico inicial de lesões precursoras, tem-se iniciado a busca por fatores que possam influenciar na progressão ou na eliminação destas manifestações iniciais. A variabilidade genética viral tem sido apontada como um dos fatores que interagem neste processo. Embora virtualmente todos os tumores da cérvice uterina apresentem o DNA viral, neoplasias em outros sítios anatômicos têm sido apenas em parte correlacionadas com a presença viral, sendo o HPV proposto como um dos agentes causadores de tumores em sítios de cabeça e pecoço. MÉTODOS: Espécimens clínicos de tumores de cabeça e pescoço, fixados em formalina e contidos em parafina (FFPE), provenientes do Instituto do Câncer do Estado de São Paulo (n=79) e da Santa Casa de Misericórida de São Paulo (n=94), tiveram seu DNA extraído, seguido de diagnóstico e genotipagem de HPV pela metodologia de Inno-LiPA. Análises de linhagens moleculares foram realizadas nas amostras HPV-16 positivas. Análise imunohistoquímica de P16INK4a foi realizada em todas as amostras. RESULTADOS: A presença do DNA viral foi encontrada em 24,1% (19/79) dentre a série de tumores provenientes do ICESP, sendo a cavidade oral o sítio em que foi observada a maior proporção de DNA viral (27,1%), enquanto que 13,8% (13/94) dentre os espécimens provenientes da Santa Casa apresentaram-se positivos para HPV, sendo a cavidade oral o sítio em que foi observada a maior proporção do DNA viral (18,1%). O HPV-16 foi o tipo mais prevalente, detectado em 73,4% das amostras HPV positivas provenientes do ICESP e 61,5% das amostras provenientes da Santa Casa. Independente da Instituição, as amostras foram alocadas no clado das linhagens Asiático-Americana e Europeia em 50%, cada uma, entre os 18 tumores HPV-16 positivos em que as análises de linhagem foram possíveis. Não foi observada, nestas séries, correlação entre a superexpressão de P16INK4a e a presença do DNA viral. CONCLUSÃO: Nas amostras analisadas, o DNA de HPV foi detectado em 18,5% dos 173 espécimens. O HPV-16 foi o tipo mais prevalente. Isolados da linhagem Europeia e da linhagem Asiatico-Americano foram detectados em 50% dos casos, cada uma, dentre as amostras HPV-16 analisadas por este estudo / INTRODUCTION: Human papillomaviruses (HPV) are widely distributed worldwide. Although the majority of infections are usually transient, asymptomatic and frequently regress spontaneously, persistent infections by high-risk HPVs are necessary for the development of cervical intraepithelial neoplasia. Once only a small proportion of infections progress to malignant lesions after a long period of time since the initial diagnosis of precursor lesions, the search for factors that might influence the progression or clearence of these early manifestations are currently under way. Viral genetic variability has been proposed as one of the factors interacting in this process. Although virtually all cervix tumors present the viral DNA, neoplasias from other anatomical sites have been only in part correlated with viral presence, and HPV has been proposed as one causative agent in tumors from head and neck sites. METHODS: Clinical specimens of formalin-fixed paraffin embedded head and neck tumors, provided by the Cancer Institute of São Paulo (n=79) and also by the Santa Casa de Misericórdia de São Paulo (n=94), were submitted to DNA extraction and further HPV diagnostic and genotyping by the Inno-LiPA methodology. Molecular lineages analyses were performed in all HPV-16 positive samples. P16INK4a immunohistochemical analyses were conducted in all samples. RESULTS: HPV DNA was detected among 24.1% (19/79) of samples provided by ICESP, tumors from oral cavity presented the highest viral positivity (27.1%), whereas 13,8% (13/94) of the samples from Santa Casa presented HPV DNA, tumors from the oral cavity also presented the highest HPV positivity with 18.1% of viral DNA presence. HPV-16 was the most prevalent type detected in 73.4% and 61.5% of HPV positive ICESP and Santa Casa samples, respectively. Irrespective of the Institution, samples submitted to lineage analyzes were allocated in the Asiatic-American and European phylogenetic branches in 50%, each one, among the 18 tumors HPV-16 positive for which lineage analysis was possible. No correlation between P16INK4a overexpression and HPV DNA presence was observed. CONCLUSION: In this study, HPV DNA was detected in 18.5% among 173 head and neck tumor specimens. HPV-16 was the most prevalent type. The European and the Asiatic-American lineage were detected in 50% of the cases, each one, among the cases HPV-16 positive analyzed

Orientadores: Anelyssa Cysne Frota D'Abreu, Iscia Teresinha Lopes-Cendes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T05:40:31Z (GMT). No. of bitstreams: 1 Piovesana_LuizaGonzaga_M.pdf: 1656872 bytes, checksum: 2a3571bb0d9dc43e8f0405aa2c799a5e (MD5) Previous issue date: 2014 / Resumo: As distonias caracterizam-se por movimentos involuntários e torsionais, que se manifestam de diferentes formas e podem afetar quaisquer músculos voluntários. Diversas mutações genéticas foram associadas às distonias primárias, destacando-se a DYT1, DYT5 e DYT6. O gene DYT1/TOR1A foi o primeiro identificado, ao apresentar uma deleção GAG que produz uma proteína mutante que altera conexões núcleo-cito-esqueléticas e o processamento proteico. O fenótipo típico inicia-se dos três aos 26 anos, tem penetrância de 30%, sendo 60% com acometimento generalizado ou multifocal. O gene DYT6/THAP1 possui diversos polimorfismos descritos e acredita-se que seja o segundo em prevalência entre as distonias hereditárias. Possui penetrância ainda menor e fenótipo de início precoce, envolvimento crânio-cervical e de fala. A frequência, etiologia e as alterações genéticas das distonias não são conhecidas na população brasileira, que por suas características específicas, não podem ter os resultados de outras populações simplesmente transpostos para a nossa. Nosso objetivo, portanto, foi caracterizar genética e clinicamente uma amostra de pacientes brasileiros com distonia. Os indivíduos foram recrutados no Ambulatório de Distúrbios do Movimento e de Distonia. Os pacientes foram avaliados por um questionário padronizado, seguido por pesquisa das mutações DYT1 e DYT6. A avaliação clínica demonstrou que a nossa coorte apresenta padrão semelhante ao internacional, com pacientes de início jovem tendendo a apresentar quadros generalizados e com história familiar positiva e pacientes adultos mantendo quadros focais ou segmentares e esporádicos. A pesquisa de mutações identificou 1 mutação missense já descrita na literatura, envolvida na penetrância da DYT1 em indivíduos com a mutação causadora clássica da doença e que pode conferir risco quando encontrada isoladamente. Também encontramos uma deleção de 6 pares de base no fim do exon 1 do gene THAP1 que em avaliações preliminares altera o sitio de splicing e acaba por abortar a tradução da proteína por meio de um stop códon precoce. Concluímos que nossos pacientes tem apresentação clinica semelhante a literatura mundial, porém com características genotípicas diferentes, pois não encontramos em nenhum individuo as mutações mais classicamente associadas as doenças. A diferenciação das distonias em subtipos e o entendimento das vias moleculares comuns devem fazer parte de investigações futuras / Abstract: Dystonia is characterized by involuntary and torsional movements, which manifest themselves in various forms and can affect any voluntary muscle. Several genetic mutations have been associated with isolated hereditary dystonia, most notably the DYT1 and DYT6. The DYT1 is caused by a GAG deletion at the TOR1A gene, which produces a protein that alters intracellular membranes connections and protein processing. The typical phenotype starts from three to 26 years of age, it has penetrance of 30 % and 60% of the subjects will progress to a generalized or multifocal involvement. The DYT6/THAP1 gene has several polymorphisms described, believed to be the second in prevalence among the hereditary dystonias. DYT6 has an even lower penetrance. It is characteristically an early-onset, dystonia with craniocervical and speech involvement. The frequency, etiology and genetic alterations of dystonia are not known in the Brazilian population, which due to its specific characteristics may not share of the same genetic background. Our aim was to evaluate genetically and clinically a sample of Brazilian patients with dystonia. Subjects were recruited from the Outpatient Movement Disorders and Dystonia Clinics. Patients were assessed by a standardized questionnaire, followed by molecular testing for DYT1 and DYT6. Our cohort showed similar results with the literature, where young, generalized and positive family history commonly group together, as focal and segmental cases that appear in adulthood tend to stabilize. Sequencing of the DYT1 gene found a known missense mutation that contributes to low penetrance in individual with the typical DYT1 mutation but might be a risk factor when found in isolation. Sequencing of the DYT6 gene showed a novel 6bp deletion at the end of exon 1, causing alternate splicing and a premature stop codon in preliminary analysis. Subdividing phenotypes and understanding common molecular pathways through new genetic data is in the future of dystonia research / Mestrado / Fisiopatologia Médica / Mestra em Ciências

Position weight matrices (PWMs) have become a tool of choice for the identification of transcription factor binding sites in DNA sequences. PWMs are compiled from experimentally verified and aligned binding sequences. PWMs are then used to computationally discover novel putative binding sites for a given protein. DNA-binding proteins often show degeneracy in their binding requirement, the overall binding specificity of many proteins is unknown and remains an active area of research. Although PWMs are more reliable predictors than consensus string matching, they generally result in a high number of false positive hits. A previous study introduced a novel method to PWM training based on the known motifs to sample additional putative binding sites from a proximal promoter area. The core idea was further developed, implemented and tested in this thesis with a large scale application. Improved mono- and dinucleotide PWMs were computed for Drosophila melanogaster. The Matthews correlation coefficient was used as an optimization criterion in the PWM refinement algorithm. New PWMs keep an account of non-uniform background nucleotide distributions on the promoters and consider a larger number of new binding sites during the refinement steps. The optimization included the PWM motif length, the position on the promoter, the threshold value and the binding site location. The obtained predictions were compared for mono- and dinucleotide PWM versions with initial matrices and with conventional tools. The optimized PWMs predicted new binding sites with better accuracy than conventional PWMs.

Analysis of gene expression has undergone a technological revolution. What was impossible 6 years ago is now routine. High-throughput DNA sequencing machines capable of generating hundreds of millions of reads allow, indeed force, a major revision toward the study of the genome’s functional output—the transcriptome. This thesis examines the history of DNA sequencing, measurement of gene expression by sequencing, isoform complexity driven by alternative splicing and mammalian piRNA precursor biogenesis. Examination of these topics is framed around development of a novel RNA-templated DNA-DNA ligation assay (SeqZip) that allows for efficient analysis of abundant, complex, and functional long RNAs. The discussion focuses on the future of transcriptome analysis, development and applications of SeqZip, and challenges presented to biomedical researchers by extremely large and rich datasets.