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Orientadores: Íscia Teresinha Lopes Cendes, Fabio Rossi Torres Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas Resumo: As epilepsias constituem um dos grupos de doenças neurológicas graves que afetam de 1,5 - 2% da população mundial. Diversos tipos de malformações do desenvolvimento cortical (MDC) estão frequentemente associados à ocorrência de crises refratárias, incluindo a Displasia Cortical Focal (DCF) que se caracteriza por alterações citoarquiteturais também observadas em outras MDC, como a Esclerose Tuberosa (ET) e a Hemimegaencefalia (HME). Uma forte associação entre ET, HME e mutações em mosaicismo tem sido demonstrada, e a identificação desse tipo de mutação pode contribuir para o entendimento dessas doenças. Portanto, o principal objetivo deste trabalho foi investigar variantes genéticas relacionadas à etiologia das DCF. Para tal, foi utilizado como estratégia o Deep Sequencing por NGS. Sequenciamos o DNA genômico de tecido cerebral proveniente de ressecção cirúrgica e do sangue periférico de pacientes com DCF. Realizamos o sequenciamento de um painel customizado de genes da via mTOR/GATOR e validamos com a técnica de PCR digital. Encontramos duas variantes em mosaico no tecido cerebral, localizadas no gene MTOR (p.Leu1460Pro e p.Ala1459Pro) em dois pacientes distintos e não aparentados com DCF do tipo IIb, correspondendo a 16,6% dos pacientes testados (2/12). A porcentagem de mosaicismo no tecido cerebral foi de 0,75% e 3,98% respectivamente nos dois pacientes. Além disso, para compararmos o perfil genético da DCF com outra MDC, realizamos o sequenciamento do mesmo painel customizado em DNA extraído do sangue de 24 pacientes com o diagnóstico clínico de ET, e encontramos mutações germinativas nos genes TSC1 e TSC2 em 54,5% dos pacientes analisados (12/22), e uma variante potencialmente patogênica no gene IRS1 em um único paciente. Completamos nosso estudo dos pacientes com DCF pesquisado também alterações cromossômicas estruturais (CNVs), utilizamos array cromossômico de citogenética molecular e validamos por qPCR. Encontramos com potencial patogênico uma CNV com deleção envolvendo o gene NOTCH2 no mesmo paciente que encontramos mutação em mosaico no gene MTOR, podendo se tratar de um caso de segundo-evento mutacional. Desse modo concluímos que 16.6% dos pacientes com DCF apresenta mutações em mosaico no tecido cerebral em genes da via mTOR/GATOR. Assim especulamos que se mutações em mosaico são realmente uma causa frequente de DCF é possível que exista o acometimento de outras vias, além da via mTOR/GATOR investigada nesse trabalho. Encontramos mutações germinativas predominantemente nos genes TSC1 e TSC2 na maioria dos pacientes com ET, o que indicaria uma etiologia genética nesses pacientes fundamentalmente diferente daquela encontrada nos pacientes com DCF. Ao encontramos alterações estruturais cromossômicas em um paciente com DCF, mostramos a importância da investigação desse tipo de mutação nesses pacientes. Desse modo, concluímos que nosso trabalho contribuiu para um melhor entendimento da etiologia genética das DCFs Abstract: Epilepsies constitute one of the groups of serious neurological diseases that affect 1.5 - 2% of the world population. Several types of malformations of cortical development (MDC) are frequently associated with the occurrence of refractory crises, including Focal Cortical Dysplasia (FCD) which is characterized by cytoarchitectural changes also observed in other MDC, such as Tuberous Sclerosis (TS) and Hemimegaencephaly (HME). A strong association between TS, HME and mosaicism mutations has been demonstrated, and the identification of this type of mutation can contribute to the understanding of these diseases. Therefore, the main goal of this work was to investigate genetic variants related to the etiology of FCD. To this end, Deep Sequencing by NGS was used as a strategy. We sequenced genomic DNA from brain tissue from surgical resection and from peripheral blood of patients with FCD. We performed the sequencing of a customized panel of genes from the mTOR/GATOR pathway and validated it with the digital PCR technique. We found two mosaic variants in brain tissue, located in the MTOR gene (p.Leu1460Pro and p..Ala1459Pro) in two distinct and unrelated patients with FCD type IIb, corresponding to 16.6% of the patients tested (2/12). The percentage of mosaicism in brain tissue was 0.75% and 3.98% respectively in the two patients. Furthermore, to compare the genetic profile of FCD with another MDC, we performed sequencing of the same customized panel on DNA extracted from the blood of 24 patients with the clinical diagnosis of TS, and we found germline mutations in the TSC1 and TSC2 genes in 54.5% of the analyzed patients (12/22), and a potentially pathogenic variant in the IRS1 gene in a single patient. We completed our study of patients with FCD, also researched structural chromosomal alterations (CNVs), used a chromosomal array of molecular cytogenetics and validated by qPCR. We found a CNV with a deletion involving the NOTCH2 gene with pathogenic potential in the same patient that we found a mosaic mutation in the MTOR gene, it may be a case of a two-hit event. Thus, we conclude that 16.6% of patients with FCD have mosaic mutations in brain tissue in genes of the mTOR/GATOR pathway. Thus, we speculate that if mosaic mutations are indeed a frequent cause of FCD, it is possible that other pathways are involved, in addition to the mTOR/GATOR pathway investigated in this work. We found germline mutations predominantly in the TSC1 and TSC2 genes in most patients with TS, which would indicate a genetic etiology in these patients fundamentally different from that found in patients with FCD. When we found chromosomal structural alterations in a patient with FCD, we showed the importance of investigating this type of mutation in these patients. Thus, we conclude that our work contributed to a better understanding of the genetic etiology of FCDs Doutorado Fisiopatologia Médica Doutora em Ciências FAPESP 2015/19768-1

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No. of bitstreams: 1 rossi_gam_dr_jabo.pdf: 3246904 bytes, checksum: 2ed62292bd3bfa8f9ec88fe41c5bbaa2 (MD5) Previous issue date: 2017-12-06 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) As bactérias pertencentes ao grupo do Bacillus cereus são importantes para indústrias processadoras de leite e produtos lácteos devido à capacidade de sobrevivência aos tratamentos térmicos utilizados durante os processamentos, e consequente deterioração dos produtos e risco à saúde pública. Assim, inicialmente, objetivou-se compreender a estrutura populacional de isolados pertencentes ao grupo do B. cereus na cadeia produtiva do leite e produtos lácteos. Por meio da utilização de amostragem estruturada e técnicas genômicas comparativas, investigou-se quais linhagens específicas e genes estão significativamente associados a estágios de produção específicos e produtos. Os genomas de 69 isolados obtidos de equipamentos, leite cru e produtos lácteos foram comparados com outros 193 disponíveis de diversas origens. A estrutura populacional incluiu os conhecidos grupos filogenéticos II, III, IV, V e VI, e quase todos os isolados dos produtos lácteos pertenceram ao grupo III. A investigação de genes específicos revelou um grande número de isolados carreando aqueles relacionados à produção de toxinas, como o cytK (53,62%), hblA (59,42%), hblC (44,93%), hblD (53,62%), nheA (84,06%), nheB (89,86%), nheC (84,06%), cesA (2,90%) e cesB (2,90%). Os isolados pertencentes aos grupos IV e V possuíram uma prevalência significativamente maior dos genes hblACD e o grupo IV do gene cytK. Os isolados obtidos de produtos lácteos tiveram uma prevalência significativamente menor dos genes cytK e hblACD comparados aos de equipamentos e leite cru/tanques de refrigeração. A análise genômica populacional demonstrou a diversidade dos isolados e a variedade de funções dentro do grupo do B. cereus da cadeia produtiva leiteira, com grande número de isolados potencialmente capazes de causar doenças alimentares. Posteriormente, objetivou-se avaliar a viabilidade do processo de tindalização de leite cru refrigerado e determinar se o processo de pasteurização do leite influencia na expressão dos genes relacionados à produção das toxinas diarreicas pelo B. cereus s.s. em leite experimentalmente contaminado. Para isso, realizou-se um processo de tindalização de leite cru refrigerado e então foi realizada a contaminação com um isolado potencialmente toxigênico, e, posteriormente, o mesmo foi pasteurizado, envasado e mantido em refrigeração durante 10 dias. Em momentos determinados, foram coletadas amostras do produto afim de avaliar a expressão de genes codificadores de toxinas (hblACD, nheABC e cytK). O protocolo de tindalização utilizado permitiu redução logarítmica da população de bactérias do grupo do B. cereus e foi possível detectar a expressão do gene hblA em amostras de leite 5 e 10 dias após a pasteurização. Conclui-se que o modelo proposto foi adequado e que a expressão do gene hblA não foi inibida após a realização da pasteurização do leite, demonstrando o potencial risco aos consumidores decorrentes do consumo de leite pasteurizado contaminado. The bacteria belonging to Bacillus cereus group are important to dairy industries due their ability to survive to thermal treatments used during processing, and consequently cause food spoilage and risk to public health. Thus, firstly, this study aimed to better understand the population structure of B. cereus group isolates in dairy production chain. Using structured sampling of B. cereus in dairy production chain and comparative genomics techniques, we investigated if specific lineages and genes are significantly overrepresented in particular production stages. The genomes of 69 isolates obtained from equipments, raw milk and dairy products were compared with others 193 avaiable from several origins. The populational structure included the known phylogenetic groups II, III, IV, V and V, and almost all isolates from dairy products belonged to group III. The genes investigation showed a high number of isolates carrying genes related to toxins production, such as cytK (53.62%), hblA (59.42%), hblC (44.93%), hblD (53.62%), nheA (84.06%), nheB (89.86%), nheC (84.06%), cesA (2.90%) and cesB (2.90%). The isolates belonging to groups IV and V had a significant higher prevalence of genes hblACD an group VI of cytK. The isolates obtained from dairy products had a significant lower prevalence of genes cytK and hblACD compared to those from equipments and raw milk/bulk tanks. The populational genomic analyses showed the diversity of isolates and variability of functions in B. cereus group in dairy production chain, with a high number of isolates potentially able to cause foodborne disease. Posteriorly, this study aimed to evaluate the viability of tyndallizating raw milk and to establish if milk’s pasteurization influences on the expression of genes related to the production of diarrheal toxins by B. cereus s.s. using a milk contamined experimentally. For this purpose, the tyndallization of raw milk was performed and later it was contaminated with a potentially toxigenic isolate, and, posteriorly, it was pasteurized, packaged and kept under refrigeration during 10 days. In established periods, samples of this product were collected in order to evaluate the expression of genes related to toxins production (hblACD, nheABC and cytK). The protocol used for tyndallization allowed a logarithmic reduction of population of B. cereus group and the expression of hblA gene was detected in samples from milk after 5 and 10 days after pasteurization. It was concluded that the proposed model was appropriate and the expression of hblA gene was not inihibited after milk pasteurization, highlighting the potential risk for consumers through consumption of contamined pasteurized milk. 2016/19214-9 2014/13104-1

Introduction: Lysosomal Storage Disorders (LSD) are a heterogenous group of rare, monogenic diseases with significant phenotypic overlap and clinical variability. For this reason, the diagnosis is difficult and time consuming, with multiple tests/samples being often required before a definitive diagnosis is reached. Next Generation Sequencing (NGS) is changing this scenario by allowing the variant assessment at a large scale in a single run. The aim of this work, was to develop an NGS-based workflow for the identification of LSD-causing variants. Methods: We designed a panel including exons and intronic flanking regions from 96 genes involved in lysosome homeostasis and function. The workflow was performed using an Agilent SureSelect QXT Target Enrichment protocol followed by sequencing in an Illumina MiSeq® platform. For alignment and variant anotation the softwares Surecall and wANNOVAR were used. This work was partially supported by N2020 (NORTE2020/DESVENDAR). info:eu-repo/semantics/publishedVersion

Background The extraordinary success of imatinib in the treatment of BCR-ABL1 associated cancers underscores the need to identify novel functional gene fusions in cancer. RNA sequencing offers a genome-wide view of expressed transcripts, uncovering biologically functional gene fusions. Although several bioinformatics tools are already available for the detection of putative fusion transcripts, candidate event lists are plagued with non-functional read-through events, reverse transcriptase template switching events, incorrect mapping, and other systematic errors. Such lists lack any indication of oncogenic relevance, and they are too large for exhaustive experimental validation. Results We have designed and implemented a pipeline, Pegasus, for the annotation and prediction of biologically functional gene fusion candidates. Pegasus provides a common interface for various gene fusion detection tools, reconstruction of novel fusion proteins, reading-frame-aware annotation of preserved/lost functional domains, and data-driven classification of oncogenic potential. Pegasus dramatically streamlines the search for oncogenic gene fusions, bridging the gap between raw RNA-Seq data and a final, tractable list of candidates for experimental validation. Conclusion We show the effectiveness of Pegasus in predicting new driver fusions in 176 RNA-Seq samples of glioblastoma multiforme (GBM) and 23 cases of anaplastic large cell lymphoma (ALCL). Contact: fa2306@columbia.edu.

TopHat is a fast splice junction mapper for Next Generation Sequencing analysis, a technology for functional genomic research. Next Generation Sequencing technology allows more accurate analysis increasing data to elaborate, this opens to new challenges in terms of development of tools and computational infrastructures. We present a solution that cover aspects both software and hardware, the first one, after a reverse engineering phase, provides an improvement of algorithm of TopHat making it parallelizable, the second aspect is an implementation of an hybrid infrastructure: grid and virtual grid computing. Moreover the system allows to have a multi sample environment and is able to process automatically totally transparent to user.