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abstract: All biological processes like cell growth, cell differentiation, development, and aging requires a series of steps which are characterized by gene regulation. Studies have shown that gene regulation is the key to various traits and diseases. Various factors affect the gene regulation which includes genetic signals, epigenetic tracks, genetic variants, etc. Deciphering and cataloging these functional genetic elements in the non-coding regions of the genome is one of the biggest challenges in precision medicine and genetic research. This thesis presents two different approaches to identifying these elements: TreeMap and DeepCORE. The first approach involves identifying putative causal genetic variants in cis-eQTL accounting for multisite effects and genetic linkage at a locus. TreeMap performs an organized search for individual and multiple causal variants using a tree guided nested machine learning method. DeepCORE on the other hand explores novel deep learning techniques that models the relationship between genetic, epigenetic and transcriptional patterns across tissues and cell lines and identifies co-operative regulatory elements that affect gene regulation. These two methods are believed to be the link for genotype-phenotype association and a necessary step to explaining various complex diseases and missing heritability. / Dissertation/Thesis / Doctoral Dissertation Biomedical Informatics 2020

Introdução: Até bem pouco tempo atrás a análise mutacional de regiões gênicas não codificantes raramente eram reportadas em estudos genéticos sobre doenças hereditárias. Geralmente, o enfoque desses estudos recaiam sobre as regiões ditas codificantes, também conhecidas como regiões exônicas. Com o progresso da tecnologia em biologia molecular, especialmente após o desenvolvimento do sequenciamento em larga escala, temos observado um avanço jamais visto antes no entendimento de como opera o genoma humano e consequentemente uma mudança de paradigmas. Estudos mais recentes tem abrangido consistentemente a análise mutacional de regiões não codificantes ou regulatórias. No caso da neuropatia de \"Charcot-Marie-Tooth\" (CMT), pouco ainda se sabe sobre os fenótipos resultantes de mutações localizadas em sequencias regulatórias dos genes relacionados a doença. Por exemplo, na CMT ligada ao cromossomo X do tipo 1 (CMTX1), que é causada por mutações no gene GJB1 que codifica para a proteína canal conexina 32, apenas 0.01% das mutações já detectadas no gene estão presentes na região 5\' UTR (Untranslated Region) (KABZINSKA et al., 2011). Objetivos: Em vista disso, o propósito deste estudo foi a analise mutacional da região não codificante 5\' UTR e região promotora P2 do gene GJB1. Mais precisamente, 600 pares de bases upstream a open Reading frame (ORF) do transcrito do gene GJB1 expresso no sistema nervoso. Métodos: Foram investigados 100 pacientes em andamento no ambulatório de neurologia do Hospital das Clinicas da Faculdade de medicina de Ribeirão preto USP (FMRP - USP) com sintomas sugestivo de CMTX do tipo 1 e que não apresentaram mutação alguma na região codificadora do mesmo gene em triagens posteriores. Para tanto foram realizadas as técnicas de PCR (para amplificação do DNA) e posteriormente o sequenciamento do produto amplificado por meio do sequenciamento de Sanger. Resultados: Três variantes foram detectadas em 4 pacientes não relacionados, duas alterações intrônicas (já descritas) e uma nova alteração, encontrada no exon 1B (não codificante) do gene GJB1. Conclusão: Os resultados obtidos em nosso estudo sugerem que mutações na região 5\' UTR do gene GJB1 na população brasileira são incomuns e aparentemente não são responsáveis por casos sugestivos de CMTX1 sem causa genética atribuída. / Introduction: Until recently mutational analysis of non-coding regions of the DNA were rarely reported on genetic studies about inherited disorders. Usually, the approach of those studies relied primarily on the analysis of the coding regions of the DNA, also known as exonic regions. However, with the progress of science in molecular biology, especially after the development of the high throughput sequencing technology, we have observed an advance never seen before regarding of how the human genome operates and consequently a paradigm shift. New studies have consistently covered these regulatory or non-coding regions of the DNA. In the case of Charcot-Marie-Tooth disease (CMT), little is known about the phenotypes resulting from mutations in the regulatory sequences of the genes related to the disease. For example, in the X linked CMT type 1 (CMTX1), caused by alterations in the GJB1 gene that encodes for the Channel like protein connexin32 (CX32), only 0.01% of the mutations ever found in the gene were actually localized in its 5\' untranslated region (5\'UTR) (KABZINSKA et al., 2011). Objectives: Considering it, the purpose of this study was the mutational analysis of the 5\' non-coding region and promoter region P2 of GJB1 gene. More specifically, 600 base pairs (bp) upstream of the open reading frame (ORF) of the transcript expressed in the nervous system (NS). Methods: 100 patients in progress in the Clinical Hospital of the Medical School at University of São Paulo presenting suggestive clinical diagnoses of CMTX1 were screened for mutations in the region of interest, after mutations in the coding region of GJB1 gene were excluded in previous analysis. Therefore, we performed a Polimerase Chain Reaction (to amplify the DNA) followed by sequencing of the amplified material via Sanger sequencing. Results: We detected 3 alterations in 4 unrelated patients, 2 intronic variants (already described) and a novel mutation in the non-coding exon 1B of the GJB1 gene. Conclusion: The obtained results in our study suggest that mutations in the 5\'UTR of the GJB1 gene in the Brazilian population are uncommon and apparently they are not a major cause of CMTX1 in genetic unassigned patients with a suggestive clinical diagnosis of CMTX1.

The complete sequence of mitochondrial DNA from Diuraphis noxia was obtained and characterized. The mitogenome contains a standard set of 13 protein-coding genes, 19 tRNA genes, 2 ribosomal RNA genes. A+T-rich and ?repets? regions in the same order as those of the other analyzed aphids. Comparison to mtDNAs from other Sternorrhyncha species obtained from GenBank revealed possible markers for studies on population differentiation. Phylogenetic analysis using parsimony and maximum likelihood confirmed the classification of Diuraphis noxia into the Aphididae.

Inflammatory Bowel Disease (IBD) is a chronic disorder that affects millions of peopleworldwide. Although the etiology behind the disease is yet unknown, current theoriespropose a complex interplay between genetic susceptibility, exposure to environmentalfactors and exacerbated immune responses. While important efforts have been made to linkgenetics and environmental factors to IBD pathogenesis, a major challenge remains to assignthem a causative role. Particularly since most of the IBD-risk genetic polymorphisms arefound in non-coding regions (NCRs) with unknown regulatory activity, and for the lack ofknowledge about how environmental factors can modulate the function of these elements invivo . A main problem to address this challenge in IBD research is the lack of an appropriatemodel system in vivo that allows for high-throughput experiments with combinations ofdifferent IBD-risk factors, while keeping the in vivo context. In this work, we sought toovercome this issue by using a zebrafish reporter for a specific human IBD-risk NCR, inorder to investigate the modulation of this element by two groups of common environmentalfactors: pollutants, such as PolyFluoroAlkyl Substances (PFASs); and diet, by activation ofdietary sensors. We found that the activity of the WT-NCR in zebrafish larvae was increased in the presenceof PFAS, while the activation of the dietary sensor PPAR δ decreased the activity. These datalead us to suggest that the function of PFAS can be counteracted by PPARδ activation.Therefore, we propose zebrafish as a suitable in vivo model in which we can screen forpotentially harmful or beneficial effects of environmental factors in the activity of humannon-coding regions. / Inflammatorisk tarmsjukdom (IBD) är en kronisk störning som drabbar miljontals människorvärlden över. Även om etiologin bakom sjukdomen fortfarande är okänd, föreslår nuvarandeteorier ett komplext samspel mellan genetisk mottaglighet, exponering av miljöfaktorer ochförvärrat immunförsvar. Även om stora ansträngningar har gjorts för att koppla genetik ochmiljöfaktorer till IBD-patogenes, återstår en stor utmaning att tilldela dem en orsakande roll.Särskilt eftersom de flesta av IBD-riskgenetiska polymorfismer finns i icke-kodande regioner(NCR) med okänd reglerande aktivitet samt för bristen på kunskap om hur miljöfaktorer kanmodulera funktionen hos dessa element in vivo . Ett huvudproblem för att möta dennautmaning i IBD-forskning är avsaknaden av ett lämpligt modellsystem in vivo som möjliggörexperiment med hög kapacitet och kombinationer av olika IBD-riskfaktorer in vivo . I dettaarbete försökte vi få svar på denna fråga genom att använda en zebrafiskreporter för ettspecifikt humant IBD-risk icke-kodande område. Detta möjliggjorde att vi kunde undersökamodulering av två gemensamma miljöfaktorer: föroreningar, såsom PolyFluoroAlkyl-ämnen(PFASs); och diet, genom aktivering av dietsensorer. Vi fann att aktiviteten i WT-NCR hos zebrafisklarver ökade i närvaro av PFAS, medanaktiveringen av dietsensorn PPARδ minskade aktiviteten. Denna data leder till att vi antyderatt funktionen för PFAS kan motverkas genom PPARδ-aktivering. Därför föreslår vizebrafisk som en lämplig in vivo -modell, i vilken vi kan screena för potentiellt skadliga ellergynnsamma effekter av miljöfaktorer i mänskligt icke-kodande DNA.

Els microsat el lits s on seq u encies d'ADN formades per repeticions en t andem de motius curts. Les curtes seq u encies repetides en t andem s on ubiq ues en els genomes dels eucariotes, tant en les regions codi cants com en les regions no codi cants. Aquestes seq u encies tenen un nivell molt elevat de polimor sme i de diverg encia interespec ca. Hem investigat si les dades obtingudes mitjan cant la seq uenciaci o de nova generaci o del Projecte Pilot dels 1000 Genomes s on utils per quanti car la variabilitat dels microsat el lits en les poblacions humanes i per descobrir nous loci hipot eticament implicats en malalties causades per l'expansi o de repeticions de trinucle otids. Hem analitzat la conservaci o ologen etica dels microsat el lits per entendre el rol que juga la selecci o en l'evoluci o dels microsat el lits. El primer estudi conclou que en els llinatges dels vertebrats, les repeticions en t andem d'amino acids estan m es conservades que altres seq u encies similars localitzades a les regions no codi cants. Aix o ens porta a concloure que l'evoluci o ha mantingut les repeticions a les regions codi cants de les prote nes. En una segona fase hem analitzat la conservaci o dels microsat el lits en diferents regions gen omiques, comparant-les amb la conservaci o dels microsat el lits a les regions interg eniques. Concloem que la selecci o no mant e nom es els microsat el lits als exons, sin o que tamb e a altres regions gen omiques. / Microsatellites are DNA sequences formed by tandem repetition of short motifs. Short sequence tandem repeats are ubiquitous in eukaryotic genomes both in coding and non-coding regions. They show a very high level of polymophism and interspeci c divergence. We investigated the use of next generation sequencing data, from the 1000 Genomes Pilot Prjects, to quantify microsatellite variability in the human population and discover putative new loci involved in trinucleotide repeat expansion diseases. We analysed microsatellites phylogenetic conservation to learn about the role of selection in shaping microsatellite evolution. The rst study con- cluded that in vertebrate lineages amino acid tandem repeats were more conserved than similar sequences located in non-coding regions. This lead us to the conclusion that evolution was preserving repeats in protein-coding regions. In a second stage we analzed the conservation of microsatellites in di erent genomic regions, comparing them with the of microsatellite in inter- genic region. We concluded that selection was not preserving microsatellites only in exons but also in other genomic regions. 1

Inactivation of tumor supressor gene BRCA1 causes a life-long risk of breast carcinoma development. Genetic screenings of indicated individuals from high-risk families help to identify large number of sequence variants in known predisposing genes. Majority of discovered variants doesn't have clinical significance yet which causes a big problem for diagnostics. Some of these variants are found within regulatory non-coding regions of gene. A part of the clinical classification of variants is their functional characterization. The goal of this thesis was to create a model system for functional characterization of variants in non-coding regions and to verify its function. Model system was based on targeted gene manipulation by co-transfecting CRISPR-Cas9 construct and donor construct that contained a portion of BRCA1 gene sequence with analyzed modifications, into U2 OS cells. The cells have stably integrated DR-GFP system which allows the activity of homologous recombination (HR) to be determined. Monoallelic modifications were induced into U2 OS cells. These modifications were in a Kozak sequence region of BRCA1 gene. Expression level of BRCA1 mRNA was determined by qRT-PCR, which showed the same levels of mRNA in all cells with analyzed alterations. Next, expression level of BRCA1 protein was...