The aim of DNA sequencing is to obtain the order of DNA composition comprising the base pairs A (adenine) T (thymine), and C (cytosine) G (guanine). The fast development of DNA sequencing technology allows us to better understand the relationships among diseases, inheritance, and individuality. Solid state nanopores have been recommended as the next generation platform for DNA sequencing due to its low-cost and high-throughput. In particular, nanopores fabricated from graphene sheets are extremely thin and structurally robust and have been extensively used in DNA detection in recent years. In DNA sequencing, the translocation of a DNA molecule through a nanopore is known to be a very complicated issue and is affected by many factors, such as ion concentration, thickness of the nanopore, and the nanopore diameter. The technique of molecular dynamic simulations has been a complementary tool to study DNA translocation through nanopores.       In this thesis, I summarize my work of computational studies of DNA sequencing using graphene nanopores. These studies include: DNA translocation through single-layer graphene nanopores of different diameters under conditions of various ion concentrations and applied voltages; DNA translocation through multilayer graphene nanopores varied from a single to a few layers; pulling out single strand DNA molecules from small graphene nanopores of different geometries. The major contributions of this work include: 1. Effects of bias voltage on DNA translocation time were investigated leading to the insight that lower applied voltages can extend the time of DNA translocation through monolayer graphene nanopores. The effect of salt concentration on the corresponding ionic current was studied. At a low ionic concentration (&lt; 0.3M), the current increases as DNA translocates through a nanopore. However, at a high ionic concentration (&gt;0.5M), the current decreases as DNA translocates through the nanopore. A theoretical model was proposed to explore the relationship between the current and the occupied nanopore area. We demonstrated that the DNA translocation time can be prolonged by narrowing the diameter of a nanopore properly and the reduction of the blockade current depends on the ratio of the unoccupied nanopore area to the total nanopore area. 2.  DNA translocation through multilayer graphene nanopores was studied by molecular dynamics simulations with the aim to achieve single-base resolution. We show that the DNA translocation time can be extended by increasing the graphene layers up to a moderate number (7) and that the current in DNA translocation undergoes a stepwise change upon DNA going through an multi-layer graphene (MLG) nanopore. A model was built to account for the relationship between the current change and the unoccupied volume of the MLG nanopore. We demonstrate that the blockade current is closely related to the unoccupied volume. The dynamics of DNA translocation depends specifically on the interaction of nucleotides with the graphene sheet. Thus, our study indicates that the resolution of DNA detection can be improved by increasing the number of graphene layers in a certain range and by modifying the surface of graphene nanopores. 3. The effect of graphene nanopore geometry on DNA sequencing has been assessed by steered molecular dynamics simulations. DNA fragments including A, T, C, G and 5-methylcytosine (MC) were pulled through graphene nanopores of different geometries with diameters down to ~1nm by steered molecular dynamics simulations. We demonstrated that the bases (A, T, C, G, and MC) can be indentified in single-base resolution by the characteristic force peak values in a circular graphene nanopore but not in graphene nanopores of other geometries. Symmetric nanopores are thus better suited to DNA sequence detection via force curves than asymmetric nanopores. This implies that the graphene nanopore surface should be modified as symmetric as possible to sequence DNA by an atomic force microscope or optical tweezers. This helps us to understand low-cost and time-efficient DNA sequencing in narrow nanopores. 4. The translocation time for different nucleotides to pass through graphene nanopores with certain diameters was investigated. It was found that the translocation times are different for different bases under a low electric field. The results indicate that DNA can be sequenced by the translocation time to pass through a graphene nanopore. 5. Inspired by the structure of K+ channel proteins, a series of oxygen doped graphene nanopores of different size were designed to discriminate the transport of K+ and Na+ ions. The results indicate that the ion selectivity of such biomimetic graphene nanopores can be simply controlled by the size of the nanopore.  Compared to K+, the smaller radius of Na+ leads to a much higher free energy barrier in the nanopore of a certain size. / <p>QC 20141212</p>

The Centromere is a vital chromosomal structure that ensures faithful segregation of replicated chromosomes to their respective daughter cells. With such an important structure, one would expect the underlying centromeric DNA sequence would be highly conserved across all species. It turns out that the underlying centromeric DNA sequences between species ranging from the yeast, fly, mouse to humans are in fact highly diverged suggesting a DNA sequence independent or an epigenetic mechanism of centromere formation. / Neocentromeres are centromeres that form de-novo at genomic locations that are devoid of highly repetitive a-satellite DNA sequences of which normal centromeres are usually comprised from. To date, the 10q25 neocentromere is the most well-characterised, fully functional human centromere that has been used previously to characterise the extent of a number of centromeric protein binding domains and characterise the properties of the underlying DNA sequence. Along with other factors, the existence of neocentromeres has given rise to a hypothesis where centromeres are defined by epigenetic or DNA sequence independent mechanisms. / The putative 10q25 neocentromere domain was recently redefined by high resolution mapping of Centromeric protein A (CENP-A) binding through a chromatin immunoprecipitation and array (CIA) analysis. The underlying DNA sequence was investigated to determine and confirm that the formation of the 10q25 neocentromere was through an epigenetic mechanism. Through a high-density restriction fragment length polymorphism (RFLP) analysis using overlapping PCR amplified DNA derived from genomic DNA representing the 10q25 region before and after neocentromere activation. No sequence polymorphisms, large insertions or deletions were detected and confirmed the epigenetic hypothesis of centromere formation. / DNA methylation is one of many epigenetic factors that are important for cellular differentiation, gene regulation and genomic imprinting. As the mechanisms and functions of DNA methylation have been well characterised, its role at the 10q25 neocentromere was investigated to try and identify the candidate epigenetic mechanism involved in the formation of centromeres. DNA methylation across the neocentromere was assessed using sodium bisulfite PCR and sequencing of selected CpG islands located across the 10q25 neocentromere. Overall, the methylation level of the selected CpG islands demonstrated no difference in DNA methylation before and after neocentromere activation. However, significant hypomethylation upon neocentromere formation was detected close to the protein-binding domain boundaries mapped previously suggesting that this may have a role in demarcating protein binding domains at the neocentromere. / Further analysis of DNA methylation investigated non-CpG island methylation at sites defined as CpG islets and CpG orphans. Interestingly, the DNA methylation level measured at selected CpG islets and CpG orphans across the 10q25 neocentromere were not completely hypermethylated as previously thought, but demonstrated variable methylation that became fully hypermethylated upon neocentromere activation in most sites investigated. These results suggested that a role for DNA methylation existed at the 10q25 neocentromere and that it occurred at sites devoid of CpG islands. / This study has found that DNA methylation at non-CpG island sites was variable contrary to popular belief and, was linked with neocentromere formation through the observation of increased DNA methylation at the 10q25 neocentromere. Inhibition of DNA methylation demonstrated increased neocentromere instability and a decrease in methylation of these CpG islets and CpG orphans confirming the importance of DNA methylation at neocentromeres. This study has characterised a new class of sequences that are involved in the maintenance of chromatin structure through DNA methylation at the 10q25 neocentromere.

[pt] Esta dissertação apresenta um estudo de caso em seqüenciamento da produção de bobinas de aço em um laminador de tiras a quente com enfornamento a frio das placas de aço. O laminador em questão é o da Companhia Siderúrgica de Tubarão, uma grande usina siderúrgica integrada localizada no estado do Espírito Santo, Brasil. Seu start up está previsto para o segundo semestre de 2002. Num primeiro momento, o problema parece ser de tratamento complexo, comportando-se como um grande problema combinatorial com restrições difíceis de serem representadas. Algumas considerações para um bom seqüenciamento são comuns tanto no forno de reaquecimento quanto no laminador desbastador. Isto permite que as placas possam ser seqüenciadas como se esses dois equipamentos fossem um único estágio (equipamento) para alguns agrupamentos de produtos. Assim, o foco pode ser deslocado para o seqüenciamento no estágio final do laminador, isto é no trem acabador. Inicialmente, a idéia era resolver um modelo da mochila compartimentada para a seleção das placas de aço a serem processadas entre duas trocas consecutivas de cilindros de trabalho do trem acabador e usar algumas heurísticas para seqüenciá-las dentro de cada compartimento (faixa de largura). Um estudo do mix de produção planejado mostrou que, devido à grande variedade e quantidade de ordens, a fase de seleção não era crítica e o seqüenciamento poderia ser feito semimanualmente com ajuda do computador. Para aumentar a vida útil dos cilindros de trabalho, assim como atender a qualidade desejada e a data de entrega prometida, no decorrer de uma boa seqüência de laminação deve ocorrer decréscimo nas larguras e na qualidade superficial, trocas suaves de espessuras e de dureza. O sistema computacional proposto implementa um algoritmo simples que seqüencia lexicograficamente o grupo de produtos a ser laminado de acordo com as prioridades dadas a cada critério de seqüenciamento pelo próprio programador da produção, que pode interagir e trocá-las de acordo com as condições prevalecentes. / [en] This thesis reports a case study on scheduling the cold charged production of coils in a hot strip mill with start- up planned for the second semester of 2002 at Companhia Siderúrgica de Tubarão, a large integrated steel plant in Espírito Santo, Brazil. At first glance, the problem seems intractably complex, being essentially a very large-scale combinatorial problem with ill-defined and difficult to represent constraints. Considerations on the requirements for good sequencing at the reheating and roughing stages showed that they could be scheduled as a single stage for some groups of products, and in doing so, the focus could be on sequencing the finishing stage. Initially, the idea was to solve a compartmented knapsack model for selecting the slabs to be processed between two consecutive changes of working rolls, and using some heuristics for sequencing within each compartment (width range). Examination of the projected product mix showed that, due to the large number and variety of orders, the selection phase was not critical, and the scheduling could be performed semi-manually with a simple computer aid. For extending the life of the working rolls, as well as attaining the desirable quality and promised dates, a good sequence must present decreasing width and surface quality, increasing promised dates, and smooth changes of thickness and hardness. The proposed computer system implements a simple algorithm that lexicographically sorts the set of products to be rolled according to priorities given to these criteria by the scheduler who can interactively change them according to the prevailing conditions.

Alzheimer's disease (AD) is a neurodegenerative condition affecting millions of individuals worldwide and is a major source of mortality in elderly populations. While it is well established that there is a strong genetic basis for the disease, the epigenetic mechanism underlying the disease is largely unknown. The main purpose of this thesis is to understand the alteration of epigenetic modifications associated with the disease and its progression. In particular, we examine how alterations in the cytosine methylation and cytosine hydroxymethylation, two epigenetic modifications that are critically important for the development and function of the brain, are associated with advancing stages of Alzheimer's disease. Eight progressive AD brain samples were examined for changes in DNA methylation and hydroxymethylation by both dot blot analysis and a new oxidative bisulfite (OXBS) deep sequencing technology. The initial results of dot blot analysis reveal a statistically significant decrease in 5hmC associated with intermediate stage AD among the samples. This data suggests that the alterations in epigenetic modifications is likely associated with the pathophysiology of Alzheimer's disease, not only shedding new light on our understanding of the epigenetics of the disease, but also providing the basis for our future investigation on the exact cause and effect relationships of these epigenetic changes and their respective stages in Alzheimer's.

Begonia is one of the most diverse genera on the planet, with a species count approaching 2000 and a distribution across tropics in South America, Africa and South East Asia. The genus has occupied a vast range of niches; many highly variable growth forms can be found across the distribution, and species exhibit very diverse morphologies, even in closely related species. A recent study has revealed a putative whole genome duplication (WGD) event in the evolutionary history of Begonia, which has prompted an interest in investigating the impact gene and genome duplication has had on the diversification of Begonia. To answer questions about phenotypic and ecological diversification in Begonia, two species from South America, B. conchifolia and B. plebeja were chosen as study species based on their close phylogenetic relationship and divergent ecology and phenotype. RNA-seq data for six tissues from B. conchifolia and B. plebeja was generated using the Illumina sequencing platform, and normalised relative expression data was obtained by mapping reads to transcripts predicted from the B. conchifolia draft genome. A bioinformatics pipeline was devised to compare expression profiles across 6 different tissues between duplicated gene pairs shared between B. conchifolia and B. plebeja. Gene duplicate pairs were selected as candidates if they showed divergent expression in one species but not in another. Such duplicate pairs are suggestive of neofunctionalization in one species, providing evidence of a potential basis for phenotypic divergence and diversification between B. conchifolia and B. plebeja. Two duplicate pairs were identified as showing such divergent expression patterns as well as being functionally ecologically relevant, Chalcone Synthase and 3-Ketoacyl-CoA synthase, involved in anthocyanin biosynthesis and wax biosynthesis respectively. Investigation of expression and duplication patterns in both gene families showed the candidate gene families to be strikingly different. While 3-Ketoacyl-CoA synthase showed deeper duplications shared with outgroup taxa, Chalcone Synthase appeared to be expanded very recently, with a burst of duplications specific to the genus. 3-Ketoacyl-CoA synthase showed examples of partitioned expression by tissue for different gene family members, with at least five members of the gene family being highly expressed in one or two tissues only. Chalcone Synthase, however, showed dominance of one basal gene family member. Other Chalcone Synthase members, though expressed at lower levels, showed some evidence of reciprocal silencing in B. plebeja, though this pattern was not observed in B. conchifolia. Further investigation of the Chalcone Synthase gene family revealed lineage specific duplication in B. plebeja, and more extensive differential duplication patterns were found across other South American Begonias. Additionally, signals of positive selection were found in two branches on the Chalcone Synthase phylogeny.

abstract: Single molecules in a tunnel junction can now be interrogated reliably using chemically-functionalized electrodes. Monitoring stochastic bonding fluctuations between a ligand bound to one electrode and its target bound to a second electrode ("tethered molecule-pair" configuration) gives insight into the nature of the intermolecular bonding at a single molecule-pair level, and defines the requirements for reproducible tunneling data. Importantly, at large tunnel gaps, there exists a regime for many molecules in which the tunneling is influenced more by the chemical identity of the molecules than by variability in the molecule-metal contact. Functionalizing a pair of electrodes with recognition reagents (the "free analyte" configuration) can generate a distinct tunneling signal when an analyte molecule is trapped in the gap. This opens up a new interface between chemistry and electronics with immediate implications for rapid sequencing of single DNA molecules. / Dissertation/Thesis / Ph.D. Physics 2012

Sequencing B and T cell receptor genes allows for detailed characterisation of the genetic diversity underlying adaptive immune responses in health and disease. In the context of infectious diseases this can act as a powerful tool for identification of pathogen-specific immune signatures and genetic determinants of immune memory, protection and response to re-exposure. As part of my PhD I developed and optimised a method for high-resolution profiling of B cell receptor (BCR) immune repertoires based on the barcoded sequencing of the human immunoglobulin genes. The use of molecular barcodes allowed for reduction of technical noise, which can lead to erroneous assignment of lymphocyte function. I applied this methodology to the study of natural infection with measles virus in unvaccinated children. Childhood measles causes a profound immune suppression, which can last for weeks to months post infection, with large reductions in numbers of circulating B cells. Interestingly, long-term consequences of measles immune suppression result in increased incidence of secondary infections up to 3 years after resolution of measles. Vaccination against measles virus with the MMR vaccine has been a major factor in reducing direct and secondary childhood morbidity and mortality. The maintenance of sufficient global vaccine coverage, however, has been challenging due to the refusal of vaccination, mainly in religious communities, resulting in increasing number of outbreaks worldwide. In addition to the overall drop in measles virus herd immunity, measles-induced immune suppression can compromise immunity to other infectious pathogens, thus complicating global vaccination and surveillance efforts. The exact mechanisms underlying the prolonged immune-suppression associated with measles remain elusive and have not been investigated in humans. I applied BCR sequencing to characterise the long-term immunological effects of natural measles virus infection in a cohort of unvaccinated children. Specifically, I addressed the restructuring of immune memory and the possible loss of immunity to non-measles pathogens. My work provided evidence for previously hypothesised depletion of B cell memory pools, referred to as ‘immunological amnesia’. Loss of clonally expanded B memory populations lead to immune re-setting and convergence in repertoire diversity between measles-infected and control groups. In addition to the loss of individual-specific variation in immune memory, a subset of measles-infected individuals exhibited dramatic collapse in the diversity of their naïve B cell compartment, despite the recovery of normal B naïve cell counts. An effect of measles on serological immunity was also demonstrated in a ferret model of measles, where lymphotropic challenge lead to significant loss of vaccine-acquired immunity to influenza virus. The work presented in this dissertation demonstrates the utility of BCR sequencing for understanding adaptive immune responses in the context of infectious diseases and highlights the potential of this approach to uncover novel mechanisms of immune (dys)function.

The study analyse presence and distribution of mutation in the LIF and IL-11 genes. The alterations were studied in peripheral blood samples by temperature gradient gel electrophoresis and sequencing.

abstract: The processes of a human somatic cell are very complex with various genetic mechanisms governing its fate. Such cells undergo various genetic mutations, which translate to the genetic aberrations that we see in cancer. There are more than 100 types of cancer, each having many more subtypes with aberrations being unique to each. In the past two decades, the widespread application of high-throughput genomic technologies, such as micro-arrays and next-generation sequencing, has led to the revelation of many such aberrations. Known types and subtypes can be readily identified using gene-expression profiling and more importantly, high-throughput genomic datasets have helped identify novel sub-types with distinct signatures. Recent studies showing usage of gene-expression profiling in clinical decision making in breast cancer patients underscore the utility of high-throughput datasets. Beyond prognosis, understanding the underlying cellular processes is essential for effective cancer treatment. Various high-throughput techniques are now available to look at a particular aspect of a genetic mechanism in cancer tissue. To look at these mechanisms individually is akin to looking at a broken watch; taking apart each of its parts, looking at them individually and finally making a list of all the faulty ones. Integrative approaches are needed to transform one-dimensional cancer signatures into multi-dimensional interaction and regulatory networks, consequently bettering our understanding of cellular processes in cancer. Here, I attempt to (i) address ways to effectively identify high quality variants when multiple assays on the same sample samples are available through two novel tools, snpSniffer and NGSPE; (ii) glean new biological insight into multiple myeloma through two novel integrative analysis approaches making use of disparate high-throughput datasets. While these methods focus on multiple myeloma datasets, the informatics approaches are applicable to all cancer datasets and will thus help advance cancer genomics. / Dissertation/Thesis / Ph.D. Biomedical Informatics 2014

Made available in DSpace on 2018-08-01T21:35:03Z (GMT). No. of bitstreams: 1 tese_12056_Dissertação_Amanda Silva Coutinho.pdf: 1166104 bytes, checksum: f4756c682c195491abc65c33b3ce87fc (MD5) Previous issue date: 2018-02-26 / A osteogênese imperfeita (OI) é uma doença genética rara do tecido conjuntivo, causada por mutações em genes que participam, em geral, da formação óssea. A maioria dos pacientes é portadora de mutações nos genes que codificam o colágeno tipo 1, mas já foram descritas mutações em mais de 17 outros genes causando OI e ainda existe uma busca constante de novos genes na área cientifica. Entre as estratégias de diagnóstico molecular destaca-se a técnica de sequenciamento de nova geração (NGS), que pode sequenciar vários genes presentes em uma plataforma customizada, gerando uma grande quantidade de dados genômicos. Esses dados se tornam preciosas fontes de informação na busca de novos genes relacionados a doenças. O objetivo desta pesquisa foi realizar a busca de novos genes potencialmente causadores de OI por meio de recursos de bioinformática. Foram utilizadas estratégias de filtragem pelo programa Microsoft Office Excel 2013, bem como análises de predição de mutação. Como referência genômica foram utilizados os bancos de dados Ensembl e National Center for Biotechnology Information. Foram selecionados quatro pacientes diagnosticados clinicamente com OI que foram submetidos à técnica de NGS e apresentaram resultados normais para os genes conhecidos. Com o intuito de selecionar uma lista de genes candidatos na plataforma customizada de NGS que estivessem relacionados com os sintomas de OI, foi realizada uma busca de genes no banco de dados Ensembl envolvidos com as vias metabólicas de formação óssea, cartilaginosa ou de colágeno, que identificou 643 genes. A lista de genes candidatos foi comparada com os genes sequenciados dos pacientes, onde foram selecionados 70 genes em comum para análise. Foram realizadas filtragens in silico de forma a selecionar alterações raras na população, preditas como patogênicas e que efetivamente codifiquem uma proteína ou uma molécula de RNA funcional. Os resultados mostraram que o paciente P.1 é portador de uma mutação em heterozigose potencialmente patogênica no gene ALX1. O paciente P.2 apresentou apenas uma alteração no gene COL6A3 que foi predita como polimorfismo. O paciente P.3 apresentou mutações patogênicas em heterozigose nos genes ALPL e FKBP10. No paciente P.4 foram encontradas mutações patogênicas em heterozigose nos genes P3H1 e RYR1. Entre os cinco genes identificados, sabe-se que dois deles, FKBP10 e P3H1, estão relacionados com a OI de herança autossômica recessiva. Também já é descrito que mutações no gene ALPL causam sintomas clínicos semelhantes a OI, podendo confundir o diagnóstico. Assim, o presente estudo identificou dois genes, ALX1 e RYR1, potencialmente causadores de OI. O gene ALX1 tem um papel importante no desenvolvimento craniano e dos membros, pois atua na formação da cartilagem. Já o RYR1 codifica a rianodina, um importante receptor de cálcio nos osteoblastos. Estudos funcionais dos genes identificados são necessários para validar esta hipótese em pesquisas futuras. Os resultados deste trabalho sugerem que ferramentas de bioinformática podem direcionar a busca por novos genes relacionados a doenças genéticas. A caracterização de novas mutações em genes relacionados com OI auxilia no planejamento de estratégias mais eficientes que permitam o diagnóstico molecular da doença e o aconselhamento genético.