Dissertation submitted in compliance with the requirements for Masters Degree in Technology in the Department of Electrical Engineering (Light Current, Technikon Natal, 2000. / The computational analysis of eukaryotic promoters are among the most important and complex research domains that may contribute to complete gene identification. The current methods for promoter recognition are not sufficiently developed. Eukaryotic promoters contain a number of short motifs that may be used in promoter recognition. Having good computational models for these motifs can be crucial for increased efficiency of promoter recognition programs. This study proposes a combined statistical and LVQ neural network system as a computational model of the TAT A box motif of eukaryotic promoters. The methodology used is universal and applicable to any short functional motif in DNA. The statistical analysis of the core TAT A motif hexamer and its neighboring haxamers show strong regularities that can be used in motif recognition. Moreover, the positional distribution of the TAT A motif in terms of its distance from the transcription start site is very regular and is used in the statistical modeling. Furthermore, the matching score of the position weight matrix for the motif was used as a part of the model. Based on these statistical properties. a novel LV Q classifier for TAT A motif recognition is developed. The characteristics of the method are that the genetic algorithm was used for finding good initial weights of the LV Q system, while fine tuning of two LVQ networks was done by the lvq? algorithm. The final computational model is developed for a recognition level of 67.8o/c correct recognition on the test set with less than 1% false recognition. This model is evaluated in the task of promoter recognition on an independent test set. The results in promoter recognition outperform three other promoter recognition programs. It is shown that the recognition of promoters based on the recognition of the TAT A motifs using this new model is superior to the recognition based on the currently used position weight matrix description of this motif. / M

As pesquisas envolvendo seres humanos geram preocupações éticas, pois os voluntários aceitam riscos e inconveniências com o objetivo de contribuir para o avanço do conhecimento científico e beneficiar outrem. A disposição para participar de pesquisas clínicas se mostra quando o paciente adere ao Termo de Consentimento Livre e Esclarecido (TCLE), compreendendo-o, assinando-o e comprometendo-se a cumprir todas as normas estabelecidas nesse documento, embora consciente de que, a qualquer momento, poderá suspender sua adesão. O TCLE aborda informações que precisam estar descritas de forma clara e de fácil compreensão, destacando riscos, possíveis benefícios e procedimentos. Além disso, garantir a participação voluntária e sua desistência em qualquer momento da pesquisa. Atualmente discute-se a possibilidade de sujeitos de pesquisa não entenderem totalmente o texto do TCLE nem seus direitos como participantes, mesmo tendo assinado o TCLE e aderido à pesquisa. A presente casuística analisa os dados de 793 pacientes, que foram convidados a participar de diferentes protocolos de pesquisa clínica, como especifica a seguir: 380 pacientes, que foram convidados a participar do grupo controle do projeto Genoma Clínico do Câncer; 365 pacientes, que foram convidados a participar do projeto Genoma Clínico do Câncer do Aparelho do Digestivo por apresentarem tumor em uma das seguintes localizações: câncer colorretal, câncer esofágico, câncer de cárdia ou câncer gástrico.; 48 pacientes que foram convidados a participar do Estudo Multicêntrico, Internacional, Randomizado, de Grupos Paralelos, Controlado por Placebo, Duplo-Cego, com subsidiária cega, para determinar o efeito de 156 semanas de tratamento com MK-966(antiinflamatório Anti-COX 2) na recorrência de pólipo adenomatoso de intestino grosso, em pacientes com histórico de adenoma colorretal ressecado por colonoscopia. Coletaram-se dados dos fichários de pesquisa científica para avaliar a aderência do sujeito de pesquisa ao protocolo, correlacionando-a com fatores demográficos (raça, sexo e idade), sociais (local de nascimento, morada atual e instituição de tratamento), relação risco/beneficio envolvida e nível de escolaridade. O grau de dificuldade dos textos que compõem os TCLE foi avaliado, aplicando-se os Índices de Legibilidade Flesch Reading Ease e Flesch- Kincaid. Aplicou-se questionário aos entrevistadores para avaliar, a posteriori, a postura do sujeito de pesquisa à adesão ao TCLE no momento de sua assinatura ou discordância. A adesão dos sujeitos de pesquisa aos protocolos propostos não teve influência dos fatores demográficos e sociais, no entanto, verificou-se maior adesão entre os pacientes de instituição de tratamento público (99,7%) em comparação com instituição de tratamento privada (93,7%). A adesão foi maior entre os pacientes que participaram de protocolos com menor risco (99,73%) em comparação com os pacientes que participaram de protocolos com maior risco (81,3%). Apesar de a adesão não ter tido influência do nível de escolaridade, este foi menor ou igual a 8 anos de estudo para 462 pacientes (58,26%), entre os quais 444 (96,1%) pacientes eram de instituição de tratamento público. Os índices de legibilidade obtidos variaram de 9.9 12 para o teste de Flesch-Kincaid e 33,1 51,3 para o teste de Flesch Reading Ease. Os resultados encontrados na aplicação dos testes de legibilidade classificaram todos os textos avaliados em nível de difícil compreensão, exigindo maior nível de escolaridade para o seu entendimento Os entrevistadores estimaram, através do questionário aplicado a eles, que 90% dos pacientes do hospital público preferem ouvir a explicação do TCLE a ler o texto. Na instituição privada esta estimativa foi de 40%. Apenas onze sujeitos de pesquisa não aderiram ao TCLE. A adesão não recebeu influência de fatores demográficos e sociais. O risco inerente aos protocolos apresentados influenciou a adesão dos sujeitos de pesquisa. Os textos avaliados não se constituíram em linguagem escrita de fácil entendimento, necessitando mais de 9 anos de estudo para sua compreensão. Esta pesquisa sugere que, apesar da alta incidência de adesão, a avaliação de novos métodos de aplicação do TCLE é necessária para que o sujeito de pesquisa menos instruído tenha condições de compreender adequadamente todo o conteúdo do texto proposto no TCLE. / Researches engaging human beings pose ethical concerns since volunteers take on risks and inconveniences aiming to contribute to advanced scientific knowledge and to benefit others. The moment patients sign the term of voluntary and informed consent TCLE (Termo de Consentimento Livre e Esclarecido) they show they are willing to participate in clinical trials and that they understand the term and commit to complying with all rules in the document, aware that they can, at any moment, withdraw acceptance. The TCLE addresses all issues in the research process and are therefore important to the study participants. The information given at the TCLE must be clearly stated and easily understood, highlighting risks, possible benefits and procedures in addition to guaranteeing volunteer participation and consent withdrawal at any time during the trial. Lately, it has been speculated that the study participants do not totally understand the TCLEs text content and their participants rights before accepting the TCLE and joining the trial. This study analyzes the data from 793 patients, invited to take part in different protocols of clinical trials, as follows: 380 patients, invited to join the Clinic Cancer Genome Project Control Group; 365 patients, invited to join the Genome Clinic Cancer Genome of the Digestive System since they had one of the four tumors: colorectal cancer, cancer of the esophagus, cardia adenocarcinoma and gastric cancer; 48 patients were invited to join the International Multicenter double-blind, randomized, parallel-group, placebocontrolled study, with undisclosed sponsor, to determine the outcome of a 156-week treatment with MK-966(anti-inflammatory Anti-COX 2) in recurrent adenomatous polyp of the large bowel, in patients with a history of colorectal resection for adenoma at colonoscopy. Data were collected from previous scientific studies to assess study participants acceptance, correlating it to demographic factors (ethnic group, gender and age), social (birthplace, home place, health institution), cost/benefit and schooling. The level of difficulty in the TCLE texts was assessed with Flesch Reading Ease and Flesch-Kincaid readability measures. Interviewers answered a questionnaire a posteriori, to evaluate the study participants attitude toward the TCLE acceptance at the moment they signed it or did not accept it. The study participants acceptance of the suggested protocols was not influenced by demographic and social factors. However, patients from public health institutions (99,7%) outnumbered those from private health institutions (93,7%). Acceptance was higher among patients taking part in low-risk protocols (99,73%) than in high-risk protocols (81,3%). Although schooling did not influence acceptance, it was 8 years or less in 462 patients (58,26%), among who 444 (96,1%) were patients from public health institutions. The indices of legibility had varied of 9.9 - 12 for the test of Flesch-Kincaid and 33.1 - 51,3 for the test of Flesch Reading Ease. The results found in the application of the legibility tests had classified all the texts evaluated in level of difficult understanding, demanding higher school level for its agreement. Interviewers reported in questionnaires that 90% of the patients from public hospitals would rather listen to an explanation of the TCLE than read the text whereas in patients from private institution the percentage dropped to 40%. Only eleven study participants did not join the TCLE. Acceptance was not influenced by social and demographic factors, but the protocols risk levels influenced the study participants decisions. The evaluated texts proved to be difficult to understand, demanding over 9 years of schooling to be understood. This study suggests that, in spite of being highly accepted, the TCLE requires new application methods so that less educated people can properly understand its text contents.

Sjèogren's syndrome (S) is a chronic autoimmune disease characterized by ocular and oral dryness and primarily affects post menopausal women. In the present study we investigated the time course of lymphocytic infiltration, apoptosis, caspase-3 activity and different cytokines levels in the lacrimal glands of both genetically predisposed and control mice to elucidate immunopathological mechanism leading to dry eye. The results of our experiments showed that ovariectomy accelerated pathological findings of SS by increasing lympocytic infiltration, cytokine production, lacrimal gland cell death and cleaved caspase-3 activity, and these effects were more pronounced and persistent in the genetically predisposed mouse model of SS. In addition, we observed that lymphocytic infiltration occurred earlier compared to apoptosis which may perpetuate immune mediated destruction of lacrimal epithelial cells. Furthermore, treatment with physioloigical doses of 17-B Estradiol (E2) or DIhydrotestosterone (DHT) prevented all these pathological events observed after ovariectomy. / by Safinaz Mostafa. / Thesis (M.S.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

Genome-wide association studies (GWAS) have identified thousands of genetic variants associated with hundreds of human traits. Yet the common variant model tested by traditional GWAS only provides an incomplete explanation for the known genetic heritability of many traits. Many divergent methods have been proposed to address the shortcomings of GWAS, including most notably the extension of association methods into rarer variants through whole exome and whole genome sequencing. GWAS methods feature numerous simplifications designed for feasibility and ease of use, as opposed to statistical rigor. Furthermore, no systematic quantification of the performance of GWAS across all traits exists. Beyond improving the utility of data that already exist, a more thorough understanding of the performance of GWAS on common variants may elucidate flaws not in the method but rather in its implementation, which may pose a continued or growing threat to the utility of rare variant association studies now underway. This thesis focuses on systematic evaluation and incremental improvement of GWAS modeling. We collect a rich dataset containing standardized association results from all GWAS conducted on quantitative human traits, finding that while the majority of published significant results in the field do not disclose sufficient information to determine whether the results are actually valid, those that do replicate precisely in concordance with their statistical power when conducted in samples of similar ancestry and reporting accurate per-locus sample sizes. We then look to the inability of effectively all existing association methods to handle missingness in genetic data, and show that adapting missingness theory from statistics can both increase power and provide a flexible framework for extending most existing tools with minimal effort. We finally undertake novel variant association in a schizophrenia cohort from a bottleneck population. We find that the study itself is confounded by nonrandom population sampling and identity-by-descent, manifesting as batch effects correlated with outcome that remain in novel variants after all sample-wide quality control. On the whole, these results emphasize both the past and present utility and reliability of the GWAS model, as well as the extent to which lessons from the GWAS era must inform genetic studies moving forward.

Genome wide studies have uncovered the existence of large-scale copy number variation (CNV) in the human genome. The human genome of different individuals was initially estimated to be 99.9% similar, but population studies on CNV have revealed that it is 12-16% copy number variable. Abnormal genomic CNVs are frequently found in subtelomeres of patients with mental retardation (MR) and other neurological disorders. Rearrangements of chromosome subtelomeric regions represent a high proportion of cytogenetic abnormalities and account for approximately 30% of pathogenic CNVs. Although DNA double strand breaks (DSBs) are implicated as a major factor in chromosomal rearrangements, the causes of chromosome breakage in subtelomeric regions have not been elucidated. But due to the presence of repetitive sequences in subtelomeres, we hypothesized that chromosomal rearrangements in these regions are not stochastic but driven by specific sequence motifs. In a collaborative effort with Dr. Rudd (Department of human genetics at Emory University), we characterized subtelomeric breakpoints on different chromosome ends in search of common motifs that cause double-strand breaks. Using a yeast-based gross chromosomal rearrangement (GCR) system, we have identified a subtelomeric breakage motif from chromosome 2 (2q SBM) with a GCR rate that is 340 fold higher than background levels. To determine if the fragility of 2q SBM was driven by the formation of secondary structures, the helicase activities of Sgs1 and Pif1 were disrupted. These helicases have been shown to destabilize DNA secondary structures such as G-quadruplex structures. Disruption of these helicases augmented chromosomal rearrangements induced by 2q SBM, indicating that these helicases are required for maintenance of this sequence. We also donwregulated replication fork components to determine if 2q SBM was imposing any problems to the replication fork machinery. Downregulation of replication fork components increased chromosomal rearrangements, indicating that intact replication fork was a critical determinant of 2q SBM fragility. Using a yeast-based functional assay, these experiments have linked human subtelomeric repetitive sequences to chromosomal breakage that could give rise to human CNV in subtelomeric regions.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (p. 156-158).

Thesis (M.S.)--Naval Postgraduate School, 2002. / Title from title screen (viewed June 18, 2003). Includes bibliographical references.

The human genome encodes information that instructs human development, physiology, medicine, and evolution. Massive amount of genomic data has generated an ever-growing pool of hypothesis. Genome editing, broadly defined as targeted changes to the genome, posits to deliver the promise of genomic revolution to transform basic science and personalized medicine. This thesis aims to contribute to this scientific endeavor with a particular focus on the development of effective human genome engineering tools.

Survival of cells and organisms requires that each of thousands of genes is expressed at the correct time in development, in the correct tissue, and under the correct conditions. Transcription is the primary point of gene regulation. Genes are activated and repressed by transcription factors, which are proteins that become active through signaling, bind, sometimes cooperatively, to regulatory regions of DNA, and interact with other proteins such as chromatin remodelers. Yeast has nearly six thousand genes, several hundred of which are transcription factors; transcription factors comprise around 2000 of the 22,000 genes in the human genome. When and how these transcription factors are activated, as well as which subsets of genes they regulate, is a current, active area of research essential to understanding the transcriptional regulatory programs of organisms. We approached this problem in two divergent ways: first, an in silico study of human transcription factor combinations, and second, an experimental study of the transcriptional response of yeast mutants deficient in DNA repair. First, in order to better understand the combinatorial nature of transcription factor binding, we developed a data mining approach to assess whether transcription factors whose binding motifs were frequently proximal in the human genome were more likely to interact. We found many instances in the literature in which over-represented transcription factor pairs co-regulated the same gene, so we used co-citation to assess the utility of this method on a larger scale. We determined that over-represented pairs were more likely to be co-cited than would be expected by chance. Because proper repair of DNA is an essential and highly-conserved process in all eukaryotes, we next used cDNA microarrays to measure differentially expressed genes in eighteen yeast deletion strains with sensitivity to the DNA cross-linking agent methyl methane sulfonate (MMS); many of these mutants were transcription factors or DNA-binding proteins. Combining this data with tools such as chromatin immunoprecipitation, gene ontology analysis, expression profile similarity, and motif analysis allowed us to propose a model for the roles of Iki3 and of YML081W, a poorly-characterized gene, in DNA repair. / text

In eukaryotic genomes ranging from plants to mammals, DNA methylation is a major epigenetic modification of DNA by adding a methyl group exclusively to cytosine residuals. In mammalian genomes such as humans, these cytosine bases are usually followed by guanine. Although it does not change the primary DNA sequence, this covalent modification plays critical roles in several regulatory processes and can impact gene activity in a heritable fashion. What is more important, DNA methylation is essential for mammalian embryonic development and aberrant DNA methylation is implicated in several human diseases, in particular in neuro-developmental syndromes (such as the fragile X and Rett syndromes) and cancer. These biological significances disclose the importance of understanding genomic patterns and function role of DNA methylation in human, as a initial step to get to know the epigenotype and its manner in connecting the phenotype and genotype. Two key papers back in 1975 independently suggested that methylation of CpG dinucleotides in vertebrates could be established de novo and inherited through somatic cell divisions by protein machineries of DNA methyltransferases that recognizes hemi-methylated CpG palindromes. They also indicated that the methyl group could be recognized by DNA-binding proteins and that DNA methylation directly silences gene expression. After almost four decades, several key points in these foundation papers are proved to be true. Take the mammalian genome for example, there are several findings indicating the epigenetic repression of gene expression by DNA methylation. These include X-chromosome inactivation, gene imprinting and suppressing the proliferation of transposable elements and repeat elements of viral or retroviral origin. In addition to these, many novel roles of DNA methylation have also been revealed. For example, DNA methylation can regulate alternative splicing by preventing CTCF, an evolutionarily conserved zinc-finger protein, binding to DNA. By using the technique of fluorescence resonance energy transfer (FRET) and fluorescence polarization, DNA methylation has also been shown to increase nucleosome compaction through DNA-histone contacts. What is more important, DNA methylation is essential for mammalian embryonic development and aberrant change of DNA methylation has been related to disease such as cancer. However, it is also notable there are several lines of evidence contradicting the relationship between DNA methylation and gene silencing. For example, comparison of DNA methylation levels in human genome on the active and inactive X chromosomes showed reduced methylation specifically over gene bodies on inactive X chromosomes. Not only in human, DNA methylation is found to be usually targeted to the transcription units of actively transcribed genes in invertebrate species. These results prove that the function of DNA methylation is challenging to be unravel. Besides, due to the development of sequencing technique, whole genome DNA methylation profiles have been detected in diverse species. Comparing genomic patterns of DNA methylation shows considerable variation among taxa, especially between vertebrates and invertebrates. However, even though extensive studies reveal the patterns and functions of DNA methylation in different species, in the mean time, they also highlight the limits to our understanding of this complex epigenetic system. During my Ph.D., in order to perform in-depth studies of DNA methylation in diverse animals as a way to understand the complexity of DNA methylation and its functions, I dedicated my efforts in investigating and analyzing the DNA methylation profiles in diverse species, ranging from insects to primates, including both model and non-model organisms. This dissertation, which constitutes an important part of my research, mainly focuses on the DNA methylation profile in primates including human and chimpanzee. In general, I will use three chapters to elucidate my work in generating and interpreting the whole genome DNA methylation data. Firstly, we generated nucleotide-resolution whole-genome methylation maps of the prefrontal cortex of multiple humans and chimpanzees, then comprehensive comparative studies for these DNA methylation maps have been performed, by integrating data on gene expression as well. This work demonstrates that differential DNA methylation might be an important molecular mechanism driving gene-expression divergence between human and chimpanzee brains and also potentially contribute to the human-specific traits, such as evolution of disease vulnerabilities. Secondly , we performed global analyses of CpG islands (CGIs) methylation across multiple methylomes of distinctive cellular origins in human. The results from this work show that the human CpG islands can be distinctly classified into different clusters solely based upon the DNA methylation profiles, and these CpG islands clusters reflect their distinctive nature at many biological levels, including both genomic characteristics and evolutionary features. Moreover, these CpG islands clusters are non-randomly associated with several important biological phenomena and processes such as diseases, aging, and gene imprinting. These new findings shed lights in deciphering the regulatory mechanisms of CpG islands in human health and diseases. At last, by utilizing the DNA methylome from human sperm and genetic map generated from the International HapMap Consortium project, we investigated the hypothesis suggesting a potential role of germ line DNA methylation in affecting meiotic recombination, which is essential for successful meiosis and various evolutionary processes. Even thought the results imply that DNA methylation is a important factor affecting regional recombination rate, the strength of correlation between these two is not as strong as the previous report. Besides, high-throughput analyses indicate that other epigenetic modifications, tri-methylation of histone 3 lysine 4 and histone 3 lysine 27 are also global features at the recombination hotspots, and may interact with methylation to affect the recombination pattern simultaneously. This work suggests epigenetic mechanisms as additional factors affecting recombination, which cannot be fully explained by the DNA sequence itself. In summary, I hope the results from these work can expand our knowledge regarding the common and variable patterns of DNA methylation in different taxa, and shed light about the function role and its major change during animal evolution.