It has been suggested that organismal complexity correlates with the complexity of gene regulation. Transcriptional control of gene expression is mediated by binding of regulatory proteins to cis-acting sequences on the genome. Hence, it is crucial to identify the chromosomal targets of transcription factors (TFs) to delineate transcriptional regulatory networks underlying gene expression programs. The development of ChIP-chip technology has enabled high throughput mapping of TF binding sites across the genome. However, there are many limitations to the technology including the availability of whole genome arrays for complex organisms such human or mouse. To circumvent these limitations, we developed the Sequence Tag Analysis of Genomic Enrichment (STAGE) methodology that is based on extracting short DNA sequences or “tags” from ChIP-enriched DNA. With improvements in sequencing technologies, we applied the recently developed ChIP-Seq technique i.e. ChIP followed by ultra high throughput sequencing, to identify binding sites for the TF E2F4 across the human genome. We identified previously uncharacterized E2F4 binding sites in intergenic regions and found that several microRNAs are potential E2F4 targets. Binding of TFs to their respective chromosomal targets requires access of the TF to its regulatory element, which is strongly influenced by nucleosomal remodeling. In order to understand nucleosome remodeling in response to transcriptional perturbation, we used ultra high throughput sequencing to map nucleosome positions in yeast that were subjected to heat shock or were grown normally. We generated nucleosome remodeling profiles across yeast promoters and found that specific remodeling patterns correlate with specific TFs active during the transcriptional reprogramming. Another important aspect of gene regulation operates at the post-transcriptional level. MicroRNAs (miRNAs) are ~22 nucleotide non-coding RNAs that suppress translation or mark mRNAs for degradation. MiRNAs regulate TFs and in turn can be regulated by TFs. We characterized a TF-miRNA network involving the oncofactor Myc and the miRNA miR-22 that suppresses the interferon pathway as primary fibroblasts enter a stage of rapid proliferation. We found that miR-22 suppresses the interferon pathway by inhibiting nuclear translocation of the TF NF-kappaB. Our results show how the oncogenic TF Myc cross-talks with other TF regulatory pathways via a miRNA intermediary. / text

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

O tema da proteção da biodiversidade e, em especial, do Genoma Humano tem suscitado grande interesse e preocupação no Direito atual, agregando-se às preocupações de profissionais de diversas áreas como bioética, genética, saúde, além de diversos outros segmentos. O artigo 225 da Constituição Federal de 1988 estabelece a proteção da biodiversidade e da integridade do patrimônio genético no país. As hipóteses desenvolvidas no presente estudo afirmam que a preservação do patrimônio genético é um dos meios eleitos pela Constituição Federal para garantir o gozo ao meio ambiente ecologicamente equilibrado; de que a preservação da diversidade do patrimônio genético humano se faz imperiosa como meio de garantir os interesses difusos, coletivos e individuais com o fim de evitar a degradação do meio ambiente e promover a garantia dos demais direitos do homem. Corroborando tais premissas além da visão jurídica destaca-se a evolução do conceito de bioética, que passou do campo médico para uma bioética global, com as conseqüências da sociedade de risco para as gerações futuras, analisando os limites éticos das intervenções no meio ambiente e no Genoma Humano. Os riscos da intervenção sobre o genoma humano são analisados de modo a considerar suas repercussões sobre as populações vulneráveis frente ao princípio da precaução e o direito à intimidade, quando do uso das informações contidas no código genético humano. Analisa-se, ainda, a responsabilidade do pesquisador tendo o princípio do poluidor-pagador como instrumento de efetivação dessa responsabilização, na perspectiva de caracterizar a tutela do patrimônio genético como um direito humano fundamental. / Submitted by Marcelo Teixeira (mvteixeira@ucs.br) on 2014-06-05T17:07:35Z No. of bitstreams: 1 Dissertacao Fernanda Martinotto.pdf: 897262 bytes, checksum: f5813eef57bd036d87c84193eb180d3e (MD5) / Made available in DSpace on 2014-06-05T17:07:35Z (GMT). No. of bitstreams: 1 Dissertacao Fernanda Martinotto.pdf: 897262 bytes, checksum: f5813eef57bd036d87c84193eb180d3e (MD5) / The issue of protecting biodiversity and in particular the human genome has sparked great interest and concern in the current law, adding to the concerns of professionals in various fields such as bioethics, genetics, health, and several other segments. Article 225 of the Constitution of 1988 provides for the protection of biodiversity and the integrity of the genetic heritage in the country. The hypotheses developed in this study say that the preservation of genetic heritage is one of the means chosen by the Federal Constitution to guarantee the enjoyment to a balanced environment, that preserving the diversity of human genetic resources becomes imperative as a means to safeguard the interests diffuse, collective and individual in order to prevent environmental degradation and promote the guarantee of other rights. Confirming these assumptions beyond the legal view highlights the evolution of the concept of bioethics, which passed the medical field for a global bioethics, with the consequences of risk society for future generations by examining the ethical limits of interventions in the environment and Human Genome. The risks of the intervention on the human genome are analyzed in order to consider its impact on vulnerable populations against the precautionary principle and the right to privacy, when the use of information contained in the human genetic code. We analyze also the responsibility of the researcher,with the polluter-pays principle as a tool for fulfillment of this responsibility in view of characterizing the genetic heritage protection as a fundamental human right.

Made available in DSpace on 2014-12-17T15:14:49Z (GMT). No. of bitstreams: 1 RicardoGS.pdf: 538526 bytes, checksum: 84faa5411061c273b56347caa03d1fac (MD5) Previous issue date: 2008-09-30 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / This dissertation analyses the influence of sugar-phosphate structure in the electronic transport in the double stretch DNA molecule, with the sequence of the base pairs modeled by two types of quasi-periodic sequences: Rudin-Shapiro and Fibonacci. For the sequences, the density of state was calculated and it was compared with the density of state of a piece of human DNA Ch22. After, the electronic transmittance was investigated. In both situations, the Hamiltonians are different. On the analysis of density of state, it was employed the Dyson equation. On the transmittance, the time independent Schr?dinger equation was used. In both cases, the tight-binding model was applied. The density of states obtained through Rudin-Shapiro sequence reveal to be similar to the density of state for the Ch22. And for transmittance only until the fifth generation of the Fibonacci sequence was acquired. We have considered long range correlations in both transport mechanism / Esta disserta??o analisa a influ?ncia do esqueleto a??car-fosfato no transporte eletr?nico na mol?cula de DNA de fita dupla, com o sequenciamento dos pares de base modelado por dois tipos de seq??ncias quasi-peri?dicas: Rudin-Shapiro e Fibonacci. Para ambas as seq??ncias, foram calculadas as densidades de estado e comparadas com a densidade de estado de um trecho do DNA humano Ch 22. Em seguida, foi investigada a transmit?ncia eletr?nica. Nos dois casos, as Hamiltonianas s?o distintas. Na an?lise da densidade de estado foi empregada a equa??o de Dyson. Na transmit?ncia foi feito uso da equa??o de Schr?dinger independente do tempo. Em ambos os casos, foi utilizado o modelo tight-binding. Os resultados para a densidade de estado foram mais satisfat?rios para a seq??ncia de Rudin-Shapiro, que forneceu um perfil muito pr?ximo do perfil da densidade de estado para o Ch22. A transmit?ncia foi calculada somente para a seq??ncia de Fibonacci at? a quinta gera??o. Nestes dois mecanismos de transporte, as correla??es s?o de longo alcance

O tema da proteção da biodiversidade e, em especial, do Genoma Humano tem suscitado grande interesse e preocupação no Direito atual, agregando-se às preocupações de profissionais de diversas áreas como bioética, genética, saúde, além de diversos outros segmentos. O artigo 225 da Constituição Federal de 1988 estabelece a proteção da biodiversidade e da integridade do patrimônio genético no país. As hipóteses desenvolvidas no presente estudo afirmam que a preservação do patrimônio genético é um dos meios eleitos pela Constituição Federal para garantir o gozo ao meio ambiente ecologicamente equilibrado; de que a preservação da diversidade do patrimônio genético humano se faz imperiosa como meio de garantir os interesses difusos, coletivos e individuais com o fim de evitar a degradação do meio ambiente e promover a garantia dos demais direitos do homem. Corroborando tais premissas além da visão jurídica destaca-se a evolução do conceito de bioética, que passou do campo médico para uma bioética global, com as conseqüências da sociedade de risco para as gerações futuras, analisando os limites éticos das intervenções no meio ambiente e no Genoma Humano. Os riscos da intervenção sobre o genoma humano são analisados de modo a considerar suas repercussões sobre as populações vulneráveis frente ao princípio da precaução e o direito à intimidade, quando do uso das informações contidas no código genético humano. Analisa-se, ainda, a responsabilidade do pesquisador tendo o princípio do poluidor-pagador como instrumento de efetivação dessa responsabilização, na perspectiva de caracterizar a tutela do patrimônio genético como um direito humano fundamental. / The issue of protecting biodiversity and in particular the human genome has sparked great interest and concern in the current law, adding to the concerns of professionals in various fields such as bioethics, genetics, health, and several other segments. Article 225 of the Constitution of 1988 provides for the protection of biodiversity and the integrity of the genetic heritage in the country. The hypotheses developed in this study say that the preservation of genetic heritage is one of the means chosen by the Federal Constitution to guarantee the enjoyment to a balanced environment, that preserving the diversity of human genetic resources becomes imperative as a means to safeguard the interests diffuse, collective and individual in order to prevent environmental degradation and promote the guarantee of other rights. Confirming these assumptions beyond the legal view highlights the evolution of the concept of bioethics, which passed the medical field for a global bioethics, with the consequences of risk society for future generations by examining the ethical limits of interventions in the environment and Human Genome. The risks of the intervention on the human genome are analyzed in order to consider its impact on vulnerable populations against the precautionary principle and the right to privacy, when the use of information contained in the human genetic code. We analyze also the responsibility of the researcher,with the polluter-pays principle as a tool for fulfillment of this responsibility in view of characterizing the genetic heritage protection as a fundamental human right.

Ce travail de thèse, réalisé dans le cadre des projets de recherche ROSIRIS (IRSN) et Geant4-DNA, porte sur la construction d’une simulation multi-échelle dédiée au calcul des dommages radio-induits précoces à l’ADN qui peuvent apparaître suite à l’irradiation d’un noyau cellulaire. L’outil développé s’appuie sur une version modifiée du code de Monte Carlo Geant4-DNA et est capable de simuler dans le détail le transport et les interactions physiques entre l’irradiation ionisante et la matière biologique (étape physique), la création d’espèces chimiques (étape physico-chimique) et les réactions et processus de diffusion de ces dernières (étape chimique). Durant la simulation de ces trois étapes, un modèle géométrique de l’ADN, décrivant l’ensemble du génome humain avec une précision moléculaire, est généré avec un nouveau logiciel développé dans le cadre de cette thèse : DnaFabric. Les premiers résultats obtenus pour des irradiations avec des protons et des ions alpha sont détaillés et comparés à des données de la littérature. Un bon accord est observés avec ces dernières illustrant ainsi la cohérence de l’ensemble de la simulation. L’influence très significative du critère de sélection utilisé pour identifier les dommages à l’ADN est également démontrée. / This work was performed in the frame of the ROSIRIS (IRSN) and Geant4-DNA research projects and describes the development of a simulation tool to compute radioinduced early DNA damages in a cell nucleus. The modeling tool is based on a modified version of the Monte Carlo code Geant4-DNA and is able to simulate the physical interactions between ionizing particles and the biological target (physical stage), the creation of chemical species within the cell nucleus (physico-chemical stage) as well as the reactions and diffusion processes of these chemical species (chemical stage). During all the simulation, a geometrical model that describes the DNA content of a human diploid cell nucleus is taken into account. This model was generated with a new software (DnaFabric) developed in the frame of this work and has a molecular level of detail.The first results (in term of DNA strand breaks) obtained with this tool are detailed and compared with experimental data from the literature. The good agreement between the simulation results and those data shows the coherence of our modeling. The significant influence of the selection criteria used to identify the DNA damages is also demonstrated.

The three dimensional structure of the human genome is known to play a critical role in gene function and expression. I used chromosome conformation capture (3C) and 3C-carbon copy (5C) techniques to investigate the three-dimensional structure of the cystic fibrosis transmembrane conductance regulator (CFTR) locus. This is an important disease gene that, when mutated, causes cystic fibrosis. 3C experiments identified four distinct looping elements that contact the CFTR gene promoter only in CFTR-expressing cells. Using 5C, I expanded the region of study to a 2.8 Mb region surrounding the CFTR gene. The 5C study shows 7 clear topologically associating domains (TADs) present at the locus, identical in all five cell lines tested, regardless of gene expression status. CFTR and all its known regulatory elements are contained within one TAD, suggesting TADs play a role in constraining promoters to a local search space. The four looping elements identified in the 3C experiment and confirmed in the 5C experiment were then tested for enhancer activity using a luciferase assay, which showed that elements III and IV could act as enhancers. These elements were tested against a library of human transcription factors in a yeast one-hybrid assay to identify potential binding proteins. Element III gave two strong candidates, TCF4 and LEF1. A literature search supported these transcription factors as playing a role in CFTR gene expression. Overall, this work represents a model locus that can be used to test important questions regarding the role of three dimensional looping on gene expression.

Indiana University-Purdue University Indianapolis (IUPUI) / The class IV alcohol dehydrogenase (ADH7, µ-ADH, σ-ADH) efficiently metabolizes ethanol and retinol. ADH7 is expressed mainly in the upper gastrointestinal tract with no expression in the liver unlike the other ADHs, and is implicated in various diseases including alcoholism, cancer and fetal alcohol syndrome. Genome wide studies have identified significant associations between ADH7 variants and alcoholism and cancer, but the causative variants have not been identified. Due to its association with two important metabolic pathways and various diseases, this dissertation is focused on studying ADH7 regulation and the effects of variants on this regulation using cell systems that replicate endogenous ADH7 expression. We identified elements regulating ADH7 transcription and observed differences in the effects of variants on gene expression. A7P-G and A7P-A, two promoter haplotypes differing in a single nucleotide at rs2851028, had different transcriptional activities and interacted with variants further upstream. A sequence located 12.5 kb upstream (7P10) can function as an enhancer. These complex interactions indicate that the effects of variants in the ADH7 regulatory elements depend on both sequence and cellular context, and should be considered in interpretation of the association of variants with alcoholism and cancer. The mechanisms governing the tissue-specific expression of ADH7 remain unexplained however. We identified an intergenic region (iA1C), located between ADH7 and ADH1C, having enhancer blocking activity in liver-derived HepG2 cells. This enhancer blocking function was cell- and position- dependent with no activity seen in CP-A esophageal cells. iA1C had a similar effect on the ectopic SV40 enhancer. The CCCTC-binding factor (CTCF) bound iA1C in HepG2 cells but not in CP-A cells. Our results suggest that in liver-derived cells, iA1C blocks the effects of downstream ADH enhancers and thereby contributes to the cell specificity of ADH7 expression. Thus, while genetic factors determine level of ADH7 transcriptional activity, iA1C helps determine the cell specificity of transcription.

Indiana University-Purdue University Indianapolis (IUPUI) / DNA double strand break (DSB) is one of the most threatening of all types of DNA damages as it leads to a complete breakage of the chromosome. The cell has evolved several mechanisms to repair DSBs, one of which is break-induced replication (BIR). BIR repair of DSBs occurs through invasion of one end of the broken chromosome into a homologous template followed by processive replication of DNA from the donor molecule. BIR is a key cellular process and is implicated in the restart of collapsed replication forks and several chromosomal instabilities. Recently, our lab demonstrated that the fidelity of DNA synthesis associated with BIR in yeast Saccharomyces Cerevisiae is extremely low. The level of frameshift mutations associated with BIR is 1000-fold higher as compared to normal DNA replication. This work demonstrates that BIR stimulates base substitution mutations, which comprise 90% of all point mutations, making them 400-1400 times more frequent than during S-phase DNA replication. We show that DNA Polymerase δ proofreading corrects many of the base substitutions in BIR. Further, we demonstrate that Pif1, a 5’-3’ DNA helicase, is responsible for making BIR efficient and also highly mutagenic. Pif1p is responsible for the majority of BIR mutagenesis not only close to the DSB site, where BIR is less stable but also at chromosomal regions far away from the DSB break site, where BIR is fast, processive and stable. This work further reveals that, at positions close to the DSB, BIR mutagenesis in the absence of Pif1 depends on Rev3, the catalytic subunit of translesion DNA Polymerase ζ. We observe that mutations promoted by Pol ζ are often complex and propose that they are generated by a Pol ζ- led template switching mechanism. These complex mutations were also found to be frequently associated with gross chromosomal rearrangements. Finally we demonstrate that BIR is carried out by unusual conservative mode of DNA synthesis. Based on this study, we speculate that the unusual mode of DNA synthesis associated with BIR leads to various kinds of genomic instability including mutations and chromosomal rearrangements.

No description available.