Global DNA Demethylation During Erythropoiesis: A Dissertation

In the mammalian genome, 5‟-CpG-3‟ dinucleotides are frequently methylated, correlating with transcriptional silencing. Genome-wide waves of demethylation are thought to occur only twice during development, in primordial germ cells and in the pre-implantation embryo. They are followed by de novo methylation, setting up a pattern that is inherited throughout development. No global methylation changes are thought to occur during further somatic development, although methylation does alter at gene-specific loci, contributing to tissue-specific patterns of gene expression. Here we studied DNA methylation in differentiating mouse erythroblasts in vivo using several approaches including genomic-scale, reduced representation bisulfite sequencing (RRBS). Surprisingly, demethylation at the erythroid-specific β-globin locus was coincident with a wave of global DNA demethylation at most genomic elements, including repetitive elements and genes silenced in erythropoiesis. Over 30% of total methylation is irreversibly lost during erythroid differentiation. Demethylation occurred through a passive mechanism, requiring the rapid DNA replication triggered with the onset of erythroid terminal differentiation. Global loss of DNA methylation was not associated with a global increase in transcription, as determined by GeneChip analysis. We propose that global demethylation is a consequence of cellular mechanisms required for the rapid demethylation and induction of β-globin and other erythroid genes. Our findings demonstrate that, contrary to previously held dogma, DNA demethylation can occur globally during somatic cell differentiation, providing a new experimental model for the study of global demethylation in development and disease

The Impact of Artificial Intelligence on the Cybersecurity Industry

As our world becomes more digitalized, cyber criminals have an increasing landscape to launch their attacks. Developments in Artificial Intelligence are being used both to attack and defend networks, therefore, what is the next step for cybersecurity companies when it comes to beating these criminals? A study was conducted that utilizes previous literature sources written on the topic of Artificial Intelligence (AI) in the cybersecurity industry. In addition, the insights of professionals in the industry today are included through a survey and interviews to dive into the details of this battle and what lays in its future. The purpose of this study is to educate the public on the current role of AI in the industry, as it is a relatively new advancement that is slowly becoming mainstreamed. Preliminary research has shown that Artificial Intelligence may be the key to defeating these criminals, but there is much to discuss in terms of the use of this technology by cyber attackers, how it is being implemented into defense mechanisms, and the issues in the industry that may prevent this technology from growing as fast as it could be. Results from this research show that AI has a positive impact on the cybersecurity industry, but there are challenges with its implementation that have prevented it from becoming truly mainstreamed. However, as cyberattacks continue to evolve, AI will be key to winning the battle in this technology arms race

Statistical expression deconvolution from mixed tissue samples

Motivation: Global expression patterns within cells are used for purposes ranging from the identification of disease biomarkers to basic understanding of cellular processes. Unfortunately, tissue samples used in cancer studies are usually composed of multiple cell types and the non-cancerous portions can significantly affect expression profiles. This severely limits the conclusions that can be made about the specificity of gene expression in the cell-type of interest. However, statistical analysis can be used to identify differentially expressed genes that are related to the biological question being studied

Compression of Structured High-Throughput Sequencing Data

Large biological datasets are being produced at a rapid pace and create substantial storage challenges, particularly in the domain of high-throughput sequencing (HTS). Most approaches currently used to store HTS data are either unable to quickly adapt to the requirements of new sequencing or analysis methods (because they do not support schema evolution), or fail to provide state of the art compression of the datasets. We have devised new approaches to store HTS data that support seamless data schema evolution and compress datasets substantially better than existing approaches. Building on these new approaches, we discuss and demonstrate how a multi-tier data organization can dramatically reduce the storage, computational and network burden of collecting, analyzing, and archiving large sequencing datasets. For instance, we show that spliced RNA-Seq alignments can be stored in less than 4% the size of a BAM file with perfect data fidelity. Compared to the previous compression state of the art, these methods reduce dataset size more than 40% when storing exome, gene expression or DNA methylation datasets. The approaches have been integrated in a comprehensive suite of software tools (http://goby.campagnelab.org) that support common analyses for a range of high-throughput sequencing assays.National Center for Research Resources (U.S.) (Grant UL1 RR024996)Leukemia & Lymphoma Society of America (Translational Research Program Grant LLS 6304-11)National Institute of Mental Health (U.S.) (R01 MH086883

Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes

Technical advances in the collection of clinical material, such as laser capture microdissection and cell sorting, provide the advantage of yielding more refined and homogenous populations of cells. However, these attractive advantages are counter balanced by the significant difficultly in obtaining adequate nucleic acid yields to allow transcriptomic analyses. Established technologies are available to carry out global transcriptomics using nanograms of input RNA, however, many clinical samples of low cell content would be expected to yield RNA within the picogram range. To fully exploit these clinical samples the challenge of isolating adequate RNA yield directly and generating sufficient microarray probes for global transcriptional profiling from this low level RNA input has been addressed in the current report. We have established an optimised RNA isolation workflow specifically designed to yield maximal RNA from minimal cell numbers. This procedure obtained RNA yield sufficient for carrying out global transcriptional profiling from vascular endothelial cell biopsies, clinical material not previously amenable to global transcriptomic approaches. In addition, by assessing the performance of two linear isothermal probe generation methods at decreasing input levels of good quality RNA we demonstrated robust detection of a class of low abundance transcripts (GPCRs) at input levels within the picogram range, a lower level of RNA input (50 pg) than previously reported for global transcriptional profiling and report the ability to interrogate the transcriptome from only 10 pg of input RNA. By exploiting an optimal RNA isolation workflow specifically for samples of low cell content, and linear isothermal RNA amplification methods for low level RNA input we were able to perform global transcriptomics on valuable and potentially informative clinically derived vascular endothelial biopsies here for the first time. These workflows provide the ability to robustly exploit ever more common clinical samples yielding extremely low cell numbers and RNA yields for global transcriptomics

A Key Commitment Step in Erythropoiesis Is Synchronized with the Cell Cycle Clock through Mutual Inhibition between PU.1 and S-Phase Progression

During red blood cell development, differentiation and cell cycle progression are intimately and uniquely linked through interdependent mechanisms involving the erythroid transcriptional suppressor PU.1 and the cyclin-dependent kinase inhibitor p57KIP2

TET family dioxygenases and DNA demethylation in stem cells and cancers

The methylation of cytosine and subsequent oxidation constitutes a fundamental epigenetic modification in mammalian genomes, and its abnormalities are intimately coupled to various pathogenic processes including cancer development. Enzymes of the Ten-eleven translocation (TET) family catalyze the stepwise oxidation of 5-methylcytosine in DNA to 5-hydroxymethylcytosine and further oxidation products. These oxidized 5-methylcytosine derivatives represent intermediates in the reversal of cytosine methylation, and also serve as stable epigenetic modifications that exert distinctive regulatory roles. It is becoming increasingly obvious that TET proteins and their catalytic products are key regulators of embryonic development, stem cell functions and lineage specification. Over the past several years, the function of TET proteins as a barrier between normal and malignant states has been extensively investigated. Dysregulation of TET protein expression or function is commonly observed in a wide range of cancers. Notably, TET loss-of-function is causally related to the onset and progression of hematologic malignancy in vivo. In this review, we focus on recent advances in the mechanistic understanding of DNA methylation-demethylation dynamics, and their potential regulatory functions in cellular differentiation and oncogenic transformation

TRATAMENTO CRÔNICO COM DECANOATO DE NANDROLONA PROMOVE REMODELAMENTO CARDÍACO E ALTERAÇÃO DA FUNÇÃO VENTRICULAR ESQUERDA EM RATAS: PAPEL DOS MODULADORES INTRACELULARES DE Ca2+

O uso dos esteróides anabólicos androgênicos (EAA) tem crescido consideravelmente nas últimas décadas, sendo utilizada tanto por homens como por mulheres, com redução significativa na idade inicial destes utilizadores. O uso abusivo e indiscriminado destes hormônios acarreta alterações cardiovasculares, como hipertrofia cardíaca associada com deposição de colágeno, considerada patológica. Este remodelamento cardíaco causado pelos EAA pode desencadear hipertensão, insuficiência cardíaca congestiva, cardiomiopatia, arritmias e até morte súbita. O uso de EAA é geralmente realizado em associação com o exercício físico, que isoladamente promove efeitos cardiovasculares benéficos, todavia em associação perde-se o efeito benéfico do exercício, com a ocorrência de adaptações cardiovasculares desfavoráveis e função ventricular comprometida, especialmente função diastólica, fibrose miocárdica e desarranjo de cardiomiócitos. Além disso, pouco se sabe sobre os efeitos cardiovasculares dessas drogas no sexo feminino. Este trabalho objetiva avaliar os efeitos do decanoato de nandrolona (DN) e do exercício físico resistido sobre a contratilidade cardíaca em ratas. Os animais foram separados em quatro grupos: C (não treinados); CE (submetidos a exercício físico resistido em água, cinco vezes por semana); ND (tratados com DN, 20 mg/kg/semana durante quatro semanas); e NDE (treinados e tratados). Os parâmetros hemodinâmicos (+dP/dtmáx, -dP/dtmin e Tau) foram avaliados no ventrículo esquerdo. O coração foi coletado para análise histológica (H&E) e deposição de colágeno (picrosírius red). A análise da expressão das proteínas relacionadas com o transiente de Ca2+ citosólico foi realizada pelo método de Western blot. Os animais tratados com DN e os animais submetidos ao treinamento físico resistido apresentaram aumento na contratilidade e relaxamento cardíaco. Além disso, o DN promoveu aumento na expressão da fosfolambam fosforilada (p-PLB) e isoforma do retículo sarcoplasmático ATPase 2 (SERCA-2a), enquanto o exercício de resistência aumentou a fosforilação de PLB e a expressão do trocador de Na+/Ca2+ (NCX). O remodelamento cardíaco patológico, característico pela hipertrofia cardíaca associada com deposição de colágeno, foi observado após o tratamento com DN. Portanto, tratamento com DN e o exercício físico resistido em fêmeas, por um período de quatro semanas, foram capazes de promover hipertrofia cardíaca e aumentar a função cardíaca, alterando proteínas responsáveis pela regulação de Ca2+ intracelular, entretanto, a hipertrofia causada pela nandrolona foi considerada patológica. Certamente, esta avaliação comparando o uso de nandrolona e a contratilidade cardíaca deve ser posteriormente investigada, especialmente com uso mais prolongado, visto que as exacerbações prolongadas desses efeitos podem desencadear complicações cardíacas graves

Global DNA demethylation during mouse erythropoiesis in vivo

In the mammalian genome, 5\u27-CpG-3\u27 dinucleotides are frequently methylated, correlating with transcriptional silencing. Genome-wide demethylation is thought to occur only twice during development, in primordial germ cells and in the pre-implantation embryo. These demethylation events are followed by de novo methylation, setting up a pattern inherited throughout development and modified only at tissue-specific loci. We studied DNA methylation in differentiating mouse erythroblasts in vivo by using genomic-scale reduced representation bisulfite sequencing (RRBS). Demethylation at the erythroid-specific β-globin locus was coincident with global DNA demethylation at most genomic elements. Global demethylation was continuous throughout differentiation and required rapid DNA replication. Hence, DNA demethylation can occur globally during somatic cell differentiation, providing an experimental model for its study in development and disease