Mechanical ventilation, a lifesaving intervention for patients with acute respiratory distress syndrome (ARDS), also unfortunately contributes to excessive mechanical stress and impaired lung physiological and structural integrity. We have elsewhere established the pivotal role of increased nicotinamide phosphoribosyltransferase (NAMPT) transcription and secretion as well as its direct binding to the toll-like receptor 4 (TLR4) in the progression of this devastating syndrome; however, regulation of this critical gene in ventilator-induced lung injury (VILI) is not well characterized. On the basis of an emerging role for epigenetics in enrichment of VILI and CpG sites within the NAMPT promoter and 5'UTR, we hypothesized that NAMPT expression and downstream transcriptional events are influenced by epigenetic mechanisms. Concomitantly, excessive mechanical stress of human pulmonary artery endothelial cells or lipopolysaccharide (LPS) treatment led to both reduced DNA methylation levels in the NAMPT promoter and increased gene transcription. Histone deacetylase inhibition by trichostatin A or Sirt-1-silencing RNA attenuates LPS-induced NAMPT expression. Furthermore, recombinant NAMPT administration induced TLR4-dependent global H3K9 hypoacetylation. These studies suggest a complex epigenetic regulatory network of NAMPT in VILI and ARDS and open novel strategies for combating VILI and ARDS.

Breast cancer it is the most common type of cancer and leading cause of cancer mortality among women worldwide. Women who inherit mutations in the breast cancer 1 susceptibility gene (BRCA1) are five times more likely to develop breast cancer than women who do not. However, only ~5-10% of breast cancer cases are due to germline mutations in tumor suppressor genes. There are currently no targeted therapies available triple negative breast cancers (TNBC), which often lack BRCA1 expression. BRCA1 is epigenetically silenced by the activated aryl-hydrocarbon receptor (AhR), suggesting that dietary antagonists of the AhR may inhibit BRCA1 silencing. Genistein is an isoflavone abundant in soy foods and its high consumption levels is thought to underlie the lower prevalence of breast cancer in Asian countries compared to Western countries. The hypothesis of this work is that genistein antagonizes AhR-dependent epigenetic silencing of BRCA1. To test this hypothesis we first determined the capacity of genistein to prevent AhR-dependent silencing of BRCA1 in estrogen receptor-alpha (ERα) expressing cells, with wild-type BRCA1 and inducible AhR (MCF-7). We also determined the effectiveness of genistein in reversing silencing of BRCA1 in ERα-negative cells with hypermethylated BRCA1 and constitutively active AhR (UACC-3199). The effect of genistein on BRCA1 promoter methylation and markers of cell proliferation was also determined in both cell lines.

Epigenetics is the study of factors that can change DNA and passed to next generation without change to DNA sequence. DNA methylation is one of the categories of epigenetic change. DNA methylation is the attachment of methyl group (CH3) to DNA. Most of the time it occurs in the sequences that G is followed by C known as CpG sites and by addition of methyl to the cytosine residue. As science and technology progress new data are available about individual’s DNA methylation profile in different conditions. Also new features discovered that can have role in DNA methylation. The availability of new data on DNA methylation and other features of DNA provide challenge to bioinformatics and the opportunity to discover new knowledge from existing data. In this research multiple data series were used to identify classes of methylation DNA to CpG sites. These classes are a) Never methylated CpG sites,b) Always methylated CpG sites, c) Methylated CpG sites in cancer/disease samples and non-methylated in normal samples d) Methylated CpG sites in normal samples and non-methylated in cancer/disease samples. After identification of these sites and their classes, an analysis was carried out to find the features which can better classify these sites a matrix of features was generated using four applications in EMBOSS software suite. Features matrix was also generated using the gUse/WS-PGRADE portal workflow system. In order to do this each of the four applications were grid enabled and ported to BOINC platform. The gUse portal was connected to the BOINC project via 3G-bridge. Each node in the workflow created portion of matrix and then these portions were combined together to create final matrix. This final feature matrix used in a hill climbing workflow. Hill climbing node was a JAVA program ported to BOINC platform. A Hill climbing search workflow was used to search for a subset of features that are better at classifying the CpG sites using 5 different measurements and three different classification methods: support vector machine, naïve bayes and J48 decision tree. Using this approach the hill climbing search found the models which contain less than half the number of features and better classification results. It is also been demonstrated that using gUse/WS-PGRADE workflow system can provide a modular way of feature generation so adding new feature generator application can be done without changing other parts. It is also shown that using grid enabled applications can speedup both feature generation and feature subset selection. The approach used in this research for distributed workflow based feature generation is not restricted to this study and can be applied in other studies that involve feature generation. The approach also needs multiple binaries to generate portions of features. The grid enabled hill climbing search application can also be used in different context as it only requires to follow the same format of feature matrix.

No description available.

Genetic analyses of increasing power are now regularly incorporated into wildlife management assessments of threatened and endangered species. Genetic data provide valuable information regarding taxonomy, kinship, and population size and structure. Recently transformed by the advent of powerful technologies that expand our view from single genes to the entire genome, the field of conservation may be on the verge of another revolution with the emergence of epigenetics as a promising means of surveying environmental response in natural populations. In this dissertation, I present my doctoral research upon population genetics and epigenetics of pronghorn (Antilocapra americana). Considerable effort has been undertaken to conserve pronghorn, particularly in the periphery of its range in the southwestern United States and northwestern Mexico. Translocation is regularly used to supplement and re-establish populations of the wide-ranging A. a. americana subspecies while captive breeding has been established for two endangered pronghorn subspecies, A. a. sonoriensis found in Arizona and Sonora, Mexico and A. a. peninsularis of the Baja Peninsula. The primary goal of my doctoral work was to provide pronghorn managers with current estimates of genetic diversity, relatedness, and structure within and between pronghorn subspecies in the desert southwest. My work shows that conservation measures for A. a. sonoriensis have successfully maintained genetic diversity within this endangered subspecies. My estimates of population structure within A. a. americana in northern Arizona reveal the influence of translocation and habitat fragmentation and demonstrate the successful reestablishment of gene flow following the removal of highway fences. With the purpose of guiding future release of captive pronghorn, I explored the subspecies status of pronghorn extirpated from a portion of their range in southern California and northern Baja California. My analyses of museum specimens indicate that the historical range of A. a. peninsularis may have extended as far north as the international border while specimens collected just north of the border share more genetic identity with A. a. sonoriensis. To follow my interests in epigenetics, I also conducted the first ever conservation epigenetics study with Arizona pronghorn. I found that pronghorn are more epigenetically than genetically diverse and this is an indicator that further epigenetic study will reveal the signature of response to environmental factors, as it has with other species demonstrating this pattern.

<p>Identifying metabolic vulnerabilities of cancer cells remains a subject of investigation for the identification of potential metabolically based therapies for cancer. It is well known that proliferating cells become largely dependent on glucose and glutamine for their growth. Interestingly, we find that lipid oxidizing genes are consistently downregulated across a wide variety of cancers while lipid synthesizing genes are elevated. This indicates that lipid oxidation may be refractory to cancer cell growth. Studies have shown beneficial effects of carbohydrate restriction in various forms in the treatment of cancer. For example, the use of ketogenic diets, which contain high levels of fat and protein with very low levels of carbohydrates, have shown efficacy in decreasing tumor growth in glioma, colon, prostate, and gastric cancers. A major challenge facing the use of these diets in cancer therapy is that the mechanism by which they show efficacy in cancer remains unclear. By using octanoate, the most well-known ketogenic fatty acid, we are able to drive fatty acid oxidation and ketogenesis to study these processes in proliferating cells. We found that supplementation of octanoate into complete culture medium causes a dramatic, dose-dependent and reversible suppression of proliferation across numerous cell lines and significant changes to anabolic cellular metabolism. Importantly, we have found that ketone production from octanoate had no effect on cell proliferation but that the overall cellular response to the lipid causes inhibition of cell growth.</p><p>Nutrients and metabolites are sensed by the cell at many levels and the cellular response to metabolites is critical to proliferation and survival of a cancer cell. One way in which the cell responds to glucose, the major fuel source in cancer cells, is by increasing histone acetylation to promote gene expression. Wellen et al., found that upon glucose addition there was a specific gene expression pattern characterized by the upregulation of genes involved in glucose metabolism. In this way the cell promotes glucose-derived fatty acid synthesis, a rate-limiting process for cancer cell proliferation. This is one way in which the metabolic response to nutrients is integrated into cellular signaling and the epigenome. Remarkably, we have found that lipids can promote a feed-forward mechanism of lipid metabolism by inducing histone acetylation and increasing gene expression of lipid metabolizing genes. We find that upon treatment of cells with octanoate there is an inhibition of both glucose and glutamine metabolism and that octanoate-derived carbon becomes the major fuel source in the cell. We then found that histones were hyperacetylated after octanoate treatment and remarkably, close to 90% of the carbon on histones was octanoate derived. In addition, octanoate is a weak HDAC inhibitor which further promotes octanoate-derived acetyl-CoA being deposited onto histones. A gene array from octanoate treated samples finds that fatty acid metabolism is the top pathway in our gene ontology analysis. This provides evidence that the cell responds to nutrient sources in a specific manner depending on the nature of the carbon source. Finally, we find the most negatively regulated pathway upon octanoate treatment is DNA replication. Consistently, we find that octanoate causes an accumulation of cells in G1 phase of the cell cycle and induction of apoptosis.</p><p>Here we describe a mechanism for how fatty acids are sensed and how they communicate with the nucleus to alter gene expression. We show that the cell responds to lipids via a coordinated response to promote lipid metabolism and induce histone acetylation. This feed-forward mechanism of lipid metabolism consists of a reprograming of anabolic metabolism, and promotion of gene expression changes culminating in inhibition of cell growth and apoptosis.</p> / Dissertation

Many haematological malignancies are associated with widespread alterations of the transcriptional and epigenetic programmes. Changes in DNA methylation provide the clearest example of epigenetic changes, but the mechanism(s) underlying such changes is unknown. To investigate this I studied DNA methylation across an ~80kb segment of the genome which is not known to be mutated in haematological malignancies. Methylation was perturbed in 35-100% of samples of DNA from individuals with a wide range of haematological malignancies but not in non-malignant haematological disorders. DNA methylation was comprehensively assessed by Southern blot analysis, classical bisulphite sequencing and using a newly developed capture bisulphite sequencing protocol. The results were also compared with analysis by MeDIP, an immunoprecipitation-based technique. These analyses provide methylation status at various levels including individual CpG resolution. This showed both gain and loss of methylation at CpG dinucleotides. Of interest, hypomethylation was most frequently seen in intergenic regions corresponding to transcription factor binding sites and areas of increased chromosome accessibility. These observations suggested that hypomethylation of the genome in haematological malignancies could arise from aberrantly expressed DNA binding proteins which, recruited to sequences in regions of open chromatin, would protect the underlying CpG dinucleotides from the methylation machinery. This, in turn, could lead to passive demethylation accumulating with increasing cell divisions. This hypothesis was tested with electrophoretic mobility shift assays using oligonucleotides representing the DNA underlying one such region. This showed that, compared to nuclear extracts from the lymphocytes of normal individuals, those from patients with CLL were enriched for a protein which binds to oligonucleotides containing the underlying sequence. Using a mass spectrometry approach, I identified a variety of proteins that may bind such regions and account for their passive demethylation in haematological malignancies.

The murine subventricular zone (SVZ) in a brain contains a population of stem cells and daily produces tens of thousands of neurons throughout lifetime. However, the mechanisms of SVZ neural stem/progenitor cell (NSPC) maintenance, differentiation and cell-fate specification are still not clear. To understand these parameters via histone methylations with bivalent mechanism, the SVZ NSPCs were first isolated by using a culture technique called neurosphere assay (NSA). It has been a challenge to culture pure cell populations of SVZ subtypes, so the NSA was initially validated. The H3K27me3 mark, which has a dominant role in the bivalent mechanism, has not been studied in postnatal and adult SVZ in vivo, yet their role has been implicated to control the shift of embryonic cortical neurogenesis to gliogenesis. Therefore, we have first investigated whether H3K27me3 marks are present in the postnatal and adult SVZ NSPC population and whether their marks have been changed after stroke or demyelination in central nervous system (CNS) by immunohistrochemistry. With the confirmation of H3K27me3 mark present in SVZ NSPCs, the presence of H3K27me3 catalyzer, called polycomb repressive complex 2 (PRC2) core components (Eed, Ezh2, Suz12) including Jarid2, was investigated and confirmed in postnatal SVZ in vitro by qRT-PCR and Western blot. To understand the role of PRC2 enzymatic activity in postnatal SVZ neurosphere self-renewal and multipotency, Eed was down-regulated by using lentiviral mediated delivery of shRNA. Also, PRC2 dependent or independent function of Jarid2 was examined via knockdown approach. The lack of Eed in the neurospheres resulted the attenuation of self-renewal and oligodendrogenesis, whereas the Jarid2 knockdown neurospheres showed the decreased proliferation with no SVZ NSPC differentiation. Based on these knockdown studies, it suggests Eed and Jarid2 might not share their function in the postnatal SVZ NSPCs to govern postnatal SVZ NSPC self-renewal and multipotency.

The African trypanosome Trypanosoma brucei evades the immune system of the mammalian host by periodically switching its surface coat which is made up of Variant Surface Glycoprotein (VSG). T. brucei shows monoallelic expression of one VSG out of a repertoire of ~1200 genes with the active VSG gene expressed from one of ~15 telomeric expression sites (ESs). The mechanism behind the monoallelic exclusion of ESs is unclear. NLP was identified as a novel and essential AT-hook protein binding transcriptionally silent simple sequence repeats in T. brucei. I depleted NLP using RNAi in various T. brucei reporter lines containing an eGFP in different transcriptionally silent areas of the genome and monitored derepression of eGFP using flow cytometry. After NLP knock-down, I observed 45-65 fold derepression of silent ESs, and up to 5 fold derepression of other transcriptionally silent areas. Using chromatin immunoprecipitation (ChIP) I found an enrichment of NLP in certain non-transcribed loci including the rDNA spacers. I also found that blocking NLP synthesis results in a rapid fall in levels of the active VSG transcript. Lastly, I discovered using tandem affinity purification (TAP) followed by mass spectrometry that NLP is a part of a novel TbISWI complex in T. brucei, which also includes two previously unidentified protein partners. The high mobility group B (HMGB) protein family constitutes a major abundant class of non-histone chromatin associated DNA-binding proteins which play a role in chromatin architecture in a wide range of eukaryotes. In T. brucei, the HMGB protein TDP1, which contains two HMG boxes and one DEK C terminal DNA-binding domain, was first identified as binding to VSG ES promoter oligomer sequences. I report that TDP1 is an essential nuclear protein enriched in the nucleolus and expression site body, and is involved in facilitating transcription. Blocking TDP1 synthesis using RNAi mediated knock-down results in approximately 40-90% reduction in transcription of RNA polymerase I transcribed genes. Using ChIP, I find that TDP1 is enriched in the rDNA and on the active VSG ES in bloodstream form T. brucei. Additionally, the relative proportion of TDP1 binding the procyclin promoter compared with the upstream spacer and downstream EP1 genes is greater in procyclic form compared with bloodstream form T. brucei. Lastly, I performed TAP experiments with TDP1 and found that TDP1 interacts with the core histones. These results indicate that TDP1 is an architectural chromatin protein important for transcription control in T. brucei.

No description available.