Transcription factor networks and chromatin remodeler function in gene regulation on the eukaryotic genome

Numerous events – from histone modification and transcription factor binding to gene expression – take place on eukaryotic chromatin, while cells are constantly exposed to dynamic stimuli ranging from spatial and temporal cues to environmental and extracellular signals. The cell’s ability to respond and adjust accordingly is directly related to cell fitness and viability. With the advent of next-generation sequencing, investigating these events has been enabled at nucleotide resolution but across the entire genome. In this dissertation, I investigate changes on eukaryotic genomes including yeast and human, which are triggered by stress and by loss of a protein of interest, by analyzing genomics data generated mainly through next-generation sequencing. In Chapter 1, I determine how yeast cells achieve transcriptional reprogramming in response to heat stress by first identifying the complete set of transcription factors that are essential for heat stress conditions. This is further explored by identifying both the target loci bound by the transcription factors under conditions of heat-stress, as well as the genes that require the function of the transcription factor for normal transcriptional response to heat stress. In Chapter 2, I study a chromatin remodeling factor, CHD1 (Chromodomain Helicase DNA binding protein 1) with regard to two aspects: first, what factors provide specificity for Chd1 positioning on chromatin, by examining the role of proteins that physically or genetically interact with Chd1, and second, what is the relationship of Chd1 with the hallmark of chromatin modifications, histone H3 tri-methylation at Lys 4 and Lys 36, by investigating changes in these histone methylation marks in the absence of Chd1. Additionally, I show a novel functional link between Chd1 and RNA splicing through analysis of intron retention in transcripts produced in the Chd1 mutant. Lastly, I investigate Chd1 role in human glioblastoma cell line by generating a Chd1 knock-out via the CRISPR/Cas9 genome editing system. Taken together, the work presented in this dissertation provides novel approaches, discoveries, and intriguing insights into how eukaryotic chromatin experiences dynamic alterations in response to various perturbations on a genome-wide scale.Cellular and Molecular Biolog

Internet Recruitment of Asian American Breast Cancer Survivors

The purpose of this paper is to identify practical issues in Internet recruitment of racial/ethnic minorities by analyzing an Internet intervention study conducted with Asian American breast cancer survivors, and to propose directions for recruitment of racial/ethnic minorities for future Internet research. Six practical issues were identified: (a) a relatively fewer number of Internet communities/groups; (b) hindrances in establishing authenticity; (c) difficulties in gaining entrée from the webmasters or website owners of Internet communities/groups; (d) the necessity of racially/ethnically matched research team members; (e) flexibility required in recruitment strategies; and (f) strategies to overcome the low response rate

Physiologic Changes during Sponge Bathing in Premature Infants

In this study, we observed physiological reactions of premature infants during sponge bathing in the neonatal intensive care unit (NICU). The infants’ body temperature, heart rate, and oxygen saturation were monitored to examine hypothermia risks during bathing. The participants of the study were 32 premature infants who were hospitalized right after their birth in the V hospital in Korea between December 2012 and August 2013. The informed consents of the study were received from the infants’ parents. The infants were randomly assigned into two-day and four-day bath cycle groups and their physiological reactions were monitored before bathing as well as 5 and 10 min after bathing. The collected data were analyzed using the SPSS statistical package through t-test. A significant drop in body temperature was noted in both groups; that is, 4-day bathing cycle and 2-day bathing cycle (p < 0.001). However, there were no significant changes in heart rate or transcutaneous oxygen levels. There was no significant change between groups at each measurement point. In order to minimize the physiological instability that may be caused during bathing, the care providers should try to complete bathing within the shortest possible time and to make bathing a pleasant and useful stimulus for infants

Analysis of transcriptional modules during human fibroblast ageing

For systematic identification of transcription signatures of human cell aging, we carried out Weighted Gene Co-expression Network Analysis (WGCNA) with the RNA-sequencing data generated with young to old human dermal fibroblasts. By relating the modules to the donor's traits, we uncovered the natural aging- and premature aging disease-associated modules. The STRING functional association networks built with the core module memberships provided a systematic overview of genome-wide transcriptional changes upon aging. We validated the selected candidates via quantitative reverse transcription PCR (RT-qPCR) assay with young and aged human fibroblasts, and uncovered several genes involved in ECM, cell, and nuclear mechanics as a potential aging biomarker. Collectively, our study not only provides a snapshot of functional changes during human fibroblast aging but also presents potential aging markers that are relevant to cell mechanics.ISSN:2045-232

Transcultural Nursing: Current Trends in Theoretical Works

Purpose: To explore the current trends in theoretical works related to transcultural nursing through an integrated literature review. Methods: The literature on theoretical works related to transcultural nursing during the past 10 years was searched through multiple databases and reviewed to determine themes reflecting the current trends in theoretical works related to transcultural nursing. Results: Through the review and analysis process, four themes reflecting the current trends in theoretical works related to transcultural nursing were found: (a) purposes of exploring, defining and clarifying, and reflecting; (b) various theorizing methods; (c) multiple sources of theorizing; and (d) clear linkages to nursing practice and research. Conclusion: Continuous efforts need to be made to further develop the theoretical works related to transcultural nursing to reflect changes in this ever-changing nursing world. Keywords: nursing research, nursing theory, transcultural nursin

Widespread misinterpretable ChIP-seq bias in yeast.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is widely used to detect genome-wide interactions between a protein of interest and DNA in vivo. Loci showing strong enrichment over adjacent background regions are typically considered to be sites of binding. Insufficient attention has been given to systematic artifacts inherent to the ChIP-seq procedure that might generate a misleading picture of protein binding to certain loci. We show here that unrelated transcription factors appear to consistently bind to the gene bodies of highly transcribed genes in yeast. Strikingly, several types of negative control experiments, including a protein that is not expected to bind chromatin, also showed similar patterns of strong binding within gene bodies. These false positive signals were evident across sequencing platforms and immunoprecipitation protocols, as well as in previously published datasets from other labs. We show that these false positive signals derive from high rates of transcription, and are inherent to the ChIP procedure, although they are exacerbated by sequencing library construction procedures. This expression bias is strong enough that a known transcriptional repressor like Tup1 can erroneously appear to be an activator. Another type of background bias stems from the inherent nucleosomal structure of chromatin, and can potentially make it seem like certain factors bind nucleosomes even when they don't. Our analysis suggests that a mock ChIP sample offers a better normalization control for the expression bias, whereas the ChIP input is more appropriate for the nucleosomal periodicity bias. While these controls alleviate the effect of the biases to some extent, they are unable to eliminate it completely. Caution is therefore warranted regarding the interpretation of data that seemingly show the association of various transcription and chromatin factors with highly transcribed genes in yeast

Isolation and characterization of five novel probiotic strains from Korean infant and children faeces.

Probiotics are dietary supplements containing viable, non-pathogenic microorganisms that interact with the gastrointestinal microflora and directly with the immune system. The possible health effects of probiotics include modulating the immune system and exerting antibacterial, anticancer, and anti-mutagenic effects. The purpose of this study was to isolate, identify, and characterize novel strains of probiotics from the faeces of Korean infants. Various assays were conducted to determine the physiological features of candidate probiotic isolates, including Gram staining, 16S rRNA gene sequencing, tolerance assays to stimulated gastric juice and bile salts, adherence ability assays, antibiotic susceptibility testing, and assays of immunomodulatory effects. Based on these morphological and biochemical characteristics, five potential probiotic isolates (Enterococcus faecalis BioE EF71, Lactobacillus fermentum BioE LF11, Lactobacillus plantarum BioE LPL59, Lactobacillus paracasei BioE LP08, and Streptococcus thermophilus BioE ST107) were selected. E. faecalis BioE EF71 and L. plantarum BioE LPL59 showed high tolerance to stimulated gastric juice and bile salts, and S. thermophilus BioE ST107 as well as these two strains exhibited stronger adherence ability than reference strain Lactobacillus rhamnosus GG. All five strains inhibited secretion of lipopolysaccharide-induced pro-inflammatory cytokines IL-6 and TNF-α in RAW264.7 macrophages in vitro. L. fermentum BioE LF11, L. plantarum BioE LPL59, and S. thermophilus BioE ST107 enhanced the production of anti-inflammatory cytokine IL-10. Overall, our findings demonstrate that the five novel strains have potential as safe probiotics and encouraged varying degrees of immunomodulatory effects

Fibroblast rejuvenation by mechanical reprogramming and redifferentiation

Over the course of the aging process, fibroblasts lose contractility, leading to reduced connective-tissue stiffness. A promising therapeutic avenue for functional rejuvenation of connective tissue is reprogrammed fibroblast replacement, although major hurdles still remain. Toward this, we recently demonstrated that the laterally confined growth of fibroblasts on micropatterned substrates induces stem-cell-like spheroids. In this study, we embedded these partially reprogrammed spheroids in collagen-I matrices of varying densities, mimicking different three-dimensional (3D) tissue constraints. In response to such matrix constraints, these spheroids regained their fibroblastic properties and sprouted to form 3D connective-tissue networks. Interestingly, we found that these differentiated fibroblasts exhibit reduced DNA damage, enhanced cytoskeletal gene expression, and actomyosin contractility. In addition, the rejuvenated fibroblasts show increased matrix protein (fibronectin and laminin) deposition and collagen remodeling compared to the parental fibroblast tissue network. Furthermore, we show that the partially reprogrammed cells have comparatively open chromatin compaction states and may be more poised to redifferentiate into contractile fibroblasts in 3D-collagen matrix. Collectively, our results highlight efficient fibroblast rejuvenation through laterally confined reprogramming, which has important implications in regenerative medicine.ISSN:0027-8424ISSN:1091-649

Example of high background signal across multiple datasets.

<p>Sequencing datasets from different factors, controls, epitope tags, transcription factors and growth conditions as indicated are represented in a browser view. Based on the read counts normalized by transcript lengths from RNA-seq data [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083506#B44" target="_blank">44</a>], PHO84 is the 82^nd most highly expressed gene under normal conditions in WT yeast.</p