1,721,049 research outputs found
LncRNA Cyrano in Neural Differentiation of Embryonic Stem Cells
Get PDFThe goal of this project is to elucidate long non-coding RNA Cyrano’s role in the neural differentiation pathway of embryonic stem cells (ESCs). Currently, long non-coding RNAs (lncRNAs) are known to be prolifically expressed, with nonprotein-coding transcripts consisting of a large portion of the human transcriptome, but their functions are understudied and not well unders tood. The studies that have been executed show that lncRNAs are impactful on gene expression and developmental processes. Further, misregulation of lncRNAs has been implicated in many diseases, including neurodegenerative disorders. It is crucial to investigate Cyrano as a key player in neural differentiation to delineate the function of a paradigmal lncRNA to build the foundation of understanding of lncRNA functions as a whole. Additionally, the implication of Cyrano in neural differentiation will further understanding of the gene expression network necessary for neural differentiation. Although further studies will be necessary, investigation of Cyrano may prove to be an informative step in understanding gene regulation of neural differentiation. Our data points to a suppressor role for Cyrano in neural differentiation, with a loss of Cyrano function resulting in an increase in neural differentiation and may suggest an interplay with miR-7 in this context.Bachelor of Art
Allele-Specific Chromosome Conformation and its Association with Allelic Expression Bias
Get PDFIt is frequently suggested in recent literature that long-range interactions, presumably between enhancer elements and promoters, are an important factor in mediating gene expression. Such chromatin loops are often described as regulatory models in which active or repressive loops are suggested to exist as predominate chromosome conformations that are tightly associated with expression states. These models are supported by numerous investigations of well-studied models such as the beta-globin locus and the Hox clusters. If correct, it follows from these models that differential chromosome conformation patterns, under various contexts, should be a widely observable property of loci that exhibit differential gene expression. To test predictions of this hypothesis, seven candidate genes exhibiting strong allele-specific expression bias were selected for analysis with allele-specific 4C-Seq assays. Conditions for chromosome conformation experiments were optimized for a trophoblast stem cell model that is ideal for interrogating allele-specific chromatin and expression regulation. Results from these experiments demonstrate that the profile of observable interactions may vary between copies. This is important because many genomics assays do not typically detect resolution to discriminate between alleles. The observations made here help to demonstrate that assumption of behavior and modification of loci with genome-wide assays may introduce errors related to inability to distinguish whether events are occurring on both alleles in similar proportions or even at all.Doctor of Philosoph
Decoding Mechanisms of Loss of Fertilization Ability of Cryopreserved Mouse Sperm
Get PDFCryopreservation of mouse sperm is an important technology for management of biomedical research resources. Dramatic progress has been made recently in the development of protocols that combat mouse-strain specific reduction of IVF after cryopreservation. Equal emphasis, however, has not been placed on investigating the biological mechanisms underlying these improvements to IVF. This dissertation broadly investigates the basic question of how mouse-strain specific reduction of IVF occurs after cryopreservation, and how recently developed protocols prevent this process. My research investigated the effects of antioxidants, the cholesterol-acceptor CD, reduced calcium media, and TYH capacitation media on sperm function and oxidative stress after cryopreservation in a variety of mouse strains. I found that reduced IVF was associated with loss of capacitation-dependent sperm function in three strains, B6/J, B6/N, and 129X1, and CD improved sperm function and IVF in all three strains. These findings suggest that cryopreservation inhibits cholesterol efflux resulting in reduced IVF of many mouse strains. I also found that cryopreservation induces uniquely high production of mitochondrial H2O2 by B6/J sperm. H2O2 present in other cellular compartments of B6/J sperm was not elevated compared to other strains. High levels of mitochondrial H2O2 were associated with lipid peroxidation of the sperm head and inability to acrosome react. Antioxidants reduced mitochondrial H2O2 production, decreased sperm head lipid peroxidation, and improved acrosome reaction. The cryopreservation-induced increase in mitochondrial H2O2 production of B6/J and B6129XF1 sperm was associated with elevation of intracellular calcium after cryopreservation and dependent on mitochondrial metabolic substrates. Reducing intracellular calcium levels or removing mitochondrial metabolic substrates decreased mitochondrial H2O2 production and increased IVF rates of cryopreserved B6/J sperm. Many of the strains I tested exhibited increased H2O2 production after cryopreservation, but cryopreservation-induced H2O2 only interfered with IVF of B6/J sperm. This dissertation describes two means to improve IVF of cryopreserved sperm, mitigation of oxidative stress in B6/J sperm and improvement of capacitation-dependent sperm function for several mouse strains.Doctor of Philosoph
Genetic regulation of epigenetic processes in mouse: DNA methylation and X chromosome inactivation
Get PDFEpigenetics is the study of inheritance not encoded by primary DNA sequence. In mammals, epigenetic processes are required for proper development, gene regulation, chromosome function (e.g., X-chromosome inactivation (XCI)), and genome stability. Misregulation of epigenetic processes is typically a hallmark of disease. Epigenetic marks vary depending on genomic position, cell type, environment, time, sex, and even between individuals within a population. Genetic variation is one source of epigenetic variability that has only recently been appreciated. It is unknown how prevalent and to what extent underlying genetic variation influences epigenetic variability, and furthermore, how this epigenetic variability contributes to phenotypic variation within a population. It has been postulated that epigenetic variation between individuals may help solve the `missing heritability' problem. In an attempt to address these questions and further characterize the influence of genetics on epigenetics, I demonstrate that DNA sequence variation in cis affects two epigenetic processes, DNA methylation and XCI. In the first section, I performed a genome-wide allele-specific methylation survey in the mouse brain to show widespread loci that influence nearby DNA methylation at CpGs. These differentially methylated CpGs tend to reside near transcription start sites and may serve a functional role. We estimate that there are roughly 13,000 of these loci genome-wide. Additionally, I show that these strain-specific cis-acting loci also influence a parent-of-origin differentially methylated region in the 3'UTR of the Actn1 gene, which suggests that genetic variation might also influence highly conserved imprinted regions as well. In the second section, I mapped a cis-acting locus called the X-chromosome controlling element (Xce) that influences XCI choice in mouse. I reduced the Xce candidate interval to a 176 kb region located approximately 500 kb proximal to Xist. I extensively characterized the genetic architecture of the new candidate interval in over 300 inbred and wild-caught mice. I conclude that each mouse taxa examined has a different functional Xce allele and there is no sharing. I identified two new Xce alleles (Xcee and (Xcef) that bring the number to six functional alleles in Mus. I propose that structural variation of segmental duplications within this interval explains the presence of multiple functional Xce alleles. Overall these results provide new insights into the genetic regulation of epigenetic processes in mouse. Furthermore, this work creates a foundation for future work to untangle the molecular mechanisms behind differential DNA methylation and X- chromosome inactivation choice.Doctor of Philosoph
Epigenetic defects in stem cells deficient in polycomb group function
Get PDFDuring development, the expression states of many genes must be maintained through cell divisions in order to ensure lineage-, time-, and dose-appropriate patterns of gene expression. This transcriptional memory is independent of permanent DNA sequences changes and instead involves reversible epigenetic mechanisms. Polycomb Group (PcG) proteins represent a conserved family of developmental regulators that mediate heritable transcriptional silencing by covalently modifying histone proteins. Here, we demonstrate that mutations in the PcG gene Embryonic ectoderm development (Eed) produce a variety of epigenetic defects in mouse embryos and stem cells. EED is a noncatalytic subunit of Polycomb Repressive Complex 2, a 600 kDa complex containing a number of proteins, including the histone H3-lysine 27 (H3K27) methyltransferase EZH2. Consistent with the role of PcG genes in transcriptional memory, Eed mutant embryos and trophoblast stem cells have defects in genomic imprinting, a process by which an allele's expression is dependent upon the gender of the parent from which it was inherited. To determine whether these gene expression defects revealed a required role for EED in PRC2 function, we characterized the status of H3K27 methylation in Eed mutant stem cells. H3K7 can be mono-, di-, or trimethylated (H3K27me1, H3K27me2, H3K27me3, respectively), but it has been unclear which of these marks are mediated by PRC2. Here, we demonstrate that EED is required for all three methylation states. Additionally, although EED is present as four distinct isoforms in mammalian cells, these isoforms are not necessary for H3K27 methylation. Instead, EED's core WD-40 motifs and histone binding domain alone are sufficient to mediate histone methylation. Finally, although the histone methylation defects in Eed mutant stem cells appear to be global, the imprinted expression defects are restricted to DNA hypomethylated, extraembryonic tissues and to genes that are imprinted normally in DNA methyltransferase 1 (Dnmt1) mutant placentas. Together, these results suggest that histone methylation and DNA methylation may have non-overlapping roles in imprinted gene regulation
Determining the Role of the Xist Non-coding RNA in Epigenetic Programming of X-chromosome Inactivation
Get PDFRepression of Xist RNA expression is considered a prerequisite to reversing X-chromosome inactivation (XCI) in the mouse inner cell mass (ICM), and reactivation of X-linked genes is thought to follow loss of Xist RNA coating and heterochromatic markers of inactivation, such as methylation of histone H3. I analyzed X-chromosome activity in developing ICMs and show reactivation of gene expression from the inactive-X initiates in the presence of Xist coating and H3K27me3. Furthermore, depletion of Xist RNA coating through forced upregulation of NANOG does not result in altered reactivation kinetics. Taken together, our observations suggest that in the ICM, X-linked gene transcription and Xist coating are uncoupled. These data fundamentally alter our perception of the reactivation process and support the existence of a mechanism to reactivate Xp-linked genes that operates independent of loss of Xist RNA and H3K27me3 from the inactive-X
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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