Dissertations / Theses on the topic 'Genetics and genomics/functional genomics'
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Burnham, Katie. "Functional genomics of the sepsis response." Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:cb98af40-1b66-4966-a643-ae8dfec2c122.
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Sepsis is defined as a dysregulated immune response to infection causing organ dysfunction, and is a major area of unmet clinical need. Although conventionally considered a unified disease with a common pathway to organ failure and death, substantial clinical and molecular heterogeneity is seen, which has limited efforts to understand pathophysiology and improve therapeutic strategies. Sepsis is associated with global changes in gene expression, and genetic variants are known to affect the response to infection. This thesis therefore uses an integrated functional genomics approach to investigate disease mechanisms and variation in the sepsis response. Data are presented for 551 patients admitted to intensive care with sepsis due to community acquired pneumonia (CAP) or faecal peritonitis (FP). The sepsis response is explored using genome-wide gene expression and proteomics data, and molecular quantitative trait loci (QTL) are mapped in the context of disease. Comparisons with cardiac surgery patients are performed to identify shared and specific aspects of the host response. The host transcriptomic response was largely shared across sources of sepsis, although some specificity relating to viral infection and interferon signalling was observed and validated in prospectively recruited patients. Expression-based sepsis response signature (SRS) subgroups previously described in CAP were validated, and were additionally observed in FP. SRS1 is associated with higher early mortality, and shows enrichment of pathways relating to T cell exhaustion, cell death, and endotoxin tolerance. Differences between SRS groups were also observed in the FP plasma proteome. Serial sampling enabled the investigation of temporal changes in gene expression and protein abundance within patients. Lastly, disease-relevant expression QTL were identified, and interactions with source of sepsis and SRS determined, highlighting the potential impact of regulatory variation on the sepsis response. This thesis demonstrates the benefit of an integrative functional genomics approach to explore heterogeneity in sepsis, and highlights opportunities for patient stratification and personalised medicine.
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Blischak, John David. "Investigating susceptibility to tuberculosis using functional genomics approaches." Thesis, The University of Chicago, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10238063.
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A major goal of human genetics is to characterize the role of genetic variation on complex, polygenic phenotypes. With the discovery from genome-wide association studies (GWAS) that many associated variants have a small effect size and are located in non-coding regions of the genome, there has been a large effort to collect functional genomics data. The hope is that a better understanding of how the genome functions in diverse developmental states and environments will provide insight into the context-specific activity of associated non-coding variants. My research applies this paradigm to the complex phenotype of susceptibility to develop tuberculosis (TB). It has been estimated that 10% of individuals infected with Mycobacterium tuberculosis (MTB) progress to active disease. Despite being heritable, very few genetic variants have been associated with susceptibility to TB. For my studies, I use RNA sequencing (RNA-seq) to interrogate genome-wide transcript levels in in vitro cellular models. In Chapter 2, I use a joint Bayesian model to identify genes which are differentially expressed in macrophages only after infection with MTB and related mycobacteria, but not other bacterial pathogens. In Chapter 3, I build a support vector machine model to classify individuals as susceptible or resistant to TB based on the gene expression levels in their dendritic cells. In Chapter 4, I characterize the technical variation introduced by batch processing of single-cell RNA-seq (scRNA-seq) and propose an effective study design that accounts for technical variation while minimizing replication. In addition to providing insight into the genes important for the innate immune response to MTB infection, my work is informative for the design and analysis of future functional genomics experiments. (Note: Supplementary tables are provided in a .zip file available online. Captions for the tables are provided within the dissertation.)
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Moustafa, Ahmed. "Evolutionary and functional genomics of photosynthetic eukaryotes." Diss., University of Iowa, 2009. https://ir.uiowa.edu/etd/311.
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My dissertation focuses on genome and functional evolution of photosynthetic eukaryotes and the design and implementation of computational methods and tools to enable genome-wide studies to investigate these taxa. The work described here is grouped into two major topics, 1) endosymbiosis and genome evolution, and 2) harmful algal blooms. I discuss my work related to endosymbiosis and genome evolution in chapters 2-4. Chapters 5-6 cover the work related to harmful algal blooms. In chapter 1, I introduce the state-of-art of what is known about the history of plastids and evolution of photosynthesis in eukaryotes, an overview of marine harmful algae, and the specific aims of my dissertation.
In chapter 2, I describe the design and implementation of the phylogenetic sorting tool, PhyloSort and the assembly of a high-throughput phylogenomic pipeline. Together, PhyloSort and the pipeline has become a key tool for multiple subsequent studies. chapter 2 also presents a case study using these tools in which we provide an estimate of the number of cyanobacterial genes that have been transferred to the nuclear genome of Plantae through primary endosymbiotic gene transfer; I use the model unicellular green alga Chlamydomonas reinhardtii for this purpose.
In chapter 3, I discuss another case of prokaryotic contribution to the nucleus of photosynthetic eukaryotes. Here, the intriguing relationship of Chlamydiae-like bacteria and plants and algae is examined in a large-scale analysis, in which we scanned all available genomes of the primary photosynthetic organisms for genes of potential Chlamydiae origin. Surprisingly, we identified more than fifty Chlamydiae-derived genes in plants and algae. Here, we propose a model for the role that a Chlamydiae-like symbiont might have played in the establishment of the primary plastid in the common ancestor of Plantae.
In chapter 4, I describe a study in which we explored the complete protein models of two diatom organisms as representative for photosynthetic chromalveolates and looked for genes that might have been acquired through endosymbiotic (secondary) or horizontal transfers from red or green algae. In contradiction of the “chromalveolate hypothesis” which states that photosynthesis in chromalveolates originated via the engulfment of a red alga symbiont, our study shows an unexpected green algal contribution that is fourfold greater than that of the canonical red algal symbiont. Our data suggest that the chromalveolate history includes a previously unrecognized green algal endosymbiont that was captured and lost prior to the more recent establishment of the red alga plastid, which is widespread in extant photosynthetic chromalveolates.
In chapter 5, I discuss the identification of the phylogenetic origin of the genes involved in the biosynthetic pathway of saxitoxin in cyanobacteria. Here, we used a pyrosequencing approach to sequence de novo genomes of two strains of Anabaena circinalis, one of which is saxitoxin-producing and the other is non-toxic. Using comparative and phylogenetic analyses, I show that, within the saxitoxin gene cluster, genes that encode the key and unique enzymes in the pathway are of foreign origin that originated via horizontal transfer from non-cyanobacterial sources. These genes introduced the ability to produce saxitoxin in the ancestor of the toxic cyanobacterial clade.
In chapter 6, I describe a gene expression study in which we used massively parallel signature sequencing (MPSS) to investigate RNA abundance patterns in the toxic dinoflagellate Alexandrium tamarense. This work provides the first clear evidence for the utilization by dinoflagellates of transcriptional to regulation. Moreover, using MPSS, we provide an estimate of the number of the distinct genes in Alexandrium tamarense; i.e., remarkably 40,000 loci. Taken together, our data indicate that dinoflagellates possess a great metabolic flexibility that allows them to efficiently toggle between photoautotrophy and heterotrophy based on the environmental conditions.
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Mizrachi, Eshchar. "Functional genomics and systems genetics of cellulose biosynthesis in Eucalyptus." Diss., University of Pretoria, 2013. http://hdl.handle.net/2263/79771.
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The globally emerging bioeconomy demands rapid advancement in the sustainable production and utilization of bio-based raw materials for a multitude of downstream applications, particularly in the areas of food, health and bioenergy and biomaterials. These needs, particularly pertaining to plant productivity, quality and stress tolerance, will need to be addressed with advanced biotechnology strategies, which accelerate progress beyond what has been achieved with traditional breeding and cultivation methods. Woody biomass is a readily available source of renewable carbon, and trees from the genus Eucalyptus, displaying superior growth and wood properties and established agricultural practices worldwide, are attractive candidates as short-rotation (5-9 years) feedstocks for biofuels and biomaterials. Guiding advanced strategies in biotechnology in Eucalyptus and other biomass feedstocks requires a sophisticated understanding of the molecular underpinnings of carbon allocation and cell wall biology. In the work presented here, we aimed to characterize the molecular biology of cellulose biosynthesis in Eucalyptus xylem (developing wood) and identify genes, processes and pathways that are linked to and possibly influence this process. We achieved this by detailed characterization of field-grown Eucalyptus hybrid trees, utilizing RNA-sequencing technology and metabolomics of xylem as well as measuring wood properties that are thought to impact the efficiency of industrial processing. Given the lack of information with regards to gene expression in Eucalyptus trees, a major aim was to characterize transcriptomes from various tissues and organs, including a cellulose-enriched form of xylem called tension wood. This involved challenging bioinformatics, which resulted in a high quality assembly and publication of a comprehensive gene catalogue for Eucalyptus, which was one of the first short-read RNA-sequencing based de novo assembly from a eukaryotic organism. We also characterized and modelled the properties of cellulose and xylan biosynthetic pathways as a biological system, the parts of which are segregating in Eucalyptus hybrid tree populations, which has generated novel insights into the allocation and partitioning of sequestered carbon between cellulose, xylan and lignin during active secondary cell wall deposition in woody stem tissues.
This research has made important contributions to the field of Eucalyptus biology, but also to the broader field of secondary cell wall biosynthesis in plants, specifically providing (i) resources for transcriptome analysis in a large woody perennial (ii) new biological insight into carbon allocation for polysaccharide biosynthesis in wood, and (iii) annotation and discovery of candidate genes and pathways that may influence wood chemical composition and structures. Importantly, we find that cellulose and xylan biosynthetic genes are transcriptionally hardwired in their co-regulation (along with other important processes for cellulose and xylan transport and deposition), likely due to the fact that they utilize a common source of sucrose-derived carbon for cell wall biosynthesis and the production of sufficient energy to do so. This co-regulation appears to be distinct from the regulation of other cell wall biopolymers. Furthermore, evidence from xylem gene expression and metabolite availability in xylem, as research has made important contributions to the field of Eucalyptus biology, but also to the broader field of secondary cell wall biosynthesis in plants, specifically providing (i) resources for transcriptome analysis in a large woody perennial (ii) new biological insight into carbon allocation for polysaccharide biosynthesis in wood, and (iii) annotation and discovery of candidate genes and pathways that may influence wood chemical composition and structures. Importantly, we find that cellulose and xylan biosynthetic genes are transcriptionally hardwired in their co-regulation (along with other important processes for cellulose and xylan transport and deposition), likely due to the fact that they utilize a common source of sucrose-derived carbon for cell wall biosynthesis and the production of sufficient energy to do so. This co-regulation appears to be distinct from the regulation of other cell wall biopolymers. Furthermore, evidence from xylem gene expression and metabolite availability in xylem, as well as from wood properties of field-grown trees, supports a model in which sucrose-derived cytosolic fructose is shunted to the production of lignin precursors during cellulose and xylan biosynthesis. This model parsimoniously explains a mechanism for trees to partition carbon between polysaccharide and lignin synthesis, and provides exciting new questions and potential strategies to influence carbon allocation in the secondary cell walls of woody plants.Thesis (PhD)--University of Pretoria, 2013.
Wood and Fibre Molecular Genetics (WFMG) Programme
National Research Foundation (NRF)
Technology and Human Resources for Industry Programme (THRIP)
Genetics
PhD
Unrestricted
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Radhakrishnan, Jayachandran. "Functional genomics of severe sepsis and septic shock." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:2b2afd65-82e0-4847-b7ae-960635b7e884.
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Sepsis is the systemic inflammatory response to an infection. Severe sepsis with multi organ failure is one of the commonest causes of admission to intensive care units, and is associated with poor early and late outcomes. The pathophysiology of sepsis is complex, and poorly understood. This is reflected in the limited and contentious treatment options for sepsis. Genetic factors have been shown to be associated with the risk of and subsequent outcomes from infection. However, clear associations with bacterial sepsis are rare, and even when associations are present their functional effects are often unknown. Gene expression signatures in sepsis are investigated in this project using serial samples obtained from patients admitted to intensive care units with community-acquired pneumonia or faecal peritonitis. The evolving gene expression signatures that define the response to sepsis were identified with large changes seen in genes coding for ribosomal proteins RPS4Y1 and RPS26P54. The differences in the sepsis response between the two diagnostic classes were examined. The gene expression predictors of mortality in sepsis were determined and include genes from the class II MHC HLA-DRB4, HLA-DRB5 and the T cell differentiation protein MAL. The effects of important covariates on gene expression were investigated and their impact on survival related expression determined. The findings were confirmed in a validation cohort. A novel clustering of samples representing distinct inflammatory patterns in a clinically homogeneous population of sepsis patients was identified and related to differences in clinical behaviour. The biological relevance of the differentially expressed genes was ascertained by identifying enriched gene sets. The gene expression changes in sepsis were examined in the context of related clinically relevant immune phenomena: the sterile systemic inflammatory response in patients undergoing elective cardiac surgery and the phenomenon of endotoxin tolerance in PBMCs derived from healthy volunteers. The results highlight the complexities of clinical sepsis and identify hypotheses for future investigations.
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Shao, Diane Donghui. "Functional Genomics Approaches to Identify and Characterize Oncogenic Signaling." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11076.
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Oncogenes drive cancer by hijacking normal cellular functions involved in proliferation and survival. Suppression of the driving oncogene is highly effective for promoting tumor regression, a phenomenon termed "oncogenic addiction." By using unbiased genetic tools to functionally probe oncogenic addiction, we can identify cancer dependencies and characterize aspects of oncogenic signaling.
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Steinberg, Julia. "Functional genomics analyses of neuropsychiatric and neurodevelopmental disorders." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:e47d1ac2-de92-47d8-864b-dac0bf6669e8.
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Recent large-scale genome-wide studies for many human disorders have identified associations with numerous genetic variants. The biological interpretation of these variants presents a major challenge. In particular, the identification of biological pathways underlying the association could provide crucial insights into the disease aetiologies. In this thesis, I used functional genomics approaches to increase our understanding of neuropsychiatric and neurodevelopmental disorders. Firstly, in an integrative analysis of autism spectrum disorder (ASD), I looked into the role of genes targeted by Fragile-X Mental Retardation Protein ("FMRP targets"). I found evidence that FMRP targets contribute to ASD via two distinct aetiologies: (1) ultra-rare and highly penetrant single disruptions of embryonically upregulated FMRP targets ("single-hit aetiology") or (2) the combination of multiple less penetrant disruptions of synaptic FMRP targets ("multiple-hit aetiology"). In particular, I developed a pathway-association test sensitive to multiple-hit aetiologies. Secondly, I carried out an integrative analysis of bipolar disorder, following up a previously identified association with long-term potentiation. The association was not consistent across independent SNP and CNV datasets. Thirdly, I addressed the difficulty in identifying functional relationships between genes by integrating different datasets into a gene functional-linkage network tuned to the nervous system ("NsNet"). NsNet identified functional links between the genes disrupted by de novo loss-of-function mutations in ASD and, separately, in schizophrenia probands more sensitively than a general functional-linkage network. Fourthly, I considered the challenge of interpreting the phenotypic impact of gene disruptions, focusing on the identification of haploinsufficient genes. I constructed a gene haploinsufficiency score based on genome-wide datasets. Compared to existing approaches, the new score performed better in identifying less-studied haploinsufficient genes. This work both extends the methodology to detect the contribution of genetic variation to neuropsychiatric disorders and also yields insights into the variant genes and the pathways that underlie them. Firstly, in an integrative analysis of autism spectrum disorder (ASD), I looked into the role of genes targeted by Fragile-X Mental Retardation Protein ("FMRP targets"). I found evidence that FMRP targets contribute to ASD via two distinct aetiologies: (1) ultra-rare and highly penetrant single disruptions of embryonically upregulated FMRP targets ("single-hit aetiology") or (2) the combination of multiple less penetrant disruptions of synaptic FMRP targets ("multiple-hit aetiology"). In particular, I developed a pathway-association test sensitive to multiple-hit aetiologies. Secondly, I carried out an integrative analysis of bipolar disorder, following up a previously identified association with long-term potentiation. The association was not consistent across independent SNP and CNV datasets. Thirdly, I addressed the difficulty in identifying functional relationships between genes by integrating different datasets into a gene functional-linkage network tuned to the nervous system ("NsNet"). NsNet identified functional links between the genes disrupted by de novo loss-of-function mutations in ASD and, separately, in schizophrenia probands more sensitively than a general functional-linkage network. Fourthly, I considered the challenge of interpreting the phenotypic impact of gene disruptions, focusing on the identification of haploinsufficient genes. I constructed a gene haploinsufficiency score based on genome-wide datasets. Compared to existing approaches, the new score performed better in identifying less-studied haploinsufficient genes. This work both extends the methodology to detect the contribution of genetic variation to neuropsychiatric disorders and also yields insights into the variant genes and the pathways that underlie them.
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Miller, Michael Ryan. "Functional Genomics of Nervous System Development and Disease." Thesis, University of Oregon, 2011. http://hdl.handle.net/1794/12102.
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xiii, 145 p. : ill. (some col.)The goal of functional genomics is to elucidate the relationship between an organism's genotype and phenotype. A key characteristic of functional genomics is the use of genome-wide approaches as opposed to more traditional single-gene approaches. Genome-wide expression profiling is used to investigate the dynamic properties of transcriptomes, provides insights into how biological functions are encoded in genomes, and is an important technique in functional genomics. This dissertation describes the use of genome-wide expression profiling and other functional genomics techniques to address a variety of biological questions related to development and disease of the nervous system. Our results reveal novel and important insights into nervous system development and disease and demonstrate the power of functional genomics approaches for the study of nervous system biology. This dissertation also describes a novel technique called TUtagging that facilitates cell type-specific RNA isolation from intact complex tissues. The isolation of RNA from specific cell types within a complex tissue is a major limiting factor in the application of genome-wide expression profiling, and TU-tagging can be used to address a wide array of interesting and important biological questions. This dissertation includes previously published and unpublished co-authored material.
Committee in charge: Dr. John Postlethwait， Chair； Dr. Chris Doe， Advisor； Dr. Bruce Bowerman， Member； Dr. Patrick Phillips， Member； Dr. Tom Stevens， Outside Member
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Butler, Heather. "Functional genomics : analysis of polytene region 38 of Drosophila melanogaster." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0016/MQ55041.pdf.
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Barona, Gómez Francisco. "Functional and structural genomics of amino acid metabolism in Streptomyces coelicolor." Thesis, University of Warwick, 2003. http://wrap.warwick.ac.uk/59424/.
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An investigation of amino acid metabolism in Streptomyces coelicolor, including the anabolism of tryptophan, histidine, the branched-chain amino acids and proline, as well as the catabolism of the latter, is reported. The experiments reported herein were conceptually conceived within a functional genomics framework. For this purpose the complete genome sequence of S. coelicolor was systematically exploited. Moreover, the current knowledge on the physiology of Streptomyces was taken onboard, as well as the prevailing and emerging notions on the evolution of proteins and metabolic pathways. Some of the results obtained using S. coelicolor as a model organism were expanded to other actinomycetes, such as Mycobacterium tuberculosis. This was aided by a comparative genomics analysis of the actinomycetes whose genomes have been sequenced. The theoretical principles that give support to this thesis are introduced in Chapter 1. This study was greatly facilitated by the development of a novel PCRtargeting mutagenesis method of which details can be found in Chapter VII. The discovery of a common isomerase for tryptophan and histidine biosynthesis is reported in Chapter II. This discovery arose from efforts aimed at reconstructing the tryptophan biosynthetic pathway of S. coelicolor, since the genome sequence project of this organism failed to identifiy a trpF gene coding for the enzyme phosphoribosyl anthranilate isomerase. The solution of this functional genomics discrepancy led to the discovery of a putative (~a)8-barrel enzyme, termed PriA, whose preliminary functional and structural characterisation is reported in Chapter III. The evolutionary implications of the discovery of PriA are discussed within Chapters III and N. A comparative genomics analysis of actinomycetes centred on the priA gene is presented in the latter Chapter, supporting the notion that this novel protein is spread across the high (0 + C) content Gram-positive organisms. Indeed, it was predicted that a priA orthologue accounts for the lack of a trpF gene from the genome of M tuberculosis, a hypothesis that proved to be correct. Finally, evidence to support the notion that the histidine and tryptophan biosynthetic pathways co-evolved is presented. In contrast to the isomerisation catalysed by PriA, in which an enzyme is shared by two amino acid biosynthetic pathways, several paralogous enzymes with the potential to account for the first step of tryptophan biosynthesis from chorismate were found on the genome of S. coelicolor. These chorismate-utilising enzymes are investigated in Chapter V. Mutational analysis of some of this paralogues is reported and it is anticipated that the analysis and results reported therein will serve to direct future experiments aimed at identifying the trpE paralogue encoding the enzyme anthranilate synthase. Chapter VI reports on the identification of the proC gene involved in the last step of proline biosynthesis in S. coelicolor. The pyrroline-5-carboxylate reductase activity of the enzyme encoded by the putative proC gene was extensively characterised, with particular emphasis on the interaction between primary and secondary metabolism. Furthermore, mutational analysis of proC suggested that paralogues of this gene are present on the genome of this organism, since its deletion did not lead to an auxotrophic phenotype. Investigation of this observation showed that two paralogous enzymes encoded by i1vC-like genes, involved in biosynthesis of the branched-chain amino acids, are capable of compensating for the lack of proC. This is the first example of a physiological link between the biosynthesis of proline and the branched-chain amino acids. To sum up, the results reported in this thesis represent an advancement towards understanding the physiology of S. coelicolor as a model actinomycete, within a functional and structural genomics framework. They also offer evidence on the evolutionary principles that lead to the appearance of novel proteins and metabolic pathways in bacteria.
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Gapp, Bianca. "Functional genomics and compound mode-of-action screening in haploid human cells." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:0c2ce8f8-15f3-447f-9117-8953329bd4ac.
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More than a decade after the completion of the human genome project, the function of a large number of genes remains to be elucidated. Forward and reverse genetic approaches have proven to be powerful tools to study gene function and have provided insights into fundamental biological processes. Furthermore, functional genetic screening can lead to a better understanding of the action of endogenous and exogenous stimuli such as hormones or drugs on biological systems. Thus far, systematic and unbiased studies have largely been limited to model organisms. However, complex disease-relevant genotypes and phenotypes cannot be studied in entirety in lower organisms creating a need for systematic approaches in human cells. This thesis describes a series of studies using forward and reverse genetic approaches combined with state-of-the-art technology in haploid human cells. The first chapter describes the development of a quantitative phenotypic read-out using a novel application of RNA-sequencing that allows the functional annotation of genes in signalling pathways. The presented data demonstrate that the employed shallow RNA-sequencing method is scalable and suitable as a read-out for reverse genetic screening. The second chapter focuses on the implementation of this method in a large reverse genetic study in human cells to functionally annotate tyrosine kinases in signalling pathways upon stimulation with a set of ten polypeptides and small molecules. The screens revealed known and unexpected interactions between different signalling molecules and pathways, validating the technical approach in a biological context. The third chapter presents a pilot study describing the set-up of a forward genetic technique for compound mode-of-action screening using a pooled human mutant cell line collection. The chemical genetic approach displayed sufficient sensitivity and allowed to monitor thousands of gene-drug interactions simultaneously. Together, this thesis combines elements to advance technological and biological aspects of functional genomics and chemical genetics.
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Wills, Quintin Frank. "The genetics of miRNA and mRNA expression in human lymphoblastoid cell lines." Thesis, University of Oxford, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.572601.
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Human clinical genome-wide association studies (GWAS) have helped identify disease trait and pharmacogenomic loci without the need for biological under- standing. Molecular GWAS - associating genetic variation with traits such as gene expression - have been slow to fill the mechanistic gaps. While tissue specificity, lack of DNA resolution, and the need for better data integration are no dou bt important bottlenecks in molecular GWAS, there is also a very poor general understanding of which molecular phenotypes are important and how best to model them. Added to this is the clear need for a greater understanding of the strengths and weaknesses facing in vitro (and ex vivo) models as hypoth- esis generating and GWAS validation tools. The studies in this work focus on RNA expression in a popular human model: lymphoblastoid cell lines (LCLs). Chapters 2 and 3 examine microRNA (miRNA) and messenger RNA (mRNA) expression in a total of 300 genotyped human LCLs. The expression of only one miRNA could be associated with a nearby genetic variant. This result was observed in both the African and European samples studied, in a separate val- idation data set, and was technically validated with quantitative PCR. While limited genotype resolution and small sample sizes are likely to be important contributors to this low hit rate, the results strongly suggest experimental con- founders. Highly expressed miRNAs reflected the transformed nature of the cells, highly correlated miRNAs enriched for EBV and malaria associated tar- get mRNA genes, and several miRNAs that were differentially expressed be- tween the European and African samples suggested differential EBV transfer- mation. Chapter 4 presents a study on single cells from some of the same samples, to test the hypothesis that the lack of tissue spatial resolution is an important limiting factor in human genetic epidemiology. Experimental con- founders were also considered: sample growth was found to associate with the expression of several genes. Cell-to-cell gene correlations and distributions made it possible to propose how genes change their expression, functionally differ from each other, and are able to alter their behaviours without altered whole-tissue expression. The results suggest which type of genes are more likely to be susceptible to genetic effects, and propose promoter behaviours altered by genetic variants located near to 13 genes. From these whole-tissue and single cell results the broad conclusion is that, while LCLs are likely to be inappropriate for the study of miRNA genetics, their functional genomics at higher spatial resolution shows promise as a more mechanistic approach for the study of germline genetics.
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Polke, James M. "Functional genomics in the stroke-prone spontaneously hypertensive rat : genome wide and candidate gene analysis." Thesis, University of Glasgow, 2008. http://theses.gla.ac.uk/258/.
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The stroke-prone spontaneously hypertensive rat (SHRSP) is an inbred model of hypertension. Renal microarrays and functional genomic strategies investigated chromosome 2 candidate hypertension genes, focussing on the oxidative-stress defence gene, glutathione s-transferse mu type 1 (Gstm1). Ingenuity pathway analysis of renal microarrays in 5 and 16-week SHRSP, normotensive Wistar Kyoto (WKY) and chromosome 2 congenic rats identified differential expression of several glutathione cycling genes. The Gstm1 promoter was investigated by luciferase and Transfac bioinformatic analysis, implicating two polymorphism clusters and several transcription factors in reduced SHRSP Gstm1 expression. Recombinant adenoviruses expressing Gstm1 and short-hairpin RNA-interference sequences to reduce Gstm family expression were produced. In-vivo overexpression of Gstm1 did not improve endothelial nitric-oxide bioavailability in SHRSP carotid arteries. Bacterial artificial chromosome and linear expression constructs were purified for production of Gstm1 transgenic rats, putative transgenic rats were screened by PCR. The strategies developed in this project are an example of thorough functional genomic analysis in experimental hypertension research.
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Davenport, Emma Elisabeth. "Functional genomics of variation in response to infection : insights into severe sepsis and common variable immune deficiency disorders." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:528edf20-f948-4a9c-aa23-1e295b11c8cc.
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Functional genomics uses high throughput genome-wide technologies to investigate the functional consequences of genetic variants on gene expression and protein products. In the context of disease, using integrative functional genomic approaches to understand the genetic variation which underlies disease susceptibility and aetiology may contribute to better diagnosis, treatment and prevention. This thesis investigated genetic determinants of variation in response to infection by applying a functional genomics approach to investigate three clinical cohorts: patients with severe sepsis, an influenza challenge study and patients with common variable immune deficiency disorders. The transcriptomic response to severe sepsis is reported here for the largest known adult severe sepsis community acquired pneumonia cohort. Two clusters within the cohort, based on gene expression signatures, which have different survival rates and identify individuals with distinct immune responses to sepsis, highlight the value of functional genomics for identifying heterogeneity within patient cohorts. This was further complemented by the resolution of gene expression signatures in healthy individuals following influenza challenge which identified individuals with moderate to severe disease. Shared gene expression signatures between the cohorts highlighted components of the immune response to viral infection important across multiple clinical settings and may assist with the identification of viral infections in the sepsis patients. Gene expression was mapped as a quantitative trait (eQTL). Comparison to data sets for healthy individuals and from innate immune stimulated cells identified regulatory variants specific to the context of sepsis. Whole genome sequencing for a cohort of patients with common variable immune deficiency disorders was performed. This identified novel variants and pathways which may play a role in the underlying immunopathogenesis of disease. Integration with RNA-seq for a small number of patients allowed prioritisation of non-coding variants based on evidence of altered gene expression. Comparison to the sepsis cohort analysis identified key immune related genes involved in rare and common responses to bacterial infection. This thesis has demonstrated the value of integrating multiple functional genomic techniques to further our understanding of the mechanisms underlying variation in the response to infection.
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Napper, Piers. "Ecological functional genomics : the genetic basis of phenotypic in Daphnia pulex." Thesis, Lancaster University, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.543998.
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Varotto, Claudio. "Genetic and molecular dissection of photosystem I functions in Arabidopsis and related functional genomics." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=96461779X.
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Treviño, Alvarado Victor Manuel. "Identifying the molecular components that matter : a statistical modelling approach to linking functional genomics data to cell physiology." Thesis, University of Birmingham, 2007. http://etheses.bham.ac.uk//id/eprint/636/.
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Functional genomics technologies, in which thousands of mRNAs, proteins, or metabolites can be measured in single experiments, have contributed to reshape biological investigations. One of the most important issues in the analysis of the generated large datasets is the selection of relatively small sub-sets of variables that are predictive of the physiological state of a cell or tissue. In this thesis, a truly multivariate variable selection framework using diverse functional genomics data has been developed, characterized, and tested. This framework has also been used to prove that it is possible to predict the physiological state of the tumour from the molecular state of adjacent normal cells. This allows us to identify novel genes involved in cell to cell communication. Then, using a network inference technique networks representing cell-cell communication in prostate cancer have been inferred. The analysis of these networks has revealed interesting properties that suggests a crucial role of directional signals in controlling the interplay between normal and tumour cell to cell communication. Experimental verification performed in our laboratory has provided evidence that one of the identified genes could be a novel tumour suppressor gene. In conclusion, the findings and methods reported in this thesis have contributed to further understanding of cell to cell interaction and multivariate variable selection not only by applying and extending previous work, but also by proposing novel approaches that can be applied to any functional genomics data.
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Avirovik, Dragana. "Transformation of a Transposon Construct into Tomato for Functional Genomics Studies." Thesis, Virginia Tech, 2014. http://hdl.handle.net/10919/24867.
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Tomato (Solanum lycopersicum) is a member of the Solanaceae family. In this research project tomato, more specifically the M82 cultivar was chosen as a model plant for Agrobacterium-mediated gene transfer by cotyledon inoculation. Our objective was to transform tomato with a T-DNA construct bearing a transposon from maize that can be used for mutagenesis when it transposes or moves around the genome of the tomato. The vector used is a two-component in-cis Ac-Ds system which needs a single transformation event. It was proved that it worked in Arabidopsis and rice according to Trijatmiko (2005). The construct consists of the BAR gene conferring resistance to herbicide Basta, hygromycin (HYG) gene conferring resistance to the antibiotic hygromycin and the green fluorescent protein (GFP) gene, which are driven by specific plant promoters. The selectable marker genes such as HYG and BAR were used to select the rare transformation events by making the transformed tomato tissue resistant to the toxic chemicals (antibiotic and herbicide) compared to the untransformed tissue in which growth was inhibited. The results described consist of developing a transformation protocol which enabled the production of transgenic tomato lines by the help of the antibiotic augmetin (amoxicillin/clavulanic acid). The transgenic lines were tested through polymerase chain reaction (PCR) and herbicide bioassays.Master of Science
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Chittaranjan, Suganthi. "A functional genomics approach identifies novel genes involved in steroid-hormove induced programmed cell death in Drosophila." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/2725.
Full textAbstract:
Programmed Cell death (PCD) is a highly conserved and genetically controlled event
that plays important roles in animal development, homeostasis and disease. Our first
objective was to discover and characterize new genes involved in PCD. Since many PCD
genes are conserved in Drosophila, and steroid-induced PCD of larval salivary glands
(SGs) is transcriptionally regulated with features of both apoptosis and autophagy, we
used this exceptionally well-suited in vivo system and performed Serial Analysis of Gene
Expression (SAGE) in three pre-death stages. SAGE identified 1244 expressed
transcripts, including genes involved in autophagy, apoptosis, immunity, cytoskeleton
remodeling, and proteolysis. Of the 1244 transcripts, 463 transcripts belonged to
knownlpredicted genes and were 5-fold differentially expressed prior to cell death.
Next, we investigated the role of differentially expressed genes from SAGE, in cell
death or cell survival, by RNA interference (RNAi ) in l(2)mbn haemocyte Drosophila
cells. l(2)mbn cells undergo morphological changes in response to ecdysone treatment,
and ultimately undergo PCD. We used cell viability, cell morphology, and apoptosis
assays to identify the death-related genes and determined their ecdysone dependency and
function in cell death regulation. Our RNAi screen identified six new pro-death related
genes, including SH3PXJ and Soxl4, and 21 new pro-survival genes including SoxN.
Identification of Soxl4 as pro-death and SoxN as pro-survival suggests that these Sox
box proteins may have opposing roles in ecdysone-mediated cell death.
Our final objective was to elucidate the function of CG409], a Drosophila
homologue of human TNF-alpha induced proteins 8 (TNFAIP8) we identified from
SAGE. We created loss-of-function and overexpression mutants of CG4091 to study
gene function in vivo and employed immunoprecipitation and mass-spectrometry assays
to identify proteins interacting with CG409] in vitro. We identified two proteins that are
involved in n-fatty acid oxidation and several cytoskeletal proteins as interaction
partners. Immunofluorescence based assays in vivo and in vitro revealed that CG409] is
necessary for cytoskeletal remodeling. Further, defects in CG4091 expression affect
cellular functions such as autophagy and lipid metabolism/trafficking that require an
intact cytoskeleton. Together, our studies provided new insights into the molecular
mechanisms involved in Drosophila SG cell death.
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Silparasetty, Shobha Lavanya. "Cloning of "Animal Cryptochrome" cDNA from the Model Organism CHLAMYDOMONAS REINHARDTII for Functional Analysis of Its Protein Product." TopSCHOLAR®, 2009. http://digitalcommons.wku.edu/theses/117.
Full textAbstract:
reinhardtii, a unicellular green alga, is a model organism to study the circadian clock. Cryptochromes are the blue light photoreceptors that entrain the clock in some organisms. The CPH1 protein of C. reinhardtii resembles the cryptochromes of the plant model Arabidopsis, but whether CPH1 entrains the circadian clock in C. reinhardtii is not yet known. Recent reports have suggested the existence of one more cryptochrome in C. reinhardtii, which resembles the cryptochromes of animals. However, the amino acid sequence of this protein shows even higher sequence similarity with the 6-4 DNA photolyase of Arabidopsis. DNA photolyases are involved in the repair of UV light-induced DNA damage using the energy of blue light. In order to determine, if the “animal cryptochrome” gene of C. reinhardtii actually encodes a 6-4 DNA photolyase rather than a photoreceptor, an experimental design was developed to test whether the protein product is able to rescue an E. coli mutant defective in its DNA photolyase gene. The design is as follows: In a first step, the coding region of the “animal cryptochrome” cDNA is cloned. In a second step, the cDNA is inserted in-frame into an E. coli expression vector. In a third step, the construct is transformed into an E. coli photolyase mutant, its expression induced, and the strain tested for better survival after UV light exposure. To accomplish the first step, the cloning of “animal cryptochrome” cDNA, total RNA was successfully extracted from C. reinhardtii 4 hrs into the light phase of a 12 h light/12 h dark cycle and reverse transcribed into cDNA using oligo(dT) primers. After initially unsuccessful attempts at amplifying animal cryptochrome from cDNA or genomic template with a variety of primers and conditions, a short fragment with the expected size of 186 bp was amplifiable with both templates. However, even this fragment was not reliably obtained in every PCR assay. Because of this difficulty, real-time PCR was finally performed in the presence of DMSO (Dimethylsulfoxide) and Betaine. These two adjuvants were reported to improve amplifications particularly for GC-rich templates. C. reinhardtii DNA is especially GC-rich with an average of 64% Gs and Cs. The improved conditions allowed the reliable amplification of the 186 bp fragment from genomic template. It also enabled the amplification of a larger fragment of 528 bp from the same template. The results suggest that a combination of 5% DMSO and 1M Betaine is optimal for the amplification of C. reinhardtii DNA and thus can serve as the basis for successful amplification of the entire 1788 bp coding region of the animal cryptochrome cDNA.
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Lu, Timothy Te Hua [Verfasser]. "Of long-tails, microarrays, and marker sets : integrative approaches in functional genomics, population genetics and genetic epidemiology / Timothy Te Hua Lu." Kiel : Universitätsbibliothek Kiel, 2009. http://d-nb.info/1019868899/34.
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Fields, Christopher J. "Functional and Expression Analysis of a Novel Basement Membrane Degrader in Drosophila Melanogaster." TopSCHOLAR®, 2016. http://digitalcommons.wku.edu/theses/1633.
Full textAbstract:
The Srivastava Lab is focused on the identification and characterization of genes that play a role in basement membrane remodeling. Previously, we identified putative basement membrane degraders through a genetic screen. One such gene has been suggested to play a role in the maintenance of the stem cell niche in Drosophila melanogaster, but no other information about the role this gene plays in development or disease has been published. Here, data are presented from experiments utilizing Drosophila genetics and immunohistochemistry that provide important insights on the biological role of this gene.
Collagenase activity was up-regulated upon overexpression of this gene, confirming it as a basement membrane degrader. Additionally, RNA in-situ hybridization experiment results showed expression in the developing imaginal discs of the 3rd instar larva tissues. Overexpression and knockdown studies further demonstrated morphological defects in a number of tissues, including the wing and the eye, and are suggestive of apoptosis. Acridine orange staining confirmed that cell death occurred when the gene was overexpressed and a cleaved caspase antibody staining indicated that process to be caspase-mediated apoptosis.
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Raza, Abbas. "Genetic And Functional Approaches To Understanding Autoimmune And Inflammatory Pathologies." ScholarWorks @ UVM, 2020. https://scholarworks.uvm.edu/graddis/1175.
Full textAbstract:
Our understanding of genetic predisposition to inflammatory and autoimmune diseases has been enhanced by large scale quantitative trait loci (QTL) linkage mapping and genome-wide association studies (GWAS). However, the resolution and interpretation of QTL linkage mapping or GWAS findings are limited. In this work, we complement genetic predictions for several human diseases including multiple sclerosis (MS) and systemic capillary leakage syndrome (SCLS) with genetic and functional data in model organisms to associate genes with phenotypes and diseases.
Focusing on MS, an autoimmune inflammatory disease of the central nervous system (CNS), we experimentally tested the effect of three of the GWAS candidate genes (SLAMF1, SLAMF2 and SLAMF7) in the experimental autoimmune encephalomyelitis (EAE) mouse model and found a male-specific locus distal to these loci regulating CNS autoimmune disease. Functional data in mouse suggests this male-specific locus modulates the frequency of immune cells including CD11b+, TCRαβ+CD4+Foxp3+, and TCRαβ+CD8+IL-17+ cells during EAE disease. Orchiectomy experiments demonstrate that this male specific phenotype is dependent on testis but not testosterone (T) or 5α-dihydrotestosterone (DHT). Using a bioinformatic approach, we identified SLAMF8 and SLAMF9 along with other differentially expressed genes in linkage with MS-GWAS predictions whose expression is testis-dependent, but not directly regulated by T or DHT, as potential positional candidates regulating CNS autoimmune disease. Further refinement of this locus is required to identify the causal gene(s) that may be targeted for prevention and/or treatment of MS in men.
Using SCLS, an extremely rare disorder of unknown etiology characterized by recurrent episodes of vascular leakage, we identified and modeled this disease in an inbred mouse strain, SJL, using susceptibility to histamine- and infection-triggered vascular leak as the major phenotypic readout. This trait “Histamine hypersensitivity” (Histh/Histh) was mapped to a region on Chr 6. Remarkably, Histh is syntenic to the genomic locus most strongly associated with SCLS in humans (3p25.3). Subsequent studies found that the Histh locus is not unique to SJL but additional mouse strains also exhibit Histh phenotype. Considering GWAS studies in SCLS are limited by the small number of patients, we utilized interval-specific SNP-based association testing among Histh phenotyped mouse strains to predict Histh candidates. Furthermore, to dissect the complexity of Histh QTL, we developed network-based functional prediction methods to rank genes in this locus by predicting functional association with multiple Histh-related processes. The top-ranked genes include Cxcl12, Ret, Cacna1c, and Cntn3, all of which have strong functional associations and are proximal to SNPs segregating with Histh.
Lastly, we utilized the power of integrating genetic and functional approaches to understand susceptibility to Bordetella pertussis and pertussis toxin (PTX) induced histamine sensitization (Bphs/Bphs), a sub-phenotype with an established role in autoimmunity. Congenic mapping in mice had earlier linked Bphs to histamine H1 receptor gene (Hrh1/H1R) and demonstrated that H1R differs at three amino acid residues in Bphs-susceptible and -resistant mice. Our subsequent studies identified eight inbred mouse strains that were susceptible to Bphs despite carrying a resistant H1R allele. Genetic analyses mapped the locus complementing Bphs to mouse Chr 6, in linkage disequilibrium with Hrh1; we have designated this Bphs-enhancer (Bphse). Similar to the approaches used for Histh, we utilized interval-specific SNP based association testing and network-based functional enrichment to predict nine candidate loci for Bphse including Atp2b2, Atg7, Pparg, Syn2, Ift122, Raf1, Mkrn2, Timp4 and Gt(ROSA)26Sor. Overall, these studies demonstrate the power of integrating genetic and functional methods in humans and animal models to predict highly plausible loci underlying QTL/GWAS data.
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Michino, Mayako. "Developing new computational methods for characterization ORFS with unknown function." Thesis, Georgia Institute of Technology, 2003. http://hdl.handle.net/1853/25208.
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Khuzwayo, Sabelo Lethukuthula. "Functional analysis of subtelomeric breakage motifs using yeast as a model organism." Thesis, Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/41119.
Full textAbstract:
Genome wide studies have uncovered the existence of large-scale copy number variation (CNV) in the human genome. The human genome of different individuals was initially estimated to be 99.9% similar, but population studies on CNV have revealed that it is 12-16% copy number variable. Abnormal genomic CNVs are frequently found in subtelomeres of patients with mental retardation (MR) and other neurological disorders. Rearrangements of chromosome subtelomeric regions represent a high proportion of cytogenetic abnormalities and account for approximately 30% of pathogenic CNVs. Although DNA double strand breaks (DSBs) are implicated as a major factor in chromosomal rearrangements, the causes of chromosome breakage in subtelomeric regions have not been elucidated. But due to the presence of repetitive sequences in subtelomeres, we hypothesized that chromosomal rearrangements in these regions are not stochastic but driven by specific sequence motifs. In a collaborative effort with Dr. Rudd (Department of human genetics at Emory University), we characterized subtelomeric breakpoints on different chromosome ends in search of common motifs that cause double-strand breaks. Using a yeast-based gross chromosomal rearrangement (GCR) system, we have identified a subtelomeric breakage motif from chromosome 2 (2q SBM) with a GCR rate that is 340 fold higher than background levels. To determine if the fragility of 2q SBM was driven by the formation of secondary structures, the helicase activities of Sgs1 and Pif1 were disrupted. These helicases have been shown to destabilize DNA secondary structures such as G-quadruplex structures. Disruption of these helicases augmented chromosomal rearrangements induced by 2q SBM, indicating that these helicases are required for maintenance of this sequence. We also donwregulated replication fork components to determine if 2q SBM was imposing any problems to the replication fork machinery. Downregulation of replication fork components increased chromosomal rearrangements, indicating that intact replication fork was a critical determinant of 2q SBM fragility. Using a yeast-based functional assay, these experiments have linked human subtelomeric repetitive sequences to chromosomal breakage that could give rise to human CNV in subtelomeric regions.
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Carnero-Montoro, Elena 1985. "Genomic and functional approaches to genetic adaptation." Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/291115.
Full textAbstract:
The genetic basis of phenotypes that have contributed to the adaptation of species and
organisms to new environments is a central question in evolutionary genetics. The
recent accumulation of genetic variability data has allowed a genome-wide search for
different signatures of positive selection which has led to the discovery of hundreds of
putative candidate genes that may have played a role in adaptation. However, such
hypothesis-free approaches do not reveal either causal variants or the actual biological
mechanisms that have made each adaptation possible. Furthermore, the detection of
molecular signatures is limited both by the complex architecture of the genome and
by the possible polygenic nature of the selected trait.
In this thesis, through different evolutionary and functional approaches, we have
disentangled the adaptive role of two non-synonymous variants in two different
candidate genes encoding for a lymphocyte receptor and a zinc transporter,
respectively. In past human adaptation, they were most likely selected as more
effective means to fight pathogens. We have also revealed differences in the action of
natural selection between different pathways and different coding and non-coding
genomic elements in the chimpanzee lineage by analyzing polymorphisms and
divergence data together. Thus, the results of this PhD thesis have contributed to
detect new instances of genetic adaptation and provide biological explanation to them.La base genética de los carácteres que han contribuido a la adaptación de los organismos y las especies ha sido siempre una pregunta central en biología evolutiva. Gracias a la acumulación masiva de datos de variabilidad genética, en los últimos años se ha podido detectar en el genoma diferentes señales de selección positiva y también localizar cientos de genes candidatos que han podido tener un papel en la adaptación de las poblaciones a diferentes ambientes. Sin embargo en estos estudios, donde no hay una hipótesis a priori, se desconoce qué variantes dentro de estos genes fueron realmente las que proporcionaron una ventaja selectiva y por qué. Además, la compleja arquitectura del genoma y la naturaleza poligénica de muchos carácteres hace que sea difícil detectar casos más complejos de adaptación. En esta tesis se intenta resolver algunos de estos problemas. En primer lugar, mediante un enfoque evolutivo y funcional, hemos descifrado el rol adaptativo de dos variantes genéticas, una en un receptor linfocitario y la otra en un transportador de zinc, que probablemente fueron seleccionadas por conferir resistencia a patógenos. En segundo lugar, mediante el análisis de datos de polimorfismo y divergencia conjuntamente, también hemos detectado distintos mecanismos de acción de la selección natural en distintos pathways y entre elementos codificantes y elementos no codificantes reguladores en chimpancé.
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Liu, Yiyong. "1, Structural and Functional Studies of Human Replication Protein A; 2 DNA Damage Responses and DNA Repair Defects in Laminopathy-Based Premature Aging." Digital Commons @ East Tennessee State University, 2007. https://dc.etsu.edu/etd/2062.
Full textAbstract:
The genome of mammalian cells is under constant attack from DNA-damaging agents. To maintain genomic integrity, cells activate an array of pathways primarily consisting of DNA repair and DNA damage checkpoints. Human replication protein A (RPA), a single-stranded DNA (ssDNA) binding protein, is essential for almost all DNA metabolic pathways. However, the role of RPA in nucleotide excision repair (NER), a DNA repair pathway for removing bulky DNA lesions, remains elusive. In this study, the binding of RPA to a battery of well-defined ssDNA substrates has been systematically examined using fluorescence spectroscopy. The results showed that RPA has a lower binding affinity for damaged ssDNA than for non-damaged ssDNA, and there was no direct contact between RPA residues and the lesion itself. These findings will help define the roles of RPA in DNA damage recognition in NER. In cells, RPA undergoes hyperphosphorylation in the N-terminus of RPA32 subunit after DNA damage. In this study, the hyperphosphorylation-induced conformational changes of RPA have been probed using mass spectrometry-based protein foot-printing, fluorescence spectroscopy and limited proteolysis. The data show that upon hyperphosphorylation RPA undergoes a subtle structural change involving its DNA-binding domain B (DBD-B), reducing its affinity for short ssDNA. These results suggest that hyperphosphorylation may modulate RPA functions by altering DBD-B-mediated RPA-DNA/protein interactions. Cellular accumulation of DNA damage has been widely implicated in premature aging. In Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD), premature aging is caused by defective maturation of lamin A and linked to accumulation of DNA double-strand breaks (DSBs). However, how lamin A dysfunction leads to genome instability and premature aging is not understood. Here evidence showed that in HGPS and RD fibroblasts DNA damage checkpoints are persistently activated and recruitment of repair factors to DSBs was impaired. Strikingly, xeroderma pigmentosum group A (XPA), a unique NER protein, formed foci and colocalized with the unrepairable DSBs in the patient cells. RNAi knockdown of XPA in HGPS cells significantly restored DSB repair. These results indicate that XPA dysfunction may play an important role in accumulating DSBs in HGPS, implicating a potential strategy for treatment of these premature aging diseases.
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Rands, Chris M. D. "Analyses of functional sequence in mammalian and avian genomes." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:27e0ac20-eb27-423c-9493-a8a1c6cc57b8.
Full textAbstract:
The first draft sequence of the human genome was published over a decade ago, yet interpreting the functional importance of nucleotides in genomes is still an ongoing challenge. I took a comparative genomic approach to identify functional sequence using signatures of natural selection in DNA sequences. Mutations that are purged or propagated by selection mark sequences of significance for biological fitness. I developed and refined methods for estimating the quantity of sequence constrained with respect to insertions and deletions (indels) between two genome sequences, a quantity I termed αselIndel. This sequence is evolving more slowly than surrounding neutral sequence due to the purging of deleterious indel variants, and thus this sequence is likely to be functional. I estimated αselIndel between diverse mammalian and avian species pairs, and found a strong negative correlation between αselIndel and the divergence between the species’ genome sequences. This implies that functional sequence turns over rapidly as it is lost and gained over time. I quantified the variable levels of sequence constraint, and rates of sequence turnover, for different types of human biochemically annotated element. Furthermore, I found that similar rates of functional turnover have occurred across mammalian and avian evolution. Finally, I identified positively selected amino acid residues that may be important for Darwin’s finch beak development, and found evidence of adaptively evolving reproductive proteins in the ancestral songbird lineage. Collectively these results demonstrate the wide-spread nature of lineage-specific functional sequence with implications for understanding species traits and the use of model organisms to inform human biology.
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Nguyen, Peter H. T. (Peter Hung Trung). "Fine-Mapping Tools : an interactive framework for dissecting disease-associated genetic loci with functional genomics data." Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/113121.
Full textAbstract:
Thesis: M. Eng., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2017.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 57-58).
Fine mapping causal SNPs from GWAS summary statistics is hard. Although many frame- works exist to support fine mapping, some of which leverage epigenomic contexts to increase predictive power, they fail to provide interactivity. Here, we introduce Fine-Mapping Tools (fm-tools), a framework for doing interactive and iterative fine mapping. Fm-tools provides a harmonized data store and implements a number of algorithms for fine mapping -- one of which is the custom RiVIERA-mini, an efficient Bayesian inference framework -- and exposes them via a rich API that can be plugged into a variety of services (e.g., web applications for visualization). Most importantly, fm-tools allows scientists to interactively and iteratively explore dynamically generated hypotheses, as demonstrated by a case study for celiac disease. In summary, fm-tools standardizes the way fine mapping is done, reduces the overhead of fine mapping for scientists and of algorithm development for researchers, and paves the way towards achieving real-time personalized medicine.
by Peter HT Nguyen.
M. Eng.
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Nadkarni, Aditi A. "Functional analysis of the Rad51d (E233G) breast cancer associated polymorphism and a pharmacogenetic evaluation of RAD51D status." Connect to full text in OhioLINK ETD Center, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=mco1222877984.
Full textAbstract:
Dissertation (Ph.D.)--University of Toledo, 2008."In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Bibliography: pages 73-77, 93-95, 109-111, 145-172.
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Liao, Rachel Grace. "Functional Studies of Candidate Oncogenes in Non-Small Cell Lung Cancer." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11173.
Full textAbstract:
Cancer is a set of complex genetic diseases driven by diverse genomic alterations. The genomic study of cancer has enabled the discovery of novel, targetable events in almost all cancer types and in turn, has led to the development of new, targeted cancer therapies benefiting patients; however, the recent explosion of genomic datasets has also resulted in huge lists of new oncogenic factors of unknown biological relevance, and uncertainty over how best to use the data appropriately to influence patient care. Some of the most pressing questions surround the use of statistical methods to identify actionable genomic alterations in cancer and the identification of driving oncogenes in the context of the genomic evolution of cancer cells, undergone before, during, and after prolonged treatment regimens.
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Carl, Sarah Hamilton. "Evolutionary patterns of group B Sox binding and function in Drosophila." Thesis, University of Cambridge, 2015. https://www.repository.cam.ac.uk/handle/1810/247430.
Full textAbstract:
Genome-wide binding and expression studies in Drosophila melanogaster have revealed widespread roles for Dichaete and SoxNeuro, two group B Sox proteins, during fly development. Although they have distinct target genes, these two transcription factors bind in very similar patterns across the genome and can partially compensate for each other's loss, both phenotypically and at the level of DNA binding. However, the inherent noise in genome-wide binding studies as well as the high affinity of transcription factors for DNA and the potential for non-specific binding makes it difficult to identify true functional binding events. Additionally, externalfactors such as chromatin accessibility are known to play a role in determining binding patterns in Drosophila. A comparative approach to transcription factor binding facilitates the use of evolutionary conservation to identify functional features of binding patterns. In order to discover highly conserved features of group B Sox binding, I performed DamID-seq for SoxNeuro and Dichaete in four species of Drosophila, D. melanogaster, D. simulans, D. yakuba and D. pseudoobscura. I also performed FAIRE-seq in D. pseudoobscura embryos to compare the chromatin accessibility landscape between two fly species and to examine the relationship between open chromatin and group B Sox binding. I found that, although the sequences, expression patterns and overall transcriptional regulatory targets of Dichaete and SoxNeuro are highly conserved across the drosophilids, both binding site turnover and rates of quantitative binding divergence between species increase with phylogenetic distance. Elevated rates of binding conservation can be found at bound genomic intervals overlapping functional sites, including known enhancers, direct targets of Dichaete and SoxNeuro, and core binding intervals identified in previous genome-wide studies. Sox motifs identified in intervals that show binding conservation are also more highly conserved than those in intervals that are only bound in one species. Notably, regions that are bound in common by SoxNeuro and Dichaete are more likely to be conserved between species than those bound by one protein alone. However, by examining binding intervals that are uniquely bound by one protein and conserved, I was able to identify distinctive features of the targets of each transcription factor that point to unique aspects of their functions. My comparative analysis of group B Sox binding suggests that sites that are commonly bound by Dichaete and SoxNeuro, primarily at targets in the developing nervous system, are highlyconstrained by natural selection. Uniquely bound targets have different tissue expression profiles, leading me to propose a model whereby the unique functions of Dichaete and SoxNeuro may arise from a combination of differences in their own expression patterns and the broader nuclear environment, including tissue-specific cofactors and patterns of accessible chromatin. These results shed light on the evolutionary forces that have maintained conservation of the complex functional relationships between group B Sox proteins from insects to mammals.
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White, Robert B. "The developmental function of Pax7 : chromatin-immunoprecipitation discovery of Pax7 target genes." Thesis, Edith Cowan University, Research Online, Perth, Western Australia, 2007. https://ro.ecu.edu.au/theses/279.
Full textAbstract:
Pax7 plays critical roles in development of brain, spinal cord, neural crest and skeletal muscle. As a sequence-specific DNA-binding transcription factor, the direct functional role played by Pax7 during development is the selection of target genes. To date, an accurate description of the function of this transcription factor has not been obtained through an understanding of its target genes. To elucidate the direct developmental functions of Pax7, this research has sought to identify genes targeted by Pax7 during mouse embryonic development.
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Schnetz, Michael Paul. "Investigation of the Molecular Function of CHD7, the Protein Implicated in CHARGE Syndrome, Using Next-Generation Genomics." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1275581022.
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Beach, Joshua S. "Functional Characterization of rai1 in Zebrafish." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3826.
Full textAbstract:
Smith-Magenis Syndrome (SMS; OMIM #182290) is a multiple congenital abnormality and intellectual disability (ID) disorder caused by either an interstitial deletion of the 17p11.2 region containing the retinoic acid induced-1 (RAI1) gene or a mutation of the RAI1 gene. Individuals diagnosed with SMS typically present characteristics such as ID, self-injurious behavior, sleep disturbance, ocular and otolaryngological abnormalities, craniofacial and skeletal abnormalities, neurological and behavioral abnormalities, as well as other systemic defects and manifestations. Previous work by Vyas in 2009 showed temporal expression of rai1 in zebrafish embryos as early as 9 hpf. We hypothesize that there is maternal rai1 expression as early as zero hours post fertilization in wild type embryos. Using end-point PCR, we found that in fact there is maternal rai1 expression is detectable as early as 2 hours post fertilization (hpf) in wild type zebrafish embryos. Furthermore, we quantified rai1 expression using qPCR and found that rai1 expression declines significantly after 6 hpf. We hypothesize that a down regulation of rai1 or loss of rai1 will lead to morphological phenotypes, especially if that loss of rai1 function occurs during the earliest stages of zebrafish embryogenesis. Using a rai1morpholino oligonucleotide (MO), we found a loss of rai1 expression did not induce a morphological phenotype in in wild type embryos; furthermore, we also found that a loss of maternal rai1 expression did not induce a morphological phenotype as well. Utilizing a mutant rai1 zebrafish line, we found that both rai1 +/fh370 progeny nor rai1 fh370/fh370 progeny exhibited a morphological phenotype and that downstream targets such as bdnf were not affected by a reduction or complete loss of rai1. Prior research has shown that retinoic acid (RA) can induce rai1 expression. We hypothesize that RA can induce expression of rai1 during zebrafish embryogenesis. Using wild type fish and a rai1 in situ hybridization probe, we found that RA treatment at 25 hpf induced expression of rai1. The construction of a rai1 overexpression vector used for overexpression studies was started. Further development of GFP expression vector and zebrafish rai1 antibody are needed to determine if the morpholino is reducing rai1 protein expression.
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Kacharia, Fenil Rashmin. "Investigating the Origin and Functions of a Novel Small RNA in Escherichia coli." PDXScholar, 2016. http://pdxscholar.library.pdx.edu/open_access_etds/3106.
Full textAbstract:
Non-coding small RNAs (sRNAs) regulate various cellular processes in bacteria. They bind to a chaperone protein Hfq for stability and regulate gene expression by base-pairing with target mRNAs. Although the importance of sRNAs in bacteria has been well established, the mode of origination of novel sRNA genes is still elusive, mainly because the rapid rate of evolution of sRNAs obscures their original sources. To overcome this impediment, we identified a recently formed sRNA (EcsR2) in E. coli, and show that it evolved from a degraded bacteriophage gene. Our analyses also revealed that young sRNAs such as EcsR2 are expressed at low levels and evolve at a rapid rate in comparison to older sRNAs, thereby uncovering a novel process that potentially facilitates newly emerging (and probably mildly deleterious) sRNAs to persist in bacterial genomes. We also show that even though EcsR2 is slightly deleterious to E. coli, it could bind to Hfq and mRNAs to regulate the expression of several genes. Interestingly, while EcsR2 expression is induced by glucose, the expression of its putative targets are regulated by the transcription factor CRP in response to glucose, indicating that EcsR2 has been incorporated into the carbon regulatory network in E. coli. Collectively, this work provides evidence for the emergence, evolution and functions of a novel "young" sRNA in bacteria.
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Arif, Km Taufiqul. "Functional association of Micrornas with molecular subtypes of breast cancer." Thesis, Queensland University of Technology, 2021. https://eprints.qut.edu.au/213110/1/Km%20Taufiqul_Arif_Thesis.pdf.
Full textAbstract:
This research study investigated the association of microRNA related single nucleotide polymorphisms (miRSNPs) with breast cancer susceptibility in Australian Caucasian women. The thesis then progressed with developing an in silico methodology for miRNA-target identification followed by the validation of miRNA-target relationships regarding the distinctive molecular subtypes of human breast cancers.
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Kamps-Hughes, Nicholas. "Massively Parallel Sequencing-Based Analyses of Genome and Protein Function." Thesis, University of Oregon, 2015. http://hdl.handle.net/1794/19234.
Full textAbstract:
The advent of high-throughput DNA and RNA sequencing has made possible the assay of millions of nucleic acid molecules in parallel. This allows functional genomic elements to be identified from background in single-tube experiments. This dissertation discusses the development of two such functional screens as well as work implementing a third that was previously developed in my thesis laboratory.
Restriction-Associated DNA sequencing (RAD-Seq) is a complexity reduction sequencing method that allows the same subset of genomic sequence to be read across multiple samples. Differences in sample collection and data analysis allow manifold applications of RAD-Seq. Here we use RAD-Seq to identify mutant genes responsible for altered phenotypes in Caenorhabditis elegans and to identify hyper-invasive alleles in trout population admixtures.
Apart from acquiring genomic sequence data, massively-parallel sequencing can be used for counting applications that quantify activity across a large number of test molecules. This dissertation describes the development of a technique for simultaneously quantifying the activity of a restriction enzyme across all possible DNA substrates by linking digest of a sequenced genome to Illumina-sequencing in an unbiased fashion. Finally, a powerful approach to analyze transcriptional activation is described. This method quantifies output from millions of potential DNA transcriptional enhancers via RNA amplicon sequencing of covalently-linked randomer tags and is used in conjunction with RNA-Seq to provide a mechanistic view of hypoxic gene regulation in Drosophila.
This dissertation includes previously published, co-authored material
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Desai, Megha. "Structural and Functional Characterization of the MBD2-NuRD Co-Repressor Complex." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3617.
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The MBD2-NuRD co-repressor complex is an epigenetic regulator of the developmental silencing of embryonic and fetal β-type globin genes in adult erythroid cells as well as aberrant methylation-dependent silencing of tumor suppressor genes in neoplastic diseases. Biochemical characterization of the MBD2-NuRD complex in chicken erythroid cells identified RbAp46/48, HDAC1/2, MTA1/2/3, p66α/β, Mi2α/β and MBD2 to comprise this multi-protein complex.
In the work presented in Chapter 2, we have pursued biophysical and molecular studies to describe a previously uncharacterized domain of human MBD2 (MBD2IDR). Biophysical analyses show that MBD2IDR is an intrinsically disordered region (IDR). Despite this inherent disorder, MBD2IDR increases the overall binding affinity of MBD2 for methylated DNA. MBD2IDR also recruits the histone deacetylase core components (RbAp48, HDAC2 and MTA2) of NuRD through a critical area of contact requiring two contiguous amino acid residues, Arg286 and Leu287. Mutation of these critical residues abrogates interaction of MBD2 with the histone deacetylase core and impairs the ability of MBD2 to repress the methylated tumor suppressor gene Prostasin in MDA-MB-435 breast cancer cells. These findings expand our knowledge of the multi-dimensional interactions of the MBD2-NuRD complex that govern its function.
In Chapter 3, we have discussed a novel mechanism for MBD2-mediated silencing of the fetal γ-globin gene. Through microarray expression analyses in adult erythroid cells of MBD2-/- mice, we identified ZBTB32 and miR-210 as downstream targets of MBD2. Over-expression of ZBTB32 and miR-210 in adult erythroid cells causes increased expression of the silenced fetal γ-globin gene. Thus, our results indicate that MBD2 may regulate γ-globin gene expression indirectly though ZBTB32 and miR-210 in adult erythroid cells.
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Picariello, Tyler August. "Meckelin Functions in the Guided Movement and Orientation of Basal Bodies Prior to Duplication in Paramecium tetraurelia." ScholarWorks @ UVM, 2015. http://scholarworks.uvm.edu/graddis/367.
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Ciliopathies are a group of disorders that arise from ciliary dysfunction. Meckelin (MKS3 or TMEM67) is a conserved transmembrane protein found at the transition zone of ciliated cells. In humans MKS3 is one of 3 genes linked to the ciliopathy Meckel Syndrome. This disease is characterized by occipital meningioencephalocoele, polycystic kidneys, fibrotic changes to the liver, postnatal polydactyly and situs inversus.
Paramecium tetraurelia is a single celled ciliated eukaryote. Its surface is organized of a meshwork of cortical units that run the length of the cell. At the center of the cortical units are either one or two basal bodies. In two basal body units only the posterior basal body is ciliated. From the ciliated basal body, three rootlets project in stereotypical orientations: the post-ciliary rootlet projects posteriorly, the transverse microtubule projects toward the adjacent basal body row and the striated rootlet projects anteriorly. Both the post-ciliary rootlet and transverse microtubule are microtubule-based structures. The striated rootlet is composed of multiple subunits that are predicted to have conserved segmented coiled coil domains known as SF-Assemblin domains.
In Picariello at al., 2014, we showed that MKS3 is present in the transition zone of Paramecium tetraurelia and that RNAi for MKS3 leads to global ciliary loss. Additionally, RNAi for MKS3 results in the disorganization of the basal body rows. Within the areas of disorganization, the basal bodies along with their striated rootlets, post-ciliary rootlets and transverse microtubules are rotated away from their expected orientation. Interestingly, the post-ciliary rootlet and transverse microtubule are still attached at the expected angles relative to each other within the areas of disorganization. Initial GST pull-down experiments using the coiled coil domain of MKS3 suggest a potential interaction between MKS3 and the striated rootlet family members KdC1 and KdB2.
To test potential interactions between MKS3 and the striated rootlet we identified 27 potential striated rootlet family members in Paramecium. Full-length sequences for 13 of these genes were marked at their N-terminus with a 3x FLAG sequence. Components with a conserved SF-Assemblin domain were distributed uniformly within the striated rootlet. Components lacking the SF-Assemblin domain were found in various cellular locations, but not within the striated rootlet. GST pull-down experiments utilizing the MKS3 C-terminus as bait were performed using cells expressing the FLAG-tagged striated rootlet family members. Unfortunately a clear interaction between MKS3 and the striated rootlet remains elusive.
The organized nature of the surface of Paramecium has allowed us to identify a previously unrealized function for MKS3. Our immunofluorescence data suggest that MKS3 functions outside the transition zone to maintain basal body row organization by potentially contributing to a link between the basal body and the striated rootlet. Without the link, the migrating basal bodies are free to rotate and project their rootlets in the wrong directions. Although the nature of the link remains elusive, the identification of disorganized basal body rows upon MKS3 reduction suggests that, in addition to ciliary dysfunction, basal body polarity defects may contribute to the development of MKS.
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Jöngren, Markus. "Gene expression in brains from red jungle fowl (Gallus gallus) that differ in fear response." Thesis, Linköping University, The Department of Physics, Chemistry and Biology, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-11830.
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The fear response of two different captive populations of red jungle fowl (rjf, Gallus gallus) was measured in three different tests, a ground predator test, an aerial predator test and a tonic immobility test. The two populations originated from Copenhagen zoo (Cop) and Götala research station (Got) but had been kept together for four generations. Earlier generations had a confirmed difference in fearfulness where the Cop birds exhibited a higher degree of fear response than Got birds (Håkansson and Jensen, 2005; Håkansson et al., 2007). The most and least fearful birds of each sex and population were identified and used in a gene expression study. The midbrain regions from the candidate birds were collected and RNA was isolated from each brain. The RNA was then reversed transcribed to cDNA which was used in a cDNA microarray experiment. 13 significantly differentially expressed genes were found between the fearful and non-fearful females. Among others were the neuroprotein Axin1, two potential DNA/RNA regulating proteins and an unknown transcript in the Quantitative Trait Locus 1(QTL 1), a well studied QTL on chromosome one with substantial effect on both behaviour and morphology during domestication (Schütz et al., 2002). This thesis succeeds in finding a difference in gene expression between fearful and non fearful female rjf but not between males. It fails in identifying gene expression differences between the two populations. Finally, the found differentiated genes suggest a potential molecular mechanism controlling the fear response in fowl.
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Häkkinen, Suvi T. "A functional genomics approach to the study of alkaloid biosynthesis and metabolism in Nicotiana tabacum and Hyoscyamus muticus cell cultures /." [Espoo, Finland] : VTT, 2008. http://www.vtt.fi/inf/pdf/publications/2008/P696.pdf.
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Squiban, Barbara. "Criblage par ARN interférence du génome complet de C. elegans pour l' identification de nouveaux gènes impliqués dans l' immunité innée." Thesis, Aix-Marseille, 2012. http://www.theses.fr/2012AIXM4056.
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Afin de caractériser les voies de signalisation du système immunitaire inné, nous étudions l'interaction entre le ver C. elegans et le champignon Drechmeria coniospora. Une des réponses du ver à l'infection consiste en une augmentation de la production de peptides antimicrobiens (PAM) dans l'épiderme. Des vers transgéniques exprimant le gène rapporteur de la GFP sous le contrôle du promoteur d'un PAM, fluorescent vert après infection. Si un gène nécessaire à l'expression des PAM est inactivé, alors les vers transgéniques ne fluorescent plus après infection. Nous avons effectué un crible pour identifier les molécules de signalisation nécessaires à l'expression des PAM en utilisant une approche quantitative et semi-automatique par ARN interference (ARNi). Deux banques d'ARNi couvrant 95% du génome, soit 20 000 gènes, ont été criblées et 360 candidats bloquant l'induction de la GFP après infection ont été obtenus, correspondant à 343 gènes. Une caractérisation phénotypique a permis de placer les candidats dans différentes catégories fonctionnelles et permis d'identifier d'une part un récepteur agissant en amont de la voie de signalisation p38 nécessaire à l'activation des gènes PAM, d'autre part une implication des granules de stress lors de l'infection. Ces analyses sont le fondement pour l'établissement d'une description compréhensive du réseau génétique régulant le système immunitaire inné du ver et permettront de révéler les interactions complexes entre l'immunité et les processus physiologiques au niveau moléculaire, cellulaire et au niveau de l'organismeTo investigate innate immune signaling, we study the interaction of C. elegans with the fungus Drechmeria coniospora. One of the responses of the worm to this infection is the up-regulation of a variety of antimicrobial peptide (AMP) genes in the epidermis. Transgenic worms carrying a GFP reporter gene under the control of an AMP promoter fluoresce green after infection by D. coniospora. If a gene required for AMP gene expression is inactivated, the reporter strain will not turn green upon infection. Using this fluorescent read-out, we have been able to screen for signaling molecules required for AMP gene expression using a quantitative semi-automated RNAi approach. We have screened two RNAi libraries that together cover 95% of the ca. 20,000 genes in the C. elegans genome and we obtained 360 high-confidence candidates that reduced the level of induction of green fluorescence after infection, and correspond to 343 genes. A further phenotypic characterization allowed the candidates to be grouped into distinct functional categories and allowed the identification of both a receptor acting upstream the p38 MAPK pathway necessary for the activation of the AMPs, and the implication of stress granules during infection. Altogether, the screen data and its analysis represent the foundation for the establishment of a comprehensive description of the signaling network regulating the innate immune system of the worm and will shed light on the complex interactions between immunity and other physiological processes at the molecular, cellular and organismal level
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Gore, Panter Shamone Robinette. "Genetic and Functional Studies of LociAssociated with Atrial Fibrillation." Case Western Reserve University School of Graduate Studies / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=case1396521127.
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Beka, Sylvia Enobong. "The genomics of Type 1 Diabetes susceptibility regions and effect of regulatory SNPs." Thesis, University of Hertfordshire, 2016. http://hdl.handle.net/2299/17200.
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Human complex diseases, like Diabetes and Cancer, affect many people worldwide today. Despite existing knowledge, many of these diseases are still not preventable. Complex diseases are known to be caused by a combination of genetic factors, as well as environmental and life style factors. The scope of this investigation covered the genomics of Type 1 Diabetes (T1D). There are 49 human genomic regions that are known to carry markers (disease-associated single nucleotide mutations) for T1D, and these were extensively studied in this research. The aim was to find out in how far this disease may be caused by problems in gene regulation rather than in gene coding. For this, the genetic factors associated with T1D, including the single point mutations and susceptibility regions, were characterised on the basis of their genomic attributes. Furthermore, mutations that occur in binding sites for transcription factors were analysed for change in the conspicuousness of their binding region, caused by allele substitution. This is called SNP (Single nucleotide polymorphism) sensitivity. From this study, it was found that the markers for T1D are mostly non-coding SNPs that occur in introns and non-coding gene transcripts, these are structures known to be involved in gene regulatory activity. It was also discovered that the T1D susceptibility regions contain an abundance of intronic, non-coding transcript and regulatory nucleotides, and that they can be split into three distinct groups on the basis of their structural and functional genomic contents. Finally, using an algorithm designed for this study, thirty-seven SNPs that change the representation of their surrounding region were identified. These regulatory mutations are non-associated T1D-SNPs that are mostly characterised by Cytosine to Thymine (C-T) transition mutations. They were found to be closer in average distance to the disease-associated SNPs than other SNPs in binding sites, and also to occur frequently in the binding motifs for the USF (Upstream stimulatory factor) protein family which is linked to problems in Type 2 diabetes.
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Cordova, Caio Mauricio Mendes de. "Desenvolvimento de plasmídeos replicativos artificiais para transformação de Mycoplasma pulmonis, M. capricolum e M. mycoïdes subsp. mycoïdes, e dirupção do gene da hemolisina A de M. pulmonis por recombinação homóloga." Universidade de São Paulo, 2002. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-20082008-112933/.
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Os micoplasmas são os menores microrganismos capazes de autoreplicação conhecidos na natureza, responsáveis por uma série de doenças no homem e nos animais, infectando ainda plantas e insetos. Constituem um grande grupo de bactérias, ordenadas em diferentes gêneros na classe Mollicutes, cuja principal característica em comum, além do genoma reduzido, é a ausência de parede celular. Mycoplasma mycoïdes subsp. mycoïdes SC, responsável pela Pleuropneumonia Contagiosa Bovina, foi o primeiro microrganismo desta classe de bactérias a ser identificado. Esta é uma doença bastante grave, com altas taxas de morbidade e mortalidade. A variedade Mycoplasma mycoïdes subsp. mycoïdes LC é responsável principalmente por casos de Pleuropneumonia Contagiosa Caprina, mastite no gado bovino, e ainda artrite em ovinos e caprinos em menor extensão. M. capricolum é um patógeno caprino, responsável principalmente por casos de artrite com grande importância econômica na medicina veterinária. M. pulmonis é um patógeno de roedores, considerado como o melhor modelo experimental para o estudo das micoplasmoses respiratórias. M. genitalium, o menor microrganismo conhecido capaz de se autoreplicar, é um patógeno humano responsável por casos de uretrite não gonocócica, cujo seqüenciamento completo do cromossomo tornou-se um marco na era da genômica. O estudo funcional do genoma destes micoplasmas, para a compreensão de sua biologia e patogenicidade, requer o desenvolvimento de ferramentas genéticas eficientes. No presente trabalho, análises in silico das seqüências na região das prováveis origens de replicação cromossômica (oriC) destes micoplasmas demonstraram a existência de possíveis DnaA boxes localizados em torno do gene dnaA. Estas regiões oriC foram caracterizadas funcionalmente após sua clonagem em vetores artificiais e a transformação dos micoplasmas com os plasmídeos recombinantes resultantes. O plasmídeo pMPO1, contendo a região oriC de M. pulmonis, sofreu integração no cromossomo do micoplasma por recombinação homóloga após poucas passagens in vitro. A redução desta oriC para o fragmento contendo somente os DnaA boxes localizados nas estremidades 5´ou 3´do gene dnaA não foi capaz de produzir plasmídeos replicativos em M. pulmonis, exceto quando estes dois fragmentos foram clonados no mesmo vetor, espaçados pelo determinante de resistência à tetraciclina tetM. Um fragmento interno do gene da hemolisina A (hlyA) de M. pulmonis foi clonado nestes plasmídeos oriC, e os vetores resultantes foram utilizados para transformar o micoplasma. A integração destes vetores por um crossing-over com o gene hlyA, causando a sua dirupção, foi documentada. Deste modo, estes plasmídeos oriC podem vir a se tornar ferramentas genéticas valiosas para o estudo do papel de genes específicos, notadamente aqueles potencialmente envolvidos na patogênese.Mycoplasmas are the smallest microorganisms capable of self replication known to date, responsible for many diseases in man and animals, infecting also plants and insects. They constitute a large group of bacteria, classified in different genera in the class Mollicutes, which main common characteristic, besides the small genome, is the absence of a cell wall. Mycoplasma mycoïdes subsp. mycoïdes SC, responsible for the Bovine Contagious Pleuropneumonia, was the first microorganism of this class of bacteria to be identified. That is a quite severe disease, with high morbidity and mortality rates. Mycoplasma mycoïdes subsp. mycoïdes LC is responsible mainly for cases of Caprine Contagious Pleuropneumonia, mastitis in cattle, and also arthritis in goats and sheep in less extension. M. capricolum is a pathogen of goats, responsible mainly by cases of arthritis with large economic impact in veterinary medicine. M. pulmonis is a rodent pathogen, considered to be the best experimental model for studying respiratory mycoplasmoses. M. genitalium, the smallest microorganism capable of self replication, is an human pathogen responsible for cases of non gonococcal urethritis, which complete chromosome sequencing has become a benchmark in the era of genomics. Functional studies of these mycoplasma genomes, for comprehension of their biology and pathogenicity, requires the development of efficient genetic tools. In the present work, in silico analysis of sequences of the putative origin of chromosome replication (oriC) region of these mycoplasmas demonstrates the existence of putative DnaA boxes located around the dnaA gene. These oriC regions were functionally characterized after cloning into artificial vectors and transformation of mycoplasmas with the resulting recombinant plasmids. The plasmid pMPO1, which contains the M. pulmonis oriC region, has integrated into the mycoplasma chromosome by homologous recombination after a few in vitro passages. Reduction of this oriC to the fragment containing only the DnaA boxes located upstream or downstream the dnaA gene could not produce plasmids able to replicate in M. pulmonis, except when these two fragments were cloned in the same vector, spaced by tetracycline resistance gene tetM. An internal fragment of the M. pulmonis hemolysine A gene (hlyA) was cloned into these oriC plasmids, and the resulting vectors were used to transform the mycoplasma. Integration of these disruption vectors by one crossing-over with the hlyA gene could be documented. Therefore, these oriC plasmids may become valuable genetic tools for studying the role of specific genes of mycoplasmas, specially those potentially involved in pathogenesis.
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Zhang, Hanshuo. "Large-scale identification of functional genes regulating cancer cell migration and metastasis using the self-assembled cell microarray." Diss., Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/49066.
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Metastasis is one of the critical hallmarks of malignancy tumor and the principal cause of death in patients with cancer. Cell migration is the basic and essential step in cancer metastasis process. To systematically investigate functional genes regulating cell migration and cancer metastasis on large scale, we developed a novel on-chip method, SAMcell (self-assembled cell microarray). This method was demonstrated to be particularly suitable for loss-of-function high-throughput screening because of its unique advantages. The first application of SAMcell was to screen human genome miRNAs, considering that more and more miRNAs had been proved to govern cancer metastasis. We found that over 20 % of miRNAs have migratory regulation activity in diverse cell types, indicating a general involvement of miRNAs in migratory regulation. Through triple-round screenings, we discovered miR-23b, which is down-regulated in human colon cancer samples, potently mediates the multiple steps of metastasis, including cell motility, cell growth and cell survival. In parallel, the second application of SAMcell was to screen human genome kinase genes, considering that more and more kinase genes had become successful diagnostic marker or drug targets. We found over 11% migratory kinase genes, suggesting the important role of kinase group in metastasis regulation. Through both functional screening and bioinformatics analysis, we discovered and validated 6 prospective metastasis-related kinase genes, which can be new potential targets in cancer therapy. These findings allow the understanding of regulation mechanism in human cancer progression, especially metastasis and provide the new insight into the biological and therapeutical importance of miRNAs or kinases in cancer.
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Gustafsson, Susanne. "Population genetic analyses in the orchid genus Gymnadenia : a conservation genetic perspective." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl [distributör], 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3305.
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Hedberg, Lilia. "Identification of obesity-associated SNPs in the human genome : Method development and implementation for SOLiD sequencing data analysis." Thesis, Linköpings universitet, Institutionen för klinisk och experimentell medicin, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-57932.
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Over the last few years, genome-wide association studies (GWAS) have been used to identify numerous obesity associated SNPs in the human genome. By using linkage studies, candidate obesity genes have been identified. When SNPs in the first intron of FTO were found to be associated to BMI, it became the first gene to be linked to common obesity. In order to look for causative explanations behind the associated SNPs, a re-sequencing of FTO had been performed on the SOLiD sequencing platform. In-house candidate gene, SLCX, was also sequenced in order to evaluate a potential obesity association. The purpose of this project was to analyse the sequences and also to evaluate the quality of the SOLiD sequencing. A part of the project consisted in performing PCRs and selecting genomic regions for future sequencing projects. I developed and implemented a sequence analysis strategy to identify obesity associated SNPs. I found 39 obesity-linked SNPs in FTO, a majority of which were located in introns 1 and 8. I also identified 3 associated intronic SNPs in SLCX. I found that the SOLiD sequencing coverage varies between non-repetitive and repetitive genomic regions, and that it is highest near amplicon ends. Interestingly, coverage varies significantly between different amplicons even after repetitive sequences have been removed, which indicates that it is affected by features inherent to the sequence. Still, the observed allele frequencies for known SNPs were highly correlated with the SNP frequencies documented in HapMap. In conclusion, I verify that SNPs in FTO are associated with obesity and also identify a previously unassociated gene, SLCX, as a potential obesity gene. Re-sequencing of genomic regions on the SOLiD platform was proven to be successful for SNP identification, although the difference in sequencing coverage might be problematic.
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Kolkailah, Naiyerah F. "Genes Encoding Flower- and Root-Specific Functions are More Resistant to Fractionation than Globally Expressed Genes in Brassica rapa." DigitalCommons@CalPoly, 2016. https://digitalcommons.calpoly.edu/theses/1586.
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Like many angiosperms, Brassica rapa underwent several rounds of whole genome duplication during its evolutionary history. Brassica rapa is particularly valuable for studying genome evolution because it also experienced whole genome triplication shortly after it diverged from the common ancestor it shares with Arabidopsis thaliana about 17-20 million years ago. While many B. rapa genes appear resistant to paralog retention, close to 50% of B. rapa genes have retained multiple, paralogous loci for millions of years and appear to be multi-copy tolerant. Based on previous studies, gene function may contribute to the selective pressure driving certain genes back to singleton status. It is suspected that other factors, such as gene expression patterns, also play a role in determining the fate of genes following whole genome triplication. Published RNA-seq data was used to determine if gene expression patterns influence the retention of extra gene copies. It is hypothesized that retention of genes in duplicate and triplicate is more likely if those genes are expressed in a tissue-specific manner, as opposed to being expressed globally across all tissues. This study shows that genes expressed specifically in flowers and roots in B. rapa are more resistant to fractionation than globally expressed genes following whole genome triplication. In particular, there appears to have been selection on genes expressed specifically in flower tissues to retain higher copy numbers and for all three copies to exhibit the same flower-specific expression pattern. Future research to determine if these observations in Brassica rapa are consistent with other angiosperms that have undergone recent whole genome duplication would confirm that retention of flower-specific-expressed genes is a general feature in plant genome evolution and not specific to B. rapa.