Dissertations / Theses on the topic 'Genetics and Genome Biology'
To see the other types of publications on this topic, follow the link: Genetics and Genome Biology.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'Genetics and Genome Biology.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Chew, Wei Leong. "Postnatal Genome Editing With CRISPR." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493352.
Full textAbstract:
Targeted genome editing holds tremendous promise for permanent correction of many genetic diseases. The recently developed CRISPR/Cas9 genome-editing tool exhibits facile programmability and robust gene-editing efficiency, and has been applied in cell cultures and animal tissues. However, multi-organ gene-editing in live mammals has not been examined or achieved. This study demonstrates genetic modification in multiple organs of postnatal mice by systemic delivery of CRISPR with adeno-associated viruses (AAVs). I resolved the AAV payload limitation by splitting Cas9 and reconstituting the native protein in vivo using scarless split-intein protein trans-splicing, which preserves full activity of Cas9. I determined that the delivery efficiency of AAV-CRISPR dictates gene-targeting rates in vivo, with the preferential gene-editing in liver and heart, and more modest editing efficiencies in skeletal muscle, brain and gonads, directly reflecting the infection profile of the virus serotype. To track CRISPR biodistribution, I established two reporter systems that apply in situ fluorescence activation to demarcate CRISPR-targeting events at single-cell resolution, identifying rare gene-edited cells that normally evade detection by sequencing. This exquisite detection sensitivity further allows evaluation of inter-generational transmission of gene-editing viruses. Finally, although Cas9 elicits host immune responses, these can be ameliorated by immunosuppression. I also identified a public Cas9-responsive T-cell clonotype and mapped the B-cell epitopes on Cas9 and AAV. Engineering tolerance to immunodominant epitopes may provide an avenue for avoiding immune rejection of AAV-CRISPR. The ability to create programmable genetic modifications in multiple organs of postnatal mammals provides a powerful tool for biological research, and foretells that the genomes of whole mammals may be rewritten at will.Medical Sciences
2
Omilian, Angela Ruggieri. "Features of Daphnia genome evolution." [Bloomington, Ind.] : Indiana University, 2006. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3243783.
Full textAbstract:
Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2006.Title from PDF t.p. (viewed Nov. 18, 2008). Source: Dissertation Abstracts International, Volume: 67-12, Section: B, page: 6862. Adviser: Michael Lynch.
3
Chung, Hattie. "Genome evolution in structured systems." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493565.
Full textAbstract:
The evolution of a genome is shaped by spatial interactions at multiple scales. At the angstrom level, structural constraints on both RNA molecules and proteins contribute to the evolution of a gene sequence. Such optimized genes are weaved together in a particular order, out of a near-infinite number of combinations, to result in a genome. The fate of a genome is intricately linked to the evolutionary fate of its host organism; in turn, the fate of an organism is governed by where it resides in space. In this dissertation, I investigate how structure shapes the evolution of a gene, genome content, and pathogen populations residing in a diseased human lung. Chapter 1 provides a brief historical overview of population genetics in structured environments. I motivate why it is important to determine the migration rate of new alleles. Chapter 2 investigates how pathogens evolve within the structure of the cystic fibrosis lung. I find that migration rate and mutation rate are on similar timescales. Selection, rather than spatial isolation, maintains diversity within a pathogen population. Chapter 3 presents a new method to probe how codon choice is optimized throughout a gene. I find that codon choice is dictated by preference for particular RNA secondary structures, rather than intrinsic properties of a codon. Chapter 4 describes an ongoing study of how rapidly P. aeruginosa populations evolve in short-term infections. Preliminary results show that gene duplication events can sweep through a population in just 11 days. Chapter 5 introduces ideas for future directions. I pose questions regarding how pathogens evolve molecular mimicry that can trigger autoimmune disease in the human host, and how cancer-inducing inflammation might be detected from mutational signatures in the microbiome.Systems Biology
4
Chen, Stacy Yen-chun. "Genome-wide analysis of yeast meiotic recombination landscape." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3390037.
Full text
5
Simonson, Matthew A. "Polygenic analysis of genome-wide SNP data." Thesis, University of Colorado at Boulder, 2013. http://pqdtopen.proquest.com/#viewpdf?dispub=3562047.
Full textAbstract:
One of the central motivators behind genetic research is to understand how genetic variation relates to human health and disease. Recently, there has been a large-scale effort to find common genetic variants associated with many forms of disease and disorder using single nucleotide polymorphisms (SNPs). Several genome-wide association (GWAS) studies have successfully identified SNPs associated with phenotypes. However, the effect sizes attributed to individual variants is generally small, explaining only a very small amount of the genetic risk and heritability expected based on the estimates of family and twin studies. Several explanations exist for the inability of GWAS to find the "missing heritability."
The results of recent research appear to confirm the prediction made by population genetics theory that most complex phenotypes are highly polygenic, occasionally influenced by a few alleles of relatively large effect, and usually by several of small effect. Studies have also confirmed that common variants are only part of what contributes to the total genetic variance for most traits, indicating rare-variants may play a significant role.
This research addresses some of the most glaring weaknesses of the traditional GWAS approach through the application of methods of polygenic analysis. We apply several methods, including those that investigate the net effects of large sets of SNPs, more sophisticated approaches informed by biology rather than the purely statistical approach of GWAS, as well as methods that infer the effects of recessive rare variants.
Our results indicate that traditional GWAS is well complemented and improved upon by methods of polygenic analysis. We demonstrate that polygenic approaches can be used to significantly predict individual risk for disease, provide an unbiased estimate of a substantial proportion of the heritability for multiple phenotypes, identify sets of genes grouped into biological pathways that are enriched for associations, and finally, detect the significant influence of recessive rare variants.
6
Wesolowska, Natalia. "Modification and nuclear organization of the Drosophila melanogaster genome." Thesis, The Johns Hopkins University, 2013. http://pqdtopen.proquest.com/#viewpdf?dispub=3575013.
Full textAbstract:
The success of Drosophila as a system for genetic analysis is closely linked to its amenability to genetic manipulation. Part 1 of the dissertation elucidates a novel scheme for long-range targeted manipulation of genes. We integrated an 80-kb genomic fragment at its endogenous locus, utilizing a targeted attP attachment site for the phiC31 integrase. We achieved single-copy reduction of the resulting region duplication by inducing recombinational DNA repair. We showed that this two-step scheme of integration and reduction is efficient and useful for delivering modifications. We established a vector configuration that facilitates the recovery of modifications. The integrating genomic fragment allowed for delivery of a new attachment site at 70 kb from the existing attP into a new locus, making it susceptible to targeted mutagenesis. We extrapolate that with this scheme, only 1 200 lines bearing att-sites throughout the genome would suffice to render all Drosophila genes amenable to targeted mutagenesis. Excitingly, this method should be readily applicable to other systems.
In Part 2 of the dissertation, I explored the question of telomere organization in Drosophila. Telomeres demarcate the ends of linear chromosomes to distinguish them from broken ends. In yeast, they cluster at the periphery of the nucleus establishing a compartment of silent chromatin. To bring insight into telomere organization in a higher organism, we followed EGFPlabeled Drosophila telomeric protein HOAP in vivo and found that the 16 telomeres cluster into 4-6 foci per nucleus in somatic tissues. Interestingly, HOAP signal intensity in the clusters doubles in interphase, potentially due to loading of HOAP to newly replicated telomeres. We tested several predictions about rules governing clustering. First, by inspecting mutant embryos that develop as haploids, we found that clustering is not mediated by associations between homologs. Second, by demonstrating clustering capability for a telomere of novel sequence, we eliminated DNA sequence homology and identity as important factors. Third, by marking both ends of a chromosome, we ruled out predominance of intra-chromosomal interactions. We propose that clustering is indiscriminate of sequence and is likely maintained by a yet undetermined factor.
7
Badhwar, AmanPreet. "Identification and characterization of rearrangements in the vervet monkey genome." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=101702.
Full textAbstract:
Several mechanisms can lead to the reorganization of genomes during speciation, including centromere repositioning, new centromere emergence or other chromosomal rearrangements. Using a comparative karyotype approach, I determined that the vervet genome contains at least 12 evolutionary young centromere locations.To study the evolutionary dynamics of centromere formation, I identified and validated the alpha-satellite repeat as a centromere-specific marker in the vervet using comparative genomics, sequence analysis and hybridization screening. I developed criteria to infer the position of vervet bacterial artificial chromosome (BAC) inserts based on alpha-satellite monomer content. In a complementary approach, I demarcated the pericentromeric boundaries in human and identified vervet BAC clones that mapped orthologously to these regions.
In addition to centromeric analyses, I developed methodologies to detect other genome rearrangements, in particular vervet deletion/human insertion and vervet translocation events. The tools and approaches developed in this thesis will prove useful in cataloguing additional vervet genome rearrangements.
8
Young, Adrian. "The Evolutionary Feedback between Genetic Conflict and Genome Architecture." Thesis, Harvard University, 2014. http://dissertations.umi.com/gsas.harvard:11482.
Full textAbstract:
The advent of separate sexes set the stage for dramatic evolutionary innovation across a wide range of taxa. Much of this innovation is attributable to divergent evolutionary interests between now distinct sub-populations of males and females. Trade-offs inherent to these divergent life histories, coupled with a common genome, conspire to limit natural selection's ability to simultaneously maximize the fitness of both sexes. Such conflict between the sexes has therefore largely shaped the history of the genomes of sexual taxa. However, various aspects of the genomic environment—including genes' spatial distributions, abilities to regulate their expression, and rates of recombination—also feed back to influence future sex-specific evolutionary trajectories. Using various genomic resources and transcriptome sequences for the lab mouse, I test several theoretical predictions regarding this feedback between genetic conflict and features of genomic organization.
9
Li, Hubo. "Genome-Wide RNAi Screens for Novel Regulators of Acute Myeloid Leukemia." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:14226105.
Full textAbstract:
Acute myeloid leukemia (AML) is a heterogeneous disease with complex molecular mechanisms. Recent advent of genomic technologies, such as copy number profiling, whole genome sequencing, and gene expression profiling has accumulated a plethora of large-scale data in AML cell lines and patient samples. However, the functional relevance of most genes identified by these methods has yet to be determined. To systematically characterize the genetic requirement in AML, we conducted genome-wide shRNA screens in 17 AML cell lines in parallel with 199 cell lines of other cancer types. We identified over 150 genes that were required for proliferation specifically by AML, but not other cancer cell lines. We further interrogated the requirements of primary screen hits in vivo with a secondary screen in a xenotransplantation model driven by the MLL-AF9 oncogenic fusion. Integrating both of the RNAi screens and additional gene expression data, we identified transcription factor ZEB2 as a top candidate for regulating AML proliferation. In human AML cells, ZEB2 inhibition impairs proliferation and promotes granulocytic differentiation. Mechanistically, we showed that ZEB2 interacts with the CtBP co-repressor complex, and transcriptionally represses genes involved in cell adhesion and migration. ZEB2’s relevance in AML is further demonstrated by its overexpression in MLL-rearranged AML, and by the epigenetic silencing of its negative regulators, miR-200 family microRNAs, in AML. Our results extend the role of ZEB2 beyond regulating epithelial-mesenchymal transition, and establish ZEB2 as a novel regulator of AML proliferation and differentiation.
MicroRNA-like off-target effect is a major caveat of RNAi screens, which often leads to false positive discoveries. However, systematic analysis of off-target effects in large-scale RNAi screen data can also lead to the discovery of microRNAs with functional relevance. By analyzing the off-target effects in our AML screen, we identified several microRNAs as candidate suppressors for AML proliferation. We show that miR-105, miR-140, miR-501, and miR-532 are novel regulators of the myeloid oncogene MYB. In particular, miR-105 inhibits AML cell growth and miR-532 is associated with myeloid differentiation. The combination of the ZEB2 and microRNA work emphasizes the power of RNAi screens in the exploration of novel cancer regulators.
10
Canver, Matthew. "Elucidation of Mechanisms of Fetal Hemoglobin Regulation by CRISPR/Cas9 Mediated Genome Editing." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493407.
Full textAbstract:
Despite nearly complete understanding of the genetics of the β-hemoglobinopathies for several decades, definitive treatment options have lagged behind. Fetal hemoglobin (HbF) reinduction represents a “silver bullet” for therapy of the β-globin disorders. Recent development of the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease system has allowed for facile manipulation of the genome for the study of genes and genetic elements. Here we developed CRISPR/Cas9-based methodology to reliably engender targeted genomic deletions ranging from 1.3 kilobases to over 1 megabase, which suggested an inverse relationship between deletion size and deletion frequency. Targeted deletion methods and Cas9-mediated in situ saturating mutagenesis were applied to the enhancer of the HbF repressor BCL11A, which revealed discrete vulnerabilities. This finding is consistent with emerging evidence in the field that large enhancers are comprised of constituent parts with some harboring the majority of the activity. The identified “Achilles heel” of the enhancer represents a promising therapeutic target. We further enhanced the resolution of the in situ saturating mutagenesis technique by using multiple Cas9 nucleases and variant-aware library design to identify functional sequences within the HBS1L-MYB intergenic region, a locus associated with elevated HbF levels. These data demonstrate the robustness of CRISPR/Cas9 mediated in situ saturating mutagenesis and targeted deletion to interrogate functional sequence within regulatory DNA. Harnessing the power of genome editing may usher in a second generation form of gene therapy for the β-globin disorders.Medical Sciences
11
Fernandes, Caroline. "Genome-wide screen for novel regulators of Parkinson's disease genes in «Drosophila melanogaster»." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103725.
Full textAbstract:
Introduction: Compromised mitochondrial integrity leads to neuronal cell death in Parkinson's disease and other neurodegenerative diseases. Cells have several quality control mechanisms in place to counteract dysfunction and preserve a healthy mitochondrial network. Recent studies have implicated the autosomal recessive Parkinson's disease genes, Parkin and Pink1, in a common pathway regulating mitochondrial quality control. Together, Parkin and Pink1 promote isolation and selective degradation of damaged mitochondria, but the mechanisms governing this process are unclear. Methods: The goal of our study was to use a genetically sensitized Drosophila model of Parkinson's disease to identify novel regulators of the Pink1/Parkin pathway. We screened deficiencies spanning the second and third chromosome for dominant enhancers and suppressors of the Pink1/parkin wing posture phenotype, which derives from mitochondrial defects in these mutants. Results: From this screen we identified several cytological regions that strongly interact with Parkin and/or Pink1. Of these, four were further dissected revealing five interacting genes. Among them, opa1 and drp1 have been previously implicated in the Pink1/Parkin pathway. The other three genes, p60, β4galNacTA, and debra, represent novel regulators of Parkin and Pink1 function. Conclusion/implications: The unbiased identification of previously known Pink1/Parkin interactors demonstrates the validity of this approach. Moreover, the discovery of novel genes involved in the Pink1/Parkin pathway will allow better understanding of the mechanisms governing mitochondrial quality control and will aid in the development of therapeutic treatments.Introduction : L'altération de la fonction mitochondriale entraîne la dégénérescence de certains neurones chez les personnes atteintes de maladies neurodégénératives dont la maladie de Parkinson. Les cellules saines sont dotées d'un système de contrôle leur permettant de faire face et de réparer les dysfonctionnements des mitochondries et ainsi de préserver leur intégrité. Des études récentes ont révélé l'implication de deux gènes à l'origine de syndromes parkinsoniens autosomiques récessifs, Pink1 et Parkin, dans une voie de signalisation commune contrôlant le maintien de la fonction mitochondriale. Pink1 et Parkin interviennent ensemble dans l'isolation et la dégradation des mitochondries défectueuses. Cependant, à l'heure actuelle, les mécanismes moléculaires contrôlant ce processus restent à élucider. Méthodes : Le but de notre étude a été d'identifier de nouveaux régulateurs de la voie de signalisation Pink1/Parkin par crible génétique dans un modèle drosophile de la maladie de Parkinson. Pour cela, nous avons criblé une collection de lignées de drosophiles déficientes sur les chromosomes deux et trois pour leur capacité à modifier (augmenter ou diminuer) le phénotype de posture de l'aile caractéristique de la mutation de Pink1/Parkin. Résultats: Nous avons identifié plusieurs régions cytologiques qui interagissent fortement avec Parkin et/ou Pink1. Quatre de ces régions ont été disséquées de façon à révéler cinq gènes. Parmi eux, opa1 et drp1 avaient déjà été impliques dans la voie de signalisation Pink1/Parkin. Les trois autres gènes p60, β4galNacTA et debra représentent de nouveaux régulateurs de la fonction de Pink1 et Parkin. Conclusion/implications : D'une part, l'identification non biaisée de gènes déjà connus comme interagissant avec Pink1/Parkin démontre la validité de cette approche. D'autre part, la découverte de nouveaux gènes candidats de la voie Pink1/Parkin pour le maintien de l'intégrité mitochondriale permettra de mieux comprendre les mécanismes moléculaires contrôlant ce processus et aidera à l'élaboration de traitements.
12
Rogers, Jameson Kerr. "Biosensing for Multiplexed Genome Engineering: Applications in Renewable Chemical Production." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467367.
Full textAbstract:
Engineered biological systems are increasingly used to produce fuels, pharmaceuticals and industrial chemicals. While transforming cells into renewable chemical factories presents an enormous opportunity, development timelines are long, costly and often uncertain. Engineering microbes for chemical production is accomplished through the biological design-build-test cycle: many designs are formulated, the corresponding organisms are constructed, and their ability to produce the desired chemical is evaluated. Designs that perform well become the starting point for the next round of the cycle. Faster design cycles result in shorter and less costly product development timelines.
Advances in DNA sequencing, synthesis and genome engineering technologies have sped up the design and build steps of the design cycle by enabling billions of organism variants to be designed and constructed simultaneously. However, evaluation of the resulting designs continues to rely on low-throughput technologies with evaluation rates on the order of thousands per day. Because the engineering process is a cycle, it can only proceed at the rate of the slowest step. A high-throughput method for design evaluation would increase the throughput of the design cycle by up to a million-fold.
This thesis describes an engineering framework that makes high-throughput design evaluation a reality. By programming cells to keep track of their own success in making a desired product, I enable screens and selections to be used for the optimization of metabolic pathways. I develop biosensors that maintain gene expression at a rate proportional to the concentration of several different chemical products and show that higher product concentration results in a higher fluorescent output. I then construct metabolic pathways for the production of the renewable plastic precursors 3-hydroxypropionate, acrylate, glucarate and muconate. I combine each pathway with the appropriate biosensor and use fluorescence to observe product formation in real-time. Next, I replace the fluorescent protein with an antibiotic resistance gene and link the level of product formation to the cell’s ability to survive an antibiotic challenge. I deploy the selection to optimize production of both glucarate and naringenin from glucose.
I further develop the characterization of these new biosensors to promote their use as genetic switches for synthetic biological circuits. Finally, I develop a device called the fluorimostat that makes long-term closed-loop programmable control of gene expression a reality.Engineering and Applied Sciences - Engineering Sciences
13
Shanmugasundram, Achchuthan. "Genome annotation and metabolic reconstruction of apicomplexan parasites." Thesis, University of Liverpool, 2014. http://livrepository.liverpool.ac.uk/2004940/.
Full textAbstract:
The apicomplexans are causative agents of human and animal infections including malaria, toxoplasmosis and theileriosis and have a huge economic and social impact. A number of apicomplexan genomes have been sequenced. However, the annotation of gene functions remains challenging. A semi-automatic approach was used to systematically assign genes to their functions within pathways/networks through the integration of genomic information with biochemical evidence from the literature. This method has resulted in the evidence-based annotation of metabolic functions and the development of organism specific metabolic pathways. A web database named Library of Apicomplexan Metabolic Pathways (LAMP, www.llamp.net) was developed to host the metabolic mappings for Toxoplasma gondii, Neospora caninum, Cryptosporidium and Theileria species and Babesia bovis at present. A comparative analysis of the overall metabolic capabilities of apicomplexan species showed that the metabolic adaptations has evolved for different ecological niches and led to the identification of putative drug targets. The identification of missing enzymes that are essential to complete the metabolic pathways and the identification of a subsection of these missing enzymes from raw genomes demonstrated probable inaccuracies in gene model predictions. The utilisation of T. gondii and N. caninum proteomic datasets and their mapping to alternative gene models showed regions of genes that require further refinement. The evaluation of the quality of two different gene model releases with this peptide evidence showed the importance of integration of RNA-Seq datasets in improving gene models and further improvements that can be made with proteomic datasets. A global post-translational modification (PTM) analysis was carried out for T. gondii and N. caninum via the utilisation of non-enriched proteomic datasets available for these species. This analysis identified proteins of functional importance that have undergone PTMs, particularly methylation, acetylation, phosphorylation and oxidative modifications. All these analyses helped in the improvement of gene models and functional annotation of genes from Apicomplexa genomes.
14
Schiavi, Alicia. "Direct assessment and validation of allele specific transcription factor binding in the human genome." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114371.
Full textAbstract:
Characterization of human genetic variation has focused on expression quantitative trait loci (eQTL) mapping; however, direct assessment of cis-regulatory variation requires allele-specific approaches. Measuring allelic expression (AE) on a genome-wide scale appears more powerful as environmental and trans-acting influences are minimized. Results indicate that allele-specific differences in transcript expression within an individual can affect up to 30% of loci. The underlying variants can be identified by mapping differences in AE on Illumina BeadChips. Over 50% of population variance in AE is explained by mapped cis-rSNPs. Studies show that these cis-rSNPs have been implicated in differences in transcription factor (TF) binding, suggesting that TF action can be further investigated using population variation as a tool. In this thesis, these approaches have been extended to explore allele-specific TF binding using the model NF-κB by monitoring the consequences of gene knockdown in a genome-wide manner. NF-κB has been shown to be involved in the immune response and the NF-κB motif is enriched in lymphoblastoid cell lines (LCLs), mainly in promoters and strong enhancer elements. We intersected mapped candidate cis-rSNPs detected in LCLs in our above experiments as well as matched control SNPs from HapMap YRI and CEU populations with publicly available NF-κB Chromatin Immunoprecipitation (ChIP)-seq experiments from the ENCODE project. Preliminary analysis of regions surrounding candidate cis-rSNPs were enriched in NF-κB binding sites versus matched controls, with 39.0 % of top SNPs overlapping at least one NF-κB ChIP-seq peak. To elucidate the impact of candidate SNPs on AE imbalances, we performed TNF- α induction coupled to inhibition of NF-κB in LCLs followed by AE analysis on Illumina HumanOmni5-Quad BeadChips. We used in house mapped cis-regulatory variants in the LCL population merged with data from the aforementioned experiment. Our data set, which consisted of loci associated to top 10 cis-rSNPs ranked by p-value (pv; top10= 10 most significant p-values) that showed diminished AE upon perturbation of NF-κB were overlapped with publicly available data. This data consisted of ENCODE ChIP-seq peaks and TRANSFAC binding sites for NF-κB and known cooperative TFs of NF-κB. Loci that had an AE change at greater than 3 SNPs upon perturbation of NF-κB and were associated to top heterozygous SNPs (rank 1, 2, 3) yielded 581 cases out of ~1700. Analysis of top 3 ¬cis-rSNPs described showed a significant difference of over 5-fold between case and control SNPs, such that 64% of loci had a top 3 heterozygous SNP that was found in an LCL specific TF ChIP-seq peak or TRANSFAC binding site for NF-κB or a known cooperative TF of NF-κB. Bioinformatics analysis suggests that identified SNPs are essential for NF-κB binding. A case study was also done in order to perturb the TF SNAI1 because we had a strong hypothesis for the association of SNAI1 and WNT4, as well as, evidence for its role in fibroblasts (FBs). We were not able to reproduce the effect of SNAI1 on WNT4 in vivo. Upon comparison of the regulatory role of NF-κB and SNAI1 in LCLs and FBs, respectively; we observed that NF-κB had a regulatory effect on approximately 33% of loci in comparison to only approximately 2% of loci for SNAI1 in FBs. This study illustrates that key regulatory TFs, such as NF-κB in LCLs, can be globally studied at a single base resolution in living cells using a combination of perturbation and sensitive measurements with allelic resolution.La caractérisation de la variation génétique a mis l'accent sur les loci d'expression de caractères quantitatifs(eLCQ); cependant, l'évaluation directe des variations régulatrices en cis nécessite des approches allèle-spécifique (cis-rSNPs). La mesure de l'expression allélique (EA) est très efficace puisque les perturbations environnementales et les influences en trans sont réduites. Les résultats indiquent que les différences d'EA peuvent affecter jusqu'à 30% des loci chez un individu. Les polymorphismes responsable de telles variations peuvent être identifiés par cartographie des différences d'EA en utilisant des puces de génotypage Illumina. Ainsi, plus de 50% de la variance en EA de la population est expliqué par la cartographie des cis-rSNPs. Des études ont montré que ces cis-rSNPs ont été impliqués dans des différences de liason de facteurs de transcription (FT). Celà suggère que l'étude du mode d'action de ces FT pourrait être approfondie par l'utilisation comme outil des polymorphismes dans la population. Nous avons appliqué cette approche au FT NF-κB et analysé les conséquences de l'inactivation de ce gène à l'échelle du genome. Des données récentes montrent l'implication de NF-κB dans la réponse immunitaire et son motif de liaison à l'ADN est retrouvé enrichi dans les cellules lymphoblastoïdes humaines (LCL), surtout au niveau des promoteurs et des activateurs transcriptionnels. Nous avons croisé les cis-rSNPs cartographiés dans des LCLs des populations HapMap YRI et CEU, ainsi que des SNPs de contrôles, avec des données d'immunoprécipitation de chromatine suivi de séquençage à haut débit (ChIP-seq) utilisant l'anticorps NF-κB du projet ENCODE et accessible au public. Des analyses préliminaires des régions contenant les cis-rSNPs candidats ont montré un enrichissement des sites de liaison pour le facteur NF-κB par rapport aux sites contrôles. En effet, 39% des sites candidats sont situés dans un site de liaison pour NF-kB. Afin d'étudier le rôle potentiel des cis-rSNPs sur l'EA différentielle, nous avons réalisé des expériences d'induction de TNF-α couplé à l'inhibition de NF-κB dans les LCLs suivie par l'analyses de l'EA sur des puces de génotypage Illumina 5M. Nous avons ensuite comparé ces données avec la cartographie de cis-rSNPs dans des LCLs générée dans notre laboratoire.Nos données sont composées de cis-rSNPs associés à l'EA différentielle de loci suite à la perturbation de NF-κB et classés par valeur p (pv; top10= les 10 valeurs les plus significatives) et ont été croisées avec des données accessibles au public. Ces données sont composées des coordonnées de pics de ChIP-seq et des sites de liaison TRANSFAC pour le facteur NF-κB et de ses co-régulateurs transcriptionnels connus. Les loci montrant des différences d'EA suite à la perturbation de NF-κB et avec 1 ou plusieurs des cis-rSNPs (« top3 » pv) hétérozygotes dans les individus étudiés étaient aux nombre de 581. La recherche de ces cis-rSNPs « top 3 » dans les sites de liaison (pics de ChIP-seq) dans les LCLs ou dans un site de TRANSFAC pour NF-κB ou pour un de ses co-régulateurs transcriptionnels montrent un enrichissement significatif (64% des loci) avec un ratio supérieur à 5 par rapport aux SNPs de contrôles.L'analyse bioinformatique suggère que les SNPs identifiés sont essentiels pour la liason de NF-κB. Une étude complémentaire à consisté à perturber le FT SNAI1 probablement associé au gène WNT4 et présentant un rôle important dans les fibroblastes (FB). Cependant, nous n'avons pas été en mesure de reproduire l'effet de SNAI1 sur WNT4 in vivo.Nous avons ensuite comparé les rôles régulateurs de NF-κB et de SNAI1 dans les LCLs et les FBs respectivement (par inactivation de ces gènes). Nous avons observé que NF-κB a un effet régulateurs sur environ 33% des loci dans les LCLs contre seulement 2 % pour SNAI1 dans des FBs.Cette étude supporte l'utilisation de perturbations de l'expression de FT pour étudier le rôle clé de FTs régulateurs dans un type cellulaire donné.
15
Yang, Joyce Lichi. "Developments in Human Pluripotent Stem Cell Genome Engineering and in Situ Sequencing Technologies." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467524.
Full textAbstract:
Technology is a key driving force in the advancement of scientific discoveries. While DNA sequencing uncovered the blueprint of life encoded in the human genome, functional roles of sequence variants remain largely unknown. This thesis focuses on developing enabling technologies with broad applications for the study of genetic variations and gene regulation.
Recent advances in CRISPR/Cas9-based genome engineering technology have revolutionized biomedical research. The facilitated genome editing system employs a programmable RNA that guides the Cas9 nuclease to its target DNA. Furthermore, gene targeting in human induced pluripotent stem cells (hiPSCs) offers the unprecedented potential for dissecting gene function and correcting disease mutations to fulfill the vision of personalized regenerative medicine. Despite phenomenal progress, the efficiency of targeted modifications remained low in hiPSCs. In part I of this thesis, we developed an efficient genome editing platform by reversibly integrating doxycycline-inducible Cas9 into the genome (iPS-Cas9). We characterized and optimized critical parameters for efficient targeting, generated precise mutations for disease modeling, and demonstrated the potential of multiplexed and continuous editing. Additionally, we initiated efforts to improve homology directed repair (HDR) frequency relative to nonhomologous end joining (NHEJ) via coupling strategies. This versatile platform enables rapid generation of mutant hiPSCs for the study of genome function and provides a test bed for further engineering of Cas9-based tools.
While DNA stores the genetic code to life, gene regulation inferred from RNA expression defines cell identity and function. Transcriptome analysis is essential for understanding developmental regulations of complex organisms by deducing gene function from expression pattern and detecting altered gene expression in disease. Traditional gene expression assays are limited by the lack of specificity, spatial context, single-cell resolution, or scalability. In part II, we explored two strategies – padlock probe (PLP) and fluorescence in situ sequencing (FISSEQ) – to develop highly multiplexed in situ RNA sequencing with single cell resolution. We concluded that PLP-based method is suitable for targeted analysis of few transcripts, while FISSEQ represents a transcriptome-wide method for in situ RNA profiling. The technologies presented will greatly accelerate the understanding of gene regulation in complex biological samples with broad applications in biology and medicine.Medical Sciences
16
Oakes, Christopher Charles. "DNA methylation in male germ cells : the acquisition and maintenance of unique genome-wide patterns." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103175.
Full textAbstract:
The development of healthy gametes is paramount to the health of progeny and to the survival of a species. Epigenetic information contained within gametic DNA in the form of DNA methylation is essential for germ cell and embryo development. DNA methylation is a genome-wide phenomenon involved in the control of gene expression and chromosome structure and stability. During germ line development, patterns of DNA methylation are established in a sex- and sequence-specific manner. The primary goal of the work presented in this thesis is to gain an understanding of the nature of the genome-wide pattern of DNA methylation in germ cells and to study its progression during germ cell development. The complexity of male germ cell development has been well studied in mice and thus makes an excellent system in which to study germ cell DNA methylation. Firstly, genome-wide patterns of DNA methylation in adult male germ cells were determined using a variety of techniques. Results from these studies demonstrate that the DNA methylation pattern in male germ cells is highly distinct from that of somatic cells. The reorganization of the germ cell pattern is associated with chromosomal features such as the chromosomal banding pattern and regional GC content. Secondly, by examining purified populations of male germ cells, we have determined that patterns of DNA methylation are being acquired during spermatogenesis. De novo methylation and demethylation events occur in a sequence-specific manner prior to the meiotic phase of germ cell development. Finally, the stability of these patterns was studied by perturbing DNA methyltransferase activity. The study of germ cells lacking a functional Dnmt3L gene demonstrates that the abnormalities displayed in these cells are associated with a failure to acquire normal levels of DNA methylation. In addition, the treatment of mice with the hypomethylating agent, 5-aza-2'-deoxycytidine, results in adverse effects on sperm function and is associated with sequence-specific hypomethylation. Collectively, these studies have uncovered several novel aspects of DNA methylation in male germ cells and contribute to our understanding of the roles) for epigenetic phenomena in the development and maintenance of healthy gametes.
17
Boettger, Linda M. "Complex Forms of Structural Variation in the Human Genome: Haplotypes, Evolution, and Relationship to Disease." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:14226090.
Full textAbstract:
Genomic mutations arise in many forms, varying from single base pair substitutions to complicated sets of overlapping copy number variants (CNVs). While each type of variation contributes to phenotype, complex structural variation, which contains multiple mutations, is difficult to type across many individuals and is largely omitted from genomic studies. This thesis presents methods to type complex structural variation, understand how it evolves, and integrate these complex variants into association studies to phenotypes.
We focused on four structurally complex regions in the human genome. The 17q21.31 region contains an inversion, previously uncharacterized overlapping copy number variants, and SNPs that associate to the female meiotic recombination rate and female fertility1. The haptoglobin (HP) gene at chromosome 16q22.2 contains a 1.7 kb tandem duplication2, previously uncharacterized paralogous gene conversion, and nearby SNPs that associate to cholesterol levels3. The haptoglobin related gene (HPR) at chromosome 16q22.2, segregates as a multi-allelic copy number variant (mCNV) specifically in African populations. Lastly, complement component 4 (C4) at chromosome 6p21.3, contains a length polymorphism, paralogous sequence variation, and copy number variation segregating in humans and non-human primates4.
We developed methods to characterize the complex structural variation in each of these four regions, type the variation at the population level and integrate it into association studies. Briefly, we determined the breakpoints of each individual structural variant, typed each variant in a population cohort, and learned which variants segregate together through trio inheritance patterns. Once these structural haplotypes were defined, we phased them with surrounding SNP haplotypes and used this data as a reference panel for imputation into disease cohorts, and to better understand their evolutionary history.
We found that two overlapping duplications in the 17q21.31 region rose rapidly and independently to high frequency within European populations, and may account for the regional association to female fertility and the female meiotic recombination rate. We also found that a recurrent deletion in the HP gene associates to total cholesterol and LDL cholesterol levels. The methods developed in this thesis enable the integration of structurally complex variation into future association studies so that we can begin to understand their effects on phenotypes.
18
Russell, Shelbi Lianne. "Mode and Fidelity of Bacterial Symbiont Transmission and Its Impact on Symbiont Genome Evolution." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493576.
Full textAbstract:
Mutualistic symbioses have enabled the colonization of novel habitats and niches in a large array of eukaryotic and bacterial taxa. Reliable mechanisms of symbiont transmission between host generations are necessary to stabilize these associations over evolutionary time. Historically, symbionts have been categorized as either vertically transmitted from the parents to offspring or horizontally transmitted through the environment. The route between hosts influences how symbiont populations are connected between hosts and between geographic localities. Over evolutionary time vertical transmission leads to gene loss and genome erosion. Growing evidence from diverse associations suggests that modes utilizing both horizontal and vertical strategies exist, raising the question of how these “mixed modes” influence symbiont genome evolution. The overarching goal of my dissertation was to determine whether the mode of symbiont transmission in an obligate mutualism is consistent over evolutionary time and what impact transmission mode fidelity has on symbiont genome evolution. To test these questions, I used the chemosynthetic symbiosis between the marine bivalve Solemya velum and its gammaproteobacterial symbionts, which has been reported to transmit its symbionts vertically through the ovary, but bears none of the genomic hallmarks of strict vertical transmission.
In this work, I used population genomics of S. velum subpopulations sampled from five localities along the New England coast, from Massachusetts to North Carolina, to test for evidence of horizontal transmission in the evolutionary history of this species. These analyses revealed that symbionts and mitochondria do not exhibit concordant genealogies, divergent symbiont lineages have come into contact and recombined, and symbiont genomes have experienced large-scale structural changes mediated by mobile elements and horizontal gene transfer. In total, these lines of evidence indicate that a substantial amount of horizontal transmission has occurred in the recent history of this association. The vertical transmission route through host tissues was characterized via in situ hybridization to symbiont 16S rRNA in adult host tissues and by qPCR of the rhlE gene in spawned eggs. These data showed that symbionts are present at low abundance in the ovary, associated with the supportive cells and mature oocytes, and each spawned egg contains approximately 50-100 copies of the symbiont genome. Lastly, I tested for evidence of mixed transmission modes in symbiont populations contained within host tissues (each host gill contains more than a billion symbiont cells), by deep-coverage whole genome sequencing. Using a novel variant-calling procedure, I detected low amounts of genetic variation among symbiont genomes within a host relative to between hosts. However, the variant sites that were present were correlated in position along the genome, present on the same chromosome, and segregating in the symbiont population at large, suggesting that these variants arose via recombination with a variant symbiont genotype introduced by horizontal transmission.
In total, this work supports the existence of mixed transmission modes in symbiotic associations and indicates they have distinct consequences for symbiont evolution. Mixed modes may provide a best-of-both-worlds strategy to ensure that hosts acquire symbionts every generation while maintaining opportunities for recombination and acquisition of novel genetic elements. These results are relevant to understanding the impact of symbiont transmission mode on genome evolution in associations ranging from mutualisms to pathogenic infections.Biology, Organismic and Evolutionary
19
Herdy, Joseph R. III. "SMALL RNA EXPRESSION DURING PROGRAMMED REARRAGEMENT OF A VERTEBRATE GENOME." UKnowledge, 2014. http://uknowledge.uky.edu/biology_etds/25.
Full textAbstract:
The sea lamprey (Petromyzon marinus) undergoes programmed genome rearrangements (PGRs) during embryogenesis that results in the deletion of ~0.5 Gb of germline DNA from the somatic lineage. The underlying mechanism of these rearrangements remains largely unknown. miRNAs (microRNAs) and piRNAs (PIWI interacting RNAs) are two classes of small noncoding RNAs that play important roles in early vertebrate development, including differentiation of cell lineages, modulation of signaling pathways, and clearing of maternal transcripts. Here, I utilized next generation sequencing to determine the temporal expression of miRNAs, piRNAs, and other small noncoding RNAs during the first five days of lamprey embryogenesis, a time series that spans the 24-32 cell stage to the formation of the neural crest. I obtained expression patterns for thousands of miRNA and piRNA species. These studies identified several thousand small RNAs that are expressed immediately before, during, and immediately after PGR. Significant sequence variation was observed at the 3’ end of miRNAs, representing template-independent covalent modifications. Patterns observed in lamprey are consistent with expectations that the addition of adenosine and uracil residues plays a role in regulation of miRNA stability during the maternal-zygotic transition. We also identified a conserved motif present in sequences without any known annotation that is expressed exclusively during PGR. This motif is similar to binding motifs of known DNA binding and nuclear export factors, and our data could represent a novel class of small noncoding RNAs operating in lamprey.
20
Fredman, David. "Computational exploration of human genome variation /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-025-7/.
Full text
21
Rios, Villanueva Xavier. "Toward Multiplex Genome Engineering in Mammalian Cells." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11179.
Full textAbstract:
Given the explosion in human genetic data, new high-throughput genetic methods are necessary for studying variants and elucidating their role in human disease. In Chapter I, I will expand on this concept and describe current methods for genetically modifying human cells. In E. coli, Multiplex Automatable Genome Engineering (MAGE) is a powerful tool that enables the targeting of multiple genomic loci simultaneously with synthetic oligos that are recombined at high frequencies in an optimized strain. MAGE as a method has two components: organism-specific optimization of oligo recombination parameters and a protein capable of increasing recombination frequencies.
22
Pettersson, Mats. "Modeling Genome Evolution : Creation, Change and Destruction." Doctoral thesis, Uppsala University, Department of Evolution, Genomics and Systematics, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8163.
Full textAbstract:
Historically, evolution has been studied either by looking at morphological traits in living organisms and the fossil record, or by using bioinformatics and comparative genomics. While highly useful for deducing evolutionary history, these approaches are not particularly well suited for studying the mechanisms of evolution. In order to address such issues, other methods are needed. Mathematical modelling is one of the most powerful options available, and it is the approach used in this thesis. By constructing models of biological systems, the work aims to resolve some of the many unresolved questions regarding evolutionary processes, such as how new genes evolve and how selection acts in fragmented populations. Some answers have been reached, and thus the thesis makes a small contribution to our overall understanding of evolution.
The creation of novel genes was studied both directly and by extension of an analogous system, which revolved around reversion of a frameshift mutant. The results pointed to gene amplification as a likely mechanism for both reversion of the frameshift mutant and creation of new genes.
Selection in fragmented populations is shown to be effective even when sub-populations, rather than individuals, are competing against each other. Modeling of a system of bacterial symbionts living in aphids indicates that, although the bacterial population within a single host is small and subject to rampant genetic drift, the bacterial population as a whole is regulated by selection on the host level. Thus, deleterious mutations do no accumulate and the population maintains its fitness over time.
23
Forgetta, Vincenzo. "Systematic search for Salmonella-susceptibility quantitative trait loci in the chicken using a whole genome scan approach." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=33758.
Full textAbstract:
The goal of this project is to identify QTL involved in Salmonella -susceptibility in the chicken. Salmonellosis is one of the most common causes of food poisoning in humans and is frequently caused by the ingestion of Salmonella-contaminated poultry products. Identification of QTL responsible for Salmonella-susceptibility may lead to more efficient control strategies, and selection against genes that may lead to increased risk of transmission. The genome scan was performed on a selection of 425 backcross progeny, (W1 x C) F1 x C, derived from C Salmonella-susceptible and W1 Salmonella-resistant chickens. The 425 backcross birds were phenotyped for their susceptibility to infection with Salmonella Typhimurium. A collection of 160 fluorolabelled microsatellite markers (FAM, TET, and HEX) was used to scan the chicken genome, which is 3800cM in size (1.2 x 109 bp) and consists of 39 linkage groups, 9 chromosomes and 30 microchromosomes. We first tested the available microsatellite markers for informativeness in C and W1 chicken lines. In addition, we also determined the allele lengths and PCR product intensity (qualitatively) to facilitate pooling of markers during electrophoresis. PCR reaction mixtures were prepared using a Packard MultiProbe II Robotic System and Minitrak to pipette DNA-PCR Master mix and primer mix into microtiter plates. PCR products were then pooled and analyzed on Perkin-Elmer ABI Prism 3700 DNA Analyzers. Analysis of informative microsatellite markers on the backcross panel resulted in the detection of two loci, one on Chromosome 7 carrying NRAMP1, and the other on microchromosome E41W17 carrying TLR4, linked to resistance to Salmonella infection in chickens.
24
Varsally, Wazeer Mohammad. "Inferring biological networks from genome-wide transcriptional and fitness data." Thesis, University of Birmingham, 2014. http://etheses.bham.ac.uk//id/eprint/5120/.
Full textAbstract:
In the last 15 years, the increased use of high throughput biology techniques such as genome-wide gene expression profiling, fitness profiling and protein interactomics has led to the generation of an extraordinary amount of data. The abundance of such diverse data has proven to be an essential foundation for understanding the complexities of molecular mechanisms and underlying pathways within a biological system. This thesis demonstrates the capabilities and applications of using biological networks to extrapolate biological information from the wealth of data available in the yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. This study marks the first time a mutual information based network inference approach has been applied to a set of specific genome-wide expression and fitness compendia. In particular, this work has generated hypotheses in S. pombe that have led to a deeper understanding of the relationship between ribosomal proteins and energy metabolism, a recently discovered pathway termed riboneogenesis. Experimental validation of this hypothesis has led to new theories on the role of energy metabolism enzymes in controlling ribosome biogenesis in S. pombe, including the novel finding that fructose-1, 6-bisphosphatase (FBP1) may have roles in both gluconeogenesis and riboneogenesis. This thesis also demonstrates how the use of multi-level data allows for comprehensive insight into nuclear functions of the S. pombe nonsense-mediated mRNA decay protein, UPF1. This study provides substantial evidence demonstrating the role of UPF1 in DNA replication. The applicability of fitness data in identifying targets of metal and metalloid toxicity in S. cerevisiae has also been investigated.
25
Söderberg, Jonas. "Surviving the ratchet : Modelling deleterious mutations in asexual populations." Doctoral thesis, Uppsala universitet, Molekylär evolution, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-157897.
Full textAbstract:
One of the most unforgiving processes in nature is that of Muller's ratchet, a seemingly irreversible accumulation of deleterious mutations that all organisms have to deal with or face extinction. The most obvious way to avoid fitness collapse is recombination, though asexual populations usually do not have the luxury of recombining freely. With the aid of computational and mathematical models, we have studied other situations where this threat is averted and the organism can survive the ratchet. The results show that a ratchet where all mutations have the same deleterious fitness effect is very effectively stalled for large effects. However, if mutations are allowed to have a broad range of effects, the fitness-loss rate can be substantial even with the same mean effect as the one-type ratchet, but we have identified parameter regions where even the broad-range effects are effectively stopped. The fitness-loss from a ratchet is very sensitive to the mutation rate and a mutation that increases the mutation rate (mutator) can easily start an otherwise stalled ratchet. Large effect mutators are heavily counter-selected, but smaller mutators can spread in the population. They can be stopped by reversals (antimutators), but even if the mutation rate is equilibrated in this way, there will be large fluctuations in mutation rate and even larger in the fitness-loss rate due to the feedback amplification in their coupling. Another way of preventing the ratchet is by reversal of the deleterious mutations themselves through back-mutations or compensatory mutations. The rate required to stop the ratchet using only back-mutations before the fitness collapses is very large. A detailed comparison between the deleterious mutations in the ratchet and in a sexual population was made and the difference was found to be greatest for large populations with large genomes. There are obviously many ways to survive the ratchet, but even more ways to drive a species to extinction by enhancing and speeding up the ratchet. By modelling and testing the ratchet for numerous different situations, we show the effects of some of these threats and benefits.
26
Gourlay, Elaine Margaret. "Strategies for human genome modification using engineered nucleases and transcription factors." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/5875/.
Full textAbstract:
VEZF1 is a highly conserved vertebrate transcription factor that is essential for mammalian development. The gene regulatory functions of VEZF1 are largely undetermined. The generation of human cells depleted or absent of VEZF1 would greatly assist the study of VEZF1 functions and mechanism of action. This study makes use of synthetic biology technologies to either repress or knock out VEZF1 gene transcription to enable further studies of VEZF1 function. This study explores various strategies to use engineered DNA-binding proteins to direct the repression or mutation of a gene of interest. Zinc Finger (ZF) and Transcription Activator Like Effector (TALE) proteins that specifically recognise DNA sequences at the VEZF1 gene promoter were constructed using modular or Golden Gate assembly methods. The ability of TALE fusion proteins to function in human cells was studied. An expression vector system was created to assemble TALE Repressor (TALER) fusion proteins. The use of TALERs allowed for the rapid assessment of TALE protein binding at their chromosomal targets in human cells. Transient expression of most of the assembled TALE repressor proteins resulted in reduced VEZF1 transcription. A subset resulted in very substantial VEZF1 repression, making them useful tools for the study of VEZF1 function. Functional TALE domains were assembled into TALE nuclease (TALEN) fusion proteins. TALEN expression vectors were developed to assemble TALEN proteins with optimised expression, cleavage activity and target specificity. Transient expression of TALEN proteins in human cells was used to direct the cleavage and error-prone DNA repair of the VEZF1 promoter. Following development of the assays used to detect TALEN-directed mutations, several functional TALEN pairs were identified. Some TALENs resulted in over 65% mutation rates, with some mutations removing the VEZF1 promoter. These TALENs will be useful for the development of VEZF1 knock out cell lines. Interestingly, our study reveals a correlation between TALE length and the activity of TALERs and TALENs that should be considered in the future application of TALE proteins.
27
Cong, Le. "Genome Engineering Technology and Its Application in Mammalian Cells." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11309.
Full textAbstract:
The advancement of high-throughput, large-scale biochemical, biophysical, and genetic technologies has enabled the generation of massive amounts of biological data and allowed us to synthesize various types of biomaterial for engineering purposes. This enabled improved observational methodologies for us to navigate and locate, with unprecedented resolution, the potential factors and connections that may contribute to biological and biomedical processes. Nonetheless, it leaves us with the increasing demand to validate these observations to elucidate the actual causal mechanisms in biology and medicine. Due to the lack of powerful and precise tools to manipulate biological systems in mammalian cells, these efforts have not been able to progress at an adequate pace.
28
Seshadri, Chitra. "Genome wide epigenetic analyses of Araptus attenuatus, a bark beetle." VCU Scholars Compass, 2016. http://scholarscompass.vcu.edu/etd/4167.
Full textAbstract:
Phylogeographic studies have relied on surveying neutral genetic variation in natural populations as a way of gaining better insights into the evolutionary processes shaping present day population demography. Recent emphasis on understanding putative adaptive variation have brought to light the role of epigenetic variation in influencing phenotypes and the mechanisms underlying local adaptation. While much is known about how methylation acts at specific loci to influence known phenotypes, there is little information on the spatial genetic structure of genome-wide patterns of methylation and the extent to which it can extend our understanding of both neutral and putatively adaptive processes. This research examines spatial genetic structure using paired nucleotide and methylation genetic markers in the Sonoran bark beetle, Araptus attenuatus, for which we have a considerable knowledge about its neutral demographic history, demography, and factors influencing ongoing genetic connectivity. Using the msAFLP approach, we attained 703 genetic markers. Of those, 297 were polymorphic in both nucleotide (SEQ) and methylation (METH) were assayed from 20 populations collected throughout the species range. Of the paired SEQ and METH locis, the METH were both more frequent (16% vs. 7%), maintained more diversity (Shannon IMeth = 0.361 vs. ISeq=0.272), and had more among-population genetic structure (ΦST; Meth = 0.035 vs. ΦST; Seq= 0.008) than their paired SEQ loci. Interpopulation genetic distance in both SEQ and METH markers were highly correlated, with 16% of the METH loci having sufficient signal to reconstruct phylogeographic history. Allele frequency variation at five loci (two SEQ and three METH) showed significant relationships with at-site bioclimatic variables suggesting the need for subsequent analysis addressing non-neutral evolution. These results suggest that methylation can be as informative as nucleotide variation when examining spatial genetic structure for phylogeography, connectivity, and, identifying putatively adaptive genetic variance.
29
Syed, Salahuddin. "Nonreplicative DNA Helicases Involved in Maintaining Genome Stability." Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6408.
Full textAbstract:
Double-strand breaks and stalled forks arise when the replication machinery encounters damage from exogenous sources like DNA damaging agents or ionizing radiation, and require specific DNA helicases to resolve these structures. Sgs1 of Saccharomyces cerevisiae is a member of the RecQ family of DNA helicases and has a role in DNA repair and recombination. The RecQ family includes human genes BLM, WRN, RECQL4, RECQL1, and RECQL5. Mutations in BLM, WRN, and RECQL4 result in genetic disorders characterized by developmental abnormalities and a predisposition to cancer. All RecQ helicases have common features including a helicase domain, an RQC domain, and a HRDC domain. In order to elucidate the role of these domains and to identify additional regions in Sgs1 that are required for the maintenance of genome integrity, a series of systematic truncations to the C terminus of Sgs1 were created. We found that ablating the HRDC domain does not cause an increase in accumulating gross chromosomal rearrangements (GCRs). But deleting the RQC domain and leaving the helicase domain intact resulted in a rate similar to that of a helicase-defective mutant. Additionally, we exposed these truncation mutants to HU and MMS and demonstrated that losing up to 200 amino acids from the C terminus did not increase sensitivity to HU or MMS, whereas losing 300 amino acids or more led to sensitivity similar to that of an sgs1∆ cell. These results suggest that the RQC domain, believed to mediate protein-protein interactions and required for DNA recognition, is important for Sgs1’s role in suppressing GCRs and sensitivity to HU and MMS, whereas the HRDC domain that is important for DNA binding is not necessary. RecQL5 is a RecQ-like helicase that is distinct from the other members through its three different isoforms, RecQL5α, RecQL5β, and RecQL5ɣ. It has a helicase domain and an RQC domain, but lacks the HRDC domain that other RecQ-like helicases possess. In contrast to Blm, Wrn, and RecQL4, no human disorder has been associated with defects in RecQL5. For this reason the role of RecQL5 in the cell has remained largely unknown. To try to elucidate the pathways RecQL5 may be involved in we performed a yeast two hybrid to identify RecQL5-interacting proteins. We found that RecQL5 interacts with Hlp2, an ATP-dependent RNA helicase, and Ube2I, a SUMO-conjugating enzyme. These novel interactions shed light on a potential role of RecQL5 in the cell as a transcriptional regulator.
Saccharomyces cerevisiae, Rrm3, is a 5’-3’ DNA helicase that is part of the Pif1 family of DNA helicases and is conserved from yeast to humans. It was initially discovered as a suppressor of recombination between tandem arrays and ribosomal DNA (rDNA) repeats. In its absence there are increased rates of extra-chromosomal rDNA circles, and cells accumulate X-shaped intermediates at stalled forks. Rrm3 may be involved in displacing DNA-protein blocks and unwinding DNA to facilitate fork progression. We used stable isotope labeling by amino acids in cell culture (SILAC)- based quantitative mass spectrometry in order to determine proteins that deal with the stalled fork in the absence of Rrm3. We found that in the absence of Rrm3 and increased replication fork pausing, there is a requirement for the error-free DNA damage bypass factor Rad5 and the homologous recombination factor Rdh54 for fork recovery. We also report a novel role for Rrm3 in controlling DNA synthesis upon exposure to replication stress and that this requirement is due to interaction with Orc5, a subunit of the origin recognition complex. Interaction of Orc5 was found to be located within a 26-residue region in the unstructured N-terminal tail of Rrm3 and loss of this interaction resulted in lethality with cells devoid of the replication checkpoint mediator Mrc1, and DNA damage sensitivity with cells lacking Tof1. In this study we describe two independent roles of Rrm3, a helicase-dependent role that requires Rad5 and Rdh54 for fork recovery, and a helicase-independent role that requires Orc5 interaction to control DNA synthesis.
Our data provides novel insight into the role of DNA helicases and their role in protecting the genome. Through yeast genetics it was possible to determine the importance of the C terminus of Sgs1 and elucidate new RecQL5 interacting partners that shed light onto roles for RecQL5 distinct from other RecQ like helicases. Quantitative mass spectrometry allowed us to take on a more global view of the cell and determine how it responds to replication fork pausing in the absence of Rrm3. Using both proteomics and yeast genetics we were able to better understand how these DNA helicases contribute to maintaining genome stability.
30
Robinson, Jason M. "Functional Significance of mtDNA Cytosine Modification Tested by Genome Editing." VCU Scholars Compass, 2016. http://scholarscompass.vcu.edu/etd/4561.
Full textAbstract:
The field of epigenetics is gaining popularity and speed, due in part to its capability to answer lingering questions about the root cause of certain diseases. Epigenetics plays a crucial role in regulation of the cell and cell survival, particularly by cytosine methylation. It remains controversial if DNMT’s which facilitate methylation are present in mammalian mitochondria and what the functional significance they may have on modification of mitochondrial DNA. CRISPR-Cas9 technology enabled genome editing to remove the MTS (mitochondrial targeting sequence) from DNMT1 of HCT116 cells, purposefully minimizing effects on nuclear cytosine methylation, while exclusively impacting mitochondrial modification. Removal of the DNMT1 MTS did not completely prevent the localization of this enzyme to the mitochondria according to immunoblot analysis. As well, deletion of the MTS in DNMT1 revealed only a small decline in transcription; not until removal of DNMT3B did we see a two-fold decrease in transcription from mitochondrial protein coding genes. No significant decline in transcription occurred when a DNMT3B knockout also lost the MTS of DNMT1; this study is evidencing that DNMT3B is possibly the more significant methyltransferase in the mitochondria. Our aim from this study and future research is to clearly characterize which enzymes in the mitochondria are controlling cytosine modifications and to understand the mechanistic complexities that accompany cause and consequence of epigenetic modifications.
31
Soleimani, Vahab D. "Genome dynamics in barley (Hordeum vulgare L) cultivars: Molecular diversity, evolution, and DNA fingerprinting." Thesis, University of Ottawa (Canada), 2005. http://hdl.handle.net/10393/29264.
Full textAbstract:
In the absence of whole genome sequencing, molecular markers are indispensable tools for the study of genome evolution, genetic diversity measurements, and genotype identification. We have used sequence-specific amplified polymorphism (S-SAP) markers that were derived from the BARE-1, an active retrotransposon of barley, to measure the contribution of this element to the evolution of barley genome among 103 cultivars that are commonly grown in Canada and the United States. The results were compared to the genome diversity measures obtained by the single nucleotide polymorphisms (SNP).
The barley populations were divided into groups based on various agronomic traits such as end use, i.e., feed versus malting, and seed morphology, i.e., naked versus covered kernel. Analysis of the genetic structure in the population using analysis of molecular variance (AMOVA) for both S-SAP and SNP attributed the largest co-variance component (90%) to the genetic diversity among cultivars within groups. Co-variance component between groups was about 6% which indicated that there was no justification for population differentiation along the set based upon agronomic traits. Genetic relationships among cultivars was assessed by cluster analysis with UPGMA and found to vary substantially between S-SAP and SNP datasets.
Quantitative analysis of BARE-1 retrotransposon with real-time PCR in a small group of cultivars showed significant differences in the copy number of the element among cultivars. Most of the BARE-1 elements were in the form of solo LTRs, indicating a high rate of homologous recombination between retrotransposon copies in the genome. Differences of up to 3000 BARE-1 copies per haploid genome were found among cultivars that have been developed and registered within the past three decades. Informative SNPs such as those with high polymorphic information content (PIC) values were used to generate identification keys to distinguish barley cultivars which were otherwise indistinguishable at the morphological and biochemical levels.
32
Ramdath, Ramona Sherry. "Aneuploidy: Using genetic instability to preserve a haploid genome?" University of Toledo Health Science Campus / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=mco1243954510.
Full text
33
Altheide, Tasha Kay. "Comparative population genetics of the Hominoidea: An investigation of locus-specific and genome-wide influences." Diss., The University of Arizona, 2002. http://hdl.handle.net/10150/279941.
Full textAbstract:
One of the central questions in population genetics is how different forces of evolution influence patterns of genetic variation within and between species. Locus-specific forces of natural selection, like selective sweeps and background selection, are expected to influence only the target of selection and any sites linked to that target. In contrast, genome-wide forces are expected to influence mane different loci simultaneously. In particular, genome-wide forces that have a sex-specific component are predicted to exert disparate influence over the four different genomic compartment, (mitochondrial DNA, non-recombining portion of the Y chromosome (NRY), X chromosome, autosomes) depending on their degree of maternal or paternal inheritance. This study compared levels of genetic variation across the four genomic compartments in the Hominoidea (humans, chimpanzees, bonobos, gorillas, and orangutans) in order to test the hypothesis that a high variance in male reproductive success, as predicted from behavioral data, may influence levels of hominoid genetic diversity. First, comparative chromosome and mitochondria) DNA diversity was quantified across species. NRY diversity was lower than mitochondria) DNA diversity in all taxa, and gorillas exhibited no NRY genetic diversity, consistent with their polygynous mating system. In order to determine whether locus-specific or genome-wide factors were responsible, diversity from all four genomic compartments was compared across species. The multilocus approach revealed patterns of variation across all hominoids that are consistent with a model of high variance in male reproductive success: relatively high mitochondrial DNA diversity, relatively low Y chromosome diversity, and intermediate X and autosomal diversity. Examination of orangutan population structure for NRY, mitochondrial DNA, and X-linked loci suggested an influence of sex-specific demographic influence, but was unclear whether other factors were involved. The study suggests that there is an influence of a high variance in male reproductive success on the hominoid genome, but does not preclude the influence of additional locus-specific factors. There are potentially important implications for population genetic models that assume a sex ratio of 1. Additional loci and additional species need to be examined to determine the generality of the pattern, as well as to further investigate the interplay between genome-wide and locus-specific factors.
34
Grandhi, Sneha. "HIGH-THROUGHPUT ANALYSIS OF THE HUMAN MITOCHONDRIAL GENOME REVEALS ITS DYNAMICS, FUNCTION, AND SIGNALS OF SELECTION IN CANCER." Case Western Reserve University School of Graduate Studies / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1525450447304917.
Full text
35
Mosberg, Joshua Adam Weintrob. "Studying and Improving Lambda Red Recombination for Genome Engineering in Escherichia coli." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10777.
Full textAbstract:
The phage-derived Lambda Red recombination system utilizes exogenous DNA in order to generate precise insertion, deletion, and point mutations in Escherichia coli and other bacteria. Due to its convenience, it is a frequently-used tool in genetics and molecular biology, as well as in larger-scale genome engineering projects. However, limited recombination frequency constrains the usefulness of Lambda Red for several important applications. In this work, I utilize a mechanism-guided approach in order to improve the power and utility of Lambda Red recombination.
36
Cook, Kristen. "Regulation of Genome-Wide Transcriptional Stress Responses in Saccharomyces cerevisiae." Thesis, Harvard University, 2011. http://dissertations.umi.com/gsas.harvard:10032.
Full textAbstract:
In response to osmotic shock in Saccharomyces cerevisiae the MAP kinase Hog1 coordinates a large-scale transcriptional stress response, rapidly producing hundreds of copies of specified transcripts. Many of the most highly induced genes are bound and regulated by a transcription factor, Sko1, but lack the canonical binding site for this factor. We use ChIP-seq to demonstrate a stress-specific binding mode of Sko1. In stress, Sko1 binds to promoters in close proximity to Hog1, and another Hog1-regulated transcription factor, Hot1. This mode of Sko1 binding requires the physical presence of Hog1, but not Hog1 phosphorylation of Sko1. We identify candidate Sko1 and Hot1 binding motifs that predict co-localization of Sko1, Hot1, and Hog1 at promoters. We then demonstrate a role for Sko1 and Hot1 in directing Hog1-associated RNA Pol II to target genes, where Hog1 is present with the elongating polymerase. We suggest a possible model for Hog1 reprogramming of transcription in the early stages of the osmotic stress response. We then determine the extent and structure of the Hog1 controlled transcriptional program in a related stress, damage to the cell wall. We find that Sko1 and Hot1 have different apparent thresholds for activation by Hog1. In addition, in cell wall damage, Hog1 regulates an additional transcription factor, Rlm1, that is not involved in other Hog1 regulated stress responses. This factor is activated by the coincidence of a signal from Hog1 with that of another MAP kinase, Slt2.
37
Wang, Xinchen Ph D. Massachusetts Institute of Technology. "Deciphering genetic associations using genome-wide epigenomics approaches." Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/111239.
Full textAbstract:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 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.
Genetic mapping of the drivers of complex human phenotypes and disease through the genome-wide association study (GWAS) has identified thousands of causal genetic loci in the human population. However, genetic mapping approaches can often only reveal a particular causal locus, not the molecular mechanism through which it acts. Biological interpretation of these genetic results is thus a bottleneck for turning results from GWAS into meaningful biological insights for human biology. Genetic mapping of complex human traits has revealed that most common variants influencing human phenotypes have weak effect sizes and reside outside protein-coding regions, complicating biological interpretation of their function. In this thesis we use computational and experimental approaches to study the non-coding genome. In particular, we focus on using epigenomic signatures to characterize non-coding transcriptional regulatory elements and predict regulatory segments of DNA disrupted by genetic variants. In Chapter 2, we describe how genome-wide maps of epigenomic modifications can be used to characterize and discover new GWAS loci. In Chapter 3, we outline an experimental method for the high-throughput assessment of putative transcriptional regulatory elements. In summary, our research highlights the value of interpreting human genetics information through an epigenomic lens, and provides a glimpse into the possible biological insights that manifest from the intersection of these two areas of research.
by Xinchen Wang.
Ph. D.
38
Liu, Yang. "Data mining methods for single nucleotide polymorphisms analysis in computational biology." HKBU Institutional Repository, 2011. http://repository.hkbu.edu.hk/etd_ra/1287.
Full text
39
Mignogna, Kristin. "Genome-Wide Systems Genetics of Alcohol Consumption and Dependence." VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/5946.
Full textAbstract:
Widely effective treatment for alcohol use disorder is not yet available, because the exact biological mechanisms that underlie this disorder are not completely understood. One way to gain a better understanding of these mechanisms is to examine the genetic frameworks that contribute to the risk for developing this disorder. This dissertation examines genetic association data in combination with gene expression networks in the brain to identify functional groups of genes associated with alcohol consumption and dependence.
The first study took advantage of the behavioral complexity of human samples, and experimental capabilities provided by mouse models, by co-analyzing gene expression networks in the mesolimbocortical system of acute alcohol-treated mice and human genetic alcohol dependence association data. This study successfully identified ethanol-responsive gene expression networks with overrepresentation of genes suggestively associated with alcohol dependence in an independent human sample, indicating that gene expression networks in mouse models are informative for identifying mechanistic networks relevant to the risk for developing dependence.
The second study aimed to identify quantitative trait loci for voluntary alcohol drinking behaviors under an intermittent ethanol access paradigm, in the genetically complex Diversity Outbred mice. After determining high heritability for alcohol consumption and dependence amongst the progenitor strains, we identified several specific genetic loci associated with these traits. One locus replicated results from a human association study of alcohol consumption, and provided insight to the potentially contributing genes. Finally, we identified alcohol consumption-correlated gene expression networks in the prefrontal cortex of these mice. We also mapped quantitative trait loci for network expression levels, some of which overlapped with the behavioral loci, indicating that the functions represented by these modules mediate the relationship between the genotypes in that region and drinking behaviors. Overall, our studies revealed neuroplastic and ubiquitin-related genes pathways involved in alcohol consumption in mice and humans, and that likely contribute to the risk for developing dependence.
40
Widmayer, Heather. "Biogeographical patterns associated with genome-wide genetic differentiation in a widespread species of South American Blepharoneura fruit flies (Tephritidae)." Thesis, University of Iowa, 2018. https://ir.uiowa.edu/etd/6337.
Full textAbstract:
Studying how new insect species originate can help us better understand the evolutionary processes that lead to the rich insect biodiversity found in the tropics. New insect species arise via the evolution of reproductive isolation, a result of the accumulation of reproductive isolating barriers. Reproductive isolating barriers can arise in the context of geographical isolation and/or can be influenced by differences in ecology. Complex evolutionary mechanisms may underlie the origins of the more than 40 species of neotropical fruit flies in the genus Blepharoneura (Diptera: Tephritidae). The immature stages of these flies are specialist feeders on plants in the Cucurbitaceae (cucurbit) family in Central and South America. Previous research focusing on six geographically widespread Blepharoneura species used 18 microsatellite loci to identify patterns of geographical and ecological divergence. While conclusions from microsatellite data are valid, these markers provide only a limited signal of genetic structure. Fine-scale, genome-wide data can reveal patterns of genetic differentiation that may help us discover and date historical and recent lineage divergence. Here I use double-digest restriction-associated DNA (ddRAD) sequencing to explore the hypothesis that geographic and ecologic barriers are restricting gene flow in one species of Blepharoneura, B. sp10. First, I use two methods to detect population structure within B. sp10 individuals. I find evidence for three distinct genetic groups characterized by geography and one genetic group defined by a difference in host plant use. Then I investigate these relationships using Mantel tests and pairwise spatial-genetic plots, which reveal distinct biogeographical genetic patterns. Finally, I infer the evolutionary history of B. sp10 using approximate Bayesian computation and find that the timing of divergence between flies collected from the Amazon Basin and those collected from the Guiana Shield coincides with the late Pleistocene, between 24,412 and 83,000 years ago. Connecting these results to historical climate data from the Late Pleistocene may help explain evolutionary processes that contributed to the patterns of genetic diversity that we see in Blepharoneura. This research provides a framework for investigating the biogeographic and evolutionary history of the more than 50 species in the Blephaorneura genus, a step towards understanding the complexity of insect diversity in the tropics.
41
Crisci, Jessica L. "On Identifying Signatures of Positive Selection in Human Populations: A Dissertation." eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/664.
Full textAbstract:
As sequencing technology continues to produce better quality genomes at decreasing costs, there has been a recent surge in the variety of data that we are now able to analyze. This is particularly true with regards to our understanding of the human genome—where the last decade has seen data advances in primate epigenomics, ancient hominid genomics, and a proliferation of human polymorphism data from multiple populations. In order to utilize such data however, it has become critical to develop increasingly sophisticated tools spanning both bioinformatics and statistical inference. In population genetics particularly, new statistical approaches for analyzing population data are constantly being developed—unfortunately, often without proper model testing and evaluation of type-I and type-II error. Because the common Wright-Fisher assumptions underlying such models are generally violated in natural populations, this statistical testing is critical. Thus, my dissertation has two distinct but related themes: 1) evaluating methods of statistical inference in population genetics, and 2) utilizing these methods to analyze the evolutionary history of humans and our closest relatives. The resulting collection of work has not only provided important biological insights (including some of the first strong evidence of selection on human-specific epigenetic modifications (Shulha, Crisci, Reshetov, Tushir et al. 2012, PLoS Bio), and a characterization of human-specific genetic changes distinguishing modern humans from Neanderthals (Crisci et al. 2011, GBE)), but also important insights in to the performance of population genetic methodologies which will motivate the future development of improved approaches for statistical inference (Crisci et al, in review).
42
Crisci, Jessica L. "On Identifying Signatures of Positive Selection in Human Populations: A Dissertation." eScholarship@UMMS, 2006. http://escholarship.umassmed.edu/gsbs_diss/664.
Full textAbstract:
As sequencing technology continues to produce better quality genomes at decreasing costs, there has been a recent surge in the variety of data that we are now able to analyze. This is particularly true with regards to our understanding of the human genome—where the last decade has seen data advances in primate epigenomics, ancient hominid genomics, and a proliferation of human polymorphism data from multiple populations. In order to utilize such data however, it has become critical to develop increasingly sophisticated tools spanning both bioinformatics and statistical inference. In population genetics particularly, new statistical approaches for analyzing population data are constantly being developed—unfortunately, often without proper model testing and evaluation of type-I and type-II error. Because the common Wright-Fisher assumptions underlying such models are generally violated in natural populations, this statistical testing is critical. Thus, my dissertation has two distinct but related themes: 1) evaluating methods of statistical inference in population genetics, and 2) utilizing these methods to analyze the evolutionary history of humans and our closest relatives. The resulting collection of work has not only provided important biological insights (including some of the first strong evidence of selection on human-specific epigenetic modifications (Shulha, Crisci, Reshetov, Tushir et al. 2012, PLoS Bio), and a characterization of human-specific genetic changes distinguishing modern humans from Neanderthals (Crisci et al. 2011, GBE)), but also important insights in to the performance of population genetic methodologies which will motivate the future development of improved approaches for statistical inference (Crisci et al, in review).
43
Ojha, Sohita. "Identifying Genetic Factors Influencing Sperm Mobility Phenotype in Chicken using Genome Wide Association Studies, Primordial Germ Cell Transplantation, and RNAseq." Thesis, University of Arkansas, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10638065.
Full textAbstract:
Sperm mobility is a major determinant of male fertility in chicken. In spite of low heritability of reproductive traits, sperm mobility has high heritability index which suggests presence of quantitative trait loci (QTLs) governing the trait. Our research focused on three objectives: i) to identify the QTLs affecting low mobility phenotype in chicken, ii) to understand the impact of Sertoli-cells and germ cells interactions in influencing the mobility phenotype and iii) to identify the genes and gene networks differentially expressed in male and female PGCs. To detect the QTLs, genome wide association studies (GWAS) was conducted which revealed the presence of multiple minor alleles influencing the trait and indicated the role of epistasis. The second section of research involved isolation, culture and transfer of primordial germ cells (PGCs) to create high line germ line chimera chicken carrying low line PGCs. We established the culture of chicken PGCs isolated from the embryonic blood in a feeder free culture conditions but could not detect the presence of low line genotype in the semen of transgenic males. Our final study involved RNA-sequencing (RNAseq) of male and female PGCs to identify differentially expressed genes from their transcriptomes. We identified five candidate genes: 3-hydroxy-3-methylglutaryl CoA reductase (HMGCA), germ cell-less (GCL), SWIM (zinc finger SWIM domain containing transcription factor), SLC1A1 (solute carrier family 1 member 1), UBE2R2L (ubiquitin conjugating enzyme) and validated their expression level in male and female PGCs by RT-qPCR. GCL was exclusively expressed in males while SLC1A1 & UBE2R2L were expressed only in female cPGCs. This present study provides novel gender specific germ cell markers in the broiler chicken. These results will help in elucidating the genetic programming of gender specific germ line development in broilers.
44
Kapoustina, Oxana. "The identification of genes regulated by Interferon Regulatory Factors (IRF) 1 and 8 in the context of pathogen challenge by ChIP on Chip and genome wide transcription profiling approaches." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=86565.
Full textAbstract:
Interferon regulatory factors (IRFs) is a family of transcriptional regulators that are essential for cell differentiation and growth, oncogenesis and the regulation of immunity. IRF1 and IRF8 play a crucial role in immunity by regulating the differentiation of the cells of myeloid lineage, particularly the development of immune cells including macrophages, NK and dendritic cells. Both transcription factors are essential for the resistance to viral and bacterial infections. We optimized the chromatin immunoprecipitation (ChIP) technique to be used with IRF8 and IRF1 antibodies in a J774 line stimulated with IFNγ/CpG. Using ChIP coupled to an exon promoter 244K microarray (ChIP on Chip) we identified 201 and 303 binding sites of high confidence for IRF1 and IRF8 respectively. Previously known IRF1 and IRF8 transcription targets (OAS1b for IRF1 and IFNb for IRF8) as well as novel transcriptional control loci were identified. One of the major gene ontology (GO) functional categories in the two gene lists was "immune response". The overlap of IRF1 and IRF8 transcription target genes from ChIP on Chip revealed an overlap of 19 genes most of which belonged to the "immune regulation" GO pathway and included targets of high confidence: Gbp6, Mx2, Tnfsf13b, H2-T24, and Ifit1. A parallel microarray transcription profiling study on mouse bone marrow macrophages (BMDMs) from an F2 generation of Balb/C IRF8-wildtype and BXH2 IRF8-mutant cross was performed. The BMDMs possessing the wildtype and the mutant IRF8 alleles were infected with Legionella pneumophila or stimulated with IFNγ/CpG to identify genes regulated by IRF8 in the context of infection. 1171 and 852 differentially regulated genes were differentially regulated in the infection and stimulation experiments respectively. Genes that displayed interaction between IRF8 allele and the infection conditions were isolated to yield high confidence gene lists of 31 and 129 genes for the Legionella and IFNγ/CpG expeLes facteurs régulateurs interférons (IRFs) sont une famille de régulateurs transcriptionnels essentiels à la différenciation et la croissance cellulaire, à l'oncogénèse et au bon maintien du système immunitaire. IRF1 et IRF8 jouent un role clé dans l'immunité de l'organisme par la régulation de la différenciation des lignées cellulaires myéloïdes, nottament le développement de cellules immunitaires comme les macrophages, les cellules tueuses naturelles (NK) et les cellules dendritiques. Ces deux facteurs de transcription sont indispensables pour la résistance aux infections virales et bactériennes. Nous avons optimisé une technique d'immunoprécipitation de la chromatine (ChIP) afin d'y utiliser des anticorps ciblant IRF1 et IRF8, et ce à partir d'une lignée cellulaire J774 suivant un stimulation par interféron gamma et par oligonucléotides CpG (IFNγ/CpG). En combinant cette technique ChIP à un microarray 244K comprenant exons et promoteurs (ChIP on Chip), nous avons identifiés respectivement 201 et 303 sites d'ancrages pour chacun de IRF1 et IRF8. Certaines cibles transcriptionnelles déjà connues pour IRF1 (OAS1b) et IRF8 (IFNb) ainsi que de nouveaux loci de control transcriptionel ont été identifiés. Parmi les catégories par fonction relevées par Ontologie de gène (GO), une des catégories majeures des deux listes de gènes générées fut pour les gènes reliés à la "réponse immunitaire''. La comparaison entre les gènes cibles de transcription pour IRF1 et IRF8 par "ChIP on Chip'' a révélé 19 gènes représentés dans les deux cas. La majeure partie de ces gènes sont reliés à la "réponse immunitaire'' par GO et ils incluent les cibles suivantes avec détection très significative: Gbp6, Mx2, Tnfsf13b, H2-T24, et Ifit1. En parallèle, des macrophages dérivés de la moëlle osseuse de souris (BMDMs) furent utilisés pour l'étude du profile transcriptionel par microarray d'une génération F2 générée avec des so
45
Martin, Kyle J. "Genomic characterization of cyclostome Dlx gene family members: Insight into the evolution of the chordate genome and body plan from the organizational and transcriptional regulatory properties of Dlx genes in the petromyzontiformes (lamprey) and the hyperotreti (Hagfish)." Thesis, University of Ottawa (Canada), 2009. http://hdl.handle.net/10393/28234.
Full textAbstract:
Gnathostome novelties include jaws, paired appendages, and true teeth. Dlx genes encode transcription factors indispensable for embryonic development of these novelties. Gnathostomes possess at least 6 Dlx genes organized in 3 bi-gene clusters, a physical arrangement which is proposed to affect their expression though shared enhancer elements. I studied the Dlx genes of Cyclostomes, the evolutionary sister group of Gnathostomes. I identified 4 novel members of the Dlx gene family in hagfish (Eptatretus stoutii), and confirmed the presence of 6 Dlx genes in lamprey ( Petromyzon marinus). I found that Cyclostomes have only 1 gene cluster and several orphan genes. This lack of conserved arrangement is coincident with an absence of conserved Dlx enhancers. Irregardless, some regulatory conservation is still apparent as lamprey non-coding DNA is able to drive dlx specific expression patterns of reporter genes in zebrafish. Therefore Cyclostome and Gnathostome Dlx are both organized and regulated differently. The causes and consequences of these changes in Chordate evolution are discussed.
46
Lyle, Suzanne McLean. "Error Correcting Codes and the Human Genome." Digital Commons @ East Tennessee State University, 2010. https://dc.etsu.edu/etd/1689.
Full textAbstract:
In this work, we study error correcting codes and generalize the concepts with a view toward a novel application in the study of DNA sequences. The author investigates the possibility that an error correcting linear code could be included in the human genome through application and research. The author finds that while it is an accepted hypothesis that it is reasonable that some kind of error correcting code is used in DNA, no one has actually been able to identify one. The author uses the application to illustrate how the subject of coding theory can provide a teaching enrichment activity for undergraduate mathematics.
47
Kim, Kieun Mohan Chilukuri K. "Parallel hierarchical adaptive genetic algorithm for genome sequencing." Related Electronic Resource: Current Research at SU : database of SU dissertations, recent titles available full text, 2003. http://wwwlib.umi.com/cr/syr/main.
Full text
48
Kohrn, Brendan F. "An Efficient Pipeline for Assaying Whole-Genome Plastid Variation for Population Genetics and Phylogeography." PDXScholar, 2017. https://pdxscholar.library.pdx.edu/open_access_etds/4007.
Full textAbstract:
Tracking seed dispersal using traditional, direct measurement approaches is difficult and generally underestimates dispersal distances. Variation in chloroplast haplotypes (cpDNA) offers a way to trace past seed dispersal and to make inferences about factors contributing to present patterns of dispersal. Although cpDNA generally has low levels of intraspecific variation, this can be overcome by assaying the whole chloroplast genome. Whole-genome sequencing is more expensive, but resources can be conserved by pooling samples. Unfortunately, haplotype associations among SNPs are lost in pooled samples and treating SNP frequencies as independent estimates of variation provides biased estimates of genetic distance. I have developed an application, CallHap, that uses a least-squares algorithm to evaluate the fit between observed and predicted SNP frequencies from pooled samples based on network topology, thus enabling pooling for chloroplast sequencing for large-scale studies of chloroplast genomic variation. This method was tested using artificially-constructed test networks and pools, and pooled samples of Lasthenia californica (California goldfields) from Whetstone Prairie, in Southern Oregon, USA. In test networks, CallHap reliably recovered network topologies and haplotype frequencies. Overall, the CallHap pipeline allows for the efficient use of resources for estimation of genetic distance for studies using non-recombining, whole-genome haplotypes, such as intra-specific variation in chloroplast, mitochondrial, bacterial, or viral DNA.
49
Li, Xiang. "STRESS-INDUCED GENETIC CHANGE IN FLAX REVEALS GENOME VARIATION MECHANISM." Case Western Reserve University School of Graduate Studies / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=case1565964370435691.
Full text
50
Burrows, Anna. "Genome-Wide Loss-of-Function Genetic Screens Identify Novel Senescence Genes and Putative Tumor Suppressors." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10191.
Full textAbstract:
During every cell cycle and upon exogenous stress, tumor suppression programs are engaged to ensure genomic stability. In response to replicative aging and oncogenic stimuli, the p53 and Rb pathways are activated to prevent the proliferation of damaged cells. Several lines of evidence suggest that escape from senescence is a crucial early step in oncogenic progression. A major challenge in the cancer field is to combine genomic information regarding cancer-associated genetic changes with high-throughput functional studies, in order to confirm genetic requirements and pinpoint biological roles of these perturbed genes in oncogenesis. Furthermore, a complete genetic understanding of replicative senescence, and how it might be bypassed, is lacking. We describe here two genome scale loss-of-function genetic screens that interrogate these tumor suppressor programs. We utilized a unique sensitization approach to isolate senescence pathways and unmask compensatory mechanisms that may have been difficult to identify in previous studies. These genetic screens have generated comprehensive and validated datasets of putative senescence and p53 pathway genes. We present this dataset as a high-quality resource for further investigation into these biological pathways. We have uncovered several genes in distinct biological pathways which have not been demonstrated to have a functional role in senescence, and which may be putative tumor suppressors. We have identified BRD7 and BAF180, two SWI/SNF components, as critical regulators of p53. BRD7 and BAF180 are required for p53 activity and p21 expression during replicative and oncogene-induced senescence, and evidence suggests that they are inactivated in human cancer. In addition, we have uncovered a role for the deubiquitinating enzyme USP28 in the regulation of p53 accumulation during senescence, such that loss of USP28 results in bypass of the senescence program. We have also investigated several other novel senescence genes including SEMA6A, SEMA3b, and TMEM154. We have found that the expression of these genes is highly regulated during senescence by distinct means, including both p53-dependent and p53-independent mechanisms. These results demonstrate the efficacy of our sensitized screening approach, and also highlight the emerging view that the senescence program requires the combined action of multiple biological pathways for its execution.