Browsing by Author "Kelleher, Erin S."
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Item A Study of Cis-Regulatory Sequences Of Wnt1 in the Disease Osteogenesis Imperfecta(2021-05) Dalibalta, Maria SaadWnt1 is known to be mutated in the inherited cases of the disease Osteogenesis Imperfecta, a bone genetic disorder also known as the brittle bone disease. The canonical Wnt signaling pathway has a known role in bone homeostasis and development and studies have shown that wnt1, an evolutionarily conserved member of the Wnt signaling pathway, is particularly important for the Wnt canonical pathway in osteoblast differentiation. This suggests a potential role of wnt1 in bone homeostasis. Understanding the regulation of wnt1 expression by cis-regulatory elements during development is important to understand the role it plays in bone development and homeostasis. This project uses ATAC seq (Assay for Transposase-Accessible Chromatin) datasets available to the public to study regions within and surrounding the wnt1 gene to find open chromatin regions, as this is associated with enhancer activity. The enhancers that are shown to be active are studied closely using the ENCODE project to obtain information on the expression profiles of the enhancers and the tissue in which they may be active. The activity of the enhancers was compared in tissues where wnt1-signaling is known to be important: hindbrain tissue, adipose tissue, and bone marrow macrophage and two enhancers with the highest activity were identified. By knowing these cis-regulatory sequences of wnt1, now it could be easier to possibly use wnt1 expression as a therapeutic target in the disease Osteogenesis Imperfecta.Item An Analysis of Errors and Discrepenices in Analyzing Single Cell RNA Sequence Data(2021-05) Modi, Paulomi SanjaykumarSingle-cell RNA sequencing (scRNA-seq) is an extremely vital sequencing technology that has enabled High-throughput mapping of cellular differentiation hierarchies. scRNA-seq has excellent sequencing potential with a wide range of applications beyond regular transcriptome profiling. scRNA-seq process involves analyzing data using 3' end counting technology, which involves sample composition and analytical processing including pre-processing, normalization, alignment and clustering. In order to accomplish this task bioinformaticians around the world have developed many computational tools. As of 2019, there exist 385 different tools that can be used to analyze scRNA-seq data, and that number is growing. Although this continuous addition of new features to single-cell data analysis confronts technical gaps with bulk RNA-seq, there have been very few attempts to standardize these practices. This study explores the various approaches to re-analyze previously published single cell RNA-cell sequencing data and discusses subsequent challenges to utilize publicly available data sets to conduct a multicenter study. Considering the differences in data publication formats, there are several methods that can be employed. 1) Analyzing BCL files 2) Analyzing FASTQ files 3) Analyzing matrix files 4) Analyzing Seurat or ScanPy objects. This thesis provides a concise overview of some of the steps, algorithms, and approaches that are currently used in the analysis of single-cell RNA-sequencing data, with an emphasis on recent developments. Hence, I propose that in order to develop reproducible algorithms and analysis software for scRNA-seq data sets, it is vital that standardization across all analysis platform exist and the software developers recognize and understand the computational challenges posed by the analysis tasks.Item Assessing The Role of Juvenile Hormone in The Blood Brain Barrier For Male Courtship in Drosophila melanogaster(2022-12-05) Reeve, Joseph Lyle; Dauwalder, Brigitte; Dierick, Herman A.; Kelleher, Erin S.; Wiernasz, DianeJuvenile hormone (JH) is one of the major insect hormones involved in many physiological processes with roles in development as well as reproduction, metamorphosis, caste determination, behavior, diapause, and polyphenisms. Previous research has shown that JH is required at normal physiological levels for proper male courtship. However, the target cells responding to JH levels modulating courtship behavior were unknown. It is expected that the JH target cells contain the JH receptor MET and/or GCE along with proteins that might interact with the JH-Met complex. RNAseq studies of isolated blood brain barrier (BBB) cells have shown that both proteins are present in the BBB. This study addresses the question of whether JH is required in the BBB for proper male courtship and whether the intracellular JH receptors Met and gce are needed in the BBB for male courtship. The first question was addressed by conditionally reducing the levels of JH in the BBB of mature males through the action of the JH degrading enzyme juvenile hormone esterase and assaying their courtship behavior. The second question was addressed by reducing the levels of Met or gce in the BBB of adult males by interfering RNA followed by assaying the courtship behavior of virgin males. Our results show that JH is required in the BBB for proper male courtship, a finding which was reinforced by rescue with the JH analog, Methoprene. We further found that Met is required in the BBB for male courtship, although gce is not. These findings suggest an important role for JH signaling through the JH receptor Met in the BBB for male courtship.Item Bruno and P-element Transposition: Positive regulator or cellular responder?(2020-09-29) Saiz, Lorissa V.Transposable elements are harmful genetic entities that can confer serious negative effects on host organisms. We are studying possible natural means of tolerance to these elements, specifically P-elements, through 3 assays.Item Drug Discovery: LXR Inverse Agonists in Pancreatic Cancer(2022-08-18) Widmann, Scott; Lin, Chin-Yo; Feng, Qin; Filgueira, Carly S.; Kelleher, Erin S.Pancreatic ductal adenocarcinoma (PDAC) is a disease for which therapeutic interventions are scarce. Currently, the effective treatments available are chemotherapies that offer minimal improvements, especially in later stages of the disease. Novel therapeutic modalities are desperately needed in the treatment of PDAC. The liver X receptor (LXR) is a ligand-dependent transcription factor that regulates the expression of genes involved in lipid metabolism, inflammation, and immune responses. Synthetic LXR agonists have been shown to be effective in reducing growth of various cancers, but their clinical use has been stymied by side-effects. Inverse agonists are a newly discovered class of LXR ligands that have been shown to possess antiproliferative effects in cancer cells and may offer a way to circumvent the side-effects of LXR agonists. A recent screening of a small molecule LXR ligand library in PDAC cells led to the discovery of two inverse agonists: GAC0001E5 (1E5) and GAC0003A4 (3A4). A multi-omic analysis, including RNA-sequencing (RNA-seq) and metabolite mass spectrometry (metabolomics) was conducted following treatments with both inverse agonists and the agonist GW3965 (GW) to ascertain the mechanisms involved in their antiproliferative effects in PDAC cells. RNA-seq results show that PDAC cells harboring mutations in the KRAS oncogene are much more responsive at the transcriptional level to LXR inverse agonists than an agonist. Lipid metabolism was extensively repressed by the inverse agonists, including cholesterol, fatty acid, and phospholipid synthesis. Metabolomics results showed perturbations in several metabolic pathways, amino acid metabolism, cholesterol metabolism, and phospholipid metabolism. Integration of transcriptomic and metabolomic data indicated disruptions in glycolysis, the TCA cycle, and fatty acid metabolism occurred at both the gene and metabolite levels. Mechanistic investigations of 3A4 revealed disruptions in cholesterol and ceramide homeostasis were involved in its antiproliferative effects. Additionally, a dual-death phenotype comprised of apoptosis and necroptosis was found to be induced by 3A4. A computational approach was taken to explore the ligand-receptor interactions of inverse agonists and the LXR ligand-binding domain. Inverse agonists were shown to cause distinct changes in receptor conformation that could be used for the design of inverse agonists with increased specificity and potency.Item Evolution of P-Element Copy Number in Drosophila melanogaster(2018-10-18) Muhammad, ShabirTransposable elements comprise a significant portion of the genomes of both plants and animals. The parasitic nature of transposable elements can modify genome structure, gene expression, and genome stability. Transposable elements are responsible for their negative impact due to their inherent tendency to insert themselves into the genome of the host organism and replicate itself throughout the genome. An example of a transposable element invasion is the invasion of the P-element transposon in D. melanogaster flies around 1950. D. melanogaster responded to the invasion via small RNAs known as piwi interacting RNA (piRNA). The piRNA pathway uses piRNAs that are transcribed in the germline in response to P-element copies located on piRNA clusters. The piRNA pathway evolved in D. melanogaster is a great model to study host tolerance and repression of transposable elements. Because the piRNA silencing pathways depends on P-element insertions into piRNA clusters, there should be a relationship between P-element copy number and evolution of the piRNA silencing pathway. As P-element copy number increases across successive generations, the probability of an insertion into a piRNA cluster should increase, which will lead to more production of piRNA and a more evolved piRNA silencing pathway. This relationship can be tested by examining the genome of experimental flies from successive generations for P-element copy numbers using relative standard qPCR.Item Evolution of P-element Repression in Drosophila Melanogaster through the Piwi-Interacting RNA Pathway(2019-08) Zhang, Shuo 1989-; Kelleher, Erin S.; Azevedo, Ricardo B. R.; Stewart, Alexander J.; Casola, ClaudioTransposable elements (TEs) are ubiquitous and selfish genetic entities whose mobilization poses a significant threat to their host. In the germline of metazoan, the Piwi-interacting RNAs (piRNAs) derived from TE-enriched loci (called piRNA clusters) regulate TE activity in a sequence specific manner. However, the emergence and dynamics of piRNA-mediated repressor alleles to an invading TE remain elusive. P-element, a DNA transposon that recently invaded the D. melanogaster genome around 1950, provides a unique opportunity to study the evolution of host repression. In this dissertation, I first adapted a targeted sequencing strategy and developed a computational pipeline to annotate P-element insertions in a sequenced Drosophila melanogaster genome. My approach precisely determined P-element insertion breakpoints and found new P-element insertions, which were undetected by previously methods. Next, I modified the pipeline to annotate P-element insertions in the Drosophila melanogaster genetic reference panel (DGRP), a panel composed of 205 fully sequenced inbred lines. I found over 90% of DGRP genomes have P-elements in ancestral piRNA clusters that are active prior to the P-element invasion. This indicates de novo mutation, in which P-elements transpose into pre-existing piRNA clusters, is the predominant mechanism for the origin of repressor alleles. Moreover, I detected no fewer than 84 independent P-element insertions in ancestral piRNA clusters. Finally, I observed that P-element insertions in piRNA clusters segregate at significant higher frequency than P-elements outside of piRNA clusters, suggesting that cluster P-elements confer a selective advantage. Taken together, my results revealed a striking example of polygenic adaptation, in which a plethora of de novo beneficial P-element insertions into multiple piRNA clusters, fueled the evolution of a ubiquitous repressive phenotype in <60 years.Item Evolution of piRNA-mediated TE Repression in Drosophila(2020-12) Wang, Luyang; Kelleher, Erin S.; Dauwalder, Brigitte; Meisel, Richard P.; Myles, KevinIn metazoan germlines, the piRNA pathway acts as a genomic immune system, employing small RNA-mediated silencing to defend host DNA from the harmful effects of transposable elements (TEs). Expression of genomic TEs is proposed to initiate self regulation by increasing the production of repressive piRNAs, thereby “adapting” piRNA-mediated control to the most active TE families. However, the mutational and epigenetic processes that give rise to piRNA-mediated silencing when new TEs invade the host also remain poorly understood. In addition, piRNA pathway proteins, which execute piRNA biogenesis and enforce silencing of targeted sequences, evolve rapidly and adaptively in animals. If TE silencing is ensured through piRNA biogenesis, what necessitates changes in piRNA pathway proteins? In this dissertation, we first explored forces that underlie the adaptive evolution of piRNA pathway proteins by performing interspecific complementation and examined functional differences between Drosophila melanogaster and D. simulans alleles of three adaptively evolving piRNA pathway proteins: Armitage, Aubergine and Spindle-E. We discovered that D. simulans alleles of aub and armi exhibit enhanced off-target effects on host transcripts in a D. melanogaster background, as well as modest reductions in the efficiency of piRNA biogenesis, suggesting that promiscuous binding of D. simulans Aub and Armi proteins to host transcripts reduces their participation in piRNA production. Avoidance of genomic auto-immunity may therefore be a critical target of selection. P-elements have invaded the genomes of three Drosophila species within the last century, providing unique opportunities to study the evolution of piRNA mediated repression. In the second part of this dissertation, we introduced P-elements into a naive D. melanogaster strain through germline transformation, and chronicled their effects on the evolution of host TE repression in 10 laboratory populations at two different temperatures. We found that populations which evolved repression are associated with accumulation of active P-elements within their genomes. Furthermore, our observation that repression correlates with P-element insertions into multiple different piRNA clusters strongly supports the “trap” model that acquisition of TE repression is through TE insertions into piRNA clusters, and indicates that the evolution of repression occurs via simultaneous emergence of numerous different repressor alleles.Item Genetic Variation in Host Tolerance of an Invading Transposon in Drosophila melanogaster(2017-10-12) Ahern, Elizabeth; Akoma, Uche; Chen, Vivian; Hubbard, Donald; Jaweria Jaweria; Mathur, Sushreyta; Ortega, Lily; Tasnim, Sadia; Tang, Wenpei; Srivastav, SatyamTransposable Elements (TEs) are both important drivers of genome evolution and genetic parasites with potentially dramatic consequences for host fitness, including insertional inactivation of functional sequences and genomic instability (Reviewed in Hedges and Deininger 2007). Although host genomes minimize these deleterious eff¬ects by regulating the activity of resident TEs, they are often invaded by new TE families, which they are unable to control (Reviewed in Wallau et al. 2012). We are taking advantage of the historical invasion of the Drosophila melanogaster genome by P-elements (Reviewed in Engels 2003), a family of autonomous DNA transposons, to understand the impact that invading TEs have on their hosts, and the mechanisms through which TE regulation evolves. Using a panel of recombinant inbred lines (RILs), which were generated from naïve genotypes isolated from nature before P-element invasion (King et al. 2012), we have identified genetic variants that allow for tolerance or control of P-element activity. Such variants could have been beneficial after P-elements invaded D. melanogaster genomes ~1950, thereby contributing to the evolution of tolerance of the transposable element by its host.Item Investigation of the Effects of Unusual Genome Architecture on the Evolution of the Ciliate Tetrahymena Using Computational Modeling(2020-05) Zhang, Hao; Azevedo, Ricardo B. R.; Zufall, Rebecca A.; Kelleher, Erin S.; Stewart, Alexander J.; Wilke, Claus O.Tetraymena is a model organism in molecular biology and has a very unusual genome architecture, including nuclear dimorphism, amitotic division of the somatic nucleus during asexual reproduction, capability to control the copy number of chromosomes during amitosis and the ability to have facultative sex when a germline nucleus is present. Recent studies also revealed that Tetrahymena has very special evolutionary characteristics, such as the prevalence of successful asexuality within the genus and the extremely low mutation rate found in the germline nucleus. How Tetrahymena evolves such unusual characteristics is still unclear, and whether and how the unusual genome architecture contributes to the unusual evolutionary characteristics remain largely untested or uninvestigated. In this study, I investigated the effect of these unusual genome features on the evolution of Tetrahymena using computational modeling, particularly on their contributions to their successful asexuality and the extremely low mutation rate. I found that compared to mitosis, amitosis of somatic nucleus, together with copy number control, can both decelerate the operation of Muller’s ratchet to a sexual-comparable extent and speed up the adaptation to changing environments. I also found that although not expressed during asexual generations, the mutations arising in the germline nucleus can also respond to selection acting on the somatic nucleus following sexual reproduction, which rejects the previous explanation for the low germline nucleus mutation rate in Tetrahymena. Instead, my results showed that the reproduction strategies adopted by Tetrahymena, including amitosis and facultative sex, can naturally promote the reduction of mutation rate under selection. This study highlighted the contribution of unusual genome architecture to the achievement of unusual evolutionary characteristics found in Tetrahymena, which both lead to a better understanding of the evolution of this organism and elucidate new mechanisms for eukaryotes to survive asexually and promote mutation rate reduction.Item Reduced insulin signaling promotes germline stem cell maintenance under P-element hybrid dysgenesis(2019) Silva, Efren; Griffiths, TerionTransposable elements (TEs) are selfish mobile DNA segments that proliferate throughout the host genome by replicating in gamete nuclei, thus allowing for vertical transmission to progeny. While often suppressed by the host, the consequences of unchecked transposition are severe, inducing deleterious mutations and disrupting the function of gametes. In the case of the P-element, a DNA transposon of Drosophila melanogaster, unregulated transposition results in atrophied ovaries that contain no germline cells. DNA damage from the P-element is hypothesized to impair germline stem cells (GSCs) in Drosophila ovaries, resulting in their permanent loss. We sought to uncover the mechanisms that promote GSC loss in the presence of P-element activity by manipulating the differentiation signals that a GSC receives. In particular, decreasing differentiation signals may suppress atrophy by stabilizing GSCs in the niche. To test this hypothesis we selected 16 genes involved GSC differentiation, and reduced their expression in dysgenic germlines using GAL4-UAS mediated RNAi. Knock-down of two genes, raptor and chico, resulted in germline rescue in dysgenic ovaries. Chico and Raptor are components of the insulin signaling pathway, which promotes the G2/M transition in GSCs. Our observations therefore suggest that G2 arrest may be critical for the recovery of GSCs from P-element induced DNA-damage.Item Satellite Repeats Are Associated with Host Tolerance of an Active TE(2017) Lama, Jyoti; Kelleher, Erin S.Item The Role of the Drosophila Dopamine 2-Like Receptor in the Blood-Brain Barrier for Male Courtship Behavior(2020-05) Love, Cameron Roger; Dauwalder, Brigitte; Kelleher, Erin S.; Roman, Gregg; Lekven, Arne C.; Bond, Richard A.Courtship in Drosophila Melanogaster is an extensively studied and well characterized complex innate behavior with known molecular determinants. We have previously shown that the G-protein Go-alpha and sex-specific signaling in the Blood-Brain Barrier (BBB) influence male innate courtship sustainment towards naïve females. Here we show that the glial cells that form the BBB modulate male courtship behavior through the dopamine receptor D2R. The subperineurial glia cells (SPG) form the BBB by providing a contiguous barrier, connected by septate junctions, at the interface between the hemolymph and the brain. While the neural circuits required to produce scripted actions in the fly, such as the steps for courtship, have been widely investigated, much less is known about how the circuitry and functions underlying the fly behavior is influenced by cell- non-autonomous molecular processes. We have previously shown that male-specific molecules in the BBB regulate male courtship. We identified the Dopamine-2 like receptor (D2R) RNA as one of a number of sex-specifically enriched BBB transcripts. D2R knockdown with RNAi or over-expression of D2R with a transgene in the SPG of adult males significantly reduces courtship. Knockdown or overexpression of D2R with ubiquitous neuronal drivers or SPG knockdown specifically during development has no effect on the courtship index. D2R mutant flies have courtship defects that can be rescued by expression of wildtype D2R in the BBB of adult males. D2R likely signals through Go and beta-arrestin, to maintain full male courtship levels. Our results indicate an expanded role for dopamine signaling in the glial cells that surround the brain and provide a critical time frame for its action in behavior.Item Understanding Host Tolerance to Transposable Elements(2021-12) Lama, Jyoti; Kelleher, Erin S.; Dauwalder, Brigitte; Bawa-Khalfe, Tasneem; Murphy, William J.Transposable elements (TE) are mobile genetic parasites, whose unregulated activity in germline causes DNA damage, and results in sterility. Host genomes can avoid these fitness costs of TEs either by regulating TE proliferation, or by altering gametogenesis to tolerate TE-induced DNA damage. Although TE regulation through piRNAs is studied extensively, little is known about mechanisms of gametogenic tolerance to TE activity. To study tolerance I take advantage of a unique phenomenon called hybrid dysgenesis in Drosophila melanogaster where naïve females devoid of P-element DNA transposon typically produce sterile offspring when mated with P-element carrying males. However, tolerant individuals are capable of producing viable gametes in spite of transposition. By performing Quantitative trait loci (QTL) mapping in a panel of highly recombinant inbred lines two genomic regions associated with natural tolerance to P-element transposition were isolated. Transcriptome analysis of multiple tolerant and sensitive genotypes shows evidence suggesting variation in the double-stranded break (DSB) repair efficiency. Tolerant genotypes show increased expression of components of the Tat interactive protein 60-kDa (TIP60) complex involved in DSB repair and also exhibited increased chorion gene expression- an indicator of enhanced DSB repair. By integrating the data from QTL mapping, gene expression as well as in-phase SNP analysis, I identified two strong candidate genes that could influence tolerance: brat and Nipped-A, a member of the TIP60 complex. Loss-of-function mutation in brat was found to promote hybrid dysgenesis by increasing ovarian atrophy in the dysgenic females. Furthermore, tolerant genotypes displayed high resilience to X-ray mediated DNA damage. These results reveal gametogenic regulation and enhanced DSB repair as two potential mechanisms of germline tolerance to TEs.