Browsing by Subject "Transcriptomics"
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Item Open Access A Next-Generation Approach to Systematics in the Classic Reticulate Polypodium vulgare Species Complex (Polypodiaceae)(2014) Sigel, Erin MackeyThe Polypodium vulgare complex (Polypodiaceae) comprises a well-studied group of fern taxa whose members are cryptically differentiated morphologically and have generated a confusing and highly reticulate species cluster. Once considered a single species spanning much of northern Eurasia and North America, P. vulgare has been segregated into approximately 17 diploid and polyploid taxa as a result of cytotaxonomic work, hybridization experiments, and isozyme studies conducted during the 20th century. Despite considerable effort, however, the evolutionary relationships among the diploid members of the P. vulgare complex remain poorly resolved, and several taxa, particularly allopolyploids and their diploid progenitors, remain challenging to delineate morphologically due to a dearth of stable diagnostic characters. Furthermore, compared to many well-studied angiosperm reticulate complexes, relatively little is known about the number of independently-derived lineages, distribution, and evolutionary significance of the allopolyploid species that have formed recurrently. This dissertation is an attempt to advance systematic knowledge of the Polypodium vulgare complex and establish it as a "model" system for investigating the evolutionary consequences of allopolyploidy in ferns.
Chapter I presents a diploids-only phylogeny of the P. vulgare complex and related species to test previous hypotheses concerning relationships within Polypodium sensu stricto. Analyses of sequence data from four plastid loci (atpA, rbcL, matK, and trnG-trnR) recovered a monophyletic P. vulgare complex comprising four well-supported clades. The P. vulgare complex is resolved as sister to the Neotropical P. plesiosorum group and these, in turn, are sister to the Asian endemic Pleurosoriopsis makinoi. Divergence time analyses incorporating previously derived age constraints and fossil data provide support for an early Miocene origin for the P. vulgare complex and a late Miocene-Pliocene origin for the four major diploid lineages of the complex, with the majority of extant diploid species diversifying from the late Miocene through the Pleistocene. Finally, node age estimates are used to reassess previous hypotheses, and to propose new hypotheses, about the historical events that shaped the diversity and current geographic distribution of the diploid species of the P. vulgare complex.
Chapter II addresses reported discrepancies regarding the occurrence of Polypodium calirhiza in Mexico. The original paper describing this taxon cited collections from Mexico, but the species was omitted from the recent Pteridophytes of Mexico. Originally treated as a tetraploid cytotype of P. californicum, P. calirhiza now is hypothesized to have arisen through hybridization between P. glycyrrhiza and P. californicum. The allotetraploid can be difficult to distinguish from either of its putative parents, but especially so from P. californicum. These analyses show that a combination of spore length and abaxial rachis scale morphology consistently distinguishes P. calirhiza from P. californicum and confirm that both species occur in Mexico. Although occasionally found growing together in the United States, the two species are strongly allopatric in Mexico, where P. californicum is restricted to coastal regions of the Baja California peninsula and neighboring Pacific islands and P. calirhiza grows at high elevations in central and southern Mexico. The occurrence of P. calirhiza in Oaxaca, Mexico, marks the southernmost extent of the P. vulgare complex in the Western Hemisphere.
Chapter III examines a case of reciprocal allopolyploid origins in the fern Polypodium hesperium and presents it as a natural model system for investigating the evolutionary potential of duplicated genomes. In allopolyploids, reciprocal crosses between the same progenitor species can yield lineages with different uniparentally inherited plastid genomes. While likely common, there are few well-documented examples of such reciprocal origins. Using a combination of uniparentally inherited plastid and biparentally inherited nuclear sequence data, we investigated the distributions and relative ages of reciprocally formed lineages in Polypodium hesperium, an allotetraploid fern that is broadly distributed in western North America. The reciprocally-derived plastid haplotypes of Polypodium hesperium are allopatric, with populations north and south of 42˚ N latitude having different plastid genomes. Biogeographic information and previously estimated ages for the diversification of its diploid progenitors, lends support for middle to late Pleistocene origins of P. hesperium. Several features of Polypodium hesperium make it a particularly promising system for investigating the evolutionary consequences of allopolyploidy. These include reciprocally derived lineages with disjunct geographic distributions, recent time of origin, and extant diploid progenitor lineages.
This dissertation concludes by demonstrating the utility of the allotetraploid Polypodium hesperium for understanding how ferns utilize the genetic diversity imparted by allopolyploidy and recurrent origins. Chapter IV details the use of high-throughput sequencing technologies to generate a reference transcriptome for Polypodium, a genus without preexisting genomic resources, and compare patterns of total and homoeolog-specific gene expression in leaf tissue of reciprocally formed lineages of P. hesperium. Genome-wide expression patterns of total gene expression and homoeolog expression ratios are strikingly similar between the lineages--total gene expression levels mirror those of the diploid progenitor P. amorphum and homoeologs derived from P. amorphum are preferentially expressed. The unprecedented levels of unbalanced expression level dominance and unbalanced homoeolog expression bias found in P. hesperium supports the hypothesis that these phenomena are pervasive consequences of allopolyploidy in plants.
Item Open Access Decoding the olfactory map: targeted transcriptomics link olfactory receptors to glomeruli(2022) Zhu, Kevin WilliamThe external world is perceived via sensory receptors arranged in highly organized systems according to functional strategies, which in turn reflect features of critical importance to both the sense and the animal. The receptor organization and functional strategies of visual, auditory, and touch sensory systems have been mapped, but such a map for olfaction, the sense of smell, has remained elusive despite a concrete understanding of the fundamental principles of the system’s architecture.Sensory processing in olfactory systems is organized across olfactory bulb glomeruli, wherein axons of peripheral sensory neurons expressing the same olfactory receptor co-terminate to transmit receptor-specific activity to central neurons. Understanding how receptors map to glomeruli is therefore critical to understanding olfaction. High-throughput spatial transcriptomics is a rapidly advancing field, but low-abundance olfactory receptor expression within glomeruli has previously precluded high-throughput mapping of receptors to glomeruli. In Chapter 2, I describe the development and application of a novel method which combines sequential sectioning along the anteroposterior, dorsoventral, and mediolateral axes with target capture enrichment sequencing to overcome low-abundance target expression. This strategy spatially mapped 86% of olfactory receptors across the olfactory bulb and uncovered a relationship between olfactory receptor sequence and glomerular position.
Item Open Access Epigenetic and transcriptional responses in circulating leukocytes are associated with future decompensation during SARS-CoV-2 infection.(iScience, 2024-01) McClain, Micah T; Zhbannikov, Ilya; Satterwhite, Lisa L; Henao, Ricardo; Giroux, Nicholas S; Ding, Shengli; Burke, Thomas W; Tsalik, Ephraim L; Nix, Christina; Balcazar, Jorge Prado; Petzold, Elizabeth A; Shen, Xiling; Woods, Christopher WTo elucidate host response elements that define impending decompensation during SARS-CoV-2 infection, we enrolled subjects hospitalized with COVID-19 who were matched for disease severity and comorbidities at the time of admission. We performed combined single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) on peripheral blood mononuclear cells (PBMCs) at admission and compared subjects who improved from their moderate disease with those who later clinically decompensated and required invasive mechanical ventilation or died. Chromatin accessibility and transcriptomic immune profiles were markedly altered between the two groups, with strong signals in CD4+ T cells, inflammatory T cells, dendritic cells, and NK cells. Multiomic signature scores at admission were tightly associated with future clinical deterioration (auROC 1.0). Epigenetic and transcriptional changes in PBMCs reveal early, broad immune dysregulation before typical clinical signs of decompensation are apparent and thus may act as biomarkers to predict future severity in COVID-19.Item Open Access High Resolution Transcriptional Profiling and Characterization of Cellular Inclusions in Arabidopsis thaliana Roots Grown in Low Sulfur Conditions(2013) Jackson, Terry LynellEnvironmental stress affects plant development and productivity. Sulfur deficiency is a key nutrient deficiency that adversely affects crop yield. The model plant Arabidopsis thaliana has played an informative role in deciphering the mechanisms involved in sulfur assimilation, as well as, the response to limited conditions. Using Arabidopsis thaliana as a model to investigate gene expression in the root, microarray data sets have been generated. These data sets consist of whole root sections for 6 time points across 72 hours, and enriched populations of 5 radial cell-types and 4 sections of 3 developmental zones of the root at 3 hrs on sulfur limited conditions. With these data it was determined which cellular tissues and developmental zones were affected most by sulfur limited conditions. Furthermore, a novel phenotype was characterized that occurs in roots after growth on low sulfur conditions. Cellular inclusions build up within the cytoplasm of mature cortical root cells. These inclusions have been termed "sulfur pox" and their composition remains to be determined.
Item Open Access Integrative Computational Genomics Defines the Molecular Origins and Outcomes of Lymphoma(2016) Moffitt, Andrea BarrettLymphomas are a heterogeneous group of hematological malignancies composed of diseases with diverse molecular origins and clinical outcomes. Derived from immune cells of lymphoid origin, lymphoma can arise from lymphoid cells present anywhere in the body, from the spleen and lymph nodes to peripheral sites like the liver and intestines. Current strategies for lymphoma diagnosis involve primarily histopathological examinations of the tumor biopsy, including cytogenetics and immunophenotyping. As more data becomes available, diagnoses may increasingly depend on genomic features that define each disease. Classification of lymphoid neoplasms is generally based on the cell of origin, or the lineage of the normal cell that the cancer is thought to arise from. Lymphomas can be classified into dozens of distinct diagnostic entities, though any two patients with the same diagnosis may have very different outcomes and molecular underpinnings, so we need to understand both the commonalities of patients with the same disease and the unique features that may require personalized treatment strategies. Patient prognosis in lymphoma depends greatly on the type of lymphoma, ranging from nearly curable diseases with over 90% five-year survival rates, to most patients dying in the first year in the worse entities. Greater clarity is needed in the role of the underlying genomics that contribute to these variable treatment responses and clinical outcomes.
Next-generation sequencing approaches allow us to delve into the molecular underpinnings of lymphomas, in order to gain insight about the origin and evolution of these diseases. High-throughput sequencing protocols allow us to examine the whole genome, exome, epigenome, or transcriptome of cancer cells in tens to hundreds of patients for each disease. As cost of sequencing is reduced, and the ability to generate more data increases, we face increasing computational challenges to both process and interpret the wealth of data available in cancer genomics. Developing efficient and effective bioinformatics tools is necessary to transform billions of sequencing reads into actionable hypotheses on the role of certain genes or biological pathways in a specific cancer type or patient.
In this dissertation, I present several strategies and applications of integrative computational genomics in lymphoma, with contributions throughout the research process, from development of initial assays and quality control strategies for the sequencing data, to joint analysis of clinical and genomic data, and finally through follow-up experimental models for lymphoma.
First, I focus on two rare T cell lymphomas, hepatosplenic T cell lymphoma (HSTL) and enteropathy associated T cell lymphoma (EATL), which are both diseases with very poor clinical outcomes and a previous dearth of knowledge on the genetic basis of the diseases. We define the somatic mutation landscape of HSTL, through application of exome sequencing and find SETD2 to be the most highly mutated gene. We further utilize the exome sequencing data to investigate copy number alterations and show a significant survival difference between cases with and without certain arm-level copy number alterations. Knockdown of SETD2 in an HSTL cell line, followed by RNA sequencing, demonstrates the role of SETD2 loss in proliferation and cell cycle changes, linking the SETD2 mutations to a potential oncogenic mechanism. Furthermore, we investigate the potentially targetable mutations in the JAK-STAT pathway and demonstrate oncogenic downstream molecular phenotypes and potential druggability of these mutations. In the enteropathy associated T cell lymphoma study, we apply exome and RNA sequencing to a large EATL cohort. Our findings show a significant role for loss of function mutations in chromatin modifiers and JAK-STAT signaling genes. EATL can be separated into two subtypes, Type I and Type II, which we show to have convergent genomic features, in the face of divergent gene expression. RNA sequencing data defines a distinct separation between the two subtypes. Delving further into the role of SETD2 in these T cell lymphomas, we generate a mouse model with a conditional knockout of SETD2 in T cells and demonstrate a role for SETD2 in altering the lineage development of T cells.
To understand more about why certain genetic abnormalities are recurrent in some disease entities and not others, we turn to the cell of origin for clues. We pair two different lymphomas, Burkitt lymphoma and mantle cell lymphoma, with their associated cells of origin, germinal center B cells and naive B cells. These closely related cell types have much in common as B cells, but from studies of their transcriptomes, we know that there are many molecular differences that distinguish the two. In this work, after looking more closely at mantle cell lymphoma genomics, we look at the underlying chromatin markers that define the epigenomes of these B cells. We test the association between chromatin markers and mutation rates of genes between these two cell types and lymphomas, and find that genes with more open chromatin may have a higher mutation rate, when comparing closely related cells and lymphomas. Finally, I present my work on developing an RNA sequencing based strategy for defining the complete transcriptome of diffuse large B cell lymphoma (DLBCL). Gene expression profiling with microarray has shown the existence of two subtypes in DLBCL, activated B cell like (ABC) and germinal center B cell like (GCB). However, the role for non-coding RNAs, alternative splicing, and mutations, in these two subtypes and the larger group is previously not well understood. We develop a strand-specific RNA sequencing strategy that will allow the investigation of the total RNA transcriptome in DLBCL, including microRNAs, lncRNAs, and other important non-coding RNAs. Furthermore, we show that RNA sequencing can be used to distinguish the two subtypes, including through RNA sequencing based mutation calls, as well as through differentially expressed lncRNAs that we define for the first time in DLBCL.
Broadly, this dissertation contributes novel findings in the field of lymphoma genomics, as well as presenting a framework for computational integrative genomics that can guide future studies. The heterogeneity of lymphoma across cases requires us to dive deep into individual diseases, even rare ones, as well as appreciate the similarities and differences across lymphomas. To improve diagnoses, prognoses, and treatment options, we need to understand the molecular origins of lymphoma. Using a range of molecular and computational approaches, we can move closer to true personalized medicine at the genomic level.
Item Open Access Metabolic Pathways of Type 2 Diabetes: Intersection of Genetics, Transcriptomics, and Metabolite Profiling(2008-07-25) Ferrara, Christine ThereseType 2 diabetes is characterized by insufficient insulin secretion to maintain euglycemia in the setting of peripheral insulin resistance. The majority of insulin-resistant diabetics are obese, yet not all insulin-resistant obese individuals develop diabetes. This obesity/diabetes dichotomy suggests that genetic factors play a pivotal role in disease pathogenesis.
Gene mapping has identified genetic quantitative trait loci (QTL) influencing disease-related phenotypes. To uncover molecular pathways leading from genotype to clinical trait, we classify phenotypes in greater depth and identify QTL that influence combinations of physiological traits, mRNA levels, and metabolite abundance. A major challenge then becomes deciphering the causal interrelationships among correlated phenotypes.
In this dissertation, we develop methods for building causal direction into an undirected network by including QTLs for each phenotype. We then apply and validate these methods in an F2 intercross between the diabetes-resistant C57BL/6 leptinob/ob (B6ob/ob) and the diabetes-susceptible BTBR leptinob/ob (BTBRob/ob) mouse strains. We show that genomic analysis can be integrated with liver transcriptional and metabolite profiling data to construct causal networks for specific metabolic processes in liver. This causal network construction led to the discovery of a pathway by which glutamine induces Phosphoenolpyruvate carboxykinase (Pck1) expression.
To investigate glutamine induction of Pck1 in the context of diabetes, we perform mRNA expression analysis and metabolic profiling in liver of the parental strains. We find glutamine is decreased with obesity in both strains; in the diabetes-resistant B6 strain, liver Pck1 expression parallels glutamine abundance, but in the diabetes-susceptible BTBR strain, Pck1 is elevated with obesity. Follow-up in vitro studies indicate that α-ketoglutarate, which is elevated nearly two fold in the livers of BTBR relative to B6 mice in vivo, may mediate the glutamine effect. We hypothesize that hepatic Pck1 is regulated by glutamine abundance in the liver of B6 animals, but in the presence of high α-ketoglutarate, Pck1 becomes uncoupled from glutamine regulation in the livers of diabetes-susceptible BTBR mice.
Our method of causal network construction led to the discovery of glutamine induction of a key hepatic gluconeogenic enzyme, a pathway potentially disrupted in the diabetes-susceptible BTBR mouse. Future studies will include identifying hepatic mediators of the glutamine effect, and applying QTL-directed networks to multiple organs to ultimately define causal relationships between tissues involved in diabetes progression.
Item Open Access Methods for Systematic Exploratory Analysis of Gene Expression Data with Applications to Cancer Genomics(2017) Wagner, FlorianAdvances in technologies for gene expression profiling have resulted in an unprecedented abundance of gene expression data. However, computational methods available for the exploratory analysis of such data are limited in their ability to generate an interpretable overview of biologically relevant similarities and differences among samples. This work first introduces the XL-mHG test, a sensitive and specific hypothesis test for detecting gene set enrichment, and discusses its algorithmic and statistical properties. It further introduces GO-PCA, a method for exploratory analysis of gene expression data using prior knowledge. The XL-mHG test serves as a building block for GO-PCA. The output of GO-PCA consists of functional expression signatures, designed to provide an interpretable representation of biologically meaningful variation in the data. The power and versatility of the method is demonstrated on heterogeneous human and mouse expression data. Finally, applications of the proposed methods to carcinoma and lymphoma expression data aim to demonstrate their clinical relevance. The effective utilization of prior knowledge in the exploratory analysis of gene expression data through carefully designed computational methods is essential for successfully harnessing the power of current and future platforms for gene expression profiling, with the aim of generating clinically relevant insights into complex diseases such as cancer.
Item Open Access Ras Post-transcriptionally Enhances a Pre-malignantly Primed EMT to Promote Invasion.(iScience, 2018-06) Bisogno, Laura S; Friedersdorf, Matthew B; Keene, Jack DEpithelial-to-mesenchymal transition (EMT) is integral to cancer progression, with considerable evidence that EMT has multiple intermediary stages. Understanding the mechanisms of this stepwise activation is of great interest. We recreated a genetically defined model in which primary cells were immortalized, resulting in migratory capacity, and subsequently H-Ras-transformed, causing malignancy and invasion. To determine the mechanisms coordinating stepwise malignancy, we quantified the changes in messenger RNA (mRNA) and protein abundance. During immortalization, we found dramatic changes in mRNA, consistent with EMT, which correlated with protein abundance. Many of these same proteins also changed following Ras transformation, suggesting that pre-malignant cells were primed for malignant conversion. Unexpectedly, changes in protein abundance did not correlate with changes in mRNA following transformation. Importantly, proteins involved in cellular adhesion and cytoskeletal structure decreased during immortalization and decreased further following Ras transformation, whereas their encoding mRNAs only changed during the immortalization step. Thus, Ras induced EMT-associated invasion via post-transcriptional mechanisms in primed pre-malignant cells.Item Open Access The Cellular Determinants of Spinal and Peripheral Pain Processing(2018) Chamessian, AlexanderChronic pain is a major public health issue, affecting over 100 million people in costing over $600 million annually in the United States. The lack of effective therapies for chronic pain have directly contributed to the ongoing epidemic of opioid abuse and addiction. Deeper understanding the pathogenesis of chronic pain is a prerequisite for remedying the status quo. To that end, in this dissertation, I have undertaken two projects that aim to elucidate the key cellular elements of mechanical pain in the periphery and spinal cord.
Mechanical allodynia is a cardinal feature of pathological pain in which innocuous mechanical stimulation such as light touch produces a painful sensation. Recent work has demonstrated the necessity of cutaneous Aβ low-threshold mechanoreceptors (Aβ-LTMRs) for mechanical allodynia-like behaviors in mice, but its remains unclear whether activation of these neurons alone is sufficient to produce pain behaviors in pathological settings. To address this question, in the first part of this dissertation, I generated and characterized a transgenic mouse line that expresses the optogenetic actuator channelrhodopsin-2 (ChR2) conditionally in Vesicular Glutamate Transporter 1 (Vglut1)-expressing sensory neurons(Vglut1-ChR2). I show that the Vglut1-ChR2 comprises a heterogeneous population of Neurofilament 200-positive, large-sized sensory neurons with cutaneous projections that terminate in Merkel Cell-Neurite Complexes, Meissner Corpuscles and Hair Follicles and with spinal projections that terminate in the deep dorsal horn (Lamina IIi-V) and ventral horn in the spinal cord. In naive Vglut1-ChR2 mice, acute transdermal photostimulation of the plantar hindpaw with blue (470nm) light produced paw withdrawal behaviors in an intensity- and frequency-dependent manner that were abolished by selective pharmacological A-fiber blockade. light-evoked nocifensive behaviors such as licking, biting, jumping and vocalization were virtually absent in Vglut1-ChR2, even at the highest stimulation intensity and frequency. Plantar photostimulation of Vglut1-ChR2 mice in a Real-Time Place-Escape/Avoidance (RT-PEA) assay did not produce aversion, in contrast to the strong aversion elicited in mice that conditionally express ChR2 in Nav1.8-positive and Npy2r-positive nociceptors. Surprisingly, in the Spared Nerve Injury model of neuropathic pain, Vglut1-ChR2 mice did not show significant differences in light-evoked withdrawal behaviors or real-time aversion despite hypersensitivity to natural mechanical stimuli. Thus, I conclude that optogenetic activation of Vglut1-ChR2 neurons alone is not sufficient to produce pain-like behaviors in neuropathic mice.
In the second part of this dissertation, I investigated the cellular determinants of mechanical pain processing in the spinal dorsal horn (SDH), which is comprised of distinct neuronal populations that process different somatosensory modalities. Somatostatin (SST)-expressing interneurons in the SDH have been implicated specifically in mediating mechanical pain. Identifying the transcriptomic profile of SST neurons could elucidate the unique genetic features of this population and enable selective analgesic targeting. To that end, I combined the Isolation of Nuclei Tagged in Specific Cell Types (INTACT) method and Fluorescence Activated Nuclei Sorting (FANS) to capture tagged SST nuclei in the SDH of adult male mice. Using RNA-sequencing (RNA-seq), I uncovered more than 13,000 genes. Differential gene expression analysis revealed more than 900 genes with at least 2-fold enrichment. In addition to many known dorsal horn genes, I identified and validated several novel transcripts from pharmacologically tractable functional classes: Carbonic Anhydrase 12 (Car12), Phosphodiesterase 11A (Pde11a), and Protease-Activated Receptor 3 (F2rl2). In situ hybridization of these novel genes showed differential expression patterns in the SDH, demonstrating the presence of transcriptionally distinct subpopulations within the SST population. Overall, my findings provide new insights into the gene repertoire of SST dorsal horn neurons and reveal several novel targets for pharmacological modulation of this pain-mediating population and pathological pain.
Item Open Access The phylogeny of extant starfish (Asteroidea: Echinodermata) including Xyloplax, based on comparative transcriptomics.(Mol Phylogenet Evol, 2017-07-27) Linchangco, Gregorio V; Foltz, David W; Reid, Rob; Williams, John; Nodzak, Conor; Kerr, Alexander M; Miller, Allison K; Hunter, Rebecca; Wilson, Nerida G; Nielsen, William J; Mah, Christopher L; Rouse, Greg W; Wray, Gregory A; Janies, Daniel AMulti-locus phylogenetic studies of echinoderms based on Sanger and RNA-seq technologies and the fossil record have provided evidence for the Asterozoa-Echinozoa hypothesis. This hypothesis posits a sister relationship between asterozoan classes (Asteroidea and Ophiuroidea) and a similar relationship between echinozoan classes (Echinoidea and Holothuroidea). Despite this consensus around Asterozoa-Echinozoa, phylogenetic relationships within the class Asteroidea (sea stars or starfish) have been controversial for over a century. Open questions include relationships within asteroids and the status of the enigmatic taxon Xyloplax. Xyloplax is thought by some to represent a newly discovered sixth class of echinoderms - and by others to be an asteroid. To address these questions, we applied a novel workflow to a large RNA-seq dataset that encompassed a broad taxonomic and genomic sample. This study included 15 species sampled from all extant orders and 13 families, plus four ophiuroid species as an outgroup. To expand the taxonomic coverage, the study also incorporated five previously published transcriptomes and one previously published expressed sequence tags (EST) dataset. We developed and applied methods that used a range of alignment parameters with increasing permissiveness in terms of gap characters present within an alignment. This procedure facilitated the selection of phylogenomic data subsets from large amounts of transcriptome data. The results included 19 nested data subsets that ranged from 37 to 4,281loci. Tree searches on all data subsets reconstructed Xyloplax as a velatid asteroid rather than a new class. This result implies that asteroid morphology remains labile well beyond the establishment of the body plan of the group. In the phylogenetic tree with the highest average asteroid nodal support several monophyletic groups were recovered. In this tree, Forcipulatida and Velatida are monophyletic and form a clade that includes Brisingida as sister to Forcipulatida. Xyloplax is consistently recovered as sister to Pteraster. Paxillosida and Spinulosida are each monophyletic, with Notomyotida as sister to the Paxillosida. Valvatida is recovered as paraphyletic. The results from other data subsets are largely consistent with these results. Our results support the hypothesis that the earliest divergence event among extant asteroids separated Velatida and Forcipulatacea from Valvatacea and Spinulosida.Item Open Access Transcriptomic and Metabolic Heterogeneity During Epstein-Barr Virus Latency Establishment(2019) Messinger, Joshua EdwardEpstein-Barr Virus (EBV) is a ubiquitous gamma-herpesvirus in the human population and highly associated with lymphomas of the immune-suppressed. EBV maintains itself within the human host via temporal regulation of viral gene expression before establishing latency in resting memory B cells. Experimentally, we can model these immune-compromised lymphomas via in vitro infection of B cells isolated from peripheral blood. This infection model is biphasic where the initial phase is characterized by hyper-proliferation and expression of the EBV nuclear antigens (EBNAs) in the absence of the latent membrane proteins (LMPs) called latency IIb before transitioning to the NFkB-dependent latency III lymphoblastoid cell line (LCL) stage characterized by full expression of the LMPs in the presence of the EBNAs. This temporal regulation manipulates host cell proliferation rates and metabolic profiles of infected B lymphocytes. Our laboratory and my studies, in particular, are focused on the latency IIb to latency III transition as these states are often observed in patient biopsies of EBV-associated malignancies.
Latent membrane protein 1 (LMP1) expression defines latency III and has been used in lymphoma samples to identify EBV latency III in vivo. While LMP1 expression is lower in latency IIb, the mechanism by which it is repressed is currently unknown. Additionally, while LMP1 expression can be used to distinguish latency IIb from latency III, LMP1 expression varies widely within latency III and a subset of LMP1lo latency III cells express LMP1 at levels similar to latency IIb making these cells hard to distinguish in vitro and in vivo. Lastly, while EBV manipulates host cell metabolism, the viral and host factors important for metabolic rewiring have not been fully elucidated.
Using RT-qPCR, ChIP-qPCR, RNA-sequencing, Western Blotting, flow cytometry, and bioinformatic approaches we have identified c-Myc as a repressor of LMP1 during latency IIb. We have also identified host mRNA biomarkers capable of distinguishing latency IIb from latency III. This discovery was additionally leveraged to develop a multiplex RNA-FISH platform capable of distinguishing latency IIb from latency III. Lastly, we have identified host and viral proteins necessary to rewire host cell metabolism and sustain EBV-mediated B cell proliferation. This work, taken together, adds new understanding to EBV latency establishment, heterogeneity, and host B cell biology to develop new, more targeted, therapeutics for EBV-associated lymphomas in the future.