Biology Theses and Dissertations
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Item CHARACTERIZING THE ROLES AND MECHANISMS OF CYTONEMES IN ASYMMETRIC SIGNALING AND ORGANIZATIONS IN THE DROSOPHILA MUSCLE PROGENITOR NICHE.(2024) Patel, Akshay Jitendrakumar; Roy, Sougata; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tissue development and homeostasis rely on the ability of embryonic or stem cells to efficiently determine whether to multiply for self-renewal or differentiate to generate a wide range of cell types that constitute an adult body. Stem cells determine these fates in the context of a specialized microenvironment or the niche that they occupy. All stem cell niches characterized to date are known to function using two key processes - adhesive interactions and asymmetric growth factor signaling between the niche and stem cells. While adhesion to the niche maintains niche occupancy and stemness, the loss of niche adhesion and occupancy initiates stem cell differentiation. Moreover, niche cells produce secreted growth factors to support stem cell self-renewal. Despite the ability of secreted growth factors to disperse across tissues over a long range, only the niche-adhering stem cells receive the self-renewal signals. The genetically identical daughter cells that lack adhesion to the niche fail to receive self-renewal signals, even when located within one or two cell diameters away, leading to the activation of their post-mitotic fates. Therefore, understanding how asymmetric signal distribution and adhesive interactions are produced and coordinated within the niche is critical to understanding how stem cells determine their identity and prime differentiation to generate or regenerate tissues. This thesis investigated and characterized a new mechanism of asymmetric signaling and cell organization in the Drosophila Adult Muscle Progenitor (AMP) niche. By employing genetic, cell-biological, and high-resolution microscopy techniques, this work discovered that AMPs extend thin polarized actin-based filopodia, called cytonemes, by orienting toward the wing disc niche. Cytonemes play a dual role. Cytonemes help AMPs to physically adhere to the wing disc niche and also directly receive a self-renewal Fibroblast Growth Factor (FGF) through the cytoneme-niche contact sites. AMP cytonemes localize the FGF-receptor (FGFR), called Heartless (Htl), and selectively adhere to the wing disc areas that express two different Htl ligands, Pyramus and Thisbe, both mammalian FGF8 homologs. Htl on these cytonemes directly receives Pyramus and Thisbe through the cytoneme-niche contact sites. Although FGFs are long-range secreted paracrine signals and Htl is the only receptor shared by Pyramus and Thisbe, these FGFs are received and restricted only to the niche-adhering AMPs due to the contact-dependent cytoneme-mediated asymmetric delivery of the signals. Moreover, despite employing a common FGF signal transduction pathway, Thisbe- and Pyramus-signaling initiates divergence of AMP fates into two distinct muscle-specific lineages. These experiments showed that cytoneme-mediated signal communication forms the basis of asymmetric signaling and organization within the AMP niche. We next asked how AMPs determine the niche-specific polarity and affinity of cytonemes. This research discovered that FGF reception and signaling activation in AMPs are required to activate polarized cytoneme formation orienting toward the wing disc niche. Without FGF signaling, AMPs cytonemes fail to polarize and adhere to the FGF-producing niche, causing them to exit the niche and start to differentiate. Thus, while target-specific asymmetric FGF distribution relies on cytonemes, activation of FGF signaling feedback maintains the polarity and adhesion of the signaling cytonemes toward the FGF-producing niche. A consequence of this interdependent relationship between niche adhesion, polarized FGF-reception, and stimulation of FGF signaling feedback is the maintenance of the self-organized niche-specific asymmetric signaling and organization via cytonemes. We next investigated whether the niche-adhering cytonemes receive additional fate-specifying cues, particularly the mechanical cues from the niche. Recent evidence suggests a critical role of mechanical and physical cues in determining stem cell fates. This work discovered that the AMP cytonemes are enriched with a common mechano-transducer, named Talin. AMP-specific genetic manipulation of talin indicates that Talin is critical for cytoneme-mediated niche occupancy and FGF signaling. Using a Talin-based force probe expressed at the physiological levels and FLIM-FRET microscopy, we discovered that Talin experiences pN level force within the cytonemes. These findings suggest that AMPs employ cytonemes not only for receiving FGFs in a restricted polarized manner but also for a mechanosensory function. In conclusion, these results strongly suggest a critical role of cytonemes in coordinating asymmetric signaling and organization in the stem cell niche. In addition, the work provides evidence that the stem cell cytonemes are critical organelles for integrating the inputs and outputs of both growth factor signaling and mechanical cues to sculpt tissues.Item The genomics of species divergence in drosophila(2023) Carpinteyro Ponce, Javier; Machado, Carlos A; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)How do new species arise and diverge? Has been a fundamental question in evolutionary biology. The process of species divergence can be studied at many different levels of biological organization. However, it is until the recent advancements of genome sequencing technologies that genome-wide signatures of species divergence have started to unveil the complex genomic landscape of speciation. In this dissertation we investigate the landscape of genomic divergence using a classic pair of Drosophila species. We generated four new high quality genome assemblies for Drosophila pseudoobscura and D. persimilis to explore the genomic differences at three different levels. We first characterized the structural variation landscape between D. pseudoobscura and D. persimilis and stablished its association with transposable elements and tested how intrinsic genomic factors, such as recombination, influence the accumulation ofstructural variation associated with transposable elements in both species. With a combination of high-quality genome assemblies and a comprehensive population genomics data set, we also explored how the contribution of recombination rate and introgression promote sequence divergence with the potential of forming species barriers. Moreover, we investigated how gene co-expression networks potentially rewiring between species contribute to the divergence landscape between D. pseudoobscura and D. persimilis. Our work highlights the complex landscape of species divergence occurring at multiple levels of organization. Moreover, the integration of potential species drivers identified at different scales shed lights on the molecular mechanisms involved in speciation.Item Assessing vertebrate biodiversity across the Chesapeake Bay using environmental DNA metabarcoding(2023) Rodriguez, Lauren Kelly; Bailey, Helen; Woodland, Ryan J; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Through the collection and sequencing of trace genomic evidence from environmental samples (e.g., water, air, and soil), environmental DNA (eDNA) metabarcoding can detect a range of vertebrates. Despite the dynamic characteristics of estuarine environments, which often hinder the persistence of genomic material, this project successfully employed metabarcoding to assess the distribution of vertebrates in the Chesapeake Bay. Primarily, the study evaluated the effects of using various eDNA sampling, laboratory, and post-hoc analysis techniques when investigating species presence and biodiversity of an area. This study also identified spatially-explicit fish communities along salinity gradients as described by a Generalized Additive Mixed Model (GAMM) and a Permutational Multivariate Analysis of Variance (PERMANOVA). Community compositions were similar to previous findings by traditional trawling and seining methods. This research supports the usefulness of eDNA metabarcoding to assess species presence across spatiotemporal extents, making it a promising tool for future biomonitoring efforts in the Chesapeake Bay.Item Systems Approaches to Immunology in Acute COVID19, Monogenic Immune Disorders, and Childhood Development(2022) Rachmaninoff, Nicholas; Johnson, Philip F; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In recent years advances in immune profiling technologies have allowed us to generate data at anunprecedented scale, and interrogate human immune systems in ways that were not previously possible. In this dissertation, I use these approaches in three different contexts. First, I explore how the cells of the immune system respond to acute COVID-19 infection and how this depends on the severity of the disease. Using CITEseq, simultaneous profiling of surface markers and RNA in peripheral blood mononuclear cells, I identify differentially expressed gene expression programs associated with COVID-19 infection and gene expression programs associated with disease severity. In addition, I explore how phenotypes of memory Tcells including the clonal nature and exhaustion signatures are associated with severity of COVID-19 infection. Second, I address what it means to be immunologically healthy through a multi-omics study of a cohort of patients at the NIH clinical center with various monogenic Immune disorders. I identify supervised and unsupervised axes of immune health that can separate disease from healthy controls, and additionally track changes to the immune system as people age, showing the parallels between disease associated inflammation and aging associated inflammation. I verify the utility of these metrics in several contexts outside of the original cohort and show that the signatures reflect broad changes to various cells of the immune system. Last, I explore the development of the immune system in childhood and the maintenance of temporally stable gene expression patterns. In a cohort of children that was tracked longitudinally over six years in Nicaragua, I utilize whole blood transcriptomics to explore both how the immune system changes as children grow older and which aspects of the immune system show large amounts of individuality or persistent inter-subject variation in their levels. I show that persistent inter-subject variation in gene expression and cellular frequencies is quite pronounced throughout childhood and attempt to identify when certain aspects of the immune system begin to stabilize in terms of their levels for an individual.Item INFORMING CONSERVATION OF THREATENED BAT SPECIES USING GENOMICS AND ACOUSTICS(2022) Nagel, Juliet Joy; Nelson, David; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bats are vital to healthy ecosystems, providing billions of dollars of services in the form of forest and crop pest control. Unfortunately, North American bat populations have faced novel pressures during the past decade that may threaten their persistence. First, several species of tree-roosting bats (primarily hoary [Lasiurus cinereus], eastern red [L. borealis], and silver-haired [Lasionycteris noctivagans] bats) are experiencing large numbers of fatalities at industrial wind-energy facilities. Second, several species of cave-dependent bats have experienced large-scale mortality as the result of infection by a fungal pathogen that causes white-nose syndrome (WNS). As bats are generally long-lived and have low reproductive rates, such increases in mortality can cause significant population declines from which they may be unable to recover. Basic questions about population trends, size and structure remain largely unanswered for these species because of challenges in applying traditional wildlife monitoring approaches to bats. This lack of understanding impedes conservation and management efforts. In my dissertation, I use genomic and acoustic survey techniques to investigate questions related to the threats that wind-energy development and WNS are posing to bat species in North America. In my first chapter, I evaluate range-wide population structure and effective population size (Ne) for hoary, eastern red, and silver-haired bats. Using genotyping-by-sequencing (GBS), I genotyped single-nucleotide polymorphism (SNP) data from 173 hoary, 113 eastern red, and 89 silver-haired bats from multiple locations spread across their geographic distributions. Hoary bats and eastern red bats showed no geographic structure in genetic diversity, whereas silver-haired bats displayed longitudinal population variation. Coalescent modeling suggested that eastern red bats have the largest evolutionary Ne, followed by hoary bats, then silver-haired bats. In my second chapter, I used GBS to assess the population structure of two federally endangered cave bat species: Indiana bats (Myotis sodalis) and gray bats (M. grisescens). Using tissue samples from 45 Indiana bats and 47 gray bats spread across their ranges, I showed that Indiana bats display no geographic genetic structure, whereas gray bats exhibit east–west population variation across the Mississippi River Valley. In my final chapter, I used acoustic surveys across the State of Maryland to investigate bat community changes in the decade following the arrival of WNS. From 2010 through 2019, I conducted annual mobile acoustic routes each summer, for a total of 344 completed routes resulting in 426 hours of recordings and 24,375 identified bat passes. I detected massive (> 92%) declines of little brown bats (M. lucifugus), northern long-eared bats (M. septentrionalis), eastern small-footed bats (M. leibii), and tricolored bats (Perimyotis subflavus), with no evidence of recovery in recent years. Trends in hoary bats and eastern red bats were non-significant during this period. Bat community composition varied among Maryland’s physiographic regions, with eastern red bats comprising a larger percentage in the east. Species composition across the state likely reflects the impact of several factors, including mortality from WNS and wind-energy development, and perhaps reduced interspecific competition. Overall, my results illustrate the unique insights, but also distinct limitations, that genomic and acoustic data can provide regarding the conservation of bats in North America.Item EXAMINING THE GENETIC BASIS AND PHYSIOLOGY OF SURVIVAL IN EXTREME LOW SALINITY TO IMPROVE AQUACULTURE OF THE EASTERN OYSTER Crassostrea virginica(2022) McCarty, Alexandra J; Plough, Louis; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The eastern oyster, Crassostrea virginica, is an important aquaculture species and supports a growing industry along the east coast of the United States. However, increases in freshwater from storm events and intentional diversions can expose coastal aquaculture operations to extreme low salinity (< 5), resulting in reduced productivity and mortality. The primary objectives of this dissertation were to investigate the biology and genetic basis of low salinity tolerance to improve eastern oyster aquaculture. In Chapter 2, I developed and conducted a series of extreme low salinity (2.5) challenges to estimate the quantitative genetic parameters of low salinity survival. A moderate narrow-sense heritability was estimated for challenge survival, h2 ≈ 0.4. In addition, osmolality of hemolymph collected from oysters during the first week of the challenge suggest that all individuals conformed to the surrounding low salinity regardless of challenge survival. In Chapter 3, I performed additional low salinity challenges to assess the importance of challenge duration (2 or 6 months) and temperature (chronic or fluctuating) on low salinity survival. I also investigated algae removal during the chronic challenge to better understand oyster response during low salinity stress. Phenotypic (rS = 0.89) and genetic (rG = 0.81) correlations between family mortality were high across the two challenges, indicating that a 30-day exposure at a constant low salinity (2.5) and temperature (27°C) is a sufficient progeny test for low salinity survival. Modest associations between algae removal metrics and survival in extreme low salinity indicate that individual feeding ability may relate to differential low salinity survival. Lastly, in Chapter 4, I performed genome mapping to investigate the genomic architecture of low salinity survival. Quantitative trait locus mapping and linkage disequilibrium analysis revealed a significant region on eastern oyster chromosome 1 and 7. Genomic prediction accuracies for survival and day to death in extreme low salinity were moderate and encouraging, 0.49 – 0.57. The results from my dissertation characterize the genetic basis of survival during low salinity events and support the incorporation of this trait into breeding efforts to improve production and enhance the resiliency of the eastern oyster aquaculture industry.Item DEVELOPMENT OF A BIOINFORMATICS PLASMID-SEARCH ENGINE FOR CRONOBACTER SPECIES.(2021) Negrete, Flavia; El-Sayeed, Najib NE; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cronobacter species. are foodborne pathogens that cause serious disease in neonates, infants, and adults. Plasmid classification lays the groundwork for understanding the stable coexistence of various extrachromosomal replicons in a single bacterium, and thus the organization of its genome. This study developed a bioinformatics plasmid-search engine to identify genomic attributes contained on Cronobacter plasmids. A database containing 32 Cronobacter plasmid sequences from all seven Cronobacter species was developed. Another database containing 683 draft and closed plasmids and genomes was also developed. Each strain’s plasmid content was sorted into six different categories based on their genetic attributes: virulence, Type-IV, heavy-metal, cryptic, multi-drug resistant, or mobilization. An in-house BLAST+-python script was used to perform a Linux-BLAST analysis to create a formatted %ID output matrix of plasmid genes. This thesis represents the first bioinformatics plasmid-search engine developed for Cronobacter. Understanding the role of plasmids in virulence and persistence underpins future mitigation strategies to be developed for controlling this pathogen.Item What makes a successful invader? Population genomics and adaptation to novel environments in the invasive Japanese white-eye (Zosterops japonicus)(2021) Venkatraman, Madhvi; Gruner, Daniel S; Fleischer, Robert C; Behavior, Ecology, Evolution and Systematics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Invasive species face many obstacles when colonizing new habitats. Yet, many overcome these hurdles and successfully establish populations. Therefore, understanding how invasive species cope with novel stressors while colonizing new environments is a fundamental goal of evolutionary biology. Additionally, broadening our understanding of how birds adapt to novel environments can help us predict how species will respond to habitat degradation and stressors resulting from climate change in the future. Here, we focus on the Japanese white-eye (Zosterops japonicus), an East Asian bird that was introduced into Hawaii in the early 1900s and is now the most abundant land bird in the archipelago. First, we sequenced and assembled a high-quality Z. japonicus genome and compared genome annotation pipelines. We found that AUGUSTUS was more conservative with gene predictions when compared to BRAKER2, but the final number of annotated gene models was similar between the two workflows. Additionally, we found that while adding more data did not significantly change the number of annotated genes using AUGUSTUS, using BRAKER2 the number increased substantially. Next, we compared whole genomes of Z. japonicus individuals from both their native and introduced ranges to characterize genetic diversity and population history and divergence and to identify genes potentially under selection between the two populations. We saw evidence of mixed ancestry in the introduced population, supported by drastically different demographic histories in Hawaii. This suggests that admixture could have contributed to increased genetic diversity in the introduced population and therefore to overall invasion success. Lastly, we conducted one-, three-, and six-week one-way transplants of individuals from near sea level to 2,790m, with individuals kept at sea level as controls, and later a six-week reciprocal transplant from high to low elevation and vice versa. We assessed morphological and physiological traits as well as gene expression using RNA-seq on heart and lung tissues. We found strong evidence for phenotypic plasticity in hematological and cardiac response to hypoxia and cold stress and some evidence of maladaptive plasticity in pulmonary circulation. We identified two genes potentially under divergent selection in the high elevation population that could be indicative of early-stage genotypic specialization in response to hypoxia and cold stress. Our results suggest that the population of Z. japonicus on Mauna Kea is able to persist at high elevation because of ancestral plasticity, which also could have contributed to its remarkable success as an invasive species.Item EXAMINING HIBERNATION IN THE BIG BROWN BAT THROUGH DNA METHYLATION(2021) Sullivan, Isabel; Wilkinson, Gerald S; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hibernation allows individuals to conserve energy during seasonal low temperatures. As the physiological regulation of hibernation is inadequately understood, I examine hibernation using DNA methylation (DNAm). DNAm is the addition of a methyl group to cytosine at cytosine guanine dinucleotide (CpG) sites in the genome. DNAm in promoters can repress gene expression and be influenced by histone modifications. Using the big brown bat, Eptesicus fuscus, I examined how hibernation influences DNAm, independent of age, through comparing DNAm from bats that differed in hibernation history and comparing DNAm from the same individual between hibernating and active seasons. Both comparisons found evidence of differential enrichment of genes near significant CpG sites resulting from hibernation. The latter analysis found evidence consistent with a histone mark, associated with active transcription, is likely enriched in hibernating bats. These results suggest that DNAm and histone modifications associated with transcription factor binding regulate gene expression during hibernation.Item INTERNAL AND EXTERNAL ENVIRONMENTAL REGULATION OF OPSIN EXPRESSION IN AFRICAN CICHLIDS(2021) Yourick, Miranda Rose; Carleton, Karen L; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Evolutionary biology has long sought to understand the evolution of adaptive phenotypes. While purely genetic factors are often emphasized, environmental influences also significantly contribute to phenotypic variation. In this dissertation, we investigate the environmental regulation of visual sensitivity in cichlids. Cichlid fishes exhibit a diversity of color vision facilitated by differential expression of opsin proteins, including light-induced phenotypic plasticity which was known to occur during development. To determine whether cichlids retain plasticity into adulthood, adult fish were moved between different light environments. Adult fish not only exhibited plasticity of opsin expression, but it was rapid, reversible, and shifted in a direction that would maximize light input to the retina. In order to observe the effect of diurnal variation of light on opsin cycles, we collected retinae from a continuous 25 hour time-series. This enabled us to develop guidelines for qRT-PCR normalization and determined that opsins were most highly expressed in the late afternoon. Opsins also cycled together, suggesting some common diurnally-sensitive regulatory mechanism. Given that there are species differences in plasticity, we sought to determine genetic loci that were affected by light. To determine how quantitative trait loci (QTL) changed between light environments, we raised all generations of a previously studied hybrid cross in a new light environment. One QTL was significant in both environments, indicating it is universally important for opsin expression. However, another QTL was only significant in one light environment, indicating it may be environmentally-sensitive. We propose a possible candidate gene for this locus. Plasticity is prevalent during development, with thyroid hormone (TH) being a common cue for developmental changes. Previous studies have found TH causes opsin expression shifts from short- to long-wavelength sensitivity as organisms age. To see its effect on species of cichlids with different developmental opsin progressions, we treated fish with TH. Interestingly, TH shifted fish to longer-wavelength sensitivities regardless of species. We also uncovered a possible interaction between TH and light-induced plasticity. Characterization of environmental influences on vision will continue to enhance our understanding of the evolution of visual diversity in these incredible fishes.