Biology Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2749
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Item SENSORY AND HORMONAL MECHANISMS OF EARLY LIFE BEHAVIOR IN A SOCIAL CICHLID FISH(2024) Westbrook, Molly; Juntti, Scott; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Studying the ontogeny of animal behavior is fundamental to ethology and allows understanding how behaviors in early life may affect later life success. The social cichlid Astatotilapia burtoni is an excellent model for examining the mechanisms of early life aggression due to the robust social hierarchy enforced by stereotyped, measurable social behaviors. We examine how hormonal signaling affects early life aggression through pharmacology and CRISPR-Cas9 mutants. We test which sensory pathways convey aggression-eliciting stimuli through sensory deprivation experiments. And we identify kinematic features that predict aggression through machine-learning video tracking algorithms. We observe that aggressive behaviors emerge around 17 days post fertilization (dpf), correlating with when the animals transition to free swimming away from the mother. We find that sex steroids subtly organize behavioral circuits for aggression and suggest that unknown additional mechanisms play a leading role. We show that thyroid hormone is not necessary or sufficient for the transition to aggressive behavior. We show that visual signals are necessary for the full expression of aggression, but in the absence of visual signal, low levels of aggression remain. We show that ciliated olfactory receptor signaling maintains low levels of aggression, as mutant animals display higher levels of aggressive behavior between 17 and 24 dpf. Finally, we demonstrate that swimming velocity has potential to predict aggressive instances of behavior. Together, we find multiple levels of control for early life aggressive bouts from sensory input to hormonal organization of brain circuits.Item Becoming a Father: Disentangling the Causes and Consequences of Caregiving Behavior in California Mouse Males(2024) Colt, Maria; Carleton, Karen L; Fisher, Heidi S; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In mammals, parental care is critical to offspring survival, however, it can also lead to measurable changes in parents. In the biparental California mouse, Peromyscus californicus, fathers actively care for their offspring, including grooming, huddling, retrieving their pups, and building nests. When fathers engage in caregiving behavior, they also experience increased memory, decreased anxiety-like behavior, and structural neuroplasticity of the hippocampus. However, the trigger, reproduction or caregiving experience, and the molecular pathways that regulate these behavioral and neurological changes, remain unclear. In the first chapter, I compared caregiving behaviors in fathers who have cared for their pups and pup-sensitized non-fathers who have cared for unrelated pups, and I found that pup-sensitized non-fathers were slower to approach pups but eventually spent more time grooming pups, whereas fathers spent more time nest-building. I then compared recognition learning, anxiety-like behaviors, and reproductive investment in fathers, pup-sensitized non-fathers, non-fathers with no caregiving experience, and virgins that were socially housed but had no caregiving experience. I found that experienced fathers exhibited increased recognition memory and decreased anxiety-like behavior compared to virgins and non-fathers, and that virgins had smaller testes and fewer sperm compared to non-fathers yet that first-time fathers had larger testes compared to non-fathers. In the second chapter, I first show that hippocampal dendritic spine density is positively associated with males’ caregiving experience. Then, I compared hippocampal gene expression in fathers, non-fathers, and pup-sensitized non-fathers and performed gene ontology, network, and pathway analyses to identify suites of RNA expression patterns associated with caregiving experience. I found that fathers exhibit an upregulation of genes associated with neurogenesis, glutamatergic synapses, neuronal signaling, cellular components of dendritic spines, and some biological pathways previously linked to maternal care, such as regulation of actin cytoskeleton. Together, my results suggest that caregiving behavior induces important behavioral, structural, and transcriptional changes in the brains of males, even if they are caring for offspring that are not their own.Item SEROTONIN REGULATES AN OLFACTORY CRITICAL PERIOD IN DROSOPHILA(2024) Mallick, Ahana; Araneda, Ricardo; Gaudry, Quentin; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Serotonin (5-HT) is known to modulate early development during critical periods when experience drives heightened levels of plasticity in sensory systems. Studies in the somatosensory and visual cortices implicate multiple target points of serotonergic modulation, yet the underlying cellular and molecular mechanisms of 5-HT modulation of critical period plasticity remain elusive. Here, we take advantage of the genetically tractable olfactory system of Drosophila to investigate how 5-HT modulates critical period plasticity (CPP) in the CO2 sensing circuit of fruit flies. During the critical period, chronic exposure to CO2 has been shown to increase the volume of the CO2 sensing V glomerulus. We found that 5-HT release by serotonergic neurons in the antennal lobe (AL) is required for increase in the volume of the V glomerulus. Furthermore, signaling via the 5-HT1B, 5-HT2B and 5-HT7 receptors in different neuronal populations is also required during the critical period. Olfactory CPP is known to involve local inhibitory networks and consistent with this we found that knocking down 5-HT7 receptors in a subset of GABAergic local interneurons was sufficient to block CPP, as was knocking down GABA receptors expressed by olfactory sensory neurons (OSNs). Additionally, 5-HT2B expression in the cognate OSNs sensing CO2 is also essential for CPP indicating that direct modulation of OSNs also contributes to the olfactory CPP. Furthermore, 5-HT1B expression by serotonergic neurons in the olfactory system is also required during the critical period. Our study reveals that 5HT modulation of multiple neuronal targets is necessary for experience-dependent structural changes in an odor processing circuit. Finally, we wanted to isolate the neuromodulatory effects of individual serotonergic neurons. To achieve this, we combined a state-of-the-art technique to sparsely label serotonergic neurons and a computer algorithm to search against 10,000 Gal4 promoter lines and identify candidate lines that would allow individual manipulation of the 110 serotonergic neurons.Item EVOLUTION OF THE CRISPR IMMUNE SYSTEM FROM ECOLOGICAL TO MOLECULAR SCALES(2024) Xiao, Wei; Johnson, Philip LF; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacteria and archaea inhabit environments that constantly face viral infections and other external genetic threats. They have evolved an arsenal of defense strategies to protect themselves. My research delves into the CRISPR immune system, the only known adaptive immune system of prokaryotes. My work explores three different dimensions of the CRISPR immune system, ranging from ecological to molecular scales.From an evolutionary perspective, CRISPR is widely distributed across the prokaryotic tree, underscoring its immune effectiveness. However, the CRISPR distribution is uneven and some lineages are devoid of CRISPR. Here, I identify two ecological drivers of the CRISPR immune system. By analyzing both 16S rRNA data and metagenomic data, I find the CRISPR system is favored in less abundant prokaryotes in the saltwater environment and higher diverse prokaryote communities in the human oral environment. On the molecular level, the CRISPR system selects and cleaves its “favorite” DNA segments (also known as “spacers”) from invading viral genomes to form immune memories. I explore how the spacer sequence composition affects its acquisition rate by the CRISPR system. I develop a convolutional neural network model to predict the spacer acquisition rate based on the spacer sequence composition in natural microbial communities. The model interpretation reveals that the PAM-proximal end of the spacer is more important in predicting the spacer abundance, which is consistent with previous findings from controlled experimental studies. Combining these scales, CRISPR repeat sequences coevolve with the rest of the genome. Thus, I explore the potential of utilizing CRISPR repeat sequences for taxonomy profiling. I find a strong relationship between unique repeat sequences and taxonomy in both the RefSeq database and a human metagenomic dataset. Then I show high accuracy when utilizing repeat sequences in taxonomy annotation of human metagenomic contigs. This novel method not only aids in annotating CRISPR arrays but also introduces a novel tool for metagenomic sequence annotation.Item INSIGHTS IN ECOLOGY, BEHAVIOR, AND REPRODUCTION FROM VISUAL MODELS OF AFRICAN CICHLIDS(2024) Gonzalez, Zeke Martin; Carleton, Karen L; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Sexual selection has long been proposed to have played an important role in the explosive speciation of east African cichlids. Further, it is known that visual signals are the most salient ones to cichlids when it comes to reproduction. However, studies examining visual signals such as egg spots and size have been historically difficult to conduct due to the relationship between such phenotypes and confounding variables like age. In addition, the results from such studies often conflict and do not highlight clear patterns and hypotheses. In this dissertation, I use a receptor noise limited (RNL) visual model of increasing complexity to examine the discriminability of important visual signals in cichlid ecology, behavior, and evolution. In determining whether cichlid egg spots are truly mimics of cichlids eggs, I quantified fish and egg reflectance and found that two cichlid species are unable to distinguish the colors of eggs and egg spots in the lighting of their natural habitat. In order to bring together these quantitative methodologies with behavioral data, I tested the viability of using virtual stimuli displayed on a monitor to robustly examine how various visual signals affect conspecific male aggression. I found that although the cichlid Metriaclima zebra responds to virtual stimuli with equal aggression as towards live fish, it also responds with equal aggression towards virtual stimuli that differ in egg spot presence, body color, movement, and size. This suggests that virtual stimuli are not useful for behavioral tests in this species. Finally, in order to examine the salience of egg spots and body color in the wild, I calculated chromatic distance as a function of viewing distance for cichlid body colors against biologically-relevant backgrounds, conspecific body colors, and heterospecific body colors. The study shows that M. zebra body colors are discriminable from the space light at up to 5 m, but from the rocks at shorter distance, though distances that are comparable to the spacing of male territories. This suggests that males should be able to discriminate potential conspecific rivals on their breeding territories. Additionally, the visual model shows that M. zebra is highly discriminable from yellow heterospecifics but not so from blue heterospecifics. This dissertation emphasizes the importance of avoiding human biases in studies of cichlid color vision and behavior.Item Reactivation of plasticity in the adult visual cortex by control of neuronal excitability(2023) Borrell, Andrew; Quinlan, elizabeth; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Amblyopia is a highly prevalent form of monocular vision loss that impacts between 1-4% of the worldwide population. Amblyopia is characterized by decreased visual acuity in a single eye and is highly refractory to treatment past a “critical period” of heightened plasticity during early adolescence (>5 years of age). The time course of this critical period is due to the developmental regulation of experience-dependent synaptic plasticity in the primary visual cortex (V1). During early development, visual experience drives activity-dependent changes in NMDA-R subunit composition, refines the convergence of binocular inputs, and promotes the maturation of inhibitory circuits in V1. The transient conditions in V1 that permit the refinement of cortical circuits during the critical period also render V1 vulnerable to the detrimental impacts of amblyopia.The expression of critical period plasticity requires visual experience: dark-rearing delays the onset and closure of the critical period and prevents the experience- dependent change in NMDA-R subunit composition. It is now understood that visual experience in adulthood is also important for the expression of plasticity: sensory deprivation via prolonged dark exposure (DE) rejuvenates the V1 circuit to a juvenile-like state via a homeostatic increase in spontaneous excitatory in V1. Subsequent visual experience during light reintroduction (LRx) enables the expression of critical period plasticity and the functional rewiring of thalamocortical inputs to V1. Here I asked how the homeostatic increase in spontaneous activity induced during DE is regulated by visual experience immediately following LRx (LRxi), and during one day of subsequent day of LRx (LRxs). I demonstrate that the homeostatic increases in spontaneous excitatory neuron activity is maintained during LRxi and is accompanied by increased evoked excitatory neuron activity. These increases in averaged spontaneous and evoked activity returned to baseline by LRxs. Next, I asked whether decreased spontaneous activity following one day of LRx was necessary for the reactivation of critical period plasticity. Using the mouse model of ocular dominance plasticity (ODP) and cell-type specific expression of inhibitory chemogenetic Gi-DREADD receptors in fast spiking Parvalbumin-expressing interneurons, I demonstrated that prolonged disinhibition of spontaneous V1 activity during LRx occludes the reactivation of ODP, but not the reactivation of the plasticity of acuity. These results demonstrate the differing contribution of cortical mechanisms to ocular dominance versus acuity in the regulation of the critical period plasticity, and the necessity of the decrease in average spontaneous activity for the re-expression ODP.Item AN INVESTIGATION ON THE MOLECULAR BASIS FOR DIMER FORMATION OF A BACTERIOPHAGE ENDOLYSIN POSSESSING ANTIMICROBIAL ACTIVITY AGAINST STREPTOCOCCUS PNEUMONIAE(2023) Alreja, Adit Bipin; Nelson, Daniel C; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The global rise of antibiotic resistance casts a shadow on treating infectious disease. An alternative to the use of antibiotics is bacteriophage-derived peptidoglycan hydrolases called endolysins. Endolysins, produced at the end of a bacteriophage replication cycle, cause bacterial cell lysis and virion release. When applied exogenously as recombinant proteins, they are also capable of cleaving the Gram-positive bacterial peptidoglycan. Various studies conducted in vitro and in vivo showcase the therapeutic potential of endolysins as the next generation of antimicrobials. Streptococcus pneumoniae is the most common cause of a variety of infections ranging from otitis media to invasive bloodstream infection (bacteremia) and meningitis (brain infection). While pneumococcal vaccination programs have proven to be effective, the high rates of antibiotic resistance reported for S. pneumoniae has led to the CDC classifying it as a “serious” threat. One of the most studied endolysins targeting S. pneumoniae is Cpl-1. This thesis represents an investigation into the molecular basis for dimer formation of the Cpl-1 endolysin which displays antibacterial activity against S. pneumoniae. In addition to disproving a long-accepted mechanism of dimerization of Cpl-1 in the presence of choline, we have conclusively identified the residue involved in the formation of the Cpl-1 dimer. Our findings led to the discovery of a novel C-terminal consensus sequence shared by all pneumococcal endolysins that informs their propensity to form dimers in the presence of choline. Next, through a bioinformatics approach we identified a new endolysin containing this C-terminal consensus sequence, produced it, named it SP-CHAP, and showed that it forms a dimer in the presence of choline, indicative of the widespread dimerization phenomenon associated with pneumococcal endolysins. Of interest, SP-CHAP is the first endolysin with antimicrobial activity against S. pneumoniae that possesses a cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain. SP-CHAP was subsequently characterized for its biochemical and antimicrobial properties and benchmarked against Cpl-1. SP-CHAP is active in all physiologically relevant conditions (pH, temperature) against various S. pneumoniae strains and displays no activity towards oral/nasal commensal organisms. This enzyme also displays pneumococcal biofilm eradication ability to a greater extent than Cpl-1, as visualized by confocal microscopy. To further translate the antimicrobial potential of this enzyme, the antimicrobial efficacy of SP-CHAP was validated in a S. pneumoniae mouse nasopharyngeal colonization model. Our results demonstrate the therapeutic potential of SP-CHAP as an attractive endolysin to treat S. pneumoniae infections and warrant further translational development of this enzyme.Item Evolutionary history and consequences of gene flow in bearded manakins(2023) Bennett, Kevin Faulkner Philipson; Braun, Michael J; Wilkinson, Gerald S; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Recent advances in DNA sequencing have revolutionized evolutionary biology by allowing for genome-scale studies of non-model organisms. We can now readily connect genotype with phenotype, i.e., identify the genetic basis of particular traits, a key goal in the study of evolution. In addition, genome-scale sequence analysis has shifted our understanding of the frequency and magnitude of gene flow in nature. Once viewed as important only for its role preventing divergence, the common view now is that in many taxa gene flow occurs among many lineages in the early stages of divergence. My dissertation focuses on gene flow in bearded manakins (genus Manacus), which are notable for their intense tandem courtship display, high degree of reproductive skew among males, and bright male plumage. In western Panama, yellow-collared M. vitellinus and white-collared M. candei interbreed in a narrow hybrid zone. Male vitellinus secondary sexual traits, including the yellow collar, have introgressed roughly 50 km west across the hybrid zone into candei populations and then stalled at the east bank of the Río Changuinola, the region’s largest river. Evidence from studies of male-male interaction and female choice implicate positive sexual selection for yellow collars as a driver of introgression. For thirty years since this situation was first described in detail, several key issues have remained unresolved, including why introgression has not continued across the river and what gene or genes are responsible for yellow coloration. In the first chapter, I reviewed the current state of knowledge of the Manacus hybrid zone system and proposed new hypotheses for some of the patterns exhibited by these populations. In the second chapter, I used reduced-representation genome sequencing to investigate whether reduced gene flow across the Río Changuinola alone can explain stalled trait introgression. I found that, although advantageous plumage traits have not introgressed far beyond the river, substantial gene flow is occurring, implicating an additional selective force or forces in preventing trait introgression. In the third chapter, I used whole-genome sequencing of all major Manacus lineages, including unpigmented M. manacus and pigmented M. aurantiacus, to explore the evolution and genetic basis of collar coloration. I identified the carotenoid metabolism gene beta-carotene oxygenase 2 (BCO2) as responsible for collar color differences between vitellinus and candei and uncovered evidence of past introgression introducing aurantiacus BCO2 alleles into vitellinus. I argue that gene flow is likely to be a more common mechanism than previously appreciated for spreading sexual traits among species.Item SYSTEMS IMMUNOLOGY OF IMMUNE IMPRINTS INDUCED BY ACUTE VIRAL INFECTIONS(2023) Liu, Can; Johnson, Philip L.F.; Tsang, John S.; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Upon encountering perturbations such as viral infections, the immune system initiates a cascade of molecular and cellular responses. These alterations may persist even after recovery, resulting in enhanced or diminished response to subsequent stimuli compared to the naïve state. Such persistent changes, referred to as immune imprints or long-term non-specific memory, indicate an incomplete resolution from immunological perturbations. The primary focus of this dissertation is to systemically investigate the immune imprints resulting from acute infections and how they shape the baseline immune status to future heterologous challenges.First, we employed cutting-edge single-cell multi-omics and computational approaches to assess the immune response during the COVID-19 disease course and severity correlates at an unprecedented resolution. We identified gene expression profiles – apoptosis in plasmacytoid dendritic cells and IL-15-linked increase of fatty acid (FA) metabolism in CD56dimCD16hi NK cells – as primary correlates of disease severity. This increase of FA signature with disease severity was also concomitant with an attenuated inflammation, indicating a dysfunctional or exhaustion-like state of these NK cells. While the depressed inflammation signature in severe patients was also found in different cell types near hospitalization, it increased temporally at later time points, indicating a critical late-stage juncture in the disease course. Next, we took the opportunity of the period following the first wave of COVID-19 pandemic to study immune imprints in human cohorts who had recovered from COVID-19 before widespread vaccination and reinfection occurred. We demonstrated that individuals who recovered from mild COVID-19, exhibit distinct immune signatures through single-cell transcriptomic profiling. Male recoverees also showed heightened responses to the seasonal influenza vaccine compared to healthy individuals without a history of COVID-19 and female recoverees. These sex dimorphic imprints highlight the interplay between intrinsic factors like sex and non-intrinsic factors such as prior SARS-CoV-2 infection, in shaping an individual's immune system over time. Lastly, we also investigated the immune imprints after acute viral infection using a controlled experimental mouse model of influenza infection. After examining cellular and gene expression profiles in various organs after the infection, we found persistent changes in both adaptive and innate immune components across multiple organs. Moreover, these changes affected subsequent local IL-17 inflammatory response and secondary heterologous vaccinations in anatomically distinct organs. Together, both human and mouse studies here are important pieces toward an improved understanding of long-term immune imprints after perturbations, which can be leveraged to develop more effective and personalized vaccines and disease treatments.Item ANALYTICAL APPROACHES FOR COMPLEX MULTI-BATCH -OMICS DATASETS AND THEIR APPLICATION TO NEURONAL DEVELOPMENT(2023) Alexander, Theresa Ann; Speer, Colenso M; El-Sayed, Najib M; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)High-throughput sequencing methods are extremely powerful tools to quantify gene expression in bulk tissue and individual cells. Experimental designs often aim to quantify expression shifts to characterize developmental trajectories, disease states, or cellular drug responses. Experimental and genetic methods are also rapidly evolving to capture specific aspects of gene expression such as in targeting individual cell types, regulatory stages, and spatially resolved cell subcompartments. These studies frequently involve a variety of experimental conditions that require many samples to guarantee sufficient statistical power for subsequent analyses. These studies are frequently processed in multiple batches due to limitations on the number of samples that can be collected, processed, and sequenced at once. To eliminate erroneous results in subsequent analyses, it is necessary to deconvolve non-biological variation (batch effect) from biological signal. Here, we explored variational contributions in multi-batch high throughput sequencing experiments by developing new methods, evaluating heterogeneity-contributors in an axon-TRAP-RiboTag protocol case-study, and highlighting biological results from this protocol. First, we describe iDA, a novel dimensionality reduction method for high-throughput sequencing data. High-dimensional data in complex, multi-batch experiments often result in discrete clustering of samples or cells. Existing unsupervised linear dimensionality reduction methods like PCA often do not resolve discreteness simply with projections of maximum variance. We show that iDA can produce better projections for separating discrete clustering that correlates with known experimental biological and batch factors. Second, we provide a case study of special considerations for a complex, multi-batch high throughput experiment. We investigated the multi-faceted heterogenic contributions of a study using the axon-TRAP-RiboTag translatomic isolation protocol in a neuronal cell type. We show that popular batch-correction methods may reduce signal due to true biological heterogeneity in addition to technical noise. We offer metrics to help identify biological signal-driven batch-differences. Lastly, we employ our understanding of variational contributions in the intrinsically photosensitive retinal ganglion cell (ipRGC) -omics case study to explore the biological transcriptomic and translatomic coordination. Our analysis revealed ipRGCs participate in subcompartment-specific local protein translation. Genetic perturbations of photopigment-driven neuronal activity led to global tissue transcriptomic shifts in both the retina and brain targets, but the ipRGC axonal-specific translatome was unaltered.