Biology Theses and Dissertations
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Item MODULATION OF SIGNALING IN THE ANTERIOR CINGULATE CORTEX AND ITS IMPACT ON DECISION-MAKING(2024) Vazquez, Daniela; Roesch, Matthew R; Neuroscience and Cognitive Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Attentional deficits are defining hallmarks of some of the most prevalent and disruptive neuropsychiatric disorders—including attention deficit hyperactivity disorder (ADHD) and substance abuse disorders. The anterior cingulate cortex (ACC) is a brain region that is highly implicated in shifting attention allocation towards relevant stimuli after unexpected events or outcomes occur. Importantly, increases in attention facilitate flexible learning, as attention allows you to dynamically filter relevant and necessary information during decision-making. My dissertation work seeks to identify the ACC as a novel point of intervention for the treatment of neuropsychiatric and addiction disorders by providing an in-depth perspective on its involvement in cognitive control and attentional processes.My research explores the neural correlates of decision-making by using electrophysiology to record single unit activity while rats perform a complex reward-based decision-making task, and employing chemical, optogenetic, and epigenetic manipulations to modulate attentional correlates in the ACC. I explored the ACC’s role in attention—and how it is impacted by drug use—using electrophysiology to record from ACC neurons as both cocaine-exposed and drug-naïve rats performed a reward-guided decision-making task. Using this task, we found a dose-dependent attenuation of ACC signaling after cocaine self-administration, which was correlated with decreases in task performance and attention to the task. Rats that had self-administered large amounts of cocaine had diminished neural responsiveness to cues, which translated into reductions in behavioral measures of attention, disruptions in cognitive flexibility, and decision-making impairments. These results both supported previous findings establishing the ACC’s role in attentional allocation, and revealed an intake-dependent effect of drugs on decision-making and neural encoding. In aim 2, we wanted to be able to precisely modulate ACC activity in order to better interrogate the role of the ACC in the absence of confounding variables (e.g. cocaine use results in the dysregulation of various neural circuits), and conduct within-subject analyses. Thus, in our next experiment we used optogenetics to inactivate the ACC, and found that ACC inhibition severely impaired task engagement, as evinced by reductions in trial initiations, and trial and session completions—resulting in overall impaired session performance. In order to disambiguate whether these behavioral deficits resulted from ACC impairment dysregulating downstream action-outcome encoding, we performed chemical lesions of the ACC, and recorded neural activity from the dorsomedial striatum (DMS)—a downstream brain region that is importantly involved in goal-directed behavior—as rats performed the previously mentioned decision-making task. Again, we found that ACC lesions resulted in disrupted attention to the task, and similar behavioral deficits to the ones we observed following cocaine use. Interestingly, we found that DMS encoding was minimally impacted, reinforcing that the observed decision-making deficits stem from disruptions in attentional signaling and not dysregulations in downstream action-outcome encoding. In the aforementioned experiments, we employed an array of techniques to dissect how disrupting ACC signaling in a variety of manners impacted task performance and engagement, so for our final experiment we sought to explore a therapeutically relevant way to potentially repair signaling disruptions that lead to the breakdown in attentional signaling. Thus, we turned to epigenetics—specifically, decreasing the expression of HDAC5, an enzyme that is involved in negatively regulating gene expression—to explore whether epigenetic changes might map onto specific alterations of neural activity and behavior. Surprisingly, we found that HDAC5 knockdown in the ACC dysregulates attentional signals that are necessary for flexible and adaptive decision-making. Together, these studies established that signaling in the functional ACC is importantly involved in attention, and that dampening these signals leads to decision-making impairments and decreased task engagement, notably characterized by significant reductions in the proportion of initiated and completed trials, and prolonged periods of inattention.Item INVESTIGATION OF A NOVEL O-GLCNAC MODIFICATION OF A VACCINIA VIRUS CORE PROTEIN(2024) Zhang, Yunliang; Scull, Margaret; Moss, Bernard; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Vaccinia virus (VACV) is a large, complex, enveloped virus that is the prototypic member of the genus Orthopoxvirus of the Poxviridae family and is well known as the live-virus vaccine that eradicated smallpox. It has a linear, double-stranded DNA genome of approximately 190 kbp that encodes about 200 proteins some of which undergo various post-translational modifications. These modifications are crucial for regulating protein function and influencing the virus behavior within the vertebrate and insect cells. Among these, O-GlcNAcylation is notable for its reversible modulation of protein function, like phosphorylation. Although over 5,000 human proteins have been documented as O-GlcNAcylated, the prevalence and function of this modification in viral proteins remain underexplored.Early studies from the Moss laboratory demonstrated the presence of a 40-kDa protein that contained N-acetylglucosamine in purified virions. The small size of the pronase-digestion product and the absence of other sugars suggested one or few glucosamines. The current study advances this understanding by pinpointing the novel O-linked β-N-acetylglucosamine (O-GlcNAc)-modified protein in VACV infectious particles. Enzymatic labeling of purified virions was performed using the mutant β-1,4-galactosyltransferase (GalT1 (Y289L)) to specifically transfer azido-modified galactose (GalNAz) from UDP-GalNAz to O-GlcNAc residues. Following copper catalyzed azide-alkyne cycloaddition (CuAAC) of biotin or an infrared dye, the candidate O-GlcNAc proteins were detected by SDS-polyacrylamide gel electrophoresis and identified by mass spectrometry (MS). Then using strain-promoted cycloaddition (SPACC) chemistry to attach a polyethylene glycol mass tag of 10 kDa to the O-GlcNAc protein, a significant shift in the electrophoretic mobility of the VACV A4 protein was documented by western blotting. The presence of O-GlcNAc in A4 was confirmed by MS and by binding to specific antibodies. Multiple modification sites were pinpointed using higher-energy collisional dissociation induced electron-transfer dissociation in MS. Further evidence linking cellular protein O-GlcNAc transferase (OGT) to the modification of A4 was derived from experiments conducted with an A4-expressing cell line. Disruption of OGT activity, either through chemical inhibition or knock-down techniques, reduced A4 O-GlcNAc modification without impairing VACV infectivity. This finding suggests that the O-GlcNAc modification of A4 does not play an essential role in VACV infectivity, which is not correlated with the A4 deletion phenotype. Therefore, the specific effects of O-GlcNAc modification on the VACV lifecycle remain elusive, indicating further studies are required to determine the potentially subtle effects of O-GlcNAcylated A4 on the VACV life cycle.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 MICROBIAL COMMUNITIES IN COASTAL ECOSYSTEMS(2024) Kim, Carol; Malkin, Sairah Y; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Firstly, I examined microbial community succession along a chronosequence of constructed salt marshes using the Poplar Island restoration project site as a case study. By comparing 16S rRNA gene amplicon sequences across 6 constructed low marshes spanning a chronosequence of 1-16 years at Poplar Island (Chesapeake Bay) and a nearby natural reference marsh, I found strong evidence that the development of soil microbial communities is on a trajectory towards natural marsh conditions following marsh restoration with successional rates within timescales expected for soil development. Results from this study showed the value of microbial communities to serve as effective bioindicators for monitoring the recovery of microbially mediated biogeochemical processes in restored or newly constructed salt marshes, as well as potentially for assessing the marsh inundation period and by extension marsh health and resiliency. Secondly, I conducted a manipulation experiment to explore microbial communities associated with cable bacteria using RNA stable isotope probing (RNA-SIP). I traced the uptake of isotopically labeled bicarbonate and acetate in sediments with baseline and with stimulated cable bacteria activity, to test the hypothesis that cable bacteria activity can stimulate chemoautotrophic bacteria in anaerobic sediments. I used 16S rRNA sequencing to identify the active “incorporators” of bicarbonate (as a tracer of chemoautotrophy) and acetate (as a tracer of heterotrophy). I found that estuarine cable bacteria activity stimulated the chemoautotrophic activity of Gammaproteobacteria (Nitrosomonas, Thioalkalispira-Sulfurivermis) and Campylobacterota (Sulfurovum, Sulfurimonas) at anaerobic depths. This result is not explainable with conventional understanding of chemoautotrophic activity. Rather, this study contributes to the emerging concept that cable bacteria activity stimulates metabolic activities at suboxic sediment depths, potentially by serving as an electron sink for other microbes. Furthermore, I found that heterotrophic activity, measured as 13C-acetate assimilation into RNA, was stimulated amongst known chemoautotrophic sulfur oxidizers at depth, highlighting that metabolic flexibility, and specifically mixotrophy, may be widespread in complex natural sediment environments. Lastly, I characterized the composition and metabolic potential of microbial communities in estuarine sediment enriched with cable bacteria. By using metagenomic and 16S rRNA sequencing, I constructed 23 medium- to high-quality metagenome-assembled genomes (MAGs) that span across 9 phyla. I retrieved MAGs exhibiting mixotrophy and a range of capabilities for extracellular electron transport. This study revealed a diverse range of metabolically flexible communities of microbes that contribute to the biogeochemical cycling of carbon, nitrogen, and sulfur.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 Fish Bioacoustics: From Basic Science to Policy(2024) Colbert, Benjamin; Bailey, Helen R; Popper, Arthur N; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Sound is critically important to fishes. Sound is used to communicate with conspecifics, to detect predators and prey, or to otherwise understand the world around them. Within this dissertation, I used a variety of methods to investigate multiple aspects of fish bioacoustics, including hearing, hearing in noise, the effects of anthropogenic sound, and the morphology of peripheral auditory structures.In Chapter 2, I reviewed international policy on the regulation of underwater sound and the effects of underwater sound on marine and aquatic habitats. I found that while there are increasing efforts to regulate underwater noise, the policy efforts are hampered by a lack of quantifiable metrics associated with impacts of anthropogenic sound in aquatic habitats and species. In Chapter 3, I measured auditory sensitivity of cyprinids using physiological methods. Auditory evoked potentials, a physiological measure of auditory sensitivity, have been used in previous studies to measure hearing sensitivity. However, while physiological methods have their place, they are measuring the sensitivity of the ear rather than the entirety of the auditory pathway. Therefore, I further measured hearing sensitivity of goldfish using behavioral methods that encompass the full auditory pathway. I found that physiological methods tend to underestimate actual hearing sensitivity at frequencies less than 1000 Hz. In Chapter 4, I investigated cyprinid hearing in noise, using both physiological and behavioral measures. Critical ratios were measured for four species of carp and goldfish using auditory evoked potentials. Behavioral methods were also used to measure critical ratios for goldfish. These data represent the first measurements of critical ratios for carp and the first comparative analysis between critical ratios measured using both physiology and behavior. I found that critical ratios for carp increase by as much as 25 dB between 300 Hz and 1500 Hz. I also found that physiological methods likely overestimate actual critical ratios for fish. In Chapter 5, I used micro-computed tomography (micro-CT) and three dimensional geometric morphometrics to compare the peripheral auditory structures of three species of carp. Three dimensional models of the tripus ossicle, the posterior most Weberian ossicle, and the sagitta otolith were created and the shape of these structures for silver carp (Hypophthalmichthys molitrix), bighead carp (H. noblis), and grass carp (Ctenopharyngodon idella) quantified and contrasted. I found that the shape of the tripus differed between the Hypophthalmichthys genus (i.e., silver and bighead carp) and Ctenopharyngodon (grass carp), demonstrating a possible phylogenetic signal in the shape of the Weberian ossicles. In Chapter 6, I studied the response of wild oyster toadfish (Opsanus tau) to underwater radiated noise from boats. I used passive acoustic monitoring to record toadfish vocalizations and vessel passages in the Chesapeake Bay, U.S.A. The effect of acute vessel passage was determined by comparing the number of calls after a vessel had passed to a control period. The effect of both aggregate vessel passage over an hour and environmental variables were investigated using generalized additive mixed models. I found that there was no significant effect on toadfish call rates from acute vessel passage but when vessel generated sound was higher over an hour long period (i.e., aggregate effect), call rate declined.Item SPATIOTEMPORAL DISTRIBUTION OF CHESAPEAKE BAY MYSIDS IN THE CHOPTANK AND PATUXENT RIVERS, MARYLAND(2024) Quill, Danielle; Woodland, Ryan; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The importance of mysids as trophic links in key Chesapeake Bay food webs has been well documented. However, their abundance, distribution, and demographics haven’t been examined in Chesapeake Bay since 1930. The goal of this study was to examine patterns of mysid abundances and demographic dynamics between and within two key Chesapeake Bay tributaries (the Choptank and Patuxent rivers). I hypothesized that mysid abundances would be greater in the Choptank River due to its historically better water quality (particularly dissolved oxygen saturation) than the Patuxent River. Secondarily, I hypothesized that Neomysis americana (hereafter, Neomysis) would be the most abundant mysid species in both the Chopank and Patuxent rivers. Six stations in each river were sampled monthly from May to September of 2018. Numerical dominance of the mysid assemblage in both rivers shifted from Neomysis in the early summer to a mixed-species group belonging to the genus Americamysis (Americamysis spp.) between August and September. Total abundance across genera and abundance of Neomysis were significantly greater in the Choptank River in early summer, then did not differ from Americamysis spp. abundance thereafter. Neomysis abundance was greater than Americamysis spp. from May through June, did not differ from Americamysis spp. abundance in July, and was less abundant than Americamysis spp. from August through September in the Patuxent River. The Patuxent River displayed overall lower dissolved oxygen saturation in the summer, which correlated with lower mysid abundances, providing support for my hypothesis. Understanding the intricacies of mysid population dynamics within nursery areas for ecologically and economically important predators should strengthen ecosystem-based management strategies for those areas.Item NITROGEN, MICROBES, PARTICLES AND OXYGEN DEFICIENT ZONES(2024) Huanca Valenzuela, Paulina Alejandra; Fuchsman, Clara A.; Cram, Jacob A.; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Single-celled microbes mediate most biogeochemical cycling in the ocean. Ammonium is generally the preferred reduced nitrogen form microbes use for assimilation and growth. However, ammonium is often removed to undetectable levels from offshore waters. Microorganisms can metabolize alternative organic reduced nitrogen forms in the absence of ammonium, if they possess genes encoding for the enzymes cyanase (cynS), and urease (ureC), which catalyze the decomposition of cyanate and urea respectively. It is unknown which microbes contain these genes in the environment.In my first chapter, I quantified the microbes that can use cyanate and/or urea in oxic and anoxic (ODZ) environments by using a phylogenetic read placement technique. First, I explored depth profiles of metagenomes from two Pacific Ocean regions: an oxic region represented by the nutrient limited Hawaii Ocean Time series, and two ODZ environments represented by the Eastern Tropical South and the North Pacific. A larger proportion of N2 producing anammox bacteria in ODZs have the ability to utilize cyanate than urea, while a larger proportion of nitrite oxidizing Nitrospina have the ability to utilize urea than cyanate. Ammonia-oxidizing Thaumarchaeota had the ability to use urea in deep oxic waters. Contrastingly, the majority of heterotrophic SAR11 bacteria had the ability to use urea in surface waters, but none did in deep waters. This structuring of who can utilize which reduced N form could reflect competition between microbes and N availability. For my second chapter, I examined microbial ability to use urea and cyanate across time and space using metagenomes from two oceanic Geotraces transects in the North Atlantic; GA02 a North-South spring transect, and GA03, a Fall West to East transect. The two transects differed in nutrient concentrations, affecting the composition of phytoplankton communities. Though eukaryotic phytoplankton were abundant on the spring GA02 transect, they did not have the ability to use urea or cyanate, probably because ammonia was present. However, the ability to use urea was still common in SAR11. Cyanobacteria Synechococcus was abundant on this transect and had the ability to use cyanate. In the nutrient limited fall GA03 transect, the results were similar to oxic waters in chapter 1 except that towards the east, cyanobacteria Prochlorococcus gained the ability to utilize cyanate. Both seasonal and spatial changes were observed in the distribution of ureC and cynS genes in microbial groups in the North Atlantic. My third chapter focuses on organisms living on suspended particles. Marine particles constitute a niche that provides ample nutrient and carbon sources. Large particles have been postulated to support anaerobic metabolism that cannot occur in the surrounding water. We examined how microbial diversity changes among a range of 7 different particle sizes in a depth profile at the East Pacific Rise, an area of the ocean with a distinct oxygen minimum. By combining a quantitative 16S rRNA amplicon sequencing dataset with size fractionated organic matter concentrations, we estimated numbers of each microbial taxa per gram of carbon. Results show differences in microbial composition at different particle sizes and depths.Item EXPLORING NEURAL REPRESENTATIONS IN MACAQUE PRIMARY VISUAL CORTEX THROUGH DATA-DRIVEN MODELS(2024) Bartsch, Felix; Butts, Daniel A; Neuroscience and Cognitive Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The study of the primary visual cortex (V1) holds profound significance for our understanding of the neural underpinnings of visual perception. Computational models have emerged as invaluable tools to decode the intricate computations occurring within V1 neurons. This dissertation embarks on a comprehensive exploration of V1 by fitting statistical models to electrophysiological data and scrutinizing model properties. My approach not only provides direct insights into how V1 neurons represent information but also furnishes mathematical descriptions of V1 computations, thereby contributing to the construction of a unified model of V1 function.I begin in Chapter 2 by employing state-of-the-art statistical and machine learning techniques to unravel the high-resolution components of V1 receptive fields as they respond to random bar stimuli. I demonstrate how these models not only replicate classical findings but also offer superior explanations of the computations V1 undertakes. These results highlight how the simultaneous processing of multiple overlapping inputs enables cells to represent high-resolution information while also responding to full-field inputs, an intricate organization unattainable using conventional stimuli. In chapter 3, I expand this modeling approach by adding mechanisms for binocular integration and apply them to data obtained from random bar stimuli that also vary in binocular disparity. This approach reveals that V1 disparity selectivity is enhanced and well characterized using spatial convolutions. Finally, I further modify the approach in chapter 4 to map spatiotemporal receptive fields in luminance and color using data recorded from the fovea and present the first spatiochromatic measurements illustrating the scale of V1 processing at the fovea. I find that color signals operate at lower spatial scales compared to luminance signals, and that receptive field substructure can allow even cells with large receptive fields to represent fine-scale information throughout the fovea.Item Mental Workload Assessment During Upper Limb Prosthetic Training and Task Performance(2023) Gaskins, Peter Christopher; Gentili, Rodolphe J; Neuroscience and Cognitive Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mental workload, defined as the recruitment and allocation of cortical resources in response to task demands, is an integral underlying mechanism of cognitive-motor learning and performance. Although widely examined in individuals without motor impairment, the study of mental workload in a clinical context of motor rehabilitation is limited. In particular, it is not well understood how the cortical processes underlying mental workload adapt over time as individuals learn to operate upper-limb prosthetic devices. In this work, mental workload assessment using electroencephalography (EEG) along with other ancillary measurement tools were employed to examine the recruitment of cognitive-motor processes as individuals learned to operate either a body-powered (BP) or myoelectric (MYO) bypass prosthesis during a ten session upper limb prosthetic training program. The first two studies examined changes in mental workload and cognitive-motor performance as prosthesis users executed tasks requiring transport of objects with the same or different shape/size during a prosthetic training program. Then, these newly trained prosthesis users engaged in activities which manipulated contextual demands to examine how real-world scenarios affect mental workload and cognitive-motor performance. Finally, a preliminary validation of a novel mental workload self-report measure aimed to address the paucity of mental workload measurement tools in upper-limb rehabilitation was implemented. Although these four studies offer a rich and complex pattern of results, the main findings suggested that i) while both prosthetic groups experienced similar levels of cognitive-motor performance by the end of training, the BP group exhibited more refined cortical dynamics and better cognitive-motor efficiency when compared to the MYO group, thus indicating a more advanced progression of learning; ii) contextual demands degrade mental workload and cognitive-motor performance similarly in both prosthetic groups and; iv) the preliminary assessment of reliability and validity of the novel mental workload self-report measure shows promise for capturing changes in mental workload during cognitive-motor performance in a rehabilitation context. Although more research is warranted to confirm and extend the findings of this work to clinical upper-limb prosthesis users, this work has the potential to inform the cognitive-motor processes in this population and inform prescriptive decisions for patient device selection, prosthetic device design and rehabilitation program development.Item Sex and the Evolution of a Double Hermaphrodite(2023) Ficklin, John Alexander; Haag, Eric S; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Kryptolebias marmoratus species complex contains the only known self-fertile hermaphroditic vertebrates. There are three species in this clade and all three live in the mangrove forests across the tropical Americas. All three have individuals with both testis and ovarian tissue in their gonads with two using self-fertility as their main mode of reproduction, and all three have apparent different sex determination and sexual modes. In this dissertation, I explore aspects of sex in these species. K. marmoratus is the androdiecious and self-fertile member of the species complex with sequential hermaphroditism. In this species, the control of sex change from hermaphrodite to male is poorly understood. Individuals that were believed to be genetically identical could be raised in the same environment and change sex at drastically different times or not at all. Small fluctuations and variance in the hormonal profiles of individuals was thought to be a potential cause and while androgen dosing can lead to masculinization of both the gonad and the soma, it was not enough to maintain a permanent transition like what is seen in nature. In K. ocellatus, the obligate outcrosser of the K. marmoratus species complex, it was believed that they were using genetic sex determination to differentiate between males and the females that had hermaphroditic gonads. While we found strong evidence against heteromorphic sex chromosomes, all tests for homomorphic sex chromosomes came back inconclusive due to apparent K. hermaphroditus DNA contaminating the dataset. K. hermaphroditus, the self-fertile hermaphrodite species with exceptionally rare males, appears to be extending its range further and further south and/or hybridizing with K. ocellatus at rates previously underappreciated. The hermaphrodites of the Kryptolebias genus still hold many evolutionary and physiological secrets but can potentially be revolutionary to the understanding of vertebrate sexual development and evolution.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 REFINING METAGENETIC ENVIRONMENTAL DNA TECHNIQUES FOR SENSITIVE BEE COMMUNITY MONITORING(2023) Avalos, Grace; Richardson, Rodney T; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Molecular taxonomic detection is now widespread across the sciences, because of advances in direct PCR, improved marker selection, and increases in sequencing throughput. Facilitated by these advances in sequencing, methodological sensitivity of sample identification has improved substantially. Metagenetic techniques to infer what species are present in a sample by sequencing unknown samples and comparing them to known references has the potential to advance our understanding of biodiversity. Metagenetic analysis of environmental DNA (eDNA) represents a novel, non-lethal method for characterizing floral-associated arthropod communities. Diverse arthropod assemblages interact with flowers, and floral surfaces have been shown to harbor arthropod DNA. We performed metagenetic sequencing on eDNA isolated from flower samples and honey bee-collected pollen samples using multiple markers and compared the frequency and taxonomic breadth of eDNA detections across these genetic markers and substrate types. Understanding which markers and substrates are most effective for eDNA characterization of floral-associated arthropod communities will guide future research and enable low-risk detection of threatened or endangered arthropods.Item EFFECTS OF ENVIRONMENTAL VARIABLES AND CHANGES IN SEASONAL PATTERNS ON SPATIAL DISTRIBUTIONS OF JONAH CRABS (CANCER BOREALIS) AND ATLANTIC ROCK CRABS (CANCER IRRORATUS) IN GEORGES BANK AND THE MID-ATLANTIC BIGHT, USA(2023) Wade, Kaitlynn Jean; Wilberg, Michael J; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The economic and commercial importance of Jonah crabs (Cancer borealis) and Atlantic rock crabs (Cancer irroratus) has increased greatly in the USA. The objectives of my research were to determine spatial distributions, habitat preferences, and potential seasonal movements of both species. Data were obtained from the offshore Northeast Fishery Science Center bottom trawl surveys. Analyses included kernel density estimates, generalized additive models, empirical cumulative distribution functions, and ANOVAs. The spatial distributions of Jonah and Atlantic rock crabs changed over time during the 1970s – 2000s. Compared to Atlantic rock crabs, Jonah crabs preferred slightly warmer temperatures, deeper depths, and muddier sediments. Seasonally, Jonah crabs were found farther offshore in the winter and closer to shore in the fall and spring. Atlantic rock crabs were found closer inshore in the winter and spring and more offshore in the fall. Both species were found to have different seasonal patterns in the Mid-Atlantic BightItem MEGAPOOLS: VEGETATION DIEBACK AND RESTORATION POTENTIAL OF A DITCHED COASTAL SALT MARSH(2023) Stahl, Katherine A.; Baldwin, Andrew H; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In many ditched coastal salt marshes, megapools, or large ponded areas of vegetation dieback,have formed. In combination with sea level rise, this interior marsh loss can decrease wildlife habitat suitability, resilience to storms, and other ecosystem services. However, mechanisms of megapool formation are poorly understood, hampering restoration efforts. Here, we explored differences in environmental characteristics between megapools in different stages of formation (Fully Formed, Partially Formed and Nonformed/Control) and between Elevations within megapools (High, Medium, Low). Using IRIS Films (Indicator of Reduction in Soil), we found that Fully Formed megapools had higher sulfide concentrations than Partially formed, which in turn were greater than Nonformed megapools. We additionally found that lower elevations correlated with higher sulfides, lower plant coverage, lower belowground biomass, lower Carbon Density, and predicted megapool type. We noted that in terms of elevation, vegetative cover, and biomass, Nonformed and Partially formed were more similar as were High and Medium elevations. Whereas in terms of soil characteristics, Fully Formed and Partially formed were more similar as were Medium and Low Elevations. To combat megapools and dieback, we will assess the effectiveness of two restoration techniques, the first of which is assessing the survival and growth of plantings at different spacings, elevations, and megapool formation levels. We found survival and growth was higher in Partially formed megapools than Fully formed, and no impact by spacing or elevation. Our second restoration technique is runnels, or 15” channels that reconnect megapools to ditches, which were installed in January of 2023. The data collected above will act as baseline data, repeated again. These baseline results support a close relationship between pool stages of formation, carbon storage, elevation, vegetation health, biomass production, and sulfide levels (Graphical Abstract).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 INTERACTIONS BETWEEN NITROGEN AND TEMPERATURE ON THE METABOLISM OF THE RED-TIDE MIXOTROPHIC DINOFLAGELLATE KARENIA SPP. IN SUPPORT OF PREDICTIVE MODELS: IMPLICATIONS FOR BLOOM DYNAMICS ON THE WEST FLORIDA SHELF(2023) Ahn, So Hyun; Glibert, Patricia; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The toxic mixotrophic dinoflagellate Karenia spp. forms blooms almost annually in the Gulf of Mexico, especially on the West Florida Shelf (WFS). Blooms typically initiate in early fall but can persist from months to years. Daily, Karenia vertically migrates to the surface water during the day, possibly experiencing changes in temperature, light, nitrogen (N), and prey type and availability. Therefore, this dissertation aimed to examine the interplay between Karenia’s photo-autotrophic and phago-mixotrophic metabolism and the short-term fluctuations in environmental conditions to understand how these factors may relate to the conditions under which Karenia spp. are found in the WFS.Title of Dissertation: INTERACTIONS BETWEEN NITROGEN AND TEMPERATURE ON THE METABOLISM OF THE RED-TIDE MIXOTROPHIC DINOFLAGELLATE KARENIA SPP. IN SUPPORT OF PREDICTIVE MODELS: IMPLICATIONS FOR BLOOM DYNAMICS ON THE WEST FLORIDA SHELF So Hyun (Sophia) Ahn, Doctor of Philosophy, 2023 Dissertation directed by: Professor Patricia M. Glibert, Marine Estuarine Environment Sciences A culture of K. mikimotoi balanced photon flux pressure (light availability) with consumption in overall metabolism when pulsed with 15N-NO3-, 15N-NH4+, or 15N-urea over the range of 15-25°C as shown by photosynthetic fluorescence. However, when shifted to 30°C, cells were significantly stressed, but urea-enriched cells showed a smaller decline in fluorescence, implying that urea might induce a photoprotective mechanism by increasing metabolic “pull.” Studies conducted with natural K. brevis winter and summer populations during 2021 showed that thermal history played a critical role. Unusually, summer blooms had higher biomass but were stressed photosynthetically and nutritionally. However, 15N-urea enriched summer cells had higher uptake rates as well as carbon (C) and N cell-1, especially in warmer waters, showing differential thermal responses based on N forms. Mixotrophy grazing measurements showed that K. brevis grazed both the picoplankter Synechococcus as well as the cryptophyte Rhodomonas. Grazing did not selectively target specific qualities of Synechococcus (based on differing N and P of the prey growth media), but ingestion rates were a function of prey-to-grazer ratios (R2=0.76) as well as prey amounts (R2=0.71). NanoSIMS confirmed 15N incorporation from Synechococcus in K. brevis. In natural communities of K. brevis, ingestion rates were also significantly related to prey-to-grazer ratios (p < 0.01) and by temperatures (p < 0.05) to a lesser degree (R2= 0.75) when incubated at ambient (24°C) and ambient temperature ± 5°C (19, 29°C). The grazer effects on the photosynthetic performance of grazer and prey were also examined. Grazing on Synechococcus indirectly reduce the photosynthetic performance of prey, especially at warmer temperatures but had little or no effect on the photosynthesis of K. brevis itself.Item Application of advanced machine learning strategies for biomedical research(2023) Chou, Renee Ti; Cummings, Michael P.; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Biomedical research delves deeply into understanding individual health and disease mechanisms. Recent advancements in technologies have further transformed the field with large-scale data sets, enabling data-driven approaches to identify important patterns and relationships from large data sets. However, these data sets are often noisy and unstructured. Moreover, missing values and high dimensionality further complicate the analysis processes aimed at yielding meaningful results. With examples in ocular diseases and malaria, this dissertation presents novel strategies employing machine learning to tackle some of the challenges in biomedical research. In ocular diseases, sustained ocular drug delivery is critical to retain therapeutic levels and improve patient adherence to dosing schedules. To enhance the sustained delivery system, we engineer peptide sequences as an adapter to impart desired properties to ocular drugs. Specifically, we develop machine learning models separately for three properties–melanin binding, cell-penetration, and non-toxicity. We employ data reduction techniques to reduce the number of features while maintaining the machine learning model performance and apply interpretable machine learning techniques to explain model predictions on the three properties. Experimental validation in rabbits show two-fold increase in drug retention time with the selected peptide candidate. The developed machine learning framework can be further tailored to engineer other properties in molecular sequences with a wide variety of potential in biomedical applications. Malaria is an infectious disease caused by protozoan of the genus Plasmodium and has been a burden in global health. Developing malaria vaccines is challenging due to the diversity in parasite antigen sequences, which may lead to immune escape. To facilitate the vaccine development process, we leverage the wealth of systems data collected from various sources. For facile data management, a database is constructed to store the structured data processed from the results of the bioinformatics tools. Due to the small fraction of Plasmodium proteins labeled as known antigens, and the remaining proteins unknown of being antigens or non-antigens, a positive-unlabeled machine learning method is applied to identify potential vaccine antigen candidates. Beyond malaria, our approach provides a promising framework for identifying and prioritizing vaccine antigen candidates for a broad range of disease pathogens.Item STUDIES ON THE GNRH/GTH SYSTEM OF FEMALE STRIPED BASS (Marone saxatilis): EFFECTS OF GNRH AGONJST THERAPY AND COMPARISON OF REPRODUCTIVE ENDOCRINE PARAMETERS BETWEEN WILD AND CAPTIVE FISH(1999) Steven, Colin R.; Zohar, Yonathan; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, MD)In the striped bass (Marone saxatilis), and many other commercially important fish species, captivity results in an inability to complete final oocyte maturation (FOM), ovu lation , and spawning. We hypothesize that this effect is mediated by a disruption of the hypothalamo-pituitary-gonadal (HPG) axis at the level of the gonadotropin -releasing hormones (GnRHs). To confirm this hypothesis research was conducted focused on three objectives: First, to complement the battery of assays previously developed for analysis of the GnRH/GtH system in striped bass, an RNAse protection assay was developed to measure specific expression of the three forms of GnRH in striped bass (salmon GnRH, chicken GnRH-If and seabream GnRH). Secondly, effects of GnRH agonist-induced ovulation on the HPG axis of captive striped bass was examined by comparison of several reproductive endocrine parameters between females sampled at four stages of oocyte development. Finally, differences were examined between the reproductive endocrine status of wild and captive female striped bass. We conclude that sbGnRH is the most important form for the preovulatory release of pituitary GtH-II in striped bass. We suggest that captive females synthesize levels of GnRH mRNA that are comparable to their wild counterparts, however fail to release adequate quantities of bioactive GnRH within the pituitary to stimulate completion of FOM. This data may indicate that regulation of sbGnRH in striped bass occurs via post-transcriptional/ translational mechanisms. Furthermore, the presence of salmon GnRH in the pituitaries of captive fema les may be indicative of a possible role for salmon GnRH in the regulation of FOM.