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Acute and Sublethal Impacts of Crude Oil Photo-Induced Toxicity in an Early Life Stage Marine Fish (Sciaenops ocellatus) and Invertebrate (Americamysis bahia): We investigated the modifying effects of ultraviolet (UV) light and chemical dispersant (Corexit 9500A) on crude oil toxicity in juvenile mysids (≤ 24 h) (Americamysis bahia) and larval red drum (24-72 hpf) (Sciaenops ocellatus). These results demonstrate that crude oil toxicity significantly increases with co-exposure to environmentally relevant UV levels in both species, indicating photo-induced toxicity. This toxicity was further exacerbated by the application of chemical dispersants which increased the dissolution and concentration of oil-derived polycyclic aromatic hydrocarbons (PAHs) in test solutions. To better understand the mechanisms and initiating events of this observed photo-induced toxicity, the incidence of apoptotic cell death and global transcriptomic changes were assessed in larval red drum (24-72 hpf) following co-exposure to crude oil and UV. These results showed that co-exposure to UV and low concentrations of crude oil (<1 µg/L ∑PAH50) induced apoptotic cell death in skin and eye tissue and altered transcriptomic pathways related to visual processing and dermatological disease. To link these cellular and molecular impacts of photo-induced toxicity to apical endpoints of ecological performance, sublethal impacts to growth, metabolic rate, and visually mediated behaviors were explored in larval red drum at 2 developmental stages. These results suggested that earlier life stages may be more sensitive to photo-induced toxicity and that growth and development, particularly of sensory systems, can be sensitive targets of photo-induced toxicity. Together, these studies provide novel insights into the photo-induced toxicity of crude oil in aquatic organisms and can be used to inform future ecological risk assessments.
Avian Community Response to Riverby Ranch Restoration Reconstruction: Anthropogenic activities have caused many wildlife spices to decline in populations worldwide. The grassland bird communities are especially being impacted by these land use changes. Breeding success is closely tied to functional habitats for most grassland bird species in North Texas. Restoring these degraded habitats is an important component to aid in conserving wildlife biodiversity. We surveyed the bird population at Riverby Ranch Mitigation site by conducting point count sampling. This site consists of recently restored grassland, wetland, and forested habitat. This research was focused on conducting post restoration monitoring of the bird community in the early succession of the restored habitats. We set out to use the bird community as biomonitors to help assess if the restoration practices could be considered successful. We found that density estimates were more than double at the restored site when comparing to three different references sites under different management practices. This included an unrestored working ranch, a wildlife management area, and a conservation managed prairie site. We found that biodiversity metrics were as high or higher than the reference sites. In addition, we also found that there were more observations of species of high conservation concern present at the restored stie. This research supports that the reconstructive restoration that took place at Riverby Ranch was highly effective at restoring a diverse and abundant grassland and wetland bird community.
Bottom-Up Impacts of Grazing Disturbance on Ground-Nesting Bee Assemblages: Do They Dig It?: In my thesis, I examined impacts of nesting and floral resources on ground-nesting bee assemblages at nine ranch sites with differing grazing histories. Because ecological disturbances can strongly affect the availability of foraging and nesting resources over time, I also examined the impacts of grazing history on nesting and floral resources for ground-nesting bees. I sampled bee assemblages, a comprehensive array of vegetation and soil measures, and floral abundance and richness. I used these data to determine (1) the importance of different nesting habitat resources and livestock grazing history for ground-nesting bees and (2) how livestock grazing history influenced the availability of nesting and floral resources. I found that sites with sandier soils had greater abundance and richness of ground-nesting bees, and sites with less compacted soils had greater bee abundance. Contrary to many other studies, the availability of bare ground was not important for ground-nesting bee abundance or richness. Grazing history did not affect any measures of nesting or floral habitat, and had negligible effects on ground-nesting bee abundance, richness, and community composition. My results suggest that the availability of sandier or less compacted soils may be limiting for ground-nesting bee abundance or richness, especially in areas with predominately clay soil types. In this context, the availability of such nesting resources may be more limiting than floral resource availability. More research is necessary to tease apart the effects of different nesting and floral resources for bees, and how different grassland management types affect bees through those resources. My findings highlight the importance of considering nesting habitat resources in bee conservation and restoration efforts.
The Effect of Developmental Hypoxia on Cardiac Physiology in Three Species: Alligator mississippiensis, Chelydra serpentina, and Danio rerio: In this dissertation, I explored the effects of developmental hypoxia on heart contractility in three separate species of ectotherms: the common snapping turtle (Chelydra serpentina), the American alligator (Alligator mississippiensis), and the zebrafish (Danio rerio). I began with the common snapping turtle and tested whether the utilization of the sarcoplasmic reticulum was altered in response to developmental hypoxia. In the next two chapters, developmental hypoxia of the American alligator was explored studying how the cardiac tissue was affected, specifically in physiological stressors, sarcoplasmic reticulum utilization and sensitivity to pharmacological increases in contractility. The last chapter explored how zebrafish heart contractility was altered in response to chronic hypoxia from egg to adult. Findings from these chapters suggest that while developmental hypoxia did alter cardiac contractility, it did not alter the response of the heart to physiological stressors such as increased heart rate or under hypoxia. Overall, these findings contribute to increasing the current understanding of how developmental hypoxia alters the cardiovascular system but with an emphasis on the cardiac tissue level.
Factors Affecting MeHg Contamination of Spiders and Insect-Mediated MeHg Flux from Human-Made Ponds: The present study focused on methylmercury (MeHg) in emergent aquatic insects and spiders from human-made ponds. This dissertation addresses two main topics: (1) factors affecting variation in spider MeHg concentrations around human-made ponds and (2) the magnitude of MeHg transported out of human-made ponds by emergent aquatic insects (insect-mediated MeHg flux). Spiders were specifically targeted in this study because they have been proposed as sentinels of MeHg contamination (organism whose tissue concentrations reflect the level of MeHg in the environment). Spider MeHg concentrations were related to spider diet, size, and proximity to waterbody, but affected individual spider taxa differently. In a second study, I found that only "large" spiders within a taxa had tissue concentrations positively related to prey MeHg concentrations. These results indicate that the relationship between spider and prey MeHg could be size-dependent and that "large" spiders within a taxa may better reflect ambient MeHg contamination. Finally, I tested a conceptual model hypothesizing insect-mediated MeHg flux from human-made ponds is controlled by pond permanence and fish presence. In agreement with the conceptual model, insect-mediated MeHg flux from ponds was suppressed by the presence of fish, likely due to fish predation on emergent insect larvae. I found the mean aggregate MeHg flux was approximately 6 times higher from ponds without fish than from ponds with fish. The suppression of insect flux by fish was stronger for large insect taxa than small insect taxa. Results of this study indicate that community structure can influence the cross-system transport of contaminants, like MeHg, from ponds to terrestrial food webs.
Fatty Acid Amide Hydrolases in Upland Cotton (Gossypium hirsutum L.) and the Legume Model Medicago truncatula: Fatty acid amide hydrolase (FAAH) is a widely conserved amidase in eukaryotes, best known for inactivating the signal of N-acylethanolamine (NAE) lipid mediators. In the plant Arabidopsis thaliana, FAAH-mediated hydrolysis of NAEs has been associated with numerous biological processes. Recently, the phylogenetic distribution of FAAH into two major branches (group I and II FAAHs) across angiosperms outside of Arabidopsis (and in other Brassicaceae), suggests a previously unrecognized complexity of this enzyme. Although A. thaliana has long been used to assess biological questions for plants, in this case it will fall short in understanding the significance of multiple FAAHs in other plant systems. Thus, in this study, I examined the role (s) of six FAAH isoforms in upland cotton (Gossypium hirsutum L.) and two FAAHs in the legume Medicago truncatula.
Glucose-Induced Developmental Delay is Modulated by Insulin Signaling and Exacerbated in Subsequent Glucose-Fed Generations in Caenorhabditis elegans: In this study, we have used genetic, cell biological and transcriptomic methods in the nematode C. elegans as a model to examine the impact of glucose supplementation during development. We show that a glucose-supplemented diet slows the rate of developmental progression (termed "glucose-induced developmental delay" or GIDD) and induces the mitochondrial unfolded protein response (UPRmt) in wild-type animals. Mutation in the insulin receptor daf-2 confers resistance to GIDD and UPRmt in a daf-16-dependent manner. We hypothesized that daf-2(e1370) animals alter their metabolism to manage excess glucose. To test this, we used RNA-sequencing which revealed that the transcriptomic profiles of glucose-supplemented wildtype and daf-2(e1370) animals are distinct. From this, we identified a set of 27 genes which are both exclusively upregulated in daf-2(e1370) animals fed a glucose-supplemented diet and regulated by daf-16, including a fatty acid desaturase (fat-5), and two insulin-like peptides (ins-16 and ins-35). Mutation of any of these genes suppresses the resistance of daf-2(e1370) to GIDD. Additionally, double mutation of ins-16 and ins-35 in a daf-2(e1370) background results in an increase in constitutive dauer formation which is suppressed by glucose supplementation. Further investigation of the insulin-like peptides revealed that ins-16 mutation in a wild-type background results in upregulation of ins-35 and DAF-16 nuclear translocation regardless of diet; however, unlike daf-2(e1370), this translocation is not associated with resistance to GIDD. Taken together, these data suggest that glucose-supplemented daf-2(e1370) animals maintain developmental trajectory in part through upregulation of specific insulin-like peptide genes and fatty acid desaturation and contribute to a deeper understanding of the mechanisms underlying the resistance of daf-2(e1370) animals to GIDD. We also showed another fascinating aspect of GIDD: it becomes more pronounced in subsequent generations exposed to a glucose-supplemented diet, suggesting that the parental glucose diet has an impact on the developmental progression of their offspring.
Investigating Novel Streptomyces Bacteriophage Endolysins as Potential Antimicrobial Agents: As antibiotic resistance has become a major global threat, the World Health Organization has urgently called scientists for alternative strategies for control of bacterial infections. Endolysin, a protein encoded by a phage gene, can degrade bacterial peptidoglycan (PG). Currently, there are three endolysin products in the clinical phase. We, thus, are interested in exploring novel endolysins from Streptomyces phages as only a few of them have been experimentally characterized. Using bioinformatics tools, we identified nine functional domain groups from 250 Streptomyces phages putative endolysins. NootNoot gp34 (transglycosylase; Nt34lys), Nabi gp26 (amidase; Nb26lys), Tribute gp42 (PGRP; Tb42lys), and LazerLemon gp35 (CHAP; LL35lys) were selected for experimental studies. We hypothesized that (1) the proteins of interest will have the ability to degrade PG, and (2) the proteins will be potential antimicrobial agents against ESKAPE safe relatives. The results showed that LL35lys, Nb26lys and Tb42lys exhibit PG-degrading activity on zymography and hydrolysis assay. The enzymes (400 µg/mL) can reduce PG turbidity to 32-40%. The killing assay suggested that Tb42lys possess a boarder range (Escherichia coli, Pseudomonas putida, Acinetobacter baylyi and Klebsiella aerogenes). While Nb26lys can attack Gram-negative bacteria, LL35lys can only reduce the growth of the Gram-positive strains with an MIC90 of 2 µg/mL. A higher concentration (≥300 µg/mL) of Nb26lys is needed to treat P. putida and K. aerogenes. Therefore, endolysins from Streptomyces phage have potential as possible antimicrobial agents against ESKAPE bacteria.
Investigating the Molecular Framesworks of Phloem-Cap Fiber Development in Cotton (Gossypium hirsutum): The current study focuses on the vascular cambium and the reiterative formation of phloem fiber bundles in cotton stems. The role of the TDIF-PXY-WOX pathway was examined in regulating cambial activity and the differentiation of phloem fibers. A study was conducted to identify and characterize the cotton WOX family genes, focusing on WOX4 and WOX14, aiming to identify and analyze their phylogenetic relationships, tissue-specific expression profiles, functional roles, and metabolic consequences. Through a sequence analysis of the Gossypium hirsutum genome, 42 cotton loci were identified as WOX family members. GhWOX4 exhibited a close homology to 7 loci, while GhWOX14 displayed homology with 8 loci. Tissue-specific expression analysis revealed prominent expression patterns of GhWOX4 and GhWOX14 in cotton internodes and roots, suggesting their involvement in vascular tissue development. Functional studies utilizing VIGS (virus-induced gene silencing) demonstrated that the knockdown of GhWOX4 and GhWOX14 resulted in a significant reduction in stem diameter and bast fiber production. This result suggests that secondary phloem fiber development is regulated by GhWOX4 and GhWOX14 genes in cotton. Additionally, the metabolic profiling of VIGS plants revealed significant alterations in amino acids, organic acids, and sugars, with implications for primary metabolic pathways. These findings suggest that GhWOX4 and GhWOX14 play pivotal roles in cotton plant development, including vascular tissue growth and phloem fiber production, and metabolic regulation.
Proteomic-Based Assessment of Estrogenic Endocrine Disruption in Hyalella azteca: In our studies, we used the environmentally important crustacean Hyalella azteca (H. azteca) as an invertebrate model and 17β-estradiol (E2) as a representative of environmental estrogenic endocrine disrupting compounds (EDCs) for proteomics-based investigations of endocrine disruptions in an aquatic ecosystem. Using liquid chromatography coupled with tandem mass spectrometry, our investigation focused for the first time on the recognition of biological and molecular events affected by E2 exposure with the long-term goal of identifying panels of potential biomarkers for environmental estrogenic endocrine disruption. We analyzed E2-induced changes in protein expressions in female and male H. azteca using label-free quantitative proteomics. With discovery-driven shotgun approach, we identified over 50 proteins that were affected by E2 in a sex-specific manner in our model organism. We selected four E2-regulated proteins (vitellogenin, cuticle protein CPR RR, titin and clumping factor A-like protein) for validation by parallel reaction monitoring-based targeted proteomics. Altogether, our proteomics studies have characterized for the first time E2-triggered endocrine disruption in H. azteca and recognized sex-specific changes in the male and female H. azteca's proteome after aquatic exposure to this estrogen. Through targeted proteomics, we were also able to quantitatively characterize a panel of selected proteins that showed distinctive sex-specific responses to this estrogen. We also mapped differentially expressed proteins to protein interaction networks and biological processes through Ingenuity® Pathway Analysis to indicate the future applicability of a bioinformatics-supported strategy focusing on potential environmental biomarker discovery and development using H. azteca as a model.
Suburban Succession and Stream Dynamics: Increasingly higher numbers of people are moving into urbanizing environments, yet our understanding of ecosystem consequences of rapid urbanization is still in its infancy. In this dissertation, I assessed dynamics of residential landscapes during suburban succession and consequences for ecosystem functioning. First, I used a space-for-time approach to quantify more than a century of suburban succession in the Dallas – Fort Worth metroplex (DFW). Attributes of residential landscape plant diversity and habitat complexity were quantified for 232 individual properties nested within 14 neighborhoods constructed between 1906 and 2020. Suburban succession progressed from simple turf lawns with limited habitat complexity to landscapes dominated by deciduous trees and high habitat complexity, but homeowner decisions related to landscape management affect the rate of that transition and the number of plants and taxa present. Next, I used the novel spatial construct of "neighborhoodsheds" to test for effects of suburban succession on carbon export, and found that the proportion of carbon derived from C3 vs. C4 plants was affected by neighborhood plant community structure (i.e. greater proportion of trees and shrubs primarily in later stages of suburban succession). Finally, I conducted a mesocosm experiment to test effects of changes in allochthonous inputs during suburban succession on aquatic ecosystem functioning. The proportion of carbon from C3 vs. C4 sources interacted with time to affect abundance of emergent mosquitoes and chironomids. This work sets the stage for further research on urban ecology in DFW and provides conceptual advances for the study of urban ecosystems more broadly.
Air Breathing Fish: Development of Air Breathing in Bristlenose Plecos (Ancistrus cirrhosus): The bristlenose pleco (Ancistrus cirrhosus) is a species of armored catfish in the Loricariidae family that breathes air facultatively when the aquatic environment becomes hypoxic. The bristlenose pleco uses its highly vascularized stomach as an air breathing organ. The two main goals of this developmental study were to determine the size of onset of air breathing and to determine the frequency of air breathing behavior in bristlenose plecos from juveniles to adults. Developing juveniles reach functional maturity within four to six months of hatching and grow to an adult size of eight to ten cm in length. To examine the developmental timing for the onset of air breathing, we tested different sized juveniles beginning at one cm up until 8 cm in length. The developmental timing for the onset of air breathing was measured by exposing each fish to a slowly decreasing aquatic oxygen content from 100% air saturation down to 8% air saturation. Fish were first able to breathe air at just over 2 cm and 1 gram in mass. There was a weak negative correlation between fish length and % air saturation at which air breathing began. When exposed to 15% air saturation, frequency of air breathing was negatively correlated with fish length. Armored catfish are becoming an invasive species in the southern US, outcompeting local fauna potentially because of this adaptation. This research provides important insight into the development of the air breathing adaptation that may allow these fish to outcompete the others.
Anti-S2 Peptides and Antibodies Binding Effect on Myosin S2 and Anti-S2 Peptide's Ability to Reach the Cardiomyocytes in vivo and Interfere in Muscle Contraction: The anti-S2 peptides, the stabilizer and destabilizer, were designed to target myosin sub-fragment 2 (S2) in muscle. When the peptides are coupled to a heart-targeting molecule, they can reach the cardiomyocytes and interfere with cardiac muscle contraction. Monoclonal antibodies, MF20 and MF30, are also known to interact with light meromyosin and S2 respectively. The MF30 antibody compared to anti-S2 peptides and the MF20 antibody is used as a control to test the central hypothesis that: Both the anti-S2 peptides and antibodies bind to myosin S2 with high affinity, compete with MyBPC, and possibly interact with titin, in which case the anti-S2 peptides have further impact on myosin helicity and reach the heart with the aid of tannic acid to modulate cardiomyocytes' contraction in live mice. In this research, the effects of anti-S2 peptides and antibodies on myosin S2 were studied at the molecular and tissue levels. The anti-myosin binding mechanism to whole myosin was determined based on total internal reflectance fluorescence spectroscopy (TIRFS), and a modified cuvette was utilized to accommodate this experiment. The binding graphs indicated the cooperative binding of the peptides and antibodies with high affinity to myosin. Anti-myosin peptides and antibodies competition with Myosin Binding Protein C (MyBPC) was revealed through the super-resolution expansion microscopy using wildtype skeletal and cardiac myofibrils, and MyBPC knock-out cardiac myofibril. This new emerging technique depends on using the regular confocal microscope in imaging expanded myofibril after embedding in a swellable hydrogel polymer and digestion. A decrease in the fluorescent intensity at the C-zone was observed in myofibrils labeled with fluorescently labeled anti-S2 peptides or antibodies supporting the competition with MyBPC, which further was confirmed by the absence of this reduction at the C-zone in the knockout MyBPC cardiac tissue. The anti-S2 peptide's ability to reach inside the cardiomyocytes was tested by …
Detection and Classification of Cancer and Other Noncommunicable Diseases Using Neural Network Models: Here, we show that training with multiple noncommunicable diseases (NCDs) is both feasible and beneficial to modeling this class of diseases. We first use data from the Cancer Genome Atlas (TCGA) to train a pan cancer model, and then characterize the information the model has learned about the cancers. In doing this we show that the model has learned concepts that are relevant to the task of cancer classification. We also test the model on datasets derived independently of the TCGA cohort and show that the model is robust to data outside of its training distribution such as precancerous legions and metastatic samples. We then utilize the cancer model as the basis of a transfer learning study where we retrain it on other, non-cancer NCDs. In doing so we show that NCDs with very differing underlying biology contain extractible information relevant to each other allowing for a broader model of NCDs to be developed with existing datasets. We then test the importance of the samples source tissue in the model and find that the NCD class and tissue source may not be independent in our model. To address this, we use the tissue encodings to create augmented samples. We test how successfully we can use these augmented samples to remove or diminish tissue source importance to NCD class through retraining the model. In doing this we make key observations about the nature of concept importance and its usefulness in future neural network explainability efforts.
Developmental Effects of a Non-Dioxin-Like Polychlorinated Biphenyl Mixture on Zebrafish (Danio rerio): PCBs are synthetic organic compounds known for their toxicity to many organisms and are notorious for having large discrepancies between measured and nominal concentrations. Historically thought to be less toxic, non-dioxin-like (NDL) PCBs represent the majority of congeners and are capable of eliciting neurotoxic effects. NDL-PCBs remain understudied, including their effects on aquatic organisms. In the first study, I collected extensive chemistry data and data on neurobehavioral and cardiac endpoints to test the acute effects of exposure to an NDL-PCB mixture on early life stage zebrafish. Neurobehavioral effects observed in the first study indicated a potential for longer term behavioral effects in these fish. In the second study, I collected data on feeding, social, and memory behavior of zebrafish at time points beyond the acute exposure from the first study. Acute and longer-term behavioral endpoints in the first and second studies demonstrated effects from PCB exposure but did not indicate mechanisms. In the third study, I collected untargeted and targeted metabolomic data on amino acid, sugar, anionic compound, and neurotransmitter profiles to determine the specific pathways affected by exposure to an NDL-PCB mixture. These combined data from these studies provide a unique insight into the chemical profile of an NDL-PCB mixture in biological applications and synthesize acute, longer-term, and mechanistic effects on developing zebrafish. These data fully illustrate an adverse outcome pathway from toxicokinetic to population level effects.
Hypoxia-Induced Cardiac Arrest Alters Central Nervous System Concentrations of the GLYT2 Glycine Transporter in Zebrafish (Danio rerio): Hypoxia as a stressor has physiological implications that have been a focal point for many physiological studies in recent years. In some studies, hypoxia had large effects on the organ tissue degeneration, which ultimately effects multiple ecological processes. These organ tissue studies played a part in the development of new fields like neurocardiology, a specialty that studied the relationship between the brain and the heart. This thesis focuses on how hypoxia-induced cardiac arrest alters the amounts of GLYT2, a glycine reuptake transporter, in the central nervous system of zebrafish, Danio rerio. At 7 days post-fertilization (dpf), zebrafish were exposed to acute, severe hypoxia until they lost equilibrium, and minutes later, subsequent cardiac arrest occurred. Zebrafish were then placed into recovery groups to measure the GLYT2 levels at multiple points in zebrafish recovery. Fish were then sacrificed, and their brains dissected. Using immunofluorescence, the outer left optic tectum of the zebrafish was imaged, and mean image pixel fluorescent intensity was taken. There were significant changes (one-way ANOVA) in the levels of GLYT2 compared to that of the control groups during the course of recovery. GLYT2 levels continued to rise through the 24-hour recovery mark but did not show significant difference after 3 hours of recovery. This suggest that GLYT2 levels increased rapidly in the first 3 hours of recovery and continued to increase through 24 hours at a slower rate. Changes in GLYT2 levels may affect motor and sensory information, movement, visualization, and audition in these zebrafish. Further research should be conducted to determine how long it takes for GLYT2 levels to return to baseline, as well as behavioral measurements through each recovery period as it relates to glycine function.
Identification and Characterization of Genes Required for Symbiotic Nitrogen Fixation in Medicago truncatula Tnt1 Insertion Mutants: In this dissertation I am using M. truncatula as a model legume that forms indeterminate nodules with rhizobia under limited nitrogen conditions. I take advantage of an M. truncatula Tnt1 mutant population that provides a useful resource to uncover and characterize novel genes. Here, I focused on several objectives. First, I carried out forward and reverse genetic screening of M. truncatula Tnt1 mutant populations to uncover novel genes involved in symbiotic nitrogen fixation. Second, I focused on reverse genetic screening of two genes, identified as encoding blue copper proteins, and characterization of their mutants' potential phenotypes. Third, I further characterized a nodule essential gene, M. truncatula vacuolar iron transporter like 8 (MtVTL8), which encodes a nodule specific iron transporter. I characterized the expression pattern, expression localization and function of MtVTL8. Additionally, I characterized several residues predicted to be essential to function using a model based on the known crystal structure of Eucalyptus grandis vacuolar iron transporter 1 (EgVIT1), a homologous protein to MtVTL8. I identified several potential essential residues of the MtVTL8 protein, mutagenized them, and through complementation experiments in planta and in yeast assessed functionality of the resulting protein. This helped us to better understand the potential mechanism by which MtVTL8 functions.
Linkage of the Nitrilase-Encoding Nit1C Gene Cluster to Cyanotrophy in Acinetobacter haemolyticus: The Nit1C cluster is a conserved gene cluster of seven genes that confers bacterial growth on cyanide as the sole nitrogen source. Bacteria with this ability are referred to as cyanotrophs. To date, the linkage between Nit1C and cyanotrophy has only been demonstrated for environmental isolates but the cluster also exists in certain medically related bacteria. In this study, a nosocomial isolate, Acinetobacter haemolyticus ATCC 19194, carrying Nit1C also displayed the ability to grow on cyanide. Growth on cyanide was accompanied by the induction of the cluster as was the mere exposure of cells to cyanide. Expression of the cluster was determined by measuring the activity of the nitrilase (NitC) coded for by the cluster and by transcriptional analysis (qRT-PCR). However, a disconnect between nitC message and NitC protein was observed depending on the phase of the growth cycle, the disconnect being related to proteolytic digestion of the NitC protein. Ironically, the cluster was also discovered to be upregulated in the absence of cyanide under nitrogen starvation conditions paralleling biofilm formation. The basis of the genetic linkage to cyanotrophy is not understood but taken together with results showing that nitrogen starvation and biofilm formation are also physiologically associated with Nit1C expression, points to a critical role for the cluster in stress-induced adaptation.
Neurotoxic Effects of Polycyclic Aromatic Hydrocarbons in Vertebrates, from Behavioral to Cellular Levels: Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental toxicants found in anthropogenic mixtures such as crude oil, air pollution, vehicle exhaust, and in some natural combustion reactions. Single PAHs such as benzo[a]pyrene (BaP) also impact fish behavior when animals are exposed in early life stages and for short periods of time. Aquatic animals such as fish may encounter BaP through road runoff and oil spills, but few studies have examined the impact of aqueous exposure on adult fish, and fewer have examined the resulting fitness-relevant behavioral consequences of BaP and PAH mixtures and their long-term persistence. This dissertation targets this gap in the literature by examining how aqueous exposure to BaP influences anxiety-like behavior, learning, and memory in adult zebrafish, and how parental exposure to the PAH mixture, crude oil, combined with hypoxia affects social and exploratory behavior in unexposed larval zebrafish. We found that learning and memory were not affected by 24 hour exposure to BaP, that anxiety-like behavior was minimally affected, and that locomotor parameters such as distance moved and times spent in darting and immobile states were significantly altered by exposure to BaP. Additionally, we found that parental exposure to crude oil and hypoxia decreased larval velocity. Additionally, we examined how crude oil, BaP, and the detergent COREXIT influence a monolayer of mouse and fish endothelial cells, as an in vitro blood-brain barrier (BBB) model. We found that exposure to BaP in particular caused significant damage to both fish and mammal in vitro BBB models, and damage to the BBB is one potential mechanism by which neural integrity and behavior may be influenced. Understanding how these toxicants influence fish brains and behavior will give insight into how fish populations explore and interact with their environment and with predators, and how these interactions persist even when toxicants are no …
Secondary Production of Dragonflies: Comparing Ecosystem Function of Ponds within an Urban Landscape in North Central Texas: The change of land use to include more urban areas is considered one of the main threats to biodiversity worldwide. Urban stormwater retention ponds have been built to collect storm runoff intensified by the increase in impervious surfaces. Although subject to environmental pressures like habitat degradation and pollution, these stormwater retention ponds are diversity hotspots by providing habitat for several aquatic and semi-aquatic species, including dragonflies. Previous research in Denton, Texas, has demonstrated that urban stormwater retention ponds support high taxa richness of adult dragonflies, but not for the aquatic nymphs. The current study builds on what we have seen by focusing on the immature aquatic stage as nymphs using secondary production of dominant dragonfly taxa and community structure to compare ecosystem function in three ponds with differing intensities of land use. Comparing communities and secondary production resulted in specific conductivity, dissolved oxygen, complex vegetation, and abundance explaining the differences between dragonfly communities. Secondary production was dependent on abundance which followed the intensity of urban land use surrounding the pond. This study supports that urban land use does have an effect on the functioning of the ponds and shows the importance of studying the communities over a year to get a clearer picture of what is happening in the ponds.
Combined Effects of Polycyclic Aromatic Hydrocarbons and Ultraviolet Light on Benthic and Pelagic Macroinvertebrates: Crude oil commonly enters freshwater aquatic ecosystems as thin sheens forming on the water surface. Oil contains mixtures of toxic compounds called polycyclic aromatic hydrocarbons (PAHs), some of which are known to be photodynamic, increasing toxicity when combined with ultraviolet radiation. Benthic macroinvertebrate communities are commonly utilized as bioindicators, and as such rely on abundant data in literature concerning benthic macroinvertebrates' relative tolerances to a wide range of pollutants. A series of 10 plastic traps, half of which were filtered from UV radiation, were deployed in an urban pond for 27 days to determine colonization preferences of benthic macroinvertebrates to UV exposure. Results of this in situ experiment indicated that the majority of aquatic insects collected from traps inhabited the UV exposed treatment group, particularly the nonbiting midge, Chironomidae. A series of bioassays were then completed to investigate the sensitivities of a Chironomidae species to thin sheens of crude oil in the presence and absence of UV radiation. All bioassays were conducted using 10 day old Chironomus dilutus larvae cultured in the lab. The series of C. dilutus bioassays were all conducted under the same water quality parameters, temperatures, and oil sheen dosing methods, under a 16:8 photoperiod and exposed to 16 h UV per day. Five replicates (n = 20) were loaded into 350 mL glass crystallizing dishes and exposed to four treatments for 96 hours: no UV/with sheen, with UV/no sheen, both UV and oil sheen, and a control. Three assays with 175 mL water volume were completed, one with no sediment, one with silica sand, and one with fine sand. Sediment type had a significant effect on mortality (p < 0.0001), but significant effects of UV or PAHs were not found. Two more C. dilutus assays were completed with identical parameters as the latter two with sediment, …
Conservation, Connectivity, and Coexistence: Understanding Corridor Efficacy in Fragmented Landscapes: Conservation corridors, areas of land connecting patches of natural land cover, are frequently cited and implemented as a restorative strategy to counteract fragmentation. Current corridor ecology focuses on experimental corridor systems or designed and built conservation corridors to assess functionality. Such systems and designs are typically short, straight swaths of homogenous land cover with unambiguous transitions between patches. Quantifying the degree to which amorphous landscape configurations, tortuosity, and heterogeneity of land cover and land uses within the corridor has on functional connectedness is a crucial yet overlooked component of corridor efficacy studies. Corridor literature lacks a robust and repeatable methodology for delineating existing landscape elements, recognizing arbitrary edges, and identifying the start and end of ambiguous transitions between the patches and corridor. Using a set of landscapes being studied as part of a global assessment of corridor efficacy, I designed a workflow that standardizes the boundary of corridor-patch interfaces. The proposed method is a quantitative and repeatable approach that minimizes the subjectivity in corridor delineations. This research investigates the degree to which the existence of a corridor modifies the structural and functional connectivity between patches connected by a corridor compared to an intact reference area.
Greater, Lesser, Guessers: A Look into the Hybridization of Greater and Lesser Prairie-Chickens: My thesis focuses on the conservation consequences of the hybridization of Lesser Prairie-Chickens in Kansas. Specifically, examining how past land management practices altering the species ranges impact the distinctiveness of Lesser Prairie-Chickens. Each chapter is an individual publication that addresses if the Greater and Lesser Prairie-Chicken are distinct when applying the morphological and biological species concepts. Chapter 2 compares the evolutionary history and morphological construct of Lesser Prairie-Chickens and other Galliformes using morphometric analysis. Chapter 3 uses low-resolution microsatellite data to reflect recent changes at the population level. This study aims to observe the Greater and Lesser Prairie-Chicken using the morphological and biological species concepts, two of the many species concepts, to determine the distinctiveness and rate of hybridization for these closely related species.
The Impact of Invasive Salmonids on Ecosystem Functioning in South America's Sub-Antarctic Inland and Marine Waters: Invasions from coho salmon were first reported in the Cape Horn Biosphere Reserve (CHBR) in 2019 which is the most southern distribution registered to date. The CHBR is known for its high number of endemic species and unique biodiversity, such as the native fishes Galaxias maculatus and Aplochiton taeniatus. There are now three invasive salmonid species in the rivers of CHBR and are a potential threat to the native fish taxa. Stable isotope and gut content analysis were used to understand resource utilization by both native galaxiid and invasive salmonid taxa, as well as aquatic macroinvertebrates and riparian spiders. The natural laboratory study approach applied to this research, allowed for comparisons of differences within streams that contain conditions in which fish do not occur naturally, to sites in which high densities of invasive salmonid exist. Analysis of the trophic niche and diet in this study showed the importance of marine resource use by the native galaxiid and coho salmon juveniles supported with elevated δ15N and δ34S ratios. Diet analysis also confirmed there was the highest similarity between the coho salmon juveniles and the native fish. Altered behavior and habitat use was shown through the isotope and diet analysis for the galaxiid in snow melt streams which could be due to the high density of invasive salmonids in these streams. The invasive salmonids were found to impact aquatic macroinvertebrate populations, specifically larger bodied Trichoptera abundance. Aquatic insect emergence was negatively correlated to salmonid densities. The aquatic insect emergence revealed alterations with significantly higher biomass of aquatic insect emergence in upstream sites without invasive fish. Lastly, aquatic insect predator (Hydrobiosidae: Rheochorema sp.) exhibited a suppressed trophic position in rivers with invasive salmonids. An unexpected finding from the study was the refugium stream habitat conditions that has been shown to be important for …
Role of MicroRNAs and Their Downstream Targets in Zebrafish Thrombopoiesis: Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development, respectively. However, there is limited information on microRNAs' role in zebrafish thrombopoiesis. Zebrafish thrombocytes could be used as a model to study their role in megakaryocyte maturation and platelet function because thrombocytes have both megakaryocyte features and platelet properties. In our laboratory, I identified 15 microRNAs in thrombocytes using single-cell RNA sequencing. Knockdown of three microRNAs, mir-7148, let-7b, and mir-223, by the piggyback method in zebrafish led to an increase in the percentage of thrombocytes. Functional thrombocyte analysis using plate tilt assay showed no modulatory effect of the three microRNAs on thrombocyte aggregation/agglutination. I then verified these findings in zebrafish larvae after the knockdown of the above microRNAs followed by an arterial laser thrombosis assay. I concluded mir-7148, let-7b, and mir-223 are repressors for thrombocyte production. Furthermore, I explored let-7b downstream genes in thrombocytes detected by RNA-seq analysis and chose 14 targets based on their role in cell differentiation (rorca, tgif1, rfx1a, deaf1, zbtb18, mafba, cebpa, spi1a, spi1b, fhl3b, ikzf1, irf5, irf8, and lbx1b) that are transcriptional regulators. The qRT-PCR analysis of expression levels the above genes following let-7b knockdown showed significant changes in the expression of 13 targets. I then studied the effect of the 14 targets on thrombocytes production and identified 5 genes (irf5, tgif1, irf8, cebpa, and rorca) that showed thrombocytosis and one gene ikzf1 that showed thrombocytopenia. Furthermore, I tested whether mir-223 regulates any of the above 13 transcription factors after mir-223 knockdown using qRT-PCR. Six of the 13 genes showed similar gene expression as observed with let-7b knockdown and 7 genes showed opposing results. Thus, our results suggested a possible regulatory network in common with both let-7b and mir-223. I also identified that tgif1, cebpa, …
Alterations in the Expression of Proteins Associated with Non-Alcoholic Fatty Liver Disease Observed in the Liver of the C57Bl/6 Wild-Type Male Mouse in Response to Exposure of Mixed Vehicle Emissions and/or High Fat Diet Consumption: Recent epidemiological studies have demonstrated a correlation between the manifestation of non-alcoholic fatty liver disease (NAFLD) and ambient air pollution levels, which is exacerbated by the presence of other risk factors, such as diabetes, dyslipidemia, obesity, and hypertension. We investigated the hypothesis that exposure to a mixture of gasoline and diesel engine emissions (MVE) coupled with the concurrent consumption of a high-fat (HF) diet promotes the development of a NAFLD phenotype within the liver. Three-month-old male C57Bl/6 mice were placed on either a low fat or HF diet and exposed via whole-body inhalation to either filtered (FA) air or MVE (30 µg PM/m3 gasoline engine emissions + 70 µg PM/m3 diesel engine emissions) 6 hr/day for 30 days. Histology revealed mild microvesicular steatosis and hepatocyte hypertrophy in response to MVE exposure alone, compared to FA controls, yielding a classification of "borderline NASH" under the criteria of the modified NAFLD active score (NAS) system. As anticipated, animals on a HF diet exhibited moderate steatosis; however, we also observed inflammatory infiltrates, hepatocyte hypertrophy, and increased lipid accumulation, with the combined effect of HF diet and MVE exposure. Immunofluorescence staining and RT-qPCR of the liver revealed the presence of lipid peroxidation, altered expression of inflammatory markers, induction of hepatic stellate cell activation biomarkers, and conversion to pyroptosis in response to MVE exposure and/or consumption of a HF diet. Our results indicate that inhalation exposure to traffic-generated air pollution initiates hepatocyte injury within the liver, exacerbates lipid accumulation and hepatocyte injury induced by the consumption of a HF diet, and alters molecular pathways associated with inflammation, fibrogenesis, and cell death, thereby contributing to the progression of NAFLD-related pathologies.
Data Mining Using Direct Injection Triple Quadrupole Mass Spectrometry, Infrared Spectroscopy, Inductively Coupled Plasma Optical Emission Spectroscopy, and Polymerase Chain Reaction for the Rapid Identification of Nutraceuticals and Contaminants: There has been a rapid surge toward "organic" products devoid of GMOs, MSGs, and other common compounds found in processed foods that continue to indicate an association with an increased risk for disease. These consumers seek nutrients and vitamins that are lacking in their diet and lifestyle in the form of nutraceuticals for disease prevention and treatment as well as overall lifestyle enhancement. However, these products generally lack clinical evidence as well as legal definition. Due to this ambiguity, nutraceuticals are neither considered a food product nor a pharmaceutical product. Furthermore, due to their alleged natural properties allowing for safe, therapeutic effects, nutraceuticals are being eagerly sought after by consumers in the place of pharmaceuticals. Additionally, since nutraceutical substances are "naturally" derived, there is a general lack of regulation regarding the manufacturing and distribution process. This mismanagement leads to lack of quality assurance (QA) and quality control (QC) protocols strictly implemented to define appropriate production and storage parameters. Without these critical measures, consumers are subjected to contamination of their products resulting from improper storage conditions and unmanaged production. These contaminants often include heavy metal impurities, pesticides, bacterial activity, and may also be adulterated with illicit drugs, all leading to detrimental health and environmental effects.
Developing a Generalizable Two-Input Genetic AND Logic Gate in Arabidopsis thaliana for Multi-Signal Processing: With effective engineering using synthetic biology approaches, plant-based platforms could conceivably be designed to minimize the production costs and wastes of high-value products such as medicines, biofuels, and chemical feedstocks that would otherwise be uneconomical. Additionally, modern agricultural crops could be engineered to be more productive, resilient, or restorative in different or rapidly changing environments and climates. To achieve these complex goals, information-processing genetic devices and circuits containing multiple interacting parts that behave predictably must be developed. A genetic Boolean AND logic gate is a device that computes the presence or absence of two inputs (signals, stimuli) and produces an output (response) only if both inputs are present. Here, we optimized individual genetic components and used synthetic protein heterodimerizing domains to rationally assemble genetic AND logic gates that integrate two hormonal inputs in whole plants. These AND gates produce an output only in the presence of both abscisic acid and auxin, but not when either or neither hormone is present. Furthermore, we demonstrate the AND gate can also integrate two plant stresses, cold temperature and bacterial infection, to produce a specific response. The design principles used here are generalizable, and therefore multiple orthogonal AND gates could be assembled and rationally layered to process complex genetic information in plants. In addition to bioproduction, these layered logic gates may also be used in circuits to probe fundamental questions in plant biology such as hormonal crosstalk.
Developing Informatics Tools and Methods Utilizing Whole Genome Sequencing and Transcription Data to Aid Gene Discovery in Medicago truncatula: Research into the mechanism of symbiotic nitrogen fixation between legumes and rhizobia involves a complex interaction between the organisms, and many genes involved in this remain either uncharacterized or undiscovered. Using forward genetics, mutant plant lines are screened to find new genes without reliance on software-based gene prediction. A large population of Tnt1-mutagenized Medicago truncatula lines is used for this purpose. Herein, the aid of tools like whole genome sequencing (WGS) in this process is explored so that new methods and tools are elucidated. The use of WGS data allows for rapid prediction of all insertions in the genome and has been shown to predict insertion locations that were missed by the TAIL-PCR-based Tnt1 mutant database already in existence. This WGS strategy has been successfully used to find the causal mutations in multiple plant lines. Two WGS strategies are used to analyze insertions in nine sequenced lines and compared with each other and the existing Tnt1 mutant database. It appears that using either WGS method will yield similar results, but the TAIL-PCR-based predictions have much less overlap. The use of the latest R108 genome appears to decrease the degree of disagreement between the methods, while the correlation in the A17 genome update is less clear. There is also a demonstration of the use of other tools in addition to the WGS prediction output. Combining transcription data from previous experiments with the predicted insertions allowed for the creation of more holistic tables, which could better assist in screening the predictions made for the most likely candidate by highlighting those with expression profiles consistent with the observed mutation phenotype. Each of these tools and methods has been shown to be effective in screening Tnt1-mutagenized M. truncatula lines to find novel genes. Without further experimental data, determining the most accurate method is not …
Identification and Characterization of Two Putative Sulfate Transporters Essential for Symbiotic Nitrogen Fixation in Medicago truncatula: The process of symbiotic nitrogen fixation (SNF) in legume root nodules requires the channeling and exchange of nutrients within and between the host plant cells and between the plant cells and their resident rhizobia. Using a forward genetics approach in the Medicago truncatula Tnt1 mutant population followed by whole genome sequencing, two putative sulfate transporter genes, MtSULTR3;5 and MtSULTR3;4b, were identified. To support the hypothesis that the defective putative sulfate transporter genes were the causative mutation for the mutants' phenotypes, the M. truncatula Tnt1 population was successfully reverse screened to find other mutant alleles of the genes. The F2 progeny of mutants backcrossed with wildtype R108 demonstrated co-segregation of mutant phenotypes with the mutant alleles confirming that the mutated mtsultr3;5 and mtsultr3;4b genes were the cause of defective SNF in the mutant lines mutated in the respective genes. This finding was further established for mtsultr3;4b by successful functional complementation of a mutant line defective in the gene with the wildtype copy of MtSULTR3;4b. A MtSULTR3;4b promoter-GUS expression experiment indicated MtSULTR3;4b expression in the vasculature and infected and uninfected plant cells of root nodules. MtSULTR3;4b was found to localize to the autophagosome membrane when expressed in Nicotiana benthamiana. A transcriptomics study on the mutant nodules revealed the probable impact of mutated mtsultr3;5 and mtsultr3;4b on expression of genes involved in N fixation and on other biological processes, including possible effects of the mutated genes on the transcriptional regulation of sulfate assimilation pathway in the respective mutants' nodules. The RNAseq study also demonstrated the mis-regulation of nodule zone-specific genes in mtsultr3;5 and mtsultr3;4b mutants. A PCR-based approach was used to study the transcription of MtSULTR3;5 and MtSULTR3;4b in the respective mutant lines. The study demonstrated formation of readthrough chimeric gene-Tnt1 transcripts in mtsultr3;5 mutant alleles and truncated chimeric gene-Tnt1 transcripts and aberrantly …
Investigating the Spatial Relationship between Suicide and Race/Ethnicity: The Case for Alternate Rate Adjustment Techniques in Medical Geography: This work explores potential distortions created by race and ethnicity on the visualization, interpretation, and understanding of the spatial distribution of suicide in the United States. Due to radically different suicide rates among racial/ethnic groups, traditional crude or age-adjusted rates may introduce statistical confounding in both linear and spatial models. Using correlation, choropleth mapping, hot spot analysis, and location-allocation modeling, this work shows how traditional methods of health system planning may unintentionally overlook elevated risk in minority-dominated areas like inner cities, the Texas/Mexico border region, and the Deep South. The final chapter introduces a simulation protocol for examining potential distortions in datasets to identify spatial and non-spatial distortions created by the underlying population composition. Methodologically, this dissertation contributes to the discourse on place context versus population composition. More generally, this research points to potential hazards to creating a more inclusive and equitable healthcare system.
Medicago truncatula NPF1.7: Structure-Function Assessment and Potential as a Phytohormone Transporter: In Medicago truncatula, the MtNPF1.7 transporter has been shown to be essential for root morphology and nodulation development. The allelic MtNPF1.7 mutants, Mtnip-1 (A497V), Mtnip-3 (E171K), and Mtlatd (W341STOP), show altered lateral root growth and compromised legume-rhizobium symbiosis. To assess the role of a series of distinct amino acids in the transporter's function, in silico structural predictions were combined with in planta complementation of the severely defective Mtnip-1 mutant plants. The findings support hypotheses about the functional importance of the ExxE(R/K) motif including an essential role for the first glutamic acid of the motif in proton(s) and possibly substrate transport. The results also question the existence of a putative TMH4-TMH10 salt bridge, which may not form in MtNPF1.7. Results reveal that a motif conserved among MFS proteins, Motif A, is essential for function. Hypothetically, the Motif A participates in intradomain packing of transmembrane helices and stabilizing one conformation during transport. The mutated valine (A497V) in Mtnip-1 may interfere with the lateral helix. Mutating a residue (L253) on the lateral helix with reduced side chain restored Mtnip-1 function. The predicted residue (Q351) for substrate binding is not essential for protein function. To probe the possibility that MtNPF1.7 transports auxin, two heterologous assay systems were attempted. The first was a Xenopus laevis oocyte assay. However, MtNPF1.7 expressed in oocytes failed to show substrate transport, which may due to low expression levels of proteins on the membrane or may be caused by other factors. Second, yeast (Saccharomyces cerevisiae) strains expressing MtNPF1.7 were constructed. They showed an increased flux of radiolabeled IAA and differential susceptibility to 5-fluoroindole-3-acetic acid (F-IAA), a toxic IAA-like compound. These results suggested that MtNPF1.7 may function as an auxin transporter in yeast. Unexpectedly, the Mtnip-1 (A497V) and Mtnip-3 (E171K) proteins when expressed in yeast also showed influx of F-IAA transport …
Migration Tracking, Survival, and Pairing Behavior of American Kestrels Wintering in North Central Texas: The American Kestrel (Falco sparverius) is the smallest and most abundant falcon in North America with a wide geographic range. Unfortunately, surveys have suggested that some kestrel populations have been in decline since the 1950s, though the nominal causes of this decline are unknown. Migratory movement patterns and connectivity have yet to be established for any population of migratory kestrels. In Chapter 2, I investigated methods for attaching migration trackers to kestrels. Specifically, I showed that leg-loop style harnesses may have negatively affected return rates whereas backpack harnesses did not. Based on these results, I recommend that backpack-style Teflon harnesses is the safest and most effective method for attaching tracking devices to small raptors. In Chapter 3, I quantified survivorship for kestrels wintering in north Texas to identify the timing of kestrel mortality. Notably, I found that juvenile kestrels had similar annual survival rates as adults (81.6% versus 79.5%). High overwintering survival in north Texas indicated that once kestrels arrived on their wintering grounds, they were highly likely to survive to spring migration. In Chapter 4, I investigated pairing behaviors previously undocumented in wintering kestrels. I found that winter pairing was relatively common, but more prevalent in urban environments than rural. My data suggested that pairing during the winter could be a coping mechanism to increase kestrel survival in stressful anthropic landscapes. Altogether, this research highlights that the documentation of migratory connectivity is essential to understanding how many processes affect kestrel population dynamics, as I found evidence of potential seasonal carryover effects.
Seeing in the Light: Using Expansion Microscopy to Achieve Super-Resolution in Transmitted Light: Light microscopy is inherently limited in resolution by properties of light such as diffraction and interference to 170-250 nm. Expansion microscopy is a quickly-developing method which achieves super-resolution by using a swellable hydrogel to physically expand biological samples themselves, rather than depending on the properties of fluorophores. This thesis demonstrates that expansion microscopy is a feasible means for achieving super-resolution in transmitted light microscopy modes. Though it has only been used for fluorescence imaging in the past, here I show that samples prepared for expansion microscopy—including liver tissue slices and myofibrillar bundles—are observable using transmitted light. While the majority of the original sample material is removed in the expansion process, the hydrogel retains visible evidence of these samples. These demonstrate increased detail under brightfield microscopy that is useful for characterization. Sarcomeric regions are identifiable by this method and are confirmed by fluorescence imaging. Thus, expansion microscopy is a means to bring super-resolution to transmitted light imaging and is entirely compatible with fluorescence for the localization of proteins of interest.
Space Use, Microhabitat and Macrohabitat Use of the Three-Toed Box Turtle (Terrapene carolina) in North Texas: Box turtle (Terrapene carolina) populations are steadily declining due their unique natural history, effects of climate change, and anthropogenic land use change. There is a need for updated information on box turtle space and micro and macro-habitat use to inform conservation efforts. This study used VHF radiotelemetry and GPS data loggers to examine box turtle space and habitat use in North Texas. Box turtle home range sizes averaged 6.6ha (range = 0.79 - 18.08, n = 23), and males (n = 9) had larger home ranges than females (n = 14; W = 31.5, P = 0.05). Home range size was best explained by a combination of variables including sex and body size, but overall, home ranges that consisted of higher percentages of suitable box turtle habitat were smaller. Box turtles used deciduous forest more than expected and wetlands less than expected by chance (Fisher's exact test, P < 0.0001). The most informative variable for box turtle macrohabitat selection was NDVI. Box turtles selected microhabitats with a higher percent litter (t = -2.16, P < 0.05) and understory cover (t = -5.03, P < 0.05). The results of CART analysis showed the nested importance of macro- and microhabitat and identified NDVI as the most important variable for predicting suitable box turtle habitat. Given these results, we postulate that NDVI can be used to identify suitable box turtle habitat at landscape scales to aid in management and conservation efforts. We found that three-toed box turtles are using habitat differently than what has been reported in eastern box turtles, providing support for the theory that three-toed box turtles should be classified as a separate species.
Acute Toxicity of Crude Oil Exposures to Early Life Stage Teleosts: Contribution of Impaired Renal Function and Select Environmental Factors: Oil spills are well-known adverse anthropogenic events, as they can induce severe impacts on the environment and negative economic consequences. Still, much remains to be learned regarding the effects of crude oil exposure to aquatic organisms. The objectives of this dissertation were to fill some of those knowledge gaps by examining the effects of Deepwater Horizon (DWH) crude oil exposure on teleost kidney development and function. To this end, I analyzed how these effects translate into potential osmoregulatory impairments and investigated the interactive effects of ubiquitous natural factors, such as dissolved organic carbon (DOC) and ultraviolet (UV) light, on acute crude oil toxicity. Results demonstrated that acute early life stage (ELS) crude oil exposure induces developmental defects to the primordial kidney in teleost fish (i.e., the pronephros) as evident by alterations in: (1) transcriptional responses of key genes involved in pronephros development and function and (2) alterations in pronephros morphology. Crude oil-exposed zebrafish (Danio rerio) larvae presented defective pronephric function characterized by reduced renal clearance capacity and altered filtration selectivity, factors that likely contributed to the formation of edema. Latent osmoregulatory implications of crude oil exposure during ELS were observed in red drum (Sciaenops ocellatus) larvae, which manifested reduced survival in hypoosmotic waters, likely due to defective pronephros development and function. Finally, DOC-UV co-exposure slightly reduced acute crude oil photo-enhanced toxicity in red drum larvae. This dissertation provided novel information regarding crude oil toxicity that can be incorporated into environmental risk assessment and management for future oil spills.
Assessing Student Perceptions in Short Research Experiences and Course Research Experiences in Undergraduate Biology Laboratories: This study examined students' perception between short research experiences (SRE) courses and full-semester course research experiences (CRE) using the Persistence in the Sciences (PITS) survey and the interview questionnaire. The study also aimed to correlate the influence of student's demographic as a predictive indicator for Project Ownership Scores (POS) and Quantitative Literacy (QL) score means. The three courses studied at the University of North Texas were Biology for Science Majors Laboratory (BIOL 1760 SRE), Microbiology with Tiny Earth (BIOL 2042 Tiny Earth SRE), and Introductory Biology Research Laboratory I (BIOL 1750 SEA-PHAGES CRE). The mean scores for the PITS categories leaned favorably towards the research component of each laboratory course assessed in this study. The interview questionnaire showed 66% of the students in the SRE courses and 90% of the students in the CRE course preferred the research component of the lab. Paired survey demographic analysis for BIOL 1760 SRE showed significance for the Science Community Values with associate/bachelor's degree. BIOL 1750 SEA-PHAGES CRE showed significance in three of the six categories when comparing means for Project Ownership Emotion, Self-Efficacy, and Science Identity with Gender. Binary logistics was used to build a regression model to predict demographics with approximately 65% to 75% accuracy for each course. When analyzing students' QL score, the demographic category "Ethnicity" showed significance for BIOL 2042 Tiny Earth SRE. Categorizing the correct response into two categories for the QL test scores, the SRE and CRE courses, and analyzing the PITS scores for paired data sets showed that there was significance in the Networking category for the question "I have discussed my research in this course with professors other than my course instructor." The validated PITS, POS, and interview questionnaire could be a tool for use to analyze laboratories at UNT that offer a SRE or CRE …
Inferring a Network of Horizontal Gene Flow among Prokaryotes Using Complementary Approaches: Horizontal gene transfer (HGT), a mechanism that facilitates exchange of genetic material between organisms from different lineages, has a profound impact on prokaryotic evolution. To infer HGT, we first developed a comparative genomics-based tool, APP, which can perform phyletic pattern analysis using completely sequenced genomes to identify genes are unique to a genome or have sporadic distribution in its close relatives. Performance assessment against currently available tools on a manually created 18-genome dataset and 2 benchmarking datasets revealed the superior accuracy of APP over other methods. We then utilized a parametric method to construct a gene exchange network. The composition-based method, Jenson-Shannon Codon Bias (JS-CB), groups genes into clusters based on similar codon usage bias. These clusters were analyzed using APP and examined for the enrichment HGT associated marker genes, then annotated as of native or alien origin based on these multiple lines of evidence. Intergenome clustering enabled identification of genes mobilized across alien components of the genomes (alien-alien transfer) and from native components of donor genomes to the recipient genomes (native-alien transfer). Functional classification of alien gene clusters revealed that metabolism associated genes are most frequently mobilized, in concurrence with previous reports, and additionally, a large number of genes with yet unknown functions were found to have been horizontally transferred, a important finding that needs to be further investigated.
Multi-Level Effects of Oxygen Exposure in Endothermic Insects: This dissertation examined the phenotypic plasticity of endothermic, flight and respiratory physiology in response to developmental oxygen exposure in the moth Manduca sexta. Development in both 10% O2 hypoxia and 30% O2 hyperoxia treatments were used to look at the physiological consequence on both ends of the oxygen spectrum. Hypoxic insects reached smaller sizes as adults and had longer pupation lengths than controls. Hyperoxic insects were larger at the end of the larval stage, had increased larval growth rates, but also had longer developmental larval developmental times and pupation lengths than controls. There was a decrease in both metabolic rate and thorax temperatures of hypoxic reared insects at normoxic levels. In flight trials hypoxic insects had the lowest critical flight PO2, and the hyperoxic insects had the highest PO2. There was an increase in hypoxic insect flight muscle mitochondria oxygen consumption in permeabilized fibers, but this did not translate to the isolated flight muscle mitochondria metabolic rates. Rearing oxygen level did not significantly affect mitochondrial density and size; myofibril density and size, or tracheal density and size in flight muscle. Overall, I found that higher levels of organization were more susceptible to the effects of chronic oxygen exposure and found more effects of hypoxia than hyperoxia.
Regulation of Receptors in Neuronal Cilia with Development, Seizures, and Knockouts: Implications for Excitability: Neurons commonly have a primary cilium, which is a non-motile organelle extending from the centrosome into the extracellular space. In most brain regions, neuronal cilia are enriched in either somatostatin receptor type 3 (SstR3) or melanin concentrating hormone receptor type 1 (MCHR1), or both. The present immunohistochemical study provides novel evidence that primary cilia regulate neuronal excitability via G-protein coupled receptors (GPCRs), and that their identity is governed by brain region and by competition, both in adulthood and in postnatal development. The hippocampus, which is particularly vulnerable to seizures, has opposing gradients of SstR3(+) and MCHR1(+) ciliary GPCRs. We hypothesized that there is a competition between these two ciliary GPCRs, which might take place on any level from gene expression to presence in the cilium. We examined whether receptor colocalization occurs transiently in development before ciliary GPCR dominance is established in neurons in the CNS. In postnatal CA1 and CA3, the first GPCR to appear in cilia was the one that will dominate in adults: MCHR1 in CA1 and SstR3 in CA3. Some days later, the second GPCR was expressed along with the first; dual-receptor cilia were the exclusive type until single-receptor cilia emerged again around P14. Single-receptor cilia then increased in numbers through adulthood. By identifying ciliary receptors that modulate seizure activity in mice, the present study lays a foundation for therapeutic approaches to reduce neuronal excitotoxicity underlying cell death in epilepsy, CNS injury, and neurodegenerative diseases.
A Sensitive and Robust Machine Learning-Based Framework for Deciphering Antimicrobial Resistance: Antibiotics have transformed modern medicine in manifold ways. However, the misuse and over-consumption of antibiotics or antimicrobials have led to the rise in antimicrobial resistance (AMR). Unfortunately, robust tools or techniques for the detection of potential loci responsible for AMR before it happens are lacking. The emergence of resistance even when a strain lacks known AMR genes has puzzled researchers for a long time. Clearly, there is a critical need for the development of novel approaches for uncovering yet unknown resistance elements in pathogens and advancing our understanding of emerging resistance mechanisms. To aid in the development of new tools for deciphering AMR, here we propose a machine learning (ML) based framework that provides ML models trained and tested on (1) genotypic AMR and phenotypic antimicrobial susceptibility testing (AST) data, which can predict novel resistance factors in bacterial strains that lack already implicated resistance genes; and (2) complete gene set and AST phenotypic data, which can predict the most important genetic loci involved in resistance to specific antibiotics in bacterial strains. The validation of resistance loci prioritized by our ML pipeline was performed using homology modeling and in silico molecular docking.
Temperature Change and Its Consequences for the Physiology of the Eurythermic Sheepshead Minnow (Cyprinodon variegatus): The estuarine sheepshead minnow (Cyprinodon variegatus) is the most eurythermic fish species, with a thermal tolerance window between 0.6°C and 45.1°C. However, little is known about the physiological mechanisms that allow this species to survive this temperature range. In order to understand how sheepshead minnow physiology is affected by temperature acclimation and acute changes in temperature, I conducted research on this species using a multi-level approach. I began at the organismal level, and examined the effects of these temperature changes on the sheepshead minnow's metabolic rate and swimming performance. The next chapter investigated the effects of changing temperatures on cardiac function (i.e., tissue/organ specific effects). In the final chapter, I conducted research at the sub-cellular level, and determined how mitochondrial bioenergetics / function is impacted by changing temperatures. This research shows that while sheepshead minnows are able to sustain heart function and mitochondrial respiration over a broad range of temperatures; they also display a plastic temperature response which is associated with the downregulation of standard metabolic rate and cardiac remodeling to maintain force generation. Collectively, these physiological responses may contribute to the sheepshead minnow's ability to maintain physiological and organismal function across a large temperature range.
Correlation of Watershed NDVI Values to Benthic Macroinvertebrate Biodiversity in Eight North American Wadeable Streams: Water quality of a stream or river is influenced by the surrounding landscape and vegetation. The Normalized Difference Vegetation Index (NDVI) is commonly used to characterize landcover and vegetation density. Benthic macroinvertebrates are ubiquitous in freshwater streams and are excellent indicators of the quality of freshwater habitats. Data from one NDVI remote sensing flight and one macroinvertebrate sampling event for eight wadeable stream study sites in the National Ecological Observatory Network (NEON) were acquired. Proportions of high, moderate, and sparse vegetation were calculated for each stream watershed using ArcGIS. Functional feeding groups and tolerance values were assigned to macroinvertebrate taxa. The Fourth-corner and RLQ methods of analysis, available in the ade4 package for R software, were used to evaluate the relationships of macroinvertebrate traits with environmental variables. Hypothesis testing using Model 6 in the ade4 package resulted in p-values of 0.066 and 0.057 for global (overall) significance. Mean NDVI values of moderately vegetated areas and proportion of sparse vegetation were found to be significant to percent shredders at alpha ≤ 0.05. Results of these methods of analysis, when combined with traditional macroinvertebrate sampling metrics, show that NDVI can be a useful, additional tool to characterize a watershed and its effects on macroinvertebrate community composition and structure.
Ecosystem Services and Sustainability: A Framework for Improving Decision-Making in Urban Areas: Ecosystem services are the varied goods and benefits provided by ecosystems that make human life possible. This concept has fostered scientific explorations of the services that nature provides to people with the goal of sustaining those services for future generations. As the world becomes increasingly urban, ecosystems are reshaped, and services are degraded. Provisioning and regulating ecosystem services, landscape planning, decision making, and agricultural systems and technologies play a distinctive role in feeding and sustaining the expanding urban population. Hence, the integrated assessment of these coupled components is necessary to understand food security and sustainable development. Nevertheless, frameworks that incorporate ecosystem services, urbanization, and human wellbeing are still scarce due to several conceptual and methodological gaps that challenge this assessment. As a consequence, these frameworks are not operationalized, and ecosystem services rarely receive proper attention in decision making. This dissertation seeks to improve our understanding of the role of ecosystem services at the landscape level and provides an approach for operationalizing decisions that affect sustainable practices and human wellbeing.
Investigation of Gene Functions in the Cyanotrophic Bacterium Pseudomonas fluorescens NCIMB 11764: Pseudomonas fluorescens NCIMB 11764 (Pf11764) is one of a group of bacteria known as cyanotrophs that exhibit the unique ability to grow on toxic cyanide as the sole nitrogen source. This ability has previously been genetically linked to a conserved cluster of seven genes (Nit1C), the signature gene (nitC) coding for a nitrilase enzyme. Nitrilases convert nitriles to ammonia and a carboxylic acid. Still, for the Pf11764 NitC enzyme (Nit11764), no in vivo substrate has been identified, and the basis of the enzyme's requirement for cyanide growth has remained unclear. Therefore, the gene was cloned and the enzyme was characterized with respect to its structure and function. These efforts resulted in the unique discovery that, aside from its nitrilase activity, Nit11764 exhibits nuclease activity towards both DNA and RNA. This ability is consistent with computer analysis of the protein providing evidence of a preponderance of amino acids with a high probability for RNA binding. A Nit11764 knock-out mutant was shown to exhibit a higher sensitivity to both cyanide (KCN) and mitomycin C, both known to induce chromosomal damage. Thus, the overall conclusion is that Nit11764, and likely the entire Nit1C gene cluster, functions as a possible repair mechanism for overcoming the damage inflicted on Pf11764 nucleic acids by toxic cyanide. Towards a further investigation of the Nit1C gene cluster in Pf11764, a second gene (nitH) annotated as a monooxygenase was also investigated. Interestingly, computer-based analysis shows that NitH also harbors a preponderance of RNA-binding amino acids. The nitH gene was cloned into an expression vector with the long-range goal of defining its role in CN utilization.
Metacommunity Dynamics of Medium- and Large-Bodied Mammals in the LBJ National Grasslands: Using metacommunity theory, I investigated the mechanisms of meta-assemblage structure and assembly among medium- to large-bodied mammals in North Texas. Mammals were surveyed with camera-traps in thirty property units of the LBJ National Grasslands (LBJNG). In Chapter II the dispersal and environmental-control based processes in community assembly were quantified within a metacommunity context and the best-fit metacommunity structure identified. A hypothesis-driven modelling approach was used in Chapter III to determine if the patterns of species composition and site use could be explained by island biogeography theory (IBT) or the habitat amount hypothesis (HAH). Islands were defined as the LBJNG property unit or the forest patch bounded by the property unit. Forest cover was selected as the focal habitat for the HAH. Seasonal dynamics were explored in both chapters. Metacommunity structure changed with each season, resulting in quasi-nested and both quasi and idealized Gleasonian and Clementsian structures. Results indicated that the anthropogenic development is, overall, not disadvantageous for this assemblage, that community assembly receives equal contributions from spatial and environmental factors, and that the metacommunity appears to operate under the mass effects paradigm. The patterns of species composition and site use were not explained by either IBT or HAH. Likely because this assemblage of generalist, dispersal-capable mammals are utilizing multiple habitat types both in the protected land and in the private land. This research highlights the versatility of these species and the potential value of rural countryside landscapes for wildlife conservation.
Role of DEFECTIVE IN SYSTEMIC DEFENSE INDUCED BY ABIETANE DITERPENOID 1 (DSA1), a Putative O-Fucosyltransferase, in Plant Systemic Acquired Resistance (SAR): Dehydroabietinal (DA), an abietane diterpenoid, was previously demonstrated to be a potent activator of systemic acquired resistance (SAR). DA also promotes flowering time in Arabidopsis thaliana by repressing expression of the flowering repressor FLOWERING LOCUS C (FLC) while simultaneously upregulating expression of FLOWERING LOCUS D (FLD), FLOWERING LOCUS VE (FVE) and RELATIVE OF EARLY FLOWERING 6 (REF6), a set of flowering time promoters. To further understand the mechanism underlying signaling by abietane diterpenoids, Arabidopsis mutants exhibiting reduced responsiveness to abietane diterpenoids were identified. One such mutant plant, ems2/7, exhibited SAR-deficiency and delayed flowering, which were found to be associated with two independent, but linked loci. The gene responsible for the SAR defect in ems2/7 was identified as DEFECTIVE IN SYSTEMIC DEFENSE INDUCED BY ABIETANE DITERPENOID 1 (DSA1). Similar to the missense mutant dsa1-1 identified in the mutant screen, the T-DNA insertion bearing null allele dsa1-2 exhibited SAR deficiency that could be complemented by a genomic copy of DSA1. The gene responsible for the delayed flowering phenotype of ems2/7 remains to be identified. DSA1 encodes a protein that is homologous to human protein O-fucosyltransferase 2. DSA1 is required for long-distance transport of the SAR signal. It is hypothesized that DSA1 is involved in the O-fucosylation-facilitated channeling through the ER/Golgi network of a protein involved in long distance SAR signaling. In a yeast two-hybrid screen, all the DSA1-interacting proteins identified are chloroplast-localized proteins, thus raising the interesting possibility of ER interaction with chloroplast and its potential role in SAR signaling.
CO2 Transport and Acid-Base Status during Fluctuations in Metabolic Status in Reptiles: Reptiles can often experience perturbations that greatly influence their metabolic status (e.g., temperature, exercise, digestion, and ontogeny). The most common cause of fluctuations in metabolic status in post-embryonic reptiles is arguably digestion and physical activity (which will be further referred to as exercise). The objective of this thesis is to determine the mechanisms involved in CO2 transport during digestion, determine the mechanisms that allow for the maintenance of acid-base homeostasis during digestion, and observing the effect of an understudied form of exercise in semi-aquatic reptiles on the regulation of metabolic acidosis and base deficit. This dissertation provided evidence for potentially novel and under investigated mechanisms for acid-base homeostasis (e.g., small intestine and tissue buffering capacity; Chapters 3 & 4), while also debunking a proposed hypothesis for the function of an anatomical feature that still remains a mystery to comparative physiologist (Chapter 2). This thesis is far from systematic and exhaustive in its approach, however, the work accomplished in this dissertation has become the foundation for multiple distinct paths for ecologically relevant investigations of the regulation of metabolic acidosis/alkalosis in reptiles.
Influence of Hypoxia on Acute Lead Toxicity and Calcium Homeostasis in Early Life Stage Zebrafish (Danio rerio): The purpose of this study was to investigate the effects of Pb and hypoxia co-exposure on Pb toxicity and Ca homeostasis in early life stage (ELS) zebrafish (Danio rerio). Previous evidence indicates that exposure of ELS zebrafish to hypoxia (~20% air saturation) reduces Ca uptake, likely through down-regulation of the apical epithelial Ca channel (ECaC). Considering that Pb and Ca are known antagonists and compete for uptake pathways, it was hypothesized that co-exposure of Pb with hypoxia would decrease Pb toxicity by reducing Pb uptake (likely mediated through a reduced number of ECaCs). However, it was shown that at 96 hpf, whole body accumulation of both Pb and Ca was lower at 40% air saturation compared to 100% and 20% air saturation. This result closely aligned with the 96h LC50 results which showed the highest mortality of zebrafish at 40% compared to the other air saturation levels. This suggests that toxicity is likely the result of exacerbated hypocalcemia at 40% air saturation due to both Pb competition for Ca binding to Ca uptake channels/transporters, such as ECaC, and potentially reduced expression of such channels/transporters in response to this level of hypoxia. Overall, it appears that ELS zebrafish respond differentially to the 40% and 20% hypoxia levels when co-exposed with Pb. Further investigation is needed to illustrate the physiological and molecular mechanisms underlying this response.
Investigating the Effects of Inhaled Diesel Exhaust Particles on Gut Microbiome, Intestinal Integrity, Systemic Inflammation, and Biomarkers of Cardiovascular Disease in Wildtype Mice: We investigated the hypothesis that exposure to inhaled diesel exhaust PM can alter the gut microbiome and intestinal integrity, thereby promoting systemic inflammatory response and early CVD risk, which are exacerbated by HF diet. Furthermore, we investigated whether the observed exposure and diet-mediated outcomes could be mitigated through probiotic treatment. We performed an exposure study on C57Bl/6 male mice, placed on either a low fat (LF) diet or a high-fat (HF) diet, and exposed via oropharyngeal aspiration to 35 μg diesel exhaust particles (DEP) suspended in 35 μl of sterile saline or sterile saline controls (CON) twice a week for four weeks. A subset of mice on HF diet were dosed with 0.3 g/day (PRO, ~7.5x108 CFU/day) of probiotic Ecologic® Barrier 849 (Winclove Probiotics) in drinking water during the course of the study. For our first aim, we investigated the alterations in the gut microbiome, measured circulating cytokines and lipopolysaccharide (LPS), and measured CVD biomarkers in the heart. Our results revealed that exposure to inhaled DEP results in gut dysbiosis characterized by expansion of the phyla Verrucomicrobia and Proteobacteria and reduction in Actinobacteria, which was exacerbated by HF diet. Probiotics mitigated the DEP-mediated expansion of Proteobacteria and re-established Actinobacteria in the intestine of HF animals. Furthermore, we determined that exposure to inhaled DEP increases systemic LPS and inflammatory markers IL-1α, IL-3, G-CSF, and TNF-α. Furthermore, we found that inhaled DEP exposure results in increased CVD biomarkers sICAM-1, sP-selectin, and thrombomodulin in the heart. Probiotic treatment was effective in attenuating LPS, inflammatory responses, and CVD biomarkers in HF animals, validating the involvement of the microbiome in mediating inhaled DEP-mediated responses. Considering the effects we observed in the microbiota and systemically of the HF and probiotic treatment animals, we investigated the effects of inhaled DEP on intestinal integrity and inflammation in HF …

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