Soil-borne oomycetes include devastating plant pathogens that cause substantial losses in the agricultural sector. To better manage this important group of pathogens, it is critical to understand how they respond to common agricultural practices, such as tillage and crop rotation. Here, a long-term field experiment was established using a split-plot design with tillage as the main plot factor (conventional tillage (CT) vs. no till (NT), two levels) and rotation as the subplot factor (monocultures of soybean, corn, or wheat, and corn–soybean–wheat rotation, four levels). Post-harvest soil oomycete communities were characterized over three consecutive years (2016–2018) by metabarcoding the Internal Transcribed Spacer 1 (ITS1) region. The community contained 292 amplicon sequence variants (ASVs) and was dominated by Globisporangium spp. (85.1% in abundance, 203 ASV) and Pythium spp. (10.4%, 51 ASV). NT decreased diversity and community compositional structure heterogeneity, while crop rotation only affected the community structure under CT. The interaction effects of tillage and rotation on most oomycetes species accentuated the complexity of managing these pathogens. Soil and crop health represented by soybean seedling vitality was lowest in soils under CT cultivating soybean or corn, while the grain yield of the three crops responded differently to tillage and crop rotation regimes.

Ten samples of tropical lichens collected from Doi Inthanon, Thailand, were explored for the diversity of their bacterial microbiomes through 16S rRNA-based metagenomics analysis. The five predominant lichen-associated bacteria belonged to the phyla Proteobacteria (31.84%), Planctomycetota (17.08%), Actinobacteriota (15.37%), Verrucomicrobiota (12.17%), and Acidobacteriota (7.87%). The diversity analysis metric showed that Heterodermia contained the highest bacterial species richness. Within the lichens, Ramalina conduplicans and Cladonia rappii showed a distinct bacterial community from the other lichen species. The community of lichen-associated actinobacteria was investigated as a potential source of synthesized biologically active compounds. From the total Operational Taxonomic Units (OTUs) found across the ten different lichen samples, 13.21% were identified as actinobacteria, including the rare actinobacterial genera that are not commonly found, such as Pseudonocardia, Kineosporia, Dactylosporangium, Amycolatopsis, Actinoplanes, and Streptosporangium. Evaluation of the pretreatment method (heat, air-drying, phenol, and flooding) and isolation media used for the culture-dependent actinobacterial isolation revealed that the different pretreatments combined with different isolation media were effective in obtaining several species of actinobacteria. However, metagenomics analyses revealed that there were still several strains, including rare actinobacterial species, that were not isolated. This research strongly suggests that lichens appear to be a promising source for obtaining actinobacteria.

Characterizing the diversity of genes associated with virulence and transmission of a pathogen across the pathogen's distribution can inform our understanding of host infection risk. Borrelia burgdorferi is a vector-borne bacterium that causes Lyme disease in humans and is common in the United States. The outer surface protein C (ospC) gene of B. burgdorferi exhibits substantial genetic variation across the pathogen's distribution and plays a critical role in virulence and transmission in vertebrate hosts. In fact, B. burgdorferi infections that disseminate across host tissues in humans are associated with only a subset of ospC alleles. Delaware has a high incidence of Lyme disease, but the diversity of ospC in B. burgdorferi in the state has not been evaluated. We used PCR to amplify ospC in B. burgdorferi-infected blacklegged ticks (Ixodes scapularis) in sites statewide and used short-read sequencing to identify ospC alleles. B. burgdorferi prevalence in blacklegged ticks varied across sites, but not significantly so. We identified 15 previously characterized ospC alleles accounting for nearly all of the expected diversity of alleles across the sites as estimated using the Chao1 index. Nearly 40% of sequenced infections (23/58) had more than one ospC allele present suggesting mixed strain infections and the relative frequencies of alleles in single infections were positively correlated with their relative frequencies in mixed infections. Turnover of ospC alleles was positively related to distance between sites with closer sites having more similar allele compositions than more distant sites. This suggests a degree of B. burgdorferi dispersal limitation or habitat specialization. OspC alleles known to cause disseminated infections in humans were found at the highest frequencies across sites, corresponding to Delaware's high incidence of Lyme disease.

Non-human primates (NHPs) are a preferred animal model for optimizing adeno-associated virus (AAV)-mediated CNS gene delivery protocols before clinical trials. In spite of its inherent appeal, it is challenging to compare different serotypes, deliv- ery routes, and disease indications in a well-powered, compre- hensive, multigroup NHP experiment. Here, a multiplex barcode recombinant AAV (rAAV) vector-tracing strategy has been applied to a systemic analysis of 29 distinct, wild- type (WT), AAV natural isolates and engineered capsids in the CNS of eight macaques. The report describes distribution of each capsid in 15 areas of the macaques’ CNS after intrapar- enchymal (putamen) injection, or cerebrospinal fluid (CSF)- mediated administration routes (intracisternal, intrathecal, or intracerebroventricular). To trace the vector biodistribution (viral DNA) and targeted tissues transduction (viral mRNA) of each capsid in each of the analyzed CNS areas, quantitative next-generation sequencing analysis, assisted by the digital- droplet PCR technology, was used. The report describes the most efficient AAV capsid variants targeting specific CNS areas after each route of administration using the direct side-by-side comparison of WT AAV isolates and a new generation of ratio- nally designed capsids. The newly developed bioinformatics and visualization algorithms, applicable to the comparative analysis of several mammalian brain models, have been devel- oped and made available in the public domain.

The high mortality, clinical heterogeneity, and relatively high incidence of triple negative breast cancer (TNBC) emphasizes the urgency for a clearer understanding of tumor development and identification of novel therapeutic targets. Recent findings suggest miRNAs play an integral role in breast tumor development, posing as important post-transcriptional regulators of cell growth, differentiation, proliferation, migration, and apoptosis. Specifically, the miR-200 family which is down regulated in TNBC, has demonstrated a significant role in the regulation of the epithelial to mesenchymal transition (EMT), cellular proliferation, migration, stem/progenitor cell characteristics, and immune regulatory molecules. However, the mechanisms by which the miR200 family regulates TNBC tumorigenesis and metastasis remain poorly understood. Therefore, this thesis aimed to uncover the functional characteristics of the miR-200 family clusters through miRNA re-expression studies to identify potential mechanisms by which they regulate the highly aggressive TNBC phenotype. This work utilized miRNA re-expression studies and confirmed that the miR-200 miRNA family, specifically the miR-200c/141 cluster can suppress mammary tumor initiation and metastasis of human TNBC breast cancer. Additionally, our studies identified SUZ12 and subsequent downregulation of MXRA8 as potential mechanisms by which the miR-200s regulate these processes. Further, our MXRA8 knockout resulted in delayed mammary tumor initiation, reduced growth, and inhibited metastasis of human TNBC. As the first studies to identify and characterize a functional role of MXRA8 in breast cancer, this thesis serves as a foundation for future inquiries into the role and mechanism of MXRA8 in breast cancer, and its potential relationship with the miR-200s, and SUZ12-related genes.

Despite extensive clinical testing, mesenchymal stem/stromal cell (MSC)-based therapies continue to underperform with respect to efficacy, which reflects the paucity of biomarkers that predict potency prior to patient administration. Previously, we reported that TWIST1 predicts inter-donor differences in MSC quality attributes that confer potency. To define the full spectrum of TWIST1 activity in MSCs, the present work employed integrated omics-based profiling to identify a high confidence set of TWIST1 targets, which mapped to cellular processes related to ECM structure/organization, skeletal and circulatory system development, interferon gamma signaling, and inflammation. These targets are implicated in contributing to both stem/progenitor and paracrine activities of MSCs indicating these processes are linked mechanistically in a TWIST1 dependent manner. Targets implicated in extracellular matrix dynamics further implicate TWIST1 in modulating cellular responses to niche remodeling. Novel TWIST1 regulated genes identified herein may be prioritized for future mechanistic and functional studies.

Achieving metabolic homeostasis is necessary for survival, and many genes are required to control organismal metabolism. A genetic screen in Drosophila larvae identified putative fat storage genes including Arc1. Arc1 has been shown to act in neurons to regulate larval lipid storage; however, whether Arc1 functions to regulate adult metabolism is unknown. Arc1 esm18 males store more fat than controls while both groups eat similar amounts. Arc1 esm18 flies express more brummer lipase and less of the glycolytic enzyme triose phosphate isomerase, which may contribute to excess fat observed in these mutants. These results suggest that Arc1 regulates adult Drosophila lipid homeostasis.

Sustained inflammation after a traumatic spinal cord injury (TSCI) triggers oxidative stress and neuronal apoptosis, hindering functional recovery. Ezetimibe (EZE) has been reported to have anti-inflammatory and antioxidative properties in hepatology-related diseases, but its potential role in SCI remains unclear. In this study, we evaluated the therapeutic effect of EZE on inflammatory microglia and in an SCI model and elucidated the underlying mechanism. First, we stimulated the BV2 microglia cell line with LPS, and we also induced moderate spinal cord injuries in adult male C57BL/6 mice. Both the cells and mice were treated with EZE, and we investigated inflammation, oxidative stress, neurologic damage, and motor function in vitro and in vivo, respectively. Our findings demonstrated that EZE administration attenuates inflammation in microglia by regulating the AMPK/Nrf2 axis. Furthermore, EZE treatment reduced inflammation and oxidative stress levels in the injured spinal cord. Additionally, treatment with EZE decreased glial scarring and improved motor function recovery, indicating the protective role of EZE in SCI. EZE was found to have anti-inflammatory and antioxidative effects on SCI, and it modulated the AMPK/Nrf2 pathway in microglia. Moreover, EZE prevented histological destruction of the spinal cord tissue. In conclusion, EZE shows promise as a drug to protect neurologic integrity following post-SCI.

Background Sleep disturbances and fatigue are common in individuals undergoing inpatient rehabilitation following stroke. Understanding the relationships between sleep, fatigue, motor performance, and key biomarkers of inflammation and neuroplasticity could provide valuable insight into stroke recovery, possibly leading to personalized rehabilitation strategies. This study aimed to investigate the influence of sleep quality on stroke recovery by utilizing wearable technology to obtain objective sleep measurements. Additionally, we explored the relationships between sleep, fatigue, and motor performance. Lastly, the study aimed to determine if salivary biomarkers of stress, inflammation, and neuroplasticity were associated with fatigue or motor function post-stroke. Methods Eighteen individuals who experienced a stroke and were undergoing inpatient rehabilitation participated in a cross-sectional observational study. Following consent, participants completed questionnaires to assess sleep patterns, fatigue, and quality of life. Objective sleep was measured throughout one night using the wearable Philips Actiwatch. Upper limb motor performance was assessed on the following day and saliva was collected for biomarker analysis. Correlation analyses were performed to assess the relationships between variables. Results Participants reported poor sleep quality, frequent awakenings, and difficulties falling asleep following stroke. We identified a significant negative relationship between fatigue severity and both sleep quality (r=-0.539, p = 0.021) and participants experience of awakening from sleep (r=-0.656, p = 0.003). A significant positive relationship was found between grip strength on the non-hemiplegic limb and salivary gene expression of Brain-derived Neurotrophic Factor (r = 0.606, p = 0.028), as well as a significant negative relationship between grip strength on the hemiplegic side and salivary gene expression of C-reactive Protein (r=-0.556, p = 0.048). Additionally, there was a positive relationship between gene expression of Interleukin-1beta and stroke severity (r = 0.78, p = 0.003) as well as between days since stroke and gene expression of C-reactive Protein (r = 0.615, p = 0.025). Conclusion The findings of this study emphasize the importance of considering sleep quality, fatigue, and biomarkers in stroke rehabilitation to optimize recovery and that interventions may need to be tailored to the individual. Future longitudinal studies are required to explore these relationships over time. Integrating wearable technology for sleep monitoring and biomarker analysis can enhance monitoring and prediction of outcomes following stroke, ultimately improving rehabilitation strategies and patient outcomes.

Genetic variation is a fundamental resource for evolution, but its timing of origin and availability for selection are not completely understood. I examined evolutionary and developmental mechanisms that influence the availability of genetic variation in Arctic charr (Salvelinus alpinus). My first research focus was to determine if large-scale chromosomal rearrangements (CR) influence genetic and morphological divergence among different populations. I characterized the diversity of CR in Canadian and Icelandic populations from two glacial lineages and inferred when this variation arose relative to the post-glacial evolutionary history of the species. I detected several types of polymorphic CR including Robertsonian fusions/fissions through comparisons of sex-specific genetic linkage maps. The observation that CR variation is largely shared across populations and between sympatric morphs suggests that standing (historical) genetic variation in CR plays a limited role in genetic divergence and contemporary patterns of phenotypic divergence in Arctic charr. My second research focus was to determine if developmental processes have an effect on the genetic architecture of important morphological traits underlying adaptive divergence within populations. Here, the goal was to determine if the genetic architecture of morphological traits related to resource use change over ontogeny (age) and between environments (dietary specialization). To address this goal, I conducted quantitative trait locus (QTL) analysis on morphological traits in families from sympatric morphs originating from two Icelandic lakes at two ages and reared on two ecologically relevant diet treatments. I found that age and diet influence the values of morphological traits and that QTL change over age and across diet. This suggests that developmental processes greatly influence the genetic variation of morphological traits available for selection. I also assessed whether the genetic architecture of morphological traits depends on allometric relationships between body shape and size. The observation that the retention or minimization of the allometric component of shape variation has little effect on genetic architecture suggests that there is limited genetic variation for allometry, thus reducing variation available for selection. Overall, my thesis illustrates that the availability of genetic variation for evolutionary change is influenced by evolutionary history and developmentally mediated effects on genetic architecture.

Regulatory T (Treg) cells and cancer-associated fibroblasts (CAFs) jointly promote tumor immune tolerance and tumorigenesis. The molecular apparatus that drives Treg cell and CAF coordination in the tumor microenvironment (TME) remains elusive. Interleukin 33 (IL-33) has been shown to enhance fibrosis and IL1RL1+ Treg cell accumulation during tumorigenesis and tissue repair. We demonstrated that IL1RL1 signaling in Treg cells greatly dampened the antitumor activity of both IL-33 and PD-1 blockade. Whole tumor single-cell RNA sequencing (scRNA-seq) analysis and blockade experiments revealed that the amphiregulin (AREG)–epidermal growth factor receptor (EGFR) axis mediated cross-talk between IL1RL1+ Treg cells and CAFs. We further demonstrated that the AREG/EGFR axis enables Treg cells to promote a profibrotic and immunosuppressive functional state of CAFs. Moreover, AREG mAbs and IL-33 concertedly inhibited tumor growth. Our study reveals a previously unidentified AREG/EGFR-mediated Treg/CAF coupling that controls the bifurcation of fibroblast functional states and is a critical barrier for cancer immunotherapy.

Congenital cytomegalovirus (cCMV) is the most common perinatal infection, the leading cause of nongenetic sensorineural hearing loss, and one of the leading causes of neurodevelopmental impairment in the developed world. Early identification via newborn screening (NBS) would benefit the many undiagnosed infants who are either asymptomatic or mildly to moderately symptomatic, of whom 20% develop sequelae. The sensitivity of a recently developed PCR-based method to detect CMV in dried blood spots (DBS) is less than 80% and requires significantly more specimen than any other NBS test. We sought to improve the analytical sensitivity of the screening method by using droplet digital PCR and direct PCR and decreasing the amount of specimen utilized. The methods were tested with CMV-spiked filters, DBS from CMV-spiked cord blood, and DBS from neonates with cCMV. The results showed that the analytical sensitivity of all modified methods was equivalent to that of the reference method, with consistent CMV detection at high viral loads and inconsistent detection at low viral loads.

Genetic variation is a fundamental resource for evolution, but its timing of origin and availability for selection are not completely understood. I examined evolutionary and developmental mechanisms that influence the availability of genetic variation in Arctic charr (Salvelinus alpinus). My first research focus was to determine if large-scale chromosomal rearrangements (CR) influence genetic and morphological divergence among different populations. I characterized the diversity of CR in Canadian and Icelandic populations from two glacial lineages and inferred when this variation arose relative to the post-glacial evolutionary history of the species. I detected several types of polymorphic CR including Robertsonian fusions/fissions through comparisons of sex-specific genetic linkage maps. The observation that CR variation is largely shared across populations and between sympatric morphs suggests that standing (historical) genetic variation in CR plays a limited role in genetic divergence and contemporary patterns of phenotypic divergence in Arctic charr. My second research focus was to determine if developmental processes have an effect on the genetic architecture of important morphological traits underlying adaptive divergence within populations. Here, the goal was to determine if the genetic architecture of morphological traits related to resource use change over ontogeny (age) and between environments (dietary specialization). To address this goal, I conducted quantitative trait locus (QTL) analysis on morphological traits in families from sympatric morphs originating from two Icelandic lakes at two ages and reared on two ecologically relevant diet treatments. I found that age and diet influence the values of morphological traits and that QTL change over age and across diet. This suggests that developmental processes greatly influence the genetic variation of morphological traits available for selection. I also assessed whether the genetic architecture of morphological traits depends on allometric relationships between body shape and size. The observation that the retention or minimization of the allometric component of shape variation has little effect on genetic architecture suggests that there is limited genetic variation for allometry, thus reducing variation available for selection. Overall, my thesis illustrates that the availability of genetic variation for evolutionary change is influenced by evolutionary history and developmentally mediated effects on genetic architecture.

Species within the dinoflagellate genus Dinophysis can produce okadiac acid and dinophysistoxins leading to diarrhetic shellfish poisoning. Since the first report of D. ovum from the Gulf of Mexico in 2008, reports of other Dinophysis species across US have increased. Members of the D. cf. acuminata complex (D. acuminata, D. acuta, D. ovum, D. sacculus) are difficult to differentiate due to their morphological similarities. Dinophysis feeds on and steals the chloroplasts from the ciliate, Mesodinium rubrum, which in turn has fed on and captured the chloroplasts of its prey, the cryptophyte Teleaulax amphioxeia. The objective of this study was to generate de novo transcriptomes for new isolates of these mixotrophic organisms. The transcriptomes obtained will serve as a reference for future experiments to assess the effect of different abiotic and biotic conditions and will also provide a useful resource for screening potential marker genes to differentiate among the closely related species within the D. cf. acuminata-complex. The complete comprehensive detailed workflow and links to obtain the transcriptome data are provided.