Source-separating urine for nutrient recovery may provide multiple benefits with regards to wastewater management, water conservation, and an impending phosphorus fertilizer shortage. Municipal wastewater systems are designed to treat the combination of urine, feces and graywater produced in household applications. Urine accounts for 1% of wastewater by volume, but provides 70-90% of nitrogen, 35-70% of phosphorus and 50% of the contaminants of emerging concern entering municipal wastewater treatment (Larsen and Gujer 1996). Research has shown managing source-separated urine for nutrient recovery is a more cost effective and less treatment intensive method than using traditional systems found in municipal wastewater plants. Phosphorus fertilizer shortages are projected as current sources diminish and become increasingly difficult to extract and refine. Phosphorus based-fertilizer recovery, in the form of 99.9% pure struvite (MgNH4PO4•6H2O), has been demonstrated successfully in full-scale sidestream treatment using dewatering liquor from anaerobically digested solids (centrate) processed through upflow fluidized bed reactor technologies (Britton et al. 2005). Prior research determined the influence of pH, magnesium to phosphorus (Mg:P) molar ratio, and age of urine on purity, pharmaceutical content and pathogen inclusion in struvite precipitated from source-separated urine. This is the first known example of an attempt to produce a commercially viable struvite product from source-separated urine in a fluidized bed reactor of a design that has been used successfully for struvite recovery in conventional wastewater applications. In order to assess the feasibility of nutrient recovery of phosphorus-based fertilizer recovery from source-separated urine, the first office-based urine separation and collection building was implemented in the U.S. Urine was collected, in a 400 gallon capacity underground sealed manhole, from HRSD's Main office building beginning in March 2015 from 5 men's waterless urinals and one women's separating toilet. Urine was collected from the manhole on a monthly basis in 275 and 330 gallon plastic totes stored at the HRSD Nansemond WWTP in Suffolk, VA. Collected urine was allowed to age while in storage to encourage the precipitation of excess multivalent cations that may interfere with struvite precipitation and inactivation of pathogens that may be present. An upflow fluidized bed reactor (UFBR) was used to recover struvite as a slow-release phosphorus based fertilizer (prill), the reactor was loaned to HRSD by the University of British Columbia. A magnesium solution was injected at the bottom of the reactor to facilitate precipitation along with the recycle urine stream and feed urine as shown. Prill production design for the reactor was 0.5 kilograms per day, but while using centrate to determine best operations practices, under loading the reactor to 0.25 kilograms per day maximized struvite recovery while minimizing particulate phosphorus loss. Urine was fed into the reactor for struvite removal based on phosphorus loading with recovery determined through removal of orthophosphate and harvesting of the struvite product. Consistency, size and quality of product including compactness, crystal structure, purity and presence of pharmaceuticals and pathogens were assessed. The UFBR was run for 50 days total; 10 days for a short term run to compare to operation of the reactor under the same conditions with centrate from anaerobically digested solids as a feed source, 30 days to assess consistency of operations over long term with respect to struvite recovery, and a 10 day test with urine spiked with pharmaceuticals and bacteriophage to evaluate inclusion of trace organics and viruses in recovered struvite. In total 2,040 gallons of urine were fed to the reactor targeting 12.45 kilograms of struvite recovery, a mass of 7.54 kilograms of prills were harvested from the reactor with 1.90 kilograms of phosphorus lost as particulate struvite (representing an recovery efficiency of 60.5%). Overall reactor operation using urine as a feed solution behaved similar to centrate, with slightly less removal of phosphorus. Urine-derived prills were lower in quality due to the lack of compact density seen in struvite recovered during full scale operation but had a visible orthombic pattern seen in precipitated struvite. Pharmaceuticals that were present in urine feed solution were found in struvite but at less than 1% of the feed mass. Some of this inclusion may have occurred due to porous characteristics of the small-scale UFBR recovered struvite rather than through actual inclusion in the mineral crystal itself. Spiking of caffeine and ibuprofen to high concentrations in the urine yielded no statistical difference from the non-spiked tote. Urine was non-detect for bacteriophage pathogen indicators leading to the assumption that no pathogens were present in urine-derived struvite. Spiking the urine with double-stranded DNA (T3) and single-stranded RNA (MS2) bacteriophage capable of infecting bacterial cells such as Escherichia coli yielded 10^6 plaque forming units per milliliter in source separated urine. Creating urine-derived struvite prills with minimal inclusion of pharmaceuticals using upflow fluidized bed technology is feasible on a small scale. Large-scale application, recovering 500 kilograms per day of struvite or more, will most likely create a higher quality prill with regards to compactness and diminished presence of pharmaceuticals and virus inclusion. Pretreatment of urine and post-treatment of prills with heat will aid in inactivation of virus that may be present. ' / Master of Science

Mitogen-activated protein kinases (MAPKs) are a family of highly conserved serine/threonine protein kinases which link upstream receptors to their downstream targets. These targets can be localized in the cytoplasm or the nucleus. Pathogens produce pathogen-associated molecular patterns (PAMPs) that are known to trigger the activation of MAPK cascades. In plants, MAPK signaling cascades regulate development and cellular processes such as stress responses, immunity, and apoptosis by means of the phosphorylation of specific targets. Phosphoproteomics analysis of PAMP-induced Arabidopsis plants led to the identification of several putative MAPK targets. USP1 (Universal Stress Protein A) (At1g11360) and WLIM2A (At2g39900) are two potential phosphorylation targets of MAPKs, and are the focus of this thesis. So far, little is known about their role in plant immunity. CRISPR-Cas9 generated knockout usp1 mutant lines enhanced resistance to infection by Pst DC3000, usp1 mutant showed a reduced level of apoplast reactive oxygen species accumulation and upregulation of defense marker genes such as WRKY29 and FRK1. Transcriptome analyses revealed that immune hormone signaling genes such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are differentially regulated. These hormones are responsible for primary defense responses against biotrophic and necrotrophic pathogens. Although the physiological role of USP1 has been established, the biochemical and molecular functions are unknown. We discovered a new role for USP1, demonstrating that it functions as a molecular chaperone and is involved in thermal priming. Overall, these data show that phosphoprotein USP1 plays an important role in orchestrating plant immunity. CRISPR-Cas9 generated knockout wlim2a mutant showed susceptibility to infection by Pst DC3000. wlim2a mutants showed a reduced level of apoplast reactive oxygen species accumulation and upregulation of defense marker genes such as WRKY29 and FRK1. Transcriptome analyses revealed that immune hormone signaling genes such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are differentially regulated in wlim2a mutants. These hormones are responsible for primary defense responses against biotrophic and necrotrophic pathogens. wlim2a mutants show enhanced fungal infection by Botrytis cinerea. Overall, the data shows that WLIM2A phosphorylation is important during plant immunity.

<p>Over the last two decades, mycoflorostic studies undertaken in Guyanese forests have uncovered hundreds of new fungal species and genera. One of the recovered fungal families was the Xylariaceae, although most were not identified to species. Members of this family play ecological roles as decomposers, endophytes, and pathogens of vascular plants and grass species. In addition, this group is increasingly recognized as a significant source of novel metabolic products with potential for applications in medicine, agriculture, and industrial biofuel. Given its potential, we took a more targeted approach to the documentation of this group. Our sampling efforts drawing on more than two decades of field collections yielded ca. 90 species in 12 genera, including a putative pathogen, <em>Xylaria karyophthora</em> of the seeds of <em>Chlorocardium</em> spp (Greenheart). Despite the significance of Greenheart to the Guyanese economy, little is known about the biology and ecology of this fungus. Due to the lack of available resources to study this fungus, our objectives were two-fold: first, to sequence and annotate the genome of <em>X. karyophthora</em> to provide a resource for genome-centric explorations, and to use this genome to infer the biology and ecology of this fungus. We focused on identifying and characterizing secretomes, viz. carbohydrate-active enzymes (CAZymes) and secondary metabolites biosynthetic gene clusters (SMBGCs) to infer the nutritional strategy of this fungus. Our results suggest that <em>X. karyophthora</em> has the capacity to act as both an endophyte and a pathogen. To make further inferences about the population, we used SSR markers to elucidate the genetic diversity and population structure of <em>X. karyophthora</em>. <em>X. karyophthora</em> populations have high genetic diversity, potentially exploiting both outcrossing and inbreeding reproductive strategies, and demonstrate a pattern consistent with human-mediated spread. This work will contribute information on new species and records of Xylariaceous fungi and their allies from Guyana with particular emphasis on unraveling the epidemiology, genetic diversity, and population structure of <em>X. karyophthora.</em></p>

<p dir="ltr">The Cdc14 phosphatase family is highly conserved in fungi. In Saccharomyces cerevisiae, Cdc14 is essential for down-regulation of cyclin-dependent kinase activity at mitotic exit. However, this essential function is not broadly conserved and requires only a small fraction of normal Cdc14 activity. In general, few conserved functions of Cdc14 phosphatase have been defined. Here, I present mechanistic biochemical and phenotypic characterization of Cdc14 phosphatases in fungi. I have demonstrated that fungal Cdc14 phosphatases possess an invariant motif in the disordered C-terminal tail that is required for full enzymatic activity. This motif, termed substrate-like catalytic enhancer (SLiCE), functions during the rate-limiting step of Cdc14-directed catalysis, by binding to the active site and supporting phospho-enzyme hydrolysis. Adjacent to the SLiCE motif exists a conserved minimal Cdk consensus motif that likely serves a regulatory function as phosphorylation of this site inhibits Cdc14 activity in vitro. Vertebrate Cdc14 enzymes also possess a distinct, but mechanistically similar SLiCE motif, which may be the first described biochemical difference between Cdc14 enzymes. Moreover, the vertebrate SLiCE motif lacks an adjacent Cdk consensus motif, which may point to differences in how Cdc14 activity is regulated in higher eukaryotes.</p><p dir="ltr">Mutation of this motif in vivo served as a tool to discover biological processes that require high Cdc14 activity. In S. cerevisiae strains expressing this hypomorphic mutant allele (cdc14hm), I discovered a novel sensitivity to cell wall stresses, including chitin-binding compounds and echinocandin antifungal drugs. This sensitivity was also observed in the distantly related fungi Schizosaccharomyces pombe deletion strain and the human fungal pathogen Candida albicans hypomorphic and deletion strains, suggesting that this phenotype reflects a conserved function of Cdc14 orthologs in mediating fungal cell wall integrity. I also revealed that high Cdc14 activity is required for C. albicans ability to develop hyphae, which is an important virulence trait. This led to our determination that high Cdc14 activity is critical for virulence in two animal models of invasive candidiasis. Together, these results argue that Cdc14 would be an excellent antifungal drug target for the treatment of invasive Candida infections and sensitization to existing antifungal drugs.</p><p dir="ltr">Lastly, I implemented the auxin-inducible degradation system in C. albicans. Using this system, we were able to deplete Cdc14 and other target protein levels to >95% within minutes. Depletion of Cdc14 was robust enough to phenocopy gene deletions, confirming previous results and demonstrating the utility of rapid target protein inactivation. This system will serve as a powerful tool for future functional characterization of Cdc14 in C. albicans and other pathogenic fungal species.</p>

Emerging wildlife diseases represent a serious threat to conservation efforts. Impacts of emerging multi-host pathogens can vary greatly among species as well as geographically, and understanding which populations will be at greatest risk is essential for conserving biodiversity. Snake fungal disease (SFD), caused by the fungal pathogen Ophidiomyces ophidiicola, is responsible for lethal infections in snakes and has contributed to the decline of multiple North American snake populations. However, which species are most affected by this disease and how infections vary regionally remains unknown. Here we sampled 44 different species across 14 sites throughout the Southeastern and Mid-Atlantic United States. We found a strong effect of latitude on both pathogen prevalence and severity, with more severe infections at more northern latitudes. We also found high variability in pathogen prevalence and infection severity among species. There was a strong positive relationship between pathogen prevalence and disease severity, suggesting that SFD is not just highly prevalent in some populations but also highly virulent. More broadly, our results support that SFD likely has continued impacts on snake populations with some species experiencing greater disease than others attributed to spatial and host variation. / Master of Science / Conserving biodiversity is a significant challenge. Wildlife species are under multiple threats including habitat loss, changing climate, species introductions, pollution, and infectious diseases. Emerging wildlife diseases can pose a major problem for wildlife as they often go undetected until they cause substantial declines for the affected species, sometimes leading to population extirpations and extinction events. Snake fungal disease (SFD) is an emerging disease caused by the fungal pathogen Ophidiomyces ophidiicola, which has contributed to the decline of some North American snake populations. However, little is known about differences in infection, transmission, and host responses to SFD in a broader community context. To investigate the dynamics of this pathogen, we collected swab samples from 44 species from a total of 14 sites in New Jersey, Virginia, North Carolina, South Carolina, Georgia, Florida, and Louisiana. We sampled individual snakes to examine variation over a geographic gradient and among species. We found high variability among sites with more severe disease at northern sites. There was also high variability among species and some populations experienced both high pathogen prevalence and disease severity. Our results show that SFD is highly variable within snake communities and may still be causing population level effects.

Salmonella enterica is a facultative intracellular pathogen capable of eliciting severe, systemic disease necessitating antibiotic intervention. Systemic infection is facilitated by intracellular replication within host immune cells, which is enabled by complex regulatory networks governed by two-component systems (TCSs). Intracellular-active TCSs sense antimicrobial chemical cues in the microenvironment and respond adaptively through transcriptional regulation to support intracellular survival. SsrA/SsrB and PhoQ/PhoP are two essential TCSs that elicit a robust defense against host immunity by regulating clusters of virulence genes and integrating novel targets to support regulon expansion and enhance pathogenicity. Metabolic adaptation is critical to bacterial survival and can initiate host-pathogen interactions that influence infection outcome. Further, mitigation of host immunity by manipulation of arginine metabolism has been documented in intracellular pathogens. Herein, I investigated TCS-mediated regulatory evolution pertaining to arginine metabolism, hypothesizing that adaptations to metabolic regulation might confer a fitness advantage to Salmonella replicating intracellularly. I explored intracellular regulation of de novo biosynthesis and extracellular import of arginine, establishing PhoP-mediated regulation of arginine transport. I determined that arginine transport contributes to bacterial fitness in macrophages and began to investigate the mechanism by which arginine importation enriches for intracellular replication. This work informs on evolutionary mechanisms that serve to enhance virulence in Salmonella and provides further insight into our understanding of the intracellular lifestyle of infection. / Thesis / Master of Science (MSc) / Salmonella enterica is an intestinal pathogen that survives within host immune cells and causes systemic disease. These bacteria replicate within antimicrobial cells by using sensory networks to detect harmful immune factors and respond adaptively by eliciting change in gene expression to defend against immune-based killing. The amino acid arginine is an important component of host immunity, as well as bacterial antimicrobial defenses; therefore, I hypothesized that bacterial metabolism might be adapted to the host immune cell environment in order to mitigate arginine-dependent antimicrobial activity. Here, I establish that arginine metabolism is controlled by intracellular-specific sensory networks, and demonstrate that this regulation is important for bacterial survival. This work provides evidence for the importance of this amino acid in Salmonella infection, which informs on our overall understanding of systemic disease.

Catfish, the "king" of the U.S. aquaculture, is threatened by a severe, systemic bacterial disease known as enteric septicemia of catfish (ESC). This disease causes high mortality and massive economic losses in cultured channel catfish (Ictalurus punctatus) in the United States. E. ictaluri penetrates catfish intestinal epithelia quickly and establishes a systemic infection rapidly. However, our knowledge on catfish intestine and E. ictaluri interaction is very limited. In Particular, catfish intestinal immune responses and virulence genes needed by E. ictaluri to evade host defenses are not well understood. Hence, our long-term goal is to identify the molecular mechanisms of E. ictaluri-host interactions. The overall objectives of this study were to understand catfish immune responses to E. ictaluri infection and determine essential genes of E. ictaluri during the intestinal invasion. To accomplish the overall objectives of this research, intestinal ligated loops were constructed surgically in live catfish and loops were injected with wild-type E. ictaluri and two live attenuated E. ictaluri vaccine strains developed recently by our research group. We first determined catfish intestinal immune responses against E. ictaluri wild-type and live attenuated vaccine strains. Then, we analyzed the global gene expression patterns of wild-type E. ictaluri and vaccine strains during catfish intestinal invasion using high throughput RNA-Seq technology. Results showed a moderate level of neutrophil and B cell infiltration correlated with significantly lower expression of TNF-α, CD4-1, and CD8-α in the vaccine injected intestinal tissue compared to that of wild-type injected intestinal tissue. Further, RNA-Seq data analysis showed the prominent expression of genes related to bacterial secretion systems, ATP production processes, and multidrug resistance (MDR) efflux pumps in wild-type E. ictaluri. In contrast, the prominently expressed genes in vaccine strains were related to the phosphotransferase system and sugar metabolism processes. All these data suggest that our live attenuated vaccines are capable of triggering effective immune responses in catfish without causing damage to the host.

Aspergillus flavus exemplifies a pathogenic fungus that remains a significant contributor to the loss of corn (Zea mays) crops. The production of carcinogenic aflatoxins renders the crop hazardous for consumption and causes significant loss to farmers. Therefore, the prevention of A. flavus contamination continues to persist as a topic for research intervention. Host resistance to this pathogen provides a promising source of defense for the corn plant. Corn inbred line Mp313E was previously identified to exhibit significant resistance against the A. flavus fungal infection and aflatoxin contamination. Quantitative trait loci (QTL) mapping has previously established four major QTL locations associated with aflatoxin resistance in the corn inbred line Mp313E. Near-isogenic lines were developed containing these previously identified QTLs from backcrossing of inbred lines Mp313E (resistant donor parent) and Va35 (susceptible recurrent parent). Quantitative RT-PCR (qRT-PCR) was used to study gene expression patterns of 17 genes selected from plant-pathogen interaction pathways. Furthermore, genomic primer analysis was used for establishment of 15 candidate genes for marker- assisted breeding.

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