Role of Light, Humidity, and Nutrition in Sporulation of Phytophthora Species

Oomycetes, a class in the kingdom Stramenopila, are filamentous eukaryotic microorganisms that are known informally as water molds, but have adapted to many environmental niches. Phytophthora, an oomycete genus of approximately 100 plant-pathogenic species, has specifically adapted to several environmental niches. Environmental conditions play an influential role in the development of Phytophthora, particularly in sporangium formation. Sporangia, the main driver of Phytophthora epidemics, are especially susceptible to damage by environmental conditions, such as light-induced DNA lesions or desiccation by drying. Variable by species, light can stimulate (e.g. P. infestans) or repress (e.g. P. capsici) the production of sporangia. Light may have two developmental roles: an early pre-sporangiophore induction signal, and a later post-sporangiophore repression signal. In this work, I found that although many types of photoreceptors are described in eukaryotes, Phytophthora genomes encode only cryptochrome photoreceptors. The foliar-adapted species P. infestans encodes three cryptochromes, while the soilborne species P. capsici encodes two cryptochromes. Silencing of the P. infestans gene encoding cryptochrome PITG_01718, which lacks an ortholog in P. capsici, inhibits sporulation. While cryptochromes have traditionally been described in other organisms as blue light receptors, each red and blue light represses P. infestans sporulation. In contrast, P. capsici sporulation responds to blue light but not red light. This work also describes the use of RNA-seq to study the genetic control of sporulation by light and humidity. Constant light was found to repress the expression of genes induced in the post-sporangiophore stage by P. infestans, while constant darkness repressed the expression of genes in the pre-sporangiophore stage in P. capsici. Humidity also had a strong effect on repressing gene expression in the pre-sporangiophore stage of sporulation in P. infestans. This work also describes that specific types of nitrogen starvation induce sporulation. In addition to advancing our knowledge of sporulation control in oomycetes, this work also provides a better understanding of the homology-based gene silencing method. While this technique is an effective method for functional genomics in Phytophthora, work in this dissertation shows that the technique must be used with prudence due to the frequency of off-target effects

Identifying Pathogens of Switchgrass and Investigating Antimicrobial Activity of Switchgrass-Derived Extractives

Switchgrass is an increasingly important biofuel crop, but knowledge of switchgrass fungal pathogens is not extensive. The purpose of this research was to identify the fungal pathogens that decrease crop yield of switchgrass grown in Tennessee and to investigate a potential value-added by-product of the switchgrass biofuel conversion process. The specific objectives were 1) to identify and characterize prevalent fungal pathogens of switchgrass in Tennessee, 2) assess switchgrass seed produced in the United States for seedborne fungal pathogens, and 3) evaluate switchgrass extractives for antimicrobial activity against plant pathogens. Diseased switchgrass samples were collected from several locations in East Tennessee. Tissue was surface-sterilized and plated on potato dextrose agar (PDA), or water agar. Emergent fungi were isolated and identified based on colony, physiological, and molecular characteristics. Pathogenicity was confirmed with Koch\u27s postulates in growth chamber studies. Nine pathogenic species were identified, several of which had not been reported on switchgrass previously. To assess presence of seedborne pathogens, seed of seven cultivars from 11 commercial seed production companies located in the United States were tested, including multiple entries of ‘Alamo’, ‘Blackwell’, ‘Cave-in-Rock’, and ‘Kanlow’. Three hundred surface-sterilized seed per lot were plated on PDA. Rates of fungal infection among the 30 sampled seed lots varied from less than 1 % to 87%. The most frequently observed pathogens were Bipolaris oryzae, Alternaria alternata, and Fusarium graminearum/ pseudograminearum. Additional species of Bipolaris and Fusarium were present with less frequency. To test the switchgrass extractives for antimicrobial activity, six ethanol-soluble extractives treatments, extracted from switchgrass collected from three farms at two different plant ages, were tested in a 3 × 2 factorial design for activity against four bacterial plant pathogens. Significant differences were shown for the main effect of bacteria (P=0.09) and the interaction effect of farm × plant age (P=0.04). Xanthomonas perforans was more sensitive to the extractives treatments than Pseudomonas syringae pv. tomato and extractives from 112 day-old switchgrass from farm C04 inhibited bacterial growth more than the extractives from 112 day-old switchgrass from farms C19 and C33

Toward precision medicine with nanopore technology

Currently, when patients are diagnosed with cancer, they often receive a treatment based on the type and stage of the tumor. However, different patients may respond to the same treatment differently, due to the variation in their genomic alteration profile. Thus, it is essential to understand the effect of genomic alterations on cancer drug efficiency and engineer devices to monitor these changes for therapeutic response prediction. Nanopore-based detection technology features devices containing a nanometer-scale pore embedded in a thin membrane that can be utilized for DNA sequencing, biosensing, and detection of biological or chemical modifications on single molecules. Overall, this project aims to evaluate the capability of the biological nanopore, alpha-hemolysin, as a biosensor for genetic and epigenetic biomarkers of cancer. Specifically, we utilized the nanopore to (1) study the effect of point mutations on C-kit1 G-quadruplex formation and its response to CX-5461 cancer drug; (2) evaluate the nanopore\u27s ability to detect cytosine methylation in label-dependent and label-independent manners; and (3) detect circulating-tumor DNA collected from lung cancer patients\u27 plasma for disease detection and treatment response monitoring. Compared to conventional techniques, nanopore assays offer increased flexibility and much shorter processing time

Synergistic effects of nucleating agents and plasticizers on the crystallization behavior of Poly(lactic acid)

The synergistic effect of nucleating agents and plasticizers on the thermal and mechanical performance of PLA nanocomposites was investigated with the objective of increasing the crystallinity and balancing the stiffness and toughness of PLA mechanical properties. Calcium carbonate, halloysite nanotubes, talc and LAK (sulfates) were compared with each other as heterogeneous nucleating agents. Both the DSC isothermal and non-isothermal studies indicated that talc and LAK were the more effective nucleating agents among the selected fillers. Poly(D-lactic acid) (PDLA) acted also as a nucleating agent due to the formation of the PLA stereocomplex. The half crystallization time was reduced by the addition of talc to about 2 min from 37.5 min of pure PLA by the isothermal crystallization study. The dynamic mechanical thermal study (DMTA) indicated that nanofillers acted as both reinforcement fillers and nucleating agents in relation to the higher storage modulus. The plasticized PLA studied by DMTA indicated a decreasing glass transition temperature with the increasing of the PEG content. The addition of nanofiller increased the Young's modulus. PEG had the plasticization effect of increasing the break deformation, while sharply decreasing the stiffness and strength of PLA. The synergistic effect of nanofillers and plasticizer achieved the balance between stiffness and toughness with well-controlled crystallization

Improving Low-Resource Question Answering using Active Learning in Multiple Stages

Neural approaches have become very popular in the domain of Question Answering, however they require a large amount of annotated data. Furthermore, they often yield very good performance but only in the domain they were trained on. In this work we propose a novel approach that combines data augmentation via question-answer generation with Active Learning to improve performance in low resource settings, where the target domains are diverse in terms of difficulty and similarity to the source domain. We also investigate Active Learning for question answering in different stages, overall reducing the annotation effort of humans. For this purpose, we consider target domains in realistic settings, with an extremely low amount of annotated samples but with many unlabeled documents, which we assume can be obtained with little effort. Additionally, we assume sufficient amount of labeled data from the source domain is available. We perform extensive experiments to find the best setup for incorporating domain experts. Our findings show that our novel approach, where humans are incorporated as early as possible in the process, boosts performance in the low-resource, domain-specific setting, allowing for low-labeling-effort question answering systems in new, specialized domains. They further demonstrate how human annotation affects the performance of QA depending on the stage it is performed.Comment: 16 pages, 8 figure

Cellulose Acetate Blends – Effect of Plasticizers on Properties and Biodegradability

Cellulose acetate (CDA) cannot be processed as raw material because it starts to decompose before melting. Triacetin and diacetin were tested to improve CDA processing versus conventional phthalate as environmentally sustainable plasticizers, because of their low toxicity and fast biodegradability. The addition of triacetin and diacetin allowed melt processing of CDA and the results of tensile tests outlined their effect as plasticizers. The values of mechanical properties were compatible with the requirements for applications in rigid packaging. From the results of biodegradation tests it can be concluded that for pure cellulose acetate, complete biodegradation was obtained within 200 days of testing after reinoculation. Incomplete biodegradation was observed for test items with 20% triacetin or with 30% phthalate. After 46 days of incubation, the test samples with 30% plasticizer based on triacetin or triacetin-diacetin were completely biodegraded. These formulations can be selected for the production of compostable blends and/or biocomposites

Non-zinc mediated inhibition of carbonic anhydrases: coumarins are a new class of suicide inhibitors.

The X-ray crystal structure of the adduct between the zinc metalloenzyme carbonic anhydrase II (CA, EC 4.2.1.1) with the recently discovered natural product coumarin derivative 6-(1S-hydroxy-3-methylbutyl)-7-methoxy-2H-chromen-2-one showed the coumarin hydrolysis product, a cis-2-hydroxy-cinnamic acid derivative, and not the parent coumarin, bound within the enzyme active site. The bound inhibitor exhibits an extended, two-arm conformation that effectively plugs the entrance to the enzyme active site with no interactions with the catalytically crucial zinc ion. The inhibitor is sandwiched between Phe131, with which it makes an edge-to-face stacking, and Asn67/Glu238sym, with which it makes several polar and hydrogen bonding interactions. This unusual binding mode, with no interactions between the inhibitor molecule and the active site metal ion is previously unobserved for this enzyme class and presents a new opportunity for future drug design campaigns to target a mode of inhibition that differs substantially from classical inhibitors such as the clinically used sulfonamides and sulfamates. Several structurally simple coumarin scaffolds were also shown to inhibit all 13 catalytically active mammalian CA isoforms, with inhibition constants ranging from nanomolar to millimolar. The inhibition is time dependent, with maximum inhibition being observed after 6 h.No Full Tex