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 …

This dissertation aimed to explore the S2 region with an attempt to modulate its elasticity in order to tune the contraction output. Two peptides, the stabilizer and destabilizer, showed high potential in modifying the S2 region at the cellular level, thus they were prepared for animal model testing. In this research, (i) S2 elasticity was studied, and the stabilizer and destabilizer peptides were built to tune contraction output through modulating S2 flexibility; (ii) the peptides were attached to heart homing adducts and the bond between them was confirmed; and (iii) it was shown that minor changes were imposed on the modulating peptides' functionality upon attaching to the heart homing adducts. S2 flexibility was confirmed through comparing it to other parts of myosin using simulated force spectroscopy. Modulatory peptides were built and computationally tested for their efficacy through interaction energy measurement, simulated force spectroscopy and molecular dynamics; these were attached to heart homing adducts for heart delivery. Interaction energy tests determined that tannic acid (TA) served well for this purpose. The stoichiometry of the bond between the TA and the modulating peptides was confirmed using mass spectroscopy. The functionality of the modulating peptides was shown to be unaltered through expansion microscopy …

Fusarium head blight (FHB) is an important disease of small grain cereals including wheat that affects grain quality and yield. The fungus Fusarium graminearum (Fg) is the major agent of this disease. Lack of natural resistance has limited ability to control wheat losses to this disease. Developing new approaches is critical for increasing host plant resistance to this fungus. This work has identified four processes that can be targeted for enhancing host plant resistance to FHB. The first involves targeting the pattern-triggered immunity mechanism to promote host plant resistance. Two other approaches involved reducing activity of susceptibility factors in the host to enhance plant resistance. The susceptibility factors targeted include accumulation of the phytohormone jasmonic acid and the 9-lipoxygenase pathway that oxidizes fatty acids. Besides suppressing host defenses against Fg, jasmonic acid also directly acts on the fungus to promote fungal growth. 9- lipoxygenases similarly suppress host defenses to promote fungal pathogenicity. Another approach that was developed involved having the plant express double stranded RNA to target fungal virulence genes for silencing. This host-induced gene silencing approach was employed to target two fungal virulence genes, the lipase encoding FGL1 and salicylate hydroxylase encoding FgNahG, which the fungus secretes into the …

This study shows that Arabidopsis thaliana WRKY45 gene has an important role in limiting green peach aphid (GPA; Myzus persicae Sülzer) infestation. WRKY45 belongs to the WRKY family of transcription factors, which is one of the largest transcription factor family in plants. In response to GPA infestation, expression of WRKY45 was systemically upregulated in leaves and roots, with highest expression in the vascular tissues, which are the site of aphid feeding. GPA colonization was better on the wrky45 mutant compared to the wild-type (WT) plant. In contrast, GPA poorly colonized plants that were overexpressing (OE) WRKY45, thus confirming an important role for WRKY45 in plant defense to the GPA. A WRKY45-dependent process adversely impacted the reproductive rate of GPA and feeding from the sieve elements. RNA-seq experiments indicated a major impact of WRKY45 overexpression on expression of genes associated with dehydration and abscisic acid biosynthesis and signaling. In agreement with the RNA-seq data, ABA content was also higher in WRKY45-OE plants. However, genetic studies with an ABA-insensitive mutant (abi2-2) indicates that the WRKY45-OE conferred resistance to GPA is mediated through an ABA-independent mechanism. WRKY45-OE plants showed enhanced tolerance to drought and salt stresses. Genetic studies indicate that ABA signaling is …

Microtubules (MT) are regulated by multiple categories of proteins, including proteins responsible for severing MTs that are therefore called MT-severing proteins. Studies of katanin, spastin, and fidgetin in animal systems have clarified that these proteins are MT-severing. However, studies in plants have been limited to katanin p60, and little is known about spastin or fidgetin and their function in plants. I looked at plant genomes to identify MT-severing protein homologues to clarify which severing proteins exist in plants. I obtained data from a variety of eukaryotic species to look for MT-severing proteins using homology to human proteins and analyzed these protein sequences to obtain information on the evolution of MT-severing proteins in different species. I focused this analysis on MT-severing proteins in the maize and Arabidopsis thaliana genomes. I created evolutionary phylogenetic trees for katanin-p60, katanin-p80, spastin, and fidgetin using sequences from animal, plant, and fungal genomes. I focused on Arabidopsis spastin and worked to understand its functionality by identifying protein interaction partners. The yeast two-hybrid technique was used to screen an Arabidopsis cDNA library to identify putative spastin interactors. I sought to confirm the putative protein interactions by using molecular tools for protein localization such as the YFP system. …

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 …

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 early signaling mechanism(s) that control oxidant perception and signal transduction leading to activation of the antioxidant defense response and survival mechanisms tailored toward specific oxidative insult remains unknown. Here, we identified early changes in metabolome and proteome of S. cerevisiae in response to hydrogen peroxide, menadione, cumene hydroperoxide, and diamide. Firstly, global untargeted LC–MS/MS analysis allowed us to identify 196 proteins in response to hydrogen peroxide, 569 proteins in response to cumene hydroperoxide, 369 proteins in response to menadione and 207 proteins in response to diamide that were significantly regulated at 3 min after exposure. We revealed that each oxidant triggered unique signaling mechanisms associated with survival and repair mechanisms as early as 3 minutes of post treatment with a set of proteins that uniquely responded to the particular oxidant. In addition, our comprehensive pathway analysis revealed signaling pathways and the molecular players that are regulated globally by all oxidants at early time points namely Ran, mTOR, Rho, and eIF2. Additionally, we analyzed metabolic response using targeted GC-MS/MS that allowed us to identity 35 metabolites that were consistently detected in all samples at 3 min of exposure. These metabolites showed distinct response to the four oxidants in carbohydrate metabolism, …